Concept Based Learning

Video Companion on Each Chapter

Next Generation

Comprehensive Review Series

“MICROBIOLOGY”
Active Recall Based
Integrated Edition
Published by Delhi Academy of Medical Sciences (P) Ltd.

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First Published 1999, Delhi Academy of Medical Sciences

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Contents
Chapter 1 Bacterial Structure and Pathogenesis 01 – 24
Chapter 2 Bacterial Growth and Genetics 25 – 40
Chapter 3 Bacterial Culture and Sterilization 41 – 60
Chapter 4 Immunology 61 – 136
Chapter 5 Systematic Bacteriology 137 – 226
Chapter 6 Virology 227 – 278
Chapter 7 Mycology 279 – 316
Chapter 8 Parasitology 317 – 356
Bacterial Structure and
1 Pathogenesis

CONCEPTS
 Concept 1.1 Microbes - Introduction
 Concept 1.2 Microscopes and its Applications
 Concept 1.3 Staining methods
 Concept 1.4 Bacterial anatomy
 Concept 1.5 Epidemiology of Infectious diseases
2 | Microbiology
Concept 1.1: Microbes – An Introduction.
Learning Objectives
• What is Medical microbiology ?
• Classification of Microbes
• Comparison of Prokaryotes and Eukaryotes
• Comparison of various medically important microbe groups.

Time Needed
1st reading 15 mins
nd
2 look 5 mins

Medical Microbiology:
• Study of living organisms of microscopic size.
• Causative agents of infectious diseases of man.
• Response generated against them.
• Methods of diagnosis, treatment and prevention.
• Microorganisms were originally divided into plant and animal kingdoms. Because this
was unsatisfactory, they were then classified under a third kingdom, protista.
• Based on cellular organizations and biochemistry they are further classified into:
Prokaryotes: Bacteria and Blue Green Algae.
Eukaryotes: Other Algae, Fungi, Slime moulds, Protozoa.

Characteristics of Prokaryotic and Eukaryotic Cells:

Characteristic Prokaryotes Eukaryotes
Genetic Material
Nuclear membrane Absent. Present.
Nucleolus Absent. Present.
Histones Absent. Present.
Chromosome One (circular) and supercoiled Multiple (linear).
except Borrelia burgdorferi.
Replication Binary fission. Mitosis/Meiosis.
Extra chromosomal DNA Plasmids. Mitochondria.
Cytoplasm
Cytoplasmic streaming Absent. Present.
Pinocytosis Absent. Present.
Mitochondria Absent. Present.
Lysosomes Absent. Present.
Bacterial Structure and Pathogenesis | 3

Golgi apparatus Absent. Present.

E.R. Absent. Present.
Ribosomes 70s (50s and 30s). 80s (60s and 40s).
Chemical Composition of Cell Envelope
Sterols Absent*. Present.
Muramic acid Present. Absent.
* Present in Mycoplasma

Comparison of Medically Important Organisms:

Characteristic Viruses Bacteria Fungi Protozoa /
Helminths
Cells No. Yes. Yes. Yes.

Approx Diam. 0.2–2.0 1–5 3–10 (yeast). 15–25 (trophozoite).

(mm)
Nucleic Acid DNA or RNA. DNA and RNA. DNA and RNA. DNA and RNA.

Type of None. Prokaryotic. Eukaryotic. Eukaryotic.

Nucleus
Ribosomes Absent. 70s (50s + 30s) 80s (60s + 40s) 80s (60s + 40s)

Mitochondria Absent. Absent Present. Present.

Outer Surface Capsid. Rigid wall Rigid wall chitin. Flexible Membrane.
peptidoglycan.
Motility None. Some. None. Most.

Replication – Binary fission. Budding, mitosis. Mitosis.

4 | Microbiology
Concept 1.2: Microscopy and its applications
Learning Objectives
• Various types of Microscopes
• Light source and resolution power
• Applications

Time Needed
st
1 reading 15 mins
nd
2 look 05 mins

A. Light Microscope:
• Living state/ after fixation and staining.
• Ordinary white transmitted light as source of illumination.
• Wet film/hanging drop: arrangement, motility, size.
• Resolving power: (1/2 the wave length): 0.1µm - 0.2µm.
• Disadvantage: no internal details can be made out.

B. Phase - Contrast Microscope:

• Light waves passing through transparent objects such as cells, emerge in different
phase.
• Special optical system converts the difference in phases into difference in intensity.
• Reveals details of internal structure of living cells.

C. Dark-Field Microscope:
• Reflected light is used instead of
transmitted light.
• Essential part: Dark-ground
condenser.
• For visualization of very thin
organisms: e.g. Spirochetes
(diameter < 0.2µm).

Fig. 1.1: Light Microscope

Bacterial Structure and Pathogenesis | 5
D. Fluorescence Microscope:
• When ultraviolet/invisible light fails on a fluorescent substance, the wavelength of the
invisible light increases so that it becomes luminous.
• Cells stained with fluorescent dyes: become luminous and seen as bright object
against dark background.
• Some fluorescent dyes have selective action for particular cell constituent
e.g.: Fluorochrome.
Auramine and Rhodamine: Mycolic acid, mycobacteria appear bright yellow/orange
against greenish background.
Acridine orange: Binds to nucleic acid (bright orange).
Calcofluor white: Binds to cell wall (chitin) of fungi (blue white).
• Immunofluorescence: Direct and Indirect.

E. Electron Microscope:
• Beam of electrons is used instead of beam of light.
• Beam is focused by circular magnets
instead of lenses.
• The object is held in the path of the
beam scatters the electrons.
• Image focused on fluorescent viewing
screen.
• Wavelength of electrons: 0.005nm;
visible light : 500nm.
• Theoretically EM should be 100,000
times more powerful.
• In practice maximum resolution
that can be obtained: 0.3–0.5nm
(hundred times better than light
microscope).
• Examination under vacuum.
• Fixatives commonly used:
Glutaral­dehyde, Formaldehyde,
Osmium tetroxide, Uranyl acetate,
Acrolein.
• Two types :
ƒ Transmission Electron Microscope
(To visualize internal structures).
ƒ Scanning Electron Microscope
(To visualize Surface -3D
Fig. 1.2: Electron Microscope
structure).

F. Interference Microscope:
• Reveals cell organelles.
• Enables quantitative measurements of the chemical constituents of cell eg: lipids,
proteins, nucleic acid.
6 | Microbiology
G. Polarisation Microscope:
• Enables the study of intracellular structures using differences in birefringence.

Microscope Type Resolution Power

Light microscope

Inverted microscope

Phase contrast microscope

0.2 µM
Dark Ground Microscope

Fluorescent Microscope

Confocal Microscope

Transmission Electron microscope 0.2 nM

Scanning Electron Microscope 20 nM

Bacterial Structure and Pathogenesis | 7
Concept 1.3: Staining Methods
Learning Objectives
• Various types of staining methods
• Gram stain – Steps and interpretation
• Acid fast structures
• Special stains for bacterial structures.

Time Needed
1st reading 20 mins
nd
2 look 5 mins

Simple Stains:
• Methylene blue, Basic fuchsin
• Provide color contrast, impart same color to all bacteria.

Negative Stains:
• India ink, Nigrosin.
• Provide uniform colored background against which the unstained bacteria stand out
in contrast.
• Demonstration of bacterial capsule.
• Demonstration of slender bacteria (spirochetes).

Impregnation Methods:
• Very slender structures not visible by ordinary microscope are thickened to render
them visible by impregnation of silver on their surface.
• Levaditi’s and Fontana’s stain for Spirochetes.
• Leifson’s and Ryu’s stain for flagella.

Differential Stains:
• Impart different colors to different bacteria/bacterial structures.

Gram Stain:
• Originally devised by Christian Gram.
• Primary staining with a pararosaniline dye: Crystal violet, Methyl violet, Gentian
violet.
• Application of a dilute solution of iodine
• Decolorization with an organic solvent eg: ethanol, acetone, aniline.
• Counterstaining with safranin.
• Gram positive: Violet, more acidic protoplasm, relative impermeability of the bacterial
cell wall and cytoplasmic membrane to the dye iodine complex.
• Gram negative: Red.
• Not all or none phenomenon: Gram positivity lost due to prolonged treatment with
decolorizer or damage to cell wall.
8 | Microbiology

Fig.1.3: Gram stain

Bacteria not stained by Gram stain

1. Chlamydia
2. Rickettsia
3. Spirochetes
4. Mycoplasma

Acid Fast Stain:

• Discovered by Ehrlich.
• Tubercle bacilli resist decolorization with acids after staining with aniline dyes.
• Modified by Ziehl and Neelsen.
• Smear stained with carbol fuchsin with application of heat.
• Decolourised with acid.
• Counterstained with methylene blue.
• Acid fast bacteria appear red.
• Due to high content of fatty acids, alcohols.
• Acid fast structures: mycobacteria (M. tuberculosis and atypical mycobacteria
20% H2SO4; M. leprae 5% H2SO4); Nocardia 1% H2SO4 (in Tissues);
Rhodococcus; Legionella micdadei; Cryptosporidium, Isospora, Cyclospora,
T. saginata egg : 0.5% H2SO4; bacterial spores 0.25% H2SO4.
Bacterial Structure and Pathogenesis | 9
Concept 1.4: Bacterial Anatomy
Learning Objectives
• Structure of bacteria and its funcrons
• Medical applications of the structures
• Bacterial endospores
• Different type of toxins

Time Needed
1st reading 45 mins
nd
2 look 15 mins

Morphology of Bacteria:
Shape of bacteria:
• Cocci: Staphylococcus, Streptococcus.
• Bacilli: Salmonella, E. coli, Corynebacterium.
• Vibrio: Comma shaped; Vibrio cholerae.
• Spirilla: Non flexuous spiral forms eg.: Spirillum minus.
• Spirochetes: Slender, flexuous: Treponema, Leptospira.
• Actinomycetes (branching, filamentous rods).
• Myloplasma: Cell wall deficient bacteria, no definite shape.
Group patterns.
• Diplococci: N. gonorrhoeae.
• Chains: Streptococci (S. pyogenes) /Streptobacilli (S. moniliformis).
• Tetrads: Micrococcus.
• Octads: Sarcina.
• Clusters: S. aureus.
• Cuneiform / Chinese letter pattern (C. diphtheriae).
10 | Microbiology

Anatomy of Bacterial Cell:

Cell Wall:
• Complex rigid structure which gives bacteria shape.
• 10-20 nm in thickness, gram negative thinner.
• Strength due to peptidoglycan/mucopeptide / murein.
• Peptidoglycan:
Backbone : N-acetylglucosamine and N-acetyl muramic acid (NAG and NAM).
Tetrapeptide side chain.
Pentapeptide cross bridges.
Except in Bacillus anthracis, in which it is a polypeptide of D-glutamic acid.

Gram-Positive Cell Wall:

• 80nm thick.
• Composed mostly of several layers of peptidoglycan.
• Also contains teichoic acid (cell wall, membrane).
Bacterial Structures
Structure Chemical Function
Composition
Essential components
Cell Wall
Peptidoglycan Sugar backbone with Gives rigid support, protects against osmotic pressure,
peptide side chains is the site of action of penicillins and cephalosporins,
that are cross-linked. and is degraded by lysozyme.
Outer membrane of Lipid A. Toxic component of endotoxin.
gram-negative bacteria
Polysaccharide. Major surface antigen used frequently in laboratory
diagnosis.
Surface fibers of Teichoic acid. Major surface antigen but rarely used in laboratory
gram-positive diagnosis.
bacteria
Plasma membrane Lipoprotein bilayer Site of oxidative and transport enzymes.
without sterols.
Ribosome RNA and protein in Protein synthesis; site of action of aminoglycosides,
50S and 30S subunits. erythromycin, tetracyclines, and chloramphenicol.
Nucleoid DNA. Genetic material.
Mesosome Invagination of Participates in cell division and secretion.
plasma membrane. Bacterial respiratory centers.
Periplasm Space between plasma Contains many hydrolytic enzymes, including
membrane and outer -lactamases.
membrane.
Bacterial Structure and Pathogenesis | 11

Nonessential components
Capsule Polysaccharide. Protects against phagocytosis.
Glycocalyx Polysaccharide. Mediates adherence to surfaces.
Flagellum Protein. Motility.
Pilus or fimbria Glycoprotein. Two types: (1) mediates attachment to cell surfaces;
(2) sex pilus mediates attachment of two bacteria
during conjugation.
Spore Protein Provides resistance to dehydration, heat, and
Keratin like coat, chemicals.
dipicolinic acid.
Plasmid DNA. Contains a variety of genes for antibiotic resistance
and toxins.

Gram Negative Cell Wall:

• Thinner than gram positive bacteria.
• Structurally more complex.
• Peptidoglycan layer: Single unit, THIN.
• Outside to peptidoglycan: Outer membrane; phospholipid bilayer in which other
large molecules embeded (outer membrane protein, porins ).
• Relatively high antibitic resistance of gram negative bacteria.
• Outer membrane anchored to peptidoglycan by lipophilic lipoprotein.
• Lipopolysaccharide (LPS) unique to gram negative outer membrane.
• Complex lipid: Lipid ‘A’, to which is attached polysaccharide made up of core and a
terminal series of repeat unit
• Firmly bound to the cell surface, released only when cells are lysed.
• Endotoxic acitivity: Lipid–A.
• Polysaccharide : Major surface antigen, O antigen.
• Periplasmic space: Between inner and outer membrane; contains peptidoglycan layer
and penicillin binding proteins.

Acid-Fast Cell Wall:

• Certain genera : Myobacteria, Nocardia.
• Gram positive cell wall structure + waxy layer of glyolipids and fatty acids (mycolic
acids) bound the exterior of cell wall.
• Difficult to stain with gram stain.
ƒ Cell wall cannot be seen by light micro­scopy, does not stain by simple dye.
ƒ Can be demonstrated by plasmolysis, microdissection, reaction with specific anti-
body, mechanical rupture, differential staining and electron microscopy.
ƒ Protoplast: cytoplasmic membrane and its contents left following action of
lysozymes on gram positive cell.
ƒ Spheroplast: Some cell wall attached after action of lysozyme on gram negative
cell.
12 | Microbiology

L-FORMS
• Cell wall deficient bacteria.

Mycoplasma/Ureaplasma:
• Derived from normal bacteria in laboratory / spontaneously.
• Stable: special conditions are not required for preventing their reversion.
• Capable of growing and multiplying on a suitable medium.
• 0.1 – 20 µm in diameter.
• Solid media: Fried egg colony.
• Could account for bacterial persistence during therapy with certain antibiotics

Protoplast Spheroplast
Gram positive. Gram negative.
No cell wall remnants. Cell wall remnants present.
Grow in size but do not multiply. Mutiply by budding or fission.
Osmotically more sensitive. Osmotically less sensitive.
Cannot revert to parent bacterial form. Can revert to parent bacterial form.
Also called unstable ‘L’ forms.

Fig. 1.4: Bacterial Cell Wall

Cytoplasmic Membrane / Cell Membrane/Inner Membrane:

• 5-10 nm, limits bacterial protoplast externally.
• Elastic, phospholipid bilayer, lacks sterols.
Bacterial Structure and Pathogenesis | 13
• Semipermeable.
• Passive diffusion: water, small molecules.
• Selective transport: specific enzymes (permeases).

Cytoplasm:
• Viscous watery solution of soft gel.
• Organic and inorganic solutes, ribosomes.
• Organelles (-), cytoplamic steraming (-), amoeboid movement (-).

Ribosomes:
• Ribosomal RNA + ribosomal proteins.
• 70s divided into 50s and 30s.
• Polysomes: Thousands of ribosomes strung together on strands of mRNA.
• Site of protein synthesis.
• Different from ribosomes of eukaryotes: basis of selective action of amino­
glycosides and tetracyclines (act on 30s); chloramphemicol, macrolides and
lincosamides (act on 50s).

Mesosomes:
• Convoluted body, develops by invagination of cytoplasmic membrane.
• ↑Membrane surface, respiratory enzymes (analogous to mitochondria).
• Formation of cross wall during cell division.
• More prominent in Gram positive bacteria.

Intracytoplasmic Inclusions:
• Volutin granules: Babe-earnst/ Metachromatic/Polymetaphosphate/ Polar bodies.
ƒ Stained by Albert’s, Neisser’s, Ponder’s stain.
ƒ C.diphtheriae, diphtheroides.
• Lipid granules: Polymerised β-hydroxy butyric acid.
ƒ Stained by sudan black.
• Polysaccharide granules: Stained with iodine.
ƒ Glycogen (red brown), strach (blue).

Bacterial DNA:
• Single, circular, double stranded DNA molecule.
• Absence of nuclear membrane, nucleolus and basic proteins.
• Plasmids are extrachromosomal pieces of circular DNA that encode both exotoxins
and many enzymes that cause antibiotic resistance.
• Transposons are small pieces of DNA that move frequently between chromosomal
DNA and plasmid DNA. They carry antibiotic-resistant genes.
14 | Microbiology
Bacterial Capsule:
• Gelatinous layer.
• Diffuses into the medium : slime layer.
• Too thin to be seen with light microscope: microcapsule.
• Chemically:
• Usually Polysaccharide: Klebsiella, Hemophilus, S. pneumoniae, Cryptoco-
ccus neoformans.
• Polypedtide/protein: Bacillus anthracis (poly D-glutamate).
• Hyaluronic acid : Streptococcus pyogenes.
• Not demonstrated by Gram stain but by india ink, nigrosin staining, Quellung reaction
(swelling of capsule when specific antiserum is added).
• Protects from antibacterial agents, inhibits phagocytosis, lost on repeated subculture.

Flagella:
• Organs of locomotion.
• 3-20 µm long, 10-20 µm thick.
• Chemically : Protein called flagellin.
• Three parts : Filament, Hook, Basal body: Rod, Rings ≥ 2 (M [cytoplasmic membrane],
S [periplasmic space above cytoplasmic membrane], P [peptidoglycan], L [outer mem-
brane]).
• Rings P and L are absent in gram positive bacteria (S and M present).
Arrangement Example
Monotrichous Vibrio
Lophotrichous Helicobacter
Amphitrichous Spirilla
Peritrichous E.coli, Serratia, Proteus, Salmonella
Atrichous Shigella, Klebsiella

• Demonstrated by: dark ground microscopy, impregnation, hanging drop, U tube,

craigie’s tube, Electron microscopy.
• Antigenic, serodiagnosis.

Fig. 1.5:
Bacterial Structure and Pathogenesis | 15
Fimbriae / Pili:
• Hair like, 1-1.5 µm length, 4-8 nm thick.
• Straigher, thinner, shorter than flagella.
• Present on many Gram-negative bacteria: adherence.
• More numerous than flagella.
• Originate from cytoplasmic membrane.
• Composed of protein pilin.
• Function: Adhesion, agglutinate RBC’s of various species, sex pili (F pili) involved in
conjugation.

Endospores:
• Bacillus, Clostridium.
• Each bacterium forms one endospore which germinates into a single vegetative cell.
• Method of preservation. not replication.
• Highly resistant resting stage formed during nutrient depletion.
• LAYERS (from inside out): spore cytoplasm, spore wall (inner membrane), spore
cortex, spore coat (outer membrane), exosporium.

Fig. 1.6: Bacterial Endospore

• Round, oval, elongated; terminal, subterminal, central; bulging, non bulging.

• Dormant for many years.
• Extremely resistant to chemical, physical agents may be due to dipicolinic acid, a
calcium ion chelator found only in bacterial spores.
• Demonstrated by: Modified ZN staining, unstained in gram stain, unstained
preparation: Refractile.
16 | Microbiology
Important Features of Spores and their Medical Implications:
Important Features of Spores Medical Implications
• Highly resistant to heating; spores are not killed • Medical supplies must be heated to 121°C for at
by boiling (100°C), but are killed at 121°C. least 15 minutes to be sterilized.
• Highly resistant to many chemicals, including • Only solutions designated as sporicidal will kill
most disinfectants, due to the thick, keratin-like spores.
coat of the spore. • Wounds contaminated with soil can be infected
• They can survive for many years, especially in with spores and cause diseases such as tetanus
the soil. (C. tetani) and gas gangrene (C. perfringens).
• They exhibit no measurable metabolic activity. • Antibiotics are ineffective against spores because
• Spores form when nutrients are insufficient but antibiotics act by inhibiting certain metabolic
then germinate to form bacteria when nutrients pathways of bacteria. Also, spore coat is
become available. impermeable to antibiotics.
• Spores are produced by members of only two • Spores are not often found at the site of infections
genera of bacteria of medical importance, because nutrients are not limiting. Bacteria rather
Bacillus and Clostridium, both of which are than spores are usually seen in Gram-stained
gram-positive rods. smears.
• Infections transmitted by spores are caused
by species of either (endotoxin) Bacillus or
Clostridium.

Fig. 1.7: Stages of Spore Formation

Bacterial Structure and Pathogenesis | 17
Toxins:
• Substances produced or present in bacteria, which have direct toxic action on the
tissue cells. Two main types viz. exotoxins and endotoxins
Exotoxins Endotoxins
Proteins. Lipopolysaccharide.
Heat labile. Heat Stable.
Highly antigenic. Weakly antigenic.
Actively secreted by the cells. Integral part of cell wall.
Can be converted into toxoid. Cannot be converted into toxoid.
Acts on specific receptors, different exotoxins have All endotoxins have similar non-specific action.
different actions.
High potency. Low potency.
Produced by gram positive and negative bacteria. Produced by gram negative bacteria only.
Frequently coded by plasmids. Coded by chromosomal genes.

Exotoxins:
• Having lethal action.
ƒ C. botulinum toxin: Neuromuscular junction.
ƒ Tetanus toxin: Voluntary muscle.
ƒ Diphtheria toxin: Heart.

Important Mechanisms of Action of Bacterial Exotoxins:

Mechanism Of Action Exotoxin
ADP-ribosylation Diphtheria toxin, cholera toxin, Escherichia coli heat-labile toxin, and
pertussis toxin.
Superantigen Toxic shock syndrome toxin, staphylococcal enterotoxin, and erythrogenic
toxin.
Protease Tetanus toxin, botulinum toxin, lethal factor of anthrax toxin, and scalded
skin toxin.
Lecithinase Clostridium perfringens alpha toxin.

Exotoxins that Increase Intracellular cyclic AMP:

Bacterium Exotoxin Mode Of Action

Vibrio cholerae Cholera toxin. ADP-ribosylates Gs factor, which activates it, thereby
stimulating adenylate cyclase.

Escherichia coli Labile toxin. Same as cholera toxin.

18 | Microbiology

Bordetella pertussis Pertussis toxin. ADP-ribosylates Gi factor, which inactivates it, thereby
stimulating adenylate cyclase.

Bacillus anthracis Edema factor of anthrax Is an adenylate cyclase.

toxin.

• PYROGENIC EFFECT: Toxic shock syndrome toxin of S. aureus, S. pyogenes (super

antigens)
• ENTEROTOXIN: Cholera enterotoxin, LT and ST of E. coli, C. diffcile, C. perfringens,
B. cereus
• TOXINS ACTING ON SKIN: Epidermolytic toxin of S. aureus, erythrogenic toxin of
S. pyogenes.
• EXTRACELLULAR ENZYMES: Urease, hyaluronidase, coagulase, collagenase,
stretokinase.

Endotoxin:

Fig. 1.8: Effects of Endotoxin

Bacterial Structure and Pathogenesis | 19
Concept 1.5: Epidemiology of infectious diseases
Learning Objectives

Time Needed
1st reading 30 mins
nd
2 look 10 mins

Pathogenesis of Bacterial Infections:

Pathogenicity The ability of any bacterial species to cause disease in a susceptible human host.
Pathogen A bacterial species able to cause such disease when presented with favorable
circumstances (for the organism).
Virulence A term which presumes pathogenicity but allows expression of degrees from low to
extremely high, for example.
Low virulence Streptococcus salivarius is universally present in the oropharyngeal flora of humans.
On its own, it seems incapable of disease production, but if during a transient
bacteremia it lands on a damaged heart valve, it can stick and cause slow but steady
destruction.
Moderate Escherichia coli is universally found in the colon, but if displacement to other sites
virulence such as adjacent tissues or the urinary bladder regularly causes acute infection.
High virulence Bordetella pertussis, the cause of whooping cough, is not found in the normal
flora, but if encountered it is highly infectious and causes disease in almost every
nonimmune person it contacts.
Extremely high Yersinia pestis, the cause of plague, is also highly infectious, but in addition leads to
virulence death in a few days in over 70% of cases.

Sources of Infection:
Endogenous infection E. coli, E. faecalis: UTI; Viridans streptococci: Infective endocarditis.
Exogenous infections Human cases and carriers.
Carriers Harbors the pathogenic microorganisms without suffering from it.
Healthy: Harbors but never suffered from the disease.
Convalescent: Recovered from disease but harbors the pathogen.
Paradoxical: Who acquires the pathogen from another carrier.
Contact: Who acquires pathogen from a patient.
Animal cases and Contact with animal, animal bite, ingestion of milk and meat.
carriers Zoonoses.
Bacterial: Bovine tuberculosis, bubonic plague, anthrax.
Viral: Rabies.
Helminthic: Hydatid disease.
Fungal: Microsporum canis.
20 | Microbiology

Man generally acts as a Exception: Pneumonic plague.

dead end host.
Insects: Called as vectors.
Mechanical vectors: Carry the organisms on their legs, wings, and body. Transmit them onto
eatables, which act as source of infection e.g.: salmonellosis and shigellosis by
domestic fly.
Biological vectors: Those in which pathogen multiplies or undergoes developmental changes with
or without multiplication.
Propagative: When pathogen only multiplies: plague bacilli in rat fleas.
Cyclo-propagative: Pathogen undergoes developmental change and multiplies: malarial parasite in
female anopheles mosquito.
Cyclo-developmental: Pathogen undergoes development without multiplication: filarial parasite in
culex mosquito.
Extrinsic incubation Time required for the biological vector to become infective from the time of
period: entry of the pathogen into it.
Environment: Soil, water, food.
Soil: Tetanus, gas gangrene, mycetoma.
Water: Shigella, Salmonella, Vibrio cholerae, Polio virus, Hepatitis A virus.
Food: Organisms causing food poisoning, diarrhoea, dysentery.

Dose of Microorganisms Required to Produce Infection in Human Volunteers:

Microbe Route Disease-Producing Dose

Salmonella serotype Typhi Oral 105

Shigella spp. Oral 10–100

Vibrio cholerae Oral 108

Vibrio cholerae Oral + HCO3– 104

Mycobacterium tuberculosis Inhalation 1–10

Modes of Spread of Infection:

Inhalation Respiratory infections.
Common cold, influenza, measles, mumps, tuberculosis, whooping cough.
Droplet infection, dust.
Ingestion Intestinal infections.
Enteric fever, cholera, dysentery, food poisoning, polio, hepatitis A.
Eatables, water supply, contamination by insects.
Bacterial Structure and Pathogenesis | 21

Contact Direct or indirect contact with patient.

Direct contact/ contagious disease: sexually transmitted diseases.
Indirect contact/ infectious disease: through fomites; inanimate objects like clothing,
pencil, toys etc.; diphtheria, trachoma.
Inoculation Tetanus, Rabies.
Congenital Vertical transmission; abortion, miscarriage, still birth, congenital malformation
(Teratogenic infections), TORCH, syphilis, HIV.
Iatrogenic Injections, blood transfusion, dialysis etc.; HIV, HBV, HCV.

Important Modes of Transmission:

Mode of Transmission Clinical Example Comment
I. HUMAN TO HUMAN
A. Direct contact Gonorrhea. Intimate contact, e.g., sexual or passage through
birth canal.
B. No direct contact Dysentery. Fecal–oral, e.g., excreted in human feces, then
ingested in food or water.
C. Transplacental Congenital syphilis. Bacteria cross the placenta and infect the fetus.
D. Blood-borne Syphilis. Transfused blood or intravenous drug use can
transmit bacteria and viruses; screening of blood
for transfusions has greatly reduced this risk.
II. NON-HUMAN TO HUMAN
A. Soil source Tetanus. Spores in soil enter wound in skin.
B. Water source Legionnaire’s disease. Bacteria in water aerosol are inhaled into lungs.
C. Animal source
1. Directly Cat-scratch fever. Bacteria enter in cat scratch.
2. Via insect vector Lyme disease. Bacteria enter in tick bite.
3. Via animal excreta H e m o l y t i c - u r e m i c Bacteria in cattle feces are ingested in
syndrome caused by E. undercooked hamburger.
coli O-157.
D. Fomite source Staphylococcal skin Bacteria on an object, e.g., a towel, are transferred
infection. onto the skin.

Microbial Pathogenicity:
Pathogenicity: Capacity of an organism to initiate disease.

• It requires the following attributes: • Transmissibility: Communicability from one host to

a fresh host.
• Infectivity: Ability to breach the new host’s defenses.
• Virulence: Capacity of the pathogen to harm the host.
22 | Microbiology

Determinants of Virulence:
Adhesion:
Fimbriae : Responsible for tissue tropism important in certain bacteria like E. coli, N. gonorrhoeae.
Teichoic acids: Streptococcus, Staphylococcus.
CURLI : Some strains of E. coli and Salmonella have surface proteins called curli, which
mediate binding of the bacteria to endothelium and to extracellular proteins such as
fibronectin. Curli also interact with serum proteins such as factor XII–a component of
the coagulation cascade. Curli, therefore, are thought to play a role in the production
of the thrombi seen in the disseminated intravascular coagulation (DIC) associated
with sepsis caused by these bacteria.

Avoidance of Host Defence Mechanisms:

Capsules: Inhibits phagocytosis. H. influenzae, S. pneumoniae, N. meningitidis.
Streptococcal M protein: Inhibits phagocytosis.

Resistance to Killing by Phagocytic Cells:

M. tuberculosis: Inhibits phagolysosome fusion.
S. aureus and N.gonorrhoeae Catalase negates the effects of toxic oxygen radicals.

Antigenic Variation: Trypanosoma brucei, N. gonorrhoeae, B. recurrentis.

Iga1 Proteases: N. meningitides, H. influenzae, Streptococcus pneumoniae.
Serum Resistance: Able to resist lysis due to deposition of compliment; smooth strains
resistant than rough strains.
Collagenase and hyaluronidase, which degrade collagen and hyaluronic acid, respectively, thereby allowing
the bacteria to spread through subcutaneous tissue; they are especially important in cellulitis caused by
Streptococcus pyogenes.
Coagulase, which is produced by Staphylococcus aureus and accelerates the formation of a fibrin clot from
its precursor, fibrinogen (this clot may protect the bacteria from phagocytosis by walling off the infected area
and by coating the organisms with a layer of fibrin).
Leukocidins, which can destroy both neutrophilic leukocytes and macrophages.
Bacterial Structure and Pathogenesis | 23

Worksheet
• MCQ OF “BACTERIAL STRUCTURE AND PATHOGENESIS” FROM DQB

• EXTRA POINTS FROM DQB

1.

2.

3.

4.

5.

6.

7.

8.

9.

10

11.

12.

13.

14.

15.
24 | Microbiology
Active Recall from Tables
Prokaryote Eukaryote

Endotoxin Exotoxin
2 Bacterial Growth and Genetics

CONCEPTS
 Concept 2.1 Bacterial Growth curve
 Concept 2.2 Nutritional requirements
 Concept 2.3 Bacterial genetics basics & transfer
methods
 Concept 2.4 Molecular microbiology
26 | Microbiology
Concept 2.1: Bacterial Growth Curve
Learning Objectives:
• Different phases of growth curve
• Bacterial generation time

Time Needed
1 reading
st
15 mins
2 look
nd
5 mins

Growth and Physiology of Bacteria:

• Bacteria divide by binary fission.
• Nuclear division precedes cell division.

Bacterial Growth Curve:

It has got following four phases:
Lag phase: Multiplication does not begin
immediately. Bacteria adapt
themselves. There is increase in
size and metabolic activity.
Log phase: Bacteria multiply at their
maximal rate, number increases
exponentially, limited duration.
Uniform in Gram staining.
Stationary Number of viable cells remains
phase: constant. Spore formation,
storage granules and bacteriocin
production.
Phase of Death rate exceeds the rate of
Fig. 2.1: Bacterial Growth Curve decline: reproduction. Involutional forms.

Generation Time:
• Time required for a bacterium to give rise to two daughter cells.
• E. coli: 20 minutes, M. tuberculosis: 16 –18 hours, M. leprae: 14 days.
Total Count:
Total number of bacteria present in a specimen irrespective of whether they are living
or dead.
Viable Count:
This measures only living cells which are capable of growing and producing a colony on
a suitable medium.
Bacterial Growth and Genetics | 27
Concept 2.2 . Bacterial Nutritional requirements
Learning Objectives
• Different nutritional requirement for the bacteria
• Examples for each group

Time Needed
1 reading
st
30 mins
2 look
nd
10 mins

Bacterial Nutrition:
All bacterial have 3 major nutritional needs.
• Source of carbon (cellular components).
• Source of nitrogen (proteins).
• Source of energy (synthesis of macromolecules and to maintain chemical gradients).

Basic building blocks required for growth are the same for all cells;
bacteria vary widely in their ability to use different sources of these molecules
Phototrophs: Derive energy from sunlight.
Chemotrophs: Derive energy from chemical oxidation.
Autotrophs: Can synthesize all organic compounds: Utilize atmospheric CO2 and N2.
Heterotrophs: Depends on preformed organic compounds: Cannot utilize CO2 as sole
source of carbon.
Organotrophs: Requires organic sources of hydrogen donor.
Lithotrophs: Use inorganic sources of hydrogen (ammonia, hydrogen sulphide).
Vast majority of medically Chemo-organotrophs.
important organisms:

Physical Conditions Required for Growth:

Oxygen
Obligate aerobes (Strict aerobes) Require oxygen. e.g. Pseudomonas spp.
Obligate anaerobes (Strict anaerobes) Require complete absence of O2. e.g. B. fragilis.
Facultative anaerobe Grow better in oxygen but still able to grow in its absence. e.g.
E. Coli, S.aureus.
Aerotolerant anaerobe Anaerobic, but tolerates exposure to oxygen. e.g. C. perfringens.
Microaerophilic organism Requires or prefers reduced oxygen levels. (5%). e.g.
Campylobacter spp., Helicobacter spp.
Capnophilic Requires or prefers increased carbon dioxide levels. e.g.
Neisseria spp., Brucella.
28 | Microbiology

Fig. 2.2: Oxygen requirement

Temperature:
Psychrophile Grows best at low temperature (<20°C). e.g. Flavobacterium spp.

Thermophile Grows best at high temprature (>60°C). e.g. Geobacillus stearothermophilus, Thermus
aquaticus.

Mesophile Grows best between 20 – 40°C; most bacterial pathogens.

pH:
Acidophile Grows best at acidic pH (<7.0). e.g. Lactobacillus

Neutrophile Grows best at neutral pH (7.0-7.2) most bacterial pathogens.

Alkalophile Grows best at alkaline pH (>7.0). eg : Vibrio cholerae

Iron Metabolism:
• Iron, in the form of ferric ion, is required for the growth of bacteria because it is an
essential component of cytochromes and other enzymes.
• To obtain iron for their growth, bacteria produce iron-binding compounds called
siderophores, which have very high binding affinity. These compounds, such as
enterobactin produced by E. coli, are secreted by the bacteria, capture iron by
chelating it, then attach to specific receptors on the bacterial surface, and are actively
transported into the cell where the iron becomes available for use.
Bacterial Growth and Genetics | 29
Concept 2.3: Bacterial genetics basics & transfer methods
Learning Objectives
• Basics of bacterial genetics
• Different gene transfer methods
• Examples and medical applications

Time Needed
1 reading
st
30 mins
2 look
nd
10 mins

Bacterial Genetics:
• Study of genes, their structure, function, heredity and variation.
• Bacteria obey the laws of genetics.
• Genetic information contained in DNA.
• Central dogma of molecular biology.
DNA RNA Polypeptide
• Structure of DNA molecule:
ƒ Pyrimidine bases (Thymine, Cytosine).
ƒ Purine bases (Guanine, Adenine).
ƒ Pair A=T; G ≡ C.
ƒ Dexoyribose sugar.
• Structure of RNA:
ƒ Ribose sugar.
ƒ Uracil instead of Thymine.
ƒ rRNA: Synthesis of proteins.
ƒ tRNA : Accepts single amino acid and transfers it to a ribosome.
ƒ mRNA: Template for translation.
• Genetic information stored as code: codon.
Triplet of Bases:
Each code specifies for a single amino acid
More than one code may exist for a single amino acid.
Code is therefore said to be degenerate.
• Start codon: AUG.
• Nonsense codons: UAA, UAG, UGA.
• Shine dalgarno sequence: Present upstream of start codon, required correct
alignment on mRNA in within the two subunits of ribosome.
• Cistron/gene: Segment of DNA carrying a number of codons specifying for a particular
polypeptide is known as cistron/gene.
30 | Microbiology
• Locus: Large number of genes.
• Genome: All the loci.

