Microns Microbiology Simplified [3 ed.] 9789389941982, 9885175004, 9334159340, 9623451994, 9000660880, 9555590180

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3rd Edition

Malathi Murugesan

MD (Microbiology), DTM&H (RCP London), PGDID Consultant Microbiologist Vellore, Tamil Nadu

CBS Publishers & Distributors Pvt Ltd • New Delhi • Bengaluru • Chennai • Kochi • Kolkata • Mumbai • Hyderabad • Nagpur • Patna • Pune • Vijayawada

DISCLAIMER This book contains questions based on important topics frequently asked in previous years National Level PG Entrance Examinations and State Level Examinations in India. Often repeated topics and sub-topics have been included for students’ benefit. We do not claim that these questions are exact or similar to questions asked in any recent examinations in India. If any such similarity is found, it is purely coincidental and by chance.

ISBN: 978-93-89941-98-2 Copyright © Author & Publishers Third Edition: 2020 Second Edition: 2019-20 All rights reserved. No part of this book 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 author and the publishers. Published by Satish Kumar Jain and produced by Varun Jain for CBS Publishers & Distributors Pvt Ltd 4819/XI Prahlad Street, 24 Ansari Road, Daryaganj, New Delhi 110 002, India. Ph: +91-11-23289259, 23266861, 23266867 Website: www.cbspd.com Fax: 011-23243014 e-mail: [email protected]; [email protected]. Corporate Office: 204 FIE, Industrial Area, Patparganj, Delhi 110 092 Ph: +91-11-4934 4934 Fax: 4934 4935 e-mail: [email protected]; [email protected]

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Dedication

This book is dedicated to my parents Mrs M Thamayanthi DCE Mr S Murugesan MA

who molded and guided me in all my tough times...

Preface To my dear PG aspirants, Microbiology and infectious diseases need regular updates because of their emerging and re-emerging trends. With the recent novel corona virus panicking whole world, the question which has been haunting every heart is: “Are we standing on the threshold of a third world war”—Man versus Microbes? Pandemic and Epidemic outbreaks make a huge impact on health, economy, travel and also our day to day life gets affected on a large scale. There is a difference in understanding microbiology during your UG days and your PG preparation. This book MICRONS—Microbiology Simplified will make you feel the difference. To prepare for a PG entrance examination, you need to be smarter than being intelligent. The most important chapters are Sterilization and Disinfection, Antimicrobial resistance, Mycobacterium, Spirochaetes, Hepatitis, HIV, Influenza, recent outbreaks and parasitology images. When you are writing your exam papers, if you feel the questions are tough, you are not alone who think like this. There are a number of others who are competing with you. Common questions never decide the seats. The rare and exceptions make the difference between you and your close competitor. While reading, you better highlight the most common facts, exceptions and recent updates. This book makes it easier for you because the important facts are highlighted well to make a quicker revision. I am happy to share that Microns – Microbiology Simplified – 3rd edition is released with added features and has been updated with recent questions and notes on novel corona virus. As I always say, students are most important for us and their opinions and ideas about a book are highly valuable for every teacher/author. My book is not an exception. I would heartily welcome your feedback and work accordingly to improve my book. Please share your feedback on my mail id or facebook page. Thank you dear students ! With Love Malathi Murugesan MBBS MD DTM&H PGDID [email protected]

Join author`s Facebook page for further updates and active discussion with author MICRONS - Microbiology Simplified by Malathi M

From the Publisher’s Desk Dear Readers, I extend my warm welcome and convey my heartfelt thanks for appreciating the CBS Exam Books for another successful year. It has been an amazing journey so far and I am highly grateful for your support and cooperation to help us achieve various milestones in this whole span of time. The mission with which we started in the year 2015 was to bring nothing but the best of everything to our target audience and today I can proudly say that we have maintained that standard and are committed to continue the same in future as well. Every single title under the banner of CBS Exam Books has been developed and nurtured like an infant. The authors and our entire team work day and night to bring the best in everything for you. Be it content, presentation, social media contests and offers, we strive to meet your expectations with every passing year. Your trust has motivated us to maintain and upgrade ourselves during this period. I am extremely thankful to all our authors who are the real pillars of the complete series of CBS Exam Books. The contributions of our esteemed authors have laid the foundations of CBS Exam Books.

At this juncture, I can recall these lines by Drake, “Sometimes it’s the journey that teaches you a lot about your destination”.

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I would like to sum up with these eternal lines of Robert Frost: Woods are lovely dark and deep, But I have promises to keep. And miles to go before I sleep, And miles to go before I sleep! Wishing you success in all your endeavors!

Bhupesh Arora Vice President – Publishing & Marketing (PGMEE and Nursing Division) Email: [email protected] Mobile: (+91) 9555590180

Acknowledgements My gratitude to the contributors who helped me in preparing this manuscript: Dr M Jane Esther, MD (micro), FID, Consultant and Head of microbiology, Doctors' Diagnostic Centre, Trichy, Tamil Nadu Dr K Deepika, MD (Microbiology), Consultant Microbiologist, NG Hospital and Research Centre, Coimbatore for her contribution on chapter Gastrointestinal infections ™ Dr M Muniraj, MD DVL, Final year postgraduate, Madras Medical College, Chennai for his contribution on STD ™ Dr P Divya, DGO, Consultant Gynaecologist, WCF Hospital, Kolathur and GIFT clinic, Chennai for her contribution on Infections related to Obstetrics and Gynecology ™ Dr J Divya John Stephy MD (Pharmacology), Assistant Professor of Pharmacology, Government Vellore Medical College and Hospital for her contribution on Antimicrobial Chemotherapy—A Short Review Dr FM Zafar, DLO, ENT specialist, Asst. Surgeon, Cheyyur GH for his contribution on Infections of Ear, Nose and Throat ™ ™ Dr Kenny Robert J MS (GS), Consultant General and Laparoscopic Surgeon, Assistant Professor of General Surgery, Government Royapettah Hospital, Kilpauk Medical College, Chennai for his contribution on surgical site and related infections ™ Dr Karthik Jayachandran, MS Orthopedics, Assistant Professor, Trichy SRM Medical College Hospital and Research Center, Irungalur, Trichy for his contribution on Infections of Bones and Joints ™ Dr MJ Felix Emerson, D. ortho, DNB Orthopaedics (II year), Ganga Orthopaedic Hospital and Research Centre, Coimbatore, Tamil Nadu Dr R Gokul, MD (Anesthesiology), 2nd year postgraduate, Government Vellore Medical College, Vellore, Tamil Nadu ™ Dr Neha Singh, II year MD (micro), Rajendra Institute of Medical Sciences, Ranchi, Jharkhand ™ ™ Dr V Srinidhi MBBS, Chengalpattu Government Medical College, Chengalpattu, Tamil Nadu S Suganya, CRRI, Chengalpattu Government Medical College, Chengalpattu, Tamil Nadu ™ ™ S Abirami, final year MBBS, Chengalpattu Government Medical College, Chengalpattu, Tamil Nadu ™ T Karthick, final year MBBS, Chengalpattu Government Medical College, Chengalpattu, Tamil Nadu ™ M Meena, final year MBBS, Chengalpattu Government Medical College, Chengalpattu, Tamil Nadu ™ N Dhanushya, final year MBBS, Chengalpattu Government Medical College, Chengalpattu, Tamil Nadu GR Manoj Kumar, final year MBBS, Chengalpattu Government Medical College, Chengalpattu, Tamil Nadu ™ D Rishaba Sri, Pre-final year MBBS, Tagore Medical College and Hospital, Chennai, Tamil Nadu ™ ™

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My sincere acknowledgement to my mentors, friends and relatives who motivated and stood on my side in all my tough times: I would like to express thanks and happiness to my pillar of support, my husband Dr R Jagadish, MD (Biochemistry), Assistant Professor, Christian Medical College, Vellore, Tamil Nadu Dr A Vijayalakshmi, MD (Microbiology), Professor & HOD, Chengalpattu Government Medical College, Chengalpattu, Tamil Nadu ™ ™ Dr B Palani Kumar, MD (General medicine), Associate Professor, Government Thoothukudi Medical College, Tuticorin, Tamil Nadu ™ Dr S Jamuna Rani, MD (Pathology), Associate Professor, Tagore Medical College and Hospital, Chennai, Tamil Nadu Dr K Shakthesh, MS(ENT), Hopkins ENT Clinic, Tambaram, Chennai, Tamil Nadu ™ ™ Dr K Bharani Raj Kumar, MS (General Surgery), Professor, Tagore Medical College and Hospital, Chennai, Tamil Nadu Dr AVM Balaji, MD (Microbiology), Sr Assistant Professor of Microbiology, Stanley Medical College, Chennai, Tamil Nadu ™ Dr R Rajamahendran, MS, MRCS (Edinburgh), MCh (Surgcial Gastroenterology, FMAS), Consultant Gastrointestinal Surgeon, ™ Director/Founder: KONCPT PG Medical Coaching Center, Tamil Nadu, India, Director: RRM Gastrosurgical and Research Center, Villupuram, Tamil Nadu Dr T Antan Uresh Kumar, MS, MCh Urology, FMAS, Founder KONCPT, laparoscopic transplant surgeon, Madras Kidney Foundation, ™ Chennai, Tamil Nadu ™ My sister: M. Abirami ™ My father-in-law Mr KM Ramu, mother-in-law Mrs J Krishnaveni and to all my maternal and paternal relatives. ™ Last but not least, I have to thank my twin children Master MJ Aathreyan and Master MJ Aaruthran, who strengthened my motherhood and gifted me the memories of maternity with this wonderful piece of book. ™

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I would also like to thank Mr Satish Kumar Jain (Chairman) and Mr Varun Jain (Managing Director), M/s CBS Publishers and Distributors Pvt Ltd for providing me the platform in bringing out the book. I have no words to describe the role, efforts, inputs and initiatives undertaken by Mr Bhupesh Arora Vice President – Publishing & Marketing, PGMEE and Nursing Division for helping and motivating me. I must admit that I have been highly demanding on the precision of expression of the content from the staff members of CBS publishers. I thank Dr Mrinalini Bakshi (Editorial Head & Content Strategist) for her editorial support and Ms Nitasha Arora (Production Head & Content Strategist), Dr Anju Dhir (Project Manager & Senior Scientific Coordinator), Mr Shivendu Bhushan Pandey (Senior Editor), Mr Ashutosh Pathak (Senior Proof Reader) and all the production team members, Mr Chaman Lal, Mr Prakash Gaur, Mr Phool Kumar, Mr Bunty Kashyap, Mr Chander Mani, Ms Tahira Parveen, Ms Babita Verma, Ms Manorama Gupta, Mr Raju Sharma, Mr Manoj Chaudhary, Mr Vikram Chaudhary, Mr Manoj Malakar and Mr Rahul Negi for devoting laborious hours in designing and typesetting of the book.

Contents

 



Preface ------------------------------------------------------------------------------------------------------------------------------------------- iii Acknowledgements----------------------------------------------------------------------------------------------------------------------------------------vii Recent Outbreak 2020--------------------------------------------------------------------------------------------------------------------------------------xi Latest Exam Questions 2020–2019--------------------------------------------------------------------------------------------------------------------- xiii Sample Video Questions----------------------------------------------------------------------------------------------------------------------------- xxxvii Image-Based Concept Zone--------------------------------------------------------------------------------------------------------------------xli

 



 





UNIT 1

GENERAL MICROBIOLOGY

Chapter 1. Chapter 2. Chapter 3. Chapter 4. Chapter 5.

Introduction, History and Microscopes---------------------------------------------------------------------------------- 3–10 Morphology and Physiology of Bacteria--------------------------------------------------------------------------------11–25 Sterilization and Disinfection--------------------------------------------------------------------------------------------26–38 Culture Media and Culture Methods------------------------------------------------------------------------------------39–48 Bacterial Genetics, Resistance and Susceptibility Testing-------------------------------------------------------------49–59

UNIT 2 BACTERIOLOGY Chapter 6. Chapter 7. Chapter 8. Chapter 9. Chapter 10. Chapter 11. Chapter 12. Chapter 13. Chapter 14. Chapter 15. Chapter 16. Chapter 17. Chapter 18. Chapter 19. Chapter 20. Chapter 21. Chapter 22. Chapter 23.

Staphylococcus-----------------------------------------------------------------------------------------------------------63–72 Streptococci---------------------------------------------------------------------------------------------------------------73–84 Pneumococcus------------------------------------------------------------------------------------------------------------85–89 Neisseria-------------------------------------------------------------------------------------------------------------------90–95 Corynebacterium------------------------------------------------------------------------------------------------------- 96–102 Bacillus----------------------------------------------------------------------------------------------------------------- 103–108 Clostridium------------------------------------------------------------------------------------------------------------- 109–118 Enterobacteriaceae---------------------------------------------------------------------------------------------------- 119–134 Vibrio------------------------------------------------------------------------------------------------------------------- 135–141 Pseudomonas, Acinetobacter and Burkholderia-------------------------------------------------------------------- 142–146 Haemophilus, Francisella and Pasteurella--------------------------------------------------------------------------- 147–151 Brucella and Bordetella----------------------------------------------------------------------------------------------- 152–155 Mycobacterium-------------------------------------------------------------------------------------------------------- 156–166 Spirochaetes------------------------------------------------------------------------------------------------------------ 167–175 Rickettsia and Chlamydia--------------------------------------------------------------------------------------------- 176–186 Helicobacter and Campylobacter------------------------------------------------------------------------------------ 187–189 Mycoplasma and Legionella------------------------------------------------------------------------------------------ 190–193 Miscellaneous Bacteria------------------------------------------------------------------------------------------------ 194–202

UNIT 3 VIROLOGY Chapter 24. Chapter 25. Chapter 26. Chapter 27. Chapter 28. Chapter 29. Chapter 30. Chapter 31. Chapter 32. Chapter 33. Chapter 34. Chapter 35. Chapter 36.

Introduction and General Properties of Viruses-------------------------------------------------------------------- 205–213 Bacteriophages--------------------------------------------------------------------------------------------------------- 214–217 Poxviruses-------------------------------------------------------------------------------------------------------------- 218–221 Herpesviruses---------------------------------------------------------------------------------------------------------- 222–230 Adenovirus------------------------------------------------------------------------------------------------------------- 231–232 Picornaviruses--------------------------------------------------------------------------------------------------------- 233–238 Orthomyxoviruses----------------------------------------------------------------------------------------------------- 239–243 Paramyxovirus--------------------------------------------------------------------------------------------------------- 244–249 Arthropod- and Rodent-Borne Viral Infections--------------------------------------------------------------------- 250–256 Rhabdovirus------------------------------------------------------------------------------------------------------------ 257–261 Hepatitis Virus--------------------------------------------------------------------------------------------------------- 262–271 Human Immunodeficiency Virus------------------------------------------------------------------------------------ 272–279 Miscellaneous Viruses------------------------------------------------------------------------------------------------- 280–286

   

 

 Contents



 



 



UNIT 4 PARASITOLOGY Chapter 37. Chapter 38. Chapter 39. Chapter 40. Chapter 41. Chapter 42. Chapter 43. Chapter 44. Chapter 45. Chapter 46. Chapter 47.

UNIT 5 MYCOLOGY Chapter 48. Chapter 49. Chapter 50. Chapter 51. Chapter 52.

 

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Characteristics and Laboratory Diagnosis of Fungi----------------------------------------------------------------- 357–363 Superficial Mycoses---------------------------------------------------------------------------------------------------- 364–370 Endemic/Systemic Mycoses------------------------------------------------------------------------------------------ 371–375 Opportunistic Mycoses------------------------------------------------------------------------------------------------ 376–383 Miscellaneous Fungi--------------------------------------------------------------------------------------------------- 384–386

UNIT 6 IMMUNOLOGY Chapter 53. Chapter 54. Chapter 55. Chapter 56. Chapter 57. Chapter 58. Chapter 59. Chapter 60. Chapter 61. Chapter 62. Chapter 63. Chapter 64.

Immunity -------------------------------------------------------------------------------------------------------------- 389–392 Structure and Functions of Immune System ------------------------------------------------------------------------ 393–400 Antigens ---------------------------------------------------------------------------------------------------------------- 401–403 Antibodies ------------------------------------------------------------------------------------------------------------- 404–410 Complement System-------------------------------------------------------------------------------------------------- 411–415 Antigen-Antibody Reactions------------------------------------------------------------------------------------------ 416–421 Immune Response ---------------------------------------------------------------------------------------------------- 422–426 Hypersensitivity ------------------------------------------------------------------------------------------------------- 427–431 Immunodeficiency Diseases ----------------------------------------------------------------------------------------- 432–435 Autoimmunity --------------------------------------------------------------------------------------------------------- 436–437 Transplantation and Tumor Immunology--------------------------------------------------------------------------- 438–440 Immunohematology -------------------------------------------------------------------------------------------------- 441–444

UNIT 7

APPLIED MICROBIOLOGY

Chapter 65.



Introduction to Parasitology------------------------------------------------------------------------------------------ 289–295 Flagellates–I------------------------------------------------------------------------------------------------------------ 296–299 Hemoflagellates-------------------------------------------------------------------------------------------------------- 300–304 Leishmania------------------------------------------------------------------------------------------------------------- 305–308 Apicomplexa----------------------------------------------------------------------------------------------------------- 309–317 Toxoplasma, Ciliate Protozoa----------------------------------------------------------------------------------------- 318–321 Coccidian Intestinal Parasites---------------------------------------------------------------------------------------- 322–325 Helminthology Cestodes---------------------------------------------------------------------------------------------- 326–332 Trematodes------------------------------------------------------------------------------------------------------------- 333–338 Nematodes------------------------------------------------------------------------------------------------------------- 339–347 Filarial Nematode------------------------------------------------------------------------------------------------------ 348–353

UNIT 8

Applied Microbiology------------------------------------------------------------------------------------------------- 447–450

INFECTIOUS DISEASES

Chapter 66. Introduction------------------------------------------------------------------------------------------------------------ 453–456 Chapter 67. Fever of Unknown Origin--------------------------------------------------------------------------------------------- 457–458 Chapter 68. Infections of Ear, Nose and Throat----------------------------------------------------------------------------------- 459–466 Chapter 69. Infections of Eye------------------------------------------------------------------------------------------------------- 467–470 Chapter 70. Infection in Lower Respiratory Tract – Pneumonia----------------------------------------------------------------- 471–472 Chapter 71. Gastrointestinal Infections-------------------------------------------------------------------------------------------- 473–478 Chapter 72. Cardiovascular Infection – Endocarditis----------------------------------------------------------------------------- 479–481 Chapter 73. CNS Infections--------------------------------------------------------------------------------------------------------- 482–484 Chapter 74. Skin and Soft Tissue Infections--------------------------------------------------------------------------------------- 485–486 Chapter 75. Infections of Bones and Joints---------------------------------------------------------------------------------------- 487–489 Chapter 76. Sexually Transmitted Infections-------------------------------------------------------------------------------------- 490–491 Chapter 77. Urinary Tract Infections----------------------------------------------------------------------------------------------- 492–493 Chapter 78. Infections Related to Obstetrics and Gynecology------------------------------------------------------------------- 494–496 Chapter 79. Surgical Site and Related Infections---------------------------------------------------------------------------------- 497–500 Chapter 80. Infections in Special Hosts-------------------------------------------------------------------------------------------- 501–503 Chapter 81. Antimicrobial Chemotherapy – A Short Review -------------------------------------------------------------------- 504–507 Chapter 82. Multiple Choice Questions-------------------------------------------------------------------------------------------- 508–510 Self-Assessment ------------------------------------------------------------------------------------------------------------------------- 511–527

Ref: CDC

CORONA VIRUS

 

Coronaviruses are named for the crown-like spikes on their surface. There are four main sub-groupings of coronaviruses, known as alpha, beta, gamma, and delta.

 Latest Questions Papers 2020–2019

Recent Outbreak 2020

HUMAN TYPES 1. 2. 3. 4. 5. 6. 7.

229E (alpha coronavirus) NL63 (alpha coronavirus) OC43 (beta coronavirus) HKU1 (beta coronavirus) MERS-CoV (the beta coronavirus that causes Middle East Respiratory Syndrome, or MERS) SARS-CoV (the beta coronavirus that causes severe acute respiratory syndrome, or SARS) 2019 Novel Coronavirus (2019-nCoV)

Electron microscopic image of corona virus Courtesy: National Institute of Allergy and Infectious Diseases (NIAID)

NOVEL CORONA VIRUS yy

yy

yy

yy

yy

2019 Novel Coronavirus (2019-nCoV) is a virus identified as the cause of an outbreak of respiratory illness first detected in Wuhan, China. Incubation period: 2 – 14 days Route of transmission: Droplets (respiratory secretions) Precautions: N95 Mask, gloves, fluid repellent gowns, goggles Clinical features:  Fever, cough, shortness of breath  In few people it develops pneumonia  No specific age predilection seen Diagnosis: Sequencing (other methods yet to develop); Testing done only in NIV, Pune for India. No antiviral drugs/vaccines currently not available. Antiviral combinations like lopinavir and ritonavir give promising improvement. But efficacy of the drug for novel corona virus is not proven by clinical trials. 





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LATEST EXAM QUESTIONS 2020–2019

1. Which of the following is the vector for Zika virus? a. Aedes aegypti b. Culex tritaeniorhynchus c. Phlebotomus papatasii d. Anopheles 2. A 30-year-old man reports to the OPD with a history of clean-cut wound before four hours. He had already received a dose of TT before 12 years. What is the ideal vaccination regimen for him? a. Single dose of TT b. Full dose of TT c. Simultaneous administration of TT and TIG d. No need of vaccine 3. What is the immunological method depicted in the following rapid test assay for Hepatitis B?













































7. The following fungal infection occurs most commonly due to thorn prick: a. Blastomycosis b. Sporotrichosis c. Coccidioidomycosis d. Paracoccidioidomycosis 8. An 8-year-old female is diagnosed with meningococcal meningitis. What advice should be given to children of household and school contacts with the primary case to avoid outbreak? a. Single dose of meningococcal Men4B vaccine b. Erythromycin prophylaxis c. Chemoprophylaxis and two doses of vaccine d. Single dose of PCV vaccine 9. An HIV patient with CD4 count less than 200/Cu.mm came to OPD with whitish lesions in the mouth as given in the image. Mucosal biopsy on HPE shows yeast and pseudohyphae. What is the diagnosis? a. Oral hairy leukoplakia b. Oral candidiasis c. Lichen planus d. Kaposi sarcoma 10. Which among the following is a category A bioterrorism agent? a. Coxiella b. Brucella c. Nipah virus d. Bacillus anthracis 11. Which of the following organelle in a bacterium helps in adhesion? a. Cytoplasmic membrane b. Mesosomes c. Fimbriae d. Lipopolysaccharide 12. Identify the following immunoglobulin





























 

 Latest Exam Questions 2020–2019

RECENT PATTERN QUESTIONS 2020





























































a. ELISA b. Immunochromatography c. CLIA d. Immunofluorescene 4. Identify the species from the following peripheral smear of a man who had fever with chills.

















a. IgM b. IgA c. IgG d. IgD 13. Stool examination of a child is showing the following soil transmitted helminthic egg. What is the diagnosis?













Courtesy: CDC/Dr. Mae Melvin



a. Plasmodium vivax b. Plasmodium falciparum c. Plasmodium ovale d. Plasmodium knowlesi 5. The active disinfectant component in bleaching powder is: a. Hypochlorous acid b. Hypochlorite c. Chlorine d. Hydrogen 6. What is the etiological agent for donovanosis? a. Klebsiella granulomatis b. Hemophilus ducreyi c. Leishmania donovani d. Treponema pallidum







































xiv



Courtesy: CDC/B.G. Partin













a. Ascaris lumbricoides c. Trichiuris trichiura







b. Hook worm d. Taenia solium



 

18. HIV patient presented with diarrhea. On stool examination, following acid fast organisms was seen. What is the drug of choice in this patient?

 Latest Exam Questions 2020–2019



14. The mechanism of resistance to penicillin by production of beta lactamases results in: a. Drug efflux b. Alteration in penicillin binding proteins c. Breaks the drug chemical bonding d. Decreases MIC 15. An eight-year-old child presented to casualty with acute febrile illness and petechial rash. What is the most likely diagnosis?





a. Meningococci b. Pneumococci c. Hemophilus influenza d. Malaria

RECENT PATTERN QUESTIONS 2019 16. True about Congenital Rubella syndrome is: a. It will become a chronic infection b. Virus can be isolated only upto 6months after birth c. Triad of CRS are cataract, cardiac defects, cerebral palsy d. Infection is most serious after five months of pregnancy

17. A 9 years old child presented to OPD with complaints of high grade fever, vomiting, one episode of seizure. CSF examination was done and Gram staining of the culture showed the following finding. What is the probable causative agent?



a. b. c. d.

Hemophilus influenzae Neisseria meningitidis Streptococcus pneumoniae Escherichia coli

a. TMP-SMX b. Nitazoxanide c. Primaquine d. Niclosamide 19. Diagnostic method of choice for leptospirosis: a. Cold agglutination test b. MSAT c. MAT d. Latex agglutination test 20. Investigation of choice for neurosyphilis: a. VDRL b. FTA-ABS c. RPR d. TPI 21. A person working in an abattoir presented with malignant pustule on hand; What is the causative agent? a. Clostridium botulinum b. Clostridium perfringens c. Bacillus anthracis d. Streptococcus pyogenes 22. Ideal dose of Diphtheria antitoxin given for treatment is: a. 10,000 to 1,00,000 units b. 20,000 to 1,00,000 units c. 10,000 to 2,00,000 units d. 20,000 to 2,00,000 units 23. Infection that causes acute febrile illness with jaundice and conjunctivitis is: a. Malaria b. Leptospirosis c. Pertussis d. Typhoid 24. A neonate was found to have cataract, deafness and cardiac defects. Which group of viruses does the mother was infected with: a. Togaviridae b. Flaviviridae c. Bunyaviridae d. Arenaviridae 25. Which vaccine is contraindicated in pregnancy? a. Hepatitis A b. Hepatitis B c. Rabies d. Chicken pox 26. Which vaccine strain is changed every yearly? a. Influenza b. Rabies c. Hepatitis d. Ebola 27. A 5 years old child presented to the OPD with complaints of rectal prolapse; On examination stunting and growth retardation was documented; What is the parasitological cause for this clinical feature? a. Trichuris trichiura b. Trichinella spiralis c. Giardia lamblia d. Enterobius vermicularis 28. Flask shaped ulcers seen in a dysentry patient is diagnostic of: a. Shigellosis b. Amoebiasis c. Giardiasis d. Typhoid

xv

 

 Latest Exam Questions 2020–2019

29. A 35 years old man presented with dry cough and rusty colored sputum; He has history of eating in chinese restaurant very often with consumption of crabs often; What is the probable causative agent in this condition? a. Diphyllobothrium latum b. Pneumocystis jirovecii c. Paragonimus westermani d. Strongyloides stercoralis 30. Culture media for Legionella: a. BCYE agar b. Baird Parker medium c. Macconkey agar d. PLET medium 31. Special stain for Cryptococcus: a. Ziehl-Neelsen stain b. Mucicarmine stain c. Malachite green d. Albert stain 32. Disk diffusion method is also known as: a. Kirby Bauer method b. E test-method c. MIC method d. Stokes method 33. A child is suffering from recurrent chronic infections with encapsulated bacteria; Which subclass of IgG does the child has deficiency? a. IgG1 b. IgG2 c. IgG3 d. IgG4

34. Contact isolation is done for: a. Mumps b. MRSA c. Diphtheria d. Typhoid 35. Coxsackie virus A causes: a. RMSF b. HFMD c. Yellow fever d. Pleurodynia 36. Infection of following organism has clinical features resembling erythroblastosis foetalis? a. Cytomegalovirus b. Ebstein Barr virus c. Toxoplasmosis d. Herpes virus 37. All are true about congenital toxoplasmosis except: a. Chorioretinitis b. Jaundice c. Macrocephaly d. Cerebral calcification 38. An AIDS patient presented to OPD with dyspnoea and respiratory illness; Which of the following is suitable to diagnose the opportunistic infection commonly seen in AIDS patient? a. Sputum microscopy b. Broncho alveolar lavage (BAL) c. Chest Xray d. CT scan

39. Identify the following life cycle:

xvi



a. Plague

b. Japanese encephalitis



c. Influenza

d. Nipah virus

AIIMS NOVEMBER 2019

 

 Latest Exam Questions 2020–2019

40. Which of the following toxin acts by this mechanism as shown in the figure:



a. Tetanus

b. Botulism



c. Neuroborreliosis

d. Neurosyphilis







41. The CLED medium is preferred over other media for the culture of the organisms in case of UTI because:a. It inhibits proteus swarming b. It differentiates Lf from NLF c. It helps growth of candida and Staphylococcus d. It identifies pseudomonas 42. Which of the following organism cause multiple alveolarlike mass in liver? a. Echinococcus multilocularis b. E. granulosus c. Amoebic liver abscess d. Cysticercus cellulose 43. A 25-Year-old man presents with urethral discharge for the last three days. A gram stained smear of the discharge is shown in the figure. All of the following are true about the likely etiology except:-

44. A Giemsa stain of a thin peripheral blood smear is prepared. Which of the following cannot be diagnosed? a. Bartonella henselae b. Coxiella burnettii c. Toxoplasma gondii d. Ehrlichia chaffeensis 45. Mechanism of action of toxin produced by the bacterium shown in the figure:





a. Virulence factor Pili b. Intracellular c. Both catalase and oxidase positive d. Show twitching motility

a. Increase in cAMP b. Increase in cGMP c. ADP-ribosylation of ribosyl transferase d. ADP-ribosylation of Gs protein 46. All can be seen intracellularly in hepatocytes except: a. Plasmodium b. Toxoplasma gondii c. Leishmania d. Babesia 47. Beta-1, 3-D glucan assay test is used to diagnose all except: a. Aspergillosis b. Mucormycosis c. Candidiasis d. Pneumocystis

xvii

AIIMS MAY 2019

48. True about γδ T cell; except: a. It is type of helper T cells b. It is an antigen presenting cell c. It resides in gastrointestinal tract epithelium d. It shows direct cytotoxicity.







51. A 40-year-old male from Himachal Pradesh has presented with verrucous lesions in the skin with following microscopic findings from the tissue biopsy. What is the diagnosis?











49. Assertion: Anaphylactoid reaction can be caused by 1st exposure. Reason: Involves mast cell degranulation but not IgE. a. Both Assertion and Reason are independently true/ correct statements and the Reason is the correct explanation for the Assertion b. Both Assertion and Reason are independently true/ correct statements, but the Reason is not the correct explanation for the Assertion c. Assertion is independently a true/correct statement, but the Reason is independently a false/incorrect statement d. Assertion is independently a false/incorrect statement, but the Reason is independently true/correct statement e. Both Assertion and Reason are independently false/ incorrect statements









 

 Latest Exam Questions 2020–2019







 



























a. b. c. d.

Anti HbC Anti HbS HBsAg Anti HBe



















a. Diluted Carbol Fuchsin, 20% H2SO4 and Methylene blue b. Strong Carbol Fuchsin, 0.5% H2SO4 and Loeffler’s methylene blue c. Strong Carbol Fuchsin, 1% H2SO4, iodine and methylene blue d. Diluted Carbol Fuchsin, 5% H2SO4 and methylene blue 53. Arrange the order of Gram staining from below: I. Mordant II. Acetone alcohol III. Crystal violet IV. Diluted carbol fuschin





















50. Patient has recovered from a hepatitis B infection. Identify the serological marker shown by the arrow in the image below:



a. Chromoblastomycosis b. Sporotrichosis c. Rhinosporidiosis d. Mycetoma 52. An HIV patient with fever, chronic diarrhea presented to OPD. His stool was examined which showed the following oocysts. Identify the components used for staining the stool?



















a. I, II, III, IV b. III, I, II, IV c. IV, II, III, I d. III, II, I, IV 54. Match the following with regards to Biomedical waste management:













Column-A

Column-B

1. Yellow 2. Red 3. Blue 4. White transparent

a. Glassware globes b. Scalpel blade c. Chemical waste d. syringe wrapper















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a. 1-C, 2-D, 3-A, 4-B c. 1-A, 2-B, 3-C, 4-D







b. 1-D, 2-A, 3-B, 4-C d. 1-B, 2-C, 3-D, 4-A



 

61. A 22-year-old college going adult has H/o sexual exposure with commercial sex worker, now presenting with painful genital lesions and lymphadenopathy in right inguinal region. Identify the diagnosis?

 Latest Exam Questions 2020–2019

55. A 48-year-old man, hospitalized for 17 days and on IV antibiotics followed by oral course of drugs for Acute febrile illness with neutropenia. He had a history of dysuria while admitted and now complaints of loose stools and abdominal pain. Which antibiotic usage has the highest risk for developing pseudomembranous colitis? a. Carbapenems b. Aminopenicillin c. Macrolides d. Fluoroquinolones 56. Which among the following is a subcutaneous fungal agent? a. Cryptococcus neoformans b. Histoplasma capsulatum c. Sporothrix schenckii d. Talaromyces marneffi 57. Choose the wrong statement about Plasmodium species: a. Drug resistance falciparum not seen in India b. Presence of Duffy blood group antigen is protective for vivax c. Cerebral malaria with shock by P.falciparum is called algid malaria d. Relapse is seen in P.ovale 58. Mw vaccine extracted from which bacteria: a. M. indicus pranii b. M. Welchii c. M. leprae d. M. bovis 59. A hospital has reported an outbreak of MRSA infection. On investigation, it was found that staff nurses and doctors had nasal carriage of MRSA. Which of the following drug helps in removal of colonization? a. Oral vancomycin b. Inj cephalosporin c. Topical bacitracin d. Inhaled colistin 60. The following vector is not involved in transmission of this disease:



a. Syphilis b. Chancroid c. LGV d. Gonorrhea 62. Which among the following occupation is a risk factor for this presenting illness?



a. A lifeguard in swimming pool b. A poultry worker c. Farmer d. A kennel worker 63. Transmission assessment survey is carried out for: a. P. vivax b. P. falciparum c. Filariasis d. Leishmania 64. Which immunoglobulin is elevated in a case of chronic allergy? a. IgA b. IgM c. IgE d. IgG 65. A patient who got exposed to Hepatitis B infection; which of the following markers will always be present in the patient even he becomes chronic or recurrent? a. HbsAg b. Anti HBs Ab c. HbcAB d. HbeAg

JIPMER MAY 2019

a. b. c. d.

Kyasanur forest disease Kala azar Chandipura encephalitis Carrion`s disease



66. Auto infection is not seen in: a. Hymenolepis nana b. Hymenolepis diminuta c. Strongyloidosis stercoralis d. Taenia solium

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 Latest Exam Questions 2020–2019

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67. The smallest helminth among the following is: a. Hymenolepis nana b. Hymenolepis diminuta c. Diphyllobothrium latum d. Balantidium coli 68. Tungiasis is a. Ectoparasitic skin infection b. Ectoparasitic intestinal infection c. Endoparasitic skin infection d. Endoparasitic intestinal infection 69. The gold standard investigation for Zika virus is: a. PCR b. Plaque reduction neutralization test c. IgM antibodies d. RT – PCR 70. Gene responsible for semi synthetic penicillin resistance in Methicillin Resistant Staphylococcus aureus (MRSA): a. Mec A b. Mec B c. Mec C d. Mec D 71. Panton Valentine leukocidin is produced by a. Staphylococcus epidermidis b. Pseudomonas c. Vibrio cholerae d. Staphylococcus aureus 72. Which of the following is the amplifying host for Japanese encephalitis? a. Pig b. Heron c. Horse d. Egrets 73. Transovarian transmission is seen among: a. Ricketssia akari b. Ricketssia ricketssii c. Ricketssia prowazekki d. Ricketssia typhi 74. A retroviral positive patient presents with frequent diarrheal episodes; Stool examination revealed acid fast oocyst of size 8-10 uns. What could be the probable diagnosis? a. Toxoplasma gondii b. Cryptosporidium parvum c. Cyclospora cayetanensis d. Sarcosystis hominis 75. Encrusted cystitis is caused by: a. Corynebacterium xerosis b. C. urealyticum c. C.renale d. C.pseudotuberculosis 76. Elek’s gel precipitation test is used as a confirmatory test for: a. Corynebacterium jeikeium b. C.diphtheriae c. Clostridium tetani d. Cl.perfringens 77. Which among the following is a cocco bacilli: a. Escherichia coli b. Listeria monocytogenes c. Haemophilus influenzae d. Acinetobacter baumannii 78. All are true about immune re constitution inflammatory syndrome (IRIS) except: a. It occurs only when CD4 cell count is less than 50 b. Develops after initiation of anti retro viral therapy c. Associated with delayed type of hypersensitivity d. Does not require specific antimicrobial therapy for recover

79. Which among the following is a bile esculin positive organism and also shows growth in 6.5% Sodium Chloride? a. Streptococcus agalactiae b. Streptococcus pneumoniae c. Enterococcus faecalis d. Streptococcus viridans 80. All of the following Mycobacteria are pigmented except: a. M.kansasii b. M.simiae c. M.avium d. M.scrofulaceum

PGI MAY 2019 81. True about Chikungunya virus: a. It is a DNA virus b. Aedes aegypti is the vector responsible for transmission c. Commonly presents as fever and arthralgia d. In acute phase of illness, virus cannot be isolated from blood e. Persistent virus presence in muscle and joint space may be cause of chronic arthralgia 82. True about Dengue fever: a. Also called as Saddle back fever b. Symptoms appear within 48 hours of infection c. Serum aminotransferase level may be increased d. Rat is the reservoir of the virus e. Maculopapular rashes over trunk and limbs 83. Which of the following organism can be cultured on Lowenstein-Jensen (LJ) medium: a. Mycobacterium tuberculosis b. Mycobacterium leprae c. Mycobacterium bovis d. Mycobacterium fortuitum e. Nocardia 84. True statement(s) about latent TB infection is/are: a. Significant chest x-ray findings b. Rifampicin and INH are given for treatment c. Standard TB regimen is used for treatment d. Interferon Gamma Release Assay (IGRA) test is used for diagnosis e. Mantoux skin test is negative 85. All are true about Diphtheria in children except: a. Incubation period is 1-6 days b. Nasal form is most fatal form c. Pharyngotonsillar diphtheria usually present as sore throat d. Membrane can cover pharynx, tonsils, soft and hard palate e. Tetracycline is given for treatment 86. True about serology in an individual with acute hepatitis B which is highly infectious: a. HBsAg b. Anti HBs c. IgM anti HBc d. HBeAg e. IgG anti HBc 87. Type II hypersensitivity reaction(s) is/are: a. Rheumatoid arthritis b. Arthus reaction c. Serum sickness d. Autoimmune haemolytic anemia e. Good pasture syndrome









92. True about Mucor: a. Unicellular organism having sporangium b. Aseptate hyphae c. (1–3)-β-D-glucan assay is helpful in diagnosis of invasive mucormycosis d. Colony grow very rapidly and changes color e. Filamentous fungus found in decaying fruits and vegetables 93. Reverse algorithm for the diagnosis of syphilis is/are: a. VDRL then RPR b. RPR then VDRL c. TPPA followed by VDRL d. Enzyme immunoassay (EIA) followed by RPR e. RPR followed by Enzyme immunoassay (EIA) 94. Among the following pathogenic protozoa, which is/are the member of mastigophora; a. Toxoplasma gondii b. Trypanosoma cruzi c. Balantidium Coli d. P. vivax e. Giardia lamblia 95. Which of the following dyads of HIV virus structural proteins/components is/are correct: a. gp 120: Core Ag b. gp 41: Envelop Ag c. p17: Shell Ag d. p15: Core Ag e. p24: polymerase Ag

 



 Latest Exam Questions 2020–2019

88. Gold standard investigation for brucellosis: a. Radioimmunoassay b. ELISA c. Blood culture d. Standard agglutination test e. RT-PCR 89. Which of the following statement(s) is/are true about Melioidosis: a. Caused by parasite found in soil and water b. Main source of infection is contaminated water or inhalation c. Presents with pneumonia and skin abscess d. May takes years before becoming symptomatic e. Eradication treatment is required with cotrimoxazole or doxycycline for at least 12 weeks 90. Which of the following can be used to culture Ureaplasma urealyticum: a. PPLO broth b. MacConkey agar c. Blood agar d. Alkaline peptone water e. SP4 medium 91. Mycoplasma genitalis causes: a. Cervicitis b. Non-gonococcal urethritis c. Bacterial vaginosis d. Urinary stones e. Pneumonia

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 Latest Exam Questions 2020–2019

ANSWERS AND EXPLANATIONS RECENT PATTERN QUESTIONS 2020

4. Ans.  (b) Plasmodium falciparum Ref: T.B. of medical parasitology – S.C. Parija – 4th edition – Page 114;

1. Ans.  (a) Aedes aegypti Ref: Ananthanarayan and Paniker’s Textbook of Micro­ biology – 10th ed – Page 530 Vector and arboviral illness Mosquito borne

Chikungunya—Aedes Dengue—Aedes Yellow fever—Aedes Zika virus—Aedes JE—Culex

Tick borne

KFD Crimean Congo hemorrhagic fever Colorado tick fever

Rodent borne

Hanta virus Lassa fever Hemorrhagic fever with renal syndrome

P.vivax

P.falciparum P.malariae

Ref: Ananthanarayan and Paniker’s Textbook of Micro­ biology – 10th ed – Page 266 Wound less than 6 hours, old, clean, non penetrating, with negligible tissue damage

Other wounds

A. Complete course of toxoid or booster in previous 5 years

Nothing needed

Nothing

B. Complete course of toxoid or booster in previous 5 -10 years

Toxoid 1 dose

Toxoid 1 dose

C. Complete course of toxoid or booster dose in > 10 years of age

Toxoid 1 dose

D. Has not had a complete course of toxoid or unknown status of vaccination

Toxoid complete course

Diagnostic forms seen in peripheral smear

Trophozoites Schizonts Gametocytes

Ring forms Trophozoites Trophozoites Gametocytes Schizonts Schizonts (banana Gametocytes Gametocytes shaped)

Infected RBC

Enlarged

Normal

Dots Others

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Ziemann’s dots

James’ dots

Relapse

-

Relapse

Recrudescence

• Bleaching powder is Sodium hypochlorite in powdered form • The active form of all chlorinated compounds are hypochlorous acid •

•

• Earlier called as Calymmatobacterium granulomatis • Gram negative rod; It has characteristic safety pin appearance • It causes a venereal disease named as donovanosis or granuloma inguinale • Lab diagnosis: Demonstration of donovan bodies in Giemsa stain from lesions in the genital area • DOC: Tetracycline •

•

Toxoid 1 dose and human tetanus IG Toxoid complete course + human tetanus Ig

Immunochromatographic test: • Rapid test method • Done in a small cassette which is already impregnated with antibody/antigen with colloidal gold dye conjugate • When antigen – antibody reaction occurs – it is visible as colored product • Used in diagnosis of HIV, HBV, HCV and many other infections •

Schuffner’s Maurer’s dots dots

Ref: Park T.B. of Social and preventive medicine

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7. Ans.  (b) Sporotrichosis Ref: Ananthanarayan and Paniker’s Textbook of Micro­ biology – 10th ed – Page 606 Sporotrichosis: • Causative agent: Sporothrix scheckii (Thermally dimorphic) • Fungal infection affecting cutaneous, subcutaneous and lymphatic tissue • Most commonly affects gardeners, forest workers → After a minor trauma (Rose thorn prick) → nodules formed → ulceration → necrosis → Fixed cutaneous sporotrichosis → spread to lymphatics → lymphocutaneous sporotrichosis → systemic spread to the bones, joints and meninges • DD: Nocardiosis, Tularemia, Non-TB mycobacterial infection and leishmaniasis • Lab diagnosis: KOH/HPE – shows asteroid body • Culture: Yeast phase in 37deg C and mycelia phase in 25deg C •

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Enlarged

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Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 111

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Normal

6. Ans.  (a) Klebsiella granulomatis

3. Ans.  (b) Immunochromatography

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P.ovale

5. Ans.  (a) Hypochlorous acid

2. Ans.  (a) single dose of TT

Categories

Features

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11. Ans.  (c) Fimbriae Ref: Ananthanarayan and Paniker’s Textbook of Micro­ biology – 10th ed – Page 12

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• Fimbriae – Organ of adhesion (also called as Pili) • Flagella – Organ of locomotion •

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12. Ans.  (b) Ig A

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Ref: Ananthanarayan and Paniker’s Textbook of Micro­ biology – 10th ed – Page 237 CDC recommends post exposure prophylaxis for contacts and vaccination for children and adolescents: Vaccination: • Vaccination available for Groups A,C,Y and W-135 • Group B – vaccine available only in European union – named as 4CmenB (not in India) •

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 100 IgA: • Immunoglobulin seen in saliva, tears, colostrum, respiratory and GI secretions • Two forms are seen: •

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Serum IgA

Monomer Seen in serum

Secretory IgA

Dimer United by J chain It has a secretory component – that protects IgA from denaturation by bacterial proteases

•

Age group

Type of vaccine

6 weeks to 18 months Hib-Men-TT (combined) four doses 9 – 23 months

2-55 years

Tetravalent conjugate vaccine–two doses (conjugation with diphtheria toxoid – Menactra vaccine Tetravalent conjugate vaccine–Menveo

• Chemoprophylaxis and Treatment for carrier eradication – Rifampicin or Ciprofloxacin •

9. Ans.  (b) Oral Candidiasis

 

8. Ans.  (c) Chemoprophylaxis and two doses of vaccine

 Latest Exam Questions 2020–2019

• Serology: Slide latex agglutination test – Antigen is peptido – L- rhamno – D –mannan - ≥1:4 titre is positive (mainly helpful in pulmonary sporotrichosis) • Treatment: KI – Potassium iodide, Itraconazole, fluconazole, cryotherapy • Itraconazole is the DOC for lympho cutaneous sporotrichosis (IDSA guidelines) • Severe pulmonary or CNS spreads needs AmB lipid therapy

13. Ans.  (c) Trichiuris trichiura Ref: T.B. of medical parasitology – S.C.Parija – 4th edition – Page 265 Trichuris Trichiura: • Definitive host: Humans (the only host) • Infection is acquired by ingestion of soil with embryonated eggs (has rhabditiform larvae) • It usually affects children and remains asymptomatic; Heavy infection causes rectal prolapse in children; appendicitis; • Lab diagnosis: ƒ Demonstration of eggs in feces; Characteristics of egg are: ○ Barrel shaped ○ Bile stained egg ○ Mucous plugs at both ends – which are not bile stained •

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Ref: Ananthanarayan and Paniker’s Textbook of Micro­ biology – 10 th ed – Page 578 • Image has DD of all of the above options; But microscopy has confirmed as yeast and pseudohyphae which denotes it is oral candidiasis. • Oral hairy leukoplakia is caused by Epstein-Barr virus (EBV) infection of the oral mucosa. It most often occurs in association with  HIV infection. It has been less frequently described in immunosuppressed patients, especially following organ transplantation, and is rare in immune competent individuals. •

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Categories of Most common Bioterrorism Agents that can be Used as an Warfare Categories Features

Organisms

A

Anthrax Botulism Plague Small pox Tularemia Ebola Marburg

B

C

Easy to disseminate but low mortality

Mass dissemination is possible

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14. Ans.  (c) Breaks the drug chemical bonding Ref: Ananthanarayan and Paniker’s Microbiology – 10th ed – Page 202

10. Ans.  (d) Bacillus anthracis

High risk and gives a threat to national security

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Brucellosis Glanders Q fever Ricin toxin Nipah virus

Textbook

of

• Penicillinases are beta lactamases which are plasmid mediated.They can be transmitted by transduction or conjugation. These enzymes from the bacteria breaks the drug and inactivates it. • Another mechanism is alteration in the penicillin binding protein PBP2a and changes in bacterial surface receptors reduces binding affinity of beta lactam antibiotics to cells. •

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15. Ans.  (a) Meningococci Ref: Ananthanarayan and Paniker’s Textbook of Micro­ biology – 10th ed – Page 235 • Most common form of infection is asymptomatic carriage of organism in nasopharynx. • A non blanching petechial rash is seen in >80% of the cases. •

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• Meningococcal meningitis presents as fever, vomiting, irritability and head ache. The classical signs like neck rigidity and photobhobia are usually not seen in infants and children. • Along with meningitis, features of septicemia are also seen in 20-40%. • Septicemia can go for shock and death. • Chronic meningococcemia can present as rash, fever, joint pain, arthritis and splenomegaly. • Post meningococcal sequelae is seen in some patients which manifest as arthritis, serositis and pericarditis.

 

 Latest Exam Questions 2020–2019

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Acid fast oocysts Cryptosporidium parvum Isospora belli Cyclospora cayetanensis

Treatment of choice In AIDS patients: Nitazoxanide Others: Paromomycin and Spiramycin TMP-SMX Self limiting; severe cases – TMP-SMX

19. Ans.  (c) MAT Ref: Harrisons T.B of internal medicine - 19th edition – page 1144 • A definitive diagnosis of leptospirosis is based on: ƒ Isolation of the organism from the patient or ƒ A positive result in the polymerase chain reaction (PCR) or ƒ Seroconversion or a rise in antibody titer. (MAT) • In cases with strong clinical evidence of infection, a single antibody titer of 1:200–1:800 (depending on whether the case occurs in a low- or high-endemic area) in the microscopic agglutination test (MAT) is required. •

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Recent Pattern Questions 2019 16. Ans.  (a) It will become a chronic infection

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Ref: Jawetz TB of medical microbiology – 27th edition – page 597 • Rubella infection gets transmitted by vertical transmission and birth defects are more common when acquired during first trimester of pregnancy. • Birth defects are uncommon when infection is acquired after 20 weeks of gestation. • Congenital Rubella syndrome leads to cardiac defects, cataract and deafness; (Classical triad). Other manifestations are hepatosplenomegaly, thrombocytopenic purpura, myocarditis and bone lesions. • Intrauterine infection of Rubella is associated with chronic persistence of disease. • Viral detection can be done in all fluids and it is excreted upto 12-18 months of age. •

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17. Ans.  (c) Streptococcus pneumoniae Ref: Ananthanarayan and Paniker T.B of microbiology – 10th ed – page 225 • Gram staining clearly shows Gram positive cocci (violet/purple colored) in pairs – classical image of Streptococcus pneumoniae or pneumococci • Clinical features also helpful to confirm the diagnosis • Infections caused by Pneumococcus: ƒ Pneumonia ƒ Meningitis ƒ Otitis media ƒ Bacteremia ƒ Septic arthritis • It is the most important cause of infections in splenectomy patients, chronic alcoholics, sickle cell anaemia patients. •

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20. Ans.  (a) VDRL Ref: Harrisons T.B of internal medicine - 19th edition – page 1136 • Sample of diagnosis for neurosyphilis is CSF • Only tests that can be done for neurosyphilis are: ƒ VDRL ƒ FTA-ABS • The diagnosis of asymptomatic neurosyphilis is made in patients who lack neurologic symptoms and signs but who have CSF abnormalities including mononuclear pleocytosis, increased protein concentrations, or CSF reactivity in the VDRL test. • When VDRL is negative, FTA-ABS is done to confirm the test. • Ideally both tests here are used in diagnosis; But when you have to choose single best option – best is VDRL according to CDC site and Mandells ID book. •

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21. Ans.  (c) Bacillus anthracis Ref: Ananthanarayan and Paniker’s Textbook of Micro­ biology – 10th ed – Page 250 • Clinical clue here are: abattoir who has contact with animal products; • Classical malignant pustule is seen in anthrax. It occurs usually in face and neck • A papule is seen at the site of entry followed by vesicle and necrotic ulcer. • The lesion characteristic of cutaneous anthrax is central black eschar; This lesion is called as malignant pustule •

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18. Ans.  (b) Nitazoxanide Ref: T.B of medical parasitology – S.C.Parija – 4th edition – page 151 • HIV patient with diarrhea having acid fast organisms shows that organism may be: ƒ Cryptosporidium parvum ƒ Isospora belli ƒ Cyclospora cayetanensis • Image shows 4-5um sized oocysts – suggestive of Cryptosporidium •

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Central black eschar in cutaneous anthrax (Courtesy: CDC/F. Marc LaForce, MD)

22. Ans.  (b) 20,000 to 1,00,000 units

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of

• Specific treatment of diphtheria consists of antitoxic and antibiotic therapy. • Antitoxin should be given immediately when diphtheria is suspected, as the fatality rate increases with delay in starting antitoxic treatment. • Antibiotic treatment only supplements and does not replace antitoxic therapy. • Diphtheria antitoxin should be given in respiratory diphtheria – 20,000 to 1,00,000 units •

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23. Ans.  (b) leptospirosis Ref: Harrisons T.B of internal medicine - 19th edition – page 1143

27. Ans.  (a) Trichuris trichiura Ref: T.B. of medical parasitology – S.C.Parija – 4th edition – page 265 • Clinical features of rectal prolapse is a direct clue for diagnosis: Trichuriasis • This infection is acquired by ingestion of soil with embryonated eggs (has rhabditiform larvae) • It usually affects children and remains asymptomatic • Heavy infection causes rectal prolapse in children; appendicitis; • Lab diagnosis: Demonstration of barrel shaped eggs with mucous plugs in feces •

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Textbook

 Latest Exam Questions 2020–2019

Ref: Ananthanarayan and Paniker’s Microbiology – 10th ed – Page 245

• Because of these variations, vaccines should be modified according to the current prevalent strain.

Differential diagnosis for acute febrile illness are: Infections Dengue Malaria Chikungunya Leptospirosis Scrub typhus Enteric fever

Classical symptoms Fever + Arthralgia + Rash Intermittent fever + Splenomegaly + chills Fever + Arthralgia Fever + Jaundice + Conjunctivitis Fever + Eschar Fever + Splenomegaly

24. Ans.  (a) Togaviridae Ref: Jawetz TB of medical microbiology – 27th edition – page 597 • Congenital Rubella syndrome leads to cardiac defects, cataract and deafness; (Classical triad). Other manifestations are hepatosplenomegaly, thrombocytopenic purpura, myocarditis and bone lesions. • Rubella belongs to Togaviridae

Figure 3: Egg of Trichuris trichiura (Courtesy: CDC/B.G. Partin) 28. Ans.  (b) Amoebiasis

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25. Ans.  (d) Chicken pox Ref: Harrisons T.B of internal medicine - 19th edition – page 1143 • All live attenuated vaccines are contraindicated in pregnancy. • Examples for live attenuated vaccines are: a. OPV b. Yellow fever vaccine c. Varicella zoster vaccine (chicken pox) d. MMR e. Influenza (attenuated vaccine) •

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26. Ans.  (a) Influenza

Ref: T.B. of Medical Parasitology–S.C.Parija–4th edition– Page 33 • Clinical features of amoebiasis are: a. Intestinal amoebiasis – characteristic flask shaped ulcers b. Amoebic liver abscess – Anchovy sauce pus c. Lung abscess d. Brain abscess •

29. Ans.  (c) Paragonimus westermani Ref: TB of Medical Parasitology–S.C.Parija–4th edition– Page 235 A patient with history of crab eating and respiratory symptoms gives clinical clue for Paragonimus westermani infection • First intermediate host–Snails • Second intermediate host–Fresh water crab or crayfish • Infective form–Metacercariae • It causes paragonimiasis in pulmonary system; it causes a granulomatous reaction that leads to blood mixed sputum–consists of golden brown eggs; a fibrous tissue that may go for cavitation in some cases; • Treatment: Praziquantel •

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Ref: Harrisons T.B of internal medicine - 19th edition – page 1209 • Influenza virus has two important antigens: Haemagglutinin (H) and Neuraminidase (N) • These antigens undergo periodic antigenic variations Major antigens variations are seen only with influenza A viruses and may be associated with pandemics – called as antigenic shifts. • Minor variations causing outbreaks are called as antigenic drifts. •

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30. Ans.  (a) BCYE agar Ref: Harrisons T.B of medicine – 19th ed – page 1018 • Legionella isolation is done from respiratory secretions – culture media is BCYE agar • Buffered Charcoal Yeast Extract Agar (BCYE) •

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• Baird Parker agar – Staphylococci • MacConkey agar – Urine sample – to differentiate LF and NLF • PLET medium – Bacillus cereus 31. Ans.  (b) Mucicarmine stain •

38. Ans.  (b) Broncho alveolar lavage (BAL)

 

 Latest Exam Questions 2020–2019

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Ref: Paniker T.B of microbiology – 10th ed – page 617 • Stains for Cryptococcus: India Ink stain (negative stain) done for capsule demonstration and Mucicarmine stain in HPE • Mucicarmine stain is also used for Rhinosporidium. •

Ref: Greenwood – medical microbiology – 18th ed – page 570 • Pneumocystis jirovecii pneumonia is more common in AIDS patients. • Fever, unproductive cough, progressive shortness of breath are presenting complaints. • Diagnosis is confirmed by: ƒ Fungal cysts (Toluidine blue) from BAL ƒ Fungal DNA dectection by PCR •

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32. Ans.  (a) Kirby-Bauer method Ref: Paniker T.B of microbiology – 10th ed – page • Kirby Bauer method is the conventional method where antibiotic disks are kept in equal distance in a lawn culture of bacterium in Mueller Hinton agar. • E test is Epsilometer test used for MIC detection •

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39. Ans.  (b) Japanese encephalitis Ref: Greenwood – medical microbiology – 18th ed – page 530 • From the image, it is understood that: ƒ Vector – mosquito (Culex) ƒ Host and reservoir – Pigs (amplifiers) and Herons ƒ Accidental host – Humans • The agent is Japanese encephalitis •

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33. Ans. (b)  IgG2 Ref: Review of medical microbiology and immunology – 13th ed – page 1127 • IgG has four subclasses: IgG1, IgG2, IgG3 and IgG4 • IgG2 antibody is directed against polysaccharide antigens and hence it is most important defence against encapsulated bacteria. •

AIIMS NOVEMBER 2019 40. Ans.  (b) Botulism

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34. Ans.  (c) Diphtheria Ref: Greenwood – medical microbiology – 18th ed – page 202 • Strict isolation is must for patient diagnosed with diphtheria. • Even when clinically suspected, patient must be isolated. • A staff who has known immunisation history should nurse the patient. •

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35. Ans.  (b) HFMD Ref: Greenwood – medical microbiology – 18th ed – page 487 Coxsackie virus group

Clinical features

A (1-24)

• Aseptic meningitis • Febrile illness • Herpangina • Hand, foot and mouth disease (HFMD) •

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Ref: Ananthanarayan and Paniker’s TB of Microbiology – 10th Ed – Page 269 • Clostridium botulinum is a Gram positive exotoxin producing organism. • Eight types of botulinum toxins – A,B,C1, C2, D,E,F,G • All are neurotoxins except C2 (which is an enterotoxin) • Action of toxin: Powerful exotoxin will be released by the organism after death → causes proteolysis of SNARE proteins → blocks the production or release of acetyl choline at the neuromuscular junction and synapses. •

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41. Ans.  (c) It helps growth of Candida and Staphylococcus Major difference between MacConkey agar and CLED agar in urine culture is: CLED has additional advantage of: • Candida growth • Staphylococcus and other Gram-positive cocci growth •

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• Neonatal disease • Bornholm disease • Myocarditis • Hepatitis • Aseptic meningitis

B (1-6)

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36. Ans.  (a) Cytomegalovirus Ref: Greenwood – medical microbiology – 18th ed – page 439 • Clinical features of congenital CMV infection is similar to those of Erythroblastosis fetalis • Symptoms are: ƒ IUGR ƒ Hepatosplenomegaly ƒ Jaundice ƒ Thrombocytopenia ƒ Microcephaly

42. Ans.  (a) Echinococcus multilocularis Ref: T.B. of parasitology – Chatterjee – 12th Edition – page 201 • Echinococcus multilocularis causes multilocular cyst in liver – it spreads to other sites like brain and called as malignant hydatid disease • Definitive host – Foxes • Intermediate host – Rodents • Accidental host – Humans •

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•

•

•

•

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37. Ans.  (c) Macrocephaly Ref: Q.21

43. Ans.  (d) Show twitching motility Ref: Ananthanarayan and Paniker’s TB of Microbiology – 10th Ed – Page 211 • Image showing Gram staining of urethral discharge – Gram negative diplococci – Neisseria gonorrhea • Intracellular, non-motile, non-sporing organism • It is catalase and oxidase positive; Ferments glucose not maltose •

•

•

50. Ans.  (a) Anti HbC Ref: Ananthanarayan and Paniker’s TB of Microbiology – 10th Ed – Page 548

44. Ans.  (b) Coxiella burnetti Ref: Ananthanarayan and Paniker’s TB of Microbiology – 10th Ed – Page 413

HBsAg HBeAg Anti HBc Anti HBs Anti HBe Interpretation +

+

IgM

–

–

Acute HBV infection; highly infectious

+

+

IgG

–

–

Late/chronic HBV infection or carrier state; highly infectious

Ref: Ananthanarayan and Paniker’s TB of Microbiology – 10th Ed – Page 241

+

–

IgG

–

+/–

• Diphtheria toxin causes ADP ribosyl transferase action – which inactivates of ribosomal elongation factor eEF2 resulting from ADP ribosylation during protein synthesis which leads to cell death • Mechanism of other toxins are given in chapter 14 Vibrio cholerae

Late/Chronic HBV infection or carrier state; low infectivity

–

+/–

IgM

–

+/–

Seen rarely in early acute HBV infection; infectious

–

–

IgG

+/–

+/–

Remote HBV infection; infectivity nil or very low

–

–

–

+

–

Immunity following HBV vaccine

• Coxiella burnetti caused Q Fever. It belongs to Rickettsiaceae family which are obligate intracellular organisms. • They cannot be seen in routine staining; •

•

45. Ans.  (c) ADP ribosylation

•

•

46. Ans.  (d) Babesia Ref: T.B. of medical parasitology – S.C. Parija – 4th edition – page 138 • Haemo flagellates (Leishmania), Plasmodium and Toxoplasma has its life cycle in hepatic stages of human. • Babesia is seen inside RBCs. Rupture of these RBCs cause generates cell debris which gets accumulated in kidney causing renal failure and anemia. It has no cycle in liver. •

•

 

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 Latest Exam Questions 2020–2019

• Most important virulence factor is pili – outer membrane protein

AIIMS MAY 2019 51. Ans.  (a) Chromoblastomycosis

47. Ans.  (b) Mucormycosis Ref: T.B of Mycology – Jagdish Chander – 3 Edition – Page 353

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th ed – page 605

• Invasive fungal infections especially due to invasive candidiasis and aspergillosis can be detected by 1,3 beta D glucan assay. It is also called as G test. • The test is helpful in detection of pneumocystis.

• Chromoblastomycosis is a fungal infection caused by pigmented fungi (also called as dematiaceous fungi) • It most commonly affects the agricultural workers and woodcutters, and usually follows a trauma. • It produces verrucous, warty, cutaneous nodules. Hence also called as verrucous dermatitis. • Agents: ƒ Fonsecaea spp., ƒ Exophiala spp., ƒ Cladophialophora • Lab diagnosis: KOH mount or tissue biopsy and staining of the lesions shows – irregular, dark brown bodies with septae (yeast like bodies) – called as Sclerotic bodies (seen in image as brown colored) • Treatment: Amphotericin B, Itraconazole

rd

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•

•

48. Ans.  (c) It resides in gastrointestinal epithelium Ref: Ananthanarayan and Paniker’s TB of Microbiology – 10th Ed – Page 133 • T cell has two types of receptors – αβ or γδ • Most common receptors are αβ T cell type; T cells are seen in the white pulp of the spleen and periarterial lymphoid collection; It is not seen in the gastrointestinal tract. •

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49. Ans.  (b) Both Assertion and Reason are independently true/correct statements, but the Reason is not the correct explanation for the Assertion

52. Ans.  (b) Strong Carbol Fuchsin, 0.5% H2SO4 and Loeffler’s methylene blue

Ref: Ananthanarayan and Paniker’s TB of Microbiology – 10th Ed – Page 167

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th ed – page 12

Anaphylactoid reaction: • Some drugs and chemicals can simulate anaphylaxis • The reaction is due to direct activation of complements which releases anaphylatoxins; It is not IgE mediated. •

•

•

• HIV patient with diarrhea, image showing blue background with pink oocysts clearly suggests acid fast staining showing coccidian parasites (Cryptosporidium or Cyclospora) •

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 Latest Exam Questions 2020–2019

Ziehl-Neelsen staining or acid fast staining: • This method is mainly to visualize the acid-fast organisms. Organism is seen in pink color with a blue background. Procedure: • Strong carbol fuchsin with heat – primary stain • 20% sulphuric acid – decolorization (Standard method); RNTCP suggests 25% sulphuric acid • Methylene blue – Counter stain •

•

•

•

List of acid-fast organisms and % of H2SO4: Acid Fast Organism

Concentration of Sulphuric Acid

Mycobacterium tuberculosis

20% H2SO4 (WHO); 25% H2SO4 (RNTCP)

Mycobacterium leprae

5%

Nocardia

1%

Spores

0.5%

Oocyst of Coccidian parasites

0.5%

53. Ans.  (b) III, I, II, IV Ref: Ananthanarayan and Paniker’s Microbiology – 10th ed – Page 13

Textbook

of

Gram staining procedure: • Primary staining with Gentian violet, Crystal violet and Methyl violet • Mordant—iodine • Decolorization—acetone, acid alcohol, alcohol • Counterstain—diluted carbol fuschin (1:10/1:20) •

• The following table shows the risk factors for CDI • Treatment for CDI: Vancomycin and or metronidazole •

•

Table 1:  Risk factor for clostridium difficle infection (Ref: Mandells TB of infectious diseases 8th edition, page 2749) Any antibiotic versus no antibiotic: Number of antibiotics (risk increases with number) Days of antibiotics (increased risk with increased days) Type of antibiotic: Highest risk: Clindamycin, fluoroquinolones, cephalosporins of second generation and higher Moderate risk: Penicillins, macrolides, penicillin β-lactamase inhibitors, carbapenems, vancomycin, metronidazole Lower risk: Aminoglycosides, tetracyclines, trimethoprim, sulfonamides, rifampin Proton-pump inhibitors and histamine type 2 blockers Patient age (increased risk with age of the patient) Prior hospitalization Severity of underlying illness Abdominal surgery Nasogastric tube Duration of hospitalization Long-term care residency 56. Ans.  (c) Sporothrix schenckii Ref: Ananthanarayan and Paniker’s Textbook of Micro­ biology – 10th ed – Page 593 Classification of fungi based on pathogenesis

•

•

Superficial mycoses

Dermatophytosis, Candidiasis, Tinea, Piedra, Pityriasis versicolor

Cutaneous mycoses

Candidiasis

Subcutaneous mycoses

Mycotic mycetoma Chromoblastomycosis Rhinosporidiosis Sporotrichosis

Deep or systemic or visceral mycoses

Blastomycosis Cryptococcosis Paracoccidioidomycosis Coccidioidomycosis Histoplasmosis Candidiasis

Opportunistic mycoses

Mucor, Aspergillus, Candida, Penicillium

Mycotoxicoses

Aspergillus, Claviceps purpura

•

54. Ans.  (a) 1-C, 2-D, 3-A, 4-B Ref: BMW 2016 guidelines, Ministry of health and family welfare

55. Ans.  (d) Fluoroquinolones Ref: Mandells’ infectious diseases – 8th edition – page 2749

Ref: Harrison’s T.B. on internal medicine – 19 th ed – page 1371

• History of antibiotic exposure for longer period of time (even more than 7 days) is a risk factor for developing Clostridium difficle infection. This CDI is called as pseudomembranous colitis. • It is one of the most common health care associated infections in the world.

Host defenses against falciparum malaria are: • The geographic distributions of sickle cell disease • Hemoglobins C and E • Hereditary ovalocytosis • Thalassemias • Glucose- 6-phosphate dehydrogenase (G6PD) deficiency

•

•

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57. Ans.  (b) Presence of Duffy blood group antigen is protective for vivax

•

•

•

•

•

•

58. Ans.  (a) M.indicus pranii Ref: Khullar G et al., (2017), Generalized granulomatous dermatitis following Mycobacterium w (Mw) immuno­therapy in lepromatous leprosy, Dermatol Ther. 2017 Mar;30(2) • Mycobacterium w (Mw) vaccine is a heat-killed suspension derived from a non-pathogenic, cultivable, atypical mycobacterium named Mycobacterium indicus pranii. • Mw immunotherapy has been reported to be efficacious as an adjunct to multidrug therapy multibacillary regimen in leprosy patients with high bacillary index. • Cutaneous reactions are predominant adverse effects associated with the administration of vaccines. •

Table 2:  Arthropod-borne diseases Arthropod Borne Diseases Sandfly

•

•

•

Rat flea

Kala azar Sand fly fever – three days fever Oriental sore Oroya fever or Carrion’s disease Chandipura virus

• Bubonic plague • Endemic typhus • Intermediate host for H.nana •

•

•

Louse

• Epidemic typhus • Trench fever • Epidemic relapsing fever •

•

•

Trombiculid mites

• Scrub typhus

Itch mites

• Scabies

Hard tick (Ixodid)

• • • • •

•

•

•

•

•

•

•

Soft tick

Babesiosis KFD Tick typhus Q fever Tularemia

• Endemic relapsing fever •

Chandipura virus (genus Vesiculovirus, family Rhabdoviridae) causes outbreaks and leads to encephalitis. Chandipura virus encephalitis may be transmitted by sandfly bites and has been identified in Aedes aegypti mosquitoes. 

59. Ans.  (c) Topical bacitracin Ref: Harrison TB of internal medicine – 19th ed – page 959 • The anterior nares are the most common site of staphylococcal colonization in humans. • It is because of attachment of S. aureus to keratinized epithelial cells of the anterior nares. • Factors that contribute to colonization include the ƒ Influence of other resident nasal flora and their bacterial density, ƒ Host factors ƒ Nasal mucosal damage (use of inhalational drugs)

•

•

•

•

• • • • •

 

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 Latest Exam Questions 2020–2019

Host defenses against vivax malaria are: • Absence of Duffy blood group antigen; Individuals with the Duffy-negative phenotype are resistant to P. vivax invasion, and the molecular mechanism that gives rise to the phenotype Fy(a - b -) in black individuals has been associated with a point mutation - 33TC expressed in homozygosity in the FYB allele; It is most commonly seen in Africa • Duffy antigen – gene – DARC (Duffy antigen/chemokine receptor)

•

61. Ans.  (b) Chancroid

•

•

Disease

Ulcer

Lymphnodes

Primary syphilis

Chancre painless

Painless-bilateral

Chancroid

Painful

Painful-usually unilateral

Donovanosis

Painless (beefy red)

Pseudobubo (lesion appearing in groin may present like lymphadenopathy prior to rupture)

LGV

Transient

Painful usually

Herpes

Painful

Painful

ƒ

ƒ

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Colonization areas are: • Anterior nares • Damaged skin • Groin (umbilicus) • Oropharynx •

•

•

•

• Primary prevention of S. aureus infections in the hospital setting involves hand washing and careful attention to appropriate isolation procedures. •

To eradicate the colonization: • Decolonization is with topical agents (e.g., mupirocin) to eliminate nasal colonization • Chlorhexidine baths to eliminate cutaneous colonization with S. aureus •

•

60. Ans.  (c) Kyasanur forest disease

Chancroid Bubo

LGV Bubo

90% unilateral

2/3 unilateral

Matted

Not matted

Unilocular

Multilocular

Rupture with single opening

Rupture with multiple opening

Accompanied by genital ulcer

Not accompanied by genital ulcer

Groove sign negative

Groove sign positive

Ref: Harrison TB of internal medicine – 19th ed – page 13061308;

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• It is of two types: ƒ Cutaneous larva migrans ƒ Visceral larva migrans

Chancre

Chancroid

Single Painless

Multiple Painful

Clean base Bilateral inguinal nodes

Necrotic base Unilateral inguinal nodes

No exudates

Yellow exudates

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Table 3: Cutaneous larva migrans and visceral larva migrans  

62. Ans. (a) A lifeguard in swimming pool

 

Cutaneous larva migrans (CLM) is a clinical syndrome consisting of an erythematous migrating linear or serpiginous cutaneous track; It is also called as creeping eruptions Individuals at greatest risk include travelers, children, swimmers in lakes, and laborers whose activities bring their skin in contact with contaminated soil Larva migrans: • It is caused by nematodes that usually cause infection to animals • When they accidentally enter humans – it cannot complete the cycle and hence gets arrested in the body causing larva migrans •

•

63. Ans. (c) Filariasis

 

Ref: https://www.who.int/lymphatic_filariasis/global_ progress/transmission_assessment_survey/en/ • Filariasis elimination needs mass drug administration. • WHO recommends the transmission assessment survey (TAS) to determine when infections have been reduced below these target thresholds and MDA can stop. •

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 Latest Exam Questions 2020–2019

Primary syphilis – presents as chancre (Hunterian Chancre)

•

Cutaneous Larva Migrans

Visceral Larva Migrans

• • • • • • • • •

• • • •

•

•

•

•

•

•

•

•

•

Ancylostoma braziliense (M/c) Ancylostoma caninum (second M/c) Necator americanus Ancylostoma duodenale Gnathostoma spinigerum Strongloides Loa loa Fasciola Paragonimus

•

•

•

•

Toxocara canis Toxocara cati Gnathostoma Anisakis

• The question is quite a tricky one. A lifeguard in swimming pool has risk comparatively, as poultry has no link with these organisms. Most of the organisms are dog and canines’ sources. •

• Once MDA has stopped, TAS is used as a surveillance tool to determine that infection • A transmission assessment survey (TAS) is designed to evaluate whether transmission of lymphatic filariasis have reached lower level or not, based on that MDA can be stopped. •

•

Technical Aspect

Guidance

Geographical area

Evaluation unit (EU)

When survey should be conducted

• When all the eligibility criteria are met • At least 6 months after the last round of MDA

Target population

Children aged 6–7 years

Diagnostic tests

W. bancrofti areas: ICT Brugia spp. areas: Brugia RapidTM

Survey design

Cluster sampling of systematic sampling in schools or the community, or a census

•

•

A TAS is a simplified version of the ‘stopping-MDA survey’ protocol. 64. Ans. (c) IgE  

 



 Latest Exam Questions 2020–2019

Transmission assessment Survey (TAS) A TAS is the basis for a decision to move from MDA to post-MDA surveillance.

Ref: Owen Kuby – Immunology – 8th ed – page 487 • Allergy (Type I hypersensitivity) is always IgE mediated, both in acute and chronic. • E.g. Hay fever, Asthma •

•

Figure 1: Pathogenesis and mechanism of type I hypersensitivity (Ref: Kuby)

Different examples for allergies (Ref: Kuby) Disorder

Symptoms

Common trigger

Wheal and flare swellings triggered by ingestion of skin contact

Multiple foods

Notes about mechanism

IgE mediated (acute) Hives (urticaria)

Contd...

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 Latest Exam Questions 2020–2019

Disorder

Symptoms

Common trigger

Notes about mechanism

Oral allergy

Itchiness, swelling of mouth

Fruits, vegetables

Due to sensitization by inhaled pollens, producing IgE that cross-reacts with food proteins

Asthma, rhinitis

Respiratory distress

Inhalation of aerosolized food proteins

Mast-cell mediated

Anaphylaxis

Rapid, multiorgan inflammation that can result in cardiovascular failure

Peanuts, tree nuts, fish, shellfish, milk, etc.

Exercise-induced anaphylaxis

As above, but occurs when one exercises after eating trigger foods

Wheat, shellfish, celery (may be due to changes in gut absorption associated with exercise)

Atopic dermatitis

Rash (often in children)

Egg, milk, wheat, soy, etc.

May be skin T cell mediated

Gastrointestinal inflammation

Pain, weight loss, edema, and/ or obstruction

Multiple foods

Eosinophil mediated

Most often seen in infants: diarrhea, poor growth, and/or bloody stools

Cow’s milk (directly of via breast TNF-α mediated milk), say, grains

IgE and cell mediated (chronic)

Cell mediated (chronic) Intestinal inflammation brought about by dietary protein (e.g. enterocolitis, proctitis)

Adapted from S.H. Sicherer and H. A. Sampson, 2009, Food allergy, Annual Review of Medicine 60:261-277 65. Ans.  (c) HbcAB Ref: Ananthanarayan and Paniker’s Textbook of Micro­biology – 10th ed – page 550 • • • •

•

•

•

•

HbcAg – cannot be demonstrable in blood The earliest antibody marker to be identified is Anti HbcAg – IgM After six months IgG starts appearing Question dint mention IgM or IgG (hence that’s the answer)

Serological markers for diagnosing HBV infection HBsAg

HBeAg

Anti HBc

Anti HBs

Anti HBe

Interpretation

+

+

IgM

–

–

Acute HBV infection; highly infectious

+

+

IgG

–

–

Late/chronic HBV infection or carrier state; highly infectious

+

–

IgG

–

+/–

Late/Chronic HBV infection or carrier state; low infectivity

–

+/–

IgM

–

+/–

Seen rarely in early acute HBV infection; infectious

–

–

IgG

+/–

+/–

Remote HBV infection; infectivity nil or very low

–

–

–

+

–

Immunity following HBV vaccine

JIPMER MAY 2019 66. Ans.  (b) Hymenolepis diminuta

67. Ans.  (a) Hymenolepis nana Ref: Medical parasitology – Arora – 3rd ed – page 209 • Hymenolepis nana is the smallest intestinal cestode that infects human – named as Dwarf tapeworm • H.diminuta – size of egg is larger than nana •

Ref: Medical parasitology – Arora – 3rd ed – page 261 • Auto infection is caused by certain parasites, when the eggs or larvae gets entered into the body from one cavity to another. • Parasites causing auto infection are: (Mneumonic: CS – TECH) ƒ Cryptosporidium parvum ƒ Strongyloides stercoralis ƒ Taenia solium ƒ Enterobius vermicularis ƒ Capillaria philippinensis ƒ Hymenolepis nana •

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68. Ans.  (a) Ectoparasitic skin infection Ref: https://emedicine.medscape.com/article/231037treatment • Tungiasis is a neglected tropical disease caused by the permanent penetration of the female sand flea (also called jigger flea) • After penetration, most commonly on the feet, the flea undergoes an impressing hypertrophy •

•

Reservoir host →

Herons

Amplifier host →

Pigs

Vector →

Culex tritaeniorhynchus and vishnui

73. Ans.  (d) Ricketssia typhi Ref: Ananthanarayan and Paniker’s TB of microbiology – 10th Ed – page 417

Figure 2: Showing the skin lesions of tungiasis Any skin lesion is a traveller or from endemic areas, the following differentials are diagnosed: • Cutaneous larva migrans • Pyoderma • Arthropod-reactive dermatitis • Myiasis • Tungiasis • Urticaria • Fever and rash • Cutaneous leishmaniasis •

Transovarian transmission of R.typhi occurs in ticks – which acts as both vector and reservoir – caused Endemic typhus 74. Ans.  (c) Cyclospora cayetanensis

 

Humans  Latest Exam Questions 2020–2019

Host and Dead end →

Ref: T.B of medical parasitology – S.C. Parija – 4th Ed – page 155

•

•

•

•

Coccidean Parasites

Identification Features

Cyclospora cayetanensis

Oocysts are of spherical or oval; Size is around 8 – 10um; It has two sporocyts; Each sporocyts has 2 sporozoites Acid fast oocyst; Partially acid fast

Cryptosporidium parvum

Oocysts are of spherical or oval; Size is around 4-5um; It has 4 sporozoites Acid fast oocyst

Cystisospora belli

Oocyst looks ellipsoidal (boat shaped); 20-33*10-19um; Each sporocyts has 2 sporozoites; Acid fast

•

•

•

69. Ans.  (d) RT PCR Ref: https:/www.cdc.gov/zika/ • Diagnosis of zika virus – samples: Whole blood, urine, CSF, amniotic fluid, semen and saliva • Gold standard method – RT PCR • < 7 days of illness only RT PCR can detect; >7days of illness – serological tests can help. •

•

•

70. Ans.  (a) Mec A Ref: Ananthanarayan and Paniker’s TB of microbiology – 10th Ed – page 202 Resistance in Staphylococcus aureus: 1.  Production of beta lactamases 2.  Alteration of penicillin binding protein (PBP2a) 3.  Chromosomally mediated resistance – SCC mec – Mec A gene 

71. Ans.  (d) Staphylococcus aureus Ref: Ananthanarayan and Paniker’s TB of microbiology – 10th Ed – page 204 Enzymes

Toxins

• • • •

• • • • •

•

•

•

•

Coagulase Lipid hydrolases Hyaluronidase Nuclease

•

•

•

•

•

Cytolytic toxins – hemolysis Panton Valentine leukocidin Enterotoxin TSST Epidermiolytic toxin

72. Ans.  (a) Pig Ref: Ananthanarayan and Paniker’s TB of microbiology – 10th Ed – page 528

75. Ans.  (b) Corynebacterium urealyticum Ref: https://www.ncbi.nlm.nih.gov/pmc/articles/ PMC6442166/ • Encrusted cystitis is a condition caused by urea splitting bacteria. • Among the given options, C.urealyticum is urease producer and causes rarely encrysted cystitis. •

•

76. Ans.  (b) Corynebacterium diphtheriae Ref: Ananthanarayan and Paniker’s TB of microbiology – 10th Ed – page 242 Lab diagnosis of C.diphtheriae 1. Staining techniques to demonstrate the metachromatic granules – Albert staining, Neisser’s staining, Ponder’s staining 2. Growth medium – Loefflers serum slope (selective medium) 3. Toxin demonstration can be done only by: i. Animal inoculation ii. Elek’s gel precipitation test – confirmatory method iii. PCR

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 Latest Exam Questions 2020–2019

77. Ans.  (d) Acinetobacter baumannii Ref: Ananthanarayan and Paniker’s TB of microbiology – 10th Ed – page 321 Gram-positive cocci

Gram-negative cocci

Gram-positive bacilli

Gram-negative bacilli

Gram-negative cocco bacilli

Staphylococci Streptococci Enterococci

Neisseria

Corynebacterium Clostridium

E.coli Proteus Klebsiella Pseudomonas Citrobacter Enterobacter Vibrio Salmonella Shigella

Acinetobacter Haemophilus Gardnerella Chlamydia

Ref: https://www.uptodate.com/contents/immunereconstitution-inflammatory-syndrome/ • IRIS - a collection of inflammatory disorders associated with paradoxical worsening of pre-existing infectious processes following the initiation of antiretroviral therapy (ART) in HIV-infected individuals  • Increases in T lymphocytes after initiation of ART are also accompanied by increased in vitro lymphocyte proliferation responses, increased markers of immune activation, and pathogen-specific delayed hyper­ sensitivity • IRIS usually develops 1-2 days after initiation of anti retro viral therapy especially in a patient with Tuberculosis. • Patient who developed IRIS with opportunistic infections can continue ART; if no opportunistic infection, ART can be discontinued and re started again. •

•

•

•

79. Ans.  (c) Enterococcus faecalis

81. Ans.  (b) Aedes aegypti is the vector responsible for transmission; (c) Commonly presents as fever and arthralgia; (d) In acute phase of illness, virus cannot be isolated from blood Ref: Ananthanarayan and Paniker’s T.B of microbiology – 10th Ed – page 524 • Chikungunya virus is an RNA virus belongs to the family Togaviridae. • Vector is Aedes aegypti; No animal reservoir • An abrupt onset of fever with joint pain is the feature. Reactive arthritis may persist for a year. • RT PCR can be used; Viral isolation can be done but difficult. • Ideal tests: Serological ELISA techniques. •

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•

•

•

82. Ans.  (a) Also called as Saddle back fever; (b) Symptoms appear within 48 hours of infection; (e) Maculopapular rashes over trunk and limbs

Ref: Ananthanarayan and Paniker’s TB of microbiology – 10th Ed – page 219

Ref: Ananthanarayan and Paniker’s T.B of microbiology – 10th Ed – page 529

• Enterococcus species are Gram positive cocci that are arranged at an angle to each other. • Special characteristics: ƒ Temperature for growth – 10 to 45 deg C ƒ Bile esculin hydrolysis – positive ƒ Grows in 6.5% Sodium chloride ƒ Intrinsically resistant to Penicillin, Cephalosporins and low-level gentamicin

• Dengue virus – belongs to flaviviridae family. It is RNA virus with four serotypes – DEN 1-4 • Vector is Aedes aegypti; Day biting mosquitoes • Incubation period – 3 to 14 days; Causes fever, arthralgia in the first phase – Break bone fever. • Maculopapular rash appears in 3-5 days, followed by critical phase of plasma leakage – shock syndrome (especially in secondary infection)

•

•

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80. Ans.  (c) Mycobacterium avium Ref: Ananthanarayan and Paniker’s Textbook of Micro­ biology – 10th ed – Page 367

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PGI MAY 2019

78. Ans. (a) It occurs only when CD4 cell count is less than 50

Runyon Classification

Species

Photochromogens

M. kansasii, M. marinum

Scotochromogens

M. scrofulaceum, M. gordonae

Non-photochromogens

M. avium, M. intracellulare, M. ulcerans, M. xenopi

Rapid growers

M. fortuitum, M. chelonae, M. smegmatis, M. phlei

•

•

•

•

83. Ans.  (b) All except leprae Ref: Ananthanarayan and Paniker’s T.B of microbiology – 10th Ed – page 352 Lowenstein Jensen medium – composed of coagulated hen’s eggs, mineral salt solution, asparagines and malachite green; Malachite green inhibits the growth of other bacteria and makes the media selective for Mycobacterium; It allows growth of M.tuberculosis, atypical Mycobacteria and Nocardia.

WHO LTBI guidelines Latent TB infection: • A state of persistent immune response to stimulation by Mycobacterium tuberculosis antigens with no evidence of clinically manifest active TB. • There is no gold standard test for direct identification of Mycobacterium tuberculosis infection in humans. • The vast majority of infected people have no signs or symptoms of TB but are at risk for active TB disease. • Mantoux test will be positive. IGRA cannot differentiate active and latent infection.

–

–

IgG

+/–

+/–

Remote HBV infection; infectivity nil or very low

–

–

–

+

–

Immunity following HBV vaccine

•

•

87. Ans.  (d) Autoimmune haemolytic anemia; (e) Good pasture syndrome Ref: Ananthanarayan and Paniker’s TB of Microbiology – 10th Ed – Page 168

•

•

85. Ans.  (a) Incubation period is 1-6 days; (c) Pharyngotonsillar diphtheria usually present as sore throa; (d) Membrane can cover pharynx, tonsils, soft and hard palate

Type

Typical Time of Onset

Clinical Manifestation or Disease

I (Immediate, anaphylactic)

Minutes

• • • • • • •

•

•

•

•

Ref: Ananthanarayan and Paniker’s T.B of microbiology – 10th Ed – page 239 Corynebacterium diphtheriae – Gram positive rods that have characteristic metachromatic granules; Incubation period – 2 to 5 days with a range of 1-10 days Toxin production is the major pathogenetic virulence factor which causes necrosis and pseudomembrane formation in the tonsillar area Pharyngeal diphtheria is the commonest type. When involving the regional lymph nodes, can cause respiratory obstruction. Treatment: Erythromycin and Antitoxin

•

•

•

II (Cytotoxic)

Hours to days

 

Ref: Ananthanarayan and Paniker’s T.B of microbiology – 10th Ed – page359

HBsAg HBeAg Anti HBc Anti HBs Anti HBe Interpretation  Latest Exam Questions 2020–2019

84. Ans.  (a) Significant chest X-ray findings and (c) Standard TB regimen is used for treatment

Systemic anaphylaxis Urticaria Asthma Hay fever Allergic rhinitis Allergic conjunctivitis, Angioedema

• • • • •

Hemolytic anemia, Neutropenia, Thrombocytopenia, ABO transfusion reactions, Rh incompatibility (erythroblastosis fetalis, hemolytic disease of the newborn) • Rheumatic fever • Goodpasture’s syndrome • Grave’s disease •

•

•

•

•

•

86. Ans.  (a) HBsAg; (c) IgM anti HBc; (d) HBeAg

•

•

Ref: Ananthanarayan and Paniker’s TB of Microbiology – 10th Ed – Page 548

III (Immune Complex)

2 to 3 weeks

HBsAg HBeAg Anti HBc Anti HBs Anti HBe Interpretation

• Systemic lupus erythematosus • Rheumatoid arthritis • Poststreptococcal glomerulonephritis • IgA nephropathy • Serum sickness • Hypersensitivity pneumonitis (e.g., farmer’s lung) •

•

•

+

+

+

–

+

+

–

+/–

IgM

IgG

IgG

IgM

–

–

–

–

–

–

+/–

+/–

Acute HBV infection; highly infectious Late/chronic HBV infection or carrier state; highly infectious

•

•

•

IV (Delayed)

2 to 3 days

Contd...

•

•

Late/Chronic HBV infection or carrier state; low infectivity Seen rarely in early acute HBV infection; infectious

• Contact dermatitis, • Tuberculin skin test reaction, • Stevens Johnson Syndrome, • Toxic epidermal necrolysis, • Erythema multiforme •

•

•

88. Ans.  (c) Blood culture Ref: Ananthanarayan and Paniker’s TB of Microbiology – 10th Ed – Page 349 Brucellosis: • Blood culture is the most definitive method of diagnosis – Castaneda method; Automated methods with prolonged incubation (Average 7 days) •

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• Serological methods – Standard agglutination test and ELISA; SAT method is most sensitive method.

 

 Latest Exam Questions 2020–2019

•

89. Ans.  (c) Presents with pneumonia and skin abscess; (d) May takes years before becoming symptomatic; (e) Eradication treatment is required with cotrimoxazole or doxycycline for at least 12 weeks Ref: Ananthanarayan and Paniker’s TB of Microbiology – 10th Ed – Page 323 • Melioidosis – caused by Gram negative bacilli bacteria – Burkholderia pseudomallei • It is most commonly seen in vegetations, rice paddy fields (Thailand and South East Asian countries) • Infection occurs by inoculation, inhalation, ingestion causing pneumonia, septicemia and acute pulmonary infection, also cutaneous lesions. • Incubation period: Average 1-21 days. Melioidosis may also remain latent for months or years before symptoms develop. • Treatment: Ceftazidime followed by carbapenem or TMP-SMX for a minimum of 8 weeks •

•

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90. Ans.  (a) PPLO broth Ref: Ananthanarayan and Paniker’s TB of Microbiology – 10th Ed – Page 393 • Mycoplasma does not have cell wall and cannot be culture in routine cultivation methods. • Culture needs PPLO broth and growth is very slow. • PPLO – Pleuro pneumonia like organisms (Mycoplasma family) •

•

•

92. Ans.  (b) Aseptate hyphae Ref: Ananthanarayan and Paniker’s TB of Microbiology – 10th Ed – Page 614 • Septate hyphae and Filamentous – Aspergillus • Aseptate hyphae – Mucor, Rhizopus • Beta D glucan assay is helpful only for invasive candidiasis and aspergillosis • Most common environmental fungi that grows in fruits and vegetables • Rapid grower; Produces thick cottony fluffy colonies •

•

•

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•

93. Ans.  (d) Enzyme immunoassay (EIA) followed by RPR • The traditional testing algorithm for syphilis begins with a screening nontreponemal test such as rapid plasma reagin (RPR), with positive results followed by a confirmatory treponemal test such as fluorescent treponemal antibody or  T. pallidum  particle agglutination assay (TP-PA) • Because of cost issues and rapid testing kits, a reverse testing algorithm which first screens with a treponemal test (e.g., syphilis IgG); reactive samples are then tested by RPR which is used to assess disease activity  •

•

94. Ans.  (e) Giardia lamblia The protozoa that are infectious to humans can be classified into four groups based on their mode of movement: • Sarcodina – the ameba, e.g., Entamoeba • Mastigophora – the flagellates, e.g., Giardia, Leishmania • Ciliophora – the ciliates, e.g., Balantidium • Sporozoa – organisms whose adult stage is not motile e.g., Plasmodium, Cryptosporidium •

 

•

 

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•

 

91. 11. Ans.  (a) Cervicitis; (b) Non-gonococcal urethritis; (c) Bacterial vaginosis Ref: Ananthanarayan and Paniker’s TB of Microbiology – 10th Ed – Page 393 Organism

Site of infection

Disease that are caused

M.pneumoniae

Respiratory infection

• • • • • • • • •

•

•

•

•

•

•

•

•

•

M.genitalium

Genito urinary infection

Pharyngitis Tracheobronchitis Pneumonia Arthritis Pericarditis Hemolytic anaemia GBS Skin lesions Encephalitis

• Non gonococcal urethritis (NGU) • PID •

•

M.hominis

Both • Pyelonephritis respiratory and • Postpartum fever genitourinary • Systemic infections infection •

•

•

U.urealyticum

Both

• • • •

•

•

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•

•

NGU Pyelonephritis Spontaneous abortion Premature birth

 

 

 

95. Ans.  (b) gp 41: Envelop Ag Ref: Ananthanarayan and Paniker’s TB of Microbiology – 10th Ed – Page 575 Gene product Description gp160

Precursor of envelope glycoproteins

gp120

Outer envelope glycoproteins of virion

p66

Reverse transcriptase and RNase H from polymerase gene product

p55

Precursor of core proteins, polyprotein from gag gene

p51

Reverse transcriptase, RT

gp41

Transmembrane envelope glycoproteins, TM

p32

Integrase, IN

p24

Nucleocapsid core protein of virion

p17

Matrix core protein of virion

1. In a war conflict zone, 150 patients got acute watery diarrhea. On stool examination, following motility pattern is seen. Which culture media is ideal for identifying the organism?

3. Identify the following organism visualised by wet mount of vaginal discharge?





a. b. c. d.



a. DCA agar c. MacConkey agar











b. TCBS agar d. Blood agar



















2. A 28 year old newly married female presented with complaints of intense itching per vagina and greenish discharge. On wet mount of vaginal discharge, following organism is seen. What morphological stage is this?









a. Cyst c. Tachyzoites







b. Trophozoites d. Oocysts









Candida albicans Cryptococcus neoformans Trichomonas vaginalis Gardnerella vaginalis

4. Vector for this African eye worm is:





  Sample Video Questions

Sample Video Questions



a. b. c. d.

Simulium Chrysops Anopheles Xenopsylla











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  Sample Video Questions

5. A patient who had undergone renal transplantation three months before has presented with bloody diarrhea. Stool sample shows the following organisms on culture measuring 280 um. Which of the following is true among the following:



a. It causes Loeffler's pneumonia b. The video shows the filariform larvae of the parasite c. It is acquired by contamination of food/water d. They are monoecious since they undergo parthenogenesis

7. An asymptomatic man on routine stool examination for health check up showed the following finding. What is the organism seen?



6. A 43 years old man came with acute abdomen to casualty. Immediate laparatomy and bowel resection showed following findings. What is the causative agent?



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a. b. c. d.

Ancylostoma duodenale Necator americanus Ascaris lumbricoides Enterobius vermicularis

a. b. c. d.

Entamoeba coli Balantidium coli Balamuthia mandrillaris Dientamoeba fragilis

8. Following staining method is used to demonstrate:



a. Cell wall b. Spores c. Capsule d. Flagella

9. What type of culture streaking method is shown here:

10. Identify the organism:

  Sample Video Questions



a. Stab culture c. Lawn culture













b. Streak culture d. Liquid culture







a. Strongyloides c. Borrelia







b. Hookworm d. Wucheria



For video, scan this QR Code

ANSWERS FOR VIDEO QUESTIONS 1. Ans. (b) TCBS agar

 

• Video shows typical darting motility of Vibrio cholerae • Vibrio cholerae has polar flagella and exhibits fast motility • Clinical features shown as acute watery diarrhea causing cholera outbreak in a camp • Vibrio cholerae is a Gram negative bacilli which grows in selective medium called Thiosulfate-citrate-bile salt – sucrose agar (TCBS). Colonies of V.cholerae are yellowish in colour because of sucrose fermentation.

3. Ans. (a) Candida albicans

 

• • • •

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•

Vaginal discharge shows yeast cells with pseudohyphae True yeasts : Cryptococcus Yeast with pseudohyphae: Candida albicans Hyphae are seen in other fungi like dermatophytes, Aspergillus, Rhizopus, Mucor

•

2. Ans. (b) Trophozoites

 

• A sexually active female with greenish discharge indicates Trichomoniasis • Video showing twitching motility of Trichomonas vaginalis • Trichomonas vaginalis has only trophozoite stage; it do not have cystic stage. •

•

•

4. Ans. (b) Chrysops

 

• Loiasis, called African eye worm by most people, is caused by the parasitic worm Loa loa. • It is transmitted to humans by the repeated bites of deerflies of the genus Chrysops. • These flies are seen in West and Central Africa. • Infection with the parasite can also cause repeated episodes of itchy swellings of the body known as Calabar swellings. • In some patients, visible movement of worm is seen inside the eye; but it wont cause much effects to the eye. •

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•

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  Sample Video Questions

5. Ans.  (b) The video shows the filariform larvae of the parasite • The clinical clue to identify the organisms are: 1) renal transplantation 2) diarrhea • The microbiological clue is stool shows larvae (only in two organisms – stool shows larvae, i.e., They are 1) Strongyloides stercoralis 2) Hook worm • Infections are more common in HIV, immunosuppressed and post-renal transplantation patients • Diagnosis: By demonstration of rhabditiform larvae in freshly passed stool; Filariform larvae can be demonstrated by the culture of the stool by Harada Mori filter paper method. • Treatment is Ivermectin and Thiabendazole •

•

•

•

•

6. Ans.  (c) Ascaris lumbricoides • Infection with  Ascaris  starts with the ingestion of embryonated eggs which hatches into larva to pierce through the duodenal wall to reach portal circulation, and via liver they migrate to lungs where they change into larva. • Larvae of Ascaris reach alveoli and with the secretions move up to upper respiratory tract, and with the swallowed sputum completes the journey in small bowel. • In small bowel, they develop in an adult and usually live asymptomatically, but in cases with high worm load they can also produce intestinal obstruction or perforation peritonitis;  • In tropical countries with acute abdomen, intestinal obstruction with Ascaris lumbricoides must be kept in mind. •

•

7. Ans.  (b) Balantidium coli • Balantidium coli, a large ciliated protozoan parasite. • Cysts are the parasite stage responsible for transmission of balantidiasis. • The host most often acquires the cyst through ingestion of contaminated food or water. • Most cases are asymptomatic. • Clinical manifestations, when present, include persistent diarrhea, occasionally dysentery, abdominal pain, and weight loss. Symptoms can be severe in debilitated persons. •

•

•

•

•

8. Ans.  (c) Capsule • India Ink staining is shown in the video • It is mainly used to stain capsule for Cryptococcus neoformans. • CSF sample are directly stained with India ink stain. •

•

•

9. Ans.  (c) Lawn culture • Lawn culture is plating the culture media plate all the sides and it is mainly helpful for antimicrobial susceptibility testing. • Streak culture is done with bacteriological loop and used to isolate organism. •

•

•

•

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10. Ans.  (c) Borrelia • Typical spiral structure which are motile in dark ground microscopy describes that the organism is a spirochaete. • Among the options, Borrelia is the spirochaete. •

•

Image-Based Concept Zone Parasitology

•

•

•

•

• Peripheral blood film shows Trypanosoma brucei gambiense • It occurs in three forms: Long slender with flagellum, intermediate form and short stumpy form without flagellum. • Metacycylic trypomastigote is the infective form for man • Short stumpy form is the infective form for tsetse fly

• • •

4. Amastigotes of Leishmania donovani

• Image shows smear from bone marrow • Many amastigotes are seen invading the REC and macrophages nearby • Amastigotes seen near macrophages suggest L. donovani •

3. Trypanosoma brucei

• Ellipsoidal in shape • Mature cyst has four nuclei • Identification is by correlation with clinical features like malabsorption, steatorrhea and by seeing cysts in feces.

•

•

•

•

• E. histolytica is an intestinal protozoan • Mature cyst has four typical nuclei (Quadrinucleate) • One nucleus is clearly visible here DD: Cyst of Entamoeba coli-it has 8 nuclei

2. Cysts of Giardia lamblia

•

1. Cyst of Entamoeba histolytica

6. Schizont stage of P. vivax

• • • •

• • • • •

P. vivax causes benign tertian malaria Prominent feature is presence of Schuffner’s dots Infected RBC is bigger than normal Trophozoite stage divides to form a Schizont stage Image shows Schizont which has merozoites inside it

•

•

•

•

•

•

• Forms seen in peripheral smear are rings and crescents (gametocytes)–Banana shaped • Infected RBC–normal in size and possesses 6–12 maurer’s dots • Duration of erythrocytic schizogony–36 to 48 hours • No exo erythrocytic cycle • Most pathogenic; highest level of parasitemia • Causes black water fever and cerebral malaria

•

 

 

Image-Based Concept Zone  Parasitology

5. Gametocyte of Plasmodium falciparum

•

•

• Babesia infections are transmitted by ticks • The peripheral smear image shown here shows rings at opposite to each other • When four rings are seen like this–it gives typical Maltese cross pattern of Babesiosis • But you cannot see in all the smears • With clinical correlation–diagnosis has to be made •

Large round-shaped cyst It has macro nuclei and micro nuclei Macro nuclei is seen in this image Micro nuclei is not seen here Tiny cilia is seen near the edge of the cysts (ciliated protozoa)

•

•

•

•

•

•

• • • • •

8. Babesia microti

•

7. Cyst of Balantidium coli

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• • • •

• • • • •

Oocysts are of spherical or oval Size is around 8–10 mm It has two sporocyts Each sporocyts has 2 sporozoites Acid fast oocyst; Partially acid fast

•

•

•

•

•

• Oocysts are of spherical or oval; • Size is around 4–5 mm; • It has 4 sporozoites (can be seen only under microscope–cannot be seen in this image) • Acid fast oocyst • Identification is with help of clinical features and size is smaller than Cyclospora

•

 

 

10. Oocysts of Cyclospora cayetanensis

Image-Based Concept Zone  Parasitology

9. Oocysts of Cryptosporidium parvum

• • • • • •

•

•

•

•

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•

Bile stained Outer–thin layer Inner–oncosphere Embryo–Hexacanth embryo Egg of T. saginata and T. solium cannot be differentiated  Infective eggs: ƒ Taenia saginata–infective to cattle only; ƒ T. solium–infective to pigs and human beings • Egg of Taenia saginata is acid fast ƒ

Oocyst looks ellipsoidal (boat shaped); Bigger in size Each sporocyts has 2 sporozoites; Acid fast

ƒ

•

•

•

•

• • • •

12. Egg of Taenia

•

11. Oocysts of Cystisospora belli

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14. Egg of Schistosoma

 

 

Image-Based Concept Zone  Parasitology

13. Egg of Hymenolepis nana

A

C

ƒ

ƒ

ƒ

(A) Schistosoma haematobium ƒ Terminal spine (B) Schistosoma mansoni ƒ Lateral spine (C) Schistosoma japonicum ƒ Lateral small knob

Smallest cestode infecting humans Egg is Spherical which has two lining membranes It has three pairs of hooklets Polar filaments seen Non bile stained egg

•

•

• • • •

•

Bile stained egg Oval shaped egg Operculated with a large unsegmented ovum It is difficult to differentiate egg of Fasciola hepatica and Fasciolopsis buski

•

•

•

• • • •

16. Fertilized egg of Ascaris lumbricoides

•

15. Egg of Fasciola

•

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•

•

•

•

• • • • •

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B

Round to oval in shape Bile stained with unsegmented ovum Has a thick albuminous coat It has a clear crescentic area between the ovum and egg shell

 

 

18. Egg of Enterobius vermicularis

Image-Based Concept Zone  Parasitology

17. Egg of hookworm

•

•

• Also called as thread worm, pin worm, seat worm (Nematode) • Eggs are colorless, not bile stained, plano convex (Flattened on one side) • Surrounded by a thin, smooth, transparent shell and usually contain well developed larvae inside it (a coiled tad pole like larva) • Identification is by characteristic eggs and clinical history that affects mostly children causing auto infection and nocturnal itching •

Egg is oval shaped Nonbile stained egg It has a thin transparent hyaline shell Ovum is segmented with four blastomeres It is not possible to differentiate egg of A. duodenale and N. americanus

•

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•

• • • • •

19. Larvae of Strongyloides stercoralis

•

•

•

•

•

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•

• Called as whipworm • Lives in large intestine: Caecum and vermiform appendix • Eggs have characteristic mucous plugs at both poles; Eggs are bile stained • Embryonated eggs are the infective form • Causes mucoid diarrhea, malnutrition • In children it causes rectal prolapse • DD for egg: Capillaria phillipensis •

•

•

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•

•

• The Smallest Nematode • Opportunistic pathogen in immunocompromised hosts • Most commonly seen form in stols–Rhabditiform larvae • Infective form is Filariform larvae • No intermediate host • Causes larva currens • Pulmonary lesions, intestinal symptoms, hyperinfection and disseminated infections in immunocompromised • Baermann technique–for culture

20. Egg of Trichuris trichiura

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21. True Yeast–Cryptococcus sp.,

22. Yeast-like fungi-Candida spp

 

 

Image-Based Concept Zone  Mycology

Mycology

•

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• •

• Microscopy of the clinical specimen shows: Broad, aseptate hyphae • Sporangiophores arising randomly along the aerial mycelium; No rhizoids • Same image–if rhizoids are seen–then it is Rhizopus spp. •

•

• Image showing Septate hyphae with acute angle branching at 45°C • Typical dichotomous acute angled branching is seen in Aspergillus spp.,

24. Aseptate molds-Mucor spp

•

23. Septate Molds-Aspergillus spp

• Image showing Gram staining of the specimen which has Gram-positive budding yeast cells with pseudo hyphae or hyphae • Only fungi that can be seen by Gram staining is Candida–since pseudo hyphae is seen–the species is Candida albicans

•

•

•

• Image showing wet mount with India Ink stain shows capsulated yeast cells • Only fungi that is capsulated is Cryptococcus neoformans

 

 

26. Dermatophytes-Microsporum canis

Image-Based Concept Zone  Mycology

25. Dermatophytes-Trichophyton rubrum

•

•

• Image showing thin septate hyphae, Abundant spindle shaped macroconidia with no microconidia • This feature suggests of Microsporum canis

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•

• Image showing septate thin hyphae with abundant tear drop shaped micro conidia (Bird on Fence pattern) • This features suggests Trichophyton rubrum

•

• Image showing Spores with tubercles (finger-like projections) • This projections are characteristic of Histoplasma

•

•

• Image showing: Body tissue or Culture isolate at 37°C showing Spherules which are thick double layered wall filled inside with endospores • Spherules are characteristic of Coccidioides spp

27. Histoplasma capsulatum

•

27. Coccidioides spp

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29. Chromoblastomycosis

 

 

Image-Based Concept Zone  Mycology

28. Rhinosporidium seeberi

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•

• Image showing irregular, dark brown with a septate yeast like bodies–Sclerotic bodies • Sclerotic bodies are unique for Chromoblastomycosis •

•

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• Image showing Spherules that contain numerous endospores are seen • These endosporulation is seen in Rhinosporidiosis

GENERAL MICROBIOLOGY

1

Unit Outline

1

Chapter 1 Introduction, History and Microscopes Chapter 2 Morphology and Physiology of Bacteria Chapter 3 Sterilization and Disinfection Chapter 4 Culture Media and Culture Methods Chapter 5 Bacterial Genetics, Resistance and Susceptibility Testing

Introduction, History and Microscopes y

y This core is surrounded by a protein coat y Viruses can reproduce only with the help of cellular environment of other organisms (intracellular). y

INTRODUCTION General Microbiology Medical microbiology is the study of microbial organisms that infect humans. Based on structures, replication and pathogenesis microbes are divided into Bacteria, Virus, Fungi and Parasites.

