Textbook of diagnostic microbiology [7 ed.] 9780323829977

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Textbook of Diagnostic Microbiology

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Textbook of Diagnostic Microbiology SEV ENTH EDI TI ON

Edited by

Connie R. Mahon, MS, MT(ASCP) Director, Organization Development (Retired) Health Resources and Services Administration Learning Institute Rockville, Maryland; Adjunct Assistant Professor Biomedical Laboratory Sciences Department The George Washington University Ashburn, Virginia

Donald C. Lehman, EdD, MLS(ASCP)CM, SM(NRCM) Professor Medical and Molecular Sciences Health Profession Advisor Center for Health Profession Studies University of Delaware Newark, Delaware

Elsevier 3251 Riverport Lane St. Louis, MO 63043 TEXTBOOK OF DIAGNOSTIC MICROBIOLOGY, SEVENTH EDITION

ISBN: 978-0-323-82997-7

Copyright © 2023 by Elsevier Inc. All rights reserved The contribution made by Kalavati Suvarna and Sumathi Nambiar is in public domain. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notice Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds or experiments described herein. Because of rapid advances in the medical sciences, in particular, independent verication of diagnoses and drug dosages should be made. To the fullest extent of the law, no responsibility is assumed by Elsevier, authors, editors or contributors for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein.

Previous editions copyrighted 2019, 2015, 2011, 2007, 2000, and 1995.

Content Strategist: Heather Bays-Petrovic/Kelly Skelton Senior Content Development Manager: Lisa Newton Senior Content Development Specialist: Danielle M. Frazier Publishing Services Manager: Catherine Jackson Specialist: Kristine Feeherty Design Direction: Maggie Reid Printed in India Last digit is the print number:

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To my husband Dan, for his love and continued support and understanding; my son Sean, who inspires me; my daughter Kathleen, for showing me courage; and my granddaughters Kelly Amelia, Natalie Page, and Madeline Belle, who have given us so much pleasure. CRM

To my wife Terri, for her constant support and encouragement, and whose love makes me realize anything is possible, and my grandchildren Shane, Athena, Jordan, and Vincent, of whom I am so proud. DCL

To George Manuselis, a dedicated microbiologist, educator, and mentor, who inspired all.

Reviewers S. Travis Altheide, PhD, MLS(ASCP)CM Assistant Professor, Medical Laboratory Science Eastern Kentucky University Richmond, Kentucky

Michele G. Harms, MS, MLS(ASCP) Program Director, Medical Laboratory Science Program UPMC Chautauqua Jamestown, New York

Mary T. Emes, MMedSc (Pathology), Certicate in Adult Education, MLT, ART (CC, MI, TS) Professor, Medical Laboratory Science The Michener Institute of Education at University Health Network Toronto, Ontario, Canada

Daniel J. Harrigan, MS, MB(ASCP) Chair, Laboratory Sciences Medical Laboratory Technology Program Blackhawk Technical College Monroe, Wisconsin

Shawn Froelich, MS, MLS(ASCP)CM Associate Professor, Medical Laboratory Science Allen College—UnityPoint Health Waterloo, Iowa

Guylaine Michaud, MLT, Ed. cert. Professor, Program Coordinator Collège Communautaire du Nouveau-Brunswick Dieppe, Nouveau-Brunswick, Canada

Julie Gardner, MS, MBA, MLS(ASCP)CMSMCM Director of Medical Laboratory Technician Program Assistant Professor of Biology University of Saint Francis Crown Point, Indiana

Karla K. Sampselle, MS, MT(ASCP)BB Program Director, Instructor Medical Laboratory Technician Program Northeast Wisconsin Technical College Green Bay, Wisconsin

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Contributors Yousif Barzani, MD, MA ed & HD, MLS (ASCP)CM Assistant Professor and Program Director School of Medicine and Health Sciences The George Washington University Washington, DC Connie F. Cañete-Gibas, PhD Clinical Research Project Manager Fungus Testing Lab, Department of Pathology University of Texas Health Science Center at San Antonio San Antonio, Texas Nina M. Clark, MD Professor Department of Internal Medicine Division of Infectious Diseases Co-Director Infectious Disease & Immunology Research Institute Loyola University Stritch School of Medicine Maywood, Illinois James L. Cook, MD Clinical Professor Division of Infectious Diseases Medicine Loyola University Chicago Maywood, Illinois; Staff Physician and Research Scientist Medicine Edward Hines, Jr. VA Hospital Hines, Illinois Jed M. Doxtater, MS, MLS(ASCP)CM Associate Professor and Program Director Medical Laboratory Sciences University of Wyoming Casper, Wyoming Brandon C. Ellis, MBA, MLS(ASCP)CM Laboratory Manager Department of Pathology Division of Medical Microbiology Johns Hopkins Hospital Baltimore, Maryland Tori Enomoto, BS, M(ASCP) Microbiologist Microbiology and Infectious Diseases Diagnostic Laboratory Services, Inc. Aiea, Hawaii CM

Nancy Gouin, MPH, MT(ASCP) Adjunct Instructor Biomedical Laboratory Sciences Department George Washington University Washington, DC

Steven D. Mahlen, PhD, D(ABMM) Clinical Microbiologist Microbiology Sanford Bismarck Bismarck, North Dakota

Amanda T. Harrington, PhD, D(ABMM) Professor Pathology and Laboratory Medicine Loyola University Chicago Maywood, Illinois

Connie R. Mahon, MS, MT(ASCP) Director, Organization Development (Retired) Health Resources and Services Administration Learning Institute Rockville, Maryland; Adjunct Assistant Professor School of Medicine and Health Sciences The George Washington University Ashburn, Virginia

Michelle M. Jackson, PhD Senior Microbiologist Division of Nonprescription Drugs II U.S. Food and Drug Administration, Center for Drug Evaluation and Research Silver Spring, Maryland Deborah A. Josko, PhD, MLT(ASCP)M,SM Associate Professor and Director - Medical Laboratory Science Program Clinical Laboratory and Medical Imaging Sciences Rutgers, The State University of New Jersey School of Health Professions Newark, New Jersey Olga Kochar, MS, CSSGB Division Director Laboratory and Transfusion Services The George Washington University Hospital; Adjunct Instructor School of Medicine and Health Sciences The George Washington University Washington, DC Donald C. Lehman, EdD, MLS(ASCP)CM, SM(NRCM) Professor Medical & Molecular Sciences Health Profession Advisor Center for Health Profession Studies University of Delaware Newark, Delaware Denene Loand, PhD, FACSc, MT(ASCP) Associate Professor Thread Director, Microbiology and Immunology Department of Microbiology and Immunology Drexel University College of Medicine Reading, Pennsylvania

Kevin M. McNabb, MBA, PhD Director of Microbiology and Immunology Pathology and Area Laboratory Services New Hanover Regional Medical Center; Director of Microbiology Microbiology Wilmington Pathology Associates Wilmington, North Carolina Alfredo J. Mena Lora, MD Assistant Professor Department of Medicine University of Illinois at Chicago Chicago, Illinois Paula C. Mister, MS, MLS, (ASCP)CM Educational Coordinator Clinical Microbiology Johns Hopkins Hospital; Adjunct Instructor Biology Community Colleges of Baltimore County Baltimore, Maryland Sumathi Nambiar, MD, MPH Senior Director Child Health Innovation and Leadership Department Johnson & Johnson Raritan, New Jersey David H. Nielsen, MS, BSAS, AAS PHSS Plant Protection and Quarantine U.S. Department of Agriculture Lincoln, Nebraska; Medical Entomologist AR-MEDCOM Army Topeka, Kansas

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Contributors

Lindsey E. Nielsen, PhD, D(ABMM) Director, Clinical Services Clinical Laboratory LN Laboratory Consulting Wood River, Nebraska Susan M. Pacheco, MD Physician Department of Medicine Division of Infectious Diseases Edward Hines, Jr. VA Hospital Hines, Illinois; Associate Professor Department of Medicine Division of Infectious Diseases Loyola University Medical Center Maywood, Illinois Vijay Parashar, PhD Assistant Professor Medical and Molecular Sciences University of Delaware Newark, Delaware Gail Reid, MD, MS-CTS Associate Professor Medicine Loyola University Medical Center Maywood, Illinois; Section Chief Division of Infectious Diseases Edward Hines, Jr. VA Hospital Hines, Illinois Lauren Roberts, BS, MS, MLS(ASCP) Microbiology Supervisor (Retired) Microbiology Laboratory St. Joseph’s Hospital & Medical Center Phoenix, Arizona; Associate Clinical Professor (Retired) School of Life Sciences, CLS Program Arizona State University Tempe, Arizona; Adjunct Faculty Medical Laboratory Sciences, Public Health, and Nutrition Science Tarleton State University Fort Worth, Texas Kalavati Suvarna, PhD Microbiologist Center for Drug Evaluation and Research US. Food and Drug Administration Silver Spring, Maryland Marie Ciacco Tsivitis, MT(ASCP), MPH, CIC, FAPIC Research Scientist/Regional Representative Bureau of Healthcare Associated Infections New York State Department of Health Central Islip, New York; Clinical Assistant Professor Clinical Laboratory Sciences Stony Brook University Stony Brook, New York

A. Christian Whelen, PhD Vice President and Technical Director Microbiology and Molecular Laboratories Diagnostic Laboratory Services Inc. and The Queen’s Health Systems Aiea, Hawaii; Adjunct Professor Pathology and Public Health Afliate Graduate Faculty Microbiology University of Hawaii Honolulu, Hawaii Nathan P. Wiederhold, PharmD Professor Pathology University of Texas Health Science Center at San Antonio San Antonio, Texas Christopher J. Woolverton, BS, MS, PhD Professor Epidemiology Kent State University Kent, Ohio; Faculty National Biosafety and Biosecurity Training Program National Institutes of Health Bethesda, Maryland; Faculty Medical Technology Training Program Akron Children’s Hospital Akron, Ohio Amy M. Woron, MS, MPH, PhD Director Department of Microbiology and Molecular Diagnostic Laboratory Services, Inc. Aiea, Hawaii; Adjunct Professor College of Health and Society Hawaii Pacic University Honolulu, Hawaii; Adjunct Instructor National Center for Biomedical Research and Training/ACE Louisiana State University Baton Rouge, Louisiana

Previous Edition Contributors Máximo O. Brito, MD, MPH Associate Professor of Medicine Division of Infectious Diseases University of Illinois; Chief of Infectious Diseases Department of Medicine Jesse Brown VA Medical Center Chicago, Illinois Robert C. Fader, PhD, D(ABMM) Section Chief (Retired) Microbiology at Baylor Scott and White Health Temple, Texas

George Manuselis, MA, MT(ASCP) Emeritus (Retired) Medical Technology Division The Ohio State University Columbus, Ohio Fred Marsik, PhD Team Leader, Clinical Microbiology (Retired) Division of Antiinfective and Ophthalmology Products Center for Drug Evaluation and Research U.S. Food and Drug Administration Silver Spring, Maryland Linda S. Monson, MS, MT(ASCP) Supervisory Microbiologist (Retired) Brooke Army Medical Center Fort Sam Houston, Texas

Test Bank Writer Lorna Ruskin, EdD, MT(ASCP) Associate Professor Medical Laboratory Sciences Center for Allied Health Programs University of Minnesota Minneapolis, Minnesota

PowerPoint Writer Elizabeth A. Gockel-Blessing, PhD, MLS(ASCP)CM Associate Dean for Student and Academic Affairs Associate Professor of Medical Laboratory Science Department of Clinical Health Sciences Doisy College of Health Sciences Saint Louis University St. Louis, Missouri

Laboratory Manual Writer Paula Denise Silver, PharmD, MEd, BS Medical Instructor ECPI University School of Health Science Newport News, Virginia

Case Studies and Review Questions Writer Joanna R. Ellis, MS, MLS(ASCP), CHWI Clinical Associate Professor and Clinical Coordinator Study Abroad Academic Program Director and Service-Learning Fellow Clinical Laboratory Science Program Texas State University San Marcos, Texas

Preface While we were preparing the seventh edition of the Textbook of Diagnostic Microbiology, a never-before-seen virus was reported in China in December 2019 and spread quickly around the world, reaching Europe and the United States in January 2020. This novel virus was identied as the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the cause of coronavirus disease 2019. This disease emphasizes how quickly infectious agents can spread and the signicance of the work of microbiologists and other health care professionals. Newly recognized pathogens continue to emerge while previously known pathogens evolve to plague society. The eld of diagnostic microbiology continues to change dramatically and becomes more complex. Hence, we look ahead to the future needs of the next generation of medical laboratory practitioners. Methods for the detection and identication of infectious agents continue to evolve, and more laboratories are replacing traditional methods (e.g., biochemical identication of bacteria and cell cultures for viruses) with molecular biology assays. Therefore we continue to update and expand this topic in Chapter 11, where we discuss matrix-assisted laser desorption/ionization–time-of-ight mass spectrometry and nucleic acid–based identication methods that include probes, amplication assays, and gene sequencing. This also includes nanomedicine in diagnosing infectious diseases. Bacteria continue to become more drug resistant, so science must keep pace by rapidly determining a bacterium’s susceptibility pattern and nding better ways to treat life-threatening infections. In Chapter 12, we discuss common means by which bacteria develop resistance to agents and present the most used and cutting-edge antimicrobial agents. We follow this in Chapter 13 by examining the best methods to assay for drug sensitivity and resistance. With the advent of these emerging molecular technology innovations, scientic discoveries, and gene-based techniques, medical microbiology education in medical laboratory science programs has also undergone dramatic changes. In these highly specialized and rapidly advancing areas of science and biotechnology, we have taken the lead to constantly look for innovative ways to effectively deliver the textbook contents in ways that they are current, appropriate, absorbable, and applicable. As in previous editions, the seventh edition maintains the characteristic features of a well-designed and organized textbook. We maintain the building-block approach to learning, critical thinking, and problem solving, attributes that students of medical laboratory science and medical laboratory technology, entry-level clinical laboratory scientists, and others have found valuable and effective. The primary goal of the Textbook of Diagnostic Microbiology is to provide a strong foundation for medical laboratory science students, entry-level practitioners, and other health care professionals; therefore discussions on organisms are limited to those that are medically

important and commonly encountered, as well as new and re-emerging pathogens. A feature that sets our textbook apart is the large number of quality full-color photographs and photomicrographs. The text also provides students and other readers with valuable learning tools, such as summary tables, owcharts, and descriptive illustrations, to help them comprehend the vast amount of information and reinforce learning. In response to our readers’ needs, we continued our efforts to enhance these features that have made this textbook user-friendly. All our contributing authors are experts in the eld of microbiology, either practicing laboratory scientists, infectious disease specialists, researchers, or educators. Their expertise helps us to have current and accurate information.

Organization Part I remains the backbone of the textbook, providing important background information; Part II focuses on laboratory identication of etiologic agents organized taxonomically; and Part III on the organ system approach—the clinical and laboratory diagnoses of infectious diseases at various body sites. Part 1 presents basic principles and concepts of diagnostic microbiology, including quality assurance, providing students with a rm theoretic foundation. Chapters 7 (Microscopic Examination of Materials From Infected Sites) and 8 (Use of Colony Morphology for the Presumptive Identication of Microorganisms) still play a vital role in this text. These two chapters help students and practitioners who may have difculty recognizing bacterial morphology on direct smear preparations and colony morphology on primary culture plates develop these skills with the use of color photomicrographs of stained direct smears and cultures from clinical samples. These two chapters also illustrate how microscopic and colony morphology of organisms can aid in the initial identication of the bacterial isolate. Chapter 9 introduces the student/reader to the principles behind various biochemical methods for identication of gram-negative bacteria. This chapter contains several color photographs to help our readers understand the principles and visualize interpretations of these important tests. Part 2 highlights methods for the identication of clinically signicant isolates that include bacteria, fungi, parasites, and viruses. Although diseases caused by the infectious agents are discussed, the emphasis is on the characteristics and methods used to isolate and identify each group of pathogens. Numerous tables summarize the major features of organisms and use schematic diagrams to show the relationships and differences among similar or closely related species. Chapters devoted to anaerobic bacterial species, medically important fungi, parasites, and viruses afrm the ix

x

Preface

signicance of these agents. Chapter 29 includes a discussion on viral pathogens, including newly discovered Zika virus, SARS-CoV-2, and avian inuenza virus. Chapter 31 describes biolm—an increasingly complex entity. It has become evident that microbial biolms are involved in the pathogenesis of several human diseases and may be a contributing factor of antimicrobial therapy failure. The organ system approach in Part 3 has been the foundation of the Textbook of Diagnostic Microbiology and provides an opportunity for students and other readers to “pull things together.” The chapters in Part 3 begin with the anatomic considerations of the organ system to be discussed and the role of the usual microbiota found at a particular site in the pathogenesis of a disease. It is important for students to be knowledgeable about the usual inhabitants at a body site before they can appreciate the signicance of the infectious agents they are most likely to encounter. These chapters also discuss proper specimen collection and processing.

Pedagogic features As in previous editions, the “Case in Point” case study found in all chapters introduces the reader to an important pathogen, infectious disease, concept, or principle that is discussed in the chapter text and is used to lead the learner to the main context discussed in the chapter. The Case in Point is followed by “Issues to Consider.” These points are presented in a bulleted format, and learners are asked to think about them as they read the chapter. We use case studies to enhance problem-solving and critical-thinking skills. The case studies describe clinical and laboratory ndings, providing students with opportunities to correlate these observations with possible etiologic agents. In most cases, the cause of the illness is not disclosed in the case study; rather, it is presented elsewhere in the chapter to give students the opportunity to independently determine the explanations. In Part 3, the case studies also help students apply the knowledge they acquired from Parts 1 and 2. “Case Check” boxes aim to reinforce understanding of the content or concept within the context of the Case in Point at the beginning of the chapter or case study at the beginning of a section within the chapter. The Case Check highlights a

specic point in the text and intends to help the learner connect the dots between the points under discussion, as illustrated by the case study. To further reinforce learning, identication tables, owcharts, and featured illustrations have been updated, and new ones have been added. Learning objectives and a list of key terms are also provided at the beginning of each chapter. The list of key terms includes abbreviations used in the text so that students can easily nd them. At the end of each chapter, readers will nd “Points to Remember” and “Learning Assessment Questions,” which help reinforce comprehension and understanding of important concepts. Points to Remember includes a bulleted list of important concepts and highlights that the reader should have learned from the chapter. We also include answers to all the learning assessment questions found in each chapter.

Ancillaries for instructors and students As in the case of previous editions, we continue to offer a variety of instructor ancillaries specically geared for this book. For instructors, the Evolve website includes a test bank containing more than 1200 questions. It also includes an electronic image collection and PowerPoint slides. For students, the Evolve website will include a laboratory manual like it always has, which contains new case studies and student review questions. It also includes appendices, which provide information on laboratory media commonly used for the cultivation of bacteria and fungi as well as detailed protocols for selected clinical microbiology laboratory tests.

Acknowledgments We are grateful to all contributing authors, students, instructors, and many other individuals, who have all made invaluable suggestions and comments on ways to improve this edition. Connie R. Mahon Donald C. Lehman

Contents Part 1: Introduction to clinical microbiology 1. Bacterial cell structure, physiology, metabolism, and genetics 2 Connie R. Mahon

2. Host-parasite interaction 25 Donald C. Lehman

3. The laboratory role in infection control 50 Marie Ciacco Tsivitis and Connie R. Mahon

4. Control of microorganisms: disinfection, sterilization, and microbiology safety 65 Michelle M. Jackson

5. Performance improvement in the microbiology laboratory 102 Connie R. Mahon and Olga Kochar

6. Specimen collection and processing 123 Lauren Roberts

7. Microscopic examination of materials from infected sites 139 Connie R. Mahon

8. Use of colony morphology for the presumptive identication of microorganisms 169 Connie R. Mahon

9. Biochemical identication of gram-negative bacteria 183 Donald C. Lehman

10. Immunodiagnosis of infectious diseases 199 Donald C. Lehman

11. Applications of molecular diagnostics 227 Steven D. Mahlen, Vijay Parashar, and Donald C. Lehman

12. Antibacterial mechanisms of action and bacterial resistance mechanisms 250 Brandon C. Ellis and Donald C. Lehman

13. Antimicrobial susceptibility testing 271 Paula C. Mister and Donald C. Lehman

16. Aerobic gram-positive bacilli 347 Steven D. Mahlen and Amanda T. Harrington

17. Neisseria species and Moraxella catarrhalis 371 Lauren Roberts

18. Haemophilus, HACEK group, Legionella, and other fastidious gram-negative bacilli 390 Tori Enomoto and A. Christian Whelen

19. Enterobacterales 417 Denene Loand, Connie R. Mahon, and Donald C. Lehman

20. Vibrio, Aeromonas, Campylobacter, and Campylobacter-like species 455 Deborah A. Josko

21. Nonfermenting and miscellaneous gram-negative bacilli 474 Yousif Barzani

22. Anaerobes of clinical importance 496 Nancy Gouin

23. The spirochetes 533 Amy M. Woron and A. Christian Whelen

24. Chlamydia, Rickettsia, and similar organisms 543 Donald C. Lehman

25. Mycoplasma and Ureaplasma 558 Donald C. Lehman and Connie R. Mahon

26. Mycobacterium tuberculosis and nontuberculous mycobacteria 570 Donald C. Lehman

27. Medically signicant fungi 597 Connie F. Cañete-Gibas and Nathan P. Wiederhold

28. Diagnostic parasitology 639 Lauren Roberts and Connie R. Mahon

29. Clinical virology 708 Kevin M. McNabb and Vijay Parashar

30. Agents of bioterror and forensic microbiology 753 Christopher J. Woolverton and Donald C. Lehman

31. Biolms: architects of disease 776 Donald C. Lehman

Part 2: Laboratory identication of signicant isolates 14. Staphylococcus and similar organisms 308 Lindsey E. Nielsen and Jed M. Doxtater

15. Streptococcus, Enterococcus, and other catalase-negative, gram-positive cocci 324 Kalavati Suvarna and Connie R. Mahon

Part 3: Laboratory diagnosis of infectious diseases: an organ system approach to diagnostic microbiology 32. Upper and lower respiratory tract infections 791 Susan M. Pacheco and James L. Cook

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Contents

xii

33. Skin and soft tissue infections 832 Nina M. Clark

34. Gastrointestinal infections and food poisoning 869 Alfredo J. Mena Lora and Connie R. Mahon

40. Zoonotic diseases 984 David H. Nielsen and Lindsey E. Nielsen

41. Ocular infections 1001 Gail Reid

35. Infections of the central nervous system 889 Sumathi Nambiar and Kalavati Suvarna

36. Bacteremia and sepsis 904 Paula C. Mister and Donald C. Lehman

37. Urinary tract infections 922 Lindsey E. Nielsen

38. Genital infections and sexually transmitted infections 942 Denene Loand

39. Infections in special populations 974

Appendix A Answers to learning assessment questions 1023 Appendix B Selected bacteriologic culture media* 1044.e1 Appendix C Selected mycology culture media and stains* 1044.e19 Appendix D Selected procedures* 1044.e23 Glossary 1045 Index 1074

Paula C. Mister and Donald C. Lehman

*Available online on the Evolve Resources site (http://evolve.elsevier.com/ Mahon/microbiology/).

PA R T

1

Introduction to clinical microbiology

1

1 Bacterial cell structure, physiology, metabolism, and genetics Connie R. Mahon

CHAPTER OUTLINE

Overview of the microbial world, 4 Bacteria, 4 Parasites, 4 Fungi, 4 Viruses, 6 Classification/Taxonomy, 6 Nomenclature, 6 Classication by phenotypic and genotypic characteristics, 7 Classication by cellular type: prokaryotes, eukaryotes, and archaea, 7 Comparison of prokaryotic and eukaryotic cell structure, 7 Prokaryotic cell structure, 7 Eukaryotic cell structure, 10 Cytoplasmic structures, 7 Bacterial morphology,11 Microscopic shapes,11 Common stains used for microscopic visualization, 11 Microbial growth and nutrition, 13 Nutritional requirements for growth, 14 Environmental factors inuencing growth, 15 Bacterial growth, 15 Bacterial biochemistry and metabolism, 16 Metabolism, 16 Fermentation and respiration, 16 Biochemical pathways from glucose to pyruvic acid, 17 Anaerobic utilization of pyruvic acid (Fermentation), 17 Aerobic utilization of pyruvate (Oxidation), 18 Carbohydrate utilization and lactose fermentation, 19 Microbial genetics, 19 Anatomy of a DNA and RNA molecule, 19 Terminology, 21 Genetic elements and alterations, 21 Mechanisms of gene transfer, 22 Bibliography, 24

2

OBJECTIVES

After reading and studying this chapter, you should be able to: 1. Differentiate among archaeal, prokaryotic (bacterial), and eukaryotic cell types. 2. Describe microbial classication (taxonomy). 3. Accurately apply the rules of scientic nomenclature for bacterial names. 4. List and dene ve methods used by epidemiologists to subdivide bacterial species. 5. Contrast prokaryotic and eukaryotic cytoplasmic and cell wall structures and functions. 6. Compare the cell walls of gram-positive and gram-negative bacteria. 7. Explain why acid-fast bacteria do not stain well with the Gram stain. 8. Justify the use of the following stains in the diagnostic microbiology laboratory: Gram stain, acid-fast stains (Ziehl-Neelsen, Kinyoun, auramine-rhodamine), acridine orange, methylene blue, calcouor white, lactophenol cotton blue, and India ink. 9. Describe the nutritional requirements for bacterial growth, and dene the categories of media used for culturing bacteria in the laboratory. 10. Dene the atmospheric requirements of obligate aerobes, microaerophiles, facultative anaerobes, obligate anaerobes, aerotolerant anaerobes, and capnophilic bacteria. 11. Differentiate an aerotolerant anaerobe from facultative and obligate anaerobes. 12. Describe the stages in the growth (i.e., growth curve) of bacterial cells. 13. Explain the importance of understanding microbial metabolism in clinical microbiology. 14. Differentiate between fermentation and oxidation (aerobic respiration). 15. Compare the three biochemical pathways that bacteria use to convert glucose to pyruvate. 16. Differentiate the two types of glucose fermentation that explain positive results with the methyl red and Voges-Proskauer tests. 17. Dene the following genetic terms: genotype, phenotype, constitutive, inducible, replication, transcription, translation, genome, chromosome, plasmids, insertion sequence element, transposon, point mutations, frameshift mutations, missense mutations, nonsense mutations, and recombination. 18. Discuss the development and transfer of antimicrobial resistance in bacteria.

Overview of the microbial world

19. Differentiate among the mechanisms of transformation, transduction, and conjugation in the transfer of genetic material from one bacterium to another. 20. Dene the terms bacteriophage, lytic phage, lysogeny, and temperate phage 21. Dene the term restriction endonuclease enzyme and explain how it applies to the clinical microbiology laboratory. KEY TERMS

Acid fast Aerobic respiration (oxidation) Aerotolerant anaerobes Anticodon Archaea Autotroph Bacteria Bacteriophage Capnophilic Capsule Codon Competent Conjugation Differential media Dimorphic Eukarya Eukaryote Facultative anaerobes Family Fermentation Fimbriae Flagella Fusiform Genotype Genus gram-negative gram-positive Halophile Heterotroph Hyphae Krebs cycle Lysogeny Mesophiles

Microaerophilic Minimal medium Mycelia Nomenclature Nutrient media Obligate aerobes Obligate anaerobes Pathogenic bacteria Phenotype Phyla Pili Plasmids Pleomorphic Prokaryote Protein expression Psychrophile Replication Restriction enzymes Selective media Species Spore Strains Taxa Taxonomy Temperate Thermophile Transcription Transduction Transformation Translation Transport medium Virion

Case in point A 4-year-ol female patient presents with symptoms of reness, burning, an light sensitivity in both eyes. She also complains of her eyelis sticking together because of exuative ischarge. A Gram stain of the conjunctival exuates (prouct of acute inammation with white bloo cells an ui) shows gram-positive intracellular an extracellular, faint-staining, coccobacillary bacteria. The organisms appear to have small, clear, nonstaining “halos” surrouning each cell. This clear area is note to be between the staine organism an the amorphous (no enite form; shapeless) backgroun material. The Gram stain of the quality control organisms Staphylococcus aureus (gram-positive) an Escherichia coli (gram-negative) reveals gram-positive reactions for both organisms.

3

Issues to consider After reading the patient’s case history, consider: • Role of microscopic morphology in presumptive ientication • Signicance of observable cellular structures • Importance of quality control in assessing an interpreting irect smear results • Unique characteristics of organisms, such as cellular structures, in initiating infection an isease in hosts

Microbial inhabitants have evolve to survive in various ecologic niches (place or location) an habitats (organism’s location an where its resources may be foun). Some grow rapily, an others grow slowly. Some can replicate with a minimal number of nutrients present, whereas others require complex, enriche nutrients to survive. Variation exists in atmospheric growth conitions an temperature requirements. This iversity exists in microorganisms that inhabit the human boy as normal biota (ora), as opportunistic pathogens, or as true pathogens. Each microbe has its own physiology an metabolic pathways that allow it to survive in its particular habitat. A main role of the clinical microbiologist is to isolate, ientify, an analyze the bacteria that cause isease in humans (pathogens). Recent avances in molecular biology methos, such as nucleic aci amplication, nucleic aci sequencing, an matrix-assiste laser esorption/ionization–time-of-ight (MALDI-TOF) mass spectrometry, have shifte ientication away from traitional biochemical testing for ientication of bacterial isolates. Nevertheless, knowlege of microbial structure an physiology is extremely important to clinical microbiologists in the following areas: • Microscopic characterization of organisms • Recovery of organisms in culture from patient specimens • Characterization an ientication of organisms after they have been isolate • Interpretation of antimicrobial susceptibility patterns The rst step in bacterial ientication is often microscopic characterization—escribing the shape of the organism an its staining characteristics, which are base on the cell wall structure. Unerstaning the growth requirements of a particular bacterium enables the microbiologist to select the appropriate meium for primary culture an optimize the recovery an isolation of the pathogen. This is followe by observation of the metabolic biochemical ifferences among organisms that form the basis for many bacterial ientication systems in use toay. The cell structure an biochemical pathways of an organism also etermine its susceptibility to various antimicrobial agents. The ability of microorganisms to change rapily, acquire new genes, an unergo mutations presents continual challenges to clinical microbiologists as they isolate an characterize microorganisms associate with infectious iseases in humans. This chapter provies a review of the structure, physiology, metabolism, an genetics of prokaryotic an eukaryotic cells. It also gives examples of common stains use to microscopically visualize microorganisms. Each topic in this chapter emphasizes to clinical microbiologists the inherent importance of their efforts to culture, ientify, an characterize the microbes that cause isease in humans.

PART 1

4

1

Bacterial cell structure, physiology, metabolism, and genetics

Overview of the microbial world The stuy of microorganisms by the Dutch biologist an lens maker Anton van Leeuwenhoek has evolve immensely from its early historic beginnings. Because of Leeuwenhoek’s iscovery of what he affectionately calle wee beasties an animalcules in a water roplet with his homemae microscope, the scientic community acknowlege him as the “father of protozoology an bacteriology.” Toay, we know that there are enormous numbers of microbes in, on, an aroun us in our environment. The vast majority of these microbes o not cause isease. This textbook focuses on microbes that are associate with human isease.

Bacteria Bacteria are unicellular organisms that are classie as prokaryotes (Greek: before kernel [nucleus]). They lack a nuclear membrane an a true nucleus, mitochonria, an enoplasmic reticulum (ER), an Golgi boies. The absence of the preceing bacterial cell structures ifferentiates them from eukaryotes (Greek eu: well or goo; Greek karyon: kernel). Table 1.1 compares prokaryotic an eukaryotic cell organization; Fig. 1.1 shows both types of cells.

Parasites Certain eukaryotic parasites (organisms that live at the expense of their hosts) exist as unicellular organisms of microscopic size, whereas others are multicellular organisms. Protozoa are unicellular organisms within the kingom Protista that obtain their nutrition through ingestion. Some are capable of locomotion (motile), whereas others are nonmotile. They are categorize by their locomotive structures: agella (Latin: whiplike), pseuopoia (Greek: false feet), or cilia (Latin: eyelash). Many multicellular parasites can be quite large; for example, tapeworms may be 7 to 10 m long (see Chapter 28).

Fungi Fungi are heterotrophic (cannot prouce all of its nutrients) eukaryotes that obtain nutrients through absorption. Most fungi are multicellular, an many can reprouce sexually an asexually. Multicellular fungi are compose of laments calle hyphae that interweave to form mats calle mycelia. Yeasts are unicellular fungi that reprouce asexually. “True” yeasts o not form hyphae or mycelia. Mols are lamentous forms that can reprouce asexually an sexually. Certain fungi can assume both morphologies (yeast an hyphae/mycelial

Table 1.1 Comparison of prokaryotic and eukaryotic cell organization Characteristic

Prokaryote

Eukaryote

Typical size

0.4–2 µm in diameter

10–100 µm in diameter

0.5–5 µm in length

>10 µm in length

No nuclear membrane; nucleoid region of the cytosol

Membrane-bound nucleus

Nucleus Genome Location

In the nucleoid, at the mesosome

In the nucleus

Chromosomal DNA

Circular; complexed with RNA

Linear; complexed with basic histones and other proteins

Extrachromosomal circular DNA

Plasmids, small circular molecule of DNA in the cytoplasm containing accessory information; most commonly found in gram-negative bacteria; each carries genes for its own replication; can confer resistance to antimicrobial agents

In mitochondria, chloroplasts, and cytoplasm

Reproduction

Asexual (binary ssion)

Sexual and asexual

Membrane-bound organelles

Absent

All

Golgi bodies

Absent in all

Present in some

Lysosomes

Absent in all

Present in some; contain hydrolytic enzymes

Endoplasmic reticulum

Absent in all

Present in all; lipid synthesis, transport

Mitochondria

Absent in all

Present in most

Chloroplasts for photosynthesis

Absent in all

Present in algae and plants

Ribosomes: site of protein synthesis (nonmembranous)

Present in all

Present in all

70 S consisting of 50 S and 30 S subunits

80 S consisting of 60 S and 40 S subunits

In the cell membrane; no mitochondria present

In the inner membrane of mitochondria and chloroplasts

Size Electron transport for energy

Overview of the microbial world

Table 1.1 Comparison of prokaryotic and eukaryotic cell organization—cont’d Characteristic

Prokaryote

Eukaryote

Sterols in cytoplasmic membrane

Absent except in Mycoplasmataceae

Present

Plasma membrane

Phospholipid bilayer; lacks carbohydrates

Phospholipid bilayer; also contains glycolipids and glycoproteins

Cell wall, if present

Peptidoglycan in most bacteria

Cellulose, phenolic polymers, lignin (plants), chitin (fungi), other glycans (algae)

Glycocalyx

Present in many as an organized capsule or unorganized slime layer

Present; some animal cells

Cilia

Absent

Present; see description of agella

Flagella, if present

Simple agella; composed of polymers of agellin; movement by rotary action at the base; spirochetes have MTs

Complex cilia or agella; composed of MTs and polymers of tubulin with dynein connecting MTs; movement by coordinated sliding MTs

Pili and mbriae

Present

Absent

MT, Microtubule.

Division septum Outer membrane Peptidoglycan Mesosome (Capsule) layer

(Pili)

(Capsule) Cytoplasmic membrane

Inclusion body

Inclusion body Peptidoglycan layer

Cytoplasmic membrane Ribosome Ribosome (Flagellum) Surface proteins Chromosome

A

GRAM-POSITIVE

Porin proteins Periplasmic space (Flagellum)

GRAM-NEGATIVE

Centrosome Ribosomes Centrioles

Smooth endoplasmic reticulum

Mitochondria Smooth endoplasmic reticulum Cilia

Mitochondrion Lysosome Rough endoplasmic reticulum Peroxisome

B

Free ribosomes Golgi apparatus Vesicle Nuclear Nucleus Nucleolus envelope

Fig. 1.1 Comparison of prokaryotic and eukaryotic cell organization and structures. A, Prokaryotic gram-positive and gram-negative bacteria. B, Structure of the generalized eukaryotic cell. (A, From Murray, P. R., et al. (2009). Medical microbiology [6th ed]. Philadelphia: Mosby; B, from Thibodeau, G. A., & Patton, K. T. (2007). Anatomy and physiology [6th ed]. St Louis: Mosby.)

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PART 1

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Bacterial cell structure, physiology, metabolism, and genetics

forms), growing as yeast at human temperature (37° C) an as the lamentous form at room temperature (22° C). These fungi are calle dimorphic. Some systemic fungal iseases in human hosts are cause by imorphic fungi (see Chapter 27).

Viruses Viruses are the smallest infectious particles an cannot be seen uner an orinary light microscope. Often, we can see their effects on cell lines grown in the laboratory, such as inclusions, rouning up of cells, an syncytium (fusion of host cells into multinucleate infecte forms), where these characteristics become iagnostic for many viral iseases. These visible changes are calle cytopathic effect. Viruses are acellular (not compose of cells) an are therefore neither prokaryotic nor eukaryotic. They are istinguishe from living cells by the following characteristics: • Viruses consist of eoxyribonucleic aci (DNA) or ribonucleic aci (RNA), but rarely both. Their genome can be ouble-strane DNA (sDNA), single-strane DNA (ssDNA), ouble-strane RNA (sRNA), or single-strane RNA (ssRNA). • Viruses lack cytoplasmic membranes an are surroune by a protein coat. • Viruses are obligate intracellular parasites that cannot self-replicate. They require host cells for replication (increase in number oes not involve mitosis, meiosis, or binary ssion) an metabolism. Because they lack ribosomes an other metabolites, they “take over” host cell function using the host cell machinery to reprouce. Growth (increase in size) oes not occur in viruses. Viruses are mostly host or host cell specic. For example, human immunoeciency virus infects T-helper lymphocytes, not muscle cells, in humans, whereas other viruses, such as the rabies virus, can infect ogs, skunks, bats, humans, an other animals. A virus that infects an possibly estroys bacterial cells is known as a bacteriophage (Greek phage: to eat). Viruses are classie an ientie by their genome (DNA or RNA), host range, host isease signs an symptoms, chemical makeup, geographic istribution, the presence or absence of an envelope, their resistance to changes in pH an temperature, their antigenicity (serologic methos), how the virus replicates, an the virion (a complete virus outsie a cell) size (see Chapter 29).

Classication/Taxonomy Taxonomy (Greek taxes: arrangement; Greek nomos: law) is the orerly classication an grouping of organisms into taxa (categories). Taxonomy involves three structure, interrelate categories: classication/taxonomy, nomenclature, an ientication. It is base on similarities an ifferences in genotype (genetic makeup of an organism, or combinations of forms of one or a few genes in an organism’s genome) an phenotype (observable physical an functional features of an organism expresse by its genotype). Examples of genotypic characteristics inclue base sequencing of DNA or RNA an DNA base composition ratio to measure the egree of relateness

of two organisms (see later in this chapter an Chapter 11). Examples of microbial phenotypic characteristics inclue macroscopic (colony morphology on meia) an microscopic morphology (size, shape, arrangement into groups or chains of cells), staining characteristics (gram-positive or gram-negative), nutritional requirements, physiologic an biochemical characteristics, antigenic markers, an susceptibility or resistance to antimicrobial agents or chemicals. See Chapters 7, 8, 9, 12, an 13 for more etaile information. Taxa (plural of taxon), for example, the levels of classication, are the categories or subsets in taxonomy. The formal levels of bacterial classication in successively smaller taxa or subsets are omain, kingom, ivision (or phylum in kingom Animalia), class, orer, family, tribe, genus, species, an subspecies. Below the subspecies level, esignations such as serotype or biotype may be given to organisms that share specic minor characteristics. Protists (protozoans) of clinical importance are name similarly to animals; instea of ivisions, phyla (plural of phylum) is use, but the names of the other classications remain the same. Prokaryotes are place in the omains Bacteria an Archaea (Greek: ancient, origin from the earliest cells), separate from the animals; plants an protists are place in the omain Eukarya. The omains Bacteria an Archaea inclue unicellular prokaryotic organisms. Clinical microbiologists traitionally emphasize placement an naming of bacterial species into three (occasionally four or ve) categories: the family (similar to a human “clan”), a genus (equivalent to a human last name), an a species (equivalent to a human rst name). The plural of genus is genera. For example, there are many genera in the family Staphyloccocceae. The proper wor for the name of a species is an epithet. For example, Staphylococcus (genus) aureus (species epithet) belongs to the family Staphylococcaceae. Although orer an tribe may be useful for the classication of plants an animals, these taxa are not always use for the classication of bacteria. In aition, there are usually ifferent strains within a given species of the same species. As an example, there are many ifferent strains of S. aureus. If the S. aureus isolate from one patient is resistant to penicillin an another S. aureus isolate from a ifferent patient is susceptible to penicillin, the two isolates belong to ifferent strains of the same species.

Nomenclature Nomenclature provies naming assignments for each organism. The following stanar rules for enoting bacterial names are use. The family name is capitalize an has an “-aceae” ening (e.g., Micrococcaceae). The genus name is capitalize an followe by the species epithet, which begins with a lowercase letter; both the genus an species shoul be italicize in print but unerline when written in script (e.g., Staphylococcus aureus or Staphylococcus aureus). Often the genus name is abbreviate by use of the rst letter (capitalize) of the genus followe by a perio an the species epithet (e.g., S. aureus). The genus name followe by the wor species (e.g., Staphylococcus species) may be use to refer to the genus as a whole. Species are abbreviate “sp.” (singular) or “spp.” (plural) when the species is not specie. When bacteria are referre to as a group, their names are neither capitalize nor unerline (e.g., staphylococci).

Comparison of prokaryotic and eukaryotic cell structure

Classication by phenotypic and genotypic characteristics The traitional metho of placing an organism into a particular genus an species is base on the similarity of all members in numerous phenotypic characteristics. In the iagnostic microbiology laboratory, this classication is accomplishe by testing each bacterial culture for various metabolic or molecular characteristics an comparing the results with those liste in establishe tables or atabases. In many rapi ientication systems, a numeric taxonomy is use in which phenotypic characteristics are assigne a numeric value, an the erive number inicates the genus an species of the bacterium by consulting a atabase of known organisms. Epiemiologists constantly seek means of further subiviing bacterial species to follow the sprea of bacterial infections. Species are subivie into subspecies (abbreviate “subsp.”) on the basis of phenotypic ifferences, serovarieties (abbreviate “serovar”) on the basis of serologic (antigenic) ifferences, or biovarieties (abbreviate “biovar”) on the basis of biochemical test result ifferences. Phage typing (base on susceptibility to specic bacterial phages) is also use for this purpose. Current technology allows the analysis of genetic relateness (DNA an RNA structure an homology) for taxonomic purposes. The analysis of ribosomal RNA (rRNA) gene sequencing is particularly useful for this purpose. The information obtaine from these stuies resulte in the reclassication of some bacteria.

Classication by cellular type: prokaryotes, eukaryotes, and archaea Base on cell organization an function, organisms fall into one of three istinct groups: prokaryotes, eukaryotes, or archaea. Taxonomists place all organisms into three omains that replace some kingoms: Bacteria, Archaea, an Eukarya. These three omains are the largest an most inclusive taxa. Each omain is ivie into kingoms on the basis of the similarities of RNA, DNA, an protein sequences. The group prokaryotes inclues the omains Archaea an Bacteria (Eubacteria), whereas fungi, algae, protozoa, animals, an plants are eukaryotic in nature an are place in the omain Eukarya. The omain Archaea (formerly Archaeobacteria) cell type appears to be more closely relate to eukaryotic cells than to prokaryotic cells an is foun in microorganisms that grow uner extreme environmental conitions. Archaeal cell walls lack peptioglycan, a major reason they are place in a omain separate from bacteria. These microbes share some common characteristics with bacteria; they too can stain gram-positive or gram-negative. Gram-positive archaea have a thick wall an stain purple. Gram-negative archaeal cells, in contrast with the typical gram-negative bacterial lipi membrane, have a layer of protein covering the cell wall an stain pink. See the “Gram Stain” section later in this chapter. The structure of the cell envelope an enzymes of archaea allows them to survive uner stressful or extreme (extremophiles; lovers of the extreme) conitions. Examples inclue

7

halophiles (salt-loving cells) in Utah’s Great Salt Lake, thermophiles (heat-loving cells) in hot springs an eep ocean vents, an the anaerobic methanogens that release swamp gas an inhabit the intestinal tracts of animals. Because archaea are not encountere in clinical microbiology, they are not iscusse further in this textbook. In general, the interior organization of eukaryotic cells is more complex than that of prokaryotic cells (see Fig. 1.1). The eukaryotic cell is usually larger an contains membraneencase organelles (“little organs”) or compartments that serve specic functions, whereas the prokaryotic cell is noncompartmentalize. Various structures are unique to prokaryotic cells (see Fig. 1.1). Differences also exist in the processes of DNA synthesis, protein synthesis, an cell wall synthesis an structure. Table 1.1 compares the major characteristics of eukaryotic an prokaryotic cells. Pathogenic (isease-causing) bacteria are prokaryotic cells that infect eukaryotic hosts. Antimicrobial agents targeting unique prokaryotic structures an metabolism inhibit bacterial growth without harming eukaryotic host cells. This is one reason that pharmaceutical companies have been successful in eveloping effective antimicrobial agents against bacterial pathogens. However, ning rugs effective against parasites an fungi, which are eukaryotic an resemble their human hosts, an viruses, which use host cells for replication, is less successful. In aition, as a result of genetic changes, bacteria continue to acquire rug resistance.

Comparison of prokaryotic and eukaryotic cell structure Prokaryotic cell structure Cytoplasmic structures Bacteria o not contain a membrane-boun nucleus. Their genome consists of a single circular chromosome. This appears as a iffuse nucleoi or chromatin boy (nuclear boy) that is attache to a mesosome, a saclike structure in the cell membrane. Bacterial ribosomes, consisting of RNA an protein, are foun free in the cytoplasm an attache to the cytoplasmic membrane. They are the site of protein synthesis. They are 70 S in size an issociate into two subunits: 50 S an 30 S (see Table 1.1). The S stans for Svedberg units, which refer to seimentation rates (unit of time) uring high-spee centrifugation. The Sveberg unit is name for Theoor Sveberg, Nobel Prize winner an inventor of the ultracentrifuge. Larger particles have higher S values. The S value is not additive. When the previously mentione two subunits 50 S an 30 S bin together, there is a loss of surface area, an the two subunits prouce a complex 70 S in size. The same occurs in the eukaryotic cell, where the two subunits 60 S an 40 S combine to form an 80 S complex. Staine bacteria sometimes reveal the presence of cytoplasmic granules. These granules are storage eposits an may consist of polysaccharies such as glycogen, lipis such as poly β-hyroxybutyrate, or polyphosphates. These granules are sometimes referre to as metachromatic granules an can be visualize with the methylene blue stain.

PART 1

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1

Bacterial cell structure, physiology, metabolism, and genetics

Certain genera, such as Bacillus an Clostridium, prouce enospores in response to harsh environmental conitions. Enospores are small, ormant (inactive), asexual spores that evelop insie the bacterial cell as a means of survival, not reprouction. Their thick protein coat makes them highly resistant to chemical agents, temperature change, starvation, ehyration, ultraviolet an gamma raiation, an esiccation. Uner harsh conitions, each vegetative cell (active, capable of growing an iviing) prouces one enospore (inactive) that later germinates uner favorable environmental conitions into one vegetative cell. Enospores shoul not be confuse with the reprouctive spores of fungi (see Chapter 27). Spores appear as highly refractile boies in the cell an are visualize with Gram stain as unstaine areas in a cell because of their thick protein coat. With the Schaeffer-Fulton stain, the most commonly use enospore stain, enospores appear green (malachite green is use as the primary stain), an the bacterial cells are re (safranin is the counterstain). The size, shape, an interior location of the spore, for example, at one en (terminal), subterminal, or central, can be use as ientifying characteristics. For instance, the terminal spore of Clostridium tetani, the etiologic (causative) agent of tetanus, causes swelling of the thin ro-shape cell giving the organism a characteristic tennis racquet–shape or lollipop-shape appearance.

o contain sterols) oes not contain sterols. This is in contrast with eukaryotic plasma membranes, which contain sterols. The plasma membrane acts as an osmotic barrier (prokaryotes have a high osmotic pressure insie the cell) an is the location of the electron transport chain, where energy is generate. The general functions of the prokaryotic plasma membrane are ientical to functions in eukaryotes, except for energy generation (Fig. 1.2).

Cell wall The cell wall of prokaryotes is a rigi structure that maintains the shape of the cell an prevents bursting of the cell from the high osmotic pressure insie it. Cell walls in bacteria have been traitionally categorize accoring to their staining characteristics. The two major types of cell walls are gram-positive an gram-negative (see Fig. 1.1A). They can be ifferentiate by the Gram stain.

Gram-positive cell wall The gram-positive cell wall is compose of a very thick peptioglycan (murein) layer. The peptioglycan layer consists of glycan (a polysaccharie) an chains of alternating N-acetyl-d-glucosamine (NAG) an N-acetyl-d-muramic aci (NAM) (Fig. 1.3). Short pepties, each consisting of four amino aci resiues, are attache to a carboxyl group on each NAM resiue. The chains are then cross-linke to form a thick network via a peptie brige (iffering in number of pepties) connecte to the tetrapepties on the NAM. Because the peptioglycan layer is the principal component of the gram-positive cell wall, antimicrobial agents that target gram-positive organisms (e.g., penicillins) are effective by preventing synthesis of peptioglycan. Gram-negative bacteria, which have a ifferent cell wall structure, are less affecte by these agents. Other components of the gram-positive cell wall that penetrate to the exterior of the cell are teichoic aci, anchore to the peptioglycan, an lipoteichoic aci, anchore to the plasma membrane. These two components are unique to the gram-positive cell wall. Other antigenic polysaccharies may be present on the surface of the peptioglycan layer.

Cell envelope structures The cell envelope consists of the membrane an structures surrouning the cytoplasm. In bacteria, these are the plasma membrane an the cell wall. Some species also prouce capsules an slime layers.

Plasma (Cell) membrane The plasma membrane is a phospholipi bilayer with embee proteins that surrouns the cytoplasm. The prokaryotic plasma membrane is mae of phospholipis an proteins an (except for those in the family Mycoplasmataceae, which

Carbohydrate chains

External membrane surface

Glycolipid Polar region of phospholipid

Phospholipid bilayer

Internal membrane surface

Cholesterol Membrane channel protein

Protein

Glycoprotein

Nonpolar region of phospholipid

Fig. 1.2 Structure of the plasma membrane. (From Thibodeau, G. A., & Patton, K. T. (2007). Anatomy and physiology [6th ed]. St Louis: Mosby.)

Comparison of prokaryotic and eukaryotic cell structure

9

CH2OH O

(NAG) OH

CH2OH (NAM) CH2OH O

(NAG) OH

O

O

O

NH CH2OH C O O CH3 (NAM)

O

NH OH CH2OH C O O O O CH3 (NAG) NH NH HC CH3 OH C O C O C O O CH3 L-Alanine NH CH3 HC CH3 C O D-Glutamate C O CH3 L-Alanine D-Alanine D-Glutamate Meso-diaminopimelate D-Alanine

Fig. 1.3 The structure of the peptidoglycan layer in the cell wall of Escherichia coli. The amino acids in the cross-linking tetrapeptides may differ among species. NAG, N-acetyl-d-glucosamine; NAM, N-acetyl-d-muramic acid. (From Neidhardt, F. C., et al. (1990). Physiology of the bacterial cell: a molecular approach. Sunderland, MA: Sinauer Associates.)

Gram-negative cell wall The cell wall of gram-negative bacteria comprises two layers: the inner peptioglycan layer, which is much thinner than in gram-positive cell walls, an an aitional outer membrane unique to the gram-negative cell wall. The outer membrane, sometimes calle an envelope, contains proteins, phospholipis, an lipopolysaccharie (LPS). LPS contains three regions: an antigenic O–specic polysaccharie, a core polysaccharie, an an inner lipi A (also calle endotoxin). The lipi A moiety is responsible for proucing fever an shock in animals infecte with gram-negative bacteria. The toxicity varies among ifferent bacterial species. The outer membrane has the following functions: • It acts as a barrier to hyrophobic compouns an harmful substances. • It acts as a sieve, allowing water-soluble molecules to enter through protein-line channels calle porins • It provies attachment sites that enhance attachment to host cells.

Case check 1.1 The differential ability of the Gram stain makes it useful in classifying a bacterium as gram-positive or gram-negative. As in the Case in Point, correct interpretation and assessment of the Gram-stained smear results are critical in the presumptive identification of the organism present. See also Procedure 12 in Appendix D on Evolve. The use of quality control organisms with known Gram stain reactions ensures that the Gram stain procedure is performed correctly. Bacteria with a thick peptidoglycan layer in the cell wall containing teichoic acid cross-linkages retain the crystal violet–iodine dye complex after decolorization and appear deep blue. They are gram-positive (e.g., S. aureus). Bacteria that possess an outer membrane, a lipid bilayer, and a thin layer of peptidoglycan do not retain the dye complex when exposed to a decolorizer. They are gram-negative (e.g., E. coli). The alcohol-acetone decolorizer damages the outer membrane of gram-negative bacteria and allows the stain complex to wash out. All unstained elements, such as gram-negative bacteria and products of inflammation, are subsequently counterstained red by the dye safranin. Older, dying, or dead gram-positive bacterial cells, having lost the integrity of the thick peptidoglycan layer, are not able to retain the crystal violet–iodine complex and will also appear red.

Between the outer membrane an the inner membrane an encompassing the thin peptioglycan layer is an area referre to as the periplasmic space. Within the periplasmic space is a gel-like matrix containing nutrient-bining proteins an egraative an etoxifying enzymes. The periplasmic space is absent in gram-positive bacteria.

yes an makes Mycobacterium spp. ifcult to stain with the Gram stain. Mycobacteria are best seen with an aci-fast stain, in which carbol fuchsin is the primary stain, followe by an aci-alcohol ecolorizer. See “Aci-fast Stains” later in this chapter an also Proceure 12 in Appenix D on Evolve.

Acid-fast cell wall

Absence of cell wall

Certain genera, such as Mycobacterium an Nocardia, have a gram-positive cell wall structure an are classie as gram-positive bacteria. However, because they contain a waxy layer of glycolipis an fatty acis (mycolic aci) boun to the exterior of the cell wall, they stain poorly with the Gram stain. More than 60% of the cell wall is lipi, an the major lipi component is mycolic aci, a strong hyrophobic molecule that forms a lipi shell aroun the organism an affects its permeability. This exclues water-soluble

Prokaryotes that belong to the genera Acholeplasma, Mycoplasma, an Ureaplasma are unique in that they lack a cell wall an contain sterols in their plasma membranes. Because they lack the rigiity of the cell wall, they are seen in various shapes microscopically referre to as being pleomorphic. Some gram-positive an gram-negative cells can lose their cell walls an grow as L-forms in meia supplemente with serum or sugar to prevent osmotic rupture of the cell membrane.

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Bacterial cell structure, physiology, metabolism, and genetics

Surface polymers Various pathogenic bacteria prouce a iscrete organize covering terme a capsule. Capsules are usually mae of polysaccharie polymers, although they may also be mae of polypepties. Capsules act as virulence factors in helping the pathogen evae phagocytosis. During ientication of certain bacteria by serologic typing, capsules sometimes must be remove to etect the somatic (cell wall) antigens present unerneath them. Capsule removal is accomplishe by boiling a suspension of the microorganism. Salmonella Typhi must have its capsular (Vi) antigen remove for the laboratory scientist to observe agglutination with Salmonella somatic (O) antisera. The capsule oes not orinarily stain with use of common laboratory stains, such as Gram stain or Inia ink. Instea, it appears as a clear area (“halo like”) between or surrouning the staine organism an the staine amorphous backgroun material in a irect smear from a clinical specimen. Slime layers or a glycocalyx are similar to capsules but are more iffuse layers surrouning the cell. They also are mae of polysaccharies an serve either to inhibit phagocytosis or, in some cases, to ai in aherence to host tissue or synthetic implants. Glycocalyx prouction can be the rst step in the formation of a biolm (see Chapter 31).

Case check 1.2 The most common mechanism for evading phagocytosis used by many microorganisms is having a surface polysaccharide capsule. Microorganisms possessing a capsule are generally highly virulent, as in the Case in Point, until removal of the capsule, at which point virulence becomes extremely low. Encapsulated strains of Streptococcus pneumoniae and Haemophilus influenzae are associated with highly invasive infections and are known to be more virulent than nonencapsulated strains. See also the discussion on the ability to resist phagocytosis in Chapter 2. Antibodies produced against the capsule often lead to phagocytosis and immunity against that bacterial strain.

Cell appendages Flagella are exterior protein laments that rotate an cause bacteria to be motile. Bacterial species iffer in their possession of agella from none (nonmotile) to many (Fig. 1.4). Flagella that exten from one en of the bacterial cell are polar. Polar agella may occur singly at one en (monotrichous) or both ens (amphitrichous) or multiply in tufts at one en (lophotrichous). Flagella that occur all aroun the bacterium are peritrichous. The number an arrangement of agella are sometimes use for ientication purposes. Flagella can be visualize microscopically with special agellum stains. Pili (plural of pilus) an mbriae (plural of mbria) are nonagellar, proteinaceous, hairlike appenages extening a short istance from the surface of some bacterial cells that ahere cells to one another an to host cells. Conjugation pili are protein tubes that connect two bacterial cells an meiate DNA exchange.

Lophotrichous

Polar

Peritrichous

Fig. 1.4 Three agellar arrangements found in bacteria. Other variations can occur.

Eukaryotic cell structure The following structures are associate with eukaryotic cells (see Table 1.1 an Fig. 1.1). In the iagnostic microbiology laboratory, meically important fungi an parasites have eukaryotic cells.

Cytoplasmic structures The nucleus of the eukaryotic cell contains the DNA of the cell in the form of iscrete chromosomes—structures in the nucleus that carry genetic information (genes)—containe within a nuclear membrane. Chromosomes are covere with basic proteins calle histones. The number of chromosomes in the nucleus iffers accoring to the particular organism. A roune, refractile boy calle a nucleolus is also locate within the nucleus. The nucleolus is the site of rRNA synthesis. The nucleus is boune by a bilayere lipoprotein nuclear membrane. The ER is a system of membranes foun throughout the cytoplasm. It occurs in two forms. The “rough” ER is covere with ribosomes an is the site of protein synthesis. The ribosomes give the ER the rough appearance. The smooth ER oes not have ribosomes on the outer surface of its membrane, hence its smooth appearance. Smooth ER oes not synthesize proteins, but it oes synthesize phospholipis. The major function of the Golgi apparatus or complex is to moify an package proteins an lipis sent to it by the ER. Eukaryotic ribosomes, where protein synthesis occurs, are 80 S in size an issociate into two subunits: 60 S an 40 S. They are attache to the rough ER. Eukaryotic cells contain several membrane-enclose organelles. Mitochonria are the main site of energy prouction. They contain DNA an the electron transport system that prouces energy in the form of aenosine triphosphate (ATP). Lysosomes contain hyrolytic enzymes for egraation of macromolecules an microorganisms within the cell. Peroxisomes contain protective enzymes that break own hyrogen peroxie an other peroxies generate within the cell. Chloroplasts, foun in plant cells, are the sites of photosynthesis. Chloroplasts are the sites where light energy is converte into chemical energy (ATP). Photosynthesis prouces oxygen from carbon ioxie an water. Fungi are not plants an have no chloroplasts.

Bacterial morphology

Cell envelope structures

11

Microscopic Morphology of Bacteria

Plasma membrane

COCCI

The plasma membrane (see Fig. 1.2) is a phospholipi bilayer with embee proteins that envelops the cytoplasm an regulates transport of macromolecules into an out of the cell. A substantial amount of cholesterol is foun in the plasma membrane of animals. Cholesterol has a stabilizing effect an helps keep the membrane ui. The polar heas of the phospholipis are hyrophilic (water loving) an lie on the intracellular an extracellular sies of the membrane; their nonpolar tails are hyrophobic (water hating) an avoi water by lining up in the center of the plasma membrane “tail to tail.” This type of hyrophobic makeup of the interior of the plasma membrane makes it potentially impermeable to water-soluble molecules. Proteins, embee in the membrane, perform several important functions. They can act as enzymes, hormone receptors, pore channels, an carriers.

In clusters

In chains

In pairs

In tetrads

BACILLI Coccobacilli

Cell wall The function of a cell wall is to provie rigiity an strength to the exterior of the cell. Most eukaryotic cells o not have cell walls. However, fungi have cell walls principally mae of polysaccharies, such as chitin, mannan, an glucan. Chitin is a istinct component of fungal cell walls.

Bacilli of various sizes

Fusiform bacilli

Motility organelles Cilia are short projections (3 to 10 µm), usually numerous, that exten from the cell surface an are use for locomotion. They are foun in certain protozoa an in ciliate epithelial cells of the respiratory tract. Flagella are longer projections (>150 µm) use for locomotion by protozoa an animal cells such as spermatozoa.

Palisading

SPIROCHETES

Fig. 1.5 The microscopic shapes and arrangements of bacteria.

Bacterial morphology Microscopic shapes The largest bacterium known, Thiomargarita namibiensis, is foun in ocean seiment an generally has a iameter of 0.1 to 0.3 mm. Most bacteria range in size from 0.4 to 2 µm. They occur in three basic shapes (Fig. 1.5): • Cocci (spherical) • Bacilli (ro-shape) • Spirochetes (spiral) Iniviual bacterial cells may form characteristic groupings. Cocci (plural of coccus) can occur singly, in pairs (iplococci), in chains (streptococci), or in clusters (staphylococci). Bacilli (plural of bacillus) vary greatly in size an length from very short coccobacilli to long lamentous ros. The ens may be square or roune. Bacilli with tapere, pointe ens are terme fusiform; these cells are sometimes curve. When a species iffers in size an shape within a pure culture, the bacterium is terme pleomorphic. Bacilli can occur as single ros or in chains or may align themselves sie by sie (palisaes) or be branching. Spirochetes vary in length an in the number of helical turns; not all helical bacteria are calle spirochetes

Common stains used for microscopic visualization Stains that impart color or uorescence are neee to visualize microscopically bacteria. The microscopic staining characteristics (i.e., shapes an groupings) are use in the classication an ientication of microorganisms (Fig. 1.6).

Gram stain The Gram stain is the most commonly use stain in the clinical microbiology laboratory. It places bacteria into one of two groups: gram-positive (blue to purple) or gram-negative (pink or re; see Fig. 1.6A–B). Some organisms are gram-variable or o not stain at all. As mentione previously, the cell wall structure etermines the Gram-staining characteristics. The Gram stain consists of gentle heat xing (methyl alcohol may be use instea for xation) of the smear an the aition of four sequential components: crystal violet (the primary stain, 1 minute), ioine (the morant or xative, 1 minute), alcohol or an alcohol-acetone solution (the ecolorizer, a quick rinse), an safranin (the counterstain, 30 secons). The time frames liste are not exact an vary with the organism; rinsing with water between each

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Bacterial cell structure, physiology, metabolism, and genetics

A

B

C

D

E

F

G

H

Fig. 1.6 A, Gram stain of Lactobacillus species illustrating gram-positive bacilli, singly and in chains. A few gram-negative–staining bacilli are also present. B, Gram stain of Escherichia coli illustrating short gram-negative bacilli. C, Acid-fast stain, carbol fuchsin–based. Sputum smear demonstrating the presence of acid-fast Mycobacterium species (arrow) stained by the Kinyoun or Ziehl-Neelsen carbol fuchsin method. D, Acid-fast stain, uorochrome-based. Mycobacterium species stained with the acid-fast uorescent auramine-rhodamine stain. This stain is useful for screening for the presence of acid-fast bacteria in clinical specimens. E, Acridine orange stain. Fluorescent stain demonstrating the presence of staphylococci in a blood culture broth. This stain is useful for detecting bacteria in situations in which debris may mask the bacteria.F, Methylene blue stain. Methylene blue stain demonstrating the typical morphology of Corynebacterium diphtheriae (arrows). G, Lactophenol cotton blue stain. Lactophenol cotton blue–stained slide of macroconidia and hyphae of the fungal dermatophyte Microsporum gypseum. H, India ink. An India ink wet mount of the yeast Cryptococcus neoformans demonstrating the presence of a capsule (arrow). (A and B, Courtesy Dr. Andrew G. Smith, Baltimore, MD; D, courtesy Clinical Microbiology Audiovisual Study Units, Health and Education Resources, Inc., Bethesda, MD; E, courtesy Dr. John E. Peters, Baltimore, MD; and H, courtesy Dr. Andrew G. Smith, Baltimore, MD.)

Microbial growth and nutrition

step is important. The bacteria are initially staine purple by the crystal violet that is boun to the cell wall with the ai of ioine. When the ecolorizer is applie to bacteria with a gram-negative cell wall, the crystal violet washes out of the cells, which then take up the pink counterstain, safranin. For this reason, gram-negative bacteria appear pink uner the light microscope. Bacteria with a gram-positive cell wall retain the primary crystal violet stain uring the ecolorizing treatment an appear purple. Cells in a irect smear from a patient specimen, such as epithelial cells, white bloo cells, re bloo cells, an amorphous backgroun material, shoul appear pink if the Gram stain proceure was performe correctly.

Case check 1.3 Review of quality control slides is important in detecting errors in the performance of the Gram stain procedure and in interpreting results. As illustrated in the Case in Point, the gram-positive control organism, S. aureus, stained gram-positive, which is an acceptable result. However, the gram-negative control organism, E. coli, also appeared gram-positive, which is an unacceptable result and indicative of an error in performing the Gram stain procedure. When such an error occurs, the results cannot be reported until the discrepancy is resolved, and the procedure is repeated with acceptable quality control results.

13

Acridine orange Acriine orange is a uorochrome ye that stains gram-positive an gram-negative bacteria, living or ea. It bins to the nucleic aci of the cell an uoresces a bright orange when a uorescent microscope is use. Acriine orange is use to locate bacteria in bloo cultures an other specimens in which iscerning bacteria might otherwise be ifcult (see Fig. 1.6E). Acriine orange will stain eukaryotic cells as well.

Calcouor white Calcouor white is a uorochrome that bins to chitin in fungal cell walls. It uoresces a bright apple-green or blue-white, allowing visualization of fungal structures with a uorescent microscope. Calcouor white was the original “bluing” use in high-volume launries to whiten yellow-appearing white cotton an other fabrics. See also Proceure 5 in Appenix D on Evolve.

Methylene blue Methylene blue traitionally has been use to stain Corynebacterium diphtheriae for observation of metachromatic granules (see Fig. 1.6F). It is also use as a counterstain in aci-fast staining proceures. It is sometimes use as a simple stain to etect white bloo cells, such as in stool samples.

Lactophenol cotton blue Acid-fast stains Aci-fast stains are use to stain bacteria that have a high lipi (mycolic aci) an wax content in their cell walls an o not stain well with traitional bacterial stains. Carbol fuchsin, a re ye, is use as the primary stain (see Fig. 1.6C). Mycolic aci makes the bacterial cell resistant to acialcohol ecolorization an hence the bacteria retain the primary stain. Mycobacteria, because they retain the stain, are esignate aci-fast bacteria. The cell wall is treate to allow penetration of the ye either by heat (Ziehl-Neelsen metho) or by a etergent (Kinyoun metho). Aciie alcohol is use as a ecolorizer, an methylene blue, or sometimes malachite green, is the counterstain. Aci-fast bacteria retain the primary stain an are pink. Bacteria that are not aci fast are blue or green epening on the counter stain. Two other gram-positive genera, Nocardia an Rhodococcus, may stain aci-fast by a moie metho that utilizes a weaker ecolorizer. They are generally consiere partially acifast when staine with a moie aci-fast stain. In aition, the nocariae give a arker blue appearance on Gram stain compare with the faint blue for mycobacteria. Acifast staining is also use to ientify Saccharomyces, a yeast, an cocciian parasites, such as Cystoisospora belli (formerly known as Isospora belli), Cryptosporidium, an other cocciia like boies. A uorochrome (i.e., uorescent) stain, auraminerhoamine, is also use to screen specimens for aci-fast bacteria (see Fig. 1.6D). This stain is selective for the cell wall of aci-fast bacteria. Aci-fast bacteria appear yellow or orange uner a uorescent microscope, making them easier to n an the staining metho more sensitive than carbol fuchsin– base stains. Non–aci-fast bacteria are unstaine.

Lactophenol cotton blue is use to stain the cell walls of meically important fungi grown in slie cultures (see Fig. 1.6G). It utilizes the blue ye aniline.

India ink Inia ink an nigrosin are negative stains use to visualize capsules surrouning certain yeasts, such as the yeast Cryptococcus spp. (see Fig. 1.6H). The ne ink particles are exclue from the capsule, leaving a ark backgroun an a clear capsule surrouning the yeast cells.

Endospore stain The Schaefer-Fulton spore stain is use to stain bacterial spores. The primary stain, malachite green, is applie (ooe) to a heat-xe smear an heate to steaming for about 5 minutes. Then the preparation is washe for about 30 secons to remove the primary stain. Next, the counterstain safranin is applie to the smear. The enospores appear green within pink- or re-appearing bacterial cells.

Microbial growth and nutrition All bacteria have three major nutritional nees for growth: • A carbon source for making cellular constituents. Carbon represents 50% of the ry weight of a bacterium. • A nitrogen source for making proteins an nucleic acis. Nitrogen makes up 14% of the ry weight. • An energy source (ATP) for performing cellular functions.

PART 1

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Bacterial cell structure, physiology, metabolism, and genetics

Smaller amounts of molecules, such as phosphate for nucleic acis an phospholipis of cell membranes an sulfur for protein synthesis, make up an aitional 4% of the weight. Various metals an ions for enzymatic activity must also be present in trace amounts. Important mineral ions, such as Na+, K+, Cl−, Fe2+, an Ca2+, are also require. Although the basic builing blocks require for growth are the same for all cells, bacteria iffer wiely in their ability to use ifferent sources of these molecules.

Nutritional requirements for growth Bacteria are classie into two groups accoring to how they meet their nutritional nees. The autotrophs (lithotrophs) are able to grow simply, using carbon ioxie as the sole source of carbon, in aition to the require water an inorganic salts. Autotrophs obtain energy either photosynthetically (phototrophs) or by oxiation of inorganic compouns (chemolithotrophs). Autotrophs occur in environmental milieus. The secon group of bacteria, the heterotrophs, require more complex substances for growth. These bacteria require an organic source of carbon, such as glucose, an obtain energy by oxiizing or fermenting organic substances. Often, the same substance (e.g., glucose) is use as both the carbon source an the energy source. All bacteria that inhabit the human boy are heterotrophs. However, nutritional nees iffer greatly within this group. Bacteria such as E. coli an Pseudomonas aeruginosa can use a wie variety of organic compouns as carbon sources an grow on most simple laboratory meia. Other pathogenic bacteria, such as H. inuenzae an the anaerobes, are fastiious, requiring aitional metabolites such as vitamins, purines, pyrimiines, an hemoglobin supplie in the growth meium. Some pathogenic bacteria, such as Chlamydia spp., cannot be culture on laboratory meia an must be grown in cell culture or etecte by other means.

Types of growth media Various types of growth meia are use in the iagnostic microbiology laboratory to cultivate an recover bacteria from clinical samples. Culture meia are categorize accoring to their function an ability to support growth. Those

A

that contain nutrients that support the growth of most nonfastiious organisms are calle nonselective or nutritive type of meia. Examples of nutrient meia inclue trypticase soy agar or broth an nutrient agar. A growth meium that contains ae growth factors, such as bloo, vitamins, an yeast extract, is referre to as an enriched meium (e.g., sheep bloo agar an chocolate agar). The ae factors allow fastiious organisms to grow. Meia that contain aitives such as yes, bile salts, alcohols, acis, an antimicrobial agents that inhibit the growth of some bacteria but allow others to grow are calle selective media. MacConkey (MAC) agar is an example of a selective meium for gram-negative bacteria. It contains bile salts an crystal violet that inhibit gram-positive bacteria but allow the growth of gram-negative bacteria. Columbia agar with colistin an naliixic aci, alternatively, is selective for gram-positive organisms by inhibiting the growth of gram-negative bacteria. Meia that contain ingreients that allow visualization of metabolic ifferences between groups or species of bacteria are calle differential media. MAC agar, a selective meium, is also a ifferential meium because it istinguishes between lactose fermenters (pink colonies) an nonlactose fermenters (clear colonies) (Fig. 1.7A–B). Sheep bloo agar, an enriche, nonselective type of meium, is also ifferential because it istinguishes between hemolytic an nonhemolytic organisms. Fig. 1.8 shows a sheep bloo agar plate containing beta-hemolytic colonies. Therefore some meia can be selective an ifferential (e.g., MAC agar) or nonselective an ifferential (e.g., sheep bloo agar). Broth media, such as thioglycollate broth, can be use to supplement agar meia to recover small numbers of organisms that may be present in a clinical sample. Enrichment broth isesigne to encourage the growth of small numbers of a particular organism while suppressing other bacteria that might be present in the specimen. An example is Lim broth (To Hewitt with colistin an naliixic aci) use to enhance the growth of group B streptococci. Enrichment broths are incubate for a specic time an then must be subculture to soli meia to isolate the organism of interest. When a elay between collection of the specimen an culturing is necessary, a transport medium is use. A transport meium is a holing meium esigne to preserve the

B

Fig. 1.7 A, Lactose-fermenting, gram-negative rods producing pink colonies on MacConkey (MAC) agar. B, Nonlactose fermenting, gram-negative rods producing colorless colonies on MAC agar.

Microbial growth and nutrition

Fig. 1.8 Blood agar plate showing beta-hemolytic colonies.

viability of microorganisms in the specimen but not allow multiplication. Stuart broth an Amies an Cary-Blair transport meia are commonly use examples.

Environmental factors inuencing growth The following three important environmental factors inuence the growth rate of bacteria an must be consiere when bacteria are culture in the laboratory: • pH • Temperature • Gaseous composition of the atmosphere Most pathogenic bacteria grow best at a neutral pH. Diagnostic laboratory meia for bacteria are usually ajuste to a nal pH between 7.0 an 7.5. Temperature inuences the rate of growth of a bacterial culture. Microorganisms are categorize accoring to their optimal temperature for growth. Bacteria that grow best at col temperatures are calle psychrophiles (optimal growth at 10° to 20° C). Bacteria that grow optimally at moerate temperatures are calle mesophiles (optimal growth at 20° to 40° C). Bacteria that grow best at high temperatures are calle thermophiles (optimal growth at 50° to 60° C). Psychrophiles an thermophiles are foun environmentally in places such as the Arctic seas an hot springs, respectively. Most bacteria that have aapte to humans are mesophiles, which grow best near the human core boy temperature of 37° C. Diagnostic laboratories routinely incubate cultures for bacterial growth at 35° C. However, some pathogenic species prefer a lower temperature for growth; when these organisms are suspecte, the meia from these specimens are incubate at a lower temperature. Fungal cultures are incubate at 30° C. The ability to grow at room temperature (22° C) or at an elevate temperature (42° C) is use as an ientication characteristic for some bacteria. Bacteria that infect humans iffer in their atmospheric requirements for growth. In the iagnostic microbiology laboratory, bacterial atmospheric growth requirements are optimize to ensure recovery an isolation of pathogenic organisms from clinical samples. Microorganisms that require oxygen for growth are calle obligate aerobes, an those that cannot grow in the presence of oxygen are

15

obligate or strict anaerobes. Obligate anaerobes must be grown in an atmosphere either evoi of oxygen or with signicantly reuce oxygen content. Aerotolerant anaerobes can survive in the presence of oxygen but grow poorly an o not use oxygen in metabolism; facultative anaerobes can grow either with or without oxygen. If oxygen is present, the facultatively anaerobic bacteria will utilize it via aerobic respiration an grow faster than without oxygen. Facultative anaerobes like E. coli are routinely culture in an aerobic atmosphere because aerobic culture is easier an less expensive than anaerobic culture, an the bacteria grow more rapily. Microaerophilic bacteria require a reuce level of oxygen to grow. An example of a pathogenic microaerophile is Campylobacter jejuni, which requires 5% to 6% oxygen. The atmosphere require by microaerophiles can be generate in culture jars or pouches with a commercially available microaerophilic atmosphere–generating system. Capnophilic organisms are those that require an atmosphere enriche with carbon ioxie (5% to 10%); an example is Neisseria gonorrhoeae. The concentration of carbon ioxie in ambient air is about 400 parts per million. Air contains approximately 21% oxygen. When the carbon ioxie content of an aerobic incubator is increase to 10%, the oxygen content of the incubator is ecrease to approximately 18%. Obligate aerobes must have oxygen to grow; incubation in air or an aerobic incubator with 10% carbon ioxie satises their oxygen requirement. Because many bacteria grow better in the presence of increase carbon ioxie, iagnostic microbiology laboratories often maintain their aerobic incubators at a 5% to 10% carbon ioxie level, in what is commonly referre to as a carbon dioxide incubator

Bacterial growth Generation time Bacteria replicate by binary ssion, with one cell iviing into two cells. The time require for one cell to ivie into two cells is calle the generation time or doubling time. The generation time of a bacterium in culture can be 20 minutes for a fast-growing bacterium such as E. coli or 24 hours for a slow-growing bacterium such as Mycobacterium tuberculosis

Growth curve If bacteria are in a growth state with enough nutrients an no toxic proucts present, the increase in bacterial numbers is proportional to the increase in other bacterial properties, such as mass, protein content, an nucleic aci content. Measurement of any of these properties can be use as an inication of bacterial growth. When the growth of a bacterial culture (number of live cells) is plotte over time, the resulting growth curve shows four phases of growth: (1) a lag phase in which bacteria are preparing to ivie; (2) an exponential phase in which bacterial numbers increase logarithmically; (3) a stationary phase in which nutrients are becoming limite an the numbers of bacteria remain constant (although viability may ecrease); an (4) a eath phase in which the number of nonviable bacterial cells excees the

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Bacterial cell structure, physiology, metabolism, and genetics

number of viable cells. An example of such a growth curve is shown in Fig. 1.9

Bacterial biochemistry and metabolism

Determination of cell numbers

Metabolism

In the iagnostic laboratory, the number of bacterial cells present is etermine in one of three ways:

Microbial metabolism consists of the biochemical reactions bacteria use to break own organic compouns (catabolism) an the reactions they use to synthesize new molecules (anabolism) from smaller subunits. Energy for anabolism is generate uring catabolism of a substrate. The occurrence of all biochemical reactions in the cell epens on the presence an activity of enzymes. Thus metabolism can be regulate in the cell either by regulation of the prouction of an enzyme itself (a genetic type of regulation in which prouction of the enzyme can be inuce or suppresse by molecules present in the cell) or by regulation of the activity of the enzyme (via feeback inhibition in which the proucts of the enzymatic reaction or a succeeing enzymatic reaction inhibit the activity of the enzyme). Bacteria iffer wiely in their ability to use various compouns as substrates an in the en proucts generate. Many biochemical pathways exist for catabolism in the microbial worl, an the particular pathway use etermines the en prouct an resulting pH of the meium (Fig. 1.10). Microbiologists use these metabolic ifferences as phenotypic markers in the ientication of bacteria. Diagnostic schemes analyze each unknown microorganism for (1) utilization of various substrates as a carbon source, (2) prouction of specic en proucts from various substrates, an (3) prouction of an aci or alkaline pH in the test meium. Knowlege of the biochemistry an metabolism of bacteria is important in ientifying microorganisms in the clinical laboratory.

• Direct counting under the microscope: This metho can be use to estimate the number of bacteria present in a specimen. It oes not istinguish between live an ea cells. • Direct plate count: By growing ilutions of broth cultures on agar plates, one can etermine the number of colony-forming units per milliliter (CFU/mL). This metho provies a count of viable cells only. It is use in etermining the bacterial cell count in urine cultures. • Density measurement: The ensity (referre to as turbiity) of a bacterial broth culture can be correlate to the number of CFU/mL of the culture. This metho is use to prepare a stanar inoculum for antimicrobial susceptibility testing.

log10 Number of bacteria

Stationary 9 7

Death phase

Exponential 5 3

Fermentation and respiration

Lag

Bacteria use biochemical pathways to catabolize carbohyrates an prouce energy by two mechanisms—fermentation an aerobic respiration (commonly referre to as oxidation). Fermentation is an anaerobic process carrie out by obligate, facultative, an aerotolerant anaerobes. In fermentation, the

1 4

8

12

16

20

Fig. 1.9 Typical growth curve of a bacterial culture.

Ethanol Propionic acid

L– or D–Lactic acid

+ 2H H2 + CO2

Acetaldehyde Succinic acid

+ 2H

+ 4H PYRUVIC ACID

Oxaloacetic acid Acetolactic acid

Acetyl CoA

+ 4H Acetoin

+ 2H 2,3–Butanediol

Ethanol

Acetic acid

Formic acid Acetoacetyl CoA + 4H Acetone

Butyryl CoA

+ 4H

+ 2H Isopropanol

Butyric acid

Butanol

Fig. 1.10 The fate of pyruvate in major fermentation pathways of microorganisms. (From Zinsser, H., et al. [1992]. Zinsser microbiology [20th ed]. Norwalk, CT: Appleton & Lange.)

Bacterial biochemistry and metabolism

electron acceptor is an organic compoun. Fermentation is less efcient in energy generation than respiration because the beginning substrate is not completely reuce; therefore all of the energy in the substrate is not release. Besies allowing growth in the absence of atmospheric oxygen, fermentation is also important because it generates nicotinamie aenine inucleotie (NAD), a molecule necessary for maintaining the Krebs cycle. When fermentation occurs, a mixture of en proucts (e.g., lactate, butyrate, ethanol, an acetoin) accumulates in the meium. Analysis of these en proucts is particularly useful for the ientication of anaerobic bacteria. En-prouct etermination is also use in the Voges-Proskauer (VP) an methyl re tests, two important tests use in the ientication of members of the orer Enterobacterales. The term fermentation is often use loosely in the iagnostic microbiology laboratory to inicate any type of utilization—fermentative or oxiative—of a carbohyrate (sugar) with the resulting prouction of an aci pH. Aerobic respiration (oxidation) is an efcient energy-generating process in which molecular oxygen (O 2) is the nal electron acceptor. Obligate aerobes an facultative anaerobes unergo aerobic respiration. Some anaerobes can carry out anaerobic respiration in which molecules other than molecular oxygen, such as nitrate an sulfate, act as the nal electron acceptors. Anaerobic respiration is less energy yieling than aerobic respiration.

Biochemical pathways from glucose to pyruvic acid The starting carbohyrate for bacterial fermentation or oxiation is glucose. When bacteria use other sugars as a carbon source, they rst convert the sugar to glucose, which is processe by one of three pathways. These pathways are esigne to generate pyruvic aci, a key three-carbon intermeiate. The three major biochemical pathways bacteria use to break own glucose to pyruvic aci are (1) the EmbenMeyerhof-Parnas (EMP) glycolytic pathway (Fig. 1.11), (2) the pentose phosphate pathway (Fig. 1.12), an (3) the EntnerDouoroff pathway (see Fig. 1.12). Pyruvate can be further catabolize either fermentatively or oxiatively. The three major metabolic pathways an their key characteristics are escribe in Box 1.1.

Anaerobic utilization of pyruvic acid (fermentation)

D–Glucose ADP D–Glucose – 6 – PO4 D–Fructose – 6 – PO4

ATP

ATP

D–Fructose – 1,6 – di PO4 Dihydroxyacetone–PO4

D–Glyceraldehyde–3–PO4

1,3–Diphosphoglycerate ADP 3–Phosphoglycerate

2 NADH

2 ATP

2–Phosphoglycerate Ethanol Lactate Acetaldehyde

Phosphoenolpyruvate ADP

2 ATP

Pyruvate CO2

Fig. 1.11 Embden-Meyerhof-Parnas glycolytic pathway. (From Zinsser, H., et al. [1992]. Zinsser microbiology [20th ed]. Norwalk, CT: Appleton & Lange.)

•

•

•

•

Pyruvic aci is a key metabolic intermeiate. Bacteria process pyruvic aci further using various fermentation pathways. Each pathway yiels ifferent en proucts, which can be analyze an use as phenotypic markers (see Fig. 1.10). Some fermentation pathways use by the microbes that inhabit the human boy are as follows: • Alcoholic fermentation: The major en prouct is ethanol. This is the pathway use by yeasts when they ferment glucose. • Homolactic fermentation: The en prouct is almost exclusively lactic aci. Members of the genus Streptococcus an

17

•

many members of the genus Lactobacillus ferment pyruvate using this pathway. Heterolactic fermentation: Some lactobacilli use a mixe fermentation pathway, of which, in aition to lactic aci, the en proucts inclue carbon ioxie, alcohols, formic aci, an acetic aci. Propionic acid fermentation: Propionic aci is the major en prouct of fermentation carrie out by Cutibacterium acnes (formerly Propionibacterium acnes) an some anaerobic non–spore-forming, gram-positive bacilli. Mixed acid fermentation: Members of the genera Escherichia, Salmonella, an Shigella within the orer Enterobacterales use this pathway for carbohyrate fermentation an prouce a number of acis as en proucts: lactic, acetic, succinic, an formic. The strong aci prouce is the basis for the positive reaction on the methyl re test exhibite by these organisms an the aciic pH on laboratory meia. Butanediol fermentation: Members of the genera Klebsiella, Enterobacter, an Serratia within the orer Enterobacterales use this pathway for carbohyrate fermentation. The en proucts are acetoin (acetyl methyl carbinol) an 2,3-butaneiol. Detection of acetoin is the basis for the positive VP reaction characteristic of these microorganisms. Little aci is prouce by this pathway. Thus organisms that have a positive VP reaction usually have a negative methyl re test, an vice versa. Butyric acid fermentation: Certain obligate anaerobes, incluing many Clostridium, Fusobacterium, an Eubacterium species, prouce butyric aci as their primary en prouct along with acetic aci, carbon ioxie, an hyrogen.

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Bacterial cell structure, physiology, metabolism, and genetics

Glucose ATP ADP Glucose–6–PO4 NAD NADH2 6–Phosphogluconic acid

2–Keto–3–deoxy–6–phosphogluconic acid

NAD NADH2 Pentose PO4 + Glyceraldehyde–3–PO4

Acetyl PO4

Acetaldehyde + CO2

NADH2 2 ATP Lactate

Glyceraldehyde–3–PO4

Pyruvic acid

CO2

NADH2 NAD

NAD Acetaldehyde

Ethanol

(Via EMP pathway)

2 ADP 2 ATP 2 NAD 2 NADH2

Pyruvic acid

NADH2

(Via EMP pathway)

NAD Ethanol

Acetaldehyde + CO2 NADH2 NAD Ethanol

Fig. 1.12 Alternative microbial pathways to the Embden-Meyerhof-Parnas (EMP) pathway for glucose fermentation. The pentose phosphate pathway (left) and the EntnerDoudoroff pathway (right) are shown. (From Zinsser, H., et al. [1992]. Zinsser microbiology [20th ed]. Norwalk, CT: Appleton & Lange.)

BOX 1.1 Three major metabolic pathways found in bacteria EMP glycolytic pathway (see Fig. 1.11) • • • • •

Major pathway in conversion of glucose to pyruvate Generates reducing power in the form of NADH2 Generates energy in the form of ATP Anaerobic; does not require oxygen Used by many bacteria, including all members of the order Enterobacterales

Pentose phosphate (phosphogluconate) pathway (see Fig. 1.12) • Alternative to EMP pathway for carbohydrate metabolism • Conversion of glucose to ribulose-5-phosphate, which is rearranged into other 3-, 4-, 5-, 6-, and 7-carbon sugars • Provides pentoses for nucleotide synthesis • Produces glyceraldehyde-3-phosphate, which can be converted to pyruvate

• Generates NADPH, which provides reducing power for biosynthetic reactions • May be used to generate ATP (yield is less than with EMP pathway) • Used by heterolactic fermenting bacteria, such as lactobacilli, and by Brucella abortus, which lacks some of the enzymes required in the EMP pathway

Entner-doudoroff pathway (see Fig. 1.12) • Converts glucose-6-phosphate (rather than glucose) to pyruvate and glyceraldehyde phosphate, which can be funneled into other pathways • Generates one NADPH per molecule of glucose, but uses one ATP • Aerobic process used by Pseudomonas, Alcaligenes, Enterococcus fecalis, and other bacteria lacking certain glycolytic enzymes

ATP, Adenosine triphosphate; EMP, Embden-Meyerhof-Parnas; NADH2, nicotinamide adenine dinucleotide dehydrogenase; NADPH, nicotinamide adenine dinucleotide phosphate.

Aerobic utilization of pyruvate (oxidation) The most important pathway for the complete oxiation of a substrate is the Krebs cycle or tricarboxylic aci cycle. In this cycle, the starting molecule, pyruvate, is oxiize, carbon skeletons for biosynthetic reactions are create,

guanosine-triphosphate (GTP) is generate, an electrons are onate to form NADH an FADH 2, which ultimately enter the electron transport chain to generate energy in the form of ATP. This cycle results in the prouction of aci an the evolution of carbon ioxie uring aerobic respiration (Fig. 1.13).

Microbial genetics

Pyruvate

Microbial genetics

Acetyl–CoA CO2

2H

19

Citrate Oxaloacetate cis–Aconitate

No iscussion of microbial genetics is complete without rst escribing DNA an RNA. DNA was rst iscovere by Freerick Miescher in 1869. In the 1920s, Phoebus A. T. Levine iscovere that DNA containe phosphates, ve-carbon sugars (cyclic pentose), an nitrogen-containing bases. Later, Rosalin Franklin iscovere the helical structure by x-ray crystallography. Most everyone is familiar with James Watson an Francis Crick, who escribe the three-imensional structure of the DNA molecule in the 1950s.

Anatomy of a DNA and RNA molecule

Malate Isocitrate CO2

Fumarate

–Ketoglutarate CO2

Succinate

2H

2H

2H

2H

Electron transport and oxidative phosphorylation

ATP Fig. 1.13 Krebs tricarboxylic acid cycle allowing complete oxidation of a substrate. (From Zinsser, H., et al. (1992). Zinsser microbiology [20th ed]. Norwalk, CT: Appleton & Lange.)

Carbohydrate utilization and lactose fermentation The ability of microorganisms to use various sugars (carbohyrates) for growth is an integral part of many ientication schemes. The fermentation of the sugar is usually etecte by aci prouction an a concomitant change of color resulting from a pH inicator present in the culture meium. Bacteria generally ferment glucose preferentially over other sugars, so glucose must be absent if the ability to ferment another sugar is being teste. The microorganism’s ability to ferment lactose is an important step in classifying members of the orer Enterobacterales. These bacteria are classie as either lactose fermenters or nonlactose fermenters. Lactose is a isaccharie consisting of one molecule of glucose an one molecule of galactose linke by a galactosie bon. The utilization of lactose by a bacterium requires two steps. The rst step requires the enzyme β-galactosie permease for the transport of lactose across the cell wall into the cytoplasm. The secon step occurs insie the cell an requires the enzyme β-galactosiase to break the galactosie bon, releasing glucose, which then can be fermente. Thus all organisms that ferment lactose can also ferment glucose.

DNA is a ouble-helical chain of eoxynucleoties. The helix is a ouble stran twiste together, which many scientists refer to as a spiral staircase (resembling the hanrail, sies, an steps of a spiral staircase). A nucleotie is a complex combination of the following: • A phosphate group (PO4) • A cyclic ve-carbon pentose (the carbons in the pentose are numbere 1′ to 5′) sugar (eoxyribose), which makes up the “hanrails an sies” • A nitrogen-containing base, or the “steps,” either a purine or a pyrimiine A purine consists of a fuse ring of nine carbon atoms an nitrogen. There are two purines in DNA: aenine (A) an guanine (G). A pyrimiine consists of a single ring of six atoms of carbon an nitrogen. There are two pyrimiines in DNA: thymine (T) an cytosine (C). A nucleotie is forme when the 5′ carbon of the sugar an one of the nitrogenous bases attaches to the 1′ carbon of the pentose sugar. These are the basic builing blocks of DNA (Fig. 1.14). In the chain of eoxynucleoties, bons form between the phosphate group of one nucleotie an the 3′ sugar of the next nucleotie. The base extens from the sugar. Aenine of one chain always pairs with thymine of the other chain, an cytosine of one chain pairs with guanine of the other chain. The bases are hel together by hyrogen bons. The information containe in DNA is etermine by the sequence of letters along the “staircase.” The sequence ACGCT represents ifferent information than the sequence AGTCC. This woul be like taking the wor stops an using the same letters to form the wor spots or posts, which have ifferent meanings but all of the same letters. The two complementary sugar phosphate strans run in opposite irections (antiparallel), 3′ to 5′ an 5′ to 3′, similar to one train with its engine going one way alongsie a caboose of a train going the opposite irection (Fig. 1.15). The irection is base on what is foun at the ens of the strans; for example, phosphate attaches to the 5′ carbon of the sugar, an the OH group is attache to the 3′ carbon of the sugar. DNA is use as a template to prouce the complementary messenger RNA (mRNA) stran. In RNA, the nitrogenous base thymine is replace by uracil, another pyrimiine. In contrast with DNA, RNA is single-strane an short, not ouble-strane an long, an it contains the sugar ribose, not eoxyribose.

20

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Bacterial cell structure, physiology, metabolism, and genetics

Pyrimidines NH2

Purines

O H 3C

N

N H Cytosine C

O

N O

O

N H Uracil U

DNA only

O

N

N

N H Thymine T

DNA and RNA

NH2

O

N

N

N H

N H

N

Adenine A

RNA only

N NH2

N

Guanine G DNA and RNA

Fig. 1.14 Molecular structure of nucleic acid bases. Pyrimidines: cytosine, thymine, and uracil. Purines: adenine and guanine.

3’ hydroxyl 5’ phosphate Base-pair Base

O P=O

H

H

HO

H

H

Nucleotide

Deoxyribose 3’ sugar

H

O O

H2C5’

A

T

H2C

O

O

1’ H

O P=O

H O

3’

3’

H

H

O

H

H

Phosphodiester bond 5’

H

O P=O

H

O O

H2C

C

G

H2C

O

O H

H

H

O

H

O P=O H

H

H H

O P=O

A

O H

T

G

C

A

H

T

C

O

O

H2C

A

H2C

T

G T

A

O H

H

H

O

H

O P=O

H

H

O P=O

H

O

H

H H

O O

H2C

C

H2 C

G

O H

H

H

O

H

O P=O

H

H

O P=O O

A

3’ O

H

H

3’

H 5’

O

H2C

H

T

A

H2C5’

B

O

H

H OH

O P=O 5’ phosphate

H

3’ hydroxyl

Fig. 1.15 A, Molecular structure of DNA showing nucleotide structure, a phosphodiester bond connecting nucleotides, and complementary pairing of bases (A, adenine; T, thymine; G, guanine; C, cytosine) between antiparallel nucleic acid strands. B, 5′ and 3′ antiparallel polarity and “twisted ladder” conguration of DNA.

Microbial genetics

Humans are 99.9% ientical. In a human genome of 3 billion “letters,” even one tenth of 1% translates into 3 million separate lettering ifferences, an important characteristic useful in forensic science. Microbial genetics is increasingly important in the iagnostic microbiology laboratory. Ientication tests have been evelope that are base on ientifying unique RNA or DNA sequences present in each bacterial species an viruses. The polymerase chain reaction technique is a means of amplifying specic DNA sequences an etecting very small numbers of bacteria present in a specimen. Genetic tests circumvent the nee to culture bacteria, proviing a more rapi metho of ientifying pathogens. An unerstaning of microbial genetics is also necessary to unerstan the evelopment an transfer of antimicrobial resistance by bacteria an mutations in viruses. Viruses constantly change, as in the case of the recently ientie SARS-CoV-2 (severe acute respiratory synrome–coronavirus-2), the virus that causes COVID-19 (Coronavirus Disease 2019). COVID-19 is the respiratory illness responsible for the COVID-19 panemic that begin in 2020. The occurrence of mutations can result in a change in the expecte phenotypic characteristics of an organism an provies an explanation for atypical results sometimes encountere on iagnostic biochemical tests. For example, mutations in the SARS-CoV-2 virus present in a patient specimen can potentially affect test performance inuence by the sequence of the variant, the esign of the test, an the prevalence of the variant in the population. To learn more about the newly ientie SARS-CoV-2 virus, see Chapter 29 This section briey reviews some of the basic terminology an concepts of microbial genetics. For a etaile iscussion of DNA an molecular iagnostics, see Chapter 11

Terminology The genotype of a cell is the genetic potential of the DNA of an organism. It inclues all of the characteristics that are coe for in the DNA of a cell an that have the potential to be expresse. Some genes are silent genes, expresse only uner certain conitions. Genes that are always expresse are constitutive. Genes that are expresse only uner certain conitions are inducible. The phenotype of a cell consists of observe characteristics expresse by the genome. The aim of a cell is to prouce proteins that are neee for cellular structure an function an to transmit that information for accomplishing this to the next generation of cells. Information for protein synthesis is encoe in the DNA an transmitte in the chromosome to each generation. The general ow of information in a cell is from DNA (which contains the genetic information) to mRNA, which acts as a blueprint for protein construction. Replication is the uplication of chromosomal DNA for insertion into a aughter cell. Transcription is the synthesis of mRNA by the enzyme RNA polymerase using one stran of DNA as a template. Translation is the synthesis of a specic protein from the mRNA coe. The term protein expression also refers to the synthesis of a protein. Proteins are polypepties compose of amino acis. The number an sequence of amino acis in a polypeptie an the character

21

of that particular protein are etermine by the sequence of coons in the mRNA molecule. A codon is a group of three nucleoties in an mRNA molecule that signies a specic amino aci. During translation, ribosomes containing rRNA sequentially a amino acis to the growing polypeptie chain. These amino acis are brought to the ribosome by transfer RNA (tRNA) molecules that “translate” the coons. The tRNA molecules temporarily attach to mRNA using their complementary anticoon regions. An anticodon is the triplet of bases on the tRNA that bin the triplet of bases (coon) on the mRNA. It ienties which amino aci will be in a specic location in the protein.

Genetic elements and alterations Bacterial genome The bacterial chromosome, also calle the genome, consists of a single, close, circular piece of sDNA that is supercoile to t insie the cell. It contains all of the information neee for cell growth an replication. Genes are specic DNA sequences that coe for the amino aci sequence in one protein (e.g., one gene equals one polypeptie), but this may be slice or combine with other polypepties to form more than one protein. In front of each gene on the DNA stran is an untranscribe area containing a promoter region, which the RNA polymerase recognizes an bins to for transcription initiation. This area may also contain regulatory regions to which molecules may attach an cause either a ecrease or an increase in transcription.

Extrachromosomal DNA elements In aition to the genetic information encoe in the bacterial chromosome, many bacteria contain aitional information on small circular pieces of extrachromosomal sDNA calle plasmids. They are not essential for bacterial growth, so they can be gaine or lost. Genes that coe for antimicrobial resistance (an sometimes toxins or other virulence factors) are often locate on plasmis. Antimicrobial therapy selects for bacterial strains containing plasmis encoing rug-resistance genes; this is one reason antimicrobial agents shoul not be overprescribe. The number of plasmis present in a bacterial cell may range from one (low copy number) to hunres (high copy number). Plasmis are locate in the cytoplasm of the cell an are self-replicating an passe to aughter cells, similar to chromosomal DNA. They also may sometimes be passe from one bacterial cell to another through conjugation (horizontal transfer of genetic material by cell-to-cell contact). This is one way rug resistance is acquire.

Mobile genetic elements Some fragments of DNA are mobile an can jump from one place in the chromosome to another place. These are sometimes referre to as jumping genes. The simplest mobile piece of DNA is an insertion sequence (IS) element. It is approximately 1000 base pairs long with inverte repeats on each en. Each IS element coes for only one gene, a transposase enzyme that allows the IS element to pop into an out of

22

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Bacterial cell structure, physiology, metabolism, and genetics

DNA. Bacterial genomes contain many IS elements. The main effect of IS elements in bacteria is that when an IS element inserts itself into the mile of a gene, it isrupts an inactivates the gene. This action can result in loss of an observable characteristic, such as the ability to ferment a particular sugar. Transposons are relate mobile elements that contain aitional genes. Transposons often carry rug-resistance genes an are usually locate in plasmis.

Bacterial chromosome

A Viral DNA being injected into the cell

Mutations Mutations are changes that occur in the DNA coe an often result in a change in the coe protein or in the prevention of its synthesis. Some mutations are silent, in which a change in the DNA sequence oes not result in the substitution of a ifferent amino aci in the resulting protein. This is cause by reunancy in protein synthesis; more than one coon coes for the same amino aci. A mutation may be the result of a change in one nucleotie base (a point mutation) that leas to a change in a single amino aci within a protein (missense mutation) or may be the result of insertions or eletions in the genome that lea to isruption of the gene or a frameshift mutation, or both. A gene sequence must be rea in the right “frame,” or series of three coons, for the correct protein to be prouce. This is because every set of three bases in mRNA (a coon) species a particular amino aci, an when the reaing frame is shifte, the coons are also shifte. Incomplete, inactive proteins are often the result of frameshift mutations. Some coons o not coe for an amino aci; they terminate translation an are calle stop codons. If a point mutation prouces a stop coon, this is referre to as a nonsense mutation. Spontaneous mutations occur in bacteria at a rate of about 1 in 109 cells. Mutations also occur as the result of error uring DNA replication at a rate of about 1 in 10 7 cells. Exposure to certain chemical an physical agents can greatly increase the mutation rate.

Genetic recombination Genetic recombination is a metho by which genes are transferre or exchange between homologous (similar) regions on two DNA molecules, forming new combinations of genes on a chromosome. This provies a way for organisms to obtain new combinations of biochemical pathways an aapt to changes in their environment.

Mechanisms of gene transfer Genetic material may be transferre from one bacterium to another in three ways: • Transformation • Transuction • Conjugation

Plasmid DNA ready to be taken into the cell

B

Bacterial chromosome

Bacterial chromosome

Plasmid DNA containing the F factor entering the F– cell

F– cell

F+ cell

C

Bacterial chromosome

Fig. 1.16 Methods of gene transfer into bacterial cells. A, Bacterial transformation. Free or “naked” DNA is taken up by a competent bacterial cell. After uptake, the DNA may take one of three courses: (1) it is integrated into existing bacterial genetic material; (2) it is degraded; or (3) if it is a compatible plasmid, it may replicate in the cytoplasm. B, Bacterial transduction. A phage injects DNA into the bacterial cell. The phage tail binds to a receptor of the bacterial cell wall and injects the DNA into the bacterium. One of two courses may then occur. In the lytic cycle, replication of the bacterial chromosome is disrupted; phage components are formed and assembled into phage particles. The bacterial cell is lysed, releasing a mature phage. In the lysogenic cycle, the phage DNA is incorporated into the bacterial genetic material, and genes encoded by the phage DNA can be expressed. At a later time, the phage may be “induced,” and a lytic cycle then ensues. C, Bacterial conjugation. An F+ cell connects with an F− cell via sex pili. DNA is then transferred from the F+ cell to the F− cell.

an the recipient cell is compatible, the plasmi can replicate in the cytoplasm an be transferre to aughter cells uring cell ivision. Cells that can take up nake DNA are referre to as being competent. Transformation is not an efcient process an has been reporte to occur naturally in only a few bacterial species, such as S. pneumoniae, N. gonorrhoeae, Helicobacter pylori, an H. inuenzae. Bacteria in biolms have an increase rate of transformation compare with bacteria in the planktonic state. Bacteria can be mae competent in the laboratory, an transformation is the main metho use to introuce genetically manipulate plasmis into bacteria, such as E. coli, uring cloning proceures.

Transformation Transformation is the uptake an incorporation of free or nake DNA into a bacterial cell (Fig. 1.16A). Once the DNA has been taken up, it can be incorporate into the bacterial genome by recombination. If the DNA is a circular plasmi

Transduction Transduction is the transfer of bacterial genes by a bacteriophage from one cell to another (see Fig. 1.16B). A bacteriophage consists of genetic material (DNA or RNA) surroune

Microbial genetics

by a protein coat. When a phage infects a bacterial cell, it injects its genome into the bacterial cell, leaving the protein coat outsie. The phage can then take a lytic pathway in which the bacteriophage DNA irects the bacterial cell to synthesize the phage genome an proteins an package it into new phage particles. The bacterial cell eventually lyses (lytic phase), releasing new phage particles that can infect other bacterial cells. In some instances, the phage DNA instea becomes incorporate into the bacterial genome, where it is replicate along with the bacterial chromosomal DNA when the cell ivies. This state is known as lysogeny, an the phage is referre to as being temperate. During lysogeny, genes present in the phage DNA may be expresse by the bacterial cell. An example of this in clinical microbiology is C. diphtheriae. Strains of C. diphtheriae that are lysogenize with a temperate phage carrying the gene for iphtheria toxin cause isease. Strains lacking the phage DNA o not prouce the toxin an o not cause isease. Uner certain conitions, a temperate phage can be inuce, the phage DNA is excise from the bacterial genome, an a lytic state occurs. During this process, ajacent bacterial genes may be excise with the phage DNA an package into the new phages. The bacterial genes may be transferre when the phage infects a new bacterium. In the el of biotechnology, phages are often use to insert clone genes into bacteria for analysis.

Conjugation Conjugation is the transfer of genetic material from a onor bacterial strain to a recipient strain (see Fig. 1.16C). Close contact is require between the two cells. In E. coli, the onor strain (F+) possesses fertility factor (F factor) on a plasmi that carries the genes for conjugative transfer. The onor strain prouces a hollow surface appenage calle a sex or conjugation pilus, which bins to the recipient F− cell an brings the two cells in close contact. Transfer of DNA then occurs. Both plasmis an chromosomal genes can be transferre by this metho. When the F factor is integrate into the bacterial chromosome rather than a plasmi, there is a higher frequency of transfer of ajacent bacterial chromosomal genes. These strains are known as high-frequency recombination strains

Restriction enzymes Bacteria have evolve a system to restrict the incorporation of foreign DNA into their genomes. Restriction enzymes are prouce that cut (restrict) incoming, foreign DNA at specic DNA sequences. The bacteria methylate their own DNA at these same sequences, so the restriction enzymes o not cut the DNA in their own cell. Many restriction enzymes with various recognition sequences have been isolate from various microorganisms. The rst three letters in the restriction enonuclease name inicate the bacterial source of the enzyme. For instance, the enzyme EcoRI was isolate from E. coli, an the enzyme Hind III was isolate from H. inuenzae type . These enzymes are use in the el of biotechnology to create sites for insertion of new genes.

23

POINTS TO REMEMBER

• Many microorganisms inhabit the environment, an most are nonpathogenic. • Prokaryotes, such as bacteria, o not have membraneenclose nuclei an organelles. • Eukaryotes iffer from prokaryotes in that they have membrane-enclose nuclei an organelles. • Viruses cannot be seen uner an orinary light microscope, although their cytopathic effects on cell cultures are visible. They are obligate parasites, an antimicrobial agents are ineffective for treatment of viral infections. Viruses have DNA or RNA, but rarely both, in contrast with prokaryotes an eukaryotes. • In the iagnostic microbiology laboratory, the rst step in ientifying bacteria is by the Gram stain reaction. Whether an organism is gram-positive (blue or purple) or gram-negative (pink or re) epens on its cell wall structure. A thick peptioglycan layer is present in gram-positive bacteria, while an outer membrane an thin peptioglycan layer are foun in gram-negative bacteria. • Bacterial spores are forme because of harsh environments. They are a means of survival, not reprouction. • The LPS containe in the outer membrane of gram-negative bacteria consists of three regions: an antigenic O–specic polysaccharie, a core polysaccharie, an an inner lipi A (also calle endotoxin). The lipi A moiety is responsible for proucing fever an shock conitions in patients infecte with gram-negative bacteria. • Base on atmospheric requirements, bacteria are classie as obligate aerobes, obligate anaerobes, facultative anaerobes, aerotolerant anaerobes, or microaerophiles. • Bacteria utilize two biochemical pathways, fermentation an oxiation (aerobic respiration), to catabolize carbohyrates to prouce energy. • The three mechanisms by which genetic material may be transferre from one bacterium to another are transformation, transuction, an conjugation.

LEARNING ASSESSMENT QUESTIONS

1. Explain why the laboratory scientist in the Case in Point shoul repeat the Gram stain proceure on the exuate. 2. What might have occurre to make the Gram stain results invali? 3. Differentiate the role of pili from the role of agella. 4. What is the role of the bacterial capsule in the pathogenesis of infectious iseases? 5. Why is lipopolysaccharie (LPS) a signicant outer-membrane structure in gram-negative bacteria? 6. A bacterium that grows only on plates incubate in the absence of oxygen woul be categorize as a(n): a. Aerotolerant anaerobe. b. Facultative anaerobe. c. Obligate anaerobe. d. Obligate aerobe. 7. Fimbriae present on the outer surface of bacteria are use for: a. Aherence to surfaces. b. Antimicrobial resistance. c. Sexual reprouction. d. Bacterial motility.

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PART 1

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Bacterial cell structure, physiology, metabolism, and genetics

8. All of the following are characteristic of fermentation except: a. It begins with the breakown of pyruvic aci. b. It follows glycolysis an prouces reuce nicotinamie aenine inucleotie (NADH). c. It prouces acis, alcohols, an gases. d. It can occur in the presence of oxygen. 9. Why are oler bacterial cells more easily ecolorize than cells from younger colonies? 10. Why are spore-forming organisms more resistant to environmental stress than non–spore-forming species? 11. Explain the three ways in which genetic material can be transferre from one bacterium to another. 12. For the following DNA, write the complementary sequence. Inclue labeling the 3′ an 5′ en. 3′ TTACGGACAAC 5′: ________________. 13. In RNA, thymine is replace by ________________. 14. In bacteriophage, how oes lysogeny iffer from the lytic cycle? 15. Nutrient an trypticase soy broths are culture meia that are use primarily to satisfy the growth requirements of bacteria an support the growth of most nonfastiiousorganisms. This type of meia is classie as: a. Nutritive. b. Selective. c. Differential. d. Enriche. 16. A patient was seen in the emergency epartment with fever an symptoms of isseminate intravascularcoagulation, sepsis, an shock. Bloo cultures taken from the patient grew gram-negative bacteria. Which of the following cellular structures mightbe responsible for this conition? a. Pili b. Capsule c. Enotoxin d. Spore

17. Which of the following best escribes an organism that cannot use oxygen but will not be kille by oxygen if expose to it? a. Aerotolerant anaerobe b. Obligate anaerobe c. Facultative anaerobe d. Obligate aerobe 18. An encapsulate organism is consiere to be virulent because it can meiate which of the following? a. Form spore b. Inhibit phagocytosis c. Increase mutation rate d. Enhance motility

BIBLIOGRAPHY Baumann, R. W. (2017). Microbiology (5th e.). San Francisco: Pearson. Centers for Disease Control an Prevention. (2021). SARS-CoV-2 variant classi cations and denitions. U.S. Department of Health an Human Services. Available at: https://www.cc.gov/coronavirus/2019-ncov/ variants/variant-classi cations.html. (Accesse 19 February 2022). Murray, P. R., et al. (2021). Medical microbiology (9th e.). St Louis: Mosby. Pommerville, J. C. (2022). Fundamentals of microbiology: an introduction (12th e.). Burlington: Jones & Bartlett. U.S. Foo an Drug Aministration. (2021). SARS-CoV-2 viral mutations: impact on COVID-19 tests. U.S. Department of Health an Human Services. Available at: https://www.fa.gov/meical-evices/ coronavirus-covi-19-an-meical-evices/sars-cov-2-viral-mutationsimpact-covi-19-tests. (Accesse 19 February 2022).

2 Host-parasite interaction Donald C. Lehman

CHAPTER OUTLINE

Origin of microbial biota, 26 Characteristics of indigenous microbial biota, 26 Factors that determine the composition of the usual microbial biota, 27 Composition of microbial biota at different body sites, 27 Normal microbiota of the skin, 27 Normal microbiota of the oral cavity, 28 Normal microbiota of the respiratory tract, 28 Normal microbiota of the gastrointestinal tract, 29 Normal microbiota of the genitourinary tract, 29 Role of the microbial biota in the pathogenesis of infectious disease, 30 Role of the microbial biota in the host defense against infectious disease, 30 Microbial factors contributing to pathogenesis and virulence, 31 Pathogenesis, 31 Routes of transmission, 32 Virulence, 35 Ability to resist phagocytosis, 35 Structures that promote adhesion to host cells and tissues, 35 Ability to survive intracellularly and proliferate, 37 Ability to produce extracellular toxins and enzymes, 37 Host resistance factors, 39 Physical barriers, 39 Cleansing mechanisms, 39 Antimicrobial substances, 40 Phagocytosis, 41 Inammation, 42 Immune responses, 42 Mechanisms by which microbes may overcome host adaptive defenses, 47 Bibliography, 49

OBJECTIVES

After reading and studying this chapter, you should be able to: 1. Dene the following terms: parasitism, indigenous biota, commensal, symbiont, opportunist, transient biota, carrier, true pathogen, opportunistic pathogen, virulence, and zoonosis 2. Explain how the following factors determine the composition of the microbial biota at various body sites: • Amounts and types of nutrients available in the environment • pH • Oxidation-reduction potential • Resistance to antibacterial substances 3. List the predominant microbiota of various body sites in a healthy individual. 4. Evaluate the role of the indigenous microbiota in the pathogenesis of infectious disease. 5. Evaluate the role of the indigenous biota in host defense against infectious diseases. 6. Differentiate the mechanisms of infections caused by true pathogens from infections caused by opportunistic pathogens. 7. Name the routes of transmission that microorganisms use to initiate infection in a host, and give examples of each. 8. Discuss the conditions that must be present or events that must occur for a microorganism to cause disease. 9. Describe the characteristics of infectious agents that enable them to cause disease in the host. 10. Describe the innate and adaptive factors and mechanisms by which the human host is protected from microbial invasion. 11. Discuss the sequence of events in the phagocytosis and killing of an infectious agent. 12. Correlate a specic immune deciency with the outcome to the host in preventing infectious diseases. KEY TERMS

Adaptive immunity Adhesin

Anamnestic immune response Antibody

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26

PART 1

Antigen Bacteriocin Carrier Carrier state Cell-mediated immune response Chemotactic agent Chemotaxis Colonization Commensalism Complement system Diapedesis Dissemination Endotoxin Exotoxin Fimbriae Fomites Humoral immune response Iatrogenic infection Immune response Immune system Immunogen Immunoglobulin A (IgA) Immunoglobulin D (IgD) Immunoglobulin E (IgE) Immunoglobulin G (IgG) Immunoglobulin M (IgM) Immunoglobulins Indigenous microbiota

2

Host-parasite interaction

Innate immunity Interferon Lactoferrin Leukocidin Lymphocyte Lymphokine Lysozyme Mutualism Opportunists Opsonin Opsonization Parasite Parasitism Pathogen Pathogenicity Pattern recognition receptor Phagocyte Phagocytosis Pili Receptor Resident microbial biota Respiratory burst Symbiosis Toll-like receptor Transient microbial biota Virulence Zoonoses

Case in point A 71-year-old male patient was treated for right lower extremity cellulitis with a 10-day course of the antibiotic cephalexin. A few days after completing the antibiotic course, he started having loose, watery diarrhea. The patient described having many episodes of diarrhea per day; after 3 days of diarrhea, he came to the emergency department. He also said that he had developed right quadrant abdominal pain over the past 24 hours. While in the emergency department, the patient had ve episodes of loose, watery diarrhea. A bacterial culture of the stool was negative for Salmonella, Shigella, Campylobacter, Yersinia, and Vibrio species, but a polymerase chain reaction assay for the Clostridioides difcile toxin B gene was positive. Issues to consider: After reading the patient’s case history, consider: • Factors that predisposed this patient to his current condition • Clues that indicate the source of the infection • Signicance of the microbiota in protecting the host against pathogenic organisms • Role of the host’s innate and acquired immunity in protecting the host from infection • Signicance of microbial virulence factors in promoting infection

The outcome from the interactions between host and pathogen, a disease-causing agent, is inuenced by numerous factors. The status of the host’s immune system and ability of the host to defend itself from microbial and viral invasion, combined

with microbial and viral factors inherent to the invading pathogen, often determine whether disease occurs. To appreciate and understand the concepts involved in the pathogenesis of infectious diseases, knowledge and understanding of the host-pathogen relationship is important. It is beyond the scope of this book to provide a detailed discussion of the complexities of the immune response. This chapter provides an overview by describing the interactions between the host and infectious agents in the pathogenesis of disease. The rst part of the chapter describes the origin of the indigenous (resident) microbial biota (microbiota) and the composition at different body sites. It presents the role of the microbial biota at each body site in the host immune defense and as a source of opportunistic infections. Factors that determine the composition of the microbiota at different body sites are described. The second part of the chapter discusses the virulence factors that contribute to the invasiveness of organisms, protective mechanisms the host employs, and how microbes can evade the host’s defenses. Lastly, this chapter describes factors that can make the host more susceptible to infections and how microbes are transmitted.

