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Microbiology
Essentials of
Microbiology
Surinder Kumar
MD DNB MNAMS
Director Professor
Department of Microbiology
Maulana Azad Medical College
New Delhi, India
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Essentials of Microbiology
First Edition: 2016
ISBN 978-93-5152-380-2
Printed at
Dedicated to
My father
Late Shri Lachhman Das
My mother
Late Smt Bal Kaur
My wife
Dr (Prof) Savita Kumari
and my sons
Dr Sourabh Kumar and Sanchit Kumar
whose love and affection make everything I do possible.
Preface
Microbiology is an extremely diverse discipline and can be a bewildering field to the novice. The
traditional books, seem to be exhaustive of microbiologic facts, are too voluminous and detailed to be
read by a typical medical student, who is trying to keep up with simultaneously several classes of other
subjects. One of the ways to fulfill the goals of the book is to concentrate on understanding concepts—
important fundamental ideas or themes that form a framework of the subject. Essentials of Microbiology
is the result of the author’s 28 years of experience in teaching medical students. The book is readable and
complete enough to meet the students’ needs. The overall objective of this book is to provide students
the basics of medical microbiology as well as a complete coverage of the subject. It contains all of the
information that is pertinent to the medical students who are studying microbiology keeping in mind
their examination.
Although the text has been designed to teach undergraduate and postgraguate medical students, it
would also serve as a review tool for the individuals who are taking medical examinations and persons
working in health-related professions, physicians, and infectious disease scientists.
Microbiology has expanded beyond recognition with various medical specialty, and it is not possible
for any one book to cover all aspects of medical microbiology in depth. This book is divided into
the following seven sections, based on the major disciplines included within microbiology: General
Bacteriology, Immunology, Systemic Bacteriology, Virology, Medical Mycology, Miscellaneous, and
Diagnostic Medical Microbiology. The chapters themselves are comprehensive yet free of unnecessary
detail and provide the reader with a framework for understanding. Mycology and parasitology have
continued to flourish and have blossomed into fields of study of their own rights. Therefore, parasitology
has not been included in this book which has a sturdy independence.
I would be thankful for any comments or suggestions from students, teachers, and all the readers of
this book for further improvements in its future editions.
Surinder Kumar
Acknowledgments
This book took years in writing but a lifetime in preparation. I would like to thank those whose example,
teaching, and prodding helped me develop a thirst for knowledge as well as methods for quenching
that thirst. I am greatly indebted to a number of my mentors, friends and colleagues who encouraged
me and gave valuable suggestions for improving the text. I am always indebted to my late brother Sant
Swaran Dev whose advice, guidance and true life philosophy gave me strength and courage to continue
my work in all odds. I would like to thank my family for patiently enduring the writing of this book,
which seemed at times to be an endless process. I am especially grateful to my wife Dr Savita Kumari,
Professor, Department of Internal Medicine, Post Graduate Institute and Medical Education and Research
(PGIMER), Chandigarh, for her support and encouragement and to my two sons, Dr Sourabh Kumar
and Mr Sanchit Kumar (medical student) who gave me their valuable moments without any complaint
for completing this book, so I could retain my sanity.
My special thanks is to Dr Sanjeev R Saigal, my PhD student and now my Research Associate Indian
Council of Medical Research (ICMR) for his constant help for this book. Sincere appreciation also goes
to all staff of M/s Jaypee Brothers Medical Publishers (P) Ltd., New Delhi, for their guidance and support
in this project.
Contents
Section 2: Immunology
12. Immunity 81
∙∙ Definition 81
∙∙ Classification 81
∙∙ Mechanisms of Innate Immunity 82
∙∙ Local Immunity 86
∙∙ Herd Immunity 86
13. Antigens 87
∙∙ Types of Antigens 87
∙∙ Antigenic Determinant or Epitome 87
∙∙ Determinants of Antigenicity 87
∙∙ Superantigens 89
14. Antibodies—Immunoglobulins 90
∙∙ Antibody Structure 90
∙∙ Immunoglobulin Classes 92
15. Complement System 96
∙∙ Principle Pathways of Complement Activation 96
∙∙ Quantitation of Complement (C) and its Components 99
∙∙ Biosynthesis of Complement 99
∙∙ Complement Deficiencies 99
16. Antigen–Antibody Reactions 101
∙∙ Antigen–Antibody Interactions 101
∙∙ General Characteristics of Antigen–Antibody Reactions 102
∙∙ Antigen and Antibody Measurement 102
∙∙ Parameters of Serological Tests 102
∙∙ Serological Reactions 102
∙∙ Types of Antigen and Antibody Reactions 102
∙∙ Applications of Agglutination Reaction 106
∙∙ ELISA 112
17. Structures and Functions of the Immune System 117
∙∙ Types of Immune Response 117
∙∙ Organs and Tissues of the Immune System 117
∙∙ Cells of the Lymphoreticular System 119
∙∙ Major Histocompatibility Complex 122
∙∙ Mhc Restriction 123
Contents | xiii
18. Immune Response 125
∙∙ Type of Immune Response 125
∙∙ Humoral Immunity 125
∙∙ Fate of Antigen in Tissues 126
∙∙ Production of Antibodies 127
∙∙ Cell-mediated Immune Responses 130
∙∙ Cytokines 131
∙∙ Immunological Tolerance 134
∙∙ Theories of Antibody formation 135
19. Immunodeficiency Diseases 137
∙∙ Classification of Immunodeficiency Diseases 137
∙∙ Primary Immunodeficiencies 137
∙∙ Disorders of Specific Immunity 137
∙∙ Disorders of Complement 140
∙∙ Disorders of Phagocyte 140
∙∙ Secondary Immunodeficiencies 140
20. Hypersensitivity Reactions 142
∙∙ Classification of Hypersensitivity Reactions 142
∙∙ Type I Hypersensitivity (IgE Dependent) 143
∙∙ Type II Hypersensitivity: Cytolytic and Cytotoxic 146
∙∙ Type III Hypersensitivity: Immune Complex-mediated 147
∙∙ Type IV Hypersensitivity—Delayed Hypersensitivity 148
∙∙ Type V: Hypersensitivity (Stimulatory Type) Jones–Mote Reaction or
Cutaneous Basophil Hypersensitivity 149
∙∙ Schwartzman Reaction 149
21. Autoimmunity 151
∙∙ Mechanisms of Autoimmunity 151
∙∙ Classification of Autoimmune Diseases 152
22. Immunology of Transplantation and Malignancy 154
∙∙ Definition 154
∙∙ Types of Transplants 154
∙∙ Allograft Reaction 154
∙∙ Histocompatibility Testing 155
∙∙ Fetus as an Allograft 156
∙∙ Graft-versus-host Reaction 156
∙∙ Immunology of Malignancy 156
∙∙ Tumor Antigens 156
∙∙ Immune Response in Malignancy 157
∙∙ Immunological Surveillance 157
∙∙ Immunotherapy of Cancer 157
23. Immunohematology 159
∙∙ Other Blood Group Systems 160
∙∙ Medical Applications of Blood Groups 160
∙∙ Complications of Blood Transfusion 160
∙∙ Hemolytic Disease of the Newborn 160
Section 4: Virology
53. General Properties of Viruses 378
∙∙ Main Properties of Viruses 378
∙∙ Morphology of Viruses 378
∙∙ Structure and Chemical Composition of the Viruses 379
∙∙ Susceptibility to Physical and Chemical Agents 380
∙∙ Viral Hemagglutination 381
∙∙ Viral Replication 381
∙∙ Eclipse Phase 383
∙∙ Abnormal Replicative Cycles 383
∙∙ Cultivation of Viruses 383
∙∙ Detection of Virus Growth in Cell Culture 385
∙∙ Viral Assay 386
∙∙ Viral Genetics 387
∙∙ Classification of Viruses 388
∙∙ Viroids 390
∙∙ Prions 390
54. Virus–Host Interactions: Viral Infections 392
∙∙ Interactions between Viruses and Host Cells 392
∙∙ Pathogenesis of Viral Diseases 393
∙∙ Transmission of Human Virus Infections 393
∙∙ Spread of Virus in the Body 394
∙∙ Significance of the Incubation Period 394
∙∙ Host Response to Virus Infections 395
55. Laboratory Diagnosis, Prophylaxis and Chemotherapy of Viral Diseases 397
∙∙ Laboratory Diagnosis of Viral Infections 397
∙∙ Immunoprophylaxis of Viral Diseases 399
∙∙ Chemoprophylaxis and Chemotherapy of Virus Diseases 400
56. Bacteriophages 402
∙∙ Role of Bacteriophages 402
∙∙ Morphology 402
∙∙ Life Cycle 402
∙∙ Significance of Phages 404
57. Poxviruses 406
∙∙ Morphology 406
∙∙ Physical and Chemical Properties 407
∙∙ Other Poxvirus Diseases 408
58. Herpesviruses 409
∙∙ Structure 409
∙∙ Classification 409
∙∙ Herpes Virus Simiae: B Virus 411
∙∙ Varicella-Zoster Virus 411
Contents | xvii
∙∙ Herpes Zoster (Shingles, Zona) 412
∙∙ Cytomegalovirus 413
∙∙ Epstein–Barr Virus 413
∙∙ Human Herpesviruses 6 (Hhv6) 415
∙∙ Human Herpesvirus 7 (Hhv7) 415
∙∙ Human Herpesvirus 8 (Hhv8) 416
59. Adenoviruses 418
∙∙ Morphology 418
∙∙ Resistance 418
∙∙ Pathogenesis 418
∙∙ Laboratory Diagnosis 419
∙∙ Treatment, Prevention and Control 420
60. Papovaviruses 421
∙∙ Papillomaviruses 421
∙∙ Polyomaviruses 422
61. Parvoviruses 424
∙∙ Parvovirus 424
∙∙ Dependovirus 424
∙∙ Erythrovirus 424
62. Picornaviruses 426
∙∙ Important Properties of Picornaviruses 426
∙∙ Enteroviruses 426
∙∙ Poliovirus 427
∙∙ Coxsackievirus 429
∙∙ Echoviruses 430
∙∙ Other Enterovirus Types 431
∙∙ Rhinoviruses 432
63. Orthomyxovirus 434
∙∙ Influenza Viruses 434
64. Paramyxoviruses 440
∙∙ Morphology and Structural Proteins of Paramyxoviruses 440
∙∙ Classification 440
∙∙ Parainfluenza Viruses 441
∙∙ Genus Rubulavirus 442
∙∙ Genus Morbillivirus 443
∙∙ Nipah and Hendra Viruses 444
∙∙ Genus Pneumovirus 444
∙∙ Metapnemovirus 445
65. Arboviruses 446
∙∙ Classification 446
∙∙ Properties 446
∙∙ Laboratory Diagnosis 447
∙∙ Pathogenesis 447
∙∙ Families of Arboviruses 447
∙∙ Ungrouped Arboviruses 455
∙∙ Arbovirus Known to Be Prevalent in India 455
66. Rhabdoviruses 457
∙∙ Rabies Virus 457
∙∙ Rabies-related Viruses 463
67. Hepatitis Viruses 465
∙∙ Hepatitis A Virus (Infectious Hepatitis) 465
∙∙ Hepatitis B Virus—Serum Hepatitis 467
∙∙ Hepatitis C Virus (HCV) 471
∙∙ Hepatitis D Virus (HDV) (Delta Agent) 472
∙∙ Hepatitis E Virus (HEV) 473
∙∙ Hepatitis G Virus 474
68. Retroviruses: Human Immunodeficiency Virus (HIV) 476
∙∙ Retroviruses 476
∙∙ Human Immunodeficiency Virus 476
xviii | Essentials of Microbiology
69. Slow Virus and Prion Diseases 488
∙∙ Characteristics of Slow Viruses 488
∙∙ Classification 488
70. Miscellaneous Viruses 492
∙∙ Rubivirus 492
∙∙ Rubella (German Measles) 492
∙∙ Viral Hemorrhagic Fevers 493
∙∙ Arenaviruses 493
∙∙ Filoviruses 494
∙∙ Coronaviruses 494
∙∙ Reoviridae 496
∙∙ Norwalk Virus 497
71. Oncogenic Viruses 499
∙∙ Properties of Cells Transformed by Viruses 499
∙∙ Types of Tumor Viruses 499
∙∙ Oncogenic Viruses 499
∙∙ Viruses Associated with Human Cancer 501
∙∙ Oncogenes 501
∙∙ Antioncogenes 502
∙∙ Mechanisms of Viral Oncogenesis 502
Section 6: Miscellaneous
77. Normal Microbial Flora of the Human Body 530
∙∙ Role of Normal Microbial Flora 530
78. Infective Syndrome 533
∙∙ Bacteremia, Septicemia and Infective Endocarditis 533
∙∙ Bacteremia and Septicemia 533
∙∙ Meningitis 535
∙∙ Purulent Meningitis (Acute Pyogenic Meningitis) 535
∙∙ Aseptic Meningitis 537
∙∙ Tuberculous Meningitis 538
Contents | xix
∙∙ Urinary Tract Infection 538
∙∙ Types of UTI 538
∙∙ Sore Throat 541
∙∙ Pneumonia 542
∙∙ Diarrhea and Dysentery 544
∙∙ Diarrhea 544
∙∙ Dysentery 546
∙∙ Food Poisoning 547
∙∙ Sexually Transmitted Diseases (STDs) 547
∙∙ Wound Infection 550
∙∙ Pyrexia of Unknown Origin (PUO) 551
79. Hospital-acquired Infection 555
∙∙ Sources of Infections 555
∙∙ Factors Influencing Hospital-associated Infections 555
∙∙ Microorganisms Causing Hospital Infection 555
∙∙ Routes of Transmission 556
∙∙ Common Hospital-acquired Infection 556
∙∙ Diagnosis and Control of Hospital Infection 557
∙∙ Infection Control Policy 557
∙∙ Prevention 557
80. Laboratory Control of Antimicrobial Therapy 559
∙∙ Antibiotic Sensitivity Tests 559
∙∙ Kirby–Bauer Disk Diffusion Method 560
∙∙ Stokes Disk Diffusion Method 561
∙∙ Dilution Methods 563
∙∙ Antibiotic Assays in Body Fluids 564
81. Antimicrobial Chemotherapy 565
∙∙ Antimicrobial Agent 565
∙∙ Antibiotic 565
∙∙ Chemotherapeutic Agents 565
∙∙ Antibacterial Agents 565
∙∙ Mechanisms of Action of Antibacterial Drugs 566
∙∙ Antibiotic Resistance 569
82. Immunoprophylaxis 571
∙∙ Vaccines 571
∙∙ Immunization 572
83. Bacteriology of Water, Milk and Air 575
∙∙ Bacteriology of Water 575
∙∙ Bacterial Flora in Water 575
∙∙ Factors Determining the Number of Bacteria in Water 575
∙∙ Water-borne Pathogens 576
∙∙ Collection of Water Samples 576
∙∙ Bacteriological Examination of Water 576
∙∙ Bacteriology of Milk 577
∙∙ Examination of Air 578
84. Hospital Waste Management 580
∙∙ Universal Precautions 580
∙∙ Definition of Biomedical Waste (BMW) 580
∙∙ Categories of Biomedical Waste 581
∙∙ Waste Segregation 581
∙∙ Treatment and Disposal Technologies for Healthcare Waste 581
∙∙ Disposal 582
85. Vehicles and Vectors 584
86. Emerging and Re-emerging Infectious Diseases 586
∙∙ Re-emerging, or Resurging Diseases 586
Index 615
Section 1: General Bacteriology
1
Chapter
Learning Objectives
Contd...
Microscopy
Learning Objectives
After reading and studying this chapter, you should ∙∙ E xplain the principles and describe the uses of the
be able to: following: darkfield microscopy; phase-contrast;
∙∙ Discuss microscopic methods microscopy; fluorescent microscopy and electron
microscopy.
Morphology of Bacteria
LEARNING OBJECTIVES
After reading and studying this chapter, you should be ∙∙ Discuss capsule or bacterial capsule
able to: ∙∙ Describe bacterial flagellae
∙∙ Differentiate between prokaryotes and eukaryotes ∙∙ Describe fimbriae or pili
∙∙ Describe anatomy of bacterial cell ∙∙ Discuss bacterial spores or endospores
∙∙ Describe cell envelope ∙∙ Explain L-forms of bacteria.
∙∙ Describe bacterial cell wall
Shape of Bacteria
Depending on their shape, bacteria are classified
into several varieties (Fig. 3.1):
1. Cocci: Cocci (from kokkos meaning berry) are
spherical, or nearly spherical.
2. Bacilli: Bacilli (from baculus meaning rod)
are relatively straight, rod-shaped (cylindrical)
cells. In some of the bacilli, the length of the
cells may be equal to the width. Such bacillary
forms are known as coccobacilli and have to be
carefully differentiated from cocci.
3. Vibrios: Vibrios are curved or comma-
shaped rods and derive the name from their
characteristic vibratory motility.
ii. Microdissection thick), elastic and can only be seen with electron
iii. Exposure to specific antibody microscope. It is a typical ‘unit membrane’,
iv. Mechanical rupture of the cell composed of phospholipids and proteins.
v. Differential staining procedures Chemically, the membrane consists of
vi. Electron microscopy. lipoprotein with small amounts of carbohydrate.
Enzymes that attack cell walls With the exception of Mycoplasma, bacterial
i. Lysozyme: The enzyme lysozyme, which is cytoplasmic membrane lacks sterols.
found in animal secretions (tears, saliva, nasal Functions of cytoplasmic membrane:
secretions) as well as in egg white, is a natural i. Semipermeable membrane: Controlling the
body defence substance which lyses bacteria inflow and outflow of metabolites to and from
of many species. the protoplasm.
ii. Autolysins: Bacteria themselves possess ii. Housing enzymes : Involved in outer
enzymes, called autolysins, able to hydrolyse membrane synthesis, cell wall synthesis,
their own cell wall substances. and in the assembly and secretion of extra
Protoplasts and spheroplasts cytoplasmic and extracellular substances.
Protoplasts iii. Housing many sensory and chemotaxis
These are derived from gram-positive bacteria. proteins
The gram-positive cell wall is almost completely iv. Generation of chemical energy (i.e. ATP)
destroyed by lysozyme. They contain cytoplasmic v. Cell motility
membrane and cell wall is totally lacking. Typically, vi. Mediation of chromosomal segregation
a protoplast is spherical and is still capable of carry during replication
ing on metabolism. Protoplasts cannot revert to
normal bacterial morphology.
b. Cellular Appendages
Spheroplasts I. Bacterial capsule or slime layer
When lysozyme is applied to gram-negative cells, Structure: Many bacteria synthesize large amount of
usually the wall is not destroyed to the same extent extracellular polymer in their natural environments.
as in gram-positive cells. Spheroplasts retain outer When the polymer forms a condensed, well-defined
membrane and entrapped peptidoglycan from layer closely surrounding the cell, it is called the
gram-negative cell. It differs from the protoplast in capsule as in the Pneumococcus. If the polymer
that some cell wall material is retained. It is also a is easily washed off and does not appear to be
spherical structure. They are capable of reverting associated with the cell in any definite fashion, it
to parent bacterial form when cell wall inhibitor is is referred to as a slime layer as in leuconostoc.
removed from the culture medium. A glycocalyx is a network of polysaccharide
2. Cytoplasmic (plasma) membrane extending from the surface of bacteria and other
Structure: The cytoplasmic (plasma) membrane cells. Capsules too thin to be seen under the light
limits the bacterial protoplast. It is thin (5–10 nm microscope are called microcapsules.
Fig. 3.7: Pneumococci negatively stained with India ink to Fig. 3.8: The structure of bacterial flagellum
show capsule
3. Mesosomes (Chondroids)
These are convoluted or multilaminated
membranous bodies formed as invaginations of
the plasma membrane into the cytoplasm. These
are of two types:
Fig. 3.9: Arrangement of flagella i. Septal mesosomes; ii. Lateral mesosomes.
Germination
Germination is the process of conversion of a spore
into vegetative cells under suitable conditions. It
occurs in three stages: activation, initiation and Fig. 3.12: Types of spores. 1. Central, bulging; 2. Central, not
outgrowth. Once activated, a spore will initiate bulging; 3. Subterminal, bulging; 4. Subterminal, not bulging;
germination if the environmental conditions are 5. Terminal, spherical; 6. Terminal, oval
favorable.
ii. Modified Ziehl-Neelsen (ZN) staini ng:
Shape and Position of the Spore Spores are slightly acid-fast. Ziehl-Neelsen
The shape and position of the spore and its size staining with 0.25–0.5% sulfuric acid (instead
relative to the parent cell are species’ characteristics. of 20% sulfuric acid as used in conventional
Spores may be central (equatorial), subterminal method) as decoloring agent is used for spore
(close to one end), or terminal (Fig. 3.12). The staining.
appearance may be spherical, ovoid or elongated,
and being narrower that the cell, or broader and Uses of Spores
bulging it. The diameter of spore may be same 1. Importance in food, industrial, and medical
or less than the width of bacteria (Bacillus), or microbiology
may be wider than the bacillary body producing a 2. Sterilization control: For proper sterilization,
distension or bulge in the cell (Clostridium). spores of certain species of bacteria are
e mp l oye d a s i n d i cat o r, e. g. B a cillus
Resistance stearothermophilus which is destroyed at a
These structures are extraordinary resistant to temperature of 121°C for 10–20 minutes. Proper
environmental stresses. Spores of all medically sterilization is indicated by the absence of the
important species are destroyed by autoclaving at spores after autoclaving.
120°C for 15 minutes. Endospore heat resistance 3. Research
probably is due to several factors: calcium-
dipicolinate and acid-soluble protein stabilization
PLEOMORPHISM AND INVOLUTION FORM
of DNA, protoplast dehydration, the spore coat,
DNA repair, the greater stability of the cell proteins During growth, bacteria of a single strain may show
in bacteria adapted to growth at high temperatures considerable variation in size and shape. This is
and others. known as pleomorphism and occurs most readily
in certain species. The abnormal cells are generally
Demonstration regarded as degenerate or involution forms.
i. Gram-staining: Spores appear as an unstained Pleomorphism and involution forms are often
refractile body within the cell. due to defective cell wall synthesis. Involution
Physiology of Bacteria
Learning Objectives
Learning Objectives
After reading and studying this chapter, you should be ∙∙ Describe filtration—uses and types
able to: ∙∙ Discuss types of radiations and their uses
∙∙ Define the following terms—sterilization, dis ∙∙ Give the mechanism of action for each type of
infection and antisepsis chemical agent commonly used in antiseptics and
∙∙ Describe various agents used in sterilization disinfectants
∙∙ Describe sterilization by moist heat ∙∙ Describe the following—aldehydes as disinfectants,
∙∙ Describe the different heat methods and their uses of formaldehyde and glutaraldehyde as
respective applications disinfectants
∙∙ Describe the following—pasteurization, tyndaliz ∙∙ Explain vapor-phase disinfectants or gaseous
ation or intermittent sterilization or fractional sterilization and discuss the role of ethylene oxide
sterilization, inspissation or serum inspissator, hot in sterilization of disposable items
air oven ∙∙ Describe various tests used for testing of
∙∙ Explain the principle and functioning of autoclave disinfectants.
Precautions
i. Air escape from the chamber : Since
temperature of air-steam mixture is lower than
that of pure steam, the air must be allowed to
Fig. 5.3: A simple autoclave escape from the chamber.
Chapter 5: Sterilization and Disinfection | 29
ii. Arrangement of the materials: This should been used widely for purification of water for
be done in such a manner which ensures free industrial and drinking purposes. They are of
circulation of steam inside the chamber. two types:
Uses a. Unglazed ceramic filters, e.g. Chamberland,
i. For sterilizing culture media and other and Doulton filters.
laboratory supplies, aqueous solutions, b. Compressed diatomaceous earth filters,
rubber material, dressing materials, gowns, e.g. the Berkefeld and Mandler filters.
dressing, linen, gloves, instruments and ii. Asbestos filters (Seitz filter): They are made
pharmaceutical products. up of a disc of asbestos (magnesium trisilicate).
ii. For all materials that are water-containing, It is supported on a perforated metal disc
permeable or wettable and not liable to be within a metal funnel. It is then fitted on to a
damaged by the process. sterile flask through a silicone rubber bung.
iii. Particularly useful for materials which cannot Sterilized fluid is collected from the flask and
withstand the higher temperature of hot air filter disc is discarded after use. These discs are
oven. available with different grades of porosity.
Examples: Seitz filter, Carlson and Sterimat
Sterilization Controls filters.
A. Biological control (Bacterial spores): iii. Sintered glass filters: They are prepared
An envelope containing a filter paper strip by size grading powdered glass followed by
impregnated with 10 6 spores of Bacillus heating.
stearothermophilus is placed with the iv. Membrane filters: Membrane filters consist
load sterilization is over, the strip is removed of a variety of polymeric materials such
and inoculated into. No growth of B. stea as cellulose nitrate, cellulose diacetate,
rothermophilus indicates proper sterilization. polycarbonate and polyester. They are
Spores of this organism withstand 121°C for up manufactured as discs from 13 to 293 mm
to 12 minutes and this has made the organism diameter and with porosities from 0.015 to
ideal for testing autoclaves. 12 mm. They come in a wide range of average
B. Chemical control: A Browne’s tube containing pore diameters (APD), the 0.22 mm size being
red solution changes to green when exposed most widely used for sterilization because the
to temperature of 121°C for 15 minutes in pore size is smaller than that of bacteria.
autoclave. It indicates proper sterilization.
Uses
C. Autoclave tapes
1. They are used routinely in water analysis
D. Thermocouples: These may also be used which
and purification.
record the temperature by a potentiometer.
2. Sterilization and sterility testing.
4. Filtration 3. For preparing sterile solutions for
Filtration is the principal method used in the parenteral use.
laboratory for the sterilization of heat labile 4. Bacterial counts of water:
materials, e.g. sera, solutions of sugars or antibiotics v. Syringe filters: Membrane of different
used for the preparation of culture media. diameters are commonly fitted in syringe-like
holders of stainless steel or polycarbonate.
Uses
For sterilization, the fluid is forced through
1. Heat-sensitive solutions: For sterilization of
the disc (membrane) by pressing the piston
pharmaceuticals, ophthalmic solutions, culture
of the syringe.
media, oils, antibiotics and other heat-sensitive
vi. Vacuum and ‘in-line’ filters: They are
solutions.
suitable for the sterilization or disinfection of
2. For separation of bacteriophages and
large volumes of liquid or air.
bacterial toxins from bacteria.
3. Isolation of organisms which are scanty in vii. Pressure filtration: It may be used for the pro
fluids. duction of very pure water for laboratory use.
4. Concentration of bacteria from liquids, e.g. viii. Air filters: They are widely used in air
in testing water samples for cholera vibrios or filtration.
typhoid bacilli.
5. For virus isolation. 5. Radiation
Two types of radiations are used:
Types of Filters I. Non-ionizing radiations : Infrared and
i. Earthware filters: These are manufactured in ultraviolet rays are of non-ionizing type.
several different grades of porosity and have The effectiveness of UV light as a lethal and
30 | Section 1: General Bacteriology
mutagenic agent is closely correlated with its Factors that determine the potency of dis
wavelength. The most effective bactericidal infectants
wavelength is in the 240–280 nm range, with the 1. The concentration and stability of the agent
optimum being about 260 nm, the wavelength 2. Nature of the organism
most effectively absorbed by DNA and this 3. Time of action
interfers with DNA replication. 4. pH
Microbial sensitivity to UV radiation 5. Temperature
i. Bacterial spores are generally more resistant 6. The presence of organic (especially protein) or
to UV light than are vegetative cells. other interfering substances
ii. Viruses are also inactivated. 7. Nature of the item to be disinfected.
iii. To disinfect drinking water.
iv. Disinfection of enclosed areas such Categories of Disinfectants
as entryways, hospital wards, operating Disinfection processes have been categorized as
theatres, laboratories and in ventilated high level, intermediate level and low level.
safety cabinets in which dangerous 1. High-level disinfection: High-level disinfections
microorganisms are being handled. can generally approach sterilization in
II. Ionizing radiations: These include X-rays, g effectiveness, whereas spore forms can survive
(gamma) rays and cosmic rays. These have very intermediate-level disinfection, and many
high penetrative power and are highly lethal to microbes can remain viable when exposed to
all cells including bacteria. Ionizing radiations low-level disinfection.
damage the DNA by various mechanisms. High-level disinfectants are used for items
involved with invasive procedures that cannot
Applications withstand sterilization procedures (e.g. certain
i. For sterilization in pharmacy and medicine. types of endoscopes, surgical instruments with
ii. Sterilization of packaged disposable plastic or other components that cannot be
articles such as plastic syringes, intravenous autoclaved).
lines, catheters and gloves that are unable to
withstand heat. Since there is no appreciable Examples:
increase in temperature in this method, it is i. Treatment with moist heat.
known as cold sterilization. ii. Use of liquids such as glutaraldehyde,
iii. Use for antibiotics, hormones, sutures, and hydrogen peroxide, peracetic acid, chlorine
vaccines and to prevent food spoilage. dioxide, and other chlorine compounds.
2. Intermediate-level disinfectants: Intermediate-
B. Chemical Agents level disinfectants are used to clean surfaces
Germicidal chemicals can be used to disinfect and, or instruments in which contamination with
in some cases, sterilize. bacterial spores and other highly resilient
organisms is unlikely. These include flexible
Characteristics of a Disinfectant fiberoptic endoscopes, laryngoscopes, vaginal
An ideal antiseptic or disinfectant should: specula, anesthesia breathing circuits, and other
∙∙ Have a wide spectrum of activity and must be items. These have been referred to as semi
effective against a wide variety of infectious critical instruments and devices.
agents (Gram-positive and gram-negative Examples: Alcohols, iod ophor compounds,
bacteria, acid-fast ; bacteria, bacterial phenolic compounds.
endospores, fungi, and viruses) 3. Low-level disinfectants: Low-level disinfectants
∙∙ Be active at high dilutions and in the presence are used to treat noncritical instruments
of organic matter; and devices such as blood pressure cuffs,
∙∙ Be effective in acid as well as alkaline media; electrocardiogram electrodes and stethoscopes.
∙∙ Have speedy action; They do not penetrate through mucosal surfaces
∙∙ Have high penetrating power; or into sterile tissues, although these items come
∙∙ Be stable; into contact with patients.
∙∙ Be compatible with other antiseptics and
disinfectants; Examples: Quaternary ammonium compounds.
∙∙ Not corrode metals;
∙∙ Not cause local irritation or sensitization; Mechanisms of Antimicrobial Action
∙∙ Not interfere with healing; The main modes of action are follows:
∙∙ Not be toxic if absorbed into circulation; A. Agents that damage the cell membrane
∙∙ Be cheap and easily available; 1. Surface active disinfectants
∙∙ Be safe and easy to use. 2. Phenolic compounds
Such an ideal chemical is yet to be found. 3. Alcohols
Chapter 5: Sterilization and Disinfection | 31
B. Agents that denature proteins active at acid pH and are active against gram-
1. Acids and alkalies positive organisms but are relatively ineffective
2. Alcohols against gram-negative species.
C. Agents that modify functional groups of c. Ampholytic (amphoteric) compounds:
proteins and nucleic acids: Known as ‘Tego’ compounds, these possess
1. Heavy metals and their compounds detergent properties of anionic and antimi
2. Oxidizing agents—Halogens crobial activity of cationic compounds. They
– Hydrogen peroxide are active over a wide range of pH but organic
3. Dyes—Aniline dyes matter markedly reduces their activity.
– Acridine dyes Uses: They are effective against a wide range
4. Alkylating agents—Aldehydes-formal of gram-positive and gram-negative organisms
dehyde, glutaraldehyde and some viruses at a concentration of 1% in
– Ethylene oxide water.
2. Phenols and phenolics
A. Agents that Damage the Cell Membrane Phenol: Phenols are obtained by distillation of coal
1. Surface-active agents tar between temperatures of 170°C and 270°C. It
Substances that alter the energy relationships is now rarely used as an antiseptic or disinfectant
at interfaces, producing a reduction of surface because it irritates the skin and has a disagreeable
or interfacial tension, are referred to as surface- odor. Phenol is bactericidal at a concentration of
active agents or surfactants. They possess both 1%.
hydrophobic (water-repelling) and hydrophilic Phenolics: Derivatives of phenol are called
(water-attracting) groups. phenolics.
Classification: These surfactants are classified Phenol derivatives: Certain phenol derivatives
into anionic, cationic, nonionic and ampholytic like cresol, chlorhexidine, chloroxylenol
(amphoteric). Of these, the cationic and anionic and hexachlorophane are commonly used as
compounds have been the most useful antibacterial antiseptics.
agents. i. Cresols: Cresols, obtained industrially by the
a. Cationic agents: These act on phosphate distillation of coal tar, are emulsified with green
groups of cell membrane phospholipids soap and sold under the trade names of Lysol
and also enter the cell. This leads to loss of and Creolin. ‘White fluids’ such as Lysol are
membrane semipermeability and leakage from effective but are irritant to the skin. They are
the cell of nitrogen and phosphorus-containing active against a wide range of organisms. They
compounds. The agent which enters the cell are most commonly used for sterilization of
denatures its proteins. Quaternary ammonium infected glasswares, cleaning floors, disinfection
compounds (quats) are the most important of excreta. They are not readily inactivated by
cationic compounds. Examples of quaternary the presence of organic matter.
ammonium compounds include cetrimide ii. Chlorhexidine: Chlorhexidine is a member
(cetavalon), benzalkonium chloride (Zephiran, of the biguanide group with a broad spectrum
a brand name) and cetylpyrimidium chloride of activity. They are more active against
(Cepacol, a brand name). Antimicrobial activity gram-positive than gram-negative bacteria.
is affected greatly by organic matter and by pH, They are biocidal against most vegetative
most active at alkaline pH and acid inactivates bacteria and fungi; mycobacteria are relatively
them. They are inactivated by hard water and resistant; endospores and protozoan cysts
soap. are not affected. The only viruses affected are
Uses certain enveloped (lipophilic) types.
i. They are primarily active against gram- Uses: Savlon (chlorhexidine and cetrimide)
positive, non-sporing bacteria; lethal is widely used in wounds, preoperative
to gram-negative organisms at high disinfection of skin, as bladder irrigant, etc.
concentrations. However, contact with the eyes can cause
ii. They are also fungistatic and active against damage.
viruses with lipid envelopes (e.g. herpes iii. Chloroxylenol: It is an active ingredient of
and influenza) and much less against dettol. It is less toxic and less irritant.
nonenveloped viruses (e.g. enteroviruses). iv. H e x a c h l o r o p h a n e : G r a m - p o s i t i v e
b. Anionic agents: These include soap and fatty staphylococci and streptococci, which
acids. Anionic surfactants such as common can cause skin infections in newborns, are
soaps usually have strong detergent but weak particularly susceptible to hexachlorophane.
antimicrobial properties. These agents are most So it is used notably for prophylaxis against
32 | Section 1: General Bacteriology
staphylococcal infection in nurseries. However, a. Iodine: Iodine compounds are the
it can cause neurotoxicity (brain damage), most effective halogens available for
especially in infants, and its use is now severely disinfection. It is actively bactericidal, with
restricted. moderate action against spores. It is active
against the tubercle bacteria and viruses.
B. Agents that Denature Proteins Uses
Acids and alkalies: Many aliphatic and aromatic i. Skin disinfectant: Iodine in aqueous and
acids are employed as preservatives, especially alcoholic solution has been used widely
in the food industry, and, to some extent, in as a skin disinfectant. Iodine often has
pharmaceutical and cosmetic products. They are been applied as tincture of iodine, 2% or
not sporicidal. more iodine in a water-ethanol solution of
potassium iodide.
Alcohols ii. Iodophors: Mixtures of iodine with various
Ethyl alcohol (ethanol) and isopropyl alcohol surface-active agents that act as carriers
are the most frequently used. They rapidly kill for iodine are known as iodophors (iodo,
bacteria including tubercle bacilli but they have ‘iodine’; phor ‘carrier’). Povidine-iodine
no action on spores and viruses. However, human (Betadine) for wounds and Wescodyne for
immunodeficiency virus (HIV) is susceptible to skin and laboratory disinfection are some
ethyl alcohol (70%) and isopropyl alcohol (35%) in popular brands.
the absence of organic matter. They must be used at b. Chlorine: In addition to chlorine itself, there
a concentration of 60 to 70% in water to be effective. are three types of chlorine compounds—
They are most frequently used as skin disinfectants hypochlorites and inorganic and organic
and act by denaturing bacterial proteins. chloramines. The disinfectant action
Isopropyl alcohol is preferred. of all chlorine compounds is due to
Methyl alcohol is effective against fungal the liberation of free chlorine. When
spores and is used for treating cabinets and elemental chlorine or hypochlorites are
incubators affected by them. Methyl alcohol added to water, the chlorine reacts with
vapour is toxic and inflammable. water to form hypochlorous acid (HOCL),
which in neutral or acidic solution is a
C. Agents that Modify Functional Groups of strong oxidizing agent and an effective
Proteins and Nucleic Acids disinfectant.
1. Heavy metals: For many years the ions of heavy The activity of chlorine is markedly
metals such as mercury, silver, arsenic, zinc influenced by the presence of organic
and copper were used as germicides. matter.
i. Mercuric chloride: It is very toxic and at Uses: The usual disinfectant for water
present has limited use supplies, swimming pools, dairy product
ii. Silver nitrate: The most commonly and food industries.
employed of the silver salts. c. Hypochlorites: The hypochlorites have
Use a bactericidal, fungicidal, virucidal and
1. Highly bactericidal for the gonococcus. rapidly sporicidal action.
2. Routinely used for the prophylaxis of i. Bleaching powder or hypochlorite
the ophthalmic neonatorum in newborn solution is most widely used for human
infants in a 1% solution. immunodeficiency virus (HIV) infected
3. To prevent infection of burns. material. Hypochlorite solution decays
iii. Copper derivatives are used as algicides, rapidly and should be prepared daily.
fungicides, wood, paint, cellulose and Chloramines are used as antiseptics for
fabric preservation. dressing wounds.
ii. Hydrogen peroxide: It is used to
2. Oxidizing Agents disinfect plastic implants, contact lenses
The most useful antimicrobial agents in this group and surgical prostheses.
are the halogens and hydrogen peroxide. They
inactivate enzymes by converting functional-SH 3. Dyes
groups to the oxidized S-S form. Aniline dyes and acridine dyes are two groups of
i. Halogens: Chlorine and iodine are among dyes which are used extensively as skin and wound
our most useful disinfectants. They are antiseptic. Both are bacteriostatic in high dilution
bactericidal and sporicidal. They are active but are of low bactericidal activity.
in very high dilutions and their action is very i. Aniline dyes: Of the aniline dyes, derivatives
rapid. of triphenylmethane, especially brilliant
Chapter 5: Sterilization and Disinfection | 33
green, malachite green and crystal violet Vapor-phase disinfectants
have many uses. 1. Ethylene oxide: This is a colorless liquid
They are highly selective for gram-positive with a boiling point of l0.7°C and is a highly
than against gram-negative organisms and penetrating gas with a sweet ethereal smell. It
have been used in the laboratory in the is highly inflammable and in concentrations
formulation of selective culture media. They in air greater than 3%, highly explosive. It is
have no activity against tubercle bacilli, and, unsuitable for fumigating rooms because of its
hence, the use of malachite green in the explosive property.
Lowenstein-Jenson medium. It is highly lethal to all kinds of microbes
ii. Acridine dyes: They are more active against including spores and tubercle bacilli.
gram-positive organisms than against gram- Uses
negative but are not as selective as the aniline a. Sterilization of articles liable to be
dyes. The more important dyes are proflavine, damaged by heat: It is specially used for
acriflavine, euflavine and aninacrine. They sterilizing heart-lung machines, respirators,
show no significant differences in potency. sutures, dental equipment, books and
clothing.
4. Alkylating Agents
b. Sterilization of a wide range of materials
i. Formaldehyde: Formaldehyde is lethal to
such as glass, metal and paper surfaces,
bacteria and their spores, viruses and fungi.
clothing, plastics, soil, some foods and
It is employed in the liquid and vapor states.
tobacco.
Formaldehyde is commercially available in
Disadvantages of ethylene oxide
aqueous solutions containing 37% formaldehyde
i. It is irritant, and personnel working with it
(formalin) or as paraformaldehyde, a solid
have to take strict precautions.
polymer that contains 91% to 99 % formaldehyde.
ii. Its use as a disinfectant presents a potential
Uses
toxicity to human beings, including
a. Formalin
mutagenicity and carcinogenicity. Baci
i. Used for preserving fresh tissues for
llus globigi, a red-pigmented variant of
histological examination and is the
B. subtilis, has been used to test ethylene
major component of embalming fluids.
oxide sterilizers.
ii. Used extensively to inactivate viruses in
2. Formaldehyde gas: It is used for fumigation of
the preparation of vaccines.
complex heat-sensitive equipment, including
b. Formaldehyde
anaesthetic machine and baby incubators and
i. Used for preser ving anatomical
for periodic decontamination of laboratory safety
specimens.
cabinets.
ii. Used for destroying anthrax spores in
Fumigation of operation theaters and other
hair.
rooms: This is also widely employed for
iii. Used as an antiseptic mouthwash.
fumigation of operation theaters and other
iv. Used for the disinfection of membranes
rooms (such as isolation rooms). After sealing
in dialysis equipment.
the windows and other outlets, formaldehyde
v. Used as a preservative in hair shampoos.
gas is generated by adding KMnO4 to formalin.
ii. Glutaraldehyde: This has an action similar The reaction produces considerable heat, and
to formaldehyde. It has a broad-spectrum so heat-resistant vessels should be used. After
action against vegetative bacteria including starting generation of formaldehyde vapour, the
mycobacteria, fungi and viruses, but acts more doors should be sealed and left unopened for
slowly against spores. It is more active and 48 hours.
less toxic than formaldehyde. It is used as 2% Formaldehyde has an extremely unpleasant
buffered solution. It can be used for delicate odor and is irritant to mucus membranes.
instruments having lenses. It is available 3. B e ta p r o p i o l a c t o n e ( B P L ) : T h i s i s a
commercially as ‘cidex’. condensation product of ketane and formal
Uses dehyde with a boiling point of 163°C. It also
i. Cold sterilant: It has been used increasingly destroys microorganisms more readily than
as a cold sterilant for surgical instruments ethylene oxide but does not penetrate materials
and endoscopes such as cystoscopes, well and may be carcinogenic. For sterilization
endoscopes and bronchosope. of biological products, 0.2% BPL is used. It is
ii. Used safely to sterilize corrugated rubber capable of killing all micro-organisms and is
anesthetic tubes and face-masks, plastic very active against viruses.
endotracheal tubes, metal instruments and Use: In the liquid form, it has been used to
polythene tubing. sterilize vaccines and sera.
34 | Section 1: General Bacteriology
RECOMMENDED CONCENTRATIONS OF Table 5.3 Various procedures of sterilization/
VARIOUS DISINFECTANTS disinfection of some important materials
Culture Media
Learning Objectives
After reading and studying this chapter, you should be ∙∙ Differentiate between the following: enriched
able to: media and enrichment media; indicator media and
∙∙ Describe classification of media differential media; selective media and differential
media with suitable examples.
v. Differential Media
A medium, which has substances incorporated in
it, enabling it to bring out differing characteristics
of bacteria and thus helping to distinguish between
them, is called a differential medium. Fig. 6.6: MacConkey agar
Example
MacConkey agar (Fig. 6.6): MacConkey agar is
both differential and selective. It contains peptone,
meat extract, NaCl, bile salt, lactose and neutral
red indicator.
Lactose fermenters (LF) form pink or red color
colonies and nonlactose fermenters (NLF) form
colorless or pale colonies.
Culture Methods
Learning Objectives
After reading and studying this chapter, you should be ∙∙ Explain the principle and describe uses of the
able to: following: Mclntosh and Fildes anaerobic jar;
∙∙ Discuss anaerobic culture methods cooked meat broth (CMB).
Introduction
In the clinical laboratory, the indications for
culture are mainly to:
1. Isolate bacteria in pure culture
2. Demonstrate their properties
3. Obtain sufficient growth for preparation of
antigens and for other tests.
4. Type isolates
5. Determine sensitivity to antibiotics
6. Estimate viable counts
7. Maintain stock cultures.
Identification of Bacteria
LEARNING OBJECTIVES
After reading and studying this chapter, you should reduction test; 7. Urease test; 8. Catalase production;
be able to: 9. Oxidase test; 10. Phenylalanine deaminase test;
∙∙ Explain principle and discuss interpretation of 11. Hydrogen sulfide production; 12. Triple-sugar
the following biochemical reactions: 1. Sugar Iron (TSI)
fermentation; 2. Indole production; 3. Methyl ∙∙ List various examples of bacteria giving positive
red (MR) test; 4. Voges-Proskauer (VP) test for biochemical reactions for mentioned above.
acetoin production; 5. Citrate utilization; 6. Nitrate
METHODS USED TO IDENTIFY BACTERIA nature of the culture medium, temperature and
time of incubation, age of the culture and the
Once a bacterium has been obtained in pure culture, number of subcultures it has undergone. The
it has to be identified. Identification schemes are characteristics noted are shape, size, side ends,
classified into one of the two categories (Table 8.1): arrangement, and irregular forms, motility, flagella
1. Phenotypic characteristics fimbriae, spores, capsule and staining.
2. Genotypic characteristics.
B. Staining Reactions
1. PHENOTYPIC CHARACTERISTICS
A number of staining techniques for the identifi
A. Microscopic Morphology cation of bacteria, are available. Of these, Gram
The morphology of the bacterium depends on stain and Ziehl-Neelson stain are most important.
a number of factors, such as the strain studied, Gram stain: The Gram stain divides bacteria into
gram-positive and gram-negative.
Table 8.1 Methods used to identify bacteria
Ziehl-Neelsen staining: With Ziehl-Neelsen stain
Phenotypic characteristics Genotypic characteristics ing divides bacteria into acid fast and non-acid fast.
A. Microscopic Nucleic acid hybridization Numerous other stains are used for special
morphology PCR-amplifying specific purposes, such as demonstration of flagella,
B. Staining reactions DNA sequences capsule, spores, and metachromatic granules. The
C. Metabolism differences Sequencing rRNA genes
fluorescent antibody technique enables one to
i. Macroscopic
morphology identify them according to their surface antigens.
or cultural
characteristics C. Metabolic Differences
ii. Fermentation and
The requirements of oxygen, the need for carbon
other biochemical
reactions dioxide, the capacity to form pigments, and the
D. Serology production of hemolysis help in classification.
E. Antibiotic tolerance
(resistance) tests, dye i. Cultural Characterstics or Macroscopic
tolerance, and other Morphology
inhibition tests
F. Bacteriophage and These provide additional information for the
bacteriocin typing identification of the bacterium. The characteristics
G. Pathogenicity revealed in different types of media are noted.
2. Indole Production
Principle: This test demonstrates the ability of
certain bacteria to decompose the amino acid
tryptophane to indole, which accumulates in the
medium. Tryptophan is decomposed by an enzyme
tryptophanase produced by certain bacteria.
Fig. 8.5: Indole test
Method: Indole production is detected by
inoculating the test bacterium into peptone water
(tryptophan rich) and incubating it at 37°C for 48–96
hours. Add 0.5 mL Kovac’s reagent and shake gently.
Tryptophan Tryptophanase
→ Indole
Kovac’s reagent consists of:
∙∙ Paradimethylaminobenzaldehyde 10 g
∙∙ Amyl or isoamyl alcohol 150 mL
∙∙ Concentrated hydrochloric acid 50 mL.
Fig. 8.7: Voges-Proskauer (VP) test Fig. 8.8: Citrate utilization test
Bacterial Taxonomy
Learning Objectives
After reading and studying this chapter, you should be able to:
∙∙ Discuss bacterial classifications.
Bacterial Genetics
Learning Objectives
After reading and studying this chapter, you should and conjugation in the transfer of genetic material
be able to: from one bacterium to another
∙∙ Explain Lac operon ∙∙ Discuss resistance transfer factor (RTF)
∙∙ Discuss regulation or control of gene expression ∙∙ Differentiate between mutational and plasmid-
mediated drug resistance
∙∙ Discuss structure and functions of the plasmids
∙∙ Discuss transposons
∙∙ Describe mutations ∙∙ Describe principle and clinical applications
∙∙ Discuss various methods of gene transfer of the following: Nucleic acid probes; genetic
∙∙ Differentiate among the mechanisms of trans engineering; polymerase chain reaction
formation, transduction, lysogenic conversion ∙∙ Describe gene therapy.
A. Phenotypic Variation
The phenotype (‘Phaeno’; display) is the physical
expression of various characters by bacterial cells
in a given environment. These properties are
determined not only by its genome (genotype), Fig. 10.4: Frameshift mutation
but also by its environment. Phenotypic variations
are reversible. Also important, but not part of the operon, is a
distant regulatory gene (in this case is LacI) which
codes for a repressor protein. It is a protein molecule
Examples of Environmental Influence on
which can combine with either operator region on
Bacteria
the chromosome or with the inducer (lactose).
1. Synthesis of flagella: The typhoid bacillus is
normally flagellated. But the flagella are not Effect of Lactose on the Control of the Lactose
synthesized when grown in phenol agar and is Operon
reversed when subcultured from phenol agar For transcription to occur as the first stage in
into broth. protein synthesis, RNA polymerase has to attach to
2. Synthesis of enzyme: Another example of DNA at a specific promoter region and transcribe
environmental influence is the synthesis the DNA in a fixed direction. In resting stage when
by Escherichia coli of the enzyme beta- lactose (inducer) is not present in the medium,
g a l a c t o s i d a s e, n e c e s s a r y f o r l a c t o s e repressor molecule is bound to the operator,
fermentation but the actual synthesis takes preventing the passage of RNA polymerase from
place only when it is grown in a medium promoter to the structural genes. The repressor
containing lactose. molecule has an affinity for lactose, in the presence
Such enzymes which are synthesized only of which it leaves the operator region free enabling
when induced by the substrate are called the transcription to take place. When lactose
induced enzymes. The enzymes which are present is completely metabolized, the repressor
synthesized irrespective of the presence or again attac hes to the operator, switching off
absence of the substrate are called constitutive transcription. Lactose acts both as an inducer and
enzymes. the substrate for the enzyme.
Infection
Learning Objectives
After reading and studying this chapter, you should ∙∙ Name and define various types of carriers
be able to: ∙∙ Discuss modes of spread of infection giving suitable
∙∙ Define the terms saprophytes, parasite, commensal, examples
pathogen ∙∙ List the differences between exotoxins and
∙∙ Describe classification of infections endotoxins.
∙∙ Define and differentiate primary, secondary,
opportunistic and reinfections
Adhesins: The initial event in the pathogenesis is 9. Highly antigenic 9. Weakly antigenic
the attachment of the bacteria to body surfaces. 10. Action specifically neu 10. Neutralization by
This attachment is not a chance event but a specific tralized by antibody antibody ineffective
reaction between surface receptors on host cells 11. Usually do not produce 11. Usually produce
and adhesive structures (ligands) on the surface fever fever by release of
interleukin-1
of bacteria. These adhesive structures are called
adhesins. 12. Produced by both 12. Produced by gram-
gram-positive bacteria negative bacteria only
Adhesions may occur as organized structures, and gram-negative
such as fimbriae or fibrillae and pilli, or as bacteria
colonization factors. 13. Frequently controlled 13. Synthesized directly by
Adhesins as virulence factors: Adhesins are by extrachromosomal chromosomal genes
usually made of protein and are antigenic in nature. genes (e.g. plasmids)
Specific immunization with adhesins has been 14. Disease examples- 14. Gram-negative
attempted as a method of prophylaxis in some Botulism diphtheria infections,
tetanus meningococcemia
infections.
Chapter 11: Infection | 79
in minute amounts and include some of the most d. Antigenic Variation
poisonous substances known. Variation in surface antigen composition during
Treatment with formaldehyde converts the course of infection provides a mechanism of
exotoxin into toxoids, are thus useful in preparing avoidance of specific immune responses directed
vaccines. at those antigens.
They exhibit specific tissue affinity and Examples: (i) Pathogenic Neisseria; (ii) The borreliae
pharmacological activities. generate antigenic variation.
They are associated with specific diseases and
have specific mechanisms of action.
e. Serum Resistance
They are easily inactivated by formaldehyde,
iodine, and other chemicals to form immunogenic To survive in the blood, bacteria must be able to
toxoids. resist lysis.
Exotoxins are generally formed by gram-
positive bacteria but may also be produced by some f. Siderophore and Iron Acquisition
gram-negative organisms such as Shiga’s dysentery Siderophores can acquire iron from the host’s iron
bacillus, cholera vibrio and enterotoxigenic E. coli. binding proteins.
b. Endotoxins 6. Enzymes
These are heat-stable, lipopolysaccharide (LPS) Many species of bacteria produce tissue-degrading
components of the outer membranes of gram- enzymes that play important roles in the infection
negative. Their toxicity depends upon the the process.
component (lipid A). i. Coagulase: Coagulase is produced by
They are released into the host’s circulation Staphylococcus. aureus. This thrombin-like
following bacterial cell lysis. enzyme prevents phagocytosis by forming a
They are toxic only at high doses. fibrin barrier around the bacteria and walling
They cannot be toxoided. off the lesion.
They are poor antigens and weakly immunogenic.
ii. Lecithinase-C and collagenase: Clostridiun.
They do not exhibit specific pharmacological
perfringens produces lecithinase-C and
activities.
collagenase promoting spread of infection in
Intravenous injections of large doses of
tissue.
endotoxin and massive gram-negative septicemias
cause endotoxic shock marked by fever, leukopenia, iii. Hyaluronidases: Hyaluronidases split hyalu
thrombocytopenia, siignificant fall in blood ronic acid and thus facilitate the spread of
pressure, circulatory collapse and bloody diarrhea infection along tissue spaces, e.g. Streptococcus.
leading to death (Table 11.1). iv. Streptokinase (fibrinolysin): Many haemolytic
streptococci produce streptokinase (fibri
5. Avoidance of Host Defence nolysin) which promotes the spread of
Mechanisms infections.
v. Cytolysins: These include hemolysins capable
a. Capsules of destroying erythrocytes and leukocidins
Some bacteria such as Streptococcus pneumoniae, damage polymorphonuclear leukocytes.
Neisseria meningitidis, and Haemophilus influenzae vi. IgA 1 proteases: These enzymes specifically
can produce a slippery mucoid capsule that cleave immunoglobulin IgA which protects
prevents the phagocyte from effectively contacting at mucosal surfaces.
the bacterium.
12
Chapter
Immunity
Learning Objectives
After reading and studying this chapter, you should ∙∙ Differentiate between active and passive immunity.
be able to:
∙∙ Describe innate immunity, artificial active immunity,
natural passive immunity, herd immunity
Antigens
Learning Objectives
Antigens: Antigens (antibody generators) are the 2. Complex haptens: Complex haptens can
substances that can stimulate an immune response precipitate with specific antibodies. Complex
and, given the opportunity, react specifically by haptens are polyvalent.
binding with the effector molecules (antibodies)
and effector cells (lymphocytes). Antigenic determinant or epitome
The smallest unit of antigenicity is known as the
Types of antigens antigenic determinant or epitope. Each antigen
can have several antigenic determinant sites or
Based on the ability of antigens to carry out these two
epitopes. The combining area on the antibody
functions, they may be classified into different types:
molecule, corresponding to the epitope, is called
the paratope.
A. Complete Antigen
Complete antigen is able to induce antibody Valence: The number of antigenic determinant
formation and produce a specific and observable sites on the surface of an antigen is its valence. The
reaction with the antibody so produced. antigen is monovalent or multivalent.
Antibodies—Immunoglobulins
Learning Objectives
After reading and studying this chapter, you should ∙∙ Describe structure and functions of IgG, IgA and
be able to: IgM
∙∙ Define antibody and draw labeled diagram of ∙∙ Discuss properties of IgM, IgG, IgA, IgD and IgE
immunoglobulin ∙∙ Draw labeled diagram of IgG, IgM and IgA.
ANTIBODY STRUCTURE
Porter, Edelman, Nisonoff and their colleagues Fig. 14.1: Basic structure of an immunoglobulin molecule and
poineered studies involving the cleavage of the the fragments obtained by the cleavage by papain and pepsin
Chapter 14: Antibodies—Immunoglobulins | 91
A B
Fig. 14.2: The four-peptide chain structure of the IgG molecule composed of two identical heavy (H) and two identical light
(L) chains linked by interchain disulfide bonds. Loops formed by intrachain disulfide bonds are domains (shown stippled).
Each chain has one domain in the variable region (VH and VL). Each light chain has one domain in the constant region (CL)
while each heavy chain has three domains in the constant region (CH1, CH2 and CH3). Between CH1 and CH2 is the hinge region
4. Fc Fragment
The Fc fragment is composed of the carboxyterminal
portion of the H-chains. It can be crystallized, and
is, therefore, called Fc (fragment-crystallizable).
Functions of Fc
Fig. 14.3: Secretory IgA. 1. Heavy chain; 2. Light chain; 3. J
Binds complement leading to complement fix chain; 4. Secretory component; 5. Disulfide bond
ation.
∙∙ Binds to cell receptors (FcRs) fluid, and it is distributed nearly equally
∙∙ Determines passage of IgG across the placental between extra- and intravascular spaces.
barrier 7. Four subclasses of IgG (Ig1, Ig2, Ig3, Ig4) have
∙∙ Determines skin fixation and catabolic rate. been recognized.
8. Catabolism of IgG is unique in that it varies with
5. Immunoglobulin Domain its serum concentration. When its level is raised,
Each immunoglobulin peptide chain has internal as in chronic malaria, kala-azar or myeloma,
disulfide links in addition to interchain disulfide the IgG synthesized against a particular antigen
bonds which bridge the H- and L-chains. These will be catabolized rapidly and may result in
interchain disulfide bonds form loops in the peptide the particular antibody deficiency. Conversely,
chain, and each of the loops is compactly folded in hypogammaglobulinemia, the IgG given for
to form a globular domain, each domain having treatment will be catabolized only slowly.
a separate function. The variable region domains, Functions of IgG: IgG is a very versatile molecule.
VL and VH, are responsible for the formation of a It may be considered a general-purpose antibody,
specific antigen-binding site. The CH2 region in IgG protective against those infectious agents, which
binds C1q in the classical complement sequence, are active in the blood and tissues.
and the CH3 domain mediates adherence to the
monocyte surface. The areas of the H-chain in the Examples of its Functions
C-region between the first and second C-region i. Transfer from mother to fetus: IgG is the only
domains (CH1 and CH2) is the hinge region. It is class of Igs that can cross the placenta and is
more flexible and is more exposed to enzymes and responsible for the protection of the infant
chemicals. Papain acts here to produce one Fc and during the first few months of life.
two Fab fragments (Figs 14.3). ii. Opsonization: IgG binds to microorganisms
and enhances their phagocytosis.
IMMUNOGLOBULIN CLASSES iii. Fixing to guinea pig skin.
Human serum contains five classes of immuno iv. Immunological reactions: IgG participates
globulins—IgG, IgA, IgM, IgD and IgE in the in complement fixation, precipitation and
desending order of the concentration. Table14.2 neutralization of toxins and viruses.
shows their differentiating features. v. Immobilize bacteria.
vi. Suppresses the homologous antibody synthesis:
1. Immunoglobulin G (IgG) ( Fig. 14.3) Passively administered IgG suppresses the
1. This is the major immunoglobulin in human homologous antibody synthesis by a feedback
serum, accounting for about 80% of the total process.
immunoglobulin pool.
2. It has a sedimentation coeffient of 7S and a 2. Immunoglobulin A (IgA)
molecular weight of 150,000. 1. It is the second most abundant class, cons
3. It contains less carbohydrate than other tituting about 10–13 per cent of serum immuno
immunoglobulins. globulins.
4. The normal serum concentration of IgG is 2. The normal serum level is 0.6–4.2 mg per mL.
about 8-16 mg per mL. 3. It has a half life of 6–8 days.
5. It has a half-life of 23 days—the longest of all of 4. IgA is the primary immunoglobulin found in
the immunoglobulin isotypes. external secretions, such as mucus, tears, saliva,
6. IgG is the predominant immunoglobulin in gastric fluid, colostrum and sweat. It exists in
blood, lymph, peritoneal fluid and cerebrospinal different forms in these various solutions.
Chapter 14: Antibodies—Immunoglobulins | 93
Table14.2 Physical, physiologic, and biologic properties of human serum immunoglobulins
Property lgG IgA* lgM lgD IgE
A. Physical properties
1. Sedimentation coefficient(s) 7 7 19 7 8
2. Molecular weight in 150,000 160,000 900,000, 180,000 190,000
kilodaltons
3. Carbohydrate (%) 3 8 12 13 12
4. Number of four-chain units 1 1–3 5–6 1 1
per molecule
B. Physiologic properties
1. Normal adult serum 12 2 1.2 0.03 0.00004
concentration (mg/mL)
2. Half-life (in days) 23 6 5 2–8 1–5
4. Daily production (mg/kg) 34 24 3.3 0.4 0.0023
5. Intravascular distribution (%) 45 42 80 75 50
C. Biologic properties
1. Complement fixation
Classical ++ – +++ – –
Alternative – + – – –
2. Placental transport to fetus + – – – –
3. Present in milk + + – – –
4. Selective selection by – + – – –
submucus glands
5. Anaphylactic – – – – ++++
hypersensitivity
6. Heat stability + + + + –
D. Major characteristics Most Protects Very efficienct Mainly Initiates
abundant Ig; mucosal against lymphocyte inflammation;
Longest half surfaces bacteremia receptor; raised in helminth
life: Crosses major surface infection; causes
placenta components of allergy symptoms
opsonizes B cells
antigen
*
IgA may occur in 7S, 9S and 11S forms
5. IgA occurs in two forms. Serum IgA and The secretory piece is believed to protect IgA
secretory IgA (SIgA). from denaturation by bacterial proteases in sites
Serum IgA: Serum IgA is monomeric (one four- such as the intestinal mucosa which have a rich
chain unit) 7S molecule (MW about 160,000). and varied bacterial flora.
Subclasses: There are two subclasses of IgA in
Secretory IgA (SIgA): In contrast, IgA found on
humans: IgA1 and IgA2.
mucosal surfaces and in secretions is a dimer
formed by two monomer units joined together at Functions of IgA
their carboxy terminals by a glycopeptide termed
i. Local immunity: Secretory IgA (SIgA) is
as the J chain (J for joining). This dimeric form
believed to play an important role in local
is more important form, known as secretory IgA
immunity against respiratory and intestinal
(SIgA). Dimeric SIgA is synthesized by plasma
pathogens.
cells situated near the mucosal or glandular
epithelium. J chains are also found in other ii. Prevention of organisms’ entry into body tissues.
polymeric immunoglobulins such as IgM. iii. Newborn protection: IgA present in breast milk
provides the newborn with protection against
Secretory component: Secretory IgA (SIgA)
infection.
contains another glycine-rich polypeptide called
the secretory component or secretory piece. This is iv. Agglutination.
not produced by lymphoid cells but by mucosal or v. Alternative pathways activation.
glandular epithelial cells. vi. Phagocytosis and intracellular killing.
94 | Section 2: Immunology
3. Immunoglobulin M (IgM) 4. Immunoglobulin D (IgD)
1. About 10% of normal serum Igs consists of this 1. IgD has a monomer structure similar to IgG.
class. 2. Its molecular weight is 180,000 daltons.
2. It is a heavy molecule (19S; MW 900,000 to 4. IgD is an immunoglobulin found in trace
1,000,000 daltons, hence called ‘millionaire amounts in the blood serum (0.03 mg/mL).
molecule’). 5. Half-life is about 3 days.
3. The normal serum level of IgM is 1.2 mg/mL. 6. IgD antibodies are abundant in combination
4. It has a half-life of about 5 days. with IgM on the surface of B cells and bind
5. IgM is the first immunoglobulin to appear antigens, thus signaling the B cell to start
after exposure to an antigen. antibody production.
6. In the circulation, IgM exists as a pentamer 7. Two subclasses of IgD (IgD1 and IgD2) are known.
of five four-chain units. The five identical IgM
monomers are connected to each other by 5. Immunoglobulin E (IgE)
a polypeptide joining (J) chain (Fig. 14.4). 1. It resembles IgG structurally and also known
Polymerization of the subunits depends upon as reagin antibody.
the presence of the J chain as with IgA (Fig. 14.3). 2. IgE is an 8S molecule (MW 19,000) and half-
7. Most of IgM (80%) is intravascular in life of two days.
distribution. 3. It is present in extremely low amounts in serum.
8. IgM is the first class of antibody produced 4. It exhibits unique properties such as heat
during the primary immune response. It is also lability (inactivated at 56°C in one hour).
the earliest to be synthesized by fetus beginning 5. It is susceptible to mercaptoethanol.
by about 20 weeks of age. As it cannot cross 6. It does not pass the placental barrier.
the placental barrier, the presence of IgM in 7. IgE does not activate complement nor
the fetus or newborn indicates intrauterine agglutinate antigens.
infection. 8. Allergic reactions: IgE may be elevated in allergic
9. They are relatively shortlived, hence their (atopic) individuals, and is responsible for
demonstration in the serum indicates recent many of the symptoms of allergies, bronchial
infection. asthma, and even systemic anaphylaxis.
10. Treatment of serum with 0.12 M 2-mercaptoe Allergy mediated by IgE is termed as type I
thanol selectively destroys IgM without hypersensitivity response.
affecting IgG antibodies. 9. Immunity against helminthic parasites:
11. Isohemagglutinins (anti-A and anti-B) and anti Children living in insanitary conditions, with
bodies to S. Typhi O antigen and Wassermann a high load of intestinal parasites, have high
reaction antibodies in syphilis are usually IgM. serum levels of IgE.
12. IgM agglutinates bacteria activates complement 10. Extravascular: It is mostly found extra
by the classical pathway, and enhances the vascularly in the lining of the respiratory and
ingestion of pathogens by phagocytic cells. IgM intestinal tracts.
is normally restricted to the intravascular space 11. Protection against pathogens: The physiological
because of its high molecular weight. role of IgE appears to be protection against
pathogens by mast cell degranulation and
release of inflammatory mediators.
Abnormal Immunoglobulins
Other structurally similar proteins are seen in
serum in many pathological processes, and
sometimes even in healthy persons apart from
antibodies in the following pathological conditions:
A. Multiple myeloma
Fig. 14.4: Pentameric IgM molecule, composed of five B. Heavy chain disease
identical monomers C. Cryoglobulinemia
Chapter 14: Antibodies—Immunoglobulins | 95
A. Multiple Myeloma An antibody molecule consists of two identical
The abnormal plasma cells are myeloma cells light chains and two identical heavy chains, which
which also collect in the solid part of the bone. are linked by disulfide bonds. Each heavy chain has
The disease is called “multiple myeloma”. Myeloma an amino-terminal variable region followed by a
constant region
is a plasma cell dyscrasia in which there is
Within the amino-terminal variable domain of each
unchecked proliferation of one clone of plasma heavy and light chain are three complementarity-
cells, resulting in the excessive production of the determining regions (CDRs). These polypeptide
particular immunoglobulin synthesized by the regions contribute the antigen-binding site of an
clone. Such immunoglobulins are, therefore, called antibody, determining its specificity
Abnormal immunoglobulins are multiple myeloma,
monoclonal.
heavy chain disease C and cryoglobulinemia.
Waldenstrom’s macroglobulinemia: Multiple
myeloma may affect plasma cells synthesizing
IgG, IgA, IgD or IgE. Myeloma involving IgM- Important questions
producing cells (lympho-plasmacytoid cells) is
known as Waldenstrom’s macroglobulinemia. In this 1. What is an antibody? Draw a labeled diagram of IgG.
condition, there occurs excessive production of the 2. Name various classes of immunoglobulins and
respective myeloma proteins (M proteins) and of describe structure and functions of IgG, IgA and
their light chains (Bence-Jones proteins). IgM.