Bacteria and Archaea

Eukaryotes

Fungi, Protozoa and Algae

SCIENTISTS AND THEIR DISCOVERIES Louis Pasteur

y

y

y

y

y He is known as the father of microbiology. y After Leeuwenhoek discovered the invisible world of microbes, the dispute about spontaneous generation theory started. y Pasteur conducted an experiment and disproved the theory of spontaneous generation. y He then proposed Germ theory of disease by stating that invisible microbes in the air causes infection. y He introduced many techniques in sterilization namely:  Steam sterilizer  Hot air oven  Autoclaving  Pasteurization of milk y His contributions in vaccine designing are:  Anthrax  Cholera in fowls  Rabies y He introduced liquid media (nutrient broth) for the growth of microbes.   

y

y

y

y Belong to Eukaryotes y Cell walls are made up of chitin y Most of the typical fungi grow as mold.

y

Fungi





y

y

y

y Prokaryotic cells y Do not have peptidoglycan y They are classified into three main groups namely: Methanogens, Extreme halophiles and Extreme thermophiles.

y

Archaea





y

y

y

y

y

y

y Bacteria are simple and unicellular organisms y They come under prokaryotes y All the bacteria are covered by cell walls which are composed of proteoglycans y They reproduce by a process called binary fission by which — bacteria divide into two equal cells y Most of the bacteria derive food by photosynthesis (on its own) and some obtain nutrition by inorganic substances y Many bacteria have the capacity to mobilize with the help of flagella.

Prokaryotes

y

Types of Microorganisms Bacteria

1

Protozoa

y

y

y

y They are unicellular, eukaryotic microbes y They can move with the help of pseudopodia, flagellum or cilia y They can reproduce sexually or asexually.

Algae y

y Algae are photosynthetic eukaryotes which have characteristic presence of cellulose.

Viruses y

y

y They are smaller than other microbes and acellular organisms y Virus particle has a core that is made up either of DNA or RNA

Figure 1: Father of microbiology – Louis Pasteur

Unit 1     General Microbiology

Robert Koch

Exception to Koch’s Postulates

y He is known as father of medical microbiology y His contributions in identification of microbial organisms are as follows:  Solid media (Agar) for the growth of organisms  Methods to isolate organisms from pure culture  Hanging drop method to test the motility of an organism  Staining techniques y He discovered the following organisms:  Anthrax bacilli  TB bacilli  Cholera bacilli

y Mycobacterium leprae – cannot be cultured in vitro y Treponema pallidum – cannot be cultured in vitro y Neisseria gonorrhea – no animal model for experimental inoculation y Partially satisfied Koch’s postulates by Escherichia coli – it showed bacterial pathogenicity in an in vitro model (Tissue cultures) rather than in an animal model. y Rickettsia spp and Chlamydia spp – It can be grown only in cell culture media and very difficult to grow in agar plates.

y

y









y



y

y

y

y

y

Remember





Koch's Phenomenon • It is seen in tuberculosis. • Koch observed that guinea pig that has already got infected with TB bacillus will produce a hypersensitivity reaction when it is injected with TB bacilli or protein – this is called Koch’s phenomenon. •

•

Organism Not satisfying the Organism Partially Koch’s postulates satisfying the Koch’s postulates • Mycobacterium leprae • Treponema pallidum • Neisseria gonorrhoeae

• Escherichia coli

•

•

•

•

Figure 2:  Father of medical microbiology – Robert Koch • • • •

Remember •

•

•

•

Vaccination of Anthrax bacilli Organism per se Anthrax bacilli Liquid media Solid media

– – – –















Louis Pasteur Robert Koch Louis Pasteur Robert Koch

Koch Postulates y Bacterium should be constantly associated with the lesions of the disease y It should be possible to isolate the bacterium in pure culture from the lesions y Inoculation of the pure culture into suitable lab animals should produce lesions of the disease y It should be possible to reisolate the bacterium in pure culture from the lesions produced in the lab animals y Specific antibodies to the bacterium should be demonstrable in the serum of the patients with the disease. y

y

Figure 3: Father of aseptic surgery – Joseph Lister

y

Remember

y

y

Molecular Koch Postulates y Additional postulates added to original Koch’s postulate by Stanley Falkow. y The phenotype or property under investigation should be significantly associated with pathogenic strains of a species and not with non pathogenic strains. y Specific inactivation of the gene or genes associated with the suspected virulence trait should lead to a measurable decrease in pathogenicity or virulence y Reversion or replacement of the mutated gene with the wildtype gene should lead to restoration of pathogenicity or virulence.

• • • •

•

•

•

•

Father of Microbiology Father of Medical Microbiology Father of Aseptic Surgery Father of Chemotherapy







– – – –









Louis Pasteur Robert Koch Joseph Lister Paul Ehrlich

y

y

y

y

4

Table 1: Scientists and their contributions  

Scientists

Contributions

Louis Pasteur (Father of Microbiology)

• • • •

Vaccine for rabies Vaccine for anthrax Technique of sterilization Disproved theory of spontaneous generation • Proposed germ theory of disease • Coined the term vaccine contd... •

•

•

•

•

•

Contributions

Robert Koch (Father of Medical Microbiology)

• • • • • •

Joseph Lister (Father of aseptic surgery)

•

•

•

•

•

•

Mycobacterium tuberculosis Vibrio cholera Staining techniques Pure culture on solid media Koch’s postulates Koch’s phenomenon

Aseptic precautions by using phenol for disinfection

Paul Ehrlich • Father of chemotherapy (Father of chemotherapy) • Standardization of toxin and antitoxins •

•

Anton Van Leeuwenhoek

Microscope

Table 3:  Golden Age of Microbiology – Discoveries Years

Scientists

Discoveries

1857

Pasteur

Fermentation

1861

Pasteur

Disproved spontaneous generation

1864

Pasteur

Pasteurization

1867

Lister

Aseptic surgery

1879

Neisser

Neisseria gonorrhea

1881

Koch

Pure cultures

1881

Finley

Yellow fever

1882

Koch

Mycobacterium tuberculosis, Agar

1883

Koch

Vibrio cholerae

1884

Metchnikoff

Phagocytosis

1884

Hans Gram

Gram staining

1884

Escherich

Escherichia coli

1887

Petri

Petri dish

Edward Jenner

Small pox vaccine

Kary B Mullis

PCR

1889

Kitasato

Clostridium tetani

Barbara McClintock

Transposons

1890

Von Bering

Diphtheria antitoxin

Ernst Ruska

Electron microscope

1890

Ehrlich

Theory of immunity

Ignaz Semmelweis

Handwashing

1898

Shiga

Shigella dysenteriae

Karl Landsteiner

Human blood groups

1908

Ehrlich

Syphilis

Niels Jerne

Natural theory of antibody synthesis

1910

Chagas

Trypanosoma cruzi

Frank Burnet

Clonal selection theory

Alexander Fleming

Penicillin – antibiotic (1929)

Chapter 1     Introduction, History and Microscopes

Scientists

MICROSCOPES y Microbial organisms are tiny to see by naked eye; hence to visualize the microbes – we need microscope y Leeuwenhoek was the person who first used microscope to visualize bacteria y Limit of resolution (ability to distinguish two different objects). Resolution is the ability of the lenses to distinguish fine details and structure.  For unaided eye – 200 microns  Light microscope – 300 nm  Electron microscope – 0.1 nm (or) 0.5 nm (according to Prescott) y Refractive index is a measure of the light-bending ability of a medium y Microscopes are classified based on the presence of number, types of lenses, and light source as follows. y

y

Nobel Prize Winners of 2015 y William C Campbell and Satoshi Õmura for discovering novel therapy against infections caused by roundworm parasites y Tu Youyou with William C Campbell and Satoshi Õmura for discovering a novel therapy against malaria y

y

y







Table 2:  Name of a few organisms that have been kept after their discoverers Name after Discoverer

Organism

Klebs-Löffler bacillus

Corynebacterium diphtheriae

Preisz-Nocard bacillus

Corynebacterium pseudotuberculosis

y

y

Light Microscope y Any type of microscope that uses visible light to observe specimens is called as light microscope. y A modern compound microscope has many lenses and light to have illumination. y Finely grounded lenses are arranged in an order that when light rays from an illuminator passes through the condenser, it gets passed through the specimen. y From the specimen the light rays pass through the objective lenses which is the lens that is closest to the specimen. y Then the image of the specimen gets magnified higher times that is being visualised in ocular lens. y

Koch-weeks bacillus

Haemophilus aegyptius

Johne’s bacillus

Mycobacterium paratuberculosis

Gaffky Eberth bacillus

Salmonella Typhi

Whitmore bacillus

Burkholderia pseudomallei

Battey bacillus

Mycobacterium intracellulare

Eaton’s agent

Mycoplasma pneumoniae

Pfeiffer’s bacillus

Haemophilus influenzae

y

y

y

y

5

Unit 1     General Microbiology

Magnification of Lens

y Dyes used are: FITC, Auramine rhodamine, Acridine orange y The same principle can also be used for diagnostic techniques like:  Fluorescent antibody technique  Immunofluorescence y Antibodies are tagged with dyes and this will help to bind with the antigen, which can be detected by the color produced. y

Total magnification of lens is calculated by multiplying ocular lens magnification and objective lens magnification, i.e. Total magnification = Ocular lens magnifications × Objective lens magnification

y





y

Table 4:  Total magnification of a lens Objective Lens

Ocular Lens

Total Magnification

Low power objective lens - 10X

10X

100 X

High power objective lens - 45X

10X

450 X

Oil immersion lens - 100X

10X

1000 X

Table 5:  Types of Microscopes Microscopes

Characteristics

Light microscope

• With the help of fixation and staining – bacteria is visualized under compound lenses • Universally used microscopy to study bacteria • To visualize: ƒ Gram staining ƒ Acid fast staining ƒ Motility by hanging drop preparation ƒ Slide agglutination test ƒ Slide culture for fungi ƒ Capsule staining •

•

Types of Microscopes Bright Field Microscopes Under the routine conditions, the field of vision in a compound light microscope is brightly illuminated. By focusing the light, the condenser produces a brightfield illumination — termed as Bright Field Microscope.

Dark-field Microscope

•

ƒ

ƒ

ƒ

ƒ

ƒ

ƒ

Phase contrast microscope

• Principle used is structures within the cells differ in thickness or refractive index; • This phase difference are converted into differences in intensity of light, which produces light and dark contrasts in the images • Mainly used for: ƒ Studying microbial motility ƒ Shape of living cells ƒ To detect endospores ƒ To see inclusion bodies ƒ To study eukaryotic cells •

•

y A dark field microscope is used for examining live microorganisms that are:  Not visible in ordinary light microscope  Cannot be stained by ordinary methods  Or its morphology gets distorted by staining methods y The only difference between bright field and dark field microscope is the condenser. y In this condenser, the light that is reflected off, i.e. it gets turned away from the specimen that enters the objective lens. y This technique helps to examine the unstained organisms in liquid y Mainly helpful in identification of Treponemes. y



•



ƒ



ƒ

y

ƒ

ƒ

y

y

ƒ

Fluorescent microscope

•

•

y

Phase Contrast Microscope y The principle of this type is based on waving nature of light rays. y Because of waving pattern we have ups and downs in light rays that gives a phase difference. y When two sets of light rays are brought together this forms an image of the specimen on the ocular lens based on phase differences between the two lights. y This will give a contrast where the internal structures of a cell can be seen more sharply defined.

• By using fluorescent dyes tagged to the target bacteria – diagnosis is done; • Immunofluorescence is a method where dye is conjugated with antibody – this reacts with antigen and produces color • Dyes mainly used are: ƒ Acridine orange ƒ Fluorescein isothiocyanate (FITC) ƒ DAPI ƒ Rhodamine •

y

ƒ

ƒ

y

ƒ

ƒ

y

y

Fluorescence Microscope y The ability of a substance to absorb shorter wavelength of light and to emit longer wavelength is called as fluorescence. y Some organism has in built nature for fluoresce and called as autofluorescence. y The dyes that are used for visualising organisms is called as Fluorochromes. y

y

y

6

Dark-field microscope

• Very slender organisms like spirochetes that cannot be seen in light microscope needs dark field; • The difference between light-and dark-field microscope is a presence of condenser (dark-field stop underneath the condenser) • This dark-field stop will produce a cone of light so that only the light entering the objective comes from the specimen alone making the surrounding dark • Helps to see the motility of spirochaetes •

•

•

•

contd...

Characteristics

Inverted microscope

• Helps in cell culture

Electron microscope

• Instead of light, a beam of electrons is used; • Organism is freezed (Freeze etching) – cellular ultrastructure is studied; • Mainly for direct visualization of virus from specimens; Eg.: Rota virus from stools • Techniques used in electron microscopy are: ƒ Shadow casting ƒ Negative staining ƒ Freeze etching

Chapter 1     Introduction, History and Microscopes

Microscopes

•

•

•

•

•

ƒ

ƒ

ƒ

Interference microscope

• Helps in quantitative measurement of chemical constituents of cells (lipids, proteins, nucleic acids) • Differential interference microscope gives a three-dimensional image •

•

Polarization microscope

• To study the intracellular structures using differences in birefringence

Confocal microscope

• Gives three-dimensional image that is studied through a computer connected with microscopy • Mainly helps in study of biofilms that is attached with surfaces like joint replacements

•

•

Figure 4: Inverted microscope (Courtesy: CDC/ Dr. Paul Fernhoff)

•

Remember

Light Microscope To visualize: • Gram staining • Acid fast staining • Motility by hanging drop preparation • Slide agglutination test • Slide culture for fungi • Capsule staining

Phase Contrast Microscope Mainly used for: • Studying microbial motility • Shape of living cells • To detect endospores • To see inclusion bodies • To study eukaryotic cells

Dark Field Microscope Helps to see the motility of spirochetes

Electron Microscope Mainly for direct visualization of virus from specimens; Eg: Rota virus from stools; Instead of light, a beam of electrons is used;

Polarization Microscope To study the intracellular structures using differences in birefringence

Fluorescent Microscope Dyes mainly used are: • Acridine orange • Fluorescein isothiocyanate (FITC) • DAPI • Rhodamine

•

•

•

•

•

•

•

•

•

•

•

•

•

•

•

7

Unit 1     General Microbiology

MULTIPLE CHOICE QUESTIONS History 1. Germ theory of disease was proposed by: (Recent Pattern Nov15) a. Louis Pasteur b. James Lind c. Aristotle d. Pattenkoffer 2. Technique of sterilization was introduced by: (Recent Pattern July 15) a. Robert Koch b. Edward Jenner c. Louis Pasteur d. Lister 3. Mycobacterium tuberculosis was discovered by: (Recent Pattern Dec 13) a. Louis Pasteur b. Robert Koch c. Lister d. Jenner 4. Microscope was invented by. (Recent Pattern Aug 13) a. Ronald Ross b. Robert Koch c. Antonie van Leeuwenhoek d. Louis Pasteur 5. Who discovered electron microscope? a. Robert Koch b. Paul Ehrlich c. Elie Metchnikoff d. Ernst Ruska 









12. Side chain theory for antibody production is proposed by (Recent Pattern 2017) a. Robert Koch b. Paul Ehrlich c. Elie Metchnikoff d. Louis Pasteur 13. Medium of travel in electron microscope 



a. Air c. High vacuum

(Recent Pattern 2017) b. Water d. Oil

14. Which method of diagnosis is shown here?  (AIIMS May 2018)

























Microscopes 6. Arrangement of lens from eye to source of light, in light microscope: (Recent Pattern Dec 13) a. Ocular lens: objective lens: condenser lens b. Subjective lens: ocular lens: condenser lens c. Condenser lens: objective lens: ocular lens d. Subjective lens: condenser lens: ocular lens 7. Light microscopy resolution: (PGI May 12) a. 200 nm b. 20 nm c. 0.2 nm d. 300 nm 8. Dark ground microscopy is used to see: a. Refractile organism  (Recent Pattern Dec 14) b. Flagella c. Capsule d. Fimbriae 9. Dye used for direct immunofluorescence:  (Recent Pattern Dec15) a. India ink b. Nigrosine c. Rhodamine d. Basic fuschin 10. Shadow casting is used in: (Recent Pattern Dec 16) a. Light microscopy b. Electron microscopy c. Optical microscopy d. Fluorescence microscopy 11. Bifringence polarization microscopy is used for: (Recent Pattern Dec 15) a. Flagella b. Intracellular structures c. Capsule d. Spores

a. Dark ground microscopy b. Phase contrast microscopy c. Flourescent microscopy d. Electron microscopy 15. Identify the condenser in the following microscope image:  (AIIMS May 2018)





8



a. b. c. d.

Above the stage Below the stage Near the eyepiece Above the objective lenses

1. Ans.  (a) Louis Pasteur

8. Ans.  (b) Flagella

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology,10th ed, Page 3

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 11

• Louis Pasteur – Disproved the theory of spontaneous generation and postulated germ theory of disease.

• Dark-field microscope is used to see very slender organisms like spirochaetes and to see the flagella • While fimbriae can be demonstrated by hemagglutination

•

2. Ans.  (c) Louis Pasteur Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 3 • Pasteur is the one who introduced techniques of sterilisation like steam steriliser, hot air oven and autoclave •

•

•

9. Ans.  (c) Rhodamine Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 11 • Fluorescent dyes used in immunofluorescence are ƒ Auramine ƒ Rhodamine ƒ Lissamine ƒ FITC (M/c used) – Fluorescein isothiocyanate •

3. Ans.  (b) Robert Koch Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 4 • Robert Koch discovered Mycobacterium tuberculosis and Vibrio cholerae

ƒ

ƒ

ƒ

ƒ

•

4. Ans.  (c) Antonie van Leeuwenhoek Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 3 • Antonie Van Leeuwenhock – first time observed and reported bacteria with his simple hand made microscope •

5. Ans.  (d) Ernst Ruska Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 4 • Ernst Ruska in 1934 developed the electron microscope – to visualise viruses •

Chapter 1     Introduction, History and Microscopes

ANSWERS AND EXPLANATIONS

10. Ans.  (b) Electron microscopy Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 12 • To visualize the organism under electron microscope – two types of techniques are followed ƒ Shadow casting ƒ Negative staining with phosphotungstic acid •

ƒ

ƒ

11. Ans.  (b) Intracellular structures Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 12 • Polarization microscope: To study the intracellular structures using differences in birefringence •

6. Ans.  (a) Ocular lens: objective lens: condenser lens Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 10 • Arrangement of lenses in bright field or light microscope is ocular lens in eye piece which is of 5X or 10X • Objective lens which has low power (10X) , high power (45X) and oil immersion (100X) lenses • Condenser lens is located in the diaphram which helps in focussing of lights from light rays • Ocular lens → Objective lenses → Condenser lens •

•

•

•

7. Ans.  (d) 300 nm Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 11 • Resolving power of a microscope is the ability to differentiate two different objects as different • It is limited by the wavelength of the light • Limit of resolution of a light microscope is 300 nm •

•

•

12. Ans.  (b) Paul Ehrlich Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 3 • Father of microbiology → Louis Pasteur • Germ theory of disease, Nutrient broth, pasteurization of milk, autoclaving, hot air oven and steam sterilizer Louis Pasteur • Father of antiseptic surgery → Joseph Lister • Introduction of solid media, pure culture techniques, anthrax bacilli, tubercle bacilli, cholera bacilli, Koch's phenomenon → Robert Koch • Father of chemotherapy → Paul Ehrlich • Acid fast nature of tubercle bacillus, toxin-antitoxin interaction called as Ehrlich phenomenon, side chain theory for antibody production • Phagocytosis → Elie Metchnikoff •

•

•

•

•

•

•

9

Unit 1     General Microbiology

13. Ans.  (c) High vacuum

15. Ans.  (b) Below the stage

Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 12

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 10

• In light microscope the medium of travel is air, here it is high vacuum •

Features

Light microscope

Electron microscope

Magnification

1000 – 1500

100,000

Best resolution

0.2 um

0.5 nm

Radiation source

Visible light

Electron beam

Medium travel

Air

High vacuum

Specimen mount

Glass slide

Metal grid

Type of lens

Glass

Electromagnet

14. Ans.  (a) Dark ground microscopy Ref: Ananthanarayan and Paniker’s Microbiology – 10th ed – Page 211 


Textbook

of

• The above image has a dark background with refractile organism – clearly states that it is an image of dark ground microscopy • Wet films are prepared from the exudates and it is examined under dark ground illumination. • T. pallidum is identified by its slender spiral structure and its slow motility •

•

•

• A modern compound microscope has many lenses and light to have illumination • Finely grounded lenses are arranged in an order that when light rays from an illuminator pass through the condenser, it gets passed through the specimen. • From the specimen the light rays passes through the objective lenses which is the lens that is closest to the specimen. • Then the image of the specimen gets magnified higher times that is being visualised in ocular lens. • Ocular lens → Objective lenses → Condenser lens •

•

•

•

•

10

2

Morphology and Physiology of Bacteria

A

B

Figures 1A and B: Eukaryotic and Prokaryotic cells

Table 1:  Difference between Prokaryotic and Eukaryotic cells Characteristics

Prokaryote

Eukaryote

Chromosome

Circular, one

Linear, many

All cellular organelles

Absent

Present

Chemical nature

Muramic acid

Sterols

Nuclear membrane and nucleolus

Absent

Present

y

Cell Wall y

y

y Cell wall— gives shape and rigidity of the cell y Cell wall is made up of mucopeptide (peptidoglycan)— formed by N-acetyl glucosamine and N-acetyl muramic acid molecules — linked with peptide pentaglycine bridges











y

y Demonstration of cell wall is by:  Plasmolysis  Microdissection  Reaction with specific antibodies  Mechanical rupture of cell  Different staining methods  Electron microscopy 

y

y

y

y

y

y

Bacteria are prokaryotes They do not have all the cell organelles to carry out life process Divide by binary fission Contain both DNA and RNA Even Chlamydia and Rickettsiae come under prokaryotes Mitochondria is absent, hence the respiratory enzymes are not located in mitochondria in bacteria; they are in mesosomes (Chondroids) y Principal sites of respiratory enzymes in bacteria— Mesosomes y y y y y y

Table 2:  Characteristic of cell wall in Gram-positive and Gram-negative Bacteria Cell Wall

Gram-Positive

Gram-Negative

Thickness

Thicker

Thinner

Amino acids

Few

Many

Lipopolysaccharides (LPS)

Absent

Present

Teichoic acid

Present

Absent

y

y

y

LPS – endotoxin nature Protoplasts: When a Gram-positive bacterium is kept in hypertonic solution, it becomes protoplast (Remember as P-P) Spheroplasts: When a Gram-negative bacterium is lysed by lysozyme it becomes spheroplasts Sterols are present in the cell membrane in Mycoplasma

y

Unit 1     General Microbiology

y y y y

Cell Wall Deficient Forms

Figure 2: Gram-positive and Gram-negative cell wall

y

y

y

y

y

y Β1 → 4 linkage of the peptidoglycan backbone is hydrolyzed by the enzyme lysozyme – causes cell wall deficiency y L forms are formed in certain species of bacteria spontaneously or because of cell wall attacking drugs (Penicillin group) y L forms are produced more readily with penicillin than with lysozyme, suggesting the need for residual peptidoglycan. y L forms revert back to normal cell when we remove the stimulus y Mycoplasma are cell wall deficient bacteria that have sterols

The difference between L forms and mycoplasmas is that when the murein is allowed to reform: •

•

• L forms revert to their original bacteria shape • But mycoplasmas never do

Gram Staining

y

y

y

Hans Christian Gram (1884) devised a method of staining for the following purposes: y To differentiate Gram-positive and Gram-negative organisms y To identify the organism y To see the size, shape and arrangement of the microbe.

Procedure

y

12

y

y

y

y Primary staining: Pararosaniline dyes, Crystal violet, Methyl violet, Gentian violet y Mordant: Iodine y Decolorisation: Alcohol, Acid-alcohol, Aniline, Acetone y Counter staining: Carbol fuchsin, safranine and neutral red

Figure 3: Procedure of Gram staining

• • • • •

Table 3: Characteristics of bacteria

Figure 5: Gram-negative organism—pink color (Courtesy: CDC/ Dr. W.A. Clark)

 

Staphylococci

Gram-positive cocci in chains

Streptococci

Gram-positive cocci in capsules

Pneumococci

Gram-negative diplococci, adjacent sides flattened

Neisseria

Gram-positive, club-shaped pleomorphic rods

Corynebacteria

Gram-negative rods with pointed ends

Fusobacteria

Gram-negative curved rods

Comma-shaped vibrios

Gram-negative straight rods with rounded ends

Enterobacteriaceae

Spiral rods

Spirilla

Gram-negative curved rods

Helicobacter

Remember

Organisms which show bipolar staining or safety pin appearance are: • Calymmatobacterium granulomatis • Burkholderia mallei • Burkholderia pseudomallei • Vibrio parahemolyticus • Yersinia pestis • Haemophilus ducreyi •

•

•

•

•

•

Gram-positive cocci in grape-like clusters

Chapter 2     Morphology and Physiology of Bacteria

Remember

Gram Staining • Gram positive looks violet • Gram negative looks pink • Only fungus that is visualized by Gram staining is Candida (Yeast cells) • Gram staining is the most important method of diagnosis in Neisseria gonorrhea (Intracellular diplococci) • Gram staining helps to differentiate pathogenic and commensal bacteria (by looking at the number of pus cells)

Cell Wall of Mycobacteria

y

y

y

y Cell wall is different from that of other bacteria, hence it cannot be seen in Gram staining y It has high concentration of mycolic acid which are lipids y Acid-fast organism: because of their resistance to decolorization with acid-alcohol

Figure 4: Gram-positive organism—violet color (Courtesy: CDC/ Dr. Richard Facklam)

Ziehl-Neelsen Staining or Acid Fast Staining y

y This method is mainly used to visualize the acid fast organisms. Organism is seen in pink color with a blue background.

y

y y y y

y

Procedure

y

•

•

•

•

•

•

Bacteria that cannot be seen in Gram staining: (Mneumonic: Tiny Rascals Lack Colour in Microscope) • Treponema pallidum • Rickettsiae • Legionella pneumophila • Chlamydiae • Mycobacteria • Mycoplasma pneumonia

y

Remember

Strong carbol fuchsin with heat – primary stain 20% sulphuric acid – decolorization (Standard method) RNTCP suggests 25% sulphuric acid Methylene blue – Counter stain

Figure 6: Sputum sample with acid-fast bacilli seen inside an epithelial cell (Pink colored) – Mycobacterium tuberculosis (Courtesy: CDC/ Ronald W Smithwick)

13

Nocardia

0.5%

Oocysts of coccidian parasites

0.25 to 0.5%

Bacterial spores Eggs of Taenia saginata Rhodococcus Legionella micdadei

1%

 

5% (less acid fast)



Mycobacterium leprae



20% (Standard method), 25% in RNTCP



Mycobacterium tuberculosis

y







Percentage of H2SO4



y

Acid fast organisms

Haemophilus influenzae Bacillus anthracis  Clostridium perfringens  Cryptococcus (fungi)  Yersinia pestis y Demonstration of capsule is by:  Negative staining  Quellung reaction in Pneumococcus  Serological methods y Capsule cannot be seen in Gram staining because it has less affinity for basic dyes 

• When the layer is well organized and not easily washed off, it is called a capsule. • A slime layer is a zone of diffuse, unorganized material that is removed easily. • A glycocalyx is a network of polysaccharides extending from the surface of bacteria and other cells. •

Unit 1     General Microbiology

Table 4:  How will you differentiate acid-fast bacilli?



Flagella y

y

y Fimbriae: Organ of adhesion y Flagella: Organ of locomotion y Types of flagella: (They are given in figure below) y







y

y

y

y It is a protective covering seen in few bacteria y Made up of polysaccharide except Bacillus anthracis – made up of polypeptide y Capsulated organisms: (Mneumonic: Some Nasty Killers Have Big Capsule Protection)  Streptococcus pneumoniae  Neisseria meningitidis  Klebsiella spp

•

•

Capsule

Figure 7: Different types of flagella

14

Remember

 

Lophotrichous – Tufts of flagella at one side

Spirilla, Bartonella bacilliformis

Amphitrichous – Flagella at both ends

Alcaligenes faecalis

Peritrichous – Flagella all over the cell

E. coli, Salmonella, Proteus

Amphi lophotrichous – Tufts of flagella at both sides

Spirillum serpens

•

Vibrio cholera, Pseudomonas, Campylobacter

•

Monotrichous – single flagella at one side

•

Lactobacillus

Pili and Fimbriae y Many Gram-negative bacteria have short, fine, hair-like appendages that are thinner than flagella and not involved in motility; called as fimbriae y Sex pili are genetically determined by sex factors or conjugative plasmids and are required for bacterial mating y Pili are basically organ of attachment y

Atrichous bacteria – no flagella

Organisms that are motile at 25°C but non-motile at 37°C: • Listeria monocytogenes • Yersinia pseudotuberculosis • Yersinia enterocolitica

y

Examples

y

Numbers of flagella present

Remember

Demonstration of Flagella

Spores Few bacteria produce spores which are nothing but highly resistant forms of bacteria which gets formed under nutritionally deficient conditions

y

y

y

y

y

y Flagella cannot be seen under light microscope y Only motility of the bacilli is seen in microscope by hanging drop preparation y Demonstration of flagella is done by Leifson method and Ryu method y Seen under dark ground illumination y Special staining like silver staining y Electron microscopy y U tube method, Cragie tube method in semisolid medium

•

•

• Organ of locomotion — Flagella • Organ of attachment — Pili

Chapter 2     Morphology and Physiology of Bacteria

Table 5: Types of flagella

y

Remember

•

y

• Aerobic spore-bearing organism: Bacillus • Anaerobic spore-bearing organism: Clostridium •

Three Parts of Flagella

Demonstration of Spores

y

y

y Modification of Ziehl-Neelsen technique with 0.25% to 0.5% H2SO4 y In Gram staining – spores look as unstained areas y Schaeffer and Fulton’s method of spore staining – using malachite green y

y

y

y

There are three parts: y Filament, cell surface to the tip. Filament is made of protein called Flagellin. y A basal body is embedded in the cell y A short, curved segment, the hook, links the filament to its basal body and acts as a flexible coupling.  

Table 6: Types of motility Name of motility

Examples

Actively motile

Pseudomonas

Lashing motility

Borrelia

Darting motility

Vibrio cholerae

Tumbling motility

Listeria monocytogenes

Falling leaf like motility

Giardia lamblia

Football motility

Balantidium coli

Serpentine motility

Salmonella

Gliding motility

Mycoplasma

Swarming

Proteus, Clostridium

Corkscrew type

Treponema

Flexion, and extension

Leptospira

Figure 8: Spores in Gram staining (look at the unstained area in the bacilli – they are the spores) (Courtesy: CDC/ Dr. William A. Clark)

15

Bacterial forms Features Cocci

Occur in clusters – E.g. Staph.aureus Occur in chains – E.g. S. pyogenes Occur in pairs (diplococci) – E.g. Neisseria Occur in tetrads – E.g. Micrococci

Straight rods

Uniform thickness, rounded ends, pointed ends, club form E.g.: Enterobacteriaceae

Curved rods

Comma shaped, spiral, screw shaped E.g. Vibrio, Spirochaetes

Mycoplasmas

Bacteria without a rigid cell wall; coccoid cells, long threads

Chlamydiae

Two forms: spherical /oval elementary bodies;spherical/oval reticular bodies

Rickettsiae

Short coccoid rods

ƒ

ƒ

ƒ

•

•

Albert Staining • It is the staining method used for metachromatic granules of Corynebacterium diphtheriae. • Albert stain has: ƒ Malachite green ƒ Toluidine blue ƒ Iodine and potassium iodide • Body of bacilli stains green with the metachromatic granules looking bluish black at the ends •

Unit 1     General Microbiology

Table 8:  Morphological forms of bacteria

High Yield

Inclusion Bodies

y

y

y

y Inclusion bodies are granules of organic or inorganic material that often are clearly visible in a light microscope. y These bodies usually are used for storage of carbon compounds, inorganic substances and energy – hence they are also called as storage granules. y Organic inclusion bodies usually contain either glycogen or poly- β-hydroxybutyrate. y Examples are:  Poly-β-hydroxybutyrate granules  Glycogen and sulfur granules  Carboxysomes  Gas vacuoles.







y

y

y

y

y

Volutin or metachromatic granules are common in: (Mneumonic: Cellphone-GSM) y Corynebacterium diphtheria y Gardenella vaginalis y Mycobacteria y Spirillum volutans



Intracytoplasmic Inclusions

y









y

PHYSIOLOGY OF BACTERIA y Generation time for bacteria – time between two cell divisions or the time required from a bacterium to give rise to two daughter cells (20-20-20)  E. coli – 20 minutes  TB – 20 hours  Lepra bacilli – 20 days y Bacterial growth curve  When organisms are cultured from another broth to new liquid medium, the period that is taking to enter into new culture medium and replicates can be drawn phase by phase termed as bacterial growth curve.

Table 7:  Staining methods and their uses

16

Gram staining

Universal staining method for most bacteria and yeast cells

Ziehl-Neelsen staining

Mycobacterium species

Modified ZN staining

Spores, Coccidian oocysts, Nocardia

Albert staining Neisser’s staining Ponders staining

Corynebacterium diphtheria granules

Schaeffer and Fulton’s method

Spores

Negative staining Indian ink staining

Capsule demonstration – Cryptococcus

Fontana staining

Leptospirosis, Treponemes

Silver impregnation staining

Treponemes

Warthin-Starry silver staining

Helicobacter pylori

Giemsa stain

Inclusion bodies of Chlamydia trachomatis

Periodic acid schiff stain, Gomori methenamine stain

HPE for fungi

Toluidine blue staining

Pneumocystis jirovecii

Figure 9: Bacterial growth curve y Lag phase: When microorganisms are introduced into fresh culture medium, there will be no immediate increase in cell number occurs, and therefore this period is called the lag phase y Log phase: During the exponential or log phase, microorganisms are growing and dividing at the maximal rate possible given their genetic potential, the nature of the medium, and the conditions under which they are growing. Their rate of growth is constant during the exponential phase; y

Uses

y

Staining methods

Cell size is maximum

Log phase

Cell size is smaller – Staining is uniform

Stationary phase

Sporulation occurs – Staining is not uniform; Metabolic products like exotoxins are produced

Table 11: Classification of bacteria based on oxygen requirement

Phase of decline

Involution forms occurs; Death of cells (due to autolytic enzymes)



y



Nutritional Requirement for Microbes

Obligate aerobes

Pseudomonas, Mycobacterium tuberculosis, Bacillus, Brucella and Nocardia

Obligate anaerobes

Clostridium

Facultative aerobes

Lactobacillus

Facultative anaerobes

E. coli, S. aureus

Aero tolerant anaerobes

Clostridium histolyticum

Microaerophilic

Campylobacter Helicobacter sp.,

Capnophilic (CO2)

Brucella

y

y

y Microbial cell composition shows that over 95% of cell dry weight is made up of carbon, oxygen, hydrogen, nitrogen, sulfur, phosphorus, potassium, calcium, magnesium and iron. These are called macroelements or macronutrients. y Certain micronutrients like manganese, zinc, cobalt, molybdenum, nickel and copper are also needed by most cells.

 

y



y

 



y

y

Table 9: Bacterial growth phase

 

Table 10: Classification of microbes based on modes of sources of energy

Chapter 2     Morphology and Physiology of Bacteria

Events

Lag phase

y

Growth phase

y Obligate aerobes: Grow only in the presence of oxygen E.g. Pseudomonas, Mycobacterium tuberculosis, Bacillus, Brucella and Nocardia y Facultative anaerobes: Aerobes that can grow anaerobically also E.g. E. coli and S. aureus y Facultative aerobes: Anaerobes that can also grow aerobically E.g. Lactobacillus y Obligate anaerobes: Grow only in the absence of oxygen E.g. Clostridium y Aero tolerant anaerobes: Tolerate oxygen for some time, but do not use it, e.g. Cl. histolyticum

y

y Stationary phase: Eventually population growth ceases and the growth curve becomes horizontal. This is to limitation of nutrition and organisms go to starvation. At this phase, sporulation starts. y Phase of decline: Nutrient deprivation and the buildup of toxic wastes lead to the decline in the number of viable cells is characteristic of the death phase.

Autotrophs

Only autotrophs can use CO2 as their sole or principal source of carbon.

Heterotrophs

Organisms that use reduced, preformed organic molecules as carbon sources are heterotrophs

Phototrophs

Phototrophs use light as their energy source

Chemotrophs

Chemotrophs obtain energy from the oxidation of chemical compounds (either organic or inorganic)

Lithotrophs

Lithotrophs (“rock-eaters”) use reduced inorganic substances as their electron source

Organotrophs

Organotrophs extract electrons from organic compounds

Energy sources

Electron sources

Table 12: Classification of bacteria based on temperature requirement  

Carbon sources

Based on Temperature Requirement y Growth below 20°C: Psychrophiles; E.g. Saprophytes y Growth between 25°C to 40°C: Mesophiles E.g. Most of the pathogenic bacteria y Growth above 55°C–80°C: Thermophiles E.g. Bacillus stearothermophilus y

Definition

y

Classification

y

Source

CLASSIFICATION Based on Oxygen Requirement



y

y Microaerophilic: Grows in the presence of low oxygen tension (5–10%) E.g. Campylobacter and Helicobacter sp.,

17

y

y Many medical conditions including indwelling medical devices, dental plaque, peritonitis, urogenital infections and upper respiratory tract infections are associated with formation of biofilms. y Bacteria commonly involved include Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus viridans, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis and Pseudomonas aeruginosa.

 

Table 13: Bacteriocins produced by certain strains of bacteria Escherichia coli

Pyocin

Pseudomonas aeruginosa

Megacin

Bacillus megaterium

Diphthericins

Coryne.diptheriae

Remember

Biofilm Producing Bacteria • CONS – Staphylococcus epidermidis (m/c) • Enterococcus faecalis • Streptococcus viridans • E. coli • K. pneumonia • Proteus mirabilis • Pseudomonas aeruginosa •

•

•

•

•

Colicin

y

y Group of highly specific antibiotic like substances which are produced by certain strains of bacteria active against other strains which belong to same or different species y

Unit 1     General Microbiology

BACTERIOCINS

•

•

BIOFILM PRODUCTION







18

y

y

y

y

y The nature of the biofilm makes a barrier to eradication and confers high level of resistance to antimicrobial agents. y So the microorganisms growing in a biofilm exhibits intrinsically more resistant to antimicrobial agents. y Prevention of CoNS infection has largely concentrated on prevention of indwelling catheter associated infection due to biofilms. y Catheters should be inserted with meticulous attention to aseptic practices. Staff should adhere to appropriate aseptic protocols in caring out the indwelling catheterization. y Prophylactic antibiotics will help in slightly slow progression of biofilms in biomaterials. y



y

y

y

y

y Biofilms are microbially derived sessile communities which are characterized by the cells that are attached to a substratum in an irreversible form. y They produce a matrix of extracellular polymeric substances (EPS) and are embedded in the matrix exhibiting an altered phenotype with respect to growth rate and gene transcription. y Biofilm mechanism is basically some chemotactic particles or pheromones which make the bacteria communicate with each other within the biofilm, a phenomenon called quorum sensing. y Factors influencing biofilm formation:  Availability of key nutrients  Surface chemotaxis  Bacterial motility  Surface adhesins



   





9. Bacteria with safety pin appearance: (Recent Pattern Dec 16) a. H. influenzae b. Vibrio parahemolyticus c. Vibrio vulnificus d. Salmonella paratyphi 10. Safety pin appearance of bacteria is seen in: (Recent Pattern Aug 13) a. Vibrio cholerae b. Chlamydia c. H influenza d. Y.pestis 11. Correct order of gram staining is: (Recent Pattern Dec 12) a. Gentian violet—Iodine – carbol fuchsin b. Iodine – Gentian violet – carbol fuchsin c. Carbol fuchsin – Iodine – Gentian violet d. Carbol fuchsin – Gentian violet – Iodine

12. Not a component of gram stain: (Recent Pattern Dec 12) a. Methylene blue b. Ethanol c. Iodine d. Gentian violet 13. Gentian violet colouration of gram positive bacteria is due to: (Recent Pattern Nov 15) a. Peptidoglycan b. Capsule c. Cell membrane d. None of the above 14. Which of the following are AFB positive with 5% sulphuric acid: (PGI May 15) a. M. avium b. M. leprae c. M. tuberculosis d. Nocardia e. Rhodopus 15. Modified Ziehl-Neelsen staining is used for: a. M. leprae  (Recent Pattern Dec 16) b. M. bovis c. Nocardia d. All of the above 16. Decolourization in ZN staining is done by: a. 1% sulphuric acid  (Recent Pattern Jun 14) b. 5% sulphuric acid c. 10% sulphuric acid d. 20% sulphuric acid 17. The difference between gram +ve and gram –ve organism is that gram +ve organism contains: (Recent Pattern July 15) a. Teichoic acid b. Muramic acid c. N-acetyl neuraminic acid d. Aromatic amino acids 18. Periplasmic space is seen in: (Recent Pattern Dec 15) a. Gram positive bacteria b. Gram negative bacteria c. Acid fast bacteria d. All 19. Mesosomes in bacteria are functional unit for (Recent Pattern Dec 14) a. Lipid storage b. Protein synthesis c. Respiratory enzymes d. None 20. Which of the following is not capsulated: (Recent Pattern Dec 13) a. Pneumococcus b. Cryptococcus c. Meningococcus d. Proteus 21. Metachromatic granules are found in: (Recent Pattern Dec 14) a. Diphtheria b. Mycoplasma c. Gardenella vaginalis d. Staphylococcus 22. Metachromatic granules are stained by: (Recent Pattern Dec 14) a. Ponder’s stain b. Negative stain c. Gram’s stain d. Leishman stain 







1. Eukaryotes are different in causing infection because (Recent Pattern Dec 12) a. Divide by binary fission b. Highly structured cell with organized cell organelles c. Don’t have all organelles d. Evolutionally ancient 2. Which is eukaryote: (PGI Nov 10, May 15) a. Mycoplasma b. Bacteria c. Fungus d. Chlamydia 3. True about bacteria: (PGI May 15) a. Mitochondria always absent b. Sterols always present in cell wall c. Divide by binary fission d. Can be seen only under electron microscope 4. Which of the following organism contain both DNA and RNA  (AIIMS May 14) a. Bacteria b. Plasmid c. Prion d. Viroid 5. Porin present in: (Recent Pattern July 15) a. Cell wall of gram positive bacteria b. Cell membrane of gram positive bacteria c. Cell wall of gram negative bacteria d. Outer membrane of gram negative bacteria 6. Wet India ink preparation is used to demonstrate: (Recent Pattern 10) a. Flagella b. Capsule c. Spore d. Fimbriae 7. Silver impregnation technique is used in the identification of: (Recent Pattern Aug 13) a. Spirochetes b. Leptospira c. Borrelia d. All of the above 8. All organism shows bipolar staining, except: (Recent Pattern Dec 13) a. Calymmatobacterium granulomatis b. Y. pestis c. Pseudomonas mallei d. H. influenzae

Chapter 2     Morphology and Physiology of Bacteria

MULTIPLE CHOICE QUESTIONS

19

37. Generation time for Mycobacterium tuberculosis: a. 20 minutes b. 14 -16 hours c. 12 – 13 days d. 20 days 38. Capsulated organisms are all except: a. Bacillus anthracis b. Cryptococcus neoformans c. Klebsiella pneumonia d. Proteus mirabilis 39. Identify the following staining method:



a. Spore staining b. Albert staining c. Acid fast staining d. Sudan black lipid staining 40. Whitmore bacillus is a. Hemophilus influenza b. Burkholderia pseudomallei c. Corynebacterium pseudotuberculosis d. Burkholderia mallei 41. Growth below 20°C – bacteria are grouped as a. Mesophiles b. Thermophiles c. Psychrophiles d. Capnophiles 42. Gram positive cocci arranged in tetrads a. Staphylococcus sp., b. Micrococcus c. Sarcina d. Enterococcus 43. Which organism shows following type of flagellum (electron microscopic image)







Unit 1     General Microbiology

23. Albert’s stain is used for: (Recent Pattern Dec 13) a. Staphylococcus b. Corynebacterium diphtheriae c. C. perfringens d. C. tetani 24. Peritrichous flagella are seen in(Recent Pattern Dec 15) a. Vibrio cholera b. Proteus c. Campylobacter d. Legionella 25. Non-motile organism: (Recent Pattern Nov 13) a. Escherichia coli b. Vibrio cholera c. Proteus mirabilis d. Shigella dysenteriae 26. Cell wall deficient organism are:(Recent Pattern Dec 13) a. Chlamydia b. Mycoplasma c. Streptococcus d. Anaerobes 27. Maximum cell size in bacterial cell growth cycle: (Recent Pattern 15) a. Lag phase b. Log phase c. End of plateau phase d. Early stage of decline 28. True regarding lag phase is: (Recent Pattern Aug 13) a. Time taken to adapt in the new environment b. Growth occurs exponentially c. The plateau in lag phase is due to cell death d. It is the 2nd phase in bacterial growth curve 29. Thermophile bacteria grow at: (Recent Pattern Dec 15) a. 20°C b. 20-40°C c. 40-60°C d. 60-80°C 30. Which of the following is microaerophilic  (Recent Pattern July 15) a. E. coli b. Bacteriodes c. Clostridium d. H. pylori 31. Continuous cell culture of bacteria: (Recent Pattern Nov 15) a. U tube b. Craige tube c. Chemostat device d. Agar dilution method 32. Concentration of sulfuric acid in acid fast staining for Nocardia a. 20% b. 5% c. 1% d. 0.5% 33. Which is not present in Gram negative bacteria? a. Peptidoglycan b. Teichoic acid c. LPS d. Porin channels 34. Which of the following is microaerophilic: a. Campylobacter b. Vibrio c. Bacteroides d. Pseudomonas 35. Cells are uniformly stained and active in which phase? a. Lag phase b. Log phase c. Stationary phase d. Decline phase 36. Tumbling motility is seen in: a. Listeria monocytogenes b. Mycoplasma c. Clostridium tetani d. Campylobacter jejuni

20



a. Vibrio cholera b. Spirillum minus c. Escherichia coli d. Campylobacter sp., 44. All are obligate aerobes except: a. Brucella abortus b. Bacillus anthracis c. Lactobacillus sp., d. Pseudomonas sp., 45. Cell wall of gram negative bacteria contains:  (PGI May 2018) a. Teichuronic acids b. Peptidoglycan c. Lipopolysaccharide d. Teichoic acid e. Lipoprotein

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 8,9

•

•

• When compared to prokaryotes, Eukaryotes have a complexed cell structure with all the organelles that are necessary for life • Hence the presentation of infection is different

•

8. Ans.  (d) H. influenzae Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 317, 337 Organisms which show bipolar staining or safety pin appearance are: • Calymmatobacter granulomatis • Burkholderia mallei • Burkholderia pseudomallei • Vibrio parahemolyticus • Yersinia pestis • Haemophilus ducreyi

Eukaryotes

Fungus, Algae other than blue green algae, Protozoa

• •

Bacteria, Chlamydiae, Mycoplasma, Blue green algae, Archaea

•

Prokaryotes

•

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 8

•

•

2. Ans.  (c) Fungus

• Mainly used for Spirochetes • Organism which comes under spirochetes are Treponema, Borrelia, Leptospira •

1. Ans.  (b) Highly structured cell with organized cell organelles

•

•

•

•

• • • •

Bacteria belongs to prokaryotes Do not have any cellular organelles Sterols are absent in cell wall exception is Mycoplasma Bacteria divides by binary fission

4. Ans.  (a) Bacteria Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 8   Bacteria has both DNA and RNA 5. Ans.  (d) Outer membrane of gram negative bacteria

• Already explained Q.8 •

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 8,9

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 317, 337

10. Ans.  (d) Y. pestis Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 317, 337 • Already explained Q.8 •

3. Ans.  (a) Mitochondria always absent; (c) Divide by binary fission

9. Ans.  (b) Vibrio parahemolyticus

11. Ans.  (a) Gentian violet—Iodine – carbol fuchsin Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 13 Gram staining procedure:

Primary staining with Gentian violet, Crystal violet and Methyl violet

Ref: Jawetz Medical microbiology – 27th ed – Page 26

Mordant - iodine

• Outer membranes are seen only in the Gram negative cell wall • Through this outer layer – many LPS molecules are seen called as porins • These are actually channels which helps in the passage of nutrients, proteins and antibiotics

Decolourisation – Acetone, Acid alcohol, alcohol Counterstain – Diluted Carbol Fuschin (1:10/1:20)

•

•

Chapter 2     Morphology and Physiology of Bacteria

ANSWERS AND EXPLANATIONS

•

12. Ans.  (a) Methylene blue

• Methylene blue is a counterstain that is used in acid fast staining • Methyl violet is the primary stain that is used in Gram staining

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 18

13. Ans.  (a) Peptidoglycan

•

•

Demonstration of capsule is by: • Negative staining in wet films with India Ink • Quellung reaction

•

•

6. Ans.  (b) Capsule

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 12

•

• Silver impregnantion technique is used to see delicate organisms

• Peptidoglycan layer is thicker in Gram positive bacteria • Hence the primary stain is getting trapped inside this PG layer which cannot get washed off with decolourisation •

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 377

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 12

•

7. Ans.  (d) All of the above

21

•

16. Ans.  (d) 20% sulphuric acid Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 13 • Already explained Q.14 •

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 13

• Spores • Coccidian parasites •

•

14. Ans.  (b) M. leprae

•

•

• Standard AFB techniques used 20% H2SO4 • Modifications of AFB are done for few acid fast organisms • 5% H2SO4 is used for M.leprae as it is less acid fast •

Unit 1     General Microbiology

• While in Gram negative, the PG layer is thin and trapping of the dye is less and it also gets washed off with decolourisation

15. Ans.  (d) All of the above Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 13

•

•

•

Modified Ziehl Neelsen staining is used for: • Mycobacterium leprae • Atypical Mycobacteria • Nocardia

17. Ans.  (a) Teichoic acid Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 16

Properties in cell wall

Gram positive

Gram negative

Thickness

Thicker

Thinner

Amino acids

Few

Many

Lipopolysaccharides (LPS)

Absent

Present

Teichoic acid

Present

Absent

18. Ans.  (b) Gram negative bacteria Ref: Jawetz Medical microbiology – 27th ed – Page 26

•

•

• A small space that is located between cytoplasmic membrane and cell wall is called as periplasmic space • It is seen in Gram negative bacterial cell wall

22

Figure: Gram negative cell wall (Adapted from Jawetz medical microbiology)

25. Ans.  (d) Shigella dysentriae

• Mitochondria is absent – hence the respiratory enzymes are not located in mitochondria in bacteria; they are in mesosomes (Chondroids) • Principal sites of respiratory enzymes in bacteria – Mesosomes

• Shigella sp are non motile GNB Ref: Q.24

•

26. Ans.  (b) Mycoplasma Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 21 • Mycoplasma has no cell wall; It has sterols in cell membranes •

20. Ans.  (d) Proteus

•

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 20

•

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 17

27. Ans.  (a) Lag phase Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 22

•

•

• Initial period of adaptation time into a new culture medium is called as Lag phase • In this phase – there is no increase in the number of cells • There is only increase in the size of the cells

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 18

• Already explained Q.1 29. Ans.  (d) 60-80°C Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 25 • Thermophilic bacteria grow at higher temperatures • Growth above 55°C – 80°C – Thermophiles   Eg.: Bacillus stearothermophilus •

•

•

•

•

Volutin or metachromatic granules are common in: • Corynebacterium diphtheria • Mycobacteria • Gardnerella vaginalis • Spirillum volutans

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 22 •

21. Ans.  (a) Diphtheria; (c) Gardenella vaginalis

28. Ans.  (a) Time taken to adapt in the new environment

•

•

•

•

•

•

•

•

•

Capsulated organisms: • Pneumococcus • Bacillus anthracis • Meningococcus • Klebsiella sp • H.influenza • Yersinia • Cl.perfingens • Cryptococcus (Fungi)

•

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed

Chapter 2     Morphology and Physiology of Bacteria

19. Ans.  (c) Respiratory enzymes

23. Ans.  (b) Corynebacterium diphtheriae Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 14

•

• Microaerophilic – grow in the presence of low oxygen tension (5-10%)   Eg.: Campylobacter, Helicobacter sp., 31. Ans.  (c) Chemostat device Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 22 • Continuous culture of bacteria is needed for industrial or research purposes • Usually done as liquid cultures • Mechnism used by Chemostat or Turbidostat • This constantly gives new medium and removes old medium and maintains the nutrition •

•

•

•

• Albert stain has two steps which stains the granules of C. diphtheriae • Bluish purple coloured - metachromatic granules • Also called as polar bodies, Volutin granules, Babes Ernst granules

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 25

•

•

•

•

Metachromatic granules are seen by: • Albert stain • Neisser’s stain • Ponder stain

30. Ans.  (d) H. pylori

•

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 14, 18,241

•

22. Ans.  (a) Ponder’s stain

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 19,287 •

• Peritrichous flagella – Flagella all around the bacilli E.g. E. coli, Proteus, Listeria, Salmonella except gallinarum-pullorum

32. Ans.  ( c) 1% Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 16 • Mycobacterium tuberculosis → 20-25% •

24. Ans.  (b) Proteus

23

• •

•

•

• Swarming motility → Proteus, Cl. tetani • Corkscrew, lashing, flexion extension → Spirochete

•

37. Ans.  (b) 14 -16 hours

33. Ans.  (b) Teichoic acid

Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 26 • Generation time: time required for a bacterium to give rise to two daughter cells under optimum condition • E. coli and other pathogenic bacteria–20 minutes • Mycobacterium tuberculosis: 14 to 16 hours • Myobacterium leprae: 12 to 13 days

Gram positive cell wall

Gram negative cell wall Thinner

Pentaglycine bridge Present

Absent

Lipid content

Nil or scanty

Present

Lipopolysaccharide

Absent

Present

Teichoic acid

Present

Absent

Aromatic aminoacids

Absent

Present

•

Peptidoglycan layer Thicker

•

Characters

•

•

Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 18

38. Ans.  (d) Proteus mirabilis Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 21

•

Capsulated bacteria: • Pneumococcus • Meningococcus • Haemophilus influenza • Klebsiella pneumoniae • Pseudomonas aeruginosa • Bacillus anthracis • Cryptococcus (fungus) Composition of most of the capsulated organisms is polysaccharide Exception: Bacillus anthracis – Polypeptide Strep. pyogenes – Hyaluronic acid •

Unit 1     General Microbiology

• Mycobacterium leprae → 5% • Nocardia →1% • Acid fast parasites → 0.25% to 0.5%

•

•

•

•

• Microaerophilic: grow in the presence of low oxygen tension (5-10%), E.g. Campylobacter, Helicobacter sp., • Obligate aerobes: grow only in the presence of oxygen E.g. Pseudomonas, M.tb, Bacillus, Brucella and Nocardia • Facultative anaerobes: aerobes that can grow anaerobically also, E.g. E. coli, Staph aureus • Facultative aerobes: anerobes that can also grow aerobically, E.g. Lactobacillus • Obligate anaerobes: grow only in the absence of oxygen, Eg: Clostridium • Aerotolerant anerobes – tolerate oxygen for some time, but do not use it, E.g. Cl. histolyticum

•

Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 28

•

•

•

34. Ans.  (a) Campylobacter

Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 16 •

• Gram stain: Gram positive (violet colour), Gram negative (Pink) • Acid fast stain: Blue background with pink curved bacilli (Acid fast bacilli) • Albert stain: Differentiaties metachromatic granules from those with and without (to diff Corynebacterium sp.,) Positive in C. diphtheria; Green bacilli with brown granules • Lipids are stained by Sudan black stain • Spores are stained by Malachite green stain •

•

•

•

39. Ans.  (b) Albert staining

Characters Lag phase

Log

Stationary Decline

Important features in each phase

Uniformly stained Small size Metabolically active

Gram variable Produces granules, spores, exotoxin, antibiotics, bacteriocin

Accumulation of enzymes and metabolites Maximum size

Produce involution forms

36. Ans.  (a) Listeria monocytogenes Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 23

•

24

40. Ans.  (b) Burkholderia pseudomallei Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 5 Kleb Loeffler bacillus

C. diphtheriae

Preisz nocard bacillus

C. pseudotuberculosis

Koch week bacillus

Haemo. aegypticus

Johne`s bacillus

Myco. Paratuberculosis

Gaffky eberth bacillus

S. Typhi

Whitmore bacillus

Burk. pseudomallei

Battey bacillus

Myco. intracellulare

Eatons agent

Myco. pneumoniae

Pfeiffer`s bacillus

Haemophilus influenzae

•

•

•

Types of motility: • Tumbling motility → Listeria • Gliding motility → Mycoplasma • Stately motility → Clostridium • Darting motility → Vibrio cholera, Campylobacter

•

Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 27

•

•

35. Ans.  (b) Log phase

•

44. Ans.  (c) Lactobacillus sp., Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 28 • Microaerophilic: grow in the presence of low oxygen tension (5-10%), E.g. Campylobacter , Helicobacter sp., • Obligate aerobes: grow only in the presence of oxygen, E.g. Pseudomonas, M. tuberculosis, Bacillus, Brucella and Nocardia • Facultative anaerobes: aerobes that can grow anaerobically also, E.g. E. coli, S. aureus • Facultative aerobes – anerobes that can also grow aerobically, E.g. Lactobacillus • Obligate anaerobes: grow only in the absence of oxygen, E.g. Clostridium • Aerotolerant anerobes – tolerate oxygen for some time, but do not use it, E.g. Cl. histolyticum

43. Ans.  (b) Spirillum minus Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 23

•

•

• Monotrichous (Single polar flagellum): Vibrio cholera, Pseudomonas and Campylobacter • Lophotrichous (Multiple polar flagella): Spirillum

• • • •

45. Ans.  (b,c,e) b. Peptidoglycan; c. Lipopolysaccharide; e. Lipoprotein Ref: Jawetz medical microbiology – 27th ed – Page 26 • Both Gram positive and Gram negative has peptidoglycans • Amino acids and lipids (lipoproteins) are plenty in Gram negative • LPS are present only in Gram negative •

•

•

•

•

•

•

Gram positive cocci arranged in • Clusters: Staphylococcus • Chain: Streptococcus • Pairs: Lanceolate shaped – Pneumococcus • Tetrads: Micrococcus • Octate: Sarcina • Pair with spectacle eye shaped: Enterococcus

•

Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 17

•

42. Ans.  (b) Micrococcus

•

•

•

•

•

• Growth below 20°C – Psychrophiles; Eg.Saprophytes • Growth between 25°C to 40°C – Mesophiles E.g.; Most of the pathogenic bacteria • Growth above 55°C– 80°C– Thermophiles E.g.: Bacillus stearothermophilus

Chapter 2     Morphology and Physiology of Bacteria

Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 29

• Peritrichous (over the entire cell surface): Salmonella Typhi, Escherichia coli • Amphitrichous (Single flagellum at both the ends): Alcaligenes faecalis, Spirillum •

41. Ans.  (c) Psychrophiles

25

3

Sterilization and Disinfection

STERILIZATION y

y

y Methods that are used to kill the microorganisms are called as sterilization methods. y There are various methods which cover different spectrum of microbes and different mechanism of action

Table 1:  Agents of sterilization Physical agents

Chemical agents

Dry heat

Flaming Incineration Hot air oven

Alcohols

Ethyl alcohol Isopropyl alcohol

Moist heat

Pasteurization Inspissation Boiling Arnold steamer Tyndallization Autoclaving

Aldehydes

Formaldehyde Glutaraldehyde Orthophthalaldehyde Peracetic acid Hypochlorous acid

Filtration

Candle filters Sintered glass filters Membrane filters

Dyes

Aniline dyes Acridine dyes

Radiation

Nonionizing radiation Ionizing radiation

Halogens

Iodine Chlorine

Phenols

Carbolic acid Lysol Cresol

Surface active agents

Cetrimide or cetavlon Benzalkonium chloride

Gases

Ethylene oxide Formaldehyde gas Betapropiolactone Hydrogen peroxide fogging

Miscellaneous

Metallic salts Plasma sterilization

Table 2:  Temperature at which organisms are killed Organisms

Temperature at which it is killed

All nonsporing bacteria

60°C for 30 minutes

S. Aureus and S. faecalis

60°C for 60 minutes

Yeasts, molds and vegetative bacteria

80°C for 5-10 minutes

Spores of Clostridium botulinum

120°C for 4 minutes or 100°C for 30 minutes

All viruses except polio and HBV

60°C

Polio and HBV

60°C for 30 minutes and 10 hours respectively

y Alteration of membrane permeability y Damage to proteins y Damage to nucleic acids y

y

y

Remember

Organisms in decreasing order of resistance Prions ↓ Endospores of Bacteria ↓ Mycobacteria ↓ Protozoal Cysts ↓ Vegetative Protozoa ↓ GNB ↓ Fungi ↓ Nonenveloped Viruses ↓ GPC/GPB ↓ Enveloped Viruses

Physical Agents Dry Heat

Table 3: Holding period used in hot air oven  

Temperature (°C)

Holding Time (Minutes)

160

120

170

60

180

30

Moist Heat Moist heat kills microbes by coagulation of proteins, i.e. denaturation. Most commonly employed method in moist heat is boiling.

Table 4: Pasteurization of milk  

Holder method

63°C for 30 minutes Coxiella burnetti is heat resistant and survives holder method

Flash method

72°C for 15-20 seconds followed by quickly cooling to 13°C

Ultra high temperature method

74 to 140°C in 5 seconds and cooled back to 74°C

Chapter 3     Sterilization and Disinfection

Mechanism of Action of Sterilizing Agents

Inspissation y A method that is used for media that contains egg or serum y Because if such media are autoclaved it leads to coagulation and the property of solidification is lost y Mainly used for Lowenstein Jensen (LJ) media and Loeffler’s serum slope y Temperature 80-85°C /half an hour for three successive days y

y

y

Mechanism of action of dry heat is: Protein denaturation, damage by oxidizing molecules and destroying the cellular constituents by elevated level of electrolytes getting accumulated causing toxicity y Flaming is the method that is used for sterilizing inoculating loop or wire, tips of forceps y Incineration is used in disposal of biomedical wastes y Hot air oven:  Mechanism: conduction of hot air  Holding period: 160°C/ 2 hours (M/c)  Used to sterilize glassware, forceps, scissors, scalpels, glass syringes, swabs, liquid paraffin, dusting powder, fats and grease  Precaution while using hot air oven are: Š Overloading should not be done Š The items should be properly arranged with space to have free flow of air circulation Š Before keeping inside hot air oven – the items should be completely dry Š Flasks and test tubes (glass wares) should be wrapped well to avoid breakage  Disadvantages of hot air oven: Š It is a bad conductor of heat and hence low penetration Š A fan is needed inside to allow even distribution of air Š Rubber material cannot be sterilized Š Culture media cannot be sterilized y

y

y







y

Tyndallization y Also called as intermittent sterilization y A method that is used for media that contains sugars or gelatin y Temperature 100°C /20 minutes for three successive days. y

y

y

Remember



Š

Š

Š

Š



Š

Š

Š

Š

27

Unit 1     General Microbiology

Autoclaving

Remember

y Mechanism is: Steam under pressure y When water inside the autoclave boils, its vapor pressure is equal to that of surrounding atmospheric pressure – the pressure inside a closed vessel gets increased. This steam has the highest penetration power. y Holding period that is usually used are 121°C for 15 minutes – 15 lb/inch2 pressure y The most effective method to kill spores y Used for sterilization of culture media, clothes, aprons, gloves, surgical trays and instruments except sharps

Sterilization Controls

y

y

To check the efficiency of sterilization – certain controls are used Controls used in the process of sterilization

y

y

y

Physical control

Check the temperature by thermocouples

Chemical control

Browne’s tube is used – Green color seen – it means effective sterilization

Biological control

Spores are used in form of tapes or packets and checked for color change or growth to know the sterilization has occurred properly

Table 5: Holding period used in autoclaving  

Temperature (°C)

Holding Time (Minutes) 15

Filtration

126

10

134

3

y Method used to remove bacteria from liquids like serum, sugar solutions, urea solutions and antibiotics. y Types of filters:  Candle filters: used for purification of water for drinking purposes; Two types – (1) Unglazed ceramic filters (Chamberland and Doulton), (2) Diatomaceous earth filters (Berkefeld and Mandler) Š Asbestos filters Š Sintered glass filters Š Membrane filters – Best method; Average pore diameter which is most common used is 0.22 nm y HEPA – High efficiency particulate air filters – removes 99.97% of particles that have a size of 0.3 um or more.

121

y

y



Š

Š

Š

y

Radiation y Ionizing radiation: X-rays, Gamma rays and cosmic rays; These have high penetrating power; also called as cold sterilization; mainly used for sterilization of plastics, syringes, swabs, catheters, animal feeds, card board, oils, greases, fabric and metal foils y Nonionising radiation  UV rays: Mainly used for sterilization of biosafety cabinets, entry ways in ICU and OT  Infrared rays: Used for prepacked items like syringes and catheters y

y





Table 6: Comparison between ionizing and Non-ionizing Radiation  

Figure 1: Laboratory autoclave (Courtesy: Dr.Jane Esther, Doctor’s Diagnostic centre, Trichy)

28

Ionizing Radiation

Nonionizing Radiation

There is no heat – hence it is called as cold sterilization

It has heat liberation

E.g. X-rays, Gamma rays and cosmic rays

E.g. UV rays and IR rays

Helpful for sterilization of packed items

Helpful for disinfection of ICU and closed cabinets like biosafety cabinets

Remember

Alcohols y Ethyl alcohol or ethanol: used as skin antiseptics y Isopropyl alcohol: for disinfection of clinical thermometers y

y

Remember

Chapter 3     Sterilization and Disinfection

Chemical Agents

Phenols y Distillation of coal tar yields phenol y It damages the cell membranes and causes protein denaturation y These are not readily inactivated by presence of organic matter y For surface disinfection – lysol and cresol are used y Chlorhexidine or hibitane is used as skin antiseptic for wound dressing y It is not sporicidal. y

y

y

Aldehydes

y

y

y Formaldehyde: for fumigation of wards, isolation rooms and laboratories; Gas is irritant and toxic on inhalation y Glutaraldehyde: Effective against TB bacilli, fungi and viruses; used to sterilize endoscopes, cystoscopes, rubber anesthetic tubes, plastic endotracheal tubes and polythene tubing; Concentration used is 2% - Cidex; y Orthophthalaldehyde: Used to clean endoscopes y Peracetic acid: Effective against MRSA, VRE and Clostridium difficile y Hypochlorous acid: Generated from sodium hypochlorite and Hydrogen peroxide; active against biofilms y

y

y

y

y

y

Gases y Ethylene oxide  Colorless, Highly explosive gas: effective against all microbes including spores  Mainly used for sterilizing Š Heart lung machines Š Respirators Š Sutures Š Dental equipment Š Glass Š Metal surfaces  It has two cycles: Cold cycle at 37±5°C; Warm cycle at 54±5°C y Beta propiolactone: Carcinogenic; not used now; previously used for fumigation and for inactivation process in vaccines y Hydrogen peroxide fogging: Nowadays formaldehyde fumigation has been replaced by H2O2 fogging; it is not toxic y





Š

Š

Š

Table 7: Aldehydes used in the process of sterilization  

Formaldehyde

Glutaraldehyde

Š

Š

Š



• Bactericidal, Virucidal and Sporicidal •

• Not sporicidal •

y

• Used to preserve anatomical • Used to disinfect specimens endoscopes, cystoscopes • Used to sterilize OT in gaseous form •

•

•

• More toxic •

• Less toxic

y

Surface Active Agents

•

y These agents produce a reduction in surface tension y Cationic compounds are also called as Quaternary ammonium compounds y Action is on bacteria; no action on spores, TB bacilli and most viruses y Agents are:  Acetyl trimethyl ammonium bromide – Cetavlon or cetrimide  Benzalkonium chloride y

Halogens y Iodine: Compounds of iodine with nonionic wetting or surface active agents is known as iodophores – these have more action than solutions of iodine; Povidone iodine (10%) is an effective skin disinfectant y Chlorine: mainly used for disinfection of town water supplies, swimming pools, food and dairy industries. y

y

y

y

y





29

Unit 1     General Microbiology

Table 8: Classification of surface active agents  

Anionic

Sulfide, Fluoride, Bromide, Iodide

Cationic

Sodium, Iron, lead

Non ionic

Polyoxyethylene (Tween/triton)

Amphoteric

Tego compounds

Plasma Sterilization y Plasma is the fourth state of matter y Using a radio frequency energy – an electromagnetic field is created y Hydrogen peroxide vapors are introduced into this which generates free radicals — plasma y Used to sterilize arthroscopes and urethroscopes y

y

y

y

Table 9: Method of sterilization and their biological control  

Methods

Sterilized from

Hot air oven

Clostridium tetani nontoxigenic strain

Autoclave

Geobacillus stearothermophilus

Filtration

Serratia marcescens

Ionizing radiation

Bacillus pumilus

Ethylene oxide

Bacillus globigii

Plasma sterilization

Bacillus subtilis subsp.,niger Geobacillus stearothermophilus

DISINFECTION Table 11: Levels of disinfection  

Low level disinfectant

Quaternary ammonium compounds (Benzalkonium chloride)

Intermediate level disinfectant

Isopropyl alcohol, Phenol

High level disinfectant

Glutaraldehyde, Formaldehyde, Hydrogen peroxide and Chlorine

Disinfection of Sputum y y y y

y

y

y

y

Treating with 5% phenol or cresol Boiling Incineration or burning Autoclaving (most efficient method)

Disinfection of Blood Spills WHO guidelines on blood spillage management state the following steps: y Wear gloves y Use disposable paper towels to absorb as much of the body fluids as possible. y Wipe the area with water and detergent until it is visibly clean. y Saturate the area again with sodium hypochlorite 0.5% (10 000 ppm available chlorine). This is a 1:10 dilution of 5.25% sodium hypochlorite bleach, which should be prepared daily. y Rinse off the tongs, brush, and pan, under running water and place to dry. y Remove gloves and discard them. y Wash hands carefully with soap and water, and dry thoroughly with single-use towels. y Record the incident in the incident book if a specimen was lost, or persons were exposed to blood and body fluids. y

y

y

y

y

y

y

Table 10: Spaulding’s classification  

y

Spaulding’s classification

Examples

Critical device

Surgical instruments, cardiac and urinary catheters, implants, eye and dental instruments

Remember

Prions are killed by: • Autoclaving at 134°C for 1 to 1.5 hours • Treatment with 1 N Sodium hydroxide (NaOH) for 1 hour • Treatment with 0.5% Sodium hypocholorite (NaOCl) for 2 hours •

•

Semicritical device

Respiratory therapy equipment, anesthesia equipment, endoscopes, laryngoscopes and probes

Noncritical device

BP cuff, ECG electrodes, bed pans, crutches, stethoscope, thermometer

•

Testing of Disinfectants y Rideal-Walker test (Phenol coefficient)  The disinfectant that need to be tested is checked for sterilization of a known suspension of S. typhi in a given period of time divided by the dilution of phenol, which sterilizes the suspension in the same time.  Phenol coefficient value >1 is considered as satisfactory  This test does not give proper result when there is a presence of organic matter y Chick-Martin test: This test can be done even in the presence of organic matter y Kelsey-Sykes or in use test: This test helps the capacity of a disinfectant to retain its activity when repeatedly using in sterilization. y

Noncritical environmental surfaces

Surfaces of medical equipment, Examination table, computers

Sterilization of Operation Theaters







y Formaldehyde fumigation is done by adding formalin to potassium permangante for the desired area. The room should be sealed with electrical supply cut for 48 hours. To neutralize the toxic gas after 48 hours – ammonia is used. But due to toxicity nowadays it is not used y Hydrogen peroxide fogging. y

30

y

y

y

Method Used

Material Sterilised

Isopropyl alcohol

Clinical thermometers

Hot air oven

Paraffin, Glass syringe, Flask, Grease, Fat

Formaldehyde

Operation theatre, lab fumigation

Orthophthaldehyde and glutaraldehyde

Cystoscope, Bronchoscope

Ethylene oxide

Heart lung machine

Filtration

Vaccine, Sera, Antibiotic, Sugar

Cresol

Sharp instrument

Pasteurisation

Milk

Ionising radiation

Plastic syringe, Catgut, Catheter

Autoclaving

Culture media, all sutures except catgut

Chapter 3     Sterilization and Disinfection

SUMMARY OF STERILISATION

31

Unit 1     General Microbiology

MULTIPLE CHOICE QUESTIONS 1. All are methods of sterilization, except: (Recent Pattern July16) a. Ionizing radiation b. Ethylene oxide c. Formaldehyde d. Chlorhexidine 2. The best skin disinfectant is: (Recent Pattern Dec 14) a. Alcohol b. Savlon c. Betadine d. Phenol 3. Most commonly used antiseptic: (Recent Pattern Dec 15) a. Povidone iodine b. Crystal violet c. Hibitane d. H2O2 solution 4. Which of the following can be reliably used for hand washing? (PGI Nov 15) a. Chlorhexidine b. Isopropyl alcohol c. Lysol d. Cresol e. Glutaraldehyde 5. True about hand hygiene: (Recent Pattern July 16) a. Betadine can cause irritation b. Alcohol based preparation are used c. Hot water is best d. Glutaraldehyde is used 6. Sterilization of culture media containing serum is by: (Recent Pattern Dec 12) a. Autoclaving b. Micropore filter c. Gamma radiation d. Centrifugation 7. All are methods of sterilization by dry heat, except: (Recent Pattern Dec 14) a. Flaming b. Incineration c. Hot air oven d. Autoclaving 8. Lethal effect of dry heat is due to: (Recent Pattern Dec 15) a. Denaturation of proteins b. Oxidative damage c. Toxicity due to metabolites d. All of the above 9. Tyndallization is a type of: (Recent Pattern Dec 12) a. Intermittent sterilization b. Pasteurisation c. Boiling d. Autoclaving 10. Glass vessels and syringes are best sterilised by   (Recent Pattern Dec 13) a. Hot air oven b. Autoclaving c. Irradiation d. Ethylene dioxide 









32

11. Autoclaving is done in: (Recent Pattern Dec 14) a. Dry air at 121°C and 15 lb pressure b. Steam at 100°C for 30 minutes c. Steam at 121°C for 15 minutes d. Dry air at 160°C for 30 min 12. Temperature required for holding period of 60 minutes in hot air oven: (Recent Pattern Dec 15) a. 160°C b. 170°C c. 120°C d. 130°C 13. Operation theaters are sterilized by: (DPG 10) a. Carbolic acid spraying b. Washing with soap and water c. Formaldehyde d. ETO gas 14. Glutaraldehyde is used to sterilize: (Recent Pattern July16) a. Endoscopes b. Corrugated rubber anesthetic tube c. Plastic endotracheal tubes d. All of the above 15. Endoscope tube is sterilized by:(Recent Pattern Dec 13) a. Glutaraldehyde b. Formalin c. Autoclaving d. Boiling 16. Percentage of glutaraldehyde used: (Recent Pattern Dec 13) a. 1% b. 2% c. 3% d. 4% 17. Ionizing radiation commonly used for disinfection: (Recent Pattern Dec 15) a. UV rays b. Infrared c. X-rays d. Gamma rays 18. Gamma irradiation used for all of the following, except: (Recent Pattern July 16) a. Syringes b. Catgut suture c. Grafts d. Endoscope 19. Irradiation can be used to sterilize A/E:  a. Bone graft  (AIIMS May 10) b. Suture c. Artificial tissue graft d. Bronchoscope 20. Inspissation is used for: (Recent Pattern July 16) a. Sputum b. Protein containing culture medium c. Serum containing culture medium d. Plasma sterilization 21. All are sporicidal agents, except: (Recent Pattern Dec 13) a. Formaldehyde b. Glutaraldehyde c. Ethylene oxide d. Isopropyl alcohol 























35. Temperature used for Pasteurisation is: (Recent Pattern 2018) a. 72°C for 20 min b. 63°C for 30 min c. 100°C for 10min d. 94°C for 20 min 36. Blood spill in operation theatre is cleaned with: (Recent Pattern 2017) a. Chlorine compound b. Phenolic compound c. Quartnery ammounium compounds d. Alcoholic compounds 37. Bacterial indicator for dry heat sterilization is? (PGI Pattern 2017) a. Bacillus subtilis b. Bacillus pumilis c. Bacillus stearothermophilus d. Coxiella burnetti e. Bacillus anthracis 38. All of the following are killed in holders method of pasteurization except: a. Mycobacteria b. Brucellae c. Salmonellae d. Coxiella burnetti 39. Tyndallization is steaming for 3 consecutive days at: a. 100°C for 10 minutes b. 100°C for 20 minutes c. 80°C for 15 minutes d. 85°C for 20 minutes 40. Biological indicator for autoclaving: a. Geobacillus stearothermophilus b. Clostridium perfringens c. Bacillus subtilis d. Clostridium tetani 41. HEPA filter removes particle of size: a. 0.5 um b. 0.3 um c. 0.8 um d. 1 um 42. Sterilisation of endoscopes and cystoscopes is by a. Glutaraldehyde b. Ortho-phthaladehyde c. Formaldehyde d. ULPA filters 43. Prions are best killed by: a. Autoclaving at 121°C b. 5% Formalin c. Sodium hydroxide for 1hr d. Sodium hypochloride for 10min 44. Low level of disinfectant is: a. Benzalkonium chloride b. Isopropyl alcohol c. Glutaraldehyde d. Hydrogen peroxide 45. Surgical blade is sterilized by: a. Autoclave b. Gamma radiation c. Hot air oven d. Steaming 46. Which of the following is used for inactivation of vaccines? a. Ethylene oxide b. Isopropyl alcohol c. Rectified sprit d. Beta propiolactone 47. According to Spaulding’s classification, ECG electrodes comes under: a. Critical device b. Semi critical device c. Non critical device d. Non critical environmental surface 





Chapter 3     Sterilization and Disinfection

22. Spores of bacteria are destroyed by: (Recent Pattern Dec 13) a. Alcohol b. Lysol c. Halogen d. Ionizing radiation 23. According to Spaulding classification system of sterilization, following is true except:(Recent Pattern Nov 10) a. “Non-critical devices” come into contact with intact skin b. Semi-critical equipment need low level sterilization c. “semi-critical devices” come into contact with nonsterile mucous membrane or nonintact skin d. Cardiac catheter is critical equipment 24. High level disinfectant are used for: (Recent Pattern July 16) a. Stethoscopes b. Electronic thermometers c. Bronchoscope d. Surgical instruments 25. Which of the following is an intermediate level disinfectant: (Recent Pattern Dec 13) a. 2% glutaraldehyde b. Ethylene oxide c. Hypochlorite d. None 26. Best virucidal disinfectant is: (Recent Pattern Nov 15) a. Phenol b. Hypochlorite c. BPL d. Formaldehyde 27. Best indicator for sterilization by autoclaving: (Recent Pattern Dec 15) a. Bacillus subtilis b. Geobacillus c. Bacillus pumilus d. Clostridium 28. Plasma sterilization accuracy is assessed by using (Recent Pattern Nov 10) a. Bacillus subtilis b. Bacillus stearothermophilus c. Staphylococcus aureus d. Clostridium tetani 29. Browne’s tube is used for: (Recent Pattern Dec 12) a. Steam sterilization b. Radiation c. Chemical sterilization d. Filtration 30. Heat labile liquids are sterilized by: (Recent Pattern Dec 15) a. Hot air oven b. Autoclaving c. Membrane filter d. Moist heat 31. Sputum can be disinfected by all, except: (AIIMS May 12, Nov 10) a. Autoclaving b. Boiling c. Cresol d. Chlorhexidine 32. Phenol coefficient indicates: (Recent Pattern July15) a. Efficacy of a disinfectant b. Dilution of a disinfectant c. Quantity of a disinfectant d. Purity of a disinfectant 33. HEPA filter is used to disinfect: (Recent Pattern Dec15) a. Water b. Air c. Culture d. Blood 34. Quaternary ammonium compound disinfectants are: (Recent Pattern July 15) a. Anionic b. Cationic c. Neutral d. Gases

33

Unit 1     General Microbiology

48. Most commonly used pore size of membrane filters is: a. 0.22 um b. 0.24 um c. 0.26 um d. 0.20 um 49. Method of disinfection of contact lenses: a. Tincture iodine b. Isopropyl alcohol c. Hydrogen peroxide d. Chlorine 50. Sterilization of serum is done by: a. Filtration b. IR radiation c. Boiling d. Pasteurisation 51. Quaternary ammonium compounds kill all except: a. Escherichia coli b. Pseudomonas sp c. Staphylococcus aureus d. Mycobacterium tuberculosis



52. Ionizing radiation includes all except: a. X-rays b. Cosmic rays c. Y rays d. UV rays 53. Plasma sterilisation is done by:  (AIIMS May 2018) a. Hydrogen peroxide b. Ethylene oxide c. Gamma rays d. UV rays 54. An intern while doing phlebotomy spilled blood in the floor accidentally. What is the next ideal step in the disinfection of blood?  (AIIMS Nov 2018) a. Pour 1% hypochlorite solution b. Cover with a cloth/material c. Mop the floor d. Call infectious control unit 





ANSWERS AND EXPLANATIONS 1. Ans.  (d) Chlorhexidine Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 34 • Chlorhexidine or hibitane is a skin antiseptic and it’s a disinfectant • All other methods come under sterilization

• Culture media containing serum, amino acids or other thermolabile constituents gets decomposed during autoclaving • Hence filtration is the ideal method •

•

•

•

2. Ans.  (c) Betadine Ref: Greenwood – Medical microbiology – 16th ed – Page 81 • The best skin disinfectant is Betadiene – 10% Povidone iodine (M/c used) • Because it is less irritant and causes less staining. •

•

3. Ans.  (a) Povidone iodine Ref: Greenwood – Medical microbiology – 16th ed – Page 81 • Already explained in Q.2 •

4. Ans.  (a) Chlorhexidine; b) Isopropyl alcohol

7. Ans.  (d) Autoclaving Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 29 • Methods of dry heat sterilization are flaming, incineration and hot air oven • Autoclaving used moist heat with steam under pressure •

•

8. Ans.  (d) All of the above Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 29 • Mechanism of action of dry heat is: ƒ Protein denaturation ƒ Damage by oxidizing molecules ƒ Destroying the cellular constituents ƒ Elevated level of electrolytes causing toxicity •

ƒ

ƒ

ƒ

Ref: Greenwood – Medical microbiology – 16th ed – Page 80 • Best disinfectants for hand washing are: ƒ Povidone iodine ƒ Chlorhexidine ƒ Isopropyl alcohol •

ƒ

9. Ans.  (a) Intermittent sterilization

ƒ

ƒ

ƒ

5. Ans.  (b) Alcohol based preparation are used

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 31 • Tyndallization is also called as intermittent sterilization • Media containing sugars or gelatin are sterilized by heating at 100°C/20 minutes on 3 successive days •

•

Ref: Greenwood – Medical microbiology – 16th ed – Page 81 • The most effective preparations are aqueous and alcohol based solutions • Povidone iodine preparation are most commonly used •

•

6. Ans.  (b) Micropore filter

10. Ans.  (a) Hot air oven Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 30 • Hot air oven is used for sterilization of Glass ware, forceps, scissors, scalpels, glass syringes, swabs, liquid paraffin, dusting powder, fats and grease •

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 32

34

18. Ans.  (d) Endoscope

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 31

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 32

• Steam under pressure • Holding period that is usually used are 121°C for 15 minutes

• Already explained in Q.14 and Q.17

•

•

•

19. Ans.  (d) Bronchoscope Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 32

12. Ans.  (b) 170 °C Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 31

Temperature (°C)

Holding Time (Minutes)

160

120

170

60

180

30

13. Ans.  (c) Formaldehyde Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 34 • Formaldehyde fumigation is the ideal choice for sterilization of OT • But the drawback is because it is irritant and toxic •

•

14. Ans.  (d) All of the above Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 33 Glutaraldehyde is used for disinfection of: • Cystoscopes • Endoscopes • Bronchoscopes • Corrugated rubber anesthetic tubes • Face masks • Plastic endotracheal tubes • Polythene tubing •

•

•

•

•

•

•

• Already explained Q.14 and Q.17 •

20. Ans.  (b) Protein containing culture medium Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 30 • Inspissation: A method that is used for media that contains egg (Protein) • Because if such media are autoclaved it leads to coagulation and the property of solidification is lost. • Mainly used for Lowenstein Jensen (LJ) media and Loeffler’s serum slope • Temperature 80-85°C /half an hour for three successive days • The question is controversial as we used Loeffler’s serum slope also in inspissation which contains serum; But the most commonly used media is LJ in inspissation hence I have chosed option B •

•

Chapter 3     Sterilization and Disinfection

11. Ans.  (c) Steam at 121°C for 15 minutes

•

•

•

21. Ans.  (d) Isopropyl alcohol Ref: Jawetz medical microbiology – 25th ed – page 63-67 • List of sporicidal agents: ƒ Glutaraldehyde ƒ Formaldehyde ƒ Beta propiolactone ƒ Ethylene oxide ƒ Halogens ƒ Peracetic acid ƒ Hydrogen peroxide •

ƒ

ƒ

ƒ

ƒ

ƒ

ƒ

15. Ans.  (a) Glutaraldehyde Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 33 • Already explained Q.14

ƒ

22. Ans.  (c) Halogen • Already explained Q.21 •

•

16. Ans.  (b) 2%

23. Ans.  (b) Semi critical equipment need low level sterilization

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 33

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 35

• 2% Cidex – Glutaraldehyde is used.

• According to Spaulding classification – semi critical items are the one that comes in contact with the mucous membranes or nonintact skin • These need high level of disinfection • Glutaraldehyde is the most commonly used disinfectant

•

17. Ans.  (d) Gamma rays Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 32 • X rays, Gamma rays and cosmic rays comes under ionizing radiation • Gamma rays are most commonly used for sterilizing of plastics, syringes, swabs, catheters, animal feeds, card board, oils, greases, fabric and metal foils, bone and tissue grafts. •

•

•

•

•

24. Ans.  (c) Bronchoscope Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 35 • Bronchoscopes comes under semi critical items and need high level of disinfection •

35

Unit 1     General Microbiology

• Stethoscopes and thermometers need low level of disinfection •

25. Ans.  (c) Hypochlorite Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 37 • Among level of disinfectants – glutaraldehyde and ethylene oxide comes under high level • Iodophores and hypochlorite are intermediate level of disinfectants

32. Ans.  (a) Efficacy of a disinfectant Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 35 • Phenol coefficient is used term in Rideal Walker test where phenol is taken as standard for checking the efficiency of a disinfectant •

•

•

33. Ans.  (b) Air Ref: Handbook of modern hospital safety – page150 • HEPA – High efficiency particulate air filter • Used in operation theatres and laboratories • It removes >95% particles with a diameter of more than 0.3um •

26. Ans.  (d) Formaldehyde Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 34 • Among all the options – formaldehyde is the highest level of disinfectant which covers all organisms including spores •

27. Ans.  (b) Geobacillus Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 32 • Geobacillus stearothermophillus is the biological indicator for sterilization by autoclaving

•

•

34. Ans.  (b) Cationic Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 9th ed – 34 • Surface active agents produce a reduction in surface tension • Among them cationic compounds are also called as quaternary ammonium compounds •

•

•

28. Ans.  (b) Bacillus stereothermophilus Ref: Gas plasma sterilization in microbiology – page 71 • Bacillus stearothermophilus is the biological indicator for plasma sterilization (Ref: the above table in text) •

35. Ans.  (b) 63°C for 30 min Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 30 • Pasteurisation is a method for sterilization of milk • Two methods are commonly used for pasteurisation ƒ Holder method → 63 °C for 30 minutes ƒ Flash method → 72°C for 20 seconds •

•

ƒ

29. Ans.  (c) Chemical sterilization Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 30 • Chemical control of sterilization - Browne’s tube is used • When green color is seen – it means effective sterilization •

•

ƒ

36. Ans.  (a) Chlorine compound Ref: https://www.cdc.gov/infectioncontrol/pdf/guidelines/ disinfection-guidelines.pdf • Chlorine is mainly used for disinfection of water – swimming pools, tanks • Its compound sodium hypochlorite is the best disinfectant for surface blood spills • Concentration used is 0.5 to 5% depend upon the contamination • Immediately seeing a blood spill, cover the area with tissue paper and pour 1% sodium hypochlorite over it for 15-30 minutes and then discard with universal precautions •

30. Ans.  (c) Membrane filter

•

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 32 • Autoclaving and hot air oven is not suitable for hot labile items as they use higher temperatures • Membrane filters are ideal to sterilize serum, heat labile liquids, oils, antimicrobials, solutions and liquid culture media •

•

31. Ans.  (d) Chlorhexidine Ref: NCA review for the laboratory clinical sciences – page 112 • Chlorhexidine is a skin disinfectant which is not suitable for sputum disinfection • Disinfection of sputum: ƒ Treating with 5% phenol or cresol ƒ Boiling ƒ Incineration or burning ƒ Autoclaving (most efficient method) •

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36

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37. Ans.  (a) Bacillus subtilis Ref: Ananthanarayan and paniker’s textbook of microbiology – 9th edition p30 • EXPL: Biological indicator in HOT AIR OVEN-The spores of non toxigenic strain of Clostridium tetani or Bacillus subtilis are used as microbiological test of dry heat efficiency. • Paper strips impregnated with 106 spores are placed in envelopes and inserted into suitable packs. • After sterilization, the strips are removed and inoculated in thioglycollate or cooked meat media and incubated •

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38. Ans.  (d) Coxiella burnetti Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 32 • Method for sterilization of milk is pasteurization • Methods: Holder method – 63°C for 30 minutes • Flash method – 72°C for 20 seconds followed by rapid cooling to 13° or lower) • All non sporing pathogen are killed except Coxiella burnetti which may survive •

43. Ans.  (c) Sodium hydroxide for 1hr Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 34 • Most resistant of all is prions; which is killed only by ƒ 0.5% hypochlorite for 2 hr ƒ 1N sodium hydroxide for 1 hour ƒ Autoclaving 134°C for 1-1.5 hours •

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39. Ans.  (b) 100°C for 20 minutes Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 33 • Tyndallization also called as intermittent sterilization – steaming at 100°C for 20 min for three consecutive days •

40. Ans.  (a) Geobacillus stearothermophilus Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 40 Hot air oven

Clostridium tetani non toxigenic strain

Autoclave

Geobacillus stearothermophilus

Filtration

Brevundimonas diminuta, Serratia

Ionising radiation

Bacillus pumilus

Ethylene oxide

Bacillus globigi

Plasma sterilization

Geobacillus stearothermophilus, Bacillus subtilis subsp.,niger

44. Ans.  (a) Benzalkonium chloride Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 36 • Low level disinfectant – Quaternary ammonium compounds (Benzalkonium chloride) • Intermediate level disinfectant – Isopropyl alcohol, Phenol • High level disinfectant – Glutaraldehyde, Formaldehyde, Hydrogen peroxide, Chlorine •

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Chapter 3     Sterilization and Disinfection

for sterility test under strick anaerobic conditions for five days at 37oC.

45. Ans.  (b) Gamma radiation Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 41 • Best method for sterilizing critical medical and surgical instruments which are not damaged by heat, steam, pressure or moisture is STEAM • Best method for metal instruments and surgical blade – Gamma radiation (especially packed items) •

•

46. Ans.  (d) Beta propiolactone Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 40 • BPL (beta propiolactone – 0.2%) – active against all microorganisms including spores • Disadvantage: Low penetrating power, Carcinogenic • Not used for fumigation • Used for inactivation of vaccines •

41. Ans.  (b) 0.3 um Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 35 • HEPA – High efficiency particulate air filters – removes 99.97% of particles that have a size of 0.3 um or more • ULPA filters – ultra low particulate/penetration air – remove from the air at least 99.999% of dust, pollen, mold, bacteria and airborne particles with a size of 0.12um or larger

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42. Ans.  (a) Glutaraldehyde Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 37 • 2% cidex – glutaraldehyde – helps in disinfection of endoscopes and cystoscopies • Ortho-phthalaldehyde: used for sterilization of endoscopes and cystoscopies; • Advantages of Ortho-phthalaldehyde over cidex: ƒ Does not require activation ƒ Low vapour property ƒ Better odour ƒ More stable during storage ƒ Increased mycobactericidal activity •

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47. Ans.  (c) Non critical device Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 42

Spaulding’s Examples classification Critical device

Surgical instruments, cardiac and urinary catheters, Implants, eye and dental instruments

Semi critical device

Respiratory therapy equipments, anaethesia equipments, endoscopes, laryngoscopes, probes

Non critical device

BP cuff, ECG electrodes, Bed pans, Crutches, Stethescope, Thermometer

Non critical Surfaces of medical equipments, environmental Examination table, computers surfaces

37

Unit 1     General Microbiology

48. Ans.  (a) 0.22um

52. Ans.  (d) UV rays

Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 35

Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 36

• Membrane filters: most widely used for bacterial filtration • Size – 0.22um; removes most of the bacteria; allows viruses to pass through • Filters of size 0.45um – retain coliform bacteria in water microbiology; • 0.8um filters – used to remove airborne microorganisms in clean rooms and for the production of bacteria free gases.

• Ionizing radiations – include X rays, Gamma rays, Cobalt 60 source • Excellent agent for sterilization; also called as cold sterilization • Used for sterilization of ƒ Disposable plastic supplies, disposable rubber or plastic syringes, infusion sets and catheters ƒ Catgut sutures, bone and tissue grafts, adhesive dressings ƒ Irradiation of food • Biological control: Bacillus pumilus

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49. Ans.  (c) Hydrogen peroxide Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 38 • Hydrogen peroxide – high level disinfectant; chemical sterilant • Used to disinfect ventilator, soft contact lenses, tonometer biprisms • Used for plasma sterilization • Environmentally safe; not carcinogenic nor mutagenic •

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53. Ans.  (a) Hydrogen peroxide Ref: Ananthanarayan and Paniker’s Textbook of Micro­ biology – 10th ed – Page 35 Plasma sterilisation: • Plasma is the fourth state of matter • Using a radio frequency energy – an electromagnetic field is created • Hydrogen peroxide vapours are introduced into this – that generates free radicals – plasms • Used to sterilise arthroscopes, urethroscopes •

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50. Ans.  (a) Filtration Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 35 • Filters – Depth and membrane filters • Filtration used for ƒ Sterilization of sera, sugar and antibiotic solutions ƒ Separation of toxins and phages from bacteria ƒ To obtain bacteria free filtrates of clinical samples for virus isolation ƒ Purification of water •

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51. Ans.  (d) Mycobacterium tuberculosis Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 39 • Quaternary ammonium compounds – cationic disinfectants • Kill Gram positive bacteria more than Gram negative bacteria • Does not kill spores or M. tuberculosis •

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54. Ans.  (b) Cover with a cloth/material Ref: WHO Guidelines on Drawing Blood: Best Practices in Phlebotomy. WHO guidelines on blood spillage management state the following steps: • Wear gloves • Use disposable paper towels to absorb as much of the body fluids as possible. • Wipe the area with water and detergent until it is visibly clean. • Saturate the area again with sodium hypochlorite 0.5% (10 000 ppm available chlorine). This is a 1:10 dilution of 5.25% sodium hypochlorite bleach, which should be prepared daily. • Rinse off the tongs, brush, and pan, under running water and place to dry. • Remove gloves and discard them. • Wash hands carefully with soap and water, and dry thoroughly with single-use towels. • Record the incident in the incident book if a specimen was lost, or persons were exposed to blood and body fluids. •

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38

Culture Media and Culture Methods

y Concentration of agar:  Solid agar preparation: Japanese agar – 2%, New Zealand agar 1.2%  Semisolid agar – 0.5%  Solid agar to inhibit swarming by Proteus – 6% 

Peptone Water

y

y

y

y

It is the basal liquid medium It is prepared by peptone + NaCl + distilled water It is the basal media for preparation of all media It is helpful in culture of organisms and to check for motility testing y It is used in indole test y y y y

y





y

y

y

y Microorganisms need proper growth constituents for its multiplication. y Culture media is prepared for meeting their requirements and also helpful in a way using their properties for identification. y Media is divided into solid and liquid media:  Liquid media is mainly used for bulk cultures of organisms as needed for vaccine production or industrial purposes.  Solid media is agar based, where agar acts as solidifying agent. Gelatin was once used as solidifying agent. But it gets melted at very low temperature (24°C) and cannot withstand autoclaving.





y

CULTURE MEDIA

4

BHI broth, peptone water, nutrient broth

Solid

Nutrient agar, blood agar, chocolate agar

Simple

Nonnutrient agar, nutrient agar

Complex

TCBS agar

Synthetic or defined

Hank’s balanced salt solution

Enriched

Blood agar, BHI agar, Chocolate agar, Todd Hewitt broth

Enrichment

Tetrathionate broth, Selenite F medium

Selective

Salmonella Shigella agar, Thayer-Martin medium, LJ medium

Indicator

MacConkey agar

Differential

Mannitol salt agar, MacConkey agar, CLED

Transport

Stuart’s transport medium

Anaerobic media

Thioglycollate medium, RCM

y y y y

y

Liquid

Basal Solid Medium y

Example

y

Culture media type

y

Table 1:  Examples of various culture media 

Figure 1: Nutrient agar plate showing colonies (Courtesy: Dr Deepika K, NG Hospital and Research Centre, Coimbatore)

Agar-Agar

Blood Agar

y

y

y

y It is an example for enriched medium y It is prepared by adding sterile blood to nutrient agar – then melted and cooled to 50°C for solidification y Concentration of blood that is added is usually around 5–10% y Usually sheep blood is added; other bloods are of human and horse. y

y

y

y

y

y

y It is a long chain polysaccharide y It is prepared from seaweeds y Agar as such has no nutritive value; it just acts as solidifying agent y It does not interfere with the growth of any bacteria y The melting temperature of agar is 95°C and it usually sets at 42°C

Nutrient broth–meat extract + peptone + NaCl Nutrient agar–Nutrient broth + agar Promotes the growth of most of the non-fastidious bacteria It is the media for most of the biochemical tests, to study the morphology of colony and to isolate the pure culture of organisms.