Origin of microbial biota The fetus is in a sterile environment. During delivery and the rst few days of life, the newborn is introduced to the many and varied microorganisms present in the environment. Many organisms can nd an area on or in the infant into which they can adapt. Microorganisms that nd their niche colonize various anatomic sites and become the predominant organisms. Colonization is growth of microbiota in or on a body site without the production of damage or notable symptoms. Other microorganisms are transient or fail to establish themselves at all. As the infant grows, the microbial biota eventually resemble the microbiota seen in older individuals. Once established onto or into a particular body site of the host, microorganisms develop a particular relationship, or symbiosis, with that host. Symbiosis is dened as the association of two organisms of different species living together. The organisms are called symbionts. The host-microbe relationship, depending on the circumstance, may be one of commensalism, mutualism, or parasitism. Symbiosis as a biological relationship in which both (host and organism) benet from one another may be described as mutualism. Lactobacilli in the urogenital tract of women offer a mutual association: the lactobacilli provide the host protection by preventing colonization of pathogenic species at that site while they derive nutrients from the host. In the relationship in which the organism benets but there is no benecial or harmful effect on the host, the association is called commensalism. Klebsiella pneumoniae is a commensal species in the gastrointestinal tract of humans. In parasitism, one species (parasite) benets at the expense of the other (host). The parasite Entamoeba histolytica is a pathogenic intestinal amoeba that derives nutrients from the host at the expense of the host, causing intestinal ulcers and amebic dysentery.

Characteristics of indigenous microbial biota Microorganisms that are commonly found on or in body sites of healthy persons are called normal or indigenous microbiota. The different body sites may have the same or different microbiota,

Composition of microbial biota at different body sites

depending on conditions. Local conditions select for organisms that are suited for growth in a particular area. For example, the environment found on the dry skin surface is different from the environment found on the moist surfaces in the oral cavity; therefore the microbiota are different at the two sites. Microorganisms that colonize an area for months or years represent resident microbiota, whereas microorganisms that are present at a site temporarily, for a shorter period, represent transient microbiota. Transient microbiota come to “visit” but do not stay. These microorganisms are eliminated either by the host-inherent immune defenses or by competition with the resident biota. Some pathogenic organisms may establish themselves in a host without manifesting symptoms. However, these hosts, called carriers, can transmit the infectious agent. The condition of these hosts is called the carrier state. The carrier state may be acute (short-lived) or chronic (lasting for months, years, or permanently). An example of a chronic carrier state is found in post–Salmonella Typhi infection. This organism can establish itself in the bile duct and can be excreted in the stool over years. In contrast, Neisseria meningitidis can be found in the nasopharynx of asymptomatic individuals during an outbreak of meningitis. After a few days or weeks at most, these individuals may no longer harbor the organism, in which case the carrier state would be termed acute. The most transient of carrier states is the inoculation of a person’s hands or ngers with an organism, for example, Staphylococcus aureus that has colonized the person’s anterior nares and is transmitted to the hands, that is carried only until the hands are washed. The organisms colonizing different body sites play a signicant role in providing host resistance to infections. The efciency of the microbial biota in providing protection to the human host is indicated by the relatively small number of infections caused by these organisms in immunocompetent individuals. Nevertheless, these organisms may cause signicant, often serious, infections or may exacerbate existing infections in individuals who are immunocompromised. Knowing the benet of the normal microbiota, individuals can ingest probiotics, a suspension of live bacteria that normally colonize the gastrointestinal tract, to reestablish the microbiota. This is useful in some patients who have taken oral broad-spectrum antimicrobial agents that have reduced the number of resident bacteria in the intestines.

Factors that determine the composition of the usual microbial biota Which microorganisms are present at a particular body site is inuenced by nutritional and environmental factors, such as the amount and types of nutrients available at the site. For example, more organisms inhabit moist areas than dry areas. Moist skin areas are dominated by diphtheroids, nonpathogenic corynebacteria. Although lipids and fatty acids are bactericidal to most bacteria, Propionibacterium spp. colonize the ducts of hair follicles because these bacteria can break down the skin lipids to fatty acids. The afnity of microorganisms for a specic site depends on the ability of the organisms to resist the antibacterial effects of substances such as fatty acids, bile, or lysozyme. The composition of the microbial biota is also affected by pH. For example, the female genital tract microbiota depends on the pH of that environment, which in women

27

of childbearing age is approximately 4.0 to 5.0. Many bacteria do not survive at this extreme pH range. Another example is the fecal biota found in infants who are breast-fed, which differs from the fecal biota in infants fed with cow’s milk. Human milk has a high lactose concentration and maintains a pH of 5.0 to 5.5, an environment supportive of Bidobacterium spp. Cow’s milk has a greater buffering capacity and is less acidic. Infants fed with cow’s milk do not have the high colonization rate by Bidobacterium spp. found in breast-fed infants but have instead a colon microbiota similar to that seen in older children and adults (see Box 2.5). In areas of the body that have a low oxidation-reduction potential, the environment supports only organisms capable of fermentation, such as is seen in the gingival crevices colonized with Bacteroides and Fusobacterium The previously described environmental conditions may change with age, nutritional status, disease states, and drug or antimicrobial therapy use. These changes can predispose an individual to infection by the indigenous biota, a type of infection referred to as an opportunistic infection. For example, two groups at increased risk for gram-negative bacillus pneumonia are diabetics and alcoholics. Also, antimicrobial agents can reduce a particular population of resident bacteria, allowing the proliferation of others. An increase in age brings with it a decrease in the effectiveness of the immune response. As a result, the incidence of infection caused by opportunistic organisms increases.

Case check 2.1 The patient in the Case in Point developed diarrhea following a course of antimicrobial therapy. As described in the text, antimicrobial therapy can alter the usual biota of nearly any body site. In this case, antimicrobial therapy altered the normal bacterial biota of the gastrointestinal tract and allowed C. difficile to cause an infection. In this case C. difficile may have been a component of this patient’s bowel biota, or, more likely, the patient acquired C. difficile from the environment.

Composition of microbial biota at different body sites Human microbiome studies using molecular sequencing strategies to determine which organisms reside in or on the human body have shown that the human host is colonized by many different species of microorganisms. For example, in the oral cavity alone, approximately 500 different species have been characterized. The effectiveness of the various host defenses is evidenced by the relatively low incidence of infection in immunocompetent individuals by members of the usual or indigenous microbiota. However, infections caused by members of the microbial biota are frequently encountered among immunocompromised patients. The clinical microbiologist must be able to recognize and identify the types of microorganisms found at the various body sites.

Normal microbiota of the skin Normal skin has numerous mechanisms to prevent infection and protect the underlying tissue from invasion by potential

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pathogens. These mechanisms include a physical barrier separating microorganisms from the tissues, presence of fatty acids that inhibit many microorganisms, excretion of lysozyme by sweat glands, and desquamation of the epithelium. The skin contains a wide variety of resident microorganisms, most of which are found on the most supercial layers of skin cells and the upper parts of hair follicles. Scrubbing and washing may reduce the number of bacteria present on the skin by about 90% but do not eliminate completely the organisms present, and their numbers return to normal within a few hours. The three types of sweat glands in humans are eccrine, apocrine, and apoeccrine. Eccrine glands are distributed abundantly in the skin and secrete mainly water and electrolytes. Apocrine glands, found mostly in the armpits and anogenital regions, produce viscous secretions. Sebaceous glands are connected to hair follicles and secrete sebum. The composition of the microbiota on the skin depends on the activity of the sebaceous or sweat glands. Organisms concentrate the most in moist areas, such as the armpit, groin, and perineum. The apocrine glands in these areas secrete substances metabolized by the skin bacteria, releasing odorous amines—the source of “body odor.” Aerobic diphtheroids are usually found in moist areas such as the axillae and between the toes. Staphylococcus epidermidis, Cutibacterium (Propionibacterium) acnes, and Propionibacterium spp. reside in hair follicles and metabolize products secreted by sebaceous glands. They are resistant to skin lipids and fatty acids as well as to supercial antiseptic agents commonly used to cleanse the skin. The presence of skin bacteria benets the host by inhibiting the growth of more pathogenic bacterial species by competing for nutrients. In addition, S. epidermidis and C. acnes secrete bacteriocins and induce the production of antimicrobial peptides by host cells, such as keratinocytes, sebocytes, and immune cells. Bacteriocins are peptides produced by bacteria to inhibit the growth of similar or closely related bacteria. Microorganisms such as C. acnes colonize the deep sebaceous glands. Supercial antisepsis of the skin does not eliminate this organism, which may be found as a contaminant in culture specimens obtained by invasive procedures (e.g., blood, cerebrospinal uid) because of contamination of the needle. Box 2.1 lists the microorganisms most commonly found on the skin. Other organisms have been isolated from the skin but are found only occasionally or rarely and are not listed. BOX 2.1 Microorganisms found on the skin Common residents Candida spp. Cutibacterium acnes Micrococcus spp. Staphylococcus spp. Propionibacterium spp. Diphtheroids (Corynebacterium spp.)

Less common or transients Streptococcus spp. Acinetobacter spp. Gram-negative rods (fermenters and nonfermenters) Moraxella spp.

Normal microbiota of the oral cavity The mouth contains large numbers of bacteria, with the genus Streptococcus predominating. Many organisms bind to the buccal mucosa and tooth surface. Unlike skin, teeth do not have a shedding epithelium. Bacteria on teeth and along the gums form a multispecies biolm (plaque), allowing longterm colonization. Without proper oral hygiene, this can lead to dental caries and periodontitis. The bacterial plaque that develops on teeth may contain 10 11 streptococci per gram. Plaque also results in a low oxidation-reduction potential at the tooth surface; this supports the growth of strict anaerobes, particularly in crevices and in the areas between the teeth. Box 2.2 provides a partial list of microorganisms found in the oral cavity.

Normal microbiota of the respiratory tract The respiratory tract, commonly divided into the upper and lower respiratory tract, is responsible for the delivery of air from outside of the body to the pulmonary tissues responsible for exchange of oxygen and carbon dioxide. The upper respiratory tract is composed of the mouth, nasopharynx, oropharynx, and larynx; the lower respiratory tract is composed of the trachea, bronchi, and pulmonary parenchyma. The trachea, bronchi, and lungs are protected by the action of ciliary epithelial cells and by the movement of mucus along the trachea. The tissues of these structures are normally sterile because of this protective action. The mouth, nasopharynx, and oropharynx are colonized predominantly with viridans streptococci such as Streptococcus mitis group, S. mutans group, S. anginosus group, and S. salivarius group. Moraxella catarrhalis, Neisseria spp., and diphtheroids also colonize the upper respiratory tract. Obligate anaerobes reside in the gingival crevices, where the anaerobic environment supports these organisms. The organisms found in the mouth, nasopharynx, oropharynx, and nose, although similar, show some differences. Box 2.3 lists common microorganisms encountered in the nose and

BOX 2.2 Microorganisms found in the oral cavity Common residents Staphylococcus epidermidis Streptococcus anginosus group Streptococcus mitis group Streptococcus salivarius group Streptococcus mutans group Peptostreptococcus spp. Actinomyces israelii Bacteroides spp. Prevotella/Porphyromonas Treponema denticola Treponema refringens Veillonella spp.

Less common or transients Candida albicans Enterococcus spp. Eikenella corrodens Fusobacterium nucleatum Staphylococcus aureus

Composition of microbial biota at different body sites

BOX 2.3 Microorganisms found in the nose and nasopharynx

BOX 2.4 Microorganisms found in the oropharynx

Common residents

Common residents

Diphtheroids (Corynebacterium spp.) Haemophilus parainfluenzae Staphylococcus aureus Staphylococcus epidermidis Streptococcus spp.

α-Hemolytic and nonhemolytic streptococci Diphtheroids (Corynebacterium spp.) Staphylococcus aureus Staphylococcus epidermidis Streptococcus pneumoniae Streptococcus mutans group Streptococcus mitis group Streptococcus anginosus group Streptococcus salivarius group Haemophilus parainfluenzae Moraxella catarrhalis Bacteroides spp. Prevotella/Porphyromonas Fusobacterium necrophorum

Less common or transients Haemophilus influenzae Moraxella spp. Neisseria meningitidis Streptococcus pneumoniae

nasopharynx. Attachment to host cells by streptococci and staphylococci is initially caused by hydrophobic or electrostatic interactions via bacterial surface polymers, such as pili and teichoic acids. Pili are found in the pathogenic S. pyogenes, S. agalactiae, and S. pneumoniae but are rare among the oral streptococci. Opportunistic pathogens such as S. aureus, found in approximately 30% of healthy individuals, colonize the anterior nares. The population of the nasopharynx mirrors that of the nose, although the environment is different enough from the environment of the nose to select for several additional organisms. Haemophilus inuenzae, S. pneumoniae, and N. meningitidis, all potential pathogens, can also be found occasionally in the nasopharynx of healthy individuals. Patients hospitalized for several days might become colonized in the upper respiratory tract by gram-negative bacteria, particularly members of the order Enterobacterales. The normal biota of the oropharynx is listed in Box 2.4

Normal microbiota of the gastrointestinal tract The gastrointestinal tract comprises the esophagus, stomach, small intestine, and colon. The gastrointestinal tract is equipped with numerous defenses and effective antimicrobial factors. Because intestinal pathogens are usually acquired by ingestion of organisms contained in contaminated food or drink, host defenses against infections are present throughout the intestinal tract. Despite the presence of antimicrobial factors, the intestinal tract is thought to be colonized by over 35,000 bacterial species. The relation between the gut microbiota and human health is being increasingly recognized. The microorganism population is lowest in the esophagus, about 10 microbes per gram of content. Some microorganisms colonize the esophagus, and others are present in ingested food as transient biota. The stomach contains gastric juices, acids (pH 2), and enzymes that help protect the stomach from microbial attack. Many microorganisms are susceptible to the acid pH of the stomach and are destroyed, except for the spore-forming bacterial species in their spore phase andthe cysts of parasites. Even with the hostile environment of the stomach, some bacteria belonging to the genera Streptococcus, Enterococcus, and Prevotella as well as the opportunistic pathogen Helicobacter pylori can inhabit the stomach. These organisms associate themselves with the stomach lining,

29

Less common or transients Haemophilus influenzae Neisseria meningitidis Gram-negative rods

protected by the layer of mucus that lines the stomach. Organisms that are pH-susceptible and survive are generally protected by being enmeshed in food as they move to the small intestine. The stomach acidity reduces the number of viable organisms that reach the small intestine. The small intestine contains fewer microorganisms compared with those usually present in the colon. Microorganisms prevalent in the colon may produce a count of 10 12 bacteria per gram of solid material. In fact, the colon contains over 70% of all microbes found in the body. Obligate anaerobes, such as Bacteroides, Bidobacterium, Clostridium, Prevotella, and Porphyromonas, far outnumber the facultative gram-negative bacilli, making up more than 90% of the microbial biota of the large intestine. Gram-positive cocci belonging to the genera Streptococcus and Enterococcus and yeasts are also present in the large intestine. A single-layer epithelium, renewed every 5 days, lines the intestine. The tight junction spaces between epithelial cells make it difcult for bacteria to penetrate the epithelium. Many of the bacteria colonizing the intestines do so using pili. The gastrointestinal tract population may be altered by antimicrobial agents. In some cases, certain populations or organisms are eradicated or suppressed, and other members of the indigenous biota can proliferate. For example, C. difcile or the yeast Candida albicans can ourish in the intestinal tract of some people who are taking an oral broad-spectrum antimicrobial agent. This alteration can be the cause of a severe necrotizing enterocolitis (C. difcile), diarrhea (C. albicans, S. aureus), or other superinfection. The bacteria constituting the usual intestinal biota also carry out various metabolic degradations and nutrient production that appear to play a role in the health of the host. The organisms found in the gastrointestinal tract are summarized in Box 2.5

Normal microbiota of the genitourinary tract The kidneys, bladder, cervix, and fallopian tubes are normally sterile, although a few organisms originating from the perineum can be found in the distal urethra, particularly in women.

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BOX 2.5 Microorganisms found in the gastrointestinal tract

BOX 2.6 Microorganisms found in the genitourinary tract

Bacteroides spp. Bifidobacterium spp. Clostridium spp. Enterobacterales Enterococcus spp. Eubacterium spp. Fusobacterium spp. Lactobacillus spp. Peptostreptococcus spp. Peptococcus spp. Porphyromonas spp. Prevotella spp. Streptococcus spp.

Common residents Bacteroides spp. Clostridium spp. Enterococcus spp. Lactobacillus spp. Peptostreptococcus spp. Staphylococcus epidermidis Diphtheroids (Corynebacterium spp.)

Less common or transients Group B streptococci Acinetobacter spp. Candida albicans Enterobacterales Staphylococcus aureus

Case check 2.2 In the Case in Point, antimicrobial therapy led to an alteration of the patient’s normal gastrointestinal tract biota. As shown in Box 2.5, Clostridium spp. and C. difficile can be part of the normal gastrointestinal tract microbiota in humans. In this case, eliminating some of the bacterial gut biota allowed C. difficile organisms to grow unchecked and cause an infection called C. difficile– associated disease that is often associated with prolonged antimicrobial therapy, as discussed in Chapter 22.

The urethra is colonized in its outermost segment by organisms found on the skin. The composition of the vaginal microbiota is consistent with hormonal changes and age. Before puberty and in postmenopausal women, vaginal biota primarily consists of yeasts, gram-negative bacilli, and gram-positive cocci. During childbearing years, high estrogen levels promote the deposition of glycogen in vaginal epithelial cells. Lactobacilli metabolize glycogen from vaginal epithelial cells to maintain a low pH, creating an environment that is inhibitory to many organisms. However, the low pH encourages colonization of the vagina with lactobacilli, anaerobic gram-negative bacilli, and gram-positive cocci. Microorganisms usually isolated from the genitourinary tract are listed in Box 2.6

Role of the microbial biota in the pathogenesis of infectious disease Some organisms that make up the microbial biota live off the host’s nutrients, but in most cases, they provide some benet to the host, creating a symbiotic relationship with the host, as mentioned earlier. However, certain members of the normal microbiota are opportunists; they cause disease when their habitat is altered or when the host’s immune system is compromised. In the case of trauma, either accidental or surgical, enough of the normal microbiota found in the traumatized area may reach sterile or other areas in the body where these organisms are not part of the microbial biota. For example, patients who undergo surgery become susceptible to infections caused by organisms that colonize the surgical site (e.g.,abdominal cavity); leakage following perforation of the colon spills the contents of the colon into the peritoneal cavity, leading to an infection by the colon biota.

The host’s immune response may be reduced or altered because of suppression by immunosuppressive drugs, chemotherapy, or radiation. Individuals with lymphoma, leukemia, or other blood disorders in which there is a functional defect in phagocytic activity or a decrease in the number of functioning cells or in which chemotactic activity is impaired also may have a reduced immune response. Members of the microbial biota also may initiate an infection or make an infection more serious in patients with chronic illnesses, including diabetes or severe hepatic disease such as cirrhosis.

Role of the microbial biota in the host defense against infectious disease The microbial biota provide benecial effects, and the development of immunologic competence depends on this biota. Vitamins and other essential nutrients are synthesized by certain bacteria in the intestine and appear to contribute to the overall health of the host. The immune system is constantly primed by contact with microorganisms. In the gut, normal microbiota interact with host Toll-like receptors (TLRs), such as TLR5 found on epithelial cells and dendritic cells. Activation of TLR5 recruits T and B lymphocytes (T and B cells), leading to the production of immunoglobulin A (IgA) to limit the overcolonization of gut microbiota. Animals born and raised in a germ-free environment have a poorly functioning immune system. Exposure to otherwise innocuous organisms can be fatal to such animals. Likewise, consider how a sterile environment might affect a newborn. Antibody production would not be stimulated, and the mononuclear phagocyte system would remain undeveloped. Serum immunoglobulin G (IgG) and other antibodies effective against microorganisms would be suppressed, which would make the individual more susceptible to pathogenic microorganisms. Without activation by microorganisms and the supporting action of antigen-presenting cells and cytokines, cell-mediated immunity would not develop normally. The microbial biota produce conditions at the microenvironmental level that block colonization by extraneous pathogens (e.g., competition for nutrients and secretion of bacteriocins). These proteins are produced by a variety of

Microbial factors contributing to pathogenesis and virulence

gram-positive and gram-negative bacteria and appear to give the secreting bacterium an advantage because they can eliminate other bacteria that would compete for nutrients and space. Some species of bacteria produce metabolic byproducts that result in a microenvironment that is hostile to potential pathogens. When the composition of the indigenous biota is altered (e.g., by broad-spectrum antimicrobial therapy), other organisms capable of causing disease may ll the void. For example, gastroenteritis caused by Salmonella is generally not treated with antimicrobial agents and is better eliminated by natural exclusion by the colon biota. If the microbial biota are eliminated, such as in patients receiving antimicrobial therapy, resistant or more pathogenic species may be able to establish infection. For example, the yeast C. albicans can multiply and cause diarrhea or infections in the mouth or vagina when the normal biota have been eliminated. The microbial biota play an important role in both health and disease. Although reduction of the usual biota can have profound negative effects, many common infections are caused by members of the resident biota. Knowledge of the role of these organisms in the pathogenesis of an infectious disease is helpful in assessing their signicance when they are isolated from clinical samples.

Microbial factors contributing to pathogenesis and virulence Pathogenesis Pathogenicity is the ability of a microbe to produce disease in an individual. An organism may be described as a true pathogen or an opportunistic pathogen. True pathogens are organisms recognized to cause disease in healthy immunocompetent individuals a high percentage of the time. Bacterial species such as Yersinia pestis and Bacillus anthracis are pathogenic in nearly all situations; when these species are recovered in clinical samples, they are clinically signicant. Today, individuals with disease are living longer and are more likely to undergo invasive medical procedures, organ transplantation, and insertion of prosthetic devices, making them more susceptible to infections. As a result, organisms of the normal biota are being seen with increasing frequency in those patients and individuals who are immunosuppressed. H. inuenzae colonizes the upper respiratory tract of healthy individuals without causing disease, but given the opportunity, it can rapidly produce a life-threatening infection involving the lower respiratory tract. Organisms such as S. epidermidis do not cause disease under usual conditions but can induce an infectious process in patients with prosthetic devices. H. inuenzae and S. epidermidis are called opportunistic pathogens, and the infections they cause are called opportunistic infections. Table 2.1 lists common opportunistic microorganisms and the conditions with which they are commonly associated. Because of compromised hosts, our denition of pathogen must be expanded to apply to virtually any microorganism when conditions for infection are met. In deciding whether a particular organism that has been isolated is a pathogen, we also must consider the human host from whom the organism was isolated and whether that host has underlying disease

31

Table 2.1 Abbreviated list of opportunistic microorganisms Conditions compromising host defenses

Organism(s)

Foreign bodies (catheters, shunts, prosthetic heart valves)

Staphylococcus epidermidis Cutibacterium acnes Viridans streptococci Serratia marcescens Pseudomonas aeruginosa Aspergillus spp. Candida albicans

Alcoholism

Streptococcus pneumoniae Klebsiella pneumoniae

Burns

Pseudomonas aeruginosa Acinetobacter baumanniicalcoaceticus complex Staphylococcus aureus

Hematoproliferative disorders

Cryptococcus neoformans Varicella-zoster virus

Cystic brosis

Pseudomonas aeruginosa Burkholderia cepacia

Immunosuppression (drugs, congenital disease)

Aspergillus spp. Candida albicans Pneumocystis jirovecii Diphtheroids (Corynebacterium spp.) Pseudomonas spp. Staphylococcus spp. Cytomegalovirus Herpes simplex virus Varicella-zoster virus

that may increase susceptibility to infection. For example, the potential pathogen list for a healthy 20-year-old individual is much shorter than that for a healthy 90-year-old individual, a transplant recipient, or a 20-year-old individual with acquired immunodeciency syndrome (AIDS). Almost any organism in the right place can cause an infection, and this must be considered when performing invasive procedures because an inadvertent result of intervention can be the transfer of an organism from where it is present as part of the indigenous biota to a place where it can replicate and cause infection. Contamination by resident skin biota of a surgical incision can lead to a serious wound infection. An iatrogenic infection is an infection that occurs as the result of medical treatment or procedures. For example, many patients who have indwelling urinary catheters develop a urinary tract infection. Although placement of the catheter was a necessary procedure in the medical treatment of the individual, its use may result in an infection. Patients who are given immunosuppressive drugs because they have received an organ transplant are more susceptible to infection. Because any infection in such a patient would probably be the result of the physician-ordered drug therapy, it would be considered an iatrogenic infection.

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Routes of transmission The rst step in initiating an infection is for the infectious agent to gain access to the host. The route by which a pathogen can be transmitted to a susceptible host is an important factor in the establishment of infection. The agent must be able to evade host defenses and colonize the tissue at the point of entry. Although some organisms may be naturally transmitted by more than one route, most have a preferred portal of entry. These routes can be characterized as through air (inhalation), via food and water (ingestion), through close contact (includes sexual transmission), through cuts and bites, and via arthropods; animal diseases that can infect humans are transmitted through animal contact (zoonoses). The routes of transmission are summarized in Table 2.2. Fig. 2.1 shows the routes of entry to and exit of microbes from the body.

Airborne transmission Respiratory spread of infectious disease is common and is generally an efcient way to enter a host. Often, the respiratory secretions from an infected host are aerosolized by coughing, sneezing, and talking. Very small particles, referred to as aerosols, are the residue from the evaporation of uid from larger droplets and are light enough to remain airborne for long periods. Large respiratory droplets fall to the ground before they evaporate contaminating surfaces. This results in droplet-contact spread in which disease transmission occurs because of touching the contaminated surface. Pathogens that are spread through the air generally must be resistant to drying and inactivation by ultraviolet light. Some infectious agents may be transmitted by dust particles that have become airborne. As discussed earlier in this chapter, the body has many defenses against airborne infectious agents. The nasal turbinates, oropharynx, and larynx provide a twisting, mucus-lined passageway that makes direct access to the lower respiratory tract mechanically difcult. In addition, the lower portions of the respiratory tract contain ciliary epithelium that sweeps organisms upward. For a microorganism to cause disease, it must circumvent these defenses, penetrate the mucous layer, and attach to the epithelium. The host also produces secretory IgA, lysozyme, alveolar macrophages, and other factors that act on the pathogen that manages to get beyond the physical barriers. Respiratory tract infections are the most common reason that patients of all ages seek medical attention. Although most upper respiratory tract infections are self-limiting and can be treated with over-the-counter medications, some are more serious. Streptococcal sore throat, sinusitis, otitis media, acute epiglottitis, and diphtheria can be serious and even life-threatening. Viral diseases causing the common cold and infectious mononucleosis are usually not life-threatening but can result in much discomfort and absenteeism from work or school. Although all of the diseases mentioned can be spread via aerosols, some may also be transmitted via the ngers and hands; this is especially true of the common cold–causing rhinoviruses. The ngers and hands are contaminated with infectious nasal secretions because of hand-to-nose contact. The infectious viral particles are passed from the infected individual to a susceptible recipient via hand-to-hand or hand-toface contact. The recipient transmits the virus picked up from

the infected individual by touching the face and nose. In this case, the disease is transmitted via the respiratory route but not in the normal, classic manner of respiratory transmission. Transmission may also result from contact with inanimate objects contaminated with the infectious agent (fomites). For example, a doorknob is contaminated by the hand and ngers of an infected individual, and the virus is transmitted to a susceptible person’s hand and ngers when that person opens the door. Control of such transmission is often as simple as frequent handwashing. Infections of the lower respiratory tract are less common but more serious than infections of the upper respiratory tract. The organisms causing these infections have managed to bypass host defenses, or the host defenses have been compromised (e.g., by alcoholism, heavy smoking), allowing the pathogen access to the normally sterile, deeper portions of the respiratory tract. The most common microorganism causing lower respiratory tract infection in individuals older than 30 years of age is S. pneumoniae. Risk increases with age. Although the pneumococcus is the most common cause of community-acquired pneumonia, it is also often seen in aspiration pneumonia, a common type of hospital-acquired pneumonia. Pneumococcal pneumonia begins suddenly and is a serious, life-threatening disease, particularly in older patients. The bacteria produce a large capsule that resists phagocytosis. In chronic lower respiratory tract infections, the survival of the infecting agent within phagocytes plays a role in the pathogenic mechanism. As the agent of tuberculosis, a chronic debilitating infection, M. tuberculosis is the classic example of an intracellular pathogen. This organism is highly virulent, is invasive, survives well, and multiplies within phagocytes. Infections are often chronic and result in granuloma formation.

Transmission by food and water Transmission of gastrointestinal infections is usually a result of ingestion of contaminated food or water. In some situations, infection occurs via the fecal-oral route. The digestive tract is colonized with vast numbers of different microorganisms. Under usual conditions, the gut biota maintain a harmless relationship with the host. Gastric enzymes and acids in the stomach prevent survival of most organisms, but many survive and colonize the small intestine and colon. Gastrointestinal infections result from organisms that survive the harsh conditions of the stomach and competition with the microbial biota and then produce damage to the tissues of the gastrointestinal tract. This damage is a result of either ingestion of a preformed toxin or disruption of the normal functioning of the intestinal cells by invasion of the pathogen or production of a toxin within the intestine. Organisms that can cause disease by means of a preformed toxin, produced outside the body, include Clostridium botulinum, Bacillus cereus, and S. aureus. The severity of disease ranges from mild diarrhea to rapidly fatal intoxication. Food poisoning by B. cereus and S. aureus is relatively common and is self-limiting. Botulism, caused by C. botulinum, although rare, can be life-threatening. Other bacteria produce a toxin after infecting the intestinal tract. Generally, to be effective as a pathogen, an organism must survive, adhere to, and colonize the intestinal mucosa and either produce a toxin or invade deeper tissues. A commonly seen cause of diarrhea and intestinal infection is

Microbial factors contributing to pathogenesis and virulence

33

Table 2.2 Common routes of transmissiona Route of exit Respiratory

Route of transmission

Example

Aerosol inhalation

Inuenza virus, tuberculosis, Neisseria meningitidis

Nose or mouth (droplets) → hand or object → nose

Common cold (rhinovirus)

Salivary

Direct salivary transfer (e.g., kissing)

Oral-labial herpes; infectious mononucleosis

Gastrointestinal

Stool → hand → mouth and/or stool → object → mouth

Enteroviruses; hepatitis A

Stool → water or food → mouth

Salmonellosis; shigellosis

Skin discharge → air → respiratory tract

Varicella; poxvirus infection

Skin to skin

Human papillomavirus (warts); syphilis

Transfusion or needle prick

Hepatitis B; cytomegalovirus infection; malaria; HIV

Insect bite

Malaria; relapsing fever; West Nile virus

Urethral or cervical secretions

Gonorrhea; herpes simplex; Chlamydia infection

Skin Blood Genital secretions Urine Eye Zoonotic

a

Semen

Cytomegalovirus infection

Urine → hand → catheter

Hospital-acquired urinary tract infection

Urine → aerosol (rare)

Tuberculosis

Conjunctival

Adenovirus

Animal bite

Rabies, Pasteurella multocida

Contact with carcasses

Tularemia

Arthropod

Lyme disease, Rocky Mountain spotted fever, plague

The examples cited are incomplete, and in some cases more than one route of transmission exists.

HIV, Human immunodeciency virus.

Infection

Influenza Diphtheria Shigella dysentery Ascending pyelonephritis

Skin, throat, lung, intestine, urinary tract

Lymph node

Dengue Malaria Typhus

Lymphatic

Bloodstream Brain

Poliomyelitis Liver Skin

Lungs

Kidney

Salivary gland

Hepatitis B Yellow fever Mumps Rabies Chickenpox Yaws

Measles Rubella

Hematogenous pyelonephritis

Fig. 2.1 Routes of entry and exit. (Reprinted from Mims, C.A., et al. (2001). Mims’ pathogenesis of infectious disease [5th ed.]. San Diego: Elsevier Ltd. With permission from Elsevier.)

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Escherichia coli. This organism is a member of the intestinal resident biota; however, some strains of E. coli produce virulence factors that cause alterations in the biochemical activity of the intestinal epithelial cells, resulting in problems with uid and electrolyte control by the intestinal cells. These strains of E. coli, referred to as diarrheagenic, are a common cause of traveler’s diarrhea and other intestinal problems. Vibrio cholerae, the cause of cholera, produces an enterotoxin that causes an outpouring of uid from the cells into the lumen of the intestine. Massive amounts (20 L per day) of uid can be lost. Other intestinal pathogens include C. difcile (see Case in Point), Shigella spp., Aeromonas hydrophila, Campylobacter jejuni, and Salmonella spp. The infective dose, severity, and incidence of disease vary with the agent. Numerous viruses also cause diarrheal disease. They multiply within the cells of the intestinal mucosa and affect the normal function of the cells. Viral agents in this category include rotavirus, adenovirus, coxsackievirus, and Norovirus spp. The incidence of diarrhea caused by these agents is high, especially when people are in close contact (e.g., in daycare centers, nursing homes, military camps, cruise ships). Some viruses enter via the gastrointestinal tract and infect the liver, such as hepatitis A and E viruses. Numerous parasites, such as Cryptosporidium spp., Giardia lamblia, E. histolytica, and Balantidium coli, also infect the gastrointestinal tract.

ea, or mite bite is a common occurrence in many parts of the world. Diseases spread by arthropods include malaria, relapsing fever, plague, Rocky Mountain spotted fever, Lyme disease, West Nile fever, and untold numbers of regional viral hemorrhagic fevers. In most cases, the infectious agent multiplies in the arthropod, which then transmits the agent while feeding on a human host. This mode of transmission is much more common in the tropics because arthropods are active year-round. However, a signicant number of arthropod-borne diseases occur in temperate zones.

Zoonoses The route of transmission known as zoonosis depends on contact with animals or animal products. Certain organisms causing disease in animals may also infect humans who have contact with them. These diseases may be passed by animal bites (rabies), arthropod vectors (plague), contact with secretions (brucellosis), and contact with animal carcasses and products (tularemia, listeriosis). The diseases are transmitted by the routes already discussed. The common factor is that, regardless of the route, the disease is a disease of animals that is transmitted to humans. A partial list of zoonotic diseases and infecting organisms is provided in Table 2.3

Close contact

Table 2.3 Zoonoses

Typically, the routes of transmission of infectious diseases require close contact. For a respiratory pathogen to be transmitted via aerosols, the susceptible host must be relatively close. However, aerosolized pathogens can stay airborne for some time and be carried by gentle air currents. For this discussion, close contact refers to passage of organisms by salivary, skin, and genital contact. Two prominent infections passed by direct transfer of saliva (e.g., kissing) are herpes simplex virus and Epstein-Barr virus. Skin-to-skin transfer of infectious disease is not as common as for some of the other routes, but diseases such as warts (human papillomavirus), syphilis, and impetigo result when material from infectious lesions inoculates the skin of a susceptible host. The list of sexually transmitted diseases is a long one. In North America, the most commonly transmitted venereal agents are human papillomavirus, Chlamydia trachomatis, Neisseria gonorrhoeae, herpes simplex virus, Treponema pallidum subsp. pallidum (syphilis), Trichomonas spp., and human immunodeciency virus (HIV).

Disease

Organism

Anthrax

Bacillus anthracis

Brucellosis

Brucella spp.

Erysipeloid

Erysipelothrix rhusiopathiae

Leptospirosis

Leptospira interrogans

Tularemia

Francisella tularensis

Ringworm

Trichophyton spp. Microsporum spp.

Lyme disease

Borrelia burgdorferi

Plague

Yersinia pestis

Rocky Mountain spotted fever

Rickettsia rickettsii

Cuts and bites The classic example of a bite-wound infection is rabies. However, human rabies is relatively rare. Of more concern with animal bites, and especially human bites, is infection by the mouth biota. Dog-bite and cat-bite infections often yield Pasteurella multocida, but the possibilities are extensive. Human bites are dangerous because the human oral biota comprises many different organisms in extremely high numbers, including obligate anaerobic bacteria.

Yellow fever

Flavivirus

Encephalitis

Alphavirus

Rabies

Rhabdovirus

Blastomycosis

Blastomyces dermatitidis

Tuberculosis

Mycobacterium bovis

Q fever

Coxiella burnetii

Ornithosis

Chlamydophila psittaci

Gastroenteritis

Campylobacter spp. Salmonella spp.

Listeriosis

Listeria monocytogenes

Giardiasis

Giardia lamblia

Toxoplasmosis

Toxoplasma gondii

Tapeworms

Taenia saginata Taenia solium Diphyllobothrium latum Echinococcus spp.

Trichinosis

Trichinella spiralis

Arthropods Infectious agents can enlist the help of arthropods for transmission among hosts. Infection following a mosquito, tick,

Microbial factors contributing to pathogenesis and virulence

Virulence Virulence is the relative ability of a microorganism to cause disease or the degree of pathogenicity. It is usually measured by the numbers of microorganisms necessary to cause infection in the host. Organisms that can establish infection with a relatively low infective dose are considered more virulent than organisms that require high numbers for infection. For example, because Shigella spp. cause disease with a relatively low infective dose (100 organisms), Shigella is considered a highly virulent organism. This generalization is misleading because the severity of disease caused by different organisms varies from one to another. If a microorganism requires a relatively high infective dose but the disease it causes is often fatal, we tend to think of the microorganism as highly virulent. A different organism may require a low infective dose but produce a relatively mild disease.

Microbial virulence factors Infectious organisms have a wide variety of mechanisms or virulence factors that allow them to persist in a host and cause disease. Some virulence factors, such as capsules and toxins, are used by many organisms. Other virulence factors tend to be specialized and specic to one particular organism, such as the tissue tropism of N. gonorrhoeae. Virulence factors allow the pathogen to evade or overcome host defenses and cause disease, and they encompass functions such as facilitating adhesion to host cells or tissues, inhibiting phagocytosis, enhancing intracellular survival after phagocytosis, and damaging tissue through the production of toxins and extracellular enzymes. Many virulence factors are well dened, such as the diphtheria and cholera toxins, the capsule of S. pneumoniae, and the mbriae of N. gonorrhoeae. Certain microorganisms produce extracellular factors that appear to aid in infection, but the exact role of these factors is unknown.

Ability to resist phagocytosis Phagocytes, or phagocytic cells, such as macrophages and polymorphonuclear cells, play a major role in defending the host from microbial invasion. These cells ingest bacteria and destroy them. The lack of functioning phagocytic cells leaves the host susceptible to overwhelming infection. Therefore an extremely important event in the life of an invading pathogen is avoiding phagocytosis. There are many ways by which microbial species evade phagocytosis; some are listed in Table 2.4 The most common mechanism for evading phagocytosis used by microorganisms is a polysaccharide capsule. Many microorganisms possessing a capsule are highly virulent until removal of the capsule, at which point virulence becomes extremely low. Encapsulated strains of S. pneumoniae and H. inuenzae are associated with highly invasive infections and are known to be more virulent than non-encapsulated strains. Host antibodies directed against the capsule can enhance phagocytosis and also decrease the microbe’s virulence, a process called opsonization. The capsule is usually composed of polysaccharides but can also be made of proteins or a combination of proteins and carbohydrates. The capsule inhibits phagocytosis primarily by masking the cell surface structures that are recognized by receptors on the surface of the

35

phagocytic cell and in the same manner inhibits activation of complement by masking structures to which complement proteins bind. Another bacterial structure that protects organisms from phagocytosis is protein A. Protein A in the cell wall of S. aureus helps the organism avoid phagocytosis by interfering with the binding of the host’s antibodies to the surface of the organism. Antibodies bind to antigens via their fragment antigen-binding (Fab) portion. Protein A binds to the Fc (crystalline fragment) portion of IgG (at the opposite end of the Fab sites), preventing opsonization and phagocytosis by turning the antibody around on the surface. Some organisms evade phagocytic cell killing by releasing toxins in tissues that kill phagocytes. Streptococci produce hemolysins that lyse red blood cells and induce toxic effects on white blood cells and macrophages. Pathogenic staphylococci release leukocidins that cause lysosomal discharge of white blood cells into the cytoplasm. A staphylococcal leukocidin, called Panton-Valentine leukocidin, is lethal to leukocytes and may contribute to the invasiveness of the organism. Other organisms inhibit chemotaxis, which is the movement of white blood cells to sites of tissue damage, and the host is less able to direct polymorphonuclear neutrophils (PMNs) and macrophages into the site of infection.

Structures that promote adhesion to host cells and tissues Most infectious agents must attach to host cells before infection occurs. In intoxication diseases caused by exotoxins (e.g., botulism, staphylococcal food poisoning), microbial adherence is not important. However, in virtually all other cases, the bacterium, virus, or fungus requires adherence to host cells before infection and disease can progress. Without adhering to host cells, the pathogen can be carried away in uid (e.g., urine or mucus). The microbial or viral surface structures that mediate attachment are called adhesins. Host cells must possess the necessary receptors for the adhesins, generally a carbohydrate. If the host or the infectious agent undergoes a mutation that changes the structure of the adhesin or the host receptor, adherence can be less efcient or not take place, and the virulence of the infectious agent is affected. Fig. 2.2 illustrates surface bacterial structures that are involved in the pathogenesis of disease. The main adhesins in bacteria are surface proteins and polysaccharides. Extending from the surface of many human bacterial pathogens are mbriae (pili). On the tips of mbriae are adhesion proteins that bind to receptors on cell surfaces. Fimbriae enable bacteria to adhere to host cell surfaces, increasing the organism’s colonizing ability and providing resistance to phagocytosis. Once bacteria are attached to nonphagocytic cells, phagocytosis by white blood cells is less likely to occur. For example, the strains of E. coli that cause traveler’s diarrhea use their mbriae to adhere to cells of the small intestine, where they secrete a toxin that causes the disease symptoms. Similarly, mbriae are essential for N. gonorrhoeae to infect the epithelial cells of the genitourinary tract. Antibodies to the mbriae of N. gonorrhoeae are protective by preventing the organism from attaching to the epithelial cells. Similarly, antibodies to viral adhesions prevent infection.

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Table 2.4 Microbial interference with phagocytic activities Microorganismsa

Type of interferenceb

Streptococcus pyogenes

Kill phagocyte

Streptolysin induces lysosomal discharge into cell cytoplasm

Inhibit neutrophil chemotaxis Resist phagocytosis

Streptolysin M and M-like proteins Hyaluronic acid capsule

Kill phagocyte Inhibit opsonized phagocytosis

Leucocidin induces lysosomal discharge into cell cytoplasm SpA binds Fc portion of Ab

Resist killing Block chemotaxis

Polysaccharide capsule in some strains

Staphylococcus aureus

Mechanism (or responsible factor)

Kill phagocyte

Lethal factor of tripartite toxin

Resist killing

Capsular polyglutamic acid

Haemophilus inuenzae

Resist phagocytosis (unless Ab present)

Polysaccharide capsule

Streptococcus pneumoniae

Resist phagocytosis (unless Ab present)

Polysaccharide capsule

Klebsiella pneumoniae

Resist phagocytosis

Polysaccharide capsule

Pseudomonas aeruginosa

Kill phagocyte

Exotoxin A kills macrophages; also four type III toxins: exoenzyme (Exo)

Resist phagocytosis

Alginate exopolysaccharide

Resist phagocytosis (unless Ab present) Kill macrophages

O antigen (smooth strains) K antigen (acid polysaccharide)

Bacillus anthracis

Escherichia coli Salmonella spp.

Resist phagocytosis (unless Ab present)

Vi antigen

Resist killing; survival in macrophages

Secreted products of SPI-2

Kill phagocyte

Secreted products of SPI-1

Clostridium perfringens

Inhibit chemotaxis Resist phagocytosis

α-toxin Capsule

Cryptococcus neoformans

Resist phagocytosis

Capsular polyuronic acid

Yersinia pestis

Kill phagocyte

Yersinia outer protein (Yop)

Mycobacteria

Resist killing Digestion Inhibit lysosomal fusion

Cell wall substance

Brucella abortus

Resist killing

Cell wall substance

Toxoplasma gondii

Inhibit attachment to neutrophil Inhibit lysosomal fusion

Ab, Antibody; SpA, staphylococcal protein A; SPI-1, Salmonella pathogenicity island 1; SPI-2, Salmonella pathogenicity island 2; Yop, Yersinia outer protein. a Often only the virulent strains show the type of interference listed. b Sometimes the type of interference listed has been described only in a particular type of phagocyte (polymorph or macrophage) from a particular host, but it generally bears a relationship to pathogenicity in that host. Modied from Mims, C. A. (2015). The pathogenesis of infectious disease (6th ed.). New York: Academic Press.

Adherence protein

Fibrilla (e.g., M protein)

Pilus Protein F

Lipopolysaccharides

Non-pili adhesins

Lipotechoic acid Capsule

Outer membrane

Peptidoglycan

Peptidoglycan Inner membrane

Cell membrane GRAM-NEGATIVE

GRAM-POSITIVE

Fig. 2.2 Surface bacterial structures that are involved in the pathogenesis of disease. (From Kumar, V., et al. (2005). Robbins and Cotran pathologic basis of disease [7th ed.]. Philadelphia: Saunders.)

Microbial factors contributing to pathogenesis and virulence

Some human pathogens express adhesion proteins not associated with mbriae. When encountering host tissue, several gram-positive bacteria, like S. aureus, use molecular adhesion molecules that include teichoic acid, lipoteichoic acid, and lipoproteins. The primary tissue receptor is TLR2 found on epithelial cells, endothelial cells, and leukocytes. However, binding to TLR2 on keratinocytes leads to the release of antimicrobial peptides and proinammatory cytokines and neutrophil-attracting chemokines, an integral part of our innate defense mechanism. An additional adherence mechanism used by bacteria is a biolm. Some bacteria secrete a sticky polysaccharide that binds bacterial cells together and to host cells or nonliving surfaces. Biolms, discussed in Chapter 31, can also protect bacteria from phagocytosis and antibodies.

Ability to survive intracellularly and proliferate Bacteria are engulfed by some host cells, such as macrophages, into a cytoplasmic vacuole called a phagosome. Phagosomes fuse with lysosomes producing phagolysosomes, resulting in microbial killing. Lysosomes contain several antimicrobial factors. The process of phagocytosis is discussed in more detail later in this chapter. After phagocytosis, some pathogens survive within the phagocytic cell. Microorganisms have developed several methods to prevent being killed intracellularly. Some organisms prevent fusion of phagosomes and lysosomes, others escape from the phagosome into the cytoplasm, and still others are resistant to the effects of the lysosomal contents. Bacteria that prevent phagolysosomal formation can survive and multiply inside the macrophage. Bacterial species, such as Chlamydia, Mycobacterium, Brucella, and Listeria species, are easily engulfed by macrophages and phagocytes. However, these species are not only able to grow inside the macrophages, but they are protected from the host’s other immune defenses and are called intracellular parasites. The bacterium Coxiella burnetii can grow inside the harsh environment of a phagolysosome. To establish itself and cause disease, a pathogen must be able to replicate after attachment to host cells. Numerous host factors work to prevent proliferation: secretory antibody acts as an opsonin, enhancing phagocytosis; lactoferrin binds iron (an essential trace element); and lysozyme lyses bacteria. To be successful in establishing infection, infectious agents must be able to avoid or overcome these local factors. Lactoferrin competes with bacteria for free iron; N. meningitidis can use lactoferrin as a source of iron and is therefore not inhibited by its presence. The nonpathogenic Neisseria spp. are usually unable to use the iron in lactoferrin and are inhibited by its presence. Several pathogens (H. inuenzae, N. gonorrhoeae, and N. meningitidis) produce an IgA protease that degrades IgA found at mucosal surfaces. Other pathogens (inuenza virus, Borrelia spp.) circumvent host antibodies by changing key surface antigens. The host produces antibodies against the “old” antigens, which are no longer effective because the agent now has “new” antigens that do not bind to antibodies made against the old antigens. Pathogens exhibit an ability to penetrate and grow in tissues, a process called invasion. With some organisms, the invasion is localized and involves only a few layers of cells. For example, N. gonorrhoeae attaches to cells in the genital

37

area, penetrates into the cells, and multiplies. The bacteria pass through the cells into the subepithelial space and establish an infection. With some organisms, such as Salmonella spp., the organisms spread to distant sites (organs and tissues), often via the bloodstream. This is called dissemination. Other organisms, such as Corynebacterium diphtheriae, do not spread beyond their initial site of infection, yet the disease they produce is serious and often fatal because the exotoxins they produce are disseminated. Certain organisms that survive phagocytosis can be disseminated rapidly to many body sites, but the organisms themselves are not invasive. The phagocyte carries the organism to other body sites, but the bacterium itself is incapable of penetrating tissues.

Ability to produce extracellular toxins and enzymes Generally, disease from infection is noticeable only if tissue damage occurs. This damage may be from toxins, either exotoxins or endotoxins, or from inammatory substances that cause host-driven, immunologically mediated damage. The ability of organisms to produce exotoxins and extracellular enzymes is another major factor that contributes to the virulence and invasiveness of organisms. Toxins are poisonous substances produced by organisms that interact with host cells, disrupting normal metabolism and causing harm. Exotoxins are produced by both gram-negative and gram-positive bacteria and are secreted by the organism into the extracellular environment, or they are released on lysis of the organism. Exotoxins can mediate direct spread of the microorganisms through the matrix of connective tissues and can cause cell and tissue damage. Some organisms produce soluble substances, such as proteases and hyaluronidases that liquefy the hyaluronic acid of the connective tissue matrix, helping bacteria to spread in tissues, promoting the dissemination of infection. Endotoxins are a constituent, the lipopolysaccharide (LPS), of the outer cell membrane of gram-negative bacteria exclusively. Endotoxins, in contrast with exotoxins, do not have enzyme activity, are secreted in only very small amounts, do not have specicity in their activity on host cells, are generally not very potent, and are not destroyed by heating. Endotoxins are released in large amounts when the bacterial cell lyses.

Exotoxins Most bacterial exotoxins are composed of two subunits: one is nontoxic and binds the toxin to the host cells and the other has toxic activity. The toxin gene is commonly encoded by phages, plasmids, or transposons. Only the organisms that carry the DNA coding for the toxin gene produce toxin. Isolates of C. difcile, for example, must be tested for the presence of toxin genes, such as in the Case in Point. Diphtheria toxin inhibits protein synthesis and affects the heart, nerve tissue, and liver. Botulinum toxin is a neurotoxin that blocks nerve impulse transmission, causing accid paralysis, especially in infants. S. pyogenes and S. aureus both produce exfoliatin, which causes a rash and massive skin peeling or exfoliation. Table 2.5 lists many bacterial exotoxins that are important in disease.

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Table 2.5 Examples of exotoxins of pathogenic bacteria Bacterium

Disease caused in humans

Toxins

Bacillus anthracis

Anthrax

Lethal, edema-producing toxins

Bordetella pertussis

Whooping cough (pertussis)

Lethal, necrotizing toxin

Clostridium botulinum

Botulism

Six type-specic lethal neurotoxinsa

Clostridioides difcile

Antibiotic-associated diarrhea, pseudomembranous colitis

Toxin A, enterotoxina

Gas gangrene

Alpha, lethal, necrotizing

Clostridium novyi

Toxin B, cytotoxina Beta, lethal, necrotizing, hemolytic Gamma, lethal, necrotizing, hemolytic Delta, hemolytic Epsilon, lethal, hemolytic Zeta, hemolytic

Clostridium perfringens

Gas gangrene, food poisoning, enteritis necroticans

Alpha, lethal, necrotizing, hemolytica Beta, lethal, necrotizing Gamma, lethal, necrotizing, hemolytic Delta, lethal Epsilon, lethal, necrotizing Iota, lethal, necrotizing Theta, lethal, cardiotoxic, hemolytic Kappa, lethal, proteolytic Enterotoxina

Clostridium septicum

Gas gangrene

Alpha, lethal, hemolytic, necrotizing

Clostridium sordellii

Gas gangrene

Lethal toxina Hemorrhagic toxina

Clostridium tetani

Tetanus

Tetanospasmin, lethal, neurotoxica Neurotoxin, nonspasmogenic Tetanolysin, lethal, cardiotoxic, hemolytic

Corynebacterium diphtheriae

Diphtheria

Diphtheria toxin, lethal, necrotizinga

Escherichia coli (unique strains)

Diarrhea

Heat-labile enterotoxina Heat-stable enterotoxin Shiga toxin

Pseudomonas aeruginosa

Pyogenic infections

Exotoxin A

Staphylococcus aureus

Pyogenic infections, enterotoxemia

Alpha, lethal, necrotizing, hemolytic Beta, lethal, hemolytic Gamma, lethal, hemolytic Delta, hemolytic Exfoliating toxina Enterotoxina

Streptococcus pyogenes

Pyogenic infections, scarlet fever, rheumatic fever

Erythrogenic, nonlethal Streptolysin O, lethal, hemolytic, cardiotoxic Streptolysin S, lethal, hemolytic

Vibrio cholerae

Cholera

Cholera toxin, lethal, enterotoxica

Salmonella Typhimurium

Enteritis

Enterotoxin?a

Shigella spp.

Dysentery

Enterotoxina

Yersinia pestis

Plague

Murine toxin, cytotoxic pore-forming toxinsa

a

Toxins that produce harmful effects of infectious disease.

Host resistance factors

Endotoxins Endotoxins are composed of the LPS portion of the outer membrane on the cell wall of gram-negative bacteria. The cell wall of gram-negative microorganisms is composed of two layers—the inner peptidoglycan layer and an outer membrane. The LPS is contained in the outer membrane along with proteins and phospholipids (see Fig. 2.2). LPS contains three regions: an inner lipid A, a core polysaccharide, and an antigenic oligosaccharide (O region). Lipid A is hydrophobic and anchors LPS in the outer membrane, while the hydrophilic O-region extends a short distance from the cell. The lipid A portion of LPS is responsible for the toxic activity of endotoxin. Its chemical structure varies among gram-negative bacteria, making some species more virulent than others. LPS stimulates the release of proinammatory cytokines (e.g., tumor necrosis factor and interleukin 1), chemokines, and other inammatory mediators that aid in mounting an innate immune response. These are the chemical mediators that produce the effects of endotoxin that consist of dramatic changes in blood pressure, clotting, body temperature, circulating blood cells, metabolism, humoral immunity, and cellular immunity. Endotoxins stimulate the fever centers in the hypothalamus, increasing body temperature within 1 hour after exposure. Endotoxins can produce severe hypotension within 30 minutes. Septic or endotoxic shock is a serious and potentially life-threatening condition. In contrast with shock caused by uid loss, such as shock seen in severe bleeding, septic shock is unaffected by uid administration. Endotoxins also initiate coagulation, which can result in disseminated intravascular coagulation. This process depletes clotting factors and activates brinolysis so brin-split products accumulate in the blood. These fragments are anticoagulants and can cause serious bleeding. Another feature of patients with endotoxic shock is severe neutropenia, which can occur within minutes after exposure. It results from sequestration of neutrophils in capillaries of the lung and other organs. Leukocytosis follows neutropenia because neutrophils are released from the bone marrow. Endotoxins also produce a wide variety of effects on the immune system. They stimulate proliferation of B lymphocytes in some animal species, activate macrophages, activate complement, and have an adjuvant effect with protein antigens. They also stimulate interferon production and cause changes in carbohydrate and lipid metabolism as well as sensitivity to epinephrine. A severe infection with gram-negative bacteria can lead to serious and often life-threatening situations. Bacterial exotoxins and endotoxins are compared in Table 2.6

Case check 2.3 In the Case in Point, diarrhea caused by C. difficile was diagnosed in a patient who had been treated with antimicrobial therapy for several days. C. difficile produces potent virulence factors, including an enterotoxin called toxin A and a cytotoxin called toxin B. C. difficile is a significant cause of diarrhea in patients who have received prolonged courses of antimicrobial therapy in both outpatient and inpatient settings.

39

Table 2.6 Differences between bacterial exotoxins and endotoxins Characteristic

Exotoxins

Endotoxins

Organism type

Gram-positive and gram-negative

Gram-negative

Chemical nature

Simple protein

Protein-lipidpolysaccharide

Stability to heating (100° C)

Labile

Stable

Detoxication by formaldehyde

Detoxied

Not detoxied

Neutralization by homologous antibody

Complete

Partial

Biological activity

Individual to toxin

Same for all toxins

Host resistance factors Physical barriers Humans have evolved a complex system of defense mechanisms to prevent infectious agents from gaining access to and replicating in the body. Healthy skin is an effective barrier against infection. The stratied and cornied epithelium presents a physical barrier to penetration by most microorganisms. Only a few microorganisms can enter the body by way of intact skin. Some of these microorganisms and others that normally enter when the skin barrier is compromised are listed in Table 2.7. Most of the organisms listed in Table 2.7 require help in penetrating the skin barrier (e.g., animal or arthropod bite) or microscopic tears. Healthy, intact skin is clearly the primary mechanical barrier to infection. The skin also has substantial numbers of microbial biota that are usually nonpathogenic organisms that contribute to a low pH by their metabolism, compete for nutrients, and produce bactericidal substances (e.g., bacteriocins). In addition, long-chain fatty acids secreted by sebaceous glands are directly toxic to some bacteria and contribute to an acid pH ensuring that relatively few organisms can survive and prosper in the acidic environment of the skin. These conditions prevent colonization by transient, possibly pathogenic organisms. Because mucous membranes interact with the environment, they are more easily penetrated by pathogenic agents. However, mucous membranes are covered by mucus, a viscous mixture of mucin, glycoproteins, antibodies (primarily IgA), and other antibacterial factors. For a pathogen to colonize mucous membranes and potentially cause an infection, it must overcome these factors. In addition, the epithelium lining mucous membranes is periodically shed, taking away any bacteria attached to the cells. Underlying the mucous membranes are several cells of the immune system including macrophages, dendritic cells, T and B lymphocytes, and innate lymphoid cells.

Cleansing mechanisms Normally, the term cleansing suggests a liquid. However, one of the most effective cleansing mechanisms humans have is

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Microorganisms

Disease

Comments

Arthropod-borne viruses

Various fevers, encephalitides

150 distinct viruses, transmitted by infected arthropod bites

Rabies virus

Rabies

Bites from infected animals

Rickettsiaceae

Typhus, spotted fevers

Bites from infected arthropods

Leptospira

Leptospirosis

Contact with water containing infected animal urine

found in the stomach. However, some bacteria can survive and pass into the small intestine. The number of bacteria in the intestine increases as the distance from the stomach increases. The distal portion of the colon contains the highest number of organisms. The gastrointestinal tract is protected by mucous secretions and peristalsis that prevent the organisms from attaching to the intestinal epithelium. The genitourinary tract is cleansed by voiding urine. Consequently, only the distal portion of the urethra typically has a microbial population. The vagina contains a large population of organisms as part of the indigenous biota. The acidity of the vagina, resulting from the breakdown of glycogen by the resident biota, tends to inhibit transient organisms from colonizing. The cervix is normally sterile.

Staphylococcus aureus

Boils, impetigo

Most common skin invaders

Antimicrobial substances

Streptococcus pyogenes

Impetigo, erysipelas

Bacillus anthracis

Cutaneous anthrax

Systemic disease following local lesion at inoculation site

Treponema pallidum subsp. pallidum

Syphilis

Warm, moist skin is more susceptible

Yersinia pestis

Plague

Bite from infected rat ea

Plasmodium spp.

Malaria

Bite from infected mosquito

Dermatophytes

Ringworm, athlete’s foot

Infection restricted to skin, nails, and hair

Table 2.7 Microorganisms that infect skin or enter the body via the skin

the desquamation of the skin surface. The keratinized squamous epithelium or outer layer of skin is being shed continuously. Many of the microorganisms colonizing the skin are disposed of with the sloughing of the epithelium. More obvious is the cleansing action of the uids of the eye and the respiratory, digestive, urinary, and genital tracts. The eye is continually exposed to microorganisms, which means this organ has some highly developed antimicrobial mechanisms. Tears bathing the cornea and sclera not only lubricate the eye but also wash foreign matter and infectious agents away from the surface. Additionally, tears contain IgA and lysozyme. The respiratory tract is also continuously exposed to microorganisms and is protected by nasal hairs, ciliary epithelium, and mucous membranes. A continuous ow of mucus emanates from the membranes lining the nasopharynx, which traps particles and microbes and sweeps them to the oropharynx, where they are either expectorated or swallowed. The trachea is lined with ciliary epithelium. These cells have hairlike extensions (cilia) that sweep particles and organisms upward toward the oropharynx. This material is expectorated or swallowed. Heavy smokers have a signicant reduction in ciliated epithelial cells and therefore are more susceptible to respiratory infections. The purpose of these mechanisms is to prevent infectious agents and other particles from reaching the bronchioles and lungs. Under normal conditions, they are very effective, and the air moving into and out of the lungs is sterile. Bacteria are swallowed into the gastrointestinal tract either as part of the mouth biota and upper respiratory tract or in liquids and food. Most bacteria are destroyed by the low pH

Various substances produced in the human host have antimicrobial activity. Some are produced as part of the phagocytic defense and are discussed later. Others, such as fatty acids, hydrogen chloride in the stomach, lysozyme, and secretory IgA have already been mentioned. Lysozyme, a low-molecular-weight (approximately 14 kDa) enzyme found throughout the animal kingdom, hydrolyzes the peptidoglycan layer of bacterial cell walls causing lysis. In some bacteria, the peptidoglycan layer is directly accessible to lysozyme. These bacteria are killed by the enzyme alone. In other bacteria, the peptidoglycan layer is exposed after other agents have damaged the cell wall (e.g., antibody and complement, hydrogen peroxide). In these cases, lysozyme acts with the other agents to cause death of the infecting bacteria. Through evolution, some bacteria exhibit chemical modication of peptidoglycan, increasing resistance to lysozyme. In fact, S. aureus is nearly totally resistant to its effects. Lysozyme is found in serum, mucus, tissue uids, tears, breast milk, saliva, and sweat. The complement system is composed of about 20 proteins, mostly produced by the liver. Many of the proteins are proenzymes, released in an inactive form but ultimately metabolized into an active enzyme. During metabolism, the proenzymes are cleaved into an “a” and “b” component, each with different functions. The outcomes of complement activation are production of opsonins, chemotaxins, and other inammatory functions. In addition, the proteins form a membrane attack complex that can lyse plasma membranes, such as those of bacteria. Antibodies, especially secretory IgA, are found in saliva and mucous secretions of the respiratory, genital, and digestive tracts. They may serve as opsonins, enhancing phagocytosis, or they may x complement and neutralize the infecting organism. Serum also contains low-molecular-weight cationic proteins, termed β-lysins. These proteins are lethal against gram-positive bacteria and are released from platelets during coagulation. The site of action is the cytoplasmic membrane. These antimicrobial substances and systems work best together. A combination of antibody, complement, and lysozyme is signicantly more effective in killing bacteria than each alone or than any combination in which one or more are missing. Interferons inhibit viral replication and are arguably the most important initial mechanism to control viral infections. Interferons are a group of cellular proteins induced by

Host resistance factors

eukaryotic cells in response to viral infection or other inducers. Interferons play a signicant role in the innate response against foreign invaders. Interferons bind to interferon receptors on cells and produce an antiviral state by activating antiviral genes in both infected and uninfected cells. These gene products can block viral penetration and inhibit viral replication. Uninfected cells that have been exposed to interferon are refractory to virus infection. Interferons also activate macrophages and natural killer cells, enhancing their antiviral activity. Some interferons play a role in the adaptive immune response by stimulating dendritic cells—which are antigen-presenting cells—and T cells. Numerous bacteria, viruses, and their products induce interferon production. Interferon is so named because it can interfere with viral replication. There are instances in which viruses escape the effects of interferons, either because they are resistant to the antiviral effects or because the induction of interferon in the host does not take place. For example, the vaccinia virus can resist the effects of interferons by inactivating interferon gamma. Other viruses can produce persistent infections because these viruses do not induce interferon production.

41

Table 2.8 Tissue distribution of monocytes/macrophages Cell name

Tissue distribution

Monocyte

Blood

Kupffer cell

Liver

Alveolar macrophage

Lung

Histiocyte

Connective tissue

Microglial cell

Central nervous system

Mesangial cell

Kidney

Macrophage

Spleen, lymph nodes

Skin surface

Injury

Diapedesis

Phagocytosis Phagocytosis is an essential component in the resistance of the host to infectious agents. It is the primary mechanism in the host defense against extracellular bacteria and numerous viruses and fungi. The PMNs, macrophages, and monocytes are the body’s second line of defense. The stem cells for neutrophils arise in the bone marrow, where they differentiate to form mature neutrophils. During this maturation, the cells synthesize myeloperoxidase, proteases, cathepsin, lactoferrin, lysozyme, and elastase. These products are incorporated into membrane-bound vesicles called lysosomes. The lysosomes appear as azurophilic granules on Wright’s stain and contain enzymes, oxygen-reactive molecules, and other substances necessary for the killing and digestion of engulfed particles. The PMN also has receptors on the cell membrane for some complement components that stimulate cell migration, the metabolic burst, and secretion of the lysosome contents into a phagosome. The PMN, an endstage cell, has a circulating half-life of 2 to 7 hours and makes up the majority of white blood cells in circulation of healthy individuals. It can migrate to the tissues, where its half-life is less than 1 week. Macrophages also originate in the bone marrow from stem cells. They circulate as monocytes for 1 to 2 days and then migrate through the blood vessel walls into the tissues and reside in specic tissues as part of the mononuclear phagocyte system. These cells are widely distributed in the body and play a central role in specic immunity and nonspecic phagocytosis (Table 2.8).

Chemotaxis Four activities must occur for phagocytosis to take place: (1) migration of the phagocyte to the area of infection (chemotaxis), (2) attachment of the particle to the phagocyte, (3) ingestion, and (4) killing. The PMNs circulate through the body followed by movement into the tissues, an action called diapedesis, which is the movement of the neutrophils

Bacteria Chemotaxins PMNs

Lumen of blood vessel

Fig. 2.3 Phagocytosis: chemotaxis migration of phagocytes PMNs, Polymorphonuclear neutrophils.

between the endothelial cells of the blood vessels into the tissues. The body is under constant surveillance by these and other phagocytic cells. When an infection occurs, massive numbers of PMNs accumulate at the site. This accumulation is not a random event; rather, it is a directed migration of PMNs into the area needing their services. This migratory process is called chemotaxis. Several substances serve as chemotactic agents (chemotaxins), which attract phagocytic cells. These include certain complement proteins, several bacterial products, products from damaged tissue cells, and products from responding immune cells. As the organism triggers tissue damage and inammation, chemotaxis of phagocytes occurs (Fig. 2.3). The speed and magnitude of this response are easily visualized by recalling how quickly a splinter or similar injury causes inammation and how much pus is produced.

Attachment The initial contact of the PMN with an invading organism may be random. One of the most effective defenses bacteria have against phagocytosis is the capsule. This structure prevents attachment of the neutrophil’s membrane to the organism, which must occur before ingestion can take place. Attachment is facilitated by the binding of specic antibodies or complement proteins to the microorganism. The neutrophil membrane has various receptors, including receptors for the Fc portion of IgG1 and IgG3, and the C3b component of complement. These three factors can bind to the invading

42

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Host-parasite interaction

microorganism, resulting in the microorganisms being coated with one or more of these factors. The receptor on the PMN for the specic factor coating the bacterium binds to the factor and forms a bridge that brings the bacterium into close physical contact with the leukocyte membrane. The coating of the bacterium with antibody or complement components results in enhanced phagocytosis by the PMN. This process or phenomenon is called opsonization

Ingestion and killing The next step of phagocytosis is ingestion. This process occurs rapidly after attachment. The cell membrane of the phagocytic cell invaginates and surrounds the attached particle. The particle is taken into the cytoplasm and enclosed within a vacuole called a phagosome (Fig. 2.4). The phagocytosis of a particle triggers a signicant increase in the metabolic activity of the neutrophil or macrophage. This increase is termed a metabolic or respiratory burst. The cell demonstrates increases in glycolysis, the hexose monophosphate shunt pathway, oxygen use, and production of lactic acid and hydrogen peroxide. The phagosome is acidied by the formation of a proton (H+) gradient. Highly toxic oxygen-reactive intermediates (e.g., hydrogen peroxide) and reactive nitrogen intermediates (e.g., nitric oxide) are secreted into the phagosome contributing to pathogen killing. Fusion with cytoplasmic granules (lysosomes), producing phagolysosomes, delivers a variety of antimicrobial factors. Lysosomes are vacuoles containing enzymes and other antibacterial components. The list of

enzymes found within the lysosomes is long—more than 20 enzymes, including proteases, lipases, RNase, DNase, and acid phosphatase. Other antimicrobial agents include lactoferrin, which chelates iron and prevents bacterial growth; lysozyme; and several basic proteins. The usual result is that a phagocytosed organism is quickly killed and digested. Neutrophils naturally contain many lysosomes and are effective at killing phagocytized pathogens. Alternatively, macrophages and monocytes require activation by factors such as interferon gamma and interaction with pattern recognition receptors, discussed later. Organisms that are intracellular pathogens survive phagocytosis and can multiply within the phagocyte, most notably mononuclear leukocytes. Because PMNs have more lysosomes, intracellular pathogens are much less likely to survive in these cells. Other defense mechanisms must play a major role in immunity to these intracellular organisms. The importance of phagocytosis is seen in patients with defects in the numbers or function of phagocytic cells. Such patients have frequent infections despite possessing high levels of serum antibody. Many of the organisms listed in Table 2.4 are common isolates, which is not surprising because they have developed a means to interfere with phagocytosis, increasing their pathogenicity.

Inammation Inammation is the body’s nonspecic response to injury or foreign body. Fig. 2.5 illustrates the components involved in acute and chronic inammatory responses. A hallmark of inammation is the accumulation of large numbers of phagocytic cells. These leukocytes release mediators or cause other cell types to release mediators. The mediators cause erythema because of greater blood ow, edema from an increase in vascular permeability, and continued phagocyte accumulation via chemotaxins, resulting in pus. The enzymes released by the phagocytes digest the foreign particles, injured cells, and cell debris. After the removal of the invader, the injured tissue is repaired.

Immune responses

Fig. 2.4 Transmission electron micrograph of engulfed bacterial cells inside phagosomes (arrows).

The immune system response to infection is briey discussed in this chapter to provide the reader with an appreciation of its role and complexity. The balance between health and infectious disease is complex and mediated by humoral and cellular factors. The relative importance of each factor depends on the microbe, route of infection, condition and genetic makeup of the host, and other factors yet to be clearly characterized. For example, a patient with AIDS becomes more susceptible to opportunistic organisms as the immune system deteriorates. Table 2.9 summarizes the defenses used by the human host against infection. Classically, the term immunity has been dened as a complex mechanism whereby the body is able to protect itself from invasion by disease-causing organisms. This mechanism, known as the immune system, consists of numerous cells and protein molecules that are responsible for recognizing and removing foreign substances. This general denition has been broadened over the years to mean a reaction to any foreign substance, including proteins and polysaccharides as well as invading microorganisms. Research advances over the years have dramatically increased our

Host resistance factors

43

Mast cell CONNECTIVE TISSUE CELLS

Smooth muscle

Basophil

Platelets VESSELS

Endothelium

Polymorphonuclear leukocyte Lymphocyte

Monocyte

Clotting factors, kininogens, and complement components

Eosinophil

Basement membrane

CONNECTIVE TISSUE MATRIX

Fig. 2.5 Components involved in acute and chronic inammatory responses. (From Kumar, V., et al. (2005). Robbins and Cotran pathologic basis of disease [7th ed.]. Philadelphia: Saunders.)

understanding of the immune response and given us an appreciation of the cellular interrelationships. The immune response can be divided into two broad categories: (1) innate or natural immunity with little or no specicity and (2) adaptive or specic immunity, which is highly specialized.

Innate, or natural, immunity Innate immunity, also referred to as natural or nonspecic immunity, consists of several components. These include (1) physical and chemical barriers such as the skin and mucous membranes, (2) blood proteins that act as mediators of infection, and (3) a cellular mechanism capable of phagocytosis such as neutrophils and macrophages and other leukocytes such as natural killer cells. Fig. 2.6 shows examples of the innate immune defenses located at different body sites. This rst line of defense has a limited capacity to distinguish one organism from another, and previous exposure to a particular foreign substance is not required. Physical barriers, mentioned earlier, may be as simple as the keratinized outer layer of the skin. Also, the secretions along the mucous membranes and the ciliated epithelial cells of the respiratory tract promote trapping and removal of microorganisms. In addition, many secretions provide a chemical barrier, such as the acidic pH of the stomach and vagina. Saliva and tears contain enzymes such as lysozyme, and the sebaceous glands of the skin contain oils and fatty acids capable of inhibiting invasion by pathogenic organisms. The normal biota of these sites adds another dimension to the host’s ability to resist invading pathogens. Once the physical and chemical barriers to infection have been penetrated, nonspecic mechanisms of innate immunity become involved—an animal’s second line of defense. Innate

immune system cells have receptors called pattern recognition receptors (PRRs) that recognize conserved sequences on the surfaces of microorganisms. The rst PRR discovered was the Toll-like receptor (TLR). These low-molecular-weight transmembrane proteins contain an extracellular domain that binds the pathogen-associated molecular patterns (PAMPs) that are present on pathogens. Eleven different TLRs (TLR1 to TLR11) have been identied in humans, and each has specicity for components of different microorganisms. For example, TLR4 recognizes LPS from many species of gram-negative bacteria. Recognition of these microbial components by PRRs allows innate immune system cells to initiate the innate immune response. PRRs on phagocytes can recognize PAMPs on pathogens and initiate phagocytosis. Phagocytic cells ingest and kill microorganisms, while activated complement components contribute to a wide variety of immunologic events, including opsonization and chemotaxis, discussed previously. Collectively, these immunologic defense mechanisms, along with host tissue damage caused by the invading organisms, combine to produce an acute inammatory response by the host. The importance of natural immune mechanisms rests in their rapid response to invading organisms. However, these mechanisms are effective primarily against extracellular bacterial pathogens, playing only a minor role by themselves in immunity to intracellular bacterial pathogens, viruses, and fungi.

Adaptive, or specic, immunity Adaptive immunity enhances the protective capability of innate immunity. This arm of the immune response is specic for distinct molecules, responding in particular ways to

PART 1

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Host-parasite interaction

Table 2.9 Summary of defenses of the human or animal host to infection and evasion mechanisms attributed to various microorganisms Host defense

Mechanism of evasion

Microbial example

Hydrodynamic ow

Attachment

Fimbriae, surface proteins, lipoteichoic acid

Mucous barrier

Attachment Penetration

Mannose-sensitive mbriae, surface proteins Mucinase

Deprivation of essential nutrients (e.g., transferrin)

Systems of high-afnity uptake

Siderophores

Lysozyme in secretions

Resistance to lysis

Modication of peptidoglycan

Surface Igs

Absent or low immunogenicity Antigenic heterogeneity Masking of antigens Destruction of immunoglobulins

Hyaluronic acid, capsules Fimbriae, capsules, LPS, M protein Capsules, IgA-binding proteins IgA protease

Mucous membrane

Penetration

Neisseria gonorrhoeae, Shigella spp.

Recognition by antibody

Antigenic heterogeneity Masking of antigen Destruction of antibody Antigenic variation

Fimbriae, capsules, LPS, M protein Capsules, Ig-binding proteins Borrelia

Complement system

Failure to activate alternative pathway Inactivation of complement components Resistance to bacteriolysis Formation of abscess

Sialic acid capsules Cleavage of C3b CoIV plasmid Bacteroides fragilis capsule

Fibrin tapping

Fibrinolysis

Streptococcus

Abscess formation

Serum defenses

Localization Collagenase, elastase

Pseudomonas, Clostridium

Adaptive immune response

Nonspecic B-cell activation Inhibition of cell-mediated immune response Rapidly fatal (toxin)

LPS, lipoprotein Anergy of miliary tuberculosis and T-cell destruction by HIV Anthrax, plague, Clostridium

Phagocytosis

Inhibition of chemotaxis

Brucella, Salmonella, Neisseria, Staphylococcus, Pseudomonas

Inhibition of attachment and ingestion

Capsules, M protein, Ig-binding proteins, gonococcal pili

Inhibition of metabolic burst

Salmonella Typhi

Prevent phagosome-lysosome fusion

Mycobacteria

Resistance to permeability-inducing cationic protein

Gram-positive cell wall, smooth LPS, polyanionic capsules

Resistance to oxidative attack

Catalase, superoxide dismutase, carotenoid pigments

Escape from phagosome

Mycobacterium bovis, Legionella pneumophila

Destruction of phagocyte

Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Pseudomonas aeruginosa

HIV, Human immunodeciency virus; Ig, immunoglobulin; IgA, Immunoglobulin A; LPS, lipopolysaccharide.

different types of foreign substances and developing memory, which allows for a more vigorous response on repeated exposures to the same invader. Lymphocytes and their products, such as antibodies, are the major cellular mediators of the adaptive immune response. Antibodies are produced in response to immunogens, substances that can induce an adaptive immune response. An antigen is a molecule that can bind specically to an antibody or T-cell receptor. An interrelationship exists between the mechanisms of the innate and adaptive responses. For instance, inammation, a nonspecic response of the innate system, provides a signal that triggers an adaptive immune response. In addition, the activity of complement (a component of innate immunity) to invading bacteria is enhanced by the presence of specic antibodies

(components of adaptive immunity). This activation leads to phagocytic clearance and elimination of the bacteria. The adaptive immune response adds a high degree of specialization to the passive mechanisms of the innate response. The nature of the adaptive immune response varies according to the type of organism and is designed to eliminate it. For example, antibodies are produced by B cells and plasma cells in response to extracellular blood-borne organisms and aid in their elimination. However, the response to intracellular parasites is primarily by T cells that produce chemicals that enhance the activities of the phagocytic cells and a subset of T cells called cytotoxic T cells. The nature and origin of these cells are described later. Most importantly, the specic response remembers each time it encounters a particular foreign immunogen.

Host resistance factors

Microbial biota, cilia, mucus Mucus, ciliated cells Skin, fatty acids, microbial biota

Urogenital tract, microbial biota, lower pH

Mucus and other secretions Saliva, lysozyme

Gastrointestinal biota, gastric acid, mucus Emptying of the bladder

Fig. 2.6 Innate immune defenses located at different body sites.

This is called immunologic memory. Subsequent exposure to that immunogen stimulates an increased and specic defense. The adaptive immune response is the third line of defense and improves signicantly the host’s defense to infection. Lymphocytes originate in the bone marrow from stem and progenitor cells. Lymphocytes mature and take up residence in various body tissues and organs, including the thymus, lymph nodes, and spleen. They are a diverse group of cells that can be classied into two major types based on cell surface markers: T (thymus-derived) cells and B (bone marrow–derived) cells. T cells mature in the thymus, while B cells leave the bone morrow as transitioning B cells and complete maturation in the spleen. The uniqueness of lymphocytes lies in the presence of specic cell surface receptor molecules that recognize and bind a unique immunogen, activating the cell to divide, differentiate, and secrete numerous effector substances. During embryogenesis, the millions of lymphocytes found in the body have been pre-engineered to recognize a vast array of substances as foreign while learning which substances constitute self. Thus the result of an encounter with the antigen is an expanded clone or clones of activated lymphocytes.

Nature of the immune response to infectious agents Although both humoral-mediated immunity and cellmediated immunity are important in protecting humans from a wide variety of infectious agents, each contributes differently according to the type of pathogen and virulence

45

mechanisms. While B cells play the predominant role in the humoral immune response, T cells mediate cellular immunity and play an important role in the humoral immune response. Antigen-presenting cells, like dendritic cells, process antigen and present it to T-helper cells with T-cell receptors specic for each antigen. Some T-helper cells become TH2 cells. Each B cell has surface receptors that recognize only one unique antigen. After antigen binding, the B cell processes the antigen, similar to the dendritic cell, and interacts with a TH2 cell with a T-cell receptor specic for the processed antigen the B cell is carrying. The TH2 cell secretes lymphokines that activate the B cell, triggering multiple cellular divisions and differentiation into plasma cells that actively secrete proteins called immunoglobulins, or antibodies. All antibody molecules derived from a single clone of B cells are of a single specicity (recognize a unique antigen), identical to the antibody receptor molecule on the original B cell. These antibody molecules circulate in the bloodstream and lymphatics, bathe tissues, and bind to infectious agents or substances to aid the host in eliminating them from the body. The detection and quantication of these antibody molecules, obtained from a patient’s serum, constitute the primary goal of diagnosing infectious diseases through serologic methods.

Classication and characteristics of antibodies Antibody molecules, found in serum and other body uids and secretions, may be classied into one of ve distinct immunoglobulin groups or classes. The classes differ from one another in several ways, including chemical structure, serum concentration, half-life, and activity. The immunoglobulin G (IgG) antibody class constitutes about 70% to 75% of the total serum immunoglobulin pool. Their half-life in serum is 3 to 4 weeks. IgG can cross the placenta to the fetus, conferring some protection in both the prenatal and the postnatal periods. Structurally, IgG is a protein with a molecular weight of about 150 KDa consisting of four polypeptides (two identical light chains and two identical heavy chains) bridged by several disulde bonds (Fig. 2.7). Although the amino acid sequence of some regions of the polypeptides is nearly identical among all IgG molecules (conserved regions), one of the ends of each polypeptide is highly variable. These variable regions create two active fragment of antigen binding (Fab) sites on each IgG molecule (see Fig. 2.7). Thus IgG antibodies are said to be bivalent—capable of binding two antigen molecules. Antibodies of the immunoglobulin M (IgM) class account for 10% to 15% of serum immunoglobulins. Their half-life in serum is about 5 days, and IgM cannot cross the placenta. A developing fetus in the second or third trimester as well as a newborn may respond to an infectious agent with an IgM antibody response of its own. IgM molecules are large; the molecule has a molecular weight of about 900 KDa and consists of a pentamer or ve basic subunits—each composed of two heavy chains and two light chains (similar to an IgG molecule) and linked to another polypeptide chain (J chain) by disulde bonds (Fig. 2.8). The IgM molecule has up to 10 antigen-binding sites available. IgM is the rst antibody class produced in response to an immunogen. Both IgG and IgM antibodies are commonly assayed in a variety of serologic tests. The differences in size and conguration of IgG and

2

PART 1

46

Host-parasite interaction

Table 2.10 Immunoglobulin G versus immunoglobulin M Immunoglobulin class

Light chain

Property Disulfide bonds

s

s

s s

s s

Heavy chains

Fab fragment

s

s

Fc fragment

Light chain

IgG

IgM

Molecular weight

150 KDa

900 KDa

Number of 4-polypeptide subunits

1

5

Number of antigen-binding sites

2

10

Serum concentration (mg/dL)

800–1600

50–200

Percentage of total immunoglobulin in serum

75

10

Ability to cross placenta

+

−

Half-life (days)

23–25

5–8

IgG, Immunoglobulin G; IgM, immunoglobulin M; KDa, kilodalton.

Fig. 2.7 Immunoglobulin G. Disulfide bonds

a few parasitic agents have been developed. IgE also plays a role is some allergic (hypersensitivity) reactions, and elevated levels can be detected in these situations. The role of serum IgD during infection is unknown, except that it functions as a receptor on B lymphocytes for antigen.

Primary and secondary antibody responses

IgG-like subunit

J chain

Fab fragment

Fig. 2.8 Immunoglobulin M (IgM).

IgM molecules result in differences in functional activity of the molecules in serologic tests (Table 2.10). Immunoglobulin A (IgA) antibodies represent 15% to 20% of the total serum immunoglobulin pool. IgA constitutes the predominant immunoglobulin class in certain body secretions, such as saliva, tears, and intestinal mucosa. Because of this association of IgA with mucosal surfaces, it provides protection against microorganisms invading at those sites. Serum IgA occurs primarily as a dimer composed of two subunits (each similar to an IgG molecule) linked together by a J chain. However, when found in secretions, the molecule also contains a secretory component that stabilizes the molecule. Although signicant increases in serum IgA levels may occur in association with certain infections, the function of serum IgA is unclear, and few serologic tests for the diagnosis of infectious disease are designed specically to detect IgA antibody. The remaining two immunoglobulin classes, immunoglobulin D (IgD) and immunoglobulin E (IgE), are normally found in very low concentrations in serum (500) were reported but not cultured. The Salmonella spp. isolates recovered were all the same serotype. Well water, food, and food preparation sites were cultured. Another example involves the investigation of a potential measles outbreak involving an international ight from Russia to the United States. The patient suspected of being the index case ew from Russia to London, where she changed planes for a ight to New York. She then changed planes in New York for a ight to Chicago, where she lived. The patient was a college student who had visited her parents in Russia

PART 1

58

3

The laboratory role in infection control

Outbreak of Diarrhea State Fair

Table 3.4 Steps of an outbreak investigation Steps

Description

1. Verify diagnosis of suspected cases.

Establish a case denition.

2. Conrm that an outbreak exists.

Be certain that all suspected cases meet the denition.

3. Find additional cases.

Investigate to determine whether additional cases exist.

4. Characterize cases.

Collect as much information as possible about the cases, including people, place, and time elements. Develop an epidemiologic curve.

4 5 6 7 8 9 10 11 12 13 14 15 Day cases were identified

5. Form a hypothesis.

Establish a “best guess” hypothesis to explain the outbreak.

Fig. 3.3 The epidemiologic curve of an outbreak investigation of diarrhea over a 15-day period at a state fair. The number of cases includes suspected cases (black bars) and culture-proven cases (colored bars). Infection control interventions were implemented on day 8 of the outbreak, and the number of cases decreased.

6. Test the hypothesis.

Evaluate the hypothesis epidemiologically with control groups and data collected.

7. Institute control measures.

Implement intervention activities to prevent and control the outbreak.

8. Evaluate the effectiveness of control measures.

Determine whether the implemented activities have an impact on the outbreak. Does the number of cases diminish or disappear?

9. Communicate the ndings.

Document the investigation, and communicate with all involved parties.

180 Intervention

160

Number

140 120

Culture confirmed

100 80 60 40 20 0

Fair started 1

2

3

Fair ended

for the summer. While in Russia, she had contact with several young children who had measles. On her return ight to the United States, she complained of a fever but did not develop a rash until she had been home in Chicago for a day. Imagine the complexity of an investigation of this case. This scenario was recognized as a potential outbreak and not an actual outbreak when the investigation was undertaken. The recognition of measles by the microbiology laboratory set the investigation into motion, not the occurrence of any additional cases. Several issues must be examined. • • • •

Did the student have measles? Were passengers on the plane immune? Where did the passengers go after their ight landed? How does the health department follow up with them?

Because of routine use of a vaccine most, but not all, people in the United States are immune to measles. However, vaccination may not have been successful or given to some individuals. The incubation period for measles is approximately 10 days (8 to 13 days for the fever, 14 days for the rash). People are contagious before the development of the fever and rash. Those who are not immune are susceptible. All three of these examples (Acinetobacter spp., Salmonella spp., measles) represent scenarios of potential infectious disease outbreaks. The investigation of these outbreaks depends heavily on the support of the microbiology laboratory to assist in ruling in or ruling out the infection and in identifying cases and sources. Laboratory support is described in detail later.

Pandemic First identied in December 2019 in Wuhan, China, COVID19 caused by SARS-CoV-2, a new human pathogen, is a respiratory illness that can be spread from person to person. Cases of COVID-19 spread rapidly around the world. On March 11, 2020, TheWHO declared the COVID-19 outbreak apandemic. To date (19 February 2022), over 422 million cases and nearly 6.55 million deaths have been reported worldwide.

In the United States, the rst case was identied in a resident of Washington state who had traveled to Wuhan. A remarkable number of cases followed in Seattle and occurred primarily among older adults living in congregant settings such as independent or assisted living communities, long-term care, and skilled nursing facilities. Across the United States, the COVID-19 epidemic has caused several outbreaks that overwhelmed the entire health care system, with the problem growing in scope until mitigation strategies were identied. There have been over 78 million cases and over 900,000 deaths reported in the United States as of February 2022.

Steps of an outbreak investigation When an outbreak is suspected, steps are taken to investigate the event. The laboratory is integral in several of the steps. Table 3.4 lists the steps that are followed in an outbreak investigation. The rst step is to establish a case denition. This step ensures that the rest of the investigation is based on a single denition. This may involve the microbiology laboratory in the search for a specic pathogen or the recovery of several pathogens. The second step is to conrm that an outbreak exists. One needs to be certain that all of the suspected cases match the case denition and that there is more than an expected number of cases. At this point, the investigator seeks as much consultative assistance as possible. The laboratory is frequently asked for additional input. The third step is to nd additional cases that might be added to the initial number of cases. Additional suspected cases may be discovered by more detailed investigation or by the new occurrence of cases. The laboratory might be asked

Outbreak investigation

to review microbiology data from a previous period to determine whether unrecognized cases have occurred. The fourth step is to gather as much information as possible about the cases with respect to person, place, and time. Persons suspected of being part of the outbreak should be interviewed to nd what the victims have in common. An epidemiologic curve may be constructed to assist in the visualization of the outbreak numbers over time. The fth and sixth steps are to form a hypothesis about the event and then to test that hypothesis. In the fth step, a tentative hypothesis is established as a best guess about the likely reservoir, source, and means of transmission. In testing that hypothesis, a control group is established; then the incident and control groups in the event are compared. Again, the microbiology laboratory may provide insight into the hypothesis and its relationship to the control group. The seventh step in the investigation may actually occur at any point along the investigation timeline. The establishment of interventions to stop the outbreak probably occurs from the initial recognition and heightened awareness of a problem. The formal steps of the intervention process might not be developed until after the hypothesis is developed and tested. Undoubtedly, interventions of some type (e.g., increased hand hygiene) are introduced early in the investigation. The eighth step, which comes after the development of interventional strategies, is to evaluate the effectiveness of the interventions. Did the outbreak cease or at least decrease in intensity? The ninth step, the nal step and one sometimes overlooked, is to communicate the ndings of the investigation. This must include a written report that is kept on le and provided to all responsible individuals. It is not unusual for an outbreak to end before all data have been collected and analyzed. This is probably because of an early intervention. However, an early end to the outbreak does not ameliorate the need for communication and a written report. The information collected can be useful in future outbreaks caused by the same agent.

Investigation support from the laboratory The microbiology laboratory plays a crucial role in providing investigative support in an outbreak investigation and in the creation of routine surveillance information. The availability of culture reviews, which may result in the initiation of an outbreak investigation, was discussed earlier. Other types of laboratory support are often important as well.

Cultures and serology In an outbreak investigation and collection of surveillance data, the collection, processing, reporting, and reviewing of pertinent cultures becomes critical. In the Salmonella spp. outbreak at the state fair discussed earlier, consider the number of fecal specimens that the health department laboratory processed, although patient cultures are only one component of the investigative activity. Cultures from other sites may provide additional signicant information. Well water and food were cultured in addition to specimens from the patients with diarrhea and food handlers. In the outbreak of infections caused by Acinetobacter spp., the laboratory may have cultured respiratory therapy equipment and water samples.

59

One of the major difculties in a large outbreak (e.g., the state fair outbreak) is the reduced ability to collect and transport specimens from persons from out of state. Individuals may have cultures processed in their home state, but information about these results may be difcult to track and retrieve. In the state fair example, only a few of the affected individuals had cultures processed and the results included in the investigation—565 suspected cases and only 47 cultures. Reasons for this low number of cultures include the following: (1) the diarrhea lasts 24 to 48 hours, and people may not seek medical help; (2) people who live outside the immediate area may not know of the potential outbreak and the need to provide culture material; (3) people do not want to bother with the expense and time spent to collect cultures; and (4) the specimens may be collected too late in the infection or not transported properly, so no organisms are recovered. In addition to the culture results, the laboratory may be asked to determine the serologic relationship of the isolates. • Are all the Salmonella spp. of the same serotype? • What is the epidemiologic prole of the serotype? Both the isolate identication and serologic relatedness may be important determinants in an outbreak investigation.

Antibiograms Antibiograms, patterns of sensitivity and resistance to antimicrobial agents in bacteria, can often be used in the investigation of an outbreak. As discussed in Chapter 13, there are times when antibiograms must be viewed with suspicion. Although these laboratory data are not always as precise as other results, they may provide guidance about the microbial relatedness of the isolates. Table 3.5 demonstrates comparisons of two antibiograms of isolates with the antibiogram of the index case. If the isolates all had identical antibiograms (e.g., isolate 1) or if some of the isolates were different in their susceptibility patterns (isolate 2), the inclusion or exclusion from the case denitions might be affected. For example, relatively susceptible S. aureus can be distinguished from MRSA in an outbreak of skin infections in prisoners.

Table 3.5 Comparison of antibiograms from microbial isolates Isolate Antimicrobial agent

Index case

1

2

Ampicillin-sulbactam

R

R

S

Piperacillin

R

R

S

Cefepime

R

R

S

Imipenem

S

S

S

Ciprooxacin

R

R

S

Gentamicin

R

R

S

Tobramycin

R

R

R

Amikacin

R

R

I

I, Intermediate; R, resistant; S, sensitive.

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Molecular epidemiology Molecular epidemiology is the analysis of molecules, such as proteins and nucleic acids, for the detection, identication, and characterization of microorganisms to generate isolate-specic markers to assess epidemiologic relatedness. In identifying SARS-CoV-2 infections, the WHO has endorsed the identication of viral RNA in a patient’s biological materials through nucleic acid amplication tests (NAATs) such as reverse-transcription polymerase chain reaction (RT-PCR). A positive result of NAATs targeting at least two different genes, one of which is specic for SARS-CoV-2, provides a nal diagnosis of COVID-19. The RT-PCR test remains the “gold standard” for identifying acute SARS-CoV-2 infection. The laboratory has been able to provide a signicant and critical contribution to diagnosing an acute SARS-CoV-2 infection through molecular testing. In addition, with serologic testing, the laboratory has been able to detect the presence and extent of an immune response against the virus. Pulsed-eld gel electrophoresis (PFGE) is a strain-typing technique that can be an important adjunct to epidemiologic investigations. In this method, enzyme-digested chromosomal fragments of bacteria are separated electrophoretically. The patterns of the fragments are compared among strains of microbes recovered in a possible outbreak. Strains with dissimilar patterns would be determined to be unrelated. However, if the patterns are similar, the strains can be identied as possibly related. This potential relatedness is an additional epidemiologic tool that can be incorporated into the investigation of an outbreak. PFGE has been used since the 1980s, and it has remained the reference method because of its high discriminatory power, reproducibility, and nearly 100% typeability. A number of other techniques involving primarily amplication of genomic sequences by polymerase chain reaction and gene sequencing are also used to determine relatedness of species in an outbreak.

Environmental culturing Case check 3.3 As part of an effective IPC program, the microbiology laboratory may be called on to perform cultures of various environmental sites. Recommendations for environmental infection control have been extensively discussed in a CDC document, “Guidelines for Environmental Infection Control in Health-Care Facilities.” The environment is rarely implicated in disease transmission, except with immunosuppressed patients. Although environmental cultures are generally to be avoided, as in the Case in Point, there are times when they become an important and often required element of an IPC program.

Air Usually, infections traced to air quality occur during construction activities in a health care setting. Because microbes in the air can be incriminated in HAIs, cultures of the air can be a component of air quality investigations. Initially, an infection control risk assessment should be conducted to determine whether the air is the likely source of infectious particles. Such an assessment is necessary in construction activities and must be done before any decision to culture the air can

be made. The CDC makes no recommendation regarding routine microbiological air sampling before, during, or after construction. If a fungal infection such as aspergillosis occurs during or immediately after construction, an outbreak investigation may be initiated and control measures implemented. Such an investigation may involve collecting environmental samples (e.g., searching for sources of airborne fungi). Highvolume air samplers are the preferred method for collection, although settle plates may also be used. The results of these microbiological cultures must be reported to the IPC team and evaluated.

Water Water is incriminated in outbreaks in many of the settings for which microbiology laboratories provide service. Outbreaks can occur in various environmental situations, such as those associated with contaminated drinking water (e.g., hospitals, ECFs, prisons) or recreational water (e.g., swimming pools, whirlpools, lakes, streams). They may take place in homes, aboard a plane or ship, in a city or state, or in a foreign country. In the United States, an average of 46 waterborne outbreaks were reported each year between 1971 and 2013, resulting in 642,782 outbreak-associated illnesses from 50 states and 6 territories. Waterborne pathogens often cause diarrheal illness. Other waterborne diseases include respiratory illnesses via inhalation of aerosols (e.g., legionellosis), hepatitis (hepatitis A or hepatitis E), skin infections (from Pseudomonas spp. or mycobacteria), and central nervous system infections via nasal aspiration (Naegleria spp.). Because of these infection control implications, the laboratory must be prepared to offer diagnostic services or recommend laboratories that do offer those services for waterborne pathogens. Table 3.6 lists examples of waterborne pathogens. Some waterborne agents can be recovered by routine microbiology procedures such as cultures, but others may require specialized techniques. When asked to perform environmental cultures of water, the microbiology laboratory must determine which specic pathogens are sought if this information is known. If legionellosis is suspected, for example, the laboratory must have standard operating procedures for recovering this microbe. If the outbreak involves diarrheal diseases of an unknown cause or causes, the recovery techniques must be broader and may require the use of a specialized laboratory. The IPs involved in the outbreak investigation must be consulted before routine culturing of environmental water is undertaken.

Table 3.6 Pathogens related to waterborne infections Viruses

Bacteria

Parasites

Norovirus Rotavirus Hepatitis A Hepatitis E

Salmonella spp. Campylobacter spp. Yersinia enterocolitica Escherichia coli (O157:H7) Legionella spp. Pseudomonas spp. Mycobacterium spp. Aeromonas spp.

Entamoeba histolytica Giardia lamblia Cryptosporidium spp. Naegleria spp. Acanthamoeba spp.

Outbreak investigation

In some settings, routine water cultures must be performed because of specic guidelines. For example, for chronic dialysis centers, the CDC recommendations include performing bacteriologic assays of water and dialysis uids at least once a month with standard methods. The IP or managers of the specic area of concern must be familiar with regulations and guidelines addressing water cultures. The laboratory must be aware of the standard methods to ensure that proper procedures and proper media are used. The laboratory scientist, IP, and manager should maintain a close working relationship to ensure compliance with culturing requirements.

Table 3.7 Examples of reportable diseasesa Class

Description

Examples

A1

Diseases of major public health concern—reported immediately on recognition of a case, suspected case, or positive laboratory results

Anthrax, botulism (foodborne), cholera, diphtheria, Ebola virus, plague, rabies, smallpox, meningococcal disease, measles, SARS-CoV-2, tularemia, yellow fever

A2

Diseases of public health concern needing timely response—reported by the end of the next business day after recognition of a case, suspected case, or positive laboratory results

Encephalitis (viral), foodborne disease outbreaks, hepatitis A, Legionnaires’ disease, pertussis, syphilis, tuberculosis, typhoid fever, vancomycin-resistant Staphylococcus aureus, vancomycinintermediate S. aureus, tetanus

A3

Diseases of signicant public health concern— reported by the end of the work week

Brucellosis, giardiasis, hepatitis B, hepatitis C, Lyme disease, Rocky Mountain spotted fever, trichinosis

B

Diseases reported only by the number of cases— reported by the end of the work week

Chickenpox (varicella), inuenza

C

Report of outbreak, unusual incidence, or epidemic—reported by the end of next work day

Blastomycosis, inuenza in a health care setting, histoplasmosis, scabies, staphylococcal skin infections, toxoplasmosis

Surfaces The culturing of environmental surfaces should be performed only under the combined direction of the laboratory and IP. In an outbreak investigation, surface culturing may be needed; however, such cultures should not be routinely obtained. The laboratory must be consulted before environmental surface cultures are undertaken to ensure that proper procedures and media are used. The laboratory scientist should be instrumental in the interpretation of the results.

Reporting The role of the microbiology laboratory does not stop with providing culture results. Depending on statutory requirements, the laboratory might also be responsible for the reporting of certain infectious diseases to public health jurisdictions. Other groups may expect reports as well, such as committees, persons managing specic programs, and the news media.

61

These are reporting regulations from the Ohio Administrative Code and may vary from state to state. a

Reporting to public health There is a requirement to report the identication or suspicion of certain infectious diseases to local, state, or federal public health entities. As shown in Table 3.7, diseases designated as class A1 are considered public health concerns and must be reported as soon as they are suspected or identied. Some of these requirements are federally mandated, whereas others may be designated by the state. It is imperative that the laboratory scientist knows which infectious diseases are reportable to what agency and in what time frame they are to be reported.

Reporting to committees and programs Depending on the setting served by the microbiology laboratory, there may be expectations of reports of infectious diseases to committees in that setting. For example, in an acute care hospital, IPC committees may expect reports with various types of microbiological information. Annual antibiograms, lists of reportable diseases, pathogens recovered in certain hospital units, isolates recovered from certain sites, and blood culture contamination rates are examples of reports that might be requested. In other settings, physicians may expect periodic updates, including antibiograms and pathogen prevalence. They may expect these to be delineated by ofce practice, physician, site, or patient type. Those making these requests may be

in home health care, extended care, or communal living settings. Schools and businesses may request updated reports, especially those related to outbreaks that affect their operation. These reports must be tempered by public health needs and individual condentiality restrictions. Insurance companies and community advocacy groups recently have expressed interest in knowing about infection control rates. Some states require periodic reporting of these rates. The microbiology laboratory may be involved in providing data for these reports.

Reporting to the media Among the activities of a microbiology laboratory, discussing microbiology and infection control activities with the media (e.g., television, radio, newspapers) may become necessary. Media relations for the laboratory should be discussed with the risk management area associated with the laboratory. Media relations represent an educational opportunity that might be investigated before the laboratory scientist speaks to media personnel. One must balance the public’s need for knowledge as perceived by the news media with privacy restraints for the patient, laboratory, and setting.

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Education Laboratory scientists and infection prevention and control practitioners The role of the microbiology laboratory in education is a further extension of its activities related to infection control. Laboratory scientists must not only keep themselves educated in their contribution to the IPC team, but also keep the infection control personnel educated regarding the laboratory’s contribution to the team. Seminars, scientic articles and books, computer-based learning, and discussion with infection control personnel are ways in which laboratory scientists can maintain their knowledge of infection control and laboratory techniques that can aid the infection control program. The laboratorian should continuously educate the IP regarding the abilities of the laboratory in contributing meaningful information to the IPC committee. As new techniques become available or old techniques are replaced, the laboratory scientist must relay this information to the IP. Similar information must be provided for others associated with the health care setting and IPC program. Ancillary personnel, such as housekeeping and maintenance personnel, benet from knowing the laboratory perspective of an IPC program: • What cleaning and disinfecting agents work against viruses? • How long does the agent need to be in contact with the environment to inactivate the virus? • When do the maintenance personnel need to worry about fungi? • What does mold growing on wet drywall look like? These and other questions arise among ancillary health care personnel, and the laboratory scientist must be prepared to respond. Consultation with the microbiology laboratory often is sought when construction is anticipated. Sometimes this education must be provided, even when it is not sought. • Which microbes may be harbored in standing water? • When should high-efciency particulate air (HEPA) lters be used? • What infectious agents might spread through a facility if proper barriers are not used to control demolition dust and debris? These are some questions that might be addressed by the microbiology laboratory scientist while acting as a consultant to the infection control program.

Safety The infection control program affects the microbiology laboratory by emphasizing the need for laboratory safety. As discussed in Chapter 4, safety in the microbiology laboratory encompasses the IPC program. Hand hygiene is a critical part of laboratory safety. Hand hygiene involves handwashing when hands are soiled or the use of alcohol hand rubs

Case check 3.4 Whereas the investigation could not conclusively determine why the outbreak in the Case in Point occurred, the use of contact precautions and improved hand hygiene stopped new cases. It is likely that the causative agent, A. baumannii, was in the hospital environment and was spread among the patients by improper hygiene. The presence of a ventilator in the affected patients bypassed host defenses in the compromised patients.

when hands are not soiled. The practice of standard precautions further extends infection control to the microbiology laboratory. Gloves must always be worn when blood or body uids are handled. Proper disposal of microbiological waste, according to state or local regulations and national guidelines, is another critical component of the infection control program in the microbiology laboratory. Receiving available vaccines and testing for diseases such as hepatitis B, inuenza, meningococcus, and tuberculosis are steps to protect laboratory personnel. All of these functions connect the microbiology laboratory safety program and the infection control program.

Response plans With the potential use of biological agents in terrorism and emerging pathogens associated with pandemics, the development of emergency response plans is paramount. The microbiology laboratory is an integral part of this infection control activity. Safety, specimen collection, agent identication, and agent control are components of the response plan to which the laboratory can contribute its input. Whether in everyday activities or activities in response to an outbreak or emerging diseases, the microbiology laboratory and laboratory scientists constitute a critical component of an efcient and successful infection control program.

POINTS TO REMEMBER

• The microbiology laboratory interacts with the infection preventionist and infection prevention and control committee in many different health care settings. • Surveillance is important to establish baseline data and recognize the need to investigate potential outbreaks. • The microbiology laboratory supports outbreak investigations by providing consultative services including epidemiologic correlation of isolates. • Although infrequently performed, environmental cultures may play a role in outbreak investigation. • Microbiology laboratory scientists must recognize their role in providing reports to health departments, committees, the infection prevention and control program, and on occasion, the public. • The microbiology laboratory must maintain competencies in new techniques and the identication of reemerging and emerging infectious diseases.

Response plans

• In anticipation of potential bioterrorism events and pandemics, the microbiology laboratory must be equipped to participate in emergency preparedness programs.

LEARNING ASSESSMENT QUESTIONS

1. Surveillance is dened as: a. The systematic collection and analysis of data b. The review of healthcare-associated infections in laboratory personnel c. The recognition of emerging pathogens d. The development of an infection control risk assessment 2. Microbes commonly encountered in healthcare-associated infections in hospitals include: a. Salmonella spp., Shigella spp., hepatitis C virus, Neisseria meningitidis b. Staphylococcus aureus, Pseudomonas aeruginosa, MRSA, Escherichia coli c. Pseudomonas aeruginosa, Salmonella spp., hepatitis C virus, Giardia spp. d. All of the above 3. Pulsed-eld gel electrophoresis (PFGE) might be performed to: a. Identify staphylococcal species b. Assist in an outbreak investigation c. Develop a new isolation precaution d. All of the above 4. The occurrence of surgical site infections is generally calculated as: a. The rate of infections in 1000 device-related events b. The percentage of infections in 100 device-related events c. The percentage of infections in surgical sites or procedures d. The rate of infections per 100 hospital days 5. Health departments frequently require the reporting by the laboratory of: a. Diseases of major health concerns (e.g., smallpox) b. Diseases needing timely response (e.g., foodborne outbreaks) c. Outbreaks of public health concern (e.g., scabies) d. All of the above 6. Microbial pathogens of potential bioterrorism activity include: a. Bacillus anthracis, Staphylococcus aureus, West Nile virus b. Yersinia pestis, Staphylococcus aureus, hepatitis C virus c. Bacillus anthracis, Yersinia pestis, Francisella tularensis d. Bacillus anthracis, Escherichia coli, coronaviruses 7. Environmental cultures are usually to be avoided, except in: a. An outbreak investigation b. The occurrence of infections following construction c. Compliance with specic regulatory requirements d. All of the above 8. The formal steps in an outbreak investigation include: a. Establishing a case denition and culturing air and water b. Establishing a case denition, forming and testing a hypothesis, and communicating ndings c. Forming and testing a hypothesis, performing PFGE, and calculating an infection rate d. Conrming an outbreak exists, calculating an infection rate, and performing serology and culture tests

63

9. Infection prevention and control programs rely on microbiology laboratory support in: a. Public health settings b. Acute care facilities c. Home care settings d. All of the above 10. The microbiology laboratory interacts with the infection prevention and control program by providing: a. Culture results b. Antibiograms and pathogen prevalence reports c. Environmental cultures when appropriate d. All of the above

BIBLIOGRAPHY Baron, E. J., et al. (2005). Blood cultures IV. Cumitech 1C. Washington, DC: American Society for Microbiology Press. Centers for Disease Control and Prevention. (2016). Foodborne (1973–2013) and waterborne (1971–2013) disease outbreaks—United States. Morbidity and Mortality Weekly Report, 63, 79. Centers for Disease Control and Prevention. (2021). 2020 national and state healthcare-associated infections (HAI) progress report. Available at: https://arpsp.cdc.gov/prole/national-progress/united-states. (Accessed 19 February 2022). Centers for Disease Control and Prevention. (2012). Principles of epidemiology in public health practice: lesson 6, section 1. Available at: https://www.cdc.gov/csels/dsepd/ss1978/lesson6/section1.html. (Accessed 19 February 2022). Centers for Disease Control and Prevention. (2020). Managing investigations during an outbreak. Available at: https://www. cdc.gov/coronavirus/2019-ncov/php/contact-tracing/ contact-tracing-plan/outbreaks.html. (Accessed 19 February 2022). Centers for Disease Control and Prevention. (2020). Infection control guidance for healthcare professionals about coronavirus (COVID-19). Available at: https://www.cdc.gov/coronavirus/2019-nCoV/ hcp/infection-control.html. (Accessed 19 February 2022). Carrico, R., et al. (2009). APIC text of infection control and epidemiology (3rd ed.). Washington, DC: Association for Professionals in Infection Control and Epidemiology. Edwards, J. R., et al. (2007). National Healthcare Safety Network (NHSN) Report, data summary for 2006. American Journal of Infection Control, 35, 290. Lippi, G., et al. (2020). Updates on laboratory investigations in coronavirus disease 2019 (COVID-19). Acta bio-medica : Atenei Parmensis, 91, e2020030. https://doi.org/10.23750/abm. v91i3.10187 (Accessed 19 February 2022). Katella, K. (2021). Our pandemic year—a COVID-19 timeline. Available at: https://www.yalemedicine.org/news/covid-timeline. (Accessed 19 February 2022). Magill, S. S., et al. (2014). Multistate point-prevalence survey of health care–associated infections. The New England Journal of Medicine, 370, 13. Nolte, F. S. (2019). Molecular epidemiology. In K. C. Carroll, et al. (Eds.), Manual of clinical microbiology (12th ed., p. 86). Washington, DC: ASM Press. Murray, C. J. L., et al. (2022). Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. The Lancet, 399, 629. Roxby, A. C., et al. (2020). Outbreak investigation of COVID-19 among residents and staff of an independent and assisted living community for older adults in Seattle, Washington. JAMA Internal Medicine, 180, 1101. Sehulster, L., et al. (2003). Guidelines for environmental infection control in health-care facilities. Recommendations of CDC and the Healthcare Infection Control Practices Advisory Committee (HICPAC). MMWR Recommendations and Reports, 52, 1. Siegel, J. D., et al. (2019). Guideline for isolation precautions: preventing transmission of infectious agents in healthcare settings (2007). Available at: https://www.cdc.gov/hicpac/pdf/ isolation/Isolation/updates.html. (Accessed 19 February 2022).

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U.S. Department of Health and Human Services. (2021). National HAI targets & metrics. Available at: https://www.hhs.gov/oidp/topics/ health-care-associated-infections/targets-metrics/index.html. (Accessed 19 February 2022). World Health Organization. (2020). Surveillance strategies for COVID-19 human infection, coronavirus (COVID-19) update no. 29, 5 June 2020.

Available at: https://www.who.int/docs/default-source/ coronaviruse/risk-comms-updates/update-29-surveillance-strategies-for-covid-19-human-infection.pdf?sfvrsn=3c2cab92_2. (Accessed 19 February 2022).

4 Control of microorganisms: disinfection, sterilization, and microbiology safety Michelle M. Jackson*

CHAPTER OUTLINE

Disinfection and sterilization, 66 Sterilization versus disinfection, 66 Factors that influence the degree of killing, 67 Types of organisms, 67 Number of organisms 67 Concentration of disinfecting agent, 68 Presence of organic material, 68 Nature of the surface to be disinfected, 68 Contact time, 68 Temperature, 68 pH, 68 Biolms, 68 Compatibility of disinfectants, 68 Methods of disinfection and sterilization, 69 Physical methods, 69 Chemical methods, 70 Disinfectants versus antiseptics, 71 Alcohols, 71 Aldehydes, 72 Halogens, 72 Chlorine and chlorine compounds, 72 Detergents: quaternary ammonium compounds, 73 Phenolics, 73 Heavy metals, 74 Gases, 74 EPA regulations on chemical surface disinfectants, 75 FDA regulations on chemical skin antiseptics, 75 Hygienic handwashing and waterless handrubs, 76 Surgical hand scrub and waterless surgical handrubs, 77 Presurgical skin disinfection, 77 Microbiology laboratory safety, 77 *This chapter was prepared by the author in her private capacity. No ofcial support or endorsement by the Food and Drug Administration is intended or implied.

General laboratory safety, 78 Safety program for the clinical laboratory, 78 Hazardous waste, 85 Chemical safety, 85 Fire safety, 96 Storage of compressed gases, 97 Electrical safety, 97 Miscellaneous safety considerations, 97 Safety training, 97 Bioterrorism and the clinical microbiology laboratory, 98 Laboratory response network, 98 Safety during a possible bioterrorism event, 98 Packaging and shipping of infectious substances, 98 Bibliography, 100

OBJECTIVES

After reading and studying this chapter, you should be able to: 1. Dene the following terms: sterilization, disinfection, and antiseptic 2. Differentiate the functions and purposes of a disinfectant and an antiseptic. 3. Describe the general modes of antimicrobial action. 4. Describe the way each physical agent controls the growth of microorganisms. 5. Give the mechanism of action for each type of chemical agent commonly used in antiseptics and disinfectants. 6. Describe the different heat methods and their respective applications. 7. Describe Environmental Protection Agency regulations on chemical surface disinfectants and Food and Drug Administration regulations on chemical skin antiseptics. 8. Discuss the appropriate use of the following skin antiseptics in health care settings by health care personnel: handwash or handrub, surgical hand scrub or surgical handrub, and patient preoperative skin preparation. 9. Describe the hazards that can be encountered in a microbiology laboratory. 10. List the elements included in an exposure control plan.

65

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Control of microorganisms: disinfection, sterilization, and microbiology safety

11. Discuss the practice of standard precautions. 12. Differentiate standard precautions and transmission-based precautions. 13. Dene and give examples of engineering controls, work practice controls, and personal protective equipment. 14. Discuss the World Health Organization classication of infectious microorganisms by risk group. 15. Compare the three types of biological safety cabinets. 16. Differentiate the four categories of biosafety levels. 17. Given an infectious agent and test procedure, justify the appropriate safety precautions to use to ensure that an exposure event does not occur. 18. Explain the information that must be included in safety data sheets. 19. Describe the components of basic re safety and electrical safety within the microbiology laboratory. 20. Discuss the special safety considerations that must be addressed in the clinical microbiology laboratory during a possible bioterrorism event.