Extra Chromosomal Genetic Elements:

• Plasmids: Extrachromosomal genetic elements that can replicate autonomously
and can maintain in the cytoplasm of a bacterium for many generations.
• Circular piece of DNA.
• Not essential for normal life, confer on it additional properties: drug resistance,
bacteriocin production, toxigenicity etc.
ƒ Information for controlling its replication.
ƒ May get integrated into host genome: Episome.
ƒ Some confer ability to conjugate (F Plasmid): Conjugative/self transmissible plas-
mid.
Plasmid Determined Properties:
• Resistance to:
ƒ Antibiotics.
ƒ Heavy metal ions.
ƒ Metabolic functions.
ƒ Citrate utilization.
ƒ Fermentation of lactose, raffinose, sucrose.
• Virulence factors:
ƒ Entero toxins.
ƒ Colicin.
ƒ Exfoliativetoxin etc.
Genotypic/ Phenotypic Variations:
• Phenotype: Characteristics expressed by a cell in a given environment.
• Genotype: Collection of genes encoding these characteristics.
• Bacteria are very adaptable, may alter their phenotype in response to environmental
change, while genotype remains unchanged.
• Not all genes of bacteria express all the time:
ƒ Typhoid bacillus: No Flagella when growth in phenol agar.
ƒ Synthesis of b galactosidase: Produced only when lactose is present in the medium
(induced enzymes).
ƒ Economy of nature.
Acquisition of New Genes:
• Mutation.
• Acquiring new DNA from external source.
• Transformation.
• Transduction.
• Conjugation.
Bacterial Growth and Genetics | 31
Comparison of Conjugation, Transduction, and Transformation:
Transfer Procedure Process Type of Cells Involved Nature of DNA
Transferred
Conjugation DNA transferred from Prokaryotic. Chromosomal or
one bacterium to another. plasmid.
Transduction DNA transferred by a Prokaryotic. Any gene in generalized
virus from one cell to transduction; only
another. certain genes in
specialized transduction.
Transformation Purified DNA taken up Prokaryotic or Any DNA.
by a cell. eukaryotic (e.g. human).

Transformation:
• Acquisition of DNA from environment and incorporation in its genome.
• First demonstrated in S. pneumoniae then also demonstrated in other bacteria like B.
subtilis, H. influenzae, N. gonorrhoeae.
• DNA must be derived from closely related strain.
• Cells that can take up naked DNA are called as competent.
• First demonstrated by Griffith 1928.
• Transformation can be inhibited by DNAases.
• When purified DNA is injected into the nucleus of a eukaryotic cell, the process is called
transfection. Transfection is frequently used in genetic engineering procedures.

Fig. 2.3: Griffith experiment

32 | Microbiology
• Genetic transformation is recognized as a major force in microbial evolution. Natural
transformation is an active process demanding specific proteins produced by the
recipient cell. For Neisseria and Haemophilus spp. specific DNA sequences (uptake
sequences) are required for uptake of the DNA. These uptake sequences are species
specific, thus restricting genetic exchange to a single species.
Transduction:
• Transfer of a portion of DNA from one bacterium to another by bacteriophage
Generalized Transduction:
• Due to phages that lyse host cell: Virulent phages.
• During assembly, phage head is filled with host cell DNA (packaging error).
• Such a phage infects a second bacterium, DNA enters.
• Can transduce any gene.
• Size similar to phage genome.
• B/w closely related strains.
• Chromosomal DNA as well as plasmid DNA.
• E.g. penicillin resistance in S. aureus.
Specific Transduction:
• Due to phages incorporated into host genome: Temperate phages (Lysogenic)
• When infected with temperate phage: small proportion get incorporated into host
cell genome.
• In some lysogenic cell lytic cycle is resumed → The prophage (integrated phage
DNA) is excised → In some cells prophage is excised inaccurately so that neighboring
portion of bacterial DNA is also removed → Such piece of DNA is transduced into the
second cell and integrates to the specific site.
• The size of DNA in transducing particles is usually no more than several percent of the
bacterial chromosome and therefore co-transduction i.e. transfer of more than one
gene at a time-is limited to linked bacterial genes.
• This process is of particular value in mapping genes that lie too close together to be
placed in map order using the gross method of conjugal transfer. Mutant phages can
be identified on the basis of the morphology of the plaque they form by lysis of a
lawn of bacteria growing on solidified agar medium.
• Pathogenicity islands are transported by phages. E.g.: two phages transport
pathogenicity islands and convert a benign form of Vibrio cholerae into the
pathogenic form responsible for epidemic cholera. These phage encode genes for
cholera toxin and bundle forming pili which function in attachment.

Fig. 2.4: Generalized Transduction

Bacterial Growth and Genetics | 33
Lysogenic Conversion:
• In lysogenic bacteria, prophage codes for new characters.
• Corynebacterium diphtheriae, diphtheria toxin is coded by b phage.
• S. pyogenes: Dick toxin is coded by a bacteriophage.
• C. botulinum: Botulinum toxin.
• V. cholera: Cholera toxin.
• S. dysenterae: Shiga’s toxin.

Fig. 2.5: Lytic and Lysogenic cycle

Conjugation:
• Transfer of DNA that occurs during contact between bacterial cells.
• Common among gram negative bacteria.
• Plasmid – codes for Sex Pilus, 1–2 mm length.
• Pilus attaches to the surface of recipient cell and holds the two cells together.
• The plasmid DNA replicates and a copy of it passes through the sex pilus.
• F-Factor: Genetic information for synthesis of sex pilus, which is required for self
transfer.
• F-Factor has an ability to integrate in the host chromosome: Hfr cells.
• F-Factor with host chromosomal genes: F’ Factor (transferred by sexduction).
• R- Plasmids: RTF + r, Resistance to eight or more drugs may be transferred simulta-
neously, most common method of resistance transfer.
• E. coli, K. pneumoniae, Salmonella, Shigella, Pseudomonas.
34 | Microbiology

Fig. 2.6: Conjugation

Fig. 2.7: High Frequency Recombination

Bacterial Growth and Genetics | 35
Mutation:
• Random, undirected, heritable variation caused by an alteration in the nucleotide
sequence.
• Addition/deletion/substitution of one/more bases.
• One per 102-1010 cell divisions.
• Mutants will outnumber and overgrow if their new character makes them better fitted
to grow under the prevailing conditions: Selection of mutants.
• Frequency increased: Nitrogen mustard, acriflavine, mitomycin-C, 5- bromouracil,
2- aminopurine.
• Missense mutation: Triplet code altered, codes for amino acid different from that
normally located at a particular position.
• Nonsense mutation: Premature polypeptide chain termination.
• Transversion: Substitution of purine for pyrimidine and vice versa.
• Lethal mutation: Involve vital functions, conditional lethal mutants: Live under
permissive conditions but not under non permissive conditions.
• Temperature-sensitive mutant (T.S.) : Live at 35°C; dies at 39°C.
• Most mutations are unrecognized.
• Test to determine whether a chemical is mutagen or carcinogen: Ames test.
Transposable Genetic Elements:
• Transposition requires no homology between transposable elements and its site of
insertion.
• Jumping Genes: Move from one plasmid to another, to a phage or to the bacterial
chromosome.
• Do not contain genetic information necessary for their own replication.
• IS (insertion sequences): Small, cryptic (1–2 kb).
• Transposon: Large (4–25 kb), encode at least one function, has a segment of DNA
coding for one/more genes at the centre and two ends carrying inverted repeat
sequences. Single stranded loop and double stranded stem.
• The genes may code for such properties as antimicrobial resistance, substrate
metabolism, or other functions.
• Composite transposons translocate by what is called simple or direct transposition,
in which the transposon is excised from its original location and inserted in a simple
cut-and-paste manner into its new site without replication.
• Another mechanism called replicative transposition leaves a copy of the replicative
transposon at its original site.
36 | Microbiology
Concept 2.4: Molecular microbiology
Learning Objectives
• Genetic recombinant technology
• Nucleic acid amplification technology
• Applications

Time Needed
1 reading
st
20 mins
2 look
nd
10 mins

Recombination DNA Technology:

• Insertion of foreign DNA molecule into DNA of a vector, which can replicate
autonomously in a suitable host.
• DNA endonucleases / restriction endonucleases: Cut DNA at specific sequences.
• Produced as part of defense against the insertion of foreign DNA.
• Target site comprise of four-six base pairs which are ‘Palindromic’.
• Blunt ends; Sticky ends.
• Rejoined with ligase.
• Uses:
ƒ Vaccine production: Hepatitis B Vaccine.
ƒ Antigen production: gp 120 of HIV.
ƒ Proteins: Human growth hormones, insulin, interferons.
ƒ Gene therapy.
Polymerase Chain Reaction:
• Discovered by Kary B Mullis.
• Primer mediated temperature dependent technique for enzymatic amplification of a
target sequence to such an extent that it can be detected.
• Target DNA.
• Primers.
• Taq Polymerase.
• Nucleotides.
• PCR Steps.

ƒ Denaturation 94°C 
ƒ Annealing 55°C One cycle


ƒ Primer extension 72°C 

Detection of Amplified Sequence by:

ƒ Gel electrophoresis.
ƒ Southern blotting.
ƒ Colorimetric method.
Bacterial Growth and Genetics | 37
Typing of Bacteria:
• A population of bacteria presumed to descend from a single bacterium as found in
a natural habitat, in primary cultures from the habitat and in subcultures from the
primary cultures, is called strain.
• Each primary culture from a natural source is called an isolate.
• Culture of typhoid bacilli isolated from 10 different patients should be regarded as 10
different isolates unless epidemiological evidence indicates that the patient have been
infected from a common source with the same strain.
• Strains with demonstrable ancestral lineage have been termed as clones (this term
should be used with caution).
Biotyping: Differences in biochemical reaction which are not universally + or − with in a species
e g. classical, and eltor Vibrio cholerae.
Serotyping: Many surface structures of bacteria (LPS, omp, flagella, capsule etc.) are antigenic
and antibodies raised against them can be used to group isolates e.g. S. pneumoniae,
Shigella spp.
Phage typing: Difference in susceptibility to bacteriophage: S. aureus, S. typhi, V. cholerae.
Bacteriocin typing: Difference in production/susceptibility to bacteriocins e.g. Proteus spp.
Protein typing: Differences in protein composition following disruption of cells and electrophoresis
in gel.
Antibiogram: Differences in susceptibility to a set of antibiotics.

Restriction Endonuclease Typing:

• Chromosomal.
• Plasmid.
• Frequent cutter endonuclease.
• Rare cutter endonuclease (PFGE).
Gene Probe Typing:
• Cloned specific.
• Random / universal sequences.
• Detect restriction site heterogeneity in the target DNA.
• Detect variation in rDNA gene loci: ribotyping.
• Some genera like Mycoplasma cannot be typed as they have single copy of rRNA
operon.
PCR Typing:
• Specific DNA sequences amplified.
• Specific primers.
• Random primers.
• Random ampified polymorphic DNA typing.
• Arbitrarily primed PCR.
38 | Microbiology

Fig. 2.8: Polymerase Chain Reaction

Bacterial Growth and Genetics | 39

Worksheet
• MCQ OF “BACTERIAL GROWTH AND GENETICS” FROM DQB

• EXTRA POINTS FROM DQB

1.

2.

3.

4.

5.

6.

7.

8.

9.

10

11.

12.

13.

14.

15.
40 | Microbiology
Active Recall from Tables
Growth phase Changes

Typing methods Definitions

3 Bacterial Culture and
Sterilization

CONCEPTS
 Concept 3.1 Bacterial Culture media
 Concept 3.2 Bacterial Culture methods
 Concept 3.3 Sterilization and disinfection
 Concept 3.4 Antimicrobial susceptibility testing
42 | Microbiology
Concept 3.1: Bacterial culture media
Learning Objectives
• Different types of culture media
• Applications

Time Needed
1 reading
st
20 mins
2 look
nd
5 mins

Culture Media:
• Initially liquids (meat broth etc.) were used as culture media.
• Disadvantages:
ƒ May not exhibit specific characteristics for identification.
ƒ Difficult to isolate different types of bacteria from mixed populations.
• Advantages:
ƒ Used when large volumes used as inoculum (Blood, Water).
ƒ Preparing bulk cultures for antigens and vaccines.
ƒ Preparation of inoculum for biochemical reactions and antibiotic susceptibility
testing.
• Solid media introduced by Robert Koch:
ƒ Pieces of potato.
Gelatin (15%): liquefies at 24°C or by proteolytic bacteria.
ƒ
Agar-agar:
ƒ
▫ Prepared from seaweed.
▫ Remains solid at all incubation temperatures.
▫ Bacteriologically inert.
▫ Does not add to nutritive properties of the medium.
▫ Long chain polysaccharide, D-galactopyranose units.
▫ Concentration used: 1-2%.
▫ Melting point: 95°C and Solidifying Point: 42°C.
• Peptone:
ƒ Water soluble products obtained from pertinacious material e.g. lean meat, heart
muscle casein, fibrin, soya flour.
ƒ By digestion with proteolytic enzymes pepsin, trypsin and papain.
ƒ Peptones, proteases, amino acids, inorganic salts, accessory growth factors.

Types of Culture Media:

Simple (basal), complex, synthetic or defined, special media: enriched, enrichment, selective, indicator,
differential, sugar, transport media.
Solid, liquid, semisolid, liquid media.
Aerobic, anaerobic media.
Bacterial Culture and Sterilization | 43

Simple/basal media Includes peptone water, nutrient broth and nutrient

agar.
Nutrient broth: Three types.
Meat infusion broth: Aqueous extract of lean meat to which
peptone is added.
Meat extract broth: Mixture of meat extract (commercially
prepared, aqueous extract of lean meat concentrated by evaporation)
with peptone.
Digest broth: Aqueous extract of lean meat that has been
digested with proteolytic enzyme so that additional peptone is not
required.
Complex medium Have added ingredients for special purposes, bringing out certain
characteristics, providing special nutrients.
Synthetic/defined media Prepared from pure chemical substances and exact chemical
composition of the medium is known; semisynthetic media: e.g.
Simple peptone water, 1% peptone with 0.5% nacl.
Enriched media Substances such as blood, serum, egg is added to the basal medium to
grow fastidious bacteria. E.g. Blood agar, chocolate agar, LSS.
Enrichment media: When substances are added to liquid medium which inhibit the
growth of the unwanted bacteria and favor the growth of the wanted
bacteria. E.g. Tetrathionate broth, Selenite F broth,
GN broth, alkaline peptone water. (Enrichment
medium is always liquid medium).
Selective media: When substances are added to solid medium which inhibit the
growth of the unwanted bacteria and permit the growth of the wanted
bacteria. E.g. MacConkey’s agar, DCA, Wilson Blair
medium, XLD. (Selective medium is always solid
medium).
Indicator media Contain an indicator which changes colour when bacteria grows in
them. E.g. MacConkey’s agar, Christensen’s urea agar, Potassium
tellurite blood agar.
Differential media Has substances incorporated in it, enabling to bring out differing
characteristics of bacteria and thus helping to distinguish between
them. E.g. Macconkey’s agar, blood agar.
Sugar media Fermentation of sugars are used for identification of various bacteria.
Monosaccaride (arabinose, dextrose), disaccharide (lactose, sucrose),
trisaccharide (raffinose), polysaccharide (starch), alcohols (glycerol),
glucosides salicin, aesculin), noncarbohydrates (inositol). Durham’s
tube used to detect gas production.
Transport media: Used to maintain the viability of a pathogen and to avoid overgrowth of
other contaminants during transit from the patient to the laboratory. E.g.
Stuart’s & Amies transport medium (Gonococci),
V.R. medium (V. cholerae).
44 | Microbiology

Storage media Used for maintenance of bacterial cultures. E.g. Semisolid nutrient
agar stabs, cooked meat broth, nutrient agar, blood agar, heated blood
agar slopes.
Anaerobic media Used to grow anaerobic organism. Robertson’s cooked meat
medium, thioglycollate broth.
Nutrient broth can be solidified by addition of 1-2% agar (Nutrient agar; solid medium), Semisolid agar: agar
concentration 0.2-0.5%, Hard agar: agar concentration: 6%.

Fig. 3.1: Gaspak – Anaerobic culture Fig. 3.2: McIntosh – Fildes’ Anaerobic Jar

Fig. 3.3: RCM medium

Bacterial Culture and Sterilization | 45
Commonly used Bacteriologic Agars and their Function:
Name of Agar Bacteria Isolated Function or Properties
on this Agar of the Agar
Blood Various bacteria. Detect hemolysis.
Bordet-Gengou Bordetella pertussis. Increased concentration of blood allows
growth.
Buffered charcoal- Legionella pneumophila. Increased concentration of iron and cysteine
yeast extract allows growth.
Chocolate Neisseria meningitidis and Heating the blood inactivates inhibitors of
Neisseria gonorrhoeae growth.
from sterile sites.
Chocolate agar plus Haemophilus influenzae. X and V factors are required for growth.
X and V factors
Egg yolk Clostridium perfringens. Lecithinase produced by the organism degrades
egg yolk to produce insoluble precipitate.
Eosin-Methylene Various enteric gram- Selects against gram-positive bacteria and
Blue negative rods. differentiates between lactose fermenters and
non-fermenters.
Lowenstein-Jensen Mycobacterium Selects against gram-positive bacteria in
tuberculosis. respiratory tract flora and contains lipids
required for growth.
MacConkey Various enteric gram- Selects against gram-positive bacteria and
negative rods. differentiates between lactose fermenters and
nonfermenters.
Potassium Tellurite Corynebacterium Tellurite metabolized to tellurium, which has
diphtheriae. black color.
Thayer-Martin N. gonorrhoeae from Chocolate agar with antibiotics to inhibit
nonsterile sites. growth of normal flora.
Triple sugar iron Various enteric gram- Distinguishes lactose fermenters from
(TSI) negative rods. nonfermenters and H2S producers from
nonproducers.
46 | Microbiology
Concept 3.2: Bacterial culture methods
Learning Objectives
• Different types of culture methods
• Applications

Time Needed
1 reading
st
15 mins
2 look
nd
5 mins

Culture Methods
• Streak culture
ƒ Useful method for obtaining discrete colonies and pure cultures.
• Lawn culture
ƒ Useful for carrying out antibiotic sensitivity testing by disc diffusion method (Kirby
– Bauer Method)
ƒ For bacteriophage typing
ƒ Producing large amount of bacterial growth required for preparation of bacterial
antigens and vaccines
• Pour-plate culture
ƒ Used to quantitate bacteria in urine cultures
ƒ To estimate viable bacterial count in a suspension.
ƒ Miles – Misra method
• Stroke culture
ƒ Provides pure growth of bacteria for carrying out diagnostic tests.
• Stab culture
ƒ Used for maintaining stock cultures.
ƒ For demonstration of oxygen requirement of the bacteria
• Anaerobic culture methods
ƒ Obligate anaerobes
ƒ Robertson cooked meat broth (RCM), Thioglycolate broth & neomycin blood agar.
ƒ Environment is free of oxygen, followed by incubation at 35°C for at least 48
hours.
ƒ McIntosh-Fildes anaerobic jar, Gas pack system & Anaerobic glove box
Bacterial Culture and Sterilization | 47
Concept 3.3: sterilization & Disinfection
Learning Objectives
• Definitions
• Classification
• Physical & Chemical methods
• Applications
• Spaulding classfication

Time Needed
1 reading
st
30 mins
2nd look 10 mins

Definitions :
Sterilization and Disinfection:
• Death/killing as it relates to microbial organisms is defined in terms of how we detect them in
culture. Operationally, it is a loss of ability to multiply under any known conditions.

• Sterilization is complete killing, or removal, of all living organisms from a particular location or
material. It can be accomplished by incineration, nondestructive heat treatment, certain gases, exposure
to ionizing radiation, some liquid chemicals, and filtration.

• Pasteurization is the use of heat at a temperature sufficient to inactivate important pathogenic

organisms in liquids such as water or milk, but at a temperature lower than that needed to ensure
sterilization. For example, heating milk at a temperature of 72°C for 20 seconds or 63°C for 30 minutes
kills the vegetative forms of most pathogenic bacteria that may be present without altering its quality.
Obviously, spores are not killed at these temperatures.

• Disinfection is the destruction of pathogenic microorganisms by processes that fail to meet

the criteria for sterilization. Pasteurization is a form of disinfection, but the term is most commonly
applied to the use of liquid chemical agents known as disinfectants, which usually have some degree of
selectivity. Bacterial spores, organisms with waxy coats (e.g. Mycobacteria), and some viruses may show
considerable resistance to the common disinfectants.

• Antiseptics are disinfectant agents that can be used on body surfaces such as the skin or vaginal
tract to reduce the numbers of normal flora and pathogenic contaminants. They have lower toxicity than
disinfectants used environmentally, but are usually less active in killing vegetative organisms.

• Sanitization is a less precise term with a meaning somewhere between disinfection and cleanliness.
It is used primarily in housekeeping and food preparation context

• Asepsis describes processes designed to prevent microorganisms from reaching a protected

environment. It is applied in many procedures used in the operating room, in the preparation of therapeutic
agents, and in technical manipulations in the microbiology laboratory. An essential component of aseptic
techniques is the sterilization of all materials and equipment used.
48
|
Microbiology

Fig. 3.4: Microbial Control Methods

Bacterial Culture and Sterilization | 49

Physical Agents Chemical Agents

Sunlight Phenols/ cresols.
Drying Halogens.
Heat Metallic salts.
Filtration Aldehydes.
Radiation Alcohol.
Dyes.
Vapour-phase disinfectants.
Surface active disinfectants.

Heat:
• Most reliable and rapid method of sterilization.
• Easily controlled, no harmful residue.
Dry heat:
• Kills by oxidation and protein
denaturation.
• Red heat: Wires, loops, points of forceps.
• Flaming: Scalpel blades, glass slides, mouth
of culture tubes.

Fig. 3.5: Bacteriological loop sterilizer – Dry heat

Hot Air Oven: 160°C for 2 hrs or 170°C

for 1 hour:
• Sterilization of glassware: Glass syringe, test
tube, petri dishes, pipettes, flasks.
• Metal instruments: Forceps, scissors, scalpels.
• Oils, jellies, powders, fats.
• Controls: Biological: Bacillus atropheus 106
spores (filter paper strip).
• Chemical control: Browne's tubes (red-green).

Fig. 3.6: Hot air oven – Dry heat

50 | Microbiology
Moist heat:
• Kills by denaturation and coagulation of proteins.
• Temperature below 100°C.
• Temperature at 100°C: Boiling water; free steam.
• Temperature above 100°C.

Temperature below 100°C:

• Heat labile fluids disinfected.
• Pasteurization:
ƒ 63°C for 30 min (Holder method).
ƒ 72°C for 20 sec (Flash method).
• Serum 56°C – 1hr
• Vaccine 60°C – 1hr.
• House hold utensils, clothing 70–80°C several minutes.
• Low Temprature Steam- Formaldehyde sterilization (LTSF):
ƒ Method of sterilization.
ƒ Steam at subatmopheric pressure.
ƒ Temp: 75°C

Moist heat, Temp at 100°C:

• Boiling at 100°C: 10-30 minutes.
• Some bacterial spores not killed.
• Metal, glass, rubber items.

Free steam at 100°C:

• Latent heat.
• Culture media containing sugar, gelatin.
• Some spores not killed.
• Tyndallization/ Intermittent sterilization: 100°C
• 20 minutes for three consecutive days.

Moist heat, Temp >100°C:

• Steam under pressure (autoclaving).
• 15 psi, 121°c, 15-20 min.
• Dressing material, linen, gloves.
• Culture media, aqueous solution.
• Method of sterilization.
• Controls: Geobacillus stearother­mophilus 106 spores.
• Chemical: Browne's tube.

Fig. 3.7: Autoclave – Moist heat

Bacterial Culture and Sterilization | 51
Comparison of Times and Temperatures to Achieve Sterilization with
Moist and Dry Heat:
Temperature Time to Sterilize
Moist Heat 121°C 15 min
125°C 10 min
134°C 3 min
Dry Heat 121°C 600 min
140°C 180 min
160°C 120 min
170°C 60 min

Filteration:
• Liquids such as sera, solutions of heat labile substances – sugars, urea sterilized by
filtration.
• Mycoplasma, viruses cannot be kept back by the bacterial filters.
• Control: Brevundimonas diminuta.
Earthenware filters • (Kieselguhr) fossil diatomaceous earth
• Chamberland made of unglazed porcelain
Asbestos (seitz) filters • Disc of magnesium trisilicate
• Vaccines

Fig. 3.8: Seitz filter

Sintered glass filters – Viruses.

Membrane filters – Cellulose esters (0.015- 12µm), most commonly used filters.
Syringe filters.
Air filters: HEPA remove particles more than 0.3µm.
52 | Microbiology
Radiation:
• Non-ionizing: UV radiation 250-260 nm, spores resistant, HIV not inactivated. Induce
thymidine/ pyrimidine dimers, disinfection of clean surfaces.
• Ionizing: α rays, gamma rays, cosmic rays, sterilization of pre packed disposable
items (cold sterilization) control: Bacillus pumilis spores; ↑cost.

Chemical Agents:
Phenols and cresols:
• Cause cell membrane damage.
• Resistant to inactivation by organic matter.
• Active against Gram +ve, Gram −ve, moderately active against Mycobacteria.
• Little activity against spores and viruses.
• Use: Discarded cultures, pipettes, other infected material.
• Phenol: Bactericidal 3–5%.
Halogens:
• Chlorine, hypochlorite, inorganic/organic chloramines.
• Bactericidal, sporicidal, viricidal, little activity against M. tuberculosis.
• Release of free chlorine → strong oxidizing agent.
• Activity decreased by presence of organic matter.
Iodine:
• Alcoholic/aqueous solutions: skin antiseptic.
• Also, active against M. tuberculosis.
• Iodophors: Mixture of iodine with surface active agents, one of the best antiseptic.
Metallic salts:
• Mercury: Combines with -SH group of bacterial proteins e.g. Merthiolate:
preservation of sera.
• 1% silver nitrate: Prophylaxis for gonococcal ophthalmia neonatorum.

Aldehydes:
Formaldehyde:
• Irritant, water soluble gas.
• Lethal to bacteria, spores, fungi, viruses.
• Cheap.
• Sterilization of rooms, furniture, clothing blankets, mattresses.
• Less effective in presence of organic matter.
Glutaraldehyde:
• More effective, less irritant.
• 2% solution: Sterilization of heat sensitive instruments cystoscopes, bronchoscopes
(Fiber optic scopes), thermometer.
Bacterial Culture and Sterilization | 53
Alcohols:
• Kill Bacteria, no action on spores, viruses.
• 60–70%; presence of water essential.
• Isopropyl alcohol: Better fat solvent, more bactericidal, less volatile.

Vapour Phase Disinfectants:

Ethylene oxide:
• Colorless gas soluble in water.
• Sterilization of plastics, rubber articles etc.
• Explosive mixture when > 3% present in air.
• Alkylating action on proteins.
• Control: Bacillus globigi.
Surface active disinfectants:
• Reduction of surface tension.
• Possess both hydrophobic and hydrophilic groups.
• Anionic, cationic, non-ionic, amphoteric.
• Cationic, quaternary ammonium compounds, most important.
ƒ Bacteriostatic.
ƒ Weak detergent.
ƒ Gram +ve > gram −ve.
ƒ Active against enveloped viruses.
• Anionic, common soap:
ƒ Strong detergent.
ƒ Weak antimicrobial.
• Amphoteric, tego compounds:
ƒ Detergent properties of anionic compounds.
ƒ Antimicrobial activity of cationic compounds.

Methods of Disinfection and Sterilization:

Method Activity Level Spectrum Uses/Comments
Heat
Autoclave Sterilizing All General
Boiling High Most pathogens, some General
spores
Pasteurization Intermediate Vegetative bacteria Beverages, plastic
hospital equipment
Ethylene oxide gas Sterilizing All Potentially explosive;
aeration required
54 | Microbiology

Radiation
Ultraviolet Sterilizing All Poor penetration
Ionizing Sterilizing All General, food
Chemicals
Alcohol Intermediate Vegetative bacteria,
fungi, some viruses
Hydrogen peroxide High Viruses, vegetative Contact lenses;
bacteria, fungi inactivated by organic
matter
Chlorine High Viruses, vegetative Water; inactivated by
bacteria, fungi organic matter
Iodophors Intermediate Viruses, vegetative Skin disinfection;
bacteria, fungi inactivated by organic
matter
Phenolics Intermediate Some viruses, vegetative Handwashing
bacteria, fungi
Glutaraldehyde High All Endoscopes, other
equipment
Quaternary ammonium Low Most bacteria and fungi, General cleaning;
compounds lipophilic viruses inactivated by organic
matter

Fig. 3.9: Spaulding classification

Bacterial Culture and Sterilization | 55
Testing the Efficacy of Disinfectants:
• Minimum inhibitory concentration (MIC): Lowest concentration of the disinfectant
that inhibits the growth of Salmonella typhi.
• Phenol coefficient test:
ƒ Similar quantities of organisms added to rising dilutions of phenol and the
disinfectant to be tested.
ƒ Rideal-Walker: No organic matter added.
ƒ Chick Martin: Organic matter added (Candida, dried feces).
ƒ Dilution of disinfectant which kills the organisms divided by dilution of phenol.
ƒ Phenol coefficient:
▫ 1.0 Equal.
▫ <1.0 Less.
▫ >1.0 More.
• Capacity test (Kelsey and Sykes test).
• In use tests Levels of Disinfection.

Fig. 3.10: General Microbiology

56 | Microbiology
Concept 3.4: Antimicrobial susceptibility testing
Learning Objectives
• Types of antibiogram
• Applications

Time Needed
1 reading
st
25 mins
2 look
nd
10 mins

Laboratory Testing of Antimicrobial:

Susceptibility:
A unique feature of laboratory testing in bacteriology is that the individual patient’s
isolate. Is routinely tested against a battery of antimicrobial agents. These tests are
built around the common theme of placing the organism in the presence of varying
concentrations of the antimicrobial in order to determine the MIC. The methods used
are standardized, including a measured inoculum of the bacteria and controlled growth
conditions (eg, medium, temperature, atmosphere, and time).
In selecting therapy, clinicians must consider more than the results of laboratory tests.
The clinical pharmacology of the drug, the cause of the disease, the site of infection,
and the pathology of the lesion must be taken into account as well. For example, the
antimicrobial must reach the subarachnoid space and cerebrospinal fluid in meningitis.
Similarly, treatment may be ineffective for an infection that has resulted in abscess
formation unless the abscess is surgically drained. Previous clinical experience is also
critical. In typhoid fever, for instance, azithromycin may be effective while aminoglycosides
are not, even though the typhoid bacillus may be equally susceptible to both in vitro.
This is due to the aminoglycosides’ failure to achieve adequate concentrations inside the
macrophages where Salmonella enterica serovar Typhi multiplies.
Dilution Tests:

Fig. 3.11: Broth dilution susceptibility test.

The stippled tubes represent turbidity produced by bacterial growth. The MIC is 2.0 µg/ml
Bacterial Culture and Sterilization | 57
Dilution tests determine the MIC directly by using serial dilutions of the antimicrobial
agent in broth that span a clinically significant range of concentrations. The dilutions
are prepared in tubes or microdilution wells, and by convention, their concentrations
are doubled using a base of 1 ìg/mL (0.25, 0.5, 1, 2, 4, 8, and so on). The bacterial
inoculum of the patient’s isolate is adjusted to a standard (105 to 106 bacteria/mL) and
added to the broth.
After incubation overnight (or other defined time), the tubes are examined for turbidity
produced by bacterial growth. The first tube in which visible growth is absent (clear) is
the MIC for that organism.
Automated Tests:
Instruments are now available that carry out rapid, automated variants of the broth
dilution test. In these systems the bacteria are incubated with the antimicrobial in
specialized modules that are read automatically on a frequent basis. The multiple
readings and the increased sensitivity of determining endpoints by turbidimetric or
fluorometric analysis makes it possible to generate MICs in as little as 4 hours. In
laboratories with sufficient volume, these methods are no more expensive than manual
methods, and the rapid results have enhanced potential to influence clinical outcome,
particularly when interfaced with computerized hospital information systems.
Diffusion Tests:
In diffusion testing (often called the Kirby-Bauer technique), the inoculum is seeded
onto the surface of an agar plate, and filter paper disks containing defined amounts of
antimicrobials are applied. While the plates are incubating, the antimicrobial diffuses
into the medium to produce a circular gradient around the disk. After incubation
overnight, the size of the zone of growth inhibition around the disk can be used as
an indirect measure of the MIC of the organism. It is also influenced by the growth
rate of the organism, the diffusibility of the drug, and other technical factors. The
diameters of the zones of inhibition obtained with the various antibiotics are converted
to “susceptible,” “intermediate,” or “resistant” categories by referring to a table. This
method is convenient and flexible for rapidly growing aerobic and facultative bacteria
such as the Enterobacteriaceae, Pseudomonas, and staphylococci. Another diffusion
procedure uses gradient strips to produce elliptical zones that can be directly correlated
with the MIC. This method, the epsilometer test, can also be applied to slow-growing,
fastidious, and anaerobic bacteria. This approach is slower and more laborious than
automated broth systems, but it has the advantage of revealing the presence of multiple
colony morphologies, mixed infections, or resistant subpopulations that appear as “inner
colonies” within an otherwise clear zone of inhibition.
Molecular Testing:
The molecular techniques of nucleic acid hybridization, sequencing, and amplification
have been applied to the detection and study of resistance. The basic strategy is to
detect the resistance gene rather than measure the phenotypic expression of that gene’s
product. These methods offer the prospect of automation and rapid results, but as with
most molecular methods, are not yet practical for routine use. Their application will also
have to recognize the fact that they will be limited to known genes, that some forms of
resistance do not yet have well-defined genetic causes, and that phenotypic expression
is the “bottom line.”
58 | Microbiology
Bactericidal Testing:
The above methods do not distinguish between inhibitory and bactericidal activity. To
do so requires quantitative subculture of the clear tubes in the broth dilution test and
comparison of the number of viable bacteria at the beginning and end of the test.
The least amount required to kill a predetermined portion of the inoculum (usually
99.9%) is called the minimal bactericidal concentration (MBC). Direct bactericidal
testing is important in the initial characterization and clinical evaluation of antimicrobial
agents but is rarely needed in individual cases. Most of the antimicrobials used for acute
and life-threatening infections (e.g. -lactams, aminoglycosides) act by bactericidal
mechanisms.

Fig. 3.12: Diffusion tests.

A. Disk diffusion. The diameter of the zone of growth inhibition around a disk of fixed antimicrobial content is
inversely proportional to the minimum inhibitory concentration (MIC) for that antimicrobial, that is , the larger
the zone, the lower the MIC. B. The E test. A strip containing a gradint of antimicrobial content creates an
ellitical zone of inhibition. The conditons are empirically adjusted so that the MIC endpoint is where the growth
intersects the strip

Antimicrobial Assays:
For antimicrobials with toxicity near the therapeutic range, monitoring the concentration
in the serum or other body fluid is sometimes necessary. Therapeutic monitoring may
also be required when the patient’s pharmacologic handling of the agent is unpredictable,
as in renal failure. A variety of biologic, immunoassay, and chemical procedures have
been developed for this purpose. The drugs most commonly measured are vancomycin
and the aminoglycosides.
Bacterial Culture and Sterilization | 59

Worksheet
• MCQ OF “BACTERIAL CULTURE AND STERILIZATION” FROM DQB

• EXTRA POINTS FROM DQB

1.

2.

3.

4.

5.

6.

7.

8.

9.

10

11.

12.

13.

14.

15.
60 | Microbiology
Active Recall from Tables
Physical methods Temperature

Sterilization & Disinfection methods Quality control agents

4 Immunology

CONCEPTS
 Concept 4.1 Types of Immunity
 Concept 4.2 Structure of Immune system
 Concept 4.3 Immune responses
 Concept 4.4 Antigen (Ag), Antibody (Ab)
& Ag-Ab reactions
 Concept 4.5 Complement system
 Concept 4.6 Hypersensitivity reactions
 Concept 4.7 Autoimmunity
 Concept 4.8 Immune deficiency disorders
 Concept 4.9 Transplant immunology
 Concept 4.10 Important tables/images for
revision
62 | Microbiology
Concept 4.1: Types of Immunity
Learning Objectives:
• Classification of Immunity
• Innate & Adaptive Immunity
• Active & Passive immunity

Time Needed
1st reading 20 mins
2 look
nd
5 mins

Immunity:
Immunity is the host defense against foreign organisms or substances known as antigens.

