, for detailed information on microbiology books, visit our website , for detailed information on microbiology books

Apurba S$ Sastry
Sandhya Bhat faition
Covering Must Know Competencies based
on the latest MCI curriculum

Expert Knowledge, Anytime, Anywhere

4. Download on the GET ITON

G Wao)obs)ce)f= > Google Play

Scratch
Redeem
D- Ar
Coupon
EX;

*Redemption starts in January 2021

ee )
[email protected]
 _ Essentials of
Practical Microbiology
 Digitized by the Internet Archive
in 2022 with funding from
Kahle/Austin Foundation

https://archive.org/details/essentialsoforacO002sast
 Essentials of
Practical Microbiology
SECOND EDITION

Apurba S Sastry MD UIPMER) DNB MNAMS PDCR

Hospital Infection Control Officer
Officer In-charge, HICC
Antimicrobial Stewardship Lead
Associate Professor
Department of Microbiology
Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Puducherry, India

Sandhya Bhat Gold medalist) MD DNB MNAMS PDCR

Professor
Department of Microbiology
Pondicherry Institute of Medical Sciences (PIMS)
(A Unit of Madras Medical Mission)
Puducherry, India

JAYPEE BROTHERS MEDICAL PUBLISHERS

The Health Sciences Publisher
New Delhi |London
 é
dsm Jaypee Brothers Medical Publishers (P) Ltd

Headquarters
Jaypee Brothers Medical Publishers (P) Ltd
EMCA House, 23/23-B
Ansari Road, Daryagan}
New Delhi - 110 002, INDIA
Landline: +91-11-23272143,+91-11-23272703, +91-11-23282021,+91-11-23245672
Head Office : 011-43574357
@E jaypee @jaypeebrothers.com: www.jaypeebrothers.com

Corporate Office Overseas Office

Jaypee Brothers Medical Publishers (P) Ltd J.P. Medical Ltd
4838/24, Ansari Road, Daryaganj 83 Victoria Street, London
New Delhi 110 002, India SW1H OHW (UK)
Phone: +91-11-43574357 Phone: +44 20 3170 8910
Fax: +91-11-43574314 Fax: +44 (0)20 3008 6180
Email: jaypee @jaypeebrothers.com Email: [email protected]
Website: www.jaypeebrothers.com
Website: www.jaypeedigital.com
© 2021, Jaypee Brothers Medical Publishers
The views and opinions expressed in this book are solely those of the original contributor (s)/author (s) and do not
necessarily represent those of editor (s) of the book.
All rights reserved. No part of this publication may be reproduced, stored or transmitted in any form or by any means,
electronic, mechanical, photocopying, recording or otherwise, without the prior permission in writing of the publishers/
editors.
All brand names and product names used in this book are trade names, service marks, trademarks or registered
trademarks of their respective owners. The publisher is not associated with any product or vendor mentioned in this
book.
Medical knowledge and practice change constantly. This book is designed to provide accurate, authoritative
information about the subject matter in question. However, readers are advised to check the most current information
available on procedures included and check information from the manufacturer of each product to be administered, to
verify the recommended dose, formula, method and duration of administration, adverse effects and contraindications.
It is the responsibility of the practitioner to take all appropriate safety precautions. Neither the publisher nor the author
(s)/editor (Ss) assume any liability for any injury and/or damage to persons or property arising from or related to use
of material in this book.
This book is sold on the understanding that the publisher is not engaged in providing professional medical services. If
such advice or services are required, the services of a competent medical professional should be sought.
Every effort has been made where necessary to contact holders of copyright to obtain permission to reproduce
copyright material. If any have been inadvertently overlooked, the publisher will be pleased to make the necessary
arrangements at the first opportunity. The CD/DVD-ROM (if any) provided in the sealed envelope with this book is
complimentary and free of cost. Not meant for sale.
Inquiries for bulk sales may be solicited at: jaypee @jaypeebrothers.com
Essentials of Practical Microbiology
First Edition. 2018
Second Edition: 2021
ISBN: 978-81-948028-2-2
Printed at Samrat offset Pvt. Ltd.
 Dedicatea AW)

Our Beloved Parents, Family Members

And, above all, the Almighty

“Life is the most difficult exam. Many fail because they tend to copy others, not realizing that
everyone has a different question paper.’

“Gold medalists are not made up of gold. They are made up of determination and hard work
and ready to kill themselves to achieve their goals.”

“Success is not the key to happiness. Happiness is the key to success. If you love what you are
doing, you will be successful.”

“You can succeed only if the fire inside you burns brighter than the fire around.”
 Golden Rules of Goal Setting

Dear Students
Here are some important tips which will help you in setting your goals in studies:
1. Set Goals That Motivate You: This means making sure that they are important to you, and that
there is value in achieving them
2. Set SMART Goals
Specific: Your goal must be clear and well defined, not vague or generalized
Measurable: Goals must have measurable objectives
Attainable : Make sure that your goals are achievable and within your limit
Relevant: Will take you to the direction you want your life and career to go
# Time Bound: You must know when you have the deadline and can celebrate success
3. Set Goals in Writing: Written commitment in presence of your close people (parents, close
friends) will always push and remind you whenever you tend to deviate from your goal
4. Make an Action Plan: Do not focus only on the outcome, but make planning ofall small steps that
collectively take to the outcome. This is especially important if your goal is big and demanding,
or long-term
5. Monitor Yourself: Compliance to the action plan should be monitored at least weekly (for one
month goal) or monthly (for a yearly goal), depending upon your goal size.

Remember,
“Success is not final; failure is not fatal: It is the courage to continue that counts.”
—Winston S$ Churchill
“There are two types of people who will tell you that you cannot make a difference in this world:
those who are afraid to try and those who are afraid you will succeed.”
—Ray Goforth
“Success ke piche mat bhago. Kabil bano kabil. Kamyabi toh sali jhak maar ke peeche ayegi.”
—Aamir Khan
 Preface to the Second Edition

It gives us immense pleasure to announce the release of second edition

of Essentials of Practical Microbiology. This book is prepared according to
competency based medical education (CBME) MBBS curriculum and the content
of the book is modified from the traditional organism-based teaching to system-
based teaching. The content has been updated, concised and reshuffled—the -
three major types of changes incorporated in this edition. The uniqueness ofthis
book is the Problem Solving Exercises. Each chapter starts with a problem solving
exercise, i.e., clinical case scenario. The book is categorized into two sections.

Section |: General Microbiology, Immunology and Hospital Infection

Control
~2
General microbiology chapters are meticulously restructured with the a
inclusion of general virology, general parasitology and general mycology —
chapters. General bacteriology is reorganized into a single chapter with several | ox;
subchapters. a A
Immunology chapters are thoroughly updated. Various topics have been \)
updated such as MAC ELISA, Elek’s gel precipitation test, etc. have been updated. Nat
Hospital infection control (HIC) chapters need a special mention.We must say Sandhya Bhat
that this section underwent a major update. In the era of COVID-19 pandemic,
the practical application of hospital infection control is being increased to thousand folds. Every
healthcare personnel is supposed to be well verse with the finer details of HIC. Therefore, the
updated version of this section will be a key in making of a skilled Indian medical graduate. The
content has been thoroughly updated with the inclusion of new topics such as hand hygiene,
personal protective equipment with special emphasis on donning/doffing and transmission-
based precautions, needle stick injury, and biomedical waste management. The sterilization and
disinfection chapter is completely revised based on hospital use of sterilizers and disinfectants
rather than traditional ‘Microbiology use’ and aptly shifted from general microbiology section to
HIC section.

Section Il: Systemic Microbiology (Infectious Diseases)

It comprises of several chapters; each chapter comprises of several infective syndromes pertaining
to system. Each infective syndrome starts with a problem solving exercise, followed by the detailed
information about the etiological agent(s) and covers the important practical aspects of the topic.
+,
+o Bloodstream and cardiovascular system infections chapters cover topics such as infective
endocarditis, acute rheumatic fever, sepsis, enteric fever, scrub typhus, brucellosis, leptospirosis
HIV/AIDS, dengue, malaria, visceral leishmaniasis, lymphatic filariasis and systemic candidiasis
Gastrointestinal tract infections chapters cover topics such as shigellosis, nontyphoidal
salmonellosis, cholera, Clostridioides difficile diarrhea, rotavirus gastroenteritis, intestinal amoebiasis,
giardiasis, cryptosporidiosis, intestinal taeniasis, hymenolepiasis, Schistosoma mansoni infection,
trichuriasis, enterobiasis, ascariasis, hookworm infection and strongyloidiasis
Hepatobiliary system infections chapters cover topics such as viral hepatitis, amoebic liver abscess
and hydatid disease
 Essentials of Practical Microbiology

€ Respiratory tract infections chapters cover topics such as streptococcal pharyngitis, diphtheria,
pneumococcal pneumonia, Haemophilus influenzae pneumonia, Klebsiella pneumoniae pneumonia,
pulmonary tuberculosis, Pseudomonas infections, influenza, COVID-19, infectious mononucleosis,
paragonimiasis, zygomycosis, aspergillosis and pneumocystosis
* Skin, soft tissue and musculoskeletal system infections chapters cover topics such as
staphylococcal and streptococcal skin and soft tissue infections, gas gangrene, leprosy, cutaneous
anthrax, mucocutaneous herpes, measles, rubella, dermatophytoses and mycetoma
“ Central nervous system infections chapters cover topics such as pneumococcal meningitis,
Haemophilus influenzae meningitis, meningococcal meningitis, viral meningitis, HSV encephalitis,
Japanese encephalitis, rabies encephalitis, neurocysticercosis, Toxoplasma encephalitis and
cryptococcal meningitis.
“+ Genitourinary system infections chapters cover topics such as urinary tract infection (UTI)
(Escherichia coli UTI, Klebsiella pneumoniae UTI, Proteus mirabilis UTI, and enterococcal UT]), urinary
schistosomiasis, syphilis, gonorrhea, non-gonococcal urethritis, trichomoniasis, vaginal candidiasis
** AETCOM module has been added as a new annexure, which covers several case scenarios pertaining
to confidentiality in disclosing laboratory reports and demonstration of respect for patient samples
“+ There is a separate chapter for ‘university practical examination’ showing the model university
practical examination pattern and mark distribution. This will help the students to prepare for their
practical examination. This will also help the teacher to modify or update the practical examination
pattern.
As you know, human errors are inevitable; and no book is immune to it. We would request all the
readers to provide any errata found and also valuable suggestions and updates via e-mail.
This is probably the first practical book in India on ‘Clinical Microbiology’, in true sense. We are
confident and hoping that you all will fall in love with this edition of the book.

fe LomBP
Apurba S Sastry Sandhya Bhat
[email protected] [email protected]
 Preface to the First Edition
LLL
ELRELL

When there is a dozen of books available in the literature on the same subject, is it required to bring
another book? The idea to bring yet another book on Practical Microbiology was born after many
discouraging and unsatisfying experiences from several existing books, that did not fulfill the needs of
the enthusiastic students on the subject and also a strong desire to make medical microbiology more
interesting, up to date, and clinically relevant. This book has several unique approaches which make
it different from the existing books.
It is a clinical microbiology practical book. After reading this book, the students will have a bird’s
eye view on how to diagnose infectious diseases clinically followed by how to go about laboratory
investigations.
It is the first microbiology book which is written in class-wise pattern according to MBBS practical
class schedule. This will help the teacher know what to teach during a practical class, what to keep for
demonstration during the practical class and also will give the teacher a holistic approach to make the
yearly practical schedule. This will also help the students to know what to read during a practical class,
what to write in the records and what to read during MBBS university practical examination.
There is a separate chapter for university practical examination providing the pattern for various
universities. This will help the students prepare for their practical examination. This will also help the
teacher to modify or update the practical examination pattern.
The uniqueness of this book is the Problem-based Exercises. Each chapter starts with a problem-
based exercise, i.e. clinical case scenario. The whole chapter is about solving the problem covering the
important practical aspects of the topic.
There are more than 600 images, kept according to the demonstrations of practical classes, which
will help the teachers know what is needed to demonstrate in practical classes. If facility is not available,
they can just keep the image itself during the practical class. Images will help the students coordinate
and read about the items demonstrated in the class with what is given in the practical book.
Like our other books, this one also has followed the same principle, e.g. more content accommodated
in less pages so as to give handy look and saving student's time, written in a concise, bulleted format
and to-the-point text and simple and lucid language.
Several new practical class topics are covered in this book which are essential for MBBS graduates to
learn, but are often missed in practical classes of most colleges, such as demonstration of automated
culture and molecular methods; antimicrobial susceptibility testing (reading of disk diffusion and
minimum inhibitory concentration)—interpretation with reference to Clinical and Laboratory
Standards Institute (CLSI); sterilization and disinfection—according to hospital practice with special
note on Central Sterile Services Department (CSSD), enzyme-linked immunosorbent assay (ELISA)
and immunofluorescence, Western blot and rapid test; laboratory diagnosis of leptospirosis and scrub
typhus and dengue; hepatitis viruses (understanding the interpretation of algorithms of markers);
human immunodeficiency virus [understanding the interpretation of tests according to National
AIDS Control Organization (NACO) guideline]; bacteriology of water, air and surface—importance
of environmental surveillance; hospital-acquired infection—methods to perform hand hygiene
and WHO's five moments of hand hygiene; biomedical waste—concept of segregation of waste in
appropriate color-coded bags according to 2016 rule; vaccines and tables in parasitology chapters to
make understanding easy.
 Essentials of Practical Microbiology

We believe that this book would bring revolution in microbiology and help in modifying the pattern
of undergraduate teaching in practical classes across the country by incorporating newer diagnostics
and essential topics with the clinical touch.

her al
Apurba S Sastry Sandhya Bhat
[email protected] [email protected]
 Acknowledgments
ARRRRAD

We take this opportunity to extend our sincere gratitude and appreciation to the following people
without whom it would not have been possible to release the second edition of Essentials of Practical
Microbiology.
Hearty acknowledgments to our teachers, departmental staff, family members and others,
for their blessings and support.
ile We would like to sincerely thank Dr Deepashree R, Assistant Professor, Department of Microbiology,
JSS Medical College, Mysuru, Karnataka and Dr Anand B Janagond, Professor, Department of
Microbiology, S Nijalingappa Medical College, Bagalkot, Karnataka for their constant inputs during
manuscript preparation.
We express heart-felt gratitude to Dr Sujatha Sistla, Professor, Department of Microbiology, JIPMER,
for her guidance during manuscript preparation. | (Dr Apurba) am greatly indebted to you mam
for your timely support and guidance.
. We are extremely thankful to Dr Rakesh Aggarwal, Director, JIPMER, Puducherry for giving the
permission to revise this textbook.
We are grateful to Dr Renu G Boy Varghese, Director-Principal, Pondicherry Institute of Medical
Sciences (PIMS), Puducherry, for giving permission to revise this textbook.
We are extremely thankful to Dr Jharna Mandal, Additional Professor and Head, Department of
Microbiology, JIPMER, for her constant encouragement and support during the preparation of the
manuscript.
We would like to express our special word ofthanks to Dr Reba Kanungo, Dean Research, Professor
and Head, Department of Microbiology, Pondicherry Institute of Medical Sciences (PIMS). | (Dr
Sandhya) am truly grateful to you mam for your wholehearted support.
_ Other Faculty of Department of Microbiology, JIPMER—Dr Rakesh Singh (Additional Professor),
Dr Rahul Dhodapkar (Additional Professor), Dr Noyal M Joseph (Associate Professor), Dr Rakhi
Biswas (Associate Professor), Dr Nonika Rajkumari (Associate Professor) and Dr Maanasa Bhaskar
(Assistant Professor).
Other Faculty of Department of Microbiology, PIMS—Dr Shashikala (Professor), Dr Sheela
Devi (Professor), Dr Johny Asir (Professor), Dr Vivian Joseph P (Professor), Dr Sujitha E (Associate
Professor), Dr Anandhalakshmi (Associate Professor), Dr Arthi E (Associate Professor), Mrs Patricia
Anita (Associate Professor), Dr Meghna (Assistant Professor) and Mrs Desdemona Rasitha (Tutor).
Residents and postgraduates, JIPMER—Dr Ketan Priyadarshi, Dr Mugundan, Dr Sindhu,
Dr Gopichand, Dr Radha, Dr Rachna, Dr Sarumathi, Dr Anitha, Dr Kalpana, Dr Monika,
Dr Lakshmi, Dr Soundarya, Dr Kowshalya, Dr Lullu, Dr mola, Dr Symphonia, Dr Pheba, Ms Rosemary,
Ms Lakshmishree, Ms Gopika and Ms Neeshma.
Ramya and
10. HICC, JIPMER—Infection control nurses and other office staff such as Ms llaveni, Ms
Mr Venkat.
ihe For providing photographs—We are extremely thankful to all people/institutes/companies who
have agreed to provide valuable photographs.
Dr Abhishekh Mitra, Senior Resident, AlIMS, Raipur, for his inputs during manuscript preparation
of COVID-19 Chapter.
Uttar
Dr Anand Sastry, Department of Neuromedicine, Banaras Hind University (BHU), Varanasi,
Pradesh. For giving inputs during manuscript preparation of various CNS infections.
 Essentials of Practical Microbiology

14. Ex-residents from JIPMER: Dr Haritha M and others—for giving inputs in the correction of
content errors of first edition.
Each and every reader (faculty and students) from various parts of the country—for communicating
to us by email and other electronic media about the content correction and updates from time to
time.
Our friends—Dr Godfred, Dr Sadia, Dr Ramakrishna, Dr Mridula, Dr Srinivas Acharya, Dr Chaya,
Dr Manisha, Dr Ira, Dr Sreeja, Dr Wajid, Dr Kumudavathi and others.
We Family—Parents, brother, sister and other family members, maternal and paternal cousins and all
other well-wishers.

Special Acknowledgments to My Publishers

Jaypee Brothers Medical Publishers (P) Ltd, New Delhi, India
Shri Jitendar P Vij (Group Chairman)
Mr Ankit Vij (Managing Director)
Mr MS Mani (Group President)
Dr Madhu Choudhary (Publishing Head—Education): She has been a great support throughout the
manuscript preparation
Ms Pooja Bhandari (Production Head)
Ms Seema Dogra (Cover Visualizer)
Dr Astha Sawhney (Development Editor): Extremely dynamic, have lot of patience and available
24x7 to address to our queries
The Development Team: Mr Deepak Saxena (Typesetter), Mr Nitin Bhardwaj (Graphic Designer),
Ms Neelam, Mr Laxmidhar and Mr Vakil Khan (Proofreaders). These guys are simply outstanding in
their work. A special mention for Mr Deepak Saxena, we must say that he is the best operator of India
in medical publishing. The way Nitin Bhardwaj does the designing of photographs is extraordinary.
It is a treat for us to work with all of them. These guys are extremely workaholic and have a very
good team spirit. We salute them, for their professionalism.
Marketing heads from various zones: Mr Narendra Shekhawat (Vice President-Sales), Mr Venugopal
V (South Head), Mr Rishi Sharma (North Region Head), CS Gawde (Western Head) and Sandip Gupta
(Eastern Head).
Branch managers and Sales manager from various branches: Bengaluru branch (Ravi Kumar,
A Palani, E Venkatesh, Vivek Bhagawan), Chennai branch (Maran A [Adoption Head for South],
Dharani Kumar P, RK Dharani, Dharanidaran), Kochi branch (Sujeesh VS, Diffin Robin, Arun Kumar),
Hyderabad branch (Parimal Guha Neogy, Marthanda Sarma, Rajesh Malothu, Hamza Ali), Mumbai
branch (Sameer S Mulla), Nagpur branch (Rajesh Shrivas), Anmedabad branch (Ms Priyanka Kansara,
Dinesh Waghade), Delhi branch (Sujatha Puri), and Kolkata branch (Sanjoy Chakraborthy).
Lastly, we would like to keep in record that without the support of our son, parents (of both Dr
Sandhya and Dr Apurba) and other family members, it would have been impossible to continue the
spirit on, during the journey of the current edition. A special mention to our son (Master Adarsh), who
really helped us being very much cooperative. In fact, he was encouraging us to work for the book. We
deeply apologize to you as well as our parents (Mr Anooj Sastry and Ms Tarini Purohit), as we could not
give enough time and care during the manuscript preparation.

Apurba S Sastry
Sandhya Bhat
 Contents

Competency
Number (MCI
Curriculum)

Section 1: General Microbiology, Immunology and Hospital Infection Control

General Microbiology
he Introduction to Microbiology Department MI 1.1
Pop Microscopy MI 1.1, 1.2
3}, General Bacteriology
3.1. Morphology and Physiology of Bacteria MI 1.1
3.2. Specimen Collection and Transport MI 8.9, 8.10
3.3. Direct Detection 1: Simple Staining MI 1.1, 1.2
3.4. Direct Detection 2: Gram Staining MI 1.2
3.5. Direct Detection 3: Special Staining (Acid-Fast Stain, Albert MI 1.1, 1.2, 8.15
Stain) and Other Methods
3.6. Culture Media (Including Automated Culture) and Culture MI 1.1, 8.15
Methods
3.7. Identification of Bacteria (Conventional and Automated) MIG, 6.15
3.8. Antimicrobial Susceptibility
Test MI 1.6
3.9 Molecular Diagnosis MI 8.15
4. Laboratory Diagnosis ofViral Diseases MI 1.1, 8.10, 8.15
5) Laboratory Diagnosis of Parasitic Diseases MI 1.2, 8.10, 8.15
6. Laboratory Diagnosis of Fungal Diseases MI 1.1, 8.10, 8.15
Immunology
Tes Precipitation and Agglutination MI 8.15
8. ELISA, ELFA and Immunofluorescence MI 8.15
8), Western Blot, Rapid Tests and CLIA MI 8.15
Hospital Infection control
10. Standard Precautions: Hand hygiene and PPE MI 8.7
(Al. Transmission-based Precautions Mi 8.6, 8.7 101
25 Sterilization and Disinfection MI 1.5 108
(13. Biomedical Waste Management MI 8.6 117
14. Needle Stick Injury MI 8.6, 8.7 121
ils Environmental Surveillance MI 8.8 125

Note: Competency numbers highlighted bold are skill based competencies.

 Essentials of Practical Microbiology

Section 2: Systemic Microbiology (Infectious Diseases)

Bloodstream and Cardiovascular System Infections
16. Cardiovascular System Infections: Infective Endocarditis and Acute MI 2.3 133
Rheumatic Fever
We Bloodstream Infections MI 2.3, 8.15 138
18. Bacterial Infections of Bloodstream: Enteric Fever, Scrub Typhus, Ml 3.4, 8.10, 8.15 142
Brucellosis, and Leptospirosis
19. Viral Infections of Bloodstream: HIV/AIDS and Dengue MI 2.7, 8.15 eye:
20. Parasitic Infections of Bloodstream: Malaria, Visceral Leishmaniasis MI 2.6 162
and Lymphatic Filariasis
P\. Fungal Infections of Bloodstream: Systemic Candidiasis and Mitel cal 172
Systemic Mycoses
Gastrointestinal Infections
phe Bacterial Diarrhea: Shigellosis, Cholera and Others MI 3.2 176
fa. Viral Gastroenteritis: Rotaviruses and Others MI 3.2 184
24. Intestinal Protozoan Infections: Intestinal Amoebiasis, Giardiasis Mid.2, 32276215 186
and Coccidian Parasitic Infections

DS), Intestinal Helminthic Infections MI 1.2, 3.2, 8.15 192

e Intestinal Cestode Infections: Intestinal Taeniasis,
Hymenolepiasis and Others
e Intestinal Trematode Infections: Fasciolopsis buski, Schistosoma
mansoni and Others
e Intestinal Nematode Infections: Trichuriasis, Enterobiasis,
Ascariasis, Hookworm Infection, Strongyloidiasis
Hepatobiliary System Infections
26. Viral Hepatitis MI 3.8 203
Af. Parasitic Infections of Hepatobiliary System: Amoebic Liver NS ly sow 208
Abscess, Hydatid Disease and Others
Skin, Soft Tissue and Musculoskeletal System Infections
28. Staphylococcal Infections MI 4.2, 4.3, 1.2 211
2), Beta-hemolytic Streptococcal Infections MI 4.3, 1.2 215
30. Miscellaneous Bacterial Infections of Skin and Soft Tissues: MI 4.3, 1.2, 8.10, 220
Anaerobic Infections including Gas Gangrene, Leprosy and 8.15
Anthrax
Sil Viral Exanthems and Other Cutaneous Viral Infections: Herpes MI 4.3, 8.10, 8.15 227
Simplex, Measles, Rubella and Others
V2, Superficial and Subcutaneous Fungal Infections MI 4.3, 8.10, 8.15 236
Respiratory Tract Infections
38). Bacterial Pharyngitis: Streptococcus pyogenes Pharyngitis and MI 6.2, 8.10, 8.15 243
Diphtheria

Note: Competency numbers highlighted bold are skill based competencies.

 Contents Coe

34. Bacterial Pneumonia: Pneumococcal Pneumonia, Haemophilus Mi 6.3, 1.2, 8.10, 250
influenzae Pneumonia, Klebsiella pneumoniae Pneumonia and 8.15
Others .
35. Tuberculosis MI 6.3, 8.15 260
MI 6.3 268
36. Pseudomonas and Acinetobacter Infections
37. Viral Infections of Respiratory Tract: Influenza, COVID-19, Infectious MI 6.2, 6.3 272
Mononucleosis, and Others
MI 6.2, 6.3 281
38. Parasitic and Fungal Infections of Respiratory Tract: Paragonimiasis,
Zygomycosis, Aspergillosis, Pneumocystosis and Others
Central Nervous System Infections
MI 5.3, 1.2, 8.10, 288
39. Bacterial Meningitis
8.15
MI 1.1, 5.3, 8.15 295
40. Viral Meningitis and Viral Encephalitis (Enteroviruses including
Polio, Rabies, Japanese Encephalitis and Others)
MI 1.1, 5.1, 5.3, 302
41. Parasitic and Fungal Infections of Central Nervous System:
Neurocysticercosis, Free-livin g Amoebae Infections , Toxoplasm osis, 8.15
Cryptococcal Meningiti s and Others
Urogenital Tract Infections
MI 7.3, 8.10, 8.15 307
42. Urinary Tract Infections
MI 7.1, 7.2, 8.10, 316
43. Infective Syndromes of Genital Tract (Sexually-transmitted
Infections) : Syphilis, Gonorrhoea, Non-gonococcal Urethritis 8.15
(Chlamydia trachomatis), Vulvovaginitis (Trichomoniasis, Vaginal
Candidiasis) and Others
Miscellaneous
MI 8.11, 8.14 324
44. AETCOM in Microbiology
330
45. University Practical Examination
5}835)
Index

are skill based competencies.

Note: Competency numbers highlighted bold
 Further Reading

Bailey & Scott’s Diagnostic Microbiology, 14th Edition.

Harrison’s Principles of Internal Medicine, 20th Edition.
Indian Council of Medical Research, New Delhi, India.
Jawetz Melnick and Adelbergs’ Medical Microbiology, 28th Edition.
Koneman’s Color Atlas and Textbook of Diagnostic Microbiology, 7th Edition.
Kuby’s Immunology, 8th Edition.
Mackie and McCartney's Practical Medical Microbiology, 14th Edition.
National AIDS Control Organisation (NACO), India.
. Revised National Tuberculosis Control Program (RNTCP), India.
. Garcia's Diagnostic Medical Parasitology, 6th edition.
. Various national and international journals and other internet sources.
. World Health Organization (WHO).
=SOPMNDAUNAWN
WN
A . Centers for Disease Control and Prevention, Atlanta, USA.
_—
edd
 General Microbiology, Immunology
and Hospital Infection Control

_SECTION OUTLINE |
1. Introduction to Microbiology Department
2. Microscopy
3. General Bacteriology
5.15 Morphology and Physiology of Bacteria
ays, Specimen Collection and Transport
55) Direct Detection 1: Simple Staining
3.4. Direct Detection 2: Gram Staining
3.0; Direct Detection 3: Special Staining (Acid-Fast Stain, Albert Stain) and
Other Methods
3.0, Culture Media (Including Automated Culture) and Culture Methods
Se Identification of Bacteria (Conventional and Automated)
5:0: Antimicrobial Susceptibility Test
Spek Molecular Diagnosis
4. Laboratory Diagnosis of Viral Diseases
5. Laboratory Diagnosis of Parasitic Diseases
6. Laboratory Diagnosis of Fungal Diseases
7. Precipitation and Agglutination
8_ ELISA, ELFA and Immunofluorescence
9_ Western Blot, Rapid Tests and CLIA
10. Standard Precautions: Hand Hygiene and PPE
Th: Transmission-based Precautions
123 Sterilization and Disinfection
13: Biomedical Waste Management
14. Needle Stick Injury
is Environmental Surveillance
 Introduction to
Microbiology Department
ACR TA

This is the first practical class in the department

5. Nails should be kept clean and short. No artificial
of Microbiology. In this class, a formal interaction nails should be worn
between students and teachers (faculty/post- 6. Personal belongings and bags should not be
graduate students) takes place where: kept near work benches
¢ Introduction: The students and the teachers 7. No eatables should be brought into the
student's practical laboratory
introduce themselves 8. Donot put your fingers into your mouth during
~ Batch distribution: Students are divided the practical class hours
into batches and table teachers are allotted 9. Care of the microscope: Students are instructed
for each batch how to handle and take care of the microscope
(explained in Chapter 2)
“> Departmental tour: Students are taken for the 10. All the slides must be stained on staining rod
departmental tour. Activities/investigations over the wash basin
carried out in each section are briefed to the 11. Cap all the reagent bottles and place them in
students order in the rack after use
12. Incase of accidental spillage of reagents, inform
* General instructions to be followed (see
the table teacher immediately
below). 13. In case of accidental contact of cultures or
infectious materials, wash hands with the hand-
General Instructions wash solution immediately and inform the table
The following instructions to be followed during the teacher
practical classes are conveyed to the students: 14. While leaving the hall: Switch off the micro-
1. Students should wear clean ironed white coat scope, clean the lenses and discard the slide in
2. Students are warned about the importance of the discarding jar after use
attendance and coming to the class on time 15. Handwashing: Students must perform hand-
3. Practical record: Students must get the practical washing at the end of every practical class
record to the class every time; which must be before leaving the hall. Students are demon-
filled and signed by the table teacher on time strated how to perform handwash. The steps of
4. Girls should put their hair inside the white coat handwashing are demonstrated and explained
to avoid infection to them (Fig. 1.1)

Problem Solving Exercise

Rohan, a 2™ year MBBS student has attended a Explanation
Microbiology practical session on Gram staining. After No, Rohan’s decision of going to hostel and then
the class got over, he wanted to do handwashing. performing hand wash was wrong. As hands are
However, as there was a long queue of 70 students at contaminated with potential microorganisms in the
the hand wash station, he decided to go to hostel and Microbiology laboratory, students must immediately
perform handwashing. perform handwashing after the practical class.
1. Opine, if the decision of Rohan was correct? For answers to the other questions, refer text.
2. What are the steps to perform handwashing?
3. List the instructions to be followed by a 2"° year
MBBS student who has attended a Microbiology
practical session?
oO SECTION 1 © General Microbiology, Immunology and Hospital Infection Control

How to handrub?
With alcohol-based formulation

Apply a palmful of the product in a

cupped hand and cover all surfaces

Sg

Rub hands Right palm over left dorsum

palm to palm interlaced fingers and vice

Back of fingers to Rotational rubbing of

opposing palms with left thumb clasped in
fingers interlocked right palm and vice versa |

...once dry, your hands are safe

Fig. 1.1: Steps of handrub and handwash.

Source: World Health Organization,
 Microscopy

A microscope is an instrument used to see m Unaided human eye is about 0.2 mm (200
objects that are too small to be seen by the naked ym)
eye. a Light microscope is about 0.2 um
ws Electron microscope is about 0.5 nm.
@ MICROSCOPE CARE Resolution depends on refractive index of
the medium. Oil has a higher refractive
Proper care and maintenance of microscope can index than air; hence use of oil enhances the
extend its life by many years. resolution power of a microscope.
¢ Handle with care: While carrying the micro- 2. Good contrast: This can be further improved
scope, hold it in both the hands, one hand
by staining of the specimen
supporting the base and the other holding the
3. Good magnification: This is achieved by use
metal support arm. Do not pick it up by the
of lenses:
stage, as this can cause misalignment
m Objective lenses: Scanning (4X), low
* Keep lenses clear of slides: While adjusting power (10X), high power (40X) and oil
the microscope, lower the objective lens down immersion (100X)
carefully till the focus plane, without allowing = Ocular lens with a magnification power of
the lens to touch the slide
10X.
* Clean lenses: Ensure the lenses are cleaned
Total magnification of a field is the product of
immediately after use, especially if using the magnification of objective lens and ocular
immersion oil. Use only lens paper and lens
lens.
cleaner (not solvents). Rub the lens gently ina
Scanning field (40X)
circular motion to remove sticky residue
= Lowpower field (100X)
“? Care of the bulb: Turn off the bulb after use. = High power field (400X)
When turning the microscope on and off, use
= Oil immersion field (1000X).
the switch; not directly the power source. Do
not switch the microscope on/off while using
full light intensity. Never touch the bulb @ MICROMETRY
“ Store: Cover when not in use and store in a Micrometry refers to the measurement of
clean, dry place preferably in a cabinet. Do dimensions of the microorganisms under a
not remove eyepieces. microscope by using micrometers. There are
two micrometers, namely (1) ocular micrometer,
§ PROPERTIES OF A MICROSCOPE and (2) stage micrometer (Figs 2.1A and B).
“ Ocular micrometer is a circular glass disk that
A good microscope should have at least three
fits into the eyepiece of the microscope. It has
properties:
100 equally spaced divisions (Marked as 0-10,
1. Good resolution: Resolution power refers
to the ability to produce separate images of at every 10 division interval) (Fig. 2.1A)
closely placed objects so that they can be “ The stage micrometer is clipped to the stage
distinguished as two separate entities. The of the microscope. In the center of the stage
resolution power of: micrometer, a known 1 mm distance is divided
 SECTION 1 © General Microbiology, Immunology and Hospital Infection Control

In ocular micrometer, there

are = divisions marked as 0 to 10
3. AMS" 6 -7= oe S910
inuoooanoao 3a 4 525.6)
Boye ea

ihai yagi

Stage micrometer

Figs 2.1A and B: (A) Principle of micrometry [The divisions of ocular micrometer are superimposed on graduations of
stage micrometer. Distance between two graduations of stage micrometer equals to 37 divisions of ocular micrometer.
Therefore, one division of ocular micrometer (i.e., calibration factor) measures to 100/37 = 2.7 um]; (B) Stage micrometer
is removed. Slide containing bacteria is focused. Size of the bacteria is determined by number of divisions of ocular
micrometer it corresponds x 2.7 um.

into 10 divisions (0.1 mm); each is further HBTYPE OF MICROSCOPES

divided into 10 graduations of 0.01 mm or 10
uum each The following types of microscopes are in use
* Calibration: The graduations on both now:
micrometers are superimposed on each
+.a
Bright-field or light microscope
other. Then the graduations on the ocular Dark-field microscope
© Ws Phase-contrast
+, microscope
microscope are calibrated against the standard
graduations on the stage micrometer as given
+“
Fluorescence microscope
below (Fig. 2.1A):
+ >*
Electron microscope.

Calibration factor for one division on ocular Bright-Field or Light Microscope

micrometer (in um) =
Known distance between two lines on stage The bright-field or light microscope forms a dark
micrometer, i.e., 100 um image against a brighter background, hence the
Number ofdivisions on ocular micrometer name.
that lies between two lines of stage micrometer
Therefore in the above example (Fig. 2.1), one Structure
division of ocular micrometer (i.e., calibration The parts in a bright-field microscope are
factor) measures to 100/37 = 2.7 um.
divided into three groups (Fig. 2.2
Hence size of the bacteria = The number of
divisions of ocular micrometer it corresponds to
x 2.7 um
Mechanical Parts
** Base: It holds various parts of microscope,
** Measurement: After calibration, the ocular such as the light source, the fine and coarse
micrometer is used to measure the size of adjustment knobs
various microbes, such as length, breadth, ** C-shaped arm: It is used for holding the
and diameter. First, count the number of microscope and it connects the eyepiece to
divisions occupied in ocular micrometer by the objective lens
the organism. Then, multiply this number by ** Mechanical stage: The arm bears a stage
the calculated calibration factor. This value with stage clips to hold the slides and
indicates the size of the organism (Fig. 2.1B). the stage control knobs to move the slide
 CHAPTER2 © Microscopy

C-shaped Ocular lens

arm
Coarse
adjustment
Revolving
nosepiece
Objective Both reflected
lens and unreflected Only light reflected
Stage light pass by the specimen is
through the captured by the
Iris diaphragm objective objective lens
Condenser
Light source Unreflected light

Opaque disk

(ID areeae
(0)
Fig. 2.2: Bright-field microscope.
=@ &
Source: Nikon Alphaphot (with permission).
Light Light — Dark-field
Daricfield condenser
during viewing. It has an aperture at the center
to permit light to reach the object from the Fig. 2.3: Light pathways of bright-field microscopes and
dark-field microscopes.
bottom.

Magnifying Parts
magnified by the ocular lens to produce the final
“ Ocular lens: The arm contains an eyepiece magnified virtual image (Fig. 2.3).
that bears an ocular lens of 10X magnification
power. Microscopes with two eyepieces are Dark-field Microscope
called as binocular microscopes
Principle
* Objective lens: The arm also contains a
revolving nosepiece that bears three to five In dark-field microscope, the object appears
objectives with lenses of differing magnifying bright against a dark background. ‘This is made
power (4X, 10X, 40X and 100X). possible by use of a special dark-field condenser
(Fige2e3))
Illuminating Parts “ The dark-field condenser has a central opaque
“+ Condenser: It is mounted beneath the stage area that blocks light from entering the
which focuses a cone of light on the slide objective lens directly and has a peripheral
“ Iris diaphragm: It controls the light that annular hollow area which allows the light
passes through the condenser to pass through and focus on the specimen
“+ Light source: It may be a mirror or an electric obliquely
bulb “* Only the light which is reflected by the
“ Fine and coarse adjustment knobs: They specimen enters the objective lens, whereas
sharpen the image. the unreflected light does not enter the
objective. As a result, the specimen is brightly
Working Principle illuminated, but the background appears
dark.
The rays emitted from the light source pass
through the iris diaphragm and fall on the Applications
specimen. The light rays passing through the
Dark-field microscope is used to:
specimen are gathered by the objective and a
magnified image is formed. This image is further * Identify the living, unstained cells
 SECTION 1 @ General Microbiology, Immunology and Hospital Infection Control

Undeviated ray Phase plate

%)

Bacterium

Ray deviated by Deviated ray is Deviated and

specimen is 1/4 1/2 wavelength undeviated rays
wavelength out out of phase cancel each
of phase other out
Fig. 2.4: Dark ground microscopic picture demonstrating
Fig. 2.5: Principle of phase-contrast microscope.
spirally coiled bacteria (spirochete).
Source: Public Health Image Library, ID# 2043; Centers for Disease
Control and Prevention (CDC), Atlanta (with permission).

“+ Identify thin bacteria like spirochetes which

cannot be visualized by light microscopy
(Fig. 2.4).

Phase-contrast Microscope
Contrast is an important property of a
microscope to visualize the objects. Contrast can
be enhanced by staining the specimen. However,
as staining kills the microbes, the properties of
living cells cannot be studied. Fig. 2.6: Phase-contrast microscopic picture demonstrat-
The phase-contrast microscope is used to ing Naegleria fowleri trophozoites (free-living amoeba)
Source: Centers for Disease Control and Prevention (CDC), Atlanta
visualize the living cells by creating difference in
(with permission)
contrast between the cells and water. It converts
slight differences in refractive index and cell
density into easily detectable variations in light *+ The phase ring is constructed in such a way
intensity. that the undeviated light passing through it is
advanced by one-fourth of a wavelength, the
Principle deviated and undeviated waves will be about
The condenser is similar to that of dark-field half wavelength out of the phase and will
microscope, consists of an opaque central area cancel each other when they come together
with a thin, transparent ring which produces a to form an image (Fig. 2.5)
hollow cone of light. * The background, formed by undeviated light,
“+ As this cone of light passes through a cell, is bright, while the unstained object appears
some light rays are bent due to variations dark and well-defined (Fig. 2.6).
in density and refractive index within the
The light rays go through — condenser —> specimen
specimen and are retarded by about one-
(e.g., bacterium) — phase ring > objective lens >
fourth of a wavelength (Fig. 2.5) ocular lens.
* The undeviated light rays strike a phase ring
in the phase plate, (a special optical disk
located in the objective), while the deviated Applications
rays miss the ring and pass through the rest Phase-contrast microscopy is especially useful
of the plate for studying:
 CHAPTER2 © Microscopy

¢* Microbial motility * The exciting rays then get reflected by a

ee Determining the shape of living cells
2,
dichromatic mirror in such a way that they
°,
~~ Detecting microbial internal cellular com- fall on the specimen, which is stained with
ponents, such as the cell membrane, nuclei, fluorescent dye
mitochondria, spindles, chromosomes, Golgi “ The fluorescent dye absorbs the exciting rays
apparatus, endospores and inclusion bodies; of short wavelength, gets activated and in turn
which become clearly visible because they emits fluorescent rays of higher wavelength
have refractive indices markedly different * A barrier filter positioned after the objective
from that of water. lens removes any remaining UV light, which
could damage the viewer’s eyes, or blue and
Fluorescence Microscope violet light, which would reduce the image’s
The “fluorescence microscope” refers to any contrast.
microscope that uses fluorescence property to
Applications of Fluorescence Microscopy
generate an image.
Epifluorescence microscope: It is the simplest
Principle form of fluorescence microscope, which has the
following applications:
When fluorescent dyes are exposed to ultraviolet
« Auto-fluorescence: Certain microbes directly
(UV) rays, they become excited and are said to
fluoresce, i.e., they convert this invisible, short
fluoresce when placed under UV lamp, e.g.,
wavelength rays into light of longer wavelengths Cyclospora (a protozoan parasite)
(i.e., visible light) (Fig. 2.7). “ Microbes coated with fluorescent dye:
Certain microbes fluoresce when they are
“+ The source of light may be a mercury-vapor
stained by fluorochrome dyes
lamp, which emits rays that pass through an
excitation filter = Acridine orange dye is used for the
* The excitation filter is so designed that it detection of parasites, such as Plasmodium
and filarial nematodes by a method
allows only short wavelength UV light (about
called as quantitative buffy coat (QBC)
400 nm) to pass through, blocking all other
examination
long wavelength rays
= Auramine phenol is used for the detection
of tubercle bacilli (Fig. 2.8).

f-
* Immunofluorescence: It uses fluorescent
dye-tagged immunoglobulins to detect cell
surface antigens or antibodies bound to
Eyepiece cell surface antigens. There are two types—
Barrier filter

tt
Se Dichromatic
mirror
Excitation
filter
Objective

Exciting Emitted
fluorescence
light
light Fig. 2.8: Tubercle bacilli seen under fluorescence

VL PCIE microscope.
Source: Department of Microbiology, JIPMER, Puducher
ry
(with permission).
Fig. 2.7: Principle offluorescence microscope.
 SECTION 1 © General Microbiology, Immunology and Hospital Infection Control

direct and indirect immunofluorescence test Transmission Electron Microscope

(described in detail in Chapter 8). Specimen Preparation

Electron Microscope Very thin specimens (20-100 nm thickness) are

suitable for EM. To prepare the thin specimen,
An electron microscope (EM) uses accelerated
cells are first fixed by using glutaraldehyde
electrons as a source of illumination. It has
or osmium tetroxide, then dehydrated with
a much better resolving power than a light
organic solvents (e.g., acetone) and then finally
microscope; hence, it can reveal the details of
embedded in plastic polymer to form a solid
flagella, fimbriae and intracellular structures of
block. Followed which specimen is cut into thin
a cell. EMs are of two types:
slices by ultramicrotome knife and mounted on
1. Transmission electron microscope (most
a metal slide (copper).
common type) (Fig. 2.9)
2. Scanning electron microscope (SEM). Electron Pathway
Differences between light microscope and EM
Electrons are generated by electron gun, which
are listed in Table 2.1.
travel in high speed. The medium of travel in
EM should be a fully vacuum path because in
air path, electrons can get deflected by collisions
with air molecules.
“+ Electron pass through a magnetic condenser
and then bombard on the thin-sliced
specimen mounted on the copper slide
** The specimen scatters electrons passing
through it, and then the electron beam
is focused by magnetic lenses to form an
enlarged, visible image of the specimen on a
fluorescent screen (Figs 2.10 and 2.11).
Various measures are followed to increase the
contrast of EM, such as:
** Staining with heavy metal salts, such as lead
citrate and uranyl acetate
* Negative staining with heavy metals, such as
phosphotungstic acid
Final image on
photographic film
Fig. 2.9: Transmission electron microscope.
Source: David J Morgan/Wikipedia.

Table 2.1: Differences between light microscope and Projector

electron microscope. lens magnet

Light Electron
microscope microscope
Highest practical About Over 100,000
Objective
magnification 1,000-1,500
lens magnet
Best resolution 0.2 um 0.5nm
Specimen ——
Radiation source Visible light Electron beam
Condenser
Medium of travel Air High vacuum
lens magnet
Specimen mount Glass slide Metal grid
Electron gun
(usually copper)
Type of lens Glass Electromagnet
Fig. 2.10: Principle of transmission electron microscope.
 CHAPTER 2 © Microscopy

** Shadowing: Specimen is coated with a thin

film of platinum or other heavy metal at 45°
angle so that the metal strikes the micro-
organism on only one side
* Freeze-etching technique is used to view
internal organelles within the organisms
giving a three-dimensional view.

Scanning Electron Microscope

Scanning electron microscope has been used
to examine the surfaces of microorganisms
in great detail. It has a resolution of 7 nm or
less. The SEM differs from other EM in pro-
Fig. 2.11: Rotavirus (electron micrograph). ducing an image from electrons emitted by an
Source: Public Health Image Library, /ID# 15194/Dr Erskine L object’s surface rather than from transmitted
Palmer/Centers for Disease Control and Prevention (CDC), Atlanta
(with permission). electrons.

Exercise e
Solving Exercis
[Problem Solving
1. Amicroscopic photograph has been provided (Fig. Explanation
2.8). Which microscope is used for visualization? The microscopic photograph provided in Figure 2.8
Discuss its principle and applications. belongs to an image visualized under fluorescence
_2. List the various parts of bright-field microscope microscope. For answer to the other questions, refer
and discuss their functions. text.
3. How will you take care of a microscope?
 General Bacteriology:
Morphology and Physiology of Bacteria 31
@ MORPHOLOGY OF BACTERIA
Problem Solving Exercise 1
Morphology of Bacteria Q Figure 3.1.3A: Reveals Gram-negative cocci
Gram stained smears of the organisms have been arranged in pair, lens-shaped, capsulated (e.g.,
focused in Figs 3.1.2A, 3.1.3A and 3.1.4A. Identify meningococcus).
the probable organism based on the morphological Q Figure 3.1.4A: Reveals Gram-positive bacilli:
appearance in the Gram stain. (A) arranged in chain, sporing (e.g., Bacillus).

Explanation
Q Figure 3.1.2A: Reveals Gram-positive cocci
arranged in cluster (e.g., Staphylococcus).

Size and Shape of Bacteria

Bacteria are measured in micrometer (1 um = 10°
mm). Most of the bacteria of medical importance
generally measure 0.2-1.5 ttm in diameter and
O400E
Clusters Chains Tetrad | Octate
3-5 um in length. Depending on their shape, Gram-positive cocci
bacteria are classified into:
** Cocci (singular coccus, from kokkos, meaning
berry) are oval or spherical cells
* Bacilli (singular Bacillus, meaning rod-
shaped).
Cocci are arranged in groups (clusters), pair Gram-negative cocci
or chains. Similarly, bacilli can be arranged
singly or in chain or pair; some bacilli are curved, re | \y)
comma shaped, cuneiform shaped (Table 3.1.1
and Fig. 3.1.1). a ./ iO N
Both cocci and bacilli are further classified Bacilli Comma shaped Spirally coiled
based on Gram-staining property into (Table Gram-negative bacilli Spirochete
3.1.1 and Figs 3.1.1 to 3.1.5):
Fig. 3.1.1: Different morphology of bacteria and Gram-
** Gram-positive cocci (Figs 3.1.2A to C) staining property.
** Gram-negative cocci (Figs 3.1.3A and B)
* Gram-positive bacilli (Figs 3.1.4A to C) * Spirochetes (Treponema and Leptospira): Thin
* Gram-negative bacilli (Figs 3.1.5A to C) spirally coiled bacilli (Fig. 3.1.5D)
However, there are some bacteria that * Mycoplasma (cell wall-deficient free living
are weakly Gram stained and hence need bacteria)
special stains for their demonstration, such * Rickettsiae and chlamydiae are obligate
as: intracellular bacteria.
 CHAPTER 3.1 © General Bacteriology: Morphology and Physiology of Bacteria

Table 3.1.1: Classification of bacteria depending on Contd...

their morphology and Gram-staining property. Bacterial
Palisade pattern Diphtheroids
Branched and filamentous form Actinomyces and
|
Nocardia
Cluster Staphylococcus
Chain Streptococcus
Pleomorphic (various shapes) Haemophilus,
Pairs, lanceolate shaped Pneumococcus Proteus
Pair or in short chain, spectacle Enterococcus Bordetella pertussis
Thumb print appearance
shaped
Comma shaped (fish in stream Vibrio cholerae
Tetrads Micrococcus
appearance)
Octate Sarcina Campylobacter and
Curved
Helicobacter
Pairs, lens shaped Meningococcus
Pairs, kidney shaped Gonococcus Chain Streptobacillus
itiv
sa Spirally coiled, flexible Spirochetes
Chain (bamboo stick appearance) Bacillus anthracis Spirillum
Rigid spiral forms
Chinese letter or cuneiform Corynebacterium
Bacteria that lack cell wall Mycoplasma
pattern diphtheriae
Contd...

(e.g., Staphylococcus); (B) Chain (e.g., Streptococcus);

Figs 3.1.2A to C: Gram-positive cocci arranged in: (A) Cluster
(C) Pair, lanceolate-shaped (e.g., pneumococcus).
Medical Sciences, Puducherry (with permission).
Source: Department of Microbiology, Pondicherry Institute of

JA\ Pair, lens-shaped, capsulated

ID# /2108, Centers for Disease

n (CDC), Atlanta; (B) Public Health Image Library,
Source: (A) Centers for Disease Control and Preventio n).
Control and Preventio n (CDC), Atlanta (with permissio
 SECTION1 @ General Microbiology, Immunology and Hospital Infection Control

: ' . A |e nc, gale S37 este

IN. i : a bd eos S | ci {tl aed, fer aa ee. Ew) e a “

Figs 3.1.4A to C: Gram-positive bacilli: (A) Arranged in chain, sporing (e.g., Bacillus); (B) With terminal spore, drumstick
appearance (e.g., Clostridium tetani); (C) Branching, filamentous (e.g., Actinomycetes).
Source: (A) Department of Microbiology, JIPMER, Puducherry (with permission); (B) Public Health Image Library, ID# 6373, Centers for
Disease Control and Prevention (CDC), Atlanta (with permission); (C) Dr Isabella Princess, Apollo Hospitals, Chennai (with permission).

6 ae Vel KK 30
ee
qe’ ae Sy
Fae etal nS
‘ ‘Ss Tye i3|al ONY

Figs 3.1.5A to C: Gram-negative bacilli: (A) Arranged singly (e.g., Escherichia coli); Fig. 3.1.5D: Spirochete (spirally
(B) Pleomorphic (e.g., Haemophilus influenzae); (C) Comma-shaped (e.g., Vibrio cholerae). coiled bacilli) in dark ground
microscopy.
Source: (A and B) Department of Microbiology, Pondicherry Institute of Medical Sciences, Puducherry (with permission); (C and D)
(C) ID#:5324; (D) ID#:2043, Public Health Image Library, Centers for Disease Control and Prevention (CDC), Atlanta (with permission).

HB PHYSIOLOGY OF BACTERIA
| Problem
Problem Solving
Solving Exercise2
Exercise 2 |
Bacterial Growth Curve Explanation
A bacterium is inoculated intoa suitable liquid culture — Fig. 3.1.6 depicts a bacterial growth curve, which is
medium and incubated. The bacterial count of the obtained when bacterial count of the liquid culture
liquid culture is determined at different intervals and is determined at different intervals and plotted in
plotted in relation to time (Fig. 3.1.6). Answer the __ relation to time.Ithas four phases—lag, log, stationary
following questions. and decline phases.
1. Inwhich phase ofthis curve, the bacterium obtains 1. The bacterium obtains maximum size at the end of
maximum size? lag phase.
2. Which phase of this curve is ideal for performing 2. Log phase is ideal for performing Gram staining.
Gram staining? 3. The bacterium undergoes sporulation in stationary
3. In which phase, the bacterium undergoes phase.
sporulation? 4. Involution forms are seen in decline phase.
4. In which phase of this curve, involution forms are
seen?
 CHAPTER 3.1 © General Bacteriology: Morphology and Physiology of Bacteria

Bacterial Growth Curve 1. Lag phase: It is the initial period, where

bacteria do not start multiplying, but take
When a bacterium is inoculated into a suitable
some time to build-up enzymes and me-
liquid culture medium and incubated, its
tabolites
growth follows a definite course. When bacterial
2. Log phase: In this phase, bacteria divide
count of such culture is determined at different
exponentially
intervals and plotted in relation to time, a
3. Stationary phase: Here, the bacteria
bacterial growth curve is obtained comprising start dying due to exhaustion of nutrients
of four phases (Fig. 3.1.6 and Table 3.1.2).
and accumulation of toxic products. The
number of progeny cells formed is just
enough to replace the number of cells that
die
4. Decline phase: Gradually, the bacteria stop
dividing completely, while the cell death
continues.
Stationary
phase Factors Affecting Growth of Bacteria
cells
of
Number There are several environmental factors that
affect the growth of the bacteria:

Oxygen
Time On the basis of their oxygen requirements,
Fig. 3.1.6: Bacterial growth curve. classification of bacteria is shown in Table 3.1.3.

Table 3.1.2: Various phases of bacterial growth curve.

Yes Yes No
Bacteria divide No
No Yes Yes
Bacterial death No
Raises Raises Flat
Total count Flat
Raises Flat Falls
Viable count Flat
Uniformly stained, Gram variable Produce
Special features Accumulation
Metabolically active, Produce: involution forms
of enzymes and
metabolites, attains Small size Granules, spores, exotoxin,
maximum size antibiotics, bacteriocin

requirement.
Table 3.1.3: Classification of bacteria based on their oxygen

Pseudomonas, Mycobacterium,
Obligate aerobes Grow only in the presence of oxygen
Bacillus, Nocardia
(Figs 3.1.7A and B-1)
Escherichia coli,
Facultative anaerobes They are aerobes that can also grow anaerobically
Staphylococcus aureus
(Figs 3.1.7A and B-2)
Campylobacter, Helicobacter,
Microaerophilic bacteria Grow in presence of low oxygen tension (51 0% O.)
Mycobacterium bovis
Most of the clostridia spp.
Obligate anaerobes Grow only in the absence of oxygen
(Figs 3.1.7A and B-3)
Clostridium histolyticum
Aerotolerant anaerobes Tolerate oxygen for some time, but do not use it
i SECTION 1 ©@ General Microbiology, Immunology and Hospital Infection Control

Propioni-
bactenum
(no growth)
Pseudomonas
(no growth) | ‘Wf
y/

Figs 3.1.7A and B: (A) Aerobic incubation; (B) Anaerobic incubation.

Source: Department of Microbiology, Pondicherry Institute of Medical Sciences, Puducherry (with permission).

Table 3.1.4: Classification of bacteria based on

Organisms that require higher amounts of TEM BSISDIES (EQUUS aria

carbon dioxide (5-10%) for growth are called eee Cee
as capnophilic bacteria. Examples include — Psychrophiles Grow best at Most of the
Brucella abortus, Streptococcus pneumoniae, etc. temperatures —_saprophytes (e.g.,
below 20°C Pseudomonas)
Mesophiles Grow within a Most of the
: ‘ ; temperature pathogenic
Most of the pathogenic bacteria grow optimally range 25°C and _ bacteria (eg.,
at 37°C. However, the optimal temperature range 40°C Staphylococcus
varies with different bacterial species. According aureus)
to temperature requirements, bacteria can be ==Thermophiles Growatahigh Geobacillus
grouped as given in Table 3.1.4. temperature stearothermophilus
range of
55-80°C

Most pathogenic bacteria grow between pH 7.2

79

and pH 7.6. Very few bacteria (lactobacilli) can

grow at acidic pH (below pH 4), while bacteria, Bacteria (except phototrophs) grow well in
such as Vibrio cholerae are capable of growingat darkness. Photochromogenic mycobacteria
alkaline pH (8.2-8.9). produce pigments only on exposure to light.

lap ieeoccus \ i/ Staphylococcus, Geobacillus ’ a : ‘

(no growth)
th j aureus
: I ij Staphylococcus, upastearother
| eee ]
/ Geobacillus -
7 stearothermophiltis
(no growth)
*
ay

n aise err : seudomonas

Mi Ss \ no)growth

seudomonas
chai

Figs 3.1.8A to C: (A) Psychrophiles (10°C); (B) Mesophiles (37°C); (C) Thermophiles (55°C).
Source: Department of Microbiology, Pondicherry Institute of Medical Sciences, Puducherry (with permission)
 General Bacteriology:
Specimen Collection and Transport
OLN AOLRGSSROSROST

B INTRODUCTION Laboratory diagnosis of bacterial infections

comprises of several steps—specimen collection,
Laboratory diagnosis of bacterial infections is direct detection, culture, identification and
useful for the following purposes: antimicrobial susceptibility test, serology and
“+ Identification: To identify the causative molecular methods (Table 3.2.1).
bacterial agent responsible for the disease
Table 3.2.1: Overview of laboratory diagnosis of
“ Treatment: To provide accurate antimicrobial bacterial infections.
therapy
1. Specimen collection
~ Surveillance purpose: To assess the dis-
2. Direct detection
ease burden in the community by estimat- > Microscopy: Gram stain, acid-fast stain, Albert
ing the prevalence and incidence of the stain, histopathological staining, dark ground,
infections phase-contrast and fluorescence microscopy
> Antigen detection from clinical specimen
* For outbreak investigation, e.g., diphtheria
+
+,

> Molecular diagnosis: Detecting bacterial DNA or

outbreaks in the community, MRSA
RNA from clinical specimen
(methicillin-resistant S. aureus) outbreaks in 3. Culture
the hospitals > Culture media
¢ To start PEP (post-exposure prophylaxis): > Culture methods
> Colony morphology, smear and motility testing
Useful in infectious diseases, such as, anthrax
4. Identification
and plague > Biochemical identification
* To initiate appropriate infection control > Automated identification methods
measures: For example, contact precaution . Antimicrobial susceptibility testing
for MRSA infection, droplet precaution for Serology
precaution for . Molecular methods
diphtheria and airborne
. Typing methods
ONAW
tuberculosis (Chapter 11).

@ SPECIMEN COLLECTION
Exercise e _|
Solving Exercis
[problem Solving
Specimen Collection Q Urine specimen for microscopy and culture
Q Stool specimen for microscopy and culture
A young patient with history of high grade fever
with chills for two days, presents to the out patient Explanation
department. Upon detailed clinical examination, The specimen collection has been explained in the
clinician decides to admit him and requests for his following Chapters
blood and urine culture and susceptibility testing. Q Blood collection for culture: Refer Chapter 17
After two days of hospitalization he develops diarrhea Q Urine specimen collection for microscopy and
with two episodes of vomiting. His stool specimen culture: Refer this Chapter and Chapter 42
was collected and sent for culture. Discuss the method Q Stool specimen collection for microscopy and
of collection of the following specimens for culture. culture: Refer this Chapter and Chapters 5 and 22.
Q Blood for blood culture
a) SECTION 1 ©@ General Microbiology, Immunology and Hospital Infection Control

Specimen collection depends upon the type Table 3.2.2: Types of infections and various specimens
collected.
of underlying infections (Table 3.2.2). The
proper collection of specimen is of paramount Type of Specimens collected
importance for the isolation of the bacteria in infections
culture. Bloodstream Paired blood culture specimens
infection, sepsis, e Collected aseptically by two-step
General Principles endocarditis disinfection of skin; first with
The following general principles should be alcohol followed by chlorhexidine
e 8-10 mL of blood (for adults)
followed while collecting the specimen: collected in blood culture bottles
+,
“
Standard precautions should be followed
for collecting and handling all specimens Infectious e Blood (2 mL/investigation)
diseases e Collected by minimal asepsis
(Chapter 10 for details) requiring (one-step skin disinfection with
Before antibiotics start: Whenever possi- serology alcohol)
ble, culture specimens should be collected e Collected in vacutainer
prior to administration of any antimicrobial Diarrheal Stool (mucus flakes), rectal swab
agents diseases
Contamination with indigenous flora should
Meningitis Cerebrospinal fluid (CSF)
be avoided, especially when collecting urine
and blood culture specimens Infections of Sterile body fluids, e.g., pleural fluid,
other sterile synovial fluid, peritoneal fluid
Swabs are though convenient but considered body area
inferior to tissue, aspirate and body fluids
Skin and soft Pus or exudate, wound swabs,
Container: Specimens should be collected
tissue infections aspirates from abscess and tissue
in sterile, tightly sealed, leak proof, wide- bits
mouth, screw-capped containers
Anaerobic Aspirates, tissue specimens, blood
Labeling: All specimens must be appropri-
infections and sterile body fluids, bone marrow
ately labeled with name, age, gender, name (swabs, sputum not satisfactory)
of the treating physician, clinical diagnosis,
Upper Throat swab with membrane over
antibiotic history, type of specimen, and respiratory tract the tonsil, nasopharyngeal swab,
desired investigation name infections pernasal swab
Rejection: Specimens grossly contaminated
Lower Sputum, endotracheal aspirate,
or compromised or improperly labeled may respiratory tract bronchoalveolar lavage (BAL),
be rejected (see highlight box) infections protected specimen brush (PSB) and
If anaerobic culture is requested, proper lung biopsy
anaerobic collection containers with media Pulmonary e¢ Sputum—early morning and spot
should be used tuberculosis ¢ Collected in well-ventilated area
Specimen should not be sent in container ¢ Gastric aspirate for infants
containing formalin for microbiological Urinary tract Midstream urine
analysis. infections Suprapubic aspirated urine
Catheterized patient—collected
Specimen rejection criteria from the catheter tube, after
Microbiology samples that do not meet the clamping distally and disinfecting;
appropriate sample and the test request requirements not from urobag
need to be rejected, so as to prevent inaccurate data Genital Urethral swab, cervical swab—for
and to ensure the safety of patients and laboratory infections urethritis
personnel. Reasons for sample rejection may include Exudate from genital ulcers
the following:
Q Improperly labeled or unlabeled sample Eye infections Conjunctival swabs
Q Incomplete specimen-related or clinical infor- Corneal scrapings
mation on the sample and/or on the requisition Aqueous or vitreous fluid
form Ear infections Swabs from outer ear
Contd...
Aspirate from inner ear
 CHAPTER 3.2 © General Bacteriology: Specimen Collection and Transport

Contd... “+ CSF: CSF is collected by lumbar puncture

at interspace L3-L4, or L4-L5. The site is first
Q Sub-optimal sample, i.e., leaking urine and/or stool
containers, insufficient quantity or inappropriate
disinfected with antiseptics similar to that
sample for the test requested used for blood culture. After collection, CSF
Q Duplicate microbiology samples received on the is then divided into three sterile tubes (2 mL
same day, i.e., multiple stool, sputum samples each) for three diagnostic laboratories
Q Sample delayed in transit more than the accepted = Biochemistry (for total protein and
limit.
glucose)
= Bacteriology (culture and susceptibility,
COLLECTION TECHNIQUE OF Gram staining, antigen detection)
VARIOUS SPECIMENS m Cytology (for cell count).
“+ Body fluids from other sterile sites such as
Blood Specimen ascitic fluid, pleural fluid, peritoneal fluid,
In clinical microbiology laboratories, blood and synovial fluid specimens are collected
collection is indicated either for blood culture by percutaneous aspiration under aseptic
or for serological tests. precautions with syringe and needle
* Body fluid specimens can also be
Collection of Blood Specimen for Culture inoculated at bedside directly on to BacT/
Blood culture is regarded as one of the most ALERT bottles for culture. In these case, a
important culture investigation performed by portion of the specimen should be aliquoted
clinical microbiology laboratory. As there is a separately for Gram stain
high risk of contamination with skin flora, sterile “+ Transport: Specimens should be transported
aseptic precautions must be taken for collection immediately (within 15 min) to the laboratory;
and skin is disinfected with two agents—70% If any delay is expected, body fluid specimens
alcohol followed by chlorhexidine. Itis discussed can be stored at 37°C (in incubator) or at room
in detail in Chapter 17. temperature; but never refrigerated.

Collection of Blood Specimen for Serology Sterile Universal Specimen Container

Collection of blood specimen for serological (Fig. 3.2.1)
(e.g., virology or immunology) diagnostic tests These are designed for collecting biological
specimens, including urine, stool, sputum, peritoneal
is similar to as done for culture; except that skin
exudate, joint fluid, biopsy specimen, sterile body
disinfection is performed by only one agent fluids and aspirates for microbiological culture and
i.e., 70% isopropyl alcohol. This is because susceptibility testing. It is very convenient for sample
contamination with skin flora is not an issue collection at the bed side. These containers should
while performing these investigations. have the following specifications:
* The blood sample is collected in clean sterile Q They should be sterile, leak-proof, wide mouthed
and screw capped.
tubes (Vacutainers) Q Each container should bear the name of the
* Tube is filled 3/4" full and then allowed to patient, from whom the specimen was collected
stand at room temperature for a few hours to and his hospital register number.
allow a solid clot to form and retract Q Specimen type and date and time of collection
* Then the tube is centrifuged, serum is Q Acompletely filled investigation requisition form
should always accompany the specimen indicating
separated, placed in another clean tube, which the investigation requested along with the
can be used for further diagnostic testing. probable clinical diagnosis and current antibiotic
therapy.
CSF and Other Sterile Body Fluids
CSE and other sterile body fluids should be
Fecal Specimens
collected before the starting of antimicrobial
therapy, in sterile screw capped container, under A small quantity of semisolid/solid stool or one
adequate aseptic precautions. third of the container in case of liquid stool
 SECTION1 @ General Microbiology, Immunology and Hospital Infection Control

people, outside in an open well ventilated

space
= Gastric aspirate may be collected for
infants in suspected case of tuberculosis.
Early morning specimen is ideal before
eating/getting up. Transport time should
be < 15 minutes.
“+ Collected specimen has to be transported to
the laboratory as soon as possible (within two
Fig. 3.2.1: Sterile universal container.
hours); as delicate pathogens may otherwise
specimen is collected in a wide mouthed, sterile die, if there is a delay.
screw capped, leak proof container (Fig. 3.2.1);
ETA, BAL, PSB and Lung Biopsy Specimens
preferably prior to initiation of antibiotics.
“+ Rectal swabs may be collected in case of Collection of endotracheal aspirate (ETA),
asymptomatic carriers bronchoalveolar lavage (BAL), protected
“* Transport: Sample should be immediately specimen brush (PSB) and lung biopsy
transported to the laboratory. In case of delay specimens is technically demanding and requires
suitable transport media may be employed specialized techniques. These specimens should
such as Cary Blair medium or Venkatraman be transported immediately to the laboratory
Ramakrishnan (VR) medium. and cultured within one hour of collection.

Respiratory Specimens Throat Swab

Respiratory specimens are categorized into two Throat swab samples for bacterial culture is
groups. collected by depressing the tongue with a
** Upper respiratory tract specimens such as tongue depressor. The oropharyngeal swab is
throat swab, nasopharyngeal swabs, bits of rubbed on the back of the throat, over the
membrane from tonsil tonsils, and in any other area where there is
** Lower respiratory tract specimens such as redness or pus. Whenever possible, a portion of
sputum, endotracheal aspirate (ETA) and pseudomembrane may be collected.
bronchoalveolar lavage (BAL), protected
specimen brush (PSB) and lung biopsy. Nasopharyngeal Secretions
Nasopharyngeal specimens are the best
Sputum specimens which may be obtained by:
Sputum cultures are indicated to identify the ** Nasopharyngeal aspiration (best method):
pathogens causing pneumonia. Collected by inserting flexible swab through
“+ Sputum specimen that results following a nose into posterior nasopharynx and rotating
deep cough is preferable and is collected for 5 seconds; specimen of choice for
in a sterile screw capped, wide-mouthed, Bordetella pertussis
leak proof container (Fig. 3.2.1). Patient ** Per-nasal swab: Collected by using a sterile
should be instructed to place the rim of the swab on a flexible wire.
container under the lower lip to catch entire Note: For culture in a suspected case of pertussis,
expectorated mucopurulent sputum alginate swabs are the best followed by dacron
** Early morning sputum samples should be swabs. Cotton swabs are not satisfactory. It is
obtained as they contain pooled overnight recommended to collect six swabs, at 1-2 days
secretions, as they may contain increased intervals to achieve maximum yield.
concentration of pathogens
** For suspected tuberculosis Exudate Specimens
= Two sputum specimens are collected- Wound swabs (sterile cotton swab, Fig. 3.2.2) are
spot and early morning. Sputum collection recommended to identify the etiological agents
booths should be located away from other causing deep-seated wound infection.
 CHAPTER 3.2 © General Bacteriology: Specimen Collection and Transport

culture. After properly cleaning the urethral

meatus or glans with soap and water, urine
specimen is collected in a sterile, wide
mouthed, screw capped, leak proof container
Fig. 3.2.2: Sterile cotton swab. by voiding the first portion (which is likely to
be contaminated with normal urethral flora)
“+ Wound swabs are ideally collected prior to
7
(Fig. 3.2.1)
starting of antibiotic therapy and only for “ Indwelling catheter: Urine specimen should
clinically infected wounds or that fail to heal be collected from the catheter tubing (after
even after a long period clamping and disinfecting a portion of the
* For closed wounds disinfect with 70% catheter tubing with alcohol), by inserting a
alcohol or 2% chlorhexidine followed by 10% sterile syringe and needle directly into the
povidone-iodine. Remove iodine with alcohol catheter tubing. Urine must not be collected
just prior to specimen collection from the drainage bag
4" Suprapubic aspiration: It is the most ideal
G
“* Open wounds are first debrided and then
+,

thoroughly rinsed with sterile saline prior to urine specimen, as it avoids the risk of
collection contamination with urethral flora
“+ Preferably sample the viable tissue and not = However it is invasive and therefore is
the superficial debris recommended only for patients in coma
* A portion of the sample also must be placed or infants
in Robertson’s Cooked Meat (RCM) medium, ® The skin above the bladder is disinfected
if anaerobic culture is indicated and then urine is collected needle aspiration
“+ For collection of pus in the form of abscess, above the symphysis pubis through the
aspirate the deepest portion of the lesion with abdominal wall into the full bladder.
a syringe and needle Urine specimen must be transported to the
“ For burn wound swab collection, consider microbiology laboratory as soon as possible
sampling different areas of the burn, as and should be processed immediately. If delay is
organisms may not be evenly distributed in expected for more than two hours, then it can be
a burn wound stored in refrigerator or stored by adding boric
* Aspirates and tissue specimens should acid, glycerol or formate for maximum 24 hours
be delivered to the laboratory for further before plating.
processing within 30 minutes of collection
Genital Specimens
for best recovery. Tissue specimens must be
kept moist to preserve viability of organisms Common genital specimens include urethral
“ For anaerobic culture, aspirates or tissue discharge for urethritis and exudate from genital
specimens are recommended. Specimen ulcers
which are not suitable include swab, urine,
Urethral Discharge
sputum etc.
* Discharging sinus: In case of actinomycetoma, Urethral swab in men and cervical swab in
granules presentin the discharge are collected women are the preferred specimens. Vaginal
in sterile gauze or loop by pressing the sinuses swab is not satisfactory.
from the periphery to express them out. “ Method: The urethral meatus is cleaned
with gauze soaked in saline. The purulent
Urine Specimen discharge is expressed out by pressing at the
It is extremely important to collect the urine base ofthe penis and collected directly on to
specimens carefully to avoid the contamination slides or swabs
with normal urethral flora. The various type of “+ Swab: Dacron or rayon swabs are preferred,
collection of urine specimen for culture has as cotton and alginate swabs are inhibitory to
been described below. many urethral pathogens such as gonococci
“ Midstream clean catch urine: It is the most * In chronic urethritis: As discharge is
common type ofurine specimen collected for minimal, prostatic massage is done to collect
 SECTION1 © General Microbiology, Immunology and Hospital Infection Control

the secretion; alternatively, the morning drop “+ Specimens such as CSF and body fluids, ocular
of secretion may also be collected specimens, tissue specimens, suprapubic
“ Transport Media: Specimens should be aspirate and bone specimen should be
transported immediately. If not possible, transported immediately (<15 minutes)
then charcoal containing Stuart’s or Amies “+ Urine (midstream) added with preservative
transport medium can be used. (boric acid) is acceptable up to 24 hours,
otherwise should be transported within 2
Exudate from Genital Ulcers hours
Surface of the genital ulcer is cleaned with saline, * Stool culture: Stool specimen should be
gentle pressure is applied at the base of the lesion, transported within 1 hour, but with transport
and a drop of exudate is collected on a slide. media such as Cary-Blair medium or
Venkatraman Ramakrishnan (VR) medium,
Endometrial specimens: Collected by surgical
biopsy or trans-cervical aspirate via sheathed is acceptable up to 24 hours
catheter.
“+ Rectal swabs—up to 24 hours is acceptable
“+ For anaerobic culture: Specimens should
Other Specimens be put into Robertson’s cooked meat broth
* Ocular specimens: They are precious speci- or any specialized anaerobic transport
mens, should be transported to laboratory
system and transported immediately to the
within 15 min. Bedside inoculation onto blood laboratory.
agar may be considered if delay is unavoidable
Specimen Storage before Processing
a Conjunctival swab: Swabs should be pre-
moistened with sterile saline and samples Most specimens can be stored at room
should be collected from both the eyes temperature immediately after receipt, for up to
= Corneal scrapings: Clinicians should 24 hours. However, there are some exceptions.
instill local anesthetics before collection ** Blood cultures—should be incubated at 37°C
= Aqueous or vitreous fluid for endo- immediately upon receipt
phthalmitis cases. ** Sterile body fluids, bone, vitreous fluid,
“* Ear specimens: suprapubic aspirate—should be immediately
= External ear: Specimen is collected by plated upon receipt and incubated at 37°C
firmly rotating the swab into the outer ear ** Corneal scraping—should be immediately
(Fig. 3.2.2) plated at bed-side on to blood agar and
m= Inner ear specimens: If ear drum is intact, chocolate agar
material behind the drum is aspirated ** Stool culture—stool specimen for culture can
with syringe; if ear drum is ruptured, swab be stored up to 72 hours at 4°C
is used to collect material from the inner ** Urine (mid-stream and from the catheter),
ear. Ear canal should be cleaned with mild lower respiratory tract specimen, gastric
soap solution before aspiration. biopsy (for Helicobacter pylori)—can be
stored up to 24 hours at 4°C.
Specimen Transport
The specimens should reach the laboratory for Prioritizing the Specimen for Processing
further processing as soon as possible after the Certain precious specimens such as CSF and
collection. If required appropriate transport sterile body fluids, ocular specimens, tissue
media should be used (discussed subsequently specimens, suprapubic aspirate and bone
in this chapter). specimen should be processed immediately as
For most of the specimens, transport time soon as received, not more than 15 min delay.
should not exceed two hours. However, there Similarly, blood culture bottles should be
are some exceptions. immediately incubated upon receipt.
 General Bacteriology:
Direct Detection 1: Simple Staining She.
Direct detection of bacteria in the clinical Basic dyes, such as methylene blue or basic
specimen plays a very important role in early fuchsin are used as simple stains (explained
institution of antimicrobial therapy. These subsequently in this chapter).
methods include microscopic demonstration
of bacteria—staining techniques and other Differential Staining
methods, such as detection of antigen or nucleic Here, two stains are used which impart differ-
acid in the clinical specimen. ent colors to different bacteria. Gram staining is
most commonly employed staining technique in
B STAINING TECHNIQUES diagnostic microbiology laboratory. It differenti-
ates bacteria into gram-positive and gram-nega-
Structural details of bacteria cannot be seen
tive groups (discussed in detail in Chapter 3.4).
under a light microscope due to lack of contrast.
Hence, it is necessary to use staining methods Special Staining
to produce color contrast and thereby increase
Special stains used are:
the visibility. Before staining, the fixation of the
* Acid-fast stain: Differentiates bacteria into
smear to the slide is done.
acid-fast and nonacid-fast groups
Fixation “ Albert stain: Differentiates bacteria having
metachromatic granules from other bacteria
Fixation is the process by which the internal and
that do not have
external structures of cells are preserved and “ Negative staining: Example, India ink stain
fixed in position. There are two types of fixation
for demonstration of capsule
as follows:
* Impregnation methods: Example, silver
1. Heat fixation is usually done for bacterial
impregnation method for demonstration of
smears by gently flame heating an air-dried
thin organisms, such as spirochetes.
smear of bacteria. This preserves overall
These special staining techniques are discussed
morphology of cells
in Chapter 3.5.
2. Chemical fixation is done by using chemicals,
such as ethanol, acetic acid, formaldehyde,
methanol and glutaraldehyde. This is useful
SIMPLE STAINING
for examination of blood smears. They protect Basic dyes, such as methylene blue or basic
the fine internal structure of cells. fuchsin are used as simple stains (Figs 3.3.1
The fixed smear is stained by appropriate and 3.3.2). They provide a quick and easy way
staining techniques. to determine cell shape, size, and arrangement.

Common Staining Techniques Used in Procedure

Microbiology Laboratory Bacterial smear is stained with one of the simple
Simple Staining stains for one minute and then the slide is rinsed
Here, only one stain is used which imparts only with water and allowed to air dry and focused
one color to all the bacteria present in the smear. under oil immersion objective.
oe SECTION 1 © General Microbiology, Immunology and Hospital Infection Control

Exercise
Solving Exercise
[Problem Solving _|
Simple Staining 3. What are the uses of simple staining?
Smear made from a bacterial colony is provided. Explanation
1. Perform simple stain of the smear provided.
For answers to these questions, refer text.
2. Draw your observations with the help of a diagram
and give your interpretation.

Fig. 3.3.1: Methylene blue stained smear demonstrating Fig. 3.3.2: Basic fuchsin stained smear demonstrating
blue-colored cocci in cluster. pink-colored bacilli in scattered arrangement.
Source: Department of Microbiology, JIPMER, Puducherry Source: Department of Microbiology, JIPMER, Puducherry
(with permission). (with permission).

Result and Interpretation ** Methylene blue stain imparts blue color

They provide the color contrast, but impart the (Fig. 3.3.1).
same color to all the bacteria in a smear. * Basic fuchsin stain imparts pink color
(Fig. 3.3.2).
 General Bacteriology:
Direct Detection 2: Gram Staining
EASTER SIIS
3.4
| Problem
Problem SolvingExercise
Solving Exercise
Gram Staining 1. Perform Gram stain of the smear provided.
2. Draw your observations with the help of a neat
Case Scenario 1: A smear is provided, made from
labeled diagram and give your interpretation.
a sputum specimen of a 5-year-old child with acute
3. Suggest which antibiotic can be started empirically
onset of fever, productive cough and dyspnea for the
past two days. Physical examination revealed dull for the treatment of this case?
4, What are various theories of Gram staining?
note on percussion.
5. What are the various uses of Gram staining?
Case Scenario 2: A smear is provided, made from a
CSF specimen of a two year old baby presented with Explanation
high grade fever, vomiting and excessive crying for The above clinical presentations are suggestive of a
past three days. case of:
Case Scenario 3: A smear is provided, made from a Q Lobar pneumonia (case scenario 1)
pus discharge of a postoperative wound infection of Q Pyogenic meningitis (case scenario 2)
a 65-year-old man who has undergone an abdominal Q Surgical site infection (case scenario 3)
surgery. Q Diabetic foot ulcer (case scenario 4)
Case Scenario 4: A smear is provided, made from a The answers to the above questions are explained in
wound swab specimen of a foot ulcer of a diabetic this chapter.
patient.

Gram Staining technique was originally devel- Step 2 (Mordant)

oped by Hans Christian Gram (1884) and itis the Gram’s iodine (dilute solution of iodine) is
most widely used stain in diagnostic bacterio- poured over the slide for one minute. Then the
logy. slide is rinsed with water. Gram’s iodine acts as
a mordant, binds to the dye to form bigger dye-
i PROCEDURE iodine complexes in the cytoplasm.

Fixation Step 3 (Decolorization)

The smear is made on a clean glass slide from Next step is pouring of few drops of decolorizer
bacterial culture or clinical specimen. It is then to the smear, such as acetone (for 2-3 sec) or
air dried and heat fixed. ethyl alcohol (20-30 sec) or acetone alcohol (for
10 sec). Slide is immediately rinsed with water.
Step 1 (Primary Stain) Decolorizer removes the primary stain from
The smear is stained with pararosaniline dyes, gram-negative bacteria while the gram-positive
such as crystal violet (or gentian violet or methyl bacteria retain the primary stain.
violet) for one minute. Then the slide is rinsed
Note: Decolorization is the most crucial step of Gram
with water. Crystal violet stains all the bacteria
staining. If the decolorizer is poured for more time,
violet in color (irrespective of whether they are
Contd...
gram-positive or gram-negative).
 SECTION 1 © General Microbiology, Immunology and Hospital Infection Control

Contd...

even gram-positive bacteria lose color (over

decolorization) and if poured for less time, the gram-
negative bacteria do not lose the color of primary stain
properly (under decolorization).

Step 4 (Counterstain)
Secondary stains, such as dilute carbol fuchsin
or safranin are added for 30 seconds. They
impart pink to red color to the gram-negative
bacteria. Alternatively, neutral red may also be
used as counterstain especially for gonococci.
The slide is rinsed in tap water, dried, and then
examined under oil immersion objective.
The steps of Gram staining and the color of Figs 3.4.2: Gram staining demonstrating violet-colored
gram-positive cocci in clusters and pink-colored gram-
gram-positive and gram-negative bacteria after
negative bacilli in scattered arrangement.
each step are depicted in Figure 3.4.1.
Source: Department of Microbiology, Pondicherry Institute of
Medical Sciences, Puducherry (with permission).
@ INTERPRETATION
pH Theory
Smear is examined under oil immersion
Cytoplasm of gram-positive bacteria is more
objective (Fig. 3.4.2).
acidic, hence can retain the basic dye (e.g.,
** Gram-positive bacteria resist decolorization
crystal violet) for longer time. Jodine serves as
and retain the color of primary stain, i.e., violet
mordant, i.e., iodine combines with the primary
“* Gram-negative bacteria are decolorized
stain to form a dye-iodine complex which gets
and, therefore, take counterstain and appear
retained inside the cell.
pink.
Cell Wall Theory
@ MECHANISM OF GRAM STAINING
This is believed to be the most important
Though the exact mechanism is not understood, postulate to describe the mechanism of Gram
the following theories have been put forward. staining.

/ / BB Crystal violet.
faa Gram’s iodine
Py Alcohol or
Acetone
| Safranin or
Dilute carbol
fuchsin

Gram-positive
Gram-negative

Step-1: Primary stain Step-2: Mordant Step-3: Decolorization Step-4: Counterstain

Crystal violet Gram’s iodine Acetone for 2—3 seconds Dilute carbo! fuchsin or
for 1 minute for 1 minute or 95% ethy! alcohol safranin for 30 seconds.
for 20-30 sec water rinse and blot
Fig. 3.4.1: Principle and procedure of Gram staining.
 CHAPTER 3.4 © General Bacteriology: Direct Detection 2: Gram Staining

Table 3.4.1: Differences between gram-positive and gram-negative cell wall.

Gram-positive cell wall Gram-negative cell wall
Peptidoglycan layer Thicker (15-80 nm), 50-100 layers, cross Thinner (2 nm), 1-2 layers, directly linked,
linked by pentaglycine bridge no pentaglycine bridge
Lipid content Nil or scanty (2-5%) Present (15-20%)
Lipopolysaccharide Absent Present (endotoxin)
Teichoic acid Present Absent
Variety of amino acids Few Several
Aromatic amino acids Absent Present

“+ Gram-positive cell wall has a thick peptido- « Preston and Morrell’s modification: Here,
glycan layer (50-100 layers thick), which are iodine-acetone is used as decolorizer
tightly cross linked to each other ¢~ Brown and Brenn modification: This is used
“+ The peptidoglycan itself is not stained; instead for Actinomycetes.
it seems to act as a permeability barrier
preventing loss of crystal violet (Table 3.4.1) USES OF GRAM STAIN
“+ Gram-negative cell wall is more permeable,
Gram staining has the following uses:
thus allowing the outflow of crystal violet
* Differentiation of bacteria into gram-
easily (Table 3.4.1). This is attributed to:
positive and gram-negative: It is the first step
m The thin peptidoglycan layer in gram-
towards identification of bacteria
negative cell wall which is not tightly cross
“* To start empirical treatment: Gram staining
linked of clinical specimen gives preliminary clue
= Presence of lipopolysaccharide layer in
about the bacteria present (based on the
the cell wall of gram-negative bacteria gets
shape and Gram staining property of the
disrupted easily by the action of acetone
bacteria) so that the empirical treatment with
or alcohol, thus allowing the primary stain
broad spectrum antibiotics can be started
to come out of the cytoplasm.
early, before the culture and susceptibility
“ After mordanting with Gram’s iodine, bigger report is available
dye-iodine complexes are formed in the
“> For identification: Gram staining from bacte-
cytoplasm. Following decolorization, gram- rial culture gives an idea to put the required
negative bacterial cell wall (as more lipid biochemical tests for further identification of
content) gets dissolved leading to formation bacteria
of larger pores through which the dye-iodine
“ For fastidious organisms, such as Haemophilus
complexes escape. Due to less lipid in gram- which takes time to grow in culture, Gram
positive bacterial cell wall, smaller pores are staining helps in early presumptive identifica-
formed and dye-iodine complexes are retained. tion based on their morphology (pleomorphic
gram-negative bacilli)
@f MODIFICATIONS OF GRAM STAINING * Anaerobic organisms, such as Clostridium
There are few minor modifications of Gram do not grow in routine culture. Therefore,
staining which vary slightly from the method organisms detected in Gram stain, but aerobic
described earlier: culture-negative gives a preliminary clue to
perform an anaerobic culture of the specimen
* Kopeloff and Beerman’s modification:
Primary stain and counterstain used are * Yeasts: In addition to stain the bacteria, Gram
methyl violet and basic fuchsin, respectively staining is useful for staining certain fungi,
¢,*‘- Jensen’s modification: This method involves such as Candida and Cryptococcus (appear
use of absolute alcohol as decolorizer and gram-positive budding yeast cells)
neutral red as counterstain. It is useful for “ Quality of specimen: Gram staining helps in
detection of meningococci and gonococci screening the quality of the sputum specimen
* Weigert’s modification: This modification before processing it for culture. Presence
is useful for staining tissue sections. Here, of more pus cells and less epithelial cells
indicates good quality specimen.
aniline-xylol is used as a decolorizer
 General Bacteriology:
|
CHAPTERJ

3.
Direct Detection 3: Special Staining
(Acid-Fast Stain, Albert Stain)
and Other Methods

HB ACID-FAST STAIN
[ Problem Solving
Solving Exercise
Exercise11 |
Acid-fast Staining 5. Which is the conventional culture medium used
A smear is provided, made from a sputum specimen and describe the colonies grown?
of a 15-year-old boy presented with fever, productive 6. What is the recommended molecular method
cough and hemoptysis for past two weeks. available for rapid and accurate identification?
1. Perform acid-fast staining of the smear provided. Mention its advantages?
2. Draw your observations with the help of a neat Explanation
labeled diagram and give your interpretation.
The above clinical presentation is suggestive of acase
3. Suggest the treatment regimen given in this
of pulmonary tuberculosis. The answers to the above
case.
questions have been explained in this chapter and
4. What is RNTCP grading and its implications?
also in Chapter 35.

The acid-fast staining was discovered by Paul Table 3.5.1: Acid-fast organisms or structures and
Ehrlich and subsequently modified by Ziehl and percentage of sulfuric acid suitable for staining (for
Neelsen. This staining is done to identify acid-fast decolorization).

organisms, such as Mycobacterium tuberculosis Acid-fast organisms/structures Sulfuric acid

and others (Table 3.5.1). Acid-fastness is due to (%) needed for
presence of mycolic acid in the cell wall. decolorization
Mycobacterium tuberculosis 25
Ziehl-Neelsen Technique (Hot Method) Mycobacterium leprae 5
Smear Preparation Nocardia 1
Smear measuring 2 x 3 cm in size is prepared ina Acid-fast parasites, such as 0.5-1
new clean grease free, scratch free slide from the Cryptosporidium, Cyclospora,
Cystoisospora, Microsporidia*, Taenia
yellow, purulent portion of the sputum.
saginata segments, hooklets of
hydatid cyst
Heat Fixation
Bacterial spore 0.25-0.5
The smear is air dried and then heat fixed by
*Microsporidia are now considered to be evolved from fungi.
passing over the flame. Coagulation of the
proteinaceous material in the sputum will ** Care must be taken to ensure that the smear
facilitate fixing of the smear. does not dry out. To prevent drying, more
carbol fuchsin stain is added to the slide and
Step 1 (Primary Stain) then the slide is reheated
Smear is poured with carbol fuchsin (1%) for 5 ** Rinse the slide with tap water, until all free
minutes. Intermittent heating is done by flaming carbol fuchsin stain is washed away. At this
the underneath of the slide until the fumes appear. point, the smear on the slide looks red in color
Heating helps in better penetration of the stain. (Fig. 3.5.1).
 ;

CHAPTER 3.5 @ General Bacteriology: Direct Detection 3: Special Staining and Other Methods On

Faey6) Acid fast

—— Soe Age tie

v a / ae S 7g — Non-acid fast
See SS (pus cells)

Application of Intermittent heating Application of Application of

by flaming underneath 25% sulfuric acid methylene biue
strong carbol fuchsin
for 5 minutes of slide until the fumes for 2-4 minutes for 30 seconds
(primary stain) appear (mordant) (decolorizer) (Counterstain)

Fig. 3.5.1: Ziehl-Neelsen technique.

Step 2 (Decolorization)
It is done with 25% sulfuric acid for 2-4 minutes.
Repeat decolorization for 1-3 minutes if the slide
is still red. Then the slide is gently rinsed with
tap water and tilted to drain off the water. The
back of the slide is wiped clean with a swab
dipped in sulfuric acid.

Step 3 (Counterstaining)
It is done with methylene blue (0.1%) for 30
seconds. Slide is rinsed in tap water, dried,
and then examined under the binocular
microscope using low power objective (10x) to
select a suitable area and then screened under
oil immersion field (100x). Contaminated
materials/slide should be discarded in jar Fig. 3.5.2: Ziehl-Neelsen staining of sputum smear
containing 5% phenol. showing long slender, straight or slightly curved, beaded
red colored acid-fast bacilli.
Source: Department of Microbiology, JIPMER, Puducherry (with
Interpretation permission).
Mycobaterium tuberculosis appears as long
= Phenol concentration in carbol fuchsin is
slender, straight or slightly curved, beaded,
increased
less uniformly stained, red-colored acid-fast
= Duration of carbol fuchsin staining is
bacillus. Other non-acid fast organisms present
more.
in the smear, pus cells and the background take
* Decolorization can be done with acid-alcohol
up the counterstain and appear blue (Fig. 3.5.2).
(3 mL of HCl and 97 mL of ethanol)
Modifications of Acid-fast Staining “» Malachite green can be used as counterstain
“ Concentration of sulfuric acid may vary
Hot method (Ziehl-Neelsen technique) is
depending on the acid-fastness ofthe structure
the most commonly done acid-fast staining
to be demonstrated. More the content of
technique. Other modifications include:
mycolic acid in the cell wall, more is the
* Cold method (Kinyoun’s method): It differs
acid-fastness, hence more is the percentage
from Ziehl-Neelsen stain in that—
of sulfuric acid needed (Table 3.5.1).
= Heating is not required
ga) SECTION1 © General Microbiology, Immunology and Hospital Infection Control

B ALBERT STAIN
[Problem Solving
Solving Exercise2
Exercise 2
Albert Staining 3. Suggest which antibiotic can be started empirically
A smear is provided, made from a throat swab for treatment in this case
specimen of a 15-year-old boy presented to an 4. How can this clinical condition be prevented?
ENT OPD with fever, sore throat and difficulty in Explanation
swallowing for past two days. On examination, a dirty The above clinical presentation is suggestive of a
grey membrane was observed over the tonsils.
case of faucial diphtheria. The answers to the above
1. Perform a suitable staining of the smear provided. questions have been explained in this chapter and
2. Draw your observations with the help of a neat
also in Chapter 33.
labeled diagram and give your interpretation.

Albert stain is used to demonstrate the

metachromatic granules of Corynebacterium
diphtheriae.

Composition of Albert Stain

Includes:
“+ Albert I: Comprises of toluidine blue,
malachite green, glacial acetic acid, alcohol
(95% ethanol), and distilled water
* Albert II: Contains iodine in potassium
iodide.

Procedure
1. Fixation: The smear is heat fixed
2. Smear is covered with Albert I (Albert’s stain) Fig. 3.5.3: Albert stained smear of Corynebacterium
for 5 minutes, then the excess stain is drained diphtheriae showing green bacilli with blue-black
out metachromatic granules (schematic).

3. Albert II is poured over the smear so as to

cover it completely for 1 minute B NEGATIVE STAINING
4. Slide is washed in water, blotted dry and
A drop of bacterial suspension is mixed with dyes,
examined under oil immersion field.
such as India ink or nigrosin. The background
Interpretation gets stained black whereas unstained bacteria
stand out in contrast. This is very useful in the
Corynebacterium diphtheriae appears as
demonstration of bacterial or yeast capsules
green-colored bacilli arranged in Chinese
which do not take up simple stains (Refer Fig.
letter or cuneiform pattern, with bluish-
41.3A, Chapter 41).
black metachromatic granules at polar ends
(Fig. 3.5.3). These can be differentiated from
diphtheroids which do not show granules and
B IMPREGNATION METHODS
are arranged in palisade pattern. However, Bacterial cells and structures that are too
certain bacteria, such as Corynebacterium thin to be seen under the light microscope
xerosis and Gardnerella vaginalis also possess are thickened by impregnation of silver
metachromatic granules. on their surface to make them visible, e.g.,
 CHAPTER 3.5 @ General Bacteriology: Direct Detection 3: Special Staining and Other Methods a

chetes in genital specimens (Refer Fig. 2.4,

Chapter 2)
* Hanging drop preparation for stool
specimen—for demonstration of darting
motility; gives a clue about V. cholerae
(discussed in Chapter 3.7).

Antigen Detection
Various immunological methods, such as latex
agglutination test, immunochromatographic
test are available which detect antigens directly
from the clinical specimens.
* The classical example includes detection of
capsular antigen of pneumococci, meningo-
Fig. 3.5.4: Silver impregnation staining demonstrating
spirochetes. cocci, H. influenzae in CSF specimen
Source: Public Health Image Library, ID# 836, Centers for Disease “ Urinary antigen detection for pneumococci
Control and Prevention (CDC), Atlanta (with permission). and Legionella
* Direct fluorescent antibody test—for
demonstration of bacterial flagella and detection of Treponema pallidum from tissue
spirochetes (Fig. 3.5.4). sections or exudates.
Details about these antigen detection methods
OTHER METHODS OF are discussed in Chapters 7 to 9.
DIRECT DETECTION Molecular Diagnosis
Other Microscopic Techniques Bacterial DNA or RNA can be directly detected
Other microscopic techniques include: in the clinical specimens by various molecular
* Dark-ground and _ phase-contrast methods, such as polymerase chain reaction
micros copy—f or demonstration of spiro- (PCR). It is discussed in detail in Chapter 3.9.
 General Bacteriology:
Culture Media (Including Automated
Culture) and Culture Methods
PLN LRTEA LIES
6
Culture investigation is the most common Table 3.6.2: Basal media and their properties.
diagnostic method used for the detection of
bacterial infections. It involves several steps—(i) Peptone water It contains peptone (1%) + NaCl
specimens are inoculated on to various culture (Fig. 3.6.1A) (0.5%) + water
media and incubated, (ii) colonies grown are Nutrient broth It is made up of peptone water +
subjected to identification (Chapter 3.7) and meat extract (1%)
(iii) antimicrobial susceptibility testing (Chapter Nutrient agar It is made up of nutrient broth +
3.8). (Fig. 3.6.1B) 2% agar

** They serve as the base for the preparation of

@ CULTURE MEDIA
many other media
Culture media are required to isolate the “+ Nutrient broth is used for studying the
bacteria from the clinical specimens. ‘The basic bacterial growth curve
constituents of culture media are enlisted in “+ Nutrient agar is the preferred medium for:
Table 3.6.1. Various types of culture media used = Performing the biochemical tests, such as
in the laboratory are as follows: oxidase, catalase and slide agglutination
test, etc.
Simple or Basal Media = To study the colony character
They contain minimum ingredients that support = Pigment demonstration.
the growth of nonfastidious bacteria (Table 3.6.2).
The basal media are used for: Enriched Media
** Testing the nonfastidiousness ofbacteria When a basal medium is added with additional
nutrients, such as blood, serum or egg, it is
Table 3.6.1: Basic constituents of culture media. called as enriched medium. In addition to
conten
[Uae
Water Distilled water or potable water
nonfastidious organisms, they also support
the growth of fastidious nutritionally exacting
Electrolytes Sodium chloride or other electrolytes bacteria (Table 3.6.3, Figs 3.6.1C and D).
Peptone It is a complex mixture of partially
digested proteins Enrichment Broth
Agar Used for solidifying the culture media They are the liquid media added with some
e Prepared from seaweeds inhibitory agents which selectively allow certain
e Itis bacteriologically inert organism to grow and inhibit others. This is
e It melts at 98°C and usually
important for isolation of the pathogens from
solidifies at 42°C
¢ Concentration of agar used for clinical specimens which also contain normal
solid agar preparation (1—2%) flora (e.g., stool and sputum specimen) (Table
Others Meat extract, yeast extract and malt 3.6.4).
extract
Blood and Usually 5—10% of sheep blood is used Selective Media
serum for enriched media to provide extra They are solid media containing inhibitory
nutrition to fastidious bacteria
substances that inhibit the normal flora present
 CHAPTER 3.6 © General Bacteriology: Culture Media and Culture Methods

N
Figs 3.6.1A to D: (A) Peptone water;(B) Nutrient agar; (C)
7
C) Blood agar; (D)Chocolate agar.
Source: (A to D) Department of Microbiology, JIPMER, Puducherry (with permission).

Table 3.6.3: Enriched media and their properties.

Enriched media
Blood agar It is the most commonly used media
(Fig. 3.6.1C) It is used to test the hemolytic property of the bacteria
Chocolate agar It is the heated blood agar, more nutritious than blood agar
(Fig. 3.6.1D) It supports certain highly fastidious bacteria, such as Haemophilus influenzae that does
not grow on blood agar
Loeffler’s serum slope It is used for isolation of Corynebacterium diphtheriae
Blood culture media They are used for culture of blood specimen. They are either monophasic or biphasic
media
e Monophasic medium is made up of brain-heart infusion (BHI) broth (Fig. 3.6.2C)
e Biphasic medium has a liquid phase containing BHI broth and a solid agar slope made
up of BHI agar (Fig. 3.6.2D)

Monophasic Biphasic Table 3.6.5: Selective media and their uses.

medium oe
Used for isolation of

Lowenstein-Jensen (LJ) Mycobacterium

medium (Fig. 3.6.3A) tuberculosis from sputum

Thiosulfate citrate bile salt Vibrio cholerae from stool

sucrose (TCBS) agar (Fig. 3.6.3B)

Deoxycholate citrate agar Enteric pathogens, such

(DCA) (Fig. 3.6.3C) as Salmonella and Shigella

Xylose lysine deoxycholate from sta)

(XLD) agar (Fig. 3.6.3D)

Figs 3.6.2A to D: (A) Robertson's cooked meat medium; Potassium tellurite agar Corynebacterium
(B) Thioglycollate broth; (C) Brain- heart infusion broth; (D) (PTA) diphtheriae from throat
swab
Biphasic medium (Brain-heart infusion broth/agar).
Source: (A, C and D) Department of Microbiology, JIPMER,
Puducherry; B. Department of Microbiology, Pondicherry Institute
of Medical Sciences, Puducherry (with permission). in the specimen and allow the pathogens to grow
(Table 3.6.5).
Table 3.6.4: Enrichment broth and their uses.
Used for isolation of Transport Media
Tetrathionate broth Salmonella Typhi They are used for the transport of the clinical
Gram-negative broth Shigella, Salmonella specimens suspected to contain delicate
Selenite F broth Shigella organism or when the delay is expected while
Alkaline peptone water Vibrio cholerae transporting the specimens from the site of
 —

SECTION 1 @ General Microbiology, Immunology and Hospital Infection Control

Figs 3.6.3A to D: (A) Lowenstein-Jensen medium; (B) Thiosulfate citrate bile salt sucrose agar;
(C) Deoxycholate citrate agar; (D) Xylose lysine deoxycholate agar.
Source: Department of Microbiology, JIPMER, Puducherry (with permission).

Table 3.6.6: Transport media used for common the color of the colonies of a particular group of
bacteria. bacteria but not the other group.

oer” wna
Streptococcus _ Pike’s medium
| ** MacConkey agar: It is a differential and low
selective medium commonly used for the
isolation of enteric gram-negative bacteria
Neisseria Amies medium, Stuart’s medium
(Fig. 3.6.4A)
Vibrio cholerae VR (Venkatraman-Ramakrishnan) = It differentiates organisms into LF or
medium
lactose fermenters (produce pink-colored
Cary-Blair medium
colonies, e.g., Escherichia coli) and NLF
Shigella, Buffered glycerol saline
or nonlactose fermenters (produce
Salmonella Cary-Blair medium
colorless colonies, e.g., Shigella) (Fig.
3.6.4B)
collection to the laboratory (Table 3.6.6). = Most microbiology laboratories use
Bacteria do not multiply in the transport media; combination of blood agar and MacConkey
they only remain viable. agar for routine bacterial culture.
** CLED agar (Cysteine lactose electrolyte-
deficient agar): This also differentiates
These media differentiate between two groups between LF and NLF; used for the processing
of bacteria by using an indicator, which changes of urine specimens (Fig. 3.6.4C).

Figs 3.6.4A and B: (A) MacConkey agar; (B) Lactose fermenters (LF) and nonlactose fermenters (NLF) colonies on
MacConkey agar; (C) Cysteine lactose electrolyte-deficient (CLED) agar.
Source: (A and C) Department of Mic robiology, JIPMER, Puducherry (with permission); (B) Department of Microbiology,
Pondicherry Institute of Medical Sciences; Puducherry (with permission).
 CHAPTER 3.6 © General Bacteriology: Culture Media and Culture Methods

Anaerobic Culture Media risk of contamination due to opening of the

cap of the bottle every time when subcultures
Anaerobic media contain reducing substances
are made
which take up oxygen and:create lower redox
“+ From biphasic BHI broth, subcultures can
potential and thus permit the growth of obligate
be made just by tilting the bottles so that
anaerobes, such as Clostridium. Examples are:
the broth runs over the agar slope. There is
Robertson’s cooked meat (RCM) broth: It lower risk of contamination as it obviates the
contains chopped meat particles (beef heart), opening of the cap of the bottle.
which provide glutathione (a sulfhydryl group
containing reducing substance) and unsaturated Automated Blood Culture Techniques
fatty acids. It is the most widely used anaerobic Automated blood culture techniques are
culture medium (Fig. 3.6.2A). revolutionary, offer several advantages over
Other anaerobic media include: conventional blood cultures.
* Thioglycolate broth (Fig. 3.6.2B) * Continuous automated monitoring:
* BHIS agar (Brain-heart infusion agar) with Following inoculation, the culture bottles are
supplements (vitamin K and hemin) loaded inside the automated culture system
7
“Bacteroides bile esculin agar (BBE agar). m The incubated bottles are periodically
<* Anaerobic blood agar tilted automatically every 10 minutes,
7
“Neomycin blood agar which allows mixing of blood with broth
*,bd
Egg yolk agar which fastens the recovery
7
“Phenylethyl agar. = Bottles are periodically monitored for
the microbial growth once in every 10
Blood Culture Media minutes by the instrument. Once positive
for microbial growth, the instrument gives
Recovery of bacteria from blood is difficult as
a signal (producing beep or color change
they are usually present in lesser quantity in
on the screen).
the blood and many of the blood pathogens * Composition: Automated blood culture
are fastidious. Therefore, enriched media are
bottles contain:
used for isolating microorganisms from blood. a Tryptic soy broth and/or brain heart infu-
Blood culture media are available either as sion broth (as enriched media) added with
conventional or automated media.
= Polymeric resin beads which adsorb and
neutralize the antimicrobials present in
Conventional Blood Culture Media
blood specimen.
The conventional blood culture media are of two “ Specimens: In addition to blood, these bottles
types. can also be used for culture of bone marrow,
1. Monophasic medium: It contains brain-heart sterile body fluids, such as CSF, peritoneal,
infusion (BHI) broth (Fig. 3.6.2C) pleural and synovial fluid
2. Biphasic medium: It has a liquid phase * More sensitive: It gives a higher yield of
containing BHI broth and a solid agar slope positive cultures from clinical specimens
made up of BHI agar (Fig. 3.6.2D). * Rapid: It takes less time than conventional
The recovery of organisms in the blood is methods
enhanced by mixing the blood in the broth * Less labor intensive, as fully-automated.
periodically. If any growth occurs, it can be
detected by subcultures. Automated Systems

Disadvantages There are three automated systems commercially

In conventional blood culture, subcultures are available.
made manually. 1. BacT/ALERT 3D (Figs 3.6.5A and B): Its
* From monophasic BHI broth, subcultures principle is based on colorimetric detection
are made onto blood agar and MacConkey of growth. When bacteria multiply, they
agar periodically for 1 week. There is a higher produce CO, that increases the pH, which in
 SECTION1 ©@ General Microbiology, Immunology and Hospital Infection Control

Note: There is an automated culture system

available for culture of Mycobacterium tubercu-
losis from various pulmonary and extrapulmo-
nary specimens; called as Mycobacteria Growth
Indicator Tube (MGIT, Chapter 35). This works
on fluorometric principle of detection, similar
to BACTEC.
Disadvantages
Automated culture methods do have several dis-
advantages, such as (1) high cost of the instru-
ment and culture bottles, (2) inability to observe
the colony morphology as liquid medium is used.

@ CULTURE METHODS
Figs 3.6.5A and B: (A) BacT/ALERT automated blood
culture system; (B) BacT/ALERT blood culture bottle. Culture methods involve inoculating the
Source: Department of Microbiology, JIPMER, Puducherry specimen on to appropriate culture media,
(with permission). followed by incubating the culture plates in
appropriate conditions.
turn changes the color of ablue-green sensor
present at the bottom of the bottle to yellow, Selection of Media
i.e., detected by colorimetry
The first step of a culture investigation is
2. BacT/ALERT VIRTUO (Fig. 3.6.6): It is an
selection of appropriate media, which in turn
advanced form of BacT/ALERT which offers
depends upon the type of specimen to be
several advantages, such as (i) automatic
processed. In general, combination of blood
loading and unloading of bottles, (ii) faster
agar and MacConkey agar is commonly used
detection of growth, (iii) can determine the
for processing of most specimens. However,
volume of blood present in the bottle
there are few specimens for which additional or
3. BACTEC: Its principle is based on fluorometric
alternative media are used (Table 3.6.7).
detection of growth; uses an oxygen-sensitive
fluorescent dye present in the medium.
Inoculation of the Specimens
Inoculation of the specimens onto the
culture media is carried out with the help of
bacteriological loops made up of platinum or
nichrome wire (Fig. 3.6.7A).
** The inoculating loop is first heated in the
Bunsen flame by making it red hot (Fig. 3.6.7B)
and then made cool waiting for 10 seconds
“+ The entire process of bacteriological culture
method should be carried out in a biological
safety cabinet and wearing appropriate
personal protective equipment, such as
gloves, laboratory coat or gown and mask (for
respiratory specimens).

Biosafety Cabinet (BSC)

It is an enclosed, ventilated laboratory work station,
Fig. 3.6.6: BacT/ALERT VIRTUO automated used to protect the laboratory personnel while
blood culture system. working with potential infectious clinical specimens.
Source: Department of Microbiology, JIPMER, Puducherry
(with permission). Contd...
 CHAPTER 3.6 © General Bacteriology: Culture Media and Culture Methods

Contd...

Q They are especially designed in a way that the air is

blown into the cabinet away from the worker and
then exhausted outside through a duct lined with
HEPA filters (Fig. 3.6.8)
Q There are various types of BSCs, depending upon
air velocity and percentage of air recirculated. Most
of the microbiology laboratories require Class 2A
BSC. A higher class of BSCs may be required for
certain high-risk pathogens.

Table 3.6.7: Selection of media for various specimen

types.
Fig. 3.6.8: Biological safety cabinet.
|Specimens _| Recommended culture media Source: Department of Microbiology, Pondicherry Institute of
Exudate Blood agar plus MacConkey agar Medical Sciences, Puducherry (with permission).
specimens*
Sterile body Blood agar, plus MacConkey agar, plus Inoculation Methods
fluids chocolate agar or
Inoculation methods are of two types:
Automated blood culture bottles
1. Methods used for inoculating clinical
Blood Blood culture bottles
(Conventional or automated)
specimens on to the culture media
Blood agar plus MacConkey agar
2. Methods used for inoculating colonies on to
Urine
CLED agar can be used alternatively various media for further processing.
Stool Selenite-F broth plus MacConkey agar
Streak Culture
plus DCA and/or XLD agar
(if cholera is suspected—add TCBS It is the most common inoculation method;
agar) used for the inoculation of the specimens on
Respiratory Blood agar, plus MacConkey agar, plus to the solid media. It is also used for obtaining
specimens chocolate agar individual isolated colonies from a mixed
(if diphtheria is suspected—add LSS
culture of bacteria.
and PTA)
* Streaking: A loopful of the specimen is
*Exudate specimens include pus, wound swab, aspirates, and tissue
smeared onto the solid media to form round-
bits.
agar; shaped primary inoculum, which is then
Abbreviations: CLED, cysteine lactose electrolyte deficient
DCA, deoxycholate citrate agar; XLD, xylose lysine deoxychola
te; spread over the culture plate by streaking
parallel lines to form the secondary, tertiary
serum
TCBS, thiosulfate-citrate-bile salts-sucrose agar; LSS, Loeffler
slope; PTA, potassium tellurite agar.
inoculum and finally a feathery tail end (Fig.
3.6.9A)
+ Intermittent heating: The loop is flamed and
cooled in between the different set of streaks
to get isolated colonies on the final streaks
(Fig. 3.6.9B). Obtaining isolated colonies is the
prerequisite to perform tests for identification
and AST.

Liquid Culture
Liquid culture is used for culture of specimens,
such as blood or body fluids, which are
Figs 3.6.7A and B: (A) Bacteriological loop and straight inoculated by directly adding the specimen into
wire: (B) Flaming the loop (red hot).
y
the liquid medium or with the help of a syringe
Source: (A) Department of Microbiology, JIPMER, Puducherr
(with permission). or pipette.
 SECTION 1 @ General Microbiology, Immunology and Hospital Infection Control

Sterilize Sterilize Sterilize

ifelo}e) ifofe}e) ifetoye)

HfakeYesiFe1ileYa)

Figs 3.6.9A to C: (A) Streak culture (schematic representation); (B) Isolated colonies grown by following streak culture;
(C) Lawn culture of a bacterial isolate to perform the antimicrobial susceptibility testing.
Source: Department of Microbiology, JIPMER, Puducherry (with permission).

is also preferred when large yields of bacteria

are required
Surface
pellicle
* Disadvantages: (1) Liquid cultures do not
provide a pure culture from a mixed inoculum,
(2) there is no visible colonies, therefore unlike
solid media, it does not give any preliminary
clue about the bacteria.

Lawn or Carpet Culture

Lawn culture is useful to carry out antimicrobial
susceptibility testing (AST) by disk diffusion
method (Fig. 3.6.9C). Here, the uniform lawn of
bacterial growth is obtained by either swabbing
or flooding with a bacterial broth onto the
Figs 3.6.10A to C: (A) Liquid culture in test tube (turbidity culture plate (discussed in detail in Chapter 3.8).
indicates growth); (B) Stroke culture; (C) Stabbing with
inoculation wire (stab culture). Pour Plate Technique
Source: Department of Microbiology, Pondicherry Institute of Seldom used for quantifying the bacterial load
Medical Sciences, Puducherry (with permission).
present in the specimens, such as urine or blood.
Here, serial dilutions of the specimen are added
** Bacterial growth is detected by observing on to the molten agar. After being cooled and
the turbidity in the medium. Some aerobic solidified, the Petri dishes are incubated and
bacteria form surface pellicles (Fig. 3.6.10A) then the colony count is estimated.
** Uses: Liquid cultures are useful for—(1)
blood, or body fluids culture, (2) automated Stroke Culture
culture for mycobacteria (MGIT, i.e., This is carried out on agar slopes or slants by
mycobacteria growth indicator tube), (3) streaking the straight wire in a zigzag fashion
water analysis (Fig. 3.6.10B). It is used for biochemical test,
* Advantages: Liquid cultures are preferable such as urease test.
for culture of—(1) specimens containing
small quantity of bacteria, (2) specimens Stab Culture
(e.g., blood) containing antibiotics and It is made by stabbing the semisolid agar butt
other antibacterial substances, as they get by a straight wire. It is used for motility testing
neutralized by dilution in the medium, (3) it using mannitol motility medium (Fig. 3.6.10C),
 CHAPTER 3.6 © General Bacteriology: Culture Media and Culture Methods

and triple sugar iron agar test (here, both stroke

and stab cultures are made). Lid

Incubatory Conditions
Most of the pathogenic bacteria are aerobes or
facultative anaerobes; grow best at 37°C, i.e.,
body temperature of human beings. Therefore,
the inoculated culture plates are incubated at
37°C aerobically overnight in an incubator.

Bacteriological Incubator Tubes with

liquid media Petri plates
It is an equipment used to incubate the culture
plates, biochemical tests and AST plates (Fig. 3.6.11). with solid
The incubator maintains optimal temperature. Some media
incubators are especially designed to maintain other
conditions, such as humidity and CO. Fig. 3.6.12: Candle jar.

Other Incubatory Conditions * For microaerophilic bacteria, such as

Campylobacter and Helicobacter require 5%
The incubatory conditions may vary depending
oxygen for optimum growth
upon the bacteria to be isolated.
* For obligate anaerobes, anaerobic culture
“ For capnophilic bacteria: Candle jar is used.
methods are used (see below).
Here, inoculated media are placed inside a
jar, along with a lighted candle and then jar Anaerobic Culture Methods
is sealed
Obligate anaerobic bacteria can grow only in the
= The burning candle reduces oxygen to
absence of oxygen, hence for the growth of such
a point where the flame goes off (Fig. is needed.
bacteria, anaerobic environment
3.6.12). This provides an atmosphere of
The following are the methods used to create
approximately 3-5% CO,
anaerobiosis.
m ‘Thisis useful for capnophilic bacteria, such
as Brucella, Streptococcus, pneumococcus Evacuation and Replacement
and gonococcus.
This involves evacuation of the air from
jar and replacement with inert gas, such as
hydrogen followed by removal of the residual
oxygen by use of a catalyst. It is carried out
either by:
* Manual method by using McIntosh and
Filde’s anaerobic jar (Fig. 3.6.13A): It was
the most popular method for creating
anaerobiosis in the past, now not in use
+ Automated system (Anoxomat): It
automatically evacuates air and replaces
by hydrogen gas from a cylinder (Fig.
26 135i)
m The catalyst used to combust residual
oxygen is a sachet containing aluminum
pellets coated with palladium
= It is easier to operate than McIntosh jar
Fig. 3.6.11: Bacteriological incubator. method and claims to be highly effective
ry
Source: Department of Microbiology, JIPMER, Puducher for creating anaerobiosis.
(with permission).
 SECTION 1 © General Microbiology, Immunology and Hospital Infection Control

7 Pressure gauge
Inlet and outlet
t TE TELLS TOUTE

Metal lid
Metal jar

Figs 3.6.13A to C: (A) Mcintosh and Filde’s anaerobicjar; (B) Anoxomat anaerobic system; (C) Anaerobic work station
(Whitley Pvt Ltd).
Source: (A) Department of Microbiology, Pondicherry Institute of Medical Sciences, Puducherry; (B) Department of Microbiology, JIPMER,
Puducherry; (C) Dr Padmaja A Shenoy, Department of Microbiology, Kasturba Medical College, Manipal, Karnataka (with permission).