KEY TERMS

Antisepsis Antiseptic Biolms Biological safety cabinet (BSC) Biosafety level (BSL) Bloodborne pathogens Disinfectants Disinfection Employee right-to-know Engineering controls Environmental Protection Agency (EPA) Exposure control plan Fast-acting antiseptic Filtration Food and Drug Administration (FDA) Generally recognized as safe and effective (GRASE) Germ theory of disease Hazard-rating diamond Health care antiseptic drug products Health care personnel handwash Laboratory-acquired infection (LAI)

Laboratory response network Microbial load Moist heat National re protection association (NFPA) New drug application (NDA) Over-the-counter (OTC) Pasteurization Patient preoperative skin preparation Personnel protective equipment (PPE) Persistent Prions Resident biota (ora) Risk groups Safety data sheets (SDSs) Sentinel laboratories Sporicidal Standard precautions Sterilization Surgical hand scrub Transmission-based precautions Transient biota Work practice controls

Case in point Dr. Adams is a geriatrician at a busy metropolitan medical clinic. When she arrives at the clinic, the patients waiting for her in the waiting room are practicing social distancing and wearing masks because of the COVID-19 pandemic. She washes her hands immediately and then uses an alcohol-based hand sanitizer. As she opens the examining room door to see her rst patient, Mrs. Jones, the patient notices that Dr. Adams opened the door with her bare hands and is about to examine her. Mrs. Jones tells the doctor to wash her hands rst or use the

hand sanitizer before she proceeds with examining her. How should Dr. Adams respond? Issues to consider After reading the patient’s case history, consider: • Potential risks that the physician is taking in spreading infectious agents to her patients • Importance of handwashing and use of an appropriate antiseptic • Quality control plan to minimize the risks to patients

Disinfection and sterilization Safety in the laboratory cannot be overemphasized. Quantication of the risk of working with an infectious agent is difcult. Risk to an individual increases with the frequency and type of organism and level of contact with the agent, as demonstrated by the Case in Point. Each laboratory must develop and institute a plan that effectively minimizes exposure to infectious agents. This chapter provides information on standard disinfection and sterilization techniques for the clinical laboratory. It presents an overview of the following topics: • Sterilization and disinfection • Chemical and physical methods of disinfection and sterilization • Principles and application of each method • Common disinfectants and antiseptics used in health care settings • Principles and applications of disinfectants and antiseptics • Regulatory process of disinfectants and antiseptics This chapter also covers laboratory safety guidelines for the clinical laboratory practitioner to ensure proper protection and reduce risks of exposure to potentially hazardous biological agents.

Sterilization versus disinfection The scientic use of disinfection and sterilization methods originated more than 100 years ago when Joseph Lister introduced the concept of aseptic surgery using carbolic acid, now called phenol. Since then, the implementation of effective sterilization and disinfection methods has remained crucial in the control of infections in the laboratory and health care facilities (healthcare-associated infections). To understand fully the principles of disinfection and sterilization, we must have accurate denitions of certain terms. Sterilization refers to the destruction of all forms of life, including bacterial spores. By denition, there are no degrees of sterilization—it is an all-or-nothing process. Chemical or physical methods may be used to accomplish sterilization. The term sterile is relevant to the method used. For example, a solution that has been ltered through a certain pore-size lter (32 single titer might be suggestive

Not recommended

Not recommended

Immunouorescent antibody

Separately measures IgG and IgM

Not recommended

Not recommended

Enzyme immunoassay

Separately or simultaneously measures IgG and IgM

Not recommended

Not recommended

Nonserologic

Serologic

IgG, Immunoglobulin G; IgM, immunoglobulin M.

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Mycoplasma and Ureaplasma

Table 25.7 Laboratory detection of frequent respiratory pathogens Epidemiologic factors

Laboratory methods

Age

Organism Frequently Involved

Disease

Season

Specimen Source

Stain

Culture

Nonculture

Newborn

1, 3, 4, 7

Pneumonia

Year rounda

Tracheal suction

Gram

Routine, plus mycoplasmal

Grade school

2, 4

Atypical pneumonia

Fall and winter

Sputum, nasopharynx

Gram

Routine, plus mycoplasmal

Mycoplasmal serology

College student

1, 2, 8

Biphasic disease with pharyngitis and later, bronchitis

Year roundb

Sputum, nasopharynx

Nonec

B. pertussis

Mycoplasmal serology

Adult

2, 4, 5, 6

Pneumonia or atypical pneumonia

Year rounda

Sputum

Gram

Routine

Mycoplasmal serology

Bronchoalveolar lavage specimen

Gram, Gomori methenamine silver, and/ or calcouor white

Routine, plus fungal

1, Chlamydia pneumoniae; 2, Mycoplasma pneumoniae (outbreak); 3, Mycoplasma hominis; 4, viral, e.g., adenovirus, respiratory syncytial virus, inuenza virus (seasonal); 5, other—fungus, Legionella, or Pneumocystis pneumonia; 6, Streptococcus pneumoniae; 7, Streptococcus agalactiae; 8, Bordetella pertussis . a

Seasonal incidence depends on the pathogen.

b c

The greatest seasonal incidence of pertussis is spring and summer, followed by winter.

In place of direct stains, nucleic acid amplication tests for C. pneumoniae and/or B. pertussis should be performed.

most likely candidate for the disease, clinical presentation, age of the patient, and seasonality, recognizing that there is a certain predictability with selected pathogens. Table 25.7 presents laboratory methods used to diagnose infections caused by several pathogens—Mycoplasma, Chlamydia, Legionella, mycobacteria, fungi, and viruses—in various age groups. All respiratory specimens should be stored at −80° C if storage time is likely to exceed 24 hours. Acute-phase sera should also be stored frozen for subsequent antibody titer testing.

POINTS TO REMEMBER

• The mollicutes are minute organisms characterized by the lack of a cell wall. Because of the lack of a cell wall, the mycoplasmas are inherently resistant to the β-lactam antibiotics. • The most clinically signicant species of the Mycoplasmataceae are M. pneumoniae, M. hominis, M. genitalium, M. fermentans, and Ureaplasma spp., although others are beginning to be recognized as opportunistic pathogens. • M. pneumoniae is an important cause of community-acquired, atypical pneumonia. It is not considered part of the normal human microbiota. • M. hominis, M. genitalium, and Ureaplasma spp. are genital mollicutes. • Because of the difculty to isolate, M. pneumoniae infection is most often diagnosed by serologic methods. M. hominis and Ureaplasma spp. are commonly diagnosed by culture, although PCR technology is also available. M. genitalium and M. fermentans are generally detected by PCR.

LEARNING ASSESSMENT QUESTIONS

1. From which source did the neonate described in the Case in Point likely acquire the infection? a. Breast milk b. Inhalation after birth c. Passed through the birth canal d. Infectious agent crossed the placenta 2. Why was the Gram stain result negative in the Case in Point? a. Mollicutes do not Gram stain. b. The Gram stain is too insensitive. c. Mollicutes do not retain safranin; need to use a different counterstain. d. When mollicutes are suspected, the time needed to heat x the smear must be extended. 3. How does primary atypical pneumonia caused by M. pneumoniae differ from pneumonia caused by S. pneumoniae? a. Has a lower mortality rate b. Is not spread person to person c. Is seen more often in older adults d. Is characterized by a bloody, productive cough 4. Which special stain is used on suspected colonies of Mycoplasma? a. Acridine orange b. Calcouor white c. Dienes d. Silver 5. An amniotic uid is submitted to the microbiology laboratory for isolation of mycoplasma. It will be 48 hours before the specimen can be processed. At what temperature should the specimen be stored?

Interpretation of laboratory results

6. 7. 8. 9. 10. 11.

a. 22° C b. 4° C c. -20° C d. -80° C Would routine prenatal culture of the mother have yielded this organism? List the four common species of mollicutes associated with the genitourinary tracts of humans. Which culture media are used to isolate M. pneumoniae, M. hominis, and U. urealyticum? What is the signicance of isolating M. hominis from a vaginal specimen? Which current serologic assays are available to demonstrate M. pneumoniae antibodies? Why are the mollicutes universally resistant to penicillin?

BIBLIOGRAPHY Centers for Disease Control and Prevention. (2020). Mycoplasma pneumoniae infections: disease specics. Available at: https://www. cdc.gov/pneumonia/atypical/mycoplasma/hcp/disease-specics. html. (Accessed 12 June 2022). Chaudhry, R., et al. (2016). Pathogenesis of Mycoplasma pneumoniae: an update. Indian Journal of Medical Microbiology, 34, 7.

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Maselli, D. J., et al. (2018). The immunopathologic effects of Mycoplasma pneumoniae and community-acquired respiratory distress syndrome toxin. A primate model. American Journal of Respiratory Cell and Molecular Biology, 58, 253. Nakane, D., et al. (2021). Molecular ruler of the attachment organelle in Mycoplasma pneumoniae. PLOS Pathogens, 17, e1009621. https://doi. org/10.1371/journal.ppat.1009621. Seo, H. Y. (2020). Immunochromatography for the diagnosis of Mycoplasma pneumoniae infection: a systematic review and meta-analysis. PLoS One, 15, e0230338. Tang, M., et al. (2021). Comparison of different detection methods for Mycoplasma pneumoniae infection in children with community-acquired pneumonia. BMC Pediatrics, 21, 90. Tantengco, O., et al. (2021). The role of genital mycoplasma infection in female infertility: a systematic review and meta-analysis. American Journal of Reproductive Immunology, 85, e13390. Waites, K. B., & Bébéar, C. (2019). Mycoplasma and Urealyticum. In K. C. Carroll, et al. (Eds.), Manual of clinical microbiology (12th ed., p. 1117). Washington, DC: ASM Press. Zhu, X., et al. (2016). Epidemiology of Ureaplasma urealyticum and Mycoplasma hominis in the semen of male outpatients with reproductive disorders. Experimental and Therapeutic Medicine, 12, 1165.

26 Mycobacterium tuberculosis and nontuberculous mycobacteria Donald C. Lehman

CHAPTER OUTLINE

General characteristics, 571 Clinical significance of the Mycobacterium tuberculosis complex, 572 Mycobacterium tuberculosis, 572 Mycobacterium bovis, 575 Clinical significance of nontuberculous mycobacteria, 575 Slowly growing species, 575 Rapidly growing species, 579 Mycobacterium leprae, 580 Isolation and identification of mycobacteria, 580 Laboratory safety considerations, 583 Specimen collection, 583 Digestion and decontamination of specimens, 585 Concentration procedures, 586 Staining for acid-fast bacilli, 586 Culture media and isolation methods, 587 Laboratory identication, 589 Susceptibility testing of Mycobacterium tuberculosis, 594 Bibliography, 596 OBJECTIVES

After reading and studying this chapter, you should be able to: 1. Compare the general characteristics of mycobacteria with those of other groups of bacteria. 2. Discuss the clinical disease caused by Mycobacterium tuberculosis 3. Analyze patient results obtained with the tuberculin skin test and interferon-gamma release assay for the diagnosis of tuberculosis. 4. Develop a protocol for the isolation and identication of M. tuberculosis from a sputum specimen. 5. Discuss the clinical signicance of nontuberculous mycobacteria. 6. Describe the appropriate specimen collection and processing procedures to recover mycobacteria from clinical samples.

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7. Discuss the safety precautions to be followed while working in a mycobacteriology laboratory. 8. Justify the digestion and decontamination requirements of certain clinical specimens for the isolation of mycobacteria. 9. Describe the principles and procedures for the stains used to demonstrate mycobacteria in clinical samples and culture isolates. 10. Compare the different culture media used for the isolation of mycobacteria. 11. Discuss the different tests used to identify mycobacteria. 12. Compare continuous monitoring systems with those of conventional media for detecting mycobacterial species in clinical samples. KEY TERMS

Acid fastness Auramine stain Auramine-rhodamine uorochrome stain Granuloma Kinyoun stain Middlebrook 7H11 medium Miliary tuberculosis Mycobacterium avium complex (MAC)

Mycobacterium tuberculosis complex (MTBC) Nonchromogenic Nontuberculous mycobacteria (NTM) Photochromogenic Pott disease Puried protein derivative (PPD) Scotochromogenic Ziehl-Neelsen stain

Case in point A 56-year-old male patient came to the emergency department complaining of fatigue and weight loss (10 lb) over the past 12 months. The patient also complained of a cough for 3 months that produced red-tinged sputum. He indicated a history of night fever and chills but reported not having dyspnea or chest pain. The patient, who moved to the United States from Mexico, had a family history of pulmonary tuberculosis. He reported that his last puried protein derivative (PPD) skin test, performed approximately 5 years ago, was nonreactive. Vital signs included temperature of 36.5° C (97.7° F), pulse of 63 beats per minute, respirations of 15 breaths per minute, and

General characteristics

blood pressure of 96/56 mm Hg. Chest radiography revealed an inltrate in the upper lobe of the right lung. Computed tomography of the chest showed a nodular patchy opacity in the upper lobe of the right lung. The patient was admitted for further evaluation. A PPD skin test showed a 10-mm by 7-mm induration. Three sputum samples were obtained over a 3-day period for acid-fast bacilli (AFB) smear and culture. All three samples were reported as no AFB seen on direct acidfast stained smears. Processed samples were inoculated onto Löwenstein-Jensen (LJ) medium and into BACTEC 12B bottles (BD Diagnostic Systems, Sparks, MD). After 12 to 14 days of incubation, the BACTEC bottles from all three specimens were positive. Kinyoun staining of smears from the bottles revealed AFB. Polymerase chain reaction (PCR) DNA amplication for Mycobacterium tuberculosis of the BACTEC medium showed a positive result. A four-drug antituberculosis regimen comprising isoniazid, rifampin, pyrazinamide (PZA), and ethambutol was recommended. Issues to consider After reading the patient’s case history, consider: • Signicant aspects of this patient’s family history • The characteristic symptoms of tuberculosis • The typical length of time for a culture to yield pathogenic Mycobacterium

The genus Mycobacterium is composed of over 180 recognized and proposed species. The most familiar of the species are Mycobacterium tuberculosis and Mycobacterium leprae, the causative agents of tuberculosis (TB) and Hansen disease (leprosy), respectively. Both diseases have long been associated with chronic illness and social stigma. TB remains a major cause of morbidity and mortality in the world today and is the leading cause of death resulting from a single infectious agent—surpassing human immunodeciency virus (HIV). In 2020, The Global Tuberculosis Report by the World Health Organization (WHO) estimated 9.9 million new cases, and 1.5 million people died from TB, including 214,000 people with HIV worldwide. Nontuberculous mycobacteria (NTM) In addition to TB and Hansen disease, Mycobacterium spp. produce a spectrum of infections in humans and animals. A large group of mycobacteria, excluding the M. tuberculosis complex (MTBC) and M. leprae, normally inhabit the environment and can cause disease that often resembles TB in humans. These organisms are sometimes referred to as atypical mycobacteria, nontuberculous mycobacteria (NTM), or mycobacteria other than the tubercle bacillus (MOTT). The term nontuberculous mycobacteria is used here. The growing number of immunocompromised individuals worldwide has led to a resurgence of TB and diseases caused by NTM. Box 26.1 shows the usual clinical signicance of Mycobacterium spp. isolates. Epidemiologic changes have led to challenges in the mycobacteriology laboratory, including rapid identication of all clinically signicant mycobacteria and antimicrobial susceptibility testing of Mycobacterium spp. Fortunately, new developments in the eld of clinical mycobacteriology are helping meet these challenges. Rapid methods, like nucleic acid amplication tests (NAATs), can eliminate the need for lengthy culturing and protracted biochemical methods of identication. Further developments in the application of molecular biology, including matrix-assisted laser desorption/

571

BOX 26.1 Usual clinical signicance of Mycobacterium species

isolates Pathogen Mycobacterium tuberculosis Mycobacterium bovis Mycobacterium africanum Mycobacterium ulcerans Mycobacterium leprae

Often pathogen, potential pathogen Mycobacterium avium complex Mycobacterium kansasii Mycobacterium marinum Mycobacterium haemophilum Mycobacterium genavense Mycobacterium abscessus subsp. abscessus Mycobacterium abscessus subsp. massiliense Mycobacterium abscessus subsp. bolletti Mycobacterium chelonae Mycobacterium fortuitum group Mycobacterium simiae Mycobacterium szulgai Mycobacterium xenopi

Potential pathogen Mycobacterium malmoense Mycobacterium scrofulaceum

Usual saprophyte, rare pathogen Mycobacterium gordonae Mycobacterium flavescens Mycobacterium gastri Mycobacterium nonchromogenicum Mycobacterium terrae Mycobacterium phlei Mycobacterium smegmatis Mycobacterium vaccae

ionization–time-of-ight (MALDI-TOF) mass spectrometry to mycobacteriology may further diminish the time required for identication, increase accuracy and reproducibility, ease performance, and reduce cost.

General characteristics Mycobacteria are slender, slightly curved or straight, rodshaped organisms 0.2 to 0.6 µm × 1 to 10 µm in size. They are nonmotile and do not form spores. However, limited studies claim to demonstrate spores or sporelike structures in Mycobacterium marinum. The cell wall has extremely high lipid (mycolic acid) content; thus mycobacterial cells resist staining with commonly used basic aniline dyes, such as those used in Gram stain, at room temperature. Mycobacteria take up dye with increased staining time or application of heat but resist decolorization with acid-ethanol. This characteristic, referred to as acid fastness—hence the term acidfast bacilli (AFB)—distinguishes mycobacteria from most other genera. Mycolic acids are also found in other genera (e.g., Corynebacterium and Rhodococcus); however, the amount of mycolic acids in the mycobacteria is much larger than in these other bacteria.

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Mycobacteria are strictly aerobic, but increased carbon dioxide (CO2) concentration will enhance the growth of some species. The pathogenic mycobacteria grow more slowly than most other bacteria that are pathogenic to humans. Typically, mycobacteria associated with disease require 2 to 6 weeks of incubation on complex media at specic optimal temperatures, whereas the rapidly growing species generally grow on simple media in 2 to 3 days at temperatures of 20° to 40° C. One of the mycobacteria pathogenic to humans, M. leprae, fails to grow in vitro.

Clinical signicance of the Mycobacterium tuberculosis complex The Mycobacterium tuberculosis complex (MTBC) consists of M. tuberculosis, M. bovis (including the vaccination strain bacillus Calmette-Guérin [BCG]), M. africanum, M. microti, M. canetii, M. caprae, M. pinnipedii, M. mungi, M. orygis, M. mungi, and M. suricattiae. Members of the MTBC display a high degree of genetic homogeneity, although they have different phenotypic characteristics and mammalian host ranges. Within this group, most human infections are caused by M. tuberculosis and to a lesser extent M. bovis and M. africanum. M. bovis is found primarily in cattle, but it can infect other mammals, including humans. M. africanum has been associated with human cases of TB in tropical Africa, and M. microti has been linked to TB in both immunocompetent and immunocompromised individuals. M. canetii has been reportedly causing infections in children and patients with HIV infection in Africa. The remaining species are infrequently associated with human infections.

Mycobacterium tuberculosis Mycobacterium tuberculosis was rst described by Robert Koch in 1882. However, TB is one of the oldest documented communicable diseases and remains a leading cause of morbidity and mortality globally. A disease of poverty, as many as 25% of the world’s population may be infected with the bacteria causing TB. About 90% of TB cases occur in 30 TB high-burden countries. The geographic areas with the most cases are Southeast Asia (44%), Africa (25%), and the Western Pacic (18%). The United States has one of the lowest TB rates in the world. In 2021, 7860 TB cases were reported, 687 more than during 2020 (7173), a 9.4% increase in TB incidence (cases per 100,000 population) from 2.16 to 2.37. However, up to 13 million people living in the United States have latent TB infection. These individuals are infected with the bacteria causing TB but remain asymptomatic. Currently, about 71% of the cases are in foreign-born individuals from endemic areas.

Primary tuberculosis After exposure to M. tuberculosis, whether or not a person develops TB is determined by his or her cellular immune response, the infective dose, and the virulence of the strain. TB is usually a disease of the lower respiratory tract. Typical signs and symptoms include malaise, weight loss, night sweats, and cough that can produce or not produce sputum that is with or without blood. Tubercle bacilli are acquired

from persons with active disease who are excreting viable bacilli by coughing, sneezing, or talking. Airborne droplets containing bacteria enter the respiratory tract of an exposed individual and reach the lung alveoli. M. tuberculosis cells are phagocytized by alveolar macrophages but can prevent fusion of phagosomes containing bacteria with lysosomes. This permits intracellular multiplication of the bacteria. In a person with adequate cellular immunity, macrophages secrete interleukin-12 and tumor necrosis factor alpha, which recruit T cells and natural killer cells and enhance the inammatory reaction. Some of the T cells differentiate into T-helper cells type-1 (Th1) releasing lymphokines, such as interferon-γ (IFN-γ). IFN-γ stimulates macrophages in the infection site to destroy intracellular mycobacteria. This is followed by regression and healing of the primary lesion. In many exposed individuals, the immune system does not initially eliminate the bacteria, and this can result in lifelong or latent infections. The pathologic features of TB are the result of a hypersensitivity reaction to mycobacterial antigen. If there is little antigen and a strong hypersensitivity reaction, a small, hard tubercle, or granuloma, may be formed. The granuloma is an organization of lymphocytes, macrophages, broblasts, and capillaries, along with brosis encapsulation. With granuloma formation, the bacteria are contained and can ultimately be killed, and healing occurs along with calcication and scar formation as a reminder of the past infection. If the antigen load and hypersensitivity reaction are both high, tissue necrosis from the enzymes of degenerating macrophages can occur; the tissue response is less organized, and granulomas are surrounded by brin that protects the bacteria from phagocytosis by macrophages. Necrotic or caseous material can be present at the site of the primary lesion because of solid or semi-solid amorphous material deposited at the site of necrosis. After healing of the primary infection, the bacilli are not totally eradicated but can remain viable, but dormant, in granulomas for months or years. In infected individuals, there is a potential for reactivation of TB when the bacteria are released from the granulomas. This happens because of immunosuppression from old age or other causes. A small percentage of individuals who are infected with TB develop progressive (active) pulmonary disease, usually from a failed cellular immune response and, hence, a failure to stop multiplication of the bacilli. In young children or older adults with primary infection and in people with an underlying immunodeciency, massive lymphohematogenous dissemination may occur and lead to meningeal or miliary (disseminated) TB, which is discussed later. In addition, 10% of young adults may progress to active disease from their primary infection. This will resemble reactivation TB in older adults; the only way to differentiate it is a positive puried protein derivative (PPD) skin test result in a previously negative individual.

Case check 26.1 A positive PPD skin test result only indicates past exposure to M. tuberculosis; it does not imply a recent infection. In the Case in Point, the patient reported a negative skin test result obtained about 5 years ago, thus establishing a negative baseline. During his current evaluation, he had a positive skin test result, indicating exposure and subsequent immune response at some point in the past 5 years.

Clinical signicance of the Mycobacterium tuberculosis complex

Reactivation tuberculosis Approximately 5% of those infected develop active disease within 5 years, and an additional 5% to 10% develop disease within their lifetime. Progression from infection to active disease differs, depending on the patient’s age and the intensity and duration of exposure. Malnutrition, with or without other factors, such as alcoholism, homelessness, incarceration, immunosuppression, and HIV infection, can contribute greatly to progression to active TB. Reactivation TB also occurs more frequently in patients with immune-mediated inammatory diseases like systemic lupus erythematosus and rheumatoid arthritis. Reactivation TB from a latent infection occurs when there is an alteration or suppression of the cellular immune system in the infected host that favors replication of the bacilli and progression to disease. The symptoms of disease are slow in developing (insidious) and consist of fever, shortness of breath, night sweats and chills, fatigue, anorexia, and weight loss. About 20% of individuals may have no symptoms, but most patients eventually have productive cough, chest pain, and fever. Hemoptysis, indicating cavitation and necrosis, occurs in 25% of cases. In patients with reactivation TB, radiography reveals a patchy or conuent consolidation with increased linear densities extending to the hilum; thickwalled cavities without air-uid levels usually are found in the apical or posterior segments of the upper lobe or in the superior segment of the middle lobe of the lung. If there is bronchogenic spread of the bacilli, multiple alveolar densities will be seen; rarely is there enlargement of the lymph nodes. In chronic disease, brosis, loss of lung volume, and calcications will be demonstrated. The PPD skin test may yield negative results in up to 25% of these cases; diagnosis is conrmed by stained smear and culture of sputum, gastric aspirates, or bronchoscopy specimens. Fiberoptic bronchoscopy has been found to yield 95% recovery of the bacteria; postbronchoscopic sputa are also usually positive. In any case of pulmonary TB, complications could occur if diagnosis and treatment are delayed. These include empyema, pleural brosis, massive hemoptysis, adrenal insufciency (rare), and hypercalcemia (up to 25% of cases). In patients with acquired immunodeciency syndrome (AIDS) and TB with drug-resistant bacilli, the risk of progression to disease from infection is quite high, although clinical ndings may differ from those in the patient with AIDS and reactivation TB. The diagnosis is usually made by stained smears and culture, with a rate of sensitivity similar to that in patients without AIDS.

Extrapulmonary tuberculosis Disseminated or extrapulmonary tuberculosis (EPTB), infection outside the lungs, occurred much less commonly than pulmonary TB before the AIDS epidemic. In the WHO’s 2020 Global Tuberculosis Report, extrapulmonary TB represented 16% of the 7.1 million incident cases. It has been suggested that an increased prevalence of risk factors for EPTB, such as HIV infection, foreign-born status, and advanced age of the population, has contributed to an increased proportion of EPTB cases in the United States. The association between HIV infection and overall TB cases is well characterized, but the relationship between EPTB and HIV infection is less clear. EPTB is a relatively common presentation in individuals with HIV infection, although pulmonary

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disease is most often associated with HIV infection. Risk factors for EPTB are not clear. Some studies have indicated that besides HIV positivity, patients with liver cirrhosis and diabetes are also at increased risk. Genetic descent (e.g., Asia Pacic and Native American) has been proposed as a risk factor as well. Miliary tuberculosis refers to the seeding of many organs outside the pulmonary tree with AFB through hematogenous spread. This usually occurs shortly after primary pulmonary disease but can take place at any point during acute or chronic TB. The most common sites of spread of M. tuberculosis are the lymph nodes, spleen, liver, lungs, bone marrow, and kidneys. Other forms of EPTB include pleuritis, lymphadenitis, meningitis, peritonitis, and gastrointestinal and skeletal infections. Almost any organ of the body can be infected by M. tuberculosis. However, diagnosing EPTB is challenging because clinical samples must be obtained from relatively inaccessible sites, and often only a few bacteria are present, thus decreasing the sensitivity of diagnostic tests. Overall, children account for most cases of miliary TB, but it is also a common form of TB in individuals with HIV infection. The mortality is 20% or higher in most studies; the nding of meningitis is an extremely poor prognostic indicator. Up to 70% of patients with HIV infection may have EPTB alone or, usually, in combination with pulmonary disease. The most common extrapulmonary sites in this population are the lymph nodes (especially mediastinal), genitourinary tract, and abdominal cavity. Bacteremia is not uncommon. Lymphadenitis is usually a disease of children, appearing as painless head or neck swellings. Genitourinary TB can involve the kidneys and genital organs. Renal TB accounts for 2% of all cases of TB and manifests itself as typical urinary tract symptoms and sterile pyuria. Cultures may be positive in up to 80% of cases. Male genital TB usually appears as a scrotal mass and frequently occurs along with renal TB. Skeletal TB of the spine is referred to as Pott disease. Back pain is the most common characteristic. Cultures of bone and tissue are needed to conrm the diagnosis. Peripheral skeletal bones and joints also may be involved, with the hip and knee being the most common sites. Meningitis caused by M. tuberculosis is usually the result of a rupture of a tubercle into the subarachnoid space and not usually via hematogenous spread. In childhood, it occurs rarely after primary pulmonary infection. Most infections occur at the base of the brain; patients may develop very thick, gelatinous, masslike lesions there. With more chronic disease, a brous mass may surround cranial nerves. Involvement of arteries can cause infarctions. Cerebrospinal uid (CSF) examination usually reveals an elevated protein level, a decreased glucose level, and a predominance of lymphocytes.

Diagnosis of Mycobacterium tuberculosis infection Diagnosis of primary TB is usually limited to signs and symptoms, chest roentograph, and a positive PPD skin test or interferon-gamma release assay (IGRA) result. Cultures are conrmatory, but because of the slow-growing nature of M. tuberculosis, a culture result can take up to 8 weeks. Children may demonstrate a nonproductive cough and fever, with or without shortness of breath; these symptoms are unusual in adults. Chest radiography usually shows normal results, although, rarely, there may be inltrates without cavitation in the anterior segment of the upper, middle, or lower lobe,

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Mycobacterium tuberculosis and nontuberculous mycobacteria

with hilar or paratracheal lymphadenopathy. Along with these limited clinical ndings, patients with primary TB can have a paucity of bacteriologic ndings. If sputum or bronchial washings are cultured during the primary infection, the positivity rate is only 25% to 30%. The tuberculin skin test has been used for many years to determine an individual’s exposure to M. tuberculosis. Protein extracted and puried from the cell wall of culture-grown M. tuberculosis is used as the antigen (i.e., PPD). A standardized amount of antigen is injected intradermally into the patient’s forearm. The skin test detects a patient’s cell-mediated immune (CMI) response to the bacterial antigens in a type IV hypersensitivity reaction. Reactivity is read at 48 hours. In immunocompetent individuals, the presence of a raised rm area (induration) 10 mm or larger is considered reactive. A reactive tuberculin skin test only indicates past exposure to M. tuberculosis. Other Mycobacterium spp. generally result in an induration smaller than 10 mm. Immunocompromised patients with previous M. tuberculosis infection may also produce induration less than 10 mm. Some countries use attenuated Mycobacterium bovis (BCG) as a vaccine against TB. Individuals who have received this vaccine will have a reactive PPD skin test. Because some people with latent TB infection have a negative reaction when tested years after being infected, the two-step PPD skin test is often recommended. First, a routine PPD skin test is administered. If the result is reactive, no additional skin testing is needed. If the result is nonreactive, a second PPD skin test is administered in 1 to 3 weeks. If the second test is also nonreactive, the individual is considered not infected. A reactive test indicates a boosted response for an infection many years ago. The individual should be evaluated to determine if treatment is necessary. IGRAs have several advantages over skin testing and have become more frequently used. The Quantiferon-TB Gold Plus assay (Cellestis, Carnegie, Victoria, Australia) and the T-SPOT. TB (Oxford Immunotec, Oxford, England) measure the CMI response in whole-blood samples to mycobacterial antigens. These assays have a greater specicity than the PPD skin test and are just as sensitive if not more so. IGRAs have reported sensitivities and specicities of 88% to greater than 95%. Like skin testing, however, the sensitivities of IGRAs in children and those who are immunosuppressed, such as patients with AIDS, are lower compared with adults and immunocompetent persons. Unlike the tuberculin skin test, IGRAs are not affected by BCG vaccination and do not cross-react with antigens from most other mycobacterial species. They are also free from the bias that might be associated with reading and interpreting the tuberculin skin test results. In addition, patients need to be seen only once by a health care provider and do not need to return, as is necessary for reading a tuberculin skin test. Results are available in about 24 hours. However, these tests are more expensive than tuberculin skin testing. Both IGRA methods measure IFN-γ production by T cells that have been stimulated by two or three secretory, low-molecular-weight mycobacterial peptides: early-secreted antigenic target 6 (ESAT-6), culture ltrate protein 10 (CFP-10), or TB 7.7. Like the tuberculin skin test, IGRAs cannot distinguish between active and latent infections. Cultures, while time-consuming, have been considered the reference method for diagnosing most Mycobacterium infections. Isolation of mycobacteria is covered in more detail later

Fig. 26.1 Mycobacterium tuberculosis growing on Löwenstein-Jensen medium.

in this chapter. Colonies of the slowly growing M. tuberculosis are typically raised, with a dry, rough appearance. The colonies are nonpigmented and classically described as being buff colored (Fig. 26.1). Elaboration of cord factor can result in characteristic cord formation. Optimal growth occurs at 35° to 37° C. M. tuberculosis is characteristically positive for niacin accumulation, reduction of nitrate to nitrite, and production of catalase, which is destroyed after heating (heat-stable catalase negative). Isoniazid-resistant strains may not produce catalase at all. M. tuberculosis is inhibited by nitroimidazopyran or p-nitroacetylamino-β-propiophenone (NAP). This species can be distinguished from M. bovis by the inhibition of M. bovis by thiophene-2-carboxylic acid hydrazide (T2H) and pyrazinamidase activity.

Treatment The treatment of TB involves the use of more than one antimycobacterial agent in two phases for 4, 6 or 9 months. Currently, 10 drugs are approved by the U.S. Food and Drug Administration (FDA) for treating TB. Typically, phase one (intensive phase) treatment of pulmonary TB disease involves 8 weeks of therapy with isoniazid, rifampin, pyrazinamide, and ethambutol. Phase two (continuation phase) treatment consists of isoniazid and rifampin for an additional 18 weeks. In most individuals, AFB are cleared from the sputum within the rst 2 months. Although people with latent TB infection are asymptomatic and cannot spread TB, the bacteria can reactivate and progress to active TB disease. Therefore not only should people with TB disease be treated but also those individuals with latent TB infection. An estimated 80% of the people in the United States who develop TB disease do so because of untreated latent TB infection. Several regimens are used to treat latent TB infection. Short-course, rifamycin-based, 3- or 4-month regimens are recommended. To insure patient compliance, the WHO and the Centers for Disease Control and Prevention (CDC) recommend directly observed therapy.

Multidrug-resistant Mycobacterium tuberculosis Multidrug-resistant tuberculosis (MDR-TB) is dened as resistance to at least isoniazid and rifampin, drugs recognized as the primary treatments for drug-susceptible M. tuberculosis. The incidence of MDR-TB in the United States has remained relatively stable, around 1%, since 1996.

Clinical signicance of nontuberculous mycobacteria

Within any population of M. tuberculosis, resistance to a single agent can develop at a fairly well-dened rate. For example, with isoniazid and streptomycin, the chance that a resistant isolate will develop is approximately 1 in 10 6. The rate of spontaneous mutation of resistance to both drugs in one cell is the product of the rates of resistance to the individual drugs, or 1 in 1012. In a patient with pulmonary TB, the pulmonary cavity may contain 107 to 109 bacterial cells. Random drug resistance has a good likelihood of developing when only one antimycobacterial agent is used or if the patient is receiving multidrug therapy and fails to complete the course of medication. Therefore combination therapy (i.e., two or more drugs) to treat mycobacterial infections is recommended. Risk factors for drug resistance may include previous treatment for TB, residence in an area endemic for drug resistance, or close contact with an individual who has MDR-TB. Drug resistance is usually acquired by spontaneous mutations because of inappropriate use of antimicrobial agents to treat M. tuberculosis and lack of patient adherence. If adherence is an issue, directly observed therapy is recommended to ensure proper treatment. Otherwise, resistance may be assumed, and in vitro testing should be performed. MDR-TB requires an extended treatment period compared with drug-susceptible isolates. Second-line anti-TB drugs may include a regimen of four or ve drugs that include an aminoglycoside, uoroquinolone, pyrazinamide, and cycloserine/terizidone, among others. With the numbers of cases of MDR-TB increasing worldwide, newer agents are being tested in vitro to determine their efcacy. In 2016, the WHO estimated that 6.2% of MDR-TB cases were extensively drug-resistant tuberculosis (XDR-TB). The CDC denes XDR-TB as TB resulting from strains resistant to rifampin (MDR/RR-TB) and isoniazid plus any uoroquinolone and at least one of three injectable second-line anti-TB drugs (i.e., amikacin, kanamycin, or capreomycin). In 2021, the WHO published a new denition of XDR-TB, which is “TB caused by Mycobacterium tuberculosis (M. tuberculosis) strains that fulll the denition of MDR/RR-TB and which are also resistant to any uoroquinolone and at least one additional Group A drug (Group A drugs are the most potent group of drugs in the ranking of second-line medicines for the treatment of drug-resistant forms of TB using longer treatment regimens and comprise levooxacin, moxioxacin, bedaquiline and linezolid).” XDR-TB has been reported in all regions of the world but is most frequently found in countries of the former Soviet Union and in Asia. Because of the threat of MDR-TB and XDR-TB, it is important for laboratories to identify the Mycobacterium spp. rapidly and perform antimicrobial susceptibility testing so appropriate therapy can be administered as quickly as possible.

Vaccine The BCG vaccine, an attenuated M. bovis, is used in many countries with endemic TB. The vaccine is more efcacious in preventing infection when administered to children compared with adults. However, the vaccine cannot be given to immunocompromised individuals, such as those with HIV infection. It is therefore unlikely to be useful in countries with a high prevalence of HIV infection. Individuals who have received the vaccine will have a positive tuberculin (PPD) skin test result.

575

Mycobacterium bovis Mycobacterium bovis causes TB primarily in cattle but also in other ruminants, as well as in dogs, cats, swine, parrots, and humans. The disease in humans closely resembles that caused by M. tuberculosis and is treated the same way. In some areas of the world, a signicant percentage of cases of TB are caused by M. bovis, but in the United States, the number of isolates of this organism is very low. M. bovis is closely related taxonomically to M. tuberculosis and belongs to the M. tuberculosis complex. It grows very slowly on egg-based media, producing small, granular, rounded, nonpigmented colonies with irregular margins after 21 days of incubation at 37° C. On Middlebrook 7H10 medium, colonies resemble those of M. tuberculosis but are slower to mature. Most strains of M. bovis are niacin negative, do not reduce nitrate, and do not grow in the presence of 2 µg/mL T2H, characteristics that distinguish the species from most strains of M. tuberculosis.

Clinical signicance of nontuberculous mycobacteria Over 180 species of NTM are recognized, and about one half are classied as slowly growing. Most NTM are found in soil and water. They have been implicated as opportunistic pathogens in patients with underlying lung disease (e.g., cystic brosis, chronic obstructive pulmonary disease), immunosuppression, or percutaneous trauma. AIDS has contributed greatly to the incidence and awareness of NTM disease. Chronic pulmonary disease resembling TB is the usual clinical presentation associated with these organisms, although a few species are more often associated with cutaneous infections. Some species can be commensal inhabitants of humans; therefore their isolation must be evaluated with clinical history and presentation. NTM infections are not considered transmissible from person to person or animal to person.

Slowly growing species Mycobacterium avium Complex Epidemiology Currently, 11 species belong to the Mycobacterium avium complex (MAC), with Mycobacterium avium and Mycobacterium intracellulare being the most common species isolated from humans. These organisms are common environmental saprophytes and have been recovered from soil, water, house dust, and other environmental sources. Certain areas, such as coastal marshes, have higher concentrations of the organism. Environmental sources, especially natural waters, seem to be the reservoir for most human infections. M. avium is a cause of disease in poultry, cattle, and swine, but animal-to-human transmission has not been shown to be an important factor in human disease. The three subspecies found in animals are rarely associated with human infections; this may explain why animal-to-human transmission is uncommon. A fourth subspecies, M. avium subsp. hominissuis, is responsible for most human infections. MAC is the

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Mycobacterium tuberculosis and nontuberculous mycobacteria

leading causes of NTM infections in humans. A large increase in MAC infections occurred in the past primarily because of the increased number of infections in patients with AIDS. Although not a reportable disease, recently MAC infections have been decreasing among persons with HIV infection.

are insufcient for species identication of the slowly growing mycobacteria. Molecular methods are now the reference method.

Clinical infections

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne disease, an intestinal infection occurring as a chronic diarrhea in cattle, sheep, goats, and other ruminants. MAP has also been reported to cause a diarrheal/ wasting disease in nonhuman primates. MAP is found in potable water, pasteurized milk, and other foods. Researchers have found a signicantly higher percentage of anti-MAP antibodies in individuals with Crohn disease compared with individuals without irritable bowel disease. These studies suggest a link between MAP and Crohn disease. However, a causal relationship between the presence of MAP infection and autoimmune disease has not been demonstrated. MAP is difcult to cultivate because of its very slow growth rate (3 to 4 months) and its need for mycobactin-supplemented medium for primary isolation. Mycobactin is an iron-binding compound produced by other mycobacterial species.

Pulmonary disease resulting from MAC infection can have a variety of clinical presentations. The most common form is a slowly progressive, chronic cavitary disease in middle-age men with a history of smoking and other underlying pulmonary disease. The disease resembles the clinical picture of TB— cough, fatigue, weight loss, low-grade fever, and night sweats. Other presentations include lymphadenitis and tenosynovitis. Disseminated disease is common in immunocompromised patients, such as those with AIDS, or in patients with hematologic abnormalities. MAC infections are the most common systemic bacterial infection in patients with AIDS. The loss of CD4+ T cells reduces the activation of macrophages to kill MAC organisms. NTM pediatric lymphadenitis previously involved M. scrofulaceum in the 1970s; however, MAC has been associated in approximately 80% of recently reported cases. The clinical outcome of MAC lung disease is unpredictable, so management of patients can be difcult. Observation, therapy for underlying pulmonary disease (e.g., bronchodilators, broad-spectrum antimicrobials, smoking cessation), and periodic sputum cultures may be all that is required for most patients. For patients with signicant symptoms and advanced or progressive radiographic disease, multidrug therapy is indicated. For children with cervical lymphadenitis, excision surgery without chemotherapy is usually successful. A combination of surgical excision and chemotherapy is the usual treatment for adults with localized nonpulmonary disease. Most isolates of MAC are resistant to commonly used antimycobacterial drugs. However, many cases of disseminated disease in immunosuppressed patients without AIDS respond to multidrug regimens. Multidrug therapy consisting of clarithromycin or azithromycin along with ethambutol and rifampin has resulted in symptomatic reduction and clinical improvement in many, but not all, patients.

Laboratory diagnosis Because the two most common human isolates in the MAC are so similar, most laboratories do not distinguish between them but report isolates of both species as MAC. On primary isolation media, these organisms grow slowly, producing thin, transparent or opaque, homogeneous smooth colonies. A small proportion of strains may exhibit rough colonies. Usually, the colonies are nonpigmented, but they may become yellow with age. Rarely are the colonies pigmented from the onset of detectable growth. The optimal growth temperature is 37° C. On microscopic examination, the cells are short, coccobacillary, and uniformly stained, without beading or banding. Long, thin, beaded bacilli resembling Nocardia spp. may be seen in stains of very young cultures or under certain other conditions. MAC species are inactive in most physiologic tests used to identify the mycobacteria. Exceptions are the production of a heat-stable catalase and the ability to grow on media containing 2 µg/mL T2H. Nucleic acid probes are available for the identication of MAC and the two species. Nucleic acid probes for the identication of M. avium and M. intracellulare are commercially available. Phenotypic characteristics

Mycobacterium avium subsp. paratuberculosis

Mycobacterium kansasii Epidemiology Mycobacterium kansasii is second to MAC as the cause of NTM lung disease. Tap water seems to be the most signicant reservoir. The natural source of human infection is unclear but is likely by aerosol. As with other NTM, infections are not normally considered contagious from person to person.

Clinical infections The most common manifestation is chronic pulmonary disease, closely resembling TB, involving the upper lobes, usually with evidence of cavitation and scarring. Most patients with pulmonary disease have underlying conditions including chronic obstructive pulmonary disease, bronchiectasis, pneumoconiosis, alcohol abuse, malignancies, and HIV infection. Extrapulmonary infections, such as lymphadenitis, skin and soft tissue infections, and joint infection, have been reported occasionally. Disseminated M. kansasii infection rarely occurs in immunocompetent individuals but has been reported in severely immunocompromised patients, particularly those with AIDS. In fact, infections are more common in HIV-infected individuals compared with those who are HIV negative.

Laboratory diagnosis M. kansasii is a slowly growing organism that appears as long rods with distinct cross-banding. M. kansasii has an optimal growth temperature of 37° C, and colonies appear smooth to rough, with characteristic wavy edges and dark centers when grown on Middlebrook 7H10 agar. Some cording can usually be seen with low-power magnication. Colonies are photochromogenic (Fig. 26.2); that is, they form a pigment when exposed to light but are nonpigmented in the dark. With prolonged exposure to light, most strains form dark red crystals of β-carotene on the surface of and inside the colony, producing reddish-orange colonies. Scotochromogenic (produce pigment in light and dark) and nonchromogenic strains are rarely isolated. Most strains are strongly catalase positive (>45 mm in a semi-quantitative test); strains that are low catalase producers (45 mm) catalase producers. These characteristics aid in differentiating this organism from other slowly growing scotochromogens, including certain strains of MAC, M. gordonae, and M. szulgai

Mycobacterium simiae The original strains of Mycobacterium simiae were isolated from the lymph nodes of monkeys. Although the organism has been recovered from tap water, there seems to be significant geographic variation in the incidence. For example, M. simiae is rarely isolated in most parts of the United States, but in parts of Texas, it is a relatively common isolate. Infrequent cases of human infection from M. simiae have been reported and are often associated with HIV-positive patients. Infection typically manifests as pulmonary disease, but lymphadenitis, skin lesions, and other presentations have been reported. Many isolates are resistant to most anti-TB drugs and are associated with signicant morbidity and mortality. Cells of M. simiae appear as short coccobacilli. When they are grown on inspissated egg medium at 37° C, smooth colonies appear in 10 to 21 days. Colonies on Middlebrook 7H10

A rare clinical isolate, the most common manifestation of Mycobacterium szulgai is pulmonary disease resembling TB. Extrapulmonary infections, including tenosynovitis of the hand, bursitis, osteomyelitis, keratitis, cervical lymphadenitis, and renal or cutaneous infection, have also been reported. This organism has been isolated from environmental sources such as snails, tropical sh, aquarium water, swimming pools, and hospital water supplies. Person-to-person transmission has not been documented. It is susceptible in vitro to most anti-TB drugs, and combination treatment is generally successful. M. szulgai cells are medium to long rods, with some cross-barring. When cultured on egg-based medium at 37° C, the organism produces smooth and rough colonies. At 37° C, yellow-to-orange pigment develops in the absence of light and intensies with exposure to light. Colonies grown at 22° C are nonpigmented or buff in the absence of light and develop yellow-to-orange pigment with light exposure. Characteristics that differentiate M. szulgai from other slowly growing mycobacteria are slow hydrolysis of Tween 80, positive nitrate reduction, and inability to grow in the presence of 5% sodium chloride.

Mycobacterium ulcerans Mycobacterium ulcerans is a rare cause of mycobacteriosis in the United States but may be underreported because of difculty in isolation. Worldwide, M. ulcerans is the third most common Mycobacterium spp., behind M. tuberculosis complex and M. leprae. The disease manifests as a painless nodule under the skin following trauma. A shallow ulcer, also referred to as a Buruli ulcer, develops that can be quite severe. Widespread ulcerative necrosis, osteomyelitis, and disgurement can result. Patients rarely develop fever or systemic symptoms. M. ulcerans cells are moderately long, without beading or cross-banding. These bacteria are fastidious and difcult to grow in vitro. Optimal growth temperature is 28° to 30° C, with little growth at 25° C and usually none at 37° C. The organism grows slowly, often requiring 6 to 12 weeks of incubation before colonies are evident. Colonies are smooth or rough and nonpigmented or lightly buff, and they do not develop pigment with exposure to light. M. ulcerans produces a heat-stable catalase but is inert in most other conventional biochemical tests. Molecular techniques are available that may improve detection.

Mycobacterium xenopi Mycobacterium xenopi has been recovered from hot- and cold-water taps (including water storage tanks of hospitals)

Clinical signicance of nontuberculous mycobacteria

and birds. The organism was rst isolated from an African toad (Xenopus laevis) and was considered nonpathogenic for humans until 1965. Isolation of M. xenopi is relatively uncommon in the United States. Human cases of M. xenopi infection are mostly slowly progressive pulmonary infections in individuals with a predisposing condition, such as chronic obstructive pulmonary disease. The pulmonary infections present a clinical picture resembling that seen in patients with M. tuberculosis, M. kansasii, or MAC infection. Disseminated and extrapulmonary infections have also been reported, primarily in patients who are immunocompromised. Strains of M. xenopi are susceptible to the quinolones (ciprooxacin, ooxacin); some isolates are susceptible to vancomycin, erythromycin, or cefuroxime. In vitro susceptibility to anti-TB drugs is variable, with resistance only to ethambutol being the most common pattern. M. xenopi cells are long lamentous rods. Colonies of this slowly growing mycobacterium on Middlebrook 7H10 agar are small, with dense centers and lamentous edges. Microscopic magnication observation of colonies growing on cornmeal-glycerol agar reveals distinctive round colonies with branching and lamentous extensions; aerial hyphaelike elements are usually seen in rough colonies. Furthermore, young colonies grown on cornmeal agar have a “bird’s nest” appearance, with characteristic sticklike projections. Optimal growth temperature is 45° C; the organism grows more rapidly at this temperature than at 37° C and fails to grow at 25° C. M. xenopi has been classied with the nonphotochromogenic group; however, colonies are frequently bright yellow on primary isolation when incubated in the absence of light and when exposed to light. Distinctive characteristics, in addition to optimal growth at 42° C and yellow scotochromogenic pigment, are negative reactions for niacin accumulation, nitrate reduction, and positive reactions for heat-stable catalase, arylsulfatase, and pyrazinamidase.

Rapidly growing species The three most important rapidly growing mycobacteria causing human infections are Mycobacterium abscessus subsp. abscessus (formerly M. abscessus), Mycobacterium chelonae, and the Mycobacterium fortuitum group. The M. chelonae/M. abscessus complex makes up one group within the rapidly growing mycobacteria. Posttraumatic wound infections are the most common infection associated with the rapidly growing mycobacteria. The M. fortuitum group causes about 60% of localized cutaneous infections.

Mycobacterium chelonae/Mycobacterium abscessus group M. chelonae is found in the environment and is associated with many of the same opportunistic infections as those associated with the M. fortuitum group. M. chelonae is related closely to M. abscessus subsp. abscessus and is a commonly isolated rapidly growing Mycobacterium. It has caused sporadic cases of localized wound infections following medical or surgical procedures, including needle injections. The M. chelonae/M. abscessus complex is the most likely among the rapidly growing mycobacteria to be isolated from disseminated cutaneous infections in immunocompromised patients. M. chelonae has been associated with a variety of

579

infections of the skin, lungs, bone, central nervous system, and prosthetic heart valves. Approximately 80% of the cases of pulmonary disease resulting from rapidly growing mycobacteria are caused by M. abscessus. Infections by this organism have also been seen in patients with cystic brosis. Unlike with M. chelonae, tap water is an important reservoir for M. abscessus. Microscopically, young cultures of M. chelonae are strongly acid fast, with pleomorphism ranging from long and tapered to short and thick rods. This rapidly growing Mycobacterium produces rough or smooth, nonpigmented to buff colonies within 3 to 5 days of incubation at 28° to 30° C. A positive 3-day arylsulfatase test result, nitrate reduction negative, and growth on MacConkey agar without crystal violet are characteristics that help differentiate M. chelonae and M. abscessus from other nonchromogenic, rapidly growing mycobacteria.

Mycobacterium fortuitum group The M. fortuitum group contains 10 species. Common in the environment, M. fortuitum has been isolated from water, soil, and dust. The M. fortuitum group is the most common rapidly growing Mycobacterium associated with localized cutaneous infections, bone and joint infections, and abscesses at the site of puncture wounds. Infections associated with long-term use of intravenous and peritoneal catheters, injection sites, and surgical wounds following mammoplasty and cardiac bypass procedures have also been reported. Differences in susceptibility to antimicrobial agents occur among the rapidly growing species; thus in vitro susceptibility testing is often recommended for clinically signicant isolates. After 3 to 5 days of incubation at 35° C, M. fortuitum colonies appear rough or smooth and nonpigmented, creamy white, or buff. Microscopic examination of growth on cornmeal-glycerol and Middlebrook 7H11 agars after 1 to 2 days of incubation reveals colonies with branching lamentous extensions and rough colonies with short aerial elements. Cells are pleomorphic, ranging from long and tapered to short and thick rods. From most cultures, especially older ones, cells tend to decolorize and appear partially acid fast with any of the acid-fast staining techniques. Additional characteristics that distinguish M. fortuitum from other rapidly growing mycobacteria are the positive 3-day arylsulfatase test result and reduction of nitrate.

Mycobacterium smegmatis group The Mycobacterium smegmatis group contains two species: M. smegmatis and Mycobacterium goodii. Commonly considered saprophytic, M. smegmatis has been implicated in rare cases of pulmonary, skin, soft tissue, and bone infections. It has been reported to cause granulomas in the soft tissue of the web space of a previously healthy 67-year-old patient who had no recollection of trauma to the hand. Upon acid-fast staining, cells appear long and tapered or as short rods with irregular acid fastness. Occasionally, rods are curved with branching or Y-shaped forms; swollen, with deeper staining, beaded, or ovoid forms are sometimes seen. Colonies appearing on egg medium after 2 to 4 days are usually rough, wrinkled, or coarsely folded; smooth, glistening,

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Mycobacterium tuberculosis and nontuberculous mycobacteria

butyrous colonies may also be seen. Colonies on Middlebrook 7H10 agar are heaped and smooth or rough with dense centers. Pigmentation is rare or late; colonies appear nonpigmented, creamy white, or buff-to-pink in older cultures. In addition to the rapid growth rate and nonpigmented rough colonies, characteristics valuable in the identication of this organism are its negative arylsulfatase reaction, positive iron uptake, ability to reduce nitrate, and growth in the presence of 5% sodium chloride (NaCl) and on MacConkey agar without crystal violet.

Mycobacterium leprae Mycobacterium leprae is the causative agent of Hansen disease (leprosy), an infection of skin, mucous membranes, and peripheral nerves. The disease is rare in the United States and other Western countries, yet it remains a major problem in other parts of the world. At one time, the WHO estimated that 11 million people had Hansen disease. However, the WHO launched an eradication program, and since 1985 the incidence of Hansen disease has been reduced by about 90%. Since 1995, the WHO has provided treatment free of charge to all patients with Hansen disease. Globally, the annual incidence has steadily declined since 2001 to 127,558 new leprosy cases detected in 2020. Currently, India, Brazil, and Indonesia account for about 80% of all new cases; each reported over 10,000 cases. In the United States, roughly 100 cases are reported annually, which are often acquired abroad. Despite its reputation, Hansen disease is not highly contagious. The most important mode of transmission is not known, but the disease can be transmitted by direct contact and inhalation of aerosols from skin lesions. Shedding from the nasal passage is another route of transmission. The bacteria multiply slowly, producing a lengthy incubation period up to 20 years. The two extreme forms of the disease are tuberculoid leprosy, also called paucibacillary Hansen disease, and lepromatous leprosy, also called multibacillary Hansen disease. Symptoms of tuberculoid leprosy include hypopigmented or hyperpigmented skin macules and nerve involvement that can produce areas with loss of sensation (anesthesia). Patients eventually exhibit an effective CMI response. The optimal growth temperature for M. leprae is approximately 30° C, and because the patient mounts an adequate CMI response, the bacteria tend to remain in the extremities. Spontaneous recovery often occurs with tuberculoid leprosy. Conversely, patients with lepromatous leprosy have a strong humoral-mediated immune response but not an effective CMI response. The disease is slowly progressive, and if untreated, it can be life-threatening. It is characterized by disguring skin lesions and progressive, symmetric nerve damage. Lesions of the mucous membranes of the nose can lead to destruction of the cartilaginous septum, resulting in nasal and facial deformities. The current recommended therapy for lepromatous leprosy consists of a combination of diaminodiphenylsulfone (dapsone) and rifampin for a minimum of 6 months. For tuberculoid leprosy, clofazimine is added, and treatment should be continued for 12 months. The third and most common form is known as borderline or dimorphous Hansen disease. It presents as intermediate in severity. Skin lesions resemble those found in the tuberculoid form,

Fig. 26.5 Mycobacterium leprae from a skin biopsy in a patient with lepromatous leprosy (acid-fast smear stained with Ziehl-Neelsen stain, ×1000).

but they are more numerous and can be found anywhere on the body. Peripheral nerves are affected, resulting in anesthesia and weakness. The lepromin skin test utilizes a standardized suspension of inactivated M. leprae. Like the PPD test for TB, a small volume is injected just under the skin and examined at 48 hours. Although it is not recommended for primary diagnosis, it can help differentiate between tuberculoid and lepromatous leprosy. Uninfected patients and those with lepromatous leprosy will be nonreactive, indicating a weak or negative CMI response. Individuals who were previously exposed and have a CMI response will have a reactive response, suggesting tuberculoid leprosy. Laboratory diagnosis of Hansen disease depends on the microscopic demonstration of AFB from skin biopsy specimens. M. leprae has not been grown on articial media. In patients with tuberculoid leprosy, organisms are extremely rare and may not be detected in skin scrapings or biopsy specimens. However, AFB are usually abundant in samples from patients with lepromatous leprosy (Fig. 26.5). M. leprae is not as acid fast or alcohol fast as in the case of other mycobacteria; as such, a weaker decolorizer consisting of 10% sulfuric acid is recommended instead of the standard acid-ethanol decolorizer. The bacteria are rod shaped, usually 1 to 7 µm long and 0.3 to 0.5 µm wide. The entire smear should be examined under oil immersion eld (×1000) for the presence of the microorganisms. PCR assays are also available to denitively detect the bacteria.

Isolation and identication of mycobacteria Growth rate, colony morphology, pigmentation, nutritional requirements, optimal incubation temperature, and biochemical test results are traditional criteria used to differentiate species within the genus Mycobacterium (Table 26.1). Rapid techniques include broth-based culture systems, including some that monitor cultures continuously during the incubation period. However, because phenotypic identication of the slowly growing mycobacteria is not sufcient for accurate species identication, molecular-based assays are the reference method. A limited number of species-specic nucleic acid probes offer rapid identication of culture isolates.

Ureasec

Tween Opacity (1 Week)

Tween Hydrolysis + 10 Days

Growth on T2H

Tellurite Reduction

Growth on 5% NaCl

b Pyrazinamidase

Nitrate Reduction

Niacin

−(99) −(99)

Growth on MacConkey Agar

+(99)

Iron Uptake

+(98)

Heat Stable (68° C)

+(96)

Catalase Semi quantitative >45

Inositol

Mannitol

Sodium Citrate

Pigment

Growth Rate

2 Weeks

Rapid growers

Carbon Sources

Smooth 87%, rough 13%

M. peregrinum M. fortuitumd,e former third biovariant complex

N = 99%

22–35

M. chelonae M. abscessus subsp. abscessus

Arylsulfatase

3 Days

M. fortuitum

Temp Growth Range (°C)

Species (subspecies)

Colony Morphology

Descriptive term

Table 26.1 Identication of clinically important mycobacteriaa

Smooth 60%, rough 40%

−(99)

Rapid

−(98)

−(99)

−(99)

−(99) −(99)

−(74)

−(99)

−(99) −(99) +(99)

−(99)

+(99) +(99)

+(96)

−(70)

+(59) +(60) +(66)

+(63)

−(56)

+(52) −(95) −(51)

−(52) +(87) +(99) +(75)

V

TB complex

(30° C) Slow (37° C) Other rapid growers

Smooth or rough

M. tuberculosis

Rough, 33–39 Slow cords Rough 35–38 Slow 7H10 = 35–38 Slow Rough L J = Smooth

M. africanum M. bovis

−(72)

+(75)

+(66)

S = 38% N = 58% N = 99%

−(99)

−(93)

−(99)

−(99)

−(99) +(98) +(99) +(98)

−(99) −(70) +(92) +(68)

−(83) +(98)

N N = 99%

−(99) −(99)

– −(87)

− −(97)

−(99) −(92)

−(99) − − − −(99) −(95) −(94) −(98)

−(99) −(99) V − −(99) −(55) −(94) −(84)

+(99) −(75) +(99)

Isolation and identication of mycobacteria

N = 99%

M. fallaxf

581

582

Tween Opacity (1 Week)

Growth on T2H

Growth on 5% NaCl

b Pyrazinamidase

Growth on MacConkey Agar

+−(99)

+(83)

−(99) −(99) −(94) +(51)

−(99) −(59) +(99) −(99)

−(99)

M. gastri

Smooth

N = 99%

−(96)

+(99)

−(99)

−(99)

−(84) −(99) −(99) +(99) +(63) −(99) −(74) +(99) +(99) 4 days 7 days

−(99) +(79)

N = 95% S = 4% P = 1%

−(99)

−(51)

+(99)

+(96)

−(77) −(99) +(72) +(63) +(86) −(94) −(75) +(99) +(99) 4 days 7 days

−(93) −(91)

N = 99%

−(66)

+(99)

+(93)

+(99)

N = 90% S = 10% N

−(99)

−(89)

−(99)

+(69)

−

−

− −(99)

M. triviale

Rough

25–40 Slow 80% Rapid 20% 22–37 Slow 77% Rapid 23% 22–37 Slow

M. malmoense

Smooth

22–37 Slow

M. haemophilum (needs hemin) M. simiae

Rough

22–35 Slow

Smooth

22–37 Slow

M. kansasii

Smooth, rough

M. marinum

Smooth

M. asciaticum

Smooth

S. scrofulaceum

Smooth

S. szulgai

Rough, Smooth Smooth

M. terrae complex Smooth, few rough

M. flavescens

P = 91% N = 9% 25–40 Slow N = 99% 98% S = 9/eld

>36/eld

4+

Modied from Kent, P. T. & Kubica, G. P. (1985). Public health mycobacteriology: a guide for the level III laboratory . Atlanta, GA: Centers for Disease Control and Prevention.

Culture media and isolation methods Mycobacteria are strictly aerobic and grow more slowly than most bacteria pathogenic for humans. The generation time of mycobacteria is longer than 12 hours; M. tuberculosis has the longest replication time, at 20 to 22 hours. The rapidly growing species generally form colonies in 2 to 3 days, whereas most pathogenic mycobacteria require 2 to 6 weeks of incubation. The growth of M. tuberculosis is enhanced by an atmosphere of 5% to 10% CO 2. Mycobacteria have an optimal pH between 6.5 and 6.8 and grow better in higher humidity. Most human pathogenic mycobacteria grow best at temperatures of 35° to 37° C. However, when mycobacteria are suspected in skin and soft tissue specimens, a second set of culture media should be incubated at 25° to 33° C for the recovery of M. haemophilium, M. marinum, M. ulcerans, and M. chelonae. One of the mycobacteria pathogenic for humans, M. genavense, does not grow on media used routinely to isolate mycobacteria and requires extended incubation (6 to 8 weeks), whereas M. leprae fails to grow on articial media. The many different media available for the recovery of mycobacteria from a clinical specimen are variations of three general types (Table 26.3): egg-based media, serum albumin agar media, and liquid media. Within each general type, there are nonselective formulations and formulations that have been made selective by the addition of antimicrobial agents. Because some isolates do not grow on a particular agar and each type of culture medium offers certain advantages, a combination of culture media is generally recommended for primary isolation. The use of a solid-based medium, such as LJ medium, in combination with a liquid-based medium is recommended for routine culturing of specimens for the recovery of AFB. Current guidelines recommend that two or more types of media be used when attempting to recover mycobacteria.

Egg-based media The basic ingredients in an inspissated egg medium, such as LJ (the most used egg-based medium), Petragnani, and American Thoracic Society (ATS) media, are fresh whole eggs, potato our, and glycerol, with slight variations in dened salts, milk, and potato our. Each contains malachite green to suppress the growth of gram-positive bacteria. Selective media that contain antimicrobial agents, such as the Gruft

modication of LJ medium and Mycobactosel (BD Diagnostic Systems), are sometimes used in combination with nonselective media to increase the isolation of mycobacteria from contaminated specimens. The nonselective egg-based media have a long shelf life up to 1 year, but distinguishing early growth from debris is sometimes difcult.

Agar-based media Agar-based media are better chemically dened than eggbased media, and they do not readily support the growth of contaminants. Serum albumin agar media, such as Middlebrook 7H10 and 7H11 agars, are prepared from a basal medium of dened salts, vitamins, cofactors, glycerol, malachite green, and agar combined with enrichment consisting of oleic acid, bovine albumin, glucose, and beef catalase (Middlebrook OADC enrichment). Middlebrook 7H11 medium also contains 0.1% casein hydrolysate, which improves the recovery of isoniazid-resistant strains of M. tuberculosis. The addition of antimicrobial agents to 7H10 or 7H11 medium makes the media more selective by suppressing the growth of contaminating bacteria. Mitchison selective 7H11 agar contains polymyxin B, amphotericin B, carbenicillin, and trimethoprim lactate. In contrast with opaque egg-based media, clear agar-based media can be examined using a dissecting microscope for early detection of growth and colony morphology. Drug susceptibility tests may be performed on agar-based media without altering drug concentrations, as occurs with egg-based media. When specimens are inoculated onto Middlebrook 7H10 and 7H11 media and incubated in an atmosphere of 10% CO 2 and 90% air, 99% of the positive cultures are detected in 3 to 4 weeks, earlier than for those detected on egg-based media. Certain precautions should be followed in the preparation, storage, and incubation of Middlebrook media. Both excess heat and exposure of the prepared media to light can result in the release of formaldehyde, which is toxic to mycobacterial growth. A CHOC agar plate should be included in the primary isolation media for skin and other body surface specimens for the recovery of M. haemophilum, which requires ferric ammonium citrate or hemin for growth. Alternatively, a Middlebrook 7H10 agar plate supplemented with hemolyzed sheep red blood cells or another source of hemin may be used. The plate should be incubated at 30° C, the optimal temperature for recovery of this organism.

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Table 26.3 Mycobacterial culture media Medium

Composition

Inhibitory agents

American Trudeau Society

Fresh whole eggs, potato our, glycerol

Malachite green (0.02%)

Petragnani

Fresh whole eggs, egg yolks, whole milk, potato, potato our, glycerol

Malachite green (0.12%)

Middlebrook 7H9

Dened salts, vitamins, cofactors base, with pyruvate, albumin, catalase, glucose,

Middlebrook 7H10

Dened salts, vitamins, cofactors, oleic acid, albumin, catalase, glycerol, glucose

Malachite green (0.00025%)

Middlebrook 7H11

Middlebrook 7H10 medium with 0.1% casein hydrolysate

Malachite green (0.0001%) Amphotericin B Nalidixic acid Trimethoprim Azlocillin Amphotericin B Nalidixic acid Trimethoprim Azlocillin

Gruft (modication of LJ)

Fresh whole eggs, dened salts, glycerol, potato our, RNA

Malachite green Penicillin Nalidixic acid

Mycobactosel (BBL, BD Diagnostic Systems, Sparks, MD) LJ

Fresh whole eggs, dened salts, glycerol, potato our

Malachite green Cycloheximide Lincomycin Nalidixic acid

Middlebrook 7H10 (selective)

Dened salts, vitamins, cofactors, oleic acid, albumin, catalase, glycerol, glucose

Malachite green Cycloheximide Lincomycin Nalidixic acid

Mitchison’s selective 7H11

Middlebrook 7H10 medium with casein hydrolysate

Carbenicillin Amphotericin B Polymyxin B Trimethoprim lactate

LJ, Löwenstein-Jensen; SPS, sodium polyanethol sulfonate.

Liquid media Mycobacterium spp. grow more rapidly in liquid medium, and it can be used for both primary isolation and subculturing. Liquid culturing systems have been demonstrated to be superior in the recovery of mycobacteria from clinical specimens compared with conventional solid media. Middlebrook 7H9 broth and Dubos Tween albumin broth are nonselective liquid media used for subculturing stock strains, picking single colonies, and preparing inoculum for in vitro testing. Numerous automated systems that use liquid media are available. The mycobacterial growth indicator tube (MGIT) system (BD Diagnostic Systems) uses modied Middlebrook 7H9 broth with a uorescence quenching-based oxygen sensor for detecting mycobacterial growth. Oxygen present in the sterile medium quenches the uorescence. Bacterial growth consumes the oxygen, allowing uorescence when the tube is exposed to UV light. In the manual system, a Wood lamp or

transilluminator can be used. Before use, oleic acid–albumin– dextrose is added to stimulate growth of mycobacteria. To inhibit growth of nonmycobacteria, the antimicrobial agents polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin (PANTA) are added. The MGIT 960 system (BD Diagnostic Systems) is a continuous monitoring system. Other continuous monitoring systems include the MB/ BacT Alert 3D system (bioMérieux), which uses a colorimetric carbon dioxide sensor in each bottle to detect bacterial growth, and the VersaTREK culture system (VersaTREK Diagnostic Systems, Cleveland, OH). The VersaTREK culture system is based on the detection of pressure changes in the headspace above the broth medium in a sealed culture bottle resulting from gas production or consumption during microbial growth. Each manufacturer provides a mixture of antimicrobial agents to be added to each culture vial at the time of inoculation. The continuous monitoring systems have similar

Isolation and identication of mycobacteria

performance and operational features. Bottles are incubated in the instrument for the entire monitoring period, and options for electronic data management are available. The MGIT 960 and VersaTREK systems have also been approved for antimicrobial susceptibility testing. A disadvantage of the liquid systems is that no colony morphology or pigmentation is available to suggest that the growth is of a mycobacterial species and not that of a contaminant or commensal organism. Another limitation is that cultures with mycobacteria with a lower optimal temperature, such as M. haemophilum, M. marinum, M. ulcerans, and M. chelonae, may not be detected.

Isolator lysis-centrifugation system Isolator is a blood collection system that contains saponin to liberate intracellular organisms. After saponin treatment and centrifugation, the sample is inoculated onto mycobacteria agar plates or tubes. The system allows higher yields and shorter recovery times for mycobacteria than conventional blood culture methods. It offers the advantage of yielding isolated colonies and the ability to quantify mycobacteremia, which may be useful in monitoring the effectiveness of therapy in disseminated MAC infection. For maximal recovery of mycobacteria, laboratories should use a battery of media that includes a liquid medium and at least one solid medium—egg-based or agar-based— for primary isolation. An additional selective medium is often reserved for specimens in which heavy contamination is anticipated.

Case check 26.2 In the patient presented in the Case in Point, no organisms were seen on the direct smears of all three samples submitted. Nucleic acid amplification tests are a rapid, more sensitive method to directly detect mycobacteria. Cultures are more sensitive than direct smears, as well, for diagnosing TB, and broth cultures are preferred. The BACTEC broth cultures were ultimately positive in this patient.

Laboratory identication Laboratory levels or extents of service A change in the distribution of mycobacterial laboratory testing led to the development of levels of service by the ATS and extents of service by the College of American Pathologists (CAP) to maintain quality of service. According to CAP, laboratories must decide which level of mycobacterial services to offer: level 1, specimen collection only; level 2, perform microscopy and isolate and identify and sometimes perform susceptibility tests for M. tuberculosis; or level 3, perform microscopy, isolate and identify, and perform susceptibility tests for all Mycobacterium spp. (Box 26.3). A facility’s selection of a level of service depends on the volume of specimens submitted, patient populations served, ability to perform the requested tests according to comfort; biological safety level and staff training in performance of each requested test; and the time, effort, and funds allocated for the service. The procedures available in a mycobacteriology laboratory differ with the level of service of that laboratory.

589

BOX 26.3 Levels of service as dened by the American

Thoracic Society and College of American Pathologists Extent of service as dened by the American Thoracic Society • Specimen collection only; no mycobacteriologic procedures performed; all specimens sent to another laboratory • Acid-fast stain, inoculation, or both; identification by a reference laboratory • Isolation and definitive identification of Mycobacterium tuberculosis; preliminary grouping of nontuberculous Mycobacterium spp., with definitive identification at a reference laboratory • Definitive identification of all mycobacterial isolates with assistance in the selection of therapy, with or without drug susceptibility testing

College of American Pathologists levels of service • Level 1: Specimen collection only; no mycobacteriologic procedures performed; all specimens are sent to another laboratory • Level 2: Perform microscopy. Isolate, identify, and sometimes perform susceptibility tests for M. tuberculosis • Level 3: Perform microscopy. Isolate, identify, and perform susceptibility tests for all species of Mycobacterium

Preliminary identication of mycobacteria Once an isolate has been recovered in the mycobacteriology laboratory, certain characteristics can be used to classify the isolate before performing biochemical or molecular tests. The rst step is to conrm that the isolate recovered in broth or on solid media is an acid-fast organism by performing acidfast staining. Then, phenotypic characteristics, such as colony morphology, growth rate, optimal growth temperature, and photoreactivity, can help speciate the mycobacteria. These characteristics do not allow denitive identication but are presumptive and help in the selection of other, more denitive tests. Historically, the Runyon classication used the rate of growth and pigment production to place the NTM into one of four categories. However, because of variability within individual species for these two criteria, the Runyon classication is no longer used today. Figs. 26.6 and 26.7 show schematic diagrams for the identication of slowly growing and rapidly growing Mycobacterium spp., respectively. See Table 26.1 for a summary of the identication characteristics of clinically important mycobacteria.

Colony morphology Colonies of mycobacteria are generally distinguished as having a smooth and soft or rough and friable appearance. Colonies of M. tuberculosis that are rough often exhibit a prominent patterned texture referred to as cording (curved strands of bacilli); this texture is the result of tight cohesion of the bacilli. Colonies of MAC have a variable appearance, with glossy whitish colonies often occurring with smaller translucent colonies.

Growth rate Growth rate and recovery time depend on the species of mycobacteria but are also inuenced by the media, incubation temperature, and initial inoculum size. The range in recovery time is wide, from 3 to 60 days. Mycobacteria are generally categorized as rapid growers, having visible growth in fewer than 7 days, or slow growers, producing colonies in more than

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Mycobacterium tuberculosis and nontuberculous mycobacteria

Acid-fast bacillus Growth rate >7 days on LJ at 37° C Pigmentation in absence of light

Buff

Orange

Pigmentation after exposure to light Yellow

Tween hydrolysis

Buff

Nitrate reduction

+

Niacin

Nitrate reduction

+

+ Tween hydrolysis + SQ catalase + Mycobacterium kansasii

–

68° C catalase – SQ catalase – Nitrate reduction + Mycobacterium tuberculosis

SQ catalase

Photochromogen at 22° C +

+ Mycobacterium asiaticum

–

Mycobacterium gordonae

– Mycobacterium flavescens

Tween –

Tween hydrolysis

Mycobacterium scrofulaceum

+

Mycobacterium szulgai

+

–

Mycobacterium marinum (optimal growth at 30° C)

+

–

Nitrate reduction +

–

68° C catalase + Mycobacterium simiae Mycobacterium terrae-triviale complex SQ catalase +

68° C catalase weak + Nitrate reduction – SQ catalase – Mycobacterium avium complex Mycobacterium xenopi

+

–

68° C catalase + Mycobacterium nonchromogenicum

68° C catalase +

–

Mycobacterium malmoense

Mycobacterium gastri

Fig. 26.6 Schematic diagram for the identication of slowly growing Mycobacterium spp. Exceptional reactions occur. Organisms should be subjected to a battery of morphologic and physiologic tests before nal identication is made. LJ, Löwenstein-Jensen medium; SQ, semi-quantitative.

7 days. Determination of growth rate should be evaluated from the time of subculture, not from the time of detection from the clinical sample. The inoculum should be sufciently small to produce isolated colonies. Microscopic examination of agar for microcolonies allows earlier detection of growth.

Temperature The optimal temperature and range at which a mycobacterial species can grow may be extremely narrow, especially at the time of initial incubation. M. haemophilum, M. marinum, M. ulcerans, and M. chelonae grow best at 25° to 33° C and poorly, if at all, at 35° to 37° C. At the other extreme, M. xenopi grows best at 42° C.

Photoreactivity Historically, Mycobacterium spp. have been categorized into three groups according to their photoreactive characteristics (Box 26.4). Species that produce carotene pigment on exposure to light are photochromogens (see Fig. 26.2). Color

ranges from pale yellow to orange. Species that produce pigment in the light and the dark are scotochromogens (see Fig. 26.4). Growth temperature may inuence the photoreactive characteristics of a species. Pigment production is oxygen dependent, so it is important that tube caps are loose. Other species, such as M. tuberculosis, are nonchromogenic or nonphotochromogenic (see Fig. 26.1). These colonies are a buff (tan) color and are nonphotoreactive; exposure to light does not induce pigment formation.

Biochemical identication A panel of biochemical tests can identify most mycobacteria isolates, but because growth of most clinically signicant Mycobacterium spp. is so slow, accomplishing this can take several weeks. Progress in molecular technology has diminished the frequency with which biochemical tests are routinely performed in the identication of mycobacteria. Because mycobacterial species may show only quantitative

Isolation and identication of mycobacteria

591

Acid-fast bacillus Growth rate 3 to 7 days on LJ at 37° C

3-Day arylsulfatase test +

–

Nitrate reduction

Mycobacterium fortuitum

Mycobacterium chelonae Pigment

Mycobacterium smegmatis

Other rapidly growing Mycobacterium species

Mycobacterium phlei

Mycobacterium vaccae

Fig. 26.7 Schematic diagram for the identication of rapidly growing Mycobacterium spp. Exceptional reactions occur. Organisms should be subjected to a battery of morphologic and physiologic tests before nal identication is made. LJ, Löwenstein-Jensen medium.

BOX 26.4 Photoreactivity of clinically important mycobacteria Nonchromogens Slow growers M. tuberculosis M. avium complexa M. bovis M. celatum M. gastri M. genavense M. haemophilum M. malmoense M. terrae complex M. ulcerans Rapid growers M. chelonae M. fortuitum group

Photochromogens Slow growers M. asiaticum M. kansasii M. marinum M. simiae

Scotochromogens Slow growers M. gordonae M. szulgaib M. scrofulaceum M. xenopic

Rapid growers M. phlei M. smegmatis group Some M. avium complex isolates are scotochromogens. Some M. szulgai isolates are photochromogens. c Young cultures may be nonchromogenic a

b

differences in the enzymes used in biochemical identication, no single biochemical test should be relied on for the identication of a species. For expediency, all necessary biochemical tests should be set up at one time. The biochemical tests are based on enzymes that the organisms possess, the substances that their metabolism produces, and the inhibition of growth on exposure to selected biochemicals.

Niacin accumulation Most mycobacteria possess the enzyme that converts free niacin to niacin ribonucleotide. However, 95% of M. tuberculosis isolates produce free niacin (nicotinic acid) because this species lacks the niacin-connecting enzyme. Accumulation of niacin, detected as nicotinic acid, was the most used biochemical test for the identication of M. tuberculosis. Nicotinic acid reacts with cyanogen bromide in the presence of an amine to form a yellow-pigmented compound. Reagent-impregnated strips eliminated the need to handle and dispose of cyanogen bromide, which is caustic and toxic. Because of molecular assays, this test is rarely used today. The niacin test may be negative when performed on young cultures with few colonies. It is recommended that the test be done on egg agar cultures 3 to 4 weeks old (results are most consistent when the test is performed on egg-based media) and with at least 50 colonies. Tests that yield negative results may need to be repeated in several weeks. The test should not be performed on scotochromogenic or rapidly growing species because M. simiae, the BCG strain of M. bovis, M. africanum, M. marinum, M. chelonae, and M. bovis, may be positive, although this rarely occurs.

Nitrate reduction The production of nitroreductase, which catalyzes the reduction of nitrate to nitrite, is relatively uncommon among Mycobacterium spp., but a positive result may be seen in M. kansasii, M. szulgai, M. fortuitum, and M. tuberculosis. Bacteria are incubated in 2 mL of sodium nitrate at 37° C for 2 hours. Hydrochloric acid (1:1 dilution in water), sulfanilamide, and

592

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N-naphthylenediamine dihydrochloride are then added; if the bacteria reduce the nitrate to nitrite, a red coloration occurs (Fig. 26.8). When no color change develops, however, either no reaction occurred, or the reaction has gone beyond nitrite. The addition of zinc detects nitrate and results in a color change to pink in a true-negative reaction. Commercially available strips have simplied the assay. The nitrate reduction test differentiates M. tuberculosis from the scotochromogens and MAC.

Catalase Catalase is an enzyme that splits hydrogen peroxide into water and oxygen. Mycobacteria are catalase positive. However, not all strains produce a positive reaction after the culture has been heated to 68° C for 20 minutes. Isolates that are catalase positive after heating have a heat-stable catalase (Fig. 26.9). Most MTBC organisms do not produce heat-stable catalase; exceptions are strains resistant to isoniazid. Other heat-stable, catalase-negative species include M. gastri, M. haemophilum, and M. marinum. Semi-quantitation of catalase production is based on the addition of Tween 80 (a detergent) and hydrogen peroxide to a 2-week-old culture grown in an agar deep. The reaction is read after 5 minutes, and the resulting column of bubbles is measured (Fig. 26.10). The column size is recorded as greater than or less than 45 mm.

coloration. The time required for the hydrolysis is variable. Results are recorded as positive after 24 hours, 5 days, or 10 days. This test is helpful in distinguishing between scotochromogenic and nonphotochromogenic mycobacteria.

Iron uptake Some mycobacteria can convert ferric ammonium citrate to an iron oxide. After growth of the isolate appears on an eggbased medium slant, rusty brown colonies appear in a positive reaction on the addition of 20% aqueous solution of ferric ammonium citrate; coloration is the result of iron uptake (Fig. 26.11). The test is most useful in distinguishing M. chelonae, which is generally negative for iron uptake, from other rapid growers, which are positive.

45

Hydrolysis of Tween 80 Some mycobacteria possess a lipase that can split the detergent Tween 80 into oleic acid and polyoxyethylated sorbitol. The pH indicator neutral red is initially bound to Tween 80 and has an amber color. After hydrolysis of Tween 80, neutral red can no longer bind, and it is released, causing pink

Fig. 26.10 Semi-quantitative catalase test.

+

1+

C

3+

5+

+

+

+ –

Fig. 26.8 Nitrate reduction test.

C

Fig. 26.9 Catalase test.

–

Fig. 26.11 Iron uptake.

–

C

Isolation and identication of mycobacteria

593

Arylsulfatase

Urease

Most members of the genus Mycobacterium possess the enzyme arylsulfatase. This enzyme hydrolyzes the bond between the sulfate group and aromatic ring structure in compounds with the formula R–OSO 3H. Tripotassium phenolphthalein sulfate is such a molecule, from which phenolphthalein is liberated with exposure to arylsulfatase. The liberation of phenolphthalein causes a pH change in the presence of sodium bicarbonate, indicated by the change to a pink coloration. The M. fortuitum complex, M. chelonae, M. xenopi, and M. triviale, have rapid arylsulfatase activity that can be detected in 3 days. M. marinum and M. szulgai exhibit activity with 14 days of incubation.

Detection of urease activity can be used to distinguish M. scrofulaceum, which is urease positive, from M. gordonae, which is urease negative (Fig. 26.14). A loopful of test organism is grown in 4 mL of urea broth at 37° C for 3 days. A pinkto-red color is indicative of a positive reaction.

Pyrazinamidase Pyrazinamidase hydrolyzes PZA to pyrazinoic acid and ammonia in 4 days. Ferrous ammonium sulfate combines with pyrazinoic acid, producing a red pigment (Fig. 26.12). This reaction occurs in about 4 days and may be useful in distinguishing M. marinum (positive) from M. kansasii (negative) and M. bovis (negative) from M. tuberculosis (positive).

Inhibitory tests Thiophene-2-carboxylic acid hydrazide T2H distinguishes M. bovis from M. tuberculosis. M. bovis is susceptible to lower concentrations of T2H than M. tuberculosis. Variability in inhibition exists, depending on the concentration of the inhibitory agent and the temperature of incubation.

Sodium chloride tolerance High salt concentration (5% NaCl) in egg-based media (e.g., LJ medium) inhibits the growth of most mycobacteria. M. avescens, M. triviale, and most rapidly growing Mycobacterium spp. are exceptions that do grow in the presence of 5% NaCl.

Tellurite reduction

Growth on MacConkey agar

Reduction of colorless potassium tellurite to black metallic tellurium in 3 to 4 days is a characteristic of MAC (Fig. 26.13) and thus is useful in distinguishing MAC from other nonchromogenic species. In addition, all rapid growers can reduce tellurite in 3 days.

The Mycobacterium fortuitum-chelonae complex can grow on MacConkey agar without crystal violet, whereas most other mycobacteria cannot. This is not the same formulation of MacConkey agar typically used for the isolation of enteric bacilli.

Chromatography

+ C

C

–

The cell walls of Mycobacterium spp. contain long-chain fatty acids called mycolic acids, which can be detected by chromatography. The type and quantity of mycolic acids are species specic. Identication of Mycobacterium spp. uses HPLC (see Chapter 11). Enough mycolic acid can easily be extracted from small quantities of bacterial cultures. A basic saponication followed by acidication and chloroform extraction allows accurate identication of most mycobacterial species using chromatograms. Species identications made with HPLC have been shown to agree well with biochemical and nucleic acid probe identications. Chromatography is rapid and highly reproducible, but the initial equipment cost is high. This method has largely been replaced my molecular assays and MALDI-TOF mass spectrometry.

+

Fig. 26.12 Pyrazinamidase test.

C

–

–

+

C

Fig. 26.13 Test for tellurite reduction.

Fig. 26.14 Urease test.

+

–

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Mycobacterium tuberculosis and nontuberculous mycobacteria

Hybridization and nucleic acid amplication tests for Mycobacterium tuberculosis The use of nucleic acid hybridization techniques allows the rapid identication of several common mycobacterial species. The rst commercially available nucleic acid probe was the AccuProbe (Gen-Probe, San Diego, CA). It was approved for use on culture isolates to identify the MTBC, the MAC, M. avium, M. intracellulare, M. kansasii, and M. gordonae. These tests use labeled (e.g., acridine ester–labeled nucleic acid) probes specic to mycobacterial ribosomal RNA (rRNA). The rRNA is released from the cell after sonication. The DNA probe is incubated with the test solution. If specic rRNA is present, a stable DNA-RNA complex, or hybrid, is formed. Unbound probe is chemically degraded. The complex is detected by adding an alkaline hydrogen peroxide solution. The hybridbound acridine ester is available to cause a chemiluminescent reaction, resulting in the emission of light. The amount of light emitted is related to the amount of hybridized probe. The sensitivity of the assay ranges from 95% to 100%, depending on the species and species complexes. The amount of organism required for testing in the case of M. tuberculosis is a single colony of at least 1 mm in diameter, or in the case of MAC, a barely visible lm of growth on the surface of the medium. Most positive results are well above the cutoff value of 10% hybridization. When a probe is used on a contaminated specimen, the resulting hybridization percentage may incorrectly fall below accepted cutoff hybridization levels, leading to a false-negative result. DNA hybridization identication can be applied to growth on conventional agar and to growth in liquid media. The combination of brothbased growth for detection and DNA hybridization identication using the probe technology allows rapid recovery and identication. In addition to hybridization assays, many laboratories use nucleic acid amplication tests to identify mycobacteria. The INNO-LiPA Mycobacteria v2 test (Fujirebo, Malvern, PA) is a line probe assay that targets the 16S-23S rRNA spacer region of mycobacteria and has been used to directly detect and identify the MTBC, the MAC, M. kansasii, M. xenopi, M. gordonae, and M. chelonae. The GenoType Mycobacteria Direct test (Hain Lifescience, Nehrin, Germany) uses a similar format and has additional probes for M. celatum, M. malmoense, M. peregrinum, and M. fortuitum. Although these assays have not yet received FDA approval, many U.S. laboratories have validated them. Automated DNA sequencing is the most accurate method for the identication of mycobacterial isolates. A commonly used target is the gene coding for 16 S rRNA. This gene is present in all bacteria and contains conserved and variable regions. Identication is accomplished by PCR amplication of DNA, followed by sequencing of the amplicons (amplied products). The organism is identied by comparison of the nucleotide sequence with reference sequences in a quality database. Other genes targeted include the hsp65 and rpoB genes. Although this method holds great promise, sequences in some reference databases are not accurate, and procedures are not yet standardized. The FDA has not approved sequence databases, but some laboratories have veried sequencing tests as laboratory-developed tests. As molecular methods became more widely available commercially, identication and detection of mycobacteria has become faster and more specic.

Direct nucleic acid amplication tests Nucleic acid amplication assays designed to detect MBTC bacilli directly from patient sputum specimens can be performed in as little as 6 hours on processed specimens and offer the promise of same-day reporting of results for detection and identication of M. tuberculosis. This methodology is recommended by the CDC as a standard of practice. However, the test is only recommended on patients with signs and symptoms of TB. The Amplied Mycobacterium tuberculosis Direct test (Hologic, Marlborough, MA) consists of transcription-mediated amplication of a specic 16 S rRNA target performed at a constant temperature for the detection of MTBC rRNA in smear-positive and smear-negative respiratory specimens. The Cepheid Xpert MTB/RIF (Cepheid, Sunnyvale, CA) consists of PCR amplication of the 584–base pair region of the 16 S rRNA gene sequence that uses molecular beacon probes.

Mass spectrometry MALDI-TOF mass spectrometry is becoming more commonly used in microbiology laboratories (see Chapter 11) and is used in the identication of the mycobacteria. Because of the increased risk of laboratory-acquired infections when working with the mycobacteria, the processing of these isolates for MALDI-TOF mass spectrometry is different from that for other bacteria. In addition, because of the unique cell wall of the mycobacteria, an extraction step is required before the sample is added to the target plate. MALDI-TOF mass spectrometry requires a large biomass, so subculturing is often required. Because of the slow-growing nature of the MTBC, this can take 1 to 3 weeks. Another drawback to this method is that the MTBC cannot be identied to the species level. Using customized databases, some U.S. laboratories have reported the ability to speciate some of the rapidly growing mycobacteria. The two commercially available systems, VITEK MS (bioMérieux) and the MALDI Biotyper system (Bruker Daltonics, Billerica, MA), have databases that include the MTBC and NTM. The MALDI-TOF mass spectrometry system provides accurate identication of mycobacteria from solid media. Tests from liquid media have yielded variable results.

Susceptibility testing of Mycobacterium tuberculosis Along with the increased incidence of mycobacterial disease, the development of multidrug-resistant strains of mycobacteria has been observed. For patients to receive appropriate therapy, the CDC recommends that when isolated, M. tuberculosis be tested for susceptibility to isoniazid, rifampin, ethambutol, and streptomycin. PZA should also be considered. Similarly, testing should be repeated if the patient’s cultures for M. tuberculosis remain positive after 3 months of therapy. Susceptibility testing of mycobacteria requires meticulous technique and experienced personnel. Therefore laboratories with few M. tuberculosis isolates should consider sending isolates to a reference laboratory for susceptibility testing. Currently, two main methods are used for determining antimicrobial susceptibility of MTBC: culture-based drug

Susceptibility testing of Mycobacterium tuberculosis

susceptibility testing and molecular drug susceptibility testing. The culture-based drug susceptibility method includes the agar proportion test, two broth systems, and the Sensititre microtiter dilution method (Thermo Fisher Scientic, Waltham, MA). The agar proportion method is commonly used in the United States and Europe for all antimycobacterial drugs except PZA. The recommended medium is Middlebrook 7H10 agar supplemented with oleic acid–albumin-dextrose-catalase medium. The agar is prepared with two different concentrations of the drugs and dispensed into quadrant Petri dishes. A bacterial suspension equal to a McFarland #1 standard is prepared. The suspension is then diluted 10 –2 and 10–4, and the dilutions are plated to separate media. The two dilutions provide a set of plates that should be countable (i.e., 100 to 300 colony-forming units [CFU] on the control plate). Plates are incubated at 37° C. A control plate is set up for each drug tested so the number of colonies on the test quadrants can be counted and compared with the number on the control quadrant. If the test growth is less than 1% of the control growth, the organism is susceptible, and if greater, it is resistant. By this method, results can be obtained in 2 to 3 weeks, depending on the growth rate of the organism. In clinical correlations with in vitro data, if greater than 1% of a patient’s bacilli are resistant to a particular drug, treatment fails. The test is often referred to as the proportion method because it allows one to predict the probability that 1% of the cells are resistant or not. The two FDA-approved liquid methods to determine the susceptibility of M. tuberculosis to antimycobacterial agents are the MGIT 960 and VersaTREK. The broth methods use the principles of the agar proportion assay and employ a continuous monitoring system providing results more quickly than the agar method. Growth is indicated by the amount of uorescence or gas measured by the BACTEC MGIT 960 or VersaTREK culture system, respectively. For each drug tested, a standardized inoculum is added to drug-free and drug-containing vials. The growth rate in the absence or presence of drug is then compared. Because it is not necessary to wait to visually detect growth, continuous monitoring system–based susceptibility results are usually available in 3 to 13 days. This compares favorably to about 21 days for the gold standard agar proportion method. Molecular assays for drug-resistant mycobacteria can provide results in a few hours. The Cepheid Xpert MTB/RIF, discussed earlier, can be performed on unprocessed or processed sputum samples. It not only can detect the presence of MTBC, but it also can detect ve separate mutations within the rpoB gene that indicate rifampin resistance. The GenoType MTBDRplus VER 2.0 (Hain Lifescience) can be used on clinical samples and culture isolates. It too can identify MTBC and detect mutations indicating resistance to rifampin and isoniazid. When a mutation is found, the CDC recommends gene sequencing to conrm. Susceptibility testing of most NTM is not performed routinely. An exception is the rapidly growing mycobacteria. Broth microdilution, agar disk diffusion, and the Etest (bioMérieux) have been used for performing susceptibility testing on the rapid growers. The Clinical and Laboratory Standards Institute published guidelines in 2003 recommending broth dilution as the reference method for NTM.

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POINTS TO REMEMBER

• Mycobacteria are important causes of human diseases, such as TB and Hansen disease. • Mycobacteria have a unique cell wall, and special stains are required to visualize the microorganisms. • Many Mycobacterium spp. are environmental microorganisms infrequently isolated from clinical specimens. • Isolation of mycobacteria requires specic safety precautions, including laboratories with negative air pressure, biological safety cabinets, the use of respirators and other PPE, and electric incinerators instead of ame incinerators. • Most pathogenic mycobacteria are slow growers, taking up to several weeks for isolation on articial media. • Some Mycobacterium spp. produce a pigment, which can be a helpful feature in their identication. • Other tests for identication of mycobacteria include rate of growth, nitrate reduction, niacin production, presence of heat-stable catalase, and sensitivity to T2H. However, phenotypic identication is being replaced by molecular methods such as nucleic acid amplication. • Antimicrobial susceptibility testing of the mycobacteria, while important, is technically demanding and should be performed only by experienced personnel.

LEARNING ASSESSMENT QUESTIONS

1. Which uorescent stain(s) can be used to detect the mycobacteria? a. Auramine-rhodamine b. Kinyoun c. Ziehl-Neelsen d. Both b and c 2. A nonpigmented mycobacterium is isolated that reduces nitrate to nitrite and is niacin positive. Which organism would you suspect? a. Mycobacterium kansasii b. Mycobacterium xenopi c. Mycobacterium tuberculosis d. Mycobacterium avium complex (MAC) 3. Which statement is correct regarding the causative agent of Hansen disease? a. It is highly contagious. b. It readily grows on most mycobacterial media. c. It grows best at core body temperature (37° C). d. It does not grow in vitro. 4. The skin test for tuberculosis is characterized by which of the following? a. Detects antibodies to mycobacterial antigens b. Detects a cell-mediated immune response to mycobacterial antigens c. Uses the bacillus Calmette-Guérin (BCG) strain as the antigen source d. Both a and b 5. When hired to work in the mycobacteriology laboratory, a laboratory scientist had a nonreactive PPD skin test. Six months later, his PPD skin test was reactive. What does this likely indicate? a. It was a false-positive result. b. Tuberculosis was acquired before his hire.

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c. He acquired Mycobacterium tuberculosis infection between the rst and second skin test. d. Not enough information is provided to determine. A primary care provider suspects a patient has a mycobacterial soft tissue infection. At which temperature should the inoculated media be incubated? a. 4° C b. 28° C c. 35° C d. 42° C When trying to isolate Mycobacterium spp., which of the following specimens should be decontaminated before plating? a. Sputum b. Lung biopsy c. Lymph node biopsy d. Pleural uid A physician provides material from a patient with a skin infection that has spread to the bone. After 3 days of incubations at 35° C on Middlebrook 7H10 medium, small, offwhite colonies are seen. Which of the following organisms would you suspect? a. Mycobacterium ulcerans b. Mycobacterium chelonae c. Mycobacterium fortuitum group d. Mycobacterium avium complex Microscopic examination of material from a cervical lymph node taken from a child with lymphadenitis reveals acidfast bacilli. Which of the following organisms would you suspect? a. Mycobacterium bovis b. Mycobacterium kansasii c. Mycobacterium genavense d. Mycobacterium avium complex What are the current recommendations for the identication of Mycobacterium tuberculosis in the clinical laboratory? Why should mycobacterial infections be treated for 6 months or longer, and why is there a need to use multiple drugs when treating M. tuberculosis infections? How do the various levels of mycobacterial laboratory testing differ, and why should smaller-volume laboratories consider not performing full identication and susceptibility testing on mycobacterial isolates? What are the methods used to process clinical specimens for mycobacterial culture and the reasons specimens must be decontaminated and digested before culture? With respect to laboratory technique in the isolation and identication of mycobacteria, what are some causes of false-negative and false-positive results? What are the important safety considerations for laboratories attempting mycobacterial isolation and identication?

BIBLIOGRAPHY Amir, J. (2010). Non-tuberculous mycobacterial lymphadenitis in children: diagnosis and treatment. Israel Medical Association Journal, 12, 49. Available at: https://www.ima.org.il/lesupload/ imaj/0/38/19376.pdf. (Accessed 16 May 2022). Brown-Elliott, B. A., & Wallace, R. J. (2019). Mycobacterium: clinical and laboratory characteristics of rapidly growing mycobacteria. In K. C. Carroll, et al. (Eds.), Manual of clinical microbiology (12th ed., p. 612). Washington, DC: ASM Press.

Cauleld, A. J., et al. (2019). Mycobacterium: laboratory characteristics of slowly growing mycobacteria other than Mycobacterium tuberculosis. In K. C. Carroll, et al. (Eds.), Manual of clinical microbiology (12th ed., p. 595). Washington, DC: ASM Press. Centers for Disease Control and Prevention. (2016). Fact sheets: interferon-gamma release assays (IGRAs)—blood tests for TB infection. Available at: http://www.cdc.gov/tb/publications/factsheets/ testing/igra.htm. (Accessed 21 June 2022). Centers for Disease Control and Prevention. (2017). Hansen’s disease (leprosy). Available at: https://www.cdc.gov/leprosy/index.html (Accessed 14 May 2022). Centers for Disease Control and Prevention. (2021). TB screening and testing of health care personnel. Available at: https://www.cdc.gov/ tb/topic/testing/healthcareworkers.htm. (Accessed 21 June 2022). Centers for Disease Control and Prevention. (2021). Trends in tuberculosis, 2020. Available at: https://www.cdc.gov/tb/ publications/factsheets/statistics/tbtrends.htm#. (Accessed 15 May 2022). Centers for Disease Control and Prevention. (2022). Tuberculosis— United States, 2021. Morbidity and Mortality Weekly Report, 71, 441. Available at: https://www.cdc.gov/mmwr/volumes/71/wr/ mm7112a1.htm#. (Accessed 13 May 2022). Di Nuzzo, M., et al. (2018). Extrapulmonary tuberculosis among immigrants in a low-TB burden and high immigrant receiving city of northern Italy. Journal of Infection in Developing Countries, 12, 73. Gido, R. D. S., et al. (2019). Pulmonary infection with Mycobacterium szulgai: a case report. SAGE Open Medical Case Reports, 7, 1. Johnston, J. C., et al. (2017). Mycobacterium kansasii. Microbiology Spectrum, 5, 10.1128/microbiolspec.TNMI7–0011-2016. Kuenstner, J. T., et al. (2020). Presence of infection by Mycobacterium avium subsp. paratuberculosis in the blood of patients with Crohn’s disease and control subjects shown by multiple laboratory culture and antibody methods. Microorganisms, 8, 2054. Martin, I., et al. (2019). Mycobacterium: general characteristics, laboratory detection, and staining procedures. In K. C. Carroll, et al. (Eds.), Manual of clinical microbiology (12th ed., p. 558). Washington, DC: ASM Press. Sama, J. N., et al. (2016). High proportion of extrapulmonary tuberculosis in a low prevalence setting: a retrospective cohort study. Public Health, 138, 101. Warshauer, D. M., et al. (2019). Mycobacterium tuberculosis complex. In K. C. Carroll, et al. (Eds.), Manual of clinical microbiology (12th ed., p. 576). Washington, DC: ASM Press. Wilson, J. W., et al. (2019). Mycobacterium scrofulaceum disease: experience from a tertiary medical centre and review of the literature. Infectious Diseases (London, England), 51, 602. Woods, G. L., et al. (2019). Susceptibility test methods: Mycobacteria, Nocardia, and other actinomycetes. In K. C. Carroll, et al. (Eds.), Manual of clinical microbiology (12th ed., p. 1398). Washington, DC: ASM Press. World Health Organization (n.d.). Global tuberculosis programme: surveillance of drug-resistant TB. Available at: https://www.who. int/teams/global-tuberculosis-programme/diagnosis-treatment/ treatment-of-drug-resistant-tb/surveillance-of-drug-resistant-tb. (Accessed 14 May 2022). World Health Organization. (2019). Global leprosy update 2018: moving towards a leprosy-fee world. Weekly Epidemiologic Record, 91, 405. Available at: https://www.who.int/publications/i/item/ who-wer9435-36. (Accessed 21 June 2022). World Health Organization. (2021). Global tuberculosis report 2021. Available at: https://www.who.int/publications/m/item/ factsheet-global-tb-report-2021. (Accessed 13 May 2022). World Health Organization. (2021). WHO announces updated denitions of extensively resistant drug resistant tuberculosis. Available at: https://www.who.int/news/item/27-01-2021-who-announces-updated-denitions-of-extensively-drug-resistant-tuberculosis. (Accessed 14 May 2022). World Health Organization. (2022). Leprosy. Available at: https://www. who.int/news-room/fact-sheets/detail/leprosy. (Accessed 13 May 2022).

27 Medically signicant fungi Connie F. Cañete-Gibas and Nathan P. Wiederhold

CHAPTER OUTLINE

General characteristics, 598 Yeasts versus molds, 598 Hyaline versus phaeoid, 599 Dimorphism and polymorphism, 600 Reproduction, 600 Taxonomy, 600 Ascomycota, 600 Basidiomycota, 601 Mucorales, 601 Fungi imperfecti, 601 Mycoses, 601 Supercial mycoses, 602 Cutaneous mycoses, 602 Subcutaneous mycoses, 602 Systemic mycoses, 602 Clinically significant species, 603 Agents of supercial mycoses, 603 Agents of cutaneous mycoses, 604 Agents of subcutaneous mycoses, 607 Agents of systemic mycoses, 610 Agents of opportunistic mycoses, 615 Agents of yeast infections, 622 Pneumocystis, 624 Laboratory diagnosis of fungi, 626 Safety issues, 626 Specimen collection, handling, and transport, 626 Direct microscopic examination of specimens, 628 Isolation methods, 630 Fungi identication, 631 Immunodiagnosis of fungal disease, 635 Antifungal susceptibility, 635 Antifungal agents, 635 Antifungal susceptibility testing, 635 Bibliography, 637

OBJECTIVES

After reading and studying this chapter, you should be able to: 1. Describe the general characteristics and structures of fungi. 2. Compare the cellular characteristics and morphology of fungi (eucaryotes) and bacteria (procaryotes). 3. Compare asexual and sexual reproduction of fungi. 4. List the divisions of fungi. 5. Identify the major causes of fungal infections. 6. Describe the common opportunistic saprobes associated with infections in immunocompromised hosts. 7. Characterize the following different types of mycoses, including the tissues they affect: a. Supercial b. Cutaneous c. Subcutaneous d. Systemic e. Opportunistic saprobes 8. Analyze the appropriate specimen collection procedures, staining methods, and culture techniques used in the mycology laboratory. 9. Describe the key characteristics associated with the identication of the clinically signicant fungi. 10. Evaluate the methods used to identify fungi. 11. Differentiate chromoblastomycosis from eumycotic mycetoma. 12. Develop a laboratory protocol for the identication of the clinically significant yeast. KEY TERMS

Anamorph Arthroconidium Ascospores Ascus Blastoconidium Conidium Dermatophytes

Dimorphic fungi Eumycotic mycetoma Germ tube Hyphae Macroconidium Microconidium Mold

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Saprobe Sporangiophores Sporangiospores Synanamorphs Teleomorph Thrush Yeast

Case in point A 32-year-old female patient developed a fever 12 days after bone marrow transplantation. Broad-spectrum antimicrobial therapy was initiated, but the fever persisted. On day 17, the patient developed skin lesions across her body and lower extremities; a biopsy was performed. Microscopic evaluation of the tissue revealed hyphal elements. That same day, after 4 days of incubation, the patient’s blood cultures were positive with a yeastlike colony. Although antifungal therapy was initiated, the patient died on day 22. Issues to consider After reading the patient’s case history, consider: • The fungi most likely to be implicated in this patient’s infection • Steps the laboratory will use to resolve the discrepancy between what is seen in the tissue slides and what grows from the blood culture • The steps necessary to arrive at a nal identication of this agent of disease

Fungi constitute an extremely diverse group of organisms and are generally classied as molds or yeasts. Some have been recognized as true pathogens, whereas others are only known as environmental saprobes, living on nonliving material. Fungi can cause mild infections, trigger allergic reactions, including asthma, and produce serious life-threatening disease. With the widespread use of chemotherapy, radiation therapy, and diseases such as acquired immunodeciency syndrome (AIDS) that affect the immune system, the line between pathogen and saprobe has been blurred. The isolation of all organisms, especially in the immunocompromised patient, must initially be considered a signicant nding and evaluated in light of the patient’s history and physical examination results.

General characteristics The characteristics of fungi differ from those of plants or bacteria. Like plants, fungi are eukaryotic; they possess a true nucleus, with a nuclear membrane and mitochondria whereas bacteria are prokaryotic, lacking these structures. Unlike plants, fungi lack chlorophyll and must absorb nutrients from the environment. In addition, fungal cell walls are made of chitin, whereas those of plants contain cellulose. Most fungi are obligate aerobes that grow best at a neutral pH, although they tolerate a wide pH range. Moisture is necessary for growth, but spores and conidia survive in dry conditions for extended lengths of time.

Yeasts versus molds Yeasts are single vegetative cells that typically form a smooth, creamy, bacterial-like colony without aerial hyphae. Because their macroscopic and microscopic morphologies are similar, identication of yeasts is based primarily on biochemical testing and molecular diagnostic methods. Yeasts reproduce by budding or ssion. Budding involves maturation of the bud to an independent blastoconidium (daughter cell), as shown in Fig. 27.1. This process involves lysis of the yeast cell wall so that a blastoconidium can form. As this structure enlarges, the nucleus of the parent cell undergoes mitosis. Once the new nucleus is passed into the daughter cell, a septum forms and the daughter cell breaks free. During ssion, two cells of equal size are formed. These cells continue to grow from the tips of the cell and divide only after a medial ssion is formed. Most molds have a fuzzy or woolly appearance because of the formation of mycelia (Fig. 27.2). Mycelia are made up of many long strands of tubelike structures called hyphae, which are either aerial or vegetative. Aerial mycelia extend above the surface of the colony and are responsible for the fuzzy appearance. In addition, aerial mycelia support the reproductive structures that produce conidia. Conidia, in many cases, are used to identify different fungal genera. The vegetative mycelia extend downward into the medium to absorb nutrients. Microscopic appearance often aids in the identication of molds. In some species, antler, racquet, rhizoid, or spiral hyphae are formed (Fig. 27.3A). Antler hyphae have swollen, branching tips that resemble moose antlers. Racquet hyphae contain enlarged, club-shaped areas. Spiral hyphae are tightly coiled. Rhizoids (see Fig. 27.3B), rootlike structures, might be seen in some members of the order Mucorales, the causative agents of mucormycosis, and their presence and placement can assist with identication. Frequently, when fungal hyphae are being described, they are referred to as septate or sparsely septate. Septate hyphae show frequent cross-walls occurring perpendicular to the outer walls of the hyphae (Fig. 27.4A), whereas sparsely septate hyphae have few crosswalls at irregular intervals (see Fig. 27.4B). The term aseptate, which means absence of septations, has historically been used to describe the hyphae of members of the order Mucorales. Microscopic examination of hyphae associated with the

Septum formation

Mother Daughter cell cell

Fig. 27.1 Formation of blastoconidia in yeasts.

Aerial mycelia Vegetative mycelia

Fig. 27.2 Aerial mycelia give molds the “woolly” appearance. Vegetative mycelia are responsible for absorbing nutrients from the medium.

General characteristics

Antler hyphae

Racquet hyphae

Spiral hyphae

Rhizoids

A

B Fig. 27.3 A, Specialized structures formed in vegetative mycelia by certain fungal species. B, Rhizopus spp., showing rhizoids (unstained, ×200).

A

B

Fig. 27.4 A, Phaeoacremonium sp. displaying septate hyphae (unstained, ×200). B, Mucorales hyphae in tissue appears sparsely septate (Gomori methenamine silver stain, ×400).

Mucorales often reveals occasional septations; therefore these hyphae are more correctly termed sparsely septate as opposed to aseptate

Hyaline versus phaeoid Another useful characteristic in identication is pigmentation. Hyaline (moniliaceous) hyphae are nonpigmented or lightly pigmented, whereas phaeoid (dematiaceous) hyphae are darkly pigmented (Fig. 27.5) because of the presence of melanin in the cell wall. Depending on the amount of melanin present, the hyphae will appear pale to dark brown or almost black. Note that the dark hyphae seen in the tissue section in Fig. 27.4B is dark colored; however, it is because of the stain that enhances visualization of fungal elements in tissue and not because of melanin. All fungal elements appear black when Gomori methenamine silver (GMS) stain is used.

Fig. 27.5 Bipolaris sp. is an example of a phaeoid fungus. Note the dark pigmentation, which is caused by the presence of melanin in the cell wall (unstained, ×200).

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Another stain that is often used to determine hyphal pigmentation in tissue is the Fontana-Masson stain. This stain specically stains melanin, causing phaeoid hyphae to appear brown, whereas hyaline hyphae stain pink to red.

Hyphae

Arthroconidia

Dimorphism and polymorphism The term dimorphism refers to the ability of some fungi to exist in two forms, dependent on growth conditions. Dimorphic fungi include a mold phase and a yeast or spherule phase. The yeast or tissue state is seen in vivo or when the organism is grown at 37°C with increased concentration of carbon dioxide (CO 2). The mold phase is seen when the organism is grown at room temperature (22° to 25°C) in ambient air conditions. Thermally dimorphic fungal species associated with human disease include Blastomyces spp., Coccidioides immitis/posadasii, Histoplasma spp., Paracoccidioides spp., Sporothrix spp., and Talaromyces (formerly Penicillium) marneffei. Several other fungi also possess this ability but have not been described as agents of human mycoses (infections caused by fungi). Polymorphic fungi have both yeast and mold forms in the same culture. This characteristic occurs despite growth conditions and is best observed in Exophiala spp., in which the yeast phase is typically observed initially, followed by the mold phase as the colony ages.

Fig. 27.7 In another form of asexual reproduction, arthroconidia are formed by fragmentation of fertile hyphae.

Hypha 1

Hypha 2

Fusion of hyphae Fusion of nuclei

Zygospore

Reproduction Fungi can reproduce asexually (imperfect) or sexually (perfect). Asexual reproduction results in the formation of conidia (singular, conidium) following mitosis. Asexual reproduction is carried out by specialized fruiting structures known as conidiogenous cells. These structures form conidia, which contain all the genetic material necessary to create a new fungal colony. Two common conidiogenous cells are the phialides and annellides. Phialides are vaselike structures that produce phialoconidia (Fig. 27.6), whereas annellides are ringed structures that produce annelloconidia. Both form their conidia blastically (budding) like many yeasts; the parent cell enlarges, and a septum forms to separate the conidial cell. Another type of conidia is the arthroconidia (singular, arthroconidium). These conidia are formed by fragmentation of fertile hyphae as opposed to being formed by conidiogenous cells (Fig. 27.7). In the clinical laboratory, mold identications may be based on microscopically observing structures formed as a result of asexual reproduction. Sexual reproduction requires the joining of two compatible nuclei, followed by meiosis (Fig. 27.8). A fungus that reproduces sexually is known as a teleomorph.

Fig. 27.8 Sexual reproduction occurs by the fusion of compatible nuclei and subsequent production of a zygospore.

Occasionally, these fungi will also reproduce asexually. When this occurs, the asexual form is termed the anamorph. If more than one anamorph is present for the same teleomorph, the anamorphic strains are termed synanamorphs

Taxonomy There are over 100,000 named fungal species and an estimated 1 million to 10 million undiscovered species. Most causative agents of clinical infections are found in four groups of fungi. They consist of the phyla Ascomycota and Basidiomycota, subphylum Mucoromycotina, and the form division Fungi Imperfecti (Deuteromycota).

Ascomycota Conidia Phialide Hyphae

Conidiophore

Fig. 27.6 An example of asexual reproduction is the production of phialoconidia. Conidia are formed from conidiogenous cells such as phialide (a vaselike structure). Phialoconidia are “blown out” of the phialide.

Approximately 50% of all named fungi are classied in the phylum Ascomycota. Fungi associated with the phylum Ascomycota are characterized by the production of sexual spores known as ascospores. Ascospores are formed within a saclike structure known as an ascus (plural, asci). It is important to note, however, that they are usually identied on the basis of characteristic asexual structures. Representative organisms include Microsporum spp., Trichophyton spp., and Scedosporium spp. Phylogenetically, the genus Candida belongs to the phyla Ascomycota. However, many Candida species do not have a teleomorph; therefore they are categorized as Fungi Imperfecti.

Mycoses

Basidiomycota Only a few members of the phylum Basidiomycota are clinically signicant. The major pathogens are members of the Cryptococcus neoformans and C. gattii species complexes. Members of the genera Malassezia, Trichosporon, and Rhodotorula are also associated with human infections. Basidiomycetous molds are being recovered in increasing numbers in the laboratory, but their clinical signicance is not clearly understood. Close communication between the physician and the laboratory would help determine whether the isolate is an environmental contaminant or an agent of disease. When basidiomycetous molds are recovered in the laboratory, they typically remain sterile, complicating the identication process. One clue that a mold is a basidiomycete is the presence of clamp connections. Clamp connections occur at the septations in the vegetative hyphae and are easily visible under a light microscope. A portion of the hypha on one side of the septation grows out and connects to the hypha on the other side of the septation, thereby bypassing the septation. Clamp connections are not always present. Staining with diazonium blue B or growing on culture medium supplemented with benomyl can assist in the identication of a putative basidiomycete isolate.

Sporangium

Fungi imperfecti The form division Fungi Imperfecti contains the largest number of organisms that are causative agents of mycoses, including cutaneous, subcutaneous, and systemic diseases. Organisms are placed within this group when no mode of sexual reproduction has been identied. Therefore they are

Sporangiospores Columella Columella

Sporangiophore

Fig. 27.9 Asexual reproduction by Mucorales is characterized by the production of spores (sporangiospores) from within a sporangium.

Keratinized layer

Mucorales The traditional Zygomycota have undergone taxonomic changes. Taxa traditionally placed in the phylum Zygomycota are now assigned among the phyla Mucoromycota, Basidiobolomycota, and Entomophthoromycota, subphyla Mucoromycotina, Basidiobolomycotina, and Entomophthoromycotina, respectively. The most clinically signicant species belong to the subphylum Mucoromycotina consisting of the orders Mucorales, Umbelopsidales, and Endogonales and includes the genera Actinomucor, Apophysomyces, Cokeromyces, Cunninghamella, Lichtheimia (formerly Absidia), Mucor, Rhizomucor, Rhizopus, Saksenaea, and Syncephalastrum. Members of the order are rapidly growing organisms normally found in soil. They are often opportunistic pathogens in immunocompromised hosts. The clinical infection is referred to as mucormycosis Mucorales, the most clinically signicant order, generally produce profuse, gray-to-white, aerial mycelia characterized by the presence of hyaline, sparsely septate hyphae. Asexual reproduction is characterized by the presence of sporangiophores and sporangiospores. The asexual spores (sporangiospores) are produced in a structure known as a sporangium, which develops from a supporting structure termed a sporangiophore (Fig. 27.9). Although some Mucorales are capable of sexual reproduction resulting in the production of zygospores, these structures are not routinely seen in clinical laboratories.