Two Types:
Innate immunity:
First line of defense and is not specific to any one pathogen.

Types of innate immunity and their mechanisms:

Type Mechanism.

Anatomic barriers

Skin Mechanical barrier retards entry of microbes.

Acidic environment (pH 3–5) retards growth of microbes.

Mucous membranes Normal flora competes with microbes for attachment sites and nutrients.
Mucus entraps foreign microorganisms.
Cilia propel microorganisms out of body.

Physiologic barriers

Temperature Normal body temperature inhibits growth of some pathogens.

Fever response inhibits growth of some pathogens.

Low pH Acidity of stomach contents kills most ingested microorganisms.

Chemical mediators Lysozyme cleaves bacterial cell wall.

Interferon induces antiviral state in uninfected cells.
Complement lyses microorganisms or facilitates phagocytosis.
Toll-like receptors recognize microbial molecules, signal cell to secrete
immunostimulatory cytokines.
Collectins disrupt cell wall of pathogen,
Immunology | 63

Phagocytic and endocytic Various cells internalize (endocytose) and break down foreign macromolecules.
barriers Specialized cells (blood monocytes, neutrophils, tissue macrophages)
internalize (phagocytose], kill, and digest whole microorganisms.

Inflammatory barriers Tissue damage and infection induce leakage of vascular fluid, containing
serum proteins with antibacterial activity, and influx of phagocytic cells into
the affected area.

Pattern Recognition Receptors (PRR): Are present on phagocytes, recognize

pathogen associated molecular patterns (PAMP) which are molecular motifs found in
microbes and damaged associated molecular patterns (DAMP) from aging, damaged or
dead cells.
There are 4 types of PRRs:
1. TLR (Toll like receptor).
2. CLR (C-type lectin receptor).
3. RLR (RIG-I like receptor).
4. NLR (NOD like receptor) – Trypanosoma cruzi and influenza virus.

Toll Like receptors and their microbial ligands:

TLRs Ligands Microbes

TLR1 Triacyl lipopeptides. Mycobacteria and Gram negative bacteria.

TLR2 Peptidoglycans. Gram positive bacteria.

GPI linked proteins. Tryponosomes.
Lipoproteins. Mycobacteria.
Zymosan. Yeast and other fungi.
Phosphatidlyserine. Schistosomes.

TLR3 dsRNA. Viruses.

TLR4 LPS. Gram negative bacteria.

F-protein. RSV.
Mannans. Fungi.

TLR5 Flagellin. Bacteria.

TLR6 Diacyl lipopolypeptides. Mycobacteria and Gram positive bacteria.

Zymosan. Yeast and other fungi.

TLR7 and 8 ssRNA. Viruses.

TLR9 CpG dinucleotides. Bacterial DNA.

Herpes virus components. Herpes virus.
Hemozoin. Malaria parasite heme byproduct.
64 | Microbiology

TLR10 Unknown. Unknown.

TLR11 Unknown. Uropathogenic bacteria.

TLR12 Unknown. Unknown.

TLR13 Unknown. Vesicular stomatitis virus.

Adaptive immunity:
Exhibits four immunologic attributes:
1. Antigenic specificity – helps to distin- guish subtle differences among anti- gens.
2. Diversity – helps to recognize billions of unique structures on different antigens.
3. Memory – helps to induce a bigger immune response on encounter with the same
antigen.
4. Self/nonself recognition – helps to respond only to foreign antigens.
Innate and adaptive immunity operate hand in hand (interdependent). The activation of
innate immune response subsequently produces adaptive immune responses.
Characteristics Innate Adaptive

Specificity For structures shared by groups Single epitope of Ag on microbial

of microbes . and non microbial agents

Diversity Limited. High.

Memory No. Yes.

Self reactivity No. No.

Anatomic and physical barriers Skin, mucosa, chemicals Lymph nodes, spleen, MALT.
(lysozyme, IFN α and β),
temperature, pH.

Blood proteins Complement. Antibodies.

Cells Phagocytes and NK cells. Lymphocytes other than NK cells

and APC.

The high degree of specificity in adaptive immunity is due to antibodies and T-cell
receptors that recognize and bind specific antigens.

Active and Passive Immunity:

Active immunity Passive immunity

Produced actively by host immune system Received passively (Administration of Antibody).

(Infection/ Contact with Ag).

Long lasting and more effecting. Transient and less effective.

Immunology | 65

Lag period present. Effective immediately.

Negative phase No negative phase.

Immunological memory present No immunological memory.

Not applicable in immunodeficient Applicable in immunodeficient.

e.g. e.g.
Natural: Natural:
• Clinical infection. • Placenta.
• Subclinical infection. • Breast milk
Artificial: Artificial:
• Vaccination (Live and Killed). • Immune cells.
• Immune serum.

Adoptive immunity: Injection of immunologically competent lymphocytes.

Local immunity: Site of invasion/ Multiplication of Pathogen; IgA antibodies
Herd immunity: Overall level of immunity in a community.
66 | Microbiology

Worksheet
• MCQ OF “IMMUNOLOGY” FROM DQB

• EXTRA POINTS FROM DQB

1.

2.

3.

4.

5.

6.

7.

8.

9.

10
Immunology | 67
Active Recall from Tables
Innate Immunity Adaptive Immunity

Active immunity Passive immunity

68 | Microbiology
Concept 4.2: Structure of Immune system
Learning Objectives
• Cells of immune system
• Organs of immune system

Time Needed
1 reading
st
45 mins
2 look
nd
15 mins

Cells and organs of Immune System:

Lymphocytes:
• 20-40% of WBC.
• 3 types: B cells, T cells and NK cells.
B cells:
• Site of maturation: Bone marrow (Bursa of fabricius in birds).
• B cell receptor: Membrane bound surface monomeric Ab of IgM and IgD isotypes
recognizing thymus independent Ag (valency of BCR-2).
• After antigenic exposure differentiate into.
ƒ Plasma cells : Secrete Ab, die within 1-2 weeks.
ƒ Memory B cells – express membrane bound surface Ab including class switched
Ab except IgD.
T cells:
• Site of maturation: Thymus
• T cell receptor: αβ chain recognizes antigen restricted by MHC presented by APC
(Valency of TCR-1).
• After antigenic exposure differentiate into:
ƒ Effector T cells.
ƒ Memory T cells.
• Subpopulation: T helper (TH), T cytotoxic (TC) and T regulatory (Treg).
• T helper cells:
ƒ CD4 glycoprotein.
ƒ Activation of B cells, Tc cells and macrophages in immune response.
• T cytotoxic cells:
ƒ CD8 glycoprotein.
ƒ Recognition of MHC I-Ag complex initiates differentiation into effector cells called
cytotoxic T lymphocytes (CTL).
ƒ Eliminates infected cells or cancerous cells.
• T regulatory cells:
ƒ CD4 and CD25 glycoproteins.
ƒ Suppress the immune system.
Immunology | 69
• Natural Killer cells:
ƒ Large granular cells.
ƒ Recognize tumor or virus infected cells.
ƒ CD16 bind Fc portion of Ab attached to cells.
ƒ Both Ab dependent and independent killing.
• Mononuclear phagocytes:
ƒ Monocytes circulate in blood and migrate into tissue to differentiate into
macrophages.
ƒ Intestinal macrophages in gut.
ƒ Alveolar macrophages in lung.
ƒ Histiocytes in connective tissue.
ƒ Kupffer cells in liver.
ƒ Mesangial cells in kidney.
ƒ Microglial cells in brain.
ƒ Osteoclasts in bone.
Complex antigens are endocytosed to form a phagosome which fuses with the lysosome
and the digested antigen is eliminated by exocytosis; some of it is presented on
membrane on MHC.
Phagocytosis is enhanced (opsonization) when antibody or complement proteins are
attached to antigens.
• Granulocytes:
ƒ Neutrophils:
▫ Multilobed nucleus, light granules.
▫ First to arrive at site of inflammation.
▫ Phagocytose antigens.
• Eosinophils:
ƒ Phagocytose.
ƒ Levels increase in parasitic infections.
• Basophils:
ƒ Nonphagocytic.
ƒ Play a role in allergic response.
• Mast cells:
ƒ Play an important role in the development of allergies.
• Dendritic cells:
ƒ Long membranous extensions, looks like dendrites on nerve cells.
ƒ Antigen presentation.
ƒ 4 types:
▫ Langerhans DC.
▫ Interstitial DC.
▫ Monocyte derived DC.
▫ Plasmacytoid derived DC.
ƒ Follicular dendritic cells:
▫ Involved with B cell maturation.
70 | Microbiology

Property T cell B cell

Antigen recognition receptor T cell receptor mIg
Surface antigen CD3 CD19, 20, 21
Receptor for Fc piece of Ig – +
Receptor for C3 component of complement – +
EAC rosette (C3 receptor, CR2, EBV receptor) – +
ĘĊ rosette (CD2, measles receptor) + –
Thymus specific antigen + –
Blast transformation on treatment with Phytohemagglutinin, EB virus, endotoxin
concanavalin A

Unlike antibodies which recognize and interact directly with antigen, Tcell receptors
recognize antigen that is presented with either class I or class II major histocompatibility
complex (MHC) molecules.
The two major subpopulations of T lymphocytes are:
i. CD4 T helper (TH) cells which recognize antigen combined with class II MHC.
ii. CD8 T cytotoxic (TC) cells which recognize antigen combined with class I MHC.

List of Antigen presenting cells (APC):

Professional APC Non-professional APC
Dendritic cells. Fibroblasts (skin).
Macrophages. Glial cells (brain).
B cells. Pancreatic beta cells.
Thymic epithelial cells.
Thyroid epithelial cells.
Vascular endothelial cells.

Primary response Secondary response

Lag period after immunization 4-10 days 1-3 days
Peak response time 7-10 days 3-5 days
B cell involved Naïve B cell Memory B cell
Peak Ab response Depends on Ag 100-1000 times more than
primary response
Predominant Isotype IgM IgG
Antigens Both thymus dependent and Only thymus dependent
independent
Ab affinity Lower Higher

Lymphoid System:
• Cells involved in the immune response collect in tissues and organs which are collectively
referred as lymphoid system.
• Lymphoid tissue is divided into Primary and Secondary.
Immunology | 71
• Primary (central):
Sites of lymphoid development.
ƒ T cells: Thymus.
ƒ B cells: Bone marrow.
• Secondary (peripheral):
Provide environment where lymphocytes can interact with antigens.
ƒ Spleen, lymph nodes, mucosa associated lymphoid tissue.
▫ Spleen: White pulp contains lymphocytes. T–cell area (around central
arteriole in peri-arteriolar sheath); B –cell area: (primary follicle,
germinal centre and mantle layer).
▫ Lymph nodes: T cell area (para-cortex), B cell area (cortex and medullary
cords).

Fig.4.1: Lymph Node

Fig.4.2: Spleen
72 | Microbiology
B lymphocytes mature in the bone marrow, and T lymphocytes mature in an
organ called the thymus; these sites in which mature lymphocytes are produced
are called the generative lymphoid organs. Mature lymphocytes leave the generative
lymphoid organs and enter the circulation and the peripheral lymphoid organs, where
they may encounter antigen for which they express specific receptors. A normal adult
contains approximately 1012 lymphocytes in the circulation and lymphoid tissues.

Fig.4.3: Development of B-Cells

Immunology | 73

Worksheet
• MCQ OF “IMMUNOLOGY” FROM DQB

• EXTRA POINTS FROM DQB

1.

2.

3.

4.

5.

6.

7.

8.

9.

10
74 | Microbiology
Active Recall from Tables
T cells B cells

Professional APC Non Professional APC

Immunology | 75
Concept 4.3: Immune response
Learning Objectives
• Humoral Mediated Immune response
• Cell Medicated Immune response
• Primary and Secondary immune responses

Time Needed
1 reading
st
45 mins
2 look
nd
15 mins

Immune Response:
• Specific reactivity following antigenic stimulus.
• Humoral or antibody mediated.
• Cell mediated immunity (CMI).
Humoral and cell-mediated branches of the immune system:
• In the humoral response, B cells interact with antigen and then differentiate into antibody-
secreting plasma cells. The secreted antibody binds to the antigen and facilitates its
clearance from the body.
• In the cell-mediated response, various subpopulations of T cells recognize antigen
presented on self-cells. TH cells respond to antigen by producing cytokines. TC cells
respond to antigen by developing into cytotoxic T lymphocytes (CTLs), which mediate
killing of altered self-cells (e.g., virus-infected cells).
Major Histocompatibility Complex:
• Encode proteins that helps in intercellular recognition and antigen presentation to T
lymphocytes.
• Inherited as a unit from parents i.e. one haplotype from father and one from mother.
• MHC genes are polymorphic i.e. large number of alleles for each gene and they are
polygenic i.e. number of different MHC genes.
• MHC class I is telomeric and class II is centromeric.
• Genes mediating graft rejection.
ƒ MICE : called as H Loci.
ƒ Humans : Human leukocyte antigen complex.
ƒ Short arm of chromosome 6.
ƒ Co-dominantly expressed.
ƒ 4 Loci of HLA: A, B, C, D.
ƒ 3 classes of Genes in these HLA loci.
MHC CLASS I:
• HLA A,B,C.
• 1 polypeptide chain non-covalently linked to a smaller peptide b2 microglobulin.
• α3 domain binds to CD8.
• Present in virtually all nucleated cells except RBCs, and platelets.
• CD8+ cells recognize Ag presented along with MHC I molecules.
• Involved in graft rejection and cell mediated cytotoxicity.
76 | Microbiology
MHC CLASS II:
• Encoded by HLA-DP, HLA-DQ, HLA-DR.
• Two polypetide chains.
• b2 domain binds to CD4.
• Present in all the antigen presenting cells.
MHC CLASS III:
• Genes coding for complement components C2, C4, properdin, factor B, TNFa, TNF-b,
Heat shock protein 70, Enzyme 21-hydroxylase.

Fig.4.4: Structure of class I and class II MHC molecules

• Both class I and class II MHC molecules present antigen toT cells.
ƒ Class I molecules expressed on most nucleated cells present processed endogenous
antigen to CD8 T cells.
ƒ Class II molecules expressed on APC present processed exogenous antigen to CD4
T cells.
• Class III region of the MHC encodes molecules that include a diverse group of proteins
that play no role in antigen presentation.
Humoral Immune Response:
• Extracellular bacterial pathogens.
• Viruses (respiratory/ intestinal).
• Hypersensitivity I, II, II.
• Antigens presented to immunocompetent cells by antigen presenting cells (macro-
phages, dendritic cell [lymph node follicle, skin, thymus, lymphnode, spleen]).
• These cells present antigen at cell surface.
• Interact with immunocomponent B cells.
• Clonal proliferation and blast transformaion.
• Some B cells converted into memory cells.
• Antibody production stimulated by TH2 cells.
Immunology | 77
Antigen Processing and Presentation:
T Cell Activation:
• Cytosolic and endocytic pathways for processing antigen.
Cytosolic pathway - The proteasome complex contains enzymes cleaves endogenous
antigens (proteins) into peptides. The antigenic peptides from proteasome cleavage are
transported through the TAP and bind to peptide binding groove of class I MHC which
can accommodate 8-10 amino acid length peptide. This peptide class I MHC complex is
expressed on cell surface which is recognized by cytotoxic T cells (Tc) expressing CD8.
Endocytic pathway – The exogenous antigens (proteins) are phagocytosed by phagocytic
cells and bind to peptide binding groove of class II MHC within the phagolysosome after
the Invariant chain (peptide which blocks the peptide binding groove) is removed. This
peptide class II MHC complex is expressed on cell surface which is recognized by helper
T cells (Th) expressing CD4.

Fig. 4.5: Antigen Processing

B Cell Activation:
Thymus-dependent (TD) antigenscan stimulate B cells only after direct contact with
TH cells, not simply exposure to TH-derived cytokines.
Thymus-independent (TI) antigens can activate B cells in the absence of this kind
of direct participation by TH cells.
Cell Mediated Immune Responses:
• Accomplished by effector T cells and macrophages rather than B cells and antibodies.
• Protects against fungi, virus, intracellular bacterial pathogens (M.leprae, M.tubercu-
losis, Brucella, Salmonella), parasites (leishmania , trypanosomes).
• Allograft rejection.
• Delayed hypersensitivity.
• Immunological surveillance.
• Immunity against cancer.
• Primary: Initial contact with foreign Ag Ag presented by antigen presenting cells to
T cell. T cell receptor combines with foreign antigen along with self MHC molecule.
Proliferation of specific clones of effector T cells.
• Secondary: More pronounced, more rapid.
78 | Microbiology
T Cell Differentiation:
• Two modes of antigen processing.
• Processing of phagocytosed material eg bacteria. The antigen is degraded and
associated with class II molecules. The antigen MHC II complex then expressed on
the surface of antigen presenting cells.
• Processing of antigens derived from within the cell (viral infection). Antigen MHC I
complex expressed on cell surface.
• CD8+ Tcells recognize Ag in association with self MHC I.
• CD4+ Tcells recognize Ag in association with self MHC II.
Cytokines
• Soluble mediators of host defence responses, both specific and non specific.
• Same cytokine can be produced by multiple cell types and can have multiple effects
on the same cell.
• Small proteins (8-80KDa) usually acting in an autocrine or paracrine manner.
Name Major Cell Source Biological Effect
• IFN a,b Phagocytes,fibroblasts Antiviral, pyrogenic.
• IFN γ T cells , NK cells Activates mononuclear phagocytes.
• TNF b T cells , B cells Activates leucocytes, antitumor.
• TGF b T cells, macrophages Leuckocyte growth angiogenesis.
• IL1 Phagocytes Fever, Cachexia.
• IL2 T cells Tcell growth, Activation.
• IL3 T cells Hematopoesis.
• IL4 T cells Isotype switching to IgE.
• IL6 T cells Lymphocyte Growth.
• IL7 Stromal cells Maturation of T and B lymphocytes.
• IL8 Macrophages Chemotactic to Neutrophils.
• IL10 Helper Tcells Inhibits Macrophages.
• IL12 Bcells, Macrophages Activates TH1 cells and NK cells.
• IL17 Th17 cells Infiltration of neutrophils, autoimmunity.
• GM-CSF Tcells, Phagocytes Hematopolesis of Granulocyte, Monocyte lineage.
• M-CSF Macrophage Differentiation to monocyte.
• G-CSF T-cells Differentiation to granulocyte.

Chemokines:
Chemoattractant cytokines.
Four major groups based on the cysteine structure near the amino terminus.
C: Lymphotactin.
CC: MIP-a (Lymphoctes, monocytes, eosinophils, basophils).
CXC: IL 8 (Neutrophils).
CXXC: Fractalkine (Neutrophils, monocytes, T cells).
Immunology | 79

Fig.4.6: Overview of the humoral and cell-mediated branches of the immune system. In the humoral response,
B cells interact with antigen and then differentiate into antibody-secreting plasma cells. The secreted antibody
binds to the antigen and facilitates its clearance from the body. In the cell-mediated response, various
subpopulations of T cells recognize antigen presented on self-cells. TH cells respond to antigen by producing
cytokines. TC cells respond to antigen by developing into cytotoxic T lymphocytes (CTLs), which mediate killing
of altered self-cells (e.g., virus-infected cells).
80 | Microbiology

Fig.4.7: Primary and Secondary immune response

Immunology | 81

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82 | Microbiology
Active Recall from Tables
Interleukins Functions

Primary Immune Response Secondary Immune Response

Immunology | 83
Concept 4.4: Antigen, Antibody & Antigen – Antibody reactions
Learning Objectives
• Antigens and their properties
• Antibodies – Types and functions.
• Different types of Antigen – Antibody reactions
• Applications of Ag – Ab reactions

Time Needed
1 reading
st
60 mins
2nd look 20 mins

Antigens:
• Stimulate immune response.
• Immunogenicity - ability to stimulate immune response.
• React specifically with:
ƒ Effector molecules (Antibodies).
ƒ Effector cells (Lymphocytes).
• Antigenic determinant/ Epitopes bind to paratope on the antibody/T-cell receptors.

Fig.4.8: Maturation of T-cells

84 | Microbiology
• Incomplete antigen/ Hapten:
ƒ Low molecular weight.
ƒ Cannot induce immune response.
ƒ Single epitope.
ƒ Carrier molecular required.
• Determinants of antigenicity:
ƒ Size: >100 KDa (lesser size are rarely immunogenic).
ƒ Foreignness.
ƒ Chemical nature (proteins>polysaccharides > lipids).
ƒ Susceptibility to tissue enzymes–large insoluble antigens are more immunogenic
than the small soluble ones.
• Common Adjuvants and their mode of action.

Adjuvants Prolongs Ag Enhances co- Induces Stimulates

persistence stimulatory granuloma lymphocytes non-
signals formation specifically

Freunds incomplete + + + -

Freunds complete + ++ ++ _

Aluminum + ? + _
potassium sulphate
(alum)

M. tuberculosis _ ? + _

B. pertussis _ ? _ +

Note: Freunds complete adjuvant containing heat killed mycobacteria unlike Freunds incomplete is more immunogenic.

• Antigen specificity:
ƒ Varies with position of antigen determinant.
ƒ Not absolute: Cross reactions.
• Species Specificity.
• Iso-specificity:
ƒ Found in some but not all members of a species.
ƒ A, B, AB, O Blood groups.
• Histocompatibility antigens:
ƒ Present on the cells of each individual of species.
ƒ Graft rejection.
ƒ MHC proteins encoded on short arm of chromonsome 6 (6p).
• Auto-specificity:
ƒ Normally non-antigenic.
ƒ Hidden/sequestered antigens.
ƒ Lens protein/ spermatozoa.
Immunology | 85
• Organ specificity: Some organs (brain, kidney, lens protein) of different species share
same antigen:
ƒ Neuroparalytic complication following anti-rabies vaccine.
• Heterogenetic (heterophile) specificity:
ƒ Closely related antigens occurring in different biological species/classes/kingdoms.
ƒ Forssman antigen.
ƒ Serological tests based on heterophile antigens: Well-felix reaction, Paul bunnel
test, Cold agglutinin test, Agglutination of streptococcus MG.

Superantigens:
• Act on vβ of TCR.
• Don;t require antigen presentation by macrophage, directly stimulate non specific
T-cells.
• Leads to massive release of cytokines and polyclonal T-cell activation.
• Example:
Organism Superantigen Disease

S. aureus Enterotoxin Food poisoning

TSS
Multiple Sclerosis

Gr A Streptococci Pyrogenic exotoxin Shock

Psoriasis
ARF

M tuberculosis Not identified Tuberculosis

HIV Nef AIDS

Rabies virus Nucleocapsid protein Rabies

EB virus Not identified B cell lymphoma

Tolerogen:
An antigen that induces specific immunological non-reactivity.

Antibody:
g globulins produced in response to antigenic stimulation.
React specifically with Ag which stimulated their production.
All antibodies are Igs, But all Ig (Myeloma proteins) are not antibodies.

Function:
The three major effector functions by which antibodies remove antigens and kill
pathogens are:
• Opsonization: Enhanced phagocytosis by macrophages and neutrophils.
• Complement activation-perforate cell membranes.
86 | Microbiology
• Antibody-Dependent Cell-mediated Cytotoxicity (ADCC), which can kill antibody-
bound target cells.

Structure:
Y shaped 4 polypeptide chain molecule
2 heavy, 2 light chains, held together by Disulphide bonds
Heavy chains: A, D, E, G, M.
Light chains two types: k(Kappa)/λ(lamda); 2:1.

Fig.4.9: Structure of Antibody

Papain: 2 Fab (fraction antibody binding) + 1Fc:

Fc (Fraction crystallisable):
• Complement fixation.
• Binds to cell receptors (FcRs).
• Passage across placental barrier.
• Distinguish b/w different classes.
Pepsin: F(ab)2.
Immunology | 87

Fig.4.10: Action of Pepsin, Papain and Mercaptoethanol on Antibody

IgG IgA IgM IgD IGE

Molecular Wt. (KDa) 150 160, 325* 900 180 190
Sedimentation 7 7, 11* 19 7 8
Carbohydrate Content % 3 8 12 13 12
Heavy chain class g 1,2,3,4 a1,2 m d e
Light chain k/l k/l k/l k/l k/l
Serum conc. (mg/ml) 12 2 1.2 0.03 0.00004
Half life (days) 21 6 5 3 2
Complement binding Classical Alternate Classical None None
Binding tissue Heterologus None None None Homologus
Secretion from serous membranes No Yes* No No Yes
Placental passage Yes No No No No
Heat stability (56°C) Yes Yes Yes Yes No
88 | Microbiology
• VL and VH: Specific antigen binding site.
• CH2 region binds C1q.
• CH3 region mediates adherence to monocyte surface.
• Isotype:
ƒ Genetic variations or differences in the constant region of the heavy chain of the Ig
classes and subclasses.
ƒ E.g. Ig classes: IgM, IgD, IgG, IgA and IgE subclasses: IgG1, IgG2, IgG3, IgG4,
IgA1 and IgA2.
• Allotype:
ƒ Multiple alleles that exist for some genes which lead to subtle amino acid differences
that occur in some but not all members of a species.
ƒ Distinct amino acid residues located primarily in g and a α chains and k L chains.
ƒ No allotypic markers have been found for L chains or µ, δ and ε H chains.
ƒ Reflect genetic polymorphism of Igs.
• Idiotype:
ƒ Located on V regions of L and H chains.
(a) Isotypic Determinants

(b) Allotypic Determinants

(c) Idiotypic Determinants

Fig.4.11: Isotypic, Allotypic and Idiotypic Determinants

Immunology | 89

Fig.4.12: Class Switching

Fig.4.13: Activation of B-cells into Plasma cells

90 | Microbiology
Antibody diversity depends on:
• Multiple germ-line gene segments.
• Combinatorial V-(D)-J joining.
• Junctional flexibility.
• P-region nucleotide addition (P-addition).
• N-region nucleotide addition (N-addition).
• Somatic hypermutation.
• Combinatorial association of light and heavy chains.
Properties Td Ti
Ti1 Ti2
Chemical nature Soluble proteins Bacterial cell wall Capsular polysaccharides
components (LPS) Polymeric protein antigens
Humoral response
Isotypic class switching Yes No Limited
Affinity maturation Yes No No
Immunologic memory Yes No No
Polyclonal activation No Yes (high dose) No

Monoclonal Antibody:
• Normal antibody response: Polyclonal.
• Antibodies produced by a single clone and directed against single antigenic
determinant.
• Kohler and Milstein 1975, nobel prize
• Lymphocytes from mice spleen fused with mouse myeloma cells (deficient in HPRTase).
• Fused cells placed in medium containing Hypoxanthine, Aminopterin, Thymidine (HAT
medium).
• Only fused cells grow.
• These hybridomas can be grown in peritoneal cavity of mouse or tissue cultures.
Abzymes-Monoclonal antibody that can catalyse reactions.

Antigen Antibody Reactions:

• Antigen and antibody combine specifically.
Non-covalent interaction.
Bonds involved: Hydrophobic, Vanderwaals, Electrostatic and hydrogen bonds.

Precipitation:
Antigen soluble.
Sensitive for detection of antigen.
• Ring test.
• Slide test.
• Tube test.
Immunology | 91
• Immunodiffusion (precipitation in gel).
ƒ Single diffusion in one dimension (Oudin’s method).
ƒ Double diffusion in one dimension (Oakley-fulthorpe's method).
ƒ Single diffusion in two dimensions (Radial immunodiffusion).
ƒ Double diffusion in two dimensions (Ouchterlony’s procedure).
ƒ Immunoelectrophoresis.
ƒ Electroimmunodiffusion.
▫ CIEP.
▫ Rocket electrophoresis.

Fig.4.14: Zone phenomenon

Maximum Ag-Ab reaction occurs in the zone of equivalence

Agglutination:
Particulate antigen:
• Slide agglutination.
• Tube agglutination (widal, weil-felix, paulbunnell).
• Antiglobulin (coomb’s test).
ƒ Direct (baby).
ƒ Indirect (mother).
92 | Microbiology

Fig.4.15: Direct and indirect Coomb's Test

• Passive (indirect) agglutination:

ƒ Latex agglutination.
ƒ Passive haemagglutination.
• Coagglutionation (Staphylococcus aureus Cowan 1 strain having protein A).
Neutralization Tests:
• Virus neutralization.
• Toxin neutralization.
ƒ Antistreptolysin O test (ASLO) (In-vitro).
ƒ Nagler reaction (In-vitro).
ƒ Shick test (In-vivo).
ƒ Dick test (In-vivo).
Complement Fixation Test:
• Ag + Patient's serum Containing Ab + C +
Hemolytic system (Sheep erythrocytes + Anti sheep Ab)
↓
No haemolysis (Test positive)
• Ag + Patient's serum Containing no Ab + C + Hemolytic system + (Sheep
erythrocytes + Anti sheep Ab)
↓
Haemolysis (Test negative)
Immunology | 93
Other tests utilizing complement:
• Immune adherence (V. cholerae, T. pallidum) (C3b).
• Treponema pallidum immobilization Test.
• Cytolytic / cytocidal test (V. cholera).

Immunofluorescence:
1. Direct immunofluorescence: for antigen detection (e.g. Rabies, Pneumocystis jiroveci,
pertussis, syphilis)

Fig.4.16: Direct immunofluorescence

2. Indirect immunofluorescence: For antibody detection (FTA- ABS)

Fig.4.17: Indirect immunofluorescence

Enzyme Linked Immunosorbent Assay (ELISA):

Enzymes used:
• Alkaline phosphatase.
• Horseradish peroxidase.
94 | Microbiology
• β galactosidase.

Fig.4.18: ELISA

Types:
1. Direct sandwich: For detecting Ag.
2. Indirect sandwich: For detecting Ab.

Fig.4.19: Indirect ELISA

3. Competitive: For detecting Ab

Fig.4.20: Competitive ELISA

Immunology | 95
4. Capture ELISA: For detecting isotype of Ab.
Chemiluminescence: Measurement of light (luxogenic substrate) in chemiluminescence
assay instead of measurement of absorbance (chromogenic substrate) in ELISA increases
sensitivity by 10 to 200 fold.

Fig.4.21: In the Elispot

Western Blot:
Detection of Ab in patient serum by using antihuman Ab labeled with enzyme on membranes
coated with Ag after separation of Ag by gel electrophoresis and blotting of those Ag.
96 | Microbiology
Used for confirmation of HIV:

Fig. 4.22: Western Blot

Immunology | 97

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6.

7.

8.

9.

10
98 | Microbiology
Active Recall from Tables
Super antigens Organisms

T dependent Ag T Independent Ag
Immunology | 99
Concept 4.5: Complement system
Learning Objectives
• Complement pathways
• Source of complements
• Applications

Time Needed
1 reading
st
25 mins
2 look
nd
10 mins

The Complement System:

• Bacteriolysis/ Cytolysis.
• Amplification of inflammatory response (c3a, c5a, Mal).
• Hypersensitivity reaction (type II, III).
• Endotoxic shock.
• Immune adherence.
• Opsonisation (c3b).
• Autoimmune diseases (SLE, RA).

Regulation of Complement:
• Inhibitors:
ƒ Bind to complement components and halt their further action.
ƒ a Neuro aminoglycoprotein↓ C1 esterase.
ƒ S Protein ↓C567.
• Inactivators:
ƒ Destroy complement proteins.
ƒ Factor I ↓ C3 activation.
ƒ Factor H binds to C3b.
ƒ C4 binding protein.
ƒ Anaphylatoxin inactivator degrades C3a, C4a, C5a.

Biosynthesis:
• Intestinal epithelium C1
• Macrophages C2, C4
• Spleen C5, C8
• Liver C3, C6, C9
• Not known C7
100 | Microbiology

Fig.4.23: Complement Activation Pathways

Immunology | 101

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102 | Microbiology
Active Recall from Tables
Complements Functions

Complement deficiency Disease

Immunology | 103
Concept 4.6: Hypersensitivity reactions
Learning Objectives
• Types of Hypersensitivity reactions
• Mechanisms
• Examples

Time Needed
1 reading
st
30 mins
2 look
nd
10 mins

Hypersensitivity:
Abnormal immune response which produces physiological / histopathological damage
in the host.
Type I / Anaphylactic: IgE mediated.
Type II / Cytotoxic.
Type III / Immune complex: Arthus reaction/serum sickness.
Type IV / Delayed: T- cell mediated.
Immediate type: I, II, III (< 24 hrs).
Delayed type: IV (24 -48 hrs).

IgE-Mediated Hypersensitivity IgG-Mediated Cytotoxic Hypersensitivity

Ag Induces crosslinking of IgE bound to mast cells Ab directed against cell surface antigens meditates
and basophils with release of vasoactive mediators cell destruction via complement activation or ADCC
Typical Manifestations include systemic anaphylaxis Typical manifestations include blood transfusion
and localized anaphylaxis such as hay fever, asthma, reactions, erythroblastosis fetalls, and autoimmune
hives, food allergies and exzema hemolytic anemia
104 | Microbiology

Immune Complex-Mediated Hypersensitivity Cell-Mediated Hypersensitivity

Ag-Ab complexes deposited in various tissues Sensitized TH1 cells release cytokines that activate
induce complement activation and an enxuing macrophages or TC cells which mediate direct
inflammatory response mediated by massive cellular damage
infiltration of neutrophils
Typical manifestations include localized Arthus Typical manifestations include contact dermatitis,
reaction and generalized reactions such as serum tubercular lesions and graft rejection
sickness, necrotizing vasculitis, glomerulnephritis,
rheumatoid arthritis, and systemic lupus
erythematosus

Fig.4.24: Type I Hypersensitivity (Anaphylaxis):

Immunology | 105

Fig.4.25: Schematic diagrams of mechanisms that can trigger degranulation of mast cells. Note that
mechanisms (b) and (c) do not require allergen; mechanisms (d) and (e) require neither allergen nor IgE;
and mechanism (e) does not even require receptor crosslinkage

Mediator Effects
Primary
Histamine, heparin Increased vascular permeability; smooth-muscle contraction
Serotonin Increased vascular permeability; smooth-muscle contraction
Eosinophil chemotactic factor Eosinophil chemotaxis
(ECF-A) Neutrophil chemotaxis
Neutrophil chemotactic factor Bronchial mucus secretion; degradation of blood-vessel basement
(IMCF-A) membrane; generation of complement split products
Proteases
106 | Microbiology

Secondary
Platelet-activating factor Platelet aggregation and degranulation; contraction of pulmonary
smooth muscles
Leukotrienes (slow reactive substance Increased vascular permeability; contraction of pulmonary
of anaphylaxis, SRS-A) smooth muscles
Prostaglandins
Vasodilation; contraction of pulmonary smooth muscles; platelet
Brady kkiin aggregation
Cytokines Increased vascular permeability; smooth-muscle contraction
IL-1 and TNF-α Systemic anaphylaxis; increased expression of CAMs on venular
endothelial cells
IL-2, IL-3, IL-4, IL-S, IL-6, TGF-β,
and GM-CSF Various effects

Type I Hypersensitivity:
• Mediated by IgE and mast cells.
• Local/ generalized.
• Local (Hay fever, asthma).
• Systemic (shock life condition) venom, penicillin, horse serum.
Prausnitz - Kustner (PK) reaction serum from allergic person into skin of normal
person.
Serum Treatment Allergen added P-K reaction at skin
site
Atopic None
Atopic None + +
Nonatopic None + −
Atopic Rabbit antiserum to human atopic serum* −
Atopic Rabbit antiserum to human IgM, IgG, IgA, and IgD † +
*Serum from an atopic incfiviclu.il was injected into rabbits to produce antiserum against human atopic
serum. When this antiserum was reacted with human atopic serum, it neutralized the P-K reaction.
†Serum from an atopic individual was reacted with rabbit antiserum to the known classes of human antibody
(IgM, IgA, IgG, and IgD) to remove these isotypes from the atopic serum. The treated atopic serum continued
to give a positive P-K reaction, indicating that a new immunoglobulin isotype was responsible Tor this
reactivity,

Passive cutaneous anaphylaxis (PCA):

1. Used to detect the human IgG which is heterocytotropic (capable of fixing to cells of
other species).
2. Serum from anaphylactically sensitised animal to (I/D) skin of normal animal.
3. After 48 hrs challenged with allergen and evans blue dye (I/V).
4. Blueing of site of I/D injection (wheel and flare)
Immunology | 107
Anaphylaxis in vitro (Schultz-dale):
1. Strips of smooth muscle contract in vitro following exposure to antigen.
Atopy:
Chronic human allergic states.
(Hay fever, asthma, food allergies).
Common allergen associated with Type I.
Proteins • Foreign serum Foods • Nuts
• Vaccines • Seafood
Plant pollens • Rye grass • Eggs
• Ragweed • Peas, beans
Timothy grass • Birch trees • Milk
Drugs • Penicillin Insect products • Bee venom
• Sulfonamides • Wasp venom
• Local anesthetics • Ant venom
• Salicylates • Cockroach calyx
• Dust mites
Mold spores • Animal hair and dander

Fig.4.26: Procedures for assessing type I hypersensitivity. (a) Radioimmunosorbent test (RIST) can quantify
nanogram amounts of total serum IgE. (b) Radioallergosorbent test (RAST) can quantify nanogram amounts of
serum IgEspecific for a particular allergen.
108 | Microbiology
Procedures for assessing Type I hypersensitivity.
a. RIST (Radioimmunosorbent assay): Can quantify nanograms amount of total serum
IgE.
b. RAST (Radioallergosorbent assay): can quantify nanograms amount of serum IgE for
a particular antigen.
Mechanism of action of drugs used for treating Type I hypersensitivity.