3. Write shot notes on:
Bence-Jones proteins: In most patients, the
a. Immunoglobulin G (IgG)
myeloma cells also secrete excessive amounts
b. Immunoglobulin M (IgM)
of light chains. These excess light chains were
first discovered in the urine of myeloma patients c. Immunoglobulin A (IgA)
and were named Bence-Jones proteins, for their
discoverer Bence Jones (1847). Bence Jones proteins Multiple choice questions (MCQs)
can be identified in urine by its characteristic 1. The immunoglobulin that crosses the placenta is:
property of coagulation when heated to 50°C but a. IgG b. IgM
redissolving at 70°C. These proteins are the light c. IgA d. IgE
chains of immunoglobulins and so may occur as 2. The J-chain is present in the immunoglobulin:
the kappa or lambda forms. But in any one patient, a. IgG b. IgM
the chain is either kappa or lambda only, and never c. IgA d. IgE
both, being uniform in all other respects also. 3. All the following immunoglobulins are heat-stable
except:
B. Heavy Chain Disease a. IgG b. IgM
It is a lymphoid neoplasia characterized by the c. IgA d. IgE
overproduction of the Fc parts of the immuno 4. Which is the earliest immunoglobulin to be
globulin heavy chains. synthesized by the fetus?
a. IgG b. IgM
C. Cryoglobulinemia c. IgA d. IgE
It is a condition in which there is the formation 5. The immunoglobulin that mediates type I hyper
of a gel or a precipitate on cooling the serum, sensitivity reaction is:
which redissolves on warming. It may not always a. IgG b. IgM
be associated with disease but is often found in c. IgA d. IgE
myelomas, macroglobulinemias and autoimmune 6. Antibodies that are bound to mast cells and involve
conditions such as systemic lupus erythematosus. in allergic reactions are:
Most cryoglobulins consist of either IgG, IgM or a. IgG b. IgM
mixture of the two. c. IgA d. IgE
7. The first antibodies synthesized, especially against
Key Points microorganisms:
An antibody or immunoglobulin (lg) is a glycoprotein a. IgG b. IgM
that is made in response to an antigen, and can c. IgA d. IgE
recognize and bind to the antigen that caused its
production Answers (MCQs)
1. a; 2. b; 3. d; 4. b; 5. d; 6. d; 7. b.
15
Chapter
Complement System
Learning Objectives
After reading and studying this chapter, you should ∙∙ Discuss biological effects of complement
be able to: ∙∙ Describe complement deficiencies and associated
∙∙ Describe the sequence of events when the diseases.
classical pathway and the alternative pathway of
the complement system is activated
Complement: The term ‘complement’ (C) refers to 5. Complement fixation, binding or consumption:
a system of factors which occur in normal serum Complement (C) ordinarily does not bind to
and are activated characteristically by antigen- free antigen or antibody but only to antibody
antibody interaction and subsequently mediate a which has combined with its antigen.
number of biologically significant consequences. 6. Site of complement binding: The site of
complement binding is located on the Fc piece
Complement system of the Ig molecule.
The complement system belongs to the group
of biological effector mechanisms (called Complement Activation
triggered enzyme cascades), which also includes There are nine components of complement
coagulation, fibrinolytic and kinin systems. called C1 to C9. The fraction C1 occurs in serum
Pfeiffer phenomenon: It was discovered by as a calcium ion-dependent complex, which on
Pfeiffer (1894) that cholera vibrios were lysed chelation with EDT A yields three protein subunits
when injected intraperitoneally into specifically called C1q, r and s. Thus, C is made up of a total
immunized guinea pigs (bactereolysis in vivo or of 11 different proteins. C fractions are named C1
Pfeiffer phenomenon). to C9 in the sequence of the cascading reaction,
except that C4 comes after C1, before C2. The
General Properties of Complement components of the classical pathway are numbered
1. Present in the sera of all mammals and of most from C1 to C9, although they do not function in
lower animals: numerical order (C4 comes after C1, before C2).
2. Nonspecific serological reagent: Complement Activation of the complement system can be
from one species can react with antibodies from initiated either by antigen–antibody complexes or
other species. by a variety of nonimmunologic molecules.
3. Serum molecules: The complement system Complement components react in a specific
consists of approximately 30 serum molecules sequence as a cascade either through the classical or
constituting nearly 10% of the total serum alternative pathway.
proteins and forming one of the major defence
systems of the body. Principle pathways of complement
4. Heat labile: Complement as a whole is heat activation
labile, and being destroyed in 30 minutes at
56°C. A serum deprived of its complement Three principle pathways are involved in
activity by heating at 56°C for 30 minutes is then complement activation (Classical pathway, alternate
said to be ‘inactivated’. or properdin pathway, and Lectin pathway).
Chapter 15: Complement System | 97
A. Classical Complement Pathway instance of amplification. Activated Cl cleaves
The chain of events in which C components react in C4 into two pieces C4a and C4b (C4® C4a + C4b).
a specific sequence following activation of C1 and C4a is an anaphylotoxin and C4b which binds
typically culminate in immune cytolysis is known to cell membrane along with C1.
as the classical pathway (Fig. 15.1). It consists of C14b in the presence of magnesium ions
the following steps: cleaves C2 into two pieces (C2® C2a + C2b).
1. Antigen–antibody binding: The first step is the C2a remains linked to cell-bound C4b, and C2b
binding of C1 to the antigen–antibody (AB) which is released into fluid phase. The pieces
complex. The recognition unit of C1 is Clq, recombine, forming C4b2a has enzymatic
which reacts with the Fc piece of bound IgM activity and is referred to as the classical
of IgG. Clq binding in the presence of calcium pathway C3 convertase.
ions leads to sequential activation of Clr and s. 3. Production of C5 convertase: C3 convertase
In the presence of calcium ions, a trimolecular cleaves C3 into two fragments (C3® C3a + C3b).
complex (CI qrs-Ag-Ab) that has esterase C3a is soluble, and is an anahylotoxin, and
activity is rapidly formed. C3b which remains cell-bound along with
2. Production of C3 convertase: Activated Cls is C4b2a to form a trimolecular complex C4b2a3b,
an esterase (C1s esterase), one molecule of which has enzymatic activity and is called C5
which can cleave several molecules of C4—an convertase of the classic pathway.
Antigen–Antibody Reactions
LEARNING OBJECTIVES
After reading and studying this chapter, you should ∙∙ Describe applications of agglutination reactions
be able to: and their uses
∙∙ Differentiate between precipitation and agglutination ∙∙ Discuss principle and applications of agglutination
∙∙ Describe prozone phenomenon reactions
∙∙ Discuss mechanism and applications of precipit ∙∙ Describe principle of complement fixation test
ation reactions giving suitable examples
∙∙ Discuss principle and clinical applications of
∙∙ Describe types of precipitation reactions
immunofluorescence technique
∙∙ Describe principle and applications of Immuno
elec trophoresis, radial immunodiffusion, ∙∙ Discuss principle, various types and clinical
counteri mmunoelectrophoresis (CIE)), rocket applications of ELISA technique.
electrophoresis
Uses
i. To quantitate serum immunoglobulins, Fig. 16.4: Single diffusion in two dimensions (Radial
immunodiffusion—Mancini method)
complement proteins, other substances.
i. Counterimmunoelectrophoresis (CIE) or
Countercurrent eletrophoresis (CIEP)
Counterimmunoelectrophoresis can be used only
for antigens and antibody that migrate in opposite
directions in the electric field. The test is set up
similarly to that for double diffusion in agar. Two
wells are punched about 1 cm apart in an agar
slab on a glass plate. The antigen and antibody
Fig. 16.6: Immunoelectrophoresis solutions are placed in these wells in such a way
that when the electric fleld is applied across the
plate, the antigen will migrate toward the antibody,
In this procedure, antigens are separated by and the antibody will migrate toward the antigen.
electrophoresis in an agar gel. A small drop of A precipitin line will form when the antigen and
solution containing the antigens (usually proteins) antibody meet in optimal proportions resulting in
is placed into a small well punched out of solidified precipitation at a point between them (Fig 16.7).
agar on a small glass plate. The plate then is placed Advantages of Counterimmunoelectrophoresis
in an electric field to allow for the electrophoretic (CIE)
migration of the antigens. A trough is then cut 1. More sensitive: It is 10 times more sensitive than
next to the wells and filled with antibody and simple diffusion in agar.
diffusion allowed to proceed for 18–24 hours.The
2. More rapid assay: It is a much more rapid assay
antibody and the antigens diffuse toward each
(as little as 30 minutes for some antigens) than
other, resulting in the formation of precipitin bands
is double diffusion in gel.
or arcs wherever they are in optimal proportions
(Fig. 16.6).
Clinical Applications
Uses i. For detection of various antigens: such as
i. To separate many antigens as well as to hepatitis B surface antigen (HBS antigen) and
indicate the potential purity of an antigen alpha-fetoprotein in serum and meningococcal
ii. To separate the major blood proteins in serum and cryptococcal antigens in CSF.
for certain diagnostic tests. ii. To detect the presence of anti-DNA antibody in
iii. For testing for normal and abnormal proteins the serum of patients with several autoimmune
in serum and urine. disorders.
Uses
Fig. 16.8: Rocket electrophoresis i. For the identification of unknown bacterial
cultures.
ii. Very rapid: It requires only small quantities of
culture and serum.
iii. Also the method for blood grouping and cross-
matching.
2. Tube Agglutination
Fig. 16.9: Laurell’s variant of rocket electrophoresis (two- Serum from a patient thought to be infected with
dimensional electrophoresis) a given bacterium is serially diluted in a series
1. First run—Antigens are separated by electrophoretic of tubes to which the bacteria is added. The last
mobility.
tube showing visible agglutination will reflect the
2. The second run is done at right angles to the first which
drives the antigens into the antiserum containing gel serum antibody titer of the patient. The reciprocal
to form precipitation peaks. The area of the peak is of the greatest serum dilution that elicits a positive
proportional to the concentration of the antigen agglutination is known as the agglutinin titer.
Principle
A known antigen is mixed with test serum lacking
complement. When immune complexes have had
time to form, complement is added to the mixture.
If the patient’s serum contains antibody to the
Fig. 16.11: Coagglutination antigen, the resulting antigen–antibody complexes
will bind all of the complement added. In most of
Staph. aureus cell wall binds the Fc portion of the cases, fixation of complement with antigen-
the immunoglobulin molecule, leaving the Fab antibody complex causes in itself no visible effect.
portion free to bind antigen. Visible agglutination (Antigen + Antibody + Complement).
of the staphylococcal cells serves as a positive test This test consists of two separate systems and
to indicate antigen-antibody binding (Fig.16.11). these two systems are tested in sequence (Fig.
16.12). Appropriate controls should be used,
Uses including the following: antigen and serum controls
to ensure that they are not anticomplementary,
i. Coagglutination can be used for detecting the
complement control to ensure that the desired
presence of antigens in serum, urine and CSF.
amount of complement is added, and cell control
ii. Several commercial suppliers have prepared
to see that sensitized erythrocytes do not undergo
coagglutination reagents for identification
lysis in the absence of complement.
of antigens of various streptococcal groups,
including Lancefield groups A, B, C, D, F, G, and N;
Procedure
Streptococcus pneumoniae; Neisseria meningitidis;
N. gonorrhoeae; and Haemophilus influenzae types A. Test system: It consists of (i) Antigen- suspected
A to F grown in culture. of causing the patient’s disease; (ii) Patient’s
serum (antibody); (iii) Complement.
C. Complement Fixation Test (CFT) B. Indicator system: It consists of sheep red cells
(antigen) coated with anti-sheep-red cell
When complement binds to an antigen–antibody
antibody and Complement—An exogenous
complex. it bec omes “fixed” and “used up.”
source of complement (usually guinea pig
Complement fixation tests are very sensitive and
serum). Afterward, sensitized indicator cells,
can be used to detect extremely small amounts of
usually sheep red blood cells previously coated
an antibody for a suspect micro-orgamism in an
with complement-fixing antibodies, are added
individual’s serum. This can detect as little as 0.04
to the mixture. Complement lyses antibody-
µg of antibody nitrogen and 0.01 µg of antigen.
coated red cells.
Standardization of Complement and
Interpretation—Positive CF Test—Absence
Amboceptor
of Lysis
Guinea pig serum is first titrated for complement
activity. One unit or minimum haemolytic dose The specific antibodies are present in the test serum
(MHD) of complement is the highest dilution of and complement is consumed by the immune
guinea pig serum that lyses one unit volume of complexes. Insufficient amount of complement
washed sheep RBCs in the presence of excess of will be available to lyse the indicator cells.
haemolysin (amboceptor) within a fixed time
(usually 30–60 minutes) at a fixed temperature Negative CF Test Lysis of The Indicator Cells
(37°C). Similarly, amboceptor should also be Lysis of the indicator cells indicates lack of antibody
titrated. One MHD of hemolysin is defined as and a negative CF test. Lysis of the indicator cells
Examples
i. Antistreptolysin O (ASO) test: Antistreptolysin
O (ASO)-antitoxin, present in the serum of
the patient suffering from Strep. pyogenes
infection, neutralizes the haemolytic activity
of the streptococcal O hemolysin (toxin). Fig. 16.13: Opsonization
Sandwich Technique
The dyes can be conjugated to the Fc region of an
antibody molecule without affecting the specificity
of the antibody. For detection of antibodies by
immunofluorescence, the sandwich technique
can be employed. The antibody is first allowed to
B
react with unlabeled antigen, which is then treated
with fluorescent labeled antibody. A sandwich, is Fig. 16.14: Direct and indirect immunofluorescence.
thus formed, the antigen being in the middle and fluorochrome-labeled reagent. Indirect immuno
labeled and unlabeled antibodies on either side. fluorescence is used to detect the presence of
antibodies in serum following an individual’s expo
Types of Immunofluorescence sure to microorganisms.
There are two main kinds of fluorescent antibody In this technique, a known antigen is fixed
assays: direct and indirect. onto a slide. The test antiserum is then added, and
if the specific antibody is present, it reacts with
1. Direct Immunofluorescence (Fig. 16.14A) antigen to form a complex. When fluorescein-
Principle: In direct staining, the specific antibody labeled anti-immunoglobulin is added, it reacts
(the primary antibody) is directly conjugated with the fixed antibody. The slide is examined with
with fluorescein. It involves fixing the specimen the fluorescence microscope. The occurrence of
containing the antigen of interest onto a slide. fluorescence shows that antibody specific to the
Fluorescein-labeled antibodies are then added test antigen is present in the serum.
to the slide and examined with the fluorescence
microscope for a yellow-green fluorescence. The Uses
pattern of fluorescence reveals the antigen’s location. Diagnosis of syphilis: It is used to identify the
presence of Treponema pallidum antibodies in
Uses the diagnosis of syphilis (fluorescent treponemal
i. It is used to identify antigens, such as those antibody absorption (FTA-ABS).
found on the surface of group A streptococci. Advantages
ii. To diagnose enteropathogenic Escherichia i. The primary antibody does not need to be
coli, Neisseria meningitidis, Salmonella Typhi, conjugated with a fluorochrome.
Shigella sonnei, Listeria monocytogenes, ii. Increase the sensitivity of staining.
Haemophilus influenzae type b.
iii. To diagnose rabies virus. G. Radioimmunoassay (RIA)
One of the most sensitive techniques for detecting
Disadvantage antigen or antibody is radioimmunoassay
Separate fluorescent conjugates hence to be (RIA). The technique was first developed by two
prepared against each entigen to be tested. endocrinologists, SA Berson and Rosalyn Yalow, in
1960. The technique soon proved its own value for
measuring hormones, serum proteins, drugs, and
2. Indirect Immunofluorescence
vitamins at concentrations of 0.001 micrograms per
(Fig. 16.14B) milliliter or less. The significance of the technique
In indirect staining, the primary antibody is was acknowledged by the award of a Nobel Prize
unlabeled and is detected with an additional to Yalow in 1977, some years after Berson’s death.
well. Any free antibody is washed away and positive. If the antigen is not recognized by the
the presence of antibody bound to the antigen absorbed antibody, the ELISA test is negative
is detected by adding an enzyme-conjugated because the unatt ached antigen has been
secondary anti-isotype antibody (antibody 2), washed away, and no antibody-enzyme is
which binds to the primary antibody. Any free bound.
antibody 2 then is washed away, and a substrate 3. Competitive ELISA: In this technique, antibody
for the enzyme is added. The amount of colored is first incubated in solution with a sample
reaction product that forms is measured by containing antigen. The antigen–antibody
specialized spectrophotometric plate readers, mixture is then added to an antigen coated
which can measure the absorbance of all of microtiter well. The more antigen present
the wells of a 96-well plate in less than a few in the sampIe, the less free antibody will be
seconds. available to bind to the antigen-coated well.
2. Sandwich ELISA: The most frequently used Addition of an enzyme-conjugated, secondary
ELISA for detecting microbial antigen is the antibody specific for the isotype of the primary
sandwich solid-phase ELISA. In this technique, antibody can be used to determine the amount
the antibody (rather than the antigen) is of primary antibody bound to the well as in
immobilized on a microtiter well. The test an indirect ELISA. In the competitive assay,
sample is then exposed to the solid-phase however, the higher the concentration of
antibody, to which the antigen, if present, antigen in the original sample, the lower the
will bind. After the well is washed, a second absorbance.
enzyme-linked antibody specific for test
antigen is added and allowed to react with the USES OF ELISA
bound antigen. The conjugated antibody will
react with the antigen held to the solid-phase by ELISA has been used to detect antigens and
the first antibody, forming an antibody-antigen- antibodies of various microorgnisms.
antibody sandwich on the solid phase. After any
free second antibody is removed by washing, Examples
substrate is added, and the colored reaction 1. Parasites
product is measured.
If the antigen has reacted with the absorbed ∙∙ Entamoeba histolytica antigens in feces.
antibodies in the first step, the ELISA test is ∙∙ Toxoplasma antigens in the patient serum.
Learning Objectives
After reading and studying this chapter, you should ∙∙ Describe cluster of differentiation (CD)
be able to: ∙∙ Discuss the following; Natural killer cells or NK cells;
∙∙ Differentiate between T and B cells in a tabulated killer cells or K cells or ADCC cells; human leukocyte
form antigen (HLA).
White pulp: The splenic white pulp surrounds Lymphatic recirculation: Lymphopoiesis takes
the branches of the splenic artery, forming a place mainly in the central lymphoid organs where
periarteriolar lymphoid sheath (PALS) populated they differentiate and mature before entering the
mainly by T-lymphocytes. Primary lymphoid circulation and then the peripheral lymphoid organs
follicles are attached to the PALS. These follicles and tissues. These populations of lymphocytes do
are rich in B-cells and some of them contain not remain distinct but mix together in a process
germinal centers which develop following antigenic known as ‘lymphocyte recirculation’. There is
stimulation. The lymphatic sheath surrounding the a constant traffic of lymphocytes through the
central arterioles is the thymus dependent area blood, lymph, lymphatic organs and tissues. This
of the spleen. The perifollicular region, germinal recirculation ensures that following introduction of
centre and mantle layer form the bursa dependent antigen into any part of the body, lymphocytes of
(thymus-independent) areas. appropriate specificity would reach the site during
their ceaseless wandering and mount an immune
Red pulp: The splenic red pulp consists of a response. Recirculating lymphocytes are mainly
network of sinusoids. T-cells. B-cells tend to be more sessile. Chronic
Functions of spleen thoracic duct drainage will therefore result in
selective T-cell depletion.
i. Functions as the graveyard for blood cells.
ii. Mounting immune responses to antigens in the
blood stream. Differences between T- and B-Cells
Many tests help in differentiation of T- and B-cells
3. Mucosa-associated lymphoid tissue (MALT) (Table 17.1). These include:
The mucosa lining the alimentary, respiratory,
1. Thymus-specific antigens: Which are absent on
genitourinary and other lumina and surfaces are
B-cells.
constantly exposed to numerous antigens. These
vulnerable membrane surfaces are defended by 2. T-cell receptor (TCR): Which resembles but
a group of organized lymphoid tissues known differs from antibody, and CD2- and CD3-
collectively as mucosal-associated lymphoid tissue associated proteins on their surface.
(MALT). 3. Surface immunoglobulins: B-cells have
immunoglobulin on their surface.
Gut-associated lymphoid tissue (GALT): GALT 4. SRBC rosette: T-cells bind to sheep erythrocytes
includes the tonsils, adenoids, and Peyer’s patches forming rosettes (SRBC) or E rosette, through
in the intestine. CD2 molecule. B-cells do not bind.
Bronchial associated lymphoid tissue (BALT): 5. EAC rosettes: B-cells bind to sheep erythrocytes
Also occurs in the respiratory system. coated with antibody and complement,forming
EAC rosettes, due to the presence of a C3
Mucosal or secretory immune system: Mucous receptor (CR2) on the B-cell surface.
membranes are an effective barrier to the
6. Microvilli: Viewed under the scanning
entrance of most pathogens, which contributes to
microscope, T cells are generally free of
nonspecific immunity. This indicates the existence
cytoplasmic surface projections, while B-cells
of a common mucosal or secretory immune system.
have an extensively filamentous surface, with
Cells of the lymphoreticular system numerous microvilli
7. Blast transformation: T-cells undergo blast
Lymphoid Cells: Cells of the Immune System transformation, evidenced by enhanced DNA
The cells responsible for both nonspecific and synthesis, on treatment with mitogens, such as
specific immunity are the white blood cells called phytohemagglutinin (PHA) or Concanavalin
leukocytes. A (Con A), while B-cells undergo similar
120 | Section 2: Immunology
Table 17.1 Comparison of T-cells and B-cells differentiation and functional properties of the
cells. Thus a cell displaying CD1 is identified by
Property T cell B cell
the binding of antibodies against CD1. Each class
A. Origin Bone marrow Bone marrow of leucocyte displays a diagnostic pattern of CDs.
B. Maturation Thymus Bursal equivalent: Over 200 CD markers have been identified so far.
bone marrow,
Examples
Payer’s patches
∙∙ CD3 is expressed only by T-cells.
C. Location
∙∙ CD 19 is expressed only by B-cells.
1. Peripheral blood 65–85% 15–25%
∙∙ CD64 is expressed only by monocytes.
2. Lymph node 60–75% 30–35% ∙∙ CD66 is expressed only by granulocytes.
3. Spleen 25–45% 55–60% ∙∙ CD68 is expressed only by macrophages.
4. Thoracic duct 80–90% 10–20% ∙∙ On the other hand, CD18 and CD45 are expr
5. Thymus 96% Negligible essed by a variety of leucocyte types.
D. Thymus specific + – 2. Antigen recognition receptors
antigens These include membrane-bound (surface)
E. CD3 receptor + – immunoglobulins (mlgs or slgs) in B-cells and
F. Surface – + T-cell receptors (TCRs) in T-cells. In contrast to
immunoglobulins CDs, which can serve as diagnostic feature for
G. Receptor for Fc – +
all leukocytes, antigen recogn ition receptors
piece of IgG are limited to B- and T-lymphocytes only. These
H. SRBC rosette (E + –
receptors are required for B- and T-cells to be
rosette) antigen reactive. Reaction of antigens with mlgs
and TCRs activates B-cells and T-cells respectively,
I. EAC rosette (C3 – +
receptor leading to proliferation and differentiation.
J. Numerous – + b. On the Basis of Functions
microvilli, on
A. Regulatory T-cells
surface
1. Helper /inducer cell(TH): Helper/inducer
K. Blast transform + –
cell (TH), with CD 4 surface marker, MHC
ation with:
a. anti-CD 3 – + class II restriction; generally stimulating
b. anti-Ig + – and promoting the growth of T-cells and
c. PHA + – macrophages. They help B-cells make antibody
d. Concanavalin A – + in response to antigenic challenge; stimulate
e. Endotoxins
cell mediated immunity. Based on the different
profiles of cytokines produced, two subsets are
transformation with bacterial endotoxins, identified. Th1 and Th2.
Staphylococcus aureus (Cowan 1 strain) or EB Th1 cells: Th1 cells produce mainly the cytokines
virus. interferon gamma (IFN-g) and interleukin-2
(IL2) which activate macrophages and T-cells
T-lymphocytes promoting CMI, destruction of target cells and
Types of T-cells killing of intracellular microbes, such as tubercle
and lepra bacilli.
Based on their surface markers, MHC restriction,
TH2 cells: TH2 cells produce mainly the
target cells and function, the following T-cells sub
cytokines IL4, 5 and 6 which stimulate B-cells
types are recognised:
to form antibodies.
T-cells may be broadly classified into:
2. Suppressor T-cells (TS cells): These have CD8
surface marker and MHC class I restriction.
a. On the Basis of Surface Markers They can suppress B-cell and T-cell response.
Classification of lymphocytes on the basis of B. Effector cells
surface markers makes use of two important char
1. Delayed type-hypersensitivity T-cells (T-cells):
acteristics:
They are involved in delayed hypersensitivity
1. Cluster of differentiation or cluster determinant and cell-mediated immune response.
(CD) 2. Cytotoxic T-cells(TC-cells): They are also called
The term cluster of differentiation (CD) refers CD8+ cells with CD8 surface marker and MHC
families of surface glycoprotein antigens that can class I restriction. They can kill and lyse target
be recognized by specific antibodies produed cells carrying new or foreign antigens, including
against them. These markers reflect the stage of tumour, allograft and virus infected cells.
Chapter 17: Structures and Functions of the Immune System | 121
ii. B-cells and Plasma Cells i. They are not inducible by antigen.
ii. They lack the classic antigen-specificity of
B-cell Maturation
T-cells and B-cells.
The B-lymphocyte derived its letter designation iii. They are not restricted by MHC-encoded
from its site of maturation, in the bursa of Fabricius proteins.
in birds; and the bone marrow of mammals, iv. NK activity is ‘natural’ or ‘nonimmune’ as
including humans and mice. B-cell differentiation it does not require sensitisation by prior
also takes place in the fetal liver and fetal spleen. antigenic contact.
About 5–15% of the circulating lymphoid pool v. They belong to a different lineage from T- and
are B cells defined by the presence of surface B-cells.
immunoglobulin.
Mature B-cell undergoes clonal proliferation on Functions: They are considered to be important
contact with its appropriate antigen. Interaction i. Immune surveillance
between antigen and the membrane-bound ii. Natural defence against virus infected and
antibody on a mature naive B-cell, as well as maliganant mutant cells.
interactions with T-cells and macrophages, iii. NK-cells are capable of nonspecific killing
selectively induces the activation and differentiation of virus-transformed target cells and are
of B-cell clones of corresponding specificity. In involved in allograft and tumour rejection.
this process, the B-cell divides repeatedly and
differentiates, generating a population of plasma b. Antibody-dependent Cell-mediated
cells and memory cells. Plasma cells, synthesize
Cytotoxicity (ADCC)
and secrete antibody. Memory cells circulate until
activated by specific antigen. A subpopulation of LGLs possesses surface
receptors for the Fc part of Ig. They are capable
Plasma cell: They are fully differentiated
of lysing or killing target cells sensitised with
antibody-secreting effector cells of the B-cell
IgG antibodies. This is an example of a process
lineage. Antigenically stimulated B-cells undego
known as antibody-dependent cell-mediated
blast transformation, becoming successively
cytotoxicity (ADCC). This antibody dependent
plasmoblasts, intermediate transitional cells and
cellular cytotoxicity is distinct from the action of
plasma cells. Plasma cells are factories of antibody
cytotoxic T-cells, which is independent of antibody.
production. A plasma cell makes an antibody of a
ADCC-cells were formerly called killer (K)-cells but
single specificity, of a single immunoglobulin class
are now classified with NK-cells.
and allotype and a single light chain type only. An
exception is found in primary antibody response,
when a plasma cell producing IgM initiallyand later
c. Lymphokine-activated Killer (LAK)
it may switch to IgG production. Lymphocytes, Cells
lymphoblasts and transitional cells may also Lymphokine activated killer (LAK) cells are
synthesize Ig to some extent, while plasma cell is NK-lymphocytes treated with interleukin-2 (lL-2),
the best antibody producing cell. which are cytotoxic to a wide range of tumour cells
without affecting normal cells. IL-2 also acts as a
iii. Null Cells growth factor for NK-cells.
A small proportion (5-10%) of lymphocytes
that lack distinguishing phenotypic markers Phagocytic Cells
characteristic of T- or B-lymphocytes are called Phagocytic cells are the mononuclear macrophages
null cells. Because of their morphology, they are (of blood and tissues) and the polymorphonuclear
also known as large granular lymphocytes (LGL). microphages.
The member of this group is the
a. Natural killer (NK)-cells Mononuclear cells: The mononuclear phagocytic
b. Antibody dependent cellular cytotoxic (ADCC)- system consists of monocytes circulating in the
cells blood and macrophages in the tissues Both types
c. Lymphokine-activated killer (LAK)-cells. are highly phagocytic and make up the monocyte-
The term NK-cell is sometimes used as a macrophage system.
common name for all null cells. Macrophages: Monocytes leave the circulation and
reach various tissues to become transformed into
a. Natural Killer (NK) Cells macrophages, with morphological and functional
Natural killer (NK) cells are derived from large features characteristic of the tissues. Macrophages
granular lymphocytes (LGL). They differ from spread throughout the animal body and take up
K-cells in being independent of antibody. NK-cells residence in specific tissues where they are given
differ Tc cells in other properties as well: special names, e.g. alveolar macrophages in the
122 | Section 2: Immunology
lung, histiocytes in connective tissues, Kupffer cells Major Histocompatibility complex
in the liver, Mesangial cells in the kidney, microglial
cells in the brain and osteoclasts in the bone. The major histocompatibility complex (MHC) is
a remarkable cluster of genes that control T-cell
Functions of Macrophages recognition of self and nonself. MHC proteins
play a pivotal role in “presenting” antigens to
1. Phagoc ytosis: The primary function of T-cells. In fact, T-cells do not respond to foreign
macrophages is phagocytosis. peptides unless the antigen peptides are properly
2. Antigen presentation to T-cells to initiate “presented” (that is, offered in combination with
immune immune responses. a MHC molecule). In humans, the MHC is called
3. Secretion of cytokines to activate and promote the human leukoc yte antigen (HLA) complex.
innate immune response. These proteins were first detected by their effect on
Macrophages secrete interleukin-1, interleukin-6, transplant rejection (that is, tissue incompatibility).
tumor necrosis factor, and interleukin-12 In 1980, Snell, Dausset and Benacerrof were
in response to bacterial interaction, which awarded the Nobel prize for their work on MHC
stimulate immune and inflammatory responses, and the genetic central of immune response.
including fever. One of the most potent activators
of macrophages is interferon gamma (IFN-g) HLA Complex
secreted by activated TH cells.
The major antigens determining histocompatibility
Polymorphonuclear Microphages in human beings are alloantigens, characteristically
found on the surface of leukocytes. Human MHC
Microphages are the polymorphonuclear antigens are therefore synonymous with human
leucocytes of the blood.Because of the irregular- leucocyte antigens (HLA), and the MHC complex
shaped nuclei, granulocytes are also called of genes with the HLA complex.
polymorphonuclear leukocytes or PMNs. Three The HLA complex of genes is located on the
types of granulocytes exist: basophils, eosinophils, short arm of chromosome 6 (Fig. 17.1). It consists
and neutrophils. of three separate clusters of genes:
a. Neutrophils: They are actively phagocytic 1. HLA Class I comprising A, B and C loci: HLA
and form the predominant cell type in acute class I comprising A, Band C loci. Class 1
inflammation. The phagocytic property of proteins are encoded by the HLA-A, HLA-B and
neutrophils is nonspecific, except for its HLA-C genes in humans. HLA Class I antigens
augmentation by opsonins. (A, B and C) are found on the surface of virtually
b. Eosinophils: They possess phagocytic activity all nucleated cells and, in some species (i.e.
but only to a limited degree. They are found mice, but not humans), on red blood cells as
in large numbers in allergic inflammation, well.
parasitic infections and around antigen- They are the principal antigens involved in
antibody complexes. graft rejection and cell mediated cytolysis. Class
c. Basophils: Basophil leukocytes are found in the I molecules may function as components of
blood and tissues (mast cells). Their cytoplasm hormone receptors.
has large numbers of prominent basophilic 2. Class II or the D region consisting of DR, DQ
granules containing heparin, histamine, and DP loci: Class II proteins are encoded
serotonin and other hydrolytic enzymes. by the HLA-D region. There are three main
Degranulation of mast cells, with release of sets: the DP, DQ and DR-encoded molecules.
pharmacologically active agents, constitutes the HLA Class II antigens are more restricted in
effector mechanism in anaphylactic and atopic distribution, being found only on cells of the
allergy. immune system—macrophages, dendritic cells,
Dendritic cells: While macrophages are the activated T-cells, and particularly on B-cells.
major antigen presenting cells, dendritic cell also 3. Class III or the complement region: Class III or
performs this function. Dendritic cells are bone the complement region containing genes for
marrow derived cells of a lineage different from complement components C2 and C4 of the
the macrophages and T- or B-lymphocytes. They
possess MHC class II antigens.
They are more potent antigen-presenting cells
than macrophages and B-cells, both of which need
to be activated before they can function as antigen-
presenting cells (APCs). Dendritic cells are specially
involved in the presentation of antigens to T-cells
during the primary immune response. Fig. 17.1: HLA complex loci on chromosome
Chapter 17: Structures and Functions of the Immune System | 123
classical pathway, as well as properdin factor B 4. An association between HLA types and diseases:
of the alternative pathway, heat shock proteins For example, strong association has been found
and tumor necrosis factors alpha and beta. between ankylosing spondylitis and HLA-B27,
HLA loci are multiallelic, that is, the gene rheumatoid arthritis and HLA-DR4, and many
occupying the locus can be anyone of several autoimmune conditions and HLA-DR3.
alternative forms (alleles). As each allele
determines a distinct product (antigen), the MHC RESTRICTION
HLA system is very pleomorphic. For example,
at least 24 distinct alleles have been identified Both CD4+ and CD8+ T-cells can recognize antigen
at HLA locus A and 50 at B. only when it is presented by a self-MHC molecule,
an attribute called self-MHC restriction. Both class
I and class II antigens operate in this phenomenon.
HLA Molecules
Cytotoxic T-lymphocytes from immunised mice
HL A antigens are two-chain glycoprotein are able to kill and lyse virus infected target
molecules anchored on the surface membrane cells only when the T-cells and target cells are of
of cells. Class I and class II MHC are membrane- the same MHC type. Helper T-cells can accept
bound glycoproteins that are closely related in both antigen presented by macrophages only when
structure and function. the macrophages bear the same class II MHC
molecules on the surface. For T-cells participating
Role of MHC Diversity in delayed type hypersensitivity the antigen has to
1. Transplantation : The MHC system was be presented along with class II MHC
originally identified in the context of
transplantation, which is an artificial event. Key Points
2. For protecting the species from the broadest
The immune system is organized into several special
possible number of pathogens: The primary aim
tissues which are collectively termed as lymphoid or
of the MHC may be defence against microbes immune tissues
and not against the graft. The lymphoid organs, based on their functions,
3. Nonimmunological phenomena: Such as are classified into primary (central) and secondary
individual odour, body weight in mice and egg (peripheral) lymphoid organs
laving in chickens. There are three types of lymphocytes: B-cells, T-cells,
and natural killer cells (NK-cells)
HLA Typing T-cells perform two important functions: cytotoxicity
and delayed hypersensitivity
Antisera for HLA typing were obtained principally T-cells play a key role in regulating antibody
from multiparous women as they tend to production and CMI, and in suppression of certain
have antibodies to the HLA antigens of their immune responses
husbands, due to sensitisation during pregnancy. Bone marrow-derived lymphocytes are known as
Monoclonal antibodies to HLA antigens have B-lymphocytes or B-cells and perform two important
been developed. functions: First, they differentiate themselves into
Typing is done serologically by microcytotoxicity, plasma cells and produce antibodies. Second, they
can present antigen to helper T-cells
which tests for complement mediated lysis of
NK-cells have the ability to kill certain virally infected
peripheral blood lymphocytes with a standard set
cells and tumor cells without prior sensitization
of typing sera. However, serological typing is not
The MHC in humans is known as HLA complex. In
possible for HLA-DR antigens, which are detected humans, the HLA complex of genes is located on
by the mixed leucocyte reaction (MLR) and primed short arm of chromosome 6 containing several genes
lymphocyte typing (PLT), respectively. Genetic that are critical to immune function
methods are being used increasingly for HLA-typing The genes encode MHC proteins that are classified
in advanced centres. These employ restriction into three groups or classes known as the class I, class
fragment length polymorphism (RFLP) and gene II, and class III molecules
sequence specific oligonucleotide probe typing HLA typing or tissue typing are usually performed
for 1. Tissue transplantation; 2. Disputed paternity;
3. Anthropological studies; 4. An association between
Uses of HLA Typing HLA types and diseases.
1. Tissue transplantation: HLA typing is used
primarily for testing compatibility between
Important questions
recipients and potential donors before tissue
transplantation. 1. Differentiate between T- and B-cells in a tabulated
2. Disputed paternity. form.
3. Anthropological studies. 2. Give an account of lymphocytes.
124 | Section 2: Immunology
3. Write short notes on: 4. All the following statements are true for helper
a. Subsets of T-lymphocytes T-cells except that they:
b. B lymphocytes a. Carry CD4 marker
c. Null cells (or) Large granular lymphocytes (LGL) b. Help or induce immune responses
d. Natural killer cells or NK-cells c. Kill intracellular microorganisms by secreting
cytokines
e. Killer cells or K-cells or ADCC-cells
d. Recognize antigen in association with class I
4. Write briefly on:
MHC
a. Major histocompatibilty complex (MHC)
5. Class II MHC antigens are present on:
b. Human leukocyte antigen (HLA)
a. Macrophages
c. HLA typing b Monocytes
d. MHC restriction c. Activated T lymphocytes (CD4)
d. All the above
Multiple choice questions (MCQs) 6. Class I proteins are encoded by:
a. HLA-A, -B, and -C loci
1. All the following are examples of secondary- b. HLA-DR, HLA-DQ, and HLA-DP loci
lymphoid organs except: c. Complement loci that encode C2 and C4
a. Lymph nodes d. Complement loci that encode factor B
b. Thymus 7. Which of the following HLA types is associated
c. Spleen with ankylosing spondylitis?
d. Mucosa-associated lymphoid tissues a. HLA-B27 b. HLA-DR4
2. The CD4+ T-cells that recruit and activate phagocytic c. HLA-DP d. None of the
cells acting against intracellular microbes are called: above
a. Th-0 cells b. Th-1 cells 8. Which of the following HLA types is associated
c. Th-2 cells d. Antigen-present- with rheumatoid arthritis?
ing cells a. HLA-B27 b. HLA-DR4
3. The molecule expressed on surface of the mature c. HLA-Al d. None of the
T-cells is: above
a. CD 19 b. CD 8 Answers (MCQs)
c. CD 3 d. CD 1 1. b; 2. c; 3. c; 4. d; 5. d; 6. a; 7. b; 8. b
18
Chapter
Immune Response
Learning Objectives
After reading and studying this chapter, you should ∙∙ Discuss monoclonal antibodies—principle, technique
be able to: and applications
∙∙ Differentiate between primary and secondary ∙∙ Describe the following: cytokines; immunological
humoral immune responses tolerance.
on whether the humoral response results from Negative phase: If the same animal is subsequently
activation of naive lymphocytes (primary response) exposed to the same antigen already carrying the
or memory lymphocytes (secondary response). specific antibody in circulation, a temporary fall in
In both cases, activation leads to production of the level of circulating antibody occurs due to the
secreted antibodies of various isotypes, which combination of the antigen with the preexisting
differ in their ability to mediate specific effector antibody. This is known as the ‘negative phase’. It is
functions. followed by an increase in the titre of the antibody
exceeding the initial level.
a. Primary Humoral Response
The first contact of an exogenous antigen with an
Fate of antigen in tissues
individual generates a primary humoral response, The manner in which an antigen is dealt with in
characterized by the production of antibody- the body depends on factors such as the physical
secreting plasma cells and memory B-cells. and chemical nature of the antigen, its dose and
When first introduced the antigen selects the route of entry, and whether the antigenic stimulus
cells that can react with it. In all cases, however, a is primary or secondary.
primary response to antigen is characterized by a
Physical and chemical nature of the antigen:
lag phase, subsequent clonal expansion, and dif
Particulate antigens are removed from circulation
ferentiation into memory cells or plasma cells. The
in two phases. The first is the nonimmune phase
duration of the lag phase varies with the nature of
during which the antigen is engulfed by the
the antigen.
phagocytic cells, broken down and eliminated.
During a primary humoral response, IgM is
The phase of immune elimination begins with the
secreted initially, often followed by a switch to an
appearance of the specific antibody, during which
increasing proportion of IgG. Depending on the
persistence of the antigen, a primary response
can last for various periods, from only a few days
to several weeks.
2. Clonal Anergy
Parameters that Affect the Induction of
Tolerance Clones of B- and T-cells expressing receptors that
recognize self-antigen might remain but they
These include age, dose of antigen, route of tolerogen cannot be activated. This is known as clonal anergy.
administration, physical nature of the antigen, and
various treatments that reduce activation of positive 3. Suppression
regulatory cells of the immune response.
Clones of B- and T-cells expressing receptors that
recognize self-antigens are preserved. Antigen
1. Age
recognition might be capable of causing activation,
The younger the recipient, the easier it is to induce however, expression of immune response might be
tolerance, and, of course, it is easiest in utero. inhibited or blocked through active suppression.
Chapter 18: Immune Response | 135
Theories of Antibody formation development in later life by somatic mutation
could lead to autoimmune processes. Each
Theories of immunity fall into two categories: immunocompetent cell was capable of reacting
instructive and selective. with one antigen (or a small number of
antigens) which could recognize and combine
A. Instructive Theories with antigens introduced into the body. The
1. Direct template theories: According to these, result of the contact with the specific antigen
the antigen or the antigenic determinant enters was cellular proliferation to form clones
the antibody-forming cell and serves as a synthesizing the antibody. This theory is more
template against which antibody molecules are widely accepted than other theories.
synthesized so that they have combining sites
Key Points
complementary to the antigenic determinants.
These are therefore known as ‘direct template’ The immune response is the specific reactivity
theories. induced in a host by an antigenic stimulus and can
be divided into two types—the humoral (antibody-
2. Indirect template theory: This theory was mediated) and the cellular (cell-mediated) types
proposed by Burnet and Fenner (1949). The production of antibodies consists of three
According to this theory, the entry of the steps: 1. Lag phase; 2. Log phase; 3. A plateau or
antigenic determinant into the antibody steady state
producing cell induced in it a heritable change. The humoral response results from activation of
naive lymphocytes (primary response) or memory
A ‘genocopy’ of the antigenic determinant
lymphocytes (secondary response)
was thus incorporated in its genome and Monoclonal antibodies: Such antibodies produced by
transmitted to the progeny cells (indirect a single clone and directed against a single antigenic
template). determinant are called monoclonal antibodies, e.g.
plasma cell tumor (myeloma)
Cell-mediated immunity’ (CMI): The term ‘cell-
B. Selective Theories mediated immunity’ (CMI) refers to the specific
1. Side chain theory: According to side chain immune responses which involves T-Iymphocyte-
theory, immunocompetent cells (ICCs) have mediated functions
Cytokines: Cytokines are biologically active
surface receptors capable of reacting with
substances produced by cells that influence other
antigens which have complementary side cells. Interferons, growth factors and others were
chains. When foreign antigens are introduced found to have similar effects. Therefore, all of them
into the body, they combine with those cell have been grouped under the term ‘cytokines’
receptors which have a complementary Transfer factor: Transfer of CMI in man by injection of
fit. This inactivates the receptors. There extract from the leukocytes from immunized individual
Immunologic tolerance is defined as the absence of a
is an overproduction of the same type of specific immune response resulting from a previous
receptors which circulate as antibodies as a exposure to the inducing antigen. Two forms of
compensatory mechanism. tolerance are known. 1. Natural; 2. Acquired
2. Natural selection theory: This theory was Tolerance can arise through three possible
mechanisms: 1. Clonal deletion; 2. Clonal anergy;
proposed by Jerne (1955) which postulates that
3. Suppression
about a million globulin (antibody) molecules Theories of immunity fall into two categories:
were formed in embryonic life, which covered instructive and selective:
the full range of antigenic specificities. These A. Instructive theories: 1. Direct template theories;
globulins were the ‘natural antibodies’. When 2. Indirect template theory
an antigen was introduced, it combined B. Selective theories: 1. Side chain theory; 2. Natural
selection theory; 3. Clonal selection theory.
selectively with the globulin that had the
nearest complementary ‘fit’. The globulin,
with the combined antigen, homed in on the Important questions
antibody-forming cells and stimulated them 1. Discuss the primary and secondary humoral
to synthesize the same kind of antibody. immune responses.
3. Clonal selection theory: This theory was 2. Discuss briefly about:
proposed by Burnet (1957). This theory states Monoclonal antibodies—production and
that during immunological development, cells applications.
Adjuvants
capable of reacting with different antigens
Cytokines
were formed by a process of somatic mutation.
Theories of antibody production
Clones of cells that had immunological 3. Write short notes on:
reactivity with self-antigens were eliminated a. Transfer factor
during embryonic life. Such clones are b. Burnet’s clonal selection theory
called forbidden clones. Their persistence or c. Immunological tolerance
136 | Section 2: Immunology
Multiple choice questions (MCQs) 4. Development of cell mediated immunity can be
detected by:
1. The synthesis and production of antibodies a. Skin test for delayed hypersensitivity
typically is dependent on complex interaction of b. Lymphocyte transformation test
all the following cells except: c. Migration inhibiting factor test
a. Macrophages d. All the above
b. Helper T cells 5. Transfer factor shows all the following features
c. Cytotoxic T cells except:
d. B cells a. It is an extract from the leukocytes from the
2. Which animal is used for for monoclonal immnized host
antibodies production: b. Transfers humoral immunity
a. Guinea pig c. Transfers CMI
b. Mouse d. Transferred immunity is systemic
c. Rabbit 6. Clonal selection theory was postulated by:
d. None of the above a. Breinl and Haurowitz
3. Interleukin-l is a protein produced mainly by: b. Burnet and Fenner
c. Ehrlich
a. Macrophages and monocytes
d. Burnet
b. Polymorphonuclear leukocytes
c. Lymphocytes Answers (MCQs)
d. Stem cells 1. c; 2. b; 3. a; 4. d; 5. b; 6. d
19
Chapter
Immunodeficiency Diseases
Learning Objectives
After reading and studying this chapter, you should ∙∙ List primary and secondary immunodeficiency
be able to: syndromes.
∙∙ Classify and enumerate immunodeficiency diseases
Hypersensitivity Reactions
Learning Objectives
After reading and studying this chapter, you should ∙∙ Discuss type I, type II, type III, type IV hypersensitivity
be able to: reactions: Mechanism and examples.
∙∙ Compare major types of hypersensitivity reactions.
∙∙ Differentiate between immediate and delayed
hypersensitivity.
Mechanism of Anaphylaxis
The immunologic basis for hypersensitivity is
cytotropic IgE antibody. After an initial contact Fig. 20.1: Antigen-induced mediator release from mast cell
Chapter 20: Hypersensitivity Reactions | 145
muscle, increased vascular permeability, and Anaphylactoid Reaction
capillary dilatation. Intravenous injection of peptone, trypsin and
4. Chemotactic Factors certain other substances provokes a clinical reaction
i. Eosinophil chemotactic factor (ECF- resembling anaphylactic shock. This is termed
A): Eosinophil chemotactic factors of ‘anaphylactoid reaction’. The clinical resemblance
anaphylaxis (ECF-A) are acidic tetrapeptides is due to the same chemical mediators participating
released from mast cell granules which are in both reactions.
strongly chemotactic for eosinophils. These Anaphylactoid shock has no immunological
probably contribute to the eosinophilia basis and is a nonspecific mechanism involving
accompanying many hypersensitivity states. the activation of complement and the release of
ii. Neutrophil chemotactic factors (NCF): anaphylatoxins which is the only difference.
A high molecular weight chemotactic
factor has been identified, which attracts B. Localized Anaphylaxis (Atopy)
neutrophils (NCF).
The term ‘atopy’ (literally meaning out of place
5. Proteases: Enzymatic mediators, such as or strangeness) refers to naturally occurring
proteases and hydrolases are also released familial hypersensitivities of human beings. It was
from mast cell granules. These enzymes most introduced by Coca (1923) and typified by hay fever
probably are involved in the digestion of blood and asthma. The antigens commonly involved in
vessel basement membranes, resulting in atopy are inhalants (for example, plant pollen,
increased permeability to a variety of cell types. fungal spores, animal dander, and house dust mites
or other types of fine particles suspended in air) or
B. Secondary Mediators of Anaphylaxis ingestants (for example, milk, milk products, eggs,
1. Platelet-activating factor (PAF): Platelet- meat, fish or cereal). Some of them are contact
activating factor (PAF) is a low molecu lar allergens, to which the skin and conjunctiva may
weight lipid released from basophils which be exposed. The symptoms depend primarily
causes aggregation of platelets and release of on the route by which the antigen enters the
their vasoactive amines. body. These atopens are generally not good
2. Leukotrienes and prostaglandin: They antigens when injected parenterally but induce IgE
are derived by two different pathways from antibodies, formerly termed as ‘reagin’ antibodies.
arachidonic acid, which is formed from Atopic sensitization is developed spontaneously
disrupted cell membranes of mast cells and following natural contact with atopens. It is difficult
other leukocytes. A substance originally to induce atopy artificially.
demonstrated in lungs, producing slow, Predisposition to atopy is genetically deter
sustained contraction of smooth muscles, and, mined, probably linked to MHC genotypes. Atopy,
therefore, termed as slow reacting substance of therefore, runs in families. What is inherited is not
anaphylaxis (SRS-A) has since been identified sensitivity to a particular antigen, or a particular
a family of leukotrienes (LTB4, C4, D4, lM). atopic syndrome but the tendency to produce
In humans, the leukotrienes are thought to IgE antibodies in unusually large quantities. All
contribute to the prolonged bronchospasm individuals are capable of forming IgE antibodies
and buildup of muc us seen in asthmatics. in small amounts but in atopics IgE response is
Prostaglandin F2-α and thromboxane A2 are preponderant. About 10% of persons have this
powerful, but transient, bronchoconstrictors. tendency to overproduce IgE. It has been reported
3. Cytokines: Human mast cells secrete IL-4, that bottlefed infants tend to develop atopy in later
IL-5, IL-6, and TNF-α. These cytokines alter the life more often than breastfed babies.
local microenvironment, eventually leading to
the recruitment of inflammatory cells such as Mechanism of Atopy
neutrophils and eosinophils. The mechanism of development of atopy is essen
tially the same as that of systemic anaphylaxis. The
Other Mediators of Anaphylaxis symptoms of atopy are caused by the release of
Besides the products of mast cells and other pharmacologically active substances following the
leucocytes, several other biologically substances combination of the antigen and the cell fixed IgE.
are known to induce mast cell degranulation and Atopic sensitivity is due to an overproduction
have been implicated in anaphylaxis. These include of IgE antibodies. This is often associated with
split products from complement activation, C3a a deficiency of IgA. When IgA is deficient, the
and C5a and bradykinin and other kinins formed antigens cause massive stimulation of IgE forming
from plasma kininogens. cells, leading to overproduction of IgE.
146 | Section 2: Immunology
Clinical Expression of Atopic Reactions
The clinical expression of atopic reactions is
usually determined by the portal of entry of the
antigen-conjunctivitis, rhinitis, gastrointestinal
symptoms and dermatitis following exposure
through the eyes, respiratory tract, intestine or
skin, respectively. Sometimes the effects may be at
sites remote from the portal of entry, for example,
urticaria following ingestion of the allergen.
Specific desensitisation (hyposensitisation) is
often practised in the treatment of atopy. Atopic
allergies, which afflict at least 20% of the population
in developed countries, include a wide range of Fig. 20.2: Type II hypersensitivity
IgE-mediated disorders, including allergic rhinitis
(hay fever), asthma, food allergies and atopic membrane of a foreign cell, or it can mediate
dermatitis (eczema). cell destruction by antibody-dependent cell-
mediated cytotoxicity (ADCC). Type II hyper
Methods to Detect Type I sensitivity is generally called cytolytic or cytotoxic
Hypersensitivity Reactions reactions because it results in the destruction of
Formerly, measurement of reaginic antibody could host cells, either by lysis or toxic mediators. Type
be done only by an in vivo assay or by skin testing. II hypersensitive reactions involve antibody-
1. Prausnitz–Kustner (PK) reaction mediated destruction of cells (Fig. 20.2).
Prausnitz and Kustner in 1921 demonstrated
transmission of IgE-mediated type I hyper Examples
sensitivity by injecting serum containing IgE 1. Transfusion reactions: A transfusion reaction
antibodies from allergic person into the skin can occur if a patient receives erythrocytes
of a normal or nonallergic person. Serum differing antigenically from his or her own
from Kustner, who was hypersensitive to during blood transfusion.
certain species of cooked fish, was injected 2. Hemolytic disease of the newborn: If the child
intracutaneously in Prausnitz (normal) followed is Rh+, Rh– mother can become sensitized to
24 hours later by an intracutaneous injection of this antigen during birth causing the mother’s
cooked fish, to which Kustner was sensitive, into body to produce anti-Rh antibodies of the IgG
the same site in Prausnitz. This led to wheal and type. If the fetus in a subsequent pregnancy
flare reaction within 20 minutes. As IgE antibody is Rh+, her anti-Rh antibodies will cross the
is homocytotropic, the test has to be carried placenta and destroy fetal RBCs. The fetal
out on human skin. Therefore, there is risk of body responds to this immune attack by
transmission of hepatitis B virus and human producing large numbers of immature RBCs
immunodeficiency virus. call erythroblasts.
2. Skin testing: It is done by injecting small 3. Drug-induced cytotoxic reactions: Some
amounts of allergen into the skin of an allergic persons develop antibodies against their blood
individual and looking for a wheal and flare elements, resulting in autoimmune hemolytic
reaction. anemia, agranulocytosis or thrombocytopenia.
3. Radioimmunosorbent test (RIST): This Blood platelets (thrombocytes) that are
highly sensitive techn ique, based on the destroyed by drug-induced cytotoxic reactions
radioimmunoassay, can detect nanomolar in the disease called thrombocytopenic
levels of total IgE. purpura (quinine is a familiar example).
4. Radioallergosorbent test (RAST): It detects Drugs may bind similarly to white or red blood
the serum level of IgE specific for a given cells (RBCs), causing local hemorrhaging and
allergen. yielding symptoms described as “blueberry
muffin” skin mottling. Immune-caused
Type II Hypersensitivity: cytolytic destruction of granulocytic white cells is called
agranulocytosis, and it affects the body’s
and cytotoxic
phagocytic defenses. When RBCs are destroyed
These reactions involve a combination of IgG (or in the same manner, the condition is termed
IgM)antibodies with an antigenic determinant hemolytic anemia.
on the surface of cells. Antibody can activate 4. Anemia due to infectious diseases: A variety
the complement system, creating pores in the of infectious diseases due to Salmonella
Chapter 20: Hypersensitivity Reactions | 147
organisms and mycobacteria are associated of the site with neutrophils. Leukocyte-platelet
with hemolytic anemia. thrombi are formed that reduce the blood supply
and lead to tissue necrosis. The Arthus reaction
Type III Hypersensitivity: immune can be passively transferred with sera containing
complex-mediated precipitating antibodies (IgG, IgM) in high titers.
Clinical syndrome: Arthus reaction forms
Type III reactions involve antibodies against
a pathogenic component of many clinical
soluble antigens circulating in the serum. The
syndromes. Examples:
antigen-antibody complexes are dep osited in
i. Farmer’s lung.
organs and cause inflammatory damage. The
ii. “Pigeon fancier’s disease”
tissue damage that results from the deposition of
immune complexes is caused by the activation of
B. Serum Sickness (Systemic Immune
complement, platelets and phagocytes; in essence,
an acute inflammatory response (Fig. 20.3). Complex Disease)
This is a systemic form of type III hypersensitivity.
Models of immune complex-mediated disease:
This appears 7–12 days following a single injection
Two basic models of immune complex-mediated
of a high concentration of foreign serum, such as the
disease have been well characterized: the Arthus
diphtheria antitoxin. The clinical syndrome consists
reaction and serum sickness. These differ
of fever, weakness, lymphadenopathy, splenomegaly,
somewhat in both their mode of development and
arthritis, glomerulonephritis, endocarditis, vasculitis,
their clinical manifestations.
urticarial rashes, abdominal pain, nausea and
vomiting.
A. Arthus Reaction (Local Immune Pathogenesis: The pathogenesis is the formation
Complex Disease) of immune complexes (consisting of the foreign
Arthus (1903) observed that when rabbits were serum and antibody to it that reaches high
repeatedly injected subcutaneously with normal enough titers by 7-12 days) and the circulating
horse serum, the initial injections were without immune complexes deposit in the blood vessel
any local effect, but with later injections, there walls and tissues, leading to increased vascular
occurred intense local reaction consisting of permeability and thus to inflammatory diseases
oedema, induration and haemorrhagic necrosis. such as glomerulonephritis and arthritis. Antigen-
This is known as Arthus reaction and is a local antibody aggregates can fix complement leading
manifestation of generalized hypersensitivity. to inflammation and tissue damage. The plasma
The tissue damage is due to formation of local concentration of complement falls due to massive
precipitating immune complexes which are complement activation and fixation by antigen-
deposited on the endothelial lining of the blood antibody complexes. The disease is self-limited.
vessels. Antigen-antibody complexes can then The latent period of 7–12 days is required only
trigg er and activate complement leading to for serum sickness following a single injection.
release of inflammatory molecules. This leads to Serum sickness differs from other types of
increased vascular permeability and infiltration hypersensitivity reaction in that a single injection
Fig. 20.3: Immune complex-mediated hypersensitivity. (1) Immune complexes on the basement membrane of the wall of
a blood vessel, where they: (2) activate complement and attract inflammatory cells such as neutrophils to the site; (3) The
neutrophilis discharge enzymes as they react with the immune complexes, resulting in damage to tissue cells
148 | Section 2: Immunology
can serve as both as sensitizing dose and a shocking tuberculin (infection) type and the contact
dose. dermatitis type.
Diseases associated with immune complexes:
Complexes of antibody with various bacterial, 1. Tuberculin (Infection) Type
viral and parasitic antigens have been shown to This form of hypersensitivity was originally
induce a variety of type III hypersensitive reactions. described by Koch. In tuberculin hypersensitivity
These include systemic lupus erythematosus, tuberculin or purified protein derivative (PPD) is
poststreptococcal glomerulonephritis, endo injected into the skin of the forearm. intradermally
carditis, dengue haemorrhagic fever, hepatitis B, in an individual sensitized to tuberculoprotein by
malaria, etc. prior infection or immunization. An indurated
(firm and hard) inflammatory reaction, 10 mm or
Type IV Hypersensitivity—delayed more in diameter, develops at the site of injection
hypersensitivity within 48–72 hours. It is characterized by erythema
Type IV hypersensitivity reactions (delayed due to increased blood flow to the damaged
hypersensitivity) constitute one aspect of cell- area and the infiltration with a large number of
mediated immune response and are caused mainly mononuclear cells, mainly T-lymphocytes and
by T-cells. It is named delayed hypersensitivity about 10–20% macrophages into the injection site
because it appears in 24–48 hours after the are responsible for the induration. In unsensitized
presensitized host encounters the antigen, while individuals, the tuberculin injection provokes no
immediate hypersensitivity reactions develop in response.
1/2 to 12 hours. The tuberculin test therefore provides useful
indication of the state of delayed hypersensitivity
Causes of Type IV Reactions (cell-mediated immunity) to the bacilli. The
Type IV reactions occur when antigens, especially tuberculin test differs from the skin test for Type I
those binding to tissue cells, are phagocytosed hypersensitivity not only in the longer interval for
by macrophages and then presented to receptors appearance but also in its morphology and histology.
on the THI cell surface in the context of class I Tuberculin type hypersensitivity develops in
MHC. Contact between the antigen and TH I cell many infections with bacteria, fungi, viruses and
causes the cell to proliferate and release cytokines. parasites, especially when the infection is subacute
Cytokines attract lymphocytes, macrophages, and or chronic and the pathogen intracellular. A similar
basophils to the affected tissue. Extensive tissue hypersensitivity is developed in allograft reaction
damage may result (Fig. 20.4). and in many autoimmune diseases.
Delayed hypersensitivity cannot be passively
transferred by serum but can be transferred by 2. Contact Dermatitis Type
Iymphocytes or the transfer factor. Allergic contact dermatitis is caused by haptens
Types of delayed hypersensitivity: Three types that combine with proteins in the skin to form
of delayed hypersensitivity are recognized—the the allergen that elicits the immune response.
The substances involved are in thems elves
not antigenic but may acquire antigenicity on
combination with skin proteins. Subs equent
contact with allergen in a sensitized individual
leads to contact dermatitis. As most of the sub
stances involved are fat soluble, passage along
sebaceous glands may be the method of entry of
the allergens.
Examples: Examples of these haptens include
cosmetics, plant materials (catechol molecules
from poison ivy and poison oak), topical chemo
therapeutic agents, metals (nickel and chromium),
and chemicals like dyes, picryl chloride and
dinitrochlorobenzene, drugs such as penicillin and
toiletries and jewelry (especially jewelry containing
nickel).
Mechanism of action: Most of these substances are
Fig. 20.4: Type IV (delayed or cell-mediated) small molecules that can complex with skin proteins.
hypersensitivity This complex is internalized by antigen-presenting
Chapter 20: Hypersensitivity Reactions | 149
cells in the skin (e.g. Langerhans’ cells), then along with hypersensitivity reactions because of
processed and presented together with class II superficial resemblance.
MHC molecules, causing activation of sensitized Shwartzman (1928) observed that when a
TH cells. The sensitized T cells travel to the skin culture filtrate of S. Typhi (endotoxin) is injected
site, where on contacting the antigen they release intradermally in a rabbit, followed by the same
various lymphokines. Approximately 48–72 hours filtrate (endotoxin) intravenously 24 hours later, a
after the second exposure, the secreted cytokines hemorrhagic necrotic lesion develops at the site of
cause macrophages to accumulate at the site. Tissue the intradermal injection.
damage results from lytic enzymes released from
activated macrophages. Contact with the allergen in a Mechanism
sensitised individual leads to ‘contact dermatitis’. The
The first dose is called preparatory dose. The
lesions varying from macules and papules to vesicles
second dose is called provocat ive dose. The
that break down, leaving behind raw weeping areas
preparatory injection causes accumulation of
typical of acute eczematous dermatitis.
leukocytes which condition the site by release
Detection by ‘patch test’: Hypersensitivity is of lysosomal enzymes. These enzymes damage
detected by the ‘patch test’. The allergen is applied capillary walls. Following provocative dose, there
to the skin under an adherent dressing. Sensitivity occurs intravascular clotting, the thrombi leading
is indicated by itching appearing in 4–5 hours, and to necrosis of vessel walls and hemorrhage. This is
local reaction which may vary from erythema to called local form of Shwartzman reaction.
vesicle or blister formation, after 24–28 hours. When both injections are given intravenously,
the animal dies 12–24 hours after the second dose.
3. Granulomatous Hypersensitivity The animal develops disseminated intravascular
Granulomatous hypersensitivity reactions develop coagulation (DIC). Autopsy reveals bilateral
over a period of 21–28 days; the granulomas are cortical necrosis of kidneys and patchy necrosis of
formed by the aggregation and proliferation of the liver, spleen and other organs. Sanarelli (1924)
macrophages, and may persist for weeks. In terms described an essentially similar phenomenon in
of its clinical consequences, this is by far the most experimental cholera. The reaction is, therefore,
serious type of Type 1V hypersensitivity response. called the Sanarelli–Shwartzman reaction or the
Examples are leprosy, tuberculosis, leish generalized Shwartzman reaction.
maniasis, candidiasis and herpes simplex lesions.
Clinical Conditions
Type V: Hypersensitivity 1. Waterhouse–Friderichsen syndrome: Pururic
(Stimulatory Type) Jones–Mote rashes of meningococcal septicemia and the
acute hemorrhagic adrenal necrosis found
Reaction (or) Cutaneous Basophil in overwhelming infections (Waterhouse–
Hypersensitivity Friderichsen syndrome) mechanisms similar
This is an antibody-mediated hypersensitivity and to the Shwartzman reaction may operate.
is a modification of type II hypersensitivity reaction. 2. Septic shock syndrome.
Antibodies interact with antigens on cell surface
which leads to cell proliferation and differentiation Key Points
instead of inhibition or killing. Antigen-antibody
reaction enhances the activity of affected cell. Hypersensitivity is an exaggerated immune
response that results in tissue damage and is
Example: Graves’ disease: Thyroid hormones are manifested in the individual on second or subsequent
produced in excess quantity in Graves’ disease. contact with an antigen. Hypersensitivity reactions
are of 5 types: Types I, II, III, IV, and V
Long-acting thyroid stimulating (LATS) antibody
Type I hypersensitive reaction is mediated by IgE
is an autoantibody to thyroid membrane antigen. antibodies
It is presumed that LATS combines with a TSH Clinical manifestations of type I reactions include
receptor on thyroid cell surface and brings about generalized or systemic anaphylaxis or localized
the the same effect as TSH resulting in excessive anaphylactic
secretion of thyroid hormone. Type II hypersensitive reactions, or cytotoxic
reactions are caused by antibodies that can destroy
normal cells by complement lysis or by antibody-
Schwartzman reaction dependent cellular cytotoxicity (ADCC). Transfusion
reactions and hemolytic disease of the newborn are
This is not an immune reaction but rather a type II reactions
perturbation in factors affecting intravascular Type III hypersensitivity reactions are mediated
coagulation. It is, however, traditionally described by small antigen–antibody complexes that activate
150 | Section 2: Immunology
2. All are primary mediators of anaphylaxis except:
complement and other inflammatory systems,
a. Histamine
attract neutrophils, and contribute to inflammation.
Deposition of immune complexes near the site
b. Proteases
of antigen entry can induce an Arthus reaction, c. Eosinophil chemotactic factors of anaphylaxis
(localized reaction) and serum sickness (systems d. Platelet-activating factor
form) 3. Schultz-Dale phenomenon is an example of:
Type IV hypersensitive reactions involve the cell- a. Type I hypersensitivity reaction
mediated branch of the immune system. b. Type II hypersensitivity reaction
Three types of delayed hypersensitivity are: c. Type III hypersensitivity reaction
i. Tuberculin skin test; ii. Contact d. Type IV hypersensitivity reaction
Hypersensitivities; iii. Granulomatous. 4. All the following statements are true for Arthus
reaction except:
a. It is a local manifestation of generalized hyper-
Important questions sensitivity.
b. The tissue damage is due to formation of local
1. What is hypersensitivity? How do you classify precipitating immune complexes.
various types of hypersensitivity reactions? Des c. This is systemic form of type III hypersensitivity.
cribe type I hypersensitivity reactions. d. It manifests after a single injection of a high
concentration of foreign serum
2. Write short notes on:
a. Anaphylaxis 5. All the following statements are true for serum
b. Atopy sickness except:
a. It manifests after a single injection of a high
c. Type III hypersensitivity or immune complex
concentration of foreign serum
diseases
b. Disease is self-limited and clears without
d. Arthus reaction
sequelae
e. Serum sickness
c. This is systemic form of type III hypersensitivity.
f. Type IV hypersensitivity or delayed hypersensi- d. It is a localized inflammatory reaction due to
tivity. deposition of immune complexes.
6. Delayed hypersensitivity reaction is mediated by:
Multiple choice questions (MCQs) a. T-lymphocytes b. B-lymphocytes
c. Macrophages d. Basophils
1. All the following statements are true for type I 7. Shwartzman reaction is an example of:
hypersensitivity reaction except: a. Type I hypersensitivity reaction
a. It is called immediate hypersensitivity reaction b. Type II hypersensitivity reaction
b. It always involves IgE-mediated degranulation c. Type III hypersensitivity reaction
of basophils or mast cells d. None of the above.
c. This reaction is always rapid Answers (MCQs)
d. Atopy is one of the manifestations 1. b; 2. d; 3. a; 4. d; 5. d; 6. a; 7. d
21
Chapter
Autoimmunity
LEARNING OBJECTIVES
After reading and studying this chapter, you should ∙∙ Classify autoimmune diseases
be able to: ∙∙ List autoimmune diseases.
∙∙ Describe the mechanisms of autoimmunity
5. Activity of Helper and Suppressor Based on the site of involvement and nature of
lesions, autoimmune diseases may be classified
T-cells as (Table 21.1):
Enhanced helper T-cell and decreased suppressor A. Localized (or organ-specific)
T-cells functions have been suggested as causes of B. Systemic (or nonorgan-specific)
autoimmunity.
A. Localized (Organ-specific)
6. Sequestered Antigens Autoimmune Diseases (Table 21.1)
Certain self-antigens are present in closed systems The immune response is directed to a target
and are not accessible to the immune apparatus. antigen unique to a single organ or gland in an
These are known as sequestered antigens. organ-specific autoimmune disease, so that the
manifestations are largely limited to that organ.