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Epidermis

Dermis (elastic fibers, collagen fibers, hair follicle, sweat gland)

Subcutaneous layer

Fig. 27.10 The layers of skin and tissues in which fungal infections can occur.

identied on the basis of characteristic asexual reproductive structures.

Mycoses Mycoses (singular, mycosis) are diseases caused by fungi. Fungal disease is frequently categorized on the basis of the site of the infection—supercial, cutaneous, subcutaneous, and systemic mycoses. Fig. 27.10 shows the different layers of tissues, which helps to classify infections of the skin, depending on where the infection occurs. Infections not involving the skin or deeper tissues just under the skin are termed systemic. Supercial and cutaneous mycoses are caused by fungi that can degrade keratin (dermatophytes) and fungi that cannot degrade keratin.

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Supercial mycoses Supercial mycoses are infections conned to the outermost layer of skin or hair. Because infections of skin, hair, and nails were at one time believed to be the result of burrowing worms that formed ring-shaped patterns in the skin, the term tinea (Latin, meaning “worm”) was applied to each disease, along with the Latin term for the body site. An example of nondermatophytic tinea is the disease tinea versicolor (pityriasis versicolor). This disease is characterized by discoloration or depigmentation and scaling of skin and is caused by the yeast Malassezia furfur complex. The disease becomes apparent in individuals with a dark complexion or in those who do not tan normally. Another nondermatophytic supercial infection is tinea nigra. This disease is almost always caused by Hortaea werneckii and is characterized by brown or black macular patches, primarily on the palms. Biopsy and culture of the site are important to distinguish this infection from melanoma, a much more serious nonfungal disease. Another supercial infection, piedra, is conned to the hair shaft and is characterized by nodules composed of hyphae and a cementlike substance that attaches it to the hair shaft. Black piedra is caused by Piedraia hortae, and white piedra is caused by Trichosporon ovoides and T. inkin. Onychomycosis, caused by Onychocola canadensis and O. kanei, is also a nondermatophytic supercial mycosis characterized by white supercial and distal lateral subungual infections of the great toenail.

Cutaneous mycoses Dermatomycoses are dened as fungal diseases of the keratinized tissues of humans and other animals. Although nondermatophyte species are capable of causing similar infections, this syndrome is usually a result of infection with a dermatophyte—hence, the term dermatophytosis. Note that this term should not be used until the causative agent has been identied as a dermatophyte. Genera in this group include Trichophyton, Microsporum, and Epidermophyton. Dermatophytic infections usually involve a restricted region of the host; traditionally, these diseases are named with respect to the portion of the body affected. The various forms of ringworm continue to be described in these terms, as shown in Table 27.1

Each ringworm lesion is the result of local inoculation of skin with the causative agent. Lesions enlarge with time, usually with most inammation occurring at the advancing edge of the lesion. However, some cases of ringworm are subclinical, exhibiting only a dry, scaly lesion without inammation. Symptoms of cutaneous mycoses include itching, scaling, or ringlike patches on skin; brittle, broken hair; and thick, discolored nails.

Subcutaneous mycoses Subcutaneous mycoses involve the deeper skin layers as well as muscle, connective tissue, and bone. Except in certain patient populations, dissemination through the blood to major organs does not occur. Characteristic clinical features include progressive, nonhealing ulcers and the presence of draining sinus tracts. In tropical areas, some agents, such as Phialophora spp. and Cladosporium spp., cause chromoblastomycosis, which is characterized by verrucous nodules that often become ulcerated and crusted. This disease is diagnosed by the presence of characteristic lesions accompanied by microscopic sclerotic bodies, often referred to as “copper pennies” because of their shape and staining properties in tissue sections. Eumycotic mycetoma is caused by fungi and results in draining sinus tracts and tissue destruction. Grains (granules), which are tightly bound hyphae, can be collected from the uids that drain from the sinus tracks and are useful in identifying the causative agent. The disease can be cutaneous or subcutaneous. Worldwide, about 40% of mycetomas are eumycotic, and the rest are actinomycotic, caused by Actinomycetes bacteria (see Chapter 16). Sporotrichosis, caused by the Sporothrix schenckii species complex, commonly presents as a progressive lymphocutaneous infection, beginning with a single draining lesion and progressing along the limbs via the lymphatic system, forming multiple draining lesions. However, granules are not formed, thus this infection does not qualify as a mycetoma. Dissemination of these species causing systemic sporotrichosis is much more common, and Sporothrix has been implicated in pneumonia that is refractory to antifungal therapy.

Systemic mycoses Table 27.1 Various forms of dermatophytoses and their respective affected sites Type of ringworm

Site affected

Tinea capitis

Head

Tinea favosa

Head (distinctive disease)

Tinea barbae

Beard

Tinea corporis

Body (glabrous skin)

Tinea manuum

Hand

Tinea unguium

Nails

Tinea cruris

Groin

Tinea pedis

Feet

Tinea imbricate

Body (distinctive lesion)

Systemic, or disseminated, mycoses are infections that affect internal organs or deep tissues of the body. Frequently, the initial site of infection is the lung, from which the organism disseminates hematogenously to other organs, including skin. Generalized symptoms include fever and fatigue. Chronic cough and chest pain might also accompany these infections. Historically, the term systemic mycoses was used to describe diseases caused by thermally dimorphic fungi, including Histoplasma, Coccidioides, and Blastomyces spp. However, other fungal agents are also capable of causing systemic disease, including but not limited to Aspergillus, Fusarium, Scedosporium, and Curvularia spp. as well as some yeasts, such as Candida and Cryptococcus spp. It is important to note that any fungus is capable of disseminating from the primary site of infection in the immunocompromised host.

Clinically signicant species

603

Clinically signicant species Agents of supercial mycoses Supercial mycoses are fungal diseases that affect only the cornied layers (stratum corneum) of the epidermis. Patients who have supercial fungal infections do not show any overt symptoms because the fungal agents do not activate any tissue response or inammatory reaction. Patients usually seek medical attention to address cosmetic rather than medical concerns caused by these fungi.

Malassezia furfur Clinical manifestations The Malassezia furfur complex causes tinea versicolor, a disease characterized by patchy lesions or scaling of varied pigmentation. It is also thought to be a cause of dandruff. Lesions associated with tinea versicolor typically appear as pale patches in individuals with darkly pigmented skin, but they can also be described as fawn-colored liver spots in individuals with a fair complexion. Lesions become especially evident in warm months, when sun exposure is more likely. Tinea versicolor may involve any area of the body, but the most prevalent sites include the face, chest, trunk, and abdomen. Antifungal therapy is not typically indicated, but the appearance of lesions may be diminished by treatment with antidandruff shampoos. Interestingly, M. furfur complex has also been implicated in disseminated infections in patients receiving lipid replacement therapy (total parenteral nutrition), particularly in infants. Removal of indwelling feeding lines is usually sufcient to clear infections without using antifungal therapy. M. furfur is a common endogenous skin colonizer. Although the reasons for overgrowth by M. furfur resulting in clinical manifestation are still unknown, it appears to be related to squamous cell turnover rates. This is evidenced by the higher incidence of tinea versicolor among persons receiving corticosteroid therapy, which decreases the rate of squamous epithelial cell turnover. Investigators have identied genetic inuence, poor nourishment, and excessive sweating as other factors that contribute to the overgrowth of the organism on the skin. This organism is found worldwide, with the greatest prevalence in hot, humid, and tropical locations.

Fig. 27.11 The typical so-called spaghetti-and-meatballs appearance showing hypha (top arrow) and a yeast cell (bottom arrow) of Malassezia furfur in a potassium hydroxide preparation (×400).

Piedraia hortae Clinical manifestations Piedraia hortae is the causative agent of black piedra, an infection of scalp hairs. The infecting organism produces hard, dark brown to black gritty nodules that are rmly attached to the hair shaft. These nodules consist of asci (saclike structures) containing eight ascospores. The disease is endemic in tropical areas of Africa, Asia, and Latin America.

Laboratory diagnosis When infected hairs are removed and placed in 10% to 20% KOH, the nodules may be crushed open to reveal the asci. Thick-walled rhomboid cells containing ascospores are seen. P. hortae grows slowly on Sabouraud dextrose agar at room temperature. It forms brown, restricted colonies that remain sterile.

Laboratory diagnosis M. furfur can be identied by microscopic examination of skin scrapings from characteristic lesions in a potassium hydroxide (KOH) preparation or by observing yellow uorescence with a Wood lamp on examination of the infected body site. Microscopic examination of the direct smear in KOH preparations reveals budding yeasts, approximately 4 to 8 µm, along with septate, sometimes branched, hyphal elements. This microscopic appearance has gained M. furfur the nickname the spaghetti-and-meatballs fungus (Fig. 27.11). M. furfur requires lipids for growth and will not grow on routine fungal media that have not been supplemented with a lipid source. Because of its special nutritional requirements, routine fungal cultures are negative for growth. Typical yeastlike colonies may be observed only after the culture medium has been overlaid with olive oil. Colonies are cream colored, moist, and smooth.

Trichosporon spp Clinical manifestations Trichosporon beigelii was previously described as a human pathogen. However, examination of the genome of members of the genus has revealed that T. beigelii is no longer a valid species infecting humans. More than 30 distinct species exist, but Trichosporon ovoides, T. asteroides, T. inkin, and Cutaneotrichosporon (previously Trichosporon) cutaneum have been implicated in most cases of supercial mycoses. An important species in this genus is T. asahii, which is implicated in severe and frequently fatal disease in immunocompromised hosts. Although less frequently encountered, T. mucoides also causes systemic disease (meningitis) and is recovered frequently from cerebrospinal uid (CSF). The colony resembles young colonies of Cryptococcus neoformans but

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is easily differentiated on the basis of physiologic and morphologic characteristics. Apiotrichum (formerly Trichosporon) mycotoxinovorans is associated with pulmonary colonization and disease in patients with cystic brosis. Trichosporon spp. are occasionally found as part of the normal skin biota and can also be isolated from animals and soil. White piedra occurs on the hair shaft and is characterized by a soft mycelial mat surrounding hair of the scalp, face, and pubic region. Members of this genus have also been recognized as opportunistic systemic pathogens. Although rare, systemic diseases caused by these fungi are frequently fatal and occur most often in immunocompromised hosts, commonly those who have hematologic disorders or malignancies or are undergoing chemotherapy. Infections in immunocompromised patients include infections of the blood, CSF, and organs. White piedra is endemic in tropical areas of South America, Africa, and parts of Asia.

Laboratory diagnosis Trichosporon spp. grow rapidly on primary fungal media and produce arthroconidia, hyphae, and blastoconidia (Fig. 27.12). The colonies are straw- to cream-colored and yeastlike. Colonies are varied and can be smooth or wrinkled, dry or moist, and creamy or velvety in appearance. Identication to the species level is conrmed by absence of carbohydrate fermentation, use of potassium nitrate (KNO 3), assimilation of sugars, and urease positivity. Molecular and proteomic approaches, such as deoxyribonucleic acid (DNA) sequence analysis and matrix-assisted laser desorption/ionization– time-of-ight (MALDI-TOF) mass spectrometry, respectively, in conjunction with biochemical reactions, lead to accurate identication of species within this genus.

resulting in unnecessary surgical procedures, especially when confused with malignant melanoma. The disease is endemic in the tropical areas of Central and South America, Africa, and Asia.

Laboratory diagnosis Diagnosis of tinea nigra can be made by direct examination of skin scrapings placed in 10% to 20% KOH. Microscopic examination shows septate hyphal elements and budding cells (Fig. 27.13). Younger cultures are primarily composed of budding blastoconidia, whereas the older mycelial portion of the colony shows wide, profusely septate hyphae with blastoconidia in clusters. Annelloconidia are seen in older hyphal colonies. H. werneckii produces shiny, moist, yeastlike colonies that start with a brownish coloration that eventually turns olive to greenish black.

Agents of cutaneous mycoses General characteristics

Tinea nigra, characterized by brown-to-black, nonscaly macules that occur most often on the palms and soles, is caused by H. werneckii. Obsolete synonyms for H. werneckii include Phaeoannellomyces werneckii and Exophiala werneckii. This disease involves no inammatory or other tissue reaction to the infecting fungus. However, the clinical presentation is so similar to that of other conditions that misdiagnosis could occur,

Three genera of fungi—Trichophyton, Microsporum, and Epidermophyton—are causative agents of dermatophytoses. Species within these genera, referred to as dermatophytes, are keratinophilic; that is, they are adapted to grow on hair, nails, and cutaneous layers of skin that contain the scleroprotein keratin. Infection of deep tissue by these fungi is rare, but occasionally extensive inammation and nail bed involvement or disseminated disease may result. Most agents of dermatophytoses live freely in the environment (geophilic), but a few have adapted almost exclusively to living on animals (zoophilic) or human tissues (anthropophilic), and they are rarely recovered from any other source. Distribution of these species is generally worldwide, although a few are found only in restricted geographic regions. Dermatophytes typically form two sizes of reproductive cells, macroconidium or microconidium. Both of these are anamorphic or asexual conidia, and their distinctive size, shape, and surface features make them valuable structures for species identication. Some dermatophytes are known to also have teleomorphic stages in which ascospores are the reproductive cells. Teleomorphs in this group of organisms are not observed in routine laboratory studies of patient

Fig. 27.12 Microscopic appearance of Trichosporon species on lactophenol cotton blue preparation showing the presence of both blastoconidia and arthroconidia (×600).

Fig. 27.13 Microscopic structures of Hortaea werneckii, showing characteristic budding annelloconidia (×400). H. werneckii causes tinea nigra.

Hortaea werneckii Clinical manifestations

Clinically signicant species

isolates because dermatophytes are heterothallic, requiring the combination of two distinct mating types. Although a few reference laboratories perform mating tests with known tester strains, this procedure is not regularly performed in clinical laboratories. Most geophilic fungi produce large numbers of conidia and therefore are among the most readily identied species. Zoophilic dermatophytes are not commonly found living freely in soil or on dead organic substrates. They often cause infections in animals and can be spread as agents of disease in humans. Fewer conidia are produced by zoophilic fungi than by geophilic species. Although the anthropophilic species are almost always encountered as agents of human disease, the infections are seldom inammatory. Species identication may be difcult because most anthropophilic species produce few conidia. Not only are sites diverse on the host involved in infections, but certain species also cause distinctive lesions. The notable example is tinea imbricata, caused by Trichophyton concentricum. Over time, involved portions of the trunk develop diagnostically distinctive concentric rings of scaling tissue. In some forms of ringworm, a persistent allergic reaction, dermatophytid, is manifested in the formation of sterile, itching lesions on body sites distant from the point of infection. Symptoms of dermatophyte infections range from slight to moderate and occasionally severe.

Infections involving hair Different body sites manifest different symptoms of mycoses. Infections of the scalp, in which hair follicles are the initiation sites, can be among the most severe and disguring forms of mycoses. Tinea favosa, or favus, begins as an infection of the hair follicle by Trichophyton schoenleinii and progresses to a crusty lesion made up of dead epithelial cells and fungal mycelia. Crusty, cup-shaped akes, called scutula, are formed. Hair loss and scar tissue formation commonly follow. Two distinct forms of tinea capitis—gray patch ringworm and black dot ringworm—are caused by different species of dermatophytes. Gray patch ringworm is a common childhood disease that is easily spread among children. The fungus colonizes primarily the outer portion of hair shafts, the so-called ectothrix hair involvement. The lesions are seldom inamed, but luster and color of the hair shaft may be lost. Microsporum audouinii and Microsporum ferrugineum are causative agents of this disease. Black dot ringworm consists of endothrix hair involvement. The hair follicle is the initial site of infection, and fungal growth continues within the hair shaft, causing it to weaken. The brittle, infected hair shafts break off at the scalp, leaving the black dot stubs. Trichophyton tonsurans and Trichophyton violaceum are the most common fungi implicated in this form of dermatophytosis.

Infections involving nails Onychomycosis, which is infection of the nails, is most often caused by dermatophytes but also may be the result of infection by other fungi. These nail and nail bed infections may be among the most difcult dermatomycoses to treat. Longterm, costly therapy with terbinane or itraconazole has been considered the best treatment, but results are often unsatisfactory. There is a direct association between dermatophytic

605

infections of the feet or hands and infections of the nails. It is unlikely that anyone suffering from tinea pedis will escape some extent of onychomycosis. The subungual form is the most common form of onychomycosis, described as lateral, distal, or proximal. Either end of the nail is rst infected, with spread continuing to the nail plate. Nails become thick, discolored, and aky. Some common agents that infect the nails are Trichophyton rubrum, T. mentagrophytes, and T. tonsurans, as well as Epidermophyton occosum.

Tinea pedis Among the shoe-wearing human population, tinea pedis (athlete’s foot) is a common disease. Infection arises from infected skin scales coming into contact with exposed skin via a carpet, shower oors, or other shared walking/standing surfaces where shoes are not always worn. It is believed that individuals have a genetic predisposition to developing the disease because not everyone encountering infected skin scales becomes infected. Infections within a family are common. Various sites on the foot may be involved, but tinea pedis usually affects the soles and toe webs. In more severe cases, the sole may develop extensive scaling, with ssuring and erythema. The disease may progress around the sides of the foot from the sole, giving rise to use of the term moccasin foot, descriptive of the appearance of the infected area. Infections of the glabrous skin range from mild, with only minimal scaling and erythema, to severely inamed lesions.

Systemic dermatophyte infections Immunocompromised persons may suffer systemic dermatophyte infections. Disseminated disease appears in different forms. In some patients, it manifests itself as granulomas, whereas in others, pea-sized to walnut-sized nodules can develop. Biopsy of these nodules reveals fungal elements that are easily recovered in culture. This phenomenon has been seen primarily in kidney transplant recipients and is thought to have spread from athlete’s foot or onychomycosis.

Epidermophyton occosum Epidermophyton occosum produces only one size of conidia described as macroconidia. The smooth, thin-walled macroconidia are produced in clusters or singly. The distal end of the conidium is broad or spatulate and reminiscent of a beaver’s tail. Occasionally, conidia may be single celled, but usually they are separated into two to ve cells by perpendicular cross-walls. Colonies of E. occosum are yellow to yellow-tan, at with feathered edges, and remain small in diameter. Epidermophyton isolates are notorious for developing pleomorphic tufts of sterile hyphae in older cultures. Epidermophyton is distributed worldwide.

Microsporum canis Macroconidia from M. canis are spindle shaped, with echinulate, thick walls; they measure 12 to 25 µm × 35 to 110 µm and have 3 to 15 cells (Fig. 27.14). The tapering, sometimes elongated, spiny distal ends of the macroconidia are key features

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Fig. 27.14 Microsporum canis showing spindle-shaped, echinulate macroconidia with thick walls and tapered ends, which are key features in identication of this species (unstained, ×450).

Fig. 27.16 Trichophyton mentagrophytes showing globose, teardrop-shaped microconidia (Nomarski optics, ×450).

Microsporum audouinii A slow-growing anthropomorphic dermatophyte, Microsporum audouinii was responsible for most of the gray patch tinea capitis of children until a few decades ago, when T. tonsurans replaced it as the leading cause of scalp infection. In culture, conidia are only rarely produced. Some isolates form chlamydoconidium-like swellings terminally on hyphae. Colonies of M. audouinii appear cottony white and generally form little or no pigment on the reverse.

Trichophyton mentagrophytes

Fig. 27.15 Nannizzia gypsea (formerly Microsporum gypseum) showing fusiform, moderately thick-walled macroconidia containing several cells (lactophenol cotton blue preparation, ×450).

that distinguish this species. Microconidia are abundantly formed by most isolates, and these may be the only conidia maintained in cultures that have been serially transferred. Colonies are uffy and white, with the reverse side of the colony usually developing a lemon yellow pigment, especially on potato dextrose agar. This fungus has a worldwide distribution.

The microconidia (Fig. 27.16) of Trichophyton mentagrophytes are primarily globose but may appear tear shaped and measure 2.5 to 4 µm in diameter. Microconidia are found primarily in clusters described as grapelike (“en grappe”). Macroconidia are thin walled, smooth, and cigar shaped, with four to ve cells separated by parallel cross-walls. These conidia measure about 7 µm × 20 to 50 µm and are produced singly on undifferentiated hyphae. Colony morphology varies with the extent of conidia production. Granular colonies are noted when abundant microconidia are formed. In the downy form, conidia are less abundant. Compared with other dermatophytes, T. mentagrophytes is a relatively rapidly growing fungus. This species is distributed worldwide.

Trichophyton rubrum Nannizzia gypsea (formerly Microsporum gypseum) The fusiform, moderately thick-walled conidia (Fig. 27.15) typical of Nannizzia gypsea measure 8 to 15 µm × 25 to 60 µm and can have as many as six cells. In some isolates, the distal end of the macroconidium might bear a thin, lamentous tail. Abundant macroconidia and microconidia produced by most isolates of this species result in a powdery, granular appearance on colony surfaces. Fresh isolates typically form tan to buff conidial masses, but this species tends to develop pleomorphic tufts of white sterile hyphae in aging cultures and after serial transfers. Abundant brown-to-red pigment can form beneath some strains, but others remain colorless. N. gypsea is a rapidly growing geophilic species found in soils worldwide.

Although Trichophyton rubrum is known to produce three- to eight-celled cylindric macroconidia measuring somewhat smaller than those of T. mentagrophytes, these are seldom seen in clinical isolates. Typical microscopic appearance of T. rubrum reveals clavate- or peg-shaped microconidia (Fig. 27.17) formed along undifferentiated hyphae, and even these may be sparse. Colonies usually remain white on the surface but may be yellow to red. Most strains develop a red to deep burgundy wine–colored pigment on the reverse that diffuses into the agar. This species has a worldwide distribution.

Trichophyton tonsurans Trichophyton tonsurans possesses microconidia that are extremely variable in shape, ranging from a round shape to a peg shape. When grown on Sabouraud dextrose agar,

Clinically signicant species

607

Table 27.2 Microscopic morphology of fungi causing chromoblastomycosis

Fig. 27.17 Trichophyton rubrum showing clavate- or peg-shaped microconidia (lactophenol cotton blue preparation, ×450).

colonies usually form a rust-colored pigment on the colony’s reverse. T. tonsurans, which infects skin, hair, and nails, has become the leading cause of tinea capitis in children in many parts of the world, including the United States.

Organism

Microscopic morphology

Phialophora verrucosa

Conidiogenous cells, phaeoid, ask-shaped phialides, with collarettes Conidia oval, one celled, occur in balls at tips of phialides

Fonsecaea pedrosoi

Primary one-celled conidia formed on sympodial conidiophores Primary conidia function as conidiogenous cells to form secondary one-celled conidia Some conidia are similar to those seen in Cladophialophora spp., some are similar to those in Rhinocladiella spp., and some are similar to those in Phialophora spp.

Fonsecaea compactum

Considered conspecic with F. pedrosoi. Similar to F. pedrosoi but with more compact conidial heads Conidia are subglobose rather than ovoid

Cladophialophora carrionii

Erect conidiophores bearing branched chains of one-celled, brown blastoconidia Conidium close to tip of conidiophore, termed shield cell Fragile chains

Rhinocladiella aquaspersa

Conidiophores erect, dark, bearing conidia only on upper portion near the tip Conidia elliptic, one celled, produced sympodially

Agents of subcutaneous mycoses Subcutaneous mycoses are fungal diseases that affect subcutaneous tissue. These mycoses are usually the result of traumatic implantation of foreign objects into the deep layers of skin, permitting the fungus to gain entry into the host. The causative agents responsible are organisms commonly found in soil or on decaying vegetation; thus agricultural workers are most often affected. Organisms causing subcutaneous mycoses belong to a variety of genera in the form class Hyphomycetes. Although some are moniliaceous (hyaline or light colored), many are phaeoid, producing darkly pigmented colonies and containing melanin in their cell walls. The infections are commonly chronic and usually incite the development of lesions at the site of trauma. Subcutaneous fungal infections may be grouped together by the disease processes they cause or by the causative agents involved.

Chromoblastomycosis Also known as verrucous dermatitis and chromomycosis, chromoblastomycosis occurs worldwide but is most common in tropical and subtropical regions of the Americas and Africa. In the United States, most cases occur in Texas and Louisiana. Several organisms are responsible for the disease, and certain organisms appear to reside in specic endemic areas throughout the world. Chromoblastomycosis is caused by several infectious agents, namely, Fonsecaea pedrosoi, Phialophora verrucosa, Cladophialophora carrionii, and Rhinocladiella aquaspersa

Clinical manifestations Chromoblastomycosis, one of the more common subcutaneous mycoses, is a chronic infection of skin and subcutaneous tissue and develops over a period of months or, more commonly, years. It is mostly asymptomatic in the absence of secondary complications, such as bacterial infections, carcinomatous degeneration, and elephantiasis. Lesions are usually conned to the extremities, often the feet and lower legs, and are a result of trauma to these areas. Lesions of

chromoblastomycosis frequently appear as verrucous nodules that may become ulcerated and crusted. Longstanding lesions have a cauliower-like surface. Brown, round sclerotic bodies, which are nonbudding structures occurring singly or in clusters, are seen in tissues. These sclerotic bodies reproduce by dividing in various planes, resulting in multicellular forms. Occasionally, short hyphal elements are also seen. The presence of sclerotic bodies is diagnostic for this disease. Males are predominately infected, possibly indicating a role of sex hormones in disease development. Fonsecaea, Cladophialophora carrionii, and Phialophora verrucosa cause most cases of chromoblastomycosis.

Laboratory diagnosis The microscopic morphology of each of the agents is described in Table 27.2. P. verrucosa and C. carrionii are shown in Fig. 27.18. Isolates are identied on the basis of characteristic structures, such as arrangement of conidia and the manner in which conidia are borne. Organisms causing chromoblastomycosis are darkly pigmented. Growth is moderate to slow, and colonies are velvety to woolly and gray-brown to olivaceous black. Species are not differentiated by colony morphologies because they all produce similar characteristics. Molecular approaches, such as ribosomal gene sequencing, provide accurate identication to the species level.

Eumycotic mycetomas Mycetoma is a chronic, granulomatous infection of the subcutaneous and cutaneous tissues that arises at the site of inoculation. The disease is characterized by swelling, with characteristic exudate draining to the skin surface through

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A

B

Fig. 27.18 A, Conidia of Phialophora verrucosa at the tips of phialides with collarettes (Nomarski optics, ×1000). B, Conidial arrangement of Cladophialophora carrionii (lactophenol cotton blue preparation, ×1250). (Courtesy Dr. Michael McGinnis.)

Table 27.3 Description of granules seen in eumycotic mycetomas Fungus

Color

Size (mm)

Texture

Scedosporium boydii

White

0.5–1.0

Soft

Fusarium falciforme

White

0.2–0.5

Soft

Madurella mycetomatis

Black

0.5–5.0

Hard

Trematosphaeria (Madurella) grisea

Black

0.3–0.6

Soft

Exophiala spp.

Black

0.2–0.3

Soft

sinus tracts. Mycetomas can be caused by fungi or bacteria. Those caused by bacteria are referred to as actinomycotic mycetomas, and those caused by fungal agents are referred to as eumycotic mycetomas. Although clinical manifestations are similar for both types, the cause must be determined because the therapy for each is different. Mycetomas occur primarily in tropical and subtropical areas but are also seen in temperate zones. The disease is endemic in India, Africa, and South America. Although mycetoma is an uncommon mycosis in the United States, the following species (in order of occurrence) are the most commonly incriminated agents: Scedosporium boydii, Fusarium falciforme, Madurella mycetomatis, Trematosphaeria grisea (formerly Madurella grisea), and Exophiala spp. Direct microscopic examination of the granules collected from the draining lesion immediately differentiates eumycotic from actinomycotic mycetomas (Table 27.3). Fig. 27.19A shows the branching lamentous rods of actinomycetes in contrast with the hyphal elements seen in eumycotic infections (see Fig. 27.19B).

Scedosporium boydii Previously, it was thought that Scedosporium apiospermum was the anamorphic state of S. boydii, but molecular characterization has revealed that S. boydii and S. apiospermum are different species. Pseudoallescheria boydii, the teleomorph, and the anamorph, S boydii, are the same species. Similarly, P. apiosperma (teleomorph) and its anamorph S. apiospermum are the same species. S boydii produces oval conidia singly at the tips of conidiogenous cells (cells that make conidia) known as annellides (Fig. 27.20). The teleomorph form is noted by the

formation of cleistothecia containing ascospores (Fig. 27.21). This phenomenon occurs in fungi that are homothallic (ability of a single organism to undergo sexual reproduction without a mate). This isolate grows rapidly and produces colonies that are white to dark gray on potato dextrose agar at 22° and 35° C. Opinions regarding this fungus differ in relation to it being hyaline or phaeoid.

Fusarium falciforme Fusarium falciforme produces mucoid clusters of single- or two-celled, slightly curved conidia borne from phialides at the tips of long, unbranched, multiseptate conidiophores. Conidia are held together in mucoid clusters at the apices of the phialides. This isolate is a hyaline, septate, lamentous mold. Colonies grow slowly and are grayish brown, becoming grayish violet.

Madurella Madurella mycetomatis causes most cases of eumycotic mycetoma. Madurella spp. are phaeoid, septate fungi. Approximately 50% of the isolates of M. mycetomatis produce conidia from the tips of phialides, but many remain sterile. This species grows very slowly but is initially white, and becomes yellow, olivaceous, or brown, with a characteristic diffusible brown pigment with age. It grows best at 37° C, with slower growth at 40° C. DNA analysis led to the description of a new species, Trematosphaeria grisea, formerly named M. grisea

Subcutaneous phaeohyphomycosis Phaeohyphomycosis is a mycotic disease caused by darkly pigmented (phaeoid or dematiaceous) fungi. The term phaeohyphomycosis was coined to distinguish several clinical infections caused by phaeoid fungi from those distinct clinical entities known as chromoblastomycosis. In tissue, these fungi may form yeastlike cells that are solitary or in short chains or hyphae that are septate, branched, or unbranched and often swollen to toruloid (irregular or beaded). The agents responsible for these mycoses are organisms commonly found in nature, encompassing many genera of Hyphomycetes, Coelomycetes, and Ascomycetes. Fungi that appear to be regularly associated with this condition include Exophiala spp., such as Exophiala dermatitidis. A more complete list of genera

Clinically signicant species

A

609

B

Fig. 27.19 Actinomycotic mycetoma showing ne-branching, lamentous rods in tissue sample (A), compared with the hyphal elements (hematoxylin and eosin stain ×1000) (B) seen in eumycotic infections (Grocott-Gomori methenamine silver stain, ×1000).

BOX 27.1 Phaeoid genera inciting subcutaneous

phaeohyphomycosisa Alternaria

Mycocentrospora

Bipolaris

Ochroconis/Verruconis

Chaetomium

Oidiodendron

Cladosporium

Phaeosclera

Curvularia

Phialophora

Dactylaria

Phoma

Exophiala

Xylohypha

Fonsecaea Fig. 27.20 Scedosporium boydii (Nomarski optics, ×625).

Fig. 27.21 Sexual structures (cleistothecia containing ascospores) of Scedosporium boydii (teleomorph Pseudallescheria boydii) (Nomarski optics, ×325).

associated with subcutaneous phaeohyphomycosis is given in Box 27.1 With most Exophiala spp., conidia are borne from annellides, with conidia aggregating in masses at the tips of the conidiophore, as seen in Fig. 27.22. E. dermatitidis, however, forms conidia at the tips of phialides (Fig. 27.23). This group of fungi produces olivaceous to black colonies that are initially yeastlike but become velvety at maturity.

a

This list is not all-inclusive.

Fig. 27.22 Conidia of Exophiala sp. borne at the tips of annellides (Nomarski optics, ×1250).

Sporothrix schenckii species complex Clinical manifestations The most commonly seen presentation of Sporothrix schenckii species complex infection is lymphocutaneous sporotrichosis. This chronic infection is characterized by nodular and ulcerative lesions along the lymph channels that drain the primary site of inoculation. Less commonly seen disease states include

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Medically signicant fungi

xed cutaneous sporotrichosis, in which the infection is conned to the site of inoculation and mucocutaneous sporotrichosis, a relatively rare condition. Primary and secondary pulmonary sporotrichosis and extracutaneous and disseminated forms of the disease may also occur. Members of this complex, which include Sporothrix schenckii, S. mexicana, S. globosa, S. luriei, S. albicans, S. inata, and S. brasiliensis, are commonly recovered from soil and are associated with decaying vegetation. Some are distributed worldwide, especially in warm, arid areas, such as Mexico, and also in moist, humid regions, such as Brazil, Uruguay, and South Africa. In temperate countries, such as France, Canada, and the United States, most cases of sporotrichosis are associated with gardening, particularly with exposure to rose thorns (rose handler’s disease) and sphagnum moss.

Laboratory diagnosis Direct examination of tissue might reveal members of the S. schenckii species complex as small, cigar-shaped yeasts (Fig. 27.24A). Although the organism is occasionally seen in a Gramstained smear, wet mounts of material are often unrewarding because of the small numbers of organisms present. Microscopic examination from culture reveals thin, delicate hyphae bearing conidia developing in a rosette pattern at the ends of delicate

conidiophores. Dark-walled conidia are also produced along the sides of the hyphae and may be more readily viewable than the rosettes in mature cultures (see Fig. 27.24B). Because members of this complex are dimorphic, cultures are examined at 22° and 37° C. These fungi grow well on most culture media, including those containing cycloheximide. The colony morphology at 22° C can be variable. At this temperature, colonies are often initially white, glabrous, and yeastlike, turning darker and more mycelial as they mature. Demonstration of dimorphism is important for the identication of these species. To induce mycelia conversion to yeast, the fungus is inoculated onto brain-heart infusion (BHI) agar supplemented with sheep red blood cells and incubated at 37° C in a CO 2 incubator. The formation of yeast colonies may require several subcultures. Complete conversion seldom occurs, but a portion of the colony will develop yeastlike cells.

Agents of systemic mycoses Organisms that cause classic systemic fungal diseases have historically been categorized together because they share several characteristics, such as mode of transmission, dimorphism, and systemic dissemination. Although the term systemic generally refers to the organisms described here, it must be understood that any fungus, in an immunocompromised host, has the potential to become invasive and disseminate to sites far removed from the portal of entry. The morphology of the systemic dimorphic at 22°C is described in Table 27.4. Conversion to the yeast or spherule form occurs when it is incubated at 35° to 37°C on enriched media with increased concentration of CO2 (Table 27.5). Each agent has a fairly well-dened endemic area. The diseases are contracted generally by the inhalation of infectious conidia. Table 27.6 summarizes systemic mycoses, their agents, and their characteristics. All laboratory procedures to recover and identify these agents must be performed in a biological safety cabinet.

Blastomyces Clinical manifestations Fig. 27.23 Conidia of Exophiala dermatitidis borne at the tips of phialides as well as the black yeast synanamorph (Nomarski optics, ×1250).

A

Blastomycosis is most prevalent in middle-age men, as are other systemic mycoses, presumably because occupational and recreational exposure to soil is often greater among men.

B

Fig. 27.24 A, Yeast phase of Sporothrix schenckii showing cigar-shaped yeast cells typical of the species (Nomarski optics, ×625). B, Mold phase of S. schenckii revealing hyaline conidia borne at the ends of conidiophore in “rosettes” as well as phaeoid conidia along the sides of the hyphae (Nomarski optics,×625).

Clinically signicant species

611

Table 27.4 Morphology of systemic fungia Fungus

Macroscopic morphology

Microscopic morphology

Blastomyces dermatitidis, B. gilchristii

Slow to moderate growth White to dark tan Young colonies tenacious, older colonies glabrous to woolly

Oval, pyriform to globose smooth conidia borne on short, lateral hyphalike conidiophores

Histoplasma capsulatum, H. mississippiense, and H. ohiense

Slow growth White to dark tan with age Woolly, cottony, or granular

Microconidia small, one-celled, round, smooth (2–5 µm) Tuberculated macroconidia large, round (7–12 µm) Hyphalike conidiophores

Coccidioides immitis, Coccidioides posadasii

Rapid growth White to tan to dark gray Young colonies tenacious, older colonies cottony Tend to grow in concentric rings

Alternating one-celled, “barrel-shaped” arthroconidia with disjunctor cells

Paracoccidioides spp.

Slow growth White to beige Colony glabrous, leathery, at to wrinkled, folded or velvety

Colonies frequently only produce sterile hyphae Fresh isolates may produce conidia similar to those of B. dermatitidis

a

At 22° C.

Table 27.5 Mold to yeast conversion of thermally dimorphic fungia Fungus

Culture media and temperature

Yeast form

Blastomyces dermatitidis, B. gilchristii

Blood agar, 37° C

Large yeast (8–12 µm) Blastoconidia attached by broad base

Histoplasma capsulatum, H. mississippiense, and H. ohiense

Pines medium, glucose-cysteine-blood, or BHI agar–blood agar, 37° C

Small, oval yeast (2–5 µm)

Paracoccidioides spp.

BHI agar–blood agar, 37° C

Multiple blastoconidia budding from single, large yeast (15–30 µm)

BHI, Brain-heart infusion. Coccidioides immitis and C. posadasii can be converted to the spherule phase in modied Converse medium at 40° C in 5% to 10% carbon dioxide. Molecular assays are preferred to this procedure in the routine clinical laboratory. a

Table 27.6 Summary of systemic mycoses Fungus

Ecology

Clinical disease

Tissue form

Blastomyces dermatitidis, B. gilchristii

Mississippi and Ohio River valleys

Primary lung Chronic skin, bone Systemic, multiorgan

Large yeast (8–12 µm) Broad-based bud

Histoplasma capsulatum, H. mississippiense, and H. ohiensea

Ohio, Missouri, and Mississippi River valleys Bird and bat guano Alkaline soil

Primary lung Asymptomatic Immunodecient hosts prone to disseminated disease

Small, oval yeast (2–5 µm) in histiocytes, phagocytes

Coccidioides immitis, C. posadasii

Semi-arid regions—southwest United States, Mexico, Central and South America In soil

Primary lung Asymptomatic Secondary cavitary Progressive pulmonary Multisystem

Spherules (30–60 µm) containing endospores

Paracoccidioides spp.

Central and South America In soil

Primary lung Granulomatous Ulcerative nasal and buccal lesions Lymph node involvement Adrenal glands

Thick-walled yeasts (15–30 µm) Multiple buds, “mariner’s wheel”

a

Histoplasma duboisii is endemic in Central Africa, and H. suramericanum is found in South America.

612

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Although patients with primary infection may exhibit ulike symptoms, most are asymptomatic and cannot accurately dene the time of onset. When the primary disease fails to resolve, pulmonary disease may ensue, with cough, weight loss, chest pain, and fever. Progressive pulmonary or invasive disease may follow, resulting in ulcerative lesions of skin and bone. In the immunocompromised patient, multiple organ systems may be involved, and the course may be rapidly fatal. Blastomycosis is also known as Gilchrist disease, North American blastomycosis, and Chicago disease. It occurs primarily in North America and parts of Africa. In the United States, B. dermatitidis is endemic in the Mississippi and Ohio river basins, while the recently described B. helicus is found primarily in the west. Sporadic point source outbreaks have also occurred in the St. Lawrence River basin. The natural reservoir has not been unequivocally established, although the organism has been recovered from soil and from some natural environments. Apparently, only a very narrow range of conditions supports its growth. In areas in which the organism appears endemic, natural disease occurs in dogs and horses, with the disease process mimicking that seen in human infections. Several species of Blastomyces are now recognized, including B. dermatitidis, B. gilchristii, and B. helicus, among others. Morphologically, B. dermatitidis and B. gilchristii are indistinguishable. B. gilchristii can be identied by using the sequences of the internal transcribed spacer 2 of the nuclear ribosomal ribonucleic acid (RNA) gene. The teleomorph or sexual form of B. dermatitidis is named Ajellomyces dermatitidis. It occurs only in rigidly controlled environments by mating of isolates with tester strains to produce gymnothecia containing ascospores. This teleomorph does not occur in the routine laboratory because the species is heterothallic, requiring two mating strains to produce the sexual form.

range in diameter from 2 to 10µm. Because they resemble a variety of other fungi, the microconidia are not diagnostic (Fig. 27.27). An enzyme immunoassay (EIA) performed on serum and blood has a sensitivity of about 89%, but the specicity is low (79%). In culture at 22° C, the organism can produce a variety of colony morphologies—white, tan, or brown—and may be uffy to glabrous. Frequently, raised areas, termed spicules, are seen in the centers of the colonies. When grown at 37° C on suitable media, B. dermatitidis and B. gilchristii produce characteristic, broad-based, budding yeast cells. The mycelial phase of the systemic dimorphic fungi—B. dermatitidis, B. gilchristii, Cocccidioides immitis, Cocccidioides posadasii, Histoplasma spp., and Paracoccidioides spp.—requires conrmatory identication, typically by DNA probe and DNA sequencing. Because of low sensitivity and specicity, antigen detection methods are generally not used.

Coccidioides species Clinical manifestations Two very similar species that infect humans are C. immitis and C. posadasii. The inhalation of only a few arthroconidia produces primary coccidioidomycosis. Clinical infections

Laboratory diagnosis For B. dermatitidis and B. gilchristii, examination of tissue or purulent material in cutaneous skin lesions may reveal large, spherical, refractile yeast cells, 8 to 15µm in diameter, with a double-contoured wall and buds connected by a broad base (Fig. 27.25). KOH (10%) or calcouor white (a uorescent dye) may be used to enhance detection of the yeast cells (Fig. 27.26). In the mold phase, conidia are borne on short lateral branches that are ovoid to dumbbell-shaped and

Fig. 27.25 Conversion of the mold phase of Blastomyces dermatitidis to the broadbased bud yeast form (Nomarski optics, ×1250).

Fig. 27.26 Expectorated sputum, smear, calcouor white stain, uorescence microscopy. Yeast (8 to 20 µm), round, thick-walled, broad-based bud. Morphology suggests Blastomyces dermatitidis (×400).

Fig. 27.27 Mold phase of Blastomyces dermatitidis grown on potato akes agar (Nomarski optics, ×1250).

Clinically signicant species

include asymptomatic pulmonary disease and allergic manifestations. Allergy can manifest itself as toxic erythema, erythema nodosum (desert bumps), erythema multiforme (valley fever), and arthritis (desert rheumatism). Primary disease usually resolves without therapy and confers a strong, specic immunity to reinfection. In symptomatic patients, fever, respiratory distress, cough, anorexia, headache, malaise, and myalgia can be present for 6 weeks or longer. The disease might then progress to secondary coccidioidomycosis, which can include nodules, cavitary lung disease, and/or progressive pulmonary disease. Fewer than 3% of infected individuals develop systemic disease. Filipinos and Blacks run the highest risk of dissemination, with meningeal involvement being a common result of disseminated disease. The sex distribution ratio for clinically apparent disease has been reported to favor males. The exception is in pregnant women, in whom the dissemination rate equals or exceeds that for men. Coccidioides spp. reside in a narrow ecologic niche known as the Lower Sonoran life zone, which is characterized by low rainfall and semi-arid conditions. Highly endemic areas include the San Joaquin Valley of California, the Maricopa and Pima counties of Arizona, and southwestern Texas. Outside the United States, endemic areas are found in northern Mexico, Guatemala, Honduras, Venezuela, Paraguay, Argentina, and Columbia. Because they are morphologically identical, molecular evaluation is required to differentiate C. immitis from C. posadasii. It appears that the species can be traced to specic geographic locations. C. immitis is encountered in the San Joaquin Valley region of California, whereas C. posadasii is found in the desert areas of the southwest United States, Mexico, and South America.

Laboratory diagnosis After inhalation, the barrel-shaped arthroconidia, which measure 2.5 to 4 µm × 3 to 6 µm, round up as they convert to spherules. At maturity, the spherules (30 to 60 µm) produce endospores by a process known as progressive cleavage; rupture of the spherule wall releases the endospores into the bloodstream and surrounding tissues. These endospores, in turn, form new spherules (Fig. 27.28). Direct smear examination of secretions may reveal the spherules containing the endospores. Caution must be exercised when diagnosis is made by histopathologic means only. Small, empty spherules may resemble the yeast cells of B. dermatitidis, and the endospores

Fig. 27.28 Spherules of Coccidioides immitis in tissue (×300).

613

can be confused with the cells of Cryptococcus, Histoplasma, and Paracoccidioides. Direct antigen detection methods are limited. Microscopic examination of the culture shows fertile hyphae arising at right angles to the vegetative hyphae, producing alternating (separated by a disjunctor cell) hyaline arthroconidia (Fig. 27.29). When released, conidia have an annular frill at both ends. As the culture ages, the vegetative hyphae also fragment into arthroconidia. Although Coccidioides spp. do not readily convert to the spherule stage at 37° C in the laboratory, they produce a variety of mold morphologies at 22° C. Initial growth, which occurs within 3 to 4 days, is white to gray, moist, and glabrous. Colonies rapidly develop abundant aerial mycelia, and the colony appears to enlarge in a circular bloom. Mature colonies usually turn tan to brown to lavender in color.

Histoplasma Clinical manifestations Histoplasmosis is acquired by inhalation of the microconidia of Histoplasma species. The microconidia are phagocytized by macrophages in the pulmonary parenchyma. In the host with intact immune defenses, the infection is limited and is usually asymptomatic, with the only sequelae being areas of calcication in the lungs, liver, and spleen. With heavy exposure, however, acute pulmonary disease can occur. In the mild form of the disease, viable organisms remain in the host, quiescent for years, and are the presumed source of reactivation in individuals with abrogated immune systems. In immunocompromised individuals, Histoplasma can cause a progressive and potentially fatal disseminated disease. Chronic pulmonary histoplasmosis in patients with chronic obstructive pulmonary disease can also occur. Other various manifestations of the disease include mediastinitis, pericarditis, and mucocutaneous lesions. Different Histoplasma spp. cause histoplasmosis, also known as reticuloendothelial cytomycosis, Cave disease, Spelunker disease, and Darling disease. Histoplasmosis occurs worldwide. The highest endemicity in the United States is found in the Ohio, Missouri, and Mississippi River deltas. Species include H. capsulatum, H. mississippiense, and H. ohiense, while H. suramericanum is found in South America. These organisms reside in soil with a high nitrogen content, particularly in areas

Fig. 27.29 Mold phase of Coccidioides immitis, 25° C (Nomarski optics,×1250).

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Medically signicant fungi

A

B

C Fig. 27.30 A, Bone marrow aspirate stained with Giemsa stain showing the yeast cells of Histoplasma capsulatum inside the monocytes (×1200). B, Tissue phase of H. capsulatum (Gomori methylene stain, ×1200). C, Bronchoalveolar lavage, cytocentrifuge preparation, calcouor white stain, uorescence microscopy. Fluorescent yeast (2 to 4 µm), small, budding. Morphology suggests Histoplasma capsulatum (×1000).

heavily contaminated with bat and bird guano. Skin testing previously demonstrated that about 80% of the population of long-term residents in endemic areas has been infected. H. duboisii, endemic in Central Africa, causes a clinically distinct form of disease involving primarily skin and bones, whereas H. capsulatum var. farciminosum causes epizootic lymphangitis in horses and mules.

Laboratory diagnosis Careful examination of direct smear preparations of specimens for histoplasmosis frequently reveals the small yeast cells of H. capsulatum and similar Histoplasma species, particularly in infections seen in immunodecient hosts. The yeast cells measure 2 to 3 µm × 4 to 5 µm. When smears are stained with Giemsa or Wright stain, the yeast cells are commonly seen within monocytes and macrophages, occurring in significant numbers, as shown in Fig. 27.30A. The small cells, when found in tissue (see Fig. 27.30B), resemble the blastoconidia of Candida glabrata, but they can be differentiated by uorescent antibody techniques or culture (see Fig. 27.30C). Although microconidia can resemble Chrysosporium spp. and the macroconidia resemble Sepedonium spp., both saprobes do not produce two types of conidia in a single culture, nor are they dimorphic. Conversion of the mold form to the yeast form, using BHI agar incubated at 37° C, is conrmatory for H. capsulatum. Although complete conversion is seldom noted, a combination of both mycelial and yeast forms is sufcient for identication. H. capsulatum grows as a white-tobrownish mold. Early growth of the mycelial culture produces

Fig. 27.31 Large tuberculate macroconidia of Histoplasma capsulatum (Nomarski optics, ×1250).

round to pyriform microconidia measuring 2 to 5 µm. As the colony matures, large echinulate or tuberculate macroconidia characteristic for the species are formed (Fig. 27.31). Direct antigen detection and serologic procedures for the diagnosis of histoplasmosis might be adjuncts to culture methods. Using EIA methods, H. capsulatum antigen can be detected from serum, CSF, and urine with a sensitivity of about 95%. Other assays include complement xation, immunodiffusion, and latex agglutination to detect circulating antibody, along with uorescent antibody test to detect viable or nonviable fungal elements in tissue sections. Currently,

Clinically signicant species

615

the most useful test for rapid diagnosis of histoplasmosis is the combination of a DNA hybridization system and DNA sequencing. However, the cost of maintaining equipment for molecular procedures remains a challenge for regions with limited resources.

indistinguishable from those observed with the mold phase of B. dermatitidis or the microconidia of Histoplasma. On BHIblood agar, at 37° C, the mycelial phase rapidly converts to the yeast phase.

Paracoccidioides

Talaromyces marneffei

Clinical manifestations

Clinical manifestations

Although the primary route of infection for Paracoccidioides spp., which includes P. brasiliensis, P. lutzii, P. americana, P. restrepiensis, and P. venezuelensis, is pulmonary and infection is usually unapparent and asymptomatic, subsequent dissemination leads to formation of ulcerative granulomatous lesions of the buccal, nasal, and occasionally gastrointestinal mucosa. A concomitant striking lymph node involvement is also evident. Although Paracoccidioides spp. have a rather narrow range of temperature tolerance, as evidenced by its predilection for growth in cooler areas of the body (nasal and oropharyngeal), dissemination to other organs, particularly the adrenal glands, occurs with compromised host defenses. Paracoccidoides is the causative agent of paracoccidioidomycosis (South American blastomycosis, Brazilian blastomycosis, Lutz-SplendoreAlmeida disease, paracoccidioidal granuloma), a chronic, progressive fungal disease endemic to Central and South America. Geographic areas of highest incidence are typically humid, high-rainfall areas, with acidic soil conditions.

Talaromyces (formerly Penicillium) marneffei is unique among the Talaromyces spp., being dimorphic. T. marneffei is a common cause of systemic infection in immunocompromised patients who have visited the endemic region of Southeast Asia. This includes patients with AIDS, hematologic malignancies, or autoimmune disease and patients undergoing organ transplantations. Infections are usually disseminated, with multiple organ involvement. The fungus can be isolated from cutaneous lesions, which are frequently present in infected individuals. Disseminated disease is typically fatal. Studies showed that the fatality rate was about 28% among patients who did not have AIDS and received appropriate antifungal therapy.

Laboratory diagnosis Direct microscopic examination of cutaneous and mucosal lesions demonstrates the characteristic yeast cells. The typical budding yeast measures 15 to 30 µm in diameter with multipolar budding at the periphery, resembling a mariner’s wheel (Fig. 27.32). These daughter cells (2 to 5 µm) are connected by a narrow base, unlike the broad-based attachment in Blastomyces dermatitidis. Many buds of various sizes can occur, or there may be only a few buds, giving the appearance of a so-called Mickey Mouse cap to the yeast cell. Paracoccidoides produces a variety of mold morphologies when grown at 22° C. Flat colonies are glabrous to leathery, wrinkled to folded, occose to velvety, and pink to beige to brown with a yellowish-brown reverse, resembling those of B. dermatitidis. Microscopically, the mold form produces small (2 to 10 µm in diameter), one-celled conidia, generally

Fig. 27.32 Yeast phase (“mariner’s wheel”) of Paracoccidioides brasiliensis with multipolar budding (Nomarski optics, ×1250).

Laboratory diagnosis The yeastlike cells of T. marneffei can be detected in Wrightstained smears from skin lesions or biopsy specimens. The cells of T. marneffei resemble those of Histoplasma. They are oval to cylindric, measuring 3 to 6 µm long, and they may have a cross-wall. The mold form has sparse green aerial and reddish-brown vegetative hyphae and produces a red diffusible pigment. Polymerase chain reaction (PCR) tests have been described for identication conrmation. Serologic assays have been shown to be important in early diagnosis; however, they are not commercially available.

Agents of opportunistic mycoses The terms saprobe and saprophyte have been used to describe free-living microorganisms that are present in the environment but are not typically of concern with regard to human disease. The line between saprobic and pathogenic organisms is increasingly blurred. The major reason for this development is the growing number of persons with defects in their immune system. For several decades, medical science has made advances in life-sustaining and life-lengthening treatments. As a result, a serious side effect of procedures such as organ transplantation and cancer chemotherapy is the short- or long-term insult to the host defenses. The spread of AIDS has greatly magnied the problem. Persons with AIDS constitute the prime targets for infection by a wide variety of microorganisms, including the recognized pathogenic fungi and a growing list of fungi previously regarded as harmless. The types of disease caused by these fungi are as varied as the species, sometimes more so, because a given fungus can have multiple clinical presentations. Surgical wounds are ideal points of inoculation, allowing saprobes to become opportunistic agents of disease. Skin and nail bed infections as well as severe respiratory infections can be caused by a variety of fungi in patients with AIDS. A discussion of the most common saprobes that have been associated with opportunistic infections follows. These fungi are found worldwide in the environment and are often associated with decaying vegetation.

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Medically signicant fungi

Fig. 27.33 Cunninghamella sp. (lactophenol cotton blue, ×450).

Fig. 27.34 Lichtheimia sp. (Nomarski optics, ×450).

Mucorales Members of the order Mucorales are common environmental isolates associated with soil and plants. They contaminate grains, breads, and fruits and are most often associated with infections of the sinuses, lungs, and skin of immunocompromised patients. Studies have indicated an increased incidence of mucormycoses. Diabetes is a signicant risk factor for these infections.

Cunninghamella Cunninghamella spp. can be recovered from the sinuses or other organs during disseminated disease. Found worldwide, this isolate is common in the environment. Sporangiophores are erect, branching into several vesicles that bear sporangioles (Fig. 27.33), and may be covered with long, ne spines. These organisms are rapidly growing and form a cottony colony that is initially white but becomes gray with age.

Lichtheimia Lichtheimia spp. have a predilection for vascular invasion, causing thrombosis and necrosis of the tissues. This agent, along with other fungi in the order Mucorales, is usually found in patients with poorly controlled diabetes. In this patient population, the infection usually begins in the sinuses, where conidia are inhaled and take up residence. From the sinuses, infection rapidly spreads to the orbits, face, palate, and brain. This presentation is known as rhinocerebral mucormycosis. Other sites of infection have been noted in patients with cancer, in whom cutaneous, subcutaneous, and systemic diseases occur. Lichtheimia spp. are found worldwide and are often associated with soil or decomposing organic matter. Lichtheimia hyphae are broad and ribbonlike, with few septations (Fig. 27.34). Erect sporangiophores, solitary or in groups (slightly branched), terminate in an apophysis surrounded by a sporangium. Sporangiospores are smooth and ovoid. Internodal rhizoids (short, thin projections that anchor the growing cells to substratum) are present. Colonies are woolly and grow rapidly, completely covering the culture medium. Colony color is initially white, becoming gray to gray-brown with age.

Mucor As with other Mucorales, Mucor spp. are asssociated with rhinocerebral mucormycosis in addition to disseminated disease. Mucor spp. are commonly isolated from the environment

Fig. 27.35 Mucor sp. (unstained, ×450).

worldwide. Sporangiospores are formed in sporangia on erect sporangiophores (Fig. 27.35). Rhizoids, typical of some Mucorales, are absent in most Mucor spp. The sporangia frequently remain intact, as opposed to Rhizopus spp., in which the sporangia typically collapse. Mucor spp. grow rapidly and form cottony, dirty white colonies that become mousy brown to gray with age.

Rhizopus Rhizopus spp. are the most common Mucorales causing human disease. These are typically involved in patients with poorly controlled diabetes, presenting as rhinocerebral mucormycosis. Rhizopus spp. may be refractory to treatment and may be recovered from almost any source. With worldwide distribution, this isolate is easily recovered from the environment in decaying vegetation. Rhizopus spp. are rapidly growing and have erect sporangiophores terminating in dark sporangia and sporangiospores (Fig. 27.36). At the base of the sporangiophores are brown rhizoids. Separate clusters of sporangiophores are joined by stolons, arching laments that terminate at the rhizoids. The sporangia are typically fragile and are not easily retained when making slide culture preparations, resulting in an umbrella-shaped structure at the end of the conidiophores. Rhizopus spp. produce rapidly growing, woolly colonies that cover the entire surface of the culture medium. Colonies are initially white but become gray to brown with age.

Clinically signicant species

617

Fig. 27.36 Rhizopus sp. (Nomarski optics, ×450). Fig. 27.38 Expectorated sputum, smear, Gram stain, light microscopy. Gramvariable hyphae present (3 to 10 µm), septate, branched 45-degree angle. Morphology suggests Aspergillus spp. (×1000).

Fig. 27.37 Syncephalastrum sp. (Nomarski optics, ×450).

Syncephalastrum Syncephalastrum is rarely implicated in human disease but has been documented in cutaneous infections. This fungus is found in soil and decaying vegetation. Microscopically, erect sporangiophores are noted. Each sporangiophore has a large columella on which merosporangia, containing stacks of sporangiospores, are formed (Fig. 27.37). Isolates are sometimes confused with Aspergillus on initial examination. Colonies are rapidly growing and are initially white and become gray with age. The growth rate is rapid, with colonies covering the entire surface of the agar.

Septate and hyaline saprobes Aspergillus Aspergillus spp. are ubiquitous environmental saprobes and can frequently be isolated from a number of hospital sites, including ventilation systems and food. Aspergilli are the second most commonly isolated fungus after Candida spp. A. fumigatus is the species most often seen; other commonly isolated pathogenic species include A. avus, A. terreus, and A. niger. Their conidia are constantly inhaled, but they are generally readily cleared in healthy, immunocompetent individuals. Mortality from infections caused by the aspergilli remains high, especially in the immunocompromised host. Aspergillus spp. are the most frequent cause of disease in bone marrow transplant recipients, in addition to other

Fig. 27.39 Expectorated sputum, smear, Gram stain, light microscopy. Grampositive conidia (2 to 4 µm), in chain. Morphotype suggests Aspergillus spp. in cavity with air interface. Impression: cavitary aspergillosis (×1000). Care must be taken not to mistake these conidia for streptococci or for yeasts (see Fig. 27.40).

transplant recipients and those with cancer. Infection is initiated following inhalation of conidia. In the lung air spaces, conidia germinate into hyphae, which invade the tissue, including blood vessels (Figs. 27.38, 27.39, and 27.40). Fungal elements are easily seen with the uorescent calcouor white stain (Fig. 27.41). Although not all patients have chest pain, it is not uncommon for pneumonia-like symptoms to appear. The infection easily spreads hematogenously, and it is not uncommon to nd multiorgan system involvement, including the brain, liver, heart, and bone (Fig. 27.42). Aspergillus spp. also trigger allergic reactions and are a common cause of sensitivities to molds. Another frequent presentation is that of so-called fungus balls in the lungs of agricultural workers who routinely are in contact with fungal conidia from environmental sources. Chronic pulmonary aspergillosis may occur in patients with structural damage to their lungs caused by other diseases, including tuberculosis and sarcoidosis. Aspergilli may be uniseriate or biseriate. Uniseriate species are those whose phialides attach directly to the vesicle at the end of the conidiophore. Biseriate species possess

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a supporting structure called a metula. Metulae attach directly to the vesicle, and attached to each of the metulae are phialides (Fig. 27.43). Conidia are produced from the phialides. Other characteristics include an erect conidiophore arising from a foot cell within the vegetative hyphae. It is also important to note whether or not phialoconidia remain in long chains or are easily disturbed into individual phialoconidia (see Figs. 27.39 and 27.40). Chains of conidia can be aligned in very straight, parallel columns or in a radiating pattern around the vesicle, and the conidia may be rough or smooth. The color of Aspergillus spp. colonies is derived from conidia. Colors range from black to white and include yellow, brown, green, gray, pink, beige, and tan. Some species also form diffusible subsurface pigments on a variety of media. A granular texture is seen in species with abundant conidial formation. Most known pathogens in this group form green- to tan-colored colonies. Many species of aspergilli have been implicated in human disease. Although some are easily distinguished from others, molecular and proteomic tests aid in denitive identication at the species level.

Beauveria Beauveria bassiana is a rare human isolate, uncommonly associated with keratitis. This fungus is a known insect pathogen and is found worldwide on vegetation and in soil. Abundant, single-celled, tear-shaped sympoduloconidia are formed on sympodulae, which taper extremely from a rather swollen base (Fig. 27.44). Conidiophores may cluster in some isolates to form radial tufts. Colonies are hyaline, moderately rapidly growing, uffy colonies, sometimes developing a powdery surface reminiscent of T. mentagrophytes

Fig. 27.42 Brain abscess, smear, toluidine blue stain, light microscopy. Fungal hyphae present (3 to 10 µm), septate, branched 45-degree angle. Morphology suggests Aspergillus spp. (×400). Impression: cerebral aspergillosis.

Fig. 27.40 Expectorated sputum, smear, calcouor white stain, uorescence microscopy, MPV. Fungal hyphae present (3 to 10 µm), septate, branched 45-degree angle. Morphology suggests Aspergillus spp. (×400).

Fig. 27.43 Aspergillus sp. (Nomarski optics, ×450).

Fig. 27.41 Expectorated sputum, smear, Gram stain, light microscopy. Gramnegative conidia (2 to 4 µm), sporulating (arrows) (×1000). Care must be taken not to confuse these conidia with yeast germ tubes.

Fig. 27.44 Beauveria sp. (Nomarski optics, ×450).

Clinically signicant species

Fig. 27.45 Chrysosporium sp. (Nomarski optics, ×450).

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Fig. 27.46 Fusarium sp. (unstained, ×450).

Chrysosporium A rare cause of disease, Chrysosporium spp. have been recovered from nails and skin lesions. Chrysosporium zonatum has been linked to pneumonia and osteomyelitis in immunocompromised patients. These organisms are found in the environment worldwide. Microscopically simple, wide-based, single-celled conidia are produced on nonspecialized cells (Fig. 27.45). The conidiogenous cell disintegrates or breaks to release conidia. Both arthroconidia and aleurioconidia may be seen. Colonies are hyaline with a moderate growth rate and with age can develop light shades of pink, gray, or tan pigment.

Fusarium Fusarium spp. are frequently seen in mycotic keratitis. In the United States, a multistate outbreak involving more than 100 individuals wearing soft contact lenses was reported in 2006. The outbreak was associated with a particular brand of contact lens solution and occurred typically in patients who wore lenses continuously without removal for several days at a time. From 2013 to 2014, seven cases of fungemia due to Fusarium oxysporum, related to central line catheters, were reported in a pediatric cancer center. In bone marrow transplant recipients with infections caused by fusaria, the mortality rate approaches 100%. Unless a patient regains some cell-mediated immunity, mortality is certain because of the ability of these fungi to grow and continue to invade despite antifungal therapy. Patients present with high fever, possibly disseminated skin lesions, and in some patients, fungemia. Fusarium spp. can be recovered in blood culture systems. Care should be taken when reviewing positive blood cultures because isolates typically appear yeastlike on initial recovery. Normally, abundant macroconidia with fewer microconidia are produced on vegetative hyphae (Fig. 27.46). Macroconidia are banana or canoe shaped and are formed singly, in small clusters, or clustered together in mats termed sporodochia. Macroconidia typically are multicelled. Fusarium is a rapidly growing hyaline fungus that can develop various colors with age, ranging from rose to mauve to purple to yellow.

Case check 27.1 Unlike most molds, Fusarium spp. are commonly recovered from blood. As demonstrated in the Case in Point, care must be taken for proper diagnosis because colonies may initially appear yeastlike on subculture but rapidly become woolly in appearance as hyphae are formed.

Fig. 27.47 Geotrichum sp. (Nomarski optics, ×650).

Geotrichum Geotrichum has been implicated in pulmonary disease in immunocompromised patients. Microscopic evaluation reveals abundant arthroconidia formed from vegetative hyphae that occur singly or may be branched (Fig. 27.47). Colonies appear white to cream and yeastlike and can be confused with Trichosporon spp. Occasionally, aerial mycelia form, producing colonies that resemble those of Coccidioides spp.

Purpureocillium Purpureocillium lilacinum, previously Paecilomyces lilacinus, has been associated with cutaneous and subcutaneous infections, in addition to pyelonephritis, endocarditis, and pulmonary infections in immunocompromised and immunocompetent patients. It was recovered in a hospital outbreak with a high rate of associated death. Microscopically, care must be taken to avoid confusion between Purpureocillium and Penicillium spp. Phialides of Purpureocillium are generally longer and more obviously tapered, and they may be singly formed or arranged in a verticillate pattern, on which long chains of spindle-shaped or somewhat cylindric conidia are formed (Fig. 27.48). Purpureocillium grows rapidly and usually form at, granular to velvety colonies in shades of tan, brownish gold, or mauve. Green or blue-green colors are not seen. Infections with Purpureocillium are potentially serious and difcult to treat, as P. lilacinum is intrinsically resistant to amphotericin B and other polyenes; care should be taken when encountering fungi that are mauve in color. Molecular testing is recommended to get a denitive species identication.

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Fig. 27.48 Purpureocillium lilacinum (Paecilomyces lilacinus) (Nomarski optics, ×650).

Fig. 27.50 Scopulariopsis sp. (lactophenol cotton blue, ×450).

Fig. 27.49 Penicillium sp. (Nomarski optics, ×650).

Fig. 27.51 Trichoderma sp. (Nomarski optics, ×650).

Penicillium Because many Penicillium spp. are inhibited at 37° C, they do not frequently cause infections. Most reports of disease involve chronic fungal sinusitis. Ubiquitous in nature, these fungi can be recovered from any location worldwide. Conidiophores are erect, sometimes branched, with metulae bearing one or several phialides on which oval to ovoid conidia are produced in long, loose chains (Fig. 27.49). This commonly seen fungus is a rapid grower, with colonies usually in shades of green or blue-green.

Scopulariopsis and Microascus Scopulariopsis and Microascus spp. are commonly isolated from nail specimens and have been implicated in pulmonary disease in immunocompromised patients. These fungi are recovered from the environment worldwide. Conidiophores occur singularly or can be in clusters (Fig. 27.50). Conidia are formed from annellides, which increase in length as conidia are formed. The truncate-based conidia tend to remain in chains on the annellides. Scopulariopsis and Microascus spp. grow moderately rapidly and form colonies covered by tanto-buff conidia. Some species are phaeoid.

Trichoderma Trichoderma spp. are emerging as pathogens that can cause a range of infections, including pulmonary and skin infections, in the immunocompromised host. This isolate is readily recovered from the environment worldwide. Trichoderma spp.

are rapidly growing and form hyaline hyphae that give rise to yellow-green to green patches of conidia formed on clusters of tapering phialides (Fig. 27.51). Conidia may remain clustered in balls at the phialide tips. Mature colonies are intensely green and granular, with an abundance of conidia.

Septate and phaeoid saprobes Alternaria

Based on phylogenetic studies and morphology, Alternaria, which consists of several saprophytic and pathogenic species, is currently grouped into 26 sections. Although Alternaria spp. can be recovered from almost any source, they are primarily implicated in chronic fungal sinusitis. The disease is often misdiagnosed, and patients are often treated for an extended period for bacterial sinusitis. The infection rarely spreads beyond the sinuses in immunocompetent hosts but can be found systemically in those with immune suppression. Alternaria spp. are found worldwide on grasses and leaves. They have been implicated in tomato rot and are readily recovered from the environment in air-settling plates. Microscopic evaluation reveals short conidiophores bearing conidia in chains that lengthen in an acropetal fashion (Fig. 27.52). Multicelled conidia have angular cross-walls and taper toward the distal end. Alternaria spp. are phaeoid, rapidly growing fungi with colonies ranging from shades of gray to brown to black.

Clinically signicant species

Aureobasidium Infections by Aureobasidium spp. are rare but have been traced to contaminated dialysis lines, catheters, and similar devices. This organism may be recovered from blood, tissues, and abscesses. It is recovered worldwide primarily in wet environments, such as from shower tiles and water lines. Microscopic examination reveals hyaline hyphae giving rise to hyaline conidia directly from the vegetative hyphae. With age, phaeoid hyphae develop and break up into arthroconidia, which do not bear hyaline conidia. These arthroconidia are responsible for the darkening colony morphology. Aureobasidium spp. grow moderately rapidly to signicantly rapidly and have a yeastlike consistency. Young cultures are off-white to pink, but they become black with age, with the production of darkly pigmented arthroconidia.

Chaetomium Infections by Chaetomium organisms have been reported in the brains of patients with central nervous system disease. Several of these patients have been identied as intravenous drug abusers. These fungi are found in the environment and have a predilection for cellulose products. They have been known to devastate printed literature and library holdings and have been associated with problems in indoor air quality. Microscopically, numerous perithecia are typically seen (Fig. 27.53). These perithecia are pineapple shaped and are ornamented with straight or curled hairs or setae. The asci contained within the perithecia are evanescent, so at maturity, the pigmented, lemon-shaped ascospores are released within the

Fig. 27.52 Alternaria sp. (unstained, ×450).

Fig. 27.53 Chaetomium sp. (unstained, ×650).

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perithecium. Colonies are moderately rapid to rapidly growing and begin dirty gray and become phaeoid with age. Some species produce a diffusible pigment that turns the agar completely red.

Cladosporium An infrequent cause of disease, Cladosporium spp. are primarily recovered as laboratory contaminants. Infections are typically conned to the sinuses or following traumatic inoculation. Ubiquitous in nature, this isolate can be recovered from almost any location in the world. Cladosporium spp. form brown to olive to black hyphae and conidia (Fig. 27.54). Conidiophores are erect and can branch into several conidiogenous cells. Spherical to ovoid conidia form blastically on the end of each previously formed conidium. Branched conidium-bearing cells may dislodge, and the three scars on each of these cells give them the appearance of a shield. Generally, conidial chains of the saprobic species break up easily, whereas those of pathogenic species remain connected. These organisms are slowly to moderately growing phaeoid fungi, with granular velvety to uffy colonies, ranging in color from olive to brown or black.

Curvularia Curvularia spp. isolates are usually implicated in chronic sinusitis in immunocompetent patients. Found worldwide, this fungus is frequently recovered from grass, leaves, and decaying vegetation. Multicelled conidia are produced on sympodial conidiophores (Fig. 27.55). This genus is among

Fig. 27.54 Cladosporium sp. (Nomarski optics, ×650).

Fig. 27.55 Curvularia sp. (Nomarski optics, ×450).

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Fig. 27.56 Phoma sp. (Nomarski optics, ×650).

Fig. 27.58 Ulocladium (lactophenol cotton blue, ×450).

and, in some species, echinulate surfaces. These species are rapidly growing phaeoid fungi, forming colonies ranging in color from brown to olivaceous to black.

Agents of yeast infections

Fig. 27.57 Pithomyces sp. (Nomarski optics, ×450).

the easiest to identify because of the frequently seen crescent-shaped conidia with three to ve cells of unequal size and an enlarged central cell. Several species previously classied in the genus Bipolaris are now considered to be members of Curvularia. These fungi form a rapidly growing phaeoid colony that is cottony and dirty gray to black.

The escalating incidence of yeast and yeastlike fungi isolated from patient specimens has emphasized the importance of identifying yeast isolates to the species level. With greater immunosuppression, the variety of organisms implicated in disease also expands. Candida albicans is the fourth most common cause of bloodborne infection in the United States, accounting for 10% to 15% of all hospital-acquired septicemia cases. Isolation of other yeasts from clinical samples is also increasing. Infections caused by many yeasts are extremely aggressive and difcult to treat. Yeast can be classied into one of two groups: yeasts and yeastlike fungi. Isolates that reproduce sexually, by forming either ascospores or basidiospores, are truly yeasts. Most isolates that are not capable of sexual reproduction or whose sexual state has not yet been discovered are correctly termed yeastlike fungi. For ease of discussion, all isolates are referred to here as yeasts

Phoma Disease caused by Phoma spp. is usually secondary to traumatic inoculation. Phoma spp. produce pycnidia, which appear as black fruiting bodies that are globose and lined inside with short conidiophores (Fig. 27.56). Large numbers of hyaline conidia are generated in the pycnidium and ow out of a small apical pore. Phoma spp. produce a moderately rapid growing, gray-to-brown colony.

Pithomyces Disease caused by Pithomyces spp. is usually secondary to traumatic inoculation. Conidia are somewhat barrel shaped, formed singly on simple short conidiophores (Fig. 27.57). Conidia have both transverse and longitudinal cross-walls and are often echinulate. Pithomyces spp. produce rapidly growing phaeoid colonies.

Ulocladium Ulocladium spp. are now classied among the sections in Alternaria. These species are sometimes implicated in subcutaneous infections, usually following traumatic inoculation. Conidiophores bear dark, multicelled conidia on sympodial conidiophores (Fig. 27.58). Conidia have angular cross-walls

General characteristics Molds and yeasts are very different morphologically, but some of the macroscopic characteristics used as aids in identifying molds can also be used to identify yeasts. Macroscopic characteristics include color and colony texture. The color of a yeast colony ranges from white to cream or tan, with a few species forming pink- to salmon-colored colonies. Some yeast isolates, referred to as phaeoid yeasts, are darkly pigmented because of melanin in their cell walls. Phaeoid yeasts are associated with several species of the polymorphic fungi and are discussed elsewhere in this chapter. The texture of the yeast colonies also differs. For example, Cryptococcus spp. tend to be mucoid and can ow across the plate, a trait shared by some bacteria, such as Klebsiella spp. Some yeasts are butterlike, and others range in texture from velvety to wrinkled. Strain-to-strain variation in texture may be noted within a species.

Candida Candida spp. are the most notorious agents of yeast infection. Clinical disease ranges from supercial skin infections

Clinically signicant species

to disseminated disease. C. albicans is a major cause of yeast infection in the world. It is recovered as normal biota from a variety of sites, including skin, the oral mucosa, the digestive tract, and the vagina. When host conditions are altered, however, this organism is capable of causing disease in almost any site. In individuals with an intact immune system, infections are localized and limited. One of the most widely recognized manifestations of C. albicans infection is thrush (oropharyngeal candidiasis), an infection of the oral mucosa. Thrush is also recognized as an indicator of immunosuppression. Among individuals infected with human immunodeciency virus (HIV) and those receiving prolonged antibacterial therapy or other chemotherapeutic agents, thrush manifests itself as a serious infection capable of dissemination (Figs. 27.59 and 27.60). Candida glabrata (synonym Nakaseomyces glabrata) is also a common species that causes disease and accounts for many urinary yeast isolates. It may now be second only to C. albicans in supercial and invasive yeast infections in North America and Europe. Infections associated with C. glabrata tend to be aggressive in patients with multiple comorbidities and may be difcult to treat with traditional antifungal therapy (e.g., amphotericin B and uconazole). This organism has different sugar assimilation patterns, notably rapid assimilation of trehalose, from those of C. albicans and therefore can be easily differentiated.

Fig. 27.59 Bronchoalveolar lavage, cytocentrifuge preparation, Gram stain, light microscopy. Gram-positive yeast with buds. Morphotype consistent with Candida spp. (×1000).

Fig. 27.60 Expectorated sputum smear, Gram stain, light microscopy. Grampositive pseudohyphae. Morphotype consistent with Candida spp. (×1000).

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Recently, C. auris has emerged as a multidrug-resistant yeast linked to high mortality rates associated with healthcareassociated infections worldwide. The yeast is likely transmitted from patient to patient in health care settings and has caused several outbreaks in numerous countries. The Centers for Disease Control and Prevention (CDC) recommends that a contact prevention protocol be followed for residents in nursing homes colonized or infected with C. auris and that they be housed in single rooms, if available. C. auris has been misidentied as several other yeast species. MALDI-TOF mass spectrometry assays can identify this species. Other notable species of Candida include C. krusei (teleomorph Pichia kudriavzeveii) and C. tropicalis. In addition, C. parapsilosis has become a major cause of outbreaks of healthcare-associated infections. These isolates are identied by the differences in their carbohydrate assimilation patterns and other secondary testing procedures, such as MALDI-TOF mass spectrometry. Table 27.7 shows important differentiating characteristics among Candida spp. and other yeasts.

Cryptococcus Cryptococcus spp. are important causes of meningitis, pulmonary disease, and septicemia. Members of the C. neoformans species complex (composed of C. neoformans and C. deneoformans), the most notable pathogens in this genus, is a major cause of opportunistic infection in patients with AIDS. These organisms are commonly found in soil contaminated with pigeon droppings and is most likely acquired by inhalation. Members of the C. gattii species complex (C. gattii, C. bacillisporus, C. deuterogattii, C. tetragattii, and C. decagattii) are emerging pathogens, particularly in the Pacic Northwest of the United States and Canada. Infections caused by this species are similar to those caused by C. neoformans species complex, targeting primarily immunocompromised patients. However, C. gattii can also cause disease in immunocompetent hosts, and it is important to distinguish the two species because the clinical course and treatment outcomes can be different. Cryptococcus spp. produce a capsule (Fig. 27.61) that produces the characteristic mucoid colony. The capsule can be detected surrounding the budding yeast in CSF with the aid of India ink or nigrosin (Fig. 27.62). The background uid is black, and clear unstained halos are seen around individual yeast cells. Because of its low sensitivity, the use of India ink preparation is being replaced by cryptococcal antigen tests. The cryptococcal antigen assays and an easy-to-use lateral-ow assay are recommended for routine use in clinical microbiology laboratories. The antigen assays detect both C. neoformans and C. gattii species complexes in CSF and serum. Cryptococcus spp. are noted for producing blastoconidia only, without producing true hyphae or pseudohyphae on cornmeal agar. All species of the genus are urease positive, and the nitrate reaction differs. Sugar assimilations differ among the species (see Table 27.7). It is extremely difcult to distinguish members of the C. neoformans and C. gattii species complexes from each other. A key laboratory characteristic is that members of the C. gattii species complex use glycine as a sole carbon and nitrogen source in the presence of canavanine, whereas C. neoformans does not. Canavanine glycine bromothymol blue agar is commercially available for this purpose and can be used to help differentiate between these species complexes.

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Table 27.7 Differentiating characteristics of yeasts

Candida C. albicans

+

+

+

+

+

−

R

−

−

C. auris

+

+

−

−

−

−

S

−

−

C. dubliniensis

+

−

−

+

+

−

R

−

−

C. glabrata (syn. Nakaseomyces glabrata)

+

+

+

−

−

−

S

−

−

C. guilliermondii (teleomorph name Meyerozyma guilliermondii)

+

+

−

+

−

−

R

−

−

C. krusei (teleomorph name + Pichia kudriavseii)

+

−

+

−

−

S

V

C. lusitaniae (teleomorph name Clavispora lusitaniae)

+

+

+

+

−

−

V

−

−

C. parapsilosis

+

−

−

+

−

−

S

−

−

C. stellatoidea (Candida albicans var. stellatoidea)

+

+

+

+

+

−

S

−

−

C. tropicalis

+

+

+

+

−

−

V

−

−

C. albidus (Naganishia albida)

−

−

−

−

−

−

S

+

+

C. neoformans

+

−

−

−

−

−

S

+

−

C. gattii

+

−

−

−

−

−

R

+

−

Trichosporon spp.

+

V

−

+

+

+

R

+

−

Cryptococcus

+, Positive;

, negative; R, resistant; S, sensitive; syn., synonym; V, variable; current names in parentheses; var., variant.

Fig. 27.61 Bronchoalveolar lavage, cytocentrifuge preparation, Gram stain, light microscopy. Red blood cells present. Gram-variable yeast with capsules. Morphology suggests Cryptococcus spp. (×1000) (see Fig. 27.68).

Fig. 27.62 India ink preparation is used primarily to examine cerebrospinal uid for the presence of the encapsulated yeast Cryptococcus neoformans. This is an India ink preparation from an exudate containing encapsulated budding yeasts (×400).

Rhodotorula

Pneumocystis

Rhodotorula spp. are noted for their bright salmon pink color. They resemble the cryptococci because they bear a capsule and are urease positive. Some species are also nitrate positive. They are not common agents of disease but have been known to cause opportunistic infections.

Pneumocystis can inhabit the lungs of many mammals. Pneumocystis carinii was originally classied with the protozoa, but nucleic acid sequencing showed conclusively that the organism is a fungus. It is apparent from nucleic acid studies that P. carinii is not a single species. P. carinii is the

Clinically signicant species

species most commonly found in rats, and P. jirovecii is the species most often recovered from humans.

Clinical manifestations Pneumocystis infection is acquired early in life; serologic studies have shown that most humans have antibodies or antigens by 2 to 4 years of age. In immunocompetent individuals, infection is asymptomatic; however, in immunocompromised patients, serious life-threatening pneumonia can develop. Pneumocystis initially was identied as the causative agent in interstitial plasma cell pneumonia seen in malnourished or premature infants. Since the early 1980s, it has remained one of the primary opportunistic infections found in patients with AIDS. A high incidence of disease also results from the use of immunosuppressive drugs in patients with malignancies and in organ transplant recipients. Underlying defects in cellular immunity apparently make patients susceptible to clinical infection with the organism. Patients infected with Pneumocystis may have nonproductive cough, difculty breathing, and a low-grade fever. Chest x-rays can be normal or show a diffuse interstitial inltrate. The immune response to the organism after it attaches to and destroys alveolar cells is partly responsible for this radiographic pattern. When the inltrate is examined, it is found to contain cells from the alveoli and plasma cells.

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Laboratory diagnosis Traditionally, diagnosis was made by finding the cyst or trophozoite in tissue obtained through open lung biopsy. Specimens now used include bronchoalveolar lavage (BAL) fluid, transbronchial biopsy specimens, tracheal aspirate, pleural fluid, and induced sputum. Sputum, however, is the least productive specimen. Lavage and sputum specimens are often prepared using a cytocentrifuge (Fig. 27.63). Histologic stains, such as Giemsa and GMS stains, are used. With the methenamine silver stain, the cyst wall stains black. Cysts often have a punched-out ping-pong ball appearance. Fig. 27.64A shows the characteristic black-staining cyst of Pneumocystis jiroveci with methenamine silver stain. With the Giemsa stain, the organism appears round, and the cyst wall is barely visible. Intracystic bodies are seen around the interior of the organism. Fig. 27.64B shows the cyst stained with Giemsa stain. The cyst wall does not pick up the stain, but the nuclei of all forms stain pink, and the intracystic bodies can be demonstrated as a circular arrangement within the cyst. Calcofluor white can be used to screen specimens for Pneumocystis and other fungi. This stain detects any

Life cycle Pneumocystis is a nonlamentous fungus. Terminology referring to the various life cycle forms, however, is reected in the fact that it was rst considered a protozoan. The life cycle of Pneumocystis has three stages: the trophozoite, which is 1 to 5 µm in size and is irregularly shaped; the precyst, which is 5 to 8 µm in size; and the cyst, which is a thick-walled sphere of about 8 µm containing up to eight intracystic bodies. Transmission of the organism is known to occur through the respiratory route, with the cyst being the infective stage. The spores or intracystic bodies are released from the cyst in the lung, and these trophic forms multiply asexually by binary ssion on the surface of the epithelial cells (pneumocyte) lining the lung. Sexual reproduction by trophozoites also occurs, rst producing a precyst and then the cyst containing spores or intracystic bodies.

A

Fig. 27.63 Bronchoalveolar lavage, cytocentrifuge preparation, Gram stain, light microscopy, high power view. Purulence, none. Local materials, moderate. Alveolar cast composed of Gram-negative matrix and intracystic bodies (arrows) (×1000). Morphology consistent with Pneumocystis jiroveci (see Fig. 27.65).

B

Fig. 27.64 A, Pneumocystis jirovecii cysts (silver stain). B, P. jirovecii (Giemsa stain). Note the circular arrangement of intracystic bodies within a faint outline of the cyst wall in the center of the eld (A, B, ×1000).

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organism that contains chitin in its cell wall. Fungi, yeasts, and Pneumocystis will fluoresce with a blue-white color when stained and viewed microscopically with ultraviolet light (Fig. 27.65). Immunofluorescent monoclonal antibody stains are commercially available and are widely used.

Laboratory diagnosis of fungi Safety issues Because of the additional hazard of airborne conidia, a class 2 biological safety cabinet should be used to reduce exposure of personnel to fungal elements. Specimen processing and plating must be performed in a properly maintained and operating biological safety cabinet. The use of an enclosed electric incinerator is recommended to eliminate the hazards of open gas ames and to contain aerosolized particles emitted when loops or needles are incinerated. The cabinet should be checked daily to see that none of the air ow inlets or outlets is blocked by supplies, incinerators, or waste-disposal containers. Use of Petri dishes in the mycology laboratory is hazardous; screw-top tubes (slants) are recommended instead for lamentous fungi. The chance for the release of airborne conidia is less likely with tubed media. Screw-capped tubes also tend to reduce dehydration of media and are more easily handled and stored compared with Petri dishes. However, Petri dishes have a larger surface area for colony isolation and are easier to manipulate when making preparations for microscopic examination. The edges of Petri dishes can be sealed with Paralm or tape to reduce the risk of releasing airborne conidia.

Specimen collection, handling, and transport Collection of appropriate specimens is the primary criterion for accurate diagnosis of mycotic infections. All specimens for mycology should be transported and processed as soon as possible. Because many pathogenic fungi grow

slowly, any delay in processing compromises specimen quality and decreases the probability of isolating the causative agent as a result of overgrowth by contaminants. In addition, all laboratories should maintain a protocol for the rejection of unsatisfactory or improperly labeled specimens. Although almost any tissue or body uid can be submitted for fungal culture, the most common specimens are respiratory secretions, hair, skin, nails, tissue, blood, bone marrow, and CSF. Table 27.8 presents the predominant culture sites for recovery of the causative agents of fungal diseases. Fig. 27.66 presents a schematic guideline to assist in making a diagnosis of a mycosis. Hair, skin, or nails submitted for dermatophyte culture are generally contaminated with bacteria, rapidly growing fungi, or both. With these types of specimens, primary isolation medium should contain antimicrobial agents. The following procedures are recommended for collecting and processing clinical samples submitted for fungal studies.

Hair The Wood lamp emits ultraviolet light of wavelength greater than 365 nm and can be useful in identifying infected hairs. Hairs infected with fungi such as M. audouinii uoresce when light from the Wood lamp is focused on the scalp. Sterile forceps should be used to pull affected hair. A less useful method involves cutting the hairs close to the scalp with sterile scissors. Hairs are placed directly into a sterile Petri dish. A few pieces of hair are inoculated onto fungal medium and incubated at 22° to 30° C.

Skin Skin must be cleaned with 70% isopropyl alcohol before sampling. Skin samples are scraped from the outer edge of a surface lesion. A KOH wet mount is prepared with some of the scrapings; the KOH breaks down tissue, making it easier to view fungal hyphae. The remaining material is inoculated directly onto the agar.

Nails Nails are cleaned with 70% isopropyl alcohol before the surface is scraped. Nail specimens may be submitted as scrapings or cuttings and occasionally as a complete nail. Deeper scrapings are necessary to prepare a KOH preparation and inoculate media. Sterile scissors are used to cut complete nails into small thin strips, which are used to inoculate media.

Blood and bone marrow

Fig. 27.65 Bronchoalveolar lavage, cytocentrifuge preparation, calcouor white stain, uorescence microscopy. Fluorescent cysts with coccoid bodies. Morphology consistent with P. jiroveci (×1000).

Blood from septicemic patients can harbor known pathogenic and opportunistic fungi, the most common being Candida spp. The lysis centrifugation system, the Isolator tube (Wampole Laboratories, Cranbury, NJ), is a method for the recovery of molds and dimorphic fungi. However, this method has a high contamination rate. The lysis of white blood cells (WBCs) and red blood cells releases microorganisms, which are then concentrated into sediment during centrifugation. The sediment is inoculated onto solid culture media. A biphasic system (broth and agar),

Laboratory diagnosis of fungi

627

Table 27.8 Predominant culture sites for recovery of causative agentsa Infection

Respiratory

Blastomycosis

+

Histoplasmosis

+

Coccidioidomycosis Paracoccidioidomycosis Sporotrichosis

+

Skin

Mucus

Bone

+

+

+

+

+

+

+

+

+

+

+

+

Chromoblastomycosis

+

+

Eumycotic mycetoma

+

+

Phaeohyphomycosis

+

+

a

Blood

Bone marrow

+

+

Tissue

Organisms may be recovered from multiple sites in disseminated infections.

CLINICAL SPECIMEN

Culture

Direct examination Tissue  Histology stain  Fluorescent antibody (selected organisms)

Cerebrospinal fluid  India ink  Cryptococcal antigen

Growth

Skin, hair, nails, and other selected specimens

35° C (suspect dimorphic)

22° to 30° C

No growth

Yeast

Mold

KOH or KOH with calcofluor white

No hyphal Hyphal elements elements  Width  Hyaline vs. dematiaceous  Septate vs. sparsely septate

Special supplementary media GROWTH 22° to 30° C

Yeast

Germ tube

Special media

Cornmeal  Blastoconidia  Chlamydospores

Mold Biochemicals  Assimilation  Fermentation

Other  Birdseed  Caffeic acid  Urea

Microscopic (LPCB)

Conidia  Size  Shape  Arrangement

Macroscopic  Color/pigment  Texture  Growth rate No conidia

Slide culture

Hyphae  Hyaline vs. dematiaceous  Septate vs. sparsely septate  Rhizoids

Fig. 27.66 Guideline for the identication of fungal isolates. KOH, Potassium hydroxide; LPCB, lactophenol cotton blue.

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such as the Septi-Chek (BD Diagnostic Systems, Sparks, MD), can also be used. Automated and continuous monitoring blood culture systems have media designed for the recovery of fungi. As with bacteriology, blood volume and blood-to-broth ratio are important for optimal recovery of fungi. Studies indicate 20 to 30 mL of blood from adults divided among two bottles and a dilution of 5- to 10-fold is ideal. Heparinized bone marrow specimens should be plated directly onto media at the bedside; use of blood culture bottles is not recommended.

Urogenital and fecal specimens

Cerebrospinal uid

Direct microscopic examination of specimens

CSF and other sterile body uids should be concentrated by centrifugation before inoculation. Part of the concentrate is used for India ink preparation or other assays for Cryptococcus (e.g., latex agglutination assay, EIA or lateral ow assay, as described later), and the remainder is inoculated onto media. If more than 5 mL is submitted, the CSF may be ltered through a membrane lter and portions of the lter placed on media. Use of media with antimicrobial agents should not be needed because CSF is normally sterile.

Abscess uid, wound exudates, and tissue Using a dissecting microscope, abscess uid and exudate from wounds can be examined for the presence of granules. If no granules are present, the material may be plated directly onto the media. Tissue should be gently minced before inoculation. Grinding of tissue has been recommended, but this process might destroy fragile fungal elements, particularly if a mucormycete is present. Although grinding of tissue might be necessary for KOH and calcouor white preparations, it should not be done unless sufcient tissue is present for mincing and grinding. When large sections of tissue are submitted, suspicious areas, such as purulent or discolored sections, are selected for mincing and grinding before subsequent culture.

Respiratory specimens Because many fungal infections have a primary focus in the lungs, lower respiratory tract secretions (e.g., sputum, transtracheal aspirates) and lavage uids (e.g., BAL) are commonly submitted. Patients should obtain sputa from a deep cough shortly after arising in the morning. If the patient cannot produce sputum, a nebulizer may be used to induce sputum. All sputum specimens should be collected in a sterile, screw-top container. If the material is not too viscous, the specimens can be inoculated onto media with a sterile pipette. With viscous materials, such as a thick tracheal aspirate, a synthetic swab (e.g., Dacron) may be used to inoculate the material onto the media, or preferably the specimen can be digested with the mucolytic agent N-acetyl-l-cysteine and concentrated before inoculation. In addition to nonselective media, a medium with antimicrobial agents should be used to prevent bacterial overgrowth. A KOH preparation should also be made. Oropharyngeal candidiasis is easily diagnosed with direct smear and culture. Nasal sinus specimens obtained surgically can be plated directly to media containing antimicrobial agents except for cycloheximide, which can inhibit some fungi.

Laboratory scientists often receive urine, feces, and vaginal secretions as specimens for bacteriologic culture; on occasion, these specimens grow a yeast that requires identication. Urine submitted specically for fungal culture should be centrifuged and the sediment used to make smears for microscopic examination and to inoculate media. Quantication of yeast, as performed for bacteria, is not useful, and 24-hour specimens are unacceptable. A rst-morning voided urine specimen is preferred.

Direct examination of clinical material for fungal elements serves several purposes. First, it helps provide a rapid report to the primary care provider, which may result in the early initiation of treatment. Second, in some cases, specic morphologic characteristics provide a clue to the genus of the organism. In turn, any special media indicated for species identication can be inoculated immediately. Third, direct examination might provide evidence of infection despite negative culture results. Such a situation can occur with specimens from patients who are receiving antifungal therapy, which may inhibit growth in vitro even though the infection can still be present in the patient. Although Gram-stained smears of clinical samples examined in the routine microbiology laboratory often give the rst evidence of infections with bacteria and yeasts, other direct stains give more specic information about mold infections. The types of direct examination used in identication of fungal infections include wet preparations, such as KOH, India ink, and calcouor white stain. Histologic stains for tissue may also be useful.

KOH preparation A 10% to 20% solution of KOH is useful for detecting fungal elements embedded within skin, hair, nails, and tissue (Fig. 27.67). In this procedure, a drop of the KOH preparation is added to a slide. Nail scrapings, hair, skin scales, or thin slices of tissue are added to the drop, and a coverslip is placed

Fig. 27.67 Skin scales, scrapings, potassium hydroxide wet preparation, light microscopy (×400). Hyphae present, septate, thin. Morphology suggests dermatophyte.

Laboratory diagnosis of fungi

629

on top. The slide is then gently heated and then allowed to cool for approximately 15 minutes. The KOH and heat break down the keratin and skin layers, revealing more clearly fungi present in the specimen. Interpreting KOH slides remains difcult, and fungal elements can still evade detection. Modications of the KOH test can provide easier and more reliable results. These preparations incorporate dimethyl sulfoxide (DMSO) and a stain into the KOH solution. The DMSO facilitates more rapid breakdown of cellular debris without requiring heat, while the stain is taken up by fungal elements, making them readily visible on microscopic examination of the slide preparation.

Potassium hydroxide with calcouor white A drop of calcouor white can be added to the KOH preparation before adding a coverslip. Calcouor white binds to polysaccharides present in the chitin of the fungus or to cellulose. Fungal elements uoresce apple green or blue-white, depending on the combination of lters used on the microscope; therefore any element with a polysaccharide skeleton uoresces (Figs. 27.68 to 27.70). Because calcouor white stains other structures, the actual fungal structure must be seen before a positive preparation is reported (Fig. 27.71). Care must also be taken when using this process because much variability exists among manufacturers and even in different lots of the stain prepared by the same manufacturer.

Fig. 27.69 Soft tissue abscess, smear, calcouor white stain, uorescence microscopy (×1000). Fungal hyphae, septate, branched chlamydospores. Impression: mycosis. These hyphae were not clearly visible on the Gram stain smear, but they stain brightly here. A dermatophyte was isolated in culture.

India ink India ink or nigrosin preparations can be used to examine CSF for the presence of the encapsulated yeast Cryptococcus. A drop of India ink is mixed with a drop of sediment from a centrifuged CSF specimen, and the preparation is examined on high magnication (×400). With this negative stain, budding yeast cells surrounded by a large clear area against a black background are presumptive evidence of Cryptococcus (see Fig. 27.62). WBCs and other artifacts can resemble encapsulated organisms; therefore careful examination is necessary. Many laboratories, however, now use a cryptococcal antigen assay (see the “Cryptococcal Antigen” section later in this chapter) in place of the India ink examination.

Fig. 27.68 Bronchoalveolar lavage, cytocentrifuge preparation, calcouor white stain, uorescence microscopy (×1000). Fluorescent yeast, small, with capsules. Morphology suggests Cryptococcus neoformans. Impression: cryptococcosis.

Fig. 27.70 Skin scales from scrapings, calcouor white stain, uorescence microscopy (×1000). Hyphae, thin. Morphology suggests dermatophyte. Impression: dermatophytosis.

Fig. 27.71 Expectorated sputum, smear, calcouor white stain, uorescence microscopy (×400). Eosinophils. These cells are other structures that stain with calcouor white. The granules from ruptured eosinophils stain brightly and should not be interpreted as remnants of fungi or parasites.

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Table 27.9 Staining characteristics of fungi

Fig. 27.72 Bone marrow stained with Giemsa stain showing the yeast phase of Histoplasma capsulatum (×1000).

The performance of these assays has improved over the years, and they are now recommended over the India ink preparations in screening CSF for Cryptococcus spp.

Tissue stains Common tissue stains used in the histology department for detection of fungal elements include the periodic acid–Schiff (PAS), GMS, hematoxylin and eosin (H&E), Giemsa, and Fontana-Masson stains. Giemsa stain is used primarily to detect Histoplasma in blood or bone marrow (Fig. 27.72). PAS stain attaches to polysaccharides in the fungal wall and stains fungi pink. The Fontana-Masson method stains melanin in the cell wall and identies the presence of phaeoid fungi. Table 27.9 lists the characteristic fungal reactions seen with selected stains.

Case check 27.2 When hyphal elements are seen in tissue sections, the disease is frequently attributed to Aspergillus spp. Although Aspergillus is the most common mold causing disease in the immunocompromised patient, dozens of other fungi, such as Fusarium in the Case in Point, give the same appearance. To document infection, the mold must be seen in tissue and grown in culture. When hyphae are not seen in tissue, the positive culture may be caused by a contaminant, and conversely, when hyphae are seen in tissue but not grown in culture, the causative agent cannot be determined.

Isolation methods Culture media In general, fungi do not share the broad range of nutritional and environmental needs that characterize bacteria, so relatively few types of standard media are needed for primary isolation. These include Sabouraud dextrose agar, Sabouraud dextrose agar with antimicrobial agents, potato dextrose agar or potato akes agar, and BHI agar enriched with blood and antimicrobial agents. Gentamicin or chloramphenicol and cycloheximide are the antimicrobials usually included with fungal media. Gentamicin and chloramphenicol inhibit bacterial growth, whereas cycloheximide inhibits bacteria and many of the environmental fungi typically considered contaminants.

Stain

Color of fungal element

Background color

Periodic acid–Schiff

Magenta

Pink or green

Grocott-Gomori methenamine silver

Black

Green

Giemsa

Purple-to-blue yeast with clear halo (capsule)

Pink to purple

India ink

Yeast with clear halo (capsule)

Black

KOH

Refractile

Clear

KOH–calcouor white

Fluorescent

Dark

Fontana-Masson

Brown

Pink to purple

KOH, Potassium hydroxide.

The pH of the Emmons modication of Sabouraud dextrose agar is close to neutral and is a more efcient medium for primary isolation compared with the original formulation. Table 27.10 shows the expected growth results with some of the standard fungal media. Selective chromogenic agars, such as CHROMagar Candida (CHROMagar, Paris, France) and CHROMID Candida (bioMérieux, Durham, NC), provide a rapid preliminary identication. Fungal media can be poured into Petri dishes or large test tubes to make slants. Petri dishes have the advantage of a larger surface area but are more prone to dehydration because of the prolonged incubation necessary for the recovery of some fungi. Petri dishes must be poured thicker than a standard medium for bacterial growth. The plates can be sealed with tape or Paralm or sealed in semi-permeable bags to minimize dehydration and prevent the spread of fungal spores. Several examples of semi-permeable shrink seal bands are commercially available as well. Tubed media have the advantage of being safer to handle and less susceptible to drying. Fungal plates and tubes should be opened only in a biological safety cabinet.

Incubation Most laboratories routinely incubate fungal cultures at room temperature or at 30° C. Fungi grow optimally at these temperatures, whereas bacteria have a slower growth rate. If the causative agent suspected is a dimorphic fungus, cultures should also be incubated at 35° C. Cultures are generally maintained for 4 to 6 weeks and should be examined twice weekly for growth. Mucorales, such as Mucor and Rhizopus spp., grow rapidly and may ll the tube or Petri dish with aerial mycelium within a few days, whereas more slowly growing organisms, such as Fonsecaea or Phialophora spp., might require 2 weeks or longer before growth is seen. Information that should be recorded about an isolate includes the number of days until the rst visible growth and the number of days required to see fruiting structures, whether mold or yeast forms are recovered, the media on which the fungus is isolated, the temperature at which growth occurs, and the morphology of the colonies.

Laboratory diagnosis of fungi

Table 27.10 Summary of primary fungal culture media Medium

Expected growth results

Incubated at 22° C SDA

Initial isolation of pathogens and saprobes Dimorphic fungi may exhibit their mycelial phase

SDA with antimicrobialsa

Saprobes generally inhibited on this medium Dermatophytes and most of the fungi considered primary pathogens grow

BHI agar

Initial isolation of pathogens and saprobes

BHI agar with antimicrobials

Recovery of pathogenic fungi Dermatophytes not usually recovered

Inhibitory mold agarb

Initial isolation of pathogens except dermatophytes

SDA with chloramphenicol and cycloheximidec

Primary recovery of dermatophytes

At 37° C SDA

The yeast form of dimorphic fungi, and other organisms grow Dermatophytes grow poorly

BHI agar with blood

Yeasts, such as Cryptococcus, grow well The yeast form of Histoplasma takes up some of the heme pigment in the medium and becomes light tan, with a grainy, wrinkled texture

BHI, Brain-heart infusion; SDA, Sabouraud dextrose agar (4% glucose) or Emmons’ modication with 2% glucose. a The antimicrobial agents generally used are cycloheximide and chloramphenicol. b Usually contains chloramphenical and is used for cultivation of cyclohexamide sensitive fungi (e.g., Cryptooccus spp. and Candida spp.). Yeast extract and glucose provide nutrients. c Several commericial formulations are available (e.g., Mycobiotic agar [Remel, Lenexa, KS] and Mycosel [BD Diagnostic Systems, Sparks, MD]).

Fungi identication Although the number of fungal species described exceeds 100,000, the number known to cause human disease is a small fraction of this number, and although the number of species that are routinely seen causing infection is quite low, new species are continually being implicated. Most diseases are caused by a handful of species, making identication, at least to the genus level, fairly easy. The traditional starting place is to decide whether the isolate is a yeast or a mold. However, molds do not always produce structures that will facilitate identication. Often, cultures from clinical samples are sterile (hyphae only) or atypical. None of the following tests alone is sufcient for proper identication, but when used together, accurate identication is often accomplished easily. Molecular and proteomic tests, whether culture based (DNA hybridization using probes, DNA sequencing and MALDI-TOF mass spectrometry) or PCR based, when used with these test procedures, enable the laboratory scientist to identify the most commonly encountered fungal isolates.

631

color, texture, and growth rate, are initial observations that should be made. Pigment on the reverse side of the colony or in the aerial mycelium can be noted but is not always helpful, especially with the phaeoid fungi.

Microscopic examination The most common procedure for microscopic examination is direct mounting of the fungal isolate. This is achieved by preparing a tease mount or cellophane tape mount. Many fungi routinely recovered can be identied by either of these two methods. Because of the risk of airborne conidia, these slides must be prepared in a biological safety cabinet. When fungi are atypical or an uncommon species is recovered, a slide culture should be prepared. Tease and tape mounts typically disturb conidia, preventing viewing of how they are formed, whereas slide cultures provide a more intact specimen. Fruiting structures, as well as conidial arrangement, are better observed by this method. The following microscopic characteristics should be observed: • • • •

Septate versus sparsely septate or aseptate hyphae Hyaline or phaeoid hyphae Reproductive (fruiting) structures The types, size, shape, and arrangement of conidia/ ascospores

Tease mount For the tease mount, teasing needles or sterile applicator sticks are used to remove a portion of the mycelium from the middle third of the colony. The mycelia are placed in a drop of lactophenol cotton blue (LPCB) on a slide and gently teased apart. A modication of this procedure is more practical and permits retention of more of the fruiting structure. The mycelia are placed into a drop of LPCB on a slide but not teased apart, a coverslip is added, and the slide is examined microscopically at low and high magnications. LPCB is used to x and stain tease or tape mounts from cultures. The combination of lactic acid, phenol, and the dye kills, preserves, and stains the organism. The hyphae take up the LPCB, but the stain does not work well with the phaeoid fungi because they retain their dark color. The major disadvantage of this procedure is the disruption of conidia during the teasing process.

Cellophane tape preparation Cellophane tape preparations involve gently touching the surface of the colony with a piece of clear tape, sticky side down, and then removing it. The tape should not be pressed into the colony but should just gently touch the surface. Frosted tape does not work because the fungi are not visible through this type of tape. The tape is placed onto a drop of LPCB on a slide and examined. An advantage of this procedure is that the conidial arrangement is retained. Major disadvantages are the potential for contamination of the colony and the temporary nature of this preparation. Tape preparations should be read within 30minutes and then discarded. A coverslip is not needed if the cellophane tape technique is used because the tape serves as a coverslip.

Macroscopic examination of cultures

Slide culture

Once an organism has grown, colonies must be examined for macroscopic characteristics. Gross morphologic traits, such as

Slide cultures are useful for demonstrating the natural morphology of fungal structures and for encouraging conidiation

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minuscule amounts of vitamin carried over can adequately supply the requirement and thereby result in a false growth reaction.

Trichophyton agars Seven different Trichophyton agars, numbered 1 to 7, are used to determine the nutritional requirements of Trichophyton spp. Each of the media has different nutritional ingredients; after inoculation and incubation, growth is scored on a scale of 1 to 4. Based on the growth pattern, identication is made.

Growth on rice grains Fig. 27.73 A positive hair perforation test result shows penetration of the fungal agent in the hair shaft (arrow). This is the typical reaction by Trichophyton mentagrophytes, whereas Trichophyton rubrum causes only surface erosion of hair shaft (unstained, ×1000).

in some poorly fruiting fungi. The slide culture can be preserved for a prolong period, making those of known isolates useful for future comparison against isolates awaiting identication. Several methods have been devised for constructing slide cultures (see Appendix D on Evolve).

Poorly sporulating isolates of M. canis can be difcult to differentiate from M. audouinii, a species that typically forms few conidia. Sterile, nonfortied rice is inoculated lightly with a portion of a colony. After 10 days of incubation at room temperature, the medium is observed for growth. M. canis and almost all other dermatophytes grow well and usually form many conidia, whereas M. audouinii does not grow but turns the rice grains brown.

Miscellaneous tests for the identication of yeasts Germ tube production

Miscellaneous tests for the identication of molds Hair perforation test In the hair perforation test, sterile 5- to 10-mm hair fragments are oated on sterile water supplemented with a few drops of sterile 10% yeast extract. Conidia or hyphae from the dermatophyte in question are inoculated onto the water surface. Hair shafts are removed and microscopically examined in LPCB at weekly intervals for up to 1 month. T. rubrum usually causes only surface erosion of hair shafts in this test, whereas T. mentagrophytes typically forms perpendicular penetration or wedge-shaped pegs in the hair shafts (Fig. 27.73). This test may be used to distinguish penetration-capable M. canis from M. equinum, which does not penetrate hair.

Urease test Another test used to help differentiate T. mentagrophytes from T. rubrum is the 5-day urease test. Tubes of Christensen urea agar are very lightly inoculated with the dermatophyte and held for 5 days at room temperature. Most isolates of T. mentagrophytes demonstrate urease production, resulting in a color change of the medium from peach to bright fuchsia within that period, whereas most T. rubrum isolates are negative or require more than 5 days to give a positive reaction. This test is also useful for other molds and yeasts.

Thiamine requirement Some dermatophytes cannot synthesize certain vitamins and therefore do not grow on vitamin-free media. Although several vitamin deciencies are recognized in fungi, the test for thiamine requirement is perhaps the single most useful nutritional test for dermatophytes. Tubes of media with and without thiamine are inoculated with a tiny, medium-free portion of the colony and observed for growth after 10 to 14 days. Great care must be exercised to avoid transfer of culture medium with the inoculum because even

The germ tube test is a relatively easy test to perform for the identication of yeasts. Fig. 27.74 shows a schematic diagram of how the germ tube test can be used to presumptively identify yeast species. Both C. albicans and C. dubliniensis are identied with germ tube production (Fig. 27.75). The standard procedure (see Appendix D on Evolve) requires the use of serum or plasma, such as fetal bovine serum. Expired fresh-frozen plasma from the blood bank can also be used. However, this is not recommended because of the potential risk in the handling of bloodborne pathogens and because of concerns over reproducibility. Many other liquid media (e.g., BHI agar, trypticase soy broth, nutrient broth) have been used successfully as an alternative. The substrate is inoculated and then incubated at 35° C for 3 hours. Care must be taken not to incubate the test beyond 3 hours because other species are capable of forming germ tubes with extended incubation. A presumptive identication of C. albicans or C. dubliniensis can be made when true germ tubes are present. This test provides only a presumptive identication because not all strains of C. albicans will be positive, and other species, in particular C. tropicalis, can yield false-positive results. True germ tubes lack constriction at their bases, where they attach to the mother cell. If a constriction is present at the base of a germ tube, the yeast is not either species. Such constricted germ tubes, called pseudo–germ tubes, are more characteristic of C. tropicalis (Fig. 27.76). C. dubliniensis is differentiated from C. albicans by its inability to grow at 42° C.

Carbohydrate assimilation Sugar fermentation tests, although valuable, are time- and labor-intensive, making them impractical for use in the routine microbiology laboratory. Carbohydrate assimilation tests, however, can be readily performed as part of routine identication protocols. Assimilation tests identify which carbohydrates a yeast can use aerobically as a sole carbon source. Assimilation patterns can be determined from such methods

Laboratory diagnosis of fungi

633

Yeast

Germ tube

+ + Growth at 42° C Candida albicans

Carbohydrate assimilations

Candida Cornmeal dubliensis agar

Blastoconidia only

Cryptococcus sp. Candida sp.

Blastoconidia Pseudohyphae

Candida sp.

Blastoconidia True hyphae

Blastoconidia Pseudohyphae True hyphae Arthroconidia

Candida sp. Trichosporon sp.

Fig. 27.74 Schematic diagram showing how the germ tube test can be used to presumptively identify yeasts.

Fig. 27.75 Germ tube production by Candida albicans. A true germ tube has no constriction at its base (unstained, ×1000).

Several kits are available for identication of yeasts, including the API 20C yeast identication system (bioMérieux). In this assay, a series of freeze-dried sugars are placed into wells on a plastic strip. Yeast isolates are suspended in an agar basal medium, pipetted into the wells, and incubated at 30°C for 72hours. As sugars are assimilated, the wells become turbid with growth. Wells remain clear when the sugar is not assimilated. A code is derived from the assimilation patterns and matched against a computerized database. Identications are accompanied by a percentage, which indicates the probability that the identication is correct. Although this test is reliable, other auxiliary testing should accompany assimilation results before a nal identication is made. Automated systems are also available for yeast identication. Many of these systems use enzyme and assimilation reactions to aid in yeast identication.

Chromogenic substrates A number of media containing chromogenic substrates are available for the presumptive identication of yeasts. CHROMagar Candida presumptively identies C. albicans, C. tropicalis, and about 10 other species. Identication is based on different colony colors, depending on the breakdown of chromogenic substrates by the different species. About 2% to 10% of C. albicans isolates are not identied on these media because they form white colonies.

Cornmeal agar

Fig. 27.76 Candida tropicalis shows constriction at the base of the germ tube, called a pseudo–germ tube (Nomarski optics, ×1250).

as an automated identication system or various manual procedures and commercial kits. The individual laboratory should adopt the method that can be practically implemented into its particular working environment.

Yeast morphology on cornmeal agar is useful in determining identication of yeasts (see Appendix D on Evolve). Recognition of four different types of morphology is an important clue to identication: blastoconidia, chlamydoconidia, pseudohyphae, and arthroconidia. Blastoconidia are the characteristic budding yeast forms usually seen on direct mounts of yeasts. C. albicans produces chlamydoconidia (chlamydospores) along with hyphae, as shown in Fig. 27.77. A chlamydoconidium is a thickwalled conidium formed within or at the end of vegetative hyphae. Pseudohyphae (Fig. 27.78) are produced when the blastoconidia germinate to form a lamentous mat.

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temperatures as high as 45° C. C. albicans grows at 45° C, whereas C. dubliniensis does not. Similarly, C. auris is capable of growing at 40° to 42° C , whereas C. haemulonii and C. duobushaemulonii, two closely related species, are not.

Urease Yeast isolates producing the enzyme urease can be detected easily with Christensen urea agar. The slants are inoculated and incubated at room temperature for 48 hours. Almost all clinically encountered Candida spp. are urease negative, whereas all Cryptococcus and Rhodotorula organisms are urease positive. Most strains of Trichosporon spp. are urease positive. Fig. 27.77 Candida albicans on cornmeal agar showing typical chlamydoconidia (unstained, ×200).

Diagnosis using clinical specimens and surrogate markers (1,3)-β-d-Glucan

Fig. 27.78 Pseudohyphae occur when the blastoconidia germinate and form a lamentous mat (unstained, ×200).

(1,3)-β d-Glucan is an important component of the cell wall of various fungi, including pathogenic yeasts and molds. Major exceptions include Cryptococcus spp. and the Mucorales. Various assays are available, but only one, the Fungitell test (Associates of Cape Cod, Falmouth, MA), is available in the United States. This assay is a chromogenic test based on activation of the horseshoe crab coagulation cascade by (1,3)-β d-glucan and uses amebocyte enzymes from Limulus polyphemus. Clinical studies have demonstrated the utility of this assay for the diagnosis of invasive fungal infections. However, various substances as well as some bacteria can lead to false-positive results. The cryptococci lack (1,3)-β d-glucan, so they are negative. In addition, this is a panfungal assay, so a positive result does not provide information on the species that may be causing infection.

Galactomannan The cross-walls help determine whether the structures are true hyphae or pseudohyphae. Cross-walls of pseudohyphae are constricted and not true septations, whereas true hyphae remain parallel at cross-walls, with no indentation. Arthroconidia begin as true hyphae but break apart at the cross-walls with maturity. Rectangular fragments of hyphae should be accompanied by blastoconidia for an isolate to be considered a yeast.

Potassium nitrate assimilation KNO3 assimilation may provide useful information for separating the clinically signicant yeasts. Use of the modied KNO3 agar is a fairly rapid, easy, and accurate method to determine the ability of yeasts to use nitrate as the sole source of nitrogen. In a positive KNO 3 assimilation result, the medium turns blue, and in a negative result, the medium turns yellow. Control organisms that may be used include Naganishia albida (formerly Cryptococcus albidus) (positive) and C. albicans (negative).

Temperature studies Temperature studies offer information for yeast identication. Cryptococcus spp. have weak growth at 35° C and no growth at 42° C. The optimal temperature for growth is about 25° C. Several Candida spp. have the ability to grow well at

The detection of galactomannan, a component of the Aspergillus cell wall, within plasma or BAL uid is often used in the diagnosis of invasive infections caused by Aspergillus species. The detection of galactomannan is now part of the diagnostic criteria for probable invasive aspergillosis. One enzyme-linked immunosorbent assay (ELISA) method, the Platelia Aspergillus assay (Bio-Rad Laboratories, Hercules, CA), which uses a rat monoclonal antibody directed against an epitope in galactomannan in an ELISA format, was shown to be sensitive and specic in patients at high risk for invasive aspergillosis.