Unit 1     General Microbiology

Differential Media

Remember

• Blood agar helps to differentiate hemolysis and separate certain organisms: ƒ Partial or alpha hemolysis a Greenish discoloration surrounding the colonies—E.g. Pneumococci, Viridans streptococci ƒ Complete or beta hemolysis–Zone of complete clearing of blood around the colonies due to complete lysis of RBC’s – E.g. Beta-hemolytic Streptococci, Staphylococcus aureus ƒ No or gamma hemolysis: no color change E.g. Enterococcus ƒ Target hemolysis – a narrow zone of complete hemolysis followed by wide zone of incomplete hemolysis Eg: Clostridium perfringens •

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y MacConkey agar and CLED agar comes under this category y It differentiates between two groups of bacteria by using an indicator, which changes the color of the colonies of a particular group of bacteria but not the other group y Both agar media differentiate lactose and non-lactose fermenters y Both media inhibit the swarming of proteus y Pseudomonas colonies are seen as greenish color in both y The main difference between MacConkey agar and CLED agar:  CLED has additional advantage of supporting growth of GPC like Staphylococcus, Streptococcus and Candida species. y

y

y

y

y

y



Remember

• MacConkey agar is prepared by following ingredients (PLANTS): ƒ Peptone ƒ Lactose ƒ Agar ƒ Neutral red (indicator) ƒ Sodium taurocholate ƒ Water •

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Figure 2: Blood agar (Courtesy: Dr Deepika K, NG Hospital and Research Centre, Coimbatore, Tamil Nadu)

Table 2: Examples of bacteria for their respective hemolysis  

Hemolysis

Examples

Streptococcus pneumoniae, Viridans streptococci Streptococcus pyogenes, Staphylococcus aureus Enterococcus (Remember: Enterococcus can produce alpha, beta and gamma (no) hemolysis) Target hemolysis Clostridium perfringens Alpha hemolysis Beta hemolysis Gamma hemolysis

Chocolate Agar y It is a heated blood agar, prepared by adding 5–10% of sheep blood to the molten nutrient agar at 70°C, so that the RBCs will be lysed and the content of the RBCs will be released– changes the color from red to Brown y Supports growth of fastidious bacteria. E.g. Haemophilus influenzae

Figure 4: MacConkey agar with LF (pink) colonies (Courtesy: CDC)

Figure 3: Chocolate agar (Courtesy: Dr. Deepika K, NG Hospital and Research Centre, Coimbatore, Tamil Nadu)

Figure 5: MacConkey agar with NLF (Colorless) colonies (Courtesy: CDC)

y

y

40

Transport Media

y It is used for primary isolation of Mycobacterium species y Also promotes the growth of Nocardia species y The ingredients are mineral salts, asparagine, glycerol, malachite green and egg y Since egg is used as the solidifying agent—autoclaving cannot be done y Inspissation is the method of sterilization for LJ media y Mycobacterium tuberculosis produce—rough, tough, buff colored dry irregular colonies y Mycobacterium bovis produces smooth, white, emulsifiable colonies.

y Some organisms are so fragile and needs to be cautious while transporting the specimens to isolate those organisms E.g. Neisseria gonorrhoeae, Chlamydia trachomatis y Media that are used in such conditions are called as transport media

y

y

y

y

y

y

y

Table 3:  Examples for transport media

y

y

Streptococcus

Pike’s medium

Neisseria

• Amie’s medium • Stuart’s medium • Venkatraman Ramakrishnan medium • Autoclaved sea water • Cary Blair medium Buffered glycerol saline Cary-Blair medium Dacron or Rayon swab in sucrose phosphate glutamate buffer or M4 media  • Eagle’s minimum essential medium • Viral transport medium •

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Vibrio cholerae

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Shigella and Salmonella Chlamydia trachomatis Viral specimens

Chapter 4     Culture Media and Culture Methods

Löwenstein-Jensen Media

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Blood Culture Liquid Media Figure 6: LJ media (Courtesy: Dr Vanathi S, KAP Viswanathan Government Medical College, Trichy, Tamil Nadu)

y Isolation of organisms from blood needs special media and inoculation y Once the vein is selected, the skin area need to be cleaned with 70% isopropyl alcohol y Venipuncture is done–blood withdrawn and inoculated in the liquid broth y For adults  10–20 mL of blood y For children  1–5 mL of blood y Blood is mixed with the medium in the ratio of 1:10 y Blood culture media available in automated systems are:  BHI broth  Tryptic soy broth  Columbia broth  Brucella broth  Thioglycollate broth y Media should be incubated for up to 7 days to rule out brucellosis y

y

Anaerobic Media y Robertson cooked meat medium - Growth of anaerobes changes the medium into:  Red color—Saccharolytic  Black color—Proteolytic y Thioglycollate broth with hemin and vitamin K y Anaerobic broth y Smith Noguchi medium y





y

y

y

y

y

y

y

y





Media for Special Use y Sabouraud’s dextrose agar – For isolation of fungi y Cation adjusted Mueller-Hinton agar (MHA) – used for antimicrobial susceptibility testing y CHROM agar - for speciation of Candida based on the color produced by the species y

y







y

y

Table 4:  Types of culture media and its purpose Type of culture media Purpose Chemically defined media Complex media Reducing media Selective media

Differential media Enrichment media Figure 7: CHROMogenic medium – Each species produces different colors (Courtesy: Image from Author’s own thesis work)

Growth of chemo and photo autotrophs Growth of chemoheterotrophs Growth of obligate anaerobes Suppression of unwanted microbes and enhancing the growth of pathogen Differentiation of colonies from other organisms Suppression of unwanted microbes and enhancing the growth of pathogen to detectable levels within short period of time (liquid media)

41

Unit 1     General Microbiology

Anaerobic Culture Methods y McIntosh Fildes Jar: It is a metallic jar with a lid which has an inlet and outlet. This tube will suck the oxygen inside. Aluminum pellets coated with palladium serve as catalyst. The oxygen inside will be replaced with nitrogen and H2 and CO2. y GasPak: It is a commercially available envelope with pre packed mixture that liberates H2 and CO2 replacing oxygen when kept inside the anaerobic jar. y Pre-reduced anaerobic system y Anaerobic chamber y Robertson cooked meat medium y Thioglycollate broth with hemin and vitamin K Effectiveness of anaerobiosis can be checked by the following methods: y Chemical indicator: Reduced methylene blue – remains colorless in anaerobic conditions, but turns blue on exposure to oxygen. y Biological indicator: Pseudomonas incubated along with other plates. Absence of Pseudomonas which is an obligate aerobe – indicates perfect anaerobiosis. y

y

y

y

y

y



y

Figure 8: Blood culture bottles (left – uninoculated BHI broth; right – broth inoculated with blood) (Courtesy: Dr Vanathi S, KAP Viswanathan Government Medical College, Trichy, Tamil Nadu)

CULTURE METHODS Table 5: Types of culture methods  

Streak culture

Surface plating method routinely used for isolation of organisms

Stroke culture

For pure growth of organism used for slide agglutination or other diagnostic test

Lawn or carpet culture

For antimicrobial susceptibility testing

Stab culture

For biochemical reactions

Pour plate culture

For quantitative estimation of bacteria. E.g. urine culture

Liquid culture

For blood culture and sterility testing

y

Automated Culture Systems y Helps in detection of bacterial growth. Based on production of CO2 as the end product of metabolism of bacteria. Sensors will detect this. E.g.: BACTEC and BacT Alert y Identification systems that uses the biochemical characteristics or metabolism property of bacteria as the mechanism to detect. E.g.: VITEK, Phoenix y MALDI-TOF: Matrix assisted laser desorption/ionizationtime of flight mass spectrometry – This identifies the bacteria based on a unique protein that is present in the cell. y

y

y

Remember

Tests used for identification of bacteria: • IMViC tests: Indole test, Methyl red test, Voges Proskauer test, Citrate test • Kovac’s reagent used in indole test • TSI: Triple sugar iron agar contains glucose, lactose and sucrose. Its indicator is phenol red • Catalase test: For differentiation of Staphylococcus and Streptococcus • Urease test: Medium used is Christensen's urease medium •

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42

Figure 9: Colonies grown by streak culture in culture plate (Courtesy: Dr M Gomathi, Department of Microbiology, Government Chengalpattu Medical College, Tamil Nadu)

Figure 10: Battery of biochemical reactions done for a culture isolate (Courtesy: Dr M Gomathi, Department of Microbiology, Government Chengalpattu Medical College, Tamil Nadu)

Kovac’s method

Reagent that is used in Indole test: After adding the reagent in peptone water culture—if red colored ring is produced—then it means indole positive organims.

A type of Oxidase test; especially done for Neisseria colonies. A strip of filter paper soaked with oxidase reagent is placed and colonies are smeared over it. Within ten seconds, if the smeared area turns violet – it means oxidase positive

Catalase test

Coagulase test

To differentiate between Staphylococci and Streptococci (in GPC)

To differentiate between Staphylococcus aureus and CONS

• Also helpful to identify GNB • All species in Enterobacteriaceae are catalase positive except Shigella dysenteriae type 1

Two types: (i) Slide coagulase–Clumping factor or bound coagulase (ii) Tube coagulase–Free coagulase

Hydrogen peroxide is added in a slide (Slide catalase test) or tube (Tube catalase test) – colonies are taken in a wooden stick and placed over H2O2 – If the organism produces catalase – it splits the H2O2 and releases hydrogen and nascent oxygen; which in turn produces air bubbles

Few colonies are taken and mixed with rabbit or human plasma; Coagulase enzyme reacts with coagulase reacting factor in the plasma – binds to prothrombin – converts fibrinogen into fibrin – a visible clot is formed

Chapter 4     Culture Media and Culture Methods

Kovac’s reagent

Figure 11: Tube catalase test (note the bubbles seen) (Courtesy: Dr M Gomathi, Government Chengalpattu Medical College, Tamil Nadu)

43

Unit 1     General Microbiology

MULTIPLE CHOICE QUESTIONS 1. Simple basal media is:  (Recent Pattern July 15) a. Simple nutrient agar b. Alkaline peptone water c. Glucose broth d. Blood agar 2. Which of the following is a differential medium:  (Recent Pattern July 15) a. MacConkey agar b. Nutrient agar c. Deoxycholate citrate agar d. Selenite F broth 3. Which is enrichment media: (Recent Pattern Dec12) a. Selenite F broth b. Chocolate media c. Meat extract media d. Egg media 4. Chocolate agar is an example of:  (Recent Pattern Aug 13) a. Enriched medium b. Enrichment medium c. Selective medium d. Transport medium 5. Blood agar is an example of:  (Recent Pattern Dec 12) a. Enriched media b. Indicator media c. Enrichment media d. Selective media 6. Loffler’s medium is:  (MH 11) a. Indicator medium b. Selective medium c. Enrichment medium d. Enriched medium 7. Selenite F broth is an enrichment media for:  (Recent Pattern Dec 16) a. Salmonella b. Shigella c. E. coli d. Campylobacter 8. DCA media used in differentiation of which infection: (Recent Pattern Nov 15) a. Salmonella b. Staphylococcus aureus c. H. influenza d. Bordetella 9. Example of selective medium is:(Recent Pattern July 16) a. LJ medium b. Blood agar c. Selenite F broth d. Chocolate agar 10. In nutrient agar conc. of agar is: (Recent Pattern Dec 12) a. 1% b. 2% c. 3% d. 4% 11. Robert Koch assistant advised him to use agar instead of gelatin for solidifying culture media for cultivation of bacteria as: (Recent Pattern Dec 14) a. Agar has more nutrition b. Gelatin melts at 37 °C c. Gelatin is not easily available d. Agar is cheaper 12. Medium for growth of anaerobic bacteria:  (Recent Pattern Dec 12) a. SN medium b. LJ medium c. Blood agar d. None of the above 13. Lactose fermentation is seen in: (Recent Pattern Dec 13) a. Blood agar b. Chocolate agar c. MacConkey agar d. LJ medium 14. Triple iron sugar medium contains all, except: (Recent Pattern Dec 15) a. Lactose b. Sucrose c. Glucose d. Maltose 







15. In a patient with UTI, CLED (Cystine, lactose electrolyte deficient) media is preferred over MacConkeys media because: (AIIMS May 16) a. It is a differential medium b. It inhibits swarming of Proteus c. Promotes growth of pseudomonas d. Promotes growth of staphylococcus aureus and Candida 16. Which anticoagulant is used when blood is sent for blood culture:  (Recent Pattern Dec 13) a. Sodium citrate b. EDTA c. Oxalate d. SPS 17. In blood culture the ratio of blood to reagent is  (Recent Pattern Dec 14) a. 1:5 b. 1:20 c. 1:10 d. 1:100 18. Specific reason to disallow the sample for culture (Recent Pattern Dec 13) a. Sample brought within 2 hours of collection b. Sample brought in sterile plastic container c. Sample brought in formalin d. Sample obtained after cleaning the collection site 19. Which of the following transport media is used for Streptococcus? a. Amies medium b. Stuart`s medium c. Pike`s medium d. Cary Blair medium 20. Which concentration of agar is used to inhibit swarming? a. 1% b. 2% c. 0.5% d. 6% 21. Which of the following is an example of Differential media? a. CLED agar b. DCA agar c. XLD agar d. LJ medium 22. Identify the following culture method? 







44

a. Stroke culture b. Stab culture c. Lawn culture d. Streak culture 23. Indicator of anaerobiosis in GasPak system? a. Staphylococcus aureus b. Pseudomonas aeruginosa c. Bacteroides fragilis d. Clostridium perfingens

a. Oxidase test b. Coagulase test c. Catalase test d. Urease test 25. Identify the following culture media?

a. Blood agar b. Chocolate agar c. MacConkey agar d. BHI agar 26. Bacteriological loop is made up of: a. Nichrome wire b. Copper wire c. Ferric wire d. Bronze wire

27. Advantage of CLED agar over MacConkey agar is: a. Supports the growth of Gram positive bacteria b. Prevent the swarming of Proteus c. Differentiates LF and NLF d. Helpful in diagnosis of UTI 28. Indole positive organism is: a. Escherichia coli b. Klebsiella pneumoniae c. Pseudomonas aeruginosa d. Shigella sonnei 29. Which of the following type of hemolysis is seen in Pneumococci? a. alpha hemolysis b. beta hemolysis c. No hemolysis d. Alpha prime hemolysis 30. Loeffler's serum slope is used for the isolation of: a. Clostridium tetani b. Haemophilus influenza c. Streptococcus pneumoniae d. Corynebacterium diphtheria 31. Diffusible pigment is produced by: a. Pseudomonas aeruginosa b. Staphylococcus aureus c. Enterococcus sp., d. Staphylococcus albus 32. Which of the following organism(s) can not be cultured in artificial media:  (PGI May 2018) a. Mycobacterium leprae b. Klebsiella rhinoscleromatis c. Rhinosporidium seeberi d. Pneumocystis jirovecii e. Aeromonas hydrophila 33. Which organism shows pitting in blood agar?  (PGI Nov 2018) a. Eikenella corrodens b. Streptococcus pyogenes c. Pasteurella multocida d. Haemophilus influenzae e. Pseudomonas stutzeri

Chapter 4     Culture Media and Culture Methods

24. Identify the following biochemical test?



ANSWERS AND EXPLANATIONS 1. Ans.  (a) Simple nutrient agar

3. Ans.  (a) Selenite F broth

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 40

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 40

• Simple or basal media – E.g. Nutrient broth/agar • Consists of peptone, meat extract, sodium chloride, water and agar • Percentage of agar used is 2%

• Enrichment media are used to suppress commensal bacteria—it allows the growth of only pathogenic bacteria • Examples are tetrathionate broth – for Salmonella and Selenite F broth—for Shigella

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2. Ans.  (a) MacConkey agar Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 42 • Differential media helps to differentiate the organism based on a property • Examples are MacConkey medium and CLED • MacConkey medium helps to differentiate lactose fermenting and non-lactose fermenting colonies •

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4. Ans.  (a) Enriched medium Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 40 • Some organisms need the exact nutritional requirements for their growth • Hence basal media are enriched with either blood, serum or egg—called as enriched medium •

•

45

Unit 1     General Microbiology

• When blood is added to nutrient agar and heated—it gives chocolate agar • Chocolate agar is helpful for the growth of H. influenzae, N. meningitidis, N. gonorrhoeae and Pneumococcus •

•

5. Ans.  (a) Enriched media Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 40 • Already explained in Q.4 •

6. Ans.  (b) Selective medium Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 243 • Loeffler's serum slope is a selective medium for Corynebacterium diphtheriae • Selective medium helps for the growth of specific organism •

10. Ans.  (b) 2% Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 40 • Already explained in Q.1 •

11. Ans.  (b) Gelatine melts at 37°C Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 40 • Gelatin was initially used as solidyfiing agent – but the limitation is it gets melted at 42°C • Agar has no nutritive value • Agar melts at 98°C and usually sets at 42°C depending on the agar concentration. This has an additional advantage because we are using higher temperatures in autoclaving •

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7. Ans.  (b) Shigella Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 40 • Already explained in Q.3 •

8. Ans.  (a) Salmonella Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 303 • DCA – Deoxycholate citrate agar medium • Fecal samples should be plated directly on the selective medium called DCA to isolate Salmonella and Shigella •

12. Ans.  (a) SN medium Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 46 • Anaerobic broth is prepared by adding fresh animal tissues in broth – tissues used are rabbit kidney, spleen, testes or heart (Smith- Noguchi medium) •

13. Ans.  (c) MacConkey agar Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 42 • Already explained in Q.2 •

•

9. Ans.  (a) LJ medium Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 42 • Lowenstein-Jensen medium – used for primary isolation of Mycobacterium species • Also promotes the growth of Nocardia sp • The ingredients are mineral salts, asparagine, glycerol, malachite green and egg • Since egg is used as the solidifying agent – we cannot do autoclaving • Inspissation is the method of sterilization for LJ media •

14. Ans.  (d) Maltose Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 51 • TSI: Triple sugar iron agar. It contains glucose, lactose and sucrose. Indicator is phenol red • TSI is an example for composite media i.e. able to bring out many properties •

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15. Ans.  (d) Promotes growth of Staphylococcus aureus and Candida • The main difference between MacConkey agar and CLED: CLED has additional advantage of supporting growth of GPC like Staphylococcus, Streptococcus and Candida species. •

16. Ans.  (d) SPS Ref: Bailey and Scott T.B of diagnostic microbiology – 12th ed – Page 712 • SPS: Sodium polyanethol sulfonate is the anticoagulant used in blood culture medium • It has additional property of anticomplementary activity, inhibits lysozyme activity, interferes with phagocytosis and inactivates aminoglycosides •

•

46

Figure 12: LJ media (Courtesy: Dr Vanathi S, KAP Viswanathan Government Medical College, Trichy, Tamil Nadu)

Ref: Bailey and Scott T.B of Diagnostic Microbiology – 12th ed – Page 712 • • • •

•

•

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•

Blood culture medium – 50–100 mL Blood added correspondingly is 5–10 mL Ratio is 1:10 Why we have to dilute? ƒ To neutralize the anti-complementary or antimicrobial activity ƒ To match the body surface area – culture ƒ

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18. Ans.  (c) Sample brought in formalin Ref: Bailey and Scott T.B of Diagnostic Microbiology – 12th ed – Page 712 • Formalin kills all the microorganisms including spores • Hence specimens that are coming in formalin bottles are not accepted for microbiology laboratory. •

•

19. Ans.  (c) Pike`s medium Ref: Essentials of Medical Microbiology–Apurba Sastry– Page 45 • Streptococcus–Pike`s medium • Neisseria–Amie`s medium and Stuart`s medium • Vibrio cholera–Venkatraman Ramakrishnan medium, autoclaved sea water, Cary Blair medium • Shigella, Salmonella–Buffered glycerol saline, Cary Blair medium •

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•

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20. Ans.  (d) 6%

ƒ Bacteriophage typing ƒ For bacterial antigens and vaccines ƒ

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23. Ans.  (b) Pseudomonas aeruginosa Ref: Essentials of Medical Microbiology–Apurba Sastry– Page 50 • Effectiveness of anaerobiasis can be checked by the following methods: ƒ Chemical indicator: Reduced methylene blue– remains colourless in anaerobic conditions, but turns blue on exposure to oxygen ƒ Biological indicator: Pseudomonas incubated along with other plates. Absence of Pseudomonas which is an obligate aerobe–indicates perfect anaerobiasis •

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24. Ans.  (c) Catalase test Ref: Essentials of Medical Microbiology–Apurba Sastry– Page 55 • Catalase test is used to differentiate between Staphylococcus (Catalase positive) and Streptococcus (Catalase negative) • Also positive for members of families Enterobacteriaceae, Vibrio • False positive: Since blood has catalase colonies from blood agar result in false positive reactions. Use of iron wire or loop for picking up colonies may also produce false positive results • Nutrient agar is the ideal medium to perform the test •

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25. Ans.  (b) Chocolate agar

Ref: Essentials of Medical Microbiology–Apurba Sastry– Page 41

Ref: Essentials of Medical Microbiology–Apurba Sastry– Page 45

Concentration of agar:

• It is heated blood agar, prepared by adding 5–10% of sheep blood to the molten nutrient agar at 70°C, so that the RBCs will be lysed and the content of the RBCs will be released–changes the colour from red to BROWN • Supports growth of fastidious bacteria E.g.: Haemophilus influenza

• Solid agar preparation: Japanese agar–2%, New Zealand agar 1.2% • Semisolid agar–0.5% • Solid agar to inhibit swarming by Proteus–6% (Agar Melting temperature – 45°C) •

•

•

21. Ans.  (a) CLED agar Ref: Essentials of Medical Microbiology–Apurba Sastry– Page 32 • Differential media: MacConkey agar, CLED agar • It differentiates between two groups of bacteria by using an indicator, which changes the colour of the colonies of a particular group of bacteria but not the other group. •

•

22. Ans.  (c) Lawn culture Ref: Essentials of Medical Microbiology–Apurba Sastry– Page 47 • Lawn or Carpet culture: Uniformly thick surface growth of the bacterium on the solid medium • Useful in: ƒ Antimicrobial susceptibility testing by disk diffusion method •

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Chapter 4     Culture Media and Culture Methods

17. Ans.  (c) 1:10

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26. Ans.  (a) Nichrome wire Ref: Essentials of Medical Microbiology–Apurba Sastry– Page 47 • Inoculation of specimen onto the culture media is carried out with the help of loops and straight wires– made up PLATINUM or NICHROME wires • Platinum–costly–so not widely used •

•

27. Ans.  (a) Supports the growth of Gram positive bacteria Ref: Essentials of Medical Microbiology–Apurba Sastry– Page 46 • CLED agar–Cyteine Lactose Electrolyte Deficient media • Differential medium similar to MacConkey agar • Advantages over Mac–less inhibitory and supports the growth of Gram positive bacteria and Candida (Except beta hemolytic Streptococcus) • All other options fit both for CLED and Mac •

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47

Unit 1     General Microbiology

28. Ans.  (a) Escherichia coli

31. Ans.  (a) Pseudomonas aeruginosa

Ref: Essentials of Medical Microbiology–Apurba Sastry– Page 56

Ref: Essentials of Medical Microbiology–Apurba Sastry– Page 54

• Reagent used: Kovac's reagent • Indole positive: Red coloured ring–Organisms positive: E. coli, Proteus vulgaris, Vibrio cholera, Klebsiella oxytoca • Indole negative: Yellow coloured ring–Organisms: Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas, Shigella, Salmonella etc.,

• Pigment production is characteristic for certain bacteria and helps in identification of bacteria • Diffusible pigments: diffuse throughout the media plate ƒ E.g. Pseudomonas aeruginosa (Blue green colour) • Non diffusible pigments: only colonies are coloured ƒ E.g. Staphylococcus aureus, Serratia marsescens

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29. Ans.  (a) Alpha hemolysis

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32. Ans.  (a,c,d) a. Mycobacterium leprae; c. Rhinosporidium seeberi; d. Pneumocystis jirovecii

Ref: Essentials of Medical Microbiology–Apurba Sastry– Page 54

Ref: Ananthanarayan and Paniker`s T.B of microbiology – 10th ed – page 372

• Partial or alpha hemolysis–Greenish discoloration surrounding the colonies-E.g. Pneumococci, Viridans Streptococci • Complete or beta hemolysis–Zone of complete clearing of blood around the colonies due to complete lysis of RBC`s–E.g.: Beta hemolytic Streptococci, Staph aureus • No or gamma hemolysis–no colour change E.g. Enterococcus

Organisms that are non cultivable in culture media are: • Mycobacterium leprae • Treponema pallidum • Rickettsiae • Chlamydiae • Rhinosporidium seeberi • Pneumocystic jirovecii

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30. Ans.  (d) Corynebacterium diphtheria Ref: Essentials of Medical Microbiology–Apurba Sastry– Page 45 • Loeffler’s serum slope is an example for Enriched media • It contains serum • Used for isolation of C. diphtheriae (growth occurs in 4–6 hours) •

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33. Ans.  (a and e) a. Eikenella corrodens; e. Pseudomonas stutzeri Ref: https://jcm.asm.org/content/jcm/3/3/381.full.pdf • Colony morphologies help us to identify the organism but biochemical tests are must to confirm the organism • Eg: Hemolysis seen in Streptococcus group • Pigment production seen only in certain bacteria like Pseudomonas aeruginosa, Serratia marsescens • Pitting of colonies and corrosion are seen in ƒ Eikenella corrodens ƒ Pseudomonas stutzeri •

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48

Bacterial Genetics, Resistance and Susceptibility Testing

Transformation

y y

y The chromosomes of prokaryotic and eukaryotic cells have some pieces of DNA that move around the genome. Such movement is called transposition. y DNA segments that carry the genes required for this process and consequently move about chromosomes are called as mobile genetic elements or transposable elements or transposons y It was first discovered in the 1940s by Barbara McClintock.

y

y

y

y Transformation is a process of random uptake of free or naked DNA fragments from medium to bacterial cell. y Griffith experiment in Pneumococci—demonstrates this y Natural transformation has been studied in—Streptococcus, Bacillus, Haemophilus, Neisseria, Acinetobacter and Pseudomonas.

TRANSPOSONS

y







Gene transfer occurs in bacteria horizontally and vertically: Vertical transmission is from parents to offsprings; Horizontal transmission is done by three main methods namely: 1. Transformation 2. Transduction 3. Conjugation

y A plasmid called the fertility or F factor plays a major role in conjugation in E. coli.

y

BACTERIAL GENETICS

5

Conjugation

y

y y y

y

y

y Conjugation is the transfer of genetic material from one bacterium to another by forming a mating through conjugation tube y Through this method, transfer of plasmids coding for multiple drug resistance occurs (R factor) y R factor is a plasmid with two components—RTF + r determinants

y

y

y

y

y

y Transduction is defined as transmission of a portion of DNA from one bacterium to another by bacteriophage. y Generalized transduction: Transfer any part of the donor bacterial genome to recipient bacteria. y Restricted transduction: Only a particular part is transferred y Studied exclusively in lambda phage of E. coli

y Blotting technique refers to a method of transferring nucleic acid or proteins from gel onto a carrier followed by their detection by using specific nucleic acid probes y Southern blotting is done for identification of DNA fragments by DNA-DNA hybridization technique. y Northern blotting is done for identification of RNA where the RNA mixture is separated by gel electrophoresis technique. y Western blotting is a technique used for the identification of proteins; it is also called as immunoblotting. Protein antigen mixture is separated by a method SDS-PAGE. Western blot method is used in diagnosis of HIV. y Eastern blot – modification of western blot used to analyze proteins for post-translational modifications. y

Transduction

BLOTTING

PLASMID

y

y

y

y

y

y Plasmids are small double-stranded DNA molecules (dsDNA). y They are usually circular, that can exist independently of host chromosomes and are present in many bacteria. y They have their own replication origins and so they replicate on their own. y An episome is a plasmid that can exist either with or without being integrated into the host’s chromosome. y Conjugative plasmid is a type that have genes for pili and can transfer copies of themselves to other bacteria during conjugation.

 

Table 1: Blotting techniques

Unit 1     General Microbiology

Remember

Applications of Recombinant DNA Technology • Production of vaccines • Production of antigens used in diagnostic kits like ELISA • Production of proteins used in therapy like human growth hormone, insulin etc. • Transgenic animals • Gene therapy

Types of PCR

Characteristics

qPCR

Quantitative or Real time PCR: Measure the quantity of PCR product while the process is running (hence named as real time) Detection is based on: Nonspecific fluorescent dyes and specific set of DNA probes

Nested PCR

Two set of primers are used to increase the specificity; more specific

Multiplex PCR

More than one pathogen can be identified using multiple primers

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POLYMERASE CHAIN REACTION Definition y PCR is a technique used in molecular biology where many copies of DNA or a gene is amplified from a single nucleic acid. y It was first discovered by Karry B Mullis in 1983 y DNA replication occurs in a cycle where the product of first cycle become template for successive and it multiplies in exponential phase. Three main steps of PCR are: y Denaturation – the two strands of DNA are melted at high temperature to give separate strands. y Annealing – temperature is lowered for the primers to anneal to the strands y Extension: Once the primer is attached to the target – replication starts with the help of added nucleotides y

y

y

y

y

y

Table 2: Main steps of polymerase chain reaction  

Figure 1: Agarose gel electrophoresis of a PCR product (Bands are seen – positive)(Courtesy: Dr M Gomathi, Government Chengalpattu Medical College, Tamil Nadu)

Steps of PCR

Temperature

Denaturation

94 - 96°C

Annealing

60°C

NONAMPLIFICATION TESTS

Extension

72°C

y Loop mediated isothermal assay (LAMP): In PCR, different temperatures are used. Here at constant temperature the reaction is done. Hence there is no need of thermocycler. Primers used are called as loop primers. y DNA microarray: DNA spots are coated on a microarray plate and this hybridize with the cDNA in the test sample—most specific and can easily detects large number of nucleic acid sequences in the target simultaneously. Sequencing based assays: y Genetic mapping y Next generation sequencing y

y During the denaturation step, the enzyme that is added should not get denatured. Hence, thermostable DNA polymerase is added – taq polymerase (obtained from Thermus Aquaticus) y

Table 3: Polymerase chain reaction: (Amplification test)  

Types of PCR

Characteristics

Conventional PCR

PCR done with the help of thermocycler and PCR product is visualized by agarose gel electrophoresis

Reverse transcriptase RT-PCR

RNA is converted to dsDNA with the help of reverse transcriptase and then PCR is performed; Helpful for RNA viruses

y

y

y

ANTIMICROBIAL DRUG RESISTANCE Antimicrobial drugs have four main mechanism of action: 1. Inhibition of cell wall synthesis 2. Inhibition of cell membrane function 3. Inhibition of protein synthesis 4. Inhibition of nucleic acid synthesis





Contd...

50



Figure 2: Antimicrobial drugs

Beta Lactamases y These are the enzymes produced by the bacteria to open the beta lactam ring in the penicillin group of antimicrobials y They are called as penicillin destroying enzymes y Beta lactamases are mainly produced by:  Staphylococcus aureus  CONS  Enterococci sp.,  Neisseria gonorrhoeae  Haemophilus influenzae  Moraxella catarrhalis y Beta lactamases production is the most common mechanism of drug resistance y Continuous mutations in due course have led to extended profile of resistance—Extended spectrum beta lactamases, AmpC beta lactamases and Metallo beta lactamases. y Mechanism of beta lactamase production:  Inactivation of the penicillin through b-lactamase- or penicillinase-mediated hydrolysis of the b-lactam ring of the antibiotic.  Alteration of the target- intrinsic resistance involving a lowering of the affinity or the amount of the PBPs  Tolerance to the bactericidal effect of b-lactam antibiotics y Detection methods:  Penicillin disc diffusion test  Penicillin broth microdilution test  Penicillin zone edge test  PCR for blaZ gene y

y

y













y

Chapter 5     Bacteria Genetics, Resistance and Susceptibility Testing

Classification of Antimicrobial Drugs

y

y







y









51 Figure 3: Illustration demonstrating b-lactamase mechanism

Unit 1     General Microbiology

Extended Spectrum Beta Lactamases: (ESBL)

Metallo Beta Lactamase (MBL)

y ESBLs are b-lactamases capable of conferring bacterial resistance to the penicillin’s, first-, second-, and third- generation cephalosporins and aztreonam (but not the cephamycins or carbapenems) by hydrolysis of these antibiotics, and which are inhibited by b-lactamase inhibitors such as clavulanic acid.

y These enzymes confer resistance to all carbapenams (Imipenams, Meropenams, Ertapenams), all betalactams, aminoglycosides and quinolones.

y

y

Carbapenamase y Carbapenamases are b-lactamases with versatile hydrolytic capacities. y They have the ability to hydrolyze penicillins, cephalosporins, monobactams, and carbapenams. y Bacteria producing these b-lactamases may cause serious infections in which the carbapenamases activity renders many b-lactams ineffective. y KPC: Klebsiella pneumoniae carbapenamase is widely spreading and nowadays recent isolates are even resistant to colistin y

Remember

ESBL producing organisms: • Escherichia coli • Klebsiella sp. • Enterobacter sp. • Proteus sp. • Salmonella sp.

y

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y Detection methods:  Screening test: Disk diffusion test  Confirmatory test: Double disk diffusion test and Broth microdilution test. y





Figure 4: Antibiotic resistance of ESBL and MBL

Bush Jacoby classification of Beta lactamases: (Important groups)

52

Bush Jacoby Group Molecular Class Resistance To

Characteristics

1

C

Cephalosporins

Greater hydrolysis of cephalosporins than benzylpenicillin; hydrolyzes cephamycins

2a

A

Penicillins

Greater hydrolysis of benzylpenicillin than cephalosporins

2be

A

Extended-spectrum cephalosporins, monobactams

Increased hydrolysis of oxyimino-β-lactams (cefotaxime, ceftazidime, ceftriaxone, cefepime, aztreonam)

2d

D

Cloxacillin

Increased hydrolysis of cloxacillin or oxacillin

3a

C

Carbapenams

Broad-spectrum hydrolysis including carbapenems but not monobactams

Figure 5: Drug resistance gene transfer

Based on the mechanism—whether it is due to mutation or transferred from commensals or neighbor bacteria—certain characteristics are seen

Table 4: Differences between mutational and transferable drug resistance  

Mutational drug resistance

Transferable drug resistance

One drug resistance at a time

Multiple drug resistance

Low degree resistance

High degree resistance

Can be overcome by high drug doses

High doses ineffective

y MIC - Dilution tests – Agar dilution and broth dilution y Automated methods y

y

Description y Kirby Bauer method:  It is the conventional method- antibiotic disks are kept at equal distance in a lawn culture of bacterium in MHA – zone of inhibition is measured.  Only five drugs need to be kept in 100 mm plate  Equal spacing should be given  Zone of inhibition is to be measured after full incubation period  The interpretative guidelines is by – clinical laboratory standards institute (CLSI) y



Chapter 5     Bacteria Genetics, Resistance and Susceptibility Testing

Mechanism of Drug Resistance









Drug combinations can prevent Combinations cannot prevent Resistance does not spread

Spreads to same or different species

Mutants may be defective

Not defective

Virulence may be low

Virulence not decreased

Remember

MRSA active cephalosporins: • Ceftaroline • Ceftabiprole •

•

ANTIMICROBIAL SUSCEPTIBILITY TESTING y Mueller Hinton agar is the best medium to do antimicrobial susceptibility testing of most of the bacteria y It almost supports growth of all non-fastidious bacteria. y

Figure 6: Kirby Bauer method – disk diffusion method (Courtesy: Dr M Gomathi, Chengalpattu Government Medical College, Tamil Nadu)

y

Methods y Kirby Bauer disk diffusion method y Stokes method y E test y

y

y

y Stokes method: MHA is divided into three parts. Test organism inoculated on the central one third and control strain on upper and lower thirds of the plate y

53

Unit 1     General Microbiology

y Epsilometer test (E test):  A predefined antibiotic dilution level made at certain concentration is impregnated in a strip (E strip)  Commercially available  Quantitative method of detecting MIC by using principles of both dilution and diffusion of antibiotic into the medium y







y MIC tests:  Detect the minimum inhibitory concentration of antimicrobial at which the organisms are killed  Divided into agar dilution method and broth dilution method y





Figure 8: MIC test by broth dilution method (Serial dilutions of drug are taken) (Courtesy: Dr M Gomathi, Chengalpattu Government Medical College, Tamil Nadu)

Remember

Automated systems that can detect susceptibility: • VITEK • MGIT • Molecular method – PCR helps to detect the resistance genes. •

•

Figure 7: E-test strip method (Courtesy: Dr M Gomathi, Chengalpattu Government Medical College, Tamil Nadu)

54

•

Bacterial Genetics and Resistance 1. Mechanism of direct transfer of free DNA is: a. Transformation b. Conjugation c. Transduction d. Transposition 2. Phage mediated transfer of DNA from one bacterium to another is called as: a. Transformation b. Conjugation c. Transduction d. Transposition 3. Western blotting technique is used to detect: a. RNA b. DNA c. Proteins d. Nucleotides 4. Recombinant DNA technology is useful in all of the following except: a. Vaccine production b. ELISA – antigen c. Transgenic animals d. Southern blot 5. Bacterial toxins that are coded by lysogenic phage except: a. Diphtheria toxin b. Cholera toxin c. TSST d. Botulinum toxin C 6. F factor carries some chromosomal DNA, which is called as: a. F factor b. Hfr c. RTF d. F’ factor 7. Transmission of resistance by means of R factor is by: a. Transformation b. Conjugation c. Transduction d. Transposition 8. Identify the correct statement about mutational drug resistance? a. Multiple drug resistance at the same time b. High degree resistance c. Resistance can be overcome by combination of drugs d. Virulence not decreased 9. ESBL includes all except: a. Resistance to penicillins b. Resistance to 1, 2 and 3 generation cephalosporins c. Resistance to monobactam d. Resistance to carbapenems 10. Why do bacteria contain restriction enzymes? (PGI Pattern 2017) a. To cleave RNA for incorporation into ribosome b. To extend the length of bacterial chromosomes c. To prevent foreign DNA from incorporating into a bacte rial genome d. To process the exons from prokaryotic mRNA e.  To proteolytically cleave nuclear promoters 11. Which is/are statements are correct regarding transposons? (PGI Pattern 2017) a. They encode enzymes that degrade the ends of bacterial chromosomes b. They are short sequences that often mediate antimicrobial resistance c. Small transposons are called as insertion sequences d. They are short sequences of RNA that integrates with nucleic acid of host e. They cut nucleic acids at palindromic sequences

12. Unique feature of LAMP assay: (PGI Pattern 2017 ) a. Thermocycler is used b. Forward and Reverse primer are same c. Primers are not used d. Constant temperature e. Drug resistance can be detected 13. Which of the following is a quantitative type of PCR? (PGI Pattern 2017) a. Conventional PCR b. Multiplex PCR c. Nested PCR d. Real time PCR e. qPCR 14. Few of the following is/are amplication based molecular methods except: (PGI Pattern 2017 ) a. Polymerase chain reaction b. Reverse transcriptase PCR c. GenXpert d. DNA microarray e. LAMP assay

Antimicrobial Testing 15. Antimicrobial susceptibility testing is done in: a. Nutrient agar b. Mueller-Hinton agar c. Blood agar d. Chocolate agar 16. E test is a method based on: a. Diffusion b. Dilution method c. Both diffusion and dilution d. Automated 17. MRSA is mediated by: a. Plasmid b. Chromosome c. Transposons d. None 18. Identify the following antimicrobial susceptibility method?

Chapter 5     Bacteria Genetics, Resistance and Susceptibility Testing

MULTIPLE CHOICE QUESTIONS





















a. Kirby Bauer method



c. Stokes method

b. Modified Kirby Bauer method d. E test

55

Unit 1     General Microbiology

19. Which of the following is an anti-pseudomonal penicillin? a. Carbenicillin b. Cloxacillin c. Oxacillin d. Methicillin



20. Most common cause of acquired resistance is due to: a. Overuse and misuse of antimicrobials b. Evolution of resistant strains c. Inherent characteristics d. Mutation

ANSWERS AND EXPLANATIONS 1. Ans.  (a) Transformation

5. Ans.  (c) TSST

Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 70

Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 73

• Transformation is a process of random uptake of free or naked DNA fragments from medium to bacterial cell. • Griffith experiment – Pneumococci – demonstrates this • Natural transformation has been studied in – Streptococcus, Bacillus, Haemophilus, Neisseria, Acinetobacter and Pseudomonas

Phage mediated toxins are: • Diphtheria toxin • Cholera toxin • Verocytotoxin of E. coli • Streptococcus pyrogenic exotoxin (SPE) – A and C • Botulinum toxin C and D

•

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2. Ans.  (c) Transduction

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6. Ans.  (d) F’ factor

Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 71

Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 73

• Transduction is defined as transmission of a portion of DNA from one bacterium to another by bacteriophage. • Generalized transduction: Transfer any part of the donor bacterial genome to recipient bacteria • Restricted transduction: Only a particular part is transferred • Studied exclusively in lambda phage of E. coli

• When the F’ factor reverts from the integrated to free state – it sometimes carry with it some chromosomal DNA from adjacent site of its attachment. • Such an F factor which carries the chromosomal DNA is named as F’ factor

•

•

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3. Ans.  (c) Proteins Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 77 • Blotting technique refers to a method of transferring nucleic acid or proteins from gel onto a carrier followed by their detection by using specific nucleic acid probes • Blotting techniques: ƒ Southern blot – DNA ƒ Northern blot – RNA ƒ Western blot – Proteins ƒ Eastern blot – modification of western blot used to analyze proteins for post translational modifications •

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7. Ans.  (b) Conjugation Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 73 • Conjugation: Transfer of genetic material from one bacterium to another by forming a mating through conjugation tube • Through this method, transfer of plasmids coding for multiple drug resistance occurs • R factor is a plasmid with two components: RTF + r determinants •

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4. Ans.  (d) Southern blot

8. Ans.  (c) Resistance can be overcome by combination of drugs Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 82

Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 76

Mutational drug resistance

Transferable drug resistance

Applications of Genetic engineering/Recombinant DNA technology: • Production of vaccines • Production of antigens used in diagnostic kits like ELISA • Production of proteins used in therapy like human growth hormone, insulin etc. • Transgenic animals • Gene therapy

One drug resistance at a time

Multiple drug resistance

Low degree resistance

High degree resistance

Can be overcome by high drug doses

High doses ineffective

Drug combinations can prevent

Combinations cannot prevent

•

•

•

•

56

•

contd...

Transferable drug resistance

Resistance does not spread

Spreads to same or different species

Mutants may be defective

Not defective

Virulence may be low

Virulence not decreased

9. Ans.  (d) Resistance to Carbapenems Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 83 • Extended spectrum beta lactamases (ESBL) • Resistant to all penicillins and 1st, 2nd, 3rd generation cephalosporins and monobactams • Remain sensitive to Carbapenems and cephamycins •

•

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10. Ans  (c) To prevent foreign DNA from incorporating into a bacterial genome Ref: Jawetz – medical microbiology – 27th edition – Page 119 • Restriction enzymes (restriction endonucleases) provide bacteria helps to distinguish between their own DNA and DNA from other biologic sources. • These enzymes hydrolyze DNA at restriction sites determined by specific DNA sequences ranging from 4 to 13 bases. • Restriction enzymes that recognize more than 10 bases are useful for construction of a physical map and for molecular typing by Pulse field gel electrophoresis. •

•

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11. Ans.  (b and c) They are short sequences that often mediate antimicrobial resistance; Small transposons are called as insertion sequences

• In PCR, different temperatures are used; Here at constant temperature the reaction is done; Hence there is no need of thermocycler; • Primers used are called as loop primers. • There are separate forward and reverse primers • Mainly used for diagnosis of Mycobacterium tuberculosis •

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13. Ans.  (d and e) Real time PCR; qPCR Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 66 • Quantitative or Real time PCR – Measure the quantity of PCR product while the process is running (hence named as real time) • Detection is based on: Non specific fluorescent dyes and specific set of DNA probes •

•

14. Ans.  (d and e) DNA microarray; LAMP assay Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 66 • Amplification tests: ƒ PCR ƒ RT PCR ƒ Real time PCR or qPCR ƒ Nested PCR ƒ Multiplex PCR • Non-amplification tests: ƒ Loop mediated isothermal assay (LAMP) – In PCR, different temperatures are used; Here at constant temperature the reaction is done; Hence there is no need of thermocycler; Primers used are called as loop primers. ƒ DNA microarray: DNA spots are coated on a microarray plate and this hybridise with the cDNA in the test sample – most specific and can easily detects large number of nucleic acid sequences in the target •

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Chapter 5     Bacteria Genetics, Resistance and Susceptibility Testing

Mutational drug resistance

ƒ

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 63 • Transposons are short sequences of DNA that have the ability to move around in a cut and paste manner between chromosomal and extra chromosomal DNA molecules • They are also called as jumping genes • The two ends of transposons has inverted repeat sequences of nucleotides • Small transposons (1-2kb) is called as insertion sequenes or IS • Transposons are not self replicating • They depend on chromosomal or plasmid DNA for replication • Resistance determining segments of the R factor evolved from transposons–hence involved in antimicrobial resistance •

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simultaneously 15. Ans.  (b) Mueller-Hinton agar Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 82 • Mueller-Hinton agar is the best medium to do antimicrobial susceptibility testing of most of the bacteria • It almost supports growth of all non-fastidious bacteria •

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12. Ans.  (d) Constant temperature Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 66 • Loop mediated isothermal assay (LAMP) – non amplification method

16. Ans.  (c) Both diffusion and dilution Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 86 • Epsilometer test (E test) • Quantitative method of detecting MIC by using principles of both dilution and diffusion of antibiotic into the medium •

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57

Unit 1     General Microbiology

17. Ans.  (b) Chromosome Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 82

Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 79

• Methicillin Resistant Staphylococcus aureus (MRSA) • Target site of penicillin – PBP gets altered to PBP-2a • Altered protein do not bind to beta lactam antibiotics – prevent them from inhibiting the cell wall synthesis

• Anti-pseudomonal penicillin – Carbenicillin, Ticarcillin and Piperacillin

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18. Ans.  (c) Stokes method Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 85 • Stokes method – MHA is divided into three parts • Test organism inoculated on the central one-third and control strain on upper and lower thirds of the plate •

•

58

19. Ans.  (a) Carbenicillin

20. Ans.  (a) Overuse and misuse of antimicrobials Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 78 • Resistance – Intrinsic and Acquired • Acquired is mainly due to over use and misuse of antimicrobial agents • Intrinsic resistance. E.g.: Gram-negative bacteria are resistant to vancomycin •

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•

Father of microbiology

Louis Pasteur

Father of medical microbiology

Robert Koch

Side chain theory of antibody production

Paul Ehrlich

Father of antiseptic surgery

Joseph Lister

Contributions of Louis Pasteur

Principles of fermentation, sterilization techniques, pasteurization, Vaccines against anthrax and rabies, germ theory of disease, liquid media

Who introduced solid media?

Robert Koch

Koch’s phenomenon

Guinea pigs already infected with TB bacillus develops a hypersensitivity reaction when injected with tubercle bacilli or its protein

Exception to Koch’s postulates

Mycobacterium leprae, Treponema pallidum, Neisseria gonorrhoeae

Who invented electron microscope?

Ernst Ruska

Who described and coined the term phagocytosis?

Élie Metchnikoff

Who discovered PCR?

Kary B Mullis

Types of PCR

Conventional PCR, Reverse transcriptase PCR (for RNA), real time PCR (Quantitative)

Automated culture systems in diagnostic bacteriology

BACTEC, BacT/Alert, ESP culture system

Bacterial identification systems

MALDI TOF, VITEK 2, MicroScan Walkaway System

Automated system for cultivation of Mycobacterium tuberculosis

Mycobacterial Growth Indicator Tube (MGIT)

GenXpert is used for

Diagnosis of TB bacilli and for identification of Rifampicin Resistance – based on real time PCR method

Three steps of PCR

Denaturation – 95°C, Primer annealing – 55°C, Extension – 72°C

Solidifying agent in LJ media is

Egg

How to sterilize LJ media?

Inspissation

How to sterlize serum containing media

Inspissation or Tyndallization

Newer agent for OT sterilization that has replaced formaldehyde is called as:

EcoShield

Examples for indicator and differential media

MacConkey agar and CLED agar

Which culture method helps in quantitative estimation?

Pour plate culture

Vaccine preparation is done by which culture method

Liquid cultures – antigen is extracted

What are the antimicrobial susceptibility-testing methods?

Disk diffusion, dilution, epsilometer (E test), Automated method, molecular methods

Media that is used for AST

Mueller-Hinton agar

The organism broth prepared should match which of the McFarland standard unit for antimicrobial susceptibility testing

McFarland 0.5

Which of the methods can calculate minimum inhibitory concentration?

Agar dilution method, Broth dilution method, Epsilometer test

Who described transposons?

Barbara McClintock

Chapter 5     Bacteria Genetics, Resistance and Susceptibility Testing

HIGH YIELDING FACTS TO BE REMEMBERED IN GENERAL MICROBIOLOGY

59

s e t o N

BACTERIOLOGY

2

Unit Outline

1

Chapter 6: Staphylococcus Chapter 7: Streptococci Chapter 8: Pneumococcus Chapter 9: Neisseria Chapter 10: Corynebacterium Chapter 11: Bacillus Chapter 12: Clostridium Chapter 13: Enterobacteriaceae Chapter 14: Vibrio Chapter 15: Pseudomonas, Acinetobacter and Burkholderia Chapter 16: Haemophilus, Francisella and Pasteurella Chapter 17: Brucella and Bordetella Chapter 18: Mycobacterium Chapter 19: Spirochaetes Chapter 20: Rickettsia and Chlamydia Chapter 21: Helicobacter and Campylobacter Chapter 22: Mycoplasma and Legionella Chapter 23: Miscellaneous Bacteria

6 • • •

Cytolytic toxins – α,β,γ,δ hemolysins Panton valentine leukocidin Enterotoxin TSST (Toxic shock syndrome toxin) Epidermolytic toxin









y

• • • • •

•

Toxins

y

The most important organism coming under this genus are: y Staphylococcus aureus y CONS (Coagulase negative staphylococci)  Staphylococcus epidermidis  Staphylococcus saprophyticus  Staphylococcus haemolyticus  Staphylococcus lugdunensis

•

Staphylococcus

•

•

•

• Gram-positive cocci • Arrangement: Clusters • Key test for identification: Catalase test and Coagulase test (Both positive)

y

y

STAPHYLOCOCCUS AUREUS

Protein A y It binds to Fc terminal of IgG antibodies (except IgG3); it is a B cell mitogen y Coating of IgG antiserum with protein A – leads to agglutination when mixed with proper antigen – this reaction is called as coagglutination test

Coagulase

y





y

y Two types of coagulase:  Bound coagulase (also called as clumping factor)  Free coagulase y Coagulase test is helpful for speciation of Staphylococci Coagulase test positive

Staphylococcus aureus

Coagulase test negative

CONS





y

y Two types of coagulase test are done:  Slide coagulase test – detects bound coagulase  Tube coagulase test – detects free coagulase

Figure 1: Gram-positive cocci in clusters (Courtesy: CDC/ Dr Richard Facklam)

Pathogenesis

y

y

y Staphylococcus aureus causes wide variety of diseases that includes diseases caused due to direct infection and due to toxin secretion y Main pathogenicity is because of certain virulence factors:

Table 1:  Virulence factors of Staph. aureus   • Peptidoglycan (made up of polysaccharides) • Teichoic acid • Capsule (seen only in fresh strains)

Surface proteins

• Protein A • Clumping factor

Enzymes

• • • •

•

•

Cell wall factors

•

•

•

•

•

•

•

Figure 2: Tube coagulase test (Courtesy: Dr Prabha P, Kilpauk Medical College, Chennai, Tamil Nadu)

Coagulase Lipid hydrolases Hyaluronidase Nuclease

Hyaluronidase contd...

This enzyme helps in the break down of surrounding tissues and leads to spreading of infection

Unit 2     Bacteriology

Toxins y Cytolytic toxins: It has four types of hemolysins and one leucocidin:  Alpha hemolysin: this protein gets inactivated at 70°C and reactivated at 100°C  Beta hemolysin: It is responsible for hot cold phenomenon (hemolysis can be seen at 37°C)  Gamma hemolysin  Delta hemolysin  Leucocidin: Also called as Panton Valentine toxin (PVL) – also called as synergohymenotropic toxins y Enterotoxin:  Food poisoning caused by Staphylococcus aureus is because of a preformed toxin named as Enterotoxin  As this is preformed – the incubation period is very less – 1 to 6 hours after consuming the food – the symptoms starts  Food products responsible for these symptoms are – milk, meat and milk products  Types of enterotoxins are: A, B, C1-3, D, E and H  Most common toxin responsible for food poisoning – Type A y Toxic shock syndrome toxin:  Certain Staphylococcus aureus strains which have a phage coding of Group I releases TSST toxin  This toxin has an association with tampon usage and other wound infections  TSST causes systemic illness  It leads to fever, hypotension, vomiting and diarrhea  TSST – also named as Enterotoxin type F (or pyrogenic exotoxin C)

Table 2:  Clinical Spectrum of diseases caused by staphylococci

y

Type

Diseases

Skin and soft tissues

• • • • • •



•



•

•



•



•



Muscle and bones

y





• Septic arthritis (Most common cause is Staphylococcus aureus in both children and adults) • Osteomyelitis (especially vertebral osteomyelitis) • Pyomyositis •

•

Respiratory infections • VAP • Community acquired pneumonia (usually after influenza viral infection – post influenza pneumonia) •

•



Cardiac infections

• Infective endocarditis in native valve (left sided); prosthetic valve; and IV drug users (right sided)

Blood borne

• Septic shock

Toxin mediated

• Toxic shock syndrome • Food poisoning • Staphylococcal scalded skin syndrome

y





Carbuncle Furuncle Impetigo Folliculitis Mastitis Cellulitis

•





•

•

•

•

•



•





Laboratory Diagnosis y Gram staining from the specimen shows – pus cells with Gram-positive cocci in clusters y In culture:  In nutrient agar – strains produces golden yellow pigment and oil paint appearance (Fig. 4)  Pigmentation can be enhanced by adding milk in media or Ludlam’s medium  In blood agar: some strains produces beta hemolysis (Fig. 5)  Selective medium – mannitol salt agar y Unique battery of tests for S. aureus are:  Coagulase test  Heat stable thermo nuclease test  Phosphatase test  DNAse test  Mannitol salt agar media – yellow colored colonies – it is the selective media for S. aureus  Potassium tellurite agar – black colonies  Gelatin liquefaction – positive y Bacteriophage typing:  Typing by phage done by pattern method y

High Yield

y



• TSST and Staphylococcal enterotoxins are super antigens •



y Exfoliative Toxin:  Also called as epidermolytic toxin or Staphylococcal scalded skin syndrome toxin (SSSS)  Two milder forms are seen: Š Bullous impetigo Š Pemphigus neonatorum  Two severe forms are seen: Š Newborn illness – called as Ritter’s disease Š Older patients – TEN (Toxic epidermal necrolysis) y





Š

Š



Š

Š





y















y



64

Chapter 6     Staphylococcus Figure 5: Staphylococcus aureus showing hemolysis (Courtesy: Dr Prabha P, Kilpauk Medical College Chennai, Tamil Nadu)

Antimicrobial Susceptibility of Staphylococcus Aureus

Figure 3: Species identification of Gram-positive cocci

Figure 4: Golden yellow pigmented Staphylococcus aureus colonies (Courtesy: Dr J Pandian, Coimbatore Medical College, Tamil Nadu)

Figure 6: Antimicrobial resistance in Staphylococcus aureus

65

Unit 2     Bacteriology

Table 3:  Drug resistant strains of S. aureus Resistance

Points

MRSA – Methicillin resistant S.aureus

• MRSA can be detected by cefoxitin disk diffusion method • Cefoxitin acts as a surrogate marker for Methicillin resistance •

•

BORSA - Borderline resistant S.aureus

• Some strains when screened by cefoxitin disk diffusion method gets missed – but they are resistant • These organisms can be diagnosed by oxacillin screen agar

Treatment of Staphylococcus Aureus Infections Table 4:  Treatment of S. aureus Type and susceptibility of infection

Treatment

Penicillin sensitive S.aureus

Penicillin

Penicillin resistant S.aureus

Methicillin, Nafcillin, Oxacillin

MRSA

Vancomycin Alternate drugs: Teicoplanin, daptomycin, linezolid, quinupristin/dalfopristin, ceftobiprole

VRSA

Teicoplanin, daptomycin, linezolid, quinupristin/dalfopristin

Skin infections (MRSA)

Clindamycin, cotrimoxazole, doxycycline and linezolid

Nasal carriers

Mupirocin ointment

•

•

VRSA – Vancomycin • Vancomycin resistance: resistant S.aureus • VRSA – Vancomycin resistant Staphylococcus aureus – has both van A and mec A genes • VISA – Vancomycin intermediate Staphylococcus aureus – Do not carry resistant genes; But due to the presence of thick wall; antibiotics cannot penetrate and thus leads to resistance • VRSA can be detected by MIC method • No vancomycin disk diffusion method •

•

•

•

•

hVISA – heteroresistance VISA

• hVISA – heteroresistant VISA – This is used to denote for heterogeneously resistant S.aureus – which likely progress to VISA later • Can be detected by: E test and population analysis profile •

•

Inducible clindamycin resistance

• When clindamycin is kept for checking susceptibility – it may be sensitive • When an erythromycin disc is placed near clindamycin disc – it shows a special D shaped flattening towards erythromycin disc (D test) • In this case - clindamycin should be reported as resistant •

•

•

COAGULASE NEGATIVE STAPHYLOCOCCI y CONS are the most common causes for prosthetic device infections. y They are mostly present in normal human skin flora. y

y

Staphylococcus Epidermidis y Most commonly isolated CONS from clinical specimens y As S. epidermidis is a skin commensal – it is important to obtain the clinical history and to check for repeat isolation to term it as a pathogen. y Its virulence is mediated by biofilm production. y This biofilm production helps the organism adhere to implants like prosthetic devices and shunts. y It causes infective endocarditis in prosthetic patients. y

y

y

y

y

Staphylococcus Saprophyticus y Mostly causes UTI in sexually active females y Unique identification feature is Novobiocin resistant y DD: Staphylococcus xylosus which is an animal pathogen that causes rarely human infections; it is also Novobiocin resistant. y

y

y

Figure 7: Cefoxitin disc test for MRSA detection (Courtesy: Dr J Pandian, Coimbatore Medical College, Tamil Nadu)

66

1. True about Staphylococcus aureus- (Recent pattern July 2016) a. Microaerophilic b. Produce lemon yellow colonies c. Grows with 10% NaCl d. All are true 2. Mannitol salt agar is used for isolation of- (Recent pattern Nov 2015) a. Gonococcus b. Pneumococcus c. Staphylococcus d. Pseudomonas 3. Most common site of Staphylococcus carrier- (Recent pattern Dec 2016) a. Skin b. Nose c. Oropharynx d. Perineum 4. Most common staphylococcal phage strain causing infection- (Recent pattern Dec 2013) a. 80/81 b. 79/80 c. 3A/3C d. 69/70 5. Oil paint appearance on nutrient agar is seen in(Recent pattern Dec 2016) a. Streptococcus pyogenes b. Staphylococcus aureus c. Bordetella pertussis d. H. influenzae 6. Catalase positive novobiocin resistant bacteria (Recent pattern Dec 2016) a. Staphylococcus aureus b. Staphylococcus epidermidis c. Staphylococcus saprophyticus d. None of the above 7. All of the following statements about Staphylococcus aureus are true except- (All India 2010) a. Most common source of infection is cross infection from infected people b. About 30% of general population is healthy nasal carriers. c. Epidermolysin and TSS toxin are superantigens. d. Methicillin resistance is chromosomally mediated. 8. To differentiate between Staphylococcus epidermidis and Staphylococcus saprophyticus, which is used- (Recent pattern June 2014) a. Coagulase test b. Catalase test c. Novobiocin sensitivity d. None of the above 9. True about protein A of Staph aureus-  (Recent pattern June 2014) a. Causes opsonization b. Binds to Fc part of IgG c. Stimulate phagocytosis d. T-cell mitogen 























10. Staphylococcal scalded skin syndrome is caused by-  (Recent pattern Dec 2012) a. Hemolysin b. Coagulase c. Enterotoxin d. Epidermolytic toxin 11. Staphylococcal pathogenicity is indicated by- (Recent pattern Nov 2015) a. Coagulase positivity b. Hemolysis c. Lipoteichoic acid d. Endotoxin 12. Protein A of Staphylococcus binds to- (Recent pattern Dec 2016) a. IgA b. IgG c. IgD d. IgE 13. Synergohymenotropic toxins of Staphylococcus consists- (PGI Nov 2015) a. α toxin b. b toxin c. d toxin d. Panton-Valentine toxin e. g toxin 14. Incubation period of staphylococcal food poisoning are- (PGI Nov 2015) a. 4-6 hours b. 6-12 hours c. 12-18 hours d. 18-24 hours 15. Enterotoxin responsible for most of the cases of food poisoning by Staphylococcus- (Recent pattern June 2014) a. Type E b. Type C c. Type B d. Type A 16. Staphylococcus aureus causes- (Recent pattern Dec 2012) a. Erythrasma b. Chancroid c. Acne vulgaris d. Bullous impetigo 17. The most common cause of scalded skin syndrome is- (Recent pattern Dec 2013) a. Staphylococci b. Pneumococci c. Enterococci d. Meningococci 18. Staphylococcus aureus does not cause which of the following skin infection- (Recent pattern July 2016) a. Ecthyma gangrenosum b. Bullous impetigo c. Botryomycosis d. Cellulitis 19. Which organism causes toxic shock syndrome- (Recent pattern Dec 2013) a. Pneumococcus b. E. coli c. Staphylococcus aureus d. Enterococcus 

Chapter 6     Staphylococcus

MULTIPLE CHOICE QUESTIONS











67

Unit 2     Bacteriology

20. Which of the following organism is implicated in the causation of botryomycosis- (Recent pattern Dec 2014) a. Staphylococcus aureus b. Staphylococcus albus c. Pseudomonas aeruginosa d. Streptococcus pneumoniae 21. All of the following are true about Methicillin resistance in MRSA, except- (All India 2011) a. Resistance is produced as a result of altered PBP’s b. Resistance may be produced by hyperproduction of beta lactamase c. Resistance is primarily mediated/transmitted via plasmids d. Resistance may be missed at incubation temperature of 37°C during susceptibility testing 22. Blood culture is positive in which infection of Staph lococcus aureus- (Recent pattern Dec 2012) a. TSS b. SSSS c. Infective endocarditis d. Impetigo 23. Staphylococcus oxacillin resistance is best detected by- (Recent pattern June 2014) a. Cefixime MIC b. Cefoxitin disc diffusion c. Oxacillin disc diffusion d. Oxacillin agar 24. Drug of choice for MRSA- (Recent pattern Dec 2013) a. Penicillin G b. Ceftriaxone c. Vancomycin d. Cefazolin 25. All are true regarding resistance of penicillin in Staphylococcus aureus, except- (Recent pattern Dec 2015) a. Penicillinase production is transmitted by transduction b. Methicillin resistance is due to change in PBP c. Hospital strains mostly produce type D penicillinase d. Penicillinase production is plasmid mediated 26. A patient has prosthetic valve replacement and he develops endocarditis 8 months later. Organism responsible is- (AIIMS Nov 2010) a. Staph aureus b. Strep viridans c. Staph epidermidis d. HACEK 27. Which of the following statements is most correct regarding resistance to methicillin in MRSA- (Recent pattern 2011) a. Resistance is produced as a result of alteration in Penicillin Binding Protein (PBP) b. Resistance is produced by production of beta lactamase c. Resistance is mediated by plasmids d. Expression of resistance is enhanced by incubating at 37°C during susceptibility testing 





68

28. A patient developed cellulitis. Isolated organism is showing positive result in coagulase test. The causative organism is- (AIIMS Nov 2016) a. Staph aureus b. Staph epidermidis c. Strep pyogenes d. Pneumococcus 29. Catalase positive coagulase negative beta hemolytic bacteria- (Recent pattern Nov 2014) a. Strep pyogenes b. Staph aureus c. Coagulase negative staph d. Enterococci 30. Staphylococcus differs from Streptococcus by- (Recent pattern Dec 2014) a. Coagulase test b. Catalase test c. Phosphatase d. Gram-negative 31. Catalase positive beta hemolytic Staphylococcus(Recent pattern Dec 2012) a. S.aureus b. S.epidermidis c. S.saprophyticus d. None 32. Staphylococcus aureus differs from Staphylococcus epidermidis by- (Recent pattern Nov 2014) a. Is coagulase positive b. Forms white colonies c. A common cause of UTI d. Causes endocarditis in drug addicts 33. Most common cause of native valve endocarditis (Recent pattern Dec 2013) a. Staphylococcus aureus b. Coagulase negative Staphylococcus c. Streptococcus d. Enterococcus 34. Test to differentiate staphylococci from micrococci(Recent pattern Dec 2016) a. Catalase test b. Coagulase test c. Novobiocin sensitivity d. Oxidation fermentation 35. Acute bacterial prostatitis is caused by: (Recent pattern 2018) a. Enterococcus b. Streptococcus viridans c. Staphylococcus aureus d. Proteus 36. Regarding resistance in Staphylococcus aureus, is/are correct?  (PGI pattern May 2012) a. Plasmid mediated resistance is responsible for beta lactamase production b. SCC mecA type IV is associated with hospital acquired infections c. mecA and mecC genes are responsible for resistance to nafcillin and methicillin d. Methicillin can be given to Vancomycin resistance strains e. Resistance to other drugs occur in MRSA strains



































1. Ans.  (c) Grows with 10% NaCl

7. Ans.  (c) Epidermolysis and TSS toxin are superantigens

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 206

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 204

Salt milk agar and salt broth (containing 8-10% NaCl) are used as selective media for isolation of Staphylococcus aureus.

• Staphylococcal enterotoxins and Toxic shock syndrome toxin (TSST-1) are superantigens. • Epidermolysin is not a superantigen.

2. Ans.  (c) Staphylococcus

•

•

8. Ans.  (c) Novobiocin sensitivity

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 206

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 208

https://microbiologyinfo.com/mannitol-salt-agar-for-theisolation-of-staphylococcus-aureus/

• Both are coagulase test negative (CONS). S. saprophyticus is novobiocin resistant which its specific identification feature is.

Mannitol Salt Agar (MSA) is used as a selective and differential medium for the isolation and identification of Staphylococcus aureus from clinical and non-clinical specimens. • It contains mannitol and NaCl (7.5%) • S. aureus produces yellow colonies due to mannitol fermentation. •

•

3. Ans.  (b) Nose Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 205 About 10–30% of healthy persons carry Staphylococci in their nose and about 10% in perineum and hair. Vaginal carriage is about 5–10%. 4. Ans.  (a) 80/81 Ref: Essentials of Medical Microbiology – Apurba Sankar Sastry 10th ed – Page 218 • Phage type 80/81 is most commonly associated with outbreaks in hospitals. It is known as the epidemic strain of S. aureus. •

5. Ans.  (b) Staphylococcus aureus Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 206 • Staphylococcus aureus grows confluently on nutrient agar slope presenting characteristically with an oil-paint appearance. •

6. Ans.  (c) Staphylococcus saprophyticus Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 208 • Staphyloccus saprophyticus is a coagulase negative Staphylococci which can be identified by its resistance to novobiocin. • They are present in the normal human skin and periurethral area and can cause urinary tract infection in young, sexually active females. •

•

Chapter 6     Staphylococcus

ANSWERS AND EXPLANATIONS

•

9. Ans.  (b) Binds to Fc part of IgG Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 203 • Protein A of Staphylococcus aureus is anti-phagocytic, anti-complementary and a B-cell mitogen. • It binds to Fc portion of any IgG molecule, leaving Fab portion free to combine with its specific antigen. This property is made use of in co-agglutination test. •

•

10. Ans.  (d) Epidermolytic toxin Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 204. • Exfoliative (epidermolytic) toxin, also known as ET or exfoliatin is responsible for Staphylococcal scalded skin syndrome (SSSS). • The severe form of SSSS is known as Ritter’s disease in newborn and toxic epidermal necrolysis in older patients. •

•

11. Ans.  (a) Coagulase positivity Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 203 • Coagulase is an enzyme which brings about clotting of human and rabbit plasma. It acts with coagulase reacting factor present in plasma, binding to prothrombin and converting fibrinogen to fibrin. • It is the test for Staphylococcal pathogenicity. •

•

12. Ans.  (b) IgG Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 203 • Protein A of Staphylococcus binds to Fc portion of any IgG molecule, leaving Fab portion free to combine with its specific antigen. • This property is made use of in co-agglutination test. •

•

69

Unit 2     Bacteriology

13. Ans.  (d) PVL and (e) g toxin

20. Ans.  (a) Staphylococcus aureus

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 204

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 401 y and 604

• Panton-Valentine Leukocidin (PVL) is a two component toxin composed of S and F components. Such bicomponent membrane active toxins have been grouped as synergohymenotropic toxins. • PVL, gamma hemolysin and Luk-M toxins of Staph aureus belongs to this group.

• Botryomycosis is a mycetoma like lesion caused by Staphylococcus aureus and other pyogenic bacteria.

•

•

14. Ans.  (a) 4-6 hours Ref: Ananthanarayan and Paniker’s Microbiology – 10th ed – Page 204

Textbook

of

• The incubation period of Staphylococcal food poisoning is 2-6 hours. • Enterotoxin is responsible for the manifestations which include nausea, vomiting and diarrhea. • The toxin is a preformed toxin and therefore the incubation period is short. •

•

•

•

21. Ans.  (c) Resistance is primarily mediated/transmitted via plasmids Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 202 • The mechanism of Methicillin resistance in Staphyococci (MRSA) is regulated by a set of chromosomal genes called Staphylococcal cassette chromosomal mec genes (SCC mec). •

22. Ans.  (c) Infective endocarditis Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 593 • TSS and SSSS are toxin mediated diseases and impetigo is localized infection of skin and subcutaneous tissue. So the infecting organism is not cultivable from blood. • Infective endocarditis due to Staphylococcus aureus is an endovascular infection and so is blood culture positive. •

15. Ans.  (d) Type A Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 204 Type A is responsible for most of the cases. • There are 8 antigenic types of enterotoxin named A, B, C1-3, D, E and H.

•

•

16. Ans.  (d) Bullous impetigo Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 204 • Milder forms of Staphylococcal scalded skin syndrome are pemphigus vulgaris and bullous impetigo. • Epidermolytic toxin is responsible for this condition. •

•

17. Ans.  (a) Staphylococci Ref: Ananthanarayan and Paniker’s Microbiology – 10th ed – Page 204

Textbook

of

• The most common cause of scalded skin syndrome is Staphylococcus causing Staphylococcal scalded skin syndrome (SSSS). • Refer answer no.16 for further details. •

•

18. Ans.  (a) Ecthyma gangrenosum

Ref: Performance standards for antimicrobial susceptibility testing – CLSI M100S27 (2017) –Page 132 • Cefoxitin resistance testing indicates mecA mediated oxacillin resistance. • Cefoxitin is a surrogate marker for MRSA; hence disk diffusion with cefoxitin helps in the identification of MRSA strains. •

•

24. Ans.  (c) Vancomycin Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 208 • Vancomycin is the drug of choice for MRSA. • In VRSA, linezolid can be used. •

•

25. Ans.  (c) Hospital strains mostly produce type D penicillinase

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 204 and 321

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 202

• Ecthyma gangrenosum is a skin lesion caused by Pseudomonas aeruginosa. • The other three conditions given as options B, C and D are caused by Staphylococci.

• Penicillinases are b-lactamases which are plasmid mediated. They can be transmitted by transduction or conjugation. • Alteration in the penicillin binding protein PBP2a and changes in bacterial surface receptors reduces binding affinity of b-lactam antibiotics to cells. • This is the mechanism imparting resistance to all b-lactam antibiotics and has been named MRSA.

•

•

19. Ans.  (c) Staphylococcus aureus Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 204 and 205 • Toxic shock syndrome (TSS) is a common toxin mediated Staphylococcal disease. • TSS is mediated by Toxic shock syndrome toxin (TSST). •

70

23. Ans.  (b) Cefoxitin disk diffusion

•

•

•

•

30. Ans.  (b) Catalase test

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 208

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 207

• S. epidermidis is the most common cause of early prosthetic device associated infections like endocarditis.

• Staphylococci are catalase positive unlike Streptococci which are negative. • Catalase test is used to differentiate Staphylococci from Streptococci. •

•

•

Classification of endocarditis

Most common agent associated

Native valve endocarditis

Staphylococcus aureus

Prosthetic valve endocarditis

Early prosthetic valve endocarditis (within 12 months of valve replacement): S. epidermidis Late prosthetic valve endocarditis (After 12 months of valve replacement): Viridans streptococci

Endocarditis in IV drug abusers

31. Ans.  (a) S aureus Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 202 (Table 21.1) and 206 • Staphylococcus aureus produces beta hemolysis on blood agar. • The hemolysis is marked when incubated under 20-25% carbon dioxide. •

•

32. Ans.  (a) Is coagulase positive Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed –Page 202 (Table 21.1) and 208.

Staphylococcus aureus

Subacute endocarditis Viridans streptococci 27. Ans.  (a) Resistance is produced as a result of alteration in Penicillin Binding Protein (PBP) Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 202 • Resistance is produced as a result of alteration in Penicillin Binding Protein (PBP). • Methicillin resistance is chromosomally acquired. • Incubation at 30°C allows detection of methicillin resistance. •

Chapter 6     Staphylococcus

26. Ans.  (c) Staph epidermidis



 able 4:  Differences between S.aureus and T S.epidermidis Test

S.aureus

S.epidermidis

Coagulase

+

–

Mannitol fermentation

+

–

Heat-stable nuclease

+

–

Phenolphthalein phosphatase

+

–

Beta hemolysis on blood agar

+

–

Golden yellow pigment

+

–

Sensitivity to lysostaphin

+

–

•

•

28. Ans.  (a) Staphylococcus aureus Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 204 • Staphylococcus aureus is the most common cause of localized pyogenic infections. • It is coagulase test positive. •

•

29. Ans.  (c) Coagulase negative staph Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 207 • Staphylococci are catalase positive unlike Streptococci which are negative. • Catalase test is used to differentiate Staphylococci from Streptococci. • Among Staphylococci, Stapahylococcus aureus is coagulase positive whereas the other Staphylococcal species that are coagulase negative are grouped together as Coagulase negative Staphylococci (CONS). CONS are catalase positive and coagulase negative. •

•

•

• S. epidermidis is a common cause of stitch abscess. It can cause cystitis, central line associated blood stream infection and infection of mechanical devices. It causes endocarditis in drug addicts. •

33. Ans.  (a) Staphylococcus aureus Ref: Essentials of Medical Microbiology – Apurba Sankar Sastry 10th ed – Page 215 Staphylococcus aureus is the most common cause of native valve endocarditis followed by Streptococci and CONS. 34. Ans.  (d) Oxidation fermentation Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 209 Micrococci are Gram-positive cocci which are catalase positive. They can be mistaken for S. aureus. They are oxidative in Hugh-Leifon’s oxidation-fermentation test.

71

Unit 2     Bacteriology

Staphylococci and utilize glucose oxidatively and ferment glucose as well. Micrococci cannot ferment glucose but can only utilization glucose oxidatively. 35. Ans.  (c) Staphylococcus aureus Ref: Bailey T.B of surgery – Chapter 77 – Page 1357 • The usual most common organism responsible for acute prostatitis is Escherichia coli • Other organisms responsible are: (in the order) ƒ Staphylococcus aureus ƒ Staphylococcus albus ƒ Streptococcus fecalis ƒ Neisseria gonnorrhea ƒ Chlamydia trachomatis •

•

ƒ

ƒ

ƒ

ƒ

ƒ

36. Ans.  (a) Plasmid mediated resistance is responsible for beta lactamase production, (c) mecA and mecC genes are responsible for resistance to nafcillin and methicillin Ref: Jawetz Microbiology – page 204 – 27th edition

72

• Resistance to drugs occur in Staph.aureus by various mechanisms • Beta lactamase production is the most common and its plasmid mediated, transferred by conjugation. It means resistance to penicillin, ampicillin, Piperacillin • Resistance to nafcillin and methicillin – MRSA – due to SCC mec A gene and newly identified mec C gene; • SCC – Staphylococcal cassette chromosome – Type IV – responsible for community acquired infections and Type I, II, III, VI, VIII are responsible for hospital acquired infections • VISA – Vancomycin intermediate sensitive staph aureus • VRSA – Vancomycin resistant staph aureus • Drug of choice for MRSA – severe infections – Vancomycin • Drug of choice for VRSA – Linezolid, Teicoplanin •

•

•

•

•

•

•

•

Streptococci

7

y

y

y Many species comes under family of Streptococci y The species are classified based on certain properties as given below.

Table 1:  Classification of Streptococci • Alpha hemolytic - Viridans group • Beta hemolytic • Gamma (no hemolysis) – Enterococcus group

Beta hemolytic is classified based on the carbohydrate antigen (Lancefield grouping)

A to V (Except I and J) Group A – Eg: Streptococcus pyogenes

Group A Streptococci – classified based on M protein (Griffith typing)

80 types

•

•

•

Based on hemolysis in blood agar

Figure 1: Classification of streptococcus based on hemolysis  

Table 2:  Different species of streptococci Species

Lancefield group

Type of hemolysis

Laboratory tests

S. pyogenes

A

Beta

Bacitracin sensitive; PYR test positive; Ribose not fermented

S. agalactiae

B

Beta

CAMP test positive; hippurate hydrolysis

Viridans – S. mitis S. mutans S. salivarius S. sanguinis

Not typed

Alpha

Optochin resistant

Enterococcus sp

D

No hemolysisgamma

PYR positive; Growth in 6.5% NaCl

Nonenterococcus sp

D

No hemolysis -gamma

No growth in 6.5% NaCl

Figure 2: Strains of β-hemolytic streptococci

Unit 2     Bacteriology

STREPTOCOCCUS PYOGENES (GAS) • Gram-positive cocci • Arrangement: In pairs and chains • Key test for identification: Catalase test - negative, Beta type of hemolysis, Bacitracin test – sensitive.

Diseases caused by Streptococcus Pyogenes Necrotizing Fasciitis

•

•

•

Virulence Factors

y Also called as hemolytic streptococcal gangrene y Caused by M types 1 and 3 forming pyrogenic exotoxin A y Because of extensive necroses: it is called as flesh eating bacteria y Vancomycin is the DOC y

y

y

y

y Capsule: Helps in the inhibition of phagocytosis y Carbohydrate antigen: This antigen is responsible for cross reactivity leading to autoimmunity y Protein antigens: (MTR)  M proteins – Griffith typing; helpful in virulence  T proteins – not associated with virulence  R proteins – not associated with virulence y Pili – made up of M proteins and lipoteichoic acid y

y

y







y

Acute Rheumatic Fever y It is a complication or post sequel of acute pharyngitis y Occurs after 2-3 weeks of infection y This is mainly an immunological mediated injury because of cross reactivity y M protein present in S. pyogenes cross reacts with the antigens in the heart and joint tissues – leading to injury y Most important strains that cause RF are M types 5,18 and 24 y

y

y

y

y

Toxin Secreted by Streptococcus Pyogenes

Acute Glomerulonephritis

y Erythrogenic, Dick or scarlational toxin:  Intradermal injection into susceptible individuals  Produce an erythematous reaction (Dick test)  Effect of toxin is seen by induction of fever  Three types are there– A, B, C  A and C – Phage coded; B –Chromosomal y Hemolysins – Streptolysin O and S.  Streptolysin O: Antibody to this appears post infections; ASO titer helps to diagnose; oxygen labile  Streptolysin S: Responsible for hemolysis seen around streptococcal colonies on the surface of blood agar plates; oxygen stable y Streptokinase – Fibrinolysin – It is responsible for breaking down of fibrin barrier and spread of infections y Deoxyribonucleases (Streptodornase, DNAse) – depolymerization of DNA; helps to liquefy the thick pus; four types A, B, C, D; type B is the most antigenic; Demonstration of anti DNAse B antibody – retrospective diagnosis of S.pyogenes infection – especially in skin infections y Hyaluronidase helps in the spread of infection along the intercellular spaces

y It is a sequela of impetigo or sometimes pharyngitis y Post-impetigo – nephritogenic strains commonly involved are M types 49, 52, 53, 57, 58, 59, 60, 61 y Postpharyngitis – nephritogenic strains are M types 3,4,12,21,25

y











y

y

y

Table 4:  Antigenic cross reactivity

y





y

y

y

S.pyogenes

Human

Hyaluronic acid in capsule

Human synovial fluid

Cell wall proteins

Myocardium

Group A carbohydrates

Cardiac valves

Cytoplasmic membrane antigens

Vascular intima

Peptidoglycans

Skin antigens

Lab Diagnosis of Streptococcus Pyogenes (GAS) Infection y Gram staining – shows Gram-positive cocci in pairs and chains y

Table 3:  Diseases caused by Streptococcus pyogenes Suppurative diseases

Nonsuppurative diseases

• Pharyngitis – most common cause is S. pyogenes in children • Scarlet fever • Skin and soft tissue infections: ƒ Impetigo ƒ Pyoderma ƒ Cellulitis ƒ Necrotizing fasciitis • Toxic shock syndrome • Puerperal sepsis • Abscesses

• Acute rheumatic fever • Poststreptococcal glomerulonephritis

•

•

•

•

•

ƒ

Figure 3: Gram-positive cocci in chains (Courtesy: CDC)

ƒ

ƒ

ƒ

•

•

74

•

y Culture needs to be done in blood agar for identification of type of hemolysis – Streptococcus pyogenes belongs to beta type of hemolysis; y Key test is Bacitracin disk (0.04U) test – all strains of S.pyogenes are sensitive to bacitracin. y

y

y

y



High Yield

y It causes infections in the neonates to those who has born to women who carry GBS in their vagina y Two different types of infections are seen:  Early onset disease (presented 200 todd units) – detected by latex agglutination test

Poststreptococcal glomerulonephritis

Anti DNAse B antibody (>300 units); and anti hyaluronidase

Figure 5: CAMP test (Courtesy: CDC/ Dr Richard Facklam) y Drug of choice: Penicillin; Empirical therapy with ampicillin and gentamicin; y Prophylaxis need to be given for women carrying GBS:  Intrapartum antibiotic prophylaxis – 37 weeks of gestation – drugs given are ampicillin or penicillin  For those who are allergic to penicillin groups – alternative drugs are clindamycin or vancomycin y

y





Figure 4: Sensitivity testing of Streptococcus pyogenes (Courtesy: Dr J Pandian, Coimbatore Medical College, Tamil Nadu)

Table 5:  Treatment of S. pyogenes Infections

GROUP D STREPTOCOCCI – ENTEROCOCCI y Normally seen as an inhabitant of GIT y Most important pathogens are:  E. faecalis (M/c)  E. faecium  E. gallinarum  E. casseliflavus y Gram-positive cocci arranged at acute angles to each other y In blood agar – it can exhibit any type of hemolysis – alpha, beta or gamma y Characteristics are – Bile-Esculin test positive y These can grow at wide range of temperatures from 10°C to 45°C y Commonly causes UTI, bacteremia, nosocomial infections and bacterial endocarditis y This group is intrinsically resistant to many group of antimicrobials namely:  Penicillin  Cephalosporins  Gentamicin (low levels) y Acquired resistance has also evolved due to Van A, B, C, D, E genes to vancomycin – VRE– vancomycin resistant enterococci y

y

Type of infection

Treatment drug

Pharyngitis

Benzathine penicillin

Impetigo

Benzathine penicillin

Cellulitis

Penicillin G

Necrotizing fasciitis

Surgical debridement + Penicillin G /Clindamycin

Streptococcal TSS

Penicillin G + Clindamycin + IV immunoglobulin









y

y

y

y

y

GROUP B STREPTOCOCCI y Streptococcus agalactiae comes under GBS. y Main virulence factor is capsule – based on the capsular polysaccharide – many types are classified: Types Ia, Ib and II – IX y 10 to 40% of the females harbour S. agalactiae in their genital tract (GBS colonization) y

y

y

y







y

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Unit 2     Bacteriology

High Yield

High Yield

Intrinsic Resistance to Enterococci: • β-lactams (particularly cephalosporins and penicillinase resistant penicillins) • Low concentrations of aminoglycosides • Clindamycin • Fluoroquinolones • Cotrimaxazole •

•

•

•

•

Acquired Resistance to Enterococci: • High concentration of β-lactams (alteration of PBPs of production of β-lactamase) • Glycopeptides like Vancomycin and teicoplanin • High concentration of aminoglycosides • Tetracycline • Erythromycin • Fluoroquinolones • Rifampin • Chloramphenicol • Fusidic acid •

•

•

•

•

•

•

•

•

Table 6:  Vancomycin resistant enterococci Characteristics

Van A

Van B

Van C

Van D

Van E

Vancomycin MIC μg/mL

64 to >1000

4 to 1024

2 to 32

128

16

Teicoplanin MIC μg/mL

16–512

10 years of age

Toxoid 1 dose and human tetanus IG (Contd...)

Toxoid 1 dose

y











y

y

y

y

y

111

Unit 2     Bacteriology

Laboratory Diagnosis y Demonstration of bacilli or toxin in food or feces y Animal inoculation done by macerating the food material and inoculating the filtrate intraperitoneally; animals used or mice or guinea pig y Universally accepted assay for botulism is mouse bioassay. y

y

y

Treatment y Botulism antitoxin heptavalent is given for all adult forms of botulism y For infant botulism – a separate antitoxin that has A and B is also available y During outbreak a prophylactic dose of antitoxin should be given IM for all the exposed persons. y

y

y

y

y

Laboratory Diagnosis y y y y y

y

y

y

y

y

By colonic visualization – microabscesses can be seen Culture in selective media Detection of toxins by ELISA from stool samples Cell cultures – using Hep-2 and human diploid cell cultures Molecular methods (which detects genes for toxin A or B)

Treatment y It is treated by vancomycin or metronidazole y

CLOSTRIDIUM DIFFICILE

MISCELLANEOUS - CLOSTRIDIUM

y Long, slender Gram-positive bacilli y Spores are large, oval and sub-terminal y It causes infection mostly in those whose normal flora is lost – it is an opportunistic organism y It secretes two exotoxins namely A and B y Toxin A – potent enterotoxin which attaches to gut receptors y Toxin B – Cytotoxin y Toxin genes are located in the pathogenicity island in chromosomes

y Cl. septicum: Secretes four distinct toxins; it is associated with gas gangrene; Citron bodies with boat or leaf shaped pleomorphic bacilli is suggestive of Cl.septicum y Cl. novyi: Four types are present namely A, B, C and D; only type A is associated with gas gangrene y Cl. histolyticum is associated with gas gangrene, cellulitis, abscess formation and endocarditis in IV drug users y Cl. sorderlli can cause endophthalmitis, can cause infection of endometrium after childbirth or abortion and it can lead to toxic shock syndrome.

y

y

y

y

y

y

y

112

y When patients are on prolonged antimicrobial usage – due to alteration in the normal flora – Cl. difficle causes colitis – it is named as pseudomembranous colitis y Antibiotics associated diarrhea is mainly because of usage of clindamycin, ampicillin or fluoroquinolones

y

y

y

y

1. Which of the following Clostridia is non-invasive: (Recent Pattern July 2016) a. Clostridium novyi b. Clostridium botulinum c. Clostridium perfringens d. Clostridium tetani 2. Drumstick appearance is seen in: (Recent Pattern Dec 2014) a. Cl. tetani b. Cl. tetanomorphum c. Cl. sphenoids d. All 3. Tennis racket shape is seen in:  (Recent Pattern Dec 2016) a. Cl. perfringens b. Cl. botulinum c. Cl. tertium d. Cl. bifermentans 4. Saccharolytic species of Clostridia: (Recent Pattern Dec 2013) a. Cl. tetani b. Cl. cochlearum c. Cl. septicum d. None 5. Which one of the following species of Clostridium does not break carbohydrate and protein: (Recent Pattern June 2014) a. Sporogenes b. Septicum c. Cochlearum d. Novyi 6. Double zone of hemolysis is seen in: (Recent Pattern Dec 2015) a. Staphylococcus aureus b. Streptococcus pyogenes c. Clostridium perfringens d. Corynebacterium diphtheriae 7. Most common organism responsible for gas gangrene is: (Recent Pattern Dec 2014) a. Clostridium perfringens b. Clostridium difficile c. Clostridium tetani d. Clostridium septicum 8. All Clostridia cause myonecrosis except: (Recent Pattern July 2015) a. C.septicum b. C.difficile c. C.novyi d. C.welchii 9. Gas gangrene is due to: (PGI Nov 2014) a. Alpha toxin b. Theta toxin c. Beta toxin d. Delta toxin e. Epsilon toxin 10. True about Cl. perfringens are all except: (Recent Pattern July 2016) a. Invasive as well as toxigenic b. Alpha toxin is detected by Nagler’s reaction c. Beta toxin is most important in gas gangrene d. Theta toxin is perfringolysin 11. Each of the following statements concerning Clostridium perfringens is correct except:(Recent Pattern Dec 2014) a. It causes gas gangrene b. It causes food poisoning c. It produces exotoxin d. It is a Gram-negative rod that does not ferment lactose 















12. Opacity around colonies of Clostridium perfringens is due to: (Recent Pattern Nov 2014) a. Theta toxin b. Lecithinase c. Desmolase d. Cytokinin 13. True about gas gangrene are all except: (Recent Pattern Dec 2016) a. Most common cause is Cl. perfringens b. Extensive necrosis of muscles c. Cl. perfringens produce heat-labile spores d. Metronidazole is the drug of choice 14. Gas gangrene is caused by all except: (AIIMS May 2009) a. Cl. histolyticum b. Cl. novyi c. Cl. septicum d. Cl. sporogenes 15. Regarding Clostridium perfringens gas gangrene, false is: (AIIMS Nov 2010) a. Common cause of gas gangrene b. Nagler reaction positive c. Most common toxin is hyaluronidase d. Food poisoning strain of Cl. perfringens produces heat resistant spores 16. “Citron bodies” boat or leaf shaped pleomorphic organism in an exudate is: (Recent Pattern Dec 2013) a. Cl. welchii b. Cl. sporogenes c. Cl. septicum d. Cl. tetani 17. Nagler’s reaction is due to which toxin of Cl. perfringens: a. Alpha toxin  (Recent Pattern July 2016) b. Epsilon toxin c. Kappa toxin d. Delta toxin 18. Nagler’s reaction is given by: (Recent Pattern Dec 2016) a. Clostridium bifermentans b. Cl. perfringens c. Cl. sordelli d. All of the above 19. Nagler’s reaction is shown by:(Recent Pattern Aug 2013) a. Clostridium tetani b. Clostridium welchii c. Mycobacterium tuberculosis d. Mycobacterium leprae 20. Clostridium tetani is: (Recent Pattern Dec 2016) a. Gram-positive bacilli b. Gram-negative bacilli c. Gram-positive cocci d. Gram-negative cocci 21. Swarming growth on culture is characteristic of which Gram-positive organism: (Recent Pattern Dec 2013) a. Clostridium welchii b. Clostridium tetani c. Bacillus cereus d. Proteus mirabilis 

Chapter 12     Clostridium

MULTIPLE CHOICE QUESTIONS

113

Unit 2     Bacteriology

22. All are true regarding tetanus except: (AIIMS November 2010) a. Transmission through contaminated wounds and injuries b. More common in winters and dry weather c. Reservoir in soil and intestines of humans and animals d. No herd immunity or life-long immunity 23. Most common symptom of tetanus is: (Recent Pattern July 2016) a. Tonic-clonic seizures b. Hemiplegia c. Lock jaw d. Opisthotonus 24. Mechanism of action of tetanospasmin: (Recent Pattern Dec 2012) a. Inhibition of GABA release b. Inhibition of cAMP c. Inactivation of Ach receptors d. Inhibition of cGMP 25. Tetanus affects primarily: (Recent Pattern Dec 2012) a. Presynaptic terminal of spinal cord b. Postsynaptic terminal of spinal cord c. Neuromuscular junction d. Muscle fibers 26. Toxigenicity of tetanus is done on: (Recent Pattern Aug 2013) a. Rabbit b. Horse c. Mouse d. Guinea pig 27. All of the following statements regarding Clostridium tetani are true, except: (All India 2011) a. Spores are resistant to heat b. Primary immunization consists of three doses c. Incubation period is 6-10 days d. Person to person transmission does not occur 28. Botulism is a disease of: (Recent Pattern Dec 2013) a. Neural transmission caused by the toxin of the bacterium Clostridium botulinum b. Muscular transmission caused by the toxin of the bacterium Clostridium botulinum c. Neuromuscular transmission caused by the toxin of the bacterium Clostridium botulinum d. Non neuromuscular transmission caused by the toxin of the bacterium Clostridium botulinum 29. Botulinum toxin acts on: (Recent Pattern Dec 2012) a. Sympathetic system b. Parasympathetic system c. Amygdala d. Motor cortex 30. Among the toxin produced by Clostridium botulinum, the non-neurotoxic one is: (Recent Pattern Dec 2014) a. A b. B c. C1 d. C2 31. Mechanism of action of botulinum toxin: (Recent Pattern Dec 2015) a. Increased cAMP b. Increased cGMP c. Inhibition of acetylcholine release d. Inhibition of noradrenaline release 











114

32. Botulism is most commonly due to: (Recent Pattern Dec 2012) a. Egg b. Milk c. Meat d. Pulses 33. Which of the following is false about botulism? (Recent Pattern July 2015) a. It is a type of food poisoning b. Botulinum toxin is a potent neurotoxin c. It is an infection and not an intoxication d. The causative organism is Clostridium botulinum 34. Botulism causes: (Recent Pattern Dec 2012) a. Descending flaccid paralysis b. Descending spastic paralysis c. Ascending paralysis d. Ascending spastic paralysis 35. Most common organism responsible for pseudomembranous colitis: (Recent Pattern Dec 2013) a. Clostridium difficile b. Clostridium botulinum c. Clostridium histolyticum d. Clostridium butyricum 36. Cause of Clostridium difficile associated diarrhea:  (Recent Pattern Dec 2012) a. Trauma b. Dairy products c. Fried rice d. Antibiotic use 37. A patient of acute lymphocytic leukemia with fever and neutropenia develops diarrhea after administration of amoxicillin therapy, which of the following organism is most likely to be the causative agent: (AIIMS Nov 2008) a. Salmonella typhi b. Clostridium difficile c. Clostridium perfringens d. Shigella flexneri 38. Patient presenting with abdominal pain, diarrhea is taking clindamycin for 5 days. Treated with metronidazole, the symptoms subsided. What is the causative agent- (Recent Pattern Dec 2013) a. Clostridium difficile b. Clostridium perfringens c. Clostridium welchii d. Clostridium marneffi 39. Best method of diagnosis for Clostridium difficile (AIIMS Nov 2017) a. Pure strain isolation from culture b. Immunofluorescence c. Toxin detection by ELISA d. Toxin gene detection by PCR 









40. Trismus and Ophisthotonus are caused by  (CET 2018) a. Clostridium botulinum b. Clostridium tetani c. Clostridium perfringens d. Clostridium sporogenes

3. Ans.  (c) Cl. tertium

1. Ans.  (b) Closridium botulinum Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 258

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 257

• C. botulinum bacilli are virtually noninvasive and noninfectious. Botulism is caused by ingestion of preformed toxin in food. • C. tetani has little inverse property. C. perfringenes is also invasive besides toxin production.

• Oval and terminal spore resembling tennis racket is characteristic of C.tertium.

•

•

2. Ans.  (d) All

•

Spore position

Appearance

Example

Central or equatorial

Spindle shape

C. bifermentans

Subterminal

Club-shaped

C. perfringenes

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 266

Oval and terminal

Tennis-racket appearance

C. tertium

• C. tetani, C. tetanomorphum and C. sphenoides have drum stick appearance (spherical, terminal and bulging spores) and are indistinguishable.

Spherical and terminal

Drumstick appearance

C. tetani

•

Chapter 12     Clostridium

ANSWERS AND EXPLANATIONS

4. Ans.  (c) Cl. septicum Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 258 • In Robertson’s cooked meat medium, saccharolytic Clostridia turn the meat pink while proteolytic turns meat black producing foul and pervasive odors. • It is used to classify Clostridia. •

•

Both proteolytic and saccharolytic Proteolytic predominating

Saccharolytic predominating

C. bifermentans C. botulinum A,B,F C. histolyticum C. sordelli C. sporogenes

C. perfringenes C. septicum C. chauvoei C. novyi C. difficile

Slightly proteolytic but not saccharolytic

Saccharolytic but not proteolytic

Neither proteolytic nor saccharolytic

C. tetani

C. fallax C. botulinum C, D, E C. tertium C. tetanomorphum C. sphenoides

C. cochlearium

5. Ans.  (c) cochlearum Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 258 • Refer answer 4. •

6. Ans.  (c) Clostridium perfringens Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 263 • On fresh and heated blood agar, target hemolysis or double zone hemolysis is produced by C. perfringens. • A narrow zone of complete hemolysis due to theta toxin and a much wider zone of incomplete hemolysis due to alpha toxin are seen. •

•

7. Ans.  (a) Clostridium perfringens Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 261 • C. perfringens is the most important of the Clostridia causing gas gangrene (about 60%), with C. novyi and •

C. seticum next (20–40%) and C. histolyticum less often. • Other Clostridia usually found are C. sporogenes, C. fallax, C. bifermentans, C. sordellii, C. aerofoetidum and C. tertium. These may not be pathogenic by themselves. •

8. Ans.  (b) C. difficile Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 261 • C. difficile does not cause myonecrosis. • Refer to answer no.7 •

•

9. Ans.  (a) Alpha toxin Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 259 • The alpha toxin produced by C. perfringenes is responsible for the profound toxemia of gas gangrene. It is lethal, dermonecrotic and hemolytic. • It is a phospholipidase (lecithinase C). •

•

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Unit 2     Bacteriology

16. Ans.  (c) Cl. septicum

10. Ans.  (c) Beta toxin is most important in gas gangrene Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 259

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 262

• Alpha toxin is the most important toxin causing gas gangrene. • Beta toxin is associated with lamb dysentery, enteritis in animals and enteritis necroticans in human beings.

• Citron bodies are boat or leaf – shaped pleomorphic, irregularly stained bacilli in direct microscopic examination of gram stained films – suggestive of C. septicum.

•

•

11. Ans.  (d) It is a Gram-negative rod that does not ferment lactose Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 263 and 264 • Clostridia are gram-positive rods. • C. perfringenes ferment glucose, maltose, lactose and sucrose with production of acid and gas. •

•

12. Ans.  (b) Lecithinase

17. Ans.  (a) Alpha toxin Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 259 • Nagler’s reaction is seen as opalescence around bacterial colonies in serum or egg yolk media. • It is due to the alpha toxin (lecithinase) produced by C. perfringenes. •

•

18. Ans.  (d) All of the above

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 259

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 263

• Alpha toxin is a lecithinase which splits lecithin into phosphoryl choline and diglyceride in the presence of calcium and magnesium ions. • This reaction is seen as opalescence around bacterial colonies in serum or egg yolk media and is specifically neutralized by the antitoxin. • This specific lecithinase effect is known as Nagler’s reaction.

• Other Clostridia producing alpha toxin (C. novyi, C. sordelli, C. bifermentans, C. baratti) also give positive Nagler reaction. • In case of Clostridia other than C. perfringenes, the toxin is not neutralized by the antitoxin of C. perfringenes except C. bifermentans which produces a serologically related lecithinase.

•

•

•

13. Ans.  (c) Cl.perfringens produce heat-labile spores Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 258 • Spores of food poisoning strains of type A and certain strains of type C are heat resistant. Autoclaving at 121°C for 15 minutes is needed to destroy them. • Other spores are usually deatroyed by boiling within 5 minutes. •

•

14. Ans.  (d) Cl. sporogenes

•

•

19. Ans.  (b) Clostridium welchii Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 263 • Nagler reaction is produced by C. perfringenes (other names C. welchii, Bacillus aerogens capsulatus, B. phlegmatis emphysematosae.) • Other Clostridia producing alpha toxin (C. novyi, C. sordelli, C. bifermentans, C. baratti) also give positive Nagler’s reaction. •

•

20. Ans.  (a) Gram-positive bacilli

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 261

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 264

• C. perfringens is the most important of the Clostridia causing gas gangrene (about 60%), with C. novyi and C. seticum next (20–40%) and C. histolyticum less often. • C. sporogenes might be found on wound. But they are not pathogenic.

• C. tetani is a gram-positive, slender bacillus with a straight axis, parallel sides and rounded ends. • The spores are terminal, spherical and bulging giving the bacillus a drumstick appearance.

•

•

15. Ans.  (c) Most common toxin is hyaluronidase Ref: Ananthanarayan and Paniker’s Microbiology – 10th ed – Page 259

Textbook

of

• The most abundantly produced and most important toxin of C. perfringens is alpha toxin, which is a lecithinase •

116

•

•

•

21. Ans.  (b) Clostridium tetani Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 267 • C. tetani produces swarming growth. An extremely fine, translucent film of growth is produced which is visible only at the advancing edge. •

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 266 • Tetanus is common in warm climate, in developing countries where the soil is made fertile with organic manure. •

23. Ans.  (c) Lock jaw Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 266 • The first symptom of the onset of tetanus is trismus or lock jaw. •

24. Ans.  (a) Inhibition of GABA release Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 265 • Tetanospasmin specifically blocks synaptic inhibition in the spinal cord, presumably at inhibitory terminals that use GABA and glycine as neurotransmitters. The toxin acts presynaptically. • The abolition of spinal inhibition causes uncontrolled spread of impulses initiated anywhere in the CNS. This results in muscle rigidity and spasms. • Its action is similar to that od strychnine, but strychnine acts post-synaptically. •

•

•

28. Ans.  (c) Neuromuscular transmission caused by the toxin of the bacterium Clostridium botulinum Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 269 • The toxin of C. botulinum acts by blocking the production or release of acetyl choline at the synapses and neuromuscular junctions. •

29. Ans.  (b) Parasympathetic system Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 269

Chapter 12     Clostridium

22. Ans.  (b) More common in winters and dry weather

• Botulinum toxin acts on postganglionic parasympathetic nerve endings. • The manifestations of botulism are due to decreased acetylcholine in cranial nerve and parasympathetic nerve terminals – diplopia, dysphasia, dysarthria, descending symmetric flaccid paralysis ending in death by respiratory paralysis. •

•

30. Ans.  (d) C2 Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 269 • A, B, C1, C2, D, E, F and G are the eight types of C. botulinum strains based on immunological differences in the toxins produced by them. • All of these except C2 are neurotoxic. C2 toxin alone shows enterotoxic activity. •

•

25. Ans.  (a) Presynaptic terminal of spinal cord Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 265 • Tetanus toxin acts presynaptically in the spinal cord. • Its mechanism of action is similar to strychnine. But the difference is that strychnine acts postsynaptically. •

•

26. Ans.  (c) Mouse Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 267 • In vivo toxigenicity of tetanus toxin is done on mouse. A 2-4 day old- cooked meat culture (0.2 mL) is injected into the tail of a mouse. • Symptoms develop in 12-24 hours, beginning with stiffness of tail. Rigidity proceeds onto leg on inoculated side, opposite leg, trunk, forelimbs and so on. The animal dies in 2 days. • A second mouse that has received 1000 U of tetanus antitoxin acts as the control. •

•

•

27. Ans.  (a) Spores are resistant to heat Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 265 • The resistance of tetanus spores to heat appears to be subject to strain differences. Most are killed by boiling for 10-15 minutes but some resist boiling for up to 3 hours. • Autoclaving at 121°C for 20 minutes ensures destruction of spores. •

•

31. Ans.  (c) Inhibition of acetylcholine release Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 269 • The toxin of C. botulinum acts by blocking the production or release of acetyl choline at the synapses and neuromuscular junctions. •

32. Ans.  (c) Meat Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 269 • The source of botulism is usually preserved food – meat and meat products, canned vegetables, fish and other sea foods. •

33. Ans.  (c) It is an infection and not an intoxication Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 269 • The manifestations of botulism are due to botulinum toxin. • C. botulinum is virtually noninfective. •

•

34. Ans.  (a) Descending flaccid paralysis Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 269 • The characteristic pattern of botulism is symmetric descending flaccid paralysis. •

117

Unit 2     Bacteriology

• The manifestations of botulism are due to decreased acetylcholine in cranial nerve and parasympathetic nerve terminals – diplopia, dysphasia, dysarthria, descending symmetric flaccid paralysis ending in death by respiratory paralysis. •

35. Ans.  (a) Clostridium difficile

•

38. Ans.  (a) Clostridium difficile Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 270 and 271 • C. difficile is the most common cause of health care associated diarrhea. It occurs following the use of broad spectrum antibiotics like clindamycin, ampicillin or fluoroquinolones. • C. difficile disease is treated by discontinuing the antibiotics causing the disease and administering vancomycin or metronidazole. •

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 270 • C. difficile is the most common cause of pseudomembranous colitis. • It causes health care associated diarrhea in many developed countries following the use of broad spectrum antibiotic like clindamycin, ampicillin or fluoroquinolones. •

•

36. Ans.  (d) Antibiotic use Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 270 • C. difficile causes health care associated diarrhea in many developed countries following the use of broad spectrum antibiotic like clindamycin, ampicillin or fluoroquinolones. •

37. Ans.  (b) Clostridium difficile Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 270 • C. difficile causes antibiotic associated diarrhea. It is an opportunistic organism that causes disease when normal host flora is lost. •

118

• Broad spectrum antibiotics like clindamycin, ampicillin or fluoroquinolones predispose to C. difficile infection.

•

39. Ans.  (c) Toxin detection by ELISA Ref: Ananthanarayan and Paniker T.B of Microbiology – 10th ed – page 270 • Main stay of diagnosis: Detection of toxin A and/or B in stool by ELISA • Other methods are: Demonstrating toxin by Hep-2 cell cultures, Toxin gene identification, culture on selective media •

•

40. Ans.  (b) Clostridium tetani Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 265 • Toxin of tetani causes presynaptic block of glycine and GABA leads to loss of spinal inhibition • Clinically patient present as Trismus – lock jaw, muscle pain, laryngeal obstruction and in severe form it leads to ophisthotonus position •

•

13

Enterobacteriaceae

y







y













y

y A group of Gram-negative bacilli that are:  Nonsporing  Nonacid fast  Ferment sugars  Grows readily in ordinary media  Many are motile by peritrichous flagella except few  All are oxidase negative y Classification of Enterobacteriaceae:  Bergey’s classification  Based on lactose fermentation  Edward – Ewing classification y Based on Edward and Ewing, 8 tribes (I – VIII) are present in Enterobacteriaceae under which genus are described.