Examples
B. Systemic (or Nonorgan-specific)
i. Lens antigen of the eye: The lens protein is
enclosed in its capsule and does not circulate Autoimmune Disease (Table 21.1)
in the blood. Hence, immunological tolerance In systemic autoimmune diseases, the response is
against this antigen is not established during directed toward a broad range of target antigens
fetal life. The release of lens protein after eye and meshces a number of organs and tissues.
damage has been shown to lead on occasion
to the formation of autoantibodies. When Pathogenesis of Autoimmune Disease
the antigen leaks out, following penetrating Many diseases are considered to be of autoimmune
injury, it may induce an immune response origin, based on their association with cellular or
causing damage to the lens of the other eye. humoral immune responses against self-antigens.
ii. Sperm antigens: Sperms arise late in develop
ment and sequestered from the circulation. As Humoral and cellular immune processes: The
spermatozoa develop only with puberty, the relative importance of humoral and cellular
antigen cannot induce tolerance during fetal immune processes in the etiology of autoimmune
life. However, after a vasectomy, some sperm diseases is not known. Antibodies may cause dam
antigens are released into the circulation age by the cytolytic or cytotoxic (type 2) and toxic
and can induce auto-antibody formation in complex (type 3) reactions. They are obviously
some men. This is also believed to be the important in hemocytolytic autoimmune diseases.
pathogenesis of orchitis following mumps. A third mechanism of autoimmune tissue
The virus damages the basement membrane damage is by sensitized T-lymphocytes (type 4
of seminiferous tubules leading to the leakage reaction). It is likely that humoral and ceIlular
of sperms and initiation of an immune immune responses may act synergistically in the
response resulting in orchitis. production of some autoimmune diseases. For
example, experimental orchitis can be induced
only when both types of immune responses are
7. Defects in the Idiotype–Anti-idiotype operative.
Network Autoimmune disease are usually treated with
It is possible that abnormalities in the generation immunosuppressants, or drugs that interfere
of appropriate anti-idiotype antibodies, either with T-cell signaling, steroids and other anti-
at the B- or T-cell level, are responsible for inflammatory drugs.
Learning Objectives
After reading and studying this chapter, you should ∙∙ Histocompatibility antigens
be able to: ∙∙ Graft versus host (GVH) reaction
Discuss the following: ∙∙ Tumor antigens
∙∙ Types of transplants ∙∙ Immunological surveillance.
Immunohematology
Learning Objectives
After reading and studying this chapter, you should ∙∙ List various infectious agents transmitted via blood
be able to: transfusion.
∙∙ Describ Rh blood groups
∙∙ Discuss complications of blood transfusion
History H Antigen
The ABO system is the most important of all Red cells of all ABO groups possess a common
the blood group systems and its discovery made antigen, the H antigen or H substance which is the
blood transfusion possible. No other blood group precursor for the formation of A and B antigens. H
antigens were discovered for the next 25 years. antigen is not ordinarily important in grouping or
Subsequently, other blood groups (MN, P, Rh, blood transfusion due to its universal distribution.
Lutheran, Lewis, Kell, Duffy, Kidd, Diego, Yt, Kg, However, Bhende et al. (1952) from Bombay
Dombroc and Colton) were reported. reported a very rare instance in which A and B
antigens as well as H antigens were absent from the
red cells. This is known as Bombay or OH blood
ABO Blood Group System group. Sera of these individuals have anti-A, anti-B
The ABO blood group system was originally and anti-H antibodies. Therefore, they can accept
described by Landsteiner (1900) and now contains the blood only from the same rare blood group.
four blood groups. The blood group is determined A, B and H antigens are glycoproteins. They
by the presence or absence of two distinct antigens are also present in almost all the tissues and fluids
A and B on the surface of the erythrocytes. It is of the body in addition to erythrocytes. They are
these antigens (also called agglunotigens) that found in secretions (saliva, gastric juice, sweat) of
cause blood transfusion reactions. Red cells of only about 75% of all persons while these antigens
group A carry antigen A, cells of group B antigen B are always present in tissues. Such persons are
and cells of group AB have both A and B antigens, called ‘secretors’ and those who lack blood group
while group O cells have neither A nor B antigen. antigens in secretions are called ‘nonsecretors’.
The serum contains the isoantibodies specific
for the antigen that is absent on the red cell. The Table 23.1 Distribution of ABO antigens on the
serum of a group A individual has anti-B antibody, red blood cells and antibodies in the serum
group B has anti-A and group O both anti-A and Blood Antigens on Antibodies Occurrence
anti-B, while in group AB, both anti-A and anti-B group red blood cells in serum (%) in India
are absent (Table 23.1). A A Anti-B 22
The frequency of ABO distribution antigens B B Anti-A 33
differs in different people. Group O is the most
AB A and B None 5
common group and AB the rarest. In India, the
distribution is approximately O–40%, A—22%, O None Anti-A and 40
Anti-B
B—33% AB—5%.
160 | Section 2: Immunology
Rh (Rhesus) Blood Group System possessed neither A nor B antigen. Hence
It was discovered by Landsteiner and Weiner in the O group was designated as the ‘universal
1940. Their experiment was to produce an antibody donor’. The anti-A and anti-B antibodies in
to the red cells of the Rhesus monkey in rabbits the transfused O blood group do not ordinarily
and guinea pigs, but they discovered that not only cause any damage to the red cells of the A or B
did the antibody in the rodents’ serum agglutinate group recipients because they will be rendered
the Rhesus monkey red cells, it also agglutinated ineffective by dilution in the recipient’s plasma.
the red cells of 85% of the human population. If an But some O group plasma may contain
individual’s red cells were clumped together by this isoantibodies in high titers (1:200 or above)
antiserum, they were said to have the Rhesus factor so that damage to recipient cells may result.
on their red cells (i.e. Rh positive). If an individual’s This is known as the ‘dangerous O group’. The
cells were not agglutinated by the antiserum, they AB group persons were designated ‘universal
were said to lack the Rhesus factor (i.e. Rh negative). recipients’ due to the absence of isoantibodies
Rh antigens—“Rh-positive” and “Rh-negative” in plasma.
people. 2. Rh compatibility: An Rh positive person may
There are six common types of Rh antigens, safely receive either Rh positive or negative
each of which is called an Rh factor. These types are blood. But an Rh-negative individual receiving
designated as C, D, E, c, d, and e. The designations Rh-positive blood may form antibodies against
employed by the two system for the different Rh the Rh-antigen. A subsequent transfusion with
types are as follows: Rh positive blood may then cause a rapid,
serious hemolytic reaction.
The type D antigen (Rho) is widely present in the
population and considerably more antigenic than
the other Rh antigens. Rh-positive or Rh-negative Complications of blood
blood depends on the presence or absence of transfusion
D-antigen on the surface of red cells respectively. The complications of blood transfusion may be divi
It can be accomplished by testing with anti-D ded into immunological and nonimmunological.
(anti-Rh) serum. About 15% of the population have
A. Immunological complications: Immunological
no RhD antigens and thus are “Rh negative”. Among
complications may be caused by red cell,
Indians, approximately 93% are Rh positive and
leukocyte or platelet incompatibility or allergic
about 7% negative. The Rh factor can be detected
reaction to plasma components. Red cell
by testing the blood with anti-D (anti-Rh) serum.
incompatibility leads to acute intravascular
hemolysis or the red cells may be coated
Other blood group systems by antibodies and engulfed by phagocytes,
Blood group systems other than ABO and Rh are removed from the circulation and subjected to
of little clinical importance as they do not usually extravascular lysis. Hemolysis may also be due
cause transfusion reactions or hemolytic disease. to transfusion of group O whole blood or plasma
They have applications in genetics, anthropology, to group A or group B or group AB recipients.
tissue typing and forensic medicine. As blood group B. Nonimmunological complications: Non-
antigens are inherited from the parents, they are immunological complications of blood trans
often useful in settling cases of disputed paternity. fusion include transmission of infectious
agents and circulatory overload. Infectious
Lewis blood group system: It differs from other
agents which may be transmitted during
blood group systems in that the antigens are
blood transfusion may be viruses, bacteria and
present primarily in the plasma and saliva and
protozoa (Table 23.2). Massive transfusion may
antigens do not form an integral part of the red
lead to circulatory overload.
cell membrane.
MN system: The antigens are M, N, S and s. Hemolytic disease of the newborn
This system has expanded to include at least 28
antigens. When an Rh– woman and an Rh+ man produce
a child, there is a 50% chance that the child will
Medical applications of blood be Rh+. If the child is Rh +, the Rh– mother can
become sensitized to this antigen during birth,
groups when the placental membranes tear and the fetal
1. Blood transfusion: Ideally, the donor and Rh+ RBCs enter the maternal circulation, causing
recipient should belong to the same ABO group. the mother’s body to produce anti-Rh antibodies
It used to be held that O group cells could be of the IgG type. During subsequent pregnancy,
transfused to recipients of any group as they Rh antibodies of the IgG class pass from the
Chapter 23: Immunohematology | 161
Table 23.2 Nonimmunological complications of Detection of Rh-antibodies
blood transfusion Most Rh-antibodies are of the IgG class, and they
A. Transmission of Infectious Agents do not agglutinate Rh-positive cells in saline being
Viruses ‘incomplete antibodies’. IgG anti-D antibodies
• Hepatitis B virus* may be detected by the following techniques:
• Hepatitis C virus** 1. Using a colloid medium such as 20% bovine
• Human immunodeficiency virus 1 and 2*
• Human T-cell Iymphotrophic virus 1 and 2
serum albumin.
• Cytomegalovirus 2. Using red cells treated with enzymes such as
Bacteria trypsin, pepsin, ficin or bromelin, and
• Treponema pallidum* 3. By the indirect Coombs test: This is the most
• Leptospira interrogans sensitive method.
• Borrelia burgdorferi
Parasites
Plasmodium spp.* Prevention of Rh-isoimmunization
• Babesia spp.
• Trypanosoma cruzi HDNB is usually prevented today by passive immu
• Leishmania donovanii nization of the Rh– mother at the time of delivery
• Toxoplasma gondii (within 24–48 hours) of any Rh+ infant with anti-
B. Circulatory Overload Rh-antibodies, which are available commercially
* Mandatory tests in India
(Rhogam). To be effective, this should be employed
** Mandatory tests in India since June 2001 from the first delivery onwards.
24
Chapter
Staphylococcus
Learning Objectives
After reading and studying this chapter, you should ∙∙ Describe staphylococcal diseases
be able to: ∙∙ Discuss laboratory diagnosis of infections caused
∙∙ Describe species of Staphylococcus by Staphylococcus aureus
∙∙ Describe morphology and culture characteristics ∙∙ Explain methicillin-resistant staphylococci and its
of Staphylococcus aureus clinical problem
∙∙ List characteristics of Staph. aureus strains ∙∙ D escribe the following: Coagulase-negative
∙∙ Explain coagulase test staphylococci (CNS); micrococci
∙∙ List and describe toxins and enzymes of Staphylo ∙∙ Distinguish characteristics of Staph. aureus, Staph.
coccus aureus epidemidis and Staph. saprophyticus.
S. delphini, S. lutrae, and some strains of S. hyicus. to novobiocin and by its failure to ferment glucose
These are often animal-associated species and are anaerobically. It is nonhemolytic and does not
infrequently isolated from human samples. contain protein A. Table 24.3 lists the features
useful for distinguishing the major species of
Coagulase-negative staphylococci.
Staphylococci
Other Coagulase-negative Staphylococci
Coagulase-negative staphylococci (CONS) are
commonly found on the surface of healthy persons. S. haemolyticus has been reported in wounds,
They are opportunistic pathogens that cause bacteremia, endocarditis, and UTIs. Other species
infection in debilitated or compromised patients. include S. lugdunensis, S. warneri, S. capitis,
Various species are Staph. epidermidis, Staph. S. simulans, and S. schleiferi.
saprophyticus, Staph. hemolyticus, Staph. hominis,
and Staph. capitis. Sensitivity to Antibiotics
Before the introduction of penicillin, most of the
Staphylococcus epidermidis strains of S. aureus were sensitive to this antibiotic.
Staph. epidermidis is invariably present on normal Staphylococci quickly developed drug resistance
human skin. It is nonpathogenic ordinarily but after penicillin was introduced.
can cause disease when the host defences are Penicillin resistance is of three types:
breached. S. epidermidis has a distinct predilection 1. Production of beta lactamase (penicillinase):
for foreign bodies, such as artificial heart valves, Production of beta lactamase (penicillinase)
indwelling intravascular catheters, central nervous which inactivates penicillin by splitting the beta
system shunts, and hip prostheses. Their etiological lactam ring. Staphylococci produce four types
role is proved by repeated isolation. of penicillinases, A to D. Penicillinase is an
inducible enzyme and its production is usually
controlled by plasmids which are transmitted
Clinical Infection
by transduction or conjugation.
∙∙ Stitch abscesses; endocarditis of native Penicillinase plasmids are transmitted to
and prosthetic valves; intravenous catheter the sensitive staphylococci by transduction and
infections; CSF shunt infections; bacteremia; also possibly by conjugation.
osteomyelitis; wound infections, vascular graft 2. Changes in bacterial surface receptors:
infections, prosthetic joint infection, etc. Changes in bacterial surface receptors, reduc
ing binding of beta-lactam antibiotics to cells.
Staphylococcus saprophyticus This change is normally chromosomal in nature
S. saprophyticus is a common cause of urinary tract and is expressed more at 30°C than at 37°C. This
infections in sexually active young women. It may resistance also extends to cover beta lactamase-
also cause urethritis in men and women, catheter- resistant penicillins, such as methicillin and
associated urinary tract infections, prostatitis in cloxacillins. Some of these strains may show
elderly men, and rarely bacteremia, sepsis and resistance to other antibiotics and heavy metals
endocarditis. also and cause outbreaks of hospital infection.
This coagulase-negative Staphylococcus can be These strains have been called ‘epidemic
distinguished from S. epidermidis by its resistance methicillin-resistant Staphylococcus aureus’
or EMRM. (as methicillin is an unstable drug, samples. A high salt concentration (5.5% NaCl)
cloxacillin is used for sensitivity testing instead). and polymyxin B make the medium selective
3. Development of tolerance: Development of for staphylococci.
tolerance to penicillin, by which the bacterium 2. The gold standard for MRSA detection is the
is only inhibited but not killed. detection of the mecA gene by using nucleic
acid probes or polymerase chain reaction (PCR)
Methicillin-resistant Staphylococcus amplification.
aureus (MRSA) Control of MRSA: Control of MRSA requires strict
Methicillin was the first compound developed adherence to infection-control practices such as
to combat resistance due to penicillinase (beta barrier.
lactamase) production by staphylococci. Due
to the limitations in clinical use of methicillin, Micrococci
cloxacillins are used instead against penicillinase-
producing strains. But methicillin resistant strains Micrococci are catalase-positive, gram-positive,
of Staph. aureus (MRSA) became common, which coagulase-negative and usually oxidase-positive.
were resistant not merely to penicillin, but also They may ocassionally colonize the skin or mucous
to all other beta lactam antibiotics and many membrane of humans, but they are only rarely
others besides. Isolates that are resistant have associated with infections.
been traditionally termed methicillin-resistant Only two species, Micrococcus luteus and
staphylococci, with S. aureus being called MRSA Micrococcus lylae, remain in the genus. Micrococci,
and S. epidermidis referred to as MRSE. When especially M. luteus, have a tendency to produce
any staphylococcus isolated is identified as being a yellow pigmented colony. Nine species of genus
resistant to methicillin, this implies that it is Micrococcus have been described.
also resistant to nafcillin and oxacillin and to all Table 24.4 gives some differentiating features
β-lactam antibiotics, including the cephalosporins. of Staphylococcus and Micrococcus.
MRSA is also becoming more common in the
community, especially in long-stay institutions. Key Points
Staphylococcus
Treatment-glycopeptides (vancomycin or
Staphylococcus is gram-positive cocci arranged in
teicoplanin) are the agents of choice in the
clusters
treatment of systemic infection, but these agents
Species of staphylococci are classified by the
are expensive and may be toxic. Concerns with coagulase test into two groups: the coagulase-
rising resistance to glycopept ides call for the positive (Staphylococcus aureus) and coagulase-
restrictive use of these drugs. negative staphylococci. S. epidermidis and S.
saprophyticus
Laboratory Diagnosis Staphylococcus aureus
Staph. aureus strains usually exhibit following
1. For laboratory purposes, oxacillin is generally characteristics: (1) Beta hemolysis; (2) Golden
used for detection of methicillin resistance. yellow pigment; (3) Coagulase positive; (4) Greater
The use of an oxacillin-salt agar plate, such as biochemical activity, ferment mannite; (5) Liquefy
the oxacillin resistance screening agar can be gelatin; (6) Produce phosphatase; (7) Black colonies
used as a screening test for MRSA in clinical on potassium tellurite blood
S. aureus produces many virulence factors including: f. Epidermolytic toxins of Staphylococcus aureus
(1) Cytolytic or membrane-damaging toxins (alpha, g. Drug resistance in staphylococci.
beta, delta, gamma, and Panton-Valentine [P-V] h. Methicillin-resistant Staphylococcus aureus
leukocidin); (2) Exfoliative toxins: (3) Enterotoxins (MRSA)
(A-E, G-I); (4) Toxic shock syndrome toxin-1 (TSST-1) i. Coagulase-negative staphylococci (CNS)
Diseases: S. aureus causes cutaneous infections j. Micrococci.
such as folliculitis, boils, carbuncles, impetigo, and
purulent abscesses. These cutaneous infections
can progress to deeper abscesses involving other
Multiple choice questions (MCQs)
organ systems and progress to septicemia and 1. Staphylococcus aureus shows the following
bacteremia. Toxin-induced diseases, such as food characters except
poisoning, scalded skin syndrome (SSS), and toxic
a. It produces golden brown pigment on the blood
shock syndrome (TSS), are also associated with this
agar
organism
Other systemic diseases (frequently associated with b. It ferments mannitol
bacteremia) include pneumonia, empyema, septic c. It is novobiocin-sensitive
arthritis, osteomyelitis, acute endocarditis, and d. It has protein A
catheter-related bacteremia 2. Protein A is a cell wall components of
Diagnosis: It is done by microscopy, culture, antibiotic a. Staphylococcus aureus
sensitivity tests and serological tests b. Staphylococcus epidermidis
Bacteriophage typing may be done if the information
c. All of the above
is desired for epidemiological purposes
Treatment: The antibiotics of choice are oxacillin
d. None of the above
(or other penicillinase-resistant penicillin) or 3. The enzyme coagulase shows all the following
vancomycin for oxacillin-resistant strains features except
Methicillin-resistant strains (MRSA). Glycopeptides a. It has eight serotypes
(vancomycin or teicoplanin) are the agents of choice b. It is extracellular
in the treatment of systemic infection c. It is detected by tube coagulase test
Coagulase-negative staphylococci: Coagulase- d. Undiluted serum is used in the test
negative staphylococci are opportunistic pathogens
4. Scalded skin syndrome is caused by the
that cause infection in debilitated or compromised
following toxin of Staphylococcus aureus
patients. Staph. epidermidis accounts for about 75%
of all clinical isolates. Other species include Staph. a. Enterotoxins
haemolyticus, Staph. hominis, Staph. capitis and b. Toxin shock syndrome
Staph. saprophyticus c. Exfoliative toxin
Staph. saprophyticus can be distinguished from S. d. Leukocidin
epidermidis by its resistance to novobiocin and by 5. Which of the of the following Staphylococcus is
its failure to ferment glucose anaerobically novobiocin resistant?
Micrococci may occasionally colonize the skin or a. Staphylococcus aureus
mucous membrane of humans, but they are only
b. Staph. epidermidis
rarely associated with infections.
c. Staph. saprophyticus
d. None of the above
Important Questions 6. The most common cause of cystitis in a young
1. Describe the morphoplogy, cultural characteristics healthy sexually active women is:
and antigenic structure of Staphylococcus aureus. a. Staphylococcus aureus
2. Name various virulence factors of Staphylococcus b. Staphylococcus epidermidis
aureus. c. Staphylococcus saprophyticus
3. Classify staphylococci. Discuss pathogenicity and d. Staphylococcus saccharolyticus
laboratory diagnosis of Staph. aureus. 7. All are coagulase-negative staphylococci except:
4. Write short notes on: a. Staphylococcus epidermidis
a. Coagulase or staphylocoagulase b. Staphylococcus saprophyticus
b. Clumping factor c. Staphylococcus haemolyticus
c. Toxins and enzymes of produced by Staphylo d. Staphylococcus aureus
coccus aureus
d. Staphylococcal food poisoning. Answers (MCQs)
e. Toxic shock syndrome 1. a; 2. a; 3. d; 4. c; 5. c; 6. c; 7. d
Learning Objectives
After reading and studying this chapter, you should ∙∙ List and describe toxins and enzymes of Strepto
be able to: coccus pyogenes
∙∙ Classify streptococci ∙∙ Describe non-suppurative complications of Str.
∙∙ Describe antigenic structure of Str. pyogenes pyogenes infections
∙∙ List and describe toxins and enzymes of Strepto ∙∙ Discuss laboratory diagnosis of streptococcal
coccus pyogenes infections
∙∙ Discuss pathogenicity of streptococci ∙∙ Discuss group B streptococci, Group D streptococci
and viridans group.
Introduction Classification
The genus Streptococcus comprises a large and Streptococci are first divided into obligate anaerobe
biologically diverse group of gram-positive cocci and facultative anaerobes. Obligate anaerobe are
that grow in pairs or chains (Fig. 25.1). They are designated as peptostreptococci.
normal flora of humans and animals. They inhabit Three different schemes are used to classify the
various sites, notably the upper respiratory tract, organism, as shown in Figure 25.2.
and live harmlessly as commensels.
Streptococci were first described by Billroth
(1874) in exudates from erysipelas and wound
infections, who called them streptococci (streptos,
meaning twisted or coiled; coccus, a grain or
berry). Pasteur (1879) found similar organisms
in the blood of a patient with puerperal sepsis.
Ogston (1881) isolated them in acute abscesses,
distinguished them staphylococci and by animal
inoculation established their pathogenicity.
Clinical Diseases
1. Infection in the neonate
A. Early-onset disease : It develops during the
first week of life and disease is characterized by
bacteremia, pneumonia, or meninigitis and is
often fatal.
B. Late-onset disease: It develops between
second and twelfth weeks of life. The predominant
manifestation is bacteremia with meningitis, but
septic arthritis. Fig. 25.4: CAMP reaction
180 | Section 3: Systemic Bacteriology
throat, pneumonia, septicemia, endocarditis, and media and on MacConkey agar, on which it forms
bone, joint, skin and wound infections. small (0.5–1 mm), usually magenta-colored
colonies.
Group D Streptococci
Distinctive Features of Enterococci
Until the mid-1980s, the group D streptococci were
The enterococci possess several distinctive
divided into the two groups:
features separting them from sreptocooci: The
1. Enterococcus group (enterococci or fecal
enterococci grow in the presence of 6.5% NaCl,
streptococci) which have been reclassified as
40% bile, at ph 9.6, at 45°C and in 0.1% methelyne
a separate genus called Enterococcus.
blue. It survives heating at 60°C for 30 min, a
2. Nonenterococcal group, for example, Str. bovis,
feature distinguishing it from streptococci, and
Str. equinus.
also grows within a wider range of temperatures
(10–45°C). On MacConkey medium, they produce
Enterococcus deep pink colonies. Enterococci are PYRase test
The enterococci (“enteric cocci”) were previously positive. They do not hydrolyze hippurate.
classified as group D streptococci (Table 25.3). The
enterococci were reclassified into the new genus Identification
Enterococcus, and there are currently 16 species The identification of Enterococcus species is made
in this genus. on biochemical characteristics. E. aecalis can
be identified by its ability to ferment mannitol,
Species sucrose, sorbitol and aesculin, and to grow on
Enterococcus faecalis (“pertaining to feces”) is tellurite blood agar producing black colonies with
the Enterococcus most often isolated from human gas production. It is VP positive.
sources.
Enterococcus faecium (“of feces”). Clinical Infections
Other species: E. durans, E. avium, E. casseliflavus, The enterococci inhabit the gasterointestinal
E. gallinarum, and E. raffinosus are observed tract and the genitourinary tract in humans and
occasionally. other animals. Enterococci are frequent causes of
nosocomial infections and may cause urinary tract
Characteristics of Enterococci infection, bacteremia, infective endocarditis,
The enterococci are gram-positive cocci typically biliary tract infection, intra-abdominal abscess
arranged in pairs and short chains, and is nonmotile complicating diverticulitis, peritonitis and
and noncapsulate. The cocci are facultatively wound infection.
anaerobic and grow optimally at 35°C, although
most isolates can grow in the temperature range Treatment
10°C to 45°C. They grow readily on blood agar Most strains of enterococci are resistant to
media, with large, white colonies appearing penicillin. They are also resistant to sulfonamides.
after 24 hours of incubation; the colonies are Recently they have developed resistance to newer
typically nonhemolytic but can be α-hemolytic or penicillins and cephalosporins, streptomycin and
β-hemolytic. It grows readily on ordinary nutrient gentamicin.
Learning Objectives
After reading and studying this chapter, you should ∙∙ Explain C-reactive protein
be able to: ∙∙ Discuss laboratory diagnosis of pneumococcal
∙∙ Describe morphology and cultural characters of infections
pneumococci ∙∙ Differentiate between Str. pneumoniae and Str.
∙∙ Describe Quellung reaction viridans.
Pneumococcus
(Diplococcus Pneumoniae,
Streptococcus Pneumoniae)
Morphology
Pneumococci are gram-positive cocci in pairs Fig. 26.1: Str. pneumoniae in pus
(diplococci). The cocci are about 1 μm, slightly
elongated cocci, with one end broad or rounded
and the other pointed, presenting a flame-shaped
or lanceolate appearance (Fig. 26.1). They are
nonmotile and nonsporing.
All freshly isolated strains are capsulate. The
capsule encloses each pair. The capsule may
be demonstrated as a clear halo in Indian ink
preparations (Fig. 26.2) or may be stained directly
by special techniques or by use of homologous
type-specific antibody in the Quellung reaction.
Cultural Characteristics
They are aerobes and facultative anaerobes. It Fig. 26.2: Pneumococci. Indian ink preparation to show
grows best in air or hydrogen with 5–10% CO2 , capsules
184 | Section 3: Systemic Bacteriology
in enriched media. It grows on ordinary media, but Optochin Sensitivity: Pneumococci are highly
better on media with serum, blood or heated blood. sensitive to killing by optochin (ethyl hydrocuprein
On blood agar, after incubation for 18 hours, hydrochloride), and is useful in distinguishing
the colonies are small (0.5–1 mm), dome-shaped on an area of a blood agar plate inoculated with
and glistening, with an area of green discoloration pneumococcus-like colonies from the primary
(alpha hemolysis) around them. On further diagnostic plate. A growth of Pneumococcus will
incubation, the colonies become flat with raised be inhibited in a zone extending radially for at least
edges and depressed centrally, so that concentric 5 mm from the margin of the disc on incubation.
rings are seen on the surface when viewed from Viridans streptococci will grow right up to the disc.
above (draughtsman or carrom coin appearance)
which is due to autolysis of bacteria within the flat Antigenic Structure
pneumococcal colonies.
1. Capsular Antigens
Under anaerobic conditions, however, a zone of
beta hemolysis is produced around the colony by The most important antigen of the Pneumococcus
an oxygenlabile pneumolysin O. In liquid media is the type specific capsular polysaccharide. It is
such as glucose broth, growth occurs as uniform also called the ‘specific soluble substance’ (SSS)
turbidity. The cocci readily undergo autolysis in as this polysaccharide diffuses into the culture
cultures due to the activity of intracellular enzymes. medium or infective exudates and tissues. These
Autolysis is enhanced by bile salts, sodium lauryl polysaccharides are antigenic and form the basis
sulfate and other surface active agents. for the separation of pneumococci into different
serotypes. A total of 90 different capsular serotypes
have been identified. The serotypes are designated
Biochemical Reactions
by numbers, and those that are structurally related
1. Inulin fermentation: Pneumococci ferment are grouped together (I, 2, 3. 4, 5, 6A, 68, etc.).
several sugars with the production of acid and The tests may be done by :
no gas. Fermentation of inulin by pneumococci 1. Agglutination of washed capsulate cocci.
is a useful test for differentiating them from 2. Precipitation of SSS from culture supernates.
streptococci as the latter do not ferment it. 3. Quellung reaction or capsule swelling reaction
Fermentation is tested in Hiss’s serum water It was described by Neufeld (1902). In the
or serum agar slopes. capsule swelling or ‘quellung’ reaction (quellung
2. Bile solubility test: = swelling), a suspension of pneumococci is mixed
Basis: Bile solubility test for identifying on a slide with a drop of the type specific antiserum
pneum ococci is based on the presence in and a loopful of methylene blue solution and then
pneumococci, of an autolytic amidase that examined using the oil-immersion objective. The
cleaves the bond between alanine and muramic capsule becomes apparently swollen, sharply
acid in the peptidog lycan. The amidase is delineated and refractile in the presence of the
activated by surface-active agents such as bile homologous antiserum. This reaction can be used
or bile salts, resulting in lysis of the organisms. to identify the organism directly from ‘sputum, CSF,
Procedure: When sodium deoxyc holate and other sources. It used to be a routine bedside
solution is added to broth culture, the culture procedure in the past.
clears due to lysis of cocci. Pneumococci are
soluble in bile; viridans and other streptococci 2. Somatic Antigen
are not.
a. C polysaccharide: The cell wall of S. pneumoniae
Alternatively, touch a suspected pneumo
contains a species specific carbohydrate
coccal colony with a loopful of 2% sodium
antigen, referred to as C substance. C-reactive
deoxycholate solution, it disappears, leaving
protein (CRP) is an abnormal protein (beta
an area of α-hemolysis on the blood agar.
globulin) that precipitates with the somatic ‘C’
3. Pneumococci are catalase and oxidase
antigen of pneumococci. It appears in acute
negative.
phase sera of cases of pneumonia but dis
appears during convalescence. It is known as
Resistance C-reactive protein because it precipitates with
Pneumococci are delicate organisms and are killed C antigen of pneumococci. CRP is present in
by moist heat at 55°C in 10 min, and readily by most low concentrations in healthy people but in
disinfectants. Most strains are highly sensitive to elevated concentrations in patients with acute
benzylpenicillin, other penicillins. A drug resistant inflammatory diseases.
Strep. pneumoniae (DRSP) strain originating in It is an ‘acute phase’ substance, produced
Spain has spread to most parts of the world posing in hepatocytes. Its production is stimulated by
problems in treament. bacterial infections, inflammation, malignancies
Chapter 26: Pneumococcus (Diplococcus pneumoniae: Streptococcus Pneumoniae) | 185
and tissue destruction. It disappears when the pneumococcal pneumonia. In children, types
inflammatory reactions subside. It is used as 6, 14, 19 and 23 are frequent causes.
an index of response to treatment in rheumatic ii. Bronchopneumonia: It is almost always a
fever and certain other conditions. secondary infection. This may be caused by
CRP testing, by passive agglutination using any serotype of Pneumococcus. Other causative
latex particles coated with anti-CRP antibody agents responsible for bronchopneumonia
is a routine diagnostic procedure. include Staph. aureus, K. pneumoniae, Str.
b. F antigen: The lipid bound teichoic acid in the pyogenes, H. influenzae, Fusobacterium species
bacterial cytoplasmic membrane is called the and Bacteroides.
F or Forssman antigen because it can cross-
react with the Forssman surface antigens on 2. Acute Exacerbations in Chronic Bronchitis
mammalian cells. Pneumococci are commonly associated with
c. M protein: Type-specific protein antigens the acute exacerbations in chronic bronchitis.
analogous to the M protein of Streptococcus Another bacterium commonly associated with this
pyogenes. condition is Haemophilus influenzae.
Learning Objectives
After reading and studying this chapter, you should ∙∙ Describe morphology, culture characteristics,
be able to: biochemical reactions of Neisseria gonorrhoeae
∙∙ Describe morphology, culture characteristics, ∙∙ Discuss pathogenicity of N. gonorrhoeae
biochemical reactions and antigenic structure of ∙∙ Discuss laboratory diagnosis of gonorrhoea
Neisseria meningitidis ∙∙ Explain nongonococcal urethritis (or) nonspecific
∙∙ Discuss pathogenicity and lab diagnosis of urethritis
meningococcal meningitis ∙∙ Describe Morexella (Branhamella) catarrhalis.
Neisseria meningitidis
(Meningococcus; Diplococcus
intracellularis meningitidis)
Morphology
Meningococci are gram-negative oval or spherical
cocci (0.6–0.8 μm in size), typically arranged
in pairs, with the adjacent sides flattened or
concave opposing edges and the long axes parallel.
They are typically seen in large numbers inside
polymorphonuclear leukocytes (Fig. 27.1).
Most fresh isolates are capsulated. They are Fig. 27.1: Neisseria meningitidis in cerebrospinal fluid.
nonsporing and nonmotile. Inset—enlarged view showing flat adjacent side of cocci
Chapter 27: Neisseria and Moraxella | 189
culturing meningococci. Modified Thayer -Martin Stages of Meningococcal Infections
(with vancomycin, colistin and nystatin) is a useful There are three stages.
selective medium.
On blood agar after 24 hours incubation, First Stage—Nasopharyngeal Infection
colonies are 1–2 mm in diameter, round, convex, The organisms appear in nasopharynx leading
gray, translucent and nonhemolytic. Heated blood to nasopharyngeal infection, which is usually
(chocolate) agar-colonies are slightly larger on asymptomatic.
heated blood (chocolate) agar than on ordinary
blood agar. Growth is poor in liquid media, producing Second Stage—Meningococcal Septicemia
a granular turbidity with little or no surface growth. In a small percentage of cases, the meningococci enter
the bloodstream from the posterior nasopharynx.
This stage is called meningococcemia. The patient
Biochemical Reactions
develops fever, malaise and petechial skin lesions.
1. They are catalase and oxidase positive. The organisms may also cause lesions in the
Oxidase test: When 1% solution of oxidase joints and lungs and rarely cause massive bilateral
reagent (tetramethylparaphenylene-diamine- hemorrhages in the adrenals (Waterhouse–
dihydrochloride) is poured on culture media, Friderichsen syndrome). It is an overwhelming
Neisseria colonies quickly turn deep-purple. and usually fatal condition, characterized by shock,
This prompt oxidase reaction helps in the disseminated intravascular coagulation (DIC)
identification of meningococci and gonococci and multisystem failure. Meningococcal disease is
in mixed cultures. favored by deficiency of the terminal complement
The test may also be performed by rubbing components (C5–C9).
a little of the growth with a loop on a strip of
filter paper moistened with the oxidase reagent Third Stage—Meningitis
(Kovacs’ method). A deep purple color appears In the third stage of meningococcal infection, the
immediately. organisms can cross the blood–brain barrier and
2. Sugar fermentation: Meningococci ferment infect the meninges. The route of spread from the
glucose and maltose with the production of nasopharynx to the meninges is controversial. The
acid but no gas, but not lactose or sucrose spread may be directly along the perineural sheath
(gonococci ferment glucose but not maltose). of the olfactory nerve, through the cribriform plate
to the subarachnoid space, or more probably,
3. Indole and hydrogen sulfide are not produced
through the bloodstream. In certain cases, the site of
and nitrates are not reduced.
entry of the Meningococcus may be the conjunctiva.
On reaching the central nervous system, a
Antigenic Classification suppurative lesion of the meninges is set up. Case
fatality is variable but in untreated cases may be as
Based on their capsular polysaccharide antigens,
high as 80%. Survivors may have sequelae such as
meningococci are classified into at least 13
blindness and deafness.
serogroups: A, B, C, X, Y, Z, Zl (29E) and W135.
The pathogenic agent in meningococcal disease
Further serogroups H, I, K and L have also been
appears to be the endotoxin (LPS) released by
described. A group D was described but no
autolysis. The vascular endothelium is particularly
capsular polysaccharide specific for this group has
sensitive to the endotoxin.
yet been demonstrated. Groups A, B and C are the
most important. Serogroups are further classified Epidemiology
into serotypes and subtypes.
Humans are the only natural carriers for N. menin
gitidis. The oral and nasopharyngeal carriage rates are
Resistance highest for school-aged children and young adults,
are higher in lower socioeconomic populations.
Meningococci are very delicate organisms, being
The carrier rate is higher in the members of the
highly susceptible to heat, dessication, alterations
household of a patient with meningococcal disease.
in pH and to disinfectants. They die within a few
Natural infection is limited to human beings.
days at room temperature. They are killed by
heating at 55°C in 5 minutes. Laboratory Diagnosis
1. Specimens
Pathogenicity
i. Cerebrospinal fluid (CSF),
Meningococci are strict human parasites ii. Blood for culture (which may come from a
inhabiting the nasopharynx. Infection is usually patient with meningitis, a hemorrhagic rash
asymptomatic. or pyrexia of uncertain origin)
190 | Section 3: Systemic Bacteriology
iii. Aspirate from skin lesions or pus from an The oxidase test is performed on colonies on
infected joint, solid medium.
vi. Throat or nasopharyngeal swabs from vi. Antibiotic sensitivity tests
suspected cases. Swabs should be transported Set up antibiotic sensitivity tests.
in Stuart’s transport medium. All speci vii. Serogrouping is performed by slide agglutina
mens where meningococcal infection is tion with hyperimmune sera that provide
suspected must be submitted to the laboratory important epidemiological information.
immediately.
3. Blood Cultures
2. Examination of CSF Blood culture is often positive in meningococcemia
If meningitis is suspected, a lumbar puncture and in early cases of meningitis. Cultures should
should be performed as soon as possible unless be incubated for 4–7 days, with daily subcultures.
there are signs of raised intracranial pressure. Subculture to blood agar and heated blood agar.
i. Perform a cell count. The exudate in mening Incubate cultures in 5–10% CO 2 for 24 hours
ococcal meningitis is typically polymor and examine oxidase-positive colonies of gram-
phonuclear. negative diplococci as above.
ii. Centrifuge the remaining CSF. Make a
smear of the centrifuged deposit and stain 4. Pus, Aspirates and Swabs
with Gram-stain. CSF from a typical case of Gram-stained films are examined. In addition
meningococcal meningitis will show gram- to blood agar and heated blood agar, Thayer–
negative diplococci inside a limited proportion Martin selective medium is used for the culture of
of the pus cells; many are extracellular. materials expected to yield a mixture of organisms
Stain a second film with methylene blue to such as pus, aspirates, and throat, nasopharyngeal
determine the cell type; occasionally, diplococci and genital swabs.
may be seen more easily with this stain.
If fluorescein isothiocyanate coupled
5. Petechial Lesions
antiserum is available, a smear of the deposit
may be examined for the direct identification Meningococci may sometimes be demonstrated in
of the meningococcal serogroup responsible petechial lesions by microscopy and culture.
for infection.
iii. Divide the supernatant CSF into two aliquots: 6. Serological Diagnosis
One to be kept if necessary for biochemical Paired sera may be tested for the presence of
examination, the other to be examined for the complement-fixing antibodies.
presence of meningococcal polysaccharide Specific antibodies to capsular polysaccharide
antigen by counterimmunoelectrophoresis, may be demonstrated by a (i) hemagglutination
latex agglutination or coagglutination using test (ii) ELISA tests.
meningococcal antisera. Similar tests are also
available for pneumococcus, H. influenzae 7. Polymerase Chain Reaction (PCR)
type b and group B Streptococcus antigens. Group specific diagnosis of infection can be made
Antigen detection is particularly useful in by detection of meningococcal DNA sequence in
partially treated patients in whom smear and CSF or blood by PCR amplification.
culture tests may be negative.
iv. Cuture: Plate out the centrifuged deposit on Treatment
both blood and heated blood agar (chocolate
agar) and incubate at 37°C in 5–10% CO2. Penicillin is currently the antibiotic of choice.
Colonies appear after 18–24 hours which Chloramphenicol is effective, but risks of blood
may be identified by morphology and dyscrasia have limited its use. Either chloramphenicol
biochemical reactions. or a third-generation cephalosporin, such as
Subculture: Add Robertson’s cooked meat cefotaxime or ceftriaxone is used in persons allergic
broth to the remaining deposit, incubate to penicillin allergy.
overnight and subculture in the same way. In At the end of a course of therapy with penicillin,
the absence of visible meningococci, glucose it is important to give eradicative treatment with
broth may be added to the remaining sed rifampicin or ciprofloxacin.
iment of the centrifuged deposit to facilitate
the isolation. Prophylaxis
v. Biochemical reactions 1. Chemoprophylaxis: Minocycline and rifampin
Sugar utilization tests or commercial kits are have been used effectively for antibiotic-
used to identify any gram-negative diplococci. mediated chemoprophylaxis. Ciprofloxacin is
Chapter 27: Neisseria and Moraxella | 191
widely used as a prophylactic for adolescents round, translucent, convex or slightly umbonate,
and adults as a single, oral dose. with finely granular surface and lobate margins
2. Immunoprophylaxis: A polyvalent vaccine after incubation for 24 hours..They are soft and
effective against serogroups A, C, Y, and W135, easily emulsifiable. After 48 hours, the colonies are
which can be administered to children older larger (1.5–2.5 mm), sometimes with a crenated
than 2 years, has been developed. The vaccine margin and an opaque raised center.
cannot be administered to children in younger Types of gonococci: Kellogg divided gonococci
age groups because they do not respond to into four types (T1–T4) on the basis of colonial
polysaccharide antigens. The immunity is appearance, auto-agglutinability and virulence.
group specific. There is no Group B vaccine
available at present. Types 1 and 2 form small brown colonies and bear
numerous fimbriae (piliated types P1 and P2). They
are auto-agglutinable and virulent.
Neisseria gonorrhoeae
(gonococcus) Types T3 and T4 are nonpiliated (P–), form smooth
suspensions and are avirulent.
N. gonorrhoeae causes the venereal disease Tl and T2 types are also known as P+ and P++,
gonorrhea. Gonococci resemble meningococci respectively, while T3 and T4 are known as P–.
very closely in many properties.
Biochemical Reactions
Morphology
Gonococcus is oxidase positive and resembles
Morphology and staining of N. gonorrhoeae are mening ococci except in the fermentation of
identical to those of N. meningitidis. In smears maltose. Gonococci ferment only glucose and
from the urethral discharge in acute gonorrhea, not maltose and neither species ferments lactose
the organism appears as a Diplococcus with the or sucrose. This can be remembered by G for
adjacent sides concave (Fig. 27.2), being typically Gonococcus and M+G for Meningococcus.
kidney-shaped. It is found predominantly within
the polymorphs, some cells containing as many
Antigenic Structure
as a hundred cocci.
The structure of N. gonorrhoeae is typical of gram-
Cultural Characterstics negative bacteria. The surface structures include
Gonococci are more diffic ult to grow than the following:
meningococci They are aerobic but may grow 1. Pili: Pili are hair-like appendages that extend
anaerobically also. Growth occurs best at pH 7.0– up to several micrometers from the gonococcal
7.4 and at a temperature of 35–36°C. It is essential surface. They act as virulence factors by
to provide 5–10% CO2. promoting attachment to host cells and
They grow well on chocolate agar and Mueller– inhibiting phagocytosis. The pili are composed
Hinton agar. A popular selective medium is of repeating protein subunits (pilins). Pili
the Thayer–Martin medium (chocolate agar undergo antigenic and phase variation.
containing vancomycin, colistin and nystatin) 2. Por proteins (protein I): The Por proteins
which inhibits most contaminants, including (formerly protein I) are porin proteins that form
nonpathogenic Neisseria. Colonies are small, pores or channels in the outer membrane. Two
classes of Por proteins (PorA and PorB), have
been identified. Anyone strain carries only
either IA or IB but not both.
3. Opa protein (protein II): These proteins facili
tate bacterial adherence to each other and to
eukaryotic cells and also for the clumping of
cocci seen in urethral exudate smears.
4. Rmp (protein III): These proteins stimulate
antibodies that block serum bactericidal
activity against N. gonorrhoeae.
5. Lipooligosaccharide (LOS): This antigen
possesses endotoxic activity.
6. Other proteins: Other important gonococcal
proteins are IgA1 protease, β-lactamase,
Fig. 27.2: N. gonorrhoeae in urethral pus. Inset— enlarged which degrades penicillin and Fbp (iron-
view showing diplococci with adjacent surfaces concave binding protein).
192 | Section 3: Systemic Bacteriology
Resistance Once ver y common, this has been
Gonococcus is a very delicate organism, readily controlled by the practice of instilling 1%
killed by drying, soap and water, and many other silver nitrate solution into the eyes of all
cleansing or antiseptic agents at their correct use- newborn babies (Crede’s method).
dilution. Organisms may remain viable for a day or ii. Vulvovaginitis: In prepubertal girls,
so in pus contaminating linen or other fabrics. In vulvovaginitis may be caused by gonococci.
cultures, the coccus dies in 3–4 days at room tem This occurs either in conditions of poor
perature. Freeze-drying is the most reliable method hygiene or by sexual abuse.
for long-term storage of gonococci but storage at
–70°C or in liquid nitrogen may be more convenient Epidemiology
for intermediate storage. Gonorrhea occurs only in humans. It has no other
known reservoir. N. gonorrhoeae is transmitted
Pathogenesis primarily by sexual contact. A higher incidence of
Gonorrhea gonorrhea has been observed in persons belonging
to blood group B. The basis for this is not known.
Gonorrhea is a venereal disease. The disease is
acquired by sexual contact. The incubation period
is 2–8 days. Laboratory Diagnosis
A. Disease in men: The most common clinical Diagnosis can be established readily in the acute
presentation is acute urethritis in the male. stage but chronic cases sometimes present great
Dysuria and a purulent penile discharge difficulties.
make most sufferers seek treatment rapidly.
The infection extends along the urethra to the 1. Specimens
prostate, seminal vesicles and epididymis. A. Specimens in Men
Chronic urethritis may lead to stricture 1. Urethra: In men, urethral samples usually suffice
formation. The infection may spread to the (with rectal cultures in homosexual males).
periurethral tissues, causing abscesses and In acute gonorrhea, the urethral discharge
multiple discharging sinuses (‘watercan contains gonococci in large numb ers. The
perineum’). meatus is cleaned with a gauze soaked in saline
B. Disease in women: Asymptomatic carriage and a sample of the discharge collected with a
in women is common, espec ially in the platinum loop for culture, or directly on slide for
endocervical canal. The infection may extend smears. Purulent discharge may be expressed at
to Bartholin’s glands, endometrium and the anterior urethra and collected with a swab.
fallopian tubes to give rise to acute salpingitis, In chronic infections, there may not be any
which may be followed by pelvic inflammatory uret hral discharge. The morning drop of
disease and a high probability of sterility. secretion may be examined or some exudate
Peritoneal spread occasionally occurs and may may be obtained after prostatic massage. It may
produce a perihepatic inflammation (Fitz– also be possible to demonstrate gonococci in
Hugh–Curtis syndrome). the centrifuged deposits of urine in cases where
Proctitis occurs in both sexes. Gonococcal no urethral discharge is available.
pharyngitis may follow orogenital contact in 2. Anal canal: In homosexual males.
either sex. Conjunctivitis may occur usually by
B. Specimens in Women
autoinoculation with fingers.
1. Endocervical swab: In women, uret hral,
C. Disseminated gonococcal disease: Blood cervical and rectal specimens should always be
invasion may occur from the primary site of examined. A single well taken endocervical swab
infection and may lead to metastatic lesions will detect approximately 90% of gonococcal
such as arthritis, ulcerative endocarditis and infections in women. A high vaginal swab is
very rarely meningitis. not suitable. Throat infection also occurs and
D. Disease in childern should be sought where appropriate.
i. Ophthalmic neonatorum: A nonvenereal 2. Urethral
infection is ophthalmia neonatorum in 3. Anal canal and
the newborn, in which the eyes are coated 4. Throat.
with gonococci as the baby passes down
the birth canal. A severe purulent eye C. Blood, Swabs of skin lesions, or pus aspirated
from a joint.
discharge with periorbital edema occurs
within a few days of birth. If untreated, D. Conjunctival Swab: Particularly in neonatal
ophthalmia leads rapidly to blindness. ophthalmia.
Chapter 27: Neisseria and Moraxella | 193
E. Urine Specimen: Any urine specimen showing 6. Genetic Probes
gram-negative diplococci in a Gram stain should Probes specific for the nucleic acids of N. gonorr
be cultured on an appropriate selective medium. hoeae have been developed for the direct detection
of bacteria in clinical specimens.
2. Transport
For culture, specimens should be inoculated on 7. Serological Diagnosis
prewarmed plates, immediately on collection. If No serological test has been found useful for
this is not possible, specimens should be collected routine diagnostic purposes.
with charcoal impregnated swabs and sent to the
laboratory in Stuart’s transport medium. Treatment
Penicillin is no longer the antibiotic of choice for
3. Direct Microscopy treatment of gonorrhea since the development and
Do the Gram staining shows characteristic kidney- widespread use of penicillin, gonococcal resistance
shaped gram-negative diplococci lying within to penicillin has gradually risen, owing to the selec-
polymorphonuclear leukocytes with a few extra tion of chromosomal mutants, so that many strains
cellular. Approximately 95% of infected men will now require high concentrations of penicillin G for
yield a positive smear. It has to be emphasized that inhibition (MIC 2 µg/mL).
diagnosis of gonorrhea by smear examination is Penicillinase p roducing gonococci (PPNG):
unreliable in women as some of the normal genital In 1976, gonococci producing β-lactamase
flora have an essentially similar morphology. (penicillinase) have appeared, rendering penicillin
Immunologic methods employing monoclonal treatment ineffective. Penicillinase production, in
antibodies may be used for the identification of gonococci, is plasmid-mediated.
N. g onorrho eae. These methods include
Chromosomally-mediated resistance (CMRNG):
coagglutination and fluorescent antibody testing.
This chromosomally-mediated resistance
(CMRNG) is not limited only to penicillin but
4. Culture extends to tetracyclines, erythromycin, and
In acute gonorrhea, cultures can be obtained aminoglycosides.
readily on chocolate agar or Mueller–Hinton agar Empirical therapy: Currently, the Centers for Dis
incubated at 35–36°C under 5–10% CO2. In chronic ease Control and Prevention (CDC) recommends
cases, where mixed infection is usual and in the that ceftriaxone, cefixime, ciprofloxacin, or
examination of lesions, such as proctitis; however, ofloxacin be used as the initial therapy for cases
it is better to use a selective medium such as the of uncomplicated gonorrhea. Doxycycline or
Thayer–Martin medium. Examine plates after 24 azithromycin should be added for infections
hours incubation and the growth is identified by complicated by dual infections with Chlamydia.
morphology and biochemical reactions. Incubation
of primary isolation plates is continued for 48 hours. Prophylaxis
Colonies are small, round, translucent, convex
Control of gonorrhea consists of early detection of
or slightly umbonate, with finely granular surface
cases, contact tracing, health education and other
and lobate margins. They are soft and easily
general measures.
emulsifiable. After 48 hours, the colonies are larger
As even clinical disease does not confer any
(1.5–2.5 mm), sometimes with a crenated margin
immunity, vaccination has no place in prophylaxis.
and an opaque raised center.
Smear is made from the colony and Gram Nongonococcal (nonspecific)
staining is done. Gonococci are gram-negative
cocci arranged in pairs (diplococci) with adjacent
urethritis
sides concave (pear or bean shaped). Nongonococcal urethritis (NGU), also known
as nonspecific urethritis (NSU), refers to chronic
5. Identification urethritis where gonococci cannot be demonstrated.
N. gonorrhoeae is identified preliminarily on the
basis of the isolation of oxidase-positive, gram-
Causative Agents
negative diplococci that grow on chocolate blood The most important causative agents are as follows:
agar or on media that are selective for pathogenic
Neisseria species.N gonorrhoeae is oxidase positive. A. Bacterial
It ferments glucose with acid only. It does not ∙∙ Chlamydia trachomatis
ferment maltose unlike meningococci. ∙∙ Ureaplasma urealyticum
194 | Section 3: Systemic Bacteriology
∙∙ Mycoplasma hominis N. flavescens, N. catarrhalis) have been reported
∙∙ Gardnerella vaginalis occasionally as having caused meningitis. N.
∙∙ Acinetobacter wolfii sicca, N. subflava, N.cinera, N. mucosa, and N.
∙∙ Acinetobacter calcoaceticus. flavescens are also members of the normal flora of
the respiratory tract, because of its carbohydrate
B. Viral fermentation pattern.
∙∙ Herpes virus
∙∙ Cytomegalovirus. Moraxella
C. Fungi The genus Moraxella is a member of the family
Neisseriaceae.
∙∙ Candida albicans.
Species: The Moraxella spp. of medical importance
D. Protozoa are M. lacunata, M. catarrhalis, M. osloensis, M.
∙∙ Trichomonas vaginalis. phenylpyruvica, M. atlantae and M. nonliquefaciens.
Corynebacterium
Learning Objectives
After reading and studying this chapter, you should ∙∙ Discuss diphtheria toxin
be able to: ∙∙ Discuss laboratory diagnosis of diphtheria
∙∙ Describe morphology, cultural characteristics, ∙∙ Toxigenicity tests/virulence tests of C. diphtheriae.
biochemical reactions, toxin production and ∙∙ Describe the following: Schick test; DPT vaccine or
pathogenesis of diphtheria triple vaccine; diphtheroids
∙∙ Differentiate three biotypes: Gravis, intermedius ∙∙ Differentiate between C. diphtheriae and diphthe
and mitis of C. diphtheriae roids.
Corynebacterium diphtheriae
Morphology
They are, thin, slender gram-positive bacilli but
are decolorized easily, particularly in old cultures,
measuring approximately 3–6 μm × 0.6–0.8 μm.
They have a tendency to clubbing at one or both
ends. They are highly pleomorphic. They are non-
motile, non-spore forming, and nonacid fast.
The bacilli are arranged in a characteristic
fashion in smears. They are usually seen in pairs, Fig. 28.1: Corynebacterium diphtheriae showing
palisades (resembling stakes of a fence) or small metachromatic granules and Chinese letter arrangement
198 | Section 3: Systemic Bacteriology
Albert’s stain, the granules stain bluish black and of cystine to a tellurite containing medium
the protoplasm green. The granules represent (Tinsdale’s medium) has greatly helped the
accumulation of polymerized polyphosphates. isolation of diphtheria bacilli. The growth
The granules formation is best seen on Loeffler’s of diphtheria bacilli may be delayed on the
serum slope. tellurite medium and colonies may take two
days to appear.
Cultural Characteristics Based on colonial morphology on the tellurite
medium and other properties, Mc Leod and
C. diphtheriae is an aerobe and facultative anaerobe;
Anderson described three different biotypes:
the optimum temperature for growth is 37°C (range
Gravis, intermedius and mitis (Table 28.1). The
15–40°C) and optimum pH 7.2. It can grow on
names were originally proposed to relate to the
ordinary nutrient agar, but its growth is improved
clinical severity of the disease produced by the
by the presence of animal proteins such as blood
three types—gravis, causing the most serious, and
or serum. Two media are useful for this purpose:
mitis the mildest variety, with intermedius being
1. Loeffler’s serum slope: Diphtheria bacilli
responsible for disease of intermediate severity.
grow on LoeffIer’s serum slope very rapidly
and colonies can be seen in 6–8 hours, long
before other bacteria grow. Colonies are at first Biochemical Reactions
small, circular white opaque disks but enlarge C. diphtheriae ferments glucose and maltose
on continued incubation and may acquire a with the production of acid (but no gas) but not
distinct yellow tint. lactose, mannitol, trehalose or sucrose. Starch and
2. Tellurite blood agar: The addition of potassium glycogen are used for biochemical differentiation
tellurite (0.03–0.04%) makes the medium of three biotypes of C. diphtheriae (Table 28.1).
selective for Corynebacteria by inhibiting most Gravis strains utilize glycogen and starch, while
other pathogenic and commensal bacteria. On mitis and intermedius do not. Fermentation of
this medium, C. diphtheriae give grey/black, sugars are usually done in Hiss’s serum peptone
shiny or dull black colonies. The addition water medium.
Table 28.1 Differentiating features of Corynebacterium diphtheriae
Gravis Intermedius Mitis
1. Morphology i. Usually short rods, with i. Long barred forms with i. Long, curved, rods
uniform staining clubbed ends; ii. Prominent granules
ii. Few or no granules. ii. Poor granulation iii. Pleomorphic
iii. P leomorphism (some iii. Very pleomorphic
degree), with irregularly
barred, snow-shoe and
teardrop forms
2. Colony on tellurite i. In 18 hours: Colony is i. 18 hour: Colony small, I i. Size variable, shiny black.
blood agar 1–2 mm in size, greyish mm in size, misty.
black centre, paler,
semitranslucent periphery
and commencing ii. i n 48 hours Does not ii. In 2–3 days: Colonies
crenation of edge. enlarge, dull granular become flat, with
ii. In 2–3 days: 3–5 mm in centre with smoother, more a central elevation
size, flat colony with raised glistening periphery and a ‘poached egg’ colony
dark centre and crenated lighter ring near the edge -
edge with radial striation - ‘frog’s egg’ colony
‘daisy head’ colony
3. Consistency of colonies i. Brittle, moves as a whole Intermediate between (i) Soft, buttery, (ii) Easily
on the plate like ‘cold gravis and mitis emulsifiable
margarine’.
ii. Not easily picked out or
emulsifiable
4. Hemolysis Variable Nonhemolytic Usually hemolytic
5. Growth in broth Surface pellicle. Deposit Turbidity in 24 hours, Turbidity diffuse with soft
granular. clearing in 48 hours, with pellicle later
Turbidity little or no fine granular sediment
6. Glycogen and starch Positive Negative Negative
fermentation
7. Toxigenic strains Almost 100% 95–99% 80–85%
8. Virulence Severe Moderate Mild
9. Predominant strains Epidemic areas Epidemic areas Endemic areas
Chapter 28: Corynebacterium | 199
C. diphtheriae is H2S positive and reduces and mediates the entry of fragment A into the
nitrate to nitrite. It does not liquefy gelatin or cytoplasm. The antibody to fragment B prevents the
hydrolyze urea or form phosphatase. binding of toxin to cells and is thus protective. The
Pyrazinamidase (PYZ) test: In pyrazinamidase toxin is heat labile. It is extremely potent (0.0001 mg
(PYZ) test, pyrazinamide is converted into pyrazinoic kills a guinea pig of 250 g weight). It is converted into
acid by the organisms which produce pyrazinamidase toxoid by heat (at 37°C for 4–6 weeks), treatment
(PYZ). This test is helpful to distinguish ‘C. diphtheriae’ with 0.2–0.4% formalin or by acidic pH. The toxoid
(PYZ-negative) from other Corynebacterium species is a toxin that has lost its toxicity but has retained the
(mostly PYZ-positive). antigenicity. It is capable of producing antitoxin. It
has a special affinity for certain tissues such as the
myocardium, adrenals and nerve endings.
Toxin
Toxigenic strains of C. diphtheriae produce a a Mode of Action
very powerful exotoxin. The toxicity observed
The diphtheria toxin acts by inhibiting protein
in diphtheria is directly attributed to the toxin
synthesis. It inhibits polypeptide chain elongation
secreted by the bacteria at the site of infection.
in the presence of nicotinamide adenosine
dinucleotide (NAD) by inactivating elongation
Synthesis factor 2 (EF-2), an enzyme required for elongation
Almost all strains of gravis, 95-99% of intermedius of polypeptide chains on ribosomes. Inhibition of
and 80-85% of mitis produce this toxin. Strains of protein synthesis is probably responsible for both
all three types are invariably virulent when isolated the necrotic and neurotoxic effects of the toxin.
from acute cases. Avirulent strains are common NAD+ + EF–2 = ADPR–EF–2 + nicotinamide + H+
among convalescents, contacts and carriers. The Active Inactive
strain almost universally used for toxin production
is the ‘Park Williams 8’ strain, which has been Resistance
variously described as a mitis (Topley and Wilson)
and an intermedius strain (Cruickshank). They are readily killed, however, by a 1-minute
exposure to100°C or a 10 minute exposure to 58°C.
They are susceptible to most of the routinely used
Lysogeny and toxin Production
disinfectants. It remains alive for weeks in dust and
The toxigenicity of the diphtheria bacillus depends on fomites when dry and protected from sunlight.
on the presence in it of corynephages (tox +), It is susceptible to penicillin, erythromycin and
which act as the genetic determinant controlling broad spectrum antibiotics.
toxin production. Nontoxigenic strains can be
converted to tox+ by infection with the appropriate Antigenic Structure
bacteriophage. This is known as lysogenic or phage
conversion. The bacillus loses the toxigenicity Diphtheria bacilli possess three distinct antigens:
when it is cured of its phage, as by growing it in the 1. A deep-seated antigen found in all Coryne
presence of antiphage serum. bacterial species
2. A heat-labile protein (K-antigen)
3. A heat-stable polysaccharide (O-antigen).
Iron for toxin Production
Toxin production is also influenced by the Serotypes: On the basis of agglutination reaction,
concentration of iron in the medium. The optimum biotypes gravis, intermedius and mitis have been
level of iron for toxin production is 0.1 mg per liter, divided into 13, 4 and 40 serotypes, respectively.
while a concentration of 0.5 mg per liter inhibits
the formation of toxin. The toxin is released in Bacteriophage Typing
significant amounts only when the available iron The susceptibility of C. diphtheriae to bacterio
in the culture medium is exhausted. phage strains has been most comprehensively
studied; 22 phages were used to type the gravis
Properties of toxin strains into 14 types, the intermedius into 3 and the
Diphtheria toxin is an iron-free, crystalline, heat mitis into 4. An additional set of 33 phages has also
labile protein. The diphtheria toxin is a protein and been used. The only other corynebacteria reported
has a molecular weight of about 62000. It consists as susceptible to the diphtheria typing phages are
of two fragments A (active) and B (binding) of C. ulcerans and C. pseudotuberculosis.
molecular weights 24000 and 38000 respectively.
Both fragments are required for toxicity. Fragment Pathogenesis
A has all the enzymatic activity whereas fragment The organism is carried in the upper respiratory tract
B is responsible for binding the toxin to the cells and spread by droplet infection or hand-to-mouth
200 | Section 3: Systemic Bacteriology
contact. The incubation period of diphtheria is 2–5 systemic complications are less common than from
days, with a range of 1–10 days. Diphtheria, which upper respiratory infections with C. diphtheriae.
occurs in two forms (respiratory and cutaneous),
is found worldwide. Laboratory Diagnosis
Diagnostic laboratory tests serve to confirm the
A. Respiratory Diphtheria clinical impression and are of epidemiologic
The illness begins gradually and is characterized significance but not for the treatment of individual
by low-grade fever, malaise, and a mild sore throat. cases. Specific treatment should be instituted
The most common site of infection is the tonsils immediately on suspicion of diphtheria without
or pharynx. The organisms rapidly multiply on waiting for laboratory tests. Any delay may be
the epithelial cells, and the toxigenic strains of C. fatal. Laboratory diagnosis consists of isolation
diphtheriae produce toxin locally, causing tissue of the diphtheria bacillus and demonstration of
necrosis and exudate formation triggering an its toxicity.
inflammatory reaction. This combination of cell 1. Specimens
necrosis and exudate forms a tough gray to white
Swabs from the nose, throat, or other suspected
pseudomembrane, which attaches to the tissues-
lesions must be obtained before antimicrobial
commonly over the tonsils, pharynx, or larynx. Any
drugs are administered. In suspected cases,
attempt to remove the pseudomembrane results
whether of faucial or nasal dipht heria, swabs
in bleeding. In nasopharyngeal infection, the
should be taken both from the throat and from the
pseudomembrane may involve nasal mucosa, the
nose, and preferably two swabs from the site most
pharyngeal wall and the soft palate. In this form,
affected. Swabs should also be taken from skin
oedema involving the cervical lymph glands may
lesions and wounds where diphtheritic infection is
occur in the anterior tissues of the neck, a condition
suspected, and both throat and nose swabs should
known as bullneck diphtheria. Laryngeal
be taken from suspected carriers.
involvement leads to obstruction of the larynx and
lower airways. 2. Microscopy
Systemic effects Direct microscopy of a smear is unreliable since
The toxin also is absorbed and can produce a vari C. diphtheriae is morphologically similar to
ety of systemic effects involving the kidneys, heart, other coryneforms. Smears stained with alkaline
and nervous system, although all tissues possess methylene blue or Gram’s stain show beaded rods
the receptor for the toxin and may be affected. in typical arrangement. Hence smear examination
Intoxication takes the form of myocarditis and alone is not sufficient for diagnosing diphtheria
peripheral neuritis, and may be associated with but is important in identifying Vincent’s angina.
thrombocytopenia. Visual disturbance, difficulty in For this, a Gram or Leishman stained smear is
swallowing and paralysis of the arms and legs also examined for Vincent’s spirochetes and fusiform
occur but usually resolve spontaneously. Complete bacilli. Toxigenic diphtheria bacilli may be
heart block may result from myocarditis. Death is identified in smears by immunofluorescence.
most commonly due to congestive heart failure and
cardiac arrhythmias. 3. Culture
The swab should be inoculated on Loffler’s serum
Complications slope, tellurite blood agar, and blood agar. The
The common complications are as follows: cultures should be incubated aerobically at 37°C.
1. Asphyxia due to mechanical obstruction of the Unless the swab can be inoculated promptly, it
respiratory passage by the pseudomembrane should be kept moistened with sterile horse serum
for which an emergency tracheostomy may so the bacilli will remain viable.
become necessary. i. Loeffler’s serum slope: After incubation for 6
2. Acute circulatorv failure, which may be hours or overnight, make a smear of growth from
peripheral or cardiac. all parts of the slope mixed in the condensation
3. Postdiphtheritic paralysis, which typically water, stain by the Albert-Laybourn method and
occurs in the third or fourth week of the disease; look for the presence of slender green-stained
palatine and ciliary but not pupillary paralysis bacilli containing the purple-black granules
is characteristic, and spontaneous recovery is characteristic of C. diphtheriae.
the rule. ii. Tellurite blood agar: Blood tellurite agar is
4. Septic, such as pneumonia and otitis media. examined after 24 hours and after 48 hours,
as growth may sometimes be delayed.
B. Cutaneous Diphtheria iii. Blood agar: It is used for differentiating
In cutaneous diphtheria, which is prevalent in the streptococcal or staphylococcal pharyngitis,
tropics, the toxin also is absorbed systemically, but which may simulate diphtheria.
Chapter 28: Corynebacterium | 201
4. Identification Tests Advantages of the intracutaneous test:
Identification is based on carbohydrate ferment a. The animals do not die
ation reactions and enzymatic activities. C. b. As many as ten strains can be tested at a time
diphtheriae ferments glucose and maltose, on a rabbit.
producing acid but not gas, and is catalase positive. B. In vitro Test
It reduces nitrate to nitrite and is nonmotile. i. Elek’s gel precipitation test: The in vitro
Commercial kits such as the API Coryne strip diphtheria toxin detection procedure is an
provide a reliable identification. immunodiffusion test first described by Elek.
Procedure: A rectangular strip of filter paper
5. Virulence Tests impregnated with diphtheria antitoxin (1000 units/
Such tests are really tests for toxigenicity of an ml) is placed on the surface of a 20% normal horse
isolated diphtheria-like organism. Diagnosis of serum agar in a Petri dish while the medium is still
diphtheria depends on showing that the isolate fluid. When the agar has set, the surface is dried.
produces diphtheria toxin. Virulence testing may The plate should be streaked with the test strain as
be by in vivo or in vitro methods. well as the control positive and negative strains at
A. In vivo tests right angles to the strip in a single straight line and
i. Subcutaneous test parallel to each other. The plate is incubated at 37°C
and examined after 24 and 48 hours.
ii. Intracutaneous test
B. In vitro test Interpretation: Toxins produced by the bacterial
growth will diffuse in the agar and where it meets the
i. Precipitation test
antitoxin at optimum concentration will produce a
ii. Tissue culture test line of precipitation (Fig. 28.2). A negative control
iii. Enzyme-linked immunosorbent assays should be free of any line. No precipitate will form
iv. Polymerase chain reaction (PCR). in the case of nontoxigenic strains.