Drug Action

Antihistamines Block H1 and H2 receptors on target cells

Cromolyn sodium Blocks Ca++ influx into mast cells
Theophylline Prolongs high cAMP levels in mast cells by inhibiting phosphodiesterase,
which cleaves cAMP to 5’-AMP
Epinephrine Stimulates cAMP production by binding to β-adrenergic receptors on
(adrenalin) mast cells

Cortisone Reduces histamine levels by blocking conversion of histidine to histamine

and stimulates mast-cell production of cAMP

TYPE II Hypersensitivity (Cytotoxic):

• Combination of IgG and IgM with foreign antigenic components on a cell surface.
• Complement - mediated cytotoxicity (Membrane attack complex, Phagocytes)
• Antibody- Dependent cell mediated cytotoxicity (ADCC).
• Drug induced immune hemolytic anaemia:
ƒ Penicillin.
ƒ Methyldopa.

Penicillin can induce all types of hypersensitivity.

Type of reaction Antibody or lymphocytes Clinical manifestations
induced

1 IgE Urticaria, systemic anaphylaxis

II IgM, IgG Hemolytic anemia

III IgG Serum sickness, glomerulonephritis

IV TDTH cells Contact dermatitis

• Transfusion reactions.
• Rh incompatibility.
• Anaemia due to infections diseases.
• Grave's disease.
• Myaesthenia gravis.
Immunology | 109
• Pernicious anaemia.
• Non insulin dependent diabetes mellitus.

Rhogam: anti RhD

Fig. 4.27: Development of erythroblastosis fetalis (hemolytic disease of the newborn) caused when an Rh–
mother carries an Rh+ fetus (left), and effect of treatment with anti-Rh antibody, or Rhogam (right)

TYPE III (Immune Complex Mediated):

• Antigen soluble (not cell bound).
• Ag-Ab complex deposition in tissues.
• Complement activation.
• At equivalence (precipitation @ local reaction).
• Large Ag excess (systemic reaction).
• Arthus reaction (Local immune complex diseases).
• Serum sickness:
ƒ Horse antitoxin:
▫ Tetanus.
▫ Gas gangrene.
▫ Diphtheria.
ƒ 7-12 days after single dose.
110 | Microbiology
ƒ Lymphadenopathy, Fever, splenomegaly, arthritis, glomerulonephritis, endocardi-
tis, vasculitis, nausea, vomiting:
▫ SLE.
▫ Rheumatoid arthritis (IgM vs IgG Fc region).
▫ Multiple sclerosis.
▫ Serum sickness.
▫ Arthus reaction.
▫ PSGN.
▫ Meningitis.
▫ Hepatitis.
▫ Mononucleosis.
▫ Malaria.
▫ Trypanosoma.
Penicillin andsulphonamide allergies.

Fig.4.28: Serum Sickness

Immunology | 111

Fig.4.29: TYPE III Hypersensitivity:

TYPE IV (Delayed) Hypersensitivity:

• Sensitized T- cells and macrophages.
• 24-48 hrs after the presensitized host encounters Ag.
• Tuberculin (infection type):
ƒ 1-3 units of PPD/ tuberculin injected I/D.
ƒ In individual sensitised to tuberculoprotein.
112 | Microbiology
ƒ Indurated reaction > 5mm within 48-72 hrs.
ƒ Erythema: Due to b ­ lood flow.
ƒ Induration: Infiltration of mononuclear cells.
• Contact dermatitis type:
ƒ Chemicals (nickel, chromium), dyes, drugs, (penicillin) come in contact with skin.
ƒ Not antigenic by themselves but acquire antigenicity on combination with skin
proteins.
ƒ Subsequent contact: Contact dermatitis.
ƒ Macules/papules/ vesicles/acute eczema.
• SLE.

Classification of adverse drug reactions based on immune pathways:

Type Key Pathway Key Immune Mediators Adverse Drug Reaction Type
Type I lgE IgE Urticaria, angioedema,
anaphylaxis
Type II IgG-mediated cytotoxicity IgG Drug-induced hemolysis,
thrombocytopenia (e.g,
penicillin)
Type III Immune complex IgG + antigen Vasculitis, serum sickness,
drug-induced lupus
Type IVa Tlymphocyle-mediaied IFN-γ TNF-α TH1 cells Tuberculin skin test, contact
macrophage inflam­mation dermatitis
Type IVb T lymphocyte-mediated IL-4, IL-5, IL13 TH2 cells Drug-induced hypersensitivity
eosinophil inflam­mation Eosinophils syndrome (DIHS)
Morbilliform eruption
Type IVc T lymphocyte-mediated Cytotoxic T lymphocytes SJS/TEN
cytotoxic T lympho­cyte Granzyme Morbilliform eruption
inflammation Perforin
Granulysin (Stevens-Johns
on syndrome [SJS] / toxic
epidermal necrolysis [TEN]
only)
Type IVd T lymphocyte-mediated CXCL8, IL-17, GM-CSF Acute generalized
neutrophil inflam­mation Neutrophils exanthematous pustulosis
(AGEP)

Shwartzmann Reaction:
• Not an immune reaction.
• Culture filtrate of Gram –ve bacteria (I/D rabbit).
• After 24 hrs larger dose of same/ unrelated toxin (I/V).
• Within few hours at the site of I/D injection, petechial haemorrhages are seen.
• Absence of specificity, short interval.
• Localized/ generalized (Waterhouse-Friderichsen syndrome).
Immunology | 113

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114 | Microbiology
Active Recall from Tables
HSR Types Mechanism

HSR types Examples

Immunology | 115
Concept 4.7: Autoimmunity
Learning Objectives
• Mechanisms of Autoimmunity
• Examples

Time Needed
1 reading
st
30 mins
2 look
nd
10 mins

Autoimmunity:
Organ specific autoimmune diseases:
Disease Self antigen Immune response
Addison’s disease Adrenal cells Auto antibodies
Autoimmune hemolytic anemia RBC membrane protein Auto antibodies
Goodpasture’s syndrome Renal and lung basement Auto antibodies
membrane
Grave’s disease TSH receptor Stimulating Auto antibodies
Hashimoto’s thyroiditis Thyroid proteins and cells DTH and Auto antibodies
Idiopathic thrombocytopenic Platelet membrane proteins Auto antibodies
purpura
IDDM Pancreatic beta cells DTH andAuto antibodies
Myasthenia gravis Acetylcholine receptors Blocking Auto antibodies
Myocardial infarction Heart Auto antibodies
Pernicious anemia Gastric parietal cells and intrinsic Auto antibodies
factor
PSGN Kidney Immune complex
Spontaneous infertility Sperm Auto antibodies

Systemic autoimmune diseases:

Disease Self antigen Immune response
Ankylosing spondylitis Vertebrae Immune complex.
Multiple sclerosis Brain or white matter Th1 and Tc cells, Auto antibodies.
Rheumatoid arthritis Connective tissue, IgG Immune complex, Auto
antibodies.
Scleroderma Nuclei, heart, lungs, GI tract, Kidney Auto antibodies.
Sjogren’s syndrome Salivary gland, liver, kidney, thyroid Auto antibodies.
SLE DNA, nuclear protein, RBC and platelet Immune complex, Auto
membrane antibodies.
116 | Microbiology
Mechanisms:
• Release of sequestered antigens.
• Molecular mimicry.

Between proteins of infectious agent and human proteins:

Proteins of infectious agent Human proteins
1 Human cytomegalovirus IE2 HLA DR
2 Poliovirus VP2 Acetylcholine receptor
3 Papilloma virus E2 Insulin receptor
4 Rabies virus glycoprotein Insulin receptor
5 Klebsiella pneumoniae nitrogenase HLA B27
6 Adenovirus 12 E1B Alpha gliadin
7 HIV p24 IgG constant region
8 Measles virus P3 Corticotropin and Myelin basic protein

• Inappropriate expression of class II MHC:

ƒ Pancreatic beta cells in IDDM patients express high levels of both class I and II
MHC.
ƒ Thyroid acinar cells in Graves disease express class II MHC.
ƒ IFN g induces class II MHC in many cells like pancreatic beta cells, thyroid acinar
cells, intestinal epithelial cells, melanoma cells (increased IFN g in SLE patients).
• Polyclonal B cell activation:
ƒ Gram negative bacteria.
ƒ Cytomegalovirus.
ƒ EB virus.
HLA Associations with Relative Risk (RR) for Various Diseases:
• Ankylosing spondylitis–B27–90%.
• Goodpasture’s syndrome–DR2–16%.
• Gluten sensitive enteropathy–DR3–12%.
• Hereditary hemachromatosis–A3–9%; B14-–2% BUT A3/B14–90%.
• IDDM–DR4/DR3–20%.
• Multiple sclerosis–DR2–5%.
• Myasthenia gravis–DR3–10%
• Narcolepsy–DR2–130%.
• Reactive arthritis–B27–18%.
• Reiter’s syndrome–B27–37%.
• Rheumatoid arthritis–DR4–10%.
• Sjogren’s syndrome–Dw3–6%.
• SLE–DR3–5%.
Immunology | 117

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• EXTRA POINTS FROM DQB

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118 | Microbiology
Active Recall from Tables
Autoimmune disease Targets
Immunology | 119
Concept 4.8: Immunodeficiency diseases
Learning Objectives
• Classification
• Mechanism of Action
• Examples

Time Needed
1 reading
st
45 mins
2 look
nd
15 mins

Immunodeficiency:
Innate immune system:
Disease Molecular defect symptoms
Chronic Deficiency of NADPH, failure to Recurrent infections with catalase
granulomatous disease generate O2 radicals positive bacteria and fungi.
Leukocyte adhesion Absence of CD18, common b chains Recurrent infections, failure to
deficiency of integrins produce pus, Omphalitis.
Chediak Higashi Granule structural defect Recurrent infections, Chemotactic and
syndrome degranulation defects, Absent NK,
partial albinism.
G6PD deficiency Deficiency of enzymes in HMP shunt Same as CGD with associated
anaemia.
MPO deficiency Granule enzyme deficiency Mild or none.

B cell deficiency:
Bruton X linked Deficiency of tyrosine ↓Ig of all classes, No Monthly Ig globulin
hypogamma kinase, blocks B cell circulating B cells, pre replacement, antibiotics
globulinemia maturation. B cells in bone marrow for infections.
normal, normal CMI.
X-linked hyper IgM Deficiency of CD40 on ↑Serum IgM without Antibiotics and g
syndrome activated T cells. other isotypes, globulins.
Normal B and T cells,
Extracellular bacteria
and opportunists.
Selective IgA deficiency Deficiency of IgA (MC) Repeated sinopulmonary Antibiotics not Ig.
and GI infections.
Common variable hypo Unknown Onset: Late teens, Antibiotics.
gglobulinemia B cells present in
blood, ↓ Ig over time,
↑autoimmunity.
120 | Microbiology

Transient hypo Delayed onset of normal Detected in 5th-6th Antibiotics in severe

gglobulinemia of IgG synthesis month, resolves by 2½ cases, g globulin
infancy years, infections with replacement.
pyogenic bacteria.
Job syndrome TH1 cells can’t make Coarse facies, cold
IFNg, PMNs don’t abscess, retained
respond to chemotactic primary teeth, ↑IgE,
stimuli eczema.

Selective T cell deficiency:

Disease Defect Clinical features
Digeorge syndrome Failure of formation of 3rd Facial and cardiac abnormalities,
(only IgM) and 4th pharyngeal pouches, Hypoparathyroidism, ↓T cell response
thymic aplasia
MHC class I Failure of TAP 1 molecules CD8↓, CD4 normal, recurrent viral infection,
deficiency to transport peptides to ER normal DTH and Ab response
MHC class II Failure of MHC II Deficient CD4, No GVHD, ↓Ig
deficiency expression, defects in
transcription factors

Combined partial B and T cell deficiency:

Disease Defect Clinical features
Wiskott Aldrich Defect in cytoskeletal ↓ Response to bacterial polysaccharide ↓ IgM,
syndrome glycoprotein, X linked. gradual ↓ HMI and CMI, thrombocytopenia and
eczema
Ataxia Defect in kinase involved in Ataxia, telengiectasia, deficiency of IgA and IgE
telangiectasia cell cycle.

Complete functional B and T cell deficiency:

Disease Defect Clinical features
SCID Defect in common g of IL2 receptor (present in Chronic diarrhoea, skin mouth and throat
receptor of IL 4,7,9,15), X linked. lesions, OI, cells unresponsive to mitogens.
Adenosine deaminase deficiency. As above.
Rag1 or Rag 2 gene nonsense mutation. Total absence of B and T cell.
Immunology | 121

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122 | Microbiology
Active Recall from Tables
Deficiency Examples

B cells

T cells

B & T cells

Phagocytes

Complements
Immunology | 123
Concept 4.9: Transplant Immunology
Learning Objectives
• Types of Grafts
• Types of rejections and Mechanism of action

Time Needed
1 reading
st
30 mins
2 look
nd
10 mins

Transplantation Immunity:
AUTOGRAFT: One part of body to another.
ISOGRAFT: Between Genetically identical twins.
ALLOGRAFT: Between members of the same species but of different genetic constitution.
XENOGERAFT: Between members of different species.

Allograft Reaction:
Hyperacute Rejection:
• Presence of antibodies against graft.
• Prior blood transfusion, multiple pregnancies, previous transplant.
• Preformed antibodies fix complement damaging endothelial lining of blood ves-
sels (blockade) of microvasculature.
• Time: Minutes to hours.
Accelerated Rejection:
• Due to antibodies formed immediately after transplantation.
• Rare.
Acute Rejection / Accelerated: Rejection:
• Days to Weeks.
• Primary Activation of T cells with Triggering of various effector mechanisms.
• Transplant given to someone who has been presensitized→ 2° Activation of Tcells→
Accelerated second set rejection (days).
Chronic Rejection:
• Months to years.
• Walls of blood vessel thicken and eventually become blocked.
• Exact cause?:
ƒ Low grade cell mediated rejection.
ƒ Deposition of Ag – Ab complex.
ƒ Recurrence of original disease.
• Main features:
ƒ Proliferation of smooth muscle cells.
ƒ Interstitial fibrosis.
124 | Microbiology
Graft Versus Host Reaction:
• Occurs in bone marrow transplantation.
• Induced by immunologically competent T cells being transplanted into allogenic
recipients which are unable to reject them.
• Due to lack of immuno-competence of host.
• Severe damage to skin and intestine.
• Potential complication after allogenic bone marrow transplantation.
• Avoided by:
ƒ Careful typing.
ƒ Removal of mature T cells from graft.
ƒ Use of immunosuppressive drugs.
ƒ Prevention of transplant rejection.
ƒ HLA typing to identify class I Ag by microcytotoxicity tests.

Fig.4.30: HLA Typing

Immunology | 125
Class II compatibility testing by mixed lymphocyte reaction

Fig.4.31: Mixed lymphocyte reaction

126 | Microbiology

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Immunology | 127
Active Recall from Tables
Rejection types Mechanism & HSR
128 | Microbiology
Concept 4.10: Important tables/ images for revision
Learning Objectives
• CD markers
• Cytokines
• Vaccines
• Images

Time Needed
1st reading 45 mins
2 look
nd
15 mins

CD markers used to differentiate functional lymphocyte subpopulation:

CD designation Function B cell Th Tc NK

CD2 Adhesion molecule; − + + +

signal transduction

CD3 Signal transduction molecule of T cell receptor − + + +

CD4 Adhesion molecule that binds to MHC II; − + − −

Signal transduction

CD5 Unknown + + + +

CD8 Adhesion molecule that binds to MHC I; Signal − − + +

transduction

CD16 Low affinity receptor for Fc region of IgG − − − +

(FcgRIII)

CD19 Signal transduction; + − − −

CD21 co-receptor

CD21 (CR2) Receptor for complement (C3d and EB virus) + − − −

CD28 Receptor for costimulatory B7 molecule on APC − + + −

CD32 Receptor for Fc region of IgG + − − −

(FcgRIII)

CD35 (CR1) Receptor for complement (C3b) + − − −

CD40 Signal transduction + _ _ _

CD45 Signal transduction + + + +

CD56 Adhesion molecule − − − +

Immunology | 129
Cytokine function in immune response:
Innate immunity:
Cytokine Secreted by Targets and effects
IL1 Monocytes, Macrophages, Endothelial Vasculature (Inflammation).
cells, Epithelial cells Hypothalamus (fever).
Liver (induction of acute phase proteins).
TNFα Macrophage, Monocytes, Neutrophils, Vasculature (Inflammation).
Activated T cells and NK cells Liver (induction of acute phase proteins).
Loss of muscle, body fat (cachexia).
Induction of death in many cell types.
Neutrophil activation.
IL12 Macrophage, Dendritic cells NK cells.
Promotes Th1 response.
IL6 Macrophage, Endothelial cells and Th2 Liver (induction of acute phase proteins).
cells Proliferation and Ab secretion of B cell.
IFNα and Macrophage, Dendritic cells, Virus Induces antiviral state in most nucleated cells.
IFNβ infected cells Increases MHC I expression.
Activates NK cells.

Adaptive immunity:
Cytokine Secreted by Targets and effects
IL2 T cells T cell proliferation.
NK cell activation and proliferation.
B cell proliferation.
IL4 Th2 cells, Mast cells Promotes Th2 differentiation.
Isotype class switching to IgE.
IL5 Th2 cells Eosinophil activation and generation.
B cell differentiation.
TGFβ T cells, Macrophages, Inhibits T cell proliferation and effector functions.
other cell types Inhibits B cell proliferation.
Isotype class switching to IgA.
Inhibits macrophages.
IFNg Th1 cells, CD8 cells, NK Activates macrophages.
cells Increased expression of MHC I and MHC II.
Increases antigen presentation.
Promotes Th1 response.
Isotype class switching to IgG.
130 | Microbiology
Cytokine based therapies in clinical use:
Agent Nature of agent Clinical application
Enbrel Chimeric TNF-receptor/IgG Rheumatoid arthritis
constant region
Remicade or Humira Monoclonal Ab against TNFα Rheumatoid arthritis, Crohn’s disease
receptor
Roferon IFNα-2a Hepatitis B, Hairy cell leukemia, Kaposi’s
sarcoma, Hepatitis C
Intron A IFNα-2b Melanoma
Avonex IFNβ-1a Multiple sclerosis
Betaseron IFNβ-1b Multiple sclerosis
Actimmune IFNg-1b Chronic Granulomatous disease, Osteopetrosis
Neupogen G-CSF Stimulates production of neutrophils
Reduction of infection in cancer patients
treated with chemotherapy
AIDS patient
Leukine GM-CSF Stimulates production of myeloid cells after
bone marrow transplantation
Neumega or Neulasta IL11 Stimulates production of platelets
Epogen Erythropoietin Stimulates production of RBC
Ankinra Recombinant IL1 Ra Rheumatoid arthritis
Daclizumab Humanized monoclonal Ab Prevents transplant rejection
against IL2 R
Basiliximab Human/mouse chimeric Prevents transplant rejection
monoclonal Ab against IL2 R
Eculizumab (Soliris) Binds C5 and inhibits PNH
complement system Neisseria meningiditis

Vaccines:
Vaccine types Diseases Advantages Disadvantages
Whole Organisms
Live attenuated Measles Strong immune response; often Requires refrigerated
Mumps lifelong immunity with full storage; may mutate to
Polio (Sabin vaccine) dose. virulent form.
Rotavirus
Rubella
Tuberculosis
Varicella
Yellow fever
Small pox
Immunology | 131

Live NOT Adenovirus

attenuated
Inactivated or Cholera Stable; safer than live Weaker immune
killed Influenza virus vaccines; refrigerated storage response ; booster dose
Hepatitis A not required. required.
Plague
Polio (Salk vaccine)
Rabies
Pertussis (whole cell)
Purified Macromolecules
Toxoid Diphtheria Immune system becomes
Tetanus primed to recognize bacterial
toxins
Subunit Hepatitis B Specific antigens lower the Difficult to develop
Pertussis (acellular) chance of adverse reactions
Streptococcal pneumonia
Conjugate HiB Primes infant immune systems
Streptococcal pneumonia to recognize certain bacteria

Major HIV 1 vaccine trials:

Vaccine design Study name Status Result

Purified protein (gp120) VAX003, VAX004 Completed in 2003 No protection

Recombinant adenovirus vector (gag/ HVTN 502 STEP Terminated in 2007 No protection
pol/nef) HVTN 503 Phambili

Recombinant canarypox vector (env/ RV 144 Completed in 2009 30% reduction

gal/protease) + env gp120 protein boost

DNA vaccine-6-plasmids (env/gag/pol/ HVTN 505 Began in 2009 In progress

nef) + recombinant adenovirus vector
boost (same genes)

Mechanism of immune evasion by tumor cells:

1. Reduced MHC I expression in tumor cells
2. Expression of dummy molecules which mimic MHC I
3. Tumor cell inhibits apoptosis signals
4. Generates poor costimulatory signals
5. Masking of tumor Antigens with anti tumor antibodies
6. Modulation of tumor antigens by tumor antibodies
7. Inhibition of inflammatory cytokines like IFNg
132 | Microbiology

Fig.4.32: IgM – Pentamer Fig.4.33: IgA -Secretory – Dimer

Fig.4.34: Structure of MHC molecules

Fig.4.35: Rocket electerophoresis

Immunology | 133

Fig.4.36: One dimension – Single and Double diffusion tests

Fig.4.37:
134 | Microbiology

Fig.4.38: Anti Nuclear Antibodies (ANA) – SLE

Fig.4.39: Myasthenia gravis

Immunology | 135

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136 | Microbiology
Active Recall from Tables
Cells CD Markers
5 Systematic Bacteriology

CONCEPTS
 Concept 5.1 Classification of bacteria
 Concept 5.2 Gram Positive Cocci
 Concept 5.3 Gram Positive Bacilli
 Concept 5.4 Gran Negative Cocci
 Concept 5.5 Gram Negative Bacilli
 Concept 5.6 Spirochetes
 Concept 5.7 Mycoplasma, Chlamydia & Rickettsia
 Concept 5.8 Miscellaneous bacteria
 Concept 5.9 Clinical Microbiology
138 | Microbiology
Concept 5.1: Classification of Bacteria
Learning Objectives
• Classification based on Oxygen requirement
• Classification based on Gram stain

Time Needed
1 reading
st
20 mins
2 look
nd
05 mins

Gram + Gram – Non-Gram Staining

Cocci Rods Anatomically Anatomically

Staphylococcus* Gram + Gram –
Streptococcus* Mycoplasma Spirochetes*
Mycobaterium* Rickettsiae
Aerobic Anaerobic Chlamydiaceae
Bacillus Clostridium*
Listeria Actinomyces
Corynebacterium* Propionibacterium
Mycobacterium* Lactobacillus

Cocci Rods
Neisseria*
Moraxella
Veillonella

Aerobic Facultative Spirillar Intracellular

Pseudomonas Anaerobes Spirochetes* Rickettsiae*
Legionella Enterobacteriacease* Chlamydiacease*
Brucella Pasteurella
Bordetella* Haemophilus*
Francisella
Curved, Anaerobic
Microaerophilic Straight
Campylobacter Bacteroides
Helicobacter Fusobacterium
Vibrio

Bacteria can be broadly divided into:

1. Aerobic bacteria–bacteria which require O2 for growth.
2. Anaerobic bacteria – bacteria not requiring O2 for growth.
Systematic Bacteriology | 139
Oxygen requirements of different organisms:
Classification Characteristics Important Genera
• Cannot grow in absence and low tension • Mycobacterium
of O2. • Pseudomonas
Obligate aerobes • Do not ferment sugars.
• Enzyme superoxide dismutase is present.
Microaerophilic Requires oxygen but low oxygen tension. • Campylobacter
• Helicobacter
• Leptospira
• M.bovis
Prefer aerobic metabolism. • Most bacteria e.g.
Facultative anaerobes Can also grow anaerobically. Enterobacteriaceae
Aerotolerant Anaerobic bacteria that tolerate presence of • Actinomyces
anaerobes oxygen for at least short periods. • C. perfringens
• Lack superoxide dismutase.
Obligate anerobes • Generally lack catalase. • Bacteroides
• Are fermenters. • Clostridium
• Cannot use O2 as terminal electron
acceptor.

Aerobic Bacteria

Gram Positive Gram Negative

Cocci Cocci Bacilli/Rods

Bacilli/Rods
e.g. Staphylococccus e.g. Bacillus e.g. Neisseria e.g. Enterobacteriaceae
Streptococcus Corynebacterium Pseudomonads
Pneumococcus Mycobacteria Pasteurella
Enterococcus Francisella
140 | Microbiology
Concept 5.2: Gram Positive Cocci
Learning Objectives
• Classification of Gram Positive cocci
• Staphylococci & Streptococci
• Virulence factors & Pathogenesis
• Clinical features
• Diagnosis & Treatment

Time Needed
1 reading
st
60 mins
2nd look 15 mins

Gram Positive Cocci:

Can be divided into two types:
1. Catalase positive – Staphylococcus species.
2. Catalase negative – Streptococcus species, Streptococcus like (Pneumococcus, En-
terococcus).
Staphylococcus:
Grape like cluster (due to division in 3 planes):
Coagulase positive (Tube) Coagulase Negative
e.g. e.g.
• Staphyococcus aureus (Human) • S. epidermidis
• Staphyococcus intermedius (animal) • S. saprophyticus
• Staphylococcus hyicus(animal) • S. haemolyticus

S. aureus differentiated from S. epidermidis by:

Test S. sureus S. epidermidis
Coagulase + −
Mannitol fermentation + −
Heat stable nuclease + −
Phenolphthalein phosphatase + −
Beta hemolysis on blood agar + −
Golden yellow pigment + −
Sensitivity to lysostaphin + −
Staphylococci are differentiated from micrococci by Hugh Leifson’s oxidative
fermentative test. Micrococcus is oxidative and Staphylococcus is fermentative.
Systematic Bacteriology | 141

Fig.5.1: Coagulase test

Staphylococcus aureus:
Media:
• 5% sheep blood agar (SBA) – shows β-haemolysis.
• MacConkey agar: Pink colonies – means Lactose fermenting.
• Selective medium:
ƒ Selective salt media (10% NaCl).
ƒ Mannitol salt agar– selective and indicator → with 7.5% NaCl.
ƒ Ludlam’s tellurite media.
Selective media are useful for isolation of Staphylococcus from food, faeces and where
mixed flora is expected
• Media for pigment enhancement – Milk agar, Glycerol monoacetate agar, 25°C
incubation.

Fig.5.2: S. aureus – Gram stain Fig.5.3: Ludlam’s medium – S. aureus

142 | Microbiology
Antigenic Structure and Virulence Factors:
Cell wall associated Structures: Activity
Capsular polysaccharide Inhibits Opsonisation.
Peptidoglycan Confers cell rigidity and induces inflammatory response.
Teichoic acid Adhesion and inhibits complement mediated Opsonisation.
Protein A Antiphagocytic, anti-complementary, chemotactic, B cell mitogen.
Binds to Fc of IgG (except IgG3) leaving Fab free to bind to Ag.
Used for coagglutination tests for streptococcal and gonococcal
typing.
Clumping factor/ Bound Activates Factor I and inhibits complement mediated opsonisation.
coagulase: Responsible for slide coagulase reaction.
Protein receptors Binds fibronectin, fibrinogen, IgG and C1q.
Extracellular factors Activity
a hemolysin Inactivated at 70°C, reactivated paradoxically at 100°C.
Lyses rabbit, sheep and human red blood cells.
Leucocidal, Cytotoxic, dermonecrotic, neurotoxic, lethal.
β Hemolysin Sphingomyelinase.
Lyses sheep, but not human or rabbit red blood cells.
Exhibits hot-cold phenomenon (lysis is initiated at 37°C but is evident
only after cooling at 4°C).
g Hemolysin Lyses rabbit, sheep and human red blood cells.
dHemolysin Lyses rabbit, sheep and human red blood cells.
Leucocidins / Panton valentine Two components F and S.
toxin Synergohymenotropic toxins.
Damage polymorphonuclear leukocytes and macrophages.
Epidermolytic toxin (Exfoliative Mainly belong to phage group II.
toxin) Two serotypes ETA and ETB.
Serine protease that cleave desmosomal cadherins.
Responsible for Staphylococcal Scalded Skin Syndrome (SSSS).
Enterotoxins Produced by 40% of clinical isolates.
Cause food poisoning (IP < 6hrs) – meat, fish and milk.
Heat stable 100°C for 10-40min.
Fifteen antigenic type (A -E and G-P) Type A – MC.
Toxic shock syndrome toxin Most strains belong to phage group I.
22,000 daltons.
Earlier called as enterotoxin F (MC cause of TSS).
Extracellular enzymes: Activity
Systematic Bacteriology | 143

Coagulase Enzyme Clots human or rabbit plasma, NOT guinea pig plasma.
Acts with coagulase reacting factor (CRF) in plasma by binding to
prothrombin and converting fibrinogen to fibrin.
Eight types of coagulase (Type A – MC).
Responsible for tube coagulase test.
• 1:6 dilution plasma used.
• Citrate NOT used as anticoagulant for preparing plasma.
• Read at 3-6 hrs.
Staphylokinase (fibrinolysin) 13,000-15,000 daltons.
Cleaves IgG and C3b and prevents opsonisation.
Breaks fibrin clots and allows spread of infection.
Hyaluronidase >90% strains produce.
Hydrolyses hyaluronic acid; destroys connective tissue.
Deoxyribonuclease Degrades DNA, heat stable nuclease.
Lipase Degrades lipid.
Phospholipase Degrades Phospholipids.
Proteases Degrades Proteins.

Clinical manifestations:
Folliculitis, furuncle, boils, impetigo and cellulitis.
MC cause of:
• Surgical site infection.
• Infective Endocarditis (Native valve and IVDA).
• Osteomyelitis.
• Post viral pneumonia.
• Epidural abscess.
• Botryomycosis.
Methicillin resistant Staphylococcus aureus (MRSA):
(1961) – Usually multi drug resistant.
• Important hospital pathogen.
• Definition – Methicillin MIC 8 µg/ml or more, Oxacillin MIC 4 µg/ml or more.
• Resistance is chromosomally mediated (mec A gene).
• Due to altered PBP 2a (MC) > hyper production of β lactamase.
• Treatment- Vancomycin is DOC.
• Others- Teicoplanin, Linezolid, Streptogramin.
• Cipro/levofloxacin, cotrimoxazole, clindamycin, mupirocin, minocycline.
• Newer – daptomycin, ceftobiprole, tigecycline, oritavancin.
144 | Microbiology
Detection:
• Using 5mg methicillin / cefoxitin or 1mg oxacillin disc.
• On media containing 4% NaCl.
• Incubation at lower temperature of 30° / 35°C.

VISA and VRSA:

• VRSA- Vancomycin Resistant Staphylococcus aureus – MIC 16 µg/ml or more.
• VISA – Vancomycin Intermediate Staphylococcus aureus – MIC 4-8 µg/ml.
• Mechanism – due to thickening of cell wall >VanA gene.
• Treatment – same drugs given for MRSA except Vancomycin and Teicoplanin.
Coagulase Negative Staphylococcus (CoNS) → normal flora of skin:
• They produce white non-pigmented colonies and can be distinguished from S. aureus
by their failure to produce coagulase and DNAse.
• Many stains of S. epidermidis are capable of producing large no.of polysaccharide
glycocalyx known as slime (Biofilm production).
Coagulase Negative Staphylococcus (particularly S. epidermidis) are principal
pathogens of:
• Peritonitis in patients undergoing CAPD.
• Endocarditis with insertion of valvular prosthesis.
• Ventricular shunt infections.
S. saprophyticus:
• Coagulase negative Staphylococcus, causes UTI in young sexually active female.
• Honeymoon cystitis.
• S.saprophyticus is novobiocin resistant.
Streptococcus:
Catalase–ve, gram positive cocci arranged in chains discovered by Billroth.

Classification:
Streptococci: On the basis of hemolysis, divided into 3 groups:
∝ haemolytic β haemolytic γ haemolytic
Viridans 20 Lancefield groups Enterococci
group A to V except I and J

Fig.5.4: Beta hemolytic Streptococci

Systematic Bacteriology | 145
Anerobic:
e.g. Peptostreptococcus
Lancefield's Group:
Streptococci divided on the basis of group specific C carbohydrate Ag.
• Lancefield’s acid extraction method.
• Fuller’s method with formamide.
• Maxted’s method by enzyme produced by Streptomyces albus.
• Rantz and Randall’s method by autoclaving.
• Agar gel precipitation method.
Griffith Typing:
GAS (group A Streptococci) is divided into serotypes on the basis of M protein:
• Lancefield’s acid extraction method.
• DNA sequence types of the emm gene encoding the M protein.

GAS (Group A streptococci /Streptococcus pyogenes):

Media:
• CVBA (Crystal violet, nalidixic acid, colistin blood agar)
• PNF (Polymyxin Neomycin Fusidic acid)
• PIKE’S (crystal violet, sodium azide blood agar) - transport media

Antigenic structure and Virulence factors:

Virulence factor Activity
Lipoteichoic acid Mediates adherence to epithelial cells.
M protein Mediates adherence to epithelial cells, inhibits phagocytosis.
Polysaccharide capsule Inhibits phagocytosis.
containing hyaluronic acid Colonization of GAS by binding to CD44 on pharyngeal epithelial
cells.
Erythrogenic toxin (Streptococcal SPE A and C phage encoded and SPE B chromosomal encoded.
pyrogenic exotoxin SPE) SPE act as superantigens causing TSS.
Hemolysin Heat labile protein.
Streptolysin O Lysis of cells by binding to cholesterol in the cell membrane.
Also produced by some group C and G streptococci.
Inactivated by oxygen.
Strongly antigenic.
Hemolysin Leucocidal.
Streptolysin S Not inactivated by oxygen.
Due to its small size it is not antigenic.
Streptokinase Produced by group A, C and G streptococci and is antigenic.
Fibrinolytic action (preventing the formation of fibrin barrier) causes
rapid spread of infection thrombolytic agent (source group C).
146 | Microbiology

Deoxyribonuclease 4 different types have been identified (A, B,C and D).
Hydrolyze nucleic acids and nucleoproteins.
Antigenic and DNAase B (MC).
Hyaluronidase Produced by strains of group A, B, C and G streptococci.
Antigenic.
Spread of infection through the tissues.
Spy CEP A serine protease that cleaves the neutrophil chemoattractant cytokine
IL-8.
Serum opacity factor It is a lipoproteinase enzyme.
It reacts with and produces opacity in mammalian sera.
It is loosely bound to the cell and is antigenic.

Clinical manifestations:
• Pharyngitis.
• Scarlet fever:
ƒ Caused by pyrogenic (erythrogenic) exotoxin.
ƒ Pharyngitis with rash (papular-sandpaper, first on trunks).
ƒ Strawberry tongue.
ƒ Pastia’s lines.
ƒ Susceptibility testing by dick test (positive: Intradermal injection of erythrogenic
toxin producing erythematous reaction).
ƒ Diagnosis by Schultz Charlton reaction (positive: Blanching of rash after injection
of antibodies).
• Skin and soft tissue infections:
ƒ Impetigo (S. aureus MC).
ƒ Erysipelas.
ƒ Cellulitis (peau d’orange skin texture).
ƒ Necrotising fasciitis (flesh eating bacteria) -M type 1 and 3.

Non suppurative sequelae:

Acute rheumatic fever (ARF) Post streptococcal
glomerulonephritis (PSGN)

initial infection Pharyngitis Impetigo or Pharyngitis.

Onset 1-5 weeks 2-6 weeks following impetigo.

1-3 weeks following pharyngitis.