Table 1:  Enterobacteriaceae members according to fermentation of lactose

Escherichia, Klebsiella, Enterobacter, Edwardsiella, Serratia

Late lactose fermenters

Citrobacter, Shigella sonnei

Non lactose fermenters

Salmonella, Shigella, Proteus, Providencia, Morganella, Hafnia,

y Surface antigens:  Somatic antigen – O antigen  Flagellar antigen – H antigen  Capsular antigen – K antigen  Fimbrial antigen – F antigen y Colonization factor antigens (CFA) – helps in adherence of organism y P fimbriae – seen in UPEC – Uropathogenic E. coil – which binds specifically to P blood group substance on RBCs

Salmonella

Tribe IV

Citrobacter

Tribe V

Klebsiella, Enterobacter, Serratia, Hafnia, Pantoea

Tribe VI

Proteus, Providencia, Morganella

Tribe VII

Yersinia

Tribe VIII

Erwinia

y

Edwardsiella

Tribe III

‘O’ Antigen y y y y y y

y

Tribe II

y

Escherichia, Shigella

y

Tribe I

y

Genus

y

Tribes

y

Table 2:  Tribes of Enterobacteriaceae

y









Lactose fermenters

Virulence Factors Antigens y

Based on fermentation Examples of lactose

Figure 1: Gram-negative bacilli (E. coli) (Courtesy: CDC/ Dr WA Clark)

O Ag is the external part of the cell wall - LPS O antigens are resistant to heat and alcohol Antibodies to O antigens are IgM type Usually O antigens are shared commonly between same tribes or family that leads to cross reactions It is nonspecific Based on typing of ‘O’ groups – we can differentiate whether it is normal bowel flora or pathogenic Types 1, 2, 3, 4 etc. (early O groups) are normal flora Types 26, 55, 86, 111 (later O groups) are pathogenic

y y

Escherichia coli is a part of normal bowel flora in humans; it also causes numerous infections:

‘H’ Antigen

•

y y y

y y y y

y

Gram-negative bacilli Motile by peritrichate flagella Lactose fermenting Ferments all sugars with production of acid and gas IMViC = ++--

y

•

•

•

•

• • • • •

y

TRIBE I: ESCHERICHIA COLI

These antigens are located in flagella It can be denatured/removed by heat or alcohol Specific to an organism (no cross reactions) Antibodies to H antigens are IgG type

Unit 2     Bacteriology

‘K’ Antigen

If delayed: Refrigeration should be done – refrigerated sample is accepted for a maximum of 4-6 hours  Other way to collect urine is from catheters and in infants and babies by suprapubic aspiration y Microscopic examination of urine – when Gram stained smear shows one bacilli/ HPF – then it is indicative of UTI (But culture is confirmatory) y Culture of urine: Media used are:  MacConkey agar or CLED agar  Blood agar  CLED agar is preferred over MAC because: Š It supports the growth of both Gram-positive cocci like Staphylococci and Gram-negative bacilli Š It supports growth of Candida species Š We can differentiate LF and NLF y Quantitative count estimation of colonies (by Kass criteria) is must to diagnose UTI:  ≥105 colonies/mL = diagnosed as UTI  Exception: 102 colonies/mL is enough to diagnose UTI in certain conditions like Š When the organism is Staphylococcus Š Catheterized samples Š Hematogenous infections Š Patients already on antimicrobials 



y Located in the envelope or microcapsule y K antigen encloses the O antigen y

y

Toxins



y

y Hemolysins y CNF 1 – Siderophores y Enterotoxins – Heat labile toxin and heat stable toxin y

y

y

y





Table 3:  Classification of E. coli Enteropathogenic E. coli (EPEC)

Watery diarrhea in infants and children; do not produce enterotoxin; not invasive

Enterotoxigenic E. coli(ETEC)

Causes traveler’s diarrhea; produces enterotoxins – heat labile toxin or heat stable toxin; adheres to intestinal mucosa by means – fimbrial or colonization factor antigen; noninvasive

Enteroinvasive E. coli (EIEC)

Resembles that of shigella; enteroinvasive; penetrates HeLa or HEP-2 cells in tissue culture; this penetration is plasmid mediated; VMA – virulence marker antigen

Enterohemorrhagic E. coli (EHEC)

Named as verotoxigenic E. coli; produces shiga like toxin; inhibits protein synthesis; causes HUS; serotype O157:H7 – does not ferment sorbitol like others – can be detected by Sorbitol MacConkey media

Enteroaggregative E. coli (EAEC)

Causes persistent diarrhea; stacked brick adherence pattern on Hep-2 cells

Uropathogenic E. coli (UPEC)

Most common cause of UTI in a community; P fimbriae is the virulence factor



Š

Š

Š

y





Š

Š

Š

Š

Clinical Manifestations y Urinary tract infections (UTI):  Most common cause of UTI overall is E. coli  Infection usually presents as uncomplicated cystitis  It is mainly an ascending type of infection from the gut flora that causes lower UTI  In pregnant women—5 – 7% are suffering from asymptomatic bacteriuria because of E. coil y It causes diarrhea – infantile diarrhea, adult and traveler’s diarrhea y Hemolytic uremic syndrome (HUS) y Septicemia y Pyogenic infections y Meningitis in infants y

Figure 2: E. coli – lactose fermenting colonies grown in urine sample (Courtesy: Dr Prabha P, Kilpauk Medical College, Chennai, Tamil Nadu)









y

y

y Other supportive tests done in urine sample to diagnose UTI:  Catalase test  Griess nitrite test  Triphenyl tetrazolium chloride test  Dip slide culture  Leukocyte esterase test y











y

y

Sample: Feces in Diarrhea

y

y Culture of stool specimen in MacConkey agar and serogrouping y

Laboratory Diagnosis Sample – Urine in UTI y Clean voided, mid-stream urine sample need to be collected y For culture – transportation should be within ½ hour to a maximum of 2 hours y

y

120

Table 4:  Toxin demonstration tests In vivo tests

In vitro tests

Rabbit ligated ileal loop tests

Tissue culture test

Infant rabbit bowel test

Chinese hamster ovary cells (Contd...)

In vitro tests

Infant mouse intra gastric

ELISA

Adult rabbit skin permeability test

Passive agglutination test

y Invasiveness is the hallmark virulence for shigellosis y Exotoxin secreted by Shigella dysenteriae type 1 – has less role in pathogenesis; it is similar to EHEC verotoxin; y Endotoxin is LPS in cell wall – that causes irritation to bowel wall. y

DNA probes

y

y

y Cell culture to demonstrate toxins y PCR to identify the genes y

Clinical Features

y

TRIBE I: SHIGELLA SPECIES • • • •

•

•

•

•

y Infection is mainly transmitted through direct contact (feco oral route), through fomites, through flies and as a part of gay bowel syndrome in homosexuals y Incubation period is 1-2 days y Abdominal pain, fever and watery diarrhea y Tenesmus seen y Can also cause hemolytic-uremic syndrome y S. dysenteriae type 1: Causes arthritis, toxic neuritis, conjunctivitis, parotitis and intussusception y Self-limiting condition usually y

Gram-negative bacilli Nonmotile, no capsule Nonlactose fermenting (Exception: Sh. sonnei) Specific media: DCA, XLD, Selenite F broth

y

y

Chapter 13     Enterobacteriaceae

Virulence Markers

In vivo tests

y

y

y Four important species: Sh. dysentriae, Sh.flexneri, Sh.boydii, Sh.sonnei y Differentiation of Shigella species done by mannitol fermentation or ODC decarboxylation y

y

Table 5: Differentiation of Shigella species by Mannitol fermentation and ODC decarboxylation  

Group Species

Serotypes Mannitol ODC fermentation

A

S. dysenteriae

10

–

–

B

S. flexneri

6

+

–

C

S. boydii

15

+

–

D

S. sonnei

1

+

+

y

y

Laboratory Diagnosis y Stool samples collected and transported in buffered glycerol saline or Gram-negative broth. y Inoculate the stool samples in Selenite F broth for 6 hours and subculture from the broth to culture plates. y Specific culture media used are:  Deoxycholate citrate agar (DCA)  Xylose lysine deoxycholate agar (XLD)  Hektoen enteric agar (HE)  Salmonella shigella agar (SS) y All the culture isolates need to be confirmed by polyvalent and monovalent sera. y

y

y









y

Remember

Important Points to be Remembered • All are catalase positive except Shigella dysenteriae type 1 • Shigella dysenteriae type 1 – is called as Shiga bacilli • Shigella sonnei and Shigella flexneri type 6 are always indole negative • S. flexneri type 6 has three biotypes namely – Boyd 88, Manchester and Newcastle • S. flexneri is the most common species isolated in India. •

•

•

•

•

Pathogenesis y Usually the infection is limited to GIT; invasiveness in blood is rare. y Infective dose for shigellosis is 10-100 (as it can survive acid environment). y Organism invades the mucosal epithelial cells called as M cells  and escapes by phagocytic vacuoles  multiplies and spread within cell cytoplasm and pass to adjacent cells (Basolateral spread) continues spreading and leads to inflammation and cell death necrosis causes transverse ulcers. (pseudomembrane) y Invasive character of Shigella is because of plasmid that codes for protein called Virulence marker antigens (VMA).

Figure 3: Colorless (nonlactose fermenting) colonies of Shigella (Courtesy: CDC)

y

y

y

y

Treatment y Uncomplicated shigellosis is self-limiting y Those with systemic presentations and debilitated persons – treatment with antibiotics is must y First line drug: Ciprofloxacin y Second line drugs: Pivmecillinam, Ceftriaxone, Azithromycin y

y

y

y

TRIBE II: EDWARDSIELLA TARDA y Gram-negative bacilli y Motile y The name tarda means weak – because it has a weak fermentation of carbohydrates y

y

y

121

Unit 2     Bacteriology

y It is a part of normal gastrointestinal flora y Its strict pathogenesis is uncertain y But it has been isolated from clinical specimens like blood, urine, wounds. y

y

y

y H antigen:  Flagellar antigen; heat labile  More specific and helps in confirmation in serology  It has phase variation – phase I and II  S.Typhi exist in only phase 1 and hence it is monophasic y Vi antigen:  Heat labile  It is a surface polysaccharide antigen enveloping the O antigen  Vi antigen is also seen in: Š Salmonella Paratyphi C Š Salmonella Dublin Š Citrobacter (Bethesda-Ballerup group)  Poorly immunogenic  After infections, antibodies to Vi antigen is very low and hence not helpful in diagnosis  Antibody also disappears so fast in human body during convalescence. y









TRIBE III: SALMONELLA

y



• Gram-negative bacilli • Motile with peritrichate flagella • Exceptions: Salmonella Gallinarum and Pullorum are non motile • Special media: DCA, XLD, SS agar, Wilson Blair bismuth sulfate medium • H2S production occurs (black colored colonies) • Broths: Tetrathionate broth and Selenite F broth • IMViC = -+-+ •

•

•

•

•

•

•





Š

Š

Š







y Salmonellae can be classified into two groups:  Typhoidal Salmonellae: Š Typhoid bacilli Š Paratyphoid bacilli – A, B, C  Nontyphoidal Salmonellae: Salmonella gastroenteritis y Characteristics of Salmonella bacilli:  All ferment sugars and form acid and gas  Exception is S. Typhi – anaerogenic – does not produce gas  S. Typhi needs tryptophan as the growth factor  Classification of Salmonellae is done based on O and H antigens and phase variation of H antigen named as Kauffmann White scheme y



Š

Š



y









Pathogenesis y Route of infections: Contaminated food or water y Infectious dose ranges from 200 CFU to 106 CFU y When the salmonella bacilli enters the small intestine, it penetrates the mucosal layer and resides in the Peyer’s patches. y After crossing the epithelial layer in SI, the bacilli is phagocytosed by macrophages and live intracellularly y This phagocytosed bacilli travels throughout the body via macrophages to lymphatic and reaches the reticuloendothelial system y

y

y

y

y

Clinical Features Fever caused by S. Typhi  Typhoid fever Fever caused by S. Paratyphi  Paratyphoid fever Fever caused by both collectively called as Enteric fever Incubation period varies from 10–14 days Symptoms: Prolonged fever, head ache, myalgia, sweating, anorexia, abdominal pain, nausea, diarrhea, coated tongue, splenomegaly y Physical findings: Rose spots, relative bradycardia y Complications: GI bleeding, Intestinal perforation, GBS, neuritis, muttering delirium, DIC, pancreatitis, arthritis, pyelonephritis, osteomyelitis, HUS, endophthalmitis, pancreatitis, hepatic abscess y Carrier state:  Untreated patients – excrete in feces for up to 3 months  1-4% go for chronic asymptomatic carriage sheds for >1 year y y y y y

y

y

y

y

y

y

y

y





Figure 4: Typical black colored salmonella colonies in XLD agar (Courtesy: CDC)

Salmonella Typhi and Paratyphi Antigenic Structure y O antigen:  Also called as Boivin antigen  It is not affected by boiling, alcohol or acids  The antigen is not specific; it can cause cross reactions y



122





Table 6:  Carriers of Salmonella Convalescent carriers

Shed bacilli 3 weeks – 3 months

Temporary carriers

Shed bacilli 3 months – 1 year

Chronic carriers (Permanent carriers)

More than 1 year

Table 8:  Interpretation of Widal Test

y Diagnosis of enteric fever: (BASU)

Organism

O antigen

H antigen

AH antige

BH antigen

S. Typhi

+

+

–

–

S. Paratyphi A

+

–

+

–

+

–

–

+

y

Table 7:  Diagnosis of enteric fever Week of diagnosis

Diagnostic method

First week

Blood culture

S. Paratyphi B

Second week

Widal test – Antibody detection – O – 1:200 H – 1:100

• O antigen positive means - Agglutination titer should be > 1:200 • H antigen positive means - Agglutination titer should be >1:100

Third week

Stool culture

Fourth week

Urine culture

Why there is variation in titer interpretation: • Depends on the dilution method used it varies slightly • Depends on the baseline titer of the endemic area - that is - In endemic areas -most of the people have pre-existing antibodies - so when active infection is to interpreted - we need to set a range above this level - hence the titer of O above 1:200 and H above 1:100 is positive; When the titre is less than this, do serial testing of serum after two weeks to check for increase in titre. Why paratyphoid O antigens not used: • Typhoid and paratyphoid O antigens - cross react due to sharing of factor 12

•

•

•

•

y Definitive diagnosis is by isolation of S. Typhi or Paratyphi from blood, bone marrow or other sites, or from rose spots, stool specimens y Sensitivity of culture:  Blood culture is 40-80%  Bone marrow culture is 55-90% y

y





Chapter 13     Enterobacteriaceae

Laboratory Diagnosis

•

Widal Test y It is an example for tube agglutination test (serological test) y Not useful nowadays because of endemic titer difference y Two tubes are used namely Felix tube and Dreyer’s tube (i) Felix tube – O antigen (ii) Dreyer’s tube – H antigen y Antibodies present in the human body reacts with commercially prepared O and H antigens y O antigen that is common to all (as it is not specific) y H antigen of S. Typhi, S. Paratyphi A and S. paratyphi B y Serial dilutions were prepared in the ratio of 1:20, 1:40, 1:80 and so on y When antibodies meet antigens at zone of equivalence according to lattice hypothesis – agglutination occurs y H agglutination looks like loose, cotton wooly clumps y O agglutination looks like disc like pattern y The titer at which the agglutination occurred is the interpretation y Every endemic area has its own antibody levels in the human body – it should be identified by studies and only above that level of antibodies are taken as pathogenic level of exposure – it is called as baseline titer y Interpretative value: (usually)  O – 1:200  H – 1:100 y

y

y





y

y

y

y Other Test:  IgM Typhidot  PCR based tests  WBC count showing leucopenia with relative lymphocytosis y Typing of Salmonella is done at National Salmonella Phage Typing centre – located at Lady Hardinge Medical college, New Delhi y S. Typhi Phage type A and E1 are common in India y







y

y

y

Diagnosis of Carriers

y

y

y

y y y y

y

y

y

y

y

y

y





High Yield • How to make Widal a best test: ƒ Do Widal test only during second week of fever (as antibodies start appear) ƒ Do follow up sample testing – if there is rise in titer in second sample then it is infectious ƒ Ask vaccination history – vaccinated people has antibodies ƒ Compare with other blood investigations ƒ H antigens are more specific and persist longer than O antigens

Vi antigen testing – presence shows carriers state Isolation from feces or from bile, urine Sewer swab technique Filtration through millipore membrane

Treatment y Treatment: Ceftriaxone is the DOC even for MDR; alternate drugs are ciprofloxacin, azithromycin; For carriers – Ampicillin or amoxicillin y MDR Salmonella – multidrug resistance to the following drugs (CAT)  Chloramphenicol  Ampicillin  Trimethoprim y FQ’s resistance is also increasing in India y

y







y

•

ƒ

Empirical treatment for Enteric fever

Azithromycin or Ceftriaxone

Confirmed case of Typhoid fever

Ciprofloxacin

MDR Salmonella

Ciprofloxacin or Azithromycin

Carrier of typhoid

Ampicillin or Cipro for 6 weeks

ƒ

ƒ

ƒ

ƒ

123

Unit 2     Bacteriology

Typhoid Vaccines y Parenteral TAB vaccine y Parenteral Vi Polysaccharide vaccine y Typhoral vaccine y

y

y y y y

Three species are seen: C.freundii, C.koseri, C.amalonaticus C. freundii strains are called as Bethesda–Ballerup group These are part of normal intestinal flora It can cause UTI, biliary tract infections, surgical site infections, neonatal meningitis, brain abscess, bactermia in neutropenic patients

y

y

y

y

y

Typhoral Vaccine y It is an oral live attenuated vaccine from S. Typhi strain named Ty21a y It lacks the UDP-galactose-4 epimerase enzyme (Gal E mutant) y When taken orally, it gets multiplied for some period of time and then self-destructs by itself. y Hence the body recognizes it as active infection (but because of mutation – pathogenesis will not be seen) y Immunity develops after a week and may last up to 4-5 years y It is given after 6 years of age y Four doses: One capsule every other day for a week (day 1, day 3, day 5, and day 7). The last dose should be given at least 1 week before travel to allow the vaccine time to work. (If traveling to endemic areas) y

y

TRIBE V: KEHS GROUP: (KLEBSIELLA, ENTEROBACTER, HAFNIA, SERRATIA) Klebsiella Species

y

y

y

y

y

Parenteral Vi Polysaccharide Vaccine y One dose provides protection. It should be given at least 2 weeks before travel to allow the vaccine time to work. y A booster dose is needed every 2 years for people who remain at risk. y

y

• • • • •

•

•

•

•

•

Gram-negative bacilli Nonmotile Lactose fermenting Capsulated IMViC = --++

y Klebsiella pneumoniae is seen in respiratory tract and in stool samples (5% of normal individuals has commensals in feces) y It causes pneumonia and extensive hemorrhagic necrotizing consolidation of lung y It also causes UTI especially in catheterised individuals, bacteremia, pyogenic liver abscess and hospital acquired infections y Klebsiella pneumoniae has subspecies namely:  K.pneumoniae pneumoniae  K.pneumoniae ozaenae  K.pneumoniae rhinoscleromatis y

y

y

y







Nontyphoidal Salmonellosis y These are caused by:  S. typhimurium  S. enteritidis  S. newport  S. heiderlberg  S. javiana y NTS is more common in tropical countries y It is mainly associated with consumption of animal products especially chicken eggs y Hence Pasteurization of eggs is must to avoid infection y MDR Salmonella was first identified in S. typhimurium – named as ACSSuT – that is it is resistant to Ampicillin, Chloramphenicol, Streptomycin, Sulfonamides, Tetracycline y It causes gastroenteritis, bacteremia, CNS infections, UTI, bone and joint infections y Salmonella osteomyelitis occurs in sickle cell disease patients y Treatment:  Gastroenteritis – Ciprofloxacin  Systemic infections – Ceftriaxone y



Klebsiella pneumoniae

Friedlander’s bacilli – most common species – causes pneumonia

Klebsiella ozaenae

Frisch bacillus – causes atrophic rhinitis – causes foul smelling discharge

Klebsiella oxytoca

Causes nosocomial infections









y

y

y

y

y

y K. ozanae causes progressive atrophy of nose and pharynx which presents as a foul smelling discharge y K. rhinoscleromatis causes rhinoscleroma, a destructive granuloma of nose and pharynx y Another species, K.granulomatis (previously called as Calymmatobacterium granulomatis) – causes chronic ulcerative disease of genital region (STD) – named as Granuloma inguinale y K. oxytoca is another species which is also isolated from clinical specimens y

y

y

y

y

y





ENTEROBACTER SPECIES y Three species are seen:  E. cloacae  E. aerogenes  E. sakazakii (this has been included now in Cronobacter) y Gram-negative, lactose fermenter y Motile, IMViC = --++ y Normal commensal found in feces y Causes UTI, pneumonia, nosocomial infections, wound and device infections y Most of the strains are intrinsically resistant to ampicillin, first and second generation cephalosporins y





TRIBE IV: CITROBACTER SPECIES



y

y

• • • • •

•

•

•

124

•

•

Gram-negative bacilli Motile Late lactose fermenter Citrate utilizing organism H2S producer – C. freundii

y

y

y









y Gram-negative, pigment producing organism y It produces a diffusible pigment named as Prodigiosin which gives the culture pink or magenta colored y Serratia when causes respiratory infection – it produces colored sputum due to pigment production that may be mistaken from hemoptysis – hence it is termed as pseudohemoptysis y But only 10% of the isolates produce the red pigment y It causes pneumonia, bacteremia and endocarditis in narcotics addicts (usually the nonpigmented isolated are more causing hospital acquired infections) y

Dienes phenomenon – to detect swarming growth Swarming cannot be seen in MacConkey agar; it can be see in nutrient agar and blood agar

y

y

y

y

Chapter 13     Enterobacteriaceae

SERRATIA MARCESCENS

Figure 6: Swarming nature of Proteus sp., (Dienes’ phenomenon) (Courtesy: CDC/ Dr John J Farmer) y It causes UTI, bacteremia, pneumonia, focal lesions in debilitated patients and infections in burn patients y It is a powerful urease splitter; hence in UTI – it causes hydrolysis of urea and liberates ammonia – ammonia is an alkalyzer which leads to alkalinization of urine y Alkalinization leads to formation of struvite stones (Staghorn calculi) y P.mirabilis is the most common isolate in clinical specimens y P. mirabilis is sensitive to ampicillin and cephalosporins usually; in contrast, P.vulgaris is highly resistant to antibiotics y

y

y

Figure 5: Pigmented colonies of Serratia in blood agar (Courtesy: CDC/ Dr Negut)

TRIBE VI: PROTEUS, PROVIDENCIA AND MORGANELLA GROUP Proteus Species • • • • • • • •

•

•

•

•

•

•

•

•

Gram-negative bacilli Motile Nonlactose fermenter Pleomorphic, no capsule Fishy odor Powerful urease producer PPA reaction positive Swarming seen

y Proteus species are of Proteus vulgaris, Proteus mirabilis, Proteus penneri y Unique characteristic is swarming growth:  To inhibit swarming: Š Increased (6%) concentration of agar Š Incorporation of chloral hydrate (1:500) Š Sodium azide (1:500) Š Alcohol (5-6%) Š Sulfonamide Š Surface active agents Š Boric acid (1:1000)

y

y

Providencia Species y y y y

Species are Providencia rettgeri, P. alcalifaciens, P. stuartii These are seen as members of normal intestinal microbiota P. stuartii causes UTI and infections in burns Drugs sensitive usually are Amikacin and ciprofloxacin

y

y

y

y

TRIBE VII: YERSINIA • • • • •

•

•

•

•

•

Gram-negative bacilli Y. pestis has bipolar staining – safety pin appearance Pleomorphic Y. pestis – nonmotile Y. pseudotuberculosis and enterocolitica – motile at 22°C

y

y



Š

Š

Š

Š

Š

Š

Š

Yersinia Pestis y Gram-negative pleomorphic bacilli y It has characteristic bipolar staining with special stains like:  Wright stain  Giemsa stain  Wayson stain y It can be grown in basal media like nutrient agar and blood agar y Nonlactose fermenter in MacConkey agar y

y







y

y

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Unit 2     Bacteriology

Types of plague

Features

Pneumonic plague • Spreads by droplet infection during epidemics • Causes hemorrhagic pneumonia • Cyanosis occurs • Highly infectious and highly fatal •

•

•

•

Septicemic plague

• Complication of bubonic or pneumonic plague •

Figure 7: Bipolar staining of Y. pestis (Courtesy: CDC)

Antigenic Structure y Plague toxin – two toxins:  Endotoxin – LPS in cell wall  Murine toxins – toxin for animals y Heat labile protein named as Fraction I (seen in all of the virulent strains) y Antigens V and W y Bacteriocin named as Pesticin y Coagulase producer y Fibrinolysin y Unidentified surface component y





y

y

Figure 8: Gangrene seen in plague infection

y

y

y

y

Laboratory Diagnosis y Buboes are collected and made special stains to visualize the bacilli y Wayson stain characteristically shows the bipolar appearance y A colorless growth in macConkey agar with clinical history suggestive of plague should be confirmed with biochemical reactions followed by definitive identification with immunofluorescent or dialysis by a specific Y. pestis bacteriophage (CDC) y Stalactite growth in ghee broth y Serological tests showing a titer >1:16 is suggestive of plague (R/o vaccination history) y

Pathogenesis

y

y Plague is a zoonotic disease (infects rodents which are the reservoir of Y. pestis) y Transmitted to humans by rat flea (Vector) y Vector: Xenopsylla cheopis in North India and X.astia in South India y Flea gets infected when feeding on the rodents – the bacilli multiplies in the stomach and blocks the proventriculus – this is called as blocked flea. y The period between the ingestion of infected blood and blocking of proventriculus is called as Extrinsic incubation period. y When this blocked flea ferociously bites human – Y. pestis enters through the bitten wound y Organisms then multiply in the macrophages and goes for lymphatic spread and causes inflammation y It spreads systemically and causes necrosis y

y

y

y

y

y

y

y

Clinical Features

y

y

y

Treatment y 10 days course of Streptomycin is the DOC y Alternative drugs are doxycycline, levofloxacin, chloramphenicol y Plague prophylaxis → Doxycycline or cotrimaxozole y Immunoprophylaxis → killed vaccine given subcutaneously two doses at interval of 1-3 months followed by third dose around six months later y

y

y

y

y Incubation period is 2-7 days

Yersiniosis

Table 9:  Types of plague depending on clinical presentation

y It is a collective term used for zoonotic infections that are caused other than by Y. pestis y Organisms are:  Y. pseudotuberculosis  Y. enterocolitica y Yersinia pseudotuberculosis:  Not grown well in MacConkey agar  Motile at 22°C and non motile at 37°C  Causes tuberculosis like lesion in infected animals and hence the name

y

y

y

Types of plague

Features

Bubonic plague

• IP = 2–5 days • Inguinal nodes becomes enlarged (Bubo) • DIC occurs once the bacilli enters the bloodstream • Case fatality is 30–90% (Contd...) •

•

•

126

•





y







y











It can cause: Š Self-limiting gastroenteritis in young children Š Mesenteric adenitis and inflammatory terminal ileitis in older children Š Systemic disease in HLA B 27 individuals y It needs cold enrichment media for growth y Treatment: Mostly self-limited; aminoglycosides, chloramphenicol, TMP-SMX can be given 



Š

Š

Š

y

y

MULTIPLE CHOICE QUESTIONS 1. Which of the following is non – lactose fermenting bacteria: (Recent Pattern Nov 2015) a. E. coli b. Klebsiella c. Citrobacter d. Salmonella 2. E. coligives pink color with: (Recent Pattern Dec 2014) a. Chocolate agar b. L.J Medium c. MacConkey’s medium d. Saline broth 3. Which of the following is late lactose fermenter:  (Recent Pattern July 2016) a. E. coli b. Klebsiella c. Salmonella d. Shigella sonnei 4. True about Enterobacteriaceae family: (Recent Pattern Dec 2015) a. Oxidase positive b. Catalase negative c. Reduces nitrates to nitrites d. Glucose non fermenters 5. Enterobacteriaceae is classified based on: (Recent Pattern July 2015) a. Mannitol fermentation b. Catalase and oxidase reaction c. Oxygen requirement d. Lactose fermentation 6. Watery Diarrhea in children is caused by: (Recent Pattern Dec 2012) a. EHEC b. EPEC c. EIEC d. EAEC 7. Enterohemorrhagic, enterotoxic and enteroinvasive are types of: (AIIMS Nov 2014) a. E. coli b. Klebsiella c. Shigella d. Streptococcus pneumoniae 8. Regarding ETEC true is: (AIIMS Nov 2010) a. Invades submucosa b. Most common in children of developing countries c. Fomite borne and person to person d. Not a common cause of traveler’s diarrhea 9. Sereny test is positive in: (Recent Pattern July 2016) a. Enteroinvasive E. coli (EIEC) b. Enteropathogenic E. coli (EPEC) c. Enterotoxigenic E. coli (ETEC) d. Enteroaggregative E. coli (EAEC) 







10. E. coli causing hemolytic uremic syndrome: (Recent Pattern Dec 2015) a. Enteropathogenic b. Enterotoxigenic c. Enteroinvasive d. Enterohemorrhagic 11. Most common strain of E. coli giving rise to traveler’s diarrhea is: (Recent Pattern Dec 2013) a. Enteroinvasive E. coli (EIEC) b. Enteropathogenic E. coli (EPEC) c. Enterotoxigenic E. coli (ETEC) d. Enteroaggregative E. coli (EAEC) 12. In E. coli true is: (Recent Pattern Dec 2012) a. ETEC is invasive b. EPEC acts via cAMP c. Pili present in uropathogenic type d. ETEC causes HUC 13. Most important serotype of E. coli causing Hemolytic Uremic Syndrome: (Recent Pattern July 2016) a. O157: H7 of EHEC b. O107: H7 of EIEC c. O157: H7 of ETEC d. O109: H7 of EAEC 14. Culture media used for diagnosis of EHEC O157:H7 is: (Recent Pattern Dec 2015) a. O2 culture b. Sorbitol MacConkey media c. XLD agar d. Deoxycholate media 15. MC cause of diarrhea in children of developing countries is: (Recent Pattern Dec 2012) a. EHEC b. ETEC c. EAEC d. EIEC 16. Toxin acting on cGMP- (Recent Pattern Dec 2013) a. Heat stable E. coli toxin b. Heat labile E. coli toxin c. Cholera toxin d. Shiga toxin 17. Most common cause of UTI in young female is: (Recent Pattern August 2013) a. Staph. saprophyticus b. E. coli c. Klebsiella d. Proteus

Chapter 13     Enterobacteriaceae

y Yersinia enterocolitica:  Motile at 22°C and non motile at 37°C  Nonlactose fermenting colonies  It has more than 70 serotypes  Usually isolated from domestic animals; humans get infected by contamination in food or drinks  Person to person transmission does not occur







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Unit 2     Bacteriology

18. Most common organism implicated in the etiology of urinary tract infection in the community is: (Recent Pattern Dec 2013) a. E. coli b. Proteus c. Pseudomonas d. Streptococci 19. Phenylalanine deaminase test is positive in: (Recent Pattern Dec 2015) a. Salmonella b. Proteus c. Vibrio cholerae d. Helicobacter 20. Proteus antigen cross reacts with: (Recent Pattern August 2013) a. Klebsiella b. Rickettsiae c. Chlamydiae d. E. coli 21. Proteus isolated from a patient of UTI will show which biochemical reaction: (Recent Pattern Dec 2016) a. Phenyl-pyruvic acid reaction b. Bile esculin reaction c. Colchicine sensitivity d. Bacitracin sensitivity 22. Frisch bacillus affects most commonly: (Recent Pattern Dec 2015) a. Mouth b. Nose c. Eye d. Ear 23. ‘Hebra’ nose is caused by: (Recent Pattern Dec 2016) a. Frisch bacillus b. Staph aureus c. Pseudomonas d. C. diphtheria 24. Friedlander’s bacillus is: (Recent Pattern Dec 2013) a. E. coli b. Pseudomonas aeruginosa c. Klebsiella pneumoniae d. Vibrio parahemolyticus 25. Most virulent variety of Shigellosis is caused by: (Recent Pattern Dec 2014) a. S. dysenteriae b. S. sonnei c. S. flexneri d. S. boydii 26. Which of the following toxins acts by inhibition of protein synthesis: (Recent Pattern Dec 2016) a. Cholera toxin b. Shiga toxin c. Pertussis toxin d. LT of enterotoxigenic E. coli 27. True about Shiga toxin: (Recent Pattern July 2016) a. Produced by Shigella sonnei b. Chromosomal encoded c. Acts by stimulating adenylyl cyclase d. An endotoxin 28. True about Shiga toxin: (Recent Pattern Nov 2015) a. An endotoxin b. Inhibit protein synthesis c. Activate adenylyl cyclase d. Increase cGMP 29. Selective medium for Shigella: (Recent Pattern Dec 2015) a. Chocolate agar b. BYCE agar c. Hektoen agar d. EMJH medium 











128

30. Shigellosis is best diagnosed by: (Recent Pattern Dec 2013) a. Stool examination b. Stool culture c. Sigmoidoscopy d. Enzyme 31. Culture medium for transport of stools in suspected case of Shigellosis: (Recent Pattern July 2016) a. Deoxycholate medium b. Blood agar c. Nutrient broth d. Buffered glycerol saline 32. Salmonella and Shigella can be differentiated from other Enterobacteriaceae member by isolation on:  (Recent Pattern Dec 2016) a. MacConkey agar b. Mannitol salt agar c. BYCE medium d. XLD agar 33. The following are gas producing Salmonella except:  (Recent Pattern Nov 2014) a. S. typhi b. S. enteritidis c. S. cholera d. S. typhimurium 34. Pea-soup stool is characteristically seen in: (Recent Pattern Dec 2014) a. Cholera b. Typhoid c. Botulism d. Polio 35. Diagnosis of typhoid in the first week is by: (Recent Pattern Dec 2012) a. Widal test b. Stool culture c. Urine culture d. Blood culture 36. Clinical significance of Vi antigen of S.typhi is: (Recent Pattern Dec 2016) a. Helps in diagnosis b. Highly immunogenic c. Most important antigen of Widal test d. Antibody against Vi antigen is used for diagnosis of carrier 37. True about Widal test: (Recent Pattern Dec 2016) a. Anti-O antibody persists longer b. O antigen of S.paratyphi is used c. H-antigen is most immunogenic d. Felix tube is used for O agglutination 38. Most immunogenic in typhoid: (Recent Pattern Dec 2013) a. O antigen b. H antigen c. Vi antigen d. Somatic antigen 39. Absence of Vi antigen in a typhoid patient has: (Recent Pattern Dec 2016) a. Good prognosis b. Bad prognosis c. No indication with prognosis d. Widal negative 40. Not true about Vi polysaccharide vaccine of typhoid: (Recent Pattern Dec 2016) a. Single dose is given b. Revaccination at 3 years c. Given at birth d. Given subcutaneously 















51. A farmer presents to the emergency department with painful inguinal lymphadenopathy and history of fever and flu like symptoms. Clinical examination reveals an ulcer in the leg. Which of the following strains should be used to detect suspected bipolar stained organisms:  (AIIMS Nov 2012, (Recent Pattern 2011) a. Albert’s stain b. Wayson’s stain c. Ziehl Neelson stain d. Mc Fayden’s stain 52. Which is not true about yersiniosis:  (Recent Pattern Dec 2012) a. Zoonosis b. Caused by Y. pestis c. By Y. enterocolitica d. By Y. pseudotuberculosis 53. The drug of choice for chemoprophylaxis in contacts of a patient of pneumonic plague is:  (Recent Pattern Dec 2016) a. Penicillin b. Rifampicin c. Erythromycin d. Tetracycline 54. Izumi fever is caused by:  (Recent Pattern Dec 2016) a. Pseudomonas auerginosa b. Burkholderia mallei c. Yersinia pseudotuberculosis d. Pasteurella multocida 55. Appendicitis like syndrome is caused by all except: (Recent Pattern Nov 2014) a. Yersinia enterocolitica b. Yersinia pseudotuberculosis c. Pasteurella septica d. Yersinia pestis 

Chapter 13     Enterobacteriaceae

41. Temporary carrier of typhoid is infective for  (Recent Pattern Nov 2014) a. 1 year 42. Vi antigen is found in:  (PGI Nov 2016) a. Salmonella paratyphi A b. Salmonella paratyphi C c. Salmonella dublin d. Klebsiella pneumoniae e. Citrobacter fregalis 43. All are true about non typhoid salmonella except:  (AIIMS May 2011) a. Poultry is source b. Can cause infective disease in neonates c. Blood culture is more sensitive than stool culture in gasteroenteritis in adults d. Resistance to fluoroquinolone has emerged 44. Salmonella other than S. Typhi and S. Paratyphi can cause: a. Typhoid fever b. Enteric fever c. Gastroenteritis d. All of the above 45. DT 104 strain is belongs to which of the following bacteria:  (PGI Nov 2012) a. Salmonella gallinarum b. Salmonella typhi c. Salmonella enteritidis d. Salmonella paratyphi A e. Salmonella typhimurium





Yersinia

46. Causative agent of plague:  (Recent Pattern Dec 2013) a. Yersinia pestis b. Yersinia enterocolitica c. Yersinia pseudotuberculosis d. Pasteurella septica 47. Stalactite growth in ghee broth is due to:  (Recent Pattern Nov 2014) a. H. influenza b. C. diphtheria c. Y. pestis d. T. pallidium 48. Pneumonic plague is caused by:  (Recent Pattern Dec 2016) a. Bite of infected flea b. Direct contact with infected tissue c. Ingestion of contaminated food d. Droplet infection 49. Reservoir of plague is:  (Recent Pattern Dec 2015) a. Domestic rat b. Wild rat c. Rat flea d. Man 50. False statement about plague is:  (Recent Pattern Dec 2013) a. It is a gram–negative coccobacillus responding to streptomycin b. Bubonic plague is the most common form c. Pneumonic plague develops most rapidly and is most frequently fatal d. The bubo of plague is characterised by intense cellulitis

56. What type of E. coli is shown in the following image? (AIIMS May 2018) 









a. ETEC b. EIEC c. EPEC d. EAEC 57. Proteus mirabilis is intrinsically resistant to:  (PGI Nov 2018) a. Tetracycline b. Colistin c. Nitrofurantoin d. Ceftriaxone e. Cotrimoxazole 

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Unit 2     Bacteriology

ANSWERS AND EXPLANATIONS 1. Ans.  (d) Salmonella Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 279 • All the options provided belong to the group Enterobacteriaceae which are classified on the basis of lactose fermentation. • The lactose fermenters are : ƒ E. coli ƒ Citrobacter ƒ Klebsiella ƒ Enterobacter • The lactose non-fermenters are: ƒ Salmonella ƒ Shigella ƒ Proteus •

•

ƒ

ƒ

ƒ

ƒ

•

• 5 types of diarrheogenic E. coli are ƒ EPEC – causes diarrhea in infants and children ƒ ETEC – MCC of travellers diarrhea ƒ EIEC – causes illness similar to shigellosis ƒ EHEC – causes hemolytic uremic syndrome (HUS) ƒ EAEC – causes persistent diarrhea. •

ƒ

ƒ

ƒ

ƒ

ƒ

7. Ans.  (a) E. coli Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 285 • Already explained in Q.6 •

8. Ans.  (c) Fomite borne and person to person

ƒ

ƒ

ƒ

2. Ans.  (c) MacConkey’s medium Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page • MacConkeys medium is a differential media to differentiate between lactose fermenters and nonfermenters. E. coli being a lactose fermenter produces pink colored colonies in this media. •

3. Ans.  (d) Shigella sonnei

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 285 • Enterotoxigenic E. coli (ETEC) affects all age groups and is endemic in developing countries. It is the MCC of Travelers diarrhea. It is spread by contaminated water or food. The pathogenesis is through production of toxin and not by invasion. •

9. Ans.  (a) Enteroinvasive E. coli (EIEC) Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 286 • Enteroinvasive E. coli is diagnosed by Sereny test which involves inoculating suspension of bacteria into guinea pigs leading to mucopurulent conjunctivitis. •

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 279 • Though shigella are lactose non-fermenters, S. sonnei is an exception as it is a late lactose fermenter. •

4. Ans.  (c) Reduces nitrates to nitrites Ref: Jawetz book on medical microbiology, 26th edition pg 229 Members of the family Enterobacteriaceae have the following characteristics: • Gram-negative rods • Grow on peptone or meat extract media without addition of sodium chloride or other supplements • Grow well on MacConkey agar • Are facultative anaerobes • Ferment glucose often with gas production • Catalase positive and oxidase negative • Reduces nitrate to nitrite. •

•

•

10. Ans.  (d) Enterohemorrhagic Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 285 • Already explained in Q.6 •

11. Ans.  (c) Entero-toxigenic E. coli (ETEC) Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 285 • Already explained in Q.6 and 8 •

12. Ans.  (c) Pili present in uropathogenic type

•

•

•

•

5. Ans.  (d) Lactose fermentation Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 279 • The classification is already explained in Q.1 •

Ref: Jawetz book on medical microbiology 26th edition pg 233 • Option A: ETEC is not invasive and acts through its toxin • Option B: EPEC acts by disruption of brush border of intestinal mucosa • Option C: Uropathogenic E. coli elaborate a specific type of pilus which helps in adhesion, colonization and subsequent infections. • Option D: HUS is produced by EHEC. •

•

•

•

6. Ans.  (b) EPEC

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Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 285

13. Ans.  (a) O157: H7 of EHEC Ref: Jawetz book on medical microbiology 26th edition pg 234

•

20. Ans.  (b) Rickettsiae Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 289

14. Ans.  (b) Sorbitol MacConkey media

• There is sharing of alkali stable carbohydrate antigen of Rickettsia and non-motile strains of Proteus mirabilis (OX-K) and Proteus vulgaris (OX-2, OX-19) which forms the basis of the Weil-Felix reaction (a heterophile agglutination reaction).

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 286

•

• O157:H7 serotype of EHEC does not ferment sorbitol and thus sorbitol MacConkey agar is used for its diagnosis. •

15. Ans.  (b) ETEC

21. Ans.  (a) Phenyl- pyruvic acid reaction

Ref. Jawetz Book on Medical Microbiology 26 edition pg 234 th

• Though EPEC is most commonly associated with diarrhea in infants and children, since it is not provided in the option we need to go for option B as ETEC is mentioned as a very important cause of diarrhea in children in developing countries.

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 289

•

16. Ans.  (a) Heat stable E. coli toxin Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 282

Heat stable E. coli toxin

Heat labile E. coli toxin cholera toxin

Verocytotoxin or shiga like toxin

Plasmid mediated

Plasmid mediated – E. coli Chromosome medicated – Cholera

Phage coded

Acts through cAMP

Inhibits ribosome and decreases protein synthesis

Acts through cGMP

Test

Organism

Optochin sensitivity Quellung reaction

Strep. pneumonia

CAMP reaction

Strep. agalactiae

Nagler reaction

Clostridium perfringens

McFadyen’s reaction

B. anthracis

Kanagawa phenomenon

Vibrio parahemolyticus

Bile esculin test

Enterococci

Phenyl pyruvic acid reaction

Proteus

Bacitracin sensitivity

Strep.pyogenes

Neil-mooser reaction

Rickettsia typhi

Chapter 13     Enterobacteriaceae

• HUS is caused by enterohemorrhagic E. coli (EHEC) most commonly the O157:H7 serotype.

22. Ans.  (b) Nose

17. Ans.  (b) E. coli Ref: Ref. Jawetz Book on Medical Microbiology 26th edition pg 233 • E. coli is the most common cause of complicated and uncomplicated UTI and accounts for 90% of first UTIs in young women. •

18. Ans.  (a) E. coli Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 283 • Already explained in Q.17

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 287 • Frisch bacillus is the other name of Klebsiella rhinoscleromatis the causative agent of rhinoscleroma which is a granulomatous condition affecting most commonly the nose. •

23. Ans.  (a) Frisch bacillus Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 287 • The term Hebra nose is related to the appearance of the nose in nodular stage of rhinoscleroma. Tapir nose is the term during the cicatrizing stage. •

24. Ans.  (c) Klebsiella pneumoniae Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 287

•

19. Ans.  (b) Proteus Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 289 • Proteus differs from others Enterobacteriaceae by presence of enzyme phenylalanine deaminase which converts phenylalanine to phenyl pyruvic acid. This is called as phenylalanine deaminase test. •

Bacteria

Other name

Corynebacterium diphtheria

Kleb loefller’s bacillus

Corynebacterium pseudotuberculosis

Preisz-Nocard bacillus

Haemophilus aegyptius

Koch weeks bacillus

Haemophilus influenzae

Pfeiffer’s bacillus Contd...

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Unit 2     Bacteriology

Bacteria

Other name

Klebsiella pneumoniae

Friedlander’s bacillus

Klebsiella ozanae

Abel’s bacillus

Mycobacterium tuberculosis

Koch’s bacillus

Mycobacterium paratuberculosis

Johne’s bacillus

Mycoplasma

Eaton agent

Pseudomonas pseudomallei

Whitmore’s bacillus

Mycobacterium intracellulare

Battey’s bacillus

Klebsiella rhinoscleromatis

Frisch bacillus

25. Ans.  (a) S. dysenteriae Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 292 • The most virulent variety of shigellosis is caused by Sh. dysenteriae type 1, while the mildest form is caused by Sh.sonnei. •

26. Ans.  (b) Shiga toxin Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 293 Toxins that inhibit protein synthesis • Sh. dysenteriae type 1 • Diphtheria • Pseudomonas • Verotoxin = shiga like toxin of E. coli •

•

The best way to diagnose shigella is by culture of mucus flakes of stool onto selective media. 31. Ans.  (d) Buffered glycerol saline Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 294 The transport media used for shigella is Sach’s buffered glycerol saline. 32. Ans.  (a) MacConkey agar Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 294 Salmonella and Shigella are non-fermenters of lactose and thus by culturing on MacConkey agar can be differentiated from other Enterobacteriaceae. 33. Ans.  (a) S. typhi Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 297 Salmonella ferment glucose with production of acid and gas with the exception of S.typhi which is anaerogenic. 34. Ans.  (b) Typhoid Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 301 The term pea-soup stools is used in typhoidal diarrhea whereas the term rice-water stools refers to cholera stools.

•

•

27. Ans.  (b) Chromosomal encoded Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 292 • Shiga toxin is an exotoxin which is chromosomally encoded and produced by Sh. dysenteriae type 1 and acts by inhibiting protein synthesis. •

28. Ans.  (b) Inhibit protein synthesis Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 292 • Already explained in Q.27 •

29. Ans.  (c) Hektoen agar

35. Ans.  (d) Blood culture Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 303 The mnemonic BASU helps remember the order of positivity of various investigations in typhoid • B – blood culture -1st week • A – antibodies (Widal test) – 2nd week • S – Stool culture – 3rd week • U – urine culture – 4th week. •

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•

•

36. Ans.  (d) Antibody against Vi antigen is used for diagnosis of carrier Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 299 • Vi polysaccharide antigen is used for the identification of carriers of salmonella. •

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 291 The selective media for salmonella and shigella are: • Xylose-lysine decarboxylase (XLD) • Deoxycholate citrate agar (DCA) • Hektoen enteric agar • Samonella-shigella agar •

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•

•

30. Ans.  (b) Stool culture

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Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 294

37. Ans.  (c) H-antigen is most immunogenic Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 299 • • • •

•

•

•

•

H (flagellar antigen) – strongest antigen Vi (capsular antigen) – weaker than H antigen O (somatic antigen) – weakest antigen 2 types of tubes are used in Widal reaction – a narrow tube with conical bottom (Dreyer’s agglutination tube) for H agglutination with the formation of cotton wooly

38. Ans.  (b) H antigen Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 299 • Already explained in Q.37 •

39. Ans.  (a) Good prognosis

46. Ans.  (a) Yersinia pesits Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 325 • Yersinia pestis – causative agent of plague. • Yersinia pseudotuberculosis – a primary pathogen of rodents. • Yersinia enterocolitica – causes enteric and systemic disease in animals and humans. • Pasteurella spp. – hemorrhagic septicemia in animals and occasionally local or systemic infections in human. •

•

•

•

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 299 • Vi antigen is associated with virulence of the organism and thus its absence is associated with good prognosis.

47. Ans.  (c) Y . pestis

•

40. Ans.  (c) Given at birth

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 326

Chapter 13     Enterobacteriaceae

clumps and a round bottomed tube (Felix tube) for O agglutination with the formation of disc-like pattern.

• When Yersinia pestis is grown in ghee broth (flask of broth with oil or ghee floated on top), a characteristic growth which hangs down from the surface into the broth, resembling stalactite occur. •

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 306 • Vi polysaccharide vaccine is an injectable vaccine given as a single dose. Being a polysaccharide vaccine it is not given to infants and young children 10% Causes otitis, eye and wound infections DOC: Tetracycline

High Yield Recap of all Toxins Enterotoxins

Cytotoxins

Neurotoxins

Cholera toxin

Shigella dysenteriae type 1

Staph aureus

Vibrio parahaemolyticus

Enterohemorrhagic E. coli

Bacillus cereus

E. coli  LT and ST of ETEC, EAEC and EHEC

Clostridium difficile (Toxin B)

Cl. botulinum toxin

• • • •

•

•

•

•

Clostridium difficile (Toxin A) Aeromonas Rota virus (NSP4) Campylobacter jejuni

Toxins

Mechanism of action

Diphtheria toxin (Corynebacterium diphtheriae)

• ADP-ribosyl transferase. • Inactivation of ribosomal elongation factor eEf2 resulting from ADP-ribosylation during protein synthesis • Leads to cell death. •

•

•

Cholera toxin (Vibrio cholerae)

• ADP-ribosyl transferase. • ADP-ribosylation of regulatory protein G5 of adenylate cyclase, resulting in permanent activation of this enzyme and increased levels of cAMP • Increased secretion of electrolytes. •

•

•

Tetanus toxin (Clostridium tetani)

• Metalloprotease. Proteolytic cleavage of protein in components from the neuro exocytosis apparatus in the synapses of the anterior horn that normally transmit inhibiting impulses to the motor nerve terminal.

Membrane toxins Alpha toxin (Clostridium perfringens)

• Phospholipase.

Listeriolysin (Listeria monocytogenes)

• Pore formation in membranes.

Superantigen toxins Toxic shock syndrome toxin-1 (TSST-1) (Staphylococcus aureus)

• Stimulation of secretion of cytokines in T cells and macrophages.

•

•

•

•

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Unit 2     Bacteriology

MULTIPLE CHOICE QUESTIONS 1. Cholera is caused by: (PGI Nov 2014) a. Vibrio cholerae – 01 b. Vibrio cholerae – 0139 c. Vibrio parahaemolyticus d. NAG vibrio 2. True about El Tor vibrio:  (Recent Pattern Dec 2016) a. More SAR b. VP reaction (+) ve c. Low carrier rate d. More severe 3. The characteristic features of El Tor Cholera are all except: (Recent Pattern Nov 2014) a. More of subclinical cases b. Mortality is less c. Secondary attack rate is high in family d. El Tor vibrio is harder and able to survive longer 4. Not true about El Tor vibrio 01:  (All India 2010) a. Animals are the only reservoir b. Epidemiologically indistinguishable from V cholera 0139 c. Humans acts as a vehicle for spread d. The efficacy of vaccine against El Tor vibrio is great 5. Transport medium for cholera: (Recent Pattern Dec 2013) a. LJ medium b. Cary-Blair medium c. MYPA medium d. Stuart’s medium 6. Optimal percentage of NaCl for V cholerae:  (Recent Pattern Dec 2015) a. 1% b. 2% c. 3% d. 4% 7. Which organism grows in alkaline pH?  (Recent Pattern Dec 2014) a. Vibrio b. Klebsiella c. Pseudomonas d. E. coli 8. Cholera toxin is due to:  (Recent Pattern Dec 2012) a. Chromosome b. Plasmid c. Phage d. Transposons 9. Which of the following stimulate adenylate cyclase with G – protein coupled action:  (Recent Pattern Dec 2013) a. Shiga toxin b. Cholera toxin c. Diphtheria toxin d. Pseudomonas toxin 10. Cholera toxin effects are mediated by stimulation of which of the following second messengers:  (Recent Pattern 2012) a. cAMP b. cGMP c. Ca ++ – calmodulin d. IP3 / DAG 11. In the small intestine, cholera toxin acts by:  (Recent Pattern Nov 2014) a. ADP ribosylation of the G regulatory protein b. Inhibition of adenyl cyclase c. Activation of GTPase d. Active absorption of NaCl 12. Vibrio cholerae acts by disrupting which of the following structures:  (AIIMS May 2015) a. Hemidesmosome b. Gap junctions c. Zona occludens d. Zona adherens 









138

13. Cholera toxin binds to which receptors in the intestine: (Recent Pattern July 2016) a. Sphingosine through A subunit b. Sphingosine through B subunit c. GMI gangliosides through A subunit d. GMI gangliosides through B subunit 14. Phage encoded exotoxin of vibrio cholerae resembles which toxin of E. coli:  (Recent Pattern Nov 2015) a. Heat labile toxin b. Heat stable toxin c. Shiga like toxin d. Verocytotoxin 15. True about cholera:  (Recent Pattern Dec 2012) a. Gram-negative rod b. Associated with fever c. Causes painful watery diarrhea d. It is an achlorhydria which renders and individual susceptible to disease 16. The endotoxin of the following Gram-negative bacteria does not play any part in the pathogenesis of the natural disease: (AIIMS Nov 2012, Recent Pattern 2012) a. E. coli b. Klebsiella c. Vibrio cholerae d. Pseudomonas 17. Which of the following about cholera is true: a. Invasive  (Recent Pattern Dec 2014) b. Endotoxin is released c. Recent infections in India are of classical type d. Vibriocidal antibody titre measures prevalence 18. All of the following statements about El Tor vibrios are true, except:  (All India 2010) a. Humans are the only reservoir b. Can survive in ice cold water for 2 – 4 weeks c. Killed by boiling for 30 seconds d. Enterotoxin can have direct effects on other tissues besides intestinal Epithelial cells 19. Invasive infection is caused by all except:  (Recent Pattern Dec 2012) a. V. cholerae b. Neisseria c. Streptococci d. H. influenzae 20. “Darting motility” is shown by:  (Recent Pattern Dec 2013) a. Proteus b. Vibrio c. Serratia d. E. coli 21. Halophilic vibrio which causes wound infection at sea coast is: (Recent Pattern July 2016) a. Vibrio vulnificus b. Vibrio parahaemolyticus c. Vibrio mimicus d. Vibrio cholerae 22. Which of the following Vibrios is most commonly associated with ear infections:  (Recent Pattern 2012) a. V. alginolyticus b. V. parahaemolyticus c. V. vulnifcus d. V. fluvialis 23. True about vibrio vulnificus:  (Recent Pattern Dec 2015) a. Causes diarrhea commonly b. Halophilic c. Drug of choice is penicillin d. Produces Shiga toxin 







26. Which of the following halophilic vibrio: (PGI May 2018) a. V. alginolyticus b. V. parahaemolyticus c. V. cholerae d. V. damsela e. V. vulnificus

27. At a birthday party, oysters, hamburger and potato salad were served. After 24 hours, 50 people had diarrhea. Which is the most likely organism causing this type of clinical presentation?  (AIIMS Nov 2018) a. Salmonella enteritidis b. Vibrio parahemolyticus c. Yersinia enterocolitica d. Staphylococcus aureus 28. 28. A 23-year-old man presented with an erythematous edematous patch with centrally grouped vesicles on the left ankle. The skin lesion occurred following a coral injury while he was skin-scuba diving at an island 2 days prior to his presentation. Which is the causative organism?  (JIPMER Nov 2018) a. Streptococcus pyogenes b. Vibrio vulnificus c. Alkaligenes faecalis d. Pseudomonas aeruginosa

Chapter 14     Vibrio

24. Kanagawa phenomenon is seen in: a. Pseudomonas aeruginosa  (Recent Pattern Dec 2015) b. Vibrio parahemolyticus c. Shigella sonnei d. Proteus mirabilis 25. True about Vibrio parahemolyticus:  (Recent Pattern Dec 2015) a. Polar flagella b. Nonhalophilic vibrios c. Non-capsulated d. Requires NaCl

ANSWERS AND EXPLANATIONS 1. Ans. (a) Vibrio cholerae – 01; (b) Vibrio cholerae – 0139; (d) NAG vibrio Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 312 • Gardner and Venkatraman gave a serological classification in which, cholera and biochemically similar vibrios possessing a common flagellar (H) antigen were classified as Group A and rest as Group B vibrios. This Group A has 139 serogroups, which have been classified on the basis of ‘O’antigen. • Earlier, only serogroup O-1 was isolated from epidemic cholera and was called pathogenic or agglutinable vibrios. But later in 1992, serogroup O-139 (non-O-1 vibrios) earlier called as non-pathogenic/NAG/nonagglutinable vibrios was identified as the causative agent in epidemic. •

•

2. Ans.  (b) VP reaction (+) ve Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 312 (Table 32.4), Page 314 • In case of El Tor vibrio, the severity of illness is much less,with a large proportion of mild and asymptomatic infections, mortality is low and the carrier rate is high. • The El Tor vibrio is much harder than the classical vibrios, capable of surviving in the environment for much longer. •

•

3. Ans.  (c) Secondary attack rate is high in family Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 314 Ref: Already explained in Q.2 4. Ans.  (a) Animals are the only reservoir Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 313 • Natural infection with cholera occurs only in humans. There are no animal reservoir. •

5. Ans.  (b) Cary Blair medium Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 310 • Holding or transport media used for Cholera vibrios are Venkatraman-Ramakrishnan medium, Cary-Blair medium and Autoclaved sea water. •

6. Ans.  (a) 1% Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 310 • NaCl (0.5-1%) is required for optimal growth of V. cholerae, though high concentrations (6% and above) are inhibitory. •

7. Ans.  (a) Vibrio Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 310 • Growth of V. cholerae is better in an alkaline medium, the range of pH being 6.4 - 9.6 (optimum 8.2). •

8. Ans.  (c) Phage Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 314 • Vibrios multiplying on the intestinal epithelium produce an enterotoxin called Cholera toxin, the production of which is determined by a filamentous phage integrated with the bacterial chromosome. •

9. Ans.  (b) Cholera toxin Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 314 • Cholera toxin molecule of approx. 84000 MW consists of 1 A and 5 B subunits. The B (binding) units attach to GM1 ganglioside receptors on the surface of jejuna epithelial cells. A(active) fragments into A1 and A2. A2 binds A1 to B. The A1 fragment causes prolonged activation of cellular •

139

Unit 2     Bacteriology

adenylate cyclase and accumulation of cAMP, leading to outpouring of large quantities of water and electrolytes into the intestinal lumen. The toxin also inhibits intestinal absorption of sodium and chloride. • Shiga toxin acts by inhibition of protein synthesis. • Diphtheria toxin inhibits polypeptide chain elongation in the presence of nicotinamide adenine dinucleotide by inactivating the elongation factor EF-2. • Pseudomonas produces Exotoxin A, the mechanism of which is similar to diphtheria toxin. •

•

•

10. Ans.  (a) cAMP Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 314 • Already explained in Q.10 •

11. Ans.  (a) ADP ribosylation of the G regulatory protein Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 314 • Already explained in Q.10 •

12. Ans.  (c) Zona occludens

16. Ans.  (c) Vibrio cholerae Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 314 • Cholera vibrios also possess lipopolysaccharide O antigen (LPS, endotoxin), which apparently plays no role in pathogenesis but is responsible for immunity induced by killed vaccines. •

17. Ans.  (d) Vibriocidal antibody titre measures prevalence Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 313 • In the small intestine, vibrios cross the protective layer of mucus and reach the epithelial cells, to which, they adhere and colonize with the help of special fimbria called Toxin co-regulated pilus (TCP). They remain attached to the epithelium but do not damage or invade the cells. The changes induced are biochemical rather than histological. • Cholera vibrios possess lipopolysaccharide endotoxin in their cell membrane. It is not released by the organism. • Recent infections in India are of serovar O-139 type. • Serological testing is not used for diagnosis in cholera cases rather they are helpful in assessing the prevalence. The tests available are agglutination using live or killed vibrio suspensions, indirect hemagglutination, vibriocidal test and antitoxin assay. •

•

•

•

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 314 • Vibrio cholera acts by disrupting the zona occludens. •

13. Ans.  (d) GMI gangliosides through B subunit Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 314 • Already explained in Q.10 •

18. Ans.  (c) Killed by boiling for 30 seconds Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 314 • Cholera toxin (Enterotoxin) exhibits other biological effects besides effect on intestinal epithelial cells like activation of lipolysis in rat testicular tissue, elongation of Chinese hamster ovary cells in culture, histological changes in adrenal tumor cell culture and vero cells. It is called permeability factor because it increases skin capillary permeability demonstrated by skin blueing test. • Humans are the only reservoir. • Samples stored in cold may harbour vibrios for more than 2 weeks. •

14. Ans.  (a) Heat labile toxin Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 313 • Cholera toxin (CT) is very similar to heat labile toxin (LT) of E. coli in structural, chemical, biological and antigenic properties, though CT is far more potent than LT in biological activity. •

•

•

15. Ans.  (d) It is an achlorhydria which renders and individual susceptible to disease Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 313 • Vibrio cholera is a gram-negative curved rod. • Cholera is an acute diarrheal disease presenting with profuse, painless, watery diarrhea and copious effortless vomiting, which may lead to hypovolemic shock and death in less than 24hrs. Its not associated with fever. The stool has characteristic inoffensive sweetish odour, termed as rice water stools. • Vibrios are highly susceptible to acids and gastric acidity provides an effective barrier against small doses of cholera vibrios. Hence, achlorhydria predisposes to cholera.

19. Ans.  (a) V. cholerae Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 313

•

•

•

140

• Already explained in Q.18 •

20. Ans.  (b) Vibrio Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 309 • Vibrio cholera are actively motile (swarm of gnats) with a single sheathed polar flagellum. Motility is of darting type and can be seen when an acute cholera stool or a young culture is examined under microscope. •

25. Ans.  (d) Requires NaCl

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 318

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 317

• Vibrio vulnificus - halophilic, VP negative, ferments lactose but not sucrose, salt tolerance of 10 weeks 16. Tuberculin test is positive if induration is: (Recent Pattern Dec 2013) a. > 2mm b. > 5mm c. > 7mm d. > 10mm 17. Tuberculin test is: (Recent Pattern Nov 2015) a. Intramuscular b. Intradermal c. Subcutaneous d. None 18. Negative Mantoux test is when induration is: (Recent PatternJuly 2016) a. 6 months y Rate of carriers are after an infection:  5-10% of adults  30% of children  90% of neonates y

–

Immunity following HBV vaccine

y



264





Prophylaxis of HBV y Currently used vaccine is prepared by cloning the S gene in bakers yeast y

y

y

y

y

Treatment y IFN-alpha2b is given subcutaneously at a dose of 5 million units daily or 10 million units thrice weekly for 16 to 24 weeks. y Oral lamivudine is given as 100 mg/day for a period of 12 to 18 months and it is effective in reducing the HBV DNA levels.

y Complications:  Glomerulonephritis  Vasculitis  Type 2 cryoglobulinemia y Hepatitis C becomes chronic in about 80 per cent of those infected y After many years, cirrhosis of the liver occurs in 10–20 per cent of patients y HCC, which develops in 1–5 per cent of those with chronic infection has a very poor prognosis. y







y

y

y

y

y

HEPATITIS C VIRUS y HCV is a member of family Flaviviridae, which has a positive sense ssRNA. y WHO estimates that 3% of the world’s population is infected with HCV. y Occult HBV infections (33%) are seen in patients with chronic HCV liver disease, where HBsAg cannot be detectable in serum because of its lower levels. y The co-infections of Hepatitis infections with HIV are more common. y

y

y

Laboratory Diagnosis

Chapter 34     Hepatitis Virus

y Given IM – deltoid or anterolateral aspect of thigh in children – 0, 1, 6 months y It should not be given in gluteal as absorption in muscle is low leading to poor immune response y Seroconversion rate is around 90% y Immunoglobulin given for babies born to carrier mothers and for those who had exposure with known postive HBV persons

y ELISA and immunoblot tests are available for detecting antibodies to core, envelope and NS3 and NS4 non-structural proteins. y ELISA has higher sensitivity when compared to the immunoblot rapid card tests. y The limitations of immunoassays are inability to distinguish among acute, chronic or resolved infections. y Nucleic acid based assays are helpful in detecting the presence of circulating HCV RNA and for genotyping. y The viral load assays are helpful in the monitoring of antiviral therapy. y

y

y

y

y

y

Properties of HCV y Most common cause of post-transfusion hepatitis in developed countries y

Treatment y Drugs used are: Interferon alpha, Sofosbuvir, Ribavirin y Interferon-Alpha2a or 2b given at the dose of 3 million units thrice weekly for 12 to 24 months is effective. y Ribavirin can also be given orally at a concentration of 10001200 mg daily for 6 to 12 months. y

y

y

HCV – causes chronic form of hepatitis; causes hepatocellular carcinoma • Carriers – 200 million worldwide • Prevalence in India – 12.5 million cases •

•

HEPATITIS D VIRUS y Hepatitis D virus is an enveloped virus with RNA genome y Classified under Delta virus y It does not have polymerase in its virion; cannot replicate on its own; called as Defective virus (Defective RNA virus – dependent virus) y HDV can replicate only when co-infected with HBV y HDV transmitted as like HBV – blood and blood borne products, vertical transmission y It can either gets infected along with HBV (Co-infection) or after infection with HBV (Super infection) y Lab diagnosis – detection of delta antigen of IgM antibody to delta antigen in blood y Experimental model for HDV - Woodchuck y No specific therapy; no vaccination y Immunization against Hepatitis B virus will reduce the hepatitis caused by HDV y

y Six different genotypes are present y The distributions of the major genotypes are related to various risk groups, response to antiviral therapy and geographical areas of prevalence. y There is no cross-protection between the various genotypes y This genotypic variation and no cross protection makes difficulty in making vaccine development. y

y

y

y

y

y

y

y

y

Mode of Spread of HCV y Parenteral transmission y Screening of blood and blood products for HBV and HCV is important y

y

y

y

y

y

Clinical Features y The incubation period is about 8 weeks. y Only very few individuals gets symptoms that too very mild like anorexia and nausea y Jaundice is uncommon in HCV y When jaundice occurs, symptoms and biochemical changes are identical to other forms of hepatitis y Alanine aminotransferase levels begin to increase shortly before symptoms and a 10-fold elevation can be detected. y

y

HEPATITIS E VIRUS Properties

y

y

y

y HEV is a calcivirus – has a single stranded positive sense RNA y HEV – non A non B hepatitis (NANB) – enterically transmitted – causes fulminant hepatic failure in pregnant females (last trimester) y

y

265

Unit 3     Virology

y Also called as epidemic NANB; It is also a common cause of acute sporadic hepatitis in Asian countries. y Animal reservoir – Pigs y Sporadic infections are zoonotic in nature: hence it is called as Zoonotic hepatitis infections. y

y

y

Clinical Features y Infection occurs through fecal-oral route y Incubation period around 6 weeks y Infections are seen in adults than in children; person to person transmission rate is lower than HAV. y Women who are infected in the last trimester of pregnancy – develops for fulminant hepatitis leading to mortality y

y

y

y

HEPATITIS G VIRUS y Hepatitis G virus: Found in patients with acute, chronic and fulminant hepatitis, hemophiliacs, patients who had undergone multiple transfusions and hemodialysis, intravenous drug addicts and blood donors y

Miscellaneous viruses that causes hepatitis are: • Epstein Barr virus • Cytomegalovirus • Herpes simplex virus • Mumps virus • ECHO virus • Rubella • Yellow fever •

•

•

•

•

•

•

Laboratory Diagnosis y Virus like particles can be seen from feces by Immunoelectron microscope y ELISA for IgG and IgM antibodies y

y

266

1. Which hepatitis virus causes acute illness: (Recent Pattern Dec 2015) a. HBV b. HCV c. HAV d. All of the above 2. Virus causing hepatocellular carcinoma belongs to:  (Recent Pattern Dec 2015) a. Hepadnaviridae b. Enterovirus c. Calcivirus d. None 3. Which is SS RNA unenveloped virus: (Recent Pattern Dec 2012) a. HBV b. HEV c. HCV d. None 4. Hepatitis A virus is best diagnosed by: (Recent Pattern Nov 2015) a. IgM antibodies in serum b. Isolation from stool c. Culture from blood d. Isolation from bile 5. True about hepatitis A virus: (Recent Pattern Dec 2013) a. Causes carcinoma b. Helps HDV replication c. Common cause of hepatitis in children d. Causes chronic hepatitis 6. Hepatitis A is transmitted by: (Recent Pattern Dec 2014) a. Blood route b. Inhalation c. Fecal-oral route d. All 7. Which is not true about hepatitis B virus:  a. DNA virus  (Recent Pattern Dec 2013) b. Transmitted by fecal-oral route c. Can be transmitted from mother to child d. Contains reverse transcriptase 8. DNA polymerase of HBV is encoded by which of the following: (Recent Pattern Dec 2015) a. S gene b. C gene c. P gene d. X gene 9. Reverse transcriptase is a RNA dependent DNA polymerase. Which of these use it: (Recent Pattern May 2015) a. Hepatitis A virus b. Hepatitis B virus c. Hepatitis C virus d. Hepatitis D virus 10. E antigen (HBeAg) of hepatitis B virus is a product of which gene: (Recent Pattern Dec 2015) a. S b. C c. P d. X 11. Which is the longest DNA of hepatitis B virus: (AIIMS Dec 2013) a. P gene b. X gene  c. S gene d. C gene 12. HBV is associated with all of the following except: (Recent Pattern Nov 2014) a. Hepatic cancer b. Chronic hepatitis c. Hepatic adenoma d. Cirrhosis 13. Serological testing of patient shows HBsAg, IgM, HBeAg positive. The patient has: (Recent Pattern Dec 2012) a. Chronic hepatitis B with low infectivity b. Acute hepatitis B with high infectivity c. Chronic hepatitis with high infectivity d. Acute on chronic hepatitis 













14. Super carrier of HBV shows following serum markers: a. HBsAg (Recent Pattern Dec 2015) b. HbsAg + HBV DNA c. HbsAg + HBeAg + HBV DNA d. Anti-HBsAg + HBV DNA 15. First antibody to appear in hepatitis: (Recent Pattern Dec 2014) a. IgM anti - HBe b. IgG anti - HBe c. IgM anti - HBc d. IgM anti - HBs 16. The serological marker of acute Hepatitis B infection is: (Recent Pattern Dec 2013) a. HBsAg + HBeAg b. HBsAg + Core antibody c. HBsAg d. HBcAg 17. Infectivity of HBsAg is best/commonly diagnosed by: (Recent Pattern Dec 2012) a. HBeAg b. HbsAg c. HBV DNA d. Anti HBsAg 18. Active replication in Hepatitis B infection is indicated by: (Recent Pattern Dec 2013) a. HBeAg b. HBsAg c. HBcAg d. Anti - HBsAg 19. Presence of HBeAg in patients with hepatitis indicates: (Recent Pattern Dec 2014) a. Simple carriers b. Late convalescence c. High infectivity d. Carrier status 20. Which of the following does not indicate Hepatitis B replication: (Recent Pattern July 2016) a. HBsAg b. HBeAg c. HBV DNA d. Viral copies 21. Which of the following antigen is found within the nuclei of infected hepatocytes and not usually in the periphery circulation in Hepatitis B infection: (Recent Pattern Dec 2013) a. HBeAg b. HBcAg c. Anti – HBc d. HBsAg 22. Best means of giving hepatitis B vaccine is:  (Recent Pattern Nov 2014) a. Subcutaneous b. Intradermal c. Intramuscular deltoid d. Intramuscular gluteal 23. True about HCV is: (Recent Pattern July 2016) a. Most common mode of transmission is by needle puncture b. Diagnosis is established by viral isolation from blood c. Chances of perinatal transmission are directly correlated with degree of maternal viremia d. Associated with polyarteritis nodosa (PAN) 24. Most common cause of chronic hepatitis: (Recent Pattern July 2016) a. HEV b. HAV  c. HBV d. HCV 25. Hepatitis C virus is: (Recent Pattern Dec 2012) a. Togavirus b. Flavivirus c. Filovirus d. Retrovirus 26. Commonest hepatotropic virus causing increased chronic carrier state is: (Recent Pattern Dec 2013) a. HEV b. HAV c. HBV d. HCV



Chapter 34     Hepatitis Virus

MULTIPLE CHOICE QUESTIONS













267

Unit 3     Virology

27. HDV is: (Recent Pattern Dec 2014) a. SS-RNA virus b. SS-DNA virus c. DS-RNA virus d. DS-DNA virus 28. HBV and HDV false is: (Recent Pattern Dec 2012) a. Both can infect simultaneously b. HDV causes more serious infection d/t super infection c. HDV cannot infect in absence of HBV d. DNA viruses 29. A pregnant women in Bihar presents with fulminant hepatic failure. The most likely etiological agent is: a. Hep. E (AI Dec 2013) b. Hep. B c. Sepsis d. Acute fatty liver of pregnancy

30. Hepatitis E usually affects: (Recent Pattern Nov 2015) a. Children b. Adults c. Old age d. Toddlers 31. Hepatitis E clinically resembles: (Recent Pattern Nov 2014) a. Hepatitis A b. Hepatitis B c. Hepatitis C d. Hepatitis D 32. Cryoglobulinemia is associated with: (Recent Pattern 2018) a. HAV b. HBV c. HCV d. HDV





(AIIMS Nov 2017)

33. Identify the virus: 



a. HIV

b. Hepatitis

34. There are 3 to 5% healthy hepatitis B carriers in India who are asymptomatic. They have the risk of developing HCC in future because: (AIIMS Nov 2017) a. They are unable to mount inflammation against the virus. b. Virus can integrate with host DNA and form complementary DNA c. There is a risk of elevation of transaminases d. Liver parenchymal cells are in a state of high proliferation

268

c. Influenza





d. Herpes simplex

35. Regarding Hepatitis D virus (HDV), which of the following is/are correct?  (PGI pattern) a. Alpha interferon can eradicate the latent stage caused by HDV b. Immunization against Hepatitis B virus will reduce the hepatitis caused by HDV c. HDV has RNA in its genome and it has no polymerase in its virion d. Laboratory diagnosis of HDV is done by growing the cells in co-infection with HBV e. It is a non-enveloped virus 36. Which is the dangerous hepatitis virus during pregnancy?  (CET 2018) a. Hepatitis A b. Hepatitis B c. Hepatitis D d. Hepatitis E

1. Ans.  (c) HAV Ref: Ananthanarayan and Paniker’s Textbook of Microbiol­ ogy – 10th ed – Page 546; Harrison T.B of internal medicine – 18th ed – Page 2559 • Acute onset is caused by HAV and HEV • Insidious onset is by HBV, HCV, HDV •

•

2. Ans.  (a) Hepadnaviridae Ref: Ananthanarayan and Paniker’s Textbook of Microbiol­ ogy – 10th ed – page 546 • Hepatitis viruses that cause hepatocellular carcinoma are HBV and HCV • HBV – Hepadnavirus • HCV – Flavivirus

8. Ans.  (c) P gene Ref: Ananthanarayan and Paniker’s Textbook of Microbiol­ ogy – 10th ed – page 548

Gene

Coding

S gene

HBsAg

C gene – C region

HBcAg

C gene – Pre C region

HBeAg

P gene

DNA polymerase

X gene

HBxAg

Chapter 34     Hepatitis Virus

ANSWERS AND EXPLANATIONS

•

9. Ans.  (b) Hepatitis B virus

•

•

3. Ans.  (b) HEV Ref: Ananthanarayan and Paniker’s Textbook of Microbiol­ ogy – 10th ed – page 555 • Hepatitis A virus and Hepatitis E virus are the non enveloped RNA viruses

Ref: Ananthanarayan and Paniker’s Textbook of Microbiol­ ogy – 10th ed – page 546 • HBV has DNA dependent DNA polymerase and RNA dependent reverse transcriptase •

10. Ans.  (b) C

•

4. Ans.  (a) IgM antibodies in serum Ref: Ananthanarayan and Paniker’s Textbook of Microbiol­ ogy – 10th ed – page 546 • HAV – Diagnosis is by IgM anti HAV antibody which starts appearing around 2 weeks and disappears after 3-4 months •

5. Ans.  (c) Common cause of hepatitis in children

Ref: Ananthanarayan and Paniker’s Textbook of Microbiol­ ogy – 10th ed – page 549 • Already explained Q.8 •

11. Ans.  (a) P gene Ref: Ananthanarayan and Paniker’s Textbook of Microbiol­ ogy – 10th ed – page 548 • Already explained Q.8 •

12. Ans.  (c) Hepatic adenoma

Ref: Ananthanarayan and Paniker’s Textbook of Microbiol­ ogy – 10th ed – page 545

Ref: Ananthanarayan and Paniker’s Textbook of Microbiol­ ogy – 10th ed – page 547

• Type A hepatitis – infectious hepatitis • Most common cause of hepatitis in children • Children – self-limiting

• HBV infections resolves in 90% of individuals • Remaining will go for chronicity – that leads to chronic hepatitis – inflammation and fibrosis – leads to cirrhosis • Cirrhosis after years may end up in Hepatocellular carcinoma • Hepatic adenoma has not be associated with HBV

•

•

•

6. Ans.  (c) Fecal-oral route Ref: Ananthanarayan and Paniker’s Textbook of Microbiol­ ogy – 10th ed – page 545 • Transmission of HAV is only by fecal-oral route • Other hepatitis virus which gets transmitted by fecaloral route is HEV

•

•

•

•

13. Ans.  (b) Acute hepatitis B with high infectivity

•

•

7. Ans.  (b) Transmitted by feco-oral route Ref: Ananthanarayan and Paniker’s Textbook of Microbiol­ ogy – 10th ed – page 547 • Mode of transmission of HBV is through blood and blood products, needle prick, perinatal transmission and sexual route • Only HAV and HEV gets transmitted by fecal-oral route

Ref: Ananthanarayan and Paniker’s Textbook of Microbiol­ ogy – 10th ed – page 552 • IgM – denotes acute infection • HbeAg positive – denotes high infectivity – means there is active replication of virus •

•

14. Ans.  (c) HbsAg + HBeAg + HBV DNA

•

•

Ref: Ananthanarayan and Paniker’s Textbook of Microbiol­ ogy – 10th ed – page 549 • Super carriers – high titer of HBsAg along with HbeAg + HBV DNA •

269

Unit 3     Virology

15. Ans.  (c) IgM anti - HBc Ref: Ananthanarayan and Paniker’s Textbook of Microbiol­ ogy – 10th ed – page 550 • HbcAg – cannot be demonstrable in blood • The earliest antibody marker to be identified is Anti HbcAg – IgM • After six months IgG starts appearing •

•

•

23. Ans.  (c) Chances of perinatal transmission are directly correlated with degree of maternal viremia Ref: Ananthanarayan and Paniker’s Textbook of Microbiol­ ogy – 10th ed – page 553; Harrison T.B of internal medicine 19th ed – page 2009 • Transmission is based on the degree of maternal viremia – this can be detected with the help of PCR for HCV RNA • But the Most common mode of transmission is blood transfusion •

•

16. Ans.  (a) HBsAg + HBeAg Ref: Ananthanarayan and Paniker’s Textbook of Microbiol­ ogy – 10th ed – page 550 Acute hepatitis has the following serological markers: • HbsAg • IgM anti HbcAg • HbeAg •

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17. Ans.  (a) HBeAg

Ref: Ananthanarayan and Paniker’s Textbook of Microbiol­ ogy – 10th ed – page 552 • About 50-80% of the persons infected with HCV goes for chronicity •

25. Ans.  (b) Flavivirus

Ref: Ananthanarayan and Paniker’s Textbook of Microbiol­ ogy – 10th ed – page 550

Ref: Ananthanarayan and Paniker’s Textbook of Microbiol­ ogy – 10th ed – page 552

• Already explained Q.13

• HCV – belongs to hepacivirus in the family of flaviviridae

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18. Ans.  (a) HBeAg

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26. Ans.  (d) HCV

Ref: Ananthanarayan and Paniker’s Textbook of Microbiol­ ogy – 10th ed – page 550

Ref: Ananthanarayan and Paniker’s Textbook of Microbiol­ ogy – 10th ed – page 552

• Already explained Q.13

• Already explained Q.24

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19. Ans.  (c) High infectivity

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27. Ans.  (a) SS-RNA virus

Ref: Ananthanarayan and Paniker’s Textbook of Microbiol­ ogy – 10th ed – page 550

Ref: Ananthanarayan and Paniker’s Textbook of Microbiol­ ogy – 10th ed – page 553

Ref: Already explained Q.13

• HDV – ssRNA virus; Defective or dependovirus.

20. Ans.  (a) HBsAg Ref: Ananthanarayan and Paniker’s Textbook of Microbiol­ ogy – 10th ed – page 550 Markers of HBV replication are: • HbeAg • HBV DNA – viral load/viral copies •

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21. Ans.  (b) HBcAg Ref: Ananthanarayan and Paniker’s Textbook of Microbiol­ ogy – 10th ed – page 548 • HbcAg is core antigen; It is not demostrated in the circulation because it is enclosed within the HbsAg envelope •

22. Ans.  (c) Intramuscular deltoid Ref: Ananthanarayan and Paniker’s Textbook of Microbiol­ ogy – 10th ed – page 552 • Vaccine is given IM in the deltoid or anterolateral aspect of thigh in children – 0, 1, 6 months • Not to be given in gluteal - poor immune response • Seroconversion – 90% •

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24. Ans.  (d) HCV

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28. Ans.  (d) DNA viruses Ref: Ananthanarayan and Paniker’s Textbook of Microbiol­ ogy – 10th ed – page 553 • HDV - Defective RNA virus – dependent virus • Needs HBV for replication • It can either gets infected along with HBV (Co-infection) or after infection with HBV (Super infection) • HBV is a DNA virus; HDV is a RNA virus. •

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29. Ans.  (a) Hep. E Ref: Ananthanarayan and Paniker’s Textbook of Microbiol­ ogy – 10th ed – page 554 • HEV – unique feature is clinical severity and high case fatality rate around 20-40% in pregnant women • Severity is more in the last trimester. •

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30. Ans.  (b) Adults Ref: Ananthanarayan and Paniker’s Textbook of Microbiol­ ogy – 10th ed – page 554 • Hepatitis E virus affects adults whereas HAV affects children. •

Ref: Ananthanarayan and Paniker’s Textbook of Microbiol­ ogy – 10th ed – page 554 • HAV and HEV - Both gets infected by fecal-oral route • Because of clinical features – both looks same. •

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32. Ans.  (c) HCV Ref: Essentials of Medical Microbiology – Apurba Sastry – Page 116 • Cryoglobulinemia is a condition in which the blood contains cryoglobulins – that is IgM antibodies that are directed against Fc portion of IgG • It is associated with multiple myeloma and Hepatitis C virus infection. •

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34. Ans.  (b) Virus can integrate with host DNA and form complementary DNA Ref: John Carter – Virology Principles and Applications – page 290 • HBV is the most significant agent for hepatocellular carcinoma • The prevalence of HCC closely parallels the prevalence of persistent HBV infection with the highest incidences in Asia and in central and southern Africa. • In most of the tumors, HBV DNA is integrated into the cell genome, and in most cases, the virus DNA has undergone rearrangements, including deletions. • The P and C gene ORFs have been generally destroyed, but the S and X ORFs are often intact. • The only viral gene product that is consistently present in the tumor cells is the X protein •

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Chapter 34     Hepatitis Virus

31. Ans.  (a) Hepatitis A

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33. Ans.  (b) Hepatitis Ref: Review of medical microbiology – Levinson – 13th ed – Page 750 • Hepatitis B virus is the only virus that produce genome DNA by reverse transcription with mRNA as template • Another identification here is cccDNA – covalently closed circular DNA • Following entry into the host cells, the virion RC DNA is released into the nucleus for conversion into CCC DNA, which is the first viral product to be made in a newly infected cell. CCC DNA then serves as the viral transcriptional template for the synthesis of all viral RNAs by the host RNA polymerase II. • Though not integrated into the host chromosomes, the episomal HBV CCC DNA functions equivalently to that of the integrated retroviral provirus and is the molecular basis of HBV persistence.

35. Ans.  (b) Immunization against Hepatitis B virus will reduce the hepatitis caused by HDV; (c) HDV has RNA in its genome and it has no polymerase in its virion

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Ref: Review of Medical Microbiology – 13th edition – Levin­ son – page 760 • Hepatitis D virus is an enveloped virus with RNA genome • It does not have polymerase in its virion; cannot replicate on its own; called as defective virus • HDV can replicate only when co-infected with HBV • HDV transmitted as like HBV – blood and blood borne products, vertical transmission • Lab diagnosis – detection of delta antigen of IgM antibody to delta antigen in blood • No specific therapy; no vaccination • Immunization against Hepatitis B virus will reduce the hepatitis caused by HDV. •

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36. Ans.  (d) Hepatitis E Ref: Ananthanarayan and Paniker’s Textbook of Micro­ biology – 10th ed – page 554 For explanation please refer Q. 29.

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35

Human Immunodeficiency Virus

INTRODUCTION

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y Family Retroviridae has many viruses that can affect different host species y All retroviruses have an outer envelope of lipid and viral proteins y The envelope encloses the core which has other proteins y The core has two molecules of viral RNA – single stranded positive sense RNA (ss RNA)2

STRUCTURE OF HIV

y

y

y HIV is a lentivirus that infects and destroys cells in the immune system. y Lentiviruses are a subgroup of retroviruses; the word lenti means slow, since HIV produces a slow viral infection. It comes under lentiviruses y HIV is an enveloped virus y The virus envelope is composed of two phospholipid layers derived from the host cell membrane y The envelope has an envelope coated protein, glycoprotein (gp) 160. y Gp160 is composed of two subunits, gp120 and gp41. y Gp 120 is an external protein and it contains sites that bind CD4 cells and coreceptors on the surface of human CD4 T cells. y gp41 is membrane-bound protein. y Inside the viral envelope there is a layer called the matrix, which is made from the protein p17. y The viral core or capsid is made up of protein p24. y Inside the core there are three enzymes required for HIV replication:  Reverse transcriptase (RT)  Integrase  Protease y Within the core is HIV genetic material which consists of two positive strands of single-stranded Ribonucleic Acid (RNA).

GENOMIC STRUCTURE OF HIV



y

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y HIV genome has nine genes and long terminal repeat (LTR) regions at either end of the genome y There are three structural genes namely env, gag and pol  Envelope (env) coding for envelop of HIV;  Group-specific antigens (gag) coding for capsid and matrix;  DNA polymerase (pol) coding for integrase, protease and reverse transcriptase enzymes. y Regulatory genes that are needed for viral replication are tat, rev, nef y Accessory genes needed for viral replication and regulations are vif, vpr, vpx, vpu y Importance of genes:  For diagnostic testing, the detection of antibodies and viral proteins (Env, Gag and Pol) are often used  During the window period of detection, the p24 (Gag) protein is used as a diagnostic for HIV infection. 

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Figure 1: Structure of HIV

Chapter 35     Human Immunodeficiency Virus

Table 1: Types of genetic structure and genes  

Genetic structure types

Genes

Structural genes

Env, gag and pol

Regulatory genes

Tat, rev, nef

Accessory genes

Vif, vpr, vpx and vpu

Figure 3: Classification of HIV Figure 2: Genomic structure of HIV (Source: Hivbook.com 2007)

Table 2: Gene product and their description

Table 3: Subtypes of HIV virus and their geographical distribution  

 

Gene products Descriptions

Subtypes

Geographical distribution

gp160

Precursor of envelope glycoproteins

A, C, D

Africa

gp120

Outer envelope glycoproteins of virion

C

India and China

p66

Reverse transcriptase and Rnase H from polymerase gene product

E, C, B

Asia

p55

Precursor of core proteins, polyprotein from gag gene

E*

Thailand

p51

Reverse transcriptase, RT

gp41

Transmembrane envelope glycoproteins, TM

p32

Integrase , IN

p24

Nucleocapsid core protein of virion

p17

Matrix core protein of virion

CLASSIFICATION OF HIV

* E is now actually a recombinant with A – AE forms or Circulating recombinant forms (CRFs)

LIFE CYCLE OF HIV y HIV virus enters the host cells with the help of CD4 receptors present on the T lymphocytes y It can also attack other cells like macrophages and dendritic cells y HIV-1 needs coreceptor for entry into cells y Coreceptors involved are: CXCR4 and CCR5 y

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y There are two HIV types:  HIV-1  HIV-2 y HIV-1 is the most prevalent type throughout the world. y HIV-2 has limited geographic distribution especially in African countries. y HIV -1 is further subdivided into three main groups with an additional group y Groups are M, N, O and P y P is a group seen only in women living in Paris. y Group M is the most common in world. It is further subdivided into subtypes as given in the following figure. y





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• Most common type in the world is – HIV-1 • Most common group in the world is – Group M • Most common subtype seen in India – Subtype C •

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Receptor for HIV virus

CD4 in T cells

Receptor of HIV virus in gut

Integrin α-4 β-7

Coreceptor for macrophage tropic strains of HIV-1

CCR5

Coreceptor for lymphocyte tropic strains of HIV-1

CXCR4

y The first step in fusion involves the high-affinity attachment of viral gp120 to a CD4 molecule. y Fusion of the virus envelope with host cell membranes and releases the viral genetic material (RNA) in the protoplasm of the host cell occurs. y Then reverse transcription occurs where single-strand HIV RNA molecules are converted to double-strand c-DNA molecules. (by reverse transcriptase) y

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Unit 3     Virology

y These DNA molecules enter the nucleus y The HIV enzyme “integrase” integrates the HIV DNA with the host cell’s DNA. The integrated HIV DNA is called provirus. The provirus may remain inactive for several years, producing few or no new copies of HIV. (Reason for latency) y When host cell is activated, transcription occurs producing copies of HIV RNAs and mRNAs. These are moved from nucleus to cytoplasm y Then viral assembly occurs and new viral particles are expelled by budding y During budding the HIV envelope also acquires host membrane proteins and a lipid bi-layer. y

y

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Figure 4: Life cycle and replication of HIV

MODES OF TRANSMISSION OF HIV

Difference of Pathogenesis in HIV-2

y Transmission occurs through:  Sexual intercourse (vaginal, anal or oral)  Parenteral route (IV drug use)  Unsafe blood transfusion  Organ transplantation  Vertical transmission from mother to child y The virus can be found in blood and body fluids like semen, vaginal secretions, breast milk, cerebrospinal fluid, amniotic fluid and synovial fluid containing blood.

y HIV-2 is less easily transmitted and less infectious y It has a longer incubation period y Immunosuppression in HIV-2 infected persons is significantly slower than in HIV-1 infected persons. y Progression of HIV-2 is slower and infection leads to a significantly lower plasma viral load.

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CLINICAL STAGES OF HIV INFECTION y Acute infection phase/primary infection or window period:  Immediately after the infection, there is wide virus dissemination and seeding of lymphoid organs.  This period usually lasts for 3–6 weeks and terminates with appearance of an adaptive immune response to HIV. y Chronic asymptomatic phase:  Between six to twelve months after infection, the host’s immune response establishes the plasma virus load set point. The plasma virus load set point is an important determinant in the progression of the HIV disease  The chronic infection phase may last for 7–10 years.  The virus becomes largely sequestered in lymphoid tissue and continues to replicate there during the years of clinical latency y AIDS  The advanced stage of HIV infection is characterized by an increase in the plasma viral load. Dysfunction in the immune system occurs. y

PATHOGENESIS OF HIV INFECTION y After the entry of virus into the body, viral replication starts, at both the sites of entry and at the draining lymph nodes within 72 hours y Lymphocytes are immediately activated once the infection is acquired. y HIV replicates more in these activated cells. y Once there is an active infection, CMI and humoral immunity starts exhibiting leading to neutralizing HIV antibodies. y HIV antibodies are first detected by IgM ELISA around 3 weeks after infection y The period from virus entry into the cells till the detectable levels of HIV is called as window period (Antibody tests are negative) y This seronegative period may ranges from 3 weeks to 3 months. y

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Clinical AIDS is defined as a CD4 cell count of less than 200/mm3 and/or the appearance of AIDS defining illnesses.

• Cytomegalovirus disease (other than liver, spleen, or nodes), onset at age >1 month • Cytomegalovirus retinitis (with loss of vision) • Encephalopathy attributed to HIV • Herpes simplex: chronic ulcers (>1 month’s duration) or bronchitis, pneumonitis or esophagitis (onset at age >1 month) • Histoplasmosis, disseminated or extrapulmonary • Isosporiasis, chronic intestinal (>1 month’s duration) • Kaposi’s sarcoma • Lymphoma, Burkitt’s (or equivalent term) • Lymphoma, immunoblastic (or equivalent term) • Lymphoma, primary of brain • Mycobacterium avium complex or Mycobacterium kansasii, disseminated or extrapulmonary • Mycobacterium tuberculosis of any site, pulmonary, disseminated or extrapulmonary • Mycobacterium, other species or unidentified species, disseminated or extrapulmonary • Pneumocystis jirovecii (previously known as Pneumocystis carinii) pneumonia • Pneumonia, recurrent • Progressive multifocal leukoencephalopathy • Salmonella septicemia, recurrent • Toxoplasmosis of brain, onset at age >1 month • Wasting syndrome attributed to HIV •

•

Table 4:  Classification of HIV infection

•

•

Group I

Acute HIV syndrome

Group II

Asymptomatic infection

Group III

Persistent generalized lymphadenopathy

Group IV  Subgroup A  Subgroup B  Subgroup C  Subgroup C1

Other diseases Constitutional disease Neurologic disease Secondary infectious disease Specified infectious disease like P. carinii, Cryptosporidiosis, toxoplasmosis, strongyloidosis Oral hairy leukoplakia, Salmonella, bacteremia, nocardiosis, TB, thrush Kaposi sarcoma, lymphoma Other conditions

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 Subgroup C2  Subgroup D  Subgroup E

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Chapter 35     Human Immunodeficiency Virus



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PROGRESSION OF HIV TO AIDS y Based on the median time period for conversion of HIV to AIDS, the persons infected with HIV are classified as:  Typical progressors  Rapid progressors  Long term nonprogressors (LTNP)  Subset of LTNP – Elite controllers y Elite Controllers: Also known as natural controllers are a subset of LTNPs. Their immune system, despite being infected with HIV, has been able to successfully suppress the virus to an undetectable level (HIV RNA below 50 copies/mL) for many years in the absence of ART y

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LABORATORY DIAGNOSIS y Serological tests:  Enzyme linked immunosorbent assays (ELISAs)  Rapid tests  Western blots y NAATs – sensitive tests for detection of HIV infection – viral load can be detected y







y

DIAGNOSIS OF HIV IN INFANTS y Diagnosis in a child less than 18 months cannot be done using antibody based assays as maternal antibodies may be present in the infant’s circulation. y Up to the age of 18 months, the diagnosis of HIV infection can only be reliably made by DNA PCR y

y

Figure 5: Different type of progressions of HIV to AIDS AIDS defining opportunistic illness are: (Ref: Harrison’s 19th ed) • Multiple or recurrent bacterial infections • Candidiasis of bronchi, trachea or lungs • Candidiasis of esophagus • Cervical cancer (invasive) • Coccidioidomycosis, • Extrapulmonary Cryptococcosis • Cryptosporidiosis, chronic intestinal (>1 month’s duration) •

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Figure 6: Laboratory methods of HIV diagnosis

Unit 3     Virology

Table 5:  Serological markers helpful for diagnosis of HIV infection

y Non-nucleoside reverse transcriptase inhibitor (NNRTI): inhibits the HIV reverse transcriptase enzyme by binding to it y Protease inhibitors (PI): binds to the active site of the protease and prevents maturation. y

y

Early infection

P24 antigen

Acute (Seroconversion stage)

Anti HIV IgM and IgG

Partial illness

gp120, gp41, gp160 antibodies, Anti HIV IgG, western blot

Carriers and asymptomatic

P24 antigen, gp41, gp160, gp120 antibodies, Anti HIV IgG, western blot,

POST EXPOSURE PROPHYLAXIS y When an occupational exposure occurred, nature of exposure, HIV status of the individual should be known for PEP y Immediately after a needle stick injury or blood spill, wash the area with soap and water thoroughly y Contact the casualty medical officer and start PEP within 72 hours of exposure y

Persistent generalized lymphadenopathy

Anti HIV IgG, western blot

AIDS

All antibodies decline; Anti HIV IgG, western blot are positive

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Table 7:  Levels of exposure – NACO guidelines Mild exposure

Mucus membrane/non-intact skin with small volume, e.g., a superficial wound (erosion of the epidermis) with a plain or low calibre needle; contact with the eyes or mucous membranes; subcutaneous injections following small-bore needles

Moderate exposure

Mucus membrane/non-intact skin with large volumes or percutaneous superficial exposure with solid needle (e.g., a cut or needle stick injury penetrating gloves).

Severe exposure

Percutaneous with large volume, e.g. an accident with wide bore needle (>18G) visibly contaminated with blood; a deep wound (hemorrhagic wound and/or very painful); transmission of a significant volume of blood; an accidental injury with material, which has previously been used intravenously or intra-arterially.

Table 6:  NACO strategies for testing different categories of samples Strategies

Samples

Strategy I

To screen blood/blood products, organ, tissues and sperms

Strategy IIA

For surveillance

Strategy IIB

For diagnosis–to determine the HIV status of clinically symptomatic suspected AIDS cases

Strategy III

For diagnosis in asymptomatic individuals

VACCINE RESEARCH IN HIV y Modified whole virus has been tried y Subunits based on envelope glycoprotein is under development y Target cell protection by anti-CD4 antibody or genetically engineered CD4 y PEP - vaccine y

y

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TREATMENT There are three major classes of antiretroviral drugs available: y Nucleoside/Nucleotide reverse transcriptase inhibitor (NRTI): acts as a DNA chain terminator y

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Dosages of the Drugs for PEP for adults and adolescents: FDC of Tenofovir ( TDF) 300 mg plus Lamivudine (3TC) 300 mg plus Efavirenz (EFV) 600 mg once daily for 4 weeks Tests needed to be done after exposure: • HIV, Anti HCV, HbsAg • Baseline, 3rd month and 6th month •

•

1. Which of the following is a lentivirus? (Recent Pattern July 2016) a. HIV b. HBV c. HCV d. Rabies virus 2. Which HIV virus is more dangerous? a. HIV-1 (Recent Pattern Dec 2016) b. HIV-2 c. Both are same d. It depends on host factors 3. HIV envelop is formed by: (Recent Pattern Dec 2016) a. Host cell b. Virus c. Both d. None 4. Nef gene in HIV is used for: (Recent Pattern Dec 2012) a. Enhancing the expression of genes b. Enhancing viral replication c. Decreasing viral replication d. Maturation 5. Subtype of HIV most common in India: (Recent Pattern Dec 2013) a. A b. B c. C d. D 6. Mother to child transmission of HIV: (Recent Pattern Dec 2013) a. 25% b. 50% c. 60% d. 75% 7. Most common mode of transmission of HIV after sexual transmission: (Recent Pattern Dec 2016) a. Blood and blood products b. Occupational c. Perinatal d. Breast feeding 8. The chance that a health worker gets HIV from an accidental needle prick is: (Recent Pattern Dec 2015) a. 1% b. 10% c. 95% d. 100% 9. The receptor through which M-tropic HIV strains bind: (Recent Pattern Dec 2013) a. CCR5 b. CXR4  c. CXCR5 d. Any of the above 10. Function of gp120 of HIV envelop: (Recent Pattern July 2016) a. Cell fusion b. Cell penetration c. Attachment of CD4 receptor d. Integration of nucleic acid 









11. Cell fusion of HIV with target cell is done by: (Recent Pattern July 2016) a. gp 120 b. gp 41 c. p 24 d. p 18 12. Most common genital lesion in HIV patient is: (Recent Pattern Dec 2016) a. Chlamydia b. Herpes c. Syphilis d. Candida 13. Most common opportunistic infection in HIV globally: (Recent Pattern Nov 2015) a. P. jiroveci b. Candida c. M.tuberculosis d. Cryptococcus 14. True about HIV all, except: (Recent Pattern Dec 2016) a. PML caused by JC virus b. CNS lymphoma is the most common CNS tumor c. CMV is the most common cause of retinitis d. Most common cause of seizure is candida 15. Oral hairy leukoplakia is caused by: (Recent Pattern Dec 2013) a. HSV b. EBV c. CMV d. HPV 16. A person has unprotected sex 3 weeks back. To rule out HIV infection the best test is: (Recent Pattern Dec 2016) a. P24 antigen assay b. ELISA c. Western blot d. Lymphnode biopsy 17. Window period in HIV infection: (Recent Pattern Dec 2015) a. 1 – 2 weeks b. 4 – 8 weeks c. 8 – 12 weeks d. >12 weeks 18. Most sensitive test for HIV infection: (Recent Pattern Dec 2013) a. Western blot b. ELISA c. Agglutination d. CFT 19. False about p24 is: (Recent Pattern Dec 2012) a. Seen after 3 weeks of infection b. Can’t be seen in first week c. Can’t be detected after seroconversion d. Detected by ELISA 20. Which of the following is included in AIDS related complex? (Recent Pattern 2018) a. Vaginal candidiasis b. Invasive Ca cervix c. Cyclospora infection d. Herpes zoster virus 21. Mean transformation time for HIV to AIDS is: a. 7.5 years (Recent Pattern 2018) b. 10 years c. 15 years d. 12 years 





Chapter 35     Human Immunodeficiency Virus

MULTIPLE CHOICE QUESTIONS







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Unit 3     Virology

ANSWERS AND EXPLANATIONS 1. Ans.  (a) HIV Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10 th ed – Page 574 HIV, the causative agent of AIDS belongs to the lentivirus subgroup of family Retroviridae. 2. Ans.  (a) HIV-1 Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10 th ed – Page 576 HIV type 1 is more virulent. HIV 2 is less virulent than HIV1. The original isolates of HIV and the related strains prevalent all over the world belong to HIV type 1. 3. Ans.  (c) Both Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10 th ed – Page 575 The lipoprotein envelope of HIV consists of lipid part and protein part. The lipid part is derived from the host cell and the protein part is virus encoded. 4. Ans.  (c) Decreasing viral replication Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10 th ed – Page 575 Nef is negative factor gene which decreases or down regulates viral replication. Non structural and regulatory genes

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10 th ed – Page 586 The second mode of transmission of HIV next only to sexual route is through blood and blood products. Screening of blood donors is mandatory now, which must include p24 antigen screening. 8. Ans.  (a) 1% Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10 th ed – Page 586 The chance of HIV infection per exposure is 0.5-1% for needle stick injuries.

Types of exposure

Approximate chance of HIV infection per exposure

Sexual intercourse

0.1-1%

Blood and blood products transfusion >90% Tissue and organ donation (semen, cornea, bone marrow and kidney)

50-90%

Injections and injuries (needle stick/ surgical wounds)

0.5 – 1%

Mother to baby

30%

9. Ans.  (a) CCR5

Gene

Function

tat (trans activating gene)

Enhances expression of all viral genes

nef (Negative factor gene)

Down regulates viral replication

rev (Regulator of virus gene)

Enhances expression of structural proteins

vif (viral infectivity factor gene)

Influences infectivity of viral particles

vpu (only in HICV-1) and vpx (only in HIV-2)

Enhance maturation and release of progeny virus from cells.

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10 th ed – Page 577

vpr

Stimulates promoter region of the virus

Specific binding of the virus to the CD4 receptor is by the envelope glycoprotein gp120.

LTR sequences

Contains the sequences thet give promoter, enhancer and integration signals

5. Ans.  (c) C Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10 th ed – Page 575 In India and China, subtype C is the most prevalent. 6. Ans.  (a) 25%

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7. Ans.  (a) Blood and blood products

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10 th ed – Page 586 Mother to child transmission of HIV is 30%.

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10 th ed – Page 577 Binding of HIV to CD4 receptor requires the participation of a co-receptor molecule, which has been identified as CC4 for T-cell tropic HIV strains and CCR5 for macrophage tropic (M-tropic) strains. 10. Ans.  (c) Attachment of CD4 receptor

11. Ans.  (b) gp 41 Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10 th ed – Page 577 Following attachment, cell fusion is necessary for HIV infection to take place. Fusion is brought about by transmembrane gp41. 12. Ans.  (b) Herpes Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10 th ed – Page 579 Herpes simplex viral infection is the most common genital infection in HIV patients.

14. Ans.  (d) Most common cause of seizure is candida Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10 th ed – Page 579 The typical CNS opportunistic infections are toxoplasmosis and cryptococcosis. Lymphomas of the CNS are more common. 15. Ans.  (b) EBV Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10 th ed – Page 578 Oral hairy leukoplakia is caused by Epstein-Barr virus (EBV) infection of the oral mucosa. It most often occurs in association with HIV infection. It has been less frequently described in immunosuppressed patients, especially following organ transplantation, and is rare in immune competent individuals. 16. Ans.  (a) P24 antigen assay Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10 th ed – Page 580 P24 antigen is detectable as ealy as 2 weeks following HIV infection and is the earliest diagnostic marker to appear in blood. P24 antigen capture assay can be used to detect p24 antigen. 17. Ans.  (b) 4 – 8 weeks Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10 th ed – Page 580 Window period is the period of serological negativity. The patient is infected and is infectious but undetectable by the serological tests. Seroconversion refers to the appearance of IgM antibodies in the patient’s serum, following the initial period of viremia and p24 antigenemia. IgM antibodies appear in about 4-6 weeks following infection, which constitutes the window period. 18. Ans.  (b) ELISA Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10 th ed – Page 582 ELISA is the test which is most commonly used for screening of HIV in patients because it is more sensitive. 19. Ans.  (a) Seen after 3 weeks of infection Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10 th ed – Page 580 P24 antigen can be seen in 2 weeks following HIV infection. It disappears before appearance of IgM antibodies.