Absorption of Oxygen by Chemical Clamp with Palladium

clamp screw catalyst pellets
Methods
Lid with
GasPak system (BD diagnostics) works on O-ring gasket
this principle. It is the the most commonly
used method for anaerobiosis, especially for
Envelope
laboratories with less sample load. containing sodium
“* Here, the oxygen is removed by chemical bicarbonate and
reactions, instead of evacuation and sodium borohydride

replacement technique used in Anoxomat

** It uses a sachet containing sodium bicarbo- Anaerobic indicator
nate and sodium borohydride which react (methylene blue)
chemically in presence of water, to produce
hydrogen and CO, gas
** The traces of oxygen is removed by using the Petri plates
same Catalyst used for Anoxomat (aluminum
pellets coated with palladium) placed below
the jar lid (Fig. 3.6.14)
* Indicator of anaerobiosis: The effective-
+, ‘+

ness of anaerobiosis can be checked by: Fig. 3.6.14: GasPak anaerobic system.
= Chemical indicator: Reduced methy-
lene blue remains colorless in anaerobic
conditions, but turns blue on exposure to Anaerobic Glove Box and Anaerobic Work
oxygen Station
= Biological indicator using obligate These systems provide facility for easy pro-
aerobe, such as Pseudomonas: Absence cessing, incubation and examination of the
of its growth indicates that complete specimens without exposure to oxygen (Fig.
anaerobiosis has been achieved. 3.6.13C).
GENbag (bioMérieux): It consists of an airtight
transparent bag with a generator sachet, which Reducing Agents
rapidly produces carbon dioxide and creates an Oxygen in culture media can be reduced by
anaerobic environment. Its application is similar various reducing agents, such as glucose,
to that of GasPak system. thioglycollate, cooked meat pieces, cysteine and
 CHAPTER 3.6 © General Bacteriology: Culture Media and Culture Methods

ascorbic acid. Robertson cooked meat broth Pre-reduced Anaerobically Sterilized (PRAS)
is the most widely employed anaerobic culture PRAS media are prepared entirely under
medium which uses chopped meat particles oxygen-free conditions from initial sterilization
(beef heart) as reducing agent (Fig. 3.6.2A). to packaging in sealed foil packets.

Problem Solving Exercise :

Culture Media and Culture Methods common culture media used for processing anaerobic
culture?
Case scenario 1: A 72-year-old female presented
with fever (104°F), heart rate of 116 beats/minute and Explanation
respiratory rate of 25 breaths/minute. A provisional The following are the recommended culture media
diagnosis of sepsis is made and blood cultures were used.
collected. Discuss the culture medium and culture Case scenario 1: For blood culture—Conventional
method required for processing of blood culture. What blood culture (monophasic media, such as brain heart
is automated blood culture? Discuss with examples. infusion broth and biphasic media, such as brain heart
Case scenario 2: A 21-year-old female was admitted infusion broth/agar) or automated blood culture
with increased frequency and burning micturition (BACTEC or BacT/ALERT) media can be used.
for the past 2 days. The urine specimen was collected Case scenario 2: For urine culture—Combination of
and sent to the laboratory . are the culture media
What blood agar plus MacConkey agar is used. CLED agar
required for processing of urine specimen. What is can be used alternatively.
differential media, discuss with examples? Case scenario 3: For stool culture—Selenite-F broth
Case scenario 3: A 2-year-old baby was admitted plus MacConkey agar plus DCA and/or XLD agar
with complain of passing liquid stool for 10-12 times is recommended. If cholera is suspected, alkaline
since one day. The stool specimen was collected and peptone water and TCBS agar are used in addition.
sent for culture. What are the culture media required Case scenario 4: For culture of respiratory
for processing of stool specimen. What is selective specimens—Blood agar plus MacConkey agar plus
medium; how itis different from enrichment medium? chocolate agar is recommended. If diphtheria is
Case scenario 4: A 12-year-old boy was admitted suspected and throat swab is sent, Loeffler’s serum
with fever, productive cough and dyspnoea. Sputum slope (LSS) and potassium tellurite agar (PTA) are used
specimen was collected and sent to the laboratory. in addition.
What are the culture media required for processing Case scenario 5: For anaerobic culture—Robertson’s
(RCM) broth is used. Other culture
of sputum specimen. What is enriched media, discuss cooked meat
with examples? media are brain-heart infusion agar with supplements
Case scenario 5: Following a penetrating trauma (vitamin K and hemin), Bacteroides bile esculin agar
to his right thigh, a construction laborer developed (BBE agar), neomycin blood agar, egg yolk agar and
discolored tissue and fowl smelling discharge. phenylethyl agar. The common methods to achieve
Anaerobic infection was suspected. How will you anaerobiosis include Anoxomat and GasPak system.
For the answers to other questions (of all the case
collect specimen for anaerobic culture? What are the
scenarios), refer the text in this chapter.
methods used to achieve anaerobiosis. What are the
 General Bacteriology: |
CHAPT ER}
Identification of Bacteria By 7
(Conventional and Automated)
PLR GRDELLIE

[ ProblemSolvingExercise
Solving Exercise _|
Identification of Bacteria Q Hanging drop is the most common method used
Case scenario 1: A 3 year old boy presents to OPD for demonstrating motility (refer text, for detail).
with watery diarrhea resembling rice water. Stool Q Vibrio cholerae exhibits highly active motility,
specimen is sent to the laboratory. Perform a test described as darting motility.
for the presumptive identification of Vibrio cholerae Case scenario 2: Patient suffering from burning
directly from the stool specimen? micturition and increased urinary frequency is
Case scenario 2: Urine specimen collected from suggestive of urinary tract infection (UTI).
a patient suffering from burning micturition, and Q Lactose fermenting pink colonies on MacConkey
increased urinary frequency is sent to the laboratory. agar is suggestive of either Escherichia coli (most
Culture on MacConkey agar revealed lactose common cause of UT1), or Klebsiella
fermenting pink colonies with colony count >10°/ Q The conventional biochemical tests performed
mL. Perform the conventional biochemical tests for for their identification are indole test, citrate test,
identification of the etiological agent. urease test and triple sugar iron test (refer text and
Case scenario 3: Sputum specimen of a 25 old man Chapter 42, for detail)
presented with productive cough, fever and dyspnoea Case scenario 3: ~-hemolytic colonies on blood agar
is sent to the laboratory. Culture on blood agar is suggestive of either pneumococcus (pathogen
revealed a-hemolytic colonies. What is the automated in sputum) or viridans streptococci (commensal in
identification system that can identify the organism sputum). MALDI-TOF is the automated identification
from the colony within minutes. Discuss its principle? system that can identify the organism from the
colony within minutes. Its principle is discussed in
Explanation the text.
Case scenario 1: The presumptive identification of
Vibrio cholerae directly from stool specimen is made
by performing motility testing.

Accurate identification of bacteria is necessary i COLONY MORPHOLOGY

as it helps in choosing the appropriate panel
of antibiotics for performing antibiotic After overnight incubation, the culture media are
susceptibility test. This in turn will guide in removed from the incubator and are examined
instituting the accurate antimicrobial therapy. under bright illumination. The appearance
* The presumptive identification of bacteria is of bacterial colony on culture medium is
made by observing their colony appearance characteristic for many organisms; which helps
in culture media, followed by performing in their preliminary identification. The following
culture smear (by Gram stain) and motility features of the colony are studied:
testing * Size—in millimeters; e.g., pinhead size is
* The final identification can be confirmed by characteristic of staphylococcal colony,
performing either conventional biochemical whereas pinpoint size is characteristic of
tests or by automated identification systems. streptococcal colony
 CHAPTER 3.7 © General Bacteriology: Identification of Bacteria

7
~e Shape—circular or irregular Contd...
o,ee
°, Consistency—dry, moist or mucoid Q Complete or B hemolysis: Zone of complete
\7
Od Density—opaque, translucent or transparent clearing of blood around the colonies due to
7
“e Hemolysis on blood agar (see below) complete lysis of the RBCs (e.g., Staphylococcus
7
DO Color of the colony: Colonies may be aureus and Streptococcus pyogenes)
colored due to certain properties of the Q No hemolysis (y hemolysis, a misnomer): There
is no color change surrounding the colony (e.g.,
media or organisms. For example, pink
Enterococcus)
colonies produced by lactose fermenters
on MacConkey agar and black colonies by
Corynebacterium diphtheriae on potassium CULTURE SMEAR AND MOTILITY
tellurite agar due to the reduction of tellurite. TESTING
Color of the colonies may also be due to
pigment production by the bacteria Culture Smear
“» Pigment production: Bacteria may produce The colonies grown on the culture media are
two types of pigments: subjected to Gram staining and motility testing
1. Diffusible pigments, e.g., blue-green by hanging drop method.
pigments produced by Pseudomonas “+ Culture smear is prepared by emulsifying a
aeruginosa bacterial colony with a drop of saline on a
2. Non-diffusible pigments: They do not slide
diffuse into surrounding media, hence * Then the smear is allowed to air-dry and then
only the colonies are colored, not the sur- subjected to Gram staining (Refer Chapter
rounding media; e.g., S. aureus producing 3.4)
golden-yellow colonies. * The morphological appearance of the
bacterium on Gram staining of culture smear
Hemolysis on Blood Agar may sometimes give a preliminary clue about
Certain bacteria produce hemolysin enzymes that the identification of the organism (Table 3.1.1,
lyse the red blood cells surrounding the colonies on
Chapter 3.1).
blood agar, forming a zone of hemolysis (Fig. 3.7.1).
Hemolysis may be:
Q Partial or a hemolysis: Partial clearing of
Demonstration of Bacterial Motility
blood around the colonies occurs with green Bacteria can be further differentiated based on
discoloration of the surrounding medium; outline whether they are motile or nonmotile (Table
of the RBCs is intact (e.g., pneumococci, viridans can be further
3.7.1). Even motile bacteria
streptococci)
differentiated based on the type of motility they
produce (Table 3.7.2). Bacterial motility can be
Contd...

tested by various methods.

* Cragie tube method
* Semisolid medium, e.g., mannitol motility
medium
Swarming of the bacteria on agar plate
¢ hemolysis “ Dark-ground microscopy
“+ Phase-contrast microscopy.

Table 3.7.1: Examples of motile and nonmotile bacteria.

Motile bacteria Nonmotile bacteria
Escherichia coli Klebsiella
Proteus Shigella
Salmonella Acinetobacter
Fig. 3.7.1: Hemolysis on blood agar. Vibrio
of
Source: Department of Microbiology, Pondicherry Institute Pseudomonas
Medical Sciences, Puducherry (with permission).
 SECTION 1 ©@ General Microbiology, Immunology and Hospital Infection Control

Table 3.7.2: Various types of motility. the true motility exhibited by motile bacteria
which needs to be differentiated from passive
Types of motility and Brownian movements shown by nonmotile
Tumbling motility Listeria
bacteria.
Gliding motility Mycoplasma
* Active movement: Organism move in
Stately motility Clostridium
different directions and change their positions
Darting motility Vibrio cholerae
in the field
Swarming motility Proteus, Clostridium tetani
** Passive movement: Drifting of the organisms
Corkscrew motility Spirochete in the same direction along the convectional
current in the fluid
Hanging Drop Method * Brownian movement: It is an oscillatory
Hanging drop preparation is one of the most movement about a nearly fixed point
common and easiest method to demonstrate possessed by all small bodies suspended
bacterial motility. The procedure is explained in fluid and due to irregularities in their
in Figure 3.7.2. After the drop is prepared on a bombardments by molecules of water.
coverslip and kept over the cavity slide, the edge of
the drop is focussed. The edge is focused because: @ CULTURE IDENTIFICATION
** Better contrast at the edge: Due to difference
Identification of bacteria from culture is
in the refractive index of the drop and the
confirmed by performing either by conventional
cover slip
biochemical tests or by automated identification
** As the drop hangs, it thins towards the edge,
systems.
containing less number of bacteria and less
overcrowding; hence motility can be clearly Biochemical Identification
appreciated
** Live aerobic bacteria come towards the edge Based on the type of colony morphology and
to get more oxygen for respiration. Gram staining appearance observed in culture
smear, the appropriate biochemical tests are
Types of Bacterial Motility employed.
1. Initially, catalase and oxidase tests are done
Three types of movements can be noticed in a
hanging drop preparation. Active movement is
on all types of colonies grown on the media
two For gram-negative bacilli: The following
are the common biochemical tests done
1. Take a clean
grease free routinely, abbreviated as ‘ICUT’:
cavity slide = Indole test
and cover slip
= Citrate utilization test
= Urea hydrolysis test
2. Place a drop of a
= ‘Triple sugar iron test (TSI).
TEccsE broth culture of an
organism on the 3. For gram-positive cocci: The useful
Vaseline + center of a cover slip biochemical tests are as follows:
3. Apply petroleum = Coagulase test (for Staphylococcus aureus)
=
be jelly/vaseline at = CAMP (Christie-Atkins-Munch-Petersen)
a cio ET the four corners
FEET
of the cover slip test for group B Streptococcus
= Bile esculin hydrolysis test (for
4. Place the cavity slide
over the coverslip, Enterococcus)
so that the drop lies at = Inulin fermentation test and bile solubility
the center of the cavity
test (for pneumococcus)
5. Invert the slide and = Antimicrobial susceptibility tests done for
focus the edge of bacterial identification are as follows:
the drop under 10x
and then under 40x ¢ Optochin susceptibility test—done to
objective and observe differentiate pneumococcus (sensitive)
Fig. 3.7.2: Hanging drop method (procedure). from viridans streptococci (resistant)
 CHAPTER 3.7 © General Bacteriology: Identification of Bacteria

Negative Indole Test

Positive
It detects the ability of certain bacteria to
produce an enzyme tryptophanase that breaks
down amino acid tryptophan present in the
medium into indole.
“+ When Kovac’s reagent is added to an overnight
Fig. 3.7.3: Catalase test. incubated broth of a bacterial colony, it
Source: Department of Microbiology, JIPMER, Puducherry complexes with indole to produce a cherry
(with permission). red color ring near the surface of the medium
* Indole positive (Fig. 3.7.4B): A red-colored
ring is formed near the surface of the broth.
¢ Bacitracin susceptibility test—done to
Examples include Escherichia coli, Proteus
differentiate group A (sensitive) from
vulgaris, Vibrio cholerae, etc.
group B (resistant) Streptococcus.
* Indole negative (Fig. 3.7.4B): Yellow-colored
Some of the important biochemical tests
ring is formed near the surface of the broth,
are described below. Coagulase test and other
e.g., Klebsiella pneumoniae, Proteus mirabilis,
biochemical reactions for gram-positive cocci
Pseudomonas, Salmonella, etc.
are described in the respective chapters.
Citrate Utilization Test
Catalase Test
It detects the ability of a few bacteria to utilize
Whenacolony ofany catalase producing bacteria
citrate as the sole source of carbon for their
is mixed with a drop of hydrogen peroxide
growth, with production of alkaline metabolic
(3% H,O,) placed on a slide, effervescence or
products. Test is performed on Simmons citrate
bubbles appear due to breakdown of H,O, by
medium. Citrate utilizing bacteria produce
catalase to produce oxygen (Fig. 3.7.3).
growth and a color change, i.e., original green
“ Catalase test is primarily used to differenti- color changes to blue (Fig. 3.7.5A).
ate between Staphylococcus (catalase positive)
* Citrate test is positive for Klebsiella
from Streptococcus (catalase negative)
pneumoniae, Citrobacter, Enterobacter, etc.
“ It is also positive for members of the
* The test is negative for Escherichia coli,
families Enterobacteriaceae, Vibrionaceae,
Shigella, etc.
Pseudomonadaceae, etc.
Urea Hydrolysis Test
Oxidase Test
Urease producing bacteria can split urea present
It detects the presence of cytochrome
in the medium to produce ammonia that makes
oxidase enzyme in bacteria, which catalyzes the medium alkaline.
the oxidation of reduced cytochrome by “* Test is done on Christensen’s urea medium,
atmospheric oxygen.
which contains phenol red indicator that
“ When a filter paper strip or disk, soaked in
oxidase reagent is smeared with a bacterial
colony producing cytochrome oxidase | Positive Negative
enzyme, the smeared area turns deep purple
within 10 seconds due to oxidation of the Positive Negative

dye to form a purple-colored compound

indophenol blue
“ Interpretation (Fig. 3.7.4A) and examples:
m Oxidase positive (deep purple):
Examples include Pseudomonas, Vibrio,
Neisseria, Bacillus, Haemophilus, etc. Al aw
m Oxidase negative (no color change): Figs 3.7.4A and B: (A) Oxidase test; (B) Indole test.
Examples include; members of family Source: Department of Microbiology, Pondicherry Institute of
Medical Sciences, Puducherry (with permission).
Enterobacteriaceae, Acinetobacter, etc.
 SECTION 1 ©@ General Microbiology, Immunology and Hospital Infection Control

Citrate Urease Interpretation

Positive Negative Positive Negative TSI detects three properties of bacteria, which
includes fermentation of sugars to produce acid
and/or gas and production of H,S (Figs 3.7.6A to
F and Table 3.7.3).
* Acid production: If acid is produced,
the medium is turned yellow from red.
Accordingly the organisms are categorized
into three groups:
1. Nonfermenters: They do not ferment any
sugars, hence the medium (both slant and
Figs 3.7.5A and B: (A) Citrate utilization test;
butt) remain red, producing Alkaline slant/
(B) Urea hydrolysis test. Alkaline butt (K/K) reaction (Fig. 3.7.6F);
Source: Department of Microbiology, Pondicherry Institute of e.g., Pseudomonas and Acinetobacter
Medical Sciences, Puducherry (with permission). 2. Glucose only fermenters: They ferment
only glucose and produce little acid only
changes to pink color in alkaline medium at the butt, whereas the slant remains
(Fig. 3.7.5B) alkaline giving rise to Alkaline slant/
* Urease test is positive for: Klebsiella Acidic butt (K/A) reaction (Fig. 3.7.6C);
pneumoniae, Proteus species, Helicobacter e.g., Salmonella and Shigella
pylori, Brucella, etc. 3 . 22 sugars fermenters: They ferment
** Urease test is negative for: Escherichia coli, glucose and also ferment lactose and/or
Shigella, Salmonella, etc. sucrose to produce large amount of acid
so that the medium (both slant and butt)
Triple Sugar Iron (TSI) Agar Test change to yellow giving rise to Acidic
TSI is a very important medium employed widely
for identification of gram-negative bacteria. Table 3.7.3: Various reactions in TS! with examples.
TSI medium contains three sugars—glucose,
sucrose and lactose in the ratio of 1:10:10 parts.
Uninoculated TSI medium is red in color; has a Acidic slant/acidic butt 22 sugars fermented (1)
slant and a butt (Fig. 3.7.6A). After inoculation, glucose, (2) lactose or/
and sucrose
the medium is incubated at 37°C for 18-24 hours.
A/A, gas produced, no H,S Escherichia coli
(Fig. 3.7.6B) Klebsiella pneumoniae
Un- K/A K/A K/A K/K Alkaline slant/acidic butt Only glucose-fermenter
inocula- gas-, gas-, gas-, gas-, group
ted H,S- H,S+ H,St+ H,S-
K/A, no gas, no H,S Shigella
4 | My 1 (Fig. 3.7.6C)
i
K/A, no gas, H,S produced Salmonella Typhi
(small amount) (Fig. 3.7.6D)
K/A, no gas, H,S produced Proteus vulgaris
(abundant) (Fig. 3.7.6E)
K/A, gas produced, H,S Salmonella Paratyphi B
produced (abundant)
K/A, gas produced, no H,S Salmonella Paratyphi A
gas HS Alkaline slant/alkaline Non-fermenters group
A 'B b) a* @ butt
Figs 3.7.6A to F: Triple sugar iron test.
K/K, no gas, no H,S Pseudomonas,
Source: Department of Microbiology, JIPMER, Puducherry
(with permission).
(Fig. 3.7.6F) Acinetobacter
 CHAPTER 3.7 © General Bacteriology: Identification of Bacteria

slant/Acidic butt (A/A) reaction (Fig. *% MicroScanWalkAway system (Beckman

3.7.6B); e.g., E. coli and Klebsiella. Coulter) for automated identification and
2, °
Gas production: If gas is produced, the antimicrobial susceptibility test.
medium is lifted up or broken with cracks
(Fig. 3.7.6B); examples, E. coli and Klebsiella MALDI-TOF
2, * H,S production: If H,S is produced, the
* MALDI-TOF technology (Matrix Assisted Laser Desor-
ption lonization Time-of-Flight) has revolutionized
medium changes color to black (Figs 3.7.6D the identification of organisms in clinical microbiol-
and E); e.g., Salmonella Typhi and Proteus ogy laboratories.
vulgaris. Q It can identify bacteria, fungi, and mycobacteria
with a turnaround time of few minutes and with
Automated Systems for Bacterial absolute accuracy
Q Two systems are commercially available: VITEK MS
identification (bioMérieux) and Biotyper system (Bruker).
Automated identification systems are revolu-
Principle (Fig. 3.7.7)
tionary in diagnostic microbiology. They have MALDI-TOF examines the pattern of ribosomal
several advantages—(i) produce faster result, proteins present in the organism.
(ii) can identify a wide range of organisms with Sample preparation: The colony of an organism is
accuracy, which are otherwise difficult to iden- smeared onto a well of the slide and one drop of matrix
tify (e.g., anaerobes) through conventional bio- solution (composed of cyano-hydroxy-cinnamic acid)
is added to the same well and mixed; then the slide is
chemical tests.
loaded in the system.
* MALDI-TOF (Matrix-assisted laser
Steps after loading: Overall, mass spectrometry
desorption/ionization time-of-flight), e.g.,
can be divided into three steps occurring in three
VITEK MS (bioMérieux): Refer the highlight chambers ofthe system.
box and Fig. 3.7.7 for details 1. lonization chamber: Here, the wells are irradiated
*% VITEK 2 (bioMérieux) for automated with the laser beam. The matrix absorbs the laser
identification and antimicrobial susceptibi- light causing desorption and ionization of bacterial
ribosomal proteins, generating singly protonated
lity test: Refer the highlight box and Figure
ions
3.7.8 for details 2. Analyzer: These ions are then accelerated into an
“ Phoenix (BD Diagnostics) for automated iden- electric field which directs them to the analyzer
tification and antimicrobial susceptibility test Contd...

Signal processing

(C) Detector ——
|

Time-of-flight
s Spectrum generated
Separation

; One colony —<——

is smeared | oe acy
Analyzer Qe © | Flight tube
onto a well | 4 of matrix
B \ 7 solution is
= of the slide
\’ added to
SS / the same well
maa Sania ead
lons accelerated in Le S.A ES
A) lonizationc +) e®,
Pee Gay i > 8 the electric field =. pcetete: @ueugtel aietere

See lonization and

PR
Bo eee
a
oe oe se 88
a
desorption cS ie
coating Slide is loaded Sample preparation

MALDI-TOF (VITEK-MS) ions to the system

Fig. 3.7.7: MALDI-TOF and its working principle.

permission).
Source: Department of Microbiology, JIPMER, Puducherry (with
 SECTION 1 © General Microbiology, Immunology and Hospital Infection Control

Contd...

chamber. The analyzer (mass spectrometer)

separates them according to their time-of-flight
(TOF) in the flight tube. The smaller molecules
travel faster, followed by the bigger, according to
the mass to charge (m/z) ratio
Detector: It converts the received ion into an
electrical current which is then amplified and
digitized to generate a characteristic spectrum
that is unique to a species due to its conserved
ribosomal proteins.
The test isolate is identified by
comparing its spectrum with a known database. Fig. 3.7.8: VITEK 2 system with its panels (reagent cards)
for identification and antimicrobial susceptibility test.
Source: Department of Microbiology, JIPMER, Puducherry
VITEK 2 Automated System (with permission).
VITEK 2 is the most widely used automated system in
India; can perform both identification and antimicrobial Contd...
susceptibility testing (AST) of bacteria and yeast.
Principle of VITEK for identification is discussed below, Q The reaction pattern obtained from the test
VITEK for AST is discussed in Chapter 3.8. organism is compared with the database and
Q It uses colorimetric reagent card containing the identification is reported with a confidence
64 wells; each well contains an individual test level of matching (excellent matching to the
substrate unidentified organism)
Q Separate cards are available for gram-negative, Incubation:
The cards are incubated in the system
gram-positive bacteria, fastidious bacteria and at 35.5 + 1°C. The reading is taken once every 15
yeasts (Fig. 3.7.8) minutes by the optical system of the equipment,
Q Substrates in the well measure various metabolic which measures the presence of any colored
activities, such as acidification, alkalinization, en- products of substrate metabolism (by advanced
zyme hydrolysis, etc., which helps in identification colorimetry method)
of the organism The result of identification is usually available
within 4-6 hours.
Contd...
 General Bacteriology:
Antimicrobial Susceptibility Test 3.8
ANTIMICROBIAL SUSCEPTIBILITY from stool is a commensal; hence, AST is not
performed.
TEST
Antimicrobial susceptibility test (AST) is the CLASSIFICATION OF AST METHODS
most important investigation carried out by a
Microbiology Laboratory. AST methods are classified into phenotypic and
* Bacteria exhibit great strain variations genotypic methods.
in susceptibility to antimicrobial agents. “+ The phenotypic methods are further grouped into:
Therefore, AST plays a vital role to guide = Disk diffusion method, e.g., Kirby-Bauer’s
the clinician for tailoring the empirical disk diffusion (DD) test
antibiotic therapy to pathogen-directed a Dilution tests: Broth dilution and agar
therapy dilution methods
“AST is performed only for pathogenic bacteria = Epsilometer or E-test
isolated from the specimen, and not for the ws Automated AST, e.g., Vitek, Phoenix and
commensal bacteria. For example, E. coli Microscan systems.
isolated from urine specimen should be “+ Genotypic methods, such as PCR detecting
subjected to AST, whereas E. coli isolated drug-resistant genes.

— DISK DIFFUSION METHOD

- Problem Solving Exercise 1
Disk Diffusion Test Explanation
Urine culture of a 23-year old female with increased 1. Antimicrobial susceptibility test (AST) performed
frequency and burning micturition yielded lactose- here is Kirby-Bauer’s disk diffusion (DD) test and
fermenting colonies which was identified as the medium used is Mueller Hinton agar (Fig.
Escherichia coli. The antimicrobial susceptibility test 3.8.1). The zone diameters were measured using
(AST) performed is demonstrated in Figure 3.8.1. The Vernier caliper (Fig. 3.8.2).
zone size diameter recorded by using an instrument 2. The observed zone when compared with CLSI's
(Fig. 3.8.2) revealed ceftriaxone (23 mm), ciprofloxacin zone interpretation chart (Table 3.8.1) revealed
(8 mm), gentamicin (10 mm), amikacin (16 mm), that the causative urinary pathogen is resistant
to gentamicin and ciprofloxacin, intermediate
meropenem (28 mm). Interpret the result according
to amikacin and sensitive to ceftriaxone and
to CLSI zone interpretation criteria as mentioned in
Table 3.8.1. meropenem.
1. Name the AST method performed, the medium Ceftriaxone is a first-line (narrow spectrum) antibiotic
used and the instrument used to measure zone whereas meropenem is a restricted antimicrobial
diameter? agent (extended spectrum). Therefore, ceftriaxone
2. Interpret the test result and suggest the would be the antibiotic of choice.
antimicrobials recommended for treatment.
 SECTION 1 © General Microbiology, Immunology and Hospital Infection Control

Kirby-Bauer’s disk diffusion (DD) test is the most

widely used AST method. They are suitable for
rapidly growing pathogenic bacteria; however, Zone of
inhibition,
they are not suitable for slow growing bacteria. It Sensitive
is mostly performed from colony (called colony- (inter-
pretation)
DD), or performed directly from the specimens
(called direct DD).

Procedure (Colony Disk Diffusion) Resistant

(inter-
Antibiotic disks are impregnated on to a suitable pretation)
medium lawn cultured with the test isolate.
“+ Antibiotic disks: Antibiotic disks are available Fig. 3.8.1: Kirby—Bauer disk diffusion method.
Source: Department of Microbiology, Pondicherry Institute of
commercially or prepared in-house. Sterile
Medical sciences, Puducherry (with permission).
filter paper disks of 6 mm diameter are
impregnated with standard quantity of
antibiotic solution
“+ Medium: Mueller-Hinton agar (MHA) is the
standard medium used for AST. For certain
fastidious organisms, such as S. pyogenes and
S. pneumoniae, Mueller-Hinton blood agar
(MHBA) containing 5% of sheep blood is used Fig. 3.8.2: Vernier caliper.
* Inoculum: The inoculum is prepared by—(1)
the disk, which can be measured by using
directly suspending the colony in the normal
Vernier caliper (Fig. 3.8.2)
saline or (2) by inoculating into a suitable
** The interpretation of zone size into sensitive,
broth and incubating at 37°C for 2 hours
intermediate or resistant is based on the
** Turbidity: The turbidity of the inoculum is
standard zone size interpretation chart,
adjusted to 0.5 McFarland opacity standard,
provided by CLSI or EUCAST guidelines
which is equivalent to approximately 1.5 x 10°
(Table 3.8.1).
CFU/mL of bacteria
** Lawn culture: The broth is then inoculated Note: CLSI (Clinical and Laboratory Standards
on to the medium by spreading with sterile Institute) and EUCAST (European Committee
swabs on Antimicrobial Susceptibility Testing) are
* Disks impregnation: After MHA plate is dried international agencies, which provide guidelines
(3-5 min), the antibiotic disks are placed and for zone size interpretation (Table 3.8.1), and are
gently pressed on its surface. Disks should be updated annually.
placed atleast 24 mm (center to center) apart
on the MHA plate. Ordinarily, maximum up Direct Disk Diffusion Test
to 6 disks can be applied on a 100 mm plate The direct DD (or direct susceptibility test,
(Fig. 3.8.1) i.e., DST) test can be performed when results
** Incubation: The plates are then incubated at are required urgently and single pathogenic
37°C for 16-18 hours and then interpreted. bacterium is suspected in the specimen (for
positively-flagged blood culture bottle, sterile
Interpretation body fluids or urine).
The antibiotic in the disk diffuses through the * Here, the specimen is directly inoculated
solid medium, so that the concentration is highest uniformly on to the surface of an agar plate
near the site of application of the antibiotic disk and the antibiotic disks are applied
and decreases gradually away from it * The results of the direct-DD test should always
* Susceptibility to the drug is determined by the be verified by performing AST from the colony
zone ofinhibition of bacterial growth around subsequently
 ‘ayninsu| spiepueys Asoyeroge7 pue jed!ul|D ‘|S1D :uonDInaiqqy
91-8 v> vls> Ol-SL JN O€ (snd2030/a}UJ) UIDAWWODUe/A
Cox
C= 07> GANG €T=< O€ (SN320I0J42e}UJ) PIjOZeuly
8< v
od ~

85 OLS z Bee OL (sn22020/9}UJ) UljjIn1Idwuy

= > [=

OL = 4
8-7 TS = x - - (snainb °s) ulpAWoouUe/,
9L<
s

vs 07> ~ L7< OE (snainbd *S) pljozeury

=
-

8<
”nuvi) fou,

8 vs bL> 8L-SL 6L< O€ (snasnb *S) aulj2ADe4}9]

9L<
”ns

9L/v < = 8e/7 > OL> GI=LL 9L< SLEC/ST L (snasnbD *s) 3|OZeXOUW1130
CHAPTER 3.8 © General Bacteriology Antim ox =° 2 Ss

y< 6 LS SLS 8L-9L 6L< S (SNaJND *S) UIDEXOYJOAS]

v> Lc> = C7 < O€ (snaJnD *S) UIXOJaD
8< Fi
sjuabe jeiqoiiwuy —
v= Ge = = - = = Ulys!|OD
v< C L> 6L> C7-07 (ae = OL Wauedolay|
9< Ce 9L> vl> 9L-SL Zine O€ ulpexiuuy
8ZL < v9-CE 9L> LL> 0¢-81L ica OL/OOL wueydeqoze}-uUl||!De1adig
SO ScTO> Lc> SC-CC 97 < S ulpexoyodid
L<
| C L> 6L> CC-0C €7< O€ SUOXeELYSD
ve
giz Gas 8 v> AS 0¢-81 Le < O€ suwIpizeyjo)
squabe jeiqooiunuy
‘(QUIJaPINB OZOZ |S1D Jad Se) 1Sa} UOISNYIP ysSIP JO UO!e}aIdJa}U! PUL SUO!}4]UBIUOD ¥SIP pasn A|UOWUWOD 2 L°8"E FIGeL
 SECTION 1 ©@ General Microbiology, Immunology and Hospital Infection Control

Test organism is inoculated in tubes containing serial

7
Sg This test is of no use when mixed growth is
dilutions of an antibiotic
suspected in the specimen, e.g., pus, stool,
Antibiotic conc (g/mL)
sputum, etc. 64 Ps ills) 8 4 2 1 Control

@ DILUTION TESTS
Here, the antimicrobial agent is serially diluted,
each dilution is tested with the test organism for
antimicrobial susceptibility test and the MIC is
calculated.
og
+,
MIC (minimum inhibitory concentration) is Subcultures on
solid media Turbidity indicates growth
the lowest concentration of an antimicrobial
agent that will inhibit the visible growth of a MIC = 8 pg/mL
microorganism after overnight incubation MBC = 32 pg/mL
+, o
Depending upon whether the dilutions of the
antimicrobial agent are made in agar or broth,
there are two types of dilution tests. No growth

Fig. 3.8.3: Macrobroth dilution method.

Broth Dilution Method Source: Department of Microbiology, Pondicherry Institute of
Medical Sciences, Puducherry (with permission).
Itis of two types: macrobroth dilution (performed
in tubes) and microbroth dilution (performed in
microtiter plate). The procedure of macrobroth visible growth, i.e., broth appears clear (Fig.
dilution is explained below: 3.8.3)
“
+,
Serial dilutions of the antimicrobial agent in The minimum bactericidal concentration
Mueller-Hinton broth are taken in tubes and (MBC) can be obtained by subculturing
each tube is inoculated with a fixed amount from each tube (showing no growth) on to a
of suspension of the test organism. A control nutrient agar plate without any antimicrobial
organism of known sensitivity should also be agent. The tube containing the lowest
tested. Tubes are incubated at 37°C for 18 hours concentration of the drug that fails to show
The MIC is determined by noting the lowest growth, on subculture, is the MBC ofthe drug
concentration of the drug at which there is no for that test strain (Fig. 3.8.3).

| Problem SolvingExercise2
Solving Exercise 2 _|
Broth Dilution Test grown (turbidity) in the test tubes containing
Blood culture of a 77 years old man with fever, amikacin <4 g/mL; whereas it did not grow in
hypotension, increased respiratory rate and test tubes containing amikacin =>8 ug/mL (no
altered mentation yielded Klebsiella pneumoniae. turbidity). Therefore, the MIC of amikacin for the
Antimicrobial susceptibility test by macro broth test isolate is 8 ug/mL. MIC (minimum inhibitory
dilution was performed for amikacin (Fig. 3.8.3). concentration) is the minimum concentration
Observe the finding and interpret the result using of the drug that inhibits the growth of the test
CLSI interpretation criteria (Table 3.8.1). isolate
CLSI interpretation criteria for amikacin as
Explanation given in Table 3.8.1 shows that MIC of >64 tg/
History of fever, hypotension, increased respiratory mL is considered resistant, 32 ug/mL is taken
rate and altered mentation suggests that it is as intermediate and <16 ug/mL is considered
suspected case of sepsis. Blood culture yielded susceptible. Therefore, the result of amikacin
Klebsiella pneumoniae. susceptibility test by macro broth dilution is
Q AST of amikacin by macro broth dilution test susceptible (MIC 8 g/mL).
(Fig. 3.8.3) revealed that the test isolate has
 CHAPTER 3.8 © General Bacteriology: Antimicrobial Susceptibility
Test C53

_ Problem Solving Exercise 3

Agar Dilution Test D vancomycin. The MIC (minimum drug concentration
Antimicrobial susceptibility test of vancomycin _Which inhibited the growth of the organism) and the
was performed by agar dilution method for four AST interpretation (according to CLSI interpretation
isolates of Staphylococcus aureus (Fig. 3.8.4). Observe _<titeria, Table 3.8.2) is as follows.
the findings and interpret the result using CLS! © Isolate 1: The MIC is <2 ug/ml (sensitive)
interpretation criteria (Table 3.8.2). Q Isolate 2: The MIC is 16 pg/mL (resistant)
Q Isolate 3: The MIC is 8 ug/mL (Intermediate)
Explanation Q Isolate 4: The MIC is >16 g/mL (resistant).
Fig. 3.8.4 shows agar dilution for MIC detection
of four isolates of Staphylococcus aureus against

Agar Dilution Method B EPSILOMETER OR E-TEST

Here, the serial dilutions of the drug are prepared This is a quantitative method of detecting MIC
in molten Mueller-Hinton agar (MHA) and by using the principles of both dilution and
poured into Petri dishes. The test strain is spot diffusion of antibiotic into the medium.
inoculated. If growth occurs at the area of spot ¢* It uses an absorbent strip containing pre-
inoculum in a MHA plate added with a particular
defined gradient (serial dilution) of anti-
concentration of an antibiotic, suggests that
biotic concentration immobilized along
the strain is resistant to the antibiotic at that :
concentration (Fig. 3.8.4, Table 3.8.2). This its length
* Itisappliedtoalawn inoculum ofabacterium.
+,*
method is more convenient than broth dilution
and has the added advantage of: Following incubation of the test organism,
“ Several strains can be tested at the same time an elliptical zone of inhibition is produced
by using the same plate surrounding the strip
** It directly measures the MBC; there is no * The antibiotic concentration at which the
need of sub-culturing as it is done with broth ellipse edge intersects the strip, is taken as
dilution method. MIC value (Fig. 3.8.5).

Vancomycin 16 pg/mL Vancomycin 8 j1g/mL Vancomycin 4 pg/mL Vancomycin 2 ug/ml

Pn
me

us aureus against vancomycin.

Fig. 3.8.4: Agar dilution for minimum inhibitory concentration (MIC) detection of Staphylococc
,g/mL (Intermediate); Isolate 4: MIC >16 g/mL
[Isolate 1: MIC- <2 ug/ml (sensitive); Isolate 2: MIC-16 g/mL (resistant); Isolate 3: MIC-8
guideline (Table 3.8.2)].
(resistant); Interpretation is based on Clinical and Laboratory Standards Institute (CLS!)

categories and breakpoints for

Table 3.8.2: Clinical and Laboratory Standards Institute (CLSI) interpretative
vancomycin susceptibility for Staphylococcus aureus.

Minimum inhibitory concentration | Interpretation Organism is labeled as

<2 g/mL Sensitive Vancomycin-sensitive Staphylococcus aureus

4-8 g/mL intermediate Vancomycin-intermediate Staphylococcus aureus

>16 ug/mL Resistant Vancomycin-resistant Staphylococcus aureus

 SECTION 1 © General Microbiology, Immunology and Hospital Infection Control

Table 3.8.3: Antibiotic panel used in VITEK AST card

for Enterobacteriaceae.
Entero- Antimicrobial agent used in VITEK
bacteriaceae
First line Ampicillin, Amoxicillin-clavulanic acid,
Ciprofloxacin, Ceftriaxone, Ceftazidime,
Gentamicin, Cotrimoxazole
Secondline Cefoperazone-sulbactam, Piperacillin-
tazobactam, Cefepime, Amikacin
Restricted Meropenem, Doripenem, Ertapenem,
Imipenem, Colistin, Tigecycline

0.5 McFarland turbidity) is added to the wells

(Fig. 3.7.8, Chapter 3.7 and Table 3.8.3)
“+ The cards are incubated in the system at
35.5 +1°C. The reading is taken once in every
15 minutes by the optical system of the
Fig. 3.8.5: Epsilometer or E-test.
equipment. It measures the presence of any
Source: Department of Microbiology, Pondicherry Institute of
Medical Sciences, Puducherry (with permission). turbidity (by nephelometry) which indicates
the organism has grown in that antibiotic well
AUTOMATED ANTIMICROBIAL “+ The MIC is determined as the highest dilution
of the antimicrobial agent which inhibits the
SUSCEPTIBILITY TESTS
growth of organism and there is no turbidity
Several automated systems are available now, in the well
such as: “+ The results are available within 8-10 hours
** VITEK 2 identification and antimicrobial for gram-negative bacilli and 16-18 hours for
sensitivity system (bioMerieux) gram-positive cocci.
“+ Phoenix System (Becton Dickinson)
“+ Micro Scan Walk Away system. Role of MIC-based Methods
Most systems are computer assisted and have The clinical microbiology laboratory should
sophisticated softwares to analyze the growth perform a MIC-based method whenever
rates and determine the antibiotic susceptibility possible. This is because the MIC-based
report. They work by the principle of microbroth methods are much superior to disk diffusion test
dilution. They use commercially available for a number of reasons.
panels that contain antibiotic solution in serial * For confirming the AST results obtained by
dilutions. They provide more rapid results disk diffusion tests, as they are more reliable
compared with traditional methods. and accurate than the latter
** AST for bacteria for which disk diffusion
VITEK 2 Automated System for AST test is not standardized should only be
VITEK 2 is the most widely used automated AST performed by MIC testing (e.g., vancomycin
system in India; can perform AST of bacteria for S. aureus)
and yeasts; whereas other automated AST ** For performing AST for slow growing bacteria,
systems can perform AST of bacteria only, not such as tubercle bacilli
for yeasts. * To select the most appropriate antibiotic:
* It works on the principle of microbroth Lower is the MIC, better is the therapeutic
dilution efficacy
* It uses a reagent card containing 64 wells, * MIC-guided therapy: There are certain
which contain doubling dilution of antimi- situations (e.g., endocarditis), where the
crobial agents. The organism suspension (of antibiotic treatment is MIC-guided.
 CHAPTER 3.8 © General Bacteriology: Antimicrobial Susceptibility
Test

B INTERPRETATION OF AST indicated either as a first-line agent or

alternative-agent for the suspected infective
The result of AST (whether disk diffusion or syndrome and the organism isolated
MIC-based methods) is always expressed in four “* Organism isolated, for which the AST is going
interpretative categories. to be performed, and its local resistance
* Susceptible (S): Indicates that the antibiotic pattern (as per hospital antibiogram)
is clinically effective when used in standard “+ Intrinsic resistance: The antibiotics to which
therapeutic dose the isolate is intrinsically resistant must be
** Intermediate (I): Indicates that the antibiotic excluded from the test panel
is not clinically effective when used in “* Antimicrobial agent: Both oral and parenteral
standard dose; but may be active when used antibiotics should be included in the testing
in increased dose. Antibiotics reported as ‘T’ panel, as they can be administered based on
should be avoided for treatment if alternative clinical severity (i.e parenteral drugs for severe
agents are available and oral for mild illness)
“> Resistant (R): Indicates that the antibiotic * Locally available antibiotic at the hospital
is NOT clinically effective when used in “+ Site of action: Only those antibiotics should
either standard dose or increased dose; be tested which are active in the site. For
and therefore should not be included in the example, the following antibiotics should be
treatment regimen. EXCLUDED from testing, depending upon
the clinical specimen. For e.g., clindamycin,
macrolides, tetracyclines should be excluded
CHOICE OF ANTIBIOTICS TO BE
from testing.
INCLUDED IN PANEL Selective or cascade reporting aims at
The panel of the drugs to be tested against an encouraging the clinicians to use narrow
isolate depends upon various factors: spectrum antimicrobials if found susceptible
* Clinically indicated: Include only those and to reserve the overuse of broad and extended
antibiotics for testing which are clinically spectrum antimicrobials (Table 3.8.3).
 | CHAPTER |

General Bacteriology: > 9

Molecular Diagnosis

In the modern era of diagnostic microbiology, * Polymerase chain reaction (PCR)

the molecular methods play a very important * Real-time polymerase chain reaction (rt-
role in the diagnosis of infectious diseases. They PCR)
can be used for the detection of the nucleic acid “+ Automated PCR, such as Biofire FilmArray
of the organism either directly from the clinical “+ Automated real-time PCR, such as cartridge-
specimen or from the culture. based nucleic acid amplification test
Molecular methods commonly used in (CBNAAT): Used for tuberculosis, described
diagnostic microbiology laboratory are: in Chapter 35.

§ POLYMERASE CHAIN REACTION (PCR)

[Problem Solving
Solving Exercise
Exercise _
Polymerase Chain Reaction Explanation
CSF specimen of four patients with suspected 1. Figure 3.9.1Cis a photograph ofgel electrophoresis
tuberculous meningitis are sent for PCR for M. of the PCR amplified products. It reveals that
tuberculosis. The results of which have been depicted visible bands of amplified DNA in line 1 and 2;
Ha) Fike}, SAC. whereas line 3 and 4 did not show any bands.
ile Interpret the result. Therefore, test specimens 1 and 2 are positive for
De What are the steps involved in PCR? gene specific for M. tuberculosis.
3 What is the modification needed to carryout 2. For steps involved in PCR refer the text.
dengue PCR? 3. As dengue virus is an RNA virus, reverse tran-
Which modification of PCR is useful for the scriptase PCR needs to be performed to detect
diagnosis of the infectious syndromes (e.g., dengue virus specific RNA in clinical specimen.
pneumonia) that can be caused by more than one 4. Multiplex PCR is useful for the diagnosis of the
organism? infectious diseases that are caused by more than
one organism.

PCR is a technology in molecular biology used to Contd...

amplify a single or few copies of a piece of DNA
(Fig. 3.9.1A). The extracted DNA is subjected to
to generate millions of copies of DNA. repeated cycles (30-35 numbers) of amplification
which takes about 3-4 hours. Each amplification
Principle of PCR cycle has three steps, such as (1) Denaturation at
PCR involves three basic steps. 95°C (2) Primer annealing (55°C) (3) Extension of
‘le DNA extraction from the organism: This involves the primer (72°C) by Taq Polymerase enzyme.
lysis of the organisms and release of theDNA which 3. Gel electrophoresis of amplified product: The
may be done by various methods—boiling, adding amplified DNA is electrophoretically migrated
enzymes (e.g., proteinase K) or by DNA extraction according to their molecular size by performing
kit. agarose gel electrophoresis (Fig. 3.9.1B). The
2, Amplification of extracted DNA: This is carried amplified DNA forms clear band, which can
out in a special PCR machine called thermocycler be visualized under ultraviolet (UV) light (Fig.
BOG)
Contd...
 CHAPTER 3.9 © General Bacteriology: Molecular Diagnosis

tel |
ame |iM
* Lines 1 and 2 show visible bandsofamplified DNA.
+ Lines 3 and 4 are negative for the DNA band

Figs 3.9.1A to C: (A) Thermocycler machine (Eppendorf); (B) Gel electrophoresis of amplified product;
(© Visualization of amplified DNA under UV light.
Source: Department of Microbiology, JIPMER, Puducherry (with permission).

Applications of PCR may be extracted from viable or nonviable

organism
PCR is now a common and often indispensable
“* False-positive amplification: [t may occur
technique used in medical diagnostics and
due to contamination with environmental
research laboratories for a variety of applications.
DNA. Hence, strict asepsis should be
It has the following advantages compared to the
maintained in the PCR laboratory
conventional culture methods:
“+ False-negative: The PCR inhibitors present
“ More sensitive: It can amplify very few copies
in some specimens, such as blood, feces,
of a specific DNA, so it is more sensitive
etc., may inhibit the amplification of target
“ More specific: Use of primers targeting
DNA.
specific DNA sequence of the organism makes
the PCR assays highly specific Modifications of PCR
“+ PCR can be done to amplify the DNA of the
organism: (1) either directly from the sample, 1. Reverse transcriptase PCR (RT-PCR): It
is useful for for amplifying RNA. After RNA
or (2) to confirm the organism grown in culture
extraction, the first step is addition of reverse
“ PCR can also detect the organisms that
are highly fastidious or noncultivable by transcriptase enzyme that coverts RNA into
DNA. Then, the remaining steps are similar
conventional culture methods
* PCR can be used to detect the genes in the as that for conventional PCR. It is useful for
organism responsible for drug resistance detection of RNA viruses from clinical specimen
(e.g., mec A gene detection in Staphylococcus 2. Nested PCR: In nested PCR two rounds of
aureus). PCR amplification are carried out by using two
primers that are targeted against two different
Disadvantages of PCR DNA sequences ofthe same organism
w It is more sensitive (yields high quantity
Conventional PCR detects only the DNA, but
of DNA), more specific (uses two primers
not the RNA (latter can be detected by reverse
transcriptase PCR). targeting two regions of DNA of the same
“* Qualitative, not quantitative: Conventional
organisms)
= Application: It is used for detection of
PCR can only detect the presence or
absence of DNA. It cannot quantitate the Mycobacterium tuberculosis (targeting
1S6110 gene) in samples.
amount of DNA of the organism present
in the sample. This is possible by real time 3. Multiplex PCR: It uses more than one primer
PGR which can detect many DNA sequences of
several organisms in one reaction
“ Viability: PCR cannot differentiate between
viable or nonviable organisms. It only detects = Syndromic approach: Multiplex PCR is
the presence of DNA in the sample which useful for the diagnosis of the infectious
 SECTION 1 ©@ General Microbiology, Immunology and Hospital Infection Control

diseases that are caused by more than one

organism
= For example, for the etiological diagnosis
of pyogenic meningitis, different primers
targeting the common agents of pyogenic
meningitis, such as pneumococcus,
meningococcus and H. influenzae can be
added simultaneously in the same reaction
tube.

Biofire FilmArray
Biofire FilmArray (bioMérieux) is a completely
automated multiplex nested PCR system
where all the steps from sample preparation
to amplification, detection and analysis are Fig. 3.9.2: Real-time PCR.
performed automatically by the system; giving Source: Department of Microbiology, JIPMER, Puducherry
result in about one hour. It has excellent (with permission).

sensitivity and specificity.

** Contamination rate is extremely less
** Sensitivity and specificity of rt-PCR assays
@ REAL-TIME PCR (RT-PCR) are much more than the conventional
It is based on PCR technology, which is used to BERe
amplify and simultaneously detect or quantify Detection of amplification products of real-
a targeted DNA molecule on a real-time basis. time PCR: The detection of amplified nucleic
Reverse transcriptase real-time PCR formats can acid in a real-time PCR reaction is carried out by
detect and quantify RNA molecules of the test using a variety of fluorogenic molecules which
organism in the sample on a real-time basis. may be either nonspecific or specific.
It uses a different thermocycler (Fig. 3.9.2) ** Nonspecific methods: They use SYBR green
than the conventional PCR. It is very expensive, dye that stains any nucleic acid nonspecifi-
5-10 times more than the cost of conventional cally
PCR. * Specific methods: They use fluorescent
Advantages: Real-time PCR has many labeled oligonucleotide probe which binds
advantages over a conventional PCR, such as: (i.e., hybridizes) only to a particular region
* Quantitative: rt-PCR can quantitate the of amplified nucleic acid. Three types of
DNA or RNA present in the specimen; hence hybridization probes are commonly used:
can be used for monitoring the disease = TaqMan or hydrolysis probe
progression in response to treatment, e.g., = Molecular beacon
viral load monitoring in HIV or hepatitis B = Fluorescence resonance energy transfer
viral infection (FRET) probe.
* Takes less time: In rt-PCR, the amplification Post-amplification melting curve analysis is
can be visualized simultaneously during used for quantitation of the nucleic acid load.
the process of amplification unlike the Problem based exercise on Real time PCR (for
conventional PCR where there is an extra-step influenza and COVID-19) has been explained in
of gel electrophoresis to detect the amplicons detail in Chapter 37.
 Laboratory Diagnosis of Viral Diseases

SSS RES

Table 4.1; Methods available for laboratory diagnosis

BINTRODUCTION
of viral diseases.
Laboratory diagnosis of viral infections is useful . Direct Demonstration of Virus
for the following purposes: Electron microscopy
“ To start antiviral drugs for those viral Fluorescent microscopy
infections for which specific drugs are Light microscopy:
oee=

available, such as herpes, CMY, HIV, influenza > Histopathological staining:Todemonstrate

inclusion bodies
and respiratory syncytial virus (RSV) > Immunoperoxidase staining
* Screening of blood donors for HIV, hepatitis
N. Detection of Viral Antigens
B and hepatitis C helps in the prevention of By various formats, such as ELISA, direct IF, ICT, flow
transfusion-transmitted infections through assays
* Surveillance purpose: To assess the disease WwW.
Detection of Specific Antibodies
burden in the community by estimating the e Conventional techniques, such as HAI and
prevalence and incidence of viral infections neutralization test
“ For outbreak or epidemic investigation, e.g., e Newer diagnostic formats, such as ELISA, ICT, flow
through assays
influenza epidemics, dengue outbreaks—to
initiate appropriate control measures 4. Molecular Methods to Detect Viral Genes
e Nucleic acid probe—for detection of DNA or RNA by
* To start post-exposure prophylaxis of
hybridization
antiretroviral drugs to the healthcare workers PCR—for DNA detection by amplification
following needle stick injury (Refer Chapter Reverse transcriptase-PCR—for RNA detection
14) Real time PCR—for DNA quantification
“ To initiate certain measures: For example, Real time RT-PCR—for RNA quantification
if rubella is diagnosed in the first trimester 5. Isolation of Virus by
of pregnancy, termination of pregnancy is e Animal inoculation
e Embryonated egg inoculation
recommended.
e Tissue cultures: Cell line culture using primary,
The various methods available for laboratory secondary or continuous cell lines
diagnosis of viral diseases are enlisted in Table
Abbreviations: ELISA, enzyme-linked immunosorbent assay; ICT,
4.1. immunochromatographic test; PCR, polymerase chain reaction;
HAI, hemagglutination inhibition test; IF, Immunofluorescence

SAMPLE COLLECTION
assay.

Specimen collection has to be done early in “* In the correct method of collection

the patient’s illness as possible, as viruses can “ In adequate volume (Fig. 4.1)
be recovered only for a few days after the onset “ In suitable containers (sterile and chemical
of illness. The following steps should be kept in free)—e.g., viral transport media
mind during specimen collection: “* Transport in correct temperature: Specimen
Appropriate time: Specimens should be to be kept at 4°C for 3-5 days, after which the
collected as soon as possible, preferably within sample should be kept at -70°C (Fig. 4.1)
3 days after onset of symptoms “ Correctly labeled (patient details and
specimen details).
* From the correct site (Table 4.2)
 SECTION 1 © General Microbiology, Immunology and Hospital Infection Control

Table 4.2: Various types of viral infections and specimen of choice.

Systemic infections Causative viral agents Specimens to be collected
Respiratory infections e Influenza virus Swabs (nasal, throat, nasopharyngeal)
e Parainfluenza virus Bronchoalveolar lavage
e Respiratory syncytial virus Nasal washings
e Rhinovirus Gargles
e Adenovirus Aspirates (nasal or sinus)
e Coronavirus Serum
Viral encephalitis e Herpes simplex virus e Cerebrospinal fluid
e Japanese encephalitis virus e Tissue by brain biopsy (postmortem)
e Rabies virus e Throat washings
e Stool
e Serum
Viral gastroenteritis Rotavirus e Stool
Adenovirus-40, 41 e Serum
Calicivirus
Astrovirus
Exanthematous infections Herpes simplex virus e Vesicle aspirate
e Varicella zoster virus e Skin scrapping
e Human papillomavirus Skin biopsy
e Molluscum contagiosum virus
Poliomyelitis e Polioviruses (I, Il, Ill) Throat swabs
Stool
Rectal swabs
Serum
Sexually-transmitted e Herpes simplex virus e Serum
infections ° Human papillomavirus
e Human immunodeficiency virus
Teratogenic viruses e Rubella virus e Serum
e Cytomegalovirus (CMV)
e Herpes simplex virus
e Varicella-zoster virus
e Parvovirus
Conjunctivitis e Adenovirus ¢ Conjunctival swab
e Enterovirus 70
e Coxsackie virus A-24
Vector-borne diseases e Dengue virus e Serum
Chikungunya virus
e Japanese encephalitis virus
Note: Blood sample is collected for serological tests and for molecular diagnostic tests (e.g., real time polymerase chain reaction for
estimation of viral load).

Viral Transport Medium ** Protective proteins (bovine serum albumin)

Viral transport medium (VTM) is a specially ** Antibiotics and fungicides to control microbial
formulated medium for collection, transport contamination, such as streptomycin,
and long-term freeze storage of viruses. Uses of amphotericin B, vancomycin, etc.
VTM are given in Figure 4.2. ** Phenol red: It is used as a pH indicator
Composition of VTM: Viral transport consists ** Distilled water to make 1 liter.
of modified Hanks’ balanced salt solution
supplemented with: i DIRECT DEMONSTRATION OF VIRUS
* Phosphate-buffered saline (PBS) for
adequate ion concentration—NaCl, KCl,
Electron Microscopy
CaCl,, Na,HPO,, KH,PO,, Mg,PO.,. It is used Detection of viruses by electron microscopy
to control the pH (EM) is increasingly used nowadays. Specimens
 CHAPTER4 © Laboratory Diagnosis of Viral Diseases

Uses of VIM |
* To preserve viral infectivity
within the specimen
* To prevent specimen from drying
* To prevent growth of contaminants
such as bacteria or fungi

Place in sterile Place in sterile Place in sterile

tube with 1-2 mL container of container with
VT™M 2-5mL of VTM 5-10 mL of VTM

Transport of laboratory at 2-8°C Fig. 4.2: Viral transport medium and rayon-tipped swabs
used for sample collection.
If storage exceeds > 5 days,
samples should be kept at-70°C = Adenovirus—space vehicle-shaped (Fig.
Fig. 4.1: Process of specimen collection and transport. 4.3E)
Abbreviations: VTM, viral transport medium; CSF, cerebrospinal
B Hepatitis B virus—occurs in three forms:
fluid; BAL, bronchoalveolar lavage. spherical form, tubular form and Dane
are negatively stained by potassium phospho- particle (Fig. 4.3F).
tungstate and scanned under EM. = Corona virus—possesses petal-shaped
* Shape: Viruses can be identified based on peplomers (Fig. 4.3G)
= Astrovirus—possesses star-shaped
their distinct appearances; for example:
= Rabies virus—bullet-shaped (Fig. 4.3A) peplomers.
= Rotavirus—wheel-shaped (Fig. 4.3B) * Direct detection from specimens: This is
= Ebola virus—filamentous (Fig. 4.3C) useful for viruses that are difficult to cultivate;
= Poxvirus—contains dumbbell-shaped e.g., rotavirus, hepatitis A and E viruses from
capsid (Fig. 4.3D) feces and CMV from urine

Rabies; (B) Rotavirus; (©) Ebola virus; (D) Poxvirus;

Figs 4.3A to G: Electron microscopy picture of: (A)
(E) Adenovirus; (F) Hepatitis B; (G) Corona virus.
1; (B) ID #15194;
Disease Control and Prevention, Atlanta. (A) ID #561
Source: Public Health Image Library, Centers for (G) ID #10270.
#10815; (D) ID #1849; (E) ID #237; (F) ID #5631;
(©) ID
 SECTION1 @ General Microbiology, Immunology and Hospital Infection Control

“ Virus detection from tissue culture: EM can Table 4.3: Inclusion bodies and viruses producing
also be used for detection of viral growth in them.
tissue cultures Intracytoplasmic inclusion bodies
* Drawbacks: EM is highly expensive, has Negri bodies—rabies virus (Fig. 4.4A)
low sensitivity with a detection threshold of Paschen body—variola virus
10’ virions/mL. The specificity is also low. Guarnieri bodies—vaccinia virus
Bollinger bodies—fowlpox virus
Fluorescent Microscopy Molluscum bodies—molluscum contagiosum virus (Fig.
4.4B)
Direct immunofluorescence (Direct-IF)
Intranuclear inclssionoo
technique is employed to detect viral particles
in the clinical samples. Cowdry type Ai
Al nclusions ae oe
* Procedure: Specimen is mounted on slide, Torres body—yellow ine virus
stained with specific antiviral antibody tagged Lipschultz body—herpes menpiexs!
virussis 4au)
with fluorescent dye and viewed under -Cowdry type Binclusions _
fluorescent microscope Poliovirus
** Clinical applications: Adenovirus
= Diagnosis of rabies virus antigen in skin Intracytoplasmic and intranuclear inclusion bodies
biopsies, corneal smear of infected patients Owl's eye appearance—cytomegalovirus (Fig. 4.4E)
m Syndromic approach: Rapid diagnosis of Measles virus (Fig. 4.4C)
respiratory infections caused by influenza
virus, rhinoviruses, respiratory syncytial Contd...
virus, adenoviruses and herpesviruses
Role in Laboratory Diagnosis
can be carried out by adding specific
Inclusion bodies are characteristic of specific viral
antibodies to each of these viruses infections. They have distinct size, shape, location
# Detection of adenovirus from conjunctival and staining properties by which they can be
smears. demonstrated in virus infected cells under the light
microscope.
Light Microscopy Location
Light microscopy is useful in the following They may be present either in the host cell cytoplasm
or nucleus or both (Table 4.3 and Figs 4.4A to E).
situations.
Q Intracytoplasmic inclusion bodies: They are
* Inclusion bodies: Histopathological staining generally acidophilic and can be seen as pink
of tissue sections may be useful for detection structures when stained with Giemsa or eosin
of inclusion bodies which helps in the diagno- methylene blue stains (e.g., most poxviruses and
sis of certain viral infections (Table 4.3) rabies)
Q Intranuclear inclusion bodies: They are
** Immunoperoxidase staining: Tissue sections
basophilic in nature. Cowdry (1934) had classified
or cells coated with viral antigens are stained them into:
using antibodies tagged with horseradish per- >» Cowdry type A inclusions: They are variable in
oxidase following which hydrogen peroxide size and have granular appearance
and a coloring agent (benzidine derivative) >» Cowdry type B inclusions: They are more
circumscribed, amorphous or hyaline spheres;
are added. The color complex formed can be
multiple in number.
viewed under a light microscope. Q Both intracytoplasmic and intranuclear
inclusions.
Inclusion Body
Certain viruses induce characteristic changes in
the host cells, called inclusion body, which can be @ DETECTION OF VIRAL ANTIGENS
detected by histopathological staining. They are
the aggregates of virions or viral proteins and other Various formats are available for the detection
products of viral replication that confer altered of viral antigens in serum and other samples,
staining property to the host cell.
such as enzyme-linked immunosorbent
Contd... assay (ELISA), immunochromatographic test
 CHAPTER4 © Laboratory Diagnosis of Viral Diseases

(B) Histopathology of skin showing molluscum bodies;

Figs 4.4A to E: (A) Negri bodies seen in brain infected with rabies;
cell of measles; (D) Tzanck smear showing multinucleated giant cell of herpes simplex virus; (E)
(© Multinucleated giant
with owl's eye inclusion.
Histopathology of kidney showing cytomegalic host cell (D) ID
and Prevention, Atlanta. (A) ID #3377; (B) ID #860; (C) ID #859;
Source: Public Health Image Library, Centers for Disease Control
#14428; (E) ID #1155.

(ICT), flow through assays, enzyme-linked Procedure

fluorescence assay (ELFA), etc. Some important Specimen is mounted on slide, stained with
antigen detection tests include: specific monoclonal or polyclonal antibody
«+ HBsAg and HBeAg antigen detection for tagged with fluorescent dye and viewed under
hepatitis B virus infection from serum fluorescent microscope.
* NS1 antigen detection for dengue virus
infection from serum Clinical Applications
“ SARS-CoV-2 antigen (nucleocapsid protein) Direct-IF has the following applications.
detection in nasopharyngeal swabs by “* Diagnosis of rabies in skin biopsies, corneal
immunochromatographic assay smear of infected patients (Fig. 4.5)
* p24 antigen detection for HIV infected patients
from serum
“ Rotavirus antigen detection from diarrheic
stool
* CMV specific pp65 antigen detection in
peripheral blood leukocyte.

Direct Immunofluorescence for Viral

Antigen Detection Figs 4.5A and B: Direct fluorescent antibody test for
is
Direct immunofluorescence technique rabies antigen detection: (A) positive; (B) negative.
clinica l
employed to detect viral particles in the
Atlanta
Source: Centers for Disease Control and Prevention,
(with permission).
samples.
 SECTION 1 @ General Microbiology, Immunology and Hospital Infection Control

“ Syndromic approach: Rapid diagnosis of DNA ladder Test PG NC

respiratory infections caused by influenza,
600 bp Be
rhinoviruses, respiratory syncytial virus,
adenoviruses and herpes viruses can be 500 bp
carried out by adding specific antibodies to 400 bp
each of these viruses
* Detection of adenovirus from conjunctival 300 bp
smears.
200 bp
@ DETECTION OF VIRAL ANTIBODIES
Antibody detection from serum is one of the
most commonly used method in diagnostic 100 bp
virology. Appearance of IgM antibody or a four-
fold rise of titer of IgG antibody indicates recent
infection; whereas the presence of IgG antibody
(without a recent rise) indicates chronic or past
Fig. 4.6: PCR for dengue virus from serum sample. Band of
infection. Various techniques available are
290 bp appeared for the test and positive control indicates
described below: presence of dengue virus serotype-3, in the test specimen.
Techniques, such as ELISA, ELFA, ICT, flow Abbreviations: NC, negative control; PC, positive control.
through assays are widely used for antibody Source: Department of Microbiology, Pondicherry Institute of
Medical Sciences, Puducherry (with permission).
detection against most of the viral infections, for
example:
into DNA, which is then subjected to amplifi-
** Anti-HBc, Anti-HBs and Anti-HBe antibodies
cation similar to that followed in PCR (Fig. 4.6)
in serum for hepatitis B infection
* Multiplex PCR formats are available
** Anti-hepatitis C antibodies in serum
that can simultaneously detect genes of
** Antibodies against HIV-1 and HIV-2 antigens
common organisms responsible for a clinical
from serum
syndrome; for example, multiplex PCR for
“ Anti-dengue IgM/IgG antibodies from serum.
upper respiratory syndromes simultaneously
detects genes of adenovirus, influenza virus,
@ MOLECULAR METHODS
parainfluenza virus and respiratory syncytial
Advent of molecular techniques has eased virus in respiratory specimen
the diagnosis of viral infections. They have ** BioFire FilmArray: It is an automated nested
revolutionized the diagnostic virology. They are multiplex PCR commercially available. It
more sensitive, specific and yield quicker results has various syndrome specific panels, such
than culture. as respiratory, gastrointestinal, meningitis-
** Polymerase chain reaction (PCR): It is the encephalitis; each panel comprises of primers
simplest molecular assay; used to detect viral targeting 20-25 common pathogens infecting
DNA in clinical specimens (e.g., HSV DNA in the respective systems
CSF). It involves three basic steps: ** Real time-PCR (rt-PCR): It is revolutionized
1. Viral DNA extraction from the specimen the diagnostic virology; considered as the gold
2. Amplification of specific region of viral standard method for diagnosis of several viral
DNA to 10° folds infections, such as influenza, COVID-19, etc.
3. Detection of amplified products by gel It has several advantages over conventional
electrophoresis. PCR, such as:
“+ Reverse transcriptase-PCR (RT-PCR): It is = Quantifying viral nucleic acid in the
used for the detection of RNA viruses (e.g., samples, hence used to monitor the
HIV and dengue RNA in blood). After RNA treatment response and estimating viral
extraction, the viral RNA is reverse transcribed load, e.g., HIV, hepatitis B virus
 CHAPTER4 © Laboratory Diagnosis of Viral Diseases

= Takes lesser time than PCR “+ Amniotic sac: Used for the isolation of
= More sensitive and specific than PCR. influenza virus
“+ Allantoic sac: It is a larger cavity, hence is
BISOLATION OF VIRUS used for better yield of viral vaccines, such as
influenza vaccine, yellow fever (17D) vaccine
Viruses cannot be grown on artificial culture ¢ Chorioallantoic membrane: Used for the
media. They are cultivated by animal inoculation, isolation of poxviruses (e.g., vaccinia and
embryonated egg inoculation or tissue cultures. variola). They produce visible lesions over the
“+ Being labor-intensive, technically demanding chorioallantoic membrane called as pocks.
and time consuming, virus isolation is not
routinely used in diagnostic virology Tissue Culture
“+ The specimen should be collected properly
Cell line cultures are used for viral isolation.
and transported immediately to the labora- “+ Preparation of the cell lines: Tissues are
tory. Refrigeration is essential during trans- completely digested by treatment with
portation as most viruses are heat labile. Type proteolytic enzymes followed by mechanical
of specimen collected depends on the virus shaking to completely dissociate them into
suspected. individual cells. Then cells are suspended in
viral growth medium containing balanced
Animal Inoculation
salt solution added with essential amino
Because of the ethical issues related to use of acids, vitamins, fetal calf serum and
animals, animal inoculation is largely restricted antibiotics. Then cells are in tissue culture
only for research purpose. flasks (Fig. 4.8)
“* Research use: To study viral pathogenesis or = On incubation, the cells adhere to the
viral oncogenesis or for viral vaccine trials glass surfaces of the flask, divide and
“+ Diagnostic use: Primary isolation of certain form a confluent monolayer sheet of cells
viruses which are difficult to cultivate oth- within a week covering the floor of the
erwise;, such as arboviruses and coxsackie- tissue culture flask
viruses. m Tissue culture flasks are incubated
horizontally in presence of CO,, either as
Egg Inoculation a stationary culture or as a roller drum
Use of embryonated eggs for viral diagnostics culture (for rotavirus isolation).
is limited now. Embryonated hen’s egg has four “ Types of cell lines: Three types of cell lines
sites that are specific for the growth of certain are available.
viruses (Fig. 4.7). 1. Primary cell lines: They are derived
“+ Yolk sac inoculation: Used for arboviruses from normal cells freshly taken from the
(e.g., JE virus) and some bacteria, such as organs and cultured. They are capable of
Rickettsia, Chlamydia and Haemophilus maximum up to 5-10 divisions. They have
ducreyi a diploid karyosome. Common examples
Chorioallantoic
include:
membrane

Air sac

Amniotic cavity

Viral culture medium

Fig. 4.8: Tissue culture flask.
Fig. 4.7: Schematic diagram of embryonated egg.
 SECTION 1 © General Microbiology, Immunology and Hospital Infection Control

Contd...

They are easy to maintain in the laboratories by serial

subculturing for indefinite divisions. This is the reason
why continuous cell lines are the most widely used
cell lines.
Common examples include (Figs 4.10A to C)
Q HeLa cell line (Human carcinoma ofcervix cell line)
Q HEp-2 cell line (Human epithelioma of larynx cell
line) —widely used for RSV, adenoviruses and HSV
Q KBcell line (Human carcinoma of nasopharynx cell
Fig. 4.9: Human lung fibroblast cell line (normal).
line)
Source: American Type Culture Collection (ATCC), USA (with Q
McCoy cell line (Human synovial carcinoma cell
permission).
line)—useful for isolation of viruses, as well as
: : Chlamydia
M Monkey Mdniey cell line—useful for Q Vero cell line (Vervet monkey kidney cell line)—
isolation of myxoviruses, enteroviruses used for rabies vaccine production
and adenoviruses Q BHK cell line (Baby hamster kidney cell line).
¢ Human amnion cell line and chick
embryo cell line.
Detection of Viral Growth in Cell Cultures
2. Secondary or diploid cell lines: They can
divide maximum up to 10-50 divisions Following methods are used to detect the growth
before they undergo death. They are also of the virus in cell cultures.
derived from the normal host cells and Cytopathic Effect (CPE)
they maintain the diploid karyosome.
Common examples include: It is defined as the morphological change
¢ Human fibroblast cell line for recovery produced by the virus in the cell line detected
of CMV (Fig. 4.9) by a light microscope.
¢ MRC-5 and WI-38 (human embryonic Cytopathic viruses: Not all, but few viruses can |
lung cell strain): Used for preparation produce CPE and those are called as cytopathic |
of various viral vaccines (rabies, viruses. The type of CPE is unique for each virus |
chickenpox, MMR vaccines) and that helps for their presumptive identifica-
3. Continuous cell lines (see the box tion (Table 4.4).
below).
Other Methods to Detect Viral Growth
Continuous Cell Lines Other methods to detect viruses in the cell line
They are derived from cancerous cell lines, hence are include:
immortal (capable of indefinite growth). They also ** Detection of viral antigens on the surface of in-
possess altered haploid chromosome.
fected cells by direct immunofluorescence assay
Contd.. * Viral genes detection by using PCR

Weg
Figs 4.10A to C: Continuous cell lines (normal, uninfected): (A) HeLa cell line; (B) Vero cell line; (C) HEp-2 cell line.
Source: American Type Culture Collection (ATCC), USA (with permission).
 CHAPTER4 © Laboratory Diagnosis of Viral Diseases

Table 4.4: Viral cytopathic effects (CPE). ** Electron microscopy, demonstrating the
Types of cytopathic effect (CPE) Ning viruses in infected cell lines.
Rapid crenation and degeneration Enteroviruses
Shell Vial Technique
ofthe entire cell sheet
Syncytium or multinucleated giant Measles, For viruses, such as CMV which take several
cell formation RSV, HSV weeks for cytopathic effect to develop, shell
Diffuse roundening and HSV vial technique can be followed for early growth
ballooning ofthe cell line detection (1-2 days).
Cytoplasmic vacuolations SV 40 ** Itinvolves centrifugation of cell culture (mixed
Large granular clumps resembling Adenovirus with the specimen) to enhance the cell contact
bunches of grapes and viral replication, followed by
Abbreviations: RSV, respiratory syncytial virus; HSV, herpes simplex ** Detection of early viral antigen in the infected
virus; SV 40, Simian vacuolating virus-40. cells by direct fluorescence technique.

| Problem Solving
Solving Exercise
Exercise _
Case Scenario 1: A patient with history of dog bite Explanation
presents to emergency with hydrophobia, increased Case Scenario 1: Patient with history of dog bite,
salivation and hyperexcitability. After the death of and presented with hydrophobia, increased salivation
the patient, the brain biopsy was sent for histopatho- and hyperexcitability is suggestive of provisional
logical examination (Fig. 4.4A). What is the etiological diagnosis as rabies. Fig. 4.4A depicts intra-cytoplasmic
diagnosis? inclusion body, described a Negri bodies which is
Case Scenario 2: Serum specimen of a 23-year old characteristic of rabies.
man presented with fever, joint pain and myalgia is Case Scenario 2: Fig. 4.6 depicts PCR for dengue virus
sent for dengue PCR (Fig. 4.6). Interpret the result. Dis- from serum sample. Band of 290 bp appeared for the
cuss its principle? test and positive control indicates presence of dengue
virus serotype-3 in the test serum.
 Laboratory Diagnosis of
Parasitic Diseases

Laboratory diagnosis plays an important role “++, When to examine: Liquid stool specimens
in establishing the specific diagnosis of various should be examined within 30 minutes,
parasitic infections. Following are the techniques semisolid stools within 1 hr (as on storage,
used in the diagnosis of parasitic infections. trophozoites may disintegrate or become non-
motile) and formed stools up to 24 hours after
Bf EXAMINATION OF FECES collection
** For monitoring response to therapy: Repeat
As many parasites inhabit in the intestinal stool examination can be done 3-4 weeks after
tract, stool examination is the most common
the therapy for intestinal protozoan infection,
diagnostic technique used for the diagnosis of and 5-6 weeks for Taenia infection
parasitic infections. * If delay in transport: Fecal specimens
should be kept at room temperature; pre-
Specimen Collection
servatives (e.g., 10% formalin) can be used
Stool specimens should be collected in a wide- to maintain the morphology of the parasitic
mouthed, clean, leak-proof, screw capped cysts and eggs
containers and should be handled carefully ** Specimens other than stool:
to avoid acquiring infection from organisms s Perianal swabs (cellophane tape or
present in the stool (Fig. 5.1). NIH swab): Useful for detecting eggs of
** Timing: Specimen should be collected before Enterobius vermicularis deposited on the
starting anti-parasitic drugs and closer to the surface of perianal skin. It is also used for
onset of symptoms eggs of Schistosoma mansoni and Taenia
** Frequency: At least three stool specimens species
collected on alternate days (within 10 = Duodenal contents: It is very useful for
days) are adequate to make the diagnosis the detection of small intestine parasites
of intestinal parasitic diseases (except for like, Giardia intestinalis and larva of
intestinal amoebiasis, for which six specimens Strongyloides stercoralis. Duodenal fluid
may be recommended)
can be collected by endoscopy or by
Entero-test.

Macroscopic Examination
Macroscopic examination of stool may provide
clue about various parasitic infections.
** Mucoid bloody stool: Found in acute amoebic
dysentery, intestinal schistosomiasis, and
invasive balantidiasis
* Color: Dark red stool indicates upper
gastrointestinal tract (GIT) bleeding and a
bright red stool is suggestive of bleeding from
Fig. 5.1: Sample container for stool. lower GIT
 CHAPTER5 ©@ Laboratory Diagnosis of Parasitic Diseases

Formed

« |ai.
Fig. 5.3: Saline and iodine wet mount.
Loose a
Source: Department of Microbiology, JIPMER, Puducherry
(with permission).

Watery Trophozoites
Fig. 5.2: Relative
an of trophozoites and cysts in
stool specimens with various consistencies.

+» Frothy pale offensive stool (containing fat) is

usually found in giardiasis.

Stool Consistency
In liquid stool, trophozoites are usually found;
Fig. 5.4: Method of screening of slide during wet mount
whereas in semi-formed stool both trophozoites examination of stool.
and cysts are found and the cysts are mainly
found in formed specimens (Fig. 5.2). Exceptions followed by high power objective (40X) for
to this general statement include: protozoan cysts and trophozoites
* Coccidian oocysts, helminths eggs can be * Screening area: The entire coverslip
found in any type of fecal specimen preparation should be examined in a zigzag
* In cryptosporidiosis, oocysts load is higher fashion, first under low power and then
in liquid stool under high power objective, before reporting
“ Tapeworm proglottids and adult worms of a negative result (Fig. 5.4)
Enterobius and Ascaris are occasionally found * Motility: If a finding is suspected to be a
in the stool. trophozoite, then examination for at least 15
seconds should be allowed to detect motility.
Microscopic Examination Motility can be stimulated by—application of
Microscopic examination includes direct wet heat by placing a hot penny on the edge of a
mount examination and permanent staining slide or tapping on the coverslip or increasing
methods. the intensity of the light source.
The following are the structures that can be
Direct Wet Mount (Saline and lodine Mount) visualized by microscopic examination of stool
Drops of saline and Lugol’s iodine are placed on specimen, which may be confused with various
left and right halves of the slide respectively (Fig. protozoan trophozoites, cysts or helminthic eggs
5.3). and larvae (Figs 5.5A to J).
“ A small amount of feces (~2 mg) is mixed with “ Normal constituents: These include plant
a stick to form a uniform smooth suspension. fiber, starch cells (stains blue-black with
If more or less fecal material has been taken iodine), muscle fibers, animal hair, pollen
for the stool wet mount, the chance of finding grains, yeast cells, bacteria, epithelial cells,
stool parasites decreases fat globules, and air bubbles, etc.
* Cover slip is placed on the mount and * Cellular elements: Like pus cells (in
examined under low power objective (10X) inflammatory diarrhea), red blood cells (RBC)
for detection of helminths eggs and larvae;
(in dysentery) may be present
 SECTION 1 © General Microbiology, Immunology and Hospital Infection Control

Figs 5.5A to J: Normal constituents and artifacts found in the stool in wet mount examination: (A) Yeast cell resembling
Giardia cyst; (B) Fungal spore resembling oocyst of Cystoisospora; (C) Pollen grain resembling Blastocystis; (D) Plant cell
resembling helminth egg; (E) Plant hair resembling Strongyloides larva; (F) Diatoms; (G) Mite egg resembling hookworm
egg; (H) Charcot-Leyden crystals; (I) Air bubbles; (J) Fat globules.
Source: (A, B, D, E, F, G, H) DPDx Image Library, Centers for Disease Control and Prevention (CDC), Atlanta; (C) W Swierczynski G, Milanesi
B. Atlas of Human Intestinal Protozoa Microscopic Diagnosis; (I and J) Department of Microbiology, JIPMER, Puducherry (with permission).

“+ Charcot-Leyden crystals (diamond-shaped): appreciated, (ii) Bile staining property cannot

They are the breakdown products of eosino- be appreciated.
phils and may be seen in the stool or sputum
of patients with parasitic diseases, such as Non-bile Stained Eggs
amoebic dysentery, ascariasis, and allergic Eggs of most of the intestinal parasites when they pass
diseases, such as bronchial asthma (sputum). through intestine are stained by bile. The exceptions
being Enterobius, hookworm and Hymenolepis nana;
these eggs are non-bile stained.
Saline Mount
Saline mount is useful in the detection of
Permanent Stained Smear
trophozoites and cysts of protozoan parasites,
and eggs and larvae of helminths. It has the Permanent stained smears are required for
following advantages than iodine mount. accurate detection of protozoan cysts and
** Motility of trophozoites and larvae in acute trophozoites by staining their internal structures.
infection can be demonstrated Commonly used methods are:
* Bile staining property can be appreciated— ** Iron-hematoxylin stain
bile stained eggs appear golden brown and ** Trichrome stain
non-bile stained eggs appear colorless ** Modified acid-fast stain—this is useful for
“+ In stool specimen with preservatives, directly coccidian parasites, such as Cryptosporidium,
the wet mount can be prepared without using Cyclospora and Cystoisospora.
saline.
Concentration Techniques
lodine Mount If the parasite output is low in feces (egg,
* Advantages: Nuclear details of protozoan cysts, trophozoites and larvae) and direct
cysts, helminthic eggs and larvae are better examination may not be able to detect the
visualized, compared to saline mount parasites, then the stool specimens need to be
* Disadvantages: (i) lodine immobilizes and concentrated. These methods are also useful in
kills the parasites, hence motility of the epidemiological analysis and for assessing the
trophozoites and helminthic larvae cannot be treatment response. The eggs, cysts and larvae
 CHAPTER5 © Laboratory Diagnosis of Parasitic Diseases

are recovered after concentration procedures;

however, the trophozoites get destroyed.
Commonly used concentration techniques
Ether
are:
* Sedimentation techniques: The eggs and Debris/fat

cysts settle down at the bottom following

centrifugation. Example include formalin-
Formalin
ether concentration technique and formalin-
ethyl acetate concentration technique
~ Flotation techniques: It involves suspending
the specimen in a medium (e.g., saturated Sediment
salt solution) of greater density, so that the
helminthic eggs and protozoan cysts float on Figs 5.6A and B: Sedimentation (Formol-ether) technique.
the surface of the solution Source: (B) Dr Anand Janagond, $ Nijalingappa Medical College,
Other flotation methods are: Bagalkot, Karnataka, India (with permission).
a Zinc sulphate flotation concentration
technique Advantages:
= Sheather’s sugar flotation technique “> The sensitivity of detecting the ova or cysts
(useful for Cryptosporidium, Cystoisospora increases by 8-10 folds
and Cyclospora). “+ The size and shape of the parasitic structures
are maintained
Sedimentation (Formol-ether) Technique “+ Inexpensive, easy to perform
Principle: It involves concentration of stool *» Fecal odor is removed
specimen by centrifugation. The protozoan * As formalin kills the fecal parasites, no risk of
acquiring laboratory-acquired infection.
cysts and helminthic eggs are concentrated at
the bottom of the tube because they have greater Disadvantage: Trophozoite forms are killed and
density than the suspending medium. hence not detected in this method.
Procedure: About half teaspoonful of feces is
Flotation (Saturated Salt Solution) Technique
mixed thoroughly 10 mL of 10% formalin, kept
standing for 30 minutes and the filtered out with Principle: Flotation involves suspending the stool
a gauge piece. specimen in a medium of greater density than that
* The filtrate is mixed 5% with formalin and of the helminthic eggs and protozoan cysts; e.g.,
centrifuged. After discarding the supernatant, saturated salt solution. After 15 minutes waiting
the sediment is again mixed with 5% formalin period, the eggs and cysts float to the top and are
and centrifuged collected by placing a glass slide on the surface of
* Finally 4-5 mL of ether is added to the the meniscus at the top of the tube (Fig. 5.7).
sediment and the tube is closed with a stopper
and shaken vigorously to mix well. The stopper
is removed and the tube is centrifuged
“ Four layers are formed—top layer consists
of ether, second is a plug of debris, third is a
clear layer of formalin and the fourth is the
sediment (Figs 5.6A and B)
“ The debris is removed from the side of the tube
with the help of a glass rod and supernatant
is discarded
* With a pipette, the sediment is removed and
the saline or iodine mount is prepared and
Fig. 5.7: Flotation (saturated salt solution) technique.
examined under the microscope.
 SECTION1 © General Microbiology, Immunology and Hospital Infection Control

Advantage: It is easy to perform than sedimenta- 2. Examination of blood smears: Thin smear
tion method. and thick smears are examined after staining
with various Romanowsky stains, such as
Disadvantages:
¢ ULTO: Flotation technique is not useful Leishman’s stain, Giemsa stain, Field’s stain
for heavier eggs that do not float in the salt and Jaswant Singh and Bhattacharjee (JSB)
solution stain. This is the most common method of
= Unfertilized eggs of Ascaris lumbricoides microscopic examination of peripheral blood,
® Larva of Strongyloides useful for most of the blood parasites
a Taenia eggs 3. Quantitative buffy coat (QBC): This involves
a Operculated eggs of trematodes. collection of the blood in a capillary tube
> If left for more than 20 minutes, protozoan coated internally with acridine orange stain,
cysts and thin-walled nematode eggs get centrifugation and then examination of the
collapsed and become distorted due to high buffy coat region under fluorescence micros-
specific gravity of the solution. copy. This is extremely useful for the detection
Various morphological forms of parasites of the malaria parasites and microfilariae
seen in stool specimens are enlisted in Table 5.1. 4. Concentration of blood: This is useful for detec-
tion of microfilariae from the blood specimen.
B EXAMINATION OF BLOOD Various concentration methods are:
a Sedimentation technique
Blood examination is useful in the diagnosis Cytocentrifugation (cytospin)
of infection caused by blood parasites, such Knott concentration
as Plasmodium, Trypanosoma, Leishmania, Gradient centrifugation
Babesia, Wuchereria bancrofti, Brugia malayi, Membrane filtration.
Loa loa and Mansonella.
Various methods of examination of blood MICROSCOPIC EXAMINATION OF
include:
OTHER SPECIMENS
1. Direct wet mount examination: It is useful
for detection of malaria parasites and Microscopic examination of various specimens
microfilariae in lymphatic filariasis (other than stool) can also be performed to
demonstrate different morphological forms of
the parasites (Table 5.2).
Table 5.1: Morphological forms of parasites seen in
stool specimens. Table 5.2: Various morphological forms of parasites
seen in different specimens other than stool.

Trophozoite and cyst Entamoeba histolytica Morphological | Parasite

Giardia lamblia form
Adult worm Ascaris lumbricoides Peripheral Ring form, Plasmodium spp.
Enterobius vermicularis blood smear schizont,
gametocyte
Adult worm segments = Taenia species
Diphyllobothrium latum Amastigote Leishmania spp.

Diphyllobothrium latum Trypomastigote Trypanosoma spp.

Egg
Taenia species Microfilaria Filarial nematodes*
Hymenolepis nana Bone marrow, Tachyzoite Toxoplasma gondii
Schistosoma species
liver, lymph Amastigote Leishmania donovani
Fasciola hepatica node, splenic
Fasciolopsis buski aspirate
Ascaris lumbricoides
Liver aspirate Trophozoite Entamoeba
Hookworm
histolytica
Enterobius vermicularis
Trichuris trichiura Lymph node — Trypomastigote Trypanosoma spp.
aspirate
Larva Strongyloides stercoralis
Contd...
 CHAPTER5 © Laboratory Diagnosis of Parasitic Diseases

Contd... Contd...
Morphological | Parasite Morphological | Parasite
form form
Lymph node = Adult worm Wuchereria bancrofti Duodenal Trophozoite Giardia lamblia
biopsy Brugia malayi aspirate Larva Strongyloides
Cerebrospinal Trophozoite Naegleria fowleri stercoralis
fluid Acanthamoeba spp. Corneal Trophozoite Acanthamoeba spp.
Trypomastigote Jrypanosoma spp. scrapings
Urine Trophozoite Trichomonas vaginalis Skin Amastigote Leishmania spp.
Microfilaria Wuchereria bancrofti Microfilaria Onchocerca volvulus
Egg Schistosoma Larva in skin Dracunculus
haematobium ulcer fluid medinensis
Sputum Adult worm Paragonimus spp. Muscle tissue Encystedlarva _ Trichinella spiralis
Egg Paragonimus spp. Cysticercus Taenia solium
cellulosae
Larva Ascaris lumbricoides
(migrating) Strongyloides spp. Perianal area Egg Enterobius spp.
Hookworm *Filarial nematodes found in the peripheral blood smears are
Trophozoite Entamoeba histolytica Wuchereria bancrofti, Brugia malayi, Loa loa, Mansonella spp.
Contd...

IMMUNODIAGNOSTIC specimens, such as CSF (neurocysticercosis) or

pleural fluid (paragonimiasis).
METHODS
Antigen Detection Tests
Immunodiagnostic methods involve detection
of parasite specific antibodies in serum, and The antigen detection methods are available for
various parasitic diseases, such as amoebiasis,
detection of circulating parasitic antigen in the
malaria and lymphatic filariasis.
serum.

Antibody Detection Tests @ MOLECULAR METHODS

Antibodies are detected in various parasitic Molecular methods most frequently used in
infections; mainly from serum (amoebic diagnostic parasitology include polymerase
liver abscess, visceral leishmaniasis and chain reaction (PCR), real time PCR and BioFire
toxoplasmosis); sometime from other FilmArray.

Go Solving Exercise
1. A laboratory technician is examining a stool examination of stool? Discuss their advantages
specimen that has come as a screening test and disadvantages.
done under master health checkup. What are the 3. Whatare the various stool concentration methods?
structures that can be visualized by microscopic Discuss their advantages and disadvantages.
examination of stool specimen, which may be Explanation
confused with various parasitic forms ? Refer text for detail.
2. What are the various methods of wet mount
 GIG |
Laboratory Diagnosis of
Fungal Diseases
0 ESTATES

BHCLASSIFICATION body temperature (37°C). Examples include

Histoplasma, Blastomyces, Coccidioides,
Morphological Classification Paracoccidioides, Penicillium marneffei and
Based on the morphology, there are four groups Sporothrix schenckii.
of fungi (Figs 6.1A to E).
1. Yeast: They grow as round to oval cells that Systemic Classification
reproduce by an asexual process called Based on diseases, fungi are classified as follows:
budding. Examples include Cryptococcus “+ Superficial mycoses: Caused by Malassezia
neoformans (Fig. 6.1A) furfur and dermatophytes
2. Yeast-like: They are oval budding cells with * Subcutaneous mycoses: Caused by
pseudohyphae (elongated budding cells Madurella and others, Sporothrix schenckii,
remain attached to the mother cell to form and Rhinosporidium
long chain of daughter cells). They can be ** Systemic mycoses: Agents are Histoplasma,
differentiated from true hyphae as they have Blastomyces, Coccidioides, and Paracoccidi-
constrictions at the septa. Examples include oides
Candida (Fig. 6.1B) ** Opportunistic mycoses: Caused by Candida
3. Molds: They grow as long branching filaments species, Cryptococcus, Rhizopus, Mucor,
of 2-10 xm wide called hyphae (mycelium) Aspergillus species and Penicillium species,
(Fig. 6.1E). Hyphae are either septate (i.e., form and Pneumocystis jirovecii.
transverse walls) (Fig. 6.1D) or nonseptate
(Fig. 6.1C). Examples of molds include B LABORATORY DIAGNOSIS
dermatophytes, Aspergillus, Penicillium,
Rhizopus, Mucor, etc. Specimen Collection
4. Dimorphic fungi: They exist as molds at It depends on the site of infection. For example:
25°C and as yeasts in human tissues at “+ For systemic mycoses—blood
** For cryptococcal meningitis—cerebrospinal
fluid.
Details on specimen collection are described
in the respective systems, where they cause the
infections.

Microscopy

A
Following direct microscopic examination
methods can be done:
* Potassium hydroxide (KOH) preparation
(10%): Used for keratinized tissue specimens
Figs 6.1A to E: Morphological forms of fungi: (A) Budding; (skin scrapings, plucked hair samples), which
(B) Budding yeast with pseudohyphae; (C) Aseptate digests the keratin material so that fungal
hyphae; (D) Septate hyphae; (E) Mycelium. hyphae will be clearly visualized (Fig. 6.2A)
 CHAPTER6 © Laboratory Diagnosis of Fungal Diseases

Figs 6.2A and B: (A) KOH mount showing fungal elements; Figs 6.3A and B: (A) India ink staining shows clear
(B) Gram staining showing gram-positive oval budding refractile capsules surrounding round budding yeast
yeast cells with pseudohyphae. cells (Cryptococcus); (B) Calcofluor-white mount showing
Source: Department of Microbiology, Pondicherry Institute of fungal elements.
Medical Sciences, Puducherry (with permission). Source:A.Public Health Image Library/A. Dr Leanor Haley, |ID#:3771/
Centers for Disease Control and Prevention (CDC), Atlanta (with
m About 20-40% KOH is used for nail permission); B. Department of Microbiology, JIPMER, Puducherry
(with permission).
clipping (as takes longer time to dissolve)
= Biopsy specimens are usually dissolved in pink whereas the nuclei stain blue (Fig.
10% KOH in a test tube and examined after 6.4A)
overnight incubation (as they take longer = Gomori methenamine silver (GMS)
time to dissolve). stain: Fungi appear black whereas the
“ Gram stain is useful in identifying the yeast background tissue takes pale green color
(e.g., Cryptococcus) and yeast-like fungi (e.g., (Fig. 6.4B)
Candida). They appear as gram-positive mw Mucicarmine stain is used for Cryptococcus
budding yeast cells (Fig. 6.2B) and Rhinosporidium
* India ink and nigrosin stains are used as = Hematoxylin and eosin (H&E) stain.
negative stains for demonstration of capsule * Lactophenol cotton blue (LPCB) is used
of Cryptococcus neoformans (Fig. 6.3A) to study the microscopic appearance of the
“+ Calcofluor-white stain: This binds to cellulose fungal isolates grown in culture (Fig. 6.4C).
and chitin of fungal cell wall and fluoresces
Culture
under ultraviolet light (Fig. 6.3B)
* Histopathological stains are useful for Fungal culture is frequently performed for
demonstrating fungal elements (causing deep isolation and correct identification of the fungi.
mycoses) from biopsy tissues * Sabouraud’s dextrose agar (SDA) is the most
= Periodic acid-Schiff (PAS) stain is the commonly used media in diagnostic mycol-
recommend stain for detecting fungi. ogy. It contains peptone (1%) and dextrose
PAS-positive fungi appear magenta/deep (4%) and has a final pH of 5.6 (Figs 6.5A and B)

: oils
ne silver stain (fungi appear black, against
Figs 6.4A to C: (A) PAS stain showing fungal hyphae; (B) Gomori methenami
Lactophen ol cotton blue mount fungal isolate grown in culture.
pale green background); (C)
Microbiology, Pondicherry Institute of Medical Sciences,
Source: (A and B) Department of Pathology, and (C) Department of
Puducherry (with permission).
 SECTION 1 © General Microbiology, Immunology and Hospital Infection Control

= Nature of hyphae (septate or aseptate,

hyaline or phaeoid, narrow or wide) and
= Type ofsporulation (conidia or sporangio-
spores).
*7 Slide culture: It gives the most accurate in
situ (undisturbed) microscopic appearance
of the fungal colony. Procedure—sterile slide
is placed on a bent glass rod in a sterile petri
dish. Two square agar blocks measuring
around | cm? (smaller than the coverslip) are
placed on the slide. Bit of the fungal colony is
Figs 6.5A and B: (A) Sabouraud’s dextrose agar slope in inoculated onto the margins (at the center) of
bottle; (B) Sabouraud’s dextrose agar plate.
the agar block. Then the coverslip is placed on
Source: Department of Microbiology, Pondicherry Institute of
Medical Sciences, Puducherry (with permission). the agar block and the petri dish is incubated
at 25°C. After sufficient growth occurs, LPCB
**‘? Specimen inoculated SDA plates are incu- mounts are made both from the coverslip and
bated at 25-30°C (except the dimorphic fungi the underneath slide (Fig. 6.6)
that grow at both 25°C and 37°C) in biochemi- Cellophane tape mount: Impressions are
cal oxygen demand (BOD) incubators for 2-3
taken by placing the cellophane tape on the
weeks colonies present on the surface of SDA plate,
* Antibiotics, such as cycloheximide, chloram-
and then LPCB mount is made from the
phenicol and gentamicin can be added to the
cellophane tape. This is easy to perform and the
culture media to inhibit growth of bacteria
in-situ fungal morphology is also maintained.
and saprophytic fungi
* Macroscopy: Following macroscopic
appearance of the colony are noted, such
as (i) Rate of growth (yeasts and agents of
opportunistic mycoses grow within 5 days;
whereas dermatophytes take 1-4 weeks), (ii)
Pigmentation (iii) Texture of the colony (waxy/
leathery, velvety, cottony or granular/powdery),
(iv) Colony topography (colony surface)
** Microscopic appearance of fungi: A bit of
fungal colony is teased out (tease mount) from
the culture tube and LPCB mount is made
Fig. 6.6: Slide culture technique.
on a slide and viewed under microscope. Source: Department of Microbiology, Pondicherry Institute of
Identification is based on the: Medical Sciences, Puducherry (with permission).

| Problem Solving
Solving Exercise
Exercise _—|
Case Scenario 1: CSF specimen is collected from a 32- ink is recommended to demonstrate capsule
year HIV positive patient with fever and neck rigidity. of Cryptococcus. The recommended medium is
What is the stain recommended for direct microscopy Sabouraud's dextrose agar.
and what is medium to be used for fungal culture. Case Scenario 2: Patient with ring like annular skin
Case Scenario 2: A young male presented with ring lesions over the groin area is suggestive of ring worm
like annular skin lesions over the groin area. Which (Dermatophyte) infection. 10% KOH mount of the
is the recommended direct microscopy technique to skin scraping is the recommended direct microscopy
demonstrate the fungal elements and how will you technique to demonstrate the fungal hyphae. The
perform fungal culture. recommended culture medium is Sabouraud’s
dextrose agar.
Explanation
Case Scenario 1: Patient with fever and neck
rigidity is suggestive of a case of meningitis. India
 Precipitation and Agglutination

AUSED REPREATES

Contd...
B INTRODUCTION
occurs at its best only in the middle test tubes where
The antigen-antibody reactions are specific and the amount of antigen and antibody are equivalent to
observable. Therefore, they are extensively used each other (zone of equivalence). The Ag-Ab reaction
in the laboratories for the diagnosis of infectious is weak or fails to occur when the number of antigen
diseases. The diagnostic tests based on Ag-Ab and antibodies are not proportionate to each other
(Figs 7.1A to C).
reactions are called as immunoassays. Most
Q In the earlier test tubes, antibodies are excess,
immunoassays are also called serological tests hence the Ag-Ab reaction does not occur: This is
as they are performed using serum samples. called as prozone phenomenon
However, other samples can also be used, such Q In the later test tubes, antigen is excess, hence
as urine, CSE, etc. Immunoassays can be broadly the Ag-Ab reaction fails to occur: This is called as
postzone phenomenon.
categorized into two types:
Marrack (1934) proposed the lattice hypothesis to
1. Antigen detection assays: Detect antigens explain this mechanism. According to this concept the
in patient’s sample by employing specific multivalent antigens combine with bivalent antibodies
antibody in varying proportions, depending on the antigen
2. Antibody detection assays: Detect antibod- antibody ratio in the reacting mixture (Figs 7.1A to ©).
ies in patient’s sample by employing specific Q Ag-Ab reaction optimally occurs when a large
lattice is formed consisting of alternating antigen
antigen. This is possible only in the
and antibody molecules.
Immunoassays can be performed by both zone of equivalence
qualitative and quantitative methods. @ In the zones of antibody or antigen excess
* Qualitative assays: Here, the undiluted (prozone/post zone), the lattice does not enlarge,
specimen containing the antibody is directly due to inhibition of lattice formation by the excess
antibody or antigen respectively.
mixed with the suspension of antigen or vice Lattice hypothesis was described first for precipitation
versa. The exact amount of antigen or antibody reaction, but it also holds true for agglutination and
present in the specimen cannot be estimated other techniques of Ag-Ab reactions.
“ Quantitative assays: Here, the exact amount The prozone phenomenon is of great importance in
of antibody in serum can be estimated by clinical serology, as sera rich in high titer of antibody
may sometimes give a false-negative result, unless
serial dilution of the patient’s serum and serial dilutions of sera are tested.
mixing it with a known quantity of antigen.
The measurement of antibody is expressed as The antigen-antibody reactions used in
titer (defined as the highest dilution of serum diagnostic laboratories are based on various
that shows an observable reaction with the techniques which are broadly classified as
antigen). conventional techniques and newer techniques
(Table 7.1).
Marrack’s Lattice Hypothesis
When the sera containing antibody is serially diluted
(in normal saline), gradually the antibody level
@ PRECIPITATION REACTION
decreases. When a fixed quantity of antigen is added
Definition
to such a set of test tubes containing serially diluted
sera, then it is observed that the Ag-Ab reaction When a soluble antigen reacts with its antibody
Contd... in the presence of optimal temperature, pH and
 SECTION 1 @ General Microbiology, Immunology and Hospital Infection Control

Clinical Applications
Earlier, precipitation reactions were one of the
widely used serological tests. However, with the
advent of simple and rapid newer techniques
their application is greatly reduced. There are
only limited situations where precipitation
reaction is still in use; discussed below.

Optimal proportion of Slide Flocculation Test (for Syphilis)

Antibody Antigen
antigen and antibody
excess
(zone of equivalence,
excess It is used for serodiagnosis of syphilis, a sexu-
(prozone) (postzone)
lattice formation) ally transmitted disease caused by Treponema
pallidum.
Figs 7.1A to C: (A) Prozone; (B) Zone of equivalence;
(C) Postzone.
“+ Procedure: When a drop of antigen is mixed
with a drop of patient’s serum (containing
Table 7.1: Types of antigen-antibody reactions. antibody) on a slide, then the precipitates
Conventional techniques formed remain suspended as floccules
“+ Examples include VDRL (Venereal Disease
e Precipitation reaction e Agglutination reaction
Research Laboratory) and RPR (Rapid
Newer techniques
Plasma Reagin) tests. Refer Chapter 43 for
e Enzyme-linked immunosorbent assay (ELISA) detail.
e Enzyme-linked fluorescent assay (ELFA)
e Immunofluorescence assay (IFA) Elek’s Gel Precipitation Test (Detecting
e Chemiluminescence-linked immunoassay (CLIA) Diphtheria Toxin)
e Rapid tests The Corynebacterium diphtheriae strain isolated
> Lateral flow assay (immunochromatographic test) is streaked on to a medium containing a filter
> Flow through assay paper soaked with diphtheria antitoxin.
e Western blot * If the strain is toxigenic, it produces the
toxin, which diffuses in the agar, meets with
electrolytes (NaCl), it leads to formation of the the antitoxin and produces arrow-shaped
antigen-antibody complex in the form of: precipitation band
* Insoluble precipitation band when gel ** The precipitate bands of outbreak isolates
or agar containing medium is used (called (streaked adjacent) when meet with each
immunodiffusion) or other, three patterns may be observed
** Insoluble floccules when liquid medium is based on relatedness between the strains
used (called flocculation test). (Fig. 7.3, Problem Solving Exercise 2).

| Problem Solving
Solving Exercise1
Exercise 1 _|
Venereal Disease Research Laboratory 3. What type of antigen is used in this test?
A commercial sex worker with painless indurated 4. What is the principle of the test?
genital lesions, presented to outpatient department
Explanation
for screening for sexually transmitted infections.
One of the test done is demonstrated below (Figs History of commercial sex worker with painless
7.2A and B). indurated genital lesions is suggestive of syphilis.The
1. Identify the test and in which condition is it used? test shown in the picture is Venereal Disease Research
2. Interpret the result. Laboratory (VDRL) test. It is the most widely used,
 CHAPTER7 © Precipitation and Agglutination

simple, nonspecific and rapid serological test used for

diagnosis of syphilis.

Procedure
50 ul of inactivated serum is mixed with a drop of
VDRL antigen (cardiolipin antigen) in a VDRL slide
containing 12 concave rings (Fig. 7.2A). Then the slide
is rotated at 180 revolutions per minute for 4 minutes
in a VDRL rotator and examined under microscope
(10X). The results are read as follows:
Q Nonreactive: Uniformly distributed fusiform
crystals represent the presence of VDRL antigen
only, which indicates a negative result.
Q Reactive: It is indicated by formation of medium
to large clumps of antigen-antibody complexes;
visualized by focusing the slide under microscope
(10x) (Fig. 7.2B).
| Non-reactive Weakly reactive Strongly reactive | Q Weakly reactive: Slight roughness denotes
Negative control plest amc _ Positive control weakly reactive test result (as in this case). In
Figs 7.2A and B: VDRL slide and VDRL test results. such cases, the test results should be confirmed
Source: Department of Microbiology, JIPMER, Puducherry by performing specific treponemal tests (Refer
(with permission). Chapter 43).

Solving Exercise
[problem Solving 2
Exercise2_|
Elek’s Gel Precipitation Test Q Determine whether the relatedness between the
strains isolated.
A cluster of diphtheria cases occurred affecting
children in a remote village. The strains of Coryne- Explanation
bacterium diphtheriae isolated from the clinical The test performed in above case scenario is Elek’s gel
specimens were subjected for toxigenicity testing and precipitation test. This is a type of immunodiffusion in
the result is displayed in the Figure 7.3. gel described by Elek (1949)
Q Identify the test performed and interpret the Q Isolates 1 to 4 are toxigenic strains (as
result. precipitation bands are formed due to binding
of toxin produced by the strains with antitoxin
released by the filter paper)
Q Isolates 1 and 2: The precipitation bands crossed
Cross over
of bands
over, indicates the toxins are not identical and
therefore strains are unrelated
Spur Q Isolate 2 and 3: There is partial fusion of
formation precipitation bands, indicates the toxins are
Fusion of partially identical and therefore strains are
bands partially related to each other
Q Isolates 3 and 4: The precipitation bands fused
with each other, indicates the toxins are identical
filter
Antitoxin-soaked
paper
Horse serum | to each other and therefore strains are completely
agar plate
related
Q Isolate 5 is non-toxigenic strain (no precipitation
Fig. 7.3: Elek’s gel precipitation test. band is formed).
 SECTION 1 ©@ General Microbiology, Immunology and Hospital Infection Control

BfAGGLUTINATION REACTION *-Clumps*

Milky-white suspension
Definition
When a particulate or insoluble antigen is mixed
with its antibody in the presence of electrolytes
at a suitable temperature and pH, the particles
are clumped or agglutinated.

Advantage Figs 7.4A and B: Slide agglutination test: (A) Negative

Agglutination is more sensitive than precipita- result; (B) Positive result (clumps).
Source: Department of Microbiology, JIPMER, Puducherry
tion test as the clumps are better visualized and
(with permission).
interpreted than bands or floccules. Hence,
agglutination tests are widely used even in
today’s modern era of diagnosis. Bacterial colony is mixed with a drop ofsaline ona slide
to form a uniform, smooth, milky-white suspension
Applications
Agglutination reactions are classified as To this, a drop of the antiserum (serum containing
appropriate antibody) is added and the slide is
direct, indirect (passive) and reverse passive
shaken thoroughly (manually or by rotator) for few
agglutination reactions. All these agglutination seconds
tests are performed either on a slide, or in 1
tube or in card or sometime in microtiter A positive result is indicated by visible clumping with
plates. clearing of the suspension (Fig. 7.4B)
or
Direct Agglutination Test If the milky-white suspension remains unchanged,
indicates a negative result (Fig. 7.4A).
Here, the antigen directly agglutinates with the
antibody.
Tube Agglutination
Slide Agglutination This is a standard quantitative test for estimating
It is usually performed to confirm the antibody in serum. Several tests are there that
identification and serotyping of bacterial are done in various clinical conditions.
colonies grown in culture. It is also the method ** Widal test in typhoid or enteric fever (Refer
used for blood grouping and cross matching. the Problem Solving Exercise 3) (Refer
The process follows: Chapter 18 for detail).

[Problem Solving
Solving Exercise3__—|
Exercise 3
Widal Test 4. Name two other tests based on the same principle
A young adult male admitted to the hospital with (Fig. 7.5).
fever of 10 days duration with step ladder pattern. Explanation
A provisional clinical diagnosis of enteric fever
History of stepladder fever is suggestive of enteric
was made. The following serological test was
fever. This is caused by Salmonella Typhi and
performed.
Salmonella Paratyphi A and B. The test shown here is
1. Identify the test performed and in which clinical
Widal test; titer of TO 1:160 and TH 1:320.
condition this test is used?
Widal test is an example of tube agglutination
2. What type of antigen-antibody reaction occurs in
test used for quantitative estimation of antibody
this test?
titer against S. Typhi and Salmonella Paratyphi A
3. How will you interpret the positive and negative
and B.
results and report the titer?
 CHAPTER7 © Precipitation and Agglutination

The antibody titer can be estimated as the highest

dilution of the serum which produces a visible
agglutination.
Procedure of Tube Agglutination Test
A fixed volume of antigen suspension is added to an
equal volume of serial dilutions of a serum sample
(containing appropriate antibody) in test tubes and
overnight incubated at 37°C in water bath.
Q Positive test indicates agglutination (matt
formation at the bottom of the tube with clearing
of the supernatant)
Q Negative test indicates agglutination did not occur
(antigen suspension forms button at the bottom
Fig. 7.5: Widal test of the tube with no clearing of supernatant).
Abbreviations: TO and TH: Antibody titers of S.Typhi O and Hin
patient's serum.
Source: Department of Microbiology, Pondicherry Institute of
Medical Sciences (with permission).

“* Standard agglutination test in acute brucel- precipitation reaction into an agglutination

losis reaction. This is possible by coating the soluble
“+ Coombs antiglobulin test: It is performed antigen on the surface of a carrier molecule
to diagnose Rh incompatibility by detecting (e.g., red blood cells, latex or bentonite), so that
Rh antibody from mother’s and baby’s the antibody binds to the coated antigen and
serum. agglutination takes place on the surface of the
“+ Heterophile agglutination tests: carrier molecule.
= Weil-Felix reaction in typhus fever * Indirect hemagglutination test (IHA): Itis a
ms Paul-Bunnell test in infectious mono- passive agglutination test where RBCs are used
nucleosis as carrier molecules. [HA was used widely in
= Cold agglutination test in Mycoplasma the past, but is less popular at present
pneumoniae. * Latex agglutination test (LAT) for antibody
detection: Here, latex particles are used as
Microscopic Agglutination carrier molecules which are capable of adsor-
Here, the agglutination test is performed on a bing several types of antigens. It is used for
microtiter plate and the result is read under a detection of antistreptolysin O antibody (ASO)
microscope. Example is microscopic agglutina- titer (Refer the Problem Solving Exercise 4).
tion test (MAT), done for leptospirosis.
In Widal test: Antibodies against both H
(flagellar) and O (somatic) antigens of Salmonella
Typhi are detected.
“* O antigen-antibody clumps appear as chalky-
white granular dense deposits.
+ H antigen-antibody clumps appear as loose
fluffy clumps (Fig. 7.6).

indirect or Passive Agglutination Test

(for Antibody Detection)
Fig. 7.6: O and H agglutination in Widal test (reading
As agglutination test is more sensitive and taken in a mirror).
better interpreted than precipitation test, Source: Department of Microbiology, Pondicherry Institute of
hence attempt has been made to convert a Medical Sciences (with permission).
 SECTION1 © General Microbiology, Immunology and Hospital Infection Control

Problem Solving Exercise 4

Latex Agglutination Test for Antistreptolysin O Negative Test Positive
(ASO) Antibody controi control
A young child with history of recurrent sore throat was
admitted to the hospital. His serum was sent to the
laboratory and the test was performed. The test result
is displayed in the Fig. 7.7.
1. Identify the test and interpret the result?
2. What is the principle of the test?
3. What is the nature of the antigen used in the test
Fig. 7.7: ASO test by latex agglutination.
(Fig. 7.7)?
Source: Department of Microbiology, JIPMER, Puducherry
Explanation (with permission).

History of recurrent sore throat in a child raises the Positive result appears as formation of visible
suspicion of streptococcal pharyngitis. The test shown clumps; is indicative of presence of ASO antibody.
here is a latex agglutination test, done for detection Negative result remains as milky-white
of ASO. suspension with no clumps; is indicative of
Principle absence of ASO antibody.
Quantitative test: Once the test is positive, the
Here, polystyrene latex particles are used as carrier
test is repeated with serial dilution of serum.
molecules which are capable of adsorbing several
Highest dilution showing a positive result is
types of antigens. For better interpretation of result,
indicative of titer of the test. ASO titer of >200 IU/
the test is performed on a black color card.
mL is considered as the significant titre.
Q Drop of patient's serum (containing ASO antibody)
Latex agglutination test is widely used as its simple
is added to a drop of latex solution coated with the
and rapid test.
antigen and the card is rocked.

Reverse Passive Agglutination Test Latex agglutination test for antigen

(for Antigen Detection) detection: It is used widely for detection of
In this test, the antibody is coated on a carrier CRP (C-reactive protein), RA (rheumatoid
molecule which detects antigen in patient’s arthritis factor), capsular antigen detection
serum. in CSF (for pneumococcus, meningococcus
* Reverse passive hemagglutination assay and Cryptococcus) and streptococcal grouping
(RPHA): Here, the RBCs are used as carrier Coagglutination test: Here, Staphylococcus
molecules. RPHA was used in the past for aureus (protein A) acts as carrier molecule.
detection of hepatitis B surface antigen This test was used in the past to detect antigen
(HBsAg); now obsolete from clinical specimens now obsolete.
 cst
ELISA, ELFA and Immunofluorescence

TOS

sample by producing a visible effect. Most of the

NEWER TECHNIQUES newer techniques use this common principle,
The newer techniques use a detector molecule to but they differ from each other by the type of
label antibody or antigen which in turn detects labeled molecule used and the type of visible
the corresponding antigen or the antibody in the effect produced (Table 8.1).

| —__ Problem Solving

Solving Exercise
Exercise
ELISA Test Answers to all other questions are explained
A screening test for hepatitis B was performed on a below.
group of hemophiliac patients attending outpatient
department. The test done is demonstrated below.
1. Identify the test and interpret the result.
2. Name various types and their principles available
for the test given below.
3. What are the advantages and disadvantages of
this test format?
4. What are the applications of this test format (Fig.
8.1)?

Explanation
Interpretation: Sample No. 2, 4 & 5 : Positive and
The test done here is ELISA for detection of hepatitis Sample No: 1, 3 and 6 : Negative
B surface antigen (HBsAg).
Fig. 8.1: ELISA for HBsAg.
Interpretation
Source: Department of Microbiology, Pondicherry Institute of
Sample 2, 4 and 5 are tested positive. Sample number Medical Sciences, Puducherry (with permission).
1,3 and 6 are tested negative.