Serotypes Any Pyodermal M types 2, 47, 49, 55, 57

and 60.
Pharyngitis M types 1-4, 12, 25 and
49.
Systematic Bacteriology | 147

Immune response Initial binding of M protein to type Immune complex mediated cellular
IV collagen RHD and by molecular damage by SPE B (Streptococcal
mimicry of the M protein and the pyrogenic exotoxin) and NAPlr
N-acetylglucosamine of GAS (nephritis associated plasmin receptor).
carbohydrate).

Genetic susceptibility Present (HLA DR4 and 7) Not known

Complement level Unaffected Lowered

Repeated attacks Common Absent

Penicillin prophylaxis Essential Not indicated

Course 60% of ARF progress to RHD Spontaneous resolution

ASO titre >200 Low

Anti DNAase B >300 >300

Streptozyme test (passive + +

slide hemagglutination test)

Confirmation of Diagnosis:
• Bacitracin (0.04U disc) sensitivity on blood agar plate.
• PYR test.
• Serology for ARF and PSGN.

GBS (Group B Streptococcus/Streptococcus agalactiae):

• Causative agent of bovine mastitis.
• Virulence factor: Polysaccharide capsule (10 types).

Clinical Manifestations:
Infections in neonates.
Incidence: 0.6 cases per 1000 live births.
• Early onset – Acquired from maternal vagina during birth and presents as septicemia
within 1 week.
• Late onset – Acquired during vaginal delivery, later contact with mother or health
care workers, environment and presents as meningitis between 2 to 12th week of life
(GBS capsular type III – MC).
• 5-40% women are carriers of GBS.
• 50% of infant delivered vaginally by carrier mothers become colonized but 1-2%
develop clinical manifestation(prematurity, prolonged labour, obstetric complication
and maternal fever are risk factors).
• The CDC recommends screening for anogenital colonization at 35-37 weeks of
pregnancy by a swab culture of the lower vagina and anorectum for chemoprophylaxis.
• Infection in adults with underlying chronic illness (MC).
148 | Microbiology
Presumptive Identification:
• Hippurate hydrolysis test +ve.
• CAMP (Christie, Atkins and Munch-Peterson) test +ve.
ƒ CAMP factor is a phospholipase produced by GBS that causes synergistic hemolysis
with β hemolysin produced by certain strains of S. aureus demonstrated by
butterfly hemolysis by cross-streaking of the strains on blood agar.
Group C Streptococci:
• Predominantly animal pathogen.
• Upper respiratory infections in humans by Streptococcus equisimilis (MC).
• Differentiated from GAS in fermenting ribose not trehalose.
• Source of streptokinase for thrombolytic therapy (streptokinase antigenically different
from that produced by GAS).
Group F Streptococci:
• Capnophilic.
• “Minute streptococci”.
• Streptococcal MG is the alphahemolytic group F Streptococci isolated from patients of
primary atypical pneumonia (hence, used as heterophile Ag for diagnosis of primary
atypical pneumonia).

Non Enterococcal Group D Streptococci:

S. bovis reclassified into 2 species:
• Streptococcus gallolyticus subspecies gallolyticus and pasteurian.
• Streptococcus infantarius subspecies infantarius and coli.
Clinical Manifestations:
• Endocarditis associated with neoplasms of the gastrointestinal tract (MC - colon
carcinoma or polyp) and other bowel lesions.
Viridans Streptococci:
• Streptococcus salivarius, Streptococcus mitis, Streptococcus sanguis and
Streptococcus mutans are part of the normal flora of the mouth.
Clinical Manifestations:
• Endocarditis due to transient viridians streptococcal bacteremia induced by eating,
toothbrushing, flossing and other sources of minor trauma, together with adherence
to biologic surfaces.
Enterococci:
• Commensal flora of the large bowel of human adults, account for <1 % of the
culturable intestinal microflora.
• E. faecalis (MC).
• E faecium (more resistant and almost as common as faecalis in HAI).
• Grows in the presence of 40% bile.
• Grows at 9.6 pH.
Systematic Bacteriology | 149
Virulence Factors:
• Enterococcal secreted factors (Hemolysin/cytolysin, Gelatinase, Serine protease).
• Enterococcal surface components – facilitates conjugation:
ƒ Adhesin of collagen of E. faecalis (Ace) and E. faecium (Acm) recognize adhesive
matrix molecules (MSCRAMMs) involved in bacterial attachment to host proteins
such as collagen, fibronectin and fibrinogen.
• E. faecali stress protein Gls24 – resistance to bile salts.
Clinical Manifestations:
• UTI.
• Bacteremia and endocarditis.
• Meningitis.
• Intraabdominal pelvic and soft tissue infections.
Resistance:
• Penicillin resistance due to:
ƒ Mutations in the PBP5 encoding gene that decrease the protein’s affinity for
ampicillin.
ƒ Hyperproduction of PBP5.
• VRE (Vancomycin resistant enterococci) first documented in 1986 due to:
ƒ Replacement of the last alanine residue peptidoglycan precursors with D-Lactate
or D-serine with consequent high and low level resistance, respectively.
ƒ Production of enzymes that destroy the D-alanine-D-alanine ending precursors
ensuring that additional binding sites for vancomycin are not available.
High level aminoglycoside resistance (HLAR) due to:
• Genes encoding aminoglycoside-modifying enzymes.
• Gentamicin MIC of >500 μg/mL and Streptomycin MIC of >2000 119/m L.
• Linezolid resistance due to mutations in the 23S rRNA genes and presence of
rRNAmethylase.
• Daptomycin resistance due to changes in cell membrane homeostasis.
Diagnosis:
γ hemolytic on blood agar
• Growth in 6.5% NaCl and at 46°C.
• Growth in tellurite media to produce black colonies.
• Ferment mannitol, sucrose and sorbitol.
• Hydrolyse esculin in presence of 40% bile salts.
• Hydrolyze pyrrolidonyl- β-naphthylamide (PYR); this characteristic is helpful.
• In differentiating enterococci from organisms of the Streptococcus gallolyticus and
pasteurian group (earlier k/a S. bovis).
S. pneumoniae:
• Flame or lanceolate shaped capsulated gram positive diplococci.
• Draughtsman colonies due to autolysis on blood agar.
150 | Microbiology

Fig.5.5: Pneumococci – Gram stain

Virulence Factors:
• Polysaccharide capsule inhibits phagocytosis; the polysaccharide can diffuse in tissue
(specific soluble substance, SSS).
• Teichoic acid and peptidoglycan induce IL1, IL6 and TNF and activate alternate
complement pathway initiate inflammatory response.
• Pneumolysin causes cytolysis.
• Pneumococcal H inhibitor (Hic) and pneumococcal surface protein (PspC) /also k/a
Choline binding protein (CbpA) inhibits complement system.
• NanA (Neuraminidase) and PsaA (pneumococcal surface adhesion) cause adhesion.
• Autolysin.
Clinical Manifestations:
• Pneumonia (MC cause of community and hospital acquired pneumonia).
• Hospital (ventilator) acquired pneumonia after 5-6days of admission or in case of
MDR strain – Pseudomonas (MC).
• Meningitis (MC cause in adults and children).
Diagnosis:
M/E by Grams stain.
Culture: Draughtsman colonies on blood agar at 5-10% CO2.
Serology:
• Quellung reaction (capsular swelling).
• Demonstration of SSS in CSF by precipitation or latex agglutination.
• CRP by latex agglutination.

Difference between Pneumococci and Viridans Streptococci:

Pneumococci Viridans streptococci
Morphology Capsulated, lanceolate Non capsulated, chains
diplococcic
Quellung reaction + -
Systematic Bacteriology | 151

Colonies Draughtsman Dome shaped

Bile solubility test (10% Na + -
deoxycholate)
Inulin fermentation + -
Optochin sensitivity (ethyl S R
hydrocuprein) on blood agar
Intraperitoneal inoculation in Fatal infection Non pathogenic
mice

Antigenic structure:
• Capsular Ag – 95 types, SSS, important for virulence, typing by Quellung reaction.
• Somatic Ag – Carbohydrate Ag – teichoic acid.
• Pneumolysin.
• Autolysin – amidase.
Pathogenicity:
• Commensal. – 5-70% humans carry S.pneumonae in throat.
• Pneumonia – In adults – type 1 – 8 , 18:
ƒ In Children – 6,14,19,23.
ƒ Lobar pneumonia : 10 – 50yrs.
ƒ Bronchopneumonia – young children, adults > 50years.
• Meningitis.
• Others – otitis media, sinusitis, peritonitis etc.
Treatment:
Penicillin.
In S. pneumoniae , penicillin resistance has emerged but this resistance is not due to
penicillinase enzyme. It is chromosomally mediated and these are altered penicillin
binding protein, which are responsible for resistance.
PPPG - Ceftriaxone.
Vaccine:
Adults: Pneumococcal polysaccharide vaccine (PPV). 23 capsular polysaccharide types
are included.
Children less than 2 years of age: 13 valent Pneumococcal conjugate vaccine (PCV).
152 | Microbiology

Worksheet
• MCQ OF “SYSTEMATIC BACTERIOLOGY” FROM DQB

• EXTRA POINTS FROM DQB

1.

2.

3.

4.

5.

6.

7.

8.

9.

10
Systematic Bacteriology | 153
Active Recall from Tables
Organism Exotoxins
S. aureus

Strep. pyogenes

Sterp. pneumoniae

Most common cause of Pathogen

Osteomyelitis

Pustular tonsilitis

Otitis media

Infective endocarditis in IDU

Surgical site infections

Toxic shock syndrome

154 | Microbiology
Concept 5.3: Gram Positive bacilli
Learning Objectives
• Classification of Gram Positive bacilli
• Virulence factors & Pathogenesis
• Clinical manifestations
• Lab diagnosis & Treatment.

Time Needed
1st reading 120 mins
2 look
nd
30 mins

Gram Positive Bacilli:

Three general groups:
• Endospore-formers–Bacillus, Clostridium.
• Non-endospore-formers–Listeria, Erysipelothrix.
• Irregular shaped and staining properties –Corynebacterium, Propionibacterium,
Mycobacterium, Actinomyces, Nocardia.

Corynebacterium:
Gram positive bacilli, club shaped, Chinese letter arrangement, cuneiform → V and L
forms.
Stained with methylene blue/toluidine blue → metachromatic granules -Babe Earnst
granules/Polar bodies/Polymetaphosphates/Volutin granules.
Culture:
• Loeffler’s serum slope:
ƒ Earliest growth (6-8hrs).
ƒ Granules best developed.
ƒ Water of condensation can be used for animal pathogenicity.

Fig.5.6: Blood Tellurite Agar – C. diphtheriae

Systematic Bacteriology | 155
• Selective medium:
ƒ Potassium tellurite (.03 – 0.04%) – black colonies due to tellurite reduction.
ƒ Tinsdale's.
ƒ Hoyle's medium.
ƒ Mcleod’s medium.
On the basis of morphology on tellurite medium C. diphtheriae has four biotypes;
a. Gravis → Daisy head colonies.
Fermentation of starch +ve.
Toxigenic strains 100%.
Epidemic areas, Nitrate +ve.
Paralytic and Haemorrhagic manifestations occur as complication.
b. Intermedius → Frogs eggs colony.
Epidemic areas . Nitrate +ve.
Hemorrhagic manifestations.
c. Mitis → Poached egg.
Endemic areas.
Nitrate +ve.
Obstructive manifestations.
d. Belfanti – Nitrate –ve.
The C. diphtheriae group of organism include the sub type species of C. diptheriae (with four subspecies),
C. ulcerans and C. pseudotuberculosis.

Diphtheria toxin +ve:

C. diphtheriae – (urease negative).
C. ulcerans – (urease positive).
C. pseudotuberculosis – (urease positive).
Toxin is released locally but it causes systemic S/S, toxaemia by absorption in circulation.
(Exo) Toxin A and B fragments  A inhibit elongation factor  inhibits protein synthesis.
Fragment B – for transportation of fragment A inside cell.
Optimum iron concentration for toxin production is 0.1mg per litre of the medium, toxin
production stops 0.5 mg per litre of the medium.
Toxigenicity due to b phage (tox phage).
Almost all strains of gravis, 95%-99% of intermedius and 80 – 85% mitis produce toxin.
The classic park William (PW8 ) strain of C. diphtheriae is used as a source of toxin for
diphtheria toxoid (DPT vaccine).
Toxigenicity or Virulence test of C. diphtheriae:
Toxigenicity is due to tox (b) by Phage (lysogenic)Conversion
Tests for pathogenicity – Guinea pig or rabbits used, mice is resistant to diphtheria
In vivo tests:
• Subcutaneous test.
• Intradermal test.
156 | Microbiology
In vitro tests:
• Elek’s gel precipitation test.
Shick test (toxin + antitoxin = neutralization)
0.2 ml (1/50 ml) toxin
↓
I/d on ant. surface of left forearm
while right forearm is control with antitoxin
↓
Results read after 24 – 48 hrs and 5-7 Days
• Negative → Immune, No immunisation required.
• Positive → Erythema and indurations (1-5 cm) on 5th day, means susceptibility, needs
immunization.
• Pseudo → Immune and HS (reaction in test and control) and by 5th day it disappears
from both arms.
• Combined → Non-immune and HS (treatment in test and control) Reaction disappears
in 5 days in control arm.
Pathogenicity:
Natural infection only in man.
Pseudomembrane formation on tonsil, posterior pharyngeal wall.
Most clinical infections are probably contracted from carriers.
Nasal carriers are particularly dangerous because shed large number of bacilli.
Treatment:
C. diphtheriae is sensitive to penicillin, Erythromycin, Rifampicin; but antibiotics do not
neutralize circulating toxin. Therefore antibiotics are of value combined with antitoxin.
Erythromycin active for treatment of carriers.
Non Diphtheria Corynebacteria:
• Those which cause infection in immunocompetent individual.
ƒ C. ulcerans.
ƒ A. haemolyticum.
ƒ C. pseudotuberculosis (ovis).
ƒ C. minutissimum (causes erythrasma).
• Those which cause opportunistic infections:
ƒ C. jeikeium.
ƒ C. xerosis.
ƒ C. hofmanni (pseudodiphtheriticum).
ƒ C . equi.
Bacillus:
• Family Bacillaceae.
• Aerobic gram positive spore forming bacilli.
• In human and animals → 2 species.
Systematic Bacteriology | 157
• B.anthracis – Highly pathogenic, non motile, zoonotic disease.
• B .cereus – Motile.
B.anthracis:
• Historic relevance.
• Koch's postulates based on B.anthracis.
• First bacterial vaccine.
• First pathogenic bacterium seen under the microscope.
• First communicable disease transmitted by infected blood.
• First to be isolated in pure culture.
Presumptive diagnosis: Mc Fadyean reaction – capsule seen when stained with poly-
chrome methylene blue.
Spores found in soil or in culture and never in animal body.
In culture : Medusa head appearance.
Grams: Bamboo-stick appearance, chain of bacilli.
Gelatin stab: Inverted fir tree appearance.

Fig.5.7: B.anthracis – Gram stain: Fig.5.8: Medusa head colony – Banthracis

Anthrax caused by B. anthracis.

It is a zoonosis (cattle and sheep).
Man acquires infection through:
• Small cuts or skin abrasion.
• Spore inhalation.
• Ingestion of meat (rarely).
Types of anthrax:
• Cutaneous anthrax → 95% of Human anthrax:
ƒ Black eschar → Malignant pustule.
ƒ Seen in farmer’s veterinary surgeons.
ƒ Persons handling animal carcasses hides.
ƒ Hide porter’s disease/cutaneous anthrax.
ƒ Pulmonary anthrax → Wool sorter’s disease (inhalation of spores).
ƒ Intestinal.
158 | Microbiology
Bioterrorism → Recently used by Afghanistan.
Spores enclosed in paper envelopes were mailed → spore inhalation.
Bacillus cereus:
• Motile.
• Lacks capsule.
• Causes food poisoning.
• Can also cause opportunistic infections like endocarditis, bacteremia, LRTI, meningitis
and crepitant cellulitis.
Toxin 1: Emetic toxin:
• Heat stable exotoxin.
• Symptoms after 1-6 hours of ingestion → preformed toxin.
• Predominantly vomiting and abdominal cramps, NO Fever.
• Caused by eating boiled or Chinese fried rice.
Toxin 2: Enterotoxin:
• Heat labile.
• Symptoms develop after 10-12 hours.
• Predominant symptoms of profuse watery diarrhea and nausea.
• Culture medium – MYPA (Mannitol Egg yolk polymyxin phenol red agar).

Anaerobic Spore forming bacteria:

Clostridium:
Gram Positive, spore forming, Anaerobes.
Pathogens:
• C. tetani.
• C. welchii.
• C. botulinum.
Motility – stately.
Growth on RCM.
• Only proteolytic- C.tetani.
• Only saccharolytic – C.botulinum.
• Neither- C.cochlearum.
• Both – C.sporogens.
• Predominant saccharolytic – C. welchii, C. novyi, C.septicum.
A. C. perfringes (C.welchii):
Non Motile, Spores oval, subterminal.
4 Major (Lethal) Toxins - a,b, ε, i→ 5 types (A-E).
BA → Target Haemolysis i.e. due to:
• θtoxin → Complete haemolysis.
Systematic Bacteriology | 159
• atoxin → Incomplete haemolysis.
Litmus Milk reaction (+).
Naegler reaction (+) d/t a toxin → opalescence on egg yolk media (inhibited) by antisera.
Selective medium → Neomycin Blood Agar.
Infections:
• Gas gangrene.
• Food Poisoning – Type A → Enterotoxin (Similar to cholera and EPEC).
• Necrotising enteritis (Pigbel): Type C.
B. C. tetani– Motile, SWARMING:
• type VI – non motile.
• Drum Stick Appearance (Terminal Spores).
• Fur tree appearance.
Two toxins:
1. Tetanospasmin – Neurotoxin
(exotoxin) ↓
inhibit Normal inhib. reflex
↓
Spasm
2. Telanoysin
C. Clostridium botulinum:
→ 8 types (A-G), A,B,. and E → Human disease.
All are Neurotoxins except C2.
Botulinum toxin→ Most toxic known to man kind.
Min Lethal dose → 1 µg.
Neurotoxin → inhibit release of Acetylcholine.
1. Food borne bolulism
→ Preformed toxin
Canned food → bulging
After 18-36 hours
2. Wound botulism.
3. Infant botulism (spore ingesn) - Floppy Child syndrome.
D. C. diffcile:
→ Pseudomenb. Colitis, due to antibiotics like.
→ Clindamycin, Ampicilin
Rx – Vancomycin
MNZ
Toxins A → Enterotoxin – diarrhea
B → Cytotoxin
160 | Microbiology
Listeria monocytogenes:
• Gram +ve coccobacillus.
• Tumbling motility, grows at lower temperature: 4°C (cold enrichment facilitates
growth).
• It can grow in refrigerated food and can tolerate preserving agents.
Infections:
Pregnancy and neonatal infections – Before 20 wks is rare. May lead to abortion, still
birth, early onset neonatal disease –septicemic illness appear with in 2 days after
delivery (granulomatosis infantiseptica).
Late onset –mainly meningitis, hospital cross infection and symptoms appear 5days or
more after birth.
Lab diagnosis→ Catalase +ve
Beta haemolysis – produces haemolysin called listeriolysin.
Specimens inoculated on blood agar, chocolate agar.

Fig.5.9: Virulence attributed to ability to replicate in the cytoplasm of

cells after inducing phagocytosis; avoids humoral immune system

Erysipelothrix rhusiopathiae:
Clinical significance:
• Primarily a pathogen of swine, turkeys, and fresh water fish.
• In man, the disease is called erysipeloid, is the most common form.
• It is an occupation-associated disease in which a reddish-blue, edematous lesion at
the site of inoculation, primarily following trauma to the hands.
Systematic Bacteriology | 161
Actinomycetes:

Fig.5.10: Actinomyces – Gram stain

Are gram positive bacteria varying from coccoid and pleomorphic forms to branched
filaments.Human pathogenic actinomycetes belong to four genes.
(1) Actinomyces (3) Streptomyces
(2) Nocardia (4) Actinomadura
Actinomyes is non-acid fast and anaerobic or micro aerophilic
Streptomyces and Actinomadura are aerobes and non acid fast.
Actinomyces → Causes Actinomycosis caused by mainly A israelii:
• Causes lumpy jaw in cattle.
• Commensal of the mouth therefore endogenous cause of disease.
• Microscopy → sulphur granules.
• Granules consist of gram positive bacilli and surrounded by clubs (sunray
appearance) Clubs are due to Ag-Ab reaction.
• Types : MC cevicofacial (lower jaw).
• A israelii produces spidery molar teeth colony in solid media, fluffy ball at bottom
of liquid medium.
Nocardia - Strictly aerobic and acid fast (1%).

Fig.5.11: Nocardia – Acid fast stain

162 | Microbiology
Unlike Actinomyces, Nocardia are environmental saprophytes, mainly cause Mycetoma.
Nocardia – grow on Sabouraud dextrose agar, brain heart infusion agar:
• For isolation of Nocardia from soil, paraffin bait technique.
• Stains: AFB, Alcian blue, Mucicarmine, Kinyons.

Mycobacterium:
Acid fast organism:
Mycobacteria - tuberculosis/leprae/NTM.
Nocardia.
Rhodococcus.
Spore.
Sperm head.
Legionella micdadei.
Parasite – Cryptosporidium, cyclospora. Isospora, Tinea scolex , Hooklet of hydatid cyst.
M tuberculosis Complex – include species.
M. tuberculosis.
M. bovis (bovine tubercle bacillius).
M. africanum (intermediate between M tuberculosis and M bovis).
M. microti (vole tubercle bacillus).
Mycobacterium – aerobic, non spore forming, nonmotile.
Average generation time 14 – 15 hours.
M. tuberculosis (obligate aerobe) M. bovis (Microaerophilic)
Eugonic, rough, buff and tough growth. Dysgonic, white.
Glycerol enhances growth. Glycerol inhibits growth.
Susceptible to pyrazinamide. Resistance to PZM.
Resistant to thiophen 2 carboxylic acid (TCH). Sensitive.
Niacin and nitrate +ve, Neutral red (+). Niacin, Nitrate, NR (−).
Pathogenic to guinea pig. Non pathogenic to rabbit. Pathogenic to both.

Pathogenicity:
• Escape killing by macrophage and inhibits phagolysosome fusion (HS IV).
• Virulence factor- cord factor, LAM , HSP.
Clinical feature: Pulmonary Tuberculosis:
• Primary TB -Affects children, subpleural focus in lower lobe of lungs, (Ghon)+ hilar
LN'= primary complex,
Lab methods:
Digestion and decontamination procedures for sputum.
ƒ Petroff’s method (NaOH).
ƒ NALC – NaOH method (N acetyl lysine).
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Direct smear microscopy:
Ziehl-Neelsen staining (ZN staining).
Kinyoun staining.
Fluorescent staining (Rhodamine /Auromine ‘O’ stains).
5000 – 5*104bacteria/ml for sputum to be +ve.
Culture detects 10 to 100 viable organism.
Culture media:
Egg Based – LJ medium, Dorset egg medium.
Liquid media – Middle brook 7H9, Dubus Tween albumin broths.

Fig.5.12: LJ medium – M. tuberculosis

Rapid Culture Methods:

BACTEC 460 TB system.
Mycobacterium Growth Indicator Tube System (MGIT).
Septi-Chek.
ESP Culture System II.
MB/BACT ALERT System.

BATEC:
• Radiometric detection system.
• 14 C labeled substrates (palmitic acid).
• The amount of 14 C°2 is translated into growth index.
Average time to detect M. tuberculosis: 9 to 14 days.
Detection time of positive cultures is decreased to < 7 days for NTM.
Serology - Ag detection, Ab detection, and quantiferon assay.
Molecular Methods Used:
PCR - detecting IS6110 gene.
DNA Probes.
Mantoux Reaction → 0.1 ml PPD - I/D reaction read after 72 hours.
164 | Microbiology
induration³ 10 mm → positive
5-9 – doubtful
≤ 4 – Negative
Use → Active infection in infants, Prevalence of infection.
False –ve: Early/advanced TB.
Miliary TB.
Measles.
↓ Immunity.
False + → Atypical Mycobacterial infection
Conventional Sensitivity Testing.
ƒ Resistance Ratio.
ƒ Absolute concentration.
ƒ Proportion method.
ƒ Radiometric method → based on proportion method.
ƒ Molecular methods.
Newer Drug Susceptibility Testing Methods:
BACTEC 460 TB SYSTEM.
MGIT.
PHAGE Plaque Assay.
Luciferase reporter phages.
PCR.
INNO-LiPA.
DNA sequencing
GeneXpert.
DNA microarrays.
Drug Resistance- due to Mutation:
INH - Kat 6 gene, Inh A gene, ahpC.
R – rpoB gene ( RNA polymerase B).
Z – Pnc A (Pyrazinamidase).
E – emb A,B,C ( Arabinosyl transferase).
S – ribosomal protein subunit 12(rpSL).
Vaccine – (a) BCG → 0-80% efficacy
(b) Recombinant vaccines 
(c) DNA vaccines  under trial


Atypical Mycobacteria:
Atypical Mycobacteria differentiated from MTB complex by:
• Resistance to both paranitrobenzoic acid and TCH.
• Aryl sufatase test +ve.
Systematic Bacteriology | 165
• Resistant to antitubercular drugs.
• Strong Catalase +ve.
Mycobacteria Causing Johnes Disease -
M. paratuberculosis.
Mycobacteria causing opportunistic infections:
• Post trauma abscess -M chelone and M fortuitum.
• Swimming pool granuloma - M marinum
• Buruli ulcer -M. ulcerens.
• Lymphadenopathy -M avium intracellulare and M. scrofulaceum.
• Pulmonary disease - M avium intracellulare, M kansasii and M. xenopi.
• Disseminated disease -M avium intracellulare.
• Organisms requiring incubation at low temp of 25 to 33°C:
ƒ M marinum.
ƒ M ulcerans.
ƒ M chelonae.
ƒ M haemophilum.
M.bovis → microaerophillic
Saprophytic – M.smegmatis ,M.phlei

Atypical (environmental) Mycobacteria:

(Mycobacterium other than Tuberculosis -MOTT)
Runyon group NAME SPECIES
I Photochromogens M kansasii, M. mqrinum, M. simiae
II Scotochromogens M. scrofulaceum, M. suzulgai
III Non chromogens MAC, M. xenopi M. ulcerans
IV Rapid growers M. chelonei, M. fortuitum

Mycobacterium leprae:
• Non cultivable on artificial medium.
• 5% H2 SO4 as decolosiser (Acid Fast).
→ Present inside the cells as parallel bundles of 50 or more organisms bound together
by a lipid like substance, the glia.
Bacteriological index – total no of leprabacilli
→ The % of solid uniformly stained live bacilli in tissues: Morphological index.
Morphological index is more meaningful for assessing the progress of patients on
chemotherapy.
Animal model:
1. Shepard model – foot pad of mouse.
2. Nine banded armadillo (Dasypus novemcintus) is highly susceptible to leprosy, due to
low body temperature.
166 | Microbiology
Classification:
Lepromatous (LL) Tuberculoid TT
AFB + −
Granuloma − +
Lepromin Test − +
Abs + −/+
Mycobacterium lepramurium – rat leprosy, grows on Ogawa’s egg yolk medium
Lepromin reaction → 0.1ml lepromin I/D
1. Early/Fernandez – ac. local. Inflammation, congestion, edema
↓
Like tuberculin (DTH) ≥ 10mm – (48 hours) and it disappears in 3-4 days
(Infection in the past)
2. Late/Mitsuda – 3-4 weeks → nodule
↓ ↓
CMI not d/t past but necrosis, ulcer
current dose of ↓
Lepromin several weeks to heal.
Uses – classify lesions of leprosy in TT/LL, - prognosis

Lab diagnosis:
• Sample- minimum 4 skin (slit skin smear from edge , NOT centre) (buttock/forehead/
chin/cheek) + ear lobule+ nasal mucosa –.
• Grading of smear is done based on MI.
• Acid fast staining with 5% sulfuric acid.
• Mouse food pad inoculation.
• Ab to PGL1.
Treatment:
• Paucibacillary- (I ,TT, BT) – Rifampicin(monthly)+ dapsone daily – for 6m.
• Multibacillary- (BB,BL, LL)- Rifampicin(monthly)+ dapsone daily +clofazimine daily
–till 1 years or smear -ve.
• Single lesion – (ROM)- rifampicin+ ofloxacin+ minocycline – single dose.
Systematic Bacteriology | 167

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• MCQ OF “SYSTEMATIC BACTERIOLOGY” FROM DQB

• EXTRA POINTS FROM DQB

1.

2.

3.

4.

5.

6.

7.

8.

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10
168 | Microbiology
Active Recall from Tables
Toxin Mechanism of Action

Anthrax

Botulinum

Diphtheria

Tetanospasmin

Cl. perfringenes toxin

Pathogen Selective media

C. diphtheriae

B. anthracis

B. cereus

Listeria monocytogenes

M. tuberculosis
Systematic Bacteriology | 169

Atypical mycobacteria Examples

Photochromogen

Scotochromogen

Nonchromogen

Rapid growers

Colony morphology Pathogen

Rough, buff and tough colony

Medusa head colony

Daisy head colony

Molar tooth colony

Poached egg colony

170 | Microbiology
Concept 5.4: Gram Negative Cocci
Learning Objectives
• Gonococci and Meningococci
• Virulence factors and pathogenesis.
• Clinical manifestations
• Lab Diagnosis & Management

Time Needed
1st reading 30 mins
2 look
nd
10 mins

Neisseria:
Family Neisseriaceae.
N. meningitidis (Meningococcus).
Gram negative diplococci, capsulated.
Media – chocolate agar.
Biochemical recations:
Catalase and oxidase +ve.
Glucose and Maltose fermentative.

Antigenic Determinants and Virulence factors:

1. Capsular polysaccharide: Inhibits phagocytosis, are of 13 types; A, B, C, D, X, Y,
Z, 29E W 135, H, I, K, and L.
Group D → Not found naturally.
Most meningococcal disease → A B and C.
Small proportion → Y and W135.
X Z and 29 E → Rarely associated with some form of immunodeficiency.
H, I, K, and L → From carrier and have not been associated with disease.
2. Endotoxin → As organism invade and multiply they release large quantities of
lipooligosaccharides (LOS) and cause vascular necrosis and produce generalized
Shwartzman reaction and are involved in pathogenesis of Water House –
Friderichsen Syndrome.
3. IgA 1 protease → IgA1 protease cleaves subclass IgA 1 and inactivates it.
4. Pili → Allow to adhere and antiphagocytic.
5. OMP - Outer Membrane Protein →divided into 5 classes.
Class 5 has been implicated with attachment.
6. Plasmids.
Pathogenicity:
• Meningococci are normally carried in nasopharynx of 5-10% of healthy individuals.
During epidemic → Carrier rates range from 20-90%.
• Rash with petechial hemorrhage,
Systematic Bacteriology | 171
• DIC, Hypotension.
• Bilateral adrenal hemorrhage - Waterhouse Friderichsen syndrome.
• Meningitis in adolescents and adults.
• Over crowding with poor hygiene, People with Terminal complement deficiency
(C5b-C9) are more susceptible.
Lab diagnosis
• CSF gram stain – Gram negative diplococci
• Antigen detection – Latex agglutination test
Treatment:
Cefotaxime and ceftriaxone → also effective for H. influenzae and Streptococcus
pneumoniae. It is important to give Rifampicin or ciprofloxacin to eradicate N.
meningitidis from nasopharynx.
Vaccine:
Gp specific → A,C,Y ,W-135 immunity lasts for 3 years.
No vaccine for GpB: capsule is poorly immunogenic.
N. gonorrhoeae:

Fig.5.13: Gonococci – Gram stain

More difficult to grow than meningococci.

Kellogg divided gonococci into 4 types based on pili (T1-T4).
T1, T2 → Numerous pili P+, P++ and are virulent.
T3 and T4 – Non pillated or P-, avirulent, forms smooth suspension.
Selective media:
Thayer Martin (VCN).
Modified Thayer Martin (VCNT).
Modified New York city medium.
N. gonorrhoeae N. meningitidis
Utilize glucose. Utilize both glucose and maltose.
172 | Microbiology
Antigenic structure:
1. Pili: Important role in attachment, are anti-phagocytic.
2. Outer membrane protein – protein I, II, and III.
Protein I → Serves as a basis for serotyping.
Protein II → associated with adherence, present in opaque colony.
3. IgA 1 protease.
4. LOS → Toxicity is due to endotoxic effect.
Clinical manifestations
Men :
• Gonococcal urethritis, Dysuria, Stricture urethra.
• Watercan perineum (Multiple abscess with discharge)
Women :
• PID (2nd Most common, 1st is Chlamydia)
• Septic arthritis
• Endocervicitis
• Rash
• Perihepatitis (Fitz – Hugh Curtis syndrome)
Babies born to infected mother:
May result in ophthalmianeonatorum due to N. gonorrhoeae (in first week of life, 30%),
Chlamydia (2nd and subsequent week).

Lab diagnosis
Specimen:
• Men – Urethral discharge
• Women – Endocervical swab
Gram stain is sufficient to start the treatment.
Treatment:
DOC : Cefriaxone + Azithromycin (Syndromic management)
Ophthalmia neonatorum : 1% tetracycline. Or 0.5% erythromycin
Systematic Bacteriology | 173

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• MCQ OF “SYSTEMATIC BACTERIOLOGY” FROM DQB

• EXTRA POINTS FROM DQB

1.

2.

3.

4.

5.

6.

7.

8.

9.

10
174 | Microbiology
Active Recall from Tables
Gonococci Meningococci

Meningitis Glucose Proteins Cells Opening pressure

Etiology

Bacterial

Viral

Fungal

Mycobacterial
Systematic Bacteriology | 175
Concept 5.5: Gram Negative Bacilli
Learning Objectives
• Classification of Gram Negative Bacilli
• Virulence factors and pathogenesis.
• Clinical manifestations
• Lab Diagnosis & Management

Time Needed
1st reading 180 mins

2 look
nd
30 mins

Gram Negative Organisms:

Can be divided into 2 types:
1. Oxidase negative – Enterobactericeae, Stenotrophomonas maltophila, Acinetobacter.
2. Oxidase positive – Neisseria, pseudomonads, Vibrio spp., Aeromonas, Plesiomonas,
Campylobacter, Helicobacter.

Enterobacteriaceae:
The organisms included in this family are Glucose-fermenting, oxidase-negative, motile
(exception Shigella, Salmonella gallinarm, Salmonella pullorum), catalase positive
(exception is Shigella dysenteria Type I) and nitrate reducing.

Classification (Bergey’s manual) Family : Enterobacteriaceae:

Tribe I : Eschericheae.
Genus : Escherichia.
Edwardsiella.
Citrobacter.
Salmonella.
Shigella.
Tribe II : Klebsielleae.
Genus : Klebsiella.
Enterobacter.
Hafnia.
Serratia.
Tribe III : Proteae.
Genus:
Proteus.
Morganella.
Providencia.
176 | Microbiology
Tribe IV : Erwinieae.
Genus : Erwinia.
Tribe V : Yersineae.
Genus : Yersinia.
Escherichia coli:
• GNR, non-capsulated, motile by peritrichous flagella, 80% strains Fimbriae present,
some strains capsulated.
• Pathogenic strains are hemolytic on Blood agar, Lactose fermenting colonies on
MacConkey’s agar.
• Inhibited on DCA, S Sagar, Wand B agar
• Biochemical reactions.
ƒ IMViC: ++--.
ƒ PPA, Urease, H2S Liquefaction, KCN: Negative.
ƒ Glucose, Mannitol, maltose, sucrose, Lactose: A+G.
• Antigenic structure:
ƒ O: Somatic antigen, Heat stable, LPS, 170.
ƒ H: Flagellar antigen, thermolabile, 75.
ƒ K: Capsular antigen, 103.
ƒ F: Fimbrial antigen, thermolabile.
• Normal colon strains: “early “O groups (1, 2, 3, 4 etc); enteropathogenic strains,
“later” O groups ( 26,55,86,111 etc.).
Virulence Factors:
• Surface antigens:
ƒ “O” antigen (LPS): endotoxic; inhibits phagocytosis.
ƒ K antigen; protects from phagocytosis.
ƒ Fimbriae.
ƒ Mannose resistant.
ƒ CFA (I-IV) colonizing) factor antigens) in ETEC.
ƒ “P” Fimbriae in uropathogenic E.coli.
Toxins:
• Hemolysin: Exact role is not known.
• Enterotoxins:
ƒ LT (Labile).
ƒ ST (Stable).
ƒ VT/SLT (Shiga-like toxin or verocytotoxin).
LT:
• Resembles cholera toxin in structure (A1 B5), antigenic properties and mode of action
(Gm1 ganglioside receptors-activates Adenylate cyclase →cAMP → fluid accumulation).
• LT1 and LT2.
Systematic Bacteriology | 177
• Powerful antigen.
• Ligated rabbit ileal loop test (18hrs.) adult rabbit skin, steroid production in Y1 mouse
adrenal cell culture, morphological changes in chinese hamster ovary cells, latex
Agglutination, ELISA.
ST (Plasmid coded):
• Low molecular weight.
• Poorly antigenic.
• 2 types : ST-I and ST-II.
• ST-A: Activation of cGMP:
ƒ Ligated rabbit ileal loop test (6hrs.) intra gastric in suckling mouse.
• ST-B: Mechanism of action not known
VT:
• E.coli O157: H7.
• VT1, VT2.
• VT: Identical to shiga toxin, phage encoded, cytotoxic to vero cells.
• VT is not neutralized by shiga antitoxin
• Clinical infections produced by E.coli:
ƒ Pyogenic infections.
ƒ Septicemia.
Urinary Tract Infection (UTI):
• Most common cause.
• “Early” O groups 1, 2, 4, 6, 7, 18,75.
• Ascending infection: pyelonephritis, K antigen.
• Kass’ concept of “significant bacteriuria”.
• Semi quantitative culture.