20. Ans.  (b) Invasive Ca cervix Ref: Harrison’s T.B of internal medicine – 19th edition – Page 1215 • • • • • • • • • • • • • • • • • • • • • • • • • • •

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AIDS defining opportunistic illness are: Multiple or recurrent bacterial infections Candidiasis of bronchi, trachea, or lungs Candidiasis of esophagus Cervical cancer (invasive) Coccidioidomycosis, Extrapulmonary Cryptococcosis Cryptosporidiosis, chronic intestinal (>1 month’s duration) Cytomegalovirus disease (other than liver, spleen, or nodes), onset at age >1 month Cytomegalovirus retinitis (with loss of vision) Encephalopathy attributed to HIV Herpes simplex: chronic ulcers (>1 month’s duration) or bronchitis, pneumonitis, or esophagitis (onset at age >1 month) Histoplasmosis, disseminated or extrapulmonary Isosporiasis, chronic intestinal (>1 month’s duration) Kaposi’s sarcoma Lymphoma, Burkitt’s (or equivalent term) Lymphoma, immunoblastic (or equivalent term) Lymphoma, primary of brain Mycobacterium avium complex or Mycobacterium kansasii, disseminated or extrapulmonary Mycobacterium tuberculosis of any site, pulmonary, disseminated, or extrapulmonary Mycobacterium, other species or unidentified species, disseminated or extrapulmonary Pneumocystis jirovecii (previously known as Pneumocystis carinii) pneumonia Pneumonia, recurrent Progressive multifocal leukoencephalopathy Salmonella septicemia, recurrent Toxoplasmosis of brain, onset at age >1 month Wasting syndrome attributed to HIV

Chapter 35     Human Immunodeficiency Virus

13. Ans.  (a) P. jiroveci Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10 th ed – Page 578 HIV/AIDS; National Institute of Allergy and Infectious Diseases National Institutes of Health Bethesda, Maryland 20892 Pneumocystis carinii pneumonia is the most common opportunistic infection in HIV patients.

21. Ans.  (b) 10 years Ref: Zuckermann principles and practice of clinical virology – 5th edition – Page 730 • Primary HIV infection entails symptomatic fever and lymphadenopathy in about 50% of infections. • Following seroconversion, there follows an asymptomatic phase of infection • CD4 cell counts decline rapidly and patients progress clinically in 5 years (rapid progressors), or a protracted course where the CD4 cell counts remain in the normal range and viral load low or undetectable with asymptomatic disease for 15 years (long-term nonprogressors). • These differences probably represent the limits of a normal distribution • The time to progression to AIDS following infection by HIV is variable but averages about 10 years. •

•

•

•

•

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36

Miscellaneous Viruses PARVOVIRIDAE Parvo Virus B19 (Smallest Virus)

y

y

y

Non enveloped ss DNA Smallest virus with smallest genome Parvo virus B19 – causes infection in children Parvoviruses has a special tropism for erythroid progenitor cells in the adult bone marrow and fetal liver It binds to the P antigen in the blood cell surface Infection is acquired through respiratory route It causes Slapped cheek disease or fifth disease or erythema infectiosum in children Causes aplastic crisis in children with sickle cell anemia In adults it can cause arthropathy and aplastic anemia. In pregnancy, it leads to nonimmune fetal hydrops

y

y y y y

Diagnosis

y y y

y y y

y

Expanded rubella syndrome – includes hepatosplenomegaly, thrombocytopenic purpura, myocarditis and bone lesions

y y y

y

Congenital rubella syndrome: Cardiac defects, cataract and deafness

y

y

y

y

y

y

y

y

y Belongs to Togaviridae: Enveloped RNA virus y Infection is acquired from a clinical or subclinical case; no carriers y Gets transmitted by either droplet infection or vertical transmission y It will affect the children of 3–10 years y Around 40% of the females are susceptible to infection; Hence vaccination is must to avoid congenital syndrome y Depending upon the trimester – infection rate differs y Damage to fetus occurs most during first trimester

y BK virus can grow in a wide range of primary and continuous cell cultures y

RUBELLA VIRUS – GERMAN MEASLES

y

y

IgM antibody: Acute infection; IgG means that person is immune to Rubella infection y Vaccine strain: RA 27/3 y MMR vaccine: Those who are vaccinated should avoid getting pregnant for three months

PAPOVAVIRIDAE HUMAN PAPILLOMA VIRUS y

y

y Belongs to Papovaviridae y Small, nonenveloped, DNA tumor viruses

Table 1:  HPV types and clinical illness HPV 6, 11

Intraepithelial neoplasia

HPV 16, 18

Cervical cancer

HPV 1, 2, 3, 4

Verruca vulgaris

HPV 6, 11

Condyloma acuminatum

•

Disease conditions associated with parvovirus: • Erythema infectiosum or fifth disease or slapped cheek disease in children • Arthralgia or Polyarthropathy in adults • Transient aplastic crisis • Pure red cell aplasia • Hydrops fetalis • Papular purpura •

y

y

y JC virus – grows only in human fetal glial cell cultures y BK virus – isolated in kidney transplant patients -nephropathy

•

•

POLYOMAVIRUS

•

•

y







y

y Recombinant vaccine:  Bivalent vaccine: HPV 16, 18  Quadrivalent vaccine: Gardesil – HPV 6, 11, 16, 18  Nonavelent vaccine: HPV 6, 11, 16, 18, 31, 33, 45, 52 y Vaccine is to be given for female population aged 9 to 26 years

Figure 1: Slapped cheek appearance caused by Parvo virus B19 (Courtesy: CDC)

ROTAVIRUS y Member of Reoviridae y Has 11 segments of dsRNA – characteristic feature is genetic reassortment y

y

y Human rotavirus does not grow in cell cultures, except Group A few strains have been made change to grow in serial culture. y

Prophylaxis y Vaccine: Rotarix and Rotateq y Both are oral live attenuated vaccines y Vaccines should be given before 32 weeks of age – to avoid intussusception y

y

y

Rotarix (2 doses)

P1 A (8) G 1

Rotateq (3 doses)

G1, G2, G3, G4 and P (8)

Table 2:  Other gastroenteritis viruses Virus

Characteristics

Norwalk virus

• • • •

Adenovirus Figure 2: Electron microscopic appearance of rotavirus (Courtesy: CDC/ Dr. Erskine Palmer) y Double walled viruses – Cart-wheel appearance y Most common cause of diarrheal disease in infants and children y Rotaviruses can be classified into five species Group A to E based on the antigenic structural protein called VP6. y Major antigens are VP6, VP4 and VP7 y Group A affects children y Group B – Adult diarrhea rotavirus (ADRV) y It also causes – SIDS, intussusception, necrotizing enterocolitis and DM.

Astrovirus

y

y

y

Pathogenesis and Clinical Findings y Rotaviruses infect the cells in the villi of small intestine y Destruction of enterocytes leading to loss of villi and diarrhea y Secretion of Enterotoxin – NSP4 (a viral encoded protein) – leads to triggering of signal transduction pathway y This leads to activation of enteric nervous system y Diarrhea caused by rotaviruses are due to impaired sodium and glucose absorption y It will take 3–8 weeks for restoration into normal villi. y Incubation period is 1–3 days y Symptoms seen are watery diarrhea, fever, abdominal pain and vomiting. y

y

y

y

•

•

Family - calciviridae Causes epidemics Associated with consumption of raw oysters Causes Adult diarrhea

• Types 40, 41 causes diarrhea • Difficult to grow in culture •

• Star-shaped virus • Epidemics of diarrhea in children •

•

Coronavirus

• Virus isolated in human feces • Association with diarrhea unknown •

•

y

y

•

•

y

y

•

Chapter 36     Miscellaneous Viruses

REOVIRIDAE

SLOW VIRAL DISEASE y Some diseases in humans take slow or chronic persistent type of infections called as slow viral diseases. y They usually affect the central nervous system y

y

Characteristics y y y y y y

y

y

y

y

y

y

Long incubation period Course of illness last for months to years Involves CNS Absence of immune response Genetic predisposition Invariable fatal termination

y

y

y

y

Group A rota viruses – most common cause of diarrhea in children Group B viruses - causes diarrhea in adults

Laboratory Diagnosis y Direct detection of viral particles in stool by electron microscopy y IgM and IgG ELISA (Most common method employed) y Most sensitive method – RT PCR y

y

y

Classification Table 3:  Classification of slow viral diseases Group A

Lenti viruses - HIV

Group B

Prions

Group C

Subacute sclerosing polioencephalitis (SSPE Progressive multifocal leucoencephalopathy (PML)

Group A y Two animal viruses namely Visna and Maedi viruses are the agents that causes slow infections y Lentiviruses like HIV usually take up to 10 year’s time for developing disease complex named AIDS y

y

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Unit 3     Virology

Group B-Prions y Prions are proteinaceous infectious materials y They do not have DNA/RNA y The normal prion protein is PrPc gets converted to abnormal prion protein - PrPsc and causes diseases y Prions are highly resistant to routine sterilization methods y It can be disinfected only by:  90% phenol  2 N NaOH  10% Sodium dodecyl sulfate  Autoclaving at 121°C for 1 hour y

y

y

y

y

y Three distint strains: y

Zaire

CFR 90%

Sudan

CFR 50%

Reston y Recent outbreak – West Africa – 2013 to 2016 y Vaccine: rVSV – ZEBOV y

y









Prion Diseases y Subacute spongiform viral encephalopathies (Sequel to measles virus) y Creutzfeldt Jakob disease y Kuru y SSPE y Progressive multifocal leukoencephalopathy y

y

y

y

y

Basic Features for Prion Diseases Are y Neurodegeneration and spongiform changes y Amyloid plaques are seen y As the agents are not considered antigenic – the host do not mount immune or inflammatory response y Prions are excreted in urine.

Figure 3: Filamentous Ebola virus (Courtesy: CDC/ Frederick A. Murphy)

y

y

y

y

CORONA VIRUS y Enveloped RNA viruses y Two groups:  Acid labile viruses – causes cold  Acid stable viruses – causes gastroenteritis y Types of corona virus:  229E  OC43  SARS – CoV  NL63 – New Haven corona virus  HKU 1  MERS – CoV – previously named as novel coronal virus 2012 y Severe acute respiratory syndrome (SARS) – spreads by inhalation of the virus y Virus undergoes high degree of mutation, vaccine is impossible y MERS-CoV – Middle east respiratory syndrome corona virus – first detected in Saudi Arabia in 2012; It causes severe respiratory illness; Epidemics occurs; No vaccine till now; Severe cases can go for acute renal failure. y

Human Spongiform Encephalopathies y Two main encephalopathies associated with prions are  Kuru  Classic Cueutzfeldt- Jakob disease (CJD) y Kuru is not seen nowadays. It is spreaded once by old custom among tribals by eating dead relatives (Cannibalism) y CJD is developed because of transformation of normal prion to abnormal prion proteins y CJD is manifested by progressive dementia, ataxia, myoclonus and death is usually seen within 6 months to 1 year y Two variants of CJD are:  Gerstmann-Straussler-Scheinker (GSS) syndrome  Fatal familial insomnia y CJD can also be transmitted through organ transplantation. Blood there is no proof for blood transfusion. y





y

y

y

y





y

VIRAL HEMORRHAGIC VIRUS Arenavirus

Causes south American hemorrhagic fever Lassa fever (transmitted through rodents)

Filoviruses

Marburg and Ebola virus belongs to this group

EBOLA VIRUS y Named Ebola virus: after Ebola river, where the first cases were noticed y Mode of infection: person to person transmission through blood and body fluids y Causes fever with hemorrhagic illnes – highly fatal y

y

282

y

y





y













y

y

y

ONCOGENIC VIRUSES Retrovirus Human papilloma virus Polyoma virus BK and JC virus Molluscum contagiosum Adenovirus EBV HSV 1 and 2 CMV HBV and HCV

1. Parvo virus B19 has predilection for:  (Recent Pattern 2018) a. Erythroid progenitor cells b. Myeloid progenitor cells c. Haematopoietic stem cells d. Lymphoid progenitor cells 2. Which HPV types causes Condyloma acuminata? (AIIMS Nov 2016) a. 1 and 4 b. 6 and 11 c. 16 and 18 d. 31 and 33 3. Human papilloma virus is associated with all of the cancers except: (AIIMS May 2015) a. Cancer base of tongue b. Tonsillar carcinoma c. Nasopharyngeal cancer d. Recurrent respiratory papilloma 4. Oropharyngeal cancer is associated with: (AIPG 2014) a. EBV b. HPV c. HSV d. HBV 5. Quadrivalent vaccine for HPV has all except: (PGI May 2013) a. Type 7 b. Type 11 c. Type 16 d. Type 18 e. Type 26 6. Slapped cheek appearance is seen in: (PGI May 2016) a. Rubella b. Rubeola c. Parvo virus B19 d. HSV – 6 7. Parvovirus infection is associated with:  (PGI June 2008) (AIIMS May 2008) a. Hydrops fetalis b. Aplastic anemia c. Abortion d. Sixth disease e. Hemophagocytic syndrome 8. All are true about SARS except: (Recent Pattern 2012) a. Epidemic in India b. Spreads by droplet c. Diagnosed by PCR d. Caused by SARS CoV 9. SARS is a type of: (Recent Pattern 2014) a. Corona virus b. Lenti virus c. Calci viridae d. Hepadna viridae 10. Prions are: (AIPG 2004) a. Infectious proteins b. Made up of virus particles c. It is a nuclear material d. Can be cultured 11. Prions consists of: (AIIMS 2007) a. DNA and RNA b. DNA, RNA and proteins c. RNA only d. Proteins only 12. Which of the following is not prion associated disease (AIIMS 2003) a. Scrapie b. Kuru c. CJD d. Alzhiemers disease 13. True about prion diseases are all, except: (AIIMS 2010) a. Myoclonus is seen in 10% of the patients b. Caused by infectious protein c. Brain biopsy is diagnostic d. Manifests commonly as dementia 









14. True about prions: (AIIMS 2008) a. Virus coded b. Causes misfolding of proteins c. Cleaves proteins d. Defect in synthesis of proteins 15. Rota virus is detected by: (AIIMS 2002) a. Antigen in stool b. Antibody in serum c. Demonstration of virus d. Stool culture 16. Vaccination causing intussuception: (PGI pattern 2017) a. Rotavirus b. Parvovirus c. IPV d. BCG e. Measles 17. Genetic Reassortment is typically seen in: (AIIMS Nov 2010) a. Herpes virus b. Hepadna virus c. Rotavirus d. Astro virus 18. True about Human papilloma virus: (PGI May 2017) a. Belongs to family papovaviridae b. DNA virus c. RNA virus d. Enveloped e. Causes anal warts 19. All are true about Ebola virus infection except: (PGI May 2017) a. Air droplet is most common mode of transmission b. Haemorrhagic manifestation may occur c. Thai forest type – most common species in epidemics d. Presents as sudden onset of fever and sore throat e. Case fatality rate may be high as 70% 20. True about Human papillomavirus (HPV): (PGI Nov 2017) a. ssDNA containing virus b. Icosahedral symmetry c. HPV type 16 and 18 causes cervical cancer d. Recently nine valent vaccine passed phase 3 clinical trial e. 5 genera of HPV identified 21. True about Ebola virus: (PGI Nov 2017) a. Flavivirus b. Icosahedral symmetry c. Predominantly found in Africa d. Spreads by direct contact with the blood or body fluids e. Currently there is no specific treatment for this disease

Chapter 36     Miscellaneous Viruses

MULTIPLE CHOICE QUESTIONS







22. Non immune hydrops is caused by:  (CET 2018) a. Parvovirus b. HPV c. Ehrlichia d. Rubella virus 23. Laryngeal papilloma is caused by  (CET 2018) a. HPV 6 and 11 b. HPV 16 and 18 c. HPV 31 and 32 d. HPV 6 and 18

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Unit 3     Virology

ANSWERS AND EXPLANATIONS 1. Ans.  (a) Erythroid progenitor cells Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 558

• Parvo virus infection is associated with ƒ Erythema infectiosum or fifth disease or slapped cheek disease in children ƒ Arthralgia or Polyarthropathy in adults ƒ Transient aplastic crisis ƒ Pure red cell aplasia ƒ Hydrops fetalis ƒ Papular purpura •

ƒ

ƒ

• • • •

Parvo virus B19 causes Erythema infectiosum Most common route is respiratory It infects the precursors of RBC’s In children it causes rashes on the cheeks giving an appearance of slapped cheek – hence called as slapped cheek disease ; other wise called as fifth disease • It also causes transient aplastic crisis •

•

•

•

•

2. Ans.  (b) 6 and 11 Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page • HPV types 6, 11, 40, 42 have very low oncogenic potential • They are the causes for benign conditions • Eg: Anogenital condylomas, larngeal papilloma, dysplasia and intraepithelial neoplasia

ƒ

ƒ

ƒ

ƒ

8. Ans.  (a) Epidemic in India Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page • First time the outbreak of SARS happened in China in 2002 • India escaped at that time •

•

•

•

•

3. Ans.  (c) Nasopharyngeal cancer Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page • Nasopharygeal carcinoma is caused by EBV • Rest all others are associated with HPV •

•

4. Ans.  (b) HPV Ref: Harrisons T.B of medicine 18th ed, chapter 185 • Most of the head and neck cancers are caused by HPV. The most common head and neck cancer is oral cancer. •

5. Ans.  (a) Type 7, (e) Type 26 Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page • Bivalent vaccine – HPV 16, 18 • Quadrivalent vaccine - Gardesil – HPV 6, 11, 16, 18 • Nonavalent vaccine – HPV 6, 11, 16, 18, 31, 33, 45, 52 •

•

•

6. Ans.  (c) Parvo virus B19 Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page • In children, parvo virus causes rashes on the cheeks giving an appearance of slapped cheek – hence called as slapped cheek disease • It is other wise called as fifth disease •

9. Ans.  (a) Corona virus Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page • SARS is a type of corona virus •

10. Ans.  (a) Infectious proteins Ref: Jawetz medical microbiology – 27th ed – page 615 • Prions are infectious protein particles that does not have nucleic acid • Prions are highly resistant • They have abnormal folding proteins – causes diseases •

•

•

11. Ans.  (d) Proteins only Ref: Jawetz medical microbiology – 27th ed – page 615 Ref: Q.10 12. Ans.  (d) Alzhiemers disease Ref: Jawetz medical microbiology – 27th ed – page 615 • Prion causes the following diseases: ƒ Kuru ƒ CJD ƒ GSS ƒ Fatal familial insomnia ƒ Scrapie ƒ Mad cow disease ƒ Spongiform encephalopathy •

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ƒ

ƒ

ƒ

ƒ

ƒ

ƒ

13. Ans.  (a) Myoclonus is seen in 10% of the patients

•

7. Ans.  (a) Hydrops fetalis, (b) Aplastic anaemia, (c) Abortion (e) Hemophagocytic syndrome Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page

284

Ref: Harrison T.B of internal medicine – 18th ed – Page 3554 • Most of the patients of CJD has myoclonus that occurs on and off throughout the illness • Most common human prion disease is sporadic CJD •

•

Ref: Harrison T.B of internal medicine – 18th ed – Page 3443 • Prion is a infectious protein particle • The normal prion protein is PrPc gets converted to abnormal prion protein - PrPsc and causes diseases •

•

15. Ans.  (a) Antigen in stool

• Mode of infection: person to person transmission through blood and body fluids • Causes fever with hemorrhagic illnes – highly fatal. •

•

20. Ans.  (a) ssDNA containing virus; (b) Icosahedral symmetry; (c) HPV type 16 and 18 causes cervical cancer; (d) Recently nine valent vaccine passed phase 3 clinical trial

Ref: Harrison T.B of internal medicine – 18th ed – Page 1592

Ref: Jawetz medical microbiology - 27th ed - page 631

• Diagnosis of rota virus is by: ƒ Genotyping of viral nucleic acid – the most sensitive method ƒ Direct visualisation of fecal particles in electron microscopy ƒ Serological methods ƒ They do not grow in cell culture except few strains of group A

• • • • •

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•

•

•

•

•

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16. Ans.  (a) Rotavirus Ref: Harrison T.B of internal medicine – 18th ed – Page 1592 • First rotavirus vaccine was introduced in the year 1998 • It was withdrawn then because of association with intussuception • Then recently multivalent bovine human reassortment rotavirus vaccines were prepared •

HPV is a ssDNA virus with icosahedral symmetry HPV 6,11 are benign HPV 16,18 causes malignant illness - cervical cancer Nonavalent vaccine is on use now there are totally 16 genera and 82 types of HPV based on genetic homology

Chapter 36     Miscellaneous Viruses

14. Ans.  (b) Causes misfolding of proteins

21. Ans:   (c) Predominantly found in Africa; (d) Spreads by direct contact with the blood or body fluids; (e) Currently there is no specific treatment for this disease Ref: Ananthanarayan and Paniker T.B of microbiology 10th ed - page 562

•

•

17. Ans.  (c) Rotavirus Ref: Harrison T.B of internal medicine – 18th ed – Page 1591 • Genetic reassortment is characteristic of rota virus and influenza virus •

18. Ans.  (a) Belongs to family papovaviridae; (b) DNA virus; (e) Causes anal warts • Already explained Q.2 ƒ Belongs to Papovaviridae ƒ They are Small, nonenveloped, DNA tumor viruses •

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19. Ans.  (a) Air droplet is most common mode of transmission; (c) Thai forest type – most common species in epidemics

• Ebola virus belongs to filoviridae; They are predominantly seen in Africa and spreads person to person through blood and body fluids; Currently there is no specific management • Vaccines are under trail •

•

22. Ans.  (a) Parvo virus Ref: Ananthanarayan and Paniker’s Textbook of Micro­ biology – 10th ed – Page 558 • Parvovirus causes: ƒ Slapped cheek disease or fifth disease in children ƒ Non immune fetal hydrops in pregnancy ƒ Aplastic anaemia in adults •

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23. Ans.  (a) HPV 6 and 11 Ref: Jawetz medical microbiology - 27th ed - page 631 • HPV 6 and 11 causes benign lesions – papillomas • HPV 16 and 18 causes malignant tumours •

•

Ref: Ananthanarayan and Paniker T.B of microbiology 10th ed - page 562 • It is named Ebola virus: after Ebola river, where the first cases were noticed •

285

Unit 3     Virology

286

HIGH YIELDING FACTS TO BE REMEMBERED IN VIROLOGY Both intracytoplasmic and intranuclear inclusion bodies are seen in

Measles – Warthin-Finkeldey cells

DNA viruses

Hepadna, Herpes, Adeno, Parvo, Papova, Pox

Segmented nucleic acid is seen in

Bunya viridae, Orthomyxoviridae, Reoviridae, Arenaviridae

Smallest and largest DNA virus

Parvovirus and Pox virus

Smallest and largest RNA virus

Picornavirus and Paramyxovirus

Most commonly employed method for virus isolation

Cell culture

Viruses causing hemagglutination

Influenza, Measles, Rubella, Coxsackie, Rhinovirus, Rabies, Parainfluenza, Toga, Entero, Echo, Reo

Microscopy of herpes lesions is by

Tzanck smear – Giant multinucleated cells

What is Shingles?

Reactivation of latent infections from the dorsal root ganglion – VZV – Herpes zoster

Which is the reservoir of EBV

Memory B cells

Malignancies associated with EBV

Burkitt lymphoma, Hodgkin’s disease, T cell lymphoma, Nasopharyngeal carcinoma

Most common viral organism causing intrauterine infection is

Cytomegalovirus

Space vehicle shaped virus

Adenovirus

Epidemic keratoconjunctivitis is caused by

Adenovirus (Shipyard eye)

Condyloma acuminate is caused by which of the HPV serotypes

HPV 6, 11

Erythema infectiosum is caused by

Parvovirus – Slapped cheek disease or fifth disease

Outbreaks of vaccine-derived polio virus is due to

OPV type 2

Hand foot mouth disease is caused by

Coxsackie virus

Acute hemorrhagic conjunctivitis is caused by

Enterovirus 70

Unique feature of Influenza virus

Antigenic drift and antigenic shift

Hecht’s pneumonia is caused by

Measles

Most common cause of bronchiolitis

Respiratory Syncytial Virus

Major vector for the transmission of KFD – Kyasanur Forest Disease

Ticks

Earliest detection for Dengue virus illness is

NS1 antigen detection

Only live vaccine that can be given during pregnancy when needed

Yellow fever vaccine

Inclusion bodies seen in rabies and its location

Negri bodies; most abundant in cerebellum and hippocampus

Vaccination schedule for rabies

Day 0, 3, 7, 14, 28

Which group is called as adult diarrhea rotavirus?

Group B virus

The most common cause of diarrhea in infant and children

Rotavirus

Strains used in Rota teq and Rotarix

G1, G2, G3, G4, P(8); G1 P (8)

Genetic reassortment is seen in

Rota virus

The only DNA hepatitis virus is

Hepatitis B virus

Enterically transmitted non A and non B hepatitis virus is

Hepatitis E virus

Three types of particles in HBV

Dane particles – complete hep B, Spherical particle which is the most common, Filamentous or tubular particle

The first marker to appear in serum in HBV infection

Hepatitis B surface antigen HBsAg

Qualitative and Quantitative marker for viral replication in HBV

HBeAg and HBV DNA

Most common cause of post-transfusion hepatitis and chronic hepatitis

Hepatitis C virus

Examples for defective viruses

HDV and Adenoassociated viruses

PARASITOLOGY

4

Unit Outline Chapter 37 Introduction to Parasitology Chapter 38 Flagellates–I Chapter 39 Hemoflagellates Chapter 40 Leishmania Chapter 41 Apicomplexa Chapter 42 Toxoplasma, Ciliate Protozoa Chapter 43 Coccidian Intestinal Parasites Chapter 44 Helminthology Cestodes Chapter 45 Trematodes Chapter 46 Nematodes Chapter 47 Filarial Nematodes

37

Introduction to Parasitology y A parasite is an organism that lives on another organism for nutrition y Types of parasites:  Ectoparasites: The Parasites that live on the outer surface of the host E.g. Lice—this condition is called as infestation.  Endoparasites: Parasites that live within the host,–e.g. Plasmodium— this condition is called as infection.

y





y

y

Intestinal Amoebae y Group of Amoeba that attacks the intestine with unique morphological features of naked cytoplasm and lobose pseudopodium comes under intestinal Amoeba y Based on the pathogenicity: Amoebae are classified into pathogenic and nonpathogenic Amoebae

y

INTRODUCTION

Accidental parasites

They attack human as unusual host, e.g. Echinococcus

Aberrant parasites

Some parasites when entering the host (called as paratenic host)—enters a site where they cannot live or develop further—called as aberrant, e.g. Toxocara causing larva migrans

y

y Organisms that gives shelter and nutrition to parasite are called as hosts y Types of hosts:  Definitive host: Host that harbors adult parasite; Host where sexual reproduction occurs  Intermediate host: Host that has the asexual forms of parasites y Parasites are broadly classified as:  Protozoa  Helminths

ƒ

These parasites can live freely also e.g. Naegleria

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Facultative parasites

ƒ

These parasites cannot exist without host e.g. Toxoplasma

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Obligate parasites

• How to differentiate pathogenic and nonpathogenic Amoeba: ƒ By zymodeme pattern ƒ Zymodeme: It is a group of Amoeba strains that share the same electrophoretic pattern and mobility for different enzymes ƒ Enzymes used are: – L–malate – NADP+ oxidoreductase – Glucose phosphate isomerase – Hexokinase – Phosphoglucomutase ƒ Based on these–Amoeba which come under seven populations namely 2, 6, 7, 11, 12, 13, 14 are pathogenic (total zymodemes = 24) •

Table 1:  Categories of parasites

High Yield

Entamoeba histolytica

Nonpathogenic Amoeba

E. dispar, E.coli, E.hartmanni, E. moshkovskii, E. gingivalis, E. polecki

ENTAMOEBA HISTOLYTICA y

y Lives in large intestine (Caecum) of man y Three morphological forms:  Trophozoite  Precyst  Cyst y Definitive host: Human beings y Infective form: Mature quadrinucleate cyst y Source of infection: Contaminated food by ingestion (fecal oral route), anal sexual transmission, through vectors like cockroaches and flies.

y

y

y y y

y

y

y

y All protozoa are unicellular eukaryotes y Single cell performs all the functions y Most of the protozoa are < 50 μm size except Balantidium coli which is >100 μm y All the protozoan nucleus are vesicular except B. coli y Stages of protozoa are trophozoites and cyst.





PROTOZOOLOGY



y





y





y

Pathogenic Amoeba

Unit 4     Parasitology

Figure 1:  E. histolytica (Courtesy: CDC)

Figure 2: Cyst of Entamoeba histolytica (Courtesy: CDC/ Dr LLA Moore, Jr.)

Pathogenesis y Both trophozoites and cysts can be seen in the intestinal lumen but only trophozoites can invade the tissue y Trophozoites attach to the colonic mucous and epithelial cells by their Gal/GalNAc lectin and then invades the mucous membrane of large intestine–erodes and forms flask shaped ulcers y These ulcers when treated gets healed; if left untreated can go for necrosis and perforation y Rarely, intestinal infection can produce a mass lesion named as ameboma in the bowel lumen which causes obstructions. y From there, it spreads to extraintestinal sites–liver–lungs– brain and all other sites y Leads to Amoebic liver abscess (ALA), pulmonary amoebiasis, cerebral amoebiasis. y

y

y

y

y

y

Figure 3: Life cycle of Entamoeba histolytica

290

y Intestinal Amoebaisis­— the most common type is asymptomatic cyst passage. y Symptomatic amoebic colitis can present as lower abdominal pain, mild diarrhea, malaise, weight loss. y Fecal material is full blown with bad odor; no blood or scanty blood is seen. y Sometimes patients may go for toxic megacolon leading to bowel dilation. y

y

y Medium used for isolation of E. histolytica are:  Boeck and Drbohlav medium  Balamuth’s medium  Diamond’s medium  Philip’s medium  Jones medium y











y

Invasive form

Trophozoite

Infective form

Cyst

y

Complications y The most common form of extra intestinal amoebiasis is Amoebic liver abscess (ALA); patients have characteristic fever with right upper quadrant pain. Some has pleural effusion. In case of endemicity and PUO are seen, Amoebic liver abscess must be ruled out. y The most common complication of ALA is pulmonary amoebiasis (Pleuropulmonary involvement). Manifestations seen are sterile effusions, or spread from liver due to rupture. y Genital and cerebral involvement also can occur in extreme cases.

Table 2:  Identification of the morphological forms

y

y

y

Trophozoite

Cyst

• Invasive and growing stage • It is motile with the help of pseudopodium • Nucleus is eccentric in position • Karyosome is central in position • Nuclear membrane has fine chromatin granules • It has ingested RBCs (called as erythrophagocytosis)

• Infective form • Immature cyst has glycogen mass and chromatoid bodies • Mature cyst is quadrinucleate

•

•

•

•

•

•

Chapter 37     Introduction to Parasitology

Clinical Features

•

•

•

Serological Test y Mainly used to diagnose invasive amoebiasis–extra intestinal y Indirect hemagglutination test y ELISA–most sensitive and specific test: Detects stool antigen (Copro antigen)-antigen positive means recent infection y Serum showing antibody titer of 1:128 is diagnostic of ALA y But antibody detection is not helpful to diagnose recent or past infection y

y

y

y

y

Figure 4:  HPE in intestinal amoebiasis showing many trophozoites (Courtesy: CDC/Dr Mae Melvin)

Additional Lab Features y Helps to differentiate from bacillary dysentery y Stool microscopy: Few pus cells, RBCs agglutinated, Charcot Leyden crystals are present y Blood smear: No/mild leucocytosis y WHO recommendation for diagnosis of intestinal amoebiasis– specific tests needs to be done; Hence stool microscopy is not confirmative. y

y

Diagnosis by Wet Mount y Saline preparation: Motile trophozoites seen y Iodine preparation: Trophozoites (motility can not be seen) and cysts seen y

y

Culture of E. Histolytica y Polyxenic culture: Means culture is done in the presence of a bacteria—Boeck and Drbohlav egg serum medium (first medium used for culture of E. histolytica) y Axenic culture–bacteria free culture: Its uses are  Pathogenicity of Amoeba can not be studied  In vitro anti Amoeba drug susceptibility  Antigen preparation y

y

y

y

Treatment y Amoebic dysentery: Metronidazole, emetine hydrochloride y ALA: Metronidazole, Dihydroemetine, chloroquine y Asymptomatic cyst passers: Metronidazole, diloxanide furoate, tetracycline y Metronidazole is the DOC for ameobic liver abscess. y

y

y

y







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Unit 4     Parasitology

Nonpathogenic Amoebae

Remember

Table 3: Characteristics of non-pathogenic amoebae

• Entamoeba histolytica vs Entamoeba dispar Vs Entamoeba moshkovskii: • In stool examination–both looks similar morphologically • Hence always the report should be given as Trophoites or Cyst suggestive of Entamoeba histolytica/Entamoeba dispar/Entamoeba moshkovskii • Differentiation is done based on: ƒ Clinical features ƒ Zymodeme pattern ƒ Agglutination with concanavalin A (positive in E. histolytica)

 

Organisms

Characteristics

Entamoeba coli

• Largest Amoebae—commensal in large intestine • No invasion • Never contain ingested RBCs • Until recently E. coli was considered nonpathogenic–but few cases of diarrhea in children has been described now.

•

•

•

•

•

•

•

ƒ

•

Entamoeba dispar

• Asymptomatic carriers • No invasion • Morphologically trophozoites and cyst are similar to E. histolytica

ƒ

•

•

•

Entamoeba moshkovskii

• Morphologically trophozoites and cyst are similar to E.histolytica

Entamoeba hartmanni

• It is called as small E. histolytica • But it is a misnomer–it does not look morphologically like E. histolytica

Entamoeba gingivalis

ƒ

•

•

•

• • • •

•

•

•

•

First Amoeba described in humans It has only trophozoite stage No ingested RBCs Commensal that is present in the margins of gum

Entamoeba polecki

• Amoeba that infects pigs and rarely humans

Endolimax nana

• Trophozoite: Nucleus contains large irregular karyosome–eccentric in position • Spherical cyst–quadrinucleate; chromatoid bodies and glycogen masses are absent

•

Free Living Amoebae y Pathogenic free living Amoebae are seen in soil and water–it does not have human carrier state; no vector y These are neuropathogenic y Four species:  Naegleria fowleri  Acanthamoeba  Balamuthia mandrillaris  Sappinia y

y

y









Table 4: Characteristics of free-living amoebae  

Free living Amoebae

Characteristics

Naegleria fowleri

• Infection is acquired while swimming in contaminated water • Amoeboid trophozoite enters through nasal mucosa—goes to brain • It causes primary amoebic meningoencephalitis • Very fatal—patients die within a week • Wet mounts of CSF shows motile amoeboid trophozoites • This free living Amoebae can be cultures on a NNA (non-nutrient agar plate) with a lawn culture of E. coli done already (which serves as a source of food for Amoebae) • Treatment: Amphotericin B •

•

•

•

Iodamoeba butschlii

•

•

• Most common Amoeba of pigs • Trophozoites: Large, irregular, round karyosome–central in position–surrounded by refractile globules • Cyst: Large vacuole that has closely packed glycogen mass

•

•

•

•

•

•

Acanthamoeba

• Infection is acquired through skin abrasions or through cornea lens (Contact lens); inhalation • Most common species is A. culbertsoni • It causes Granulomatous amoebic encephalitis and keratitis • In GAE–wet mount of CSF shows– trophozoites • In keratitis–Corneal scrapings shows–cysts • Fatal condition–no appropriate treatment •

•

•

•

•

•

Balamuthia mandrillaris

• Infection is transmitted through respiratory tract or skin lesions • It causes GAE • Presence of multiple nucleoli in trophozoites helps in diagnosis • Not reported in India yet •

•

•

•

292

Figure 5:  Illustration showing Entamoeba coli (Courtesy: CDC)

Sappinia

• Only one case of Sappinia causing amoebic encephalitis has been reported •

Entamoeba 1. All the following amoeba live in the large intestine except: a. E. coli b. E. nana c. E. gingivalis d. I.butschlii 2. Culture media used for entamoeba histolytica a. Blood agar b. Philip’s medium c. CLED medium d. Trypticase serum 3. Non pathogenic amoeba is/are a. Entamoeba histolytica b. E. coli c. Acanthamoeba d. E. Hartmani e. Balamuthia 4. Amoebiasis is transmitted by all except a. Cockroach b. Faeco oral c. Vertical transmission d. Oro-rectal 5. The pathogenicity of Entamoeba histolytica is indicated by a. Zymodeme pattern b. Size c. Nuclear pattern d. ELISA test 6. Which is true of trophozoites of E. histolytica (PGI 2017) a. Has eccentric karyosome b. Nuclear membrane with chromatin c. Shows erythrophagocytosis d. Presence of bacteria inside cell 7. Amoebiasis is not transmitted by a. Feco-oral route b. Sexual transmission c. Blood and blood products d. Vector transmission 8. The main reservoir for Entamoeba histolytica is a. Man b. Dirty water c. Soil d. Ponds 9. Charcot-Leyden crystals are seen in: a. Bacillary dysentery b. Amoebic dysentery c. Giardiasis d. Cholera 10. Stain with parasite having Charcot-Leyden crystals but no pus cells a. Giardia b. Taenia c. E.histolytica d. Trichomonas 11. Most common extrahepatic complication of amoebic hepatitis is a. Meningitis b. Lung abscess c. Nephritis d. Encephalitis 12. Regarding amoebic liver abscess all are true except a. Trophozoite in stool are essential for clinical diagnosis b. Mostly asymptomatic c. More common in male than female d. It rarely affects brain eye and skin

13. A 23 year old male presented with abdominal pain and bloody diarrhea of one week duration. The following colonic biopsy is diagnostic of infection with



a. Giardiasis c. Enterobius

Chapter 37     Introduction to Parasitology

MULTIPLE CHOICE QUESTIONS

b. Amoebiasis d. Severe bacterial infection

Free Living Amoeba







14. Acute Primary Amoebic meningoencephalitis true is (Recent Pattern 2017) a. Meningitis caused by acanthamoeba species is acute in nature b. Diagnosis is by demonstration of trophozoite in CSF c. Caused by feco-oral transmission d. More common in tropical climate 15. A 15 year old girl residing in a village recently returned from a vacation visiting her friends in another village. she complained of severe headache and fever, was diagnosed as a case of pyogenic meningitis and admitted to the hospital. She died 5 days later. Which of the following organism should be considered in the diagnosis? (AIIMS 2017) a. Entamoeba histolytica b. Naegleria fowleri c. Toxoplasma gondii d. Falciparum malaria 16. A patient presents with headache, high fever and meningismus. Within 3 days he become unconscious. Most probable causative agent (Recent Pattern 2018) a. Naegleria fowleri b. Acanthamoeba castellani c. Entamoeba histolytica d. Trypanosoma cruzi 17. A 30 year old patient presented with features of acute meningoencephalitis in the casualty. His CSF on wet mount microscopy revealed motile unicellular microorganism. The most likely organism is (AIIMS 2017) a. Naegleria fowleri b. Acanthamoeba catellini c. Entamoeba histolytica d. Trypanosoma cruzi

293

Unit 4     Parasitology

18. Which of the following amoebae does not have neuropathogenic effect (Recent Pattern 2017) a. Naegleria b. Acanthamoeba c. Dientamoeba d. Balamuthia 19. Cerebral amoebiasis is not caused by (Recent Pattern 2017) a. Nagleria b. Acanthamoeba c. Dientamoeba d. Balamuthia



20. Which of the following is true regarding Naegleria fowleri  (Recent Pattern Nov 2018) a. Fresh water living  b. Hot springs c. Salt water living  d. Desert living

ANSWERS AND EXPLANATIONS 1. Ans.  (c) E.gingivalis Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 41 • Entamoeba gingivalis is an amoeba of human mouth – gums • It has only trophozoite stage • Rest all three are intestinal amoebae •

•

•

2. Ans.  (b) Philip’s medium Ref: T.B of Parasitology–Rajesh Karyakarte–Page 221

• Zymodeme–it is a group of amoeba strains that share the same electrophoretic pattern and mobility for different enzymes •

6. Ans.  (b) Nuclear membrane with chromatin; (c) Shows erythrophagocytosis Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 24 • Trophozoites is the invasive form of the parasite • It has peripheral chromatin and central karyosome • The cytoplasm has a clear ectoplasm and granular endoplasm • Characteristic feature is presence of ingested RBCs •

•

•

Culture media used for cultivation of E. histolytica: • Boeck and Drbohlav medium • Balamuth’s medium • Diamond’s medium • Philip’s medium • Jones medium •

•

•

•

•

•

3. Ans.  (b) E. coli ; (d) E. Hartmani Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 40 • Non pathogenic amoebae: ƒ E. coli ƒ E. hartmanni ƒ E. polecki ƒ Endolimax nana ƒ Iodamoeba botschlii ƒ Dientamoeba fragilis •

ƒ

ƒ

ƒ

ƒ

ƒ

7. Ans.  (c) Blood and blood products Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 33 • Already explained Q.4 •

8. Ans.  (a) Man Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 33 • Reservoir of infection is infected persons especially cyst carriers • There are no animal reservoirs •

•

9. Ans.  (b) Amoebic dysentery

ƒ

4. Ans.  (c) Vertical transmission Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 33 • Amoebiasis is transmitted by: ƒ Feco oral route ƒ Homosexual – anal contact ƒ Flies ƒ Cockroaches •

Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 37 • Stool microscopy in amoebiasis-Few pus cells, RBCs agglutinated, Charcot Leyden crystals are present •

10. Ans.  (c) E. histolytica

ƒ

ƒ

ƒ

ƒ

Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 37 • Already explained Q.9 •

5. Ans.  (a) Zymodeme pattern Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 24 • Pathogenic and nonpathogenic amoeba are differentiated by Zymodeme pattern •

294

11. Ans.  (b) Lung abscess Ref: Harrison’s T.B of Internal Medicine–19th edition– Page 1365 • The most common form of extra intestinal amoebiasis is Amoebic liver abscess •

•

12. Ans.  (a) Trophozoite in stool are essential for clinical diagnosis Ref: Harrison’s T.B of Internal Medicine–19th edition– Page 1366 • Most of the amoebic liver abscess cases have no symptoms and signs of colitis • When Trophozoites in the stool are searched also – most cases are not available •

•

13. Ans.  (b) Amoebiasis • The image shows flask shaped ulcer seen in intestinal amoebiasis • It clearly indicates amoebiasis •

•

17. Ans.  (a) Nagleria fowleri Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 44; Harrison T.B of Internal Medicine–19th edition– Page 1367 • Patients is diagnosed with acute meningoencephalitis with CSF shows unicellular motile organism–hence it is amoeba–trophozoite stage • This confirms the diagnosis of Naegleria fowleri •

•

18. Ans.  (c) Dientamoeba Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 43 • Free living amoeba which are neuropathogenic are: ƒ Naegleria fowlerii ƒ Acanthamoeba ƒ Balamuthia mandrillaris ƒ Sappinia •

ƒ

ƒ

ƒ

14. Ans.  (b) Diagnosis is by demonstration of trophozoite in CSF Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 44 • Acute primary amoebic meningoencephalitis is caused by Naegleria fowleri • Infection is acquired through contaminated water from pools and lakes during swimming • The trophozoites enter via the nasal mucosa • Diagnosis is by wet mount of CSF – showing trophozoites •

•

•

•

Chapter 37     Introduction to Parasitology

• The most common complication of ALA is pulmonary amoebiasis (Pleuropulmonary involvement)

ƒ

19. Ans.  (c) Dientamoeba Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 43 • Already explained Q. 5 •

20. Ans.  (a) Fresh water living  Ref: Medical parastiology – Parija – 4th edition – Page 43 • Naegleria fowleri are free living amoebae • Found in freshwater or brackish water like lakes, river or ponds and in the soil • They are sensitive to extremes of pH and certain environments like aridity • They cannot survive in sea water •

15. Ans.  (b) Nagleria fowleri Ref: T.B of medical parasitology–S.C.Parija–4th edition– Page 44 • Most fatal condition among all neuropathogenic free living amoeba infections are Naegleria fowleri–in spite of treatment–mortality rate is higher •

•

•

•

16. Ans.  (a) Nagleria fowleri Ref: T.B of medical parasitology–S.C.Parija–4th edition– Page 44 • Already explained Q.2 •

295

38

Flagellates–I

Table 1:  Flagellates Intestinal flagellate

Giardia lamblia Chilomastix mesnili Retortamonas intestinalis Enteromonas hominis Trichomonas hominis Dientamoeba fragilis

Oral flagellate

Trichomonas tenax

Genital flagellate

Trichomonas vaginalis

Hemoflagellates

Leishmania spp and Trypanosoma sp

GENERAL FEATURES y

y All flagellates except Dientamoeba fragilis (has no flagella) possess flagella y Dientamoeba and Trichomonas vaginalis–do not have cystic stage y All flagellates have both trophozoite and cyst stage except above two y

y

Figure 1:  Figure showing trophozoite of Giardia (Courtesy: CDC/ Dr Mae Melvin)

GIARDIA LAMBLIA





y

y

y

y

y

y

y

y This flagellate lives in duodenum and upper jejunum (Small intestine) y Trophozoite is tennis racket shaped and actively motile; it has a central axostyle y Cyst stage is the infective stage y Definitive host: Human beings y No intermediate host y Infective form: Mature cyst y Source of infection:  Contaminated food/water by ingestion  Person to person transmission  Homosexual-transmission y Giardia cysts are highly resistant to standard concentrations of chlorine y With the help of sucking disc: Trophozoites infect the duodenal epithelium–causes interference in absorption of fats and vitamins leads to malabsorption y The parasite causes Giardiasis: Diarrhea, abdominal pain, steatorrhea, malabsorption, flatulence y G.intestinalis or G.lamblia: One of the first intestinal pathogens to infect infants y



Figure 2: Figure showing cysts of Giardia in stool sample (Courtesy: CDC)

y Flagellate having only trophozoites; no cystic stage y It is noninvasive

y y y y

 

TRICHOMONAS VAGINALIS y

y y y

y

y Stool examination:  Direct wet mount shows falling leaf motility of trophozoites seen in liquid stools;  In formed stools – cyst can be seen

y

Diagnosis

y String test or Entero test: A gelatin capsule with a nylon string attached is inserted up to duodenum and jejunum and remained there for 4–6 hours–after removal of the capsule–it is examined for trophozoites by microscopy y ELISA: Done on stool samples to detect the antigen (Copro antigen) y Most sensitive method of diagnosis is Duodenal sampling by endoscopy–followed by staining with Giemsa or Trichrome y Treatment: Metronidazole or Tinidazole

y

y Treatment: Metronidazole; It is very important to treat the sexual partner with a complete course y

y

y

y

Table 2:  Miscellanous Flagellates Flagellate

Characteristics

Chilomastix mesnili

• Flagellate located in cecum and colon • Trophozoites and cyst can be demonstrated in formed stool • Cytostome present • It is harmless commensal • Cysts are lemon shaped

y

•

•

•

y

•

Chapter 38     Flagellates–I

y Definitive host–humans; Man is the only reservoir of infection y Lives in vagina, cervix, urethra and prostate (adheres to the mucosa) y Route of transmission: Sexual and through fomites (sharing towels, blades) y Most common sexually transmitted disease in humans y In females: It causes trichomoniasis–Strawberry vagina because of inflammation that leads to itching, greeny discharge with an abnormal odor y In males–it causes–non gonococcal urethritis

•

Diagnosis y Saline wet mount of vaginal or urethral discharge shows-Pear shaped trophozoites (Infective form) with jerky movements– twitching motility y Stain used in Giemsa stain and Acridine orange stain to look for trophozoites y

Retortamonas intestinalis

• Rare commensal located in intestines • Also known as Embadomonas intestinalis

Enteromonas hominis

• Commensal lives in intestine • Harmless • Cytostome is absent

•

•

y

•

•

•

Trichomonas tenax

• Present in oral cavity–in gums • Nonpathogenic •

•

Trichomonas hominis

• Present in lower GIT–in cecum • Nonpathogenic •

•

Dientamoeba fragilis

• Flagellate that does not have flagellum • Inhabits large intestine • No cystic stage • Transmitted by feco oral route; Enterobius vermicularis acts as a vector for this amoeba • Non invasive • Acts as a chronic irritant–causes dientamoebiasis–diarrhea, abdominal pain and oral pruritus •

•

•

Figure 3: Trophozoites of Trichomonas vaginalis (Courtesy: CDC) y Gold standard method: Culture in Diamond’s medium– most sensitive method y ELISA and DFA tests to identify the antigen y PCR y Vaginal pH estimation by pH paper and Whiff or amine odor test helps in preliminary diagnosis–normal pH excludes the diagnosis of trichomoniasis y

y

y

•

•

•

y

297

Unit 4     Parasitology

MULTIPLE CHOICE QUESTIONS Giardia Lamblia











298



Trichomonas Vaginalis

1. True about Giardiasis (Recent Pattern 2017) a. Only cyst is infective b. Reside in cecum c. only man to man transmission d. Exist in one phase 2. How many pairs of flagella does Giardia lamblia possess (Recent Pattern 2018) a. One b. Two c. Three d. Four 3. The normal habitat of giardia is (Recent Pattern 2017) a. Duodenum and jejunum b. Stomach c. Cecum d. Ileum 4. A 4 year old child presents with acute watery diarrhea and abdominal cramps. Stool microscopy reveals trophozoites with falling leaf motility. The etiological agent is ? (Recent Pattern 2018) a. Entamoeba histolytica b. Giardia lamblia c. Trichomonas tenax d. Balantidium coli 5. A patient is presenting with abdominal pain, steatorrhea and normal LFT; Most likely infecting organism (Recent Pattern 2017) a. Shigella b. E.histolytica c. Giardia d. Enterotoxigenic E.coli 6. Giardiasis true is all except (Recent Pattern 2017) a. Diarrhea with steatosis b. Bloody diarrhea c. Metronidazole is the drug of choice d. Absent fever 7. A case of giardiasis presents with (Recent Pattern 2018) a. Nausea and vomiting b. Abdominal pain c. Steatorrhea and flatulence d. All of the above 8. Identify the following egg: (Recent Pattern Nov 2017)

a. Entamoeba dispar c. Balantidium coli

b. Giardia lamblia d. Entamoeba histolytica

9. A pap smear was prepared from the vagina and it showed the following findings. Identify the organism responsible. (Recent Pattern 2017)



a. Trichomonas vaginalis b. Neisseria gonorrhea c. Chlamydia d. Treponema pallidum 10. The cystic form of all are seen in man except (Recent Pattern 2018) a. E.histolytica b. Giardia c. Trichomonas d. Toxoplasma 11. Gold standard method of diagnosis for trichomoniasis (Recent Pattern 2017) a. PCR b. Microscopy c. Giemsa stain d. Culture 12. All are true regarding trichomoniasis except: (Recent Pattern 2018) a. Man to man transmission occurs b. Reservoir for infection is lice c. Fomites act as source of infection sometimes d. Sexual mode is the most common method of transmission 13. True regarding treatment of trichomoniasis: (Recent Pattern 2017) a. It is important to give full course of treatment for partner b. Metronidazole can be given in pregnancy to avoid fetal transmission c. Diloxanide is the drug of choice d. No drug resistant strains have been isolated till now

1. Ans.  (a) Only cyst is infective

7. Ans.  (d) All of the above

Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 56

Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 59

• Intestinal flagellate that most commonly causes diarrhea is Giardia intestinalis or Giardia lamblia • There are two morphological forms namely trophozoites and cyst • Mature cyst is the infective stage • It lives in small intestine • It is transmitted through contaminated food, water and from man to man.

• Already explained Q.5

•

•

•

•

•

2. Ans.  (d) Four

8. Ans.  (b) Giardia lamblia Ref: T.B of Medical Parasitology – Parija – 4th ed – Page 173 Giardia lamblia: • Lives in duodenum and upper jejunum • Trophozoite is tennis racket shaped and actively motile; it has a central axostyle • Cyst stage – infective form; oval shaped – thick cyst wall surrounds •

•

•

Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 56 • Trophozoite of Giardia–tennis racket shaped or pear shaped • It has two axonemes, two median bodies and four pairs of flagella • Four pairs–two pairs of lateral, one pair of ventral and one pair of caudal flagella • It has two nuclei •

•

•

•

3. Ans.  (a) Duodenum and jejunum Ref: T.B of Medical Parasitology–S.C.Parija–4 Page 56

•

9. Ans.  (a) Trichomonas vaginalis

Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 69 • Pap smear shows some kite shaped trichomonas–it is very difficult to see the flagella in pap smear and so the classical pear shaped is lost during pap smear preparation; Surrounding reactive squamous cells are seen. This feature is suggestive of trichomoniasis •

10. Ans.  (c) Trichomonas edition–

Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 65

• Giardia lives in the small intestine of humans– duodenum, jejunum and upper ileum

• Flagellates that do not have cystic stage is–Trichomonas and Dientamoeba

th

•

4. Ans.  (b) Giardia lamblia

•

11. Ans.  (d) Culture

Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 56

Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 69

• Already explained Q.1 • Features of diarrhea in children with stool microscopy showing falling leaf motility is characteristic of Giardiasis

• Culture is the gold standard and most sensitive for diagnosis of Trichomonas vaginalis

•

•

5. Ans.  (c) Giardia Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 59 • Symptoms suggest features of Giardiasis–foul smelling diarrhea, bloating, abdominal cramps, flatulence, steatorrhoea, malabsorption, weight loss • Normal LFT to rule out amoebiasis–that may spread causing amoebic hepatitis •

•

6. Ans.  (b) Bloody diarrhea Ref: T.B of Medical Parasitology–S.C.Parija–4 Page 59

th

edition–

• Bloody diarrhea is a feature of invasive organisms like Entamoeba histolytica, Shigella • Giardiasis is not invasive–hence no blood is seen •

•

Chapter 38     Flagellates–I

ANSWERS AND EXPLANATIONS

•

12. Ans.  (b) Reservoir for infection is lice Ref: T.B of medical parasitology–S.C.Parija–4th edition– Page 68 • There is no reservoir for infection for trichomonas other than man • Man is the only definitive host where the trichomonas harbours the genital area •

•

13. Ans.  (a) It is important to give full course of treatment for partner Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 70 • Metronidazole is the DOC for trichomoniasis • It is must to treat the partner • Drug is contraindicated in pregnancy because of teratogenicity • In some patients, recently drug resistant strains of trichomonas has been isolated •

•

•

•

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39

Hemoflagellates • • • • • •

FEATURES OF HEMOFLAGELLATES

y

y

y

y

y They all have a kinetoplast–which is a DNA carrying organelle y They are pleomorphic–variety of stages are seen morphologically y They live in blood and tissue y Presence of undulating membrane

 

TRYPANOSOMA y Flagellate that infects humans and are found in blood and lymph nodes y They are elongated in shape with a central nucleus and kinetoplast–which contains DNA and mitochondria y An undulating membrane originates from the kinetoplast y A single flagellum is seen at the anterior end–organism is motile y

• Trypanosoma cruzi • Trypanosoma brucei gambiense • Trypanosoma brucei rhodesiense

Amastigotes and Promastigotes Leishmania Amastigote Trypanosoma cruzi Epimastigotes and Trypomastigotes Trypanosoma brucei and cruzi

y

Trypanosoma

•

• • • •

y

Leishmania donovani Leishmania tropica Leishmania mexicana Leishmania braziliensis

Leishmania

Table 2: Stages of Hemoflagellates

y

 

Table 1: Important Hemoflagellates

Trypanosoma cruzi

Chagas disease (American trypanosomiasis)

Trypanosoma brucei

Sleeping sickness (African trypanosomiasis)

Figure 1: Life cycle of Trypanosoma cruzi (Courtesy: CDC/ Alexander J. da Silva, PhD, Melanie Moser)

Chapter 39     Hemoflagellates

TRYPANOSOMA CRUZI y Zoonotic disease–seen in South America y Not seen in India y Three main developmental forms are:  Amastigotes  Trypomastigotes  Epimastigotes y Definitive host: Humans and other vertebrates y Intermediate host: Reduviid bugs y

y

y







y

y

Table 3:  Location of morphological forms Amastigotes

Reticulo endothelial system, muscles, mononuclear phagocytes

Non multiplying trypomastigotes

Peripheral blood

Multiplying trypomastigotes

Reduviid bug

Treatment

Epimastigotes

Reduviid bug

y Nifurtimox y Benznidazole y Gentian violet

Figure 2: Amastigotes of Trypanosoma cruzi (Courtesy: CDC/Dr AJ Sulzer)

y

y Infective form to man is metacyclic trypomastigotes y Apart from vector transmission, Trypanosoma is transmitted by:  Blood transfusion  Transplacental transfusion  Organ transplantation  Laboratory accidental inoculation

y

y

y

y









Clinical Features y Chaga’s disease is most commonly seen in infants and children y At the bite area–a localized edema and erythema occurs named as Chagoma y Followed by local lymphadenopathy y In Acute Chaga’s disease–when the site of inoculation is in conjunctiva–it leads to unilateral painless edema of the eye named as Romana’s sign y Complications of acute disease are:  Myocarditis  Meningoencephalitis y In Chronic Chaga’s disease–it causes: (Mega disease)  Cardiomyopathy–heart block  Mega colon  Megaoesophagus  Colopathy  Sudden death occurs due to ventricular fibrillation y

y

TRYPANOSOMA BRUCEI y Trypanosoma brucei has two subspecies: gambiense and rhodesiense y Seen in Africa; Not in India y Morphological forms are trypomastigotes and epimastigotes (No amastigotes) y

y

y

Trypomastigote Epimastigote

Humans and other vertebrates Tsetse fly

y Definitive host: Humans and other vertebrates y Intermediate host: Tsetse fly (Glossina) y Infective form: Metacyclic trypomastigote y

y

y

y

y

y





y



Clinical Features Table 4:  Clinical Features of different subspecies of T.brucei T.brucei gambiense

T.brucei rhodesiense

• Causes West African trypanosomiasis • Winterbottom sign is seen– cervical lymphadenopathy

• Causes East African trypanosomiasis • Less common lymphadenopathy

•

•

•

•









Diagnosis y Wet mount of anticoagulated blood–shows motile trypomastigotes y Thick or thin blood smear preparation is done–to see the morphology clearly y Blood culture done in NNN medium y The only confirmatory method to diagnose chronic chagas disease is xenodiagnosis (Using bugs); Serological methods also help to diagnose chronic chagas disease y Animal inoculation–intraperitoneally into mice y

y After the bite from the fly a chancre develops at the site–then leads to hemolymphatic spread followed by CNS spread y CNS stage leads to sleeping sickness y

y

Table 5:  Differentiating features of West African and East African trypanosomiasis Features

West African trypanosomiasis

East African trypanosomiasis

Causative agent Vector Reservoir Type of illness Lymphadenopathy Parasitemia

T. brucei.gambiense Tsetse flies Humans Chronic CNS disease Prominent Low

T. brucei.rhodesiense Tsetse flies Antelope and cattle Acute CNS disease Minimal High

y

y

y

y

301

Unit 4     Parasitology

Lab Diagnosis y Lymph node specimens and peripheral smear show trypomastigotes y Culture can be done in liquid media y IHA, IFA and ELISA are most commonly used method for diagnosis y

y

y

Figure 3: Trypomastigotes of T. brucei (Courtesy: CDC/ Dr. Mae Melvin)

Treatment Table 6:  Treatment of T. brucei infection Organism

Normal CSF

Abnormal CSF

T. brucei gambiense

First drug: Pentamidine Alternative drug: Suramin

Eflornithine (or) Nifurtimox-Eflornithine combination

T. brucei rhodesiense

First drug: Suramin Alternative drug: Pentamidine

Melarsoprol

New drug (November, 2018) FDA has approved orally administered fexinidazole to replace the current treatment regimen for Trypanosoma brucei gambiense infections causing sleeping sickness.

302







1. Protozoa associated with megaesophagus: (Recent pattern 2017) a. Trypanosome b. Amoeba c. Giardia d. Gnathostoma 2. Vector for T. cruzi is: (Recent pattern 2017) a. Reduviid bug b. Tsetse fly c. Sand fly d. Hard tick 3. Amastigote form of which parasite is found in human: (PGI pattern 2017) a. Trypanosoma cruzi b. Trypanosoma brucei c. Trypanosoma gambiense d. Trypanosoma rhodesinse e. Trypanosoma rangelii 4. Winter bottom sign in sleeping sickness refers to: (Recent pattern 2017) a. Unilateral conjunctivitis b. Posterior cervical lymphadenopathy c. Narcolepsy d. Transient erythema 5. Tsetse fly transmits: (Recent pattern 2017) a. Trypanosoma brucei b. T.cruzi c. Kala-azar d. Oriental sore 6. Infective form of T. brucei: (Recent pattern 2017) a. Amastigote b. Trypomastigote c. Egg d. none

7. The following vector takes part in the life cycle of African trypanosomiasis. Identify it: (AIIMS 2017) a. Glossina b. Reduviid bug c. House flea d. Mite 8. Which form of the following flagellate is infective for mammalian host? (AIIMS 2017)

Chapter 39     Hemoflagellates

MULTIPLE CHOICE QUESTIONS

a. Metacyclic trypomastigote b. Long slender form c. Short stumpy form d. Intermediate form

ANSWERS AND EXPLANATIONS 1. Ans.  (a) Trypanosome

4. Ans.  (b) Posterior cervical lymphadenopathy

Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 100

Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 104

• In Chagas disease caused by T. cruzi–esophagus, stomach and colon are dilated leading to mega esophagus, megacolon and other organs are enlarged–called as mega disease

• Cervical lymphadenopathy is a feature of west african sleeping sickness caused by T. brucei gambiense • There are enlarged, non tender and mobile posterior cervical lymph nodes called as Winter bottom sign

•

2. Ans.  (a) Reduviid bug

•

•

5. Ans.  (a) Trypanosoma brucei

Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 95

Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 95, 102

• Reduviid bug is the vector and intermediate host for T.cruzi • Unique feature of disease is R.J Chagas first discovered T.cruzi in Reduviid bug rather than in man

• Vector for trypanosomiasis: ƒ T. cruzi–Reduviid bug ƒ T. brucei–Tsetse fly

•

•

3. Ans.  (a) Trypanosoma cruzi Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 94 • Amastigote forms are mostly seen in: ƒ Leishmania ƒ Trypanosoma cruzi • T. cruzi resembles the amastigotes of Leishmania–hence it is called Leishmanial form •

•

ƒ

ƒ

6. Ans.  (b) Trypomastigote Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 102 • Infective form for trypanosoma is metacyclic trypomastigote •

ƒ

ƒ

•

303

Unit 4     Parasitology

7. Ans.  (a) Glossina Ref: Medical Parasitology–3rd edition–D.R.Arora–page 59 • The invertebrate host involved in the life cycle of African trypanosomiasis is Glossina genus termed as tsetse fly. •

8. Ans.  (a) Metacyclic trypomastigote Ref: Medical Parasitology–3rd edition–D.R.Arora–page 59 • • • •

•

•

•

•

304

The above peripheral blood film shows Trypanosoma brucei gambiense It occurs in three forms: long slender with flagellum, intermediate form and short stumpy form without flagellum. Metacyclic trypomastigote is the infective form for man Short stumpy form is the infective form for tsetse fly

40

Leishmania INTRODUCTION

y

y

y

y Leishmaniasis is a zoonotic infection y Vector is sand fly (Phlebotomus) y There are several species in Leishmania • Visceral leishmaniasis • Post kala azar dermal leishmaniasis

Leishmania tropica

• Cutaneous leishmaniasis

Leishmania mexicana

• Cutaneous leishmaniasis •

•

•

•

Leishmania donovani

•

Leishmania braziliensis • Mucocutaneous leishmaniasis (Espundia)

LEISHMANIA DONOVANI y

y Morphological forms are Amastigotes and Promastigotes Humans

Promastigotes

Sand fly

Figure 1: Amastigotes of L. donovani in microscopy CDC/Dr Francis W Chandler y Culture can be done by NNN medium y Animal inoculation done in Chinese and golden hamster (intraperitoneal inoculation) y Serodiagnosis:  Complement fixation test  Direct agglutination test  Rapid immunochromatographic test (RDT)–for detection of antibodies against rK39 antigen–rk39 dipstick test is widely used now and sensitive.  Napier`s aldehyde test–1–2ml of the serum from patient is added with 40% formalin; a milky white jellification is seen means that indicates positive test. But this test is nonspecific.  Antimony test y Leishmanin skin test: Montenegro test (used for epidemiological studies)–this test is usually positive 4 to 6 weeks after onset in case of Cutaneous and mucocutaneous leishmaniasis. 









y y

High Yield

y





y

y

y

y

y

y

y

y

y Definitive host: Humans; Intermediate host: Sand fly y Humans are the only reservoir; no animal reservoirs (Indian kala azar)–while in other countries zoonotic spread is there y Infective form: Promastigote y It causes visceral leishmaniasis or Kala azar y Clinical features are–Fever, Anemia, hepatosplenomegaly, weight loss and lymphadenopathy y Additional features are–leucopenia, thrombocytopenia, hypergammaglobulinemia y After treatment of the primary disease–due to partially treated or untreated cases–recurrence occurs as non ulcerative lesions even up to 20 years–named as post kala azar dermal leishmaniasis (PKDL) y PKDL:  Indian form–usually present after years, with nodule and no ulceration without nerve involvement  African form–presents suddenly, ulceration occurs, nerve involvement occurs y In India–endemic areas are Bihar, West Bengal, Eastern UP

y

y

Amastigotes (LD bodies)

y

y

y

y Smears prepared from spleen, BM, lymph nodes or peripheral smear shows amastigotes seen inside macrophages (LD bodies) y Most sensitive method to detect LD bodies in splenic smear y But the most common method of diagnosis is bone marrow aspiration and microscopy

•

Case definition for kala azar • A case of Kala azar is defined as a person from an endemic area who has fever for more than 14 days duration with splenomegaly and has positive RDT test or a biopsy.

Treatment y Sodium stibogluconate (SSG) y

Lab Diagnosis

Unit 4     Parasitology

y When resistant strains to SSG are seen:  Amphotericin B deoxycholate  Miltefosine y Treatment of PKDL: SSG + Rifampicin (4 months)

y Aldehyde test is negative in cutaneous leishmaniasis y Skin test is positive in cases treated for ulcers but skin test is usually negative in diffuse cutaneous leishmaniasis

LEISHMANIA TROPICA

y Most dangerous form of cutaneous leishmaniasis is mucocutaneous leishmaniasis also called as Espundia is caused by braziliensis y Amastigotes are seen in the skin and the nasal mucosal membranes y Ulcers starts progressing and may lead to perforation or severe destruction of the nasal septum y Nasal tip may collapse named as tapir nose y This condition is not seen in India y Slit skin smear helps to visualize the amastigotes y Leishmanin test is positive y Serological tests are helpful y DOC–Pentavalent antimonials

y





y

y

y

y Morphology is identical to that of donovani y L. tropica causes cutaneous leishmaniasis otherwise called as Delhi boil, Aleppo boil y The difference between tropica and donovani is tropica do not invade organs y Bite of the sandfly region develops a papule followed by ulceration and scar y This ulcer is called as oriental sore y From the localized site–amastigotes starts moving and cause satellite lesions–finally metastasise to face and extremities– leading to diffuse cutaneous leishmaniasis y

y

y

y

y

y

LEISHMANIA BRAZILIENSIS y

y

y

y

y

y

y

y

y

Lab Diagnosis y Full thickness skin biopsy–shows amastigotes y Culture is needed when no parasites are seen in microscopy y

y

LEISHMANIA MEXICANA y Zoonotic disease y Causes bay sore or Chiclero ulcer y

y

306









1. Amastigote forms are seen in (PGI 2017) a. Leishmania donovani b. Toxoplasma gondii c. Leishmania major d. Entamoeba e. T.brucei 2. Nasopharyngeal leishmaniasis is caused due to? (Recent pattern 2017) a. Leishmania brazilensis b. Leishmania tropica c. Leishmania chagasis d. Leishmania donovani 3. Drug not used in visceral leishmaniasis (AIIMS May 2018) a. Sitamaquine b. Paromomycin c. Miltefosine d. Hydroxychloroquine 4. Which of the following is most severely affected in Kala azar (Recent pattern 2017) a. Spleen b. Liver c. Adrenal gland d. Bone marrow 5. 40 year old male from Bihar complains of abdomen pain, having hepatosplenomegaly, smear on stain shows (Recent pattern 2017)



a. Plasmodium vivax b. Leishmania c. Microfilaria d. None 6. In a case of Kala azar aldehyde test becomes positive after (Recent pattern 2018) a. 2 weeks b. 4 weeks c. 8 weeks d. 12 weeks 7. Leishmania is cultured in ……………….media (Recent pattern 2017) a. Chocolate agar b. NNN c. Tellurite d. Sabourauds 8. Espundia is caused by (Recent pattern 2018) a. L.donovani b. L.tropica c. L.mexicana d. L.braziliensis 9. Oriental sore is caused by (Recent pattern 2017) a. L.donovani b. L.tropica c. L.mexicana d. L.braziliensis 10. Infective stage for kala azar (Recent pattern 2017) a. Amastigotes b. Promastigote c. Trypomastigote d. Epimastigote 11. A patient presenting from West Bengal with fever, lymphadenopathy. Serological test showed rk39 positive. What is the treatment of choice?  (AIIMS May 2018) a. Sodium stibogluconate b. Artemesinin c. Chloroquine d. Dapsone

Chapter 40     Leishmania

MULTIPLE CHOICE QUESTIONS



ANSWERS AND EXPLANATIONS 1. Ans.  (a) Leishmania donovani; (c) Leishmania major Ref: T.B of Medical Parasitology–S.C.Parija–4 Page 75

th

edition–

• Hemoflagellates that have amastigote form are: ƒ Leishmania sp (all species) ƒ Trypanosoma cruzi •

ƒ

3. Ans.  (d) Hydroxychloroquine Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 83, Harrison T.B of internal medicine 19th edition– Page 1390 • Drugs used in the treatment of kala azar or visceral leishmaniasis are: ƒ Sodium stibogluconate ƒ Amphotericin B ƒ Paromomycin ƒ Pentamidine ƒ Miltefosine ƒ Sitamaquine •

ƒ

ƒ

2. Ans.  (a) Leishmania brazilensis

ƒ

Ref: T.B of medical parasitology–S.C.Parija–4 Page 92

th

edition–

• Nasopharyngeal leishmaniasis is a part of mucocutaneous leishmaniasis • Classical features of nasal septum perforation occurs and tip of the nose gets collapsed named as Espundia • This condition is caused by Leishmania braziliensis •

ƒ

ƒ

ƒ

ƒ

•

•

307

Unit 4     Parasitology

9. Ans.  (b) L.tropica

4. Ans.  (a) Spleen Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 78

Ref: T.B of medical parasitology–S.C.Parija–4th edition– Page 86

• Splenomegaly is the most important sign seen in kala azar

• Oriental sore is caused by L.tropica • It presents as an ulcerative lesion in the skin – this ulcer is called as oriental sore

•

5. Ans.  (b) Leishmania Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 81 • Above image shows macrophages • Within the macrophages–arrow points show amastigotes of L. donovani called as LD bodies •

•

6. Ans.  (d) 12 weeks

•

•

10. Ans.  (b) Promastigote Ref: T.B of medical parasitology–S.C.Parija–4th edition– Page 76 • Bite of infected sandfly with promastigotes causes kala azar •

11. Ans.  (a) Sodium stibogluconate

Ref: T.B of parasitology–Chaterjee–12 edition–Page 61 th

• Napier’s aldehyde test principle is based on increase in the serum gamma globulins • Visceral leishmaniasis has hypergammaglobulinemia • Serum of the patient is added with two drops of 40% formalin–if jellification occurs then the test is positive • The test is positive in: ƒ Visceral leishmaniasis or Kala azar ƒ African trypanosomiasis or T.brucei infection ƒ S. japonicum infection •

•

•

•

ƒ

ƒ

ƒ

Ref: T.B of medical parasitology–S.C.Parija – 4th edition– Page 83, Harrison T.B of internal medicine 19th edition– Page 1390 • Rapid immunochromatographic test (RDT) is done for detection of antibodies against rK39 antigen of Leishmania donovani - rk39 dipstick test is widely used now and sensitive. • Leishmania donovani causes kala-azar • Drugs used in the treatment of kala azar or visceral leishmaniasis are: ƒ Sodium stibogluconate ƒ Amphotericin B ƒ Paromomycin ƒ Pentamidine ƒ Miltefosine ƒ Sitamaquine •

•

•

ƒ

7. Ans.  (b) NNN

ƒ

Ref: T.B of medical parasitology–S.C.Parija–4 Page 82

th

edition–

• Leishmania donovani is cultured in NNN medium • NNN – Novy, McNeal and Nicolle medium • The amastigote form in the blood when in culture with NNN medium gets converted into promastigote form •

•

•

8. Ans.  (d) L.braziliensis Ref: T.B of medical parasitology–S.C.Parija–4th edition– Page 92 • Already explained Q.2 •

308

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41

Apicomplexa • Plasmodium

• Babesia

• Haemoproteus

• Entopolypoides •

•

Features • But exception in P. falciparumlive in brain capillaries and other organs by forming aggregates (only young ring forms are seen in peripheral blood) • Pigments are seen in late trophozoite and schizont stage

•

Order Piroplasmida

•

Order Haemosporida

Stages of Life Cycle in human

•

INTRODUCTION

•

• Leucocytozoon

•

PLASMODIUM

• Merozoites inside the red cells develop into gametocytes (male and female) • Male gametocytes are called as microgametocytes • Female gametocytes are called as macrogametocytes

Exoerythrocytic schizogony

• Occurs in P. vivax and P. ovale • Some of the sporozoites in liver gets converted into dormant stage called as hypnozoites – this is responsible for relapse of malaria



•

 

Definitive host

Female Anopheles mosquito (Sexual cycle happens)

Intermediate host

Man (Asexual cycle happens)

•

•

•

 

Hosts

•

Gametogony

y

y

y Malarial parasites are called as Plasmodium y There are five species in Plasmodium  Plasmodium falciparum  Plasmodium vivax  Plasmodium malariae  Plasmodium ovale  Plasmodium knowlesi

y Infective form for humans–sporozoites (from mosquito) y Infective form for mosquito–Gametocyte

y When mosquito bites human–the gametocytes are ingested by them y Inside the stomach of mosquito–they are converted into gametes–zygote–ookinete–oocyst y

y

y

Cycle in Mosquito

y

Life Cycle of Malarial Parasite

• Merozoites in the blood–invade RBCs • Converted into young ring formtrophozoites–then schizonts–then merozoites • During this phase after a time period they die out (RBC life cycle) (Contd...)

•

Bite of infected female anopheles mosquito Blood transfusion Maternal to fetus Contaminated syringes

• • • •

• • • • • • •

y

Erythrocytic schizogony • P. falciparum–36 to 48 hours • P. vivax–48 hours • P. ovale–48 hours • P. malariae–72 hours

y

• Occurs in liver parenchymal cells • Sporozoites gets converted into merozoites • When the cycle is complete–these merozoites are liberated from the liver to blood

Routes of Transmission of Malaria y y y y

y

Pre erythrocytic schizogony • P. falciparum–6 days • P. vivax–8 days • P. ovale–9 days • P. malariae–13 to 16 days

•

Features

•

Stages of Life Cycle in human

y

Table 1:  Stages of life cycle in human

Table 2:  Incubation period of species Species

Incubation period

P. falciparum

12 days (9-14)

P. vivax

14 days (8-17)

P. ovale

17 days (16-18)

P. malariae

28 days (18-40)

Unit 4     Parasitology

Figure 1: Life cycle of Plasmodium

Pathogenesis of Malaria y Human infection begins with bite of female anopheline mosquito that inoculates sporozoites into man. y These sporozoites are carried via the bloodstream to the liver. They invade the hepatic parenchymal cells and starts asexual reproduction. y This amplification process is called as intrahepatic or pre-erythrocytic schizogony or merogony (sporozoites → Merozoites) y The infected hepatocytes bursts and releases these merozoites into the blood stream. y These merozoites then go and invade the RBCs and multiply inside them. y Because of multiplication - the parasite density reaches as 50/ μl of blood and that leads to symptomatic stage of infection. y In some species like P. vivax and P. ovale, some intrahepatic forms remain inside for even years and termed as Hypnozoites (dormant forms)–these forms are responsible for relapse of infection. y After entering into the blood stream, the merozoites gets converted into trophozoites. Parasite gets attached to the RBCs by erythrocyte surface receptor. y By the end of intra-erythrocytic cycle–the parasite eats 2/3rd of the hemoglobin and occupies the cells named as Schizont. The RBC then ruptures releasing the merozoites and this invades new RBC and repeats the cycle. y But some of the blood forms instead of going to schizont stage, gets entered into sexual reproduction phase and gets converted to gametocytes. y These micro and macro gametocytes are taken by mosquitoes where sexual reproduction occurs leading to oocyte.







y

y





y y

y

y

y

y

y

y

y

Benign tertian malariae–P. vivax and P. ovale Malignant tertian malariae–P. falciparum  Quartan malariae–P. malariae  Quotidian malariae–P. knowlesi Microcytic or normocytic hypochromic anemia; Pernicious anemia Splenomegaly (tropical splenomegaly or hyper reactive malarial splenomegaly seen in endemic areas) Chronic or repeated infections with P. malariae causes immune mediated injury to renal glomeruli causing quartan malarial nephropathy. P. falciparum causes:  Cerebral malaria  Black water fever 

y

y





y

y

y

y

y

Table 3:  Features of various species of Plasmodium Features P. vivax

P. falciparum P. malariae

P. ovale

Diagnostic forms seen in peripheral smear

Trophozoites Schizonts Gametocytes

Ring forms Gametocytes

Trophozoites Schizonts Gametocytes

Trophozoites Schizonts Gametocytes

Infected RBC

Enlarged

Normal

Normal

Enlarged

Dots

Schuffner’s Maurer’s dots dots

Ziemann’s dots

James’ dots

Others

Relapse

y

Clinical Features 310

y Febrile paroxysms: Based on the periodicity of malarial fever– fever is divided into: y

Recrudescence -

Relapse

It is also known as monkey malarial parasite This species is seen in South East Asia and South America The main hosts are long tailed and pig tailed macaques The fever is typically quotidian Parasite looks similar to P. malariae–hence it may be misdiagnosed y But the disease is more severe than malaria y Treatment: Chloroquine y y y y y

y

y

y

y

y

y

y

Lab Diagnosis

Chapter 41     Apicomplexa

Plasmodium Knowlesi

y Microscopy of peripheral smear–thick and thin smear method y Conventional light microscopy is the gold standard for confirmation of malaria (Ref: Parija) y Thick smear is helpful to easy screening of malarial parasite y Thin smear is useful for species identification y Fluorescence method using acridine orange staining is also helpful y QBC–Quantitative buffy coat:  Principle is based on acridine orange which stain the nucleic acid in the parasites  More sensitive method than thick blood smear–can detect as low as 3–4 parasites/uL of blood y Serodiagnosis:  Immunochromatographic tests–rapid diagnostic tests– detects HRP-2 and pLDH and aldolase  ELISA y PCR–helpful to diagnose P. knowlesi y

y

Figure 2: Schizont of P. vivax (Courtesy: CDC/ Dr Mae Melvin)

y

y

y

y





y





y

Remember Figure 3: Gametocyte (banana shaped) of P. falciparum (Courtesy: CDC/ Dr. Mae Melvin)

Remember

Manifestation of Severe Falciparum Malaria • Cerebral coma • Acidosis • Severe normochromic/normocytic anemia • Renal failure • Pulmonary edema/ARDS • Hypoglycemia • Hypotension/shock • DIC • Convulsions • Hemoglobinuria • Hyperparasitemia • Jaundice

Plasmodium vivax

Schuffner’s dots

Plasmodium falciparum

Maurer’s dots

Plasmodium malariae

Ziemann’s dots

Plasmodium ovale

James’ dots

•

•

•

•

•

•

•

•

•

•

•

•

High Yield Why Plasmodium Falciparum is most Pathogenic? • > 2,50,000 to 3,00,000/mL parasites of blood • 30–40% of total RBC’s are parasitized • Invasion of erythrocytes of all ages • Ability to do cytoadherence that leads to sequestration of the parasite • Extreme diversity of PfEMP1 antigen–which cannot be targeted by immune system • Massive cytokine production leading to end organ disease •

•

•

•

•

•

Figure 4: Schizont and ring forms of Plasmodium vivax (Courtesy: Dr S. Jamuna Rani, Associate professor of pathology, Tagore Medical College & Hospital, Chennai)

311

Unit 4     Parasitology

Chemoprophylaxis

Remember

Laboratory manifestations seen in severe falciparum malaria: • Hypoglycemia • Hyperlactatemia • Acidosis • Elevated serum creatinine • Elevated total bilirubin • Elevated liver enzymes • Elevated muscle enzymes • Elevated urate • Leukocytosis • Severe anemia • Coagulopathic features • Hyperparasitemia •

•

y For all chloroquine sensitive areas–prophylaxis with chloroquine y Chloroquine resistant areas–Atovaquone-Proguanil or Doxycycline y For P. vivax–Primaquine y

y

y

•

•

Treatment Failure

•

•

•

•

•

•

•

•

y Early treatment failure:  Development of danger signs or severe malaria on Day 1,2 or 3 in the presence of parasitemia  Parasitemia higher on Day 2 than Day 0 y Late clinical failure:  Development of danger signs on day 4 to day 28 y Late parasitological failure:  Presence of parasitemia on any day between day 7 and day 28 y





y



y



Table 4:  Diagnosis of malaria Standard methods for diagnosis of malaria

Features

Thick blood film

Sensitive method that will detect even 0. 0001% of parasitemia; but it needs experience to look the parasites.

Thin blood film

PfHRP2 dipstick test

Rapid and specific method but it can detect only 0. 05% parasitemia. Rapid and inexpensive tests. Help in field surveys. Detects only plasmodium falciparum

Plasmodium LDH dipstick test

Rapid tests; can detect all species

Microtube concentration methods (with Acridine orange staining)

Sensitive tests and superior to thick films; helps to identify and process large number of samples; but it needs fluorescence microscopy.

High Yield Initiatives to Reduce Malaria • Global Technical Strategy for malaria: (2016–2030) • Roll back malaria strategy by WHO: 1998 • National malaria control programme •

•

•

BABESIOSIS y Babesiosis is an emerging Tick borne zoonotic illness y Causative agent is Babesia; There are many species namely:  Babesia microti  Babesia bovis  Babesia divergens y Vector: Ixodid ticks; it can also be occasionally transmitted through transfusion of blood or blood products. y Babesia microti is the most common transfusion transmitted pathogen. y Morphological forms:  Merozoites  Trophozoites  Sporozoites y Sporozoites are the infective form y

y







y

y

y







y

Table 5:  Treatment Chloroquine sensitive–for all species

Chloroquine (dosage–10 mg/kg)

P. vivax and P. ovale–to avoid relapse

In addition to chloroquine + Primaquine (Primaquine should not be given in G6PD deficiency)

P. falciparum

Artesunate + Sulfadoxine/ Pyrimethamine

MDR P. falciparum

Second line drugs

312

Artemether-Lumefantrine (or) Artesunate–Mefloquine (or) Dihydroartemisinin–piperaquine Tetracycline Doxycycline Clindamycin

Definitive host: Hard ticks Intermediate host: Humans and animals

Pathogenesis y Pathogen is seen inside RBCs. Rupture of infected RBCs leads to anemia. y Ruptured RBCs generates cell debris and this gets accumulated in kidney causing renal failure. y Babesia microti infections are usually asymptomatic y Infection with Babesia bovis leads to hemolytic anemia, thrombocytic anemia an haemoglobinuria (resembling falciparum) y

y

y

y

Remember

Clinical suspicion for Babesiosis: • H/o travel to Babesia endemic area (north eastern and mid western USA) • H/o fever in late spring or summer • H/o blood transfusion within 6 months •

•

•

y y y y

y

y

y

y

Complete blood count shows anemia, thrombocytopenia, elevated reticulocyte counts and elevated LDH levels. LFT: Liver enzymes elevated Urine analysis: Hemoglobinuria, proteinuria, excess urobilinogen, elevated BUN, serum creatinine. Specific diagnosis is done by Peripheral smear–Demonstration of ring forms; pigments are absent; no gametocytes; maltese cross pattern (merozoites are arranged in tetrads) helps to differentiate from malaria

Chapter 41     Apicomplexa

Lab Diagnosis

Figure 5: Babesia in blood smear (Courtesy: CDC/Dr. George Healy) y Intraperitoneal inoculation in golden hamsters or gerbil y Serology: Indirect fluorescent antibody test y PCR (helps in confirmation) y

y

y

Table 6:  Treatment of Babesiosis Species and type of infection

DOC

Babesia microti – mild to moderate illness

Atovaquone + Azithromycin

Babesia microti – severe illness

Clindamycin + Quinine + Exchange transfusion

Babesia divergens

Immediate complete exchange transfusion + clindamycin + Quinine

313

Unit 4     Parasitology

314

MULTIPLE CHOICE QUESTIONS Plasmodium 1. Causative agent of malaria (Recent pattern 2017) a. Protozoa b. Mosquito c. Bacteria d. Virus 2. The scientist who discovered the transmission of malaria by anopheline mosquito (Recent pattern 2017) a. Laveran b. Paul muller c. Ronald ross d. Pampana 3. Malarial parasite was discovered by (Recent pattern 2017) a. Ronald ross b. Paul muller c. Laveran d. Pampania 4. In malaria, sexual cycle is (Recent pattern 2017) a. Sporozoite to gametocyte b. Gametocyte to sporozoite c. Occurs in human d. Responsible for relapse 5. In malaria pre-erythrocytic schizogony occurs in (Recent pattern 2018) a. Lung b. Liver c. Spleen d. Kidney 6. Infective form of mosquito in plasmodium falciparum (AIIMS 2017) a. Merozoites b. Sporozoites c. Gametocytes d. Trophozoites 7. Which form of the malarial parasite is present in saliva of an infective mosquito (Recent pattern 2018) a. Ring form b. Schizont c. Gametocyte d. Sporozoite 8. Transfusion associated malaria has a shorter incubation period because of: (AIIMS 2017) a. Trophozoites b. Sporozoites c. Female Gametocyte d. Merozoites 9. Malaria carriers contain (Recent pattern 2017) a. Trophozoite b. Gametocytes c. Merozoites d. Trophozoites 10. Recrudescences are seen in which malaria (Recent pattern 2018) a. P vivax b. P ovale c. P malariae d. P falciparum 11. Why are schizont and late trophozoite stages of plasmodium falciparum not seen in peripheral blood smear? (AIIMS 2017) a. They are sequestered in the spleen b. Due to adherence to the capillary endothelium,they are not seen in peripheral blood c. Due to antigen-antibody reaction and removal d. They are seen in mosquito blood

12. Which of the following is true about P. falciparum (Recent pattern 2017) a. James dots are seen b. Accole forms are seen c. Relapses are frequent d. Longest incubation period 13. Ziemann’s stippling of erythrocyte is caused by which species of plasmodium (Recent pattern 2018) a. Vivax b. Falciparum c. Malariae d. Ovale 14. Stages seen in peripheral smear of falciparum malaria (PGI May 2018) a. Schizont b. Gametocyte c. Accole d. Ring form e. Trophozoite 15. Stages of falciparum not seen in PBS is (Recent pattern 2017) a. Schizont b. Gametocyte c. Ring form d. Double ring 16. Malarial pigment is formed by (Recent pattern 2017) a. Parasite b. Bilirubin c. Hemoglobin d. Any of the above 17. A patient presents with fever. Peripheral smear shows band across the erythrocytes. Diagnosis is (AIIMS 2017) a. P. falciparum b. P. vivax c. P. ovale d. P. malariae 18. Band form of P. malariae is (Recent pattern 2017) a. Schizont stage b. Trophozoite stage c. Merozoite stage d. Gametocytic stage 19. Relapse in seen in:  (Recent Pattern Nov 2017) a. Plasmodium vivax and falciparum b. Plasmodium ovale and falciparum c. Plasmodium vivax and ovale d. Plasmodium vivax and malariae 20. All are true about Plasmodium knowlesi all except: (Recent Nov 2018) a. Monkeys are the host b. Chloroquine sensitivity c. Central Africa d. Quotidian malaria 21. Ziemann dots are seen in:  (Recent pattern Nov 2017) a. Plasmodium malariae b. Plasmodium ovale c. Plasmodium vivax d. Plasmodium falciparum 

Babesiosis 22. Babesiosis is transmitted by (Recent pattern 2017) a. Tick b. Mites c. Flea d. Mosquito 23. Maltese cross is characteristic feature of (Recent pattern 2017) a. Cryptococcus neoformans b. Babesia microti c. Blastomycosis d. Penicillium marneffei

26. Identify the vector that is involved in following diagnostic image: (Recent pattern 2018)





Chapter 41     Apicomplexa

24. Infective form of humans for babesiosis is (Recent pattern 2017) a. Sporozoites b. Merozoites c. Gametocytes d. Trophozoites 25. Which of the following is true about Malaria:  (PGI May 2017) a. Chloroquine resistance occurs in India b. Relapses is usual for vivax and ovale malaria c. Sexual cycle occurs in mosquito d. Not a public problem in India

a. Female anopheles mosquito b. Ixodid tick c. Glossina d. Reduvid bug

ANSWERS AND EXPLANATIONS 1. Ans.  (a) Protozoa

5. Ans.  (b) Liver

Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 111

Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 113

• Malaria is caused by Plasmodium–which is a protozoan parasite • Female anopheles mosquito acts as a vector

• Pre erythrocytic and exo erythrocytic schizogony occurs in liver • Erythrocytic schizogony occurs inside RBCs

•

•

2. Ans.  (c) Ronald ross

•

•

6. Ans.  (c) Gametocytes

Ref: T.B of Medical Parasitology–S.C.Parija – 4th edition– Page 109

Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 114

• Ronald Ross demonstrated the life cycle of malarial parasite in mosquito • He was given Nobel prize

• Infective form for man-Sporozoites • Infective form for mosquito – Gametocytes

•

•

•

•

3. Ans.  (a) Ronald ross Ref: T.B of Medical Parasitology–S.C.Parija–4 Page 109

th

edition–

• Already explained Q.2

7. Ans.  (d) Sporozoite Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 114 • Already explained Q.6 •

•

4. Ans.  (b) Gametocyte to sporozoite Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 114 • Asexual cycle occurs in humans • Sexual cycle occurs in female anopheles mosquito • Gametocytes are in the infective form for anopheles from man; these gets converted to sporozoites and become infective for humans

8. Ans.  (a) Trophozoites Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 120

Mosquito borne malaria

Blood transfusion malaria

• Infective stage is sporozoite

• Infective stage is trophozoite

• Long incubation period

• Short incubation period

• Pre-erythrocytic and exoerythrocytic schizogony present

• Pre-erythrocytic and exoerythrocytic schizogony absent

•

•

•

Gametocytes → Sporozoites (Mosquito)

•

•

•

Sporozoites → Gametocytes (Man)

•

•

•

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Unit 4     Parasitology

9. Ans.  (b) Gametocytes

14. Ans.  (b) Gametocyte; (c) Accole; (d) Ring form

Ref: T.B of Parasitology–Chaterjee–12th edition–Page 113 • In endemic areas, malarial parasites are residing in humans and act as as carriers (reservoirs) • Carriers harbors gametocytes •

•

Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 114,115 • The diagnostic forms of falciparum that are seen in peripheral smear are early ring forms and gametocytes • Rings forms when seen attached to the margin of a red blood cell is called as accole •

•

10. Ans.  (d) P falciparum Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 119

15. Ans.  (a) Schizont Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 114

• Relapse is seen in P.vivax and P.ovale • Recrudescence is seen in P.falciparum •

•

• Already explained Q.14 •

11. Ans.  (b) Due to adherence to the capillary endothelium, they are not seen in peripheral blood Ref: T.B of Medical Parasitology–S.C.Parija–4 Page 114;

16. Ans.  (c) Hemoglobin

edition–

Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 115

• In P. falciparum, the erythrocytic schizogony takes place within 48 hours • This takes places in the capillaries of the internal organs • Hence the schizonts and merozoites are not seen in peripheral blood • Only young ring forms and gametocytes can be seen

• Malarial parasite resides in RBC and feeds on the hemoglobin • These hemoglobin feed becomes pigments inside the parasite

th

•

•

•

•

12. Ans.  (b) Accole forms are seen Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 112 • P.falciparum parasite attaches to the margin or edge of the host cell, nucleus and near the cytoplasm too • It is known as accole form •

•

•

•

17. Ans.  (d) P.malariae Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 112 • Band form trophozites are characteristic of P malariae •

18. Ans.  (b) Trophozoite stage Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 112 • Already explained Q.17 •

19. Ans.  (c) Plasmodium vivax and ovale Ref: T.B of Medical Parasitology – Parija – 4th ed – Page 173 • True relapse – caused by hypnozoites • Specific to P.ovale and P.vivax • Due to re emergence of blood stages from latent hypnozoites •

•

•

20. Ans.  (c) Central Africa Ref: Harrison T.B of Internal Medicine – 19th edition – Page 1368-1371 • Plasmodium knowlesi is called as monkey malarial parasite • This species is particularly seen on the island of Borneo and, to a lesser extent, elsewhere in Southeast Asia, where the main hosts, long-tailed and pig-tailed macaques, are found. • It may cause severe malaria as indicated by its asexual erythrocytic cycle of about 24 hours, with an associated fever that typically occurs at the same frequency (i.e. the fever is quotidian). • Treatment of Plamodium knowlesi uncomplicated malaria is: Chloroquine (10 mg of base/kg stat followed by 5 mg/ kg at 12, 24, and 36 h or by 10 mg/kg at 24 h and 5 mg/kg at 48 h) •

 Accole form of falciparum parasite 13. Ans.  (c) Malariae Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 112 P. vivax

Schuffner’s dots

P. falciparum

Maurer’s dots

P. malariae

Ziemann’s stippling

P. ovale

James dots

•

•

•

316

24. Ans.  (a) Sporozoites

Ref: Medical Parasitology – Arora – 3rd edition – Page 75 • Infected red blood cells by the Plasmodium species undergoes alteration in size and shape • In P. malariae – the RBC are of normal size with fine stippling seen on prolonged staining – this stippling is called Ziemann’s dots •

Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 138 • Infective form for humans is sporozoites •

•

Plasmodium vivax

Schuffner’s dots

Plasmodium falciparum

Maurer’s dots

Plasmodium malariae

Ziemann’s dots

Plasmodium ovale

James’ dots

22. Ans.  (a) Tick

25. Ans: (a, b, c) (a) Chloroquine resistance occurs in India; (b) Relapses is usual for vivax and ovale malaria; (c) Sexual cycle occurs in mosquito • Already explained Q.4 and Q.10 •

26. Ans.  (b) Ixodid tick

Chapter 41     Apicomplexa

21. Ans.  (a) Plasmodium malariae

Ref: Review of Medical Microbiology and Immunology – 13th edition – Page 975 • The above figure showing maltese cross pattern of Babesia • Babesiosis is a Tick borne zoonotic illness • Pathogen is seen inside RBCs • Sporozoites are the infective form • Peripheral smear: Demonstration of ring forms; pigments are absent; no gametocytes; maltese cross pattern (merozoites are arranged in tetrads) helps to differentiate from malaria •

Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 138 • Definitive host is hard ticks (vector) • Intermediate host is humans and animals •

•

•

•

•

•

23. Ans.  (b) Babesia miroti Ref: T.B of Medical Parasitology – S.C.Parija – 4th edition – Page 141 • In peripheral smear of babesiosis – merozoites are arranged in tetrads and called as maltese cross pattern • This is not so common condition •

•

317

42

Toxoplasma, Ciliate Protozoa

INTRODUCTION

Diagnostic forms Features • These are tissue cysts or resting form in humans • Seen in chronic stage • Commonly seen in brain, skeletal and heart muscles

Oocysts

• Seen in cat and other felines • Not seen in humans

•

•

Bradyzoites

•



•





y

•

y

y

y Toxoplasma is an obligate intracellular parasite that lives in the REC and nucleated cells y Unique feature of this parasite is that all the stages are infective to humans y Morphological forms:  Tachyzoites  Tissue cysts or Bradyzoites  Oocysts

y Definitive host: Cats (Sexual cycle occurs) y Intermediate host: Humans and other mammals (Asexual cycle occurs) y Infecting form: Oocysts through cat feces y

Table 1:  Features of diagnostic forms

y

•

•

• Actively multiplying form in humans • Seen in acute stage • This form causes invasion in all the cells except RBCs •

Tachyzoites

y

Diagnostic forms Features

(Contd...)