Table 8.1: Immunoassays and the types of molecules used for labeling.
Immunoassay method Molecules used for labeling Type of visible effect

ELISA Enzyme-linked immunosorbent Enzyme-substrate-chromogen Color change is detected by

assay complex spectrophotometer

ELFA Enzyme-linked fluorescent assay Enzyme-substrate Fluorometric detection

IFA Immunofluorescence assay Fluorescent dye Emits light, detected by

fluorescence microscope

CLIA Chemiluminescence-linked Chemiluminescent compounds Emits light, detected by

immunoassay luminometer
WB Western blot Enzyme Color band (naked eye)

Rapid tests Immunochromatographic test Colloidal gold or silver Color band (naked eye)
Flow-through assay Protein A conjugate Color band (naked eye)
 SECTION1 © General Microbiology, Immunology and Hospital Infection Control

* ELISA kits are commercially available;

BELISA
contain all necessary reagents (such as
Enzyme-linked immunosorbent assay (ELISA) enzyme conjugate, dilution buffer, substrate/
is an immunoassay that detects either antigen chromogen, etc.)
or antibodies in the specimen, by using enzyme- ** The procedure involves a series of steps
substrate-chromogen system for detection. done sequentially. At each step, a reagent is
being added, and then incubated, followed
Principle of ELISA
by washing of the wells [manually or by an
ELISA is so named because ofits two components: automated ELISA washer (Fig. 8.2B)].
* Immunosorbent: Here, an absorbing material
is used (e.g., polystyrene, polyvinyl) that Types of ELISA
specifically absorbs the antigen or antibody There are several types of ELISA, which differ
present in serum from each other in their principles.
** Enzyme is used to label one of the components
of immunoassay (i.e., antigen or antibody). Direct ELISA
Substrate-chromogen system: A substrate-
It is used for detection of antigen in test serum.
chromogen system is added at the final step of
Here, the primary antibody (targeted against the
ELISA.
serum antigen) is labeled with the enzyme.
** The enzyme reacts with the substrate, which in
turn activates the chromogen to produce a color Well + Ag (test serum) + primary Ab-enzyme +
* The classical example is, horseradish substrate-chromogen — color change (Fig. 8.3A)
peroxidase used as enzyme which reacts
with its substrate (hydrogen peroxide), that
in turn activates the chromogen (tetramethyl Indirect ELISA
benzidine) to produce a color It is used for detection of antibody or less
** The color change is detected by spectro- commonly antigen in serum. It differs from the
photometry in an ELISA reader. Intensity direct ELISA in that the secondary antibody
of the color is directly proportional to the is labeled with enzyme instead of primary
amount of the detection molecule (Ag or Ab) antibody. The secondary antibody is an anti-
present in test serum. species antibody, e.g., anti-human Ig (an
antibody targeted to Fc region of any human
(Ag-Ab complex)-enzyme + substrate — activates
Ig). Indirect ELISA for antibody detection is
the chromogen -> color change — detected by
spectrophotometry (ELISA reader, Fig. 8.2A) described below (Fig. 8.3B).
** Step 1: The solid phase of the wells of
microtiter plates are precoated with the
Procedure of ELISA
Ag
ELISA is performed on a microtiter plate ** Step 2: Test serum (containing primary Ab
containing 96 wells (Fig. 8.1), made up of specific to the Ag) is added to the wells. Ab
polystyrene, polyvinyl or polycarbonate material. gets attached to the Ag coated on the well

©) Substrate e Substrate
. .Ch
Chromogen E@ 4 ASME

ae Secondary Ab
Pigs Ab conjugate
Na ated aN Primary Ab
(serum)

Figs 8.2A and B: (A) ELISA reader (Biorad); * Ag Ag coated in wells

(B) ELISA washer. Figs 8.3A and B: (A) Direct ELISA (for antigen detection);
Source: Biorad Pvt. Ltd (with permission). (B) Indirect ELISA (for antibody detection).
 CHAPTER8 © ELISA, ELFA and Immunofluorescence

7
*%
Step 3: After washing, enzyme-labeled and West Nile virus, scrub typhus, leptospirosis,
secondary Ab (anti-human immunoglobulin) toxoplasmosis, etc. (Fig. 8.4B).
is added ~
2,
It is based on capturing primary IgM Ab
fe
Step 4: After washing, a substrate- chromogen (in test serum) on a microtiter plate pre-
system is added and color is developed. coated with anti-human-IgM Ab, followed
by addition of recombinant antigen (e.g.,
Wells are coated with Ag + primary Ab (test serum) dengue antigen)
+ secondary Ab-enzyme + substrate-chromogen —> bd
+,
Subsequently, enzyme labelled secondary
development of color (Fig. 8.3B) antibody specific for the antigen is added,
followed by addition of substrate-chromogen
Sandwich ELISA system
It detects the antigen in test serum. It is so
+e
¢,
The use of avidin-biotin system helps in
named because the antigen gets sandwiched amplifying the signal generated between
between a capture antibody and a detector enzyme-antibody complex, thus increases
antibody (Fig. 8.4A). the sensitivity of the assay.
7
~
Step 1: The microtiter well is precoated
Wells coated with capture anti-lgM Ab + IgM Ab (test
with the capture antibody (monoclonal Ab serum) + recombinant antigen + secondary Ab-biotin
raised in rabbit) targeted against the test + avidin-enzyme + substrate-chromogen — color (Fig.
antigen 8.4B)
Step 2: The test serum (containing antigen)
is added to the wells. Ag gets attached to the
Competitive ELISA
capture antibody coated on the well
Step 3: After washing, an enzyme labeled Competitive ELISA is so named because, antigen
primary ‘detector antibody’ specific for the in test serum competes with another antigen
antigen is added. The detector antibody can of the same type coated on well to bind to the
be same as the capture antibody primary antibody.
+¢, Step 1: Primary antibody is first incubated in
Step 4: After washing, a substrate-chromogen
system is added and color is developed. a solution with a serum sample containing
the test antigen
Wells coated with capture Ab + Ag (test serum) +
Step 2: This antigen-antibody mixture is then
primary Ab-enzyme + substrate-chromogen > color added to the microtiter well precoated with
(Fig. 8.4A) the same type of antigen
Step 3: The free antibodies bind to the antigen
IgM Antibody Capture (MAC) ELISA coated on the well. More the test antigens
present in the sample, lesser free antibodies
This is an enzymatically amplified sandwich- will be available to bind to the antigens coated
type immunoassay. This format of ELISA is onto well
widely used for dengue, Japanese encephalitis Step 4: After washing (to remove free
antibodies and antigens), enzyme-conjugated
Al BI ce) Substrate |
secondary antibody is added
‘az §Chromogen Step 5: After washing, a substrate-chromogen
E@
system is added and color is developed.
ry & Intensity of the color is inversely proportional
®
Secondary Ab
conjugate to the amount of antigen present in the test
Chromogen
nts Recombinant serum (Fig. 8.5).
» Primary Ab Ma The competitive ELISA can also be used
Serum AI® amet Primary Ab (Serum
conjugate
YN (Anti-human IgM for the detection of antibody in serum. More
\ feCapture Ab ] (capture Ab) so, different formats of competitive ELISA are
available, such as direct, indirect and sandwich
Figs 8.4A and B: (A) Sandwich ELISA (for antigen
detection); (B) IgM antibody capture (MAC) ELISA. formats. The example given above is an indirect
 SECTION1 ©@ General Microbiology, Immunology and Hospital Infection Control

aN ENZYME-LINKED FLUORESCENT
¥ ¥ Alls
Incubate }
ASSAY (ELFA)
primary Ab ELFA is an modification of ELISA, differs from
with antigen = = J
ELISA in two ways: (i) automated system, all steps
tobe Add Ag-Ab Add enzyme- Add substrate
measured mixture to conjugated and are performed by the instrument itself, (ii) Ag-
antigen-coated secondary measure Ab-enzyme complex is detected by fluorometric
well antibody color
method. VIDAS and miniVIDAS (bioMérieux)
Fig. 8.5: Competitive ELISA for antigen detection. are commercially available systems based on
ELFA technology (Fig. 8.6).
competitive ELISA format used for antigen “+ Procedure: The solid phase receptacle (Fig.
detection (Fig. 8.5). 8.6) present in reagent strip (equivalent to
wells in microtiter plate in ELISA) serves as
Advantages of ELISA the solid phase; which is either coated with
ELISA is the method of choice for detection of capture antigen (for antibody detection) or
antigens/antibodies in serum in modern days,
antibody (for antigen detection). Alkaline
especially in big laboratories as large number of phosphatase is used as an enzyme and and
samples can be tested together using the 96 well the substrate used is a fluorescent molecule
microtiter plate. (4-methyl-umbelliferyl phosphate)
** It is economical, takes 2-3 hours for * Advantages: It has many advantages over
performing the assay ELISA: (i) an automated system, (ii) easy
** ELISA has a high sensitivity; that is why, it is to perform and user friendly, (iii) less
commonly used for performing screening test contamination chance, (iv) gives quantitative
at blood banks and tertiary care sites results and (v) more sensitive and specific
** Its specificity used to be low. But now, with use * Disadvantages: (i) Expensive, (ii) can run
of more purified recombinant and synthetic only 12-24 number oftests at a time (VIDAS),
antigens, and monoclonal antibodies, ELISA (iii) can run 2-4 types of tests at a time (mini-
has become more specific. VIDAS)
* Use: It can be used to detect numerous
Disadvantages of ELISA parameters
m Infectious diseases: Markers of hepatitis
** In small laboratories having less sample
viruses and HIV (Ag and Ab), antibody
load, ELISA is less preferred than rapid tests
as the latter can be performed on individual to TORCH infection, measles, mumps,
samples varicella, H. pylori and antigen to C.
difficile, rotavirus, etc.
* It takes more time (2-3 hours) compared to
rapid tests which take 10-20 minutes m= Other uses: Biomarkers (e.g., procalci-
** It needs expensive equipment, such as ELISA tonin), hormones (e.g., thyroid), tumor
washer and reader.
Solid phase
receptacle
Applications of ELISA
ELISA can be used both for antigen and antibody
detection.
** ELISA used for antigen detection: Hepatitis
B [hepatitis B surface antigen (HBsAg) and
precore antigen (HBeAg)], NS1 antigen for
dengue, etc. Fig. 8.6: miniVIDAS system and reagent strip (first well is
solid phase receptacle coated with Ag or Ab and other
“+ ELISA can also be used for antibody detection
wells contain various reagents).
against hepatitis B, hepatitis C, HIV, dengue, Source: Department of Microbiology, JIPMER, Puducherry (with
EBV, HSV, toxoplasmosis, leishmaniasis, etc. permission).
 CHAPTER 8 © ELISA, ELFA and Immunofluorescence

markers, cardiac markers and screening Fluorescent Antihuman IgG

label conjugated
for allergy. with fluorescein
Primary
antibody
lf IMMUNOFLUORESCENCE ASSAY (IFA)
It is a technique similar to ELISA, but differs by Antigen
some important features: (sample) :
Antibody from
“+ Fluorescent dye is used instead of enzyme for the patient serum
labeling of antibody
** It detects cell surface antigens. It is also used Antigen coated on slide
to detect antibodies bound to cell surface 'B|
antigens, unlike ELISA which detects free Figs 8.7A and B: Immunofluorescence assay:
antigen or antibody. (A) Direct; (B) Indirect.

Principle
Fluorescence refers to absorbing high energy-
shorter wavelength ultraviolet light rays by the
fluorescent compounds and in turn emitting visi-
ble light rays with a low energy-longer wavelength.
“+ The fluorescent dye is used to conjugate the
antibody and such labeled antibody can
be used to detect the antigens or antigen-
antibody complexes on the cell surface
“ The fluorescent compounds commonly used Figs 8.8A and B: Immunofluorescence assay for
detection of antinuclear antibodies: (A) Positive result;
is fluorescein isothiocyanate (FITC).
(B) Negative result.
Source: EUROIMMUN AG Pvt Ltd. (with permission).
Types
Direct Immunofluorescence Assay * Step 2: Slide is washed to remove the
unbound antibodies. A secondary antibody
“ Step 1: Sample containing cells carrying
(antihuman antibody conjugated with
surface antigens is smeared on a slide
fluorescent dye) is added
* Step 2: Primary antibody specific to the
* Step 3: Slide is washed and then viewed under
antigen, tagged with fluorescent dye is added
a fluorescence microscope (Fig. 8.7B).
* Step 3: Slide is washed to remove the
unbound antibodies and then viewed under Applications: Immunofluorescence assay has
a fluorescence microscope (Fig. 8.7A). various applications, such as:
* Detection of autoantibodies (e.g., antinuclear
Indirect Immunofluorescence Assay antibody) in autoimmune diseases (Figs 8.8A
This detects antibodies in sample. Slides and B)
smeared with cells carrying known antigens are “ Detecting microbial antigens, e.g., rabies
commercially available. antigen in corneal smear
*% Step 1: Test serum containing primary * Detection of viral antigens in cell lines
antibody is added to the slide inoculated with the specimens.
 Western Blot, Rapid Tests and CLIA

ST AER

@ WESTERN BLOT “> The Eastern blot is the latest addition to the
list; itis a modification of Western blot, which
Western blot detects specific proteins
detects the carbohydrate epitopes present on
(antibodies) in a sample containing mixture
proteins or lipids.
of antibodies each targeted against different
antigens of same microbe.
Procedure
“+ It is so named for its similarity to Southern
blot (detects DNA fragments) and Northern Western blot comprises of three basic
blot (detects mRNAs) components as follows (Fig. 9.2):

| Problem Solving
Solving Exercise
Exercise1_|
1
Western Blot 0
A commercial sex worker is presented with diarrhea
for 1 month. Screening test [enzyme-linked immuno-
gp 160
sorbent assay (ELISA)] performed for human immuno-
deficiency virus (HIV) showed inconclusive (equivo-
gp 120 gp 120
cal) result. For confirmation, the following test given p66
below was performed. p55/p51 ps5/p51 | 2
1. Identify the test and interpret the result. =
2. What is the principle of the test? 2

gp-41 gp-41 $
3. What are the other applications of the test B
(Fig. 9.1)?
oO
a
p32
Explanation
The test given in the picture is Western blot, done for p24 p24
detection of HIV antibodies.
For HIV diagnosis, if the screening test (ELISA/rapid
diagnostic test) shows inconclusive (equivocal) result, pi7
then it has to be confirmed by supplemental test.

Interpretation
Bands are positive for three antibodies against HIV
antigens gp120, gp41, p24 and p55/51. Control

HIV-2 Final report:

Final Report (gp36) 100 MM Reactive for
Reactive for HIV-1 antibodies. HIV-1
Control Test antibodies
Answers to the other questions are explained
below. Fig. 9.1: Western blot for HIV.
 CHAPTER9 © Western Blot, Rapid Tests and CLIA

(a) Add SDS-treated (b) Individual Ag 2. Nitrocellulose Membrane Blotting

Ag mixture to the fragments are
well containing gel separated by The gel is removed from the electrophoresis
. electrophoresis
apparatus and placed over a protein-binding
sheet, such as nitrocellulose or nylon and the
antigen fragments in the gel are transferred
Direction (blotted) to the nitrocellulose membrane
of
migration (NCM) sheet by the passage of an electric
current.
Protein antigens
denatured in SDS
3. Enzyme Immunoassay (to Detect the
Antibodies)
(c) Ag fragments are ¢*2 Nitrocellulose membrane strips containing
SDS-PAGE
transferred from gel to protein antigen fragments are treated with
NCM by electric current
patient’s sample containing antibodies.
2
Individual serum antibodies would bind to
the respective antigen fragments, thus making
|
a Western blot highly specific
s,
¢ Addition of enzyme-linked anti-human
, immunoglobulin (Ig), detects the individual
current
Nitrocellulose
serum antibodies bound to antigen
membrane fragments. Substrate/chromogen is added
sheet (NCM) for development of colored bands.
(d) Antibodies in sample +
bind to Ag fragments
Applications
Western blot has an excellent specificity. Hence,
itis often used as asupplementary test to confirm
(e) Enzyme-linked & the result of ELISA or other immunoassays
anti-human antibodies y having higher sensitivity. Western blot formats
are added; followed by
substrate is added to
are available to detect antibody (Ab) in various
develop color bands diseases, such as human immunodeficiency
virus (HIV) infection (discussed in Chapter 19),
Lyme’s disease, herpes simplex virus infection,
cysticercosis, hydatid disease and toxoplasmosis.
Fig. 9.2: Western blot (principle).
Abbreviations: SDS-PAGE, sodium dodecyl sulfate polyacrylamide
gel electrophoresis; Ag, antigen.
B RAPID TEST
Rapid tests are revolutionary in the diagnosis of
infectious diseases.
1. Separation of Complex Protein Antigen “* They are very simple to perform, rapid (result
Mixture by SDS PAGE obtained in 10-20 minutes), require minimal
“ SDS: The complex protein (antigen) mixture training, and do not need any sophisticated
is treated with a strong denaturing detergent instruments
called SDS (sodium dodecyl sulfate) “ These tests are also called point-of-care
* PAGE: Then the mixture is subjected to (POC) tests, because unlike ELISA and
polyacrylamide gel electrophoresis (PAGE) other immunoassays, the POC tests can
which separates the antigenic components be performed independent of laboratory
according to their molecular weight; lower equipment and deliver instant results
molecular weight components migrate farther * Two principles of rapid tests are available—
than higher molecular weight ones. lateral-flow assay and flow-through assay
 SECTION1 ©@ General Microbiology, Immunology and Hospital Infection Control

“+ Both the formats are available for the diagnosis HIV infection, leptospirosis, Helicobacter
of various diseases, such as malaria, viral pylori infection, syphilis, etc.
hepatitis by hepatitis B and hepatitis C virus,

Immunochromatographic Test (Lateral-flow Assay)

[Problem Solving
Solving Exercise
Exercise2_|
2
Immunochromatographic Test Explanation
The following test was performed for a patient The test performed here is immunochromatographic
suffering from jaundice for 2 months for the diagnosis test.
of hepatitis B virus infection.
Interpretation
1. Identify the test and interpret the result.
The positive bands are formed at test line [hepatitis B
2. What is the principle of the test?
3. Whatare the other applications of the test (Fig. 9.3)? surface antigen (HBsAg)] as well as control line.
Report
ree tabs Positive for HBsAg.
Answers to the other questions are explained
Fig. 9.3: ICT for HBsAg. below.

Immunochromatographic test (ICT) is based

on lateral-flow technique. It is widely used in
diagnostic laboratories because of its simpli-
city, economy and rapidity. It can be used
for both antigen and antibody detection in
sample. Principle of antigen detection method
cassette .
is described below.
a)
oO

Principle (Antigen Detection) $ on

Zine t T
The test system consists of NCM and an Do D
2 SS | Sample
absorbent pad. Two formats are available— Control band window
cassette or strip (Figs 9.4A and B). The NCM LN
is coated at two places in the form of lines—a iar ° . t
test line, coated with monoclonal Ab targeted ae ne es hy

against the test antigen, and a control line, Galt bi Invalid result

coated with anti-human Ig. Specific Ab against PAPA by

the target Ag labeled with chromogenic BI] GF Positive result
marker (specific Ab tagged with colloidal
Figs 9.4A and B: |Immunochromatographic test (ICT) for
gold or silver, a visually detectable marker) is hepatitis B surface antigen (HBsAg) detection. (A) Cassette
infiltrated in the sample pad lining the sample format; (B) Strip format.
window. Abbreviation: HBsAg, hepatitis B surface antigen.
* The sample (serum) containing the test Source: Department of Microbiology, Pondicherry Institute of
antigen is added to sample well, it reacts Medical sciences, Puducherry (with permission).

with Ab labeled with chromogenic marker

the monoclonal Ab in the test line to form a
(colloidal gold ox silver, a visually detectable colored band (Figs 9.4A and B)
marker) ** Control band: The free colloidal gold-labeled
* Both “Ag-specific Ab-colloidal gold complex” Ab can move further and binds to the anti-
as well as the “free colloidal gold-labeled Ab” human Ig to form a colored control band. If
in buffer moves laterally along the NCM the control band is not formed, then the test is
* Test band: At the test line, the Ag-labeled considered invalid irrespective of whether the
Ab complex is immobilized by binding to test band is formed or not (Figs 9.4A and B).
 CHAPTER9 © Western Blot, Rapid Tests and CLIA

Flow-through Assay Protein A

Control
Flow-through tests are another type of rapid conjugate
diagnostic assays which differ from ICT in two | HIV-1 Capture Immobilized
aspects—(1) protein A is used for labeling Ab | Ab HIV Ab || HIV antigen
instead of gold conjugate, and (2) the sample
eee / Ab
flows vertically through the NCM as compared
AN [E] =—Absorbent
to lateral flow in ICT.
Flow-through tests can be used for both Figs 9.5A and B: Flow-through assays: (A) HIV TRI-DOT
assay for HIV 1 and 2 antibodies detection; (B) Principle.
antigen and Ab detection. HIV TRI-DOT test is
Source: Department of Microbiology, Pondicherry Institute of
a classical example (described below, Fig. 9.5A). Medical sciences, Puducherry (with permission).
It detects antibodies to HIV-1 and 2 separately in
Chemiluminescent .
patient’s serum. substrate u,Light
“+ The test system isin acassette format, consisting 2° Ab a
of NCM and an absorbent pad. The NCM Product

is coated at three regions—two test regions

coated with HIV-1 and 2 antigens and the third
control region coated with anti-human Ig
* Sample and buffer reagents are added
sequentially from the top following which they
pass through the membrane and excess fluid Fig. 9.6: Chemiluminescence system (CLIA)
is absorbed into the underlying absorbent pad and its principle.
* As the patient’s sample passes through the Source: Department of Microbiology, JIPMER, Puducherry
(with permission).
membrane, HIV antibodies, if present, bind
Contd...
to the immobilized antigens (Fig. 9.5B)
* Test dots: Protein A conjugate (present in product + light (photons) > detected by luminometer
buffer) binds to the Fc portion of the HIV or photomultiplier.
antibodies to give distinct pinkish purple
DOT(s), separately for HIV-1 and 2 antibodies Advantages of CLIA
* Control dot: Irrespective of whether the HIV
CLIA claims to be 10 times more sensitive than
antibodies are present or not, protein A can
ELISA.
bind to any IgG present in serum and the IgG-
¢ CLIA can be further modified by using an en-
protein A complex can further bind to the anti-
hancer that potentiates the chemical reaction.
human Ig at the control dot to give a pinkish
This gives CLIA an overall improvement of 200
purple DOT.
folds over ELISA
* Most samples have no ‘background’ signal,
CHEMILUMINESCENCE-LINKED
i.e., luminol compounds do not themselves
IMMUNOASSAY (CLIA) emit light
Chemiluminescence refers to the emission of “* Measurement of chemiluminescence is nota
light (luminescence), as a result of a chemical ratio unlike the measurement of fluorescence
reaction. The principle of CLIA is similar to that (IFA) and or color (ELISA)
of ELISA; however, the chromogenic substance is % Individual specimens can be tested in CLIA in
replaced by chemiluminescent compounds (e.g., contrast to ELISA which is preferred for testing
luminol and acridinium ester) that generate light multiple samples at a time.
during a chemical reaction (luxogenic). The light
Applications
(photons) can be detected by a photomultiplier,
also called as luminometer (Fig. 9.6). Currently, CLIA is available for detection of
antigens or antibodies against various infections,
(Ag-Ab complex)-enzyme (e.g., HRP) + chemilumines- such as hepatitis viruses, HIV, TORCH infections
cent substrate (e.g., luminol and acridinium ester) > (common congenital infection causing agents)
Contd... and biomarkers, such as procalcitonin.
 [CHAPTER|

PSEC
Standard Precautions:
Hand Hygiene and PPE
STIS
10
Healthcare-associated infections (HAIs) refer
Standard Precautions
to (i) the infections acquired in the hospital by
They are indicated while handling all patients,
a patient admitted for a reason other than the specimens and sharps. Components of standard
infection in context, (ii) the infection should not precautions include:
be present or incubating at the time of admission, Q Hand hygiene (details explained later):
and (iii) the symptoms should appear at least > Wash hands promptly after contact with
after 48 hours of admission. infective material
>» Use no touch technique wherever possible.
Q Personal protective equipment (PPE): Described
PREVENTION OF HAIls later
Q Biomedical waste: All biomedical waste including
The preventive measures for HAIs can be broadly sharp should be segregated and disposed appro-
categorized into (i) standard precautions and priately (Refer Chapter 13)
(ii) transmission-based or specific precautions Q_ Spillage cleaning: Clean up spills of infective ma-
terial promptly
(discussed in Chapter 11).
Q Disinfection of patient care items: Ensure that
all patient-care items, such as instruments, devices
@ STANDARD PRECAUTIONS and linens are disinfected before reuse
Q Environmental cleaning of surface and floor
Standard precautions are a set of infection (Refer Chapter 12)
control practices (see highlight box below) Q Sharp: Safe use and disposal of sharp (Refer
used to prevent transmission of diseases that Chapters 13 and 14)
can be acquired by contact with blood, body Q Respiratory hygiene and cough etiquette (Refer
Chapter 11).
fluids, non-intact skin (including rashes),
and mucous membranes. These measures
should be followed when providing care to or Hand Hygiene
handling: Hands of the HCWs are the main source of trans-
* All individuals, whether they appear mission of infections in healthcare facilities. Hand
infectious/symptomatic or not
hygiene is therefore the most important measure
“> All specimens (blood or body fluids) whether
to avoid the transmission of harmful microbes
they appear infectious or not
and prevent healthcare-associated infections.
“+ All needles and sharps whether they appear
infectious or not. Types of Hand Hygiene Methods
Note: Universal precautions was a term used
Hand Rub
in the past to refer to the infection control
practices to avoid contact with patients’ body Alcohol based (70-80% ethyl alcohol) and
fluids, by means of wearing the nonporous chlorhexidine (0.5-4%) based hand rubs are
articles, such as medical gloves, goggles, and available. The duration of contact has to be at
face shields. Now it is replaced by the word least for 20-30 seconds (10.2).
“standard precaution” which in addition * Advantage: After a period of contact, it gets
includes contact with all body fluids regardless evaporated of its own, hence drying of hands
of whether blood is present. is not required separately
 CHAPTER 10 © Standard Precautions: Hand Hygiene and PPE

| Problem
Problem Solving
Solving Exercise?
Exercise 1 |
Hand Hygiene Hand rub: Nurse has to perform hand rub five times,
A nurse in an ICU records the pulse of a patient, 20-30 seconds in each time with 70-80% alcohol hand
records it in the case sheet and then goes back to rub.
Before recording pulse (WHO Moment 1)
nursing station. Then she draws blood of another
After recording pulse (WHO Moment 4)
patient. While transporting the specimen, a drop of
Before drawing blood (WHO Moment 2)
blood fell on her palm. Then she enters the operation
After drawing blood (WHO Moment 3)
theater to assist for a surgery.
1. How many times she has to perform hand hygiene? After recording in the case sheet (WHO Moment 5).
ooocodo

2. What are the hand hygiene methods she has to Handwash: She has to perform handwash once
perform and for how much duration? (after blood drop fell on her palm), with 2-4%
3. What are the hand hygiene products to be used? chlorhexidine hand wash for 40-60 seconds.

Explanation Hand scrub: She has to perform hand scrub once

(before assisting for surgery), with 4% chlorhexidine
Nurse has to perform totally seven times hand hygiene: hand wash for 3-5 minutes.
Five times hand rub, once hand wash and hand scrub (Refer the text below for further details.)
each (Figs 10.1 to 10.3).

can also be used. The duration of contact has to be

at least for 40-60 seconds (10.2). Handwashing is
indicated in the following situations:
“ When the hands are visibly soiled with blood,
excreta, pus, etc.
%,
bdBefore and after eating
7 After going to toilet
~~

+, Before and after shift of the duty

+e

+“ When
+,
giving care to a patient with diarrhea.
Surgical hand scrub (3-5 min): This is indicated
prior to any surgical procedure and also in
between the cases; using 4% chlorhexidine hand
Satter ~procedure // |) wash (Fig. 10.3).
or body fluid ||
xposure risk
After touching My Five Moments for Hand Hygiene
5, patient's
surroundings Indications: The WHO has published standard
guidelines describing the situations or
opportunities when hand hygiene is indicated
Fig. 10.1: My five moments for hand hygiene. in healthcare sectors (Fig. 10.1)—known as ‘My
).
Source: World Health Organization (WHO) (with permission Five Moments for Hand Hygiene’; which include:
1. Before touching a patient
2. Before clean/aseptic procedures
* Indications: Hand rub is indicated during
3. After body fluid exposure/risk
routine patient care activities or taking rounds
4. After touching a patient
in the wards or ICUs—whenever opportunity
5. After touching patient’s surroundings.
for hand hygiene arises, except when the
hands are visibly soiled with blood or other Steps of Hand Rubbing and Hand Washing
specimens.
WHO has also laid down the guidelines
Handwash describing the appropriate steps involved for an
effective hand rubbing and hand washing (Fig.
Antimicrobial soaps (liquid, gel or bars) are
10.2). The method of performing surgical hand
available containing 4% chlorhexidine. If facilities
scrub in depcited in Figure 10.3.
arenotavailable, then even ordinary soap and water
 SECTION1 © General Microbiology, Immunology and Hospital Infection Control

Thumb Back of finger on palms

(Rotational rubbing, both sides) (both the sides)

Rinse hands ~ Close the tap with same

with water use paper towel paper towel or elbow if
there is elbow handle
Scrub your nails on plams Step 9 Step 10
Step 8
(both sides)
Fig. 10.2: Steps of hand rubbing and handwashing (WHO): Hand rub step 1 to 7 (20-30 seconds);
Handwash step 1 to 10 (40-60 seconds).

| Problem Solving
Solving Exercise
Exercise22 _|
Personal Protective Equipment (PPE) Technique and the correct sequence of
A 65-year-old patient was admitted to a hospital with donning and doffing of each of the PPE is explained
complaints of dry cough, sore throat and fever. He was subsequently in the text.
kept in an isolation room. His throat swab was sent Precautions: Doffing should be performed with
for COVID-19 testing which came positive. A nurse is utmost precautions as even a minor breach in the
posted to give care to the patient. doffing procedure would subject the HCW to a huge
1. What are the personal protective equipment (PPE), risk of acquiring the infection.
the nurse should wear while giving care to the Q Do not touch the exposed/contaminated area of
patient? PPE, such as front part of the mask or goggles and
2. Demonstrate the donning and doffing technique outer surface of gloves or gown.
of each of the PPE. Q All PPEneed to be doffed in the designated doffing
3. What is the correct sequence of donning and area of the isolation room except the mask, which
doffing of PPE? need to be doffed after coming out of the isolation
4. What are the precautions need to be followed room.
while doffing of the PPE? Q_ Discard PPE into appropriate BMW bins:
>» Yellow bag: Gown/coverall, mask/respirator,
Explanation shoe cover and cap
PPE: The PPE needed for giving care to the COVID-19 » Red bag: Plastic apron, goggles/face shield,
patient are a pair of gloves, 3-ply mask, gown and gloves
goggles/face shield. If aerosol generating procedure Q Perform hand hygiene after removal of PPE
are to be performed, N95 respirator need to be worn
in place of 3-ply mask.
 CHAPTER 10 © Standard Precautions: Hand Hygiene and PPE

Put approximately 5 mL Dip the fingertips of your right {mages 3-7: Smear the handrub on
(3 doses) of alcohol-based hand in the handrub to the right forearm up to the elbow.
handrub in the palm of decontaminate under the Ensure that the whole skin area is
your left hand, using the elbow nails (5 seconds) covered by using circular movements
of your other arm to operate around the forearm until the handrub
the dispenser has fully evaporated (10-15 seconds)

Dip the fingertips of your left Smear the handrub on the left forearm
Put approximately 5 mL (3 doses) of
hand in the handrub to up to the elbow. Ensure that the whole
alcohol-based handrub in the palm of
decontaminate under the skin area is covered by using circular
your right hand, using the elbow of
nails (5 seconds) movements around the forearm until
your other arm to operate the
the handrub has fully evaporated
dispenser (10-15 seconds)

Lif) Re, A <<

Se) PUN c
rs oe
Nea Sa
ics erie Sri
= eho)

Se |ae

8 “ih 9
Put approximately 5 mL (3 doses) Perform hand rub steps as When the hands are dry,
described in Fig. 10.2
of alcohol-based handrub in the palm sterile surgical clothing and
of your left hand, using the elbow of gloves can be donned
your other arm to operate the dispenser.
Rub both hands at the same time up to
the wrists, and ensure that all the steps
represented in images 2-6 are
followed (20-30 seconds)
Fig. 10.3: Method of performing surgical hand scrub.

apron/coverall, goggles or face shield, shoe

Personal Protective Equipment (PPE)
cover and head cover (Figs 10.4A to N)
Personal protective equipment are used to “ Selection of appropriate PPE is based on:
of
protect the skin and mucous membranes « The level of risk associated with contami-
HCWs from exposure to blood and/o r body
nation of skin, mucous membranes, and
g
fluids and from the HCW to the patient durin
ures. clothing by blood and body fluids during
sterile and invasi ve proced
a specific patient care activity or inter-
“ The various PPE used in healthcare settings
vention (as a part of standard precaution)
are gloves, mask/respirator, gown/plastic
 SECTION1 © General Microbiology, Immunology and Hospital Infection Control

Table 10.1: Indications for appropriate use of glove use.

Indications for glove use

e Asa part of standard precautions
> Before a sterile procedure
> Anticipation of contact with blood or body fluid,
regardless of the existence of sterile conditions
and including contact with non-intact skin and
mucous membrane
e Asa part of contact precautions: Contact with a
patient (and his/her immediate surroundings)
e Heavy duty gloves:To protect from sharp injuries,
mainly used by biomedical waste handlers (Fig. 10.4B)
Indications for glove removal
e Assoon as gloves are damaged
e Gloves are meant for single-use, must be changed
in-between patients or patient care activities
e When there is an indication for hand hygiene
Clinical situations where use of gloves is not
recommended
e For routine patient care activities if there is no
anticipated risk to blood/body fluid or no indication
for contact precautions
e Examples: Measuring blood pressure, temperature,
and pulse, while administering medications (oral
or injections), during maintenance of IV cannula,
during dressing and transporting patient, writing in
the patient's case sheet, etc.

colonized their hands. It is used as part of

standard, contact and droplet precautions.
Gloves should be worn only when there is
an indication (Table 10.1 and Fig. 10.4A). The
use of gloves in situations when their use is
not indicated represents a waste of resources
and gives a false sense of security. Therefore,
gloves should not be used when not clinically
indicated (Table 10.1).
Figs 10.4A to N: Personal protective equipment (PPE):
(A) Gloves; (B) Heavy duty gloves; (C) Surgical mask; Hand Hygiene and Glove Use
(D) N95 respirator; (E) Plastic apron; (F) Linen gown; Glove is not a substitute for hand hygiene. In
(G) Disposable gown; (H) Coverall; (I) Goggles; (J) Face
no way does the glove use modify hand hygiene
shield; (K) Cap; (L) Shoes; (M) Gum boot; (N) Shoe cover.
indications or replace hand hygiene. The
following measures should be adapted during
® Route of transmission of suspected organ- gloves use.
isms—contact, droplet and inhalation (as * Hand hygiene before gloves use: This is
a part of transmission-based precaution). to prevent possible cross-contamination of
* The PPE must be removed immediately gloves with HCW’s flora
following the indication for which it was used. * Handwash after glove use: To prevent
cross-contamination, perform hand wash
Gloves immediately after the removal of gloves
Gloves can protect both patients and HCWs as it creates a moist, warm, and occlusive
from exposure to microorganisms that have environment between the skin and the glove
 CHAPTER 10 © Standard Precautions: Hand Hygiene and PPE

which is ‘safe haven’ for microorganisms. Surgical (3-ply) Mask and Respirators
Furthermore, microtears can occur in gloves Respiratory protection is essential when there is
which may lead to transmission of organism if a risk of transmission of droplets and aerosols.
the HCW has had contact with blood or body There are two type of PPE available for respiratory
fluid protection; surgical mask and respirators.
** Change: Gloves should be worn for a single
patient care activity and not beyond. Gloves Surgical Mask (3-ply Mask)
must be changed between patient contacts Surgical masks (also called as medical mask or
and between separate procedures on the 3-ply mask) are loose fitting, single-use item that
same patient cover the nose and mouth.
* No hand hygiene over the gloved hand: “* They are used as part of standard precautions
Gloved hands should neither be wiped with to prevent splashes or sprays from reaching
any form of handrub nor washed with soap the mouth and nose of the person wearing
and water. them
The technique for donning and doffing of * They also provide some protection from
gloves has been depicted in Figures 10.5 and respiratory secretions and are worn when
10.6. caring for patients on droplet precautions.
Composition
It has three layers (Fig. 10.4C):
1. Outer fluid repellent layer: Hydrophobic
layer that can repel water, blood and body
fluids
2. Middle filter layer: It is made up of melt-
blown material; filters bacteria/viruses and
also filters out the water droplets. In contrast
to N95 respirator, the filter pore size of a
Fig. 10.5: Steps of gloves donning (wearing).
1. Donning of the first glove: Wear by touching and pulling surgical mask is not standardized
only the edge ofthe cuff. 3. Inner hydrophilic layer: Absorbs water, sweat
2. Donning of the second glove: Avoid touching the fore- and spit; made up of non-woven fabric.
arm skin by pulling external surface of second glove by Note: 2-ply masks may look similar to 3-ply
the finger of gloved hand. mask in appearance. However, they have only
two layers (outer and inner), but no middle
filter layer. They should be used for hygienic
and sanitation purposes—in restaurants, spa
centers, food industry; but not in the hospitals.

Instructions
When using a surgical mask, the following
measures should be considered:
* Shelf-life: Disposable (single-use); should be
discarded or changed after 4-6 hours of use or
earlier if itbecomes soiled or wet
Fig. 10.6: Steps of gloves removal (doffing). * Donning: Place the mask carefully, ensuring
Do not touch the outside of the gloves (contaminated)
it covers the mouth and nose, adjust to the
+ Using a gloved hand, grasp the palm area of the other
gloved hand peel off first glove. nose bridge, and tie it securely to minimize
« Hold removed glove in gloved hand slide fingers of any gaps between the face and the mask
ungloved hand under the other glove at wrist and peel * Hanging mask syndrome: Masks should not
off second glove over first glove. be left dangling around the neck, a common
+ First glove will remain inside the pouch of the second
practice observed among doctors, doing so
glove.
+ Perform hand hygiene after removal.
may contaminate the inner side of mask
 SECTION 1 © General Microbiology, Immunology and Hospital Infection Control

“ Negative-pressure: N95 respirators are

described as “negative-pressure” because
the pressure inside the facepiece is negative
during inhalation compared to the pressure
outside the respirator
“+ Removal: N95 respirator should be removed
or changed once in 8 hours or earlier if it
gets clogged, wet or dirty on the inside, or
deformed, or torn
“+ Single-use: N95 respirator is for single-use
Fig. 10.7: Steps of mask donning (wearing).
¢ Pull the straps tight and pull the mask to below chin only, should not be reused as it cannot be
and then apply knots. cleaned or disinfected
¢ Press on the nasal bridge part of the mask to seal “+ Fit checking: After wearing the N95 respirator,
tightly and for N95 respirator, perform fit check. the HCW must perform a fit check to ensure if
it is properly fitted. No clinical activity should
be undertaken until a satisfactory fit check
has been achieved. It includes the following
steps:
= Sealing: The respirator is compressed to
ensure a Seal across the face, cheeks and
the nasal bridge
= The positive pressure seal of the
Fig. 10.8: Steps of mask removal (doffing). respirator is checked by gently exhaling.
¢ Donot touch front part of the mask. If air escapes, the respirator needs to be
« Untie the lower knot first, then the upper knot and adjusted
remove the mask by holding its straps, without = The negative pressure seal of the
touching the front; handwash after removal.
respirator is checked by gently inhaling. If
** Touching the front of the mask while wearing the respirator is not drawn in towards the
should be avoided face, or air leaks around the face seal, the
** Mask should not be worn with beard or respirator is readjusted.
unshaven face ** Fit testing: Fit testing is done to identify which
“+ Hand hygiene should be performed before size and style of N95 respirator is suitable for
donning the mask, upon touching or an individual and to train the HCW on how to
discarding a used mask. don and doff N95 respirator. It should be done
The technique of donning and doffing of surgical at the time of joining and thereafter annually.
mask has been depicted in Figures 10.7 and 10.8.
Protective Body Clothing
Respirator (N95 Respirator) Laboratory coats, plastic aprons, disposable
A respirator is a device designed to protect gowns and coverall (full body cover) are examples
the wearer from airborne microorganisms of protective body wears used in hospitals. They
(e.g., M. tuberculosis). There are many types of are worn when there is a risk that clothing may
respirators. The most common respirator used become exposed to blood or body fluids
in hospital settings is N95 respirator. * Laboratory coats: They are used as a part of
** N95 refers to ‘not resistance to oil and ability a standard precaution by all laboratory staff
to filter off 95% of airborne particles’ which protect their clothing and skin from the
** Composition: The N95 respirator is comprised splash of blood or body fluid; however, they
of four layers of material: an outer and inner are not fluid resistant
layers of spun-bond polypropylene and ** Plastic aprons: Worn when there is a low risk
middle two layers of cellulose/polyester, melt- of contamination of blood/body fluid. They
blown polypropylene filter are fluid-resistant and for single-use only, i.e.,
 CHAPTER 10 © Standard Precautions: Hand Hygiene and PPE

used for one procedure or one patient care

activity (Fig. 10.4E)
* Disposable gowns: They are long-sleeved,
fluid resistant; indicated when there is a
moderate risk of contamination with blood/
body fluid (Fig. 10.4G)
ae* Coverall: It comprises of a gown with pant

and hood, which covers the whole body

including the head. Coverall should be used
in the following situations Fig. 10.10: Steps of gown removal (doffing).
= Anticipated risk of splashing with a large Do not touch front part of the gown
volume blood/body fluid (e.g., cardiac e Unfasten gown ties, taking care that sleeves do not
touch the body when reaching for ties.
surgeries)
e Pull the gown away from neck and shoulders, touching
m Anticipated risk of extensive skin to skin inside of gown only.
contact with a patient known to harbor e Turn gown inside out and roll into a bundle and
organisms of contact transmission (e.g., discard.
lifting a patient with uncontrolled diarrhea) e Perform hand hygiene after removal.
m Handling patients infected with patho-
gens of high mortality (e.g., Nipah or
Oo
Prevents exposure to blood and/or body fluids
Ebola) or in the laboratory while handling that may be splashed, sprayed, or splattered
their specimens (Fig. 10.4H). into the face during clinical procedures
“+ Donning: Gown should be fully covered, torso “+ Eyewear must be worn during procedures
from neck to knees, arms to end of the wrist that are likely to generate droplets or aerosols
and then wrapped around the neck. It should of blood and/or high-risk body fluids (Figs
be fastened in the back of neck and waist (Fig. 10.4] and J).
10.9)
Head Cover and Shoe Cover
“+ Doffing: Once the taskis performed, the gown
must be removed immediately after use by “+ Head cover or cap (Fig. 10.4K) is used when
unfastening the gown ties taking care that spillage of blood is suspected, e.g., during
sleeves should not contact the body while major cardiac surgeries, etc.
reaching for the ties (Fig. 10.10). “ Shoe covers include: (1) Surgical shoes
(slippers) and shoe covers (Figs 10.4L
Protective Eye/Face Wear and N): Used mainly in ICUs and opera-
Protective eyewear (goggles, or face-shields) are tion theaters to protect HCWs from organ-
used to protect the mucous membranes of the isms present in floor and (2) Gumboots:
eyes, nose, and mouth Used for anticipated risk of sharp injuries
(e.g., for biomedical waste handlers, laun-
dry staff and housekeeping staff) (Fig.
10.4M).
Donning and Doffing
In order to minimize the risk of transmission
of infection, donning (wearing) and doffing
(removing) of PPE must be performed in a
particular sequence.

Donning (wearing): Gown first > Mask or respirator

Fig. 10.9: Steps of gown donning (wearing). — Goggles or face shield + Gloves
Fully cover torso from neck to knees, arms to end of wrists, Doffing (removing): Gloves first + Face shield or
and wrap around the back. Fasten it in the back of neck goggles — Gown — Mask or respirator.
and waist.
 SECTION 1 © General Microbiology, Immunology and Hospital Infection Control

Doffing is extremely important as even a few mL) should be considered infectious, and
minor breach in the doffing procedure would need to be cleaned at the earliest.
subject the HCW to a huge risk of acquiring
the infection. This could be a potential reason Steps of Spill Management (CDC)
why many HCWs got infected during COVID-19 The following steps need to be sequentially followed
pandemic. for the management of blood or body fluid spillage.
Ve Any spillage, should be attended immediately
«+ All PPE should be removed just before exit-
Pe, Mark the spill area, place the wet floor signage
ing the patient room except a respirator,
3 Wear appropriate PPE (gloves and gown) as
which should be removed after leaving the mentioned in the spill kit
patient room and closing the door Confine the spill and wipe immediately with an
** Discard into appropriate BMW bins: absorbent towel or cloth, which is spread over the
w Yellow bag: Gown/coverall, mask/ spill to solidify the blood or body fluid. Then it is
disposed as infectious waste
respirator, shoe cover and cap
Clean with hypochlorite (freshly prepared)
= Red bag: Plastic apron, goggles/face
> For large spills (>10 cm size): Use 1:10 dilution
shield, gloves. of 5% hypochlorite (5000 ppm), i.e., 0.5%
> Forsmall spills (<10 cm size): Use 1:100 dilution
Blood Spill Management of 5% hypochlorite (500 ppm), i.e., 0.05%
Spillage of blood and body fluid poses a Allow the disinfectant to remain wet on the surface
substantial risk for the transmission of blood- for at least a contact time of 10 min
Rinse the area with clean water to remove the
borne viruses, such as hepatitis B, C and HIV.
disinfectant residue.
Therefore, any spillage (small, few drops to large,
 Transmission-based Precautions

SEEEE SG NORE LLL

@ DEFINITION be discontinued later when the diagnosis is

ruled out.
Transmission-based precautions (TBPs), also
called as specific precautions are set of infection HB CONTACT PRECAUTIONS
control practices which should be followed over
and above the standard precautions. Contact precaution should be followed when
¢* TBPs should be practiced when giving there is a definitive or suspected evidence of
care for the patients who are infected with certain infectious agents that are transmitted
infectious agents having specific mode of by direct or indirect contact during patient care.
transmissions, such as contact, droplet and “+ Direct transmission occurs when infectious
airborne agents are transferred from one person to
*» Accordingly, there are three types of TBPs— another person without a contaminated
contact precautions, droplet precautions and intermediate object or person. For example,
airborne precautions direct contact through contaminated hands
** TBPs should be followed even when the (most common mode of transmission of
specific infections are suspected and may organism in healthcare settings)

[Problem Solving
Solving Exercise
Exercise11 |:
Contact Precautions Explanation
A 70-year-old woman after surgery for total knee A cluster of cases of surgical site infection occurred
replacement, is transferred to the postoperative with MRSA infection which resulted from lack of
ward. Four days later, patient develops erythema standard and contact precautions of the index
and pus discharge at the wound site. Wound swab case.
sent for culture shows growth of MRSA (methicillin- Q Inadequate staffing: Ten patients are there in the
resistant S. aureus); sensitive only to vancomycin ward and only two sisters are posted there for their
and linezolid. Total of 10 patients are housed in the care
same ward and only two nurses are posted. Hand Q Inaccessibility to handrub: Handrub are available
rub is available only at the entrance and at the only at the entrance and nursing station but not at
nursing station. There is only one stethoscope, blood the bedside
pressure (BP) apparatus and thermometer in the Q Nopatient dedicated equipment: There was only
ward. It is a practice in the ward to use same gloves one stethoscope, BP apparatus and thermometer,
continuously, due to shortage of supply. After 2 days, etc., in the ward
another patient following appendectomy develops Q Inappropriate use of gloves: HCWs are using
discharge from the wound site and MRSA grows on the same gloves in multiple occasions, without
culture with same antimicrobial sensitivity pattern. changing them when indicated
Identify the risks for transmission and the type of Q Patient placement is not followed: Patient
transmission-based precaution applicable? isolation or cohorting are not followed.
 SECTION 1 © General Microbiology, Immunology and Hospital Infection Control

“ Indirect transmission involves the transfer of = Patients with similar infections requiring
an infectious agent through a contaminated contact precautions can be placed together
intermediate object (clothes, patient-care either in the same isolation room, or in the
devices, environmental surfaces, fomite) or same cubicle or corner of a ward or
person. = Spatial separation of minimum of 3 feet
distance between the beds with privacy
Agents Transmitted Through Contact curtains.
MRSA (methicillin resistant S. aureus) “« Transfer of patients: Patient movement
CRE (carbapenem resistant Enterobacteriaceae) should be limited only to medically-necessary
VRE (vancomycin resistant enterococci)
purposes. When transport is necessary, the
OS)
BareMDR (multi-drug resistant) nonfermenting
gram-negative bacilli, such as Acinetobacter, HCW must wear PPE before transport and the
Pseudomonas, etc. infected areas of the patient’s body should be
Q Agents of conjunctivitis (e.g., adenovirus, covered to contain the infection
gonococcus, Chlamydia) * Disinfection of the rooms: Patient rooms
Q Any highly contagious skin lesions (abscess,
must be frequently cleaned and disinfected
impetigo, infected ulcers) infected with Group A
Streptococcus, Staphylococcus, HSV lesions adequately (e.g., at least daily and before use
Q_ Skin infestations (e.g., scabies) by another patient) focusing on frequently-
Q Agents of diarrhea, such as rotavirus, Vibrio, C. touched surfaces and equipment in the
difficile immediate vicinity of the patient.
Q Enterically transmitted hepatitis viruses (HAV and
HEV).
& DROPLET PRECAUTIONS
Infection Control Measures Droplet precautions when used in addition to
standard precautions are intended to prevent the
The following infection control measures
spread of infectious agents that are transmitted
should be applied in addition to other standard
through respiratory droplet via close respiratory
precaution measures.
or mucous membrane contact with respiratory
** Hand hygiene: Strict adherence to hand
secretions.
hygiene is an absolute requirement of contact
** Respiratory droplets are large-particles (>5
precaution as transmission via contaminated
um in size) that are generated by a patient
hands accounts for majority of contact
who is coughing, sneezing or talking
transmission
** Transmission via large droplets requires close
** PPE: Gloves and gown are the essential
contact (<3 feet) as droplets do not remain
personal protective equipment (PPE) that
suspended in the air and generally, only travel
the healthcare worker (HCW) should wear
shorter distances
upon entry to the patient-care area and must
* Some infectious agents transmitted by droplet
be removed before leaving the patient-care
route can also be significantly transmitted
area. Surgical mask and protective eyewear
by contact mode. This is because the larger
are optional PPEs, needed if there is a risk of
droplets settle on the surfaces and inanimate
exposure to splashes or spray of blood and
objects within l-meter distance, which
body substances into the face and eyes
subsequently spread to other individuals
* Equipment: Single-use patient-dedicated
when they touch the contaminated surfaces
equipment (e.g., blood pressure cuffs,
and then touch their eyes, nose or mouth.
stethoscopes, thermometers) must be used.
If not possible, then the equipment should
Agents Transmitted Through Droplets
be cleaned and allowed to dry before use on Q_ Diphtheria (pharyngeal)
another patient Haemophilus influenzae type b
* Patient placement: Single isolation room Neisseria meningitidis
with a bathroom facility is preferred. If not Pertussis (whooping cough)
oD Pneumonic plague
ee
available, then cohorting is recommended.
Cohorting may be carried out in various ways Contd...
 CHAPTER 11 © Transmission-based Precautions

‘Problem Solving Exercise 2

Droplet Precautions > Explanation
A cluster of cases of upper respiratory tract infection A cluster of COVID-19 (URTI) cases occurred in a long-
(URTI) occurred in a long-term care facility, following term care facility following a group activity where one
a group activity held in a common food area of the of the attendants was already suffering from URTI. The
hospital. All cases who attended the group activity factor which promoted the spread of infection include:
had food, sitting close to each other at the dining Q Overcrowding: Group activity held ina common
table. One of the individual who attended the group food area and individuals had food, sitting close to
activity was already suffering from URTI since four each other at the dining tables
days. Due to the lack of waste bins in the dining room, Q Lack of droplet precaution by the index case:
used tissues were placed on the dining room tables. The index case did not follow any measures of
The shared bathrooms were far from the dining area, droplet precaution, such as wearing surgical mask,
therefore hand hygiene was not performed during hand hygiene, etc. He should not have attended
the event. Eight individuals reported symptoms any group activity when suffering from URTI
consistent with COVID-19, which was later confirmed Q Inappropriate respiratory hygiene: Due to the
by molecular test. Discuss the infection control lack of waste bins in the dining room, used tissues
breach occurred. What type of transmission based were placed on the dining room tables
precautions needed for these cases when they are Q Inadequate hand hygiene due to hand hygiene
admitted in ward? facility was far away from the dining area.

Contd... * The primary function of surgical mask is

for ‘source control’; which prevents the
Mycoplasma pneumonia transmission of droplets from the wearer to
Streptococcal (group A) pharyngitis
the environment. N95 respirator does not
Influenza viruses, seasonal
SARS-CoV2 (COVID-19)
provide additional environmental protection
Viral hemorrhagic fevers due to
oOooovo Lassa, Ebola, and therefore, should not be used for this
Marburg, Crimean Congo Fever viruses purpose
Q Other viruses: Mumps, Parvovirus B19, Rhinovirus, * Secondary function of the surgical mask is
Rubella, Adenovirus
to protect the person wearing it from larger
droplets in the environment
infection Control Measures “ AGPs: For certain diseases like seasonal
influenza, viral hemorrhagic fever or
The following infection control measures should
be applied in addition to standard precautions.
COVID-19, the HCWs should wear N95
respirator during aerosol generating
1. Hand Hygiene procedures (AGPs), described subsequently
in this chapter.
Droplet transmission is also associated with
contact transmission (as discussed earlier). 3. Respiratory Hygiene/Cough Etiquette
Therefore, hand hygiene is an important
The following measures are recommended for
component of droplet precautions.
all individuals with respiratory symptoms (Fig.
2. Personal Protective Equipment jeehy
Healthcare workers (HCWs) should wear a sur- “ Directly coughing or sneezing on hands or
gical mask when close contact (<3 feet) with the rubbing of the nose should be strictly avoided
patient is anticipated and also upon room entry. “ Mouth and nose should be covered with a
“ Patients should wear a surgical mask (all the tissue when coughing or sneezing. Tissues
time) should be disposed into the yellow waste bins
“* HCWs should wear protective eyewear if there
after use
is a risk of splashes or spray to eye/face. Gown “ If no tissues are available, coughing or
and gloves should also be worn to prevent sneezing can be done into the inner elbow
contact transmission (sleeves), turning away from other patients
 SECTION 1 ©@ General Microbiology, Immunology and Hospital Infection Control

Do not directly Use your * Social distancing: Individuals with respiratory

cough or sleeve (inner elbow)
sneeze on hands while coughing symptoms should always maintain a distance
of least 1 meter from others.

4. Patient Placement
A single room is preferred for patients who
require droplet precautions. If not available,
alternative placement options can be looked for
similar to contact precaution, such as cohorting,
spatial separation of >3 feet and drawing the
curtain between patient beds.

5. Transfer of Patients
Transfer of patients on droplet precautions
should be limited as there is a high-risk of
transmission. If unavoidable, then the following
Wear surgical Turn your head Hand hygiene
mask to limit away from others afer coughing precautions should be undertaken.
the spread and use tissue while or sneezing ** The patient should wear a surgical mask while
coughing/sneezing
they are being transferred
Fig. 11.1: Respiratory hygiene/cough etiquette.
** Patients should follow respiratory hygiene and
cough etiquette
** Hand hygiene should be performed after “+ HCW transporting the patient should wear
having contact with respiratory secretions surgical mask, gloves, gown and protective
* Contaminated hands should be kept eyewear.
away from the mucous membranes of the
eyes 6. Disinfection of the Rooms
** In outpatient settings, patients with respiratory Patient-care items, bedside equipment, frequent-
symptoms should be segregated separately, ly touched surfaces area and environmental sur-
provided with mask and attending the cases faces should be cleaned daily with appropriate
must be fast-tracked disinfectants according to the hospital policy.

@ AIRBORNE PRECAUTIONS
[Problem Solving
Solving Exercise3_—|
Exercise 3
Airborne Precautions 3. Demonstrate the method of donning and doffing
An intern is posted in tuberculosis isolation ward, of each PPE with correct sequence.
which comprises of individual isolation rooms. His 4. What are the precautions need to be followed
nature of job is to draw blood specimen, giving while donning and doffing of each PPE?
injections, measuring blood pressure, changing the Explanation
dress of the patient etc.
M. tuberculosis is transmitted through aerosol.
1. What type of transmission-based precaution is
Therefore, aerosol precaution is needed. The
needed in tuberculosis isolation ward? Discuss its
recommended PPE to be worn before entering into
components.
an isolation room are gloves, gown, N95 respirator
2. What are the recommended personal protective
and goggles/face shield.
equipment (PPE) to be worn before he enters into
For the answers to the other questions, refer text
an isolation room?
below and also Chapter 10.

Airborne precautions when used in addition to spread of infectious agents that are transmitted
standard precautions are intended to prevent the through respiratory aerosols.
 CHAPTER 11 © Transmission-based Precautions

Aerosols are small-particles (<5 um) gener- 2. Patient Placement

ated by an infectious person during coughing, Patients should be placed in an airborne infec-
sneezing, talking or performing certain aerosol- tion isolation room (AIIR). The components of
generating procedures (e.g., intubation). These AIIR include: adequate ventilation, ultraviolet
smaller droplets remain suspended in air for germicidal irradiation (UVGI) and filtration.
long periods and may disperse to a distant place
along with the air current. 3. Ventilation
Ventilation can reduce the risk of infection
Agents Transmitted Through Aerosols through dilution and removal of room air
Mycobacterium tuberculosis
containing infectious aerosols by introduction of
Measles virus
Varicella (chickenpox and zoster) clean or fresh air into the room, either by natural
Smallpox (variola) and monkeypox virus or mechanical ventilation.
Aerosolizable spore-containing powders, such as
ooooo
Bacillus anthracis Natural Ventilation
Q Aspergillus (pulmonary aspergillosis) Natural ventilation refers to the fresh air that
enters and leaves a room through openings,
Aerosol-generating Procedures (AGPs) such as windows or doors.
* The effect of natural ventilation is maximized
AGPs are procedures that can generate much
when the door and windows are placed at
higher concentrations of aerosols as compared
opposite to each other and are kept open to
to coughing, sneezing, or speaking and are as-
maintain airflow at all times
sociated with higher risk of pathogen transmis-
* In a consultation room with natural
sion.
“ Therefore, it is recommended to follow ventilation, the seating arrangement for
patient and doctor should be made in such
airborne precautions, such as isolating
that doctor should sit away from the direction
the patient in negative pressure room and
of natural airflow, thus has a lesser risk of
wearing appropriate PPE like N95 respirator
exposure (Figs 11.2A and B)
“» Examples of AGPs include: Endotracheal intu-
bation, open respiratory and airway suction-
ing, tracheostomy care, cardiopulmonary
resuscitation, sputum induction and bron-
choscopy.

Infection Control Measures

A prudent approach is to implement infection
control measures at the earliest based on
clinical suspicion, and discontinue it later if
the patient is subsequently diagnosed with
a disease that does not require airborne
precautions.

odds 4
While giving care to a patient with airborne Figs 11.2A and B: Schematic diagram showing seating
directions of patient and doctor in a consultation room.
precaution, the HCWs must wear N95 or higher
In room (A), the seating arrangement is along with
level respirator. The HCW must perform fit direct of natural ventilation of air flow, so that the
checking every time before donning the N95 doctor has a higher risk of exposure to the potentially
respirator to ensure it is properly applied. Gloves, infected air.
gown and protective eyewear may be needed if In room (B), doctor is sitting away from the direction of
natural airflow, thus has a lesser risk of exposure.
the exposure risk is likely to be present.
 SECTION 1 @ General Microbiology, Immunology and Hospital Infection Control

ae
AER
fas)wet

—<—
i
SF

Open
windows
windows
Open

Doors
Direction of airflow
under the door
Fig. 11.3: Schematic diagram of a room with natural
ventilation.

“+ In ward set-up, the beds should be placed

away from air flow (door-window direction)
(Fig. 11.3).
Mechanical Ventilation (Negative-pressure
Room)
Negative-pressure room includes a mechanical Figs 11.5A and B: Exhaust fan: (A) Poorly-installed in an
ventilation system which maintains the pressure open window space; (B) well-installed, closely fitted.
of the room slightly lesser than the pressure
of the entry area (i.e., creating a “negative room only when it is absolutely necessary. In
pressure”), so that it allows air to flow into the such a case, the following measures should be
isolation room but not escape from the room, undertaken.
as air naturally flows from areas with higher ** The patient should wear a surgical mask
pressure to the areas with lower pressure, and follow respiratory hygiene and cough
thereby preventing contaminated air from etiquette
escaping the room. ** Any skin lesions associated with the condition
(e.g., chickenpox) should be covered
4, Ultraviolet Germicidal Irradiation ** The HCW must wear N95 respirator and other
If adequate ventilation is not possible, wall PPE as indicated.
mounted ultraviolet germicidal irradiation
(UVGI) devices can be used as in addition to 7. Respiratory Hygiene
negative pressure ventilation (Fig. 11.4). Patients must be explained in detail about cough
hygiene as described under droplet precautions
5. Filtration and Figure 11.1.
The room air directly exhausted to the outside
through an exhaust fan or through HEPA (high 8. Visitors and Staff
efficiency particulate air) filtration. Exhaust fans Entry of the visitors and staff should be absolutely
must be properly installed closely fitting to the restricted or they should wear PPE before entry
window (Figs 11.5A and B). into the room.
The infection control measures need to be
6. Transfer of Patients taken for various standard and transmission-
The patient on airborne precaution should based precautions have been summarized in
be transferred outside the negative pressure Table 11.1.
 CHAPTER 11 © Transmission-based Precautions 107

_ Table 11.1: Measures be followed during standard and transmission-based precautions.

Type Isolation | Hand Apron or Eye pro- | Handling of | Visitors

room or hygiene gown tection equipment
cohorting
Standard Not Yes As If soiling As As Single use or No additional
required required* likely’ required? required’ reprocessed precautions

Contact Essential Yes Essential Essential As As Same as Same

required’ = required* standard precautions
as for HCWs

Droplet Essential Yes As If soiling Surgical As Same as Restricted.

required* likely’ mask is required’ standard Precautions,
essential - same as for
HCWs

Airborne _ Essential Yes As If soiling N95 As Same as Same as for

(negative required* _ likely* respirator required* contact droplet
pressure) is essential

*Gloves are used when there is likely exposure to blood, body fluids and contaminated items.
specimens on face respectively.
SMask or eye protection is required during procedures likely to generate droplets or anticipated splash of
body fluids.
*Soiling is likely to occur during procedures that are expected to generate contamination from blood and
Environmental cleaning with an appropriate disinfectant is required for all type of precautions.
 Sterilization and Disinfection

OREN PEELE
2
Table 12.1: Level of sterilant/disinfectants according
B INTRODUCTION
to their microbicidal action.
The sterilization and disinfection practices 3 y =]
in a hospital is of paramount importance Level ofdis- | 2 a é «| 2
in preventing transmission of healthcare- infectant/ § 5 ‘ 3 rfs 3
associated infections. sterilant & 2 s E £sisé

Definitions Sterilant Yes Yes Yes Yes Yes Yes

Sterilization, disinfection and cleaning—aim Disinfectant

at removing or destroying the microorganisms High level +/- Yes Yes Yes Yes Yes
from materials or from body surfaces. However, Intermediate No Yes Yes Yes Yes Yes
they vary in their efficacy of destroying the level
microorganisms (Table 12.1). Low level No No +4/- +/- Yes Yes

Sterilization ** Results in reduction of 210° log colony forming

Sterilization is a process by which all living units (CFU) of microorganisms and their
microorganisms including viable spores, are spores
either destroyed or removed from an article, ** The agents which achieve sterilization are
surface or medium. called as sterilants (Table 12.2).

Table 12.2: Agents used in the hospital for achieving sterilization, disinfection and cleaning.
Physical methods Chemical methods
Sterilants
Agents of e Steam sterilizer (autoclave) e Ethylene oxide (ETO) sterilizer
sterilization e Dry heat sterilizer (hot air oven) e Plasma sterilizer
e Filtration
e Radiation: lonizing and non-ionizing (infrared)
Disinfectants
High-level No physical methods in this category e Aldehydes—glutaraldehyde, orthophthaldehyde
disinfectants e Peracetic acid
e Hydrogen peroxide
Intermediate- e Heat-based methods: Boiling e Alcohols—ethyl alcohol and isopropyl alcohol
level e Ultraviolet (non-ionizing) radiation e Phenolics—phenol, cresol, lysol
disinfectants e Halogens—iodine and chlorine
Low-level No physical methods in this category e¢ Quaternary ammonium compound (QAC)
disinfectants e Chlorhexidine
Cleaning
Agents of Automated washers, such as ultrasonic Enzymatic solution
cleaning washers, washer-disinfector and automated Detergent
cart washers Soap (antimicrobial or plain soap)
 CHAPTER 12 © Sterilization and Disinfection

Disinfection Storage Packaging

area Re
area Decontamination
It refers to a process that destroys or removes Issue
area
count
most if not all pathogenic organisms but may or
may not destroy bacterial spores.
“+ Results in reduction of 210° log CFU of most
microorganism but not spores
* Achieved by a physical agent or a chemical
Sterilization
agent and are normally used only on /Receipt
“counter
inanimate objects, not on body surfaces
* The agents which achieve disinfection are
called as disinfectants (Table 12.2) Fig. 12.1: Central Sterile Services Department (CSSD).

* Depending upon their efficacy, the disinfect- Contd...

ants are further classified into three categories
(Table 12.1)—high, intermediate and low level 1. Decontamination area: The items are collected
disinfectants. and then decontaminated/cleaned by either
manual wash or by automated machines
Note: Antiseptics are a type of disinfectants (ultrasonic washer and washer-disinfector)
which are safe to apply on body surfaces (skin 2. Packaging area: Here, the items (medical
and mucosa) resulting in the destruction of devices) are enclosed in materials or a container
organisms present on the body surfaces. This designed to allow the penetration and removal
type of disinfection is termed as asepsis. of the sterilant during sterilization and then to
protect the device from contamination and other
damage following sterilization and until the time
Cleaning (Decontamination)
of use
Cleaning refers to the reduction in the 3. Sterilization area: The packed medical devices
pathogenic microbial population to a level at received from the packaging area are subjected to
sterilization process by steam sterilizer, ethylene
which items are considered as safe without
oxide sterilizer (ETO) or plasma sterilizer
protective attire
4. Sterile storage area: After sterilization the
* Results in reduction of at least 1 log CFU of sterilized items are stored in this area. It has an
most of the microorganism but not spores issue counter to supply the items to OTs and
“* Achieved by manual or mechanical cleaning various other areas of the hospital.
by soap and detergents to eliminate debris or
organic matter from the medical devices or
surfaces.
@STERILANTS
In a healthcare facility, most of the Steam Sterilizer (Autoclave)
sterilization practices for surgical instrument Steam sterilizer functions similar to a pressure
and other critical care items are carried out cooker; by providing moist heat of >100°C. It is
in Central Sterile Supply Department (CSSD). a pressure chamber; consists of a cylinder, a lid
Therefore, it is important to understand the and an electrical heater.
workflow of CSSD. “ Pressure chamber: It consists of:
a A large cylinder (vertical or horizontal)
Central Sterile Supply Department (CSSD) made up of gunmetal or stainless steel,
CSSD is an integrated place in hospitals that performs
in which the materials to be sterilized are
sterilization of medical devices, equipment and
consumables; that are used in the operating theater placed
(OT) of the hospital and also for other aseptic = A steam jacket (water compartment).
procedures. * Lid: It bears the following:
four
The processing area of CSSD consists of = A discharge tap for the passage of air and
zones starting from an unsteril e area to
unidirectional
steam
a sterile area separated by a physical barrier (Fig. 12.1).
Decontamination area — Packaging area
—> m A pressure gauge (sets the pressure at a
Sterilization area — Sterile storage area particular level)
Contd... = Asafety valve (to remove the excess steam).
 SECTION 1 © General Microbiology, Immunology and Hospital Infection Control

: Safety valve
“> Electrical heater: It is attached to the jacket; Discharge tap
that heats the water to produce steam. ¢3— Pressure gauge

Sterilization Conditions
The steam sterilizer can be set to provide higher
temperatures by adjusting the pressure provided
Pressure chamber
to the vessel. The most commonly used sterilization
condition is 121°C for 15 min at a pressure of 15
pounds per square inch (psi).

Steam
Uses of Steam Sterilizer (Autoclave) jacket
Steam sterilizer is the most commonly used Electrical
sterilization method in the hospital. It is used for: heater
“* All critical and semi-critical items that are heat
and moisture resistant: surgical instruments,
anesthetic equipment, dental instruments,
implanted medical devices and surgical
drapes and linens
“* Culture media preparation
“+ Biomedical waste treatment of waste and
sharps.

Types of Steam Sterilizer

Steam sterilizers are available in various sizes
and dimensions.
“+ Horizontal type (large volume capacity) (Figs
12.2A and B): Itis used in CSSD, large-size lab-
Figs 12.2A to C: Steam sterilizer (autoclave): (A) Schematic
oratories and for biomedical waste treatment
diagram; (B) Horizontal autoclave; (C) Vertical autoclave.
“+ Vertical type (small volume capacity) (Fig.
12.2C): It is used for small-size laboratories. = Respirators and dental equipment
= Devices with electronic components
Sterilization Control ® Multi-lumen tubings, etc.
The effectiveness ofthe sterilization achieved by * Advantages of ETO: (i) Large chamber
steam sterilizer can be monitored by biological capacity, (ii) low temperature (55°C) is
indicator, such as spores of Geobacillus maintained, therefore suitable for heat
stearothermophilus: sensitive items, (iii) high penetration power-
ETO is highly diffusible, penetrates areas that
Ethylene Oxide (ETO) Sterilizer cannot be reached by steam, (iv) non-corrosive
Ethylene oxide (ETO) is one of the widely used to plastic, metal and rubber materials
gaseous chemical sterilants in CSSD. ** Disadvantages: (i) ETO is highly inflammable,
* Sterilization cycle: It is carried out ina special irritant, explosive and carcinogenic, (ii) long
equipment called ethylene oxide sterilizer duration of cycle (12-14 hours), (iv) high cost
(Fig. 12.3) of instrument and consumables
** Uses: ETO is used by CSSD to sterilize critical * Sterilization control: By using spores of
items (and sometimes semicritical items) that Bacillus atrophaeus.
are moisture or heat sensitive and cannot be
sterilized by steam sterilization. Examples Plasma Sterilization
include: Plasma sterilizer is a special device used to
# Heart-lung machine components create the plasma state (commercial brands,
= Sutures, catheters and stents such as Sterrad, Fig. 12.4).
 CHAPTER 12 © Sterilization and Disinfection

Fig. 12.3: Ethylene oxide sterilizer. Fig. 12.4: Plasma sterilizer (Sterrad). Fig. 12.5: Dry heat sterilizer
Source: Johnson & Johnson Pvt. Ltd. (hot air oven).
Source: 3M India Pvt. Ltd.

** Ituses hydrogen peroxide as chemical sterilant

* The cycle is run for 24-75 min, at low tempera-
ture (37-44°C)
* Sterilization control: Spores of Bacillus
stearothermophilus is used as a biological
indicator
* Uses: It is used by CSSD for sterilization of
materials and devices that cannot tolerate
high temperature and humidity of steam
sterilizer, such as some plastics, electrical
devices, and corrosion-susceptible metals,
such as arthroscope, micro and vascular
instruments, spine sets and laparoscope.

Dry Heat Sterilizer (Hot Air Oven)

mn,esrnee eTED

This method is used for materials that might be Fig. 12.6: Filter apparatus with membrane filter.
Source: Department of Microbiology, JIPMER, Puducherry.
damaged by moist heat or that are impenetrable
to the moist heat (e.g., glass wares, powders,
* Filtration of air: (1) HEPA filters (High-
petroleum products, sharp instruments) (Fig.
efficiency particulate air filters) in hospitals
12.5).
are used in biological safety cabinets, airflow
“ The most common cycle used is 160°C for
120 minutes system, operation theatre, and isolation
rooms. (2) Filters are also used in surgical (3-
* Sterilization control: Spores of Bacillus
atrophaeus. ply) mask and N95 respirators
* Filtration of liquid: (1) Used for bacteriologi-
Filtration cal examination ofwater in hospital settings,
especially dialysis water. (2) It is also used
Filtration acts by removing microorganisms,
to remove bacteria from heat labile liquids,
not by killing. Membrane filters are the most
such as sera, sugar, toxin, vaccine and anti-
widely used filters in hospitals. They retain all
biotic solutions.
the particles on the surface that are larger than
their pore size (Fig. 12.6). Membrane filtration The sterilization control of membrane filters
has two wider applications in hospital settings— includes Brevundimonas diminuta and Serratia
marcescens.
filtration of air and water.
 SECTION 1 ©@ General Microbiology, Immunology and Hospital Infection Control

Radiation over glutaraldehyde—(1) it does not require

activation, (2) better odor, (3) less eye irritation,
Radiations are of two types:
(4) acts faster (5-10 min). However, it does not
* Ionizing radiation (e.g., cobalt 60 gamma
kill spores effectively and stains skin gray.
rays): It is a low-temperature sterilization
method (called as cold sterilization) that
Peracetic Acid
has been used for a number of medical
products (e.g., tissue for transplantation, Peracetic acid is used in automated machines.
pharmaceuticals, medical devices) It is also available for manual immersion; 0.1-
* Non-ionizing radiation: Infrared radiation 0.2%, used for 5-15 min.
technology can be used for sterilization for Use: It can be used to sterilize medical (e.g.,
selected heat-resistant instruments. Ultraviolet endoscopes, arthroscopes), surgical, and dental
radiation is another example, described under instruments. Peracetic acid in combination
intermediate level disinfectant. with hydrogen peroxide has been used for
disinfecting hemodialyzers
@ HIGH-LEVEL DISINFECTANTS (HLD)
Hydrogen Peroxide (H,O.)
HLD agents are capable of killing bacterial
spores when used in sufficient concentration H,O, works by producing destructive hydroxyl
under suitable conditions. They can kill all the free radicals that can attack various cell compo-
other microorganisms. nents.
Uses: H,O, has several usages at various
Aldehyde concentrations. It is sporicidal only at >4-5%
Formaldehyde, glutaraldehyde and ortho- * 3% H,O, is used for environmental surface
phthalaldehyde are the commonly used disin- disinfection, fogging and for wound clean-
fectants. ing
* 3-6% H,O, is used to disinfect soft contact
Glutaraldehyde lens, tonometer biprisms, ventilators, fabrics,
“++, Semicritical items: It remains active in and endoscopes, etc.
the presence of organic matter and is * 6-7.5% H,O, is used as chemical sterilant in
non-corrosive to equipment. Therefore, plasma sterilization
glutaraldehyde is the most common HLD * Vaporized H,O,isused for industrial steriliza-
used for semicritical equipment, such as tion of medical devices and for decontamina-
endoscopes and cystoscopes tion of large and small area.
m It is used at 2% or 2.4% concentration
(e.g., Cidex). It disinfects objects within 20 INTERMEDIATE-LEVEL
minutes but may require longer time to kill DISINFECTANTS
spores (10-14 hours)
m It is available in inactive form; has to be Alcohol
activated by alkalinization before use. Ethyl alcohol and isopropyl alcohol are the most
Once activated, it remains active only for popular alcohols used in hospitals.
14 days. ** Uses: Alcohol (60-80%) is used for various
* Aerial disinfection and cleaning: It is also purposes
used for fogging and cleaning of floor and = Alcohol-based handrub (ABHR), e.g.,
surfaces of critical areas, such as operation Sterillium, a popular commercial product
theater (e.g., Bacillocid Extra). m Disinfecting smaller non-critical instru-
ments, such as thermometers, which are
Ortho-phthalaldehyde (0.55%) immersed in alcohol for 10-15 minutes
This can also be used for disinfection of m Disinfection of small medical items/
semicritical items, has many advantages surfaces, such as rubber stoppers of
 CHAPTER 12 © Sterilization and Disinfection

multiple-dose medication vials or vaccine “+ 5% topical solution and ointment is used for
bottles and hubs of the central line wound cleaning
s Disinfection of external surfaces of ** 10% is used for surgical skin preparation.
equipment, such as stethoscopes,
ventilators, manual ventilation bags, Chlorine and Hypochlorite
ultrasound machines, etc. Chlorine is one of the most commonly available
= Disinfection of non-critical surfaces, disinfectant in hospital.
such as laboratory bench, medication “+ Preparations: Chlorine occurs as—(1) free
preparation areas chlorine, (2) hypochlorite—it is available in
= Spirit (70% alcohol): Used a skin antiseptic. two preparations
* Disadvantages: (i) Flammable and must = Liquid form (sodium hypochlorite or
be stored in a cool, well-ventilated area, (ii) household bleach), or
Evaporate rapidly. = Powder form (calcium hypochlorite or
bleaching powder)
Phenolics = Other forms: Include sodium dichloroiso-
Phenol (carbolic acid) was the first widely used cyanurate (NaDCC) available as tablets
antiseptic and disinfectant. and chlorine dioxide.
** Used as disinfectants: Cresol, and lysol are * Uses (free chlorine): Chlorine is used for
the common phenolics used for disinfecting disinfection of municipal water supplies and
environmental surfaces (e.g., bedside tables, swimming pool water. It is also employed in
bedrails, and laboratory surfaces) and the dairy and food industries
noncritical medical devices. They are toxic to * Uses (sodium hypochlorite): It is available
skin, hence not used as antiseptics. 5% phenol at 5.25-6.15%, which is equivalent to 50,000
is mycobactericidal, used for disinfection of ppm of available chlorine. It should be used
sputum specimen in appropriate dilutions (by adding with
“ Used as antiseptics: Certain phenolics are water) for disinfection of various hospital
compatible with skin and are widely used supplies. The contact time is about 10-20
as antiseptics. The classical example is minutes
chloroxylenol (the active ingredient of the w Large blood spill: 0.5% (1:10 dilution or
commercial brand, Dettol) 5,000 ppm) is used
~ Phenolics are the only ILD that retain activity = Small blood spill: 0.05% (1: 100 dilution, or
in the presence of organic materials. 500 ppm) is used
= Pre-treatment of liquid waste before
Halogens disposal: 1% (1:5 dilution, 10,000 ppm) is
Among the halogens, iodine and chlorine have used
antimicrobial activity. They exist in free state, = Laundry items: 0.1% (1 in 50 dilution 1,000
and form salt with sodium and other metals. ppm) is used
m Surface disinfectant: 0.5% (1:10 dilution or
lodine 5,000 ppm) is used
Two preparations are available. a C. difficile (diarrheal stool): Hypocholorite
“ Tincture of iodine: It used as antiseptic for is sporicidal only >0.5% (5000 ppm).
wound cleaning. It can stain the skin * Advantages: Hypochlorites are broad
* Povidone iodine (e.g., Betadine): It is spectrum, rapid in its action, non-flammable,
prepared by complexing iodine with carrier low cost and are widely available
* Disadvantages: (1) Inactivated by organic
(povidone) which helps in sustained-release
of iodine. It is nonstaining and free of skin matter, (2) Toxic to skin and mucosa, and
toxicity. It is used as antiseptics at different carcinogenic, (3) Daily preparation is required
concentrations (4) Corrosive to fabrics and carpets.
 SECTION 1 ©@ General Microbiology, Immunology and Hospital Infection Control

Ultraviolet (UV) Radiation cellulase, protease which breakdown

proteinaceous matter present on equipment.
UV radiation has been employed for disinfection
Enzymatic cleaners are not disinfectants; they
of air and/or surfaces as in operating rooms,
only remove protein from surfaces
isolation rooms, and biologic safety cabinets.
2. Cleaning chemicals (detergents): These
It can also be used for disinfection of drinking
agents act by reducing surface tension and
water, titanium implants and contact lenses. Its
dissolving fat and organic matter.
effectiveness is influenced by the presence of
organic matter.
B ENVIRONMENTAL CLEANING
@ LOW-LEVEL DISINFECTANTS Environmental cleaning of the floor and
surface of hospitals play a vital role in
Low-level disinfectants (LLD) destroy vegetative
controlling the spread of infections. The
bacteria and enveloped viruses, variable action
on nonenveloped viruses, and fungi, but no general principles of environmental cleaning
action on tubercle bacilli and spores. are as follows.
* Cleaning followed by disinfections:
Quaternary Ammonium Compound (QAC) = Cleaning: Always cleaning with a
detergent is performed first, before
QAC are commonly used in environmental
applying disinfectant
sanitation of noncritical surfaces, such as floors,
= Disinfection: CDC recommends to use
furniture, and walls. Some products are also used
low- to intermediate-level disinfectants
for disinfecting non-critical medical equipment
that contacts intact skin (e.g., blood pressure
for environmental cleaning, such as QAC,
hypochlorite and improved hydrogen
cuffs). QAC are also good cleaning agents as they
peroxide.
have surfactant like action. Benzyl ammonium
chloride is the classical example of QAC. * Cleaning sequence: Cleaning should be
performed in correct sequence to prevent
Chlorhexidine Gluconate (CHG) recontamination
= Cleaner to dirtier: The cleaner areas are
CHG is widely used in antiseptic products, at
cleaned first, followed by the dirtier areas;
various concentrations
e.g., low-touch surfaces should be cleaned
** Hand hygiene product: Hand rub (0.5%),
handwash (4%) (e.g., Microshield, a
first followed by high-touch surfaces
= High to low: Top area should be cleaned
commercial product)
first, then proceed towards bottom (e.g.,
+,+?
Mouthwash (0.1-0.2%)
+,OG
Body wash solutions (used before surgery) bedrails + bed legs and table surfaces >
floors)
° mo
Skin disinfectant before surgery (2%)
°.od
Antiseptic for wound cleaning: Commercially = Inward to outwards: Clean the farthest
available as Savion which is a combination of point from the door first and then proceed
towards the door.
CHG 0.3%, cetrimide and isopropyl alcohol.
* Frequency of cleaning for common
situations:
@ CLEANING AGENTS
= Non-critical surfaces and floors can be
Most disinfectants act well only when the cleaned 2-3 times a day
instrument or the surface is free from organic = Mattress used for patients should be
matter, such as dirt, blood, or other specimens. cleaned weekly and after discharge
Therefore, cleaning is a very important step = Doors, windows, walls and ceiling should
which needs to be performed before the be cleaned once a month and spot-
disinfectants are applied. Broadly two types of cleaning when soiled
cleaning agents are available. = High touch areas, such as doorknobs,
1. Enzymatic (proteolytic) cleaners: They elevator buttons, telephones, bedrails,
contain enzymes, such as amylase, lipase, light switches, computer keyboards,
 CHAPTER 12 © Sterilization and Disinfection

METHODS TO TEST EFFICACY OF

STERILIZERS
The efficacy of sterilizers can be assessed
by using physical, chemical and biological
indicators.

Physical Indicator
These are the digital displays of the sterilizer
equipment showing parameters, such as
Fig. 12.7: Fogging of dental unit following construction.
temperature, time and pressure, etc.
Source: Sanitary Department, JIPMER, Puducherry.
Chemical Indicator
monitoring equipment should be cleaned They use heat or chemical sensitive materials
more frequently, every 3-4 hours. which undergo a color change if the sterilization
parameter (e.g., time, steam quality and
Disinfection of Operation Theater temperature) for which it is issued is achieved.
Environmental cleaning in operation theater Common types used are:
(OT) minimizes patients’ and HCWs’ exposure ** Class I: Also called as exposure indicator or
to potentially infectious microorganisms. external pack control. They are used on the
* Surface disinfection: Cleaning should external surface of each pack, to indicate that
be performed first with a cleansing agent, the pack has been directly exposed to the
followed by disinfection by using an aldehyde- sterilant. However, it does not assure sterility
based disinfectant. Disinfection of OT is (Fig. 12.8A)
carried out in the following situations “> Class II: It is called as Bowie-Dick test or as
1. First cleaning of the day (before cases equipment control; i.e., it checks the efficacy
begin) of air removal, air leaks and steam penetration
2. In between cases (cleaning 3 to 4 feet and ensures that the steam sterilizer is
perimeter around the OT table) functioning well
3. Terminal cleaning of OT after the last case * Class IV and V: Also called as internal pack
4. Detailed wash-down of the OT complex control indicator. It is placed inside the packs
once a week and therefore it verifies whether the critical
5. During renovation or construction of OT parameters, such as time, steam quality and
or nearby places. temperature are attained inside the pack or
* Fogging: Also called aerial disinfection,
+, ? not (Fig. 12.8B).
involves spraying of a disinfectant (e.g.,
Biological Indicator
glutaraldehyde, H,O, or QAC based
product) with the help of a fogger machine It is the most reliable indicator as it uses bacterial
(Fig. 12.7) spores to check the effectiveness of sterilization.
m The procedure takes around 1-2 hours,
during which OT should be closed down
and personnel need to be vacated
= Indication: Routine periodic fogging YY A 3M Comply™
( iy Steam

is not recommended, but is indicated eSVaz

eer
ae Chemwmal
mere
Integrator

only when any outbreak of infection is

suspected or any change in infection Ar a
control practice implemented or during Figs 12.8A and B: Chemical indicator: (A) Type | (autoclave
renovation or construction of OT or tape); (B) Type V (internal pack control indicator).
Source: Department of CSSD, JIPMER, Puducherry.
nearby places.
 SECTION 1 © General Microbiology, Immunology and Hospital Infection Control

The spores are highly resistant and will be

destroyed only when the effective condition is
achieved.
Geobacillus stearothermophilus for steam
sterilizer and gas plasma (hydrogen peroxide)
and liquid acetic acid sterilizer
Bacillus atrophaeus for ethylene oxide
sterilizer and dry heat sterilizer (hot air
oven)
Spore containing vials are incubated.
Depending upon the incubators used, the Figs 12.9A and B: Biological indicator:
result is obtained in 24 minutes to 48 hours (A) Vial; (B) Incubator.
time (Figs 12.9A and B). Source: Department of CSSD, JIPMER, Puducherry.

| Problem Solving
Solving Exercise
Exercise _—| |
What are the recommended methods for sterilization/ QO Ventilator tubes: Can be sterilized at CSSD by
disinfection offollowing items in a healthcare facility— ethylene oxide sterilizer, plasma sterilizer
endoscope, culture media, handrub, ventilator tubes, O Operation theatre disinfection: Cleaning with
operation theatre disinfection, cleaning of surgical a detergent, followed by disinfection with a high
instrument, stethoscope, wound disinfection? level disinfectant, such as glutaraldehyde or
hydrogen peroxide
Explanation
Cleaning of surgical instruments: The surgical
The recommended methods for sterilization/disinfec- instruments should be cleaned first with enzymatic
tion of following items in a healthcare facility are: (proteolytic) cleaners before sending to CSSD for
Q Endoscope: 2% Glutaraldehyde or 0.55% ortho-
sterilization
phthalaldehyde Stethoscope: The external surfaces of stetho-
a Culture media: Should be sterilized by autoclave
scope should be disinfected with alcohol 60-80%
(121°C for 15 min)
(isopropyl alcohol) after each use
Q Handrub: Hands should be disinfected with
Wound disinfection: Povidone iodine (5% topical
either alcohol-based hand rub (60-80%) or
solution and ointment) is recommended for
chlorhexidine alcohol hand rub after contact with wound disinfection.
patient or its surrounding
 Biomedical Waste Management

STEREOS

[ Problem Solving
Solving Exercise
Exercise
Biomedical Waste Segregation Audit
While examining the biomedical waste receptacles at the common collection point in a hospital, the following
items were found. Find out how many items are segregated appropriately according to biomedical waste rule
2016.

1. N95 Mask 1. Syringe 1. Microscopy slide 1. Cytotoxic drug bottle

2. Cyclophosphamide vial 2. IV set 2. Metallic implant 2. Glass ampoule
3. Nitrile gloves 3. Blood bag 3. Needle (amikacin)
4. Central venous catheter 4. Nasogastric tube 4. Scalpel 3. Stained cloth
5. Coverall 5. Syringe with fixed 5. Broken glassampoule _— 4. Stained cotton
needle 5. BacT/ALERT bottle

Explanation: The following are the correct items segregated in the biomedical waste receptacles

Correctly segregated Correctly segregated Correctly segregated Correctly segregated

1. N95 Mask 1. Syringe 1. Needle 1. Glass ampoule
2. Cyclophosphamide vial 2. IV set 2. Scalpel (amikacin)
3. Coverall 3. Nasogastric tube Not segregated correctly Not segregated correctly
Not segregated correctly Not segregated correctly 1. Microscopy slide (blue) 1. Cytotoxic drug bottle
1. Nitrile gloves (red) 1. Blood bag (yellow) 2. Metallic implant (blue) (yellow)
2. Central venous catheter 2. Syringe with fixed 3. Broken glass ampoule 2. Stained cloth (yellow)
needle (white) (blue) 3. Stained cotton (yellow)
(red)
4. BacT/ALERT bottle (red)

waste category, which may be disposed of

B INTRODUCTION with the usual domestic and urban waste
Biomedical wastes (BMW) are defined as wastes management system. They do not cause any
that are generated during the laboratory diagnosis, harm to humans. They are not considered as
treatment or immunization of human beings BMW. They should not be mixed with BMW
or animals, or in research activities pertaining 2. Biomedical waste: BMW accounts for a minor
thereto, or in the production of biologicals. proportion of the total waste generated in the
hospitals; which includes infectious waste
Waste Generated in Hospitals (10%) and chemical/radioactive waste (5%).
It is estimated that the quantity of solid waste
Hazards Associated with BMW
generated in hospitals varies from 1/2 to 2 kg/
bed; which can be divided into two categories: Inappropriate and inefficient disposal of BMW
1. General (non-hazardous solid waste, 80%): can lead to infectious hazards, malignancies,
malformations, and environmental (air, land
A large amount of waste falls in the general
 SECTION 1 @© General Microbiology, Immunology and Hospital Infection Control

and water) pollution not only to the current source in the hospital and thereby reducing the
generation but also for future generations. The risks as well as the cost of handling and disposal.
various hazards are: According to BMW Rule (2016), segregation of
“* Hazards from infectious sharps: They may waste should be done by using containers of
lead to transmission of blood borne viruses four different colors—each is designated for
(hepatitis B, C and HIV) segregation ofa particular waste category (Table
“+ Hazards from chemical wastes: They are 13.1).
toxic, corrosive, explosive and flammable; * Yellow bag—for infectious non-plastic waste
can cause chemical burns ** Red bag—for infectious plastic waste
“+ Pharmaceutical waste: Exposure to these * White or translucent sharp container
agents may cause several adverse effects (puncture-proof box)—for metal sharps
“+ Hazards from cytotoxic waste: They can be ** Blue container (puncture-proof box)—for
mutagenic, teratogenic, or carcinogenic broken glass items and metal implants.
“+ Hazards from radioactive waste: They are The following general principles need to be
genotoxic, in higher doses can cause tissue followed during segregation, transport and
destruction. storage of BMW:
“+ Waste receptacles: The waste receptacles
@ BIOMEDICAL WASTE RULE, INDIA should have the following properties
= Plastic bags must be labeled with
The Ministry of Environment and Forests
biohazard logos (Fig. 13.1) and should be
(MoEP) has formulated biomedical waste rule
non-inflammable, autoclave stable and
in 2016 with an amendment added in 2018 and
non-chlorinated
2019 (Table 13.1). = Containers should have well-fitting lids,
** Itwas implemented with a vision of simplifying
either removable by hand or preferably
categorization of BMWs, while improving
operated by a foot pedal
the ease of segregation, transportation and
= Sharp box should be puncture-proof,
disposal methods to decrease environmental
leak-proofand tamper-proofimpermeable
pollution
container.
“+ According to this new rule, there are four
** Importance of segregation: Segregation is
categories of BMWs, each is segregated by a
the most crucial step in BMW management.
single color-coded container.
Wrong segregation may lead to serious
Steps of BMW Management consequences, such as:
= Needle stick injury transmitting hepatitis
The management of BMW can overall be B or HIV (if sharp items are segregated in
summarized into five simple steps: to yellow or red bags)
1. Waste segregation (at the point of generation)
= Production of carcinogens (if plastic items
into color-coded containers
are wrongly segregated into yellow bag
2. Pre-treatment for laboratory liquid waste
3. Transport of waste from generation site to
central storage area of the hospital

Wy
4. Transport of waste from central storage area to
common biomedical waste treatment facility
(CBMWTF)
5. Treatment and/or disposal (within 48 hours
of generation).

Waste Segregation in Hospitals

Waste segregation refers to the basic separation Biohazard symbol Cytotoxic hazard symbol
of different categories of waste generated at Fig. 13.1: Logos used for segregation of biomedical waste.
 CHAPTER 13 © Biomedical Waste Management

Table 13.1: Biomedical Waste Management Rule, India, 2016 (including the amendment added in 2018 and
2019).

Type of waste Type of bag/container | Treatment/disposal

options
Yellow iA.
j
Human anatomical waste | Incineration/plasma
t —
; B. “Animal. anatomical waste
} pyrolysis/deep tburial
!C. Soiled waste | Yellow-colored non- Incineration/plasma
_chlorinated plastic bags pyrolysis/ deep burial/
|autoclaving or hydroclaving
| +shredding/mutilation _
“D.Expired/discarded |Yellow-colored Sent back to manufacturer/
medicines—pharmaceutical | containers/non- _CBMWTF for incineration
waste, cytotoxic drugs _ chlorinated plastic bags (cytotoxic drugs at
___ with cytotoxic label temperature >1200°C) _
_E. Chemical solid waste _ |Yellow-colored containers/ | Incineration or plasma
' nonchlorinated | pyrolysis or encapsulation
_plasticbags
-F. Chemical liquid waste, such ‘Tobe discharged | into Pre-treated" before | mixing
as discarded disinfectants, | separate collection system, _with other wastewater
infected body fluids and _which leads to effluent
secretions, liquid from _ treatment system
housekeeping-related _ Not to be discarded into
activities yellow bag
|G. Discarded linen waste -Non-chlorinated yellow j Non-chlorinated chemical
contaminated with blood/ plastic bags/suitable disinfection? followed by
body fluids, mask, cap, gown _ packing material | incineration/plasma pyrolysis
and shoe cover Se
_H. Microbiology, other clinical Autoclave safe plastic Pre-treat to sterilize with
laboratory waste, blood bags, |bag/container ' non-chlorinated chemicals?
live attenuated vaccines | on-site as per NACO/ WHO
'guidelines (Blue book 2014)
i+ incineration
Red Infectious plastic waste _ Red-colored non- e Autoclaving/microwaving/
Disposable items, such as chlorinated plastic bags hydroclaving + shredding
_ tubing, bottles, intravenous or containers e Mutilation/sterilization+
tubes and sets, catheters, urine shredding
bags, syringes (without needles Treated waste sent to
_and fixed needle syringes) and authorized recyclers orfor
vacutainer with their needles energy recovery
cut), gloves, plastic apron and
goggles

White (Translucent) Waste sharps including metal Puncture-proof, leak- Autoclaving/dry heat
sharps proof, tamper-proof sterilization followed by:
Needles, syringes with fixed containers e Shredding or mutilation
needles, needles from needle tip or encapsulation in metal
cutter or burner, scalpels, blades, container or cement
or any other contaminated concrete or
Sharp sharp (used or discarded) e Sanitary landfill or
e Designated concrete
waste sharp pit
Contd...
 SECTION 1 © General Microbiology, Immunology and Hospital Infection Control

Contd...
Type of waste Type of bag/container | Treatment/disposal
options
Blue |a. Glasswares: Broken or |Puncture--proof and leak- Disinfection can be carried
discarded and contaminated proof container _ out by:
|
{
glass including medicine vials —i le Soaking the washed glass
i
_ and ampoules except those i waste after cleaning with
| contaminated with cytotoxic
1
i]i detergent and sodium
| wastes, microscope slides hypochlorite treatment
b. Metallic body implants (1-2%) or
' Dental implants, other body | e Autoclaving/microwaving/
i
implants and plates hydroclaving and then it is
sent for recycling
Note:
¢ Biomedical waste rule does not specify any specific color coded bag for general waste segregation in hospital. Depending upon the local
policy, hospitals choose any color-coded bag for general waste (for e.g., JIPMER uses black bag for general waste).
¢ ‘Chemical treatment: Hypochlorite should be used at 1-2% concentration having 30% residual chlorine with contact time of 20
minutes.
?Non-chlorinated chemicals include 5% phenol, 5% cresol or 5% lysol.
The chlorinated plastic bags (except blood bags) and gloves should be phased out and replaced by non-chlorinated bags and gloves.
Every healthcare facility should have their own STP (sewage treatment plant).
Barcoding system should be introduced to monitor the segregation compliance.
Abbreviations: NACO, National AIDS Control Organization; WHO, World Health Organization; CBMWTF, common biomedical waste
treatment facility.

and subjected to incineration, leads to different types of waste should be brought for
production of carcinogenic furans). safe retention until it is treated or collected for
* Securement: All the bags used for waste transport to CBMWTF
collection need to be sealed once they are “+ PPE: HCWs handling BMW during transport
filled to 3/4th of their capacity or in the storage area should wear appropriate
* Labeling: Bags and containers should be personal protective equipment (PPE), such
labeled properly with the date and place as heavy duty gloves, 3-ply mask, gowns and
* Pre-treatment: The laboratory liquid waste gumboots (Fig. 10.4, Chapter 10).
should always be pre-treated either with
chemical (1-2% hypochlorite) or autoclave Treatment and Disposal Methods
before segregating into appropriate containers As per the mandate of theBMWM rules, 2016, the
“* Transport: The waste should be transported final disposal and recycling must be performed
within 24 hours by dedicated trolley to the at common biomedical waste treatment facility
central BMW storage facility of the hospital. (CBMWTE). Only when there is no CBMWTE
Separate routes should be used for transport within 75 km, the hospital can create its own
to prevent exposure to staff and patients and to the disposal facility. The various methods
minimize the passage of loaded carts through used for treatment/disposal of BMW are—
patient care and other clean areas. Interim stor- incineration, autoclave, chemical disinfection
age of the waste at ward is strongly discouraged (hypochlorite), microwave, effluent treatment
* Central storage area: This is a temporary plant, hydroclaving, shredder, deep burial and
storage facility present within a hospital where sharp pit.
 | Needle Stick Injury 1A

| Problem Solving
Solving Exercise1
Exercise 1
A 32-year-old staff working in biomedical waste post-exposure prophylaxis measures need to be
department reported to HICC with complaints of taken.
needle stick injury while segregating a yellow bag. The Q For HIV: First dose of ART has to be given
incident happened 18 hours back. He did not perform immediately (within 2hr of exposure); followed
any first aid measures at the time of injury. He has not by full course of ART for 28 days (Refer the text for
received any hepatitis B vaccination before. Discuss the drugs given in ART and the dosage)
the post-exposure prophylaxis measures that should Q For hepatitis B: He should receive hepatitis B
be undertaken. immunoglobulin plus hepatitis B vaccine series
(three doses); 1st dose to be taken now, followed
Explanation by 2nd and 3rd dose after 1 month and six month
The source is unknown as the prick happened while respectively.
segregating a yellow bag. Therefore, the following

Problem Solving Exercise 2 .

A 28-year-old medicine resident reported to HICC Explanation
with complaints of needle stick injury on his left The source sample was found to be positive for
thumb while recapping the needle. The incident hepatitis B surface antigen and reactive for HIV
happened 1 hr back. On enquiry, he mentioned that antibodies. Therefore, the following post-exposure
he had immediately washed his finger with running prophylaxis measures need to be taken.
tap water for 2 minutes. The source sample was Q For HIV: First dose of ART has to be given
tested and was found to be positive for hepatitis B immediately (within 2hr of exposure); followed
surface antigen and reactive for HIV antibodies. The by full course of ART for 28 days (Refer the text for
resident had received a complete course (one series) the drugs given in ART and the dosage)
of hepatitis B vaccine 10 years back, following which Q For hepatitis B: As he was vaccinated and
he had an anti-HBs titer of 550 mIU/mL. Discuss the protected with anti-HBs titer of 550 mIU/mL;
post-exposure prophylaxis measures that should be therefore no further action (post-exposure
undertaken. prophylaxis) is necessary.

_ Problem Solving Exercise 3

A 41-year-old surgeon reported to HICC with com- Explanation
plaints of surgical blade injury on his left thumb, 3 hr The source sample was found to be positive for
back. The source sample was tested positive for hepa- hepatitis B surface antigen but non-reactive for HIV.
titis Bsurface antigen, but negative for HIV antibodies Therefore, the following post-exposure prophylaxis
and HCV antibodies. The surgeon had received two measures need to be taken.
doses of hepatitis B vaccine 2 years back. Discuss the Q For HIV: No further action (post-exposure
post-exposure prophylaxis measures that should be prophylaxis) is necessary
undertaken.
 SECTION 1 © General Microbiology, Immunology and Hospital Infection Control

Q For hepatitis B: He was partially vaccinated for B immunoglobulin plus the third dose hepatitis B
hepatitis B, therefore, he should be given hepatitis vaccine.

Table 14.1: Steps of post-exposure management.

B INTRODUCTION
. First aid
An occupational exposure is defined as:
. Report to designated nodal center
“+ Percutaneous injury, e.g., needle stick injury . Take first dose of PEP for HIV
(NSD or other sharp injury . Testing for BBVs
** Splash injury: Decision on PEP for HIV and HBV
= Contact with the mucous membrane (e.g., . Documentation and recording of exposure
. Informed consent and counseling
eye or mouth)
. Follow-up testing of HCWs
= Contact with non-intact skin (abraded oO. Precautions during the follow-up period
skin or afflicted with dermatitis)
Abbreviations: PEP, post-exposure prophylaxis; HCW, healthcare
# Contact with the intact skin when the worker; BBV, blood-borne virus.
duration is prolonged (e.g., several
minutes or more). Table 14.2: First Aid: Management of exposed site.
An occupational injury is often loosely
termed as needle stick injury though it includes
injury through needle or other sharps and Earlier the first aid, lesser is the e Do not panic
splashes. chance of transmission of BBVs e Donot place
e For splash injury: Irrigate the pricked
Agents transmitted: Hepatitis B virus (HBV), thoroughly the site (e.g., eyes or finger into
hepatitis C virus (HCV) and HIV are three major mouth or other exposed area) the mouth
blood-borne viruses (BBVs) that are transmitted vigorously with water at least for reflexively
through NSI. The risk of transmission is highest 5 minutes e Donot
e Spit fluid out immediately if squeeze blood
for HBV (30%) followed by HCV (3%) and HIV
gone into mouth and rinse the from wound
(0.3%). mouth several times e Donotuse
e If wearing contact lenses, leave antiseptics and
@ POST-EXPOSURE MANAGEMENT them in place while irrigating. detergents
Once the eye is cleaned, remove
Steps of Post-exposure Management the contact lens and clean them
in anormal manner
The following are the sequential steps to be
followed following an occupational exposure
(Table 14.1): combination of five tablets; given on the
1. First aid: First aid has to be started as early as first day of exposure
possible (Table 14.2) ¢ Tenofovir 300 mg + Lamivudine 300
2. Report to the designated nodal center: mg, one tablet once daily and
Every hospital must have a nodal center for ¢ Lopinavir (200 mg) + Ritonavir (50 mg)
the management of NSI. In most hospitals, two tablets twice daily.
HICC office acts as a nodal center, other m Ifthe HIV negative status of the source is
hospitals may designate staff clinic or casualty documented in patient's case record or in
for the purpose. Nodal centers perform the the hospital information system, then the
following functions as mentioned below first dose of PEP is not required
(steps 3-9) = If test report is not available, then
3. Take first dose of PEP for HIV: administer the first dose regimen
= The first dose of PEP for HIV should immediately without waiting for the
be taken as early as possible. Effect is laboratory result.
maximum if taken <2 hours and effect is 4. Testing for BBVs: The following tests
nil if taken after 72 hours of exposure are done for both source and HCW. The
= NACO recommendation: The first test format should be a rapid method
dose regimen comprises of a fixed-dose (immunochromatographic test or flow
 CHAPTER 14 © Needle Stick Injury

through assay) and result should be available 5. Decision on post-exposure prophylaxis

within 1-2 hours (PEP) for HIV and HBV is taken based on
m Anti-HIV antibody detection standard guidelines (NACO for HIV and CDC
m HBsAg detection for HBV) as described in Tables 14.3 and 14.4
mw Anti-HCV antibody detection respectively
a Anti-HBs antibody (done for HCW if . Informed consent and counseling: Almost
previously vaccinated for HBV and titer every person feels anxious after exposure.
not tested). They should be counseled and provided with
HCW’s baseline status is determined because psychological support
later it may be difficult to attribute whether m They should be informed about the risks
the infection was acquired due to this and benefits of PEP medications
occupational exposure or any other prior m PEP is not mandatory. If the exposed
exposure. This may guide while taking person refuses to take the PEP, it should
a decision, when the HCW claims for be documented. However, he should
compensation from the health authorities. be made to understand about the risk

Table 14.3: Revised NACO Guidelines for post-exposure prophylaxis (PEP), 2018.
Exposure code (EC) | Source HIV status code (SC) | PEP Recommendation

1,2 0r3 Negative Not warranted

1 1 Not warranted
1 2 PEP is recommended
2 1 Duration of PEP: 28 days
5 5 Primary TL+LR regimen*: Regimen comprises of fixed dose
combination offive tablets to be taken every day.
3 1or2 e Tenofovir (300 mg) + Lamivudine (300 mg), one tablet
2o0r3 Unknown (in area with high once daily and
prevalence) e Lopinavir (200 mg) + Ritonavir (50 mg) two tablets twice

Alternative TLE regimen: Fixed dose combination of

tenofovir (300 mg)+ Lamivudine (300 mg) + Efavirenz (600
mg), one tablet once daily
Exposure code:
or less duration
1. EC-1 (Mild exposure): Mucous membrane/non-intact skin exposure with small volumes,
2 EC-2 (Moderate exposure):
duration OR
> Mucous membrane/non-intact skin with large volumes/splashes for several minutes or more
> Percutaneous superficial exposure with solid needle or superficial scratch
. EC-3 (Severe exposure): Percutaneous exposure with:
> Large volume transfer
> By hollow needle, wide bore needle or deep puncture
> Visible blood on device
> Needle used in patient's artery or vein
Source HIV Status Code (SC):
1. SC-1: HIV positive, asymptomatic or low viral load (<400 copies/mL)
viral load
2. SC-2: HIV positive, symptomatic (advanced AIDS or primary HIV infection), high
of the patient is unknown and neither the patient nor his/her blood is available for testing
3. SC Unknown: Status
4. HIV negative: Tested negative according to NACO strategy
The first dose of PEP
72 hours.
Should be started within 2 hours (for greater impact) and definitely within
Side effects and complian ce to PEP:
anemia and leukopenia
e Common side effects are: Nausea, diarrhea, myalgia, headache or fatigue,
PEP should never be disconti nued as complian ce of >95% to the PEP schedule is required to maximize the
e
efficacy of PEP.
of non-pregnant persons.
*Regimen for exposure in pregnant women is essentially same as that
l therapy.
Abbreviations: NACO, National AIDS Control Organization; ART, antiretrovira
124 SECTION 1 © General Microbiology, Immunology and Hospital Infection Control

Table 14.4: Post-exposure prophylaxis (PEP) for hepatitis B.

If source is positive or unknown for If source is negative for
HBsAg HBsAg
If the exposed person is completely No further treatment is required:
vaccinated and the antibody titer is e Regardless of the HBV status of the source*
protective (=10 mlU/mL) e Regardless if the titer falls down later*
If the exposed person is completely HBIG-1 dose should be started Vaccine: Start the second
vaccinated and the titer is not immediately; maximum within 7 days series (3 doses)
protective (<10 mlU/mL) Vaccine: Start the second series (3 doses)
If the exposed person is not vaccinated ~ HBIG-1 dose should be started Vaccine: Complete the
or partially vaccinated immediately; maximum within 7 days vaccine series from the last
Vaccine: Complete the vaccine series from dose given (do not restart)
the last dose given (do not restart)
Nonresponders (If the exposed person HBIG-2 doses at 1 month apart (0.06 mL/kg Nothing is required
is vaccinated for 2 series, i.e., 6 doses or 10-12 |U/kg)
and the titer is not protective)
Note:
+ HCWis said to be protected when titer rises (anti-HBs >10 mlU/mL), after three or more doses of vaccination. Rise of titer after one or
two doses of vaccine should not be considered as protective.
+ HCWs who are not protected must be checked for their HBsAg status at baseline and follow-up testing 6 months later, regardless of
their vaccination status.
+ Anti-HBs antibody titer should be checked only after 2 months of last dose of vaccine and 6 months after HBIG administration;
otherwise, it will give erratic results.
+ HBIG and HBV vaccine can be administered simultaneously but at different sites.
+ HBIG provides a temporary protection for 3-6 months.
Previous report of Anti-HBs titer is acceptable only if it is documented. Verbal reports should not be considered.
* In a previously protected person, the memory B cells will start producing antibodies soon after the antigenic challenge, hence
revaccination by booster doses is not recommended even ifthe titer falls down later.
Adapted from CDC guideline, 2013.
Abbreviations: HBIG, hepatitis B immunoglobulin; HCW, healthcare worker; HBsAg, hepatitis B surface antigen.

of acquiring infection if PEP is not =HIV testing follow-up is done: At 6 weeks,

taken. 3 months and 6 months after exposure
7. Documentation and recording of exposure: = HBV and HCV follow-up testing is done at
= A structured proforma should be used 6 months after exposure.
to collect the detail information related 9. Precautions during the follow-up period: If
to exposure, such as date, time, and place the source status is positive/unknown, then
of exposure, type of procedure done, type the following precautions should be adopted
of exposure, duration of exposure, source by the HCW during the follow-up period,
status, volume and type of specimen especially the first 6-12 weeks
involved = Refraining from blood, semen, organ
= Consent form: For prophylactic treatment, donation
the exposed person must sign a consent = Abstinence from sexual intercourse or use
form. If the individual refuses to initiate of latex condom till both baseline and 3
PEP, it should be documented. The des- months HIV tests are found negative
ignated officer for PEP should keep this = Women should not breastfeed their
document. infants
8. Follow-up testing of HCWs for BBVs should = The exposed person is advised to seek
be done if the source status is positive/ medical evaluation for any febrile illness
unknown that occurs within 12 weeks of exposure.
 |
CHAPTER}
Environmental Surveillance

The various environmental sources from which ing of microbiological quality of water, air and
microorganisms can be transmitted to patients surfaces are of paramount importance for safe
and healthcare workers include water, air and hospital environment.
environmental surfaces. Therefore, monitor-

@ WATER SURVEILLANCE
[Problem Exercise e
Solving Exercis
Solving |
Water Surveillance When matched with McCrady statistical Table
(Table 15.3), the presumptive coliform count
Water surveillance was performed in a newly construct-
(MPN) is estimated as 6 coliforms per 100 mL
ed ICU of atertiary care hospital and the result is shown
water sample. The quality of water supply is of
in Figure 15.2. Interpret the result using McCrady sta- ‘Intermediate quality’ (Table 15.4).
tistical Table (Table 15.3). Discuss the procedure of this
Q Differential Coliform Count (Eijkman Test)
test. What is the further test advised to perform.
needs to be performed to find out whether
Explanation the organism is thermotolerant E. coli. If tested
Q The test performed in Fig. 15.2 is a multiple tube positive, the quality of water supply is reported as
method, performed on an unpolluted water ‘unsatisfactory’.
sample. It shows positive result for one 50 mL tube
and two numbers of 10 mL tubes.

Microbial contamination of water in healthcare Test of Drinking Water Contaminated

settings is broadly of two types. with Enteric Pathogens
1. Enteric pathogens: This results from fecal Hospital drinking water should be regularly
contamination of drinking water supplies. tested to confirm that they are free from
Most of them cause diarrheal outbreaks. These contamination with enteric pathogens. As
include bacteria (Salmonella, Shigella, Vibrio enteric pathogens (Salmonella, Shigella)
cholerae), viruses (rotavirus, and others),
parasites (Entamoeba histolytica, Giardia and Table 15.1: Microbiological testing methods for water
others) analysis.
2. Common hospital pathogens: These include Bacteriological | Indications
multidrug resistant gram-negative bacilli, examination
legionellae, etc., commonly present in hos- Multiple-tube e Extensively used for drinking
pital environment; can contaminate various method water analysis
hospital water supplies and can lead to wa- e For highly turbid samples

terborne outbreaks in healthcare settings. Membrane e For testing dialysis water

e For testing clean water, where
The methods employed for detection filtration
the bacterial count in water is
of microbial contamination of water must
method
expected to be low
have capability to detect both the category of e For testing large volume of water
pathogens (Table 15.1).
 SECTION 1 © General Microbiology, Immunology and Hospital Infection Control

Table 15.2: Indicator organisms of fecal pollution of

water.
Indicator
organisms

Coliform (other Remote contamination—either

than Escherichia by fecal (presumptive) or soil and
coli) vegetation
Fecal Confirms recent fecal contamination
(thermotolerant) of water; most sensitive indicator Fig. 15.1: Water sampling methods.
Escherichia coli
“+ Sampling points in hospitals must represent
Other indicators Less reliable, such as Fecal
different sources from which water is obtained,
streptococci, Clostridium perfringens,
Pseudomonas aeruginosa such as portable water from pipelines,
endoscopy rinse water, dialysis water, dental
chair unit waterline, etc.
are usually present in small quantity, it is “* Sampling method from the tap: Care should
impracticable to detect the presence of all be taken while collecting water to minimize
types. Instead, the water supplies are tested extraneous contamination. Hand washing
for those microorganisms which indicate that should be performed and gloves should be
fecal contamination has taken place (indicator worn before collection (Fig. 15.1)
organisms) (Table 15.2). ** Tap swabs: Sterile swab is inserted into the
nozzle of the tap carefully, without touching
Indicator Organisms the outer tap surface. The swab is then rubbed
Indicator organisms are usually the commensal around, i.e., moved backwards and forwards
bacteria of intestine which satisfy two proper- and up and down, as much as possible, on
ties: the inside surface of the tap outlet or flow
1. They should be present in excess number than straightener (Fig. 15.1).
any pathogen so that they can be detected
easily; at the same time, they should not be Multiple-tube Method
able to proliferate in water to any extent It is the most common method used for water
2. They should be more resistant than the patho- surveillance. It is so named as it involves mixing
gens to the stresses of aquatic environment of specific volume of water samples to multiple
and disinfection processes. tubes containing a special culture medium—
Mere presence of these indicator organisms MacConkey purple broth.
does not assure the presence of water borne * Procedure: Most of the hospital water
pathogens; but their presence in water supplies supplies are non-turbid and unpolluted. As
indicates that there is a contamination of sewage per WHO guideline, for testing of unpolluted
and the water supplies needs to be disinfected. water samples the following method should
‘There are a number of intestinal commensals, be followed (Fig. 15.2)
used as indicator organisms as enlisted in Table = 50 mL of water is added to one tube having
Wy 50 mL of culture medium
= 10mLofwateris added to each offive tubes
Collection and Transport of Water Sample containing 10 mL of culture medium.
Water specimen should be collected in a screw- * Positive result: After incubated for 24-48
capped wide sterile container. hours, the medium turns to yellow from
** Volume: At least 150-200 mL of water should purple (due to lactose fermentation), along
be collected with turbidity of the medium and gas collected
** Neutralizer: Sodium thiosulfate is added to in the Durham's tube
neutralize the bactericidal effect of residual * Determination of MPN: The number of
chlorine present in water if any tubes giving positive reaction is compared
 CHAPTER
15 © Environmental Surveillance 127

Table 15.3: MPN values per 100 mL of sample for non-

polluted/treated water samples (McCrady statistical
table).
No. of tubes giving a
MPN per 100 mL
positive reaction out of
of water
TofsomL |10m
Sof
N =

Fig. 15.2: MacConkey purple broth for multiple tube

method (one 50 mL and five 10 mL tubes are needed
for testing unpolluted water); if negative, media appears
purple; if positive, media turn yellow.
Source: Department of Microbiology, JIPMER, Puducherry (with
permission).
ay
Gri
be
Ns
ee
RCS
est
hoe
Be
HOW
POG

with McCrady statistical table (Table 15.3) wow,

UW
oO
=|
NY
Bw
©WB
| Ms 0
to determine the most probable number
(MPN) of coliform count present per 100 Table 15.4: Classification of quality of drinking water
mL of water. This is called as presumptive supply.
coliform count. Most probable number
For example: As shown in Figure 15.2, Quality of (MPN)/100 mL of water
multiple tube method performed on an drinking water | Coliform Thermotolerant
unpolluted water sample gives positive supply count/ E. coli count/
result for one 50 mL tube and two numbers 100 mL
of 10 mL tubes. When matched with Table Excellent 0 0
15.3, the presumptive coliform count (MPN) Satisfactory 1-3 0
is estimated as 6 coliforms per 100 mL water 0
Intermediate 4-9
sample
Unsatisfactory =10 21
“ Quality of water supply: Depending upon
the MPN/100 mL, the quality of the water
specimen can be interpreted as excellent, Membrane Filtration Method
satisfactory, intermediate or unsatisfactory
This method is based on the filtration of a
(Table 15.4).
known volume (e.g., 100 mL) of water through a
Differential Coliform Count (Eijkman Test) cellulose membrane of pore size 0.2 or 0.45 pm.
It is the recommended method for—(1) testing
Detection of coliform bacteria does not always
dialysis water, (2) for testing clean water, where
indicate fecal contamination as some of them
Hence, it is the bacterial count in water is expected to be
may be found in environment.
low and (3) for testing a large volume of water.
further tested by differential coliform count
However, It is not suitable for turbid water.
to detect the fecal E. coli. This is done by sub-
culturing the positive tubes (from the multiple Test of Water Contaminated with
,
tube method) on lactose containing medium Healthcare Associated Pathogens
such as brilliant green bile broth for:
“ Detection of lactose fermentation with the Most of the healthcare associated pathogens
production of acid and gas at 44°C and are recovered by membrane filtration method,
+ Demonstrating a positive indole test at 44°C. followed by plating on to a suitable culture
 SECTION 1 © General Microbiology, Immunology and Hospital Infection Control

medium, e.g., Legionella, on to buffer charcoal given time and then the plates are incubated at
yeast extract (BCYE) medium. 37°C for 24 hours aerobically.
1,1, 1 method: Here, the plates are placed at
Endotoxin Detection
different locations in the OT one meter away
Dialysis water, devices, etc. contaminated with from the side walls, one meter above the floor
endotoxin can cause serious toxic effects. and for a duration of one hour.
Therefore, the dialysis water used for
hemodialysis is tested for presence of endotoxin. Active Monitoring (Slit Sampler Method)
“+ Method: Gel clot assay (Limulus amebocyte In active monitoring, a microbiological air
lysate assay) sampler (e.g., sieve impactor) is used. It has a
** Permissive level: Water used to prepare vacuum pump, and a perforated lid (Fig. 15.3B),
dialysate and to reprocess hemodialyzers in which an agar plate can be placed.
should contain endotoxin unit <0.25 EU/mL. “+ The vacuum pump physically draws a known
volume of air through the perforated lid and
BAIR SURVEILLANCE allows it to impact on the agar plate (e.g.,
Air is an important vehicle of transmission
blood agar)
of many pathogenic organisms. Therefore, * Following incubation, the quantity of
microorganisms present in the culture plate
the examination of air to detect the number
of bacteria carrying particles is important is measured in terms of CFU/m‘* of air (Fig.
particularly in critical areas, such as operation 15.3A)
** Active monitoring is applicable when the
theaters (OTs), bone marrow transplant units,
lcs
concentration of microorganisms is not very
high, such as in an operating theater, bone
Indication (CDC Recommendations) marrow transplant unit, etc.
Routine air sampling (i.e., random or periodic Air Particle Counters
sampling) is not recommended. CDC recom-
Air particle counters have been developed
mends targeted air surveillance, which should
recently, that are capable of detecting airborne
be carried out for the following indications:
particles containing microorganisms in real
** Investigation of an outbreak
time.
** For research purpose
** After reconstruction or newly constructed
Non-microbiological Parameters
buildings
» After fogging (to monitor the quality)
ae
The number of bacteria in air at any given point of
* For short-term evaluation of a change in
oe
time depends upon various non-microbiological
infection control practice. parameters, such as air changes per hour,

Evaluation of the Quality of Air in OT

Evaluation of the quality of air includes both
microbiological and non-microbiological
(physical) parameters.