Diarrhoea:
1. EPEC:
• Diarrhoea in infants and children, sporadic diarrhoea in adults.
• O26, O55,O86,O111,O114,O125,O126,O127,O128,O142.
• Adhere to intestinal mucosa in localized microcolonies and disrupt brush border
microvilli : attaching effacing lesions (A/E lesions).
• Diagnosis:
ƒ Typing.
ƒ Eae (E.coli attaching and effacing) and EAF (EPEC adherence factor) probes.
ƒ FAS (Fluorescence actin staining ) in HeLa and Hep 2 cell lines.
2. ETEC:
• Acute watery diarrhoea in infants and adults: Traveler’s diarrhea (MCC).
• O6,O8,O15,O20,O25,O27,O63,O78,O115,O148,O153,O159,O167.
178 | Microbiology
• Pathogenesis:
ƒ LT, ST (plasmid mediated).
ƒ Adhesive Fimbrial proteins (CFA I, II, III, IV).
Diagnosis:
ƒ Typing.
ƒ Demonstration of toxins in tissue culture by latex agglutination, ELISA (LT) and
RIA (ST), animal models.
3. EIEC:
• Dysentery like disease in all ages.
• O28, 112,124,136,143,144,152,164.
• Pathogenesis:
ƒ Epithelial cell invasion.
Diagnosis:
ƒ Typing.
ƒ Atypical biochemistry (biochemically similar to Shigella spp.).
ƒ Invasion Plasmid probes.
ƒ Hela and Hep2 cell invasion assay.
ƒ Sereny’s test.
4. EHEC (VTEC):
• O157:H7.
• Bloody diarrhoea in all ages: HC: HUS.
ƒ VT1 and / or VT2.
ƒ Capillary microangiopathy.

Diagnosis:
• Typing.
• Atypical biochemical (β-glucurronidase negative, do not ferment sorbitol and
rhamonose – MacConkey sorbitol agar).
• VT1 and VT2 probes.
• Demonstration of VT1 and VT2 in Vero cell lines.

5. EAEC:
• Persistent diarrhoea especially in developing countries.
• Most are “O” un-typeable but “H” type able.
• Aggregated in a “Stacked Brick” formation on Hep2 cell lines.
• EAST 1 (enter aggregative heat stable enterotoxins).

6. DAEC:
• Diffusely adherent to epithelial cells.
• Carry gene coding surface fimbria.
Systematic Bacteriology | 179
Klebsiella:

Fig.5.14: Macconkey agar with Mucoid LF colonies – Klebsiella

• Capsule, LF, NM, produce mucoid colonies, Urease +ve.

• K. pneumoniae, K. ozaenae, K. rhinoscleromatis.
FREIDLANDER PNEUMONIA → Multiple abscess formation with severe bronchopneumo-
nia and Chronic destructive lesions.

Proteus, Morganella, Providentia:

• Motile, NLF, swarming growth.
• Fishy smell.
• Phenyl alanine Deaminase – PPA test.
• Diene’s phenomenon – to know the relatedness between different strains.
P vulgaris Urease+ve

P mirabilis Most Common PPA+ve

P penneri
P myxofaciens H2S+ve

Non motile strains of Proteus – OX 19, OX2, OXK

↓
(Weil felix) Pr. vulgaris Pr. mirabilis
• Swarming inhibited on MAC, CLED.
• Swarming inhibited – by 6% agar, chloral hydrate, boric acid, alcohol, surface active agents.
180 | Microbiology

Fig.5.15: Swarming - Proteus Fig. 5.16: Diene’s phenomenon – Proteus

Shigella:
NM, NLF , causes dysentery.
MEDIA → DCA, XLD, SS, HE Agar.
Enrichment Broth – G.N broth
Selenite F broth,
A. Sh. dysentriae – 10 serotypes:
Type 1 – Shiga's bacillus (catalase negative).
Type 2 – Schmitzi.
Type 3-7 – Large and Sachs group.
• Mannitol non-fermenting.
• Type 1 causes HUS (also EHEC).
B. Sh. flexneri – Mannitol fermenting, 6+2 variants – MC IN India.
Type 6 88
New castle biotypes
Manchester
C. Sh. boydii → Mannitol fermenting, 18 Serotypes
D. Sh. sonnei → Mannitol fermenting.
LLF.
2 phases and 26 colicin types, MC in the west.
Pathogenicity:
• Endotoxin – LPS – Diarrhoea, ulcers.
• Exotoxin – Sh.dysenteriae type–I (Stx 1 and 2, cytotoxin; also neurotoxin and
enterotoxin)
Cytotoxin – acts on Vero cell lines (VT).
• Invasive – VMA (Virulence Marker Antigen).
Systematic Bacteriology | 181
Dysentery – Incubation Period – 1-7days.
Complications: Arthritis
Toxic Neuritis
Conjunctivitis, Parotitis
Intususception
HUS → associated with complement activation and DIC.
Salmonella:

Fig. 5.17: S. typhi – DCA

• Motile (by means of peritrichous flagella).

• Non motile strain → S .gallinarum and S pullorum.
• Enrichment media – Tetrathionate broth, Selenite F broth , GN broth.
• Selective media → DCA, XLD, SS, Wilson blair.
Biochemical reaction:
1. H2S +ve in TSI except strains of S. paratyphi A and S. cholerasuis.
2. Citrate + except S. typhi.
3. Salmonella do not ferment lactose (NLF).
4. Urease negative.
Antigenic structure more than 2500 serotypes (Kaufmann White Scheme):
• O (Somatic).
• H (Flagellar).
• Fimbrial (F).
H antigen:
• Heat labile, alcohol labile. Formaldehyde stable.
• Strongly immunogenic, appears late.
• Reacts to H Ab- forms large loosed fluffy clumps.
182 | Microbiology
O Ag → Heat stable formaldehyde labile polysaccharide:
• Less immunogenic, appears early, goes early.
• Reacts with O Ab- forms granular chalky clumps.
Vi Ag – Surface polysaccharide covering O Ag.
• Heat labile when present it renders the bacterium nonagglutinable by O antiserum.
• Possessed by S. typhi, S.dublin and S. paratyphi C , Citrobacter.
• Poorly immunogenic, but protective.
• Absence of Vi Ab- poorprognosis.
• Persistance in coalescent stage – carier state.
• Epidemiological typing of S.typhi- by Vi specificbacteriophage.
Lab diagnosis:
Enteric fever: step ladder-fever, rose spots, coated tongue:
1. Culture – blood, faeces, urine, bone marrow, rose spots etc.
2. Demonstration of circulating Ag.
3. Demonstration of Antibodies in serum.
Blood culture:
90% +ve   :   1st week
75% +ve   :   2nd week
60% +ve   :   3rd week
25% +ve after till the fever subsides.
Stool: During first week, organisms isolated in half the cases:
3rd to 5th week – most easily isolated.
Urine: Positive generally in the second and 3rd week in 25% cases.
Demonstration of circulating Ag:
• Coagglutination.
• ELISA.
• CIEP.
Detection of Antibodies (Widal):
Interpretation of Widal:
• Antibodies appear by 7th to 10th day of illness.
↓ Till the 3rd or 4th week after which declines.
• Demonstration of 4 fold rise is highly significant between 1st and 3rd week.
• Titre of 1: 100 of O agglutinins → significant.
1: 200 of H agglutinins → significant.
• H appears late and disappears late. Agglutinins persist after vaccinations.
• O agglutinins disappear within 6 weeks -Rise in O indicate recent infection.
Patients treated with chloramphenicol show poor agglutinin response.
Carriers: Widal is of no value in detection of carrier in endemic countries like India.
• Vi agglutinins (1:10) or more indicate carrier state.
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Salmonella septicemia – typically caused by S. cholerasuis.
Salmonella gastroenteritis:
• Zoonotic food poisoning.
• Food like meat, egg , milk.
• S. typhimurium: commonest species (30-40%).
Others are S. enteritidis, S. dublin, S. newport, S. heidelberg.
Vaccine:
1. Parenteral – (a) Killed WC 2 doses 6 wks apart (b) TABvaccine booster at 3 yrs. (c)
Purified Vi ag for vaccination.
2. Oral –Ty 21a (Gal E mutant):
• Live attenuated vaccine, available as enteric coated capsule or liquid formulation.
• It consists of a mutant virus which initiates infection but self destructs after 4-5 cell
divisions.
• Three doses 2 days apart are taken on an empty stomach.
• Elicits protection from 10-14 days of the third dose.
• 65-96% protect for 3-5 yrs.

Yersinia:
(Yersin and Kitast).
Yersinia pestis:
• Gram negative coccobacillus.
• Safety pin appearance (Bipolar staining) with Waysons’s stain.
• Pleomorphism.
• Aerobic or facultative anaerobe optimum temp. 27°C.
• Grows on ordinary media – Nutrient agar.
• In broth – stalactite growth.
Antigenic structure:
• Protein envelope fraction I or FI.
• Formed in vivo and cultures grown at 37°C– develops protecting immunity; inhibits
phagocytosis.
• V and W proteins:
ƒ Formed at 37°C.
ƒ Determined by plasmid.
• Pesticin , fibrinolysin, coagulase.
Determinants of pathogenicity:
• W and V Ag.
• F1 envelop Ag.
• Ability to synthesize purine.
• Production of pigmented colonies on medium containing haemin.
184 | Microbiology
Endotoxin is similar to GNB:
Urban plague – Man and rodents. In addition, cat, goat sheep and camel are also sus-
ceptible plague –bubonic septicemic, pneumonic.
Plague: bubone.
Wild or Sylvatic plague - Wild rodents squirrels.
Infection in man → Xenopsylla cheopsis (Rat flea bites sucks) (about 0.5 ml contain
5000 to 50,000 bacilli).
India – outbreak August to Oct.1994
876 cases – NICD.
Rat flea – Wingless hops < 2 ft.
Epicenters – Beed district (Maharashtra).
Sweat city (Gujarat)
Y. pseudotuberculosis:
• Zoonosis.
• Is motile at 22°C and not at 37°C:
ƒ In animals, mode of infection is ingestion.
ƒ Man acquires infection by skin contact with contamination.
ƒ Water or consumption of contaminated vegetables, water.
C/F - Mesentric lymphadenitis, resembling appendicitis, Erythema nodosum.
Y.enterocolitica:
Causes terminal ileitis, lymphadenitis, acute enterocolitis and septicemia in Fe overload
patients.
Acinetobacter baumanni:
• Gram negative, oxidase negative, non motile.
• MC cause of VAP.
• Non fermenter.
Stenotrophomonas maltophila
• Oxidase negative, non fermenter, motile.
• Opportunistic pathogen.

Oxidase Positive Organisms:

Pseudomonas aeruginosa.
Pseudomonas aeruginosa is a ubiquitous water and soil organism that grows to very
high titers overnight in standing water (distilled or tap).
Sources for infections include:
• Raw vegetables, respiratory, humidifiers, sink drains, faucet aerators, cut and potted
flowers, and if not properly maintained, whirlpools.
• Transient colonization of colon of about 10% of people. Bacteria get on skin from
fecal organism. Requires exquisitely careful housekeeping and restricted diets in burn
units.
Systematic Bacteriology | 185

Fig.5.18: P. aeruginosa – Cetrimide agar

Distinguishing Characteristics:
• Oxidase positive, Gram negative rods, non–fermenting.
• Pigments: Pyocyanin (blue–green) and fluorescein.
• Grape–like odor.
• Slime layer (alginate).
• Non–lactose fermenting colonies on EMB or MacConkey.
Reservoir:
• Ubiquitous in water.
Transmission:
• Water aerosols, raw vegetables, flowers.
Pathogenesis:
• Endotoxin causes inflammation in tissue and Gram–negative shock in septicemia.
• Pseudomonas exotoxin A ADP ribosylates EF–2, inhibiting protein synthesis (like
diphtheria toxin).
• Capsule/slime layer: Allows formation of pulmonary microcolonies; difficult to remove
by phagocytosis.
Compromising Condition/Opportunistic Infections.
Normal people:
• Transient GI tract colonization: loose stools (10% pop.).
• Hot tub folliculitis.
• Eye ulcers; trauma, coma, or prolonged contract water.
Burn patients:
• GI tract colonization – skin – colonization of eschar – cellulitis (blue – green pus) –
septicemia.
186 | Microbiology
Neutropenic Patients:
• Pneumonia and septicemia – often superinfections (infections while on antibiotics).
Chronic Granulomatous Disease (CGD):
• Pneumonias, septicemias (Pseudomonasis catalase positive).
Septicemia:
• Fever, shock ± skin lesions, (black necrotic center, erythematous margin (ecthyma
gangrenosum).
Catheterized patients:
• Urinary tract infections (UTI) .
Cystic fibrosis:
• Early pulmonary colonization, recurrent pneumonias. Always high slime producing
strains (alginate).
Drug Resistance in P. aeruginosa:
Knowing susceptibilities important.
Drug resistance very common.
• Inherent resistance (missing high affinity porin some drugs enter through); plasmid
mediated b - lactamases and acetylating enzymes.
Burkholderia pseudomallei:
• Causative agent of meliodosis (Glanders like disease).
• Gram negative rod, bipolar stained, motile, oxidase positive.
• Found in soil in South East Asia, North Australia.
• Occurs in rats, guinea pigs, rabbits.
• Human infection: Inhalation, inoculation, ingestion of foodstuffs contaminated with
excreta of infected animals.
Humans:
• Subclinical infection.
• Pulmonary infections : similar to TB.
• Multiple abscesses in various organs and tissues.
• Fulminating septicemia.
• Also known as “ Vietnam Time-Bomb”.
India:
Reported form Maharashtra, Tamil Nadu, Orrisa, west Bengal, Tripura.
Laboratory diagnosis:
1. Culture.
2. Serology: ELISA, IHA.
Burkholderia mallei:
Gram negative rod, non-motile, aerobe and facultative anaerobe, oxidase negative
(some positive), grows on ordinary media.
Systematic Bacteriology | 187
Causes infection in horse, mules and assess.
Glanders: Respiratory system affected, catarrhal discharge, nodules in nasal septum
which ulcerate.
Farcy: Infection through skin with involvement of lymph vessels and lymph nodes.
“Farcy pipes”.
Strauss reaction: Intraperitoneal injection in male guinea pig causes testicular swelling
in 2-3days due to bacillary invasion of tunica vaginalis.
Humans:
• Skin abarasions or wounds which come in contact with sick animal.
• Acute fulminant febrile illness.
• Chronic indolent infection.
Vibrionaceae:
• Important pathogens of man –Vibrio cholerae V.parahaemolyticus and V.vulnificus.
Non-Halophilic Vibrios:
i. V. cholerae.
ii. Non 01 V. cholerae.
iii. V.mimicus.
Halophilic Vibrios:
i. V. parahaemolyticus.
ii. V. alginolyticus.
iii. V. vulnificus.

Fig.5.19: Antigenic Classification of Vibrios

Vibrio cholerae:
• Gram negative curved rods.
• Actively motile by single polar flagellum (darting motility).
188 | Microbiology
• Catalase +, oxidase positive.
• Late lactose fermenters.
• Transport media- Venkatraman Ramakrishnan (VR) medium.
ƒ Cary Blair medium.
ƒ Taurocholate p/w.
• Enrichment – APW (alkaline peptone water).
ƒ Taurocholate p/w.
• Selective – Bile Salt Agar (BSA).
ƒ Monsurs Gelatin Taurocholate Trypticase Tellurite Agar (GTTA).
ƒ Thiosulphate citrate bile sucrose (TCBS).

Fig.5.20: String test – V. cholerae Fig.5.21: TCBS medium – V. cholerae

• Biotypes of VC O1→
Classical El tor
• VP – +
• Fowl RBC Agglutination – +
• Hemolysis of sheep RBC – +
• Sensitivity to polymyxin B and IV phage + –
• CAMP test – +
WHO classification of VC → Typical VC
Atypical VC
Non 01 VC (NAG)
Epidemic cholera – VCO1 and 0139 bengal
VCO1 Serotypes – Ogawa, Inaba, Hikojima
VCO1 Phage types → classical → five on the basis of 4 phages
El tor : Six on the basis of 5 phages
→
Systematic Bacteriology | 189
Cholera occurs only in MAN (RICE WATER STOOLS)
Cholera Toxin (CT)- resembles heat labile toxin of E.coli (LT)
↓ ↓
Chromosomal Plasmid (DNA
(DNA coding) coding)
Stimulates cAMP- ↑ secretion of Cl– , bicarbonate, water
CT:
• A1, A2.
• B- binds to ganglioside receptors.
VC 0139 → In 1992 in Madras/ Chennai (Bengal strains).
Halophilicvibrio:
• V. parahaemolyticus, V. alginolyticus, V. vulnificus.
• Causes food poisoning (sea fish).
V. parahaemolyticus – Shows Kanagawa phenomenon on Wagatsuma agar.
VC vaccines:
• Killed whole organism.
• Oral Vaccine – Killed and Live.
Aeromonas → infection in cold blooded aquatic animals like frog – red leg disease.
In humans: diarrhoea.

Causes of Gastroenteritis / food poisoning:

Organism Common Food IP

(1) Salmonella typhimurium Poultry eggs 18-36 hrs

(2) B. cereus toxin Reheated rice 1-6 hrs

(Emetic toxin – Heat stable (1-6 hrs)

Enterotoxin (10-12 hrs)

(Heat labile like cholera toxin)

(3) Clostridium perfringens toxin Reheated meat foods 8-16 hrs

(4) C. botulinum toxin (botulism) Canned food > 48 hrs

(5) S. aureus toxins Meat and dairy products 1 – 6 hrs

(6) Campylobacter jejuni Poultry, Unpasteurised milk 2-11 days

(7) EHEC (VTEC) 0157 H7 026 Meat (hamburger) unpasteurised milk 1-5 days

(8) Yersinia enterocolitica Raw or under cooked pork 4-7 days

(9) Vibrio parahaemolyticus Shellfish 8hrs-2 days

(10) Aeromonas resh water fish 1-4 days

190 | Microbiology
Dysentery:
• Protozoa – E.histolytica, Balantiduim coli, Cyptosporidium.
• Bacteria:
ƒ Shigella (dysentriae, flexneri, boydii, sonnei).
ƒ E.coli (EIEC, EHEC).
ƒ Y.enterocolitica.
ƒ Campylobacter jejuni.
Campylobacter jejuni:
• Spiral, GN; single polar flagellum, microaerophilic
• Thermophilic 42°C.
• 4-35% of fecal specimens of patients with acute diarrheal disease, faeco-oral (raw
milk, partially cooked poultry, contaminated water); present in GIT of wild and
domestic animals.
• Mechanism of diarrhoea:
ƒ Heat labile enterotoxins resembling CT.
ƒ Invasion like Shigella.
ƒ Cytotoxin.
Media:
• Transport: Cary Blair medium.
• Selective: Skirrow’s, Campy BAP, Butzler.
Helicobacter pylori:
• GN spiral rod, motile unipolar tuft of lophotrichous flagella.
• Microaerophilic, abundant urease production.
• Mild acute gastritis, peptic ulcer disease, chronic gastritis.
• Risk factor for gastric malignancy.
Invasive:
• Warthin starry silver staining.
• Culture: Skirrow’s medium, Trypticase soy and BHI with 5% SB (Gold standard).
• Biopsy urease test.
Noninvasive:
• Urea breath test.
• Serology : ELISA.
• Virulence: Urease production, CAG (cytotoxin associated gene), vac (vacoulating cy-
totoxin gene).
Pasteurella multocida:
• GN, NM, oxidase+, Indole+, Mac: NG
• Sometimes commensal in human upper respiratory tract.
• Human infections: Animal bites or trauma.
• Local suppuration following bite (wound infection, cellulitis, abscess, osteomyelitis),
meningitis following head injury, respiratory tract infection, appendicitis.
Systematic Bacteriology | 191
Fastidious organisms:
Francisella tularensis:
• Tularemia: A disease of rodents, originally described in Tulare country, California.
Human infections:
• Tick bite.
• Direct contact with rodents.
• Inhalation of aerosols.
• Ingestion of infected meat or water.
ƒ Capsulated, NM, GNR.
ƒ Found intracellularly in liver, spleen cells.
ƒ Fastidious.
ƒ Francis blood dextrose cystine agar.
ƒ Highly virulent strains: In North America, low virulence strains: Europe, Asia.
Human disease:
• Local ulceration with lymphadenitis (most common form).
• Occuloglandular.
• Typhoid like fever with glandular enlargement.
• Influenza like respiratory disease.

Haemophilus:
H. influenzae:
• Blood loving.
• Requirement for one or more of the accessory factors: X and V in blood.
• “Pfeiffer’s bacillus”.
ƒ Plemorphism.
ƒ Acute infection: Capsulated strains.
ƒ Stain: Methylene blue or dilute carbol fuchsin.
ƒ X: Hemin or porphyrin required for aerobic respiratory: heat stable.
ƒ V: Heat labile, inside RBCs, NAD or NADP.
ƒ Satellitism.
ƒ Levinthal’s agar (iridescent colonies of capsulated strains), Filde’s agar (best for
primary isolation).
ƒ 8 biotypes.
ƒ Oxidase +, Catalase +.
ƒ Refrigeration kills.
ƒ Antigenic properties:
▫ Capsules: a-f; 95% infections by type b; (PRP/ribose or ribitol).
▫ OMP.
▫ LOS.
192 | Microbiology

Fig.5.22: Satellitism – H. influenzae

Pathogenicity:
• Humans only.
• Invasive: Meningitis, Arthritis, Epiglottitis, Pneumonia, Bacteremia, Endocarditis,
Pericarditis: Capsulated; children.
• Non-invasive: Secondary: otitis media, sinusitis, COPD adults; non-capsulated
• Meningitis: Nasopharynx-blood stream; 2 months to 3 years.
• Epiglottitis: Second most common cause; blood culture.
• Pneumonia: Infants; older children and adults – lobar.
• Hib PRP vaccine: Not effective in <2 years of age.
• Ceftriaxone: Prophylaxis.
H. aegyptius (Koch’s – Week’s bacillus):
• “Pink eye”.
• Brazilian purpuric fever.
H. ducreyi:
• Chancroid: Painful, LNs.
• Safety pin appearence.
• “School of fish” or “rail road track” appearance.
• Medium used: Chocolate agar with 1% isovitalex, Vancomycin 93mg/ml.
• DOC : Azithromycin.
HACEK group of organisms:
• Fastidious slow growing bacteria, normal or oral cavity.
• Hemophilus species.
• Actinobacillus actinomycetemcomitans.
• Cardiobacterium hominis.
• Eikenella corrodens.
• Kingella kingae.
Systematic Bacteriology | 193
Bordetella:
• Gram negative coccobacillus. Thumbprint appearance.
• Culture media → Bordet Gengou (glycerol, potato extract) (colonies: Bisected pearls
or Mercury drop).
• Transport media → Regan Lowe.
Virulence factors→ Heat labile toxin, tracheal cytotoxin.
Lipopolysaccharide endotoxin, pertusis toxin, adenylate cyclase, haemolysin, filamentous
haemaglutinin.

Fig.5.23: Bordetella pertussis – Regan Lowe medium

Pathogenesis → Source of infection (whooping cough):

• Patient in early stage.
• No healthy carrier.
• Attack rate of more than 90% in non immunized individuals.
Specimen – Per nasal swab for nasopharynx.
DOC → Erythromycin for whooping cough.
Brucella:
• Zoonosis.
• Gram negative coccobacillus → Goat, Sheep, Cattle.
• Six Spp:
ƒ B. melitensis – Sheep, Goat (MC).
ƒ B. abortus – Cattle (Capnophilic).
ƒ B. suis – Pig.
ƒ B. canis – Dog.
• Undulant fever characteristic.
• Intermittent or irregular fever with variable duration.
• Nonspecific and variable symptoms.
• Headache, weakness, arthralgia, depression, weight loss, fatigue, liver dysfunction.
• Any organ or system can be affected.
194 | Microbiology
• CNS or heart disease more difficult:
ƒ Strict aerobe.
ƒ Media – Can grow on NA (slow), SDA, TSA.
ƒ Blood culture: Casteneda method – Biphasic medium.
ƒ Brucellosis – Malta/Undulant fever.
ƒ Disease of RES.

Fig.5.24: Biphasic medium

Serology:
– Castaneda method
• Standard Agglutination Test (SAT) for IgM (acute infection).
• CFT and ELISA for chronic infection.
• Blocking Abs by Coomb's test.
Brucellin Skin Test
Animals ∆ → Rose Bengal Card test
Milk Ring Test
↓
Br. abortus stained with Haematoxylin.
+
Milk (Suspected to contain Ab)
↓70°C X 40 - 50 min
Positive → Bacteria Agglutinated with milk
↓
Rise with cream
↓
Blue Ring
Legionella:
• Weakly Gram – negative.
• Pleomorphic rods requiring cysteine and iron.
• Water organisms.
Legionella pneumophila:
Distinguishing Characteristics:
• Stain poorly with standard Gram stain; gram – negative.
• Fastidious requiring increased iron and cysteine for laboratory culture (CYE, charcoal
Yeast Extract).
Systematic Bacteriology | 195
• Facultative intracellular pathogen.
• Diagnosis: Direct Fluorescent Antibody (DFA)on biopsy, (+) Dieterle silver stain.
• Antigen urine test for serogroup 1 only.
• Fourfold increase in antibody.
Reservoir:
• Rivers/streams/amoebae;air-conditioning water cooling tanks.
Transmission:
• Aspiration is the predominant mode.
• Aerosols from contaminated air – conditioners.
• No human to human transmission.
Predisposing Factors:
• Smokers over 55 years with high alcohol intake.
• Immunosuppressed patients e.g. renal transplant patients.
Pathogenesis:
• Facultative intracellular pathogen.
• Endotoxin.
Disease:
• Associated with air – conditioning systems, now routinely decontaminated.
Legionnaires’ Disease (“Atypical Pneumonia”):
• Pneumonia.
• Mental confusion.
• Diarrhea (No Legionella in gastrointestinal tract).
Pontiac Fever:
• Pneumonitis.
• No fatalities.
Treatment:
• Fluoroquinolone or azithromycin or erythromycin with rifampin for immunocompro-
mised patients.
• Drug must penetrate human cells.
Prevention
• Routine decontamination of air – conditioner cooling tanks.
196 | Microbiology

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Systematic Bacteriology | 197
Active Recall from Tables
Pathogen Selective media

Salmonella

Bordetella

Brucella

Legionella

Pseudomonas aeruginosa

Toxin MoA
Exotoxin A - Pseudomonas

LT - ETEC

ST - ETEC

VTEC
198 | Microbiology

Colony morphology Pathogen

Bisected pearl appearence

Cut glass appearence

Oil drop colonies

Fish eye colonies

Swarming
Systematic Bacteriology | 199
Concept 5.6: Spirochetes
Learning Objectives
• Classification of Spirochetes
• Virulence factors & Pathogenesis
• Clinical features
• Diagnosis & Treatment

Time Needed
1st reading 45 mins
2 look
nd
15 mins

Spirochetes:
Pathogenic spirochetes Include:
• Treponema.
• Leptospira.
• Borrelia.

Fig.5.25: Spirochete – DGM Fig.5.26: Fontana stain – Spirochetes

Borrelia:
Relapsing fever →
1. Epidemic or louse borne relapsing fever caused by B recurrentis.
2. Endemic or tick borne relapsing fever – caused by B. duttoni, B. hermsii.
Lymes disease – B. burgdorferi – Tick borne.

Relapsing Fever:
Lab diagnosis:
1. Microscopic examination → Blood – wet film under dark ground or phase contrast
microscope.
Thick and thin smears, Giemsa and Leishman stain.
2. Animal inoculation mice – intra peritoneal infection.
3. OXK may be positive.
B. burgdorferi → First identified in 1975 in Lyme Connecticut USA (Lymes Disease).
200 | Microbiology
Lab Diagnosis:
1. Diagnosis mainly on clinical ground. The presence of vector bite, Erythema
Chronicum Migrans(ECM) at site of vector bite, followed by expanding bright red
rash suggestive of positive diagnosis.
2. Isolation and identification of B. burgdorferi from skin lesions or blood.
ƒ Culture too slow, low yield.
3. Microscopic detection – Dark ground, phase contrasts, Immunofluorescence, silver
staining.
4. Antigen in urine.
5. DNA probes.
Borrelia/Treponema vincentii:
Is a normal commensal of mouth but may under predisposing conditions such as
malnutrition, viral infection may give rise to ulcerative gingivostomatitis (Vincent's
angina).
Vincent's angina – T vincentii is often associated with fusiform bacillus known as
Leptotrichia buccalis, which is also known as Fusobacterium fusiformis.
T. pallidum sub spp. pallidum – Syphilis:
Nonveneral
1. T. pertenue : Yaws
2. T. endemicum : Endemic Syphilis (Bejel)
3. T. carateum : Pinta
Can’t differentiate all these morphologically
T. pallidum → Motile (fine fibrils)
↓
Lashing Motility Flexion / Extension
Corck screw
Translatory
Wet Mount : Dark Ground Microscope
Dry : Silver Impreg.
Levaditi (tissue)
Fontanas (smear)
  ImmunoFluo (DFA)
Can not be cultivated on artificial media
Lab Diagnosis :
• In Exudates : Darkground microscope, DFA
• Serology :
ƒ Non Treponemal Test :
▫ Wasserman (CFT).
▫ Kahn Test – Tube Flocculation.
▫ VDRL – Slide Flocculation.
▫ RPR.
Ag → Cardiolipin (Diphosphatidyl Glycerol) + Lecithin + Cholesterol.
Systematic Bacteriology | 201
• Specific Treponemal test: TPI (T. pallidum Immobilisation): Live treponemes used (using
Nicholle’s).
(Killed) → TPA (agg). TPIA (Immune adherence.
(Extract) TPHA – Most specific, ELISA.
FTA, FTA-ABS (most sensitive).
• Group specific Using Reiter strain RP CFT.

Biological false positive reactions:

Acute Chronic
Last few weeks or months Last longer than 6 months
Acute infections, injuries, inflammation SLE, Collagen vascular diseases, leprosy ,malaria relapsing
and early HIV infection fever, hepatitis, Infectious mononucleosis, tropical eosinophilia

Leptospira:
Leptospira – obligate aerobes
L. interrogans –Parasitic strains.
L. biflexa – Free living saprophytic strains.
• Leptospira may be demonstrated in blood and in urine by Dark ground microscopy,
phase contrast, silver impregnation.
• Blood examination useful in early phase as disappear from blood after 8 days.
• Leptospires may be present in the urine in the second week and intermittently
thereafter for 4 – 6 weeks.
Culture media - Korthof’s medium , EMJH, Fletchers.
Serological Diagnosis:
ƒ Genus specific – CFT, ELISA.
ƒ Sero group andserovar specific → macroscopic and microscopic agglutination
tests.
202 | Microbiology

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Systematic Bacteriology | 203
Active Recall from Tables
Disease Agent

Yaws

Pinta

Lyme disease

Swamp fever

Great pox
204 | Microbiology
Concept 5.7: Mycoplasma, Chlamydia & Rickettsia
Learning Objectives
• Mycoplasma, Chlamydia, Rickettsial group of bacteria
• Virulence factors & Pathogenesis
• Clinical features
• Diagnosis & Treatment

Time Needed
1st reading 45 mins
2 look
nd
15 mins

Mycoplasma and Ureaplasma:

Very small measuring 0.2 -0.3 mm in diameter, can pass through bacterial filters. Flagella,
pili –absent but some strains show gliding mobility due to special tip like structure.
Lack rigid cell wall bounded by single trilaminar cell membrane. Mycoplasma and
Ureaplasma cannot synthesize their own cholesterol and require it as a growth factor in
culture medium.

Fig.5.27: Mycoplasma – Diene’s method of staining

• Pleomorphic.
• Stain with Dienes or Giemsa stain.
• Stain poorly with Gram stain and are gram negative.
• Fried egg colonies.
• Aerobes and facultative anaerobes.
Culture medium – PPLO broth (bovine heart infusion broth + 20% horse serum + 10%
fresh yeast extract).
200-500 mm – large colony: Mycoplasma
15-60mm- small colony: Ureaplasma.
Systematic Bacteriology | 205
Mycoplasma pneumoniae → commonly causes tracheobronchitis, pharyngitis, sinusitis,
primary atypical pneumonia (PAP) accompanied by formation of cold haemagglutinins,
Streptococcus. MG agglutinin (Heterophile agglutination test) and biological false
+ve Wasserman reaction
M pneumoniae may also cause extrapulmonary lesions:
• Arthritis, meningoencephalitis, transverse myelitis.
• Gullian Barre Syndrome – Haemolytic anemia.
• Myocarditis, Pericarditis.
Ureaplasmaurealyticum→ Cause NGU, Non chlamydial urethritis, epididymitis, Vaginitis
and cervicitis.
C. trachomatis responsible for 30-50% NGU:
• Cause chorioamnionitis, prematurity, post partum endometritis.
• Chronic lung disease of premature infant.
• Ureaplasma isolated from CNS or lower respiratory tract of sick premature new born
infants.
• M. hominis →Salpingitis, PID , septic abortion, peritonitis, brain abscess.
• → Cause PAP similar to M. pneumoniae and meningitis in new born.
Lab Diagnosis:
M pneumoniae – Throat swab, Nasopharnygeal swab, Throat washings, Sputum, BAL.
Genital Cervical swab.
Urethral swab.
Prostatic secretions, Tracheal aspirates; Urine.
• Culture – PPLO broth.
• Serological tests – Detection of antigen.
ƒ Immunofluorescence.
ƒ PCR.
Detection of Antibody:
• Cold agglutinins – M pneumoniae.
• Complement fixation test (CFT).
• ELISA for IgM, IgG and IgA.
DOC – Tetracycline, Erythromycin.
Rickettsiaceae:
• Family rickettsiaceae has four genera.
Genus Species
Rickettsia – R. prowazekii, R. rickettsi, R. akari, R. typhi
Orientia – O. tsustsugamushi
Coxiella – C. burnetti
Ehrlichia. – E. chaffensis
E. sennetsu
E. phagocytophila.
206 | Microbiology
No arthropod vector
No skin rash in Coxiella

Fig.5.28:

General Characters:
→ Small gram negative bacilli. The stains commonly used for staining giminez,
Machiavelloand Giemsa.
ƒ Possess both RNA andDNA. Requires an arthropod vector for transmission.
ƒ Large enough to be held back by bacterial filters.
Scrub typhus rickettsiae have been placed in a separate genus Orientia tsutsugamushi.
Epidemic Typhus fever → R. prowazekii vector – body louse, recrudescence is seen and
called as + Brill Zinsser disease.
Endemic Typhus fever → R. typhi (mosseri) – Ratflea, No recrudescence.
Neil mooser. Reaction is positive.
(RMS) Rocky mountain spotted fever – R.rickettsii – vector – tick – rash is peripheral.
Ricketsial pox – R. akari → Vector – mite
Scrub typhus – R. tsutsugamushi and vector is mite.
Weil Felix OX2 OX19 OXK
Epidemic typhus + ++++ –
Endemic Typhus + ++++ –
RMS Fever + to ++++ ++++ –
Scrub typhus – – +++
Systematic Bacteriology | 207
Lab diagnosis:
• Isolation – in lab animals, hens egg and cell cultures.
• Direct detection of the organisms and their antigens in clinical specimens.
• Serology.
R typhi differentiated from R prowazekii by Neil Mooser or tunica reaction. Reaction – ve
in R prowazekii.
Coxiella burnetii – It survives holders method of pasteurization of milk
→ Only rickettsial infection which can be transmitted without arthropod ie man to man
be respiratory mode
→ Ticks do not play any significant role in transmitting the infection to man.
→ No skin rash in Q fever.
Ehrlichia→ Small negative bacilli that multiply with in membrane bound vacuoles in
phagocytes
Chlamydia:
Developmental cycle:

Fig. 5.29:
208 | Microbiology
4 species :
1 C. trachomatis.
2 C. psittaci – (Levinthal Cole-Lillie bodies) (LCL).
3. C. pneumoniae, (atypical pneumonia) TWAR agent.
4. C. picorum.
C.trachomatis 3 biovars – TRIC – A-K serovars Ophthalmianeonatorum.
Genital infection
A – C – Trachoma
D –K – Inclusion conjunctivitis
LGV – L1, L2, L3 serovars
• Mouse pneumonia.
• Obligate intracellular gram negative bacteria. Lack peptidoglycan.
• Posses RNA and DNA. Can’t produce their own ATP.
• Stain by Casteneda , Macchiavello, Giminez, Culture → Yolk sac, cell culture (McCoy
Cells).
• Dev cycle – elementary body (E/C, infectious form).
ƒ Reticulate body (I/C, replicating).
• Halbestaeder prowazek (HP)body in conjunctival epithelial cells.
Treatment of chlamydial infections → Tetracycline, EM, Sulfonamides.
For LGV à skin test →Frie test
• No genital lesion (only systemic manifestations) AIDS, HBV, HCV.
→ Non gonococcal urethritis (NGU).
20-30% -Chlamydia trachomatis.
10% - Mycoplasma genitalium and hominis
ƒ Bacteroides urealyticus.
ƒ Herpes virus hominis.
ƒ Cytomegalovirus.
ƒ Trichomonas vaginalis.