Figure 1:  Life cycle of Toxoplasma

y Congenital toxoplasmosis: (Mother to fetus)  When infection occurs during pregnancy–it gets transmitted to fetus (man to man transmission occurs like this)  Classical triad is: (3Cs) y





CILIATE PROTOZOA Balantidium Coli y It is the largest human protozoa y Site of infection is large intestine of humans, pigs, monkeys y Morphological forms:  Trophozoite: Š Invasive form Š Oval shaped with cilia all around it Š It has a micro and macro nucleus Š It ingests bacteria, RBC and fat  Cyst: Š Infective form Š Round in shape Š Resting stage y Life cycle occurs in single host usually in pigs (natural host); Humans are accidental host y Humans acquire infection by ingestion of cysts contaminating food. y It causes ciliate dysentry–diarrhea with blood and mucous (acute cases); in chronic cases it presents as diarrhea alternative with constipation; y Balantidium invades the mucosa and submucosa causing ulcers named as Balantidum ulcers. They are found in cecum, ascending colon and rectum. These ulcers do not invade the muscular layer. Main complication of these ulcers is perforation. y

y

y



Š

• Chorioretinitis •

Š

Š

• Cerebral calcifications •

• Convulsions •

Š



Š

Š

y Acquired toxoplasmosis: (Ingestion of undercooked meat, ingestion of food contaminated with cat feces, blood transfusion, organ transplantation, accidental inoculation)  Usually self-limiting in immunocompetent hosts–presents as lymphadenopathy  In immunocompromised patients like in AIDS patients– presents as life threatening disease; presents as encephalitis  Ocular toxoplasmosis infection presents as choroiretinitis y







LAB DIAGNOSIS y Tissue samples like bone marrow aspiration shows tachyzoites or bradyzoites depends upon the clinical stage y Stain used is PAS or Giemsa–comma shaped tachyzoites seen y Animal inoculation–intraperitoneally into mice y Serological test: ELISA, IFA, IHA and latex agglutination tests y Sabin Feldman dye test:  Gold standard test for toxoplasmosis (highly sensitive and specific test)  Done only in reference laboratories  Test serum (contains antibodies) + live tachyzoites – inactivated and killed (because of complement mediated lysis)  This is then stained with alkaline blue–which shows dead tachyzoites as colorless and distorted–shows the test is positive y PCR helpful in detecting congenital infections in utero (by amniotic fluid sampling) y

y

y

y

y



Š

y

y

Chapter 42     Toxoplasma, Ciliate Protozoa

CLINICAL FEATURES

y

y

Lab Diagnosis y Microscopic examination of stool–wet mount–shows football revolving motility of the trophozoites with characteristic cilia around it y Balantidium grows well in:  Robinson’s medium  Balamuth’s medium  Dobell’s medium  Jone’s medium y

y















y

TREATMENT y y y y y

y

y

y

y

y

Pyrimethamine Sulfadiazine Clindamycin Atovaquone Azithromycin

Figure 2: Trophozoite of Balantidium coli (Courtesy: CDC/ Dr L.L.A. Moore, Jr.) y Biopsy from the large intestine and scrapings from ulcers also shows the trophozoite y

Treatment y Tetracycline is the DOC; alternatively metronidazole can be given. y

319

Unit 4     Parasitology

MULTIPLE CHOICE QUESTIONS











1. Intermediate host for toxoplasma are all except: (Recent pattern 2017) a. Human b. Sheep c. Cat d. Pig 2. Oocyst of toxoplasma is found in (Recent pattern 2018) a. Cat b. Dog c. Mosquito d. Cow 3. Most common clinical feature of toxoplasmosis in an immunocompetent adult: (Recent pattern 2018) a. Encephalitis b. Lymphadenopathy c. Chorioretinitis d. Glaucoma 4. Sabin feldman Dye test is used to demonstrate infection with(Recent pattern 2017) a. Filaria b. Toxoplasma c. Histoplasma d. Ascaris 5. Congenital toxoplasmosis false is (AIIMS 2017) a. IgA is better than IgM in detection b. Diagnosed by detection of IgM in cord blood c. IgG is diagnostic d. Cannot be diagnosed by antibodies 6. Which of the following is true regarding transmission of Toxoplasma(Recent pattern Nov 2017) a. Vertical b. Ingestion of uncooked food with cyst c. Organ transplantation d. All of the above 7. In Toxoplasmosis the oocyst seen in________and pseudocyst is seen in_______ respectively (Recent pattern Nov 2017) a. Tissue, felines b. Human, cats c. Cats, human d. Tissue, feces

8. Sabin Feldman dye test is used for diagnosis of: (Recent pattern 2018) a. Leishmaniasis b. Echinococcosis c. Toxoplasmosis d. Balantidiasis 

Balantidium Coli

9. All are true regarding Balantidium coli except: a. Humans get infection from pigs (Recent pattern 2017) b. Trophozoite is the infective form c. It causes ulcers in the caecum d. Only ciliate that infect humans 10. Revolving motility is seen in: (Recent pattern 2017) a. Balantidium coli b. Trichomonas vaginalis c. Balamuthia mandrillaris d. Dientamoeba fragilis 11. Largest human protozoan is (Recent pattern 2017) a. Entamoeba histolytica b. Entamoeba gingivalis c. Balantidium coli d. Naegleria fowleri 12. Fecal smear showed the following finding; Identify: (Recent pattern Nov 2017) 



a. Balantidium coli c. Cryptosporidium

b. Entamoeba dispar d. Giardia

ANSWERS AND EXPLANATIONS 1. Ans.  (c) Cat Ref: T.B of Medical Parasitology–S.C.Parija–4 Page 159

3. Ans.  (b) Lymphadenopathy edition–

Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 162

• Definitive host is cat and other felines • Intermediate hosts are humans, mouse, sheep and pigs

• Clinical presentation of toxoplasmosis is based on the immunity of the host • In immunocompetent hosts–it is benign condition and self-limiting–most common presentation is cervical lymphadenopathy

th

•

•

•

•

2. Ans.  (a) Cat Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 159 • There are three morphological forms in toxoplasma– tachyzoites, bradyzoites and oocysts • Sexual multiplication occurs in cat–oocyts are excreted in feces • Asexual multiplication occurs in humans and other animals–tachyzoites are active replicating forms • Bradyzoites are resting forms •

•

•

320

•

4. Ans.  (b) Toxoplasma Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 165 • Sabin Feldman dye test–used for the diagnosis of toxoplasmosis • After infection is acquired–antibodies appear in the body following 2–3 weeks •

•

•

•

•

•

•

5. Ans.  (c) IgG is diagnostic Ref: T.B of Medical Parasitology–S.C.Parija–4 Page 166

• Sabin Feldman dye test – used for the diagnosis of toxoplasmosis • After infection is acquired – antibodies appear in the body following 2–3 weeks • When live tachyzoites (active multiplying stage) of Toxoplasma is treated with serum of the patient (has antibodies) and alkaline methylene blue – the antibodies inactivate the live tachyzoites • This looks as thin, distorted and colourless even in presence of alkaline blue as they are dead • If more than 50% are unstained then the test is positive • Gold standard method • Done only in reference laboratories •

•

•

•

•

th

edition–

• IgG antibodies can cross the placenta and so they are not helpful in any of the congenital infections; it is from maternal blood • IgM is diagnostic of any of the congenital infections •

•

•

•

9. Ans.  (b) Trophozoite is the infective form Ref: T.B. of Medical Parasitology–S.C.Parija–4th edition– Page 171 • Natural host for Balantidium coli is pigs • Infective form is cyst and invasive form is trophozoites

Chapter 42     Toxoplasma, Ciliate Protozoa

• When live tachyzoites (active multiplying stage) of Toxoplasma is treated with serum of the patient (has antibodies) and alkaline methylene blue–the antibodies inactivate the live tachyzoites • This looks as thin, distorted and colourless even in presence of alkaline blue as they are dead • If more than 50% are unstained then the test is positive • Gold standard method • Done only in reference laboratories

•

6. Ans.  (d) All of the above Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 161 • Humans acquire infection by ingestion of food contaminated with sporulated oocysts and from uncooked meat containing tissue cysts • Humans are the intermediate host and cats are the definitive host • Other modes of acquiring infection are ƒ Blood transfusion ƒ Organ transplantation ƒ Transplacental transmission ƒ Accidental inoculation of tachyzoites in laboratory •

•

•

ƒ

•

10. Ans.  (a) Balantidium coli Ref: T.B. of Medical Parasitology–S.C.Parija–4th edition– Page 174 • Motile trophozoites are seen only in direct wet mount examination of fresh liquid stools • Rapid revolving motility (also called as football motility) is seen in Balantidium coli • In Trichomonas vaginalis it is called as twitching motility •

•

•

ƒ

ƒ

ƒ

7. Ans.  (c) Cats, human Ref: T.B. of Medical Parasitology–S.C.Parija–4th edition– Page 159 • There are three stages in toxoplasma gondii—Tachyzoites, bradyzoites and oocysts • Tachyzoites–active, multiplying trophozoites seen during acute infection–seen in skeletal muscles, cardiac muscles and brain • Bradyzoites–resting form of the parasite-called as tissue cysts • Oocysts are present only in cat and other felines–not in humans • Groups of proliferating tachyzoites inside a host cell is called as pseudocyst •

•

•

•

•

8. Ans.  (c) Toxoplasmosis

11. Ans.  (c) Balantidium coli Ref: T.B. of Medical Parasitology–S.C.Parija–4th edition– Page 171 • The only ciliate and largest human protozoan is Balantidium coli •

12. Ans.  (a) Balantidium coli Ref: T.B. of Medical Parasitology – Parija – 4th ed – Page 173 • • • • • • • •

•

•

•

•

•

•

•

•

Identification clue is: cilia all over the trophozoites Balantidium coli – only ciliate known to infect humans Two forms: Trophozoite and cyst Invasive form is Trophozoite – oval in shape – cilated – anterior end has mouth; macro and micro nucleus seen Trophozoite is capable of ingesting bacteria, RBC and fats Contains many vacuoles Causes ciliate dysentery or balantidiasis DOC: Tetracycline

Ref: T.B. of Medical Parasitology – S.C.Parija – 4th edition – Page 165

321

43

Coccidian Intestinal Parasites

COCCIDIAN PARASITES

y

y

y

y Cryptosporidium y Cyclospora y Isospora

CRYPTOSPORIDIUM











y

y

y

y Cryptosporidium parvum is an intestinal parasite that causes diarrhea (especially in immunocompromised patients) y Site–small intestine y Morphological forms of this parasite are:  Oocysts  Sporozoite  Trophozoite  Merozoite  Microgamont  Macrogamont y Infective form–Oocysts; there are two types of oocysts  Thick walled oocysts that cause infections to others  Thin walled oocysts that cause autoinfection 



y



Figure 1: Oocysts of Cryptosporidium parvum (Courtesy: Department of microbiology, Chengalpattu Medical College, Tamil Nadu)

y Feco oral route y Zoonotic infections (contamination of oocysts from animal feces in animal products) y It does not cause invasion after the mucosal layer in small intestine–hence the diarrhea is non inflammatory/profuse/ non bloody y The presentation of illness depends upon the immunity of the individual  Immunocompetent persons–self-limiting illness  Immunocompromised patients (AIDS)–life threatening diarrhea 

y y y y y

y



CYCLOSPORA y Species that cause intestinal infection is Cyclospora cayetanensis y It affects the small intestine y Humans get the infection by feco oral route through contamination with oocysts y Humans are the only source for infection y Clinical condition resembles that of cryptosporidiosis y

y y y

y

Treatment y Anti diarrheal agents have no effect y Some benefits are there for paromomycin and spiramycin y In AIDS patients–Nitazoxanide has an effect

y

Mode of Infection

y

y

y

Š

Š

Lab Diagnosis  

Stool microscopy by acid fast staining–shows oocysts Identification keys are: Š Round to ovoid shape Š 8 to 10 um size Š Variable acid fast Š Auto fluorescence Š



Š



Š

Š

Š

y

y

y

y Stool microscopic examination–oocysts are seen y Acid fast staining using 0.5% H2SO4 (modified acid fast staining) y Identification keys are: Š Oval shaped Š 4–5 um in diameter Š Acid fast oocysts Š Uniformly acid fast y ELISA to detect copro-antigen in stool y Indirect fluorescent antibody tests y PCR

Š

Diagnosis

Treatment y Self-limiting condition; if severe–TMP-SMX can be given y

Figure 3: Oocyst of Isospora Belli (Courtesy: Dr A Vijayalakshmi, HOD of microbiology. Chengalpattu Medical College, Chengalpattu, Tamil Nadu)

Treatment

ISOSPORA

y TMP-SMX is the DOC

y Cystoisospora belli is a parasite that infects the small intestine of humans y Humans gets infection by feco oral route through contamination with mature oocysts y It causes diarrhea–which is watery, profuse and foul smelling y Resembles that of cryptosporidiosis

Table 1: Identification of coccidian parasites Coccidian Identification features parasites

y

y

y

y

y

 

Cyclospora cayetanensis

Lab Diagnosis 



Microscopic examination of acid fast staining of the fecal smears shows Š Elliptical shaped oocysts; boat shaped (Variations in shape occurs) Š Acid fast oocysts Š 20–33 um* 10–19 um size Š Each oocyst contains two sporoblasts–each has four sporozoites Š Autofluorescence Š

Š

Š

Š

Chapter 43     Coccidian Intestinal Parasites

Figure 2: Oocysts of Cyclospora (Courtesy: CDC/DPDx-Melanie Moser)

Oocysts are of spherical or oval; Size is around 8–10 um; It has two sporocyts; Each sporocyts has 2 sporozoites Acid fast oocyst; Partially acid fast

Cryptosporidium Oocysts are of spherical or oval; Size is parvum around 4–5 um; It has 4 sporozoites Acid fast oocyst Cystoisospora belli

Oocyst looks ellipsoidal (boat shaped); 20-33*10-19 um; Each sporocyts has 2 sporozoites; Acid fast

Š

323

Unit 4     Parasitology

MULTIPLE CHOICE QUESTIONS Coccidian Parasites







1. In humans, cryptosporidiosis present as: (Recent pattern 2017) a. Meningitis b. Diarrhea c. Pneumonia d. Hepatitis 2. A HIV patient with malabsorption fever, chronic diarrhea, with acid fast positive organism. What is the causative agent? (AIIMS 2017) a. Giardia b. Microsporidia c. Isospora d. E. histolytica 3. A HIV positive individual having diarrheal episodes. Stool examination reveals a oocyst of size 8-10 um. What could be the diagnosis? (Recent pattern Nov 2017) a. Cyclospora b. Cryptococcus c. Cryptosporidium parvum d. Isospora 4. Drug of choice for cryptosporidiosis is: (Recent pattern 2018) a. TMP-SMX b. Nitazoxanide c. Primaquine d. Niclosamide



5. Auto infection is seen in: (Recent pattern 2017) a. Cryptosporidium b. Cyclospora c. Cystisospora d. Microsporidia 6. A kinyoun positive oocyst was isolated from a HIV patient with diarrhea; Identify the organism:  (AIIMS May 2018) 



a. Cystisospora c. Cryptosporidium

b. Cyclospora d. Microspora

ANSWERS AND EXPLANATIONS 1. Ans.  (b) Diarrhea Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 147 • Cryptosporidiosis presents the clinical manifestations depending upon the immunity of the individual • Immunocompetent individuals–it presents as selflimiting diarrhea • Immunosuppressed patients–life threatening diarrhea •

•

• In patients with HIV–self-limiting diarrheal episodes are more common with Cyclospora • While Cryptosporidium can cause life endangering diarrhea in immunocompromised patients •

•

Coccidian parasites

Identification features

Cyclospora cayetanensis

Oocysts are of spherical or oval; Size is around 8–10 um; It has two sporocyts; Each sporocyts has 2 sporozoites Acid fast oocyst; Partially acid fast

Cryptosporidium parvum

Oocysts are of spherical or oval; Size is around 4–5 um; It has 4 sporozoites Acid fast oocyst

Cystoisospora belli

Oocyst looks ellipsoidal (boat shaped); 20-33*10–19 um; Each sporocyts has 2 sporozoites; Acid fast

•

2. Ans.  (c) Isospora Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 155 • Acid fast parasites that causes intestinal infections and diarrhea in immunosuppressed patients are: ƒ Cryptosporidium parvum ƒ Cyclospora cayetanensis ƒ Cystisospora belli •

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3. Ans.  (a) Cyclospora

324

Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 146-155

4. Ans.  (b) Nitazoxanide Ref: T.B of medical parasitology–S.C.Parija–4th edition– Page 151

•

•

•

5. Ans.  (a) Cryptosporidium Ref: T.B of medical parasitology–S.C.Parija–4th edition– Page 145 • In Cryptosporidium parvum–there are two types of oocysts ƒ Thick walled oocysts that cause infections to others ƒ Thin walled oocysts that causes autoinfection •

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• The thin walled oocysts which are seen in the intestinal lumen releases the sporozoites and they are responsible for auto infection •

6. Ans.  (a) Cystisospora Ref: Medical parasitology – Arora – 3rd edition – Page 97 • Clinical picture of HIV with diarrhea clearly suggests that patient is suffering from cocciean parasite induced infection • Oocysts seen in the image is identified as Cystisospora •

•

Chapter 43     Coccidian Intestinal Parasites

• Anti diarrheal agents have no effect in the treatment of cryptosporidial diarrhea • Some benefits are there for paromomycin and spiramycin • In AIDS patients–Nitazoxanide has a symptomatic relief and effective

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44

Helminthology Cestodes y Infective form: Plerocercoid larva y Mode of infection: Ingestion of raw or inadequately cooked fish

y

y

y Helminths include worm like parasites y They are classified as:  Platyhelminths (flat)–which includes Cestodes and Trematodes  Nemathelminths (Thread)–which includes Nematodes

y

y

INTRODUCTION



Remember

•

y y y y

• • • • •

Tapeworm y

y These group of worms have a scolex or head; with a uterus having branches and genital pores y Larval forms of tapeworms are:  Cysticercus  Coenurus  Unilocular hydatid cyst  Multilocular hydatid cyst 







y

• •

y Adult worm absorbs the Vit B12 from the stomach and causes B12 deficiency anemia–pernicious anemia y Characteristics of anemia is hyperchromic, macrocyticmegaloblastic anemia with thrombocytopenia and mild leukopenia y Diagnosis is by demonstration of eggs in stool microscopy y Treatment: Praziquantel and Niclosamide

•

•

•

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• Ascaris lumbricoides • Necator americanus • Strongyloides stercoralis • Trichuris trichiura • Ancylostoma duodenale • Enterobius vermicularis • Trichinella spiralis •

•

•

•

Blood trematodes: • Schistosoma haematobium • Schistosoma mansoni • Schistosoma japonicum Hepatic trematodes: • Fasciola hepatica • Fasciola gigantica • Clonorchis sinensis • Opisthorchis sp Intestinal trematodes: • Fasciolopsis buski • Heterophyes heterophyes • Metagonimus yokogawai • Watsonius watsoni • Gastrodiscoides hominis Lung trematode: • Paragonimus westermani •

•

•

•

•

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• Diphyllobothrium latum • Taenia solium • Taenia saginata • Echinococcus granulosus • Hymenolepis nana

Nematodes

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Table 1:  Various Helminths Cestodes Trematodes

•



•

• It is the only human infecting tapeworm that has a life cycle in aquatic life • It is the only cestode that has two intermediate hosts • Only cestode which has operculated egg

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CESTODES Diphyllobothrium Latum



y

Table 2:  Features of T.saginata and T.solium Features

T.saginata

T.solium

Name

Beef or unarmed tapeworm

Pork or armed tapeworm

Habitat

Jejunum

Upper jejunum

Adult worm

Tape like 5-10 m in length

Tape like 2-3 m in length

Scolex

Large, quadrate with 4 suckers-pigmented Head without rostellum and hooklets

Small, globular with 4 suckers Head with rostellum armed with double row of hooklets





y

y

y

y

y

y

y Fish tapeworm y Longest parasite seen in the intestine (jejunum and ileum) of humans y Egg: Bile stained, operculated y Larvae: Coracidium, Procercoid larva and Plerocercoid larva y Definitive host: Human beings y Intermediate host:  First–Cyclops  Second–Fresh water fish

y Two species of Taenia are:  Taenia saginata  Taenia solium 

•

•

Taenia

(Contd...)

T.saginata

T.solium

Neck

Long and thin

Short and thick

Proglottids

1000-2000

800-900

Vaginal sphincter

Present

Absent

Ovary

Two without accessory lobes

Two with accessory lobes

Clinical manifestations

Mostly asymptomatic intestinal taeniasis; presentation may be abdominal pain and nausea

Larval form causes intestinal taeniasis; Egg causes cysticercosis;

Egg of Tapeworm y y y y y

y

y

y

y

y

Bile stained Outer–thin layer Inner–oncosphere Embryo–Hexacanth embryo Egg of T. saginata and T. solium cannot be differentiated

Figure 1:  Figure showing egg of Taenia (Courtesy: CDC/Dr. Mae Melvin)

Chapter 44     Helminthology Cestodes

Features

Infective Form y Egg of T. saginata–infective to cattle only y Egg of T. solium–infective to pigs and human beings y Larvae: Cysticercus bovis (T.saginata) and Cysticercus cellulosae (T.solium)–infective to human beings y

y

y

Figure 2:  Life cycle of Taenia solium

Cysticercosis

Diagnostic Criteria for Human Cysticercosis

y Fatal systemic condition seen in T.solium y Larvae is seen in muscle, subcutaneous tissue, eyes and brain y Neurocysticercosis–most common cause for seizures in India y Three important features of neurocysticercosis are:  Convulsions  Intracranial hypertension  Psychiatric disturbances y Other areas are: Thigh muscles, ocular cysticercosis y Diagnosis: CT, MRI-calcifications; Serological tests are most helpful y Treatment: Praziquantel, Albendazole, Niclosamide

(Source: Modified from Harrison`s principles of internal medicine–19th edition vol 2–Page 1431)

y

y

y

y





Absolute Criteria y Demonstration of cysticerci in histology or microscopy y Direct visualization of the organism in fundoscopy in the eye y Neurological features of cystic lesions as seen in radiography y

y

y



y

y

y

Major Criteria y Radiological features suggestive of Neurocysticercosis (NCC) y Demonstration of antibodies in ELISA for cysticerci y

y

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Unit 4     Parasitology

y Spontaneous resolution or resolution following therapy of the cystic lesions y

Minor Criteria y y y y

Lesions similar to NCC in imaging studies Clinical features suggestive of NCC Demonstration of antigens or antibodies in CSF for cysticerci Any evidence of cysticerci outside CNS

y

y

y

y

Table 3:  Lab diagnosis of Taenia infections Taenia solium

Taenia saginata

• Intestinal taeniasis– • Intestinal taeniasis– diagnosed by demonstration diagnosed by demonstration of gravid proglottids in the of gravid proglottids in the feces; feces; • As eggs are morphologically • As eggs are morphologically similar with T.saginata similar with T.saginata it does not help in it does not help in confirmatory diagnosis confirmatory diagnosis •

•

• Neurocysticercosis– diagnosed by imaging methods like CT and MRI which shows the number, site and stage of cysticerci (calcifications) • CT is best method • Serodiagnosis like ELISA helps to identify the antibodies in serum and CSF • Postmortem diagnosis by demonstration of cysticerci in the brain biopsy tissue

•

•

•

•

•

Eggs of both Taenia saginata and solium can be differentiating by acid fast staining

Echinococcus Granulosus y y y y

y

y

y

y

Also known as dog tape worm; it is a zoonotic disease Definitive host: Dog Intermediate host: Sheep, cattle, humans Infective form: Eggs

Hydatid Cyst: (Larval Stage of the Tapeworm) It represents the scolex of adult worm Outer layer–Ectocyst–Cuticular layer Inner layer–Endocyst–Germinal layer Brood capsules are formed from germinal layer When brood capsules are not formed–called as acephalocysts This causes unilocular hydatid disease Hydatid fluid:  Antigenic in nature and highly toxic; if when secreted into the human body by rupture of cysts–it leads to anaphylaxis  Hydatid sand indicates brood capsules and protoscolices which are floating in the hydatid fluid gives an appearance of sand grains; y Hydatid cyst can occur anywhere in the body; M/c is liver followed by lung y Lab diagnosis:  Wet mount of the hydatid fluid: Demonstration of protoscolices  Casoni’s test–immediate hypersensitivity test  Antigen detection by ELISA  Antibody detection has low sensitivity  X-ray–helps in diagnosis of lung hydatid cyst, USG–helps in diagnosis of liver and other organs y y y y y y y

y

y

y

y

y

y

y





y

y







Eggs of T.saginata are acid fast Eggs of T.solium are non acid fast





•

Treatment: • Praziquantel • Niclosamide • Albendazole • Praziquantel is the ideal DOC + needs to be given with steroids to avoid inflammatory reactions caused by dead cysticerci •

•

Treatment: • Praziquantel • Niclosamide •

•

•

•

Figure 3:  Hydatid cyst (Courtesy: CDC/Dr Mae Melvin)

Table 4:  Species of Echinococcus Species

Disease

Echinococcus Multilocularis

Echinococcus granulosus

Hydatid disease or Cystic echinococcosis

y y y y

Echinococcus multilocularis Echinococcus oligarthus

328

y Main modality of treatment is surgical removal of the cyst followed by albendazole or mebendazole for a minimum of three months y

Echinococcus

Echinococcus vogeli

Alveolar echinococcosis or malignant hydatid disease

Definitive host–foxes Intermediate host–Rodents Accidental host–Humans Mode of infection–humans ingesting food that is contaminated with feces of foxes y It causes multilocular cysts in liver–that does not have membrane and brood capsules–because of that the larvae spreading and causes liver necrosis y

y

y

y

y

Polycystic echinococcosis

y

Table 5:  Differences between E.granulosus and E.multilocularis Echinococcus granulosus

Echinococcus multilocularis

• Larvae has unilocular cyst • Definitive host–Dogs • Intermediate host–Humans, sheep, cow • Causes hydatid cyst

• Larvae has multilocular cyst • Definitive host–Fox • Intermediate host–Humans and oriental rodents • Causes malignant hydatid disease

•

•

•

•

•

•

•

•

Figure 4: Egg of Hymenolepis nana (Courtesy: CDC/Dr. Moore)

Hymenolepis Species y Two important species that cause infections in humans are:  Hymenolepis nana  Hymenolepis diminuta y





Hymenolepis Nana The smallest intestinal cestode that infects human is H.nana Hence it is called as dwarf tapeworm Definitive host: Humans, rats and mice Intermediate host: Not needed Mode of infection: Ingestion of eggs The eggs that gets released from the host may hatch out in the intestinal lumen and releases the embryo–which invades the intestinal villi–it is called as internal autoinfection; Due to bad hygiene–by feco oral route–own eggs cause infection called as external autoinfection; y It most commonly affects children; mostly asymptomatic; Heavy infections may cause diarrhea or abdominal pain y Egg of H.nana in stools is the identification method  Colorless, non bile stained  Outer membrane is thin and inner membrane is embryophore which encloses an oncosphere with three pairs of hooklets  Thread like polar filaments is present on each two poles y Treatment: Praziquantel is the DOC y y y y y y

y

y

Hymenolepis Diminuta y y y y y y

Usually affects rats and mice; humans are affected rarely It is called as rat tapeworm Definitive host: Rats, Mice, Humans Intermediate host: Cockroach, beetles, flea Infection is most common in children; asymptomatic Demonstration of eggs in stool is diagnostic:  Round in shape and larger than H.nana  Polar filaments are absent

y

y

y

y

y

y



y



y

y

Chapter 44     Helminthology Cestodes

y Because of the spread of larvae to other sites like brain–it is called as malignant hydatid disease

Table 6:  Differences between H.nana and H.diminuta

y

y

y







y

Hymenolepis nana

Hymenolepis diminuta

• Egg is smaller in size • Polar filaments are present

• Egg is larger than nana • Polar filaments are absent

•

•

•

•

Dipylidium Caninum Parasite of dogs, cats and foxes It is called as double pored dog tapeworm Definitive host: Dogs, cat and humans (accidental) Intermediate host: Flea and louse Diagnosis is by: Demonstration of egg capsules that has eggs in stools y DOC: Niclosamide y y y y y

y

y

y

y

y

y

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Unit 4     Parasitology

330

MULTIPLE CHOICE QUESTIONS 1. Definitive host for Taenia saginata: (Recent pattern 2017) a. Man b. Pig c. Cattle d. Cow 2. Man is both intermediate and definitive host for: (Recent pattern 2018) a. T. solium b. T. saginata c. D. latum d. Dicroftis hominis 3. Consumption of uncooked pork is likely to cause which of the following helminthic diseases: (AIIMS 2017) a. Taenia saginata b. Taenia solium c. Hydatid cyst d. Trichuris trichura 4. Commonest parasite of CNS in India is: (Recent pattern 2018) a. Schistosomiasis b. Cysticercosis c. Trichinella spiralis d. Hydatid cyst 5. Intermediate host for hydatid disease: (AIIMS 2017) a. Man b. Dog c. Cat d. Foxes 6. Most common site for hydatid cyst: (Recent pattern 2018) a. Lung b. Liver c. Brain d. Kidney 7. Least common site of calcified hydatid cyst is: (Recent pattern 2018) a. Lung b. Mediastinum c. Extraperitoneal site d. Liver 8. The following infection resembles malignancy: (Recent pattern 2017) a. Echinococcus granulosa b. E. multilocularis c. E. vogeli d. E. oligarthus 9. Arc C-5 in countercurrent electrophoresis of serum is diagnostic of: (Recent pattern 2017) a. Cysticercosis b. Cryptococcosis c. Hydatidosis d. Brucellosis 10. Hydatid disease is caused by: (Recent pattern 2018) a. Echinococcus b. Tapeworm c. Ascaris d. Clonorchis 11. Dwarf tapeworm refers to: (Recent pattern 2017) a. Echinococcus b. Loa loa c. Hymenolepis nana d. Schistosoma mansoni 12. All are true about Taenia solium except: (AIIMS 2017) a. Egg of T.solium causes neurocysticercosis (NCC) b. Egg of T.solium is acid fast c. Praziquantel is the DOC of NCC d. NCC is the most common cause of seizures in India 13. All the following spread by auto infection except? (Recent pattern 2018) a. Taenia solium b. Strongyloides stercoralis c. Hymenolepis nana d. Hymenolepis diminuta

14. The following is the ovum of an helminth. What is true about helminth? (AIIMS 2017)



a. Transmission is through ingestion of infected pork b. Both adult and larval stage are seen in humans c. The helminth causes a transient self-resolving infection in humans d. Drug of choice for this condition is albendazole 15. Hydatid sand contains: (Recent pattern Nov 2017) a. Protoscolices b. Brood capsule c. Ectocyst d. Germinal layer 16. Adult worm of Echinococcus is found in: (Recent pattern 2018) a. Dog b. Humans c. Domestic animals d. Felines 17. All the following spread by auto infection except? (Recent pattern Nov 2017) a. Taenia solium b. Strongyloides stercoralis c. Hymenolepis nana d. Hymenolepis diminuta 18. Drug of choice for treatment of following egg:  (AIIMS May 2018)







a. Albendazole c. Niclosamide

b. Praziquantel d. Pyranteol pamoate

1. Ans.  (a) Man

8. Ans.  (b) E. multilocularis

Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 191

Species

Definitive host

Intermediate host

Taenia saginata Humans

Cattle

Taenia solium

Pigs, humans

Humans

Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 195 • Already explained Q.1

•

9. Ans.  (c) Hydatidosis Ref: T.B of Parasitology–Chaterjee–12th edition–Page 127 • Serological tests are helpful in diagnosis of hydatid disease • Tests that are done are: ƒ Indirect haemagglutination test ƒ Immunodiffusion in gel ƒ ELISA ƒ Western blot •

•

3. Ans.  (b) Tinea solium

•

th

edition–

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• Consumption of uncooked or undercooked pork– infection of T.solium • Consumption of uncooked or undercooked beef – infection of T.saginata •

•

4. Ans.  (b) Cysticercosis Ref: T.B of Medical Parasitology–S.C.Parija–4 Page 197

• E. multilocularis causes multilocular cysts in liver–that does not have membrane and brood capsules–because of that the larvae spreads and causes liver necrosis • Because of the spread of larvae to other sites like brain– it is called as malignant hydatid disease •

2. Ans.  (a) T.solium

Ref: T.B of Medical Parasitology–S.C.Parija–4 Page 195

Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 208

Chapter 44     Helminthology Cestodes

ANSWERS AND EXPLANATIONS

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10. Ans.  (a) Echinococcus Ref: T.B of Parasitology–Chaterjee–12th edition–Page 201 • Echinococcus granulosus is the causative agent for hydatid cyst • Hence it is also called as hydatid tape worm •

th

edition–

• Cysticercosis is systemic disease caused by T.solium • Neurocysticercosis is the most serious form seen in brain • It is the most commonest cause of seizures in India •

•

11. Ans.  (c) Hymenolepis nana

•

•

Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 209 • Hymenolepis nana is the smallest cestode that causes infection in humans • Hence it is called as dwarf tapeworm •

5. Ans.  (a) Man Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 203 • Definitive host for Echinococcus is Dogs and wild carnivores • Intermediate host is humans and domestic animals

•

12. Ans.  (b) Egg of T. solium is acid fast

•

•

Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 198-199 • Egg of Taenia solium and Taenia saginata are morphologically similar • It can be differentiated by acid fast staining ƒ Egg of Taenia solium–non acid fast ƒ Egg of Taenia saginata–acid fast •

6. Ans.  (b) Liver Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 204 • Cystic Echinococcosis caused by E.granulosis presents as hydatid cyst in organs • Most common site for hydatid cyst is liver followed lung • Other sites are spleen, heart, kidney, brain and bone

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13. Ans.  (d) Hymenolepis diminuta

•

•

7. Ans.  (a) Lung • Complication of hydatid cysts are rupture and calcification • Among the cysts, mediastinal cysts undergo calcification most commonly and pulmonary cysts never undergo calcification mostly •

•

Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 213 • Eggs or larvae gets entered into the body from one cavity to another and causes auto infection • Parasites causing auto infection are: ƒ Enterobius vermicularis ƒ Taenia solium ƒ Strongyloides stercoralis •

•

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331

Unit 4     Parasitology

16. Ans.  (a) Dog

ƒ Capillaria philippinensis ƒ Hymenolepis nana ƒ Cryptosporidium parvum ƒ

Ref: T.B of Medical Parasitology – S.C.Parija – 4th edition – Page 203

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14. Ans.  (b) Both adult and larval stage are seen in humans Ref: T.B of Medical Parasitology–S.C.Parija–4 Page 209

th

edition–

• • • •

The given image shows egg of H.nana It does not need an intermediate host Infections is acquired by ingestion of eggs Both adult and larval stage are seen in definitive host– humans • Treatment is praziquantel •

•

•

•

•

• Definitive host for Echinococcus is Dogs and wild carnivores – adult worms live in definitive host • Intermediate host is humans and domestic animals •

•

17. Ans.  (d) Hymenolepis diminuta Ref: Medical Parasitology – Arora – 3rd edition – Page 261 • Eggs or larvae gets entered into the body from one cavity to another and causes auto infection • Parasites causing auto infection are: ƒ Enterobius vermicularis ƒ Taenia solium ƒ Strongyloides stercoralis ƒ Capillaria philippinensis ƒ Hymenolepis nana ƒ Cryptosporidium parvum •

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15. Ans.  (a) Protoscolices Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 202 • Hydatid cyst has three layers–Pericyst, Ectocyst and Endocyst • Pericyst–outer most layer contains blood vessels and fibroblasts • Ectocyst–acellular, laminated, hyaline layer • Endocyst–Germinal layer which gives rise to brood capsules and scolices on inside • When the embryos gets released from this layer and floats freely in the fluid–it is called as hydatid sand–it has daughter cysts with scolices. •

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•

•

•

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18. Ans.  (b) Praziquantel Ref: T.B of Medical Parasitology–S.C. Parija–4th edition– Page 209 
 • Above image clearly shows the following findings: ƒ Colorless, non bile stained ƒ Outer membrane is thin and inner membrane is 
embryophore which encloses an oncosphere with three pairs of hooklets • Thread like polar filaments is present on each two poles 
 • Hence the egg is identified as Hymenolepis nana • Treatment: Praziquantel is the DOC. •

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•

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45

Trematodes INTRODUCTION

y







y

y

y

y Trematodes have characteristic suckers y They are also called as flukes y All the trematodes have definitive host and two intermediate hosts  Definitive host: Humans and animals  First intermediate host: Fresh water snail or molluscs  Second intermediate host: Fish or Crab y Infection with trematodes is mainly due to two mechanisms

Penetration of skin by cercariae

Ingestion of metacercariae •

Figure 1: Egg of Schistosoma haematobium (Courtesy: CDC/Dr D.S. Martin)

y A pruritic rash appears at the site of penetration y Eggs get accumulated in the bladder–leads to inflammation and egg granuloma in the ureter and bladder y This inflammatory granuloma leads to fibrosis and obstruction in the urinary tract y Because of this continuous irritation–it leads to malignant tumors in the urinary bladder (Squamous cell carcinoma) y Acute infection causes–Katayama syndrome–fever, itching and lesions at the penetration site; y Chronic infection leads to terminal hematuria, dysuria named as Endemic hematuria y

All trematodes have operculated eggs except Schistosoma sp.,

Clinical Features y

•

•

•

•

• Schistosoma (all species) • Clonorchis sinensis (Fish) • Paragonimus westermani (Crab) • Fasciola hepatica (Vegetables or aquatic plants) • Fasciolopsis buski (Vegetables or aquatic plants)

y y







y

y

y All Schistosoma species are unisexual y Major species are:  S. haematobium  S. mansoni  S. japonicum

y

y

SCHISTOSOMA SPECIES

Schistosoma Haematobium

Diagnosis

y

y

y

y

y

y Nonoperculated, terminal spine egg seen in urine sediments y Most sensitive and specific is–Bladder mucosal biopsy y Serological test is helpful in chronic cases and epidemiological studies y Skin test used–Fairley`s test y DOC–Praziquantel and Metrifonate







y

y

y

y

y

y

y

y

y Major blood fluke that affects humans y The fluke live in the venous plexus that drains the urinary bladder, ureter and pelvis y Infective form is Cercariae–that enters the body through skin penetration y Definitive host: Humans y Intermediate host: Fresh water snails y After entering the epidermis–the larvae spreads to the capillaries in lung–liver–where they develop into adult worms y Adult worms moves to vesical venous plexus and lay eggs– these eggs are passed in the urine y Characteristics of Egg:  Eggs are oval shaped, yellowish brown in color  Nonoperculated  Terminal spine seen in the posterior side is the unique key for identification

Table 1:  Clinical manifestation of various Schistosoma species Schistosoma species Clinical manifestation S.haematobium S.mansoni S.japonicum S.intercalatum S.mekongi

Haematuria and dysuria Dysentery Katayama syndrome Intestinal disorders Hepatomegaly

Unit 4     Parasitology

Schistosoma Mansoni y It causes intestinal schistosomiasis; also called as bilharziasis y Adult worm lives in the mesenteric venous plexus draining the intestine y Cercariae is the infective form y Heavy infection is seen in the age group of 10 to 14 years y Clinical features are:  Katayama fever (M/c seen)  Dysentery  Symmer’s pipestem fibrosis (periportal fibrosis)  Portal hypertension  Transverse myelitis y Diagnosis is by characteristic demonstration of eggs in fecal sample or rectal biopsy (most sensitive and specific is rectal biopsy):  Nonoperculated egg  Yellowish brown  Lateral spine seen near to the rounded posterior end

y Treatment: It is more resistant for treatment compared to other species; Praziquantel is safe and effective; y

y

y

y

y

y

Table 2:  Identification of schistosoma eggs Schistosoma haematobium Schistosoma mansoni Schistosoma japonicum

Terminal spine Lateral spine Lateral small knob











y

FLUKES Fasciola Hepatica y y y y

y

y

y

y







y y

y

y

Common liver fluke Definitive host: Sheep, humans Intermediate host: Snails Infective form: Metacercariae (aquatic plants that has this metacercariae is the source of infection) Pathogenesis is due to adult worm and migrating larvae It causes fascioliasis  Hepatomegaly, biliary obstruction, jaundice occurs  Acute fascioliasis is due to migrating larvae  Chronic fascioliasis is due to adult worms in bile ducts Halzoun is a syndrome that occurs due to eating raw liver– adult worm enters the posterior pharyngeal wall and causes air way obstruction that leads to death Diagnosis is by demonstration of egg: Bile stained, operculated with a large unsegmented ovum 





y

y

y

y

Figure 2: Egg of Schistosoma mansoni (Courtesy: CDC) y DOC–Praziquantel y

Schistosoma Japonicum y It causes oriental schistosomiasis; it is the most severe form y Adult worms live in the superior mesenteric plexus y Clinical features are:  Katayama syndrome (M/c)  Periportal fibrosis  Pulmonary hypertension  CNS schistosomiasis y Diagnosis is by characteristic eggs:  Oval shaped, bile stained egg  Lateral knob seen y

y

y



Figure 4: Egg of Fasciola hepatica (Courtesy: CDC)







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y Serodiagnosis are of most helpful–ELISA is best method y Serology is helpful as antibodies are positive before the eggs can be demonstrated in the stool sample y Treatment: Bithionol; Triclabendazole y

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Table 3:  Habitat of fasciola species Organism Habitat Fasciola hepatica Fasciola gigantica Fasciolopsis buski

Bile tract Bile tract Duodenum and jejunum

Fasciolopsis Buski y Giant intestinal fluke–because it is the largest intestinal fluke y Fluke lives in the duodenum and jejunum y It is the most common intestinal trematode that causes infections in humans y Definitive host: Pigs and humans y

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y

Figure 3: Egg of Schistosoma japonicum (Courtesy: CDC/Dr Moore)

y

y y y y

First Intermediate host: Snails Second intermediate host: Fresh water plants Mode of infection: Ingestion of fresh water aquatic plants It causes fasciolopsiasis–worms causes obstruction and ulceration Vit B12 malabsorption occurs Demonstration of operculated eggs–helps in diagnosis Eggs of F.buski and F.hepatica are not distinguishable Treatment: Praziquantel; Niclosamide

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Paragonimus Westermani y y y y y y y

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Remember Vitamin B12 Malabsorption

• Diphyllobothrium latum (Cestode) • Fasciolopsis buski (Trematode)

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It is also called as Oriental lung fluke Definitive host–Man First intermediate host–Snails Second intermediate host–Fresh water crab or crayfish Infective form–Metacercariae Cases have been reported in Assam, Bengal, Tamil Nadu and Kerala in India Causes paragonimiasis in pulmonary system; it causes a granulomatous reaction that leads to blood mixed sputum– consists of golden brown eggs; a fibrous tissue that may go for cavitation in some cases; A patient with hemoptysis should be evaluated for P.westermani also Diagnosis is by demonstration of eggs in sputum Treatment: Praziquantel

Chapter 45     Trematodes

y y y y

Figure 5: Egg of Fasciolopsis buski (Courtesy: CDC/Dr Mae Melvin)

Remember Parasites causing malignancy are: • Schistosoma haematobium–Bladder cancer • Clonorchis sinensis–Cholangiocarcinoma • Opisthorchis felineus–Cholangiocarcinoma

Figure 6: Eggs of trematodes (Courtesy: CDC/Dr Mae Melvin)

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Clonorchis Sinensis Also called as Chinese or Oriental liver fluke It lives in the biliary passage and pancreatic duct Definitive host–Man, pig, dog or cat First intermediate host–Snails Second intermediate host–Fresh water fish Infective form–Metacercariae The fluke proliferates in the biliary epithelium of bile ducts– leads to dilatation of bile ducts y It is associated with:  Bile duct carcinoma (cholangiocarcinoma)  Pancreatic carcinoma  Cholangitis  Cholangiohepatitis y Lab diagnosis is by demonstration of eggs:  Eggs are bile stained  Has a convex operculum  A small knob is seen at the posterior end  But egg of C.sinensis cannot be distinguished from Š Heterophyes heterophyes Š Opisthorchis spp., Š Metagonimus yokogawai y Treatment: Praziquantel y y y y y y y

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List of operculated eggs: • Diphyllobothrium latum • Fasciola hepatica • Fasciola gigantica • Fasciolopsis buski • Clonorchis sinensis • Paragonimus westermanii • Gastrodiscoides hominis • Watsonius watsoni • Opisthorchis felineus • Opisthorchis viverrini • Heterophyes heterophyes • Metagonium yokogawai •

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Table 4:  Summary of treatment Name of trematode Blood fluke: Schistosomes (all species) Biliary fluke: Clonorchis sinensis Opisthorchis viverrini Liver fluke: Fasciola hepatica Fasciola gigantica Intestinal fluke: Fasciolopsis buski Heterophyes heterophyes Lung fluke: Paragonimus westermani

Drug of choice Praziquantel Praziquantel Ticlabendazole Praziquantel Praziquantel

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Unit 4     Parasitology

MULTIPLE CHOICE QUESTIONS





1. Painless terminal hematuria is seen as one of the manifestations in the infection caused by: (Recent pattern 2017) a. Schistosoma Japonicum b. Schistosoma mansoni c. Schistosoma haematobium d. Plasmodium falciparum 2. Katayama fever is caused by: (Recent pattern 2018) a. F. hepatica b. C. sinensis c. S. haematobium d. A. lumbricoides 3. A man on return from a country complains of pain in abdomen, jaundice, with increased alkaline phosphatase and conjugated hyperbilirubinemia. USG shows blockage in biliary tree what could be the cause? (AIIMS 2018) a. Fasciolopsis buski b. Clonorchis sinensis c. Strongyloides d. Ancylostoma







4. Which is not a liver fluke: (Recent pattern 2018) a. Paragonimus b. Whipworm c. Clonorchis sinensis d. Gnathostoma spinigerum e. Opisthotrichus 5. Clonorchis sinensis infection is due to ingestion of: (Recent pattern 2017) a. Fish b. Pork c. Snail d. Beef 6. Clonorchis sinensis is: (Recent pattern 2017) a. Tape worm b. Round worm c. Thread worm d. Fluke 7. Which of the following is a risk factor for bladder carcinoma: (Recent pattern 2017) a. Clonorchis sinensis b. Opisthorchis sp c. Schistosoma haematobium d. Fasciola hepatica

8. Which of the following parasite’s life cycle is shown below?

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a. Fasciolopsis c. Paragonimus westermani

b. Fasciola hepatica d. Clonorchis sinensis

(AIIMS 2017)

10. Following type of eggs are seen in: (Recent pattern 2017)



Chapter 45     Trematodes



9. All of the following have operculated eggs except? (Recent pattern Nov 2017) a. Clonorchis sinensis b. Diphyllobothrium latum c. Hymenolepis diminuta d. Paragonimus westermanii

a. Schistosoma haematobium b. Schistosoma mansoni c. S.japonicum d. Clonorchis sinensis

ANSWERS AND EXPLANATIONS 1. Ans.  (c) Schistosoma haematobium Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 222 • Painless terminal hematuria is a feature of urinary schistosomiasis • Urinary schistosomiasis is caused by S.haemotobium • Chronic urinary infections lead to squamous cell bladder carcinoma •

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•

2. Ans.  (c) S.haematobium Ref: T.B of Medical Parasitology–S.C.Parija–4 Page 222, 227

4. Ans.  (a) Paragonimus; (b) Whipworm; (d) Gnathostoma spinigerum Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 235 • Liver flukes are: ƒ Fasciola hepatica ƒ Clonorchis sinensis ƒ Opisthorchis felineus •

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5. Ans.  (a) Fish edition–

Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 252

• Katayama fever is caused by any of the species of Schistosoma • The most common causative pathogen in order are: ƒ S.mansoni ƒ S.japonicum ƒ S.haematobium • Fever, myalgia, arthralgia are the presenting symptoms in katayama fever

• Second intermediate host is fish • The infection is due to ingestion of metacercaria from inadequately cooked freshwater fish

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6. Ans.  (d) Fluke Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 252 • Clonorchis sinensis belongs to trematodes inturn called as flukes • It is also called as Chinese liver fluke •

3. Ans.  (b) Clonorchis sinensis Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 252 • Clonorchis sinensis is called Chinese liver fluke • The fluke proliferates in the biliary epithelium of bile ducts–leads to dilatation of bile ducts • It is associated with: ƒ Bile duct carcinoma (cholangiocarcinoma) ƒ Pancreatic carcinoma ƒ Cholangitis ƒ Cholangiohepatitis •

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7. Ans.  (c) Schistosoma haematobium Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 222 • Already explained Q.1 •

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8. Ans.  (b) Fasciola hepatica

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Ref: T.B of Parasitology–Chaterjee–Page 187 • Fasciola hepatica: Sheep liver fluke comes under Trematodes/flukes •

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Unit 4     Parasitology

• Adult worms reside in the biliary passages of the liver of sheep, goat, cattle and man • Infective form: Metacercariae encysted on water plants that are ingested by humans • Definitive host: Sheep, goat, cattle and man • First intermediate host: Snails • Second intermediate host: Aquatic vegetations

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9. Ans.  (c) Hymenolepis diminuta

ƒ

Ref: Medical Parasitology–Arora–3 edition–Page 126,151 rd

• In cestodes–the only helminth that has operculated egg is Diphyllobothrium latum; It is a bile stained egg • In trematodes–all of them lay operculated eggs except Schistosomes • List of operculated eggs: ƒ Diphyllobothrium latum ƒ Fasciola hepatica ƒ Fasciola gigantica •

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ƒ ƒ ƒ ƒ ƒ ƒ ƒ ƒ ƒ

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Fasciolopsis buski Clonorchis sinensis Paragonimus westermani Gastrodiscoides hominis Watsonius watsoni Opisthorchis felineus Opisthorchis viverrini Heterophyes heterophyes Metagonium yokogawai

10. Ans.  (a) Schistosoma haematobium Ref: Medical Parasitology–Arora–3rd edition–Page 222 • Characteristics of Egg: ƒ Eggs are oval shaped, yellowish brown in color ƒ Nonoperculated ƒ Terminal spine seen in the posterior side is the unique key for identification •

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46

Nematodes

• Wuchereria bancrofti • Brugia malayi

•

• Trichinella spiralis • Strongyloides stercoralis • Dracunculus medinensis

Conjunctival nematodes

• Loa loa

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Smallest nematode

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Lymphatic nematodes

y

y Nematodes are cylindrical worms and looks elongated they are called as roundworms.

•

INTRODUCTION

TRICHINELLA SPIRALIS

y

y Based on the habitat of the nematodes – they are divided into

y

Table 1:  Classification of Nematodes

y This worm is a common parasite of pig–causes infection to humans (zoonosis) y Life cycle gets completed in a single host–Pig; Accidental host are humans y

•

Largest nematode

Small intestine: • Ascaris lumbricoides • Ancylostoma duodenale • Necator americanus • Strongyloides stercoralis • Trichinella spiralis • Capillaria philippinensis Large intestine: • Enterobius vermicularis • Trichuris trichiura

Adult worm

Lives in intestine

Larvae

Lives in striated muscles

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Intestinal nematodes

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Dracunculus medinensis Onchocerca volvulus Loa loa Mansonella perstans Mansonella ozzardi

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Subcutaneous tissue nematodes

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y Infection is acquired by ingestion of encysted larvae which is present in raw pork or insufficiently cooked pork y This larva enters the duodenum and develop into adult male and female within two days y Gravid female directly gives rise to larva (No Egg Stage) y This larvae goes to muscles of tongue, mastication, intercostal muscle, eye muscles, thigh muscles and get encysted y After few months–it goes for calcification after death y Infection in human is a dead end (Contd...)

Figure 1: Illustration showing life cycle of Trichinella spiralis

Unit 4     Parasitology

y The larvae that are seeding in the muscles causes myositis– leading to myalgia; periorbital edema and facial puffiness y Myocarditis, bronchopneumonia, vascular thrombosis and encephalitis occurs at later stage y Lab diagnosis:  Ideal specimen: Muscle tissue by biopsy  Serological tests: ELISA, Bentonite flocculation test, Western blot  Antigen is named as Bachman antigen y

y

y







Figure 3: Egg of Trichuris trichiura Courtesy: CDC/B.G. Partin

Remember

Mucous plugs are seen in: • Trichiruis trichiura • Capillaria philippinensis • Gnathostoma spinigerum •

•

•

y Treatment: Mebendazole y DD for Trichuris is Capillaria philippensis y

y

Figure 2: Encystation of Trichinella spiralis larvae in muscle (Courtesy: CDC) y Treatment  Thiabendazole  Mebendazole y





Remember

Soil transmitted helminths (STH) are: • Ascaris lumbricoides • Hook worm • Trichuris trichiura • Strongyloides stercoralis •

•

A B Figures 4 A and B: Trichuris trichiura and Capillaria philippensis (Mucous plugs are prominent in trichuris; Size of Capillaria is smaller)

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Trichuris Trichiura y The worm has a characteristic whip like shape–hence called as Whip worm y It lives in the large intestine y Definitive host: Humans (the only host) y Infection is acquired by ingestion of soil with embryonated eggs (has rhabditiform larvae) y It usually affects children and remains asymptomatic y Heavy infection causes rectal prolapse in children; appendicitis; y Lab diagnosis:  Demonstration of eggs in feces  Characteristics of egg: Š Barrel shaped Š Bile stained egg Š Mucous plugs at both ends–which are not bile stained y

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STRONGYLOIDES STERCORALIS y This worm is also called as dwarf threadworm y Unique characteristic features:  It has both parasitic and free living forms  No male worm  Females reproduce itself and produce eggs  Eggs give rise to larvae and develop into filariform larvae y Infective form–Filariform larvae (enters by skin penetration) y Life cycle of Strongyloides:  Parasitic forms enters into human and continues its life  Free living forms enters the soil and develop themselves  Autoinfection y When the larvae enters through the skin–it causes larva currens–dermatitis y Larvae then migrates through capillaries–lungs–causing pneumonitis, bronchopneumonia, eosinophilia y Two important manifestations seen in immunocompromised (AIDS) patients are:  Hyperinfection syndrome  Disseminated strongyloidiasis y

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Hyperinfection syndrome

Remember

• After an initial infection with S.stercoralis– chronic infection persists • Suddenly when that patient goes for immunocompromised state–it leads to hyperinfection (more multiplication) • But it does not cause invasion • So no extra intestinal manifestation can be seen

Parasites causing auto infection are: • Enterobius vermicularis • Taenia solium • Strongyloides stercoralis • Capillaria philippinensis • Hymenolepis nana • Cryptosporidium parvum

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Disseminated strongyloidiasis

• When an immunosuppressed patient acquired infection with S.sterocoralis–it leads to dissemination • It affects extra intestinal organs • May lead to pulmonary and neurological complications

Chapter 46     Nematodes

Table 2:  Diseases caused by S. stercoralis

•

•

•

•

y Lab diagnosis:  Demonstration of rhabditiform larvae from the stool (Not Egg)  When eggs are hatching out in the stool–it immediately forms rhabditiform larvae–hence from stool specimens these are commonly seen  But infective form y







ASCARIS LUMBRICOIDES This worm is also called as round worm Most common infection acquired by humans Largest intestinal nematode infecting humans Adult worm lives in the small intestine Humans are the only definitive host Infection is acquired by ingestion of food with embryonated eggs (only embryonated eggs are infective) y Clinical manifestations:  Intestinal ascariasis–few worms usually means asymptomatic; Heavy infections leads to: Š Malabsorption Š Intussuception Š Intestinal obstruction  Pulmonary ascariasis: Due to migrating larvae that enters the capillaries–leads to Loeffler’s syndrome  Anemia y Lab diagnosis:  Demonstration of eggs in stool samples; y y y y y y

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Table 3:  Characteristics of of eggs in stool Fertilized egg

• Bile stained egg • Outer • Oval to spherical albuminous coat shaped is lost here • Surrounded by a thick outer albuminous coat • A clear crescentic space at each sides • Has an unsegmented ovum •

•

Figure 5: Filariform larvae of Strongyloides stercoralis (Courtesy: CDC/Dr Mae Melvin) 



Stool culture can be done to identify the larvae by following methods: Š Harada Mori filter paper method Š Baermann funnel method Š Agar plate method Enterotest can be done by checking the aspirations for rhabditiform larvae Serodiagnosis by ELISA, IHA PCR Š

Š

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Decorticated egg

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Unfertilized egg • Ellipsoidal shape • Heaviest of all helminthic eggs • When no male worm is seen in the infected person–female produces unfertilized egg •

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Remember

Enterotest is done for: • Giardia lamblia • Cystoisospora belli • Strongyloides stercoralis •

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y Treatment  Ivermectin is the first DOC for acute and strong strongyloidiasis; Second drug is albendazole  Thiabendazole is the DOC for disseminated strongyloidiasis y





Figure 6: Egg of Ascaris lumbricoides (Fertilized egg) (Courtesy: CDC/Dr Mae Melvin)

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Unit 4     Parasitology

y Demonstration of egg is difficult in following conditions:  Extra intestinal infections  When only male worms infect the individual y Treatment  DOC–Mebendazole  Pyrantel pamoate–causes spastic paralysis of the helminth  Piperazine citrate–causes flaccid paralysis of the worm– DOC for intestinal or biliary ascariasis

Remember

y



Hookworm that infect animals and accidentally humans: • Ancylostoma ceylanicum • Ancylostoma braziliense • Ancylostoma caninum



y

•



•



•



ENTEROBIUS VERMICULARIS This worm is also called as threadworm or pin worm Most commonly affects children Humans are the natural host Infection is acquired by ingestion of embryonated eggs through hands; through inhalation of ariborne eggs; y Once eggs come out of human–it cause autoinfection by hand to mouth transfer y Clinical features:  Perianal pruritus (nocturnal itching)–because the worms come out  Abdominal pain  Excoriation y Laboratory diagnosis:  Demonstration of eggs from anal swab/perianal scrapings– done by scotch tape swab method using NIH swab Š Plano convex shaped Š Not bile stained Š Very thin, hyaline egg shell Š Coiled tad pole like larvae y y y y

y

y

Ancylostoma Duodenale y y y y

It is also called as old world hook worm It lives in the small intestine–jejunum Humans are the only host Infection is acquired by penetration of the filariform larvae (third stage) through the skin–causes local reaction called as Ground itch Once the larvae enters the cavities–it causes cough, pharyngitis and dyspnea It then gets moulted to adults in small intestine–where it sucks the blood and causes hypochromic microcytic anemia; also causes hypoproteinemia Lab diagnosis:  Demonstration of eggs in stool  To estimate the quantity of eggs–Kato Katz method is helpful  For culture–Harada Mori culture method is used (Filariform larvae can be grown) Treatment:  Mebendazole  Ferrous sulfate for anemia

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Necator Americanus y Also called as new world hook worm y Manifestations are similar to Ancylostoma except that the severity of anemia is less here y

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Table 4:  Differences between Ancylostoma and Necator Ancylostoma duodenale

Necator americanus

Filariform larvae: • has a conical head • no gap seen between esophagus and intestine Adult worm: • two pairs of claw like teeth on anterior side • one pair of knob like teeth on posterior side • copulatory bursa has 13 rays Egg: • oval shaped • non bile stained • surrounded by a thin hyaline membrane • contains blastomeres Pathogenesis: • Blood loss is around 0.15– 0.26 mL/day • Eggs released are 10000– 25000 eggs/day • More severe anemia

Filariform larvae: • has a rounded head • a gap seen between esophagus and intestine Adult worm: • one pair of chitinous plates on anterior side • one pair of plates on the posterior side • copulatory bursa has 12 rays Egg: • oval shaped • non bile stained • surrounded by a thin hyaline membrane • contains blastomeres Pathogenesis: • Blood loss is 0.03 mL/day • Eggs released are 5000– 10000/day

•

•

•

Figure 7: Egg of Enterobius vermicularis Courtesy: CDC y Treatment: Pyrantel pamoate is the DOC; alternate drugs are mebendazole y All the family members and contacts need to get a course as prophylaxis y

y

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HOOK WORM

Remember

Hookworm that infect humans: • Ancylostoma duodenale • Necator americanus •

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• Less severe anemia •

Important Points in Intestinal Nematodes: • All intestinal nematodes are diagnosed by eggs in stool except Strongyloides stercoralis which passes larvae in stool • Perianal pruritus is hallmark of Enterobius vermicularis • Iron deficiency anemia is seen in hook worm • Biliary obstruction in heavy infections are seen with Ascaris lumbricoides • Small bowel infection is seen with round worm and hook worm, Strongyloides. • Large bowel infection is seen with Whip worm and Pin worm. •

•

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•

Chapter 46     Nematodes

High Yield

•

Figure 8: Egg of hookworm (Egg of Ancylostoma duodenale and Necator americanus cannot be differentiated) (Courtesy: CDC)

Table 5:  Miscellaneous nematodes Nematodes

Characteristics

Ancylostoma ceylanicum

• Smallest hookworm • Commonly seen in cats • Causes intestinal infection in humans •

•

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Ancylostoma braziliense

• Hookworm of dogs and cats • Causes creeping eruptions in humans (cutaneous larva migrans)

Ancylostoma caninum

• Dog hookworm • Causes cutaneous larva migrans

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•

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Trichostrongylus

• Also called as pseudo hook worm • Egg similar to hook worm •

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Angiostrongylus cantonensis

• Rodent or rat lung worm • Causes Eosinophilic meningitis in humans

Angiostrongylus costaricensis

• Causes abdominal angiostrongyliasis and eosinophilic gastroenteritis

•

•

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Table 6:  Differences between cutaneous and visceral larva migrans Cutaneous larva migrans

Visceral larva migrans

• Ancylostoma braziliense (M/c) • Ancylostoma caninum (second M/c) • Necator americanus • Ancylostoma duodenale • Gnathostoma spinigerum • Strongloides • Loa loa • Fasciola • Paragonimus

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Toxocara canis Toxocara cati Gnathostoma Anisakis

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EOSINOPHILIC MENINGOENCEPHALITIS y Eosinophilic meningitis is defined as the presence of more than 10  eosinophils/mm3  in the cerebrospinal fluid (CSF)  and/or eosinophils accounting for more than 10% of CSF leukocytes y Three important parasitic infections associated with eosinophilic meningitis  y

y

• Angiostrongylus cantonensis • Baylisascaris procyonis • Gnathostoma spinigerum •

•

•

343

Unit 4     Parasitology

MULTIPLE CHOICE QUESTIONS











344



1. The cause of larva currens: (Recent pattern 2017) a. Strongyloides stercoralis b. Necator americanus c. Ankylostoma duodenale d. H.nana 2. A 40 year HIV positive male patient comes with odynophagia and watery diarrhea. An endoscopy reveals esophageal and gastric candidiasis. A wet mount of the stool of the patient reveals following picture: (AIIMS 2017)

a. Filariform larvae is infective for humans as shown in the diagram b. Transmitted through contaminated food and water usually c. Females of these species show parthogenesis d. Drug of choice is Triclabendazole 3. Ankylostoma enters the human body by: (Recent pattern 2018) a. Ingestion b. Inhalation c. Penetration of skin d. Inoculation 4. Habitat of Nectator americanus: (PGI pattern 2017) a. Jejunum b. Ileum c. Colon d. Duodenum e. Caecum 5. Visceral larva migrans is associated with: (Recent pattern 2017) a. Strongyloides stercoralis b. Ancylostoma braziliensis c. Toxocara canis d. Visceral leishmaniasis 6. The hookworm thrives on: (Recent pattern 2017) a. Whole blood b. Plasma c. Serum d. RBC

7. Child is having perianal pruritus with following eggs due to: (Recent pattern 2017) a. E.vermicularis b. Ascaris c. Ancylostoma duodenale d. S stercoralis 8. Larvae of Ascaris lumbricoides most commonly causes: (AIIMS 2017) a. Cardiac symptoms b. Respiratory symptoms c. Genitourinary symptoms d. Cerebral symptoms 9. What is the most common clinical manifestation when larvae of ascaris, hookworm and strongyloides migrates through the body? (Recent pattern 2017) a. Asymptomatic b. Pneumonitis c. Acute dermal reaction d. Anemia 10. Cutaneous larva migrans is due to: (Recent pattern 2017) a. Ancylostoma braziliensis b. Wucheria bancrofti c. Brugia malayi d. Dracunculus medinensis 11. Stool examination in a patient reveals the following finding. What is the likely route of infection of this parasite? (AIIMS 2017)



a. b. c. d.

Ingestion of food contaminated with egg of larva Insect bite Improperly cooked beef Swimming in dirty water pool





15. Barrel shaped eggs is/are seen in: a. Hook worm b. Roundworm c. Pin worm d. Whipworm e. Strongyloides stercoralis















(PGI May 2017)











16. Which of the following larva is/are found in stools: (PGI May 2018) a. Strongyloides stercoralis b. Ankylostoma duodenale c. Ascaris lumbricoides d. Hymenolepis nana e. Enterobius vermicularis

























Chapter 46     Nematodes

12. The following are images of an intestinal nematode. Which of these are true about it? (AIIMS 2017)

17. A patient who had undergone renal transplantation three months before has presented with bloody diarrhea. Stool sample shows the following organisms on culture measuring 280 um. Which of the following is true among the following: (AIIMS Nov 2018 Video Question) For video scan this QR Code















a. Transmitted through percutaneous and autoinoculation b. Embryonated egg is the infectious stage for this worm c. The adult worm usually lives in the small intestine d. Triclabendazole is the drug of choice 13. Cutaneous larva migrans is caused by: (Recent pattern 2018) a. Ancylostoma braziliense b. Anisakiasis c. Necator americanus d. Ancylostoma catifera 14. A 48-year-old immunocompromised patient attended the emergency block with complaints of abdominal pain, vomiting and dyspnea. On examination and baseline investigation, he was diagnosed as gastrointestinal perforation with paralytic ileus. The most common cause of this disseminated infection in immunocompromised patient is due to the following. Identify it: (PGI pattern 2017) a. Rhabditiform larvae of S. stercoralis b. Filariform larvae of S. stercoralis c. Adult female worm of S. stercoralis d. Adult male worm of S. stercoralis e. Egg of S. stercoralis



















































a. b. c. d.









It causes Loeffler’s pneumonia The video shows the filariform larvae of the parasite It is acquired by contamination of food/water They are monoecious since they undergo parthenogenesis











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Unit 4     Parasitology

ANSWERS AND EXPLANATIONS 1. Ans.  (a) Strongyloides stercoralis

7. Ans.  (a) E.vermicularis

Ref: T.B. of Medical Parasitology–S.C.Parija–4th edition– Page 271

Ref: T.B. of Medical Parasitology–S.C.Parija–4th edition– Page 287

• Strongyloides infection is acquired by penetration of filariform larvae through skin • When it causes skin penetration–it produces localized dermatitis at that site–leading to itching–named as larva currens

• Clinical features of Enterobius vermicularis infection: ƒ Perianal pruritis (nocturnal itching) – because the worms come out ƒ Abdominal pain ƒ Excoriation

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2. Ans.  (c) Females of these species show parthogenesis Ref: T.B. of Medical Parasitology–S.C.Parija–4 Page 268

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8. Ans.  (b) Respiratory symptoms

edition–

Ref: T.B. of Medical Parasitology–S.C.Parija–4th edition– Page 291

• The above image shows Rhabdiform larvae of Strongyloides stercoralis • Identification feature: ƒ Unsheathed ƒ Short mouth with double bulbed esophagus • Unique feature of Strongyloides is it does not have male worms • Female worms reproduce themselves by parthenogentically releasing eggs

• Migrating larvae of Ascaris causes pulmonary symptoms • This is the initial stage of infection • It causes eosinophilic pneumonia–Loeffler’s syndrome

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3. Ans.  (c) Penetration of skin Ref: T.B. of Medical Parasitology–S.C.Parija–4th edition– Page 277 • Penetration of skin by filariform larvae is by: ƒ Hookworm ƒ Strongyloides •

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9. Ans.  (a) Asymptomatic Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 287–291 • Usually the infections with nematodes are asymptomatic when the larval migration occurs • The symptoms are mainly due to adult worm and when heavy infections persists •

•

10. Ans.  (a) Ancylostoma braziliensis Ref: T.B. of Medical Parasitology–S.C.Parija–4th edition– Page 294 • Cutaneous larva migrans is due to: ƒ Ancylostoma braziliense (M/c) ƒ Ancylostoma caninum (second M/c) ƒ Necator americanus ƒ Ancylostoma duodenale ƒ Gnathostoma spinigerum ƒ Strongloides ƒ Loa loa ƒ Fasciola ƒ Paragonimus •

4. Ans.  (a) Jejunum; (b) Ileum; (d) Duodenum Ref: T.B. of Medical Parasitology–S.C.Parija–4th edition– Page 275 • • • •

•

•

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Adult hookworms live in the lumen of small intestine Most common site is jejunum Less commonly in duodenum and ileum If needed to choose one option –choose jejunum

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5. Ans.  (c) Toxocara canis Ref: T.B. of Medical Parasitology–S.C. Parija–4th edition– Page 294 • Visceral larva migrans is caused by: ƒ Angiostrongylus cantonensis ƒ A.costaricensis ƒ Toxocara canis ƒ Toxocara catis ƒ Anisakine sp., ƒ Gnathostoma spinigerum •

11. Ans.  (a) Ingestion of food contaminated with egg of larva Ref: T.B. of Medical Parasitology–S.C.Parija–4th edition– Page 265

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6. Ans.  (b) Plasma Ref: T.B. of Parasitology–Chaterjee–12th edition–Page 175

346

• The main nutrition for hookworm is from plasma •

• The above image shows egg of Trichiuris trichirua • Identification is by: ƒ Barrel shaped egg ƒ Mucous plugs at both ends • Infection is acquired by ingestion of food that is contaminated with embryonated egg •

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12. Ans.  (b) Embryonated egg is the infectious stage for this worm Ref: T.B. of Medical Parasitology–S.C.Parija–4th edition– Page 265

•

15. Ans.  (d) Whip worm • Already explained Q.11 •

•

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13. Ans.  (a) Ancylostoma braziliense Ref: T.B of Parasitology – Chatterjee – 12th edition – Page 206

Cutaneous larva migrans

Visceral larva migrans

• Ancylostoma braziliense (M/c) • Ancylostoma caninum (second M/c) • Necator americanus • Ancylostoma duodenale • Gnathostoma spinigerum • Strongloides • Loa loa • Fasciola • Paragonimus

• • • •

•

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•

•

•

•

Toxocara canis Toxocara cati Gnathostoma Anisakis

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16. Ans.  (a) Strongyloides stercoralis; (b) Ankylostoma duodenale Ref: T.B of Medical Parasitology – S.C. Parija – 4th edition– page 268 • Larvae seen in the stools are characteristic for: • Strongyloides stercoralis • Hook worm – Ancylostoma duodenale and Necator americanus •

•

•

Chapter 46     Nematodes

• The above image shows adult male and female worms of Trichuris • Characteristic whip shaped hence called as whip worm • Infection is acquired by ingestion of food that is contaminated with embryonated egg

17. Ans.  (b) The video shows the filariform larvae of the parasite Ref: T.B of Medical Parasitology – S.C. Parija – 4th edition– page 268 • The clinical clue to identify the organisms are: ƒ Renal transplantation ƒ Diarrhea • The microbiological clue is shows larvae (only in two organisms – stool shows larvae, i.e.,); they are ƒ Strongyloides stercoralis ƒ Hook worm • Infections are more common in HIV, immunosuppressed and post-renal transplantation patients • Diagnosis: By demonstration of rhabditiform larvae in freshly passed stool; Filariform larvae can be demonstrated by the culture of the stool by Harada Mori filter paper method. • Treatment is Ivermectin and Thiabendazole •

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• Considering the options – most common cause is taken as choice •

14. Ans.  (a) Rhabditiform larvae of S.stercoralis

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Ref: Medical Parasitology – 3rd edition – D. R. Arora – page 180 • Patients with S.stercoralis hyperinfection presents with above features of dissemination. It occurs in immunocompromised patients • It is the smallest nematode and rhabditiform larvae is the most common form seen in the stool specimen. •

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47

Filarial Nematodes y Definitive host: Humans y Intermediate host: Mosquitoes–Culex, Anopheles, Aedes y Wuchereria has a endosymbiotic bacteria called as Wolbachia

Table 1:  Nematodes and their associated manifestations

• Wuchereria bancrofti • Brugia malayi • Brugia timori

• Loa loa • Mansonella streptocerca • Onchocerca volvulus

• Mansonella perstans • Mansonella ozzardi

y

y

y Adult worms causes inflammatory damage to the lymphatics. y Infiltration of plasma cella, eosinophils and thickening of vessel walls leads to tortous lymph vessels and lymphedema. y Clinical features of lymphatic filariasis (Bancroftian filariasis):  Lymphangitis  Obstruction of lymph nodes  Lymph varices  Hydrocele  Elephantiasis  Chyluria y Acute adeno lymphangitis (ADL) is characterized by high fever, lymphatic inflammation and local edema. Regional lymph nodes are enlarged and entire lymphatic channel gets blocked. y Genital involvement occurs manifesting as funiculitis, epididymitis, scrotal pain and tenderness. y In endemic areas, another syndrome complex presents as high fever, chills, myalgia with head ache and edematous inflammatory plaques–termed as dermatolymphangioadenitis (DLA). y Occult filariasis–means a hypersensitivity reaction that occurs to filarial antigens; In this condition microfilaria cannot be seen in peripheral blood; the most common manifestation is TPE (Tropical pulmonary eosinophilia)

Lives in peripheral blood, hydrocele fluid and chylous urine (blood and body fluids)

Third stage larva

Infective form









y

y Characteristics of microfilaria:  Key identification feature for diagnosis  Transparent and colourless with a sheath  End of the tail does not contain nuclei  Based on the periodicity of microfilaria circulation– infectivity differs–Microfilaria of Wuchereria has nocturnal periodicity

 

y

Microfilaria (First stage larva)

y

Lives in the lymphatics

y

Adult worm

y

y

y

y This worm lives in the lymphatic vessels–hence called as lymphatic filarial worm y Important morphological forms are:





WUCHERERIA BANCROFTI





y

•

• •

• •

Pathogenesis

•

Serous cavity filariasis

•

Subcutaneous filariasis

•

Lymphatic filariasis

y

y

y

INTRODUCTION

Table 2:  Differences between classical and occult filariasis • It is due to microfilariae • Hypersensitivity reaction is the feature • Microfilariae is not seen in the peripheral blood • High level of antibodies are seen (Increased IgE) • • •

• • •

y Laboratory diagnosis:  Blood microscopy–peripheral blood smear Š Collection of blood is during night (because of nocturnal periodicity) Š



y

Figure 1: Figure showing microfilariae of Wuchereria bancrofti (Courtesy: CDC/Dr Mae Melvi)

•

Occult filariasis

• It is due to adult worms • Lymphangitis and lymph nodes are affected • Microfilariae in the peripheral blood is present • Serological tests are not useful •

Classical filariasis

Š

Table 4:  Other filarial nematodes Brugia malayi

Š

• Causes lymphatic filariasis called as Brugian filariasis • Identification is by presence of two distinct nuclei in the tail tip of microfilariae • Genital involvement and chyluria are not seen •

•

Š

•







Onchocerca volvulus



• Causes river blindness • Transmitted by the bite of infected blackfly • Causes subcutaneous itchy nodules mainly over the bony prominences • Visual impairment occurs due to punctate keratitis • Microfilariae are found in the skin; not seen in the peripheral blood–it is demonstrated by skin snips • Mazzotti patch test is helpful •

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y

•





•





Chapter 47     Filarial Nematodes

Š DEC provocation test: DEC given at 2–8 mg/kg BW–after 30 minutes–the capillary blood collected and checked for microfilariae Š Sensitivity can be increased by concentration method named as Knott’s concentration technique. Š QBC  Immunodiagnosis–circulating antibodies are demonstrated  Filarial skin test  PCR–helpful in chronic and occult filariasis  The only noninvasive method helpful for detection of live adult worms–is USG of scrotum y Treatment:  Diethylcarbamazine (DEC) → 3–6 mg/kg for 3 weeks  Ivermectin  Levamisole  Mebendazole

•

TROPICAL PULMONARY EOSINOPHILIA y Syndrome that is developed in individuals who are infected with lymphatic dwelling filarial species. y Usually occurs in endemic areas y Symptoms are mostly seen at night because of nocturnal periodicity of microfilariae. y Clinical features are nocturnal cough, weight loss, low grade fever, lymphadenopathy and high blood eosinophilia. y Serum IgE levels and antifilarial antibody titers are markedly elevated. y DD: Churg Strauss syndrome, ABPA, Asthama, Loeffler`s syndrome y Treatment: DEC 4–6 mg/kg for 14 days

Loa loa

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y

y

•

y

Dracunculus medinensis

y

Filarial organism Vector Wuchereria bancrofti

Culex

Brugia malayi

Mansonia, Culex

Brugia timori

Simulum fly

Loa loa

Chrysops fly

Mansonella streptocerca

Subcutaneous nodules; River blindness Calabar swellings Mild clinical illness

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•

•

Dirofilariae

• • • •

•

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Bancroftian lymphatic filariasis

Brugian lymphatic Mansonia, Culex, filariasis Anopheles

Onchocerca volvulus Mansonella perstans

Disease

• Also called as Guinea worm; Serpent worm or Dragon worm • Definitive host: Man • Intermediate host: Cyclops • Mode of infection is through drinking water that has cyclops •

y

Table 3:  Important filarial organism

•

•

y

y

• Called as Eye worm • It produces transient subcutaneous swellings– calabar swellings • Microfilariae are sheathed and nuclei seen in the tail tip • Causes calabar swelling; conjunctival granuloma; it causes proptosis; meningoencephalopathy

•

•

Zoonotic filarial infection Primarily affects dogs, cats Humans are accidental host It usually causes solitary pulmonary nodule

Table 5:  Identification of filarial nematodes Sheathed Microfilaria

Unsheathed Microfilaria

• Wuchereria bancrofti • Loa loa • Brugia malayi

• • • •

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Mansonella perstans M.ozzardi M.streptocerca O.volvulus

Culicoides

Mansonella ozzardi

349

Unit 4     Parasitology

Microfilaria Identification First look whether sheathed or unsheathed Sheathed means: then look at tail tip

Sheathed Tail tip no nuclei

Sheathed Tail tip has two-three nuclei

Sheathed Tail tip has dence nuclei fully

Wuchereria bancrofti

Brugia malayi

Loa loa

Unsheathed Body nuclei extends up to tip

Unsheathed No tail tip nuclei

Unsheathed; Very thin among all; tail tip nuclei seen

Mansonella perstans

Mansonella ozzardi

Onchocerca volvulus

Microfilaria Identification Unsheathed means:

350



1. Wuchereria bancrofti, true is: (Recent pattern Dec 2012) a. Unsheathed b. Tail tip free from nuclei c. Non -periodic d. All



2. Which is non lymphatic filariasis: (Recent pattern Dec 2012) a. Loa loa b. Wuchereria bancrofti c. Brugia malayi d. Brugia timori



3. All the filarial worms are found in blood except: (Recent pattern Dec 2013) a. W. bancrofti b. B. malayi c. L. loa d. O. volvulus 4. This is a schematic diagram depicting body structure of which of these helminthes? (AIIMS Nov 2015)



a. Loa loa c. Brugia malayi



5. Calabar swelling is produced by: (Recent pattern Nov 2014) a. Onchocerca volvulus b. Loa loa c. Brugia malayi d. Wuchereria bancrofti



6. River blindness is caused by: (Recent pattern Dec 2013) a. Onchocera b. Loa loa c. Ascaris d. B.malayi



7. Which of the following is/are causes lymphatic filariasis: (PGI May 2018) a. Loa-Loa b. Oncocerca volvulus c. Wuchereria bancrofti d. Brugia malayi e. Brugia timori

Chapter 47     Filarial Nematodes

MULTIPLE CHOICE QUESTIONS



b. Wuchereria bancrofti d. Onchocera volvulus

351

Unit 4     Parasitology

ANSWERS AND EXPLANATIONS 1. Ans.  (b) Tail tip free from nuclei

4. Ans.  (b) Wuchereria bancrofti

Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 298

Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 298

• Wuchereria bancrofti is a filarial nematode that has nocturnal periodicity • It has sheathed microfilaria • The tail is pointed and it if free from nuclei

• Image clearly shows microfilaria of Wuchereria bancrofti • Wuchereria bancrofti is a filarial nematode that has nocturnal periodicity • It has sheathed microfilaria • The tail is pointed if free from nuclei

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2. Ans.  (a) Loa loa

5. Ans.  (b) Loa loa

Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 296 • Filariasis is classified into: ƒ Lymphatic filariasis–W.bancrofti, Brugia malayi and Brugia timori ƒ Subcutaneous filariasis: Loa loa, Mansonella streptocerca, Onchocerca volvulus ƒ Serous cavity filariasis: Mansonella perstans and Mansonella ozzardi •

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Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 316 • Loa Loa or eye worm causes transient subcutaneous swellings known as calabar swellings •

6. Ans.  (a) Onchocera Ref: T.B of Medical Parasitology–S.C.Parija–4th edition– Page 313 • Onchocerca volvulus causes onchocerciasis or river blindness • It causes cutaneous filariasis •

3. Ans.  (d) O.volvulus Ref: T.B of Medical Parasitology – S.C.Parija – 4th edition – Page 297

Habitat Microfilariae Examples Blood

• • • • •

•

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•

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Skin

Wuchereria bancrofti Brugia malayi Brugia timori Loa loa Mansonella perstans

• Onchocerca volvulus • Mansonella streptocerca •

•

Blood and skin

352

• Mansonella ozzardi •

•

7. Ans.  (c) Wuchereria bancrofti; (d) Brugia malayi; (e) Brugia timori Ref: T.B of Medical Parasitology – S.C.Parija – 4th edition – Page 297 • Lymphatic filariasis is caused by: ƒ Wucheria bancrofti ƒ Brugia malayi ƒ Brugia timori • Subcutaneous filariasis is caused by: ƒ Onchocerca volvulus ƒ Loa loa •

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Man acts as intermediate host in: • Plasmodium • Babesia • Toxoplasma • Echinococcus • Sarcocystis Man acts as both definitive and intermediate host in: • Taenia solium

Culture media used in parasitology

Important vectors in parasitology

Skin tests in parasitic diseases

Entamoeba histolytica

Cockroaches

Leishmania

Sandfly

Trypanosoma cruzi

Reduviid bugs

Trypanosoma brucei

Tsetse fly

Babesia

Ixodid ticks

Plasmodium

Female anopheles

Type I Hypersensitivity reactions: • Hydatid disease–Casoni’s test • Ascariasis • Strongyloidiasis • Filariasis • Schistosomiasis • Trichinellosis–Bachmann test

Filarial nematodes

Culex, anopheles

Dracunculus

Cyclops

•

•

•

Leishmania and Trypanosoma

NNN medium

Plasmodium

RPMI medium

Nematodes like Strongyloides, Coproculture technique hook worm Harada-Mori filter paper method Baermann technique Agar plate culture

•

•

•

Chapter 47     Filarial Nematodes

HIGH YIELDING FACTS TO BE REMEMBERED IN PARASITOLOGY

•

•

•

•

•

•

Type IV Hypersensitivity reactions: • Leishmaniasis–Montenegro test • Trypanosomiasis • Toxoplasmosis •

•

•

Modes of transmission of parasitic infections Entamoeba histolytica Giardia lamblia Ascaris lumbricoides Enterobius vermicularis Trichuris trichiura

Arthropod borne diseases

Ingestion of undercooked beef

Taenia saginata

Rat flea

Ingestion of undercooked pork

Taenia solium Trichinella spiralis

Ingestion of food, water and vegetables

Sandfly

• • • •

•

•

•

•

Kala azar Sandfly fever–three days fever Oriental sore Oroya fever or Carrion’s disease

• Bubonic plague • Endemic typhus • Intermediate host for H.nana •

•

•

Louse

• Epidemic typhus • Trench fever • Epidemic relapsing fever •

Ingestion of raw/undercooked fish

Clonorchis sinensis Diphyllobothrium latum

Ingestion of undercooked crab

Paragonimus westermani

Trombiculid mites

• Scrub typhus

Ingestion of water plants

Fasciola hepatica Fasciolopsis buski

Itch mites

• Scabies

Skin penetration

Strongyloides stercoralis Schistosoma species

Hard tick (Ixodid)

• • • • •

Sexually transmitted agents

•

•

•

•

•

•

Trichomonas vaginalis Entamoeba histolytica (homosexuals MSM) Giardia lamblia

•

•

Soft tick

Babesiosis KFD Tick typhus Q fever Tularemia

• Endemic relapsing fever •

Bile stained eggs

Culture media used in parasitology Entamoeba histolytica Balantidium coli

•

• • • • •

•

Boeck and Dr Bohlav’s medium Balamuth’s medium Robinson’s medium Jone’s medium Diamond’s medium

Giardia lamblia

Diamond’s medium

Trichomonas vaginalis

Modified Diamond’s medium TYM medium

•

•

•

•

• • • •

•

•

•

•

Fasciolopsis buski Clonorchis sinensis Opisthorchis felineus Diphyllobothrium latum

Parasitic infections not in India: (As of 2017) • • • •

•

contd...

Ascaris lumbricoides Trichuris trichiura Taenia Schistosoma japonicum Fasciola hepatica

•

•

•

Trypanosoma spp Leishmania braziliensis Balamuthia spp Plasmodium knowlesi

• Onchocerca volvulus • Capillaria philippinensis • Sappinia spp •

•

•

353

s e t o N

MYCOLOGY

5

Unit Outline

1

Chapter 48: Characteristics and Laboratory Diagnosis of Fungi Chapter 49. Superficial Mycoses Chapter 50: Endemic/Systemic Mycoses Chapter 51: Opportunistic Mycoses Chapter 52. Miscellaneous Fungi

Characteristics and Laboratory Diagnosis of Fungi CHARACTERISTICS OF FUNGI

48

Table 3:  Classification of fungi based on pathogenesis Superficial mycoses

Dermatophytosis, Candidiasis, Tinea, Piedra, Pityriasis versicolor

Cutaneous mycoses

Candidiasis

Subcutaneous mycoses

Mycotic mycetoma Chromoblastomycosis Rhinosporidiosis Sporotrichosis

CLASSIFICATION

Deep or systemic or visceral mycoses

Blastomycosis Cryptococcosis Paracoccidioidomycosis Coccidioidomycosis Histoplasmosis Candidiasis

y

y

y

y

y

y

Fungi are Eukaryotes. They have rigid cell walls. They have chitin, mannan and other polysaccharides. True nuclei with nuclear membrane, paired chromosomes are seen. y They are unicellular or multicellular y They divide sexually or asexually y y y y

Table 1:  Classification of fungi based on cell morphology Yeast like

Candida sp.

Moulds

Dermatophytes Aspergillus Mucor Penicillium Rhizopus

Dimorphic fungi (Two growth forms – Yeast and Mould)

Blastomyces dermatitidis Paracoccidioides brasiliensis Coccidioides immitis Histoplasma capsulatum Sporothrix schenckii Penicilliosis marneffei

Table 2:  Systemic classification based on sexual spore formation Aseptate hyphae, Endogenous asexual spores (Lower fungi) and sexual spores– E.g. Rhizopus, Mucor

Ascomycetes

Septate hyphae, exogenous asexual spores (Higher fungi) and sexual spores – E.g. Dermatophytes

Mycotoxicoses

Table 4:  Asexual spores with their example Conidia

Examples

Sporangiospores

Mucor, Rhizopus

Arthrospores

Coccidioides

Blastospores

Cladosporium

Chlamydospores

Candida albicans

Phialoconidia

Aspergillus fumigatus

TERMINOLOGY Terminologies used in Mycology: y Yeast – Unicellular, reproduce by budding y Yeast like – Unicellular, reproduce by budding or by fission y Moulds – Filamentous fungi – has hyphae – which is either septate or aseptate, reproduce by spore formation y Thermally dimorphic fungi – Grow as yeasts in 37°C, as moulds in 25°C y

No sexual spores E.g: Candida, Coccidiodes, Paracoccidiodes

y

Same as above E.g. Cryptococcus

Fungi imperfecti

y

Basidiomycetes

Aspergillus, Claviceps purpurea

Important points to be noted in classification: y Sexual spores: Ascospore, Zygospore, Basidiospore, Oospore y Asexual spores (Conidia): Sporangiospores, Arthrospores, Blastospores, Chlamydospores, Phialoconidia

y

Phycomycetes

Opportunistic mycoses Mucor, Aspergillus, Candida, Penicillium

y

Cryptococcus

y

Yeast

Unit 5     Mycology

y Coenocytic hyphae – Aseptate hyphae y Pseudohyphae – When yeast cell starts reproduce by budding → some of the buds fail to detach from the cells and becomes elongated → This elongated budding cells are called as pseudohyphae as they look like hyphae E.g.: Candida albicans y Mycelium – Tufts of hyphae y Anamorph – Asexual form y Telemorph – Sexual or perfect form y

y

y

y

y

MORPHOLOGY OF FUNGI

Figure 1: Morphological features of fungi

Figure 2: Budding yeast cells— oval shaped cells (Courtesy: CDC PHIL/Dr. Libero Ajello)

358

Figure 3: Septate hyphae (Courtesy: CDC PHIL/Dr. Lucille Georg)

Figure 4: Pseudohyphae

Table 5:  Endemic and opportunistic mycoses Endemic mycoses

Opportunistic mycoses

• • • • • • •

• • • • • • •

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•

•

•

•

•

•

Coccidioidomycosis Histoplasmosis Blastomycosis Phaeohyphomycosis Penicilliosis Sporotrichosis Paracoccidioidomycosis

•

•

•

•

•

•

•

Candidiasis Aspergillosis Cryptococcosis Mucormycosis Scedosporiosis Fusariosis Pneumocystosis

Culture of Fungi y Requirements for culture – Aerobic, lower pH than bacteria, Temperature range 25-30°C y Exceptions  Deep mycotic organisms – grow even in 37°C  Asp.fumigatus – grow even in 50°C y Agar used – SDA – Sabouraud dextrose agar – pH 5.4 y Emmon’s modification (Neutral) SDA – pH 7.2 y

y





y

y

Chapter 48     Characteristics and Laboratory Diagnosis of Fungi

Figure 5: Systemic classification of fungi

LABORATORY DIAGNOSIS OF FUNGI

Figure 7: A culture plate of SDA y Other culture media used in fungal culture:  Blood agar  Brain heart infusion agar/broth  Czapek Dox medium  Bird seed agar  Corn meal agar  CHROMagar y Special addition in culture media – Chloramphenicol – to suppress bacterial contamination; Cycloheximide (Actidione) – to suppress saprophytic fungi y

Figure 6: Laboratory diagnosis of fungi

Microscopy y KOH mount – Specimens are first seen microscopically; After applying 10-40% KOH in the specimen – wait for few minutes for the tissue debris to get removed so that fungal elements can be seen clearly y 0.1% Calcofluor white – Fluorescent stain that binds to the cellulose and chitin of the fungal cell wall y Gram stain – helpful for yeasts and yeast like fungi and to identify pseudohyphae (Candida sp is a Gram positive budding yeast cell) y India Ink stain – Negative staining procedure – for capsule of Cryptococcus neoformans y LPCB – Lactophenol cotton blue – for preparing mount from the fungal colonies y Tissue stains: H & E, GMS, PAS, Mayer’s Mucicarmine stain y

y













y

y

y

y

y

Serological Methods y Antigen detection – latex agglutination test – Cryptococcal antigen in CSF y Antibody detection – ELISA, Immunodiffusion test, Agglutination test, Complement fixation test y Immunohistochemistry – used in deep mycoses y

y

y

359

Unit 5     Mycology

Metabolites y Fungal metabolite detection – by Gas liquid chromatography y

Skin Tests y Some fungi elicit delayed type hypersensitivity reactions For example: Histoplasma, blastomyces, cocciodiodes, paracocciodioides, dermatophytes, sporothrix and Candida y

Special Stains Table 6:  Recommended special stains for various fungi

360

Stains

Uses

Periodic acid schiff (PAS)

Recommended stain for fungi – stains only live fungi

Gomori methenamine silver (GMS)

Stains both live and dead fungi

Mucicarmine stain

For Cryptococcus and Rhinosporidium

Masson Fontana stain

For pigmented fungi

H and E stain

For tissue stain

Toluidine blue

Pneumocystis carinii

1. Fungi are: (Recent Pattern Dec 2014) a. Prokaryotes b. Eukaryotes c. Plant d. Animalia 2. Which of the following is a yeast? a. Candida (Recent Pattern Dec 2012) b. Rhizopus c. Mucor d. Cryptococcus 3. Acute angled septate hyphae are seen in: a. Aspergillus (Recent Pattern Dec 2013) b. Mucor c. Penicillium d. Candida 4. Budding reproduction in tissue is seen in: a. Cryptococcus, Candida (Recent Pattern Nov2014) b. Candida, Rhizopus c. Rhizopus, Mucor d. Histoplasma, Candida 5. Ascospores are: (Recent Pattern Dec 2015) a. Asexual spores b. Sexual spores c. Conidia d. None of the above 6. All of the following fungi has sexual phase except: (Recent Pattern Dec 2016) a. Phycomycetes b. Fungi imperfecti c. Basidiomycetes d. Ascomycetes 7. Aseptate hyphae are not seen in: a. Rhizopus (Recent Pattern Dec 2012) b. Mucor c. Aspergillus d. Absidia 8. KOH mount is used to examine:  (Recent Pattern Aug 2013) a. Bacteria b. Virus c. Fungus d. Parasite 9. Culture medium used for isolation of fungus is: (Recent Pattern Dec 2014) a. NNN medium b. Potassium tellurite medium c. Chocolate agar d. Sabouraud’s dextrose agar 







10. All are dimorphic fungi except: (AIIMS May 2009) a. Blastomyces dermatitidis b. Histoplasma capsulatum c. Penicillium marneffei d. Phialophora 11. Systemic infection is caused by all of the fungi except: a. Cryptococcus (Recent Pattern Dec 2013) b. Histoplasma c. Dermatophytes d. Paracoccidioides 12. pH of SDA is: a. 5.4 b. 7.4 c. 7.2 d. 6.4 13. In thermally dimorphic fungi, mycelial phase occurs in which temperature: a. 25°C b. 37°C c. 45°C d. 15°C 14. Arthrospores are seen in: a. Coccidioides b. Histoplasma c. Dermatophytes d. Paracoccidioides 15. Special stain used to stain both live and dead fungi is: a. H and E b. PAS c. Gomori methenamine silver d. Toluidine blue 16. Not a dimorphic fungus (AIIMS Nov 2017) a. Blastomyces dermatitidis b. Histoplasma capsulatum c. Pneumocystis jirovecii d. Penicillium marneffei 17. Dimorphic fungi is/are: (PGI May 2017) a. Histoplasma capsulatum b. Sporothrix schenckii c. Malassezia furfur d. Cryptococcus neoformans e. Aspergillus

Chapter 48     Characteristics and Laboratory Diagnosis of Fungi

MULTIPLE CHOICE QUESTIONS



ANSWERS AND EXPLANATIONS 1. Ans.  (b) Eukaryotes Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 593 • • • •

Fungi – Eukaryotic Protista Rigid cell walls Has chitin, mannan and other polysaccharides True nuclei with nuclear membrane, paired chromosomes • Unicellular or multicellular • Divide sexually or asexually •

•

•

•

2. Ans.  (d) Cryptococcus Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 593

Classification of fungi based on cell morphology Yeast

Cryptococcus

Yeast like

Candida sp.,

•

•

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Unit 5     Mycology

3. Ans.  (a) Aspergillus

9. Ans.  (d) Sabouraud’s dextrose agar

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 613

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 597

• Septate hyphae with acute angle dichotomous branching is suggestive of Aspergillus species

• Agar used – SDA – Sabouraud dextrose agar – pH 5.4 • Emmon’s modification (Neutral) SDA – pH 7.2

•

4. Ans.  (a) Cryptococcus, Candida

•

•

10. Ans.  (d) Phialophora

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 594

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 595

• Yeast: Unicellular, reproduce by budding – E.g. Cryptococcus • Yeast like: Unicellular, reproduce by budding or by fission E.g. Candida

Examples for dimorphic fungi: • Blastomyces dermatitidis • Paracoccidioides brasiliensis • Coccidioides immitis • Histoplasma capsulatum • Sporothrix schenckii • Penicilliosis marneffei

•

•

•

•

•

•

5. Ans.  (b) Sexual spores Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 595 • Sexual spores: Ascospore, Zygospore, Basidiospore, Oospore • Asexual spores (Conidia): Sporangiospores, Arthrospores, Blastospores, Chlamydospores, Phialoconidia •

•

•

•

11. Ans.  (c) Dermatophytes Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 595 Systemic mycoses are: • Blastomycosis • Cryptococcosis • Paracoccidioidomycosis • Coccidioidomycosis • Histoplasmosis • Candidiasis •

6. Ans.  (b) Fungi imperfecti Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 595 • Based on sexual spore formation – Fungi are classified into Ascomytes, Basidiomycetes, Phycomycetes and Fungi Imperfecti • Fungi imperfecti is a special group which has no sexual spores e.g.: Coccidioides, Paracoccidiodes •

•

7. Ans.  (c) Aspergillus Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 594 • Moulds–Filamentous fungi–has hyphae – which is either septate or aseptate, reproduce by spore formation • Septate hyphae – E.g. Aspergillus • Aseptate hyphae – E.g. Zygomycetes – Mucor, Rhizopus, Absidia •

•

•

8. Ans.  (c) Fungus Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 596 • Specimens are first seen microscopically; After applying KOH in the specimen – wait for few minutes for the tissue debris to get removed so that fungal elements can be seen clearly •

362

•

•

•

•

•

12. Ans.  (a) 5.4 Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 595 • Already explained in Q.9 •

13. Ans.  (a) 25°C Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 595 • Thermally dimorphic fungi – Grow as yeasts in 37°C, as moulds in 25°C •

14. Ans.  (a) Coccidioides Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 595

Conidia

Examples

Sporangiospores

Mucor, Rhizopus

Arthrospores

Coccidioides

Blastospores

Cladosporum

Chlamydospores

Candida albicans

Phialoconidia

Aspergillus fumigatus

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 597 Periodic acid schiff (PAS)

Recommended stain for fungi – stains only live fungi

Gomori methenamine silver (GMS)

Stains both live and dead fungi

Mucicarmine stain

For Cryptococcus and Rhinosporidium

Masson Fontana stain

For pigmented fungi

H and E stain

For tissue stain

Toluidine blue

Pneumocystis carinii

16. Ans.  (c) Pneumocystis jirovecii Ref: Ananthanarayan and Paniker T.B of microbiology – 10th ed – page 593 Dimorphic fungi: • Blastomyces dermatitidis • Paracoccidioides brasiliensis • Coccidioides immitis • Histoplasma capsulatum • Sporothrix schenckii • Penicilliosis marneffei •

•

•

•

•

•

17. Ans.  (a) Histoplasma capsulatum; (b) Sporothrix schenckii • Already explained in Q. 16 •

Chapter 48     Characteristics and Laboratory Diagnosis of Fungi

15. Ans.  (c) Gomori methenamine silver

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49

Superficial Mycoses

There are two types of superficial mycoses: y Surface mycoses y Cutaneous mycoses

y y y y y

e.g. Dermatophytes, Candida albicans

SURFACE MYCOSES Pityriasis Versicolor (Tinea Versicolor)

y y y y

y It is also called as Tinea or Ringworm y Infection of skin, hair and nail (keratinized structures) are caused by dermatophytoses y Three genera:  Trichophyton  Microsporum  Epidermophyton

Table 2:  C  lassification of dermatophytes based on Morphology Genera

Site of infection

Macroconidia

Microconidia

Trichophyton

Skin, hair, nail

Very few, pencil shaped or cylindrical shaped

Abundant tear drop shaped in clusters

Microsporum

Hair and skin

Abundant spindle shaped

Rare

Epidermophyton

Skin and nail

Abundant club shaped

Absent

y

y

y

y

y

y

y

y

y It affects stratum corneum y Agent– Malassezia furfur: It causes Tinea versicolor, Folliculitis and seborrheic dermatitis y Lesions appear as macular areas of discoloration – depigmentation; DD - Vitiligo y Sites: Skin of the chest, abdomen, upper limbs and back y Seborrheic dermatitis manifests as erythematous pruritic scaly lesions seen in the eyebrows, moustache, nasolabial folds and scalp; scalp lesions seen in babies is called as cradle cap. y Diagnosis: Specimen – Skin scrapings – shows yeast like cells with short branched filaments y Culture: SDA with olive oil layer (lipophilic fungi) y Treatment: Topical creams and lotions like selenium sulfide shampoo, ketoconazole shampoo, terbinafine cream and ciclopirox cream.

CUTANEOUS MYCOSES Dermatophytoses



E.g. Pityriasis versicolor, Tinea nigra, Piedra



Inflammatory response and allergic response occurs



No inflammation (Since it does not affect living tissues)

y Irregular nodules in hair shaft y Black piedra – Piedraia hortae y White piedra – Trichosporon beigelii y

Living tissues affected

y

No contact with living tissues

Piedra

y

Affects the cornified layer of skin

y

Cutaneous Mycoses

Affects the dead layers

y

Surface Mycoses

Localized infection of Stratum corneum Agent: Exophiala werneckii, Exophiala castellanii Lesions: Brownish or black macular lesions Site: Palms Diagnosis: Skin scrapings – brownish, branched, septate hyphae (Pigmented fungi)

y

y

Table 1:  Different between surface and cutaneous mycoses

y

Tinea Nigra

y

INTRODUCTION

Figure 1: Skin lesions of Pityriasis versicolor

y

y

Figure 2: Trichophyton rubrum (Bird in fence pattern of micro conidia) (Courtesy: CDC PHIL/ Dr. Libero Ajello)

Chapter 49     Superficial Mycoses

y Ectothrix: Arthrospores are seen as a sheath surrounding the hair – caused by Microsporum, T.rubrum, T.mentagrophytes y Endothrix: Arthrospores are inside the hair shaft – caused by T.schoenleinii, T.tonsurans, T.violaceum

Table 3:  Classification of Dermatophytes based on Source of Infection Anthropophilic Human to human T.rubrum, T.tonsurans, transmission T.violaceum, T.schoenleini, M.audounii Zoophilic

Animal to human

T.mentagrophytes, T.verrucosum, M.canis

Geophilic

Soil to human

M.gypseum, M.fulvum

Figure 3: Ectothrix and Endothrix type of hair infection

Clinical Manifestations y Tinea capitis is seen mostly in children of age 3-7 years old; they have well demarcated scaly patches in the hair and that leads to alopecia:  Kerion: Boggy lesion in the scalp – usually in children  Favus: Dense crusts develop in the hair follicles – leads to alopecia y Tinea corporis has well demarcated, annular, pruritic, scaly lesions that has central clearing. y Tinea cruris is seen in men; the perineal rash also called as Jock’s itch – is erythematous and pustular without satellite lesions. y Tinea pedis is also more common in men than women with hyperkeratosis of soles. y

y Pathogenicity: They grow only in keratinized layers of skin and its appendages (Keratinophilic fungi) y Sometimes, after initiation of antifungal treatment → hypersensitivity to fungal antigens occurs → Dermatophytids (id reaction) y

y





y

Table 4:  Clinical features of dermatophytoses and their etiological agents Clinical infection Region

Causative organism

Tinea barbae (Barber’s itch)

Face and neck

T. rubrum, T. mentagrophytes, T. verrucosum

Tinea corporis

Body

T.rubrum (M/c) and any other

Tinea imbricata

Concentric rings of papulosquamous scaly patches in body

T.concentricum

Tinea capitis (Favus, kerion)

Scalp

Trichophyton and Microsporum

Tinea cruris (Jock itch)

Groin and perineum

T.rubrum, E.floccosum

Tinea pedis (Athlete’s foot)

Foot

T.rubrum, E.floccosum

Tinea manuum

Hand

T.rubrum

Tinea unguium (Onychomycosis)

Nails

T.rubrum, T.mentagrophytes, E.floccosum

y

y

Figure 4: Kerion

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Unit 5     Mycology













Sporotrichosis Rhinosporidiosis Entomophthoromycosis

Mycetoma y Fungal mycetoma also called as Maduromycosis or Madura foot y Three types: y

y

Table 5:  Types of mycetoma Types of Mycetoma

Organisms

Eumycetoma

Madurella mycetomatis, M.grisea, Exophiala sp., Acremonium sp., Aspergillus sp., Scedosporium, Fusarium sp.,

Actinomycetoma

Actinomadura sp., Streptomyces sp., Nocardia sp.,

Botryomycosis

Staphylococcus aureus and bacterial cause

Figure 5: Favus (Courtesy: CDC PHIL)

Laboratory Diagnosis y Specimens: Skin, hair, nail y KOH examination of specimen: Reveals branching hyaline septate (nonpigmented) hyphae y Wood’s lamp examination: Positive for Microsporum canis and certain Trichophyton species— Direct examination of hair with Wood’s lamp helps in visualization of affected hair (Due to the presence of metabolites) – But not 100% of dermatophytes can be diagnosed by Wood’s lamp y Culture: SDA with actidione and chloramphenicol y Hair perforation test: Positive in T.mentagrophytes y

y

y

y

y

y Triad: Swelling of the foot, Multiple sinuses, Discharge of granules y Granules or Grains are actually Micro colonies of the organisms y Color of the granules helps in presumptive identification of the organism y

y

y

Nocardia asteroides Nocardia brasiliensis Actinomadura madurae

White to yellow granules

Streptomyces somaliensis Scedosporium apiospermum Madurella mycetomatis Madurella grisea

Brown to black

Exophiala jeanselmei Actinomadura pelletieri

Red

y Treatment: Surgery – Main mode of treatment y Eumycetoma: Antifungal agents y Actinomycetoma: Dapsone, sulfonamides, cotrimoxazole, Rifampicin y

y

Figure 6: Spindle shaped macroconidia of M.canis (Courtesy: CDC/ Dr. Lucille K. Georg)

Treatment y y y y

y

y

y

y

Topical – Miconazole, Clotrimazole, Econazole Terbinafine cream Griseofulvin orally 100 mg tds Whitfield’s lotion – Benzoic acid

SUBCUTANEOUS MYCOSES y Predisposing risk factor – Trauma y Types are:  Mycotic mycetoma  Chromoblastomycosis y

y



366



y

Chromoblastomycosis y Fungal infection caused by pigmented fungi, also called as dematiaceous fungi y Called as verrucous dermatitis y Most commonly affects the agricultural workers and woodcutters → After a trauma → soil fungi → Enters the subcutaneous tissue of the feet and lower legs → causes Warty, Cutaneous nodules y Agents causing: Fonsecaea sp., Exophiala dermatitidis, Phialophora verrucosa, Cladophialophora y Laboratory diagnosis: Lesions – KOH mount - show irregular, dark brown with a septae yeast like bodies – Sclerotic bodies y Treatment: Amphotericin B, Thiabendazole, Itraconazole and Voriconazole y

y

y

y

y

y

y

y

y

y

y

y

Rhinosporidiosis y Polyps occurring in nose, mouth, eye rarely in genitals y Agent: Rhinosporidium seeberi y Pseudofungi; belongs to hydrophilic fungi – DRIP clade of aquatic protista y Endemic in Tamil Nadu, Kerala, Orissa, AP and Sri Lanka y Source of infection – Stagnant water like pool, lakes y Diagnosis – Spherules that contain numerous endospores are seen y It cannot be cultivated y Treatment – Surgery, to prevent recurrence – Dapsone y

Figure 7: Sclerotic bodies (Courtesy: CDC/ Dr. Libero Ajello)

y

Chapter 49     Superficial Mycoses

y Lab diagnosis: KOH / HPE – shows asteroid body y Culture: Yeast phase in 37deg C and mycelia phase in 25deg C y Serology: Slide latex agglutination test – Antigen is peptido – L- rhamno – D –mannan - ≥1:4 titre is positive (mainly helpful in pulmonary sporotrichosis) y Treatment: KI – Potassium iodide, Itraconazole, fluconazole, cryotherapy y Itraconazole is the DOC for lymphocutaneous sporotrichosis (IDSA guidelines) y Severe pulmonary or CNS spreads needs AmB lipid therapy

y

Phaeohyphomycosis

y

y Infections caused by pigmented fungi are called phaeohyphomycosis y Colored or melanin or pigmented fungi are called as dematiaceous fungi E.g. Phialophora, Cladosporium, Alternaria y Fungi enters the body through inoculation through the skin by prick; usually seen in tropical countries and rural workers. y Melanin is the virulence factor for all pigmented moulds. y Brain abscess is caused by pigmented fungi - Cladophialophora bantiana y Subcutaneous lesions with abscess are caused by Exophiala jeanselmei, E.spinifera, E.dermatitidis y Microscopy of the specimen or culture shows Brown colored hyphae y Surgical removal of the lesions with antifungal therapy is the treatment. y

y

y

y

y

y

y

y

y

y

y

y

Sporotrichosis y Causative agent: Sporothrix schenckii (Thermally dimorphic) y Fungal infection affecting cutaneous, subcutaneous and lymphatic tissue y Most commonly affects gardeners, forest workers → After a minor trauma (Rose thorn prick) → nodules formed → ulceration → necrosis → Fixed cutaneous sporotrichosis → spread to lymphatics → lymphocutaneous sporotrichosis → systemic spread to the bones, joints and meninges y DD: Nocardiosis, Tularemia, Non TB mycobacterial infection and leishmaniasis y

y

y

y

Figure 8: Spherules of Rhinosporidium seeberi (Courtesy: Dr.S.Jamuna Rani, Associate Professor of Pathology, Tagore Medical College & Hospital, Chennai, Tamil Nadu)

Remember

Chromoblastomycosis – Sclerotic bodies Sporotrichosis – Asteroid body Rhinosporidiosis - Spherules

367

Unit 5     Mycology

MULTIPLE CHOICE QUESTIONS 1. Tinea cruris is caused by: (Recent Pattern Dec 2015) a. Trichophyton rubrum b. M.canis c. T.verrucosum d. T.tonsurans 2. Trichophyton – Zoophilic species is: (Recent Pattern Dec 2012) a. T.tonsurans b. T.violaceum c. T.schoenleinii d. T.mentagrophytes 3. Pityriasis versicolor is caused by: (Recent Pattern Nov 2014) a. E.floccosum b. M.gypseum c. M.furfur d. T.tonsurans 4. Endothrix is caused by: (Recent Pattern Nov 2015) a. E.floccosum b. M.gypseum c. T.tonsurans d. M.canis 5. Black dot ring worm is caused by: (Recent Pattern Dec 2014) a. Microsporum b. Trichophyton c. Epidermophyton d. Candida 6. Most common cause of tinea capitis is: (Recent Pattern Dec 2013) a. M.canis b. E.floccosum c. T.tonsurans d. M.gypseum 7. Which of the following infects hair, skin and nails? (Recent Pattern Dec 2012) a. Microsporum b. Trichophyton c. Epidermophyton d. Trichosporon 8. White piedra is caused by: (MH 2011) a. Piedra hortae b. Malassasia furfur c. Hortaea werneckii d. Trichosporon beigelli 9. Color of granules produced by Actinomadura pelletieri (Recent Pattern Dec 2013) a. Black b. Yellow c. Red d. Brown 10. Granules discharged in mycetoma contains (Recent Pattern Dec 2013) a. Bone spicules b. Fungal colonies c. Pus cells d. Inflammatory cells 11. Rhinosporidium seeberi belongs to a. Fungus b. Aquatic Protista c. Protozoa d. Helminth 12. Sclerotic bodies are seen in: a. Rhinosporidiosis b. Chromoblastomycosis c. Candidiasis d. Entomophthoromycosis 13. Rose Gardner’s disease is a. Rhinosporidiosis b. Chromoblastomycosis c. Sporotrichosis d. Entomophthoromycosis 



14. Potassium iodide is used in the treatment of: a. Rhinosporidiosis b. Chromoblastomycosis c. Sporotrichosis d. Entomophthoromycosis 15. Dapsone is used to treat recurrence of: a. Rhinosporidiosis b. Sporotrichosis c. Entomophthoromycosis d. Candidiasis 16. A female from Himachal Pradesh presented with history of thorn prick, a year back has verrucous lesions in the skin with following microscopic findings; Identify the agent: (AIIMS 2017)







a. Blastomycosis c. Sporotrichosis

b. Phaeohyphomycosis d. Chromoblastomycosis

17. A 21 years old man presented with following skin lesion. Identify the causative agent for this infection: (AIIMS Nov 2018)











a. Trichophyton rubrum b. Microsporum spp c. Epidermophyton d. Aspergillus spp

ANSWERS AND EXPLANATIONS 1. Ans.  (a) Trichophyton rubrum

368

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 601 • Tinea cruris - T.rubrum, E.floccosum •

2. Ans.  (d) T. mentagrophytes Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 604 • Anthropophilic - T.rubrum, T.tonsurans, T.violaceum, T.schoenleini, M.audounii •

•

Nocardia asteroides

•

3. Ans.  (c) M. furfur

Nocardia brasiliensis Actinomadura madurae

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 601 • Agent: Malassezia furfur • Culture – SDA with olive oil layer (lipophilic fungi) •

•

Streptomyces somaliensis Scedosporium apiospermum Madurella mycetomatis Madurella grisea

4. Ans.  (c) T. tonsurans Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 601 • Ectothrix: Arthrospores are seen as a sheath surrounding the hair – caused by Microsporum, T.rubrum, T.mentagrophytes • Endothrix: Arthrospores are inside the hair shaft – caused by T.schoenleinii, T.tonsurans, T.violaceum

White to yellow granules

Brown to black

Exophiala jeanselmei Actinomadura pelletieri

Red

•

•

10. Ans.  (b) Fungal colonies

Chapter 49     Superficial Mycoses

• Zoophilic – T.mentagrophyte, T.verrucosum, M.canis • Geophilic - M.gypseum, M.fulvum

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 605 • Triad: Swelling of the foot, Multiple sinuses, Discharge of granules • Granules or Grains → Micro colonies of the organisms • Color of the granules helps in presumptive identification of the organism •

5. Ans.  (b) Trichophyton Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 601 • Another name for Dermatophytosis is Tinea or ring worm • Most common cause for black dot ring worm is Trichophyton tonsurans

•

•

•

•

6. Ans.  (a) M. canis

11. Ans.  (b) Aquatic Protista Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 607 • Pseudofungi; belongs to hydrophilic fungi – DRIP clade of aquatic Protista •

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 601 • Most common mode of presentation of Tinea capitis is Ectothrix • Most common organism causing Ectothrix is Microsporum canis •

12. Ans.  (b) Chromoblastomycosis Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 605

•

7. Ans.  (b) Trichophyton

• Chromoblastomycosis: Fungal infection caused by pigmented fungi, also called as dematiaceous fungi • Lab diagnosis: Lesions – KOH mount - show irregular, dark brown with a septae yeast like bodies – Sclerotic bodies •

•

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 601 Trichophyton

Skin, hair, nail

Microsporum

Hair and skin

Epidermophyton

Skin and nail

13. Ans.  (c) Sporotrichosis Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 606 • Causative agent: Sporothrix scheckii (Thermally dimorphic) • Most commonly affects gardeners, forest workers → After a minor trauma (Rose thorn prick) – hence called as Rose Gardner’s disease •

8. Ans.  (d) Trichosporon beigelli Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 600 • Black piedra – Piedraia hortae • White piedra – Trichosporon beigelii

•

•

•

9. Ans.  (c) Red Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 605

14. Ans.  (c) Sporotrichosis Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 606 • Treatment: KI – Potassium iodide, Itraconazole, fluconazole, cryotherapy •

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Unit 5     Mycology

15. Ans.  (a) Rhinosporidiosis Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 607 • Treatment: Surgery, To prevent recurrence – Dapsone •

16. Ans.  (d) Chromoblastomycosis Ref: Ananthanarayan and Paniker T.B of microbiology – 10th ed – page 605 • Fungal infection caused by pigmented fungi, also called as dematiaceous fungi • Called as verrucous dermatitis (because the lesions are verrucous) • Most commonly occurs after a minor trauma → soil fungi → Enters the subcutaneous tissue of the feet and lower legs → causes Warty, verrucous, cutaneous nodules •

•

•

370

• Lab diagnosis: Lesions - show irregular, dark brown with a septae yeast like bodies – Sclerotic bodies or medlar bodies or copper penny bodies •

17. Ans. (a) Trichophyton rubrum  

Ref: Ananthanarayan and Paniker`s T.B of microbiology – 10th ed – page 601 • Above image shows Tinea infection caused by dermatophytes. It is seen over the beard areas – so it is named as Tinea barbae (Barber`s itch) • All these species of Dermatophytes causes Tinea infection – species depends upon the area of infection and microbiological diagnosis • Barber`s itch is caused by Trichophyton species namely T. rubrum, T. mentagrophytes •

•

•

50

Endemic/Systemic Mycoses CHARACTERISTICS y

y

y Most of the infections are caused by soil fungi y Systemic infections are caused by dimorphic fungi. Blastomyces dermatitidis Paracoccidioides brasiliensis Coccidioides immitis Histoplasma capsulatum

y y

Figure 1: Cutaneous lesions of Blastomycosis (Courtesy: CDC/ Dr. Lucille K. Georg) y Antigen detection in urine is more sensitive than in serum. y

y y y y y y

Causative agent: Blastomyces dermatitidis Telemorph stage (sexual stage): Ajellomyces dermatitidis Region: North America (North American blastomycosis) Source of infection is soil Mode of infection is inhalation fungal spores enters the body through inhalation from the soil and then goes to lungs. Alveolar macrophages and polymorphonuclear leukocytes are most needed for phagocytosis and killing of inhaled conidia. Conidia upon entering inside the body, at body temperature (37°C) – it gets converted into thick walled yeast which are difficult to kill by phagocytosis. This conidia to yeast phase conversion leads to expression of protein called as BAD-1 (virulence factor) It causes acute pulmonary infection (resembles that of TB/ histoplasmosis) Pulmonary presentation – may be asymptomatic; sometimes consolidation and abscess can occur Fungus then spreads to the blood and to various organs (Disseminated blastomycosis) In cutaneous blastomycosis, a papule occurs followed by nodule develops – leading to ulcerative lesions; Site is usually skin of face or hands; Two types of skin diseases are seen:  Verrucous blastomycosis  Ulcerative blastomycosis It also causes osteomyelitis, which usually affects vertebrae, sacrum, pelvis, skull and ribs. CNS blastomycosis presents as brain abscess.