A. In vivo Tests ii. Tissue culture test : The toxigenicity of
i. Subcutaneous test: The growth from an diphtheria bacilli can be demonstrated by
overnight culture on Loeffler’s slope is incorporating the strains in the agar overlay of
emulsified in 2–4 mL broth and 1 mL of the cell culture monolayers. The toxin produced
emulsion injected subcutaneously into diffuses into the cells below and kills them.
two guinea pigs or rabbits, one of which has iii. Enzyme-linked immunosorb ent assays
been protected with the diphtheria antitoxin (ELISA): Rapid, enzyme-linked immunosor
(500–1000 units) 18–24 hours previously and bent assays and immunochromatographic
was used as control. If the strain is virulent, strip assays are also available for the detection
the unprotected animal will die within of diphtheria toxin.
four days. Postmortem examination would iv. Polymerase chain reaction (PCR): In addition,
show hemorrhage at the site of injection procedures for detecting the C. diphtheriae tox
and injected blood vessels, with typically gene by the polymerase chain reaction (PCR)
hemorrhagic adrenal necrosis. have been developed. The PCR assay can also
Simple and reliable subcutaneous be applied directly to clinical specimens.
toxigenicity tests in rabbits or larger guinea- Schick Test
pigs were used in the past, at a time when
laboratories had many isolates each day. Schick (1913) introduced an intradermal test
The method is not usually employed as it is (Schick test) for distinguishing between susceptible
wasteful of animals. and immune persons.
ii. Intracutaneous (Intradermal) test: The
broth emulsion of the culture is inoculated
intracutaneously into two guinea pigs (or
rabbits) so that each receives 0.1 mL in two
different sites. One animal acts as the control
and should receive antitoxin (500 units)
the previous day. After four hours the skin
test, the other is given 50 units of antitoxin
intraperitoneally in order to prevent death.
In the test animal, toxigenicity is indicated by
inflammatory reaction at the site of injection,
progressing to necrosis in 48–72 hours and in
the control animal no change. Fig. 28.2: Elek’s test
202 | Section 3: Systemic Bacteriology
Principle DPT Vaccine: Diphtheria toxoid is usually given
This test depends upon the principle of toxin- in children as a trivalent preparation containing
antitoxin neutralization, in vivo, and the test is tetanus toxoid and pertussis vaccine also as the
carried out by injecting one Schick test dose of DPT or triple vaccine.
diphtheria toxin (0.2 mL containing 1/50 MLD)
intradermally on the anterior surface of the left Schedule of primary immunization: The schedule
forearm and a control injection in the right forearm of primary immunization of infants and children
contains a heat-inactivated dose (70°C for 30 consists of DPT given at the age of 6 weeks, 10
minutes) or preferably, purified diphtheria toxoid. weeks, 14 weeks and 16–24 months followed by
booster dose DT at the age of 5–6 years (school
Results entry).
Readings are taken after 1, 4 and 7 days. Four types
of reactions may occur:
B. Passive Immunization
1. Negative reaction: There is no reaction of any
kind in either arm. This indicates that the toxin This is an emergency measure to be employed
has been neutralized by the circulating antitoxin where susceptibles (nonimmunized) are exposed
and the person is immune to diphtheria and to infection, as when a case of diphtheria is
does not need immunization. admitted to general pediatric wards. It consists of
2. Positive reaction: In the test arm, there appears the subcutaneous administration of 500–1000 units
erythema and swelling at the site of inoculation in of antitoxin (antidiphtheritic serum, ADS). As this is
24–36 hours, reaching its maximum (1–5 cm) by a horse serum, precaution against hypersensitivity
the 4th to 7th days and then fading with superficial should be observed.
scaling and persistent brownish pigmentation. On
the control arm, there is no reaction. C. Combined Immunization
A positive Schick test indicates that the This consists of administration of the first dose
individual is susceptible to diphtheria and of adsorbed toxoid on, while ADS is given on the
little or no antitoxin (less than 0.01 unit/mL) is other arm, to be continued by the full course of
present. The subject is not immune and should active immunization since protection conferred
be immunized. by passive immunization is of short duration.
3. Pseudoreaction: There is erythema occurring Ideally, all cases that receive ADS prophylactically
within 6–24 hours and disappearing within four should receive combined immunization.
days. The reaction is the same on both arms.
This indicates that the individual is immune to
Treatment
diphtheria and also that he is hypersensitive to
one or more antigens in the toxin preparation. Specific treatment of diphtheria consists of
This individual does not need immunization. antitoxic and antibiotic therapy. Antitoxin
4. Combined reaction: Here the initial picture is should be given immediately as soon as clinical
that of pseudoreaction, but while the erythema diagnosis is made to neutralize the toxin being
in the control arm fades, within four days, it produced. The dosage recommended is 20,000
progresses in the test arm to a typical positive units intramuscularly for moderate cases and
reaction. This indicates that the individual is 50,000 to 100,000 units for serious cases, half the
susceptible to diphtheria and is sensitive to dose being given intravenously.
one or more antigens in the toxin preparation C. diphtheriae is sensitive to most antibiotics,
making immunization necessary but likely to including penicillin and erythromycin for the
induce reaction. treatment of patients as well as carriers. The
antibiotics do not neutralize circulating toxin.
Prophylaxis Penicillin-sensitive individuals can be given
The methods of immunization available are active, erythromycin. Erythromycin is more active than
passive or combined. penicillin in the treatment of carriers.
f. Nondiphtheria corynebacteria
Key Points g. Diphtheroids.
Corynebacterium is gram-positive bacilli with an
irregular shape, tendency to clubbing at one or both,
highly pleomorphic with Chinese letter or cuneiform
Multiple choice questions (MCQs)
arrangement. The granules in the cell are known as 1. Corynebacterium diphtheriae is classified into three
metachromatic granules volutin granules or Babes distinct biotypes (mitis, intermedius, and gravis)
Ernst granules. With Albert’s stain, the granules stain based on the morphologies of the colonies on:
bluish black and the protoplasm green a. Tellurite blood agar
Two media are useful. 1. Loeffler’s serum slope- b. Loeffler’s serum slope
tellurite blood agar c. Blood agar
Fermentations of sugars are usually done in Hiss’s
d. All the above media
serum peptone water medium
2. Diphtheria toxin shows following features except:
C. diphtheriae is H2S positive and reduces nitrate
a. It is a protein with a molecular weight of 58,300
to nitrite
Toxin: Toxigenic strains of C. diphtheriae produce
Da
a very powerful exotoxin. The toxigenicity of the b. It consists of two functionally distinct polypep-
diphtheria bacillus depends on the presence in it of tide chain fragments, A and B protein
corynephages (tox+). Diphtheria toxin is, heat labile c. It inhibits synthesis of fatty acids
protein, and consists of two fragments. Inhibition of d. It is a very potent toxin
protein synthesis is probably responsible for both 3. Following statements are true for diphtheria
the necrotic and neurotoxic effects of the toxin antitoxin except:
Clinical diseases: Diphtheria occurs in two forms a. It is the mainstay of therapy in diphtheria
(respiratory and cutaneous) b. It is of immense value in cutaneous diphtheria
Laboratory diagnosis: Depends upon micros c. It is of no value in treatment of asymptomatic
copy,culture and virulence tests. Virulence testing carriers
may be by in vivo or in vitro methods. In vivo tests d. It is ineffective after toxin has entered into the cell
are i. Subcutaneous test; ii. Intracutaneous test. 4. Diphtheria toxin has a special affinity for which of
In vitro test include i) Precipitation test; ii) Tissue the following tissue/s?
culture test; iii) Enzyme-linked immunosorbent a. Heart muscles b. Nerve endings
assays (ELISA); iv) Polymerase chain reaction (PCR) c. Adrenal glands d. All of the above
Prophylaxis: DPT given at the age of 6 weeks, 10 5. Which of the following sites is most commonly
weeks, 14 weeks and 16–24 months followed by
affected by diphtheria bacilli?
booster dose DT at the age of 5–6 years (school entry)
a. Upper respiratory tract b. Skin
Diphtheroids: Cor ynebacteria resembling
c. Cornea d. Conjunctiva
C. diphtheriae occur as normal commensals in the
throat, skin and other areas. These may be mistaken
6. Diphtheroids show following features except:
for diphtheria bacilli and are known as diphtheroids. a. They possess few or no metachromatic granules
The common diphtheroids are C. pseudodiphth b. They are usually arranged in parallel rows
eriticum and C. xerosis. c. Most of them do not produce toxins
d. They do not ferment sucrose
7. A positive Schick test implies that the person is:
Important questions a. Immune and nonhypersensitive.
b. Susceptible and nonhypersensitive
1. Discuss morphology, cultural characteristics c. Immune and hypersensitive
and biochemical characters of Corynebacterium d. Non-immune and hypersensitive
diphtheriae. 8. Which of the following bacteria can cause infection
2. Name different species of genus Corynebacterium. in immunocompetent patients?
Discuss in detail laboratory diagnosis of diphtheria. a. Corynebacterium ulcerans
3. Write short notes on: b. Corynebacterium haemolyticum
a. Diphtheria toxin c. Corynebacterium pseudotuberculosis
b. Pathogenicity of C. diphtheriae d. All of the above
c. Toxigenicity tests/Virulence tests of C. diphtheriae
d. Schick test Answers (MCQs)
e. Prophylaxis of diphtheria 1. a; 2. c; 3. b; 4. d; 5. a; 6. d; 7. b; 8. d
29
Chapter
Bacillus
Learning Objectives
After reading and studying this chapter, you should ∙∙ Discuss laboratory diagnosis of anthrax
be able to: ∙∙ Descrbethe following: Anthracoid bacilli; Bacillus
∙∙ Describe the morphology, cultural characteristics cereus food poisoning.
and pathogenicity of Bacillus anthracis
Fig. 29.1: Anthrax bacilli Fig. 29.2: Medusa head appearance of anthrax bacilli
B. Human infection
Resistance
Based on the mode of infection, human Anthrax
The spores are resistant to chemical disinfectants
presents in one of three ways: (1) cutaneous (2)
and heat. With moist heat, the vegetative bacilli are
pulmonary, or (3) intestinal. All types leading to
killed at 60°C in 30 minutes and the spores at 100°C
fatal septicemia or meningitis.
in 10 minutes. With dry heat the spores are killed at
150°C in 60 minutes. The spores are also killed by 1. Cutaneous Anthrax
4% formaldehyde or 4% potassium permanganate Cutaneous anthrax used to be caused by shaving
in a few minutes. brushes made with animal hair. It begins 2–5 days
The bacilli are sensitive to benzylpenicillin, after infection as a small papule that develops
streptomycin, tetracyclines, chloramphenicol, within a few days into a vesicle filled with dark
ciprofloxacin, the cephalosporins and sulfonamides. bluish black fluid. Rupture of the vesicle reveals
a black eschar at the base, with a very prominent
Antigenic Structure inflammatory ring of reaction around the eschar.
(The name anthrax, which means coal, comes
Three main antigens have been characterized: from the black color of the esc har). This is
1. Capsular polypeptide: It is polypeptide con sometimes referred to as a malignant pustule.
sisting exclusively of D-glutamic acid. The lesion is classically found on the hands,
2. Somatic polysaccharide: It is a component of forearms, or head and is painless. The disease used
the cell wall. to be common in dock workers carrying loads of
3. Complex protein toxin (anthrax toxin): hides and skins on their bare backs and hence was
Anthrax toxin is a complex exotoxin consisting of known as the ‘hide porter’s disease.’ Cutaneous
three protein components: Protective antigen anthrax generally resolves spontaneously, but
(PA), lethal factor (LF), and edema factor (EF). 10–20% of untreated patients may develop fatal
Each of the separate components is serologically septicemia or meningitis.
active and distinct and is also immunogenic.
2. Pulmonary Anthrax
Pulmonary anthrax, known as ‘wool-sorter’s
Virulence Factors disease, because it used to be common in workers
The pathogenesis of B. anthracis depends on two in wool factories, due to inhalation of dust from
important virulence factors: A poly (D-glutamic infected wool. It occurs in patients who handle raw
acid) capsule and a three-component protein wool, hides, or horsehair and acquire the disease
exotoxin. Fully virulent organisms have two large by the inhalation of spores. This is a hemorrhagic
plasmids that code for these products. pneumonia with a high fatality rate. Hemorrhagic
1. Capsule: The capsule interferes with phagocytosis. meningitis may occur as a complication.
2. Anthrax toxin: Anthrax toxin consists of three 3. Intestinal Anthrax
proteins called protective antigen (PA), edema Intestinal anthrax, is rare and occurs mainly in
factor (EF), and lethal factor (LF), each of primitive communities who eat the carcasses of
which individually is nontoxic but together act animals dying of anthrax. An individual may suffer
synergistically to produce damaging effects. after a day or so from hemorrhagic diarrhea, and
The three factors have been characterized and dies rapidly from septicemia.
cloned.
Laboratory Diagnosis of Human Anthrax
Pathegenesis
In laboratories unfamiliar with the disease,
Cattle, sheep, goats and other herbivores are
additional precautions for staff safety need to be
naturally infected.
organized. All procedures connected with the
handling of B. anthracis should be carried out with
A. Animal Infection greatest care in a safety cabinet.
Anthrax is a zoonosis. Animals are infected by the 1. Specimens: Material from a malignant pustule,
ingestion of the spores present in the soil. Direct sputum from pulmonary anthrax, gastric
spread from animal to animal is rare. The disease aspirates, feces or food in intestinal anthrax and
is generally a fatal septicemia but may sometimes in the blood in the septicemic stage of all forms
208 | Section 3: Systemic Bacteriology
of the infection. Specimens should be taken antigen, a ring of precipitation will appear at the
before antibiotic therapy has been instituted. junction of the two liquids within 5 minutes at
2. Microscopy: Prepare smears of each specimen room temperature.
and stain with Gram’s method and McFadyean’s With the availability of purified anthrax toxin
method or with Giemsa stain. Gram’s stain antigen, Ascoli’s test has been replaced by highly
may show typical large gram-positive bacilli. sensitive and specific immunoassays. EIA can also
Capsule appears as a clear halo around the detect antibody in the serum of animals surviving
bacterium by India-ink staining. anthrax infection.
Direct fluorescent antibody test (DFA) for
capsule specific staining and for polysaccharide 6. Polymerase Chain Reaction (PCR)
(cell wall) antigen confirms the identification. A sensitive and specific PCR technique has been
3. Culture: Culture the exudate on nutrient agar, developed for the detection of anthrax contami
blood agar, PLET medium and nutrient broth. nation of animal and agricultural products.
Incubate at 37°C for 18 hours. Examine plates
for the medusa-head colonies characteristic Treatment
of B. anthracis, nonhemolytic on the blood Ciprofloxacin is the drug of choice. Penicillin,
agar plate. Prepare a smear, stain it by Gram’s doxycycline, erythromycin, or chloramphenicol
method and look for tangled chains of large can be used (if susceptible).
gram-positive bacilli some of which have
central, oval, non-bulging spores. In nutrient Prophylaxis
broth, look for a pellicle and a deposit.
Control of human anthrax ultimately depends on
4. Confirmatory tests
control of the disease in animals. Animals with
i. Biochemical and physiological reactions:
known or suspected anthrax should be handled
Demonstration of non-motility, gelatin
with care and carcasses of animals suspected to have
liquefaction, growth in straight chains and died of anthrax are incinerated or buried deep in
enhanced growth aerobically, as seen in the quicklime. Wool, horsehair, and hides coming from
characteristic inverted fir tree appearance areas where epidemic anthrax is present should be
in a gelatin stab, will generally identify B. gas sterilized. A vaccine is available for control of
anthracis completely. outbreaks of human anthrax in an ‘industrial setting’.
Toxin production can be demonstrated by
immunological or gene probe methods in Immunization
reference laboratories. Live-attenuated bacilli were first used by Louis
ii. Animal inoculation: Inoculate intraperi Pasteur in May 1881. Pasteur’s vaccine was the
toneally in mice a 24 hours broth culture. anthrax bacillus attenuated by growth at 42–43°C.
The animal dies in 48–72 hours. Make The original Pasteur’s anthrax vaccine is of great
smears from heart blood and spleen, stain historical importance.
by Gram’s and McFadyean’s methods, and
Sterne strain of live spore vaccine: Subsequently,
look for typical anthrax bacilli. the Sterne strain of live spore vaccine has been
The anthrax bacillus can often be isolated used for animal immunization. The Sterne vaccine
from contaminated tissues by applying contained spores of a noncapsulated avirulent
them over the shaven skin of a guinea mutant strain. Live bacterial vaccines are not
pig. It is able to penetrate through minute considered safe for man.
abrasions and produce fatal infection.
Alum-precipitated toxoid prepared from the
5. Serological diagnosis: Serological diagnosis by
protective antigen has been shown to be a safe
enzyme-linked immunosorbent assay (ELlSA)
and effective vaccine for human use. It has been
is seldom used diagnostically.
used in persons occupationally exposed to
Ascoli’s thermoprecipitin test: If the sample anthrax infection. Three doses intramuscularly at
received is putrid so that viable bacilli are intervals of six weeks between first and second,
unlikely, diagnosis may be established by Ascoli’s and six months between second and third doses
thermoprecipitin test by demonstration of the induce good immunity, which can be reinforced if
anthrax antigen in tissue extracts.The original necessary with annual booster injections. Frequent
thermoprecipitin test devised by Ascoli (191l) booster doses are necessary.
was a ring precipitation by letting the boiled
tissue extract in a test tube react with the anthrax
ANTHRACOID BACILLI
antiserum. The tissue is ground up in saline, boiled
for 5 minutes, filtered and layered over anti anthrax Nonpathogenic aerobic spore bearing bacilli having
serum in a narrow tube. If tissue contains anthrax a general resemblance to anthrax bacilli have been
Chapter 29: Bacillus | 209
collectively called pseudoanthrax or anthracoid There is a longer incubation period occurring
bacilli. The important species include B. cereus, B. 8–24 hours after ingestion, during which the
subtilis, B. stearothermophilus B. licheniformis, B. organism multiplies in the patient’s intestinal
pumilus. B. cereus has been recognized as a frequent tract and produces the heat-labile enterotoxin.
cause of foodborne gastroenteritis. Table 29.1 lists Then the diarrhea, nausea, and abdominal
the main differentiating features between Anthracoid cramps develop.
bacilli and B. anthracis. The diarrheal disease is mostly caused
by serotypes 2, 6, 8, 9, 10 or 12 of B. cereus
Bacillus cereus strains. Isolates from the diarrheal type of
B. cereus has recently assumed importance as a disease produce enterotoxin which causes
cause of food poisoning. It is widely distributed in fluid accumulation in ligated rabbit ileal loop,
nature, may be readily isolated from soil, vegetables resembling the heat labile enterotoxin of
and a wide variety of foods including milk, cereals, Escherichia coli and Vibrio cholerae.
spices, meat and poultary. 3. Ocular infection: Common cause of post-
traumatic ophthalmitis.
4. Other opportunistic infections: Intravenous
Pathogenesis catheter and central nervous system shunt
1. Emetic form (the short incubation type): The infections and endocarditis as well as
emetic form results from the consumption of pneumonitis, bacteremia, and meningitis in
contaminated rice. The heat-stable enterotoxin severely immunosuppressed patients.
that is released is not destroyed when the rice
is reheated. After ingestion of the enterotoxin Laboratory Diagnosis
and a 1–6-hour incubation period, a disease of
If food is available for testing, confirmation is easy.
short duration (less than 24 hours) develops.
High numbers of B. cereus, in the absence of other
Symptoms consist of vomiting, nausea, and
food poisoning bacteria are sufficient to make the
abdominal cramps. Fever and diarrhea are
diagnosis.
generally absent.
Large facultatively anaerobic Gram-positive
Two mechanisms of action have been
bacilIi that produce anthracoid colonies on blood
described for the enterotoxin of B. cereus, one
agar after overnight incubation at 37°C are almost
involving stimulation of CAMP system and the
certain to be B. cereus.
other independent of it.
2. Diarrheal form: The diarrheal form of B. cereus Treatment : Both the emetic and diarrheal
food poisoning results from the consumption syndromes are shortlived and no specific treatment
of contaminated meat, vegetables or sauces. is needed.
Clostridium
Learning Objectives
After reading and studying this chapter, you should ∙∙ Discuss morphology, cultural characteristics of
be able to: C. tetani
∙∙ Describe classification of clostridia and diseases ∙∙ Describe toxins produced by C. tetani
produced by different clostridia ∙∙ Describe the following: Pathogenesis of tetanus;
∙∙ Discuss morphology, cultural characteristics of prophylaxis of tetanus
C. welchii ∙∙ Discuss morphology and cultural characteristics
∙∙ Discuss Nagler reaction of C. botulinum
∙∙ Discuss lab. diagnosis and prophylaxis of gas ∙∙ Discuss lab. diagnosis of botulinum
gangrene ∙∙ Describe the following: gas gangrene; botulinum
toxin; Clostridium difficile.
Toxins
C. perfringens is one of the most prolific of toxin-
producing bacteria, forming at least 12 distinct
soluble substances or toxins, all of which are of
protein in nature and antigenic. Major lethal toxins
include: α (alpha), b (beta), e (epsilon) and i (iota)
and minor lethal toxins include: g (gamma), d
(delta), k (kappa), l (lambda), m (mu), n (nu), q
(theta) and h (eta). The four 'major toxins', alpha,
Fig. 30.2: Nagler’s reaction. C perfringens colonies on the
beta, epsilon and iota, are predominantly respon right half of the plate are surrounded by haloes, while colonies
sible for pathogenicity. on the left half (containing antiserum to alpha toxin) have no
haloes around them
Classification Interpretation: On the section containing no
C. perfringens can be divided into five types, A to antitoxin, C. perfringens colonies show surrounding
E on the basis of four major toxins. Strains of C. zone of opalescence, i.e. Nagler reaction. There
perfringens type A that produce enterotoxin are will be no opacity around the colonies on the half
associated with a mild form of food poisoning. of the plate with the antitoxin, due to the specific
neutralization of the alpha toxin (Fig. 30.2).
Alpha Toxin This reaction, however, is not totally specific
The alpha (a) toxin is produced by all types of for C. perfringens since the opalescence in the egg
C. perfringens and most abundantly by type A yolk media may be produced by other lecithinase
strains, is a lecithinase (phospholipase C) that lyses forming bacteria (C. novyi, C. bifermentans, some
erythrocytes, platelets, leukocytes, and endothelial vibrios, some aerobic spore bearers). The reaction
cells. It is lethal, dermonecrotic and hemolytic for produced by C. perfringens is specifically neutralized
the red cells of most species, except horse and by C. perfringens antitoxin, but serologically related
goat. The lysis is of the hot-cold variety, being best phospholipases of C. bifermentans and ‘C. sordellii
seen after incubation at 37°C followed by chilling and some other phospholipases are also inhibited.
at 4°C. This toxin increases vascular permeability, These organisms can be separated by other tests.
resulting in massive hemolysis and bleeding,
tissue destruction, hepatic toxicity, and myocardial Other Major Toxins
dysfunction. It is relatively heat stable. Beta (β), Epsilon (e) and iota (i) toxins have lethal
and necrotizing properties.
Nagler’s Reaction
Minor Toxins
Basis: The alpha (a) toxin is lecithinase C (or
phospholipidase C) splits lecithin into phosphoryl Gamma and eta toxins have minor lethal toxins.
choline and diglyceride, in the presence of Ca++ and Delta toxin has a lethal effect and is hemolytic for
Mg++ ions because the toxin is activated by Ca++ and the red cells of even- goat, pigs and cattle. Theta
Mg++ ions. This reaction is seen as opalescence in toxin is oxygen-labile hemolysin antigenically
serum or egg yolk media and is specifically neutral related to streptolysin O. It is also lethal and a
ized by the antitoxin. This is the basis of Nagler general cytolytic toxin.
reaction.
Enterotoxin
Procedure: For rapid detection of C. perfringens, a
C. perfringens type A strains produce a potent
culture plate containing 6% agar, 5% peptic digest
enterotoxin which causes diarrhaea and other
of sheep blood and 20% human serum or 5% egg-
symptoms of food poisoning.
yolk is prepared. The incorporation of neomycin
sulphate in the medium makes it more selective,
Pathogenesis
inhibiting coliforms and aerobic spore bearers.
On one half of the plate, 2–3 drops of C. 1. Soft Tissue Infections
perfringens antitoxin are spread and allowed to dry. Soft tissue infections caused by C. perfringens
The plate is then inoculated with the test organisms are subdivided into (1) cellulitis, (2) fasciitis or
or the exudate under investigation and incubated suppurative myositis, and (3) myonecrosis or gas
anaerobically at 37°C for 18 hours. gangrene.
214 | Section 3: Systemic Bacteriology
Clostridial myonecrosis or gas gangrene: The 5. C. perfringens Colitis
disease is characterized by rapidly spreading edema, A sporadic diarrheal syndrome, usually occurring in
myositis, necrosis of tissues, gas production and elderly patients during treatment with antibiotics,
profound toxemia occurring as a complication of has been described.
wound infection. The disease has been referred to in
the past as ‘malignant edema’. Other descriptive terms 6. Clostridial Endometritis
that have been used are ‘anaerobic (clostridial)
This condition is a grave complication of incom
myositis’ and ‘clostridial myonecrosis.
plete abortion, or the use of inadequately sterilized
Etiology: Amongst the pathogenic clostridia, instruments.
C. perfringens is the most frequently encountered
(app rox imately 60%), and C . n ov y i and Laboratory Diagnosis
C. septicum being the next common (20–40%), and
Gas gangrene is a medical emergency. The diagnosis
C. histolyticum less often. Other clostridia usually
of gas gangrene must be made primarily on clinical
found are C. sporogenes, C. fallax, C. bifermentans,
grounds, and the function of the laboratory is only
C. sordelli, C. aerofoetidum and C. tertium.
to provide confirmation of the clinical diagnosis
Mechanism of infection: Clostridial spores are as well as identification and enumeration of the
introduced into tissue, e.g. by contamination with infecting organisms.
dirt, or by endogenous transfer from the intestinal
tract. After injury there is incubation period may A. Specimens
be as short as seven hours or as long as six weeks,
(1) Edge of the affected muscles; (2) Exudates from
usually of 12–48 hours.
the wound; and (3) Necrotic tissue and muscle
When there is considerable cell injury or
fragments.
compromise of circulation, germination and
outgrowth of clostria spores occurs. Alpha toxin and
B. Microscopy
other exotoxins are secreted and extensive cell killing
ensues. The production of enzymes that break down Gram stained films give presumptive information
ground substance facilitates the spread of infection. about the species of clostridia present and their
Fermentation of tissue carbohydrates yields gas, and relat ive numbers. If gas gangrene is present,
an accumulation of gas bubbles in the subcutaneous Gram-positive rods may predominate. Thick,
spaces produces a crinkling sensation on palpation stubby, Gram-positive rods suggest C. perfringens
(crepitation), hence the name gas gangrene. or C. sordellii, ‘citron bodies’, boat- or leafshaped
pleomorphic bacilli with irregular staining, may
2. Septicemia indicate C. septicum; slender rods with round
terminal spores suggest C. tetani and large rods with
Invasion of the bloodstream may occur in associ oval sub terminal spores indicate C. novyi.
ation with malignancy and may involve a localized
myonecrosis in addition to a fulminating clostridial C. Culture
septicemia.
Fresh and heated blood agar are used for aerobic
and anaerobic cultures. To prevent swarming
3. Food Poisoning by some species of clostridia, the use of plates
The organisms usually involved are strains of type containing increased agar (5-6%) are considered. A
A. Meat, chicken, fish and their by-products are the plate of serum or egg yolk agar, with C. perfringens
most common vehicles for clostrial food poisoning. antitoxin spread on one half is used for the ‘Nagler
Clostridial food poisoning, is characterized by (i) a reaction’.
short incubation period (8–24 hours), (ii) a clinical Four tubes of cooked meat broth are inoculated
presentation that includes abdominal cramps and and heated at 100°C for 5, 10, 15 and 20 minutes,
watery diarrhea but no fever, nausea, or vomiting, incubated and subcultured on blood agar plates
and (iii) a clinical course lasting less than 24 hours. after 24–48 hours, to differentiate the organisms
The illness is self-limited and recovery occurs in with heat resistant spores. Blood cultures are
24–48 hours. often positive, especially in C. perfringens and
C. septicum infections. However, C. perfringens
4. Enteritis Necroticans (Necrotizing bacteremia may occur without gas gangrene.
Jejunitis, Necrotic Enteritis)
This is severe and often fatal enteritis known by D. Identification
different names in different countries: β-toxin- Examine plates for typical colonies. The isolates are
producing C. perfringens type C is responsible for identified based on their morphological, cultural,
this disease. biochemical and toxigenic characters.
Chapter 30: Clostridium | 215
Animal Pathogenicity
0.1 mL 24 hours growth in cooked meat broth is
injected into a healthy guinea pig by intramuscular
route. The animal dies within 24 hours. A control
animal protected with antiserum prior to test is also
included. On autopsy, bacteria can be recovered
from heart and spleen of the test animal.
Nonsporing Anaerobes
Learning Objectives
After reading and studying this chapter, you should ∙∙ List infections caused by nonsporing anaerobes
be able to: ∙∙ Discuss laboratory diagnosis of infections caused
∙∙ Describe classification of nonsporing anaerobes by nonsporing anaerobes.
hemin and vitamin K is adequate. Plates are incu 3. An indication of antimicrobial agents likely to
bated at 37°C in an anaerobic jar, with 10% CO2. The be used.
Gas-Pak system provides a convenient method of
routine anaerobic cultures. Plates are examined after E. Antibiotic Sensitivity Tests
24 or 48 hours. Some anaerobes, such as fusobacte Antibiotic sensitivity tests can be done by disc
ria, require longer periods of incubation. Since many diffusion or dilution methods.
anaerobes are relatively slow-growing, it is essential
that cultures are incubated for several days before F. Other Anaerobic Techniques
being discarded. In mixed infections, fast-growing Gloved anaerobic chambers with continuous gas
aerobic or facultatively anaerobic organisms are flow may be used for culture of specimens. Pre-
often detected within 24 hours, whereas some reduced anaerobically sterilised media (PRAS) can
anaerobes may require incubation for 7–10 days also be employed but are not essential for rountine
before their colonies can be recognized. diagnostic procedures.
Other anaerobic media, such as cooked meat
broth (CMB) and thioglycollate broth, may also Treatment of Anaerobic Infections
be used for incoculating the specimens. Parallel Treatment of mixed anaerobic infections is by
aerobic cultures (such as Pseudomonas aeruginosa surgical drainage (under most circumstances) plus
should always be set up. This is necessary as a antimicrobial therapy.
control for the growth on anaerobic plates and The most active drugs for treatment of anaerobic
also because in most anaerobic infections aerobic infections are clindamycin and metronidazole.
bacteria are also involved. Clindamycin is preferred for infections above the
D. Identification diaphragm. Penicillin G remains the drug of choice
Colony morphology, pigmentation, and fluorescence for treatment of anaerobic infections that do not
are helpful in identifying anaerobes. Biochemical involve β-lactamase-producing Bacteroides and
activities and production of short-chain fatty acids Prevotella species.
as measured by gas-liquid chromatography are used
Key Points
for laboratory confirmation. It takes time and is
difficult, but it is possible to report on the following: Many anaerobic bacteria are pathogenic for human
1. Whether the infection is solely aerobic, anaerobic beings, and they outnumber aerobic bacteria in
or mixed. many habitats
Anaerobic gram-positive cocci: Most of important
2. The identification of the more common anaero anaerobic cocci belong to the genus Peptostreptococcus
bes, particularly of B. fragilis.
226 | Section 3: Systemic Bacteriology
b. Lactobacillus
Anaerobic gram-negative cocci: Veillonella parvula c. Bacteroides
is the species frequently reported from clinical d. Fusobacterium
specimens e. Anaerobic gram-positive bacilli.
Anaerobic nonspore-forming gram-positive
bacilli: Include Eubacterium, Propionibacterium,
Lactobacillus, Bifidobacterium and Mobiluncus Multiple choice questions (MCQs)
Anaerobic gram-negative bacilli: Medically
important anaerobic gram-negative bacilli belong 1. The following is an example of gram-negative
to the family Bacteroidaceae and are classified into anaerobic cocci
the genera Bacteroides, Prevotella, Porphyromonas, a. Veillonella
Fusobacterium and Leptotrichia b. Peptococcus
Laboratory diagnosis: Specimens should be placed c. Peptostreptococcus
in an anaerobic transport device. Examination of d. Bacteroides
a Gram stained smear is very useful. The Gas-Pak 2. Lactobacilli constitute the normal flora of:
system provides a convenient method of routine a. Adult vagina
anaerobic cultures. Other anaerobic media, such b. Prepubertal vagina
as cooked meat broth (CMB) and thioglycollate c. Post-menopausal vagina
broth, may also be used. Colony morphology, d. None of the above
pigmentation, and fluorescence are helpful in
3. All the following anaerobic bacteria cause head
identifying anaerobes. Biochemical activities and
production of short-chain fatty acids as measured
and neckinfections except.
by gas-liquid chromatography are used for labora a. Bacteroides thetaiotaomicron
tory confirmation. b. Fusobacterium nucleatum
c. Fusobacterium necrophorum
d. Porphyromonas asaccharolytica
Important questions 4. Which of the following bacterial colonies fluoresce
brick-red in UV light?
1. Classify nonsporing anaerobes. Discuss the a. Bacteroides fragilis
laboratory diagnosis of infections caused by b. Bacteroides melaninogenicus
nonsporing anaerobes. c. Bacteroides gingiva/is
2. Give an account of infections caused by nonsporing d. Bacteroides levii.
anaerobes.
3. Write short notes on: Answers (MCQs)
a. Anaerobic cocci 1. (a); 2. (a); 3. (a); 4. (b)
32
Chapter
Mycobacterium tuberculosis
LEARNING OBJECTIVES
After reading and studying this chapter, you should ∙∙ Differentiate between Mycobacterium tuberculosis
be able to: and M. bovis
∙∙ Classify mycobacteria ∙∙ Describe pathogenesis of Mycobacterium tuberculosis
∙∙ Discuss morphology and culture characteristics ∙∙ Discuss the laboratory diagnosis of pulmonary
and biochemical characteristics of Mycobacterium tuberculosis
tuberculosis ∙∙ Describe the following: Koch’s phenomenon;
Tuberculin test; BCG vaccine.
MYCOBACTERIUM TUBERCULOSIS
Morphology
M. tuberculosis is a slender, straight, or slightly
curved rod with rounded ends, about 3 µm × 0.3 µm, Fig. 32.1: Mycobacterium tuberculosis in Ziehl–Neelsen
in pairs or as small clumps. The bacilli are nonmotile, stained smear
nonsporing, noncapsulated and acid-fast.
When stained with carbol fuchsin by the Ziehl–
Neelsen method or by fluorescent dyes (auramine
O, rhodamine), they resist decolorization by 20%
sulphuric acid and absolute alcohol for 10 minutes
(acid and alcohol fast). With Ziehl–Neelsen acid-
fast stain, the tubercle bacilli stain bright red, while
the tissue cells and other organisms are stained
blue (Fig. 32.1).
Acid fastness has been ascribed variously to
the presence in the bacillus of an unsaponifiable
wax (mycoloic acid) or to a semi permeable
membrane around the cell. It is related to the
integrity of the cell and appears to be a property
of the lipid-rich waxy cell wall. Staining may be
uniform or granular. In M. tuberculosis beaded or
barred forms are frequently seen, but M. bovis stains
more uniformly. M. bovis appear straighter, stouter Figs 32.2: L–J media without growth (A) and with growth (B)
and shorter with uniform stainin (Table 32.2).
other bacteria and to provide a contrasting color
Cultural Characteristics against which colonies of mycobacteria are easily
seen. In this medium egg acts as a solidifying agent.
M. tuberculosis is an obligate aerobe while M. The addition of 0.5% glycerol improves the growth
bovis is microaerophilic on primary isolation, of M. tuberculosis, but has no effect on or may even
becoming aerobic on subculture. The optimal impair the growth of M. bovis. Sodium pyruvate
growth temperature of tubercle bacilli is 35–37°C. helps the growth of both types (Figs 32.2A and B).
Optimum pH is 6.4–7.0. The bacilli grow slowly, Human tubercle bacilli produce visible growth
the generation time in vitro being 14–15 hours. on LJ medium in about 2 weeks, although on
Colonies appear in about two weeks and may primary isolation from clinical material colonies
sometimes take up to eight weeks. may take up to 8 weeks to appear. On solid media,
M. tuberculosis forms dry, rough, raised, irregular
Solid Medium colonies with a wrinkled surface. They are creamy
Tubercle bacilli are able to grow on a wide range of white, becoming yellowish or buff colored on
enriched culture media and do not have exacting further incubation. They are tenacious and not
growth requirements. The solid media contain egg easily emulsified. Mycobacterium tuberculosis has
(Lowenstein Jensen, Petragnini, Dorset), blood a luxuriant growth (eugenic growth) as compared
(Tarshis), serum (Loeffler) or potato (Pawlowsky). to Mycobacterium bovis which grows poorly on LJ
The solid medium most widely employed glycerol medium (dysgonic growth) and colonies,
for routine culture is Lowenstein–Jensen (L–J) in comparison are flat, smooth, moist, white and
medium. This consists of coagulated hens’ egg, break up easily when touched. The growth of M.
mineral salt solution, asparagine and malachite bovis is much better on LJ pyruvate medium (media
green, the last acting as a selective agent inhibiting containing sodium pyruvate in place of glycerol).
Table 32.4 Some differential characteristics of tubercle bacilli causing human disease
Species Oxygen Glycerol Niacin Nitrate TCH Pyrazinamide Pathogenicity
preference enhanced reduction
Mycobacterium leprae
Learning Objectives
After reading and studying this chapter, you should ∙∙ Describe the following:
be able to: – Lepromatous leprosy; tuberculoid leprosy;
∙∙ Describe morphology of M. leprae lepromin test
∙∙ Discuss cultivation of lepra bacilli – Differentiate between lepromatous and tuber
∙∙ Explain animal models in leprosy culoid leprosy
∙∙ Describe pathogenesis of leprosy – Discuss laboratory diagnosis of leprosy.
Nontuberculous Mycobacteria
LEARNING OBJECTIVES
After reading and studying this chapter, you should ∙∙ Name the diseases caused by nontuberculous
be able to: mycobacteria
∙∙ Classify nontuberculous mycobacteria and ∙∙ Discuss the clinical significance of nontuberculous
examples of different groups of nontuberculous mycobacteria.
mycobacteria
Table34.4 Differentiation between tubercle bacilli and some species of atypical mycobacteria
Test M. M. M. M M M. M. avium M. M. M. M.
tubercu bovis microti kansasii marinum scrofula intra fortuitum chelonei phlei smegmatis
losis ceum cellulare
complex
Growth in – – – – – – – + + + +
7 days
Growth at – – – + + + ± + + + +
25°C
Growth at + + + + ± + + + + + +
37ºC
Growth at – – – – – ± – – – + +
45°C
Pigment – – – + + + – – – + –
in light
Pigment – – – – – + – – – + –
in dark
Niacin + – ± – – – – – – – –
Nitrate + – – + – – – + – + +
reduction
Urease + + + – + + – + + + +
Actinomycetes
Learning Objectives
After reading and studying this chapter, you should ∙∙ Discuss laboratory diagnosis of actinomycosis
be able to: ∙∙ Differentiate between the genera Actinomyces and
∙∙ Describe morphology of Actinomyces Nocardia
∙∙ Explain clinical forms of Actinomyces ∙∙ Describe the following: nocardiosis, mycetoma.
Introduction Morphology
Actinomyces are Gram-positive, nonmotile, non
Actinomycetes are traditionally considered to be
sporing, nonacid-fast. They often grow in mycelial
transitional forms between bacteria and fungi.
forms and break-up into coccoid and bacillary
They form a mycelial network of branching
forms. Most show true branching.
filaments like fungi but, like bacteria, they are
thin, possess cell walls containing muramic acid,
have prokaryotic nuclei and are susceptible to
Cultural Characteristics
antibacterial antibiotics. They are therefore true They are facultative anaerobes. They grow best
bacteria, bearing a superficial resemblance to under anaerobic or micro-aerophilic conditions
fungi. Actinomycetes are related to mycobacteria with the addition of 5–10% CO2. The optimum tem
and corynebacteria. perature for growth is 35–37°C. They can be grown
They are gram-positive, nonmotile, nonsporing, on brain heart infusion agar, heart infusion agar
noncapsulated filaments that break up into supplemented with 5% defibrinated horse, rabbit
bacillary and coccoid elements. Although mostly or sheep blood. Suitable liquid media include brain
soil saprophytes, occas ionally cause chronic heart infusion broth and thioglycollate broth which
granulomatous infections in animals and man. may be supplemented with 0.1–0.2% sterile rabbit
serum. Most species show good growth after 3–4
Important genera: The family Actinomycetes days incubation however, A. israelii may take 7–14
contains three major medically important genera, days.
Actinomyces, Nocardia and Actinomadura. Another
genus, Streptomyces rarely causes disease in man.
Pathogenesis
Actinomyces is anaerobic or microaerophilic
and nonacid-fast, while Nocardia is acid-fast and Actinomycosis: The Actinomyces causes the
aerobic. Streptomyces is nonacid-fast and aerobic. disease known as actinomycosis. Actinomycosis is a
chronic disease characterized by multiple abscesses
and granulomata, tissue destruction, extensive
Actinomyces
fibrosis and the formation of sinuses. Within
A mould-like organism in the lesion of ‘lumpy jaw’ diseased tissues the actinomycetes form large
(actinomycosis) in cattle was found by Bollinger masses of mycelia embedded in an amorphous
(1877).The name Actinomyces was coined by protein–polysaccharide matrix and surrounded by
Harz to refer to the raylike appearance of the a zone of gram-negative, weakly acid-fast, club-like
organism in the granules that characterize the structures (Fig. 35.1). The mycelial masses may
lesions (Actinomyces, meaning ray fungus). This be visible to the naked eye and are called sulfur
was named Actinomyces israelii. It causes human granules, as they are often light yellow in color. The
actinomycosis. sulfur granules may be dark brown and very hard in
Chapter 35: Actinomycetes | 253
using 1% sulfuric acid for decolorization. Gram
staining shows a dense network of thin gram-positive
filaments, surrounded by a peripheral zone of
swollen radiating club shaped structures, presenting
a sun-ray appearance (Fig. 35.1). The ‘clubs’ are
believed to be antigen-antibody complexes.Acid
fast staining shows central part as non-acidfast
surrounded by acid-fast ‘clubs’. In absence of sulfur
granules, Gram’s staining of pus shows gram-positive
branching filaments.
Sulfur granules and mycelia in tissue sections
Fig. 35.1: Sulfur granule. Section of tissue showing an
actinomycotic clolony, the clubs at the periphery giving a can also be identified by direct fluorescence
‘sun-ray’ appearance microscopy.
older lesions because of the deposition of calcium
3. Culture
phosphate in the matrix.
In man, actinomycosis.is usually caused by Sulfur granules or pus containing actinomycetes are
Actinomyces israelii. Less common causes include washed and inoculated into thioglycollate liquid
A. gerencseriae, A. naeslundii, A. odontolyticus, A. medium or streaked on brain h eart infusion agar
viscosus, A. meyeri, Propionibacterium propionicum (BHI agar) blood agar and incubated anaerobically
and members of the genus Bifidobacterium. at 37°C. On solid media, A. israelii may form
so-called spider colonies that resemble molar teeth
in 48–72 hours that become heaped up, white and
Human Actinomycosis
irregular or smooth, large colonies in 10 days. Other
Human actinomycosis may take several forms: species have different types of colonies.
1. Cervicofacial: This is the most common type
and it occur mainly in cheek and submaxillary 4. Identification
regions. The disease is endogenous in origin. The identity may be confirmed by direct fluo
Dental caries is a predisposing factor, and rescence microscopy and biochemical tests or
infection may follow tooth extractions or other by gas chromatography of metabolic products of
dental procedures. carbohydrate fermentation.
2. Thoracic: Thoracic actinomycosis commences
in the lung, probably as a result of aspiration of 5. Biopsy
actinomyces from the mouth. In hematoxylin and eosin stained sections, the
3. Abdominal: The lesion is usually around the sulfur granules are deeply stained with hematoxy
cecum, with the involvement of the neighboring lin except in the periphery which is stained by
tissues and the abdominal wall. eosin, which shows short, radiate, club-like
4. Pelvic: Pelvic actinomycosis occasionally structures. On Gram staining, the filaments are
occurs in women fitted with plastic intra- gram-positive and periphery gram-negative.
uterine contraceptive devices.
5. Punch actinomycosis: It is a rare infection of Epidemiology
the hand acquired by injury of the knuckles on
Actinomycosis is an endogenous infection.
an adversary’s teeth.
Actinomycosis is more common in rural areas and
Laboratory Diagnosis in agricultural workers. Young male persons (10–30
years old) are most commonly affected. About
1. Specimens 60% of the cases are cervicofacial and some 20%
Pus, sinus discharge, bronchial secretions, sputum abdominal. Pelvic actinomyces is seen mainly in
or infected tissues are collected aseptically. women using intrauterine devices.
2. Microscopy Treatment
‘Sulfur granules’ may be demonstrated in pus Treatment for actinomycosis involves the combin
by shaking it up in a test tube with some saline. ation of surgical debridement of the involved
On standing, the granules sediment may be tissues and prolonged treatment with penicillin or
withdrawn with a capillary pipette. Granules may tetracycline.
also be obtained by applying gauze pads over the
discharging sinuses. Nocardia
Granules are crushed between two slides and Nocardia resemble Actinomycetes morphologically
stained with Gram and Ziehl–Neelsen staining but are aerobic. Most species (such as N. asetroides
254 | Section 3: Systemic Bacteriology
Table 35.1 Differences between the genera Cutaneous infection: Primary or secondary
Actinomyces and Nocardia cutaneous infection may lead to mycetoma, lymph
ocutaneous infections, cellulites, subcutaneous
Actinomyces spp. Nocardia spp.
abscesses.
• Facultative anaerobes • Strict aerobes
• Grow at 35–37°C • W
ide temperature Laboratory Diagnosis
range of growth
Diagnosis is by demonstration of branching fila
• Oral commensals • E nvironmental
ments microscopically and by isolation in culture.
saprophytes
• Nonacid-fast mycelia • Usually weakly acid-fast
1. Specimens
• E ndogenous cause of • Exogenous cause of
disease disease
Pus or purulent sputum.
LEARNING OBJECTIVES
After reading and studying this chapter, you should – Discuss various groups of E. coli producing diarrhea
be able to: – Differentiate between heat labile toxin (LT) and
∙∙ Describe general characters of the family Enter heat stable toxin (ST) of E. coli
obacteriaceae – Discuss laboratory diagnosis of urinary tract
– Classify the family Enterobacteriaceae infections caused by E. coli
– Describe morphology, culture characteristics and – Discuss morphology, culture characteristics and
biochemical reactions of E. coli biochemical reactions of Klebsiella sp
– Discuss pathogenicity of E. coli – Differentiate between E. coli and Klebsiella sp.
INTRODUCTION CLASSIFICATION OF
The family Enterobacteriaceae is the largest, most ENTEROBACTERIACEAE
heterogeneous collection of medically important
Gram-negative bacilli. Because of their normal
Lactose Fermentation
habitat in humans, these organisms are referred The oldest method was to classify these bacteria
to as the “enteric bacilli” or “enterics” into three groups based on their action on
lactose. Lactose fermenters (LF), Late lactose
CHARACTERISTICS OF THE FAMILY fermenter and nonlactose-fermenting (NLF).
ENTEROBACTERIACEAE The specimen is plated on a medium containing
lactose and neutral red indicator. (MacConkey
Members of the family Enterobacteriaceae have agar). The organisms fermenting the lactose form
the following characteristics: acid and in acidic pH, neutral red is red in colour,
i. They are gram-negative bacilli. therefore, the colonies of lactose-fermenting
ii. They are aerobes or and facultative anaerobes bacteria are red or pink and those of nonlactose-
and grow readily on ordinary laboratory media fermenting (NLF) bacteria are pale. All lactose-
including MacConkey’s lactose bile-salt agar. ferm enting enterobacteria, e.g. Escherichia,
iii. All species ferment glucose with the production Klebsiell a, Enterobacter and Citrobacter are
of acid or acid and gas. popularly known as ‘coliform bacilli’ as the
iv. They are either non-motile or motile with most common member of this group is the colon
peritrichous flagella. bacillus or Escherichia coli. The major intestinal
pathogens, Salmonella and Shigella are non-
v. They are catalase positive (except for Shigella
Iactose-fermenters (NLF). There remained a
dysenteriae type 1 which is catalase-negative)
small group which showed delayed fermentation
vi. They are oxidase-negative. of lactose (2–8 days) and with the exception of
vii. They reduce nitrate to nitrites Shigella sonnei, they were all commensals. This
viii. They are typically intestinal parasites of heterogenous group of late lactose fermenters was
humans and animals. called paracolon bacilli.
VP – – – + + + + – – – – –
Citrate – – – + + + + + + d d d
H2S – – + + – – – + + + – –
Urease – – – + d – – – – + + d
Phenylalanine – – – – – – – – – + + +
deaminase (PPA)
Arginine d – – – d – – d + – – –
dehydrolase
Lysine + – + d d + + – + – – –
decarboxylase
Ornithine d d + – + + + d + d + –
decarboxylase
(d = results different in different species or strains)
Important exceptions:
1. Sh. sonnei ferments lactose and sucrose late
2. S. Typhi does not produce gas from sugars
15-03-2016 11:15:18
Chapter 36: Enterobacteriaceae: Escherichia, Klebsiella, Proteus and Other Genera | 259
Table 36.3 K antigens (group I and group II) of colonization factor antigens (CFAs) (CFAI,
E. coli CFAII, CFA/III) expressed by enterotoxigenic E.
coli (ETEC) causing diarrheal disease in humans.
Properties Group I Group II
1. Molecular weight >100 000 <50 000 B. Toxins
2. O groups 08.09 Many 1. Exotoxins: E. coli produces two kinds of
3. Acidic component Hexuronic acid Glucuronic acid, exotoxins hemolysins and enterotoxins.
phosphate, a. Hemolysins: Hemolysins do not appear to
KDO, NeuNAc be relevant in pathogenesis.
4. Electrophoretic Low High b. Enterotoxins: Three distinct types of E. coli
mobility enterotoxins have been identified:
5. Expressed at Yes No i. Heat-labile toxin (LT): Mechanism
17-20°C of action of LT: E. coli LT is heat labile
6. Chromosome site His SerA protein and is closely related to the
KDO, ketodeoxyoctonate; NeuNAc, N-acetylneuraminic toxin produced by strains of Vibrio
acid cholerae. There are two main forms,
termed LT-I and LT-II. Different forms
of LT-1 have been described. Similarly,
the agglutinability, antigenicity and antibody two forms of LT-II (LT-IIa and LT- lIb)
binding power of bacterial strains that express have been detected.
them. Later it was shown that the B antigen was LT is a complex of polypeptide subunits-
not a separate entity. K antigens are therefore each unit of the toxin consisting of
currently classified into two groups, I and II, one subunit A (A for active) and five
(Table 36.3). subunits B (B for binding). The toxin
4. Fimbrial antigen (F antigen): These are binds to the Gm1 ganglioside receptor
thermolabile proteins. Heating the organisms on intestinal epithelial cells by means of
at 100°C leads to detachment of fimbriae. The subunit B, following which the subunit
F antigen has no role in antigenic classification A is activated to yield two fragments—A1
of E. coli. and A2. The A1 fragment activates adenyl
cyclase in the enterocyte to form cyclic
Virulence Factors
adenosine 5’ monophosphate (cAMP),
Two types of virulence factors have been recognized leading to increased outflow of water
in E. coli—surface antigens and toxins. and electrolytes into the gut lumen, with
consequent diarrhea.
A. Surface Antigens LT is a powerful antigen and can
1. Somatic antigen (O antigen): The somatic therefore, be detected by a number of
lipopolysaccharide surface O antigen, besides serological as well as biological tests.
exerting endotoxic activity, also protects the ii. Heat-stable toxin (ST): The heat stable
bacillus from phagocytosis and the bactericidal toxins of E. coli (ST) have a low molecular
effects of complement. weight which is probably responsible for
2. K antigen: Most strains of E. coli responsible their heat stability and poor antigenicity.
for neonatal meningitis and septicemia carry There are two major classes, designated
the KI envelope antigen which is a virulence ST-I (or STa) and ST-II (or STb). STa is
factor resembling the group B antigen of associated with human disease.
meningococcci. a. ST-I (or STa): STa is a small, monomeric
3. Fimbriae: Like many other members of the toxin that binds to guanylate cyclase,
Enterobacteriaceae, strains of E. coli exhibit leading to an increase in the level of
common fimbriae which are chromosomally cyclic guanosine monophosphate and
determined, present in large numbers and subsequent hypersecretion of fluids. ST-I
causing mannose sensitive hemagglutination is methanol soluble, is plasmid encoded.
and probably not relevant in pathogenesis. b. ST-II (STb)—ST-II (STb) is distinguished
Filamentous protein structures resembling from ST-I (STa) by its biological activity
fimbriae cause mannose-resistant hemagglutin and by its insolubility in methanol. It
ation and play an important part in the stimulates fluid accumulation in ligated
pathogenesis of diarrheal disease and in intestinal loops of young piglets (upto
urinary tract infection. They include the nine weeks) but not in the infant mouse
K88 antigen found in strains causing enteritis test. The mechanism of action is not
of pigs, the K99 antigen found in strains known but it appears not to act via cAMP
causing enteritis of calves and lambs, and the or cGMP.
K. rhinoscleromatis causes rhinoscleroma, a urine, pus and other pathological materials. They
chronic granulomatous hypertrophy of the nose. may cause urinary tract infections and hospital
K. oxytoca may be rarely isolated from clinical infections. They are occasionally associated with
specimens (Table 36.7). meningitis and septicemia.
Treatment: Aminoglycosides are often effective in
Laboratory Diagnosis the treatment of Enterobacter infections.
Diagnosis is made by culturing appropriate spec
imens on blood agar and Mac Conkey agar and HAFNIA
identifying the isolate by biochemical reactions.
Antibiotic sensitivity should invariably be done. These organisms are probably best regarded
Many strains carry plasmids determining multiple as non-lactose fermenter (NLF) member of
drug resistance. genus Enterobacter. This is a motile, nonlactose-
fermenting bacillus which is indole and MR
Treatment negative and VP and citrate positive. Biochemical
They are normally susceptible to cephaloporins, reactions are evident best at 22°C but at 37°C they
especially β-lactamase stable derivatives may be negative or irregular. Hafnia alvei is the
such as cefuroxime and cefotaxime, and to only species.
fluoroquinolones. They are often sensitive to Strains are isolated from feces of man and
gentamicin and other aminoglycosides., but other animals and are also found in sewage, soil,
transferable enzymic resistance to aminoglycosides water and dairy products. They are occasionally
and other antimicrobial agents has become encountered as opportunistic pathogens that has
common in strains found in some hospitals. been recovered from infected wounds, abscesses,
Klebsiella infection of the urine often responds sputum urine, blood and other sites.
to trimethoprim, nitrofurantoin, co-amoxiclav
or oral cephalosporins. Pneumonia and other SERRATIA
serious infections require vigorous treatment S. marcescens is the one most commonly
with aminoglycoside or a cephalosporin, such as encountered species in clinical specimens. It is
cefotaxime. small, motile, gram-negative bacillus. This differs
from Hafnia in forming a pink, red or magenta,
ENTEROBACTER nondiffusible pigment called prodigiosin which
Enterobacter is a motile, capsulated, lactose is formed optimally at room temperature.
fermenting bacillus which is indole and MR negative
and VP and citrate positive (IMViC – - + +). These Pathogenesis
characteristics are similar to those of Klebsiella but It is a saprophyte found in water, soil and food.
can be differentiated from Klebsiella because it is It may grow in sputum after collection and may
motile and ornithine positive. Two clinically relevant suggest hemoptysis because of the pigment formed
species are E. cloacae and E. aerogenes. (pseudohemoptysis).Nosocomial infections due to
Clinical Infections: They are normally found S. marcescens are being reported with increasing
in feces, sewage, soil and water and rarely in frequency. The bacillus has been associated with
Learning Objectives
After reading and studying this chapter, you should ∙∙ List various differences among Proteus, Morganella
be able to: and Providentia.
∙∙ Describe morphology, culture characteristics and
biochemical reactions of Proteus sp
Shigella
LEARNING OBJECTIVES
After reading and studying this chapter, you should ∙∙ Discuss the antigenic structure and toxins of
be able to: Shigella
∙∙ Describe morphology and various culture media ∙∙ Discuss laboratory diagnosis of bacillary dysentery.
for the isolation of Shigella
LEARNING OBJECTIVES
After reading and studying this chapter, you should Salmonella; antigenic variation of Salmonellae;
be able to: Kauffmann-White scheme
∙∙ Describe morphology, culture charactrestics and ∙∙ Discuss laboratory diagnosis of enteric fever
biochemical reactions of Salmonella sp ∙∙ Describe vaccination against enteric fever
∙∙ Describe the following: Antigenic structure of ∙∙ Discuss Salmonella gastroenteritis.
Biochemical Reactions
1. Salmonellae ferment glucose, mannitol, arabi
nose, maltose, dulcitol and sorbitol, forming
acid and gas except S. Typhi, Gallinarum and
rare anaerogenic variants in other serotypes form
only acid and no gas.
2. Lactose, sucrose, salicin or adonitol are not
fermented.
3. They are, indole positive, MR positive, VP
negative and citrate positive (IMViC – + – +) Fig. 39.1: Antigenic structure of salmonellae
except by S. Typhi and S. Paratyphi A which are Several strains carry fimbriae. Fimbrial antigens
citrate negative as they need tryptophan as the are not important in identification but may cause
growth factor. confusion due to their nonspecific nature and
4. Hydrogen sulfide is produced except by S. widespread sharing among enterobacteria.
Paratyphi A, S. Choleraesuis, S. Typhisuis and 1. H antigen: These antigens represent deter
S. Sendai. minant groups on the flagellar protein. They
5. Urease is not hydrolyzed. are heat-labile and alcohol-labile, but are well
6. Salmonellae decarboxylate the amino acids preserved in 0.04–0.2% formaldehyde. In most
lysine, ornithine and arginine. salmonellae, H antigen exists in two alternative
The enteric fever group may be separated phases called phase 1, and phase 2.
biochemically (Table 39.1). When mixed with antisera, H suspensions
agglutinate rapidly, producing large, loose, fluffy
Resistance clumps. The H antigen is strongly immunogenic
Salmonellae are readily killed by moist heat, at 55°C and induces antibody formation rapidly and in
in 1 hour or at 60°C in 15 minutes and most strong high titers following infection or immunization.
disinfectants. Boiling or chlorination of water and 2. O antigens: The somatic O antigen is a
pasteurization of milk destroy the bacilli. They are phospholipidprotein-polysaccharide complex
killed within five minutes by mercuric chloride (1: which forms an integral part of the cell wall.
500) or 5% phenol. The O antigen is unaffected by boiling (heat-
stable), alcohol (alcohol-stable) or weak acids.
Antigenic Structure The O antigen is less immunogenic than the H
antigen and the titer of the O antibody induced
Antigenic structure of salmonellae is shown in after infection or immunization is generally lower
Figure 39.1. Salmonellae possess the following than that of the H antibody. O agglutination takes
antigens based on which they are classified and place more slowly and at a higher temperature
identified: optimum (50–55°C) than H agglutination (37°C).
1. Flagellar antigen H. 3. Vi antigen: Almost all recently isolated strains of
2. Somatic antigen O. S. Typhi form Vi antigen as a covering layer
3. Surface antigen Vi, found in some species. outside their cell wall. This antigen is an acidic
Table 39.1 Biochemical characters of typhoid and polysaccharide.When fully developed it renders
paratyphoid bacilli the bacteria agglutinable by Vi antibody and
inagglutinable by O antibody. Felix and Pitt,
Glucose Xylose d-Tartrate Mucate who first described this antigen, believed that
S. Typhi A d A d it was related to virulence and gave it the name
S. Paratyphi A AG − − − ‘Vi antigen’ (Vi for virulence). It is analogous to
S. Paratyphi B AG AG − AG
the K antigens of coliforms.
The Vi antigen is heat-labile. It can be removed
S. Paratyphi C AG AG AG −
from the bacteria by heating a suspension for 1
A = Acid; AG = Acid and Gas; d = Delayed hour at 100°C and centrifuging the bacteria from
not found in other O groups. The O groups first more than 2400 serotypes of salmonellae, giving
defined were designated by capital letters A to Z individual names is not realistic.
and those discovered later by the number (51–67)
of the’ characteristic O antigen. It would be more Differentiation of Antigenically Similar
logical to name the serogroups according to their Strains
characteristic O antigen factor number rather than
Serotypes that share the same antigenic formula
by letters, i.e. to abandon the letters A–Z used to
may be distinguished from one another by bio
designate early O groups. Hence, O groups become:
chemical tests, e.g. Paratyphi C has the same O and
O2 (A), O4 (B), O7 (Cl), O8 (C2–C3), 09, 12 (Dl),
H antigens as Choleraesuis, and Typhisuis. Thus,
09, 46 (D2), 03, 10 (EI) etc. Some serogroups were
Paratyphi C ferments d-tartrate and trehalose,
subdivideed into subserogroups (C1–C4; E1–E4).
but not Stern’s glycerol. Choleraesuis ferments
Serogroups A–Z and 51–67 represented O antigens
d-tartrate, but not trehalose or Stern’s glycerol, and
2–50 and 51–67 respectively. Groups 02 to 03, 10
Typhisuis only trehalose.
(A–EI) contain nearly all the salmonellae that are
important pathogens in man and animals.
Within each O group the different serotypes are Bacteriophage Typing
distinguished by their particular H antigen or com Strains within a particular serotype may be differen
bination of H antigens (Table 39.3). Kauffmann– tiated into a number of phage types by their patterns
White classification gave species status to each of susceptibility to lysis by members of a series of
serotype. The species were named according to phages with different specificities. Intraspecies
the disease caused (S. Typhi), the animal source (S. classification of S. Typhi for epidemiolocal
Gallinarium), the discoverer (S. Schottmulleri), the purpose was made possible by bacteriophage
name of the patient from whom the first strain was typing, first developed by Craige and Yen (1937).
isolated (S. Thompson), or the place of isolation They observed that a bacteriophage acting on the
(S. Poona). This was satisfactory so long as the Vi antigen of the typhoid bacillus (Vi phage II) is
serotypes were not too many but now with some highly adaptable. The parent phage is called phage
Epidemiology
The typhoid and paratyphoid bacilli are essentially
human parasites. Man is the reservoir host and
most infections can be traced to a human source,
or at least to a source of human sewage. All other
salmonellae have animal hosts.
S. Paratyphi A is prevalent in India and other
Asian countries, Eastern Europe and South
America, S. Paratyphi B in Western Europe, Britain
and North America; and S. Paratyphi C in Eastern Fig. 39.3: Laboratory diagnosis of typhoid fever. The appro
Europe and Guyana. Enteric fever is endemic in all ximate percentages of tests found positive during different
parts of India. Paratyphoid B is rare and C very rare. stages of the disease (from 1st to 5th week). A. Widal aggluti
nation. B. Feces culture. C. Blood culture
The disease occurs at all ages but is probably most
common in the 5–20 year age group.
The source of infection is a patient, or far more
Table 39.4 Positivity of various specimens at
different phases of enteric fever
frequently, a carrier. Food handlers or cooks who
become carriers are particularly dangerous. The Duration Specimen % positivity
best known of such typhoid carriers was Mary 1st week Blood culture 90
Mallon (‘Typhoid Mary’), a New York cook who Feces culture –
caused at least seven outbreaks affecting over 200
Widal test –
persons over a period of 15 years.
2nd week Blood culture 75
Laboratory Diagnosis Feces culture 50
Bactriological dignosis of enteric fever consists of: Widal test Low titer
A. Isolation and identification of the bacilli. 3rd week Blood culture 60
B. Demonstration of circulating antigen.
Feces culture 80
C. Demonstration of antibodies in patient’s serum.
Widal test 80–100
D. Other laboratory tests.
LEARNING OBJECTIVES
After reading and studying this chapter, you should ∙∙ Differentiate between classical and El Tor vibrios
be able to: ∙∙ Discuss laboratory diagnosis of cholera
∙∙ Describe morphology, cuture characteristics and ∙∙ Describe the following: cholera vaccine; nonagglut
biochemical reactions of Vibrio cholerae inating vibrios; halophilic vibrios
∙∙ Discuss antigenic structure of Vibrio cholerae
∙∙ Describe the following: pathogenesis of cholera;
mechanism of action of cholera toxin
VIBRIO CHOLERAE
Morphology
These are gram-negative, short, curved, cylindrical
rods, about 1.5 μm × 0.2–0.4 μm in size. The cell
is typically comma-shaped. S-shaped or spiral
forms may be seen due to two or more cells lying
end-to-end. In old cultures, they are frequently
highly pleomorphic. The vibrios are seen arranged
in parallel rows, described by Koch as the ‘fish in
stream’ appearance in stained films of mucous
flakes from acute cholera cases. Fig. 40.1: Cholera vibrios
V. cholerae 0139
Table 40.3 Differeces between classical cholera In 1992 cases of cholera indistinguishable from that
and El Tor vibrios caused by V. cholerae 01 were reported in Madras
Test Classical cholera El. Tor (Chennai), India. By mid-January 1993 similar
isolates were found in neighboring Bangladesh,
Hemolysis − +*
and these rapidly spread north, following the
Voges-Proskauer − +* course of the major rivers and raising fears of a new
Chick erythrocyte − + pandemic.
agglutination V. cholerae 0139 may have evolved from V.
Polymyxin B sensitivity† + − cholerae 01, but with modified lipopolysaccharide
Group IV phage + − structure. V. cholerae 0139 makes a polysaccharide
susceptibility capsule like other non-O 1 V. cholerae strains, while
El Tor phage 5 − + V. cholerae 01 does not make a capsule. In the
susceptibility affected areas, this strain replaced the El Tor vibrios
* Strain isolated after 1961 give variable results. as the epidemic and environmental serovar. It also
†
50 i. u. disk. showed a tendency to be more invasive, causing
bacteremic illness in some. Both O-1 El Tor and
2. Sensitivity to polymyxin B: The organism is O-139 strains began to coexist in endemic areas.
tested by the disc diffusion method using discs
containing 50 units of polymyxin B. All strains Phage Typing
of classical cholera vibrio are sensitive and all Phage typing schemes have been standardized
strains of EI Tor vibrio are resistant. for classical and El Tor biotypes. New molecular
Learning Objectives
After reading and studying this chapter, you should ∙∙ Discuss morphology, culture characteristics and
be able to: biochemical reactions of Helicobacter
∙∙ Describe morphology, culture characteristics and ∙∙ Discuss laboratory diagnosis of Helicobacter pylori
biochemical reactions of Campylobacter infections.
Learning Objectives
After reading and studying this chapter, you should ∙∙ Describe the following: Pigments of Pseudomonas;
be able to: pathogenicity of Pseudomonas aeruginosa;
∙∙ Describe morphology, cultural characteristics, Burkholderia mallei.
biochemical reactions and laboratory diagnosis of
Pseudomonas aeruginosa
Pseudomonas aeruginosa
Morphology
It is a slender gram-negative bacillus, 1.5–3 μm ×
0.5 μm, actively motile usually with a single polar
flagellum. Occasional strains have two or three
flagella. It is nonsporing, noncapsulated but many
strains have a mucoid slime layer.
Cultural Characteristics
It is a strict aerobe but can grow anaerobically if Fig. 42.1: Pseudomonas aeruginosa on nutrient
nitrate is available. Growth occurs at a wide range agar
302 | Section 3: Systemic Bacteriology
5. Cetrimide agar is selective medium for Ps. Epidemiological Typing Methods
aeruginosa 1. Serotyping: Identification of group-specific
heat-stable lipopolysaccharide antigens by
Pigment Production agglutination forms the basis of O serotyping.
P. aeruginosa produces at least 4 distinct pigments: Typically, nine serotypes account for over 90%
i. Pyocyanin: It is a bluish-green phenazine of isolates.
pigment soluble in chloroform and water. This 2. Bacteriocin (pyocin) typing: Three types
pigment is not produced by other species of of bacteriocins (pyocins) are produced by P.
this genus. Demonstration of the presence aeruginosa which are known as R, F and S.
of the blue phenazine pigment pyocyanin Depending upon the growth inhibition of these
is absolute confirmation of a strain as P. 13 indicator strains, 105 types are recognized.
aeruginosa. Pyocin typing is easy to perform, results are
ii. Pyoverdin (fluorescein): The yellow/green available by the third day and has a reasonable
pigment pyoverdin (fluorescein) is also reproducibility and good discrimination.
produced by most strains, giving the charac 3. Phage typing: In bacteriophage typing
teristic blue-green appearance of infected pus considerable difficulties have been encounterd.
or cultures. It is insoluble in chloroform but 4. Molecular methods: Restriction endonuclease
soluble in water. typing wih pulsed field gel electrophoresis
iii. Pyorubrin: It is a bright red water soluble (PFGE) is the most reliable typing method
pigment and is insoluble in chloroform. and discriminatory of the present DNA-based
iv. Pyomelanin: It is a brown to black pigment typing methods and is considered to be the gold
and its production is uncommon. standard.