Comparison of the Genera Rickettsia, Chlamydia,

and Mycoplasma with Typical bacteria:
Typical bacteria Chlamydia Rickettsia Mycoplasma
(S. aureus)
Obligate Most no Yes Yes No
intracellular
parasite?
Make ATP Normal ATP No ATP Limited ATP Normal ATP
Peptidoglycan Normal No (Protein Normal No Peptidoglycan
layer in cell Peptidoglycan Cell Wall) Peptidoglycan
envelop Peptidoglycan
Systematic Bacteriology | 209
Ehrlichiae laden
• Vacuole is known as morula:
ƒ E sennetsu – illness resembling Glandular fever; pt develops lymphoid hyperplasia
and atypical lymphocytosis seen in Japan and Malaysia. Suspected to be ingested
with raw fish.
ƒ E. chaffeensis – Infects human monocytic cells and produce febrile illness.
Transmitted to human by ticks.
Bartonella:
• Gram negative bacilli.
• Involve RBC.
• 3 species →
1. B.bacilliformis – OROYA fever.
2. B. quintana – Trench fever, louse borne.
3. B. hensalae – Cat scratch disease, Bacillary angiomatosis.
210 | Microbiology

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Systematic Bacteriology | 211
Active Recall from Tables
Disease Vector

R. pox

Epidemic typhus

Endemic typhus

RMSF

Scrub typhus
212 | Microbiology
Concept 5.8: Miscellaneous bacteria
Learning Objectives
• Anaerobes and other miscellaneous bacteria
• Virulence factors & Pathogenesis
• Clinical features
• Diagnosis & Treatment

Time Needed
1st reading 30 mins
2 look
nd
10 mins

Genus: Bacteroides:
• Gram –negative rod.
• Anaerobic.

Bacteroides fragilis:
Distinguishing Characteristics:
• Anaerobic, gram –negative rods.
• Anaerobes are identified by biochemical test and chromatography.
Reservoir:
• Human colon; the genus Bacteroides in the predominant anaerobe.
Pathogenesis:
• Modified LPS (Missing heptose and 2-keto-3 deoxyocotnate) has reduced endotoxin
activity.
• Capsule is antiophagocytic.
Disease:
Septicemia, peritonitis (often mixed infections), and abdominal abscess.
Treatment:
• Metronidazole, Clindamycin or cefoxitin. Abscesses should be surgically drained.
• Antibiotic resistance is common (penicillin G, some cephalosporins, and
aminoglycosides).
Prevention:
• Prophylactic antibiotics for gastrointestinal or biliary tract surgery.
Bacteroides melaninogenicus = Prevotella melaninogenica
• Melanin-producing (black) Bacteriodes).
• Normal gingival flora.
• Oral abscesses.
• Heparinase leads to clotting in brain.
• Bright red fluorescence under UV light.
Systematic Bacteriology | 213
Calymmatobacterium granulomatis:
• Present name: Klebsiella granulomatis.
• Produce granuloma inguinale, Granuloma venereum or Donovanosis.
• Bipolar staining, Safety pin appearance.
• Best stained with Wright's, Giemsa.
• Donovan bodies → Body of the bacillus blue and capsule pink contained within the
cytoplasmic vacuoles of large macrophages.
• C/F : Painless ulcer without lymphadenopathy.
• Culture – Embryonated hens egg.
• Treatment: Tetracycline, EM, chloramphenicol.
Streptobacillus moniliformis:
• Gram negative, highly pleomorphic bacillus.
• String of bead appearance, non motile, fastidious.
• Exists as L form also.
• Pathogenicity → Normal inhabitant of the nasopharynx of rats, causes streptobacillary
rat bite fever also caused by Spirillum minus.
• When the organisms are acquired by ingestion of food, milk or water contaminated
by rat excrement it is known as Haverhill fever.
• Lab diagnosis → Grow on culture media containing blood serum or ascitic fluid.
• L forms – Fried egg appearance.
• Mice are susceptible to intraperitoneal inoculation of infected material.
• Treatment – penicillin.
Eikeniella corrodens:
• Lacks flagella and show jerking or twitching motility, requires haemin for aerobic
growth.
• Characteristic pitting or corroding of blood agar.
• Pathogenicity – Normal inhabitant of human mouth, intestine and upper respiratory
tract but occasionally causes opportunistic infections such as dental and periodontal
infection, sinusitis, otitis media, mastoiditis, pneumonia, lung abscess, wound
infection following human bite, septic arthritis or osteomyelitis.
• Bacteremia and endocarditis in immunocompromised hosts, I/V drug users or persons
with previous valvular damage.

Erysipelothrix rhusiopathiae:
Clinical significance:
• Primarily a pathogen of swine, turkeys, and fresh water fish.
• In man, the disease is called erysipeloid, is the most common form.
• It is an occupation-associated disease in which a reddish-blue, edematous lesion at
the site of inoculation, primarily following trauma to the hands.
• Whale finger
214 | Microbiology

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Systematic Bacteriology | 215
Active Recall from Tables
Disease Agent

Whale finger

Rat bite fever

Haverhill fever
216 | Microbiology
Concept 5.9: Clinical Microbiology
Learning Objectives
• Tables for rapid revision

Time Needed
1 reading
st
120 mins
2nd look 30 mins

Organisms and Specific Culture Media:

Organism Medium
Anaerobes Thioglycolate
Anthrax bacilli PLET Media
Bacillus cerus MYPA ( manitol egg yolk polymixin agar)
Bordetella Bordet Gangou media
Borrelia burgdorferi Kellys media
Brucella Castanada method
Campylobacter jejuni Campy Bap, Skirrows, Butzlers
Chlamydiae HeLa cells, Mcoy cells
Clostridia Robertsons cooked meat media
Corynebacterium Loffler’s serum slop, potassium Tellurite agar (D)
Legionella BYCE media
Leptospira EMJH , kellys and korthoff , Fletchers
Neisseria from sites with normal flora Thayer-Martin selective medium(S), NYC media
Shigella/ Salmonella DCA/ XLD / WB
Vibrio cholerae TCBS (Thiosulfate citrate Bile salts sucrose agar), Bile Salt Agar
MGTTTA (Monsur’s Gelatin Taurocholate Tellurite Trypticase
Agar)

Bacterial Vaccines:
Childhood Vaccines:
DtaP:
• Totally acellular.
• Components of B. pertussis.
ƒ Pertussis toxoid.
ƒ ± Filamentous hemagglutinin.
ƒ ± pertactin (adhesion).
• D – T protein are still toxoids.
Systematic Bacteriology | 217
DTP:
• Diphtheria: diphtheria toxoid.
• (= inactivated toxin that no longer causes disease but produces immunity).
• Tetanus: tetanus toxoid).
• Pertusis: killed Bordetella pertussis cells.
HIB (Haemophilius influenzae type b)
• H. influenzae capsular polysaccharide conjugated to protein (diphtheria toxoid or
Neisseria meningitidis outer membrane proteins), making it a T cell – dependent that
infants respond to.

Senior Citizens or Asplenic:

• Capsular polysaccharides of 23 different Pneumococcus strains.
Limited Usage:
• Four capsular polysaccharides: Y, W – 135, C and A.
• B serotype: capsule is sialic acid so not good immunogen.
• Used in outbreaks along with antibiotic prophylaxis.
• Routine usage in military recruits.
Salmonella typhi (ty21):
• Attenuated bacterium for travelers to endemic typhoid areas.
Yersinia pestis:
• Killed cellular vaccine (F – 1 antigen).
• Military in endemic areas and Y. pestis laboratory workers.
Bacillus anthracis:
• Supernatant of partially purified proteins.
• Military or occupational usage.
BCG = Bacille Calmette Guerin (BCG):
• Attenuated (living) strain of Mycobacterium bovis.
• Doesn’t prevent pulmonary tuberculosis but reduces dissemination .

Definitions of Immunizing Agents:

Term Definition
Vaccine A suspension of attenuated live or killed microorganisms or antigenic portions of these
agents presented to a potential host to induce immunity and prevent disease.
Toxoid A modified bacterial toxin that has been made nontoxic but retains the capacity to
stimulate the formation of antitoxin
Immune globulin An antibody-containing solution derived from human blood by cold ethanol
fractionation of large pools of plasma and used primarily for maintenance of the
immunity of immunodeficient persons or for passive immunization; intramuscular and
intravenous preparations available.
Antitoxin An antibody derived from the serum of animals after stimulation with specific antigens
and used to provide passive immunity.
218 | Microbiology
Gastrointestinal Pathogens Causing Acute Diarrhea:
Mechanism Location Illness Stool Findings Examples of Pathogens
Involved
Noninflammatory Proximal Watery No fecal leukocytes Vibrio cholerae,
(enterotoxin) small bowel diarrhea enterotoxigenic Escherichia
coli (LT and / or ST).
Clostridium perfringens,
Bacillus cereus,
Staphylococcus aureus,
Aermonas hydrophila,
Plesiomonas shigelloides,
Rotavirus, Norwalk-like
virus, enteric Adenoviruses,
Giardia lamblia,
Cryptosporidium spp.
Cyclospora spp.
Inflammatory Colon or Dysentery or Fecal Shigella spp. Salmonella
(invasion or distal small inflammatory polymorphonuclear spp. Campylobacter jejuni,
cytotoxin) bowel diarrhea leukocytes enterohemorrhagic E. coli,
enteroinvasive E. coli,
Yersinia enterocolitica,
Vibrio parahaemolyticus,
Clostridium difficile, ? A.
hydrophila, ? P. shigelloides,
Entamoeba histolytica
Penetrating Distal small Enteric fever Fecal mononuclear Salmonella typhi, Y.
bowel leukocytes enterocolitica

Causes of Traveler's Diarrhea:

Etiologic Agent % of Cases Comments
BACTERIA 50–75
ETEC 10–45 Single most important agent.
EAEC 5–35 Emerging enteric pathogen
with worldwide distribution.
Campylobacter jejuni 5–25 More common in Asia.
Shigella 0–15 Major cause of dysentery.
Salmonella 0–15
Others Including Aeromonas, 0–5
Plesiomonas, and Vibrio cholerae
VIRUSES 0–20
Norovirus 0–10 MC in children; Associated with cruise ships.
Systematic Bacteriology | 219

Rotavirus 0–5 MC in infants.

PARASITES 0–10
Giardia intestinalis 0–5 Affects hikers and campers who drink from
freshwater streams.
Cryptosporidium 0–5 Resistant to chlorine treatment.
Entamoeba histolytica <1
Cyclospora <1

Post Diarrhea Complications and Microorganisms:

Lactase deficiency and malabsorption – protozoal infections.
Irritable bowel syndrome – etiological agents of traveler's diarrhea.
Reactive arthritis (Reiter’s syndrome) - Shigella, Salmonella, Campylobacter, Yersinia.
Hemolytic-uremic syndrome (hemolytic anemia, thrombocytopenia, and renal failure)
Shigella dysenteriae type 1 and EHEC).
Guillain-Barre syndrome - Campylobacter.

Bacterial Food Poisoning:

Incubation Period, Organisms Symptoms Common Food Sources
1 to 6 hours
Staphylococcus aureus Nausea, Vomiting, Diarrhea Ham, Poultry, Potato or egg salad,
mayonnaise, cream pastries
Bacillus cereus Nausea, Vomiting, Diarrhea Fried rice
8 to 16 H
Clostridium perfringens Abdominal cramps, diarrhea Beef, poultry, legumes, gravies
(vomiting rare)
B. cereus Abdominal cramps, diarrhea Meats, vegetables, dried beans,
(Vomiting rare) cereals
> 16 H
Vibrio cholerae Watery diarrhea Shellfish
Enterotoxigenic Escherichia coli Watery diarrhea Salads, cheese, meats, water
Enterohemorrhagic E. coli Bloody diarrhea Ground beef, roast beef, salami
raw milk, raw vegetables, apple
juice
Salmonella spp. Inflammatory diarrhea Beef, poultry,eggs, dairy products
Shigella spp. Dysentery Potato or egg salad, lettuce, raw
vegetables
Vibrio parahaemolyticus Dysentery Mollusks, crustaceans.
220 | Microbiology
Sexually Transmitted and Sexually Transmissible Microorganisms:
Bacteria Viruses Othera
Transmitted in Adults Predominantly by Sexual Intercourse
Neisseria gonorrhoeae HIV (Types 1 and 2) Trichomonas vaginalis, Pthirus
Chlamydia trachomatis Human T cell lymphotropic virus pubis
Treponema pallidium type 1
Haemophilus ducreyi Herpes simplex virus type 2
Calymmatobacterium Human papillomavirus (multiple
granulomatis genotypes)
Ureaplasma urealyticum Hepatitis B Virusb
Molluscum contagiosum virus
Sexual Transmission Repeatedly Described But not well defined or not the Predominant mode
Mycoplasma hominis Cytomegalovirus Candida albicans
Mycoplasma genitalium Human T cell lymphotropic virus Sarcoptes scabiei
Gardnerella vaginalis and other type II
vaginal bacteria (?) Hepatitis C, D viruses
Group B Streptococcus Herpes simplex virus type I
Mobiluncus spp. (?) Epstein-Barr virus
Helicobacter cinaedi Kaposi’s sarcoma-associated
Helicobacter fennelliae herpesvirus
Transfusion-Transmitted virus
Transmitted by Sexual contact involving oral-fecal exposure; of declining importance in homosexual Men
Shigella spp. Hepatitis A virus Giardia lamblia
Campylobacter spp. Entamoeba histolytica

• Includes protozoa, ectoparasites and fungi

• In the west, most hepatitis B virus infections are transmitted sexually or by injection
drug use.
• Human herpesvirus type 8.

Clinical Features of Genital Ulcers:

Feature Syphilis Herpes Chancroid Lymphogranulo- Donovanosis
ma venereum
Incubation 9 –90 days 2-7 days 1-14 days 3 days- 6 weeks 1-4 weeks
period (Upto 6
months)
Early Papule Vesicle Psutule Papule, pustule, or Papule
primary vesicle
lesions
No. of lesions Usually one Multiple, may Usually Usually one Variable
coalesce multiple, may
coalesce
Systematic Bacteriology | 221

Diameter 5-15 mm 1-2 mm Variable 2-10 mm Variable

Edges Sharply Erythematous Undermined, Elevated, round Elevated
demarcated, ragged, or oval irregular
elevated, round irregular
or oval
Depth Base Superficial or Superficial Excavated Superficial or Elevated
deep smooth, Serous, Purulent, deep Variable, Red and
nonpurulent, erythematous, bleeds easily nonvascular velvety bleed
relatively nonvascular readily
nonvascular
Induration Firm None Soft Occasionally firm Firm
Pain Uncommon Frequently Usually very Variable Uncommon
tender tender
Lymphadeno- Firm, non Firm, tender, Tender, may Tender, may None:
pathy tender, often bilateral suppurate, suppurate, Pseudobuboes
bilateral with initial loculated, loculated, usually
episode usually unilateral
unilateral

Organisms Transmitted by organ Transplantation and Common Sites of

Reactivation Disease:
Site
Organism Blood Lungs Heart Brain Liver Skin
Viruses:
Cytomegalovirusa + + +/- + + +
Epstein-Barr virus + + + + + +
Herpes simplex virus + + +
Human herpesvirus 6 + + + +
Human herpervirus 8 + +
Hepatits B and C virusus +
Fungi:
Candida albicans + + + +
Histoplasma capsulatum + + +
Cryptococcus + + + +
Parasites:
Toxoplasma gondii + + +
Strongyloides stercoralisd +
Trypanosoma cruzi +
Plasmodium falciparum +
222 | Microbiology
• Cytomegalovirus reactivation is prone to occur in the transplanted organ.
• Epstein-Barr virus reactivation usually presents as a proliferation of transformed B
cells and can either be a diffuse disease or produce a mass lesion in a single organ.
• T. gondii usually causes disease in the brain. In bone marrow transplant recipients,
acute pulmonary disease may also occur.
• Strongyloides “hyperinfection” may present with pulmonary disease – often associated
with bacterial pneumonia.
• While transmission with organs has been described, it is unusual.

Recommended 7 to 14 day Regimens for the Eradication of Helicobacter

pylori:
Name Drug 1a Drug 2 Drug 3 Drug 4
OCA Omeprazole Clarithromycin Amoxicillin -
(20 mg bid) (500 mg bid) (1 g bid)
OCM Ompeprazole Clarithromycin Metronidazole -
(20 mg bid) (250 mg bid) (500 mg bid)
OBTM Omeprazole Bismuth Tetracycline HCI Metronidazole
(20 mg bid) subsalicylate (500 mg qid) (500 mg bid)
(2 tabs qid)
• In any of the three regimens, omeprazole may be replaced by lansoprazole (30 mg
bid pantoprazole (40 mg bid), ranitidine bismuth citrate (400 mg bid), or possibly
ranitidine (150 mg bid). Most available data are for omeprazole.
• These regimens may be given for 7 to 14 days; meta-analysis suggests that 14 day
regimens are slightly more effective.
• The optional dose of metronidazole is not known. Tinidazole (500 mg bid) can be
used in place of metronidazole.
• Data for this regimen are mainly from Europe and are based on bismuth subcitrase,
omeprazole is given for 10 days, and the other three agents are given on days 4
through 10.

Comparison of the Treponemes and the Diseases They Cause:

Feature Venereal Syphilis Yaws Endemic Syphilis Pinta
Organism T. pallidum T. pallidum T.pallidum subspecies T. carateum
subspecies subspecies endemicum
pallidum pertenue
Mode of Sexual, Skin-to-skin Household contacts: Skin-to-Skin
transmission transplacental mouth-to-mouth or via
shared drinking/eating
utensils; insect vector?
Usual age of Adulthood Early childhood Early childhood Late childhood
acquisition
Systematic Bacteriology | 223

Primary lesion Cutaneous ulcer Papilloma, Rarely seen Nonulcerating

(Chancre) often ulcerative papule with
satellites, pruritic.
Location Genital, oral, anal. Extremities. Oral. Extremities, face.
Secondary Mucocutaneous Cutaneous Florid mucocutaneous Pintides,
lesions lesions; papulosqua- lesions (mucous patch, pigmented. Pruritic
Condylomata lata. mous lesions; spilt papule; condyloma
osteoperiostitis. latum); osteoperiostitis.
Infectious ~25% Common Unknown None
relapses
Late Gummas, Destructive Destructive gummas of Nondestructive,
complications cardiovascular and gummas of skin, bone, cartilage. dyschromic,
CNS involvement. skin, bone, achromic macules.
cartilage.
• Because the nonvenereal treponematoses are usually acquired in childhood and
treponemal bacteremia ceases with time, only in adult onset venereal syphilis is there a
reasonable likelihood that a woman will give birth to an infected child.
• CNS involvement in the endemic treponematoses has been postulated by some
investigators.

Major Exotoxins:
Organism Toxin Mode of Action Role in Disease
Protein Corynebacte- Diphtheria toxin ADP ribosyl Inhibits eukaryotic cell
inhibitors rium diphthe- transferase; protein synthesis.
riae inactivates EF –
2; targets: heart/
nerves/ epithelium.
Pseudomonas Exotoxin A ADP ribosyl Inhibits eukaryotic cells
Aeruginosa (-) transferase; protein synthesis.
inactivates EF – 2;
Target: liver.
Shigella Shiga toxin Interferes with 60S Inhibits protein synthesis in
dysenteriae (-) ribosomal subunit. eukaryotic cells, Enterotoxic,
cytotoxic and neurotoxic.
Enterohemor- Verotoxin (a shiga Interferes with 60S Inhibits protein synthesis in
rhagic E. coli like toxin) ribosomal subunit. eukaryotic cells.
(EHEC)
Neurotoxins Clostridium Tetanus toxin Blocks release Inhibits neurotransmission in
tetani (+) or the inhibitory inhibitory synapses.
transmitters glycine
and GABA.
Clostridium Botulinum toxin Blocks release of Inhibits cholinergic synapses.
botulinum (+) acetylcholine.
224 | Microbiology

Endotoxin Staphylococ- TSST – 1 Pyogenic, Fever, increase susceptibility

Enhancers cus aureus (+) decreases liver to LPS, rash, shock, capillary
clearance of LPS, leakage.
superantigen.
Streptococcus Exotoxin (A) Similar to TSST Fever, increased
pyogenes (+) a.k.a: Erythrogenic – 1. susceptibility to LPS, rash,
or pyrogenic toxin shock, capillary leakage,
cardiotoxicity.
CAMP Enterotoxigen- Heat labile Toxin LT stimulates Both LT and ST promote
inducers ic Escherichia (LT) an adenylate secretion of fluid and
coli (-) cyclase by ADP electrolytes from intestinal
ribosylation of epithelium.
GTP binding
binding protein.

Normal Human Flora:

Skin Nasophyrynx Gastrointestinal tract Genitalia
and rectum

• S. epidermidis • S. epidermidis • S. aureus • S. epidermidis

• S. aureus • S aureus • α and Non-hemolytic • α and Non-hemolytic
• α and Non-hemolytic • α and Non-hemolytic Streptococci streptococci
Streptococci (e.g. streptococci • Diphtheroids • Corynebacterium
S. mitis) • Diphtheroids • Enterococcus • Non pathogenic
• Corynebacterium • Non pathogenic • S. bovis Neisseria
• Micrococcus Neisseria • Yeast • Lactobacillus
• Propionibacterium • Haemophilus • Anaerobes • Candida and other
• Peptostreptococcus • Neisseria • Non fermenters yeast
• Acinetobacter meningitidis • Gram negative bacilli
• Candida • Pneumococci • Anaerobes
• Pseudomonas • Gram negative bacilli
• Anaerobes
• Yeast

Mechanism of immune evasion by tumor cells:

• Reduced MHC I expression in tumor cells.
• Expression of dummy molecules which mimic MHC I.
• Tumor cell inhibits apoptosis signals.
• Generates poor costimulatory signals.
• Masking of tumor antigens with antitumor antibodies.
• Modulation of tumor antigens by tumor antibodies.
• Inhibition of inflammatory cytokines like IFNγ.
Systematic Bacteriology | 225
Agents of Bioterrorism:
Category A:
• Anthrax (Bacillus anthracis).
• Botulism (Clostridium botulinum toxin).
• Plague (Yersinia pestis).
• Smallpox (Variola major).
• Tularemia (Francisella tularensis).
• Viral hemorrhagic fevers.
Arenaviruses: Lassa, New World (Machupo, Junin, Guanarito and Sabia).
Bunyaviridae: Crimean-Congo, Rift Valley.
Filoviridae: Ebola, Marburg.
Category B:
• Brucellosis (Brucella spp.).
• Epsilon toxin of Clostridium perfringens
• Food safety threats (e.g., Salmonella spp., Escherichia coli 0157:H7, Shigella).
• Glanders (Burkholderia mallei ).
• Melioidosis (B. pseudomallei).
• Psittacosis (Chlamydophila psittaci).
• Q fever (Coxiella burnetii ).
• Ricin toxin from Ricinus communis (castor beans).
• Staphylococcal enterotoxin B.
• Typhus fever (Rickettsia prowazekii).
• Viral encephalitis [alphaviruses (e.g., Venezuelan, eastern, and western equine
encephalitis)].
• Water safety threats (e.g., Vibrio cholerae, Cryptosporidium parvum).
Category C:
Emerging infectious diseases threats such as Nipah, hantavirus, SARS coronavirus and
pandemic influenza.
226 | Microbiology

Worksheet
• MCQ OF “SYSTEMATIC BACTERIOLOGY” FROM DQB

• EXTRA POINTS FROM DQB

1.

2.

3.

4.

5.

6.

7.

8.

9.

10
6 Virology

CONCEPTS
 Concept 6.1 General virology
 Concept 6.2 DNA viruses
 Concept 6.3 RNA viruses
 Concept 6.4 Important virusese
228 | Microbiology
Concept 6.1: General Virology
Learning Objectives
• General properties
• Classification
• Multiplication
• Cultivation
• Identification
• Viral Genetics

Time Needed
1 reading
st
90 mins
2 look
nd
30 mins

General Properties:
• Smallest known infective agents.
• Obligate intracellular parasites.
• Vary in size from 10-300nm therefore, cannot be seen by light microscope.
• Carries its own genetic information in the form of either RNA or DNA, but not both.
• Viruses have no metabolic activity outside susceptible host cells. They do not possess
protein-synthesizing apparatus therefore, cannot multiply in inanimate media but
only inside living cells where it takes over the host protein-synthesizing machinery to
synthesize new virus particles.

Structure and Chemical Composition of Viruses:

Viruses consist of nucleic acid core surrounded by a protein coat called capsid.

Viral Proteins:
Proteins which are a part of the virion are called as structural proteins [capsid, surface
glycoprotein, enzymes (in some viruses)].
• Functions of structural proteins:
ƒ Facilitate transfer of viral nucleic acid from one host cell to another.
ƒ Protect the viral genome from nucleases.
ƒ Promote attachment of the virus particle to susceptible cell.
ƒ Provide structural symmetry to the virus particle.
Capsid:
• Composed of repeating protein units called as capsomeres.
• Capsid with enclosed nucleic acid is called as nucleocapsid.
Virology | 229
Capsid shows three types of symmetry:
Icosahedral/cubic symmetry: Capsomeres are arranged in such a way that the capsid appears as
an icosahedron having 20 faces and 12 vertices e.g. Adenoviruses.
There is a possibility of formation of some empty particles devoid
of nucleic acid.

Helical symmetry: The nucleic acid and capsomeres are wound together in the form of
a helix e.g. Orthomyxoviruses. It is not possible for empty helical
particles to form.

Complex symmetry: Viruses, which do not show either icosahedral or helical symmetry
but are more complicated in structure e.g. Poxviruses.

Some viruses carry enzymes, which are essential for the initiation of the viral replicative cycle when
a virion enters the host cell. For e.g. RNA polymerase carried by the viruses with negative sense RNA
genomes (Orthomyxoviruses, Rhabdoviruses).

Fig.6.1: Symmetry of viruses

230 | Microbiology
Viral Nucleic Acid:
• Viruses contain a single kind of nucleic acid, either DNA or RNA.
• The genome may be single-stranded or double-stranded, circular or linear and
segmented or unsegmented.
• It is the major characteristic used for classification of the viruses.

Lipid Envelope:
• Many viruses contain lipid envelope.
• The lipid is acquired when the viral nucleocapsid buds through a cellular membrane
(cytoplasmic or nuclear membrane) during maturation.
• Budding occurs only at sites where virus-specific proteins have been inserted in the
host membrane.
Enveloped viruses are sensitive to treatment with ether and other organic solvents

Surface Glycoproteins:
• Viral envelope contains glycoproteins (peplomers), which project as spikes from the
outer surface of the envelope.
• In contrast to the lipids in the viral membranes, the envelope glycoproteins are virus
encoded. For e.g. Hemagglutinin and Neuraminidase in Influenza virus.
Functions:
• Mediate attachment of the virus to the host cell receptor.
• Some attach to receptors on the red blood cells leading to hemagglutination.
• Some cleave neuraminic acid from the from host cell glycoproteins (neuraminidase/
receptor destroying enzyme).

Susceptibility to Physical and Chemical Agents:

• Heat and cold: Enveloped viruses are heat labile as compared to non-enveloped
viruses. Viral infectivity is generally destroyed by heating at 56°C except for Hepatitis
B virus, Papova viruses, and scrapie agent.
• pH: Some viruses (e.g. Enteroviruses) are resistant to acidic conditions. All viruses
are destroyed by alkaline conditions.
• Ether susceptibility: Enveloped viruses are susceptible to ether while non-
enveloped viruses are resistant.
• Chlorine: Larger concentrations of chlorine are required to kill viruses than to destroy
bacteria.
Families of Viruses that Infect Humans
Nucleic acid core Capsid symmetry Enveloped/ naked Physical type of Virus family
nucleic acid
DNA Icosahedral Naked ss Parvoviridae
ds circular Papovaviridae
ds Adenoviridae
Enveloped ds circular Hepadnaviridae
ds Herpesviridae
Virology | 231

Complex Complex coat ds Poxviridae

RNA Icosahedral Naked ss Picornaviridae
ss Astroviridae
ss Caliciviridae
ds segmented Reoviridae
Enveloped ss Togaviridae
Unknown or Enveloped ss Flaviviridae
complex ss segmented Arenaviridae
ss Coronaviridae
ss diploid Retroviridae
Helical Enveloped ss segmented Bunyaviridae
ss segmented Orthomyxoviridae
ss Paramyxoviridae
ss Rhabdoviridae
ss Filoviridae

Replication of Viruses:
• Attachment
ƒ Mediated by binding of virion surface structures (ligands) to receptors on the cell
surface therefore the attachment is specific.
Virus Receptor
HIV CD4
Rhinoviruses ICAM 1
Epstein-Barr virus CD 21/ complement receptor 2 (CR2)
Measles virus CD 2
Rabies virus Acetylcholine receptors
Parvovirus B19 P antigen
• Penetration:
ƒ After binding, the virus particle is taken up inside the cell.
ƒ Receptor mediated endocytosis: In some viruses (especially non-enveloped) there
is receptor-mediated endocytosis, with uptake of ingested virus particles within
endosomes.
ƒ Fusion of viral envelope with the plasma membrane: This requires the interaction
of viral fusion protein with a second cellular receptor (e.g., chemokine receptors
for HIV).
• Uncoating:
ƒ Occurs concomitantly or shortly after penetration.
ƒ It is the physical separation of the viral nucleic acid from the outer structural
components of the virion such that it can function.
ƒ Infectivity of the parental virus is lost at the uncoating stage.
232 | Microbiology
• Expression of viral genome and synthesis of viral components.
ƒ Specific mRNAs are transcribed from the viral nucleic acid.
ƒ Some viruses (negative sense RNA viruses) carry their own RNA polymerases to
synthesize mRNA (conventionally designated positive sense).
ƒ Viruses then use the cell components to translate the mRNA.
ƒ Initially early or non-structural proteins are synthesized these are enzymes which
initiate the synthesis of viral components and induce the shutdown of host protein
and nucleic acid synthesis.
ƒ This is followed by replication of viral nucleic acid.
ƒ Finally, there is synthesis of late or structural proteins which constitute daughter
virion capsids.
• Morphogenesis:
ƒ Newly synthesized viral genomes and capsid polypeptides assemble to form
progeny viruses.
• Release:
ƒ Non-enveloped viruses lyse the infected cells and are released e.g., Poliovirus.
ƒ Enveloped viruses are released by the process of budding through the special
areas of the host cell membrane, where virus encoded glycoproteins have been
inserted.
ƒ Enveloped viruses are not infectious until they have acquired their envelopes.

Productive infection: Results after infection of the permissive cells and leads to the production of
infectious virus particles.

Abortive infection: Results in failure of production of infectious progeny. It may either be due to
infection of non-permissive cell, which does not allow the expression of all the
viral genes or because of the defective virus, lacking in some functional viral gene.

Latent infection: It is characterized by persistence of viral genome in the infected cell. The infected
cell survives, as there is expression of no or very few viral genes.

Eclipse phase: Between the stage of penetration to the release of daughter virion, the infective
virion is disrupted and its measurable infectivity is lost. This phase is called as
eclipse phase.

Virus Isolation:
Laboratory Animals

Suckling mice for isolation of Arboviruses and Coxsackie viruses

● Primates for Hepatitis viruses.

● Animals observed for signs of disease or death.
● Viruses identifies by testing for neutralization of their pathogenicity for animals, by standard antiviral
sera.
Virology | 233
Chick Embryos:
• Chorioallantoic membrane: Poxviruses, Herpes simplex virus.
• Amniotic cavity: Primary isolation of Influenza virus, Mumps virus.
• Allantoic cavity: Influenza virus (for serial passages, vaccine production), Sendai vi-
rus, Newcastle disease virus.
• Yolk sac: Japanese B encephalitis virus, West Nile virus.
Cell culture:
Most commonly used method for cultivation of viruses.
• Primary cell culture:
ƒ These are normal cells freshly taken from the body and cultured.
ƒ Capable of limited growth (5-10 divisions).
ƒ Preferred for cultivating viruses for vaccine production.
ƒ E.g., Rhesus monkey kidney cell culture, human amnion cell culture, chick
embryo fibroblast cell culture.
• Semi-continuous/Diploid cell strains:
ƒ These are the cell of a single type which that have retained their normal diploid
chromosome number and karyotype. They can be passaged 3-40 times before the
cells die off. Used for isolation of viruses and production of viral vaccines.
ƒ E.g. human embryonic lung cell strain.
• Continuous cell lines:
ƒ These are cells of a single type capable of indefinite propagation in vitro.
ƒ They are derived from malignant tissue therefore have irregular chromosome
numbers.
ƒ E.g. HeLa (human carcinoma cervix), Hep-2 (human epithelioma of
larynx), Kb (human carcinoma of nasopharynx).
ƒ They are used for isolation of viruses but are not used for cultivation of viruses for
vaccine production.
Detection of Growth In Cell Culture:
• Cytopathic effect:
ƒ Many viruses produce morphological changes in the cells in which they grow.
These are called as cytopathic effects:
▫ Rounding and degeneration of cells: Picornaviruses.
▫ Rounding and aggregation of cells: Adenoviruses.
▫ Syncytium formation: Measles virus, RSV.
▫ Inclusion bodies: Intracellular or intranuclear aggregates of products of viral
replication:
f Nucleus: Herpes virus.
f Cytoplasmic: Poxvirus, Rabies virus.
f Both: Measles virus, CMV.
234 | Microbiology
• Hemadsorption:
ƒ Some Orthomyxoviruses and Paramyxoviruses code for red cell agglutinins that
are incorporated in the host cell membrane during infection. When erythrocytes
are added to the infected cell layer, they adhere to the surface of the infected
cells. This is called as hemadsorption.
• Interference:
ƒ The growth of the non-cytopathogenic virus in cell culture can be detected by
subsequent challenge with a known cytopathogenic virus. The growth of the first
virus will inhibit the infection with the second virus by interference.
• Transformation:
ƒ Oncogenic viruses induce cell transformation, so that growth of cells appears in
the piled-up fashion due to the formation of microtumors.
• Immunofluorescence:
ƒ Cells from virus infected cultures can be stained with fluorescent-conjugated
antiserum and examined under UV light for detection of viral antigen. Most
sensitive and specific method for detection of viruses in tissue culture.

Viral Genetics:
● Mutation:
ƒ Random, undirected, heritable variation.
ƒ Frequency in viruses: 104 – 108.
ƒ Conditional lethal mutants: Mutants that multiply in susceptible cells under permissive conditions
but cannot grow under non-permissive conditions. E.g., temperature-sensitive mutants: Grow at low
temperatures (31-35°C) but not at high temperatures (38°C – 42°C).

● Recombination:
ƒ When two different viruses infect same cell, genetic recombination can take place.
ƒ Intramolecular recombination: Exchange of nucleic acid sequences between different but closely
related viruses during replication. Hybrid virus so generated breeds true. ds DNA viruses and RNA
viruses (picornaviridae, coronaviruses, togaviruses).