Microbiological Parameters
There are two principle means of monitoring the
microbiological parameters present in the air,
passive monitoring and active sampling.

Passive Monitoring (Settle Plate) Method Figs 15.3A and B: (A) Air sampler method showing blood
agar with bacterial colonies; (B) Air sampler (HiMedia).
Standard Petri dishes containing culture media Source: Department of Microbiology, JIPMER, Puducherry
(e.g., blood agar) are exposed to the air for a (with permission).
 CHAPTER15 © Environmental Surveillance

air velocity, positive pressure environment, “+ Indications (CDC recommendation):

temperature and relative humidity inside OT, etc. m Surface sampling is currently indicated
Therefore, there should be periodic monitoring for research, as a part of an epidemiologic
of these parameters. : investigation, or during an outbreak
investigation
@ SURFACE SURVEILLANCE = Routine periodic surface surveillance is
not recommended.
Environmental surface sampling has been
“+ Method: Moistened sterile swabs (soaked in
used to determine (a) reservoirs of potential
environmental pathogens, and (b) the sources sterile saline) are used to collect the samples
of the environmental contamination. from high-risk areas and then inoculated on to
* Locations: It is required for high-risk blood agar for the recovery of aerobic bacteria
locations, such as operation theaters and “ Reporting: Only pathogenic organisms
ICU settings isolated are reported. A semi-quantitative
* Sites for sampling (high touch areas): report (as heavy, moderate or light growth)
Surface sampling is taken from sites where should be provided. Contaminants, such as
there is high-risk of contaminations aerobic spore bearers are not reported.
 Systemic Microbiology
(Infectious Diseases)

16 Cardiovascular System Infections: Infective Endocarditis and Acute

Rheumatic Fever
17. Bloodstream Infections
18. Bacterial Infections of Bloodstream: Enteric Fever, Scrub Typhus, Brucellosis,
and Leptospirosis
1, Viral Infections of Bloodstream: HIV/AIDS and Dengue
20. Parasitic Infections of Bloodstream: Malaria, Visceral Leishmaniasis and
Lymphatic Filariasis
2\. Fungal Infections of Bloodstream: Systemic Candidiasis and Systemic
Mycoses
22. Bacterial Diarrhea: Shigellosis, Cholera and Others
23. Viral Gastroenteritis: Rotaviruses and Others
24. Intestinal Protozoan Infections: Intestinal Amoebiasis, Giardiasis and
Coccidian Parasitic Infections
22) Intestinal Helminthic Infections
» Intestinal Cestode Infections: Intestinal Taeniasis, Hymenolepiasis and
Others
. Intestinal Trematode Infections: Fasciolopsis buski, Schistosoma mansoni
and Others
- Intestinal Nematode Infections: Trichuriasis, Enterobiasis, Ascariasis,
Hookworm Infection, Strongyloidiasis
26. Viral Hepatitis
27. Parasitic Infections of Hepatobiliary System: Amoebic Liver Abscess, Hydatid
Disease and Others
28. Staphylococcal Infections
22. Beta-hemolytic Streptococcal Infections
30. Miscellaneous Bacterial Infections of Skin and Soft Tissues: Anaerobic
Infections including Gas Gangrene, Leprosy and Anthrax
oi Viral Exanthems and Other Cutaneous Viral Infections: Herpes Simplex,
Measles, Rubella and Others
a2. Superficial and Subcutaneous Fungal Infections
33. Bacterial Pharyngitis: Streptococcus pyogenes Pharyngitis and Diphtheria
34. Bacterial Pheumonia: Pheumococcal Pneumonia, Haemophilus influenzae
Pneumonia, Klebsiella pneumoniae Pneumonia and Others
35. Tuberculosis
36. Pseudomonas and Acinetobacter Infections
57. Viral Infections of Respiratory Tract: Influenza, COVID-19, Infectious
Mononucleosis, and Others
38. Parasitic and Fungal Infections of Respiratory Tract: Paragonimiasis,
Zygomycosis, Aspergillosis, Pheumocystosis and Others
Bee Bacterial Meningitis
40. Viral Meningitis and Viral Encephalitis (Enteroviruses including Polio, Rabies,
Japanese Encephalitis and Others)
41. Parasitic and Fungal Infections of Central Nervous System:
Neurocysticercosis, Free-living Amoebae Infections, Toxoplasmosis,
Cryptococcal Meningitis and Others
42. Urinary Tract Infections
43. Infective Syndromes of Genital Tract (Sexually-transmitted Infections) :
Syphilis, Gonorrhoea, Non-gonococcal Urethritis (Chlamydia trachomatis),
Vulvovaginitis (Trichomoniasis, Vaginal Candidiasis) and Others
44, AETCOM in Microbiology
45, University Practical Examination
 Cardiovascular System Infections: CHAPTER]
| aol!

‘Infective Endocarditis and

Acute Rheumatic Fever
SETA OITA

infection) and suppurative thrombo-

B INTRODUCTION
phlebitis
Cardiovascular system infections include ¢» Autoimmune-mediated: Acute rheumatic fever.
infections of heart and blood vessels.
“* Infections of heart: This includes infection of B INFECTIVE ENDOCARDITIS
the three layers of the heart wall endocarditis,
myocarditis and pericarditis Infective endocarditis (IE) refers to microbial
* Infections of blood vessels: Infections of invasion of the heart valves or mural
blood vessels include mycotic aneurysm, and endocardium—characteristically results in
infective endarteritis formation of bulky friable vegetations, composed
* Device-related infections: These include of mass of platelets, fibrin, microcolonies of

CRBSI (catheter-related bloodstream organisms, and scanty inflammatory cells.

Exercise e11
Solving Exercis
[Problem Solving
Infective Endocarditis
A75-year-old man was hospitalized with fever (101°F),
severe back-pain and weakness in lower limbs. On
examination, few non-tender, small erythematous
nodular lesions on soles were seen. Echocardiogram
showed valvular vegetations on mitral valve. He was
diagnosed to have a cardiac valve vegetations 3
years back. Laboratory tests showed CRP 2.5 mg/dL, Ys ,
ESR 66 mm/h, leukocytes 15.6 x 10°/L and creatinine Figs 16.1A and B: Viridans streptococci: (A) Gram-
4.6 mg/dL. Two pairs of blood cultures were sent in positive cocci in long chains; (B) a-hemolytic colonies on
BacT/ALERT bottles; both flagged positive. The smear blood agar.
made from BacT/ALERT broth and subculture done on Source: Department of Microbiology, Pondicherry Institute of
blood agar have been depicted in the Figures 16.1A Medical Sciences, Puducherry (with permission).

and B. The patient was immediately started on benzyl Q The typical agents of IE include: Viridans
penicillin. streptococci, Streptococcus gallolyticus, HACEK
1. What is the probable clinical diagnosis? group, Staphylococcus aureus or enterococci
2. What are the typical etiological agents of this Q Modified Duke criteria is the diagnostic criteria
clinical condition? applied in this clinical condition—two major
3. Describe the diagnostic criteria used for this criteria and two minor criteria are fulfilled here.
condition. > Major criteria 1: Blood culture criterion is
4. How will you collect specimen for this clinical fulfilled. The smear made from BacT/ALERT
condition? broth showed gram-positive cocci in chain
(Fig. 16.1A) and subculture on blood agar grew
Explanation
minute, a-hemolytic colonies (Fig. 16.1 B). This
This is a case of Infective endocarditis, caused by finding is suggestive of viridans streptococci.
viridans streptococci
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

Optochin susceptibility test can be performed Q Collection specimen: Blood cultures should be
to rule out pneumococcus. MALDI-TOF can be collected before starting antibiotic therapy
performed for species identification > Two blood culture sets should be collected at
» Major criteria 2; Echocardiogram showed an interval of >12 hr between 1st and 2nd set
valvular vegetations on mitral valve > Alternatively, three blood culture sets can
> Twominor criteria fulfilled are: (1) Fever (101°F), be collected over one hour (e.g., 30 min gap
(2) Vascular phenomena: few non-tender, between 1st and 2nd set and 30 min gap
small erythematous nodular lesions on soles, between 2nd and 3rd set)
described as Janeway lesions Note: Blood culture set refers to ‘pair of bottles’;
collected from different venipuncture sites.

Classification Table 16.2: Agents of infective endocarditis.

Infective endocarditis can be classified into Etiological agents of infective endocarditis
acute and subacute forms based on rapidity of Staphylococcus aureus: Overall, the most common
evolution, severity of infection and virulence of cause of IE
the implicated organism (Table 16.1). Coagulase-negative staphylococci (e.g.,
Staphylococcus epidermidis): Associated with
Etiological Agents of IE prosthetic valve endocarditis
Streptococci (viridans streptococci and others): Most
The causative organisms of IE differ depending common cause of subacute bacterial endocarditis
on the underlying risk factors such as native or Enterococci: Associated with left-sided (mitral valve)
prosthetic valve IE, acute or subacute IE, other endocarditis in lV drug abusers
risk factors such as IV drug abuser (Table 16.2). e Pneumococci
Fastidious gram-negative coccobacilli (HACEK*
Clinical Manifestations group), Coxiella burnetii, Brucella species, etc.)
Enterobacteriaceae
The clinical spectrum of IE includes both cardiac Pseudomonas spp. (usually in drug users)
and noncardiac manifestations. Candida species
** Cardiac manifestations include the appear- Diphtheroids
ance of anew/worsened regurgitant murmur, *HACEK, Haemophilus species, Aggregatibacter species,
Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae.
which is more useful for the diagnosis of IE
involving a normal valve
* Noncardiac manifestations include fever, myalgia, arthralgia, arterial emboli, sple-
chills and sweats, anorexia, weight loss, nomegaly, clubbing, petechiae, neurologic
manifestations and peripheral manifesta-
Table 16.1: Differences between acute and subacute tions (Osler’s nodes, subungual hemor-
endocarditis. rhages, Janeway lesions)
Acute endocarditis Subacute endocarditis ae Laboratory manifestations such as anemia,
Evolution is rapid Evolution is slow leukocytosis, microscopic hematuria, elevated
Involves normal cardiac Involves previously erythrocyte sedimentation rate (ESR),
valve damaged heart (scarred or C-reactive protein (CRP), or rheumatoid factor.
deformed valve)
Implicated organism is Implicated organism is of Diagnosis (Modified Duke Criteria)
of high virulence, e.g., S. low virulence, e.g., viridans The diagnosis of IE is established with the help
aureus streptococci
of a highly sensitive and specific diagnostic
Causes substantial Follows a gradually
schema—known as the modified Duke criteria;
morbidity and progressive course of
mortality even with the weeks to months; most which is based on clinical, laboratory, and
appropriate antibiotic patients recover after echocardiographic findings (Table 16.3).
therapy and/or surgery antibiotic therapy
Less common type, More common type, Blood Cultures
accounts for 10-20% of accounts for 50-60% of all Isolation of the causative microorganism
all cases cases
from blood cultures is critical for diagnosis,
CHAPTER 16 @ Cardiovascular System Infections: Infective Endocarditis and Acute Rheumatic Fever 5

determination of antimicrobial susceptibility, Table 16.3: Modified Duke criteria for the clinical
and planning of treatment. Blood cultures diagnosis of infective endocarditis.
should be collected before antibiotic therapy.
* Two blood culture sets should be collected 1. Positive blood culture: Any one of the following:
at an interval of >12hr between Ist and 2nd A. Typical IE organism isolated from two
set separate blood cultures (Viridans streptococci,
** Alternatively, three blood culture sets can Streptococcus gallolyticus, HACEK group, S. aureus
be collected over one hour (e.g., 30 min gap or enterococci) or
B. Persistently positive blood culture with agents
between Ist and 2nd set and 30 min gap other than typical IE organisms:
between 2nd and 3rd set). > Blood culture sets drawn >12 h apart; or
Note: Blood culture set refers to ‘pair of bottles’; > All of 3 sets or a majority of 24 separate
collected from different venipuncture sites. blood cultures, with first and last drawn at
A major criterion can be fulfilled (Table 16.3), if: least 1 h apart
C. Single positive blood culture for Coxiella burnetii
* A typical IE organism is isolated from two or phase | IgG antibody titer of >1:800
separate blood cultures, or
2. Evidence of endocardial involvement: Any one
* Agent other than typical IE organisms is A. Positive echocardiogram
isolated persistently from blood cultures > Oscillating intracardiac mass on valve or
(Table 16.3) in the absence of an extracardiac >» Abscess, or
focus of infection. > New partial dehiscence of prosthetic valve
B. New valvular regurgitation
A minor criterion is considered to be fulfilled
(Table 16.3) if blood cultures show positive but Niayeln@ ater)
not meeting major criterion. 1. Predisposition: Predisposing heart conditions or IV
Blood culture collection technique and drug use
2. Fever = 38.0°C (=100.4°F)
processing is discussed in detail in Chapter 17.
3. Vascular phenomena: Major arterial emboli, septic
pulmonary infarcts, mycotic aneurysm, intracranial
Non-blood-culture Tests
hemorrhage, conjunctival hemorrhages or Janeway
Various non-blood-culture tests that can be used lesions
for the diagnosis of IE include: 4. Immunologic phenomena: Glomerulonephritis,
Osler’s nodes, Roth's spots or rheumatoid factor
“ Serologic tests can be used to implicate
5. Microbiologic evidence: Positive blood culture but
some organisms that are difficult to recover not meeting major criterion as noted previously’ or
by blood culture: Brucella, Bartonella, serologic evidence of active infection with organism
Legionella, Chlamydophila psittaci, and consistent with infective endocarditis
Coxiella burnetii Definite endocarditis if the followings are present:
* Isolation of the pathogens in vegetations by e Two major criteria or
culture e One major criterion and three minor criteria or
“ Direct fluorescence antibody techniques e Five minor criteria

% PCR to recover unique microbial DNA or *Excluding single positive blood cultures for coagulase-negative
staphylococci and diphtheroids, which are common culture
16S rRNA that, when sequenced, allows contaminants, and organisms that do not cause endocarditis
identification of the etiologic agent. frequently, such as gram-negative bacilli.
Abbreviation: \E, infective endocarditis.
Echocardiography
= For prosthetic valve IE: In addition to the
Echocardiography allows anatomic confirma-
tion of infective endocarditis, sizing of vegeta- above regimen, rifampin (for 6 weeks) and
tions, detection of intracardiac complications,
gentamicin (for 2 weeks) are added.
and assessment of cardiac function. 2. Regimen for Viridans Streptococci and S.
gallolyticus IE
Treatment of Infective Endocarditis = Fornative valve IE: Penicillin or ceftriaxone
is given for 4 weeks
1. Regimen for S. aureus IE:
or = For prosthetic valve IE: Gentamicin is
= For native valve IE: Cloxacillin
added for 6 weeks
vancomycin is given for 6 weeks
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

B ACUTE RHEUMATIC FEVER

Problem Solving Exercise 2

Acute Rheumatic Fever Modified Jones criteria is the diagnostic criteria
A 8-year-old female child presented to the cardiology applied in this condition—four major criteria and one
OPD with swollen, red, and/or tender joints, which minor criteria are fulfilled here.
migrates from one joint to another (knees, ankles, hips, Q Major criteria fulfilled are:
and elbows) over a period of hours. The child was having >» Migratory polyarthritis: Swollen, red, and/or
an abnormal gait. She also complained of painless, tender joints, which migrates from one joint to
small, mobile lumps beneath the skin overlying bony another (knees, ankles, hips, and elbows) over
prominences, particularly of the hands, feet, and a period of hours
elbows. On auscultation of CVS, murmur was heard Vv Subcutaneous nodules: Painless, small, mobile
over the mitral valve area. ECG showed prolongation lumps beneath the skin overlying bony
of P-R interval. On inquiry, it was found that the child prominences, particularly of the hands, feet,
had an episode of sore throat 3 weeks back. and elbows
1. What is the probable clinical diagnosis and its Vv Carditis: On auscultation, murmur was heard
etiological agent? over the mitral valve area
2. Describe the diagnostic criteria used for this > Chorea: Child has abnormal gait
clinical condition. Q Minor criterion fulfilled was: ECG showed
3. How will you prevent recurrence of such episodes? prolongation of P-R interval.
Refer text for the explanation of other questions.
Explanation
This is a case of Acute Rheumatic Fever; which
occurred as a sequel to streptococcal sore throat

Acute rheumatic fever (ARF) is a multisystem usually appear after period of ~3 weeks following
disease that occurs in people previously affected precipitating group A streptococcal infection.
with streptococcal (group A) sore throat, as a The prior streptococcal infection may be either
result of an autoimmune reaction. subclinical (more common) or presents as sore
** Autoimmune mechanism: Antibodies tar- throat.
geted against streptococcal antigens (M pro- Acute rheumatic fever affects heart, joints,
tein) during past episode of sore throat, cross skin and brain. The common manifestations in
react with human tissue antigens (e.g., heart the order of frequency include:
and joint) *
“e
Migrating polyarthritis: It is the most
“+ Although ARF may involve many parts of the common manifestation; affects the large
body, almost all the manifestations resolve joints—most commonly the knees, ankles,
completely; except the cardiac valvular hips, and elbows
damage, which is called as rheumatic heart Pancarditis, affecting endocardium,
disease (RHD). pericardium, or myocardium
Group A Streptococcus (S. pyogenes) Subcutaneous nodules: Occur as painless,
principally causes infections of skin and soft small, mobile lumps beneath the skin
tissues and pharyngitis (Chapter 29 and 33). overlying bony prominences, particularly of
the hands, feet, and elbows
Clinical Manifestations Chorea (Sydenham’s): It is an abnormal
Primary ARF is mainly a disease of children, involuntary movement disorder, mainly
of 5-14 years age. The clinical manifestations affecting head and limbs
CHAPTER 16 © Cardiovascular System Infections: Infective Endocarditis and Acute Rheumatic Fever

Table 16.4: Diagnostic criteria for rheumatic fever—modified Jones criteria (2015).

Carditis (clinical or subclinical)

Arthritis—only polyarthritis Arthritis—monoarthritis or polyarthritis
Polyarthralgia
Chorea Chorea
Erythema marginatum Erythema marginatum
Subcutaneous nodules Subcutaneous nodules
Minor criteria
|1 ie
waSEE

Polyarthralgia Monoarthralgia

Hyperpyrexia (238.5°C) Hyperpyrexia (238.0°C)

ESR >60 mm/h and/or ESR >30 mm/h and/or

CRP =3.0 mg/dL CRP =3.0 mg/dL
Prolonged PR interval Prolonged PR interval
Diagnostic criteria

Initial ARF Two major or

: One major + two minor

Recurrent ARF Two major or

(with a reliable past history of ARF/RHD) One major + two minor or
Three minor criteria
rheumatic fever; RHD, rheumatic heart disease.
Abbreviations: ESR, erythrocyte sedimentation rate; CRP, C-reactive protein; ARF, acute
evidence (of previous streptococcal infection), which was a part of previous version ofJones criteria (1992), has been
Note:The supporting
removed from the new modified Jones criteria, 2015.

* Erythema marginatum: They are pink Prevention

macular rashes that appear and disappear Primary Prevention
before the examiner’s eyes.
It includes timely and complete treatment of
Diagnosis of ARF (Jones Criteria) group A streptococcal sore throat with antibiotics
(penicillin) within 9 days of sore throat onset,
The diagnosis of ARF is made based on diagnostic
which will prevent almost all cases of ARF.
criteria known as revised Jones criteria (2015).
It is based on the presence of a combination of Secondary Prevention
typical clinical features together with ECG and
The mainstay of controlling ARF and RHD is
laboratory (ESR, CRP) findings (Table 16.4).
secondary prevention. Patients with ARF are at
Treatment much higher risk of developing recurrent ARF.
Penicillin is the drug of choice; can be given orally Therefore, long-term (5-10 yrs or even longer)
(as penicillin V for 10 days) or intramuscularly penicillin prophylaxis is indicated to prevent
(as single dose of benzathine penicillin G). recurrences.
 ,

Bloodstream Infections ],

SLATE

[Problem Solving
Solving Exercise
Exercise)1
Sepsis respiratory rate >22/min, altered mentation and
systolic blood pressure <100 mm Hg
A 42-year-old female presented with fever, chills and
SOFA scoring system: Sepsis is clinically diagnosed
rigors, confusion, anxiety, difficulty in breathing,
by SOFA (sepsis-related organ failure assessment)
malaise and vomiting. On examination, the following
signs were noticed: body temperature 102°F, heart score which in turn depends on six parameters:
rate 106 per minute and respiratory rate 24 per Respiratory system (PaO2/FiO2)
minute, blood pressure 90/60 mm of Hg. Urine output Coagulation system (platelet count)
was significantly decreased. Liver (serum bilirubin)
Cardiovascular (mean arterial pressure)
1. What is the probable clinical diagnosis?
2. What scoring system is used to assess the severity CNS (Glasgow coma scale score)
of infection and extent of organ failure? eS
US)
ey
Ren)Renal (serum creatinine and urine output).
3. How will you collect the specimen? Refer Table 17.1 for detail.
4. Describe the laboratory diagnosis in detail. Collection of blood culture and laboratory diagnosis
Explanation of sepsis is discussed in the text below.
The provisional diagnosis in this case is sepsis; as it
satisfies the bed side qSOFA (Quick SOFA) criteria—

B INTRODUCTION ** Septicemia is a condition in which bacteria

circulate and actively multiply in the
Bloodstream infections (BSI) refer to the bloodstream and may produce their products
presence of microorganisms in blood, which (e.g., toxins) that cause harm to the host
constitute one of the most serious situations
** Similarly, the presence of viruses, parasites and
among infectious diseases; as they are a threat
fungi in blood can be described as ‘viremia;
to every organ in the body.
‘parasitemia’ and ‘fungemia’ respectively.
** Microbial invasion of bloodstream can have
serious immediate consequences such
as shock, multiple organ failure, and DIC
§ CLINICAL MANIFESTATIONS
(disseminated intravascular coagulopathies) Bloodstream infections have a bacteremia stage
** Therefore, timely detection of the causative followed by a septicemic stage. The clinical
agent is one of the most important goals of manifestations are evident only in the septicemic
the microbiology laboratory. stage. In this stage, the bacteria multiply and
release their products (e.g., toxins) which travel
Terminologies to various organs affecting their functions. Based
The suffix ‘emia’ is derived from the Greek word on the severity and the extent of organ failure;
meaning “blood” and refers to the presence ofa bloodstream infection can be divided into two
substance in the blood. stages: sepsis and septic shock (Table 17.1).
“+ Bacteremia refers to the presence of bacteria * Sepsis: The common signs and symptoms
in blood without any multiplication include:
 CHAPTER17 © Bloodstream Infections

m Fever or hypothermia with/without chills Table 17.1: Definition of sepsis and the assessment of
and rigors SE verity and organ failure.

= Hyperventilation leads to excess loss of Ssepsis

CO, and subsequent respiratory alkalosis S epsis is defined as life-threatening organ dysfunction
= Skin lesions, change of mental status and caused by a dysregulated host response to infection
diarrhea. SsOFA score
Gg
+,*!
Septic shock: This is the gravest late stage S epsis is diagnosed by SOFA (sepsis-related organ
failure assessment) score which in turn depends on six
complication of septicemia and is manifested
parameters.
as—hypotension, DIC and multiorgan failure 1 . Respiratory system—PaO,/FiO,
(e.g., acute respiratory distress, renal failure, . Coagulation system—platelet count
tissue destruction, etc.). The endotoxins of . Liver—serum bilirubin
gram-negative bacteria have a direct effect . Cardiovascular—mean arterial pressure (MAP)
. Central nervous system—Glasgow coma scale score
on the pathogenesis of septic (or endotoxic) mWBWN

6 . Renal—serum creatinine and urine output

shock. Organ dysfunction can be identified as an acute
In sepsis, the severity and degree of organ change in the total SOFA score =2 points following the
failure can be determined by an assessment infection
score called as SOFA (sepsis-related organ qSOFA (Quick SOFA) Criteria
failure assessment) score Quick SOFA criteria Determination of SOFA score takes considerable time
(Table 17.1). as it depends upon a number of laboratory parameters.
However, before the result of SOFA score is available,
The common agents implicated in sepsis
sepsis can promptly be identified at the bedside with
are gram-negative organisms such as Escheri-
q SOFA score
chia coli, Klebsiella pneumoniae, Pseudomonas, Respiratory rate =22/min
Acinetobacter and gram-positive cocci such as Altered mentation
Staphylococcus aureus and Enterococcus species. Systolic blood pressure <100 mm Hg
Sseptic shock
§ LABORATORY DIAGNOSIS It is a subset of sepsis in which underlying circulatory
a nd cellular/metabolic abnormalities are profound.

Diagnosis of bloodstream infection depends on Pp.atients with septic shock can be identified with a

the isolation of the causative agent from blood clinical construct of sepsis with:
Persisting hypotension requiring vasopressors to
by performing blood culture. maintain MAP (mean arterial pressure) 265 mm Hg
and
Specimen Collection for Blood Culture Serum lactate level >2 mmol/L (18 mg/dL) despite
Extreme care should be taken while collection adequate volume resuscitation
P.atients with septic shock have a mortality of >40% in
of blood for culture, as there is a high-risk of
contrast to 10%, for sepsis cases
contamination with skin flora.
No te: PaO,,/FiO, is the ratio of arterial oxygen partial pressure to
<2
% ‘e Site: Blood for culture should always be col-
fra ctional inspired oxygen.
lected in pairs; from two separate venipunc-
tures and 2 separate skin decontamination
processes. If a central line is present, then one = The disinfectants should be applied in
sample from the central line and one from the a circular motion (5 cm in diameter),
venipuncture should be collected starting from the center to the periphery
Preparation of the site: To avoid contamina- = The area should be allowed to air dry
tion with skin flora, blood should be collected before venipuncture.
under strict aseptic conditions using sterile
“* Timing of collection: Blood should be
disposable syringe (Fig. 17.1) collected before starting antimicrobial
Skin decontamination: Skin should be therapy. If the antimicrobial agent is already
disinfected by two-step procedure—first, started, then the best time ofcollection is just
treated with 70% isopropyl alcohol and then before the next dose of the antimicrobial agent
asecond antiseptic solution such as povidone Blood volume: Blood specimen is drawn
iodine or chlorhexidine should be applied using a sterile syringe and needle. Higher
 s)
SECTION 2 ® Systemic Microbiology (Infectious Disease

ict
|ie 4
sel ||
: | i :
ys | s
I il
| |1 | ~~
Apply tourniquet, Use 70% alcohol to Use chlorhexidine/ Alcohol wipe to Collect blood
Perform hand
palpate the vein disinfect the site providone iodine clean the bottle aseptically and
hygiene and use
upside down. to disinfect the site, top inject into blood
sterile gloves and mark the area
Wait for 30 sec concentric inside out. culture bottle
(allow the skin Wait for 1 min without changing
to dry) (allow the skin to dry) the needle

Fig. 17.1: Steps of collection of blood for culture.

the volume of blood, greater is the chance of “+ Dilution: The blood is inoculated in the me-
isolation (yield increases by 3.2% per mL of dium ata dilution of 1:5 so that the antibacterial
blood cultured). At least 8-10 mL of blood per components in the blood, if any, will get diluted
bottle for an adult and 1-3 mL per pediatric “+ SPS (sodium polyanethol sulfonate) is added
bottle is recommended to the medium as an anticoagulant. It also
“+ Number of blood cultures: At least 2-3 blood counteracts the bactericidal action of blood
culture sets (each set consists of two bottles: 1 ** Incubation: Upon receipt, the bottles should
aerobic and 1 anaerobic) are required to have be directly incubated in the upright position
good isolation rate (around 65%, 80% and 95% at 37° C for up to 7 days
with one, two and three sets respectively). “+ Repeat subcultures are made from the BHI
Multiple blood cultures should be collected broth onto blood agar and MacConkey agar
for endocarditis cases = From monophasic medium: Subcultures
“+ Dispensing: Collected blood is then directly are made when the broth becomes turbid
dispensed into either blood culture bottle or periodically (blind subcultures) for one
at the bedside; either a conventional or week. There is a risk of contamination due
automated blood culture. Change of needle to opening of the cap of the bottle every
between collection and dispensing, an old time when subcultures are done
practice is no longer recommended
*,
*
* Transport of blood specimen: The collected
blood is gently mixed with the broth and then
transported immediately to the Microbiology
laboratory. In case of delay, blood culture
bottle should never be refrigerated. It can
be kept at 35°C in an incubator (if available)
or left at room temperature.

Conventional Culture Medium

The method used for the conventional blood
culture is as follows (Refer Chapter 3.6),
* Types of media: There are two types of
conventional blood culture media (Figs 17.2A Ey
and B) Figs 17.2A to C: Blood culture bottles: (A) Monophasic
= Monophasic medium: It contains 50-100 medium (BHI broth); (B) Biphasic medium (Castaneda’s),
mL of brain heart infusion (BHI) broth containing BHI broth and BHI agar slant; (C) BacT/ALERT
bottle.
m Castaneda’s biphasic medium: It consists
Source: Department of Microbiology, JIPMER, Puducherry (with
of BHI agar slope and BHI broth (50 mL). permission).
 CHAPTER17 © Bloodstream Infections

= In biphasic medium, the subcultures can either conventional biochemical reactions or

be made just by tilting the bottles so that automated identification system such as MALDI-
the broth runs over the agar slope. There is TOF or VITEK.
lower risk of contamination as it obviates
the opening of the cap of the bottle. The Antimicrobial Susceptibility Test (AST)
colonies appear over the agar slant, which Antimicrobial susceptibility test is carried
is further used for identification. out for guiding the institution of appropriate
therapy. Minimum inhibitory concentration
Automated Culture Media (MIC) based method (e.g., VITEK) is preferred
BACTEC and BacT/ALERT are the automated over disk diffusion method when testing for
blood culture systems. The most advanced blood isolates. It is ideal for endocarditis
system is Bact/ALERT Virtuo. In these systems, isolates, especially while reporting AST result
the growth is continuously monitored and of penicillin.
reading is recorded every 15-20 min (Fig. 17.2C).
When the growth is detected, the system gives Treatment of Sepsis/Bloodstream Infection
a positive signal. Then the bottle is removed and Due to higher prevalence of multidrug resistant
processed similarly as done for conventional bacteria (MDROs) and higher mortality in sepsis,
bottles. Automated systems are much superior antibiotics should be instituted at the earliest,
to conventional media in terms of faster as soon as sepsis is clinically suspected. De-
isolation and increased sensitivity. More so, escalation approach is usually followed which
they also help in diagnosing catheter related means:
bloodstream infection (CRBSI) by determining “+ Empirical treatment consists of higher class of
the differential time to positivity. antimicrobials with both gram-negative and
gram-positive coverage; e.g., carbapenem
identification such as meropenem plus vancomycin
The isolated organism is identified by colony “+ Definitive treatment can be tailored according
morphology, Gram staining, followed by to the culture sensitivity report.

[Problem Solving
Solving Exercise2__|
Exercise 2 :

Catheter-related Bloodstream Infection Explanation

(CRBSI) This is a case of CRBSI (catheter-related bloodstream
A 42-year-old male on central line presented infection) as the following criteria are met:
with fever (>103°F), altered mental status, heart Q Patient is on central line
rate of 102 per minute and respiratory rate of 24 Q Presence of signs of sepsis: Fever with altered
breaths per minute. Blood was collected both mental status, increased heart rate, respiratory rate.
from central line and venepuncture separately Q Culture criteria (differential time to positivity
in BacT/ALERT bottles and sent for culture. Both >2 hours): Culture of both central line and
venepuncture blood specimens revealed the same
central line and venepuncture bottles flagged
positive for Staphylococcus aureus after 4 hours pathogen (Staphylococcus aureus); with central line
and 7 hours of incubation respectively. What is the bottle flagged positive (at 4 hours of incubation)
>2 hours earlier than the venepuncture bottle
clinical condition and how will you diagnose this
(flagged at 7 hours of incubation).
condition?
 Bacterial Infections of Bloodstream: CHAPTER|
Enteric Fever, Scrub Typhus,
Brucellosis, and Leptospirosis 18
@ ENTERIC FEVER (TYPHOIDAL SALMONELLA)
Problem Solving Exercise 1
A 12-year-old boy was admitted with high-grade In first week, blood culture is the investigation
fever, abdominal pain, nausea, vomiting and anorexia of choice. Blood specimen should be collected
for the past 5 days. On examination, tongue was aseptically (Fig. 18.10), preferably in automated blood
coated and spleen was palpable. Blood specimen was culture bottle (if facilities are available).
collected for culture in automated culture bottle (Fig.
Identification
18.1C) and results of all the investigations done are
shown in the Figures 18.2, 18.4 to 18.7 and Table 18.3.
Q Positive blood culture bottles or conventional
Q How will you collect the blood sample and send it blood culture in biphasic media when subcultured
to the laboratory for culture? on MacConkey agar reveals NLF colonies (Fig. 18.2).
a Identify the test and interpret the result. Q Biochemical reactions (Fig. 18.5) and agglutination
Q Interpret the antimicrobial susceptibility test result with Salmonella polyvalent and O9 antisera suggests
and suggest the antibiotic to be used. the identification as Salmonella Typhi (Fig. 18.6).
Q Name two vaccines to prevent the infection. Antimicrobial Susceptibility Test
Explanation Antibiogram (Fig. 18.7 and Table 18.3) reveals the strain is
Clinical Diagnosis sensitive to ceftriaxone, chloramphenicol, azithromycin,
ampicillin and co-trimoxazole and intermediate to
Fever, abdominal discomfort for 5 days and palpable
ciprofloxacin. Ceftriaxone being the drug of choice,
spleen in endemic area, such as India is suggestive of
should be the first line of treatment given.
enteric fever.
(For answers to other questions, refer below).

Clinical Manifestations of Enteric Fever ** Complications: Gastrointestinal bleeding and

Enteric fever is a misnomer as the manifestations intestinal perforation can occur mostly in the
are more extraintestinal than intestinal. It is third and fourth weeks ofillness.
transmitted by ingestion of contaminated food * Neurologic manifestations occur rarely
or water. Various manifestations include: which include meningitis, cerebellar ataxia
* Fever: Described as step ladder pattern type and neuropsychiatric symptoms.
of remittent fever
* Other symptoms: Headache, chills, cough, Laboratory Diagnosis
sweating, myalgia and arthralgia Specimen collection largely depends on the
Rashes (called as rose spots) duration ofillness (Table 18.1).
Early intestinal manifestations, such
as abdominal pain, nausea, vomiting,
Culture
anorexia, diarrhea or constipation and loss
of appetite Blood Culture
* Important signs include hepatospleno- Blood culture is the ideal method for diagnosis
megaly, epistaxis and relative bradycardia in the first week offever, which becomes positive
 CHAPTER 18 © Bacterial Infections of Bloodstream 143°

Table 18.1: Tests used for diagnosis of enteric fever. “+ Procedure: 10-20 mL of fresh blood is directly
Duration of illness |Specimen used and test done injected at the bedside through a hole present
eae tee on the cap of the bottle rather than opening
First week
Blood the bottle
> Bone marrow aspirate * Incubation: Blood culture bottles are
> Duodenal aspirate incubated at 37°C for 24 hours
Second week and e Serum: “ Repeat subcultures:
Third week > For antibody detection by # From monophasic medium: Repeat
Widal test subcultures are made onto blood agar
> For antigen detection and MacConkey agar periodically for one
e Stool and urine culture : 6 Sen
week. There is a risk of contamination due
Fourth week Stool and urine culture to opening of the cap of the bottle every
Carriers e Stool and urine culture time when subcultures are made
° Serum: For detection of = Biphasic medium is preferred as the
antibodies to Vi antigen
ee eco cuits died way subcultures can be made just by tilting the
bottles so that the broth runs over the agar
slope. Bottle is incubated in the upright
in about 90% of cases. Thereafter, the positivity position. If colonies appear over the agar
declines to 75% in the second week and 60% in slant, it is used for further identification
the third week and 25% till the fever subsides. = From positive BacT/ALERT bottles sub-
* Culture medium: Blood culture bottles are cultures are done.
the recommended media. There are two types “* Colony appearance:
of media: = Blood agar: Nonhemolytic moist colonies
1. Conventional blood culture media: m= MacConkey agar: Colonies are round,
¢ Monophasic medium: Brain heart translucent, pale and lactose nonferment-
infusion (BHI) broth (Fig. 18.1A) ing (Fig. 18.2).
¢ Castaneda’s biphasic medium: Consists
of BHI agar slope and BHI broth (Fig. Stool and Urine Culture
18.1B) This is useful for isolation of Salmonella in the
2. Automated blood culture bottles: BACTEC third and fourth weeks of illness. They remain
or BacT/ALERT (Fig. 18.1C) positive even after antibiotic treatment. Stool
and urine culture are also done for detection of
carriers.

Figs 18.1A to C: Blood culture bottles: (A) Monophasic

medium (BHI broth); (B) Biphasic medium (Castaneda’s),
containing BHI broth and BHI agar slant; (C) BacT/ALERT Fig. 18.2: MacConkey agar showing nonlactose
bottle. fermenter colonies of Salmonella.
Source: (A to C) Department of Microbiology, JIPMER, Puducherry Source: Department of Microbiology, Pondicherry Institute of
Medical Sciences, Puducherry (with permission).
(with permission).
 Diseases)
SECTION 2 © Systemic Microb iology (Infectious

Y ms

Pa ~~ ‘4

ONY <4"
/

A J é
Figs 18.3A and B: Colonies of 5S. Typhi: (A) DCA
(Deoxycholate citrate agar) showing pale colonies with
black center; (B) XLD agar (Xylose lysine deoxycholate)
showing red colonies with black center.
4,
i leeed
4
Ke
— —
Source: Department of Microbiology, JIPMER, Puducherry (with
permission). Fig. 18.4: Gram-stained smear showing gram-negative
bacilli (Salmonella).
¢* Urine culture: Urine is centrifuged and the Source: Department of Microbiology, Pondicherry Institute of
deposit is inoculated onto MacConkey agar. Medical Sciences, Puducherry (with permission).
“+ Stool culture is done similar to that is followed
for Shigella.
a Enrichment broth, such as Selenite F Indole Citrate Urease =
negative negative negative gas-, H,S+
broth, tetrathionate broth and gram-
negative broth are used
Selective media, such as MacConkey agar
and DCA or XLD are used.
* .
¢ DCA: It produces non-lactose-
fermenting pale colonies with black
center (Fig. 18.3A)
XLD agar: It produces red colonies with
black center (Fig. 18.3B).

Other Specimens Include

“+ Bone marrow culture is useful during the first
week of illness (55-90% sensitive); when blood
culture is negative, especially when patient is Fig. 18.5: Biochemical reactions of Salmonella Typhi.
on antibiotics Source: Department of Microbiology, Pondicherry Institute of
Medical Sciences, Puducherry (with permission).
Duodenal aspirate culture is recommended
during first week of illness if both blood and Table 18.2: ICUT reaction for Salmonella.
bone marrow cultures turn negative.
P| S. Typhi S. Paratyphi A
Identification Indole —_Negative Negative

In culture smear, Salmonella appear gram- Citrate Negative Negative

negative bacilli (Fig. 18.4). Motility testing by Urease Negative Negative
hanging drop reveals that they are motile with TSI Alkaline slant/acidic Alkaline slant/acidic
peritrichous flagella. butt, gas absent, speck butt, gas present,
of H,S present H,S absent
Biochemical Identification (Fig. 18.5)
Two common enteric fever isolates (S. Typhi and Slide Agglutination Test
S. Paratyphi A) show the following biochemical Identification of Salmonella at genus level
properties (Table 18.2). can be confirmed by slide agglutination using
 CHAPTER 18 © Bacterial Infections of Bloodstream

Saline control ai t strain

Saimor polyvalent

Fig. 18.6: Bacterial agglutination with antisera: Test strain

is tested with Salmonella polyvalent antisera and then
with O9 antisera. (Presence of clumps indicates test is
positive and the stain is identified as Salmonella Typhi).
Source: Department of Microbiology, Pondicherry Institute of
Medical Sciences, Puducherry (with permission).

polyvalent O antisera. Then, the serotypes can Fig. 18.7: Antimicrobial susceptibility testing on Mueller-
be identified by using type specific O antisera. Hinton Agar for Salmonella Typhi (Refer Table 18.3 for CLS|
* S. Typhi: Agglutinates with O9 antisera (Fig. 18.6) zone interpretation).
Abbreviations: Cf, ciprofloxacin; Ci, ceftriaxone; C, chloramphenicol;
“+ S. Paratyphi A: Agglutinates with O2 antisera. Az, azithromycin; A, ampicillin; Co, cotrimoxazole; CLSI, Clinical and

Antimicrobial susceptibility testing (Fig. Laboratory Standards Institute.

Source: Department of Microbiology, Pondicherry Institute of
18.7): This is done on Mueller-Hinton agar by Medical Sciences, Puducherry (with permission).
disk diffusion method (Table 18.3).
(As the paratyphoid O antigens cross react
with the typhoid O antigen due to their
Widal test is widely used serological tests for sharing of factor 12, hence, they are not used
diagnosis of enteric fever. Also refer Problem in the test.)
Solving Exercise 3 of Chapter 7. “* Procedure of Widal test:
¢* Principle: It is an agglutination test where = Patient’s serum is serially diluted in
H and O antibodies against S. Typhi and S. normal saline in test tubes from 1 in 10 to
Paratyphi A and B are detected. 1 in 640 dilutions. Four such sets are made
* Antigens used: Four antigens are used. = To each set of diluted sera, respective four
1. O antigen of S. Typhi (TO) antigen suspensions are added
2. Hantigen of S. Typhi (TH) = Control tubes containing the antigens and
3. H antigen of S. Paratyphi A (AH) normal saline should be kept to check for
4. H antigen of S. Paratyphi B (BH). auto-agglutination

Table 18.3: Interpretative categories (CLS!) and observed zone size diameter (mm) to various antimicrobial
agents tested for Salmonella Typhi.
EE SanCRTE aE

Ciprofloxacin (Cf) 5 <20 21-30 231 21 Intermediate

30 <19 20-22 >23 28 Sensitive

Ceftriaxone (Ci)
30 <12 13-17 =18 19 Sensitive
Chloramphenicol (C)
15 <12 - 213 16 Sensitive
Azithromycin (Az)
10 S13 14-16 =17 19 Sensitive
Ampicillin (A)
1.25/23.75 <10 11-15 216 20 Sensitive
Cotrimoxazole (Co)
Abbreviation: CLSI, Clinical and Laboratory Standards Institute.
 Diseases)
SECTION 2 © Systemic Microbiology (Infectious

4:20 1:40 1:80 1:160 1:320 1:640 Saline

control

— ob (i

1:20 1:40 1:80

Figs 18.8A and B: O and H agglutination in Widal test

reading taken in a mirror.
Source: Department of Microbiology, Pondicherry Institute of
Medical Sciences, Puducherry (with permission).

= Test tubes are incubated in water bath at 4:20 1:40 1:80 1:160 1:320 1:640 Saline
control
37°C overnight.
* Results:
® O agglutination appears as compact
granular chalky clumps (disk-like pattern),
with clear supernatant fluid (Fig. 18.8A)
ue
= H agglutination appears as large loose
1:20 1:40 1:80 1:160 1:320 1:640 Saline
fluffy cotton-woolly clumps, with clear control
supernatant fluid (Fig. 18.8B)
= If agglutination does not occur, button for-
mation occurs due to deposition of antigens
and the supernatant fluid remains hazy
a Titer: The highest dilution of sera, at
SEEEE §-
Fig. 18.9: Widal test (titer
TO 1:160; TH 1: 320; AH <1:20;
which agglutination occurs, is taken as the
BH <1:20. Titer suggestive of enteric fever due to S. Typhi.
antibody titer.
Source: Department of Microbiology, Pondicherry Institute of
Medical Sciences, Puducherry (with permission).
Interpretation (Table 18.4 and Fig. 18.9):
* Significant titer: Higher titers are only (malaria, dengue) in persons who have had
significant. The cut-off varies from place to prior infection or immunization
place depending on endemicity of the disease. = Persons with inapparent infection or
Significant titer in most ofthe places in India ® Persons with prior immunization (with
is taken as: TAB vaccine).
® H agglutinin titer >200 and * Four-fold rise in antibody titer demonstrated
= O agglutinin titer >100 by testing paired sera at 1 week interval is
* False positive: Widal test may occur due to: more meaningful than a single high titer. Rise
= Anamnestic response: It refers to a tran- in titers in anamnestic responses are transient
sient rise of titer due to unrelated infections that usually fall down after 1 week whereas, in
Table 18.4: Interpretation of Widal test,

Widal test result Suggestive of

Rise ofTO and TH antibody (Fig. 18.9) Enteric fever due to S. Typhi
Rise of TO and AH antibody Enteric fever due to S. Paratyphi A
Rise ofTO and BH antibody Enteric fever due to S. Paratyphi B
Rise of only TO antibody Recent infection—due to any serotype—S. Typhi or S. Paratyphi A or B
Rise of only TH antibody ? Convalescent stage/anamnestic response
Rise of all three TH, AH, BH antibodies Post-TAB vaccination
 CHAPTER 18 © Bacterial Infections of Bloodstream 147

true infection, the titer increases by four-fold Treatment

after 1 week Treatment of enteric fever depends on the suscep-
** False negative Widal test may occur in:
tibility of the strains. The drugs usually given are:
= Early stage (first week of illness) “+ Third generation cephalosporins, e.g., ceftri-
= Late stage (after fourth week) axone
= Carriers “+ Azithromycin
= Patients on antibiotics “+ Ciprofloxacin (currently increasing resistance
m Due to prozone phenomena (antibody has been reported, hence nota preferred drug).
excess): This can be obviated by serial
dilution of sera. Vaccines for Typhoid Fever
* O agglutinins appear early and disappear
The following are vaccines available for typhoid.
early and indicate recent infection. H
“+ Parenteral Vi polysaccharide vaccine: It is
agglutinins appear late and disappear late
composed of purified Vi capsular polysaccha-
“* O antibodies are serotype nonspecific. They
ride antigen.
are raised in all infections, i.e., S. Typhi, S.
= Dosage: Single dose containing 25 pg of
Paratyphi A and B
Vi antigen is given intramuscularly or
“+ H antibodies are specific. TH, AH and BH
subcutaneously
antibodies are raised in S. Typhi, S. Paratyphi
m Vaccine confers protection for 2 years
A and B infections, respectively.
m Age: It is given only after 2 years of age.
Other Antibody Detection Tests “ Typhoral: Oral, live attenuated vaccine.
= Typhoralis a stable live attenuated mutant
Various commercial methods available are:
of S. Typhi strain Ty2 la
* Typhidot test: OMP (outer membrane protein)
= It is given orally before food, on alternate
antigen is used, detects both immunoglobulin
days 1, 3, 5 and/or 7 (total of three or four
(Ig) M and IgG antibodies
doses).
* IDL Tubex test: O9 antigen is used, detects
s Boosters are recommended every 3 years
only IgM antibodies against S. Typhi.
for people residing in endemic areas.
Demonstration of Serum Antigens
Antigens of typhoidal salmonellae are @ SCRUB TYPHUS
consistently present in the blood in the early
Weil-Felix Test
course of the disease, and also in the urine of
patients during the late phase. Several methods, It is heterophile agglutination test works on the
such as ELISA are available for antigen detection. principle of antigenic cross reactivity.
«+ Antigen: Group specific alkali stable lipopol-
Molecular Methods ysaccharide (LPS) antigen found in some
Several polymerase chain reaction (PCR)-based rickettsiae is also shared by certain strains of
methods (e.g., nested PCR) are available to Proteus (OX19, OX2 and OXK strains). Hence,
detect and differentiate typhoidal salmonellae rickettsial antibodies are detected by using
by targeting various genes, such as flagellin Proteus antigens
gene, Jro B and FIiC gene. * Procedure: It is a tube agglutination test;
serial dilutions of patient’s serum are treated
Detection of Carriers with nonmotile strains of Proteus vulgaris
Typhoid carriers are detected by: OX19 and OX2 and Proteus mirabilis OXK
“ Culture: Stool and bile culture (detects fecal “* Results:
carriers) and urine culture (detects urinary m In epidemic and endemic typhus: Sera
carriers) agglutinate mainly with OX19 and some-
* Detection of Vi antibodies times with OX2
~ Isolation from sewage is carried out to trace = In tickborne spotted fever: Antibodies to
the carriers in the communities. both OX19 and OX2 are elevated
 s)
148 SECTION 2 © Systemic Microbiology (Infectious Disease

Solvingg Exercis
[Problem Solvin e 2 —_|
Exercise2_

A 49-year-old man from village area near Puducherry Explanation

presented with vesicular rashes, lymphadenopathy Clinical Diagnosis
and eschar on upper limb. There was history of high The history of triad of an eschar (at the site of bite),
grade fever, myalgia and respiratory distress for the regional lymphadenopathy and maculopapular rash;
past 2 days. A serological test was performed as however seen only in 40-50% of cases. Patient may
shown in the picture below (Fig. 18.10): also present with non-specific symptoms, such as
1. Identify the test and interpret the result. How will fever, headache, myalgia, cough, and gastrointestinal
you confirm the diagnosis? symptoms.
2. What is the clinical diagnosis and its causative
Complication: Encephalitis and interstitial pneumo-
organism and mode of transmission?
nia may occur rarely in the late stage (due to vascular
3. What are the clinical manifestations seen in this
injury).
conditions ?
4. What are the various modalities of laboratory Etiological agent: Orientia tsutsugamushi
diagnosis? Vector: It is transmitted by trombiculid mite.
5. How will you treat this condition?
Serological test: The test shown here is Weil-Felix
test (Fig. 18.10). Raised antibody titer against OX-K
is suggestive of scrub typhus. As it is a heterophile
agglutination test, it should be confirmed by
serological test detecting specific antibody, such as
IgM ELISA for scrub typhus (using 56-kDa recombinant
major surface protein antigens) and indirect
immunofluorescence test (IFA, gold standard test).
Molecular test: PCR detecting specific genes, such
+ve +ve +ve +ve -—ve -ve Saline |
control as major 56-kDa gene, 47-kDa gene and 16S rRNA
| Interpretation: Titer is 1:160
| Patient serum is serially diluted and Proteus mirabilis
gene.
OX K antigen is added to all tubes. Clumps formation with Treatment: Doxycycline is the drug of choice.
| clearing of supernatant |fluid indicates Positive>test. a
Azithromycin is given alternatively.
Fig. 18.10: Weil Felix test. Details of the Weil-Felix test is explained below.

= In scrub typhus: Antibodies to OXK are ** B. abortus infects cattle and buffalo
raised (Fig. 18.10) * B. suis infects pigs.
= ‘The test is negative in rickettsial pox, Q
Transmission: Man usually gets infection (1)
fever, ehrlichiosis and bartonellosis.
most commonly by direct contact from infected
* False positive titer may be seen in presence
animals tissue, urine, etc.; (2) ingestion of
of underlying Proteus infection. Hence, four-
infected raw milk; and (3) inhalation of dust or
fold rise of antibody titer in paired sera is more
aerosols.
meaningful than a single high titer
* False negative result may occur due to excess
+, ?
Manifestations are of following types:
antibodies in patient's sera (prozone phenom- * Classic triad: Though the manifestations
enon). This can be obviated by testing with vary (40-50%), the classic triad of fever with
serial dilutions of patient’s sera. profuse night sweats; arthralgia/arthritis and
hepatosplenomegaly are present in most
BBRUCELLOSIS patients
* Typhoid-like illness: Overall, brucellosis
Clinical Manifestations
resembles typhoid-like illness except that
Brucella is a zoonotic pathogen; infect various it is less acute, less severe with undulating
animals. pattern of fever (remittent course) and
* B. melitensis is the most pathogenic species to more musculoskeletal symptoms (vertebral
man. Jt infects sheep, goat and camel osteomyelitis and septic arthritis).
 CHAPTER 18 © Bacterial Infections of Bloodstream

._ Problem Solving Exercise 3

A 52-year-old slaughterhouse worker presented with 3. How the disease is transmitted?
on and off fever with profuse night sweats and joint 4. What are the various modalities of laboratory
pain. On examination, hepatosplenomegaly was diagnosis?
found. A serological test performed is displayed in 5. How will you treat this condition?
Figure 18.11.
Explanation
1. Identify the serological test and interpret the
result. Clinical Diagnosis
2. Whatis the clinical diagnosis? The history is suggestive of brucellosis. Points in favor
are:
Q Slaughterhouse worker (occupational exposure).
Q Triad of fever (on and off) with profuse night
sweats, joint pain and hepatosplenomegaly.
Standard Agglutination Test
This is the serological test shown in Figure 18.11.
First tube do not show agglutination (button
formed) due to prozone phenomena.
|-ve +ve +ve +ve +ve +ve Saline control All other tubes show agglutination; clump
(prozone)
+——— Brucella abortus antigen is added ——————>
formation with clearing of supernatant.
Saline control is satisfactory (no agglutination).
Fig. 18.11: Serological test. Interpretation: The test is found be positive with a
Source: Department of Microbiology, Pondicherry Institute of titer of =>1:640 (significant).
Medical Sciences, Puducherry (with permission). (For answers to other questions, refer text below).

Laboratory Diagnosis Result:

m Positive reaction—characterized by
Culture and Identification
clumps formation with clearing of
Blood and bone marrow, CSF, joint fluid or other supernatant
tissues are the useful specimens. Blood and body w Negative reaction—characterized by
fluids are inoculated and processed in blood button formation.
culture bottles (monophasic or Castaneda’s + Significant titer:
biphasic media) or automated systems, such as w Titer of >1:160 is considered as significant
BACTEC and BacT/ALERT as discussed earlier. in nonendemic areas
“+ Subcultures are made from positively flagged = Inendemic area or following occupational
blood culture bottles onto blood agar and then exposure: Titer of 21:320 or rising titer
incubated in candle jar at 37°C overnight by repeating the test after 2-4 weeks is
“ Identification is done by—Culture Smear considered diagnostic.
(small, gram-negative coccobacilli), auto- - Interpretation: A positive SAT result indicates
mated identification systems (MALDI-TOF either acute or chronic brucellosis. As SAT
or VITEK) or conventional biochemical tests detects total antibodies (IgM + IgG); it can-
(catalase and oxidase positive, rapid urease not differentiate between acute and chronic
positive). infection
2-mercaptoethanol (2ME) SAT test: 2ME
Serological Tests (Antibody Detection) destroys IgM antibodies. Therefore, SAT
Standard Agglutination Test (SAT) performed with 2ME treated serum detects
“ Procedure: It is a tube agglutination test. only IgG and confirms chronic brucellosis
Equal volumes of serial dilutions of patient's m SAT positive and 2ME SAT negative—
sera are mixed with the killed smooth indicates acute brucellosis (IgM)
suspension of a standard strain of B. abortus m SAT positive and 2ME SAT positive—
and incubated at 37°C for 48 hours indicates chronic brucellosis (IgG).
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

* False negative SAT may occur due to: Molecular Method

= Prozone phenomenon (due to excess of Polymerase chain reaction is rapid, sensitive and
antibodies in patient’s sera) specific and can also differentiate between the
= Presence of blocking or non-agglutinating species and biovars.
antibodies.
False positive SAT may occur due to antigenic Diagnosis of Brucellosis in Animals
cross-reactions with some other gram- ¢ Isolation from milk and dairy products
negative bacteria having similar O chains. “ Antibody detection in milk, such as milk
ring test, Rose Bengal card test and whey
Other Antibody Detection Tests agglutination test.
* ELISA is a highly sensitive test; uses either
cytoplasmic proteins or LPS antigens to detect Treatment
IgM, IgG, and IgA antibodies individually. Treatment regimen used for brucellosis are:
Therefore, it is useful for diagnosis chronic “+ Standard regimen in adults: Gentamicin for
brucellosis. However, the result has to be 7 days plus doxycycline for 6 weeks.
confirmed by SAT. ELISA is also useful in * WHO regimen in adults: Rifampin for 6
diagnosis neurobrucellosis weeks plus doxycycline for 6 weeks. Relapse
“+ Dipstick assays for anti-Brucella IgM are or treatment failure occurs in 5-10% of cases
available for diagnosis of acute infection, but ** For CNS involvement: Ceftriaxone is added
is less sensitive. and treatment is prolonged for 3-6 months.

BLEPTOSPIROSIS
| Problem Solving
Solving Exercise4
Exercise 4 |
r | { | |
A young farmer presented with fever, headache, and
myalgia and yellow discoloration of skin and sclera. ©
On examination, he had conjunctival inflammation
as =
oie
« i
26 |' |
and hepatosplenomegaly. His blood count showed a ep
oS
neutrophilia with thrombocytopenia. a
|
|_peton
Liver function tests showed an elevated conjugated
bilirubin with mild elevation of transaminases. He Fig. 18.12: ICT for Leptospira antibody.
was also found to be oliguric and uremic. A rapid Source: Department of Microbiology, Pondicherry Institute of
Medical Sciences, Puducherry (with permission).
serological test was performed as shown in the
picture below (Fig. 18.12): Explanation
1. What is the clinical diagnosis and how this disease This is a case of leptospirosis. The points in favor are:
is transmitted?
Q The history of fever, conjunctival inflammation
2. What are the clinical manifestations seen in this and hepatosplenomegaly
disease?
Q Jaundice with ¢ bilirubin and liver enzymes
3. What are the various modalities of laboratory
Q The rapid serological test detected anti-Leptospira
diagnosis?
IgM antibodies (Fig. 18.12).
4. How will you treat this condition?
(For answers to other questions, refer below).

Clinical Manifestations (Leptospirosis) Mild Anicteric Febrile IlIness

Leptospirosis is zoonotic disease caused by It occurs in 90% of patients. It is biphasic:
Leptospira interrogans. It is transmitted by * Septicemic phase occurs first, presented
contact with water, moist soil and wet surfaces with fever, headache, conjunctival suffusion,
contaminated with rodent urine. Direct human- abdominal pain
to-human transmission does not occur. Disease * Followed by immune phase, presented with
is presented in two forms: meningitis, uveitis and rash.
 CHAPTER 18 © Bacterial Infections of Bloodstream

Weil’s Disease “+ EMJH (Ellinghausen, McCullough, Johnson,

It is also called as hepatorenal-hemorrhagic Harris) medium
syndrome. It is a severe form of icteric illness “* Korthof’s media with rabbit blood
with renal dysfunction, occurs in 10% patients. ** Fletcher’s semisolid media.

Laboratory Diagnosis Serology for Antibody Detection

Cerebrospinal fluid and blood (in first 10 days ** IgM antibodies appear early within one week
of infection) and urine (between 10-30 days of of illness, reach peak levels in third or fourth
infection) are useful specimens. week and then decline slowly and become
undetectable within six months
Microscopy * IgG antibodies appear later than IgM;
reach peak level after few weeks of illness
“+2, Under dark-ground microscope (Fig. 18.13):
and may persist at low level for years (Fig.
They are tightly and regularly coiled, with
characteristic hooked ends, such as umbrella
18.14).
Antibody detection tests can be broadly classified
handle
into:
“* Staining: They do not stain by ordinary stain,
“ Genus specific tests: They use broadly
but can be stained by sliver impregnation
reactive genus-specific antigen prepared
stains, such as Fontana stain and modified
from nonpathogenic Leptospira biflexa Patoc
Steiner technique
1 strain. They cannot detect the infecting
“+ Appearance: L. interrogans is 6-12 um long;
serovar. Various tests available are:
tightly and regularly coiled, with characteristic
= Macroscopic slide agglutination test
hooked ends, such as umbrella handle (hence
= Microcapsule agglutination test
the species name interrogans—resembling
= Latex agglutination test
interrogation or question mark)
= Enzyme-linked immunosorbent assay
“* Disadvantage: Microscopy is less sensitive
(ELISA)
and requires technical expertise.
# Immunochromatographic test (ICT): It
Isolation detects IgM and IgG antibodies separately
(Fig. 18.14).
Leptospira is obligate aerobe, fastidious and
grows slowly. Cultures should be incubated at
30°C for 4-6 weeks in media, such as:
Leptospira Leptospira Leptospira
IgG/IgM IgG/IgM IgG/IgM

aeQ=50
S =
ee
GeeQ=O
ciatenmniaiail
O50

Or or Gr

Fig. 18.14: Genus-specific rapid diagnostic test for

leptospirosis.
Fig. 18.13: Dark-ground microscopy demonstrating Source: Department of Microbiology, Pondicherry Institute of
Leptospira interrogans (schematic diagram). Medical Sciences, Puducherry (with permission).
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

* Serovar-specific test: Microscopic aggluti- “+ Various genes, such as 16S or 23S rRNA or
nation test detects antibodies against spe- IS1533 insertion sequence are targeted
cific serovars of L. interrogans. It is the gold “+ However, PCR is not serovar specific.
standard method and reference test for the
diagnosis of leptospirosis Treatment
7“
Cross agglutination and absorption test ** Mild leptospirosis should be treated with
(CAAT): It is done to detect the relatedness oral doxycycline (100 mg twice a day for 7
between the strains. days)
** Severe leptospirosis: Penicillin is the drug
Molecular Methods of choice.
Polymerase chain reaction (PCR) has been
found particularly useful in severe disease,
before seroconversion occurs.
 Viral Infections of Bloodstream:

‘SLRS
HIV/AIDS and Dengue 19
BHIV/AIDS

Problem Solving Exercise 1 , |

In a blood bank, samples received from six donors and 6 are not reactive for HIV. The positive (PC) and
were subjected to a serological test as shown in Figure negative controls (NC) are satisfactory.
19.1 (for HIV infection screening).
National AIDS Control Organization (NACO)
1. Interpret the test result.
2. Which test you will do further to establish the Strategy|
diagnosis according to National AIDS Control Q Testing for HIV infection in blood bank belongs to
Organization (NACO) strategy? NACO strategy I.
3. Mention the various modes of transmission of HIV Q Here, if first test comes positive, no further test
infection. is required to carry out. The blood sample is
4. What are the various generations of HIV enzyme- discarded.
linked immunosorbent assay (ELISA)? Q Result is not reported to the donor. However,
the donor is advised to attend the Integrated
Counseling and Testing Centre (ICTC) clinic for
further evaluation.

HIV Transmission
The various modes of transmission of HIV include:
Q Sexual intercourse (anal >vaginal; male to
female > female to male). It is the most common
mode of transmission (75% of total cases in the
world)
Q Parenteral: Blood transfusion, injection drug
Fig. 19.1: Screening test of six blood donors for HIV. abuse, needle stick exposure
Abbreviations: PC, positive control; NC, negative control. Q Vertical: Mother to fetus.
Source: Department of Microbiology, Pondicherry Institute of Answer to all the others questions are explained in
Medical Sciences, Puducherry (with permission). the text of this chapter.
Explanation HIV ELISA: There are four generations of HIV ELISA.
The test shown here is HIV ELISA. Out of six samples, (Details have been explained below under laboratory
sample no. 5 is reactive for HIV and samples 1, 2, 3, 4 diagnosis).

Solving Exerci
[problem Solving 2
Exercise se2_—_
The Government of a newly formed state was inter- 2. Which test you will do further to establish the
diagnosis according to NACO strategy?
ested to determine the prevalence of HIV infection
in the state. Specimens (serum) were collected from Explanation
various general populations and subjected to the fol- The test shown here (Fig. 19.2), is rapid test which
lowing serological screening test (Fig. 19.2). works on immunocomb principle. Out of 12 samples
1. Interpret the test results and what is the most tested:
probable diagnosis? Q Sample no. 9, 10 are reactive for HIV
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

Q Samples no. 1, 2, 3,4, 5, 6, 7, 8, 12 are nonreactive Q Test for sample 11 is not valid as internal control is
for HIV not satisfactory
Q The internal control is satisfactory for the
remaining samples.
Immunocomb test |@| Internal control
®) Anti-HIV-1
NACO Strategy IIA
5 On ae eo) wo Ole ts 2©
For seroprevalence or epidemiological purpose,
NACO strategy IIA is done.
Q Here confirmation of HIV-1 diagnosis is done by
a second screening test which works on either
different principle or uses different antigens than
the first screening test.
Q If both the tests are reactive; it is reported as
“REACTIVE”.
Q In case if second test is nonreactive, then the
Fig. 19.2: Serological screening test.
result is taken as negative for sentinel surveillance
Source: Department of Microbiology, Pondicherry Institute of
Medical Sciences, Puducherry (with permission). purposes.

Problem Solving Exercise 3

A 25-year-old male with history of multiple sex part- Microbiological Diagnosis
ners is admitted with complaints of unexplained Test shown here is HIV TRI-DOT test (Fig. 19.3), a
fever, progressive loss of weight, persistent diarrhea rapid test for HIV which works on flow through assay
and generalized lymphadenopathy for the past 6 principle. Both control dot and test dot at HIV-1
months. region have shown reactive, hence the test is reactive
1. Interpret the test result and what is the most for HIV-1 antibodies.
probable diagnosis?
2. What further test you will do to establish the NACO Strategy IIB
diagnosis according to NACO strategy? Q Diagnosis of HIV in symptomatic individual
| belongs to NACO strategy IIB.
Q Here, positive result of first test should be
| c confirmed by a second screening test which
works on either different principle or uses different
® antigens than the first screening test.
Q Incase sample is positive by first test, and positive

| by second test, then sample is reported as reactive

and post-test counseling should be given.
| Q Incase sample is positive by first and negative by
| second test, then third test is done.
|
{ Q_ If third test is reactive then sample is reported as
Fig. 19.3: HIV TRI-DOT test. indeterminate and repeat testing is done after 2-4
Source: Department of Microbiology, Pondicherry Institute of weeks. In case if third test is negative, sample is
Medical Sciences, Puducherry (with permission). reported as negative.
Q In case of indeterminate result, sample should be
Explanation sent to reference laboratory for confirmation by
Clinical Diagnosis western blot or reverse transcriptase polymerase
chain reaction (RT-PCR).
History of unexplained fever, progressive loss
of weight, persistent diarrhea and generalized Q 3Cs: Counseling, informed Consent and
Confidentiality are must in these cases.
lymphadenopathy for the past 6 months in a male
having multiple sex partner—most likely diagnosis
is HIV infection.
 CHAPTER 19 © Viral Infections of Bloodstream: HIV/AIDS and Dengue

[Problem
Problem Solving
Solving Exercise
Exercise44 |
A patient was waiting for appendectomy surgery. The Both control band and also test band at HIV-1 region
surgeon wanted to rule out HIV status preoperatively. had shown reactive.
A serological test was performed as shown in Figure Q Here, the confirmation of HIV-1 diagnosis is done
19.4. by performing second and third screening tests
1. Interpret the test result and diagnosis. which work on either different principle or uses
2. What further test you will do to establish the different antigens.
pacaes according to NACO atte oy If all three tests are positive, then reported as
“REACTIVE”.
i: If first and second tests are positive, still third test

- mse ———
should be done to report as HIV reactive. If third test
is negative then sample is reported as indeterminate
C 2.4 and repeat testing is done after 2-4 weeks.
In case sample is positive by first test and negative
by 2nd and 3rd tests, then:
Fig.19.4:eee test.
> If patient belongs to high-risk category,
Source: Department of Microbiology, Pondicherry Institute of
Medical Sciences, Puducherry (with permission). reported as intermediate or
> If belongs to low-risk category then reported
Explanation as non-reactive.
NACO Strategy III In case of indeterminate result, sample should be
It is a case of diagnosis of HIV in asymptomatic sent to reference laboratory for confirmation by
individual which belongs to NACO strategy III. western blot or RT-PCR.
Test show here is HIV rapid test, which works on lateral 3Cs: Counseling, informed Consent and
flow or immunochromatographic assay principle. Confidentiality are must in these cases.

Table 19.1: Laboratory diagnosis of HIV/AIDS.

LABORATORY DIAGNOSIS OF
HIV/AIDS Specific Tests for HIV Infection
e Screening tests (antibody detection):
The various modalities available for the > ELISA (takes 2-3 hours)
laboratory diagnosis of HIV/AIDS are enlisted > Rapid/Simple test (takes <30 minutes)
in Table 19.1. e Supplemental tests (antibody detection):
Diagnosis of HIV/AIDS is not like other > Western blot assay
infectious diseases. A number of moral, ethical, > Line immunoassay (LIA)
legal and psychosocial issues are associated with e Confirmatory tests
> p24 antigen detection (after 12-26 days of
a positive HIV status. The following care should
infection)
be taken (3Cs) while performing the test for HIV. > Viral culture—by Co-cultivation technique
“ Consent in written format should be taken > HIVRNA (best confirmatory method)—can be
before the test is done. The patient should be detected 10-14 days after infection
explained about the nature of the test being @ Reverse transcriptase PCR (RT-PCR)
performed ¢# Branched DNA assay
* Confidentiality of a positive test result is a @ NASBA (nucleic acid sequence-based
must. Patient name or the word “HIV positive” amplification)
4 Real time RT-PCR for estimating viral load
should not be written on the report form
> HIV DNA detection: Useful for diagnosis of
“ Counseling should be provided to motivate
pediatric HIV
the individual to tell the spouse/family and
Non-specific Immunological Methods
induce behavioral change.
Low CD4T cell count
Antibody Detection Hypergammaglobulinemia:
>» Neopterin
Detection of anti-HIV antibodies is the mainstay > B2-macroglobulin
of diagnosis of HIV. Tests to detect specific HIV Altered CD4: CD8T cell ratio
antibodies can be classified into:
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

* Dot blot assays (or Immunoconcentration

Screening Assays
or flow through method, e.g., Tridot test, Fig.
Screening assays usually take less time (2-3 19.3)
hours for ELISA, less than 30 minutes for rapid/ * Immunochromatography (or ICT, lateral flow
simple tests): assay, Fig. 19.4)
* High sensitivity and specificity: NACO * Particle agglutination assays (using latex,
recommends the use of ELISA and rapid
gelatin, RBCs)
kits which have a sensitivity of 299.5% and a
“+ Dip stick/Comb tests (Enzyme immune assay-
specificity of 298%
based tests, Fig. 19.2).
“ Should be confirmed: Results of a single
screening test should never be used as the Supplemental Tests
final interpretation of HIV status. It is always
These assays are highly specific antibody
subjected to confirmatory tests
“+ Antigens used in most of the screening tests detection methods; hence used for validation
of positive results of screening tests. They are
are:
= HIV-1 specific (p24, gp 120, gp160, gp41) expensive, labor intensive, need expertise
= HIV-2 specific gp36. to interpret, and may also give equivocal/
“+ They detect HIV-1 and 2 either separately or indeterminate results.
together. Western Blot
ELISA (Enzyme-linked Immunosorbant Assay) It is the most commonly used supplemental
ELISA is the most commonly performed test available and is also recommended by
screening test at blood banks and tertiary care NACO.
sites. It is easy to perform, adaptable to large ** It works on the principle of immunoblot
number of samples. It is sensitive, specific, and technique (described in Chapter 9)
cost effective. ** It detects individual antibodies in serum
separately against various antigenic fragments
ELISA kits: Most of the currently available ELISA
of HIV (Fig. 19.5)
kits are of two types:
= Antibody to gag gene products (p55, p40,
1. 3rd generation ELISA that uses recombinant
p24, p18)
and/or synthetic peptides as antigen to detect
= Antibody to pol gene products (p65/66,
HIV antibodies
p55/51, p31)
2. 4th generation ELISA that detects both
= Antibody to env gene products (gp 120,
HIV antibodies and p24 antigen by using
gp160, gp41).
combination of recombinant/synthetic
* The antigen antibody complexes appear as
peptides as well as monoclonal antibodies
distinct bands on nitrocellulose strip
respectively. It reduces the window period
* Reactive results are interpreted as per:
considerably.
= WHO criteria: presence of at least two
Types of ELISA: Various ELISA formats are in envelope bands (out of gp120, gp160 or
use depending on different principles, such as: gp41) with or without gag or pol bands
(i) indirect ELISA, (ii) competitive ELISA, (iii) = CDC criteria: presence of any two; out of
sandwich ELISA. p24, gp120, gp160 and gp41 bands.
Rapid/Simple Test
These assays have been developed for ease of

Lill
10d
r fANA7
performance and quick results. They generally
} Bt
~

‘al Western
require less than 30 minutes to perform and aie)
do not require special equipment. They are the OO.
=a NO
oO G8
bh DO W
o fk OA NY
most commonly used tests in India. They work oO
on various principles, such as: Fig. 19.5: Western blot test strip.
 CHAPTER 19 © Viral Infections of Bloodstream: HIV/AIDS and Dengue

| Problem
Problem Solving
Solving Exercise5
Exercise 5
A commercial sex worker presented with diarrhea Explanation
and loss of weight for 2 months. Screening tests The test given in the picture is Western blot, done for
performed for HIV showed equivocal results (Fig. detection of HIV antibodies.
19.6). For confirmation, the following test given below For HIV diagnosis, if the screening tests show
was performed. inconclusive (equivocal) result, then it has to be
1. Identify the test and interpret the result. confirmed by supplemental test.
2. What is the principle of the test?
3. Whatare the other tests can be used for confirmation? Interpretation
Colored bands are present for antibodies against HIV
antigens gp160, gp120, gp41 (env gene products),
p65, p55/51, p31 (pol gene products), p40, p24, p18
(gag gene products).
Final Report
Fig. 19.6: HIV Western blot test strip. Reactive for HIV-1 antibodies; as antibodies to p24, gp
Source: Department of Microbiology, JIPMER, Puducherry (with
120, gp160, gp41 antigens are specific to HIV-1.
permission).

Detection of p24 Core Antigen “ Reverse transcriptase polymerase chain

reaction (RT-PCR)
The p24 antigen becomes detectable after “+ Branched DNA assay
12-26 days of infection and lasts for 3-4 weeks
* NASBA: Nucleic acid sequence-based
thereafter. Again, it is elevated during the late amplification
advanced stage of AIDS. p24 Ag is detected by “ Real time RT-PCR: For estimating viral load.
4th generation ELISA (described earlier).
Apart from the routine diagnosis of HIV, RNA
* It is less sensitive (~30%) because once detection has several other uses, such as:
the antibody is formed, it binds to the p24 * It is the most sensitive and specific method,
protein and the antigen-antibody complex detects even few copies of viral RNA and is the
gets eliminated from the blood best method for confirmation of HIV
* Recently, antigen dissociation assay has * Itis the best tool for diagnosis of HIV during
been developed that involves pretreatment of window period, detects HIV earlier than
serum to an agent, that liberates p24 antigen all available methods (10-14 days post-
from the immunocomplexes. This has shown exposure)
better sensitivity * Viral load monitoring: Real time RT-PCR
* Uses of p24 antigen detection test: can quantify the viral load and is the most
= Forconfirmation of diagnosis of HIV/AIDS appropriate tool for monitoring the response
= Diagnosis of HIV during the window to antiretroviral therapy
period “* Typing: RT-PCR can successfully differentiate
= To diagnose the late stage of HIV/AIDS between HIV-1 and HIV-2 infections and can
(immune collapse) or CNS disease detect the specific genotype or subtype
= Diagnosis of HIV in infants (not reliable) “+ Detection of drug resistance genes.
= Monitoring the progress of HIV infection
= To resolve equivocal western blot results. DNA PCR
PCR detecting proviral DNA is extremely
Viral RNA Detection useful for diagnosis of pediatric HIV and to
Detection of viral RNA is the “gold standard” differentiate latent HIV infection from active
method for confirmation of HIV diagnosis. viral transcription. It is also useful during the
Various formats are available targeting pol and window period, viral load estimation (real time
env genes. PCR) and detection of genotypes.
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

isolation of the Virus

treatment should be started in all patients
regardless of CD4 T cell count
Isolation is time consuming, expensive, takes = Monitoring the response to antiretroviral
longer time (6 weeks or more) and not sensitive. therapy.
It is used only for research and not for routine “+ Abnormal proteins, such as neopterin and
diagnostics.
beta 2-microglobulin are elevated.
Co-cultivation is the method used for
virus isolation. Here, the peripheral blood NACO Strategy for HIV Diagnosis
mononuclear cells (PBMCs) obtained from
For the resource poor countries, it is
the patient are co-cultured along with the
impracticable to confirm the result of HIV
PBMCs from healthy donor; followed by
screening tests by PCR or western blot as these
detection of viral RNA or antigen in culture
assays are expensive and available only at
suspension.
limited centers.
Non-specific/Immunological Tests NACO (National AIDS Control Organization,
“ CD4 T cell count: Measurement of CD4 T India) has formulated a strategic plan (Fig. 19.7)
cell count is carried out by flow cytometry for HIV diagnosis. The guidelines are as follows:
method. It is useful for: “* Depending on the situation/condition, for
m Assessing the risk of opportunistic which the test is done, the positive result
infections of the first screening test should be either
w Initiation of antiretroviral therapy— considered as such or confirmed by another
previously used. Current guideline says one or two screening tests

Strategy/Algorithm | Strategy/Algorithm IIA

(For transfusion/transplantation safety) | (For surveillance)
1 Test kit required | 2 Test kits required
A,
|
|
ore shee tegJ
Sook mag | Report negative
Consider Consider
positive negative
| (Destroy the unit of blood as per guidelines. [ Ait Al+ | A,+ A,-
Refer to ICTC for confirmation of status after consent) Report positive Report negative
ae Ng
Strategy/Algorithm Ii B- | Strategy/Algorithm III
| (Diagnosis of an individual with AIDS | (To detect HIV infection in
| indicator disease with symptoms) asymptomatic individuals)
2 or 3 Test kits required
A,

Report negative

Report positive
with post-test Y ay
counseling [A,+ A,+ A,+|[A,+ A,+ A,-|[A,+ A,- A, + A,+ A,- A;-
| Report positive Indeterminate Indeterminate
A, 4S A, m A; es with post-test
Indeterminate Report negative counseling
High-risk Low-risk |
consider consider
indeterminate negative |
Fig. 19.7: NACO strategies/algorithms for diagnosing HIV infection.
Abbreviations: NACO, National AIDS Control Organization; ICTC, Integrated
Counselling and Testing Centre.
 CHAPTER 19 © Viral Infections of Bloodstream: HIV/AIDS and Dengue

“> The first screening test should be highly sensi- “ HIV RNA detection
tive, whereas the second and third screening ** p24 antigen detection
tests should have high specificity *: IgG ELISA only after 18 months of age.
“> The three screening tests should use different
principles or different antigens. The same kit Diagnosis of HIV in Window Period
should not be used again Window period refers to the initial time interval
“+ Supplemental or confirmatory tests should be between the exposure and appearance of
used only when the screening test(s) results detectable levels of antibodies in the serum.
are equivocal/intermediate. ** The antibodies appear in blood within 2-8
weeks after infection, but usually become
Prognosis/Monitoring of HIV detectable after 3 weeks to 12 weeks with the
Various tools available for monitoring the assays available presently. It can be as low
response to antiretroviral therapy include: as 22 days; when third generation antibody
“* CD4T cell count: Most commonly used detection kits with high sensitivity are used
“+ HIV RNA load: Most consistent and best tool ** p24 antigen detection (30% sensitive; by 4th
at present generation ELISA): It can be detected by 12-26
“+ p24 antigen detection days after infection
*» Neopterin and §2 macroglobulin level. ¢ HIV RNA detection (by RT-PCR) is the best
Note: Viral antibody levels are inconsistent method—it detects HIV RNA around 10-14
and variable during late stage due to immune days after infection.
collapse; hence not reliable for prognosis.
Treatment of HIV infection (ART
Diagnosis of Pediatric HIV Infection Guideline, NACO 2018)
to start ART: TREAT ALL; ie.,
The routine screening methods (ELISA or rapid/ Indication
simple tests) detect IgG antibodies. antiretroviral therapy (ART) has to be started in
* They cannot differentiate between baby’s IgG all patients irrespective of CD4 count, clinical
or maternally transferred IgG, hence cannot stage, age, population or associated opportun-
be used for the diagnosis of pediatric HIV istic infections (OIs).
“+ As all maternal antibodies would disappear “+ HAART: Highly active antiretroviral therapy
by 18 months; therefore IgG assays can be (HAART) is use of combination of at least three
performed after 18 months of birth. antiretroviral drugs to maximally suppress the
Various methods used for diagnosis of pediatric HIV and stop the progression of the disease
HIV include: “ TLE regimen: Tenofovir + Lamivudine +
“ HIV DNA PCR: This is the most recommended Efavirenz is used to treat HIV-1 infection in
method for diagnosis of pediatric HIV. Baby adults
* TL + LR regimen: Tenofovir-Lamivudine +
is tested for HIV DNA PCR at 6 weeks by DBS
(Dry Blood Spot) collection. If found positive, Lopinavir-Ritonavir is indicated for HIV-2
then is reconfirmed by a repeat HIV DNA PCR. infection, for HIV-1/2 co-infection and for
Then it is reported as positive and the baby is post-exposure prophylaxis for healthcare
then initiated on lifelong ART workers (Chapter 14).

§ DENGUE
Problem Solving Exercise 6 :

A 29-year-old female came to casualty with complaints over the cubital fossa demonstrated 25 petechial
of high-grade fever, severe joint pain, back pain and spots/square inch area. On inquiry, she told that she
myalgia for last 2 days. Gradually, she developed stays in area, where mosquitoes are prevalent. Her
petechial rashes over the body. On examination, she blood sample was sent for a dengue NS1 antigen
was found to have jaundice, hepatomegaly and a low enzyme-linked immunosorbent assay (ELISA). Test
platelet count (20,000/mm?). A tourniquet test done
result is displayed in Figure 19.8.
 )
SECTION 2 © Systemic Microbiology (Infectious Diseases

Interpret the test result.

What is your clinical diagnosis?
What is the pathogenesis of this disease?
eS What are the different modalities of laboratory
or
Se
diagnosis?
5. List the viruses that cause hemorrhagic fever.
Explanation
Clinical Diagnosis
The history is suggestive of dengue hemorrhagic
fever. Points in favor are:
Q High-grade fever, severe joint pain, back pain and
Fig. 19.8: ELISA for detection of NS1 antigen of Dengue.
myalgia
Q Petechial rashes all over the body with positive results in symptomatic bleeding (hemorrhage). VHFs
tourniquet test are caused by viruses of three distinct groups:
Q Low platelet count. 1. Arboviruses: Transmitted by arthropod vectors.
Confirmation Examples include dengue, yellow fever viruses
2. Filoviruses such as Ebola and Marburg viruses
Positive dengue NS1 ELISA along with other clues as
3. Rodent borne viruses such as Hantaviruses and
already described above the etiological agent can be
Arenaviruses.
identified as dengue virus.
(For answers to other questions, refer below.)
Viral hemorrhagic fevers (VHF)
VHF are as a group of illnesses caused by different
families of viruses that cause vascular damage that

Pathogenesis of Dengue Maculopapular rashes over the chest and

Primary dengue infection occurs when a upper limbs
person is infected with dengue virus for the first Severe frontal headache
time with any one serotype. Muscle and joint pains
Secondary dengue infection: It is more severe Lymphadenopathy
form of dengue illness; appear months to years Retro-orbital pain
later. It occurs due to infection with another Loss of appetite, nausea and vomiting.
second serotype which is different from the first 2. Dengue hemorrhagic fever (DHE): It is
serotype causing primary infection. characterized by:
* ADE: During secondary dengue infection, = High-grade continuous fever
there occurs an immunolgical phenomenon = Hepatomegaly
called antibody dependent enhancement = Thrombocytopenia (platelet count <1
(ADE) Lakh/mm‘)
* This in-turn leads to development of compli- = Raised hematocrit (packed cell volume)
cations, such as dengue hemorrhagic fever by 20%
(DHF) and dengue shock syndrome (DSS). m Evidence of hemorrhages which can be
detected by:
Clinical Classifications of Dengue ¢ Positive tourniquet test (>20 petechial
spots per square inch area in cubital fossa
The Traditional (1997) WHO Classification
¢ Spontaneous bleeding from skin, nose,
This classification divides dengue into three mouth and gums.
clinical stages: 3. Dengue shock syndrome (DSS): Here, all
1. Dengue fever (DF): It is characterized by: the above criteria of DHF are present, and in
m Abrupt onset of high fever (also called addition manifestations of shock are present,
biphasic fever, break bone fever or saddle such as:
back fever) m= Rapid and weak pulse
 CHAPTER 19 © Viral Infections of Bloodstream: HIV/AIDS and Dengue

= Narrow pulse pressure (<20 mm Hg) or “+ In secondary infection: IgG antibody titers
hypotension rise rapidly. IgG is often cross reactive with
m Presence of cold and.clammy skin many flaviviruses and may give false positive
= Restlessness. result after recent infection or vaccination
with yellow fever virus or JE. In contrast,
2009 WHO Classification IgM titer is significantly low and may be
This is the most recently described classification undetectable
by WHO which grades dengue into two stages * In past infection: Low levels of IgG remain
based on the severity of infection: detectable for over 60 years and in the absence
1. Dengue with/without warning signs, such as of symptoms, is a useful indicator of past
abdominal pain, persistent vomiting, mucosal infection
bleed, lethargy, liver enlargement, Increase * MAC-ELISA (IgM antibody capture ELISA):
in hematocrit and rapid decrease in platelet This is the recommended serological testing
count in India. Kits are supplied by NIV, Pune
2. Severe dengue: Criteria included are severe = Principle (Fig. 8.4B, Chapter 8): It is a
plasma leakage leading to shock and fluid double sandwich ELISA; which captures
accumulation with respiratory distress, severe human IgM antibodies on a microtiter
bleeding and severe organ involvement plate using anti-human-IgM antibody
(elevated liver enzymes, impaired conscious- followed by the addition of dengue virus
ness, etc.). four serotypes specific envelope protein
antigens (this step makes the test specific).
There is a signal enhancement due to use
Laboratory Diagnosis of Dengue
of avidin-biotin complex (ABC) which
The outline of laboratory diagnosis of dengue is makes the test more sensitive
mainly dependent on serological tests. m Cross-reactivity with other flaviviruses is a
limitation of this test.
NS1 Antigen Detection
ELISA and ICT formats are available for Molecular Method
detecting NS1 antigen in serum. They gained Detection of specific genes of viral RNA (3’-
recent popularity because of the early detection UTR region) by real time RT-PCR: It is the most
of the infection. sensitive (80-90%) and specific assay (95%),
* NS1 antigen becomes detectable from day 1 can be used for detection of serotypes and
of fever and remains positive up to 18 days quantification of viral load in blood (within -1
* Highly specific: It differentiates between to +5 days ofonset of symptoms).
flaviviruses. It can also be specific to different
dengue serotypes. Treatment
There is no specific antiviral therapy for dengue.
Antibody Detection Treatment is symptomatic and supportive such
* In primary infection: Antibody response is as:
slow and of low titer. IgM appears first after 5 ** Replacement of plasma losses
days of fever and disappears within 90 days. * Correction of electrolyte and metabolic dis-
IgG is detectable at low titer in 14-21 days of turbances
illness, and then it slowly increases “+ Platelet transfusion if needed.
 Parasitic Infections of Bloodstream:
Malaria, Visceral Leishmaniasis
and Lymphatic Filariasis

B MALARIA
Problem Solving Exercise 1
Plasmodium falciparum Explanation
A 36-year-old female from Odisha presented with This is a case of cerebral malaria due to Plasmodium
fever, chills and rigor for 5 days with anemia. The falciparum.
patient developed seizures prior to admission. She Clinical Diagnosis—Points in Favor
was started on ceftriaxone by a private medical
Q From Odisha (endemic for falciparum malaria)
practitioner and she did not improve. On physical
Q Presented with splenomegaly, anemia, and fever
examination, splenomegaly was present and signs of
with chills and rigor
meningeal irritation were absent. Her blood sample
Q Central nervous system involvement such as
was collected and sent to laboratory for peripheral
history of seizure and absence of meningeal
blood smear examination and for other laboratory
irritations-classical feature of cerebral malaria.
investigations (Figs 20.4A and B).
1. What is the etiological agent based on test Identification
performed? Peripheral thin blood smear examination: Figure 20.4A
2. What is the host, infective form, pathogenic form, shows red blood cells (RBCs) containing multiple ring
diagnostic form, habitat and mode oftransmission forms, accole forms and double dot ring forms and
of the parasite? Fig. 20.4B shows banana-shaped gametocyte; hence
3. What are the various complications seen? the diagnosis is cerebral malaria due to P. falciparum.
4, What are the various diagnostic modalities? For answers to the other questions, refer text below
5. How will you treat this clinical condition? and Table 20.1.

[Problem Solving
Solving Exercise
Exercise22 |
Plasmodium vivax Clinical Diagnosis
A 13-year-old boy from Mangaluru, presented with Q From Mangaluru (endemic for vivax malaria).
high grade fever rises every third day with chills and Q Presented with high grade fever rises every third
rigor. His serum sample was subjected to a rapid day with chills and rigor.
diagnostic test (RDT) (Fig. 20.1).
1. What is the etiological agent based on inter- Identification
pretation of the test? Rapid diagnostic test for malaria, works on immuno-
2. What is the principle of this test? chromatographic test.
3. How will you treat this condition? In Figure 20.1, bands present at panmalarial
- a (genus specific) region, but absent at P. falciparum
ay Oo Sia: i (Pf) region—indicates infection is caused by one of
2) = 0 the non-falciparum Plasmodium species. The most
= common non-falciparum Plasmodium species (in
India) is Plasmodium vivax.
Fig. 20.1: Rapid diagnostic test for malaria.
Treatment
Explanation For answers to the other questions, refer text below
This is a case of vivax malaria. and Table 20.1.
 CHAPTER 20 © Parasitic Infections of Bloodstream

Table 20.1: Features of malaria parasites. the paroxysm of fever and before taking
antimalarial drugs. Parasite density is
Features or characteristics of malaria parasites
maximum during this period
Host e Definitive host (vector): Female
“+ Frequency: Smears should be examined at
Anopheles mosquito
e Intermediate host: Man least twice daily until parasites are detected.
Infective form For human: Types of Peripheral Blood Smear
and modes of e Sporozoites when transmitted by
transmission mosquito bite It is of two types—(1) thin, and (2) thick smears.
e Trophozoites and merozoites when Both the smears are made at the same time from
transmitted by blood transfusion capillary blood either on the same or different
For mosquito—gametocytes slides (Fig. 20.2). At least two thick and two thin
Habitat in man Hepatocytes and red blood cells smears should be made.
(RBCs) ** For thick smear, a big drop of blood is spread
Manifestations e Paroxysms of fever with chills and over 1-2 cm square area on a clean glass slide.
(benign rigor The thickness of the film should be such that
malaria) e Anemia
e Splenomegaly
it allows newsprint to be read
ate> For thin smear, a small drop of blood is taken
Complications Seen in only Plasmodium falciparum
(due to sequestration of parasites in
on acorner of a slide. It is spread by another
(malignant
malaria) the blood vessels of deep viscera and spreader slide at an angle of 45° and then is
cytoadherence of infected RBCs to lowered to an angle of 30° and is pushed gently
endothelial cells) to the left, till the blood is exhausted
Cerebral malaria +,* The surface of a good thin film is: (i) even and
Blackwater fever
uniform, (ii) consists ofa single layer of RBCs,
Algid malaria (shock)
Pulmonary edema (iii) forms a “feathery tail end” near the center
Hypoglycemia of the slide, and (iv) margins of the film do not
Renal failure touch the sides of the slide
Treatment Vivax malaria—chloroquine ate> Stains: They are stained with one of the
Falciparum malaria—quinine and Romanowsky’s stains such as Leishman’s,
artemisinin Giemsa and Field’s, Wright’s or JSB (Jaswant
Singh and Bhattacharya) stain
Laboratory Diagnosis of Malaria ** Examination: Both the smears are examined.
+. o

The thin smear is screened near the feathery

The diagnostic tests for malaria can be divided
tail end. At least 200-300 oil immersion fields
into microscopic and nonmicroscopic tests.
should be examined before the smears are
considered as negative
Peripheral Blood Smear
Peripheral smear study still remains the simple
and gold standard confirmatory test for detection Blood films
of malarial parasites. (for microscopic analysis)

Specimen Thin film

Peripheral blood is the specimen of choice,
collected from earlobe or by finger prick in older Thick film

children and adults and from the great toe in

infants.
“ Blood films should be prepared directly from
the capillary blood. In case of anticoagulated
blood, smears should be made within an hour
of collection of blood
* Time for taking blood: Blood should be Fig. 20.2: Glass slide showing thin and thick blood
smears.
collected few hours after the height of
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

“» Advantages: Peripheral smear is simple, rapid of thin smear is >200 parasites per pL of
and cheap blood.
= Thick smear is useful in—(1) Detecting The speciation by thin smear is based on the
the parasites: It is 40 times more detection of the various parasitic morphological
sensitive than thin smear, can detect as forms. In falciparum malaria, only gametocytes
low as 5-10 parasites per pL of blood; and ring forms are demonstrated (but not
(2) Quantification of parasitemia; (3) schizonts and late trophozoites) (Table 20.2 and
Demonstrating the malaria pigments Figs 20.3 to 20.6).
= Thin smear is useful in speciation of
malaria parasites (Table 20.2).
Quantitative Buffy Coat Examination
“ Disadvantages: (1) It is labor intensive The quantitative buffy coat (QBC) is an advanced
and requires experienced microscopist; microscopic technique for malaria diagnosis. It
(2) Low sensitivity—the detection limit consists of three basic steps:

Table 20.2: Differences between the four malarial parasites (Figs 20.3 to 20.6).
Parasitic changes | Plasmodium Plasmodium Plasmodium Plasmodium ovale
vivax falciparum malariae
Forms seen in Trophozoites (early/ Ring forms (early Similar to that of Similar to that of P.
peripheral blood ring forms and late), trophozoites), P. vivax vivax
smear gametocytes, schizonts gametocytes
2. Ringforms (early Ring occupies 1/3rd of Rings aresmallerthan Similar to that Similar to that
trophozoites): the size of red blood in P. vivax occupying of P. vivax but of P. vivax, more
Vacuole in the cell (RBC), 1/6th of RBC (Fig. thicker compact
center, peripheral cytoplasm opposite to 20.4A). (Fig. 20.6B)
thin rim of blue the nucleus is thicker Variants of ring forms
cytoplasm, (Fig. 20.5A) are:
surrounding the e Multiple rings
red nucleus e Accole forms
¢ Double dot ring
forms
3. Latetrophozoites Large, amoeboid, Small, compact, Small, compact, Small, compact,
prominent vacuole rounded, slightly band forms rounded, coarse
amoeboid, vacuole seen, vacuole pigment, vacuole
inconspicuous, not inconspicuous inconspicuous
seen in smear (Fig. 20.6A)
4. — Schizont Large, 9-10 um, Small, 4.5—5 um size, Small, 6.5-7 um — Small, 6.2 um size,
completely fills the Fills 2/3rd of normal size, almost fills Fills 3/4th of
enlarged RBC (Fig. sized RBC a normal sized enlarged oval RBC
20.5B) RBC
5. Merozoites/ 12-24 no. 18-24 no. 6-12 no. 8-12 no.
schizonts
6. Gametocyte Spherical, almost Crescent/banana Similar to that of Similar to that of P.
occupies the RBC (Fig. shaped, larger than P. vivax vivax
20.5B) RBC size (Fig. 20.4B)

— RBC eae Young RBC RBC ofall age Old RBC Young RBC
2h, RBC size Enlarged, round Normal in size Normal in size Enlarged, oval,
fimbriated margin
3. — Stippling Schuffner’s dots Maurer’s cleft Ziemann’s dots James's dots
4. Malarial pigments Yellowish brown Dark brown Dark brown Dark yellowish
brown
Note: In falciparum malaria, only the gametocytes and ring forms are demonstrated in peripheral blood but
not schizonts and late
trophozoites (as the later stages of erythrocytic cycle occurs in deep vessels, not in peripheral blood).
 CHAPTER 20 © Parasitic Infections of Bloodstream

oe P. vivax | P. falciparum P. malariae | P. ovale

}— + 4
Early Accole form
trophozoite Double dot
ting form
Multiple
ring form
us
Late
trophozoite
Not seen in
peripheral blood
|
Me 4 Band form |
[Schizont oe ~

Not seen in
peripheral blood

|Gametocyte |

| eel
Fig. 20.3: Morphological forms of malaria parasites seen in the peripheral smear.

Ring form

aGametocyte
|
A ho Ey 2
Figs 20.4A and B: Thin smear showing Plasmodium Figs 20.5A and B:Thin smear showing Plasmodium vivax.
falciparum. (A) Accole form and double dot (headphone (A) Ring form; (B) Schizont and gametocyte.
shaped) ring forms; (B) Gametocyte (banana shaped). Source: DPDx Image Library, Centers for Disease Control and
and Prevention (CDC), Atlanta (with permission).
Source: DPDx Image Library, Centers for Disease Control
Prevention (CDC), Atlanta (with permission).

1. Blood (60 pL) is collected in a capillary tube

coated internally with acridine orange (Fig.
20.7A)
2. Capillary tube is centrifuged, which causes
separation of components of blood according
to their densities, forming discrete layers as
RBCs, WBCs, lymphocytes and platelets (Fig.
Figs 20.6A and B: Thin smear showing ring form of: (A)
20.7B) Plasmodium malariae (band form); (B) Plasmodium ovale.
3. Examination of capillary tube at the buffy coat Source: DPDx Image Library, Centers for Disease Control and
region under ultraviolet (UV) light source Prevention (CDC), Atlanta (with permission).

(Figs 20.7C and D).

RBCs do not take up the stain (as they are a
Interpretation nucleated). However, parasitized RBCs appear
Acridine orange has a property of staining the as brilliant green dots. WBCs also take up the
nuclear DNA fluorescent brilliant green. Normal stain (Figs 20.7C and D).
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

Platelets

Lymphocytes/
monocytes

Granulocytes

Red blood
cell layer

InFigs 20.7A to D: (A) QBC al tube;(B) Magnified view of QBC capillary tube after centrifugation;(C) Crescent-
shaped gametocyte of Plasmodium falciparum; (D) Ring forms of Plasmodium falciparum seen as Aneeae dots.
Source: (C and D) Department of Microbiology, Sri Siddhartha Medical College, Tumkur, Karnataka (with permission).

Advantages colloidal gold present in buffer and move

along with NCM to meet its corresponding
QBC is faster (the entire tube can be screened
antibodies at different test lines (Fig. 20.8A)
within minutes), more sensitive (at least as good
“+ Interpretation is based on immobilization
as a thick film), and quantification is possible.
of malarial antigens at test lines 1 and/or 2
Disadvantages forming colored bands (Figs 20.8A and B)
It is expensive, less specific and speciation is # If band is formed at only test line 1:
difficult. Indicates P. falciparum infection
= If band is formed at only test line 2:
Antigen Detection (Rapid Diagnostic Tests) Indicates Plasmodium species other than
Rapid diagnostic tests (RDTs) have revolution- P. falciparum infection
ized the diagnosis of malaria.
* Antigens: Several malarial antigens can be = Buffer
detected: © vs solution Various types of results seen in ICT
a § added
= pLDH (Parasite lactate dehydrogenase): Drop of blood
is added oy oe | |
It is produced by all Plasmodium species.
Sample
Currently available test kits can differenti- window |
ate pan-LDH common to all species and Antifalciparum
Pf-LDH specific to P. falciparum antibody > 11 T1
m Parasite aldolase: Produced by all four Antimalarial
antibody > T2
Plasmodium species (all species)
Control > oy
® Histidine rich protein-2 (HRP-II): It is antibody
produced only by P. falciparum. Nitro-
* Principle: Test kits currently available use a cellulose —_|—
membrane
nitrocellulose membrane (NCM) strip with
two parasite detection lines and a control line
Absorbent pad
as | z |
Components of |Nesetve Non- —P. falciparum Invalid
(Fig. 20.8A): rapid malaria test falciparum __ infection
malaria alone or mixed
m Test line-1: Coated with capture antibodies Al with other
specific for P.falciparum (e.g., HRP-II or species

Pf-LDH)
= ‘Test line-2: Coated with capture antibod-
ies common to all Plasmodium spp. (e.g.,
pan-LDH or aldolase). Figs 20.8A and B: (A) Schematic diagram of rapid
* Procedure: Drop of blood specimen is diagnostic test kit showing negative, non-falciparum, pure
or mixed infection with Plasmodium falciparum and invalid
added along with a buffer solution to sample
result of malaria; (B) Real image of rapid diagnostic test kit.
window. Malarial antigens combine with
Source: (B) Department of Microbiology, Sri Siddhartha Medical
polyclonal malarial antibody labeled with College, Tumakuru, Karnataka (with permission).
 CHAPTER 20 © Parasitic Infections of Bloodstream

s Ifbands are formed at both test lines 1 and “+ Gametocytes cannot be detected
2: Indicates infection with P. falciparum or “ Low sensitivity: The lower limit to detect
mixed infection. * HRP-II is 40 parasites/uL and pLDH is 100
Note: The band at control line must come to parasites/uL
validate the test; if it does not come, test is “+ RDT has not been developed for P. knowlesi yet.
considered invalid. Control line is coated with Comparison of peripheral smear, QBC and
antibody against polyclonal malarial antibody RDTs are described in Table 20.3.
present in buffer.
Antibody Detection
Advantages of Rapid Diagnostic Tests Antibodies persist even after the clinical cure.
Rapid diagnostic tests are simple to perform, Serology does not detect current infection,
do not need extra equipment or trained micro- but only measures past exposure. Therefore,
scopist. Government of India has banned the use of
“+ Sensitivity: Rapid diagnostic tests are more antibody detection tests for malaria diagnosis.
than 90% sensitive at >100 parasites/L. But
the sensitivity is markedly reduced at <100 Other Nonspecific Tests
parasites/pL Other nonspecific tests include normocytic
* Prognosis: pLDH is produced by the viable hemolytic anemia, leukopenia, metabolic
parasites, hence it is used to monitor the acidosis, raised ESR, and hypoglycemia.
response for treatment (microscopy is the best
to assess prognosis) Treatment
* Pregnancy: HRP-II is a reliable marker to For vivax malaria: The recommended regimen
diagnose malaria in pregnancy is chloroquine 25 mg/kg (divided over three
* Severity: Intensity of the band is directly days) and primaquine 0.25 mg/kg body weight
proportional to the parasitemia and severity
(daily for 14 days; to prevent relapse)
of the disease.
For falciparum malaria:
Disadvantages of Rapid Diagnostic Tests “ North-Eastern states: ACT-AL regimen—
RDTs have several disadvantages. artemisinin combination therapy-artemether-
~ It cannot differentiate between the non- lumefantrine for 3 days plus Primaquine
falciparum malaria species single dose on second day (to kill gametocytes
“+ Expensive than peripheral smear of P. falciparum)

tests.
Table 20.3: Comparison of peripheral smear, quantitative buffy coat and rapid diagnostic
Peripheral smear Quantitative buffy coat Rapid diagnostic tests <
|Features
Method Cumbersome Easy Easy

Time Longer, 60-120 minutes Faster, 15-30 minutes Faster, 15-30 minutes

Sensitivity Detection limit: Claimed to be more e >100 parasites/uUL, sensitivity >90%

e 5 parasites/pL in thick film sensitive, at least as good as e <100 parasites/uL, sensitivity falls
e 200 parasites/pL in thin film a thick film
False positives—artifacts False positive in RA factor (rheumatoid
Specificity Gold standard
may be reported as positive arthritis) positive cases
by nontrained technicians
Difficult Detect Plasmodium falciparum but
Speciation Accurate, gold standard
cannot differentiate non-falciparum
species
Costly equipment and Kits are costly but no extra equipment
Cost Inexpensive
consumables required. Good for field study
Not required, minimal Not required, minimal training is
Experienced Required
training is sufficient sufficient
Microscopist
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

= Other states: ACT-SP regimen—artesu- amine given on first day plus Primaquine
nate for 3 days plus sulfadoxine/pyrimeth- single dose on second day.

VISCERAL LEISHMANIASIS
ee
GanoSolving Exercise 3

A 31-year-old man from Bihar presented with spleno- (For answer to the other questions, refer Tables 20.4
megaly, anemia and fever. The bone marrow aspirate and 20.5).
collected was sent for Giemsa staining (Fig. 20.9B). ——

1. Identify the etiological agent and the clinical

diagnosis based on the smear focused.
2. What is the host, infective form, pathogenic form,
diagnostic form, habitat and mode of transmission
of the parasite?
3. What are the various diagnostic modalities?
4, How will you treat this condition?
Explanation
Figs 20.9A and B: L. donovani amastigotes: showing a
This is a case of visceral leishmaniasis. Points in favor
macrophage containing multiple Leishmania amastigotes:
are:
(A) Schematic; (B) In bone marrow smear stained with
Q Residence from Bihar
Giemsa. Note that each amastigote has a nucleus (red
Q Presented with splenomegaly, anemia and fever arrow) and a rod-shaped kinetoplast (black arrow).
Q Figure 20.9 B shows Leishman Donovan bodies (LD
Source: (B) DPDx Image Library, Centers for Disease Control and
bodies, i.e., amastigotes filled within a macrophage) Prevention (CDC), Atlanta (with permission).
Q Treatment: Amphotericin B.

Table 20.4: Features/characteristics of Leishmania donovani.

Ca
Host Man
Vector: Sandfly (Phlebotomus)
Morphological forms e Amastigotein human
¢ Promastigotein sandfly
Infective form Promastigote
Transmission Bite of sand fly
Habitat Reticuloendothelial cells of spleen, bone marrow, lymph node, liver and peripheral
blood
Pathogenic form Amastigote form
Major manifestations of kala- Due to Leishman Donovan bodies deposit in various organs:
azar ¢ Splenomegaly: most consistent sign
¢ Bone marrow involvement leads to pancytopenia and hypergammaglobulinemia
e Lymphadenopathy (African cases)
¢ Hyperpigmentation (on face, hands, feet, and abdomen) and fever are the
common presentations in Indian cases; hence the name kala-azar or black fever
Diagnostic form Amastigote forms reside inside the macrophages, called as LD (Leishman Donovan)
(Figs 20.9A and B) body
Treatment Pentavalent antimonials—drug of choice
Liposomal amphotericin B is the first-line drug in Bihar (due to resistance to
antimonials)
 CHAPTER 20 © Parasitic Infections of Bloodstream 169

Table 20.5: Laboratory diagnosis of Leishmania donovani.

Specimen e Splenic aspirate (most sensitive)

e Bone marrow aspirate (most common)
e Lymph node aspirate (in African cases)
e Peripheral blood (in HIV-infected patients)

Direct microscopy Leishman or Giemsa stain reveals LD bodies (macrophages filled with amastigote forms of
3-5 um) (Fig. 20.9A and B).

Culture ~ e NNN (McNeal, Novy, Nicolle) media or Schneider's Drosophila media are used
e Amastigotes transform into promastigote forms which are detected in culture fluid
microscopy by staining with Giemsa stain (Figs 20.10A and B).

Antibody detection Hypergammaglobulinemia detection by:

(nonspecific) e Napier’s aldehyde test
e Chopra's antimony test

Antibody detection Uses Leishmania specific antigen:

(specific) e ELISA
e |CT detecting antibody to recombinant kinesin antigen (rk-39). Recently, ICT based on
another novel antigen rKE16 (from L. donovani) has been developed
e Direct agglutination test (DAT)—100% sensitive and specific
PCR Detecting kinetoplast DNA

Montenegro skin test e |tis hypersensitivity type IV reaction, characterized by induration following injection of
L. donovani killed antigen
e {tis positive when CMI is good, ie., positive in all stages of leishmaniasis; except active VL and
diffuse CL

Animal inoculation Chinese and golden hamsters

immunochromatographic test; CMI, cell-mediated
Abbreviations: LD, Leishman Donovan; ELISA, enzyme-linked immunosorbent assay; ICT,
immunity; VL, visceral leishmaniasis; CL, cutaneous leishmaniasis; PCR: polymerase chain reaction.

|
|
}
}
|

|
|
}
|
}
|

Fine | it
||j
|
| |

eee
|
||
pe |

promastigote forms (Giemsa stain); (B) NNN medium.

Figs 20.10A and B: (A) Smear made from culture fluid shows
Health
and Prevention (CDC), Atlanta (with permission); (B) World
Source: (A) DPDx Image Library, Centers for Disease Control
(Slide22/Alvar) (with permission).
Organization, “Manual on visceral leishmaniasis control”
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

LYMPHATIC FILARIASIS
Exercise4
Solving Exercise
[Problem Solving 4

A 25-year-old male from a village in Puducherry came Explanation

to this hospital with history of fever on and offforthe — {t is a case of lymphatic filariasis due to Wuchereria
past 1 year and recent unilateral swelling of the left ggncrofti. Points in favor are:
lower limb. His blood sample was collected, and sent = Fever with unilateral lower limb swelling
for peripheral blood smear examination (Fig. 20.11A). Q Peripheral blood smear examination revealed
1, Identify the parasite. microfilaria, 240 um in length, tail tip pointed free
2. Draw a neat, labeled diagram of the etiological of nuclei (Fig. 20.11A).
agent focused. Q Neat labeled diagram of microfilaria is shown in
3. Which is the infective stage of the parasite for Figure 20.12.
man? Q Microfilaria of W. bancrofti is often confused
4. How does man acquire this infection and what is with that of Brugiya malayi (Fig. 20.11B); the
the vector involved? differentiating features of both are depicted in
What are the different modalities of diagnosis of (Fig. 20.13).
this clinical condition? For answer to other questions, refer Table 20.6.

Figs 20.11A and B: Peripheral blood smear showing microfilaria of: (A) Wuchereria bancrofti: (B) Brugiya malayi.
Source: (A) |ID# 3009/; (B) ID# 3003; Dr Mae Melvin, Public Health Image Library, Centers for Disease Control and Prevention (CDC),
Atlanta (with permission).

Sheath Microfilaria Head end | Tail end | Features

Gantt! Cephalic Sheath :

cells 2,3,4 Space +—Cephalic space (1:1)
Wuch 3] i\\g A: 240-300 mm
‘uchereria $4)- Coarse nuclei \+— No nuclei in the tail tip B: Nocturnal
Nerve ring bancrofti 133] well-separated = F C: Sheathed
res f Pointed tail tip
co io D: Blood |
==\- No nuclei in T T
the tail tip i a Sheath +
{ 7—Cephalic space (2:1) 2
Excretory f n A: 220 mm
pore Brugia ¥ Darkly stained large | Four to five nuclei B: Nocturnal
malayi z Renee overlapping y VY in the tail region C: Sheathed
3 nuclei [> Two widely spaced D: Blood
nuclei in tail tip
ae = aad
Fig. 20.12: Microfllaria of Fig. 20.13: Differences between microfilaria of Wuchereria bancrofti and
Wuchereria bancrofti. Brugiya malayi.
Key: *A: Size, B: Periodicity, C: Sheath, D: Habitat,
 CHAPTER 20 © Parasitic Infections of Bloodstream 171

Table 20.6: Characteristics of lymphatic filarial worms.

Characteristics
Host * e Definitive—humans
e Intermediate—mosquito, for example:
> Wuchereria: Culex fatigans, Aedes and Anopheles
> Brugiya: Mansonella and Anopheles
Infective form Filariform (L3) larva
Transmission Mosquito bite
Habitat Lymphatics
Pathogenic form Adult worms in lymphatics
Manifestations of lymphatic Acute filariasis (acute adenolymphangitis)
filariasis e Fever
e Transient local edema
e Dermatolymphangitis: Plaque-like lesion
Chronic filariasis: lt develops 10-15 years after infection.
e Hydrocele
e Elephantiasis (swelling of lower limb or less commonly arm, vulva or breast)
e Chyluria—excretion of chyle in urine
Occult filariasis e This is a hypersensitivity reaction to microfilarial antigens mainly affecting
lungs causing tropical pulmonary eosinophilia.
e Microfilaria will be absent from peripheral blood
Diagnostic form (Fig. 20.11) Demonstration of microfilariae by thin or thick smear stained with Giemsa or
by QBC examination—blood collected during night hours (or day time after DEC
provocation test)
Detects microfilaria; based on which Wuchereria and Brugia can be differentiated
Other methods of laboratory e Antigen detection (ELISA, ICT)—detects Ag by using monoclonal antibodies
diagnosis to Og4C3 Ag and AD12 Ag
e Antibody detection—IFA, ELISA
ELISA detecting antibody to AD12 and Og4C3 antigen
e Imaging methods—USG, X-ray
e Molecular methods—real-time PCR detecting genes such as Ssp/ repeat, pWb12
repeat, pWb-35 etc.
e Eosinophilia and elevated IgE
Treatment In India: DEC + albendazole
African cases: DEC + Ivermectin
ICT, inmunochromatographic test; USG,
Abbreviations: ELISA, enzyme-linked immunosorbent assay; IFA, immunofluorescent antibody;
ultrasonography; PCR, polymerase chain reaction; DEC, diethylcarbam azine
 Fungal Infections of Bloodstream:
Systemic Candidiasis and
Systemic Mycoses Za
B SYSTEMIC CANDIDIASIS

[Problem
Problem Solving
Solving Exercise
Exercise _|
A 29-year-old HIV-infected male presents to the clinic d. Describe the laboratory diagnosis of this clinical
with history of high-grade fever and altered mental condition in detail.
status. On examination, his blood pressure was found
Explanation:
as 90/60 mm of Hg, and respiratory rate was increased
It is a case of sepsis due to systemic candidiasis
to 28 per minute. Blood cultures were collected in
Q Low blood pressure, increased respiratory rate
BacT/ALERT bottles, which flagged positive after 22
hours of incubation. Gram stain of blood culture broth
and altered mental status is suggestive of clinical
is shown in the Figure 21.1A. diagnosis of sepsis
a. What is the clinical diagnosis and the likely Q Blood culture broth smear (Fig. 21.1A) reveals
etiological agent? gram-positive oval budding yeast cells with
b. Name the risk factors predisposing this clinical pseudohyphae—suggestive of Candida species.
condition. The explanation for the remaining questions have
c. What are the other clinical manifestations caused been discussed subsequently in the text.
by this organism?