Treatment Table 1:  Treatment of blastomycosis Patient condition Type of disease

Treatment

Immunocompetent Pulmonary patient/life threatening disease Disseminated CNS

Lipid AmB or Itraconazole

Disseminated Non - CNS

Lipid AmB or itraconazole

Lipid AmB or fluconazole

Immunocompetent Pulmonary or patient/ non-life disseminated threatening disease non CNS

Itraconazole or fluconazole or ketoconazole

Immunocompromised patient

Lipid AmB or AmB deoxycholate

All infections

y

y

y Body tissue/Culture at 37°C shows Yeast phase – single broad bud with double contoured wall y Culture at 25°C (Room temperature) shows Septate hyphae with round to oval conidia

y y y

y y y y y

y

Laboratory Diagnosis

PARACOCCIDIOIDOMYCOSIS

y

y

y

y

y





y

y

y

y

y

y

y

y

y y y y y

y

BLASTOMYCOSIS

Causative agent: Paracoccidioides brasiliensis Region: South America (South American blastomycosis) Source of infection is soil Mode of infection is inhalation Fungal spores are inhaled: Primary pulmonary infection – spreads through blood – to mucosa of nose, mouth, GIT, skin, lymphatics

Unit 5     Mycology

y Leads to ulcerative granuloma of the buccal and nasal mucosa y Laboratory diagnosis:  Body tissue/Culture at 37°C – Yeast phase – large, globose or oval cells with multiple buds surrounding the mother cell  Culture at 25°C (Room temp) – Septate hyphae with small conidia  Appearance: Mariner’s wheel or Pilot’s wheel or Captain’s wheel y

y



Š Immunodiffusion test Š EIA (most frequently used screening technique) y Organism is highly infective; bioterrorism agent; should be worked always in biological safety cabinet. Š

Š

y





COCCIDIOIDOMYCOSIS y Causative agent: Coccidioides immitis y Two species are there: C. immitis and C. posadasii y Region: USA – called as Rift valley fever or Desert Rheumatism y Source of infection is soil y Mode of infection is inhalation y Arthrospores which are present in the dust are inhaled and it enters the lungs y 60% of the infected individuals are completely asymptomatic and 40% have symptoms y

y

y

y

y

Figure 2: Spherule of Coccidioides (Tissue stage) (Courtesy: CDC/ Dr. Lucille K. Georg)

y

y

Clinical Features y Respiratory infection is mostly asymptomatic and it gives lifelong immunity y It presents as a flu like illness which is mild and self-limiting, called as Desert rheumatism y DD: Community acquired bacterial pneumonia y In very few persons, there occurs a chronic progressive disseminated disease – Coccidioidal granuloma (Fatal) y Cutaneous manifestations of primary pulmonary coccidioidomycosis are toxic erythema, erythema nodosum and arthralgia. y Rarely, Coccidioidal meningitis can occur which is always fatal. y

y

y

y

y

Figure 3: Barrel shaped arthroconidia of Coccidioides

y

Table 2:  Clinical clue for primary pulmonary coccidioidomycosis • History of residence or travel to endemic area (western hemisphere) • History of night sweats • Profound fatigue • Peripheral blood eosinophilia • Hilar or mediastinal lymphadenopathy in CXR • Failure to improve with antibiotics •

•

•

Treatment Table 3:  Treatment of Coccidioidomycosis Clinical type

Treatment

Asymptomatic infection

None

Primary pneumonia

None

Diffuse pneumonia

Amphotericin B followed by oral itraconazole for months

Pulmonary sequelae

None, but when there is chronic pneumonia start on oral itraconazole

Disseminated disease

Lifelong triazole therapy for meningitis

•

•

•

Laboratory Diagnosis 







372









Body tissue/Culture at 37°C shows Spherules which are thick double layered wall filled inside with endospores Culture at 25°C (Room temperature) in blood agar shows colonies; these colonies when seen under microscope have Hyphae that looks fragmented giving rise to arthrospores (Barrel shaped) DTH skin test with coccidioidin – positive (used in endemic areas) Serological tests play a major role in diagnosis: Š Tube precipitin test Š Complement fixation test Š

Š

HISTOPLASMOSIS y Causative agent: Histoplasma capsulatum  Two types: H.capsulatum.var.capsulatum, H.capsulatum. var.duboisii  Disease identified by Darling – hence called as Darling’s disease y





y

y

y

y

y

Clinical Features y In immunocompetent individuals with low level of exposure – infections are usually mild and self-limiting y Most of the adults in endemic area have 50-80% positive skin tests suggest that they had mild infections. y Heavy exposure leads to flu like illness with CXR showing pneumonitis with hilar or mediastinal lymphadenopathy. y Classical histoplasmosis – asymptomatic – heal with just a miliary calcification y DD: Tuberculosis y Progressive Disseminated histoplasmosis (PDH) – occurs only in immunocompromised individuals which affects Reticuloendothelial system (intracellular infection) and it is highly fatal

y Specimens from tissues shows yeast cells which are seen within phagocytic cells (intracellular) y Growth at 37°C produces yeast cells y Room temperature – Spores with tubercles (finger like projections) y Detection of histoplasma antigen in body fluids helps in diagnosis of PDH and acute diffuse pulmonary histopladmosis. y Histoplasmin skin test helps in endemic area to know the disease burden. y

y

y

y

y

y

y

y

y

y

Chapter 50     Endemic/Systemic Mycoses

y Region: USA, Africa y H.capsulatum.var.duboisii – responsible for African histoplasmosis y Source of infection is soil, rotting trees, birds y Mode of infection is inhalation of microconidia y Inhalation of spores – pulmonary entry

y

Risk factors for PDH are: • AIDS with CD4 counts less than 200/uL • Extremes of age • Immunosuppressive drug therapy • Inflammatory diseases •

•

•

•

y Lymphadenopathy, hepatosplenomegaly, fever, anaemia occurs y African histoplasmosis – affects skin, subcutaneous tissues and bones; lungs infection and dissemination is very rare. y Chronic cavitary histoplasmosis (resembles TB) is seen in smokers. y In healed histoplasmosis – the calcified mediastinal nodes or lung parenchyma erodes the airways and causes hemoptysis. This condition is called as broncholithiasis. y

y

Figure 4: Tuberculate spores (Courtesy: CDC)

Treatment Table 4:  Treatment of histoplasmosis Type of histoplasmosis Treatment Acute pulmonary illness with diffuse infiltrates

Lipid amphotericin B ± glucocorticoids for 1-2 weeks Followed by Itraconazole for 12 weeks

Chronic/Cavitary pulmonary

Itraconazole bd for at least 12 months

Progressive disseminated

Lipid amphotericin B for 1-2 weeks followed by Itraconazole for 12 weeks

Central nervous system involved

Lipid amphotericin B for 4-6 weeks followed by Itraconazole for 12 months

y

y

Laboratory Diagnosis y Gold standard for diagnosis is fungal culture y Cultures are positive in 75% cases of PDH and chronic pulmonary histoplasmosis y

y

373

Unit 5     Mycology

MULTIPLE CHOICE QUESTIONS 1. Which of the following is not an endemic mycosis: (Recent Pattern Dec 2013) a. Histoplasmosis b. Blastomycosis c. Cryptococcosis d. Candidiasis 2. Darling disease is caused by (Recent Pattern Dec 2012) a. Histoplasma b. Candida c. Cryptococcus d. Rhizopus 3. “Tuberculate spores” are characteristic feature of (Recent Pattern Dec 2014) a. Candida b. Histoplasma c. Coccidioidomycosis d. Cryptococcus 4. Fungus that infects reticuloendothelial cells is: (Recent Pattern July 2016) a. Cryptococcus b. Candida c. Aspergillus d. Histoplasma 5. Valley fever or desert rheumatism is caused by (Recent Pattern Dec 2013) a. Sporothrix b. Coccidioides c. Phialophora d. Histoplasma

6. A patient with HIV develops diarrhea n fecal examination shows Isospora belli. He was given treatment with TMPSMX. Diarrhea subsided but fever persisted. Bone marrow examination showed the following picture with an intracellular fungi. Which of the following is wrong statement:





















a. b. c. d.

It cannot be grown in SDA Spores are infective form It is intracellular budding yeast It can cause systemic disease

ANSWERS AND EXPLANATIONS 1. Ans.  (d) Candidiasis

ƒ 37°C – yeast cells ƒ Room temperature – Spores with tubercles (finger like ƒ

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 609 • • • • •

•

•

•

•

•

Blastomyces dermatitidis Paracoccidioides brasiliensis Coccidioides immitis Histoplasma capsulatum Cryptococcus sp

2. Ans.  (a) Histoplasma Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 609 • Histoplasmosis - Causative agent: Histoplasma capsulatum ƒ Two types: H.capsulatum.var.capsulatum, H.capsulatum.var.duboisii ƒ Disease identified by Darling – hence called as Darling’s disease •

ƒ

ƒ

ƒ

projections) 4. Ans.  (d) Histoplasma Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 610 • Classical histoplasmosis – asymptomatic – heal with just a miliary calcification • Disseminated histoplasmosis – occurs in few – affects Reticuloendothelial system (intracellular infection) – highly fatal • Lymphadenopathy, hepatosplenomegaly, fever, anemia occurs •

•

•

5. Ans.  (b) Coccidioides Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 611 • Coccidioidomycosis - Causative agent: Coccidioides immitis • Region: USA – called as Rift valley fever or Desert Rheumatism •

3. Ans.  (b) Histoplasma Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 610 • Lab diagnosis of Histoplasmosis: ƒ Tissues – yeast cells occurs within phagocytic cells (intracellular) •

374

ƒ

•

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th ed – page 610 • Causative agent: Histoplasma capsulatum • Mode of infection is inhalation of spores • Inhalation of spores: pulmonary entry ƒ Classical histoplasmosis: asymptomatic – heal with just a miliary calcification ƒ Disseminated histoplasmosis: occurs in few – affects Reticuloendothelial system (intracellular infection) highly fatal •

•

•

ƒ

ƒ

ƒ Lymphadenopathy, hepatosplenomegaly, fever, aneƒ

mia occurs. ƒ African histoplasmosis: affects skin, subcutaneous ƒ

tissues and bones; lungs infection and dissemination is very rare. • Lab diagnosis: ƒ Tissues – yeast cells occurs within phagocytic cells (intracellular) ƒ 37°C – yeast cells ƒ Room temperature – Spores with tubercles (finger like projections) ƒ Fungi grows in SDA and blood agar ƒ SDA – colonies are white, cottony, mycelial growth •

ƒ

ƒ

ƒ

ƒ

ƒ

Chapter 50     Endemic/Systemic Mycoses

6. Ans.  (a) It cannot be grown in SDA

375

Opportunistic Mycoses

•

• Aspergillosis •

• Penicilliosis

Table1: Microscopic appearance of various species of Aspergillus  

51

Species

Microscopic appearance

Aspergillus fumigatus

Uniseriate, Conidia covers only upper one third of vesicle,

Aspergillus flavus

Uniseriate and Biseriate, covers entire vesicle

Aspergillus niger

Biseriate, covers entire vesicle – Black colored

•

• Zygomycosis •

• Candidiasis •

• Cryptococcosis •

• Pneumocystis jirovecii

ASPERGILLOSIS

y

y

y

y

y

y

y Aspergillosis is a clinical term used to describe all diseases that are caused by species of aspergillus; y The species that grows at 37°C can cause invasive infections and others can cause only allergic manifestation. y Most common species are: Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger and Aspergillus terreus. y Aspergillus is seen ubiquitously in all the environments especially in decomposing plant materials and in bedding. y Incubation period of invasive aspergillosis after inhalation is 2 to 90 days. y Main important risk factors for aspergillosis are:  Profound neutropenia  Glucocorticoid usage 



Figure 1: Acute angled hyphae of Aspergillus (Courtesy: CDC)

Clinical Features



y

y

y

y Microscopy – Septate hyphae with acute angle branching at 45°C y Morphology of each species differs and identification is mainly by the conidial arrangement

y

y

Laboratory Diagnosis

y Serological test: Antibodies can be demonstrated by ELISA, counter current immunoelectrophoresis and immunodiffusion y Serological tests are most helpful in allergic aspergillosis y Intradermal injection of Aspergillus antigen – Immediate reaction (Type I hypersensitivity) and Arthus type reaction (Type III reaction) y Beta-D-glucan assay helps in invasive aspergillosis y













y

y Clinical diseases caused by Aspergillus are:  ABPA – Allergic Bronchopulmonary Aspergillosis – Type I (M/c) and Type III hypersensitivity reactions that occurs after inhalation of spores – spores enters inside lungs and grows within the lumen of bronchioles – occludes them  Aspergilloma – Fungus grows within and occurs as a fungal ball  Invasive aspergillosis – Pneumonia occurs then dissemination starts to involve all organs – occurs in immunocompromised individuals, and those who are taking chronic steroids.  Otomycosis, mycotic keratitis, sinusitis  Cerebral aspergillosis  Cutaneous aspergillosis  Endocarditis

y









ZYGOMYCOSIS y Zygo/Mucormycosis is a group of life threatening infections that are caused by fungus belonging to order mucorales y Infections have high morbidity and high mortality. y Other names: mucormycosis and Phycomycosis y Organisms causing: Rhizopus, Mucor, Rhizomucor, Absidia y These fungi are seen ubiquitously in the moist environment causing exposure to humans often. y But infections are caused mainly to diabetics and other immunosuppressive patients. y

y

y

y

Definitive confirmation of aspergillosis needs: • Positive culture of a sample taken directly from an ordinarily sterile site like brain abscess site • Positive results of both histologic testing and culture of a sample taken from an affected organ •

•

Treatment

y

y

Risk factors of Zygomycosis: • Diabetes mellitus especially diabetic ketoacidosis • Patients on glucocorticoid therapy • Neutropenic patients • Iron overloaded patients (end stage renal failure patients) • Haematopoietic stem cell transplantation patients •

Table 2:  Treatment of Aspergillosis

•

Chapter 51     Opportunistic Mycoses

y Criteria for diagnosing ABPA includes microbiological parameters like:  Serum precipitating antibodies  Elevated serum IgE  Elevated serum IgE and IgG antiaspergillus antibodies  Arthus (late) skin reaction

•

Type of aspergillosis

Primary treatment

Secondary treatment

Invasive aspergillosis

Voriconazole

AmB, Caspofungin, Micafungin

Prophylaxis

Posaconazole or Itraconazole

Micafungin, Aerosolized AmB

Single aspergilloma

Surgery

Itraconazole, Voriconazole

y Pathogenesis: Spores from the soil or environment enters the body – respiratory cavity – enters the orbit, sinuses and brain; systemic dissemination (occurs most commonly in diabetics and immunosuppressed individuals, long term steroids); Fungus causes vascular invasion and leads to thrombosis and infarction thus it is a fatal condition

Chronic pulmonary

Itraconazole or Voriconazole

Posaconazole, IV micafungin

ABPA

Itraconazole

Voriconazole, Posaconazole

Clinical Features

•

•

y

y Six categories of mucormycosis are seen:  Rhino-orbital-cerebral  Pulmonary  Cutaneous  Gastrointestinal  Disseminated  Miscellaneous y Rhinocerebral mucormycosis:  Most common form is Rhinocerebral form  Patient presents with eye or facial pain and numbness and conjunctival suffusion.  Severe cases present with bilateral proptosis, chemosis, vision loss and opthalmoplegia, which indicates cavernous sinus thrombosis.  When there is a sign of painful necrotic ulcerations in the hard palate – it indicates well established infection that is almost fatal y





PENICILLIOSIS





y Ubiquitous species present in the environment y Clinical features: Fever, Papulo necrotic skin lesions, weight loss, anemia, lymphadenopathy, hepatosplenomegaly y In HIV infected individuals, papulonecrotic skin lesions seen which are caused by Penicillium marneffi. It is endemic in north east India, Thailand and Myanmar. y Penicillium marneffei – Dimorphic fungi y Produces dark red colored pigment in culture media y Treatment: Amphotericin B, Oral Itraconazole y

y

y

y

y

y





y









Laboratory Diagnosis y Microscopy of the clinical specimen shows: Broad, aseptate hyphae y Culture: Thick cottony fluffy colonies y

y

Figure 2: Pigmented Penicillium marneffei (Courtesy: CDC/ Dr. Libero Ajello)

Mucor sp.,

Sporangiophores arising randomly along the aerial mycelium; No rhizoids

Rhizopus sp.,

Sporangiophores arise in groups directly above the rhizoids

377

Unit 5     Mycology

y Balanitis y Erosio interdigitalis blastomycetica – infection between the digits of hands and toes. y Chronic mucocutaneous candidiasis (CMC) – have an association with endocrine abnormalities named as APECED syndrome – autoimmune polyendocrinopathy, candidiasis, ectodermal dystrophy. This syndrome is due to mutations in the AIRE gene. y Septicemia y Meningitis y Endocarditis y

y

y

y

y

y

Table 3:  Risk factors for disseminated candidiasis Figure 3: Mucor sp., (Courtesy: CDC/Dr. Lucille K. Georg)

• History of anti bacterial agents • Indwelling intravenous or urinary catheters • Hyperalimentation fluids •

• AIDS •

y When organisms grow in the culture plate – it is must to know about the clinical condition – as these fungi are environmental contaminants of culture plates; Clinical correlation should be done to know whether the organism is a true pathogen y Most sensitive and specific modality for definitive diagnosis is Biopsy with histopathological examination. Biopsy clearly shows wide, thick walled, ribbon like, aseptate hyphal elements that branch at right angles. y MRI is most sensitive method in orbital and CNS diseases.

Laboratory Diagnosis

Treatment

y Gram staining of the specimen shows Gram positive budding yeast cells with pseudohyphae or hyphae

y

y

y

•

•

• History of surgery (abdominal) • History of immunosuppresive drugs intake • Neutropenia • Low birth weight neonates • Diabetes mellitus •

•

•

• Intake of steroids • Severe burns •

•

•

•

y

Primary antifungal therapy

AmB deoxycholate Liposomal AmB

Primary combination therapy

Caspofungin + lipid polyene Micafungin + Anidula fungin + lipid polyene

CANDIDIASIS y Candida can cause almost all of the fungal infections described except mycetoma y Most common species: Candida albicans – yeast like fungi y Other species are grouped together named as Candida non albicans – C.glabrata, C.tropicalis, C.keyft, C.krusei, C.guilliermondii, C.parapsilosis and C.stellatoidea y They are normal commensals in skin and mucosa in our body y When immunosuppression occurs, they become pathogenic y

y

y

y

y

Clinical Features y Cutaneous candidiasis: Affects mainly groin, perineum, axillae and folds y Paronychia: Painful swelling in nail and skin interface y Onychomycosis: Fungal infection of nail y Vulvo vaginitis: Presents as pruritis, pain and vaginal curdy white discharge. y Oral thrush: Characterized by white, adherent, painless, discrete pateches in mouth, tongue and in esophagus. y Intestinal candidiasis (occurs in patients who are taking chronic antimicrobial therapy) y

y

y

y

y

y

378

Figure 4: Yeast cells in vaginal smear (Courtesy: Image from author’s own thesis) y It has pseudohyphae y To differentiate whether it is a commensal or pathogen – demonstration of mycelial forms is necessary y SDA growth shows Creamy white colonies y Differentiation of Candida species is based on glucose fermentation and assimilation tests, chlamydospore production, color production in CHROM agar y Corn meal agar shows production of chlamydospores which is diagnostic of Candida albicans (called as Dalmau plate culture technique) y Germ tube production – Reynolds Braude phenomenon diagnostic of Candida albicans y CHROM agar shows the colors produced by different species which helps in identification of species. y Β –D- glucan assay helps in invasive candidiasis y

y

y

y

y

y

y

y

y Cryptococcus is a yeast fungi that is the etiological agent for cryptococcosis. y Cryptococcosis also called as European blastomycosis, Torulosis y Main agent:  Cryptococcus neoformans var neoformans  Cryptococcus neoformans var.gattii  Cryptococcus neoformans var. grubii y Capsular serotypes: A, B, C, D (four types) y Capsule is made up of polysaccharide – it inhibits phagocytosis y Source of infection: Pigeon feces (types A and D) and Eucalyptus tree bark (Type B) y

y

y







y

y

y

Figure 5: Dalmau plate culture technique done for speciation of Candida (Source: Image from author’s own thesis work)

Cryptococcus neoformans

Source is from Avian excreta (Pigeon droppings)

Cryptococcus gattii

Source is from Eucalyptus tree bark

Chapter 51     Opportunistic Mycoses

CRYPTOCOCCOSIS

y Other non neoformans species are – C. albidus, C. laurentii y Teleomorph or sexual stage – Filobasidiella neoformans y Pathogenesis: Route of entry is inhalation:  Pulmonary cryptococcosis (M/c form) – mild pneumonitis  Cryptococcal meningitis – (M/c) in AIDS patients – region affected are basal ganglia or head of caudate nucleus  It also affects skin and bone y

y

y







Clinical Features y Usually it presents as chronic meningoencephalitis y Most common cause of meningitis in AIDS patients is Cryptococcus y Clinically it shows headache, fever, lethargy, sensory deficits, memory deficits, cranial nerve palsies, vision defects and meningismus. y Meningeal cryptococcosis can lead to sudden vision loss. y Lesions are most commonly seen in basal ganglia and at the head of caudate nucleus y Skin lesion is seen in patients with disseminated cryptococcosis. It may present as papules, plaques, purpura, vesicles and rashes. y

y

Figure 6: Candida species in microscopy (Courtesy: CDC/Dr. Stuart Brown)

Treatment

y

y

y Imidazoles, Amphotericin B, 5-Fluorocytosine, Disseminated infections – Echinocandins y

y

y

Table 4:  Treatment of Candidiasis Type of Candidiasis

Prime drug

Alternative therapy

Cutaneous

Topical azoles

Topical nystatin

Vulvovaginal

Oral flucanazole + suppositories

Nystatin suppository

Clotrimazole paints

Nystatin or Flucanazole

Oral thrush Esophageal candidiasis

Fluconazole tablets or itraconazole solutions

Disseminated Candidiasis

Amphotericin B deoxycholate Azoles Echinocandins

Caspofungin, Micafungin or amphotericin B

Laboratory Diagnosis y CSF analysis on performing wet mount with India Ink or nigrosine stain shows capsulated yeast cells y Cell count for CSF shows mononuclear cell pleocytosis with increased protein levels y Serological assay that has CRAg detection in CSF and blood is helpful; it is done by Latex agglutination test – for polysaccharide antigen (most useful in meningitis – bed side test) y Special media: Bird seed agar, sunflower seed agar y

y

y

y

Treatment y Amphotericin B with Flucytosine as lifelong maintenance therapy y

379

Unit 5     Mycology

Figure 7: Capsulated yeast cells in wet mount of CSF (Courtesy: CDC/ Dr. Leanor Haley)

380







1. Aspergillus causes all except:(Recent Pattern Dec 2013) a. Bronchopulmonary allergy b. Otomycosis c. Dermatophytosis d. Keratitis 2. Allergic BPA which kind of hypersensitivity reaction  (Recent Pattern Nov 2015) a. Type I b. Type II c. Type III d. Type IV 3. Aflatoxins are produced by  (Recent Pattern Dec 2013) a. Aspergillus flavus b. Aspergillus niger c. Aspergillus fumigatus d. Candida 4. Renauld Braude phenomenon is seen in  (Recent Pattern Dec 2012) a. Candida albicans b. Candida glabrata c. Histoplasma d. Cryptococcus 5. Candida albicans causes all the following except (Recent Pattern Dec 2015) a. Endocarditis b. Mycetoma c. Meningitis d. Oral thrush 6. Latex agglutination study of the antigen in CSF helps in the diagnosis of (Recent Pattern Jul 2016) a. Cryptococcus b. Candidiasis c. Aspergillosis d. Histoplasmosis 7. Primary site of infection of Cryptococcus is (Recent Pattern Dec 2013) a. Adrenal b. Bone c. Central nervous system d. Lung 8. A patient presented with headache and projectile vomiting along the alteration in sensorium. The following parasite demonstrated on Indian Ink staining. What is the likely diagnosis? (AIIMS Nov 2015)

10. Orbital mucormycosis is a complication of a. AIDS b. Steroid therapy c. Cushing’s disease d. Diabetic ketoacidosis 11. 1,3 beta D-glucan is helpful for identification of: (AIIMS Nov 2017) a. Invasive candidiasis b. Rhizopus c. Cryptococcus d. Mucormycosis 12. Identify the following organism (AIIMS Nov 2017)





Chapter 51     Opportunistic Mycoses

MULTIPLE CHOICE QUESTIONS













a. Cryptococcus b. Blastomyces c. Histoplasma d. Coccidioides 9. A diabetic patient presents with pus from eye. Colonies of isolated organisms are black with microscopic feature of non-septate hyphae and obtuse branching. Diagnosis is (Recent Pattern Dec 2013) a. Aspergillosis b. Candidiasis c. Mucormycosis d. Histoplasmosis

a. Acute angle branching Aspergillus b. Obtuse angled Mucor c. Acute angled Penicillium d. Obtuse angle Rhizopus 13. A 20-year-old female patient with HIV antibody test shows positive result showed a CD4 count of 50. She has been diagnosed with Cryptococcal meningitis. Which of the following is/are suits this condition?  (PGI pattern 2017) a. Collection of CSF and detection of capsular polysaccharide of the organism helps in diagnosis of Cryptococcal meningitis b. On bed side, Dark field microscopy helps in diagnosis c. Mode of infection is mainly due to food contaminated with pigeon excreta d. Long term prophylactic treatment is must with Flucanozole to prevent further episodes e. Isolation is must as it leads to transmission to other patients in ward 14. Which of the following fungus gives germ tube test positive? (PGI pattern 2017) a. Mucor sp., b. Candida albicans c. Candida dubliniensis d. All candida species e. Aspergillus sp.,



15. Which of the following will give positive β-D-Glucan test: (PGI May 2018) a. Mucor b. Candida c. Pseudomonas aeruginosa d. Aspergillus e. Cryptococcus

381

Unit 5     Mycology

(AIIMS May 2018)

16. Following test is shown positive for identification of:



a. Candida albicans c. Candida parapsilosis

b. Candida glabrata d. Candida tropicalis

ANSWERS AND EXPLANATONS 1. Ans.  (c) Dermatophytosis Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 613 • ABPA – Allergic Broncho Pulmonary Aspergillosis • Aspergilloma – Fungus grows within and occurs as a fungal ball • Invasive aspergillosis • Otomycosis, mycotic kertatitis, sinusitis •

•

•

•

2. Ans.  (a) Type I Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 613

5. Ans.  (b) Mycetoma Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 616 • Clinical features of Candidiasis ƒ Cutaneous candidiasis – affects mainly groin, perineum, axillae and folds ƒ Paronychia ƒ Onychomycosis ƒ Vulvo vaginitis ƒ Oral thrush ƒ Intestinal candidiasis (occurs in patients who are taking chronic antimicrobial therapy) ƒ Septicemia ƒ Meningitis ƒ Endocarditis •

ƒ

ƒ

ƒ

ƒ

ƒ

ƒ

ƒ

• ABPA – Allergic Broncho pulmonary Aspergillosis – Type I (M/c) and Type III hypersensitivity reactions that occurs after inhalation of spores – spores enters inside lungs and grows within the lumen of bronchioles – occludes them •

3. Ans.  (a) Aspergillus flavus Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 619 • Fungal toxins contaminates the food – mycotoxicoses • Aflatoxins are mostly produced by Aspergillus flavus and Aspergillus parasiticus •

•

4. Ans.  (a) Candida albicans Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 616 • When few candida colonies are incubated in human serum for two hours - Germ tube production occurs– Reynolds Braude phenomenon - diagnostic of Candida albicans •

ƒ

ƒ

6. Ans.  (a) Cryptococcus Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 618 • Latex agglutination test – for polysaccharide antigen (most useful in meningitis – bed side test) •

7. Ans.  (d) Lung Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 618 • Pathogenesis: Route of entry is inhalation; • Pulmonary cryptococcosis (M/c form) – mild pneumonitis •

•

8. Ans.  (a) Cryptococcus Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 617 • Clinical picture with diagnostic image confirms the diagnosis of Cryptococcal meningitis •

382

•

• Cryptococcus neoformans – oval budding yeast, with polysaccharide capsule surrounding it • Grows abundantly in soil containing bird dropping – Pigeon excreta • Humans occur infection by inhalation of organism from pigeon droppings • No human to human transmission • Usually asymptomatic, or mild infection • In immunocompromised individuals – Dissemination occurs – meningitis occurs; HIV – IRIS – Cryptococcal meningitis occurs • Lab diagnosis: Spinal fluid aspiration – India ink preparation; or latex particle agglutination test for detection of capsular polysaccharide • Treatment: Liposomal amphotericin B (Treatment); Flucanozole (long-term prophylaxis) •

•

9. Ans.  (c) Mucormycosis Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 614 • Spores from the soil or environment enters the body – respiratory cavity – enters the orbit, sinuses and brain; systemic dissemination (occurs most commonly in diabetics and immunosuppressed individuals, long term steroids) • Most common form is Rhinocerebral form •

•

10. Ans.  (d) Diabetic ketoacidosis ; (b) Steroid therapy

•

•

•

•

•

•

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 614 • Already explained Q.9

Chapter 51     Opportunistic Mycoses

• CSF analysis – Wet mount with India Ink or nigrosine stain shows capsulated yeast cells

14. Ans.  (b) Candida albicans; (c) Candida dubliniensis

•

When a question like this is asked in NEET, choose DKA over steroid therapy

Ref: Review of medical microbiology – 13th edition – Levinson – page 903 • Germ tube test helpful in classification and identification of Candida species • Growth inoculated in human serum for two hours and on visualization with wet mount preparation shows elongated buds from the yeast cells – germ tube – reynold braude phenomenon • Positive only in Candida albicans and Candida dublinensis • To differentiate above both, growth at 45°C will be positive in dublinensis. •

11. Ans.  (a) Invasive candidiasis Ref: T.B of mycology – Jagdish Chander – 3rd edition – Page 353 • Antigen which is helpful in diagnosis of invasive fungal infections is 1,3-beta D glucan test • It is called as G test • Helpful to diagnose invasive candidiasis, invasive aspergillosis • But it cannot distinguish whether it is aspergillosis or candidiasis •

•

•

•

•

•

•

12. Ans.  (a) Acute angle branching Aspergillus Ref: Ananthanarayan and Paniker T.B of microbiology – 10th ed – page 613 • Most common species are: Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger • Microscopy – Septate hyphae with acute angle branching at 45°C • Morphology of each species differs and identification is mainly by the conidial arrangement • Above image is GMS stain from tissue sections •

•

•

•

15. Ans.  (b) Candida; (d) Aspergillus Ref: Ananthanarayan and Paniker’s Microbiology – 10th ed – Page 616 Q.11

Textbook

of

Textbook

of

16. Ans.  (a) Candida albicans Ref: Ananthanarayan and Paniker’s Microbiology – 10th ed – Page 616

The above image shows Germ tube production • When few candida colonies are incubated in human serum for two hours - Germ tube production occurs– Reynolds Braude phenomenon - diagnostic of Candida albicans •

13. Ans.  (a) Collection of CSF and detection of capsular polysaccharide of the organism helps in diagnosis of Cryptococcal meningitis; (d) Long term prophylactic treatment is must with Flucanozole to prevent further episodes Ref: Review of medical microbiology – 13th edition – Levinson – page 910

383

52 Miscellaneous Fungi Mycetism y This means eating fungus itself is a poison Claviceps species

Ergot poisoning

Caprine species

Coprine poisoning

Inocybe species

Muscarine poisoning

Mycotoxicosis y

Fusarium

Ochratoxin

Aspergillus and Penicillium

Ergotoxin

Claviceps purpurea

Zearalenone

Fusarium graminearum

OCULOMYCOSIS

•

• •

• • • •

•

•

• Dematiaceous fungi

•

•

•

• Fusarium solani • Aspergillus fumigatus • Paecilomyces sp • Penicillium spp

•

Table 1:  Fungi causing keratitis Candida albicans Candida tropicalis Candida parapsilosis Cryptococcus neoformans

y

y y y y y y y y

y

FUNGI CAUSING ENDOPHTHALMITIS

y

y

y

y Drug of choice: TMP-SMZ (Hence when CD4 count comes down in HIV patients – TMP-SMZ is started as prophylaxis) y Alternative drugs given are TMP + Dapsone/Atovaquone/ Clindamycin + Primaquine or pentamidine (in sulfa drug allergic patients)

Fumonisin

y

 

Treatment

Aspergillus sp.,

y



Fungus

Aflatoxin

y



Ideal specimen: Bronchoalveolar lavage (BAL) - demonstration of organisms is 100% sensitive and specific. Stains: Giemsa, Toluidine blue, Methenamine silver, Calcofluor white stain – for Trophozoites Methanamine silver stain- for Cysts Not cultivable Complement fixation test titer ≥1:4 – active disease Classic CXR findings are diffuse bilateral interstitial infiltrates that are perihilar and symmetric.

Mycotoxin

y

















Laboratory Diagnosis

y This happens due to fungal toxins that contaminates the food

y

y

y

y







y

y

y

y

y

y Pneumocystis is an opportunistic pathogen that is an important etiological agent of pneumonia in immunocompromised individuals. y Thought as a protozoan, now classified as fungi – ascomycetes y Former name: Pneumocystis carinii y Most commonly seen in AIDS patient y Morphological forms  Trophozoites  Precyst  Cyst y Route of infection is through respiratory; Source: droplets y It has a unique tropism for lung and hence it attacks the alveolar capillaries after entering through inhalation. y Usually asymptomatic: It causes fatal pneumonia only in immunosuppressed patients (HIV)

MYCOTIC POISONING

y

PNEUMOCYSTIS JIROVECII

Candida sp Aspergillus sp Fusarium sp Penicillium sp Cryptococcus neoformans Blastomyces dermatitidis Histoplamsa capsulatum Coccidioides immitis

y y y y y y

Figure 1: Pneumocystis jirovecii (methenamine silver stain) (Courtesy: CDC/ Dr. Edwin P. Ewing, Jr)

y

FUNGI CAUSING CONJUNCTIVITIS y y y y y y y

Candida sp Malassezia sp Sporothrix schenckii Blastomyces dermatitidis Coccidioides immitis Paracoccidioides brasiliensis Aspergillus niger

1. Type of pneumonia in P. jirovecii (Recent Pattern Jul 2016) a. Lobar pneumonia b. Interstitial pneumonia c. Bronchopneumonia d. Any of the above 2. P. carinii causes infection of primarily (Recent Pattern Dec 2012) a. Rats b. Mice c. Humans d. Rabbits 





3. Select the false statement about P. jirovecii (AI 2008) a. It is seen in immunocompromised individuals b. Frequently associated with CMV c. May be associated with pneumatocele d. Diagnosed with spectrum microscopy 4. A patient of acute leukemia is admitted with febrile neutropenia. On day four of being treated with broad spectrum antibiotics, his fever increases. X-ray chest shows bilateral fluffy infiltrates. Which of the following should be the most appropriate next step in the management? a. Add antiviral therapy b. Add Antifungal therapy c. Add cotrimoxazole d. Continue chemotherapy 

Chapter 52     Miscellaneous Fungi

MULTIPLE CHOICE QUESTIONS

ANSWERS AND EXPLANATIONS 1. Ans  (b) Interstitial pneumonia

3. Ans  (b) Frequently associated with CMV

Ref: Harrison’s T.B of internal medicine – 19th ed – Page 1358

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 618

• Route of infection is through respiratory; Source: droplets • Usually asymptomic; It causes fatal pneumonia only in immuno suppressed patients (HIV) • Type of pneumonia seen is Interstitial

• It causes fatal infections in immunocompromised individuals • As it causes pneumonia – pneumatocele occurs • Toluidine blue staining from biopsy is the most common method but microscopy analysis also can be done • Hence second option which says CMV is frequently associated cannot be taken as right; as it can occur but not so frequently

•

•

•

•

•

•

•

2. Ans  (a) Rats Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 618 • Pneumocystis carinii – rats • Pneumocystis jirovecii – Humans •

•

4. Ans  (c) Add cotrimoxazole Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 618 • Drug of choice: TMP-SMZ (Hence when CD4 count comes down in HIV patients – TMP-SMZ is started as prophylaxis) • TMP-SMZ is cotrimaxozole •

•

385

Unit 5     Mycology

HIGH YIELDING FACTS TO BE REMEMBERED IN MYCOLOGY Examples for dimorphic fungi (BPH – CPS)

• • • • • •

•

•

•

•

•

•

Blastomyces dermatitidis Paracoccidiodes brasiliensis Histoplasma capsulatum Coccidioides immitis Penicilliosis marneffi Sporothrix schenckii

Darling disease

• Histoplasmosis

Tuberculate spores are seen in

• Histoplasmosis

Arthrospores seen in

• Coccidioides

Valley fever

• Coccidiodomycosis

Captain wheel formation seen in

• Paracoccidiodomycosis

Genus that comes under dermatophytes are

• Trichophyton • Microsporum • Epidermophyton

•

•

•

•

•

•

•

•

Only species in Epidermophyton is

• E.floccosum

Tinea versicolor is caused by

• It is not caused by dermatophytes; it is caused by Malassezia furfur

Germ tube formation seen in

• Candida albicans • Candida dublinensis

•

•

•

•

Pseudohyphae is absent in which candida species

• Candida glabrata

Chlamydospores are seen in

• Candida albicans

Reynolds Braude phenomenon is seen in

• Candida albicans

CHROM agar is useful for

• Speciation of Candida

Acute angled dichotomous branching septate hyphae seen in

• Aspergillus sp

Broad aseptate hyphae seen in

• Mucor, Rhizopus

Capsulated fungus

• Cryptococcus neoformans

Rose Gardener’s disease is caused by

• Sporothrix schenckii

Potassium iodide is used in the treatment of which fungi

• Sporothrix schenckii

Sclerotic bodies are seen in

• Chromoblastomycosis

Pigmented fungi are called as

• Dematacious fungi

Botryomycosis is caused by

• Not fungi ; it is caused by Staph aureus and other bacteria

Entomophthromycoses is caused by

• Conidiobolus coronatus • Basidiobolus ranarum

•

•

•

•

•

•

•

•

•

•

•

•

•

•

386

Non cultivable fungi

• Rhinosporidium seeberi

Dapsone is used in treatment of which fungi

• Rhinosporidium seeberi

Which kingdom does Rhinosporidium seeberi belongs to

• Aquatic protista – DRIP CLADE

Fungi that has trophozoites and cystic stage is

• Pneumocystis jirovecii

TMP-SMX drug is given for which fungi

• Pneumocystis jirovecii

ABPA is an example of

• Type I and III hypersensitivity

Pigmented pencillium species that causes infections in AIDS patients

• Penicillium marneffei

Most common fungal infection in diabetics and steroid intake patients

• Zygomycosis

Which bird and Which tree is associated with Cryptococcal infection

• Bird – Pigeon • Tree - Eucalyptus

•

•

•

•

•

•

•

•

•

•

IMMUNOLOGY

6

Unit Outline Chapter 53 Immunity Chapter 54 Structure and Functions of Immune System Chapter 55 Antigens Chapter 56 Antibodies Chapter 57 Complement System Chapter 58 Antigen-Antibody Reactions Chapter 59 Immune Response Chapter 60 Hypersensitivity Chapter 61 Immunodeficiency Diseases Chapter 62 Autoimmunity Chapter 63 Transplantation and Tumor Immunology Chapter 64 Immunohematology

53

Immunity WHAT IS IMMUNITY?

y

y

y When human body is encountering the microorganisms – some protective mechanism are exhibited by various cells – termed as immunity y Simply, it is the resistance against infections or other foreign substances.

TYPES OF IMMUNITY

A





y

y

y

y





y

y Mainly divided into two types  Innate  Acquired or adaptive—active and passive y When a foreign substance encounters our human body the first-line of defense is intact skin and mucous membranes y When the skin or mucous membranes are breached then the innate system of immunity starts working to attack the infections (second-line of defense) y Adaptive immunity or acquired immunity is acquired by an individual after he encounters the antigen and it takes several days for the immune process y Based on the cells that are involved in the immune process – the immunity is divided into  Cell-mediated immunity  Humoral immunity

Table 1:  Cells involved in types of immunity Cells involved

Innate immunity

• • • •

•

•

•

•

Types of immunity

Figure 1: Development of immune cells

Complement Neutrophils Macrophages NK cells

•

• B cells and antibodies (Humoral immunity) • Helper T cells and Cytotoxic T cells (Cell mediated immunity) •

Acquired immunity

B

Table 2:  Innate and acquired immunity Types of Specificity Immediate Improves Memory immunity protection after exposure Innate

Non specific

Yes

No

No

Acquired

Highly specific

No

Yes

Yes

Table 3:  Differences between antibody and cell-mediated immunity Antibody-Mediated Immunity

Cell-Mediated Immunity

Exhibited by B cells

Exhibited by T cells

Host defense – by opsonised bacteria, neutralize toxin and viruses

Host defense – against M.tuberculosis, fungi and viruses (Intracellular infections)

Allergic reactions like anaphylactic shock and hay fever

Allergic reactions like delayed type reactions, contact dermatitis

Autoimmunity

Graft and tumor rejection Regulation of antibody response

y Acquired or adaptive immunity, i.e. antibody-mediated or cell mediated immunity has four characteristic features:  Antigenic specificity  Diversity  Immunologic memory  Recognition of self and nonself y Adaptive immunity is again divided into active and passive immunity based on the features given below in Table 5.    

 

Table 4: Differences between active and passive immunity











y

y y y

y

y Mainly influenced by the intactness of skin and mucosal system y Tear secretion: Contains lysozyme which is present in almost all tissue fluids except CSF, sweat and urine y Lysozyme: It splits the polysaccharide components of the cell wall of organisms y Flushing action of urine helps to eliminate bacteria y Antibacterial substance that are present in the blood and tissues helps in destruction of pathogenic bacteria  Beta lysin  Basic polypeptides: Leukins and plakins  Acidic substances: Lactic acid  Lactoperoxidase in milk  Interferon. y

Adaptive Immunity

y

Unit 6     Immunology

Innate Immunity

Remember

Passive Immunity

Produced actively by host immune system

Immunoglobulin received passively

Induced by infection and vaccination

Acquired by mother to fetus transfer and readymade antibody transfer

Long lasting

Lasts for short time

Lag period present

No lag period

Memory present

No memory

Booster doses useful

Subsequent doses – less effective

Negative phase may occur

No negative phase

Not useful in immunodeficient Useful

•

•

•

•

•

•

•

•

•

•

Acute Phase Reactants • Serum amyloid A • CRP • Complement proteins • Fibrinogen • Von willebrand factor • Proteinase inhibitors – alpha 1 antitrypsin • Alpha 1 acid glycoprotein • Mannose binding protein • Haptoglobin • Ceruloplasmin

Active Immunity

y Natural active immunity – acquired either because of a clinical or subclinical infection; A special type is called premunition immunity which is seen in syphilis. In this condition, immunity to re-infection lasts only as long as the original infection is active; if the disease is cured then the patient is susceptible to re-infection y Artificial active immunity: Induced by vaccines y Natural passive immunity: Transferred from mother to baby y Artificial passive immunity: Transferred by administration of antibodies in sera (immunoglobulin) y Adoptive immunity: Special type of immunity, an extract of immunologically competent lymphocytes is injected named as transfer factor: Used in the treatment of lepromatous leprosy y Herd immunity: Immunity for the community by vaccines like OPV

 

Table 4: Conditions in which CRP is increased Heavy exercise, common cold, pregnancy

Moderate increase of CRP

Bronchitis, cystitis, malignancies, pancreatitis, myocardial infarction

Marked increase of CRP

Acute bacterial infections, major trauma, systemic vasculitis

y y

Insignificant increase of CRP

y

y

•

•

•

C–Reactive Protein • CRP is an inflammatory marker that gets raised during many inflammations and infections; • hCRP is high sensitive CRP that is sensitive than CRP • CRP is used as a prognostic marker for sepsis; but nowadays procalcitonin is used as marker for sepsis

y

y

High Yield

Types of Active Immunity

390







7. Adoptive immunity is by:  (PGI 2017) a. Infection b. Injection of antibodies c. Injection of lymphocytes d. Immunization 8. Transfer factor used for immunization is: (Recent Pattern 2018) a. Immunoglobulin b. Vaccine c. Toxoid d. Lymphocytes 9. When transfer factor is given as treatment results in:  a. Natural active immunity  (Recent Pattern 2017) b. Artificial active immunity c. Artificial passive immunity d. Adoptive immunity 10. Active immunity is:  (Recent Pattern 2017) a. Immunization by DPT vaccine b. Transfer of antibodies from mother c. Immunoglobulin injection d. None of the above 11. First chemical barrier encountered by microorganism for common exposed sites: (AIIMS 2017) a. Lysozyme b. Acidic pH c. Skin d. Lactose 12. Vaccination is based on the principal of: (Recent Pattern 2017) a. Agglutination b. Phagocytosis c. Immunological memory d. Clonal detection 13. Which of the following is/are the components of innate immune system: (PGI May 2018) a. NK cell b. Beta defensins c. T cell d. B–cell e. Dendritic cell 









1. All are true about innate immunity, except: a. Non-specific  (Recent Pattern 2017) b. First-line of defence c. Not affected by genetic affected d. Includes complement 2. Which is specific for acquired immunity: a. Immunological memory (Recent Pattern 2017) b. Affected by genetic makeup c. No antigen exposure d. All of the above 3. Innate immunity is stimulated by which part of bacteria: (Recent Pattern 2017) a. Carbohydrate sequence in cell wall b. Flagella c. Bacterial cell membrane d. Nucleus 4. Cells involved in humoral immunity: (Recent Pattern 2017) a. B-cells b. T-cells c. Helper cells d. Dendritic cells 5. Active immunity is not acquired by: (Recent Pattern 2017) a. Infection b. Vaccination c. Transplacental immunoglobulin transfer d. Subclinical infection 6. True about active immunity: (Recent Pattern 2017) a. Less effective b. Can be given in immunodeficient states c. Immunological memory present d. No lag period

Chapter 53     Immunity

MULTIPLE CHOICE QUESTIONS

ANSWERS WITH EXPLANATIONS

•

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 81

• Lipopolysaccharides are the major outer surface component which is present in all the Gram-negative bacteria; Mannans are seen in the cell wall • These are the primary stimulator of innate immunity. •

1. Ans.  (c)  Not affected by genetic affected

•

• Innate or native immunity – basically the resistance to infection based on the individual’s genetic make up (Racial/Species)

5. Ans.  (c)  Transplacental immunoglobulin transfer Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 84 • Active immunity is mainly acquired by foreign substance like microbes or antigen, i.e. by vaccination, infection • Passive immunity is acquired by antibodies – by maternal to fetal transfer or by immunoglobulin injection.

3. Ans.  (a)  Carbohydrate sequence in cell wall Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 83, Kuby Immunology – 7th edition – Page 149

•

•

•

• Adaptive immune system has the characterisitc feature of immunological memory when it encounters the same antigen on a second time – the response will be quicker and more specific

• Humoral immunity – by B-cells • Cell mediated immunity – by T cells •

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 83

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 147

•

2. Ans.  (a)  Immunological memory

4. Ans.  (a) B-cells

391

•

• Adaptive immunity has two types active and passive immunity • Ref Q.2

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 83, Kuby Immunology – 7th edition – Page 146 • Most of the body surfaces especially mucosal surfaces – protected due to acidic pH – e.g. Skin, gastric juice, vagina, urinary tract. •

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 84

11. Ans.  (c) Skin

• •

• Adoptive immunity – a special type of immunity by which immunologically competent lymphocytes are injected (Transfer factor). 8. Ans.  (d) Lymphocytes

•

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 86 • Already explained Q.7 9. Ans.  (d)  Adoptive immunity

• Vaccination is a type of adaptive immunity—active immunity • The basic characteristic of adaptive immunity is immunological memory • Vaccination gives the specific antigenic stimulus and helps to combat the infection in a secondary response by quicker and more specific response. 13. Ans.  (a) NK cell; (b) Beta defensins Ref: Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 83, Kuby Immunology – 7th edition – Page 149 Components of Innate immunity are: • Skin and mucosa • Tears – lysozyme • Antibacterial substrances in blood like beta lysin (beta defensins) • Cells: NK cells, macrophages, complement, neutrophils •

10. Ans.  (a)  Immunization by DPT vaccine

•

•

•

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 86 • Already explained Q.7

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 83 •

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 86

12. Ans.  (c) Immunological memory

•

7. Ans.  (c)  Injection of lymphocytes

•

Unit 6     Immunology

6. Ans.  (c)  Immunological memory present

•

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 84

•

•

• Active immunity is the acquired as a result of an antigenic stimulus – either by infection or vaccination • DPT vaccine elicits active immunity.

392

Structure and Functions of Immune System

54

INTRODUCTION

y

y

y

y

y Immune system has a complex mechanism involving many cells y These cells ideally develop from the blood cell precursors in yolk sac and fetal liver during embryonic life; Later from the bone marrow y Pleuripotent stem cells gives rise to myeloid, erythroid and lymphoid series y From the lymphoid series: Two groups of population namely T cells and B cells arise:

Table 1:  Lymphoid Organs

• Thymus • Bone marrow

• Spleen • Lymph nodes • Mucosa associated lymphoid tissue (MALT) • Lymphoid tissues in gut, liver, lungs and bone marrow • •

•

•

•

Secondary or peripheral lymphoid organs

•

Primary or Central lymphoid organs

T CELLS Origin of T Cells

Differentiation of T Cells

y

y

y Double negative (DN) and Double positive (DP) cells – located in cortex of thymus y Single positive cells – located in medulla

y y y

Location of Cells

y During this process inside the thymus, the T cells that react with the self antigens are clonally deleted by apoptosis – clonal deletion – Self restriction – thus preventing autoimmunity. y Each double positive T cells acquires T cell receptor on their surface – TCR – has three genes namely variable (V), diversity (D), joining (J) genes – VDJ recombination occurs in different ways – which helps to recognize millions of antigens with greater specificity. y Among the T cells, 40% of population do not develop in thymus. It goes to gut-associated lymphoid tissue (GALT) y GALT (intraepithelial T cells) – helps protection against intestinal pathogens. y

y

y

y

y Bone marrow (Origin) stem cells gives rise to immature T cells; These cells move to the thymus (maturation) y After entering into the thymus – T cells acquire antigen receptors and various CD receptors y Initially the T cells do not express CD4/CD8 (Double negative) → then begins to express both CD4/CD8 (Double positive) → finally few cells turns CD4 + and few cells CD8 + (Single positive)

Figure 1: Differentiation of T cells

PERIPHERAL LYMPHOID ORGANS

y Helper T cells:  T cells that are having CD4 surface marker and bind to the MHC class II  Two types of helper T cells are present: } Effector cells } Memory cells  Effector CD4 T cells are again divided into : Th1, Th2, Th17 cells

Remember • Periarterial lymphoid • Malphigian corpuscle seen in white pulp

Lymph node

• Para cortical area

Spleen

• Perifollicular region • Mantle layer

Lymph node

• Cortical area • Germinal layer • Medullary cords

•

Spleen

 

Table 2: Characteristics of effector T-helper cells

• • •

Thymusindependent region (B cells)

•

•

Types of effector Characteristics T helper cells

•



}

}



Thymusdependent region (T cells)

•



y

Unit 6     Immunology

Types of T Cells

Th 17 cells

• Produces IL 17, IL 22 • Promote inflammation • Involved in autoimmune disease and cancer

•





y

•

•

•

•

•

•

•



•



• Activated by IL 4 • Produce IL 4, IL 5, IL 6 and IL 13 • Helps in synthesis of all antibodies except IgG2 • Kills helminthic parasites by the production of IgE



Th 2 cells

y Cytotoxic T cells:  These type of T cells have CD8 cell surface marker; They are MHC Class I restricted  Helpful in killing and lysing the tumor cells, virus infected cells  Involves in type II hypersensitivity reactions y Suppressor T cells:  These type of T cells have CD8 cell surface marker. They are MHC Class I restricted  Involves in downregulation of immune system y

• Activated by IFN gamma • Produce IL 2, IFN gamma, IL 12 • Involved in delayed hypersensitivity, cell-mediated immunity, macrophage activation, killing of intracellular organisms like M.tuberculosis, M.leprae •

Th 1 cells

High Yield ƒ

ƒ

•

• Do T cells have a role in antibody production? ƒ Not direct role; But indirectly it has a vital role ƒ When antigen is encountered – antigen presenting cells takes this antigen to T cells – secrete some cytokines – these cytokines go and stimulate B cells – these B cells – converted to plasma cells – produce antibodies

 

Table 3: Main function of helper T cell and respective cytokine mediator Cytokine Mediator

Activates the antigen-specific helper T cell to produce a clone of these cells

IL - 2

Activates cytotoxic T cells

IL - 2

Activates B cells

IL -4 and IL - 5

Activates macrophages

IFN gamma

B CELLS Origin of B Cells y B cells originate and mature in the Bone marrow itself y During the process of synthesis – B cells acquire IgM and IgD as surface markers in the cell y B cells also undergo clonal deletion but certain B cells that recognize self antigens may get escaped by a process called receptor editing (This is not seen in T cells) y Antigen is recognized via these surface markers y When antigen stimulates the B cells – they get transformed into plasma cells – secrete immunoglobulins y Plasma cells are antibody secreting cells y

y

y

y

y

y

Main Function of Helper T Cell

Figure 2: Cytokines produced by T-helper cells

394

Origin

Maturation

T cells

Bone marrow

Thymus

B cells

Bone marrow

Bone marrow

y When our body encounters the antigen, whole antigen cannot be directly involved, it has to be processed and presented to the T cells. This happens with the help of few cells called antigen presenting cells (APCs)

y Also called as large granular lymphocytes y Types of null cells:  Antibody-dependent cytotoxic cells (ADCC)  Natural killer cells  Lymphokine activated killer cells (LAK) 

y y y y y

MACROPHAGES

y

Table 6:  Sites of dendritic cells Types of Dendritic Cells

Sites

Langerhans cells

Skin and mucosa

Interstitial dendritic cells

Organs like lungs, liver, spleen

Interdigitating dendritic cells

Thymus

Circulating dendritic cells

Blood and Lymph

Follicular dendritic cells

Lymph nodes

MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) HLA

y

y

y Human leukocyte antigens encoded by the HLA genes which are present in the body unique to each individual, is the major determinant for organ transplantation, i.e. tissue matching y The gene is located in the maternal chromosome 6 y

y







y

y

y

y All of the immune cells come from lymphoid lineage except macrophages that come through myeloid lineage y Macrophages help in chronic inflammation y Major functions are:  Phagocytosis  Antigen processing and presentation  Cytokines secretion y IFN gamma is the major activator for macrophages y Blood macrophages is called monocytes (life span 1–3 days) and tissue macrophages called as histiocytes (life span 3 months to years)

y





y

NATURAL KILLER CELLS y Cells that do not have either CD4/CD8 receptors y These cells are LGL – large granular lymphocytes that do not pass through thymus y Their main function is to kill the intracellular cells – virus infected and tumor cells y These function are not antibody or MHC mediated y NK cells have surface markers for CD16 and CD56

y

Types of APC y Professional APCs: Macrophages, dendritic cells, B cells y Nonprofessional APCs – Fibroblasts, thymic epithelial cells, pancreatic beta cells, vascular endothelial cells, glial cells and Thyroid epithelial cells y Dendritic cells presents antigen to T cells, Whereas follicular dendritic cells presents antigen to B cells.

y

NULL CELLS

Chapter 54     Structure and Functions of Immune System

Cells

ANTIGEN PRESENTING CELLS y

Table 4:  Sites of origin and maturation of T and B cells

Table 5:  Tissue macrophages

Bone

Osteoclasts

Skin

Langerhans cells

Connective tissue

Histiocytes

Kidney

Mesangial cells

MISCELLANEOUS CELLS

y

y

y

y Eosinophils: Mediate allergic responses and parasitic infestation; The granules in the eosinophils have major basic protein which causes degranulation of mast cells y Neutrophils: First cells to involve in acute inflammation y Mast cells: Important source of histamine, involved in immediate hypersensitivity

Figure 3: HLA gene y Between class I and class II is the Class III loci which has complements y

Kupffer cells

MHC Class I y y y y y

y

Liver

y

Microglia

y

Brain

y

Alveolar macrophages

y

Lung

Located in the surface of all nucleated cells RBC do not have nucleus – they do not have MHC Consists of heavy chain bound to beta 2 microglobulin The heavy chain is highly pleomorphic MHC polymorphism is responsible for the recognition between self and nonself

395

Unit 6     Immunology

Class I MHC

Class II MHC

Composed of one peptide encoded in the HLA locus and beta 2 microglobulin

Composed of two peptides encoded in the HLA locus

Antigen binding site is between Antigen binding site is between α1 and α2 α1 and β1 CD8 T cells respond to antigen presented along with Class I MHC

CD4 T cells respond to antigen presented along with Class II MHC

 

Table 8: Surface markers of various immune cells

Figure 4: MHC class I

MHC CLASS II

y

y

y Located in certain cells like B cells, Langerhan cells, macrophages and dendritic cells y Hypervariable regions provide pleomorphism

Type of cells

Surface markers

Helper T cells

CD4, TCR, CD28

Cytotoxic T cells

CD8, TCR

B cells

IgM, IgD, B7

Macrophages, APC

Class II MHC

N K cells

CD16, CD56, receptors for class I MHC

All cells other than mature red cells

Class I MHC

 

Table 9: HLA-Associated disorders B27 B27 B27 Dw14, Dw4 DQw8 DR3 DR3 DR2

CYTOKINES y During the immune response or inflammation  T lymphocytes secretes substances called as lymphokines  Macrophages secrete – monokines  Virus infected cells secrete - interleukins y All these are called in group as cytokines

Class II MHC

Antigen presentation to CD8 positive cells

Antigen presentation to CD4 positive cells

Found on surface of all nucleated cells

Found on surface of professional APC

Encoded by genes in the HLA locus

Encoded by genes in the HLA locus

Expression of gene is codominant

Expression of gene is codominant

Multiple alleles at each gene locus

Multiple alleles at each gene locus

396

y



Class I MHC





 

Table 7: Differences between class I and class II MHC

HLA Type

Ankylosing spondylitis Reiter`s syndrome Acute anterior uveitis Juvenile rheumatoid arthritis Insulin dependent DM Grave’s disease Sjogren’s syndrome SLE

y

Figure 5: MHC class II

Disease

 

Table 10: Cytokines and their major sources Major Source

Cytokines

Macrophages Th1 cells Th2 cells Th17 cells Macrophage, T cells, B cells Virus infected cells

IL 1, IL 6, TNF, IL 12 IL 2, IFN gamma IL 4, IL 5, IL 10 IL 17 TGF beta Interleukins













13. All are mononuclear-macrophage, except: (Recent Pattern 2017) a. Histiocytes b. Microglia c. Kupffer cells d. B-cells 14. Which is not true about macrophage? a. Activation by IFN-γ (Recent Pattern 2017) b. Major cells in chronic inflammation c. M2 type involved in inflammation d. Phagocytic cells 15. All of these are antigen presenting cells [APC’S], except:  (PGI 2017) a. T cells b. B cells c. Fibroblasts d. Dendritic cells e. Langerhans cells 16. Most potent stimulator of naïve T cells:  (Recent Pattern 2017) a. Mature dendritic cells b. Follicular dendritic cells c. Macrophages d. B cells 17. Langerhans cells in skin are:  (Recent Pattern 2017) a. Antigen presenting cells b. Pigment producing cells c. Keratin synthesizing cells d. Sensory neurons 18. Follicular dendritic cells, main function is:  (Recent Pattern 2017) a. Catches antigen and presents it to T cells b. Catches antigen and presents it to B cells c. Phagocytic activity d. Produce immunoglobulin 19. Which cells cause E-rosette formation with sheep RBCs: (Recent Pattern 2017) a. T cells b. NK cells c. Monocytes d. B cells 20. EAC rosette formation is the property of one of the following type of immune cells:  (Recent Pattern 2017) a. T-cells b. B-cells c. Macrophage d. All of the above 21. Cellular immunity is induced by:  (Recent Pattern 2017) a. NK – cells b. Dendritic – cells c. TH1 – cells d. TH2 – cells 22. Which of the following features is not shared between ‘T Cells’ and ‘B Cells’:  (AIIMS 2017) a. Positive selection during development b. Class 1 MHC expression c. Antigen specific receptors d. All of the above 23. Longest life span is of:  (Recent Pattern 2017) a. Macrophage b. Memory T & B cell c. Neutrophils d. Platelets 24. MHC class 2 is coded in which region:  (Recent Pattern 2017) a. A b. B c. C d. D 

















1. All are peripheral lymphoid organs, except: a. Lymph nodes  (Recent Pattern 2017) b. Spleen c. Mucosa associated lymphoid tissue d. Thymus 2. Common between B and T cells: (PGI 2017) a. Origin from same cell lineage b. Site differentiation c. Antigenic marker d. Both humoral and cellular immunity e. Further differentiation seen 3. Thymus dependent area in spleen: (Recent Pattern 2017) a. Mantle layer b. Perifollicular region c. Malpighian corpuscle d. All of the above 4. T cell dependent region is:  (Recent Pattern) a. Cortical follicles of lymph node b. Medullary cords c. Mantle layer d. Para cortical area 5. All are true regarding development of T-cells, except: (Recent Pattern 2017) a. T-cells are formed in bone marrow  b. Maturation of T-cells take place in thymus c. T-cells are located in mantle layer of spleen d. In lymph nodes, T-cells are found in Para cortical area 6. Immunoglobulins are produced by:  (Recent Pattern 2017) a. Macrophages b. B-cells c. T-cells d. NK-cells 7. IgE is secreted by: (Recent Pattern 2017) a. Mast cell b. Basophils c. Eosinophils d. Plasma cells 8. Large granular cells belong to: (Recent Pattern 2017) a. Neutrophils b. Macrophages c. Eosinophils d. Lymphocytes 9. NK cells activity is enhanced by:  (Recent Pattern 2017) a. IL-1 b. TNF c. IL-2 d. TGF-β 10. All are true regarding NK cells:  (PGI 2017) a. CD 16 positive b. CD 56 positive c. Secrete complement like substance d. Important role in viral infected cell 11. Virus infected cells are killed by:  (Recent Pattern 2017) a. NK cells b. Plasma cells c. B-cells d. None 12. Antibody dependent cytotoxicity is seen with:  (Recent Pattern 2017) a. Cytotoxic T cells b. Natural killer cells c. ADCC d. All of the above

Chapter 54     Structure and Functions of Immune System

MULTIPLE CHOICE QUESTIONS

397





Unit 6     Immunology

25. HLA-1 is present on  (Recent Pattern 2017) a. All nucleated cells b. Only on cells of immune system c. Only on B –cells d. Only on T – cells 26. Gene component of HLA class 1 includes: (Recent Pattern 2017) a. A,B,C b. DR c. DQ d. DP 27. The role played by major histocompatibility complex proteins [MHC-1 & MHC-2] is to:  (AIIMS 2017) a. Transduce the signals to T cells following antigen recognition b. Mediate immunoglobulin class-switching c. Present antigen for recognition by T cell antigen receptors d. Enhance the secretion of cytokines 28. Peptide binding site on class 1 MHC molecule for presenting processed antigens to CD8 T cells is formed by: (Recent Pattern 2017) a. Proximal domain of α subunits b. Distal domain of α subunits c. Proximal domains of α and β subunits d. Distal domains of α and β subunits

29. T helper cells recognizes:  (Recent Pattern 2017) a. MHC class 1 b. MHC class 2 c. Processed peptides d. Surface Ig 30. True about MHC: (Recent Pattern 2017)) a. Present on chromosome 4 b. Class 2 comprises A,B,C loci c. Class 3 has complement d. Class 1 is involved in mixed leucocyte reaction 31. Cytokine that have pyrogenic activity is:  (Recent Pattern 2017) a. IL-6 b. IL -8 c. TGF beta d. IL – 3 32. HLA gene is located in: (Recent Pattern Nov 2017) a. Chromosome 6 b. Chromosome 22 c. Chromosome 11 d. Chromosome 9 33. Class II MCH molecule are expressed on: (PGI May 2018) a. Dendritic cell b. Basophils c. Eosinophils d. Activated T-cell e. Macrophages 34. Which of the following immunoglobulins are commonly present in antigen binding site of B cell: (PGI May 2018) a. IgA b. IgG c. IgD d. IgM e. IgE

ANSWERS WITH EXPLANATIONS Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 133

5. Ans.  (c)  T-cell are located in mantle layer of spleen Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 133 • T cells are seen in the white pulp (malpighian corpuscle) of the spleen • Mantle layer – B cells are seen

•

•

•

3. Ans.  (c)  Malpighian corpuscle Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 133 •

• T cell dependent (thymus) areas in spleen are periarterial lymphoid collection, malpighian corpuscle in the white pulp 4. Ans.  (d)  Para cortical area

398

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 133

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 138 • When antigen is encountered – it is presented by antigen presenting cells – to MHC – T cells – secrete cytokines – goes and stimulates B cells – gets transformed into plasma cells – secrete immunoglobulins •

•

• Both the B and T cells are derived from the same lymphoid lineage from bone marrow • Further differentiation occurred during maturation • T cells mature in thymus • B cells mature in bone marrow itself

6. Ans.  (b) B-cells

7. Ans.  (d)  Plasma cells Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 138 • Plasma cells are the antibody secreting cells • Immunoglobulins are the antibodies •

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 135

•

2. Ans.  (a, e)  Origin from same cell lineage; Further differentiation seen

•

•

•

•

• Central lymphoid organs – Thymus and Bone marrow • Peripheral lymphoid organs – Lymph nodes, spleen, MALT, bone marrow, GALT

• T cell dependent area in the lymph node is para cortical area •

1. Ans.  (d) Thymus

8. Ans.  (d) Lymphocytes Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 139

•

• IL – 2 acts as a growth factor for natural killer cells 10. Ans.  (a) CD 16 positive; (b) CD 56 positive; (c) Secrete complement like substance; (d) Important role in viral infected cell Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 139

ƒ ƒ

• Before getting an antigenic exposure the new cells are called as Naïve B and T cells • When an antigen is encountered – the langerhan cells go and capture the antigen – presents to the T cells with corresponding MHC – stimulates the naïve T cells – produce cytokines 17. Ans.  (a)  Antigen presenting cells Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 140 •

• Langerhans cells are dendritic cells; they belong to antigen presenting cells

• Follicular dendritic cells are located in the B cell containing germinal centres of the follicles in the spleen and lymph nodes • They do not produce MHC class II • Hence they do not present antigen to helper T cells • They directly capture antigen antibody complexes and presents to B cells •

•

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 139 Ref: Already explained Q.10

Ref: Review of medical microbiology and immunology –13th edition - page 1107 •

11. Ans.  (a)  NK cells

18. Ans.  (b)  Catches antigen and presents it to B cells

•

•

•

•

•

• Natural killer cells have CD16 and CD56 on their surface • They secrete perforins – that resembles that of complement C9 • Interferon acts a stimulator for NK cells • NK cells are mainly involved in immune surveillance and they are the defence against virus infected cells and cancer cells

Ref: Robbins – pathology – 8th edition – page 189 •

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 139

16. Ans.  (a)  Mature dendritic cells

•

9. Ans.  (c) IL-2

Chapter 54     Structure and Functions of Immune System

ƒ

ƒ Dendritic cells ƒ B cells ƒ Langerhan cells

•

• Apart from T and B cells, certain cells do not have the surface markers and neither fit into T and B cells – 5 to 10% are found – called as null cells/Large granular lymphocytes (LGL)

12. Ans.  (c) ADCC Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 139

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 135

13. Ans.  (d) B-cells Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 139,140

•

•

• B cells are lymphocytes • Macrophages are histiocytes (tissue macrophages), kupffer cells, osteoclasts, mesangial cells, synovial cells 14. Ans.  (c) M2 type involved in inflammation Ref: Review of medical microbiology and immunology – page 1105

•

20. Ans.  (b) B-cells Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 135 Ref: Already explained Q.19 21. Ans.  (c)  TH1 – cells Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 137

•

• M2 type of macrophages are having anti inflammatory action

• When B cells bind with the sheep erythrocytes – they form EAC rosettes due to the presence of C3 receptor (CR2) on the B cell • When T cells bind to sheep erythrocytes – they form SRBC/E rosette by the CD2 antigen •

ƒ

ƒ

ƒ

•

• There are three types of large granular lymphocytes ƒ NK cells – not antibody dependent ƒ Cytotoxic T cells – not antibody dependent ƒ ADCC – antibody dependent

19. Ans.  (a) T cells

15. Ans.  (a) T cells; (c) Fibroblasts Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 140

•

22. Ans.  (a)  Positive selection during development Ref: Review of medical microbiology and immunology –13th edition - page 1080 • During development – immature cells develop to mature cells •

ƒ

•

• Antigen presenting cells are ƒ Macrophages

• Cell mediated immunity is mediated by T helper cells – type Th1

399

•

30. Ans.  (c)  Class 3 has complement Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 142 Ref: Already explained Q.29

•

Unit 6     Immunology

• In this process - T cells first acquire double negative cells – then double positive – then single positive cells – which is acquired by process called positive selection followed by negative selection • B cells do not have these – it has only negative selection 23. Ans.  (a) Macrophage

31. Ans.  (a) IL-6

Ref: Review of medical microbiology and immunology –13th edition - page 1105 •

• Tissue macrophages has the longest life span of months upto years 24. Ans.  (d) D Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 142 MHC CLASS I MHC CLASS II MHC CLASS III

A,B,C loci D region – DR, DQ, DP loci Complement region

Function

Cytokine mediators

Pyrogenic

TNF alpha, IL-1, IL-6

Anti tumour

TNF alpha, TNF beta

Anti viral

IFN alpha and IFN beta

32. Ans.  (a) Chromosome 6 Ref: Ananthanarayan and Paniker T.B of microbiology – 10th ed – Page 143 • HLA – Human leucocyte antigen • Counterpart is MHC – Major histocopatibility complex • HLA complex of genes are located on the short arm of chromosome 6 • It has three clusters of genes:

•

•

26. Ans.  (a)  A, B, C Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 142 Ref: Already explained Q.25

Class I

A, B, C

Class II

DR, DQ, DP

Class III

C4B, C2, BF, TNF

33. Ans.  (a) Dendritic cell; (e) Macrophages Ref: Ananthanarayan and Paniker T.B of microbiology – 10th ed – Page 143 • MHC class I is located in the surface of all nucleated cells • MHC class II is located in B cells, Langerhan cells, macrophages and dendritic cells •

27. Ans.  (c)  Present antigen for recognition by T cell antigen receptors

•

• HLA CLASS I - Located in the surface of all nucleated cells • HLA CLASS II – located in the cells of immune system

•

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 142

•

•

•

25. Ans.  (a)  All nucleated cells

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 159

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 142

34. Ans.  (c) IgD; (d) IgM

•

• Main function of MHC molecule is to bind the peptide fragments of foreign proteins and present them to antigen specific T cells with the helps of APC 28. Ans.  (b)  Distal domain of α subunits Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 142

MHC CLASS I

MHC CLASS II

Antigen binding site is between α1 and α2

Antigen binding site is between α1 and β1

29. Ans.  (b)  MHC class 2 Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 142

•

•

• T helper cells has CD4 surface marker • CD4 binds with MHC Class I

400

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 136 During the process of origin and synthesis – B cells acquire two immunoglobulins on their surface – IgM and IgD

55

Antigens y Any foreign substance that enters into the human body, elicits immune response by the production of antibodies, is called as antigen y Two important attributes of antigenicity are:  Immunogenicity: Ability to produce an immune response  Immunological reactivity: Specificity of the immunological reaction with antibodies y Types of antigens:  Complete antigen: Induces antibody and produces a specific reaction  Incomplete antigen: Haptens y Haptens are the substances that cannot directly induce antibody production but they can react with the antibodies  Types of haptens: Š Simple haptens: Univalent, nonprecipitating Š Complex haptens: Polyvalent, precipitate with specific antibodies y Epitope  There is a small area in the antigen which has the specific structure. That determines the antigenicity  The smallest unit of antigenicity is known as antigenic determinant or epitope  Types of epitopes: Š Linear or sequential epitope: Presents as single chain Š Conformational epitope: Presents as folding y Paratope:  The corresponding area in the antibody in which the epitope combines is called as paratope

y Based on the immune response, antigens are divided into T cell-dependent and T cell-independent antigens

T cell Dependent Antigens (Type 1)

y

y

y

y Most of the antigens that are protein in nature are T cell dependent y When these antigens are encountered – T cells help are needed for the B cells to produce antibodies y T cells that are involved in this are T helper (Th) cells y When T-cell-dependent antigen is involved in immune response – subsequence exposure to the same antigen  Gives more rapid production of antibodies  Greater amounts of antibodies  Production of antibodies occur for longer period of time

Epitope

Antibody

Paratope

y

y A substance is made as an antigen by certain properties it possess:  Size of the molecule ≈ antigenicity (Larger sized molecules have greater antigenicity)  Chemical nature: Proteins and polysaccharides are highly antigenic; Lipids and nucleic acids are less antigenic; Protein that is not antigenic is gelatin  Susceptibility to tissue enzymes  Foreignness, i.e. body’s ability to differentiate into self and non-self  Antigenic specificity  Heterophile specificity

y

y

y

y Carbohydrate/Polysaccharide, lipids, certain nucleic acids antigens are T cell independent y These antigens directly stimulate B cells without antigen processing y No memory response is seen  

Table 1: Differences between type 1 and type 2 antigen Type 1 Antigen

Type 2 Antigen

Endotoxin, Proteins Activate B cells with the help of cytokines and complements or with other immune cells Specific immunoglobulin production

Polysaccharides, LPS Directy activates the B cells

Causes polyclonal B cell activation

Table 2: Differences between cytosolic and endocytic pathways  

Antigen

T cell Independent Antigens (Type 2)

Property

Cytosolic Pathway

Antigen processed Endogenous Antigen complexed with MHC I molecules Antigen presented to Tc Cells

Endocytic Pathway Exogenous MHC II molecules Th cells

SUPERANTIGENS y Certain antigens – irrespective of the specificity – they activate large number of T cells, they are called superantigens y

y

Š

Š

y

Š

Š

y

y y y

y

TYPES OF ANTIGENS

y

INTRODUCTION

Remember

y Normally antigens bind in the αβ heterodimer groove of the MHC molecules connecting α and β of the TCR y Superantigens directly bind outside the antibody-binding groove y It goes and bind lateral aspect of T cell Receptor β chain.

ƒ

y

•

y

Examples for Superantigens • Bacterial superantigens ƒ Staphylococcal toxins like TSST-1, exfoliative toxin and enterotoxins; ƒ Streptococcal pyrogenic exotoxin A and C ƒ Mycoplasma arthritidis mitogen – I ƒ Yersinia enterocolitica and Yersinia pseudotuberculosis • Viral superantigens ƒ EBV associated superantigens ƒ Cytomegalovirus associated superantigens ƒ Rabies nucleocapsid ƒ HIV encoded superantigen (nef) • Fungal superantigen ƒ Malassezia furfur ƒ

•

ƒ

ƒ

ƒ

ƒ

•

ƒ

ƒ

ƒ

y

Unit 6     Immunology

Mechanism of Action

Figure 1: Superantigen

MULTIPLE CHOICE QUESTIONS







































































































































































402

7. All of the following statements about carbohydrate antigen are true, except: (Recent Pattern 2017) a. It has lower immunogenicity b. Memory response is seen c. Cause polyclonal B cell stimulation d. Does not require stimulation by T cells 8. Which of the following T cell independent antigen acts through: (Recent Pattern 2018) a. T-cell b. B-cell c. Macrophages d. CD8+ T cells 9. Superantigens true is: (AIIMS 2017) a. They bind to the cleft of the MHC b. Needs to processed before presentation c. They are presented by APC’s to T cells d. Directly attached to lateral aspect of TCR beta chain 10. Super antigen causes: (Recent Pattern 2018) a. Polyclonal activation of T-cells b. Stimulation of B cells c. Enhancement of phagocytosis d. Activation of complement 11. Which of the following is super antigen: (Recent Pattern 2017) a. M. protein b. Streptodornase c. Exfoliative toxin d. Hemolysin 12. Which of the following is a superantigen: (Recent Pattern 2017) a. Cholera toxin b. Diphtheria toxin c. TSST d. Vero- cytoxin



1. Haptens are immunogenic when they covalently bind to: (Recent Pattern 2018) a. Lipid carrier b. Polysaccharide carrier c. Protein carrier d. Any of the above carrier 2. Which of the following statements is true about Hapten: a. It induces brisk immune response (Recent Pattern 2018) b. It needs carrier to induce immune response c. It is a T-Independent antigen d. It has no association with MHC 3. The exact part of the antigen that reacts with the immune system is called: (Recent Pattern 2017) a. Clone b. Epitope c. Idiotope d. Effector 4. Monoclonal antibody binds to: (Recent Pattern 2018) a. Epitope b. Paratope c. Both epitope and paratope d. None of the above 5. Which part of bacteria is most antigenic? (Recent Pattern 2017) a. Protein coat b. Lipopolysaccharide c. Nucleic acid d. Lipids 6. Activation of naïve B lymphocytes by protein antigens is: (Recent Pattern 2017) a. T cell independent b. NK cell dependent c. NK cell independent d. T cell dependent

•

•

• Epitope is the smallest part present in the antigen • Antibody binds in this part in the antigen 4. Ans.  (a) Epitope

•

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 90 • Already explained Q.3 5. Ans.  (a)  Protein coat Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 90

• • •

• Superantigens directly bind outside the antibody binding groove • It goes and bind lateral aspect of T cell Receptor β chain • Ref the image above in theory part 10. Ans.  (a)  Polyclonal activation of T-cells Ref: Ananthanarayan and Paniker T.B. of microbiology – 10th edition – Page 93 • Superantigens cause a massive proliferation of T. cells – release massive amount of cytokines 11. Ans.  (c)  Exfoliative toxin Ref: Greenwood – Medical microbiology – 16th edition – page 92 • Staphylococcal exfoliative toxin is a superantigen • Other examples are: ƒ Staphylococcal toxic shock syndrome toxin ƒ Staphylococcal enterotoxins ƒ Streptococcal TSST ƒ Yersinia pseudotuberculosis ƒ Mycoplasma arthritis

•

•

•

• There are two types of antigens based on T cell dependency and chemical nature • Protein antigens are T cell-dependent antigens • Polysaccharide antigens are T cell-independent antigens

ƒ

12. Ans.  (c) TSST Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 93 • Already explained Q.11 •

7. Ans.  (b)  Memory response is seen

ƒ

ƒ

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 92

ƒ

6. Ans.  (d)  T cell dependent

ƒ

•

•

•

• Based on the chemical nature of antigen – the immunogenicity differs • Order of immunogenicity = Protein > Polysaccharides > lipids and nucleic acids

Ref: Ananthanarayan and Paniker T.B. of microbiology – 10th edition – Page 93 •

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 90

9. Ans.  (d) Directly attached to lateral aspect of TCR beta chain

•

3. Ans.  (b) Epitope

• T cell-independent antigens directly stimulate B cells • Antigen presenting cells and T cells have no role

•

•

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 89 • Already explained Q.1

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 92

•

2. Ans.  (b)  It needs carrier to induce immune response

8. Ans.  (b) B-cell

•

•

•

•

• Haptens are the substances that cannot induce antibody • They become immunogenic when they combine with larger molecules • Proteins are the larger carrier molecules

•

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 89

• Polysaccharide (Carbohydrate) antigens are T cellindependent antigens • These antigens directly stimulate B cells • No memory response is seen •

1. Ans.  (c)  Protein carrier

Chapter 55     Antigens

ANSWERS WITH EXPLANATIONS

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 92

403

56

Antibodies

y y y y

y y y y y

y

INTRODUCTION Antibodies are the glycoprotein molecules that are produced during immune response These antibodies go and specifically bind to the epitope region of antigen Antibodies are secreted by plasma cells Most of the antibodies are γ globulins, hence named as immunoglobulins Among the total serum proteins, Immunoglobulins represent 20–25%

STRUCTURE OF AN IMMUNOGLOBULIN

Figure 1: Immunoglobulin molecule y

y Each heavy chain has a variable region and constant regions divided into three/four domains – CH1, CH2, CH3, CH4  

Table 1: Functions of different regions on light and heavy chain Light chain

y





y

y

y

y

y

y

Immunoglobulins are made up of heavy and light chains Heavy chains have a molecular weight of 50,000 to 70,000 Light chains have a MW of 20,000 Shape of the immunoglobulin is Y shaped with two heavy (H) and two light (L) chains y The four chains are linked by disulfide bonds y The H and L chains in an immunoglobulin molecule are always identical because of two reasons:  Allelic exclusion  Regulations in the B cells – synthesis of either kappa or lambda occus in one immunoglobulin y Each light chain has a constant and variable region y y y y

Heavy chain

Variable region

Antigen binding

Constant region

No biological function

Variable region

Antigen binding

Constant region - CH2 Complement binding site Constant region - CH3 IgG receptors attach to neutrophils and macrophages

Based on the type of heavy chain, immunoglobulins are divided in to five classes: IgG (gamma), IgA (alpha), IgM (mu), IgD (delta), IgE (epsilon)

Table 2:  Characteristics of various immunoglobulins Characteristics

IgG

IgA

IgM

IgD

IgE

Sedimentation coefficient

7

7

19

7

8

Valency

2

2 or 4

2 or 10

2

2

150000

160000

900000

180000

190000

Serum concentration (mg/ml)

12

2

1.2

0.03

0.00004

Half life (days)

23

6

5

2-8

1-5

Daily production (mg/kg)

34

24

3.3

0.4

0.0023

Intravascular distribution (%)

45

42

80

75

50

Carbohydrate (%)

3

8

12

13

12

Complement fixation – Classical

++

–

+++

–

–

Complement fixation – Alternative

–

+

–

–

–

Placental transport

+

+

–

–

–

Present in milk

+

+

–

–

–

Selective secretion by seromucous glands

–

+

–

–

–

Heat stability (56deg C)

+

+

+

+

–

Forms

–

Dimer

Pentamer

–

–

Molecular weight

y Produced in secondary immune response y There are four subclasses of IgG y IgG1> IgG2 > IgG3 > IgG4 – based on the concentration present in the serum y IgG1, IgG3 and IgG2 (in the order) activate classical complement pathway y IgG4 activates alternate complement pathway y The only immunoglobulin that crosses the placenta and gives passive immunity naturally to fetus y IgG production by babies starts after 6 months y y y y

y

y

y

y

y

y

y First Ig class to be synthesized by the fetus y First Ig class to be produced in primary immune response, hence identification of IgM indicates recent infection y IgM is a pentameric immunoglobulin y Blood group antibodies are belonging to IgM class y Surface of B cell carries IgM in monomeric form

y

y

IgM

Chapter 56     Antibodies

CLASSIFICATION OF IMMUNOGLOBULINS

Table 3:  Properties of IgG

Figure 2: Structure of IgM molecule

IgG1

IgG2

IgG3

IgG4

% of concentration

70

20

6

4

Half life in serum (days)

23

23

7

23

Placental passage

+++

+

+++

+++

Complement fixation

+

+

+++

–

Binding to Fc receptors

+++

+

+++

–

IgA y Immunoglobulin seen in saliva, tears, colostrum, respiratory and GI secretions y

IgG

Properties of IgG

y

y

y Most available immunoglobulin in serum is around 80% y It has the maximum half-life of 23 days

405

y

Immunoglobulin Major Functions

• Monomer • Seen in serum

IgD

Uncertain. Found on the surface of many B cells as well as in serum.

Secretory IgA

• • • •

IgE

Mediates immediate hypersensitivity by causing release of mediators from mast cells and basophils upon exposure to antigen (allergen). Defends against worm infections by causing release of enzymes from eosinophils. Does not fix complement. Main host defense against helminth infections.

•

•

•

•

•

Serum IgA

•

Unit 6     Immunology

y Two forms are seen:

Dimer United by J chain It has a secretory component – that protects IgA from denaturation by bacterial proteases

Remember

Terminologies

• Antigenic differences in the constant regions decide isotype • IgA, IgG, IgM, IgE and IgD

Allotype

• Based on the allelic differences in immunoglobulins E.g. Gm type

Idiotype

• Based on the antigenic differences in the hypervariable regions

Isotypic switching

• All B cells have IgM on their surface and upon immune response it initially produces IgM • Because of VDJ genes that gets recombined because of enzyme called Switch recombinase – isotype switching occurs • This leads to production of other isotypes, i.e. other types of immunoglobulins

IgE

y Following an infection, antibodies are produced – these antibodies are produced against multiple epitopes of antigens that leads to separate classes of clones of antibodies y Monoclonal antibody is a single antibody forming cells against a single antigenic determinant specifically y Kohler and Milstein (1975) found a method for production of monoclonal antibodies – they were awarded Nobel prize for medicine in 1984 y Medium used for monoclonal antibody production is HAT medium—hypoxanthine, aminopterin and thymidine medium y Myeloma cells are used that has deficiency of HPRT enzyme —Hypoxanthine phosphoribosyl transferase y Hybridoma – Fusion of antibody forming spleen cells with myeloma cells

406

IgM

Produced in the primary response to an antigen. Fixes complement. Does not cross the placenta. Antigen receptor on the surface of B cells.

y y

Secretory IgA prevents attachment of bacteria and viruses to mucous membranes. Does not fix complement.

y

IgA

Types of Monoclonal Antibodies y Mouse mAb: 100% mouse-derived proteins y Chimeric mAb: Recombination of mouse proteins (Variable region) and human proteins (Constant region) y Humanized mAb: Only the antigen-binding site is mouse derived ; remaining part human derived y Human mAb: 100% human derived y

Main antibody in the secondary response. Opsonizes bacteria, making them easier to phagocytize. Fixes complement, which enhances bacterial killing. Neutralizes bacterial toxins and viruses. Crosses the placenta.

y

Immunoglobulin Major Functions IgG

y

 

Table 4: Important functions of immunoglobulins

y

y

y

y Present on the surface of B cells y No specific function

y

y

IgD

MONOCLONAL ANTIBODIES y

y

y

y

y

•

y

y

Called as Reagin antibody Heat labile immunoglobulin Species specific – homocytotropic IgE is responsible for anaphylaxis and atopy (Type 1 hypersensitivity) y IgE levels are elevated in parasitic infections, helminths, atopy y Only human IgE can fix to the surface of human cells – i.e it is species specific – this is the basis of Prausnitz Kustner reaction y y y y

•

•

Figure 3: Structure of IgA

•



•

•

•

Isotype

Table 5:  Enzymatic digestion of Ig molecule Papain digestion

2 Fab fragments 1 Fc fragment

Pepsin digestion

1 F(ab)2 fragment Fragmented Fc

β mercaptoethanol treatment

All disulfide bonds broken Seperated heavy and light chains

y

y

y

y Antibody molecule when treated with proteolytic enzyme like papain cleaves the molecule into two Fab fragments and 1 Fc fragment y Hinge region broken y This helps in placental transfer, taking part in complement fixation

MULTIPLE CHOICE QUESTIONS



c. IgA has minimum concentration d. IgM has minimum concentration 13. Pentavalent immunoglobulin is: (Recent Pattern 2017) a. IgA b. IgG c. IgM d. IgE 14. Heaviest immunoglobulin is: (Recent Pattern 2017) a. IgA b. IgG c. IgD d. IgM 15. Capacity of producing IgG starts at what age: (Recent Pattern 2017) a. 6 months b. 1 year c. 2 year d. 3 year 16. Which of the following immunoglobulins can cross placenta:  (Recent Pattern 2017) a. IgA b. IgM c. IgG d. IgD 17. The serum concentration of which of the following human IgG subclass is maximum: (Recent Pattern 2017) a. IgG 1 b. IgG 2 c. IgG 3 d. IgG 4 18. Maximum half-life  (Recent Pattern 2018) a. IgG b. IgA c. IgM d. IgE 19. Molecular mass of IgG (in k Da):  (Recent Pattern 2017) a. 150 b. 400 c. 1000 d. 1500 20. The earliest immunoglobulin to be synthesized by the fetus is:  (Recent Pattern 2017) a. IgA b. IgG c. IgE d. IgM 21. IgM appears in fetus at what gestational age:  (Recent Pattern 2018) a. 10 weeks b. 20 weeks c. 30 weeks d. At birth 22. ABO isoantibodies are of which class:  (Recent Pattern 2018) a. IgG b. IgM c. IgD d. IgA 23. Which of the following immunoglobulin is responsible for opsonisation:  (Recent Pattern 2018) a. IgA b. IgG c. IgM d. IgE 24. Activation of classical complement pathway: a. IgA b. IgG (Recent Pattern 2018) c. IgM d. IgD 















1. Number of variable regions on each light and heavy chain of an antibody: (Recent Pattern 2017) a. 1 b. 2 c. 3 d. 4 2. Complement attaches to immunoglobulin at: (Recent Pattern 2017) a. Amino terminal b. Fab region c. Variable region d. Fc fragment 3. Variable portion of antibody molecule is:  (Recent Pattern) a. C-terminal b. N-terminal c. CHO moiety d. None 4. Antigen binding site on antibody is:  (PGI Model Q) a. Hinge region b. Constant region c. Variable region d. Hypervariable region e. Idiotype region 5. Which portion of antibody binds to antigen?  (Recent Pattern 2017) a. Hinge region b. Constant region c. Variable region d. Hypervariable region 6. Antigen idiotype is related to:  (Recent Pattern 2017) a. Fc fragment b. Hinge region c. C-terminal d. N-terminal 7. Idiotypic class of antibody is determined by:  (Recent Pattern 2017) a. Fc region b. Hinge region c. Carboxy end d. Amino end 8. Immunoglobulin isotype class switching is determined by:  (Recent Pattern 2018) a. Constant region of light chain b. Constant region of heavy chain c. Variable region of light chain d. Variable region of heavy chain 9. Immunoglobulin variation does not depend on: (Recent Pattern 2017) a. Light chain b. Heavy chain c. Amino acid sequence d. Constant region 10. Immunoglobulin changes in variable region: a. Idiotype b. Isotype (Recent Pattern 2017) c. Allotype d. Epitope 11. Heat labile immunoglobulin:  (Recent Pattern 2017) a. IgA b. IgG c. IgE d. IgM 12. True about immunoglobulin:  (Recent Question 2017) a. IgE has maximum concentration b. IgG has maximum concentration

Chapter 56     Antibodies

Enzymatic Digestion of Immunoglobulin Molecule

407

 

Unit 6     Immunology

25. The Fc piece of which immunoglobulin fixes Cl: (Recent Pattern 2017) a. IgA b. IgG c. IgM d. IgE 26. Heavy chain of IgA is: (Recent Pattern 2017) a. Gamma b. Mu c. Delta d. Alpha 27. Major immunoglobulin secreted by intestine: (Recent Pattern 2017) a. IgG b. IgM c. IgA d. IgD 28. The secretory component of immunoglobulin molecule is: (Recent Pattern 2017) a. Formed by epithelial cells of lining mucosa b. Formed by plasma cells c. Formed by epithelial cell and plasma cell d. Secreted by bone marrow

29. IgE binds to which cell:  (Recent Pattern 2017) a. T cells b. B cells c. Mast cells d. NK cells 30. IgE receptor present on  (AIIMS 2017) a. Mast cell b. NK cell c. B cell d. Pro monocyte 31. PK reaction detects (Recent Pattern 2018) a. IgG b. IgA c. IgE d. IgM 32. Antibody elevated in parasitic infection: a. IgA b. IgE (Recent Pattern 2018) c. IgG d. IgM 33. Regarding IgE, Which of the following is false:  (AIIMS 2018) a. Causes anaphylaxis b. Immediate reaction c. Fix complement d. Cross placenta 34. Papain acts a gamma globulin to form:  (Recent Pattern 2017) a. 2 Fc fragments b. 2Fab fragments c. 1 Fab fragments d. None

ANSWERS WITH EXPLANATIONS 1. Ans.  (a) 1 Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 97

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 97 Ref: Already explained Q.4

•

• Heavy chain has one variable region and three/four constant regions • Light chain has one variable region and one constant region

5. Ans.  (c) Variable region; (d)  Hypervariable region

•

6. Ans.  (d) N-terminal

7. Ans.  (d)  Amino end

•

•

•

• Fc fragment – carboxy terminal • This terminal has the activity such as complement fixation, placental transfer • It is the catabolic state

• Each individual antigenic determinant of the variable region i.e paratope is called idiotope • It is the specific region of the Fab portion of the Ig molecule to which antigen binds •

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 98

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 97,102

•

2. Ans.  (d)  Fc fragment

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 97

• Idiotype – varible region – hence it is amino terminal end 8. Ans.  (b)  Constant region of heavy chain

9. Ans.  (a)  Light chain Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 97 • Already explained Q.8 •

408

•

•

•

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 97, Harrison T.B of internal medicine – 19th edition - Page 372 • Amino acid sequences in the variable regions has some invariable sequences and some highly variable sequences • These hypervariable regions form the antigen binding site of the antibody molecules – complementarity determining regions (CDRs) • Idiotype is defined as the specific region of the fab portion in immunoglobulin molecule where the antigen binds

• Upon antigenic stimulation → the heavy chain VDJ unit can join any other constant heavy gene → leads to expression of particular antibody → this process is called as isotypic class switching • Because of variations in the amino acid sequence in the constant region → five classes of antibody are available – IgG, IgA, IgM, IgE, IgD •

4. Ans.  ( c) Variable region; (d) Hypervariable region; (e) Idiotype region

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 97,103

•

•

•

• Carboxy terminal (C) – constant unit • Amino terminal (N) – variable unit

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 97,102 •

3. Ans.  (b) N-terminal

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 99 • IgG has the maximum half life of 23 days •

•

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 97,102 • Already explained Q.6, Q.7

18. Ans.  (a) IgG

12. Ans.  (b)  IgG has maximum concentration Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 99

•

•

• IgG has the maximum serum concentration of 12 mg/ml • IgE has the minimum serum concentration of 0.00004 mg/ml 13. Ans.  (c) IgM Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 100

•

•

•

• IgM is a heavy molecule – Molecular weight is 1,00,000 – hence called as millionaire molecule • IgM has a theoretical valency of 10 – but the effective valency is 5 • It is a pentavalent molecule with a J chain 14. Ans.  (d) IgM

•

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 100 • Already explained Q.13 15. Ans.  (a)  6 months Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 99

Molecular weight of immunoglobulins in kDa IgG

150

IgA

160 (monomer) 400 (dimer)

IgM

1000

IgE

190

IgD

175

20. Ans.  (d) IgM Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 100 • Around 20 weeks of age – the first immunoglobulin secreted by the fetus is IgM • It is the antibody that is produced in primary immune response 21. Ans.  (b)  20 weeks Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 100 Ref: Already explained Q.20 22. Ans.  (b) IgM Ref: Kuby – immunology – 7th edition – page 812 • Blood group antibodies belong to IgM type 23. Ans.  (c) IgM

•

• Immunoglobulins that are involved in opsonisation are IgM and IgG • Comparatively IgM is more effective than IgG • IgM does opsonisation through classical pathway – C3b •

16. Ans.  (c) IgG

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 97, Harrison T.B of internal medicine – 19th edition – page 372

•

•

•

•

• IgM is the primary antibody secreted immediately after birth • Maternal IgG crosses placenta and gives passive protection • After 6 months – IgG synthesis attains steady level and body has its own capacity to meet immunity

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 97

•

•

• All of the immunoglobulins are heat stable except IgE – heat labile

19. Ans.  (a) 150

•

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 100

•

11. Ans.  (c) IgE

Chapter 56     Antibodies

10. Ans.  (a) Idiotype

•

• The only maternal immunoglobulin that crosses placenta is IgG

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 100 • Activator of classical complement pathway – IgM, IgG • Activator of alternate complement pathway – IgA •

17. Ans.  (a)  IgG 1

24. Ans.  (c) IgM

•

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 99

•

•

•

• The maximum concentration that is present in the serum is IgG • It has four subclasses IgG1-4 • Among them IgG1 is the greatest amount ; IgG4 the least

25. Ans.  (c) IgM Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 100 • C1 – involved in classical complement pathway ( will be explained in next chapter) •

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 99

409

•

• Immunoglobulin seen in saliva, tears, colostrum, respiratory and GI secretions 28. Ans.  (a)  Formed by epithelial cells of lining mucosa Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 99

•

•

• IgA has a secretory component – which is secreted by the mucosal epithelial cells • IgA as such and J chain are produced from plasma cells

• • •

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 100 • IgE is elevated in parasitic infections especially helminthic infections •

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 99

32. Ans.  (b) IgE

33. Ans.  (d) Cross placenta; (c) Fix complement Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 100 • • • •

•

27. Ans.  (c) IgA

• IgE is homocytotropic – i.e species specific • Only human IgE can fix the surface of human cells • For detecting allergy – atopy – Prausnitz and Kustner made an experiment injecting the serum of other one who had allergic preponderance

•

•

• Heavy chains are the basis for classes of immunoglobulins • IgG (gamma), IgA (alpha), IgM (mu), IgD (delta), IgE (epsilon) are the five classes

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 100

•

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 98

31. Ans.  (c) IgE

•

•

26. Ans.  (d) Alpha

•

Unit 6     Immunology

• IgM is the antibody more effective in classical complement pathway

29. Ans.  (c)  Mast cells

•

•

• IgE is involved in type 1 hypersensitivity – atopy • IgE goes and binds to mast cells and basophils and secrete lots of histamine

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 100 • Antibody molecule when treated with proteleolytic enzyme like papain cleaves the molecule into two Fab fragments and 1 Fc fragment • Hinge region broken •

30. Ans.  (a)  Mast cell

34. Ans.  (b)  2 Fab fragments

•

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 100

•

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 100 • Already explained Q.29

410

IgG is the only immunoglobulin that crosses placenta Activator of classical complement pathway – IgM, IgG Activator of alternate complement pathway – IgA Hence option C and D are wrong (question may be wrong)

Complement System

57

INTRODUCTION







y

y

y There are certain factors that occur in normal sera which are activated by antigen and antibody complexes – these factors are called complements y The three main effects of complement are:  Lysis of bacteria, allografts and tumor cells  Generation of mediators that participate in inflammation and attract neutrophils  Opsonization.

SYNTHESIS OF COMPLEMENT Table 1:  Sites of synthesis of complements Complements

Sites of synthesis

C1

Intestinal epithelium

C2, C4

Macrophages

C5, C8

Spleen

C3, C6, C9

Liver

COMPLEMENT PATHWAYS







y





y

y

y Most of the complements are present as inactive forms, which are then activated to active forms y The activators of complement are:  Antigen: Antibody complexes  Nonimmunological molecules y Activation of complement pathways occurs via three pathways:  Classic pathway  Alternative pathway  Lectin pathway Figure 1: Pathways of complement system [(Error in the image – C5 convertase in alternative pathway is C3bBbC3b – not 8 (not eight – its Bb)]

Unit 6     Immunology

Table 2:  Characteristics of pathways

Table 4:  Biological effects of complement

Pathways

Characteristics

Effects

Mediators

Classical pathway

• Antibody-dependent pathway; • Pathway is triggered by antigen-antibody complex • Involved in specific active immunity

Opsonization

C3b

Chemotaxis

C5a, C5,C6,C7 - complex

Anaphylatoxin

C5a > C3a > C4a

• Antibody-independent pathway • Pathway triggered by antigen directly • Involved in nonspecific innate immunity

Cytolysis

C5b6-9 (MAC ) complex

Enhancement of antibody production

C3b

•

•

•

Alternative pathway

•

•

•

Lectin pathway

• Antibody-independent pathway • Involved in nonspecific innate immunity •

•

y All the three pathways lead to the production of central molecule named as C3b y In classical pathway C3 convertase is C4b2a y In alternative pathway C3 convertase is C3bBb y

y

y

REGULATION OF COMPLEMENT PATHWAYS Inhibitors y Normally serum has an inhibitor of C1 esterase (C1sINH) – checks the autocatalytic prolongation y S protein is seen in normal serum – modulates the cytolytic action of the MAC y

y

Inactivators 











Figure 2: C3 and C5 convertase in classical and alternative pathways

Table 3:  Activators of pathways Pathways

Activator

Classical pathway

• Antigen-antibody complex

Alternative pathway

• Endotoxin / LPS • Teichoic acid • Fungal cells – Zymosan polysaccharide • Yeast • Trypanosomes • Viral infected cells • Tumor cells • Antibodies in complexed – IgA, IgD • Cobra venom factor • Dextran sulfate • Nephritic factor • Inulin • Agar





Factor I – controls C3 activation and alternative pathway Factor H – acts with Factor I – regulates C3 activation Anaphylatoxin inactivator degrades the anaphylatoxins (C3a, C4a, C5a) C4 binding protein controls the activity of C4b.