T2 magnetic resonance assay A relatively new assay for the diagnosis of invasive candidiasis combines nuclear magnetic resonance spectroscopy with PCR to detect Candida cells directly in blood samples. One assay, the T2Candida assay (T2Biosystems, Lexington, MA), is approved by the U.S. Food and Drug Administration (FDA) and can detect ve common Candida species: C. albicans, C. glabrata, C. krusei, C. parapsilosis, and C. tropicalis. This assay has excellent analytical sensitivity, being able to detect one to three colony-forming units of Candida per milliliter of blood. The results are rapidly available, usually within 4 hours in one study, compared with over 100 hours for traditional blood cultures, which is a major advantage. A research use only product is also available for C. auris

Immunodiagnosis of fungal disease

Cryptococcal antigen The detection of cryptococcal antigen, within either CSF or serum, has proven to be very useful in the diagnosis of cryptococcosis. Several available assays detect the glucuronoxylomannan component of the Cryptococcus antigen. These include latex agglutination assays, EIAs, and lateral ow assays. A lateral ow assay (IMMY cryptococcal lateral ow assay, Immuno-Mycologics, Norman, OK) is a rapid point-ofcare dipstick test that uses a monoclonal antibody against the cryptococcal antigen. Because this assay is sensitive, is easy to perform, and does not require specialized equipment or refrigeration, the World Health Organization recommends that it be used in resource-limited settings for screening HIVpositive individuals for cryptococcosis.

Immunodiagnosis of fungal disease Skin test reactivity to fungal antigens is sometimes used in the diagnosis of fungal allergies and infections. For example, Aspergillus spp. antigen extract is used for suspected allergic bronchopulmonary aspergillosis, atopic dermatitis, and allergic asthma. Skin testing for diagnosing fungal infection is valuable only if the patient has a history of a nonreactive skin test. A few antigen detection assays are commercially available for fungal pathogens including Cryptococcus spp. and H. capsulatum Although few are commercially available, assays to detect antibodies to fungi have also been used to help diagnose infection. Results depend on the antigen chosen and its quality. Cross-reactivity among related fungi has been reported. Double-immunodiffusion is probably the most commonly used method; however, interlaboratory discrepancies have been noted. One problem with serologic assays is the ubiquitous nature of many of the opportunistic fungi. Individuals can have detectable antibodies but not an active infection. It has also been shown that some individuals with Aspergillus spp. infections do not have detectable levels of antibody.

Antifungal susceptibility Antifungal agents Over the past few decades, fungal infections have steadily increased in incidence. This increase can be attributed to advances in medicine that have prolonged life expectancy, especially for those with chronic diseases. Advances in the area of transplantation medicine and chemotherapy, combined with immunosuppression from HIV infection, have created more patient populations susceptible to fungal infections. Primary care providers have far fewer options for treating fungal infections compared with bacterial infections. Current options for treating fungal infection include drugs primarily from four classes: polyene, azole, echinocandin, and allylamine. For many years, the primary antifungal agent was amphotericin B, a polyene. This agent has a broad spectrum of activity in addition to fungicidal activity. Not only is this agent lethal to fungi, but it is also toxic

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to humans. Patients treated with amphotericin B can experience many adverse side effects, including infusion-related reactions (fever, rigor, myalgia, and arthralgia) and renal impairment. Despite these problems, amphotericin B is frequently used for life-threatening fungal disease. Unfortunately, resistance to amphotericin B has been documented with Scedosporium spp., L. prolicans, P. lilacinum, A. terreus, and Candida lusitaniae isolates. The class that has provided the largest number of agents is the azoles. The most noteworthy compounds in this class include uconazole, itraconazole, isavuconazole, posaconazole, and voriconazole. The azoles are important because they exhibit reasonable activity against fungi while causing fewer side effects. Fluconazole is the leading agent for treating yeast infections but has limited to no activity against molds. It is widely used by many practitioners to treat all types of infections, including vaginitis and thrush. Unfortunately, its overuse or misuse has resulted in the development of resistance, most notably with C. glabrata. Most C. auris isolates are also resistant to uconazole, and C. krusei is considered intrinsically resistant. It is extremely important to determine the susceptibility pattern of a specic isolate before prescribing uconazole for severe infections in patients who have been receiving long-term antifungal therapy, The other azoles, itraconazole, isavuconazole, voriconazole, and posaconazole, are more frequently prescribed for treating mold infections. Itraconazole has been useful for treating aspergilli and phaeoid fungi infections. Isavuconazole and posaconazole have perhaps the broadest activity of the azoles against fungal species. Voriconazole has potent activity against the aspergilli, in addition to several emerging pathogens. Although prophylaxis with voriconazole is effective in decreasing infections by Aspergillus spp., it lacks activity against Mucorales. The rst agent to be released in the echinocandins group was caspofungin. This agent is lethal for yeast and, although effective against the aspergilli, is not generally lethal. This class targets cell wall synthesis, which makes it an attractive option for treating fungi that have developed resistance to other agents. Two other echinocandins are anidulafungin and micafungin. These two antifungals tend to have lower minimal inhibitory concentrations (MICs) compared with caspofungin. The CDC recommends an echinocandin for the treatment of invasive C. auris infections. Two FDA-approved allylamines are terbinane and naftine. These compounds interfere with the synthesis of ergosterol, a principal sterol in the plasma membrane of many fungi. Terbinane may be given orally or applied topically and is active against several groups of fungi, including the dermatophytes. Naftine is used only topically.

Antifungal susceptibility testing Until the past several years, many laboratories used methods that had been developed in-house to determine susceptibility patterns for the fungi. The Clinical and Laboratory Standards Institute (CLSI) in the United States has published four methods for antifungal susceptibility testing. These include M27 for yeast testing, M38 for mold testing, M44 for disk diffusion testing for yeasts, and M51 for disk diffusion testing for molds.

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Although much debate exists regarding antifungal susceptibility testing, it is gaining popularity with more laboratories. The FDA has approved a microtiter method from TREK Diagnostic Systems (Thermo Fisher Scientic, Waltham, MA), a gradient diffusion method (Etest; bioMérieux), and MIC Test Strip (MTS; Liolchem, Waltham, MA). Before these products were developed, only laboratories that could prepare their own reagents performed antifungal susceptibility testing. The largest barrier to widespread antifungal testing is the lack of established breakpoints for most of the agents. Breakpoints are now species specic by drug, with results placing the organism categorically in the same manner as bacteria. These categories are S (susceptible), I (intermediate), and R (resistant) for the echinocandins. The azoles uconazole and voriconazole are categorized as S, SDD (susceptible, dose dependent, uconazole), I (for voriconazole), and R. The SDD category for uconazole indicates isolates that may be considered susceptible when high-dose therapy, as opposed to standard therapy, is used. Until breakpoints are established, the CLSI has provided epidemiologic cutoff values (ECVs). The ECV is an end point that can be used to evaluate a given MIC when clinical breakpoints are not available. The ECV does not indicate susceptible or resistant strains rather, wild-type and non–wild-type strains, where non–wild-type strains may harbor mechanisms of antifungal resistance. The health care provider will be able to review the activity of a drug against a specic strain to determine whether the MIC result is typical for that species or high. Those strains with an MIC above the ECV have potentially acquired mechanisms of resistance. Until other drugs are studied and breakpoints are established, the usefulness of antifungal susceptibility testing will most likely remain limited.

POINTS TO REMEMBER

• Yeasts typically reproduce by budding, whereas molds often reproduce by forming conidia/spores. • A large array of fungi are capable of causing human infections. • Fungi can be isolated from almost any type of clinical specimen. • Fungal identication is based on a variety of characteristics, including macroscopic appearance, microscopic appearance, ability to grow at various temperatures, and biochemical reactions. Molecular and proteomic assays are now commonly used for fungal identication. • Phaeoid (dematiaceous) fungi produce dark pigments. • Dermatophytoses are caused by Trichophyton, Microsporum, and Epidermophyton species. • The thermally dimorphic fungi Blastomyces spp., Coccidioides immitis, Coccidioides posadasii, Histoplasma spp., Paracoccidioides spp., Sporothrix schenckii species complex, and Talaromyces marneffei are often associated with systemic mycoses. • Clinically important Mucorales include Rhizopus, Mucor, Lichtheimia, Cunninghamella, Syncephalastrum, Apophysomyces, and Saksenaea

• Geotrichum is a mold that is often initially mistaken for a yeast. • Saprobic fungi are most problematic in the immunocompromised host. • Candida albicans is the most commonly isolated yeast and the one responsible for most human infections. • Pneumocystis jirovecii is an important pathogen in patients with AIDS. • Extreme care should be taken when working with dimorphic fungi in the laboratory. Cultures for all molds should be processed in a biological safety cabinet. • Antifungal therapy is frequently ineffective when diagnosis is delayed.

LEARNING ASSESSMENT QUESTIONS

1. Which of the following organisms is the causative agent of tinea nigra? a. Microsporum canis b. Trichosporon beigelii c. Piedraia hortae d. Hortaea werneckii 2. Which of the following organisms is not a causative agent of chromoblastomycosis? a. Fonsecaea compacta b. Talaromyces marneffei c. Fonsecaea pedrosoi d. Phialophora verrucosa 3. A patient with very pale patches on his arms and legs is examined at his physician’s ofce. His physician orders a fungal culture. The fungus shows a “spaghetti-and-meatball” appearance on direct smear. Which organism would you suspect? a. Microsporum canis b. Malassezia furfur c. Trichophyton rubrum d. Epidermophyton occosum 4. Describe the characteristic microscopic appearance for each of the following dimorphic fungi when grown at 22° or 35° C. a. Blastomyces dermatitidis b. Coccidioides immitis and C. posadasii c. Histoplasma capsulatum d. Sporothrix schenckii species complex 5. What are the distinguishing microscopic characteristics for each of the following organisms? a. Nannizzia gypsea (Microsporum gypseum) b. Microsporum canis c. Trichophyton rubrum d. Trichophyton mentagrophytes 6. What are the expected results of the urease test and the hair perforation test for T. rubrum and T. mentagrophytes? 7. Which organism is one of the primary opportunistic infections in patients with acquired immunodeciency syndrome? a. Pneumocystis jirovecii b. Hortaea werneckii c. Coccidioides immitis d. Paracoccidioides brasiliensis

Antifungal susceptibility

8. What is the signicance of isolating a saprobe from an infection in an immunocompromised patient? 9. How would you compare the macroscopic and microscopic morphology of the following saprobes: Penicillium spp., Aspergillus fumigatus, Fusarium spp., and Curvularia spp.? 10. What are the differences between chromoblastomycosis and eumycotic mycetoma? 11. What are the major differences between bacteria and fungi? 12. How are hyaline fungi different from phaeoid fungi? 13. Dene teleomorph, anamorph, and synanamorph 14. What are the expected results of the germ tube test for Candida albicans? What morphology would you likely see if you inoculated the colony onto cornmeal agar? 15. Large, one-celled, smooth to tuberculate macroconidia and smooth or echinulate microconidia are typical of the mycelial-phase growth of which of the following fungi? a. Blastomyces dermatitidis b. Coccidioides immitis c. Histoplasma capsulatum d. Paracoccidioides braziliensis 16. Which of the following are the infectious structures of Coccidioides immitis? a. Hyphae b. Blastoconidia c. Arthroconidia d. Chlamydospores 17. Black-dot ringworm is an endothrix hair involvement caused by which of the following dermatophytes? a. Microsporum canis b. Trichophyton tonsurans c. Trichophyton mentagrophytes d. Epidermophyton occosum 18. A subcutaneous fungal infection described as necrotic ulcers may follow direct inoculation of fungal spores into the skin. The causative agent when cultured at 37o C forms small, cigar-shaped yeasts and at room temperature, grows as a mold with delicate hyphae and rosette-like conidia. What is the name of this disease? a. Blastomycosis b. Chromomycosis c. Sporotrichosis d. Mycetoma 19. Most cases of pulmonary infections and invasive diseases caused by Aspergillus in humans result from which of the following organisms? a. Aspergillus fumigatus b. Aspergillus avus c. Aspergillus niger d. Aspergillus terreus 20. A 10-year-old patient was admitted to the hospital with diabetic ketoacidosis. Three days later, he complained of nasal sinus blockage. Within 3 days, white uffy mold grew from the sinus drainage and showed erect sporangiophores terminating in dark sporangia and sporangiospores. Brown rhizoids appeared at the base of the sporangiophores. Which of the following organisms would you suspect to identify? a. Alternaria b. Mucor c. Rhizopus d. Curvularia

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BIBLIOGRAPHY Ahmed, A. O. A., et al. (2019). Fungi causing eumycotic mycetoma. In K. C. Carroll, et al. (Eds.), Manual of clinical microbiology (12th ed., p. 2261). Washington, DC: ASM Press. Albert, O., et al. (2011). Reactivity of (1→3)-β-D-glucan assay in bacterial bloodstream infections. European Journal of Clinical Microbiology & Infectious Diseases, 30, 1453. Angoulvant, A., et al. (2015). Old and new pathogenic Nakaseomyces species: epidemiology, biology, identication, pathogenicity and antifungal resistance. FEMS Yeast Research, 16. doi:https://doi. org/10.1093/femsyr/fov114. Bormann, A. M., & Johnson, E. M. (2019). Candida, Cryptococcus, and other yeasts of medical importance. In K. C. Carroll, et al. (Eds.), Manual of clinical microbiology (12th ed., p. 2056). Washington, DC: ASM Press. Bormann, A. M., & Summerbell, R. C. (2019). Trichophyton, Microsporum, Epidermophyton, and agents of supercial mycoses. In K. C. Carroll, et al. (Eds.), Manual of clinical microbiology (12th ed., p. 2208). Washington, DC: ASM Press. Brown, E. M., et al. (2013). Phylogenetic analysis reveals a cryptic species Blastomyces gilchristii, sp. nov. within the human pathogenic fungus Blastomyces dermatitidis. PLoS ONE, 8, e59237. Carlesse, F., et al. (2017). Outbreak of Fusarium oxysporum infections in children with cancer: an experience with 7 episodes of catheterrelated fungemia. Antimicrobial Resistance & Infection Control, 6, 93. Centers for Disease Control and Prevention. (2021). Candida auris. Available at: https://www.cdc.gov/fungal/candida-auris/index. html. (Accessed 9 July 2022). Chan, J. F., et al. (2016). Talaromyces (Penicillium) marneffei infection in non-HIV-infected patients. Emerging Microbes & Infection, 5, e19. Chen, S. C.-A., et al. (2019). Aspergillus and Penicillium. In K. C. Carroll, et al. (Eds.), Manual of clinical microbiology (12th ed., p. 2103). Washington, DC: ASM Press. Clinical and Laboratory Standards Institute. (2021). Principles and procedures for detection and culture of fungi in clinical specimens. CLSI guideline M54 (2nd ed.). Clinical and Laboratory Standards Institute. Cushion, M. T., & Rai, M. A. (2019). Pneumocystis. In K. C. Carroll, et al. (Eds.), Manual of clinical microbiology (12th ed., p. 2087). Washington, DC: ASM Press. Dalcin, D., et al. (2016). Blastomyces gilchristii as cause of fatal acute respiratory distress syndrome. Emerging Infectious Diseases, 22, 306. Available at: https://pubmed.ncbi.nlm.nih.gov/26812599. (Accessed 9 July 2022). D’Souza, C. A., et al. (2011). Genome variation in Cryptococcus gattii, an emerging pathogen of immunocompetent hosts. mBio, 2, e00342. Garcia-Hermoso, D., et al. (2019). Agents of systemic and subcutaneous mucormycosis and entomophthoromycosis. In K. C. Carroll, et al. (Eds.), Manual of clinical microbiology (12th ed., p. 2163). Washington, DC: ASM Press. Guarro, J., et al. (2019). Curvularia, Exophiala, Scedosporium, Sporothrix, and other melanized fungi. In K. C. Carroll, et al. (Eds.), Manual of clinical microbiology (12th ed., p. 2234). Washington, DC: ASM Press. Jarvis, J. N., et al. (2011). Evaluation of a novel point-of-care cryptococcal antigen test on serum, plasma, and urine from patients with HIV-associated cryptococcal meningitis. Clinical Infectious Diseases, 53, 1019. Larone, D. H. (2016). Medically important fungi: a guide to identication (5th ed.). Washington, DC: ASM Press. Lindsley, M. D., et al. (2011). Evaluation of a newly developed lateral ow immunoassay for the diagnosis of cryptococcosis. Clinical Infectious Diseases, 53, 321. Lockhart, S. R., & Berkow, E. L. (2019). Antifungal agents. In K. C. Carroll, et al. (Eds.), Manual of clinical microbiology (12th ed., p. 2319). Washington, DC: ASM Press. Lombard, L., et al. (2015). Generic concepts in Nectriaceae. Studies in Mycology, 80, 189. Luangsa-Ard, J., et al. (2011). Purpureocillium, a new genus for the medically important Paecilomyces lilacinus. FEMS Microbiology Letters, 321, 141.

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Mylonakis, E., et al. (2015). T2 magnetic resonance assay for the rapid diagnosis of candidemia in whole blood: a clinical trial. Clinical Infectious Diseases, 60, 892. Neely, L. A., et al. (2013). T2 magnetic resonance enables nanoparticle-mediated rapid detection of candidemia in whole blood. Science Translational Medicine, 5, 182ra54. Patel, R. (2019). A moldy application of MALDI: MALDI-ToF mass spectrometry for fungal identication. Journal of Fungi (Basel, Switzerland), 5, 4. Rizzato, C., et al. (2015). Pushing the limits of MALDI-TOF mass spectrometry: beyond fungal species identication. Journal of Fungi, 1, 367. Schwartz, I. S., & Kauffman, C. A. (2020). Blastomycosis. Seminars in Respiratory and Critical Care Medicine, 41, 31. Seagle, E. E., et al. (2021). Recent trends in the epidemiology of fungal infections. Infectious Diseases Clinics of North America, 35, 237. Suwantarat, N., et al. (2015). Large-scale clinical validation of a lateral ow immunoassay for detection of cryptococcal antigen in serum and cerebrospinal uid specimens. Diagnostic Microbiology and Infectious Disease, 82, 54.

Thompson, G. R., & Gómez, B. L. (2019). Histoplasma, Blastomyces, Coccidioides, and other dimorphic fungi causing systemic mycoses. In K. C. Carroll, et al. (Eds.), Manual of clinical microbiology (12th ed., p. 2187). Washington, DC: ASM Press. Walsh, T. J., & McCarthy, M. W. (2019). The expanding use of matrix-assisted laser desorption/ionization-time of ight mass spectroscopy in the diagnosis of patients with mycotic diseases. Expert Review of Molecular Diagnostics, 19, 241. Walther, G., et al. (2019). Updates on the taxonomy of Mucorales with an emphasis on clinically important taxa. Journal of Fungi, 5, 106. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/ PMC6958464/. (Accessed 9 July 2022). Woudenberg, J. H. C., et al. (2015). Alternaria section Alternaria: species, formae speciales or pathotypes? Available at: https://doi. org/10.1016/j.simyco.2015.07.001. (Accessed 9 July 2022). Wright, W. F., et al. (2011). (1–3)-β-d-Glucan assay: a review of its laboratory and clinical application. Laboratory Medicine, 42, 679. Zhang, S. X., et al. (2019). Fusarium, and other opportunistic hyaline fungi. In K. C. Carroll, et al. (Eds.), Manual of clinical microbiology (12th ed., p. 2132). Washington, DC: ASM Press.

28 Diagnostic parasitology Lauren Roberts and Connie R. Mahon

CHAPTER OUTLINE

General concepts in parasitology laboratory methods, 640 Fecal specimens, 640 Other specimens examined for intestinal and urogenital parasites, 643 Examination of specimens for blood and tissue parasites, 644 Immunologic diagnosis, 645 Quality assurance in the parasitology laboratory 646 Medically important parasitic agents, 646 Protozoa, 646 Apicomplexa, 667 Microsporidia, 681 Helminths, 682 Flukes, 682 Tapeworms, 687 Tissue infections with cestodes, 691 Roundworms, 692 Hookworms, 695 Strongyloides stercoralis, 697 Blood and tissue roundworm infections, 700 Bibliography, 706

OBJECTIVES

After reading and studying this chapter, you should be able to: 1. List the major considerations in the collection and handling of specimens for the identication of intestinal and blood and tissue parasites. 2. Describe protocols for sample collection, handling, and transport of specimens for blood and tissue parasites. 3. Describe the general procedures for performing the direct wet mount, fecal concentration, and permanently stained smears. 4. Determine the stages of parasites found during microscopic examination of fecal material with direct wet mount, fecal concentration, and permanently stained smears. 5. Compare the procedures and uses of thick and thin blood smears for the identication of blood parasites.

6. Justify performing a thick and thin blood smear for the identication of blood parasites. 7. Justify the use of molecular assays to detect parasites in clinical specimens. 8. Compare the general characteristics of the major phyla of human parasites. 9. Discriminate between pathogenic and nonpathogenic protozoa on the basis of morphologic features. 10. Justify reporting nonpathogenic intestinal parasites when they are identied in clinical specimens. 11. For the major human pathogens, describe the mechanism of pathogenesis, clinical symptoms, treatment, and prevention. 12. For each organism presented, describe the morphology; life cycle, including the infective and diagnostic stages; and usual procedure for identication. KEY TERMS

Amastigote Appliqué forms Asexual reproduction Axostyle Blackwater fever Bradyzoites Cercaria Chromatoidal bars Cyst Cysticercus Cytostome Denitive hosts Erythrocytic phase Exoerythrocytic phase Filariform larvae Gametes Gametocytes Granulomatous amebic encephalitis (GAE)

Hexacanth embryo (oncosphere) Intermediate hosts Karyosome Kinetoplast Maurer dots Merozoites Metacercaria Microlariae Miracidium Peripheral chromatin Polyvinyl alcohol (PVA) Primary amebic meningoencephalitis (PAM) Proglottids Rapid diagnostic tests (RDTs) Rhabditiform larva Schizogony Schizont Schüffner stippling

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Scolex Sexual reproduction Sporoblast Sporocysts Sporogony Sporozoites

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Diagnostic parasitology

Tachyzoites Trophozoites Trypomastigote Undulating membrane Vectors Ziemann dots

Case in point A 4-year-old male patient was brought to the public health clinic because of intermittent bouts of diarrhea lasting almost 4 weeks. The mother did not note any bright red blood in his stool. The child was pale, listless, and had a protuberant abdomen. He had several small erythematous vesicles on his feet. His mother said that he sometimes ate dirt and always had a good appetite. The family lived in a rural part of Georgia and had a well from which they obtained their drinking water. This part of the county had only recently been connected to the local city’s sanitation system. The white blood cell (WBC) count was 10.2 × 103/µL (reference range, 4.8 × 103 to 10.8 × 103/µL), and the differential showed 14% eosinophils (reference range, 0% to 4%). The hemoglobin level was 6.2 g/dL (reference range, 9 to 14 g/dL), and the reticulocyte count was 8% (reference range, 0.5% to 2%). The red blood cell (RBC) morphology was described as microcytic and hypochromic. The physician ordered a stool culture for bacterial pathogens and an ova and parasite (O & P) examination. The bacterial culture was negative for enteric pathogens, but the O & P examination revealed parasitic organisms and the presence of Charcot-Leyden crystals. Issues to consider After reading the patient’s case history, consider: • Signicant features in the patient’s history • Intestinal parasites that should be considered part of the patient’s differential diagnosis • Additional laboratory tests that could aid in the diagnosis

Parasites are an important cause of human morbidity and death in many parts of the world. However, in the United States and other developed countries, they are seldom regarded as major causes of disease. Although parasites are often associated with gastrointestinal (GI) infection or, in the case of malaria, a blood infection, other parasites can live in organs and be transmitted via organ transplants. Some can invade the central nervous system (CNS). A few can cross the placenta and cause congenital infection. Health care professionals have become increasingly aware that parasites should be considered as possible causative agents of a patient’s clinical condition. The factors that have led to this greater awareness include the rising number of immunocompromised patients susceptible to infections caused by known pathogens and opportunistic organisms, the increase in the number of people who travel to countries that have less than ideal sanitation and a large number of endemic parasites, and the growing population of immigrants from areas with endemic parasites. In addition, there is concern that ongoing climate change might affect the movement of insect vectors into new geographic areas, thus expanding the range of some diseases normally associated with tropical climates. When a health care provider encounters a case of an infection that may be caused by a parasite, the patient’s symptoms and clinical history, including travel history, are signicant

data that must be gathered and shared with the laboratory scientist. The clinician and laboratory scientist should collaborate to make sure that the appropriate specimen is properly collected, handled, and examined. Parasitic infections can be difcult to diagnose, however, because patients often have nonspecic clinical symptoms that can be attributed to a number of disease agents. Knowledge of common pathogens and nonpathogens that exist in specic geographic regions and for a given body site is necessary to ensure proper identication and, if necessary, therapy. Detection and identication of a parasite depend not only on the adequacy of the submitted specimen but also on the procedures established by the clinical laboratory, including the criteria for specimen collection, handling, and transport and for the laboratory methods used. Due to the low incidence of parasitic infections in the United States, many laboratories do not have extensive experience detecting parasites. This chapter discusses the major medically important parasites, their epidemiology and life cycle, and the clinical infections they cause. In addition, it presents the diagnostic features that characterize these agents. Readers are referred to parasitology references for detailed procedures, reagent preparation, and a comprehensive description of parasites that have been implicated in human disease.

General concepts in parasitology laboratory methods Fecal specimens Collection, handling, and transport The collection and handling of a stool specimen before laboratory examination may inuence whether organisms will be identied. The stool should be delivered to the laboratory as soon as possible after collection, or a portion should be placed immediately in a preservative. Trophozoites, the motile and reproductive form of some amebae, or eggs of some helminths may disintegrate if not preserved or examined within a short time after collection. Because many intestinal organisms are shed into the stool irregularly, a single stool specimen may be insufcient to detect an intestinal parasite. Studies have shown that only 58% to 72% of protozoa are detected with a single specimen. Traditionally, for optimal detection of intestinal parasites, a series of three stool specimens collected a day or two apart, within a 10-day period, has been recommended. This procedure of examining each specimen submitted is timeconsuming and labor-intensive. Published articles suggest that in some circumstances, pooling of the three formalin-preserved specimens gives a parasite recovery rate comparable with that of the individual examination of formalinpreserved stools. Although this method saves time, there is a risk that if organisms are present in small numbers, they may be missed on microscopic examination of wet mounts because of a dilution effect (i.e., reduced sensitivity). Another costeffective option includes examination of a second or third specimen only if the rst test is negative and the patient remains symptomatic. Many laboratories establish an algorithm that considers the types of parasitology examinations routinely performed (e.g., antigen detection, nucleic acid

General concepts in parasitology laboratory methods

amplication testing [NAAT], fecal concentration, permanently stained smear), patient population, and specic criteria (e.g., travel, symptoms, immune status, inpatient or outpatient classication) to determine if a single specimen or multiple stool specimens should be examined. It has been recommended that permanently stained smears be made and examined for each specimen individually. In addition, whether the patient is an inpatient or outpatient, the presence of symptoms should dictate whether three specimens are needed. For example, although inpatients frequently acquire healthcare-associated bacterial infections, it is highly unlikely that an inpatient would acquire a healthcare-associated parasitic infection. An immunoassay for Giardia duodenalis or Cryptosporidium may be requested on specimens from immunocompromised patients with diarrhea or children in daycare settings who are symptomatic in place of, or in addition to, the usual O & P procedure. Reex testing often begins with an immunoassay or multiplex procedure for parasite antigens of the more common organisms, such as Giardia, and then a routine O & P examination is performed, if the results are negative, and the patient is symptomatic. The collection container for feces should be clean, dry, sealed tightly, and waterproof (e.g., a plastic container with lid). Commercial systems that incorporate collection container and preservatives are also available. Stool specimens should never be collected from bedpans or toilet bowls; such practice might contaminate the specimen with urine or water, resulting in the destruction of trophozoites or introduction of free-living protozoa. As an alternative, the specimen could be collected on a clean piece of waxed paper or newspaper and transferred to the container. Another alternative is to use a disposable collection container that can be tted under the toilet bowl rim. The specimen should be submitted as soon as possible after passage. The specimen should be properly labeled as discussed in Chapter 6 Stool specimens for parasites should be collected before a barium enema, certain procedures using dyes, or the start of antimicrobial therapy. Antimicrobials can reduce the number of organisms present. If the patient has undergone a barium enema, stool examination should be delayed for 7 to 10 days because barium obscures organisms when specimens are examined microscopically, even after concentration procedures. If a purged specimen is to be collected, it is recommended that a saline or phosphosoda purgative be used because mineral oil droplets interfere with identication of parasites, especially protozoan cysts, an infective dormant form resistant to environmental stress. The second or third specimen after the purge is more likely to contain trophozoites that inhabit the cecum.

Preservation Several methods are available for stool preservation if the specimen will not be delivered immediately to the laboratory. The preservative used is determined by the procedure to be performed on the fecal sample. Regardless of the preservative used, the ratio of three parts preservative to one part feces should be maintained for optimal xation. Table 28.1 presents some of the more common preservatives and their appropriate use. The time that the stool was passed and the time it was placed in the xative should be noted on the laboratory requisition and container. A commercially available two-vial system using modied polyvinyl alcohol (PVA), a resin polymer, in

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Table 28.1 Preservatives commonly used for fecal samples Preservative

Laboratory examination method

Modied polyvinyl alcohol

Permanently stained smear, DNA-PCR

10% formalin

Formalin–ethyl acetate concentration, direct wet mount, and most immunoassays

Sodium acetate–acetic acid–formalin

Permanently stained smears, concentration, and most immunoassays

Merthiolate-iodine-formalin

Concentration and direct wet mount

Single-vial systems (EcoFix, Parasafe, PROTO-FIX)

Concentration, direct wet mount, permanently stained smears, and most immunoassays

DNA, Deoxyribonucleic acid; PCR, polymerase chain reaction.

one vial and 10% formalin in the other vial is commonly used. The system comes with patient instructions and a self-sealing plastic bag for transport. The classic PVA xative, which consists of mercuric chloride (for xation) and PVA (to increase adhesion of the stool to the slide), was traditionally used when a permanently stained smear was to be made. Because of the toxicity of mercuric chloride, modications that do not contain mercury have been developed. The modied PVA preservatives do not provide the same level of microscopic detail as the mercury-based formula, but they are safer acceptable alternatives. Schaudinn xative also contains mercuric chloride. The formalin vial is used for direct wet mount and concentration procedures. Alternative nontoxic xatives, EcoFix (Meridian Bioscience, Cincinnati, OH), Parasafe (Scientic Device Laboratory, Des Plaines, IL), and PROTO-FIX (Alpha-Tec Systems, Vancouver, WA) are single-vial xatives for wet mounts and permanently stained slides that do not use formaldehyde or mercury compounds. The specimen only needs to be added to one vial that can be used for wet mount, concentration, and permanently stained slides. Evaluation of stains made from stools preserved with these compounds has shown differing quality of the stained preparations. In some cases, the background quality was poor, and in others a less sharp morphology of the organism was observed. The xative sodium acetate– acetic acid–formalin (SAF) is another alternative that can be used for the preservation of fecal specimens when concentration procedures and permanent stains will be used.

Macroscopic examination The examination of an unpreserved stool specimen should include macroscopic (gross) and microscopic procedures. The initial laboratory procedure is the macroscopic examination. Laboratories that receive stool specimens already placed into preservative(s) are not able to perform a macroscopic examination. During gross examination, intact worms or proglottids (tapeworm segments) can be seen on the surface of the stool. Gross examination of the specimen also reveals the consistency (liquid, soft, formed) of the stool sample. Consistency may help determine the type of preservative to be used, indicate the forms of parasites expected to be present, or dictate the immediacy of examination. Fig. 28.1 shows the relationship between stool consistency and protozoan stage. Cysts (infective stage) are most likely to be found in formed stool

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Relative numbers

Cysts

Trophozoites

Liquid

Soft

Formed

Consistency of stool specimen

Fig. 28.1 Relationship of stool consistency to protozoan stage.

and sometimes in soft stool, whereas liquid stool will most likely contain the trophozoite stage. During gross examination, the color of the stool specimen is noted. A normal stool sample usually appears brown. Stool that appears black may indicate bleeding in the upper GI tract, whereas the presence of fresh blood may indicate bleeding in the lower portion of the intestinal tract. Any portion of the stool that contains blood or blood-tinged mucus should be selected for wet mount preparations and adding to preservative. A liquid stool specimen should be examined within 30 minutes of passage to detect the presence of motile trophozoites. A formed stool specimen should be examined within 2 to 3 hours of passage if held at room temperature; however, examination may be delayed up to 24 hours after passage if the specimen is placed at 4° C. The specimen should not be placed in a 37° C incubator, which increases the rate of disintegration of any organisms present and enhances overgrowth by bacteria. Following macroscopic examination, the specimen should be placed into the appropriate xative(s) for concentration and preparation of permanently stained smear.

Microscopic examination Several diagnostic methods can be used in the microscopic examination of a fecal specimen: • Direct wet mount examination (iodine-stained and unstained) of fresh stool specimens • Concentration procedures with wet mount examination of the concentrate • Preparation of permanently stained smears In general, concentration and permanent staining procedures should be performed on all specimens.

Wet mount preparations The direct wet mount of unpreserved fecal material is primarily used to detect the presence of motile protozoan trophozoites in a fresh liquid stool or from sigmoidoscopy material. A liquid stool specimen or purged specimen may contain motile protozoan trophozoites; hence, purged specimens should be examined immediately (within 30 minutes) after passage to ensure motility of the organisms. A portion should also be placed in a xative so permanently stained smears for denitive identication can be made. Formed stools are unlikely to yield motile trophozoites, thus a direct wet mount

examination is not necessary. Direct wet mounts are not performed on specimens received in xatives. The wet mount procedure uses a glass slide on which a drop of physiologic saline (0.85%) has been placed at one end and a drop of iodine (Dobell and O’Connor iodine, D’Antoni iodine, or 1:5 dilution of Lugol solution) at the other end. A small amount (2 mg) of feces is added to each drop and mixed well. Each preparation should be covered with a No. 1, 22-mm square coverslip. The preparation should be thin enough so newsprint can be read through it and should not overow beyond the edges of the coverslip. In unxed stool specimens, the saline preparation is useful for the detection of helminth eggs or larvae, motile protozoa, and refractile protozoan cysts. Iodine emphasizes nuclear detail and glycogen masses but kills trophozoites. Wet mounts are also made from a fecal specimen following a concentration procedure. This detects protozoan cysts, helminth eggs, and helminth larvae. Reading the wet mount involves thorough examination of each coverslipped preparation at low power, starting at one corner and following a systematic vertical or horizontal pattern until the entire preparation has been examined. A high-power objective is used to identify any suspicious structures. Oil immersion should not be used on a wet preparation unless the preparation has been sealed.

Concentration techniques Concentration techniques are designed to concentrate the parasites present into a small volume of uid and remove as much debris as possible. Fresh or stool specimens in an acceptable preservative may be used. The concentrate sediment may then be examined unstained or stained with iodine. Protozoan trophozoites do not survive the procedure. Protozoan cysts, helminth larvae, and helminth eggs, however, can be detected using this method. Sedimentation and otation methods, both of which are based on the difference in specic gravity between the parasites and concentrating solution, are used to concentrate parasites into a small volume for easier detection and increased sensitivity. In sedimentation methods, the organisms are concentrated in sediment at the bottom of the centrifuge tube. In otation methods, the organisms are suspended at the top of a high-density uid. Overall, sedimentation methods concentrate a greater diversity of organisms, including cysts, larvae, and eggs. The formalin–ethyl acetate sedimentation (FES) method was the standard sedimentation method. A number of manufacturers now market self-contained fecal

General concepts in parasitology laboratory methods

concentration kits that do not use the solvent ethyl acetate. Some kits incorporating collection and concentration in a single tube may not require centrifugation, making them useful in rural areas and developing countries. Although these kits offer easier disposability and a cleaner preparation because of the ltration method used, studies indicate that their use is more expensive than that of the traditional FES method. The zinc sulfate method is the usual otation procedure, although other methods such as zinc chloride are also available. While the otation methods yield less fecal debris in the nished preparation compared with the FES method, the otation liquids can cause operculated eggs to open or collapse. It also tends to distort protozoan cysts. When used, this procedure may miss infertile Ascaris lumbricoides eggs and Schistosoma spp. eggs. Because of their high density, these eggs sink to the bottom of the test tube. Most organisms tend to settle after about 30 minutes. Therefore the examination should be performed as soon as possible after the procedure has been completed to ensure optimal recovery of organisms.

Permanently stained smears Permanently stained smear preparations of all stool specimens should be made to detect and identify protozoan trophozoites and cysts. The characteristics needed for identication of the protozoa, including nuclear detail, size, and internal structures, are visible in a well-made and properly stained smear. The permanent stains commonly used include iron hematoxylin and trichrome (Wheatley modication of the Gomori stain). The stain of choice in most laboratories is the trichrome stain because results are somewhat less dependent on the technique, and the procedure is less time-consuming. Although some laboratories prepare the stain in-house, manufacturers provide prepared stains and reagents for this procedure. Trichrome staining can be performed on a smear made from a fresh stool specimen xed in Schaudinn xative or from one that has been preserved in PVA. Specimens preserved in SAF do not stain well with trichrome and should be stained with iron hematoxylin. To prepare a trichrome-stained smear of a fresh specimen, applicator sticks are used to smear a thin lm of stool across a 1- by 3-inch slide. The smear is placed immediately in Schaudinn xative; it must not be allowed to dry before xation. For PVA-xed specimens, several drops of specimen are placed on a paper towel to drain excess uid. The material on the paper towel is collected to prepare the smear in the same way as for a fresh specimen. The specimen should be allowed to air-dry thoroughly before staining. In a well-stained trichrome smear, the cytoplasm of protozoan cysts and trophozoites stains blue-green, although Entamoeba coli often stains purple. Nuclear peripheral chromatin (deoxyribonucleic acid [DNA] and proteins on the edge of the nucleus), karyosome (mass of chromatin in the nucleus), chromatoidal bars (dark-staining cytoplasmic inclusions of chromatin), and RBCs stain dark red to purple. Eggs and larvae stain red; however, they are often distorted or destroyed in the staining process. Background debris and yeasts stain green. With an iron hematoxylin stain, the parasites stain gray to black, nuclear material stains black, and background material stains light blue to gray. With either stain, poor xation of fecal material results in poorly staining or nonstaining organisms. Several modications of the trichrome staining procedure have been developed to allow the detection of a group of organisms known as the microsporidia

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All permanently stained smears should be examined by rst scanning for thick and thin areas using lower-power magnication (×10 or ×40 objective). Thin areas should be selected and examined under oil immersion (×100 objective) for identication of organisms. It should take approximately 10 to 15 minutes to adequately examine selected areas. Organisms that stain lightly and may be difcult to identify in either the cyst or the trophozoite stage include Entamoeba hartmanni, Dientamoeba fragilis, Endolimax nana, Chilomastix mesnili, and Giardia duodenalis.

Modied acid-fast stain The Kinyoun modied acid-fast stain is used to detect oocysts of Cryptosporidium spp., Cystoisospora belli (formerly Isospora belli), and Cyclospora cayetanensis. With this procedure, the oocysts appear as magenta-stained organisms against a blue background.

Other specimens examined for intestinal and urogenital parasites Cellophane tape preparation for pinworm The life cycle of the pinworm Enterobius vermicularis includes migration of the female from the anus at night to lay eggs in the perianal area. Therefore a fecal specimen is not the optimal specimen for diagnosis of infection with this organism. Instead, the cellophane tape preparation is routinely used for detection of suspected pinworm infections. This procedure involves swabbing the person’s perianal area with a tongue blade covered with cellophane tape (sticky side out). The collection should take place rst thing in the morning before the individual uses the bathroom or has bathed. After the sample has been taken, the sticky side of the tape is placed on a microscope slide and scanned at low- and high-power magnication for the characteristically shaped eggs. Adaptations of this procedure using paddles with a sticky surface are commercially available.

Duodenal aspirates Material obtained from duodenal aspirates or from the duodenal capsule Entero-Test (HDC, San Jose, CA) may be submitted in cases of suspected giardiasis or strongyloidiasis when clinical symptoms are suggestive of infection but repeated routine stool examination results are negative. In the Entero-Test, the patient swallows a gelatin capsule containing a weighted string. One end of the string is taped to the side of the patient’s mouth; the weighted end is carried into the upper small intestine. After about 4 hours, the string is brought up, and part of the mucus adhering to the surface is stripped off and examined on a wet mount for motile trophozoites. The remainder of the specimen is placed in a xative for a permanently stained smear. Eggs of Fasciola hepatica and Clonorchis sinensis as well as oocysts of Cryptosporidium and C. belli can also be recovered.

Sigmoidoscopy specimens Scrapings or aspirates obtained by sigmoidoscopy may be used to diagnose amebiasis or cryptosporidiosis. These specimens are examined immediately for motile trophozoites, and a portion of the sample is placed in PVA xative so

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permanently stained smears can be prepared for examination. If cryptosporidiosis is suspected, a smear for staining with a modied acid-fast stain or by a uorescent procedure should be prepared.

Urine, vaginal, and urethral specimens Eggs of Schistosoma haematobium and E. vermicularis and trophozoites of Trichomonas vaginalis can be detected in urine sediment. T. vaginalis can also be detected in a wet mount of vaginal or urethral discharge. Envelope culture methods for T. vaginalis, such as the InPouch TV (Biomed Diagnostics, White City, OR), are also available. In this system, the specimen is added to the medium and incubated. Growth of the organism can be microscopically observed through the pouch within 3 days. Rapid antigen detection kits and molecular assays for T. vaginalis are also available.

Sputum In cases of Strongyloides stercoralis hyperinfection, lariform larvae may be seen in a direct wet mount of sputum and bronchoalveolar lavage and washings. Larval forms of other migrating helminths, such as A. lumbricoides and hookworm, can also be detected. Eggs of the lung uke Paragonimus westermani can be identied in a wet mount. In addition, Entamoeba histolytica, Cryptosporidium oocysts, and microsporidia can be found in lower respiratory tract specimens. If the patient is suspected of having a pulmonary infection caused by these agents, the specimen should be examined as a permanently stained smear.

Examination of specimens for blood and tissue parasites Blood smears Depending on the blood parasite, whole blood or buffy coat preparations can be used. Examination of a whole blood smear stained with the Giemsa or Wright stain is the most common method of detecting Plasmodium, Babesia, Trypanosoma, and some species of microlaria. Although motile organisms, such as Trypanosoma trypomastigotes and microlariae (larval form of larial worms), can be detected on a wet preparation of a fresh blood specimen under lowand high-power magnication, identication is made on the basis of characteristics seen on a permanently stained smear. Stained smears from buffy coats are helpful in nding Leishmania spp. and Toxoplasma gondii in the cytoplasm of mononuclear cells. The yeast form of the dimorphic fungus Histoplasma capsulatum can also be seen. Concentration methods using membrane lters can be used to detect Trypanosoma spp. or microlariae, but these methods are rarely used in the clinical laboratory.

Collection and preparation Blood taken directly from a nger stick is the ideal specimen for a malarial smear because it tends to give the best staining characteristics. Blood collected in ethylenediamine tetraacetic acid (EDTA) gives adequate staining if processed within 1 hour. Distortion of the organism may occur if the time to preparation of the slide is longer than 1 hour, and organisms may be

lost if the time exceeds 4 hours. For example, the gametocytes of Plasmodium falciparum may lose their characteristic banana shape and round up. With Giemsa stain, the cytoplasm of the parasite stains bluish, and the chromatin stains red to purple red. If malarial stippling is present, it appears as discrete pinkred dots. The Giemsa stain gives the best morphologic detail but is a time-consuming procedure. Wright stain has a shorter staining period, but the color intensity for the differentiation of parasites is not as good as that with Giemsa stain.

Identication procedure For suspected cases of blood parasites, a thick lm and a thin lm should be made. Both preparations can be made on the same slide or on separate slides. Because the two preparations are treated differently before staining, however, use of two slides may be more convenient. The Giemsa stain provides the best staining of the organisms and should be used on thick and thin lms. Thin smears must rst be xed in methanol before staining with the Giemsa stain. RBCs in the unxed thick smear will lyse during the staining procedure. Unless the RBCs on the slide are rst lysed in distilled water, Wright stain cannot be used for a thick lm because the stain contains methanol, which will x RBCs. A thick lm is best for the detection of parasites (high sensitivity) because of the larger volume of blood and the fact that organisms are concentrated in a relatively small area. The thick lm is made by pooling several drops of blood on the slide and then spread into a circular area with a diameter approximately equal to 1.5 cm. A lm that is too thick peels from the slide; thickness is optimal when newsprint is barely visible through the drop of blood before it dries. The blood should be allowed to dry for at least 6 hours before staining. It should not be xed with methanol before staining; xing prevents lysis of the RBCs. The Giemsa stain releases hemoglobin by lysing unxed RBCs. Initial scanning of the stained smear at ×100 magnication detects microlariae. The thick smear is examined at ×1000 magnication for the presence of malarial organisms. In the thick lm, the RBCs are destroyed, so only WBCs, platelets, and parasites are visible. In a thick lm, the organisms may be difcult to identify, and there is no way to compare the size of infected and noninfected erythrocytes. Therefore species identication should be made from a thin lm because the characteristics of the parasite and the RBCs can be seen. The thin lm is made in the same way as that for a differential cell count. It should be xed in methanol for 1 minute and air-dried before staining with Giemsa stain. The entire smear should be scanned at low-power (×100 magnication) for detection of large organisms such as microlariae; then at least 100 oil immersion elds (×1000 magnication) must be examined for the presence of organisms such as Trypanosoma or intracellular organisms such as Plasmodium or Babesia. Because the RBCs are intact, the thin smear has a higher specicity compared with a thick smear. For a symptomatic patient, several blood smears from samples collected at approximately 6-hour intervals over 36 to 48 hours should be examined before a nal negative diagnosis is made. Parasitemia (percentage of erythrocytes parasitized) can be calculated from the thin blood smear. It is recommended that a minimum of 500 RBCs be counted. The calculation is as follows: (Number of infected RBCs / Total number of RBCs counted) × 100 ≡ Percent infected RBCs

General concepts in parasitology laboratory methods

Biopsy specimens Biopsy specimens are typically examined using stained tissue sections processed in the histology section of a clinical laboratory. Tissues that might yield parasites include skin, muscle, cornea, intestine, liver, lung, and brain. Biopsy specimens are usually needed to diagnose infections with Leishmania spp. because the organisms are intracellular. Depending on the species present, the amastigote (obligate intracellular form) can be detected in tissues, such as skin, liver, spleen, and bone marrow. Cutaneous lesions should be sampled below the edges of the ulcer; surface samples do not yield infected cells. Biopsy of striated muscle may also be performed for the diagnosis of Trichinella spiralis. T. gondii can also be identied by examination of tissue and muscle biopsy specimens.

Cerebrospinal uid Viable organisms in suspected cases of amebic meningitis or sleeping sickness can occasionally be seen in a cerebrospinal uid (CSF) specimen. The trypomastigote, an extracellular nonreplicating form, is readily visible because of the motion of the agellum and undulating membrane. It requires a skillful microscopist, however, to discern amebic motility in a eld of neutrophils. If amebic meningoencephalitis caused by Naegleria fowleri is suspected, CSF can be cultured. Nonnutrient agar is seeded with an Escherichia coli overlay, and the CSF sediment is inoculated onto the medium. The specimen is sealed and incubated at 35° C. The medium is microscopically examined daily for thin tracks made by the amebae as they feed on the bacteria.

Immunologic diagnosis Parasites that invade tissue (e.g., E. histolytica, Trypanosoma cruzi, or T. gondii) are the primary organisms that stimulate antibody production. Serologic tests are sometimes useful if invasive methods cannot be used for identication. In most cases, however, tests for antibody serve only as epidemiologic markers, especially in endemic areas. Detection of immunoglobulin M (IgM) can be useful in identifying infection during the acute phase, but this class of antibody generally declines to nondetectable levels as the infection begins to resolve. Detection of immunoglobulin G (IgG) does not distinguish between a relatively recent infection and a past infection because this class of antibody can persist for years. In some cases, however, detection of antibody is useful—for example, a patient who lives in an area nonendemic for a parasite has recently traveled to an endemic area and now shows symptoms, but the organism has not been detected in a clinical specimen. A positive test for antibody would help conrm a diagnosis. Another disadvantage of antibody tests is that they can have many cross-reactions that limit their diagnostic usefulness. In addition, it is difcult to obtain quality parasite antigen. Many serologic tests are used by reference laboratories, such as the U.S. Centers for Disease Control and Prevention (CDC), and are not commercially available. The parameters that should be considered in selecting a method to be used should therefore include not only cost but also diagnostic yield, patient population, relative incidence of the parasite in the area, and number of specimens to be processed. Classically, such methods as hemagglutination or complement xation were used to detect antibodies to

645

parasitic organisms. Newer tests use uorescent or enzyme immunoassay (EIA) techniques. Immunoassay tests for antibodies to T. gondii or E. histolytica (extraintestinal infections) are available for use in clinical laboratories.

Enzyme immunoassays The major use of EIA has been to detect parasite antigens in clinical specimens. These tests, in contrast with antibody tests, provide information about current infection. Several EIAs are available to detect the presence of Giardia duodenalis, Cryptosporidium spp., E. histolytica, and Entamoeba dispar. The sensitivity and specicity of these tests to detect intestinal parasites is excellent compared to microscopic examination. Some can nd more than one parasite in the specimen by detecting antigens of the organism in the stool using organism-specic antibodies immobilized on a membrane. Rapid EIA kits that can be used with fresh or preserved fecal specimens and kits using monoclonal antibody to detect Giardia and Cryptosporidium antigens are also available. Not all kits detecting E. histolytica, however, can differentiate between E. histolytica (pathogenic) and E. dispar (nonpathogenic). Antigen detection kits have replaced microscopic examinations in some hospital laboratories. Another growing area of EIA application testing is eld diagnosis of malaria. Although most of these tests are used in endemic areas, one has been approved by the U.S. Food and Drug Administration (FDA) for use in the United States. These tests are based on the principle of immunochromatographic antigen capture (see Chapter 10), and use whole blood to detect malarial proteins. The patient’s blood is reacted with monoclonal antibody labeled with dye or gold particles. Some tests are relatively non–species specic and detect a protein, such as parasite lactate dehydrogenase or aldolase, that is common to all four human Plasmodium spp. Other tests may detect a species-specic protein, such as histidine-rich protein, which is associated with P. falciparum. Some tests detect both types of protein to provide a more complete picture of the infective agent.

Fluorescent antibody techniques Direct uorescent antibody (DFA) techniques using monoclonal antibodies have been developed to detect Cryptosporidium oocysts in fecal specimens. Fecal material is spread on a slide, reagent containing the antibody is added, and the specimen is examined under a uorescent microscope for a characteristic apple-green structure. These methods are more expensive than the modied acid-fast procedure but demonstrate greater sensitivity, especially when only rare oocysts are present. A DFA combination reagent for G. duodenalis and Cryptosporidium antigens is also available. The monoclonal antibodies help avoid false-positive and false-negative results. Such procedures are useful in screening large numbers of specimens during epidemiologic studies. DFA assays have greater sensitivity and specicity compared with routine stains. The quantitative buffy coat (QBC; Drucker Diagnostics, Port Matilda, PA) procedure uses the uorescent dye acridine orange to stain nuclear material for detecting malarial organisms in blood. A microhematocrit tube is coated with the uorescent dye. After centrifugation, the parasites can be viewed

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in a small area at the top of the erythrocyte column. This method is more sensitive than a thick smear in demonstrating the presence of parasites, but a thin smear must still be made for denitive identication. However, the instrumentation required may not be available for use in all areas of the world.

Molecular methods Molecular methods for parasite identication are rapidly being adopted by clinical laboratories. Methods can include classic and real-time polymerase chain reaction (PCR) assays. Molecular methods of detection exist that identify several organisms, including malarial parasites. Multiplex PCR panels are commercially available for GI pathogens that can detect a signicant number of viruses and bacteria and a limited number of parasites (e.g., Giardia duodenalis, E. histolytica, Cryptosporidium spp., and in some cases Cyclospora cayetanensis). Dientamoeba fragilis, Blastocystis spp., and microsporidia do not yet have a rapid identication procedure. Several of the current molecular procedures are rapid and do not require specimen processing. Although these newer methods have eliminated the need for routine concentration for selected organisms, the downside is that if the test result is negative and the patient remains symptomatic, routine procedures then should be instituted.

Quality assurance in the parasitology laboratory Quality assurance procedures in the parasitology laboratory are like those in other laboratory sections. An updated procedure manual, controls for staining procedures, and records of centrifuge calibration, ocular micrometer calibration, and refrigerator and incubator temperatures should be available. Reagents, solutions, and kits must be properly labeled. In addition, the parasitology laboratory should have the following: • A reference book collection, including texts and atlases • A set of digital images of common parasites; many are available online • A set of clinical reference specimens, including permanently stained smears and formalin-preserved feces The department should also be enrolled in an external prociency testing program. An ongoing internal prociency testing program can be used to enhance the identication skills of the clinical laboratory scientists, especially if a fulltime parasitologist is not employed. It has been shown that approximately twice as many parasites are detected when a single laboratory scientist staffs the parasitology department compared with departments that rotate personnel through the department. One type of program might assess the reproducibility of results in the examination of fecal specimens. Preserved specimens that have been reported are reexamined as part of this program to see if the initial results (organism identication and quantication) are duplicated. Size is an important diagnostic criterion for parasites, and use of a properly calibrated ocular micrometer ensures accurate measurement of organisms. The micrometer should be calibrated for each objective on the microscope. Calibration requires two parts: (1) the stage micrometer, a 0.1-mm line

that is ruled in 0.01-mm units, and (2) the ocular micrometer, which is ruled in 100 units but has no value assigned to the units. Values for each ocular unit can be calculated by using the stage micrometer according to the procedure found in Appendix D on Evolve.

Medically important parasitic agents Historically, the human medically important eucaryotic parasites were classied as protozoan, helminth, and arthropod. More recently the microsporidia, which belong to the kingdom of fungi, were recognized as human pathogens. Due to their morphology and pathogenesis, the microsporidia are often considered with the parasites. Medically important parasites can be found in phyla representing single-celled organisms, such as the protozoa and sporozoa, and complex, multicelled organisms, such as the helminths (worms). Table 28.2 lists the characteristics of the phyla in which most medically important human parasites are found; they are described in this chapter. The protozoan parasites are classied according to their motility organelles; the ameba move by pseudopodia, the agellates move by agella, and the ciliates move by cilia. The sporozoa are nonmotile. Helminths are classied according to their shape and include atworms (ukes and and tapeworms) and roundworms. Arthropods, such as Pediculosis humanus (pediculosis) and Sarcoptes scabiei (scabies), cause human infestations. They can also sometimes be vectors for infectious pathogens. These agents are discussed in Chapter 33.

Protozoa The protozoan parasites include pathogens and nonpathogens (commensals). Some of the commensal organisms closely resemble the pathogens, so it is important for the laboratory scientist to be able to recognize and distinguish among them. This ensures that a patient will be given appropriate therapy when necessary and not be treated unnecessarily. Commensal parasites must be reported in the test results. It is important to recognize that the commensals are not considered normal biota, but their presence indicates that the patient has ingested fecally contaminated food or water. When a commensal is detected, it is important for the laboratory scientist to examine the specimen closely for the presence of a pathogen.

Intestinal amebae In general, amebae present the most difcult challenge regarding identication. Their average size range is smaller than that of most other parasitic organisms, and they must be distinguished from artifacts and cells that appear in the clinical specimen. Species identication, whether in the cyst or in the trophozoite stage, often is based on the size, number of nuclei, nuclear structure, and presence of specic internal structures. In a direct wet preparation, the motility of the trophozoite may aid in presumptive identication. Overall, however, the permanently stained smear is the best preparation for identication of the amebae. E. histolytica is recognized as a true pathogen, whereas Blastocystis spp. still have questionable status as a pathogen. Other amebae are considered nonpathogens. All of the amebic

Medically important parasitic agents

Table 28.2 Characteristics of phyla of medically important parasites Organisms

Characteristics

Phylum: Sarcomastigophora Subphylum: Sarcodina (ameba)

Subphylum: Mastigophora (agellates)

Single celled Trophozoite and cyst stages

Life cycle

Asexual reproduction

The life cycle of amebae is relatively simple, with direct fecaloral transmission in food or water via the cyst stage and no intermediate hosts. Humans ingest the infective cysts, which excyst in the intestinal tract, and the emerged trophozoites multiply by binary ssion. Trophozoites colonize the cecal area. Fig. 28.2 illustrates a generalized life cycle for amebae and the extraintestinal phase of E. histolytica

Single celled Most move by action of agella

Asexual reproduction Some blood agellates Single celled Move by action of cilia Trophozoite and cyst stages Asexual reproduction Phylum: Apicomplexa (sporozoa)

Single celled Usually inhabit tissue and blood cells Insects and other mammals are involved as part of life cycle May have both sexual and asexual life cycles

Phylum: Microsporidia True eucaryotes with membrane bound nucleus Bacterium-like ribosomes No discernible mitochondria Belong to the kingdom of fungi Phylum: Platyhelminthes (atworms) Class: Trematoda (ukes)

Multicelled and bilaterally symmetric Most are hermaphroditic Egg, miracidium, cercaria, and adult are life cycle stages Fish, snails, and crabs are involved as intermediate hosts in life cycle

Class: Cestoda (tapeworms)

Multicelled, ribbonlike body Hermaphroditic Egg, larva, and adult worm are life cycle stages Mammals and insects are involved as intermediate hosts in life cycle

Phylum: Aschelminthes Class: Nematoda (roundworms)

organisms discussed here live in the large intestine. All amebae possess a trophozoite and cyst stage. B. hominis has additional morphologic stages. The trophozoite is the motile feeding stage that reproduces by binary ssion. The cyst is an infective, environmentally resistant stage. Multiplication of nuclei in the cyst stage also serves a reproductive function.

Move by pseudopodia

Trophozoite and cyst stages for intestinal organisms, except for Dientamoeba fragilis

Phylum: Ciliophora (ciliates)

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Adults of both sexes Egg, larva, and adult worm are life cycle stages May have free-living form or may require intermediate host

Treatment Treatment is administered only for E. histolytica infections; treatment for nonpathogens is not indicated. Luminal amebicides, such as paromomycin, are given to carriers in nonendemic areas to prevent the invasive phase and reduce the risk of transmission. In endemic areas with a high risk of reinfection, treatment may not be indicated. Patients with invasive amebiases are treated with systemic drugs, such as metronidazole and luminal amebicides. In cases of Blastocystis spp. infection, treatment may be indicated if the patient is symptomatic.

Entamoeba histolytica and related organisms E. histolytica and E. dispar are morphologically identical organisms that differ in their effects on the host. E. histolytica is a recognized pathogen, while E. dispar is nonpathogenic. Recently, E. moshkovkii and E. bangladeshi, which are also morphologically identical to E. histolytica, have been identied as belonging to this complex. It is unclear if the latter two organisms are pathogenic; however, some studies indicate E. bangladeshi is an agent of diarrhea. Historically, studies demonstrated that many people were infected with an organism identied as E. histolytica. However, only about 10% of these individuals developed clinical symptoms or invasive disease. It was thought that perhaps two strains of the organism existed, one pathogenic and one nonpathogenic. For many years, this hypothesis remained unproven because there was no way to differentiate the strains morphologically. Clinical studies that took place after the emergence of human immunodeciency virus (HIV) provided the impetus to explain the discrepancy. Studies using electrophoresis identied differing isoenzyme (zymodeme) patterns between organisms that caused clinical symptoms and those found in asymptomatic persons. The studies showed that most strains in asymptomatic cyst passers and in most men who have sex with men were nonpathogenic, whereas several strains from areas with high rates of endemic disease were pathogenic. Further immunologic and DNA probe studies supplied additional evidence, such as differences in epitopes of the galactose-binding lectin, differences in surface antigens, and differences in gene expression. On the basis of this evidence, the noninvasive organism, formerly referred to as nonpathogenic E. histolytica, was named E. dispar. The pathogenic organism continued to be referred to as E. histolytica. E. moshkovkii is morphologically identical to E. histolytica and E. dispar and

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Diagnostic parasitology

Human ingestion of infective cyst in contaminated food or water Trophozoite

Excysting of organism in intestine—asexual reproduction in colon Encyst

Passing of trophozoite in liquid or soft stool —not infective

Hepatic abscess

Infective cyst in formed stool

Erosion of intestinal wall by Entamoeba histolytica only

To liver via circulatory system

Fig. 28.2 Generalized life cycle of intestinal ameba.

was thought to be a free-living organism. Subsequent studies identied it as a GI organism reported from humans in many countries. Its true signicance is unknown, but initial evidence suggests that it is nonpathogenic. The organisms are morphologically identical and must be distinguished on the basis of identication of surface antigens. However, the presence of ingested erythrocytes in the trophozoite stage will distinguish E. histolytica from the other species. E. histolytica is found worldwide, but especially in the tropics and subtropics. It is a major protozoan pathogen for humans, causing an estimated 500 million infections and 50 million cases of colitis and hepatic abscesses annually. It ranks third behind malaria and schistosomiasis as a cause of death resulting from a parasitic infection, accounting for an estimated 40,000 to 100,000 annual deaths. Due to misdiagnosis and under-reporting, the prevalence is thought to be much greater. Prevalence of infection differs according to socioeconomic levels and sanitary practices; infection is more common in developing countries. Travelers to those areas are at increased risk of acquiring infection. Other factors that may inuence susceptibility include specic human leukocyte antigen (HLA) phenotypes and mutations in the hormone leptin. Although the primary mode of transmission is fecal-oral transmission, the organism has also been identied as a sexually transmitted agent among men who have sex with men. HIV infection does not appear to increase the risk of invasive disease.

Clinical infection E. histolytica normally subsists on intestinal bacteria and partially digested food of the host. The pathogenicity of

E. histolytica is reected, however, in its ability to cause invasive intestinal amebiasis and extraintestinal amebic infections. The organism adheres to the mucous layer and cells of the intestine using surface lectins with afnity for galactose and N-acetyl-galactosamine. It invades and disrupts the mucosal barrier, produces contact-dependent killing, and induces apoptosis of the intestinal cells. A protein known as amoebapore will create a channel in the cell that allows rapid inux of calcium, resulting in cell death. The organism lyses and phagocytizes the cell. Invasion of the deeper layers of the intestinal wall is mediated by cysteine proteases that destroy collagen and bronectin. Evidence suggests that trophozoites also use trogocytosis (the process of ingesting pieces of living cells) to disrupt cell membranes. The host secretes proinammatory cytokines, leading to an acute inammatory response and migration of neutrophils and macrophages into the tissue. The organism can also secrete chemoattractants for neutrophils. It kills these cells by contact-dependent lysis, and the subsequent release of lysozymes, superoxides, and collagenases from the neutrophil granules produces additional damage to the intestinal mucosa. The host’s innate and acquired immune responses come into play to prevent colonization. Mucin in the intestinal mucus competes for attachment to the lectin and protects epithelial cells from attack. The host may have intestinal immunoglobulin A (IgA) that helps prevent colonization and repeat infection, and antigen-specic T cells secrete cytokines that have direct cytotoxicity for the trophozoites. The trophozoite is, however, resistant to complementmediated lysis.

Medically important parasitic agents

E. histolytica infection presents in several ways: asymptomatic colonization, amebic dysentery (colitis), and extraintestinal amebiasis. In almost 90% of infections, the patients are asymptomatic. Individuals who are colonized but remain asymptomatic pose a great risk to others because they are cyst passers and therefore infective. In symptomatic individuals, clinical infection may appear in an acute or chronic form. Those with increased risk for severe disease include young individuals, older adults, malnourished individuals, and those receiving immunosuppression therapy. In acute infections, the patient may experience vague abdominal symptoms, such as tenderness and cramping, fever, and up to 20 diarrheic stools per day that contain trophozoites, blood, and mucus. Some stools contain Charcot-Leyden crystals, which are breakdown remnants of eosinophils; their presence in the stool is suggestive of an intestinal parasitic infection but is not specic for E. histolytica In severe E. histolytica infection, the patient may shed pieces of intestinal mucosa. Some patients develop an ameboma (amebic granuloma), a tumorlike lesion that forms in the submucosa of the intestine. This represents an area of chronic lysis and inltration with neutrophils, lymphocytes, and eosinophils. In chronic infections, however, there may be alternating asymptomatic periods and diarrheal episodes. The characteristic lesion in the intestinal mucosa, a askshaped ulcer, is a result of lysis of the intestinal mucosa. The lesion shows a pinpoint ulceration on the mucosal surface and a gradual widening in the submucosal areas and lamina propria as the parasites invade the tissue. The organisms may completely erode the intestinal mucosa and enter the circulation. To survive and colonize the liver, they must resist both antibody and complement activity. Cysteine proteases can inactivate IgG. The trophozoite also uses surface receptor capping—a process of removing surface receptors that have been recognized by antibody. These receptors move to the posterior of the organism and are shed in vesicles. When the trophozoite avoids destruction in the circulation, extraintestinal amebiasis often results. The organ usually colonized is the right lobe of the liver because organisms enter the portal circulation and are then trapped in the venules of the liver. Patients with hepatic abscesses may have symptoms, such as fever, chills, and pain in the upper right quadrant, or they can be asymptomatic. Weight loss, increased WBC counts, or

A

649

elevated liver enzyme levels may be present. Jaundice is usually absent. Lung abscesses may be seen as the result of penetration of the diaphragm by amebae from hepatic abscesses or from hematogenous spread. Invasion of the lung can cause the patient to have chest pain, dyspnea, and a productive cough.

Laboratory diagnosis Patients with diarrhea are most likely to have trophozoites in the stool that may be seen in direct wet mounts or trichrome-stained smears. Sigmoid biopsies may be used to demonstrate the characteristic morphology of the intestinal ulcers or to identify trophozoites in tissue when none can be isolated from the stool specimen. Table 28.3 summarizes the characteristics of E. histolytica trophozoites and cysts and compares E. histolytica with other amebae. In a direct saline wet mount of a diarrheic stool, the trophozoite of E. histolytica may exhibit a progressive, directional motility by extending long, thin pseudopods. The size of the organism ranges from 10 to 50 µm, averaging 15 to 25 µm. The organism is refractile, with the characteristic “bull’s eye” nucleus, consisting of a small central karyosome. Even, ne peripheral chromatin may be only slightly visible. In a trichrome-stained smear, the cytoplasm of the trophozoite appears clean and free from ingested bacteria and vacuoles. Finely granular nuclear chromatin is evenly distributed on the nuclear membrane, and the small central karyosome stains dark purple-red. Ingested RBCs are diagnostic for E. histolytica trophozoites but usually are not seen. If there are no ingested RBCs, the organism must be reported as E. histolytica/E. dispar. The World Health Organization (WHO) and the Pan American Health Organization recommend that E. moshkovkii also be included. Immunoassays are necessary to differentiate these species. Fig. 28.3 shows trichrome-stained trophozoites of E. histolytica. The trophozoite in Fig. 28.3B contains an ingested RBC. Cysts of E. histolytica may have one to four nuclei, each with a small central karyosome and ne, evenly distributed peripheral chromatin. The cytoplasm may occasionally contain chromatoidal bars composed of ribonucleic acid. The average size of the cyst is 10 to 20 µm (Fig. 28.4). The chromatoidal bars are cigar shaped, with rounded ends. Young cysts often contain multiple bars, and mature cysts may show only one.

B

Fig. 28.3 A, Entamoeba histolytica trophozoite (trichrome stain). B, E. histolytica trophozoite. Notice the darkly staining, ingested red blood cell near the nucleus (trichrome stain). (A, ×1000; B, ×1000.)

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Table 28.3 Comparisons of amebae Trophozoite Motility

Cytoplasm

Entamoeba histolytica

15–25

Progressive, directional

Finely granular May contain ingested red blood cells

Entamoeba coli

15–50

Nondirectional

Entamoeba hartmanni

4–12

Endolimax nana

Iodamoeba bütschlii

Trophozoite and cyst nuclear structure

Cyst Size (µm) and shape

No. of nuclei in mature cyst

Chromatoidal bars

Glycogen vacuole

Small, central karyosome Fine, evenly distributed peripheral chromatin

10–20, round

Four

Rounded Elongated Usually seen

Not usually seen Diffuse in young cyst

Vacuolated Ingested bacteria

Large, eccentric karyosomes Coarse, uneven peripheral chromatin

15–25, round

Eight

Elongated Splintered ends Not always seen

Not seen

Nondirectional

Finely granular

Small, central karyosomes Fine, evenly distributed peripheral chromatin

5–10, round

Four

Rounded ends Elongated Not always present

Not seen

5–12

Nondirectional

Vacuolated May contain ingested bacteria

Large, irregularly shaped karyosome No peripheral chromatin

5–12, oval

Four

Not present

Not seen

6–20

Nondirectional

Vacuolated May contain ingested bacteria

Large karyosome surrounded by achromatic granules No peripheral chromatin

6–15, oval or irregular

One

Not present

Single, dened

Diagnostic parasitology

Size (µm)

28

Organism

Medically important parasitic agents

In an iodine wet mount, the nuclei appear as yellowish refractile bodies within the cyst; chromatoidal bars do not take up stain and appear as colorless areas. With trichrome stain, the cyst stains light green to gray; nuclear material and chromatoidal bars stain dark purple-red. Young cysts may show discrete glycogen masses that stain light brown in an iodine wet mount, but in the more mature cyst, the glycogen is diffuse. Cysts can persist for 2 to 4 weeks in a moist environment but may be killed by drying, temperatures over 55° C, superchlorination, or addition of iodine to drinking water. Amebic ulcers of the liver often are detected with ultrasonography or radiography. Subsequent aspiration of the abscess may yield motile trophozoites and necrotic material composed of lysed cells. Serologic methods of detecting antibody to E. histolytica are available; the results are positive in more than 90% of patients with extraintestinal disease. The antibody levels increase after tissue invasion but are not protective. Tests for antibodies, however, are not particularly useful in distinguishing between past and current infection because antibodies can persist for years after an infection has resolved. In addition, these tests provide limited information in patients from endemic areas. Tests that detect E. histolytica antigen in stool provide evidence of current infection. Some kits require a large number of organisms to be present for a positive result; however, antigen detection is still considered more sensitive than a microscopic examination of feces. These tests use the EIA method with

monoclonal antibodies to proteins (e.g., serine-rich antigen) or the galactose/N-acetylgalactose adhesion lectins. A number of kits detect E. histolytica antigens without excluding E. dispar; others differentiate E. histolytica from E. dispar. Point-of-care tests using immunochromatographic techniques assist with the rapid diagnosis of E. histolytica infection. Antigen detection assays can be used on other specimens besides feces. For example, testing serum can be effective in diagnosing amebic liver abscess. Multiplex molecular assays for GI syndromic testing are also available, and E. histolytica is often included.

Entamoeba hartmanni E. hartmanni, once known as small race E. histolytica, is a nonpathogen. It generally resembles E. histolytica in a trichrome-stained smear but is more likely to have an eccentric karyosome or uneven peripheral chromatin resembling that of Entamoeba coli. Size is a major determinant in differentiating E. histolytica from E. hartmanni. The average size of the trophozoites of E. hartmanni is 4 to 12 µm; trophozoites with an average size larger than 12 µm are identied as E. histolytica or E. dispar. E. hartmanni cysts measure 5 to 10 µm; those of 10 µm or larger are identied as E. histolytica, E. dispar, or E. moshkovkii. Fig. 28.5A shows the trichrome-stained trophozoite, and Fig. 28.5B shows an enlarged view of E. hartmanni demonstrating the irregular peripheral chromatin. Fig. 28.6 shows the cyst of E. hartmanni, with three nuclei visible and at least one chromatoidal bar.

Fig. 28.4 Entamoeba histolytica cyst with round-end chromatoidal bars. Two nuclei are visible (trichrome stain, ×1000).

A

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Fig. 28.6 Entamoeba hartmanni cyst (trichrome stain, ×1000).

B

Fig. 28.5 A, Entamoeba hartmanni. Identied by the arrow (trichrome stain). B, Enlarged view of E. hartmanni trophozoite. (A, ×400; B, ×1000.)

PART 2

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28

Diagnostic parasitology

Entamoeba coli Entamoeba coli is a commonly found intestinal commensal transmitted by the ingestion of cysts in fecally contaminated food or water. The average size of the trophozoite is 15 to 50 µm, with most measuring 25 µm (Fig. 28.7A). The nuclear structure is characterized by a large eccentric karyosome and coarse, uneven peripheral chromatin on the nuclear membrane. Fig. 28.7B shows an enlargement of the organism demonstrating the characteristic nuclear morphology. The motility of the trophozoite in a wet preparation is sluggish and nondirectional. In a permanently stained preparation, the cytoplasm of the trophozoite may stain a dark purplish gray and contains vacuoles and ingested materials. The mature cyst has eight nuclei; the immature cyst may have one or two large nuclei, with a large glycogen vacuole. Cysts often stain darkly and unevenly, giving an irregular appearance to the cyst. The large eccentric karyosome is visible, but peripheral chromatin surrounding the entire nucleus may be difcult to see. Chromatoidal bars, when present, have a pointed, splintered appearance. The average size of the cyst is 15 to 25 µm. Fig. 28.8A shows a merthiolate-iodine-formalin wet mount of an E. coli cyst, and Fig. 28.8B shows a trichrome-stained cyst.

Endolimax nana The trophozoite of E. nana has a large karyosome, with no peripheral chromatin on the nuclear membrane. The trophozoite ranges in size from 5 to 12 µm, with the average being less

A

than 10 µm. Fig. 28.9 shows three trophozoites. The cytoplasm is granular and vacuolated. In a wet preparation, the motility is sluggish. In a wet mount, it may be difcult to distinguish the large karyosome of E. nana from the karyosome of E. hartmanni, and the organisms may be misidentied. The cyst of E. nana is oval or spherical, 5 to 12 µm, and has up to four large nuclei (Fig. 28.10). Fig. 28.10B shows an enlargement of the cyst, with three of the characteristic large buttonhole nuclei easily visible. A fourth is partially visible. At least one of the nuclei shows evidence of the thin nuclear membrane.

Iodamoeba bütschlii Iodamoeba bütschlii is less commonly encountered compared with other nonpathogenic amebae. The nucleus is composed of a single, irregularly shaped karyosome surrounded by achromatic granules and a thin nuclear membrane, with no peripheral chromatin. The trophozoites of I. bütschlii, which are 6 to 20 µm in size, show a vacuolated cytoplasm in a permanently stained smear (Fig. 28.11). The oval cyst is 6 to 15 µm (average, 9 to 10 µm) in size and contains a single large karyosome and a large, well-dened glycogen vacuole. The vacuole stains dark brown in an iodine wet mount and appears empty in a permanently stained smear. Fig. 28.12 demonstrates an iron hematoxylin–stained cyst of I. bütschlii

Blastocystis spp. The taxonomy of Blastocystis is unresolved. While originally classied as a yeast, the genus Blastocystis has recently

B

Fig. 28.7 A, Entamoeba coli trophozoite. Notice the darkly staining, highly vacuolated cytoplasm (trichrome stain). B, Enlarged view of Entamoeba coli trophozoite to demonstrate peripheral chromatin (trichrome stain). (A, ×1000; B, original magnication ×1000.)

A

B

Fig. 28.8 A, Entamoeba coli cyst (merthiolate-iodine-formalin) wet mount. B, Entamoeba coli cyst with ve nuclei visible (trichrome stain). (A, ×1000; B, ×400.)

Medically important parasitic agents

653

undergone taxonomic reclassication. Genetically, Blastocystis spp. closely resemble Proteromonas lacerate, a agellate. However, Blastocystis is nonmotile and does not have agella. Ribosomal ribonucleic acid (rRNA) analysis indicates that it is related to the stramenopiles (brown algae and water molds). Studies indicate that at least 12 different species are found in humans and other animals, and Blastocystis hominis

is no longer considered a valid species name. Clinical microbiologists should simply report isolates as Blastocystis sp. Blastocystis is one of the most common intestinal protozoa and has a prevalence rate of greater than 50% in developing countries. Infection occurs in immunocompetent and immunocompromised individuals. Blastocystis has come to prominence as a possible cause of diarrhea in humans, although controversy concerning its pathogenicity persists because of conicting results from numerous studies. Although not considered a common cause of diarrheal disease, this organism,

Fig. 28.9 Endolimax nana trophozoites, identied by arrows (trichrome stain, ×1000).

Fig. 28.11 Iodamoeba bütschlii trophozoite, identied by the arrow (trichrome stain, ×1000).

A

B

Fig. 28.10 A, Endolimax nana cyst, identied by the arrow (trichrome stain). B, Enlarged view of E. nana cyst (trichrome stain). C, Cysts (identied by arrow). (A, ×1000; B, original magnication, ×1000, C, original magnication ×1000.)

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Diagnostic parasitology

Fig. 28.12 Iodamoeba bütschlii cyst with prominent glycogen vacuole (iron hematoxylin stain, ×1000). Fig. 28.13 Blastocystis sp. vacuolar form, identied by arrows (trichrome stain, ×1000).

nevertheless, has been found in patients who have abdominal pain and diarrhea but no other intestinal pathogens. The organism has been associated with chronic infection in some patients. Infected individuals often have a history of travel abroad and, most frequently, of consuming untreated water. Other patients have a history that involves handling of animals. Some studies linked the presence of B. hominis to colitis and irritable bowel syndrome (IBS), whereas others have not. Others have suggested that B. hominis has no role as a pathogen. Several subtypes have been identied based on ribosomal DNA (rDNA), which could explain the differences in clinical presentation. The STI-4 strain is seen primarily in humans, whereas other strains are seen in a variety of animals. In addition, the presence of specic cysteine proteases that may function as virulence factors could affect pathogenicity. These proteases are essential for host invasion and trigger proinammatory cytokines, such as interleukin (IL)-8. Some studies have suggested that certain genotypes may be part of the intestinal microbiota and contribute to intestinal homeostasis. Patients infected with Blastocystis may present with diarrhea, abdominal pain, bloating, anorexia, and atulence. Laboratories often report the presence of the organism quantitatively, with the presence of more than ve organisms per high-power eld and no other known enteric pathogens in symptomatic patients considered evidence of Blastocystis as the cause of GI symptoms. Blastocystis sp. is a polymorphic organism that exists in four forms: ameboid, granular, central vacuolar (central body form), and cystic. The central body form is the most commonly identied. The life cycle is not completely known, and revisions continue to occur. The cyst is ingested and is infective. The organism excysts in the large intestine, where it transforms into the central vacuolar form. It is not known what triggers the change into other forms. It is known that the organism encysts as stool passes through the large intestine. The average size for the vacuolar form is 5 to 15 µm, but up to 20% of organisms are smaller than 5 µm, and a few approach 100 µm. The organism is round and has a ring of cytoplasm lining the inside of the plasma membrane, with as many as four nuclei present, which are usually pushed to the side. There is a large central vacuole that occupies up to 90% of the cell volume and may play a role as a storage organelle (Fig. 28.13). The granular form is similar but possesses

granules in the central vacuole and cytoplasm. The ameboid form, which may be seen in diarrheic stools, is 3 to 8 µm and often lacks a vacuole but possesses several pseudopods. In an iodine mount, the cytoplasm and central area of the vacuolar form stain brown. With the trichrome stain, the cytoplasm stains dark green and the central area may stain pale to intensely green, with the nuclei staining dark purple to black. The cyst form can be found in stool and in culture. The cyst stage is small (3 to 5 µm), is oval to round, and possesses one to four nuclei and multiple vacuoles. Because of its small size, it may resemble fecal debris. It is resistant to the usual chlorine levels in drinking water, and water has been implicated in transmission of the organism.

Tissue amebae Free-living, thermotolerant amebae can tolerate a wide range of temperatures, pH, and salinity. They are found in soil and water (e.g., freshwater sources, domestic water supplies, sewage, swimming pools), usually feeding on bacteria in the environment, but are known to cause human disease. They can gain access to the CNS by inhalation into the upper respiratory tract followed by penetration of the nasal mucosa, which allows them to travel along the olfactory nerve to the brain, or by hematogenous spread from the lungs or skin lesions. Although the number of infections caused by these organisms is low compared with those caused by intestinal protozoans, they are very difcult to diagnose and treat and are associated with a high mortality rate. Naegleria fowleri and Acanthamoeba spp. have been recognized for many years as the amebae most commonly associated with CNS invasion in humans. The media sometimes refer to N. fowleri as the “brain-eating ameba.” The ameboagellate N. fowleri is the only one of about 30 species of Naegleria that has been identied as a human pathogen. It is the causative agent of primary amebic meningoencephalitis (PAM), a rapidly fatal condition involving the CNS. Acanthamoeba spp. have been associated with a more chronic condition, granulomatous amebic encephalitis (GAE), especially in individuals with impaired cell-mediated immunity. In addition, Acanthamoeba spp. have been linked with amebic keratitis in soft contact lens wearers and with

Medically important parasitic agents

Table 28.4 Comparison of central nervous system infections caused by amebae Parameter

Primary amebic meningoencephalitis

Granulomatous amebic encephalitis

Causative agent

Naegleria fowleri

Acanthamoeba spp. and Balamuthia mandrillarisa

Cerebrospinal uid

Trophozoite

Trophozoite

Brain biopsy

Trophozoite

Trophozoite and cyst

Characteristics

Trophozoite, 10–35 µm Large karyosome Broad pseudopods

Trophozoite, 15–45 µm Spinelike pseudopod Cyst, 15–20 µm Wrinkled double wall

Entry

Nasal passage—olfactory nerve to CNS

Lungs and skin with hematogenous spread to CNS

Clinical course

Fulminant (death within 1 week of onset)

Slow and chronic

Population at risk

Children to young adults, healthy (history of water activities in stagnant, warm water)

Immunocompromised

Stages in:

CNS, Central nervous system. The pathogenesis and morphology of Acanthamoeba spp. and Balamuthia mandrillaris are similar. In some cases, Balamuthia appears to have more than one nucleolus in tissue sections compared to one found in Acanthamoeba. a

cutaneous infections in patients with acquired immunodeciency syndrome (AIDS). A comparison of the CNS infections caused by N. fowleri and Acanthamoeba spp. is provided in Table 28.4. Balamuthia mandrillaris, another free-living ameba, is primarily associated with GAE and cutaneous lesions in humans. In addition, Sappinia spp. and Paravahlkampa francinae, also free-living amebae, have been identied in rare cases of encephalitis and meningitis.

Naegleria fowleri Although PAM has been reported from many countries worldwide, it is a relatively rare infection. However, many studies have shown the presence of antibodies directed against N. fowleri in healthy individuals. In the United States, between 1962 and 2021, there were 154 reported cases, with only four survivors. PAM is most frequently reported in Texas (40 cases), Florida (36 cases), and California (10 cases) but has been reported as far north as Minnesota. It occurs in healthy, immunocompetent children and young adults with no predisposing condition. A common factor in infection is the report of recent swimming or other water-related activities in warm, articial lakes or brackish or muddy water or exposure to bottom sediment. Waterskiing, wakeboarding, or other activities that increase the chances of forceful entry of water into the nose may facilitate infection. It is not known why only a few individuals are infected in spite of such

655

common exposure. In recent years, there have been several cases related to sinus irrigation using tap water. The life cycle of N. fowleri is relatively simple, consisting of three stages: (1) free-living amebic trophozoite, (2) transient agellate form that appears when there is a scarcity of nutrients, and (3) environmentally resistant cyst. The trophozoite enters the nasal cavity through inhalation of contaminated water or soil. The amebic form colonizes the nasal cavity, invades the nasal mucosa, attaches to olfactory nerves, penetrates the cribriform plate, moves along the olfactory nerve to the olfactory bulb, and moves into the arachnoid space. From there, it is free to spread throughout the CNS. Infections are thought to be related to inhalation of a large number of organisms or forceful entry of water into the nose (microtrauma) and to the virulence of the strain.

Clinical infection Clinically, the disease cannot be distinguished from bacterial meningitis. The incubation period is usually 2 to 3 days but may range up to 2 weeks. Initial symptoms include severe bifrontal headache, fever (38° to 41° C), stiff neck, and nausea and vomiting. CSF will show evidence of increased intracranial pressure, increased levels of neutrophils and protein, and decreased glucose levels. In the late stages, there may be increased numbers of RBCs in the CSF. The organism multiplies in brain tissue, and within 2 to 4 days, the patient can experience drowsiness, confusion, and seizures that can progress to coma. The disease is usually fatal within 1 week of the appearance of clinical symptoms. At autopsy, there will be evidence of trophozoites along with a purulent exudate, edema, and hemorrhagic and necrotic areas of infection in the brain. N. fowleri is capable of direct cell-to-cell damage resulting in destruction of erythrocytes and other cells, including nerve cells. The invasive properties of the organism are related to its ability to secrete cytotoxic enzymes (e.g., phospholipase, sphingomyelinase) and induce generation of proinammatory cytokines that facilitate tissue destruction. Macrophages and neutrophils are the primary host defense against the organism because the trophozoites are relatively resistant to the actions of host cytokines. Infection with N. fowleri has greater than 95% mortality. Because the symptoms resemble those of bacterial meningitis, specic treatment may be delayed, yet the possibility of cure depends on early diagnosis. Aggressive therapy with intravenous and intrathecal administration of amphotericin B has been used. Rifampin, miconazole, or uconazole and azithromycin have been used in addition to amphotericin B. Survival in several cases has been linked to additional procedures, including use of medically induced hypothermia and the experimental drug miltefosine.

Laboratory diagnosis Diagnosis can be made by nding motile amebic trophozoites in CSF. The trophozoite ranges in size from 10 to 35 µm and moves by explosively extending large, broad pseudopods (lobopodia). The nucleus contains a large central karyosome that is surrounded by a halo. Fig. 28.14 shows a trophozoite of N. fowleri as it appears in a CSF specimen stained with iron hematoxylin. In PAM, CSF contains many segmented neutrophils and RBCs and demonstrates elevated protein levels and normal to decreased glucose levels, which are characteristic of bacterial meningitis.

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Fig. 28.15 Cornea, scraping, calcouor white stain, uorescence microscopy. Polyhedral parasitic cyst (13 µm). Morphology consistent with Acanthamoeba cyst. Fig. 28.14 Naegleria fowleri trophozoite. Note the prominent karyosome (iron hematoxylin stain, magnication ×1000).

N. fowleri does not stain well with Gram stain, but motile trophozoites can be seen in a wet mount of CSF. They must be carefully distinguished from leukocytes. The trophozoite may also be seen in cytocentrifuged CSF specimens stained with Wright stain. The ameba can be converted to the agellate stage by adding one drop of CSF sediment to 1 mL of distilled water and incubating it at 37° C. Conversion to the agellate form occurs in 2 to 20 hours. The organisms can be cultured by overlaying nonnutrient agar with Escherichia coli and inoculating the agar with a drop of the CSF sediment and incubating it at 37° C. Clearing of the agar in thin tracks is evidence of the organism feeding on the bacteria. Trophozoites may be observed microscopically. Cysts, which are approximately 10 µm in diameter and have a round, smooth double wall and a single pore, are not seen in clinical specimens. Immunouorescent staining of CSF with monoclonal antibody can be done in some reference laboratories for detection of the trophozoite. Molecular methods are used to identify strains.

Acanthamoeba Acanthamoeba spp. have been linked to several clinical conditions, including GAE, cutaneous infections, and amebic keratitis. They have also been identied as the host for several pathogenic bacteria, such as Legionella spp., Vibrio cholerae, and Escherichia coli O157, but the role these may play—if any—in pathogenicity is unknown. Cutaneous acanthamebiasis has been associated primarily with patients with AIDS whose CD4 count is less than 250/µL. The condition is characterized by chronic nonhealing lesions that may present as nodules, papules, or ulcerations, especially on the extremities and the face. Lesions may develop at the site of inoculation or may occur as a result of hematogenous dissemination from the lungs. Trophozoites and cysts can be seen in biopsy preparations of the lesion. Although it is possible for Acanthamoeba spp. to enter the CNS through the nasal passage, the organisms are characterized by hematogenous spread to the CNS from a primary inoculation site in the lungs or skin. The organism penetrates the blood-brain barrier because of changes in the endothelial cell barrier caused by the interaction of the parasitic enzymes and host cytokines. GAE is subacute; the incubation time is unknown but may range from months to years. Symptoms may include drowsiness, seizures, loss of reex activity, hemiparesis, headache,

stiff neck, and personality disorders. The trophozoite is uncommonly seen in CSF, and diagnosis is typically made by brain biopsy demonstrating cysts, trophozoites, or both. Histologic preparations of the brain at autopsy show inammatory lesions containing many segmented neutrophils, eosinophils, and trophozoites. A specic therapeutic regimen has not been established because most infections have been diagnosed at autopsy. However, disseminated Acanthamoeba infections have been treated with amphotericin B, pentamidine isethionate, uconazole, ketoconazole, and co-trimoxazole (trimethoprim-sulfamethoxazole). Amebic keratitis, associated with Acanthamoeba spp., has been identied in immunocompetent individuals since the 1980s. Individuals who wear contact lenses, especially the soft and extended-wear types, are the primary at-risk group, with greater than 80% of the cases identied. Factors in these infections include improper storage and disinfection procedures, a history of corneal trauma, or wearing contact lenses during swimming, all of which may cause corneal trauma and subsequent colonization by the organism. The organism binds directly to corneal epithelium via acanthopodia and produces proteases and other enzymes that cause cell lysis. Patients experience photophobia, blurred vision, inammation, ring inltrates, and pain. Because of the similarity in tissue damage, the infection may initially be confused with bacterial or herpes simplex virus infection, delaying treatment. Phase-contrast microscopy of direct wet mount preparations of corneal scrapings can reveal the trophozoite or cyst. The use of calcouor white increases detection of the organisms (Fig. 28.15). Permanent stains, such as trichrome and Wright-Giemsa stains, may also demonstrate the trophozoite in clinical specimens (Fig. 28.16). Isolation of Acanthamoeba may be performed in a manner similar to that for N. fowleri. Acanthamoeba spp. have only two stages: the resistant cyst and the motile trophozoite. The trophozoite ranges from 15 to 45 µm in diameter and has a single nucleus, with a central prominent endosome. Blunt pseudopods and characteristic spinelike projections of the cytoplasm (acanthopodia) may also be seen on a wet mount. The cyst is approximately 15 to 20 µm, spherical, and double walled, with the walls having a wrinkled appearance. Topical applications of chlorhexidine gluconate and ketoconazole have been used to treat cutaneous infections. Corticosteroids are used to reduce inammation. Despite treatment, patients

Medically important parasitic agents

with systemic infections have a poor prognosis, and many patients with keratitis lose their sight in the affected eye.

Balamuthia mandrillaris B. mandrillaris is an emerging opportunistic pathogen that has been identied as a cause of skin lesions and GAE. Unlike Acanthamoeba, which has a wide distribution, this organism is found primarily in soil. From 1990 (when it was rst linked to human illness) to 2019, this free-living ameba has caused more than 200 cases worldwide and about 100 cases in the United States. The mortality rate is greater than 95%. In the Unites States, more than 50% of cases reported occurred in individuals of Hispanic origin. It is unknown if there is a genetic predisposition or if the type of work in which the individuals engage increases exposure risk. Unlike infections with Acanthamoeba spp., which occur primarily in immunocompromised hosts, infection with B. mandrillaris can occur in immunocompetent and immunocompromised individuals. Humans become infected by inhaling airborne cysts of the organism or by direct inoculation through skin lesions. There have been several reported cases of transmission via organ transplants. No person-to-person spread has been documented. Skin infections present as a relatively painless nodule. Once the encephalotropic organism has entered the body through the lungs or skin, it spreads hematogenously. Only rarely will it invade the body nasally and spread along nerve bers to

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the olfactory bulb. Once the organism reaches the blood-brain barrier, it is able to bind to microvascular endothelial cells through receptor molecules. The organism produces proteolytic enzymes such as collagenases and metalloproteases that facilitate tissue damage, and the host’s inammatory response helps increase the permeability of the barrier. In the CNS, the organism multiplies by binary ssion and causes a necrotizing hemorrhagic infection similar to that caused by Acanthamoeba spp. The onset is insidious, with fever, headache, stiff neck, vomiting, and photophobia, and progresses to personality changes and seizures. Onset of symptoms can occur weeks to months after infection, but when the brain is affected, the time to death is short. Treatment involves a multiple antimicrobial regimen, including uconazole, clarithromycin, and sulfadiazine. In most cases, infection is identied at autopsy by nding trophozoites and cysts in the tissue. The trophozoite is 30 to 60 µm in diameter, with broad pseudopods and a large single nucleus, with multiple nucleoli. The cyst is round, 10 to 30 µm in diameter, and it has a single nucleus and is multiwalled. The CSF will usually exhibit increased protein levels, normal or decreased glucose levels, and increased levels of lymphocytes. The culture methods using nonnutrient agar seeded with Escherichia coli are nonproductive because this organism will not feed on gram-negative bacteria. Immunohistochemical or nucleic acid assays are often needed to identify B. mandrillaris

Ciliates

Fig. 28.16 Cornea, scraping, Wright-Giemsa stain, light microscopy Parasitic precyst (13 µm) (arrow). Morphology consistent with Acanthamoeba spp.

A

Only one ciliate, Neobalantidium coli (formerly Balantidium coli), is considered pathogenic for humans. Pigs are the natural host for this organism, and humans serve as accidental hosts. The organism lives in the large intestine, where it may cause mucosal lesions but not extraintestinal infections. Most people with this infection are asymptomatic, but the organism can cause a self-limiting diarrhea with nausea, vomiting, and abdominal tenderness. The life cycle is similar to that of the amebae, with the cyst being the infective stage. This is the largest of the protozoa, and the trophozoite is covered with short cilia. The oval trophozoite (Fig. 28.17A) demonstrates two size ranges: (1) 45 to 60 µm × 30 to 40 µm and (2) 90 to 120 µm × 60 to 80 µm.

B

Fig. 28.17 A, Balantidium coli trophozoite. The arrow denotes the cytostome (trichrome stain). B, B. coli cyst (trichrome stain). (A, ×200; B, ×200.)

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The cilia-lined cytostome is located at the slightly pointed anterior end. The cytoplasm contains food vacuoles. A small opening at the posterior, the cytopyge, is used to expel the contents of food vacuoles. In a wet mount, the cilia are seen propelling the organism with a rotary motion. The rounded, thick-walled cyst (see Fig. 28.17B) averages 45 to 75 µm. Cilia may be seen retracted within the cyst wall. Both stages are characterized by the presence of two nuclei, a kidney bean– shaped macronucleus, and a small, round micronucleus that is usually situated in the small curvature of the macronucleus. The micronucleus is rarely visible in routinely stained smears. Although this is the largest of protozoa, it can be missed during microscopic examination because laboratory scientists are trained to search for protozoan shapes that are typically much smaller.

Pathogenic intestinal and urogenital agellates The agellates constitute another major group of parasites that can inhabit the intestinal tract. The life cycle is relatively simple, resembling that of the amebae (Fig. 28.18). Most agellates have both cyst and trophozoite stages. Dientamoeba fragilis, T. vaginalis, and Trichomonas hominis lack a cyst stage, however, and the trophozoite of these organisms serves as the infective stage. Giardia duodenalis, also known as Giardia intestinalis or Giardia lamblia, originally was considered the only pathogenic intestinal agellate. However, D. fragilis is now recognized as a pathogen. T. vaginalis is a pathogen of the genitourinary

tract in men and women. Table 28.5 shows the characteristics of the trophozoite and cyst stages of the intestinal and genitourinary agellates.

Giardia duodenalis G. duodenalis has a worldwide distribution and has frequently been identied as the causative agent of outbreaks of gastroenteritis and traveler’s diarrhea. It is the most commonly reported intestinal protozoan in the United States. The prevalence rate in underdeveloped countries ranges from 20% to 30%, and the organism is now included in the WHO Neglected Disease Initiative. The main groups at risk for infection are travelers returning from endemic areas, hikers who drink untreated water from streams, and children in daycare centers. Travelers in areas with poor sanitation are often exposed to conditions in which waterborne or foodborne (rare) cysts are ingested. Outbreaks have been reported from swimming pools contaminated with feces. Cysts may remain viable for several months in cold water. They are somewhat resistant to chlorine and iodine, but they are susceptible to desiccation and heating to 50° C. Some animals, such as beavers, may serve as reservoirs and can be a source of infection for backpackers who drink from streams or rivers. The terms beaver fever and backpacker’s diarrhea have been used to describe the condition in this group of people. Children younger than 5 years of age are at risk for infection, and G. duodenalis has caused outbreaks of diarrhea in nurseries and daycare centers as a result of person-to-person contact. Along with E. histolytica, G. duodenalis has been identied as a sexually transmitted pathogen among those who practice oral-anal sex.

Clinical infection

Human ingestion of infective cyst in contaminated food or water

Asexual reproduction

Formed stool Cyst—infective

Liquid or soft stool Trophozoite—not infective

Fig. 28.18 Generalized life cycle for intestinal agellates.

A low number of cysts, as few as 10, can initiate infection with G. duodenalis. Once the cysts are ingested, the gastric acid in the stomach triggers the beginning of excystation. The organism then responds to the slightly alkaline pH in the small intestine and completes the excystation process in the duodenal area, in which the presence of carbohydrates and bile stimulates growth of the trophozoite. Although it does not invade the mucosal surface, it does attach to the surface of columnar epithelial cells. Adherence to the intestinal mucosa is achieved by the ventral sucking disk. Pathologic mechanisms associated with G. duodenalis include a mix of human and organism factors. These result in irritation and damage to the mucosa, as well as interference with absorption of nutrients, fats, and fat-soluble vitamins. No toxins or virulence factors have been identied but, there is evidence of inammatory cell inltration. There are eight genetic assemblages—A to H—identied, with subtypes A1 and A2, and B affecting humans. Strain A has a large number of variant surface proteins (VSPs) that can be altered and thus involved in parasite evasion of the host immune response. The intestinal mucous layer; host proteases and lipases; peptides such as defensin and lactoferrin; the presence of secretory IgA; and the inux of mast cells that produce ILs, such as IL-6, help prevent colonization by the organism. The organism increases apoptosis of intestinal cells via caspases, leading to shortening of the intestinal brush border. Damage to the mucosal surface results in atrophy of the microvilli. Bacterial overgrowth may contribute to symptoms.

Table 28.5 Comparison of intestinal and urogenital agellates Organism

No. of nuclei

Other features

shape 9–21 × 5–15

Falling leaf

Two

Sucking disk, ventral surface Parabasal bodies and axonemes

8–12, oval

Four

Cytoplasm retracted from cyst wall Fibrils and agella inside cyst

Chilomastix mesnili

10–20 × 3–10

Rotary

One

Spiral groove Cytostome

6–10, lemon-shaped

One

Anterior of cyst has nipple-like protrusion

Trichomonas hominis

6–14

Jerky

One

Undulating membrane the entire length of the organism Axostyle through body

No cyst stage

Dientamoeba fragilis

5–12

Nondirectional

Two (20% have one)

Nucleus made of four to eight clustered granules Resembles ameba

No cyst stage

Trichomonas vaginalis

7–23 × 5–12; average, 15–18

Jerky, nondirectional

One

Undulating membrane half the length of the body Found in urine

No cyst stage

Medically important parasitic agents

Giardia duodenalis

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Clinical presentation ranges from asymptomatic infection to acute infection or chronic infection. In most patients, the acute infection manifests as self-limiting diarrhea, with malaise, cramps, nausea, and abdominal tenderness after an incubation period of 12 to 14 days. Explosive, foul-smelling, nonbloody diarrhea is present, while fever and evidence of inammation are absent. Symptoms last 1 to 4 weeks, and the patient may lose a signicant amount of weight. Patients with secretory IgA deciency or achlorhydria seem not only to be more apt to acquire the infection but also to develop chronic disease. In chronic giardiasis, there may be a malabsorption-like syndrome, with up to 20% weight loss, fatigue, anorexia, and steatorrhea with large amounts of gas. Fats and vitamin B12 are two of the substances that may be incompletely absorbed in chronic infection, but macrocytic anemia is not common. Children 6 months to 5 years of age are most susceptible to metabolic problems, including iron deciency, protein malnutrition, and micronutrient deciency. They may also demonstrate growth and cognitive delay or general failure to thrive. Children with preexisting malnutrition are more likely to develop long-term complications. There have been suggestions that there may also be postinfection complications, such as IBS or reactive arthritis in some individuals. Metronidazole (Flagyl), which has been the drug of choice for treating giardiasis, may be prescribed. Albendazole is another drug that may be used.

A

Laboratory diagnosis Feces serve as the usual diagnostic specimen, but shedding of the cysts is irregular, and multiple stool specimens are often required for diagnosis. The trophozoites of G. duodenalis are pear shaped or teardrop shaped, have bilateral symmetry, and measure approximately 9 to 21 µm × 5 to 15 µm. They show a characteristic “falling leaf” motility in a wet mount. In a permanently stained smear, the binucleate organism has been described as having an “old man appearance” (Fig. 28.19A). Two oval nuclei, each with a large central karyosome, are on each side of the midline. Four pairs of agella, midline axonemes, and two median bodies posterior to the nuclei are also present. A large ventral sucking disk composed of microtubules is used by the organism to attach itself to the intestinal wall. The organism often stains faintly with trichrome stain. Fig. 28.19B shows an enlargement of the trophozoite that reveals several morphologic features. Table 28.5 summarizes the characteristics of G. duodenalis and compares them with those of other agellates. Cysts of G. duodenalis are oval and measure approximately 8 to 12 µm × 7 to 10 µm. There are up to four nuclei, and the cytoplasm is often pulled away from the cyst wall. On a permanently stained smear, the retracted agella and other internal structures give the cyst a cluttered appearance. Fig. 28.20 show the presence of several cysts of G. duodenalis EIAs using monoclonal antibodies to detect soluble antigens of G. duodenalis in stool are available. Newer lateral ow

B

Fig. 28.19 A, Giardia duodenalis trophozoite, identied by the arrow (trichrome stain). B, Enlarged view of G. duodenalis trophozoite (trichrome stain). (A, ×1000; B, original magnication, ×1000.)

A

B

Fig. 28.20 A, Giardia duodenalis cysts (arrow). B, Giardia duodenalis cysts (arrows). (A, Trichrome stain, ×1000; B, trichrome stain, ×1000.)

Medically important parasitic agents

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immunochromatographic tests that may detect E. histolytica and/or Cryptosporidium spp. provide results in 15 minutes. DFA tests can be used to identify the cyst of Giardia in a stool specimen. Several of these DFA tests also contain a monoclonal antibody that identies Cryptosporidium. The antigen detection assays have a sensitivity of 63% to 100% and a specicity from 90% to 100%. When clinical symptoms persist and the organism cannot be demonstrated in feces or when the patient does not respond to treatment, duodenal aspiration or the Entero-Test has been used to detect the organism.

Dientamoeba fragilis D. fragilis has a worldwide distribution, with prevalence ranging from less than 1% to greater than 40%. For many years, there were questions about the pathogenicity of D. fragilis. The organism was isolated from the stools of patients who were asymptomatic and from those who had GI symptoms, such as abdominal pain or tenderness and diarrhea. Evidence now suggests that the organism has a role as a pathogen in either acute or chronic infections. As with Blastocystis infections, links to IBS or irritable bowel disease have been suggested. Case reports show that treatment with metronidazole results in the clearance of the organism and alleviation of symptoms. Molecular methods show that there are at least two genotypes. While their signicance is unknown, genotype 1 is more common. The organism was historically described as lacking a cyst stage, but recent animal studies have identied a cystlike structure in feces. The life span of the trophozoite outside the body is very short, so direct transmission is unlikely. Foodborne or waterborne outbreaks are not common, but some infections have been seen in family groups. Controversy about transmission exits in research studies. Some studies suggest that the trophozoite may be transmitted to humans through ingestion of helminth eggs, especially those of Enterobius vermicularis, because co-infection of D. fragilis and E. vermicularis is more common than co-infection of D. fragilis and other organisms. Some, but not all, molecular studies have shown evidence of D. fragilis DNA within the pinworm egg. Whether the cystlike structure can be conrmed as the transmission stage remains to be determined. The morphology of D. fragilis closely resembles that of the amebae, hence the name, but electron microscopy studies of ultrastructure, small-subunit rRNA gene analysis, and the lack of a cyst stage all indicate that it is closely related to the trichomonads. D. fragilis has apparently permanently lost its agella. The organism is characteristically binucleate, with 50% to 80% of organisms demonstrating this characteristic. The delicate nuclear membrane has no peripheral chromatin, and the karyosome consists of four to eight discrete granules, one of which is often larger than the others. The size of the trophozoite ranges from 5 to 12 µm, and the cytoplasm contains many food vacuoles and bacteria (Fig. 28.21). This organism degenerates within hours after excretion, and rapid preservation of the stool is important. Since trophozoites do not usually survive the concentration technique, they will only be detected on a permanent stained smear. D. fragilis can be difcult to see on a trichrome-stained smear because its outline is often indistinct and blends into the background. No commercial tests are available for D. fragilis antigen in stool, so microscopy remains the method of choice.

Fig. 28.21 Dientamoeba fragilis binucleate trophozoite, identied by the arrow (trichrome stain, ×1000).

Trichomonas vaginalis T. vaginalis, a pathogen of the urogenital tract in men and women, causes trichomoniasis, one of the most common, nonviral, sexually transmitted diseases (STDs), with an estimate of 180 million cases worldwide. Approximately 3 million to 5 million new cases are reported in the United States each year, with up to two thirds of these occurring in women 15 to 24 years of age. Humans are the only host, and the presence of infection with the organism is associated with other STDs, especially gonorrhea. Trichomoniasis has been increasingly associated with adverse pregnancy outcomes, including transmission of the organism to the newborn, in addition to cervical neoplasia and pelvic inammatory disease. The most signicant complication is increased risk of HIV transmission primarily because of the inammatory response that compromises the mucosal barriers to HIV. T. vaginalis lacks a cyst stage, and the trophozoite stage is infective through sexual contact. The pear-shaped trophozoite assumes an ameboid form on contact with epithelial cells of the genital tract. Adhesion molecules, known as lipoglycans, mediate this binding. The organism is susceptible to rapid drying in the environment, but a few cases of nonsexual transmission have been reported. In women, the infection is primarily localized to the vagina, resulting in itching and the production of a frothy, creamy, mucopurulent vaginal discharge as well as dysuria. About one third of infected women, however, are asymptomatic. A chronic state can develop, with mild symptoms of pruritus and scanty discharge. Women with chronic infection are often a major source of transmission to sexual partners. Men infected with T. vaginalis are usually asymptomatic and serve as carriers, although they can develop nonspecic urethritis with a milky discharge that lasts up to 4 weeks. T. vaginalis is now considered an important cause of nongonococcal urethritis. Long-term immunity is not developed after an acute infection, and reinfection can occur. The diagnosis in women is typically made by nding the trophozoite in vaginal discharge or occasionally in urine; in men, the trophozoite is seen in urine or prostatic secretions. The organism is 5 to 18 µm in diameter and has four anterior agella and an undulating membrane that extends one half the length of the body. In a wet mount, the trophozoite has a characteristic jerky motility, and the motion of the agella and undulating membrane may be seen. The preparation should be examined

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immediately because the organism loses viability quickly. In a Giemsa-stained preparation, the pear-shaped organism shows the presence of an axostyle extending the length of the organism, a single nucleus near the anterior end, and chromatic granules extending the length of the axostyle. Fluorescent staining of exudates using acridine orange will demonstrate a typical uorescent morphology with a yellow to green nucleus. Although most clinicians rely on wet mount preparations, the method has relatively low sensitivity (30% to 70%). Until the advent of molecular methods, culture of the organism from exudate was considered the most sensitive and specic method of detection, although an inoculum of 300 to 500 organisms is necessary. Commercial systems using a plastic pouch containing culture medium are available for detection of the organisms. The specimen should be inoculated into the pouch within 30 minutes of collection to ensure optimal viability of the organism. Culture specimens should be held for up to 5 days and then examined microscopically through the pouch. PCR methods and antigen detection assays are also commercially available. Rapid antigen detection tests are based on immunochromatographic EIA procedures using monoclonal antibodies. These tests have relatively high sensitivity compared with examination of a wet preparation and often test for more than one agent of vaginal infection. PCR tests are especially useful in detecting infection in asymptomatic men. Infections are usually treated with metronidazole. Treatment of sexual partners is suggested to obtain optimal cure and prevent reinfection.

Nonpathogenic intestinal agellates Chilomastix mesnili and Pentatrichomonas (Trichomonas) hominis are intestinal nonpathogens that must be differentiated from pathogenic agellates. C. mesnili trophozoites are pear shaped and approximately 10 to 20 µm long × 3 to 10 µm wide. The cytostome and nucleus are prominent in the anterior of the organism, and a spiral groove encircles the body of the organism. The nucleus has a small central karyosome and is surrounded by brils that curl around the cytostome to give a so-called “shepherd’s crook” appearance. The cytostome is elongated and rounded at the anterior and posterior. The cyst of C. mesnili, which measures 6 to 10 µm, is lemon shaped and has an anterior nipple. The nucleus, cytostome, and curved brils are visible in a stained smear (Fig. 28.22).

The P. hominis trophozoite is 6 to 14 µm long, with a prominent axostyle extending through the posterior of the organism, four anterior agella, and an oval nucleus with a small karyosome. The undulating membrane extends the length of the organism and is joined to the body along the costa. Trophozoites stain weakly, making them difcult to detect on stained-smears. Enteromonas hominis and Retortamonas intestinalis are two additional nonpathogenic agellates occasionally found in clinical specimens.

Case check 28.1 The intermittent bouts of diarrhea in the young patient in the Case in Point could be caused by any organism—viral, bacterial, or parasitic. The culture results were negative, so that would seemingly rule out a bacterial cause. Viral agents are not routinely cultured, nor would we expect viral causes to extend for such a long period. Stools from patients with parasitic infections caused by G. duodenalis are usually characterized as having a pale, frothy, gassy, and greasy appearance. Stools from patients with symptomatic amebiasis caused by E. histolytica often demonstrate blood. Neither organism is associated with pica (eating dirt or other nonfood material). In general, infections with protozoa do not cause eosinophilia.

Blood and tissue agellates The hemoagellates in the genera Leishmania and Trypanosoma differ in several ways from the intestinal agellates. First, they are transmitted by insect vectors, organisms that can transmit disease-causing agents. These vectors are necessary for completion of the life cycle. Second, these organisms have different life cycle stages for diagnosis. Fig. 28.23 shows the four life cycle stages of the hemoagellates. The trypomastigote and amastigote are the diagnostic stages found in humans. The amastigote is an obligate intracellular organism, 2 to 5 µm in diameter, found within macrophages and liver, spleen, or bone marrow cells in diseases caused by Leishmania spp. In addition, the amastigote stage may be seen in cardiac or GI cells of patients infected with Trypanosoma cruzi. The trypomastigote, a agellated form measuring 15 to 20 µm in size, is found in the blood, lymphatic uid, and CSF of patients infected by Trypanosoma spp. The epimastigote and promastigote stages are seen in the insect vectors.

Leishmania The genus Leishmania contains several complexes of at least 20 species that cause disease in humans: Leishmania tropica, Leishmania mexicana, Leishmania braziliensis, and Leishmania donovani complexes. Leishmaniasis is a zoonotic infection in which dogs and rodents serve as the primary reservoir hosts for all species, and humans serve as an accidental host. The insect vectors are sand ies of the genera Phlebotomus and Lutzomyia

Clinical infections

Fig. 28.22 Chilomastix mesnili cyst, identied by the arrow (trichrome stain, ×1000).

Clinical infections range from single, self-healing skin ulcers to visceral disease affecting multiple organs. L. tropica complex, the cause of cutaneous leishmaniasis, or Oriental sore, is found primarily in the Far East and North and Central Africa. The condition is characterized by the presence of a crusted

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HUMAN Tissue (Leishmania spp. and Trypanosoma cruzi) Kinetoplast

Blood/cerebrospinal fluid (Trypanosoma spp.)

Nucleus

Amastigote

Trypomastigote  Free flagellum  Undulating membrane attached posterior to nucleus

 Retracted flagellum

INSECT

Sand fly (vector for Leishmania spp.)

Promastigote  Free flagellum

Tsetse fly Reduviid bugs

Epimastigote  Free flagellum  Undulating membrane attached anterior to nucleus

Fig. 28.23 Life cycle stages of the blood and tissue agellates.

circular lesion on any exposed body surface, especially the face and extremities. The lesion begins as a small, red papule at the site of the insect bite and progresses to a lesion with an elevated indurated margin that may reach 8 cm. The lesion resolves in several months and provides incomplete immunity against future infection. L. mexicana complex, the cause of New World cutaneous leishmaniasis, is found in South and Central America. Another form of the disease, chiclero ulcer—often associated with L. mexicana complex—is characterized by lesions on the ear. The infection is self-limiting and does not invade mucosal surfaces, but secondary bacterial infection can occur. L. braziliensis complex is the causative agent of mucocutaneous leishmaniasis, or espundia. This infection manifests itself as an initial lesion that may increase in size, invading and destroying the mucosal surfaces of the nose and mouth. It may also destroy cartilage, leaving the patient with signicant disgurement. L. braziliensis complex is found primarily in Mexico and Central and South America. The most severe infection, visceral leishmaniasis, or kalaazar, is endemic in parts of South America, Africa, southern Europe, and Asia, and it commonly affects children. The causative agents are organisms of the L. donovani complex. In this disease, organisms spread through the lymphatics and invade organs of the reticuloendothelial system, including the liver, spleen, lymph nodes, and bone marrow. Patients with kalaazar exhibit malaise, anorexia, weight loss, headache, and fever. In addition, they may show splenomegaly and hepatomegaly with elevated liver enzyme levels, hypogammaglobulinemia, and hypoproteinemia. Leishmania-containing macrophages invade the bone marrow. The kidneys and heart may also be affected. A few patients experience a nodular or macular rash, known as post-kala-azar dermal leishmaniasis, on the face, trunk, and limbs months to years after treatment. If untreated, visceral leishmaniasis is often fatal within 2 years. Cases of cutaneous leishmaniasis and visceral leishmaniasis have been

seen in several military personnel serving in Middle Eastern countries, such as Iraq and Afghanistan. Visceral leishmaniasis has also been reported to be transmitted through solid organ transplants. Previously, standard therapy for all leishmanial infections was pentavalent antimony compounds. However, liposomal amphotericin B and paromomycin were used in the past few years. A new drug, miltefosine, has been successful in treating infections.

Life cycle Fig. 28.24 shows the generalized life cycle for Leishmania spp. When the female insect takes a blood meal, she ingests the amastigote stage. The parasite develops as a promastigote in the gut of the insect and migrates to the salivary glands when mature. The promastigote is transmitted to humans through the salivary glands of the insect when it takes a blood meal. The promastigote is inoculated into the dermal-epidermal junction and taken up via receptor-mediated phagocytosis by a macrophage. Within the macrophage, it converts to the amastigote stage and multiplies within the cell. When the cell ruptures, amastigotes are released and invade other macrophages. Parasite and immune cell interactions may lead to an inammatory response that controls the parasite but also causes tissue damage.

Laboratory diagnosis The amastigote is the diagnostic stage in humans. It is a small intracellular stage found in macrophages or histiocytes around the periphery of the skin lesions (L. tropica or L. braziliensis) or within cells of a bone marrow aspirate or liver or spleen biopsy specimen (L. donovani). Wright staining of bone marrow aspirate reveals an oval organism 2 to 5 µm in diameter, with pale blue cytoplasm, a large red nucleus, and a rodlike kinetoplast (a structure rich in DNA associated with the basal body near the base of the agellum) within the cytoplasm (Fig. 28.25). The amastigote has also been reported extracellularly in cases of cutaneous leishmaniasis.