Introduction ** Lowimmunity: Patients on steroid or immu-

Candidiasis is the most common fungal disease nosuppressive drugs, post-transplantation,
in humans, affecting the skin, mucosa, and malignancy, HIV-infected people
various internal organs; caused by Candida, a * Patients on broad spectrum antibiotics—
yeast-like fungus that produces pseudohyphae. suppress the normal flora
Pseudohypha is an important virulence factor, ** Others: Diabetes mellitus, febrile neutropenia
helps in invasion. Various species of Candida and zinc or iron deficiency.
include:
Clinical Manifestations
* Candida albicans: It is the most pathogenic
species of Candida infecting humans Candida species produce a spectrum of
** Other Candida species which can also cause infections ranging from skin and mucosal
infection are C. tropicalis (most common infection to invasive and allergic infections.
species), C. glabrata, C. krusei, C. parapsilosis, ** Invasive candidiasis: It results from hemato-
C. dubliniensis, C. kefyr, C. guilliermondii, C. genous or local spread of the fungi. Various
viswanathii and C. auris. forms are:
m Urinary tract infection
Predisposing Factors = Pulmonary candidiasis
Predisposing factors that are associated with = Septicemia (mainly by C. albicans and C.
increased risk of infection with Candida include: glabrata)
* Physiological state: Extremes of age (infancy, m Arthritis and osteomyelitis
old age), pregnancy Meningitis
 CHAPTER 21 © Fungal Infections of Bloodstream: Systemic Candidiasis and Systemic Mycoses 173

=
Ocular—keratoconjunctivitis and endo- culture media, such as blood agar. Blood for
phthalmitis culture can be inoculated into blood culture
= Hepatosplenic candidiasis bottles (conventional or automated blood
= Disseminated candidiasis culture bottles, such as BacT/ALERT).
= Nosocomial candidiasis (mainly by C. “+ Colonies appear in 1-2 days and described as
glabrata). creamy white, smooth, and pasty with typical
** Mucosal candidiasis: The various mucosal yeasty odor (Fig. 21.1B)
manifestations include oropharyngeal * Gram staining of the colonies shows
candidiasis (oral thrush), vulvovaginitis, etc. gram-positive budding yeast cells with
Cutaneous candidiasis: The cutaneous mani-
2%,
“~ pseudohyphae except for C. glabrata which
festations seen in candidiasis are intertrigo does not show pseudohyphae.
(pustules in the skin folds) and nail infections
such as paronychia and onychomycosis Tests for Species Identification
* Allergic candidiasis, such as candidid “+ Germ tube test: It is a specific test for C.
reaction albicans; also called Reynolds Braude
* Candida auris has recently emerged as a phenomenon
potentially multi-drug resistant species of = Colonies are mixed with human or
Candida known to cause severe disease in sheep serum and incubated for 2 hours.
healthcare setting. Wet mount preparation is examined under
microscope
Laboratory Diagnosis = Germ tubes are formed, described as long
Specimen Collection tube-like projections extending from the
yeast cells
Depending on the site of infection, various spec- = Itis differentiated from pseudohyphae as
imens can be collected, such as urine or blood. there is no constriction at the origin (Fig.
21.1D, Table 21.1)
Direct Microscopy
Gram staining reveals gram-positive oval Table 21.1: Differences between pseudohyphae and
budding yeast cells (4-6 pm size) with pseudo- true hyphae.
hyphae (Fig. 21.1A). It has to be differentiated
|Features | Pseudohyphae True hyphae
from true hyphae (Table 21.1).
Morphology Elongated chains of Elongated
Culture budding yeast cells branching
filaments
Specimens can be inoculated onto SDA with Budding Apical elongation
Grows by
antibiotic supplements and then incubated at No constriction
Septa Constricted
37°C. Candida can also grow in bacteriological

9
° ed
% — ¢ &in 9, %&
jae 7,0aSa
‘ 8 { * >
0

bf oshime Se ING
- 7a fee.8 ; &%
thes bi ibaa, é
aye Ms mss. ee
D paatiity ain a
cells with pseudohyphae; (B) Candida albicans
Figs 21.1A to E: (A) Candida albicans—gram-positive oval budding yeast
of various Candida species producing different
on SDA shows creamy white colonies; (C) CHROMagar showing colonies
( Candida albicans shows positive germ tube test (arrow
colors (e.g., light-green color by C. albicans, red arrow); D)
spores (arrow showing).
showing); , (E) Candida albicans shows thick walled chlamydo
of Medical Sciences, Puducherry; (E) ID#:2917/Centers for Disease
Source: (A to D) Department of Microbiology, Pondicherry Institute
Control and Prevention (CDC), Atlanta (with permission ).
174 SECTION 2 © Systemic Microbiology (Infectious Diseases)

e Though the test is specific for C. albicans, Histoplasmosis

it may also be positive for C. dubliniensis. Histoplasmosis is a systemic granulomatous dis-
* Dalmau plate culture: Culture on cornmeal ease caused by dimorphic fungus, Histoplasma
agar can provide clue for species identifi- capsulatum.
cation. C. albicans produces thick walled
chlamydospores (Fig. 21.1E) Pathogenesis
* CHROMagar: Different Candida species H. capsulatum is transmitted by inhalation of
produce different colored colonies on spores (i.e., microconidia) which usually circu-
CHROMagar (Fig. 21.1C) late in the air after contaminated soil is disturbed.
“ Automated systems, such as MALDI-TOF and
VITEK can also be used for speciation Clinical Manifestations
“ Molecular methods, such as PCR using
Clinically, the classical histoplasmosis ranges
species specific primers are useful for species
from asymptomatic infection (in immuno-
identification.
competent people) to life-threatening illness
Immunodiagnosis seen in people with low case mix index. The
various clinical types include:
“+ Antibody detection: Various formats, such as
“ Pulmonary: It is the most common form
ELISA, latex agglutination tests are available
** Mucocutaneous: Skin and oral mucosal
detecting serum antibodies against cell wall
lesions seen
mannan antigen
** Disseminated form: most commonly invol-
« Antigen detection: Candida specific antigen,
ving the bone marrow, spleen, liver, eyes and
such as cell wall mannan and cytoplasmic
adrenal glands, in immunocompromised host.
antigens can be detected by ELISA
** B-d-Glucan assay: It is a marker of invasive Laboratory Diagnosis
fungal infections, (including invasive candi-
Histoplasmosis can be diagnosed by:
diasis) and can also be used for monitoring
response to treatment. It can be detected in ** Specimens helpful for establishing the
blood by colorimetric enzyme immunoassay. diagnosis include sputum, aspirate from bone
marrow and lymph node, blood and biopsies
Treatment from skin and mucosa
* The antifungal drugs recommended for sys-
* Direct microscopy: Histopathological
staining (such as periodic acid-Schiff,
temic candidiasis are liposomal amphotericin
Giemsa or Gomori methenamine silver) ofthe
B or caspofungin or voriconazole/fluconazole
* C. glabrata and C. krusei exhibit resistance specimens reveal tiny oval yeast cells (2-4 um
size) with narrow based budding within the
to azoles and are refractory to treatment with
azoles. macrophages and underlying granulomatous
response (Fig. 21.2A)
* Culture: Histoplasma is a dimorphic fungus,
HSYSTEMIC MYCOSES
hence:
There are four agents that cause deep or m At 25°C: It produces mycelial colonies
systemic mycoses. These fungi are also thermally which on LPCB mount appears as tubercu-
dimorphic. late macroconidia (thick walls and finger-
* Histoplasma capsulatum causes histoplasmo- like projections) and microconidia (small-
sis or Darling’s disease er, thin and smooth-walled) (Fig. 21.2B)
* Blastomyces dermatitidis causes blastomyco- m At 37°C: It produces yeast form (creamy-
sis ' white colonies).
** Coccidioides immitis causes coccidioido-
mycosis Treatment
* Paracoccidioides brasiliensis causes paraco- Liposomal amphotericin B is the antifungal of
ccidioidomycosis. choice in acute pulmonary and disseminated
 CHAPTER 21 © Fungal Infections of Bloodstream: Systemic Candidiasis and Systemic Mycoses 175

Figs 21.2A to C: (A) H. capsulatum [yeast cells with narrow-based budding (Giemsa stain)]; (B) Mold form of
H. capsulatum, septate thin hyphae with tuberculate macroconidia (arrows showing); (C) Blastomyces [histopathological
stain- shows broad-based budding yeast cells (figure of 8appearance)].
Source: (A) Dr Lucille K. Georg/ID#:15365; (B) Dr Libero Ajello/ID#:15364; (C) ID#:493/Centers for Disease Control and Prevention (CDC),
Atlanta (with permission).

histoplasmosis. Itraconazole is recommended shaped arthrospores with alternate cells

for chronic cavitary pulmonary histoplasmosis. distorted (empty cells) (Fig. 21.3B).

Blastomycosis Paracoccidioidomycosis
This disease is caused by Blastomyces This disease is caused by Paracoccidioides
dermatitidis; endemic in North America. brasiliensis; endemic in South America.
“* Clinical manifestations: Acute pulmonary “ Clinical manifestations: It occurs in two forms
form is the most common. Extrapulmonary = Acute form (or juvenile type): It is less
manifestations include verrucose skin lesions, common but more severe form, affects
osteomyelitis and rarely CNS involvement in young adults; manifests as disseminated
AIDS patients infection involving multiple viscera and is
* Itis diagnosed by histopathological staining refractory to treatment
of the tissue biopsy specimens which reveals = Chronic form (or adult form): It is more
thick-walled round yeast cells of 8-15 um size common, less severe form, affects older
with single broad-based budding (figure of 8 men, manifests as progressive pulmonary
appearance) (Fig. 21.2C). disease.
* Laboratory diagnosis: Histopathological
Coccidioidomycosis staining of pus, tissue biopsies or sputum
reveals round thick-walled yeasts, with
Also called as desert rheumatism or Valley fever;
multiple narrow-necked buds attached
caused by Coccidioides immitis.
“+ Clinical manifestations: Most patients are circumferentially giving rise to Mickey Mouse
or pilot wheel appearance (Fig. 21.3C).
asymptomatic (60%). In remainders, pulmo-
nary form is the most common followed by
skin lesions and arthritis. Disseminated form
occurs in AIDS patients with low CMI
“+ Laboratory diagnosis:
= Histopathological staining of sputum or
tissue biopsy specimens demonstrates ce
spherules. Spherules are large sac, such feet Se

as structures (20-80 tum size), have thick, Figs 21.3A to C: Coccidioides (A) Spherule (PAS staining);
with arthroconidia (LPCB mount)]; (©)
doubly refractive wall, and are filled with (B) Hyphae
Paracoccidioidomycosis [Methenamine silver staining
endospores (Fig. 21.3A)
shows yeast from (pilot wheel appearance).
= Cultures on SDA produces mycelial Source: Public Health Image Library/(A) ID#:14499; (B) ID#:12196;
growth which on LPCB mount reveals (C) Dr Lucille K Georg/ID#:527/Centers for Disease Control and
fragmented hyphae consisting of barrel- Prevention (CDC) Atlanta (with permission).
 Bacterial Diarrhea:
Shigellosis, Cholera and Others 22]
LER

¢ Enteropathogenic E. coli
B INTRODUCTION
¢ Enterotoxigenic E. coli
The diarrheal diseases are one of the leading ¢ Enteroaggregative E. coli.
cause of illness globally; cause significant Clostridium perfringens
morbidity and mortality. Diarrhea and dysentery Bacillus cereus
are the two important clinical types: Staphylococcus aureus
1. Diarrhea is defined as passage of three or Aeromonas hydrophila
more loose or liquid stools per day, in excess Plesiomonas shigelloides.
than the usual habit for that person. It may be 9,* Predominantly inflammatory diarrhea:
watery type of diarrhea (non-inflammatory) Non-typhoidal salmonellae
or stool mixed with pus (inflammatory type) Yersinia enterocolitica
2. Dysentery is characterized by diarrhea with Listeria monocytogenes
increased blood and mucus, often associated Clostridioides difficile
with fever, abdominal pain, and tenesmus Plesiomonas shigelloides.
(feeling of constant need to pass stools, +,oo
redominantly dysentery:
despite an empty colon). Shigella species
Campylobacter jejuni
Bacterial Etiology Be
Hee
whe
BeeDiarrheagenic E. coli
Bacterial agents account for a significant ¢ Enterohemorrhagic E. coli
proportion of diarrheal diseases. ¢ Enteroinvasive E. coli.
* Bacterial agents causing diarrhea: Mostly = Vibrio parahaemolyticus.
enterotoxin mediated Important bacterial pathogens of GIT are
w Vibric cholerae discussed here. The viral and parasitic agents
a Diarrheagenic Escherichia coli are discussed in subsequent chapters.

BSHIGELLOSIS
| Problem Solving
Solving Exercise)
Exercise 1
A6-year-old child presented with tenesmus, abdominal Q How will you treat this condition?
pain and passage of blood-tinged stool 10 times for
Explanation
one day. Stool specimen was subjected to culture
(Fig. 22.1), Gram staining of culture smear (Fig. 22.2), Clinical Diagnosis
biochemical reactions (Fig. 22.3) and agglutination The history of passage of blood-tinged stool,
test with specific antisera (Fig. 22.4). The antimicrobial tenesmus and abdominal pain is suggestive of
susceptibility test (AST) is demonstrated in Figure 22.5 dysentery.
with zone interpretation chart in Table 22.1. The common agents of dysentery include Shigella,
Q What is the clinical diagnosis and its causative enteroinvasive Escherichia coli, enterohemorrhagic
organism? E. coli, Campylobacter jejuni, Vibrio parahemolyticus
Q What are the various modalities of laboratory and parasites, such as Entamoeba histolytica and
diagnosis? Balantidium coli.
 CHAPTER 22 © Bacterial Diarrhea: Shigellosis, Cholera and Others

Identification Antimicrobial Susceptibility Test and Treatment

Based on the culture on MacConkey agar (Fig. Antimicrobial susceptibility test on Mueller-Hinton
22.1A), Gram staining of culture smear (Fig. 22.2), agar (Fig. 22.5) with zone interpretation chart
biochemical reactions (Fig. 22.3), and positive (Table 22.1) shows that the pathogen is sensitive to
agglutination with Shigella polyvalent antisera (Fig. ciprofloxacin, ampicillin and co-trimoxazole. As the
22.4), the causative organism can be identified as patient is 6-year-old boy, preferred treatment option
Shigella. would be ampicillin or co-trimoxazole.
(For answers to other questions, refer text below).

Shigella is one of the important agent of bacillary specimens should be transported in a suitable
dysentery; characterized by frequent passage medium, such as Sach’s buffered glycerol saline
of bloody mucopurulent stools with increased “+ Wet mount preparation of feces shows large
tenesmus and abdominal cramps. There are four number of pus cells, red blood cells and
species or serogroups which are further divided macrophages.
into serotypes:
1. Serogroup A: S. dysenteriae (15 serotypes) Culture
2. Serogroup B: S. flexneri (8 serotypes) To inhibit the commensals, fecal specimen is
3. Serogroup C: S. boydii (19 serotypes) inoculated simultaneously onto enrichment
4. Serogroup D: S. sonnei (one serotype). broth and selective media.
¢« Enrichment broth, such as Selenite F broth,
Pathogenesis tetrathionate broth and Gram-negative broth
* Mode of transmission: Infection occurs by are used. Turbidity indicating growth appears
ingestion through contaminated fingers (most in 18-24 hours, from which again subcultures
common), food, and water or rarely flies are made onto selective media
“+ Infective dose: As low as 10-100 bacilli are “+ Selective media, such as:
capable of initiating the disease = Mildly selective media: On MacConkey agar,
** Invasion: Bacilli enter the mucosa via M cells Shigella produces translucent non-lactose
(spread cell to cell) and are then engulfed fermenting (NLF) colonies (Fig. 22.1A)
by macrophages and induce recruitment of = Highly selective media: They contain of bile
inflammatory cells releasing cytokines, which salts as inhibitory agent
cause acute colitis—the hallmark of shigellosis ¢ Deoxycholate citrate agar (DCA): It
“ Exotoxin production: Shigella produce produces translucent NLF colonies
exotoxins, such as enterotoxins and shiga (Fig. 22.1B)
toxin (causes local vascular damage of ¢ Xylose lysine deoxycholate agar (XLD):
intestine, kidney and brain). Colonies of Shigella appear red without
black center.
Clinical Manifestations
¢ Incubation period: It usually lasts for 1-4 days
* Dysentery: Frequent passage of bloody
mucopurulent stools with increased tenesmus
and abdominal cramps
* Complications: Theses are toxic megacolon,
perforations and rectal prolapse.

Laboratory Diagnosis A 4 B
Figs 22.1A and B: Shigella producing translucent non-
“» Specimen collection: Fresh stool sample is lactose fermenters colonies on (A) MacConkey agar; (B)
collected. Rectal swabs are not satisfactory Deoxycholate citrate agar.
* Transport media: Specimens should be Source: Department of Microbiology, Pondicherry Institute of
transported immediately. If delay is inevitable, Medical Sciences, Puducherry (with permission).
 SECTION2 © Systemic Microbiology (Infectious Diseases)

§ CHOLERA
2
Exerceise2_
Solving Exercis
[Problem Solving —_ .

A 8-year-old boy developed severe watery diarrhea Identification

(10-12 times) and vomiting for 2 days. Stool collected Q Based on the curved (comma shaped) gram-
has a rice water type of appearance. The specimen was negative bacilli (Fig. 22.6), yellow-colored colonies
subjected to Gram staining (Fig. 22.6), culture (Fig. 22.7B) on thiosulfate-citrate-bile salt-sucrose agar (TCBS
and biochemical reactions (Fig. 22.8), agglutination with agar; Fig. 22.7B), and biochemical reactions (Fig.
antisera (Fig. 22.11). The antimicrobial susceptibility 22.8), the identification is Vibrio cholerae
test (AST) is demonstrated in Figure 22.12 with zone of Q Based on agglutination with specific antisera—the
interpretation chart in Table 22.3. serotype is identified as “ogawa’ (Fig. 22.11).
Q What is the clinical diagnosis and its causative
Antimicrobial Susceptibility Test
organism?
Q List the virulence factors and its mechanism of Antimicrobial susceptibility test on Mueller-Hinton
action. agar (Fig. 22.12) with zone of interpretation (Table
OQ What are the various modalities of laboratory 22.3) shows that the isolate is sensitive to tetracycline,
diagnosis? ampicillin, chloramphenicol and resistant to
Q How will you treat this condition? ciprofloxacin.
(For answers to other questions, refer text below).
Explanation
Clinical Diagnosis
The history of severe watery diarrhea (rice water stool)
and vomiting is suggestive of cholera.

Clinical Manifestations (Cholera) Laboratory Diagnosis

Vibrio cholerae is the causative agent of cholera Specimens
characterized by painless watery diarrhea.
“+ Freshly collected watery stool is the specimen of
** The stool is watery (rice water in appearance) choice; collected before starting the antibiotics
with mucus flakes without blood or pus cells ** Rectal swab is preferred for carriers.
with a fishy, nonoffensive odor. Vomiting and
muscle cramps are present but fever is usually Transport/Holding Media
absent
Specimens should be transported immediately.
** Complications: Occurs late, include increased
If delay is expected, transport media are used,
thirst, postural hypotension, tachycardia, such as:
decreased skin turgor and renal failure. ** Venkatraman-Ramakrishnan (VR) medium
** Cary-Blair medium
Virulence Factor
** Autoclaved sea water.
The following are the important virulence factors
of V. cholerae: Direct Microscopy
* Toxin coregulated pilus: [t helps in adhesion
* Gram staining of fecal smear (mucus flakes)
of Vibrio to intestinal mucosa reveals short curved comma-shaped gram-
* Cholera toxin: It has two fragments. Fragment
negative rods, typically arranged in parallel
A is active, causes ADP-ribosylation of G rows, described by Koch as “fish in stream”
protein, which in turn up regulates the appearance (Fig. 22.6)
activity of adenylate cyclase results in intra- * Motility testing by hanging drop method:
cellular accumulation of cyclic adenosine They are actively motile, described as darting
monophosphate (cAMP), leads to accumula- motility. V. cholerae becomes nonmotile after
tion of sodium chloride in the intestinal stool specimen treated with flagellar (H)
lumen, further leads to severe diarrhea. antiserum.
 CHAPTER 22 © Bacterial Diarrhea: Shigellosis, Cholera and Others

Culture smear and Motility Testing

** Culture smear of the colonies reveals short
curved gram-negative bacilli
* Hanging drop shows typical darting motility.

Bacterial Identification
Identification is made either by automated
systems, such as MALDI-TOF or VITEK; or
by conventional biochemical tests. The key
biochemical properties include:
** Catalase and oxidase positive
* ICUT test—shows the following reactions
Fig. 22.6: Vibrio cholerae (Gram stain): Curved comma-
shaped gram-negative rods (fish in stream appearance). (Fig. 22.8):
Source; Public Health Image Library, ID#:5324/Centers for Disease Indole test—positive
Control and Prevention (CDC) (with permission). Citrate test—variable
Urease test—negative
Culture TSI (triple sugar iron agar test)—Being
Vibrio cholerae is nonfastidious, strongly sucrose fermenter, it shows acid/acid, gas
aerobic. Stool specimen is inoculated onto absent, H,S absent.
enrichment broth and selective media. * Hemodigestion: On blood agar, it causes
nonspecific lysis of blood cells, seen as
Enrichment Broth greenish clearing around the main inoculum
* Alkaline peptone water (APW) (Fig. 22.7A)
** Monsur’s taurocholate tellurite peptone water. “+ String test: When a colony of Vibrio is mixed
with a drop of 0.5% sodium deoxycholate on
Selective Media a slide, the suspension loses its turbidity, and
* Alkaline bile salt agar (BSA) becomes mucoid. When tried lifting the sus-
* Monsur’s gelatin taurocholate trypticase pension with a loop, it forms a string (Fig. 22.9).
tellurite agar (GTTT)
*,Dd TCBS
Typing of Vibrio cholerae (Fig. 22.10)
agar: This medium is widely in use
at present (Fig. 22.7B). V. cholerae produces Serogrouping
yellow-colored colonies due to sucrose Specific serogroups can be identified by using
fermentation group-specific antisera. First the colony is tested
“ MacConkey agar: V. cholerae produces
translucent non-lactose fermenting (NLF) TSI
Indole Citrate | Urease K/A
colonies. positive negative negative gas—, H,S—

Oxidase
positive

y
Figs 22.7A and B: (A) Vibrio cholerae on blood agar (he-
modigestion); (B) TCBS agar with yellow-colored colonies
of Vibrio cholerae. Fig. 22.8: Biochemical reactions of V. cholerae.
Source: Department of Microbiology, Pondicherry Institute of Source; Department of Microbiology, Pondicherry Institute of
Medical Sciences, Puducherry (with permission). Medical Sciences, Puducherry (with permission).
 )
SECTION 2 © Systemic Microbiology (Infectious Diseases

B CHOLERA
Gi Solving Exercise 2
A 8-year-old boy developed severe watery diarrhea Identification
(10-12 times) and vomiting for 2 days. Stool collected Q Based on the curved (comma shaped) gram-
has a rice water type of appearance. The specimen was negative bacilli (Fig. 22.6), yellow-colored colonies
subjected to Gram staining (Fig. 22.6), culture (Fig. 22.7B) on thiosulfate-citrate-bile salt-sucrose agar (TCBS
and biochemical reactions (Fig. 22.8), agglutination with agar; Fig. 22.7B), and biochemical reactions (Fig.
antisera (Fig. 22.11). The antimicrobial susceptibility 22.8), the identification is Vibrio cholerae
test (AST) is demonstrated in Figure 22.12 with zone of Q Based on agglutination with specific antisera—the
interpretation chart in Table 22.3. serotype is identified as “ogawa’ (Fig. 22.11).
Q What is the clinical diagnosis and its causative
Antimicrobial Susceptibility Test
organism?
Q List the virulence factors and its mechanism of Antimicrobial susceptibility test on Mueller-Hinton
action. agar (Fig. 22.12) with zone of interpretation (Table
Q What are the various modalities of laboratory 22.3) shows that the isolate is sensitive to tetracycline,
diagnosis? ampicillin, chloramphenicol and resistant to
Q How will you treat this condition? ciprofloxacin.
(For answers to other questions, refer text below).
Explanation
Clinical Diagnosis
The history of severe watery diarrhea (rice water stool)
and vomiting is suggestive of cholera.

Clinical Manifestations (Cholera) Laboratory Diagnosis

Vibrio cholerae is the causative agent of cholera Specimens
characterized by painless watery diarrhea. ** Freshly collected watery stool is the specimen of
“+ The stool is watery (rice water in appearance) choice; collected before starting the antibiotics
with mucus flakes without blood or pus cells ** Rectal swab is preferred for carriers.
with a fishy, nonoffensive odor. Vomiting and
muscle cramps are present but fever is usually Transport/Holding Media
absent
Specimens should be transported immediately.
** Complications: Occurs late, include increased
If delay is expected, transport media are used,
thirst, postural hypotension, tachycardia, such as:
decreased skin turgor and renal failure. ** Venkatraman-Ramakrishnan (VR) medium
** Cary-Blair medium
Virulence Factor
** Autoclaved sea water.
The following are the important virulence factors
of V. cholerae: Direct Microscopy
* Toxin coregulated pilus: It helps in adhesion * Gram staining of fecal smear (mucus flakes)
of Vibrio to intestinal mucosa reveals short curved comma-shaped gram-
* Cholera toxin: It has two fragments. Fragment
negative rods, typically arranged in parallel
A is active, causes ADP-ribosylation of G rows, described by Koch as “fish in stream”
protein, which in turn up regulates the appearance (Fig. 22.6)
activity of adenylate cyclase results in intra- * Motility testing by hanging drop method:
cellular accumulation of cyclic adenosine They are actively motile, described as darting
monophosphate (cAMP), leads to accumula- motility. V. cholerae becomes nonmotile after
tion of sodium chloride in the intestinal stool specimen treated with flagellar (H)
lumen, further leads to severe diarrhea. antiserum.
 CHAPTER 22 © Bacterial Diarrhea: Shigellosis, Cholera and Others

Culture smear and Motility Testing

** Culture smear of the colonies reveals short
curved gram-negative bacilli
** Hanging drop shows typical darting motility.

Bacterial Identification
Identification is made either by automated
systems, such as MALDI-TOF or VITEK; or
by conventional biochemical tests. The key
biochemical properties include:
“* Catalase and oxidase positive
** ICUT test—shows the following reactions
Fig. 22.6: Vibrio cholerae (Gram stain): Curved comma-
shaped gram-negative rods (fish in stream appearance). (Fig. 22.8):
Source: Public Health Image Library, ID#:5324/Centers for Disease = Indole test—positive
Control and Prevention (CDC) (with permission). Citrate test—variable
= Urease test—negative
Culture = TSI (triple sugar iron agar test)—Being
Vibrio cholerae is nonfastidious, strongly sucrose fermenter, it shows acid/acid, gas
aerobic. Stool specimen is inoculated onto absent, H,S absent.
enrichment broth and selective media. * Hemodigestion: On blood agar, it causes
nonspecific lysis of blood cells, seen as
Enrichment Broth greenish clearing around the main inoculum
“* Alkaline peptone water (APW) (Fig. 22.7A)
“* Monsur’s taurocholate tellurite peptone water. ** String test: When a colony of Vibrio is mixed
with a drop of 0.5% sodium deoxycholate on
Selective Media a slide, the suspension loses its turbidity, and
“+ Alkaline bile salt agar (BSA) becomes mucoid. When tried lifting the sus-
“+ Monsur’s gelatin taurocholate trypticase pension with a loop, it forms a string (Fig. 22.9).
tellurite agar (GTTT)
Typing of Vibrio cholerae (Fig. 22.10)
** TCBS agar: This medium is widely in use
at present (Fig. 22.7B). V. cholerae produces Serogrouping
yellow-colored colonies due to sucrose Specific serogroups can be identified by using
fermentation group-specific antisera. First the colony is tested
“ MacConkey agar: V. cholerae produces
translucent non-lactose fermenting (NLF) TSI
Indole Citrate Urease K/A
colonies. positive negative negative gas—, H,S—

Oxidase
positive

f
Figs 22.7A and B: (A) Vibrio cholerae on blood agar (he- S
modigestion); (B) TCBS agar with yellow-colored colonies
of Vibrio cholerae. Fig. 22.8: Biochemical reactions of V. cholerae.
Source: Department of Microbiology, Pondicherry Institute of Source: Department of Microbiology, Pondicherry Institute of
Medical Sciences, Puducherry (with permission). Medical Sciences, Puducherry (with permission).
 SECTION2 © Systemic Microbiology (Infectious Diseases)

Clumps Milky white suspension

Fig. 22.11: Agglutination positive with O1 antisera and

Ogawa antisera.
Source: Department of Microbiology, Pondicherry Institute of
Fig. 22.9: String test (positive).
Medical Sciences, Puducherry (with permission).
Source: Department of Microbiology, Pondicherry Institute of
Medical Sciences, Puducherry (with permission).
strains are hybrid variety showing mixed results
of both classical and El Tor biotypes.

Antimicrobial Susceptibility Testing

y y It is done on Mueller-Hinton agar by disk
Groups Binks cholerae ||Other Vibrio species
diffusion test. Interpretation is based on Clinical
il and Laboratory Standards Institute (CLSI)
guideline (Fig. 22.12 and Table 22.3).
Subgroups/ : Non Aa
serogroups/ O01 ag :O2 to >O 200) |
serovars
Table 22.2: Differences between classical and El Tor
Vibrio cholerae.
Classical ElTor || Biotypes | Biotypes of V. cholerae O1 |Classical |
Hemolysis on sheep blood agar Negative Positive
Polymyxin B (50 IU) Susceptible Resistant

| Ogawa | |Inaba | Hikojima | |Serotypes | VP test Negative Positive

Cholera toxin gene CTX-1 CTX-2
Fig. 22.10: Typing of V. cholerae (Gardner and
Abbreviation: VP, Voges-Proskauer test
Venkatraman classification).

with O1 antisera> If found negative, then tested

with 0139 antisera (Fig. 22.11).

Serotyping
If agglutinated with O1 antisera, then the
serotyping is done by testing simultaneously
with Ogawa and Inaba antisera (Fig. 22.11).
* If agglutinated with Ogawa antisera, it is
designated as Ogawa serotype
* If agglutinated with Inaba antisera, it is
designated as Inaba serotype
* If agglutinated with both Ogawa and Inaba
antisera, it is designated as Hikojima
serotype. Fig. 22.12: Antimicrobial susceptibility testing on Mueller-
Hinton Agar for Vibrio cholerae (Table 22.3 for CLSI zone
Biotyping interpretation).
Vibrio cholerae serogroup O1 can be further Abbreviations: C, chloramphenicol; A, ampicillin; Cf, ciprofloxacin;
T, Tetracycline; CLSI, Clinical and Laboratory Standards Institute.
differentiated to biotypes classical and El Tor by
Source: Department of Microbiology, Pondicherry Institute of
various tests (Table 22.2). However, most of the Medical Sciences, Puducherry (with permission).
 CHAPTER 22 © Bacterial Diarrhea: Shigellosis, Cholera and Others

Table 22.3: Interpretative categories (CLSI) and observed zone size diameter (mm) to various antimicrobial
agents tested for Vibrio cholerae.
Antimicrobial agents Disk CLSI interpretative criteria for Vibrio | Observed zone Interpretation
strength cholerae species (in mm) size (Fig. 22.12)
(ug) |Resistant | Intermediate |Sensitive | (inmm)
Ciprofloxacin (Cf) 5 <15 16-20 >21 6 Resistant
Ampicillin (A) 10 <13 14-16 21 Sensitive
Tetracycline (T) 30 <11 12-14 El> 19 Sensitive
Chloramphenicol (C) 30 <12 13-17 218 26 Sensitive
Abbreviation: CLSI, Clinical and Laboratory Standards Institute.
Note: Doxycycline should be tested by a MIC based method only (as there is no disk diffusion break point).

Treatment Vaccine Prophylaxis

** Fluid replacement is crucial. Oral rehydration
+,
Oral cholera vaccines are currently in practice.
solution is given in mild cases. In severe cases, ** Killed whole-cell vaccine and whole-cell
intravenous fluid replacement with Ringer’s recombinant B subunit cholera vaccine (WC/
lactate (or normal saline) is advised rBS) (Dukoral); given two oral doses
** Antibiotics have a minor role as the patho- +,
“2 Oral live attenuated vaccines: They use
genesis is toxin mediated; recommended only mutant strains that lack the gene encoding
in severe cases. Azithromycin or erythromycin for cholera toxin, e.g., CVD 103-HgR vaccine
are the drugs of choice. Alternatively doxycy- (Orochol).
cline or tetracycline can be given.

@ CLOSTRIDIOIDES DIFFICILE DIARRHEA

Problem Solving Exercise 3
A seriously-ill patient on ventilator in ICU developed antibiotics raises the suspicion of C. difficile diar-
diarrhea after 10 days of hospitalization. He was on rhea.
ceftriaxone and clindamycin for 10 days. Stool sample Q Identification: The detection of toxin and gluta-
was subjected to the following test shown in Figure mate dehydrogenase (GDH) antigen by the immu-
PA NAC. nological test done on fecal specimen confirms the
1. What is the clinical diagnosis and its causative diagnosis (Fig. 22.13C)
organism? Q Test Principle: C. DIFF QUIK CHEK test: It is a rapid
2. Write the principle of the test. cassette assay that simultaneously detects both
3. How will you treat this condition? organism specific glutamate dehydrogenase (GDH)
antigen and toxins of C. difficile in fecal specimens
Explanation (Figs 22.13A to C).
Q Clinical Diagnosis: The history of severe diarrhea Q Treatment: Oral vancomycin and/or oral metronida-
in a hospitalized patient after prolonged intake of zole are the recommended antibiotics for treatment.
]|
Cc \ Cc || c \

Ag be Tox Ag Tox
||
i Ag e Tox i]
GDH ° A&B GDH A&B | GDH - ° A&B ||

C. DIFF COMPLETE
i
C. DIFF COMPLETE C. DIFF COMPLETE i

a E>" Bo SD |

Figs 22.13A to C: C. DIFF QUIK CHEK test: (A) Only control band positive indicate test is negative; (B) Control band
and glutamate dehydrogenase (GDH) band positive indicates nontoxigenic Clostridioides difficile present in stool
(commensal); (C) Control band and GDH band positive indicates toxigenic C. difficile present in stool.
 “‘ ;&P&n!..—
eeeE_--—

Viral Gastroenteritis:
Rotaviruses and Others 23
SNe ANE

Exercise e
Solving Exercis
[Problem Solving
A 4-year-old child was admitted to the pediatrics’ positive for rotavirus antigen in stool confirms the
ward with abdominal distress, diarrhea and mild diagnosis as rotavirus gastroenteritis.
dehydration, fever and vomiting for 2 days. Stool (For answers to other questions, refer below).
sample was for rotavirus antigen detection by ELISA
(Fig. 23.1).
Q Interpret the test result.
Q What are the other viral agents that may cause
similar illness?
Q Name the mode of transmission of this infec-
tion.
Q What are the different modalities of laboratory
pay Tae BS A ates
diagnosis?
Q How will you prevent such disease in future?

Explanation
A 4-year-old child with symptoms of acute Fig. 23.1: Rotavirus antigen detection by ELISA.
gastroenteritis, most common etiological agents Source: Department of Microbiology, Pondicherry Institute of
would be gastroenteritis causing viruses. ELISA Medical Sciences, Puducherry (with permission).

VIRAL GASTROENTERITIS * Adenovirus (type 40 and 41): Second most

common viral agent of endemic diarrheal
Viral etiology accounts for the most of the acute
illness of infants and young children world-
infectious gastroenteritis worldwide. The mode
wide.
of transmission of infection is by consumption
Rotaviruses are transmitted by fecal-oral
of contaminated food and water. The viral agents
route, then they progress further to destroy
are:
enterocytes of small intestine to cause diarrhea.
** Rotavirus: Most common cause of severe
diarrheal illness in children worldwide Rotavirus Diarrhea
* Caliciviruses (norovirus and sapovirus)
m Norovirus causes outbreaks of vomiting Laboratory Diagnosis
and diarrheal illness in all ages Rotavirus diarrhea can be diagnosed by:
m Sapovirus causes sporadic cases and * Direct detection of virus: Feces collected
occasional outbreaks of diarrheal illness early in the illness is the most ideal specimen.
in infants, young children, and in elderly. Rotaviruses can be demonstrated in stool
“* Astrovirus: It causes outbreaks of diarrheal by:
illness in infants, young children, and in a Immunoelectron microscopy: Rotaviruses
elderly have sharp-edged, triple-shelled capsids;
 CHAPTER 23 © Viral Gastroenteritis: Rotaviruses and Others

Andhra Pradesh, Assam, Haryana, Himachal

Pradesh, Jharkhand, Madhya Pradesh,
Odisha, Rajasthan, Tamil Nadu, Tripura and
Uttar Pradesh
“ Three doses (5 drops/dose): Administered
orally at 6, 10 and 14 weeks along with DPT
and OPV
“+ Overall efficacy in first 2 years of life is about
55%
* Side effects (25%): Crying, irritability,
fever and diarrhea. No vaccine induced
intussusception has been reported.

Rotarix
Fig. 23.2: Rotavirus (electron micrograph).
Source: Public Health Image Library, ID# 15194; Centers for Rotarix contains live attenuated G1P[8]strain;
Disease Control and Prevention (with permission). also provides cross protection against G3, G4
and G9. It has to be reconstituted before use.
look-like spokes grouped around the hub Given as two doses: Ist at 6 week and 2nd dose
of a wheel (Fig. 23.2) is given 4 weeks later.
= Isolation of rotavirus is difficult. Rolling
General Preventive Measures
of tissue cultures may be attempted to
enhance viral replication. It includes—(1) measures to improve hygiene
~ Detection of viral antigen in stool—by ELISA and sanitation in the community, and (2)
and latex agglutination based methods contact precautions, such as strict hand hygiene
“ RT-PCR is the most sensitive detection to prevent transmission from infected persons
method for detection of rotavirus from (Chapter 11).
stool
Norwalk Virus
* Serologic tests (ELISA) can be used to detect
the rise in antibody titer. This may be useful It is the most important cause of epidemic viral
for seroprevalence purpose. gastroenteritis in adults
“* It is common in winter months in temperate
Treatment climates; therefore called as winter vomiting
Treatment is mainly supportive, to correct the disease or gastric flu
loss of water and electrolytes, such as oral or “ Symptoms begin 12-48 hours after the
parenteral fluid replacement. exposure; characterized by diarrhea,
abdominal pain, nausea and vomiting
Vaccine “+ Common food sources include contaminated
Two brands of rotavirus vaccine are available:
salad, fresh fruits, shellfish (such as
oysters), or water. Other sources include
Rotavac the infected person; touching contaminated
It contains live attenuated rotavirus 116E surfaces.
(G9P[10] strain). It provides cross protection
Sapoviruses
against many types including G1P[8] type.
“ Jt is manufactured by Bharat Biotech, India They cause sporadic cases and occasional
¢ Jt is introduced under national immunization outbreaks of diarrheal illness in infants, young
schedule of India (2020), in selected states— children, and the elderly.
 a eeeee eEE~-,-,.,,st“
rr

Intestinal Protozoan Infections: WesLtaiis

Intestinal Amoebiasis, Giardiasis,
Coccidian Parasitic Infections
OVE EAOTRE

B INTRODUCTION
The protozoan parasites which cause intestinal * Others: Balantidium coli, Blastocystis
infection include: hominis, Sarcocystis and Microsporidia (now
“+ Intestinal amoebae: Entamoeba histolytica considered as fungi).
“+ Intestinal flagellate: Giardia lamblia Entamoeba histolytica and Balantidium coli
“+ Opportunistic intestinal coccidian parasites: produce dysentery, whereas the other intestinal
Cryptosporidium, Cyclospora, and Cystoisospora protozoans produce diarrheal disease.

B INTESTINAL AMOEBIASIS
[Problem Solving
Solving Exercise
Exercise11 |
Intestinal Amoebiasis Q History of bloody diarrhea with mucus and pus
A 13-year-old girl presented with bloody diarrhea with cells, colicky abdominal pain
mucus and pus cells, colicky abdominal pain, fever and Q The structure focused (Fig. 24.1A) is round,
prostration. The wet mount examination of the stool 12-15 um size, with 3 to 4 nuclei and a central
sample has been focused (Fig. 24.1A). karyosome—suggestive of cyst of Entamoeba
1. Identify the structure focused and the etiological histolytica or E. dispar
agent. Q Cysts of E. histolytica and E. dispar are morpho-
2. What is the host, infective form, pathogenic form, logically indistinguishable. To differentiate,
diagnostic form, habitat and mode of transmission multiplex polymerase chain reaction (PCR) should
of the parasite? be performed targeting specific genes
3. What are the various diagnostic modalities? Q Treatment: Metronidazole 750 mg thrice a day
4. How will you treat this condition? given for 5-10 days (Table 24.1).
(For answers to the other questions, refer Tables 24.1
Explanation and 24.2).
This is a case of amoebic dysentery. Points in favor are:

Laboratory Diagnosis of Intestinal ** ‘The cyst and trophozoites of E. histolytica are

Amoebiasis morphologically similar to that of E. dispar,
Stool microscopy is the mainstay of diagnosis of
intestinal amoebiasis.
* Repeated stool microscopy is done by
wet mount and permanent stains. If fails,
then examination is done following stool
concentration technique
* The cyst and trophozoites of E. histolytica
+, ?

must be differentiated from that of Enta-

moeba coli which is a harmless commen-
pd
Figs 24.1A and B: Cyst of (A) Entamoeba histolytica; (B) E. coli.
sal of gut (Table 24.3 and Figs 24.1 and
Source: Swierczynski G, Milanesi B. Atlas of human intestinal.
24,2) protozoa microscopic diagnosis (with permission).
 CHAPTER 24 © Intestinal Protozoan Infections

Table 24.1: Features/characteristics of Entamoeba Table 24.2: Laboratory diagnosis Intestinal amoebiasis.
histolytica. |Methods | Salient features
lHost | Humans are the only host Stool Detects cysts (most common) and
microscopy trophozoites (rarely)
Morphological Trophozoites, precyst and cyst
Histology Intestinal biopsies stained with PAS or
forms
H&E stains reveal trophozoites
Infective form Mature quadrinucleated cyst Stool culture Polyxenic and axenic culture
Transmission Feco-oral route (contaminated food Stool antigen ELISA and ICT detecting lectin antigen
and water) detection
Large intestine
(coproantigen)
Habitat
Serology Amoebic antigen—ELISA (170-kDa of
Pathogenic e Trophozoites lectin Ag)
form e Surface lectin antigen helps in Amoebic antibody—ELISA
invasion This is more useful in amoebic liver
Major mani- Intestinal amoebiasis: abscess
festations e Amoebic dysentery Nested PCR Can differentiate E. histolytica and E.
e Amoebic appendicitis dispar
e Amoeboma
Abbreviations: ELISA, enzyme-linked immunosorbent assay; ICT,
e Fulminant colitis immunochromatographic test; PCR, polymerase chain reaction.
Extraintestinal amoebiasis:
e Amoebic liver abscess—anchovy
sauce pus and abscess formation
e Other forms—pulmonary, CNS,
cutaneous, etc.
Diagnostic e Cysts (both mature and immature
form cyst) are common
e Trophozoites are rare
Treatment For intestinal amoebiasis: ——

e Tissue agents— metronidazole (5-10 Figs 24.2A and B:Trophozoite of (A) Entamoeba coli;
days) or tinidazole (3 days) plus (B) E. histolytica.
e Luminal agents— lodoquinol (20 Source: Swierczynski G, Milanesi B. Atlas of human intestinal
days) or Paromomycin (10 days) protozoa microscopic diagnosis (with permission).

Table 24.3: Differentiating features between Entamoeba histolytica and E. coli (Figs 24.1 to 24.4).
Entamoeba histolytica Entamoeba coli

15-20 um 20-25 um
Size
Pseudopodia Pseudopodia with finger-like projection Blunt pseudopodia
Very active movement Sluggish, aimless motility

Differentiated to ectoplasm and Not differentiated

Cytoplasm
endoplasm
Red blood cell (RBC), leukocytes, tissue Same except it does not contain RBC
Cytoplasmic inclusions
debris and bacteria
Karyosome is small and central Karyosome is large and eccentric
Nucleus
(Figs 24.3A and 24.4A) Nuclear membrane is thin and lined by Nuclear membrane is thick and lined
fine chromatin granules by coarse chromatin granules

Cyst (Figs 24.3C and 24.4C)

12-15 um 15-25 um
Size
Same as trophozoite Same as trophozoite
Nucleus
1-4 1-8
No. of nuclei
Thick bars with rounded ends Filamentous and thread-like ends
Chromatoid body
 s)
188 SECTION 2 © Systemic Microbiology (Infectious Disease
Pseudopodium
Ectoplasm

White blood cell

Endoplasm
Karyosome
Chromatid body
Rood (central) Glycogen mass
vacuoles Peripheral
chromatin Uninucleated cyst Quadrinucleated cyst
granule (fine) (Immature) (mature)

Red blood cell [J

Ectoplasm Glycogen
mass
Karyosome
(eccentric)
Peripheral chromatin
granules (coarse)
Filamentous
Food vacuole
chromatid body
Endoplasm Quadrinucleated cyst Octanucleated
E (Immature) Mature cyst
Figs 24.4A to C: Entamoeba coli: (A) Trophozoite; (B) Immature cyst; (C) Mature cyst.

which is again a harmless commensal of gut. E. = Presence of red blood cells (RBCs) inside
histolytica can be differentiated by E. dispar by: the trophozoite (sign of invasion)
m Detection of lection antigen in stool m Isoenzyme (zymodeme analysis) detecting
= Polymerase chain reaction specific isoenzyme.

BGIARDIASIS
| Problem Solving
Solving Exercise
Exercise |
A 3-year-old boy presented with recurrent episodes Explanation
of foul smelling diarrhea, foul flatus, sulfurous This is a case of giardiasis, caused by Giardia lamblia or
belching and profound weight loss. The wet mount G. intestinalis. Points in favor are:
examination of the stool sample has been focused Q History of recurrent episodes of foul smelling
(Figs 24.5B and C). diarrhea, foul flatus, sulfurous belching and
1. Identify the structure focused and the etiological profound weight loss
agent. The structure focused (Figs 24.5B and C) is oval
2. What is the host, infective form, pathogenic form, cysts measuring 12-15 um size, having 1-4
diagnostic form, habitat and mode oftransmission number nucleus with central axoneme (axostyle),
of the parasite?
suggestive of Giardia lamblia.
3. What are the various diagnostic modalities? Q Treatment: Metronidazole or tinidazole.
4. How will you treat this condition? (For answers to the other questions, refer Tables 24.4
to 24.5).

Laboratory Diagnosis of Giardiasis ** Repeated stool examination (at least three

Stool microscopy is the mainstay of diagnosis of consecutive samples) should be done
intestinal amoebiasis; carried out by wet mount following concentration techniques for better
(saline and iodine) preparation: positivity
 CHAPTER 24 © Intestinal Protozoan Infections

poreetiea
Table 24.4: pe diagnosis of Giardia. Table 24.5: Trophozoite and cyst of Giardia lamblia.

|Methods —_| Salient features [Forms Characteristics

Stool Detects cysts (most common) and Trophozoite e Front view: Pear-shaped (or tear
microscopy trophozoites (rarely) (Figs 24.5A and drop or tennis racket-shaped)
e Sensitivity is 60% to 80% with one 24.6A) e Lateral view: Spoon shaped
stool and >90% after three stools e Measures 10-20 um in length and
examination 5-15 um in width
e Zinc sulfate flotation or formalin e Contains two adhesive disks, one
ether sedimentation concentration pair of nuclei and four pairs of
methods are used to increase the flagella
chance of detection e Has falling leaf-like motility
e Presence of trophozoites indicates
Duodenal Indicated when stool examination is active stage ofthe disease
sampling negative. Direct duodenal samples,
such as aspirates (obtained by entero- Cyst e Oval, measuring 11-14 um in
(Figs 24.5B and length and 7-10 pm in width
test) or biopsy (done by endoscopy)
Cand 24.6B) e Contains 1-4 nuclei and central
used (Fig. 24.7)
axoneme or axostyle (remnant of
Permanent Trichrome stain can be used to flagella)
staining demonstrate cysts and trophozoites e Cysts cannot differentiate active
in stool disease from carriers.
Stool culture In axenic media, such as Diamond's
media, not routinely used
Stool antigen Coproantigen detection stool by
detection ELISA, IFA and ICT methods
ICT (triage parasite panel) available
that simultaneously detects antigen
of Entamoeba histolytica, Giardia and
Cryptosporidium in stool
Serology Formats: ELISA and IFA
(antibody Cannot differentiate recent and past
detection) infection
PCR Most sensitive and specific

Abbreviations: ELISA, enzyme-linked immunosorbent assay; ICT, Figs 24.5 A to C: Giardia lamblia: (A) Trophozoites in saline
immunochromatographic test; IFA, indirect fluorescent antibody mount; (B) Cyst in iodine mount; (C) Cysts in saline mount.
test; PCR, polymerase chain reaction.
Source: Giovanni Swierczynski, Bruno Milanesi. “Atlas of Human
Intestinal Protozoa Microscopic Diagnosis” (with permission).

* If still fails, direct examination of duodenal con-

tent (by performing string test or Entero-test).
Sucking disk (two)
Entero-test (or String Test) Nucleus (two)
It uses a gelatin capsule attached to a thread Central karyosome
containing a weight (Fig. 24.7).
Q One end of the thread is attached to the outer Flagella (four pairs)
aspect of the patient's cheek, and then, the capsule
Parabasal body
is swallowed
Q Capsule gets dissolved in stomach releasing the
thread which is carried to the duodenum, gets
Thick wall
unfolded and takes up the duodenal samples
Q Four hours later, the thread is withdrawn and Axoneme
shaken in saline to release trophozoites which .
can be detected microscopically by wet mount or é Op Nucleus (four)
permanent stained smear
O The entero-test is also useful for other upper El
intestinal parasites, such as Strongyloides, Fig. 24.6: Giardia lamblia (schematic diagram):
Cryptosporidium and Clonorchis. (A) Trophozoite; (B) Cyst.
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

COCCIDIAN PARASITIC
Gelatin capsule containing the thread
with a weight attached at the other end INFECTIONS
Cryptosporidium parvum, Cyclospora cayetan-
ensis and Cystoisospora belli (earlier known as
Isospora belli) are the coccidian parasites that
cause diarrhea in immunocompromised host,
End of the thread which will be
attached to the patient's cheek such as HIV-infected patients.
Differences between properties of Crypto-
a sporidium, Cyclospora and Cystoisospora is
eile
ee ie given in Table 24.6 and shown in Figures 24.8 to
24.11. Laboratory diagnosis of intestinal coccid-
Fig. 24.7: Entero-test equipment showing duodenal capsule. ian parasites is given in Table 24.7.

Problem Solving Exercise

A 61-year-old female presented with severe profuse oocyst (4-6 um in size), containing four sporozoites
diarrhea (20 times a day) for more than 10 days, (Figs 24.8A and B).
weight loss and abdominal pain. The stool specimen Treatment: Nitazoxanide 500 mg, twice a day for 3 days.
was sent to microbiology laboratory for investigation For answer to the other questions, refer Tables 24.6
(Fig. 24.8A and B.). and 24.7.
1. Identify the stain and etiological agent based on
the smear focused.
2. What is the host, infective form, pathogenic form,
diagnostic form, habitat and mode oftransmission
of this parasite?
3. How will you treat this clinical condition?
Explanation
This is a case of cryptosporidiosis, caused by
Cryptosporidium parvum. Figs 24.8A and B: Cryptosporidium species: (A) Modified
Points in favor are: acid-fast staining shows red colored (round) oocyst against
Q HIV-infected patient blue background; (B) Direct fluorescent antibody staining
Q Profuse diarrhea shows brilliant green fluorescent oocysts.
Q Modified acid-fast stain and direct fluorescent Source: Swierczynski G, Milanesi B. Atlas of human intestinal
antibody staining showing round sporulated protozoa Microscopic diagnosis (with permission).

Cyst wall a Sporozoites —~ -

Figs 24.9A to C: Sporulated oocysts (schematic diagram) of (A) Cryptosporidium; (B)

Cyclospora; (C) Cystoisospora.
 CHAPTER 24 © Intestinal Protozoan Infections

Fig. 24.10: Cyclospora species in modified acid-fast staining—shows variable acid-fast oocysts.
Figs 24.11A and B: Cystoisospora belli: (A) Saline mount shows unsporulated oocyst (left) and
sporulated oocyst (right); (B) Modified acid-fast staining shows unsporulated oocysts.
Source: (Figs. 24.10 and 24.11A) Swierczynski G, Milanesi B. Atlas of human intestinal protozoa microscopic diagnosis (with permission).
Source: (Fig. 24.11B) Dr Anand Janagond, Department of Microbiology, S Nijalingappa Medical College, Bagalkot, Karnataka
(with permission).

Table 24.6: Differences between properties of Cryptosporidium, Cyclospora and Cystoisospora.

Cryptosporidium Cyclospora
Infective form Sporulated oocyst Sporulated oocyst Sporulated oocyst

Diagnostic form Sporulated oocyst Unsporulated oocyst Unsporulated oocyst

Oocyst size 4-6 um 8-10 um 23-36 um

Oocyst shape Round Round Oval

Oocyst contains 4 sporozoites 2 sporoblasts, each having 2 sporoblasts, each having

2 sporozoites 4 sporozoites (Fig. 24.11A)
(Fig. 24.9)
Uniformly acid-fast Variable acid-fast Uniformly acid-fast
Oocyst acid fastness
(Fig. 24.8A) (Fig. 24.10) (Fig. 24.11B)
No, but can be stained with Autofluorescence ++ Autofluorescence +/-
Autofluorescence
fluorescent dye (Fig. 24.8B)
Occurs inside the host cells Occurs in soil Occurs in soil
Sporulation of the
(enterocytes) (environment) (environment)
oocyst
Nitazoxanide Cotrimoxazole Cotrimoxazole
Treatment

Table 24.7: Laboratory diagnosis of intestinal coccidian parasites.

Cea ES
Stool examination It is carried out to detect oocyst. Various methods:
e Direct wet mount
e Wet mount after stool concentration (Sheather’s sugar floatation)
e Modified acid-fast stain of stool (1% sulfuric acid as decolorizer)
e Direct fluorescent antibody technique

Antigen detection in e Antigen detection in stool by ICT or ELISA

of Giardia,
stool e Triage Micro Parasite Panel is an ICT used for simultaneous detection
Entamoeba histolytica and Cryptosporidium
Antibody detection e ELISA and IFA format available
e Used for seroepidemiological purposes
Molecular method Polymerase chain reaction detecting specific genes
Histopathology Histopathology of intestinal biopsy specimen detecting oocyst
rescence assay; ICT, immunochromatographic test.
Abbreviations: ELISA, enzyme-linked immunosorbent assay; IFA, immunofluo
nn

CHAPTER|
|
Intestinal Helminthic Infections 2 5

Cestodes exist in three morphological forms:

B INTRODUCTION
1. Adult (tapeworm): Divided into head (scolex),
Intestinal helminthic infections contribute a neck and segments called as proglottids or
significant proportion of gastrointestinal (GI) strobila. Some adult worms bear hooklets in
infections. Nevertheless, majority of helminths scolex and are called as armed tapeworm, €.g.,
(cestodes, trematodes and nematodes) cause T. solium, Echinococcus, H. nana. Proglottids
infections of GIT. are further grouped into immature, mature
“+ Intestinal cestodes: Diphyllobothrium, and gravid segments (Fig. 25.1A)
Taenia saginata, T. solium, Hymenolepis nana 2. Eggs: All cestodes eggs have an embryophore
and Dipylidium caninum and an embryo with three pair of hooklets
“* Intestinal trematodes: (Fig. 25.1B). The only exception is eggs of
= Intestinal fluke, e.g., Fasciolopsis buski Diphyllobothrium latum which are oper-
= Blood flukes, suchas Schistosoma mansoni culated (Fig. 25.4)
and S. japonicum reside in mesenteric 3. Larva: Eggs develop into larva which are
venous plexus of GIT, and cause various called as:
GI symptoms including dysentery. mw Cysticercus—larval stage of Taenia (T.
** Intestinal nematodes: Include saginata—Cysticercus bovis, T. solium—
# Small intestinal nematodes—Ascaris, Cysticercus cellulosae)
hookworm and Strongyloides = Hydatid cyst—larval stage of Echinococcus
= Large intestinal nematodes, such as m Cysticercoid—larval stage of Hymenolepis.
Trichuris and Enterobius.

Hooklets
INTESTINAL CESTODE on rostellum

INFECTIONS “8*— sucker

&ya ~— Neck region
Cestodes, or tapeworms, are segmented worms. 4 Immature )
Based on habitat, cestodes are classified into: f4 proglottids
** Intestinal cestodes:
=X >
® Diphyllobothrium spp. ~>
a Mature
m Taenia saginata and Taenia solium
}\ proglottids Strobila
= Hymenolepis nana.
** Tissue cestodes:
m= Echinococcus—causes hydatid disease; — Gravid
mainly affecting liver (Chapter 27) yw proglottids |
a Taenia solium—causes cysticercosis, A 'B|
mainly infecting CNS (Refer Chapter Figs 25.1A and B: (A) Adult worm of cestode (schematic
41). diagram); (B) Egg of cestode (schematic diagram).
 CHAPTER 25 © Intestinal Helminthic Infections

B INTESTINAL TAENIASIS
| _ Problem Solving
Solving Exercise
Exercise1_|
1
A 10-year-old child came to the pediatric OPD with his- oval, containing an embryo with three pairs of
tory of passing segments of a worm. The stool examina- hooklets, surrounded by an embryophore. The eggs
tion revealed segments (Fig. 25.2C) and ova (Fig. 25.1B). of T. saginata and T. solium are indistinguishable,
1. Identify the disease and the causative agent. except that T. saginata eggs are acid-fast.
2. Name the different species which can infect man _ 7. saginata and T. solium can be differentiated by:
and how to differentiate them. Q Proglottid: The proglottid of T. saginata has 15-20
lateral branches from uterus in comparison to that
3. Which are the hosts, mode of transmission?
of T. solium which has 7-13 lateral branches from
4. Name the larval stage of the species found in man
and complications caused by the same. eters
; Q Scolex: The scolex of T. saginata is quadrangular
Explanation in shape bearing 4 suckers and with no hooklets
This is the case of intestinal taeniasis. (Fig. 25.2A). This can be differentiated from that of
Q There are two species of Taenia infecting man, T. T. solium (globular, 4 suckers, armed with hooklets
saginata and T. solium. arising from rostellum (Fig. 25.2B).
Q Figure 25.2C shows Taenia adult worm segments, _ Refer Table 25.1 for other details.
tape-like segments.

Figs 25.2A to C: (A) Carmine-stained scolex of T. saginata; (B) Carmine-ree scolex of T. solium; (C) Adult worm of
Taenia species.
Source: (A) DPDx Image Library, Centers for Disease Control and Prevention (CDC), Atlanta (with permission); (B) Public Health Image
Library, ID#: 5262, Centers for Disease Control and Prevention (CDC), Atlanta (with permission); C. Head, Department of Microbiology,
Meenakshi Medical College, Chennai (with permission).
Q Figure 25.1B shows Taenia egg which is round to

@ HYMENOLEPIASIS
Exercise2 2___|
Solving Exercise
[Problem Solving
A 6-year-old child came to the OPD with history of — Polar thickening
anorexia, addominal pain, dizziness and diarrhea. On Yolk granules
‘
examination, the child was malnourished. The stool Inner membrane
ee Hooklets
specimen was sent for microscopic examination (Fig. |
J
rd
(three pair)
25.3): Outer membrane
1. Identify the parasite based on stool microscopy. y Embryo 2
2. Which is the infective stage for man? An iam Polar filament Bi

3. How the disease is transmitted?

Figs 25.3A and B: Egg of ee eolcoe nana:
Re: (A)
4, Which are the parasites that are transmitted by Schematic; (B) In saline mount (non-bile stained).
autoinfection? Source: (B) DPDx Image Library, Centers for Disease Control and
5. What are the examples of non-bile stained eggs? Prevention (CDC), Atlanta (with permission).
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

Explanation Autoinfection is seen in:

Cryptosporidium parvum
The causative agent is Hymenolepis nana. The points
Hymenolepis nana
in favor are:
Enterobius vermicularis
Q Gastrointestinal symptoms—anorexia, abdominal
Strongyloides stercoralis
pain, headache, and diarrhea.
s Taenia solium
esos
Oe)
Q Figure 25.3B reveals non-bile stained eggs, with
Examples of non-bile stained eggs:
three pairs of hooklets, polar filaments filling
between two membranes (Fig. 25.3A).
Q Hymenolepis nana
Q_ Enterobius vermicularis
Q Infective form: Eggs
OQ Mode of transmission: (i) Ingestion of contami- Q Hookworm.
nated food and water containing eggs; (ii) Auto- Refer Table 25.1 for other details.
infection.

@ DIPHYLLOBOTHRIASIS
| Problem Solving
Solving Exercise
Exercise3_
3
A 16-year-old boy from Russia came to the OPD Q Evidence of megaloblastic anemia
with complain of abdominal discomfort, diarrhea, Q Stool microscopy revealed oval, operculated eggs
vomiting, weakness and weight loss. Peripheral with knob at the other end; measuring 70 um x 50
blood smear examination revealed increased mean uum in size (Fig. 25.4).
corpuscular volume, increased mean corpuscular Refer Table 25.1 for other details.
hemoglobin concentration, and enlarged red blood
cells. The stool specimen was sent for microscopic r
||
examination (Fig. 25.4).
1. Identify the parasite based on the stool microscopy.
2. Which is the infective stage for man?
3. How will you diagnose this condition in the
laboratory?
4. Mention two complications caused by the adult |
worm.
Explanation
This is a case of megaloblastic anemia due to
Diphyllobothrium latum. Points in favor are:
rn ewee as
Q Patient from Russia—endemic for D. latum
infection Fig. 25.4: Egg of Diphyllobothrium latum.
Q Gastrointestinal symptoms Source: DPDx Image Library, Centers for Disease Control and
Prevention (CDC), Atlanta (with permission).

INTESTINAL TREMATODE INFECTIONS

Trematodes (also called as flukes) include = Schistosoma mansoni, Schistosoma
the helminths that are unsegmented, flat (flat japonicum—resides in rectal venous
worms) and leaf like. They are classified based plexus and portal venous plexus.
on their habitat into: * Hepatic flukes: Fasciola hepatica, Clonorchis
** Blood flukes: spp., Opisthorchis spp (Refer Chapter 27)
a Schistosoma haematobium—tresides in ** Intestinal flukes: Fasciolopsis buski
vesical venous plexus of bladder (Refer * Lung flukes: Paragonimus westermani (Refer
Chapter 42) Chapter 38).
 CHAPTER 25 © Intestinal Helminthic Infections

Table 25.1: Features or characteristics of intestinal cestodes.

Properties Diphyllobothriasis Hymenolepiasis

Taenia saginata/T. solium (Diphyllobothrium latum) (Hymenolepis nana)

Definitive host Man Man Man

Intermediate host —_T. saginata—cattle 1st—cyclopes No
T. solium—pig 2nd—fish
Infective form Larva (cysticercus bovis or L3 larva (pleurocercoid larva) Eggs
, cysticercus cellulosae)

Transmission by Contaminated beef or pork meat Fish containing pleurocercoid Contaminated food and
ingestion of: (partially/uncooked) larva (partially cooked/ water or autoinfection
uncooked)

Habitat Intestine Intestine Intestine

Pathogenic form Adult Adult Adult
Manifestations Intestinal symptoms only Intestinal symptoms, Intestinal symptoms only
megaloblastic anemia
Diagnostic form Demonstration of eggs in feces Eggs (Fig. 25.4): Eggs (Fig. 25.3B):
(Fig. 25.1B) e Operculated e Non-bile stained
e Knob at the other end e Round to oval
e Shape: Oval e Polar filaments
e Size: 70 um x 50 um

Treatment e Praziquantel (DOC) e Praziquantel e Praziquantel

: e Niclosamide e Niclosamide e Niclosamide
e Parenteral vitamin B,,

Abbreviations: ELISA, enzyme-linked immunosorbent assay; DOC, drug ofchoice.

B SCHISTOSOMIASIS

[Problem Solving
Solving Exercise
Exercise44
Schistosoma mansoni Infection Q Stool microscopy reveals nonoperculated oval
A 62-year-old man from Africa came to the OPD with elongated eggs with lateral spine (Fig. 25.5)
maculopapular rash, fever and hepatosplenomegaly. 1 Drug of choice is praziquantel.
Stool sample was sent for wet mount examination For further details, refer Tables 25.2 and 25.3.
(Fig. 25.5).
1. Identify the causative agent based on the stool
microscopic findings.
2. Which is the infective stage, host and mode of
transmission of the parasite?
3. Mention two complications produced by infection
with this parasite.
4. What is the drug of choice for treatment?

Explanation
The causative agent is Schistosoma mansoni. Points
in favor are: Fig. 25.5: Schistosoma mansoni (egg).
Q From Africa—endemic for schistosomiasis
Source: ID# 4841. Public Health Image Library, Centers for Disease
Q Presented with maculopapular rash, fever and Control and Prevention (CDC), Atlanta (with permission).
hepatosplenomegaly
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

Bi FASCIOLOPSIS BUSKI INFECTION

Problem Solving Exercise 5 7

A 6-year-old child came to the OPD with complaints lateral branches from intestinal caeca are the
of vague abdominal pain, passage of greenish-yellow identifying features.
stool. On examination, the child was malnourished. For further details, refer Tables 25.2 and 25.3.
Stool specimen was sent for wet mount examination
for detection of ova (Fig. 25.6). The adult form of the
causative agent ofthe condition is also displayed (Fig.
259/).
1. Identify the causative agent based on the
microscopic findings.
2. Which is the infective stage, mode of transmission,
hosts for the parasite?

Explanation
The causative agent is F. buski. Points in favor are:
Q Passage ofgreenish-yellow stool. Fig. 25.6: Fasciolopsis buski Fig. 25.7: Carmine stained
Q Stool microscopy reveals large (140 um x 80-85 (egg). adult worm of Fasciolopsis
um), oval, operculated, bile-stained eggs (Fig. 25.6). buski.
Q Figure 25.7: Adult worm of F.buski. Oral and ventral Source: DPDx Image Library, Centers for Disease Control and
suckers are close to each other and absence of Prevention (CDC), Atlanta (with permission).

Table 25.2: Features or characteristics of intestinal Table 25.3: Eggs of intestinal trematodes (diagnostic
trematodes. forms).
Properties Fasciolopsis Schistosomes (Nonoperculated eggs)
buski
Schistosoma mansoni
Definitive host Man
e Size: 110-175 umin
Intermediate — Snail 1st—Snail length and 45-70 um in
host 2nd—Aquatic breadth
plant e Shape: Elongated oval
Infective form Cercaria larva Metacercaria e¢ Nonoperculated
larva e Lateral spine at the
Transmission — Skin penetration Ingestion of posterior end
aquatic plant
containing Schistosoma japonicum
metacercaria e Size: 70-100 um in
Habitat Blood vessels of Blood vessels of length and 50-70 um in
bladder/intestine small intestine breadth
e Shape: Spherical
Manifesta- S. mansoni/ Malabsorption
e Nonoperculated
tions japonicum and protein
e Rudimentary lateral
e Cercarial losing
knob*
dermatitis enteropathy
e Katayama fever with profuse Other trematodes (Operculated eggs)
e Egggranuloma _ yellowish-green
formation in stool Fasciolopsis buski
intestine, liver e Large, oval eggs
and spleen ¢ Operculated
Diagnostic e¢ Measuring 130-140 um
Eggs (nonopercu- —_Eggs (opercu-
form (Table long and 80-85 um in
lated) in stool lated) in stool
breadth
253)
Treatment Praziquantel Praziquantel “me on
*S. hematobium resides in blood vessels of urinary bladder, “Source: |D#4842. Public Health Image Library, Centers for Disease
discussed in Chapter 42.
Control and Prevention (CDC), Atlanta (with permission).
 CHAPTER 25 © Intestinal Helminthic Infections 197

INTESTINAL NEMATODE INFECTIONS

Nematodes are classified into intesti- = Hookworm (Ancylostoma duodenale and
nal and tissue or somatic nematodes. In- Necator americanus)
testinal nematodes are further grouped = Strongyloides stercoralis.
into: * Large intestinal nematodes:
“* Small intestinal nematodes: = Enterobius vermicularis (pinworm)
w Ascaris lumbricoides (roundworm) a Trichuris trichiura (whipworm).

BTRICHURIASIS
Problem Solving Exercise 6
A 3-year-old child was brought to the OPD with Q Figure 25.8A: Stool microscopy showed barrel-
history of dysentery and growth retardation. The stool shaped egg with mucus plugs.
specimen was sent for direct microscopy (Fig. 25.8A). Q Figure 25.9: Gross specimen revealed whip-shaped
The gross specimen of the causative agent obtained adult worms of 3-5 cm long, anterior 3/5th is thin,
through proctoscopy is displayed (Fig. 25.9). hair like, coiled (like a whip), and posterior 2/5th
1. Identify the causative agent based on microscopic is short and thick (like a handle of a whip). Hence,
findings and gross morphology. Trichuris is also called as whipworm.
2. Drawalabeled diagram of the ova ofthe causative Q Labeled diagram of ova is shown in Figure 25.8B.
agent. Q Infective form: Embryonated egg
3. Whatis the infective form and mode of transmission? Q Mode of transmission: Ingestion of contaminated
food and water containing embryonated egg.
Explanation For further details, refer Tables 25.4 and 25.5.
It is a case of trichuriasis; caused by Trichuris trichiura.
Points in favor are:

Barrel shaped
|
Ovum unembryonated |
when freshly passed in |
stool |

Figs 25.8A and B: Trichuris eggs: (A) In saline mount;

(B) Schematic diagram.
Fig. 25.9: Trichuris trichiura (adult worm).
Source: (A) Dr Anand Janagond, Department of Microbiology,
Source: Dr Mae Melvin, Public Health Image Library, Centers for
S. Nijalingappa Medical College, Bagalkot, Karnataka (with
Disease Control and Prevention (CDC), Atlanta (with pemission).
permission).

@ ENTEROBIASIS
Problem Solving Exercise7 —s|
A5-year-old child was brought to the OPD with history 2. Drawa labeled diagram ofthe ova ofthe causative
of repeated episodes of nocturnal enuresis and itching agent.
over the perianal region. Perianal swab [NIH (National 3. Howwill you collect the specimen to diagnose this
Institutes of Health) swab] was collected and sent for clinical condition?
direct microscopy (Figs 25.10A and 25.11). 4. What is the infective form and mode of transmis-
1. Identify the causative agent based on the sion?
microscopic findings.
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

Explanation Q Labeled diagram of ova is shown in Figure 25.10B.

0 Infective form: Embryonated egg.
It is a case of enterobiasis; caused by Enterobius
Q Mode of infection: Ingestion of contaminated
vermicularis. Points in favor are:
food and water containing embryonated egg
Q History of repeated episodes of nocturnal enuresis
containing larva or by autoinfection.
and itching over the perianal region.
Q Specimen collection: Perianal sampling is collected
Q Figure 25.10A: Perianal swab and stool microscopy
by cellophane tape (Fig. 25.11A) or specialized
showed non-bile stained egg, planoconvex
swab called as NIH swab (Fig. 25.11C).
shaped, containing a tadpole shaped larva inside.
For further details, refer Tables 25.4 and 25.5.
Q Figure 25.11B: Gross specimen revealed small,
white and thread-like worm (hence, named |Cellophane tape NIH swab
as threadworm) with posterior end pointed
and tapering (looks like a pin, hence called as {}

pinworm). Rubber stopper |

Planoconvex |
in shape Glass rod
a Test tube
Non-bile
11 |
stained
Rubber band |
Embryonated ovum Cellophane |
with a larva inside _|

Figs 25.10A and B: Enterobius eggs: (A) In saline mount; Figs 25.11A to C: Enterobius vermicularis: (A) Cellophane
(B) Schematic diagram. tape technique; (B) Adult worms; (C) NIH swab (schematic).
Source: (A) DPDx Image Library, Centers for Disease Control and Source: (B) Head, Department of Microbiology, Meenakshi
Prevention (CDC), Atlanta (with permission). Medical College, Chennai (with permission).

BHASCARIASIS
Problem Solving Exercise 8
A 4-year-old child came to the OPD with history Q Labeled diagram of ova is shown in Figure 25.12B.
of severe acute abdominal pain associated with O Infective form: Embryonated egg
nausea and vomiting. On examination, the child was Q Mode of transmission: Ingestion of contaminated
malnourished. The stool specimen was sent for direct food and water containing embryonated eggs.
microscopy (Fig. 25.12A). The gross specimen of the Q Complications: Small-bowel obstruction and
causative agent is displayed (Fig. 25.13). intussusception,
1. Identify the causative agent based on microscopic For further details, refer Tables 25.4 and 25.5.
findings and gross morphology.
2. Drawalabeled diagram of the ova of the causative
agent.
=e Mamillated
albuminous
3. Which is the infective stage and mode oftransmis-
ee coat

sion? Unsegmented
4. Howwill you diagnose this condition in the labora- ovum
tory?
5. Mention two complications caused by the adult
Se ey Crescentic

worm.
A iiakalieamit =a0 B| space
a |

Figs 25.12A and B: Ascaris fertlized eggs: (A) In saline

Explanation mount; (B) Schematic diagram.
It is a case of ascariasis; caused by A. lumbricoides. Source: DPDx Image Library, Centers for Disease Control and
Points in favor are: Prevention (CDC), Atlanta (with permission).
Q History of severe acute abdominal pain, vomiting
and malnutrition. |
Q Figure 25.12A: Stool microscopy showed round
to oval bile-stained egg with thick albuminous
coat (mammillated)— indicates fertilized egg of A.

Q
lumbricoides.
Figure 25.13: Gross specimen revealed cylindrical
J)
Fig. 25.13: Adult worm of Ascaris lumbricoides.
worm (hence called as roundworm); 15-31 cm Source: Head, Department of Microbiology, Meenakshi Medical
long with tapering ends. College, Chennai (with permission).
 “Apoqijue JUSdSONJOUNWUI ‘Y/4] ‘YIEAH JO SeINISU| |EUONeN ‘HIN ‘Resse JUaquOSOUNWIW! paxUl|-ewAZu—a ‘YS}1F ‘SUONDIAZIqGY
ajozepuaqye ajozepuaqye
UNIIUUIA| JO ajozepuege;w ajozepuaq|y ajozepuaq|e 10 ajozepueqey\ JO ajozepuaqew quawjyealy
uaping WOM ay} MOU (GL 1°S7'b14) Ajases paqdaqep
0} WAOMYOOY JO} aUOP (pouUjaW Z}ey-07ey)) BuyUNOD 66 aq osje Aew sajewiay piIAei
poyjew ajejd seby (11) ‘enbiuydeai WOW—epeieH (I) spouyoy) asodind jesibojoiwapide (D pue VLL'SZ
(9°SZ a]qeL) WOMYooY WO saplojAbuoys ayelusia}ip O| pue aseyd Ayeuow nd sBl4) GEMS HIN Palje> qems
(7) ease] Uosely 10J—(W4I ‘WSI14) ABojosas z — jeinads 10 ade} aueydojja2 sisoubeip
Buruiwexa hq pwojsojAouy pue JojDIaN ayenuaiayig e (Aei-X ea WiNHeq) ausajul —-Aq pa}da|jod sqems |euelied © Asoyesoge| jo
:0] DUOP $184N}jNI}O0}S —_-UU] WOM YNPe Jo U0!}99}9q "| sajdwes — spoyjeaw JeuiO
saJaWojse|q Ino} s6ba pezi|ijuajyun pue paz! ease jeueled ul s6hb3 (SSz a|qeL)
uw
(9°Sz aiqeL) (7) ease] WuOyIIpqeyy yum sh6a pajuauibas —-|1aj—sHHa payeuoAiquiauy paysodap shba payeuoAiquy pajyeuokiquiauy) Wo} 1s0ubeig
S
v uonwinujeyy e uojouny
= sjuaijed pa}dajul-A|H Ul UOWWWWOD ejwwaue eed aAaj—JIHJal|y_ aAIUH0) peaseduu!
¥ —uo!}Dajul Waysks sNOAJaU Adualyap ud] e D109 Aueijig pue uoljepiejas
Ss jeujuad ‘payeulwassip—awopuds !pbo] Wom AADa}{ uoljdadsnssnju| e YIMOID
s uondesuedky—pvgy DAD] AavayyY eayLelp Auojewweyul uawopge uled e sso] 1yHIam asdejoid
£ sso| poojq pue uled duuysebidq e uO!}DNAYsqO Jamo ||eWIS e pue ued jeulwopqy e je]De1 JUSIINIDY
= ‘eayueip ‘easneu ‘ued d1jsebide PDO] WOM pjl|N uolepiejal sisainua |PUINIION e elwaue
w 32npoid—ppo] W10M pili aseyd jeuljse}uj ymosb pue uoNnujeW e uolbai jeueliad ul Adualdyap Uudl| e
s aseud jeuljse}uy syoejj snoulbidias e aseydjeunsaju; s6be Huljesaq!) Wi0M ayy awoipus
g yses jeuedin saysel (sniuownaud s,jayyja0q) jo UoNesBiuW yeusNjDOU 34} Ajayuashp snyoul
= snoulbidias—suawind DAADT = 1YWANId—D1! punoly e ejuouUNaud dI|iydoulsoqZ jo ynsai e se yyHiu je asiOM eixaioue
} aseyd Aso} e161y aseyd Asojesbiyy aseyd Asojesbiw uayo snyunadjeueliag e = ‘uledjeulWopqy e suole}sasiuey)
in (Ul}s
(uns ‘sBun}) ease] Bunesbiyy ‘sBun}) ease] Huei) (sbun}) ease; Bunesbiw (euisejul) UOJ
=
ud (@uI}Sa}U!) WOM INpY (@UI}SOzU!) WOM IjNpY (@uI}se}U!) WOM INpY (@uIgsayul) WOM YNpy WOM yNpY diuaboujed
= JUasald JUISAId JUasald juasgy juasqy aseud bun
a : SUIISO}U! |JLLWUS SUI}S9IU! ||ELUS SUIJSIU! |JELUS auljsaju! a61e7 auljsaju! a61e7 yeuqey
uoldajuloyne shba payeuoAiquia
shba payeuoAiquia Jo H6a payeuohiqua Hujureyuod JayeM pue
uoNdajuIOjNe Aq 40 BAIL] BAIR] (J) WOJe]Y Hujule}Uuod 19}eM pue poo} Hulujeyuod 13}eM Pue poo} poo} pa}eulwue}uod
(7) wuoyely Aq uonedjauad uns Aq uojje1j39Ued Uys payeurwejuod Jo uoNsabu| payeulwejuod jo uoNsabu| jo uonsebu; uOIssiWSUeL|
RAIL]
BAIR] (J) WHOJe|I4 BAIR] (J) WOJUE|I4 s66a payeuokiquuy =Bulujeyuod Hba payeuokiquig == sbba payeuoAiquig = WH0J BAND A4U]
sueWUNH suenH sueLUNH suewunH SUeLUNH JSOH
Saploziquiny siupdsy SIIDINIIUWAA $Nigosa}UZ
“SOPOJEWAU [CUIJSOJU! JO SDIFSHSIDEILYD JO SOINIe{ *H"°ST 9IGeL
200] SECTION2 © Systemic Microbiology (Infectious Diseases)

HB HOOKWORM INFECTIONS
Problem Solving Exercise 9
A 10-year-old child came to the pediatric OPD for What is the mode oftransmission?
school health check-up. On examination, he had What further laboratory diagnosis you would like
pallor. Peripheral blood smear revealed microcytic, to perform?
hypochromic anemia. The stool specimen was sent Explanation
for direct microscopy (Fig. 25.14).
It is a case of hookworm infection; caused by A.
a Identify the causative agent based on stool
duodenale or N. americanus. Points in favor are:
microscopy findings.
Q History of pallor and microcytic, hypochromic
Q Which is the infective form of the parasite?
anemia.
Q Figure 25.14: Stool microscopy showed round
to oval non-bile stained egg with segmented
ovum (four blastomeres)—indicates egg of
hookworm.
Q Infective form: Filariform larva
Q Mode of transmission: Skin penetration by
filariform (L,) larva.
Q As eggs of Ancylostoma and Necator are
indistinguishable, hence stool culture (e.g.,
Harada—Mori) should be performed. Eggs hatch
ace out to develop to filariform larvae (L,), which can
Fig. 25.14: Hookworm: Egg with four blastomeres. be used to differentiate between Necator and
Source: DPDx Image Library, Centers for Disease Control and Ancylostoma.
Prevention (CDC), Atlanta (with permission). For further detail, refer Tables 25.4 and 25.5.

i STRONGYLOIDIASIS
| Problem Solving
Solving Exercise
Exercise 10
10 _|
A 7-year-old child was brought to the OPD with Q Infective form: Filariform larva
history of epigastric pain, nausea, diarrhea, andblood Q Mode of transmission: Skin penetration by
loss. On examination, a serpiginous urticarial rash was filariform larva or by autoinfection.
observed in his lower limb. The stool specimen was Q_ Treatment: Ivermectin is the drug of choice.
sent for direct microscopy (Fig. 25.15). For further details, refer Tables 25.4 and 25.5.
1. Identify the causative agent based on the stool
microscopy finding. [
2. Name the infective form and modes of transmission
of infection by this parasite.
3. How will you treat this condition?
Explanation
It is a case of strongyloidiasis; caused by Strongyloides
stercoralis. Points in favor are:
Q History of epigastric pain, nausea, diarrhea and
blood loss.
QO Serpiginous urticarial rash, i.e., larva currens
Q Figure 25.15: Stool microscopy revealed larva of a ee —
250 um X 16 Um in size, with short buccal cavity Fig. 25.15: Strongyloides stercoralis (rhabditiform larva).
and prominent large genital primordium— Source: Department of Microbiology, Meenakshi Medical
indicates rhabditiform larva of Strongyloides. College, Chennai (with permission).
 CHAPTER 25 © Intestinal Helminthic Infections

Table 25.5: Characteristics or identifying features of eggs of intestinal nematodes.

Diagnostic form
Trichuris eggs Mucus plug
e Size: 50 um x 22 um Barrel shaped
e Shape: Barrel shaped
e Unembryonated ovum Ovum unembryonated
e Mucus plugs at both ends Wer lesy occa
e Bile stained Se |

Planoconvex in|
Enterobius eggs shape
e Size: 50-60 um x 20-30 um
e Shape: Planoconvex
e Embryonated ovum with a larva inside Non-bile stained
e Non-bile stained
Embryonated ovum |
with a larva inside |

nena “|
eee
Ascaris fertlized eggs
Mamillated
e Size: 50-70 um x 40-50 um albuminous
e Shape: Round to oval coat
e Surrounded by a thick mammillated,
albuminous coat Unsegmented
Crescentic space at poles ovum }}
e |}
Bile stained it
{
Crescentic space
Floats in saturated salt solution |
Lromate
Unembryonated large ovum
Ascaris unfertilized eggs |
e Size: 90 um x 45 um
Thin/scanty
e Shape: Elongated |
albuminous coat
e Albuminous coat is thin/absent |
e Nocrescentic space at poles
Ovum atrophied |
a Be : with refractile granules
e Does not float in saturated salt |
solution
—— _ ee
e Ovumis atrophied with a mass of
highly refractile granules
Hookworm eggs
e Size: 60 um x 40 um
Oval shaped
Non-bile stained, colorless
Surrounded by thin, hyaline shell
Clear space between the egg shell and Four blastomeres
ovum
e Egg is segmented with four
blastomeres Egg shell |
Eggs of both Ancylostoma duodenale and
Necator americanus are morphologically
indistinguishable; hence stool culture is
done for species identification. Both are
differentiated by their filariform larva
grown in culture.
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

Table 25.6: Differences between rhabditiform larva of hookworm and Strongyloides stercoralis.
Rhabditiform Strongyloides stercoralis
larva
Size 108-380 um long x 14-20 pm width 100-150 um long x 16 um width
Buccal cavity Shorter Three times longer
Genital Prominent and large Less prominent, small
primordium
Anal pore 50 um from the posterior end 80 um from the posterior end
Stool microscopy

——— tas io

Source: Department of Microbiology, Source: Department of Microbiology,

Meenakshi Medical College, Chennai Pondicherry Institute of Medical Sciences,
(with permission). Puducherry (with permission).
Schematic Genital primordium Double bulb Double bulb
diagram esophagus Intestine esophagus
Intestine

Buccal cavity Anal Genital Buccal cavity]

(smaller) pore — primordium (larger)
 Viral Hepatitis 6
9)

B HEPATITIS B VIRUS INFECTION

Exercise 1 e?
Solving Exercis
[Problem Solving |

A 40-year-old male presented with history of loss of Explanation

appetite, malaise and jaundice of 2 months duration. Clinical Diagnosis
On examination, there was icterus, hepatomegaly History of jaundice, hepatomegaly and tenderness in
and tenderness in the right hypochondriac region. the right hypochondriac region is suggestive of a case
He gave a history of blood transfusion in the past. of hepatitis.
Liver enzymes were found to be elevated. His serum
specimen was sent for hepatitis B surface antigen Laboratory Diagnosis
(HBsAg) tests as shown in Figures 26.1A and B. It is a case of hepatitis due to hepatitis B virus (HBV).
Q What are the test formats used and interpret the TestA is sandwich enzyme-linked immunosorbent
results? assay (ELISA) for detection of HBsAg, and test B
Q What are the investigations you would like to is immunochromatographic test (ICT) for rapid
advice further? detection of HBsAg. In both the tests, the sample was
Q List other hepatitis viruses and their modes of found to be positive for HBsAg.
transmission.
Interpretation
Q Describe the three morphological forms of this
Hepatitis B surface antigen may be present at any stage
agent seen under electron microscopy.
of hepatitis B infection ranging from carrier to acute and
chronic hepatitis. Hence, to determine the stage of the
disease further investigations should be done such as:
QO Hepatitis B precore antigen (HBeAg): If present,
indicates active viral replication and high infectivity.
Q Anti-hepatitis B core (HBc) antibody: Immuno-
globulin (Ig) M indicates acute and IgG indicates
chronic infection.
Modes of Transmission
There are five hepatitis viruses (Table 26.1).
Q Hepatitis A virus (HAV) and hepatitis E virus (HEV)
are transmitted by consumption of contaminated
food and water (feco-oral route).
Q Hepatitis B virus (HBV), hepatitis C virus (HCV) and
hepatitis D virus (HDV) are transmitted by blood,
sexual and vertical route.

Morphological Forms of HBV

Figs 26.1A and B: Test for detection of hepatitis
B Hepatitis B has three morphological forms; spherical
surface antigen. form, tubular form and complete form or Dane
Abbreviations: PC, positive control; NC, negative control particles (Fig. 26.2) These forms can be visualized
Source: Department of Microbiology, Pondicherry Institute
of under electron microscopy.
Medical Sciences, Puducherry (with permission). (For answers to other questions, refer text below).
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

Table 26.1: Features of hepatitis viruses.

HAV and HEV HBV, HCV and HDV

Both HAV and HEV are HBV is a DNA virus

RNA viruses HCV and HDV are RNA
viruses

Feco-oral transmission Blood (common), sexual,

and vertical
Incubation period: 15-50 Incubation period: 30-180
days,onset-abrupt days, insidious onset
No carriers Carriers seen
No chronicity Chronicity seen
Not oncogenic Oncogenic
Fulminant rare (<1%); Fulminant rare (1-2%)
except HEV in pregnancy except HDV (10-20%) B virus showing: 1, spherical form; 2, tubular form and
(10-20%) 3, Dane particle.
Non-enveloped; Enveloped; spherical Source: |ID# 5631/Public Health Image Library/Centers for Disease
icosahedron symmetry symmetry Control and Prevention (CDC), Atlanta (with permission).

| Problem Solving
Solving Exercise2__|
Exercise 2
Acute Hepatitis Explanation
In a patient with jaundice and elevated liver enzymes The stage of the disease is acute HBV infection with
for 2 months, the following serological investigations active viral replication and high infectivity.
are done with their results displayed below. Interpret Q_ IgM Anti-HBc antibody indicates—acute hepatitis
the diagnosis. Q HBeAg indicates—active viral replication and high
[HBsAg |Anti-HBs |Anti-HBc [HBeAg |Anti-HBe | infectivity.
= - IgM + -

Problem Solving Exercise 3

Chronic Hepatitis Explanation
In a patient with history of jaundice and elevated A: The stage of the disease is chronic active HBV
liver enzymes for 6 months, the following serological infection with high infectivity (immunoreactive
investigations are done. chronic hepatitis).
Two patterns of results are displayed below » IgG anti-HBc antibody indicates—chronic
(A and B). Interpret the diagnosis. hepatitis.
>» HBeAg indicates—active viral replication and
high infectivity.
A:
The same set of markers in an asymptomatic indi-
CI se vidual would suggest the stage as “supercarrier”,
+ - IgG + - B: The stage of the disease is chronic hepatitis B with
low infectivity (chronic inactive hepatitis).
» IgG anti-HBc antibody indicates—chronic HBV
B:
infection.
» HBeAg absent indicates—low infectivity.
[HBsAg |Anti-HBs |Anti-HBc |HBeAg |Anti-HBe | The same set of markers in an asymptomatic
+ = IgG = + individual would suggest the stage as “simple
carrier”.
 CHAPTER 26 © Viral Hepatitis

Problem Solving Exercise 4

Vaccination/Recovery Explanation
A patient is asymptomatic, with normal liver enzymes. A: The diagnosis here is post-hepatitis B vaccination.
He attended a master health check-up; the following Presence of anti-HBs antibody in the absence of
serological investigations were done. Two patterns of any other viral markers indicates vaccination with
results are displayed below (A and B). Interpret the HBsAg. Details about the vaccination—discussed
diagnosis. later in this chapter.
A: B: The diagnosis here is post-recovery from hepatitis

[HBsAg |Anti-HBs |Anti-HBc |HBeAg |Anti-HBe

— + — — ES
B infection. Along with anti-HBs antibody, anti-
HBc IgG indicates post-recovery..

B:

FHBsAg |Anti-HBs [Anti-HBc [HBeAg |Anti-HBe|

2S + + os a

[problem Solving
SolvingExercise5
Exercise 5 |
Carriers/Incubation Period Explanation
A person came to donate blood in a blood donation A: Presence of HBsAg in the absence of any other
camp. As a part of routine screening he was tested hepatitis B viral marker in an asymptomatic
for HBsAg, which came positive. He is asymptomatic, individual with normal liver enzymes suggests
with normal liver enzymes. The following serological that he is in incubation period of infection with HBV
investigations were done and their results shown (early acute hepatitis).
below. Interpret the results. B: Presence of HBsAg and anti-HBc IgG in the
A: absence of other hepatitis B viral marker in
an asymptomatic individual with normal liver
HBsAg |Anti-HBs |Anti-HBc |HBeAg |Anti-HBe | enzymes suggests that he is simple carrier of HBV
infection. In additon if HBeAg is also positive, then
= — i
a —

B: he will be called as supercarrier.

HBsAg |Anti-HBs |Anti-HBc |HBeAg |-Anti-HBe |
+ - IgG -

Laboratory Diagnosis of Hepatitis B Hepatitis B Surface Antigen

Definitive diagnosis of hepatitis B depends It is the first marker to be elevated following

infection; within 8-12 weeks.
on the serological demonstration of the viral
markers, which can be classified as: * It appears during incubation period.
“ Antigen markers: HBsAg and HBeAg * Presence of HBsAg indicates onset of infectivity.
* Antibody markers: Anti-HBs, anti-HBe and
* It remains elevated in the entire duration
anti- HBc of acute hepatitis; rarely persists beyond 6
months if the disease progresses to chronic
“+ Molecular markers: HBV DNA
hepatitis or in carrier state.
* Nonspecific markers: Elevated liver enzymes
and serum bilirubin. * It is used as an epidemiological marker of
hepatitis B infection.
The most useful detection method for HBV
antigens and antibodies is ELISA; although Hepatitis B Precore Antigen and HBV DNA
various rapid test formats such as ICT are also
available. They appear concurrently with or shortly after
Viral DNA can be detected by polymerase appearance of HBsAg in serum. They are the
chain reaction (PCR); real-time PCR is very
markers of active viral replication and high viral
useful for quantification of HBV DNA. HBV does infectivity. HBV DNA load is used to monitor the
response to treatment.
not grow in any conventional culture system.
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

Hepatitis B Core Antigen Anti-hepatitis B Precore Antibody

Hepatitis B core antigen (HBcAg) is a hidden Anti-HBe antibodies appear after the clearance
antigen, nonsecretory in nature; hence, it cannot of HBeAg and its presence signifies diminished
be detected in blood. However, can be detected viral replication and decreased infectivity.
in hepatocytes by immunofluorescence.
Anti-hepatitis B Surface Antibody
Anti-HBc IgM (Hepatitis B Core Antibody) It appears after the clearance of HBsAg and
Anti-HBc IgM is the first antibody to elevate remains elevated indefinitely.
following infection: “+ Its presence indicates recovery, immunity and
“ It appears within the first 1-2 weeks after noninfectivity (i.e., stoppage of transmission).
the appearance of HBsAg and lasts for 3-6 “+ It is also the marker of vaccination if rest all
months. markers are negative (Fig. 26.3).
“ Its presence indicates acute HBV infection. Various outcomes following HBV infection and
markers for diagnosis is depicted in Fig. 26.3.
Anti-HBc IgG (Hepatitis B Core Antibody)
Anti-HBc IgG appears in late acute stage and Treatment
remains positive indefinitely whether the patient Most acute hepatitis B infections are self-limiting;
proceeds to—chronic stage or carrier state or do not require any specific treatment. In contrast,
recovery. It can also be used as epidemiological treatment of chronic hepatitis B may require
marker of HBV infection. antiviral drugs such as tenofovir and telbivudine.

|| Following | =| Following ~~-—> Incubation period

| hepatitis Bo | hepatiisB = = + HBsAg +ve
| | vaccination | ___ infection | ~—+ All other markers —ve
+ Symptoms —ve
+ Liver enzymes —ve

| Acute hepatitis
* HBsAg +ve
* IgM HBc +ve
* HBeAg +ve (usually)
* Symptoms ++
+ Liver enzymes ++

ee * _ ¥90-95% |Rare (5%) 4 Rare (5%)

Post-vaccination | ___ Recovery —«—— Chronic hepatitis > Carrier
* HBsAb +ve * HBsAb +ve * HBsAg +ve * HBsAg t+ve
* Symptoms —ve * Symptoms —ve * IgG HBc +ve * IgG HBc +ve
- Liver enzymes —ve * Liver enzymes —ve * Symptoms ++ + Symptoms —ve
* History of vaccination *lgG HBc +ve » Liver enzymes +ve * Liver enzymes —ve
* Rest all markers: —ve
i

| Chronic active
7 Franag
Immunotolerant fren
| hepatitis Chronic inactive chronic HBV Cron eC |
| (Immunoreactive hepatitis infection | HBV lnfection ||
| hepatitis) (super carriers) — bes WE a ||
| In addition In addition In addition In addition
| HBeAg +ve HBeAg —ve HBeAg +ve ||
HBeAg —ve

Fig. 26.3: Various outcomes following hepatitis B infection and markers for
diagnosis.
 CHAPTER 26 © Viral Hepatitis

Prevention protection is warranted; given at dose of 0.06 mL/

kg or 10-12 IU/kg.
Hepatitis B Vaccine (Active Immunization)
*» Acutely exposed to HBsAg positive blood, e.g.,
Hepatitis B vaccine is a recombinant subunit surgeons, nurses, laboratory workers
vaccine of HBsAg, prepared in Baker’s yeast by “+ Sexual partners of acute hepatitis B patients
DNA recombinant technology. “+ Neonates borne to hepatitis B carrier mothers
“+ Route: Administered by intramuscular route * Post-liver transplant patients who need
over deltoid (in infant—anterolateral thigh) protection against HBV infection
* Dosage: 10-20 yg/dose (half of the dose is “+ Following accidental exposure.
given to children below 10 years) The post-exposure prophylaxis for HBV is
“+ Schedule: Three doses are given at 0, 1 and discussed in Chapter 14.
6 months. Under National Immunization
Schedule, it is given at 6, 10, 14 weeks (along
@ OTHER HEPATITIS VIRUSES
with DPT vaccine)
“ Marker of protection: Recipients are said to Both ELISA and rapid tests (e.g., immunochro-
be protected if the anti- HBsAg antibody titer matographic or flow through assay) formats are
is above 10 mIU/mL. available for detection of anti-HAV, anti-HCV
and anti-HDV antibodies. Apart from molecular
Hepatitis B Immunoglobulin methods like PCR and real-time PCR are available
Hepatitis B immunoglobulin (HBIG) is used in for detection of viral RNA in blood (for HCV and
the following situations where an immediate HDV) and from stool specimen (HAV and HEV).

Gini eee
Solving Exercise 6
A patient with history of jaundice and elevated liver done. Three patterns of results are displayed below
enzymes, the following serological investigations are (A, Band C). Interpret the diagnosis in all test patterns.
A:

A i CCC
HBsAg —ve Anti-HCV IgG +ve Anti-HDV —ve Anti-HEV —ve
Anti-HAV IgG +ve

HBsAg —ve Anti-HCV —-ve Anti-HDV —ve Anti-HEV —ve

Anti-HAV IgM +ve
Cc:

mv YL Cis
IgM +ve
HBsAg +ve Anti-HCV -ve Anti-HDV Anti-HEV —ve
Anti-HAV —ve
Anti-HBs —ve
Anti-HBc IgM +ve
HBeAg +ve
Anti-HBe —ve

Explanation
A. Anti-HCV IgG +ve in a patient with jaundice > Positive for anti-HDV lgM—indicates acute
hepatitis D infection
indicates chronic hepatitis C virus infection. Please
note that there is no chronic infection in HAV and > Positive for HBsAg, anti-HBc IgM and HBeAg—
HEV; hence detection of anti-HAV IgG indicates acute hepatitis B infection with high infectivity.
past infection or recovery. Note: However, in case of superinfection (HDV
B. Anti-HAV IgM +ve in patient with history of jaundice
infects a hepatocyte which is already infected by
indicates acute hepatitis A virus infection. HBV)—Positive for HBsAg, anti-HBc (IgG) and anti-
HDV (IgM).
C. It is a case of acute HDV coinfection with acute
hepatitis B infection with high infectivity. Points in
favor are:
 Parasitic Infections of Hepatobiliary
System: Amoebic Liver Abscess, 2 7
Hydatid Disease and Others
PEEING

* Nematode: Toxocara causing visceral larva

BINTRODUCTION
migrans.
Parasites causing hepatobiliary infections
include: @ AMOEBIC LIVER ABSCESS
* Protozoa: Entamoeba histolytica, causes
amoebic liver abscess (ALA) Invasion of trophozoites lead to various
* Cestode: Echinococcus, causes human extraintestinal complications. Amoebic liver
echinococcosis (e.g., hydatid disease) abscess is the most common complication;
** Trematodes such as Fasciola hepatica, F. others are amoebic appendicitis, ameboma,
gigantica, Clonorchis and Opisthorchis fulminant colitis and ruptured liver abscess
leading to peritonitis.

| Problem
Problem Solving
Solving Exercise
Exercise1_|
1
A patient with history of dysentery and jaundice for dysentery and jaundice, suggest ruptured
2 months had a sudden episode of high-grade fever amoebic liver abscess, leading to acute peritonitis
and acute pain in the right hypochondrium was and death of the patient.
brought to the emergency department. Ultrasound Q The causative agent for this clinical condition is
scan of the abdomen revealed enlarged liver with Entamoeba histolytica.
acute peritonitis. He succumbs to death before any Me answers to other Keene refer below).
intervention. A postmortem sample from the patient ine
| rer. wee —
——ss=—“‘i~“C
_ {

is displayed in Figure 27.1. | |

1. Identify the clinical condition based on the lesion

displayed and the most probable causative
agent.
2. Name the laboratory tests that can be done to
confirm the diagnosis.
3. What are the complications of intestinal
amoebiasis?

Explanation
This is a case of ruptured amoebic liver abscess with
acute peritonitis. Points in favor are: Fig. 27.1: Cross section of liver showing amoebic liver
Q Acute onset of pain in the right hypochondrium abscess.
with high-grade fever, in a patient with chronic Source: Head, Department of Pathology, Meenakshi Medical
College, Chennai (with permission).

Laboratory Diagnosis of ALA Trophozoites measure 15-20 um, possess a

* Microscopy of liver pus (Anchovy sauce single nucleus; are actively motile, with finger-
pus)—trophozoites of E. histolytica are seen like pseudopodia (Fig. 24.2B, Chapter 24)
* Trophozoites may be found only in the last ** Lectin antigen detection and antibody detec-
portion of the aspirated material from the tion methods like indirect hemagglutination
abscess wall, not in the necrotic debris test, indirect fluorescent antibody test, ELISA,
obtained from the center of the abscess. etc., are useful.
 CHAPTER 27 © Parasitic Infections of Hepatobiliary System

** Histopathological staining of pus aspirate — amoebicidal agent to eradicate the intestinal

demonstrates amoebic.trophozoites carriage
“* Ultrasonography of liver shows the site and m Tissue agents: Metronidazole (for 5-10
extension of abscess. Posterior superior days) or tinidazole/ornidazole (once) and
area of right liver is the most common site = Luminal agents: lodoquinol (for 20 days)
affected. or paromomycin (for 10 days).
** Aspiration of the liver abscess content is
Treatment indicated—(1) risk of impending rupture,
* Amoebicidal agents: Treatment of ALA (2) left lobe liver abscess of >10 cm, (3) no
consists of a tissue amoebicidal agent (acts on improvement after anti-protozoan therapy
trophozoites in the liver), followed by luminal for 5-7 days.

B HYDATID DISEASE

| Problem Solving
Solving Exercise2_
Exercise 2
A 40-year-old man presented with complaints of Q The causative agent—Echinococcus granulosus
pain in the right hypochondrium. Ultrasonography Q Host: Definitive (dog) and intermediate host
revealed a single space-occupying lesion in the right (sheep, man)
lobe ofthe liver. This was surgically removed (Fig. 27.2) Q Infective form: Eggs
and subjected to histopathological examination (Figs Q Mode of transmission: Ingestion of food and water
27.3A and B). contaminated with dog's feces containing eggs.
1. Identify the specimen. For answer to the other questions, refer Table 27.1.
2. Draw a neat, labeled diagram of the structure
focused in the slide.
3. What is the causative agent of this condition?
Name the definitive and intermediate hosts.
5. Which is the infective form ofthe parasite for man
and how does man acquire this infection?
6. What are the various diagnostic modalities?
Explanation
This is a hydatid cyst specimen (Fig. 27.2), obtained
surgically from a patient suffering from right
hypochondrial pain and cystic lesion in liver detected
by ultrasound scan.
Q Figures 27.3A and B show histopathological
section of hydatid cyst: (A) All three layers of cyst
wall are seen—pericyst, ectocyst and endocyst; (B)
Endocyst with attached brood capsules. Fig. 27.2: Surgically resected hydatid cyst from liver.
Q Labeled diagram of section of hydatid cyst (Fig. Source: Head, Department of Pathology, Meenakshi Medical
College, Chennai (with permission).
27,38).
Brood capsules |

Ch
Figs 27.3A to C: Histopathological section of hydatid cyst (hematoxylin and eosin stain). (A) Cyst wall—pericyst,
ectocyst and endocyst; (B) Endocyst with attached brood capsules; (C) Schematic diagram ofsection of hydatid cyst.
Source: Head of Department (Pathology), Meenakshi Medical College, Chennai (with permission).
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

Table 27.1: Hydatid disease (cystic echinococcosis).

Hydatid diseas
Agent Echinococcus granulosus
Hosts Definitive host: Dog, Intermediate host: Sheep, man (accidental)
Infective form Eggs
Transmission Consumption of contaminated food and water (ingestion)
Habitat Tissues: Liver (most common), lungs, brains and others
Pathogenic form __ Larva (hydatid cyst)
Manifestations e Pressure effect of the enlarging cyst: leads to palpable abdominal mass, hepatomegaly,
abdominal tenderness, portal hypertension and ascites
e Secondary bacterial infection
e Anaphylactic reactions due to cyst leakage or rupture
Diagnosticform Hydatid fluid microscopy (direct mount or staining with acid-fast stain)—detects brood
capsules and protoscolices
e Histological examination (H & E)—demonstrates cyst wall and attached brood capsules
e Antibody detection—ELISA (using B2t antigen), DIGFA (dot immunogold filtration assay) and
western blot
e Imaging methods—x-ray, USG (demonstrates Water lily sign), CT scan, MRI
e Molecular methods—PCR, PCR-RFLP and molecular typing (10 genotypes, most common in
India is type 1)
e Skin test (Casoni test)—demonstrates type | hypersensitivity reaction (obsolete now)
Treatment e Albendazole
e Surgery (resection of affected part of liver)
PAIR (percutaneous puncture, aspiration, injection and re-aspiration)

@ TREMATODE INFECTIONS OF LIVER of the bile duct, leading to fibrosis and

cholangiocarcinoma. Demonstration of the
Fasciola hepatica, Fasciola gigantica, Clonorchis, characteristic flask-shaped eggs (measuring
and Opisthorchis are together called as liver 28-35 tum x 12-19 jim) in the stool establishes
flukes. the diagnosis (Fig. 27.4B)
**Oo Hosts: They have three hosts. Humans are the * Treatment of liver fluke infections:
definitive host, snails are the first intermediate = Triclabendazole is the drug of choice for
host, whereas the second intermediate host is fascioliasis
aquatic plant (for Fasciola) and cray fish (for
= Praziquantel is the drug of choice for
Clonorchis, and Opisthorchis)
clonorchiasis and opisthorchiasis.
* Transmission: Man gets infection by
ingestion of second intermediate host carrying
metacercaria larvae (infective form) =
* Fasciola hepatica and F. gigantica: They
infect liver; cause fever, right upper quadrant
pain, and hepatomegaly. F hepatica is
diagnosed by detection of characteristic
operculated eggs, measuring 130-150 jum x
63-90 jum in size in stool microscopy (Fig.
27.4A). Eggs of F gigantica are morphologically
Figs 27.4A and B: Saline mount showing operculated
similar to that of Fhepatica, but larger in size
eggs of: (A) Fasciola hepatica; (B) Clonorchis or Opisthorchis.
* Clonorchis and Opisthorchis: They infect
Source; DPDx Image Library, Centers for Disease Control and
bile duct and cause mechanical obstruction Prevention (CDC), Atlanta (with permission).
 Staphylococcal Infections

STEREO

| __ProblemSolvingExercise
Solving Exercise
A 55-year-old male diabetic patient admitted to Identification
the hospital with complaints of severe calf pain and Gram-positive cocci in cluster with pus cells in Gram-
pus discharge from the ingrown hair. On physical stained smear of pus discharge (Fig. 28.1A), golden
examination, the local area was found to be red, yellow hemolytic colonies on blood agar (Fig. 28.2B),
warm and tender. Pus aspirated was subjected to the mannitol fermented (Fig. 28.3A) and tube coagulase
following tests: Gram stain (Fig. 28.1A), culture (Fig. test positive (Fig. 28.4A) indicate that the causative
28.2B), biochemical reactions (Figs 28.3A and 28.4A) organism is Staphylococcus aureus.
and antimicrobial susceptibility testing (Fig. 28.5 and
Table 28.3) Antimicrobial Susceptibility Testing
1. What is the clinical diagnosis and its causative Antimicrobial susceptibility testing on Mueller Hinton
organism? agar (Fig. 28.5 and Table 28.3) showed resistance to
2. Interpret the antimicrobial susceptibility testing. penicillin and cefoxitin, and sensitive to clindamycin,
3. List the infections caused by this organism. doxycycline and linezolid.
4. What antibiotic you would like to prescribe? As the isolate is resistant to cefoxitin; this isolate is
5. What are the different modalities of laboratory methicillin-resistant S. aureus (MRSA).
diagnosis?
Treatment
6. What is MRSA? How will you detect, treat and
Methicillin-resistant S. aureus is resistant to all beta-
prevent its transmission?
lactams; hence all beta-lactam drugs should be
Explanation avoided in this case. Doxycycline, clindamycin or
Clinical Diagnosis linezolid are the useful options available for this
Diabetic patient with clinical features of severe calf patient (skin and soft tissue infection).
pain with pus discharge with clinical signs of local (For answers to other questions, refer below)
inflammation indicates skin and soft tissue infection.

CLINICAL SPECTRUM Clinical spectrum of S. aureus infections

Staphylococcus aureus produces a range of Skin and soft tissue infections
Q Folliculitis, furuncle, carbuncle, impetigo, mastitis
manifestations; which is attributed to production
and breast abscess (in nursing mothers), surgical
of several virulence factors by the organisms site wound infections, cellulitis, etc.
such as: Musculoskeletal infections
+,
Cell wall factors such as peptidoglycan, Q Septic arthritis, osteomyelitis, pyomyositis, psoas
abscess and epidural abscess
protein A, clumping factor and teichoic acid
Respiratory tract infections
Hemolysins and leukocidin (panton-valentine Q Ventilator-associated pneumonia, post-viral pneu-
toxin) monia (e.g., influenza), empyema, pneumatocele, etc.
Toxins: Epidermolytic toxin, enterotoxin and Bacteremia and its complications
toxic shock syndrome toxin Q Sepsis, central line associated bloodstream
infection, infective endocarditis and urinary tract
Enzymes: Coagulase, nucleases, deoxyribonu- infection (UTI)
clease (DNase), etc.
Contd...
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

Contd... pigment), 1-3 mm in size, circular, convex and

opaque (Fig. 28.2B). However, pigmentation
Toxin-mediated illness is better appreciated on nutrient agar (Fig.
Q Toxic shock syndrome, food poisoning and
staphylococcal scalded-skin syndrome 28.2A).
“ Selective media are useful when staphylococci
are expected to be scanty or outnumbered by
LABORATORY DIAGNOSIS other bacteria in the sample (e.g., swabs from
carriers, feces); e.g., mannitol salt agar (Fig.
Sample Collection
28.2C).
It depends on the site affected (Table 28.1). The
discussion in this chapter will be confined to Culture Smear Microscopy
skin and soft tissue (suppurative) infections. Gram staining from the colonies shows gram-
positive cocci (1 xm), arranged in clusters (Fig.
Direct Smear Microscopy
28.1B).
Gram staining of pus or wound swab reveals
pus cells with gram-positive cocci in clusters Biochemical Tests for identification
(Fig. 28.1A).
Staphylococci are catalase positive which
Culture differentiates them from streptococci (catalase
negative). Once genus identification is done,
Specimens are inoculated into various media
the following tests should be carried out to
and incubated overnight at 37°C aerobically. The
differentiate S. aureus from other Staphylococcus
colony morphology observed is as follows:
** Blood agar: Colonies are B-hemolytic with species (coagulase-negative staphylococci,
CoNS).
golden yellow in color (due to non-diffusible +,od
Coagulase test (tube and slide)
Table 28.1: Specimen collection for S. aureus infections. o“
Deoxyribonuclease test
ce oo +,“
Phosphatase
Suppurative lesion Pus, wound swab +,“~
Golden yellow pigmentation
Respiratory infections Sputum °“
Hemolysis on blood agar
Urinary tract infection Mid-stream urine +,“
Mannitol fermentation: S. aureus ferments
Pyrexia of unknow origin Blood mannitol, indicating change in the color of
(PUO), bacteremia
the medium from pink to yellow (Fig. 28.3A)
Food poisoning Feces, vomitus, food
** Protein A detection.
Carriers Nasal and skin swab
Coagulase Test
RRL
| iv, Oe .
ee
se oe ry A It is the most commonly used biochemical
reaction for identification of S. aureus (Table
28.2). There are two types of coagulase tests.
1. Tube coagulase test: It detects free coagulase.
= Colony of S. aureus is emulsified in a broth
of S. aureus and incubated for 4 hours at
ih Gre
Rech
PSee
= Positive testis indicated by formation ofa
clot (Fig. 28.4A).
= The negative test is indicated by no clot
formation (Fig. 28.4B).
Figs 28.1A and B: (A) Direct smear: arrow showing gram-
positive cocci in clusters with pus cells; (B) Culture smear 2. Slide coagulase test: It detects clumping
showing gram-positive cocci in clusters. factor (i.e., bound coagulase).
Source: Department of Microbiology, JIPMER, Puducherry (with = A colony of S. aureus is emulsified with a
permission).
drop of normal saline to form a milky white
 CHAPTER 28 © Staphylococcal Infections

he eee AS
Figs 28.2A to C: Colonies of 5. aureus: (A) Nutrient agar—shows golden-yellow pigmented colonies; (B) Blood agar—
arrow shows zone of beta hemolysis surrounding the colonies; (C) Mannitol salt agar shows yellow-colored colonies of S.
aureus due to fermentation of mannitol.
Source: Department of Microbiology, Pondicherry Institute of Medical Sciences, Puducherry (with permission).

Positive Negative
ee
noses

Positive Negative
|Ae

Figs 28.4A to C: Coagulase test: (A) Tube coagulase test

(positive); (B) Tube coagulase test (negative); (C) Slide
showing coagulase test.
Source: Department of Microbiology, Pondicherry Institute of
Medical Sciences, Puducherry (with permission).
A |
Figs 28.3A and B: Mannitol fermentation test; (A) Positive
(mannitol fermented); (B) Negative (mannitol non-fermented).
Source: Department of Microbiology, JIPMER, Puducherry (with
permission).

Table 28.2: Tube coagulase and slide coagulase test.

Tube coagulase Slide coagulase
Due to coagulase enzyme Due to clumping factor
Requires CRF in plasma Does not require CRF in
plasma
Done in tube Done in slide
Positive if clot is formed Positive if clumps are formed Fig. 28.5: Antimicrobial susceptibility testing on Mueller
Abbreviation: CRF, coagulase reacting factor. Hinton Agar [Refer Table 28.3 for Clinical and Laboratory
Standards Institute (CLSI) zone interpretation].
suspension. Then a drop of undiluted Source: Department of Microbiology, Pondicherry Institute of
Medical Sciences, Puducherry (with permission).
plasma is added and mixed properly.
= Positive result is indicated by formation of
Antimicrobial Susceptibility Test
coarse clumps (Fig. 28.4C).
Staphylococci are also identified up to species As S. aureus develops resistance to antibiotics
level by automated identification systems such readily, drugs should be prescribed according
as MALDI-TOF or VITEK, which detect more to the antimicrobial susceptibility test (disk
accurately and faster than biochemical tests. diffusion) done on Mueller Hinton agar (Fig. 28.5)
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

to various antimicrobial
Table 28.3: Interpretative categories (CLSI) and observed zone size diameter (mm)
agents tested for Staphyloc occus aureus isolate.

Disk (ug) CLSI interpretative criteria for Observed Interpretation

Antimicrobial
agents strength | S. aureus (inmm) zone size (in

|Sensitive |Tis:Fig. 28.5 “0

[Resistant |intermediate
Benzyl penicillin(P) 1Ounits <28 229 12 Resistant

Cefoxitin (Cn) 30 <21 - >22 15 Resistant (MRSA)*

Clindamycin (Cd) 2 <14 15-20 221 22 Sensitive

Linezolid (Lz) 30 <20 - 221 24 Sensitive

Doxycycline (Do) 30 <12 13-15 216 18 Sensitive

Note:
* Methicillin-resistant S. aureus (MRSA) is resistant to all beta-lactam antibiotics.
Note: Disk diffusion is not recommended for susceptibility testing of vancomycin to S. aureus isolates (as there are no standard
interpretative criteria); therefore should only be reported based on MIC testing (e.g., VITEK).

and zone interpretation is done as per Clinical and on S. aureus cell membrane to PBP-2a. The
Laboratory Standards Institute (CLSI) guidelines altered PBP-2a of MRSA strains has less
(Table 28.3). affinity for beta-lactam antibiotics; hence
MRSA strains are resistant to all beta-lactam
Treatment antibiotics.
For MSSA (methicillin sensitive S. aureus): Beta- ** Detection of MRSA is carried out by:
lactams such as cloxacillin, cefazolin (for serious m Cefoxitin disk diffusion or MIC testing
systemic infections) or cephalexin (for local = Oxacillin screening agar or MIC testing
skin and soft tissue infections) are the preferred ® Polymerase chain reaction (PCR)
drugs. Vancomycin is inferior to beta-lactams for detecting mec A gene
the treatment of MSSA infections. ** Prevention: As it is transmitted by contact
For MRSA: MRSA strains are resistant to all beta-
from HCWs and environment; thorough
lactams (except fifth generation cephalosporins handwashing and other methods of contact
such as ceftaroline); hence beta-lactams are
precaution (Chapter 11) are the important to
contraindicated. Vancomycin is the drug of prevent its spread.
choice. Alternatively linezolid, daptomycin,
Coagulase-negative Staphylococcus
clindamycin, doxycycline, etc. are also useful.
Most of the coagulase-negative Staphylococcus
Staphylococcus aureus shows resistance to beta-
(CoNS) are harmless skin commensals; however,
lactam antibiotics; mainly by two mechanisms.
recently their role as pathogens is increasingly
* Production of penicillinase enzyme (90% of
been reported.
S. aureus).
* Staphylococcus epidermidis: It causes stitch
* By alteration of penicillin-binding protein
abscess and prosthetic device-related
(PBP): It is shown by MRSA strains, which
infections such as endocarditis with insertion
accounts for 30-40% of S. aureus isolates.
of valvular prosthesis and ventricular shunt
Methicillin-Resistant Staphylococcus infections.
aureus (MRSA) ** Staphylococcus saprophyticus: It causes UTI
in sexually active young women.
MRSA is mediated by mec A gene; which is
chromosomally coded. It alters PBP present
 |
CHAPTER|
Beta-hemolytic Streptococcal Infections 29

BINTRODUCTION observed with viridans streptococci (Chapter

16) and pneumococci (Chapter 34)
Streptococci are catalase negative gram-positive
“: B-hemolysis or clearing of blood surrounding
cocci arranged in pairs or chains (Fig. 29.1A).
the colonies: Itis due to complete lysis of RBCs.
They can be classified based on the hemolysis
produced on 5% sheep blood agar into a, 8B and It is observed with Streptococcus pyogenes
y-hemolytic streptococci. (group A) and S. agalactiae (group B)
“* a-hemolysis: It is due to partial lysis of | Y-hemollysis: There is no hemolysis surround-
red blood cells (RBCs); there is greenish ing the colonies; e.g., Enterococcus (Chapter
discoloration surrounding the colonies. It is 42).