Legionella
Learning Objectives
After reading and studying this chapter, you should ∙∙ Describe the following: Legionella pneumophilia;
be able to: diseases caused by Legionella.
∙∙ Describe morphology, culture characteristics and
biochemical reactions of Legionella
Introduction Culture
In the summer of 1976, public attention was Legionellae are nutritionally fastidious. Their
focused on an outbreak of severe pneumonia that growth is enhanced with iron salts and depends
caused many deaths in members of the American on the supplementation of media the L-cysteine.
Legion convention in Philadelphia. The disease They have fastidious requirements and grow on
was characterized by fever, cough and chest pain, complex media such as buffered charcoal, yeast
leading on to pneumonia and often ending fatally. extract (BCYE) agar, with L-cysteine and antibiotic
The causative agent has been called Legionella supplements, with 5% CO2, at pH 6.9, 35°C and 90%
pneumophila. Subsequent studies found this humidity. Growth is slow and colonies take 3–6
organism to be the cause of multiple epidemic and days to appear.
sporadic infections. It is now recognized to be a
ubiquitous aquatic saprophyte. Biochemical Reactions
The organisms are nonfermentative. Most species
Species: Taxonomic studies have shown that are motile and catalase-positive, liquefy gelatin,
the family Legionellaceae consists of one genus, and do not reduce nitrate or hydrolyze urea.
LegionelIa, with 40 species and more than 60
serogroups. The original isolate in this genus is Epidemiology
designated L. pneumophila serogroup 1 (SG1),
The bacteria are commonly present in natural
which accounts for nearly all severe infections.
bodies of water, such as lakes and streams, as well
Examples of other species that cause human
as in air conditioning cooling towers and condens
infection less often are L. micdadei, L. bozemanii,
ers and in water systems (e.g. showers, hot tubs).
L. dumoffii and L. gormanii.
Legionellae survive and multiply inside free-living
amebae and other protozoa. They also multiply in
LEGIONELLA PNEUMOPHILA some artificial aquatic environments, which serve
Morphology as amplifiers.
Legionellae are thin, noncapsulated bacilli, 25 mm Human infection is typically by inhalation of
× 0.3–0.1 mm coccobacillary in clinical material aerosols produced by cooling towers, air conditioners
and assuming longer forms in culture. Most are and shower heads which act as disseminators. Man-
motile with polar or subpolar flagella. They are to-man transmission does not occur.
gram-negative but stain poorly, particularly in
smears from clinical specimens. They stain better Pathogenesis
by silver impregnation, but are best visualized by Respiratory tract disease caused by Legionella
direct fluorescent antibody (DFA) staining with species develops in susceptible people who inhale
monoclonal or polyclonal sera. infectious aerosols.
308 | Section 3: Systemic Bacteriology
Clinical Diseases Treatment
Asymptomatic Legionella infections are relatively For treatment, the newer macrolides,ciprofloxacin,
common. Symptomatic infections primarily affect and tetracyclines are effective. Rifampicin is
the lungs and present in one of two forms: employed in severe cases.
1. Pontiac fever: An influenza-like illness
2. Legionnaires’ disease: A severe form of Prevention
pneumonia Prevention of legionellosis requires identification of
1. Pontiac fever: Pontiac fever is a milder, nonfatal the environmental source of the organism and reduc
‘influenza-like’ illness with fever, chills, myalgia tion of the microbial burden. Hyperchlorination of
malaise, and headache but no clinical evidence the water supply and the maintenance of elevated
of pneumonia. Outbreaks with high attack rates water temperatures have proved moderately
may occur. successful. Howe ver, complete elimination of
2. Legionnaire’s disease: The incubation period Legionella organisms from a water supply is often
is 2–10 days. ‘The disease presents with fever, difficult or impossible. Because the organism
nonproductive cough and dyspnea, rapidly has a low potential for causing disease, reducing
progressing, if untreated, to pneumonia. Multi the number of organisms in the water supply is
organ disease involving the gastrointestinal frequently an adequate control measure. Hospitals
tract, central nervous system, liver and kidneys with patients at high risk for disease should monitor
is common. their water supply on a regular basis for the presence
Case fatality may be 15–20%. All age groups are of Legionella and their hospital population for
susceptible, though more cases have occurred in disease.
the elderly. Key Points
Legionella pneumophilia serogroup 1 is most
Laboratory Diagnosis common human pathogen
1. Microscopy L. pneumophilia are small, slender, pleomorphic,
Gram-negative bacilli
Legionellae in clinical specimens stain poorly Human infection is typically by inhalation of aerosols
with Gram stain. Nonspecific staining methods, produced by cooling towers, air conditioners and
such as those using Dieterle’s silver or Gimenez’s shower heads
stain, can be used to visualize the organisms. L. pneumophilia causes Legionnaire’s disease
The most sensitive way of detecting legionellae Laboratory diagnosis depends on direct fluorescent
antibody (DFA) test, culture, detection of antigen and
microscopically in clinical specimens is to use the demonstration of serum antibodies
direct fluorescent antibody (DFA) test, in which Hyperchlorination of the water and superheating of
fluorescein-labeled monoclonal or polyclonal water is of value in preventing the disease.
antibodies directed against Legionella species are
used.
Important questions
2. Culture 1. Discuss pathogenicity and laboratory diagnosis of
The medium most commonly used for the isolation Legionnaire’s disease.
2. Write short notes on:
of legionellae is buffered charcoal-yeast extract
a. Legionella pneumophilia
(BCYE) agar. Antibiotics can be added to suppress b. Legionnaire’s disease
the growth of rapidly growing contaminating c. Pontiac fever.
bacteria. Legionellae grow in air or 3% to 5% carbon
dioxide at 35°C after 3–5 days. Their small (1–3 mm) Multiple choice question (mCQs)
colonies have a groundglass appearance. 1. The causative agent of Pontiac fever is:
a. Legionella pneumophila
3. Antigen Detection b. Pseudomonas putida
c. Yersinia pseudotuberculosis
Enzyme-linked immunoassays, radioimmuno
d. Francisella tularensis
assays, the agglutination of antibody-coated latex 2. The natural habitat of Legionella pneumophila is:
particles, and nucleic acid analysis studies have a. Water b. Sputum
all been used to detect legionellae in respiratory c. Faeces d. Urine
specimens and urine. 3. The antibiotic of choice in Legionella infections is:
a. Erythromycin b. Cephalosporins
4. Serology c. Penicillins d. All of the above
Detection of serum antibody is done by ELISA or Answers (MCQs)
indirect immunofluorescent assay. 1. a; 2. a; 3. a
44
Chapter
Learning Objectives
After reading and studying this chapter, you should ∙∙ Describe laboratory diagnosis of plague
be able to: ∙∙ Describe the following: prophylaxis against plague;
∙∙ Describe morphology, culture characteristics and Yersinia enterocolitica; Yersinia pseudotuberculosis;
biochemical reactions of Yersinia pestis Pasteurella multocida; Francisella tularensis.
Pathogenesis Pathogenesis
The bacillus is usually normal inhabitant of the
Y. enterocolitica has been isolated from a wide
upper respiratory tract of a variety of animals
range of domestic and wild animals. Human
such as dogs, cats, cattle and sheep. Pasteurella
disease usuaIly results from ingestion of contam
infections are considered zoonoses. The most
inated food or from contact with the environment.
common presentation is a history of animal bite.
Blood transfusion is a significant hazard.
Human infections usually present as:
Types of Disease in Humans 1. A local abscess at the site of a cat or dog bite;
2. Infections of the respiratory system.
1. Gastroenteritis or enterocolitis, which is self-
3. Meningitis or cerebral abscess (usually follow
limited and occurs in young children.
ing head injury), endocarditis, pericarditis or
2. Mesenteric lymphadenitis and terminal ileitis
septicemia.
in older children that may mimic appendicitis
3. Septicemia, which is often fatal. Animals and birds: P. multocida can be extremely
4. Pneumonia and meningitis are rare present virulent to many species of animals and birds,
ations. causing fowl cholera; hemorrhagic septicemia. It
5. Postinfectious complications include erythema respiratory infections and atrophic rhinitis in pigs.
nodosum, polyarthritis, Reiter’s syndrome and
thyroiditis. Laboratory Diagnosis
1. Culture: Swabs from bite wounds, from blood,
Laboratory Diagnosis from CSF in cases of meningitis and from
The laboratory diagnosis of Y. enterocolitica secretions in suppurative conditions of the
consists of isolation of the organism and indirectly respiratory tract are cultured on blood agar
Chapter 44: Yersinia, Pasteurella, Francisella | 315
plates incubated at 37°C for 24 hours. The Prophylaxis
organisms are identified by various cultural and A vaccine based on the live-attenuated LVS strain
biochemical tests. confers some protection. It can be administerd by
2. Serology: Serology is of no value in diagnosis. scarification to persons who are subject to high
3. Polymerasrs chain reaction (PCR): PCR is risk of infection.
potentially useful but rarely available. F. tularensis has been developed as a biological
warfare agent and has potential application in
Francisella tularensis bioterrorism.
(Pasteurella tularensis, Brucella Key Points
tularensis) Yersinia are gram-negative bacilli that are facultative
Francisella tularensis produces tularaemia in man anaerobes, positive by the catalase test and negative
by the oxidase test
and certain small mammals, notably rabbits, hares,
The genus Yersinia consists of 10 species, with Y. pestis,
beavers and various rodent species. Tularemia was Yersinia enterocolitica, and Yersinia pseudotuberculosis
originally described in Tulare county, California. It the well-known human pathogens
can be transmitted by direct contact, by biting flies, Y. pestis is a gram-negative, shows bipolar staining
mosquitoes and ticks, by contaminated water or (safety pin appearance), pleomorphic, nonmotile,
meat, or aerosols. and is capsulated
F-l antigen or envelope antigen has been considered
a virulence determinant
Morphology Plague is a zoonotic infection in humans. Disease is
It is a very small, nonmotile, non-sporing, capsulate, spread by flea bites or direct contact with infected
tissues or person-to-person by inhalation of infectious
Gram- negative coccobacillus, about 0.3–0.7 µm ×
aerosols from a patient with pulmonary disease
0.2 µm in size. Diseases: Yersinia pestis causes plague, which
manifests in one of three forms: Bubonic plague;
Cultural Characters pneumonic plague; septicemic plague
Diagnosis: For diagnosis, material is aspirated
F. tularensis is strictly aerobic. It will not grow on from the bubo, for demonstration of bipolar
ordinary nutrient media, but grows well on blood staining short rods (coccobacilli) using Wright’s or
agar containing 2.5% glucose and 0.1% cysteine Gram stain, for demonstration of the bacteria by
hydrochlor ide. Minute droplet-like colonies immunofluorescence. Culture of the bacteria on
blood agar. Serology can be used to detect antibody
develop in 72 hours. to capsular antigen
Yersinia pseudotuberculosis: It causes pseudotuber
Pathogenesis culosis, a zoonotic disease
Yesinia enterocolitica
The infection, which is a typical zoonosis, is mainly
It produces in humans-gastroenteritis or enterocol
spread by insects or ticks among lagomorphs and itis, mesenteric lymphadenitis and terminal ileitis
rodents. It is transmitted to man through handling in older children, septicemia, pneumonia and
of infected animals. meningitis, erythema nodosum, polyarthritis, Reiter’s
In human beings, tularemia may present as a syndrome and thyroiditis
local ulceration with lymphadenitis, a typhoid like Pasteurella multocida
fever with glandular enlargement or an influenza Pasteurella infections are considered zoonoses
like respiratory infection. Francisella tularensis
Francisella tularensis produces tularemia in man and
certain small mammals. The infection, is a typical
Laboratory Diagnosis zoonosis.
Diagnosis may be made by culture or by inoculation
into guinea pigs or mice. A PCR has been Important questions
described, but is not widely available. Serology is
most likely to be positive after 3 weeks. Rising titers 1. Describe the pathogenesis and laboratory diagnosis
of plague.
of agglutinins to F. tularensis or individual titers
2. Write short notes on:
of 160 are diagnostic. An intradermal delayed
a. Prophylaxis against plague
hypersensitivity test has been used in the past but
b. Yersinia pseudouberculosis
the antigen is not readily available.
c. Yersinia enterocolitica
d. Pasteurellosis
Treatment e. Pasteurella multocida
Streptomycin or gentamicin are the antibiotics of f. Francisella tularensis
choice in tularemia and are usually curative. g. Tularemia
316 | Section 3: Systemic Bacteriology
Multiple choice questions (Mcqs) 5. Which of the following vaccines is/are recommended
for immunization against plague?
1. Bipolar staining is characteristic of: a. Live b. Killed
a. Yersinia pestis c. Subunit d. All of the above
b. Yersinia enterocolitica 6. Yersinia enterocolitica shows all the following
c. Yersinia pseudotuberculosis characteristics except:
d. Proteus mirabilis a. They are motile at 22°C
2. Bubonic plague is transmitted by: b. They produce hemolytic colonies on blood agar
a. Inoculation b. Inhalation c. They ferment sucrose and cellobiose
c. Ingestion d. All of the above d. They decarboxylate ornithine
routes 7. Which of the following diseases is/are caused by Y.
3. Pneumonic plague is transmitted to humans by: enterocolitica in man?
a. Rat flea b. Droplet infection a. Gastroenteritis
c. Ingestion d. Inoculation b. Mesenteric lymphadenitis
c. Septicemia
4. The name black death is given to which of the d. All of the above
following diseases?
a. Tuberculosis b. Diphtheria Answers (MCQs)
c. Plague d. AIDS 1. a; 2. a; 3. b; 4. c; 5. b; 6. c; 7. d
45
Chapter
Haemophilus
Learning Objectives
After reading and studying this chapter, you should ∙∙ Discuss laboratory diagnosis of infections caused
be able to: by Haemophilus influenzae
∙∙ Describe morphology and culture characteristics ∙∙ Discuss Haemophilus influenzae biogroup aegyptius
of Haemophilus influenzae ∙∙ Describe morphology and culture characteristics of
∙∙ Describe the following: X and V factors; Satellitism; Haemophilus ducreyi
Antigenic structure of Haemophilus influenzae ∙∙ Discuss Haemophilus ducreyi or chancroid or soft
∙∙ Describe pathogenicity of H. influenzae sore and its laboratory diagnosis.
Bordetella
Learning Objectives
After reading and studying this chapter, you should ∙∙ Discuss pathogenesis of pertussis
be able to: ∙∙ Discuss laboratory diagnosis of pertussis
∙∙ Describe various factors which determine the ∙∙ Describe the following: Cough plate method;
virulence of Bordetella pertussis vaccination against pertussis
∙∙ Describe culture media for Bordetella pertussis
Brucella
Learning Objectives
After reading and studying this chapter, you should ∙∙ Discuss pathogenesis of brucellosis
be able to: ∙∙ Discuss laboratory diagnosis of brucellosis
∙∙ Discuss classification of Brucella ∙∙ Describe Castaneda method of blood culture.
∙∙ Describe culture characteristics and biochemical
reactions of Brucella sp.
Thionin 1:50,000
CO2 requirement
H2S production
RTD x 104
Biotypes
Species
RTD
A M R
B. melitensis 1 − − − − + − + − + Sheep,
2 − − − − + − + + − − goats
3 − − − − + − + + + −
B. abortus 1 + + ± + + − − + − − Cattle
2 + + + + − − − + − −
3 + + ± + + + + + − −
4 + + ± + + − − − + −
5 + + − − + + + − + −
6 + + − − + + + + − −
9 + + ± ± + + + − + −
B. suis 1 − + − + − − + + − − Pigs
2 − + − − − − + + − − Pigs, hare
3 − + − − + + + + − − Pigs
4 − + − − + + + + + − Reindeer
B. neotoma'e − + − + − − − + − − Wood rat
B. ovis − − + − + + + − − + Sheep
B. canis − − − − − + + − − + Dogs
RTD: Routine test dilution
A—abortus; M—melitensis; R—rough
Chapter 47: Brucella | 329
inoculation. Person-to-person spread does not goat, sheep, cattle, buffaloes, and swine. Infection
ordinarily occur, but very rarely transmission has is transmitted among animals directly or through
been reported through the placenta, breast feeding blood-sucking arthropods, particularly ticks.
and sex. Brucellosis is a zoonosis of worldwide impor
i. Ingestion: The most important vehicle of tance, particularly in developing countries.
infection is raw milk. Brucellosis may also be potentially transmitted
ii. Contact: Contact infection is especially in association with biowarfare, bioterrorism, or
important as an occupational hazard in agri biocriminal activities.
cultural workers, veterinarians, butchers, Almost all human infections in various parts of
animal handlers, and others in occupations that India are due to B. melitensis acquired from goats
involve handling of animals or uncooked animal and sheep. Animal brucellosis is reported from
tissues are at higher risk for direct inoculation. practically every state in India.
iii. Inhalation: Infection is transmitted by inhal
ation of dried material of animal origin such Laboratory Diagnosis
as dust from wool. The clinical manifestations of human brucellosis are
Course of disease: Brucellosis is primarily an variable, so clinical diagnosis is almost impossible
intracellular pathogen affecting reticuloendothelial and laboratory aid is therefore essential. Laboratory
system. Brucellae have a special predilection for methods include culture of brucellae, serology,
intracellular growth and may be demonstrated polymerse chain reaction and hypersensitivity type
inside phagocytic cells. The brucellae spread skin tests.
from the initial site of infection through lymphatic
channels to the local lymph glands, in the cells of 1. Specimens
which they multiply. They then spill over into the
Blood culture is most important. Material from
bloodstream and are disseminated throughout the
bone marrow or liver biopsy, is also cultured, lymph
body. Once liberated into the bloodstream, they
nodes, cerebrospinal fluid, urine and abscesses, and
may infect a variety of organs but are most often
on occasion, also from sputum, breast milk, vaginal
localized in the reticuloendothelial system, where
discharges and seminal fluid.
they reside within phagocytic cells. Granulomas or
abscesses most often develop in the bone marrow,
2. Culture
liver, spleen, lymph nodes, or lungs. Other sites
of infection may include subcutaneous tissue, a. Blood culture
testes, epididymis, ovary, gallbladder, kidneys, and Blood culture is the most definitive method for
brain. Meningitis and endocarditis are commonly the diagnosis of brucellosis. When brucellosis is
reported complications. suspected, blood culture should be attempted
repeatedly, not only during the febrile phase.
Types of Human Infection Because the organisms may be scanty, at least
10 mL of blood should be withdrawn on each
The incubation period is usually about 10–30 days.
occasion, 5 mL being added to each of two blood
Human infection may be of three types:
culture bottles containing serum dextrose (SD)
1. Latent or subclinical infection
broth. One of these bottles should be incubated in
There is no clinical evidence of disease but is
an atmosphere containing 10% carbon dioxide at
detectable only by serological tests.
37°C. Subcultures are made on solid media (serum
2. Acute brucellosis: Acute brucellosis is mostly
dextrose agar) every 3–5 days, beginning on the
due to B. melitensis. It is associated with
fourth day. Growth may often be delayed and blood
prolonged bacteremia and irregular fever. It is
cultures should be retained for 6–8 weeks before
also known as undulant fever or Malta fever
being discarded as negative. Preliminary lysis and
because of the periodic noctural fever that may
centrifugation of the blood improves the isolation
occur over weeks, months or years particularly
rate. Automated blood culture systems may also
in untreated cases.
be used.
3. Chronic brucellosis: Chronic brucellosis is a low
grade infection with periodic exacerbations. It is b. Castaneda’s method of blood culture (Fig.
usually nonbacteremic. The illness lasts for years. 47.1)
Advantages: The Castaneda method of blood
Epidemiology culture has several advantages and is recommended.
Brucella organisms are distributed throughout i. A two-phase Castaneda culture system, in
the world. Human brucellosis is acquired from which the broth is periodically allowed to flow
animals, directly or indirectly. The animals that over agar contained within the blood culture
commonly act as sources of human infection are bottle, may be used. The blood is inoculated
330 | Section 3: Systemic Bacteriology
usually persist throughout the active phase of the
disease and in some cases long thereafter. As the
disease progresses, IgM antibodies decline, while
the IgG antibodies persist or increase in titer.
In chronic infections, IgM may often be absent
and only IgG can be demonstrated.
The agglutination test identifies mainly the
IgM antibody, while both IgM and IgG can fix
complement. However, as IgG and IgA antibodies
are formed during the course of the infection
some of them bind with antigen, thus preventing
its agglutination by larger IgM molecule. These
IgG and IgA antibodies are known as blocking
or non-agglutinating antibodies which may
prevent agglutination. It is thus evident that the
agglutination test is usually posit ive in acute
infection but may be only weakly positive or even
negative in chronic cases.
Fig. 47.1: Castaneda's method of blood culture
Brucella antibodies can be detected by a variety
into the broth and the bottle incubated in the of serological tests.
upright position. For subculture, it is sufficient i. Standard agglutination test (SAT): This is a
if a bottle is tilted so that the broth flows tube agglutination test in which equal volumes
over the surface of the agar slant. It is again of serial dilutions of the patient’s serum and the
incubated in an upright position. Colonies standardized antigen (a killed suspension of a
appear on the slant. standard strain of Br. abortus) are mixed and
ii. This method minimizes materials and incubated at 37°C for 24 hours or 50°C for 18
manipulation. hours. It detects antibodies against B. abortus,
iii. It reduces chances of contamination during B. suis, and B. melitensis but not B. canis. A titer
the period of incubation and risk of infection of 160 or more is considered significant.
to laboratory workers. Prozone phenomenon: Sera often contains
Blood cultures are positive only in about 30–50 ‘blocking’ or ‘nonagglutinating’ antibodies. A
per cent cases, even when repeated samples blocking factor may interfere with agglutination at
are tested. B. melitensis and B. suis are more low serum dilutions (the prozone phenomenon)
frequently isolated from blood than are B. although positive in higher dilutions.
abortus or B. canis. The blocking effect may sometimes be removed
c. Bone marrow or liver biopsy by prior heating of the serum at 55°C for 30 minutes
Isolation rates can be markedly improved if material or by using 4% saline as the diluent for the test. The
from bone marrow or liver biopsy is also cultured. most reliable method for obviating the blocking
Identification: Depends upon biochemical effect and detecting the ‘incomplete’ antibodies is
tests and may be classified into different species the antiglobulin (Coombs) test.
and biovars, based on CO 2 requirements, H 2S Cross-reactions: Cross-reactions may be observed
production, sensitivity to dyes (basic fuchsin and with antibodies directed against Francisella
thionin), agglutination with monospecific sera, tularensis, Vibrio cholerae, or Yersinia enterocolitica
lysis by specific phage and oxidative metabolic tests or immunization. Cholera induced agglutinins may
with amino acids and carbohydrates. be differentiated by the agglutinin absorption test
and also as they are removed by treatment with
3. Serological Tests 2-mercapto-ethanol. In order that results from,
In the absence of positive cultures, the diagnosis different laboratories are comparable; it is the
of brucellosis usually depends on serological tests, practice to express agglutinin titers in International
the results of which tend to vary with the stage of Units. This is done by using a standard reference
the infection. serum for comparison.
Antibodies appear within 7–10 days of onset ii. Mercaptoethanol test: The mercaptoethanol
of the disease. IgM antibodies appear first which test is carried out simultaneously and in
are rapidly followed and superseded by IgG and the same manner as the standard aggluti
to lesser extent IgA antibodies. These antibodies nation test except that the saline diluent
reach their maximum titers in the third or fourth contains 0.05 M 2-mercaptoethanol. The
week of disease and then slowly decline but they agglutinating ability of IgM, and sometimes
Chapter 47: Brucella | 331
IgA, is destroyed by 2-mercaptoethanol, and Result
therefore agglutination in this test is indicative Positive test: The bacilli are agglutinated and rise
of the continuing presence of IgG and the with the cream to form a blue ring at the top, leav
likelihood of persisting infection. ing the milk unstained.
iii. Complement fixation test: The complement
fixation test is more useful in chronic cases as Negative: No colored ring is formed and the milk
it detects IgG and IgM antibody also. remains uniformly blue.
iv. Enzyme-linked immunosorbentassay
(ELISA) and radio immunoassay (RIA): These ii. Whey Agglutination Test
tests are very sensitive useful for differentiation It is another useful method for detecting the
between the acute and chronic phases of antibodies in milk.
brucellosis. ELISA is sensitive, specific and can
detect IgM and IgG antibody separately. Prophylaxis
v. Rose Bengal plate test: This is a rapid slide
1. Prevention consists of checking brucellosis in
agglutination test. It is widely used as a
dairy animals.
screening test in farm animals.
2. Control of this disease in cattle has been
vi. Rapid dipstick assay: The rapid dipstick
achieved by serologic surveillance, vaccination
assay, a relatively simple screening procedure
(B. abortus strain 19), and elimination of
for Brucella specific IgM, shows promise as a
reactor cattle.
field test in areas without direct access to a
reference laboratory. 3. Pasteurization of infected milk or milk products.
4. Vaccines have been developed for use in animals.
Polymerase Chain Reaction (PCR) The live-attenuated B. abortus strain S19 vaccine
has been is now being replaced by the rough
Promising results have been obtained in clinical
strain B. abortus RB51, which gives comparable
studies.
protection, but does not induce interfering
Hypersensitivity Test (Brucellin Skin Test) antibodies and is less hazardous to man.
The live-attenuated smooth strain B. melitensis
Delayed hypersensitivity type skin tests with
Rev I is used to protect sheep and goats from B.
brucella antigens (‘brucellins’) are not useful in
melitensis infection. Strain 2 has been used in
diagnosing acute brucellosis. This test, similar to
China.
the tuberculin test, is no longer recommended.
They parallel the tuberculin test in indicating only 5. Human vaccination is not recommended.
prior sensitization with the antigens, and may
remain positive for years. Treatment
Brucella infections respond to a combination of
Detection of Animal Infection streptomycin or gentamicin and tetracycline or to
The methods used for the laboratory diagnosis rifampicin and doxycycline. Tetracycline alone is
of human brucellosis may also be employed for often adequate in mild cases. Treatment should be
the diagnosis of animal infections. In addition, continued for at least 6 weeks. Cotrimoxazole and
brucellae may be demonstrated microscopically rifampicin can be used in children.
in pathological specimens by suitable staining or
Key Points
by immunofluorescence.
Several rapid methods have been employed for The genus Brucella consists of very small, nonmotile,
the detection of brucellosis in herds of cattle. These aerobic, Gram-negative coccobacilli. They are
essentially pathogens of goats, sheep, cattle and pigs
include the rapid plate agglutination test and the
Six species are currently recognized and three major
Rose Bengal card test. For the detection of infected species of the genus Brucella are B. melitensis, B.
animals in dairies, pooled milk samples may be abortus, B. suis
tested for bacilli by culture and for antibodies by They grow best on media employed currently is
several techniques such as milk ring test. serum dextrose agar, serum potato infusion agar,
trypticase soy agar, or tryptose agar. They are
i. Milk Ring Test catalase and oxidase positive
Animal reservoirs are goats and sheep, cattle, swine,
1. In the milk ring test a sample of whole milk is and dogs. Individuals at greatest risk for disease are
mixed well with a drop of the stained brucella people who consume unpasteurized dairy products,
antigen (a concentrated suspension of killed Br. people in direct contact with infected animals, and
abortus stained with hematoxylin). laboratory workers
Human brucellosis is primarily a zoonotic bacterial
2. It is incubated in a water bath at 70°C for 40–50 infection
minutes.
332 | Section 3: Systemic Bacteriology
3. Brucellae are transmitted to humans by:
Laboratory diagnosis: Depends on the culture of
brucellae serology. Detection of Brucella species. Milk a. Direct contact with animal tissues
ring test is a screening test used for demonstration b. Ingestion of contaminated meats
of antibodies in the milk of animals c. Ingestion of raw infected milk
Human disease is controlled by eradication of the d. All of the above
disease in the animal reservoir; pasteurization of 4. The most common Brucella species causing human
dairy products; and use of proper safety techniques infection in India is:
in clinical laboratories working with this organism. a. Brucella melitensis b. Brucella abortus
c. Brucella suis d. Brucella canis
Important questions 5. The specimen of choice for isolation of Brucella is:
a. Blood b. Bone marrow
1. Discuss laboratory diagnosis of brucellosis. c. Urine d. Stool
2. Write short notes on: 6. Rose Bengal Card’ test is employed for which of the
a. Castaneda method of blood culture following infections?
b. Serodiagnosis of brucellosis a. Brucellosis
c. Diagnosis of brucellosis in animals. b. Salmonellosis
c. Tularensis
Multiple choice questions (Mcqs) d. None of the above
7. Milk ring test is used for diagnosis of:
1. All the following statements are true regarding
a. Bovine tuberculosis b. Brucellosis
cultural characteristics of Brucella except:
c. Salmonellosis d. Anthrax
a. They are strict aerobes
b. They require 5–10% of CO2 for better growth 8. Vaccination of cattle against brucellosis is done
c. They produce detectable colonies within 24–48 with:
hours a. Attenuated B. abortus vaccine
d. Erythritol has stimulatory effect on growth of the b. Attenuated B. melitensis vaccine
bacteria. c. Attenuated B. ovis vaccine
2. Humans acquire Brucella melitensis infection from: d. Attenuated B. neotome vaccine
a. Sheep b. Cow Answers (MCQs)
c. Pig d. Wood rat 1. c; 2. a; 3. d; 4. a; 5. a; 6. a; 7. b; 8. a
4848
Chapter
Spirochetes
LEARNING OBJECTIVES
∙∙ Describe diseses caused by different spirochetes ∙∙ Describe the following: fluorescent treponemal
∙∙ Name various pathogenic treponemes antibody-absorption (FTA-ABS) test; TPHA (or) T.
∙∙ Describe morphology of Treponema pallidum pallidum hemagglutination test
∙∙ Discuss BFP (Biological false-positive) reactions
∙∙ Discuss pathogenesis of syphilis
∙∙ Describe the following: i. Endemic syphilis (or)
∙∙ Describe diseses caused by Treponema pallidum
Bejel; ii. Yaws; iii. Treponema pertenue; iv. Pinta; v.
their laboratory diagnosis
Borrelia recurrentis; vi. Borrelia vincentii; vii. Borrelia
∙∙ Explain serological tests for syphilis burgdorferi or Lyme disease; viii. Leptospirosis; ix.
∙∙ Discuss standard tests for syphilis (STS) Weil’s disease.
Pathogenesis
Treponema pallidum subsp. pallidum causes
syphilis. Venereal syphilis is acquired by sexual
contact. Syphilis can also be acquired by nongeni
tal contact with a lesion (e.g. on the lip) or
transplacental transmission to a fetus, resulting in
congenital syphilis. T. pallidum enters tissues by
penetration of intact mucosae or through abraded
skin. Clinical disease sets in after an incubation
period of about a month (range 10–90 days). The
Fig. 48.2: Treponema pallidum-dark ground illumination natural course of syphilis can be divided into
primary, secondary, and tertiary stages based
passage in rabbits for many decades. One such
on the clinical manifestations.
strain (Nichol’s strain) was isolated in 1912 from a
patient of general paralysis of the insane. i. Primary Disease
Cultivable treponemes such as T. phagedenis
The bacteria multiply at the initial entry site and
(Reiter’s treponeme) and T. refringens (Noguchi
the primary lesion in syphilis is the chancre.
strain) are nonpathogenic. They can be grown
The chancre is a painless, relatively avascular,
under strict anaerobic conditions.
circumscribed, indurated, superficially ulcerated
lesion. It is covered by a thick, glairy exudate very
Resistance
rich in spirochetes. It is known as ‘hard chancre’.
T. pallidum is very delicate, being readily inactivated The chancre is most frequently on the external
by drying or by heat (41–42°C in one hour). It is genitalia.
inactivated by contact with oxygen, distilled water, The regional lymph nodes are swollen,
soap, arsenicals,mercurials, bismuth, common disc rete, rubbery and nontender. The chan
antiseptic agents and antibiotics. It is killed in 1–3 cre invariably heals in about 10–40 days, even
days at 0–4°C, so that transfusion syphilis can be without treatment, leaving a thin scar. Chancres
prevented by storing blood for at least four days in usually occur singly but multiple or persistent
the refrigerator before transfusion. Stored frozen chancres may develop in immunocompromised
at –70°C in 10% glycerol, or in liquid nitrogen individuals, such as those infected with the human
(–130°C), it remains viable for 10–15 years. immunodeficiency virus (HIV).
Antigenic Structure ii. Secondary Syphilis
The antigenic structure of T. pallidum is complex. Secondary syphilis sets in 1–3 months after healing
Treponemal infection induces at least three types of primary lesion. The secondary lesions are due
of antibodies. On the basis of these antibodies, to widespread multiplication of the spirochetes
the treponemal antigens may be divided into and their dissemination through the blood. In this
nonspecific and specific antigens. stage, patients typically experience a “flu-like”
syndrome, lymphadenopathy, and a generalized
A. Nonspecific Antigen mucocutaneous rash. Characteristic lesions
The first is the reagin antibody in which a hapten are roseolar or papular skin rashes, mucous
extracted from the beef heart is used as the antigen. patches in the oropharynx and condylomata at
This lipid hapten is known as cardioliom and is the mucocutaneous junctions. Condylomata lata
chemically a diphosphatidyl glycerol. This lipid occur around moist areas, such as the anus and
has been detected in T. pallidum but it is not vagina. The patient is most infectious as with the
known whether the reagin antibody is induced primary chancre. There may also be ophthalmic,
by cardiolipin that is present in the spirochete or osseous and meningeal involvement.
released from damaged host tissues.
iii. Latent Syphilis
B. Specific Antigens After the secondary lesions disappear there is a
1. Group-specific antigen: It is a protein anti period of quiescence known as ‘latent syphilis’. No
gen present in T. pallidum as well as in clinical manifestations are evident and diagnosis
includes Wassermann, Kahn, Venereal Diseases Serum is inactivate heated to it prior to the test,
Research Laboratory (VDRL) and the rapid plasma whereas CSF need not be heated.
reagin (RPR) tests. All these tests are flocculation 2. Inactivated patient serum (0.05 mL) is pipetted
tests except Wassermann reaction which is a into the paraffin ring on the glass slide. Each
complement fixation test (CFT). The Wassermann (0.05 mL) of positive and negative control sera
reaction is no longer in use. Similarly Kahn test is are pipetted into other paraffin rings.
rarely done these days. 3. One drop of working antigen suspension is
The two nontreponemal tests widely used today added to each of these paraffin rings from a
are the Venereal Disease Research Laboratory syringe delivering 60 drops in 1 mL.
(VDRL) and rapid plasma reagin (RPR) tests.
4. Mix with wooden sticks and rotate slide at 180
Veneral Disease Research Laboratory revolutions per minute for four minutes on a
(VDRL) Test mechanical VDRL or manually. Flocculation
occurs in a positive reaction and is observed
It is more rapid test which gives more quantitative
microscopically.
results (VDRL, for Veneral Disease Research
Laboratory, USPHS, New York, where the test was
developed). These tests are cheaper, more rapid Interpretation
and simpler to perform and control. The results of qualitative test are reported as
VDRL is the most widely used simple and rapid ‘reactive’, ‘weak reactive’ or ‘nonreactive’.
test which requires only a small quantity of serum. Reactive’ means positive (large clumps of antigen
The VDRL test uses a cardiolipin antigen that is with marked background clearing are obtained.)
mixed with the patient’s serum or CSF. Flocculation while ‘nonreactive’ is negative.The reciprocal of
occurs in a positive reaction and is observed the end point is given as the titer for reporting
microscopically. of quantitative test, e.g. reactive in 1:4 dilution is
reported as ‘reactive 4 dilution’ or R4.
Method Sometimes VDRL test may give false-negative
1. The test is done in a specially prepared slide, reaction due to high titers of antibody in patient’s
with depressions of 14 mm diameter each. serum (prozone phenomenon). The test is
Epidemiology
The etiologic agent of louse-borne epidemic
relapsing fever is B. recurrentis. The vector is
the human bodylouse, and humans are the only
reservoir. The infection is transmitted not by the
bite of lice but by their being crushed and rubbed
into abraded skin.
Louse-borne relapsing fever tends to occur as
epidemics whenever poverty, overcrowding and
lack of personal hygiene encourage louse infesta
Fig. 48.3: Borrelia recurrentis in peripheral blood smear tion.
survival in water or soil depends on tempera Duration of the illness varies from less than
ture, acidity, salinity and nature and amount of 1 week to 3 weeks. Late manifestations may be
pollution, dying rapidly in acid urine, nonaerated caused by the host immunologic response to the
sewage, saltish or brackish water. They can survive infection (Table 48.4).
for days in moist conditions at pH 6.8–8. Serious cases of leptospirosis are caused most
often by serotype icterohemorrhagiae, though
Antigenic Properties they m ay also be due to coenhageni and less
Leptospires exhibit considerable antigenic cross often batavitae, gripotyphosahosa, pyrogenes
reaction. A genus specific somatic antigen is and some others. Aseptic meningitis is common
present in all members of the genus. Classification in canicola infection and abdominal symptoms in
into serogroups and serotypes (now referred to grippotyphosa infections.
as sero-varieties or serovars) is based on surface
antigens. Laboratory Diagnosis
Diagnosis may be made by 1. Demonstration of
Pathogenesis the leptospires microscopically in blood or urine;
In natural reservoir hosts, leptospiral infection is 2. Isolation in culture; 3. Animal inoculation; 4.
asymptomatic. It is transmitted to humans when Serological tests.
the leptospires in water contaminated by the urine
of carrier animals enters the body through cuts or 1. Demonstration of Leptospiras in Blood
abrasions on the skin or through intact mucosa of
or Urine
mouth, nose or conjuctiva. During the acute phase
of the disease, leptospires are seen in the blood but Microscopy
can seldom be demonstrated after 8–10 days. They As leptospires disappear from the blood after the
persist in the internal organs, and most abundantly first week, blood examination is helpful only in
in the kidneys, so that they may be demonstrated in the early stages of the disease. Leptospires may be
the urine in the later stages of the disease. demonstrated by examination of the blood under the
Diseases: Mild virus-like syndrome. The incubation dark field microscope or by immunofluorescence
period is usually about 10 days (range 2–26 days). but this is of little practical value.
The onset of clinical illness is usually abrupt, Leptospires may be found in the urine during
with nonspecific, influenza-like constitutional the second week and intermittently for 4–6 weeks
symptoms such as fever, chills, headache, severe or even longer. Centrifuged deposit of the urine
myalgia, and malaise. may be examined under dark ground illumination.
Systemic leptospirosis with aseptic meningitis.
The patient may develop a more advanced disease- 2. Culture
including aseptic meningitis. Three or four drops of blood are inoculated into
Severe systemic disease (Well’s disease) each of several bijou bottles containing EMJH
includes renal failure, hepatic failure, and intra or similar medium. The bottles are incubated
vascular disease, and may result in death. at 37°C for two days and left thereafter at room
LEARNING OBJECTIVES
After reading and studying this chapter, you should pneumoniae, Mycoplasma hominis, and Ureaplasma
be able to: urealyticum
∙∙ Describe the general characteristics of the ∙∙ Analyze the diagnostic methods appropriate for
Mycoplasma spp. and how they differ from other detection of mycoplasmal and ureaplasmal infections
bacterial species ∙∙ Discuss the use of serologic tests for diagnosing M.
∙∙ Describe morphology and characteristies of pneumoniae infections
Mycoplasma spp ∙∙ Compare mycoplasmas and L forms
∙∙ Compare the clinical diseases caused by Mycoplasma ∙∙ Describe Ureaplasma urealyticum.
Cultural Characteristics
Most mycoplasmas are facultatively anaerobic but,
since organisms from primary tissue specimens
frequently grow only under anaerobic conditions,
an atmosphere of 95% nitrogen and 5% carbon
dioxide is preferred for primary isolation. They
grow within a temperature range of 22–41°C, the
parasitic species growing optimally at 35–37 °C and
the saprophytes at lower temperatures.
Media for cultivating Mycoplasma are enriched
with 20% horse or human serum and yeast extract. Fig. 49.1: Typical large Mycoplasma colony showing ‘fried
The high concentration of serum is necessary egg” appearance
1. Specimens
M. pneumoniae may be recovered from throat
swabs, nasopharyngeal swabs, sputum, throat
washings, bronchoalveolar lavage, tracheal
aspirate and lung tissue specimens.
2. Culture
In the laboratory, if inoculation is not possible
immediately, then the specimen may be held up
to 24 hours at 4°C. If delay more than 24 hours is
expected, then the specimen should be frozen at
Fig. 49.2: Typical large Mycoplasma pneumoniae colony
– 70°C. A widely used isolation medium contains showing “mullberry shaped” appearance. (Courtesy
bovine heart infusion (PPLO broth) with fresh Dr Surinder Kumar, Director Professor, Department of
yeast extract and horse serum supplemented with Microbiology, Maulana Azad Medical College, New Delhi, India)
penicillin (to inhibit other bacteria), thallium
ii. Species identification: These colonies may
acetate, glucose and with phenol red as a pH
be identified by:
indicator because mycoplasmas do not produce
turbidity in broth media. For genital Mycoplasma, a. Hemadsorption test: The characteristic
polymymin B and amphotericin B and lincomycin of guinea pig red blood cells is adhering to
are also added to mycoplamal broth. colonies of M. pneumoniae.
b. Tetrazolium reduction test: Colonies of
1. Isolation and Identification M. pneumoniae appear red when these
In general, the growth of M. pneumoniae from are flooded with solution oftetrazolium
clinical specimens is detected by the ability of compound which is colourless. M.
these organisms to produce acid from glucose. pneumoniae reduces tetrazolium (colour
Most broth media are diphasic such as methylene less) to red coloured compound.
blue-glucose diphasic medium. Broth cultures are c. Serological techniques: Identification of
incubated at 35°C with the caps tightened. Tubes isolates can be confirmed by inhibition of
are inspected daily for color changes (from salmon their growth with specific antisera.
to yellow) in the medium. A slight, gradual shift in
the pH indicator over an 8–15 day period without 2. Polymerase Chain Reaction (PCR)
gross turbidity suggests a true-positive culture. Amplification
The broth must be subcultured to appropriate agar
M. pneumoniae in respiratory exudates or secretions
medium as soon as color changes in the medium
is detected by polymerase chain reaction (PCR)
are apparent. Inspection of the agar surface under
amplification of a chosen sequence in its genome.
the low power of the microscope will reveal small
colonies of the organisms. In the absence of
obvious color change in diphasic media, a blind 3. Antigen Detection Techniques
subculture to agar media should be performed after Detection of antigen in respiratory exudates by
1 and 3 weeks of incubation. direct immunofluorescence and counterimmuno
i. Colonies: M. pneumoniae may take 21 days or electrophoresis techniques, immunoblotting
more, while M. hominis colonies may appear with monoclonal antibodies and antigen capture
within 2–4 days. Colonies of M. pneumoniae are enzyme immunoassay (EIA).
small, betahemolytic and have a homogeneous
granular appearance (“mulberry shaped”), 4. DNA Probes
unlike the fried-egg morphology of other
DNA probes can be used to detect M. pneumoniae
mycoplasmas. Because the organisms do not
RNA in sputum specimens.
stain well with gram or acridine orange stains,
Mycoplasma like colonies are stained with the
Dienes or methylene blue stains. M. hominis 5. Serological Tests
colonies have a typical large “fried egg” Serologic tests are available only for M. pneumoniae
appearance (Fig. 49.2 ). Serological diagnosis may be made by:
Miscellaneous Bacteria
LEARNING OBJECTIVES
After reading and studying this chapter, you should ∙∙ Discuss rat-bite fever
be able to: ∙∙ Describe Klebsiella granulomatis and disease caused
∙∙ Describe morphology and culture characteristics by it
of Listeria ∙∙ Acinetobacter spp.
∙∙ Describe infections caused by Listeria monocytogenes
and their laboratory diagnosis
A. lowffi Pathogenesis
This forms yellow colonies on MacConkey medium In humans, it causes streptobacillary rat-bite fever.
and does not acidify sugars. Some strains are Another type of clinically indistinguishable, rat-bite
oxidase positive. fever is caused by Spirillum minus.
LEARNING OBJECTIVES
After reading and studying this chapter, you should ∙∙ Discuss the laboratory diagnosis of rickettsial
be able to: infections
∙∙ Describe diseases caused by different rickettsiae ∙∙ Describe Weil–Felix reaction
∙∙ Describe Brill–Zinsser disease ∙∙ Discuss Q fever, Trench fever and Oroya fever.
ehrlichiosis; It is transmitted by ticks. Leukopenia year or more at 4°C and in meat at least for one
and thrombocytopenia are seen in patients. month. It is not completely inactivated at 60°C or
by 1% phenol in one hour. In milk it may survive
3. Anaplasma phagocytophilum pasteurization by the holding method, but the
It infects human granulocytic cells. Anaplasma flash method is effective. It can survive in dust
phagocytophilum, causes human granulocyte and aerosols, therefore, can be transmitted as an
anaplasmosis (HGE). The human pathogens in airborne infection. It can be inactivated by 2%
the group have animal reservoirs and can cause formaldehyde, 5% hydrogen peroxide and 1% lysol.
disease in animals as well
Antigenic Variation
4. Neorickettsia sennestu An important characteristic of Coxiella infections
It was previously called Ehrlichia sennestu, an is the ability to undergo antigenic variation in
intraleukocytic bacterium. It causes sennestu expression of the cell wall LPS antigen. Cox. bumetii
ehrlichiosis, an illness resebling glandular fever shows phase variation. Fresh isolates are in Phase I.
(sennetsu being the Japanese name for glandular It becomes Phase II on repeated passage in yolk sac,
fever). No insect vector has yet been implicated in its but reverts to Phase I by passaging in guinea pigs.
transmission. It is associated with ingestion of raw fish
infested with infected flukes. Similar cases have been Pathogenesis
reported in Malaysia and Philippines. Coxiella burnetii causes Q fever which has a world
wide distribution, as a zoonosis solidly established
Laboratory Diagnosis in domestic livestock.
1. Identification of characteristic morulae: Typical Cycles of infection: In nature there are two cycles
morulae in white blood cells is diagnostic in of infection of C. burnetii. One involves arthropods
Giemsa stained peripheral blood smear. (especially ticks) and a variety of vertebrates. The
2. Specific antibodies can be demonstrated by other cycle is maintained among domestic animals
indirect immunofluorescence methods. (cattle, sheep and goats).
3. Polymerase chain reaction (PCR) for ehrlichial
DNA. Reservoirs of the disease: The primary reservoirs
of the disease are wild and domestic ungulates,
including cattle, sheep, goats, rabbits, cats and dogs.
GENUS COXIELLA: Q FEVER It is transmitted among them and to cattle, sheep
The name Q fever (Q for “query”) was first used when and poultry by ixodid ticks.
Derrick (1935) was investigating an outbreak of Animal infection: Domestic animals have
typhoid-like fever in abattoir workers in Australia. Q inapparent infections but may shed large quantities
fever is caused by Coxiella burnetit. Coxiella burnetii, of infectious organisms in their urine, milk, feces,
is an obligately intracellular prokaryote. and especially, their placental products.
LEARNING OBJECTIVES
After reading and studying this chapter, you should ∙∙ Discuss morphology and the unique growth cycle
be able to: of Chlamydia, describing elementary body (EB) and
∙∙ Describe differences between chlamydiae and viruses reticulate body (RB) bodies
∙∙ Describe serotypes of various chlamydiae and ∙∙ Discuss laboratory diagnosis of chlamydial infections
diseases produced by them ∙∙ Describe the following: TRIC agents, inclusion
∙∙ Associate each of the major diseases with the three conjunctivitis lymphogranuloma venereum (LGV),
most important human species of Chlamydia and Frei’s test.
Chlamydophila
INTRODUCTION CLASSIFICATION
Chlamydiaceae is a family of obligate intracellular Genus Chlamydia is in the order Chlamydiales
bacterial parasites, small, nonmotile and gram- and the family Chlamydiaceae. The proposed new
negative with a tropism for columnar epithelial taxonomic classification for the family Chlamy
cells lining the mucous membranes. diaceae consists of two genera: (1) Chlamydia to
Based on the human diseases they were then include C. trachomatis and (2) Chlamydophila to
known to cause, they were called psittacosis- include C. pneumoniae, C. psittaci, and C. pecorum.
lymphogranuloma-trachoma (PLT) viruses, or Other species have been placed into the two genera,
noncommittally as ‘PLT agents’ or TRIC (trachoma- but they are uncommon human pathogens and are
inclusion conjunctivitis) organisms. not discussed in this chapter (Table 52.1).
Chlamydia Species
Differences between Chlamydiae and Chlamydia trachomatis: C. trachomatis is divi
Viruses ded into two biovars—those causing trachoma,
The Chlamydiaceae were once considered viruses. inclusion conjunctivitis (the so-called TRIC agents)
However, they differ from viruses in many respects and oculogenital infection.
and the organisms have the following properties
Chlamydophila Species
of bacteria:
1. Chlamydophila psittaci: It may lead to severe
1. They possess inner and outer membranes and sometimes fatal pneumonia in man (psittacosis
similar to those of gram-negative bacteria. or ornithosis).
2. They contain both DNA and RNA.
Table 52.1 Revised classification of the family
3. They possess prokaryotic ribosomes.
Chlamydiaceae
4. They synthesize their own proteins, nucleic
acids, and lipids. Genus Chlamydia Genus Chlamydophila
5. They multiply by binary fission. C. trachomatis C. pneumoniae
C. muridarum C. psittaci
6. They do not have ‘eclipse phase’ following C. suis C. pecorum
cellular infection. C. abortus
7. They are susceptible to numerous antibacterial C. caviae
antibiotics. C. felis
5353
Chapter
Learning Objectives
After reading and studying this chapter, you should ∙∙ List various cell cultures
be able to: ∙∙ Discuss detection of virus growth in cell cultures
∙∙ Describe size, shape and symmetry of viruses ∙∙ List DNA and RNA viruses
∙∙ Describe cultivation of viruses ∙∙ Describe prions and viroids.
3. Complex Symmetry
Viruses (e.g. poxviruses) which do not show either
icosahedral or helical symmetry due to complexity
of their structure are referred to have complex
symmetry.
A B
C. Viral Envelope
Virions may be enveloped or nonenveloped
(naked). Enveloped Virus: The envelope or
outer covering of virus containing lipid is derived
from the plasma membrane of the host cell during
their release by budding from the cell surface. The
envelope is glycoprotein in nature. The lipid is largely
C D of host cell origin while the protein is virus-encoded.
Fig. 53.1: Schematic diagram illustrating the components of Peplomers: In mature virus particle, the glyco
the complete virus particle (the virion). (A) Naked icosahedral proteins often appear as projecting spikes on the
virus, consisting of an inner core of nucleic acid enclosed by a outer surface of the envelope. These are known as
capsid, which is made of capsomers; (B) Enveloped virus; (C)
Naked helical virus composed of capsomers wound round the
peplomers. A virus may have more than one type
nucleic acid to form a tubular structure; (D) Envelope helical of peplomers, e.g. the influenza virus carries two
virus in which the tubular capsid is pliable and is enclosed kinds of peplomers, the hemagglutinin which is
within an envelope a triangular spike and the neuraminidase which
Fig. 53.2: Shapes and relative sizes of animal viruses of families that infect vertebrates
is a mushroom-shaped structure. Envelope confer information necessary for replication of the virus.
chemical, antigenic and biological properties on The genome may be single-stranded or double-
viruses. stranded, circular or linear, and segmented or
nonsegmented. The type of nucleic acid, its
Functions of Peplomers strandedness, and its size are major characteristics
i. Mediate attachment of the virus to the host- used for classifying viruses into families.
cell receptors.
ii. Attach to receptors on red blood cells.
Susceptibility to physical and
iii. Enzymatic activity chemical agents
iv. Major antigens: For protective immunity. 1. Heat and Cold
i. Heat-labile: With few exceptions, viruses are
D. Viral Nucleic Acids very heat-labile. They are inactivated within
Viruses contain a single kind of nucleic acid— seconds at 56°C, minutes at 37 °C and days at
either DNA or RNA—that encodes the genetic 4°C.
Viral hemagglutination
A large number of viruses contain hemagglutinin
spikes (peplomers) on the capsid or envelope
which can agglutinate red cells of different Fig. 53.3: Viral hemagglutination. Virus containing fluid is
diluted in doubling dilutions and 0.5% suspension of chick
species. Hemagglutinin of influenza virus is due
red cells added. There is diffuse widespread even pattern
to the presence of hemagglutinin spikes on the on the bottom of the wells in the plastic plate where virus is
surface of the virus. When RBCs are added to present. The cells settle down to a button like aggregate with
serial dilutions of viral suspension, the RBCs and sharp edges where no virus is present
A B
C D
Figs 53.5A to D: A. Normal vero cell monolayer. B. Vero cell monolayer infected with Coxsackie B virus C. HeLa cell
monolayer. D. HeLa cell monolayer infected with Coxsackie virus B3, stained after 48 hours
B. Recombination
When two different, but related, viruses infect a
Fig. 53.6: Plaque formation in monkey kidney cells by cell simultaneously, genetic recombination may
poliovirus take place. The two viruses exchange segments
of nucleic acid between them so that a hybrid
ii. Pock Assay is formed possessing genes from both parents.
Certain viruses, e.g. herpes and vaccinia, form Recombinants may occur between (1) two active
pocks when inoculated onto the chorioallantoic (infectious) viruses, (2) one active and one inactive
membrane of an embryonated egg. Such viruses virus, and (3) two inactive viruses.
can be assayed by counting the number of pocks i. Recombinants between two active (infect
formed on CAM by appropriate inocula of virus. ious) viruses: When two inactive viruses
This is known as pock assay. markers (such as pock morphology or antigenic
properties) are grown together, recombinants
Viral genetics may be derived that possess the distinctive
Viruses obey the laws of genetics like all other ‘living properties of both parents. Thus, if a human
beings’. Several properties of viruses, such as virulence and an avian strain of influenza virus (whose
and antigenicity are under genetic control. Main hemagglutinin and neuraminidase antigens
mechanisms for genetic modification in viruses are: are different and easily identifiable) are grown
A. Mutation. together, a hybrid may be obtained with
B. Recombination. the hemagglutinin of one parent and the
C. Nonheritable variations: In addition, viruses neuraminidase of the other. This may be one
may exhibit many nonheritable variations due of the ways by which the pandemic strains of
to gene product interactions. the influenza virus originate in nature.
ii. Recombinants between one active and one
A. Mutation inactive virus: Cross-reactivation or marker
rescue
It is a random, undirected and heritable variation. When a cell is ‘infected’ with an active virus
The frequency of mutation in viruses is about 10-4 and a different but related inactivated virus,
to 10-8, approximately the same as in bacteria. progeny possessing one or more genetic traits
Mutations, therefore, occur during every viral of the inactivated virus may be produced. This
infection. Many mutations are lethal, because phenomenon is known as cross-reactivation
the mutated virus is unable to replicate. A mutant or marker rescue.
becomes evident only if the mutation confers some iii. Recombinants between two inactive viru
readily observable property or affords the mutant ses: Multiplicity reactivation: When a cell is
virus some selection or survival advantage. Mutation ‘infected’ with a large dose (high multiplicity)
may occur spontaneously or may be induced by of a single virus inactivated by UV irradiation,
mutagens, physical agents, such as UV light or live virus may be produced. The different
irradiation or chemical agents such as 5-fluorouracil. virions that cause multiple infection of a
cell may have suffered damage to different
Conditional lethal Mutant genes. Thus from the total genetic pool it may
Mutations in essential genes are termed lethal be possible to obtain a full complement of
mutations. A class of mutants that are of great undamaged genes. This phenomenon is called
Learning Objectives
Learning Objectives
After reading and studying this chapter, you should ∙∙ Describe immunoprophylaxis of viral diseases
be able to: ∙∙ Describe the following: Live viral vaccines; killed
∙∙ Describe laboratory diagnosis of viral infections viral vaccines.
Bacteriophages
Learning Objectives
After reading and studying this chapter, you should ∙∙ Discuss lytic and lysogenic cycle of bacteriophage
be able to: ∙∙ Describe phage typing.
∙∙ Describe morphology of bacteriophage
Morphology
Certain bacteriophages that infect E. coli, called the
T even phages (T2, T4, T6), have been studied in
great detail and traditionally serve as the prototypes
in describing the properties of bacteriophages.
Most phages are tadpole-shaped, possess a
hexagonal head and a cylindrical tail.
1. Head: The head consists of a tightly packed
core of nucleic acid (double stranded DNA) Fig. 56.1: Morphology of bacteriophage. A. hexagonal head,
surrounded by a protein coat or capsid. The B. DNA core, C. demarcation between head and tail, D. tail,
head of phage T4 has a diameter of 65 nm and E. base plate, F. tail fibers, G. prongs; right, process of injection
is 100 nm long. of phage DNA into host cell
Chapter 56: Bacteriophages | 403
phage DNA becomes integrated with the bacterial protein capsid outside. The process of penetration
genome, replicating synchronously with it, causing resembles injection through a syringe. Following
no harm to the host cell (Fig. 56.2). adsorption, six tail pins make contact with the host
cell surface and firmly attach the phage plate to it.
1. Lytic Cycle The contractile tail sheath then contracts forcing
Replication of a virulent phage can be considered the hollow interior tail tube into the bacterial cell
in the following stages adsorption, penetration wall. The phage DNA then passes through the
and synthesis of phage components, assembly, hollow interior tail tube. The empty head and tail
maturation and release of progeny phage particles. of the phage remain outside the bacterium as the
shell or ‘ghost’ after penetration.
When bacteria are mixed with phage particles at
i. Adsorption
high multiplicity (that is very large number of phages
By random collision, phage particles attach to per bacterial cell), multiple holes are produced on
virus-specific receptors on the host cell by its tail. the cell with the consequent leakage of cell contents.
Adsorption is a specific process and depends on Bacterial lysis occurs without viral multiplication.
the presence of complementary chemical groups This is known as ‘lysis from without’.
on the receptor sites on the bacterial surface and on
the terminal base plate of the phage. The infection iii. Synthesis of Phage Nucleic Acid and Proteins
of a bacterium by the naked phage nucleic acid is The synthesis of the phage components is initiated
known as transfection. immediately after penetration of the phage nucleic
acid. The first products to be synthesized (called
ii. Penetration early proteins) are the enzymes necessary for the
After adsorption, most phages inject their nucleic building of the complex molecules peculiar to the
acid into the bacterial cytoplasm and leave their phage. Subsequently, late proteins appear which
Fig. 56.2: Lytic and lysogenic life of bacteriophage. 1. Adsorption, 2. Injection of phage DNA, 3. Circularization of phage
DNA, 4. Replication of phage DNA, 5. Production of phage components, 6. Assenbly of phage, 7. Release of progeny phage,
8. Integration of phage DNA with host chromosome, 9. Binary fission of lysogenic bacterium, 10. Daughter bacteria carrying
prophage, 11. Excision of prophage, 12. Same stage as 3 (1 to 3 injection; 4 to 7 lytic; 8 to 10 lysogenic cycle; 11 and 12 induction)
404 | Section 4: Virology
include the protein subunits of the phage head and bacillus and nontoxigenic strains can be made
tail. During this period, the synthesis of bacterial toxigenic by lysogenization.
protein, DNA and RNA ceases and the cell is forced ii. Clostridium botulinum types C and D produce
to make viral constituents. toxin only if these are infected with phage CE
b and DE b, respectively.
iv. Assembly and Maturation iii. A wide variety of temperate phages of Salmo
Phage DNA, head protein and tail protein are nella can modify the antigenic properties of
synthesized separately in the bacterial cell. The somatic O antigen. When Salmonella is infected
DNA is condensed into a compact polyhedron by an epsilon phage, the structure of its outer
and ‘packaged’ into the head and, finally, the tail lipopolysaccharide layer may be modified.
structures are added. This assembly of the phage The prophage may become ‘excised’ from
components into the mat ure infective phage occasional cells during the multiplication of
particle is known as maturation. lysogenic bacteria. The excised prophage initiates
lytic replication and the daughter phage particles
v. Release are released, which infect other bacterial cells
Release of the mature progeny phages typically and render them lysogenic. This is known as
occurs by lysis of the bacterial cell. Phage enzymes act spontaneous induction of prophage. While this is
on the weakened cell wall causing it to burst or lyse a rare event, all lysogenic bacteria in a population
resulting in the release of mature daughter phages. can be induced to shift to the lytic cycle by
exposure to certain physical and chemical agents.
Such inducing agents include UV rays, hydrogen
Eclipse Phase
pero xide and nitrogen mustard. A lysogenic
The interval between the entry of the phage nucleic bacterium is resistant to reinfection by the same
acid into the bacterial cell and the appearance of or related phages. This is known as superinfection
the first infectious intracellular phage particle is immunity.
known as the eclipse phase. The interval between
the infection of a bacterial cell and the first release Significance of phages
of infectious phage particles is known as the latent
period (20–40 minutes). Immediately following the 1. Virulent Phage
latent period, the number of phage particles released i. Phage Assay
increases for a few minutes till the maximum number
of daughter phages is attained. This period, during When a phage is applied on the lawn culture of
which the number of infectious phages released a susceptible bacterium, areas of clearing occur
rises, is known as the rise period. The average yield after incubation. These zones of lysis are called
of progeny phages per infected bacterial cell is plaques. The size, shape and nature of plaques are
known as the burst size (100–300 phages). characteristic for different phages. Plaque assay
can be employed for titrating the number of viable
phages in a preparation.
2. Lysogenic Cycle
Unlike virulent phages which produce lysis of the
host cell, temperate phages enter into a symbiotic ii. Phage Typing
relationship with their host cells without destroying Bacteriophage (phage) typing is based on the
them. Following entry into the host cell, the specificity of phage surface receptors for cell surface
temperate phage nucleic acid becomes integrated receptors. The limited host range of many phages
with the bacterial chromosome. The integrated enables them to be used as an epidemiological
phage nucleic acid is known as the prophage. marker to discriminate between bacterial strains
The prophage behaves like a segment of the that are biochemically or serologically indisting
host chromosome and replicates synchronously uishable.
with it. This phenomenon is called lysogeny The strain to be typed is inoculated on a plate
and a bacterium that carries a prophage within of nutrient agar to produce a lawn culture. After
its genome is called a lysogenic bacterium or drying, the phages are applied over marked squares
lysogen. in a fixed dose (routine test dose). The highest
The prophage confers certain new properties dilution of the phage preparation that just produces
on the lysogenic bacterium. This is known as lyso confluent lysis known as the routine test dose
genic or phage conversion. (RTD). After overnight incubation, the culture will
i. Toxin production in Corynebacterium be observed to be lysed by some phages but not by
diphtheriae is determined by the presence in others. The phage type of the strain is expressed by
it of the prophage b. The elimination of this the designation of the phage/phages that lyse it.
prophage abolishes the toxigenicity of the Area of lysis caused by phage is known as plaque.
Chapter 56: Bacteriophages | 405
The most important application of phage typing
(virulent) cycle, intracellular multiplication of the
is for intraspecies typing of bacteria, as in the phage phage ends in lysis of the host bacterium and release
typing of Salmonella Typhi and staphylococi. of progeny virions
Phages are available that lyse all members of In the lysogenic (temperate) cycle, phage DNA
a bacterial genus (for example genus-specific becomes incorporated within the bacterial genome
bacteriophage for Salmonella), all members of and then replicates synchronous with it, causing no
harm to the host cell
a species (for example specific bacteriophage
Phage typing has been used for Staphylococcus
for B. anthracis), and all members of a biotype aureus, Vibrio cholerae, Salmonella, and many other
or subspecies (for example Mukerjee’s phage IV bacteria.
which lyses -all strains of classical V. cholerae but
not V. cholerae biotype EI Tor).
Important questions
1. Draw a labeled diagram of a T-even phage.
2. Temperate Phage 2. Discuss the life cycle of bacteriophages.
i. Transduction: Bacteriophages may act 3. Write short notes on:
as carriers of genes from one bacterium a. Lysogenic cycle of phages
to another. This is known as transduction. b. Lysogenic conversion
Two types of transduction are recognized: c. Phage typing
d. Significance of phages.
generalized transduction, in which any
portion of the donor DNA can be transferred,
and specialized transduction, in which only Multiple choice questions (Mcqs)
a specific set of genes can be carried to a 1. T-even bacteriophages possess
recipient cell. a. Single-stranded RNA
ii. Toxin production: Toxin production in b. Double-stranded RNA
Corynebacterium diphtheriae and Clostridium c. Single-stranded DNA
botulinum are determined by genes carried in d. Double-stranded DNA
prohage DNA. 2. All the following statements for stage of adsorption
iii. Antigenic property: A wide variety of in lytic cycle of bacteriophage are true accept
a. It is a specific process
temperate phages of Salmonella can modify
b. It depends upon the susceptibility of the bacte-
the antigenic properties of somatic O antigen. rium to the specific phages
Such acquisition of new properties by c. It occurs by random collision
bacterial cells following phage infection is d. It is a very slow process
called “phage conversion”. 3. All the following are the examples of phage
iv. Cloning vector: Bacteriophages have been used conversions except
as cloning vectors in genetic manipulations. a. Phage-mediated conversion of somatic anti-
gens of Salmonella spp.
Key Points b. Toxigenicity of Corynebacterium diphtheriae
Bacteriophages are bacterial viruses. They are c. Toxicity of Clostridium botulinum
associated with transmission of genetic material d. Toxigenicity of Vibrio cholerae
from one bacterium to another 4. Which of the following bacteria can be typed by
Morphology: Most phages are tadpole-shaped, phage typing method?
possess a hexagonal head and a cylindrical tail. Most a. Staph. aureus
of the phages usually consist of single, linear, and b. Salmonella typhi
doublestranded DNA molecule genome. The phages c. Vibrio cholerae
show high host specificity d. All of the above
Life cycle: Phages exhibit two different types of
life cycle: lytic cycle and lysogenic cycle. In the lytic Answers (MCQs)
1. d; 2. d; 3. d; 4. d.
57
Chapter
Poxviruses
Learning Objectives
After reading and studying this chapter, you should ∙∙ Describe molluscum contagiosum.
be able to:
∙∙ Describe structure of vaccinia virus
A B
Fig. 57.1: Structure of vaccinia virus. The nucleic acid is Fig. 57.2: Variola and vaccinia pocks on cam. A. Variola,
contained within a dumb-bell shaped core. Fitting into the showing small, uniform pocks; B. Vaccinia, showing large,
concavities of the core are two lateral bodies. The virion is irregular pocks
enclosed within a protein shell which has an irregular surface
Herpesviruses
Learning Objectives
Classification
The family Herpesviridae is divided into three
subfamilies based on biological, physical and
genetic properties (Table 58.1).
Eight different types of herpesviruses are known
whose primary hosts are human (Table 58.1).
serologically. They can be differentiated by the Once HSV has established itself in a ganglion in
following features: vivo, reactivations are liable to take place at inter
1. Antigenic differences can be made out using vals of weeks or months thereafter triggered by a
type specific monoclonal antibodies. variety of stimuli. The virus follows axons back to
2. On chick embryo CAM type 2 strains form the peripheral site, and replication proceeds at the
larger pocks resembling variola. skin or mucous membranes.
3. Types 2 strains replicate well in chick embryo HSV-1 is usually associated with infections
fibroblast cells, while type 1 strains do so above the waist, and HSV-2 with infections below
poorly. the waist, consistent with the means of spread for
4. The infectivity of type 2 is more temperature these viruses.
sensitive than that of type 1.
5. Type 2 strains are more neurovirulent in labora Clinical Features
tory animals than type 1. 1. Cutaneous infections: (i) The most common
6. Type 2 strains are more resistant to antiviral site is the face—on the cheeks, chin, around
agents like IUDR and cytarabine in culture. the mouth or on the forepead; (ii) Napkin
7. Restriction endonuclease analysis of viral DNA rash; (iii) ‘Fever blister’ or herpis febrilis. In
enables differentiation between the two types some sensitive persons, very minor stimuli, like
as well as between strains within the same type. common cold, exposure to sun or even mental
strain or menses may bring on such reactivation;
Pathogenesis (iii) Herpetic whitlow- an infection of the
finger; (iv) Herpes gladiatorum an infection
The mechanisms involved in the pathogenesis of of the body; (v) Eczema herpeticum.