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Multiplication of amastigotes intracellularly

Ingestion of amastigote by sand fly

Promastigote phagocytosis by macrophage and transformation into amastigote

Transformation of amastigote into promastigote in midgut of fly

Fly’s biting of human and regurgitation of promastigote

Fig. 28.24 Life cycle of Leishmania spp.

and more chronic of the two diseases. A long asymptomatic stage is followed by death years later. East African sleeping sickness, caused by T. brucei rhodesiense, is characterized by a rapid course, often resulting in death within 6 months after the onset of symptoms. East African sleeping sickness is a zoonosis, with game animals serving as important reservoirs of T. brucei rhodesiense. Humans are the main reservoir for T. brucei gambiense, but this species can sometimes be found in animals. T. cruzi, the agent of American trypanosomiasis or Chagas disease, is discussed in the next section.

Clinical infections

Fig. 28.25 Amastigotes of Leishmania spp., identied by arrows (Giemsa stain, ×1000).

Promastigotes (the insect form of the parasite) have been identied in some patient samples. Serologic tests, such as direct agglutination and indirect uorescent assays, can be used to detect antibodies in individuals from nonendemic areas.

Trypanosoma Trypanosomes are blood and CSF agellates that require an insect vector. Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense are the causative agents of human African trypanosomiasis, commonly called sleeping sickness, which is seen primarily in Central Africa. The tsetse y (genus Glossina) serves as the insect vector. West African sleeping sickness, caused by T. brucei gambiense, is the more common, milder,

Initial symptoms of African sleeping sickness include a local inammatory reaction with tenderness, edema, and erythema at the site of the insect bite. This occurs within 2 to 3 days and can last up to 4 weeks. During this time, the organisms are reproducing and beginning to enter the bloodstream. The rst stage of the disease is the hemolymphatic phase, which occurs about 1 to 3 weeks after infection. Trypomastigotes enter the blood and lymphatics, and the patient experiences generalized symptoms, including fever, headache, joint and muscle pain, weakness, and lymphadenopathy. Enlargement of the lymph glands in the posterolateral triangle of the neck is known as the Winterbottom sign. Edema in the legs and arms and around the eyes is possible. While circulating, the organisms can resist antibody-mediated destruction as a result of continuous antigenic variation. A protective glycoprotein layer helps the organism resist direct lysis by complement. The second (meningoencephalitic) stage begins as the trypomastigotes invade the CNS; the patient develops severe headaches, mental dullness, and apathy, and may experience

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Tsetse fly’s biting of human and ingestion of trypomastigotes

Development in gut of fly

Trypomastigotes in blood and cerebrospinal fluid

Trypomastigote injection into human by bite

Possible migration of amastigotes to choroid plexus of brain

Epimastigote in salivary gland of fly

Formation of metacyclic trypomastigote

Fig. 28.26 Life cycle of the agents of sleeping sickness (Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense).

coordination problems, altered reexes, and paralysis. Eventually, the patient has convulsions, lapses into a coma, and dies.

Life cycle The tsetse y is the biological vector for agents of sleeping sickness. Fig. 28.26 shows the generalized life cycle of these agents. The y ingests the trypomastigote stage when it takes a blood meal from a human. The organisms migrate to the insect gut and multiply. The organisms later migrate back to the salivary gland where they transform into the epimastigote form. The epimastigotes multiply and develop into infective metacyclic trypomastigotes, which are transmitted to humans in saliva when the y takes another blood meal. The trypomastigote form rst circulates in the blood and lymphatic system, ultimately invading the CNS.

Laboratory diagnosis The diagnostic stage in humans is the trypomastigote, which is usually seen in a Wright-stained blood smear, although wet lms may be used to detect motile trypomastigotes. Blood concentration methods are often needed because of the small

number of organisms present. The organism can also be detected in lymphatic uid and CSF. In infected individuals, CSF will often show increased numbers of WBCs (primarily lymphocytes) and elevated protein levels. The trypomastigote is 15 to 20 µm long, with a single large nucleus and a posterior kinetoplast to which is attached the agellum of the undulating membrane (Fig. 28.27). Trypanosoma spp. cannot be differentiated on a blood smear; therefore the organism is reported as Trypanosoma sp. Final determination of species may be made on the basis of clinical symptoms and the geographic area. The QBC method, which has been used in the detection of malarial parasites, has also been adapted for the detection of trypomastigotes. Field diagnosis of infection is important in endemic areas, and a serologic method using a card agglutination test for trypanosomiasis (CATT) and a micro-CATT have been developed. The test uses lyophilized T. brucei gambiense antigens to detect antibodies in blood. The test cannot distinguish current infection from past infection; therefore microscopy should be used to conrm the presence of the organisms in blood. It cannot be used as a test of cure because antibodies can persist for years after treatment. A latex agglutination test

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Fig. 28.27 Trypanosoma trypomastigote in a blood smear. The anterior agellum is not visible in this organism. (Giemsa stain, ×1000.)

that uses three surface antigens is considered more specic than the CATT. There is no comparable method for detecting T. brucei rhodesiense

Trypanosoma cruzi Chagas disease is a zoonotic infection found primarily in rural areas of Mexico and Central and South America. It is caused by the hemoagellate T. cruzi, transmitted by the triatomid bug, also known as the reduviid or kissing bug (Triatoma sp. or Panstrongylus sp.). It has been estimated that up to 20 million people in these regions are infected. Reduviid bugs live in the mud walls or thatch walls of a dwelling during the day and come out at night to take a blood meal from the human inhabitants. Some animals, such as armadillos and opossums, may also be infected by the organism and serve as reservoirs. Sporadic cases of infection have been reported among nonimmigrants in the United States because several of the insect vectors exist there. Although insect transmission is most common, the organism has been transmitted via blood transfusion (especially platelet concentrate), transplanted organs, and congenital infection. In endemic areas of the world, transmission via transfusion is relatively common, and in North America, several cases of transfusion-transmitted Chagas disease have been reported. Donors in these cases were asymptomatic and in the latent or chronic state of the disease. There are estimates that greater than 300,000 immigrants living in the United States are chronically infected and could be capable of transmitting Chagas disease through transfusions or solid organ transplantations. Screening of blood donors for antibodies to T. cruzi in some countries of Central and South America has decreased the rate of transfusion-related transmission. Screening tests for blood donors in the United States have been implemented.

Clinical infections Infections may be symptomatic or asymptomatic. The infection is divided into three phases: acute, intermediate (latent), and chronic. After the insect bites, there is an incubation period that ranges from 2 to 4 weeks. As the organisms enter the blood, the acute phase begins and lasts 4 to 8 weeks. Individuals in the acute phase may be asymptomatic or have local symptoms, including the presence of a chagoma (ulcerative skin lesion at the site of the insect bite) or unilateral

edema around the eye (Romaña sign) if the bite is near the ocular conjunctiva. Systemic manifestations include fever, lymphadenitis, hepatosplenomegaly, malaise, muscular pains, and diarrhea and vomiting. Adults often have a milder form of the disease, and the most severe form of acute infection usually occurs in children. This may manifest itself as myocarditis or meningoencephalitis. If untreated during the acute phase, the patient will most likely remain chronically infected. The latent period develops as the trypomastigotes disappear from the circulation and invade the cells of the cardiac or GI system. The latent phase can last 10 to 40 years after infection, but only about 30% to 40% of patients develop the chronic form of Chagas disease—primarily cardiomyopathy. Chronic myocarditis develops with damage to all heart chambers and the conduction system. This may progress to tachycardia and eventually congestive heart failure. In some patients, the chronic form may manifest itself as megacolon, megastomach, or megaesophagus, which results in difculty swallowing and disturbances to intestinal motility. Damage to the organs is caused by a combination of continuous lowlevel parasitemia and immune-mediated tissue damage, including high levels of TNF-α and IFN-γ. Polyclonal B-cell activation in the acute phase produces antibodies that have a weak afnity for the organism and may cross-react with heart tissue. Controversy exists about the role of these autoantibodies in tissue damage. When the patient enters the chronic stage of the disease, there are periods of intermittent parasitemia of trypomastigotes, during which time the organism may be transmitted.

Life cycle Transmission of the organism to humans occurs when the insect vector defecates in the area surrounding its bite site. Metacyclic trypomastigotes in the feces are scratched into the bite site and invade the bloodstream. The trypomastigotes circulate in the bloodstream and eventually enter the cells, where they transform into amastigotes. Cells of the cardiac muscle and skeletal muscle are most commonly infected. Within the cell, the amastigote multiplies and causes the cell to rupture. Free amastigotes then invade other cells. A few of the released amastigotes will transform into trypomastigotes in the bloodstream and can be ingested by the insect to continue the life cycle. Fig. 28.28 shows the life cycle of T. cruzi

Laboratory diagnosis The primary diagnostic stage in blood during acute infection is the trypomastigote. It is an elongated structure 15 to 20 µm long that often appears in a C or U shape. Like the other trypomastigotes, it shows a single, large-nucleus midbody; a single anterior agellum; and a posterior kinetoplast to which the undulating membrane is attached. In most Wright-stained smears, the nucleus and kinetoplast and parts of the undulating membrane are well stained, but only the suggestion of a agellum can be seen at the anterior end. In the chronic stage, the organism can be seen as an amastigote in a cardiac or other tissue biopsy specimen. The morphology of all trypomastigotes is similar (see Fig. 28.27); therefore the complete patient history must be obtained to determine the species present. If the history is not available, then the organisms are reported as trypomastigotes of Trypanosoma spp. Diagnosis of chronic infection with T. cruzi is based on patient history, clinical symptoms, and

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Development in gut

Trypomastigote circulation in blood of host

Reduviid bug’s biting of human and ingestion of trypomastigote Epimastigote

Some amastigote transformation back to trypomastigotes

Bursting of cell, which releases amastigotes

Intestine of bug

Metacyclic trypomastigotes

Bite

Feces

Reinfection Feces deposition near site of bug bite Trypomastigote entry into bloodstream through bite

Trypomastigote transformation into amastigote and multiplication

Trypomastigote entry into tissue cells (liver, heart, macrophage)

Fig. 28.28 Life cycle of Trypanosoma cruzi.

presence of IgG antibodies to the organism. Serologic testing for antibodies to T. cruzi is also used in screening blood donors in the United States and other countries. Xenodiagnosis is used in Central and South America as a diagnostic method. A laboratory-raised triatomid bug is allowed to feed on patients suspected of having T. cruzi infection. The insect’s feces are examined on a regular basis for the presence of the parasite. Presence of the parasite in the insect’s feces indicates that the patient was infected. This method is most helpful for diagnosing infections in the chronic stage, when fewer parasites are present. Rapid diagnostic tests (RDTs) using whole blood are under evaluation for testing individuals in remote areas.

Apicomplexa The phylum Apicomplexa includes blood and tissue parasites. This group of organisms shows a diversity of morphology and transmission methods and can infect many different body sites. The life cycles are complex, characterized by sexual reproduction and asexual reproduction (reproduction when offspring arise from a single parent) phases. In addition, some may require an insect vector or intermediate host for completion of the life cycle. Humans can serve as denitive hosts

when sexual reproduction (offspring arise from the exchange of genetic material between two parents) occurs in human tissues and as intermediate hosts when asexual reproduction occurs.

Plasmodium Plasmodium spp., which cause malaria, remain endemic throughout the world in tropical and subtropical countries, with almost one half of the world’s population residing in an endemic area. Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium falciparum are the causative agents of human malaria. A fth species, Plasmodium knowlesi, which originated in simian species, is now associated with cases of human malaria and has been reported in most countries of Southeast Asia. Along with schistosomiasis and amebiasis, malaria is a major cause of death in people in underdeveloped countries. In 2020, there were an estimated 241 million cases worldwide with 627,000 deaths. The majority of infections occur in sub-Saharan Africa, where P. falciparum is the predominant species. Children under 5 years of age are the most at risk and accounted for 77% of malaria deaths in 2020. In the United States, approximately 2000 cases are reported annually, with P. falciparum being the causative agent in more

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than 50% of cases. Most cases are seen in persons who have traveled to endemic areas. P. vivax has the widest geographic distribution and is the species most likely to be found in temperate climates. P. vivax and P. falciparum cause more than 95% of infections. P. ovale is primarily conned to western parts of Africa; P. falciparum and P. malariae have similar distributions throughout Africa and in tropical countries. In general, infections caused by P. vivax, P. ovale, and P. malariae are less severe than those caused by P. falciparum.

Clinical infections Although malaria is usually transmitted through the bite of an anopheline mosquito, transmission through blood transfusion and infected needles has been reported. Transplacental infection also has been documented. In these cases, the organism is sequestered in the placenta and can compromise fetal development because it affects transplacental transport of nutrients. The malarial paroxysm, which begins 10 to 15 days after the bite of an infected mosquito, is associated with the growth of the parasites in RBCs and release of high levels of cytokines. Symptoms are linked to the rupture of RBCs and release of merozoites (parasitic forms produced from multiple ssion), malarial metabolites, and endotoxin-like substances into the bloodstream. The malarial paroxysm has three phases. The prodromal phase involves headache, bone pain, nausea, and/or ulike symptoms. In the rst phase the patient experiences a shaking chill lasting 15 to 60 minutes. The second phase is characterized by a fever up to 40o C along with headache, myalgia, and nausea lasting 2 to 4 hours. In the third and nal phase the fever nally breaks. Over the course of 2 to 4 hours, the patient begins to sweat profusely and is left exhausted and sleepy. This cycle repeats itself at regular intervals, depending on the species of malarial organism present, as parasites exit infected RBCs. The anemia seen in malaria is the result of lysis of erythrocytes, removal of the infected cells by splenic macrophages, and in some cases immune clearance of erythrocytes coated with immune complexes of malarial antigens and antibodies. In small children and in those with heavy infections, severe anemia can develop. Long-term infection with any malarial organism may result in damage to the liver and spleen caused by deposits of malarial pigment (hemozoin). P. vivax and P. ovale infections generally do not have the range of complications seen with other species. Because the organism invades reticulocytes, there is generally low-level parasitemia (2% to 5%). However, the persistence of hypnozoites that remain dormant in the liver can lead to relapses within 3 years of initial exposure. P. malariae infections can lead to nephrotic syndrome, which arises from the deposition of circulating immune complexes of malarial antigens and antibodies on the basement membrane of the glomeruli, causing an autoimmune-like reaction. This organism has the most chronic course, and recrudescence occurs even decades after the initial infection. The most severe form of malaria is caused by P. falciparum. Up to 50% of erythrocytes may be infected, and the primary complication is development of cerebral malaria. Between 20% and 50% of deaths caused by P. falciparum are the result of CNS complications. The parasite-infected RBCs demonstrate

sticky knobs that mediate adhesion to the endothelial cells of the capillary walls. The parasite protein P. falciparum erythrocyte membrane protein 1 is involved in mediating this attachment. In addition, complement receptor 1 on the surface of erythrocytes may help infected cells bind with other erythrocytes to form rosettes that can obstruct small capillaries. Blood ow is slowed in the microcirculation, reducing oxygen delivery to the tissues, with resultant tissue anoxia. The patient has severe headaches, may be confused, and ultimately lapses into a coma. A second but less common complication of infection with P. falciparum is blackwater fever, a condition characterized by hemoglobinuria (hence the term blackwater). It usually develops in patients with repeated infections and those undergoing quinine therapy. Blackwater fever may be mediated by an antigen-antibody reaction caused by the development of an autoantibody to the RBCs. The black appearance of the urine is the result of massive intravascular hemolysis and resulting hemoglobinuria that appears black in acid urine. In addition, the high-level parasitemia that often accompanies P. falciparum infection can lead to anemia and deposits of malarial pigment in some organs, such as the spleen. Renal complications, such as nephrotic syndrome or even renal failure, can occur with P. falciparum infection as a result of tubular necrosis brought about by tissue anoxia. Although complete immunity to malaria does not exist, individuals in endemic areas develop antibodies against the asexual stages and merozoites, which helps reduce the parasite load and severity of illness. Merozoite-specic antibodies may help block invasion of the RBCs or enhance opsonization and induce phagocytosis by macrophages. In addition, reports of antibodies directed against sporozoites and gametes (sexual reproductive cells that unite with another cell to form a zygote) indicate that these also may help reduce the rate of infection and the severity of illness. Patients with hemoglobinopathies, such as sickle cell disease, hemoglobin C disease, and glucose-6-phosphate dehydrogenase deciency, are somewhat protected against severe malaria because the parasite cannot replicate or, in some cases, exist in these RBCs.

Treatment Chloroquine remains the primary drug for prophylaxis and treatment of malaria. It is effective against all asexual stages of malarial organisms and all gametocytes, except those of P. falciparum. Many strains of P. falciparum have become resistant to the drug. Recent reports indicate that strains of P. vivax in Southeast Asia and Papua New Guinea also show diminished response to treatment with chloroquine. P. falciparum has also demonstrated resistance to sulfadoxine-pyrimethamine (Fansidar) and meoquine (Lariam) in parts of South America, Southeast Asia, and Africa. Doxycycline, atovaquone-proguanil (Malarone), or quinine use has been reestablished in some cases of multidrugresistant P. falciparum. Chloroquine-resistant P. vivax may be treated with meoquine or quinine sulfate with doxycycline. Primaquine phosphate, which is effective against hypnozoites that persist in the liver, is used to treat individuals infected with P. vivax and P. ovale to prevent relapses of these infections. There is also evidence that some strains of P. vivax in the geographic areas of chloroquine resistance may be refractory to primaquine. Artemisinin-based drugs have been used in

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Ingestion during blood meal

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Zygote Exflagellation Gametocytes Fertilization Ookinete

Formation of gametocytes infective in mosquito

Oocyst

Gut wall of mosquito

Rupture Mature trophozoite Mature schizont

Immature schizont

Sporozoites

Migration to mosquito's salivary gland

Early trophozoite Erythrocytic cycle Mosquito’s biting of human and injection of sporozoite

Red blood cells invaded

Via bloodstream to liver cells Release of merozoites

Schizogony

Exoerythrocytic cycle

Fig. 28.29 Life cycle of Plasmodium spp.

combination with classic drugs in areas in which highly resistant strains of P. falciparum have emerged. The level of parasitemia should be calculated as a percentage before and during treatment to follow effectiveness of treatment. In cases of high-level parasitemia, an RBC exchange may be used before treatment to decrease the level of parasitemia and increase effectiveness of the drug.

Life cycle The life cycle of Plasmodium involves sexual reproduction (sporogony) and asexual reproduction (schizogony), as shown in Fig. 28.29. Sporogony is the formation of oocysts containing sporozoites that results from the division of a zygote. Schizogony produces merozoites by the process of multiple ssion. The female Anopheles mosquito, in which sexual reproduction occurs, serves as the biological vector and denitive host. Asexual reproduction, which occurs in humans, has an exoerythrocytic phase that takes place in the liver and an erythrocytic phase that takes place in erythrocytes. In human infections, the earliest stage is the ring-form trophozoite (Fig. 28.30), in which the organism has a prominent, red-to-purple chromatin dot and a small blue ring

of cytoplasm surrounding a vacuole (in a Giemsa-stained blood smear). The growing trophozoite is characterized by an increase in the amount of cytoplasm, the disappearance of the vacuole, and the appearance of malarial pigment in the organism’s cytoplasm. The immature schizont (multinucleate stage) is characterized by a splitting of the chromatin mass. The mature schizont contains merozoites, which are individual chromatin masses, each surrounded by cytoplasm. Microgametocytes (male) have pale blue cytoplasm and a diffuse chromatin mass that stains pale pink to purple. The chromatin may be surrounded by a clear halo. Macrogametocytes (female) show a well-dened, compact chromatin mass that stains dark pink; the cytoplasm stains a darker blue than in microgametocytes. The chromatin mass often is set eccentrically in the organism. Pigment is distributed throughout the cytoplasm except in P. falciparum, in which it is often clumped near the chromatin mass.

Exoerythrocytic phase Humans serve as intermediate hosts and acquire the infection when the female mosquito takes a blood meal and injects the infective sporozoites with salivary secretions. The sporozoites

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Chromatin mass Ring-form trophozoite Red blood cell

Malarial cytoplasm Chromatin Mature trophozoite

Pigment granules

Malarial cytoplasm Chromatin masses Immature schizont

Pigment

Merozoites

is composed of iron deposits, is formed in the growing trophozoite as a result of incomplete metabolism of hemoglobin. Once the organism has reached the mature trophozoite stage, the chromatin begins to divide (developing schizont). When the chromatin split has been completed (mature schizont), each chromatin mass is surrounded by its own small amount of malarial cytoplasm (merozoite). Each malarial species has a typical number of merozoites, which may be used as an identifying characteristic. When the RBC ruptures, merozoites are released to invade other RBCs. At this point, two outcomes are possible. One is that the merozoite enters a cell and repeats development into a schizont, and the other is that it enters a cell and develops into one of the sexual stages, the microgametocyte or macrogametocyte.

Sexual phase Mature schizont

Chromatin mass (loosely organized) Microgametocyte Pigment

Cytoplasm Macrogametocyte

Pigment

Chromatin (condensed)

Fig. 28.30 Life cycle stages of Plasmodium spp.

enter the human circulation and take approximately 60 minutes to reach the liver, where they begin the exoerythrocytic phase by penetrating parenchymal cells. Maturation through the trophozoite and schizont phases results in the production of hepatic merozoites. Each schizont produces a large number of merozoites. The release of mature merozoites from liver cells and invasion of RBCs signal the beginning of the erythrocytic phase. Generally, only one cycle of merozoite production occurs in the liver before RBCs are invaded. P. vivax and P. ovale, however, may persist in the liver in a dormant stage known as hypnozoites, which accounts for the relapse (recurrence) of the disease within 1 to 3 years after the primary infection. Neither P. malariae nor P. falciparum has a persistent liver phase, although recrudescence in untreated individuals with either of these organisms may be the result of a continued subclinical erythrocytic infection.

Erythrocytic phase Merozoites have structures and proteins (e.g., erythrocyte-binding antigen) that selectively adhere to receptors on the RBC membrane. P. vivax and P. knowlesi use antigens of the Duffy blood group as receptors for attachment to and internalization into the RBCs, whereas P. falciparum may simply attach to receptors that are integral parts of the RBC membrane itself. Organisms may evade antibody recognition by varying their antigenic makeup. When merozoites have attached, endocytic invagination of the RBC membrane allows the organism to enter the RBC within a vacuole. Once inside the RBC, the organism feeds on hemoglobin and initiates the erythrocytic phase. Malarial pigment, which

Sporogony, which takes place in the mosquito, results in the production of sporozoites infective for humans. Both the microgametocyte and the macrogametocyte are infective for the female mosquito when she takes a blood meal. In the insect’s stomach, exagellation by the male microgamete and subsequent fertilization of the female macrogamete result in formation of an ookinete that migrates through the gut wall and forms an oocyst on the exterior gut wall. Sporozoites are produced within the oocyst. Mature sporozoites are released into the body cavity of the mosquito and migrate to the salivary glands. The female then injects sporozoites into a human as she takes her blood meal.

Laboratory diagnosis History of travel to an endemic area and the presence of classic clinical symptoms, including the malarial paroxysm of fever and chills, should alert a health care provider to request Giemsa-stained thick and thin smears for malaria. Examination of these blood smears remains the classic method of diagnosing malaria. Laboratory identication of malarial species involves examination of the morphology of infected RBCs and characteristic morphology of the parasite. The thick smear is used to detect malarial parasites; however, distortions of parasite morphology and the lack of intact RBCs require that a thin smear be examined to identify the species. Trophozoites, schizonts, and gametocytes may be seen in the blood smear. It is recommended that at least 200 to 300 oil immersion elds be examined on either type of smear before the smear is considered negative. In addition, more than one set of smears should be made within a 36-hour period because one set is inadequate to completely rule out the presence of malarial organisms. Table 28.6 gives the general characteristics of trophozoites and schizonts of the human malarial species. The QBC system using the uorescent dye acridine orange is a sensitive method for demonstrating the presence of parasites; however, a thin smear must still be examined for denitive identication of Plasmodium spp. Nucleic acid amplication tests have been proposed as another method for diagnosing malaria. They have good sensitivity in detecting low levels of P. falciparum infection. However, the extended time and costly equipment required do not make the procedure cost-effective or efcient, especially for eld work. Several different immunoassays, collectively referred to as RDTs, use malarial antibody–impregnated dipsticks to

Medically important parasitic agents

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Table 28.6 Comparisons of malarial species Plasmodium species

Red blood cell morphology

Trophozoite

No. of merozoites in schizont

Reproductive cycle (hours)

Other characteristics

P. vivax

Enlarged (to ×2) Schüffner dots

Ameboid Large vacuoles Golden-brown pigment

12–24 Average: 16

48

Wide range of stages in peripheral blood

P. malariae

Normal size May have dark hue Ziemann dots (rare)

Compact May assume band form across cell Coarse, dark brown pigment

6–12; average, 8 with daisy petal–like arrangement around clumped pigment

72

Wide range of stages in peripheral blood

P. ovale

Enlarged, oval Fringed edge Schüffner dots

Compact Golden-brown pigment (resembles P. vivax)

6–14 Average: 8

48

Wide range of stages in peripheral blood

P. falciparum

Normal size Multiple infections

Small, delicate Double chromatin dots Appliqué forms Dark pigment

8–24 Average: 20–24

Irregular, 36–48

Crescent-shaped gametocyte Ring and gametocyte stages only in peripheral blood

detect Plasmodium infection. These use an antigen capture immunochromatographic principle to detect soluble proteins from malarial organisms in blood. Some tests use a monoclonal antibody directed against an antigen specic to P. falciparum, histidine-rich protein 2. Other tests use an antibody that detects a protein common to all species, parasite lactate dehydrogenase or Plasmodium aldolase. One commercial rapid detection kit is approved for use in the United States. Serologic tests for antibody to malaria are not useful in an endemic area but may be useful for diagnosis in those who have traveled to an endemic area and have clinical symptoms of malaria. Testing for the presence of hemozoin in blood is an emerging area of malaria diagnostics.

Plasmodium vivax Plasmodium vivax has a tertian life cycle pattern; that is, it takes approximately 48 hours for the life cycle to be completed. The invasion of a new group of RBCs begins on the third day. P. vivax usually invades young RBCs (reticulocytes) and therefore is characterized by enlarged infected RBCs, often up to double the normal size. A ne pink stippling known as Schüffner stippling (or dots) may be present in the infected cell. The young trophozoite is characterized by its ameboid appearance; by maturity, it usually lls the RBC, and golden-brown malarial pigment is present. The mature schizont contains 12 to 24 merozoites, with an average of 16. Gametocytes are rounded and ll the cell. Macrogametocytes are often difcult to differentiate from mature trophozoites. Fig. 28.31 shows several stages of P. vivax

Plasmodium malariae Plasmodium malariae usually invades older RBCs, perhaps accounting for the occasional darker appearance of the invaded RBC. The life cycle is characterized as quartan, with reproduction occurring every 72 hours and invasion of new RBCs every fourth day. The trophozoite is compact and may assume a characteristic band appearance, in which it stretches across the diameter of the RBCs (Fig. 28.32A). Note the presence of dark, coarse, brown-to-black pigment in the band form. Occasionally, a few pink cytoplasmic dots, called

Ziemann dots, may be seen. The mature schizont contains 6 to 14 merozoites (see Fig. 28.32B), with an average of eight. Merozoites may be arranged in a characteristic “loose daisy petal” arrangement around the clumped pigment; however, they may also be randomly arranged.

Plasmodium ovale Plasmodium ovale, the least commonly seen species, resembles P. vivax. In P. ovale infections, the RBC is enlarged and may assume an oval shape with mbriated or fringelike edges. Schüffner dots are less commonly seen than with P. vivax. The parasite remains compact, has a golden-brown pigment, and has a range of 6 to 14 merozoites in the mature schizont. It also exhibits a tertian life cycle. Fig. 28.33A shows trophozoites of P. ovale. The Schüffner stippling in Fig. 28.33B has stained almost a bluish pink, but the compact organism and mbriated cell are characteristic.

Plasmodium falciparum Although identied as having a tertian life cycle, P. falciparum often demonstrates an asynchronous life cycle, with rupture of RBCs taking place at irregular intervals, ranging from 36 to 48 hours. The life cycle stages seen in peripheral blood are usually limited to the ring-form trophozoite and gametocyte. Other stages mature in the venules and capillaries of the major organs. P. falciparum invades RBCs of any age and, for this reason, often exhibits the highest level of parasitemia, reaching 50% in some cases. The ring forms of P. falciparum (Fig. 28.34A) are more delicate than those of other species and often have two chromatin dots. Appliqué forms, which are parasites at the edge of the RBC, and multiple ring forms in a single RBC are common. In this gure, the appliqué form is seen in the lower left and upper right of the photograph. Occasionally, small comma-shaped, red dots, referred to as Maurer dots, can be seen in the cytoplasm of infected cells. The mature trophozoite is small and compact and may have a dark brown pigment. The schizont (which is rarely seen in peripheral blood) has 8 to 24 merozoites, with an average of 20 to 24. Gametocytes have a characteristic banana or crescent shape (see Fig. 28.34B). P. knowlesi has early life cycle stages

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A

B

C

D

Fig. 28.31 A, Plasmodium vivax trophozoite. B, P. vivax mature schizont. C, P. vivax macrogametocyte. D, P. vivax microgametocyte (Giemsa stain, ×1000).

A

B

Fig. 28.32 A, Plasmodium malariae band form trophozoite. B, P. malariae schizont (Giemsa stain, ×1000).

that resemble those of P. falciparum and mature trophozoites and gametocytes that resemble those of P. malariae. Therefore pertinent information, including travel history to an endemic area, is important in making a diagnosis.

Babesia spp. Babesiosis is a zoonotic intraerythrocytic infection transmitted by the bite of a tick. Babesia spp. have been known to infect cattle and other vertebrates, but the rst cases reported in humans occurred in the 1950s. These initial cases were limited to patients who had undergone splenectomy, but since then,

cases of babesiosis have been reported in patients who do not have asplenia. The hard tick (Ixodes spp.) serves as the vector, and white-footed mice and white-tailed deer are the reservoirs. There also have been reports indicating simultaneous transmission of B. microti and Borrelia burgdorferi (the causative agent of Lyme borreliosis) because these two organisms have the same tick vector. In the United States, most cases occur in the Northeast and upper Midwest. Patients often manifest clinical symptoms of one disease but show a concurrent increase and decrease in antibody titers to both Babesia spp. and B. burgdorferi There are over 100 species of Babesia, and several species have been reported to infect humans. B. microti in the United

Medically important parasitic agents

A

673

B

Fig. 28.33 A, Plasmodium ovale trophozoite. The arrow denotes the mbriated edge of the red blood cell. B, Schüffner stippling of P. ovale clearly visible (Giemsa stain, ×1000).

A

B

Fig. 28.34 A, Plasmodium falciparum ring-form trophozoites. B, P. falciparum gametocyte (Giemsa stain, ×1000).

States and B. divergens in Europe are the most common. Several cases of Babesia duncani and B. duncani–like organisms have been reported in areas from the state of Washington to California. Sporadic cases in the states of Missouri, Kentucky, and Washington occurred with an organism that more closely resembles Babesia divergens, a parasite of cattle in Europe. As with malaria, cases of congenital babesiosis have been reported. The incidence of babesiosis in the United States increased from 1,761 cases in 2013 to 2,418 in 2019. The disease is a potential threat to the U.S. blood supply. B. microti is the most common parasite transmitted in transfused blood. There were 159 cases of transfusion-transmitted babesiosis reported between 1979 and 2009, with 122 cases (77%) occurring between 2000 and 2009. Through 2014, the number increased to over 200 transfusion-transmitted cases. Most cases were linked to RBC components. Currently, there are no FDA-approved tests for screening donor blood units for antibodies to Babesia spp.

Clinical infections Most cases of babesiosis occur from May to September, when outdoor activities peak and exposure to ticks is highest. Infection is often asymptomatic, but the very young, older adults, and immunosuppressed patients are at greatest risk of symptomatic infection. Symptoms are related to the asexual reproductive cycle, lysis of RBCs, and the level of parasitemia. The time from infection to development of

symptoms is 1 to 6 weeks. Patients with babesiosis may have malaria-like symptoms, such as malaise, fever, chills, sweating, and myalgia; however, many patients are asymptomatic. The fever is not cyclic as in malaria. Anemia may develop if hemolysis is severe or prolonged. The clinical course tends to be more severe or have complications if the patient has undergone splenectomy, is immunosuppressed, or is older. Complications may include respiratory, liver, or renal failure or disseminated intravascular coagulation. Treatment includes atovaquone plus azithromycin or clindamycin plus quinine. In very severe infections, exchange transfusion may be used.

Life cycle As with Plasmodium spp., the life cycle of Babesia spp. has alternating sexual and asexual reproduction stages. Production of the infective stage (sexual reproduction) takes place in the tick. The zygote migrates to the salivary glands of the tick, where sporozoites are formed. These sporozoites enter vertebrates during a blood meal and infect erythrocytes by attaching to a sialoglycoprotein on the erythrocyte membrane. Inside the cell, they become trophozoites and divide by asexual reproduction (budding) to form pairs and tetrads. Unlike malaria, in which the reproductive life cycle in the RBC may take 48 to 72 hours, the reproduction time in Babesia infection is approximately 4 to 6 hours. The resulting merozoites are released to infect other RBCs, where they use merozoite

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surface proteins to attach to glycophorin A and glycophorin B on the RBC membrane. Some merozoites differentiate into gametocytes (larger, more variably shaped), but this is not an easily identiable stage from the trophozoite. One difference in this life cycle from that of malaria is the absence of the exoerythrocytic cycle in humans. Transovarian transmission can occur in the tick, allowing the life cycle to persist without an intermediate host.

Laboratory diagnosis The diagnosis of babesiosis is made from Wright- or Giemsastained thin blood smears. The organisms appear as small, delicate, pleomorphic ring-form trophozoites, about 1 to 2 µm in length, with a prominent chromatin dot and faintly staining cytoplasm. More mature trophozoites may appear as pyriform organisms in single, double, or the classic tetrad (Maltese cross) formation within the RBC. Fig. 28.35A shows several small, compact, ringlike trophozoites of B. microti; Fig. 28.35B demonstrates the irregularly arranged tetrad formation characteristic of the organism. Although they are characteristic for babesiosis, the tetrad forms are not always seen. Fig. 28.35C demonstrates a cell with a pair of trophozoites as well as cells with single organisms. The morphology initially may be confused with that of P. falciparum. Lack of pigment, presence of extraerythrocytic organisms, and absence of other life cycle stages serve as keys to differentiate Babesia from P. falciparum.

A

Patients with a normocytic hemolytic anemia may have decreased haptoglobin and elevated indirect bilirubin levels and reticulocytosis. Some patients may demonstrate elevated levels of liver enzymes and lactate dehydrogenase, and decreased numbers of platelets. Serologic studies, such as immunouorescence assays, can be used to detect circulating antibodies (IgM and IgG). The levels of IgM antibodies are usually elevated during the rst 2 weeks and then decline. IgG titers greater than 1:1024 are generally considered indicative of recent or active infection. Although transfusion-transmitted babesiosis has been reported, there is no screening test for blood donors; obtaining a detailed donor history is the only method used to screen donors. Molecular methods to detect parasite DNA have been developed and may be used when there is a high suspicion of babesiosis but the smears remain negative.

Toxoplasma gondii T. gondii is an obligate intracellular parasite found in mammals worldwide. Serologic studies have indicated that the rate of infection often differs among counties, but, overall, up to one third of individuals worldwide may be infected. More than 20% of the U.S. population shows serologic evidence of infection, but in parts of Europe, the proportion may be as high as 80%. Toxoplasmosis can present with a wide range of

B

C

Fig. 28.35 A, Babesia microti trophozoites (Giemsa stain). B, B. microti tetrad form identied by the arrow (Giemsa stain, ×1000). C, Paired trophozoites and single trophozoites identied by arrows.

Medically important parasitic agents

clinical symptoms and complications that are often related to the immunologic status of the patient. Most immunocompetent individuals do not have serious infection, whereas HIVpositive individuals, transplant recipients, and those with defects in T cell–mediated immunity are found to have the most serious forms of infection. In addition, infants may contract the disease in utero and present with various degrees of severity.

Clinical infections The clinical presentation of toxoplasmosis differs based on the immunologic status of the individual. Immunocompetent patients with acute infections may be asymptomatic or may have mild ulike or mononucleosis-like symptoms, including low-grade fever, lymphadenopathy, malaise, and muscle pain. Once the acute infection has resolved, the organism enters a relatively inactive stage, in which tissue cysts containing many slow-growing forms of the organism are found. The organism may also be transmitted congenitally because of a transient parasitemia in pregnant women who have primary infection. Congenital transmission occurs when the tachyzoites, the motile, rapidly dividing forms in maternal circulation, cross the placenta and enter fetal circulation and tissues. Children with congenital toxoplasmosis may have a range of serious complications, including intellectual disability, microcephaly, seizures, hydrocephalus, retinochoroiditis, and blindness. If the fetus is exposed to the infection early in the pregnancy, more severe complications are likely to result. Infections acquired later in pregnancy may result in the child being asymptomatic at birth but developing complications, especially ocular problems, later in childhood. Immunosuppressed patients, particularly those with leukemia or lymphoma and those undergoing chemotherapy, may experience a serious primary infection or reactivation of a latent infection that can manifest itself as a fulminating encephalitis and result in rapid death. Up to 10% of the deaths of patients with AIDS are caused by reactivation of latent toxoplasmosis, resulting in encephalitis. Computed tomography (CT) may demonstrate lesions in the brain that represent Toxoplasma spp. cysts. Pulmonary toxoplasmosis may be present in conjunction with CNS infections or can be the presenting condition; however, the organism can spread to any organ of the body.

A

675

The organism can also be transmitted via organ transplantation. In this case, the organ may contain bradyzoites, which are slow-growing encysted forms of the parasite responsible for chronic infections that are reactivated in the immunocompromised recipient. This frequently occurs when the organ recipient is seronegative for T. gondii (lacks antibodies to T. gondii) and the organ donor is seropositive. In these patients, the infection usually occurs within the rst 3 months after transplantation and may spread to multiple organs. In the case of a seropositive transplant recipient, immunosuppressive treatment to prevent organ rejection may reactivate a latent infection in the transplant recipient. In general, toxoplasmosis in the immunocompetent host is not treated. When treatment is necessary, a combination of antimicrobials, such as trimethoprim-sulfamethoxazole, is used for treating the infection in the tachyzoite stage. The drugs are not effective against bradyzoites in cysts located in muscle and brain tissues.

Life cycle T. gondii has two life-cycle stages in humans: tachyzoite and bradyzoite. Tachyzoites can invade any nucleated cell and are the actively motile and reproducing forms. They are crescent shaped, are 4 to 6 µm long, and have a prominent nucleus (Fig. 28.36A). Fig. 28.36B shows lung tissue containing free tachyzoites and a number of intracellular forms. Bradyzoites are the slow-growing asexual forms found in a cystlike structure during the dormant phase of infection. As shown in the life cycle illustrated in Fig. 28.37, humans can acquire T. gondii infection in several ways, such as through ingestion or inhalation of the oocyst from cat feces, soil, or water; through ingestion of undercooked meat containing the cystlike structure with bradyzoites; and through congenital transmission. Primary risk factors for acquiring the infection include cleaning a cat litter box, gardening without gloves, and eating raw or undercooked meat or unwashed vegetables and fruit. The household cat and other members of the family Felidae serve as denitive hosts for the organism. Sexual and asexual reproduction occur in the intestine of the cat, whereas only asexual reproduction occurs in humans and other intermediate hosts. The result of sexual reproduction is the production of an oocyst, which is passed in cat feces. The oocyst requires 2 to 5 days in the environment to mature and become infective.

B

Fig. 28.36 A, Toxoplasma gondii tachyzoites, identied by the arrow (hematoxylin and eosin stain). B, T. gondii tachyzoites in lung tissue (hematoxylin and eosin stain, ×1000).

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Human ingestion of raw meat containing cyst with bradyzoites

Human ingestion of infective oocyst from cat feces

Release of sporozoite, which penetrates intestinal cell Congenital toxoplasmosis

Formation of “cyst” containing bradyzoites in various organs

Infection of fetus

Tachyzoite formation

Tachyzoites crossing placental barrier

Immune system response

Asexual reproduction in cells

Invasion of other tissue cells by tachyzoites Pregnant woman

Rupture of cells

Hematogenous spread

Fig. 28.37 Life cycle of Toxoplasma gondii.

When a human ingests the infective oocyst, changes in pH and temperature induce release of sporozoites in the oocyst into the intestine. The sporozoites penetrate the intestinal wall, gain access to the circulation, and migrate to the organs. Migration of dendritic cells infected with tachyzoites is one of several proposed methods that contribute to dissemination. These tachyzoites, as they are now called, are responsible for tissue destruction. They invade cells by attaching to surface proteins (using receptors, such as proteoglycans) on nucleated cells, replicate intracellularly within a parasitophorous vacuole formed by the host cell plasma membrane, cause cells to rupture, and release tachyzoites to invade other cells. The immune system, in particular, T cells, responds strongly when tachyzoites invade the tissues. This TH1 immune response includes the production of IL-12 and IFN-γ. Natural killer (NK) cells may also play a role in control of the organism. As a result of this immune response, tachyzoites are converted to bradyzoites, and eventually the cystlike structure contains hundreds to thousands of the slowly growing and reproducing bradyzoites. At this point, the infection generally remains dormant for the life of the host unless the immune system is compromised. In the immunocompromised patient, the bradyzoites are reactivated and released from the cyst and become active tachyzoites that invade multiple organs, resulting in disseminated infection. If a human ingests raw or undercooked meat containing the tissue cysts, the cyst wall is dissolved and bradyzoites are liberated. These organisms are resistant to digestive tract

enzymes for about 6 hours, during which time they convert to tachyzoites and invade the intestinal wall. They then gain access to the circulation. All types of meat, including lamb, pork, beef, and that of game animals, such as deer and bear, have been implicated in this mechanism.

Laboratory diagnosis Identication of the tachyzoite or pseudocysts with bradyzoites in tissue is very difcult because no single organ is invaded. Finding free tachyzoites in CSF, bronchoalveolar lavage specimens (Figs. 28.38 and 28.39), or specimens from other sites, such as lymph nodes, is unusual unless the infection is disseminated. In disseminated toxoplasmosis, histologic stains of biopsy materials may demonstrate the cyst or, in some cases, tachyzoites. Noninvasive techniques, such as magnetic resonance imaging (MRI) and CT, may be used in the diagnosis for patients suspected of having disseminated toxoplasmosis. The levels of antibodies to the organism show a rapid increase during infection, and tests for antibodies are most commonly used for diagnosis. Indirect uorescent antibody tests and EIAs with T. gondii organisms as the antigen are routinely used for diagnosis. Because most patients have an antibody titer to the organism, interpretation of the titer must be linked to clinical symptoms and the patient’s history. A signicant rise in titer (fourfold) between acute-phase and convalescent specimens may indicate acute infection. An IgMspecic test may also be used to diagnose acute infections and can be useful in the diagnosis of primary toxoplasmosis

Medically important parasitic agents

Fig. 28.38 Bronchoalveolar lavage cytocentrifuge preparation, Wright-Giemsa stain, Crescent-shaped cells with central nucleus (arrows). Morphology consistent with trophozoites (tachyzoites) of Toxoplasma gondii (see Fig. 28.39).

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have been associated with ingestion of raw foods or unpasteurized drinks. Sexual and asexual reproduction occur in the human intestinal tract. Over 30 Cryptosporidium species are recognized; Cryptosporidium was initially identied as causing zoonotic infections in veterinarians and other animal handlers. The parasites can infect a range of hosts including mammals, birds, and reptiles. Cows are an important reservoir for human disease. Now, over 20 species have been found in humans; the most common are Cryptosporidium hominis and Cryptosporidium parvum. Cryptosporidiosis is the leading cause of waterborne disease outbreaks in the United States. Outbreaks in daycare centers and from environmental contamination of municipal water supplies have been reported. In the 1980s, cryptosporidiosis was identied as a major opportunistic infection in patients with AIDS and classied as an AIDS-dening illness. In a 2019 report, the CDC estimated that 823,000 cases of cryptosporidiosis occur annually.

Clinical infections

Fig. 28.39 Bronchoalveolar lavage. Cytocentrifuge preparation, acridine orange stain, uorescence microscopy, Crescent-shaped cells composed of RNA. Morphology consistent with trophozoites (tachyzoites) of Toxoplasma gondii.

in pregnant women suspected of having been exposed to the organism or in neonates in whom congenital infection is suspected. IgM titers usually peak within the rst month of infection. Antibody titers may be unreliable in immunocompromised patients because these patients lack the ability to produce sufcient antibody to cause a signicant increase in titer. PCR assays to detect T. gondii DNA may be used for CSF or amniotic uid and are useful in detecting congenital infection in utero. Molecular methods are also used in bone marrow transplant recipients to detect infection.

Opportunistic intestinal Apicomplexa species Cryptosporidium spp. are recognized for causing self-limiting GI infection in immunocompetent hosts. However, they have also been identied as opportunistic pathogens causing severe infection in immunocompromised hosts, particularly in those with AIDS. Cryptosporidium spp. are obligate intracellular parasites transmitted by ingestion of the infective oocyst. These organisms are transmitted by the fecal-oral route—ingestion of contaminated water or food or, for some, by direct contact with fecal material containing oocysts. The oocysts are infective at a low dose (as few as 10 oocysts) and are resistant to common chlorine- and ammonia-based disinfectants. Most infections are reported in summer, when there is increased recreational water activity. Foodborne infections

In immunocompetent patients, the organisms cause a transient profuse, watery diarrhea along with mild to severe nausea and vomiting, headache, and cramps. The mucosa is inamed, and there is an inux of segmented neutrophils, macrophages, and lymphocytes. The onset is rapid (within 3 to 7 days after ingestion of the oocyst), but the infection is self-limiting. Symptoms resolve in several weeks, although infections can last up to 1 month. Infection begins in the small intestine but may spread to the large intestine. In endemic areas, the rst infection occurs in early childhood and is symptomatic with profuse, watery diarrhea, but subsequent infections are mild to asymptomatic. Antibodies (IgG, IgA, and IgM) are produced, but it is not clear how much of a protective role they play. Antibodies developed against one of the organisms can provide protection against infection by the same species, but antibodies developed against one species may provide only incomplete protection against another species. In addition, the host mounts a cell-mediated immune response (T cells and NK cells) that includes production of several cytokines, including IFN-γ and IL-12. The organism alters osmotic pressure in the gut, with a resulting inux of uid. The diarrhea is cholera-like, with bits of mucus and little fecal material. Fluid loss has been reported to range from 3 to 6 L/day to as much as 17 L/day. In addition to weight loss, patients show signs of dehydration and electrolyte imbalance. In patients with AIDS whose cell-mediated immunity is compromised and whose CD4 count is less than 50 cells/μL, the infection is more likely to become fulminant or life-threatening or spread to extraintestinal sites. In chronic cases, the intestinal villi may show signs of atrophy caused by inammation, and the brush border of the cells is disrupted because of invasion by the organism. This damage alters intestinal permeability and can result in decreased uptake of uids, electrolytes, and nutrients. Malabsorption syndrome may occur. No antimicrobial is completely effective against this infection. Paromomycin and azithromycin, which can suppress the infection, have been used, with mixed results. Nitazoxanide, a relatively new agent, has been used for treating Giardia and Cryptosporidium infections in immunocompetent children and adults. It shortens the duration of diarrheal episodes and appears to be the only one of the drugs tested that has even partial effectiveness in immunocompromised individuals.

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into a microgamete or macrogamete. Fertilization of the macrogamete results in formation of the oocyst, which contains four sporozoites. Two types of oocysts may be formed, the thin-walled oocyst, which ruptures within the intestine and results in autoinfection, and the thick-walled, fully sporulated oocyst, which is infective when passed in feces. Key factors in the life cycle of this organism that contribute to its pathogenicity include the following:

Life cycle The sexual and asexual life cycles of Cryptosporidium occur in the same host. Life cycle stages, as shown in Fig. 28.40, develop under the brush border of the intestinal mucosal epithelial cells. Oocysts are infective when passed and may be ingested in contaminated water or food or passed by person-to-person contact. Ingestion of the infective oocyst initiates the asexual cycle (sporogony) with the release of sporozoites. Sporozoites attach to receptors on the intestinal mucosal border, penetrate an intestinal epithelial cell and create a parasitophorous vacuole between the cell membrane and cytoplasm, and mature into trophozoites in this intracellular but extracytoplasmic location. Once trophozoites have matured, the development of meronts with merozoites begins. The nucleus and cytoplasm divide to form individual organisms known as merozoites. When the meront ruptures, merozoites are released and penetrate other cells to continue asexual reproduction or to transform

Thick-walled oocyst ingested by host

• The oocysts are infective when passed in feces. • Rupture of thin-walled oocysts in the intestine creates the potential for continual autoinfection. • Patients may remain infective and continue to shed oocysts for a time after the diarrhea ceases.

Laboratory diagnosis The small size (4 to 6 µm), refractile appearance, and round shape of the oocyst make detection difcult with routine

Sporozoite release Entry into intestinal cell to form type I meront

Contamination of food and water with oocysts

Autoinfection

Rupture in lumen of intestine OR

Thick-walled oocyst (sporulated) exits host

Merozoite release

Maturation into thick-walled oocyst (4 sporozoites) Maturation into thin-walled oocyst Maturation into thin-walled oocyst (2 sporozoites)

Type II meront

OR Merozoite release Zygote Macrogamont

Microgamete release

Fig. 28.40 Life cycle of Cryptosporidium sp.

Microgamont

Medically important parasitic agents

concentration procedures because the organism may resemble a yeast or an RBC. More oocysts are seen in liquid stool than in formed stool. Trichrome and iron hematoxylin stains are not useful permanent stains for identication of Cryptosporidium spp. The recommended detection methods for Cryptosporidium infection are the modied acid-fast stain, an antigen detection test, and a DFA test using a monoclonal antibody directed against Cryptosporidium spp. These methods do not distinguish the species. With the acid-fast stain, the organism appears as a bright red sphere, which distinguishes it from yeasts, which stain green. Figs. 28.41, 28.42, and 28.43 show Cryptosporidium oocyts stained with modied acid-fast stain. Studies indicate that antigen detection by monoclonal antibody tests demonstrates greater sensitivity and specicity than the modied acid-fast stain. EIA methods can also be used but are less sensitive (sensitivity of 70%) than uorescent antibody methods. More recently, lateral-ow immunochromatographic assays have become available. These assays are highly specic (>99%), but the sensitivity ranges from 68% to 100%. Antigen-detection methods have become popular in clinical laboratories for their ease of use—they do not require an experienced microscopist.

Cystoisospora belli Cystoisospora belli (formerly Isospora belli) is an opportunistic organism seen less frequently than Cryptosporidium spp. It is

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known to infect only humans. Most patients infected with C. belli are asymptomatic, but symptoms, such as low-grade fever, headache, diarrhea, and colicky abdominal pain, may be present. Acute infections with C. belli are usually clinically indistinguishable from those with Cryptosporidium spp. The infection is self-limiting and usually resolves in several weeks in an immunocompetent host. The infection is often more serious in immunocompromised patients, including those with AIDS and those with conditions such as Hodgkin disease, lymphoproliferative disorders, or lymphoblastic leukemia. These patients may have watery diarrhea and concurrent weight loss, or chronic infection may develop. Treatment with trimethoprim-sulfamethoxazole has been effective in eliminating diarrhea, but patients often show recurrence of infection when therapy is discontinued. The life cycle of C. belli is similar to that of Cryptosporidium spp. in that it requires only one host but occurs within the cytoplasm of epithelial cells of the small intestine. The oocyst of this organism is not infective when passed in the feces and requires 24 to 48 hours outside the body to become infective. There is no thin-walled oocyst to cause continued autoinfection. The mature oocyst of C. belli is oval, 20 to 33 µm by 10 to 19 µm, with a hyaline cell wall. The immature oocyst usually shows a single sporoblast (an early stage in the development of a sporocyst, before differentiation of the sporozoites). The mature oocyst has two sporocysts, which are walled bodies. Each sporocyst contains four elongated sporozoites. Oocysts can be seen in wet mounts. The modied acid-fast stain has been used to detect C. belli. The oocyst wall does not stain but often shows a faint outline because of precipitated stain, and the sporoblasts or sporocysts stain dark red. Like oocysts of Cyclospora cayetanensis, the oocysts of C. belli will autouoresce a bluish color at 330 to 365 nm and a bright green at 450 to 490 nm. The size and shape of the oocyst serve as the identication characteristics. Fig. 28.44 shows the characteristic appearance of an acid-fast stain of the oocyst of C. belli

Cyclospora cayetanensis

Fig. 28.41 Cryptosporidium oocysts (modied acid-fast stain, ×1000).

Fig. 28.42 Watery, frothy diarrheic stool, smear, acid-fast stain, (×1000). Acidfast oocysts (4 to 6 µm). Sporulated oocysts containing four sporozoites (arrow) Morphology consistent with Cryptosporidium sp.

C. cayetanensis is a foodborne and waterborne organism causing endemic and epidemic diarrheal disease. Humans are the only known host for this organism, but other Cyclospora spp. are found in animals. The organism was rst linked to human

Fig. 28.43 Watery, frothy diarrheic stool, smear, acid-fast stain. Acid-fast oocysts (4 to 6 µm) (arrows) (×1000). The measurement is taken to differentiate this oocyst from the 8- to 10-µm oocysts of Cyclospora spp. (see Fig. 28.45B).

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A

Fig. 28.44 Cystoisospora belli oocysts (modied acid-fast stain, ×1000).

disease in the 1990s, when it was found in stool specimens. It was originally thought to be a Cyanobacterium-like body, bluegreen alga, or large C. parvum C. cayetanensis is endemic in Nepal, Peru, Guatemala, and Haiti, but outbreaks have been reported in many countries, including those in Central and South America, parts of the Caribbean, India, and Europe. The usual mode of transmission is ingestion of fecally contaminated water or food. Most outbreaks in endemic areas occur during the rainy season. Persons living in these areas may acquire some level of immunity that increases resistance to future infection. Individuals who travel to endemic areas are susceptible to traveler’s diarrhea caused by C. cayetanensis. The organism has also been linked to several large foodborne outbreaks in North America. Most of the outbreaks were associated with the consumption of contaminated imported fresh produce, especially raspberries and strawberries from endemic areas of Central America. Subsequently, there have been isolated outbreaks associated with imported vegetables, such as mesclun lettuce, basil, and snow peas. The infective dose is not known, but it is thought to be relatively low, between 10 and 100 oocysts. Although the organism is most commonly encountered in immunocompetent patients, it may be considered an opportunistic infection in patients with AIDS. Symptoms of infection with C. cayetanensis differ based on age, infective dose, and host immune status. The incubation period is approximately 1 week. The organism infects cells in the upper portion of the small intestine and causes frequent, watery, but nonbloody stools that may alternate with bouts of constipation. Symptoms, which can mimic those of cryptosporidiosis or cystoisosporiasis, include anorexia, fatigue, weight loss, abdominal cramping and bloating, vomiting, low-grade fever, and nausea. Patients often report a ulike syndrome before the onset of diarrhea. In immunocompetent hosts, the symptoms are self-limiting and persist for several weeks but may occur in a relapsing pattern for up to 2 months. Immunocompromised patients have prolonged course and can be symptomatic for as long as 4 months. Some cases of malabsorption caused by inammation and damage to the intestinal villi and a few cases of extraintestinal infection have occurred in immunocompromised patients. In endemic areas, infections are common in children 2 to 10 years of age, and most infections resolve spontaneously. Trimethoprimsulfamethoxazole is used to treat the infection; when treatment is started, symptoms usually abate within several days.

B

Fig. 28.45 Cyclospora cayetanensis oocyst. A, Wet mount. B, Modied acid-fast stain. (A, original magnication ×1000; B, ×1000.)

The organism shares the general life cycle characteristics of other intestinal Apicomplexa species. Once the mature oocyst has been ingested, the presence of bile and trypsin in the small intestine helps trigger release of sporozoites, which invade the intestinal cells. Two types of meronts develop: type I, which produce merozoites that infect other intestinal cells (asexual cycle), and type II, which progress to the sexual stage. Once fertilization occurs, a zygote forms and develops into an immature oocyst that is passed in feces. Unlike Cryptosporidium, however, immediate person-to-person transmission is unusual because these oocysts require about 1 to 2 weeks outside the body to mature and become infective. The oocyst is similar to that of human Cryptosporidium spp. but larger, with an average size of 8 to 10 µm. In wet mounts, the oocyst appears nonrefractile, spherical, and unsporulated, with multiple internal globules or granules. Sporulation occurs after several days, resulting in the production of two sporocysts with two sporozoites each. The organism does not stain with traditional trichrome or iron hematoxylin stains. The modied acid-fast stain demonstrates variably staining organisms, from dark pink to almost colorless (ghost forms), with no visible internal structures. Fig. 28.45A shows an oocyst of C. cayetanensis in a wet mount, and Fig. 28.45B shows an oocyst in an acid-fast stain. For comparison, Fig. 28.43 shows acid-fast Cryptosporidium oocysts that measure 4 to 6 µm (arrows) One distinguishing characteristic of C. cayetanensis is autouorescence, exhibiting a bright blue uorescence (330 to 365 nm) or mint green uorescence (450 to 490 nm) under ultraviolet light. Nucleic acid detection methods developed in reference and research laboratories are often used for

Medically important parasitic agents

identication during outbreaks. Serologic methods to detect antibodies and antigen detection assays are not available.

Microsporidia In the mid-1980s, a group of organisms were linked to infections in patients with HIV infection. These organisms, collectively referred to as microsporidia, are obligate intracellular, parasites common to invertebrates and other animals. Originally considered protists, they have been reclassied as fungi based on chitin present in the spore wall and rRNA sequencing. There are more than 200 genera and 1500 species identied, with many being reclassied based on molecular sequencing. At least nine genera (Anncaliia, Encephalitozoon, Endoreticulatus, Enterocytozoon, Nosema, Pleistophora, Trachipleistophora, Tubulinosema, Vittaforma, and the unclassied collective group Microsporidium) have been implicated in human infections. Although infection with the organisms is often linked to HIV infection, microsporidia have been identied in organ transplant recipients, older adults, and patients with traveler’s diarrhea. The organisms are capable of infecting a wide variety of human organs, including intestine, eyes, muscles, liver, kidneys, and CNS.

Clinical infections Symptoms differ with the species and organ infected. Intestinal infections are most often caused by Enterocytozoon bieneusi or Encephalitozoon intestinalis, and infection in immunocompetent hosts is characterized by diarrhea, cramps, loss of appetite, and fatigue. Dehydration and weight loss are sometimes seen. Infected patients have four to eight liquid or loose stools a day, and symptoms may persist for up to 8 months, with spontaneous exacerbations and remissions. In patients with AIDS, infection is chronic, and the diarrhea may persist for several years, with malabsorption of fats and vitamin B12 and cachexia as additional complications. Risk factors in patients with AIDS include sexual transmission, a CD4 cell count of less than 100/μL, exposure to water during swimming, and contact with animals. Dissemination of E. intestinalis, especially to the urinary tract, gallbladder, or respiratory tract, has been seen. Dissemination to muscles may result in weakness and pain. Patients with AIDS can develop encephalitis, nephritis, or keratoconjunctivitis. Contact lens wear is a risk factor for corneal infection. Albendazole has been used to treat infections with Encephalitozoon spp., but most other microsporidial organisms are resistant to this and other drugs. Metronidazole may provide some relief, but the symptoms recur when use of the drug is discontinued. Disseminated infections have also been reported.

polar tube and receptors on the cell membrane. The contents of the spore (sporoplasm containing a nucleus, ribosomes, Golgi apparatus) are then transferred into the cell. Within the host cell, the sporoplasm divides and develops into meronts. These structures subsequently develop into sporoblasts that differentiate into sporonts. As these structures mature into spores, they develop a thick membrane and the polar tube. The host cell ruptures, and spores are released to penetrate other host cells to repeat the reproductive cycle or be passed from the body. In the case of intestinal infection, spores are passed in feces; in infection of the urinary tract, they can be detected in urine.

Laboratory diagnosis Microsporidia can be found in stool, bronchoalveolar lavage uid, sputum, CSF, conjunctival swabs, corneal scrapings, and other specimens. Initially, identication methods were limited to nding the small spores in Giemsa-stained tissue sections or in electron microscopic examination of biopsy specimens. Electron microscopy must be used to identify the species of the organism. Speciation is based on the number of coils in the polar tubule, septations in the spore, and size. Routine O & P examination will not detect the spores in a fecal specimen. However, staining of formalin-preserved feces using the Weber modication of the trichrome stain and the Ryan trichrome blue stain can be used to detect microsporidial spores. The small size of the spores, however, makes them easy to overlook in clinical specimens. A thin smear of feces must be used so debris does not obscure the small, faintly staining spores. Fig. 28.46 shows the small spores (1.5 to 4.0 µm) in a chromotrope stain of a fecal specimen. Spores stain pink to red and may have a diagonal or equatorial band that helps distinguish them from bacteria or yeasts. Background staining in the Weber stain is pale green (Fig. 28.47), whereas in the Ryan stain it is blue. A Gramchromotrope stain will produce spores that are dark violet and demonstrate an equatorial band. Calcouor white, a uorescent stain used for the detection of fungi, can also be used to screen specimens for microsporidial spores. The stain is a chemouorescent agent, such as the Remel Calcouor White Stain Kit (Thermo Fisher Scientic, Waltham, MA) or Fungi-Fluor (Polysciences, Warrington, PA).

Life cycle The infective stage for humans is the environmentally resistant spore, which can survive outside the host for as long as 1 year. Once the infective spore is ingested, shifts in pH and ionic concentration in the intestinal tract trigger infection. The organism gains access to host cells by emergence of a polar tube through the spore wall and its insertion through the cell membrane. This insertion is thought to be mediated by a series of molecular interactions between the tip of the

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Fig. 28.46 Microsporidia spores identied by the arrow (chromotrope stain, ×1000). (Courtesy Texas Department of State Health, Austin, TX.)

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present, commonly referred to as the worm burden. Patients with only a few adult worms are usually asymptomatic, whereas a patient with a large number of adults often shows clinical symptoms. Most of the parasites inhabit the intestinal tract, but species also infect the liver, lungs, lymphatics, and blood vessels.

1

Flukes 2

Fig. 28.47 Diarrheic stool, smear, modied Weber stain, light microscopy, ×2000 oil. Parasite spores, small (1.5 × 0.9 µm). Morphology consistent with Enterocytozoon sp. (arrow 1) (see Fig. 28.48). Compare with size of the yeast (arrow 2).

Fig. 28.48 Diarrheic stool from concentration, transmission electron microscopy, ×57,000. Endospore layer and polar tubes are present. Morphology is consistent with Enterocytozoon bieneusi

The stain is nonspecic and binds to chitin in the spore wall. Therefore the presence of spores should be conrmed by using one of the modied trichrome stains. Species-specic indirect uorescent antibody staining is useful to identify spores in uids, such as urine, or in biopsy specimens. Species-specic PCR assays are also available for identication. The transmission electron microscope micrograph in Fig. 28.48 shows an endospore layer and polar tubes. The morphology is consistent with Enterocytozoon bieneusi

Helminths Helminth infections in humans are caused by ukes, tapeworms, or roundworms. Humans become infected by directly ingesting eggs (ova), by ingesting larvae in an intermediate host, or through direct larval penetration of the skin. Adult forms do not multiply in the human body; therefore the number of adult worms present is related to the number of eggs or larvae ingested. The pathologic consequences and severity of infection are related to the number of adults

Flukes (trematodes) are members of the phylum Platyhelminthes, or atworms. Most infections are seen in people from East Asia, Africa, South America, and some areas of the Caribbean. Adults can range in size from a few millimeters to almost 8 cm. With the exception of the blood ukes, adult ukes are dorsoventrally attened and have an oral sucker at the anterior end and a ventral sucker located midline, posterior to the anterior sucker. Except for the blood ukes, ukes are hermaphroditic, possessing male and female reproductive organs. Fig. 28.49 shows the generalized life cycle of the liver, lung, and intestinal ukes. In all species, eggs must reach water to mature, and all have a snail species as the rst intermediate host. The miracidium (rst-stage larva) is ingested by a snail while within the egg or is released from eggs and penetrates the snail. Within the snail, a complex development of germinal tissue occurs, resulting rst in sporocysts, which contain undifferentiated germinal structures, and then in rediae, which contain partially differentiated germinal material. The cercaria (second-stage larva) develops within the redia and is released into the water. Then, depending on the species, the cercaria attaches to aquatic vegetation or invades the esh of aquatic organisms. At this stage, the organism is referred to as a metacercaria and is infective for humans. Except for the schistosomes, which infect humans by direct cercarial penetration, infection occurs when an individual ingests the metacercaria in raw or undercooked aquatic animals or on water vegetation. Prevention includes adequate cooking of water vegetation, sh, and crustaceans. In the case of the blood ukes, individuals should wear clothing and shoes to prevent cercarial penetration. Praziquantel is the recommended treatment for intestinal ukes, the liver uke Clonorchis sinensis, the lung uke Paragonimus westermani, and blood ukes. The liver uke Fasciola hepatica is treated with triclabendazole. The egg is the primary diagnostic stage. It is best detected on a wet mount of a concentrated specimen. Routine concentration procedures for feces, such as FES, may be used. The zinc sulfate method is not satisfactory, however, because all eggs except those of schistosomes are operculated. With the zinc sulfate method, the operculum might open and release the contents or cause the egg to sink. Table 28.7 compares the characteristics of uke eggs.

Intestinal ukes Fasciolopsis buski, known as the giant intestinal uke, is found in the Far East, including China, Vietnam, and India. Dogs and pigs serve as reservoir hosts. Humans acquire the infection by ingesting metacercariae on freshwater vegetation, such as bamboo shoots and water chestnuts. Adults of F. buski live in the duodenum, where they cause mechanical and toxic damage. Inammation and ulceration of the mucosa

Medically important parasitic agents

Adults in specific organs

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Eggs in feces or sputum Freshwater

Fasciolopsis buski adults in intestine

Penetration of or ingestion by snail

Metacercariae encystment on aquatic vegetation— ingestion by humans

Opisthorchis sinensis adults in liver

First intermediate host

Metacercariae ingestion in raw or undercooked fish

Penetration of appropriate host Emergence of cercariae (second larval stage) from snail

Heterophyes heterophyes, Metagonimus yokogawai adults in intestine

Paragonimus westermani adults in lungs

Miracidia (first larval stage) release

Fasciola hepatica adults in liver

Metacercariae ingestion in raw or undercooked crabs

Development into sporocyst and redial generations

Fig. 28.49 Life cycle of liver, lung, and intestinal ukes.

may be present. Heavy infections result in persistent diarrhea, anorexia, edema, ascites, nausea and vomiting, and/or intestinal obstruction. Finding the adult or egg is diagnostic, although the egg is more commonly seen. The adult is attened, is 2 to 7 cm long, and lacks the cephalic cone seen in Fasciola hepatica. Adults are usually not seen in a stool sample unless it is a purged specimen. Eggs are yellow to brown, average 130 to 140 µm by 80 to 85 µm in size, and have a small, relatively inconspicuous operculum. They are unembryonated when passed (Fig. 28.50). These eggs are identical to those of F. hepatica and, when seen, should be reported as Fasciolopsis buski/Fasciola hepatica eggs. Metagonimus yokogawai and Heterophyes heterophyes are two small ukes found in the Far East and Middle East. Humans acquire infection with these organisms by ingesting the metacercariae in undercooked or raw sh. Adults live in the small intestine and produce few symptoms. A patient with a heavy worm burden may have diarrhea, colic, and loose stools, with a large amount of mucus. Adults of both species are small (1 to 2 mm) and delicate. Eggs serve as the primary diagnostic stage. They are 28 to 30 µm long and have a vase or ask shape. They are embryonated and operculated, with inconspicuous shoulders at the operculum. Eggs of these species resemble each other and those of Clonorchis sinensis

Liver ukes Fasciola hepatica, the sheep liver uke, is seen in the major sheep-raising areas of the world, including parts of the southwestern United States. In addition, the organism is found in some cattle-raising areas. In sheep, the organism causes a disease known as liver rot, which is characterized by liver destruction. Humans acquire the infection by ingesting metacercaria on raw water vegetation, especially watercress. The larvae reach the liver by migrating through the intestinal wall and peritoneal cavity. Adults live in the biliary passages and gallbladder and rarely cause overt symptoms because infections are light. Tissue damage during migration through the liver can result in eosinophilia, an inammatory reaction, secondary bacterial infection, and brosis in the biliary ducts. Heavy infections induce diarrhea, upper right quadrant abdominal pain, hepatomegaly, cirrhosis, and liver obstruction, with resulting jaundice. Chronic infections are usually asymptomatic. Adults are approximately 3 cm long and have a prominent cephalic cone. The unembryonated, operculated eggs are carried in the bile to the intestinal tract and are passed in the feces. The size is 130 to 150 µm × 60 to 90 µm. They are almost indistinguishable from eggs of F. buski and are reported as F. buski/F. hepatica eggs (see Fig. 28.50).

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Table 28.7 Comparisons of uke eggs Organism

Average size (µm) and shape

Other identifying features

Fasciola hepatica

130 × 60–90, ellipsoidal

Small, indistinct operculum Yellow-to-brown color Unembryonated when passed

Fasciolopsis buski

130 × 80–85, ellipsoidal

Cannot be distinguished from F. hepatica Unembryonated when passed

Paragonimus westermani

80–115 × 48–60, oval

Brown, thick shell Slightly attened operculum Shoulders at operculum Unembryonated when passed

Clonorchis sinensis

29–35 × 12–19, vase shaped

Domed operculum Prominent shoulders Knob at end opposite operculum Embryonated when passed

Heterophyes heterophyes and Metagonimus yokogawaia

28–30 × 15–17, vase shaped

Operculated Shoulders not distinct Small knob Embryonated Similar to C. sinensis

Schistosoma mansoni

115–175 × 45–75, oval

Lateral spine No operculum Embryonated when passed

Schistosoma haematobium

110–170 × 40–70, oval

Rounded anterior Terminal spine Embryonated when passed

Schistosoma japonicum

60–95 × 40–60, round to slightly oval

Small, inconspicuous, hooked lateral spine Embryonated when passed

a

The eggs of these two species, C. sinensis, and Opisthorchis, are almost indistinguishable.

The Chinese or Oriental liver uke, Clonorchis sinensis, is geographically limited to the Far East, where dogs and cats serve as reservoir hosts. Opisthorchis spp. are additional liver ukes found in humans. Opisthorchis viverrini is known as the Southeast Asian liver uke, and Opisthorchis felineus is known as the cat liver uke. The adults live in the distal portion of the bile ducts. As with F. hepatica, light infections produce few or no symptoms. Repeated or heavy infections cause inammation because of mechanical irritation, fever, diarrhea, pain, brotic changes, or obstruction of the bile duct. Humans acquire the infection by ingesting the metacercariae in raw, undercooked, or pickled fresh-water sh. The diagnosis is made by nding ova in feces or, occasionally, in duodenal aspirates. Adults are thin, tapered at both ends, and 1 to 2.5 cm long. It is difcult to

Fig. 28.50 Fasciolopsis buski/Fasciola hepatica egg (unstained, ×400).

Medically important parasitic agents

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Fig. 28.52 Paragonimus westermani egg (unstained, ×400). Fig. 28.51 Clonorchis sinensis egg (unstained, ×1000).

Blood ukes distinguish the ova of Clonorchis, Opisthorchis, Heterophyes, and Metagonimus. The egg is 29 to 35 µm long, embryonated when passed, ask shaped, and operculated, with prominent shoulders at the operculum and a knob at the opposite end (Fig. 28.51).

Lung ukes Organisms of the genus Paragonimus usually infect tigers, leopards, dogs, and foxes. About 30 species of Paragonimus have been named, and 9 have been identied as human parasites. An estimated 20 million people suffer from paragonimiasis. Paragonimus westermani, the lung uke, is found primarily in Southeast Asia and in focal areas of Latin America and Africa. It is the most common species affecting humans. Paragonimus kellicoti is occasionally found in North America. Humans acquire the infection by ingesting metacercariae in raw, pickled, wine-soaked, or undercooked freshwater crabs or craysh. The metacercaria excysts in the small intestine and burrows through the duodenal wall into the peritoneal cavity. It eventually penetrates the diaphragm and enters the lung. The host shows few symptoms during this migration but may exhibit intermittent coughing and chest pain. The parasite induces an inammatory response in the lung characterized by the presence of neutrophils and eosinophils. Major symptoms associated with lung habitation are nonspecic and often include persistent cough, chest pain, and hemoptysis. Adults, which are reddish brown and approximately 1 cm long, live within capsules in the bronchioles. Sputum is the primary diagnostic specimen. Eggs are expelled from the capsule into the bronchioles and carried upward in the sputum. Eggs may be found in feces if they have been coughed up and subsequently swallowed. P. westermanni eggs are yellowish-brown, are broadly oval, are 80 to 115 µm × 48 to 60 µm in size, and have a attened operculum and slight shoulders. They are unembryonated when passed. The shell thickens at the end opposite the operculum (Fig. 28.52). These eggs can appear similar to those of Diphyllobothrium latum and must be carefully examined when seen in the feces. A wet mount of sputum demonstrates the egg in some patients.

Blood ukes, Schistosoma spp., differ from other ukes in the following manner: • Both male and female forms exist; the female lives in an involuted chamber, the gynecophoral canal, which extends the length of the male. • The eggs are unoperculated. • Humans are infected by direct cercarial penetration of skin. • The ukes have a cylindric shape, rather than being dorsoventrally attened. The three primary species of schistosomes pathogenic to humans are Schistosoma mansoni, Schistosoma haematobium, and Schistosoma japonicum. Male adult schistosomes measure 7 to 20 mm, and adult females measure 7 to 26 mm. S. mansoni, which is commonly found in Africa, parts of South America, the West Indies, and Puerto Rico, lives in venules of the mesentery and large intestines. S. japonicum, which is commonly found in the Far East, including Japan, China, and the Philippines, lives in venules of the small intestine. This species, unlike the other two, has many mammalian reservoir hosts. S. haematobium, which is primarily found in the Nile Valley, the Middle East, and East Africa, lives in the veins surrounding the bladder. Two additional species pathogenic for humans are S. intercalatum and S. mekongi

Clinical infections Schistosomiasis (bilharziasis) affects approximately 200 million people worldwide. Symptoms are related to the phases of the uke’s life cycle and location of the adults. Cercarial penetration may cause a self-limiting local dermatitis, including irritation, redness, and rash, that persists for approximately 3 days. Larval migration through the body causes generalized symptoms, such as urticaria, fever, and malaise, which can last up to 4 weeks. The presence of the migrating larvae and adults in the veins causes little inammatory damage seemingly because they acquire host HLAs and ABO blood group antigens on their surface that diminish the host’s immune response. Egg production and egg migration through the tissues are responsible for most of the immediate damage because the eggs are highly immunogenic. After release by the adult female, the eggs secrete enzymes and begin to penetrate

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vessel walls and tissue. Eggs subsequently nd their way into the lumen of the intestines or bladder. The egg spines cause trauma to the tissues and walls of the vessels during the early stage of acute infections and can result in gross or microscopic hematuria (S. haematobium) or diarrhea (S. mansoni and S. japonicum). In some individuals, especially those heavily infected with S. japonicum, an acute serum sickness–like illness (Katayama fever) occurs during the initial egg-laying period. This is induced by hypersensitivity response to egg antigens and is characterized by increased circulating levels of immune complexes and eosinophils. In chronic infections, the eggs remaining in the tissue induce an immune response, resulting in granuloma formation, which leads to thickening and brotic changes. Scarring of the veins, development of ascites, pain, anemia, hypertension, hepatomegaly, and splenomegaly are also seen. In urinary schistosomiasis, microscopic bleeding into urine is present during the acute phase. In chronic stages, dysuria, urine retention, and urinary tract infections occur. Penetration of humans by cercariae of the ukes of birds and other mammals causes schistosomal dermatitis, commonly referred to as swimmer’s itch. Foreign proteins from these cercariae elicit a tissue reaction characterized by small papules 3 to 5 mm in diameter, edema, erythema, and intense itching. Symptoms last about 1 week and disappear as cercariae die and degenerate.

Life cycle The life cycles of all three schistosomes are identical (Fig. 28.53). The eggs are embryonated when excreted, and the miracidium is released when the egg reaches water. After

the miracidium penetrates a snail (the rst intermediate host), sporocysts and then cercariae are produced during a 6-week period. Cercariae migrate from the snail into water. Cercariae, with the help of enzymes, penetrate intact human skin. Once in the vasculature, they shed their forked tails and are referred to as schistosomula. They circulate until they reach the lungs or enter the liver, where maturation and pairing of the female and male are completed. The adults use oral and ventral suckers for attachment. The paired adult ukes use the portal system to reach veins of the intestine or bladder. Adults living in the veins can be killed with praziquantel, but the drug has no effect on the eggs in tissues.

Laboratory diagnosis Diagnosis is made by nding embryonated eggs in feces (S. mansoni and S. japonicum) or in urine (S. haematobium). The egg of S. mansoni (Fig. 28.54A) is yellowish, elongated, 115 to 175 µm × 45 to 75 µm in size, and has a prominent lateral spine. S. haematobium eggs (see Fig. 28.54B) are elongated, 110 to 170 µm × 40 to 70 µm in size, and have a terminal spine. S. japonicum eggs (see Fig. 28.54C), which resemble S. mekongi eggs, are round, 60 to 95 µm × 40 to 60 µm in size, and have a small, curved, rudimentary spine that might be obscured. The best time to collect eggs in urinary schistosomiasis is during peak excretion time in the early afternoon (noon to 2 pm). Biopsy may also be used in the diagnosis of schistosomiasis. Serodiagnosis can be useful to diagnose infection in patients from nonendemic countries who develop symptoms after visiting endemic areas. In areas of endemicity, serum antibody testing cannot differentiate between active and nonactive infections. A urine-based, point-of-care, rapid test strip

Eggs passing in feces, urine

Adults in specific organs

Freshwater

First larval stage—miracidia Veins of small intestine Schistosoma japonicum

Penetration or ingestion by snail

Circulation Penetration of appropriate host

Veins of large colon Schistosoma mansoni

Veins of bladder Schistosoma haematobium

Fig. 28.53 Life cycle of blood ukes (Schistosoma spp.).

Cercariae emerging from snail

Development of sporocyst and redial generations

Medically important parasitic agents

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Rostellum and hooklets

for the detection of parasite antigen (circulating cathodic antigen) is commercially available and useful for eld screening in endemic areas.

Suckers

Tapeworms

Scolex —means of attachment

Neck—origin of proglottids

Tapeworms (cestodes) are another group of human parasites in the phylum Platyhelminthes. They show extensive size variation, ranging from 3 mm to 10 m, generally require intermediate hosts in their life cycle, and are hermaphroditic. They are ribbonlike organisms, whose method of growth involves the addition of segments, termed proglottids. Each proglottid, when mature, produces eggs infective for the intermediate host. Fig. 28.55 shows a general diagram of the tapeworm. The anterior headlike segment of a tapeworm, or scolex, has suckers and, in some species, hooklets as a means of attachment to the intestinal mucosa. The neck, located directly behind the scolex, is where proglottid production occurs. Treatment is targeted at detaching the scolex from the mucosa. Gravid proglottids at the distal end of the organism discharge eggs into feces. Eggs of most of the tapeworms contain a hexacanth embryo or oncosphere (tapeworm embryo with three pairs of hooks that is infective for the intermediate host). Transmission to humans involves ingestion of a larval stage, called the cysticercus (larva consisting of a uid-lled sac containing an invaginated scolex), cysticercoid, or plerocercoid larva (depending on the genus) in raw or undercooked meat or sh or of insects harboring the larval stage. The larval stage contains an invaginated scolex inside a protective membrane.

A

Immature

Mature (male and female sex organs)

Strobila —all proglottids Gravid (producing eggs)

Fig. 28.55 A tapeworm.

B

C

Fig. 28.54 A, Schistosoma mansoni egg. B, Schistosoma haematobium egg. C, Schistosoma japonicum egg. (Unstained wet mounts, ×400.)

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Table 28.8 Comparisons of tapeworm eggs Organism

Average size (µm) and shape

Other identifying features

Diphyllobothrium latum

58–76 × 40–50, oval

Inconspicuous operculum Small knob at end opposite operculum Unembryonated when passed

Taenia spp.

30–35, round

Thick, brown, radially striated shell Embryonated, with six-hooked oncosphere when passed

Hymenolepis nana

30–47, oval

Two membranes—inner has two polar knobs, from which four polar laments extend into space between inner and outer membranes Embryonated, with six-hooked oncosphere when passed

Hymenolepis diminuta

50–75, round to slightly oval

Two membranes—inner has very slight polar knobs No polar laments Embryonated, with six-hooked oncosphere when passed

Dipylidium caninum

20–40 (each egg), round; resembles Taenia spp.

Eggs passed in packet of 15–25 Eggs embryonated, with six-hooked oncosphere when passed

The diagnosis of tapeworm infection is usually made by nding eggs in feces, although proglottids can be used if they are passed intact. Intestinal tapeworm infections are usually treated with praziquantel; alternatively, they can be treated with niclosamide, albendazole, or nitazoxanide. Table 28.8 compares the characteristics of tapeworm eggs.

Diphyllobothrium latum D. latum, the sh tapeworm, is found worldwide in areas in which the population eats pickled or raw freshwater sh. In the United States, it is primarily seen in the areas around the Great Lakes. Fish-eating mammals in endemic areas may also be infected. Humans usually harbor only a single worm, which attaches in the jejunum and can reach a length of up to 10 m. Most infected individuals demonstrate no clinical symptoms; others have vague GI symptoms, including nausea and vomiting and intestinal irritation. The organism may cause a vitamin B12 deciency, especially in persons of northern European descent, and long-term infection may lead to megaloblastic anemia. The life cycle of D. latum is somewhat of a hybrid between that of the ukes and that of the tapeworms (Fig. 28.56). The operculated, unembryonated egg, passed in human feces, must reach water to mature. The rst larval stage (coracidium) is ingested by a copepod and develops into a procercoid larva within the copepod. When the infected copepod is ingested by a sh, the larva leaves the sh’s intestine and invades the esh, where it develops into a plerocercoid larva, which consists of a scolex with a thin, ribbonlike portion of tissue. Humans ingest the plerocercoid larvae by eating raw or undercooked sh. The scolex is released in the intestine, where it develops into an adult worm.

The scolex, proglottid, and egg are diagnostic structures that can be found in fecal specimens. The egg, however, is usually detected. The egg is unembryonated when passed, operculated, and yellow to brown (Fig. 28.57). It is about 58 to 76 µm × 40 to 50 µm in size and has a small, knoblike protuberance at the end opposite the operculum. The knob may not be seen on all eggs, so size and lack of shoulders must be used to distinguish the egg from that of Paragonimus westermani. The proglottid is wider than it is long, with a characteristic rosette-shaped or coiled uterus. The scolex, which is 2 to 3 mm long, is elongated and has two sucking grooves, one located on the dorsal surface and the other on the ventral surface, and no hooks.

Taenia Two Taenia spp. infect humans: (1) Taenia saginata, the beef tapeworm, which is found primarily in beef-eating countries of the world, and (2) Taenia solium, the pork tapeworm, which is found in areas of the world with a high consumption of pork, such as Latin America. Both organisms attach to the intestinal mucosa of the small intestine. The adult T. saginata can reach a length of 10 m, whereas the adult T. solium may reach only 7 m. Infection with the adult tapeworm of either species usually causes few clinical symptoms, although vague abdominal pain, indigestion, and loss of appetite may be present. The major complication of infection with T. solium is cysticercosis, in which the infected individual becomes the intermediate host and harbors the larvae in tissues. This is discussed in the section on tissue infections with cestodes (see the “Cysticercosis” section later in this chapter). As noted, the life cycles of the two Taenia spp. Fig. 28.58) are identical except that humans may also serve as intermediate

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Eggs in feces Adults in intestine of human Eggs in water

Coracidium release

Ingestion by Cyclops (crustacean)

Ingestion of Cyclops (crustacean) by fish

Release of larvae and maturation in intestine

Human ingestion of raw fish

Procercoid larva in Cyclops (crustacean)

Plerocercoid larva development in tissue

Fig. 28.56 Life cycle of Diphyllobothrium latum

Fig. 28.57 Diphyllobothrium latum egg (unstained, ×400).

hosts for T. solium. Embryonated eggs are passed in human feces and ingested by the intermediate host. The oncosphere is freed in the intestinal tract, migrates through the intestinal wall, and gains access via the circulatory system to the muscles of the host, where it transforms into a cysticercus. When humans ingest raw or undercooked meat, the scolex in the cysticercus is freed, attaches in the human small intestine, and matures into the adult tapeworm within 10 weeks. The proglottids are motile and, if broken off in the intestinal tract, may actively migrate out via the anus.

Laboratory diagnosis of Taenia infection can be made by nding the egg, scolex, or proglottid in the feces. The egg, which is the most common stage found, is yellow to brown, round, and surrounded by a thick wall with radial striations; it measures 30 to 35 µm in diameter. The egg is embryonated, with a six-hooked oncosphere when passed in feces (Fig. 28.59). Eggs of these species are indistinguishable and must be reported as Taenia sp. eggs. Gravid proglottids may be seen in the stool specimen and can be used to differentiate the two species. Proglottids of T. solium have 7 to 13 primary uterine branches on each side of the main uterine trunk, whereas proglottids of T. saginata show 15 to 20 per side. The scolex, if found, can also be used to distinguish the organisms. The scolex of T. saginata is less than 5 mm long and has four suckers, whereas that of T. solium has a rostellum, with a double row of 25 to 30 hooklets in addition to the four suckers.

Hymenolepis The dwarf tapeworm, Hymenolepis nana, is found worldwide and is a common tapeworm in children, whereas Hymenolepis diminuta, the rat tapeworm, is seen less frequently. Light infections are usually asymptomatic; large numbers of worms may cause abdominal pain, diarrhea, irritability, and headache. Infections with H. nana are easily transmitted among children because an intermediate host is not required. Direct fecal-oral transmission of the egg, development of the cysticercoid in

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Eggs in feces Ingestion by specific intermediate host

Hexacanth embryo release in intestine of host Penetration of mucosa to tissue

Taenia solium only Development into cysticercus

Human ingestion of eggs

Cow Taenia saginata

Embryo released in intestine

Hog Taenia solium

Development into adult

To tissue

Cysticercus in eye, brain, muscle, bone

Scolex release

Attachment to intestine

Human ingestion of undercooked meat

Dissolution of cysticercus in intestine

Fig. 28.58 Life cycle of Taenia spp.

The primary method of diagnosis is nding the egg in a stool specimen. The egg is spherical to oval, measures 30 to 47 µm, and has a grayish color. The hexacanth embryo is contained within an inner membrane, and the area between the inner membrane and egg wall contains two polar thickenings from which four to eight polar laments extend (Fig. 28.61). Infection with H. diminuta is acquired by ingesting eas that contain the infective cysticercoid. The adult tapeworm is 20 to 60 cm long. The egg, which must be distinguished from that of H. nana, is 50 to 75 µm, gray or straw colored, and oval. An inner membrane with inconspicuous polar thickenings but no polar laments surround the oncosphere (Fig. 28.62).

Fig. 28.59 Taenia sp. embryonated egg (unstained wet mount, ×400).

the intestinal tissue of the host, and reentry into the lumen for development into an adult characterize the life cycle (Fig. 28.60). This autoinfective life cycle is most common, although an insect vector may serve as an intermediate host in an alternative form of the life cycle. The adult H. nana is approximately 40 mm long and has a small scolex, with four suckers and a rostellum with spines.

Dipylidium caninum Humans serve as accidental hosts for Dipylidium caninum, the dog tapeworm. Children are usually infected by ingesting eas containing the larval stage. The resulting infections generally do not cause any symptoms. The proglottid may be seen in human feces and is characterized by its pumpkin-seed shape, twin genitalia, and the presence of two genital pores, one on each side of the proglottid. The eggs are characteristically seen in packets of 15 to 25 eggs. Individual eggs are 20 to 40 µm in size and resemble those of Taenia spp. (Fig. 28.63).

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Eggs in human feces

Adults in intestine Hymenolepis nana or Hymenolepis diminuta

Insect ingestion of infective egg Hymenolepis nana autoinfection

Scolex release

Human ingestion of egg

Migration back to intestine

Hexacanth embryo release in intestine

Penetration into tissue to form cysticercoid

Embryo release into intestinal tissue Migration to bowel

Human ingestion of insect Cysticercoid formation

Freeing of scolex in intestine and attachment to wall

Fig. 28.60 Life cycle of Hymenolepis spp.

Fig. 28.61 Hymenolepis nana egg (iodine wet mount, ×400).

Tissue infections with cestodes Cysticercosis, sparganosis, and hydatid cyst disease are the major diseases caused by the tissue stage of a tapeworm. They originate when a human accidentally becomes the intermediate host for the parasite.

Fig. 28.62 Hymenolepis diminuta egg (iodine wet mount, ×400).

Cysticercosis Cysticercosis results when a human ingests the infective eggs of T. solium, the pork tapeworm, thus becoming an intermediate host. The disease is endemic in areas of rural Latin America, Asia, and Africa and is reemerging as a zoonosis in the United States as a result of immigration of persons from

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animals containing the plerocercoid larva; or through invasion by the plerocercoid larva when the raw tissue from the second intermediate host is used as a poultice. The disease is most common in Southeast Asia. Infection is often seen in the eye after a poultice has been applied to relieve an infection. The organism may also cause migratory subcutaneous nodules, itching, and pain. The diagnosis of sparganosis is made by nding a small, white, ribbonlike organism with a rudimentary scolex. Size ranges from a few millimeters to 40 cm. The organism may be removed surgically.

Echinococcosis Fig. 28.63 Dipylidium caninum egg packet (unstained, ×400).

endemic areas. Contributing factors for infection include poor hygiene and sanitary habits that result in ingestion of food containing an infective egg as well as residence in rural areas and hog farming. Once the egg is ingested, the hexacanth embryo is released into the intestines, penetrates the intestinal wall, and enters the circulation to develop as a cysticercus in any tissue or organ. The larva can live up to 7 years and elicits a host tissue reaction, resulting in production of a brous capsule. Once the organism dies and releases larval antigens, there is an intense host inammatory reaction that leads to tissue damage in the area. Eventual calcication of the cysticercus will occur. The most commonly infected sites are the striated muscle, eye, and brain. Light infections usually cause no clinical symptoms. When present, symptoms depend on the organ affected and the size and number of cysticerci present. Muscular pain, weakness, and cramps characterize infections of the striated muscle. A cysticercus can form in the vitreous or subretinal space of the eye. Retinal detachment, intraorbital pain, ashes of light, and blurred vision may occur. Neurocysticercosis, which is the most serious manifestation, is the causative agent for up to 30% of epilepsy cases seen in patients in countries where the disease is endemic. In the United States, the condition is often seen in Hispanic immigrants from endemic areas. Infection may be manifested by headaches, symptoms resembling those seen in meningitis or a brain tumor, convulsions, or a variety of motor and sensory problems. The cysticercus is oval, translucent, and about 5 to 18 mm in size. It contains an invaginated scolex containing four suckers and a circle of hooklets on the rostellum. The infection may be diagnosed by various methods, including radiography to detect calcied cysts, ophthalmoscopic examination of the eye to detect cysticerci, imaging techniques (CT and MRI) to locate larvae in the brain, and biopsy and histologic staining of tissue. Serologic tests, including immunoblot techniques, have been developed to detect antibodies against specic cysticercal antigens. These are most useful in epidemiologic studies.

Sparganosis Human infection with the plerocercoid larva (sparganum) of a dog or cat tapeworm can result in sparganosis. Humans acquire the infection by ingesting a copepod containing the procercoid larva; by ingesting reptiles, amphibians, or other

Echinococcosis (hydatid cyst disease) is an infection by Echinococcus granulosus that normally involves the dog or another member of the family Canidae as the denitive host. Sheep and other herbivores are the usual host of the larval stage (hydatid cyst). The disease is primarily seen in sheep-raising areas of the world, including Australia, southern South America, and parts of the southwestern United States. The adult worm is approximately 5 mm long and contains only three proglottids. The eggs are found in the feces of dogs or other denitive hosts and resemble those of Taenia spp. A human becomes an intermediate host by accidentally ingesting the eggs of E. granulosus containing the hexacanth embryo. The oncosphere is liberated in the intestine, penetrates the mucosa, enters the circulation, and usually lodges in the liver. The embryo develops a central cavity–like structure lined with a germinal membrane, from which brood capsules and protoscolices (hydatid sand) develop. The hydatid cyst’s size is limited by the organ in which it develops. In bone, a limiting membrane never develops, so the cyst lls the marrow and eventually erodes the bone. Symptoms differ, depending on the organ infected. Pressure from the increasing size of a cyst may cause necrosis of surrounding tissue. Rupture of the cyst liberates large amounts of foreign protein (allergen) that may elicit an anaphylactic response. In addition, freed germinal epithelium may serve as a source of new infection. The diagnosis can be made by radiologic examination, ultrasonography, or other imaging techniques. Aspiration of the cyst contents usually reveals the presence of protoscolices.

Roundworms Human roundworms include those that infect the intestinal tract and blood and tissue. These organisms, found worldwide, are transmitted by the ingestion of the embryonated egg or by direct penetration of skin by larvae in the soil, or they may require an insect vector. Intestinal roundworms are the most common of all the helminths that cause human infections in the United States. Infected individuals are found in highest numbers in warm, moist areas of the Southeast and in areas with poor sanitation. Roundworms are characterized by the presence of two sexes and a life cycle that may involve larval migration throughout the body. The adults obtain nourishment by absorbing nutrients from partly digested intestinal contents or by sucking blood. Patients may be asymptomatic or symptomatic; the severity of the symptoms is related to the worm burden, host’s nutritional status and age, and duration of

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Table 28.9 Comparisons of intestinal roundworm eggs and larvae Organism

Average size (µm) and shape

Other identifying features

Ascaris lumbricoides Fertile

45–75 × 35–50, oval

Bile-stained shell Bumpy, mammillated In one-cell stage when passed Some eggs may be decorticated (lack mammillated coat)

Infertile

85–95 × 43–47, oval (some bizarrely shaped)

Mammillated Thin shell Undifferentiated internal granules

Enterobius vermicularis

50–60 × 20–30, oval, attened on one side

Colorless shell Usually embryonated with C-shaped larva

Trichuris trichiura

50–55 × 22–23, barrel shaped

Bile-stained, thick shell Hyaline polar plugs Unembryonated when passed

Hookworm egg

50–60 × 35–40, broadly oval

Thin shell, colorless In four- to eight-cell stage when passed

Rhabditiform larva

250–300

Long buccal capsule Inconspicuous genital primordium

Filariform larva

500

Pointed tail Esophageal-to-intestinal ratio 1:4

Strongyloides stercoralis

Egg rarely seen; resembles that of hookworm

Rhabditiform larva

200–250

Short buccal capsule Prominent genital primordium

Filariform larva

500

Notched tail Esophageal-to-intestinal ratio 1:1

infection. Most roundworm infections can be treated with oral administration of albendazole or mebendazole. Table 28.9 compares the diagnostic characteristics of the eggs and larvae of intestinal roundworms.

Enterobius vermicularis E. vermicularis, often called the pinworm, is a worldwide parasite commonly detected in children, especially those 5 to 10 years of age. It is estimated that 20 million to 40 million individuals are infected in the United States alone. Key risk factors for this infection are inadequate personal and community hygiene. Enterobiasis is frequently found in families, kindergartens, daycare centers, or crowded conditions in which the eggs can be easily transmitted. The eggs are resistant to drying and are easily spread in the environment. Adult worms live in the large intestine (cecum), although they have occasionally

been found in the appendix or vagina. Ectopic infections have also caused endometritis, urethritis, and salpingitis. There is evidence that the organism may also be associated with urinary tract infections in young girls. Although patients with E. vermicularis infection are often asymptomatic, they may experience loss of appetite, abdominal pain, loss of sleep, and nausea and vomiting. Anal pruritus is caused by migration of the female worm to the perianal area. The life cycle of this organism (Fig. 28.64) is characterized by migration of the female out through the anus at night to lay eggs in the perianal area. The eggs are infective with a third-stage larva within several hours of being laid. Typically, transmission involves inhalation or ingestion of the infective eggs. Direct anal-oral transmission occurs in children as a result of poor hand washing or ngernail biting. Autoinfection, in which the hatched larvae reenter the intestine to mature into an adult, may also occur.

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Deposition of eggs in perianal areas Human ingestion of infective eggs

Female migration from anus at night Egg maturation within several hours

Adults in intestine

Reentry into intestine

Hatching of larvae

Autoinfection

Complete maturation

Release of larvae in intestine

Fig. 28.64 Life cycle of Enterobius vermicularis

Treatment for pinworm includes mebendazole, pyrantel pamoate, or albendazole. Treatment requires an initial dose followed by an additional treatment 2 weeks later to prevent reinfection caused by ingestion of eggs remaining in the environment. Because eggs are laid outside the body in the perianal area and are rarely present in the stool, a fecal specimen is unsatisfactory for diagnosis. The cellophane tape preparation or commercially available sticky paddle is considered the diagnostic method of choice. The procedure must be done as soon as the child arises in the morning. The perianal area is touched with the sticky side of the tape or paddle. The adult female can occasionally be seen in this preparation. Because the gravid female can migrate into the vagina, eggs can also be seen in vaginal specimens. The adult female measures 8 to 13 mm long and has a long, pointed tail and three cuticle lips, with alae at the anterior end. The less commonly seen male is 2 to 5 mm long, with a curved posterior. The egg is oval, colorless, and slightly attened on one side. It measures approximately 50 to 60 µm × 20 to 30 µm. The egg is usually seen embryonated, with a C-shaped larva (Fig. 28.65).

Trichuris trichiura Ascaris lumbricoides, T. trichiura, and two genera of hookworms, Ancylostoma and Necator, are the most common soil-transmitted helminths. These organisms have a worldwide distribution and are major causes of morbidity, rather than death, in developing areas of the world. Estimates indicate that 25% of the world’s population is infected with

Fig. 28.65 Enterobius vermicularis egg (unstained, ×400).

one or more of these organisms. Chronic infection caused by these helminths, especially hookworm, can adversely affect physical and mental development in children. A heavy worm burden is more likely to result in complications. Some of the risk factors for infection include poor sanitation (personal and community), poverty, occupation, and climate (necessary for maturation and survival of the eggs in the soil). Strongyloides stercoralis is also a soil-transmitted helminth but does not have the broad geographic distribution of the other organisms. T. trichiura, referred to as the whipworm, is found worldwide, especially in areas with a moist, warm climate. It is found in the southeastern United States, often as a co-infection

Medically important parasitic agents

Fig. 28.66 Trichuris trichiura egg (unstained, ×400).

with A. lumbricoides. Light infections with T. trichiura rarely cause symptoms; heavy infections result in intestinal bleeding, weight loss, abdominal pain, nausea and vomiting, and chronic diarrhea. As the adults thread themselves through the intestinal mucosa, inammation develops. Prolonged heavy infection can result in colitis or diarrhea with bloodtinged stools. Rectal prolapse can be the result of repeated heavy infections in undernourished children. Hypochromic anemia may occur in children with inadequate iron and protein intake in the presence of constant, low-level bleeding with chronic infection. Treatment includes mebendazole or albendazole. Eggs are passed in feces and require at least 14 days in warm, moist soil for embryonation to occur. Humans acquire infection by ingesting the infective egg. The larva is released in the small intestine and undergoes several molts before maturing into an adult worm in the cecum. The egg and occasionally the adult of T. trichiura may be seen in fecal specimens. The adult male measures 30 to 45 mm and has a thin anterior and a thick, coiled posterior. The female is 30 to 50 mm long, with a thin anterior and thick, straight posterior. Eggs are the typical diagnostic form. They are brown, barrel-shaped, unembryonated when passed, and 50 to 55 µm × 22 to 23 µm in size, with a thick wall and hyaline polar plugs at each end (Fig. 28.66).

Ascaris lumbricoides An estimated 1 billion people worldwide are infected with A. lumbricoides. The organism is most common in tropical and subtropical areas, and in areas of poor sanitation. Children are most commonly infected. Infections are uncommon in the United States, but the organism is most frequently seen in rural parts of the Southeast. Transmission is primarily by the fecal-oral route, and clinical symptoms may be related to the different phases of the life cycle. The organism is often found concurrently with whipworm. Abdominal discomfort, loss of appetite, and colicky pains are caused by the presence of adult worms in the intestine. There is evidence that heavy infections may contribute to lactose intolerance and malabsorption of some vitamins, including vitamin A. In children, large numbers of adult worms can cause intestinal obstruction. Because the worms feed on liquid intestinal contents, chronic infection with A. lumbricoides

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in children may hamper growth and development. Larvae migrating through the lungs can cause an immune response in the host characterized by asthma, edema, pneumonitis, and eosinophilic inltration. Rarely, larvae are seen in the sputum in heavy infections. Occasionally, fever or other disease conditions cause the adults to migrate from the intestine and invade other organs, resulting in peritonitis, liver abscess, or secondary infection in the lungs. Adults may also exit through the mouth, tear duct, or nose and have been reported to enter and block catheters. Eosinophilia may be present. Albendazole or mebendazole are the drugs of choice for treating infection with Ascaris. Eggs that are deposited in warm, moist soil become infective within about 2 weeks. After the egg is ingested, larvae hatch in the duodenum, penetrate the intestinal wall, and gain access to the hepatic portal circulation. They break out from the capillaries into the lungs, travel up the bronchial tree and trachea and over the epiglottis, and are swallowed. Maturation is completed in the intestine. The life cycle (Fig. 28.67) takes about 50 days after infection until adults are mature. The usual diagnostic stage is the egg. Fertile Ascaris eggs are oval, measure 45 to 75 µm × 35 to 50 µm, and have a thick hyaline wall surrounding a one-cell–stage embryo. Most eggs have a brown, bile-stained, mammillated outer layer (Fig. 28.68). Some eggs, described as decorticated, lack the mammillated outer coat. Infertile eggs, up to 90 µm in length, are often elongated and contain a mass of highly refractile granules. Adults, measuring 15 to 35 cm long and about the diameter of a lead pencil, may be seen in stool samples. The female has a straight posterior, and the male has a curved posterior. Both have three anterior lips with small, toothlike projections.

Hookworms Worldwide, about 740 million people are estimated to be infected with hookworms. The greatest incidence is in the tropics and subtropics, particularly Asia, Latin America, the Caribbean, and sub-Saharan Africa. In the United States in the 1950s, hookworm infection was very common, particularly in the South. Improved sanitation has greatly reduced the incidence. Unlike other helminths, in which infection peaks in childhood and adolescence, the hookworm burden often increases with age. Classically, two species of hookworm, Necator americanus (New World) and Ancylostoma duodenale (Old World), were known to infect humans. Recently an animal parasite, Anclyostoma ceyanicum (prevalent in Southeast Asia, Australia, and the Pacic Islands) was recognized as a human pathogen. A. duodenale is seen in southern Europe and northern Africa along the Mediterranean as well as in parts of Southeast Asia and South America. N. americanus has a geographic distribution in Africa, Southeast Asia, and South and Central America and is endemic in rural areas of the southeastern United States. Adults of the three species can be differentiated by the morphology of the buccal capsule or, in the male, the copulatory bursa. The eggs, however, are identical. These worms live in the small intestine and attach to the mucosa by means of teeth (A. duodenale) or cutting plates (N. americanus). They digest the tissue plug and pierce capillaries. Once attached, they continue to ingest blood as a source of nourishment by secreting anticoagulants, platelet inhibitors, and substances

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Mating of adults in intestine Maturation in soil

Passage of eggs in stool Complete maturation in intestine Human ingestion of infective eggs

Migration up trachea, over epiglottis Breaking of larvae into alveoli of lungs Release of larvae into intestine and penetration of intestinal wall

Into circulation

Swallowed

Fig. 28.67 Life cycle of Ascaris lumbricoides.

Fig. 28.68 Ascaris lumbricoides egg, fertile (unstained, ×400).

that interfere with the tissue factor–factor VIIa complex. The organisms also secrete substances that interfere with the action of digestive enzymes and inhibit host absorption of nutrients. Clinical symptoms differ according to the phase of the life cycle and the worm burden. A small, red, itchy papule,

referred to as ground itch, develops at the site of larval penetration. If large numbers of larvae are present during the lung phase of migration, the patient may have bronchitis, but unlike with Ascaris larvae, no host sensitization occurs. The most severe symptoms are associated with the adult, including nonspecic symptoms such as diarrhea, fever, and nausea and vomiting. Eosinophilia is often present. A few patients may experience pica and then ingest dirt (geophagia). Blood loss, ranging from 0.03 to 0.2 mL per worm per day, is primarily the result of the ingestion of blood by the adult worm. Hemorrhages at the site of attachment, however, also contribute to total blood loss. Chronic heavy hookworm infection can lead to microcytic hypochromic anemia, especially in children whose diet is inadequate in iron and protein. A chronic heavy infection may affect the mental and physical development of a child because of the complications of anemia and malnutrition. Infection is usually treated with albendazole or mebendazole. Supportive therapy, including iron and protein supplements, may be needed in severe cases, especially if the child shows evidence of anemia or if the infection occurs in a pregnant woman. Vaccine development using hookworm antigens is being targeted as a way to help control infections.

Medically important parasitic agents

after skin penetration by the lariform larva. Fig. 28.69 shows the life cycle of the hookworm. Adult hookworms are rarely seen in stool specimens; the egg and the rhabditiform larva are the usual diagnostic stages. The eggs and rhabditiform larvae of the three species are indistinguishable; therefore the laboratory report can only state “hookworm” when a characteristic egg or larva is found in a stool specimen. The egg is oval, colorless, thin shelled, and 50 to 60 µm long and usually contains an embryo in the four- to eight-cell stage of cleavage (Fig. 28.70). The rhabditiform larva must be differentiated from that of S. stercoralis because treatment is different. The hookworm rhabditiform larva is 250 to 300 µm long and has a small, inconspicuous, genital primordium (Fig. 28.71A) and a long buccal capsule (see Fig. 28.71B). The lariform larva also must be distinguished from that of S. stercoralis. Hookworm lariform larvae are about 500 µm long, with a pointed tail and an esophageal-intestinal ratio of 1:4.

Case check 28.2 The patient in the Case in Point shows several characteristics associated with hookworm infection. The vesicular lesions on the foot (ground itch) represent sites at which filariform larvae have penetrated skin and entered the blood circulation. In the case of other roundworms, the egg is usually ingested; in the case of tapeworms, there could possibly be a history of eating raw or undercooked meat.

The presence of a low hemoglobin level and microcytic hypochromic anemia is common in severe or long-term hookworm infection because the organism attaches to the intestinal mucosa and uses blood as a source of nourishment. The long-term, low-level blood loss will cause this type of anemia, characteristic of iron-deciency anemia, in individuals who are malnourished or lack adequate dietary iron. When the eggs are deposited in warm, moist soil, the noninfective, feeding, rst-stage rhabditiform larva develops within 1 to 2 days and feeds on bacteria in the soil. A nonfeeding, infective, lariform larva develops within 1 week. Humans are infected when the lariform larvae penetrate skin. The organisms enter the circulation and break out of the capillaries into the lung and then migrate up the bronchial tree, over the epiglottis, and into the digestive tract. After additional larval molts, the worms attach to the mucosa in the small intestine. Eggs are produced within 6 to 8 weeks

Strongyloides stercoralis S. stercoralis, known as the threadworm, inhabits the small intestine but is also capable of existing as a free-living worm. It is endemic in the tropics and subtropics, including Southeast Asia, Latin America, and sub-Saharan Africa but has been found on all continents except Antarctica. It is estimated that 30 million to 100 million people are infected worldwide. S. stercoralis can persist in the host for decades after initial infection, and infection may progress to hyperinfection if the

Adults in intestine Maturation in soil

Eggs in feces

Attachment to intestinal wall

Emergence of rhabditiform larvae

Maturation in lumen of small intestine Circulation Migration up trachea, over epiglottis Breaking of larvae into alveoli of lungs Penetration of skin

Swallowed

Fig. 28.69 Life cycle of the hookworm.

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host becomes immunocompromised. In the United States, the prevalence rate ranges from 0.4% to 4%, with most cases found in people living in Appalachia, people living in rural areas of the Southeast, or immigrants from endemic areas. Although many patients with S. stercoralis infection are asymptomatic, some may exhibit fever, nausea and vomiting, tracheal irritation, and sharp, stabbing pains that resemble those of an ulcer or other GI diseases, including pancreatitis. Chronic mild diarrhea may be present. The eosinophil count is often elevated—in some cases, up to 40%. Unlike hookworms, S. stercoralis larval penetration of skin does not cause a prominent papule, and migration through the lungs rarely elicits pneumonitis, but the patient may have wheezing and a mild cough. In contrast with the mild symptoms in an immunocompetent host, patients with a drug-induced immunocompromised state (corticosteroids), lymphoma, human T-lymphotropic virus 1 infection, malignancy, or other conditions that cause T-cell depletion can develop severe infections, referred to as disseminated strongyloidiasis or hyperinfection. In this population, large numbers of the lariform larvae develop in the intestine in an autoinfective cycle and migrate from the intestine into the lungs and other organs, such as the liver, heart, and CNS, causing a fulminating, often fatal infection. Shortness of breath and coughing are common symptoms in

Fig. 28.70 Hookworm egg (unstained, ×400).

A

this group of patients. Fig. 28.72 is a Gram-stained smear of aspirated sputum showing coiled nematode larvae (arrows), the morphology of which is consistent with S. stercoralis Most infections in organ transplant recipients are caused by reactivation of latent or chronic infection, although some cases may result from primary infection. Individuals undergoing transplantation and who have a history of travel to or residence in endemic areas may need to be screened for S. stercoralis. Secondary bacterial infections that occur because of massive larval migration may be seen in up to 40% of patients with disseminated strongyloidiasis and can delay diagnosis of the underlying infection. The mortality rate in immunocompromised patients is over 80%; the usual causes of death are complications resulting in respiratory failure. Disseminated strongyloidiasis, however, is not common in patients with advanced AIDS, despite their immunocompromised status. Research into this seeming contradiction has shown that progression from intestinal to hyperinfection is dependent on an intact mucosal immune response that allows rhabditiform larvae to develop into infectious larvae in the gut leading to autoinfection. It is hypothesized that immune suppression in HIV prevents the development of the larvae in the gut, and autoinfection is halted. The life cycle of S. stercoralis can take one of three phases (Fig. 28.73): (1) direct, which is similar to that of the

Fig. 28.72 Aspirated sputum, Gram stain, light microscopy, (×200). Coiled nematode larvae (arrows). Morphology consistent with Strongyloides stercoralis

B

Fig. 28.71 A, Hookworm rhabditiform larva. Note the long buccal capsule and lack of prominent genital primordium (iodine wet mount, ×200). B, Hookworm rhabditiform larva, buccal capsule (iodine wet mount, ×200).

Medically important parasitic agents

Adults in small intestine

Eggs in small intestine

699

Rhabditiform larvae in feces

Hatching of eggs

Feces deposition in soil

1 Autoinfection

2 Free-living cycle

3 Direct route

Filariform larvae development in intestine

Complete development in intestine

Free-living male and female adult development

Penetration of intestinal wall Entry into circulation

Egg production

Filariform larvae development in soil (infective for humans)

Hatching of rhabditiform larvae

Migration up trachea

Entering circulation Breaking through to alveoli

Penetration of human skin Swallowed

Fig. 28.73 Life cycle of Strongyloides stercoralis.

hookworm; (2) indirect, which involves a free-living phase; or (3) autoinfection. In the direct life cycle, the fertile egg hatches in the intestine and develops into the rhabditiform larva (noninfective form), which is passed in feces. In the soil, the rhabditiform larvae develop into lariform larvae, which are infective for humans by direct penetration. Once the larva has penetrated skin, it enters the blood circulation, breaks out from the capillaries in the lung, migrates up the bronchial tree and over the epiglottis, and enters the digestive tract, where it matures into the adult worm. In the indirect life cycle, the rhabditiform larvae in the soil develop into free-living males and into females that produce eggs. At any point, the free-living cycle may revert and result in the production of infective lariform larvae. In most individuals, the autoinfective life cycle allows the initial infection to persist at low levels for years and is the underlying cause of hyperinfection. In this cycle, the rhabditiform larvae develop into the lariform larvae in the intestine rather than being passed in feces. These lariform larvae then penetrate the mucosa, enter the blood circulation, and return to the intestine to develop into adults. The parasitic female threadworm is small (2.5 mm) and rarely seen in a stool specimen. No male has been identied in intestinal infections. The primary diagnostic stage in humans is the rhabditiform larva. It is 200 to 250 µm long, with a short buccal capsule (Fig. 28.74A), large bulb in the esophagus, and

prominent genital primordium located in its posterior half to posterior third (see Fig. 28.74B). The egg, which is rarely seen except in cases of severe diarrhea, resembles that of a hookworm. It is thin shelled, measures 54 × 32 µm, and often is segmented. The lariform larva has a notched tail, is 500 µm long, and has an esophageal-to-intestinal ratio of 1:1. Filariform larvae may be identied in the sputum of patients with hyperinfection (Fig. 28.72). Fig. 28.75 shows a sheep blood agar plate inoculated with sputum, after 24-hour incubation with 5% carbon dioxide in air. Note heavy bacterial growth in the area of primary inoculation. The thin trails of colonies (arrows) lacing the surface of the agar are due to motile larvae. If clinical symptoms suggest Strongyloides infection but multiple stool specimens test negative for the larvae, a duodenal aspirate or biopsy specimen may be used for diagnosis because the organism lives in the upper small intestine. There are no antigen-specic tests for Strongyloides infection, and diagnosis relies on identication of the larvae in stool. Antibody tests cannot distinguish current infection from past infection and are prone to yielding false-positive results in infections with other helminths. Ivermectin is the recommended therapy with albendazole used as an alternative. In hyperinfection or disseminated infections, it is recommended that immunosuppressive therapy be reduced if possible.

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A

B

Fig. 28.74 A, Strongyloides stercoralis rhabditiform larva, buccal capsule (iodine wet mount, ×200). B, S. stercoralis rhabditiform larva. Note the short buccal capsule and prominent genital primordium (unstained, ×200).

Fig. 28.75 Sheep blood agar plate inoculated with expectorated sputum, 24-hour incubation with 5% carbon dioxide in air. Note heavy bacterial growth in the area of primary inoculation with thin trails of colonies (arrows) lacing the surface of the agar (see Fig. 28.72).

Blood and tissue roundworm infections Trichinella spiralis Trichinosis is the infection of muscle tissue with the larval form of T. spiralis, a helminth whose adult stages live in the human intestine. Humans acquire the infection by eating undercooked meat, particularly pork that contains the larval forms. In recent years, ingestion of wild game has led to infection by other species of Trichinella. The larvae are released from the tissue capsule in the intestine and mature into adults. The female produces liveborn larvae that penetrate the intestinal wall, enter the circulation, and are carried throughout the body. Once the larvae enter the striated muscle, they begin a maturation cycle that is completed in about 1 month. The larvae coil and become encapsulated. Although larvae remain viable for many years, eventually the capsules calcify, and the larvae die. During the intestinal phase, infected individuals have few symptoms, although diarrhea and abdominal discomfort may be present. Most symptoms occur during the migration and encapsulation periods; the severity of symptoms depends on the number of parasites, the tissues invaded, and the person’s general health. Symptoms that occur during the larval phase are the result of an intense inammatory response by the host.