F Problem Solving
Solving Exercise1
Exercise 1 _|
Necrotising fasciitis (Group A Streptococcus) and crepitus is suggestive of a case of necrotising
A 51-year old male, presented with right foot pain and fasciitis.
swelling over 2 weeks, with 10-year history of poorly |gentification
controlled diabetes mellitus. Clinical examination
Gram-positive cocci in chain and pus cells seen on
revealed a gangrenous lateral two toes, with pus
direct smear from specimen, pinpoint colonies on
discharge and associated warmth and crepitus on
blood agar with wide zone of beta-hemolysis (Fig.
the affected area. Following wound debridement,
29.2A), gram-positive cocci in chain in culture smear
tissue cultures were taken perioperatively and sent
(Fig. 29.1B), Bacitracin sensitive (Fig. 29.2B) are
to the microbiology laboratory for Gram stain (Fig. indicative that the causative organism is Streptococcus
29.1 A), culture (29.2 A), culture smear (Fig. 29.1B) and
antimicrobial susceptibility testing (AST) (Fig. 29.2B, heels
29.3 and Table 29.3). Antimicrobial Susceptibility Testing
1. Identify the causative agent based on the Antimicrobial susceptibility testing on Mueller Hinton
microbiological investigations performed. blood agar (MHBA) Fig. 29.3 and Table 29.3 showed
2. List the infections caused by this organism. sensitive to penicillin, azithromycin and clindamycin
3. Interpret the AST. and intermediate to erythromycin.
4. What antibiotic you would like to prescribe?
5. What are the different modalities of laboratory Treatment
diagnosis? Surgical debridement (most crucial) along with anti-
’ biotics, such as penicillin G plus clindamycin (standard
Explanation treatment given for necrotising fasciitis).
Clinical Diagnosis (For answers to other questions, refer below).
A diabetic patient with gangrenous lateral two
toes, with pus discharge and associated warmth
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

STREPTOCOCCUS PYOGENES BR
INFECTIONS
Clinical Manifestations of S. pyogenes
Group A Streptococcus (GAS) produces both
suppurative and non-suppurative manifesta-
tions (Table 29.1).

Laboratory Diagnosis
Specimen Collection and Transport
¢
Commonly collected specimens are throat swab,
Figs 29.1A and B: Streptococci: (A) In Gram stained smear
pus swab, exudates and blood, based on the of pus; (B) In culture smear showing gram-positive cocci
clinical manifestation. in chains.
Source: Department of Microbiology, Pondicherry Institute of
Direct Smear Microscopy Medical Sciences, Puducherry (with permission).

Gram staining of pus or wound swab reveals pus

cells with gram-positive cocci in short chains are small, pinpoint, circular, with a wide zone of
(Fig. 29.1A). However, direct microscopy is not B-hemolysis (Fig. 29.2A).
much useful when S. pyogenes is a part of normal
flora in the sample (e.g., throat swab). Culture Smear Microscopy
Gram-stained smear from the colonies shows
Culture
gram-positive spherical cocci, arranged in short
Streptococcus pyogenes is fastidious, does not chains (Fig. 29.1B).
grow in basal media. On blood agar colonies
Biochemical Tests for Identification
Table 29.1: Suppurative and nonsuppurative manifes- Catalase test: Streptococci are catalase negative;
tations of Streptococcus pyogenes. in contrast to staphylococci which are catalase
positive.
Respiratory infections Acute rheumatic fever
(Refer Chapter 33): (Refer Chapter 16)
e Pharyngitis/sore throat
Acute glomerulonephritis
e Scarlet fever
e Others (rare):
Pneumonia, empyema,
quinsy, sinusitis and
otitis media
Skin and soft tissue Guttate psoriasis
infections (superficial):
Reactive arthritis
e Impetigo (pyoderma)
e Cellulitis and erysipelas Bacitracin
Deep soft tissue PANDAS a. |
infections: (Pediatric Autoimmune
e Necrotizing fasciitis Neuropsychiatric
e Streptococcal myositis Disorders Associated with
Bacteremia leading Streptococcal infections)
to endocarditis,
osteomyelitis, septic
Figs 29.2A and B: Streptococcus pyogenes: (A) Growth on
arthritis, meningitis, etc.
blood agar with wide zone of beta-hemolysis around the
Toxic shock syndrome pin point colonies; (B) Bacitracin sensitive.
Puerperal sepsis (rare) Source: Department of Microbiology, Pondicherry Institute of
Medical Sciences, Puducherry (with permission).
 CHAPTER 29 © Beta-hemolytic Streptococcal Infections

Table 29.2: Tests to differentiate Streptococcus

pyogenes and Streptococcus agalactiae.

Lancefield group A B
Bacitracin Sensitive Resistant
CAMP test* Negative Positive
B-hemolytic 0.5-1 mm, Mucoid, larger
colonies pinpoint (2 mm)
*CAMP, Christie-Atkins-—Munch-Peterson test.

Bacitracin sensitivity testing: GAS is sensitive

to bacitracin 0.04 U disk (any zone of inhibition
around the disk is considered as sensitive),
which differentiates it from group B streptococci
(resistant) (Fig. 29.2B and Table 29.2). Fig. 29.3: Antimicrobial susceptibility testing on Mueller
Hinton blood agar (zone interpretation is given in Table
Serology 29.3).
Abbreviations: P, penicillin; E, erythromycin; Cd, clindamycin; Az,
Nonsuppurative streptococcal infections such azithromycin.
as acute rheumatic fever and glomerulone- Source: Department of Microbiology, Pondicherry Institute of
phritis [post-streptococcal glomerulonephritis Medical Sciences, Puducherry (with permission).
(PSGN)] usually develop in patients with a past
history of streptococcal sore throat and skin
infection (pyoderma), respectively. Hence, in Antimicrobial Susceptibility Test
these conditions antibodies in patient’s serum It is carried out on MHBA by disk diffusion test
is detected to establish retrospective diagnosis (Fig. 29.3) and reported as per Clinical and
of past streptococcal infections. Laboratory Standards Institute (CLSI) zone
“ ASO (antistreptolysin O) antibodies titer is interpretation criteria (Table 29.3).
elevated >200 Todd unit/mL in most of the
Treatment
streptococcal infections except pyoderma
and PSGN Penicillin is the drug of choice for pharyngeal
* Anti-DNase-B antibodies: Titer >300- infections as well as for suppurative complica-
350 units/mL is diagnostic of PSGN and tions. Resistance to penicillin is not reported
pyoderma. yet.

various antimicrobial
Table 29.3: Interpretative categories (CLSI) and observed zone size diameter (mm) to
agents tested for Streptococcus pyogenes isolate.

Disk strength CLSI interpretative criteria for Observed Interpretation

Antimicrobial
Streptococcus pyogenes zone size
agents Ut)
(in mm) (inmm)

PResstant [Intermediate [Sensitive |(8-22-)

a se >24 30 Sensitive
Penicillin (P) 10 units

15 <15 16-20 221 18 Intermediate

Erythromycin (E)
2 <15 16-18 219 30 Sensitive
Clindamycin (Cd)
15 3, 14-17 =18 19 Sensitive
Azithromycin (Az)
Abbreviation: CLSI, Clinical and Laboratory Standards Institute.
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

* For outpatients with upper respiratory “ Surgical debridement (most crucial) +

infections, oral cefepime or azithromycin can Penicillin G + Clindamycin is the standard
be given treatment given for necrotizing fasciitis.

§ GROUP B STREPTOCOCCI (STREPTOCOCCUS AGALACTIAE)

| Exercise 22
Solving Exercise
problem Solving |

Group B Streptococcal Cellulitis Identification

A 13-year-old boy presented to surgery OPD with Beta hemolytic colonies (29.4A), CAMP test positive
tender, bright red, subcutaneous swelling on malar (Fig. 29.5), bacitracin resistant (Fig. 29.6). These
area of the face with indurated peau d’orange texture findings are indicative that the causative organism is
of involved skin along with fever and chills. A clinical Streptococcus agalactiae.
diagnosis of cellulitis was made. The aspirated pus
Antimicrobial Susceptibility Testing
specimen was sent to microbiology laboratory
Antimicrobial susceptibility testing on MHBA (Fig.
for culture (Fig. 29.4A and 29.5) and antimicrobial
29.6 and Table 29.5) showed sensitive to penicillin,
susceptibility testing (Fig. 29.6 and Table 29.5).
1. Identify the causative agent based on the cefotaxime and resistant to erythromycin.
microbiological investigations performed. Treatment
2. List the infections caused by this organism. Penicillin would be preferred choice of treatment for
3. Interpret the AST. streptococcal cellulitis. (if patient is not allergic to
4. What antibiotic you would like to prescribe? penicillin).
Explanation (For answers to other questions, refer below)
Clinical Diagnosis
History of tender, bright red, subcutaneous swelling
on malar indicates that it is a case of cellulitis.

Clinical Manifestations Table 29.4: Early and late onset group B Streptococcus
disease in neonates.
Approximately 30% of women are vaginal or
rectal carriers of group B Streptococcus (GBS). Characteristics | Early-onset Late-onset
Hence, the GBS infection is common in neonates disease disease
and in pregnancy. Common infections caused by Age of onset 0-6 days of birth 7-90 days of
GBS are: birth
* Neonatal sepsis and meningitis: two types— Obstetric risk Prematurity and Not associated
early onset and late onset type (Table 29.4) prolonged labor with obstetric
risk
** Peripartum fever in women
Mode of During or before = Contact with
* Cellulitis and soft tissue infections, urinary
transmission to — birth from a colonized
tract infection, pneumonia, and endocarditis, the baby the colonized mother and
(commonly occurs among diabetics and maternal genital nursing
elderly people). tract personnel
Common Pneumonia and/ = Bacteremia
Laboratory Diagnosis clinical or respiratory and meningitis
manifestations distress syndrome (most common)
GBS is catalase negative and B-hemolytic like followed by
GAS, but differs from it by (Table 29.2): meningitis
** B-hemolytic colonies are mucoid and slightly
larger (Fig. 29.4A) ** CAMP positive: CAMP factor (named after the
* Bacitracin resistant (Figs 29.4B and 29.6) discoverers: Christie-Atkins-Munch-Petersen)
 CHAPTER 29 © Beta-hemolytic Streptococcal Infections

is a phospholipase produced by GBS

that causes synergistic hemolysis with
B-hemolysin produced by S. aureus. When
GBS is streaked on blood agar plate per-
pendicular to S. aureus, an enhanced arrow
head-shaped hemolysis is produced at their
junction (Fig. 29.5)
“+ AST: Antimicrobial susceptibility test: It is
Bacitracin carried out on MHBA by disk diffusion test
(Fig. 29.6) and reported as per CLSI zone
interpretation criteria (Table 29.5).

Penicillin is the drug of choice for all GBS infec-

tions. GBS is less sensitive to penicillin than GAS,
Figs 29.4 A and B: Streptococcus agalactiae: (A) Growth on hence a higher dose of penicillin is recommended.
blood agar with wide zone of beta-hemolysis around the
colonies; (B) Bacitracin resistant.
Source: Department of Microbiology, JIPMER, Puducherry (with
permission).

PA” oi ons
otreels [ine
S, EeEIEES

Fig. 29.6: Antimicrobial susceptibility testing on Mueller

Hinton blood agar (for zone size interpretation, refer Table
29.5).
Fig. 29.5: CAMP test positive, indicates the test isolate is Abbreviations: P, penicillin; E, erythromycin; Ce, Cefotaxime; Ba,

Streptococcus agalactiae. bacitracin.

Source: Department of Microbiology, Pondicherry Institute of
Source: Department of Microbiology, JIPMER, Puducherry
(with permission). Medical Sciences, Puducherry (with permission).

(mm) to various antimicrobial

Table 29.5: Interpretative categories (CLSI) and observed zone size diameter
agents tested for Strepto coccus aga lactiae isolate.

antimicrobial ; "

i Death

— — >24 27 Sensitive
Penicillin (P) 10 units
<15 16-20 221 15 Resistant
Erythromycin (E) 15
— — >24 28 Sensitive
Cefotaxime (Ce) 2
Abbreviation: CLSI, Clinical and Laboratory Standards Institute.
— |

Miscellaneous Bacterial Infections

of Skin and Soft Tissues: Anaerobic |CHAPTER |
Infections including Gas Gangrene, 3O
Leprosy and Anthrax
LPL SAO

B ANAEROBIC INFECTIONS
>,ba Foul smelling putrid pus
S od
Abscess formation
Obligate anaerobes cannot grow in presence of +,% Septic thrombophlebitis
oxygen. They need special requirements to grow *,a*
Toxemia and fever not marked
in culture such as: +,bo Failure to respond to antibiotics that do not
“ Anaerobic condition: This can be achieved by have significant anaerobic activity
various methods such as (Refer Chapter 3.6): “ Organisms are seen under Gram stain, but fail
e McIntosh and Filde’s anaerobic jar to grow in routine aerobic culture
w GasPak system “+ Special features may be observed such as:
s Anoxomat system
= Gas in specimen (gas gangrene)
s Anaerobic glove box workstation = Black pigment that fluoresce (Prevotella
m Pre-reduced anaerobically sterilized
melaninogenica)
(PRAS) media.
= Sulfur granules (Actinomyces).
>,Sd
Medium with low redox potential: This can
be achieved by adding to the media with Laboratory Diagnosis
reducing substances such as unsaturated
fatty acid, ascorbic acid, glutathione, cysteine, Specimens
glucose, sulfites and metallic iron. All clinical specimens for anaerobic culture
must be handled meticulously as brief exposure
Examples of obligate anaerobes include:
to oxygen may kill obligate anaerobes and result
“+ Spore bearing anaerobes (e.g., Clostridium):
in failure to isolate them in the laboratory.
Clostridia are gram-positive bacilli, having
** Accepted specimens: Tissue bits, necrotic ma-
bulging spores; found as saprophytes in
environment as well as commensal in human terials, aspirated body fluids or pus in syringes
and animal gut. However, few members are ** Unacceptable specimens: All swabs, sputum
pathogenic to man such as C. perfringens, C. or voided urine
tetani, C. botulinum and C. difficile * Specimens should be immediately put into
“* Non-sporing anaerobes (e.g., Bacteroides). RCM broth or other anaerobic transport
media and brought to the laboratory as soon
Clinical Presentations as possible.
Anaerobic infections are associated with various
clinical clues, such as: Robertson's cooked meat (RCM) broth
It is the most commonly used anaerobic media. It
“+ Infections adjacent to mucosal surfaces that
contains chopped meat particles (beef heart), which
bear anaerobic flora provide glutathione and unsaturated fatty acids, which
“* Predisposing factors such as ischemia, take up oxygen and create lower redox potential and
tumor, penetrating trauma, foreign body, or thus permit the growth of obligate anaerobes (Fig.
perforated viscus 30.1A). C. perfringens turns meat particles pink and
“ Spreading gangrene involving skin, subcuta- broth turbid (30.1B); whereas C. tetani turns the color
black and turbid (30.1C).
neous tissue, fascia, and muscle
 CHAPTER 30 © Miscellaneous Bacterial Infections of Skin and Soft Tissues

“* Culture: Various culture media can be used

for the isolation of anaerobes, such as:
m Anaerobic blood agar and neomycin
blood agar
m Egg yolk agar and phenylethyl agar
(PEA)
= BHIS agar: Brain-heart infusion agar
added with supplements, such as vitamin
K and hemin
= Bacteroides bile esculin agar (BBE agar).
~ Identification of anaerobes is based on:
= Biochemical tests
= Susceptibility to antibiotic disks
= Gas liquid chromatography
= Automated systems such as MALDI-
Figs 30.1A to C: Robertson cooked meat broth: (A) Unin-
oculated; (B) Pink and turbid (C. perfringens); (C) Black and TOR.
turbid (C. tetani).
Source: Department of Microbiology, JIPMER, Puducherry (with Treatment
permission). Common antibiotics given for anaerobic
infections are:
Microscopy “+ Metronidazole plus penicillin- DOC for odon-
All clinical specimens from suspected anaerobic togenic infections
infections should be Gram stained and examined “ Carbapenems (e.g., meropenem)
for the characteristic morphology. “ B-lactam/f-lactamase inhibitor combina-
tion (amoxicillin-clavulanate, piperacillin-
Cultural Identification tazobactam)
Samples should be processed immediately “ Clindamycin (in case of penicillin allergy).
under anaerobic condition which can be created Antimicrobial resistance in anaerobic
by various methods as described earlier. bacteria is an increasing problem.

Problem Solving Exercise 1

Explanation
Gas gangrene (Clostridium perfringens)
Clinical Diagnosis
Three days after a road traffic accident, a 30-year-
old man presented to hospital with sudden onset The history is suggestive of gas gangrene. Points in
favor are:
of excruciating pain at left leg with wound oozing
Road traffic accident, wound heavily contaminated
foul-smelling thin serosanguineous discharge. On
Q
d with soil
examination, the wound was found to be bandage
Q Five days later, develops painful wound oozing foul-
with a soiled gauze, appeared to be heavily contami- smelling serosanguineous discharge
nated with soil, there was edema and pain at the On examination, edema and pain at the site and
Q
site and crepitus was felt on palpation. Gram-stained crepitus on palpation.
the
smear of exudate specimen from deeper part of
wound is shown in Figure 30.3. Identification
1. What is the clinical diagnosis and its causativ
e Thick, boxcar-shaped gram-positive bacilli without
organism? spore in direct smear from deeper exudate speci-
2. List other infections caused by this organism? men (Fig. 30.3) indicates that causative agent is
3. What are the various modalities of laboratory C. perfringens.
diagnosis? (For answers to other questions, refer below).
4. How will you treat this condition?
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

Gas Gangrene
Gas gangrene is defined as a rapidly spreading,
edematous myonecrosis, occurring in
associat ion with severely crushed wounds
contaminated with pathogenic clostridia,
particularly with C. perfringens.

Etiological Agents
Gas gangrene is always polymicrobial and is
caused by many clostridial species.
* Established agents: Clostridium perfringens
(most common, 60% of the total cases) and Fig. 30.2: Gas gangrene of the right leg showing swelling
and discoloration ofthe right thigh with bullae, and palpable
C. novyi and C. septicum (20-40%)
crepitus.
* Probable agents: They are less commonly Source: Wikipedia/Cases/Engelbert Schropfer, Stephan Rauthe and
implicated, e.g., C. histolyticum, C. sporogenes, Thomas Meyer (with permission).
C. fallax, C. bifermentans, C. sordellii, C.
“+ Brawny edema and induration
aerofoetidum, and C. tertium.
“+ Such gangrenous tissues later may become
Pathogenesis liquefied and sloughed off
“+ Shock and organ failure develop later
The development of gas gangrene requires:
“+ Associated with higher mortality rate (50%).
* Anaerobic environment: Crushing injuries
of muscles such as road traffic accidents, Laboratory Diagnosis of Gas Gangrene
bullet injuries leads to interruption in the
blood supply and tissue ischemia Based on the clinical diagnosis of gas gangrene,
** Contamination of wound with clostridial treatment should be started as early as possible.
spores present in the soil (during war or road Laboratory diagnosis has role only for (1)
traffic accident) or clothes confirmation ofthe clinical diagnosis, (2) species
* Toxin production: Once introduced, C. identification.
perfringens proliferates locally and elaborates Specimen
exotoxins, chiefly a-toxin and 0-toxin. a-toxin
is the principle virulence factor. It has both Ideal specimens are necrotic tissues, muscle
phospholipase C and sphingomyelinase fragments and exudates from deeper part of the
activities. wound, where the infection appears to be more
active.
Clinical Manifestations “+ Blood culture may be positive for C. perfringens
The incubation period is variable. Depending and C. septicum. However, C. perfringens
upon the nature ofinjury, the amount of wound bacteremia can occur even in the absence of
contamination and the type of clostridial species gas gangrene
involved, the incubation period varies. For ** Swabs rubbed over the wound surface or
example: soaked in exudates are not satisfactory
“+ 10-48 hours for C. perfringens ** Specimens should be put into Robertson’s
“+ 2-3 days for C. septicum cooked meat broth and transported
* 5-6 days for C. novyi. immediately to the laboratory.

Various manifestations include: Direct Microscopy

* Sudden onset of excruciating pain at the Gram stained films provide clues about the spe-
affected site cies of clostridia present. Absence of neutrophils
* Rapid development of a foul-smelling thin in the infected tissues is a characteristic feature.
serosanguineous discharge * Thick, stubby, boxcar-shaped, gram-positive
¢* Gas bubbles (crepitus) in the muscle bacilli without spore—suggestive of C.
planes (Fig. 30.2) perfringens (Fig. 30.3)
 CHAPTER 30 © Miscellaneous Bacterial Infections of Skin and Soft Tissues

serum (due to lecithinase activity of a-toxin).

Opalescence can be inhibited by incorporating
anti-c-toxin to the medium (30.4B). The test
is also positive for C. bifermentans, C. baratti
and C. sordellii (all produce a-toxin)
“ Reverse CAMP test: C. perfringens is
streaked over the center of blood agar plate
and Streptococcus agalactiae is streaked
perpendicular to it. Presence of enhanced
zone of hemolysis (arrow-shaped) pointing
towards C. perfringens indicates the test is
positive (Fig. 30.4C).
Fig. 30.3: Gram stained smear of Clostridium perfringens.
Automated methods such as MALDI-TOF
Source: Public Health Image Library/ID# 11196, Don Stalons/
Centers for Disease Control and Prevention (CDC), Atlanta (with is currently the method of choice of rapid and
permission). accurate identification of various clostridia
species.
“+ Spore bearing gram-positive bacilli suggest
Treatment (Gas Gangrene)
other clostridia species
= Citron bodies (boat or leaf-shaped * Early surgical debridement is the most
+,

pleomorphic irregularly stained bacilli crucial step in the management of gas

with spores)—suggest C. septicum gangrene. All devitalized tissues should be
= Large rods with oval sub-terminal spores— widely resected so as to remove conditions
suggest C. novyi. that produce anaerobic environment. Closure
of wounds should be delayed for 5-6 days until
Identification the sites are free from infection
C. perfringens can be further identified by the “ Antibiotics: Combination of penicillin and
following properties. Culture plates should be clindamycin is recommended for 10-14 days
incubated anaerobically at 37°C for 2 days. “» Hyperbaric oxygen: It may kill the obligate
* Target hemolysis (double zone hemolysis, anaerobic clostridia such as C. perfringens;
Fig. 30.4A): On blood agar, C. perfringens however, it has no effect on aerotolerant
produce an inner narrow zone of complete clostridia (C. septicum)
* Passive immunization with anti-o-toxin
hemolysis (due to §-toxin), surrounded by a
much wider zone of incomplete hemolysis antiserum.
(due to the alpha toxin)
Other pathogenic clostridia include:
* Nagler’s reaction: C. perfringens produces an
opalescence surrounding the streak line on * ©. tetani: Causes tetanus; mediated by a
neurotoxin (tetanus toxin) which causes
egg yolk agar or media containing 20% human

Egg yolkcaga EGg,yolk agar with Gs pertringens

amti-ctoxin, oO

\
S) Eleeleleitels

of incomplete hemolysis (blue arrow) and zone of complete

Figs 30.4A to C: (A) Target hemolysis of C. perfringens-zone test.
hemolysis (black arrow); (B) Nagler’s reaction; (C) Reverse CAMP
Microbiology, Kasturba
Puducherry; (C) Dr Padmaja A Shenoy, Department of
Source: (A and B) Department of Microbiology, JIPMER, ).
Medical College, Manipal, Karnataka (with permission
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

skeletal muscle spasm and autonomic clinical forms—food poisoning, wound

nervous system disturbance infection and infant botulism
* C. difficile: Causes pseudomembranous
* C. botulinum: Produces a powerful
neurotoxin called botulinum toxin that colitis in hospitalized patients who have a
causes botulism; characterized by flaccid history of prolonged intake of broad spectrum
paralysis of muscles. It presents in three antibiotics (Chapter 22).

BLEPROSY
Exercise 2 e2_|
Solving Exercis
[Problem Solving
A 37-year-old village lady presented with complaints singly or in cigar-like bundles. This suggests that the
of numerous hypopigmented skin lesions on her causative agent is Mycobacterium leprae.
arms, cheeks, abdomen, back and legs for last 5 (For answers to other questions, refer below).
years. Her eyebrows had started thinning and she
had numbness in her forearm. Specimen collected
from the skin lesion was subjected to microscopy
(Fig. 30.5).
Q What is the clinical diagnosis and its causative
organism?
Q What are the various modalities of laboratory
diagnosis?
Q How will you treat this condition?
Explanation
Clinical Diagnosis
The history of numerous hypopigmented skin lesions,
numbness in her forearm and eyebrows started
thinning is suggestive of leprosy.
Fig. 30.5: Microscopy of specimen (slit skin smear)
Identification
collected from the skin lesion.
The modified acid-fast smear (Fig. 30.5) demonstrat- Source: Dr Isabella Princess, Apollo Hospital, Chennai
ing globi (arrow) filled with acid-fast bacilli arranged (with permission).

Laboratory Diagnosis of Leprosy cigar bundle appearance). The globi are present
Smear Microscopy inside the foamy macrophages called as Vir-
chow's “lepra cells” or “foamy cells” (Fig. 30.5)
** Specimen collection:
= Live bacilli will be uniformly stained with
= Total six samples are collected; four from parallel sides and round ends and length
skin (forehead, cheek, chin and buttock), is five times the width
one from ear lobe and nasal mucosa by = Dead bacilli are less uniformly stained
nasal blow or scraping and have fragmented and granular
m Slit skin smear is the technique followed appearance.
to collect the skin and ear lobe specimens * Grading of the smear: The smears are graded,
from the edge ofthe lesion. based on the number of bacilli
* Procedure: The smears are stained by ZN = Bacteriological index (BI) is based on the
technique by using 5% sulfuric acid for total number of bacilli (live and dead)
decolorization seen per oil immersion field
* Interpretation: Under oil immersion objec- = Morphological index (MI) is expressed
tive, red acid-fast bacilli are seen, arranged as the percentage of uniformly stained
singly or in groups, bound together by lipid- bacilli out of the total number of bacilli
like substance, the glia to form globi (called as counted.
 CHAPTER 30 © Miscellaneous Bacterial Infections of Skin and Soft Tissues

mice keeping at 20°C for 6-9 months. Other

1-10 bacilliin 100 OIF . =1+
animals such as nine-banded armadillo can
1-10 bacilli in 10 OIF =2+
1-10 bacilli per OIF =3+ also be used
10-100 bacilli per OIF =4+ 2,~e
Lepromin test: It demonstrates the delayed
100-1,000 bacilli per OIF =5+ hypersensitivity reaction and an intact
>1,000 bacilli or bacilli in cell-mediated immunity against the lepra
clumps and globiin each OIF =6+
antigen.
Other tests include:
** Mouse foot pad cultivation: M. leprae is not Treatment
cultivable either in artificial culture media or WHO recommends multidrug therapy for
in tissue culture. It can be cultivated only by treatment of leprosy. Dapsone, rifampicin and
inoculating the specimens into foot pad of clofazimine are the recommended drugs.

B ANTHRAX
Problem Solving Exercise 3
A 35-year-old male who is a worker in a wool factory, stained sputum. Sputum specimen was collected and
was admitted into hospital with prolonged fever, with was subjected to Gram staining (Fig. 30.6).
chills, night sweats and chest discomfort with blood- 1. What is the clinical diagnosis and its causative
organism?
2. List the virulence factors and other infections
caused by this organism?
3. What are the various modalities of laboratory
diagnosis?
4. What antibiotic you would like to prescribe?
Explanation
Clinical Diagnosis
History of fever and blood-stained sputum in wool
factory worker is suggestive of respiratory anthrax
(wool sorter’s disease). Occupational exposures,
leading to infection among farmer, butcher, abattoir,
etc., are common in anthrax.

\ eo
yd
ed ‘=
Identification
Direct Gram staining (Fig. 30.6) shows gram-positive
Fig. 30.6: Gram stain of B. anthracis showing gram-
large rectangular rods arranged in chain. This suggests
positive, large rectangular bacilli and pus cells.
that the causative agent as Bacillus anthracis.
Source: A. Public Health Image Library/ID#: 1811, Centers for
(For answers to other questions, refer below).
Disease Control and Prevention (CDC), Atlanta (with permission).

Clinical Manifestations surrounded by non-pitting indurated edema,

called as malignant pustule
Anthrax is primarily a zoonosis affecting “ Pulmonary anthrax or Wool sorter’s disease:
herbivorous animals such as cattle and sheep. It is characterized by hemorrhagic pneumonia
Human beings acquire infection by: (1) direct with blood stained sputum
contact by spores entering through the abraded “+ Intestinal anthrax.
skin among people with occupational exposure
Virulence factors of B. anthracis are:
to animals; (2) by inhalation of spores and (3)
ingestion of carcasses. “ Anthrax toxin: Tripartite toxin consists of
edema factor, protective factor and lethal
Types of human anthrax are:
factor
* Cutaneous anthrax (hide porter’s disease):
It is characterized by necrotic eschar * Anthrax polypeptide capsule.
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

me

Figs 30.7A to C: (A) McFadyean's reaction—amorphous purple capsule surrounding blue bacilli (polychrome
methylene blue stain); (B) Medusa head colonies of Bacillus anthracis on nutrient agar (10x magnification);
(C) Non-hemolytic dry wrinkled colonies of Bacillus anthracis on blood agar.
Source: (A) Department of Pathobiology, University of Guelph, Canada; (B) Dr J Glenn Songer, lowa State University, USA; (C) Public Health
Image Library/ID#: 1897/Dr Larry Stauffer, Centers for Disease Control and Prevention (CDC), Atlanta (with permission).

Laboratory Diagnosis
As there is high-risk of laboratory-acquired
infection of anthrax, hence specimens should
be processed in appropriate biological safety
cabinets.

Specimen
The following specimens to be collected—pus
or swab from malignant pustule, sputum (in
pulmonary anthrax), blood (in septicemia) and
CSF (in hemorrhagic meningitis).

Direct Demonstration
Fig. 30.8: Gram stained culture smear-shows gram-
“* Gram staining: Reveals gram-positive, large positive bacilli with spores (bamboo stick appearance).
rectangular rods; spores are usually not seen Source: Department of Microbiology, JIPMER, Puducherry.
in clinical samples (Fig. 30.6)
* McFadyean’s reaction: Polypeptide capsule interlacing chains of bacilli, appears as locks
can be demonstrated by staining with of matted hair (Fig. 30.7B)
polychrome methylene blue stain for 30 * Blood agar: Dry wrinkled, nonhemolytic
seconds. Capsule appears as amorphous colonies are produced (Fig. 30.7C)
purple material surrounding blue bacilli * Culture smear: Gram staining reveals
(Fig. 30.7A). bamboo stick appearance, i.e., long chain of
gram-positive bacilli with nonbulging spores
Culture (appear as empty space) (Fig. 30.8).
Bacillus anthracis is obligate aerobic and non-
fastidious. Treatment
** Medusa head appearance: When colonies Treatment should be started early; consists of
are viewed under low power microscope, ciprofloxacin or doxycycline plus clindamycin
the edge of the colony is composed of long for 60 days.
 CHAPTER |
SA RRR

=
Viral Exanthems and
Other Cutaneous Viral Infections
SERA TRD

B INTRODUCTION Table 31.1: Viral exanthems and other cutaneous viral

infections.
An exanthem is an eruption or rash on the
Viral exanthems/other skin
skin, that may be associated with fever or other esions
systemic symptoms. Majority of exanthems
have an infectious etiology; most commonly DEER hnie Gitiaas ABoes He 5

Herpes simplex virus Vesicular lesions

by viruses. Exanthems may also be seen due
to drug reactions. The viruses that can cause Varicella-zoster virus Chickenpox and zoster
exanthematous and other types of skin lesions Epstein-Barr virus Following ampicillin therapy
are enlisted in Table 31.1. Human herpesvirus-6 Roseola infantum (exanthem
subitum or sixth disease)

HERPES SIMPLEX VIRUS

i Hite ar DI ARV ERS NS

INFECTIONS Poxviruses Smallpox

Molluscum contagiosum*
Introduction Parvovirus Erythema infectiosum (fifth
Herpes simplex viruses belong to «-subfamily of disease)
Papular-purpuric gloves and
Herpesviridae.
socks syndrome*
“+ They are extremely widespread and exhibit a
Human papillomavirus Warts*: Common warts,
broad host range; can infect many types of cells
(HPV) flat warts, plantar warts,
and different animals. However, the human anogenital warts (condyloma
herpesviruses infect exclusively man acuminatum)
*» They replicate fast (12-18 hours cycle), spread
LEONG ACRE ta IT NRE

oi 1 se LES a
fast and are cytolytic Measles virus Rashes, Koplik’s spots
“ They can cause a spectrum of diseases,
Rubella virus Rashes
involving skin, mucosa and various organs
Coxsackie viruses Hand-foot-mouth disease
* They undergo latency in nerve cells;
reactivate later causing recurrent lesions. Agents of viral Dengue, Ebola and others
hemorrhagic fever
Herpes simplex viruses (HSV) are of two
distinct types: HSV-1 and HSV-2. They differ *These viruses produce non-exanthematous skin lesions.

from each other in many aspects (Table 31.2). * Site of infection: HSV replicates at the
local site of infection and produces lesions
Pathogenesis anywhere, but more commonly in:
Primary Infection m HSV-1 lesions are confined to areas above
“ Transmission occurs through abraded skin or the waist (most common site—around
mucosa from any site, but more commonly by: mouth)
= HSV-1: Oropharyngeal contact with = HSV-2 produces lesions below the waist
infected saliva or direct skin contact (most common site—genital area).
= HSV-2: Sexual contact or rarely vertical * Spread via nerve: Virus then invades the local
mode (from mother to fetus). nerve endings and is transported by retrograde
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

Problem Solving Exercise 1

Herpes Explanation
A 7-year-old boy had developed multiple painful
It is a case of Herpes labialis and the etiological agent
vesicles over the lips, tongue and buccal mucosa is herpes simplex virus
(Fig. 31.1A). His parents revealed that two children of Q Multiple painful vesicles over the lips, tongue and
his school had a similar presentation few days back. buccal mucosa (Fig. 31.1A).
Scrapings obtained from the base of the lesion is Q Scrapings obtained from the base of the
subjected to staining with Wright's or Giemsa (Tzanck lesion is subjected to staining with Wright's or
preparation) (Fig. 31.2A) Giemsa [Tzanck preparation)which revealed
a. What is the most probable diagnosis and multinucleated giant cell (Tzanck cell) in the center
etiological diagnosis? (arrow showing) (Fig. 31.2A)].
b. What are the various cutaneous and mucosal (For answers to other questions, refer below).
infections produced?
c. How is this infection diagnosed in the laboratory?

Table 31.2: Differences between HSV-1 and HSV-2.

Properties Herpes simplex Herpes simplex

virus 1 virus 2
Common Direct contact Sexual mode or
modes of with mucosa or vertical mode
transmission abraded skin
Latency in Trigeminal ganglia Sacral ganglia
Age affected Young children Young adults
Common e Oral-facial e Genital a 31.2A and B: (A) Tzanck smear of a tissue scraping
manifestations mucosal lesions lesions showing multinucleated giant cell (Tzanck cell) in the
e Encephalitis e Skin lesions— center (arrow showing); (B) Indirect IF for HSV1/2 antibody
and meningitis below the detection.
e Ocular lesions waist
Source: (A) Public Health Image Library, ID# 14428/Centers for
e Skin lesions— e Neonatal Disease Control and Prevention (CDC), Atlanta; (B) Euroimmun
above the waist herpes (with permission)

organ involvement and systemic manifesta-

tions.

Clinical Manifestations
Both HSV-1 and 2 have been isolated from nearly
all mucocutaneous sites and viscera; however,
in general, oral-facial infections are common
Figs 31.1A and B: (A) Vesicular lesions on lips and tongue with HSV-1, whereas HSV-2 frequently causes
due to HSV-1 infection; (B) Periocular vesicular lesions due genital infections and intrauterine infections.
to HSV-1 infection.
The incubation period ranges from 1 to 26 days
Source; Public Health Image Library, (A) ID# 12616 (Robert FE
Sumpter); (B) ID# 6492 (Dr KL Hermann)/Centers
(median, 6-8 days).
for Disease
Control and Prevention (CDC), Atlanta (with permission).
Oral-facial Mucosal Lesions
axonal flow to the dorsal root ganglia, where it Oral-facial mucosal lesions are the most
replicates further, and then undergoes latency common manifestation of HSV infections (Figs
“+ Primary HSV infections are usually mild: in 31.1A and B).
fact, most are asymptomatic ** Most common affected site is buccal mucosa
** However, in immunocompromised hosts, * Most frequent primary lesions are gingivo-
ote

viremia occurs that leads to widespread stomatitis and pharyngitis

 CHAPTER 31 © Viral Exanthems and Other Cutaneous Viral Infections

** Most frequent recurrent lesion is herpes preparation), or Papanicolaou stain. Sensitivity

labialis (painful vesicles near lips) (Fig. 31. of staining is low (<30% for mucosal swabs). It
1A) cannot differentiate between HSV-1, HSV-2, and
* Other lesions produced are—ulcerative varicella-zoster virus; as all of them produce
stomatitis, tonsillitis and vesicular lesions on similar but characteristic cytopathological
the eyelids (Fig. 31.1B) changes such as:
“* Many cases are asymptomatic but can * Production of Cowdry type A intranuclear
predispose to secondary bacterial infection. inclusion bodies (Lipschultz body)
“+ Formation of multinucleated giant cells with
Cutaneous Lesions faceted nuclei and ground glass chromatin
HSV usually infects through abraded skin and (Tzanck cells) (Fig. 31.2A)
causes various cutaneous lesions. “+ Ballooning of infected cells, margination of
“ Herpetic whitlow: Lesions present on the chromatin.
fingers of dentists and hospital personnel
“+ Febrile blisters (herpes febrilis): Fever due to Virus Isolation
any other cause can provocate HSV to cause It remains the most definitive tool for HSV
recurrent blisters diagnosis. McCoy cell lines are preferred for
“+ Herpes gladiatorum: Mucocutaneous lesions isolation of HSV. Viral growth can be detected
present on the body of wrestlers in 2-4 days by:
“* Skin lesions are often severe on underlying “ Characteristic cytopathic effect: Diffuse
eczema or burns which permit extensive local rounding and ballooning of the infected
viral replication and spread cells
+,*Oo Viral antigen detection by neutralization test
“ Eczema herpeticum: Caused by HSV-1 in
patients with chronic eczema or immunofluorescence staining with specific
* Erythema multiforme: HSV is commonly antiserum
associated with this condition. “* Shell vial technique can be followed to
decrease the detection time to <24 hours.
Other infective Syndromes of HSV
encephalitis,
Viral Antigen Detection by
“ CNS infections: Such as
meningitis
Immunofluorescence
* Ocular manifestations: such as keratocon- Viral antigen detection (targeting cell surface
junctivitis, corneal ulcer and blindness glycoprotein antigens) by direct IF is also a
“ Genital lesions: described as bilateral, sensitive and specific assay. It can differentiate
painful, multiple, tiny vesicular ulcers (HSV- HSV-1 from HSV-2.
2. is more common than HSV-1)
HSV DNA Detection
Visceral and disseminated herpes
+.
DC

+,
Neonatal herpes. Molecular methods such as polymerase chain
reaction (PCR), real-time PCR (can quantitate
Laboratory Diagnosis the viral load in specimens) and BioFire
The sensitivity of all the methods to diagnose FilmArray (automated nested mulitplex PCR)
HSV infection depends on the type of specimen, are useful; can also be used to differentiate
as well as the type of infection. The sensitivity is between HSV-1 and 2.
more for vesicular lesions and primary infection
Antibody Detection
than for ulcerative lesions and recurrent
infections. Antibodies appear in 4-7 days after the infection
and peak in 2-4 weeks. IgM appears first and is
Cytopathology replaced by IgG, which persists for life.
“ Most available tests usually detect IgG or
Scrapings obtained from the base of the lesion
total antibodies, hence cannot differentiate
can be stained with Wright’s or Giemsa (Tzanck
 SECTION 2 @ Systemic Microbiology (Infectious Diseases)

between recent and past infections. Sero- Treatment

conversion or arise in titer is more meaningful For mucocutaneous infections, acyclovir,
“ Serologic assays (e.g., ELISA) based on the famciclovir and valacyclovir have been the
type-specific antigens such as glycoprotein mainstay of treatment.
G antigens (gG1 and gG2) can differentiate
Prevention
between HSV-1 and HSV-2. Western blot
is more accurate, with 98% sensitivity and Infection control measures: Patients with
specificity mucocutaneous herpes in hospitals, should be
+ Both ELISA and indirect IF formats are kept on contact precautions until lesions are dry
available (Fig. 31.2B). and crusted (Refer Chapter 11).

@ VARICELLA-ZOSTER VIRUS INFECTIONS

[Problem Solving
Solving Exercise
Exercise22
A child presents with vesicular rashes (Fig. 31.3), which Q Fever appears with each crop of rashes.
appeared first on the face and trunk, spread rapidly Refer text below for the answer to other questions.
to involve flexor surfaces; sparing distal part of the
limbs. Rashes are bilateral and diffuse in distribution,
appear in multiple crops. Fever appears with each
crop of rashes.
Q Whatis the clinical diagnosis?
Q What are the complications seen?
Q Discuss about the infection control measures to be
taken when giving care to this patient.
Explanation

The clinical presentation is suggestive of chickenpox:

Q Vesicular rashes (Fig. 31.3), on the face and trunk
of child, which spread rapidly to involve flexor
Fig. 31.3: Vesicular rashes of Chickenpox.
surface.
Source: Public Health Image Library, |D#/2882/JD Millar/
Q Rashes are bilateral and diffuse in distribution, Centers for Disease Control and Prevention (CDC),
appear in multiple crops. Atlanta (with permission).

Introduction common) and contact transmission. From

Varicella-zoster virus (VZV) produces vesicular the local site, it spills over to blood and then is
eruptions (rashes) on the skin and mucous carried through the infected mononuclear cells
membranes in the form oftwo clinical entities: to target sites such as:
1. Chickenpox: It is characterized by general- “+ Skin (produces rashes)
ized diffuse bilateral vesicular rashes which ** Respiratory tract (shed in respiratory secretions)
occur following primary infection, usually “+ Neurons (undergoes latency).
affecting children
2. Zoster or shingles: It occurs following reacti- Clinical Manifestations
vation of latent VZV, present in the trigeminal * Incubation period is about 10-21 days (2-3
ganglia that occurs mainly in adult life. Vesicular weeks)
rashes are unilateral and segmental (confined to * Rashes are the main manifestation of
the skin innervated by a single sensory ganglion). chickenpox
= Rashes are vesicular (Fig. 31.3)
Chickenpox
= Centripetal in distribution: Usually start
Pathogenesis on the face and trunk, spread rapidly to
VZV enters through the upper respiratory involve flexor surfaces; sparing distal part
mucosa or the conjunctiva by aerosol (most of the limbs
 CHAPTER 31 © Viral Exanthems and Other Cutaneous Viral Infections

= Bilateral and diffuse in distribution age; 2 doses, first dose is given at 12-15 months
= Rashes appear in multiple crops: Lesions and second at 4-6 years.
in various stages of evolution, such as
maculopapules, vesicles, pustules, and Varicella-zoster Immunoglobulin (VZlg)
scabs can be found in one area at the same * Itis useful for post-exposure prophylaxis;
time given within 96 hours (preferably within 72
m Fever appears with each crop of rashes. hours) of exposure
* Chickenpox is a disease of childhood * It is also indicated for neonates born to
*“*» When occurs in adults, it is more severe with mothers suffering from chickenpox.
bullous and hemorrhagic rashes leaving
behind pitted scars on skin after recovery. Infection Control Measures
Patients infected with VZV should be kept in
Complications isolation. Airborne precautions (e.g., negative
Complications are more common in adults and air-flow rooms, and PPE such as N95 respirator)
in immunocompromised individuals. plus contact precautions must be followed until
* Most common complications: Secondary lesions are dry and crusted (Refer Chapter 11).
bacterial infections of the skin and CNS For localized zoster in an immunocompetent
involvement (cerebellar ataxia, encephalitis host, contact precaution alone need to be
and aseptic meningitis) followed.
** Other complications: (1) Varicella pneumo-
nia—serious complication, seen in adults and BHPV INFECTIONS
(2) Reye’s syndrome—fatty degeneration of
Human papillomavirus (HPV) is a DNA virus,
liver, occurs following aspirin intake
belongs Papillomaviridae family. It has selective
“ Chickenpox in pregnancy: Chickenpox in
tropism for epithelium of skin and mucous
pregnancy can affect both mother and the fetus
membranes. It has >100 serotypes, which
= Mothers are at high-risk of developing
produce an array of infections ranging from
varicella pneumonia
benign warts, to malignant neoplasia of cervix.
= Fetus is at higher risk of developing con-
genital varicella syndrome (in early preg- Benign warts: They are small, hard, rough growth
nancy); characterized by cicatricial skin on the skin (Fig. 31.4). They are offollowing types:
lesions and limb hypoplasia. “ Common skin warts (verruca vulgaris) and
flat warts (verruca plana) are common in
Zoster or Shingles or Zona children (seen with serotypes 2, 4, 27, 57)
usually occurs due to reactivation “ Plantar warts (verruca plantaris)—benign
Zoster
lesion, widely prevalent among adolescents
of latent VZV in old age (>60 years of age),
(seen with serotype 1)
in immunocompromised individuals or
“ Anogenital warts (condyloma acuminatum):
occasionally in healthy adults.
It is a sexually transmitted infection, seen
* It usually starts with severe pain in the area
of skin or mucosa supplied by one or more
groups of sensory nerves and ganglia
“ Rashes: They are unilateral and segmental,
confined to the area of skin supplied by the
affected nerves
“ Most common nerve involved is ophthalmic
branch of trigeminal nerve. Head, neck and
trunk are the most common affected sites.

Vaccine
Live attenuated vaccine using Oka strain of VZV Fig. 31.4: Plantar warts.

is available. It is given to children after 1 year of Source: Wikipedia (with permission).

 SECTION 2 ©@ Systemic Microbiology (Infectious Diseases)

among adults and is associated with HPV

serotypes 6 and 11.

Molluscum Contagiosum
Molluscum contagiosum virus is an obligate
human poxvirus that produces characteristic
skin lesions.
* Lesions: It produces dome-shaped, pink
pearly wart-like lesions (2-5 mm size),
umbilicated, with a dimple at the center
(Fig. 31.5A). Lesions are found singly or in
clusters, anywhere on the body except on the a

palms and soles. Genital lesions are seen in Figs 31.5A and B: (A) Molluscum contagiosum lesions
on skin; (B) Histopathology of skin showing molluscum
adults
bodies.
* Transmission: Children are commonly
Source: (A) CDC/ L Sperling, MD, Walter Reed Army Medical Center;
affected, acquire infection by direct and (B) Public Health Image Library, ID# 860/Centers for Disease
indirect contact Control and Prevention (CDC), Atlanta/Dr Edwin P Ewing, Jr (with
permission).
“+ Self-limiting but more generalized and severe
in HIV-infected patients
¢ Laboratory diagnosis: Molluscum bodies 1. Prodromal Stage
are the intracytoplasmic eosinophilic This stage lasts for 4 days (i.e., from 10th to
inclusions seen in skin scrapings stained with 14th day of infection) and is characterized by
histopathological stains (Fig. 31.5B) manifestations such as:
* Treatment: Surgical removal of the lesions “+ Fever is the first manifestation, occurs on day
by ablation (by cryotherapy or laser therapy) 1 (i.e., on 10th day of infection)
is the mainstay of treatment. *+ Koplik’s spots are pathognomonic of measles,
appear after two days following fever (i.e., on
@ MEASLES 12th day of infection) and are characterized
by:
Clinical Manifestations = White to bluish spot surrounded by an
Measles is an acute, highly contagious childhood erythema
disease, characterized by fever and respiratory m Appear first on buccal mucosa near
symptoms, followed by rashes. It is transmitted second lower molars (Fig. 31.6A)
by droplet and aerosol routes. m Rapidly spread to involve the entire buccal
Incubation period is about 10 days. Disease can mucosa and then fade with the onset of
be divided into three stages. rash.

~ Problem Solving Exercise 3

Measles 3. How the disease can be prevented?
A 3-year-old child developed fever, cough, coryza Explanation
and conjunctivitis followed by the appearance
Clinical Diagnosis
of a maculopapular rash behind the ears, soon
progressed all over the body (Fig. 31.6B). On The symptoms of fever, cough, coryza and
examination, small bluish white spot surrounded conjunctivitis, maculopapular rash (first to appear
by erythema was found in the buccal mucosa behind ears, then spreads; Fig. 31.6B), with a Koplik
(Fig. 31.6A) and on inquiry mother gave history of spot (small bluish white spot in the buccal mucosa,
incomplete vaccination. Fig. 31.6A) in a 3-year-old child with incomplete
1. What is the most probable of clinical diagnosis? vaccination, are all suggestive of measles as first
2. What are the different modalities of laboratory differential diagnosis.
diagnosis? (For answers to other questions, refer below.)
 CHAPTER 31 © Viral Exanthems and Other Cutaneous Viral Infections

“+ Non-specific symptoms may be present such as Antigen Detection

cough, coryza, nasal discharge, redness of eye, Measles antigens in the infected cells can be
diarrhea or vomiting. detected directly by using anti-nucleoprotein
antibodies (direct immunofluorescence test).
2. Eruptive Stage
Maculopapular dusky red rashes appear after 4 Virus Isolation
days of fever (i.e., on 14th day of infection). Monkey or human kidney cell lines are used.
“+ Rashes typically appear first behind the ears Cytopathic effect observed—multinucleated
— then spread to face, arm, trunk and legs giant cells (Warthin-Finkeldey cells) containing
— then fade in the same order after 4 days of both intranuclear and intracytoplasmic inclusion
onset (Fig. 31.6B) bodies (see Fig. 31.6C).
* Rashes are typically absent in HIV infected
people. Antibody Detection
* Detection of measles-specific IgM antibody
+,

Fever (10th day) —> Koplik’s spot (12th day) — rash in serum or oral fluid or four-fold rise
(14th day)
of IgG antibody titer between acute and
convalescent-phase sera is taken as significant
3. Post-measles Stage “+ Demonstration of raised titers of anti-measles
It is characterized by weight loss and weakness. antibody in the CSF is diagnostic of SSPE
“ ELISA is the most recommended test that
There may be failure to recover and gradual
deterioration into chronic illness. uses recombinant measles nucleoprotein
(NP) antigen.
Complications
Reverse-transcription PCR
Complications following measles include:
* Secondary bacterial infections such as otitis RT-PCR is extremely sensitive and specific;
media measles RNA can be detected in specimens up
to 10-14 days post rashes.
+,
Giant-cell pneumonitis (Hecht’s pneumonia)
“

7
Acute laryngotracheobronchitis (croup)
“
Prevention
+,*
Diarrhea, leads to malnutrition
+

7
“ Subacute sclerosing panencephalitis (SSPE): General Preventive Measures
Rare, but most severe. Airborne precaution such as isolation in
negative pressure room, use of PPEs such as N95
Laboratory Diagnosis
respirator, etc., must be followed while handling
Specimens measles cases (Refer Chapter 11 for detail).
Nasopharyngeal swab, conjunctival swab, Measles Vaccine
blood, respiratory secretions, and urine are
the ideal specimens. Synthetic swabs are Live attenuated vaccine is available for measles;
using Se strain. Under national
recommended.

ry 7
i. "eee Se,
Ry: >
a
(B) Measles rashes (on face);
Figs 31.6A to C: (A) Koplik spot in buccal mucosa (measles) (arrow deine
(arrow showing).
(C) Multinucleated giant cell of measles infected cell lines
(B) ID# 17980; C. ID# 859/Cente rs for Disease Control
Source: (A) Public Health Image Library, ID# 6111;
and Prevention (CDC), Atlanta (with permission).
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

immunization schedule of India, measles-rubella 12 months along with vitamin A supplements

(MR) vaccine is given at 9 completed months to and second dose of MR vaccine at 16-24 months.

B RUBELLA
Problem Solving Exercise 4
A 5-month-old infant was brought to the neonatal
care unit with history of preterm delivery, low
birth weight, hearing and visual impairment.
On detail investigations baby was found to have
hearing impairment, congenital heart defects,
hyperpigmented skin lesions, bilateral cataract and
cerebral edema. The blood sample of the infant was
sent for rubella-specific IgM ELISA and the test result
is displayed in Figure 31.7.
1. Interpret the test result and etiological agent.
2. What are the different modalities of laboratory Fig. 31.7: Rubella-specific IgM ELISA test.
diagnosis? Source: Pondicherry Institute of Medical Sciences, Puducherry
3. How can you prevent this clinical condition? (with permission).

Introduction
Rubella virus produces a childhood exanthema
similar to that of measles. Therefore, rubella
is also known as German measles. However,
unlike measles, it is highly teratogenic; can
cause congenital rubella syndrome. Rubella ”
may present in two clinical forms—postnatal
infection and congenital infection.
Figs 31.8A and B: (A) Child with rubella rash; (B) Cataract
Postnatal Rubella seen in congenital rubella infection (arrows showing).
Postnatal rubella may occur during neonatal Source; Public Health Image Library, A. |ID# 10146; B.|D#4284/Centers
for Disease Control and Prevention (CDC), Atlanta (with permission).
age, childhood, and adult life. It spreads from
person-to-person by respiratory droplets via
upper respiratory mucosa. Laboratory Diagnosis
** Isolation of virus: Nasopharyngeal or throat
+.

Clinical Manifestations swabs taken 6 days before and after the onset
* Incubation period is about 14 days (range, of rash. Monkey or rabbit origin cell lines are
12-23 days) preferred
* Rashes are generalized and maculopapular in * Serology (antibody detection):
nature, start on the face, extend to trunk and m ELISA is the preferred method for rubella
extremities, and disappear in 3 days (Fig. 31.8A) diagnosis (Fig. 31.7). It detects both IgM
* Lymphadenopathy (occipital and postau- and IgG separately. Various antigens
ricular) is the most striking feature employed are whole virus lysate or
* Forchheimer spots may be seen in some recombinant E1/E2 antigens
cases. They are pin-head sized petechiae; = Results need to be confirmed by IgG
develop on the soft palate and uvula; usually avidity test to differentiate active infection
start with the onset of rash. from the past infection or vaccination.
 CHAPTER 31 © Viral Exanthems and Other Cutaneous Viral Infections

* Molecular test: RT-PCR is available for (1-14 years). Under national immunization
detecting rubella specific RNA (nucleoprotein schedule, rubella vaccine is given along with
N gene) in clinical specimens. measles (MR vaccine) at 9-12 months of age
and second dose at 16-24 months in selected
Congenital Rubella Syndrome states
The most serious consequence of rubella virus
+,+o
Precautions:
infection is congenital rubella syndrome. Rubella = Vaccine is contraindicated in pregnancy
is highly teratogenic; affects ear (deafness), eyes = As it is teratogenic, pregnancy should
(cataract, Fig. 31.8B), and heart (patent ductus be avoided at least for 4 weeks (28 days)
arteriosus). The severity is maximum in first following vaccination
= Infants below 1 year should not be
trimester.
vaccinated due to possible interference
Prevention from persisting maternal antibody.

General Preventive Measures

HAND-FOOT-AND-MOUTH (HFM)
Airborne precaution must be followed while
DISEASE
handling rubella cases (Refer Chapter 11).
HEM disease usually affects children; is
Rubella Vaccine characterized by ulcerations on oral and
RA 27/3 is a live attenuated vaccine for rubella, pharyngeal mucosa and vesicular rashes on the
prepared from human diploid fibroblast cell palms and soles, which heal without crusting
line. (Figs 31.9A to C). Fever and sore throat with flu-
* It is available singly or in combination with like symptoms are the other manifestations.
vaccines of mumps and measles (MMR “+ Agents: Itis mainly caused by Coxsackieviruses,
vaccine) rarely by other enteroviruses. Coxsackievirus
¢ Indication: In India, rubella vaccine is A16 is the most common cause
indicated to all women of reproductive age * Transmission occurs through contact (direct
(first priority group) followed by all children or indirect) and droplets.

(HFM) disease: (A) Hand; (B) Foot; (C) Mouth.

Figs 31.9A to C: Vesicular eruptions in hand-foot-and-mouth
(with permission); C. Wikipedia, Mr Midgley (with permission).
Source: A and B. Centers for Disease Control and Prevention
 Superficial and
Subcutaneous Fungal Infections 32
hair and nail; caused by a group of related fungi
SUPERFICIAL MYCOSES
(called dermatophytes).
Dermatophytoses “+ Trichophyton species: Infect skin, hair and nail
Dermatophytoses (tinea or ringworm) is the “+ Microsporum species: Infect skin and hair
commonest superficial mycoses affecting skin, ** Epidermophyton species: Infect skin and nail.

[Problem Solving
Solving Exercise
Exercise?1
Dermatophytoses caused by either Trichophyton species or Microsporum
A 6-year-old girl presented to the dermatology clinic species.
with scaly patches on her scalp for one month. On Identification
speaking with her parents, it was discovered that SDA culture revealed cottony growth, with orange
there were several pets in the household. Her scalp
pigment on reverse (Fig. 32.1A).
hair and scaly scrapings were sent for fungal culture
LPCB mount showed abundant, thick-walled, spiny,
and identification (Figs 32.1A and B).
spindle-shaped (up to 15 septa), pointed ends
1. What is the clinical diagnosis and its probable
macroconidia and hence the causative agent is
causative organisms?
Microsporum canis (Fig. 32.1B).
2. Identify the causative agent based on fungal
(For answers to the other questions, refer below).
culture on Sabouraud’s dextrose agar (SDA) and
lactophenol cotton blue (LPCB) mount.
3. What are the clinical features seen in this disease?
4. What are the various modalities of laboratory
diagnosis?
5. Mention about the treatment for the same clinical
condition.

Explanation
Clinical Diagnosis
A 6-year-old child with scaly lesions on scalp Figs 32.1A and B: Microsporum canis: (A) Culture on
(involving hair and scalp), with contact history Sabouraud's dextroseagar (SDA); (B) Lactophenol cotton
of pets at residence, is suggestive of tinea capitis blue (LPCB) mount.
(dermatophytic infection of scalp) due to a zoophilic Source: Public Health Image Library, Centers for Disease Control
dermatophyte. Infection of hair and skin can be and Prevention (CDC), Atlanta; (A) ID #15474, (B) ID #15472.

Pathogenesis ** Nails: They invade the nails through the lateral

or superficial nail plates
Dermatophyte infection is acquired by direct
* Hair shafts: Hairs become brittle and areas of
contact of soil, animals or humans infected with
alopecia may appear.
fungal spores.
** Skin: Characteristic well-demarcated annular Clinical Types
or ring-shaped pruritic scaly skin lesions with Depending on the site of involvement, various
central clearing and raised edges clinical types of tinea/ringworm infections
 CHAPTER 32 © Superficial and Subcutaneous Fungal Infections

Table 32.1: Clinical types of dermatophytoses/tinea Direct Examination

infection. ; KOH mount examination with 10% for skin
scrapings or hair, 20% for nail clippings or
Tinea capitis (Fig. 32.2A) Infection of the scalp
calcofluor-white mount shows presence of
thin septate hyaline hyphae with arthroconidia;
Tinea faciei (Fig. 32.2B) _ Infection of the nonbearded
area of face
arthroconidia can be found on the periphery
of the hair shaft (ectothrix) or within the shaft
Tinea pedis (Athlete Infect first the webs between
foot) (Fig. 32.2C) the toes, then spread to the (endothrix).
sole in a“moccasin” pattern
Culture
Tinea corporis Infection of the nonhairy
(Fig. 32.2D) skin of the body Specimens should be inoculated onto SDA
Tinea cruris (or jock itch) Infection of the groin area containing cycloheximide (actidione) and
incubated at 26-28°C for 4 weeks. Potato dextrose
agar is used to stimulate the sporulation.
Identification is made by:
“* Macroscopic appearance of the colonies
such as rate of growth, texture, pigmentation,
colony topography
“+ Microscopic appearance:
s Hyphae: Dermatophytes possess thin
septate hyaline hyphae
= Two types of spores are observed: Small
unicellular microconidia and large
septate macroconidia; both are used for
identification of species (Table 32.2) (Fig.
32.3).
Identification features (macroscopic and
microscopic) of commonly encountered
Figs 32.2A to D: Ringworm infections (Tinea): (A) Tinea dermatophytes are given in Table 32.3.
capitis; (B) Tinea faciei; (C) Tinea pedis; (D) Tinea corporis.
Other Methods of Diagnosis
Source: Public Health Image Library, Centers for Disease Control
(C) ID
and Prevention (CDC), Atlanta; (A) ID #2936, (B) ID #4807, * Hair perforation test: It is positive
#2939 and (D) ID #2938 (with permission).
for Trichophyton mentagrophytes and
Microsporum canis
are produced (Table 32.1) (Figs 32.2A to D). * Urease test: Trichophyton mentagrophytes is
Incubation period is about 1-2 weeks. urease positive
“+ Dermatophyte test medium and dermato-
Laboratory Diagnosis
phyte identification medium
Wood's Lamp Examination
Certain dermatophytes fluoresce when the Table 32.2: Distribution of conidia of dermatophytes.
infected lesions are viewed under Wood's lamp
(due to presence of pteridine pigment in cell wall).
It is usually positive for various Microsporum Trichophyton Rare, thin, walled, |Abundant,
smooth, pencil, round to tear
species and Trichophyton schoenleinit. shaped drop shaped

Specimen Collection Microsporum Numerous, thick Rare

or scaly walled, rough,
Skin scrapings, hair plucks (broken spindle shaped
ones) and nail clippings are obtained from the
in folded Epidermophyton | Numerous, Absent
active margin of the lesion and are kept smooth, walled,
black paper. Hairs should be plucked and not
club-shaped
cut.
 s)
SECTION 2 © Systemic Microbiology (Infectious Disease

Figs 32.4A and B: Trichophyton mentagrophyte:

(A) Colony on SDA; (B) LPCB mount.
Source: Public Health Image Library, Centers for Disease Control
and Prevention (CDC), Atlanta; (A) ID #14717, (B) ID #15105
(with permission).

Figs 32.3A to C: Macroconidia of various dermatophyte

species: (A) Trichophyton mentagrophyte; (B) Microsporum
canis; (C) Epidermophyton floccosum.

Table 32.3: Identification features of commonly

encountered dermatophytes.

Dermatophytes | Macroscopic | Microscopic

appearance | appearance
Trichophyton White totan Microconidia—
mentagrophytes powdery numerous, round to
(Figs 32.4A Pigment pyriform Figs 32.5A and B: Epidermophyton floccosum: (A) Culture
and B) variable Macroconidia— on SDA; (B) LPCB mount.
cigar-shaped Source: Public Health Image Library, Centers for Disease Control
Spiral hyphae seen and Prevention (CDC), Atlanta; (A) Dr Lucille KG/ID #2937, (B) ID
#14588 (with permission).
Microsporum Cottony, Macroconidia—
canis orange abundant, thick
(Figs 32.1A pigmenton walled, spiny, “+ Alternatively: Oral griseofulvin or topical
and B) ovielte spindle shaped, up lotion such as Whitfield ointment or tolnaftate
to 15 septa, pointed can be applied.
ends
Microconidia— Mucocutaneous Candidiasis
rare Candida species (Chapter 21) are the most
Epidermophyton Powdery, Macroconidia—club common fungal agent to cause lesions of skin
floccosum folded, or clavate shaped in and mucosa.
(Figs 32.5A yellowish- clusters, 4-6 septa
and B) green Microconidia— Mucosal Candidiasis
absent
The various mucosal manifestations include:
** Oropharyngeal candidiasis (oral thrush): It
presents as white, adherent, painless patches
** Molecular methods: PCR can be used to in the mouth
detect species specific genes (e.g., chitin * Vulvovaginitis
synthase gene). * Balanitis and balanoposthitis (occurring in
uncircumcised males)
Treatment
** Esophageal candidiasis
“+ Oral terbinafine or itraconazole, used for ** Angular stomatitis and denture stomatitis
1-2 weeks for skin lesions, 6 weeks for hair ** Chronic mucocutaneous candidiasis: It is
infection, 3 months for onychomycosis seen in children with deficient CMI.
 CHAPTER 32 © Superficial and Subcutaneous Fungal Infections

Cutaneous Candidiasis Table 32.4: Agents of mycetoma and the types of

grains they produce.
The following cutaneous manifestations are
produced in candidiasis:
“+ Intertrigo: It is characterized by erythema Black granules: White to yellow granules:
and pustules in the skin folds; associated with e Madurellamycetomatis ¢ Nocardia species
e Madurella grisea Streptomyces somaliensis
tight fitting undergarments and sweating
e Exophiala jeanselmei e Actinomadura madurae
* Paronychia (involving nail-skin interface) e Curvularia species :
and onychomycosis (fungal infection of nail)
White granules: Pink to red granules:
* Diaper candidiasis: Pustular rashes, e Pseudallescheria boydii ¢ Actinomadura pelletieri
associated with use of diapers in infants e Aspergillus nidulans
“+ Perianal candidiasis e Acremonium species
* Erosio interdigitalis blastomycetica: It is e Fusarium species
an infection affecting the web spaces of
hands or toes Table 32.5: Clinical manifestations of eumycetoma
“ Generalized disseminated cutaneous and actinomycetoma.
candidiasis, seen in infants.
For laboratory diagnosis, refer Chapter 21. Tumor Single, well Multiple tumor masses,
defined margins _ ill-defined margins
§ SUBCUTANEOUS MYCOSES Sinuses Appear late, few Appear early,
in number numerous with raised
The agents of subcutaneous mycoses usually inflamed opening
inhabit soil and they enter the skin by traumatic Discharge Serous Purulent
inoculation with contaminated material and
Grains Black/white White/red
tend to produce the granulomatous lesions in
the subcutaneous tissue. Grains Fungal hyphae __ Filamentous bacteria
contain (>2 um) (<2 um)

Mycetoma
Mycetoma is a chronic, slowly progressive
bacterial agents (actinomycetoma). They differ
granulomatous infection of the skin and sub- from each other by various properties (Tables
cutaneous tissues. 32.4 and 32.5).
Clinical Manifestations
Types of Mycetoma and Causative Agents
Mycetoma can be of two types. It can be caused Clinical triad consists of (Fig. 32.6):
“ Tumor-like swelling, i.e., tumefaction
by either fungal agents (eumycetoma) or

Solvinng
[Problem Solvi Exercse
g Exerci 2
ise2 | |
Explanation
Mycetoma
Clinical Diagnosis
A 35-year-old farmer, with history of thorn prick
injury 20 days back presented to the hospital with A farmer, with thorn prick injury developed swelling
swelling of the right foot, 2-3 sinuses, discharging of the right foot, 2-3 discharging sinuses, gives a clue
black granules for 1 week (Fig. 32.6). Granules were to the diagnosis of eumycetoma (Fig 32.6).
collected in a sterile gauge and sent to the laboratory Identification
the
for diagnosis. Histopathogical examination of
As the discharging granules were black in color
granules is given in Figure 32.7A.
probable causative agents are Madurella mycetomatis,
1. What is the clinical diagnosis?
Madurella grisea, Exophiala jeanselmei and Curvularia
2. List the probable causative organisms.
to (Fig. 32.7A).
3. What are the various modalities available
(For answers to the other questions, refer below.)
confirm the diagnosis?
4. Mention about the treatment for the same clinical
condition.
 SECTION2 © Systemic Microbiology (Infectious Diseases)

* If eumycetoma is suspected then grains are

subjected to KOH mount, which reveal fungal
hyphae
* Ifactinomycetoma is suspected, grains are
subjected to Gram staining which reveals
filamentous gram-positive bacilli (0.5-1 pm
wide). Modified acid-fast stain is performed
if Nocardia is suspected, as it is partially
acid-fast
“~ Histopathological staining of the gran-
ules
= Eumycetoma: Reveals granulomatous
reaction with palisade arrangement of
Fig. 32.6: Mycetoma of foot hyphae in the cement substance (Fig.
Source: Public Health Image Library, Centers for Disease Control 32.7A)
and Prevention (CDC), Atlanta; ID #14816 (with permission). = Actinomycetoma: Shows granulomatous
reaction with filamentous bacteria at the
* Discharging sinuses
2,
margin (Fig. 32.7B).
* Discharge oozing from sinuses containing
granules. Treatment
Treatment of mycetoma consists of surgical
Laboratory Diagnosis
removal of the lesion followed by use of:
Specimen Collection “* For eumycetoma, itraconazole or ampho-
The lesions should be thoroughly cleaned with tericin B for 8-24 months, or
antiseptics and the grains should be collected ** For actinomycetoma, amikacin plus cotri-
on sterile gauze by pressing the sinuses from moxazole.
periphery or by a loop.
Sporotrichosis
Direct Examination
Sporotrichosis or rose gardener’s disease is
Granules are thoroughly washed in sterile saline; characterized by subcutaneous noduloulcerative
crushed between the slides and examined. lesions along the lymphatics; caused by a
* Macroscopic appearance of granules such thermally dimorphic fungus, Sporothrix
as color and size schenckii.

aA SSN wS ee
eh oe art 4Oe
es wea,
Taran’

Bie we bu ae
Figs 32.7A and B: (A) Eumycetoma (black grain and cement-like substance); (B) Actinomycetoma caused by
Nocardia brasiliensis (hematoxylin-eosin staining).
Source: (A) ID: #4331; (B) ID: #15055, Public Health Image Library, Centers for Disease Control and Prevention
(CDO),
Atlanta (with permission).
 CHAPTER 32 © Superficial and Subcutaneous Fungal Infections

Laboratory Diagnosis and occasionally in ears, larynx, bronchus and

Direct Microscopy genitalia.
** Agent: Itis caused by Rhinosporidium seeberi,
** Specimens such as pus, aspirate from nodules,
a lower aquatic fungus
are subjected to KOH mount or calcofluor
** Source: Fungal spores are inhaled while
staining which demonstrate elongated yeasts
taking bath in contaminated ponds and rivers
cells of 3-5 uum in diameter
** Diagnosis is made by histopathology of the
** Histopathological staining of tissue sections
polyps that demonstrates spherules (large
reveals cigar-shaped asteroid bodies. Asteroid sporangia up to 350 um size that contain
body consists of a central basophilic yeast
numerous endospores, each 6-9 tm in
cell surrounded by radiating extensions of size) (Fig. 32.9). It is stained better with
eosinophilic mass, composed of antigen- mucicarmine stain. R. seeberi has not been
antibody complexes (Fig. 32.8A). cultivated yet
“+ Treatment: Radical surgery with cauterization
Culture
is the mainstay of treatment. Dapsone has
The most definitive tool for diagnosis is culture.
been found to be effective.
Specimens are inoculated on SDA and blood
agar in duplicate and incubated at 25°C and 37°C Penicillium marneffei
as S. schenckii is a dimorphic fungus.
Penicillium marneffei is a thermally dimorphic
“* At 25°C: It produces mycelial form, consisting
fungus that causes opportunistic infection in
of hyphae with conidia arranged in flower-like
HIV-infected patients. It is endemic in Thailand,
pattern (Fig. 32.8B) Vietnam and India (Manipur).
“+ At37°C: It produces yeast form, with creamy-
white colonies. Clinical Manifestations

Treatment
P. marneffei produces two types of clinical
manifestations:
Itraconazole is the drug of choice for all forms ** Systemic infection mimicking that of
of sporotrichosis, except for disseminated form
disseminated histoplasmosis—such as fever,
where amphotericin B is recommended. weight loss, dyspnea, lymphadenopathy and
hepatosplenomegaly
Rhinosporidiosis
“+ Skin lesions: Warty lesions mimicking that of
Rhinosporidiosis is a chronic granulomatous molluscum contagiosum are seen.
disease, characterized by large friable polyps
in the nose (most common site), conjunctiva

ea
Figs 32.8A and B: Sporothrix schenckii. (A) Yeast form
(asteroid body); (B) Mold form showing thin septate
hyphae with flower-like sporulation. Fig. 32.9: Rhinosporidium seeberi—spherules containing
sporangia filled with endospores.
Source: (A) Dr Manoj Singh and Dr M Ramam, All India Institute
of Medical Sciences, New Delhi; (B) Public Health Image Library, Source: Public Health Image Library, Centers for Disease Control
and Prevention (CDC), Atlanta; Dr Martin Hicklin, ID #3107 (with
Centers for Disease Control and Prevention (CDC), Atlanta; Dr Libero
Ajello, ID #4208 (with permission). permission).
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

Figs 32.10A and B: Penicillium marneffei: (A) Methenamine silver staining shows yeast cells with central septations;
(B) Red pigmented colony (mold form).
Source: Public Health Image Library/Dr Libero Ajello, (A) ID#: 11959; (B) ID#: 11967/Centers for Disease Control and Prevention (CDC),
Atlanta (with permission).

Laboratory Diagnosis ** The mold form has a characteristic brick red

pigment, but the microscopic appearance is
** Histopathological staining of tissue sections
similar to other Penicillium species.
shows oval or elliptical yeast cells with central
septation (Fig. 32.10A) Treatment
* Culture: P. marneffei being dimorphic Patients with HIV/AIDS suffering from severe
produces yeast-like colonies at 37°C and mold penicilliosis are treated with amphotericin B and
form at 25°C (Fig. 32.10B) in mild cases, itraconazole is recommended.
 Bacterial Pharyngitis: Streptococcus
pyogenes Pharyngitis and Diphtheria
{ATES

B INTRODUCTION “+ Other rare causes include:

= Other B-hemolytic streptococci (group C
Pharyngitis (or sore throat) is one of the most and G)
common upper respiratory tract infections Arcanobacterium hemolyticum
(URTI). Viral pharyngitis accounts for the vast Fusobacterium necrophorum
majority of cases, and is usually self-limited. Mycoplasma pneumoniae
Bacteria are also important etiologic agents of Neisseria gonorrhoeae.
pharyngitis, require specific antibiotic treatment;
if not given, can lead to serious complications
@ STREPTOCOCCAL PHARYNGITIS
and sequelae. The common etiological agents of
bacterial pharyngitis include: Streptococcus pyogenes (or group A
* Streptococcus pyogenes (most common) Streptococcus) is the most common bacterial
“+ Corynebacterium diphtheriae cause of pharyngitis in children and accounts

Problem Solving Exercise 1

Streptococcal pharyngitis Identification
A 7-year-old child with history of pain in the throat Pinpoint colonies on blood agar with wide zone of
and fever for 2 days was brought to ear, nose, and beta-hemolysis (Fig. 29.2A), gram-positive cocci in
throat (ENT) out-patient department (OPD). On chain in culture smear (Fig. 29.1B), bacitracin sensitive
examination, he was febrile and throat examination (Fig. 29.2B and 29.3) are indicative that the causative
revealed pustules over the tonsils (Fig. 33.1). His throat organism is Streptococcus pyogenes.
swab was sent to the microbiology laboratory and Antimicrobial susceptibility testing
was subjected to culture (Fig. 29.2A, Chapter 29), and
Antimicrobial susceptibility testing on Mueller Hinton
antimicrobial susceptibility testing (AST) (Figs 29.2B,
blood agar (MHBA) (Fig. 29.3 and Table 29.3) showed
29.3 and Table 29.3, Chapter 29).
sensitive to penicillin, azithromycin and clindamycin,
1. Identify the causative agent based on the
and intermediate to erythromycin.
microbiological investigations performed.
List the infections caused by this organism. Treatment
Interpret the AST. As patient is a child and from OPD, azithromycin can
What antibiotic you would like to prescribe? be used for treatment.
wR
WN What are the different modalities of laboratory (For answers to other questions, refer below).
diagnosis?
Explanation
Clinical Diagnosis
Child with sore throat and fever for 2 days, with
enlarged tonsils covered with white fibrinous exudate
(Fig. 33.1) indicate acute pharyngitis.
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

(with sandpaper feel), strawberry tongue. It

has become less common now a days.

Laboratory Diagnosis
Culture
Throat (oropharyngeal) swabs should be collected
by vigorous rubbing of a sterile cotton swab over
both tonsillar pillars. Two swabs may be collected,
one for direct smear and the other for culture.
“+ Culture: On blood agar, S. pyogenes forms
small pinpoint colonies of 0.5-1 mm size with
a wide zone of B-hemolysis (Fig. 29.2A, Refer
Chapter 29)
Fig. 33.1: Streptococcal sore throat, with enlarged
“> Culture smear: Gram-stained smear from the
tonsils covered with white fibrinous exudate.
Source: James Heilman, Wikipedia (with permission).
colonies show gram-positive spherical cocci
(0.5-1 tm), arranged in short chains (Fig.
for 5-15% of all sore throats in adults. Infection 29.1B, Refer Chapter 29)
occurs through respiratory droplets. In addition, * Identification: S. pyogenes is identified by
it causes skin and soft tissue infections (Refer conventional biochemical tests (catalase
Chapter 29). negative and susceptible to bacitracin, Fig.
29.2B) or by automated identification systems
Clinical Manifestations such as MALDI-TOF or VITEK.
Streptococcal sore throat presents as either
Rapid Antigen Detection Test
localized (tonsillitis) or diffuse (pharyngitis).
“+ Symptoms: Manifests as erythema and swell- Rapid antigen detection test (RADT) is available
ing of pharyngeal mucosa with purulent exu- commercially, which detects group A carbohy-
date formation (Fig. 33.1); commonly affects drate antigens from throat swab by latex aggluti-
children nation or enzyme immunoassay for the diagnosis
** Suppurative complications: May occur of pharyngitis.
due to direct extension to deeper tissues
causing peritonsillar abscess (or quinsy) and ASO Titer
retropharyngeal abscess, or spillover to blood Anti-streptolysin O (ASO) antibodies appear
(bacteremia) late; therefore, a retrospective diagnosis of
** Non-suppurative complications may develop streptococcal infection may be established by
2-3 weeks following streptococcal sore throat, detecting ASO antibodies in patient’s serum.
such as acute rheumatic fever (ARF) and post- ASO titer is elevated >200 Todd unit/mL in most
streptococcal glomerulonephritis (PSGN) of the streptococcal infections. This was used in
* Differential diagnosis of streptococcal sore the past for establishing rheumatic fever.
throat include:
= Diphtheria: It can be differentiated from Molecular Test
streptococcal pharyngitis by presence of Commercial PCR assay is available for the
membrane over the tonsil detection of S. pyogenes in throat swab. It is a
m Viral pharyngitis: It is differentiated from rapid point of care test (turnaround time of 15
streptococcal pharyngitis by presence minutes), claims to be more sensitive.
of concomitant rhinorrhea, oral ulcers,
cough and/or hoarseness. Treatment
* Scarlet fever: It is mediated by streptococcal Streptococcal pharyngitis is treated with
pyrogenic exotoxins (e.g., SPE-A, B, and C) benzathine penicillin G, IM single dose or oral
It is characterized by pharyngitis and rashes penicillin V for 10 days.
 CHAPTER 33 © Bacterial Pharyngitis: Streptococcus pyogenes Pharyngitis and Diphtheria

HB DIPHTHERIA frequently shows club-shaped swellings (Fig.

33.2A) (Greek word koryne, meaning club)
introduction “> It has several species; the most important
Diphtheria is a highly infectious childhood dis- pathogenic being C. diphtheriae. The
ease caused by the bacterium Corynebacterium other Corynebacterium species (called
diphtheriae, which primarily infects the throat as diphtheroids) are usually found as
and produces toxin (diphtheria toxin) that commensals of throat and skin in man, can
causes an exudative pharyngitis and membra- occasionally cause infections
nous tonsillitis. If not promptly treated, it can “+ Corynebacterium diphtheriae typically shows
be life-threatening. two characteristic features, which differentiates
* The genus Corynebacterium is a gram- it from other Corynebacterium species
positive, non-capsulated, non-sporing, non- 1. Chinese letter or cuneiform arrangement:
motile bacillus. It is irregularly stained and They appear as V- or L-shaped in smear,

[ Problem Solving
Solving Exercise2,
Exercise2 _
Diphtheria of childhood immunization is suggestive of respiratory
A 2-year-old baby presented with pain in throat. On diphtheria. However, streptococcal pharyngitis and
examination a white patch over the tonsil was found candidalpharyngitis are other possibilities.
which bled on touch (Fig. 33.3A). His mother revealed Identification
that the baby has not taken immunization properly. Gram-positive bacilli in cuneiform arrangement
Throat swab collected was subjected to staining (Figs on direct Gram staining (Fig. 33.2B), green bacilli
33.2B and 33.2C) and culture (Fig. 33.4B). with metachromatic granules at the ends on Albert
1. What is the clinical diagnosis and its causative staining (Fig. 33.2C) and black colonies on potassium
organism; and what further test you would like to tellurite agar (Fig. 33.4B)—all confirms the causative
do? agent as C. diphtheriae.
2. List the virulence factors and other infections Toxigenicity test should be carried out further to
caused by this organism. find out the C. diphtheriae isolated is just acommensal
3. What are the various modalities of laboratory or is responsible for the clinical condition (Fig. 7.3,
diagnosis? Chapter 7).
4. What antibiotic you would like to prescribe? (For answers to other questions, refer below).
5. Howwill you prevent this condition?
Explanation
Clinical Diagnosis
History of white patch over the tonsil (Fig. 33.3A),
which bled on touch (pseudomembrane) and absence

smear; (B) Gram-stained

Figs 33.2A to C: Corynebacterium diphtheriae: (A) Club-shaped bacilli in methylene blue-stained
shows dark blue metachromatic
smear shows V- or L-shaped bacilli with cuneiform arrangement; (C) Albert's stain
granules at the ends of the green bacilli (schematic).
for Disease Control and Prevention (CDC), Atlanta (with
Source: Public Health Image Library, (A) ID# /7323/P.B. Smith; (B) ID# /1943, Centers
permission).
246 SECTION 2 ©@ Systemic Microbiology (Infectious Diseases)

because the bacterial cells divide and

daughter cells tend to lie at acute angles to
each other. This type of cell division is called
snapping type of division (Fig. 33.2B)
2. Metachromatic granules: They are
present at ends or poles of the bacilli.

Metachromatic Granules
Also called polar bodies or Babes-Ernst bodies or
volutin granules).
Q They are storage granules of the organism,
composed of polymetaphosphates
Q Granules are stained strongly gram-positive Figs 33.3A and B: (A) Pseudomembrane covering
compared to remaining part of the bacilli. The the tonsils classically seen in diphtheria; (B) Bull neck
granules take up bluish purple metachromatic appearance (arrow showing).
color when stained with Loeffler’s methylene blue Source: (A) Department of Microbiology, JIPMER, Puducherry; (B)
Public Health Image Library/ID#5325, Centers for Disease Control
Q However, they are better stained with special
and Prevention (CDC), Atlanta (with permission).
stains, such as Albert's, Neisser’s and Ponder’s stain
(Fig. 33.2C) and systemic complications (except the skin
Q Granules are well developed on enriched media,
such as blood agar or Loeffler’s serum slope
lesions, which is caused due to the organism,
Q Volutin granules can also be possessed by other not toxin).
organisms such as—by Corynebacterium xerosis
and Gardnerella vaginalis. Respiratory Diphtheria
This is the most common form of diphtheria.
Virulence Factors (Diphtheria Toxin) Tonsil and pharynx (faucial diphtheria) are the
most common sites followed by nose and larynx.
Diphtheria toxin (DT) is the primary virulence
Incubation period is about 3-4 days.
factor responsible for diphtheria. It is encoded
* Faucial diphtheria: Characterized by
by genome of a bacteriophage.
formation of a tough leathery greyish white
** Toxin is a polypeptide chain, comprises of two
pseudomembrane over tonsil (Fig. 33.3A).
fragments—A (active) and B (binding)
It is so named as it is adherent to the mucosal
“+ Fragment B binds to the host cell receptors
base and bleeds on removal, in contrast to the
and helps in entry of fragment A
true membrane which can be easily separated
** Fragment A gets internalized into the cell and
** Extension of pseudomembrane: In severe
then acts by the mechanism given below.
cases, it may extend into the larynx and
Mechanism of Diphtheria Toxin bronchial airways, which may result in fatal
Q Fragment A is the active fragment, which causes airway obstruction leading to asphyxia. This
ADP ribosylation of elongation factor 2 (EF-2) > mandates immediate tracheostomy
leads to inhibition of EF-2 — leads to inhibition of * Bull-neck appearance: It is characterized by
translation step of protein synthesis massive tonsillar swelling and neck edema.
Q Exotoxin A of Pseudomonas has a similar
mechanism like that of DT.
Patients present with foul breath, thick speech,
and stridor (noisy breathing) (Fig. 33.3B).

Pathogenicity and Clinical Features Laboratory Diagnosis

Pathogenesis of diphtheria is toxin mediated. The diagnosis of diphtheria is based on the
** Diphtheria is toxemia but never a bacteremia clinical signs and symptoms plus laboratory
* Bacilli are noninvasive, present only at local confirmation.
site (pharynx), secrete the toxin which spreads ** Because ofthe risk of respiratory obstruction,
via bloodstream to various organs specific treatment should be instituted
* Itis the toxin which is responsible for all types immediately on clinical suspicion without
of manifestations including local (respiratory) waiting for laboratory reports
 CHAPTER 33 © Bacterial Pharyngitis: Streptococcus pyogenes Pharyngitis and Diphtheria

** Laboratory diagnosis is necessary only for:

= Confirmation of clinical diagnosis
® Initiating the control measures
= Epidemiological purposes.
Laboratory diagnosis consists of isolation of
the bacilli and toxin demonstration.

Isolation of Diphtheria Bacilli

Specimen
Figs 33.4A and B: (A) Loeffler’s seruin slope; (B) Potassium
Useful specimens include: (1) throat swab (one
tellurite agar shows black colonies.
or two) containing fibrinous exudates, (2) a
Source: (A) Department of Microbiology, JIPMER, Puducherry; (B)
portion of pseudomembrane, (3) nose or skin Department of Microbiology, Pondicherry Institute of Medical
specimens (if infected). Sciences, Puducherry (with permission).

Direct Smear Microscopy Selective Medium

Selective media are best for isolation of C.
* Gram stain: C. diphtheriae appear as
diphtheriae from cases as well as from carriers,
irregularly stained club-shaped gram-
as the normal flora will be inhibited.
positive bacilli of 3-6 um length, typically
arranged in Chinese letter or cuneiform Potassium tellurite agar (PTA): C. diphtheriae
arrangement (V- or L-shaped). It is difficult reduces tellurite to metallic tellurium which
to differentiate them from other commensal gets incorporated into the colonies giving
coryneforms found in the respiratory tract them black color (Fig. 33.4B). C. ulcerans and
(see Fig. 33.2B) C. pseudotuberculosis can also grow on PTA
“ Albert’s stain: It is more specific for C. producing black colored colonies.
diphtheriae; they appear as green bacilli “+ Advantage: Throat commensals are inhibited
with bluish black metachromatic granules “+ Disadvantage: Colonies appear only after 48
at the poles. Details of Albert’s staining hours of incubation.
procedure is given in Chapter 3.5 (see Fig.
Identification
23 2G):
Identification of C. diphtheriae is made by:
Culture Media “ Biochemical tests such as—(i) Sugar
Enriched Medium fermentation test using Hiss’s serum sugar
C. diphtheriae is fastidious, aerobe and media, (ii) Pyrazinamidase test, and (iii)
facultative anaerobe; does not grow on ordinary Urease test. The latter two tests are negative
medium. It grows best in enriched medium for C. diphtheriae
such as blood agar, chocolate agar and Loeffler’s “ Automated identification systems such as
serum slope. Plates are incubated at 37°C MALDI-TOF or VITEK.
aerobically.
Toxin Demonstration
* Blood agar: Colonies are small circular, white
and sometimes hemolytic (mitis biotype) As the pathogenesis is due to diphtheria toxin,
* Loeffler’s serum slope: Colonies appear as mere isolation of bacilli does not complete the
small, circular, glistening, and white with a diagnosis. Toxin demonstration should be done
yellow tinge in 6-8 hours (Fig. 33.4A) following isolation, which can be performed by
m Advantages: (1) Growth can be detected in vivo and in vitro methods.
as early as 6-8 hours. (2) Best medium for
In Vivo Tests (Animal Inoculation)
metachromatic granules production
m Disadvantages: Being an enriched In vivo toxin demonstration can be done by
medium, if incubated beyond 6-8 inoculation of culture broth into guinea pig.
hours, it supports growth of other throat With the advent ofother techniques, this method
commensals also. is rarely followed nowadays.
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

In Vitro Test
= Ifgiven early (<6 h of infection), before the
toxin release
* Elek’s gel precipitation test: This is a type = Prevent further release of toxin by killing
of immunodiffusion in gel described by Elek
the bacilli
(1949)
= Treatment of cutaneous diphtheria
= The strain isolated is streaked onto a
= Treatment of carriers: Drug of choice is
media containing a filter paper soaked
erythromycin.
with antitoxin
w If the strain is toxigenic, it liberates the
Prophylaxis
toxin which diffuses in the agar and meets
with the antitoxin to produce an arrow- Infection Control Measures
shaped precipitation band Patient should be placed in isolation room and
= This test can also be used to know the all the steps of droplet precaution should be
relatedness between the strains isolated followed for the prevention of transmission of
during an outbreak. The precipitate bands C. diphtheriae in hospitals (Refer Chapter 11).
of outbreak isolates (streaked adjacent)
when meet with each other, three patterns Post-exposure Prophylaxis
may be observed (Fig. 7.3, Chapter 7) For close contacts (e.g., household), booster dose
1. Cross-over with each other—indicates of diphtheria vaccine + penicillin G (single dose)
unrelated strain or erythromycin (7-10 days) is recommended.
2. Spur formation—indicates partially
related strain Vaccination
3. Fused with each other—indicates Diphtheria toxoid is used for vaccination as it
identical strain. induces antitoxin production in the body. A
“+ Other in vitro tests include: protective titer of more than 0.01 Unit/mL of
= Detection of Tox gene by PCR antitoxin can prevent all forms of diphtheria.
® Detection of diphtheria toxin by ELISA or However, vaccine is not effective for:
immunochromatographic test (ICT) ** Prevention of cutaneous diphtheria
= Cytotoxicity produced on cell lines. ** Elimination ofcarrier state.

Typing of C. diphtheriae Types of Vccine

Typing methods are useful for epidemiological ** Single vaccine: Diphtheria toxoid (DT) is
os

studies, to know the relatedness between the prepared by incubating toxin with formalin
isolates. Biotyping was in use in the past, based on and then the toxoid is adsorbed on to alum.
which there are four biotypes of C. diphtheriae— Alum acts as adjuvant and increases the
gravis, intermedius, mitis and belfanti. They vary immunogenicity of toxoid
in virulence and toxin production; gravis being * Combined vaccine: Various vaccines
100% toxigenic and more virulent. available are:
= DPT: Contains DT (diphtheria
Treatment
toxoid), Pertussis (whole cell) and TT
Treatment should be started immediately on (tetanus toxoid). Pertussis component
clinical suspicion of diphtheria. acts as adjuvant and increases the
* Antidiphtheritic serum or ADS (antitoxin): immunogenicity of DT and TT
Passive immunization with antidiphtheritic = DaPT: Contains DT, TT and acellular
horse serum is the treatment of choice as it pertussis (aP)
neutralizes the toxin = Td: It contains tetanus toxoid and adult
* Antibiotics: Penicillin or erythromycin is the dose (2 Lf)of diphtheria toxoid
drug of choice. Antibiotic plays a minor role m Pentavalent vaccine: DPT can also
as it is of no use once the toxin is secreted. be given along with hepatitis B and
However, antibiotics are useful: Haemophilus influenzae type b.
 CHAPTER 33 © Bacterial Pharyngitis: Streptococcus pyogenes Pharyngitis and Diphtheria

Administration of Diphtheria Vaccine booster doses of DPT at 16-24 months and

** Schedule: Under National Immunization 5 years and another two booster doses of
Schedule (NIS) of India 2020: Td at 10 years and 16 years
® Children: Total seven doses are given; = Pregnant woman also should receive two
three doses of pentavalent vaccine at 6, doses of Td at one month interval.
10 and 14 weeks of birth, followed by two * Site: DPT is given deep intramuscularly (IM)
at anterolateral aspect of thigh.
LC
OSES
aR ols
’ |

wt ose.
Diphtheroids
Diphtheroids or coryneforms are the
nondiphtherial corynebacteria, that usually
exist as normal commensals in the throat, skin,
conjunctiva and other areas. However, they
“ have been associated with invasive disease,

' | ct.F particularly in immunocompromised patients.

They can be differentiated from C. diphtheriae
by many features such as:
> “+ Stains more uniformly than C. diphtheriae
o-
¢* Palisade arrangement: Arranged in parallel
Fig. 33.5: Diphtheroids—palisade arrangement
rows rather than cuneiform pattern (Fig. 33.5)
of gram-positive bacilli.
Source: Department of Microbiology, JIPMER, Puducherry
* Absence of metachromatic granules (except
(with permission). C. xerosis).
 Bacterial Pheumonia 3A

B INTRODUCTION lobar pneumonia, otitis media in children and

meningitis in all ages.
Bacterial pneumonia is classified into two
groups; typical (lobar) and atypical or Clinical Manifestations
(interstitial) pneumonia. Pneumococci first colonize the human
* Lobar or typical pneumonia: It involves nasopharynx, which usually occurs at an early
infection of the lung parenchyma and its age. From the nasopharynx, the bacteria spread
alveoli. It is characterized by consolidation either via the bloodstream to distant sites (e.g.,
(gives a dull note on percussion) and brain, joint, bones and peritoneal cavity) or spread
productive cough with purulent sputum. It is locally to cause otitis media or pneumonia.
mostly caused by pyogenic organisms, such as:
ws Streptococcus pneumoniae Table 34.1: Differences between Streptococcus
= Haemophilus influenzae pneumoniae and viridans streptococci.
= Staphylococcus aureus S. pneumoniae Viridans
= Gram-negative bacilli. streptococci
** Interstitial
: : or: atypical pneumonia occurs: Arrangement Gram-positive Gram-positive
in the interstitial space of lungs. Cough is cocci in pairs cocci in long
characteristically non-productive. It is mostly chains
caused by bacteria, such as Mycoplasma, Morphology Lanceolate shaped Round/oval
Chlamydia, Legionella species, etc. Capsule Present Absent
On blood Draughtsman or Minute colony
fl PNEUMOCOCCAL PNEUMONIA a stro are
Introduction Bile solubility Soluble in bile Insoluble in bile

Streptococcus pneumoniae (commonly referred D ESHinenes Nor Fernenies

to as pneumococcus) is the leading cause of OPtechin _ Sensitive ae

| Problem
Problem Solving
Solving Exercise
Exercise1 _|
Pneumococcal Pneumonia (Figs 34.2B and D) and antimicrobial susceptibility
A 15-year-old boy presented to emergency _ test (AST) (Fig. 34.3 and Table 34.2).
department with complaints of high grade fever, |: What is the clinical diagnosis and its causative
productive cough, chest pain and dyspnea for past organism?
3 days. Physical examination revealed dull note on 2: List the virulence factors and infections caused by
percussion. Chest X-ray revealed consolidation over this organism.
right lower lobe. 3. Briefly discuss the laboratory diagnosis.
Sputum sample was collected and sent to “4. Interpret the AST.
microbiology laboratory for Gram stain (Fig. 34.1), 5 What antibiotic you would like to prescribe?
culture (Figs 34.2A and C), biochemical reactions
 CHAPTER 34 © Bacterial Pneumonia

Explanation Antimicrobial Susceptibility Testing

Clinical Diagnosis AST on Mueller Hinton blood agar (Fig. 34.3 and Table
High grade fever, productive cough, chest pain 34.2) showed resistant to levofloxacin, ofloxacin and
and dyspnea with dull note on percussion and sensitive to penicillin, erythromycin and vancomycin.
consolidation over right lower lobe (on chest X-ray) Oxacillin is a surrogate marker of penicillin susceptibil-
are clinically suggestive of lobar pneumonia. ity for pneumococcus.

Identification Treatment
Gram-positive cocci in pair (lanceolate shaped) (Fig. As it is a pneumonia isolate, oral erythromycin can
34.1), a-hemolytic colonies on blood agar (Fig. 34.2A), be given for treatment. If it was a meningitis isolate,
bleaching on chocolate agar (Fig. 34.2C), soluble in penicillin or other beta-lactam antibiotics, such as
bile (Fig. 34.2D), sensitive to optochin (Figs 34.2B and ceftriaxone would have been the drug of choice.
34.3) are suggestive ofidentification ofthe etiological (For answers to other questions, refer below).
agent as Streptococcus pneumoniae.

Various manifestations include: collected. Automated blood culture is useful

“> Lobar pneumonia: S. pneumoniae is the most for invasive infection. Other specimens include
common cause of lobar (alveolar) pneumonia. aspiration of sinus material and ear discharge/
Patients present with productive purulent aspirate.
cough, fever and chest pain. Important
signs are dullness on percussion due to Direct Smear Microscopy
consolidation and crackles on auscultation Direct microscopy of smears made from
‘» Empyema and parapneumonic effusion specimens show numerous pus cells and
may occur as complications of pneumococcal lanceolate or flame-shaped gram-positive
pneumonia cocci (1 4m) in pairs, surrounded by a clear
* Invasive pneumococcal disease (IPD): halo (due to capsule). Direct microscopy is
Defined as an infection confirmed by isolation extremely useful especially for meningitis, as
of pneumococci from a normally sterile site. empirical treatment (antibiotics) can be started
Various examples include: early (Fig. 34.1).
= Bloodstream infection
m Pyogenic meningitis: S. pneumoniae is
the leading cause of meningitis in all ages
(Refer Chapter 39)
m= Other invasive manifestations: S.
pneumoniae can cause osteomyelitis,
septic arthritis, endocarditis, pericarditis,
primary peritonitis; rarely, brain abscess
and hemolytic-uremic syndrome.
* Noninvasive infections—otitis media (most
common) and sinusitis.

Laboratory Diagnosis
General laboratory diagnosis of various
pneumococcal infections is discussed below.
Fig. 34.1: Pheumococci in gram-stained smear of sputum
Specimen Collection
[lanceolate shaped gram-positive cocci in pair surrounded
Depending on the site of infection, specimens, by clear halo (capsule)].
such as sputum, cerebrospinal fluid (CSF), Source: Department of Microbiology, JIPMER, Puducherry (with

pleural fluid and other sterile body fluids are permission).

 SECTION 2 © Systemic Microbiology (Infectious Diseases)

Antigen Detection * Chocolate agar: It produces greenish

discoloration (described as bleaching effect)
* Capsular antigens: Detection of capsular (Fig. 34.2C).
antigens in CSF is more sensitive than
microscopy. It is done by latex agglutination Culture Smear
test using latex beads coated with anticapsular
Gram stained smear of the colonies reveals
antibodies
lanceolate or flame-shaped gram-positive cocci
“ C-antigens: Detection of C-polysaccharide
(1 pm) in pairs.
antigen in urine by immunochromatographic
test (ICT) is useful for diagnosis of pneumonia; Identification
however, it gives positive results even for
Pneumococci are catalase negative and can
nasopharyngeal carriers. Therefore, it is not
be differentiated from viridans streptococci
useful in children where the carriage rate is
(which are also a-hemolytic, found as oral
high. This ICT is also available for antigen
commensals in sputum specimens) in various
detection from CSF in meningitis cases.
ways (Table 34.1).
** Bile solubility: Pneumococci are soluble
Culture
in bile (sodium deoxycholate) due to their
S. pneumoniae is fastidious, does not grow in enhanced autolytic activity in presence of
basal media like nutrient agar or nutrient broth. bile. Viridans streptococci are insoluble in
Specimens are inoculated onto enriched media, bile (Fig. 34.2D)
such as blood agar, and chocolate agar and * Optochin sensitivity: Pneumococci are
incubated for 24 hours at 37°C in presence of sensitive to optochin disk and produce wider
5-10% of CO,,. zone of inhibition (14 mm or more) (Fig.
* Blood agar: Colonies are initially small 34.2B). Viridans streptococci are resistant to
dome shaped, surrounded by green zone optochin
of a-hemolysis. Colonies on prolonged * Inulin fermentation: Pneumococci can
incubation, undergo autolysis, and therefore ferment inulin to form acid, but not viridans
have a central depression with an elevated streptococci
rim; giving rise to draughtsman-shaped or ** Automated methods, such as MALDI-TOF
carrom coin-shaped appearance (Fig. 34.2A) and VITEK can also be used for early and

Capsule (before
adding antiserum)

Ee capsule
(after adding
antiserum)
Figs 34.2A to E: Properties of pneumococci: (A) a-hemolytic draughtsman-shaped colonies on blood agar;
(B) Sensitive
to optochin; (C) Bleaching effect on chocolate agar; (D) Bile solubility test (left-viridans streptococci,
not soluble in bile;
right—pneumococcus, soluble in bile); (E) Quellung reaction.
Source: (A) Department of Microbiology, f Pondicherry Institute of Medical Sciences, Puducherry; ; (B to D) D epartment of M i i
JIPMER, Puducherry (with permission). : wae at esta
 CHAPTER 34 © Bacterial Pneumonia

accurate identification. This is very useful in consolidation is seen in upper part of the lower
early institution of pathogen-directed therapy. lobe, called round pneumonia).

Typing of S. pneumoniae Antimicrobial Susceptibility Test (AST)

“+ Quellung or Neufeld reaction: This test was AST is necessary for institution of appropriate
routinely done in the past at bedside, directly antibiotic treatment. It can be performed by
from sputum samples from pneumonia Cases. disk diffusion test on Mueller Hinton blood agar
When specimen is treated with type-specific (Fig. 34.3) or by automated MIC detection
antiserum, along with methylene blue dye; method by microbroth dilution (e.g., VITEK). The
capsule becomes swollen, sharply delineated latter is the preferred method as disk diffusion
and refractile (Fig. 34.2E) break points of several antibiotics (e.g., penicillin,
* Currently, serotyping is done by latex ceftriaxone) are not available for pneumococcus.
agglutination test using type specific antisera. AST should be reported according to CLSI
interpretation criteria (Table 34.2).
Molecular Methods
Molecular methods, such as PCR are highly
sensitive, more useful when organism load is
scanty (e.g., CSF), detect earlier than culture
and also help in serotype identification.
* Real-time PCR is even more sensitive,
specific, takes less time and is quantitative
* Multiplex PCR (e.g., BioFire FilmArray,
bioMérieux) and Multiplex real-time PCR
can be used for simultaneous detection of
common agents of lobar pneumonia from
sputum or bronchoalveolar lavage
“ Common genes targeted include: lytA
(autolysin gene), ply (pneumolysin) and psaA
(pneumococcus surface antigen A).

Nonspecific Findings Fig. 34.3: Antimicrobial susceptibility testing on Mueller

Hinton blood agar (Pneumococcus) (Refer Table 34.2 for
* Elevated acute phase reactant proteins, such CLSI zone interpretation).
as C-reactive protein, procalcitonin Abbreviations: Op, optochin; Ox, oxacillin; Le, levofloxacin; Of,
** Leukocytosis ofloxacin; E, erythromycin;
Va,vancomycin.
* Chest X-ray shows infiltrates and lobar Source: Department of Microbiology, Pondicherry Institute of

consolidation (In children-distinctly spherical Medical Sciences, Puducherry (with permission).

zone size diameter (mm) to various antimicrobial

Table 34.2: Interpretative categories (CLSI) and observed
agents tested for pneumococcus isolates .
Observed zone
Antimicrobial Disk strength | CLSI interpretative criteria for
Streptococcus pneumoniae (in mm) size (in mm) (Fig.
agents
FResistant [intermediate [Sensitive |°0™)
14 Sensitive
<14 = 214
Optochin (Op)
— 220 22 Sensitive
Oxacillin (Ox)* 1 _
14-16 6 Resistant
Levofloxacin (Le) 5
6 Resistant
5 13-15
Ofloxacin (Of)
16-20 221 25 Sensitive
Erythromycin (E) 15 <15
— 217 WA Sensitive
Vancomycin (Va) 30 —
susceptibility for pneumococcus.
*Note: Oxacillin is a surrogate marker of penicillin
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

Treatment Prevention and Vaccination

Measures to prevent pneumococcal disease
The treatment regimen for pneumococci
varies depending upon the type of infective include vaccination, treatment of underlying
syndrome. diseases (that increase the risk of pneumococcal
“ Pneumonia: Oral therapy with amoxicillin disease), infection control measures (droplet
for five days remains the standard treatment precautions, refer Chapter 11), and prevention
among outpatients. Alternative drugs include of antibiotic overuse.
IV penicillin or ceftriaxone, oral quinolone
(levofloxacin or moxifloxacin), clindamycin Pneumococcal Vaccines
or azithromycin There are two vaccines available for
* Meningitis: As mortality is very high pneumococcus: (i) 23-valent pneumococcal
(~20%), treatment should be initiated as polysaccharide vaccine (PPSV23), and (ii)
early as possible. Ceftriaxone/cefotaxime + pneumococcal conjugate vaccine (PCV13). The
vancomycin is given for 10-14 days indications include:
* Other invasive infections: For stable ¢ After birth: PCV13 is given in infants after
children, penicillin, cefotaxime or ceftriaxone birth; three primary doses at 6th, 10th and
can be instituted. In critically ill children, 14th week of age and a booster at 15 months
the treatment should be same as that of “+ In adults (265 year, or in presence of risk
meningitis factors): Either PPSV23 only can be given or
“+ Otitis media: Oral amoxicillin for 7-10 days PCV13 is given first followed by PPSV23 at a
is the drug of choice. gap of 21 year.

B HAEMOPHILUS INFLUENZAE PNEUMONIA

| Problem Solving
Solving Exercise2
Exercise 2 |
Haemophilus influenzae Pnuemonia Explanation
A 4-year-old orphan girl was brought to the Clinical Diagnosis
emergency room by her parents due to an acute Fever, productive cough and dyspnea with dull note
onset offever, productive cough and dyspnea for past on percussion and consolidation over left lower lobe
two days. Physical examination revealed dull note (on chest X-ray) are clinically suggestive of lobar
on percussion. Chest X-ray showed consolidation pneumonia.
over left lower lobe. Sputum sample was sent to
microbiology laboratory for Gram stain (Fig. 34.5A) Identification
and culture (Fig. 34.5B). The causative agent here is H. influenzae. Points in
1. What is the clinical diagnosis and its causative favor are:
organism? Q Pleomorphic gram-negative bacilli shown in
2. List the clinical conditions caused by this sputum Gram stain (Fig. 34.5A)
organism. Q Presence of satellitism surrounding Staphylococcus
3. How will you treat this condition? aureus streak line on blood agar (Fig. 34.5B).
4. How this disease can be prevented? (For answers to other questions, refer below).

Introduction Serotyping
Haemophilus species are oxidase positive, Based on the capsular polysaccharide of H. influ-
capsulated pleomorphic gram-negative bacilli enzae, it can be typed into six serotypes (a to f).
that require special growth factors present in However, some strains lack capsule and are re-
blood, such as factor X and V. H. influenzae is ferred to as nontypeable strains. H. influenzae se-
the most pathogenic species, which causes rotype b (Hib) is the most virulent among all types
pneumonia and meningitis in children. and accounts for most of the invasive infections.
 CHAPTER 34 © Bacterial Pneumonia

Clinical Manifestations “ Parameningeal focus of infection is often

present in adults (such as sinusitis and
H. influenzae Type b (Hib) otitis), from which the infection may spread
H. influenzae type b is the most common to meninges.
and most invasive serotype of H. influenzae.
It causes systemic disease by invasion and Nontypeable H. influenzae
hematogenous spread from the respiratory Next to Hib, nontypeable strains are the most
tract to distant sites, such as the meninges, common group encountered clinically. They
bones, and joints. cause disease by local invasion of mucosal
« Central nervous system infections: It can surfaces, such as otitis media, sinusitis etc.
cause pyogenic meningitis and subdural
effusion. Mortality rate is high if untreated Laboratory Diagnosis
(Refer Chapter 39) Specimen Collection and Transport
* Epiglottitis: Affects children, can lead to acute
airway obstruction Depending upon the site of infection, various
specimens may be collected, such as CSF, blood,
* Community acquired bacterial pneumonia:
Hib causes pneumonia in infants, which is sputum, pus, aspirates from joints, middle ears
or sinuses
clinically similar to other types of bacterial
* As H. influenzae is highly sensitive to low
pneumonia except that, pleural involvement
is more common in Hib infection temperature, the specimens for culture
should never be refrigerated
“* The specimens should be transported to the
S.aureus laboratory without any delay and processed
streak line immediately.

Direct Detection
Colonies of
| H. influenzae * Gram staining of CSF and other specimen
shows pleomorphic gram-negative
coccobacilli (Fig. 34.5A)
* Capsule detection (Quellung reaction):
Capsular swelling occurs when a drop of CSF
is mixed with type b antiserum and methylene
blue and observed under the microscope
* Antigen detection: The type b capsular
Fig. 34.4: Satellitism of Haemophilus influenzae
(schematic diagram). antigen can be detected in CSE, urine or other

*, 7 * $, Zz rave
je * 2s
i? ; ' ey ’ x |

a influenzae WN i gt * ts
lewy4 Kal
ye ve
° |
*g|

= S.aureus

e
hic gram-negative bacilli; (B) Satellitism of H. influenza
Figs 34.5A to D: (A) Gram-stained smear showing pleomorp of H. influenzae showing
chocolate agar; (D) Colony smear
around S. aureus streak line; (C) Colonies of H. influenzae on
pleomorphic gram-negative bacilli. Institute of Medical
y; (B to D) Department of Microbiology, Pondicherry
Source: (A) Department of Microbiology JIPMER, Puducherr
Sciences, Puducherry (with permissi on).
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

body fluids by latex agglutination test using

* It is largely aerobic and growth is poor
latex particles coated with antibody to type anaerobically. Culture plates are incubated
b antigen. at 37°C ii candle jar. Growth is enhanced by
5-10% CO,,.
Culture
Culture Smear and Motility Testing
H. influenzae is highly fastidious, requires two
accessory growth factors (factor X and V) in Gram staining of culture isolates reveals
blood for their growth. pleomorphic gram-negative bacilli (Fig. 34.5D)
“> Factor X is a hemin, present freely in blood and hanging drop reveals nonmotile bacilli.
* Factor Vis nicotinamide adenine dinucleotide
(NAD), which is present inside RBCs. It is Biochemical Tests
also produced by some bacteria, such as “+ Catalase positive and oxidase positive
Staphylococcus aureus. * Disk test for X and V requirement:
The growth of H. influenzae may vary in different Haemophilus species vary in their X and V
media depending upon the availability of X and requirement. This property can be exploited
V factors. for speciation by inoculating the isolate onto
“+ No growth on basal media: Nutrient agar and medium lacking X and V factors and then
peptone water lack X and V factors, therefore, placing the X, V, and combined XV disks on
they do not support the growth of H. influenzae the medium for demonstrating the growth
** Growth is scanty on blood agar: It is because requirements. H. influenzae produces growth
only factor X is available freely in blood agar surrounding combined XV disk, but fails to
and factor V is largely intracellular, present grow surrounding individual X and V disks
only inside the RBCs. It is available in very (Fig. 34.6).
minute quantities freely in the medium
** Grows well on chocolate agar: While Molecular Method
preparing chocolate agar, blood is poured ** Multiplex PCR: It is useful in simultaneous
a

into molten agar at 75°C which lyses RBCs detection of common agents of pyogenic
releasing excess of factor V. Hence, it supports meningitis, such as pneumococcus,
the growth ofH. influenzae (Fig. 34.5C) meningococcus, Listeria and H. influenzae
*‘¢ Grows well on blood agar with S. aureus (targeting conserved capsular gene bexA)
streak line: Colonies of H. influenzae * BioFire FilmArray: It is an automated
grow adjacent to S. aureus streak line—a multiplex PCR; the respiratory panel can
phenomenon called as satellitism simultaneously detect 33 pathogens including
H. influenzae.
Satellitism
H. influenzae can grow on blood agar if the source
of V factor is provided (Figs 34.4 and 34.5B)
Q When S. aureus is streaked across a blood agar
plate perpendicular to the H. influenzae streak
line, factorV is released from S. aureus
Q Therefore, it forms larger colonies adjacent to
S. aureus streak line and size of the colonies
decreases gradually away from the S. aureus
streak line
Q This phenomenon is called satellitism, a
property that is routinely employed for the
isolation of H. influenzae.

* Special media, such as Fildes agar and

Levinthal’s agar; containing factor X and V
Fig. 34.6: H. influenzae showing growth around
* Haemophilus selective medium containing
combined XV disk only, but not around X and V disks.
bacitracin and sucrose, is useful for sputum
Source: Department of Microbiology, Pondicherry Institute
specimen of Medical Sciences, Puducherry (with permission).
 CHAPTER 34 © Bacterial Pneumonia

Antimicrobial Susceptibility Testing (AST) § STAPHYLOCOCCAL PNEUMONIA

AST is performed by disk diffusion method (on Staphylococcus aureus causes pneumonia in
Haemophilus test medium or chocolate agar) selected clinical settings.
or by automated MIC detection method by * Infant pneumonia: S. aureus is a leading
microbroth dilution (e.g., VITEK). cause of pneumonia in newborns and infants;
presents with dyspnea, fever, and respiratory
Treatment
failure
“+ For invasive infections due to H. influenzae = Chest X-ray reveals characteristic pneu-
type b, cephalosporins, such as ceftriaxone, matocele (shaggy, thin-walled cavities)
cefotaxime (for 1-2 weeks) are the drug of = Complications, such as pneumothorax
choice and empyema may be developed subse-
“+ Nontypeable strains of H. influenzae are often quently.
resistant to B-lactams. Those strains are usually * VAP in adults: S. aureus can cause
susceptible to quinolones (levofloxacin) and pneumonia in hospitalized patients who are
macrolides (azithromycin). on mechanical ventilation, called ventilator-
associated pneumonia (VAP). Patients who
Hib Conjugate Vaccine are nasal carriers are at increased risk to
The capsular antigen of H. influenzae type develop VAP
b is used for vaccination. To increase its * Post-viral pneumonia: S. aureus can cause
immunogenicity, the capsular antigens are pneumonia secondary to viral infections—
conjugated with adjuvants, such as diphtheria. most commonly influenza
Schedule: Under national immunization ¢ CA-MRSA: Community-associated MRSA
program, Hib vaccine is given in combination strains can be more virulent and can cause
with DPT, hepatitis B (pentavalent vaccine) at 6, necrotizing pneumonia.
10 and 14 weeks of birth. It is administered in IM Staphylococcus aureus mainly causes skin and
route, at anterolateral side of mid-thigh. soft tissue infections, discussed in Chapter 28.