HSV-1 and HSV-2 are very similar. Both viruses 2. Oral infection: Acute gingivostomatitis,
initially infect and replicate in mucoepithelial cells herpetic stomatitis, pharyngitis, tonsillitis and
and then establish latent infection of the innervating localized lymphadenopathy may occur.
neurons. Skin and mucous membranes are the 3. Ophthalmic: Severe keratoconjunctivitis,
portals of entry in which the virus also multiplies, follicular conjunctivitis with vesicle formation
causing lysis of cells and formation of vesicles. on the lids, dendritic keratitis or corneal
Soon after replication is under way in the skin ulcers or as vesicles on the eyelids, corneal
or a mucous membrane, virions travel to the root scarring and impairment of vision.
ganglia via the sensory nerves supplying the area. 4. Nervous system: (i) HSV encephalitis; (ii)
Primary infections of the orofacial and genital Sporadic encephalitis; (iii) HSV meningitis;
areas involve respectively the trigeminal and (iv) Sacral autonomic dysfunction; (v) rarely
lumbosacral dorsal root ganglia. The virus then transverse myelitis or the Guillain–Barre
becomes latent in the ganglia. syndrome and Bell’s palsy.
Chapter 58: Herpesviruses | 411
5. Visceral: (i) HSV esophagitis; (ii) tracheo be used. Typical cytopathic changes may appear
bronchitis and pneumonitis; (iii) hepatitis; (iv) as early as in 24–48 hours but cultures should be
Erythema multiforme; (v) disseminated HSV observed for two weeks before being declared
infection. negative.
6. Genital infections: Genital disease is usually HSV isolates can be typed by biochemical,
caused by HSV-2. Genital herpetic ulcers are biologic, nucleic acid, or immunologic methods.
known to increase the risk of transmission of The restriction endonuclease cleavage patterns
infection with human immunodeiciecy virus of the DNA of HSV-1 and HSV-2 are unique allow
(HIV). unequivocal typing of the isolates. HSV type-
(i) In male patients: The lesions typically specific DNA probes, specific DNA primers for PCR
develop on the glans or shaft of the penis and and antibodies are also available for detecting and
occasionally in the urethra; (ii) In female differentiating HSV-1 and HSV-2.
patients: The lesions may be seen on the vulva,
vagina, cervix, perianal area, or inner thigh; C. Serology
(iii) Inguinal lymphadenopathy: In patients
of both sexes; (iv) Herpetic proctitis: in Rise in titer of antibodies may be demonstrated by
homosexuals; (v) Association between HSV-2 ELISA, neutralization or complement fixation tests.
and carcinoma of the cervix uteri: There have
been several reports of an association between D. Polymerase Chain Reaction
HSV -2 and carcinoma of the cervix uteri but a
causal relationship has not been established. Polymerase chain reaction is useful for detecting
viral DNA in cerebrospinal fluid when herpetic
7. Neonatal Herpes: Transplacental infection with
HSV1 or 2 can lead to congenital malformations. infection of the CNS is suspected.
8. Infections in immunocompromised hosts.
Treatment
Laboratory Diagnosis Idoxuridine used topically in eye and skin infections
was one of the first clinically successful antiviral
1. Specimens
agents. Acyclovir and vidarabine enabled the effective
2. Microscopy management of deep and systemic infections.
i. Tzanck smear: Characteristic cytopathologic Intravenous, oral, and topical preparations are
effects (CPEs) can be identified in a Tzanck available. Drug-resistant virus strains may emerge.
smear, Papanicolaou smear, or biopsy Early treatment with intravenous acyclovir has
specimen. CPEs include syncytia, “ballooning” improved the outcome of encephalitis.Valaciclovir
cytoplasm, and Cowdry type A intranuclear and famciclovir are more effective oral agents.
inclusions. When resistance to these drugs develop, drugs like
The Tzanck smear is a rapid, fairly sensitive foscarnet which are independent of viral thymidine
and inexpensive diagnostic method. Smears kinase action may be useful.
are prepared from the lesions, preferably
from the base of vesicles and stained with Herpes virus simiae: B virus
1% aqueous solution of toluidine blue ‘0’ for
15 seconds. Multinucleated giant cells with Herpes B virus of Old World monkeys is highly
faceted nuclei and homogeneously stained pathogenic for humans. This virus was isolated
‘ground glass’ chromatin (Tzanck cells) by Sabin and Wright (1934) from the brain of a
constitute a positive smears. laboratory worker who developed fatal ascending
myelitis after being bitten by an apparently healthy
ii. Electron microscopy: The virus particle may
monkey. It came to be known as the ‘B’ virus from
also be demonstrated under the electron
the initials of this patient.
microscope.
The virus is transmitted to humans by monkey
iii. Fluoroscent ant ibody technique: The
bites or saliva or even by tissues and cells widely
fluorescent antibody test on brain biopsy
used in virology laboratories.
specimens provides reliable and speedy
diagnosis in encephalitis. Virus isolation or serologic tests can be used to
establish the diagnosis of B-virus infections.
B. Virus Isolation
Varicella-zoster virus
Primary human embryonic kidney, human amnion
and many other cells are susceptible, but human Varicella-zoster virus (VZV) causes chickenpox
diploid fibroblasts are preferred. Specimen such (varicella) and, with recurrence, causes herpes
as vesicle fluid, spinal fluid, saliva and swab may zoster, or shingles. Varicella (chickenpox) and
412 | Section 4: Virology
herpes zoster are different manifestations of the residual immunity. It is believed that years after the
same virus infection. Thus, chickenpox is ‘caught’ initial infection, when the immunity has waned,
but not zoster. the virus may be reactivated, and triggered by
some precipitating stimulus, travel along the sen
Properties of the Virus sory nerve to produce zoster lesions on the area of
Varicella-zoster virus is morphologically identical the skin or mucosa supplied by it. It usually starts
to herpes simplex virus. It can be grown in cultures with severe pain in the area of skin or mucosa
of human fibroblasts, human amnion or HeLa cells. supplied by one or more groups of sensory nerves
Cytopathic changes are more focal and spread and ganglia. The most common sites are the areas
much more slowly than those induced by HSV. Only innervated by spinal cord segments D3 to L2 and
one antigenic type of VZV is known. the trigeminal nerve, particularly, its ophthalmic
branch. Within a few days after onset, a crop of
vesicles appears over the skin supplied by the
Varicella (Chickenpox) affected nerves.
Varicella (chickenpox) is one of the mildest highly
communicable and most common of childhood Complications
infections. 1. Postherpetic pain: In the affected area is
It is usually a mild disease of childhood and is frequent, particularly in the elderly.
normally symptomatic, although asymptomatic 2. Ophthalmic zoster.
infection may occur. The portal of entry of the 3. Generalized zoster
virus is the respiratory tract or conjunctiva. After an 4. Ramsay Hunt syndrome: It is a rare form of
incubation period of about two weeks (7–23 days) zoster affecting the facial nerve, with eruption
the lesions begin to appear. on the tympanic membrane and external
In children, there is little prodromal illness and auditory canal, and often a facial palsy.
the disease is first noticed when skin lesions appear.
Initially macular, the rash rapidly evolves through
papules to the characteristic clear vesicles. The
Immunity
rash is characteristically centripetal in distribution, Previous infection with varicella is believed to
affecting mainly the trunk, and is very superficial confer lifelong immunity to varicella.
without involving the deeper layers of the skin. The The development of varicella-zoster virus-
rash appears in successive crops, so that all stages specific cell-mediated immunity is important
of the eruption can be seen at the same time. It in recovery from both varicella and zoster.
matures very quickly, beginning to crust within 48 Appearance of local interferon may also contribute
hours.Recovery is usually uneventful. to recovery.
Complications: Primary infection is usually more
severe in adults than in children. The rash may Laboratory Diagnosis
become hemorrhagic. Varicella pneumonia is Diagnosis is usually clinical.
more common in adults. A variety of organs may 1. Microscopy: Multinucleated giant cells and type
be affected with complications like myocarditis, A intranuclear inclusion bodies may be seen
nephritis, acute cerebellar ataxia, meningitis in smears prepared by scraping the base of the
and encephalitis. Secondary bacterial infections, early vesicles (Tzanck smear) and stained with
usually due to staphylococci or streptococci, may toluidine blue, Giemsa or Papanicolou stain.
occur. Reyes’ syndrome may follow varicella in Direct examination by electron microscopy will
some cases with a history of administration of reveal herpes particles.
salicylates. 2. Virus isolation: Virus isolation can be
attempted by inoculating human amnion,
Herpes zoster (Shingles, Zona) human fibroblast, HeLa or Vero cells.
3. Virus antigen: The virus antigen can be
The name is derived from Heroein, meaning to detected in scrapings from skin lesions by
creep; Zoster, meaning girdle. immunofluorescence, and in vesicle fluid by
While varicella is typically a disease of child counterimmunoelectrophoresis with zoster
hood, herpes zoster is one of old age. immune serum. ELISA and PCR techniques
are also in use.
Pathogenesis 4. Serological diagnosis: A rise in specific
Herpes zoster usually occurs in persons who antibody titer can be detected in the patient’s
had chickenpox several year earlier. The virus serum by various tests, including fluorescent
remaining latent in the sensory ganglia, may leak antibody, latex agglutination, and enzyme
out at times but is usually held in check by the immunoassay.
Chapter 58: Herpesviruses | 413
Prophylaxis and Treatment and the reticuloendothelial system. Clinical
features—include intrauterine growth retar
Active Immunization
dation, jaundice, hepatosplenomegaly, throm
A live-attenuated varicella vaccine was developed bocytopenia, microcephaly, chorioretinitis and
by Takahashi in Japan in 1974 by attenuating a cerebral calcification resembling congenital
strain of varicella virus (Oka strain, so named after toxoplasmosis.
the patient) by serial passage in tissue culture.
Perinatal infection may be acquired from the
This vaccine, given by subcutaneous injection,
infected mother through genital secretions or
has been found to be immunogenic with good
breast milk.
antibody response but it was very labile and had
to be stored frozen. A modified lyophilized form of D. Postnatal infection: This can be acquired in
the vaccine is now available, which can be stored many ways-such as saliva, sexual transmission,
between 2°C and 8°C. It is recommended as a single blood transfusion and donated organs
subcutaneuos dose for childern 1–12 years old, and
for those older as 2 doses 6–10 weeks apart. It is safe Laboratory Diagnosis
and effective. It is not considered safe in pregnancy. A. Specimens: CMV can be isolated from the
urine, saliva, breast milk, semen, cervical
Passive Immunization secretions and blood leucocytes.
Varicella-zoster immunoglobulin (VZIG) prepared B. Demonsration of cytom egalic cell: The
from the patients convalescing from herpes zoster histologic hallmark of CMV infection is the
seems to be of some use in preventing or modifying cytomegalic cell, which is an enlarged cell
severe disease in immunodeficient patients. that contains a dense, central, “owl’s-eye,”
basophilic intranuclear inclusion body. The
Laboratory Diagnosis inclusions are readily seen with Papanicolaou
or hematoxylin-eosin staining
Diagnosis is easily made clinical. Laboratory
C. lsolation of virus: Human cytomegaloviruses
diagnosis is as for chickenpox.
can be grown in human fibroblast cultures.
Cultures have to be incubated for prolonged
Treatment periods as the cytopathic effects (swollen
It is as for chickenpox. refractile cells with cytoplasmic granules) are
slow in appearance.
Cytomegalovirus D. DNA probes: DNA probes are used to directly
detect the CMV antigens in tissues or fluids.
Cytomegaloviruses (CMV), formerly known as
salivary gland viruses, are a group of ubiquitous E. Polymerase chain reaction (PCR): To directly
herpesviruses of humans and animals. detect the genome in tissues or fluids.
F. Serology: IgM antibodies suggests a current
infection and can be detected in serum by
Pathogenesis
ELISA.
The congenital, oral, and sexual routes, blood
transfusion, and tissue transplantation are the major Treatment and Prevention
means by which CMV is transmitted. Congenitally
infected infants have viruria for upto 4–5 years. They Ganciclovir (dihydroxypropoxymethyl guanine)
are highly infectious in early infancy. and foscarnet (phosphonoformic acid) have been
approved for the treatment of CMV infections.
A. Normal hosts: Primary cytomegalovirus
infection of older children and adults is Prevention is indicated only in high risk
usually asymptomatic but occasionally causes cases. Screening of blood and organ donors and
a spontaneous infectious mononucleosis administration of CMV immunoglobulins have
syndrome. been employed in prevention.
B. Immunocompromised hosts: This occurs No vaccine is available.
in transplant recipients, cancer patients on
chemotherapy, and more particularly in the
Epstein–Barr virus
HIV infected. Epstein–Barr virus (EBV) is in some respects the
C. Congenital and perinatal infections: Intra most sinister herpesvirus, for its association with
uterine infection leads to fetal death or malignant disease is now well established. Only
cytomegalic inclusion disease of the newborn human and some subhuman primate B cells have
which is often fatal. Cytomegalic inclusion receptors for the virus. EBV infected B cells are
disease of newborns is characterized by transformed so that they become capable of con
involvement of the central nervous system tinuous growth in vitro.
414 | Section 4: Virology
Pathogenesis A. Infectious Mononudeosis (Glandular Fever)
Epstein–barr virus (EBV) is commonly transmitted This is an acute self-limited illness usually seen
by infected saliva and initiates infection in the in nonimmune young adults. The incubation
oropharynx. The virus enters the pharyngeal period is 4–8 weeks.Infectious mononucleosis is
epithelial cells through CR2 (or CD21) receptors. characterized by high fever, malaise, pharyngitis,
It multiplies locally, invades the bloodstream lymphadenopathy (swollen glands), and, often,
and infects B lymphocytes in which two types hepatosplenomegaly. A mild transient rash may
of changes are produced. In most cases, the virus be present. Some patients treated with ampicillin
becomes latent inside the lymphocytes, which may develop a maculopapular rash due to immune
become transformed or ‘immortalized so that complex reaction to the drug. In most patients
they become capable of indefinite growth in vitro. the spleen is palpable and there is some liver
Lytic infection is a second type of effect, shown by dysfunction, occasionally with frank jaundice. The
a few infected B cells with cell death and release of typical illness is self-limited and lasts 2–4 weeks.
mature progency virions. Complications are rare but some are serious:
The classic lymphocytosis associated with i. Acute airway obstruction.
infectious mononucleosis results mainly from the ii. Splenic rupture.
activation and proliferation of T cells. These appear iii. Neurological complications include meningitis,
as atypical lymphocytes (also called Downey encephalitis and the Guillain-Barre syndrome.
cells).
Intermittent reactivation of the latent EB virus B. Oral Hairy leukoplakia
leads to clonal proliferation of infected B cells. In This lesion is a wart-like growth that develops
immunocompetent subjects, this is kept in check on the tongue in some HIV-infected persons and
by activated T cells. In the immunodeficient, B transplant patients. It is an epithelial focus of EBV
cell clones may replicate unchecked, resulting in replication.
lymphomas. Hyperendemic malaria prevalent in
Africa is believed to be responsible for the immune C. Chronic Disease
impairment in children with Burkitt’ lymphoma. Epstein-barr virus (EBV) can cause cyclic recurrent
The frequency of lymphomas seen in many types of disease in some people. This disorder is different
immunodeficiencies, most typically in AIDS, may from chronic fatigue syndrome, which has another
have a similar pathogenesis. etiology.
Nasopharyngeal carcinoma seen in men of
Chinese origin. Genetic and environmental factors D. Burkitt’s lymphoma
are said to be important in the and EB virus DNA
Epstein–barr virus (EBV) is associated with the
is regularly found in the tumour cells.
development of Burkitt’s lymphoma (a tumor of
the jaw in African children and young adults).
Epidemiology Most African tumors (>90%) contain EBV DNA
Epstein–Barr (EB) virus is ubiquitous in all human and express EBNA1 antigen. Malaria, a recognized
populations. Infection with EBV is transmitted by cofactor.
saliva, and requires intimate oral contact.
The source of infection is usually the saliva E. Nasopharyngeal Carcinoma
of infected persons who shed the virus in This cancer of epithelial cells is common in males
oropharyngeal secretions. Saliva sharing between of Chinese origin. It mainly affects people aged
adolescents and young adults often occurs during 20–50 years, males preponderating. EBV DNA is
kissing; thus, the nickname “kissing disease” for regularly found in nasopharyngeal carcinoma
infectious mononucleosis. Children can acquire (NPC) cells. Genetic and environmental factors
the virus at an early age by sharing contaminated are believed to be important in the development
drinking glasses. Infection may also follow blood of nasopharyngeal carcinoma.
or marrow transfusion but these are rare events.
F. Lymphoproliferative Diseases in
Clinical conditions
Immunodeficient Hosts
1. Infectious mononucleosis.
Immunodeficient patients are susceptible to EBV
2. EBV associated malignancies:
induced lymphoproliferative diseases that may
a. Burkitt’s lymphoma. be fatal. AIDS patients are susceptible to EBV-
b. Lymphomas in immunodeficient persons. associated lymphomas and hairy oral leukoplakia
c. Nasopharyngeal carcinoma. of the tongue.
Chapter 58: Herpesviruses | 415
Laboratory Diagnosis IgG anti-VCA antibody indicates past or recent
infection and persists throughout life.
1. Differential White Cell Count
Early antigen (EA) antibodies are often found in
Blood examination during the initial phase may patients with Burkitt’s lymphoma or nasopharyngeal
show leucopenia. Later there is a prominent carcinoma.
leucocytosis with the appearance of abnormal Antibodies to the EB nuclear antigen (EBNA)
mononuclear cells. These atypical mononuclear reveal past infection with EBV, though detection
cells are lymphoblasts derived from T cells reactive of a rise in anti-EBNA antibody would suggest a
to the virus infection. The blood picture may primary infection.
sometimes resemble lymphocytic leukemia.
4. Antigen Detection
2. Paul–Bunnell Test
Epstein-barr virus (EBV) antigen can be detected
B cells transformed by EBV undergo polyclonal by immunofluorescence using monodonal
expansion. These antib odies include an IgM antibodies.
heterophile antibody. Agglutination of horse or
sheep red cells by serum absorbed to exclude a 5. Nucleic Acid Hybridization
natural antibody is the basis of this test. It is the most sensitive means of detecting EBV in
patient materials.
Procedure
Inactivated serum. 56°C for 30 minutes in doubling 6. Virus Isolation
dilutions is mixed with equal volumes of a 1% Epstein-barr virus (EBV) can be isolated from
suspension of sheep erythrocytes. After incubation saliva, peripheral blood, or lymphoid tissue by
at 37°C for four hours the tubes are examined for immortalization of normal human lymphocytes,
agglutination. An agglutination titer of 100 or above usually obtained from umbilical cord blood.
is suggestive of infectious mononucleosis.
7. Polymerase Chain Reaction.
Confirmation
For confirmation, differential absorpt ion of Human Herpesviruses 6 (HHV6)
agglutinins with guineapig kidney and ox red cells Human herpesviruses 6 was first isolated from the
is necessary. The Forssman antibody induced blood of patients with AIDS and grown in T-cell
by injection of horse serum is removed by cultures. HHV6 is lymphotropic and ubiquitous. It
treatment with guineapig kidney and ox red cells. is present in the saliva of most adults and is spread
Normally occurring agglutinins are removed by by oral secretions. Two variants are recognized,
guineapig kidney, but not by ox red cells. Infectious A and B. Variant B is the cause of the mild but
mononucleosis antibody is removed by ox red cells common childhood illness ‘exanthem subitum’
but not guinea pig kidney (Table 58.2). (roseola infantum or ‘sixth disease’). In older age
groups, it has been associated with infectious
3. Epstein–Barr Virus-specific Antibodies mononucleosis syndrome, focal encephalitis and,
Tests are also available for the demonstration of in the immunodeficient, with pneumonia and
specific EB virus antibodies. Immunofluorescence disseminated disease.
and ELISA are commonly employed. Laboratory dignosis: HHV6 can be isolated
from peripheral blood mononuclear cells in early
The IgM antibody to VCA (virus capsid antigen)
febrile stage of the illness by co-cultivation with
appears soon after primary infection and disappears
lymphocytes.
in 1–2 weeks and indicative of current infection. The
Virus antigen-can be detected by immuno
fluroscence using monoclonal antibodies. ELISA
Table 58.2: Differential absorption test for Paul- is used for detecting both antigen and antibidies
Bunnell antibody in patient serum.
Result of absorption Ox red cells
on guinea pig kidney Human Herpesvirus 7 (HHV7)
Normal serum Absorbed Absorbed
Like HHV6, HHV7 also appears to be widely distri
Antibody after Absorbed Not absorbed buted and transmitted through saliva. However,
serum therapy
HHV7 remains an orphan virus with no disease
Infectious Not absorbed Absorbed association. It shares with HIV the same CD4
mononucleosis
receptor on T cells.
416 | Section 4: Virology
It may cause an illness resembling infectious
Laboratory diagnosis—Atypical lymphocytes are
mononucleosis, some cases of exanthem subitum. probably the earliest detectable indication of an
EBV infection
Human Herpesvirus 8 (HHV8) Paul-Bunnell test is most frequently used test
to detect heterop hile antibodies in infectious
A new herpesvirus, also called Kaposi’s sarcoma- mononucleosis patients
associa ted herpesvirus (KSHV) is the cause Human Herpesvirus 8 (HHV8)
of Kaposi’s sarcomas, vascular tumors of mixed It is the cause of Kaposi’s sarcomas, vascular tumors
cellular composition, and is involved in the of mixed cellular composition, body cavity-based
lymphomas occurring in AIDS patients and of
pathogenesis of body cavity-based lymphomas multicentric Castleman’s disease.
occurring in AIDS patients and of multicentric
Castleman’s disease.
It appears to be sexually transmitted among Important questions
men who have sex with men. Infections are
common in Africa with infections acquired early in 1. Name various viruses of the family Herpesviridae.
life by nonsexual routes, possibly through contact Discuss the various infections caused by herpes
with oral secretions. simplex virus types 1 and 2.
Diagnosis depends mainly on detection of viral 2. Classify human herpesviruses. Discuss briefly
DNA by PCR. their pathogenesis and laboratory diagnosis.
3. Describe the lesions caused by herpes simplex
virus and their laboratory diagnosis.
Key Points
4. Write short notes on:
Herpesviruses are DNA viruses a. Varicella-zoster virus
Human herpesviruses include human herpesvirus b. Varicella or chickenpox
1 (HV1) to human herpesvirus 8 (HV-8). HHV 3, HHV c. Cytomegalovirus
4 and HHV 5 are varicella-zoster virus, Epstein-Barr d. Epstein-Barr virus (or) EB virus
(EB) and cytomegalovirus (CMV) respectively. Herpes e. Infectious mononucleosis
simplex virus type 1 and 2 are designated as HHV 1 f. Paul-Bunnel test
and HHV 2
g. Human herpesvirus 6 (HHV6)
Clinical syndromes
HSV-l is generally transmitted orally; HSV-2 is gener
ally transmitted sexually Multiple choice questions (mcqs)
HSV-1 causes acute herpetic gingivostomatitis, acute
herpetic pharyngotonsillitis, herpes labial is, herpes 1. Which of the following infection/s is/are caused by
encephalitis, eczema herpeticum, and herpetic herpes simplex virus type I?
whitlow. HSV-2 causes genital herpes, neonatal a. Acute gingivostomatitis
infection, and aseptic meningitis b. Keratoconjunctivitis
Laboratory diagnosis: (A) Direct microscopic examin c. Encephalitis
ation of cells from base of lesion; (B) Cell culture; d. All of the above
(C) Assay of tissue biopsy, smear, or vesicular fluid 2. All the following infections are caused by HSV-2
for HSV antigen; (D) HSV type distinction (HSV-l vs. except:
HSV-2) type a. Neonatal infection
Varicella Zoster Virus (VZV)—causes chickenpox b. Aseptic meningitis
(varicella) and herpes zoster or shingles, two distinct
c. Herpetic whitlow
clinical entities in humans
d. Genital herpes
Virus causes lifelong infection
3. All the following infections are caused by HSV-2
Cytomegalovirus
except:
Virus is transmitted orally and sexually, in blood
a. Neonatal infection
transfusions, in tissue transplants, in utero, at birth,
and by nursing b. Aseptic meningitis
Clinical syndromes—Congenital CMV infection,
c. Herpetic whitlow
acquired CMV infection, CMV infec t ion in d. Genital herpes
immunocompromised patients, and CMV infection 4. Ramsay–Hunt syndrome can be caused by:
in immunocompetent adult hosts. CMV generally a. Herpes simplex virus
causes subclinical infection b. Herpes-zoster virus
Epstein–Barr Virus c. Cytomegalovirus
EBV causes heterophile antibody-positive infectious d. Epstein–Barr virus
mononucleosis and has been causally associated
5. Shingles is caused by:
with Burkitt’s lymphoma, Hodgk in’s disease, and
a. Varicella–zoster virus
nasopharyngeal carcinoma
b. Cytomegalovirus
Transmission occurs via saliva, close oral contact
(“kissing disease”), or sharing of items c. Epstein–Barr virus
d. Herpes simplex virus type 1
Chapter 58: Herpesviruses | 417
6. Owl’s eye is the characteristic feature of the cell 8. Paul–Bunnell test is used for serodiagnosis of:
infected by: a. Infectious mononucleosis
a. Herpes simplex virus b. Genital herpes
b. Epstein-Barr virus c. Neonatal infection
d. Aseptic meningitis
c. Cytomegalovirus
9. Which of the following viruses is associated in
d. Human herpesvirus 8
causation of Kaposi’s sarcoma?
7. Which of the following malignancies is/are associated a. Herpes simplex virus
with Epstein–Barr virus? b. Herpesvirus simiae
a. Burkitt’s lymphoma c. Human herpesvirus 8
b. Nasopharyngeal carcinoma d. Human herpesvirus 6
c. B cell lymphoma Answers (MCQs)
d. All of the above 1. d; 2. c; 3. c; 4. b; 5. a; 6. c; 7. d; 8. a; 9. c.
59
Chapter
Adenoviruses
Learning Objectives
After reading and studying this chapter, you should ∙∙ Describe diseases associated with adenovirus
be able to: ∙∙ Describe adenovirus-associated viruses (AAV).
∙∙ Describe morphology of adenovirus
C. Gastrointestinal Disease
Diarrhea: Some fastidious adenoviruses can cause
Fig. 59.1: Morphology of advenovirus diarrheal disease in children (for example, types
40 and 41).
A. Respiratory Diseases
1. Pharyngitis: Adenoviruses are the major cause D. Other Diseases
of nonbacterial pharyngitis and tonsillitis,
Mesenteric adenitis and intussusception in children,
presenting as febrile common cold. Types 1–7
pertussis-like illness, acute hemorrhagic cystitis with
are commonly responsible.
dysuria and hematuria in young boys, musculoskeletal
2. Pneumonia: Adenovirus types 3 and 7 are
disorders, genital and skin infections.
associated with pneumonia in adults resembling
primary atypical pneumonia. Adenoviruses,
E. Systemic Infection in Immunocompromised
particularly types 3, 7, and 21 are thought to be
responsible for about 10–20% of pneumonias Patients Include Pneumonia and Hepatitis
in childhood. In infants and young children
type 7 may lead to more serious and even fatal Laboratory Diagnosis
pneumonia.
3. Acute respiratory diseases: Adenoviruses are A. Specimens
the cause of an acute respiratory disease (ARD) Depending on the clinical disease, virus may be
syndrome among military recruits. Serotypes 4, recovered from stool or urine or from a throat,
7 and 21 are the agents commonly isolated. conjunctival, or rectal swab.
Papovaviruses
Learning Objectives
After reading and studying this chapter, you should be able to:
∙∙ Describe the following: Papillomaviruses; Polyomaviruses.
Parvoviruses
Learning Objectives
After reading and studying this chapter, you should be able to:
∙∙ Describe Parvoviruses.
Picornaviruses
LEARNING OBJECTIVES
After reading and studying this chapter, you should ∙∙ Differentiate coxsackie A and coxsackie B viruses
be able to: ∙∙ Describe diseases caused by coxsackie viruses
∙∙ Describe enteroviruses ∙∙ D escribe the following: acute hemorrhagic
∙∙ Discuss prophylaxis against poliomyelitis conjunctivitis; Rhinoviruses.
∙∙ Differentiate live and killed polio vaccines
Orthomyxovirus
Learning Objectives
After reading and studying this chapter, you should ∙∙ D escrbe the following: Hemagglutinin (H) and
be able to: neuraminidase (NA); Antigenic variation in Influenza
∙∙ Differentiate between orthomyxoviruses and virus; Antigenic shift and antigenic drift
paramyxoviruses ∙∙ Discuss laboratory diagnosis of influenza
∙∙ Describe morphology of influenza virus ∙∙ D escribe the following: Influenza pandemics;
∙∙ Discuss types and subtypes of orthomyxoviruses prophylaxis against influenza; influenza vaccines.
Antigenic Variation
Influenza viruses are remarkable because of the
frequent ‘antigenic changes that occur in HA and
NA. This is of great importance in the epidemiology
of the disease. Antigenic variability is highest in
influenza virus type A and less in type B, while it
has not been demonstrated in type C.
The internal RNP antigen and M protein
Fig. 63.1: Diagrammatic representation of influenza virus antigen are stable but both the surface antigens,
436 | Section 4: Virology
hemagglutin in and neuraminidase, undergo hemagglutinin titers, but low infectivity. This has
independent antigenic variations, which may be of been called the von Magnus phenomenonand is
two types a
ntgenic drift (minor antigenic changes) due to the formation of incomplete virus particles
and antigenic drift (major antigenic changes) in HA lacking nucleic acid.
or NA result in the appearance of a new subtype.
Antigenic drift: Antigenic drift is the gradual
Pathogenesis
sequential change in antigenic structure occurring Influenza virus spreads from person to person
regu larly at frequent intervals. Here the new by airborne droplets. The viral neuraminidase
antigens, though different from the previous facilitates infection by reducing the viscosity
antigens, are yet related to them. Antigenic drift of the mucous film lining the respiratory tract
is due to mutation and selection. Antigenic drift and exposing the cell surface receptors for virus
accounts for the periodical epidemics of influenza. adsorption. The ciliated cells of the respiratory
tract are the main sites of viral infection. Within a
Antigenic shift: Antigenic shift is an abrupt, short time, many cells in the respiratory tract are
drastic, discontinuous variation in the antigenic infected and eventually killed. Influenza infections
structure, resulting in a novel virus strain unrelated cause cellular destruction and desquamation of
antigenically to predecessor strains. Such changes superficial mucosa of the respiratory tract. This
may involve hemagglutinin, neuraminidase or both. renders the respirator y tract highly vulnerable
The mechanism for shift is genetic reassortment to bacterial invasion, especially staphylococci,
between human and avian influenza viruses. streptococci, and Haemophilus influenzae. Viral
Antibodies to predecessor viruses do not neutralise pneumonia is seen only in more severe cases.
the new variants and can, therefore, spread widely
in the population causing major epidemics or Clinical Features
pandemic. Influenza B and C viruses do not exhibit
A. Uncomplicated Influenza
antigenic shift because few related viruses exist in
animals. The incubation period is 1–3 days. The disease
varies in severity from a mild coryza to fulminating
and rapidly fatal pneumonia. Most infections are
Antigenic Classification
subclinical.
Antigenic differences exhibited by two of the inter Symptoms of classic influenza usually appear
nal structural proteins, the nucleocapsid (NP) and abruptly and include chills, headache and dry
matrix (M) proteins, are used to divide influenza cough, followed closely by high fever, generalized
viruses into types A, B and C. These proteins muscular aches, malaise and anorexia. The fever
possess no cross reactivity among the three types. usually lasts 3–5 days, as do the systemic symptoms.
Antigenic variations in the surface glycoproteins,
HA and NA, are used to subtype the viruses. Only
Complications
type A has designated subtypes. Influenza virus
type A strains can be classified into subtypes based Complications of influenza include primary viral
on variations in their surface antigens. pneumonia, secondary bacterial pneumonia,
myositis and cardiac complicat ions, such as
congestive failure or myocarditis and, neurological
Host Range
involvement, such as Guillain–Barre syndrome,
1. Animals: Intranasal inoculation in ferrets encephalopathy, encephalitis and Reye’s syndrome
produces an acute respiratory disease. may occur.
2. Egg inoculation: The virus grows well in
the amniotic cavity of chick embryos. After Reye’s Syndrome
a few egg passages, the virus grows well in
Influenza, particularly infection with type B, has
the allantoic cavity also, except for the type
been associated with Reye’s syndrome. It is an
C virus which does not generally grow in the
acute encephalopathy of children and adolescents,
allantoic cavity. Virus growth is detected by the
usually between 2 and 16 years of age and is
appearance of hemagglutinin in the allantoic
characterized by acute degenerative changes in
and amniotic fluids.
the brain, liver and kidneys. Type B infections
3. Cell culture: The virus grows in primary may sometimes cause gastrointestinal symptoms
monkey kidney cell cultures, as well as in some (gastric flu).
continuous cell lines.
von Magnus phenonomenon: When passaged Laboratory Diagnosis
serially in eggs, using as inocula undiluted infected Diagnosis of influenza relies on isolation of the
allantoic fluid, the progeny virus will show high virus, identification of viral antigens or viral nucleic
Chapter 63: Orthomyxovirus | 437
acid in the patient’s cells, or demonstration of a and convalescent sera are necessary. A fourfold
specific immunologic response by the patient. or greater increase in titer must occur to indicate
influenza infection.
1. Demonstration of the Virus Antigen Neutralization tests are the most specific and
Rapid diagnosis of influenza may be made by the best predictor of susceptibility to infection.
demonstration of the virus antigen on the surface of ELISA test is more sensitive than other assays.
the nasopharyngeal cells by immunofluorescence. It is possible to estimate the neuraminidase
This test is rapid but is not as sensitive as viral antibody by enzyme neutralization tests.
isolation.
Detection of influenza RNA by reverse trans Immunity
criptase polymerase chain reaction may be more Immunity to influenza is long-lived and subtype-
sensitive than antigen detection but is not widely specific. Antibodies against HA and NA are
available in diagnostic laboratories. important in immunity to influenza, whereas
antibodies against the other virus-encoded proteins
2. Isolation of the Virus are not protective.
Nasal washings, gargles and throat swabs are the An attack of influenza confers protection
best specimens for viral isolation and should be effective for about one or two years. The apparent
obtained within 3 days after the onset of symptoms short duration of immunity is due to the antigenic
but less often in later stages. Throat garglings variation that the antigenic variation that the virus
are collected using broth saline or other suitable undergoes frequently. Following infection and
buffered salt solution. The sample should be held at immunization, circulating antibodies are formed
4°C until inoculation into cell culture, or if the delay against the various antigens of the virus. However,
is long, at –70°C. The specimen should be treated it the local concentration of anthaemagglutinin
with antibiotics to destroy bacteria. Isolation may and, to a smaller extent, of anineuraminadase
be made in eggs or in monkey kidney cell culture. antibodies (mainly IgA) in the respiratory tract that
The material is inoculated into the amniotic is more relevant in protection.
cavity of 11–13 day-old eggs, using at least six eggs When an individual experiences repeated
per specimen. After incubation at 35°C for three infections with different antigenic variants of
days, the eggs are chilled and the amniotic and influenza virus type A, he responds by forming
allantoic fluids harvested separately. The fluids antibodies not only against each infecting strain
are tested for hemagglutination using guinea pig but also against the strain that he first comes into
and fowl cells in parallel, at room temperature and contact with. The dominant antibody response
at 4°C. Some strains of the influenza virus type A will be against the strain that caused the earliest
agglutinate only guinea pig cells on initial isolation. infection. This phenomenon called the doctrine
The type B virus agglutinates both cells, while type of “original antigenic sin”.
C strains agglutinate only fowl cells at 4°C. Subtype Influenza virus infection induces cell-mediated
identification is made by hemagglutination immunity also but its role in protection has not
inhibition test. Some of the recent type A strains been clarified.
can be isolated by direct allantoic inoculation of the
clinical specimen into 9–11-day-old eggs. However, Epidemiology
type Band C viruses will be missed if only allantoic Influenza viruses occur worldwide and occurs
inoculation is used. sporadically, as epidemics or in pandemic form.
For primary isolation the most suitable cells are The source of infection is an infected individual.
primary monkey kidney or human embryo kidney Influenza infection is spread readily via small
cells, but most laboratories now use secondary airborne droplets expelled during talking,
baboon kidney cells or Madin–Darby canine breathing, and coughing. Influenza C is least
kidney cells. Incubation at 33°C in roller drum significant; it causes mild, sporadic respiratory
is recommended. The presence of virus may be disease but not epidemic influenza. Influenza B
detected by hemadsorption with human O group, sometimes causes epidemics, but influenza type A
fowl or guineapig red blood cells. Rapid results can sweep across continents and around the world
can be obtained by demonstrating virus antigen in massive epidemics called pandemics.
in infected cell cultures by immunofluoroscence. What makes influenza an important and
challenging disease is its propensity for causing
3. Serology pandemics. Domestic birds like ducks can get
Complement fixation tests (CFTs) and hemaggluti infected from wild birds and carry the infection to
nation inhibition (HI) tests are employed for the pigs.The genetic reassortment may take place in
serological diagnosis of influenza. Paired acute pigs that have receptors for both human and avian
438 | Section 4: Virology
and rimantadine which block the viral M2 protein
which functions as an ion channel. These act only
with type A virus and not with type B, which lacks
the M2 components.
B. Influenza Vaccines
Influenza vaccines have been in use for many
decades. The aim of immunization is to produce
hemagglutinat ion inhibiting or neutralizing
antibody in all vaccinees. However, cert ain
characteristics of influenza viruses make prevention
and control of the disease by immunization
especially difficult. Existing vaccines are continually
being rendered obsolete as the viruses undergo
antigenic drift and shift. Vaccines are of two types
as follow.
Paramyxoviruses
Learning Objectives
Subfamily Paramyxoviridae
1. Respirovirus (para-influenza viruses 1, 3).
2. Rubulavirus (mumps virus, para-influenza
viruses 2, 4a, 4b).
3. Morbillivirus (Measles).
4. Henipavirus—Nipah virus and Hendra virus.
Subfamily Pneumovirinae
1. Pneumovirus (respiratory syncytial virus (RSV)
Fig. 64.1: Schematic diagram of a paramyxovirus showing 2. Metapneumovirus—Human metapneumo
major components virus.
Chapter 64: Paramyxoviruses | 441
Table 64.1: Characteristics of genera in the subfamilies of the family Paramyxoviridae
Paramyxovirinae Pneumovirinae
Property Respirovirus Rubulavirus Morbillivirus Henipavirus 1
Pneumovirus Metapneumovirus
Human Parainfluenza Mumps, Measles Hendra, Respiratory Human
viruses 1,3 parainfluenza Nipah syncytial metapneumovirus
2, 4a, 4b virus
Serotypes 1 each 1 each 1 ? 2 ?
Diameter of 18 18 18 ? 13 13
nucleocapsid
(nm)
Membrane + + + + + +
fusion
(F protein)
Hemolysin2 + + + ? 0 0
Hemaglutinin +3 +3 +4 0 0 0
Hemadsorption + + + 0 0 0
Neuraminidase +3 +3 0 0 0 0
Inclusions 5
C C N, C ? C ?
1
Zoonotic paramyxoviruses
2
Hemolysin activity carried by F glycoprotein
3
Hemagglutination and neuraminidase activities carried by HN glycoprotein
4
Hemagglutination of monkey erythrocytes only, by H glycoprotein that lacks neuraminidase activity
5
C, cytoplasm; N, nucleus
Arboviruses
LEARNING OBJECTIVES
After reading and studying this chapter, you should ∙∙ List the arboviruses prevalent in India
be able to: ∙∙ Describe the following: Chikungunya; Japanese B
∙∙ Classify arboviruses encephalitis; Dengue fever (or) Break bone fever;
∙∙ Describe laboratory diagnosis of arboviruses Kyasanur forest disease (KFD).
∙∙ List mosquito-borne and tick-borne arboviruses
cultures of primary cells and in cultures of reverse transcriptase polymerase chain reaction
appropriate insect tissues. (RTPCR).
4. Many arboviruses multiply in continuous tissue
cultures of mosquito cells when incubated at D. Serology
34°C or lower temperatures. Diagnosis may also be made serologically by
5. Mosquito-borne arboviruses multiply after oral demonstrating rise in antibody titer in paired
feeding or intrathoracic injection of several serum samples by hemagglutination inhibition,
Aedes and Culex mosquito species. complement fixation or neutralization tests. Virus-
6. Tick-borne arboviruses multiply after oral specific IgM antibody may be detected within I day
feeding to larval or nymphal ixodes ticks. of onset of clinical symptoms using an IgM capture
7. In general, arboviruses are labile, being readily ELISA test.
inactivated at room temperature and by bile
salts, ether and other lipid solvents. PATHOGENESIS
The virus enters the body through the bite of the
LABORATORY DIAGNOSIS insect vector. After multiplication in the reticulo
A. Specimen endothelial system, viremia of varying duration
ensues and, in some cases, the virus is transported
As all arbovirus infect ions are viremic, blood
to the target organs, such as the central nervous
collected during the acute phase of the disease may
system in encephalitides, the liver in yellow fever
yield the virus. Isolation may also be made from
and the capillary endothelium in hemorrhagic
the CSF in some encephalitic cases but the best
fevers. Arboviruses cause the following clinical
specimen for virus isolation is the brain.
syndromes (Table 65.3).
∙∙ Fever with or without rash and arthralgia
B. Virus Isolation ∙∙ Encephalitis
i. Suckling Mice ∙∙ Hemorrhagic fever
Specimens are inoculated intracerebrally into suck ∙∙ The characteristic systemic disease, yellow
ling mice. The animals develop fatal encephalitis. This fever
is the most sensitive method for isolation of viruses. Arboviruses are maintained in natural trans
mission cycles involving reservoir hosts and
ii. Tissue Cultures arthropod vectors, typically: ticks, mosquitoes and
Some viruses may also be isolated in tiss ue other biting flies.
cultures or, less readily, in eggs. Specimens are
inoculated into Vero, BHK-21 and mosquito cello FAMILIES OF ARBOVIRUSES
lines. Isolates are identified by hemaglutination A. Family Togaviridae
inhibition, complement fixation, gel precipitation,
Togaviruses
immunofluorescence, immunochromatography,
ELISA or neutralization with appropriate antisera. Morphology
Togaviruses are spherical enveloped viruses with
iii. Virus Isolation from Insect Vectors and a diameter of 50–70 nm. The genome is single
Reservoir Animal stranded RNA. The name Togavirus is derived from
‘toga’, meaning the Roman mantle or cloak, and
C. Arbovirus-specific RNA Detection refers to the viral envelope.
Viral RNA is extracted from serum or suspensions
of tissues from patients, or from tissue culture cells Classification
or mosquito homogenates. This is amplified by The family Togaviridae contains two genera:
Alphavirus Rubivirus
The Alphavirus genus consists of about 32 viruses Rubivirus, which is not arthropod-borne and which
of which at least 13 are known to infect humans. causes rubella.
All of them are mosquitoborne.
The disease has been recognised in Japan since Control of Japanese Encephalitis
1871 and was named Japanese ‘B’ encephalitis to
distinguish it from ‘encephalitis A (encephalitis Preventive measures include mosquito control and
lethargica, von Economo’s disease) which was locating piggeries away from human dwellings.
then prevalent. The virus was first isolated in Japan
during an epidemic in 1935. i. Formalin Inactivated Mouse Brain Vaccine
A formalin inactivated mouse brain vaccine
Problem in India using the Nakayama strain has been employed
Recognition of JE was first made in 1955 when the successfully for human immunization in Japan
virus was isolated from mosquitoes of the Culex and, in a small scale, in India also. For primary
vishnui complex from Vellore. From 1976, there have immunization, 2 doses of 1 ml each (0.5 mL for
been periodical outbreaks of the disease in various children under the age of 3 years) should be
parts of India—Dibrugarh (Assam), Gorakhpur (Uttar administered subcutaneously at an interval of
Pradesh) and Haryana and Goa and Maharashtra, 7–14 days. A booster injection of 1 mL should be
Kolar in Karnataka, Andhra Pradesh, in Tamil given after a few months (before one year) in order
Nadu, in Pondicherry and lately in Kerala. Japanese to develop full protection. Protective immunity
encephalitis has become a major public health develops in about a month’s time after the second
problem of national importance in India. dose. Revaccinations may be given after 3 years.
Rhabdoviruses
Learning Objectives
After reading and studying this chapter, you should ∙∙ Discuss laboratory diagnosis of rabies
be able to: ∙∙ Discuss prophylaxis against rabies
∙∙ Describe morphology of rabies virus ∙∙ Describe neural and non-neural vaccines against
∙∙ Describe the following: Street virus vs. fixed virus; rabies.
pathogenesis and clinical features of rabies; Negri
bodies
Rabies virus
Morphology
1. Virion: The rabies virion consists of a helical
nucleocapsid contained in a bullet-shaped
lipoprotein envelope 180 × 75 nm, with one
end rounded or conical and the other plane or
concave (Fig. 66.1).
2. Proteins: Protruding from the lipid envelope
are approximately 200 glycoprotein (G) spikes
of the virus, hemagglutinin activity. Spikes do
not cover the planar end of the virion.
3. Membrane or matrix (M) protein: Beneath Fig. 66.1: Rabies virus: Bullet-shaped virion, showing tightly
the envelope is the membrane or matrix (M) wound helix of ribonucleoprotein in the core, and bilayered
protein and is the major structural protein membranous envelope carrying glycoprotein spikes
458 | Section 4: Virology
Resistance Street Virus
Rabies virus is sensitive to ethanol, iodine prepar The rabies virus isolated from natural human
ations, quaternary ammonium compounds, or animal infection is termed the street virus.
soap, detergents and lipid solvents such as ether, Following inoculation by any route, it can
chloroform and acetone. It is inactivated by phenol, cause fatal encephalitis in laboratory animals
formalin, beta propiolactone, ultraviolet irradiation, after a long and variable incubation period of
sunlight and heat at 50°C for 1 hour or 60°C for 5 about 1–12 weeks (usually 21–60 days in dogs).
minutes. Rabies virus survives storage at 4°C for Intracytoplasmic inclusion bodies (Negri bodies)
weeks and at –70°C for years or by lyophilization It can be demonstrated in the brain of animals dying
is inactivated by CO2 so on dry ice it must be stored of street virus infection.
in glass-sealed vials.
Fixed Virus
Antigenic Properties After several serial intracerebral passages in rabbits,
There is a single serotype of rabies virus. the virus undergoes certain changes and becomes
A. Glycoprotein G: The surface spikes are what is called the fixed virus that no longer multiplies
composed of glycoprotein G, which is important in extraneural tissues. The fixed (or murant) virus
in pathogenesis, virulence and immunity. is more neurotropic, though it is much less infective
Purified spikes containing the viral glycoprotein by other routes. After intracerebral inoculation, it
elicit neutralizing antibody in animals. The produces fatal encephalitis after a short and fixed
purified glycoprotein may therefore provide a incubation period of 6–7 days. Negri bodies are
safe and effective subunit vaccine. usually not demonstrable in the brain of animals
Hemagglutinating activity: Rabies virus dying of fixed virus infection. The fixed virus is used
possesses hemagglutinating activity, optimally for vaccine production.
seen with goose erythrocytes at 0–4 °C and
pH 6.2. Hemagglutination is a property of B. Chick Embryos
the glycoprotein spikes. The hemagglutinin The rabies virus can be grown in chick embryos.
antigen is species specific and distinct from the The usual mode of inoculation is into the yolk sac.
antigens on rabies related viruses. Serial propagation in chick embryos has led to the
B. Nucleocapsid protein: Complement fixing development of attenuated vaccine strains like
antibodies are induced by the nucleocapsid Flury and Kelev.
protein and are not protective. This antigen
is group specific and cross-reactions are seen C. Tissue Culture
with some rabies related viruses. Antiserum pre
The rabies virus can grow in chick embryo
pared against the purified nucleocapsid is used
fibroblast, porcine or hamster kidney cells human
in diagnostic immunofluorescence for rabies.
diploid cell and vero cell cultures.
C. Other antigens: Other antigens identified
include two membrane proteins, glycolipid
and RNA dependent RNA polymerase.
Pathogenesis
Rabies infection usually results from the bite of
Host Range and Growth Characteristics rabid dogs or other animals. The virus can also be
transmitted following nonbite exposures through
A. Animals the inhalation of aerosolized virus (as may be found
Rabies virus has a wide host range. All warm- in bat caves), in transplanted infected tissue (e.g.
blooded animals, including humans, can be cornea), and by inoculation through intact mucosal
infected. All mammals are susceptible to rabies membranes. The virus present in the saliva of the
infection, though differences in susceptibility animal is deposited in the wound.
exist between species. Susceptibility varies among The virus appears to multiply in the muscles,
mammalian species, ranging from very high connective tissue or nerves at the site of deposition.
(foxes, coyotes, wolves) to low (opossums); those The virus remains at the site for days to months
with intermediate susceptibility include skunks, (Fig. 66.2) before progressing to the central
raccoons, and bats. Humans and dogs occupy an nervous system (CNS). Rabies virus travels by
intermediate position. Pups are more susceptible retrograde axoplasmic transport to the dorsal root
than adult dogs. Experimental infection can be ganglia and to the spinal cord. Once the virus gains
produced in any laboratory animal but mice are access to the spinal cord, the brain becomes rapidly
the animals of choice. They can be infected by any infected. The virus then disseminates from the CNS
route. After intracerebral inoculation, they develop via afferent neurons to highly innervated sites, such
encephalitis and die within 5–30 days. as the skin of the head and neck, salivary glands,
Chapter 66: Rhabdoviruses | 459
b. Acute neurologic phase: During the acute
neurologic phase, which lasts 2–7 days, patients
show signs of nervous system dysfunction such
as nervousness, apprehension, hallucinations,
and bizarre behavior.
Hydrophobia: The pathognomonic feature
is difficulty in drinking, together with intense
thirst. Patients may be able to swallow dry solids
but not liquids. Attempts to drink bring on
such painful spasms of the pharynx and larynx
producing choking or gagging that patients
develop a dread of even the sight or sound of
water (hydrophobia). Generalized convulsions
follow.
c. Coma: Patients who survive the stage of acute
neurological involvement lapse into coma.
Death usually occurs within 1–6 days due to
respiratory arrest during convulsions.
B. Rabies in Dogs
Clinical Picture
Fig. 66.2: Pathogenesis of rabies virus infection Rabies in dogs may manifest itself in two forms—
Furious rabies and Dumb rabies.
retina, cornea, nasal mucosa, adrenal medulla, a. Furious rabies: This is the typical “mad dog
renal parenchyma, and pancreatic acinar cells. syndrome”, characterized by (i) A change in
The presence of the virus in the saliva and behavior; (ii) Running amuck; (iii) Change in
the irritability and aggression brought on by the voice; (iv) Excessive salivation and foaming at
encephalitis ensure the transmission and survival the angle of the mouth; (v) Paralytic stage—
of the virus in nature. The virus ultimately reaches Paralysis, convulsions and death follow.
virtually every tissue in the body, though the
b. Dumb rabies: In this type, the excitative or
centrifugal dissemination may be interrupted at
irritative stage is lacking. The dumb form is as
any stage by death. The virus is almost invariably
infectious as the furious type.
present in the cornea and the facial skin of patients
because of their proximity to the brain. The virus
may also be shed in milk and urine. Laboratory Diagnosis
With rare exception (three known cases), rabies 1. Diagnosis of Human Rabies
is fatal once clinical disease is apparent.
Tests are performed on samples of saliva, serum,
spinal fluid, and skin biopsies of hair follicles at the
Clinical Features nape of the neck.
A. Humans
Rabies is primarily a disease of lower animals and A. Rabies Antigens by Immunofluorescence
is spread to humans by bites of rabid animals or
The method most commonly used for diagnosis
by contact with saliva from rabid animals. The
is the demonstration of rabies virus antigens by
infection has also been acquired from aerosols in
immunofluorescence. The specimens tested are
bats’ caves.
corneal smears and skin biopsy (from face or neck)
The incubation period in humans is typically
or saliva antemortem, and brain postmorterm.
1–2 months but may be as short as 1 week or as long
Direct immunofluorescence is done using antirabies
as many years (up to 19 years). It is usually shorter
serum tagged with fluorescein isothiocyanate. The
in children than in adults.
use of monoclonal antibody instead of crude
antiserum makes the test more specific.
Phases of Clinical Spectrum
The clinical spectrum can be divided into three B. Virus Isolation
phases:
a. Prodromal phase: The prodrome, lasting a. Mouse Inoculation
2–10 days, may show any of the nonspecific Samples of brain tissue, saliva, CSF, or urine may
symptoms. be injected intracerebrally into newborn mice for
460 | Section 4: Virology
isolation of the virus. Infection in mice results in Italian physician who first discovered them. This is
encephalitis and death. The inoculated mice are still the method most commonly used as facilities
examined for signs of illness and their brains are for immunofluorescence and biological tests are
examined at death or at 28 days postinoculation not available in many laboratories.
for Negri bodies, or by immunofluorescence rabies Impression smears of the brain are stained by
antigen. Seller’s technique (basic fuchsin and methylene
blue in methanol), which has the advantage that
b. Isolation in Cell Culture fixation and staining are done simultaneously.
Negri bodies are seen as intracytoplasmic, round
A more rapid and sensitive method is isolation
or oval, purplish pink structures with characteristic
of the virus in tissue culture cell lines (WI38,
basophilic inner granules. Negri bodies vary in size
BHK21,CER). Virus isolations is identified by
from 3–27 µm. Other types of inclusion bodies may
immunofluorescence. A positive IF test can be
sometimes be seen in the brain in diseases such
obtained as early as 2–4 days after inoculation.
as canine distemper but the presence of inner
The identity of the isolate can be established by the
structures in the Negri bodies makes differentiation
neutralization test with specific antirabies antibody.
easy. Failure to find Negri bodies does not
exclude the diagnosis of rabies. The microscopic
C. Serology examination for Negri bodies identifies 75–90% of
Rabies antibodies can be detected in the serum and cases of rabies in dogs. Failure to find Negri bodies
CSF of the patient by ELISA. High titer antibodies does not exclude the diagnosis of rabies.
are present in the CSF in rabies but not after Negri bodies contain rabies virus antigens and
immunization. can be demonstrated by immunofluorescence.
Both Negri bodies and rabies antigen can usually
D. Detection of Nucleic Acid be found in animals or humans infected with
rabies, but they are rarely found in bats.
Reverse transcription-polymerase chain reaction
(RT-PCR) testing can be used to amplify parts If impression smears are negative, the tissue
of a rabies virus genome from fixed or unfixed should be sectioned and stained by Giemsa or
brain tissue for detection of rabies virus RNA. This Mann’s method.
technique can confirm dFA results and can detect 3. Isolation of the rabies virus (biological test)
rabies virus in saliva and skin biopsy samples. This is done as described above, for human rabies
diagnosis.
2. Animal Rabies 4. Corneal test
The head of the animal is cut off and sent to the Rabies virus antigen can be detected in live animals
nearest testing laboratory, duly packed in ice in an in corneal impressions or in frozen sections of skin
air-tight container. Alternatively, the brain may be biopsies by the flueroscent antibody test.
removed with antiseptic precautions and sent in
50% glycerol-saline for examination and the other Prophylaxis
in Zenker’s fixative, sent for biological test and
microscopy, respectively. The portion of brain sent This may be considered under:
should include the hippocampus and cerebellum
as Negri bodies are most abundant there. The A. Postexposure Prophylaxis
following tests are done in the laboratory: B. Preexposure Prophylaxis
1. Demonstration of rabies virus antigen by A. Post-exposure Prophylaxis
immunofluorescence This consists of: a. Local treatment , b. Antirabic
This is a highly reliable and the best single test vaccines, c. Hyperimmune serum.
currently available for the rapid diagnosis of rabies a. Local treatment of wound:
viral antigen in infected specimens. This test i. Cleansing: Immediate flushing and
can establish a highly specific diagnosis within washing the wound(s), scratches and the
a few hours. Examination of salivary glands by
adjoining areas with plenty of soap and
immunofluorescence is useful.
water, preferably under a running tap, for
2. Demonstration of inclusion bodies (Negri at least 5 minutes as soap inactivates virus
bodies) by destroying its envelope.
A definitive pathologic diagnosis of rabies can be ii. Chemical treatment: After cleansing
based on the finding of Negri bodies in the brain wound should be inactivated by irrigation
or the spinal cord. Negri bodies, named after the with virucidal agents - either alcohol
Chapter 66: Rhabdoviruses | 461
(400–700 ml/liter), tincture or 0.01% 3. Infant brain vaccines
aqueous solution of iodine or povidone The encephalitogenic factor in brain tissue is a
iodine. basic protein associated with myelin. It is scanty
iii. Suturing: Bite wounds should not be or absent in the nonmyelinated neural tissue of
immediately sutured. newborn animals. So vaccines were developed
iv. Antirabies Serum: The local application of using infant mouse, rat or rabbit brain.
antirabies serum or its infiltration around
the wound has been shown to be highly II. Non-neural Vaccines
effective in preventing rabies. 1. Egg vaccines
v. Antibiotics and antitetanus measure:
a. Duck embryo vaccine (DEV): It was disconti
When indicated should follow the local
nued because of its poor immunogenicity. (b)
treatment recommended above.
Live attenuated chick embryo vaccines—These
b. Antirabic vaccines: Antirabic vaccines fall into vaccines were used for vaccination of animals.
two main categories: neural and non-neural Two types of vaccines were developed with the
(Table 66.2). The former are associated with Flury strain.
serious risk of neurological complications and
i. Low Egg Passage (LEP) vaccine: at 40–50 egg
have been replaced by the latter.
passage level for immunization of dogs.
I. Neural Vaccines ii. High Egg Passage (HEP) vaccine: at 180
passage level for cattle and cats. These are not
These are suspensions of nervous tissues of
in use now.
animals infected with the fixed rabies virus.
2. Cell culture vaccines
1. Semple Vaccine
Semple (1911) developed this vaccine at the Central The cell culture vaccines are of two types:
Research Institute, Kasauli (India), had been the
i. Human origin
most widely used vaccine for over half a century.
It is a 5% suspension of sheep brain infected with Human diploid cell vaccine (HDCV)
fixed virus and inactivated with phenol at 37°C, The first cell culture vaccine was the human diploid
leaving no residual live virus. cell (HDC) vaccine developed by Koprowsky,
Wiktor and Plotkin. It is a purified and concentrated
2. Beta propiolactone (BPL) vaccine preparation of fixed rabies virus (Pitman–Moore
This is a modification of the Semple vaccine, strain) grown on human diploid cells (WI 38 or
in which beta propiolactone is used as the MRC 5) and inactivated with beta propiolactone
inactivating agent instead of phenol. It is prepared or tri-n-butyl phosphate. It is highly antigenic and
from fixed virus grown in the brains of adult sheep free from serious side effects. Its only disadvantage
(Semple type) or other animals. It is believed to be is its high cost. HDC vaccine is now licensed for use
more antigenic. in a number of countries including India for both
pre- and postexposure immunization.
Hepatitis Viruses
Learning Objectives
After reading and studying this chapter, you should modes of transmission of hepatitis B virus; hepatits
be able to: B carriers
∙∙ Classify various hepatitis viruses ∙∙ Describe laboratory diagnosis of hepatitis B virus
∙∙ Tabulate differences between various hepatitis ∙∙ Discuss prophylaxis of hepatitis B infections or
viruses hepatitis B vaccine
∙∙ Compare various features of hepatitis A virus (HAV) ∙∙ Describe the following:Hepatitis C virus or Type C
and hepatitis B virus (HBV) hepatitis; Hepatitis D virus or Delta agent; Hepatitis
∙∙ Describe the following: Morphology of hepatitis E virus; Hepatitis G virus.
B virus; antigenic structure of hepatitis B virus;
Fig. 67.1: The picornavirus structure of hepatitis A virus. The Laboratory Diagnosis
icosahedral capsid is made up of four viral polypeptides (VP1– Etiological diagnosis of type A hepatitis may be
VP4). Inside the capsid is a single-stranded, positive-sense RNA made by demonstration of the virus or its antibody.
single stranded RNA (ssRNA) that has a genomic viral protein
(VPg) on the 5’ end A. Demonstration of the virus: The virus can be
visualized by immune electron microscopy
(IEM) in fecal extracts during the late incubation
Natural infection with HAV is seen only in period and the preicteric phase.
humans. Though primates, such as chimpanzees B. Demonstration of antibody: The best way
have been shown to acquire the infection from to demonstrate an acute HAV infection is by
humans and transmit it to human contacts, there finding anti-HAV immunoglobulin M (IgM), as
is no evidence of any extrahuman source of the measured by an enzyme-linked immunosorbent
virus in nature. assay (ELISA) or radioimmunoassay. Antibody
IgM anti-HAV antibody appears during the
Clinical Features late incubation period, reaches peak levels in
The incubation period is 2–6 weeks. Disease in 2–3 weeks and disappears after 3–4 months.
children is generally milder than that in adults IgG antibody appears at about the same time,
and is usually asymptomatic. The clinical disease peaks in 3–4 months and persists much longer,
consists of two stages: the prodromal or preicteric perhaps for life (Fig. 67.2). Demonstration of
and the icteric stages. The onset may be acute or IgM antibody in serum indicates current or
insidious, with fever, malaise, anorexia, nausea, recent infection, while IgG antibody denotes
Chapter 67: Hepatitis Viruses | 467
Fig. 67.2: Typical course of hepatitis type A Fig. 67.3: Hepatitis B virus structure
recent or remote infection and immunity. ELISA virus (HBV) infects the liver and, to a lesser extent,
kits for detection of IgM and IgG antibodies are the kidneys and pancreas of only humans and
available. chimpanzees.
C. Virus isolation: It is not routinely performed.
Classification
Prophylaxis
HBV is assigned to a separate family Hepadnaviridae
A. General Measures (Hepatitis DNA viruses) which consists of two
The spread of HAV is reduced by interrupting the genera:
fecal–oral spread of the virus by avoiding potentially i. Orthohepadnavirus: It containing HBV as
contaminated water or food, water. well as the woodchuck and ground squirrel
hepatitis viruses. HBV is Hepadnavirus type 1.
B. Immunization ii. Avihepadnavirus: It containing the Pekin
1. Passive protection: Specific passive prophylaxis duck and gray heron hepatitis viruses.
by pooled normal human immunoglobulin
(16% solution in a dose of 0.2–0.12 mL/kg body Structure
weight) intramuscular (IM), before exposure
The HBV is a 42 nm DNA virus with an outer
or in early incubation period, can prevent or
envelope and an inner core, 27 nm in diameter,
attenuate clinical illness, while not necessarily
enclosing the viral genome and a DNA polymerase
preventing infection and virus excretion.
(Fig. 67.3) which is circular double stranded DNA.
2. Hepatitis A vaccine
i. Formalin inactivated, alum conjugaged Australia antigen: In 1965, Blumberg, studying
vaccine: A safe and effective formalin human serum lipoprotein allotypes, observed in
inactivated, alum conjugated vaccine the serum of an Australian aborigine, a new antigen
containing HAV grown in human diploid which gave a clearly defined line of precipitation
cell culture is available for use in children with sera from two hemophiliacs who had received
and adults at high risk for infection, multiple blood transfusions. This was named the
especially travelers to endemic regions. A Australia antigen. By 1968 the ‘Australia antigen’
full course consists of two intramuscular was found to be associated with serum hepatitis.
injections of the vaccine. Protection begins It was subsequently shown to be the surface
4 weeks after injection and lasts for 10–20 component of HBV. Therefore, the name Australia
antigen was changed to hepatitis B surface antigen
years.
(HBsAg).
ii. Live HAV vaccine: It has been developed
in China. Types of particles: Under the electron microscope,
sera from type B hepatitis patients show three types
Treatment of particles (Fig. 67.4).
Treatment is symptomatic. No specific antiviral i. Spherical particle: The predominant form is
drug is available. a small, spherical particle with a diameter of
22 nm.
ii. Tubular particle: The second type of particle
Hepatitis B Virus—serum hepatitis
is filamentous or tubular with a diameter of 22
Type B hepatitis is the most widespread and the nm and of varying length. The particles carry
most important type of viral hepatitis. Hepatitis B the hepatitis B surface antigen (HBsAg).
468 | Section 4: Virology
iii. Dane particle: The third type of particle, far 2. Hepatitis B core antigen (HBcAg): The antigen
fewer in number, is a double walled spherical expressed on the core is called the hepatitis B
structure, 42 nm in diameter. This particle core antigen (HBcAg).
is the complete hepatitis B virus. It was first 3. Hepatitis B e antigen (HBeAg): Hepatitis Be
described by Dane in 1970 and so is known antigen (HBeAg) is a soluble nonparticulate
as the Dane particle.The outer surface, or nucleocapsid protein. The HBeAg and HBcAg
envelope, contains HBsAg and surrounds a proteins share most of their protein sequence.
27-nm inner nucleocapsid core that contains 4. Viral genes and antigens: The genome has
HBcAg. The variable length of a single- a compact structure with four overlapping
stranded region of the circular DNA genome genes. These include structural proteins of the
results in genetically heterogeneous particles virion surface and core, a small transcriptional
with a wide range of buoyant densities. transactivator (X), and a large polymerase (P)
Genome: The nucleocapsid encloses the viral protein that includes DNA polymerase, reverse
genome consisting of two linear strands of DNA held transcriptase, and RNase H activities (Table
in a circular configuration. One of the strands (the 67.2, Fig. 67.5).
plus strand) is incomplete, so that the DNA appears
partially double stranded and partially single HBV Subtypes
stranded. Associated with the plus strand is a viral The particles containing HBsAg are antigenically
DNA polymerase, which has both DNA-dependent complex. It contains two different antigenic
DNA polymerase and RNA-dependent reverse components—the common group reactive antigen
transcriptase functions. Although a DNA virus, a, and two pairs of type specific antigens d-y and
it encodes a reverse transcriptase and replicates w-r, only one member of each pair being present at
through an RNA intermediate. This polymerase a time. HBsAg can thus be divided into four major
can repair the gap in the plus strand and render the antigenic subtypes: adw, adr, ayw and ayr.
genome fully double stranded (Fig. 67.3). They show a distinct geographical distribution.
(Table 67.3).
Antigenic Structure
1. Hepatitis B surface antigen (HBsAg): The Cultivation
envelope proteins expressed on the surface
HBV does not grow in any conventional culture
of the virion and the surplus 22 nm diameter
system. However, limited production of the virus
spherical and filamentous particles constitute
and its proteins can be obtained from several cell
the hepatitis B surface antigen (HBsAg).
lines transfected with HBV DNA. HBV proteins have
been cloned in bacteria and yeast. The chimpanzee
is susceptible to experimental infection and can be
used as a laboratory model.
Stability
The HBV is a relatively heat-stable virus. It remains
viable at room temperature for long periods. Heating
to 60°C for 10 hours inactivates virus by a factor of
100–1000-fold. Exposure to hypochlorite (10,000
ppm available chlorine) or 2% glutaraldehyde for 10
Fig. 67.4: Different types of particles of HBV: Spherical 22 minutes will inactivate virus 100,000-fold, though
nm particle, double shelled 42 nm particle (Dane particle), HBsAg may not be destroyed by such treatment.
tubular 22 nm particle
Epidemiology
The HBV is worldwide in distribution. Natural
infection occurs only in humans. There is no animal
reservoir. The virus is maintained in the large
pool of carriers whose blood contains circulating
virus for long periods, in some even lifelong.
The prevalence of HBV infection varies widely in
different parts of the world.
India falls in the intermediate group, with
Fig. 67.5: The HBV genes and gene products higher carrier rates in the southern part of the
country and lower rates in the northern part. The
Table 67.3: Antigenic types of HBsAg rich and the poor countries also differ in the age
and modes of infection.
Antigenic types Distribution
adw Worldwide Mode of Transmission
adr Asia
The HBV is a blood borne virus and there are three
ayw India, Africa, Russia important modes of transmission:
ayr India, Africa, Russia 1. Parenteral transmission
2. Perinatal transmission
Clinical Syndromes 3. Sexual transmission.