Fig. 28.76 Trichinella spiralis larva (biopsy specimen) (hematoxylin and eosin stain, ×200).

Common symptoms include periorbital edema, fever, muscular pain or tenderness, headache, and general weakness. Muscle enzyme levels may be elevated. Splinter hemorrhages beneath the nails can be seen in many patients. A 40% to 80% increase in the number of eosinophils is common. Patients with symptoms should be treated with analgesics and general supportive measures. Steroids are given only in rare cases. The use of albendazole or mebendazole is effective when administered early in the infection, before the release of larvae. If diagnosis is delayed, the impact of antiparasitic drugs is less effective. Because it is difcult to recover adults or larvae in a stool specimen, the diagnosis is often based on clinical symptoms and the patient’s history. Biopsy of muscle tissue and identication of the encapsulated, coiled larva is the denitive diagnostic method. Fig. 28.76 shows a biopsy specimen of a muscle containing a larva of T. spiralis. Specimens from large muscles, such as the deltoid and gastrocnemius, should be stained for histologic examination (Fig. 28.77). The presence of calcied larvae on a radiographic lm indicates infection. Serologic tests are available.

Larva migrans Two forms of larva migrans exist in humans: cutaneous (creeping eruption) and visceral. In both cases, humans are the accidental host for nonhuman nematode larvae that are unable to

Medically important parasitic agents

Fig. 28.77 Muscle tissue, directly viewed. Encysted calcied larvae (arrow). Morphology consistent with Trichinella spiralis.

complete their life cycle in humans. In the United States, cutaneous larva migrans occurs primarily in the Southwest, MidAtlantic, and Gulf Coast areas, and it is most commonly caused by the lariform larva of the dog or cat hookworm (Ancylostoma braziliense). The larva penetrates skin through a hair follicle, a break in skin, or unbroken skin. Once inside the body, it does not enter the circulation but wanders through the subcutaneous tissue, creating long, winding tunnels. Secretions from the larva create a severe allergic reaction, with intensely itchy skin lesions that are vesicular and erythematous. Secondary bacterial infections can result from scratching. The infection resolves within several weeks when the larva dies. Diagnosis is based primarily on history and clinical symptoms. In visceral larva migrans, a human accidentally ingests the eggs of the dog roundworm (Toxocara canis) or cat roundworm (Toxocara cati). The larvae hatch in the intestine, penetrate the intestinal mucosa, and wander through the abdominal cavity and can enter the lung, eye, liver, or brain. The infection is seen primarily in children 1 to 4 years of age. Clinical symptoms include malaise, fever, pneumonitis, and hepatomegaly. An increase in the number of eosinophils ranging from 30% to 50% and as high as 85% has been reported. CNS complications may develop. Eye invasion is referred to as ocular larva migrans, and in these cases, eosinophilia is usually absent. The diagnosis is made on the basis of clinical ndings and results of serologic tests using Toxocara-specic antigens.

Filarial worms Filarial worms are roundworms of blood and tissue found primarily in tropical areas of the world. At least eight species infect humans. Those considered most pathogenic are Brugia malayi, Wuchereria bancrofti, Onchocerca volvulus, and Loa loa. Lymphatic lariasis (caused by W. bancrofti, B. malayi, and Brugia timori) is the second most common mosquito-borne disease, after malaria. In addition, the nonpathogens Mansonella ozzardi, Mansonella perstans, and Mansonella streptocerca may be seen in clinical specimens. W. bancrofti is the most common species and has the greatest geographic distribution being found throughout the tropics and subtropics. The adult larial worms give birth to liveborn larvae referred to as microlariae. Identication of the various species depends on the morphology of the microlaria, periodicity (microlaria migration), and location of the worms in

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the host. Important microlaria morphologic characteristics include the presence or absence of a sheath (the remnant of the egg from which the larva hatched) and the presence and arrangement of nuclei in the tail. Table 28.10 compares the species of microlariae commonly found in humans. Adult worms, which may range in size from 2 to 50 cm, live in human lymphatics, muscles, or connective tissues. Mature females produce microlariae that are the infective stage for the insect during the insect’s blood meal. Once ingested, microlariae penetrate the insect’s gut wall and develop into infective third-stage (lariform) larvae. These larvae enter the insect proboscis and are introduced into human circulation when the insect feeds. Fig. 28.78 illustrates a generalized life cycle for microlariae.

Wuchereria bancrofti W. bancrofti is the causative agent of bancroftian lariasis and elephantiasis. Several genera/species of mosquito serve as the insect vector. The adult larial worm lives in the lymphatics and lymph nodes, especially those in the lower extremities. The presence of the adults initiates an immunologic response consisting of cellular reactions, edema, and hyperplasia. A strong granulomatous reaction with production of brous tissue around dead worms ensues. The resulting reaction causes the small lymphatics to become narrowed or closed, causing increased hydrostatic pressure, with subsequent leakage of uid into the surrounding tissue. During this period, the patient may experience generalized symptoms, such as fever, headache, and chills, as well as localized swelling, redness, and lymphangitis, primarily at sites in the male and female genitalia and extremities. Elephantiasis, a debilitating and deforming complication, occurs in less than 10% of infections, usually after many years of continual larial infection. Chronic obstruction to the lymphatic ow results in lymphatic varices, brosis, and proliferation of dermal and connective tissue. The enlarged areas eventually develop a hard, leathery appearance. Diagnosis of W. bancrofti should include the examination of a blood specimen obtained at night (10 pm to 2 am) for the presence of microlariae. Blood specimens may be examined immediately for live microlariae or may be pooled on a slide and stained. Filtration of up to 5 mL of blood through a 5-µm nuclepore lter can detect light infections. The microlariae of W. bancrofti are sheathed, and the nuclei do not extend to the tip of the tail (Fig. 28.79). Three antigen detections assays are available, but none have U.S. FDA approval. No antibody detection tests are available.

Brugia malayi B. malayi, another nocturnal microlarial species, is limited to the Far East, including Korea, China, and the Philippines. Mosquitoes of the genera Mansonia and Aedes have been shown to transmit the organism. The pathologic aspects of the disease and the clinical symptoms are the same as those seen with W. bancrofti infections. The distinguishing characteristics of the microlariae are the presence of a sheath and the arrangement of tail nuclei—the nuclei extend to the tip, but a space separates the two terminal nuclei.

Loa loa Infection with L. loa, the eye worm, is limited to the African equatorial rainforest, where the y vector (Chrysops) breeds. Adult worms, which may live for as long as 15 years in

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Table 28.10 Comparisons of microlariae Organism

Arthropod vector

Periodicity

Location of adult, microlaria

Tail morphology

Wuchereria bancrofti

Mosquito (Culex, Aedes, Anopheles spp. and others)

Nocturnal

Lymphatics, blood

Sheathed Nuclei do not extend to tip of tail

Brugia malayi

Mosquito (Aedes, Mansonia spp.)

Nocturnal

Lymphatics, blood

Sheathed Terminal nuclei separated

Loa loa

Fly (Chrysops sp.)

Diurnal

Subcutaneous tissue, blood

Sheathed Nuclei extend to tip of tail

Onchocerca volvulus

Fly (Simulium sp.)

Nonperiodic

Subcutaneous nodule, subcutaneous tissue

Unsheathed Nuclei do not extend to tip of tail

Mansonella ozzardi

Midge (Culicoides sp.)

Nonperiodic

Body cavity, blood, skin

Unsheathed Nuclei do not extend to tip of tail

Mansonella perstans

Midge (Culicoides sp.)

Nonperiodic

Mesentery, blood

Unsheathed Nuclei extend to blunt tip of tail

Mansonella streptocerca

Midge (Culicoides sp.)

Nonperiodic

Subcutaneous, skin

Unsheathed Nuclei extend to tip of hooked tail

humans, migrate through the subcutaneous tissue, causing temporary inammatory reactions called Calabar swellings. These characteristic swellings can cause pain and pruritus that last about 1 week before disappearing, only to reappear in another part of the body. The adult worm can often be seen as it migrates across the surface of the eye. Diagnosis can be based on the presence of Calabar swellings or of the adult worm in the conjunctiva of the eye. Microlariae may be seen in a blood specimen if it is taken during the day, especially around noon, when migration peaks. The microlaria is sheathed, and nuclei extend to the tip of the tail.

Onchocerca volvulus Infection with O. volvulus is referred to as onchocercosis, or river blindness. The organism can be found in Africa and South and Central America; transmission occurs by the bite of the black y (Simulium). Adult worms are encapsulated in brous tumors in the subcutaneous tissues of humans. Microlariae can be isolated from the subcutaneous tissue, skin, and the nodule itself, but are rarely found in blood or lymphatic uid. The nodules in which adults live may measure up to 25 mm and can be found on most parts of the body. They are the result of an inammatory and granulomatous reaction around the adult worms. Fig. 28.80 depicts a cross-section of

tissue containing these organisms. Blindness, the most serious complication, results when microlariae collect in the cornea and iris, causing hemorrhage, keratitis, and atrophy of the iris. The presence of endosymbiotic bacteria of the genus Wolbachia has been linked to stimulation of the host immune response and may contribute to the inammatory tissue reaction. Diagnosis involves clinical symptoms, such as the presence of nodules, and microscopic identication of microlariae. The diagnostic method used is the skin snip, in which a small slice of skin is obtained and placed on a saline mount. Microlariae with no sheath and with nuclei that do not extend into the tip of the tail are characteristic of this organism. Because the skin snip is painful and poses a risk of infection, researchers are trying to develop alternate diagnostic methods. NAATs are being used in research laboratories. In addition, serologic assays using recombinant antigens have demonstrated high sensitivity and specicity.

Mansonella spp. M. ozzardi, M. streptocerca, and M. perstans are larial worms not usually associated with serious infections. They are transmitted by midges belonging to the genus Culicoides. The microlariae of M. streptocerca are found in skin.

Medically important parasitic agents

703

Birth of live microfilariae via female

Microfilariae in blood and lymphatics or subcutaneous tissue

Adult worms in respective tissues

Biting of human and ingestion of microfilariae by insect

Larvae migration Larvae infection of human when insect bites Microfilariae development in insect

Infective filariform larvae migration to insect salivary gland

Fig. 28.78 Generalized life cycle of microlariae.

Fig. 28.79 Wuchereria bancrofti microlaria. Note the faintly staining sheath extend from both ends of organism (Giemsa stain, ×1000).

Fig. 28.80 Cross-section of tissue infected with Onchocerca volvulus (hematoxylin and eosin stain, ×100).

They are unsheathed and have nuclei that extend to the end of the so-called shepherd’s crook tail. Microlariae of M. ozzardi and M. perstans are found in blood as unsheathed organisms. M. ozzardi microlariae have tails with nuclei that do not extend to the tips, whereas the nuclei in the tail of an M. perstans microlaria extend to the tip.

caused serious infections in the Middle East, parts of Africa, and India. It is often found in areas where step-down wells are used. Several health agencies, including the WHO and the United Nations Children’s Fund, launched an eradication program in 1986. At that time, an estimated 3.5 million people were infected. By 2012, only 542 cases were reported, and dracunculiasis was limited to four African countries. In 2020, only 27 cases were reported, raising hopes that the disease may be totally eradicated. Adult worms mature in the deep connective tissue, and the gravid females migrate to the subcutaneous tissue. Initially, a

Dracunculus medinensis D. medinensis, known as the guinea worm (also called the ery serpent of the Israelites described in the Bible), historically

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painful, blisterlike, inammatory papule appears on the leg in the area where the gravid female is present. The papule ulcerates, and when the person’s body comes in contact with water, the female worm exposes her uterus through the ulceration and releases larvae into the water. Patients may experience nausea and vomiting, urticaria, and dyspnea before the rupture of the worm’s uterus. If the worm is broken during an attempt to remove it, the patient may experience a severe inammatory reaction and secondary bacterial infection. Humans acquire the infection by ingesting a copepod (cyclops) that contains an infective larva. The larva is released in the intestine, penetrates the intestinal wall, and migrates to the body cavity, in which males and females mature. When mature and gravid, the female migrates through the subcutaneous tissue to the arm or leg to release liveborn larvae into the water. The rhabditiform larvae are then ingested by the copepod. The diagnosis is made from the typical appearance of the lesion. Although organism-specic antibodies develop with infection, immunity does not develop, and an individual can be infected multiple times. Treatment is based on the extraction of the adult worm. The worm can be wound around a stick and eased out a few centimeters each day. Analgesics and topical agents, such as metronidazole or thiabendazole, can help in the removal process, although they do not kill the adult worm. Because this method can rupture the worm, surgical excision is preferred.

POINTS TO REMEMBER

• Protozoan cysts, helminth eggs, and helminth larvae can be identied on a wet mount preparation of a fecal concentrate. The identication of protozoan trophozoites and cysts is conrmed on a permanently stained smear. • Rapid diagnostic tests based on antigen detection (e.g., immunochromatographic) are routinely used for detection of Giardia duodenalis, Cryptosporidium spp., and Entamoeba histolytica. • Multiplex nucleic acid assays for intestinal pathogens include several organisms—usually including the parasites G. intestinalis, Cryptosporidium spp., and E. histolytica. • Fecal-oral transmission is the route of infection for enteric protozoa. The cyst is the infective stage, and the trophozoite is the stage that divides and causes tissue damage. • The major intestinal protozoan pathogens include E. histolytica and G. duodenalis. D. fragilis and B. hominis cause symptomatic infections in some patients. There are several commensal protozoa that must be differentiated from the pathogens to ensure appropriate therapy when necessary. • E. histolytica, E. dispar, E. moshkovkii, and E. bangladeshi are morphologically identical, and specic immunoassay techniques must be performed to differentiate the pathogenic E. histolytica from the other three nonpathogenic species. If ingested RBCs are present in the trophozoite, the organism can be reported as E. histolytica. • N. fowleri, Acanthamoeba spp., and B. mandrillaris are freeliving amebae that can infect humans. N. fowleri causes PAM, which is rapidly fatal. Acanthamoeba spp. cause keratitis, skin infections, or granulomatous amebic encephalitis, whereas B. mandrillaris is associated with GAE and skin infections.

• The genera Leishmania and Trypanosoma are blood agellates of humans transmitted by insect bites. • Human malaria can be caused by ve different Plasmodium spp.: P. vivax, P. ovale, P. malariae, P. falciparum, and P. knowlesi. P. vivax is the most prevalent. • The life cycle of malaria is complex, with asexual reproduction taking place in human RBCs (intermediate host) and sexual reproduction occurring in the gut of the mosquito (denitive host). • Identication of Plasmodium spp. is made by observing characteristics of the infected RBCs and the malarial organism on a Wright- or Giemsa-stained peripheral blood smear. • One rapid diagnostic test (P. vivax and P. falciparum) is approved for use in the United States for diagnosing malaria. Microscopic conrmation is still performed. • B. microti, an intraerythrocytic parasite, morphologically resembles P. falciparum on blood smears. • The intestinal Apicomplexa species includes C. parvum, C. belli, and C. cayetanensis; the infective stage for humans is the acid-fast–positive oocyst. • The microsporidia are small intracellular organisms that can infect the gastrointestinal tract and other tissue. Even though they are classied as fungi, the microsporidia are often discussed with eucaryotic parasites because of their morphology and pathogenesis. • T. gondii causes a tissue infection that is usually asymptomatic in immunocompetent hosts. In patients with AIDS, a latent infection can reactivate and cause encephalitis or pneumonia. Congenital transmission can result in serious complications. • For most ukes, diagnosis is made by nding the egg in a fecal specimen. In the case of the lung uke (P. westermani), the egg may be found in sputum; in the case of S. haematobium, the egg is found in urine. • Humans are the denitive hosts, and animals or insects serve as intermediate hosts for most tapeworms infecting humans. • Eggs of the beef tapeworm, T. saginata, and eggs of the pork tapeworm, T. solium, are identical and must be reported as Taenia sp. • The diagnostic stage for a roundworm may be an egg or a larval form, depending on the species. • Pinworm (E. vermicularis) infection is common in children and is diagnosed by nding eggs on a cellophane tape preparation or pinworm paddle. Eggs are laid on the perianal area when the female migrates out through the anus at night. • Human hookworm infections are generally caused by N. americanus and A. duodenale. Recently, a third species (A. ceyanicum) pathogenic for humans was identied. The eggs are identical and are reported as “hookworm eggs.” • Eggs of S. stercoralis are not usually present in the stool; the typical diagnostic stage is the rhabditiform larva. • T. spiralis is acquired when humans ingest raw or undercooked pork containing the larval form. Diagnosis of trichinosis is made through biopsy of tissue to identify the coiled larval stage. • Diagnosis of larial worm infection is made by observing the microlariae in blood or tissue specimens. Larval characteristics include the presence or absence of a sheath and the location and arrangement of nuclei in the tail of the microlariae.

Medically important parasitic agents

LEARNING ASSESSMENT QUESTIONS

1. A trichrome-stained smear of a patient’s fecal specimen shows the presence of cysts that are oval and approximately 11 µm in size and have four nuclei containing large karyosomes with no peripheral chromatin and a cluttered appearance in the cytoplasm. What is the most likely identication of the organism? Is the organism considered a pathogen? 2. A patient with a history of travel to Africa has fever and chills. The physician suspects malaria and orders a blood smear for examination. Why should you do both a thin lm and a thick lm? Why would nal species identication be made from the thin smear? 3. Give the major characteristics (including size) that you would use to identify eggs of the following organisms: Taenia spp., Ascaris lumbricoides, Trichuris trichiura, and hookworm. 4. Describe the diagnostic method you would use to detect Enterobius vermicularis eggs that would not be used with the other types of eggs of intestinal helminths. Explain your answer. 5. Describe the microscopic characteristics you would use to differentiate the oocysts of Cyclospora cayetanensis and Cryptosporidium parvum. Include size, appearance on routine wet mount or trichrome stain, and appearance with special stains. 6. You identify a trophozoite that is approximately 22 µm in diameter on a trichrome-stained smear of a stool sample. There is a single nucleus that shows even peripheral chromatin and a small central karyosome. The cytoplasm is relatively clean, but ingested red blood cells are seen. What is the most likely identication of the organism? Is the organism considered a pathogen? If yes, describe the typical patient symptoms and possible complications. 7. For both Cryptosporidium spp. and Strongyloides stercoralis, explain the mechanism of autoinfection in the life cycle and why this phase contributes to increased severity of infection. 8. You are examining a blood smear and nd an extracellular structure that is approximately 18 µm long. It is tapered at both ends and has an anterior agellum. An undulating membrane extends the length of the body. What is the genus of this organism? What is the morphologic stage of this organism? With which two diseases do you see this stage in blood? 9. Compare primary amebic meningoencephalitis and granulomatous amebic encephalitis. Include the following in your discussion: causative organism, population usually infected, route of infection, clinical symptoms, and method of diagnosis. 10. For Toxoplasma gondii, Cryptosporidium spp., and S. stercoralis, compare the clinical presentation in the immunocompetent host and in the immunocompromised host. 11. Which of the following are remnants of eosinophils in the stool of some patients with an intestinal parasite infection? a. Ameboma b. Charcot-Leyden crystals c. Morulae d. Weil-Felix granules

705

12. An international male student from Egypt comes to the campus clinic complaining of blood in his urine. A urinalysis reveals an elongated structure that measures about 110 to 170 µm × 40 to 70 µm in size and has a terminal spine. Which of the following should be suspected? a. Schistosoma japonicum egg b. Trichuris trichiura egg c. Schistosoma haematobium egg d. Enterobius vermicularis egg 13. A patient with recent history of camping has explosive, foul-smelling, nonbloody diarrhea. An ova and parasite examination of the stool shows oval structures, about 12 × 8 µm, with four oval nuclei, each with a large central karyosome, midline axonemes, and two median bodies. Which of the following matches this description? a. Giardia duodenalis cyst b. Entamoeba histolytica cyst c. Cryptosporidium parvum oocyst d. Chilomastix mesnili cyst 14. Several children at a daycare center are having trouble sleeping and are showing increasing signs of restlessness. One child was recently diagnosed with an intestinal parasite, so the parents are informed that other children should be tested. The parasite egg found during examination is described as embryonated, containing a C-shaped larva, oval, colorless, and slightly attened on one side. It measures approximately 50 to 60 µm × 20 to 30 µm. Which of the following matches this description? a. Enterobius vermicularis b. Hookworm c. Ascaris lumbricoides d. Trichuris trichiura 15. Although uncommon, which intestinal parasite is known to leave the colon and cause metastatic diseases, especially liver abscesses? a. Giardia lamblia b. Entamoeba histolytica c. Cryptosporidium parvum d. Microsporidia 16. What is the most commonly identied intestinal protozoan parasite in the United States? a. Cryptosporidium parvum b. Entamoeba histolytica c. Giardia duodenalis d. Microsporidia 17. This pathogen can be seen in the sputum and bronchoalveolar uid of patients with a hyperinfestation syndrome. What is the name of this pathogen? a. Ascaris lumbricoides b. Entamoeba histolytica c. Giardia duodenalis d. Strongyloides stercoralis 18. Which parasite is responsible for causing a form of blindness in West Africa? a. Whipworm b. Hookworm c. Onchocerca volvulus d. Wuchereria bancrofti

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PART 2

28

Diagnostic parasitology

19. A corneal scraping from a patient with keratitis is submitted to the laboratory for examination. Which parasite would you look for in this type of clinical sample? a. Naegleria sp. trophozoites b. Acanthamoeba sp. cysts and trophozoites c. Trichinella spiralis larvae d. Toxoplasma gondii oocyts 20. While examining a peripheral blood smear stained with Giemsa-Wright stain, the hematologist notices several red blood cells that contain delicate malarial ring-forms. Further examination shows gametocytes that are banana- or crescent-shaped. Which Plasmodium sp. demonstrates these characteristics? a. Plasmodium vivax b. Plasmodium malariae c. Plasmodium falciparum d. Plasmodium ovale

BIBLIOGRAPHY Anuradha, R., et al. (2016). Systemic cytokine proles in Strongyloides stercoralis infection and alterations following treatment. Infection and Immunity, 84, 425. Baig, A. M. (2015). Pathogenesis of amoebic encephalitis: are the amoeba being credited to an “inside job” done by the host immune response? Acta Tropica, 148, 72. Bloch, E. M., et al. (2012). The third described case of transfusion-transmitted Babesia duncani. Transfusion, 52, 1517. Branda, J. A., et al. (2006). A rational approach to the stool ova and parasite examination. Clinical Infectious Diseases: An Ofcial Publication of the Infectious Diseases Society of America, 42, 972. Cama, V. A., & Mathison, B. A. (2015). Infections by intestinal coccidia and Giardia duodenalis. Clinics in Laboratory Medicine, 35, 423. Caravedo, M. A., et al. (2022). Cryptosporidiosis medication. Available at: https://emedicine.medscape.com/article/215490-medication. (Accessed 20 July 2022). Centers for Disease Control and Prevention. (2022). Naegleria fowleri—primary amebic meningoencephalitis (PAM)—amebic encephalitis. Available at: https://www.cdc.gov/parasites/ naegleria/index.html. (Accessed 20 July 2022). Centers for Disease Control and Prevention. (2019). Parasites— Balamuthia mandrillaris—granulomatous amebic encephalitis (GAE). Available at: https://www.cdc.gov/parasites/balamuthia/index. html. (Accessed 16 May 2022). Centers for Disease Control and Prevention. (2020). Parasites— cyclosporiasis (cyclospora infection). Available at: https://www.cdc. gov/parasites/cyclosporiasis/index.html. (Accessed 20 July 2022). Centers for Disease Control and Prevention. (2021). Cryptosporidiosis NNDSS summary report for 2019. Available at: https://www.cdc. gov/healthywater/surveillance/cryptosporidium/cryptosporidium-2019.html. (Accessed 20 July 2022). Centers for Disease Control and Prevention. (2022). DPDx—laboratory identication of parasites of public health concern. Available at: https://www.cdc.gov/dpdx/index.html. (Accessed 20 July 2022). Chang, T., et al. (2020). Morphological and molecular diagnosis of Necator americanus and Ancylostoma ceylanicum recovered from villagers in Northern Cambodia. The Korean Journal of Parasitology, 58, 619. Chiang, E., & Haller, N. (2011). Babesiosis: an emerging infectious disease that can affect those who travel to the northeastern United States. Travel Medicine and Infectious Disease, 9, 238. Clark, C. G., et al. (2016). Transmission of Dientamoeba fragilis: pinworm or cyst? Trends in Parasitology, 30, 136. Colley, D. G., & Secor, W. E. (2014). Immunology of human schistosomes. Parasite Immunology, 36, 347. Cope, J. R., et al. (2015). The rst association of a primary amebic meningoencephalitis death with culturable Naegleria fowleri in tap water from a US treated public drinking water system. Clinical Infectious Diseases: An Ofcial Publication of the Infectious Diseases Society of America, 60, e36.

Couturier, B. A., et al. (2015). Clinical and analytical evaluation of a single-vial stool collection device with formalin-free xative for improved processing and comprehensive detection of gastrointestinal parasites. Journal of Clinical Microbiology, 58, 2539. Demogines, A., et al. (2012). Species-specic features of DARC, the primate receptor for Plasmodium vivax and Plasmodium knowlesi. Molecular Biology and Evolution, 29, 445. Deng, L. et al. (2021). New insights into the interactions between blastocystis, the gut microbiota, and host immunity. PLoS Pathogens, 17. Available at: https://journals.plos.org/plospathogens/ article?id=10.1371/journal.ppat.1009253. (Accessed 20 July 2022). Dunn, A. L., et al. (2016). Naegleria fowleri that induces primary amoebic meningoencephalitis: rapid diagnosis and rare case of survival in a 12-year-old Caucasian girl. Laboratory Medicine, 42, 149. Dupont, H. L. (2013). Giardia: both a harmless commensal and a devastating pathogen. The Journal of Clinical Investigation, 123, 2352. Dye-Braumuller, K. C., & Kanyangarara, M. (2021). Malaria in the USA: how vulnerable are we to future outbreaks? Current Tropical Medicine Reports, 8, 43. Elbashier, M. M., et al. (2020). Evaluation of a rapid diagnostic test for Schistosoma mansoni infection based on the detection of circulating cathodic antigen in urine in Central Sudan. PLoS Neglected Tropical Diseases, 14, e0008313. doi.org/10.1371/journal.pntd.0008313. El-Taweel, H. A. (2015). Understanding drug resistance in human intestinal protozoa. Parasitology Research, 114, 1647. Emery, S. J., et al. (2014). Proteomic analysis in Giardia duodenalis yields insights into strain virulence and antigenic variation. Proteomics, 14, 2523. Esper, L., et al. (2015). Molecular mechanisms of myocarditis caused by Trypanosoma cruzi. Current Opinion in Infectious Diseases, 28, 246. Garcia, H. H., et al. (2018). Laboratory diagnosis of neurocysticercosis (Taenia solium). Journal of Clinical Microbiology, 56, e00424–18. Garcia, L. S. (2021). Practical guide to diagnostic parasitology (3rd ed.). Washington, DC: ASM Press. Gryseels, B. (2012). Schistosomiasis. Infectious Disease Clinics of North America, 26, 383. Halliez, M. C. M., & Buret, A. G. (2013). Extra-intestinal and long term consequences of Giardia duodenalis infections. World Journal of Gastroenterology, 19, 8974. Han, B., & Weiss, L. M. (2017). Microsporidia: obligate intracellular pathogens within the fungal kingdom. Microbiology Spectrum, 5. http://doi.org/10.1128/microbiolspec.FUNK-0018-2016. Hirt, R. P. (2013). Trichomonas vaginalis virulence factors: an integrative overview. Sexually Transmitted Infections, 89, 439. Hirt, R. P., & Sherrard, J. (2015). Trichomonas vaginalis origins, molecular pathology and clinical considerations. Current Opinion in Infectious Diseases, 26, 72. Jones, M. K., et al. (2019). Trematodes. In K. C. Carroll, et al. (Eds.), Manual of clinical microbiology (12th ed., p. 2590). Washington, DC: ASM Press. Linam, W. M., et al. (2015). Successful treatment of an adolescent with Naegleria fowleri primary amebic meningoencephalitis. Pediatrics, 135, e744. Lopez, C., et al. (2012). Primary amebic meningoencephalitis: a case report and literature review. Pediatric Emergency Care, 28, 272. Machado, C. M., & Levi, J. E. (2012). Transplant-associated and blood transfusion–associated tropical and parasitic infections. Infectious Disease Clinics of North America, 26, 225. Makker, J., et al. (2015). Strongyloidiasis: a case with acute pancreatitis and a literature review. World Journal of Gastroenterology, 21, 3367. Matthews, K. R., et al. (2015). The within-host dynamics of African trypanosome infections. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences, B370 20140288. http://doi.org/ 10.1098/rstb.2014.0288. Maycock, N. J. R., & Jayaswal, R. (2016). Update on Acanthamoeba keratitis: diagnosis, treatment, outcomes. Cornea, 35, 713. Mogk, S., et al. (2014). The lane to the brain: how African trypanosomes invade the CNS. Trends in Parasitology, 30, 470. Molina, J. A. P., et al. (2015). Old and new challenges in Chagas disease. The Lancet Infectious Diseases, 15, 1347. Munasinghe, V. S., et al. (2013). Cyst formation and faecal-oral transmission of Dientamoeba fragilis—the missing link in the life cycle of an emerging pathogen. International Journal for Parasitology, 43, 870.

Medically important parasitic agents

Nair, G. V., & Variyam, E. P. (2014). Noninvasive intestinal amebiasis: Entamoeba histolytica colonization without invasion. Current Opinion in Infectious Diseases, 27, 465. Novak-Weekley, S., & Leber, A. L. (2019). Intestinal and urogenital amebae, agellates, and ciliates. In K. C. Carroll, et al. (Eds.), Manual of clinical microbiology (12th ed., p. 2497). Washington, DC: ASM Press. Nunes, M. C. P., et al. (2013). Chagas disease: an overview of clinical and epidemiological aspects. Journal of the American College of Cardiology, 62, 767. Pace, D. (2014). Leishmaniasis. The Journal of Infection, 69, S10. Pirnstill, C. W., & Cote, G. L. (2015). Malaria diagnosis using mobile phone polarized microscope. Scientic Reports, 5, 13368. Rossouw, I., et al. (2015). Morphological and molecular descriptors of the developmental cycle of Babesia divergens in human erythrocytes. PLoS Neglected Tropical Diseases, 9, e0003711. doi:10.1371/ journal.pntd.0003711. Russell, E. S., et al. (2014). Short report: prevalence of Strongyloides stercoralis antibodies among a rural Appalachian population – Kentucky 2013. The American Journal of Tropical Medicine and Hygiene, 91, 1000. Schafer, K. R., et al. (2015). Disseminated Balamuthia mandrillaris infection. Journal of Clinical Microbiology, 53, 3072. Sheorey, H., et al. (2019). Nematodes. In K. C. Carroll, et al. (Eds.), Manual of clinical microbiology (12th ed., p. 2551). Washington, DC: ASM Press. Szumowski, S. C., & Troemel, E. R. (2015). Microsporidia-host interactions. Current Opinion in Microbiology, 26, 10.

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Thanh, P. V., et al. (2015). Conrmed Plasmodium vivax resistance to chloroquine in Central Vietnam. Antimicrobial Agents and Chemotherapy, 59, 7411. U.S. Food and Drug Administration (FDA). (2019). Recommendations for reducing the risk of transfusion-transmitted babesiosis: guidance for industry. Available at: https://www.fda.gov/media/114847/ download. (Accessed 20 July 2022). Weber, R., et al. (2019). Microsporidia. In K. C. Carroll, et al. (Eds.), Manual of clinical microbiology (12th ed., p. 2305). Washington, DC: ASM Press. Wong, S. S. Y., et al. (2014). Molecular diagnosis in clinical parasitology: when and why? Experimental Biology and Medicine, 239, 1443. World Health Organization. (2021). World malaria report 2021. Available at: https://www.who.int/teams/global-malariaprogramme/reports/world-malaria-report-2021. (Accessed 20 July 2022). World Health Organization. (2022). Dracunculiasis (guinea-worm disease). Available at: https://www.who.int/news-room/fact-sheets/detail/ dracunculiasis-(guinea-worm-disease). (Accessed 19 May 2022). Xiao, L., & Cama, V. (2019). Cryptosporidium. In K. C. Carroll, et al. (Eds.), Manual of clinical microbiology (12th ed., p. 2536). Washington, DC: ASM Press. Zimmerman, P. A., & Howes, R. E. (2015). Malaria diagnosis for malaria elimination. Current Opinion in Infectious Diseases, 28, 446. Zulqar, H., et al. (2022). Amebiasis. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing. Available at: https://www.ncbi. nlm.nih.gov/books/NBK519535/. (Accessed 20 July 2022).

29 Clinical virology Kevin M. McNabb and Vijay Parashar

CHAPTER OUTLINE

Characteristics of viruses, 710 Structure, 710 Taxonomy, 710 Viral replication, 710 Laboratory diagnosis of viral infections, 713 Specimen selection, collection, and transport, 713 Appropriate specimens for maximum recovery, 713 Methods in diagnostic virology, 714 Double-stranded DNA viruses, 718 Adenoviridae, 718 Herpesviridae, 719 Papillomaviridae, 726 Poxviridae, 726 Single-stranded DNA viruses, 727 Parvoviridae, 727 Double-stranded RNA viruses, 728 Reoviridae, 728 Single-stranded RNA viruses, 729 Arenaviridae, 729 Bunyavirales, 729 Caliciviridae, 730 Coronaviridae, 731 Filoviridae, 732 Flaviviridae, 733 Orthomyxoviridae, 734 Paramyxoviridae, 736 Picornaviridae, 738 Retroviridae, 739 Rhabdoviridae, 742 Togaviridae, 742 Hepatitis viruses, 743 Hepatitis A virus, 744 Hepatitis B virus, 744 Hepatitis D virus, 745

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Hepatitis C virus, 747 Hepatitis E virus, 748 Other hepatitis viruses, 748 Prions, 749 Treatment and management of viral infections, 749 Antiviral therapy, 749 Vaccines, 749 Bibliography, 751 OBJECTIVES

After reading and studying this chapter, you should be able to: 1. Describe the characteristics of viruses and how they differ from bacteria. 2. Describe how viruses replicate. 3. Describe the proper procedures for collection and transport of specimens for viral detection. 4. Name the appropriate specimen for maximum recovery of the suspected viral agent. 5. Compare the different methods used in the diagnosis of viral infections. 6. Explain the advantages and limitations of cell cultures for diagnosing viral infections. 7. Explain the advantages and limitations of rapid viral antigen detection methods. 8. Discuss the indications and limitations of serologic assays in the diagnosis of viral infections. 9. Describe how cytopathic effect is used to presumptively identify viral agents. 10. Evaluate the vaccination program for inuenza. 11. List common opportunistic infections and other indicators of acquired immunodeciency syndrome. 12. Create an algorithm for the serologic diagnosis of human immunodeciency virus infection. 13. Compare the genomes and modes of transmission of the human hepatitis viruses. 14. Develop an algorithm for the serologic diagnosis of viral hepatitis.

Characteristics of viruses

15. Interpret the results of a hepatitis serologic prole. 16. For each of the viral agents presented in this chapter, discuss how the virus is transmitted, how the infection is produced, and the most effective method of laboratory diagnosis. 17. Discuss common treatment and prevention strategies for viral infections. KEY TERMS

Aneuploid Antigenic drift Antigenic shift Arboviruses Capsid Cell cultures Continuous cell cultures Cytopathic effect Diploid Envelope Hemagglutinin Heteroploid

Koplik spots Nucleocapsid Nucleoside reverse transcriptase inhibitors (NRTIs) Obligate intracellular parasites Pandemic Primary cell cultures Prions Syncytia Tissue culture Vaccinia virus Virion

Case in point On March 2, 2020, a 65-year-old male patient with a 14-year history of chronic lymphocytic leukemia along with hypogammaglobulinemia, chronic leukocytosis, and severe anemia presented to the emergency department of a local community hospital with lower back, lower hip, and lower extremity pain. He underwent surgery for a hip fracture related to his cancer on March 5, 2020, and was subsequently transferred to a rehabilitation facility. He was readmitted to the hospital for anemia 2 weeks later. A chest x-ray was normal, and other tests were unremarkable. A chest computed tomography (CT) was also unremarkable. The patient had no respiratory or systemic symptoms. He could not return to the rehabilitation center because of a conrmed outbreak of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection, the cause of coronavirus disease 2019 (COVID-19), at the facility. Because he lived in the rehabilitation facility around the time of the COVID-19 outbreak, he was tested and found positive for SARS-CoV-2 on March 25, 2020. After the SARS-CoV-2 diagnosis, the patient was kept in an isolation unit in a single room with negative air pressure. Because of acquired hypogammaglobulinemia caused by his cancer, the patient received intravenous (IV) immunoglobulin every 4 weeks as part of his treatment regimen. In early May, he was transfused with 200 mL of SARS-CoV-2 convalescent plasma under a U.S. Food and Drug Administration (FDA) emergency investigational new drug protocol. The infection persisted, and 1 week later, he received another 200-mL dose of convalescent plasma from a different donor. Infectious SARS-CoV-2 was successfully cultured from the nasopharyngeal swabs collected on day 35 and day 72. For the next 18 weeks, he was tested for SARS-CoV-2 another 20 times using an in-house real-time reverse-transcription polymerase chain reaction (RT-PCR) test and remained positive through July 16, 2020 (113 days after the initial positive RT-PCR test). Subsequently, he tested negative on six consecutive swabs from July 22 to August 10, indicating that the infection had nally cleared. While the patient remained asymptomatic for the entire duration of his hospitalization, culturable virus was obtained on day 25, day 60, and day 80 of his admission.

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Issues to consider After reading the patient’s case history, consider: • How the patient’s medical history affected his viral infection • What information is obtained from the laboratory, radiograph, and CT results • Could this patient have been returned to the rehabilitation facility sooner? If yes, why? If no, why not? • Other testing for SAR-CoV-2 includes antigen testing and testing for immunoglobulin G (IgG) and IgM antibodies. Would the patient’s diagnosis or treatment have improved with any of these assays?

Clinical virology continues to be a challenging and exciting area of clinical microbiology. It has evolved over the years from viral diagnostic testing performed in only very few, highly specialized laboratories to being available in many modern laboratories of today. Most of the older, traditional diagnostic methods were time-consuming, cumbersome, and required signicant expertise because they were primarily based on cell culture, serology, and microscopy (both brighteld and electron). Often, results were too slow to be clinically useful and were perhaps even irrelevant to the clinician. With the recent emergence of molecular diagnostic testing for viral infections, detection is much faster, more sensitive, and more specic, resulting in earlier intervention, quicker treatment, and more favorable patient outcomes. The development of increasingly larger molecular panels to detect viral infections from blood, cerebrospinal uid (CSF), and respiratory samples has made the diagnosis of these infections more clinically useful. Molecular technology is becoming more cost-effective and more common in the routine clinical microbiology laboratory. Viruses have been ominous clinical threats since documented medical history. Viral infections that begin in local communities as endemic disease could eventually spread globally, leading to pandemics (as we are currently facing with COVID-19) that burst into our daily lives, disrupting all facets of life. The most deadly viral pandemic of the last century was caused by inuenza A virus (HINI) in 1918. This disease, known as the Spanish u, resulted in at least 500 million infections and 50 million deaths worldwide. There have been several deadly viral pandemics in the last 40 years, including the following: • The human immunodeciency virus (HIV), identied as the causative agent of acquired immunodeciency syndrome (AIDS), was rst identied in 1981 and is still causing an ongoing epidemic in many parts of the world. HIV has infected over 79 million people and resulted in 36 million deaths. The development of antiretroviral therapy (ART) has helped to decrease mortality and improve the quality of life of infected individuals. • The West Nile virus (WNV) was introduced into North America in 1999 and within a few years became endemic to most regions of the United States. In 2012 there was a resurgence, resulting in double the yearly cases seen before that time. • Dengue virus, the causative agent of dengue fever (DF), is one of the most concerning members of the family Flaviviridae. Spread by mosquito bites, it is emerging as a pandemic in many countries. More than 1 billion people are at risk of dengue infection worldwide.

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PART 2

29

Clinical virology

Brazil has been plagued with outbreaks of DF for over 30 years, and currently over 1 million cases are reported annually. The rst coronavirus pandemic was caused by severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1). The infections began in late 2002 and spread rapidly worldwide in 2003. Although the mortality rate was high, the global outbreak was quickly contained. In 2007, a Zika virus outbreak was reported on the island of Yap (Micronesia), where nearly 75% of the population was infected. In Brazil, a Zika virus outbreak started in November 2015 and continued into 2016 with subsequent spread to the Americas, including the southern United States. In this outbreak, there was a link to microcephaly in neonates. Inuenza A virus subtype H1N1 (2009) rst emerged in Mexico and rapidly spread to other countries, resulting in thousands of deaths worldwide. Since then, several variants of inuenza virus subtypes appeared epidemically. One variant, inuenza A (H3N2), affected humans and swine throughout the United States and was implicated in infections in visitors to county fairs. The key to control the spread of inuenza viruses is to develop new vaccines based on the subtypes identied in the population every year. Annual u vaccines have been successful in preventing u pandemic outbreaks. Middle East respiratory syndrome coronavirus (MERSCoV) emerged in Saudi Arabia in 2012 and later spread to 27 countries, resulting in about 2000 cases and 866 deaths. The spread of chikungunya virus to Caribbean countries and territories of the United States was rst seen in late 2013 and reported in Florida, Puerto Rico, and the U.S. Virgin Islands. Prior to 2013, viral outbreaks were limited to countries in Africa, Asia, Europe, and the Indian and Pacic Oceans. In West Africa, an Ebola outbreak started in 2014 and lasted well into 2016. Over 28,000 cases in 10 countries were reported. During the outbreak, 11 patients were treated in the United States. Two cases were travel associated, and two cases involved health care workers caring for one of the patients. An additional 7 patients were transported by chartered aircraft to hospitals for treatment. Additional outbreaks were reported in 2017, 2018, 2020, and 2021. SARS-CoV-2 was rst identied in China in late 2019 as cases of atypical pneumonia. The infection spread rapidly across the world, leading to global shutdowns and travel restrictions. As of July 2022, the World Health Organization (WHO) reported 544,324,069 conrmed cases of COVID-19, including 6,332,963 deaths (and still counting). The rapid development of effective mRNAbased vaccine against the virus helped to bring a state of some normalcy and decline in mortality, but the virus is still mutating rapidly and the pandemic is far from over. In 2022, monkeypox caused outbreaks in several nonendemic countries, including the United States. Sporadic outbreaks of monkeypox virus have been reported in many African countries since 2003. The rst outbreak of monkeypox in the United States occurred in 2003. Monkeypox virus is closely related to smallpox virus, and the smallpox vaccine has been effective in preventing monkeypox infections.

Viruses continue to thrive in a small subset of hosts (human and sometimes animal reservoirs) even after a pandemic ends. For example, HIV continues to devastate entire continents, effectively reducing large portions of each generation. Mosquitoes continue to spread dengue virus throughout the world with signicant impact. Over the years, there has been a rise in infections by enterovirus 71 (EV71), which has killed hundreds of children throughout parts of the Asian continent. Every year about 5% to 15% of the world’s population is infected with inuenza virus. Despite inuenza surveillance programs, reliable annual vaccines, and dependable antiviral medications, several of these infected individuals develop severe disease. Inuenza virus and other viruses lead to hundreds of thousands of deaths globally. This chapter discusses basic virology, including the advances and challenges in clinical virology in the modern clinical laboratory and how the laboratory helps diagnose viral illnesses.

Characteristics of viruses Structure All viruses contain nucleic acid and a protein capsid, with some viruses having up to 90% of their mass from the protein component. Typically, viruses contain a viral genome of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). The genome can be double-stranded (ds) or single-stranded (ss). The genome and its protein coat together are referred to as the nucleocapsid. The entire virus particle is called the virion. Some viruses also have a phospholipid, labile envelope surrounding the virion. Enveloped viruses are usually more susceptible to inactivation by high temperature, extreme pH, and chemicals compared with non-enveloped (or naked) viruses. The envelopes are of host origin, derived from the plasma membrane, but contain virus-encoded proteins. The morphology of virions is helical, polyhedral (e.g., icosahedral, a geometric shape with 20 triangular sides), or complex. The envelope masks the shape of the virion, so most enveloped viruses are variably shaped or pleomorphic. The poxviruses are the largest viruses (260 × 450 nm), and the smallest human virus is the poliovirus, which is 25 nm in diameter.

Taxonomy Originally, viruses were classied by the diseases they caused and their host range. Now, viruses are classied in orders, families, genera, and species based on genome type (RNA or DNA), number of strands in the genome (ds or ss), morphology, and presence or absence of an envelope. Nucleotide sequence is also a valuable tool for the taxonomic placement of viruses. A summary of the clinically signicant viruses is shown in Table 29.1.

Viral replication Viruses are obligate intracellular parasites, that is, they must be inside a living cell and use the host cell machinery to replicate. Virions absorbing or attaching to the cell surface is the rst step in infecting a cell. The absorption is specic for certain cell receptors via viral adhesion molecules. Most host

Characteristics of viruses

711

Table 29.1 List of viruses causing human disease, based on nucleic acid characteristics and taxonomy Genome strand

Family (subfamily)

Genus

Species

dsDNA

Adenoviridae

Mastadenovirus

Human mastadenoviruses A to G

Herpesviridae (Alphaherpesvirinae)

Simplexvirus

Human alphaherpesviruses 1 and 2, macacine herpesvirus 1 (B virus)

(Betaherpesvirinae)

Varicellovirus

Human alphaherpesvirus 3 (varicella-zoster virus)

Cytomegalovirus Roseolovirus

Human betaherpesvirus 5 (cytomegalovirus) Human betaherpesvirus 6 Human betaherpesvirus 7

(Gammaherpesvirinae) Papillomaviridae

Polyomaviridae

Poxviridae (Chordopoxvirinae)

Lymphocryptovirus

Human gammaherpesvirus 4 (Epstein-Barr virus)

Rhadinovirus

Human gammaherpesvirus 8 (Kaposi sarcoma–associated virus)

Alphapapillomavirus

Alphapapillomavirus 1 (human papillomavirus 32)

Betapapillomavirus

Betapapillomavirus 1 (human papillomavirus 5)

Gammapapillomavirus

Gammapapillomavirus 1 (human papillomavirus 4)

Mupapapillomavirus

Mupapapillomavirus 1 (human papillomavirus 1)

Nupapapillomavirus

Nupapapillomavirus 1 (human papillomavirus 41)

Alphapolyomavirus

Human polyomavirus 5, 8, 9, 12, and 13

Betapolyomavirus

Human polyomavirus 1 (BK polyomavirus), Human polyomavirus 2 (JC polyomavirus)

Deltapolyomavirus

Human polyomavirus 6, 7, 10, and 11

Molluscipoxvirus

Molluscum contagiosum virus

Orthopoxvirus

Cowpox virus, Monkeypox virus, Vaccinia virus, Variola virus

Parapoxvirus

Orf virus

Yatapoxvirus

Yaba monkey tumor virus

dsDNA, ssDNA

Hepadnaviridae

Orthohepadnavirus

Hepatitis B virus

ssDNA

Parvoviridae (Parvovirinae)

Bocaparvovirus

Primate bocaparvovirus 1 and 2 (human bocaviruses)

Dependoparvovirus

Adeno-associated dependoparvovirus A and B

Erythroparvovirus

Primate erythroparvovirus 1 (human parvovirus B19)

dsRNA

ssRNA

Picobirnaviridae

Picobirnavirus

Human picobirnavirus

Reoviridae (Sedoreovirinae)

Rotavirus

Rotavirus A, B, C, and H

Orbivirus

Changuinola virus, Great Island virus, Lebombo virus, Orungo virus

Seadornavirus

Banna virus

Reoviridae (Spinareovirinae)

Coltivirus

Colorado tick fever virus

Orthoreovirus

Mammalian orthoreovirus

Arenaviridae

Mamma renavirus

Lymphocytic choriomeningitis mammarenavirus, Lassa mammarenavirus, Junín mammarenavirus, Lujo mammarenavirus, Machupo mammarenavirus

Astroviridae

Mamastrovirus

Mamastrovirus (human astroviruses 1, 6, 8, and 9)

Peribunyaviridae

Orthobunyavirus

California encephalitis orthobunyavirus, Bunyamwera orthobunyavirus, Madrid orthobunyavirus

Hantaviridae

Orthohantavirus

Hantaan orthohantavirus, Sin Nombre orthohantavirus, Puumala orthohantavirus

Nairoviridae

Orthonairovirus

Crimean-Congo hemorrhagic fever orthonairovirus

Phenuivirus

Phlebovirus

Rift Valley fever phlebovirus, Punta Toro phlebovirus

Caliciviridae

Norovirus

Norwalk virus

Sapovirus

Sapporo virus Continued

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Table 29.1 List of viruses causing human disease, based on nucleic acid characteristics and taxonomy—cont’d Genome strand

Family (subfamily)

Genus

Species

Coronaviridae (Coronavirinae)

Alphacoronovirus

Human coronavirus 229E, human coronavirus NL63

Betacoronavirus

Betacoronavirus 1, human coronavirus HKU1, human coronavirus OC43, MERS-related coronavirus, SARS-related coronavirus

(Torovirinae)

Torovirus

Human torovirus

Filoviridae

Marburgvirus

Marburg marburgvirus, Ravn virus

Ebolavirus

Zaire ebolavirus, Tai Forest ebolavirus, Reston ebolavirus, Sudan ebolavirus, Bundibugyo ebolavirus

Flavivirus

Yellow fever virus, West Nile virus, Dengue virus, Zika virus, Japanese encephalitis virus, Langat virus, Murray Valley encephalitis virus, Omsk hemorrhagic fever virus, Powassan virus, St. Louis encephalitis virus, Tick-borne encephalitis virus

Flaviviridae

Hepacivirus

Hepacivirus C (hepatitis C virus)

Hepeviridae

Orthohepevirus

Orthohepevirus A (hepatitis E virus)

Orthomyxoviridae

Inuenzavirus A

Inuenza A virus

Inuenzavirus B

Inuenza B virus

Paramyxoviridae

Pneumoviridae Picornaviridae

Rhabdoviridae

Inuenzavirus C

Inuenza C virus

Inuenzavirus D

Inuenza D virus

Respirovirus

Human respirovirus 1 and 3 (human parainuenza viruses 1 and 3)

Morbillivirus

Measles virus

Rubulavirus

Human rubulavirus 2 and 4 (human parainuenza viruses 2 and 4), Mumps rubulavirus

Henipavirus

Hendra henipavirus, Nipah henipavirus

Orthopneumovirus

Human orthopneumovirus (human respiratory syncytial virus)

Metapneumovirus

Human metapneumovirus

Enterovirus

Enterovirus A (includes some coxsackieviruses) Enterovirus B (includes echoviruses and some coxsackieviruses) Enterovirus C (includes polioviruses and some coxsackieviruses) Enterovirus D Rhinovirus A, B, and C

Parechovirus

Parechovirus A (human parechovirus)

Hepatovirus

Hepatovirus A (hepatitis A virus)

Lyssavirus

Rabies lyssavirus

Lentivirus

Human immunodeciency virus 1 and 2

Deltaretrovirus

Primate T-cell lymphotropic virus 1 and 2

Alphavirus

Chikungunya virus, eastern equine encephalitis virus, Venezuelan equine encephalitis virus, western equine encephalitis virus

Rubivirus

Rubella virus

Retroviridae (Orthoretrovirinae) Togaviridae

CoV, Coronavirus; dsDNA, double-stranded deoxyribonucleic acid; MERS, Middle East respiratory syndrome; SARS, severe acute respiratory syndrome; ssDNA, single-stranded deoxyribonucleic acid; ssRNA, single-stranded ribonucleic acid.

cell receptors are glycoproteins, some of which include the immunoglobulin superfamily molecules (for poliovirus), acetylcholine (for rabies virus), sialic acid (for inuenza virus), CD4 (for HIV), complement receptor C3d (for Epstein-Barr virus [EBV]), and angiotensin-converting enzyme 2 (for SARS CoV-2). The adhesion molecule receptor interaction determines the viral tissue tropism in the host. The next step in viral replication is penetration. Viruses can penetrate the cell by several mechanisms. Naked virions can penetrate the cell membrane directly. Enveloped viruses can enter the cell by fusion with the plasma membrane, or by endocytosis, whereby the enveloped virus enters the cell in a cytoplasmic vacuole. Once inside the cell, the virus loses

its protein coat, releasing the genome. This process is called uncoating. RNA viruses usually release the genome into the cytoplasm, whereas most DNA viruses release their genome into the host nucleus. The viral genome then directs the host cell to make viral proteins and replicate the viral genome. Depending on the virus, the metabolism of the host cell may be completely stopped (as with polioviruses) or may continue at a reduced scale, as seen with inuenza viruses. The next step is the assembly or maturation of the virus particles. The capsid protein subunits aggregate to form capsomers, and the capsomers combine to form the capsid. The capsid and genome associate to form the nucleocapsid. The new virions are then released by lysis if they are

Laboratory diagnosis of viral infections

naked viruses or by budding if they are enveloped viruses. During budding, part of the host cell plasma membrane surrounds the viral capsid and becomes the viral envelope.

Laboratory diagnosis of viral infections Laboratories provide different levels of service, depending on the mission, nancial resources, and patient populations. All of these must be balanced to provide the most cost-effective and complete diagnostic methods that meet the needs of the clinical staff. Full-service virology laboratories provide viral culture and identication by using mammalian cell cultures to support the growth of viruses in clinical specimens. Although not all clinical diagnostic facilities provide full virology services, laboratories can still provide information about viral infections by utilizing rapid tests that detect specic viruses in clinical specimens. Tests can involve the detection of viral antigens by using methods such as immunouorescence (IF) or enzyme immunoassay (EIA) and detection of viral RNA or DNA using polymerase chain reaction (PCR) assays. Low-complexity tests with waivers from the Clinical Laboratory Improvement Act (CLIA) bring viral identication services into physicians’ ofces and clinics. Some laboratories limit their virology services to viral serology—determining the patient’s immune response to viruses—rather than detecting viruses directly. Although this is sometimes useful, it usually takes several weeks after infection before these antibodies are produced, which may mean that treatment would be too late or not needed before the diagnosis is made. Molecular methods based on nucleic acid detection and amplication are being used by more clinical laboratories. This technology can detect viral infections very early in infection, and many tests are completed in less than 1 hour. Over the past 10 years, this technology has improved such that larger, more complete syndromic panels are available that can quickly identify infectious agents from many types of specimens to provide a quick, accurate diagnosis of many viral and bacterial infections at once (e.g., multiplex PCR). Since viruses tend to undergo frequent mutations, the molecular test also helps to identify different variants of the same virus to identify the virulent and more dominate strains.

Specimen selection, collection, and transport Several different clinical specimens are suitable for the diagnosis of viral diseases. The clinical signs and symptoms of diseases often point to the target organ(s) involved, which can help determine the most appropriate specimen(s) to collect. This, combined with a basic understanding of viral pathogenesis, can help in specimen selection for each specic virus. It is important to ensure, however, that the specimen collected can be used to isolate a wide range of viral pathogens because different viruses can have overlapping clinical symptoms. Since viral shedding is usually greatest during the early stages of infection, the best specimens are those collected as early as possible, which, in many infections, is even before symptoms occur. The sensitivity of viral culture can decrease rapidly 3 days after the acute onset of symptoms, so care must be taken to collect specimens appropriately to maximize detection and identication. Specimens should be collected aseptically.

713

Depending on the anatomic site and the method of collection, specimens can be sterile or nonsterile (i.e., contaminated with bacteria and/or fungi). This will determine how much specimen processing is required before viral culture. Non–culture-based test methods are typically not affected by contamination, but this varies with the system. In a normally sterile specimen, such as blood, CSF, or tissue, identication of a virus without bacterial isolates usually means that the virus is the cause of the disease. Nonsterile specimens are obtained from sites that contain normal biota, such as the respiratory tract, genital tract, skin, or stool. These specimens may require processing to reduce contaminants and promote viral growth. Aspirated secretions are often preferable, but swabs are easier to use for collection. Swabs must be made of Dacron or rayon. Calcium alginate swabs inhibit the replication of some viruses and can interfere with nucleic acid amplication tests (NAATs). Tissue samples must be kept moist and must not be placed in transport media unless it is specically designed for viral preservation. Often it is preferred for tissue specimens to be sent to the laboratory in a sterile container. Viral transport medium, saline, or trypticase soy broth can be added to sterile containers to keep tissues from drying. Several viral transport media are commercially available, and most consist of a buffered isotonic solution with a protein, such as albumin, gelatin, or serum, to protect more fragile viruses. Often, antibacterial and antifungal agents are added to viral transport media to inhibit contamination by microorganisms. Samples that can be collected with viral transport media are respiratory, swab, and tissue samples. Samples that should be collected without viral transport media include blood, bone marrow, CSF, amniotic uid, urine, pericardial uid, and pleural uid. The transport container should be unbreakable and able to withstand freezing and thawing. It is optimal to process viral specimens for culture immediately. Some viruses, such as respiratory syncytial virus (RSV), are much more difcult to recover, even a few hours after collection. If specimens cannot be processed immediately after collection, they should be stored at 4 C. Specimens should not be frozen unless a signicant delay (>4 days) in processing is anticipated. In this case, specimens should be frozen and held at–70 C. Specimens should never be stored at −20 C because this temperature facilitates the formation of ice crystals that will disrupt the host cells and result in loss of viral viability. Repeated freeze-thaw cycles must be avoided because they can also result in loss of viral viability. Freeze-thaw cycles are less of a problem for molecular testing since nucleic acids are able to withstand temperature changes. As a rule, repeat free thaw cycles should always be avoided and the laboratory scientists must follow the guidelines provided by the manufacturer of the specic test.

Appropriate specimens for maximum recovery For optimal recovery, specimens for viral isolation should be collected from the affected site. For example, secretions from the respiratory mucosa are most appropriate for viral diagnosis of respiratory infections. Aspirates, or surface swabs, are usually appropriate for lesions. If the intestinal mucosa is involved, a stool specimen is appropriate. However, if systemic, congenital, or generalized disease is involved,

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Table 29.2 Tests available for common viral pathogens and specimens for culture Body system affected

Antigen detection

Virus isolation

Serology

Culture specimens

Molecular testing

Respiratory tract

Adenovirus, coronavirus-2, herpes simplex virus (HSV), cytomegalovirus (CMV), inuenza virus types A and B, parainuenza virus, respiratory syncytial virus (RSV)

Adenovirus, coxsackievirus group A, coxsackievirus group B, echovirus, HSV, CMV, inuenza virus types A and B, parainuenza virus, RSV, reovirus, rhinovirus

Adenovirus, coxsackievirus group A, coxsackievirus group B, echovirus, HSV, CMV, inuenza virus types A and B, parainuenza virus, RSV

Nasal aspirate, nasopharynx (NP) or throat swabs, bronchoalveolar lavage, lung biopsy

Single marker molecular testing available or panels for typical respiratory pathogens available

Gastrointestinal tract

Adenoviruses 40 and 41, rotavirus

Adenoviruses 40 and 41, coxsackievirus group A, reovirus

Adenoviruses 40 and 41, coxsackievirus group A

Stool, rectal swab

Panels for multiple markers available

Liver

Hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), hepatitis E virus (HEV), Epstein-Barr virus (EBV)

Cutaneous

HSV, adenovirus, varicella-zoster virus (VZV)

HSV, adenovirus, coxsackievirus group A, coxsackievirus group B, echovirus, enterovirus, measles virus, VZV, reovirus, rubella virus, vaccinia virus

HSV, adenovirus, coxsackievirus group B, dengue virus, echovirus, human herpesvirus 6 (HHV-6), measles virus, VZV, parvovirus B19, rubella virus, vaccinia virus

Vesicle aspirate, NP aspirate and stool, lesion swab

Molecular testing is available for HSV-1 and HSV-2

Central nervous system

HSV-1 and HSV-2, mumps virus, CMV, enterovirus, HHV-6, human parechovirus, VZV

Coxsackievirus group A, coxsackievirus group B, echovirus, enterovirus, poliovirus, HSV-1 and HSV-2, mumps virus

Coxsackievirus group A, coxsackievirus group B, echovirus, poliovirus, HSV, HHV-6, mumps virus

Cerebrospinal uid (CSF), brain biopsy, NP swabs, stool

Molecular panel available for CSF that includes CMV, enterovirus, HSV-1 and HSV-2, HHV-6, human parechovirus, and VZV

Ocular

Adenovirus, HSV

Adenovirus, HSV, coxsackievirus group A, enterovirus

HSV, coxsackie group A

Corneal swabs, conjunctival scrapings

Genital

HSV

HSV

HSV

Vesicle aspirate, vesicle swab

specimens from multiple sites, including blood (buffy coat), CSF, and the portals of entry (oral or respiratory tract) or exit (urine or stool) are appropriate. For example, enteroviruses that cause respiratory infections could also be recovered from the stool after the respiratory shedding has ceased. In addition, enteroviruses are a major cause of aseptic meningitis and can also be isolated from urine specimens. Table 29.2 lists recommended specimens to be collected for viral diagnosis according to the body site affected. Incorrect or poor specimen collection can result in a false-negative diagnostic result.

Methods in diagnostic virology The clinical laboratory uses three major methods to diagnose viral infections: • Direct detection in clinical specimen • Microscopy to detect cytopathic effect

Molecular testing available for HSV-1 and HSV-2

• Detection of virus antigens • Nucleic acid detection • Isolation of viruses in cell cultures • Serologic assays to detect antibodies to virus Each laboratory must decide on the methods to offer based on the spectrum of infections encountered, population of patients served, nancial resources, and stafng. In most laboratories, a combination of several methods is used to optimize detection and reduce cost.

Direct detection In general, direct detection methods are not as sensitive as culture methods but can offer quick results to allow rapid therapy. Many of these tests can be performed in a few minutes. Viral detection allows clinicians to make relevant decisions about therapy, infection control measures, and

Laboratory diagnosis of viral infections

hospitalization. In many cases, virology results may be available before routine bacteriology culture results are.

Microscopy Because of their extremely small size, bright-eld light microscopy is not useful for visualizing viruses. The poxviruses, however, are the largest of the human viruses (about the size of chlamydiae) and can be seen. Electron microscopy has a greater magnication and can be used to visualize virions, and it is useful to detect nonculturable viruses, such as Norwalk virus, in stool ltrates. However, electron microscopy is expensive, labor-intensive, and not a very sensitive method of detecting viruses. Therefore electron microscopy is rarely used in clinical laboratories and is more suited for large teaching or research institutions. Many viruses produce distinctive visual changes in infected cells referred to as a cytopathic effect (CPE). Although virus particles cannot be visualized, the CPE can be detected in cells collected from infected sites with bright-eld microscopy. The most common method used is the Tzanck smear, which is the scraping of an ulcer base to look for Tzanck cells. Tzanck cells are large round keratinocytes that are degenerated as a result of viral infection. A Tzanck smear can detect Cowdry type A bodies from herpes simplex virus (HSV) and varicella-zoster virus (VZV) lesions. Papanicolaou (Pap) smears can reveal human papillomavirus (HPV)-associated koilocytes, which are squamous cells with an enlarged nucleus surrounded by a nonstaining halo. Rabies is sometimes diagnosed by detecting Negri bodies, which are eosinophilic cytoplasmic inclusions in neurons. However, this is an insensitive method for diagnosing rabies.

Antigen detection IF can be a valuable tool to detect viral antigens in clinical specimens. Fluorophore-labeled antibodies against viral antigens allow direct visualization of viruses and infected cells, and some tests can amplify signals, which enhance sensitivity. In the direct uorescent antibody (DFA) tests, cells from a patient are xed to a microscope slide, and uorescence-labeled antibodies are added. If viral antigens are present in the sample, the labeled antibody will bind, and uorescence will be seen using uorescent microscopy (see Chapter 10 for a more detailed description). This test is reasonably easy to

A

715

perform and is highly specic, and can be completed in 2 to 6 hours. DFA assays are available for numerous viruses, including adenovirus, inuenza A and B viruses, measles virus, parainuenza viruses (PIVs) 1 to 4, and respiratory syncytial virus (RSV) from respiratory specimens; herpes simplex virus (HSV)-1, HSV-2, and varicella zoster virus (VZV) from cutaneous lesion material; and cytomegalovirus (CMV) from blood. The key caveat is cross-reactivity of closely related viruses to the antibodies used. Therefore it can be difcult to precisely identify the viral strain using DFA. Many EIA tests for viral antigen detection are commercially available, and most use multi-well microtiter plate formats, which offer variable testing capacity to meet the needs of small or large laboratories. These tests can detect RSV and inuenza A virus from respiratory specimens, hepatitis B virus (HBV) and HIV-1 from serum or plasma, enteric adenoviruses from feces, and HSV from cutaneous lesions and conjunctival swabs. Other tests are packaged in single-test platforms, with positive specimens detected by colorimetric or optical density changes on membrane or silicon surfaces (Fig. 29.1). Often the biggest problem with this testing is in the interpretation of a color change or the detection of a line. Some vendors now supply optical readers for these types of assays to help standardize interpretation of the results. EIA is often less sensitive than cell cultures or IF, so negative results are conrmed with cell culture, IF, or nucleic acid–based tests. These assays are the most popular viral testing methods in hospital-based laboratories, but most EIA testing will likely be replaced by nucleic acid–based detection as it continues to become cheaper, is easier to perform, and offers the ability to detect multiple viruses simultaneously (multiplex testing).

Nucleic acid–based detection Nucleic acid–based detection assays have shifted the focus of clinical virology. Not only can the presence or absence of a virus be determined with nucleic acid–based analysis but, depending on the assay used, a quantitative result can also be obtained. Advantages of nucleic acid–based detection assays include a much faster turnaround time (TAT); better sensitivity compared with cell culture and DFA; assays that can be quantitative; detection of nonculturable viruses such as norovirus (NoV) and hepatitis viruses; multiplex testing; and

B

Fig. 29.1 A, Card format rapid immunochromatographic membrane assay, BinaxNOW (Abbott Laboratories, Chicago, IL), for three common respiratory viruses: inuenza A and B and respiratory syncytial virus. B, Examples of positive and negative results.

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potentially genetic characterization of the virus (genotype). Overall, molecular assays lead to faster treatment and more favorable patient outcomes. Disadvantages include detection of both active and inactivated virus, higher cost, need for specialized training and more complex facilities, and lack of assays approved by the FDA. Smaller clinical laboratories often rely on sending these tests to reference laboratories at a higher cost and longer TAT. Examples of nucleic acid–based assays include the polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR) assays; branched DNA assay; nucleic acid sequence– based amplication (NASBA); and a combination of PCR and ow cytometry, such as the Luminex system (Luminex Molecular Diagnostics, Austin, TX) for multiplex detection. Nucleic acid hybridization assays can detect viruses from various clinical specimens. Assays are available to detect many viruses, including HPV from endocervical specimens, and classify them into types that have a high risk or a low risk for cancer. Other hybridization tests can detect CMV from blood and HBV from plasma and serum. Numerous gene amplication techniques are available for detection of viral genomes, primarily bloodborne pathogens, such as HIV-1, HBV, hepatitis C virus (HCV), and WNV. With a dramatic increase in the incidence of West Nile fever in the United States, there has been an increased demand for WNV testing by PCR. Detection of inuenza A virus by PCR assay was shown to be not only more sensitive than traditional cell culture and shell vial methods, but it also allowed earlier administration of antiviral therapy to patients, resulting in more favorable patient outcomes. A microarray assay for rapid subtyping of inuenza A virus isolates is available and would be valuable in an outbreak or pandemic. A Luminex assay to detect and type or subtype 20 different viral pathogens within 5 hours has also been described. These types of systems help epidemiologists, infectious disease physicians, and others in the public health community by rapidly identifying viral pathogens during an outbreak. Newer isothermal nucleic acid amplication technology is now becoming more prevalent (Alere, Waltham, MA); it does not require temperature cycling and can deliver results in as soon as 20 minutes with vast performance improvement over slower PCR-based assays. In the case of global pandemics, rapid and continuous monitoring is of utmost importance. For example, a number of nucleic acid–based assays were deployed to monitor the spread of COVID-19. These include quantitative real-time PCR assays, which provide results in 2 to 3 hours, as well as isothermal assays such as the Abbott assay, which can provide a result within 5 minutes. With the emergence of mutations, it is desirable to screen for multiple variants of virus in a single assay. Several multiplex assay systems have been developed. For example, Cephid’s GenXpert, which identies several viruses including Zika, Ebola, SARS-COV-2, and others, is widely used in clinical laboratories for a rapid and automated way of screening a large number of samples.

Isolation of viruses in cell cultures In clinical virology, isolating viruses is still the “gold standard” against which all other methods are compared. Three methods are used for the isolation of viruses in diagnostic virology:

cell culture, animal inoculation, and embryonated eggs. The method most commonly used by clinical virology laboratories is cell culture. Animal inoculation is extremely costly and used only as a special resource in reference or research laboratories. For example, certain coxsackie A viruses require suckling mice for isolation of the virus. Embryonated eggs are rarely used; isolation of inuenza viruses is enhanced in embryonated eggs, but this is accomplished more easily in cell culture. Establishing at least a limited clinical virus isolation capability in routine laboratories can be justied, provided qualied personnel and space are available. Most of the clinical workload is for the detection of HSV in genital specimens and respiratory viruses. A signicant number of common clinical viruses can often be identied within 48 hours of inoculation, including HSV, inuenza A and B viruses, PIVs 1 to 4, RSV, adenovirus, and many enteroviruses.

Cell culture The term cell culture is technically used to indicate culture of cells in vitro; the cells are not organized into a tissue. The term tissue culture or organ culture is used to denote the growth of tissues or an organ so that the architecture or function of the tissue or organ is preserved. Many clinical virologists use these terms interchangeably; however, cell culture is the correct term. Cell cultures can be divided into three categories based on their longevity in cultures: primary, low passage (or nite), and continuous. Primary cell cultures are obtained from tissue removed from an animal. The tissue is nely minced and then treated with an enzyme, such as trypsin, to disperse individual cells further. The cells are then seeded onto a surface, such as in a ask or a test tube, to form a monolayer. With primary cell lines, only minimal cell division occurs. Cell viability is maintained by periodically removing cells from the surface, diluting them, and placing them into a new container. This process is referred to as splitting or passaging. Primary cell lines can only be passaged a few times before new cells must be obtained. An example of commonly used primary cell culture is one with primary monkey kidney (PMK) cells. Finite cell cultures can divide, but passage is limited to about 50 generations. Finite cell lines, like primary cell lines, are diploid, that is, they contain two copies of each chromosome. Diploid is the normal genetic makeup for eukaryotic cells. As the number of passages increases, these cells become less sensitive to viral infection. Human neonatal lung is an example of a standard nite cell culture used in diagnostic virology. Continuous cell cultures are capable of innite passage and are heteroploid, that is, they have an abnormal and variable number of chromosomes. Sometimes the number of chromosomes is not a multiple of the normal haploid number; this is referred to as aneuploid. HEp2 (derived from a human laryngeal epidermoid carcinoma), A549 (derived from a human lung carcinoma), and Vero (derived from a monkey kidney) are examples of continuous cell lines used in diagnostic virology. Both HEp2 and A549 were developed from cancer tissue obtained from patients during treatment. Each laboratory must decide which cell lines to use based on the spectrum of viral sensitivity, availability, and cost. Optimally, several different cell lines will be used for a single specimen to recover different viruses that may be present, analogous

Laboratory diagnosis of viral infections

to the strategy used with media for the recovery of bacteria. Table 29.3 lists some cell culture lines commonly used in clinical virology. Mixed or engineered cell cultures are cell lines that contain a mixture of two different cell types or are made up of cells genetically modied to make identication of viral infection easier. Mixed cell lines have been developed by combining two cell lines susceptible to certain types of viruses, such as respiratory or enteric viruses. The mixed line can have greater sensitivity to a wider range of viruses and therefore reduce the number of culture vials that must be incubated. Interpreting these mixed cell cultures is sometimes difcult, but this is well worth the effort when dealing with fastidious viruses.

717

PMK cells, is presumptive evidence for the identication of HSV. HSV is one of the few viruses that can grow on rabbit kidney cells (Fig. 29.2); therefore it is a useful cell line for HSV detection. CMV produces an HSV-like CPE (Fig. 29.3) but grows much more slowly and only on diploid broblasts. VZV grows on several types of cells, including diploid broblasts, A549 cells, and Vero cells. Enteroviruses characteristically produce rather small, round infected cells that spread diffusely on PMK cells, diploid broblasts, human embryonal rhabdomyosarcoma (RD) cells, and A549 cells. Adenoviruses also produce cell rounding (Fig. 29.4) on many cell types, including diploid broblasts, HEp2 cells, A549 cells, and PMK cells, but this is usually larger than that caused by enteroviruses. The rounding may be diffuse or focal, appearing like a cluster of grapes. The respiratory viruses may not produce characteristic CPE. RSV can produce classic syncytial formation in HEp2 or MKC cells. Syncytia are giant multinucleated cells resulting from cell fusion in response to the RSV infection. PIV type 2, and to a lesser extent PIV type 3, can also produce syncytia. Inuenza virus commonly does not exhibit a well-dened CPE. Specimens submitted for inuenza virus cultures are usually inoculated onto PMK cells, LLC-MK2 (a continuous line derived from rhesus monkey kidney), or MDCK (MadinDarby canine kidney epithelial cells) cells. Because inuenza

Cytopathic effect on cell cultures Some viruses produce a very characteristic CPE that can provide a presumptive identication of a virus isolated from a clinical specimen. For example, HSV grows rapidly on many different cell lines and frequently produces a CPE within 24 hours. A predominantly cell-associated virus, HSV produces a focal CPE (in which adjacent cells become infected) and plaques, or clusters of infected cells. The combination of rapid growth, plaque formation, and growth on many different cell types, such as MRC-5 (Medical Research Council cell strain 5), human broblasts, Vero, HEp2, mink lung, and

Table 29.3 Cell cultures commonly used in the clinical virology laboratory Virus

PMK

HDF

HEp2

Herpes simplex virus

−

+++

+++

Cytomegalovirus

−

+++

−

Varicella-zoster virus

−

+++

−

Enterovirus

+

+

Adenovirus

+

Respiratory syncytial virus

±

Inuenza virus, parainuenza virus

+++

RK

A549

CPE

+++

+++

Large, rounded cells

−

−

Large, rounded cells

−

±

Foci or rounded cells; possible syncytia

++

−

+

Refractile, round cells in clusters

++

+++

−

++

Large, rounded cells in clusters

±

+++

−

++

Syncytia

±

−

−

−

Variable—none to granular appearance

, Negative; +, acceptable; ++, good viral recovery; +++, recommended; ±, positive or negative; A549, human lung carcinoma cell line; CPE, cytopathic effect; HDF, human diploid broblasts; HEp2, human laryngeal carcinoma cell line; PMK, primary monkey kidney; RK, rabbit kidney. Modied from Costello, M. J., et al. (1993). Guidelines for specimen collection, transportation, and test selection, Lab Med, 24, 19.

A

B

Fig. 29.2 A, Herpes simplex virus (HSV) from the skin, showing the cytopathic effect (CPE) in less than 1 day on rabbit kidney cells. B, HSV showing the CPE in less than 1 day on HeLa cells (unstained, ×400).

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and suspected viruses, a variety of uorescent-labeled antibodies can be used. A modication of this procedure is to use at-bottomed microtiter plates. Although this is better than looking for CPE, in many cases it can be labor-intensive, and often cultures are done in duplicate, which results in reading at 24 hours then again at 48 hours, thus increasing the TAT.

Serologic assays to detect antibodies to virus

Fig. 29.3 Cytomegalovirus from cerebrospinal uid forming cytopathic effect on diploid broblast cells (unstained, ×400).

Fig. 29.4 Cytopathic effect of adenovirus on HeLa cells (unstained, ×400).

viruses typically do not produce CPE, a hemagglutination or hemadsorption test is done to detect these viruses. Cells infected with inuenza virus express viral hemagglutinin (H) protein on their surface that binds red blood cells (RBCs). In the hemadsorption test, a suspension of RBCs is added to the infected cell monolayer. If inuenza virus is present, the RBCs will adsorb or stick to the infected cells. In the hemagglutination assay, supernatant from the infected monolayer containing inuenza virus is mixed with a suspension of RBCs. Inuenza viruses also have the H protein on their surface; therefore the RBCs will visibly agglutinate. Fluorescent antibody stains that detect viral antigen, such as those used directly on clinical specimens, can also be used to screen cell cultures before a nal negative result is reported. IF, EIA, and NAATs can also be used to detect and identify viruses in cell cultures to ensure that true positives are not missed.

Centrifugation-enhanced shell vial culture The shell vial culture technique can more rapidly identify viruses than the traditional cell culture method. Cells are grown on a round coverslip in a shell vial. A shell vial is a small, round, at-bottomed tube, generally with a screw cap. The shell vial, containing a cell monolayer on the coverslip, is inoculated with the clinical sample and then centrifuged to promote viral absorption. The shell vial is incubated for 24 to 48 hours, after which the coverslip is removed, and an IF assay is performed. Based on the type of clinical specimen

Viral serology detects circulating antibodies to viruses after exposure. This method provides limited information and has certain inherent problems (see Chapter 10). First, serologic assays measure the host response rather than directly detecting the virus. Second, the antibody-producing capabilities of human hosts differ widely from person to person. For example, despite being actively infected, immunocompromised individuals may not produce enough antibodies to be detected, and the antibodies produced may be very shortlived. This is typically seen in patients with HIV infection. Third, the antibody level does not necessarily correlate with the acuteness or activity level of the infection because this is also host dependent. With few exceptions, paired sera (acute and convalescent) demonstrating seroconversion or a fourfold rise in titer are required to establish a diagnosis of recent infection. Therefore serologic studies are usually retrospective. Some assays can distinguish between immunoglobulin M (IgM) and immunoglobulin G (IgG); the presence of IgM indicates an acute (recent) infection. Cross-reactions with nonspecic antibodies can occur, which makes interpretation of results difcult. Interpretation is also difcult because of passive transfer of antibodies, such as in transplacental or transfusion transmission. The following are indications for serologic testing: • Diagnosis of infections with nonculturable agents, such as hepatitis viruses • Distinguishing between a past (IgG) or acute (IgM) viral infection • Determination of immune status regarding rubella virus, measles virus, VZV, hepatitis A virus (HAV), and HBV • Monitoring of patients who are immunosuppressed or have had transplantations • Epidemiologic or prevalence studies that are critical during outbreaks to assess overall spreading and herd immunity status

Double-stranded DNA viruses In this chapter, viruses are discussed in groups based on nucleic acid types: double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), double-stranded RNA (dsRNA), and single-stranded RNA (ssRNA) viruses. Hepatitis viruses are the only exception, and they will be discussed as one group because they do not all have the same type of nucleic acid.

Adenoviridae Adenovirus was rst isolated from adenoid tissue of children and was thus named for the initial isolation location. Human adenoviruses belong to the family Adenoviridae and

Double-stranded DNA viruses

Fig. 29.5 Transmission electron micrograph of adenovirus (×60,000). (Courtesy Dr. G. William Gary, Jr., Centers for Disease Control and Prevention, Atlanta, GA.)

the genus Mastadenovirus. Adenoviruses are naked icosahedral viruses with dsDNA (Fig. 29.5). Adenovirus has over 60 distinct serotypes (seven subgenera, A to G), and the different serotypes are associated with numerous common clinical manifestations. The clinical manifestations seen are dependent on the age and immune status of the person infected. Adenovirus is shed in secretions from the eyes and respiratory tract. Viral shedding in feces and urine can occur for days after the symptoms have disappeared. The viruses are spread by aerosols, fomites, the oral-fecal route, and personal contact. Most infections are mild and require no specic treatment. Effective infection control measures, including adequate chlorination of swimming pools, prevent adenovirus infections, such as adenovirus-associated conjunctivitis. The most common serotypes are 1 to 8, 11, 21, 35, 37, and 40. Although one half of all adenovirus infections are asymptomatic, the virus causes about 10% of all cases of pneumonia and 5% to 15% of all cases of gastroenteritis in children. Adenovirus infections affect the respiratory tract, eye, and gastrointestinal (GI) tract, with lesser involvement of the urinary tract, heart, central nervous system (CNS), liver, pancreas, and genital tract. The viruses can also cause epidemic keratoconjunctivitis, acute hemorrhagic cystitis, and pharyngoconjunctival fever. Adenovirus infections occur throughout the year and affect every age group. Adenovirus serotype 14 is rarely reported but causes severe and sometimes fatal acute respiratory disease or distress (ARD) in patients of all ages. In the United States, an outbreak of adenovirus 14 was reported in four states from 2006 to 2007. The outbreak included one infant in New York and 140 additional cases from the states of Oregon, Texas, and Washington. Although no link could be found between the New York case and the other cases, all isolates were identical by hexon and ber gene sequencing. Since 2007, adenovirus has been associated with outbreaks of ARD in U.S. military recruits and the general public. Adenovirus types 3, 4, and 7 are most associated with ARD and can be fatal. A vaccine for types 4 and 7 became available in October 2011 and is used by the U.S. military. Adenovirus types 40 and 41 are called enteric adenoviruses because they cause epidemics of gastroenteritis, usually in young children, with diarrhea being a prominent feature of the

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Fig. 29.6 Bronchoalveolar lavage, cytocentrifuge preparation, immunouorescent antigen adenovirus stain, uorescence microscopy showing prominent uorescence of virus-infected cells (×200).

illness. There is far less vomiting and fever than with rotavirus infections. Enteric adenoviruses have a worldwide, endemic distribution, and the number of cases increases during the warmer months. These adenoviruses can be identied but not serotyped by EIA. Commercial antigen detection kits are available, and although inexpensive, they lack sensitivity. There is a molecular panel (FilmArray, BioFire Diagnostics, Salt Lake City, UT) that is specic for adenovirus types 40 and 41. Adenoviruses are quite stable and can be isolated in human embryonic kidney and many continuous epithelial cell lines. They produce a characteristic CPE, with swollen cells in grapelike clusters. Isolates can be identied by uorescent antibody (Fig. 29.6) and EIA methods, along with nucleic acid tests. Serotyping is accomplished by serum neutralization or hemagglutination inhibition. Electron microscopy has been used in several epidemiologic studies, but it is not routinely used as a clinical tool and is mostly only used in research settings.

Herpesviridae The herpesviruses belong to the family Herpesviridae. The herpesviruses have a genome of linear dsDNA, an icosahedral capsid, an amorphous integument surrounding the capsid, and an outer envelope. All herpesviruses share the property of producing latency and life-long persistence in their hosts. The virus is latent between active infections. It can be activated from latency by various stimuli, including stress, physical exertion, fever, comicrobial infection, hormonal imbalance, and sunlight. Reactivation tends to be milder than the primary infection. Eight species of human herpesvirus (HHV) are currently known: • • • • • • • •

HSV-1, also known as HHV-1 HSV-2, also known as HHV-2 VZV, also known as HHV-3 EBV, also known as HHV-4 CMV, also known as HHV-5 HHV-6 HHV-7 HHV-8, also known as Kaposi sarcoma (KS) herpesvirus

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There are other herpesviruses that infect only primates, except for herpes B virus, which has produced fatal infections in animal handlers and researchers working with primates.

Herpes simplex viruses HSV-1 and HSV-2 belong to the genus Simplexvirus. HSV infections are very common. By adulthood, about 80% of Americans have been infected with HSV-1. Approximately 20% of Americans have had HSV-2 infections. These gures indicate that about one in six persons in the United States has had HSV infection, and most infections are asymptomatic. Disease caused by HSV infection is generally divided into two categories: primary (rst or initial infection) and recurrent (reactivation of the latent virus). Infections are generally spread by contact with contaminated secretions. Lesions usually occur on mucous membranes after an incubation period of 2 to 11 days. Infected individuals are most infectious during the early days of a primary infection. Virus-infected cells are usually found at the edge and in the base of lesions; however, the virus can be transmitted from older lesions as well as from asymptomatic patients.

Types of infections HSV infections occur worldwide, and infections can cause a wide spectrum of clinical manifestations. HSV-1 and HSV-2 cause the same diseases. Skin vesicles are lled with clear uid and have a red base. The vesicles progress to pustular lesions, ulcerate, and then nally crust over.

Oral herpes Most oral herpes infections, herpes labialis, are caused by HSV1, but many cases are caused by HSV-2. The incubation period ranges from 2 days to 2 weeks. Primary infections are usually asymptomatic, but when apparent, they commonly manifest as mucosal uid-lled vesicles inside the mouth or as ulcerations (gingivostomatitis) that can be widespread and involve the buccal mucosa, posterior pharynx, and gingival and palatal mucosae. In young adults, a primary HSV infection can involve the posterior pharynx and produce acute pharyngitis. Recurrent, or reactivation, HSV infection usually occurs on the border of the lips at the junction of the oral mucosa and skin. An early symptom of burning or pain followed by vesicles, ulcers, and crusted lesions is the typical pattern. These eruptions can result from stress or sunlight exposure and are often quite painful.

Genital herpes Genital herpes, or herpes genitalis, infections are usually caused by HSV-2, although HSV-1 can cause as many as one third of the infections. Many individuals with antibodies to HSV-2 have not been diagnosed with genital herpes. The infection manifests itself in females as vesicles on the mucosa of the labia, vagina, or both. Involvement of the cervix and vulva is not uncommon. In males, the shaft, glans, and prepuce of the penis are the most affected sites. The urethra is commonly involved in both men and women. Recurrent herpes infections involve the same sites as primary infections, but the urethra is less commonly involved. The symptoms are usually less severe in recurrent disease and have a shorter duration. Herpes genitalis can as much as double the risk of sexual transmission of HIV.

Neonatal herpes Transmission of HSV from infected mothers to neonates is less common than might be expected, but the risk of mother-to-infant transmission is 10 times higher when mothers have an unrecognized primary infection during labor and delivery. Infection can be acquired in utero (during birth) or postnatally (after birth) from hospital personnel or family members. The infection is usually transmitted during a vaginal delivery and is more severe when HSV-2 is involved compared with HSV-1. The rate of transmission is about 50% when the mother has a primary infection. Most newborns are infected by mothers who are asymptomatically shedding the virus during a primary infection. The risk of transmission is very low when the mother has recurrent herpes. Cesarean delivery or suppressive antiviral therapy at delivery signicantly reduces the risk of transmission. Mortality associated with disseminated neonatal disease is about 60% in treated neonates and exceeds 70% in untreated neonates.

Herpes simplex virus encephalitis HSV encephalitis is a rare but devastating disease with a mortality rate of about 70%. In the United States, HSV encephalitis may account for up to 20% of all encephalitis cases. HSV is the leading cause of fatal sporadic encephalitis in the United States. Encephalitis is usually caused by HSV-2 in neonates and HSV-1 in older children and adults. HSV encephalitis is also associated with an immunocompromised status. Survival rates and clinical outcomes are greatly improved with IV antiviral treatment.

Ocular herpes A herpes simplex infection of the conjunctiva can manifest itself as swelling of the eyelids associated with uid-lled vesicles. Corneal involvement, herpes keratitis, can result in destructive ulceration and perforation of the cornea, leading to blindness. HSV is the most common cause of corneal infection in the United States. Fortunately, most infections involve only the supercial epithelial layer and heal completely with treatment.

Diagnosis Diagnosis of HSV infection is best made by antigen detection or viral isolation in culture. However, smaller laboratories may perform a Tzanck smear from the lesion and look for CPE typical of HSV, and, if present, this can be conrmed using specic uorescent antibodies to HSV-1 or HSV-2 (Figs. 29.7 and 29.8). Often, other specimen types can also be stained and observed for CPE consistent with HSV infection (Fig. 29.9). However, culture is still considered the gold standard. The best specimens for culture are aspirates from vesicles, open lesions, or host cells collected from infected sites. Culture of CSF is usually not productive. To make a culture-conrmed diagnosis of encephalitis, brain biopsy material is required. The preferred diagnostic method for HSV in CSF is PCR. In many studies, gene amplication for HSV in CSF approaches 100% sensitivity with almost the same specicity. Some of the newer nucleic acid assays are becoming easier to perform and less costly, so it is expected that they will continue to increase in use in clinical laboratories. A diagnostic panel is available for the detection of HSV-1, HSV-2, and several other viruses and bacteria that cause meningitis (FilmArray, BioFire Diagnostics), and this test is frequently performed to reduce

Double-stranded DNA viruses

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Fig. 29.7 Skin vesicle uid, Tzanck preparation, hematoxylin and eosin stain. Multinucleated epithelial cells present (arrow). Intranuclear inclusions present, morphology consistent with herpes virus inclusions. (×400). Fig. 29.10 Advanced cytopathic effect in an A549 cell line caused by herpes simplex virus infection (unstained, ×400). (Courtesy Sarah Pierson.)

Fig. 29.8 Skin vesicle uid, Tzanck preparation, uorescent antibody stain for herpes simplex virus (×400).

Fig. 29.9 Bronchoalveolar lavage, centrifuge preparation, rapid Wright-Giemsa stain. Multinucleated epithelial cells and intranuclear inclusions present. Morphology consistent with herpes simplex virus infection (×400).

the need for antiviral therapy, especially in infants. It is rapidly becoming the standard of care for rapid identication of viral encephalitis. There are also several HSV-1/HSV-2 standalone assays that use isothermal amplication, another form of molecular assay that does not need a thermocycler. Diagnosis can be made in a few hours, and appropriate therapy can be initiated quickly, resulting in more favorable patient outcomes.

In culture, HSV replicates rapidly, and CPE can be seen within 24 hours (Figs. 29.2 and 29.10). Diagnosis in cell cultures is quicker than for many other viruses, but it is not as rapid as molecular assays. HSV can be isolated in numerous cell lines, including human embryonic lung, rabbit kidney, MRC-5, HEp2, and A549 cells. HSV is one of the most frequently isolated viruses in the clinical virology laboratory. Once isolated, monoclonal antibodies can be used to type the virus. Typing genital lesion isolates can be prognostic in that HSV-2 reactivation occurs more readily than HSV-1. In addition, typing genital lesions from children has been used to provide legal evidence supporting potential sexual abuse. Commercially available engineered cell lines improve the detection of HSV. In the Enzyme Linked Virus Inducible system (ELVIS; BioWhittaker, Walkersville, MD), a gene for the enzyme β-galactosidase linked to a virus-induced promoter has been inserted into baby hamster kidney cells. If HSV-1 is present in the cell line, a viral protein will activate the promoter, resulting in β-galactosidase expression. Detection is accomplished by addition of a chromogenic reagent, which is cleaved by the enzyme produced in virus-infected cells and results in the formation of a blue color, which is easily seen by light microscopy. Several FDA-approved, type-specic assays that differentiate antibody response to HSV are available. The tests come in a variety of formats, including EIA, strip immunoblot, and even simple membrane-based, point-of-care assays. Newer tests use recombinant or afnity-puried, type-specic glycoprotein G-1 or G-2, giving the tests the ability to distinguish between HSV-1 and HSV-2. Older-generation tests used crude antigen preparations from lysed cell culture of the virus and were shown to have cross-reactivity rates of as much as 82% in positive specimens. With this change, antigen testing has become more specic with much better results in a rapid, low cost, and easy-to-perform assay. However, because of the latent, life-long infections, many adults have antibodies to HSV-1 and HSV-2. Serologic assays can be of value in diagnosing new infections in patients without a history of infection, particularly women who are pregnant.

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Cytomegalovirus CMV is in the genus Cytomegalovirus, and the name originates from the enlargement of infected cells (from Latin cyto, meaning cell, and mega, meaning large). CMV is typically spread by close contact with an infected person. Most adults demonstrate antibodies against the virus, with a prevalence rate in the United States of 55% among adult women and 32% among adult men. The seroprevalence of CMV increases with age in all populations; it is highest among lower socioeconomic groups living in crowded conditions. Persons who live in overcrowded conditions can acquire CMV at an early age. The virus is shed in saliva, tears, urine, stool, and breast milk. CMV infection can also be transmitted sexually via semen and cervical and vaginal secretions and through blood and blood products. CMV infection is the most common congenital infection in the United States. Most CMV infections are asymptomatic in the immune-competent host but can manifest themselves as a self-limiting, infectious mononucleosis-like illness, with fever and hepatitis. In immunocompromised hosts, such as transplant recipients and patients with HIV infection, CMV infection can become a signicant, life-threatening, systemic disease involving almost any organ, including the lungs, liver, intestinal tract, and retina, as well as the CNS. Congenital infections are often symptomatic and can present with serious clinical manifestations if the mother acquires the primary infection during pregnancy. Congenital infection, however, is unlikely to occur if the mother was seropositive at the time of conception. Symptomatic congenital infection is characterized by petechiae, hepatosplenomegaly, microcephaly, and chorioretinitis. Other manifestations are reduced birth weight, CNS involvement, mental impairment, deafness, and even death. CMV infection is one of the leading causes of deafness and intellectual impairment. The diagnosis of CMV infection is best conrmed by isolation of the virus from normally sterile body uids, such as the buffy coat of blood or other internal uids or tissues. The virus can also be cultured from urine or respiratory secretions, but because shedding of CMV from these sites is common in normal hosts, isolation from these sources must be interpreted with extreme caution. Typically, this is done using standard stains on respiratory specimens looking for inclusions typical of CMV infection (Figs. 29.11 and 29.12).

Fig. 29.12 Bronchoalveolar lavage, centrifuge preparation, rapid Wright-Giemsa stain. Enlarged pneumocyte consistent with cytomegalovirus (CMV). The large regular-sized, magenta cytoplasmic inclusions (arrow), when present, are characteristic of CMV (×400).

A viral antigenemia test is widely used by clinical virology laboratories. The antigenemia assay is specic, sensitive, rapid, and relatively easy to perform. The test is based on the immunocytochemical detection of the 65-kilodalton (kDa), lower-matrix phosphoprotein (pp65) in the nuclei of infected peripheral WBCs. The antigenemia test is more sensitive and rapid than cell culture, and it can prove helpful in assessing the efcacy of antiviral therapy. However, several newer NAATs may replace this test. Molecular-based testing is widely used to detect virus particles in clinical samples. PCR, branched DNA, and hybridization assays are all used for diagnostic applications and blood donor screening. Meningitis panels that include CMV are available and are suitable for use in pediatric populations. A congenital infection is best conrmed by isolation of CMV from the infant within the rst 2 weeks of life. Isolation after the rst 2 weeks does not conrm congenital infection. Urine is the most common specimen submitted for viral detection in these patients. NAATs are the preferred method for determining viral loads. CMV is a typical herpesvirus, but it replicates only in human cells and more slowly compared with HSV or VZV. CMV can be isolated in cell culture only by using human diploid broblast cell lines, such as human embryonic lung or human foreskin broblasts (see Fig. 29.3). The virus replicates slowly, so it may take up to 3 weeks for the CPE to appear in culture. However, the use of shell vials can reduce the time for detection to as little as 1 day. CMV produces characteristic CPE, which can sometimes be seen in clinical specimens (Fig. 29.13). Serology is not as helpful as a culture in diagnosing the infection.

Epstein-Barr virus

Fig. 29.11 Bronchoalveolar lavage, centrifuge preparation, rapid Wright-Giemsa stain. Enlarged pneumocyte with intranuclear inclusion. Morphology consistent with cytomegalovirus (CMV). Observe the characteristic nuclear changes for CMV. The cell and nucleus are enlarged, the nucleus is granular, and the nuclear membrane is indistinct (arrow). (×400).

EBV, in the genus Lymphocryptovirus, causes infectious mononucleosis (Fig. 29.14). Up to 95% of adults between 35 and 40 years of age have been infected. Many children become infected with EBV and show few signs of infection. When infection with EBV occurs in adolescence, it presents as infectious mononucleosis 35% to 50% of the time. The signs and symptoms of EBV infection include sore throat, fever, lymphadenopathy, hepatomegaly, splenomegaly, and general malaise. These usually resolve within a few weeks, although

Double-stranded DNA viruses

malaise can be prolonged in some cases. Complications of EBV infection include splenic hemorrhage and rupture, hepatitis, thrombocytopenia purpura with hemolytic anemia, Reye syndrome, encephalitis, and other neurologic syndromes. EBV can be recovered from the oropharynx of symptomatic

Fig. 29.13 Active cytomegalovirus lung infection in a patient with acquired immunodeciency syndrome. Lung histopathology shows cytomegalic pneumocyte containing characteristic intranuclear inclusions (hematoxylin & eosin ×1000). (Courtesy Edwin P. Ewing, Jr., Centers for Disease Control and Prevention, Atlanta, GA.)

Fig. 29.14 Negatively stained transmission electron micrograph revealing the presence of numerous Epstein-Barr virions (×40,000). (Courtesy Fred Murphy, Centers for Disease Control and Prevention, Atlanta, GA.)

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as well as healthy persons, who can transmit the virus to susceptible individuals via infected saliva. The incubation period for EBV infection ranges from 2 weeks to 2 months. As with the other herpes group viruses, infection is very common and results in latency, and most adults demonstrate antibodies against the virus. Young children with the infection are almost always asymptomatic. As the age at the time of infection increases to young adulthood, a corresponding increase occurs in the ratio of symptomatic to asymptomatic infections. Some cancers have been associated with EBV, including Burkitt lymphoma, Hodgkin disease, and nasopharyngeal carcinoma (NPC). Burkitt lymphoma is a malignant disease of the lymphoid tissue seen most commonly in African children. The virus has also been increasingly recognized as an important infectious agent in transplant recipients. The most signicant clinical effect of EBV infection in these patients is the development of a B-cell lymphoproliferative disorder or lymphoma. Viral culture for EBV requires human B lymphocytes and is beyond the capabilities of most clinical virology laboratories. Therefore laboratory diagnosis of EBV infection is often accomplished with serologic tests. EBV infects circulating B lymphocytes and stimulates them to produce multiple heterophile antibodies, including antibodies to sheep and horse RBCs. The Paul-Bunnell heterophile antibody test is an excellent rapid screening test for these antibodies, although some false-positive reactions do occur. Many rapid test kits, generally based on EIA or latex agglutination, are commercially available for detecting heterophile antibodies. These tests are 80% to 85% effective. Some false-positive test results represent patients who have had an EBV infection and still have low antibody levels. Young children can have false-negative results with the heterophile test; performing an EBV-specic antibody test on these individuals is appropriate. EBV-specic serologic tests (Table 29.4; Fig. 29.15) include the following: • Anti-VCA (antibodies to the viral capsid antigen): IgM to the VCA occurs early in the infection and disappears in about 4 weeks, so its presence indicates current infection. IgG often appears in the acute stage and will persist for life at lower titers. • Anti-EA IgG (IgG antibody to early antigen): IgG to EA can appear in the acute phase, and its presence indicates current or recent infection. The antibody usually cannot be detected after 6 months. • Anti-EA/D (antibody to early antigen, diffuse): Antibodies to EA/D appear in the acute phase, and their presence indicates current or recent infection. The antibodies

Table 29.4 Interpretation of Epstein-Barr virus serologic markers PB

Anti-VCA IgM

Anti-VCA IgG

Anti-EA IgG

Anti-EBNA

Interpretation

−

−

−

−

−

No previous exposure to Epstein-Barr virus

+

+

+

±

−

Acute infectious mononucleosis

±

±

+

±

+

Recent infection

−−

−

+

−

+

Past infection

−, Negative; +, positive; ± positive or negative; Anti-EA IgG, immunoglobulin G antibodies to early antigen; anti-EBNA, antibodies to Epstein-Barr virus nuclear antigen; anti-VCA IgG, immunoglobulin G antibodies against the viral capsid antigen; anti-VCA IgM, immunoglobulin M antibodies against the viral capsid antigen; PB, Paul-Bunnell antibody.

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Antibody titer

Clinical illness Anti-VCA IgG Anti-EBNA Anti-EA

Anti-VCA IgM Heterophil (PB) antibody 1

Fig. 29.15 Serologic evaluation of Epstein-Barr virus infection (infectious mononucleosis) showing the increase and decrease of detectable antibodies.Anti-EA, Antibody to early antigen; anti-EBNA, antibody to Epstein-Barr virus nuclear antigen; anti-VCA IgG, immunoglobulin G antibody to the viral capsid antigen; anti-VCA IgM, immunoglobulin M antibody to the viral capsid antigen; PB, Paul-Bunnell heterophile antibody.

usually cannot be detected after 6 months. Patients with NPC often have elevated levels of IgG and IgA anti-EA/D antibodies. • Anti-EA/R (antibody to early antigen, restricted): Antibodies to EA/R appear in the acute phase and disappear soon after anti-EA/D, but they can persist for up to 2 years and may be life-long in some patients. The anti-EA/R IgG antibody level is elevated in patients with Burkitt lymphoma. • Anti-EBNA (antibody to the EBV nuclear antigen): Antibodies appear about 1 month after infection, with titers peaking in 6 to 12 months. Several molecular assays that use real-time PCR to detect and quantitate viral load are coming to market and will be key to evaluating treatment effectiveness for patients who are EBV-positive. This will especially be critical in patients who have other medical conditions that lower immune response, such as HIV infection, diabetes, or cancer.

Varicella-zoster virus

Fig. 29.16 Electron micrograph of a varicella virus (×100,000). (Courtesy Erskine Palmer and B.G. Partin, Centers for Disease Control and Prevention, Atlanta, GA.)

VZV is in the genus Varicellovirus. VZV spreads by droplet inhalation or direct contact with infectious lesions. Cell-free virus is produced at very high levels in the skin vesicles, thus the uid from these vesicles is highly infectious. The virus causes two different clinical manifestations: varicella (chickenpox) and zoster (shingles). In the United States, more than 90% of adults have antibodies to VZV. Varicella is the primary infection and is highly contagious (Fig. 29.16). In contrast with infections caused by other herpesviruses that do not usually manifest symptoms, varicella is generally clinically apparent. It commonly appears in childhood and includes symptoms such as a mild febrile illness, rash, and vesicular lesions. Usually, the lesions appear rst on the head and trunk and then spread to the limbs. The lesions dry, crust over, and heal in 1 to 2 weeks. Painful oral mucosal lesions may develop, particularly in adults. Herpes zoster, or shingles, is the clinical manifestation caused by reactivation of VZV; it usually occurs in adults. Approximately one in three adults will develop herpes zoster in their lifetime. It is thought that the virus remains latent in the dorsal root or cranial nerve ganglia after primary

infection. In a small proportion of patients, the virus becomes reactivated, travels down the nerve, and causes zoster. The most common presentation is rash, followed by vesicular lesions in a unilateral dermatome pattern. These lesions may be associated with prolonged disabling pain that can remain for months, long after the vesicular lesions disappear. VZV infection is usually diagnosed based on characteristic clinical ndings. Some smaller laboratories use smears made with standard stains from lesion specimens to detect cellular inclusions typical of VZV (Fig. 29.17). In atypical cases, such as in immunocompromised patients, the diagnosis may be more difcult or questionable. In such patients, culture of fresh lesions (vesicles) or the use of uorescent-labeled monoclonal antibodies against VZV conrms the diagnosis. VZV can be cultured on human embryonic lung or Vero cells. Cytopathic changes may not be evident for 3 to 7 days. Recently, NAATs, such as PCR assays, have become the standard for the diagnosis of VZV disease. These assays have

Double-stranded DNA viruses

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of latent infections can become clinically signicant in immunocompromised individuals. HHV-6 has also been proposed as having some involvement in the development of progressive multifocal leukoencephalopathy and multiple sclerosis. The diagnosis of HHV-6 infection is usually made clinically. Isolation of the virus is most sensitive with lymphocyte cell culture, which is not practical for routine diagnosis. Serology may not be helpful unless paired sera are available. Patients do not usually have a positive IgM result until about 5 days after infection; IgG appears several days later. PCR and viral load testing offer the most sensitive and specic means of diagnosing primary HHV-6 infection. Fig. 29.17 Skin vesicle uid, Tzanck preparation, Wright-Giemsa stain. Multinucleated epithelial cells present. Intranuclear inclusions present. Morphology consistent with herpes viral inclusions, varicella-zoster virus infection (×400).

revolutionized the diagnosis of VZV disease of the CNS and of disseminated VZV infection, especially in immunocompromised patients (e.g., HIV infection, diabetes), and the identication of herpes zoster in patients who do not develop the rash associated with VZV. The advantages of these molecular assays are that they require small specimen volumes and are highly sensitive, specic, and rapid. An attenuated vaccine to prevent chickenpox was approved for use in the United States in 1995. Before routine use of the vaccine in children, an estimated 4 million to 5 million cases occurred annually. The vaccine is expected to give life-long immunity. In 2006, a single-dose, attenuated VZV vaccine for shingles, Zostavax (Merck, Whitehouse Station, NJ), was approved. In 2017, Shingrix (GlaxoSmithKline, Philadelphia, PA), a recombinant subunit adjuvanted vaccine, was approved; this is a two-dose vaccine. It is currently the only FDA-approved vaccine available to prevent shingles. The shingles vaccine is recommended for individuals 50 years of age and older. Antiviral treatment of VZV infection and reactivation is possible and is quite effective in reducing the infection time for patients, especially if coupled with more rapid molecular diagnostics.

Human herpesvirus 6 HHV-6 is in the genus Roseolovirus. The two variants of the virus, A and B, are indistinguishable serologically, but variant B appears to be the cause of disease. HHV-6 is a common pathogen. About 95% of young adults are seropositive. Studies have shown that the virus persists in the salivary glands and has been isolated from stool specimens, but most evidence indicates that saliva is the most likely route of transmission. Inhalation of respiratory droplets from and close contact with infected individuals is the primary portal of entry. HHV-6 has been associated with the childhood disease roseola, which is also called roseola infantum, exanthem subitum, and sixth disease, reecting its role as the sixth childhood rash. Children are protected by maternal antibodies until approximately 6 months of age. Seroconversion occurs in 90% of children between 6 months and 2 years of age. In immunocompetent individuals, most infections are mild or asymptomatic. When symptoms occur, the disease is acute and febrile; a maculopapular rash appears as the fever resolves. About 30% to 40% of infected children with symptoms experience seizures. As with all members of the family Herpesviridae, reactivation

Human herpesvirus 7 HHV-7 is in the genus Roseolovirus with HHV-6. The CD4 molecule serves as a receptor for HHV-7 to infect T lymphocytes. It also uses other receptors and has a broad range of host cells. Like HHV-6, HHV-7 is extremely common and is shed in the saliva of 75% of adults. The virus causes roseola, which is clinically identical to that caused by HHV-6. HHV-7 causes latent infections in T lymphocytes. Despite the similarities between HHV-6 and HHV-7, their antigenic diversity is such that antibodies to one virus do not protect against infection from the other. In addition, exposure to HHV-7 seems to occur later in life than exposure to HHV-6. Most 2-year-olds are seronegative for HHV-7, but most children are seropositive by 6 years of age. HHV-7 can be isolated in culture in peripheral blood lymphocytes or in cord blood lymphocytes. Although the virus can be isolated from the saliva of healthy individuals, it is rarely isolated from peripheral blood mononuclear cells. PCR assay can detect the virus, but the ubiquitous nature of the virus can lead to difculties in interpreting the results. Serologic results can be confusing because of cross-reactions, but patients with increasing levels of antibody to HHV-7 but not to HHV-6 may have an active HHV-7 infection.

Human herpesvirus 8 HHV-8, in the genus Rhadinovirus, can be detected in all forms of KS, including acquired immunodeciency syndrome (AIDS)-related, Mediterranean, and HIV-1–negative KS, which is endemic to Africa, as well as posttransplantation KS. This association has earned it as the more common name Kaposi sarcoma–associated herpesvirus. It has also been shown to play a role in the development of primary effusion lymphomas and multicentric Castleman disease. In North America and much of Europe, HHV-8 appears to be transmitted primarily through sexual contact, but studies in Africa and some Mediterranean populations suggest transmission by more casual means. The pattern of infection is like that of HSV-2, although men who have sex with men (MSM) seem to be more susceptible than heterosexuals. Prevalence ranges from close to zero in a study of Japanese blood donors to more than 50% in some parts of Africa. In HIV-positive persons, the seroprevalence can be as much as 20% to 50% higher than that of the surrounding healthy population. In the United States, as many as 20% of normal adults have antibodies to HHV-8, as do 27% of patients with HIV-1 who do not have KS and 60% of patients with HIV and KS. Currently, the virus cannot be recovered in cell culture. PCR assay, although considered very sensitive, was shown

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to be less sensitive than some immunologic assays. However, PCR assay has been used to detect the virus in various specimens, including tissue, blood, bone marrow, saliva, and semen. HHV-8 DNA or antigens are rarely detected in immunocompetent individuals, even if they are seropositive. In situ hybridization can detect HHV-8–affected tissue. The availability of commercially prepared monoclonal antibodies has made the identication of HHV-8–infected cells in various types of lesions by immunohistochemistry more common. Serologic tests are being evaluated and may soon be available.

Papillomaviridae HPVs, the cause of papillomas (or warts), are clustered in ve genera (see Table 29.1). Most clinically signicant HPVs are found in the genus Alphapapillomavirus, which includes types infecting the genital and nongenital mucosa and genital cutaneous surfaces as well as types most often seen in human cancers. Although associated with the common wart, some HPV types are linked to cancers, including cervical cancer. There are more than 100 types of these small dsDNA viruses; more than 40 types are sexually transmitted and are known as the genital types. HPV 1, 2, 3, and 4 are thought to infect all children and young adults universally, with no signicant consequences. Different HPV types exhibit different tissue tropism based on the type of epithelial cells that the viruses preferentially infect: cutaneous or mucosal. The genital HPVs are further categorized as low, intermediate, or high risk based on their association with genital tract cancers. Table 29.5 lists some HPV types and their clinical signicance. Cervical HPV lesions typically consist of at areas of dysplasia and are often difcult to see. Rinsing the area with 5% acetic acid, which turns the lesion white, makes the lesions more visible; however, due to low sensitivity this method is not used for diagnosis. Some types of HPV will result in genital wart formation (condylomata acuminata) that can easily

be identied. Lesions can be removed by several methods, including surgery, cryotherapy, and laser. The HPVs cannot be grown in cell cultures; therefore laboratory diagnosis of HPV infection often involves cytology sections. Cytotechnologists and cytopathologists examine Pap smears looking for koilocytes, cells with perinuclear clearing accompanied by an increased density of the surrounding rim of cytoplasm, which are indicative of HPV infection. Additional testing, such as nucleic acid probe tests, can help detect HPV DNA in endocervical cells and identify the HPV type. PCR techniques are more sensitive and have shown that HPV is present in 95% or more of invasive cervical cancers, but the presence of the virus alone is not the sole factor in cancer development. As many as one third of all college-age women are infected with HPV, and most develop only subclinical infections. Because nding HPV in cervical tissue is not the sole predictor of invasive disease, there is some debate about whether it is useful to look routinely for the virus in cervical specimens. A quadrivalent vaccine, Gardasil (Merck, Kenilworth, NJ), against HPV types 6, 11, 16, and 18 to prevent cervical cancer was approved by the FDA in 2006 for females 9 to 26 years of age; the vaccine was approved for use in males in 2009. A second vaccine that protects against HPV types 16 and 18, Ceravix (GlaxoSmithKline), was approved for use in women in 2009. HPV types 16 and 18 are linked to most cervical cancers and other HPV-associated cancers. There has been controversy about the use of these vaccines because it is recommended that they be administered at a young age, preferably before sexual activity.

Poxviridae Poxviruses belong to the family Poxviridae, and they are among the largest of all viruses. They are about 225 to 450 nm long and about 140 to 260 nm wide. These viruses have a characteristic brick shape and contain a dsDNA genome. Variola

Table 29.5 Human papillomaviruses and their clinical signicance Human papillomavirus type

Clinical manifestation

Association with malignancy

Plantar warts

None

Cutaneous 1 2–4

Common warts

None

5, 8, 9, 12, 14, 15, 17, 19-25, 36–38

Flat and macular warts

>30% of patients with epidermodysplasia verruciformis (a rare autosomal disease) with types 5, 8, 14, 17, and 20 develop malignancy

26–29, 34

Common and at warts

Frequent, especially in immunocompromised patients

6, 11

Papillomatosis, primarily laryngeal, also upper respiratory tract and condylomata acuminata (genital warts)

Low risk

42, 43, 44

Condylomata acuminata

Low risk

31, 33, 35, 51, 52

Condylomata acuminata

Intermediate risk

16, 18, 45, 56, 58, 59, 68

Condylomata acuminata

High risk

Mucosal

Modied from Gravitt, P. E., & Ginocchio, C. C. (2011). Human papillomaviruses. In J. Versalovic, et al. (Eds), Manual of clinical microbiology (10th ed., p. 1612). Washington, DC: ASM Press; 2011.

Single-stranded DNA viruses

virus belongs to the genus Orthopoxvirus. Members of the genus include vaccinia virus (the smallpox vaccine strain), monkeypox virus, cowpox virus, among others.

Variola virus Variola virus causes smallpox, a disease that was common throughout early history (Fig. 29.18). Edward Jenner demonstrated the efcacy of vaccination against smallpox in 1796, which ultimately led to control and eradication of the disease. The last reported case of smallpox in the United States was in 1949, and the last case of smallpox worldwide was in Somalia in 1977. After decades of aggressive efforts toward vaccination, education, and eradication, the WHO ofcially declared the world smallpox free in May 1980. Although extinct in nature, smallpox virus is known to be maintained in at least two locations: in the United States at the Centers for Disease Control and Prevention (CDC) and in Russia. These viruses are maintained under strict security measures, and their very existence remains a point of contention between scientists who advocate their destruction and those who wish to continue studying the virus. The two countries continue to defend maintaining these stocks on the grounds that further study is needed to produce better vaccines and as countermeasures to bioterrorism. Typically, smallpox is characterized as a synchronous progressive rash accompanied by fever. The incubation period is approximately 10 to 17 days. The patient becomes febrile, and oral lesions can appear. At this point, the patient is infectious. Within 24 to 48 hours, a faint macular rash develops on all parts of the body. However, lesions are present in greater concentration on the head and limbs (centrifugal distribution), including the palms and soles. The macular rash progresses into papules, then vesicles, and nally into pustules that resemble chickenpox lesions. Pustules are deeply embedded into tissues. All lesions change at the same time—hence the term synchronous. Infected persons with dark skin tones typically scar as a result of this infection because of the depth of the pustules. The mortality rate for smallpox was, on average, 30%. Other forms of smallpox, including at and hemorrhagic smallpox, occurred rarely and were almost always fatal.

727

Routine vaccination against smallpox in the United States ended in 1972, and other countries have also stopped their vaccination programs. Two vaccines are approved for use in the United States: ACAM2000, manufactured by Sano Pasteur Biologics (Canton, MA), and JYNNEOS, produced by Bavarian Nordic (Denmark). Although smallpox was eradicated with the use of a relatively safe vaccine, most of today’s population is now susceptible to infection because vaccination is thought to offer protection for only up to 10 years. Because most humans are now susceptible to variola virus, it remains a threat as a bioterrorism weapon. Most military personnel moving into areas of conict receive the vaccine, with little to no ill effects. If an outbreak of smallpox were to occur, there is sufcient stock of smallpox vaccine to mount an effective vaccination program in the United States. The antiviral compounds cidofovir and brincidofovir have been shown to be effective in combating disease in humans. More information on smallpox as an agent of bioterrorism can be found in Chapter 30

Monkeypox virus Monkeypox was rst described in primates in 1958, with the rst case of human monkeypox in 1970. In 2003, a multistate outbreak of monkeypox occurred in the United States. It is thought that the virus was introduced into the country by rodents imported from Africa (Gambian rats). Human monkeypox infection occurs primarily in central and western Africa. Human infections are rare but result in a vesicular, pustular febrile illness that is very similar to smallpox. Monkeypox infections are less severe in humans compared with smallpox, and mortality rates are signicantly lower. In 2022, an outbreak of monkeypox occurred in 75 countries. The WHO reported 18,095 laboratory conrmed and suspected cases and 5 deaths between January 1 and July 22, 2022. The majority of the cases (72%) were reported in the European region, although the United States reported more cases than any other country. In July, the WHO declared monkeypox a global emergency. It has been suggested that monkeypox virus might have been circulating below detectable levels and maintained undetected human-to-human transmission for a period of time. It is unknown how people in this outbreak were exposed. Data suggest it is transmitted by close personal contact, and a number of cases have been found in gay, bisexual, and other men who have sex with men. Vaccination for smallpox provides some degree of protection against monkeypox virus. There has been a concerning rise in the number of cases of monkeypox infection in nonendemic countries in recent years, indicating a weakening of protective immunity or emergence of mutant strains that are not protected by the smallpox vaccination.