Deficiencies of Complement y C2 deficiency is the most common complement defect y

Table 5:  Syndromes resulting from complement deficiency

•

Group Deficiency

Syndrome

I

Cl Inhibitor

Hereditary angioneurotic edema

II

Early components SLE and other collagen vascular of classical diseases pathway C1, C2, C4

III

C3 and its regulatory protein C3b inactivator

Severe recurrent pyogenic infections

IV

C5 to C8

Bacteremia, mainly with Gramnegative diplococci, toxoplasmosis

V

C9

No particular disease

DAF – Decay accelerating factor

PNH – Paroxysmal nocturnal hemoglobinuria

•

•

•

•

•

•

•

•

•

•

•

•

•

Lectin pathway

• Mannose (Carbohydrate) residues on microbes • Salmonella • Neisseria • Listeria • Cryptococcus • Candida albicans •

•

•

•

•

•

412

1. Total number of complement factors in complement 12. Anaphylotoxin in complement system:  system:  (Recent Pattern 2017) (Recent Pattern 2017) a. 7 b. 9 a. C3b b. C5-9 b. 13 d. 20 c. C5a d. C1-3 2. Complement components are:  13. Chemoattractant is:  (Recent Pattern 2017) (Recent Pattern 2017) a. C5a b. C1 a. Lipids b. Proteins c. C3 d. C2 c. Lipoproteins d. Polysaccharide 14. In cell, lysis by complement:  (Recent Pattern 2018) 3. Complement components are:  a. They activate cyclise (Recent Pattern 2017) b. Inhibits elongation factor P a. Lipoproteins b. Glycoproteins c. Destruction of cell wall c. Polysaccharides d. Lipid d. Increased permeability of cell membrane 4. Complement formed in liver:  (Recent Pattern 2017) 15. MAC in complement system is:  a. C2, C4 b. C3, C6, C9 (Recent Pattern 2017) c. C5, C8 d. C1 a. C3a b. C3b 5. Activator of alternative complement pathway:  c. C5-9 d. C4b (Recent Pattern 2017) 16. Function of complement include all, except:  a. Antigen-antibody complex (Recent Pattern 2018) b. Mannose-binding lectin a. Chemotaxis b. Opsonisation c. Microbial surface polysaccharide c. Cell lysis d. Antigen presentation d. All of the above 17. Hereditary angioneurotic edema is due to:  6. Which complement component is involved in both (Recent Pattern 2018) classical and alternative pathways:  a. Deficiency of C1 inhibitor (Recent Pattern 2017) b. Deficiency of NADPH oxidase a. C1 b. C2 c. Deficiency of MPO c. C3 d. C4 d. Deficiency of properdin 7. Center of complement pathway:  (Recent Pattern 2017) 18. Neisseria infection is predisposed by:  a. C3 b. C1 (Recent Pattern 2017) c. C5 d. C2 a. Properdin b. Factor D 8. True regarding C4b2a: (Recent Pattern 2017) c. C5-C9 component d. All of the above a. C3 convertase in classical pathway 19. Recurrent Neisseria infection are predisposed by:  b. C3 convertase in alternative pathway (Recent Pattern 2017) c. C5 convertase in classical pathway a. Early complement component deficiency d. C5 convertase in alternative pathway b. Late complement component deficiency 9. C3 convertase in alternate complement pathway:  c. C1 esterase deficiency (Recent Pattern 2017) d. Properdin deficiency a. C4b2a b. C3b 20. Complement deficiency has not been implicated in c. C3bBb d. C3a causing:  (Recent Pattern 2018) 10. C3b is converted to C3 convertase by:  a. SLE (Recent Pattern 2017) b. PNH a. Factor B b. Factor P c. Hereditary angioedema c. Factor H d. Factor I d. Membranous nephritis 11. Byproducts of complement system anaphylactic-toxins: 21. Which is the opsonin in complements: (Recent Pattern 2017) (Recent Pattern 2017) a. C3b b. C4a  a. C3b b. C3a c. C5a d. C5678 c. C5a d. C4a 





Chapter 57     Complement System

MULTIPLE CHOICE QUESTIONS





















413

Unit 6     Immunology

ANSWERS AND EXPLANATIONS 1. Ans.  (b) 9

8. Ans.  (a) C3 convertase in classical pathway

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 123

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 126

• Complement pathway has nine distinct factors from C1 to C9 • C1 has three subunits

• In classical pathway C3 convertase is C4b2a • In alternative pathway C3 convertase is C3bBb

•

•

•

•

2. Ans.  (b) Proteins Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 123

9. Ans.  (c) C3bBb Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 126 • Already explRecent Patternned Q.8 •

• Complement system consists of effector and regulatory proteins • At least a minimum of 30. •

•

3. Ans.  (b) Glycoproteins Ref: Medical immunology – Owen Kuby • Most of the complement components are glycoproteins. •

4. Ans.  (b) C3, C6, C9

10. Ans.  (a) Factor B Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 126 • In alternative pathway C3b is converted to C3Bb by Factor B • C3Bb is the C3 convertase •

•

11. Ans.  (c) C5a

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 128

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 125

• Complements are synthesized from various parts of the body ƒ Intestine – C1 ƒ Macrophages – C2, C4 ƒ Spleen – C5, C8 ƒ Liver – C3, C6, C9

• C5a, C3a are the potent anaphylatoxins • C4a has also feature of anaphylatoxin • But the answer need to be chosen as one choice – go for the most potent—it is C5a • If it is PGI question – choose both C5a and C4a

•

ƒ

ƒ

ƒ

•

•

•

•

ƒ

5. Ans.  (c) Microbial surface polysaccharide Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 125

12. Ans.  (c) C5a Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 125 • Already expl Answer 11 •

• Alternative complement pathway is activated by zymosan – which is a polysaccharide from the yeast cell wall • Other activators are bacterial endotoxins, IgA, IgD, cobra venom fator, nephritic factor •

•

13. Ans.  (a) C5a Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 125 • C5a – involved in chemotaxis. •

6. Ans.  (c) C3 Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 123 • There are C3 convertases in both the pathways which act on C3 and splits it into C3a and C3b • Hence C3 is the complement seen in both pathways. •

•

14. Ans.  (d) Increased permeability of cell membrane Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 126 • The final by product of complement pathways is MAC complex which is C5-C9 complements • This MAC – membrane attack complex forms channels in the lipid cell membrane and leads to cell lysis. •

•

7. Ans.  (a) C3 Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 126 • All the three complement pathways meet at the center molecule – C3 convertase – which act on C3 – the major component of complement pathways. •

414

15. Ans.  (c) C5-9 Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 126 • Already explRecent Patternned answer 14. •

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 127 • Biological effects of complement are: ƒ Phagocytosis – opsonization ƒ Chemotaxis ƒ MAC – cell lysis ƒ Inflammatory response ƒ Hypersensitivity reactions ƒ Autoimmune disease ƒ Endotoxic shock ƒ Immune adherence ƒ Conglutination

that is not formed – MAC cannot be formed – indirectly it also predisposes to Neisseria infection • But specifically MAC complex deficiency leads to Neisseria infection. •

•

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ƒ

ƒ

ƒ

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ƒ

19. Ans.  (b) Late complement component deficiency Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 126,128 • Already explained Q.18 •

20. Ans.  (b) PNH

ƒ

ƒ

ƒ

17. Ans.  (a) Deficiency of C1 inhibitor Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 128 • Deficiency of C1 inhibitor leads to hereditary angioneurotic odema • Characterized by episodic angioedema of the subcutaneous tissues or of the mucosa of alimentary and respiratory tracts •

•

18. Ans.  (d) All of the above Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 128 • Terminal components of complements namely C5 to C9 forms the membrane attack complex (MAC) • Deficiency of any of these componenets – leads to inability to form the complex – that leads to predisposition of infection with Neisseria sp • Neisseria meningitidis infections are more common • Considering other options – Properdin and Factor D are needed to activate the alternative pathway – hence if •

•

•

•

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 128 • Genetic deficiencies of complement components leads to: • Hereditary angioneurotic edema • SLE • Severe recurrent pyogenic infections • Gram-negative septicemia • Toxoplasmosis infection

Chapter 57     Complement System

16. Ans.  (d) Antigen presentation

•

•

•

•

•

•

21. Ans.  (a) C3b Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 125

Effects

Mediators

Opsonisation

C3b

Chemotaxis

C5a, C5, C6, C7 - complex

Anaphylatoxin

C5a > C3a > C4a

Cytolysis

C5b6-9 (MAC ) complex

Enhancement of antibody production

C3b

415

58

Antigen-Antibody Reactions Examples

Tube precipitation

Kahn test for syphilis

Precipitation in gel—Immuno diffusion • Oudin procedure

• Double diffusion in one dimension

• Oakley Fulthorpe procedure

• Single diffusion in two dimension

• Radial immunodiffusion • For screening antibodies in sera for influenza virus

• Double diffusion in two dimensions

• Ouchterlony procedure • Elek’s gel test for Diphtheria bacilli

Precipitation Reaction

• • • •

•

•

•

Precipitation reactions Agglutination reactions Complement fixation tests Neutralization tests Radioimmunoassay Enzyme linked immunoassay Chemiluminescence immunoassay Immunoelectrophoresis blot Immunochromatographic tests Immunoflourescence

Electroimmunodiffusion •

• For cryptococcal and meningococcal antigens in the CSF •

• One dimensional single • Rocket electrophoresis electroimmunodiffusion • Laurell’s electrophoresis •

•

• Two-dimensional electrophoresis

y

y

y

y

y Precipitation reaction is very sensitive in the detection of antigens y Precipitation reaction is based on lattice hypothesis y Depending upon the amount of antigen and antibody – the precipitate formation differs y When the amount of precipitate is plotted on a graph vs antigen and antibody concentration, three phases of curve is obtained

• Counterimmunoelectrophoresis •

Soluble antigen + antibody (in the presence of NaCl) → antigenantibody complex → Precipitate (insoluble)

•

y y y y y y y y y

y y y y y y y y y y

y

TYPES OF ANTIGEN-ANTIBODY REACTIONS

•

• Single diffusion in one dimension •

y

y

y Antigen and antibody form complexes in the body which is the important event of immune system y These reactions can be done in vitro and this helps in the diagnosis of immunological reactions.

Types

•

INTRODUCTION

Table 1:  Marrack’s Lattice Hypothesis

Agglutination Reaction Particulate antigen + antibody (in the presence of NaCl) → antigen-antibody complex → visible clumps (agglutinated)

Excess antibody Visible reaction is not seen

Zone of equivalence

Equal antigen and antibody Lattice formation and visible reaction seen

Post-zone phenomenon

Excess antigen No visible reaction

y Agglutination reaction is more sensitive for the detection of antibodies y Incomplete antibodies do not cause agglutination but they will combine with antigen and blocks further reaction with a complete antibody—blocking antibodies y These blocking antibodies can give false negative results by blocking the antigen; thereby true antibodies cannot bind and cause agglutination y Blocking antibodies are commonly seen in brucellosis y Blocking antibodies can be detected by:  Hypertonic 5% saline  Albumin saline  Antiglobulin (Coomb’s) test y

y

y

Prozone phenomenon

Table 2:  Types of precipitation tests Examples

Slide flocculation test

VDRL test for syphilis

Ring precipitation

Ascoli’s thermoprecipitin test Lancefield Grouping of Streptococci

 

contd…



y

y

Types

Types

Examples

Slide agglutination test

Blood grouping

Tube agglutination test

• Widal test • Standard agglutination test for Brucellosis •

•

Heterophile agglutination test

• Weil-Felix test • Streptococcus MG agglutination test • Paul Bunnell test •

•

•

Antiglobulin (Coomb’s) test Direct Coomb’s test

Rh incompatibility

Indirect Coomb’s test

Rh incompatibility

Passive agglutination test: (Soluble antigens are used) Hemagglutination test

Rose Waaler test

Latex agglutination test

• • • •

•

•

•

•

• Coagglutination test (S.aureus – Protein A is used) •

ASO titer detection CRP estimation RA factor HCG

• Salmonella • Legionella •

•

y Source of complement is guinea pig serum y Most sensitive test, e.g. Wassermann complement fixation test for syphilis, Treponema pallidum immobilization test y

y

Neutralization Test y Virus neutralization test, e.g. Plaque inhibition test y Toxin neutralization test:  In vitro – Nagler reaction  In vivo – Dick test, Schick test y

y





Coomb’s Test Table 4:  Differences between direct and indirect coomb’s test Direct Coomb’s test

Indirect Coomb’s test

• In vivo test

• In vitro test

• Detects the antibodies bound to RBCs

• Detects free antibodies

•

•

•

•

Chapter 58     Antigen-Antibody Reactions

Table 3:  Types of agglutination reaction

Enzyme-Linked Immunosorbent Assay (ELISA) y Most commonly used serological technique for diagnosis of infections y Types of ELISA:  Indirect ELISA → Antigen – Antibody – Antibody to antibody (Conjugate)  Sandwich ELISA → Antibody – Antigen – Antibody to antigen (Conjugate)  Competitive ELISA → Antibody – Known antigen + Unknown antigen  Cassette ELISA or Cylinder ELISA → done in disposable cassettes – quick test – E.g. Dot blot assay y Enzyme used in ELISA: Horse radish peroxidase (HRP) y Substrate used in ELISA: O-phenylene diamine dihydrochloride y

y









y

y

Figure 1: Rapid plasma reagin test (Courtesy: Image from Author’s own thesis work)

Figure 2: Tube agglutination test – Widal test (Courtesy: Dr. Vanathi S, KAP Vishwanathan Govt Medical College, Trichy)

Figure 3: ELISA plate with wells (Courtesy: Image from Author’s own thesis work)

Immunochromatographic Test y Rapid test method y Done in a small cassette which is already impregnated with antibody/antigen with colloidal gold dye conjugate y When antigen–antibody reaction occurs, it is visible as colored product y

Complement Fixation Test y Antigen and antibody complexes fix complement and this principle is used for diagnosis y

y

y

417

Unit 6     Immunology

y Used in diagnosis of HIV, HBV, HCV and many other infections y

Figures 4: Immunochromatographic test (Courtesy: Dr.S.Vanathi, Asst Prof of Microbiology, Govt KAP Vishwanathan Medical College, Trichy, Tamil Nadu)

FLOW CYTOMETRY y Based on laser emission – forward and side scattering from the cells are measured y Cell count, size, shape and certain markers can be estimated y Helps for CD4 count y

y

y

418

1. Rose waaler test: (Recent Pattern 2018) a. Complement fixation test b. Precipitation in gel c. Ring precipitation d. Passive haemagglutination test 2. Precipitation in comparison to agglutination requires:  (Recent Pattern 2017) a. Less PH b. High temperature c. Specific enzyme d. Soluble antigen 3. VDRL is an example of:  (Recent Pattern 2017) a. Slide agglutination b. Tube agglutination c. Slide flocculation d. Tube flocculation 4. Which of the following is agglutination test:  (Recent Pattern 2017) a. Widal test b. VDRL c. Ascoli’s test d. Kahn test 5. Paul Bunnell reaction is a type of: (Recent Pattern 2017) a. Agglutination b. CF c. Precipitation d. Flocculation test 6. Weil-Felix reaction is:  (Recent Pattern 2018) a. Ring test b. Tube precipitation test c. Slide agglutination test d. Tube agglutination test 7. Nagler reaction is a type of:  (Recent Pattern 2018) a. Neutralization reaction b. CFT c. Precipitation d. Agglutination 8. Coomb’s test is:  (Recent Pattern 2018) a. Precipitation test b. Agglutination test c. CFT d. Neutralization test 9. Post-zone phenomenon is seen in:  (Recent Pattern 2017) a. Antigen excess b. Antibody excess c. Equivalence zone d. None of the above 10. Prozone phenomenon is seen with:  (Recent Pattern 2018) a. Same concentration of antibody and antigen b. In antigen excess to antibody c. Antibody excess to antigen d. Hyperimmune reaction 





11. Prozone phenomenon is responsible for   (Recent Pattern 2017) a. False negative test b. False positive test c. May cause any of the above d. Has no relation with accuracy of test 12. Prozone phenomenon is a feature is:  (Recent Pattern 2017) a. Tularaemia b. Legionnaire’s disease c. Plague d. Secondary syphilis 13. Antigen-antibody precipitation is maximally seen in which of the following:  (AIIMS 2017) a. Excess of antibody b. Excess of antigen c. Equivalence of antibody and antigen d. Antigen Hapten interaction 14. Lattice phenomenon is seen in:  (Recent Pattern 2017) a. Neutralization reaction b. Complement fixation test c. Precipitation test d. All of the above 15. Maximum lattice formation occurs in:  (Recent Pattern 2017) a. Zone of antibody excess b. Zone of antigen excess c. Zone of equivalence d. Can occur in any zone 16. Indirect coomb’s test detect:  (Recent Pattern 2017) a. Incomplete antibody bound to RBC b. Free antibody c. Free agglutinable RBCs d. None Of the above 17. Heterophile agglutination test is: (Recent Pattern 2017) a. Widal test b. Streptococcus MG agglutination c. VDRL d. Kahn test of syphilis 18. Heterophile antibody is found in: (Recent Pattern 2018) a. Weil Felix test b. Widal test c. VDRL d. All 19. Oakley- fulthorpe procedure is:  (Recent Pattern 2017) a. Agglutination test b. Precipitation test c. Single diffusion in one dimension d. Double diffusion in one dimension 

Chapter 58     Antigen-Antibody Reactions

MULTIPLE CHOICE QUESTIONS



419

Unit 6     Immunology

ANSWERS AND EXPLANATIONS 1. Ans.  (d) Passive haemagglutination test

8. Ans.  (b) Agglutination test

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 112

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 111

• Rose-Waaler test – passive agglutination test for RA • RA factor – is an autoantibody

• Coomb’s test is an example for agglutination test • Also called as antiglobulin test • Helpful in diagnosis of Rh incompatibility.

•

•

•

•

•

2. Ans.  (d) Soluble antigen Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 107 • Soluble antigen is used for precipitation reactions • Particulate antigen is used for agglutination reactions. •

•

9. Ans.  (a) Antigen excess Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 107 • Lattice hypothesis states three zones • Pro zone phenomenon – excess antibody • Zone of equivalence – equal amounts of antigen and antibody • Post zone phenomenon – excess antigen. •

•

3. Ans.  (c) Slide flocculation Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 107 • In precipitation reactions – sedimentation occurs • Instead of that – if the precipitate remains suspended as floccules – the reaction is known as flocculation E.g. for slide flocculation test is diagnosis for syphilis – VDRL •

•

4. Ans.  (a) Widal test

•

•

10. Ans.  (c) Antibody excess to antigen Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 107 • Already explained Q.9 •

11. Ans.  (a) False negative test

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 111

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 107

• Example for tube agglutination test is Widal test • All other tests are precipitation reactions.

• Prozone phenomenon has excess antibody which gives false negative reactions as the antibodies forms complexes and do not react – no agglutination or precipitation occurs visibly • To avoid this – serial dilutions are done and checked.

•

•

5. Ans.  (a) Agglutination Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 111 • Paul Bunnel test is helpful in diagnosis of Infectious mononucleosis • It is an example of heterophile agglutination test using RBCs as antigens •

•

6. Ans.  (d) Tube agglutination test Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 111 • Examples of tube agglutination test: ƒ Widal test ƒ Standard agglutination test for Brucellosis ƒ Weil-Felix test ƒ Streptococcus MG agglutination test ƒ Paul Bunnell test •

ƒ

ƒ

ƒ

•

•

12. Ans.  (d) Secondary syphilis Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 381 • Antibodies starts to raise during secondary stage of syphilis • This leads to high titre sera – false negative • Serial dilutions are done to avoid this. •

•

•

13. Ans.  (c) Equivalence of antibody and antigen Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 107 • Already explained Q.9 •

ƒ

ƒ

7. Ans.  (a) Neutralization reaction Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 114 • Nagler’s reaction is for identification of alpha toxin of Clostridium perfringens • It is an example for in vitro neutralization test •

420

•

14. Ans.  (c) Precipitation test Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 107 • Marrack’s lattice hypothesis is the basic principle for precipitation and agglutination •

19. Ans.  (d) Double diffusion in one dimension

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 107

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 109

• Already explained Answer 9

Immunodiffusion types:

•

16. Ans.  (b) Free antibody Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 111 • Direct Coomb’s test – detects antibodies bound to RBC • Indirect Coomb’s test – detects free antibodies. •

Single diffusion in one dimension

Oudin procedure

Double diffusion in one dimension

Oakley Fulthorpe procedure

Single diffusion in two dimensions

Radial immunodiffusion For screening antibodies in sera for influenza virus

Double diffusion in two dimensions

Ouchterlony procedure Elek’s gel test for Diphtheria bacilli

•

17. Ans.  (b) Streptococcus MG agglutination Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 111 • Examples for heterophile agglutination test: ƒ Weil-Felix test ƒ Streptococcus MG agglutination test ƒ Paul Bunnell test •

ƒ

Chapter 58     Antigen-Antibody Reactions

15. Ans.  (c) Zone of equivalence

ƒ

ƒ

• Option b: Precipitation is also correct; Because immunodiffusion is nothing but precipitation in gel • But since it is a NEET pattern type – choose the specific answer •

18. Ans.  (a) Weil-Felix test

•

Ref: Ananthanarayan and Paniker T.B of microbiology – 10th edition – Page 111 • Already explained Q.17 •

421

59

Immune Response

y

y y

y When an antigen is encountered by the human body → series of events occur in the body based on the nature of antigen → called as immune response

y Rejection of grafts: Graft vs host reaction y Immunological surveillance: Immunity against cancer y Delayed type hypersensitivity (Type 4) y

IMMUNE RESPONSE

Antibody-Mediated Immunity (Humoral Immunity)

y

y

y







y

y Antibody-Mediated immunity needs three cells  Antigen presenting cells  T helper cells  B cells y Like in cell mediated immunity—Microbes are processed into peptide antigen (T cell-dependent antigens) along with MHC Class II it is presented to the receptor in T helper cells y Activation of these T helper cells—leads to synthesis and secretion of interleukins—IL2, IFN gamma y These cytokines go and act on B cells that will produce plasma cells in turn produce specific antibodies (Immunoglobulins) to that antigen

Functions y

y Defence against extracellular bacterial pathogens y Defence against viruses that infect through respiratory or intestinal tracts y Prevents recurrence of virus infections y Participates in the pathogenesis of type 1, 2, 3 hypersensitivity

y y

y

TYPES OF IMMUNE RESPONSE There are two types of immune response y Cell-mediated immunity (CMI) y Antibody mediated immunity

y

y

FIGURE 1: Adaptive immunity

Production of Antibodies y

y Antibody production occurs in characteristic phases

Cell-Mediated Immunity

Table 1:  Phases of antibodies production Phases

Characteristics

Lag phase

Antigen stimulates – no antibody in circulation

Log phase

Titre of antibody rises steadily

Plateau phase

Equilibrium between antibody synthesis and catabolism

Phase of decline

Catabolism exceeds production and titre falls

y

y

y

y

y

y When a bacterium, e.g. Mycobaterium tuberculosis → enters the body → it splits into fragments → peptides → called as antigens → occurs in macrophages along with MHC class II y This antigen along with MHC Class II is presented to the receptor in T helper cells y Activation of these T helper cells leads to clonal proliferation y In turn leads to synthesis and secretion of interleukins—IL2, IFN gamma y The same process happens through MHC Class I when viral cells or tumor cells are processed

Function y

y Protects against fungi, viruses, facultative intra cellular bacterial pathogens

Features

Primary response

Secondary response

Lag period

4–10 days

1–3 days

Type of B cells involved

Naïve B cell

Memory B cell

Peak response

7–10 days

3–5 days

Peak antibody response

Antigen dependent

Usually more intense than primary response

Type of antibody seen

IgM

IgG

Antibody affinity

Lower

Higher

Type of antigens

Thymus independent as well as thymus dependent

Only thymus dependent

Immunization

Needed preferably protein antigens with adjuvants

Adjuvants not required

Chapter 59     Immune Response

Table 2:  Features of primary and secondary immune response

THEORIES OF IMMUNE RESPONSE Figure 2: Immune response

Side chain theory: Postulated by Ehrlich Direct template theories Indirect template theory Natural selection theory: Postulated by Jerne Clonal selection theory: Postulated by Burnet  Clones of cells which react with self-antigens are destructed during embryonic life – such clones are called as forbidden clones  If they persist in adult life they lead to autoimmune disease y Split gene theory: Postulated by Susumu Tonegawa. y y y y y

y

y

Other Types Based on certain other characteristics, immune response can be classified as: y Primary response y Secondary response y

y

y

y

y





y

423

Unit 6     Immunology

MULTIPLE CHOICE QUESTIONS 1. Antibody mediated immunity helps in: a. Graft versus host reaction (Recent Pattern 2017) b. Immunological surveillance c. DTH d. Type 2 hypersensitivity 2. Feature of secondary immune response: (Recent Pattern 2018) a. Naïve B cell is involved b. IgM mediated c. Only thymus dependent antigens d. Lower antibody immunity 3. Polysaccharide antigens may persist in the body up to (Recent Pattern 2018) a. 10 days b. 10 weeks c. 10 months d. 10 years 4. Polysaccharide antigens are: (Recent Pattern 2017) a. T cell dependent antigens b. T cell independent antigens c. MHC I dependent antigens d. MHC II dependent antigens 5. CD8 cells are activated by: (Recent Pattern 2017) a. Antigens with MHC Class I b. Antigens with MHC Class II c. Antigens with MHC Class III d. Antigens with complement 6. Example for an adjuvant: (Recent Pattern 2017) a. Corynebacterium diphtheria b. Bordetella pertussis c. Brucella abortans d. Clostridium tetani 7. All of the following are lymphokines affecting lymphocytes, except: (Recent Pattern 2017) a. Blastogenic factor b. T cell growth factor c. B cell growth factor d. Chemotactic factor 8. Vaccination is based on the principle of : a. Agglutination (Recent Pattern 2017) b. Phagocytosis c. Immunological surveillance d. Immunologic memory 



9. IL-1 is produced by: (Recent Pattern 2017) a. Macrophages b. T cells c. B cells d. Lymphocytes 10. Transfer factor mediates: (Recent Pattern 2017) a. Cell-mediated immunity b. Adoptive immunity c. Humoral immunity d. Natural immunity 11. Growth factor for bone marrow stem cells: (Recent Pattern 2018) a. IL -3 b. IFN gamma c. IL -1 d. TNF 12. Cytokine responsible for septic shock: (Recent Pattern 2018) a. IL -3 b. IFN gamma c. IL -2 d. TNF 13. Which theory of immune response was proposed by Paul Ehrlich? (Recent Pattern 2018) a. Side chain theory b. Direct template theory c. Natural selection theory d. Clonal selection theory 14. Distinct amino acid sequences at the antigen combining site is called: (Recent Pattern 2018) a. Idiotype b. Allotype c. Epitope d. Paratope 15. Who got Nobel Prize for the discovery of split genes? (Recent Pattern 2017) a. Burnet b. Susumu Tonegawa c. Niels K Jerne d. Paul Ehrlich 







16. Antibody dependent cytotoxic cell killing is mediated by: (AIIMS Pattern Nov. 2018) a. NK cells only b. NK cells and macrophages c. NK cells and neutrophils d. NK cells, macrophages and neutrophils 17. Naïve T cell can be stimulated by: (AIIMS Pattern Nov. 2018) a. NK cells and macrophages b. NK cells only c. B lymphocytes d. Antigen presenting cells



ANSWERS AND EXPLANATIONS 1. Ans.  (d) Type 2 hypersensitivity Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 147 Antibody mediated immunity: • Primary defence against extracellular bacterial pathogens • Defence against viruses that infect via respiratory and intestinal • Prevents recurrent viral infections • Type 1, 2, 3 hypersensitivity • Certain autoimmune diseases

2. Ans.  (c) Only thymus dependent antigens Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 148

Features

Primary response

Secondary response

Lag period

4–10 days

1–3 days

Type of B cells involved

Naïve B cell

Memory B cell

Peak response

7–10 days

3–5 days

•

•

•

•

424

•

Contd…

Primary response

Secondary response

Peak antibody response

Antigen dependent

Usually more intense than primary response

Type of antibody seen Antibody affinity Type of antigens

Immunization

IgM

IgG

Lower Thymus independent as well as thymus dependent

Higher Only thymus dependent

8. Ans.  (d) Immunologic memory Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 86 • Introduction of inactivated pathogen, i.e. Antigen into the body – elicits immune response by a process called immunologic memory • When the same antigen is encountered later  a much greater response will be produced without any active illness •

•

Needed Adjuvants not preferably required protein antigens with adjuvants

3. Ans.  (d) 10 years Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 149 • Protein antigens – eliminated within days to weeks • Polysaccharide antigens – persist from months to years; E.g. Pneumococcal polysaccharide may persist up to 20 years in humans following a single injection •

9. Ans.  (a) Macrophages Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 156 • • • • • • •

•

•

•

•

•

•

•

Chapter 59     Immune Response

Features

IL -1: Macrophages IL -2, 3, 9, 12, 13: T cells IL-4, 5, 6: Th cells IL-7: Spleen, BM stromal cells IL-10: T, B cells, macrophages IL-11: BM stromal cells IL-17: Th 17 cells

10. Ans.  (b) Adoptive immunity

•

4. Ans.  (b) T cell independent antigens

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 158 • Transfer factor – Extracts of leucocytes • Passive transfer of CMI is achieved by Transfer factor • Helps in the treatment of T cell deficiency, lepromatous leprosy •

•

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 149 • First step in antigen processing – Capture of Ag by APC – present to appropriate MHC molecules – to T cells • This happens for all T cell dependent antigens Eg: Proteins, erythrocytes • For T cell independent antigens E.g. Polysaccharides – they do not require T cell participation •

•

•

•

11. Ans.  (a) IL -3 Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 157 • IL -3 – growth factor for bone marrow stem cells • Called as multicolony stimulating factor – as it stimulates the multilineage hematopoitec cells •

•

5. Ans.  (a) Antigens with MHC Class I Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 149 • CD8 cells – MHC class I with Ag • CD4 cells – MHC class II with Ag •

•

12. Ans.  (d) TNF Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 157 • Tumor Necrosis Factor (TNF) • Responsible for hematological changes in septic chok • Enhances the initial meningeal inflammation in bacterial meningitis •

6. Ans.  (b) Bordetella pertussis Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 153 • Adjuvant – increases the immunogenicity of an antigen • Bordetella pertussis has a lymphocytosis promoting factor – which acts on the B and T cells – acts as a good adjuvant in DPT vaccine •

•

•

13. Ans.  (a) Side chain theory

•

7. Ans.  (d) Chemotactic factor

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 160 • Side chain theory – proposed by Paul Ehrlich • First theory of immune response • Other theories are – Direct template theory, Indirect template theory, Natural selection theory, Clonal selection theory •

•

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 154 • All of the above are lymphokines affecting the lymphocytes except chemotactic factor which affects granulocytes

•

•

425

Unit 6     Immunology

14. Ans.  (a) Idiotype Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 161

• It is driven by immune cells mostly NK cells but also by macrophages and neutrophils •

• Distinct amino acid sequences at the antigen combining site and near the parts of variable region – IDIOTYPES • It induces anti idiotypic antibodies •

•

15. Ans.  (b) Susumu Tonegawa Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 161 • One gene – one protein concept was in the past • It was overcome by the discovery of split genes for immunoglobulins • Discovered by Susumu Tonegawa – awarded Nobel prize in 1987 •

•

•

17. Ans.  (d) Antigen presenting cells Ref: Ananthanarayan and Paniker T.B of microbiology – 10th ed – page 149 • A naïve T cell is the one that has completed the maturation process in thymus and already has undergone positive and negative selection. • It is a mature T cell but not activated. • Once it is activated by antigen presenting cells (Dendritic cells, B cells, and macrophages) with antigens – it becomes effector T cells. • All naïve T cells have an expression of IL-7 receptors. •

16. Ans.  (d) NK cells, macrophages and neutrophils • Antibody-dependent cellular cytotoxicity (ADCC), also called as antibody-dependent cell-mediated cytotoxicity, is an immune mechanism through which Fc receptor-bearing effector cells can recognize and kill antibody-coated target cells expressing tumor or pathogen on their surfaces. •

426

•

•

•

60

Hypersensitivity

y Cutaneous anaphylaxis: Small dose injected intradermally leads to local wheal and flare reaction, this basis is used for skin tests to diagnose hypersensitivity y Passive cutaneous anaphylaxis: Used to detect human IgG antibody, which is heterocytotropic but not IgE which is homocytotropic y Anaphylaxis in vitro: Called as Schultz Dale phenomenon. y y

Mediators of Anaphylaxis

y

y

y

y

y

y

y y y y y y y

y





y











y





y

y

Types of Anaphylaxis

y

INTRODUCTION y When our body encounters specific antigens, certain undersirable reactions and consequences occur in the sensitized host called hypersensitive reactions y Based on the time required for the sensitized host to develop reactions, hypersensitivity is divided into:  Immediate hypersensitivity  Delayed hypersensitivity y Immediate hypersensitivity: It is B cell or antibody-mediated  Anaphylaxis  Atopy  Antibody-mediated cell damage  Arthus phenomenon  Serum sickness y Delayed hypersensitivity: It is T cell mediated  Tuberculin test  Contact dermatitis

Histamine Serotonin Chemotactic factors Enzyme mediators like proteases and hydrolases Prostaglandins Leukotriens Platelet activating factor

TYPE I HYPERSENSITIVITY

Remember

Anaphylactoid Reaction • Some drugs and chemicals can simulate anaphylaxis • But this is not IgE mediated • Directly activates the complement and releases anaphylatoxins

Anaphylaxis

Schwartzmann Reaction • Perturbation in coagulation factors • Not actually an immune-mediated reaction • E.g. Waterhouse Friderichsen syndrome by N. meningitidis, DIC caused by S. aureus in septicemia (TSS), Gram-negative septicemia

y

• •

y

y

y First dose that sensitizes the host is called sensitizing dose y Next dose which causes the reaction, is called as shocking dose y The interval between both doses should be at least 2–3 weeks to produce the reaction

•

•



•



•

y

y

y IgE dependent y Two forms of type 1 hypersensitivity:  Acute, fatal: Anaphylaxis  Chronic, non fatal: Atopy

 

Table 1: Immunologic aspects of hypersensitivity reactions Antibody or Cell Mediated Immunologic Reaction

I (Immediate, anaphylactic)

Antibody (IgE)

• Antigen (allergen) induces IgE antibody that binds to mast cells and basophils. • When exposed to the allergen again, the allergen cross-links the bound IgE on those cells. • This causes degranulation and release of mediators - histamine

II (Cytotoxic)

Antibody (IgG)

• Antigens on a cell surface combine with IgG antibody. • This leads to complement-mediated lysis of the cells (e.g., Transfusion or Rh reactions) or autoimmune hemolytic anemia.

III (Immune Complex)

Antibody (IgG)

• Antigen-antibody immune complexes are deposited in tissues, complement is activated and polymorphonuclear cells are attracted to the site. • They release Iysosomal enzymes, causing tissue damage. •

•

•

•

•

•

•

Type

Contd…

Unit 6     Immunology

Type

Antibody or Cell Mediated Immunologic Reaction

IV (Delayed)

Cell

• T lymphocytes activated/sensitized by an antigen release lymphokines upon second contact with the same antigen. • The lymphokines induce inflammation and activate macrophages, which, in turn, release various inflammatory mediators. •

•

Table 2:  Clinical manifestations of hypersensitivity reactions Type

Typical Time of Onset

Clinical Manifestation or Disease

I (Immediate, anaphylactic)

Minutes

• • • • • • •

Systemic anaphylaxis Urticaria Asthma Hay fever Allergic rhinitis Allergic conjunctivitis, Angioedema

• • • • • • • •

Hemolytic anemia, Neutropenia, Thrombocytopenia, ABO transfusion reactions, Rh incompatibility (erythroblastosis fetalis, hemolytic disease of the newborn) Rheumatic fever Goodpasture’s syndrome Grave’s disease

• • • • • •

Systemic lupus erythematosus Rheumatoid arthritis Poststreptococcal glomerulonephritis IgA nephropathy Serum sickness Hypersensitivity pneumonitis (e.g., farmer’s lung)

• • • • •

Contact dermatitis, Tuberculin skin test reaction, Stevens Johnson Syndrome, Toxic epidermal necrolysis, Erythema multiforme

•

•

•

•

•

•

•

II (Cytotoxic)

Hours to days

•

•

•

•

•

•

•

•

III (Immune Complex)

2 to 3 weeks

•

•

•

•

•

•

IV (Delayed)

2 to 3 days

•

•

•

•

•

High Yield • Type V hypersensitivity (Stimulatory hypersensitivity): ƒ It is a modified form of type II reaction; E.g. LATS antibody, i.e long acting thyroid stimulator antibody against thyroid cells and certain drug reactions like SJS, sulphonamide allergies. ƒ Here the antibody activates the receptors and causes increased activity of the cells • Skin Tests that are involved with hypersensitivity: Casoni’s test ƒ Type I → ƒ Type III → Schick’s test ƒ Type IV → Tuberculin test and lepromin test •

ƒ



ƒ

•

ƒ

ƒ

ƒ

JERISH HERHEXIMER REACTION When Cell wall antibiotics e.g. Penicillin are used in the treatment - it causes rapid cell wall lysis leading to release of pro-inflammatory cytokines or toxic bacterial products - a condition called Jerish Herheximer reaction; It occurs in treatment of primary and secondary syphilis.

JONES MOTE REACTION 428

y It is cutaneous basophil hypersensitivity. It is delayed form of response which involves basophil leukocytes. y It can be tested by skin tests. y

y

1. B cell mediated hypersensitivity reaction is: a. Arthus phenomenon (Recent Pattern 2017) b. Contact dermatitis c. Delayed type hypersensitivity reaction d. Tuberculin reaction 2. Type I hypersensitivity is mediated through: a. IgM (Recent Pattern 2017) b. IgG c. IgE d. IgA 3. Hemolytic anemia belongs to: (Recent Pattern 2018) a. Type I hypersensitivity b. Type II hypersensitivity c. Type III hypersensitivity d. Type IV hypersensitivity 4. Passive cutaneous anaphylaxis detects: a. Heterocytotropic antibody (Recent Pattern 2018) b. Wheal and flare response c. Atopy d. Delayed type hypersensitivity 5. Schultz Dale phenomenon is: (Recent Pattern 2018) a. Anaphylaxis in vivo b. Anaphylaxis in vitro c. Atopy d. Cutaneous hypersensitivity 6. Prausnitz –Kustner reaction is mediated by: a. IgE b. IgG (Recent Pattern 2017) c. IgM d. IgA 7. Stimulatory hypersensitivity is: (Recent Pattern 2017) a. Modified type I hypersensitivity b. Modified type II hypersensitivity c. Modified type III hypersensitivity d. Modified type IV hypersensitivity 8. Erythroblastosis fetalis is an example of which type of hypersensitivity: (Recent Pattern 2017) a. Type I b. Type II c. Type III d. Type IV 9. Which one the following statement is false: a. Theobald smith phenomenon is a type I sensitivity reaction (Recent Pattern 2017) b. Serum sickness is a type II reaction c. Allograft rejection is a type IV reaction d. Transfusion reaction is a type II reaction

10. Which of the following cells plays an important role in contact dermatitis? (Recent Pattern 2017) a. T cells b. B cells c. Langerhan cells d. Macrophages 11. Reaction due to lysis of bacterial cell wall and necrotic cell product is: (Recent Pattern 2017) a. Arthus reaction b. Serum sickness c. Jarisch–Herxheimer reaction d. Infectious mononucleosis ampicillin reaction 12. Delayed hypersensitivity involves: (Recent Pattern 2017) a. Neutrophils b. RBCs c. Eosinophils d. Lymphocytes 13. Type III reaction is: (Recent Pattern 2017) a. Antibody mediated b. Immunocomplex mediated c. Cell mediated d. None 14. Frie test belongs to which type of hypersensitivity: (Recent Pattern) a. Type I b. Type II c. Type III d. Type IV 15. Mechanism similar to Shwartzman reaction occurs in: a. Fitz Hugh Curtis syndrome (Recent Pattern 2017) b. Waterhouse Friderichsen syndrome c. Eichwald silmser effect d. Anaphylactoid reaction 16. Prausnitz kustner [PK] reaction is which type of hypersensitivity:  (Recent Pattern 2017) a. Type 1 b. Type 2 c. Type 3 d. Type 4 17. Myasthenia gravis is which type of hypersensitivity: (Recent Pattern 2017) a. Type 1 b. Type 2 c. Type 3 d. Type 4 18. Which of the following is/are examples of type 3 hypersensitivity reaction(s): (PGI May 2018) a. Serum sickness b. Arthus reaction c. Erythroblastosis fetalis d. Farmer’s lung e. Goodpasteur’s syndrome

Chapter 60     Hypersensitivity

MULTIPLE CHOICE QUESTIONS





429

Unit 6     Immunology

ANSWERS AND EXPLANATIONS 1. Ans.  (a) Arthus phenomenon Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 163 B cell mediated hypersensitivity (Immediate ) • Anaphylaxis • Atopy • Antibody mediated cell injury • Arthus phenomenon • Serum sickness •

•

•

•

•

a small quantity of cooked fish antigen is injected in the same site • But this is not in use as it carries the risk of transmission of infections •

7. Ans.  (b) Modified type II hypersensitivity Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 164 • Stimulatory hypersensitivity – Type V • Modified form of Type II reaction • Antibodies recognize and bind to the cell surface receptors instead of cell surface components •

•

2. Ans.  (c) IgE Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 165 • Type I hypersensitivity – IgE mediated • Two forms : Anaphylaxis and atopy •

•

3. Ans.  (b) Type II hypersensitivity Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 164 • Type II hypersensitivity – Cytolytic and Cytotoxic • It causes antibody mediated damage • E.g. Thrombocytopenia, Agranulocytosis, Hemolytic anemia

•

8. Ans.  (b) Type II Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 168 Examples of Type II hypersensitivy: • Lysis of RBC: because of anti-erythrocytic antibodies (Autoimmunity) • Hemolytic diseases of newborn. •

•

•

•

•

4. Ans.  (a) Heterocytotropic antibody Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 166 • • • •

Passive cutaneous anaphylaxis – in vivo method For detection of antibodies Antigen injection intradermally Then same antigen with a dye injected i.v. → after 4 to 24 hours  Wheal and Flare reaction occurs in intradermal site • This helps to detect IgG antibody which is heterocytotropic • Not for IgM which is homocytotropic (Species specific) •

9. Ans.  (b) Serum sickness is a type II reaction Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 168 Examples of Type III hypersensitivity: • Arthus reaction • Serum sickness • SLE • Farmer’s lung •

•

•

•

•

•

•

•

•

5. Ans.  (b) Anaphylaxis in vitro Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 166 • Uterine muscle strips from a sensitized guinea pigs → kept in a Ringer’s solution with the specific antigen → vigorously contracts → Schultz Dale phenomenon • Sensitization elicited in vitro (Anaphylaxis) •

•

10. Ans.  (a) T cells Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 169 • Type IV hypersensitivity – DTH – cell-mediated immune response • Eg.: Tuberculin infection – reaction, Cutaneous basophil hypersensitivity (Jones –Mote reaction), Contact dermatitis •

•

11. Ans.  (c) Jerish herheximer reaction Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 384 • Cell wall antibiotics e.g. Penicillin causes rapid cell wall lysis • Release of proinflammatory cytokines or toxic bacterial products • Occurs in treatment of primary and secondary syphilis. •

•

6. Ans.  (a) IgE Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 167 • IgE – homocytotropic (Species specific) • Serum collected from Kustner who has a atopy to cooked fish → when injected intracutaneously into Prausnitz → 24 hours later → Wheal and flare reaction occurs when •

•

430

•

12. Ans.  (d) Lymphocytes Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 169 • Type IV hypersensitivity – DTH – cell-mediated immune response - T lymphocytes •

•

13. Ans.  (b) Immuno complex mediated Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 164 • Type III is immune complex mediated • Mediators are IgG, IgM, leucocytes

16. Ans.  (a) Type 1 Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 167 • PK reaction is based on the principle that IgE is homocytotropic – i.e species specific – human IgE can fix only human cells • Using cooked fish – atopy was tested in vivo •

•

•

•

14. Ans.  (d) Type IV Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 169 • Intracellular microbes / haptens – like chemical dyes – applied on the skin → causes mixed cellular reaction – involves lymphocytes and macrophages

17. Ans.  (b) Type 2 Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 168 • Myasthenia gravis belongs to antibody mediated Type II hypersensitivity

Chapter 60     Hypersensitivity

• E.g. Tuberculin infection – reaction, Cutaneous basophil hypersensitivity (Jones–Mote reaction), Contact dermatitis

•

•

15. Ans.  (b) Waterhouse Friderichsen syndrome Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 170 • Factors affecting intravascular coagulation is affected • Not actually an immune mediated reaction • Similar reaction occurs in Waterhouse-Friderichsen syndrome – reason for purpuric rashes and acute hemorrhagic adrenal necrosis •

•

•

18. Ans.  ( a) Serum sickness; (b) Arthus reaction ; (d) Farmer’s lung Ref: Ananthanarayan and Paniker’s Microbiology – 10th ed – Page 168

Textbook

of

Examples for Type III hypensensitivity are: • Arthus reaction • Serum sickness • SLE • PSGN • Rheumatoid arthritis • IgA nephropathy • Serum sickness • Hypersensitivity pneumonitis – Farmer’s lung •

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•

•

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•

431

61

Immunodeficiency Diseases y y

DiGeorge Syndrome y Leads to aplasia/hypoplasia of thymus due to defect in 3rd and 4th pharyngeal pouches y Primary defect is thymus-dependent lymphocytes are depleted y Circulating T cells are reduced y Neonatal tetany, Fallot’s tetralogy it is associated with. y

Table 1:  Primary immunodeficiency disorders

y

PRIMARY IMMUNODEFICIENCIES

y

y

y

y

y

y Certain conditions of the body, which has defects in immune pathways leading to more prone for infections are called immunodeficiency diseases y It can be either primary or secondary y Primary immunodeficiency diseases are inherited; Secondary occurs in adult life due to an disease, drugs or nutritional defects.

y More chance of pyogenic infections and increased reports of associated autoimmune disease y B cells may be normal, but defective, hence immunoglobulin levels are low y Malabsoprtion and Giardia infection is most common y

INTRODUCTION

y There is a defect in T cell specifically for Candida infections y Associated with endocrinopathies.

Ataxia Telangiectasia

• • • • •

•

y y

Wiskott-Aldrich Syndrome y

y

y Wiskott-Aldrich syndrome: Combined immunodeficiency y X linked y Triad: Eczema, thrombocytopenic purpura and recurrent infections y Death due to infections, hemorrhage or lymphoreticular malignancy IgM levels low; IgG , IgA normal

Bruton’s Agammaglobulinemia

y

y

y Known as late onset hypogammaglobulinemia y It manifests by the age of 15–33 years



y

y

y Autosomal recessive (mostly) and X linked y Three forms of (SCID):  Swiss type agammaglobulinemia  Reticular dysgenesis of de Vaal  Adenosine deaminase (ADA) deficiency (First immunodeficiency to be associated with an enzyme) 

Common Variable Immunodeficiency

Severe Combined Immunodeficiency (SCID)



y

y

y

y

y

y

y Bruton’s agammaglobulinemia: X linked (seen only in male infants) y First immunodeficiency to have recognized y It is a type of humoral immunodeficiency y Recurrent pyogenic infections by bacteria occurs y For viral infections – immune response is normal because T cells are involved in viral infections (Here only B cell is defect) y All Ig classes levels are low.

y

y

•

•

•

•

•

•

Nezelof syndrome Ataxia telangiectasia Wiskott – Aldrich syndrome Immunodeficiency with thymoma Immunodeficiency with short limbed dwarfism • Severe combined immunodeficiency (SCID)

y

y

•

y

B and T cell defects

y

• Thymic hypoplasis – DiGeorge syndrome • Chronic mucocutaneous candidiasis • Purine nucleoside phosphorylase (PNP) deficiency •

T cell defect

y Ataxia telangiectasia: Combined immunodeficiency y Autosomal recessive AR y Ataxia, telangiectasia, ovarian dysgenesis, chromosomal abnormalities y Death is due to sinopulmonary infection y Serum and secretory IgA – absent y IgE deficiency is also seen y CMI is also defective. y

•

•

•

•

•

Chronic Mucocutaneous Candidiasis y

• X linked agammaglobulinemia • Transient hypogammaglobulinemia of infancy • Common variable immunodeficiency • Selective immunoglobulin deficiencies • Immunodeficiencies with hyper IgM • Transcobalamin II deficiency

y

Immunodeficiency

B cell defect

•

Disorder

y

y

y

y

Chronic Granulomatous Disease y Due to defect in the intracellular microbicidal activity of neutrophils because of lack of NADPH oxidase y X linked (most common), Autosomal y Defect in killing the microbes by forming peroxides during phagocytosis y Widespread association of granulomas with this condition gives this unique name y Lab diagnosis is by Nitroblue tetrazolium dye reduction test. y

y

y

y

HYPER IGM SYNDROME y An inherited deficiency in CD40 ligand (CD40L or CD154) leads to impaired communication between T cells and antigen-presenting cells (APCs) y The B-cell response to T-independent antigens, however, is unaffected, accounting for the presence of IgM antibodies in these patients, which ranges from normal to high levels and give the disorder its common name, hyper IgM syndrome (HIM)

y

y

y

Chédiak–Higashi Syndrome y Due to failure of lysosomes of neutrophils to fuse with phagosomes y Recurrent Staphylococci and Streptococcal infections y

y

Job’s Syndrome

Chapter 61     Immunodeficiency Diseases

y X linked have defect is IL-2 receptor y Recticular dysgenesis of de Vaal defects is at multipotent hemopoietic stem cell y Some patients with autosomal form have either mutation in ZAP-70, Janus Kinase 3, RAG-1, RAG-2 y Immunity is profoundly depressed; hence no live vaccines in this condition should be strictly followed

y Hyper IgE syndrome y Recurrent cold abscess by Staphylococci, eczema, skeletal defects with increased levels of IgE. y

DISORDERS OF PHAGOCYTOSIS

Remember

Following are the disorders of phagocytosis • Chronic granulomatous disease • Myeloperoxidase (MPO) deficiency • Chediak-Higashi syndrome • Leukocyte G6PD deficiency • Job’s syndrome • Tuftsin deficiency • Lazy leukocyte syndrome • Hyper IgE syndrome • Actin binding protein deficiency • Shwachman’s disease •

•

•

•

•

•

y

Lazy Leucocyte Syndrome y Lazy leucocyte syndrome is due to defect in chemotaxis and neutrophil mobility y In bone marrow there is normal neutrophil count y But peripheral neutropenia is seen y Increased susceptibility to bacterial infection y Recurrent stomatitis, gingivitis and otitis. y

y

y

y

y

•

•

•

•

433

Unit 6     Immunology

MULTIPLE CHOICE QUESTIONS 1. Bruton’s agammaglobulinemia is:(Recent Pattern 2017) a. Seen only in male infants b. Autosomal dominant c. Recurrent viral infections d. No T cell mediated immunity 2. Following are the T cell defects except: (Recent Pattern 2017) a. DiGeorge syndrome b. Chronic mucocutaneous candidiasis c. Transcobalamin deficiency d. PNP deficiency 3. Lazy leukocyte syndrome is: (Recent Pattern 2017) a. T cell defect b. B cell defect c. Combined defect d. Disorder of phagocytosis 4. Ataxia telangiectasia patients have a deficiency of: (Recent Pattern 2017) a. IgA b. IgM c. IgD d. IgG 5. All of the following are clinical features of Wiskott Aldrich syndrome except: (Recent Pattern 2017) a. Eczema b. Recurrent infections c. Atopy d. Thrombocytopenic purpura 6. The defect in reticular dysgenesis of de Vaal is: a. Multipotent hemopoietic stem cells b. Lymphoid stem cells (Recent Pattern 2017) c. Myeloid stem cells d. Erythroid stem cells 7. Multiple cold staphylococcal abscesses are features of: a. Tuftsin deficiency (Recent Pattern 2017) b. Job syndrome c. Chediak Higashi syndrome d. Myeloperoxidase deficiency 





8. The NBT (nitro blue tetrazolium) reduction assay is used to:  (AIIMS 2017) a. Evaluate granulocyte function b. Evaluate T-cell function c. Determine whether polymorph nuclear leucocytes can produce superoxide d. Stain B –lymphocytes 9. SCID which is true: (Recent Pattern 2017) a. Adenosine deaminase deficiency b. Decreased circulating lymphocytes c. NADPH oxidase deficiency d. C1 esterase deficiency 10. CD59 deficiency leads to: (Recent Pattern 2017) a. Hereditary angioneurotic odema b. Paroxysmal nocturnal hemoglobinuria c. Chediak Higashi syndrome d. Job syndrome 11. Deficiency of both T and B Lymphocyte involved in all except: (PGI May 2017) a. Chronic mucocutaneous candidiasis b. Wiskott-Aldrich syndrome c. DiGeorge Syndrome d. Ataxia Telangiectasia e. Common variable immunodeficiency 12. True about Severe Combined Immunodeficiency: (PGI may 2018) a. Autosomal dominant transmission b. Both B & T cell defect c. Decreased lymphocyte count d. More common in boys e. Absent tonsils

ANSWERS AND EXPLANATIONS 1. Ans.  (a) Seen only in male infants Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 173 • Bruton’s agammaglobulinemia – X linked (seen only in male infants) • First immunodeficiency to have recognized • Humoral immunodeficiency • Recurrent pyogenic infections by bacteria • Viral infections – immune response is normal because T cells are involved in viral infections • All Ig classes levels are low

T cell defects – Cellular immunodeficiencies: • Thymic hypoplasis – DiGeorge syndrome • Chronic mucocutaneous candidiasis • Purine nucleoside phosphorylase (PNP) deficiency •

•

•

•

•

•

•

•

•

3. Ans.  (d) Disorder of phagocytosis Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 178 • Lazy leucocyte syndrome – defect in chemotaxis and neutrophil mobility • BM – normal neutrophil count • Peripheral neutropenia is seen • Increased susceptibility to bacterial infection • Recurrent stomatitis, gingivitis and otitis •

•

•

2. Ans.  (c) Transcobalamin deficiency

434

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 174

•

•

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 176 • Ataxia telangiectasia: Combined immunodeficiency • Autosomal recessive AR • Ataxia, telangiectasia, ovarian dysgenesis, chromosomal abnormalities • Death due to sinopulmonary infection • Serum and secretory IgA – absent • IgE deficiency also seen • CMI is also defective •

•

•

•

•

•

•

5. Ans.  (c) Atopy Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 176 • Wiskott-Aldrich syndrome – Combined immunodeficiency • X linked • Triad: Eczema, Thrombocytopenic purpura and Recurrent infections • Death due to infections, hemorrhage or lymphoreticular malignancy • IgM levels low; IgG , IgA normal •

•

•

•

• The principle is: normally phagocytic cells produce superoxide free radicals with the help of NADPH oxidase and kill the organisms • These superoxides reduce nitroblue tetrazolium → formazan (insoluble) → means the test is positive (normal individual) • If there is no superoxide formation → no reduction → negative test (means chronic granulamatous disease) •

•

•

9. Ans.  (a) Adenosine deaminase deficiency Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 176 • Enzyme deficiency that is first identified with an immunodeficiency disorder is Adenosine deaminase – SCID •

10. Ans.  (b) Paroxysmal nocturnal hemoglobinuria Ref: Wintrobe`s clinical hematology – Volume I – Page 1001

Chapter 61     Immunodeficiency Diseases

4. Ans.  (a) IgA

• CD59 is expressed in most of the hematopoeitic cells and endothelial cells • It helps in protection from the intra vascular complement mediated lysis • CD59 is called as membrane inhibitor of reactive lysis • Deficiency of CD59 causes PNH •

•

•

•

•

6. Ans.  (a) Multipotent hemopoietic stem cells Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 176 • SCID – Three forms are there ƒ Swiss type agammaglobulinemia ƒ Reticular dysgenesis of de Vaal ƒ Adenosine deaminase (ADA) deficiency • Recticular dysgenesis of de Vaal – defect is at multipotent hematopoietic stem cell •

ƒ

11. Ans:  ( a) Chronic mucocutaneous candidiasis; (c) DiGeorge Syndrome; (e) Common variable immunodeficiency Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 174 Combined B and T cell defects are:

ƒ

ƒ

•

7. Ans.  (b) Job syndrome Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 178 • Job syndrome – Disorder of phagocytosis • Multiple large cold staphylococcal abscesses • Ig levels normal – EXCEPT IgE elevated •

Nezel of Syndrome • • • • •

•

•

•

•

•

Ataxia telangiectasia Wiskott – Aldrich syndrome Immunodeficiency with thymoma Immunodeficiency with short limbed dwarfism Severe combined immunodeficiency (SCID)

12. Ans.  ( b) Both B & T cell defect; (c) Decreased lymphocyte count; (d) More common in boys

•

•

8. Ans.  (c) Determine whether polymorph nuclear leucocytes can produce superoxide Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 177

Ref: Ananthanarayan and Paniker’s Microbiology – 10th ed – Page 176

Textbook

of

• SCID is a X linked – AR disorder • It has both B and T cell defects • Hence lymphocytes will be less •

•

•

• Nitroblue tetrazolium test is used for diagnosis of chronic granulomatous disease – which is a disorder of phagocytosis •

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62

Autoimmunity

INTRODUCTION

Trypsin Nystatin  Mycoplasma  EB virus  Malarial parasite  Infectious mononucleosis Forbidden clones: Break down of immunological homeostasis – leads to cessation of tolerance Altered B or T cell function Defects in idiotype, antiidiotype network Defective Ir or immunoglobulin genes. 

 



y







y



y When the immunological reaction occurs against the body’s own component that leads to structural or functional damage, it is called as autoimmunity y Postulated as horror autotoxicus by Ehrlich

 

Table 1: Cross-reacting antigens Sheep brain antigen in Anti-rabies immunization

Heart muscles

Streptococcal M proteins

Renal glomeruli

Nephritogenic strains of Streptococci



AUTOANTIBODIES Cold Autoantibodies Complete agglutinating antibodies— IgM y Agglutinate erythrocytes at 4°C / not at 37° y Seen in autoimmune hemolytic anemia, primary atypical pneumonia, trypanosomiasis, black water fever

Warm Autoantibodies Incomplete, non agglutinating antibodies—IgG y Seen in patients taking drugs like sulfonamide and methyldopa y

Human brain antigens

7. 8. 9.

y











1. Antigenic alterations: Sometimes cells may undergo some alteration because of physical, chemical damage leads to autoimmunity 2. Sequestered antigens: Antigens in closed system during embryonic life when exposed later leads to autoimmunity; Examples are:  Lens protein  Sperm antigen 3. Cross-reacting foreign antigens: Similarities between foreign and self-antigens are the basis of many of the auto immune diseases

6.

y

MECHANISM OF AUTOIMMUNITY

 

• Hashimoto’s thyroiditis • Grave’s disease • Addison’s disease • Autoimmune orchitis • Myasthenia gravis • Sympathetic ophthalmia • Pernicious anemia • Guillain Barre syndrome • Pemphigus vulgaris • Bullous pemphigoid • Dermatitis herpetiformis

• SLE • Rheumatoid arthritis • Polyarteritis nodosa • Sjögren’s syndrome

Shigella flexneri

Joint membranes

Mycobacterium tuberculosis

Myocardium

Coxsackie B



• • •

• • • • • •







5. Polyclonal B cell activation: Some stimuli may cause nonspecific activation of B cells – leads to IgM production of autoantibodies - Stimuli is caused by  2- Mercaptoethanol  Purified protein derivative  Lipopolysaccharide

•

•

HLA B 27

•

•

 

Table 2: Molecular mimicry

•

• Autoimmune hemolytic anemia • Autoimmune thrombocytopenia • Autoimmune leucopenia

•

Systemic autoimmune diseases

•

Localised autoimmune diseases

•



4. Molecular mimicry: This is due to presence of epitopes with identical peptide sequences in both the infecting micro organisms and self-antigens—leads to autoimmune reaction (note the difference between this and cross reacting antigens)

Hemocytolytic autoimmune diseases

•

Remember

Table 3: Autoimmune diseases

1. All are example for molecular mimicry except: (Recent Pattern 2017) a. Shigella flexneri and HLA B27 b. Mycobacterium tuberculosis and joint membranes c. Coxsackie B virus and myocardium d. Salmonella typhi and HLA DRQ 2. Virus that may trigger autoimmunity is: a. Ebstein Barr virus (Recent Pattern 2017) b. Herpes simples virus c. Human papilloma virus d. Human immunodeficiency virus 3. Cold autoantibodies are: (Recent Pattern 2018) a. IgM b. IgG c. IgA d. IgD 4. Warm autoantibodies are: (Recent Pattern 2018) a. IgM b. IgG c. IgA d. IgD 

5. Streptococcal M protein is an example of: (Recent Pattern 2017) a. Cross-reacting foreign antigen b. Sequestered antigen c. Molecular mimicry d. Forbidden clone 6. Lens protein is an example of: (Recent Pattern 2017) a. Cross reacting foreign antigen b. Sequestered antigen c. Molecular mimicry d. Forbidden clone 7. Rheumatoid factor is: (Recent Pattern 2017) a. IgM against Fc portion of IgG b. IgG against Fc portion of IgM c. IgE against Fc portion of IgM d. IgD against Fc portion of IgG 8. Long acting thyroid stimulator (LATS) is: (Recent Pattern 2017) a. IgM b. IgG c. IgA d. IgD 



Chapter 62     Autoimmunity

MULTIPLE CHOICE QUESTIONS



ANSWERS AND EXPLANATIONS 1. Ans.  (d) Salmonella typhi and HLA DRQ Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 181 • Molecular mimicry – presence of similar peptide sequences in microorganisms and self-antigens • It is seen in all of the above options except S. Typhi which has no role in molecular mimicry •

•

2. Ans.  (a) Ebstein Barr virus Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 181 • Certain stimuli can activate multiple B cells - polyclonal B cell activation – reason for autoimmunity, E.g. 2-mercaptoethanol, LPS, Trypsin, Nystatin, Mycoplasma, EBV, Malaria •

3. Ans.  (a) IgM Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 181 • Cold autoantibodies: Complete agglutinating antibodies – IgM • Agglutinate erythrocytes at 4°C / not at 37°C • Seen in autoimmune hemolytic anemia, primary atypical pneumonia, trypanosomiasis, black water fever •

•

•

4. Ans.  (b) IgG

• Seen in patients taking drugs like sulphonamides, methyldopa •

5. Ans.  (a) Cross-reacting foreign antigen Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 181 • Similarity between foreign (microbe) antigens and selfantigens • Streptococcal M protein and cardiac muscle • Nephritogenic strain of Streptococci and renal glomeruli •

•

•

6. Ans.  (b) Sequestered antigen Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 180 • Self-antigens in closed systems – when exposed in adult life – immunological damage • E.g. Lens protein and sperms •

•

7. Ans.  (a) IgM against Fc portion of IgG Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 183 • Rheumatoid factor – autoantibody • IgM or IgG or IgA RF • Against Fc fragment of IgG •

•

•

8. Ans.  (b) IgG

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 181

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 182

• Warm autoantibodies: Incomplete, nonagglutinating antibodies—IgG

• Thyrotoxicosis – presence of autoantibody – LATS • IgG antibody to thyroid membrane antigen

•

•

•

437

63

Transplantation and Tumor Immunology

INTRODUCTION

y

y

y When an organ is damaged permanently or defective since birth, it needs grafting / transplantation y The person who donates the organ is called donor and who receives the organ is called recipient

Table 2:  Graft rejection Days

First Set Response (Mediated by T lymphocytes)

1–3 days

Vascularization occurs

4 day

Inflammation occurs (Graft is surrounded by lymphocytes and macrophages)

5–9 days

Blood vessels occluded by thrombi lead to necrosis

10 day

Graft becomes a slough

th

HISTOCOMPATIBILITY TESTING

y

HISTOCOMPATIBILITY ANTIGENS y When the recipient do not have that specific antigen that is present in the grafted tissue, it leads to immune reactions and graft rejection, those antigens are called as histocompatibility antigens y When transplantation occurs between different sex chromosome:  Grafts from female (XX) to male (XY) is accepted  Grafts from male (XY) to female (XX) is rejected because of Y chromosome—this unilateral sex linked histoincompatibility is called as Eichwald-silmser effect

y











y





y

y

y Before any organ transplantation, tissue matching is must y Tests need to be done are:  Blood grouping  HLA compatibility y HLA compatibility is tested by:  Microcytotoxicity test  Mixed leucocyte reaction  Mixed lymphocyte culture  RFLP  PCR y Sera for micro cytotoxicity test is obtained from multigravidae, placental fluid and from multiple blood transfusion recipients.

y After rejection, when another graft from the same donor is applied – these above said reactions occur more rapidly called second set response (mediated by antibodies) – also called white graft response (as the graft is rejected fastly and makes it pale)

y

Xenograft

th

y

Allograft

Transplant from an individual to himself Between same genetic constitution E.g. Identical twins Between two genetically nonidentical members of same species Between members of different species



Autograft Isograft



Table 1:  Types of grafts

y y y

   

y

y

y

y

y Any organ that is transplanted is considered as a foreign body by the immune system and it tries to reject it; Hence immunosuppression is must before transplantation y The most effective drug is cyclosporine A, azathioprine and steroids y Rate of allograft rejection varies according to the tissue involved; skin grafts are generally rejected faster than other tissues, such as kidney or heart. y After organ transplantation, certain series of reactions occur in some cases called Allograft reaction:

y Certain areas in the body accepts the graft without rejection, they are called as privileged sites y Areas which lack circulation or lymphatic drainage are mostly privileged sites y Examples:  Uterus – Fetus (it does not reject fetus as foreign body because of protection by placenta, which acts as an immunological barrier)  Corneal transplantation (due to lack of vascularity)  Cartilage  Brain  Testes 

GRAFT REJECTION

Privileged Sites

IMMUNOTHERAPY OF CANCER y Passive immunotherapy:  A special type of anti sera was given previously – that had de-blocking activity – that did regression of tumors y Active immunotherapy:  Specific active immunotherapy: Purified tumor cell antigens was made as vaccine and trial was done, But not in use  Nonspecific active immunotherapy: BCG, Corynebacterium parvum, Dinitrochlorobenzene, Glucan, Levamisole, Interferons y Specific adoptive immunotherapy:  Lymphocytes  Transfer factor  Immune RNA

y

y



Host attacks graft → allograft reaction Graft attacks host →GVH reaction

y



y GVH reaction occurs due to following conditions:  Graft contains immunocompetent T cells  Recipient possess transplantation antigens that are absent in graft  Recipient should not reject the graft (Allograft reaction) y Clinical features of GVH reaction are growth retardation, diarrhea, HSM, anemia and lead to fatal condition called as Runt disease y







y



y







MULTIPLE CHOICE QUESTIONS 1. Graft between identical twins is called as: (Recent Question 2017) a. Autograft b. Isograft c. Allograft d. Xenograft 2. First set response of allograft rejection is by: (Recent Question 2017) a. B cells b. T lymphocytes c. Macrophages d. Phagocytes 3. Immunological enhancement in graft rejection is mediated by: (Recent Question 2017) a. Antibodies b. T lymphocytes c. Macrophages d. Antigens 4. Eichwald – Silmser effect is: (Recent Question 2018) a. Autosomal dominant histoincompatibility b. Unilateral sex linked histoincompatibility c. Autosomal recessive histoincompatibility d. None of the above 5. Method to test for histocompatibility before transplantation: (Recent Question 2018) a. Flow cytometry b. Microcytotoxicity test c. ELISA d. Latex agglutination test 



6. Graft versus Host reactions occurs only if: (Recent Question 2017) a. Graft contains immunocompetent B cells b. Graft contains macrophages c. Recipient must not reject the graft d. Recipient is not on immunosuppressive drugs 7. All of the following are Tumor associated antigens except: (Recent Question 2016) a. CEA b. CA 125 c. PSA d. HRP 8. Example for an immunomodulator: (Recent Question 2016) a. Corynebacterium diphtheria b. Corynebacterium parvum c. Corynebacterium xerosis d. Corynebacterium minutissimum 9. Nude mice is able to accept xeno graft because they lack? (Recent Question 2017) a. T cells b. B cells c. NK cells d. LAK cells 

Chapter 63     Transplantation and Tumor Immunology

GRAFT VERSUS HOST REACTION y This reaction is opposite to that of allograft reaction



439

Unit 9     Immunology

ANSWERS AND EXPLANATIONS 1. Ans.  (b) Isograft Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 185

Histocompatibility testing is done by: • Micro - cytotoxicity test • RFLP • Southern blotting • PCR • Mixed lymphocyte reaction/culture •

•

•

Autograft

Transplant from an individual to himself

Isograft

Between same genetic constitution E.g. Identical twins

Allograft

Between two genetically nonidentical members of same species

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 188

Xenograft

Between members of different species

GVH reaction occurs only if: • Graft – has immunocompetent T cells • Recipient has transplantation antigens; which is absent in graft • Recipient must not reject the graft • Recipient should be on immunosuppression

•

•

6. Ans.  (c) Recipient must not reject the graft

•

2. Ans.  (b) T lymphocytes Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 186 • Allograft rejection – After transplantation series of reactions occurs • First set of response – reactions occurring from Day 1 up to Day 10 – exclusively done by T lymphocytes • Second set of response – When graft is rejected in first set of response, similar graft kept again will lead to drastic rejection is a fastest manner – mainly due to antibodies along with CMI

•

•

•

•

•

7. Ans.  (d) HRP Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 189

•

Tumor-associated antigens: • Alpha feto protein (AFP) • Carcino embryonic antigen (CEA) • Prostate specific antigen (PSA) • CA 125    (HRP – Horse Radish Peroxidase enzyme used in ELISA) •

•

•

3. Ans.  (a) Antibodies Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 186 • CMI – responsible for graft rejection • Humoral antibodies may act in opposition with CMI – thus prevent the graft rejection – Immunological enhancement •

•

4. Ans.  (b) Unilateral sex linked histoincompatibility Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 187 • Grafts from male to female – rejected • Grafts from female to male – accepted • Because of presence of antigens determined in Y chromosome • This unilateral sex linked histoincompatibility is called Eichwald – Silmser effect

•

8. Ans.  (b) Corynebacterium parvum Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 190 • BCG and C. parvum are used as immunotherapeutic agents for the treatment of cancer • They act as immunomodulators. •

•

9. Ans.  (a) T cells Ref: The Mouse in Biomedical Research: Diseases – Page 400

•

•

•

•

• A nude mouse is a laboratory mouse from a strain with a genetic mutation in FOXN1 gene • This causes a deteriorated or absent thymus, resulting in an inhibited immune system due to a greatly reduced number of T cells. • This mouse has no body hair that gives the name – nude mouse • It is valuable in research because it can receive many different types of tissue and tumor grafts, as it mounts no rejection response •

•

•

5. Ans.  (b) Microcytotoxicity test Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 187

440

•

Immunohematology

Table 1:  Blood groups RBC

Serum

A

A

Anti B

B

B

Anti A

AB

A and B

None

O

None

Anti A and Anti B

MATERNOFETAL ABO INCOMPATIBILITY y Anti A, Anti B – IgM type – it wont cross the placenta y Anti O – IgG type – it will cross the placenta y If mother is O blood group; Baby is whether A/B – chance of ABO incompatibility occurs y But it is a milder disease and not fatal y Diagnosis:  Direct Coomb’s test: Negative  Indirect Coomb’s test: Positive y y y

 

y

y

y

y These antibodies (isoantibodies) are called as natural antibodies because they seem to arise from genetic control without any apparent antigenic stimulation y Anti A and anti B antibodies – blood group antibodies – IgM type (natural); anti O isoantibodies are IgG type; Following any blood transfusion reactions or Rh incompatibility – antibodies occur—those are of IgG type y Red cells of all ABO groups has a common antigen – called as H antigen y Some individuals lack A, B and H antigen – named OH blood or Bombay Blood Group.

y

y

Blood group

y

y

y

y

y ABO blood group system was first described by Landsteiner y Based on the antigen present in the red cells and antibodies present in the serum – blood groups are identified

has this variant; to overcome this Direct Coomb’s test should be performed y Rh compatibility testing is must only when the recipient is Rh negative y When Rh-negative person receives Rh-positive blood → not all of them mounts immune reaction → some accepts → the reason is unknown but they are termed as non responders y Fetal Rh incompatibility: Most common in second pregnancy because of maternal sensitization in the primigravidae forms enormous amount of antibodies and that easily attacks in subsequent pregnancies. y

ABO BLOOD GROUP SYSTEM

64

y

TRANSFUSION TRANSMITTED INFECTIONS y y

y

y

y

y

y y y y

y

y

y When Rh negative mother gives birth to Rh-positive child (father is Rh-positive), Child may develop hemolytic disease. Through these findings Rh incompatibility was identified y This unique antigen reacts with rabbit antiserum to Rhesus monkey erythrocytes (hence called as Rh factor) y To identify whether a person is Rh-positive or negative → antigen D (Rho) presence/absence is identified y Among Indians : 93% are Rh-positive and 7% Rh negative y A variant of D antigen called Du is present – problem with this subtype is not all Du antigens reacts with anti D serum – hence diagnosis of Rh antigen will be difficult when person

y

y

RH BLOOD GROUP SYSTEM

Human immunodeficiency virus Hepatitis B virus Hepatitis C virus Cytomegalovirus Syphilis Malaria CJD prions West nile virus Toxoplasmosis  When the red blood cells in blood bag are contaminated with bacteria like Pseudomonas → it leads to agglutination of blood cells with all type of antisera (A,B,O) → this is due to unmasking of a hidden antigen that is normally present in RBCs → T antigen → this is called Thomsen Friedenreich phenomenon 

y y y y y y y y y

Unit 6     Immunology

MULTIPLE CHOICE QUESTIONS 1. Serum of blood group A patients has: (Recent Pattern 2017) a. Anti A b. Anti B c. None d. Anti A and Anti B 2. Bombay blood group has absence of: (Recent Pattern 2017) a. A antigen b. A and B antigen c. A, B and H antigen d. H antigen alone 3. Rh antibodies responsible for hemolytic disease of newborn are: (Recent Pattern 2018) a. IgD b. IgG c. IgA d. IgE 4. Rh compatibility testing should be considered only if: a. Donor is Rh negative (Recent Pattern 2018) b. Recipient is Rh negative c. Both are Rh negative d. None of the above 



5. The most sensitive method for the detection of Rh antibodies is: (Recent Pattern 2017) a. Direct Coomb’s test b. Indirect Coomb’s test c. Precipitation test d. Hemagglutination test 6. All of the following infections are transmitted through blood transfusion except: (Recent Pattern 2017) a. Cytomegalovirus b. Variant CJD Prion c. Hepatitis A virus d. Malaria 7. Thomsen Freidenreich phenomenon is due to: a. A antigen (Recent Pattern 2017) b. B antigen c. T antigen d. H antigen

ANSWERS AND EXPLANATIONS 1. Ans.  (b) Anti B

• But Rh negative people should receive only negative blood group. •

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 193

Blood group

RBC

Serum

A

A

Anti B

B

B

Anti A

AB

A and B

None

O

None

Anti A and Anti B

5. Ans.  (b) Indirect Coomb’s test Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 196 IgG anti-D (Rh) antibodies can be detected by: • Colloid medium with 20% BSA (bovine serum albumin) • RBC treated with trypsin, pepsin or other enzymes • Indirect Coomb’s test (most sensitive method). •

•

2. Ans.  (c) A, B and H antigen

•

6. Ans.  (c) Hepatitis A virus

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 194

Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 197

• Absence of A, B, H antigens in the RBCs – Bombay blood group • They have anti A, anti B, anti H in the serum.

Transfusion transmitted infections: • Bacterial contamination, E.g. Pseudomonas • HIV • HBV B, C,D • CMV • Prions – Variant CJD • Syphilis • Malaria • Toxoplasmosis

•

•

3. Ans.  (b) IgG Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 196 • Most of the Rh antibodies are – Complete IgG type • Minority can be incomplete – IgM type. •

•

•

•

•

•

•

•

•

•

4. Ans.  (b) Recipient is Rh negative Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 195 • Rh-positive person can receive blood irrespective of the recipient’s Rh status •

442

7. Ans.  (c) T antigen Ref: Ananthanarayan and Paniker’s Textbook of Microbiology – 10th ed – Page 197 • RBC contaminated with Pseudomonas (Blood bag) – agglutinates with all the blood group sera – due to the unmasking of hidden antigen seen in normal human RBC - T antigen - Thomsen Freidenreich phenomenon •

Components of Acquired immunity

T cell, B cell, Classical complement pathway, Antigen presenting cells, Cytokines

What are the acute phase reactants?

Serum Amyloid A, CRP, Complement factors, Fibrinogen, von Willebrand factor, alpha 1 antitrypsin, Mannose-binding protein, haptoglobin, ceruloplasmin

Herd immunity is seen in which of the vaccinations

Diphtheria, Pertussis, MMR, OPV and Small pox vaccine

What is adoptive immunity?

Transfer of CMI from one individual to another (Transfer factor) – used in treatment of lepromatous leprosy

Define epitope, paratope and haptens?

Epitope – Smallest unit of antigenicity; Paratope – Specific site in antibody that reacts with the antigen; Haptens – low molecular weight molecule that lack immunogenicity but retain antigenicity

Name some examples for adjuvants?

Alum, Mineral oil, Freund’s incomplete adjuvant, Freund’s complete adjuvant, LPS of Bordetella pertussis, Mycobacterium bovis, Toxoid of DT and TT

Heterophile antigens – uses

Weil-Felix reaction, Paul Bunnell test, Cold agglutination test, Streptococcus MG test, Forssman antigen

Examples of superantigens

Staphylococcal toxin – TSST, Exfoliative toxin, Enterotoxins; Streptococcal toxin – Streptococcal pyrogenic exotoxin A and C ; Mycoplasma arthritidis mitogen – 1, Yersinia enterocolitica, Y. pseudotuberculosis; EBV, CMV, Rabies nucleocapsid, HIV , Malassezia furfur

Chromosomes linked with different immunoglobulin classes

Heavy chain – Chromosome 14; Light chain kappa – Chr 2; Light chain lambda – Chr 22;

Immunoglobulins and its enzymatic digestion – List out the enzymes and fragments

Papain digestion – generates two Fac and one Fc fragment; Pepsin digestion causes one F(ab’)2 and many small fragments; Mercaptoethanol caused four fragments – two H and two L chains

Chapter 64     Immunohematology

HIGH YIELDING FACTS TO BE REMEMBERED IN IMMUNOLOGY

Name some abnormal immunoglobulins Bence Jones proteins – Multiple myeloma; Excess IgM – Waldenstrom’s macroglobulinemia; and diseases alpha chain – Seligmann’s disease ; Gamma – Franklin’s disease; Cryoglobulinemia – multiple myeloma and Hep C infection What is the difference between T-independent and T-dependent antigen?

T-independent antigen: No memory, no antigen processing, slowly metabolised, activate B cells polyclonally, Activate both mature and immature B cells; T-dependent antigen— Memory present, antigen processing step is needed, rapidly metabolized, Activate B cells monoclonally and activate mature B cells only

What are all the types of Monoclonal antibodies?

Mouse mAb – 100% mouse derived proteins; Chimeric mAb – Recombination of mouse proteins (Variable region) and human proteins (Constant region); Humanized mAb – only the antigen binding site is mouse derived ; remaining part human derived; Human mAb – 100% human derived

What is lattice hypothesis?

Zone of equivalence – Both antigen and antibody equal; Prozone phenomenon – Antibody excess; Post-zone phenomenon – Antigen excess;

Examples of Precipitation reaction

Lancefield grouping, Ascoli’s thermoprecipitation test , VDRL, RPR, Kahn test

Examples of agglutination tests

Widal test, Standard agglutination test for Brucellosis, Coomb’s test, Blood grouping, Heterophile agglutination test

Complement fixation test is used in

Treponema pallidum immobilization test, Sabin Feldman dye test for Toxoplasma antibodies, Vibriocidal antibody test

MHC Class I and MHC class II location

Class I located in all nucleated cells and platelets ; Class II located in antigen presenting cells Contd...

443

Unit 6     Immunology

What are professional and nonprofessional antigen presenting cells

Professional APCs – Macrophages, Dendritic cells and B cells; Nonprofessional APCs – Fibroblasts, Thymic epithelial cells, Pancreatic beta cells, Vascular endothelial cells, Glial cells and thyroid epithelial cells

What are the CD markers for B cell, T cell and natural killer cell?

B cell – CD19,21,24; T cell – CD3,4,8; NK cell – CD16, CD56

What is IPEX syndrome?

Deficiency of Foxp3 receptors leads to autoimmune disease ; Immune dysregulation, Polyendocrinopathy, Enteropathy and X linked syndrome

Diseases due to humoral immunodeficiency (B cell defects)

X linked agammaglobulinemia – Bruton disease ; Common variable immunodeficiency; Isolated IgA deficiency; Hyper IgM syndrome; Transient hypogammaglobulinemia of infancy

Diseases due to cellular immunodeficiency (T cell defects)

DiGeorge syndrome (Thymic hypoplasia), Chronic mucocutaneous candidiasis; Purine nucleoside phosphorylase deficiency;

Name the combined immunodeficiencies

SCID, Wiskott-Aldrich syndrome, Ataxia telangiectasia, Nezelof syndrome

Disorders of phagocytosis

Chronic granulomatous diseases, Myeloperoxidase deficiency, Chediak-Higashi syndrome , Leukocyte adhesion deficiency, Lazy leukocyte syndrome, Job’s syndrome, Tuftsin deficiency, Shwachman’s disease

Different type of Grafts in transplant immunology

Autograft – one part of body site to another; Isograft – between genetically identical E.g. monozygotic twins; Allograft – genetically nonidentical (M/c); Xenograft – different species;

Lymph node and immune cells

B cells located in follicle T cells located in para cortex

Site of differentiation and maturation of T cells from Thymus immune cells B cells from Bone marrow Splenic dysfunction or postsplenectomy leads to infections of:

• • • • • • •

•

•

•

•

•

•

•

444

Streptococcus pneumoniae H. influenzae type b Neisseria meningitidis E. coli Salmonella sp Klebsiella pneumoniae GBS

Antibody structure – Fab fragment

Antigen binding fragment

Antibody structure – Fc fragment

Constant Carboxy terminal Complement binding Carbohydrate side chains It determines the isotype

Complements and their functions

C3b – opsonization C345a – anaphylaxis C5a – neutrophil chemotaxis C5-9 – cytolysis by MAC

APPLIED MICROBIOLOGY

7

Unit Outline

1

Chapter 65 Applied Microbiology

65

Applied Microbiology BACTERIOLOGY OF AIR, WATER AND MILK Water

Coliforms

Marker of fecal contamination, E.g. E. coli

Fecal streptococci

If fecal streptococci are present–then it means recent contamination of water

Clostridium perfringens If only Clostridium perfringens is seen without E. coli or fecal streptococci– then it means the fecal contamination is not recent

Air y Most of the infectious diseases are through inhalation y Two types of infection:  Airborne infection: Infection that occurs by respiratory droplets that are ≤5 μm in size  Droplet infection: Infection that occurs by respiratory droplets that are >5 μm in size

Plate count

• Water is cultured by pour plate culture method • It is incubated under two different temperatures: • 37°C • 22°C • Organisms that grow in 37°C are mostly from human or animal origin • Organisms that grow in 22°C are saprophytic in nature

Table 3:  Organisms transmitted through air Droplet infection

Airborne infection

• Streptococcus pyogenes • Neisseria meningitidis • Corynebacterium diphtheriae • Haemophilus influenzae B • Bordetella pertussis • Yersinia pestis • Mycoplasma pneumoniae • Influenza virus • Rubella virus • Mumps virus • Adenovirus • Parvovirus

• • • •

• • • •

• • • •

Mycobacterium. tuberculosis VZV Measles virus Influenza virus

Membrane filtration method

• A known volume of water is filtered through membrane filter–pore size is 22 μm • Bacteria if present–it gets filtered through the membrane • This membrane is then cultured in a media • Colonies are identified

• •

•

•

• • • • • •

y

•

•

y

• •

• It is called as Eijkman test • Test is done following presumptive coliform count test • Test to check that whether the organism is E. coli or not •

Differential coliform count

y Quality of air is must in operation theaters, ICU to avoid nosocomial infections y The bacteria carrying particles per cubic meter should not exceed 35 in empty theater and 180 during operative procedures y For ultra clean OT’s–the particle should be less that 1 for empty theater and less than 10 during operation y

•

• Multiple tubes are taken at different dilutions of broth medium • Water is inoculated in it • When coliform count is more than 10/ mL–then the water is unsatisfactory • Probable numbers of coliforms/100 mL–is read from the probability table of McCrady •

Presumptive coliform count

•

•

•

•

•

•

•



Features •

Method to test water



Table 1:  Bacteriological methods for water analysis

y

y

y











y

y

y Water is the most potential source of infection in many diseases. y For example  Salmonella  Shigella  Vibrio  Poliovirus  Hepatitis A y Hence, constant examination of drinking water and proper decontamination is must to ensure safe drinking.

Table 2:  Organisms needed to be tested

Table 4:  Amount of particles in OT Operation theater Empty theater During operative procedure Regular theater

100°F) with other signs pertaining to systemic illness is called acute febrile illness y Fever of unknown origin (FUO) is a term coined when it meets the following criteria:  Fever higher than 38.3ºC on several occasions  Duration of fever for at least three weeks (21 days)  Uncertain diagnosis after 1 week of study in the hospital y Most common reasons for FUO/PUO are  Infections  Malignancies  Connective tissue diseases (e.g., vasculitis, rheumatoid arthritis) y Fever should be diagnosed along with other presenting complaints or by other systemic manifestations; commonly encountered presentations are:  Viral fevers  Pneumonia  Abscess  Bacterial endocarditis  Meningitis  Hemorrhagic fevers

67

Bacterial meningitis Suppurative intracranial infections Brain abscess Cerebral malaria (caused by P. falciparum) Lemierre’s disease (caused by Fusobacterium necrophorum)

Unit 8     Infectious Diseases

DIAGNOSIS OF PYREXIA OF UNKNOWN ORIGIN A thorough history should include the following information: Travel, animal exposure (e.g., pets, occupational, living on a farm), immunosuppression (with the degree noted), drug and toxin history, including antimicrobials and localizing symptoms. Since all the other investigations would have been completed till 21 days – some unique tests are needed to diagnose the cause for PUO.

DIAGNOSTIC TESTS INCLUDE y y y y y

y y y y y y

458

y

y

y

y

y

y

y

y

y

y

y

Basic blood counts including ESR/CRP Serum lactate dehydrogenase Tuberculin skin test or IGRA HIV testing (antibody testing and viral load assay) Three routine blood cultures drawn from different sites over a period of at least several hours without administering antibiotics (if not done already) Rheumatoid factor Creatine phosphokinase Heterophile antibody test in children and young adults Antinuclear antibodies Serum protein electrophoresis Computed tomography (CT) scan of abdomen

y CT scan of chest y Molecular methods to identify bacterial infection include polymerase chain reaction (PCR) and detection of bacterial 16S ribosomal ribonucleic acid (RNA) genes or host RNA signatures. y

y

Table 3:  Empirical therapy in common infectious diseases with fever Infections

Empirical Treatment

Bacterial meningitis

Ceftriaxone + Vancomycin

Brain abscess

Vancomycin + Metronidazole + Ceftriaxone

Cerebral malaria

Artesunate (or) Quinine

Acute bacterial endocarditis

Ceftriaxone + Vancomycin

Septic shock

Vancomycin + Gentamycin + PT (or) cefepime

Meningococcemia

Penicillin (or) Ceftriaxone

Necrotizing fasciitis

Vancomycin + Clindamycin + Gentamycin

Clostridial myonecrosis Penicillin + Clindamycin

68

Infections of Ear, Nose and Throat  



Oral Streptococci (Viridians)

y

y

y

y

y y y y y

y

y





y



y

y

y Colonization occurs in 48 hours of birth y Normal Flora  Gram positive and negative aerobes like, oral streptococci, Neisseria, bacteriophages, diphtheroids and anaerobes. y Asymptomatically carried pathogens:  Streptococcus pneumoniae,  H. influenza, M. catarrhalis, N meningitidis.  Patients on antibiotic therapy y Enterobacteriaceae (aerobic Gram-ve bacilli)

Example of group A.- S. pyogenes. most important Suppurative (tonsillitis TSS septicemia) and nonsuppurative (AGM, RF) diseases y B not in nasopharynx- not in ENT disease; cause neonatal sepsis. e.g. S. agalactiae. 

y

NORMAL BACTERIAL FLORA OF NASOPHARYNX

Alpha hemolysis (incomplete) Normal oral and nasopharynx flora Dental abscess and periodontal disease Native wall endocarditis Use as bacteriotherapy beneficial in recurrent otitis media

S. pneumoniae

Prevention of adhesion of pathogens by the normal oral streptococci.

y Alpha hemolysis y Draughtsman like colonies that are bile soluble and optochin sensitive y Normal nasopharynx flora in 0-2 years age y Most common cause of otitis media sinusitis and pneumonia in all ages y Meningitis from hematogenous spread from respiratory infection y Vaccine available. y y y y y

Enterococci = fecal streptococci  y

y

y Group D lancefield y Resistant to bile and antibiotics y Normal fecal flora → UTI abdominal sepsis

Corynebacteria





Gram positive, catalase + Divide in 2 planes → clusters Grow in 18-40° and in NaCl → presence on skin and mucosa Divided on the basis of coagulase test  Positive: S. aureus presents in anterior nares. Has many virulence factors- Coagulase, CPS, FBP (fibrinectin binding protein), Protein A, Staphylokinase Hemolytic toxin, Other toxins: TSST enterotoxin  Negative: not significant in ENT.

y

y y y

y y y y

y

BACTERIAL CAUSES OF ENT INFECTIONS Gram-positive Bacteria Staphylococci

y

Bacterial Interference

Š Š Š

y

y

y

y Beta hemolysis (complete) y Divided into lancefield Groups A B C G y A, C, G -in nasopharynx.

y

y

y

y

y

Pyogenic Streptococci



y

y

y

y G+ catalase -ve y Divide in single plane. y Divided on the basis of - hemolysis on blood agar, Lancefield antigen, Growth

G+ pleomorphic bacilli Chinese letter like on gram stain. Daisy head appearance on tellurite containing media Mostly bacterial Flora. Only few pathogens  C. diptheriae  Disease due to exotoxin (produced only by bacteria infected with lysogenic phage)  Exotoxin → inhibits protein synthesis → pharynx damage → into blood stream → myocardial and neurologic toxicity  Three biotypes  Š Gravis: severe disease Š Intermedius: severe disease  Š Mitis 

y y y y y



Streptococci

Unit 8     Infectious Diseases

Gram–Negative Bacteria Neisseria y G -ve diplococci y 10 sps - 2 are the main pathogens. Adapted to survive in man.. needs CO2 37°C enriched media.  N. gonorrhoeae: genital and urinary infections sexually acquired pharyngitis  N. meningitidis serotypes. A, B, C, W135, Y  Š Normal flora in 10%. Disease only in small percentage  Š Meningitis, septicemia and septic arthritis y

y

Pseudomonas y y y y

y

y

y

y







Š

Š

Moraxella Catarrhalis y y y y

y

y

y

y

Morphologically like Neisseria Third most common cause of otitis media Acute exacerbation of COPD Resistant to penicillins (produces beta lactamase)

Haemophilus  y G -ve pleomorphic coccobacilli y Major part of normal resp flora y Has specific growth requirements factors V and X, hemin, NAD y H influenzae  Poor growth in blood agar coz needs factors V and X  Forms 10% of nasopharynx flora  Mainly noncapsulated. One of common cause of otitis media and sinusitis  Capsular types a-f.  Type b severely invasive,  Meningitis, epiglottitis, septic arthritis  Hib vaccine available. y

Thin G –ve bacilli Resistant to many antimicrobial and disinfectants. Opportunist infections P aeruginosa   Produce pyocyanin pigment.  Malignant otitis externa, surgical wounds and leg ulcers 

Anaerobes  y Only in strict absence of oxygen. Even while transporting the pus (large volume of pus and long incubation needed). y Resistance to metronidazole used in culture. y Includes many G + and -ve microbes y Part of normal and large bowel flora. y

y

y

y

Bacteroides Fragilis y G -ve bacilli y Small % of oral and bowel flora y Most common anaerobes in infections—usually polymicrobial y

y

y

y

y

y





Fusobacterium Necrophorum y Fusiform G -ve y Normal oral flora y Necrobacillosis - Rapidly progressive disease y

y

y



Microaerophilic Bacteria







Bordetella Pertussis y G -ve coccobacilli y Fastidious growth needs. 5 days incubation in charcoal containing media  y Virulence factors.  FHA -cell surface-associated adherence protein   Pertussis toxin causes cough and lymphocytosis. y Disease in nonvaccinated infants (refusal / “too young ‘) y Reservoir-adult carriers, subclinical cases

y Very low concentrations of O2, slow growing, difficult to isolate y Part of normal oral and large bowel flora y E.g. Actinomyces (A. israelii,→ clumps of G +ve bacilli, Sulfur containing granules y

y

y

Mycobacteria

y

y

y





y

y

Enterobacteriaceae y y y y y y y

y

y

y

y

y

y

y

100 + species like E. coli, Klebsiella, pseudomonas Aerobic G -ve bacilli Grow in both aerobic and anaerobic condition and bile salts   Divided based on lactose fermentation  Normal Flora in bowel Not in nasopharynx normally; only in hospitalized patients. UTI, abdominal sepsis and rarely respiratory infection.

Klebsiella Rhinoscleromatis y Chronic granuloma in upper respiratory tract y Endemic in South America, East Europe and Asia y

y

460

y Acid and alcohol fast, grow slowly in aerobic conditions. Not isolated on routine agar y 25 species divided into M. tuberculosis and others (atypical mycobacteria) y Mycobacterium tuberculosis  Grows slowly on agar containing fatty acids (egg containing L-J medium → buff colored bread crumbs like appearance)  4-6 weeks for colonies to appear  Decrease isolation time → use of liquid media with automated detection of growth and DNA probes  Samples to be handled in category 3 lab using safety cabinets. y

y

y









Atypical Mycobacteria y M. avium-intracellulare, M. kansasii, M. scrofulaceum and M. malmoense y Cervical lymphadenitis in frontal y

y

CT Scan

y

y

Investigations y y y y

y

y Partial/ total opacification, with flocculent calcification, bone thickening and sclerosis. y No air fluid levels (seen more commonly with bacterial sinusitis) y

y

y

y

y

Endoscopy: Allergic mucin Mucus microscopy and HPE CT scan Lab: Eosinophilia, total IgE, specific IgE against fungi, fungal culture.

Management

HPE y Dense matted conglomeration of hyphae separate from but adjacent to mucosa y No e/o allergic mucin or granulomatous reaction in mucosa y No invasion of mucosa/ blood vessels/ bone. y

y

y

Treatment

Surgery: Remove polyp and mucin-containing fungi Postoperative oral steroids Adjunctive Allergen immunotherapy, nasal steroids, antihistamines, antileukotrienes and sinonasal saline lavage y Antifungals not needed Recurrence common: Needing regular follow-up and repeat surgery y y y y

y

y

y

y

y

y Surgical removal y No local or systemic antifungals needed. y

Chapter 68     Infections of Ear, Nose and Throat

Clinical Features

y

Allergic Fungal Sinusitis Noninvasive disorder in immunocompetent persons y Criteria  CT- chronic rhinosinusitis – serpiginous sinus opacification of >1 sinus, mucosal thickening and erosion of bone  Presence of allergic mucin with clusters of eosinophils  Presence of noninvasive fungal elements in the mucin  Type 1 hypersensitivity to cultured fungi  Nasal polyposis  Fungal cultures: variable yield hence not used as criteria for diagnosis y Cause – ABCD  A– Alternaria,  B– Bipolaris  C – Cladosporium, Curvularia  D- Drechslera y



Mycotic Diseases of Throat Oropharyngeal Candidiasis Causes y MCC – C. albicans y C. glabrata, C. krusei, C. tropicalis, and C. parapsilosis y

y





Table 2:  Predisposing factors





General

Local

Broad spectrum antibiotics, steroids, DM, nutritional deficiency, immunosuppressive disease HIV (one of the earliest manifestation in asymptomatic patients, candidiasis was most common opportunistic infection before antiretrovirals)

Unhygienic / ill-fitting dentures Tobacco smoking



y









Clinical Features y Young immunocompetent adults, in warm humid areas y h/o CRS, with H/o multiple surgeries y

Oropharyngeal (not life threatening but discomfort) → esophageal (life threatening)

y

Table 3:  Clinical forms of oral candidiasis Pseudomembranous

Erythematous/atrophic

Hyperplastic / hypertrophic

• Most common in HIV, extremes of age, and immunosuppressed – malignancies • White raised lesions on tongue, palate, buccal mucosa, tonsils • Can be wiped off to reveal eroded erythematous bleeding base • Painless in oral • Painful if spreads to throat

• Associated with broad spectrum antibiotics, chronic steroids, HIV • Consequence of persistent pseudomembranous • Red flat lesion on dorsum of tongue or palate • Depapillated area on tongue • premalignant

• Premalignant lesion • Small palpable translucent areas to large opaque, white, dense hard and rough mass • Inner surface of cheek • Cant be wiped off unlike pseudomembranous • Usually asymptomatic

•

•

•

•

•

•

•

•

•

•

•

•

•

•

•

463

Unit 8     Infectious Diseases

Other Forms of Candidiasis

Viruses Causing Respiratory Illness

y Chronic atropic denture candidiasis y Laryngeal candidiasis: Can obstruct airway if untreated y Investigations  Scraping and microscopy in oral and oropharyngeal  Fiberoptic laryngoscopy and biopsy for laryngeal

y Pharyngitis: PIV, CMV, In-A and B, HSV, Coxsackie, enterovirus y Common cold – rhinovirus, corona virus and PIn-4 y Croup – In, Pin -1, 2, 3, RSV y Flu – In – A and B

Treatment

Diagnosis

y Infants: nystatin oral suspension (100,000 units/mL) or Amp B oral suspension (100 mg/mL) 6 hourly × 2 weeks y Older children and adults- clotrimazole troche (like candy) – 10 mg troche 5 times a day, nystatin or Amp B oral suspension / miconazole gel y HIV patients: Oral fluconazole (100–200 mg/ day – 1–2 weeks) or itraconazole oral solution ( 200–400 mg day for 1–2 weeks) y For recurrent and disabling cases (HIV, Malignancy) – flucomazole long term y Refractory: iv Amp B 0.3-0.5 mg / kg/day or Caspofungin – 50 mg/day × 1 week y Laryngeal candidiasis: iv Amp B 0.7-1.0 mg / kg/day, oral fluconazole after recovery, ET intubation.

Nasopharyngeal aspirates used instead of nose and throat swabs due to better yield. y Traditional: Inoculation of secretions of tissue culture - time consuming y Recent:  Immunoassays and immunofluorescent labeled antibody assay – rapid  PCR- sensitive and not needed for virus to be present.

y

y

y



y

y

y

y



y

y

y

y

y





y

y

y

Other Mycotic Infections of Pharynx and Larynx y y y y

y

y

y

y

Histoplasmosis and paracoccidiodomycosis Sore throat, dysphagia, dyspnea and cough Ulcer/ granuloma on vocal cords/ laryngeal epithelium Biopsy and HPE with culture -to rule out malignancy

Treatment y Itraconazole (better tolerated): 200–400 mg/day × 6–18 months or ketoconazole 200–400 mg/day  Delay in diagnosis → fibrosis → laryngeal stenosis – permanent speech and breathing difficulties. y



Corona Virus y Largest human RNA virus y Aerosol spread y 2 human strains – 229E and OC4 – epithelium of trachea, nasal mucosa, alveoli of lungs y 10 % of common cold esp in winter y Nasal discharge, malaise and exacerbation of chronic obstructive pulmonary disease (COPD) y Lymphadenitis cough and fever less common y

y

y

y

y

y

Adenovirus y Double standard DNA virus originally isolated from human adenoid tissue. y 45 serotypes y Children asymptomatically infected with types 1 and 2 in early childhood and type 3 and 5 in later life y Spread, Aerosol, feco-oral y Outbreaks of URTI and LRTI in small communities and institutions. y Swimming pool outbreaks of pharyngoconjunctival fever y Exudative tonsillitis with high fever pharyngitis and pneumonia. y Each type specific features  Type 2 – milder  Type 3 and 7 – severe disease – pneumonia in children. y

y

y

y

Colonization of TE Prosthesis y Biofilm formation and invasion of the silastic by Candida sps. C. glabrata, C. krusei, C. tropicalis. y Occur in 2-4 months of implantation – requires replacement y Radiation shortens this time y Prevention  Buccal bioadhesive slow release miconazole nitrate tablet  Metal coating of prosthesis y

y

y

y

y

y

y

y









VIRAL CAUSES OF ENT INFECTIONS

Rhinovirus Small nonenveloped RNA virus, >100 serotypes Replicates only in URT. Optimal temperature – 33° Aerosol and contact with fomites Symptoms partly due to bradykinin release – nasal congestion, sneezing, sore throat, headache and cough. y Infections throughout year, several serotypes. y y y y

y

y

Respiratory Tract Infections

y

y

y Most common disease of humans y 30% of all childhood deaths worldwide y Seasonal activity: Parainfluenza type 1 and 2- autumn, 3 in spring and RSV in winter y Pathogenesis: Virus binds with host cell receptors, e.g. Rhinoviruses: ICAM-1 (intercellular cell adhesion molecule) y Interferon response → systemic symptoms y Bradykinin → local symptoms y

y

y

y

Influenza

y

464

y

y

y RNA orthomyxovirus y Subtypes – A,B,C y Droplets → incubate in 1–3 days y

y

y

y

y

y Diagnosis by indirect immunoflourscence of antigen and PCR of nasopharyngeal aspirates. y

y

y

y

y

Mumps RNA virus Incubation period: 2–3 weeks Transmitted via droplet and close contact Fever → parotid swelling → subsides VII nerve swelling/orchitis (1 in 5 men) and atrophy of testis Sterility uncommon y Encephalitis and aseptic meningitis with residual deafness y Live attenuated trivalent MMR vaccine very effective. y y y y y

y

y

y

y

y

y

Herpes Simplex Virus y Double standard DNA y Types -1 and 2 – both cause primary infection and reactivation y HSV 1 – oral secretions → 2 days – 2 weeks → sore throat, pharyngitis, painful vesicles on oropharynx → extensive gingivostomatis, extending to lips and cheeks y Complications: Encephalitis, meningitis, erythema multiforme, eczema herpeticum, Bell’s palsy. y

y

y

y

Recurrent Herpes y y y y y y

y

y

y

y

y

y

y

Measles Incubation period of 2 weeks Children 3–6 years age Around 1 million child deaths in developing countries Virus replicates in respiratory and conjunctival epithelium → reticuloendothelial cells y Symptoms- rhinorrhea, cough, fever, Kopliks spot on mucus membrane of cheeks → rash after few days (face → trunk → limbs), bronchitis and diarrhea y Complications y Croup, otitis media, conjunctivitis, enteritis, febrile fits, rarely Subacute sclerosing pan encephalitis (SSPE). y y y y

y

y

y

y

Epstein-Barr Virus y y y y y

y

y

y

y

y

y

y

y y y

y

y

y

y

Respiratory Syncytial Virus (RSV) y y y y y y y y y y y y

y

y

y

y

y

y

y

y

y

y

y

y

RNA virus Two serotypes: A, B Winter outbreaks in infants and epidemics lasting for months Million deaths in developing countries Highly contagious: Contacts with respiratory secretions (not aerosol) Incubation: 2–8 days Age mostly 6 weeks to 6 months Coryzal symptoms → ARDS in 24 hours → death Necrosis of bronchiolar epithelium and lung parenchyma → bronchiolitis → recurrent wheezing Severe systemic symptoms due to greater interferon response Vaccine not available → preventive measures crucial (limiting aerosol, cohorting and hand wash) Ribavarin in high-risk infants.

Parainfluenza Virus y y y y y y

y

y

y

y

y

y

One-third of all RTIs 1 and 2 – autumn croup outbreaks every 1–2 years 3- endemic – infantile bronchilolitis, pneumonia 4- less common both URI and LRI Exacerbation of COPD, asthma, cystic fibrosis Transmitted: Person to person

Itching, burning, tingling and pain lasting for 6 hours – 2 days. Lesion on lips and perioral skin Multiple lesions on same site Some secondarily infected with S. aureus. Complete healing