Acute Infection 1. Parenteral transmission: HBV is transmitted
The clinical presentation of HBV in children is less only in blood and other body fluids, including
severe than that in adults, and infection may even cervical secretions, semen, and breast milk. Many
be asymptomatic. Clinically apparent illness occurs other therapeutic, diagnostic, prophylactic and
in as many as 25% of those infected with HBV. even nonmedical procedures are now the main
1. Preicteric phase: HBV infection is characterized modes of infection. HBV is very highly infectious
by a long incubation period (about 1–6 months) far more that HIV.
and an insidious onset. Symptoms during the 2. Perinatal transmission: Vertical transmission
prodromal period may include fever, malaise, from mother to child is one of the most
and anorexia, followed by nausea, vomiting, important routes. HBV can be transmitted to
abdominal discomfort, and chills. babies through contact with the mother’s blood
2. Icteric phase: The classic icteric symptoms of at birth and in mother’s milk.
liver damage (e.g. jaundice, dark urine, pale 3. Sexual transmission: Since HBV is present in
stools) follow soon thereafter. semen and vaginal secretions, therefore, it can
3. Convalescent phase: About 90–95% of adults be transmitted by sexual contact. The risk of
with acute hepatitis B infection recover within transmission by heterosexual and homosexual
1–2 months of onset and eliminate the virus from contact increases with the number of partners
the body within about six months, remaining and the duration of such relationships.
immune thereafter. Mortality is about 0.5–2%.
Chronic infection: A proportion of cases (1–10%) Hepatitis B Carriers
remain chronically infected. They may be asympto Carriers are of two types:
matic carriers or may progress to recurrent or chronic 1. Super carriers: They have HBeAg, high titters
liver disease or cirrhosis. A few of them may develop of HBsAg and DNA polymerase in their blood.
hepatocellular carcinoma after many decades. HBV may also be demonstrable in their blood.
Very minute amount of serum or blood from
Pathogenesis such carriers can transmit the infection. About a
The pathogenesis of hepatitis appears to be immune quarter of the carriers in India are HBeAg positive.
mediated. HBV replicates in the hepatocytes, 2. Simple carriers: These are more common
reflected in the detection of viral DNA and HBcAg types of carriers who have low titer of HBsAg
in the nucleus and HBsAg in the cytoplasm and at in blood, with negative HBeAg, HBV and DNA
the hepatocyte membrane. polymerase. They transmit the infection only
470 | Section 4: Virology
when large volumes of blood are transferred indicative of viral replication. HBcAg is not
as in blood transfusion. Many super carriers in demonstrable in circulation. It is the earliest
time become simple carriers. antibody marker to be seen in blood, long
before anti-HBe or anti-HBs. As anti-HBc
HBV Markers remains lifelong, it serves as a useful indicator
The main antigens HBsAg, HBcAg, and HBeAg of prior infection with HBV; even after all the
each induce corresponding antibodies. With other viral markers become undetectable.
the exception of HBcAg, all these antigens and Initially, anti-H Bc is predominantly IgM,
antibodies, together with the viral DNA polymerase, but after about 6 months, it is mainly IgG.
can be detected in the blood at various times after Selective tests for IgM or IgG anti-HBc
infection and are referred to as ‘markers’ (Table therefore enable distinction between recent
67.4). HBcAg is readily detectable only in the or remote infection respectively.
hepatocyte nuclei. iii. HBeAg: HBeAg presence denotes high
infectivity and its absence, along with the
Laboratory Diagnosis presence of anti-HBe, indicates low infectivity.
HBeAg appears in blood concurrently with
Specific Diagnosis HBsAg, or soon afterwards. Circulating HBeAg is
Specific diagnosis of hepatitis B tests on serological an indicator of active intrahepatic viral replication,
demonstration of the viral markers and can be and the presence in blood of DNA polymerase,
carried out by detection of HBsAg, anti-HBs, HBV DNA and virions, reflecting high infectivity.
HBeAg, anti-HBe, IgM anti-HBc, IgG anti-HBc Before HBsAg disapp ears, HBeAg is replaced
and HBV DNA in the serum. The sequence of by anti-HBe, signaling the start of resolution of
appearance of viral markers in the blood is shown the disease. Anti-HBe levels often are no longer
in Figure 67.6. These can be detected by sensitive detectable after 6 months.
and specific tests like ELISA and RIA.
2. Viral DNA Polymerase
1. Detection of Viral Markers
DNA polymerase activity, HBV DNA, and HBeAg,
i. HBsAg: HBsAg is the first marker to appear which are representative of the viremic stage of
in blood after infection. HBsAg is usually hepatitis B, occur early in the incubation period,
detectable 2–6 weeks in advance of clinical concurrency or shortly after the first appearance
and biochemical evidence of hepatitis and of HBsAg.
persists throughout the clinical course of
the disease. In the typical case, it disappears
3. Polymerase Chain Reaction (PCR)
within about 2 months of the start of clinical
disease, but may sometimes last for 6 months Like HBeAg, HBV DNA is also an indicator of viral
and even beyond but typically disappears by replication and infectivity. Molecular methods,
the sixth month after exposure. such as DNA:DNA hybridization and PCR, at
ii. HBcAg: High levels of IgM-specific anti-HBc present used for HBV DNA testing are highly
are frequently detected at the onset of clinical sensitive and quantitative. HBV DNA level in serum
illness. Because this antib ody is directed reflects the degree of viral, replication in the liver
against the 27 nm internal core compo and so helps assess the progress of patients with
nent of HBV, its appearance in the serum is chronic hepatitis under antiviral chemotherapy.
Prophylaxis
Only general prophylaxis, such as screening of
blood and blood products prior to transfusion, is
possible. No specific active or passive immunizing
agent is available.
Treatment
Recombinant interferon-alpha, alone or with
ribavarin is the only known treatment for HCV. Fig. 67.7: Delta hepatitis virion
Chapter 67: Hepatitis Viruses | 473
Types of infection: Two types of infection are young to middle aged adults (15–40 years old). The
recognized: symptoms and course of HEV disease are similar
1. Coinfection: In coinfection, delta and HBV are to those of HAV disease; it causes only acute dis
transmitted together at the same time. ease. However, the symptoms for HEV may occur
2. Superinfection: In superinfection, delta later than those of HAV disease, and response
infection occurs in a person already harboring to serum immunoglobulin G may be poor. The
HBV. More rapid, severe progression occurs in mortality rate associated with HEV disease is 1–2%.
HBV carriers superinfected with HDV than in HEV infection is especially serious in pregnant
people coinfected with HBV and the delta agent. women and during pregnancy may cause a high
No association has been noted between HDV rate of abortion, intrauterine death and increased
and hepatocellular carcinoma. In simultaneous perinatal mortality in babies born to women with
acute HBV and HDV infections, IgM anti-HBc fulminant hepatitis.
will be detectable, while in acute HDV infection
superimposed on chronic HBV infections, anti- Epidemiology
HBc will be of IgG class. HEV is predominantly spread by the feco-oral
route, especially in contaminated water. Hepatitis
Laboratory Diagnosis E has been shown to occur in epidemics, endemics
The only way to determine the presence of the and sporadic forms almost exclusively in the
agent is by detecting the delta antigen or antibodies. less developed parts of the world. A substantial
ELISA and radioimmunoassay procedures are proportion of cases of acute viral hepatitis occurring
available for doing this. Anti-delta antibodies in young to middle-aged adults in Asia and the
appear in serum and can be identified by ELISA. Indian subcontinent appear to be caused by HEV.
The IgM antibody appears 2–3 weeks after infection The largest such epidemic occurred in Delhi
and is soon replaced by the IgG antibody in acute during the winter of 1955–56, following a breakdown
delta infection. in the water supply and sewage systems of Delhi,
For the rapid identification of delta particles in affecting over 30,000 persons. The cause was
circulation, RNA sequences have been cloned and eventually identified as a new agent, HEV. A similar
DNA probes have been developed. The woodchuck epidemic of hepatitis E occurred between December
has been found to be a suitable experimental 1975 and January 1976 in Ahmedabad city, India.
model for the study of HDV infection.
Laboratory Diagnosis
Treatment Several antibody assays are being developed,
There is no known specific treatment for HDV mostly based on ELISA methods.
hepatitis. 1. Exclusion of hepatitis A and hepatitis B:
Exclusion of hepatitis A by IgM serology and
Prophylaxis hepatitis B by absence of HBsAg and IgM anti-
Because the delta agent depends on HBV for repli HBc.
cation and is spread by the same routes, prevention 2. Immunoelectron microscopy: Immunoelectron
of infection with HBV prevents HDV infection. microscopic examination of patient feces for
Immunization with HBV vaccine protects against aggregated calicivirus-like particles using
subsequent deltavirus infection. monoclonal antibodies.
3. ELISA tests: for IgM and IgG anti-HEV.
Hepatitis E Virus (HEV) 4. Western blot assay: A Western blot assay for
IgM and/or IgG antiHEV.
Hepatitis E virus (HEV) has been provisionally
5. Polymerase chain reaction (PCR): Polymerase
classified in the genus Hepevirus under the family
chain reaction (PCR) assay for the detection of
Caliciviridae. HEV is a spherical nonenveloped
HEV RNA (as cDNA) in patient feces or in acute-
virus, 32–34 nm in diameter, with a single
phase sera.
stranded RNA genome. The surface of the virion
shows indentation and spikes. HEV (E-NANBH)
(The E stands for “Enteric” or “epidemic”) is Prophylaxis
predominantly spread by the feco-oral route, General measures: These depend on the mainten
especially in contaminated water. ance of a clean water supply, and generally
resemble those used to control HAV.
Clinical Features Immunization: Vaccines based on recombinant
The incubation period ranges from 2–9 weeks with antigens are under development, and show some
an average of six weeks. Most cases occur in the promise.
474 | Section 4: Virology
Hepatitis G Virus encloses an inner icosahedral 27 nm nucleocapsid
(core), which contains hepatitis B core antigen
Two flavivirus-like isolates were’ obtained in 1995
(HBcAg). Inside the core is the genome, a circular
from Tamarin monkeys inoculated with blood from double stranded DNA and a DNA polymerase
a young surgeon (GB) with acute hepatitis. It was Hepatitis B surface antigen (H BsAg) is also named
termed GB, the patient’s initials. A similar virus was as Australia antigen
isolated from another human specimen the same Mode of transmission: HBV is a blood borne
year. These isolates were called GB viruses A, B virus and there are three important modes of
and C respectively. transmission: 1. Parenteral transmission; 2.
Perinatal transmission; 3. Sexual transmission
In 1996, an isolate closely resembling GBV-C Laboratory diagnosis: Specific diagnosis of hepatitis
was obtained from a patient with chronic hepatitis. B rests on serological demonstration of the viral
This has been called hepatitis G virus (HGV). markers and can be carried out by detection of
Hepatitis G virus resembles HCV in many ways. HBsAg, anti-HBs, HBeAg, anti-HBe, IgM anti-HBc, IgG
HGV is a flavivirus, is transmitted in blood, and has anti-HBc and HBV DNA in the serum. These can be
detected by sensitive and specific tests like ELISA
a predilection for chronic hepatitis disease. It has not
and RIA. Molecular methods such as DNA: DNA
been grown, but its RNA genome has been cloned. hybridization and PCR are used for HBV DNA testing
The HGV RNA has been found in patients with are highly sensitive and quantitative
acute, chronic and fulminant hepatitis, hemophiliacs, Prophylaxis: General prophylaxis consists in
patients with multiple transfusions and hemodialysis, avoiding risky practices
intravenous drug addicts and blood donors. HGV Immunization: I. Passive immunization: Hyper
appears to be a blood borne virus resembling HCV. immune hepatitis B immune globulin (HBIG)
administered 1 M in a dose of 300–500 iu soon after
Its role in hepatitis is yet to be clarified. exposure to infection
The virus is present worldwide. Majority of the II. Active immunization: such as plasma-derived
individuals with HGV infection have no detectable hepatitis B vaccine, recombinant yeast hepatitis
evidence of liver disease. There have been, however, B vaccine (Three doses given at 0, 1 and 6 months
cases of acute, fulminant and chronic hepatitis constitute the full course), recombinant Chinese
hamster ovary (CHO) cell hepatitis vaccine,
where HGV is presently the only explanation for
synthetic peptide vaccines and hybrid virus vaccine
their liver disease. There is no evidence of a causal
Hepatitis C Virus
relationship between HGV infection and hepato
HCV can cause acute HCV infection, chronic HCV
cellular carcinoma. HGV infection results frequently infection, and cirrhosis and other complications
in chronic viraemia. It often subsides after several induced by hepatitis
years and anti-HGenv antibody develops. Blood or blood products and also organs of infected
patients are the major sources of infection
For diagnosis, antibody to HCV antigen can be
Laboratory Diagnosis detected by enzyme immunoassay
1. HGV is identified by detection of the genome by The HCV RNA can be amplified by RT-PCR
reverse transcriptase polymerase chain reaction Hepatitis D Virus
(RT-PCR) or other RNA detection methods. The hepatitis D virus (HDV) is unique in being an
2. An immunoassay has been developed to detect incomplete virus, that requires hepadnavirus helper
anti-HG env. Serum HGV RNA indicates viremia, functions for propagation in hepatocytes
Transmission of HDV occurs parenterally
whereas anti-HGenv is associated with recovery.
HDV superinfections in patie nts with HBV results in
Key Points chronic HDV infection
Vaccination with HBV vaccine protects against
At least six viruses, A through G (A, B, C, D, E and G.) subsequent HDV infection
are hepatitis viruses Hepatitis E virus (HEV)
All the hepatitis viruses are RNA viruses, except Hepatitis E virus is the primary cause of enterically
the hepatitis B virus (HBV), which is a DNA virus transmitted non-A, non-B hepatitis
belonging to the family Hepadnaviridae It usually causes an acute, self-limiting disease similar
Hepatitis A virus (HAV) to HAV. Pregnant women—high mortality associated
Hepatitis A virus (HAV) can be demonstrated in the with HEV
stool by immunoelectron microscopy (IEM). ELISA is Hepatitis G virus (HGV) is a flavivirus, is transmitted
the method for detection of IgM and IgG antibodies in blood, and has a predilection for chronic hepatitis
in the serum disease.
Prevention of HAV infection depends on: vaccines
containing formalin-inactivated HAV
Hepatitis B Virus
HBV is associated with hepatocellular carcinoma Important questions
Hepatitis B virus (HBV) is a double-walled spherical
1. Name the hepatitis viruses. Describe the morphology
structure and measures 42 nm in diameter (Dane
and antigenic structure of hepatitis B virus.
particle). The outer surface or envelope of virus
2. Classify hepatitis viruses. Discuss the laboratory
contains hepatitis B surface antigen (HBsAg). It
diagnosis of infections caused by hepatitis B virus.
Chapter 67: Hepatitis Viruses | 475
3. Write short notes on: 8. Which of the following viral markers is first marker to
a. Hepatitis A virus (HAV) or type A hepatitis or appear in biood after infection with hepatitis B virus?
infectious hepatitis. a. HBsAg
b. Hepatitis B virus or Dane’s particle. b. HBcAg
c. Hepatitis B surface antigen (HBsAg) or Austral c. HBeAg
ia antigen. d. Antibody to HBsAg
d. Draw a neat labeled diagram of hepatitis B virus. 9. Which of the following viral markers is/are positive
e. Hepatitis B virus markers. in simple carriers of hepatitis B?
f. Hepatitis C virus or type C hepatitis. a. HBsAg
g. Hepatitis D virus or Delta agent. b. HBeAg
h. Non-A, Non-B hepatits c. Both of the above
i. Hepatitis E virus d. None of the above
j. Hepatitis G virus 10. In super carriers of hepatitis B which of the following
k. Pathogenesis of HBV or type B hepatitis or viral markers is/are positive?
serum hepatitis or serum jaundice or transfu a. HBsAg
sion hepatitis. b. HBeAg
l. Laboratory diagnosis of type B hepatitis. c. Both of the above
m. Prophylaxis of hepatitis B or hepatitis B vaccine. d. None of the above
11. High infectivity of hepatitis B virus is indicated by
which of the following markers when positive?
Multiple choice questions (mcqs) a. HBsAg
1. Which of the following hepatitis viruses is DNA b. HBeAg
c. HBcAg
virus?
d. None of the above
a. Hepatitis A virus
12. For preparation of recombinant hepatitis B vaccine
b. Hepatitis B virus
which of the following hepatitis B genes are used?
c. Hepatitis C virus
a. HBsAg gene b. HBeAg gene
d. Hepatitis E virus
c. HBcAg gene d. All of the above
2. Which of the following hepatitis viruses can be
13. The carrier state in hepatitis B virus infection is
transmitted by sexual route?
demonstrated by:
a. Hepatitis A virus a. Persistent presence of HBsAg in blood for at
b. Hepatitis C virus’ least 6 months
c. Hepatitis E virus b. Persistent presence of H and Ab in blood for at
d. All of the above least 6 months
3. Which of the following hepatitis viruses may cause c. Persistent presence of HBeAg in blood for at
hepatic carcinoma? least 6 months
a. Hepatitis A virus d. Persistent presence of H and Ag in blood for at
b. Hepatitis C virus least 6 months
c. Hepatitis E virus 14. All the following statements are true for hepatitis C
d. Hepatitis G virus virus except:
4. Which of the following hepatitis viruses is non a. It is closely related to hepatitis D virus
enveloped? b. It is a leading cause of cirrhosis
a. Hepatitis A virus c. Blood transfusion is the most important mode
b. Hepatitis B virus of transmission of HCV
c. Hepatitis C virus d. A live vaccine is available against HCV
d. Hepatitis D virus 15. Which statement is true for hepatitis D virus:
5. All the following statements are true for hepatitis A a. An incomplete virus
virus (HA V) except: b. Related to hepatitis B
a. It is most commonly transmitted by fecal-oral c. A single-stranded DNA
route d. Transmitted by fecal-oral route
b. It causes chronic disease or fatal disease 16. Hepatitis E virus is:
c. t has a short incubation period of 3–4 weeks a. Related to hepatitis C virus
d. There is only one serotype of HAV b. Yet to be cultured
6. Which of the following methods is preferred for c. Associated with chronic liver infection
diagnosis of hepatitis A virus (HAV) infection? d. A major cause of hepatitis transmitted by blood
a. Alanine aminotransferase levels 17. Which of the following statement is true for hepatitis
b. Detection of IgM antibody to HAV by ELISA G virus (HGV):
c. Detection of IgG antibody to HAV by ELISA a. HGV is a flavivirus
d. Cell cultures for growing HAV b. Hepatitis G virus resembles hepatitis C virus
(HCV)
7. Which of the following antigens is present in
c. Hepatitis G virus is transmitted in blood
the envelope of hepatitis B virus?
d. All of the above
a. HBsAg
b. HBcAg Answers (MCQs)
c. HBeAg 1. b; 2. b; 3. b; 4. a; 5. b; 6. b; 7. a; 8. a; 9. a; 10. c; 11. b; 12.
d. None of the above a; 13. a; 14. d; 15. a; 16. b; 17. d
68
Chapter
Retroviruses: Human
Immunodeficiency Virus (HIV)
Learning Objectives
After reading and studying this chapter, you should ∙∙ D iscuss opportunistic infections associated with
be able to: human immunodeficiency virus infection
∙∙ Classify retroviruses. ∙∙ D escribe the laboratory diagnosis of human
∙∙ Describe morphology of human immunodeficiency immunodeficiency virus infection
virus ∙∙ D escribe the laboratory tests for detection of
∙∙ Describe the antigenic structure of human immuno specific antibodies in human immunodeficiency
deficiency virus (HIV) and draw labeled diagram of virus infection
HIV-I ∙∙ Describe strategies of human immunodeficiency
∙∙ Describe modes of transmission of human immuno virus testing
deficiency virus ∙∙ Describe the following: Antiviral therapy for HIV;
∙∙ Describe types of exposure and their relative risks postexposure prophylaxis.
B. Nonspecific Tests
Immunological Tests
Fig. 68.3: Diagrammatic representation of Western blot The following parameters help to establish the
test for HIV immunodeficiency in HIV infection.
484 | Section 4: Virology
a. Total leukocyte and lymphocyte count to tests are negative during the early stage of HIV
demonstrate leukopenia and a lymphocyte infection when the individual is infectious,
count usually below 2000/mm3. screening may not detect all dangerous donors
b. T cell subset assays. Absolute CD4+ T cell count but can still eliminate a large majority of them.
will be usually less than 200/mm3. T4:T8 cell Screening for p24 antigen can detect those in
ratio is reversed. the window period also.
c. Platelet count will show thrombocytopenia. A person found positive for HIV antigen or
d. Raised IgG and IgA levels. antibody should never donate blood or other
e. Diminished CMI as indicated by skin tests. biological materials. As the infection can be
f. Lymph node biopsy showing profound transmitted from mother to baby before, during
abnormalities. or after birth, antenatal screening is useful.
2. Seroepidemiology: Antibody surveys have been
C. Tests for Opportunistic Infections and most useful in identifying the geographical extent
Tumors of HIV infection and in other epidemiological
Apart from diagnosing HIV infection, the laboratory studies.
would be called upon to identify the opportunistic 3. Diagnosis: Serology is almost always positive
infections that are a feature of AIDS. Routine in persons with clinical features of AIDS. It
microbiological methods would suffice for this. may, however, be negative in acute illness
However, serological diagnosis of infections may and sometimes in the very late cases where
not always be reliable in AIDS as antibody formation the immune system is nonreactive. Routine
may be affected by the immune deficiency. serology may also be negative when the
infection is with a different AIDS virus. For
Applications of Serological Tests (Table example, HIV-2 infections are likely to be
68.11) missed if antibody testing is done with the HIV-l
antigen alone.
Serological tests for HIV infection are employed in
4. Prognosis: In a person infected with HIV,
the following situations:
loss of detectable anti-p24 antibody indicates
1. Screening: Screening is defined as the systematic
clinical deterioration. This is also associated
application of HIV testing, whether voluntary or
with HIV antigenemia and increased virus titer
mandatory, to entire populations or selected
in circulation.
target groups. It should be mandatory that all
donors of blood, blood products, semen, cells,
tissues and organs be screened. As antibody Laboratory Monitoring of HIV Infection
Some laboratory tests are important in monitoring
Table 68.11: Potential antiviral therapies the course of HIV infection.
for HIV infection 1. CD4+ T cell count: When the count falls below
500, it is an indication of disease progression
Nucleoside analog reverse transcriptase inhibitors
Azidothymidine (AZT) (Zidovudine) and the need for antiretroviral therapy. Counts
Dideoxycytidine (ddC) (Zalcitabine) below 200 denote risk of serious infections.
Dideoxyinosine (ddI) (Didanosine) 2. Direct measurement of HIV RNA: Direct
d4T (Stavudine) measurement of HIV RNA becomes necessary,
3TC (Lamivudine)
ABC (Abacavir) particularly in the course of treatment. This
is done usually by two methods, the reverse
Non-nucleoside reverse transcriptase inhibitors
Nevirapine (Viranune)
transcriptase PCR (RT-PCR) assay and the
Delavirdine (Rescriptor) branched DNA (bDNA) assay.
Efavirenz (Sustiva) 3. Beta-2-microglobulin and Neopterin: Beta-2-
Protease inhibitors microglobulin and Neopterin can be measured
Saquinavir (Invirase/Fortovase) in serum or urine. Their concentrations are
Ritonavir (Norvir) low in asymptomatic infection and rise with
Indinavir (Crixivan)
advancing disease.
Nelfinavir (Viracept)
Amprenavir (Agenerase)
Highly active antiretroviral therapy (HAART) Epidemiology
(combination) Routes of Transmission
Indinavir/AZT/3TC
Ritonavir/AZT/3TC HIV is spread only by three modes—sexual
Nelfinavir/AZT/3TC contact with infected persons; by blood and blood
Nevirapine/AZT/ddI products; and from infected mother to babies
Nevirapine/indinavir/3TC (intrapartum, perinatal, postnatal).
Chapter 68: Retroviruses: Human Immunodeficiency Virus | 485
Geographic distribution: HIV -1 infections are non-nucleoside reverse transcriptilse inhibitors, or
spreading worldwide, with the largest number protease inhibitors (Table 68.11). Other anti-HIV
of AIDS cases in sub-Saharan Africa but with drugs being developed
a growing number of cases in Asia, the United In the current guidelines, AZT is recommended
States, and the rest of the world. HIV-2 is more for the treatment of asymptomatic or mildly
prevalent in Africa (especially West Africa) than symptomatic people with CD4 counts of less than
in the United States. Heterosexual transmission is 500/µL and for the treatment of infected pregnant
the major means of spread of HIV-1 and HIV-2 in women to reduce the likelihood of transmission
Africa, HIV-2 produces a disease similar to but less of the virus to the fetus. Unfortunately, the high
severe than AIDS. mutation rate of HIV promotes the development
AIDS in the developing countries differs from of resistance to these drugs.
the disease in the western countries clinically too. A cocktail of several antiviral drugs (e.g. AZT,
In Africa, the major manifestation is pronounced 3TC, protease inhibitor) termed highly active
wasting so that it has been called the ‘slim disease’. antiretroviral treatment (HAART), each with
The high prevalence of tuberculosis and parasitic different mechanisms of action, has less potential to
infections complicate the clinical picture. breed resistance and has become a recommended
HIV infection in India: HIV infection was detected therapy. Multidrug therapy can reduce blood levels
rather late in India, the first cases having been of virus to nearly zero and reduce morbidity and
found in female sex workers in Madras (Chennai) mortality in many patients with advanced AIDS.
in 1986 and the first AIDS patient the same year Although HAART is a difficult drug regimen, many
from Bombay (Mumbai). Since then in every high patients return to nearly normal on this therapy.
risk group, the rate of infection has been mounting. Apart from specific antiretroviral therapy,
By the end of 2003, India is believed to have about other measures in the treatment of AIDS include
5 million HIV-infected people, the second largest (i) treatment and prophylaxis of opportunistic
such population after South Africa. infections and tumors (ii) general management
and (iii) immunorestorative measures.
Control
i. Sexual transmission: The best method of Postexposure Prophylaxis (Pep)
checking sexual transmission of infection is If an accidental exposure occurs, any wound
health education The use of condoms and should be washed with soap and water, or mucous
vaginal antiseptics could have an impact. membranes flushed with water. The accident must
ii. Exposure to blood: Drug injectors can avoid be reported so that, if necessary, prophylaxis can
risk by not injecting, or can reduce risk by be started as soon as possible. Knowledge of the
using only clean equipment. status of the source patient is essential.
iii. Mother to child transmission: This can be Exposure to blood, body fluid, other potentially
reduced by identifying infected mothers and infected material or an instrument contaminated
giving specific therapy in the later stages of with one of these materials may lead to risk of
pregnancy and to the baby after birth. All acquiring HIV infection. The risk of infection varies
women who have been potentially exposed with the type of exposure and other factors. Most
should seek HIV antibody testing before exposures do not result in infection. Health workers
becoming pregnant and, if the test is positive, are normally at very low risk of acquiring infection
should consider avoiding pregnancy. HIV during management of infected patients. Following
infected mothers should avoid breastfeeding exposure, postexposure prophylaxis (PEP) may be
to reduce transmission of the virus to their required depending upon the category of exposure
children. and HIV status of exposure source (Table 68.12).
Prophylaxis Basic PEP regimen consists of two drug
combination while expanded PEP regimen is a
The prevention of AIDS rests at present on general
combination of three drugs. Zidovudine 300 mg
measures, such as health education, identification
BD and Lamivudine 150 mg BD are used in basic
of sources and elimination of high risk activities. No
two drug regimen. In expanded three drug PEP
specific vaccine is available.
regimen, a protease inhibitor is added to this
Vaccine development: No vaccine against HIV is combination of drugs. Among protease inhibitors,
available despite several trials. lopinavir 400 mg BD or 800 mg OD or ritonavir 100
mg BD or 200 mg OD are preferred as third drug.
Treatment To be effective these drugs must be started within
The anti-HIV drugs approved can be classified as the first 72 hours and ideally within 2 hours. The
nucleoside analog reverse transcriptase inhibitors, PEP should be continued for a period of four weeks.
486 | Section 4: Virology
Table 68.12: PEP regimen according to exposure and status of source
Category of exposure HIV positive and HIV positive HIV staus not known
asymptomatic and clinically
symptomatic
i. Mild exposure (Mucous Consider two drug PEP regimen Start two drug PEP Usually no PEP or
membrane/non-intact skin with regimen consider two drug
small volumes). PEP regimen
ii. Moderate exposure (Mucous Start two drug PEP regimen Start three drug PEP Usually no PEP or
membrane/ nonintact skin with regimen consider two drug
large volume or percutaneous PEP regimen
superficial exposure with solid
needle)
iii. Severe exposure (Percutaneous Start two drug PEP regimen Start three drug PEP Usually no PEP or
with large volume) regimen consider two drug
PEP regimen
Both risk of infection and possible side-effects of Laboratory diagnosis of HIV infection includes specific
antiretroviral drugs should be carefully considered tests for HIV as well as tests for immunodeficiency.
when deciding to start PEP. Besides PEP, injured Specific tests include antigen (P24) detection, virus
isolation, detection of viral nucleic add and antibody
site on the wound should be thoroughly washed
detection
with soap and water. Antiseptics may also be used.
The p24 antigen is the earliest virus marker to appear
Therapy should be continued for 4 weeks and in the blood. HIV infected persons remain negative
the victim followed with testing for virus for the for antibodies during window period. Viral isolation,
next 6 months. Exposed persons should have post- detection of viral nucleic acid by polymerase chain
PEP HIV testing, at three months and at 6 months. If reaction (PCR) and p24 antigen detection are useful
for diagnosis in window period
the test at six months is negative, no further testing
HIV can be cultured by co-cultivation of lymphocytes
is required. with potentially infected and uninfected mono
Key Points nuclear cells
The diagnosis of HIV infection is made by detecting
Human immunodeficiency virus (HIV) causes AIDS, serum antibodies to viral proteins. There are two
belongs to retroviruses types of serological tests for anti-HIVantibodies are of
HIV is a spherical enveloped virus measuring up to two types—screening tests and supplementary tests
120 nm in diameter and consists of two identical Screening tests—ELISA, rapid test, and simple test
copies of single-stranded positive sense RNA (E/R/S) are usually highly sensitive tests
genome There are three strategies (strategy I to III) for HIV
The three important enzymes contained in the virion testing in India
are reverse transcriptase, protease and integrase.In Prevention and control include health education,
association with viral RNA is the reverse transcriptase screening of blood and blood products for HIV
enzyme antibodies or antigens, and infection control
Important structural components of the virus include Antiretroviral treatment (ART) is the mainstay in HIV
the surface antigen gp120, the transmembrane treatment
antigen gp41, the matrix protein p17 and the capsid
Postexposure prophylaxis (PEP) may be required
antigen p25
when there is exposure to blood, body fluid, other
The genome of HIV contains the three structural potentially infected material or an instrument
genes (gag, pol and env) characteristic of all contaminated with HIV.
retroviruses, as well as other nonstructural and
regulatory genes specific for the virus (tat, rev, nef,
vif, vpr and vpu). both Important questions
HIV shows two distinct antigenic types—HIV-1 and
HIV-2 1.
Describe the structure and laboratory
There are three modes of transmission of HIV diagnosis of human immunodeficiency virus.
infection. Sexual contact, parenteral and perinatal 2. Describe the antigenic structure of human
HIV infects principally the CD4 lymphocytes. immunodeficiency virus and draw labeled
The infection causes damage to T helper (T4)
lymphocytes. T4 cells are depleted in numbers and
diagram of HIV-1.
the T4:TB (helper: suppressor) ratio is reversed 3. Discuss the modes of transmission and
HIV causes acute infection, AIDS related complex pathogenesis of human immunodeficiency
(ARC), and AIDS virus.
When CD4+ cells fall below 200 per mm3, the titer 4. Write short notes on:
of virus increases markedly and there is irreversible a. Human immunodeficiency virus.
breakdown of immune defence mechanisms
b. Antigens of human immunodeficiency virus.
Chapter 68: Retroviruses: Human Immunodeficiency Virus | 487
c. Opportunistic infections associated with hu- 7. Which type of cells are most often infected by HIV?
man immunodeficiency virus infection. a. CD4+T lymphocytes
d. Strategies of human immunodeficiency virus b. CD8+T lymphocytes
testing. c. Null cells
e. Control HIV d. None of the above
f. Antiviral therapy for HIV 8. Which of the following opportunistic parasitic
infections is/are associated with HIV infection?
g. Postexposure prophylaxis
a. Toxoplasmosis
b. Cryptosporidiosis
Multiple choice questions (mcqs) c. Isosporiasis
d. All of the above
1. All of the following statements are true for the 9. Which of the following tests may be used for
viral genome in HIV except that they: diagnosis of HIV infection?
a. Are diploid a. P24 antigen detection
b. Consist of DNA-dependent DNA polymerase b. Virus nucleic acid detection
activity c. Antibody detection
Consist of three major genes gag, pol, and env;
c. d. All of the above
characteristic of all retroviruses 10. All of the following are the examples of screening
d. Are most complex of human retroviruses tests in HIV except:
2. What is the characteristic feature of viruses a. Western blot
b. ELISA
belonging to family Retroviridae?
c. HIV spot test
a. Presence of enzyme reverse transcriptase d. Latex agglutination test
b. Presence of envelope
11. Which of the following tests is/are confirmatory
c. Presence of nucleic acid
test for HIV infection?
d. Presence of lipid
a. Virus isolation.
3. Which structural genes are present in the genome b. Detection of p24 antigen.
of human immunodeficiency virus-I and not in c. Detection of viral nucleic acid.
immunodeficiency virus-2? d. All of the above.
a. gag gene 12. In HIV testing in India, the strategy I is used for:
b. pol gene a. Diagnosis of HIV infection in asymptomatic
c. env gene persons
d. All of the above b. Diagnosis of HIV infection in symptomatic per-
4. Which is the principal envelope spike antigen of sons
HIV-1? c. HIV surveillance
a. gp120 d. Ensuring safety of blood, organ, and tissue do-
b. gp140 nations
c. gp41 13. Which of the following antiviral agents has been
d. gp38 most widely used to inhibit HIV replication?
5. Which is the transmembrane pedicle antigen of a. Azidothymidine b. Zintevir
HIV-1? c. Nevirapine d. Indinavir
a. gp120 14. All of the following statements are true for highly
b. gp140 active antiretroviral therapy (HAART) except:
c. gp41 a. Inhibition of HIV replication
d. gp38 b. Improved efficacy of the therapy
c. Prevents emergence of drug resistance
6. HIV cannot be transmitted by: d. Minimize toxicity following thearpy)
a. Sexual contact
b. Kissing Answers (MCQs)
c. Intravenous drug abuse 1. b; 2. a; 3. d; 4 a; 5. c; 6. b; 7. a; 8. d; 9; d; 10. a; 11. d; 12.
d. Blood transfusion d; 13. a; 14. c
69
Chapter
Learning Objectives
After reading and studying this chapter, you should (CJD); Kuru; Bovine spongiform encephalopathy
be able to: (BSE) or mad cow disease; subacute sclerosing
∙∙ Describe slow virus and prion diseases panencephalitis (SSPE); progressive multifocal
∙∙ Describe the following: Creutzfeldt-Jakob Disease leukoencephalopathy (PML).
The virus can be grown in sheep choroid plexus ii. Transmissible mink encephalopathy: Trans
tissue cultures, from the CSF, blood and saliva of missible mink encephalopathy is a scrapie-
affected animals. like disease of farm-raised mink. It is believed
to have spread to mink by feeding them on
Maedi (Progressive Pneumonia) scrapie infected sheep meat.
Maedi (progressive pneumonia) is a slowly pro iii. Bovine spongiform encephalopathy (BSE)
gressive fatal hemorrhagic pneumonia of sheep, or mad cow disease:
with an incubation period of 2–3 years. A disease similar to scrapie, designated bovine
Visna and maedi (progressive pneumonia) spongiform encephalopathy (BSE), or “mad
viruses are closely related and are classified as cow disease,” emerged in cattle in Great
retroviruses (genus Lentivirus). Britain in 1986. This outbreak was traced to the
use of cattle feed that contained contaminated
Group B (Prion Diseases) bone meal from scrapie-infected sheep and
BSE-infected cattle carcasses.
Transmissible Spongiform Encephalopathies The epidemic of “mad cow disease” peaked
(Prion Diseases) in Great Britain in 1993.
Prion diseases of animals It is now accepted that the new variant forms
i. Scrapie: Scrapie is the prototype prion disease of CJD and BSE are caused by a common
and shows marked differences in susceptibility agent, indicating that the BSE agent had
of different breeds of animal. The incubation infected humans. Through 2002, of 129 people
period is about two years. The affected animals who had been diagnosed with new variant CJD
are irritable and develop intense pruritus, in England, 121 had died.
scraping themselves against trees and rocks, iv. Chronic wasting disease: A scrapie-like
hence the name scrapie. Emaciation and disease, designated chronic wasting disease, is
paralysis set in, leading to death. found in mule deer and elk in the United States.
The causative agent has been maintained in Human prion diseases
brain tissue explant cultures through several 1. Kuru: Kuru was a fatal neurologic disease
serial passages. with cerebellar signs exclusive to the Fore
490 | Section 4: Virology
tribes in Papua New Guinea. Kuru (means 3. Gerstmann–Strauss ler–Scheinker (GSS)
“shivering” or “trembling,”) was first described disease: It is rare neurologic diseases of humans
by Gajdusek and Zigas in 1957 as a mysterious due to prion-type agent.
disease seen only in the Fore tribe inhabiting 4. Fatal familial insomnia (FFI): It is also a rare
the eastern highlands of New Guinea. The dis neurologic diseases of humans are due to prion-
ease had an incubation period is 5–10 years type agent. In fatal familial insomnia there is loss
and led to progressive cerebellar ataxia and of ability to sleep and death within 1 to 2 years.
tremors, ending fatally in 3 to 6 months. A 5. Alzheimer’s disease: There are some neuro
total of 3700 cases occurred in a population of pathologic similarities between CJD and
35,000. Alzheimer’s disease. However, the disease has
Transmission from human to human was not been transmitted experimentally to primates
associated with ritual cannibalism involving or rodents, and the amyloid material in the
the consumption of the body of a dead member brains of Alzheimer’s patients does not contain
of the family after ritual nonsterilizing cooking. Prpsc protein.
When Gajdusek began his study, he noted
that women and children in particular were
the most susceptible to the disease, and he
Group C
deduced that the reasons were that the women i. Subacute Sclerosing Panencephalitis (SSPE)
and children prepared the food and they were
given the less desirable viscera and brains to Subacute sclerosing panencephalitis (SSPE) is an
eat. Their risk for infection was higher because extremely serious, very late neurologic sequela
they handled the contaminated tissue, making of measles. This disease occurs when a defective
it possible for the agent to be introduced measles virus persists in the brain and acts as a
through the conjunctiva or cuts in the skin, and slow virus. It has been reported that SSPE may
they ingested the neural tissue, which contains also develop as a very rare late complication of
the highest concentrations of the kuru agent. live measles virus vaccination. A similar picture
No cases have been seen in those born since has also been described as a rare complication of
1957, when cannibalism ceased. rubella infection.
Carlton Gajdusek was awarded the Nobel The SSPE is most prevalent in children who
Prize for Medicine in 1976 for his important were initially infected when younger than 2 years.
contributions on Kuru. The disease begins insidiously 5 to 15 years after a
case of measles. It is characterized by progressive
2. Creutzfeldt–Jakob disease(CJD): Creutzfeldt-
mental deterioration, involuntary movements,
Jakob disease is a rare chronic encephalopathy
muscular rigidity, and coma. Death occurs 1 to 3
of humans with associated dementia. CJD in
years after onset of symptoms.
humans develops gradually, with progressive
dementia, ataxia, and myoclonus, and leads to
death in 5–12 months. The disease was known ii. Progressive Multifocal
as both sporadic and inherited forms. leukoencephalopathy (PML)
The natural mode of transmission of Progressive multifocal leukoencephalopathy (PML)
Creutzfeldt–Jakob disease (GJD) is unknown, is a rare, degenerative CNS infection that usually
and it does not appear to be acquired from occurs in persons with severe immunodeficiency
sheep. Iatrogenic CJD has been transmitted disorders. The disease is characterized by
accidentally by contaminated growth hormone, memory loss, difficulty in speaking, and a loss of
by corneal transplant, by contaminated surgical coordination. Death occurs in 3–4 months.
instruments, and by cadaveric human dura
mater grafts. Causative agent: The causative agent is a human
A protein very similar to scrapie PrPsc is polyomavirus (JC virus), a member of the family
present in brain tissue infected with classic CJD. Polyomaviridae. PML has been seen in several
It has been speculated that the agent of CJD patients with AIDS-associated neurologic compli
was derived originally from scrapie-infected cations.
sheep and transmitted to humans by ingestion
of poorly cooked sheep brains. Key Points
An epidemic of mad cow disease in the United
Kingdom and the unusual incidence of CJD The term ‘slow virus disease’ is applied to a group
of infections in animals and human beings,
in younger people (younger than 45 years) characterized by a very long incubation period and
prompted concern that contaminated beef was a slow but relentless course, terminating fatally
the source of this new variant of CJD.
Chapter 69: Slow Virus and Prion Diseases | 491
d. Subacute sclerosing panencephalitis (SSPE)
Slow virus diseases are classified into: e. Progressive multifocal leukoencephalopathy
– Group A; such as visna and medi. (PML)
– Group B; prion diseases of the CNS (subacute
spongiform viral encephalopathies). Human
diseases include kuru, Creutzfeldt–Jakob disease Multiple choice questions (mcqs)
(CJD), Gerstmann–Straussler–Scheinker (GSS)
1. The prion-mediated disease in humans is
disease and fatal familial insomnia (FFI) Scrapie,
bovine spongiform encephalopathy (BSE) (mad cow a. Progressive multifocal leukoencephalopathy
disease), chronic wasting disease, and transmissible b. Subacute sclerosing panencephalitis
mink encephalopathy occur in animals. c. Gerstmann–Straussler–Scheinker syndrome
– Group C diseases include subacute sclerosing d. Bovine spongiform encephalopathy
panencephalitis and progressive multifocal 2. Variant Creutzfeldt–Jakob disease is transmitted by
leukoencephalopathy. a. Cuts in the skin
SSPE is a very rare delayed sequel to infection with
b. Transplantation of contaminated cornea
measles virus, many years after the initial infection.
Papova virus is responsible for PML. c. Ingestion of infected tissue
d. Blood transfusion
3. All the following are slow diseases transmitted by
Important Questions conventional viruses in humans except
a. Subacute sclerosing panencephalitis
1. Write briefly on slow virus diseases. b Progressive multifocal leukoencephalopathy
2. Write short notes on: c. Acquired immunodeficiency syndrome
a. Creutzfeldt–Jakob disease d. Creutzfeldt–Jakob disease
b. Kuru
c. Bovine spongiform encephalopathy (BSE) or Answers (MCQs):
mad cow disease 1. c; 2. d; 3. d
70
Chapter
Miscellaneous Viruses
LEARNING OBJECTIVES
After reading and studying this chapter, you should ∙∙ Describe structure of rotavirus
be able to: ∙∙ Discuss laboratory diagnosis of rotavirus infections
∙∙ Describe Rubella virus ∙∙ List the viruses causing diarrhea.
∙∙ Describe the following: Lassa fever; severe acute
respiratory syndrome (SARS)
CORONAVIRUSES
A group of spherical or pleomorphic enveloped RNA
viruses, carrying petal or club shaped peplomers on
their surface has been classified as coronaviruses
(Corona, meaning crown) resembling the solar
corona (Fig. 70.2). It has been classified in the
family coronaviridae with two genera: Torovirus
and Coronavirus.
Torovirus: The toroviruses are widespread in ungul
ates and appear to be associated with diarrheas.
Coronavirus: There seem to be two serogroups of
human coronaviruses. Coronaviruses of domestic
animals and rodents are included in these two
Fig. 70.1: Ebola virus (long thin filamentous form) groups. There is a third distinct antigenic group
Oncogenic Viruses
LEARNING OBJECTIVES
After reading and studying this chapter, you should ∙∙ Describe oncogenes and mechanism of viral
be able to: oncogenesis
∙∙ Describe oncogenic viruses ∙∙ List of viruses associated with human cancers.
∙∙ Classify oncogenic viruses
72
Chapter
General Properties, Classification and
Laboratory Diagnosis of Fungi
Learning Objectives
After reading and studying this chapter, you should be ∙∙ Classify fungi
able to: ∙∙ Describe laboratory diagnosis of fungal infections
∙∙ Differntiate between fungi and bacteria ∙∙ Discuss diseases caused by fungi.
B. Direct Microscopy
I. Potassium Hydroxide (KOH) Preparation
A portion of the treated specimen should be
examined microscopically to determine whether
hyphal elements are present. Most specimens
can be examined satisfactorily in wet mounts
after partial digestion of the tissue with 10–20%
potassium hydroxide. The alkali digests cells
and other tissue materials, enabling the fungus
elements to be seen clearly. Newer preparations
for the KOH test may provide easier and more
reliable results.
III. Gram-staining
It is used for the diagnosis of yeast infections of
mucous membranes.
C. Culture
1. Culture Media: The commonest culture media
used in mycology are Sabouraud’s dextrose agar
(SDA, pH 5.4), SDA with antibiotics, potato
dextrose or the slightly modified potato flakes
agar (PFA), and brain heart infusion (BHI) agar
with blood and antibiotics. The antimicrobials
usually included in SDA with antibiotics are
Fig. 72.4: Aerial spores
chloramphenicol and gentamicin to inhibit
bacterial growth and cycloheximide (actidione)
Laboratory Diagnosis to inhibit saprophytic fungi. The pH of Emmons’
modification of SDA is close to neutral and is
A. Collection and Processing of more efficient medium for primary isolation
Specimens than the original formulation.
The sampling procedures vary according to the 2. Incubation: Cultures are routinely incubated
area and type of tissue involved. Causative agents in parallel at room temperature 25°C (room
506 | Section 5: Medical Mycology
temperature for weeks) and at 37°C for days.
Cultures should be retained for at least 2-3
weeks before being discarded.
D. Identification of Fungi
Once an organism has grown, it is examined for
characteristic gross and microscopic structures,
so that identification can be made (Fig. 72.5).
1. Gross or macroscopic examination of
cultures: Growth characteristics useful for
identification are the rapidity of growth, colour
and morphology of the colony on the obverse
and pigmentation on the reverse.
2. Microscopic examination
i. Tease mount: For microscopic examination
slide mounts should be made in lactophenol Fig. 72.5: Asexual forms and asexual spores of fungi
cotton blue (LCB) from fungal colony (in
teased mounts or slide cultures) to study
the morphology of hyphae, spores and other Mycoses (Fungus Infections)
structures. Teased mounts are prepared Infection caused by fungus is known as mycosis
in lactophenol cotton blue (LCB) which (plural mycoses).
contains lactic acid, phenol and cotton blue.
Microscopic characteristics that should be Classification of mycoses
obtained as the following:
a. Septate versus sparsely septate hyphae Classification of fungal disease according to
b. Spores or conidia primary sites of infections is as follows (Table 72.1):
ii. Cellophane tape preparation: It should be A. Superficial mycoses: These infections are
read within 30 minutes and then discarded. limited to the outermost layers of the skin and
iii. Slide culture: Slide culture provides a hair. These include:
useful technique for demonstrating the a. Infection of skin caused by Malassezia
natural morphology of fungal structures. furfur (pityriasis versicolor) and Exophiala
3. Tests for the identification of molds: werneckii (tinea nigra), and
i. Hair perforation test: It is done to differtiate b. Infection of hair caused by Piedraia hortae
T. rubrum, from T. mentagrophytes,. (black piedra) and Trichosporon beigelii
ii. Urease test: It is done to help differentiate T. (white piedra).
mentagrophytes from T. rubrum B. Cutaneous mycoses: Infections that extend
iii. Thiamine requirement deeper into the epidermis as well as invasive
iv. Trichophyton agars hair and nail diseases.
v. Growth on rice grains.
4. Tests for the identification of yeasts Examples
Germ tube production, carbohydrate assimilation, a. Infection of skin, hair and nail caused by
chromogenic substrates, cornmeal agar, potassium dermatophytes.
nitrate assimilation, temperature studies and urease. b. Infection of skin, nail and mucous membrane
caused by C. albicans and other Candida
E. Serologic Tests specicies.
The most common tests for C. Subcutaneous mycoses: These infections
a. Fungal antibodies: involve the dermis, subcutaneous tissues,
1. Immunodiffusion; 2. countercurrent immuno- muscle, and fascia.
electrophoresis (CIE); 3. whole cell agglutination; Examples: Mycetoma, chromomycosis, sporotri
4. complement fixation; 5. Enzyme-linked chosis and rhinosporidiosis.
immunosorbent assay (ELISA). D. Systemic mycoses: Systemic mycoses-infections
b. Antigen detection: that originate primarily in the lung but that
1. Latex particle agglutination; 2. ELISA. may spread to many organ systems. Systemic
mycoses are caused by inhalation of airborne
F. Polymerase Chain Reaction (PCR) spores.
Polymerase chain reaction (PCR) for detection of Examples: Blastomycosis, histoplasmosis, cocci
fungal DNA in clinical material. dioidomycosis and paracoccidioidomycosis.
Chapter 72: General Properties, Classification and Laboratory Dignosis of Fungi | 507
Table 72.1 The major mycoses and causative fungi
Type of mycosis Mycosis Causative Fungal Agents
A. Superficial Pityriasis versicolor Malassezia species
Tinea nigra Hortaea werneckii
White piedra Trichosporon species
Black piedra Piedraia hortae
B. Cutaneous Dermatophytosis Microsporum species, Trichophyton species, and
Epidermophyton floccosum
Candidiasis of skin, mucosa, or nails Candida albicans and other candida species
C. Subcutaneous Sporotrichosis Sporothrix schencki
Chromoblastomycosis Phialophora verrucosa, Fonsecaea pedrosoi, others
Mycetoma Pseudallescheria boydii, Madurella mycetomatis, others
Phaeohyphomycosis Exophiala, bipolaris, exserohilum, and others
D. Systemic (prlmary, Coccidioidomycosis Coccidioides immitis, C. posadasii
endemic) Histoplasmosis Histoplasma capsulatum
Blastomycosis Blastomyces dermatitidis
Paracoccidioidomycosis Paracoccidioides brasiliensis
E. Opportunistic Systemic candidiasis Candida albicans and other candida species
Cryptococcosis Cryptococcus neoformans
Aspergillosis Aspergillus fumigatus and other aspergillus species
Mucormycosis (zygomycosis) Species of Rhizopus, Absidia, Mucor, and other
zygomycetes
Penicilliosis Penicillium marneffei
LEARNING OBJECTIVES
After reading and studying this chapter, you should ∙∙ Describe causative agets of ectothrix and endothrix
be able to: ∙∙ Describe the following: mycetoma; chromoblasto-
∙∙ List superficial, cutaneous and subcutaneous mycosis; sporotrichosis; rhinosporidiosis.
mycoses
A. SUPERFICIAL MYCOSES
These infections are limited to the outermost layers
of the skin and hair. These include:
a. Infection of skin: Caused by Malassezia furfur
(pityriasis versicolor) and Exophiala werneckii
(tinea nigra)
b. Infection of hair: Caused by Piedraia hortae
(black piedra) and Trichosporon beigelii (white
piedra).
a. Infection of Skin
Pityriasis Versicolor (Tinea Versicolor) Fig. 73.1: Morphology of pityriasis versicolor
Pityriasis versicolor (Tinea versicolor) is a chronic, Creamy colony develop within 5–7 days at 37°C.
usually asymptomatic, involvement of the stratum Lactophenol cotton blue mount of the colonies
corneum, characterized by discrete or confluent show budding yeast cells along with a number
macular areas of discoloration or depigmentation of bottleshaped cells. Hyphae are occasionally
of the skin. The areas involved are mainly the chest, seen in culture.
abdomen, upper limbs and back. The disease is
worldwide in distribution. Tinea Nigra
Causative agent: A lipophilic, yeast-like fungus Tinea nigra (or tinea nigra palmaris) is a localized
Pityrosporum orbiculare (Malassezia furfur). infection of the stratum corneum, particularly of
the palms, producing black or brownish macular
Laboratory Diagnosis lesions. It is caused by the dematiaceous fungus
A. Direct microscopy: The diagnosis is confirmed Hortaea (Exophiala) werneckii.
by direct microscopic examination of scrapings
of infected skin, treated with 10–20% KOH or Laboratory Diagnosis
stained with calcofluor white. Demonstration of A. Direct microscopy: Microscopic examination
clusters of the characteristic round yeast cells with of skin scrapings from the periphery of the
short, stout hyphae, which may be curved and lesion will reveal brownish, branched, septate
occasionally branched, is diagnostic (Fig. 73.1). hyphae and budding cells (Fig. 73.2).
B. Culture: The fungus can be grown on Sabour B. Culture: On Sabouraud agar the fungus
aud’s agar, covered with a layer of olive oil. develops as grey, yeast-like colonies, which
M. canis
M. canis forms numerous thick-walled, 8–15 celled
macroconidia with curved or hooked spiny tips. A
Fig. 73.3: Characteristic macroconidia of dermatophytes. (1) lemon-yellow or yellow-orange pigment develops
Cylindrical in Trichophyton; (2) Fusiform in Microsporum; and on the reverse of the colony. It is zoophilic.
(3) Club-shaped in Epidermophyton
M. gypseum
characters, such as spiral hyphae, racquet mycelium M. gypseum colonies grow rapidly producing a flat,
and favic chandeliers (Fig. 73.4). spreading, powdery surface that is cinnamonbuff to
brown which consists of abundant macroconidia.
Important Species They are boat-shaped, rough walled with 4 to 6
T. rubrum, T. mentagrophyte, T. tonsurans, T. septa. It is geophilic.
schoenleinii, T. violaceum.
Pathogenicity
T. rubrum: The typical colony of T. rubrum has a Dermatophytes grow only on the keratinised
white, cottony surface and a deep red, nondiffusible layers of the skin and its appendages and do not
pigment when viewed from the reverse side of the ordinarily penetrate the living tissues. Lesions vary
colony. The microconidia are small and piriform considerably according to the site of the infection
(pear-shaped) and clubshaped thinwalled macro and the species of fungus involved.
conidia (scanty).
Dermatophytids (or ‘id’ reaction): Hypersensitivity
T. mentagrophyte: T. mentagrophyte has grape- to fungus antigens may play a role in pathogenesis
like clusters of microconidia (Fig. 73.4) and cigar and is probably responsible for the sterile
shaped macroconidia.It has no red pigment and vesicular lesions, sometimes seen in sites distant
urease positive. Hair perforation test is positive. from the ringworm. These lesions are called
T. tonsurans: T. tonsurans produces a flat, dermatophytids (or ‘id’ reaction). Hypersensitivity
powdery to velvety colony on the obverse surface can be demonstrated by skin testing with the fungus
that becomes reddish-brown on reverse. The antigen, trichophytin.
microconidia are mostly elongate (Fig. 73.4). Types of hair infections: Three types of hair
infection can be seen in 10% KOH wet mounts:
Microsporum 1. Ectothrix: In this, the hyphae are sparsely
Colonies are cotton-like, velvety or powdery, with distributed within hair shaft with a sheath
white to brown pigmentation. Microconidia are of arthrospores on the surface of hair shaft.
relatively scanty and are not distinctive. Macroconidia It is caused by M. audouinii, M. canis and T.
are the predominant spore form. They are large, mentagrophytes (Fig. 73.5A).
multicellular, spindle shaped structures, borne singly 2. Endothrix: In this, the arthrospore formation
on the ends of hyphae. Both types of conidia are occurs entirely within the hair completely filling
borne singly in these genera. Microsporum species the hair shaft. This is caused by T. tonsurans and
infect only hair and skin but usually not the nail. T. violaceum (Fig. 73.5B).
Important species are M. audouinii, M. canis, 3. Favus: In this, there is sparse hyphal growth and
M. gypseum formation of air spaces within the hair shaft.
This is caused by T. schoenleinii (Fig. 73.5C).
M. audouinii
M. audouinii rarely forms conidia in the culture but Clinical Findings
many thick-walled chlamydospores (chlamydo Dermatophyte infections were mistakenly termed
conidia) may be present. It is anthropophilic. ringworm or tinea. Table 73.1 lists the clinical
Diagnosis can be established by demonstration on delicate sterigmata (Fig. 73.7). Conidia are
of these sclerotic bodies in KOH mounts or in also produced along the sides of the hyphae.
sections, and by culture on Sabouraud’s agar. C. Serology: A latex agglutination test is of value
B. Culture: Culture on Sabouraud agar at 25–30°C for the diagnosis of the extracutaneous forms of
yields slow-growing, greenish grey to black, sporotrichosis.
compact, folded colonies. Cultures should be D. Skin test: A skin test with sporotrichin antigen
incubated for 4–6 weeks. is positive in almost all patients with cutaneous
sporotrichosis.
Phaeohyphomycosis E. Animal inoculation: Rats are highly susceptible
Phaeohyphomycosis is a term applied to infections and can be infected by intraperitoneal or
characterized by the presence of darkly pigmented intratesticular inoculation.
septate hyphae in tissue. The sites of lesions may be
cutaneous, subcutaneous, deeper tissues, or organs 4. Rhinosporidiosis
like the brain or lung. Sclerotic cells or granules are Rhinosporidiosis is a chronic granulomatous
not found. The fungi appear in lesions as distorted disease characterized by the development of large
hyphal strands. polyps or wart-like lesions in the nose, conjunctiva
and occasionally in ears, larynx, bronchus, penile
3. Sporotrichosis urethra, vagina, rectum and skin. More than 90% of
Sporotrichosis is a chronic, pyogenic granulomatous the cases have been reported from India, Sri Lanka
infection of the skin and subcutaneous tissues and South America.
which may remain localized or show lymphatic
Etiology: The causative fungus Rhinosporidium
spread. The disease is worldwide and is rare in
seeberi.
Europe.
Causative agent: Sporothrix schenckii, a saprophyte Mode of infection: The mode of infection is not
in nature. known, though infection is believed to originate
from stagnant water or aquatic life. It is believed
Pathogenesis that fish may be the natural hosts of R. seeberi.
The fungus is a saprophyte found widely on plants,
thorns and timber. Infection is acquired through Laboratory Diagnosis
thorn pricks or other minor injuries. It has not been cultured and animal inoculation is
also not successful.
Laboratory Diagnosis
Diagnosis is made by culture as frequently the Demonstration of Sporangia
fungus may not be demonstrable in pus or tissues. Diagnosis depends on the demonstration of
A. Microscopic examination: Direct microscopy sporangia. Direct examination of the surface of
is of little value. the polypoid growth and histologic examination
B. Culture: SDA or blood agar are the media used. are the only ways to make a diagnosis. R. seeberi
S. schenckii is a dimorphic fungus occurring can be identified in hematoxylin and eosin-stained
in the yeast phase in tissues and in cultures at sections, but sometimes one may need special
37°C, and in the mycelial phase in nature and stains. Histologically, the lesion is composed of
in cultures at room temperature. The septate large numbers of fungal spherules embedded in
hyphae are very thin (1-2 mm diameter) and a stroma of connective tissue and capillaries. The
carry flower-like clusters of small conidia borne spherules are 10–200 mm in diameter and contain
IMPORTANT QUESTIONS
1. Discuss the morphology, cultural characters and
Fig. 73.8: Rhinosporidiosis: Sporangium with numerous pathogenicity of dermatophytes.
endospores 2. Describe the etiology, pathogenesis and laboratory
diagnosis of mycetoma.
thousands of endospores (6–7 μm in diameter) 3. Discuss different kinds of subcutaneous mycoses.
(Fig. 73.8). These spores develop into new 4. Write short riotes on:
sporangia when released. a. Superficial mycoses
b. Dermatophytes
5. Subcutaneous Phycomycosis c. Mycetoma or Madura foot or maduramycosis
In this condition, a painless subcutaneous nodule d. Chromoblastomycosis or verrucous dermatitis
develops which enlarges to involve a whole limb e. Sporotrichosis
or large areas of the body. f. Rhinosporidiosis.
Causative agent: is Basiodiobolus haptosporus,
a saprophytic phycomycete. MULTIPLE CHOICE QUESTIONS (MCQs)
Key Points 1. Black piedra is a superficial infection of the hair
caused by:
Superficial Mycoses a. Malassezia furfur
Pityriasis versicolor or tinea versicolor is caused by
b. Cladosporium werneckiib
Malassezia furfur (Pityrosporum orbiculare)
Tinea nigra is an infection of keratinized layer of skin
c. Trichosporon beigelli
caused by Hortaea (Exophiala) werneckii d. Piedraia hortae
Black piedra is a superficial infection of the hair 2. All the following are anthropophilic dermatophytes
caused by Piedroiohortae, a dematiaceous fungus except:
White piedra is an infection of the hair caused by a. Trichophyton violaceum
yeast-like organism, Trichosporon beigelii b. Trichophyton rubrum
Cutaneous Mycoses c. Epidermophyton floccosum
The dermatophytes infect only superficial keratinized d. Microsporum audouinii
structure such as skin, hair and nail but not deeper 3. Urease test is positive by:
tissues
a. Trichophyton violaceum
Dermatophytes belong to three genera: Trichophyton,
Microsporum and Epidermophyton
b. Trichophyton rubrum
Clinical findings: Dermatophyte infections were c. Trichophyton mentagrophytes
mistakenly termed ringworm or tinea because of d. Epidermophyton floccosum
the raised circular lesions 4. The major causative agent of mycetoma in India is:
Laboratory diagnosis is based on microscopy and a. Actinomadura madurae
culture. The differentiation of the three genera is b. Streptomyces somaliensis
based mainly on nature of macroconidia c. Nocardia brasiliensis
Subcutaneous Mycosis d. Actinomyces pelletieri
Mycetoma is a chronic, granulomatous infection
5. The sclerotic bodies are useful for diagnosis of:
of the skin, subcutaneous tissues, fascia and bone,
a. Sporotrichosis b. Mycetoma
which most often affects the foot or the hand
It can be divided into three types, eumycetomas,
c. Chromoblastomycosis d. Rhinosporidiosis
actinomycetomas and botryomycosis 6. Which of the following fungi has not been cultured:
Chromoblastomycosis or chromomycosis is caused a. Sporothrix b. Rhinosporidium
by fungi collectively called dematiaceous fungi c. Blastomyces d. Acremonium
Sporotrichosis is caused by Sporothrix schenckii, a
saprophyte in nature
ANSWERS (MCQs)
1. d; 2 a; 3. c; 4. a; 5. c; 6. b
Systemic Mycoses
Learning Objectives
After reading and studying this chapter, you should ∙∙ Descrbe histoplasmosis
be able to:
∙∙ List of fungi causing systemic infections
Introduction
These are the include blastomycosis, histoplasmosis,
coccidioidomycosis and paracoccidioidomycosis.
1. Blastomycosis
Blastomycosis is a chronic infection of the lungs
which may spread to other tissues, particularly
skin, bone and genitourinary tract.
It is caused by Blastomyces dermatitidis, a
dimorphic fungus. The disease has been called Fig 74.1: Blastomyces dermatitidis: yeast and mycelial
North American blastomycosis, because it is forms
endemic and most cases occur in the United States filamentous with septate hyphae and many round or
and Canada. oval conidia, and in older cultures, chlamydospores
also.
Pathogenesis
Laboratory Diagnosis
Soil is considered to be the source of infection,
which is acquired by inhalation. Human infection A. Specimens: Sputum or pus, exudates, urine
is initiated in the lungs. and biopsy lesions.
B. Microcopic examination: Potassium hydroxide
1. Asymptomatic
(10%) mount of specimens may show char
2. Chronic pneumonia acteristic thick-walled yeast cells with a single
3. Disseminated diseases: They form multiple broadbased bud.
abscesses in various parts of the body. C. Culture: Colonies usually develop on Sabouraud’s
4. Cutaneous blastomycosis: The cutaneous dextrose agar or enriched blood agar at 30°C
disease is usually on the skin of the face or other within 2 weeks.
exposed parts of the body. D. Serology: Antibodies can be measured by
complement fixation (CF) test, immunodiffusion
Morphology (ID) tests and enzyme immunoassay (EIA).
Blastomyces dermatitidis is a dimorphic fungus. In Treatment: Severe cases of blastomycosis are
tissue and in cultures at 37°C, the fungus appears treated with amphotericin B.
as budding yeast cells, which are large (7–20 μm)
and spherical, with thick, double-contoured walls. 2. Paracoccidioidomycosis
Each cell carries only a single broad-based bud This is a chronic granulomatous disease of the skin,
(Fig. 74.1). At room temperature, the culture is mucosa, lymph nodes and internal organs. As the
516 | Section 5: Medical Mycology
A B
Figs 74.3A and B: Coccidioides immitis: (A) Arthrospores
formation; (B) Spherule formation with endospores
Fig. 74.4: H. capsulatum: yeast cells in macrophage Fig. 74.5: H. capsulatum: mycelial form
518 | Section 5: Medical Mycology
identical to H. capsulatum in its mycelial phase but Important Questions
differs in the yeast phase both in vivo and in vitro 1. Enumerate dimorphic fungi. Describe the morphology,
and H. duboisii is morphologically identical to H. pathogenicity and lab diagnosis of dimorphic fungi.
capsulatum in its mycelial phase but the yeast phase 2. Write short notes on:
has larger cells (12–15 μm diameter). a. Blastomycosis
African histoplasmosis is mainly restricted to b. Coccidioidomycosis or Coccidioides immitis
c. Histoplasmosis or Darling’s disease
the continent of Africa. It involves mainly the skin,
d. Paracoccidioidomycosis or Paracoccidioides
subcutaneous tissues and bones. The lungs are not brasiliensis
commonly affected and the disseminated disease
is infrequent. Multiple choice questions (MCQs)
1. All the following statements are true for spherule
of Coccidioides immitis except:
Key Points
a. It is infective stage of the fungus
Blastomycosis: Blastomycosis is caused by Blasto b. It reproduces byendosporulation
myces dermatitidis, a dimorphic fungus. It is also c. It contains endospores
known as North American blastomycosis. d. It is not found in culture
Coccidioidomycosis is primarily an infection of the
2. Which of the following fungi infects reticuloendo
lungs caused by Coccidioides immitis, a dimorphic thelial system?
fungus. Infection is acquired by inhalation of dust a. Aspergillus fumigatus
containing arthrospores of the fungus. b. Histoplasma capsula tum
c. Trichophyton rubrum
Histoplasmosis: It is primarily a disease of reticulo
d. All the above
endothelial system caused by an intracellular fungus
3. The diagnostic form of Histoplasma capsulatum is:
Histoplasma capsulatum, a dimorphic fungus.
a. Arthrospore b. Spherule
H. capsulatum causes acute pulmonary histoplasmosis, c. Macroconidia d. Microconidia
chronic pulmonary histoplasmosis, and progressive
disseminated histoplasmosis. Answers (MCQs)
1. a; 2. b; 3. c
75
Chapter
Opportunistic Mycoses
LEARNING OBJECTIVES
After reading and studying this chapter, you should ∙∙ Discuss cryptococcosis
be able to: ∙∙ Discuss laboratory diagnosis of Cryptococcus
∙∙ Describe diseases caused by Candida albicans neoformans
∙∙ Describe the following: thrush or oral thrush; Germ ∙∙ Describe species of Asperillus
tube test or Reynaulds–Braude phenomenon ∙∙ Describe the following: Aspergillosis; mucormycosis;
∙∙ Discuss laboratory diagnosis of candidiasis pneumocystosis; otomycosis; mycotic keratitis.
∙∙ List opportunistic fungi
Morphology
In culture or tissue, Candida species grow as oval, Fig. 75.1: Sputum specimen illustrating budding yeast cells
budding yeast cells (3–6 μm in size). They also form and pseudohyphae of Candida species
Fig. 75.2: Candida albicans: yeast form and pseudohyphae Fig. 75.3: Gram stained smear of Candida albicans
Fig. 75.4: Candida albicans showing germ tubes Fig. 75.5: Formation of chlamydospores by Candida
albicans when cultured on cornmeal agar at 25°C
Treatment
Management of candidosis is mainly by removing the
predisposing causes. All Candida strains are sensitive
to Nystatin. Amphotericin B, 5-fluorocytosine
and clotrimazole may be used for disseminated
candidosis.