Single-stranded DNA viruses Parvoviridae Fig. 29.18 Negatively stained transmission electron micrograph of the smallpox (variola) virus (×100,000). (Courtesy J. Nakano, Centers for Disease Control and Prevention, Atlanta, GA.)

The smallest of the DNA viruses are the family Parvoviridae, which are naked ssDNA viruses that measure about 22 to 26 nm in diameter. Parvovirus B19 is the principal human pathogen in the family. It is classied in the genus Erythrovirus. Parvovirus B19 was named after the serum sample (number 19 of panel B)

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in which the initial viral isolate was observed by electron microscopy. Infections range from asymptomatic to potentially fatal. The most recognized syndrome is erythema infectiosum, more commonly referred to as fth disease. Patients with erythema infectiosum experience a prodrome of fever, headache, malaise, and myalgia, with respiratory and GI symptoms (nausea and vomiting). The prodromal phase lasts a few days, after which a rash often appears. The rash gives a slapped cheek appearance and then spreads to the trunk and limbs. The rash occurs more commonly in children than in adults, lasts up to 2 weeks, and can recur after exposure to heat and sunlight. Adults may also experience arthralgia, arthritis, or both. In some cases, the connective tissue manifestation occurs without the prodrome or rash stage. Most infections occur in children and adolescents, and 80% of adults are seropositive by 65 years of age. Parvovirus B19 viremia can cause transient aplastic crisis, a self-limiting erythropoietic arrest. Erythroid precursor cells contain a receptor for the virus, allowing viral infection and replication. The disease is characterized by a decrease in RBC production in bone marrow. In otherwise normal individuals, this decrease results in a short-lived anemia. The disease can be severe; complications include viremia, thrombocytopenia, granulocytopenia, pancytopenia, ulike symptoms, and congestive heart failure. Within about 1 week, reticulocytosis occurs, and the patient recovers. Persons with weakened immune systems caused by HIV infection, organ transplantation, cancer, or leukemia are at risk for serious complications from fth disease. Parvovirus B19 viremia creates a risk for fetuses and blood transmission in blood units. In utero infection can cause hydrops fetalis resulting from anemia. Although the most vulnerable period for the fetus is the third trimester, most women exposed to the virus do not develop an acute disease, and few infections result in loss of the fetus. The infection usually does not require therapy other than relief of symptoms, such as anti-inammatory agents for painful joints and generalized aches. A novel parvovirus was described in 2005. It was named the human bocavirus (HBoV) and causes a variety of upper and lower respiratory tract illnesses. Other viruses related to the rst HBoV isolate, namely HBoV2, HBoV3, and HBoV4, have been detected principally in human feces. All of these viruses have been found in patients with gastroenteritis and in healthy controls, with HBoV2 found most often. HBoV is closely related to the bovine parvovirus and canine minute virus, both members of the genus Bocavirus in the family Parvoviridae. Clinical symptoms of HBoV infection include cough, rhinorrhea, fever, difculty breathing, diarrhea, conjunctivitis, and rash. HBoV has been increasingly present as a co-infection with RSV and human metapneumovirus (HMPV). Some studies have indicated a potential link to HBoV respiratory illness and gastroenteritis. However, researchers concluded that HBoV is shed in high quantities in stool, but its link to gastroenteritis has not been demonstrated. HBoV infection is highest during the winter months and has been detected worldwide in 5% to 10% of children 7 to 18 months of age with upper and lower respiratory tract infections. Since its rst description, HBoV has been described in at least 19 countries on 5 continents. HBoV was detected less frequently than common respiratory agents (e.g., inuenza virus, parainuenza virus, adenovirus, RSV) in South African

children. Detection of HBoV has improved with the development of sensitive and specic RT-PCR assays.

Double-stranded RNA viruses Reoviridae Rotaviruses Rotaviruses are naked viruses about 75 nm in diameter, with two protein layers surrounding the capsid. They belong to the genus Rotavirus. Rotaviruses are the most common cause of viral gastroenteritis in infants and children. Gastroenteritis is a major cause of infant death and failure to thrive. Rotaviruses have a worldwide distribution and in 2016 caused an estimated 258 million cases of diarrhea and 128,500 deaths in children less than 5 years of age. Death rates have declined due to increased availability of a vaccine. However, rotavirus accounted for 29% of all diarrhea deaths in children younger than 5 years age. Most outbreaks occur in the winter months in the temperate zones and year-round in subtropical and tropical regions. Rotaviruses are spread by the fecal-oral route and have an incubation period of 1 to 4 days. Symptoms generally occur suddenly and include vomiting, diarrhea, fever, and in many cases, abdominal pain and respiratory symptoms. Vomiting and diarrhea can cause rapid loss of uids and fatal dehydration. The rotavirus replicates in the epithelial cells in the tips of the microvilli of the small intestine. The microvilli are stunted, and adsorption is reduced. The virus is shed in large quantities in the stool and can cause healthcare-associated outbreaks in the absence of good hygiene. Although the rotavirus is present in large numbers in feces, it can be isolated only with special procedures. Enzyme-linked immunosorbent assay (ELISA) and latex agglutination tests detect the viral antigens in fecal material. Rapid membrane-bound colorimetric tests are also available. Electron microscopy examination of stool samples can be used; however, as mentioned previously, this method is not very sensitive. With the introduction in 2006 of a human-bovine rotavirus vaccine, RotaTeq (RV5; Merck), a delay in the onset of rotavirus season was seen from mid-November to late February. RotaTeq protects against ve strains of RSV and is a series of three oral vaccines administered beginning at age 6 to 12 weeks. A second vaccine, Rotarix (RV1; GlaxoSmithKline), was approved in June 2008. In clinical trials, both vaccines were shown to be safe and effective. During approximately the rst year of an infant’s life, the rotavirus vaccine prevented 85% to 98% of severe rotavirus illness episodes and prevented 74% to 87% of all rotavirus illness episodes, which was a signicant reduction in both populations.

Colorado tick fever virus The genus Coltivirus contains the Colorado tick fever virus that causes a dengue-like infection in the western United States and Canada. It is an 80-nm spherical particle with two outer shells containing 12 RNA segments. Because Colorado tick fever is not reportable, actual numbers of cases are unknown. It is thought to be one of the most common diseases transmitted by ticks in the United States. Viruses transmitted by arthropods, such as ticks and mosquitoes,

Single-stranded RNA viruses

are referred to as arboviruses. The vector for the infection is Dermacentor andersoni, which has many hosts in nature, including deer, squirrels, and rabbits. Infected individuals develop fever, photophobia, myalgia, arthralgia, and chills. As with dengue fever, patients can also have a biphasic fever with a rash, and children can experience hemorrhagic fever. No commercially produced laboratory tests are available, but recombinant immunoassays to detect Colorado tick fever IgG have been developed. Some nucleic acid–based assays using RT-PCR in research laboratories are becoming available and may be helpful in the future. Many primary health care providers rely on ruling out other tickborne diseases to diagnose Colorado tick fever.

Single-stranded RNA viruses Arenaviridae The arenaviruses get their name from the Latin arena, meaning “sand.” Under an electron microscope, arenaviruses appear sandy and granular. There are 43 named arenaviruses; the family includes many species that cause hemorrhagic fever. Arenaviruses are commonly divided into two complexes: Old World and New World. The New World complex includes the common human pathogens Tacaribe, Junín, and Machupo viruses, among several others. The Old World complex contains the arenaviruses commonly associated with human infection, the lymphocytic choriomeningitis (LCM) virus and the Lassa viruses, and also the benign Lassa-like Mopeia, Mobala, and Ippy viruses. Recently, a new arenavirus was identied from humans, the Lujo virus, during an outbreak of human fatal hemorrhagic fever, in which person-to-person transmission was documented. Some of the arenaviruses have not yet been isolated and are known only from molecular sequencing data. The rst arenavirus to be described was LCM virus in 1933. The rst arenavirus found to cause hemorrhagic fever was Junín virus, which causes Argentine hemorrhagic fever. In 1969, Lassa virus was isolated in Africa, and it became the basis of the novel Fever by John Fuller. The book details the emergence of Lassa fever, which in retrospect is eerily like the emergence of other hemorrhagic fevers, including Ebola virus (EBOV) disease, which would be identied later in Africa. The arenaviruses infect rodents, and humans are exposed to the disease by zoonotic transmission. All have been isolated from rodents of the family Muridae. The rodents are infected for long periods and typically do not become ill, but they shed virus in urine, feces, and saliva. In some parts of the United States, as many as 20% of Mus musculus mice carry LCM virus. Pet hamsters are also reservoirs. Humans become infected when they inhale the aerosolized virus or contact fomites. LCM virus causes ulike illness; about 25% of infected individuals develop meningitis. Lassa virus is the most well-known of the arenaviruses. Most exposed individuals develop an asymptomatic infection, but some patients experience fever, headache, pharyngitis, myalgia, diarrhea, and vomiting. Some patients develop pleural effusions, hypotension, and hemorrhaging. CNS involvement includes seizures and encephalopathy. The mortality rate is about 15% for patients needing hospitalization. West African nations are most affected, with more

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than 200,000 cases and approximately 3000 deaths occurring annually. Spread of the virus through airline travel by people from endemic areas highlights the vulnerability of large populations; this combined with the resurgence of the infection in Nigeria has prompted vaccine development efforts. Most cases of Lassa fever are community acquired, primarily through contact with excretions from the multimammate rat Mastomys natalensis, which, once infected, sheds the virus throughout its life. Humans inhale the aerosolized virus or contract the virus directly through breaks in skin. Lassa virus is present in throat secretions, can be transmitted from person to person, and can also be transmitted through sexual contact and nosocomially. If therapy begins within the rst 6 days of exposure, Lassa virus infection can be effectively treated with the antiviral drug ribavirin. Diagnosis of Lassa virus infection is typically made with ELISA to detect IgM and IgG antibodies.

Bunyavirales The order Bunyavirales includes the families Peribunyaviridae (genus Orthobunyavirus), Nairoviridae (genus Orthonairovirus), and Phenuiviridae (Phlebovirus), all of which are classied as arboviruses. These viruses replicate initially in the gut of the arthropod vector and eventually appear in saliva. The arthropod transmits the virus when feeding on the blood of vertebrate hosts, including humans. After a few days, the infected host usually develops an asymptomatic viremia; however, some hosts become febrile, which is far less common. Most members of the order Bunyavirales cause a febrile illness, hemorrhagic fever, or encephalitis. Rift Valley fever virus targets the brain and liver to cause encephalitis and hepatitis. La Crosse virus (LACV) and California encephalitis virus cause encephalitis, and Crimean-Congo hemorrhagic fever (CCHF) virus infects the vascular endothelium and liver. The hantaviruses, which belong to the family Hantaviridae (genus Orthohantavirus) within the order Bunyavirales, do not infect arthropod hosts. They are rodent-borne viruses. Hantaviruses typically affect the peritoneal cavity, thoracic cavity, kidneys, or lungs. The CCHF virus causes a high-mortality infection in humans. Infection begins with fever, myalgia, arthralgia, and photophobia. Patients exhibit mental status changes, ranging from confusion and agitation to depression and drowsiness. Petechiae and ecchymoses can form on mucosal surfaces and the skin. The patient may bleed from the bowel, nose, and gums. About 30% of patients die. Others begin recovering after about 10 days of illness. Healthcare-associated transmission of CCHF virus has been reported. In the United States, LACV is a reportable disease. About 50 to 150 severe CNS diseases are reported annually. The incidence is underestimated because the disease manifests as a nonspecic fever, headache, nausea, vomiting, and lethargy. The disease is commonly found in children and usually develops in the summer, frequently referred to as the summer u or summer cold. Because serologic tests for LACV are not offered in most laboratories and because this disease has very low mortality (about 1%), a denitive diagnosis is not often made. The genus Orthohantavirus includes Hantaan virus, Seoul virus, Puumala virus, and Dobrava virus, which cause the disease hemorrhagic fever with renal syndrome (HFRS). These viruses are present in Asia and Europe, except for Seoul virus,

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which is found worldwide. Hantaviruses endemic to Europe and Asia are called Old World hantaviruses. Puumala virus is the most common member of this genus in Europe and causes a mild form of HFRS, called nephropathia epidemica. Viruses causing HFRS target the kidneys. Patients develop a febrile prodrome and enter a phase of fever and shock, accompanied by oliguria. As the patient recovers, the kidneys gradually regain function. The mortality rate for HFRS is 1% to 15%. In 1993, two adults from the same household in New Mexico died of an unusual respiratory illness. Serologic testing indicated that these patients had been exposed to an unknown agent that was antigenically related to one of the Asian hantaviruses despite the different clinical presentation. Serology surveys also determined that 30% of the deer mice tested in the New Mexico area were seropositive for the same unknown virus. The virus was ultimately characterized as a new hantavirus and was named the Sin Nombre (“without a name”) virus (SNV). The disease caused by this virus was named hantavirus pulmonary syndrome (HPS). Molecular techniques were subsequently developed to detect many new hantaviruses in the Americas (Table 29.6), sometimes called the New World hantaviruses SNV is transmitted through inhalation of contaminated aerosolized mouse urine, saliva, and feces. Generally, person-to-person transmission does not occur with hantavirus. Patients with HPS have a 3- to 5-day febrile prodrome, with fever, chills, and myalgia. Patients then enter a phase of hypotensive shock and pulmonary edema. The patient develops tachycardia, hypoxia, and hypotension. In severe cases, the patient can develop disseminated intravascular coagulation. The mortality rate for HPS is about 50%. Treatment for HPS is primarily supportive. No FDA-approved laboratory tests for the identication of a hantavirus infection are available; however, some European tests are being evaluated. Some Table 29.6 Hantaviruses that cause hantavirus pulmonary syndrome Hantavirus

Host

Location

Sin Nombre

Peromyscus maniculatis (deer mouse)

United States, western Canada

Black Creek Canal

Sigmodon hispidus (cotton rat)

United States, South America

Bayou

Oryzomys palustris (marsh rice rat)

Southeastern United States

Monongahela

Peromyscus maniculatis (deer mouse)

Eastern United States

New York

Peromyscus leucopus (white-footed deer mouse)

New York

Oran

Oligoryzomys longicaudatus (long-tailed pygmy rice rat)

Argentina

Andes

Oligoryzomys longicaudatus

Argentina, Chile

Lechiguanas

Oligoryzomys avescens (yellow pygmy rice rat)

Argentina

Laguna Negra

Calomys laucha (vesper mouse)

Paraguay, Bolivia

state health laboratories and the CDC perform EIAs to detect anti-SNV IgM and IgG antibodies, which may provide some useful information on the infection. Immunohistochemistry is a sensitive method used to detect hantavirus antigens in the capillary endothelium; a high concentration of antigens is found in capillary tissue specimens from the lung.

Caliciviridae The family Caliciviridae contains 10 genera; Norovirus and Sapovirus are the most clinically signicant NoVs (Fig. 29.19) are the most common cause of infectious gastroenteritis in the United States, accounting for as many as 19 to 21 million cases annually and 900 deaths. Most deaths occur in adults 65 years of age and older. Until recently, these small, round ssRNA viruses, 27 to 30 nm in diameter, were called Norwalklike viruses, caliciviruses, and small round structured viruses. They are currently placed in the genus Norovirus. They cause outbreaks of acute gastroenteritis in schools, colleges, nursing homes, and families as well as on cruise ships and in resort areas. NoVs have been found in drinking water, swimming areas, and contaminated food. Transmission is most commonly foodborne, although waterborne and person-to-person transmission can be signicant. The incubation period is 24 to 48 hours; the onset of severe nausea, vomiting, diarrhea, and low-grade fever is abrupt. The infection rate can be as high as 50%. The illness usually subsides within 72 hours. Immunity may be short-lived, leading to the potential for multiple infections throughout life. The viruses cannot be grown in culture, so diagnosis relies on electron microscopy, immune electron microscopy, and real-time PCR. RT-PCR is now the most used diagnostic assay for detecting NoV. This assay detects viral RNA and can be used to test stool, vomitus, and environmental samples. Stool is the best sample to use to detect NoV. It should be collected when a person has acute illness, within 48 to 72 hours after onset of symptoms. In some cases, NoV can be detected in stool samples collected 2 weeks after recovery. Several EIAs for detecting NoV in stool samples are available. The FDA has approved an EIA for detecting NoV during outbreaks. However, at this time, EIAs are not sensitive enough for diagnosing individuals suspected of being infected.

Fig. 29.19 Transmission electron micrograph revealing the ultrastructure morphology of norovirus virions (×100,000). (Courtesy Charles D. Humphrey, Centers for Disease Control and Prevention, Atlanta, GA.)

Single-stranded RNA viruses

Sapporo viruses are small (30–35 nm in diameter) diarrheagenic viruses distinguished by a cup-shaped morphology. They usually cause diarrhea and vomiting in infants, young children, and older adults. Originally discovered in Sapporo, Japan, in 1977, these viruses are detected by electron microscopy, molecular methods (e.g., RT-PCR), and/or immunologic methods (e.g., ELISA).

Coronaviridae Coronaviridae is one of seven families within the order Nidovirales. Of the 30 recognized species within the subfamily Coronavirinae, six are found in humans. Two human species are found in the genus Alphacoronavirus, and the remaining four belong to the genus Betacoronavirus, along with many other mammalian coronaviruses (CoVs). Many new CoVs have been discovered recently and analyzed genetically. This has resulted in hundreds of new CoV genome sequences, which has led to reorganization of the taxonomic structure and the current phylogenetic relationship of the species. CoVs have large, linear, positive-stranded RNA genomes, ranging from approximately 25 to 32 kilobases (kb) in size, and they are enveloped, helical viruses. They were rst identied by electron microscopy and were named because the distinctive club-shaped projections on their surface resemble a crown (Fig. 29.20). CoVs infect several different animals; however, most individual strains of virus typically infect only a single animal species. Some can infect more than one related species. Four strains of CoV are considered endemic to the United States. These strains are associated with upper respiratory tract infections and occasionally lower respiratory tract infections in people of all ages. One strain in particular has been linked to croup in children. CoVs may be responsible for 15% of coldlike infections in adults, but higher seroconversion rates have been seen in children. A few CoVs are responsible for a small percentage of pediatric diarrhea cases. In general, the illness lasts about 1 week, and blood may appear in the stool.

Fig. 29.20 Transmission electron micrograph of the coronavirus. This virus derives its name from the fact that under electron microscopy, the virion is surrounded by a corona, or halo (×100,000). (Courtesy Fred Murphy and Sylvia Whiteld, Centers for Disease Control and Prevention, Atlanta, GA.)

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A novel CoV was the causative agent of a pandemic respiratory disease that emerged from Hong Kong in late 2002. During a 6-month period, the infection spread rapidly to 26 countries in Asia, Europe, South America, and North America. The virus infected at least 8000 people and resulted in a mortality rate of approximately 10%. The disease was characterized by high-grade fever, pneumonia, and in some patients, acute respiratory distress syndrome (ARDS). The disease was ultimately termed severe acute respiratory syndrome (SARS), and the causative agent was designated as the SARSassociated CoV (SARS-CoV). No vaccine or antiviral agent was available to ght the pandemic, which was ultimately ended through intense public health intervention, including massive screening programs, voluntary quarantine, and travel restrictions. This human infection apparently started as a CoV that jumped from its natural animal host, possibly a civet cat, to humans. SARS-CoV highlights the public health risk that can occur when animal viruses suddenly appear in a susceptible human population. In 2012, a SARS-like virus was linked to severe respiratory tract infections in the Middle East. This virus was named the Middle East respiratory syndrome CoV (MERS-CoV). By August 2013, MERS-CoV was associated with 94 infections and 46 deaths, primarily in Saudi Arabia. MERS-CoV is unlike any other CoV that infects humans. In 2014, another outbreak of MERS-CoV occurred in Saudi Arabia, with 14 people infected and 4 reported deaths. As of July 2022, over 2600 cases and 858 conrmed deaths of MERS-CoV infection in 27 countries have been reported. The 2002 SARS outbreak created much interest in understanding the epidemiology, reservoir-host relationship, and vaccination possibilities associated with this CoV. In 2007, SARS-CoV antibodies were detected in 47 of 705 South African and Democratic Republic of the Congo bat sera samples collected from 1986 to 1999. Researchers tested bats from the Rocky Mountain region in 2006 and detected CoV RNA in two species of bats: Eptesicus fuscus and Myotis occultus. Animals, such as civets, can acquire infections with SARS-like CoV from contact with infected bats. Current data indicate that bats, primarily horseshoe bats, are the most likely reservoir for SARS-CoV, although the bat CoVs are species specic. More than 10 mammalian species have been identied as being susceptible to SARS-CoV by natural or experimental infection. Infections of these secondary hosts may give rise to strains that could potentially infect humans. On December 31, 2019, the existence of patients with pneumonia of an unknown etiology was reported to the WHO by national authorities in China. It was rst seen in Wuhan, China, and was thought to have emerged in a food market. This virus was ofcially identied by the coronavirus study group as SARS coronavirus 2 (SARS-CoV-2), and the resulting outbreak of a coronavirus-associated acute respiratory disease was named coronavirus disease 19 (COVID-19). From China, COVID-19 rapidly spread into Europe. The rst reported case in the United States was on January 21, 2020, in Washington state in a 35-year-old male patient who had just returned from Wuhan, China. On February 20, 2020, the CDC announced that the rst person in the United States had died from COVID-19. By July 2022, the WHO reported that the COVID-19 pandemic resulted in over 577,000,000 conrmed cases and over 6,400,000 deaths worldwide. For the same time period, the CDC reported over 90,000,000 U.S. cases with over 1,000,000 deaths.

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This virus is highly infectious, and although most individuals are asymptomatic or have mild symptoms, those with certain medical conditions often progress to severe, life-threatening illness. High-risk conditions include diabetes, overweight or obesity, history of smoking or cancer, and heart and kidney disease. Severe illness is linked to two primary causes: secondary pneumonia and an overexuberant proinammatory cytokine response. This cytokine response is referred to as a cytokine storm or hyperinammation. Cytokine storm syndrome is an uncontrolled activation and amplication of the host immune response resulting in the release of several cytokines such as tumor necrosis factor, interleukin-1 (IL-1), IL-6, and IL-18. The cytokine response appears to be related to the severity of ARDS, which is a type of respiratory failure characterized by widespread inammation in the lungs. SARS-CoV targets the epithelial cells of the respiratory tract and is transmitted from person to person by direct contact, droplet, or airborne routes. Other organ systems affected by SARS include the spleen, lymph nodes, digestive tract, urogenital tract, CNS, bone marrow, and heart. The virus can also be isolated from urine and feces, suggesting other potential routes of transmission. The receptor for SARS-CoV-2 is angiotensin-converting enzyme 2. This enzyme is expressed on many human cells including those in the respiratory airways, intestine, kidney, heart, and pancreas. Certain individuals may be genetically more susceptible to SARS than others. CoVs are extremely fragile and difcult to culture, but it is

Case check 29.1 History of leukemia and hypogammaglobulinemia and advanced age placed the patient in the Case in Point at high risk for severe COVID-19. Fortunately, the two treatments of SARS-CoV-2 convalescent plasma he received helped him to remain asymptomatic. However, he was culture positive for the virus several weeks after his diagnosis.

possible to detect viral antigens from some species directly by IF or ELISA (EUROIMMUN Medical Laboratory Diagnostics, Lübeck, Germany). Viral particles can also be detected by electron microscopy in stool samples. However, sensitivity is generally lower with electron microscopy and antigen detection than RT-PCR. The most common diagnostic approach for the identication of CoVs is amplication and detection of virus-specic RNA using RT-PCR. RT-PCR assays have demonstrated greater sensitivity and specicity and a much shorter TAT. Many viral panels are now available for the detection of the four endemic CoVs found in the United States—CoV 229E, CoV HKU1, CoV NL63, and CoV OC43— from respiratory specimens (FilmArray, BioFire Diagnostics). Other nucleic acid–based tests include isothermal amplication methods and loop-mediated amplication assays. These are typically used in research or larger reference laboratories. Antibodies can be detected through western blot analysis and ELISA. Although serologic assays have largely been replaced by NAATs, they still have a role in epidemiologic studies. The COVID-19 pandemic has resulted in an expansion of testing for CoV-2. However, at time of publication, all testing for detection and exposure has EUO authorization from the FDA and CDC. The treatment of COVID-19 has been based on providing basic life support and mitigating the effects on the respiratory

system caused by ARDS, which is the leading cause of mortality. Modulating cytokine levels is key in preventing widespread organ damage, respiratory failure, and death. Several drugs with antiinammatory activity in other illnesses were evaluated in respect to their potential utility in the treatment of the hyperinammation induced by SARS-CoV-2 infection, and these included interferon, steroids, atazanavir, favipiravir, lopinavir-ritonavir, anakinra, baricitinib, and remdesivir. The current preferred therapy in most cases is ritonavir-boosted nirmatrelvir (Paxlovid). The anti-inammatory agent dexamethasone is used in severe cases. Heparin is added in cases with coagulopathy. Multiple COVID-19 vaccines were developed, and two successful vaccines were approved for EUO authorization in December 2020. These vaccines are unique in that they use messenger RNA (mRNA) coding for a viral protein. After injection, cells in the recipient use the mRNA to express the viral spike protein, which initiates the immune response to the virus. Although these vaccines are new, mRNA vaccine technology began in the 1980s.

Filoviridae The family Filoviridae includes eight genera; Marburgvirus and Ebolavirus are the most clinically signicant. The genus Marburgvirus contains one species (Marburg marburgvirus) and two viruses: Marburg virus (MARV) and Ravn virus. The genus Ebolavirus contains ve viruses: EBOV, Sudan virus (SUDV), Reston virus (RESTV), Tai Forest virus, and Bundibugyo virus. Both Marburg virus and EBOV share a common morphology (Fig. 29.21) and similar genome and structural proteins. These viruses have many similarities; they rarely cause human infections, they cause infections with high mortality rates, and they have unknown reservoirs in nature, although human infection can result from contact with infected monkeys. Lake Victoria Marburg virus, now known as MARV, was named after the location of one of the rst outbreaks: Marburg, Germany. In 1967, 32 people from Marburg and Frankfurt, in Germany, and from Belgrade, Serbia, and Yugoslavia contracted an unknown infection, and 7 died. Epidemiologists noted that the deceased individuals all worked in vaccine-producing facilities and had contact with African green monkeys

Fig. 29.21 Transmission electron micrograph of an Ebola virus (×100,000). (Courtesy Fred A. Murphy, Centers for Disease Control and Prevention, Atlanta, GA.)

Single-stranded RNA viruses

that had arrived recently from Uganda. The virus had been transmitted to 24 other people through healthcare-associated transmission, casual contact, and sexual contact. Patients with secondary infection had a milder illness, and all survived the infection. Outbreaks of MARV are rare. The EBOVs are named after the Ebola River in the Democratic Republic of the Congo, where the infection rst emerged in 1976. The virus emerged almost simultaneously in Sudan. In Zaire, a patient treated at a village hospital for a bloody nose probably introduced the virus into the hospital, where it was then transmitted nosocomially and via contact with individuals at home. Nuns in the hospital routinely reused syringes without sterilizing them. Therefore the hospital amplied the number of cases. Infections were also passed to the victims’ families, often during a process in which the intestines of infected deceased males were cleansed to prepare the bodies for funerals. The two simultaneous outbreaks of Ebola fever were caused by two different viruses, Ebola Zaire, now called Ebola virus, and Ebola Sudan, now called Sudan virus. Of the two, EBOV is the more virulent. In the initial outbreak, 318 individuals were infected in Zaire; the mortality rate was 88%. In the Sudan outbreak, 284 people were infected, and a mortality rate of 53% was reported. After the initial two large outbreaks, smaller outbreaks occurred. In Sudan, 34 cases occurred in 1979, and 65% of the 34 patients died. The virus seemed to retreat into the jungles for the next 15 years. An unusual sequence of events occurred in 1989, when monkeys imported to Reston, Virginia, from the Philippines were aficted by an epidemic of infection by what eventually became the third type of EBOV, RESTV. Four workers in the animal facility developed antibodies to RESTV but did not develop the disease. RESTV was isolated from the bloodstream of one of the workers. EBOV continued to emerge periodically in the Democratic Republic of the Congo (2003 and 2007), southern Sudan (2004), and Uganda (2007). In 2012, several more outbreaks were reported. Beginning in March 2014, West Africa had the largest outbreak of EBOV in history. Multiple countries were involved, with Liberia, Sierra Leone, and Guinea affected the most through early 2016. According to the CDC, there were 28,652 probable cases, with 15,261 laboratory conrmed cases and 11,325 deaths during this outbreak. This outbreak resulted in precautions in both Europe and the United States to handle suspected cases of infected people traveling from West Africa. Many clinical laboratories have now established protocols to handle suspected cases, ensuring safely for both staff and other patients. Newer molecular testing is being developed to detect EBOV from serum, and this will likely obtain rapid FDA review and approval. Symptoms of EBOV and MARV disease are similar, producing a hemorrhagic fever. After an incubation period of 4 to 16 days, there is sudden onset of fever, chills, myalgia, and anorexia. Patients then develop a sore throat, abdominal pain, diarrhea, vomiting, and a maculopapular rash, and they begin to bleed from injection sites and the GI tract. Hemorrhaging in skin and the internal organs may occur as well. Diagnosis of the infection can be made using RT-PCR, IF, or viral culture. However, because of the risk of laboratory infection, biosafety level 3 laboratories are required. Sometimes electron microscopy of clinical samples will yield the characteristic long, rodlike virions.

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Flaviviridae The family Flaviviridae contains several important human pathogens, many of which are zoonotic arboviruses, including Japanese encephalitis virus, dengue virus, yellow fever virus, St. Louis encephalitis (SLE) virus, Zika virus, Kyasanur Forest disease virus, Langat virus, Löuping ill virus, Murray Valley encephalitis virus, Omsk hemorrhagic fever virus, Powassan virus, tickborne encephalitis virus, Wesselsbron virus, and WNV. Japanese encephalitis virus is a major cause of encephalitis in Asia and is the most common cause of arboviral encephalitis in the world. Because it is being reported in regions previously free from Japanese encephalitis, including Australia, Japanese encephalitis virus is considered an emerging pathogen. Currently, nearly 68,000 cases of Japanese encephalitis are reported annually. Because most patients are asymptomatic, cases are likely under-reported. Disease ranges from a ulike illness to acute encephalitis. Children are mostly affected by this infection, with mortality as high as 30%; mortality in adults is much lower. Another important member of the family Flaviviridae is dengue virus, which causes two distinct diseases: classic dengue fever (DF) and dengue hemorrhagic fever (DHF). Worldwide, tens of millions of cases of DF and approximately 500,000 cases of the more serious DHF occur annually. The average mortality associated with DHF is 5%, accounting for 22,000 deaths each year. Most deaths occur in children younger than 15 years. The virus is transmitted by Aedes mosquitoes, including Aedes aegypti and Aedes albopictus. These mosquitoes infest more than 100 countries and bring the risk of DF to 2.5 billion people. Dengue virus has four serotypes (1 to 4); DF is a relatively mild infection. Patients with DF develop fever, headache, myalgia, and bone pain (resulting in the nickname breakbone fever). Some patients also develop a rash. The disease is self-limiting and often resolves in 1 to 2 weeks. Although classic DF is a mild disease, DHF is not. Patients develop DHF after previous exposure to one serotype of dengue virus and are then exposed to one of the other three serotypes. Exposure to two different serotypes of dengue virus appears to be necessary for development of DHF. Patients with DHF develop the symptoms of classic DF, along with thrombocytopenia, hemorrhage, shock, and sometimes death. Yellow fever, caused by yellow fever virus, is also considered an emerging infection. Although a safe vaccine has been available for decades, about 200,000 cases of yellow fever with 30,000 resulting deaths are reported annually worldwide. The actual incidence may greatly exceed these numbers. The emergence results from increased spread of the mosquito vectors, deforestation of Africa and South America, and increased travel to endemic regions. The vaccine has greatly reduced or eliminated the transmission of yellow fever in some countries. However, yellow fever is still epidemic in parts of Africa and South America, where about 80% of the population must be vaccinated to reduce the impact of the disease. Patients bitten by mosquitoes carrying yellow fever virus can develop an asymptomatic or acute infection involving fever, myalgia, backache, headache, anorexia, nausea, and vomiting. Most patients experiencing acute disease recover after about 4 days. However, about 15% enter a systemic toxic phase in which fever reappears. The patient develops jaundice (hence the name yellow fever) as well as bleeding from the mouth, eyes, nose, stomach, or other areas. The kidneys

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may fail, and about 50% of patients in the toxic phase die. The other 50% recover without serious sequelae. The three different transmission cycles for yellow fever virus are sylvatic, urban, and intermediate cycles. In the sylvatic cycle, yellow fever virus is maintained in monkey populations and transmitted by mosquitoes. Monkeys become sufciently viremic to pass the virus to mosquitoes as they feed on the monkeys, thus keeping the transmission cycle active. Humans are not the usual hosts when they enter jungle areas in which the sylvatic cycle exists. The urban cycle occurs in larger towns and cities when infected A. aegypti mosquitoes transmit the infection to humans. Because infected humans can continue the transmission when bitten by uninfected mosquitoes, large outbreaks can occur from a single case of yellow fever. The intermediate transmission cycle of yellow fever occurs in smaller villages in Africa. In the intermediate cycle, humans and monkeys are reservoirs, while mosquitoes serve as reservoirs and vectors in the high-morbidity, low-mortality outbreaks. In the intermediate cycle, mosquitoes can transmit yellow fever virus from monkeys to humans, and vice versa. If patients who develop yellow fever from the intermediate cycle travel to larger cities and are bitten by mosquitoes, they can trigger an outbreak of urban yellow fever. From 2011 to 2020, a total of 103 SLE neuroinvasive disease cases were reported in the United States. Patients with SLE are most likely to be asymptomatic; hence total cases are much higher. While cases are seen from Canada to Argentina, most human cases occur in the United States. Symptomatic patients may develop a fever only, whereas some patients develop meningoencephalitis. The mortality rate of symptomatic patients is 3% to 20%. Unlike many of the arboviral infections, SLE is milder in children than in adults; older patients have the greatest risk of serious illness and death. SLE is transmitted to humans by the bird biting Culex mosquitoes. Most infections occur in the summer months. First isolated and identied in 1937 from a febrile patient in the West Nile district of Uganda, WNV is an ssRNA virus and member of the Japanese encephalitis antigenic complex, like SLE. The virus is transmitted by a mosquito vector between birds and humans. The virus replicates actively inside the avian host; however, the virus does not replicate well in humans, making humans a dead-end host for infection. The incidence of WNV infection in the United States increased during the mid-1990s, prompting the development of a national surveillance system in 1999. In 2003, the CDC documented 9862 human cases of WNV infection in 44 states, the most ever reported in one year. A decline followed in 2011 to 712, the lowest number of cases ever reported. The number of cases has uctuated since then. In 2019, a total of 971 cases were reported, and 633 were described as neuroinvasive. A total of 60 patients (9%) with neuroinvasive disease died. From 1999 to 2019, overall, about 16% of the cases were in California and 13% in Texas. Approximately 80% of individuals infected with WNV are asymptomatic. The remaining 20% display symptoms of what is termed West Nile fever, which includes fever, headache, fatigue, occasional rash on the trunk, swollen lymph glands, and/or eye pain. The primary risk factor for serious neuroinvasive disease is age greater than 50 years. Neuroinvasive disease typically manifests as meningitis or encephalitis.

Laboratory tests approved for the detection of WNV include IgG and IgM ELISA, including a rapid WNV ELISA assay and an indirect IF assay for antibodies. WNV ELISA assays can cross-react with other aviviruses and should be conrmed by antibody neutralization. IgM antibody does not cross the blood-brain barrier; therefore the presence of IgM in CSF strongly suggests CNS infection. WNV can be present in tissues, blood, serum, and CSF of infected humans or animals. Several RT-PCR, TaqMan, and nucleic acid sequence– based amplication assays have been used for conrmation of WNV infection. There is no specic treatment for WNV infection, but in severe cases requiring hospitalization, supportive care, including IV uids, respiratory support, and prevention of secondary infection, may be warranted. Zika virus is an insect vector–borne disease that is most commonly transmitted through Aedes (A. aegypti and A. albopictus) mosquitoes. Zika virus can also be transmitted by exposure to infected blood or sexual contact. Less commonly, Zika virus can be transmitted from a mother to a child during pregnancy. Zika virus is typically endemic to parts of Africa and Asia. However, in 2015 to 2016, an epidemic occurred across South, Central, and North America, where the disease was previously unreported. Almost 500,000 cases of Zika virus infection were reported in 38 countries throughout the Americas. Almost 50,000 of these cases were laboratory conrmed, and 360,000 cases were suspected to be positive. In 2016, the CDC reported more than 5000 cases of Zika virus infection, with 95% of the cases in travelers returning from affected areas. Reported cases have dropped signicantly since then, with only 4 cases found in the United States and 57 cases in U.S. territories in 2020. Symptoms of Zika virus infection in most people resemble those of infections with other arboviruses, such as chikungunya virus (primarily fever, headache, and fatigue). The epidemic in Brazil between 2015 and 2016, resulting in over 300,000 cases, was marked by the detection of the virus in fetal amniotic uid and an increased reporting of cases of microcephaly (small head size) in newborns.

Orthomyxoviridae The inuenza viruses are enveloped and belong to the family Orthomyxoviridae. These viruses are distinguished by using two major structural proteins: matrix protein (M) and nucleoprotein (NP). This places the inuenza viruses into four genera within the family: Inuenzavirus A, Inuenzavirus B, Inuenzavirus C, and Inuenzavirus D. Inuenza viruses have a worldwide distribution and originate as zoonotic infections, carried by several different species of birds and mammals. The inuenza season in the southern hemisphere is from May to October and in the northern hemisphere is from November to April. Inuenza A virus remains one of the most crucial health problems worldwide. In the pandemic of 1918 and 1919, inuenza infected an estimated 500 million people, about one third of the world’s population, and killed at least 50 million people, including more than 600,000 in the United States. However, since that event, the world has only been able to react to the threat of inuenza, rather than vanquish it. A typical inuenza season in the United States hospitalizes almost 700,000 and kills about 36,000 people.

Single-stranded RNA viruses

Inuenza viruses are enveloped; types A and B have eight segments of ssRNA, while types C and D have seven. Inuenza A viruses are classied into subtypes using the two major surface glycoproteins: hemagglutinin (H) and neuraminidase (N). The H antigen is used to bind to host cells, and the N antigen cleaves budding viruses from infected cells. The H protein is the major antigenic determinant detected by antisera. There are 17 H antigens (H1 to H17), although human infections usually occur only with H1, H2, and H3. There are 10 N antigens (N1 to N10); human infections usually occur with N1 and N2. The key to the persistence of inuenza virus is its antigenic variation. Each year, antigenic drifts are caused by RNA replication errors of the virus. Antigenic drift is a minor change in antigenic structure as mutations accumulate. Antigenic drift occurs with all four inuenza viruses—A, B, C, and D. The surface antigens sometimes can change drastically, causing an antigenic shift, resulting in a new H or N antigen. Antigenic shift is associated only with inuenza A virus. There are two mechanisms of antigenic shift. The rst is genetic reassortment of the eight ssRNA strands of two separate inuenza strains. Pigs have receptors for both avian and human inuenza viruses as well as swine inuenza viruses and can be co-infected with all three types of viruses. Reassortment occurs when the genomes of different inuenza viruses combine into a single virion, resulting in a new strain of inuenza virus. The second mechanism is an adaptive mutation, in which a novel virus slowly adjusts and becomes transmissible from a mammalian (including human) host. Shifts result in novel strains of inuenza virus, so the human population is likely to have little or no historic exposure or resistance to the new strain, which greatly increases the risk of pandemics. Three major shifts occurred during the 20th century. Infection with inuenza A virus (H1N1), the Spanish u, appeared in 1918 to 1919. H1N1 was the predominant strain until a shift to inuenza A virus (H2N2) occurred in 1957 to 1958. This shift resulted in the Asian u pandemic. In 1968, another shift occurred, and a pandemic strain of inuenza A virus (H3N2) resulted in the Hong Kong u. The dominant strains of inuenza A virus since 1977 have been inuenza A viruses H1N1 and H3N2. In 1998, an outbreak of infection with inuenza A virus (H5N1), the avian u, appeared in poultry in Hong Kong. At least 18 humans acquired the disease via contact with birds that year, and 6 deaths were reported. H5N1 is a highly pathogenic avian inuenza (HPAI) virus; historically, most human infections are acquired after direct or close contact with infected birds. The H5N1 inuenza virus appeared in ocks in parts of Asia in 2004 and again in 2005, resulting in large poultry losses in Japan, Cambodia, China, Vietnam, and nearby countries. Some humans were infected during these poultry outbreaks. Again, the incidence of human disease was low, but the mortality rate was high, although it is possible that some mild cases went undiagnosed. Studies documented wide dissemination of inuenza A virus (H5N1) throughout Asia because of migrating birds. By early 2006, inuenza A virus (H5N1) had been isolated from birds in Turkey, Greece, Italy, Germany, Iran, Iraq, Nigeria, and many other countries. In April 2013, a novel avian u caused by inuenza A virus (H7N9) was reported in China. Only a few cases were reported initially, but 5 of the 11 human cases were fatal. Human-to-human transmission was not

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documented. The potential for the avian viruses to adapt to the human host, through genetic reassortment or adaptive mutation, remains an important concern for future inuenza seasons. In the spring of 2009, a highly infectious novel form of inuenza, swine u, caused by inuenza A virus (H1N1) emerged. It was rst reported throughout Mexico and forced school closings and cancellation of sporting events. By October 2009, the WHO had received reports of more than 375,000 human cases and 4525 deaths. Because many countries stopped counting individual cases, the actual number was much larger. The outbreak quickly reached phase 6, which is the WHO denition of a pandemic. This was the rst inuenza pandemic in 40 years. In April 2010, the WHO announced that the H1N1 inuenza virus had moved into the post-pandemic period. H1N1 was found to contain RNA from avian, human, and porcine strains of the virus. In 2011, human infections caused by inuenza A H3N2v, a nonhuman inuenza virus variant normally found in swine, were reported. When viruses that normally circulate in animals infect humans, they are termed variant viruses. This H3N2v virus was rst detected in January 2011, and it had genes from avian, porcine, and human viruses and the 2009 H1N1 pandemic virus M gene. Because the H and N antigens of inuenza A virus continually change, the CDC and the WHO make recommendations for the composition of the quadrivalent inuenza vaccine several months before the inuenza season begins. The current U.S. vaccine usually contains two different inuenza A virus strains, H1N1 and H3N2, and two inuenza B virus strains. Inuenza B virus infections, which also can occur seasonally, are usually less common than inuenza A virus infections, although epidemics of inuenza B virus infections can occur every few years. It is highly recommended that persons with reduced immune status (e.g., those with HIV, those with diabetes, and older adults) be vaccinated annually with the inactivated virus vaccine to prevent infection, because it can be rapidly fatal in these populations. A pediatric version for children 6 to 35 months is available. The use of an attenuated quadrivalent intranasal vaccine is only recommended for individuals between 2 and 49 years of age who are in good health. Inuenza C virus can cause mild upper respiratory tract illness in humans, indistinguishable from the common cold. Its genome consists of seven ssRNA segments, lacking the gene coding for neuraminidase, as in inuenza A and B viruses. Studies have shown inuenza C virus to be more stable genetically compared with inuenza A virus, and although reassortment does occur in the former, it is less prone to major changes in infectivity. The inuenza D virus was isolated from a pig in Oklahoma in 2011. Subsequently, it has been isolated from other animals. Little is known about its likelihood to cause human infections. Inuenza viruses are spread through aerosol inhalation. Infection may also be spread by handling fomites. Viruses attack the ciliated epithelial cells lining the respiratory tract, causing necrosis and sloughing of the cells. The incubation period is 1 to 4 days. Although asymptomatic infections can occur, infections are usually characterized by cough, sore throat, rapid onset of malaise, fever, chills, myalgia, and often a nonproductive cough. The fever can be as high as 41° C (105.8° F). Infected patients are ill for as long as 7 days, and

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convalescence may require more than 2 weeks. Inuenza can also cause a fatal viral pneumonia. Complications include secondary bacterial pneumonia. The best specimens for diagnosis are nasopharyngeal swabs, washes, or aspirates collected early in the course of the disease. Flocked swabs (Copan Diagnostics, Corona, CA) are reported to collect signicantly more epithelial cells from the nasopharynx compared with rayon swabs. Specimens should never be frozen. Several rapid kits are commercially available for the diagnosis of inuenza in about 30 minutes. Some of these kits are of low complexity and are waived by the CLIA. Some kits can detect and distinguish between inuenza A and B viruses, but some cannot distinguish between them or detect only inuenza A virus. These tests are valuable in outpatient settings, and their low cost and ease of use make them ideal for quick screening. Inuenza virus can also be identied in respiratory secretions by using DFA, EIA, and immunochromatographic assays. Nucleic acid–based assays are also used for the detection of inuenza viruses, with the most common method being RT-PCR. These assays are fast becoming the method of choice for detection. Some can be completed in less than 30 minutes, have a low cost, and are easy to perform. They can be used as point-of-care tests, thus improving patient care. Inuenza viruses grow in the amniotic cavity of embryonated chicken eggs and various mammalian cell culture lines, such as PMK and MDCK cells. Inuenza-infected culture cells adsorb RBCs, a feature that can be used to detect positive cell cultures. Positive cultures can be detected rapidly using IF staining of infected monolayers grown in shell vials or at-bottomed wells of microtiter plates. The antiviral drugs amantadine and rimantadine can prevent infection or reduce the severity of symptoms if administered within 48 hours of onset of infection. These antiviral drugs are effective only against inuenza A virus. A newer class of antivirals, termed neuraminidase inhibitors, is available. These agents, such as zanamivir (Relenza) and oseltamivir (Tamiu), are more expensive compared with amantadine but provide coverage against infections by inuenza A virus and inuenza B virus. Current CDC guidelines highly recommend both zanamivir and oseltamivir as primary treatments in conrmed cases of inuenza. There has been signicant resistance to amantadine by inuenza A virus (H3N2), so it is not recommended for treatment of infection by this virus. There have been some reports of resistance to oseltamivir by inuenza A virus (H1N1), but use of this agent is still recommended as a primary treatment.

Paramyxoviridae Parainuenza viruses Several genera belong to the family Paramyxoviridae, including Morbillivirus, and Rubulavirus. Four types (1 to 4) of PIVs cause disease in humans. Human PIV-1 and PIV-3 belong to the genus Respirovirus; PIV-2 and PIV-4 belong to the genus Rubulavirus. PIVs are enveloped helical RNA viruses with two surface antigens: hemagglutinin-neuraminidase (HN) antigen and fusion (F) antigen. HN antigen is the viral adhesion molecule; F antigen is responsible for the fusion of the virus to the cell and of one infected cell to another infected cell. PIV-1 infections occur most often in the fall every other year, and the incidence of PIV-2 is generally lower than that of

PIV-1 and PIV-3. PIV-2 is seen every 2 years alternating with PIV-1. PIV-3 occurs almost every year in spring and summer and can been seen yearlong in temperate climates. PIV-4 is seldom isolated, but routine testing is not readily available. PIVs are a primary cause of respiratory disease in young children. PIV-1 and PIV-2 cause the most serious illnesses in children between 2 and 4 years of age. PIV-1 is the primary cause of croup (laryngotracheobronchitis) in children. PIV-3 causes bronchiolitis and pneumonia in infants and is second in importance only to RSV. PIV-4 generally causes mild upper respiratory tract infections. The viruses are spread through respiratory secretions, aerosol inhalation, and direct contact. Infection of the cells in the respiratory tract leads to cell death and an inammatory reaction in the upper and lower portions of the respiratory tract. Rhinitis, pharyngitis, laryngotracheitis, tracheobronchitis, bronchiolitis, and pneumonia can result. Direct examination of nasopharyngeal secretions by IF can give rapid results. Some newer nucleic acid assay panels now include PIV-1, PIV-2, and PIV-3, which aid in treatment and epidemiology efforts. Most of these newer PCR technologies are on panels that include several other respiratory pathogens. Serologic assays are more valuable for epidemiologic studies than for diagnostic purposes. The best specimens for viral culture are aspirated secretions and nasopharyngeal washes. Specimens for viral isolation should be taken as early in the illness as possible, kept cold, and inoculated into PMK cells or LLC-MK2 cells. The viruses can be identied by using hemadsorption, IF, or EIA techniques. Aerosolized ribavirin can be used to treat infection. No vaccines are available, and infection control measures like those for RSV are used to prevent spread in health care facilities.

Mumps virus Mumps virus is related to PIVs and is in the genus Rubulavirus. It is an enveloped virus with HN and F surface antigens. Mumps virus, which has a global distribution, is spread through droplets of infected saliva. It causes an acute illness producing unilateral or bilateral swelling of the parotid glands, although other sites, such as the testes, ovaries, and pancreas, can be infected. The virus infects primarily children and adolescents and usually results in long-lasting immunity. The primary infection of the ductal epithelial cells in the glands results in cell death and inammation. A vaccine effective in controlling the disease is available. Two doses are recommended for better immunity—the rst at 12 to 15 months and the second at 4, 6, 11, or 13 years of age. The annual incidence of mumps in the United States varies. From 2000 to 2020, cases ranged from about 200 to over 6000. Only 154 cases were reported in 2021. Large multistate outbreaks of over 6000 mumps cases were documented in 2006, 2016, and 2017. The reasons for the outbreaks remain largely unknown. They have occurred in communities of individuals who received one or two vaccines. Outbreaks are generally seen in close-contact settings. During the outbreak of 2016 to 2017, most cases (nearly 3000) occurred in a close-knit community in northwest Arkansas. From 2009 to 2010, another outbreak, with 3502 cases in a New York camp, was reported and seemed to conrm waning immune protection by the vaccine. The mumps virus can be isolated from infected saliva and swabbing of the Stensen duct, from 9 days before onset of symptoms until 8 days after parotitis appears. The virus, which is relatively fragile, can also be recovered from urine

Single-stranded RNA viruses

and CSF. Specimens may be examined directly by using IF and EIA methods. Studies have shown viral isolation using shell vial cultures of Vero or LLC-MK2 cells to be more successful than those with HEp2 or HeLa cell lines. Isolates can be identied by hemadsorption inhibition, IF, and EIA tests. Paired sera can be tested for mumps antibody by EIA, IF, and hemagglutination inhibition tests. Paired sera taken at as small an interval as 4 to 5 days can demonstrate a diagnostic or fourfold increase in titer. Cross-reactions between soluble and viral antigens can confuse the interpretation of serologic results. Virus isolation is preferable, although primary health care providers rarely have trouble recognizing mumps clinically.

Measles virus The measles virus is an enveloped virus classied in the genus Morbillivirus. It is found worldwide; in temperate zones, epidemics occur during winter and spring. At one time, measles (rubeola) was the most common viral disease in children in the United States. An average of 500,000 cases of measles were reported annually in the 1950s, with an average of 500 deaths. Immunization programs began in the United States in 1963, and the reported number of cases decreased to fewer than 1500 by 1983. A reemergence of measles occurred in 1989 to 1991, attributed to immigration and lack of vaccination. The decision to administer a second dose of vaccine to school-age children has drastically reduced the incidence of measles in the United States. From 2010 to 2021, the number of cases has ranged from 13 to 667. The WHO reported a 50% surge in measles cases from 2016 to 2019, with 869,770 total cases and 207,500 deaths. This is the highest number of reported cases since 1996. Measles is highly contagious and spreads by aerosol. Initial replication takes place in the mucosal cells of the respiratory tract. The measles virus then replicates in the local lymph nodes and spreads systemically. The virus circulates in T and B cells and monocytes until eventually the lungs, gut, bile duct, bladder, skin, and lymphatic organs are involved. After an incubation period of 7 to 10 days, there is an abrupt onset, with symptoms of sneezing, runny nose and cough, red eyes, and rapidly increasing temperature. About 2 to 3 days later, a maculopapular rash appears on the head and trunk. Koplik spots, lesions on the oral mucosa consisting of irregular red spots with a bluish-white speck in the center (Fig. 29.22),

Fig. 29.22 Patient presenting on the third pre-eruptive day with Koplik spots indicative of the onset of measles. (Courtesy Centers for Disease Control and Prevention, Atlanta, GA.)

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generally appear 2 to 3 days before the rash and are diagnostic. Complications such as otitis, pneumonia, and encephalitis may occur. A progressive, highly fatal form of encephalitis can occur but is rare. In developing countries with malnutrition and poor hygiene, measles can have a high fatality rate. Infection confers life-long immunity. An effective attenuated vaccine is available and recommended for all children. Measles is easily diagnosed clinically, so few requests for laboratory identication are made. The virus is fragile and must be handled carefully. The specimens of choice are from the nasopharynx and urine, but the virus can be recovered from these sources only in the early stages of infection. The virus grows in PMK cells, causing the formation of distinctive spindle-shaped or multinucleated cells. Virus isolates can be identied by using serum neutralization, EIA, or IF tests. Serologic diagnosis of measles is accomplished by demonstrating measles-specic IgM in the specimens collected during the acute phase of the disease. Nucleic acid testing should be considered for diagnostic use if IgM testing is compromised by the recent use of measles virus–containing vaccine as part of a routine vaccination or in response to a suspected outbreak.

Respiratory syncytial virus RSV, a member of the genus Orthopneumovirus, causes croup, bronchitis, bronchiolitis, and interstitial pneumonia. It is the most common cause of severe lower respiratory tract disease among infants and young children worldwide. Almost one half of all infants are infected by RSV during their rst year of life, and almost all have been exposed to RSV by 2 years of age. The CDC estimates 2.1 million outpatient visits among children younger than 5 years of age, 58,000 hospitalizations, and 14,000 deaths annually. Because infection does not confer complete immunity, multiple infections can occur throughout life and can be severe in older adults, the immunocompromised, and those with cardiac and respiratory problems. It is estimated that 24 of 1000 children with RSV infection will be hospitalized. For this reason, healthcare-associated RSV is a problem in many medical facilities. Recommendations to reduce the risk of healthcare–associated spread include testing hospital personnel and infants with upper respiratory tract infections for RSV, isolating infants with RSV infection, following good handwashing and personal protective equipment practices, limiting visitation, and organizing patients and staff members into cohorts. RSV can be a signicant cause of morbidity and death in older patients. With a rapidly growing aging population, RSV in elder care facilities is becoming a signicant problem in the United States. Unlike the bronchiolitis caused by RSV in children, pneumonia often develops in adults. The virus spreads mostly through large-particle droplets and contact with fomites rather than through inhalation of small aerosols. The virus may be carried in the nares of asymptomatic adults. RSV infections occur in yearly outbreaks that last 2 to 5 months and usually appear during winter or early spring in temperate zones. RSV can be identied in specimens from nasopharyngeal swabs and washes by using DFA or EIA. Rapid antigen detection kits are also available for RSV, but they have a relatively low sensitivity. Several NAATs are available for RSV testing, with most taking less than 1 hour to provide results. These are normally part of a multiplex assay for other respiratory

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pathogens. Because the virus is extremely fragile, recovering it from cultures is difcult, but culture remains the gold standard. It is important to collect samples within the rst few days of illness. In about 50% of cases, antigen detection and viral cultures are negative at 1 week. Specimens for culture must be kept cold but cannot be frozen. RSV grows readily in continuous epithelial cell lines, such as HEp2, forming syncytia. It also grows in PMK and human diploid fetal cells. Once the CPE is detected, RSV can be identied by using IF, EIA, and serum neutralization tests. The antiviral compound ribavirin is approved for treatment of RSV infection. However, some controversy regarding the efcacy of ribavirin therapy still exists. A monoclonal antibody, palivizumab (PVZ), which blocks RSV entry into the host cell, is also approved for RSV treatment and prophylaxis in high-risk children. Often, ribavirin is recommended to be used in combination with PVZ. No vaccine for RSV infection is available.

Human metapneumovirus Human metapneumovirus (HMPV) was rst described in children with previously virus-negative cultures in 2001. Infected children display many clinical symptoms that mimic infections with RSV, inuenza virus, and PIV. Often, these diseases are ruled out early in diagnosis, which leads to a presumptive identication of HMPV. Serologic and RNA sequence studies have shown that the virus is found worldwide in all age groups. Most children have been exposed by the time they reach 5 years of age. One half of the lower respiratory tract infections seen in children in the rst 6 months of life are caused by this virus. Clinical disease ranges from mild upper respiratory tract infection to acute lower respiratory tract infection and includes fever, nonproductive cough, sore throat, wheezing, congestion, shortness of breath, and lethargy. HMPV infections usually occur in the winter months, but outbreaks have been documented during summer. HMPV infections usually occur in children. A survey in Finland among 1338 children younger than 13 years of age found that 47 (3.5%) with respiratory illness were positive for HMPV. The highest concentration of illness (7.6%) was seen in children younger than 2 years of age. Co-infections with another virus, including enterovirus, rhinovirus, inuenza virus, and PIV, were detected in eight (17%) of the infected children. HMPV has also been documented to cause outbreaks in long-term care facilities. In the summer of 2006, HMPV affected 26 residents and 13 staff members in a 171bed California long-term care facility. All affected residents had an underlying medical condition; two were hospitalized, but none died. This outbreak indicates a year-round risk of infection in institutionalized older adults. Treatment for HMPV infection is mostly supportive. Specimens for detecting HMPV can be collected from the nasopharynx by using ocked swabs placed in transport media and transported to the laboratory for culture or molecular analysis. Specimens should be processed immediately but can be stored at 4°C for up to 72 hours. DFA and RT-PCR assays are used for identication, although DFA has lower sensitivity. The respiratory viral panel assay from Luminex Molecular Diagnostics (Toronto, Canada) claims 100% sensitivity and 98.2% specicity for identication of HMPV in clinical specimens. HMPV grows slowly in standard cell culture lines, such as monkey kidney and A549 cell lines.

BOX 29.1 Genera within the family Picornaviridae of human

signicance Genus Aphthovirus Foot and mouth disease virus Genus Enterovirus Enteroviruses species A to D (about 106 types) Polioviruses types 1 to 3 Echoviruses Human coxsackieviruses Rhinovirus species A to C (about 165 types) Genus Parechovirus Parechovirus species A and B (about 24 types) Genus Hepatovirus Hepatitis A virus

Picornaviridae Picornaviridae is one of the largest families of viruses, with more than 280 members. It contains many important human and animal pathogens. Four genera with human clinical signicance belong to the family Picornaviridae: Aphthovirus, Enterovirus, Hepatovirus, and Parechovirus (see Box 29.1). The genus Hepatovirus includes HAV. This virus is discussed in detail in the “Hepatitis Viruses” section later in this chapter.

Enteroviruses These small naked viruses cause various conditions, including fever of unknown origin, aseptic meningitis, paralysis, sepsis-like illness, myopericarditis, pleurodynia, conjunctivitis, exanthemas, pharyngitis, and pneumonia. Enteroviruses have also been implicated in early-onset diabetes, cardiomyopathy, and fetal malformations. Most serotypes of the enteroviruses are distributed worldwide. In temperate zones, enterovirus epidemics occur in summer and early fall. Enterovirus infections are more prevalent in areas with poverty, overcrowding, poor hygiene, and poor sanitation. Viruses are spread via aerosol inhalation, the fecal-oral route, and fomites. The portal of entry is the alimentary canal via the mouth. The viruses replicate initially in the lymphoid tissues of the pharynx and gut. Viremia can result in the virus spreading from these locations to the spinal cord, heart, and skin. The clinical disease caused by enteroviruses can be neurologic, respiratory, or cardiac, depending on viral spreading and the immune status of the host. Enterovirus infections most often cause mild nausea and diarrhea in adults. However, disease can be much more severe in neonates because of their immature immune system. The polioviruses tend to infect the CNS and cause paralysis in a small percentage of infected individuals. The viruses destroy their host cells. In the intestines, damage is temporary because the cells lining the gut are rapidly replaced. In contrast, neurons are not replaced, which results in neuron death and permanent paralysis. No vaccines are available for enteroviruses other than poliovirus. Good personal and health care facility hygiene and proper sanitation can reduce the incidence of enterovirus infections. Poliovirus vaccines of attenuated or inactivated viruses are available. Since 1988, the polio vaccine program has been crucial to the WHO’s effort to eradicate polio worldwide. In countries where polio is considered to have endemic rates of incidence, there has been a steady decrease since

Single-stranded RNA viruses

the program began. In 1988, 125 countries reported endemic rates; by 1999, the number of such countries had decreased to 30, and in 2013 to only 3: Afghanistan, Nigeria, and Pakistan. In 2020, only two countries were considered endemic for wildtype poliovirus: Afghanistan and Pakistan. Unfortunately, interruptions in vaccine programs has caused an increase in cases. The WHO reported 175 cases in 2019, up from only 33 in 2018; transmission continues to be widespread in Pakistan. In 2020, there were 1107 cases of circulating vaccine-derived poliovirus infections; 443 were in the two endemic countries, and 664 were in nonendemic countries. Enteroviruses can be cultured from pharyngeal specimens immediately before the onset of symptoms and for 1 to 2 weeks afterward; the viruses can be isolated from feces for as long as 6 weeks thereafter. However, ideally, specimens should be obtained early in the course of the infection. Specimens from the throat, feces, rectum, CSF, and conjunctiva are recommended. Polioviruses, type B coxsackieviruses, and echoviruses grow readily in several cell lines, including PMK, continuous human and primate, and human fetal diploid broblast lines. The high-numbered enteroviruses (68 to 71) require special handling. The CPE appears quickly and is readily identiable. Enteroviruses have no group antigen, so they must be identied individually by a serum neutralization test. The WHO distributes pools of enterovirus antisera that allow identication by neutralization patterns in the antisera. The CPE and resistance to detergent, acid, and solvents constitute a presumptive diagnosis of enterovirus infection. Hand, foot, and mouth disease (HFMD) is caused primarily by coxsackievirus types A5, A10, and A16 and occasionally by enterovirus type 71. HFMD generally occurs in young children. Outbreaks of HFMD caused by EV71 have been reported, mostly in children in East and Southeast Asia, with some cases seen in Cambodia and China, resulting in the death of hundreds. It is spread by fomites or via the oral-fecal route. A mild prodromal phase can develop, with malaise, headache, and abdominal pain. Small, painful sores suddenly appear on the tongue, buccal mucosa, and soft palate. Simultaneously, a maculopapular rash appears on the hands, feet, and buttocks, followed by bullae on the soles of feet and the palms of hands. The lesions regress in about 1 week. If a rash develops, it is transient. The virus can be isolated from specimens from swabs of the mouth and bullae. Coxsackievirus A16 grows in PMK and human diploid broblast cells and can be identied by serum neutralization tests. More than 150 serotypes of rhinoviruses exist, and they are the major cause of the common cold. Most people experience two to ve colds each year, and almost 50% of these colds are caused by the rhinoviruses. Rhinovirus infections occur throughout the year, but their incidence increases in winter and spring. Transmission is primarily via aerosols, but contact with secretions and fomites can also cause infection. Rhinoviruses infect the nasal epithelial cells and activate the mediators of inammation. Symptoms include a profuse watery discharge, nasal congestion, sneezing, headache, sore throat, and cough. In severe cases, bronchitis and asthma can result. Unfortunately, no cure for the common cold has been found. Treating symptoms and reducing the spread of the virus in the household is the typical response. Both natural and recombinant interferons have been shown to be effective in preventing infection and illness when given intranasally over short periods. However, prolonged administration has resulted in adverse effects, such as nasal irritation, ulceration, and bleeding.

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Case in point A 36-year-old man was admitted to the hospital after presenting at the emergency department with a self-reported, 7-month history of numbness and weakness in his right leg. He had lost 25 lb, was experiencing bowel incontinence, and had been unable to urinate for 3 days. Two years previously, the patient had been diagnosed with HIV infection. A physical examination demonstrated bilateral lower extremity weakness, and his reexes were slowed throughout his body. KS lesions were noted, especially on the lower extremities, along with thrush and herpes lesions in the perianal region. The patient had no fever, and magnetic resonance imaging ruled out spinal cord compression. The patient had a history of intravenous drug abuse, chronic diarrhea for 1.5 years, KS for 2 years, and pancytopenia for several weeks. The patient had large right arachnoid cysts of congenital origin. No previous laboratory report indicated infectious agents in the CSF. Meningitis was suspected, and the patient was admitted with a diagnosis of polyradiculopathy (neuropathy of the spinal nerve roots) secondary to AIDS. Blood and CSF were collected. Although numerous white blood cells were found, the CSF produced no growth on routine bacteriologic culture. The blood cultures were also negative. Acyclovir was administered after culture results were received.

Retroviridae The family Retroviridae contains several subfamilies, including Oncovirinae and Lentivirinae. The retroviruses have a unique mode of replication; they require an RNA-dependent DNA polymerase (reverse transcriptase) to synthesize DNA from the RNA genome. The human T-lymphotropic viruses HTLV-1, HTLV-2, and HTLV-5 belong to the subfamily Oncovirinae. These viruses are not cytolytic but are associated with several leukemias, sarcomas, and lymphomas. HIV belongs to the subfamily Lentivirinae. Although some groups of individuals, mostly in West Africa, are infected by HIV-2, it is the impact of HIV-1 that continues to be felt around the world. HIV causes AIDS. HIV-1 was identied rst and is responsible for the AIDS pandemic. The WHO estimates about 37.7 million people were living with HIV/AIDS at the end of 2020 and approximately 1.5 million new cases were diagnosed. Approximately 680,000 HIV/AIDS–related deaths occurred in 2020. The region most severely affected by HIV/AIDS is sub-Saharan Africa, which has approximately 25.4 million patients with HIV infection, accounting for 67% of the total worldwide incidence. Children share this burden; in 2020, approximately 1.7 million children younger than 15 years of age had HIV/AIDS, and about 150,000 cases of newly infected children were reported. It was reported that at the end of 2019, an estimated 1.2 million people 13 years of age and older had HIV/AIDS in the United States, and there were 36,801 new HIV cases. Approximately 14% are not aware of their infection. Of the new cases, approximately 81% were men. The greatest number of persons who contract HIV/AIDS are MSM. This group accounted for 70% of the new cases. The virus is transmitted via blood and exchange of other body uids. HIV is cell associated, so fewer viruses are found in cell-free plasma than in whole blood, and even fewer viruses are found in saliva, tears, urine, or breast milk. HIV is not highly contagious, and normal, social, nonsexual contact

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29 gp41

Clinical virology

p18 Lipid envelope

BOX 29.2 Common opportunistic infections and cancers in

patients with acquired immunodeciency syndrome • • • • • • • • • •

p24 RNA Reverse transcriptase

Fig. 29.23 Human immunodeciency virus (HIV).

• • • • • •

poses no threat to individuals. High risk for contracting the virus includes unprotected sex with multiple partners, IV drug abuse, infusion of blood and blood products, and presence of the virus in a pregnant woman who passes it to the fetus during pregnancy. Individuals with ulcerative sexually transmitted infections (STIs), e.g., syphilis, genital herpes, chancroid, are at greatest risk. Donor blood and blood products are screened for HIV, which substantially reduces the risk. HIV is a spherical virus, with a three-layer structure (Fig. 29.23). In the center are two identical copies of ssRNA and reverse transcriptase surrounded by an icosahedral capsid. The nucleocapsid is enclosed by a matrix shell to which an envelope of host cell origin is attached. Inserted into the viral envelope are viral glycoprotein (gp) trimers or spikes. The diagnostically important HIV antigens are the structural proteins p24, gp4l, gpl20, and gpl60. Once HIV enters the body, the primary target cells are the CD4+ T cells, monocytes, and macrophages. Acute infections are generally mild and can resemble infectious mononucleosis. The individual will then enter a period of clinical latency, and even though the virus is replicating rapidly in lymphoid tissues, the virus is not detectable in the bloodstream, and the patient remains asymptomatic. Eventually lymphopenia results, with the greatest loss in the CD4+ T-cell population. Healthy individuals have CD4+ T-cell counts of at least 1000/mm 3, whereas patients with HIV/AIDS can have counts lower than 200/mm3. Lymph nodes become enlarged and hyperplastic. The virus destroys the T-helper cells, which are critical in host immune response to infectious agents. The patient begins experiencing several chronic and recurrent infections (Box 29.2). As the disease progresses, the CD4+ cell count continues to decline, and the severity of opportunistic infections increases. The patient can also develop virus-induced cancers, such as KS. Death usually occurs because of opportunistic infections, although HIV-1 itself can directly cause encephalitis and dementia. In adult patients living in developed countries, the average length of time from HIV infection to development of AIDS is about 10 years. About 20% develop AIDS within 5 years, and fewer than 5% have an asymptomatic HIV infection for

Candidiasis of the respiratory tract Coccidiomycosis Cryptococcal meningitis Cryptosporidiosis with persistent diarrhea Cytomegalovirus infections of organs other than the liver, spleen, or lymph nodes Histoplasmosis Persistent herpes simplex virus infections including the respiratory tract Kaposi sarcoma or lymphoma of the brain in patients older than 60 years of age Oral hairy leukoplakia Lymphoid interstitial pneumonia, pulmonary lymphoid hyperplasia, or both in children younger than 13 years of age Invasive cervical cancer Mycobacterium avium complex, Mycobacterium kansasii infection Pneumocystis jirovecii pneumonia Progressive multifocal leukoencephalopathy Toxoplasmosis of the brain in infants younger than 1 month of age Wasting disease

Case check 29.2 Clinical manifestations of HIV infection include CNS involvement, opportunistic infections, and tumors. CNS involvement is often seen in HIV-associated dementia complex but can manifest itself as other neurologic problems, such as loss of bowel or bladder control and weakness in the extremities. KS develops from the cells lining the lymph or blood vessels. The resulting lesions are red, purple, or brown blotches or tumors on skin. Other than their appearance, the skin lesions of KS often cause no symptoms. However, in some cases, they may cause painful swelling and be more painful when found in the legs, groin, or skin around the eyes. KS in such sites as the liver, lungs, or digestive tract may be life-threatening because of abnormal bleeding or difficulty breathing.

periods longer than 10 years. The rate at which the virus multiplies in the host is related to the onset of AIDS. This rate can be measured with an HIV viral load assay, a quantitative gene amplication technique that measures the amount of HIV-1 RNA in the plasma. Laboratory diagnosis of HIV infection is generally based on demonstration of anti-HIV antibodies and, in some cases, detection of viral antigens and RNA. Serologic tests suffer from the disadvantage of the window period, the time between infection and the ability to detect antibodies (seroconversion). HIV antibodies are normally produced within a few weeks after infection. The window period varies depending on the assay, but the fourth-generation assays have a median window period of 18 days. Several assays are commercially available as screening tests using different methods, including EIA and IF. The early diagnostic kits, referred to as rst-generation screening tests, used puried viral lysate as antigens. The second-generation tests used recombinant viral proteins, thus improving performance. The third-generation tests relied on the double-antigen sandwich assay. In this procedure, viral

Single-stranded RNA viruses

antigen attached to a solid-phase bound antibody to HIV from the patient’s serum. Labeled HIV antigen was then added, captured by the patient’s antibody, and measured. Fourthgeneration kits detect antibody and HIV-1 p24 antigen. The rst fth-generation assay is a multiplexed screening test that detects and differentiates three HIV analyte markers: antiHIV-1 antibodies, anti-HIV-2 antibodies, and the p24 antigen. The immunoassays are generally based on EIA or chemiluminescent immunoassay methods. By detecting antigen, early infections could be identied before antibody is produced. These newest assays are considered the standard of care for diagnosis of HIV infection. Several rapid assays screen for HIV infection by using serum, plasma, and saliva. By 2012, the FDA had approved six rapid tests for the diagnosis of HIV infection: OraQuick Advance rapid HIV-1/2 antibody test (OraSure, Bethlehem, PA); Uni-Gold Recombigen HIV test (Trinity Biotech, Wicklow, Ireland); Reveal G2 rapid HIV-1 antibody test (MedMira, Halifax, Canada); Multispot HIV-1/2 rapid test (Bio-Rad Laboratories, Hercules, CA); and Clearview HIV1/2 and Clearview Complete HIV-1/2 (Inverness Medical Professional Diagnostics, Princeton, NJ). OraQuick for whole-blood and oral uid specimens, Clearview Complete for whole blood, and Uni-Gold for whole-blood samples have waivers from the CLIA. Currently, the FDA has approved one home collection kit, the Home Access HIV-1 test system (Home Access Health Corporation, Maria Stein, OH). In 2012, the rst FDA-approved in-home HIV test kit was the OraQuick In-home HIV Test (OraSure Technologies), and by 2016, the FDA had approved multiple tests in each category. Current CDC guidelines (published in 2014 and revised in 2018) recommend using a screening test that detects HIV-1 and HIV-2 antibodies and p24 antigen. No further testing is required if the test result is negative. Any reactive result on a screening test should be tested with a supplemental antibody immunoassay that differentiates HIV-1 antibodies from HIV-2 antibodies. If both tests are reactive, the specimen should be interpreted as positive for HIV. A specimen positive in the initial screening test and negative in the supplemental test should be tested with a NAAT. A positive NAAT indicates an acute HIV infection. A specimen with a negative NAAT should be referred for testing with a different validated supplemental HIV-2 test (antibody test or NAAT) or repeat testing in 2 to 4 weeks. Other immunologic markers of HIV infection are listed in Box 29.3 The western blot has been the reference method for HIV diagnosis for many years. Viruses grown in cell cultures are lysed, and the proteins are separated by electrophoresis and transferred to nitrocellulose. Antibodies to several HIV proteins and glycoproteins can be detected. For a positive result, CDC guidelines require detection of at least two antibodies

BOX 29.3 Important immunologic markers for acquired immu-

nodeciency syndrome • Steady decline in number of CD4+ T cells • Depression of the CD4+-to-CD8+ cell ratio to less than 0.9 (reference value, ≥1.5) • Functional impairment of monocytes and macrophages • Decreased natural killer cell activity • Anergy to recall antigens in skin tests

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to the three proteins p24, gp41, and gp120/160. Western blot results should also be followed by tests for anti-HIV-2 antibodies (Fig. 29.24). Because these viruses readily develop resistance to drugs, HIV infection is often treated with combination therapy. Highly active antiretroviral therapy (HAART) involves aggressive combination therapy soon after HIV infection is diagnosed, ideally within 7 days. In hospital exposures, a similar strategy is used if a health care worker is accidentally exposed to the virus. Aggressive therapy is initiated after the exposure, and this signicantly reduces the risk of contracting an infection. Despite aggressive therapy, a cure is not achieved. In 2008, scientists reported that an American living in Berlin (the Berlin patient) was cured of HIV infection. The patient received a bone marrow transplant from a donor with a CCR5 mutation (CCR5Δ32). CCR5 is a coreceptor for HIV adhesion. The mutation prevents virus infection. Subsequently, two additional patients receiving similar treatment were also reported to be cured, the most recent in 2022. Additional reports of cures in patients receiving HAART have been made; however, these have not been substantiated. Several classes of antiviral drugs are approved for treatment. Nucleoside reverse transcriptase inhibitors (NRTIs) were the rst class of retroviral drugs developed and are incorporated into viral DNA; these include adefovir, azidothymidine, dideoxyinosine, d4T (stavudine), 3TC (lamivudine), and tenofovir. NRTIs inhibit the conversion of nucleoside analogues in the body to nucleotide analogues. Non-NRTIs attach to reverse transcriptase, preventing conversion of RNA to DNA; examples include delavirdine, nevirapine, and

Fig. 29.24 Human immunodeciency virus immunoblot. Reactive protein (p) and glycoprotein (gp) bands appear as purplish lines across the strip. Proteins with higher molecular weights appear at the top of the strip. Structural and nonstructural proteins are given RNA structural genome codes: GAG for group-specic antigens; POL for polymerase; and ENV for envelope. ENV codes for glycoprotein precursors: gp160, gp120, and gp41 to gp43. POL codes for p65, p51, and p31. GAG codes for p55, p24, and p17. Results are negative, indeterminate, or positive based on the pattern on the strip. Positive corresponds to reactivity to two or more of the following antigens: p24, gp41, or gp120/gp160. Indeterminate corresponds to the appearance of one or more bands in a pattern that does not satisfy the positive criteria. Negative corresponds to the absence of any band on the strips. (Courtesy Patricia A. Cruse.)

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efavirenz. Other classes of antiviral drugs include protease inhibitors (e.g., ritonavir, saquinavir, indinavir, amprenavir), fusion inhibitors (e.g., enfuvirtide), integrase strand transfer inhibitors (e.g., dolutegravir, elvitegravir, raltegravir), and chemokine receptor antagonists (e.g., maraviroc). HAART includes combinations, such as two NRTIs combined with a protease inhibitor. HIV viral load assays can predict therapeutic efcacy. In different studies, suppression of HIV RNA levels to less than 5000 copies of RNA/mL for up to 2 years was correlated with an increase in CD4+ cell counts, up to 90/mm 3. In contrast, patients with HIV RNA loads of more than 5000 copies of RNA/mL generally showed a decrease in CD4 + cell counts. Usually, these assays are performed monthly so therapy can be adjusted on an individual basis.

Case check 29.3 In HIV infection, the immune system is severely compromised, giving rise to many opportunistic infections caused by viruses, bacteria, fungi, and protozoa, as well as neoplastic disease, wasting syndrome, progressive multifocal leukoencephalopathy, and HIV encephalopathy. These complications often result in death. Meningitis occurs frequently in patients with HIV infection and can be caused by a variety of agents. In the Case in Point, the patient was treated with acyclovir for a presumed HSV meningitis and/or encephalitis. Acyclovir is an inactive nucleoside analogue that is metabolized by viral thymidine kinase into an active form, which results in premature termination of DNA during synthesis.

Rhabdoviridae Rabies is caused by several strains of viruses belonging to the genus Lyssavirus in the family Rhabdoviridae. An estimated 55,000 persons die as a result of rabies worldwide annually, with 40% of these being children younger than 15 years of age. Dogs are implicated in 99% of rabies deaths among humans. In the United States, human cases of rabies are rare, with approximately two per year. However, rabies is an emerging infection in animals, with all mammals being susceptible. Most rabies infections 40 years ago occurred in dogs, with some occurring in cats, foxes, and skunks. However, in the United States, rabies is now more closely associated with wild animals (92%), such as raccoons, skunks, foxes, and bats. Programs to vaccinate domestic animals have reduced the number of rabies cases in dogs and cats, which has, in turn, decreased the risk to humans. Humans usually acquire the rabies virus when they are bitten or scratched by animals with rabies. With the number of endemic areas increasing among wildlife, the risk of human exposure to rabies increases because of the increased likelihood of encountering a wild animal with rabies or a domestic animal that has contracted rabies from wildlife. Humans infected with the rabies virus experience a brief prodromal period of pain at the exposure site and have vague ulike symptoms. Mental status changes, such as anxiety, irritability, and depression, can also occur. After the prodromal period, patients suffer additional CNS changes, including hallucinations, paralysis, excessive salivation, hydrophobia, bouts of terror, seizures, respiratory and cardiac abnormalities, and hypertension. These symptoms are followed ultimately by

coma and death. In 2004, a Texas hospital encountered ve cases of rabies. This occurrence was a result of transplanting organ and tissues from a person who was later discovered to have been a victim of a bat bite into four patients, all of whom subsequently died because of the disease. Rabies virus is not considered a bloodborne pathogen; therefore it is likely that the spread was caused by infected nerve tissue that came with the new organs. Laboratory diagnosis of rabies involves determining whether an animal that has bitten a human has rabies. The animal is killed, and its head is removed and sent to a reference laboratory. The fastest and most sensitive method of identifying rabies virus in a specimen is by using a direct IF technique. Impression smears should be made from various areas of the brain, primarily the hippocampus, pons, cerebellum, and medulla oblongata. In living patients suspected of having rabies, skin biopsy, especially at the hairline at the back of the neck, and an impression of the cornea may be performed. The presence of rabies virus in these specimens is diagnostic, but its absence merely means that no virus is present in those specic specimens, not that the patient does not have rabies. EIAs are currently the most sensitive assays to use for serologic tests. Rabies virus can be grown in suckling or young adult mice, murine neuroblastoma, or related cell lines. Rabies cannot be successfully treated once symptoms appear. However, postexposure prophylaxis is 100% effective in preventing the disease if the bite victim is treated immediately after exposure. Postexposure prophylaxis includes vigorously cleansing the wound site, providing human anti-rabies immunoglobulin, and administering a three-injection series of the rabies vaccine. Each year, more than 15 million people worldwide receive postexposure preventive treatment. This is estimated to prevent 327,000 rabies deaths annually. Two approved human vaccine preparations are available in the United States: IMOVAX (Sano Pasteur Biologics, Canton, MA) and RabAvert (GlaxoSmithKline, Philadelphia, PA). The vaccine can be given to persons who may have been exposed to rabies, such as veterinarians, laboratory personnel, people who explore caves, and those visiting high-risk countries for longer than 30 days. Only one unvaccinated person with rabies has ever survived. A female teenager in Wisconsin developed rabies about 1 month after being bitten by a rabid bat. She was put into a coma and treated with several antiviral compounds. However, the reason for her survival is still not completely understood. Her survival resulted in a new method of rabies treatment called the Milwaukee Protocol.

Togaviridae The family Togaviridae contains the genera Alphavirus, Rubivirus, and Arterivirus. No member of the genus Arterivirus is known to infect humans. Many of the viruses in the genus Alphavirus are mosquito-borne and cause encephalitis. Eastern equine encephalitis (EEE), which causes disease in horses and humans, occurs primarily in the eastern half of the United States. From 2010 to 2020, 120 human cases were reported, with a mortality rate of about 45%. Infections can cause a range of effects, from mild ulike symptoms to encephalitis. Of those who survive, almost 50% suffer permanent CNS

Hepatitis viruses

damage. Birds are the natural reservoirs of the virus, which is spread to humans and horses via bites of mosquitoes. Because horses and humans are dead-end hosts, EEE in horses can be a predictor of human EEE cases. The western equine encephalitis (WEE) virus also causes disease in humans and horses. WEE virus causes a milder disease compared with EEE virus, and patients develop an asymptomatic or mild infection consisting of fever, headache, nausea, and mental status changes. Of young children and infants who survive, 5% to 30% will suffer permanent CNS damage. Mortality is about 3%. Since 1964, 639 cases were reported in the United States, but none have been reported since 1994. Venezuelan equine encephalitis (VEE) has caused large outbreaks of human and equine encephalitis in the Americas. Outbreaks of epizootic strains have affected up to 75,000 people during a single epidemic. Death is much less common in patients with VEE than in patients with WEE or EEE. Between 4% and 14% of infected individuals will develop neurologic disease. Infected adults often develop a ulike illness, whereas encephalitis is more commonly seen in children with VEE. Rubella virus is an enveloped virus belonging to the genus Rubivirus. It causes the disease rubella, or German measles, a mild febrile illness accompanied by an erythematous, maculopapular, discrete rash with postauricular and suboccipital lymphadenopathy. Like measles, rubella occurs most frequently in winter and spring. The diseases are so similar that as many as 50% of suspected measles cases are diagnosed as rubella. The rubella virus is transmitted via droplets. The virus is present in nasopharyngeal secretions or any secretion of infected infants, who shed the virus in large amounts for long periods. A rash starts on the face and spreads to the trunk and limbs. No rash appears on the palms and soles. About 50% of those infected with rubella virus are asymptomatic. Transient polyarthralgia and polyarthritis can occur in children and are common in adults. Rubella would be of little concern if it did not cross the placenta and spread to the fetus, which results in congenital rubella syndrome. The syndrome can cause effects ranging from birth of a normal infant to birth of a severely impaired infant, fetal death, or spontaneous abortion. Because the

743

rubella virus halts or slows cell growth, the impact on the embryo is worse when the infection develops in the earliest stages of pregnancy. An effective attenuated vaccine is available and should be administered to all children and to young women before they become sexually active. In 2004, the CDC declared the United States rubella free, meaning an absence of continuous disease transmission for 12 months or more. Currently, fewer than 10 cases are reported annually, and since 2012, all cases were acquired outside the country. Before the vaccination program begin in 1969, about 12.5 million people contracted rubella annually. It is important to collect specimens (e.g., throat swabs) the day the rash appears. Direct examination of specimens with RT-PCR has been attempted. However, not all are signicantly sensitive to be used on clinical specimens, and no protocol for detecting viral RNA has been established. Several molecular tests are being evaluated. Isolation procedures are difcult because of the lack of a cell line that exhibits cytopathic effect. Infected cells can be detected by RT-PCR. Serologic procedures are effective because any rubella antibody is presumed to be protective. The most sensitive serologic assays are the solid-phase and passive hemagglutination tests. Latex agglutination and antigen-coated RBC tests are useful but less sensitive.

Hepatitis viruses The hepatitis viruses are grouped together, not because of their structural or genetic similarities but because they share the same tissue tropism—the liver. Before the 1970s, patients with hepatitis were classied as having infectious hepatitis or serum hepatitis. Infectious hepatitis was transmitted from person to person via the fecal-oral route, and serum hepatitis resulted from transfusion of or exposure to infected blood and blood products. During the past several years eight different human hepatitis viruses have been recognized. The most clinically signicant are HAV, HBV, HCV, and hepatitis D virus (HDV) (see Table 29.7). Four additional viruses have been described: hepatitis E virus (HEV), hepatitis G virus (HGV), SEN virus, and transfusion-transmitted virus (TTV).

Table 29.7 Clinical and epidemiologic differences among HAV, HBV, HCV, and HDV Clinical Features

Hepatitis A (HAV)

Hepatitis B (HBV)

Hepatitis C (HCV)

Hepatitis D (HDV)

Incubation (days)

15–45

30–120

40–50

21–90

Type of onset

Acute

Insidious

Insidious

Usually acute

Fecal-oral

Usual

Not likely

Not likely

Not likely

Parenteral

Increasing

Usual

Likely

Usual

Other

Foodborne, waterborne

Intimate contact, transmucosal transfer

Vertical transmissionIntranasal cocaine use

Intimate contact, less efcient than for hepatitis B virus

Carrier

No

Yes

Yes

Yes

Chronic hepatitis

No

Yes

Yes

Yes

Mortality (%)

0.1–0.2

0.5–2.0

0.2–0.3

30 (chronic form)

Mode of transmission

Sequelae

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HAV and HEV are transmitted via the fecal-oral route; HBV, HCV, HDV, HGV, SEN virus, and TTV are transmitted via contact with infected blood and blood products. HBV, TTV, and SEN virus have DNA genomes, whereas the others have an RNA genome. Despite the biological and morphologic differences among the hepatitis viruses, many of the clinical symptoms caused by them are similar. Therefore differentiation based on clinical ndings should not be relied on for diagnosis. The most common signs and symptoms are fatigue, headache, anorexia, nausea, vomiting, abdominal pain (right upper quadrant or diffuse), jaundice, and dark-colored urine.

can result. The incubation period for HAV infection is approximately 1 month. After infection, individuals experience a transient viremia, after which the virus reaches the liver and replicates in hepatocytes. The virus passes into the intestine, and viral shedding begins and can persist for months. Infections in more than 90% of children younger than 5 years of age tend to be asymptomatic. In adults, symptoms can range from mild to severe prolonged hepatitis. The onset is abrupt, and patients experience fever, chills, fatigue, malaise, aches, pains, and, in some cases, jaundice. The infection is self-limiting, with convalescence possibly lasting weeks. Complete recovery can take months. HAV infection has a low mortality rate and no persistence and does not cause chronic liver damage. The most common method for laboratory diagnosis of HAV infection is to demonstrate IgM to HAV (Fig. 29.25). Isolation of HAV is not practical because it is difcult to grow in culture and tends to mutate drastically. Some reference laboratories offer in-house RT-PCR assays that can detect HAV infection before liver enzymes are elevated. Studies comparing antibody detection with RT-PCR have shown that HAV RNA can be detected much earlier after infection. Vaccination of children has the potential to reduce the incidence of HAV infection. Other vaccination target groups include people who travel to countries with endemic HAV, MSM, drug abusers, and patients with chronic liver disease. Persons who have not been vaccinated and have been exposed to HAV can receive immunoglobulin therapy, which is 80% to 90% effective in preventing infection when administered soon after exposure. Immunoglobulin therapy can also be used as preexposure prophylaxis.

Hepatitis A virus HAV is a small, icosahedral, naked ssRNA virus, the sole member of the genus Hepatovirus in the family Picornaviridae. HAV infects people of all ages. In the United States, reported cases declined from the 1990s, when the annual average was about 25,000, to 2014, when 1239 cases were reported. In 2015, cases began increasing and reached 18,846 in 2019. The 30- to 39-year age group had the highest rate of infection, 14.5/100,000 population. In 2022, the CDC reported an outbreak of HAV infection linked to fresh organic strawberries. As of June 2022, 18 cases were conrmed. The WHO estimates that 1.5 million clinical cases of HAV infection occur each year. HAV is almost always transmitted via the fecal-oral route and is usually acquired through close personal contact or via contaminated food. The risk factors for HAV infection include sexual or household contact with an infected person, daycare contacts, foodborne or waterborne outbreaks, IV drug use, and international travel. However, almost 50% of the cases in the United States have no established risk factor. The virus is shed in large amounts in feces during the incubation period and early prodromal stage, and food and water contamination

Hepatitis B virus HBV is an enveloped, partially dsDNA virus that belongs to the family Hepadnaviridae. The virus contains the hepatitis B surface antigen (HBsAg), which circulates in the bloodstream

Antibody titer

Abnormal LFT results

Anti-HAV IgG

HAV in feces

Anti-HAV IgM

Level of detection

Weeks

1

2

3

4

5

6

8

12

24

Fig. 29.25 Serologic evaluation of hepatitis A virus (HAV) infection showing the increase and decrease of detectable antibodies. IgG, Immunoglobulin G; IgM, immunoglobulin M; LFT, liver function test.

Hepatitis viruses

as 22-nm particles. The whole virus has a total diameter of about 45 nm. The virion also contains a hepatitis B core antigen (HBcAg) and hepatitis B envelope antigen (HBeAg). Eight genotypes of HBV have been identied (A to H), and several studies have shown a difference in clinical outcome based on the genotype. Almost one half the world’s population lives in areas with endemic HBV, and more than 8% of the population is positive for HBsAg. According to the WHO, about 296 million people worldwide are infected with HBV. In the United States, the prevalence rate among adults decreased from 5.7/100,000 in 1999 to 1.0 in 2019. The incidence of acute HBV infection is estimated to be about 20,700 cases annually. HBV is primarily a bloodborne pathogen. Infected individuals can have as many as 1 million infectious particles per milliliter of blood. Lower concentrations of virus appear in semen, vaginal uid, and saliva. Many other body uids (e.g., tears, urine, sweat, breast milk) contain HBsAg but do not seem to be infective. The main modes of transmission are through sexual, perinatal, and parenteral routes. In the United States, heterosexual and male homosexual contacts are the most common routes of transmission. High-risk groups include IV drug abusers, MSM, individuals from endemic areas, persons with household or sexual contacts with HBV carriers, health care personnel, people with tattoos or body piercings, and infants born to HBV-positive mothers. Almost one third of the patients who become infected, however, have no known risk factor. The human cost of infection is high. An estimated 820,000 deaths per year worldwide are related to HBV infection. Once HBV enters the host, it travels from the bloodstream to the liver and infects the hepatocytes. Cytotoxic T cells then attack the HBV-infected hepatocytes. The incubation period for HBV infection ranges from 2 to 6 months, with an insidious onset that includes symptoms of fever, anorexia, and hepatic tenderness. Jaundice occurs in only about 10% of children who are younger than 5 years of age and is much more common in older children and adults (32% to 54%). As the immune response is activated, the virus is slowly cleared from the system, and most patients become noninfectious. In adults, about 50% of infections are asymptomatic; 20% to 30% of patients exhibit clinical jaundice but have a benign resolution of the infection. Therefore about 80% of infections do not cause serious sequelae. The risk for chronic infection is inversely proportional to the age at the time of infection, with approximately 90% of infants and only 3% of adults developing chronic infection. Individuals with chronic infection have a higher risk of liver disease, such as cirrhosis or hepatic carcinoma. A safe and effective recombinant vaccine is available for preventing HBV infection. The U.S. Advisory Committee on Immunization Practices recommends that the series begins at birth and be completed by 6 to 18 months. Diagnosis of HBV infection is based on clinical presentation and demonstration of specic serologic markers for HBV (Box 29.4). Serum aminotransferase levels also increase in infected patients. The presence of HBsAg in a patient’s serum indicates that the patient has an active HBV infection, is a long-term carrier, or is in an incubation period. IgM anti-HBc appears early in the course of the disease and indicates an acute infection. In patients in whom HBsAg is not detected and anti-HBs has not yet appeared, detection of IgM anti-HBc conrms the diagnosis of acute HBV infection. The period

745

BOX 29.4 Serologic markers for the diagnosis of hepatitis B

virus infection • HBsAg—hepatitis B surface antigen, the envelope protein consisting of three polypeptides • Anti-HBs—antibody to hepatitis B surface antigen • Anti-HBc—antibody to hepatitis B core antigen • HBeAg—antigen associated with the nucleocapsid, also found as soluble protein in serum • Anti-HBe—antibody to hepatitis B envelope antigen

between the inability to detect HBsAg and the detection of anti-HBs antibodies is referred to as the core window. The detection of anti-HBs antibodies in serum indicates convalescence or immune status. When the infection resolves, IgG anti-HBc and anti-HBs antibodies become detectable in the patient’s serum. The presence of HBsAg after 6 months of acute infection is a strong indication that the patient is a long-term carrier; the appearance of HBeAg in this case is indicative of a chronic infection and high infectivity. Table 29.8 shows the interpretation of HBV serologic markers. Fig. 29.26 depicts the increase and decrease in the levels of detectable serologic markers during acute HBV infection and resolution and presentation of chronic HBV infection. Several molecular assays are available that detect viral DNA and provide a short TAT and very sensitive detection.

Hepatitis D virus HDV, also known as the delta hepatitis virus, is a defective 1.7-kb ssRNA virus that requires HBV for replication. HDV requires the HBV HBsAg for its envelope. HDV is the sole member of the genus Deltavirus, and the genus is not currently assigned to a family. HDV is transmitted primarily via parenteral routes, although transmission through mucosal contact has been implicated in epidemics in endemic areas. At-risk groups in the United States are primarily IV drug users, although limited numbers of MSM in certain parts of the country are also at risk. Because of overlap in the clinical presentation, presumed low incidence of infection, and lack of an effective surveillance mechanism, current epidemiologic data on HDV are minimal. HDV infection is rare but usually severe and results in acute disease, with a fatality rate of 5%, or chronic symptoms progressing to cirrhosis in two thirds of those infected. The infection can occur in one of two clinical forms: co-infection or superinfection. In co-infection, the patient is simultaneously infected by HBV and HDV. In superinfection, HDV infection develops in a patient with chronic HBV infection. Patients with superinfection have a more severe acute infection and have a higher risk of fulminant hepatitis compared with patients with co-infection. Long-term carriers of HBV who become superinfected with HDV also develop chronic HDV infection, which increases their chance of developing cirrhosis. Diagnosis of HDV infection requires serologic testing for specic HDV antibody. Commercial tests are available for IgG anti-HDV antibody. Reference laboratories may offer IgM anti-HDV antibody testing and PCR assay for HDV. Table 29.9 presents the interpretation of HDV serologic

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Table 29.8 Interpretation of hepatitis B virus serologic markers HBsAg

HBeAg

Anti-HBc

NA

−

NA

+

NA +

Anti-HBc IgM

Anti-HBs

Anti-HBe

Interpretation

−

NA

No previous infection with HBV or early incubation

−

±

NA

Convalescent or past infection

−

−

±

NA

Immunization to HBsAg

−

−

±

−

−

Acute infection

+

+

±

+

−

−

Acute infection, high infectivity

+

−

±

+

−

+

Acute infection, low infectivity

+

+

+

−

−

−

Chronic infection, high infectivity

+

−

+

−

−

+

Chronic infection, low infectivity

, Negative; +, positive; ±, positive or negative; anti-HBc, antibody to hepatitis B core antigen; anti-HBe, antibodies against hepatitis B envelope antigen; anti-HBs, antibody to hepatitis B surface antigen; HBeAg, hepatitis B envelope antigen; HBsAg, hepatitis B surface antigen; IgM, immunoglobulin M; NA, not applicable.

Antibody titer

Abnormal LFT results HBsAg

Window period of immunity

HBeAg

Anti-HBc total

Anti-HBs Anti-HBe total Level of detection Anti-HBc IgM A

Months 1

2

3

4

5

6

12

24

Antibody titer

Abnormal LFT results HBsAg HBeAg Anti-HBc total Anti-HBe Anti-HBc IgM Level of detection

B

Months 1

2

3

4

5

6

12

24

Fig. 29.26 Serologic evaluation of hepatitis B virus infection showing the increase and decrease in levels of detectable antibodies. A, Serologic presentation in acute hepatitis infection with resolution. B, Serologic presentation in chronic hepatitis infection with late seroconversion. Anti-HBc, Antibodies against hepatitis B core antigen; anti-HBe, antibodies against hepatitis B envelope antigen; anti-HBs, antibodies against hepatitis B surface antigen; HBeAg, hepatitis B envelope antigen; HBsAg, hepatitis B surface antigen; LFT, liver function test.

Hepatitis viruses

markers; Fig. 29.27 depicts serologic presentations of HDV co-infection and superinfection. Detection of HDV RNA is done with RT-PCR, and diagnosis using this method is becoming more common from liver tissue.

Hepatitis C virus After methods for diagnosing HAV and HBV became available, it was apparent that these two viruses were not Table 29.9 Interpretation of hepatitis D virus serologic markers Clinical variant

Serologic markers Anti-HBc IgM

HBsAg

Anti-HDV

Anti-HDV IgM

Co-infection

+

+

+

+

Superinfection

−

+

+

NA

Anti-HBc, antibody to hepatitis B core antigen; HBsAg, hepatitis B surface antigen; HDV, hepatitis D virus; IgM, immunoglobulin M; NA, not applicable.

747

responsible for all hepatitis cases, especially those related to blood transfusions. The resulting disease was termed nonA, non-B (NANB) hepatitis. The diagnosis of NANB hepatitis was primarily one of exclusion. In 1974, without any direct evidence, scientists predicted that a type C hepatitis virus must exist. Then, 15 years later, with the aid of molecular and cloning techniques, the genomic sequence of HCV was determined before the virus was ever seen with an electron microscope. HCV is an ssRNA virus in the genus Hepacivirus, family Flaviviridae; it accounts for about 90% of all previous cases of NANB hepatitis. There has been a gradual increase in the annual incidence of acute HCV infections in the United States since 2012. In 2019, 4136 cases were reported, but the CDC estimates that 57,500 acute cases occurred. Worldwide, as many as 1.5 million new cases may develop each year, and an estimated 58 million people have chronic HCV. Although perinatal and sexual transmission of infection occur and parenteral transmission has been identied as a major route for infection, HCV antibodies have been detected in patients in

Abnormal LFT results

Antibody titer

HBsAg

Anti-HBc total Anti-HDV IgM

Anti-HBc IgM

Level of detection

A

Anti-HDV

Months

Abnormal LFT results

Antibody titer

HBsAg

Anti-HDV

Level of detection

B

Anti-HDV IgM

Months

Fig. 29.27 Serologic evaluation of hepatitis D virus (HDV) infection showing the persistence of detectable antibodies. A, Hepatitis B virus (HBV)-HDV co-infection. B, HBVHDV superinfection. Anti-HBc, Antibodies against hepatitis B core antigen; HBsAg, hepatitis B surface antigen; IgM, immunoglobulin M; LFT, liver function test.

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Clinical virology

whom the routes of transmission are poorly understood or who have no evidence of identiable risk factors. Symptoms can be subtle and may take time to become apparent. About 50% of HCV-positive patients become longterm carriers, and about 20% to 30% of patients with chronic infections develop cirrhosis. Cirrhosis is a major risk factor for hepatocellular carcinoma. In 2019, over 100,000 new chronic HCV infections were recorded in the United States. It is estimated that the number of U.S. citizens living with chronic HCV is 2.4 million, but it might be as many as 4.7 million. Among the three hepatitis viruses—HAV, HBV, and HCV—HCV has the highest mortality rate. Gene amplication tests prove that HCV RNA appears in newly infected patients in as little as 2 weeks. However, most infections are detected by serologic testing. HCV is less immunogenic than HBV. Antibodies to HCV appear in about 6 weeks in 80% of patients and within 12 weeks in 90% of patients. HCV infection does not produce persistent, life-long levels of antibody; rather, persistence of anti-HCV antibody is linked to the presence of replicating HCV. EIAs that detect serum antibodies to HCV proteins are available as screening tests; however, they can have a low sensitivity. Second-generation immunoblot assays use recombinant and/or synthetic proteins to detect anti-HCV antibodies. Currently, third-generation tests are more sensitive and accurate and are commonly used. These assays also employ recombinant proteins but have an improved version of NS3 and have added NS5. Conrmation currently relies on nucleic acid amplication testing. Figure 29.28 depicts the immunologic prole of HCV infection. Patients with chronic HCV infection were previously treated with interferon, with or without ribavirin. First approved in 2011, direct-acting antiviral (DAA) therapy has proven highly effective in curing HCV infection. DAAs are molecules that target specic nonstructural proteins of the virus, which results in disruption of viral replication and infection. There are four classes of DAAs dened by their mechanism of action and therapeutic targets.

Hepatitis E virus HEV is a small (32 to 34 nm), naked, ssRNA virus classied in the genus Hepevirus, family Hepeviridae. HEV is transmitted via the fecal-oral route, particularly through contaminated drinking water. HEV has been identied as the cause of epidemics of enterically transmitted hepatitis in developing countries in Asia, Africa, and Central America. Although the virus has not been associated with outbreaks in the United States, it has been linked to sporadic cases in travelers returning from endemic areas. HEV causes an acute, self-limiting disease with clinical symptoms like those of HAV. The incubation period is 2 to 9 weeks. Signs and symptoms of HEV infection include fever, malaise, nausea, vomiting, jaundice, and dark-colored urine. No specic antiviral therapy is available; patients receive supportive therapy. Viral shedding in feces has been shown to persist for several weeks. The mortality rate is 1% to 3% overall, with a higher likelihood of death in women who are pregnant (15% to 25%). Epidemics affect primarily young to middle-age adults. An ELISA test is available to detect IgG and IgM antiHEV antibodies, although HEV testing is not currently performed in diagnostic laboratories in the United States.

Other hepatitis viruses Evidence for HGV was derived originally from a patient with NANB hepatitis. This RNA virus, now known as GB virus type C (GBV-C), is a member of the family Flaviviridae. GBV-C viremia has been demonstrated worldwide in 0.6% to 14% of blood donors, depending on the geographic location. However, infection does not seem to be common in the United States. The clinical signicance of the virus is still under investigation. Experimental RT-PCR tests can be performed to detect the virus, but routine testing is not yet recommended. The most recently identied hepatitis viruses are SEN virus and TTV. SEN virus has a circular DNA genome. It is

Antibody titer

Abnormal LFT results

Anti-HCV

Level of detection

Months

1

2

3

4

5

6

12

24

Fig. 29.28 Serologic evaluation of hepatitis C virus (HCV) infection showing the persistence of detectable antibodies, indicating the presence of replication HCV. LFT, Liver function test.

Treatment and management of viral infections

bloodborne, and although originally suspected to be a cause of hepatitis, it has not been denitively linked to any human disease. Transmission appears to be linked to blood transfusion. About 30% of patients with HIV infection have antibodies to the SEN virus. TTV was rst identied in the serum of a Japanese patient in 1997. It is an ssDNA virus related to animal circoviruses. The role of TTV in human disease is unknown but may be associated with some cases of posttransfusion hepatitis.

Prions Prions are not viruses but are proteinaceous infectious particles that cause a group of diseases in mammals called transmissible spongiform encephalopathies (TSEs). The name prion (from the words protein and infection) was coined by Stanley B. Prusiner in 1982. Examples of TSEs include kuru and Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle, and chronic wasting disease in deer and elk. BSE, also referred to as mad cow disease, was responsible for the loss of hundreds of thousands of cattle in the United Kingdom, most notably during the 1980s. An increase in a variant form of CJD (vCJD) in the United Kingdom was noted at about the same time as the surge of BSE. It appears that the agent of scrapie crossed species and infected cows and that it is the causative agent in BSEinfected humans, causing vCJD. Between 1993 and 2018, 26 cases of BSE were discovered in North America: 6 cases in the United States and 20 in Canada. TSEs are known to be acquired via ingestion, although some prions may also enter the body by other routes. Sheep offal used in cattle feed was the likely cause of the prion moving from sheep to cattle. TSEs are characterized by progressive, relentless degeneration of the CNS, which is ultimately fatal. Typical histopathology is neuronal vacuolation and eventual loss of neurons, accompanied by proliferation and hypertrophy of brous astrocytes. The prion protein (PrP) found in animals with TSE is often referred to as PrP Sc, named after the prion found in sheep with scrapie. Animals, including humans, have a similar but normal protein found on cells of the CNS, referred to as PrPC. Ingested PrPSc is absorbed into the bloodstream and makes its way into the CNS. PrP Sc converts PrPC into PrP Sc , which is released by neuronal cells. PrP Sc accumulates in the CNS, producing amyloid plaques and the characteristic histopathology. The diagnosis of TSE is often based on clinical ndings. Routine analysis of CSF is nonrevealing. Many patients with CJD will have 14-3-3 proteins in CSF; however, these proteins are not specic for CJD. The presence of these proteins in CSF is a marker for rapid neuronal cell death, present not only in CJD but also in encephalitis and conditions with CNS hemorrhaging. Histopathology staining and PrP Sc immunostaining remain the most specic diagnostic methods. Other detection methods include antigen detection, serologic testing, and nucleic acid sequencing for inheritable forms. Recent ndings have suggested the excretion of TSEs in urine. The risk of infection to laboratory personnel is low, but material suspected of containing these agents must be handled carefully. Prions are extremely resistant to inactivation; even 2 hours in a steam autoclave might not inactivate all

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prions in a specimen. Exposure to household bleach with more than 20,000 parts per million available chlorine or 1 M sodium hydroxide is recommended. It is not recommended to test any specimens from a suspected case of prion disease until it is ruled out because instrumentation cannot be sufciently decontaminated, and it presents a risk of infection to staff.

Treatment and management of viral infections Viruses have evolved to replicate inside host cells. Since the virus uses host machinery to replicate, most drugs targeting viruses have the potential to affect the host cell as well. Viruses with very high mortality rates (e.g., Ebola virus) are often eradicated equally rapidly as the survival of virus relies on effective transmission to new hosts. Therefore most pathogenic viruses eventually mutate to milder infections and become more contagious. This is the trend being observed in the ongoing COVID-19 pandemic. Most mild viral illnesses are not treated; instead, supportive care is provided for the viral cycle to be completed, and quarantine of infected individuals is recommended to break the transmission cycle.

Antiviral therapy Some viral infections are treatable, especially if the laboratory can rapidly identify the pathogen. Antiviral compounds must target an essential viral replicative mechanism without destroying or damaging uninfected host cells. Several antiviral agents resemble nucleosides used in viral replication. The viruses insert these nucleoside analogues into their own nucleic acid, resulting in disruption of viral replication. Other antivirals are non-NRTIs, which also disrupt viral replication. Phosphonoformic acid (foscarnet) is an analogue of pyrophosphate that acts directly as a DNA polymerase inhibitor. Other antiviral compounds inhibit viral replication by targeting key viral proteins (e.g., protease inhibitors). Some commonly used antiviral agents are given in Table 29.10. A number of antiviral drugs have been developed, and several are in clinical trials for the treatment of COVID-19. Just as antibacterial agent use increases the risk of drug resistance in bacteria, the use of antiviral agents can result in viruses that become resistant to therapy. As more antiviral agents become available, antiviral susceptibility testing will become increasingly important. For example, foscarnet is currently being used to treat infections caused by HSV strains resistant to acyclovir and to treat CMV strains resistant to ganciclovir.

Vaccines Development of vaccines for highly contagious viruses is a top priority in the management of clinically relevant viral infections. A vaccine is composed of a highly weakened attenuated virus or virus component that can trigger an immune response. When the body encounters the actual infection, the host will mount a heightened (anamnestic) immune response, ultimately leading to clearance of the infectious agent. It is important to note that vaccines prevent disease

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Table 29.10 Examples of antiviral compounds Antiviral

Inhibits

Active against

Acyclovir

DNA polymerase

HSV, VZV

Cidofovir

DNA polymerase

CMV (retinitis)

Famciclovir

DNA polymerase

HSV-2

Ganciclovir

DNA polymerase

CMV (retinitis)

Valacyclovir

DNA polymerase

HSV-2

Idoxuridine, triuridine

DNA synthesis (DNA base analogue)

HSV (keratitis)

Amantadine, rimantadine

Uncoating

Inuenza A (treatment and prophylaxis)

Interferon-α

Viral replication (multiple mechanisms)

HPV (genital warts); chronic HCV, Kaposi sarcoma

Ribavirin

Viral replication (multiple mechanisms)

RSV, CCHF

AZT or ZDV

Reverse transcriptase

HIV

ddI

Reverse transcriptase

HIV

ABC

Reverse transcriptase

HIV

3TC

Reverse transcriptase

HIV

d4T

Reverse transcriptase

HIV

ddC

Reverse transcriptase

HIV

FTC

Reverse transcriptase

HIV

TDF

Reverse transcriptase

HIV

Indinavir

Proteases

HIV

Nelnavir, ritonavir

Proteases

HIV, CoV-2

Saquinavir

Proteases

HIV

Lamivudine

Chronic HBV

Adefovir Remdesivir

Chronic HBV Adenosine analog

CoV-2

ABC, Abacavir sulfate; AZT, azidothymidine; d4T, stavudine; CCHF, CrimeanCongo hemorrhagic fever; CMV, cytomegalovirus; CoV-2, coronavirus 2, ddC, zalcitabine; ddI, didanosine; FTC, emtricitabine; HCV, hepatitis C virus; HIV, human immunodeciency virus; HPV, human papillomavirus; HSV, herpes simplex virus; RSV, respiratory syncytial virus; 3TC, lamivudine; TDF, tenofovir; VZV, varicella-zoster virus; ZDV, zidovudine.

but not infection. Those infected after vaccination are usually asymptomatic, or have a milder form of disease, and eliminate the infectious agent quickly. They are much less likely to transmit the agent to other hosts. Once a signicant percent of the population develops immunity, either by vaccination or surviving the disease, the infection rate becomes very low. This concept is referred to as herd immunity Unfortunately, due to the high rate of mutations, effective vaccines cannot be developed for all viruses. A number of

successful vaccines like smallpox vaccine and polio vaccine were tremendously successful, leading to eradication or near eradication of these viral diseases worldwide. Most recently, three COVID-19 vaccines were developed, two of which (Moderna [Cambridge, MA] and Pzer [New York, NY]BioNT [Germany]) used mRNA coding for a surface protein of SARS-COV-2. The third, from Johnson and Johnson (New Brunswick, NJ), used an attenuated virus. These vaccines have been instrumental in curbing the spread and mortality of COVID-19. Global efforts to invest in research and development of effective vaccines as well as increasing awareness to the public about the safety and efcacy of vaccinations is a key step to prevent future pandemics.

POINTS TO REMEMBER

• Viruses have been responsible for several pandemics including inuenza viruses and coronaviruses. • Clinical virology services can consist of rapid antigen detection, NAATs, antibody detection, or cell cultures. • Clinically signicant viruses can be isolated from patients with signs and symptoms commonly thought to be associated with bacterial infections, including pneumonia, GI disorders, cutaneous lesions, sexually transmitted infections, and sepsis. • Members of the family Herpesviridae produce life-long, latent infections. • Most cases of cervical cancer are linked to HPV, the causative agent of genital warts. • Some viruses mutate rapidly, resulting in new strains, which can be challenging to contain or treat. • Retroviruses (e.g., HIV) replicate with reverse transcriptase, which uses viral RNA as a template to make a complementary DNA strand. • Arboviruses are those viruses transmitted by the bite of arthropods, such as mosquitoes. • Many emerging infections are caused by zoonotic viral agents that are unexpectedly transplanted into a susceptible human population. • The hepatitis viruses are a diverse collection of viruses grouped together because they all infect primarily the liver. Laboratory diagnosis is based on serologic markers. • Antiviral compounds can treat numerous viral infections, but resistance has been seen. • Vaccines against viruses is an effective way to manage viral infections and severity.

LEARNING ASSESSMENT QUESTIONS

1. Which of the following serologic markers should be positive following administration of the HBV vaccine? a. HBeAg b. HBcAg c. Anti-HBs d. Anti-HBc 2. Which of the following conditions is most often associated with rotavirus? a. Infant diarrhea b. Paralysis in children

Treatment and management of viral infections

3.

4.

5.

6.

7.

8.

9.

10.

11.

12. 13. 14.

c. Infant respiratory infections d. Infectious mononucleosis–like symptoms in young adults Which virus causes fth disease? a. Molluscum contagiosum b. Parvovirus B19 c. Measles virus d. Varicella-zoster virus During the “core window” of acute hepatitis B virus infection, what is the primary marker for diagnosis? a. HBsAg b. HBeAg c. Anti-HBs d. Anti-HBc Reactivation of the virus causing chickenpox results in which condition? a. Shingles b. Infectious mononucleosis c. Dengue fever d. Polio The presence of Kaposi sarcoma in a young adult is suggestive of which illness? a. West Nile fever b. Ebola hemorrhagic fever c. Acquired immunodeciency syndrome d. Coronavirus disease A patient suspected of having hepatitis has a positive anti-hepatitis C virus (HCV) antibody test. Which of the following should be done next? a. Report the patient as HCV positive. b. Perform an anti-HBs test to rule out a false positive c. Request a second blood sample to repeat the test. d. Conrm the result with a nucleic acid amplication test for HCV. What is the greatest risk for developing dengue hemorrhagic fever? a. History of dengue fever with a different serotype b. Older than 50 years of age c. Diabetes d. Obesity What is the most common complication of West Nile fever? a. Pneumonia b. Neuroinvasive disease c. Chronic hepatitis d. Osteomyelitis Which of the following specimens is best to recover cytomegalovirus? a. Nasopharyngeal swabs b. Bronchial wash c. Buffy coat d. Saliva Which opportunistic infections or conditions are used as indicators of acquired immunodeciency syndrome (AIDS)? Which immunologic markers are used to diagnose human immunodeciency virus (HIV) infection? What disease does Epstein-Barr virus (EBV) produce? What complications can result from EBV infections? How is acute hepatitis B virus (HBV) infection differentiated from chronic infection? Which markers indicate resolution of the infection?

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15. What are the differences between classic dengue fever and dengue hemorrhagic fever? 16. What are the methods commonly used to diagnosis rabies? 17. Which types of infections are caused by human papillomavirus (HPV)? 18. Which viruses have the potential for latency? 19. Why are vaccines for inuenza not always effective? 20. What type of vaccines showed highest success against SARS-CoV-2 virus?

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