HB GRAM-NEGATIVE BACILLI PNEUMONIA

ro
GiSolving Exercise 3
Klebsiella pneumoniae Pneumonia Q Short, plump, straight capsulated gram-negative
rods in sputum Gram stain (Fig. 34.7A)
A 70-year-old male is admitted with high grade
Q Presence of large dome shaped mucoid sticky,
fever and productive cough. Chest X-ray showed
pink color, lactose fermenting colonies on
consolidation over left lower lobe. Sputum sample
MacConkey agar (Fig. 34.7B)
was collected and sent to microbiology laboratory for
Q Biochemical reaction (Fig. 34.8) shows indole
Gram stain (Fig. 34.7A), culture (Fig. 34.7B), biochemical
(negative), citrate (positive), urease (positive), TSI
reactions (Fig. 34.8) and AST (Fig. 34.9 and Table 34.3).
shows acid/acid, gas(+) and no H,S.
1. What is the clinical diagnosis and its causative
organism? Antimicrobial Susceptibility Testing
2. What is hypervirulent strains of this organism. Antimicrobial susceptibility test on Muller Hinton
3. How will you treat this condition? agar (Fig. 34.9) with zone interpretation chart
(Table 34.3) shows that the pathogen is resistant to
Explanation
ceftriaxone, gentamicin, ciprofloxacin and sensitive
Clinical Diagnosis
to amikacin, meropenem, or piperacillin-tazobactam
Fever, productive cough with consolidation over left and ceftazidime.
lower lobe (on chest X-ray) are clinically suggestive of The drug of choice would be ceftazidime as it
lobar pneumonia. is the narrowest spectrum among the susceptible
Identification antibiotics.
The causative agent is Klebsiella pneumoniae. Points (For answers to other questions, refer below).
in favor are:
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

Gram-negative bacilli are increasingly associated Contd...

to cause lobar pneumonia, particularly VAP in Pyogenic liver abscess (most common)
the healthcare facility. Most of these agents are Metastasize from liver to distant sites, such as
multidrug resistant (MDR) pathogens found in eye, lung and CNS
> Itcanalso cause primary extrahepatic infections
the hospital environment.
including bacteremia, pneumonia and soft
* Non-fermenters, such as Pseudomonas
tissue infections
aeruginosa, Acinetobacter, Burkholderia Q Identification: hvKp strains are hypermucoid—
cepacia. They are the major cause of ventilator- a viscous string is formed when a loop is used to
associated pneumonia stretch the colony on an agar plate (Fig. 34.7C).
“* Enterobacteriaceae: Various members of
the family Enterobacteriaceae can cause Laboratory Diagnosis
pneumonia, such as Escherichia coli,
Klebsiella pneumoniae, Enterobacter species
Klebsiella belongs to the family Enterobacte-
and Serratia marcescens. riaceae. Similar to E. coli, Klebsiella species are
also lactose fermenters; however, they differ in
Klebsiella pneumoniae Pneumonia being non-motile and capsulated (possess cap-
K. pneumoniae causes various infections.
sular polysaccharide).
** Itis responsible for severe lobar pneumonia, Specimens collected depend upon the site
urinary tract infections, meningitis (neo- of infection, such as sputum, endotracheal
nates), septicemia and pyogenic infections,
aspirate, urine, blood, exudate specimen.
such as abscesses and wound infections K. pneumoniae shows the following properties:
** It frequently colonizes the oropharynx of the ** Gram staining: It is short, plump, straight
hospitalized patients and is a common cause capsulated gram-negative rods (Fig. 34.7A)
of nosocomial infections. Most of the hospital ** Culture: On MacConkey agar, it produces
strains are multidrug resistant large dome-shaped mucoid (due to capsule)
** Pneumonia tends to be destructive with sticky, pink color, lactose fermenting colonies
production of thick, mucoid, brick red (Fig. 34.7B)
sputum. Some time, the sputum has a thin * Identification: Identification of K.
and currant jelly-like appearance.
pneumoniae from colonies is made either
Hypervirulent Strains of K. pneumoniae (hvKp) by automated identification systems, such
It is a strain of K. pneumoniae, which is recently as MALDI-TOF or VITEK; or by conventional
emerged as a pathogen of global concern. biochemical tests as described below
Q It is more virulent than the classical type, = It is catalase positive and oxidase nega-
capable of causing various community-acquired tive
infections, such as:
= ICUT tests: Indole test (negative), citrate
Contd... test (positive), urease test (positive) and

Figs 34.7A to C: (A) Direct smear (sputum) of Klebsiella pneumoniae; showing pus cells with gram-negative
bacilli with
clear halo (capsule) (arrows showing); (B) Klebsiella on MacConkey agar (Mucoid dome-shaped pink-colored
lactose-
fermenting colonies); (C) Hypervirulent Klebsiella showing string test positive.
Source: (A) Department of Microbiology, JIPMER, Puducherry (with permission); (B and C) Department
of Microbiology, Pondicherry
Institute of Medical Sciences, Puducherry (with permission).
 CHAPTER 34 © Bacterial Pheumonia

Indole Citrate Urease

negative positive negative

Fig. 34.8: Biochemical reactions of Klebsiella species. Fig. 34.9: Antimicrobial susceptibility testing on Mueller-
Source: Department of Microbiology, JIPMER, Puducherry Hinton Agar for Klebsiella [observed zone size diameter
(with permission). (mm) and zone interpretation to various antimicrobial
agents tested are given in Table 34.3].
Abbreviations: Cf, ciprofloxacin; Ak, amikacin; G, gentamicin; Ci,
TSI (triple sugar iron agar) test shows acid/ ceftriaxone; Pt, piperacillin/tazobactum; M, meropenem; Ca,
acid, gas present, H,S absent (Fig. 34.8). ceftazidime.
“* AST: It is carried out by disk-diffusion test Source: Department of Microbiology, Pondicherry Institute of
Medical Sciences, Puducherry (with permission).
(on Mueller-Hinton agar, Fig. 34.9) or by
automated MIC based method (by VITEK)
and reported according to CLSI interpretation antimicrobial. The therapy can be tailored
criteria (Table 34.3). based on the susceptibility report
“ Carbapenems, amikacin or B-lactam/f-
Treatment lactamase inhibitor combinations (BL/
Treatment of Klebsiella is global challenge owing BLIs), such as piperacillin-tazobactam or
to marked drug resistance, which is much higher cefoperazone-sulbactam are usually the
than that of E. coli. agents of choice for hospital acquired MDR
~ In healthcare facilities with high prevalence infections. Polymyxins or tigecycline are the
of MDR K. pneumoniae, empirical treatment next line antimicrobials, used for carbapenem
should be started with higher spectrum resistant isolates.

antimicrobial
Table 34.3: Interpretative categories (CLSI) and observed zone size diameter (mm) to various
agents tested for Klebsiella pneumoniae.

Antimicrobial Disk CLSI interpretative criteria for Observed zone | Interpretation

agents strength | Enterobacteriaceae (in mm) size (in mm)
(ng) Fig. 34.9

Ciprofloxacin (Cf) 5 <21 22-25 226 16 Resistant

Amikacin (Ak) 30 <14 15-16 217 24 Sensitive

10 <12 13-14 215 6 Resistant

Gentamicin (G)
30 <21 20-24 225 6 Resistant
Ceftriaxone (Ci)
100/10 <17 18-20 221 22 Sensitive
Piperacillin/
Tazobactam (Pt)
(M) 10 <19 20-22 223 28 Sensitive
Meropenem
30 <17 18-20 221 21 Sensitive
Ceftazidime (Ca)
Abbreviation: CLSI, Clinical and Laboratory Standards Institute.
 —— LL
——————————————

Tuberculosis aI
35

in infant) or consumption of unpasteurized

§ TUBERCULOSIS
(infected) milk] and inoculation.
Tuberculosis is one of the oldest disease of
mankind affecting lungs and other organs. It Clinical Manifestations
is caused by M. tuberculosis complex, which Tuberculosis (TB) is classified as pulmonary and
includes several species, of which the most extrapulmonary forms.
important ones are M. tuberculosis (the most +,
DG Pulmonary tuberculosis (PTB): It accounts
common species) and M. bovis. for 60-90% of all cases of tuberculosis (TB).
It can be further categorized into primary or
Mode of Transmission postprimary (secondary) types (Table 35.1)
“ Air-borne: M. tuberculosis is mainly transmit- Extrapulmonary tuberculosis (EPTB): It
ted by inhalation of aerosols, generated while results from hematogenous dissemination of
coughing, sneezing, or speaking of infected tubercle bacilli to various organs
patients. At least 10* bacilli/mL in sputum is Though EPTB constitute about 10-40% of
required for an effective transmission all cases of TB. In HIV-positive patients, the
*: Other modes of transmission are rare, such frequency is much higher accounting up to
as ingestion [swallowing of sputum (seen two-thirds of all cases of tuberculosis

Problem Solving Exercise

A 29-year-old lady was admitted with complaints of Q Cough with expectoration for 1 month
cough with expectoration, chest pain, evening rise Q Chest pain, evening rise in temperature and loss of
of temperature and loss of weight for more than 1 weight for 1 month
month. Clinical examination revealed crepitations and Q Crepitations and rales with reduced breath sounds
rales with reduced breath sounds over left upper lobe over left upper lobe of lungs
of lungs on auscultation. Chest X-ray showed cavity Q Chest X-ray showed cavity measuring 18 mm x 11
measuring 18 mm X 11 mmin the left upper lobe. Her mm in the left upper lobe.
sputum was collected and subjected to microscopy
Identification
(Fig. 35.1A) and culture (Fig. 35.2A).
1. What is the clinical diagnosis and its causative The causative agent is Mycobacterium tuberculosis.
organism? Points in favor are:
2. What are the newer modalities of laboratory Q The acid-fast stained sputum smear (Fig. 35.1A)
diagnosis? showing pus cells and long slender and beaded
How will you perform drug susceptibility test? acid-fast bacilli. Revised National Tuberculosis
4. What are the infection control measures need to Control Program (RNTCP) grading is found to be
be followed while giving care to this patient? grade 3+.
Culture on Lowenstein-Jensen (LJ) medium
Explanation shows rough, tough and buff colored colonies
Clinical Diagnosis (Fig. 35.2A).
The history is suggestive of pulmonary tuberculosis (For answers to other questions, refer text below).
(PTB). Points in favor are:
 CHAPTER 35 © Tuberculosis

Table 35.1: Comparison of primary and secondary pulmonary tuberculosis.

—_ Primary pulmonary tuberculosis | Postprimary (adult-type)/secondary pulmonary
tuberculosis
Results due to Initial exogenous infection with e Exogenous reinfection
tubercle bacilli e Endogenous—reactivation of the latent primary lesion
Age group affected Children Adults
Parts of the lungs Subpleural lesion affecting, middle Apical and posterior segments of the upper lobes (areas
commonly affected and lower lung lobes of high oxygen tension)
Lesions formed at Fibrotic nodular lesions are formed Hematogenous seedling in the apex of lungs called
the initial sites (Ghon focus) Simon’s focus ‘
Reactivated Simon focus with central caseation
(Assmann focus)
Lymph node Ghon focus with associated hilar Lymph node involvement is unusual
lymphadenopathy is common
(called primary complex)
Clinical features It may be asymptomatic or may Lesions undergoing necrosis and tissue destruction,
present with fever, productive leading to cavity formation
cough (with or without hemoptysis) Symptoms are similar, but more pronounced
and occasionally pleuritic chest
pain, night sweating, weight loss

“ Virtually all organ systems may be affected spit the sputum into the wide mouthed screw
however, the sites commonly involved (in capped container
order of frequency) are lymph node (most * Quality: Sputum should be at least 3-5 mL
common), pleura, genitourinary form, skeletal in quantity; thick and purulent (yellowish
form, CNS, etc. mucus). Salivary specimens that appear
watery should be rejected.
Laboratory Diagnosis The extrapulmonary specimens vary
Laboratory diagnosis of active tuberculosis can depending on the site involved, which can be
be established by various methods described divided into two categories (Table 35.2).
below. The diagnosis of latent tuberculosis is Digestion, Decontamination and
explained later in this chapter.
Concentration
Specimen Collection Sputum and specimens from non-sterile sites
In PTB, two sputum samples are recommended— subjected to smear microscopy and culture
spot sample (collected on the same day under need prior treatment for digestion (to liquefy
supervision) and early morning sample the thick pus cells and homogenization),
(collected on the next day). Alternatively 2 spot decontamination (to inhibit the normal
samples at least one hour apart can be collected. flora) and concentration (to increase the
* Sputum collection booths should be yield). However, this step is not required for
located away from other people, outside in molecular methods and also for processing of
an open well-ventilated space; as air dilutes extrapulmonary specimens collected aseptically
the aerosols generated during coughs from sterile sites. Commonly used methods are:
*% Modified Petroff’s method (4% NaOH):
* Early morning sputum specimen should
1

be collected in empty stomach, after rinsing Sputum is mixed with 4% sodium hydroxide,
the mouth well; so as to remove any food
centrifuged and the sediment is neutralized
remnants, as they interfere with smear
with phosphate buffer saline. This method is
examination
recommended for LJ culture
“ Inhale deeply: Patient should be advised to * NALC(N-acetyl-L-cysteine) + 2% NaOH: This
inhale deeply (2-3 times) and cough out deep is superior to Petroff’s method for isolation.
from the chest during exhalation and then to NALC liquefies the sputum and NaOH kills the
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

normal flora. This method is more compatible Direct Microscopy by Acid-fast Staining
with automated culture systems. ZiehI—-Neelsen (ZN) Technique (Hot Method)
Smears are prepared from thick mucopurulent
Table 35.2: Extrapulmonary specimens.
part of sputum or with the sediment obtained
Sterile site specimens collected aseptically after concentration. Optimum thickness of the
Optimum CSF, pericardial fluid, synovial fluid and smear can be assessed by placing the smear on
specimens _ ascitic fluid printed matter. The print should be just readable
Suboptimal Pleural fluid (20-50 mL is collected and through the smear. Then the smear is stained by
specimens centrifuged) acid-fast stain (for procedure, refer ‘acid-fast
(organism Blood (indicated only for disseminated
staining’ in Chapter 3.5).
loadisless) TB and co-infected with HIV)
“+ Interpretation
Specimens containing normal flora = Negative result: At least 100 oil immersion
Swabs Considered suboptimal specimen. fields should be examined for 10-15
The only recommended swabs are: minutes before giving a negative report
e Laryngeal swabs: Collected early
ws Positive result: M. tuberculosis appears
morning in empty stomach or
e Swab from discharging sinus as long slender, beaded, less uniformly
stained red colored acid-fast bacilli (AFB)
Urine Three early morning specimens
collected (500 mL/specimen,
(Fig. 35.1A).
centrifuged) on different days as TB “+ Presumptive diagnosis: Microscopy provides
bacilli in urine are shed intermittently only presumptive diagnosis. If typical beaded
Stool For disseminated TB in HIV infected appearance is seen, then it should be reported
patients and infants as ‘acid-fast bacilli resembling M. tuberculosis
Other Bronchial secretions (2-5 mL) are seen by smear microscopy by ZN stain’
respiratory Bronchoalveolar lavage (20-50 mL) “+ Advantages: Smear microscopy is rapid, easy
specimens — Transbronchial and other biopsies to perform at peripheral laboratories and is
(collected in sterile normal saline) cheaper
Gastric Recommended for children (tend ** Disadvantages: (i) Smear microscopy is less
lavage to swallow sputum), or ICU patients sensitive than culture, (ii) low sensitivity
(aspiration)
with a detection limit of 10,000 bacilli/mL of
Early morning lavage should be
collected and processed early (<4 hours)
sputum, (iii) it cannot determine the viability
of bacilli
Note: Samples for culture should never be collected in formalin. If
histopathological examination is required, two samples should be = Itis difficult to differentiate M. tuberculosis
collected. from saprophytic mycobacteria present in
tap water or even as commensal in clinical
ames t
samples such as gastric aspirate, and urine
= if . 2 = Acid alcohol (3% hydrochloric acid + 95%
‘ —
ethyl alcohol) can be used to differentiate
i«w M. tuberculosis (acid and alcohol-fast)
~~ ‘ + ha!
~~
»
AY
a
f t from M. smegmatis (only acid-fast, but not
© s Aha x cad alcohol-fast) in urine sample.
BG aS
RNTCP Guidelines for Grading of Sputum
Smear
Figs 35.1A and B: (A) ZN staining of sputum smear Revised National Tuberculosis Control Programme
showing long, slender and beaded red colored acid-fast
(RNTCP) of India has given guidelines for grading of
ZN stained sputum smears (Table 35.3).
bacilli; (B) Auramine phenol staining of sputum smear—
RNTCP grading is useful for:
tubercle bacilli appear bright brilliant green against the
dark background. Q Monitoring the treatment response ofthe patients
Q Assessing the severity of disease
Source: Department of Microbiology, JIPMER, Puducherry (with
permission). Contd...
 CHAPTER 35 © Tuberculosis

Contd... Table 35.3: RNTCP guidelines for grading of ZN

stained sputum smears.
Q Assessing the infectiousness ofthe patient: Higher
the grade more is the infectiousness. Smear No. of AFB seen OIF to be
negative patients (<10,000 bacilli/mL of sputum) screened
are less infectious. No AFB in 100 OIF 100 0 Negative
However, grading of the sputum smear depends upon 1-9/ 100 OIF 100 Scanty* Positive
the quality of sputum collected.
10-99/100 OIF 100 1+ Positive
Note: In 2020, RNTCP has been renamed as National Tubercu-
losis Elimination Programme (NTEP). 1-10/ OIF 50 2+ Positive
>10/ OIF 20 3+ Positive
Abbreviations: AFB, acid-fast bacilli; OIF, oil immersion fields; ZN,
Kinyoun’s Cold Acid-fast Staining Ziehl-Neelsen.
It differs from ZN stain in that—(i) heating is not *Record the actual no. of bacilli seen in 100 fields, e.g., “Scanty 8”

required, (ii) phenol concentration in carbol Conventional Solid Media (Lowenstein-Jensen

fuchsin is increased, and (iii) duration of carbol Medium)
fuchsin staining is more.
Lowenstein-Jensen (LJ) medium has been the
Fluorescence Staining most widely used and recommended by RNTCP.
“ It is composed of coagulated hen’s eggs,
It is a fluorescent
staining technique, uses mineral salt solution, asparagine and
auramine-phenol solution (for 7-10 min) as malachite green (as a selective agent)
primary stain, 0.5% acid alcohol (for 2 min,
* Inoculated media are incubated for a
twice) as decolorizer and 0.1% potassium prolonged duration of 6-8 weeks. This is
permanganate (for 30 sec) as counter stain. Then because of the slow-growing nature of
the slide is examined under fluorescent LED tubercle bacilli (long generation time of 10-15
(light-emitting diode) microscope. hours)
“ The bacilli appear brilliant yellow against the * Colonies: M. tuberculosis produces typical
dark background (Fig. 35.1B) rough, tough and buff-colored colonies (Fig.
* Smears are screened by using 20X or 25X 35.2A). Incontrast, M. bovis produces smooth,
objective, hence can be screened faster (2 moist and white colored colonies that break
min for 100 fields) up easily when touched.
* It is more sensitive than ZN staining and has Conventional liquid media are Kirchner’s
been the recommended screening method medium and Middlebrook 7H9 medium. They
by RNTCP are not routinely used.
“ However, artifacts may confound with the
interpretation. Hence the reading should be Automated Liquid Culture
taken by an expert. Automated culture systems monitor the growth
continuously and offer a faster turnaround time
Culture Methods
compared to conventional culture.
Culture is traditionally considered as the gold “ Positive growth (99%) gets detected within
standard method of diagnosis of TB. It offers 3-4 weeks. However, the negative result is
several advantages: reported after 6 weeks of incubation
* Ttis more sensitive than microscopy with the “* They use liquid broth such as Middlebrook
detection limit of 10-100 viable bacilli 7H9 medium supplemented with enriched
* Indicates viability: TB bacilli growing on growth media and antibiotic mixture (to
culture indicates that they are viable inhibit other organisms).
“: Drug susceptibility testing can be performed.
Various automated systems available are:
RNTCP recommended culture media include
* BACTEC MGIT (Mycobacteria growth
both conventional solid media (Lowenstein-
indicator tube): This is the automated system
Jensen medium) and automated liquid culture,
endorsed by WHO and RNTCP (Figs 35.2B
such as Mycobacteria Growth Indicator Tube
and C)
(MGIT).
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

bottle are first subjected to acid-fast stain. If

found AFB positive, then further tests are done
for species identification.
* MPT 64 antigen detection by rapid
immunochromatographic test: MPT64, a
28 Da antigen is specific for M. tuberculosis
complex (M. tuberculosis, M.bovis and
M. africanum) and negative for NTM
(nontuberculous mycobacteria)
“ Automated identification system such as
MALDI-TOF
“ Biochemical tests such as niacin test and
Rabbit pathogenicity tests were in use in the
past; now obsolete.

Molecular Methods
Molecular methods are extremely useful as:
“+ They take less time than culture
“+ They are more sensitive than culture. This is
very much useful for extrapulmonary samples
that are usually paucibacillary
“+ They can also detect the genes coding for drug
resistance
“+ Used for epidemiological typing of strains.
Figs 35.2A to D: Culture media/culture systems for M.
There are several molecular methods
tuberculosis: (A) LowensteinJJensen medium (arrow available as described below.
showing rough, tough and buff-colored colonies);
(B) BACTEC MGIT; (C) MGIT liquid culture medium; Polymerase Chain Reaction
(D) GeneXpert system with cartridge. Nested polymerase chain reaction (PCR)
Source: Department of Microbiology, JIPMER, Puducherry (with
permission).
targeting /S6110 gene was the most common
molecular test used earlier. Other genes which
= Uses: (i) It detects growth of mycobacte- were targeted by using PCR—MPT64 gene, 65
ria, and (ii) also performs the drug sus- KDa and 38 KDa genes.
ceptibility testing against first-line and
Automated Real Time PCR
second-line antitubercular drugs
m Principle: It uses an oxygen sensitive With the advent of automated real time PCR
fluorescent compound, dissolved in systems, the diagnosis of TB has been completely
the broth. Initially, the large amount of revolutionized. In addition, these systems can
dissolved oxygen in the medium que- be used for diagnosis of other diseases such as
nches emissions from the fluorescent COVID-19. Methods available are:
compound. Later, actively respiring ** Cartridge-based nucleic acid amplification
microorganisms consume the oxygen; test (CBNAAT): GeneXpert
the quenching effect is lost which allows “+ Chip-based real-time PCR: Truenat.
the fluorescence to be detected. GeneXpert (CBNAAT)
** Other automated systems include BacT/ GeneXpert (Cepheid’s) is the CBNAAT system
ALERT systems. endorsed by WHO and is used in India under
RNTCP (Fig. 35.2D).
Culture Identification
** Rapid: It has the lowest turnaround time (2
The colonies grown on LJ media and the broth hours) among all the diagnostic methods
from a positively flagged automated culture currently available for TB
 CHAPTER 35 © Tuberculosis

* Principle: It is based on real-time PCR RNTCP Diagnostic Algorithm

technique; simultaneously detects: (i) MTB RNTCP recommended diagnostic algorithm
complex DNA, and (ii) rifampicin resistance should be followed for the diagnosis and further
(mutations of the rpoB gene). It uses five management of tuberculosis.
probes targeting various sequences of rpoB * For pulmonary TB in adult (Flowchart 35.1):
gene First sputum smear examination is performed
“+ EPTB: WHO recommends GenexXpert as the = Ifsmear positive—diagnosis is confirmed
initial test for diagnosis of EPTB; especially m If smear negative, but chest X-ray is
for CSF, lymph nodes and other tissue suggestive or in presence of high clinical
specimens suspicion—CBNAAT is performed to
* Diagnostic utility: The detection limit confirm the diagnosis.
of GeneXpert is about 131 bacilli/mL of * For pediatric pulmonary TB: CBNAAT is
specimen directly performed
* Disadvantages: (i) Very expensive, (ii) cannot “> For EPTB: EPTB specimens are paucibacil-
further speciate MTB complex. lary, therefore, CBNAAT is directly performed.
Chip-based NAAT (Truenat) If not available, liquid culture (MGIT) is per-
Truenat is a chip-based real-time PCR system, formed.
validated recently by Indian Council of Medical
Diagnosis of Latent Tuberculosis
Research (ICMR), in 2017; also endorsed by
Latent tuberculosis is diagnosed by
WHO in 2019. It is used in India under RNTCP.
demonstration of delayed or type IV hyper-
It has been developed by the Indian firm Molbio
Diagnostics. sensitivity reaction against the tubercle bacilli
* Advantages: It is an automated battery antigens. Two methods are available, (1)
operated device; can be used at level of tuberculin skin test (also called Mantoux test),
(2) IFN-y release assay.
primary health center where GeneXpert
cannot be used as it needs uninterrupted DST (Drug Susceptibility Testing)
power supply and air conditioning. The
turnaround time is around one hour Several methods of DST (drug susceptibility
“» Disadvantages: (i) Very expensive, (ii) cannot testing) are available which can be grouped into:
further speciate MTB complex, and (iii) it tests
Phenotypic Methods
only limited samples (1-4) at a time.
* MGIT (used for 1st and 2nd line drugs):
Line Probe Assay (LPA) Resistance is determined by growth of TB
Line probe assay involves probe-based detection bacilli in drug containing tube as compared
of amplified DNA in the specimen. to the control tube (drug free) within 4-21
days of incubation
“ Uses: LPA in TB diagnostics has the following
uses: * Proportion method (used for Ist and 2nd line
= Identification of MTB complex drugs): An isolate is considered resistant to
= Detection of resistance to first-line a given drug when growth of 1% or more is
antitubercular drugs (ATDs) observed in the drug containing LJ medium
compared to the control LJ medium without
= Speciation of MTB complex and NTM
drug after 42 days of incubation.
= Detection of resistance to second line
ATDs.
Genotypic Methods
* Limitation: LPA can be performed only on
“ GeneXpert: It is used for detection of
positive cultures or smear positive clinical
resistance to rifampicin, targeting five
specimens. It is not recommended for smear
different sequences of rpoB gene. Turnaround
negative specimens as the sensitivity is low
time is <2 hours
“ LPA is useful particularly in isoniazid mono-
“+ Line probe assay (LPA): It detects resistance
resistant cases of TB, which are not diagnosed anti-
by GeneXpert. to both first-line and second-line
 SECTION 2 ©@ Systemic Microbiology (Infectious Diseases)

Flowchart 35.1: Diagnostic algorithm for pulmonary tuberculosis.

PLHA Presumptive TB patient

Pediatric
EPTB

ona
Pp mptive Z
oR
Smear positive Smear negative Smear and CXR
regardless of but CXR either negative or not
CXR report suggestive of TB available with high
clinical suspicion

Directly

MTB MTB not detected Clinically

detected or CBNAAT result suspicion
not available high

Consider Clinically
Rif Rif Rif alternate diagnosed
sensitive indeterminate resistant diagnosis TB

Repeat CBNAAT Refer to management

on 2nd sample of Rif resistance

Microbiologically If indeterminate on 2nd sample,

| confirmed TB collect fresh sample for liquid culture/LPA |
|
| All presumptive TB cases should be offered HIV counseling and
|
|| testing: however diagnostic work up for TB must not be delayed
L

Source: Adapted from RNTCP guideline 2016.

Note: Presumptive pulmonary TB refers to a person with any of the following—(i) cough >2 weeks, (ii) fever >2 weeks, (iii) significant
weight loss, (iv) hemoptysis, (v) any abnormalities in the chest X-ray.
Abbreviations: Rif, Rifampicin; CBNAAT, cartridge-based nucleic acid amplification test; PLHA, people living with HIV/AIDS; DRTB, drug
resistant tuberculosis; CXR, chest X-ray; LPA, line probe assay.

tubercular drugs; with a turnaround time of The treatment regimens are of various types,
2-3 days. depending upon the resistance pattern.
Standard regimen for drug sensitive-TB: It is a
U-DST
six-month course; comprises of two phases.
Universal-Drug Susceptibility Testing (U-DST) refers
to testing all TB patients for resistance to at least
1. Intensive phase, with four drugs [HRZE-
rifampicin (by performing CBNAAT). U-DST program isoniazid (H), rifampicin (R), pyrazinamide
has been rolled out across India since January 2018. (Z), ethambutol (E)| for two months; followed
by
2. Continuation phase, with three drugs (HRE)
Treatment
for four months.
Multidrug therapy is recommended for FDC: All drugs must be given in fixed dose
tuberculosis, for rapid and effective killing combination (FDC) tablets as per appropriate
of tubercle bacilli. The treatment should be weight bands. The FDC tablets should be taken
planned only after the result of DST is available. orally, once in a day.
 CHAPTER 35 © Tuberculosis

Drug Resistance Prevention

*« MDR-TB: Multidrug-resistant tuberculosis “
7
Infection control: Airborne precautions
(MDR-TB) is defined as resistance to both need to be followed while giving care to a
isoniazid and rifampicin with or without TB patient; various components include
resistance to other first-line drugs use of N95 respirator, negative pressure
* XDR-TB: Extensively drug-resistant room and others (Refer Chapter 11, for
tuberculosis (XDR-TB) is defined as MDR-TB detail)
case which is also resistant to: BCG Vaccine: Bacillus Calmette-
= Fluoroquinolones (levofloxacin/ Guerin (BCG) vaccine is available. It is
moxifloxacin/gatifloxacin), and a live-attenuated vaccine, made up of
m Atleast one second-line injectable agents Mycobacterium bovis strain. It is given at birth,
(amikacin/capreomycin/kanamycin). administered as single dose, intradermally.
OO

Pseudomonas and
Acinetobacter Infections

Pseudomonas aeruginosa
BINTRODUCTION
Burkholderia cepacia
Non-fermenting gram-negative bacilli (NF- Acinetobacter baumannii
GNB) do not ferment any sugars, but they utilize Stenotrophomonas maltophilia
the sugars oxidatively. Elizabethkingia meningosepticum.
“+ Most of the NF-GNB exist as environmental However, there are some non-fermenters
commensals in hospitals that inhabit in moist which are principally community associated
environments, detergents and IV fluids. They pathogens. Classical example is Burkholderia
are resistant to multiple antibiotics. However, pseudomallei, which causes melioidosis,
they cause various infections in hospitalized characterized by infections of various systems,
patients, of which respiratory infections are of which the notable are respiratory, skin and
noteworthy. Examples include: soft tissue, and bloodstream infections.

& PSEUDOMONAS INFECTIONS

| Problem Solving
Solving Exercise
Exercise _—|
A 32-year-old man with 30% burn injury developed burn Q Burn wound infection
wound infection after 5 days of hospitalization. Exudate Q Hospitalized patient
specimen was sent for culture (Fig. 36.1B), biochemical Q MacConkey agar showing blue green pigmented
reaction (Fig. 36.4) and AST (Fig. 36.5 and Table 36.1). nonlactose fermenting (NLF) colonies with
Q What is the causative organism? metallic sheen (Fig. 36.1B)
Q List the clinical conditions caused by this organ- Biochemical reactions are typical of Pseudomonas
ism. (Fig. 36.4): Indole test (negative), Citrate test
OQ What are the various modalities of laboratory (positive), Urease (negative), TSI test showing
diagnosis? alkaline slant/alkaline butt (no change) with no
Q How will you treat this condition? gas and no HS, and Oxidase positive.
Q How is this disease prevented in hospitals?
Antimicrobial Susceptibility Test
Expianation Antimicrobial susceptibility test on Muller—-Hinton
Clinical Diagnosis agar (Fig. 36.5 and Table 36.1) reveals the organism
It is a case of burn wound surface infection. is sensitive to ceftazidime, amikacin, ciprofloxacin,
Nosocomial pathogens such as Pseudomonas and piperacillin/tazobactam and meropenem. The drug
Staphylococcus aureus, etc. frequently colonize and of choice would be one of the narrow spectrum
cause infection on burn wound surface. antibiotic such as ceftazidime, or ciprofloxacin.

Identification (For answers to other questions, refer below).

The causative organism is Pseudomonas aeruginosa.

Points in favor are:

Clinical Manifestations
being lungs, skin and soft tissues. Most of the
Pseudomonas aeruginosa is notorious to cause infections are encountered in hospitalized
infections at almost all sites, most common patients who get colonized with the organisms
 CHAPTER 36 © Pseudomonas and Acinetobacter Infections

either from heavily contaminated hospital

environment or from the hospital staff (through
contaminated hands). Colonized patients
develop disease in the presence of underlying
risk factors such as burn wounds, patients with
immunosuppression and post-surgeries. The
common infections are:
“+ Ventilator-associated pneumonia
¢,“e Chronic respiratory tract infections in patients

with cystic fibrosis (mucoid strains)

“+ Ear infections: Swimmer’s ear (in children) and
malignant otitis externa (in elderly diabetics)
“* Eye infections such as corneal ulcers (in
contact lens wearers) and endophthalmitis
+,
+9 Shanghai fever (typhoid like illness)
¢,>% Skin and soft tissue infections
¢,
“9 Wound infection in burns patients
+,~~ Ecthyma gangrenosum
7
“% Cellulitis (characterized by blue green pus)
Figs 36.1A and B: Pseudomonas aeruginosa on: (A) Blood
G
+>
Urinary tract infection in catheterized patients.
agar showing hemolytic colonies; (B) MacConkey agar
showing NLF colonies with metallic sheen.
Source: Department of Microbiology, Pondicherry Institute of
Specimen: Various specimens such as pus, Medical sciences, Puducherry (with permission).

wound swab, urine, sputum, blood or cerebro-

spinal fluid (CSF) are collected, depending up
on the site infected.
Direct smear: Gram staining of the specimen
shows plenty of pus cells and slender gram-
negative bacilli, occasionally capsulated.
Calftimro

Pseudomonas is nonfastidious, can grow in

ordinary media but it is obligate aerobe.
“ Nutrient agar: It produces opaque, irregular
colonies with a metallic sheen with greenish
pigmentation (commonly) (Fig. 36.2)
= Diffusible pigments are produced by
most strains which produce blue green
Fig. 36.2: Large, opaque, irregular colonies of Pseudo-
(pyocyanin) or yellow green (pyoverdin)
monas aeruginosa with a metallic sheen and green color
pigmentation pigmentation on nutrient agar.
= Most colonies have a characteristic sweet Source: Department of Microbiology, Pondicherry Institute of
ether or alcohol-like fruity odor Medical sciences, Puducherry (with permission).

= Morphotypes: Colonies show various

“» Selective media such as cetrimide agar can
morphological appearances such as large
be used to isolate the organism from mixed
spreading type, mucoid type, small round
growth in purulent specimens
type, minute type, etc.
agar: It produces hemolytic colonies * Culture smear and motility testing: Gram
“* Blood
staining of the culture smear reveals gram-
on blood agar (Fig. 36.1A)
negative bacilli (Fig. 36.3). They are motile
“ MacConkey agar: Produce pale NLF colonies
with single polar flagellum.
with metallic sheen (Fig. 36.1B)
270 SECTION 2 © Systemic Microbiology (Infectious Diseases)

Po ee Sn)
/ ee FF}
| TSI
1||
PRES
[
} ~~tras
hye
‘ Indole Citrate Urease K/A
|
{ ate
z o pl ead | negative positive negative gas-,H,S—
: J 4 “£ "
} Pat > ‘ mae |
g ACA: ||
/ Oxidase
y
}
if t~ ; positive
1 ; }

|
| # + \ t.# L |i
| é : x |
| : |
y 2 |
|
\*
|
|
£ ra
|
|
| i salt
$7 | 1
| 4 “i
i
. & ae ;
|

| 4
|
| - , bs
WI
i — E a Se enn Fig. 36.4: Biochemical reactions of Pseudomonas.
Fig. 36.3: Gram-stained culture smear of Pseudomonas Source: Department of Microbiology, JIPMER, Puducherry
(with permission).
showing gram-negative bacilli.
Source: Department of Microbiology, JIPMER, Puducherry
(with permission).

Biochemical Properties
Pseudomonas shows the following biochemical
properties (Fig. 36.4):
“* Oxidase and catalase—positive
** Nonfermenter—OF test (oxidative fermenta-
tive test) shows oxidative pattern
2,sO
Indole test—negative
2,“Se
Citrate test—variable
©,
Urease—negative
>
+ *'
TSI test shows alkaline slant/alkaline butt (no
change) with no gas and no HS.
Identification of Pseudomonas from colonies
can also be made by automated identification
Fig. 36.5: Antimicrobial susceptibility testing on Mueller-
systems such as MALDI-TOF or VITEK. Hinton Agar (Pseudomonas) (Refer Table 36.1 for CLS! zone
interpretation).
Antimicrobial Susceptibility Testing (AST) Abbreviations: Cf, ciprofloxacin; Ca, ceftazidime; Ak, amikacin;
Pt, piperacillin-tazobactam; M, meropenem; CLSI, Clinical and
AST is essential to administer proper antibiotics.
Laboratory Standards Institute.
It is done on Mueller-Hinton agar by disk Source: Department of Microbiology, Pondicherry Institute of
diffusion method (Fig. 36.5 and Table 36.1). Medical sciences, Puducherry (with permission)

Table 36.1: Interpretative categories (CLSI) and observed zone size diameter (mm) to various antimicrobial
agents tested for Pseudomonas aeruginosa.
Antimicrobial agents Disk Observed zone Interpreta-
strength | Pseudomonas aeruginosa (in mm) size (in mm) tion

Ceftazidime (Ca) 30
|intermediateant
(49) [Resist [sensitive|8-36
<14 15-17 218 26 Sensitive
Amikacin (Ak) 30 <14 15-16 217 22 Sensitive
Ciprofloxacin (Cf) 5 <18 19-24 225 30 Sensitive
Piperacillin/Tazobactam (Pt) 100/10 <14 15-20 221 26 Sensitive
Meropenem (M) 10 <15 16-18 >=19 21 Sensitive
Abbreviation: CLSI, Clinical and Laboratory Standards Institute,
 CHAPTER 36 & Pseudomonas and Acinetobacter Infections

Treatment
Pseudomonas species are widely distributed and
inherently resistant to most of the antibiotics.
Only limited antimicrobial agents have anti-
pseudomonal action such as:
** Penicillins: Piperacillin, mezlocillin and
ticarcillin
“* Cephalosporins: Ceftazidime, cefoperazone
and cefepime
“+ Carbapenems: Imipenem and meropenem
* Monobactam: Aztreonam
** Aminoglycoside: Tobramycin, gentamicin and
amikacin Fig. 36.6: Lactose non-fermenting colonies (with faint
* Quinolones: Ciprofloxacin and levofloxacin pink tint) of Acinetobacter on MacConkey agar.
** Polymyxins: Polymyxin B and colistin. Source: Department of Microbiology, JIPMER, Puducherry
(with permission).

Drug Resistance
Pseudomonas possesses a number of drug- o,1 Central line associated bloodstream infection
resistant plasmids which confer multiple 2,“ Post-neurosurgical meningitis
drug resistance. Many strains are producers +,+e Catheter- associated UTI
of B-lactamases such as extended spectrum 2,¢
+ Wound and soft tissue infections
B-lactamases, carbapenemases and AmpC Infections in burn patients.
+
+,*

B-lactamases. Many strains are resistant to

aminoglycosides and quinolones. Laboratory Diagnosis
It is an obligate aerobe, grows well on ordinary
Prevention medium. Specimens can be inoculated onto
Infection control measures such as contact blood agar (non-hemolytic colonies) and
isolation precautions and improved hand MacConkey agar (lactose non-fermenting
hygiene are essential to prevent nosocomial colonies with faint pink tint) (Fig. 36.6).
infections (Refer Chapter 11). Important identification features ies’
baumannii include:
BfACINETOBACTER INFECTIONS * Gram staining: They are gram-negative
Acinetobacter baumannii is another non- coccobacilli
fermenter like Pseudomonas which has attracted
+,bgNon-motile
attention recent days as nosocomial pathogen.
o,XaOxidase negative and catalase positive
Hospital environment is heavily contaminated
+,“ Non-fermenter of sugars
with these organisms. They are also commensals
+,
ICUT tests (Fig. 36.4): Indole test (negative),
in skin, oral cavity and intestine of hospitalized citrate test (positive), urease test (negative)
patients. They are resistant to most of the and TSI (triple sugar iron agar) test shows
available antibiotics; pose a huge challenge for alkaline slant/alkaline butt with no gas and
treatment. no H,S
“ Automation: Species identification can also
Clinical Manifestations be made by automated identification systems
hospital such as MALDI-TOF or VITEK.
A. baumannii causes widespread
infections such as: Treatment for Acinetobacter infections is
similar to that of Pseudomonas infections.
* Ventilator associated pneumonia
——

Viral Infections of Respiratory Tract:

Influenza, COVID-19 and Others
AAO ATT

* Other respiratory viruses include:

B INTRODUCTION
= Enteroviruses: Coxsackie A virus (causes
Viruses account for vast majority of respiratory vesicular pharyngitis or herpangina),
illness. Common respiratory viruses include: echoviruses, enterovirus 71
* Myxoviruses: Influenza viruses, parainfluenza m Adenoviruses: Cause upper respiratory
viruses, respiratory syncytial virus (RSV) and tract infection in children and pneumo-
mumps nia
¢ Coronaviruses including the virus causing = Rhinoviruses: Cause common cold syn-
COVID-19 drome
“+ Epstein-Barr virus: Causes infectious mono- = Herpes simplex virus
nucleosis mw HIV (as acute retroviral syndrome).

BINFLUENZA
| Problem Solving
SolvingExercise1
Exercise 1 _—|
In month of December, a 53-year-old man from for real-time polymerase chain reaction (PCR) and
Puducherry presented with fever with chills, myalgia, result is shown here (Fig. 37.1).
dry cough and sore throat and running nose. A clinical 1. Mention the ideal method of specimen collection
diagnosis of influenza-like illness (ILI) was made. The and transport.
treating physician revealed that always there has been 2. What is the reason behind repeated outbreaks of
an increase in number of such cases during winter this clinical condition?
season. A nasopharyngeal swab was collected, sent 3. What are the precautions to be taken to prevent
the spread of the disease?
4. What are the different modalities of laboratory
Sample positive diagnosis?
for Influenza
A/H,N, Explanation
Clinical Diagnosis
The symptoms of cough, sore throat, myalgia, etc., in
a winter season is suggestive of influenza-like illness.
Etiological Diagnosis
Real-time PCR (Fig. 37.1) revealed that there is
emission of fluorescence during the cycles for
Influenza A (matrix gene), H1N1 (HA gene) and the
Cycles internal control RNP (ribonucleoprotein) gene. But
there is no emission of fluorescence for H3N2 (HA
Fig. 37.1: Real-time RT-PCR showing the result (amplifi- gene) and Influenza B (HA gene). Therefore, it is
cation curves) of specimen tested for Influenza types A/ identified as Influenza A/H1N1.
H1N1, A/H3N2 and B. (For answers to other questions, refer below).
 CHAPTER 37 © Viral Infections of Respiratory Tract

Introduction and sneezing. This mode can infect only those

Influenza viruses are the members of people who are within 1-meter distance, (ii)
Orthomyxoviridae family. They are one of the via direct contact (e.g., touching the droplets)
major causes of morbidity and mortality and or indirect contact with surfaces or fomites
have been responsible for several epidemics infected with respiratory droplets and then
and pandemics of respiratory diseases in the last touching nose, eyes or mouth
two centuries. It has four types—A, B, C and D.
Uncomplicated Influenza (Flu Syndrome)
Influenza type A can be further subtyped based
on hemagglutinin (HA) and neuraminidase Incubation period is about 18-72 hours. Majority
of the individuals are either asymptomatic or de-
(NA) peplomer antigens present in its envelope.
velop minor upper respiratory symptoms such
Antigenic Variation as chills, headache, and dry cough, followed by
high-grade fever, myalgia and anorexia. It is a
Antigenic variation is an unique property of
self-limiting condition, indistinguishable from
influenza viruses, which is responsible for
the infections caused by other upper respiratory
causing epidemics and pandemics. Antigenic
tract pathogens.
variation is due to the result of antigenic changes
occurring in HA and NA antigens. It is of two Complications
types. Occasionally, patient may develop complications.
“+ Pneumonia: It occurs either as a result of
Antigenic Variations
secondary bacterial infection (most common)
1. Antigenic Drift or as a primary influenza pneumonia
It is a minor change occurring due to point mutations * Other respiratory tract complications
in the HA/NA gene, resulting in alteration of amino include worsening of chronic obstructive
acid sequence of the antigenic sites on HA/NA, such
that virus can partially escape recognition by the
pulmonary disease, exacerbation of chronic
host's immune system. The new variant must sustain bronchitis, etc.
two or more mutations to become epidemiologically
significant. Influenza A (H1N1) pdmo9
Q Seen in both influenza virus type—A and B It has caused the most recent pandemic of
Q Results in outbreaks and minor periodic epidemics
influenza in 2009, affecting several countries
Q Antigenic drift occurs more frequently, every 2-3
years. including India. However, thereafter it is
circulating in the community and has been
2. Antigenic Shift
causing sporadic infection and periodic
It is an abrupt, major drastic, discontinuous variation
in the sequence of a viral surface protein (HA/NA), outbreaks of seasonal flu.
that occurs due to genetic reassortment between
genomes of two or more influenza viruses infecting Seasonal Flu
the same host cells; resulting in a new virus strain, un- Influenza viruses cause outbreaks of seasonal
related antigenically to the predecessor strains. Thus, flu worldwide during winter season almost every
antibodies developed against previous strain (due to
infection or vaccination) become ineffective. year, however, they differ widely in severity and
Q Occurs only in influenza A virus the extent of spread. The common serotypes
Q Results in pandemics and major epidemics, e.g., currently circulating in India to cause seasonal
H1N1 pandemics of 2009 influenza include influenza A/H1N1, influenza
Q Antigenic shift occurs less frequently every 10-20 A/H3Nz2 and influenza B.
years.
Laboratory Diagnosis
Clinical Manifestations Specimen Collection
Transmission * Ideal specimens are nasopharyngeal swab or
Influenza is transmitted by (i) inhalation of lavage fluid, nasal aspirate or to a less extent
respiratory droplets generated by coughing throat swab
 SECTION 2 @ Systemic Microbiology (Infectious Diseases)

Yes aie
Sample positive 2.87 Sample positive RNP
2.8
for Influenza RNP = 9.54 for Influenza ia
25
InfA 22 HN,
2.2
68 1.9 HN, 2 19
5 16 o™ 8 16
81.3 $13
°
2 4.0
z 1.0
0.7 0.7
Fig. 37.2: Viral transport medium and swab. 0.4 04
Source: Department of Microbiology, JIPMER, Puducherry
(with permission).
'B Bid! (1500) 25 Si Stas
34 Cycles 347 Cycles

** Swabs with a synthetic tip (e.g., polyester

+, +
2.8t Sample positive 28t Sample
2.5 4for Influenza 2 | negative
or Dacron swabs) are best for specimen
collection (Fig. 37.2). Cotton or alginate swabs
are unsatisfactory
** Transport: Swabs are immediately put inside Fluorescence
the viral transport media, kept at 4°C during
transport up to 4 days, thereafter at -70°C.
5 05 15 25 35 45
Isolation of Virus Cycles BD) Cycles

Embryonated eggs (amniotic cavity) and Figs 37.3A to D: Real-time RT-PCR showing the result
primary monkey kidney cell lines have been (amplification curves) of specimen(s) tested positive
used (in past); viral growth was detected by for Influenza type: (A) A/H,N,;ta
(B) A/H,N.; (C) Type B;
(D) Negative (Refer Table 37.1).
hemadsorption or hemagglutination test.
Source: Department of Microbiology, JIPMER, Puducherry (with
permission).
Direct Immunofluorescence Test
Viral antigens coated onto epithelial cells can Antibody Detection (Serology)
be directly detected in nasal aspirates by using Various assays are available such as ELISA,
fluorescent tagged antibodies. This is rapid, but neutralization test, and previously used HAI
less sensitive than viral isolation. (hemagglutination inhibition) test, to detect
subtype specific serum antibodies by using
Molecular Methods
specific influenza antigens. It is mainly useful
Molecular methods have revolutionized the for sero-epidemiology purpose, not for clinical
diagnosis of influenza. diagnosis.
* RT-PCR (reverse transcriptase polymerase
chain reaction): It is highly sensitive, specific
and rapid (turnaround time of<1 day). It can Table 37.1: Real-time RT-PCR for seasonal influenza
types.
also detect the specific type and subtype of
influenza virus Simultaneously Specimens positive Influenza
* Real-time RT-PCR: It is currently the gold detects five with influenza type negative
standard method for influenza diagnosis. genes
A/ Al Type
It has higher sensitivity and specificity than H1N1 | H3N2 |B
RT-PCR with turnaround time of 2-3 hours. Influenza A (matrix 4 + - =
It simultaneously detects the three common gene)
seasonal flu strains (A/HIN1, A/H3N2 and H1N1 (HA gene) + = & =
type B). The result is expressed as the emission H3N2 (HA gene) = + = cs
of fluorescence during the cycles as described Influenza B (HA = = ss a
in Figure 37.3 and Table 37.1 gene)
* BioFire FilmArray Respiratory Panel (RP)
RNP (ribonucleo- + + + +
tests simultaneously 20 respiratory pathogens, protein)*
including Influenza A, Influenza A/H1, Influen-
*RNP (ribonucleoprotein) is used as internal control in real-time
za A/H3, Influenza A/H1-2009 and Influenza B. RT-PCR; if it is not detected, then the test is considered invalid.
 CHAPTER 37 © Viral Infections of Respiratory Tract

Treatment * Serotypes: Vaccines contain the com-

Neuraminidase inhibitors (such as zanamivir mon influenza types/subtypes currently
or oseltamivir) are effective for influenza. circulating—A/H1N1, A/H3N2 and influ-
** Itis the drug of choice for A/H1N1 2009 flu, enza B
A/H5N1 avian flu and influenza-B “* Indication: Given annually (once ina year) to
** Oseltamivir (75 mg tablets): Given twice a day people at higher risk of infection.
for 5 days (for treatment) or once daily for 7
days (for chemoprophylaxis). Prevention
Infection control measures such as droplet
Vaccine precautions and contact precautions need to
Both injectable (inactivated) and nasal spray be followed while giving care to patients with
(live attenuated) vaccines are available. influenza (Refer Chapter 11, for detail).

@ COVID-19
| Problem Solving
Solving Exercise2
Exercise2 |
Laboratory Diagnosis of COVID-19 > The internal control RNP (ribonucleoprotein)
A 72-year-old patient with complaints of dry cough, gene.
sore throat and fever visited a hospital. He was kept Q The threshold (Ct value) of the run (the point at
in an isolation room. His throat swab was sent for which the fluorescence starts emitting) is found
COVID-19 testing by real-time RT-PCR. The result is to be satisfactory; 25th cycle (for E gene) and 30th
shown Fig. 37.4. cycle (for RdRP gene).
1. Interpret the test result. Therefore, the serotype is identified as SARS-CoV-2.
2. Discuss the method of specimen collection. Refer text below, for answer to other questions.
3. Discuss the laboratory diagnosis of this disease. | 3.1
got

Explanation 2.8
25 RNP |
Clinical diagnosis | 2.2 E gene
Dry cough, sore throat and fever suggests that it is a 8 1.9
case of Influenza like illness (ILI). i ae
8 4.3
Interpretation of test 3 1.0
Throat swab was subjected to COVID-19 testing by real- 0.7
time RT-PCR. Fig. 37.4 shows the following findings. | 0.4
Q There is emission offluorescence during the cycles 0.1 |
for: |
> Envelope (E) gene (for screening, specific for |
Cycles |
beta coronavirus) Sree ee ee

> RdRp gene (for confirmation, specific to SARS- Fig. 37.4: Real-time RT-PCR showing the result (amplifi-
CoV-2 ) and cation curves) of specimen tested for SARS-CoV-2.

Clinical Manifestations * Common features: Fever, cough with

expectoration, fatigue, shortness of breath,
COVID-19 (Coronavirus disease-2019) is
myalgia, rhinorrhea, sore throat, diarrhea.
caused by severe acute respiratory syndrome
Loss of smell or taste sensation may
coronavirus 2 (SARS-CoV-2). It is primarily
occasionally occur preceding the onset of
transmitted via respiratory droplets and contact
respiratory symptoms
routes (same, as discussed for influenza).
“+ Atypical symptoms: Particularly seen in older
The incubation period for COVID-19 is on an
average of 5-6 days, but can be as long as 14 people and immune-suppressed patients—
days. COVID-19 patients may present with such as fatigue, reduced alertness, reduced
following signs and symptoms: mobility, diarrhea, loss of appetite, delirium,
276 SECTION 2 © Systemic Microbiology (Infectious Diseases)

and absence of fever. Children might not Gene targets for screening are genus specific; i.e.,
develop fever or cough as frequently as adults specific for Sarbecovirus (Betacoronavirus):
* Clinical severity: Based on the clinical Q Spike protein (S)
severity, the disease may be classified into Q Envelope protein (E)
Q Membrane protein (M)
the three clinical stages
Q Nucleocapsid protein (N).
1. Mild disease (ILI or influenza-like illness) Gene targets for confirmation are species
2. Moderate disease: Pneumonia with no specific; i.e., specific for SARS-CoV-2
signs of severe disease OQ RNA-dependent RNA polymerase (RdRp)
3. Severe disease: Called as severe acute Q Open reading frames (ORF1a/b)
respiratory illness (SARI), characterized Q N2 nucleocapsid.
by severe pneumonia, acute respiratory
“+ Principle: Most commercial kits available are
distress, sepsis or septic shock.
based on qualitative real-time PCR
Laboratory Diagnosis = The target gene/s in the specimen is
amplified in the thermocycler
Specimen Collection and Transport = When the amplicon binds with the probe,
* Preferred specimens: Throat (i.e., a fluorescence is generated. The point at
oropharyngeal) and nasal swabs are the which the fluorescence starts is the cycle
preferred specimens. Dacron or polyester threshold (Ct) of the run
flocked swabs are used, dipped in viral = Asample is considered positive when both
transport media (VTM) after collection screening, as well as confirmatory genes,
* Alternative specimens include: Naso- are detected with a Ct value <40 cycles
pharyngeal swab, bronchoalveolar lavage (Fig. 37.4)
(BAL) or endotracheal aspirate (in ventilated m Detectable: NAAT becomes positive as
patients) early as day 1 of onset of symptom (usually
** PPE: Appropriate PPE should be used for after 5 days of infection) and starts to
specimen collection such as gloves, gown, decline by 3rd week and subsequently
N95 respirator and face shield becomes undetectable (Fig. 37.5).
“+ Specimen transport and packing: Samples
collected should be properly labelled, packed Automated Real-time RT-PCR
in three layers (triple packaging method) and Several automated real-time PCR are
transported to the laboratory maintaining an commercially available such as—Truenat
adequate cold chain. and CBNAAT (cartridge-based nucleic acid
amplification test, e.g., GeneXpert). Both these
Nucleic Acid Amplification Testing (NAAT)
Real-time RT-PCR | <+—Window—>
1
+ Decline -»<- Convale- —»>
1
period t ; } scence i
Patient begins
Real-time reverse transcriptase PCR is the gold
I t
t ' 1 t
' ; to recover '
standard test for diagnosis of COVID-19. '
(
1
!

t
'
* The average time taken is around 4-5 hours '
'
'
1
1 1
from receipt of sample to generation of the ae -
t ss oe ot
' IgG remains in ;
'
result ‘oma ic ' detectable ' blood and provides
\
stage |' '
1 long-term immunity
* The advantage of this platform lies in its
'
! '
! '
accuracy of detection as well as the ability to
'
'
1 Onset of : '
1symptoms! !

run up to 90 samples in a single run. Therefore, 1

production
!
1

if available, this platform should be used as a & \ begins 4 isappeatsn

~ 1

frontline test for the diagnosis of SARS-CoV-2 0 5 7 14 21 28

Days since infection
* Gene targets: Most of the commercial kits
target two genes, performed in a single | — SARS-CoV-2 RNA — IgM antibody — |gG antibody
and antigen
reaction—one for screening and other for
confirmatory Fig. 37.5: Course of the diagnostic markers in COVID-19.
 CHAPTER 37 © Viral Infections of Respiratory Tract

systems are already in use for the diagnosis of

tuberculosis. ;
** Advantages of these systems include:
= These platforms have widespread avail-
ability even at district and primary health
center level as these systems are alreadyin
use for the diagnosis of tuberculosis and
other infectious diseases Fig. 37.6: Antigen detection test for COVID.
Source: Department of Microbiology, JIPMER, Puducherry
m They have a quick turnaround time (30-60
(with permission).
minutes)
= Fully-automated, involves minimal han-
dling; therefore poses minimum biosafety and last for several months. Therefore, IgG
hazard. Safety is further augmented by the test should not be used for clinical diagnosis.
closed nature of these platforms. ELISA, chemiluminescence and ICT formats
* Gene targets used are: are available. They may be useful for sero-
a CBNAAT: Two targets are used; E gene for surveillance purpose and to conduct survey
screening and N2 gene for confirmation among high-risk or vulnerable populations.
= Truenat: Two targets are used; E gene for
screening and RdRp gene for confirmation. Nonspecific Tests
** However, disadvantages of these systems *+, Prognostic markers: There are several
include: Only 1-4 samples can be tested in one prognostic markers which can be used in the
run, therefore, suitable only for laboratories setting of ARDS, include:
with less sample load (24-48 samples/day). = Elevated IL-6 level: Indicates cytokine
storm
Antigen Detection =Elevated D-dimer: Indicates the presence
A rapid chromatographic immunoassay is of high level of fibrin degradation
commercially available (SD Biosensor) for products, thus suggesting an underlying
qualitative detection of specific antigens coagulopathy
(nucleocapsid protein) to SARS-CoV-2. m Elevated serum ferritin: Indicates
* Nasopharyngeal swab, after collection inflammation
should be immersed and squeezed in the = Severe lymphopenia
viral extraction buffer, provided with the kit. m Elevated C-reactive protein: Marker of
This buffer inactivates the virus, releasing the acute inflammation.
antigen * CT scan of lungs shows ground glass
* It is a point-of-care test, conducted at the appearance (Fig. 37.7) and/or consolidation.
bedside within one hour, as the antigen in
Treatment
the extracted buffer is stable only for an hour
“ Performance: It is highly specific (99-100%), Currently, there is no definitive therapy available
with moderate sensitivity (50-84%). Therefore, for COVID-19; however many drugs are under
symptomatic but negative patients should be research such as remdesivir, favipiravir,
essentially referred for a real-time RT-PCR test lopinavir/ritonavir, hydroxychloroquine, plasma
(Fig. 37.6). therapy and anti-cytokines such as tocilizumab
There are various other antigen detection (IL-6 receptor blocker).
kits under validation, which may be marketed
Prevention of COVID-19
in near future.
Infection prevention and control (IPC) is the
Antibody Detection most effective method currently available for the
IgG antibodies start appearing after two prevention of COVID-19. The following are the
weeks of the onset of infection, after recovery key IPC measures need to be strictlyfollowed.
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

Solvingg Exercis
blemm Solvin
[ProProble 3
Exerceise3_ _|

Prevention of COVID-19 Explanation

A 72-year-old patient (without wearing any mask) Influenza like illness (fever, dry cough,malaise and
presented to casualty with complaints of fever, dry throat pain) with throat swab confirmed for COVID-19,
cough, malaise and throat pain. The security guard suggests that it is case of COVID-19
(without mask) guided him to go to the casualty. Q Infection control breaches: Patient was not
The resident doctor (without mask) took history, wearing mask. Security staff and resident doctor
examined the patient. His throat swab was sent for were not wearing mask or any other PPE. Patient
COVID-19 testing which came positive. Subsequently was not kept in isolation room. No hand hygiene
the security staff and the resident doctor were also was performed
turned positive for COVID-19. Q PPE: 3-ply mask, pair of gloves, gown and eye
1. Identify the infection control breaches protection (goggle or face shield) need to be worn
2. What are the personal protective equipment (PPE) while giving care to this patient
need to be worn while giving care to this patient? Q Donning (wearing) sequence: Gown first > Mask
3. What is the correct sequence of donning and or respirator + Goggles or face shield — Gloves
doffing of PPE Q Doffing (removing) sequence: Gloves first — Face
4. Discuss the infection control measures to prevent shield or goggles Gown —> Mask or respirator.
the transmission. Refer text below, for answer to other questions.

** Hand hygiene needs to be performed when

opportunity arises (as per WHO's ‘my five
moments of hand hygiene; refer Chapter 10)
** Hand hygiene must be performed by the cor-
rect technique, and for appropriate duration
(20-30 sec for hand rub).

Personal Protective Equipment

The following are the current recommendations:
* HCWs giving care to the COVID-19 suspects:
—_ i EE Should wear a medical (3-ply) mask, a pair
of gloves, gown, and a face shield. Medical
Fig. 37.7: A case of COVID-19 pneumonia with CT scan
lungs showing ground-glass opacity seen in both lungs, mask should be replaced by a N95 respirator
predominantly subpleural in distribution. Ground-glass if AGPs are carried out (Fig. 37.8)
opacity is a feature of interstitial pneumonia. * HCWs working in non-COVID areas: Should
Source: Dr SunithaV, Department of Radiology, JIPMER, Puducherry wear a medical (3-ply) mask. This referred to
(with permission).
as ‘targeted continuous medical mask use’
= Should wear masks during all routine
IPC Measures at Healthcare Facility activities throughout the entire shift
IPC measures ofdroplet and contact precautions = Masks are only changed if they become
need to be followed by the healthcare workers soiled, wet or damaged, or at the end of
while handling COVID-19 cases, except for shift
aerosol-generating procedures (AGPs) when = Front part of the mask should never be
airborne precautions need to be followed (Refer touched
Chapter 11). = Mask should never be hanged around the
neck.
Hand Hygiene
* Anyone entering into a healthcare facility:
As contact mode appears to be an important Must wear a face mask (e.g., cloth/fabric
mode of transmission, absolute hand hygiene mask), regardless of the activities he is
is probably the most effective method for the involved in. This is referred to as ‘universal
prevention of COVID-19. masking’ in healthcare facilities.
 CHAPTER 37 © Viral Infections of Respiratory Tract

“COVID-19 waste’, and disinfecting outer bag

Face shield
or goggles with hypochlorite before handing over
* Laundry: All linens used for COVID-19
Medical (3-ply) mask
(Change with N95 patients should be washed at 60-90°C with
respirator if aerosol risk) laundry detergent followed by soaking in 0.1%
sodium hypochlorite for approximately 30
minutes and dried.

One pair of IPC Measures for General Public

clean gloves
Handwash
Frequent handwash is necessary after contact
with other individuals, high-touch area, public
places, after receipt of any items, or after blowing
nose, coughing, or sneezing. Touching of eyes,
nose, and mouth with unwashed hands must be
avoided.
Fig. 37.8: Personal protective equipment recommended
for healthcare workers when giving care to COVID-19 Social Distancing
patients. Ideally, people should stay at home as much as
possible. Ifnot possible, 1 meter (2 arms) distance
Environmental Cleaning should be strictly maintained from other people
SARS-CoV-2 may survive on surface and floor for at all times. As droplets can travel a maximum of
a variable period, ranging from few hours to as 1-meter distance, therefore, the social distance of
long as 9 days. This may be a potential source of 1 meter would prevent the droplet transmission.
transmission by indirect contact. Therefore, the
following measures need to be followed. Environmental Cleaning
“+ Floor and surfaces: Should be cleaned with Frequently touched surfaces should be
a detergent, followed by disinfected with disinfected daily. This includes tables, doorknobs,
sodium hypochlorite (0.5%) light switches, countertops, handles, desks,
“+ Cleaning of equipment or patient care items phones, keyboards, toilets, faucets, and sinks. If
such as stethoscope, BP apparatus, etc. should surfaces are dirty, then it should be cleaned with
be done by using alcohol (70%) detergent or soap and water prior to disinfection.
* High touch surfaces such as lift button, rail
Cloth Mask (Non-medical Masks)
of the staircase, patient trolley, bed rails, bed
frames, bedside tables, door handles, etc. Everyone should wear a cloth face mask when
should be frequently disinfected with alcohol they have to go out in public, e.g., for the grocery
* Terminal disinfection in patient care room store. It primarily aims at source control, i.e., pre-
after discharge/transfer of patients. venting transmission from the wearers to others).
“+ Cloth masks are made from a variety of fabrics,
Other IPC Measures such as polypropylene. It should comprise of
* Respiratory hygiene and cough etiquette at least two layers:
such as wearing a medical mask by all = Internal layers are made up of water-
individuals who are symptomatic, hand wash absorbing (hydrophilic) fabrics to readily
immediately after sneezing or coughing, etc. absorb droplets
(Refer Chapter 11) = Anexternal synthetic material (hydropho-
* Biomedical waste management should be bic), which does not easily absorb liquid.
carried out as per the 2016 guideline (Refer “+ They should not be used in hospital settings,
Chapter 13). However, additional precautions as there is no filter used
are taken such as use of double bag, use of “+ They can be washed and reused
dedicated trolley and collection bins, label as “+ Should not be shared between individuals.
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

B INFECTIOUS MONONUCLEOSIS
Exerci4se4_|
Solving Exercise
[problem Solving
An 18-year-old young boy presented with headache, Q Young patient with pharyngitis, rashes, hepato-
fever, malaise, pharyngitis and rashes. On examination, splenomegaly and enlarged cervical lymph nodes.
he was found to have hepatosplenomegaly and Q Atypical lymphocytes in peripheral smear.
enlarged cervical lymph nodes. Peripheral blood
Identification
examination shows presence of atypical lymphocytes.
Infectious mononucleosis (IM) is caused by Epstein-
1. What is the clinical diagnosis and the most
Barr virus. However, IM-like syndrome may be seen
common causative organism?
in various infections such as CMV following blood
2. What is the next investigation you would like to
transfusion, toxplasmosis, HIV infection, etc.
advise?
Paul-Bunnell test is the next investigation to be
3. List the clinical conditions caused bythis organism.
done as it can differentiate IM (Paul-Bunnell test
4. What are the different modalities of laboratory
positive) from mononucleosis like syndrome (PB test
diagnosis?
negative).
Explanation For answers to other questions, refer below.
Clinical Diagnosis
It is a case of infectious mononucleosis (IM). Points
in favor are:

Clinical Manifestations of EBV antibodies in patient’s serum. Agglutination

titer of >256 is considered as significant. It is a
Infectious Mononucleosis
heterophile agglutination test; false positive test
It is also called as kissing disease (transmitted results (normal individuals, following serum
through salivary contact) or glandular disease, therapy) can be confirmed by differential
affecting young adults. It is characterized by: absorption test (e.g., monospot test).
Re* Headache, fever, malaise and pharyngitis
+,DO
Cervical lymphadenopathy Other Tests
+,“e
Hepatosplenomegaly
Other tests for diagnosis of EBV include:
Rashes following ampicillin therapy
** Specific antibody detection such as:
se Atypical lymphocytosis
7+,
(CD8 T cells)
= Antibody to viral capsid antigen (VCA)
*,“
Autoantibodies reactive to sheep RBC antigens
= Antibodies to early antigen
(detected by Paul-Bunnell test).
ms Antibodies to EBNA (Epstein-Barr nuclear
EBVAssociated Malignancies antigen).
* EBV antigen detection by immunofluores-
Epstein-Barr virus is associated with several
cence
malignancies such as Burkitt’s lymphoma,
** EBV DNA (by PCR) detection
nasopharyngeal carcinoma, Hodgkin's and non-
** EBER RNA (EBV encoded small RNAs, by
Hodgkin’s lymphoma.
reverse transcriptase PCR)
Laboratory Diagnosis ** Real-time PCR.

Heterophile Agglutination Test Treatment

Paul-Bunnell test is a tube agglutination test Acyclovir is not useful for IM cases; symptomatic
that uses sheep RBCs to detect heterophile treatment is needed.
 Parasitic and Fungal Infections of

SRST EE
Respiratory Tract 38
B INTRODUCTION = Pneumocystis jirovecii pneumonia.
* Fungi causing systemic mycoses: They
Parasitic infections of respiratory tract include: involve multiple organs. They are saprophytic
* Paragonimus westermani: This human fungi present in the environment. Human
parasite is a primary pathogen of lungs infection occurs by inhalation of spores
“+ Parasites that pass through lungs during their leading to pulmonary infection. From lungs,
life cycle, e.g., intestinal nematodes, such as they disseminate to cause various systemic
Ascaris, hookworm, and Strongyloides manifestations (discussed in detail in Chapter
“~ Parasites causing hypersensitivity in 21). There are four agents of systemic mycoses,
lungs: Filarial nematodes causing tropical all are dimorphic fungi:
pulmonary eosinophilia (TPE) 1. Blastomyces dermatitidis
“ Parasites that infect elsewhere, rarely 2. Histoplasma capsulatum
infect lungs: E. histolytica, Toxoplasma, 3. Paracoccidioides brasiliensis
Balantidium coli, Cryptosporidium parvum 4. Coccidioides immitis.
and Echinococcus granulosus. * Yeast: Cryptococcus neoformans: It also
Fungal infections of respiratory tract include: causes meningitis, (Refer Chapter 41).
** Opportunistic fungal agents: They are major Note: Isolation of Candida species in sputum
fungal agents that cause respiratory infections: culture is a common finding; but it represents
= Zygomycoses colonization. It is almost never indicative of
= Aspergillosis underlying pulmonary candidiasis and therefore
= Penicillosis does not warrant antifungal treatment.

B PARAGONIMIASIS
Exerciese11
Solvingg Exercis
[problem Solvin
Paragonimiasis Q Sputum microscopy reveals large operculated oval
eggs, golden-brown in color, measuring 80-120
A 12-year-old girl from Manipur presented with um x 45-65 um in size (Fig. 38.1).
productive cough with blood-tinged, rusty sputum For further details, refer Table 38.1.
with offensive fishy odor. Sputum specimen was sent BE ae ie
for microscopic examination (Fig. 38.1).
1. Identify the causative agent based on sputum
microscopy.
2. Which is the infective stage for man?
3. Mention two complications caused by the adult
worm.
Explanation
The causative agent is Paragonimus westermani.
Points in favor are:
Q From Manipur—area endemic for paragonimiasis Fig. 38.1: Paragonimus westermani (egg).
Q Productive cough with blood-tinged, rusty sputum Source: DPDx Image Library, Centers for Disease Control and
Prevention (CDC), Atlanta (with permission).
with offensive fishy odor
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

Table 38.1: Features of paragonimiasis. Risk Factors

Properties Paragonimus westermani Agents of mucormycosis require iron as growth
Definitive host Man factor. Hence, conditions with increased iron
1st—Snail
load are at higher risk of developing invasive
Intermediate
hosts 2nd—Cray fish/carb mucormycosis, such as:
Infective form Metacercaria larva “ Diabetic ketoacidosis (DKA) is the most
Transmission Ingestion of cray fish/carb containing important risk factor. Acidosis causes release
metacercaria of iron from the sequestered proteins in serum
Habitat Lungs * End-stage renal disease
Manifestations Endemic hemoptysis: “+ Patients taking iron therapy or deferoxamine
e Granuloma formation in lungs and (iron chelator)
extrapulmonary sites, such as liver, ** Defects in phagocytic functions.
spleen, brain
Blood tinged rusty sputum with Clinical Manifestations
offensive fishy odour
Agents of mucormycosis are angioinvasive in
Diagnostic Eggs (operculated) in sputum (Fig. 38.1)
form e Large oval eggs nature.
e Operculated * Rhinocerebral mucormycosis: Occurs
e Measures 80-120 um long and commonly in patients with DKA. It starts as
45-65 um in breadth eye and facial pain, may progress to cause
e Golden brown in color
orbital cellulitis, proptosis and vision loss
Treatment Praziquantel ** Pulmonary mucormycosis
** Cutaneous mucormycosis
BZYGOMYCOSIS
* Gastrointestinal mucormycosis
Zygomycosis represents group of life-threatening “+ Disseminated mucormycosis.
infections caused by aseptate fungi called as
zygomycetes. The important causative agents of Laboratory Diagnosis
zygomycosis are Rhizopus, Mucor and Absidia. ** Histopathological staining of tissue biopsies
Zygomycosis caused due to these agents is shows broad aseptate hyaline hyphae with
known as Mucormycosis. wide angle branching (Fig. 38.2)

[Problem SolvingExercise2
Solving Exercise 2
Mucormycosis bridge collapse with black eschars on nasal mucosa
A 40-year-old female with uncontrolled diabetes and bilateral loss of vision give clue to the diagnosis
mellitus was referred to a teaching hospital with severe of “Rhinocerebral mucormycosis”.
eye pain and facial rash for 4 days. Facial rash progressed Identification
to extensive ulceration of the midface and bilateral
loss of vision. She had nasal bridge collapse, with black Histopathological examination also showed broad
eschars on the nasal mucosa and markedly elevated aseptate hyaline hyphae with wide angle branching,
fasting blood sugar. She had surgical debridement and gives presumptive identification of one of the agents
tissue was sent for histopathological examination (Fig. causing mucormycosis (Fig. 38.2).
38.2) and fungal culture (Fig. 38.3A and C). Culture of the tissue (nasal mucosa) on Sab-
1. Whatis the clinical diagnosis and the likely etiologi- ouraud's dextrose agar (SDA) grew cottony woolly
cal agent based on the histopathological finding? brown-black colonies with tube filling growth (Fig.
2. Howcan you confirm the diagnosis in the laboratory? 38.3A) and lactophenol cotton blue (LPCB) tease
3. What are the other clinical manifestations caused mount (Fig. 38.3C) of the colony revealed broad asep-
by this organism? tate hyaline hyphae, from which sporangiophore arise
4. Mention the treatment option for this clinical which ends at sporangium-containing numerous
condition. sporangiospores, which are characteristic identifica-
tion features of fungi Rhizopus.
Explanation For answers to the other questions, refer below.
Clinical Diagnosis
Female patient with uncontrolled diabetes, with acute
presentation of progressive facial ulceration, nasal
 CHAPTER 38 © Parasitic and Fungal Infections of Respiratory Tract 283

rise to salt and pepper appearance seen in

Rhizopus (Fig. 38.3A)
= White cottony colony seen in Mucor (Fig.
38.3B).
* Microscopic appearance: LPCB mount of
the colonies reveals broad aseptate hyaline
hyphae, from which sporangiophore arise
which ends at sporangium-containing nu-
merous sporangiospores (Figs 38.3C and D)
“+ Rhizoid: Some species beara unique root like
growth arising from hyphae called rhizoid,
which provides initial clue for identification
i es 5 = ¥ “ of the fungus. Species can be differentiated
Fig. 38.2: Zygomycosis—histopathology oftissue section depending on the position of the rhizoids
shows aseptate broad hyphae. with respect to sporangiophore (Figs 38.4A
Source: Public Health Image Library, Centers for Disease Control to C)
dP ion (CDC), Atlanta; Dr Libero Ajello,ID ith ' Aint :
eee EG), Alanis OL bo Nell saat = Rhizopus bears nodal rhizoids (Fig. 38.3C,
38.4A)
“+ Culture on SDA at 25°C reveals characteristic = Lichtheimia bears internodal rhizoids
cottony woolly colonies with tube filling growth (Fig. 38.4B)
= White cottony colonies initially, later turn = Mucor: Rhizoids are typically absent (Fig.
to brown-black due to sporulation giving 38.3D and 38.4C).

ery
fi“ °*
es Ro, Sie Sporangium
sy ea

ey

A a
ia 3

eg D|
™ Sh

colonies with black spores (salt and pepper

Figs 38.3A to D: (A) Rhizopus colony on SDA shows white cottony woolly shows
cottony woolly colonies; (C) LPCB mount of colonies of Rhizopus
appearance); (B) Mucor on SDA—white
sporangium (absence of rhizoid).
sporangium with rhizoid present; (D) LPCB mount of colonies of Mucor shows
of Medical Sciences, Puducherry; (D) Public Health Image Library/Dr
Source: (A to C) Department of Microbiology, Pondicherry Institute
nters for Disease Control and Prevention (CDC), Atlanta (with permission).
Lucille K Georg, ID#:3960/Ce
Sporangium
-2., __— Sporangiospore
Columella

Sporangiophore

Aseptate ea?
i
= =
hyphae

Nodal lent
Internodal— SS cellel
Sa
rhizoids __ saan z
'B) rhizoid
A)
; (C) Mucor.
Figs 38.4A to C: Microscopic schematic diagram: (A) Rhizopus; (B) Lichtheimia
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

Treatment localized skin lesions in immunocompetent

Amphotericin B remains the drug of choice patients, which can be removed surgically. Azoles
for all forms of mucormycosis, except the mild are usually ineffective, except posaconazole.

HBASPERGILLOSIS
[Problem Solving
Solving Exercise3_
Exercise 3
Aspergillosis Explanation
A 46-year-old lady complaints of hemoptysis for 2-3 Clinical Diagnosis
episodes in 2 days and cough for 1 month. History of hemoptysis, chronic cough, past history
She gave past history of pulmonary tuberculosis of pulmonary tuberculosis and chest X-ray showing
10 years back. On chest examination, bilateral fungal ball in previous cavitary lesion suggests fungal
breath sounds were reduced. Chest X-ray revealed invasion into previously formed cavitary lesions in the
fungal ball in previous cavitary lesion in right upper lung. These clues suggest fungal ball due to Aspergillus
lobe of lung. Sputum and lung biopsy were sent for infection in lung (Aspergilloma).
fungal culture and identification (Figs 38.5, 38.7A
and 38.8A). Identification
1. What is the clinical diagnosis and the likely Histopathological staining reveals narrow septate
etiological agent based on the chest X-ray finding? hyphae (Fig. 38.5). Soutum culture on SDA showed
2. How can you confirm the diagnosis in the smoky green, velvety to powdery colonies (Fig. 38.7A),
laboratory? and LPCB mount of the colonies showed conical-
3. What are the other clinical manifestations caused shaped vesicle, uniseriate phialides, and conidia arise
by this organism? from upper third of vesicle; hence, final identification
4. Mention the treatment option for this clinical of the causative agent is Aspergillus fumigatus (Fig.
condition. 38.8A) and for the labeled diagram of the agent (see
5. Draw a neat labeled diagram of microscopic Fig. 38.6A).
appearance in LPCB mount? For answers to the other questions, refer below.

Introduction Allergic bronchopulmonary aspergillosis

Aspergillosis refers to the invasive and allergic (ABPA)
d iseases caused by a hyaline mold named
+.“
Severe bronchial asthma
A spergillus. Important species are Aspergillus Extrinsic allergic alveolitis
fumigatus, Aspergillus flavus and Aspergillus
+,
o CeOAspergilloma (fungal ball)
n iger. Transmission of infection occurs by
<+ >
Acute angioinvasive pulmonary aspergil-
inhalation of airborne conidia. losis
+ ?
Chronic cavitary pulmonary aspergillosis.
R isk Factors
Other Types of Aspergillosis
Risk factors for invasive aspergillosis are: *
Invasive sinusitis
Glucocorticoid use
= Invasive sinusitis (acute and chronic from)
Profound neutropenia
Neutrophil dysfunction = Chronic granulomatous sinusitis
Underlying pneumonia or chronic obstruc- = Allergic fungal sinusitis
tive pulmonary disease, tuberculosis or sar- Cardiac aspergillosis
coidosis Cerebral aspergillosis
Antitumor necrosis factor therapy. Ocular aspergillosis—keratitis and endoph-
thalmitis
+
Clinical Manifestations
DOU
Ear infection—otitis externa
ot
« Cutaneous aspergillosis
Pulmonary Aspergillosis +
BOG
Nail bed infection—onychomycosis.
It is the most common form; appears in various Clinical manifestations also depend on the
fo rms, such as: species involved:
 CHAPTER 38 © Parasitic and Fungal Infections of Respiratory Tract

* A. fumigatus causes commonly—acute Table 38.2: Identification features of Aspergillus species.

pulmonary and allergic aspergillosis Aspergillus | Macroscopic | Microscopic
“+ A. flavus causes more commonly sinus, skin appearance | appearance (LPCB
and ocular infections of colony mount)
“+ A. niger can cause invasive infection and A.fumigatus Colonies: Vesicle is conical-shaped
causes Otitis externa. (Figs 38.6A Smoky green, Phialides are arranged
and 38.7A velvety to in single row
Laboratory Diagnosis and 38.8A) powdery, Conidia arise from
reverse is upper third of vesicle
Specimens, such as sputum and tissue biopsies white Conidia are hyaline
may be collected.
A. flavus Colonies: Vesicle is globular-
(Figs 38.6B Yellow green, shaped
Direct Examination and 38.7B velvety, Phialides in one or two
Ten percent KOH mount or histopathological and 38.8B) reverse is rows
staining of specimens reveal characteristic white Conidia arise from
upper two-third to
narrow septate hyaline hyphae with acute angle entire vesicle
branching (Fig. 38.5). Conidia are hyaline
A. niger Colonies: Vesicle is globular
Culture (Figs 38.6C Black, shaped
Specimens are inoculated onto SDA and and 38.7C cottony type, Phialides in two rows
Conidia arise from
incubated at 25°C. Species identification is done and 38.8C) reverse is
white entire vesicle
based on macroscopic and microscopic (LPCB Conidia are black
mount) appearance of the colonies (Table 38.2).
* Colonies consist of hyaline septate hyphae
“+ Conidia arise from the vesicles either on their
from which conidiophores arise which end at
vesicles. Vesicles are either tubular or globular entire surface or only on the upper half (Figs
38.6A to C, 38.8A to C, Table 38.2).
in shape
* From the vesicle, finger-like projections Antigen Detection
of conidia-producing cells arise called as
phialides or sterigmata. Phialides are arranged “» §-d-Glucan antigen assay: It is a marker
of invasive fungal infections, raised in most
either in one or two rows, the first row is called
as metulae invasive fungal infection including invasive
aspergillosis
* Galactomannan antigen: This is an
Aspergillus specific antigen, can be detected
by ELISA in patient’s sera or urine. It is useful
for establishing early diagnosis.

Antibody Detection
* Detection of serum antibodies is very
useful for chronic invasive aspergillosis and
aspergilloma, where the culture is usually
negative. Titer falls rapidly following clinical
improvement
“+ Inallergic syndromes such as ABPA and severe
asthma, specific serum IgE levels are elevated.
lung section
Fig. 38.5: Hematoxylin-eosin stained (H&E) Treatment
invasive asper-
shows septate narrow hyphae—confirms
gillosis. * For invasive aspergillosis, voriconazole is the
Forces Institute of
Source: Public Health Image Library/Armed drug of choice
Prevention (CDC),
Pathology, Centers for Disease Control and
“ For ABPA, itraconazole is the drug of choice
Atlanta; Dr Hardin, ID #15630 (with permission).
286 | SECTION 2 © Systemic Microbiology (Infectious Diseases)

Conidia
Phialides

Phialides Vesicie

Vesicle

Conidiophore Conidiophore
Septate hypha Septate
hypha

1B) a
Figs 38.6A to C: Conidiation of various Aspergillus species (schematic diagram): (A) A. fumigatus; (B) A. flavus; (C) A. niger.

“+ For chronic pulmonary aspergillosis, itracona-

zole or voriconazole is the drug of choice
“+ For prophylaxis, posaconazole is indicated.

BPENICILLIOSIS
Penicilliosis denotes the group of infections
caused by pathogenic Penicillium species.

Clinical Disease
Penicillium species are most often environmental
contaminants. Occasionally, they cause human
infections.
Penicillium marneffei (a dimorphic fungus):
Figs 38.7A to C: Aspergillus (colonies on Sabouraud’s
Produces skin lesions
dextrose agar): (A) Aspergillus fumigatus; (B) Aspergillus
flavus; (C) Aspergillus niger. ** Mycotoxicoses
: is caused by Penicillium
Source: Department of Microbiology, Pondicherry Institute of cyclopium, Penicillium verrucosum and
Medical Sciences, Puducherry (with permission). Penicillium puberulum

Figs 38.8A to C: Aspergillus microscopic picture (LPCB mount): (A) Aspergillus fumigatus; (B) Aspergillus flavus;
(C) Aspergillus niger.
Source: Department of Microbiology, Pondicherry Institute of Medical Sciences, Puducherry (with permission).
 CHAPTER 38 © Parasitic and Fungal Infections of Respiratory Tract

3 £8— Conidia

Brush border
appearance
Sterigmata
(phialides)

Metulae

Conidiophore

Septate

a
hyphae
Al =
Figs 38.9A to C: Penicillium species: (A) Colonies on SDA; (B) Microscopic picture (LPCB mount);
(C) Schematic microscopic picture.
Health Image Library/Lucille
Source: (A) Department of Microbiology, Pondicherry Institute of Medical Sciences, Puducherry; (B) Public
Georg, ID#:8398/Centers for Disease Control and Prevention (CDC), Atlanta (with permission).

“ Endophthalmitis and endocarditis

“ Otomycosis, keratitis and onychomycosis
* Allergic disease—asthma and allergic
pneumonitis.

Laboratory Diagnosis
* Penicillium species occur as molds on SDA
at 25°C (except for P. marneffei which is a
dimorphic fungus)
* Colonies are rapid growing, flat with velvety
to powdery texture and greenish in color
(Fig. 38.9A)
“ Microscopic appearance: LPCB mount of the
a ee
Cysts of Pneumocystis jirovecii in an AIDS
colonies reveal hyaline thin septate hyphae, Fig. 38.10:
patient (methenamine silver stain).
vesicles are absent, and conidiophore directly
Source: Public Health Image Library, Centers for Disease Control
divides into elongated metulae, from which and Prevention (CDO), Atlanta; Dr Edwin P Ewing, Jr., ID #960 (with
flask-shaped phialides originate which bear permission).

chain of conidia. Such an arrangementis called

as brush border appearance (Figs 38.9B and G). Laboratory Diagnosis
* Histopathological examination of lung tissue
§ PNEUMOCYSTIS PNEUMONIA or fluids or open lung biopsy reveals cysts
pneumonia (PcP) has been “ Gomori’s methenamine silver (GMS) staining
Pneumocystis
demonstrates the cysts of P_jirovecii (Fig. 38. 10).
increasingly reported after the discovery of
The cysts resemble black-colored crushed ping-
human immunodeficiency virus (HIV) or AIDS.
pong balls, against the green background
Causative agent is Pneumocystis jirovecit.
“ Pneumocystis is not cultivable and there is no
Pathogenesis serological test available
e “* Polymerase chain reaction assays have been
“ Pneumocystis exists in cyst and trophozoit
developed for detection of P. jirovecii specific
forms
are inhaled, carried to the lungs, genes.
* Cysts
transformed into trophozoite which induces Treatment
an inflammatory response that leads to for
Cotrimoxazole is the drug of choice
recruitment of plasma cells resulting in
Pneumocystis pneumonia.
formation of frothy exudate filling the alveoli.
 nl

Bacterial Meningitis 39

(~50%). Other agents include meningococcus

B ACUTE BACTERIAL MENINGITIS
(~25%), Streptococcus agalactiae (~15%),
Acute bacterial meningitis (also called as Listeria (~10%) and Haemophilus influenzae
pyogenic meningitis), is an acute purulent (<10%)
infection within the subarachnoid space. It is * Neonates: The common agents of neonatal
characterized by elevated polymorphonuclear meningitis include Streptococcus agalactiae,
cells in CSF. gram-negative bacilli, such as Escherichia coli
The agents implicated in pyogenic meningitis and Klebsiella, and Listeria monocytogenes
may vary according to the age. * Elderly (>60 years): Common agents
“ Overall: Streptococcus pneumoniae is the are Streptococcus agalactiae and Listeria
most common cause of pyogenic meningitis monocytogenes.

Problem Solving Exercise 1

Meningococcal meningitis 2. Describe the laboratory diagnosis in detail.
A 7-year-old girl was admitted to the hospital with 3. What is the preferred treatment of choice in this
complaints of high-grade fever, headache, vomiting, case?
altered mental status, seizure and neck rigidity. CSF Explanation
sample was collected by lumbar puncture in a sterile
Clinical Diagnosis
container and sent to the laboratory for Gram staining
(Fig. 39.1). Biochemical analysis of CSF revealed CSF History of high-grade fever, headache, vomiting,
pressure (200 mm of water), protein (260 mg/dL) altered mental status, seizure and neck rigidity is
and glucose (20 mg/dL). Cytological analysis of CSF suggestive of meningitis.
revealed the total leukocyte count (2000 per mm?) CSF analysis is characteristic of pyogenic (acute
which is predominantly neutrophilic. bacterial) meningitis.
1. What is the probable clinical diagnosis and the OQ Biochemical analysis: Elevated CSF pressure
etiological agent? (>180 mm of water), markedly elevated protein
(>250 mg/dL)and decreased glucose (<40 mg/dL)
Q Cytological analysis: Increased total leukocyte
count which is predominantly neutrophilic.
Etiological Diagnosis
Gram staining of CSF (Fig. 39.1) reveals capsulated
gram-negative diplococci, with adjacent sides flat-
tened. This is suggestive of presumptive identification
as meningococcal meningitis (Neisseria meningitidis).
Treatment
Third-generation cephalosporins, such as ceftriaxone
or cefotaxime is the drug of choice for meningococcal
Fig. 39.1: Meningococci in CSF smear (gram-negative meningitis; given for 7 days. Penicillin can also be
diplococci, lens-shaped) (arrows showing). given; however, reduced meningococcal sensitivity
Source: Centers for Disease Control and Prevention (CDO), Atlanta to penicillin has been reported from few countries.
(with permission). For answers to the other questions, refer text.
 CHAPTER 39 © Bacterial Meningitis

Problem Solving Exercise2 _

Pneumococcal meningitis
A 24-year-female was brought to the emergency
room by her parents due to an acute onset of fever,
neck rigidity and altered sensorium for the past 2
days. Physical examination showed that when her
neck was passively flexed, her legs also flexed. Direct
examination of the CSF is depicted in Figure 39.2.
1. Identify the clinical diagnosis of this condition and
the most likely etiologic agent?
2. How will you confirm the etiological diagnosis in
the laboratory?
3. What is the preferred treatment of choice in this
case?
Fig. 39.2: Pneumococci in gram-stained smear of
Explanation sputum [lanceolate-shaped gram-positive cocci in pair
Clinical Diagnosis surrounded by clear halo (capsule)].
History of fever, neck rigidity and altered sensorium Source: Public Health Image Library, ID#/2896/Dr Mike Miller/
Centers for Disease Control and Prevention (CDC), Atlanta (with
along with positive Brudzinski’s sign (flexion of legs
permission).
when neck was passively flexed) is suggestive of
meningitis. Treatment
Etiological Diagnosis As mortality is very high (~ 20%) in pneumococcal
Gram staining of CSF (Fig. 39.2) showed gram-positive, meningitis, treatment should be initiated as early as
lanceolate-shaped diplococci surrounded by a clear possible. Ceftriaxone + vancomycin is given for 10-14
halo (capsule). This is suggestive of presumptive iden- days.
tification as pneumococcal meningitis (Streptococcus For answers to the other questions, refer text.
pneumoniae).

Ge Solving Exercise
eee3 : |
H. influenzae meningitis 1. What is the probable clinical diagnosis and the
etiological agent?
A 7-year-old girl was admitted to the hospital with
2. What are the various tests you will perform from
complaints of high-grade fever, altered mental status
the CSF specimen for this case?
and neck rigidity. On examination, it was found that
3. What is the preferred treatment of choice in this
there was inability to straighten the leg when the
case?
hip is flexed to 90°. CSF sample was collected by
lumbar puncture in a sterile container and sent to the Explanation
laboratory for culture (Fig. 39.3). Clinical Diagnosis
History of fever, neck rigidity and altered sensorium
along with positive Kernig's sign (severe stiffness of
the hamstrings causes an inability to straighten the leg
when the hip is flexed to 90°) is suggestive of meningitis.
Etiological Diagnosis
S. aureus
Culture on blood agar (Fig. 39.3). reveals larger colonies
formed adjacent to S. aureus streak line and the size
of the colonies decreased gradually away from the S.
aureus streak line. This property is known as satellitism,
which is a feature of Haemophilus influenzae.
Treatment
Fig. 39.3: Satellitism of H. influenzae around Third-generation cephalosporins, such as ceftriaxone
S. aureus streak line. or cefotaxime is the drug ofchoice.
Source: Department of Microbiology, Pondicherry Institute of For answers to the other questions, refer text.
Medical Sciences, Puducherry (with permission).
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

Pathogenesis
The bacteria that cause acute meningitis are
transmitted from person-to-person through
droplets of respiratory secretions from cases or
nasopharyngeal carriers. Close and prolonged
contact—kissing, sneezing or coughing on
someone, or living in close quarters with an
infected person facilitate the spread of the disease.

Routes of Infection
Organisms may gain access to the meninges by
€A Involuntary hip and
several routes: ¢V knee flexion
“ Hematogenous spread: This is the most
common route, where entry into the
subarachnoid space is gained through the
choroid plexus or through other blood vessels
of the brain
* Direct spread from an infected site present
Figs 39.4A and B: Signs seen in meningitis:
close to meninges—otitis media, mastoiditis,
(A) Kernig's sign; (B) Brudzinski’s sign.
sinusitis, etc.
“* Anatomical defect in central nervous system
(CNS): It may occur as a result of surgery, Babies usually present with fever, irritability
trauma, congenital defects, which can allow and bulging fontanelle
organisms for ready and easy access to CNS. “+ Complications: In due course, the disease
may involve brain parenchyma leading to
Clinical Manifestations meningoencephalitis—that may result in
The average incubation period is 4 days but decreased consciousness, seizures, raised
can range between 2 and 10 days. Patients with intracranial pressure, and stroke
meningitis develop various manifestations, such * Organism specific finding, e.g., purpuric
as: rashes seen in meningococcal meningitis.
** Important symptoms include fever, vomiting,
Laboratory Diagnosis
intense headache, altered consciousness and
occasionally photophobia Specimen Collection and Transport
* Signs of meningism (meningeal irritation), CSF is the most ideal specimen for pyogenic
such as: meningitis. Blood culture is another useful
= Nuchal rigidity (“stiff neck”) is the patho- specimen for culture.
gnomonic sign of meningeal irritation and ** CSF collection: CSF is obtained by lumbar
is present when the neck resists passive puncture under strict aseptic conditions. It
flexion is divided into three sterile containers; one
= Kernig’s sign: Severe stiffness of the ham- each for cell count, biochemical analysis and
strings causes an inability to straighten bacteriological examination
the leg when the hip is flexed to 90° (Fig. «+ CSF transport: CSF being the most precious
39.4A) specimen should be examined immediately
= Brudzinski’s sign: When the neck is m= When the bacteriological examination (cul-
passively flexed, results in spontaneous ture) is required, CSF should never be re-
flexion of the hips and knees (Fig. 39.4B). frigerated as delicate pathogens, such as H.
** Ininfants: Pyogenic meningitis in infants may influenzae, pneumococci or meningococci
have a slower onset, signs may be nonspecific, may die. Therefore if a delay is expected, it
and neck stiffness may not be present. may be kept in an incubator at 37°C
 CHAPTER 39 © Bacterial Meningitis

Table 39.1: Cytological and biochemical parameters in CSF of normal individuals and in different types of
meningitis.

Characteristics Normal Pyogenic meningitis Tuberculous Viral meningitis

individual meningitis
CSF pressure (mm of Normal (50-150) Highly elevated (>180) Moderately elevated Slightly elevated/
water) normal
Total leukocyte count 0-5 100-10,000 10-500 25-500
(per mm?)
Predominant cell type Lymphocytes Neutrophils Lymphocytes Lymphocytes
Glucose (mg%) 40-70 <40 mg/dL (decreasedto 20-40 mg/dL Normal
absent) (slightly decreased)
Total proteins (mg%) 15-45 >45 mg/dL (usually >250; 100-500 mg/dL 20-80 mg/dL
markedly increased) (moderate to (normal or slightly
markedly increased) elevated)

= However for molecular diagnosis, CSF can Table 39.2: Preliminary clue about the etiological
be kept inside the freezer. agents of pyogenic meningitis based on CSF Gram
Other useful specimens for isolation of stain.
etiological agents of pyogenic meningitis are: Appearance in CSF Gram stain Suggestive of
“+ Blood culture: Blood should be collected Gram-positive diplococci, flame or Streptococcus
in automated blood culture bottles, such as lanceolate-shaped with clearhalo | pneumoniae
BacT/ALERT (capsulated) (Fig. 39.2)
“+ For suspected meningococcal meningitis: Gram-negative diplococci, Neisseria
Other useful specimens are nasopharyngeal capsulated, with adjacent sides meningitidis
flattened (lens or half-moon
swabs, pus or scrapings from rashes; which
shaped) (Fig, 39.1)
should be carried in transport media (such
Pleomorphic gram-negative Haemophilus
as Stuart’s medium). These specimens are
coccobacilli (Fig. 34.5A) influenzae
inoculated onto selective media, such as Thayer
Gram-negative bacilli, arranged Escherichia coli or
Martin medium or New York City medium, to
singly others
suppress the growth of normal flora.
Gram-positive cocciin short chain Streptococcus
agalactiae
Cytological and Biochemical Analysis
Gram-positive short bacilli, often Listeria
Biochemical analysis and cell count of CSF give confused with diphtheroids monocytogenes
a preliminary clue about the type of meningitis
(Table 39.1) “+ This helps in early initiation of appropriate
In acute bacterial (pyogenic) meningitis: empirical antimicrobial therapy
* CSF usually contains >1000 leukocytes/pL “+ Heaped smear: As the bacterial load in CSF
and predominantly neutrophils (90-95%). may be very low, to increase the sensitivity,
However in Listeria meningitis, there is several drops of CSF should be placed at
increased lymphocyte count in CSF the same spot on the slide, each drop being
* The total protein content is elevated and the allowed to air dry before the next is added
glucose level is markedly diminished or even “+ Centrifugation: Alternatively, CSF can be
absent centrifuged (by cytospin) and the deposit is
“» CSF pressure is highly elevated. examined for Gram staining.

CSF Microscopy (Gram Staining) Direct Antigen Detection

Microscopic examination of gram-stained smear From CSF: After centrifugation of CSF, the
may give a preliminary clue about the etiological supernatant can be used for antigen detection.
agent of pyogenic meningitis based on the Latex agglutination test is performed using latex
morphology of the bacteria (Table 39. 2). beads coated with anti-capsular antibodies.
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

“ [tis available for detection ofcapsular antigens * Bloodculture canbe collected in conventional
of common agents of meningitis, such as S. blood culture bottles, such as BHI broth/agar
pneumoniae, S. agalactiae, N. meningitidis, or preferably in automated blood cultures
H. influenzae or E. coli : (e.g., BacT/ALERT)
“ Detection of capsular antigens inCSFismore * Culture plates (blood agar and chocolate
itive than CSF microscopy. agar) are incubated at 37°C, preferably in
Sau: Zt candle jar (provides 5% CO,) for 48 hours
From urine: Antigen detection in urine is use- » Identification: Colonies grown on solid
ful for pneumococcal meningitis. Immuno- media are processed for identification of the
chromatographic test (ICT) is available to detect organism either by automated identification
the C-polysaccharide antigen of S. pneumoniae system, such as MALDL-TOF or VITEK, or by
in urine. conventional biochemical tests (Table 39.3)
* Antimicrobial susceptibility test should
Culture be done to initiate definitive antimicrobial
Ideal media for bacteriological culture of CSF therapy. It is carried out by disk diffusion
are enriched media, such as chocolate agar test or preferably by automated MIC-based
and blood agar, and differential media, such as methods, such as VITEK
MacConkey agar * Sensitivity: CSF and blood cultures may take
* Enriching: As the bacterial load is very low, a >48 hours for organism identification and are
part of the CSF can be inoculated into enriched positive in 70-85% of patients with bacterial
media, such as blood culture bottles at the bed meningitis. However, sensitivity drops rapidly
side (preferred) or brain heart infusion (BHI) in case of prior antimicrobial therapy or delay
broth in the laboratory in processing

Table 39.3: Culture and identification properties of common bacterial agents of pyogenic meningitis.

Common bacterial agents of pyogenic meningitis and their culture and identification properties
Streptococcus pneumoniae (Chapter 34)
e Culture: It produces a-hemolytic colonies on blood agar, described as draughtsman-shaped or carrom coin appearance
¢ Biochemical identification: It shows bile soluble, ferments inulin and sensitive to optochin
Neisseria meningitidis
It produces non-hemolytic colonies on blood agar, which on smear shows gram-negative diplococci (Fig. 39.1)
¢ Biochemical identification: Meningococci are catalase and oxidase positive. They ferment glucose and maltose
but not sucrose
e Serogrouping: Slide agglutination serogrouping (SASG) test is done to identify the serogroups of meningococci
isolates by using appropriate antisera
Haemophilus influenzae (Chapter 34)
e Culture: Blood agar with S. aureus streak line shows satellitism.
¢ Biochemical identification: Disk test for X and V factor requirement shows growth surrounding combined XV disk
Streptococcus agalactiae (Chapter 29)
e Culture: It produces B-hemolytic colonies on blood agar, which on smear shows gram-positive cocci in short chain
e Biochemical identification: It shows CAMP test positive and resistance to bacitracin
¢ Serogrouping with group specific antisera shows Lancefield group B
Gram-negative bacilli meningitis
e Escherichia coli and Klebsiella produce lactose-fermenting colonies on MacConkey agar; identified by ICUT tests
¢ Non-fermenters: Pseudomonas is oxidase positive, whereas Acinetobacter is oxidase negative. They produce non-
lactose fermenting colonies; identified by ICUT tests (Chapter 36)
Listeria monocytogenes
¢ Motility: It shows tumbling type of motility at 25°C but non-motile at 37°C (called differential motility, which is
due to temperature dependent flagella expression)
e Culture: It grows on blood agar (B-hemolytic colonies), and chocolate agar.
Note: Selective media, such as PALCAM agar (containing mixture of antibiotics) may be useful for isolation of Listeria
from specimens, such as food and environmental samples.
Abbreviations: |CUT tests, indole, citrate, urease and triple sugar iron agar test; CAMP, Christie, Atkins, and Munch-Peterson test.
 CHAPTER 39 © Bacterial Meningitis

** Therefore, rapid diagnostic tests, such as ** For neonates: IV ampicillin plus gentamicin
antigen detection or molecular test should be is the recommended regimen
considered to determine the bacterial etiology ** IV dexamethasone is added to the regimen
of pyogenic meningitis. to reduce intracranial pressure.
Definitive therapy: After the culture report
Serology
is available, the empirical therapy is modified
Antibodies to capsular antigens of meningococci based on the organism isolated and its
can be detected in patient’s serum by ELISA. This antimicrobial susceptibility pattern.
is useful to study seroprevalence and to know
the response to vaccination; not for diagnosis.
@ CHRONIC BACTERIAL MENINGITIS
Molecular Methods Several bacterial meningitis present as chronic
Molecular tests are highly sensitive, detect stage, characterized by persistence of signs and
even few bacteria in CSF with less turnaround symptoms as well as the CSF abnormality for
time than culture and also help in serogroup >4 weeks. The bacterial agents causing chronic
identification. meningitis include the following.
“* Formats: Multiplex PCR and multiplex real-
4,e
Partially treated pyogenic meningitis
time PCR can be used for simultaneous
+,“e Parameningeal infections (e.g., otitis media)
detection of common agents of pyogenic
+,CG
* Mycobacterium tuberculosis
meningitis
+,ee
Borrelia burgdorferi (Lyme disease)
“* BioFire FilmArray is an automated nested
o,OC Treponema pallidum (tertiary syphilis)
multiplex PCR commercially available, which
+,“ Rare bacterial agents, such as Nocardia,
can simultaneously detect 14 common agents Actinomyces, Tropheryma whipplei, Leptospira
of meningitis (both pyogenic and viral) in CSF, and Brucella.
with a turnaround time of 1 hour
Tuberculous Meningitis (TBM)
“* Common genes targeted include:
= For meningococcus: ctrA (capsule trans- TBM results from the hematogenous spread
port gene) and sodC (Cu-Zn superoxide of primary or post-primary pulmonary TB.
dismutase gene) Typically, the disease evolves over 1-2 weeks
= For pneumococcus: lytA (autolysin or longer, which differentiates it from bacterial
gene), ply (pneumolysin) and psaA meningitis.
(pneumococcus surface antigen A)
Clinical Features
m For H. influenzae: Conserved capsular
gene bexA. TBM often presents subtly as headache, slight
mental changes, low-grade fever, malaise, night
Treatment of Pyogenic Meningitis sweat, anorexia, and irritability.
** Subsequently, it may evolve acutely with
The mortality of pyogenic meningitis is very
severe headache, confusion, lethargy, altered
high (~20% for pneumococci) and among the
sensorium, and neck rigidity
survivors, up to 50% develop complications.
“* Cranial nerves paresis (ocular nerves in
Therefore, treatment should be initiated as early
particular) is a frequent finding. Stroke
as possible.
may occur due to involvement of cerebral
The choice of antimicrobial agent is based on
arteritis
the type of organism suspected and good CSF
“+ Ultimately, it progresses towards coma, with
penetration ability of the agent.
hydrocephalus and intracranial hypertension.
Empirical therapy comprises of:
* Adult: IV cefotaxime or ceftriaxone and Laboratory Diagnosis
vancomycin is the recommended regimen.
* CSF analysis: Examination of CSF reveals
If Listeria is suspected, IV ampicillin can be
added to the regimen (Table 39.1):
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

= High leukocyte count (up to 1,000/pL), “+ Culture of CSF is diagnostic in up to 80% of

mostly lymphocytic. However, neutrophils cases and remains as the gold standard test.
may be elevated in the early stage However, culture is time consuming, takes 4-8
s Protein content of 100-800 mg/dL weeks by Lowenstein-Jensen medium and 2-3
= Low glucose concentration. weeks by automated liquid culture, such as
However, it should be noted that any of these Mycobacteria Growth Indicator Tube (MGIT)
three parameters can be within the normal “* GeneXpert assay: It is an automated real-
range. time PCR, has a sensitivity of up to 80% and
Cobweb coagulum: When CSF is kept in a is the preferred initial diagnostic option. In
tube for 12 hours, a coagulum forms in the addition to detection of M. tuberculosis, it can
form of a cobweb due to higher fibrin content also detect resistance to rifampicin
in the fluid * Imaging studies (CT and MRI) may show
io%
Acid-fast staining of CSF: Direct smear of hydrocephalus and abnormal enhancement
CSF sediment may reveal long slender beaded of basal cisterns. In more than half of cases,
acid-fast bacilli evidence of old pulmonary lesions or a miliary
= However, the sensitivity of acid-fast stain pattern is found on chest X-ray.
is very low (10-40%) and may require
repeated lumbar punctures to increase Treatment
the yield If unrecognized, TBM is invariably fatal.
® Acid-fast staining of cobweb may give Treatment should be initiated immediately
better yield as TB bacilli may be trapped upon a positive GeneXpert MTB/RIF result.
in the cobweb. However, fibrin strands in It responds well to anti-tubercular therapy, if
the cobweb may be mistaken as fungi. started early.
 Viral Meningitis and Viral Encephalitis AQ

aE

BVIRAL MENINGITIS
Problem Solving Exercise1
Viral Meningitis Q Biochemical and cytological analysis of CSF
A 12-year-old girl was admitted to the hospital with sample is suggestive of viral meningitis.
complaints of high-grade fever, headache, vomiting, > Biochemical analysis: Normal glucose level
altered mental status, seizure and neck rigidity. (40 mg/dL), mildly elevated CSF pressure (100
Biochemical and cytological analysis of CSF sample mm H,O)and slightly elevated protein (60 mg/
revealed glucose level (40 mg/dL), CSF pressure (100 dL)
mm H,O), protein (60 mg/dL) and cell count (25/uL), > Cytological analysis: Mildly increased cell count
which is predominantly lymphocytic. No organisms (25/uL), which is predominantly lymphocytic.
were detected in Gram staining and Indian ink staining
This is suggestive of viral meningitis
>» Noorganisms were detected on Gram staining
of CSF.
1. What is the probable clinical diagnosis and the and Indian ink staining of CSF.
Q Etiological agents of viral meningitis include
probable etiological agent(s)?
enteroviruses (most common cause, accounting
2. Describe the laboratory diagnosis in detail.
for >85% of cases), herpes simplex and arbo-
3. What is the preferred treatment of choice in this
Viruses
case?
Q Molecular methods: Multiplex PCR and multi-
Explanation plex real-time PCR can be used for simultaneous
Clinical Diagnosis detection of common agents of viral meningitis.
For answers to the other questions, refer text.
History of high-grade fever, headache, vomiting,
altered mental status, seizure and neck rigidity is
suggestive of meningitis.

Viral meningitis, inflammation of subarachnoid “ Transmission: Close contacts with infected

space due to viral etiology. It is the second patients is necessary for the transmission to
most common type of meningitis, next to acute set in
“ Seasonality: Most cases of viral meningitis
bacterial meningitis. However, it is often less
occur from nonwinter months (from late
severe than bacterial meningitis and has a better
prognosis. spring to fall), the time when enteroviruses
and arbovirus spread most often.
Epidemiology
Clinical Manifestations
“ People at risk: Although people of any
age can get viral meningitis, some have Common symptoms in children and adults
include fever, headache (frontal or retro-orbital),
a higher risk of acquiring the disease,
including: stiff neck (milder than bacterial meningitis),
= Children (<5 years old) and old age photophobia, sleepiness or trouble in waking up
= Immunocompromised individuals, from sleep, nausea, irritability, vomiting, lack of
appetite (poor eating in babies), and lethargy.
chemotherapy or transplant recipients.
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

sensitive and specific, with a turnaround time

Note: Seizures or focal neurologic signs, or
profound alterations in consciousness, such of 1 hour.
as coma, or marked confusion does not occur
Viral Culture
in viral meningitis and suggest the presence of
encephalitis or other alternative diagnoses. The sensitivity of CSF cultures for the diagnosis
of viral meningitis is generally poor.
Laboratory Diagnosis * In addition to CSF, specific viruses may also
Laboratory diagnosis of viral meningitis includes be isolated from throat swabs, stool, blood,
the following investigations. and urine
* However, isolation of enteroviruses from
CSF Analysis (Cytological and Biochemical) stool is not diagnostic as it may also result
Examination of CSF reveals the following (Table from residual fecal shedding from a previous
39.1, Chapter 39). infection.
“+ Normal or slightly elevated protein level (20-
80 mg/dL) Antibody Detection
“+ Normal glucose level (rarely low glucose level Antibody detection is important for the diagnosis
may occur, as in cytomegalovirus meningitis) of less prevalent arboviruses, such as West Nile
“+ Normal or mildly elevated CSF pressure (100- virus, however, it is of less useful for viruses
350 mm H,O) that have a high seroprevalence in the general
“+ Cell count is typically 25-500/uL, although population, such as HSV and VZV.
in some viral meningitis (e.g., LCM virus and
mumps) the cell counts of several thousands/ Treatment of Viral Meningitis
uL may be seen Treatment of almost all cases of viral meningitis
* Pleocytosis: Lymphocytes are typically the is primarily symptomatic, which includes use
predominant cell type, although neutrophils of analgesics, antipyretics, antiemetics and fluid
may predominate in the first 48 h of illness and electrolyte replacement. Antivirals may be
in some viral meningitis (e.g., West Nile
useful for certain viral agents.
virus) ** Oral or intravenous acyclovir may be of benefit
** Organisms are not seen on Gram staining of
in patients with meningitis caused by HSV-1 or
CSE
2 and in cases of severe EBV or VZV infection
Molecular Methods ** Patients with HIV meningitis should receive
highly active antiretroviral therapy.
Molecular methods have become the
gold standard method for diagnosing viral Agents Causing Viral Meningitis
meningitis, appears to be more sensitive than
Non-polio Enterovirus Infections
viral cultures.
* Amplification of specific viral DNA or RNA Non-polio enteroviruses are the most common
from CSF by PCR-based method provides cause of viral meningitis, accounting for >85% of
definitive diagnosis cases in which a specific etiology can be identified.
* PCR of throat washings or stool specimen ** Cases may either be sporadic or occur in
may assist in the diagnosis of enterovirus clusters
infections ** Although cases can occur throughout the year,
* Formats: Multiplex PCR and multiplex real- affecting any age, but majority occur in the
time PCR can be used for simultaneous summer and rainfall, especially in children
detection of common agents of viral meningitis ** Examples include Coxsackieviruses, echo-
* BioFire FilmArray is an automated nested viruses, parechoviruses and Enterovirus 71.
multiplex PCR commercially available that
can simultaneously detect 14 common agents HSV Meningitis
of meningitis in CSE, which include agents of Herpes simplex viruses (HSV) are the second
pyogenic and viral meningitis. It is extremely most common cause ofviral meningitis, next to
 CHAPTER 40 © Viral Meningitis and Viral Encephalitis

enteroviruses; accounting for 5% of cases. Adults Table 40.1: Agents of viral encephalitis and
are commonly affected than children. encephalopathy.
“> HSV-2 is a more frequent cause of meningitis Viral encephalitis
than HSV-1; in contrast to HSV encephalitis, Herpesviruses
where HSV-1 accounts for >90% of cases e Herpes simplex virus (HSV-1>HSV-2): The most
* History of genital herpes may be an common cause of sporadic encephalitis
important clue as HSV meningitis can occur Cytomegalovirus (in immunocompromised host)
in ~25-35% of women and ~10-15% of men Human herpesvirus 6
Varicella-zoster virus
at the time of an initial (primary) episodeof Epstein-Barr virus
genital herpes.
Arboviruses: Important ones in India are:
e Japanese encephalitis virus (the most common
Mollaret Meningitis
cause of epidemic encephalitis in India)
HSV typically produces a chronic recurrent e West Nile virus (the most common cause of epidemic
lymhocytic meningitis, called as Mollaret encephalitis in USA)
meningitis; characterized by repeated episodes Rabies virus: Causes encephalitis following dog bite
of meningitis, typically lasting two to five days; Nipah and Hendra viruses
occurring weeks to years apart. It can also be Rare causes: Enteroviruses and mumps virus
caused by EBV.

B VIRAL ENCEPHALITIS behavioral disorders, and, at times, a frankly

psychotic state
Encephalitis is an acute inflammation of the * Focal or diffuse neurologic signs: The common
brain parenchyma, caused by invasion of focal findings are:
infectious agents—most often viruses; rarely m Aphasia, ataxia, upper or lower motor
by parasites. The common etiological agents neuron patterns of weakness
of viral encephalitis is enlisted in Table 40.1. # Involuntary movements (e.g., myoclonic
The parasitic causes of encephalitis (e.g., jerks, tremor)
Toxoplasma) is discussed in Chapter 41. m Cranial nerve deficits (e.g., ocular palsies,
facial weakness).
Clinical Manifestations * Features of meningitis (if involved), such as
In addition to the acute febrile illness, the neck rigidity
patients with encephalitis commonly present “ Involvement of the hypothalamic-pituitary
with the following features depending upon the axis may result in temperature dysregulation,
site of involved: diabetes insipidus, etc.
* Altered level of consciousness (confusion) or
a depressed level of consciousness ranging CSF analysis: The characteristic CSF profile in
from mild lethargy to coma encephalitis is indistinguishable from that of viral
meningitis; typically consists of a lymphocytic
“ Seizures: Focal or generalized seizures
pleocytosis, a mildly elevated protein level, and
“ Neuropsychiatric manifestations, such as
, a normal glucose level.
hallucinations, agitation, personality change

2 2_—_
cise :
lem Solvi
Probem
[probl Exerise
ng Exerc
Solving
chain reaction (PCR) for vial etiology (see below) (Fig.
HSV Encephalitis
fever, 40.1).
A 7-year-old boy presented with high-grade 1. What is the clinical diagnosis?
seizures,
vomiting altered consciousness (confusion),
for 3 2. Identify the causative organism based on the test
personality change, aphasia, ataxia, and tremor
ted by result.
days. Cerebrospinal fluid (CSF) specimen collec
rase 3. List the clinical conditions caused by this organism.
lumbar puncture was sent for a multiplex polyme
 SECTION 2 @ Systemic Microbiology (Infectious Diseases)

4. How this clinical condition can be diagnosed in the | DNA ladder Test NC PC
laboratory? H

5. Howwill you treat this condition? 700 bp

Explanation
| 600 bp
Clinical Diagnosis 500 bp — = HSV-2 (469 bp)|
History of high-grade fever, altered consciousness
400 bp = - HSV-1 (391 bp)
(confusion), seizures, personality change, and focal 300 bp
or diffuse neurologic signs (aphasia, ataxia, and
tremor)—suggestive of encephalitis. | 200 bp
Etiological Diagnosis
The multiplex PCR done here (Fig. 40.1) is for herpes | 100 bp
simplex virus (HSV)-1 and 2. Band of 391 bp appeared
|
for test region which coincides with HSV-1 band in
positive control. This confirms the diagnosis as HSV-1 |
| A RO SEeen nnghana APA AORTA PRES
ESS SMe NEE OATESBeA
infection.
For answers to other questions, refer below. Fig. 40.1: Multiplex PCR for herpes meningitis.
Abbreviations: HSV, herpes simplex virus; NC, negative control;
PC, positive control.

Problem Solving Exercise 3

Japanese Encephalitis 4. What are the vaccines available?
A 10-year-old boy is presented to causality with fever, Explanation
mental confusion, disorientation, delirium and seizures.
Clinical and Etiological Diagnosis
The CSF specimen collected was sent to microbiology
laboratory for molecular diagnosis by real time PCR, History of fever, mental confusion, disorientation,
which detected JE virus specific envelope (E) gene. delirium, seizures in a 10-year-old boy is suggestive
1. What is the clinical diagnosis and the etiological of acute encephalitis syndrome.
agent? Real time PCR detected JE virus specific envelope (E)
2. Whatis the mode oftransmission and amplifier host? gene in CSF; confirms the etiology to be Japanese
3. Which district of India reported highest endemicity encephalitis virus.
for this disease? For answers to other questions, refer below.