Cryptococcosis
Cryptococcosis (torulosis, European blastmycosis,
Busse–Buschke disease) is subacute or chronic
infection caused by the capsulate yeast Cryptococcus
neoformans. It is most frequently recognized as a
disease of the central nervous system (CNS), although
the primary site of infection is the lungs. The disease
Fig. 75.6: Cryptococcus neoformans: India ink preparation of
occurs sporadically throughout the world but it is now spinal fluid showing yeast cells surrounded by a large capsule
seen most often in patients with AIDS.
do not appear to become infected, probably
Morphology because of their high body temperature. In addition
In culture, C. neojormans produces a whitish mucoid to patients with AIDS or hematologic malignancies,
colony in 2–3 days. Microscopically, in culture patients being maintained on corticosteroids are
or clinical material, C. neoformans is a spherical highly susceptible to cryptococcosis.
budding yeast (5–10 μm in diameter), surrounded
by a thick polysaccharide capsule (Fig. 75.6). Pathogenesis
Infection is usually acquired by inhalation but may
Serotypes sometimes be through skin or mucosa. The primary
Adsorbed antisera have defined five serotypes pulmonary infection may be asymptomatic or may
(A–D and AD) and three varieties—C. neoformans mimic an influenza-like respiratory infection, often
vargrubii (serotype A), C. neoformans var neofor resolving spontaneously. Pulmonary cryptoccoc
mans (serotype D), and C. neoformans var gattii osisis may lead to a mild pneumonitis. In patients
(serotype B or C). Most infections are caused by C. who are compromised, the yeasts may multiply
neoformans var. neoformans, which is commonly and disseminate to other parts of the body but
found in the excreta of wild and domesticated birds preferent ially to the central nervous system,
throughout the world. causing cryptococcal meningoencephalitis. Other
common sites of dissemination include the skin,
Epidemiology eye, and prostate gland.
Cryptococcosis is worldwide in distribution.
Bird droppings (particularly pigeon droppings) Cryptococcal meningitis
enrich for the growth of C. neoformans and serve Cryptococcal meningitis is the most serious type
as a reservoir of infection. The organism grows of infection and can resemble tuberculous or other
luxuriantly in pigeon excreta. The birds themselves chronic types of meningitis.
Fig. 75.8: Zygomycetes: (A) Mucor; (B) Rhizopus; (C) Absidia Fig. 75.9: Penicillium
Morphology
Treatment
P. carinii has three stages:
Treatment consists of aggressive surgical debride Trophozoites thinwalled (1–4 μm).
ment, rapid administration of amphotericin B, and Precyst: It is 5–8 μm.
control of the underlying disease.
Cysts: Cysts are thick-walled and spherical (4–6 μm)
and contains 4 to 8 nuclei.
Penicilliosis
Pathogenesis
There are more than 150 known species of the
genus Penicillium. Infections are caused by P. jiroveci is normally a commensal in the lung, spread
Penicillium marnefei. by respiratory droplets. In immunocompetent
individuals, infection is asymptomatic. However,
Pathogenesis in inimunocompromised patients, serious life-
threatening pneumonia can develop (Fig. 75.10).
It causes penicillosis, keratitis, otomycosis and Until the AIDS epidemic, human disease was
rarely deep infections. Penicillium marneffei causes confined to interstitial plasma cell pneumonia
serious disseminated disease with characteristic in malnourished or premature infants and
papular skin lesions in AIDS patients in South- immunosuppressed patients. Since the early 1980s,
East Asia. Cutaneous lesions and subcutaneous it has remained one of the primary opportunistic
abscesses have been reported. infections found in patients with AIDS.
The multiplication of the parasite in the lungs
Identification induces a hyaline or foamy alveolar exudate. In
Fungi belonging to this genus are characterized by stained sections, the exudate filling the alveoli
producing conidiophores at the tips of branching shows a characteristic honeycomb pattern. Chest
septate hyphae, which in turn may produce radiographs may be normal or show a diffuse
secondary structures termed metulae, from which interstitial infiltrate.
flask-shaped structures called phialides bearing
smooth or rough shaped conidia are produced in Laboratory Diagnosis
chains, giving the entire structure a brush-like or 1. Specimens: Traditionally, diagnosis was made
broom-like appearance (Fig. 75.9). by finding the cyst or trophozoite in tissue
Mycotoxicosis
Learning Objectives
After reading and studying this chapter, you should ∙∙ Describe the following: mycotoxicosis; aflatoxin
be able to:
∙∙ Describe mycotic poisoning
77
Chapter
Learning Objectives
After reading and studying this chapter, you should ∙∙ L ist organisms present as normal microbial flora
be able to: in normal flora of upper respiratory tract and
∙∙ Describe the role of normal flora in a human body gastrointeinal tract
Infective Syndromes*
Learning Objectives
After reading and studying this chapter, you should ∙∙ List microbial pathogens that cause pneumonia.
be able to: ∙∙ Define diarrhea and dysentery
∙∙ Define bacteremia, septicemia, pyemia and ∙∙ List causative organisms of diarrhea and dysentery
endotoxemia ∙∙ Discuss laboratory diagnosis of dirrhea
∙∙ List causative organisms of septicemia, infective ∙∙ Discuss laboratory diagnosis of dysentery
endocarditis and subacute endocarditis ∙∙ List causative organisms of food poisoning
∙∙ Discuss laboratory diagnosis of subacute endocarditis ∙∙ Discuss the laboratory diagnosis of food poisoning
∙∙ List causative organisms of meningitis ∙∙ List microbial pathogens that cause pneumonia
∙∙ Discuss the laboratory diagnosis of acute pyogenic ∙∙ List causative organisms of (i) sexually transmitted
meningitis diseases (STDs); (ii) painless genital ulcer; painful
∙∙ Describe characterstics features of cerebrospinal genital ulcer; urethral discharge
fluid (CSF) in different forms of meningitis ∙∙ Discuss the laboratory diagnosis of gonorrhea
∙∙ List causative organisms of urinary tract infection ∙∙ Describe the following; Non-gonococcal urethritis
∙∙ Discuss the laboratory diagnosis of urinary tract (NGU)
infection ∙∙ Discuss the laboratory diagnosis of syphilis
∙∙ Describe characterstics features of cerebrospinal ∙∙ List causative organisms of wound infection
fluid (CSF) in different forms of meningitis ∙∙ Discuss the laboratory diagnosis of wound infection
∙∙ Describe the following: aseptic meningitis; tuber ∙∙ List causative organisms of pyrexia of unknown origin
culous meningitis (PUO)
∙∙ List causative organisms of sore throat ∙∙ Discuss the laboratory diagnosis of pyrexia of
∙∙ Discuss the laboratory diagnosis of sore throat unknown origin (PUO)
*This chapter was contributed by Dr Savita Kumari, Professor, Department of Internal Medicine, Postgraduate
Institute and Medical Research (PGIMER), Chandigarh-160 012, India.
534 | Section 6: Miscellaneous
Table 78.1 Causative organisms of septicemia Table 78.3 Causative agents of subacute endocar
ditis
A. Gram-negative bacilli (60–70% cases)
Salmonella Typhi (A) Bacteria
S. Paratyphi A Viridans group of streptococci- responsible for 60–80%
S. Paratyphi B of cases?
S. Paratyphi C Streptococcus sanguis
Brucella spp. Str. mutans
Haemophilus influenzae Str. mitis
Escherichia coli Enterococcus faecalis
Klebsiella pneumoniae Staph. epidermidis
Proteus spp. Coxiella burnetii
Enterobacter spp. Chlamydia psittaci
Bacteroides spp. (B) Fungi
Pseudomonas spp. Candida albicans
B. Gram-positive bacilli Aspergillus spp.
Listeria monocytogenes
C. Gram-negative cocci
Neisseria meningitidis vegetations comprising of dense fibrin, platelet
D. Gram-positive cocci (20–40% cases) aggregates with bacterial colonies are formed.
Staphylococcus aureus It runs a chronic course. It is more common
Staph. epidermidis and comprises of almost 70% cases of bacterial
Streptococcus pyogenes
Str. pneumoniae
endocarditis. Causative agents of subacute
bacterial endocarditis are shown in Table 78.3.
2. Meningitis
Meningitis is an inflammation of the membranes 1. Purulent meningitis (acute
surrounding the brain and spinal cord. Most cases pyogenic meningitis)
of meningitis fall into one of the two categories:
purulent meningitis and aseptic meningitis. The In purulent meningitis, the CSF is typically turbid
causative agents of these types are given in Table due to the presence of large numbers of leukocytes,
78.4. from 100 to several thousands/mm3, most of which
536 | Section 6: Miscellaneous
Table 78.4 Causative agents of purulent and B. Laboratory Examination of CSF for Cells
aseptic meningitis and Microorganisms
Purulent meningitis Aseptic meningitis 1. Naked eye examination: Naked eye examin
• Neisseria meningitidis A. Viruses ation is done for the presence of turbidity and
• Streptococcus • Enteroviruses any sign of contamination with blood from the
pneumoniae (echoviruses, polioviruses, puncture wound.
• Haemophilus coxsackieviruses)
2. Cell count: The leucocytes in the CSF are
influenzae • Mumps
• Escherichia coli • Herpes simplex counted by microscopical observation. The
• Group B streptococci • Varicella-zoster relative numbers of polymorphs and lympho
• Pseudomonas • Measles cytes should be noted, and the number of
• Salmonellae • Adenoviruses erythrocytes in specimens contaminated with
• Staphylococcus aureus • Arboviruses
blood (Table 78.5).
• S. epidermidis B. Spiral bacteria
• Listeria • Syphilis (Treponema A wet film of centrifuged CSF deposit mixed
monocytogenes pallidum) with India ink will, when examined under oil-
• Klebsiella • Leptospira immersion, demonstrate the characteristic
• Anaerobic cocci Interrogans serovars capsulate yeast cells of Cryptococcus neofor
• Bacteroides Icterohaemorrhagiae and
mans and a wet film examined on a warm
In neonates and canicola
infants C. Other bacteria stage will show the slowly motile trophozoites
E. coli Tuberculosis (Mycobacterium of Acanthamoeba (Hartmanella) or Naegleria.
Group B streptococci tuberculosis)
Pseudomonads, Partially treated with Dilution
salmonellae antibiotics
Staph. aureus Brain abscess
CSF that is clear or only slightly turbid should be
H. influenzae D. Fungi examined undiluted but when the specimen is
Listeria monocytogenes Cryptococcus neoformans highly turbid and its cell count very high, it may
Streptococcus Candida albicans be necessary to dilute it 1 in 10 or 1 in 100 before
pneumoniae E. Protozoa examination.
Klebsiella spp. • Acanthameba
• Naegleria
• Toxoplasma gondli Gram Film of CSF
F. Noninfective After taking some CSF for the cell count, the
Lymphoma remaind er should be centrifuged to deposit
Leukemias
Metastatic and primary
any cells and bacteria and a film of the deposit
neoplasms should be stained by Gram’s method. The finding
Collagen-vascular disease of bacterial forms resemb ling meningococci,
pneumococci, haemophili, coliform bacilli,
streptococci or listeriae be reported.
The supernatant from the centrifuged CSF should
are polymorphs. The majority of cases are caused be tested for its content of glucose and protein.
by one or other of three bacteria: Meningococcus,
Pneumococcus and Haemophilus influenzae. In Culture
neonates and infants, coliform bacilli, group B i. CSF Culture
streptococci and, less commonly, pseudomonads, Immediately after centrifugation of the CSF and the
salmonellae and Listeria monocytogenes may be the removal of some of the deposit for the Gram film, the
cause (Table 78.4). remainder of the deposit should be seeded heavily
on to culture media, blood agar and chocolate agar
Laboratory Diagnosis for incubation in humid air plus 5–10%, CO2 and
A. Collection of Specimens a-tube of cooked-meat broth. A further blood agar
The principal specimen to be examined is of CSF plate should be seeded for incubation for 2–5 days
collected by lumbar puncture under strict aseptic in an anerobic atmosphere with 5-10% CO2.
conditions. Only 3–5 mL of fluid should be collected The cultures should be inspected after overnight
in a fresh sterile screw-capped containers. The incubation. The isolated organisms are identified
specimen must be dispatched to the laboratory as by colony morphology, Gram staining from
quickly as possible, for delay may result in the death colonies, biochemical reactions and/or serological
of delicate pathogens, such as meningococci, and the tests. Tests with appropriate antibiotics should be
disintegration of leukocytes. It should not be kept in a done on the isolate.
refrigerator, which tends to kill H. influenzae. If delay If no growth is apparent after overnight
for a few hours is unavoidable, the specimen is best incubation, the plates should at once be reincubated
kept in an incubator at 37°C. for another day and then again inspected for growth.
Chapter 78: Infective Syndromes | 537
Table 78.5 Typical CSF findings in different types of meningitis
CSF in
Characteristic Normal CSF Acute pyogenic Tuberculous meningitis Viral meningitis
meningitis
I. Pressure Normal Highly increased Moderately increased Slightly increased
II. Direct examination
A. Cell count
1. Total cell 1–3 1,000–20,000 50–500 10–500
(per cu. mm
2. Predominant Lymphocytes Neutrophils (90–95%) Lymphocytes (90%) Lymphocytes
B. Biochemical analysis
1. Total 30–45 100–600 (Highly 80–120 (moderately 60–80 (slightly
proteins increased) increased) increased)
(mg%)
2. Sugars 40–80 Diminished or absent Diminished (30–50) Normal
(mg%) (10–20)
III. Bacteriological examination
A. Microsocopy
1. Gram Nil Gram-negative cocci, — —
staining Gram-positive cocci,
Gram-negative bacilli
or gram-positive bacilli
may be found depending
upon the causative agent
responsible.
2. Ziehl– Nil Nil Acid-fast bacilli (AFB) —
Neelsen may be found
staining
B. Culture Nil According to the M. tuberculosis may grow Viruses may be
causative agent, specific on L–J media grown on cell
organism may grow on cultures
appropriate media.
If the plate cultures remain free from growth, tests are used for rapid diagnosis of meningitis and
and turbidity develops in the cooked-meat broth, are particularly useful in partially treated patients
the broth should be filmed and subcultured in whom smear and culture may be negative. These
on to blood agar and heated-blood agar plates, are available for N. meningitidis, Str. pneumoniae,
incubated aerobically and anerobically. H. influenzae type b and Group B streptococcus.
Laboratory Diagnosis
Important Questions
1. Specimen
1. Name the various organisms causing meningitis.
CSF is collected by lumbar puncture in a sterile
Discuss the laboratory diagnosis of acute pyogenic
container under aseptic conditions. When CSF meningitis.
is allowed to stand, a fibrin web (cobweb) often 2. Write short notes on:
develops. Cell count and biochemical analysis can a. Aseptic meningitis
be done as described earlier. b. Tuberculous meningitis.
II. Filter Paper Method Differentiation of Upper UTI and Lower UTI
This method of semiquantitative culture is rapid The antibody-coated bacteria test has been
and very economical in the use of culture medium. employed for the localization of the site of urinary
infection. This is based on the assumption that
bacteria coated with specific antibodies are present
III. Dip-slide Method
in the urine only when the kidneys are infected
The dip-slide is small plastic tray carrying a layer (upper UTI) and not when the infection is confined
of an appropriate agar culture medium. Opposite to the bladder (lower UTI). Antib ody-coated
sides of the tray may carry different media, e.g. bacteria are detected by immunofluorescence
cysteine lactose electrolyte-deficient (CLED) agar using fluorescent-tagged antihuman globulin or
medium on one side and MacConkey, brainheart by staphylococcal coagglutination.
infusion or pseudomonas selective agar on
the other. The dip-slide is withdrawn from the Tuberculosis of Kidney and Urinary Tract
container and briefly immersed in the urine. It is Tuberculosis of kidney is a blood-borne infection.
then incubated at 37°C overnight and examined The patient presents with frequency and painless
for a growth of colonies. This method is relatively hematuria and routine urine culture does not show
expensive. any pathogen. Tuberculosis must be considered in
cases where pyuria is present without bacteriuria.
Screening Techniques
Laboratory Diagnosis
Several screening techniques have been introduced
for the presumptive diagnosis of significant 1. Specimen
bacteriuria. These include the following: Excretion of M. tuberculosis from kidney is
i. Griess nitrite test: It is based on nitrite- intermittent. Hence, midstream urine specimen
reducing enzymes produced by bacteria is not useful. Early morning urine specimens
present in urine. The presence of nitrite, should be collected in sterile container on three
detectable by a simple test, indicates the consecutive days.
presence of nitrite-reducing bacteria in urine.
ii. Catalase test: The presence of catalase as 2. Direct Ziehl–Neelsen Staining
evidenced by frothing on addition of hydrogen Smear made from centrifuged deposit of urine
peroxide indicates bacteriuria, though a is stained with Ziehl–Neelsen staining and may
positive result is obtained also in hematuria. reveal acid-fast bacilli. Saprophytic mycobacteria
Chapter 78: Infective Syndromes | 541
(e.g. M. smegmatis) may be present in normal urine
Laboratory diagnosis: For specimen collection,
which may be excluded by using acid-alcohol various methods used are: 1. Midstream specimen
as decolorizing agent in staining procedure. M. of urine (MSU); 2. Catheter specimen of urine (CSU);
tuberculosis is acid-alcohol-fast while M. smegmatis 3. Suprapubic stab; 4. Noninvasive method; 5. Early
is only acid-fast. morning urine (EMU); 6. Initial flow of urine
Part of the specimen is used for bacteriological
culture and the rest is examined immediately under
3. Culture the microscope
Culture is performed on Lowenstein–Jensen Culture: Semi-quantitative methods (Standard loop
medium and incubated for 6–8 weeks. Growth technique) are used for culture
is identified by Ziehl–Neelsen staining and Count more than 105 bacteria of single species per ml
is called significant bacteriuria. It indicates active UTI
biochemical tests. None of the screening methods is as sensitive or
reliable as a culture.
Key Points
Urinary tract infection (UTI) may be defined as the
presence of bacteria undergoing multiplication in Important Questions
urine within the urinary drainage system
1. Name the various organisms causing urinary tract
Lower UTI includes) Urethritis, cystitis and prostatitis infection. Discuss the laboratory diagnosis of this
and due to ascending infection condition.
Upper UTI includes acute pyelitis and acute pyelone
2. Write short notes on:
phritis.
Midstream specimen of urine (MSU).
4. Sore throat
Sore throat is essentially an acute tonsillitis Viral pharyngitis or other causes of pharyngitis/
or pharyngitis. It is characterized by redness tonsillitis must be differentiated from that caused
and edema of mucosa, exudation of tonsils, by Streptococcus pyogenes since it is treatable with
pseudomembrane formation, edema of uvula, penicillin whereas viral infections are not.
grey coating of tongue and enlargement of cervical
lymph nodes. Causative agents of sore throat are A. Specimen
given in Table 78.7. For bacteriological sampling, a plain, albumen-
Pseudomembrane formation: Corynebacterium coated or charcoal-coated cotton-wool swab should
diphtheriae, Candida albicans, β-hemolytic group A be used to collect as much exudate as possible from
Streptococcus, Treponema vincentii and Leptotrichia the tonsils, posterior pharyngeal wall and any other
buccalis may lead to pseudomembrane formation. area that is inflamed or bears exudate. Two throat
swabs are collected. If it cannot be delivered to the
Laboratory Diagnosis laboratory within about 1 hour, it should be placed
in a refrigerator at 4°C until delivery.
The signs and symptoms of sore throat (pharyngitis)
caused by streptococci and viruses are similar.
B. Direct Microscopy
From one swab, make two smears for Gram
Table 78.7 Causative agents of sore throat staining and Albert-staining.
A. Bacteria 1. Gram Staining
• Streptococcus β-hemolytic group A and occasionally
groups C and G
It is not helpful unless Vincent’s organisms or
• Corynebacterium diphtheriae
Candida albicans are suspected. Vincent’s infection
• Haemophilus influenzae
shows gram-negative spirochaetes (Borrelia
• Bordetella pertussis
vincentii) and gram-negative fusiform bacilli
• Neisseria gonorrheae
(Fusobacterium spp.). When Candida albicans is
• Treponema vincentii
suspected, it appears as gram-positive oval budding
• Leptotrichia buccalis
yeast cells.
B. Fungi 2. Albert Staining
• Candida albicans It shows green-colored, V and L-shaped (Chinese
C. Viruses letter pattern) bacilli with bluish-black metachromatic
• Epstein–Barr virus granules in infection due to C. diphtheriae. Albert-
• Adenoviruses staining is helpful for presumptive diagnosis of C.
• Coxsackievirus A diphtheriae.
542 | Section 6: Miscellaneous
C. Culture Elek’s gel precipitation test .
Culture media are selected according to the Animal inculation test
organism suspected to be the causative agent iii. For Candida albicans
of sore throat. Following media may be used for a. Germ tube test
culture. b. Carbohydrate fermentation and assimil
ation tests.
Blood agar: All the organisms will grow on this iv. For other causative agents
medium. Special culture media and different biochemical
reactions or serological tests may be required
Crystal violet blood agar: It is selective for Str.
as described in respective chapters. For
pyogenes especially when incubated anaerobically.
details of tests mentioned above, refer to the
Loeffler’s serum slope: For isolation of C. corresponding chapters.
diphtheriae, grow very rapidly (in 6–8 hours).
Potassium tellurite blood agar: Selective media E. Antibiotic Sensitivity
for isolation of C. diphtheriae. All β-hemolytic group A streptococci are sensitive to
Sabouraud’s dextrose agar (SDA): When penicillin G, and most are sensitive to erythromycin.
suspecting Candida albicans, SDA should be C. diphtheriae is sensitive to penicillin.
included.
These culture media should be incubated at 37°C Pneumonia
for overnight. In case of potassium tellurite blood
agar, it should be incubated for 48 hours. After 6–8 Pneumonia may be defined as inflammation and
hours, a subculture should be made from Loeffier’s consolidation of the lung substance. Bacterial
serum slope onto potassium tellurite blood agar, causes for pneumonia are listed in Table 78.8.
which is then incubated at 37°C for 48 hours.
Table 78.8 Microbial pathogens that cause pneu
D. Identification monia
1. Morphology A. Community-acquired pneumonia
i. Str. pyogenes: Colnies are small (pin-point), Common organisms
circular, semitransparent, low convex discs Streptococcus pneumoniae
having β-hemolysis. Chlamydophila pneumoniae
ii. C. diphtheriae: On potassium tellurite blood Mycoplasma pneumoniae
agar, gray or black coloured round colonies Legionella pneumophila
Uncommon organisms
are seen.
Haemophilus influenzae
iii. Candida albicans: White or cream colored
Staphylococcus aureus
colonies may be seen.
Chlamydophila psittaci
Coxiella burnetii
2. Gram Staining or Albert Staining Kleb. pneumoniae
i. Gram Staining Actinomyces israillae
Primary viral pneumonia
∙∙ Small Gram: Positive spherical cocci occurring Influenza, parainfluenza virus and measles
in chains is characteristic of Str. pyogenes. Respiratory syncytial virus in infancy
∙∙ Candida albicans reveals gram-positive budding Varicella (chickenpox)
yeast cells. B. Hospital-acquired pneumonia
C. Diphtheriae is seen as gram-positive bacilli. Gram-negative bacilli (60%)
Escherichia, Pseudomonas, Klebsiella species
ii. Albert Staining Gram-positive organisms (16%)
Staph. aureus
Diphtheriae shows green-colored V-or L-shaped
Anaerobes
bacilli with bluish-black metachromatic granules.
Legionella spp.
C. Pneumonia in immunocompromised patients
3. Other Tests for Confirmation Pnemocystis carinii
i. For β-hemolytic streptococci Gram-negative bacteria—Ps. aeruginosa
a. Bacitracin sensitivity test—for Str. pyogenes Mycobacteruim tuberculosis
b. Lancefield grouping—for all β-hemolytic Streptococcus pneumoniae
streptococci Haemophilus influenzae
ii. For C. Diphtheriae Candida albicans
Aspergillus fumigatus
a. Biochemical tests
Viruses–Cytomegalovirus
b. Toxigenicity tests
Chapter 78: Infective Syndromes | 543
1. Classification 3. Giemsa staining: Giemsa stain of sputum.
is useful to detect cysts and trophozoites of
A. Community-acquired Pneumonia Pneumocystis carinii.
Community-acquired pneumonia has been thought
to present as either of the two syndromes—typical C. Culture
or atypical. Sputum, blood and pleural fluid can be cultured
on blood agar and chocolate agar. Purulent portion
Typical Pneumonia Syndrome of sputum is best for culture. If the sputum is too
Typical pneumonia syndrome is usually caused viscid, it may be homogenized.
by the most bacterial pathogen in community- 1. Blood agar: It is incubated aerobically at 37°C
acquired pneumonia, Streptococcus pneumoniae, under 5–10% CO2.
but can also be due to other bacterial pathogens. 2. Chocolate agar: It is also incubated at 37°C
with 5–10 percent CO2.
Atypical Pneumonia Syndrome 3. Lowenstein–Jensen (L–J) medium: Three
Atypical pneumonia is classically produced by specimens of sputum are collected on three
Mycoplasma pneumoniae, Legionella pneumophila. successive days for culture on L–J media which
Chlamydophila pneumoniae. There is patchy are then incubated at 37°C for 6–8 weeks.
consolidation of lungs. 4. Selective media are required for culture of L.
pneumophila (see Chapter 37).
Lobar Pneumonia 5. Isolation of chlamydia can be done on cell-lines.
It is an acute inflammation characterized by homo
geneous consolidation of one or more lobes. It is D. Detection of Bacterial Antigens
caused by Streptococcus pneumoniae.
Direct Immunofluorescence Test
Bronchopneumonia Direct immunofluorescence examination of sputum
It is almost always a secondary infection and is done to detect antigens in L. pneumophila.
generally follows viral infections of the respiratory
tract. It is an acute inflammation of bronchi and E. Serology
the consolidation is scattered. It is caused by 1. Mycoplasma pneumoniae
Streptococcus pneumoniae Haemophilus influenzae –– Complement fixation test (CFT)
(rarely Kleb. pneumoniae, Staph. aureus). –– Cold agglutinin test
Respiratory syncytial virus, Mycoplasma 2. Chlamydia pneumoniae
pneumoniae, Chlamydophila pneumoniae and
–– Microimmunofluorescence
Bordetella pertussis cause bronchitis and bronchiolitis.
–– TWAR antigen
B. Hospital-acquired Pneumonia –– CFT
–– Immunofluorescent antibody test
Hospital-acquired or nosocomial pneumonia
4. Coxiella burnetii
refers to a new episode of pneumonia occurring
–– CFT
at least two days after admission in the hospital.
Key Points
C. Pneumonia in lmmunocompromized
Sore throat is essentially an acute tonsillitis or
Patients pharyngitis
The common causative agents include Pneumocystis Laboratory diagnosis of sore throat caused by
carinii, Staph. aureus, Ps. aeruginosa, viral bacteria depends on direct microscopy and culture.
infections (CMV and herpes) and M. tuberculosis. For bacteriological sampling, two throat swabs are
collected
Pneumonia may be defined as inflammation and
Laboratory Diagnosis consolidation of the lung substance, which may
A. Specimen be classified as community-acquired pneumonia,
hospital-acquired pneumonia and pneumonia in
Sputum; blood; pleural fluid; blood for serological immunocompromized patients
tests. Laboratory diagnosis of pneumonia depends on
direct microscopy and culture.
B. Direct Microscopy
1. Gram Staining: Adequate number of pus Important Questions
cells alongwith the presence of predominant 1. Name the various organisms causing sore throat.
organisms gives a clue to the probable pathogen. How will you diagnose it in the laboratory?
2. Ziehl–Neelsen staining: Presence of acid-fast 2. Name the various bacterial causes of pneumonia.
bacilli (AFB) gives a presumptive diagnosis of Discuss the laboratory diagnosis of pneumococcal
tuberculosis. pneumonia.
544 | Section 6: Miscellaneous
1. Entamoeba histolytica
Key Points
2. Giardia lamblia
3. Cryptosporidium parvum Diarrhea is defined as the passage of loose, liquid or
watery stools. These liquid stools are usually passed
4. Balantidium coli: This is a rare cause of chronic more than three times a day
recurrent diarrhea, with dysenteric episodes in Gastroenteritis may be defined as inflammation of
some. the mucous membrane of stomach and intestine,
resulting in frequent loose motions with or without
Laboratory Diagnosis mucus and with or without blood, pain abdomen
and with or without fever. It is often used as a
i. Specimen: Feces synonym for acute diarrhea, especially when
associated with vomiting
ii. Microscopy
Bacterial diarrhea Vibrio cholerae, E. coli, Salmonellae
are some important bacterial causes of diarrheal
a. Saline Preparation and Iodine Mount diseases. Rotavirus is the most important viral
etiology of diarrhea
In saline preparation, motility of trophozoites Dysentery is a disease marked by frequent
can be observed while cysts take up iodine and watery stools, often with blood and mucus, and
appear distinct in iodine mount. Cysts and motile characterized clinically by cramping abdominal pain,
trophozoites of E. histolytica can be observed in feces tenesmus, fever and dehydration
of amebic dysentery. Giardia lamblia cysts in formed Laboratory diagnosis of diarrhea, and dysentery
stools, or trophozoites in fresh diarrheal stools can be depends on isolation of organism from the relevant
specimen.
seen in giardiasis. Trophozoites of Balantidium coli
are found in liquid stool of this parasitic infection.
Rarely cysts may be seen in formed stools. Important questions
1. Enumerate the different causes of diarrhea. How will
b. Acid-fast Staining you diagnose a case of diarrhea in the laboratory.
2. Enumerate the etiological agents of dysentery.
Feces smear shows acid-fast oocyst of Crypto
Discuss in detail the laboratory diagnosis of
sporidium parvum.
dysentery.
iii. Serological tests: Indirect hemagglutination 3. Write short notes on:
assay (lHA) and ELISA are used to detect a. Traveller’s diarrhea
antibody titer in sera of patients with amebiasis. b. Viral diarrhea
Chapter 78: Infective Syndromes | 547
6. Food poisoning
The term bacterial food poisoning is restricted to 8–24 hours. The typical example of this type of food
acute gastroenteritis due to the presence of bacteria, poisoning is by salmonellae.
usually in large numbers, or their products in food.
It is of three types (Table 78.11). B. Toxic Type
In this type, the disease follows ingestion of food
A. Infective Type with preformed toxin. Incubation period is short
In this type, multiplication of bacteria occurs in (2 to 6 hours).Example is staphylococcal food
vivo when infective doses of microorganisms are poisoning.
ingested with food. Incubation period is generally
C. Infective-toxic Type
Table 78.11 Causative agents of food poisoning In this type, bacteria release the toxin in the bowel.
The incubation period is 6–12 hours. The typical
1. Infectlve type example is Cl. perfringens food poisoning.
• Salmonella Typhimurium For the laboratory diagnosis, refer to the corres
• S. Enteritidis ponding chapters. It has also been described under
• S. Heidelberg “Laboratory diagnosis of diarehea”.
• S. Indiana
Key Points
• S. Newport
The term ‘bacterial food poisoning’ acute in is of
• S. Dublin
three types A. Infective type, B. Toxic type, and
• Vibrio parahaemolyticus Infective-toxic type.
• Campylobacter jejuni Laboratory diagnosis of food poisoning depends on
isolation of organism from the relevant specimen.
2. Toxic type
• Staphylococcus aureus
Bacillus cereus Important questions
• Clostridium botulinum 1. Name the various organisms causing food
poisoning. Describe briefly the laboratory diagnosis
3. Infectlve-toxic type
of this condition.
Clostridium perfringens
2. Write short notes on food-borne botulism.
E. Herpes genitalis
Herpes simplex virus (HSV), types 1 and 2, is the
b. FTA-ABS (fluorescent treponemal antibody etiological agent but type 2 strains are more com
absorption test). monly associated.
c. TPI (Treponema pallidum immobilization 1. Specimens
test) i. Scrapings from base of the lesions
VDRL and TPHA are two most commonly
ii. Blood for serology
used tests.
2. Microscopy: Intranuclear type A inclusion
bodies may be seen in Giemsa-stained smears.
B. Lymphogranuloma Venereum (LGV) The virus particle may also be demonstrated
It is caused by C. trachomatis serotypes L1, L2 and L3. under the electron microscope.
1. Direct microscopy: Smears of material aspirated 3. Virus isolation: Diagnosis is confirmed by
from the bubos may show the elementary tissue culture in human diploid fibroblast cells.
bodies (Miyagawa’s granulocorpuscles). The Typical cytopathic changes may appear as early
sensitivity of microscopy is very low. as in 24–48 hours.
Chapter 78: Infective Syndromes | 549
4. Serology: ELISA, neutralization or complement immunofluorescence test with a mono
fixation tests are used for antibody detection clonal antibody or by ELISA.
and are useful in the diagnosis of primary 3. Culture: The exudate is inoculated on McCoy or
infection. HeLa cell cultures treated with cycloheximide.
Intracytoplasmic glycogen-rich inclusions
F. Gonorrhea are detected by Giemsa stain or by immuno
It is caused by Neisseria gonorrhoeae. fluorescence. These are suggestive of C.
1. Specimens: Specimens used are: (i) Urethral trachomatis.
discharge; (ii) An endocervical swab; (iii) In 4. Serology
chronic cases; (iv) Rectal swab. i. Complement fixation test (CFT)
2. Direct Gram-staining: Gram-stain of the penile ii. Microimmunofluorescence or ELISA is useful
exudate shows characteristic and diagnostic for detection of serovar-specific antibody.
gram-negative intracellular diplococci (GNID)
in granulocytes.
H. Trichomoniasis
3. Culture: Commonly used selective media are
modified Thayer–Martin and Martin–Lewis It is caused by Trichomonas vaginalis.
plates. Inoculated plates should immediately 1. Specimen: Swab of vaginal discharge is exami
be placed in an incubator at 37°C for 24–48 ned freshly. Specimen should be collected in
hours in the presence of CO2. Cultures with no stuart’s transport medium if delay in transport
visible growth must be held for 72 hours before is inevitable.
discarding. 2. Direct microscopy: Direct wet film shows
4. Identification: Identification of an organism motile trichomonads. Direct microscopy is at
is based on colony morphology, Gram staining least 80 percent as positive as culture.
from colonies and biochemical reactions. 3. Culture: Fineberg’s medium is used for culture
i. Colony morpholo g y: Small, gray, of specimen and it is incubated for 5 days and
translucent, raised colonies. examined for motile protozoa.
ii. Gram-staining: Gram-negative diplococci
iii. Biochemical reactions: They are oxidase I. Bacterial Vaginosis-associated Organisms
positive and produce acid from glucose but The diagnosis of bacterial vaginosis does not depend
not lactose, maltose or sucrose. on the isolation of a particular microorganism, e.g.
iv. Direct detection methods, such as Gardnerella vaginalis or Mycoplasma hominis, but on
fluorescent antibody and agglutination the replacement of the predominantly lactobacillary
tests, are also available. flora with a mixture of aerobes; and anaerobic
v. Nucleic acid probes for the direct detection organisms, a shift of pH to neutral or alkaline, and
of N. gonorrhoeae from clinical specimens. the presence of ‘clue’ cells. These changes are most
easily assessed from the Gram stained film.
G. Nongonococcal Genital Infection For diagnosis of Shigella sp, Campylobacter
Symptoms of discharge and dysuria clinically sp, Group B streptococci refer to corresponding
indistinguishable from gonorrhea caused by chapters.
organisms other than N. gonorrhoeae is called
nongonococcal urethritis (NGU). Causative agents J. Vulvovaginal Candidiasis
are shown in Table 78.13. A significant proportion It is caused by various species of Candida but C.
of nongonococcal genital infection in women is albicans accounts for 80% of cases.
generally due to chlamydia trachomatis. 1. Specimen: Swab from vaginal secretions
2. Direct microscopy
Laboratory Diagnosis
i. KOH mount: It shows yeast cells.
1. Specimens
(i) Swabs from exudate of urethra; (ii) Cervical ii. Gram-staining: Gram staining shows
discharge. characteristic gram-positive budding yeast
2. Direct examination cells and pseudohyphae.
i. Giemsa stain: It shows intracytoplasmic 3. Culture: Sabouraud’s dextrose agar (SDA) is
inclusion bodies suggestive of C. tracho inoculated with the specimen and incubated
matis. at 37°C for 48 hours.
ii. Antigen detection: For detection of ele 4. Identification
mentary bodies of C. trachomatis, smears i. Colony morphology: Colonies are creamy
are made from exudate and examined by white and smooth.
550 | Section 6: Miscellaneous
ii. Gram staining: Gram-stained smear shows Key Points
budding gram-positive yeast cells.
The sexually transmitted diseases (STDs) are a group
Germ tube formation: C. albicans forms
iii.
of communicable diseases which are transmitted
germ tube within two hours when incubated predominantly or entirely by sexual contact. The
in human serum at 37°C. causative organisms include a wide range of
iv. Chlamydospores formation: On cornmeal bacterial, viral. protozoal and fungal agents
For laboratory diagnosis and treatment according
agar C. albicans forms chlamydospores.
to the suspected organism responsible for the
manifestations of that particular STD.
K. Genital Warts
Genital warts, also known as condyloma acuminata, Important Questions
are common in sexually active adults. These are 1. Name the various organisms causing sexually
usually due to human papillomavirus (HPV) types transmitted diseases. Discuss the laboratory
6 and 11. diagnosis of syphilis.
For detection of inclusion bodies of HPV, 2. Write short notes on:
cytological or histological examination of cells in a. Laboratory diagnosis of gonorrhea
b. Nongonococcal urethritis (NGU)
urine is used.
c. Chancroid
For diagnosis of Shigella sp, Campylobacter spp, d. Lymphogranulum venereum (LGV)
Group B streptococci, refer to the corresponding e. Donovanosis
chapters. f. Vulvovaginal candidiasis.
8. Wound Infection
Wound infections may be endogenous or exogenous. Gas chromatography may be performed directly
It may be caused by a variety of aerobic and on liquid specimens to indicate the presence of
anerobic species of bacteria (Table 78.13). anerobes.
C. Viral Infections
4. Identification
Peripheral blood smear may be helpful in infectious
Organisms may be identified on the basis of
mononucleosis. Viral infections may be detected
colony morphology, Gram-staining, biochemical
either by tissue culture or by serology. Paul–Bunnel
reactions and agglutination, etc. Ziehl–Neelsen
test is useful in infectious mononucleosis.
(Z–N) staining is performed to detect acid-fast
bacilli (AFB) for M. tuberculosis. This is further
confirmed by culture and biochemical reactions. D. Fungal Infections
For details of individual organisms, refer to Specimens may be cultured on Sabouraud’s
corresponding chapters. dextrose agar or brain–heart infusion agar.
Chapter 78: Infective Syndromes | 553
E. Other Tests for Diagnosis 4. The most common viral agent causing aseptic
meningitis is:
1. Skin Tests a. Enteroviruses
Mantoux test: A tuberculin test and a chest X-ray b. Adenoviruses
should be done to detect tuberculosis. c. Cytomegalovirus
Skin tests for histoplasmosis, coccidioido d. Parainfluenza virus
mycosis, sarcoidosis should also be done. 5. Which of the following bacteria is the most
comrnon cause of UTI:
2. Hematological Investigations a. E. coli
Hematological investigations should be done b. Ps. aeruginosa
to detect leukocytosis, suggestive of a cryptic c. Staph. aureus
abscess; eosinophilia, suggestive of helminthiasis; d. Staph. saprophyticus
and atypical lymphocytes, suggestive of infectious 6. All the following are causative agents of sore throat
mononucleosis. except:
a. Streptococcus pyogenes
3. Immunologic Tests b. Haemophilus influenzae
LE cell phenomenon and antinuclear antibody c. Borrelia vincenti
test in SLE. d. Staphylococcus aureus
7. Which of the following organisms may lead to
4. Biopsy pseudomembrane formation in throat:
a. C. diphtheriae b. Strep. pyogenes
Biopsy of liver and bone marrow and other tissues
c. Candida albicans d. All the above
such as skin, lymph nodes and kidney.
8. Which of the following agents can cause diarrhea?
Key Points a. Rotavirus b. Vibrio cholerae
c. S. Typhimurium d. All the above
Pyrexia of unknown origin (PUO) may be defined
as (a) any febrile illness (body temperature greater 9. Which of the following protozoa can cause diarrhea:
than 38°C) on several occasions, (b) duration of a. Entamoeba histolytica
fever of more than 3 weeks, and (c) failure to reach a b. Cryptosporidium parvum
diagnosis despite 1 week of inpatient investigation.
c. Giardia lamblia
It is also known as ‘fever of unknown origin’ (FUO)
d. All the above
The causes of PUO include infections bacterial, parasitic
and viral) neoplasms, connective tissue disorders, 10. Which of the following agents can cause dysentery:
granulomatous diseases and drug reactions a. Escherichia coli (EIEC)
Tests should first be done for the more likely infec b. Shigella dysenteriae
tions and then, if these are negative, tests for the less
c. Entamoeba histolytica
likely should be done.
d. All the above
11. Which of the following bacteria can cause infective
Important Question type of food poisoning:
Define and enumerate the causes of pyrexia of unknown a. S Enteritidis
origin (PUO). Discuss the laboratory diagnosis of PUO. b. Clostridium botulinum
c. Staph. aureus
Multiple choice questions (MCQs) d. All the above
12. Infective-toxic type of food poisoning is caused by:
1. Presence of bacteria in blood without multiplication
a. Staph. aureus
is known as:
b. Salomonella Enteritidis
a. Bacteremia b. Septicemia
c. Clostridium perfringens
c. Pyemia d. Endotoxemi
d. Salmonella Dublin
2. Which of the following bacteria can cause purulent
meningitidis: 13. Ulcer/s is/are painless I n which of the following
a. H. influenzae sexually transmitted diseases:
b. Strep. pneumoniae a. Syphilis b. Chancroid
c. N. meningitis c. Herpes genitalis d. All of the above
d. All the above 14. Genital ulcer is painful in:
3. Which of the following agents cause/s aseptic a. Syphilis
meningitis? b. Chancroid
a. Herpes simplex b. Enteroviruses c. lymphogranuloma venereum
c. Arboviruses d. All of the above d. Donovanosis
554 | Section 6: Miscellaneous
15. Which of the following bacteria is/are implicated 17. Which of the following anerobes can cause wound
as causative agents of nongonococcal urethritis: infection:
a. C. trachomatis a. Bacteroides spp
b. Peptostreptococci
b. Ureaplasma urealyticum
c. Clostridium perjringens
c. Mycoplasma hominis. d. All the above
d. All of the above 18. Which of the following conditions can result in PUO:
16. Urethral discharge is present in: a. Urinary tract infection b. Enteric fever
a. Chancroid c. Malaria d. All the above
b. Gonorrhea Answers (MCQs)
c. Herpes genitalis 1. a; 2. d; 3. d; 4. a; 5. a; 6. d; 7. d; 8. d; 9. d; 10. d; 11. d;
d. Lymphogranuloma venereum 12. c; 13. a; 14. b; 15. d; 16. b; 17. d; 18. d.
79
Chapter
Hospital-acquired Infection
Learning Objectives
After reading and studying this chapter, you should ∙∙ Describe common hospital-associated infections
be able to: and causative organisms responsible for these
∙∙ Define hospital-associated infection conditions
∙∙ List of routes of transmission of hospital-associated ∙∙ Describe diagnosis and control of hospital-
infections associated infections.
LEARNING OBJECTIVES
After reading and studying this chapter, you should ∙∙ Explain the meaning of sensitive, intermediate and
be able to: resistant applied to antimicrobial susceptibility
∙∙ List different methods of antibiotic sensitivity test results
testing ∙∙ Describe the following: Epsilometer or E-Test;
∙∙ Describe disk diffusion methods Minimum inhibitory concentration of antimicrobial
∙∙ Explain Stokes disk diffusion method and its reporting agents;Minimum bactericidal concentration of
∙∙ Differentiate between Stokes disk diffusion and antimicrobial agents.
modified Stokes disk diffusion method
INTRODUCTION Medium
It is essential to determine the susceptibility of Mueller–Hinton broth and agar may be used for
isolates of pathogenic bacteria to antibiotics that testing aerobic and facultative anaerobic isolates.
are likely to be used in treatment. As strains of most The medium is and poured to a depth of 4 mm
pathogenic organisms differ from one another (25 mL medium) in flat-bottomed 9 cm Petri dishes
within their species in their antibiotic sensitivities, on a level surface. When set, the plates may be
sensitivity tests are required as a routine. stored for up to a week at 4°C and their surfaces
should be dried with their lids ajar before use. The
ANTIBIOTIC SENSITIVITY TESTS pH of the medium must be close to 7.3. A more acid
Antibiotic sensitivity tests are of two types: reaction decreases the activity of aminoglycoside
A. Diffusion methods and macrolide antibiotics. A more alkaline pH
1. Kirby–Bauer disk diffusion method favors the action of tetracyclines, novobiocin
2. Stokes disk diffusion method and fusidic acid, but interferes seriously with the
B. Dilution methods activity of nitrofurantoin.
1. Broth dilution method The addition of 5% lysed horse blood is needed
2. Agar dilution method to support the growth of fastidious species such
Diffusion methods: Here the drug is allowed as Haemophilus influenzae. Lysed horse blood
to diffuse through a solid medium so that a should also be added for tests with sulfonamides
gradient is established, the concentration and trimethoprim; its content of thymidine
being highest near the site of application of phosphorylase is needed to neutralize the inhibitory
the drug and decreasing with distance. The effect of thymidine in the medium on the action of
test bacterium is seeded on the medium and these drugs. Low levels of free Ca2+ and Mg2+ ions in
its sensitivity to the drug determined from the the medium increase the action of aminoglycoside
inhibition of its growth. Several methods have antibiotics against Pseudomonas aeruginosa.
been used for the application of the drug. The The addition of 5% NaCl to the medium
method most commonly employed is to use is needed in one of the methods for detecting
filter paper disks, impregnated with antibiotics. resistance to methicillin in strains of staphylococci .
Disk Diffusion Methods Inoculum: Prepare the inoculum from material
These methods are suitable for organisms that picked up with a loop from five to ten colonies
grow rapidly overnight at 35°C. of the species to be tested. Inoculate them in a
3. Reference points in the evaluation and com in controls against many variables and therefore
parison of new and existing antimicrobial provides dependable results.
agents. Dilution tests: There are two types of dilution tests:
Broth dilution method and agar dilution method.
Agar Dilution Method
Here, serial dilutions of the drug are prepared in IMPORTANT QUESTIONS
agar (Mueller–Hinton agar) and poured into plates.
1. Name different methods of antibiotic sensitivity
The ‘agar dilution’ method is more convenient when testing. Discuss in detail Kirby–Bauer disk diffusion
several strains are to be tested at the same time. The method for antimicrobial sensitivity testing.
advantage is that many strains can be inoculated on 2. Write short notes on:
each plate containing an antibiotic dilution. a. Kirby–Bauer disk diffusion method
Automated versions of sensitivity tests are b. Stokes disk diffusion method
available and are in use in large laboratories. c. Minimum inhibitory concentration of antimi
crobial agents.
ANTIBIOTIC ASSAYS IN BODY FLUIDS d. Minimum bactericidal concentration of antimi-
crobial agents.
These are required to verify whether adequate drug
concentrations are achieved in blood and other MULTIPLE CHOICE QUESTIONS (MCQs)
body fluids. Likewise, new drugs must be tested to
determine achievable levels in the blood, urine, or 1. Which of the following media is most suitable for
antibiotic sensitivity testing?
other body fluids.
The assays are generally done by making serial a. Mueller–Hinton medium b. Nutrient agar
dilutions of the specimen and inoculating standard c. Blood agar d. MacConkey agar
suspensions of bacteria of known MIC. Assays 2. The pH of the medium for antibiotic sensitivity
by the agar diffusion method can also be done. testing should be:
A technique called the diffusion assay is used to a. 6.0–6.2 b. 6.8–7.0
measure the concentration of an antimicrobial c. 7.2–7.4 d. 7.6–7.8
in a fluid specimen. The test relies on the same 3. In Stokes disk diffusion method, if the zone size
principle as the Kirby–Bauer test, except in this case is larger than, equal to or not more than 3 mm
it is the concentration of drug, not the sensitivity of smaller than the control a strain is considered:
organism, being assayed. This depends on the direct a. Sensitive b. Intermediate
relationship between antibiotic concentration c. Resistant d. None of the above
and the diameter of the zone of inhibition with a 4. Addition of 5% salt in the medium is done when
standard sensitive strain of bacterium. testing antibiotic susceptibility of :
a. Methicillin resistant staphylococci
Key Points b. Pneumococci
Antibiotic susceptibility tests are of two types: c. Coliforms
Diffusion tests and dilution tests d. Non fermenting gram-negative bacilli
Diffusion tests consists of Kirby–Bauer and Stokes
disk method. Stokes disk method incorporates built- ANSWERS (MCQs)
1. a; 2. c; 3. a; 4. a
Antimicrobial Chemotherapy
LEARNING OBJECTIVES
After reading and studying this chapter, you should ∙∙ Describe mechanism of drug resistance
be able to: ∙∙ List cephalosporins of first, second, third and fourth
∙∙ Describe mechanism of action of antibacterial generation.
drugs
Penicillins
Penicillins are a group of antimicrobial substances,
all of which possess a common chemical nucleus
(6-aminopenicillanic acid) which contains a
b-lactam ring essential to their biologic activity. A
side chain is attached to the b-lactam ring which
determines many of the antibacterial and pharma
cological characteristics of a particular type of
penicillin.
All b-lactam antibiotics inhibit formation of
bacterial cell wall. They particularly block the
final transpeptidation reaction in the synthesis of
cell wall peptidoglycan and also activate autolytic
enzymes in the cell wall.
Penicillins can be divided into several groups:
1. Those with highest activity against gram-posi
tive organisms but susceptible to hydrolysis by Figs 81.1A and B: The β-lactam ring of penicillins and
b-lactamases: cephalosporins the core chemical structure of (A) a penicillin
(B) a cephalosporin. The β-lactam rings are marked by an
• Penicillin-G orange circle. The R groups vary among different penicillins
• Penicillin-V and cephalosporins
pathogens. Second generation drugs act against subunit of DNA gyrase, an enzyme that engineers
many gram-negative as well as Gram-positive the breaking and rejoining of super coiled DNA.
pathogens. Third-generation drugs are particularly Nalidixic acid and its early congeners are narrow-
effective against gram-negative pathogens, and spectrum agents active only against gram-negative
often also reach the central nervous system. bacteria.
Newer quinolones like ciprofloxacin, norflox
Other b-Lactam Antibiotics acin, ofloxacin, pefloxacin and lomefloxacin are
Two other groups of β-lactam drugs, carbapenems broad spectrum quinolones. These have been
and monobactams, are very resistant to β-lactamases. successfully used in a wide variety of infections, but
resistance is becoming more prevalent.
Carbapenems
The carbapenems are effective against a wide range Rifamycins
of gram-negative and gram-positive bacteria. Two Rifamycins inhibit bacterial growth by binding
types are available, imipenem and meropenem. strongly to the DNA-dependent RNA polymerase of
Glycopeptides bacteria thus inhibiting transcription of RNA from
DNA. This group of antibiotics is characterized
Two glycopeptides, vancomycin and teicoplanin,
by excellent activity against mycobacteria.
are in clinical use. Their chief importance resides
Staphylococci in particular are exquisitely sensitive.
in their action against gram-positive cocci with
Rifampicin and rifabutin are most widely used.
multiple resistance to other drugs. They are mainly
used in serious infections with staphylococci and
Nitroimidazoles
enterococci that are resistant to other drugs.
Azole derivatives have wide-ranging antimicrobial
Other Inhibitors of Bacterial Cell Wall activity against fungi, protozoa, helminths, as well
Synthesis as bacteria. Those that exhibit antibacterial activity
These include bacitracin, cycloserine, fosfomycin are 5-nitroimidazoles which are active only against
and isoniazid. anaerobic bacteria and anaerobic protozoa. The
representative of the group most commonly used
2. Inhibition of Bacterial Cytoplasmic clinically is metronidazole, but similar derivatives
Membrane Function include tinidazole, ornidazole and nimorazole.
They are primarily antiprotozoal agents, but they
Only polymyxins have been regularly used
exhibit potent activity against anaerobic bacteria.
systemically among membrane active agents used
in human medicine. Two members of the family Nitrofurans
are in therapeutic use: polymyxin B and colistin
(polymyxin E). Various nitrofuran derivatives are in use around
They exhibit potent antipseudomonal activity, the world as antibacterial agents. These include
but toxicity has limited their usefulness, except in nitrofurantoin and furazolidone.
topical preparations and bowel decontamination Nitrofurantoin: It is bactericidal to most urinary
regimens. pathogens at concentrations achievable in urine.
Furazolidone: Furazolidone is used in enteric
3. Inhibitors of Nucleic Acid Synthesis
infections. The mode of action of nitrofurans
Quinolones has not been elucidated, but it is probable that
The quinolones are synthetic drugs that contain a reduced metabolite acts on DNA in a manner
the 4-quinolone ring. Quinolones act on the α analogous to that of the nitroimidazoles
ANTIBIOTIC RESISTANCE
Understanding the mechanisms and the spread
of antimicrobial resistance is an important step in
curtailing the problem.
Immunoprophylaxis
Learning Objectives
After reading and studying this chapter, you should ∙∙ Explain immunization schedule
be able to: ∙∙ Discuss national immunization schedule
∙∙ List of immunizing agents ∙∙ Describe passive immunization.
∙∙ Describe the following: live attenuated vaccines;
killed vaccines; toxoids
Learning Objectives
After reading and studying this chapter, you should ∙∙ Describe the following: Water-borne pathogens;
be able to: presumptive coliform count; Eijkman test
∙∙ Describe bacteriological examination of water ∙∙ Describe settle plate method and slit sampler
∙∙ Discuss bacteriological examination of milk method for bacteriology of air.
LEARNING OBJECTIVES
After reading and studying this chapter, you should waste; Waste egregation; Waste treatment and
be able to: disposal
∙∙ Describe universal precautions ∙∙ Describe treatment and technologies for health
∙∙ Describe the following: Categories of biomedical care waste.
Let the wastes of “the sick” not contaminate the lives of the healthy”
Table 84.1 Color coding and type of container for disposal of biomedical wastes
Color coding Type of container Waste category Treatment option as per
schedule 1
Yellow Plastic bag 1, 2, and 5, Cat. 6 Incineration/deep burial
Red Disinfected container/plastic 3, 4, 7 Autoclaving/microwaving/
bag chemical treatment
Blue/white translucent Plastic bag/puncture proof Autoclaving/microwaving/
container chemical treatment and
destruction/shredding
Black Plastic bag Cat. 5 and Cat. 9 and Cat. Disposal in secured landfill
10 (solid)
Learning Objectives
Introduction ∙∙ Cholera
∙∙ Poliomyelitis
To maintain an active infectious disease in a human ∙∙ Hepatitis A virus infection
population, the pathogen must be transmitted ∙∙ Food poisoning
from one host or source to another. Transmission ∙∙ Intestinal parasitic infestation.
is the third link in the infectious disease cycle and
occurs by four main routes: airborne, contact, B. Diseases Transmitted by Blood
vehicle, and vector-borne.
1. Viruses
Vehicles and vectors ∙∙ Hepatitis B
∙∙ Human immunodeficiency viruses (HIV)
The agents of transmission that bring the ∙∙ Human T cell lymphotropic viruses
microorganism from the reservoir to the host may ∙∙ Infectious mononucleosis
be a living entity, in which case they are called ∙∙ Cytomegalovirus
vectors, or they may be a nonliving entity referred
to as a vehicle or fomite. 2. Bacteria
Modes of transmission ∙∙ Syphilis
A. Direct: Transmitted by direct contact between ∙∙ Brucellosis
reservoir and host.
B. Indirect: Transmitted to host (human host) 3. Parasites
via intervening agent(s) such as vectors ∙∙ Malaria
(animals, insects, other humans and vehicles ∙∙ Trypansomiasis (Chaga’s disease)
(water, food, air, medical devices, various other ∙∙ Trypanosoma cruzi.
inanimate objects).
2. Vector-borne
1. Vehicle-borne In infectious disease epidemiology, vector is
Vehicle-borne transmission implies transmission defined as an arthropod or any living carrier (e.g.
of the infectious agent through the agency of water, snail) that transports an infectious agent to a
food, ice, blood, serum plasma or other biological susceptible individual. Transmission by a vector
products such as tissues and organs. Of these may be mechanical or biological. In the latter case,
water and food are the most frequent vehicles of the disease agent passes through a developmental
transmission, because they are used by everyone. cycle multiplication in the vector.
Learning Objectives
After reading and studying this chapter, you should be able to:
∙∙ Describe the following: Emerging infectious diseases; re-emerging infectious diseases.
87
Chapter
Learning Objectives
After reading and studying this chapter, you should ∙∙ Describe the following: Nucleic acid probes,
be able to: polymerase chain reaction (PCR); Transcription
∙∙ Describe molecular methods for microbial mediated amplification ( TMA); Nucleic acid
identification sequence based amplification (NASBA); Ligase
chain reaction (LCR).
Staining Methods
Learning Objectives
After reading and studying this chapter, you should ∙∙ Describe the following: Simple stains; Differential
be able to: stains; Gram stain; Acid fast stain (Ziehl–Neelsen
∙∙ Describe common staining techniques staining of acidfast bacilli); Albert’s stain.
LEARNING OBJECTIVES
After reading and studying this chapter, you should ∙∙ Describe the following: Gram staining; Ziehl–
be able to: Neelsen staing; Alber staining
∙∙ Describe various spots for practical examination ∙∙ Discuss identification of bacterial culture
with identification with relevant questions to answer ∙∙ Describe stool/feces examination for isolation and
∙∙ Prepare exercises for practical in microbiology for identification of pathogenic findings.
undergraduate students
Table 89.1 Specimen Practical question paper of Microbiology for MBBS 2nd Professional examination
Total Marks:25
Ques. No. 1 Identify the spots displayed. (2 × 2 + 3 × 1) = 7
Ques. No. 2 (a) Stain smears “A” by Ziehl–Neelsen technique. Draw and comment on this smear and show your findings
to the examiner. (3)
(b) Stain smear “B” by Albert’s technique. Draw and comment on this smear and show your findings tto the examiner. (2)
Ques. No. 3 You have been provided with a pure bacterial culture on plate and in broth. Do Gram’s staining from plate
and hanging drop preparation for motility from broth. Comment on the growth and mention other tests needed to
identify this bacterium. Show your findings to the examiner. 9(3+3+3)
Ques. No. 4 Identify and draw two abnormal findings (ova and cysts) in the given specimen of feces. Show your findings
to the examiner. 4(2+2)
Contd...
B. Albert’s stain
A fixed smear is provided for Albert’s staining. If the Fig. 89.2: Albert’s stain
smear is unfixed, it is fixed by passing the dried slide,
film downwards, three times slowly through the Gram Staining (See Also Chapter 88)
flame, or by heating through the glass slide (the slide Gram staining is done for bacterial culture (solid
is held, film upwards) in the top of the Bunsen flame material, such as cultures on agar) which is provided
for a few seconds so that the slide becomes hot. to the student along with liquid culture of the same
material in a test tube which is used for hanging
Staining Solution drop preparation to observe the motility of bacteria.
A. Albert 1 or Albert stain Preparing film or Smear for Staining
1. Toluidine blue 1.5 g 1. Film preparations are made on clean and free
2. Malachite green 2g from grease glass slides.
3. Glacial acetic acid 10 mL 2. Place a loopful of clean water on the slide and
4. Alcohol (95% ethanol) 20 mL the loop is then sterilized.
5. Distilled water 1 liter 3. A minute quantity of material, obtained by
just touching the growth (with solid material,
B. Albert II or Albert’s iodine
such as cultures on agar), is transferred to the
Iodine 6g
drop, thoroughly emulsified to make a smooth
Potassium iodide 9g suspension, and the mixture is spread evenly
Distilled water. 900 mL on the slide. A thin film of material containing
the microorganisms is spread over the surface
Procedure of the slide with the help of wire loop. This film,
1. Make film, dry in air, and fix by heat. called a smear, is allowed to air dry.
2. Cover slide with Albert’s stain (Albert’s solution 4. The slide is then held in the palm of the hand
A) and allow to act for 3–5 min. high over a Bunsen flame and dried. The
Identification Scheme
The following scheme of identification should be
adopted.
A. Culture Plate
Culture medium is identified and cultural
characteristics of bacterial growth are noted.
1. Culture Medium
Culture media provided may be nutrient agar,
blood agar or MacConkey’s agar. A
i. Cultural characteristics: Then the cultural
characteristics (Table 89.3) of bacterial
growth is described. This may help in some
provisional identification of the bacteria.
ii. Gram staining: Gram staining is done from
culture plate and notes the following:
i. Gram reaction—Gram-positive or gram- B
negative Figs 89.4: (A) Hanging drop preparation;
ii. Morphology—Cocci, bacilli or coccobacilli, (B) Hanging drop examination
approximate size. If bacilli, then describe
Various characterstics are noted such as degree,
whether these are thin, stout, long or short
turbidity, deposit and surface growth (Table 89.4).
bacilli.
Hanging drop preparation—is done to find out.
i. Motility—Motile or non-motile.
B. Liquid Medium ii. Morphology—Cocci, bacilli or coccobacilli.
Liquid medium is prepared from the same culture
plate which is given for examination and has the same Hanging Drop Preparation
bacteria. Therefore, first identify the liquid culture
This experiment is done to demonstrate motility
medium and describe the characteristics of growth
and to study morphology of bacteria (Fig. 89.4). It
(Table 89.4). Hanging drop preparation is made
is prepared from the bacterial growth provided in
from this liquid media for motility and morphology
liquid culture medium in a test tube.
of the bacteria.
Requirements: 1. A clean cavity (depression) slide;
i. Liquid Culture Medium 2. A clean coverslip; 3. Young broth culture; 4. Wire
Liquid culture medium may be in peptone water loop; 5. Bunsen burner/spirit lamp; 6. Microscope.
or glucose broth. Glucose broth is darker and more
yellowish in color as compared to peptone water. Procedure
Generally, streptococci are provided in glucose broth 1. A ‘hollow ground’ slide, (a glass slide with a
whiles all other bacteria are given in peptone water. shallow, circular concavity in its center) is used
Fig. 89.6: Growth of Staphylococcus aureus showing beta Fig. 89.7: Growth of Streptococcus pyogenes showing beta
hemolysis and golden pigment on blood agar hemolysis on blood agar
3. MacConkey’s Agar
1. Gram-negative rods are better described on
MacConkey agar because these organisms
produce similar-looking colonies on blood agar
plate and chocolate agar media. MacConkey
is best used, however, to differentiate lactose
fermenters (LF) from nonlactose ferment
ers (NLF). Therefore, on MacConkey’s agar,
Fig. 89.8: Growth of Proteus sp. showing swarning on colonies may be either pink (lactose fermen
blood agar ter) or pale (non-lactose fermenter).
Fig. 89.9: MacConkey agar with smooth, lactose fermenter Fig. 89.10: MacConkey‘s agar with lactose fermenter
(non-mucoid) colonies of Escherichia coli (mucoid) colonies of Klebsiella sp.
2. The LF colonies may be E. coli or Klebsiella Escherichia coli: Red or pink, nonmucoid colony,
sp., while NLF colonies generally given are gram-negative straight, rod and motile.
Salmonella sp. or Shigella sp. Klebsiella sp: 2 Red or pink, mucoid colonies, short
3. Examine the colony characters on solid and plump bacilli on Gram staining and nonmotile.
medium and growth in liquid medium.
4. Gram staining from culture plate and hanging F. Confirmatory Tests by Various
drop preparation from liquid broth are to be Biochemical Reactions (Table 89.5)
made.
Escherchia coli: Biochemical reactions
5. The following features may be observed on
i. E. coli ferments glucose, lactose, mannitol,
Gram’s staining and hanging drop preparation.
maltose and many other sugars with the
production of acid and gas. Typical strains do
Lactose Fermenter (LF)
not ferment sucrose.
A. Solid Media ii. Indole and MR positive, and VP and citrate
1. Culture medium: MacConkey’s agar (Figs negative (IMViC + + – –)
89.9 and 89.10). iii. It is negative for phenylalanine deaminase
2. Colony morphology: red or pink, nonmucoid test, urease test, H 2S production, gelatin
colony—Escherichia coli. liquefaction, growth in the presence of KCN,
Large, mucoid pink colonies—Klebsiellaj sp. and malonate utilization.
B. Klebsiella sp: Biochemical reactions (Table 89.3)
B. Liquid Broth They ferment sugars (glucose, lactose, sucrose,
Growth in broth: General turbidity and a
i. mannitol) with production of acid and gas, urease
heavy deposit or uniform turbidity. positive, indole negative, MR negative, VP positive
and citrate positive (IMViC – – ++). These reactions
C. Gram Staining and Hanging Drop are typical of K. pneumoniae subsp. aerogenes.
Preparation Final diagnosis of Esch. coli or Klebsiella sp. can be
made based on the above-mentioned biochemical
i. Gram staining: Gram-negative straight rod- reactions. Further tests, such as serotyping should
Escherichia coli; gram-negative short and be done for confirmation. Typing mehods, such as
plump bacilli Klebsiellaj sp. bacteriocin typing, phage typing, resistotyping and
ii. Hanging drop preparation: Motile or non molecular typing methods (Plasmid analysis, DNA
motile. profiling by random amplified polymorphic DNA
(RAPD) and pulsed-field gel electrophoresis) can
D. Identification be used for Klebsiella strains.
The bacteria may be identified by correlating the
colony characters, Gram staining and hanging B. Nonlactose Fermenter (NLF) Colonies
drop preparation and then by various biochemical 1. For undergraduate practical examination the
reactions. students are provided either of two important
NLF bacteria—Salmonella sp. or Shigella sp
E. Presumptive Diagnosis (nonlactose-fermenting bacteria are pale).
Presumptive diagnosis of Esch. coli or Klebsiella sp. Liquid broth of the same bacteria is also given
can be made on the basis of above features. alongwith it.
Fig. 89.11: Nonlactose fermenter colonies of Salmonella sp Fig. 89.12: Nonlactose fermenter colonies of Shigella sp
on MacConkey‘s agar on MacConkey‘s agar
Table 89.7 Distinguishing features of Shigella vi. They reduce nitrates to nitrites, do not form
species H2S and are inhibited by KCN.
Subgroup A B C D vii. Catalase is produced, except by S. dysenteriae
type 1 which are catalase negative.
Species Sh. Sh. Sh. Sh.
dysenteriae flexneri boydii sonnei viii. Members of groups A, B and C fail to decar
Mannitol – A A A boxylate lysine and ornithine. S. sonnei decar
boxylates ornithine but not lysine.
Lactose – – – A (Late)
Salmonella sp. Biochemical Reactions (Table
Sucrose – – – A (Late)
89.8)
Dulcitol – – d
Salmonellae will be indole and urease negative,
Indole d d d
catalase positive, oxidase negative and ferment
Ornithine – – – + glucose, mannitol and maltose but not lactose or
decarboxylase
sucrose. S. Typhi will be anaerogenic and ferments
Serotypes 10 6+ 15 Only glucose and mannitol with production of acid only,
variants one
while paratyphoid bacilli (S. Paratyphi A, B and C)
A = Acid; d = Variable will form acid and gas from sugars. Identification
of the isolate is by slide agglutination.
biochemical reactions are very important to
F. Show your findings to the examiner after
differentiate these Salmonella sp. and Shigella sp.
drawing well labeled diagrams of your observations
up to species level (Tables 89.6 to 89.8).
of Salmonella sp. or Shigella sp.
Shigella sp: Biochemical reactions (Table 89.7)
i. The shigellae are divided into four groups, or IV. Bacterial Growth on any Other Medium
species, by their biochemical reactions and
antigenic structure (Table 89.7). The groups Usually bacterial growth is given on the medium
A, B, C and D correspond to the species S. such as nutrient agar, blood agar and MacConkey’s
dysenteriae, S. flexneri, S. boydii and S. sonnei. agar. If the media other than nutrient agar, blood
agar and MacConkey’s agar is provided for
ii. Glucose is fermented with the production of
examination, then proceed using the same basic
acid, without gas, except for two serotypes:
method of processing, i.e. colony morphology,
Sh. flexneri serotype 6 (Newcastle and
liquid broth, Gram staining, hanging drop
Manchester varieties) and S. boydii serotype
preparation, presumptive diagnosis and further
14. Fermentation of mannitol in Sh. flexneri,
confirmatory tests. In vivo examiner may ask any
Sh. boydii and Sh. sonnei
question related to the specific bacteria along with
Mannitol nonfermenting—Sh. dysenteriae questions related to culture media, Gram staining,
iii. Lactose and sucrose are not fermented, except hanging drop preparation.
by Sh. sonnei which ferments them late.
iv. Dulcitol is not fermented by the majority of
shigellae. Adonitol, inositol and salicin are 4. STOOL/FECES EXAMINATION
not fermented. In this exercise, stool specimen is provided to
v. IMV, C––– + +–– (S. dysenteriae serotype 1, S. isolate two pathogenic parasitic findings. Stool
flexneri serotype 6 and S. sonnei are always examination is done to find out ova and cysts of
indole negative). different parasites.
B
Figs 89.15A and B: (A) Fertilized egg of Ascaris lumbricoides; Fig. 89.16: Egg of Trichuris trichiura
(B) Unfertilized egg of Ascaris lumbricoides (Roundworm)