HSV Encephalitis Japanese Encephalitis

HSV is the most common cause (10-20%) Japanese encephalitis virus is the leading cause
of acute sporadic viral encephalitis, most of vaccine preventable viral encephalitis in
frequently involving temporal lobe. HSV-1 is Asia, including India. It is an arbovirus, belongs
more common (95%) than HSV-2. to family Flaviviridae. It is an enveloped virus,
* Children generally get primary HSV infection, containing ssRNA.
acquired exogenously and invades CNS * Vector: JE virus is transmitted by bite of Culex
via the olfactory bulb; whereas adults get mosquito. C. tritaeniorhynchus is the major
recurrent infections due to reactivation of HSV vector worldwide including India. C. vishnui
in trigeminal nerve is the next common vector found in India
* Clinical manifestation is same as for viral * Animal hosts: JE virus has several animal
encephalitis described earlier hosts. Pigs have been incriminated as the
* Laboratory diagnosis: Detection of viral DNA major vertebrate host; considered as the
in CSF remains the mainstay of diagnosis amplifier host for JE. Cattle and buffaloes
* Treatment: IV acyclovir (10 mg/kg q8h) may act as mosquito attractants
is given for 10 days or until HSV DNA is no * Geographical distribution: Currently, JE is
longer detected in CSR endemic in Southeast Asian region. In India,
 CHAPTER 40 © Viral Meningitis and Viral Encephalitis

JE is endemic in 15 states; Uttar Pradesh sandwich ELISA, uses JERA (JE recombinant
(Gorakhpur district) accounted for the largest antigen) to detect JE-specific IgM antibody in
burden in past. serum. Refer Figure 8.4B (Chapter 8) for detail
«+ Molecular methods: Reverse-transcriptase
Clinical Manifestations (RT) PCR and real time RT-PCR have been
JE is the most common cause of epidemic developed to detect JE virus specific envelope
encephalitis. The incubation period is 5-15 days. (E) gene in blood.
Majority of infections are asymptomatic (iceberg Treatment of JE is only by supportive
phenomenon). Clinical course of the disease measures; no specific antiviral drugs are
can be divided into three stages: available.
1. Prodromal stage of febrile illness
2. Acute encephalitis stage: JE is the most Vaccine Prophylaxis
common cause of acute encephalitis syndrome The following JE vaccines are licensed in India.
(AES) in India; characterized by an acute onset * Live attenuated SA 14-14-2 vaccine (pre-
of fever, mental confusion, disorientation, pared from SA 14-14-2 strain of JE virus)
delirium, seizures (among children), or = It is cell line-derived; primary hamster
coma kidney cell lines are commonly used
3. Late stage and sequelae: It is the convalescent Under National Immunization Program, it
stage in which the patient may be recovered is given to 231 endemic districts of states,
fully or retain some neurological deficits such as—UP, Bihar, Assam, West Bengal
permanently (up to 50%). and Karnataka
= Schedule: Two doses; lst at 9 completed
Laboratory Diagnosis months-12 months of age and 2 nd at 16-
“* IgM capture antibody (MAC) ELISA supplied 24 months.
“> Inactivated JE vaccine: It is inactivated, Vero
by NIV, Pune has been the recommended
method for diagnosis of JE. It is a two-step cell culture-derived vaccine.

B RABIES ENCEPHALITIS
Problem Solving Exercise4 _ |
Rabies Encephalitis Q Dog brain biopsy: Showed Negri bodies (Fig. 40.2A)
Q Electron micrograph: Showed bullet-shaped rabies
A 25-year-old man is bitten by a street dog, which virus (Fig. 40.2B).
was barking excessively and very agitated in behavior. (For answers to other questions, refer below).
Four days later, the dog was found dead. Brain biopsy
of the dog was done and sent for histopathological
staining (Fig. 40.2A). The electron micrograph of the
causative agent is also shown in Figures 40.4A and B.
Q What is the most probable diagnosis based on
histopathology and electron micrograph given in
Fig. 40.28.
Q Whatare the various modalities of diagnosis of this
disease?
Q How will you manage this case of dog bite? Figs 40.2A and B: (A) Histopathological biopsy from
brain and (B) Electron micrograph ofthe causative agent.
Explanation
Source: Public Health Image Library, (A) ID# 3377; (B) ID# 5611;
Clinical Diagnosis Centers for Disease Control and Prevention, Atlanta (with

This is proven case of Rabies. Points in favor are: permission).

ation)
Q History of dog bite (dog bite without provoc
mal behavi or and dog died within 4
with abnor
days.
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

Clinical Manifestations of Rabies Negri Body Detection

Rabies is transmitted by—(1) animal bite (mainly It is useful for postmortem diagnosis of rabies.
dog, but can be any animals); (2) exposure to bat “ They are intracytoplasmic eosinophilic
(inhalation); and (3) corneal transplantation. inclusions with characteristic basophilic inner
Symptoms appear after an incubation period of granules
1-12 weeks; categorized into two types. “+ Sharply demarcated, spherical to oval, and
1. Encephalitic rabies: Most common (80%); about 2-10 pm in size (Fig. 40.2A)
characterized by— * Most common sites of Negri bodies are
= Hyperexcitability and agitation neurons of cerebellum and hippocampus;
= Autonomic dysfunction features however, they can also be less frequently seen
« Hydrophobia (fear of water) or aerophobia. in cortical and brainstem neurons
2. Paralytic or dumb rabies: Occurs in 20% of * Commonly used stains are: Histological
cases. Hydrophobia will be absent. Paralytic stains, such as hematoxylin and eosin and
features predominate. Sellers stains (basic fuchsin and methylene
blue in methanol)
Laboratory Diagnosis * Negri body is pathognomonic of rabies.
Rabies Antigen Detection However, it may not be detected in 20% of
cases. Therefore, the absence of Negri bodies
Direct immunofluorescence test is performed
does not rule out the diagnosis of rabies.
to detect rabies nucleoprotein antigens in
specimens (hair follicle of nape of neck or Prevention of Human Rabies (WHO
corneal impression smear) by using specific Guideline 2018)
monoclonal antibodies tagged with fluorescent
Rabies is prevented by providing prophylactic
dye.
measures, such as post-exposure prophylaxis
Viral Isolation (PEP) and pre-exposure prophylaxis (PrEP).
** Intracerebral inoculation into suckling PEP For Individuals not Received Vaccine
mice
Post-exposure prophylaxis (PEP) consists of
** Cell lines: Mouse neuroblastoma cell lines
three components—local wound care, rabies
and baby hamster kidney cell lines are the
vaccine and rabies immunoglobulin (RIG).
preferred cell lines.
Inclusion of these components in PEP depends
Antibody Detection upon the risk of exposure. WHO has classified
the exposures into three categories (Table 40.2).
Detection of cerebrospinal fluid antibodies is
* For category I exposures: Require only
more significant than serum antibodies. Various
wound care. Vaccine or RIG are not required
antibody detection tests include:
** For category II exposures: Require local
* Mouse neutralization test (MNT)
wound care and rabies vaccine. RIG is
* Rapid fluorescent focus inhibition test
not required except for immunodeficient
(RFFIT)
individuals who need RIG in addition
** Fluorescent antibody virus neutralization
* For category III exposures: All three
(FAVN)
components of PEP are required, such as local
* Indirect fluorescence assay (IFA).
wound care, rabies vaccine and RIG.
Viral RNA Detection Local Wound Care
Reverse transcriptase PCR can be used to It consists of the following measures:
amplify genes of rabies virus RNA from fixed or * Physical cleansing of bite wounds with soap
unfixed brain tissue. It is the most sensitive and and water, followed by antiseptics, such as
specific assay available presently. povidone iodine or alcohol
 CHAPTER 40 © Viral Meningitis and Viral Encephalitis

Table 40.2: Risk categorization and recommended anti-rabies prophylaxis (WHO, 2018).

Category of risk Type of exposure Recommended prophylaxis (WHO**)

Category | (No risk) e Touching, or feeding of animal e Notreatment needed if history is reliable
e Licks on intact skin
Category Il (Minor risk) Minor scratches or abrasions without e Wound management
bleeding or nibbling of uncovered skin ° Rabies vaccine
e Observe the dog for 10 days*
Category Ill (Major risk) Single or multiple transdermal bites with Wound management
oozing of blood Rabies immunoglobulin
e Licks on broken skin (fresh wounds) or Rabies vaccine :
mucous membrane Observe the dog for 10 days*
e Direct contact with bats or wild animals
*Vaccine may be discontinued if animal (dogs and cats) is healthy after 10 days of bite. Other animals are humanely killed and tissue is
examined for detection of rabies antigen/Negri body in brain biopsies.
**In India post-exposure prophylaxis is indicated following exposure to any animal bite except rodents and bat bite.

* Suturing: To be avoided as it causes local * Dose: The maximum dose is 20 IU (hRIG) or

tissue damage, which may help in spreading 40 IU (eRIG) per kg body weight. There is no
of the virus minimum dose.
“* Debridement of devitalized tissues
PEP for Individuals Previously Vaccinated
“+ Tetanus prophylaxis.
For individuals who previously received
Rabies Vaccine rabies vaccine (either PEP or PrEP), RIG is not
A series of rabies vaccine injections should be necessary regardless of exposure category.
administered promptly after the exposure. They need local wound care and an accelerated
“* Type of vaccines: Cell line derived non- vaccine regimen; consisting of one of the
neural vaccines are recommended. Three following schedules.
vaccines are available “ 1-site ID vaccine given on days 0 and 3 or
1. Purified chick embryo cell (PCEC) vaccine: * 4-site ID vaccine given on day 0 only (left
It is prepared from chicken fibroblast cell and right deltoids, thigh and suprascapular
line areas).
2. Purified Vero cell (PVC) vaccine: It is Pre-exposure Prophylaxis (PrEP)
prepared from Vero cell line
PrEP is recommended in two conditions.
3. Human diploid cell (HDC) vaccine: It is
* For individuals at higher occupational
derived from WI-38 (human embryonic
lung fibroblast cell line). risk, such as laboratory staff handling the vi-
rus and infected material, clinicians attend-
* Schedule of PEP regimen: Intradermal (ID)
o
+,

ing to human rabies cases, veterinarians,

regimens are cost-effective; dose-sparing
animal handlers and travelers to endemic
and time-sparing and therefore are preferred
areas
over intramuscular (IM) regimens. 2-site ID
* For sub-populations in remote endemic
vaccine (six doses) is given on days 0, 3 and 7.
areas, which have limited access to PEP and
Rabies Immunoglobulin (RIG) if annual dog bite incidence is > 5% or vampire
by bat exposures prevail
RIG provides passive immunization,
ng the virus. “* Schedule: 2-site ID vaccine (four doses) is
neutralizi
given on days 0 and 7. Boosters are usually
“ Preparations: It is available in two forms;
not necessary as the protection is long-term.
human RIG (hRIG) and equine RIG (eRIG)
 CHAPTER]
Parasitic and Fungal Infections
of Central Nervous System
"SLES SAR RLM A TET TILA

cause opportunistic infections in humans.

B INTRODUCTION
Among the many free-living amoebae that exist
A number of parasites and fungi can infect in nature, only four genera have an association
central nervous system (CNS). with human disease.
* Major parasitic infections of CNS include: IE Naegleria fowleri: It is a causative agent
= Free-living amoebae of primary amoebic meningoencephalitis
= Toxoplasmosis (PAM)
se Neurocysticercosis
. Acanthamoeba species: It causes granulo-
= Cerebral malaria
matous amoebic encephalitis (GAE) in pa-
* Major fungal infections of CNS include:
tients with HIV/AIDS and amoebic keratitis
Cryptococcal Meningitis.
in contact lens wearers
ee Balamuthia mandrillaris: It causes
B FREE-LIVING AMOEBAE INFECTIONS granulomatous amoebic encephalitis
Free-living amoebae are small, freely living, (GAE)
widely distributed in soil and water and can 4. Sappinia species: Causes encephalitis.

@ TOXOPLASMOSIS
| Problem Solving
Solving Exercise1
Exercise 1 _|
Toxoplasma Encephalitis Q Giemsa stained smear showing crescent-shaped
A 55-year-old person reactive for HIV, presented with tachyzoites (6 x 2 um in size)—confirms the
altered mental status, seizures, sensory abnormalities. etiological agent to be Toxoplasma gondii (Fig. 41.1).
The bone marrow aspirate collected was sent for Q Treatment: Cotrimoxazole is drug of choice in HIV
Giemsa stain (Fig. 41.1). infected patients.
Q Identify the etiological agent and diagnose For answers to the other questions, refer Tables 41.1
the clinical condition based on the smear and 41.2.
focused.
Q What is the host, infective form, pathogenic form,
diagnostic form and modes oftransmission of the
parasite?
OQ What are the various diagnostic modalities?
Q How will you treat this condition?
Explanation
This is a case of Toxoplasma encephalitis. Points in ha SE vette
favor are:
Fig. 41.1: Giemsa stained smear showing comma-
Q HlVinfected patient
shaped tachyzoites of Toxoplasma gondii.
Q Presented with altered mental status, seizures,
Source: DPDx Image Library, Centers for Disease Control and
sensory abnormalities—encephalitis Prevention (CDC), Atlanta.
 CHAPTER 41 © Parasitic and Fungal Infections of Central Nervous System

Table 41.1: Features or characteristics of Toxoplasma. Table 41.2: Laboratory diagnosis of Toxoplasma gondii.
Characteristics of Toxoplasma
Host e Definitive host—cat and other Direct microscopy Blood smear—Crescent- or
feline animals (Giemsa stain) comma-shaped tachyzoites
e Intermediate host—man and (indicates acute infection) (Fig.
other mammals (sheep, goat) 41.1).

Morphological e Tachyzoites in blood (man) Smear from biopsy from

forms e Tissue cyst containing organs—tissue cyst with
bradyzoites in organs (man) bradyzoites (indicates chronic
or past infection)
e Oocyst found in cat (feces)
All the three morphological forms Antibody detection Antibody detection remains
Infective form
the most widely used
Transmission e Ingestion of tissue cyst from method for diagnosis of
undercooked meat (most acute toxoplasmosis in
common route) immunocompetent individuals.
e Ingestion of sporulated oocysts e Capture ELISA and
from contaminated soil, food, immunosorbent
or water agglutination assay (ISAGA).
e By blood transfusion, organ Acute toxoplasmosis is
transplantation, or mother- diagnosed by:
to-fetus: Tachyzoites are the > Four-fold rise in IgG titer,
infective form low IgG avidity
Habitat Reticuloendothelial cells of spleen, > Detecton of IgM or IgA
bone marrow, lymph node, liver e Sabin-Feldman dye test:
and peripheral blood This is the gold standard
antibody detection method,
Pathogenic form Tachyzoites usually done in the reference
Manifestations e Asymptomatic—most common laboratories. As it is
(Adult) e Cervical lymphadenopathy technically difficult, seldom
e 1st trimester: More severe used nowadays in routine
Manifestations
infection diagnostics
(congenital
toxoplasmosis) e 3rd trimester: More chance of PCR Detects T. gondii specific genes
transmission of infection
Animal inoculation Intraperitoneal inoculation into
e Manifestation: chorioretinitis, mice
cerebral calcification, convulsion,
microcephaly and mental Imaging CT and MRI scan for encephalitis
retardation

Manifestations e Encephalitis, pulmonary

in HIV infections and chorioretinitis * Pathogenesis: P. falciparum exhibit unique
e Occurs if CD4 T-cell count <100/pL property of being sequestered inside the
Diagnostic form e Tachyzoites in blood brain capillaries by:
e Tissue cyst containing = Expressing a protein called PfEMP (P.
bradyzoites in organs
falciparum erythrocyte membrane
Treatment e Immunocompetent adults—no protein-1), by virtue of which, the
treatment required parasitized RBCs adhere to vascular
e Toxoplasmosis during
pregnancy—spiramycin endothelium and also adhere to non-
e InHlV infected patients— parasitized RBCs
cotrimoxazole = This results in plugging of brain capillaries
by the rosettes of sequestered parasitized
RBCs leading to vascular occlusion and
— CEREBRAL MALARIA cerebral anoxia.
of Clinical manifestations: Cerebral
Cerebral malaria occurs as a complication
falci parum infect ion, not by any malaria manifests as diffuse symmetric
Plasmodium
encephalopathy characterized by repeated
other Plasmodium species (Chapter 20).
304 SECTION 2 @ Systemic Microbiology (Infectious Diseases)

seizures, reduced muscle tone and tendon = Peripheral blood smear examination is
reflexes the gold standard method to identify P.
= Seizures are more common in children falciparum
(up to 50%) than in adults (10%) ¢ Banana-shaped gametocytes
= Other defects are retinal hemorrhages, ¢ Ring forms-multiple ring forms, accole
neurologic sequelae, and rarely deep forms and head-phone shaped ring forms
coma ¢ Schizonts are not seen.
= Signs of focal neurologic and meningeal ® Quantitative buffy coat examination
irritations are absent = Rapid diagnostic tests: ICT detecting P.
= Associated with high mortality rate of falciparum specific HRP-II antigen.
15-20%. “+ Treatment: Artesunate, artemether, arteether
“+ Laboratory diagnosis: The various diagnostic and quinine are the drugs used for treatment
methods include (Chapter 20): of cerebral malaria (Chapter 20).

HBCYSTICERCOSIS
[ Problem Solving
Solving Exercise2
Exercise 2
Cysticercosis Q Vegetarian diet (cysticercosis can also occur among
A 32-year-old vegetarian male presented with vegetarians, in contrast to intestinal taeniasis
recurrent episodes of seizure, headache vomiting which occurs only to beef/pork eaters).
and vertigo. MRI scan of brain was done (Fig. 41.2A), CNS involvement: Presented with recurrent epi-
following which surgery was performed. The sodes of seizure, headache, vomiting and vertigo.
surgically removed cysts have been focused in Figure MRI scan shows cystic lesion in subarachnoid
41.2B, which were subjected to histopathological space (arrow) (Fig. 41.2A).
examination (Fig. 41.2C). Figure 41.2B shows cysticercus cellulosae
ls Identify the etiological agent and the clinical (surgically removed):
condition based on the investigations done. > Yellowish white, 0.5-1.5 cm size, slightly oval
What is the host, infective form, pathogenic form, > Bladder-like sac filled with vesicular fluid
diagnostic form and mode of transmission of the >» White spot which represents the future
parasite? growing scolex.
By, What are the various diagnostic modalities? Histopathological biopsy from the brain
4. How will you treat this condition? (hematoxylin and eosin stain) shows entire
cysticercus seen within the bladder walls. Arrow
Explanation showing the extensive folding of the spiral canal
This is a case of neurocysticercosis. Points in favor and one sucker of the scolex (Fig. 41.2C).
are: For answer to the other questions, refer Table 41.3.

ic
Figs 41.2A and B: (A) MRI of brain—arrow shows cystic lesion in Fig. 41.2C: Cysticercus cellulosae in biopsy
subarachnoid space; (B) Cysticercus cellulosae (surgically removed). from the brain (hematoxylin and eosin stain).
Source: (A) Dr A Subathra. Department of Radiodiagnosis, JIPMER, Puducherry Source: Head of Department (Pathology), Meenakshi
(with permission); (B) Head of Department (Microbiology), Meenakshi Medical Medical College, Chennai (with permission)
College, Chennai (with permission).
 CHAPTER 41 © Parasitic and Fungal Infections of Central Nervous System

Table 41.3: Differences between intestinal taeniasis and cysticercosis.

Agent Taenia saginata, T. solium Taenia solium

Definitive host Man Man
Intermediate host T. saginata-cattle Man
T. solium-pig
Infective form Larva (cysticercus bovis or Eggs of T. solium
cysticercus cellulosae)
Transmission by ingestion Contaminated beef or pork meat —_1. Contaminated food and water
of (partially /uncooked) 2. Autoinfection (contaminated fingers)
Habitat Intestine Tissue (CNS, eye, muscle)
Pathogenic form Adult Larva (Cysticercus cellulosae)
Manifestations Intestinal symptoms only CNS- Seizure, hydrocephalus, pressure effect etc
Diagnostic form Demonstration of eggs in feces Demonstration of larvae in tissues—by CT or MRI
scan
Antibody detection—ELISA or Western blot-
detecting antibody against highly specific 50-13
kDa lentil lectin-purified seven glycoprotein (LLGP)
antigenic fractions
Treatment Praziquantel (DOC), Niclosamide = Drugs: Albendazole, Praziquantel
Surgery

@ CRYPTOCOCCAL MENINGITIS
Problem Solving Exercise 3

Cryptococcal Meningitis 5. Mention the treatment of choice in this clinical

A 45-year-old HIV-infected male presented to the condition.
emergency department with on and off fever Explanation
and severe headache for 1 month duration. On
Clinical diagnosis
examination, he had signs of meningeal irritation.
HIV-infected adult patient with intermittent fever
His cerebrospinal fluid (CSF) sample was collected
and chronic headache (1 month) gives clue that
by lumbar puncture and sent for microbiological
probably the patient is suffering from chronic men-
investigation, such as CSF smear (India ink preparation)
ingitis.
(Fig. 41.3A) and culture on SDA (Fig. 41.3B).
1. Identify the etiological agent based on the tests Identification
done. India ink staining showed clear refractile capsules
2. What is the clinical diagnosis? surrounding spherical budding yeast cells (Fig.
3. Name the virulence factors responsible for this 41.3A) and creamy-to-white mucoid colonies on
clinical condition. SDA (Fig. 41.3B), hence the final identification is C.
4. Describe the various modalities of laboratory neoformans.
diagnosis of this clinical condition.

Cryptococcosis is caused by capsulated yeast “ Polysaccharide capsule: It is the princi-

called Cryptococcus neoformans. pal virulence factor, antiphagocytic and
also inhibits the host’s local immune
Pathogenesis responses
Virulence Factors “+ Ability to make melanin by producing
* Production of other enzymes, such as
Cryptococcus has ability to cross blood-brain
barrier and can cause fatal meningitis with help phospholipase, urease and pheny! oxidase
of various virulence factors, such as: enzyme (help in melanin synthesis).
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

Risk Factors Culture

Individuals at high risk for cryptococcosis include: Cerebrospinal fluid is inoculated onto SDA
“ Patients with advanced HIV infection with without antibiotics, blood agar or chocolate
CD4 T cell counts less than 200/pL agar and incubated at 37°C. Colonies appear as
“+ Patients with hematologic malignancies mucoid creamy-white yeast-like colonies (Fig.
“+ Transplant recipients 41.3B).
¢* Patients on immunosuppressive therapy. Confirmation of Cryptococcus species is
made by:
Clinical Manifestation “+ Niger seed agar and bird seed agar is used to
Various clinical manifestations of cryptococcosis demonstrate melanin production (brown-
include: colored colonies)
“ Pulmonary cryptococcosis: It is the first and “* Growth at 37°C
the most common presentation “+ Urease test positive.
“ Cryptococcal meningitis: It presents as
chronic meningitis, with headache, fever, Treatment
sensory and memory loss, cranial nerve ** Cryptococcosis without central nervous
paresis and loss of vision (due to optic nerve system (CNS) involvement—fluconazole is
involvement) the drug of choice
“+ Skin lesions and osteolytic bone lesions. “+ HIV-infected patients with CNS involvement,
the recommended regimen is induction phase
Laboratory Diagnosis for 2 weeks (Amphotericin B + flucytosine)
Specimens such as CSF, blood or skin scrapings followed by lifelong maintenance therapy with
can be collected in cryptococcosis, depending fluconazole.
upon the site affected.

Direct Detection Methods

“* Negative staining by modified India ink stain
(added with 2% mercurochrome) and nigrosin
stain are used to demonstrate the capsule,
which appears as refractile clear space
surrounding the spherical budding yeast cells
against black background (Fig. 41.3A). India
ink stain is less sensitive (60-70%)
“+ Other stains that can be used are:
m™ Mucicarmine stain
® Masson-Fontana stain Figs 41.3A and B: Cryptococcus neoformans: (A) India ink
m Alcian blue stain (to demonstrate the staining shows clear refractile capsules surrounding round
capsule). budding yeast cells (arrow showing); (B) Growth on SDA at
* Capsular antigen detection from CSF or 37°C—shows creamy white mucoid colonies.
Source; Public Health Image Library/(A) Dr Leanor Haley, ID#:3771;
serum by latex agglutination test is a rapid, (B) Dr William Kaplan, ID#:3199/Centers for Disease Control and
sensitive and specific method. Prevention (CDC), Atlanta (with permission),
 Urinary Tract Infections 4A)
LUE

B INTRODUCTION so that there is more chance of introduction

of endogenous bacteria into the urinary tract
Urinary tract infection (UTI) is defined as a
“+ Age: UTI is common during adult life (10-20%
disease caused by microbial invasion of the in females)
urinary tract that extends from the renal cortex * Pregnancy: Anatomical and hormonal
of the kidney to the urethral meatus. UTI is changes in pregnancy favor the development
the leading cause of morbidity and healthcare of UTIs. Most females develop asymptomatic
expenditures in persons of all ages. bacteriuria during pregnancy. In some cases,
it can lead to serious infections in both mother
Classification
and fetus
** UTIs may be broadly classified into two * Structural and functional abnormality of
types—lower UTI and upper UTI (Table 42.1) urinary tract may obstruct the urine flow, that
depending upon the anatomical sites involved can lead to urinary stasis; which predisposes
** Depending upon the source of infection,
2%, O
to infection
UTI can be of two types: healthcare- ** Bacterial virulence, such as expression of pili
associated (e.g., catheter-associated UTI) and helps in bacterial adhesion to uroepithelium
community-acquired. ** Vesicoureteral reflux: If the normal valve-
like mechanism at the vesicoureteric junction
Predisposing Factors is weakened, it allows urine to flow from the
“+ Prevalence: About 10% of humans develop bladder up into the ureters and sometimes
UTI in some part of their life into the renal pelvis
“+ Gender: UTI is predominantly a disease of ** Genetic factors: Genetically determined
females. The higher prevalence in females receptors present on uroepithelial cells may
is due to the anatomical structure of female help in bacterial attachment.
urogenital system—(1) short urethra, and (2)
close proximity of urethral meatus to anus; Etiology
Escherichia coli (uropathogenic E. coli) is by far
Table 42.1: Comparison between lower and upper UTIs. the most common cause of all forms of UTIs (i.e.,
Lower UTI Upper UTI community-acquired and healthcare-associated
Sites Urethra, and bladder Kidney and ureter UTI and upper and lower UTI); accounting for
involved 70% of total cases.
Symptoms Local Local and systemic * The endogenous flora, such as gram-negative
manifestations: manifestations bacilli (e.g., E. coli, Klebsiella, Proteus, etc.)
dysuria, urgency, (fever, vomiting, and enterococci are the important agents
frequency abdominal pain) “+ In healthcare-associated UTIs, the agents
Route of Ascending route Both ascending are often multidrug resistant. In addition to
spread (common) and the members of Enterobacteriaceae, other
descending route
organisms, such as staphylococci, Pseudomonas,
Occurrence More common Less common Acinetobacter are also increasingly reported.
 SECTION 2 @ Systemic Microbiology (Infectious Diseases)

Table 42.2: Common microorganisms causing UTIs. Direct Examination

Bacterial agents Other agents The screening tests done are as follows:
Gram-negative bacilli: Fungus:
“ Wet mount examination: It is done to
Enterobacteriaceae Candida albicans demonstrate the pus cells in urine. Pyuria
e Escherichia coli: Most of more than 8 pus cells/mm? is taken as
common (70%) significant
e Klebsiella pneumoniae . “ Leukocyte esterase test: It is a rapid and
: Parasites:
e Enterobacter species :
;
e Proteus species
e Schistosoma
:
cheaper method that detects leukocyte
: : haematobium esterases secreted by pus cells present in urine
e Serratia species :
e Trichomonas
Non-fermenters ene “+ Nitrate reduction test (Griess test): Nitrate
vaginalis
e Pseudomonas aeruginosa reducing bacteria, such as E. coli gives a
e Acinetobacter species * Dioctephymie renale positive result
Gram-positive cocci: Viruses: “* Gram staining of urine is not a reliable
e Enterococcus species e BK virus
indicator as—(1) the bacterial count in urine
e Staphylococcus e Adenovirus
saprophyticus* types-11 and 21 is usually low, (2) pus cells rapidly deteriorate
e Staphylococcus aureus in urine and may not be seen well. Gram
e Staphylococcus epidermidis staining may be limited to pyelonephritis and
e Streptococcus agalactiae invasive UTI cases and a count of 21 bacteria/
Abbreviation: UTI, Urinary tract infection. oil immersion field is taken as significant.
*Common in sexually active females.
Culture
Bacterial pathogens are the major cause of ** Culture media: Urine sample should be
UTI. In general, viruses, parasites and fungi inoculated onto CLED agar (cysteine lactose
infrequently infect the urinary tract; discussed electrolyte deficient agar) or combination of
subsequently in this chapter (Table 42.2). MacConkey agar and blood agar. CLED agar
is preferred in laboratories with higher sample
Laboratory Diagnosis load
Specimen Collection ** Kass concept of significant bacteriuria: This
Urine should be collected in a wide mouth is based on the fact that, though the normal
screw capped sterile container by various urine is sterile, it may get contaminated during
methods. voiding, with normal urethral flora. However,
* Clean voided midstream urine: It is the the bacterial count in contaminated urine
most common specimen for UTI; collected would be lower than that caused by an infection
after properly cleaning the urethral meatus Significant bacteriuria
or glans Q A count of 210° colony forming units (CFU)/
* Suprapubic aspiration of urine from the mL of urine is considered as significant—
bladder: It is the most ideal specimen. It indicates infection (referred as ‘significant
is recommended for patients in coma or bacteriuria’ developed by Kass)
infants Q Count between 10% to 10° CFU/mL indicates
doubtful significance; should be clinically
* In catheterized patients, urine should correlated
be collected from the catheter tube (after Q Low count of <10* CFU/mL is due to presence
clamping distally and disinfecting); but not of commensal bacteria (due to contamination
from the uro bag. during voiding) and is of no significance.
However, low counts can be significant in the
Transport following conditions:
» Patient on antibiotic or on diuretic treatment
Urine sample should be processed immediately. >» Infection with some gram-positive
If delay is expected for more than 1-2 hours, organisms, such as S. aureus
then it can be stored in refrigerator or stored by » Pyelonephritis and acute urethral syndrome
adding boric acid for maximum 24 hours. Contd...
 CHAPTER 42 © Urinary Tract Infections

Contd... e infections and also commonly isolated from

>» Sample taken by suprapubic aspiration various hospital-acquired infections.
» In catheterized patients: If the patient is
symptomatic, then a count of >103 CFU/ Clinical Manifestations
mL is considered significant Escherichia coli is associated with the following
manifestations:
** Quantitative culture: This is done to count
the number of colonies. Each colony on
“+ Urinary tract infection—caused by uro-
plate corresponds to one bacterium in urine pathogenic E. coli (UPEC)
sample. Quantitation is done by: ** Diarrhea—caused by six types diarrheagenic
= Semi-quantitative method, such as
E. coli (Chapter 22)
standardized loop technique ** Other infective syndromes include:
® Quantitative method, such as pour plate
s Abdominal infections—bacterial peri-
method. tonitis occurs secondary to intestinal
* Colony appearance: It depends upon the or-
*, ¢ perforation leading to spillage of
ganism grown. For example, lactose ferment- commensal E. coli from intestine
ers, such as E. coli and Klebsiella produce = Pneumonia (especially in hospitalized pa-
tients—ventilator-associated pneumonia)
pink colonies on MacConkey agar and yellow
colonies on CLED agar; whereas non-lactose
= Meningitis (especially neonatal meningitis)
« Wound and soft tissue infections, such
fermenters, such as Proteus, Pseudomonas
and Acinetobacter produce pale colonies
as cellulitis and infection of ulcers and
* Identification: The colonies grown are wounds especially in diabetic foot
# Osteomyelitis
identified either by automated identification
® Endovascular infection and bacteremia.
systems, such as MALDI-TOF or VITEK, or by
** It is responsible for severe lobar pneumonia,
conventional biochemical tests
UTIs, meningitis (neonates), septicemia and
+,~
AST: Antimicrobial susceptibility test is
essential to guide the appropriate treatment. It pyogenic infections, such as abscesses and
is performed conventionally by disk diffusion wound infections
test (on Mueller-Hinton agar) or by automated * It frequently colonizes the oropharynx of
MIC-based methods, such as VITEK. hospitalized patients and is a common cause
of nosocomial infections. Most of the hospital
Treatment strains are multidrugresistant.
Treatment of UTI should be based on Klebsiella pneumoniae UTI
antimicrobial susceptibility testing report.
Quinolones (e.g., norfloxacin), nitrofurantoin, Klebsiella pneumoniae is usually found as
cephalosporins, and aminoglycosides are commensal in human intestines; causes
among the preferred drugs. infections similar to E. coli such as urinary tract
Higher antibiotics, such as carbapenem (e.g., infections, meningitis (neonates), septicemia and
meropenem), B-lactam/f-lactamase inhibitor pyogenic infections such as abscesses and wound
combinations (e.g., piperacillin/tazobactam) or infections. Detail is discussed in Chapter 34.
fosfomycin are used for treatment of healthcare-
Proteus UTI
associated UTIs caused by multidrug resistant
gram-negative bacilli. Proteus mirabilis and P. vulgaris are the most
commonly encountered species from the
AGENTS OF UTI clinical specimens.
COMMON
“+ Proteus species are widely distributed in nature
Escherichia coli UTI and also commensal of human intestine
Escherichia coli is a commensal harbored in “* They are opportunistic pathogens, commonly
intestine. At the same time, it is also one of the responsible for urinary, wound and soft tissue
most common organism associated with human infections and septicemia
 SECTION 2 @ Systemic Microbiology (Infectious Diseases)

Problem
[Probl Exercise 1
Solving Exercise1
em Solving | ‘

Urinary Tract Infections (E. coli/Klebsiella/ Proteus) deficient agar showing significant growth of flat
A 24-year-old female was admitted with fever, dysuria
LF colonies
and frequency of micturition for the past 3 days. Q Fig. 42.2: Gram-stained culture smear showing
Urine microscopy revealed pyuria. Urine specimen gram-negative bacilli
was further subjected to cculture, Gram staining of
Q Biochemical tests (ICUT tests, Fig. 42.3):
culture smear and biochemical reactions. Result of > Indole test—positive (cherry red ring is formed)
antimicrobial susceptibility test (AST) is demonstrated > Citrate test—negative (citrate is not utilized)
in Figure 42.4 and Table 42.3. » Urease test—negative (urea is not hydrolyzed)
Exercise 1: Figures 42.1 to 42.3 > TSI (triple sugar iron test)—shows acid slant/
Exercise 2: Figures 42.5 to 42.7 acid butt, gas present, H,S absent.
Exercise 3: Figures 42.8 to 42.11 Exercise 2: Figures 42.5 to 42.7 (Klebsiella
1. What is the clinical diagnosis and its causative pneumoniae)
organism in all three exercises?
The identification is Klebsiella pneumoniae, as it shows
2. List the clinical conditions caused bythis organism.
the following properties:
Q What are the various modalities of laboratory Q Fig. 42.6: Mucoid dome-shaped pink-colored
diagnosis? colonies on MacConkey agar
Q How will you treat this condition? Q Fig. 42.5: Gram-stained smear showing short
Explanation stout gram-negative bacilli (Klebsiella)
Q Biochemical tests (ICUT tests, Fig. 42.7):
Clinical Diagnosis
> Indole test—negative
The history of fever, dysuria and frequency of > Citrate test—positive (citrate is utilized)
micturition in a female of reproductive age is >» Urease test—positive (urea is hydrolyzed)
suggestive of urinary tract infection (UTI). The > TSI (triple sugar iron test)—shows acid slant/
common agents of UTI are the gram-negative bacilli, acid butt, gas present, HS absent.
such as E. coli (most common), K. pneumoniae and
Proteus and gram-positive cocci, such as enterococci. Exercise 3: Figures 42.8 to 42.1 (Proteus)
The identification is Proteus mirabilis, as it shows the
Identification
following properties:
Based on the culture on MacConkey agar, Gram Q Fig. 42.9: Swarming growth on blood agar
staining of culture smear and biochemical reactions, Q Fig. 42.10: Non-lactose fermenting colonies on
the identification in all three exercises is as follows: MacConkey agar
Exercise 1; Figures 42.1 to 42.3 (Escherichia coli) Q Fig. 42.8: Gram-stained smear showing
The identification is Escherichia coli, as it shows the pleomorphic gram-negative bacilli (Proteus)
following properties: Q Biochemical tests (ICUT tests, Fig. 42.11):
» Indole test—negative (therefore, P. mirabilis)(P.
Q Figs 42.1A to C: (A) Blood agar shows moist
colonies; (B) MacConkey agar shows flat lactose vulgaris gives positive reaction)
» Citrate positive (citrate is utilized)
fermenting (LF) colonies; (C) Semi quantitative
urine culture on cysteine lactose electrolyte » Urease positive (urea is hydrolyzed)

Figs 42.1A to C: Escherichia coli on (A) Blood agar (moist colonies); (B) MacConkey agar [flat lactose fermenting (LF)
colonies]; (C) Semiquantitative urine culture on cysteine lactose electrolyte deficient agar showing significant growth of
flat LF colonies.
Source: Department of Microbiology, Pondicherry Institute of Medical Sciences, Puducherry (with permission).
 CHAPTER 42 © Urin
Tractary
Infections

» TSI shows alkaline slant/acid butt, gas present to ceftriaxone, gentamicin, ciprofloxacin and sensitive
and H,S present to amikacin, meropenem, piperacillin or tazobactam
Antimicrobial Susceptibility Test and Treatment and nitrofurantoin; hence one of the sensitive
drug would be the antibiotic of choice. However,
Antimicrobial susceptibility test on Muller Hinton agar
nitrofurantoin is not given for Proteus infections as it
(Fig. 42.4) with zone interpretation chart (Table 42.3)
is intrinsic resistant.
shows that the causative urinary pathogen is resistant
(For answers to other questions, refer below).

Fig. 42.2: Gram-stained culture smear showing gram-

negative bacilli (Escherichia coli).
Source: Department of Microbiology, JIPMER, Puducherry
(with permission).

Indole Citrate Urease A Fig. 42.4: Antimicrobial susceptibility testing on

positive negative negative gas+, H,S- Mueller-Hinton Agar for Escherichia coli/Klebsiella/
Proteus (observed zone size diameter (mm) and zone
interpretation to various antimicrobial agents tested are
given in Table 42.3).
Abbreviations: Cf, ciprofloxacin; Ak, amikacin; G, gentamicin;
Ci, ceftriaxone; Pt, piperacillin/tazobactam; M, meropenem; Nf,
nitrofurantoin.
Source: Department of Microbiology, Pondicherry Institute of
Medical Sciences, Puducherry (with permission).

F
- yt ey ee La
a fute ee
oF
B ~ 3
4

| oe See os
i 77. *
Fig. 42.3: Biochemical reactions of Escherichia coli.
Source: Department of Microbiology, JIPMER, Puducherry
(with permission). bs “ “ee ate ~ Sih. *
2% |
pe 3h ‘ <4 ne we =
“* Proteus species are often involved in nosoco-
mial outbreaks Py # : ig? x e . om
“ They can cause Struvite stones in bladder in | P| RL « % . ak |
alkaline urine
* Basis of Weil-Felix reaction: Certain Proteus
ra a , ae
a ~.
SO tS
es
antigens cross react with antigen of some Fig. 42.5: Gram-stained smear showing short stout gram-
Rickettsia species and hence are used to negative bacilli (Klebsiella).
detect antibodies in various rickettsial Source: Department of Microbiology, JIPMER, Puducherry
diseases. (with permission).
312) SECTION 2 @ Systemic Microbiology (Infectious Diseases)

Table 42.3: Interpretative categories (CLSI) and observed zone size diameter (mm) to various antimicrobial
agents tested for Enterobacteriaceae.
Antimicrobial Disk CLSl interpretative criteria for Observed zone | Interpretation
agents strength Enterobacteriaceae (in mm)* size (in mm)

(us) | Resistant |intermediate [Sensitive |09° Resistant

Ciprofloxacin (Cf) 5 <21 22-25 226 16
Amikacin (Ak) 30 <14 15-16 217 24 Sensitive

Gentamicin (G) 10 <12 13-14 215 6 Resistant

Ceftriaxone (Ci) 30 <19 20-22 >23 6 Resistant

Piperacillin/ 100/10 <17 18-20 >21 22 Sensitive
Tazobactam (Pt)
Meropenem (M) 10 <19 20-22 >23 28 Sensitive
Nitrofurantoin (Nf)** 300 <14 15-16 >17 21 Sensitive
Abbreviations: CLSI, Clinical and Laboratory Standards Institute.
Note*: CLSI interpretative categories and zone size diameter (mm) to various antimicrobial agents tested for Klebsiella and Proteus are
same as that for E. coli.
**Proteus species are intrinsically resistant to nitrofurantoin.

| tier \ t™ a a

| =A = 3\
— af . SL iW,

\- DN! =e
—- 9 SS \

Fig. 42.8: Gram-stained smear showing pleomorphic

Fig. 42.6: Klebsiella species on MacConkey agar (Mucoid gram-negative bacilli (Proteus).
dome-shaped pink-colored lactose-fermenting colonies). Source: Department of Microbiology, JIPMER, Puducherry
(with permission).
Source: Department of Microbiology, Pondicherry Institute of
Medical Sciences, Puducherry (with permission).

Indole Citrate Urease AIA

negative positive negative

>» Gas
: . Fig. 42.9: Proteus on blood agar, showing swarming
Fig. 42.7: Biochemical reactions of Klebsiella species. growth (arrow showing).
Source: Department of Microbiology, JIPMER, Puducherry Source; Department of Microbiology, JIPMER, Puducherry
(with permission). (with permission).
 CHAPTER 42 © Urinary
Tract Infections

Indole Citrate Urease Len

negative positive negative gast+ H,S+
2

Fig. 42.10: Proteus species on MacConkey agar

(nonlactose fermenting colonies).
Source: Department of Microbiology, Pondicherry Institute of
Medical Sciences, Puducherry (with permission). HS
2

Enteroco :
Fig. 42.11: Biochemical reactions of Proteus mirabilis.
erococcal UTI
Clinical Manifestations Source: Department of Microbiology, JIPMER, Puducherry
: ‘ ’ (with permission).
Enterococci produce various manifestations:
“+ Urinary tract infections (cystitis, urethritis, «, MacConkey agar: It produces minute magenta
pyelonephritis and prostatitis) pink colonies (Fig. 42.12B)
* Bacteremia and mitral valve endocarditis (in + Enterococci are gram-positive oval cocci
intravenous drug abusers) arranged in pairs (spectacle eyed appear-
* Intra-abdominal, pelvic and soft tissue ance)—both in direct smear and colony smear
infections (Fig. 42.12C)
“+ Late-onset neonatal sepsis and meningitis. * Bile esculin hydrolysis test is positive (Fig.
42.13A)
Laboratory Diagnosis “+ Growth occurs in presence of 6.5% NaCl, 40%
They show the following characteristics that help bile, pH 9.6, and at 45°C and 10°C
in the identification: “+ Heat tolerance test: They are relatively heat
* Blood agar: It produces nonhemolytic, resistant, can survive 60°C for 30 minutes
translucent colonies (rarely produces a or “ Pathogenic species are two: E. faecium and
B-hemolysis) (Fig. 42.12A) E. faecalis. They can be differentiated based

Enterococcal UTI Identification

A 25-year-old female admitted to the hospital with Translucent nonhemolytic colonies on blood agar
complaints of burning micturition, fever, vomiting (Fig. 42.12A), magenta pink colonies on MacConkey
and abdominal pain. Urine specimen was collected agar (Fig. 42.12B), gram-positive oval cocci in pair
tests (Fig.
and sent to microbiology laboratory for culture — (Fig. 42.12C), positive bile esculin hydrolysis
(Figs 42.12 and 42.13) and AST (Fig. 42.13A) and arabinose nonfermentati on (Fig. 42.13B)
identification
42.14 and Table 42.4). together reveal the identification as Enterococcus
1. What is the clinical diagnosis and its causative faecalis.

oeGas : ; Antimicrobial Susceptibility Testing and

2. List the infections caused by this organism. wrentnicnt
eAE RET ee ‘ ;
3. Briefly discuss the laboratory diagnosis.
4. Interpret the AST. Antimicrobial susceptibility testing on Mueller Hinton

5. What antibiotic you would like to prescribe? agar (Fig. 42.14 and Table 42.4) showed resistant to
ampicillin, high level gentamicin, nitrofurantoin and
Explanation sensitive to ciprofloxacin, linezolid and vancomycin.
Clinical Diagnosis So any of the sensitive antibiotics should be given
for treatment.
Burning micturition, fever, vomiting and abdominal
(For answers to other questions, refer below).
pain in a patient are clinically suggestive of urinary
tract infection (UTI).
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

Figs 42.12A to C: Enterococcus: (A) Translucent nonhemolytic colonies on blood agar; (B) Magenta pink colonies on
MacConkey agar; (C) Colony smear showing gram-shaped oval cocci in pair (spectacle-eyed appearance).
Source: Department of Microbiology, Pondicherry Institute of Medical Sciences, Puducherry (with permission).

Antimicrobial Susceptibility Test

Positive Negative
Positive Test Negative It is carried out on MHBA by disk diffusion
control Test contro!
test (Fig. 42.14) and reported as per CLSI zone
interpretation criteria (Table 42.4).

Treatment
Most strains of enterococci are resistant to
penicillins, aminoglycosides and sulfonamides.
They show intrinsic resistance to cephalosporins
and cotrimoxazole.
* For UTI, monotherapy with ampicillin
or nitrofurantoin is sufficient, if isolate is
sensitive
Figs 42.13A and B: Enterococcus faecalis: (A) Bile esculin
* Life-threatening infections—combination
agar test (test-positive); (B) Arabinose fermentation (test
not fermented). therapy with penicillin and aminoglyco-
Source: Department of Microbiology, JIPMER, Puducherry (with side (due to synergistic effect). Resistance
permission). to this combination therapy may also be
developed
on arabinose fermentation (Fig. 42.13B); E. ** Vancomycin is usually indicated in resistant
faecium ferments arabinose and E. faecalis is cases; but resistance to vancomycin has also
arabinose nonfermenter. been reported.

Table 42.4: Interpretative categories (CLSI) and observed zone size diameter (mm) to various antimicrobial
agents tested for Enterococcus faecalis isolates.

Antimicrobial Disk strength | CLSI interpretative criteria for Observed zone Inter-
agents (yg) Enterococcus (in mm) size (in mm) (Fig. pretation

Ampicillin (A) 10
Resistant [Intermediate [Sensitive |42"4
<16 - 217 6 Resistant
High level 120 6 7-9 2 6 Resistant
gentamicin (HLG)
Nitrofurantoin (Nf) 300 <14 15-16 217 6 Resistant
Ciprofloxacin (Cf) 5 <15 16-20 221 pm) Sensitive
Linezolid (Lz) 30 <20 21-22 223 25 Sensitive
Vancomycin (Va) 30 <14 15-16 217 17 Sensitive
Abbreviation: CLSI, Clinical and Laboratory Standards Institute.
 CHAPTER 42 © Urinary Tract Infections

Table 42.5: Schistosoma haematobium infection.

Schistosoma haematobium
Definitive host Man
Intermediate host Snail
Infective form Cercaria larva
Transmission Skin penetration
Habitat Blood vessels of bladder
Manifestations Dysuria, hematuria
Hydroureter, hydronephrosis
Bladder carcinoma
Laboratory diagnosis
Diagnosticform Elliptical, non-operculated eggs
(in urine) of 112-170 x 40-70 um in size,
Fig. 42.14: Antimicrobial susceptibility testing on Mueller with a sharp terminal spine
Hinton agar for Enterococcus faecalis (Refer Table 42.4 for Antibody detec- |By HAMA-FAST-ELISA; uses S.
CLSI zone interpretation). tion (in serum) hematobium microsomal antigen
Abbreviations: A; ampicillin; HLG, high level gentamicin; Nf, Antigen Antigens such as CCA and CAA
nitrofurantoin; Cf, ciprofloxacin; Lz, linezolid; Va, vancomycin; CLSI, can be detected by ELISA or dip
detection (in
Clinical and Laboratory Standards Institute.
urine and serum) _ stick assays
Source: Department of Microbiology, PIMS, Puducherry (with
permission). Treatment Praziquantel
Abbreviations: HAMA-FAST-ELISA, S. haematobium microsomal
antigen-Falcon assay screening test-ELISA; CCA, Circulating
cathodic antigen; CAA, circulating anodic antigen.

Problem Solving Exercise 3 |

Schistosoma haematobium Infection Q Figure 42.15B shows the adult worms of
schistosomes.
The thin female worm resides in the
Atraveler from Africa came to the OPD with abdominal
gynecophoric canal of the thicker male.
pain, hematuria and dysuria. Urine culture was
sterile. Urine was collected and sent for wet mount Urine Collection
examination (Fig. 42.15A). The adult form of the same The terminal hematuria portion of urine should be
parasite has been focused in Figure 42.15B. collected and concentrated by centrifugation or by
1. Identify the causative agent based on the membrane filtration. Refer Table 42.5 for details.
microscopic findings. $j

How will you collect and process urine specimen?

Which is the infective stage, host and mode of
transmission of the parasite?
Adult female
Mention two complications produced by infection
with this parasite.
Explanation
Clinical Diagnosis and Identification
The causative agent is Schistosoma haematobium. Adult male
Points in favor are:
ie From Africa—endemic for schistosomiasis
Q Presented with abdominal pain, hematuria and
dysuria
Q Bacterial UTI can be ruled out as urine culture is
Figs 42.15A and B: (A) Schistosoma haematobium
(egg); (B) Adult worms of schistosomes.
sterile
Urine microscopy reveals oval nonoperculated Source: (A) ID# 4843. Public Health Image Library;
(B) DPDx Image Library, CDC, Atlanta (with permission).
elongated eggs with terminal spine (Fig. 42.1 5A)
 | CHAPTER |

Infective Syndromes of Genital Tract

(Sexually-transmitted Infections) 43
rye

B INTRODUCTION = Lesionscommon to both sexes:, such as gen-

ital ulcers, urethritis, and anorectal lesions
The sexually transmitted infections (STIs) are = Female genital tract infections:, such as
a group of communicable diseases which are vulvovaginitis, cervicitis and others
transmitted by sexual contact. Causative agents sw Male genital tract infections:, such as
of STIs may be classified into two groups (Table prostatitis, epididymitis, and orchitis.
43.1): 2. Agents causing systemic manifestations
1. Agents causing local manifestations—called without producing local manifestations (e.g.,
genital tract infections HIV, hepatitis B and C).

BSYPHILIS
Problem Solving Exercise 1
A 35-year-old male with a history of extramarital test serum with saline to know the titer. As VDRL is
sexual contact presented with localized, painless a nonspecific test, the result has to be confirmed by
indolent ulcers with hard base on the penis. On a specific/treponemal test, such as TPHA (7. pallidum
examination, the inguinal lymph nodes were found hemagglutination test).
to be enlarged, hard, and nontender. The serum (For answers to other questions, refer below).
collected from patient was subjected to a serological r

test given below (Fig. 43.1):

1. Identify the test and interpret the result.
2. What is the clinical diagnosis and its causative
organism?
3. What tests do you recommend to confirm this
diagnosis?
4. What are the clinical features seen in this disease?
5. What are the various modalities of laboratory
diagnosis?
6. How will you treat this condition?

Explanation
Clinical Diagnosis
Painless, hard genital ulcers with painless non-
indurated inguinal lymph nodes in a man with history
of sexual contact is suggestive of primary syphilis.
The causative agent is Treponema pallidum.
Serological Test (VDRL) ‘Bp
Non-reactive Weakly reactive Strongly reactive |
The serological test shown in the picture is venereal |

| Negative control Test Positive control

disease research laboratory (VDRL) test (Fig. 43.1). ee

The test serum is weakly reactive. Quantitative VDRL Figs 43.1A and B: (A) VDRL slide; (B) VDRL test done on
test should be repeated on serial dilution of the a test sample with positive and negative controls.
 CHAPTER 43 © Infective Syndromes of Genital Tract (Sexually-transmitted Infections)

Table 43.1: Causative agents of sexually transmitted condylomata lata (mucocutaneous papules)
infections. and mucosal patches
. Agents causing local manifestations (genital tract * Late or tertiary syphilis develops several
infections) decades after, characterized by:
e Skin lesions called gumma
SIE erence
= Neurosyphilis—general paresis of insane
I. Genito-ulcerative disease:
and tabes dorsalis
Syphilis: Caused by Treponema pallidum
Chancroid: Caused by Haemophilus ducreyi ® Cardiovascular syphilis—such as aneu-
Genital herpes: Caused by herpes simplex viruses rysm of ascending aorta and aortic regur-
Lymphogranuloma venereum: Caused by gitation.
Chlamydia trachomatis
e Donovanosis: Caused by Klebsiella granulomatis Laboratory Diagnosis of Syphilis
The above genito-ulcerative diseases can be differen-
tiated from each other by several properties (Table Microscopy
43.2). “+*, Specimen collection: Surface of the chancre
Il. Urethritis: is cleaned with saline, gentle pressure is
e Gonococcal urethritis: Caused by Neisseria
gonorrhoeae
applied at the base of the lesion, and a drop
e Non-gonococcal urethritis (NGU): Caused by of exudate is collected on a slide
> Chlamydia trachomatis (D-K) * Dark-ground microscopy: T. pallidum
> Genital mycoplasmas: Ureaplasma urealyticum, appears as slender, flexible, spirally coiled
Mycoplasma genitalium, M. hominis bacilli with tapering ends (Fig. 43.2A)
> Herpes simplex virus
~ DEA-TP: Direct fluorescent antibody staining
> Candida albicans
> Trichomonas vaginalis for T. pallidum (Fig. 43.2B)
Ill. Other genital tract infections common to both * Silver impregnation method, such as Levaditi
sexes stain (for tissue section) and Fontana stain (for
e Genital tuberculosis: Caused by M. tuberculosis smear) (Fig. 43.2C).
e Anorectal lesions:
> Proctitis: Caused by HSV, gonococcus, C. Serological Test (Antibody Detection)
trachomatis
> Anogenital warts: Caused by human papilloma Serological tests are grouped into treponemal
and nontreponemal tests.

Nontreponemal or Nonspecific Tests

and candidiasis
These are also called as standard tests for
e Mucopurulent cervicitis caused by gonococcus, G syphilis. Here, the reagin antibodies are detected
trachomatis by using nonspecific cardiolipin antigen derived
e Pelvic inflammatory disease from bovine heart muscle. Examples include:
“ Venereal disease research laboratory (VDRL)
*» Rapid plasma reagin (RPR) test.
Prostatitis, epididymitis, and orchitis
Note: Agents sexually-transmitted, but cause only systemic
1. Venereal disease research laboratory
manifestations—HIV, Hepatitis B virus, Hepatitis C virus. This test was named after Venereal Disease
Research Laboratory, New York, where the
Clinical Manifestations of Syphilis test was developed. It is the most widely used,
simple and rapid serological test. VDRL antigen
Syphilis is a sexually transmitted disease caused is a cardiolipin antigen to which cholesterol and
by T. pallidum.
lecithin are added.
“» Primary syphilis develops after incubation
period of 9-90 days and is characterized by Procedure:
painless hard chancre (genital ulcer) and 1. VDRL antigen is reconstituted by mixing
painless indurated lymphadenopathy with a buffer. Then the patient's serum is heat
* Secondary syphilis usually develops 4-8 inactivated at 56°C for 30 minutes to remove
the nonspecific inhibitors
weeks later, characterized by skin rashes,
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

Table 43.2: Comparison of manifestations of genito-ulcerative diseases.

Incubation 9-90 days 2-7 days 1-14 days 3 days—6 1-4 weeks (up to 6
period weeks months)

Genital ulcer Painless, Painful, multiple, Painful, soft, usually Painless, firm Painless, single/
single, bilateral, tiny multiple, purulent, single lesion multiple, beefy-red
indurated vesicular ulcers bleeds easily ulcer, bleeds readily

Lymphadenopa- Painless, Painful, firm, Painful, soft, marked Painful Absent (pseudobubo
thy non-indurated often bilateral swelling leads to and soft, may be present due
(firm), bilateral with initial bubo formation, unilateral to subcutaneous
episode unilateral swelling)
Doxycycline Azithromycin

Vs
Treatment Penicillin Acyclovir Azithromycin
(single dose) (7-14 days) (single dose) (21 days) (7 days)

Figs 43.2A to C: Direct microscopy of T. pallidum.(A) Dark-ground microscope; (B) Direct fluorescent antibody
staining (DFA-TP); (C) bia impregnation method.
Source: Public Health Image Library, (A) ID# 2043; (B) ID# 14967/Dr Russell; (C) ID# 836, Centers for Disease Control
and Prevention (CDC), Atlanta (with permission).

2. VDRL slide containing 12 concave rings is onto separate reaction circles of the disposable
used (Fig. 43.1A) slide. The slide is rotated either manually or
3. Qualitative test: 50 uL of inactivated serum on a mechanical rotor at 180 rpm for at least 8
is mixed with a drop of VDRL antigen and the minutes (Table 43.3).
slide is rotated at 180 revolutions per minute ** Reactive: Indicated by aggregates in the
for 4 minutes in a VDRL rotator and examined center or the periphery of the test circle (Fig.
under microscope (10X). The results are read 43.3)
as follows: “+ Nonreactive: Indicated by a smooth, even
® Nonreactive: Uniformly distributed light grey appearance with no aggregates
fusiform crystals represent the presence visible.
of VDRL antigen only, which indicates a Advantages of nontreponemal tests
negative result ** Used to monitor the response to treatment
® Reactive: Presence of medium to large +,
** Neurosyphilis: VDRL can also be used to
clumps signifies antigen antibody com- detect cerebrospinal fluid (CSF) antibodies.
plexes, hence indicates a positive result
Disadvantages of nontreponemal tests
(Fig. 43.1B).
* Biological false positive (BFP) reactions:
4. Quantitative test: Ifthe test is found reactive,
= Biological false positive reactions are
antibody titer is determined by performing
defined as positive results in non-
the test with serial dilutions with saline.
treponemal tests, with negative results
2. Rapid plasma reagin test in treponemal tests, in the absence of
A drop of RPR reagent is added to a drop of syphilis and not caused by technical
the test serum, positive and negative controls faults
 CHAPTER 43 © Infective Syndromes of Genital Tract (Sexually-transmitted Infections)

Table 43.3: Differences between venereal disease * Prozone phenomenon: If antibody titer
research laboratory (VDRL) and rapid plasma reagin in patient’s sera is high, it may lead to false
(RPR).

Be
negative result; hence, it is essential to test
sera in dilutions.
Results read Results read Specific/Treponemal Serological Test
microscopically as clumps macroscopically. Finely
are smaller in size divided carbon particles As the nontreponemal tests are nonspecific,
coated cardiolipin the result has to be confirmed by a specific/
antigens are used so that treponemal test which detects antibodies by
larger visible clumps are using T: pallidum antigens.
formed
“+ FTA-ABS (Fluorescent treponemal antibody
Preheating of serum is Preheating of serum is not absorption test)
required required * TPHA (T. pallidum hemagglutination test)
Blood, plasma, serum, and Blood, plasma and serum *% TPPA (T. pallidum particle agglutination
cerebrospinal fluid can be can be tested but not test)
tested cerebrospinal fluid
“+ Western blot
Rotation of slide is done Rotation of card is done “ Enzyme immunoassay (EIA)
for 4minutes for 8 minutes * TPI(T. pallidum immobilization test).
Sensitivity (78%) More sensitive (86%)

RPR is expensive than

Testing Algorithm
It is cheaper. It is preferred
when sample load is high VDRL. It is preferred when CDC recommends to use a testing algorithm
sample load is less comprising of non-treponemal test (as
screening test), followed by treponemal test (for
confirmation) for serodiagnosis of syphilis.
Testing for syphilis in pregnancy: Every
pregnant woman should undergo a non-
treponemal screening test at her first antena-
tal visit and, if there is high-risk of exposure,
Negative control Test Positive control again retested at the third trimester and at
delivery.
Fig. 43.3: Rapid plasma reagin test done on a test sample
with positive and negative control.
Treatment of Syphilis
= Cardiolipin antigen being nonspecific may ¢ Penicillin is the drug of choice for all the stages
react with the sera of patients suffering of syphilis
from unrelated diseases but not having * Alternate drug is used in patients with
syphilis penicillin allergy:
= Biological false positive antibody is = Primary, secondary, latent, cardiovascular
system or benign tertiary syphilis—
usually of IgM type, while reagin antibody
in syphilis is mainly IgG tetracycline is recommended
= Types of BFP: Conditions in which BFP = Neurosyphilis or pregnancy or associated
reactions occur can be classified into: human immunodeficiency virus infec-
¢ Acute BFP reactions persist for less than tion—desensitization to penicillin has
6 months and are usually associated to be done following which penicillin is
with acute infections, injuries or administered.
inflammatory conditions
¢ Chronic BFP reactions last for more HBGONORRHOEA
than 6 months and are typically seen
Clinical Manifestations
in systemic lupus erythematosus (SLE)
and other collagen diseases, such as Gonorrhea is a venereal disease, caused by
rheumatoid arthritis. Neisseria gonorrhoeae.
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

Problem
[prob Solving Exerci
lem Solving 2
Exercise se2_| :

A 32-year-old male with history of frequent sexual 2 List the infections caused by this organism.
contact with a commercial sex worker presented 33 What are the various modalities of laboratory
with urethral discharge. Gram staining of the urethral diagnosis?
discharge is shown in Figure 43.4. 4. What antibiotic you would like to prescribe?
1. What is the clinical diagnosis and its causative
Explanation
organism?
Clinical Diagnosis
History of urethral discharge from a male who has a
relationship with sex worker is suggestive of sexually
transmitted disease, such as gonorrhea or Chlamydia
infection.
Identification
Direct Gram staining of urethral discharge (Fig. 43.4)
shows gram-negative cocci in pair (kidney shaped).
at on AO fom ty ¥ This is suggestive of Neisseria gonorrhoeae.
Fig. 43.4: Gram staining ofthe urethral discharge.
(For answers to other questions, refer below).
Source: Public Health Image Library, ID# /2108, Centers for
Disease Control and Prevention (CDC), Atlanta (with permission).

Males present as acute urethritis, epididymi- In chronic urethritis—as discharge is minimal,

tis and abscess in periurethral region with prostatic massage is done to collect the
sinus formation (known as water-can peri- secretion; alternatively the morning drop of
neum) secretion may be collected
** In females, infection is less severe; more Blood and synovial fluid in DGI cases.
asymptomatic carriage than males. Symp-
tomatic females present with mucopurulent Transport Media
cervicitis and pelvic inflammatory disease Specimens should be transported immediately.
“+ In pregnant women, it causes premature If not possible, then urethral discharge should
delivery, chorioamnionitis and sepsis in the be collected in charcoal coated swabs kept in
infant Stuart's transport medium or alternatively,
In neonates (ophthalmia neonatorum) charcoal-containing medium (Amies medium)
Disseminated gonococcal infection (DGI). can be used.

Laboratory Diagnosis Gram-staining of Urethral Exudates

Specimen Collection It reveals gram-negative intracellular kidney-
U rethral swab in men and cervical swab in shaped diplococci (Fig. 43.4). Gram staining is
women are the preferred specimens. Vaginal more useful in males than in females because
and supravaginal swabs are not satisfactory. of the presence of commensal Neisseria
“+,
The urethral meatus is cleaned with gauze species in female genital tract confound with
soaked in saline. The purulent discharge is interpretation. Hence, culture is recommended
expressed out by pressing at the base of the for diagnosis of gonorrhea in women.
penis and collected directly on to slides or
swabs Culture
Dacron or rayon swabs are preferred, as As cervical swabs contain normal flora, hence
cotton and alginate swabs are inhibitory to selective media are preferred, such as:
gonococci +,
DU
Thayer Martin medium
 CHAPTER 43 © Infective Syndromes of Genital Tract (Sexually-transmitted Infections)

** Modified New York City medium * Bacteria: These agents are discussed below:
ate

«* Martin Lewis medium. = Chlamydia trachomatis: Most common

Colonies on Thayer Martin media are gray, agent of NGU
convex, with crenated margin and raised opaque m Urogenital Mycoplasma: Ureaplasma
center. urealyticum and Mycoplasma hominis.
“+ Viruses: Herpes simplex virus
Biochemical Tests ** Fungi: Candida albicans
** Gonococci are catalase and oxidase positive ** Parasites: Trichomonas vaginalis.
** They ferment only glucose, but not maltose Differences between gonoccccal and non-
and sucrose. gonococcal urethritis are given in Table 43.4.

Treatment B VULVOVAGINITIS
** Drug of choice: Third generation cephalo- Vulvovaginitis refers to inflammation of the vagi-
sporins: nal mucosa (called vaginitis) and the external
= Ceftriaxone (250 mg given intramuscularly, genitalia vulva (called vulvitis). It is the most
single dose) common genital tract infection in females.
= Cefixime (400 mg given orally, single ** Women present with vaginal symptoms,
dose). such as abnormal discharge with/without
“> Both the sexual partners should be treated offensive odor or itching
“+ Azithromycin is added if coexisting chlamydial “+ The three most common causes of vaginitis in
infection is suspected. premenopausal women are trichomoniasis,
bacterial vaginosis and vaginal candidiasis;
NON-GONOCOCCAL URETHRITIS can be differentiated from each other as given
(NGU) in Table 43.5.

Chronic urethritis where gonococci cannot be

i TRICHOMONIASIS
demonstrated has been labeled as non-gono-
coccal urethritis. NGU is more common than Trichomonas vaginalis is a genital flagellate,
gonococcal urethritis. Several agents are impli- causes trichomoniasis; the only parasitic sexually
cated in NGU, such as: transmitted disease (Table 43.6 and Fig. 43.5).

Table 43.4: Differences between gonococcal and non-gonococcal urethritis.

Features | Gonococcal urethritis Non-gonococcal urethritis
Onset 48 hours Longer (>1 week)

Urethral Purulent (flow of seed-resembling semen) Mucous to mucopurulent

discharge
Complication DGI (polyarthritis and endocarditis) Reiter's syndrome: Characterized by conjunctivitis,
Water-can perineum urethritis, arthritis and mucosal lesions

Diagnosis e Gram stain e For Chlamydia (most common agent): Culture on

e Culture on Thayer Martin media McCoy and HeLa cell lines
e For Trichomonas—detection of trophozoite
e For Candida—detection of budding yeast cells in
discharge
e For PCR—can be done for HSV or Chlamydia
Treatment Ceftriaxone e For Chlamydia—Doxycycline
e For Trichomonas—Metronidazole
e For Candida—Clotrimazole (as vaginal cream or
tablet)
chain reaction.
Abbreviations: DG!, disseminated gonococcal infection; HSV, herpes simplex virus; PCR, polymerase
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

Exercise 3 ___|
Solving Exercise3
Problem Solving
[Problem ;
Trichomoniasis Etiology
A 28-year-old female sex worker presents to the Figure 43.5B reveales permanent staining of vaginal
STD clinic with history of thin profuse foul smelling discharge showing trophozoites of Trichomonas
purulent vaginal discharge, dysuria and lower vaginalis.
abdominal pain. The wet mount examination of Q Pear-shaped, measures 7-23 um, possesses one
vaginal discharge is depicted in Figure 43.5B. What nucleus, axostyle and five flagella:
is the etiologial diagnosis and how will you treat this Q Four anterior flagella
case? Q One posterior recurrent flagellum supported by
undulating membrane and costa
Explanation
Clinical Diagnosis Treatment
History of thin profuse foul smelling purulent vaginal Treatment of Trichomonas vaginalis includes metro-
discharge, dysuria and lower abdominal pain is nidazole 2 g, single dose; given to both sexual part-
suggestive of vulvovaginitis. ners

Table 43.5: Differential diagnosis of vulvovaginitis.

Feature =—s——sd Vulvovaginal candidiasis Trichomonal vaginitis Bacterial vaginosis
Etiology Candida albicans Trichomonas vaginalis Gardnerella vaginalis, various
anaerobic bacteria
Typical symptoms Vulvar itching and/or irritation Profuse purulent Malodorous, slightly increased
discharge; vulvar itching discharge
Discharge Scanty, white, thick and cheesy Profuse, white or yellow Moderate, thin, white to gray
pH of vaginal fluid Usually <4.5 Usually >5 Usually >4.5
Fishy odor with 10% None May be present Present
KOH
Vaginal inflammation May be present Colpitis macularis None
(erythema) (strawberry appearance)
Microscopy of vaginal T Leukocytes, epithelial T Leukocytes; trophozo- Clue cells, few leukocytes, no/
discharge cells; budding yeast cell with ites seen in 80-90% of few lactobacilli
pseudohyphae symptomatic patients (Nugent's score >7)
Other laboratory Isolation of Candida spp. Antigen detection or PCR Culture, broad-range PCR
findings
Treatment of the Azole cream, tablet Metronidazole or Metronidazole (tablet) and
patient tinidazole clindamycin cream
Treatment of sexual None; topical treatment Usually treatment needed None
partner needed in case of Candida
dermatitis of penis

Table 43.6: Features of Trichomonas vaginalis.

Characteristics of Trichomonas vaginalis

Host Humans are the only host
Infective form Trophozoites
Transmission Sexually transmitted
Habitat Vagina
Pathogenic form Trophozoites
Major manifestations Vulvovaginitis—characterized by:
(trichomoniasis) * Vaginal discharge with profuse foul smelling, increases by adding 10% KOH (Whiff test)
e Raised pH (>4.5)
e Strawberry appearance of vaginal mucosa (colpitis macularis)
Contd...
 CHAPTER 43 © Infective Syndromes of Genital Tract (Sexually-transmitted Infections)

Contd... z

Characteristics of Trichomonas vaginalis

Diagnostic form e Trophozoites detected by wet mount or permanent staining of vaginal discharge
(Fig. 43.5) e Pear-shaped, measures 7-23 um, possesses one nucleus, axostyle and five flagella:
» Four anterior flagella
> One posterior recurrent flagellum supported by undulating membrane and costa
Other methods of e Antigen detection (ICT)
diagnosis’ e PCR detecting tubulin gene
Treatment Metronidazole 2 g, single dose; given to both sexual partners
Abbreviations: ICT, immunochromatographic test; PCR, polymerase chain reaction.

Free flagellae
anteriorly (two pairs)
|

Recurrent te
flagellum
Food vacuole
Costa Sinal
ingle nucleus
Undulating 2
membrane Siderophoric
granules
Food vacuole
with bacteria

Figs 43.5A and B: Trichomonas vaginalis trophozoite: (A) Schematic diagram; (B) Giemsa staining.
Source: DPDx Image Library, CDC, Atlanta (with permission).

B VAGINAL CANDIDIASIS
Grou
Solving Exercise 4
A 32-year-old female sex worker presents to the STD identification can be made by conventional (e.g., germ
clinic with complain of vulvar itching and/or irritation tube test, growth on CHROM agar, cornmeal agar, etc.)
and scanty, white, thick and cheesy vaginal discharge. or automated methods (VITEK or MALDI-TOF).
Vaginal discharge was sent for Gram staining and For, answer to other questions, refer Chapter 21.
culture (Figs 43.6A and B). What is the etiological
“|
diagnosis and what further tests are needed for
accurate species identification.
|
|
Explanation
|
Clinical Diagnosis
History of vulvar itching and/or irritation and scanty,
|
white, thick and cheesy vaginal discharge is suggestive = |
of vulvovaginitis, probably candidiasis.
a |
Etiology Figs 43.6A and B: Candida albicans: (A) Gram-positive
Gram staining (Fig. 43.6A) vaginal discharge oval budding yeast cells with pseudohyphae; (B) On SDA
reveals gram-positive oval budding yeast cells with shows creamy-white colonies.
pseudohyphae and culture on Sabouraud’s dextrose Source: (A) Public Health Image Library, Centers for Disease
agar (Fig. 43.6B) showed dry creamy white colonies. Control and Prevention (CDC), Atlanta; ID #2934; (B) Pondicherry
The genus identification is Candida species. Species Institute of Medical Sciences, Puducherry (with permissicn).
 | CHAPTER |}

AETCOM in Microbiology AA

STREP LALIT

B AETCOM interactions, practicing informed decision

making, breaking bad news (new diagnosis
AETCOM refers to the soft skills ‘Attitude, Ethics of chronic disease, malignancy, death, etc.),
and Communication, that an IMG needs to learn communication and documentation
along with the knowledge and clinical skills to “+ Lack of effective communication has direct
provide holistic healthcare. There should also bearing on medical errors, mistakes in
be an assessment system in place to ensure that diagnosis, inaccurate treatment, compromised
the learning about AETCOM has actually taken patient safety, and patient noncompliance
place. The essential components of AETCOM are leading to stressful legal and sociocultural
as follows. issues
“+ Attitude: It is a settled way of thinking or ** AETCOM competencies are essential to
feeling about something address issues, such as growing distrust in
“* Ethics: Means moral principles that govern healthcare, violence on doctors, litigations
a person’s behavior or the conducting of an on doctors, etc.
activity. Ethics in MBBS curriculum basically
means bioethics or more specifically medical AETCOM Learning in Current vs Previous
ethics, which has the 4 pillars— Curriculum
1. Patient autonomy: Allowing a capable
AETOM in earlier MBBS curriculum: Before
person to exercise the right to make own
CBME was introduced, medical education
decisions and only facilitating in such
focussed mainly on gaining knowledge and
decision-making, e.g., informed consent
clinical skills. AETCOM competencies were
2. Beneficence: Patients’ right to improve-
ment or restoration of their health neither formally taught nor assessed. Some
3. Non-maleficence: An obligation not to learning used to happen by observing seniors
(role modelling) or with self-experience.
inflict harm on others
4. Social justice: Fair and non-discrimi- AETCOM in current curriculum: AETCOM
natory distribution of the limited resources modules in current MBBS curriculum aim at
and, prioritising the need and distribution acquisition of minimum essential skills, such as
proportionate to the need. communicating effectively and sympathetically
** Communication: It is imparting or exchanging with patients and their relatives by all the IMGs
of information by speaking, writing, or using uniformly.
some other medium. * Longitudinally spread: AETCOM competen-
cies relevant to each subject are defined in
Need for AETCOM Competencies the CBME and will be taught longitudinally
Patients and members of the healthcare team spread over all the phases of MBBS
value AETCOM skills as much as the clinical skills. * Increasing complexity: The topics/compe-
“+ Proficiency in AETCOM competencies is tencies are interlinked and are of increasing
very much essential in carrying out routine complexity as one moves from the first year to
healthcare activities including doctor-patient the final professional year of MBBS.
 CHAPTER 44 © AETCOM in Microbiology

Teaching-Learning Methods for AETCOM themselves to develop the following essential

Competencies skills:
m= To elicit, observe and record data
Similar to gaining subject knowledge and
= To reflect on the data at a higher level
learning clinical skills, AETCOM competencies
of thinking and derive opinions and
can also be taught and learnt, though the
conclusions
approach needs appropriate modifications.
= To communicate the observations and
Teaching-Learning sessions are planned in
conclusions in a written and verbal form
such a way that the students are provided
and expand on and defend the conclusions
with opportunities to learn the basic essential
with colleagues and teachers, and
background knowledge, opportunities to learn
= To form new experiences and conclusions
by experiencing (mostly simulated) and reflect
based on this discussion.
on the experiences. Innovative teaching-
learning (TL) methods can be used for AETCOM Assessment of AETCOM Competencies
competencies which will be more engaging and
In the CBME, AETCOM competencies will be
effective. assessed formatively as well as summatively.
Problem-based Learning (PBL) Formative Assessment
Problem-based learning (PBL) is suggested by Formative assessment is done during and along
the MCI as the main TL method for AETCOM. with day-to-day TL sessions and its purpose is to
PBL helps students explore the various facets of provide feedback to the students and help them
“real life issues” that will confront them in their improve. Formative assessment is done based
careers, develop problem solving skills. Case on the student participation in small group
discussions promote collaborative learning and discussions, performance in assignments or
team work, reflection and self-directed learning. internal assessment tests, etc.
*» In this, first a case scenario is introduced to
the students and they are guided to explore Summative Assessment
ethical, legal and sociocultural aspects Summative assessment is qualifying examination
involved in that case and prepare learning which decides pass or fail status.
objectives * There will be questions on AETCOM
~ Then the students are divided into groups competencies in the theory and practical
and assignments are given for self-directed examinations conducted at the end of each
learning (SDL) by using various teacher- professional year
suggested or self-explored resources. “* Student needs to maintain a logbook as a
Student learning may also be helped by record of his performance and acquisition of
an anchoring learning activity to increase essential competencies
relevant background knowledge (e.g., lecture, * Objective structured clinical/practical
panel discussion) or experience (e.g., visit to examination (OSCE/OSPE) or Kalamazoo
hospital/laboratory, observation of working skill rating scale may be used to make the
pattern of different types of healthcare assessment less subjective and to include all
workers, discussion with patients or their relevant aspects in the assessment.
family members, etc.)
“ After some days of learning time through Communications Skill Rating Scale
SDL, the same case or another related case is It is adapted from Kalamazoo consensus. The
discussed. The students provide suggestions parameters assessed are: (i) builds relationship, (ii)
opens the discussion, (iii) gathers information, (iv)
and alternatives on the approach for doctors understands the patient's perspective, (v) shares
to follow and conclusions are drawn information, (vi) manages flow, (vii) overall rating.
“ Finally the students write narrative about their Rating: 1-3 Poor, 4-6 Satisfactory, 6-10 Superior.
learning experience which is expected to help
 SECTION 2 @ Systemic Microbiology (Infectious Diseases)

SUGGESTED AETCOM TOPICS IN 1. Demonstration of confidentiality per-

taining to patient identity on laboratory
MICROBIOLOGY ae
MClIhas specified 8AETCOM modules and37TL 2. Demonstration ofrespect for patient samples
hours in the second professional year for all the sent to the laboratory for performance of
subjects concerned. Following 2 competencies laboratory tests in the detection of microbial
are specified in the Microbiology subject. agents causing infectious diseases.

COMPETENCY-1
DEMONSTRATE CONFIDENTIALITY PERTAINING TO PATIENT IDENTITY ON
LABORATORY RESULTS

Competency: Demonstrate confidentiality pertaining to patient identity on laboratory results

Domain: Attitude
Level of learning: Knows how

Sample Case Scenarios

Case scenario 1 (Disclosing HIV result)
A lady aged 20 years admitted for fever and breathlessness jumps from the 4th floor of the hospital and dies. On
enquiry, it was revealed that she was recently diagnosed to be HIV reactive (one week ago) and “Retro positive”
labels were put on her case file and bed. Her family members had come to know about her HIV status when they had
enquired with a junior resident about the “Retro positive” label. They became stressed out and shouted at the patient.
Except for her mother, all other family members had stopped visiting her then onwards.
Case scenario 2 (Not disclosing HIV result)
A 33-year-old man is admitted to the emergency ward with multiple limb fractures. With ongoing medical treatment,
his vitals are stabilized, he is conscious and oriented. Emergency surgery is planned. His relative comes to the
laboratory to collect the investigation reports, he is given all the reports except HIV results. He is suspicious and is
specifically asking if the laboratory is not issuing only HIV report because it is reactive. Technician gets a call from the
ward asking for the HIV report immediately over the phone, as the patient is being shifted to the operating room.
Case scenario 3 (Patient refuses to provide samples)
A nurse caring for an admitted patient gets a needle stick injury with a syringe used for the patient. The patient's HIV
and HBV infection status unknown.To decide the need for post-exposure prophylaxis the nurse wants to get patient
tested. The patient asks the nurse not to worry, refuses saying he has no such infection, hence no need to test and has
financial constrain for performing the tests. The nurse is anxious. The nurse decides to use the patient's blood sample
collected for some other tests for testing for HIV and HBsAg and bear the cost by herself.
Case scenario 4 (Social stigma in COVID-19)
A patient with influenza, such as illness (ILI) has come to the screening OPD. He had exposure to a confirmed COVID-19
case five days back. He wants to get tested and treated, as he is anxious. However, he does not want to be quarantined
or discriminated, and has a fear of social stigma and losing the job. He is also the only caretaker ofold parents at home.
Case scenario 5 (Occupational exposure)
A medical intern comes to the infection control division with a history of needle stick injury 1 hour back. The source
patient’s blood sample is collected and tested for HIV, hepatitis B and C. The intern is waiting in the reception of the
infection control division to know about the test result of source sample. The test result shows it is reactive for HIV
but negative for hepatitis B and C. The infection control officer discloses the source result to the intern immediately
and provides appropriate counseling and post-exposure prophylaxis. He also forwards the source result to ICTC. The
ICTC calls the source patient on next day and informs about the test result after providing counseling.

Essential Background Knowledge

Principles of medical ethics
Medicolegal aspects of confidentiality
Confidentiality and privileged communication related to laboratory results
Modes of transmission and diagnostic approach for HIV or COVID-19, etc.
Sociocultural issues related to sensitive infections, such as HIV or COVID-19 or needle stick injury
ANRWN>
Post-exposure prophylaxis for needle stick injury when the source turns out to be positive for HIV or hepatitis B.
 CHAPTER 44 © AETCOM in Microbiology

Specific Learning Objectives

Discuss the rights and responsibilities of patients (or healthcare worker in case scenario 5)
Discuss the rights and responsibilities of the laboratory with respect to the confidentiality of laboratory results
Analyze the ethical issues involved in confidentiality pertaining to patient identity
Describe the medicolegal consequences of a breach in confidentiality
OD Demonstration of sympathy when breaking through of result to healthcare workers, providing counseling and
BIS)
maintaining confidentiality pertaining to occupational hazards, such as needle stick injury (in case scenario 5).

Teaching-Learning Method
il Introductory session: Introduction of paper case in small group discussion, identification of various aspects
involved, framing learning objectives and deciding assignments along with learning resources
SDL: Self-directed learning by the students
Anchoring learning sessions: This involves one or more of the following depending upon the case scenario:
> Interaction with laboratory technician and counselor of Integrated Counseling and Testing Center (ICTC)
> Interaction with Microbiology laboratory technician involved in HIV/COVID-19 testing and report dispatch
> Interaction with infection contro! officer involved in management of needle stick injury.
Concluding session: Small group discussion of various possible approaches for the case, their pros and cons, and
justification for the best approach selected by each student. However, it may be possible that there may not be
single best approach.
Ds Writing narratives by the students about their learning experiences.
Resources: Standard medical microbiology and forensic medicine textbooks, NACO guidelines.

Assessment
Formative: Participation in the group discussion, assignments, reflection writing, MCQs to assess relevant background
knowledge, OSPE, etc.
Summative (Theory): Short notes and short answer questions.
Summative (Practical): Includes OSPE with a simulated patient-HIV pre-test/post-test counseling, counseling
following needle stick injury, informing positive COVID-19 report to the patient and informing needle stick injury
test result to the healthcare worker.

Key Learning Points

1. Confidentiality: It is part of the professional secrecy, where a patient's personal/health information (history,
clinical diagnosis, lab results, etc.) is not shared with others, including close relatives and employers, without the
all
patient’s consent. The person with HIV has the right to privacy, and the right to exercise informed consent in
decisions about disclosure of his/her HIV status except in circumstances when disclosure to another person is
required by law or ethical or health considerations
Informed consent: The patient gives written permission to carry out HIV testing after understanding the potential
implications, risks and advantages of the possible test results
of
Counseling is confidential communication limited to the patient and a counselor with the purpose
the risk of HIV infection, to help cope with the stress and to make personal decisions related to
understanding
HIV/AIDS
confidentiality)
Privileged communication: It is the unbiased, bonafide communication by a doctor (bypassing
The doctor first tries
to an individual or an authority who has corresponding legal, social and moral obligations.
risky behavior
to convince the patient to disclose the information himself to the person(s) at risk and to avoid
potentially affected/autho rity directly when in doubt. Such disclosures are made to the people
and, informs the
at risk, such as the spouse, healthcare workers involved in patient care, etc.
with privacy. Pre-
The method followed in ICTC for HIV testing: Only the counselor interacts with the patient
Report will
test counseling is done and written informed consent is taken before sample collection and testing.
etc.), age and gender
not have the patient's name written, instead will have identification marks (moles, scars,
counseling and
mentioned for patient identification. Irrespective of the result, the counselor does the post-test
HIV infection status
hands over the report directly to the patient. Confidentiality regarding patient identity and
is maintained throughout
patient identity
Method followed for other STDs (Syphilis, Gonorrhea): Confidentiality is maintained regarding
tested and both advised to
and laboratory results. Patient is counseled to avoid risky behavior, get partner also
get treated simultaneously if required
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

name. Details
7. Method followed for COVID-19: Patient is identified with an alphanumeric code instead of the
identity.
are released in the media by the Government authorities with the code and not disclosing the patient
Labels are put on the patient house to identify and alert other people.

COMPETENCY-2
DEMONSTRATION OF RESPECT FOR PATIENT SAMPLES
ee

Competency: Demonstration of respect for patient samples sent to the laboratory for performance of laboratory
tests in the detection of microbial agents causing infectious diseases
Domain: Attitude
Level of learning: Shows how

Sample Case Scenarios

Case scenario 1 (Rejection due to improper transport)
Sequestrum from a chronic osteomyelitis case was debrided and sent for culture and sensitivity. The sample was
rejected by the laboratory mentioning that it was received in formalin, hence unsuitable for culture. There is no more
sample available for culture now.
Case scenario 2 (Specimen did not reach laboratory)
A critically ill 5-year-old child’s CSF report is awaited for 3 days. On enquiry, laboratory says it did not receive the
sample. On further probing, it was found that the nursing staff had kept the small bottle with the sample in his pocket
and mistakenly taken it outside the hospital and had dropped it somewhere, and did not submit it to the laboratory
for testing. Now, the baby needs to undergo lumbar puncture again, results may not be the same as antibiotics are
given and need to wait for some more days for the culture report.
Case scenario 3 (Misguided report due to inadequate information in requisition form)
Urologist calls up the laboratory to discuss about “Insignificant bacteriuria” culture report of a pyelonephritis
patient. He says it was a percutaneous nephrostomy sample and asks for the organism and antimicrobial sensitivity.
Microbiologist says it was written as urine sample on the request form, some gram-negative bacillus had grown
and the count was less than 10,000 CFU/mL, so it was thought to be a periurethral commensal and the isolate was
discarded, and hence further testing cannot be done.
Case scenario 4 (Specimen kept at wrong place)
Junior resident gets angry and yells at the patient on noticing a stool sample kept on the bedside table. The patient's
attendant tries to explain that the container is covered in a plastic cover and all these days the junior resident herself
used to keep collected blood and swab samples in that very same place, and he was not informed that stool sample
was not to be kept on the side table.
Case scenario 5 (Rejection due to improper collection)
A suspected pulmonary tuberculosis patient, who would travel 30 km from his village to the private hospital with the
attached laboratory in the city, had submitted spot sputum sample the previous day and an early morning sample
today for acid-fast staining. Reports of both the samples mentioned “many epithelial cells suggestive of excessive
salivary contamination. Repeat with the proper sample”. Blood culture was also collected from the patient by the
clinical team, the result of which came as contaminated blood culture specimen with patient's skin flora.
The doctor found it very difficult to convince the patient to submit proper samples again and pay for them too.
Case scenario 6 (Sample collected for culture and sensitivity in unsterile container)
Paired blood specimen (5 mL each) was sent to the laboratory in two vacutainers for blood culture. The laboratory
rejected the specimen. The patient screams that he cannot allow to draw another set of blood specimen for
investigation.
Case scenario 7 (Rejection due to lack of patient informations)
Microbiology laboratory rejects a bunch of specimens because one or the other relevant informations were missing in
those specimens—patient’s name, age or gender, ward, hospital number, sample type, clinical diagnosis, or treatment
history. The clinical team screams at the laboratory that they could have called the ward or the patient's attendant
and verified the details instead of rejecting.
Case scenario 8 (Rejection due to mismatch of name)
Microbiology laboratory rejects a blood culture specimen collected in BacT/ALERT bottle because the patient's name
written on the bottle did not match with that of the requisition form. The clinical team asks the patient to pay again
 CHAPTER 44 © AETCOM in Microbiology

for a repeat blood culture investigation. The patient complains that he cannot afford the price for another test and
neither he can give consent to draw another specimen.
Case scenario 9 (Prioritising a sample requiring immediate processing and reporting over the others)
In the midnight, the Microbiology laboratory receives three specimens (urine, sputum, CSF) from a patient for culture.
The technician was already processing a huge load of investigations, therefore he informed the clinical team that
these specimens can only be processed on the next day.

Essential Background Knowledge

1. Appropriate sample for the test planned: Sample type, amount, collection procedure, preservative if any, container
type used—and its transportation and storage
Appropriate labeling for correct sample identification
Accompanying Clinical information for correlation
Possible medicolegal issues following incomplete/incorrect sample identification
wn Sociocultural issues following incomplete/incorrect sample identification, relevant clinical information for
we
correlation, improper storage or transportation
6. Ethical issues following incomplete/incorrect sample identification, relevant clinical information for correlation,
improper storage or transportation.

Specific Learning Objectives

Choose an appropriate container for sample collection
Demonstrate an appropriate procedure for temporary storage and transportation of clinical sample
legibly
Discuss the information that shall be written in the request form and the sample container, completely and
Discuss the judicious application of sample rejection criteria in the best interest of patient care
Discuss the importance of prioritising the specimen as relevant to the clinical situation
Oe
ON
oe Discuss medical, ethical and socio-economical considerations of errors in sample collection and submission
process.

Teaching-Learning Methods
Introduction of scenarios with the help of paper case/role plays/videos
issues involved
Small group discussion: Identification of clinical, medicolegal, sociocultural and ethical
Writing learning objectives
Writing narratives by the students about their learning experiences
collection, transportation and
Ml
=
EeeAnchoring lecture and demonstration of appropriate procedure of sample
technicians to gather first-
reception at the laboratory. Discussion with nursing staff, phlebotomist, laboratory
hand information
6. Closing session with small group discussion
es.
7. Writing narratives by the students about their learning experienc Dr
Medicine, Communicate-care-Cure by
Resources: Sample collection (refer in this book), Textbook of Forensic
Alexander Thomas.

Assessment
writing, MCQs to assess relevant background
Formative: Participation in the group discussion, Assignments, reflection
knowledge and OSPE.
s.
Summative (Theory): Short notes and short answer question
l): OSPE can be conducte d covering the following aspects
Summative (Practica
on appropriate sample collection (e.g., urine,
Q Sample collection with care and empathy, instructing patients
sputum , blood culture, etc.)
clinical scenario provided.
Q Labeling sample containers and filling request form for the

Key Learning Points

Microbiology investigations, the various specimen
The method of collection and transport of various specimens for in Chapter
ng the specimen for processing have been discussed in detail
rejection criteria and the criteria for prioritizi
Bes
 CHAPTER |
SS RD

University Practical Examination A5

SLATE PARA

@ PRACTICAL ASSESSMENT PATTERN * Topic distribution under practical assessment

is up to the department to decide
The Competency-based Medical Education * Departments are advised to have OSPE
has been implemented in MBBS since 2019. stations for assessment of practical skills.
The salient features of university practical
examination according to this new MBBS
B AUTHOR’S SUGGESTION
curriculum is as follows.
“+ Total mark allotted for practical examination Authors suggest the following model of topic
is 100, which is divided into: distribution as given below in the Table 45.1.
= Practical (80 marks) and “+ Sample collection and transport: Authors
m Viva voce (20 marks): Viva voce marks are have suggested to keep ‘’Sample collection
to be added to practical, not to theory and transport’ as a separate exercise as this

Table 45.1; Practical assessment pattern.

Practical 80 marks
Problem-based exercise on Gram-staining 15
(Smears made from clinical specimens-Pus, sputum, body fluids, etc.)
e Staining quality (5)
e Microscopic finding with report writing (5)
e Knowledge testing particularly application part (5)
Problem-based exercise on acid-fast staining of sputum smears 15
e Staining quality (5)
e Microscopic finding with report writing (5)
e Knowledge testing particularly application part (5)
Sample collection and transport 10
Hospital infection control 10
Clinical Microbiology Applied Exercise 10 marks x
(Based on clinical infective syndromes) 3 exercises= 30
Viva voce 20 marks
Viva voce-I: General Microbiology, immunology, infections of bloodstream and 10 marks
cardiovascular system, gastrointestinal tract and hepatobiliary system
Viva voce-Il: Infections of skin, soft tissue and musculoskeletal system, respiratory 10 marks
system, central nervous system, genitourinary and sexually-transmitted infections,
hospital infection and control, and miscellaneous
Overall total in practical assessment (including Viva) 100
Note: This is just an author's suggestion. Medical colleges can use this as a template and modify according to the departmental
consensus and university policy.
 CHAPTER 45 © University Practical Examination

is the most important part of microbiology Indian medical graduate to know the detail of
that an Indian medical graduate should infection control practices
know “+ Stool examination for parasites need not be
Hospital infection control: Authors have kept as a separate exercise, but can be asked as
suggested to keep ‘Hospital infection control’ problem based exercise under applied exercise.
as a separate exercise. In lieu of COVID-19 Exercises included in practical assessment
pandemic, MCI hasimplementedapandemic and chapter in which the topics are discussed
module and has made it mandatory forevery are enlisted in Table 45.2.

Table 45.2: Exercises included in practical assessment and chapter in which the topics are discussed.

Exercises included in practical assessment and chapter in which the topics are discussed
1 Problem-based exercise on Gram staining (Chapter 3.4)
(Smears made from clinical specimens——pus, sputum, body fluids, etc.)
e Staining quality
e Microscopic finding with report writing
e Knowledge testing particularly application part
Problem-based exercise on acid-fast staining of sputum smears (Chapter 3.5)
e Staining quality
e Microscopic finding with report writing
e Knowledge testing particularly application part
Sample collection and transport (Any one ofthe following exercise)
Urine specimen for microscopy and culture (Chapters 3.2 and 42)
Blood specimen for culture (Chapters 3.2 and 17)
Blood specimen for serology
22)
Stool specimen for microscopy (for parasites) and bacteriological culture (Chapters 3.2, 5 and
Sterile body fluid specimens for microscopy and culture (Chapter 3.2)
Pus and wound swab specimens for microscopy and culture (Chapter 3.2)
Respiratory specimens for microscopy, culture or for molecular test (Chapter 3.2)
Anaerobic specimens for microscopy and culture (Chapters 3.2 and 30)
Hospital infection control (Any one of the following exercise)
e Hand hygiene (methods and indications) (Chapter 10)
of their use) (Chapter 10)
e Personal protective equipment (donning and doffing of PPE and indications
e Biomedical waste (segregation compliance) (Chapter 13)
B) (Chapter 14)
e Needle stick injuries (post-exposure prophylaxis for HIV and hepatitis
Clinical Microbiology Applied Exercise (Any three of the following exercise)

Bloodstream and cardiovascular system infections Gastrointestinal tract infections

Infective endocardit is (Chapter 16) Shigellosis (Chapter 22)
Acute rheumatic fever (Chapter 16) Cholera (Chapter 22)
Bloodstream infections (sepsis) (Chapter 17) Clostridioides difficile diarrhea (Chapter 22)
Enteric fever (Chapter 18) Rotavirus diarrhea (Chapter 23)
Scrub typhus (Chapter 18) Intestinal amoebiasis (Chapter 24)
Brucellosis (Chapter 18) Giardiasis (Chapter 24)
Leptospirosis (Chapter 18) Cryptosporidiosis (Chapter 24)
HIV/AIDS (Chapter 19) Intestinal taeniasis (Chapter 25)
Dengue (Chapter 19) Hymenolepiasis (Chapter 25)
Malaria (Chapter 20) Schistosoma mansoni infection (Chapter 25)
Visceral leishmaniasis (Chapter 20) Trichuriasis (Chapter 25)
Lymphatic filariasis (Chapter 20) Enterobiasis (Chapter 25)
Systemic candidiasis (Chapter 21) Ascariasis (Chapter 25)
Hookworm infection (Chapter 25)
Strongyloidiasis (Chapter 25)
Contd...
 SECTION 2 © Systemic Microbiology (Infectious Diseases)

Contd...
ce Exercises included in practical assessment and chapter in which the topics are discussed
Hepatobiliary system infections Respiratory tract infections
e Viral hepatitis (Chapter 26) e Streptococcal pharyngitis (Chapter 33)
e Amoebic liver abscess (Chapter 27) e Diphtheria (Chapter 33)
e Hydatid disease (Chapter 27) e Pneumococcal pneumonia (Chapter 34)
Skin, soft tissue and musculoskeletal system infections ° a ophilus influenzae pneumonia (Chapter
e Staphylococcal skin and soft tissue infections (Chapter 28) . .

e Streptococcal skin and soft tissue infections (Chapter 29) ° tere ee aay dee pobaieen ee
e Gas gangrene (Chapter 30) e Pulmonary tuberculosis (Chapter 35)
¢ Leprosy (Chapter 30) e Pseudomonas infections (Chapter 36)
e Cutaneous anthrax (Chapter 30) ¢ Influenza (Chapter 37)
e Herpes simplex (cutaneous and mucocutaneous) : COVID "19 (Chapter 37) :
(Chapter 31) e Infectious mononucleosis (Chapter 37)
° Measles (Chapter 31) e Paragonimiasis (Chapter 38)
e Rubella (Chapter 31) % eee Sie
e Dermatophytoses (Chapter 32) e Aspergillosis (Chapter 38)
* ~Mycetoma (Chapter 32) e Pneumocystosis (Chapter 38)

Central nervous system infections Genitourinary system infections

e Pneumococcal meningitis (Chapter 39) e Urinary tract infection (Escherichia coli)
e Haemophilus influenzae meningitis (Chapter 39) (Chapter 42)
e¢ Meningococcal meningitis (Chapter 39) e Urinary tract infection (Klebsiella pneumoniae)
e Viral meningitis (Chapter 40) (Chapter 42)
e Hsv encephalitis (Chapter 40) e Urinary tract infection (Proteus mirabilis)
e Japanese encephalitis (Chapter 40) (Chapter 42)
e Rabies encephalitis (Chapter 40) e Urinary schistosomiasis (Chapter 42)
e Neurocysticercosis (Chapter 41) e Syphilis (Chapter 43)
e Toxoplasma encephalitis (Chapter 41) ¢ Gonorrhea (Chapter 43)
e Cryptococcal meningitis (Chapter 41) e Non-gonococcal urethritis (Chapter 43)
e Trichomoniasis (Chapter 43)
e Vaginal candidiasis (Chapter 43)
 Index

Page numbers followed by / refer to figure and / refer to table.

A Beta-hemolytic streptococcal Cryptococcal meningitis 306

infections 215 Cryptosporidium 186, 190
Acanthamoeba 302 Bile esculin agar test 314/ Cyclospora 186, 190, 190/
Acid-fast stain 28, 191/, 262 Bile solubility test 252, 252/ Cystic echinococcosis 210/
Acinetobacter baumannii 271 Biomedical waste management Cystoisospora 186, 190, 190f
Acquired immunodeficiency 117, 119¢, 279
syndrome 153 Bloodstream infection 18, 138 D
Adenovirus 61/ Bordetella pertussis 20
AETCOM 324 Dalmau plate culture 174
Bright-field microscope 7/ Dark-ground microscopy 31, 317
Agar dilution method 53 Broth dilution method 52
Agglutination reaction 80 Dengue 153, 159, 160
Brown and Brenn modification 27 Diarrheagenic Escherichia coli
Air surveillance 128 Brucellosis 148
Albert stain 28, 30, 245/, 247 infections 176, 179
Brugiya malayi 170, 170/ Dimorphic fungi 74
Amoebic liver abscess 208 Burkholderia cepacia 258, 268
Amoebic meningoencephalitis, Diphtheria 102, 243-247
Burkholderia pseudomallei 268 Diphyllobothriasis 194
primary 302
Anaerobic culture 21, 22 Direct immunofluorescence assay
Cc 87, 274
Anaerobic glove box 40
Animal inoculation 65, 247 Candida albicans 172, 173/, 321, 323/ Donovanosis 317, 318
Anoxomat system 220 Cellophane tape technique 198/ Duke criteria, modified 134, 135/
Anthrax 225 Central sterile services department
Antigen-antibody reactions, types 109, 109/
of 78 Cerebral malaria 303 Echinococcus granulosus 281
Antigenic variation 273 Cerebrospinal fluid analysis 293, Eijkman test 127
Antimicrobial susceptibility test 17, 296, 297 Elek’s gel precipitation test 78, 79,
48/, 49, 54 Cestode 192/ 79f, 248
Anti-rabies prophylaxis 301/ Chancroid 317, 318 Entamoeba histolytica 125, 186,
Antiretroviral therapy 123, 159 Chemiluminescence-linked 187/, 188/, 191, 208
Antistreptolysin O 217 immunoassay 91 Enteric fever 142
Ascaris lumbricoides 198 Chickenpox 230 Enterobiasis 197
Aspergillosis 105, 281, 284 Chlamydia trachomatis 321 Enterococcus faecalis 314f
Automated blood culture Chlamydophila psittaci 135 Entero-test 189
techniques 35 Chocolate agar 33/, 252, 255/ Enzyme-linked immunosorbent
Automated identification system Cholera 180 assay 59, 83, 84, 84/, 86, 156
247, 264 Citrate utilization test 45, 46/ Epstein-Barr virus 272, 280
Clonorchis 210, 210f Escherichia coli 14/, 34, 45, 126, 139,
B Clostridioides difficile 176 258, 288, 307-310
Clostridium perfringens 126, 176,
Bacillus cereus 176 Ethylene oxide sterilizer 108
221 Extrapulmonary tuberculosis 260
Bacitracin sensitivity testing 217
Coagulase negative Staphylococcus
Bacterial agglutination test 145/,
214 F
178/ Coccidian parasitic infections 190
Bacterial growth curve 14, 15
Coccidioides immitis 174, 281 Fasciola hepatica 194, 208, 210, 210/
Bacterial meningitis 288 Fasciolopsis buski 192, 194, 196/
Bacterial motility 43 Congenital rubella syndrome 235
Corona virus 61/, 272 Flotation technique 71, 71/
Bacterial pharyngitis 243 Fluorescence microscope 9, 62
Bacterial pneumonia 250 Corynebacterium diphtheria 30, 33,
43, 79, 243, 245, 245/ Formol-ether technique 71/
Bacterial vaginosis 322
COVID-19 272, 275, 276/ Free-living amoebae infections 302
Balantidium coli 186, 281
 Essentials of Practical Microbiology

G J
Gas gangrene 220-222 Japanese encephalitis 298 Naegleria fowleri 302
GeneXpert 264, 265 Jones criteria 137 Nagler’s reaction 223, 223f
Genito-ulcerative disease 317 National AIDS Control Organization
Giardia lamblia 186, 189/ K 120,123, 153
Giemsa stain 169/, 175/, 302 Needle stick injury 121, 122
Kinyoun’s cold acid-fast staining
Gonococcal urethritis 317, 321 Negri body 63/, 300
263
Gram-staining 25, 75, 247, 255, 271, Neisseria gonorrhoeae 243
Kirby-Bauer’s disk diffusion test
291, 2911, 308 Neisseria meningitidis 102, 292
49, 50
Group A Streptococcus 136, 216 Nipah virus 297
Klebsiella 310
Nitrate reduction test 308
Kopeloff and Beerman’s
H Nocardia 240
modification 27
Non-gonococcal urethritis 317, 321,
Haemophilus ducreyi 65
321t
Haemophilus influenzae 14/, 250, L
Nontreponemal tests 317
256f, 288, 292 Lactophenol cotton blue 75 Non-typhoidal salmonellosis 179
Hand hygiene 92, 93, 96, 102 Latex agglutination test 81, 82, 151 Norwalk virus 185
Hand-foot-and-mouth disease 235, Leishmania donovani 168 Nutrient agar 33, 213/, 269
235/ Lepromin test 225
Hanging drop method 44, 44/
Helicobacter pylori 22
Leprosy 220, 224 Oo
Leptospira 151
Hepatitis viruses 203, 205, 206 Listeria monocytogenes 176, 288, Optochin sensitivity 252
Herpes simplex virus 227, 296 292; Orientia tsutsugamushi 148
Heterophile agglutination test 81, Loeffler’s serum slope 37, 247 Oxidase test 45, 45/, 178
280 Lowenstein-Jensen medium 33, 34/,
Histoplasmosis 174 263, 264/ P
Hookworm infections 200 Lung flukes 194 Paracoccidioides brasiliensis 174,
Hospital infection control 331 Lyme disease 293 175, 281
Hot air oven 111 Lymphatic filariasis 162, 170 Paragonimiasis 281, 282/
Human echinococcosis 208 Lymphogranuloma venereum 317 Passive agglutination test 81
Human immunodeficiency virus
Paul-Bunnell test 280
153, 158 M Penicilliosis 281, 286
Human papillomavirus 231
Malaria 162 Peptone water 33/
Hydatid cyst 209f
McCrady statistical table 127/ Periodic acid-Schiff stain 75
Hymenolepiasis 193
McFadyean’s reaction 226/ Peripheral blood smear 163, 170/
McIntosh and Filde’s anaerobic jar Personal protective equipment 94,
40f, 220 95, 96f, 102, 103, 120, 27
ICUT tests 178, 258, 271 Measles 232 Pertussis 102
Immunochromatographic test 59, Melioidosis 268 Plasma sterilization 110
90, 90/, 151, 169, 171, 187 Membrane filtration method 127 Plasmodium falciparum 165f, 166/,
Immunoelectron microscopy 184 Meningitis 18, 172, 254, 290/ 303
Immunofluorescence assay 9, 59, Meningococcal meningitis 288 Plasmodium vivax 162, 165/
87, 87/, 191 Metachromatic granules 246 Pneumococcal meningitis 250, 289
Impregnation methods 23, 30 Microsporidia 186 Pneumocystis pneumonia 287
Inclusion bodies 62 Microsporum canis 236/, 237 Pneumonic plague 102
Indole test 45, 45/ Molecular test 148, 244 Polymerase chain reaction 56, 59/,
Infectious mononucleosis 280 Molluscum bodies 63/, 232 64, 147, 152, 169, 171
Infective endocarditis 133, 135 Motility, types of 44, 44/7 Post-exposure prophylaxis 122, 123,
Influenza 272, 273 Mucormycosis 282 1247, 248
Intestinal cestode infections 192 Mucosal candidiasis 238 Post-streptococcal
Intestinal coccidian parasites, Mueller-Hinton agar 50, 179/ glomerulonephritis 217,
laboratory diagnosis of 191/ Multiple tube method 126, 127/ 244
Intestinal flukes 194 Mycetoma 239 Pour plate technique 38
Intestinal taeniasis 193, 305/ Mycobacterium tuberculosis 29, 33, Poxvirus 61/
Intestinal trematode 192 36, 104, 105, 260, 293 Proteus 45, 147, 309
Invasive aspergillosis 285/ Mycoplasma pneumonia 81, 103, Pseudomonas 126, 258, 268, 269/,
Invasive pneumococcal disease 251 243 270t
lodine mount 70 Myxoviruses 272 Psychrophiles 16/
 Index

Pulmonary tuberculosis, primary Staphylococcal pneumonia 257 Urease test 46, 237
261, 261¢ Staphylococcus aureus 53/, 531, 82, Urethritis 21, 313, 317, 320
Pyogenic meningitis 291 OZ SS) Urinary tract infection 18, 172, 211,
Steam sterilizer 109, 110/ 307-310, 313
Q Sterile universal container 19
Quellung reaction 253, 255
Sterilization 108 Vv
Streptococcus agalactiae 217/, 218,
Quick SOFA criteria 139 Vaginal candidiasis 323
219/, 2191, 288, 292
Varicella-zoster virus 230
Streptococcus gallolyticus 133
R Vector-borne diseases 60
Streptococcus pneumoniae 16, 250,
Venereal disease research
Rabies 299, 300, 301 2501, 288, 292
laboratory 78, 316/, 317,
Rapid diagnostic test 166, 293 Streptococcus pyogenes 215, 216,
3191
Rapid plasma reagin test 318, 319/, 216f/, 216, 217%, 243
319¢
Venkatraman-Ramakrishnan
String test 182/, 189
Revised National Tuberculosis medium 34, 180
Strongyloides 192
Control Program 260 Vibrio cholerae 14/, 16, 33, 34, 45,
Strongyloidiasis 200
Rheumatic fever 133, 136, 244 176, 180, 181, 181/, 182
Syphilis 78, 316-318
Rhinosporidiosis 241 Viral encephalitis 60, 295, 297
Viral exanthems 227
Robertson cooked meat broth 35, T
Al, 220, 221f Viral hemorrhagic fevers 160
Rotavirus 11/, 61/, 125, 184 Taenia 192 Viral isolation 300
Thayer Martin medium 320 Viral meningitis 291, 295, 296
Rubella 234
Tissue culture 65 Viral transport medium 61, 274
Toxocara 208 Viridans streptococci 250!
Ss
Toxoplasmosis 302 Visceral leishmaniasis 162, 168
Sabin-Feldman dye test 303 Transmission electron microscope Voges-Proskauer test 182
Sabouraud’s dextrose agar 75, 76/, 10, 10f Vulvovaginal candidiasis 322
236/, 286/ Treponema pallidum 31, 293
Salmonella 45, 143f Treponemal serological test 319 WwW
Saturated salt solution technique Trichomonas vaginalis 321, 3221
T, Tit, Trichomoniasis 321, 322 Water surveillance 125
Schistosomiasis 194, 195/ Trichophyton mentagrophyte 237, Weigert's modification 27
Scrub typhus 142, 147, 148 Weil's disease 151
238/
Sedimentation technique 71 Trichophyton schoenleinti 237 Weil-Felix test 147, 311
Sexually transmitted infections 60, Trichuris 192, 197 Western blot 88, 89/, 156
316 Triple sugar iron agar test 46, 46/, 292 Whooping cough 102
Shigella 144, 176 Tube coagulase test 212, 213/, Widal test 80, 81, 81/, 145
Silver impregnation method 31/, 317 Tuberculosis 18, 260 Wood's lamp examination 237
Slide agglutination test 80/, 144 Tuberculous meningitis 291 Wool Sorter’s disease 225
Slide coagulase test 212, 213/ Typhoid fever 147 Wuchereria bancrofti 170f
Slide culture technique 76/ Tzanck smear 63/, 228/
Specimen 163, 180, 220, 222 Z
Sporothrix schenckii 240, 241/ U Ziehl-Neelsen technique 28, 29,
Sporotrichosis 240
Sporulated oocysts 190/ Ultraviolet radiation 114 29/, 262
Urea hydrolysis test 45, 46/ Zygomycoses 281, 282, 283/
Staphylococcal infections 211
 ~ st
Se ie 2)
i = a ee. She
(im G= Ca ionlt

a « 6 eee Gemle=od by) a iJ

= - ee ) eae
aa a uae L i J toss ww

no ° 4 $i ee
 Other Best-selling Books

ESSENTIALS OF MEDICAL
MICROBIOLOGY

”seHUE883
TWIIGAIN
ADOTOIPGOYIIN

Forewords
Pallab Ray
Sujatha Sistla
one sandhiya Bhat

Apurba S Sastry, et al.

Full Colour | Soft Cover | 3/e, 2021
8.5"x 11" | 934 Pages | 9788194709015
This is the only textbook in ‘Clinical Microbiology’, in accordance with the MCI’s competency based medical education curriculum for MBBS,
System-based approach, rather than traditional organism-based approach -Divided into two parts: (A) General Microbiology, Immunology and Hospital infection
control and(B) Systemic microbiology
Systemic Microbiology (Infectious Diseases there are eight sections, each comprises of a first chapter on clinical infective syndrome followed by several chapters
covering detailed information about the etiological agents
Parasitology has been incorporated—obviates the reading of aseparate book
Hospital Infection Control section—thoroughly updated
General Microbiology section—meticulously restructured with the inclusion of general virology, general parasitology and general mycology chapters
Overview chapters—incorporated which will help in better understanding of individual organisms when discussed under systemic microbiology
Sterilization and Disinfection chapter—completely revised based on hospital use of sterilizers and disinfectants; shifted to Hospital infection control section
COV|D-19 chapter—a completely new chapter has been added
AETCOM module—added as anew annexure

JAYPEE
j,h m6 « Please visit our website
re
The ; egitt Sdences Publisher www.jaypeebrothers.com or Scan the QR Code [=]
for detailed information on microbiology books, visit our website www.jaypeebrothers.com, for detailed information on microbiology books, visit our websit

Essentials of
Practical Microbiology

This is the only practical textbook in ‘Clinical Microbiology’ based on infective syndromes, in accordance with the MCI's competency-based medical education
(CBME) curriculum for MBBS.
+ First microbiology book written in class-wise pattern according to undergraduate practical schedule
- Will help the teacher: To know what to teach during a practical class, what to keep for demonstration during the practical class and also will give the
teacher a holistic approach to make the yearly practical schedule
- Will help the student: To know what to read during a practical class, what to write in the records and what to read during MBBS university practical
examination
Divided into two sections—(|) General Microbiology, Immunology and Hospital Infection Control and (Il) Systemic Microbiology
Each chapter begins with problem-solving exercise (PSE). After reading this book, the students will have a bird’s eye view of how to diagnose infectious
diseases
+ Parasitology has been incorporated—obviates the reading of aseparate book
+ — Hospital Infection Control section—thoroughly updated with donning/doffing of PPE, needle stick injury, etc.
General Microbiology section—meticulously restructured with the inclusion of general virology, general parasitology and general mycology chapters.
General bacteriology is reorganized into a single chapter with several subchapters
+ — Sterilization and Disinfection chapter—completely revised based on hospital use of sterilizers and disinfectants; shifted to Hospital Infection Control section
+ COVID-19 chapter has been included which covers its laboratory diagnosis in detail
+ _AETCOM module is added that covers topics pertaining to confidentiality in disclosing laboratory reports and demonstration of respect for patient samples
+ Separate chapter for university practical examination showing the pattern of various universities
+ Images: >700 images, kept according to the demonstrations of practical class
+ — Like any other book of the authors; this book also has the same principle:
- More content, less pages—handy look, saves student's time
- Concise, bulleted format and to-the-point text—easy to read during MBBS examination
- Simple and lucid language—makes the understanding easy.

Apurba S Sastry MD (JIPMER) DNB MNAMS PocR has done his MBBS from MKCG Medical College, Berhampur, Odisha, India and residency from Jawaharlal Institute of
Postgraduate Medical Education and Research (JIPMER), Puducherry, India. He is currently working as an Associate Professor, Department of Microbiology in the
same institute. He is also the Hospital Infection Control Officer and Antimicrobial Stewardship Lead of JIPMER. He is the course coordinator of the most popular
HICC workshop of India— ‘National Workshop on Hospital Infection Control, conducted annually by HICC, JIPMER; in which more than 600 infection control
practitioners across India have been trained. He is a field expert in the core area of HICC and also is zestful in implementing antimicrobial stewardship program
in Indian scenario. He has developed ‘Clinical Microbiology Reporting Software’ based on CLS! and EUCAST guidelines, which will be revolutionary in
upgrading the diagnostic stewardship to the next level. He has guided and co-guided more than 40 student dissertations and contributed more than 40 research
publications. He is passionate about teaching and has been a popular teacher among the students. He has introduced many innovative teaching methodologies
at JIPMER such as pen tablet and clickers.

Sandhya Bhat (Gold medalist) MD DNB MNAMS PDCR Wife of Dr Apurba, has done her graduation from Father Muller Medical College, Mangaluru, Karnataka, India and
postgraduation from Sri Siddhartha Medical College, Tumkur, Karnataka, India. She was awarded FMC| President's Gold Medal Award for the best outgoing MBBS
graduate. Currently, she is working as Professor, Department of Microbiology, Pondicherry Institute of Medical Sciences (PIMS), Puducherry, India. She has
contributed more than 30 research publications. She has undergone training in medical education, NABH and NABL internal auditor's training course.

They are also authors of other popular books which are extremely appreciated among faculty and students:
1. Essentials of Medical Microbiology, 3rd edition for MBBS
2 Essentials of Hospital infection control, 1st edition (Co-authored with Dr Deepashree R)
3. Essentials of Medical Parasitology, 2nd edition for MBBS
4. Review of Microbiology and Immunology, 9th edition for PG entrance examination
5. Microbiology for FMGE, 2nd edition for foreign medical graduate entrance examination

lldlalisem lam larelie]

Available at all medical bookstores Shelving Recommendation

or buy online at www.jaypeebrothers.com oan ticactaaal
JAYPEE BROTHERS ISBN 978-81-948028-2-2
Medical Publishers (P) Ltd.
EMCA House, 23/23-B, Ansari Road,
Daryaganj, New Delhi - 110 002, INDIA
JAYPEE
www.jaypeebrothers.com.
Join us on facebook.com/JaypeeMedicalPublishers 9°? 88194"802822