● Reassortment: Occurs in viruses having segmented genome (Arenaviridae, Bunyaviridae, Influenza

A virus, Rotavirus). In a single cell infected by two related viruses there is exchange of segments with
the production of reassortants.

● Reactivation: Is of two types:

ƒ Cross-reactivation/marker rescue: When a cell is infected with an active virus and a related inactive
virus, progeny possessing one or more genetic traits of inactive virus may be produced. When epidemic
strain (A2) of influenza virus is grown with the standard strain (AO) inactivated by UV irradiation a
progeny may be produced which has antigenic character of A2 but growth character of AO. Used for
vaccine production for influenza virus.
ƒ Multiplicity reactivation: When a cell is infected with two or more virus particles of the same stain,
each of which has suffered a lethal mutation in a different gene, live virus may be produced. Different
virions suffer from damage to different genes by UV radiation, so that from total genetic pool it may
be possible to obtain full complement of undamaged genes. Therefore, UV radiation not used for
production of inactivated virus vaccines.
Virology | 235
Non-Genetic Interactions:
Phenotypic mixing:
When two different viruses such as Influenza virus and a paramyxovirus infect the same cell, some mix-
up may take place during the assembly so that progeny genome of one virus is surrounded by the capsid
belonging entirely or partly to other virus. This is called as phenotypic mixing. This is not a stable variation
as on subsequent passage capsid of the original type will be formed. In nonenveloped viruses, when nucleic
acid of one is surrounded by entire capsid of another it is called as transcapsidation. In enveloped viruses,
phenotypic mixing results in haring of surface glycoproteins resulting in the formation of mosaics.
Polyploidy:
Viruses that mature by budding (e.g., paramyxoviruses) from the plasma membrane, several nucleocapsids
may be enclosed within a single envelope. This is called as polyploidy.
Complementation:
Protein encoded by one virus allows a different virus to replicate in doubly infected cells. E.g., HDV
(defective virus) and HBV (helper virus).
236 | Microbiology
Concept 6.2: DNA viruses
Learning Objectives
• Herpes virus
• Pox virus
• Adeno virus
• Parvo virus

Time Needed
1st reading 90 mins
2 look
nd
30 mins

Herpesviruses:
Spherical ds DNA virus, 120-200nm in diameter.
Comprise of four structural elements: envelope, tegument, capsid and core.
Envelope is outermost; tegument is present between envelope and capsid. Inner to
tegument is icosahedral capsid, which contains double stranded DNA.
These groups of viruses establish latent infections which get reactivated in
immunosuppressed hosts.
Classification of Human Herpesviruses
Subfamily Growth Cytopathology Site of latent Official Common name
cycle infection name
Alpha Short Cytolytic Neurons HHV1 HSV1
herpesvirinae HHV2 HSV2
HHV3 Varicella-zoster virus
Beta Long Cytomegalic Glands, HHV5 CMV
herpesvirinae kidneys
Lymphoproliferative Lymphoid HHV6 HHV6
tissue HHV7 HHV7
Gamma Variable Lymphoproliferative Lymphoid HHV4 EBV
herpesvirinae tissue HHV8 Kaposi’s sarcoma
associated herpes virus

Herpes Simplex Virus:

• Two types HSV1 and HSV2.
• HSV1: Infects mouth, eye, CNS (regions above
the waste), but is also responsible for few cases
of genital herpes.
• HSV2: Infects genital and anal regions.
• Infections caused by herpes simplex can be
divided into primary infection, latent infection
and reactivation.
Fig.6.2: Herpes labialis
Virology | 237
Primary infections:
• Transmitted by contact through damaged skin or mucosa, classic lesion is a vesicle:
ƒ HSV1 causes the following primary infections:
▫ Acute gingivostomatitis.
▫ Herpetic whitlow.
▫ Keratoconjunctivitis.
▫ Eczema herpeticum.
▫ Encephalitis (Involves temporal lobes, M/C cause of sporadic encephalitis).
▫ Generalized infections.
ƒ HSV2 causes following primary infections:
▫ Genital herpes (painful vesicles, fever, malaise, swollen lymph nodes).
▫ Aseptic meningitis:
▫ Neonatal infection.
• Acquired during passage of the baby through infected birth canal, abortion, congenital
defects.
ƒ Disseminated disease: Mortality rate: 80%.
ƒ Encephalitis.
ƒ Localized infection (skin, eye, mouth).
• Latent infection:
ƒ HSV1: Cranial (trigeminal) ganglia.
ƒ HSV2: Sacral ganglia.
ƒ Within the sensory ganglia, viral DNA exists as a free circular episome.
• Reactivation:
ƒ Provoked by various stimuli like common cold, fever, stress etc.
ƒ Virions migrate back to the nerve endings, where infection of the epithelial cells
results in cluster of vesicles commonly found at the mucocutaneous junctions of
the lips, nose, eyes.
• Laboratory diagnosis:
ƒ Direct examination: Electron microscopy (virions), fluorescence microscopy (viral
antigens), light microscopy (Tzanck smear: Multinucleated giant cells with
faceted nuclei, homogeneously stained ground glass chromatin with eo-
sinophilic intranuclear inclusions).
ƒ Culture: Human fibroblast/ vero cell line, swollen rounded cells (CPE),
immunofluorescent staining of the infected cells.
ƒ Serological tests: CFT, NT, Immunofluorescence, ELISA.
• Treatment: Acyclovir.
Varicella Zoster:
2 distinct clinical entities: Varicella / Chicken pox and Herpes Zoster / Shingles.
Mode of infection: Respiratory route.
Vesicles: Corium + dermis - ballooning, multinucleated giant cells, Eosinophilic
intranuclear IB.
• Involve blood vessels of skin: Necrosis + epidermal hemorrhage
238 | Microbiology
Pulmonary Involvement: Interstitial pneumonitis:
ƒ Multinucleated giant cells.
ƒ Intranuclear inclusions and
ƒ Pulmonary hemorrhage.
CNS involvement: Perivascular cuffing.
Chicken Pox:
• Humans are only reservoir for VZV.
• Secondary attack rate: 90% ( 70 - 90%).
• Late winter + Early spring.
• Age : 5 - 9yrs, I.P. 10 - 21 days.
• Infectious : 48 hrs prior to the onset of vesicular rash, during the period of
vesicular formation (4- 5days) and until all vesicles are erupted and scab is
formed.
• Skin lesions: maculopapules / vesicles/ scabs.
• Immunocompromised patients: Have more lesions and greater risk of visceral
complications (30 - 50%) and fatal (15%).
• Most common complication: Infection = S. pyogenes / S. aureus.
• Most common extracutaneous site involved: CNS (acute cerebellar ataxia + meningeal
irritation):
ƒ CSF: Increased protein + lymphocytes.
ƒ Others CNS manifestations: Aseptic meningitis, Reye’s syndrome, GBS and
Transverse myelitis.
• Varicella pneumonia: is serious complication, more common in adults.
• Perinatal varicella: ↑ mortality, if maternal disease occurs 5 days before delivery or
48hrs thereafter (Mortality = 30%).
• Congenital Varicella: Limb hypoplasia, cicatricial skin lesions, microcephaly.
Herpes Zoster:
• 6th - 8th decade.
• T3 - L3, if ophthalmic division of trigeminal nerve: Zoster opthalamicus.
• Zoster associated pain: Continuation of pain from onset to resolution.
• 1st dermatomal pain (48 - 72 hrs), then rash ( 3 - 5days).
In Ramsay Hunt syndrome:
• Pain and vesicles in external auditory canal.
• Lose taste in anterior 2/3rd of tongue and develop ipsilateral facial palsy:
ƒ (Geniculate ganglion of sensory branch of VII nerve involved).
• Most debilitating complication: Pain associated with neuritis and post herpetic
neuralgia.
• Patient with Hodgkin’s, Non-hodgkin’s lymphoma and bone marrow transplant are at
increased risk of HZV.
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Lab. Investigations:
1. TZANCK smear.
2. Isolation of VZV in susceptible tissue culture cell.
3. Demonstration of seroconversion:
Demonstration of a 4 fold / greater rise in antibody titre between convalescent and
acute phase sera by ELISA, CFT, NT, IFA.
4. PCR.
Prophylaxis:
• VZIG and ZIG: within 96hrs (pref. 72 hrs).
• Vaccine: OKA strain (life attenuated vaccine).
Treatment:
• Acyclovir, vidarabine, valacyclovir.
• Acute Neuritis / Neuralgia: Analgesics / Glucocorticoids.
Cytomegalovirus:
• Beta herpes virus group.
• ds DNA.
• Protein capsid.
• Lipoprotein envelope.
• Icosahedral symmetry.
Virus replicates in epithelial cells in salivary glands, kidneys and respiratory epithelium.
Virus may be present in milk, saliva, faeces and urine.

Fig.6.3: CMV – owl eye inclusion bodies

• Transmission:
ƒ Infection is transmitted by close contact between individuals.
ƒ Blood transfusion.
ƒ Vertical transmission (prenatal, perinatal and postnatal).
ƒ Once infected, an individual probably carries the virus for life.
ƒ The infection usually remains latent.
240 | Microbiology
ƒ CMV reactivation syndromes develop frequently when T-lymphocyte mediated
immunity is compromised.
• Pathology : Cytomegalic cells in vivo, “Owl’s Eye“ appearance.
• Clinical manifestations:
ƒ Prenatal CMV infection:
▫ Most common agent responsible for intrauterine infection.
▫ Primary maternal infection or reactivation during pregnancy.
▫ Most common presenting features:
f Growth retardation, hepatosplenomegaly, jaundice, thrombocytopenia,
microcephaly, encephalitis, chorioretinitis, deafness, mental retardation.
ƒ Lab abnormalities in decreasing order of frequency:
f Serum IgM level > 20mg/dl.
f Atypical lymphocytosis.
f Elevated liver aminotransferases.
f Thrombocytopenia.
f Hyperbilirubinemia.
f CSF protein level > 20 mg/dl.
ƒ Perinatal CMV infection:
▫ Acquired either from infected maternal genital secretions or from breast milk.
ƒ Postnatal infection:
▫ Acquired by kissing, sexual intercourse, blood transfusion or organ
transplantation.
▫ Generally subclinical.
▫ CMV Mononucleosis:
f Most often involves sexually active young adults.
f I.P.: 20 - 60days.
f Illness lasts for 2 - 6 weeks.
f Characteristic laboratory abnormality - Relative lymphocytosis in peripheral
blood with > 10% atypical lymphocytes.
ƒ Most common cause of heterophile negative mononucleosis syndrome.
ƒ CMV infection in Immunocompromised host.
▫ MC and important viral pathogen complicating organ transplantation.
▫ Period of maximum risk between 1 and 4 months after transplantation
f Patients with CD4+ T cell count below 100/µl are highly susceptible to CMV.
f Disseminated infection leading to interstitial pneumonia, retinitis, hepati-
tis, arthritis, encephalitis, GBS, transverse myelitis.
ƒ Laboratory diagnosis:
Direct detection: Inclusion bodies, viral antigen (immunofluorescence), virus
(electron microscopy):
f Culture: Virus grows slowly taking 1-2 weeks for appearance of cytologic
changes (multinucleate giant cells with perinuclear cytoplasmic and intranu-
clear inclusions).
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f Nucleic acid detection: DNA probe, PCR.
f Serology: NT, CF, IFA, RIA.
f Treatment: Drug of choice is ganciclovir.
Epstein Barr Virus (EBV):
• Family Herpesviridae, double stranded DNA virus.
• EBV replicates in epithelial cells of nasopharynx and salivary glands.
• Latent infection is established in B cells (EBV receptor: CD21 on surface of B
cells).
• Infection is acquired through kissing or contaminated utensils.
• It is associated with following diseases:
1. Heterophile +ve infectious mononucleosis.
2. Burkitt’s lymphoma (equatorial Africa, children).
3. Hodgkin’s disease (mixed cellularity).
4. Non-Hodgkins’s lymphoma.
5. Primary B cell lymphomas (in immunocompromised).
6. Oral hairy leukoplakia (not a malignancy).
7. X linked lymphoproliferative syndrome (DUNCAN’S DISEASE).
8. Anaplastic nasopharyngeal carcinoma (southern provinces of China,
adults).
9. Post transplant lymphomas.
• Bimodal peak.
1. Early childhood (asymptomatic).
2. Late adolescence (symptomatic, infectious mononucleosis or glandular fever):
Pathogenesis: Infects epithelium of orophraynx and salivary glands. Prolifer-
ation of EBV infected B - cell, reactive T cell, enlargement of lymphoid tissue,
Inverted CD4+ / CD8+ T cell ratio
Clinical Manifestation: I.P.: 4 - 6 weeks, patient presents with sore throat, gener-
alized lympadenopathy, fever, malaise, myalgia.
Complications : Most cases of IM are self limited but few develop GBS, Bell’s
palsy, meningoencephalitis, transverse myelitis, thrombocytopenia, carditis, ne-
phritis, pneumonia, splenic rupture.
• Laboratory diagnosis:
ƒ WBC - 10,000 - 20,000 / µl.
ƒ Lymphocytosis with > 10% atypical lymphocytes (CD8 lymphocytes).
ƒ Neutropenia and thrombocytopenia.
ƒ Serological testing:
▫ Isolation: by immortalization of normal human lymphocytes.
▫ Nucleic acid detection: DNA hybridization, PCR.
▫ Antibody detection.
▫ Early in the acute disease, a transient rise in IgM antibodies to viral capsid
antigen occurs, replaced within weeks by IgG antibodies to this antigen, which
persist for life, Slightly later, antibodies to the early antigen develop and persist
for several months. Several weeks later, antibodies to EB nuclear antigen (EBNA)
and the membrane antigen arise and persist throughout life.
242 | Microbiology
▫
IgM anti viral capsid antigen: Current infection.
▫
IgG anti viral capsid antigen: Past infection and immunity.
▫
Antibodies to early antigen: Current infection.
▫
Antibodies to EBNA: Past infection.
Heterophile antibody test (Paul Bunnel test/ Monospot test): Agglutination
▫
of sheep RBCs (less specific).
• Treatment:
ƒ Patients of IM - Supportive measures with rest and analgesics.
ƒ Prednisolone (40 - 60mg /d) in complicated IM like airway obstruction, autoim-
mune haemolytic anemia or severe thrombocytopenia.
ƒ Acyclovir: Decreases shedding from oropharynx, no effect on symptoms of IM.
Human Herpes Virus 6:
• Infect CD4+ T cells.
• Salivary glands major reservoir and saliva main route of transmission.
• Clinical features:
ƒ Most infections asymptomatic.
ƒ Exanthem subitum/roseola infantum/ sixth disease: mild facial rash
occurring between 6 months to 3 years of age with fever.
ƒ Mononucleosis with cervical lymphadenopathy: adults.
Human Herpes Virus 7:
• Like HHV 6, it may also cause roseola infantum.
• Infect CD4+ T cells.
Human Herpes Virus 8:
• Also called as Kaposi’s sarcoma associated herpes virus.
• Detected in over 90% of Kaposi’s sarcomas.
• Also associated with Castleman’s Disease and Primary Effusion Lymphoma.
Herpes B virus:
• Cercopithecine herpesvirus 1/ herpes virus simiae.
• Infects macaque monkeys causing vesicular eruptions on the tongue and buccal
mucosa.
• Human infection acquired from bite or handling of infected monkeys.
• Local inflammation at the site of entry followed by fatal ascending paralysis.

Smallpox, Vaccinia and Other Poxviruses:

Poxviruses:
• Characterized by brick shaped morphology.
• Double stranded DNA genome.
• Only DNA viruses that replicate in cytoplasm where viral particles accumulate to form
eosinophilic inclusions or Guarneri bodies, visible by light microscope.
• Poxviruses associated with human diseases:
ƒ Variola.
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ƒ Vaccinia.
ƒ Molluscum contagiosum.
• Poxviruses associated with zoonotic infection - Monkey Pox.
Small Pox: Last case reported in 1977 in Somalia.
WHO officially declared in 1980 that small pox has been eradicated completely.

Factors contributing to this accomplishment:

1. Universal interest in eliminating this costly disease with high mortality and morbidity.
2. Infection’s long incubation and low communicability.
3. Ease of diagnosis of skin lesions by histology or antigen detection.
4. Humans - Sole reservoir of infection.
5. No carrier state.
6. Availability of live virus vaccine: which was resistant to temperature changes and
drying.
Only known remaining repositories of small pox virus are in research labs in USA and
Russia.
Variola Virus: Existed in 2 strains:
• Variola Major (Small pox)[Case mortality-20-50%].
• Variola Minor (Alastrim) [Milder form of smallpox, case fatality less than 1%].
Smallpox: Transmission requires close contact.
IP: 12 days.
C/F: Fever, macular rash - progressing to typical vesicular and pustular lesion over 1
-2weeks.
Classified as Class A Bioterrorism agent and most dangerous bioterrorism agent.
Vaccinia:
• Virus used for vaccination against small pox.
• Probably derived from cowpox, variola virus or hybrid of the two.
• Now a laboratory virus, no natural host
• Percutaneous administration of vaccinia virus vaccine results in protective cellular and
humoral immune response in more than 95% of primary vaccinee.
• Formation of pustule and scab at the site of inoculation is indicative of immunity.
• Immunity wanes after 10 - 20yrs.
• Revaccination after 10yrs - recommended.
• Routine vaccination for small pox discontinued in 1971.
• Now recommended for health care employees and lab employees who work directly
with virus vectors and selected groups.
• Exposed to pox viruses (e.g. Military personnel and individualy who work with
animals).
Complications:
1. Generalized vaccinia - in otherwise healthy individualy, generally self-limited.
2. Eczema vaccinatum - Disseminated cutaneous lesions in highly susceptible patients
with eczema or other chronic skin diseases and is occasionally fatal.
244 | Microbiology
3. Progressive vaccinia (vaccinia necrosum) - severe, potentially fatal illness in individ-
uals with immunodeficiency - acquired / congenital, iatrogenic (glucocorticoid thera-
py), HIV individuals.
4. Post infectious encephalitis - Rare (3 cases in 1million) - Fatal in 15 - 25% cases with
25% patients with permanent neurological sequelae.
5. Vaccinia can be transmitted to susceptible individuals by vaccine thus vaccination
is contraindicated in those whose household contacts who have eczema, are
immunocompromised or pregnant.
Molluscum Contagiosum: Benign disease characterized by
• Pearly, flesh colored umblicated skin lesions 2 - 5mm in diameter.
• Infection transmitted by close contact including sexual intercourse.
• Can occur anywhere, but mainly genital region; palms and soles spared.
• Disease self-limited, no systemic complication.
• Associated with advanced stages of HIV infection. Disease more generalized, severe
and persistent in AIDS patients. Involves face and upper body.
• Diagnosis: By histological demonstration of cytoplasmic eosinophilic inclusions (Mol-
luscum bodies/Henderson Peterson bodies).
• The virus has not been cultured so far.
• Virus identification by electron microscopy and molecular methods.
Treatment:
• No specific systemic treatment.
• Only physical ablation techniques used.

Fig.6.4: Molluscum contagiosum

Monkey pox viruses:

• Infect non-human primates.
• Humans infected if come in direct contact with infected animal.
C/F:
• Human disease is rare.
• Vesicular rash, like small pox.
• Occasionally fatal in children and adults who have not received small pox vaccine.
Virology | 245
Other pox viruses: Cause localised vesicular lesion when humans come in direct
contact with infected animal (zoonotic infection):
1. Cowpox virus.
2. Pseudo cowpox virus (Milker’s nodule).
3. Buffalopox.
4. Bovine papular stomatitis virus.
5. Orf virus (contagious pustular dermatitis).

Adenovirus:
DNA virus: Double stranded DNA, non-enveloped, icosahedral symmetry.
47 distinct antigenic types have been isolated from humans.
Human adenoviruses are divided into six groups (A-F) based on their physical, chemical
and biological prperties.
Pathogenesis:
Adenoviruses infect and replicate in the epithelial cells of respiratory tract, eye,
gastrointestinal tract, urinary bladder and liver.

Clinical features:
Group Principal types Disease
B 3,7,14 Pharyngoconjunctival fever (swimming pool conjunctivitis).
3,7,14,21 Acute respiratory disease.
3,7 Pneumonia, acute febrile pharyngitis in small children.
11, 21 Acute haemorrhagic cystitis.
34,35 Pneumonia with dissemination (AIDS patients).
C 1,2,5,6 Acute febrile pharyngitis in small children.
1,2,5 Hepatitis in children with liver transplant.
D 8,19,37 Epidemic keratoconjunctivitis (ship yard eye).
E 4 Acute respiratory disease with fever.
F 40,41 Infantile gastroenteritis ( difficult to cultivate).

Laboratory Diagnosis:
Culture: primary human embryonic kidney cells,
Hep-2, HeLa.
Rounding and clustering of swollen cells:
Antigen detection: Immunofluorescence, ELISA,
latex agglutination, electron microscopy.
Antibody detection: CFT, NT, HAI.
Treatment: Symptomatic.
Vaccine - Live attenuated / killed, for prevention of
acute respiratory disease, useful for military recruits.
Fig.6.5: Adenovirus – Satellite
246 | Microbiology
Parvoviruses:
Parvoviruses group includes several species-specific viruses of animals.
B - 19:
• Is a human pathogen - Named so after code number of human serum in which it was
discovered:
ƒ Icosahedral.
ƒ Non enveloped.
ƒ Single stranded DNA virus with outer capsid made of two structural proteins.
ƒ Individual virus particles contain DNA strands of +ve and -ve polarity.
ƒ Thermostable, retains infectivity after incubation at 60° C for 16 hrs.
ƒ Fails to grow in conventional cell culture lines and animal model.
ƒ Only replicates in vitro in erythroid progenitor cells derived from human bone
marrow, umbilical cord, peripheral blood or fetal liver.
• Causes variety of disorders:
ƒ Erythema infectiosum (Most Common).
ƒ Acute arthropathy.
ƒ Transient aplastic crisis and pure red cell aplasia in immune compromised.
ƒ Fetal infections - manifested by death or hydrops fetalis, risk of
transplacental transmission 30%.
B 19 binds specifically to cellular receptors, erythrocyte P antigen therefore, explains the
tropism of B19 for erythroid precursor cells particularly pronormoblast and normoblasts.
Clinical manifestations:
Erythema Infectiosum:
• Most common manifestation of Parvovirus B19 infection.
• Also called Fifth disease/ Erythema infectiosum.
C/F:
• Mild illness, facial rash (typical presentation) with a “Slapped cheek” appearance
preceded by low grade fever.
• Rash:
ƒ On arms, legs and has a lacy, reticular, erythematous appearance.
ƒ Trunk, palm and soles less commonly involved.
• Also, rash can be maculopapular, morbilliform, vesicular, purpuric or pruritic.
• Rash resolves in about a week but recurs intermittently for several weeks, after stress
exercise, exposure to sunlight, bathing and change in environment temperature.
• Overall most common manifestation.
Arthropathy:
Most common manifestation in adults: Arthralgia and Arthritis.
Arthritis: Symmetrical, peripheral involving wrists, hands, knees most frequently.
• Usually resolves in 3 weeks, non-destructive, may last for many months, even years.
Virology | 247
Transient Aplastic Crisis: Parvovirus B19 causes transient aplastic crisis in patients
with chronic hemolytic disease.
• B19 can cause aplastic crisis in all hemolytic conditions e.g.: Sickle cell disease,
Hereditary spherocytosis, Thalassemias etc.
C/F:
• Weakness, lethargy, pallor, severe anemia preceded by non specific symptoms.
• Patients have intense reticulocytopenia.
• Patients with transient aplastic crisis unlike others like arthritis and arthropathy can
transmit B19 infection to other people.
Chronic anemia in immunodeficient patients: Unable to eliminate B19 infection due
to inadequate production of IgG antibodies, thus persistence of infection.
Fetal and congenital infections: Maternal B19 infections generally do not adversely
affect the fetus:
• Rate of transplacental transmission >30%. However, < 10% of maternal B19 infection
usually lead to fetal death.
• If infected, usually death due to development of non immune fetal hydrops, wherein
fetus has severe anemia and CHF.
• For prevention - pregnant women with known exposure to B19 should have IgM
monitored and check for elevated level of α AFP, USG for Hydrops fetalis.
Diagnosis:
Parvovirus B19 specifies IgM and IgG antibodies.
Acute infection shows raised IgM.
Bone marrow biopsy: Giant pronormablast and Hyperplasia.
Fetal infection: Presence of B19 DNA in amniotic fluid or Fetal blood.
Treatment:
Erythema infectiosum - Self immunity.
Arthritis and Arthropathy - NSAIDS.
Aplastic crisis - Erythrocyte transfusion.
Immunodeficient patient - I/V Ig contain anti Parvovirus IgG.
No vaccine for Parvovirus B19 is currently available - A baculo virus infected insect cell
line that expresses non infectious immunogenic B19 capsid proteins is being evaluated
as a vaccine candidate.
248 | Microbiology
Concept 6.3: RNA Viruses
Learning Objectives
• Orthomyxoviridae
• Paramyxoviridae
• Picornaviridae
• Rhabdoviridae
• Arboviridae
• Retroviridae

Time Needed
1 reading
st
90 mins
2 look
nd
30 mins

Orthomyxoviridae:
• Spherical, pleomorphic, 80-120 nm.
• Single stranded RNA, negative sense, segmented (A,B: 8 segments, C: 7 segments).
• Enveloped.
• Infuenza virus A, B and C.

Influenza Virus:
• Affects both upper and lower respiratory tracts.
• Outbreaks in winter.
Etiological agents:
• Orthomyxoviridae family.
• Divided into A, B and C depending on antigenic characteristics of nucleoprotein
(NP) and Matrix (M).
• Influenza A further divided on the basis of haemagglutinin (H) and
neuraminidase (N).
• Virions are irregularly spherical, 80 - 120nm in diameter and contain lipid envelope,
from the surface of which H and N glycoprotein projects.
• Hemagglutinin is the site at which virus binds to cell receptor. Antibodies to H Ag are
major determinants of immunity to Influenza virus.
• Neuraminidase degrades the receptor and also causes release of virus from infected
cells. Antibodies to N Ag limits viral spread.
• M protein present on surface of lipid envelope causes virus assembly and stabilizes
lipid envelope.
• Genome is 8, single stranded RNA segments.
• Segmentation of genome leads to increased chances of genetic Reassortment.
• Seasonal flu: H3N2, H1N1.
• Pandemic influenza: A H1N1/ Swine Flu.
• Bird flu virus/ Highly pathogenic Avian Influenza Virus (HPAI): H5N1.
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Epidemology:
• Localized epidemics occur every 1 - 3yrs.
• Pandemics occur every 10 - 15yrs.
• Epidemics occur mainly due to Influenza A virus due to antigenic variation in H and
N Ag.
• Antigenic Shift: Major antigenic variation, due to genetic reassortment. They
are associated with pandemics and are restricted to Influenza A.
• Antigenic drift - Minor antigenic variations due to point mutation.
• Influenza B causes less severe outbreaks.
Pathogenesis:
• Infection spreads by aerosols.
• Virus multiplies mainly in ciliated columnar respiratory epithelium. Virus is not found
in any extrapulmonary site.
• Severity of illness is related to quantity of virus shed in secretions. Virus shedding
lasts for 2 - 5days after appearance of symptoms.
• Incubation period 18 - 72 hrs.
• Serum antibodies appear after 2 weeks of primary infection.
• IgA antibodies are found in respiratory secretions.
• Interferons appear in respiratory secretion shortly after virus shedding begins and
rise of interferons coincides with decrease in virus shedding.
Clinical features:
• Abrupt onset of symptoms.
• Moderate fever, sore throat, myalgia.
• Ocular signs e.g.: Photophobia and burning of eyes.
• Mild cervical lymphadenopathy.
• Acute illness lasts for 2 - 5days.
Complications:
1. Pneumonia - Most common complication.
Primary viral pneumonia is severe with scanty sputum production. Secondary bacterial
pneumonia is commonly caused by Streptococcus pneumoniae, Staphylococcus
aureus and Haemophilus influenzae.
Mixed bacterial and viral pneumonia commonly occurs.
2. Worsening of COPD and exacerbation of chronic bronchitis and asthma.
3. Reye’s Syndrome: Mainly associated with Influenza ‘B’, especially with the use
of aspirin for viral infection.
4. Myositis, myoglobinuria, rhabdomyolysis:
ƒ Myositis leads to increase in CPK and aldolase.
ƒ Myoglobinuria may cause renal failure.
5. Pericarditis, Myocarditis.
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Laboratory diagnosis:
• Demonstration of viral antigen: Immunofluorescence staining.
• Virus is isolated from throat swabs, nasopharyngeal washes or sputum through tissue
culture or amniotic cavity of chick embryo within 48 - 72hrs of inoculation.
• Antibody detection: CFT, HAI, NT.
Treatment:
• Salicylates and acetaminophen are used for symptomatic relief.
• Amantadine and Rimantadine are active only against influenza A.
• Ribavirin is effective against both Influenza A and Influenza B.
• Oseltamivir – currently in practice.
• Maintenance of oxygenation and fluid is done in viral pneumonia.
• Secondary bacterial pneumonia is treated with antibacterial drugs.
Prophylaxis:
• Vaccines against Influenza A and B are effective in 50 - 80% cases.
• Live attenuated vaccine against Influenza A is given intranasally.
• Commercially available vaccine is the killed vaccine.
• Amantadine and Rimantadine are 70 - 100% effective in prophylaxis of illness.
• During outbreaks, amantadine and vaccine can be given simultaneously.

Sequential Changes in Influenza A Virus Antigens due to Antigenic Drift:

Year Subtype

1890 H2N2

1900 H3N8

1918 H1N1 Spanish influenza

1957 H2N2 Asian influenza

1968 H3N2 Hong Kong influenza

1977 H3N2 and H1N1 Russian influenza

1990 H3N2 and H1N1

2009 Pandemic influenza A H1N1 2009

Paramyxoviridae:
Spherical, pleomorphic, 150-300nm.
Single stranded RNA, linear, nonsegmented, negative sense.
Enveloped, contains two different transmembrane glycoproteins.
HN/ H: May possess both haemagglutinin and neuraminidase activities.
F: Membrane fusion and hemolysin activities.
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Characteristics of Genera in the Subfamilies of the Family Paramyxoviridae:
Property Paramyxoviridiae Pneumovirinae
Paramyxovirus Rubulavirus Morbillivirus Pneumovirus
Parainfluenza 1,3 Mumps, Measles RSV
parainfluenza 2,4
Serotypes 4 1 1 1
Diameter of nucle- 18 18 18 13
ocapsid (nm)
F protein + + + +
Hemolysin + + + -
Hemagglutinin HN HN H -
Hemadsorption + + + -
Neuraminidase HN HN - -
Inclusions C C N,C C

Parainfluenza Virus:
• Infection acquired by droplets.
• All parainfluenza viruses produce respiratory tract infections.
• In infants and children: Lower respiratory tract infection, pneumonia.
• In older children (6 months to 5 years): Mainly type1 and in some cases type 2 cause
layryngotracheobronchitis or croup.
• Fever, cough, respiratory distress, emergency tracheostomy.
• Laboratory diagnosis:
ƒ Demonstration of viral antigen: Immunofluorescence staining.
ƒ Virus is isolated from throat swabs, nasopharyngeal washes or sputum through
tissue culture, viral growth detected by immunofluorescent staining.
ƒ Antibody detection: CFT, HAI, NT, ELISA.
MUMPS:
• Disease of childhood.
• Transmitted by respiratory secretions.
• Virus multiplies in the upper respiratory tract and local lymph nodes, viremia occurs
and virus spreads to many organs.
• Major manifestation: Painful swelling of one or both salivary glands 14-18 days after
exposure.
• Complications: Aseptic meningitis (most common in children), orchitis (most
common in post-pubertal males), oophoritis, pancreatitis, arthritis, myocarditis,
renal dysfunction.
• Natural mumps confers life long immunity.
• Some patients may not develop parotitis.
• Sterility (due to orchitis) is not a common complication.
252 | Microbiology
• Laboratory diagnosis:
ƒ Isolation: tissue culture.
ƒ Serology: ELISA, IgM antibody detection.
ƒ Prophylaxis.
ƒ Live attenuated vaccine (Jeryl –Lynn strain).
Measles (Rubeola) Virus:
• Contagious childhood disease spread by respiratory secretions.
• Pathogenesis:
Virus gains access through respiratory route→multiplies locally→spreads to the regional
lymph nodes →primary viraemia→dissemination of virus→multiplies in reticuloendothelial
system→secondary viremia→seeds epithelial surfaces of the body (skin, respiratory
tract, conjunctiva).
• Clinical features:
ƒ Incubation period: 10-12 days.
ƒ Upper respiratory tract infection, fever, rhinitis, cough, conjunctivitis.
ƒ Koplik’s spots: small, 1-3mm diameter, bluish white spots surrounded by
erythema seen on buccal mucosa.
ƒ After 1-2 days symptoms decline with the appearance of maculopapular rash
which appears first on the cheek and then spreads to the rest of the body. In next
10-14 days rash fades with the desquamation of the skin.
• Complications:
ƒ Most common: Acute otitis media.
ƒ Most common cause of death: pneumonia and diarrhea.
ƒ Giant cell pneumonia in patients with impared cell mediated immunity.
ƒ Acute post infectious encephalitis (1/1000 cases of measles).
ƒ Subacute sclerosing pan encephalitis (1/300,000 cases of measles).
ƒ It occurs 5-15 years after a an attack of measles.
ƒ Progressive mental deterioration, involuntary movements, muscular rigidity, coma.
ƒ Invariably fatal.
ƒ High titres of measles antibody in CSF, defective measles virus in brain cells.
• Laboratory diagnosis:
ƒ Direct detection: Multinucleated giant cells, antigen (immunofluorescence) in
nasopharyngeal aspirates.
ƒ Isolation: Tissue culture, multinucleated
giant cells containing eosinophilic
inclusions in cytoplasm and
nucleus, detection of growth by
immunofluorescence.
ƒ Serology: IgM antibody by ELISA, CFT.
• Prophylaxis:
Live attenuated vaccine, Edmonston-Zagreb
strain.
Fig.6.6: Measles rash
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Respiratory Syncytial Virus (RSV):
• Lacks both haemagglutinin and neuraminidase activities.
• Contains fusion (F) protein that results in the formation of multinucleated syncytia.
• Responsible for 50% cases of bronchiolitis and 25% cases of pneumonia
during first few months of life.
• Infection is spread through respiratory secretions.
• Lower respiratory infection (bronchiolitis, pneumonia) in infants less than 6 months
of age.
• Upper respiratory tract infection (cold) in older children and adults.
• Immunocompromised transplant recipients, it may cause severe pneumonia.
• Laboratory diagnosis:
ƒ Detection of antigen in nasopharyngeal aspirates by immunofluorescence, ELISA.
ƒ Isolation: Tissue culture, syncytia formed in 2-10 days, definitive identification by
immunofluorescence.
ƒ Serology: Immunofluorescence, ELISA, CFT.
• Treatment: Supportive care, ribavarin.
Rubella Virus:
• German measles/ 3-day measles.
• Genus: Rubivirus.
• Belongs to Togaviridae family, not a paramyxovirus.
• Spherical, 70nm diameter, positive sense, single stranded RNA, enveloped.
• Not transmitted by arthropods.
• Mild childhood fever.
• May also be acquired congenitally or postnatally.
• Postnatal rubella.
• Virus excreted in oropharyngeal secretions and infection is acquired by inhalation.
• Virus multiplies locally in upper respiratory tract and cervical lymph nodesviraemia,
dissemination throughout the body.
• Incubation period: 2-3 weeks, fine pink macules first appearing on the face and then
spreading to involve trunk and limbs.
• Enlargement of postauricular, suboccipital, posterior cervical lymph nodes.
• Recovery within 3-4 days of appearance of rash.
• Complications: Polyarthritis (adult women, thrombocytopenic purpura, encephalopathy,
panencephalitis).
• Congenital rubella:
ƒ Virus can cross the placental barrier and infect the fetus.
ƒ Congenital anomalies/ death of the fetus.
ƒ Congenital defects: deafness, cataract, glaucoma, microphthalmia, retinopathy,
congenital heart disease (patent ductus arteriosus, pulmonary artery stenosis),
microcephaly, mental retardation, thrombocytopenic purpura, hepatosplenomegaly.
ƒ Timing of fetal infection determines the extent of teratogenic effect:
▫ Infection in first trimester: Abnormalities in 85% cases.
▫ Infection in second trimester: Abnormalities in 16% cases.
▫ Infection after 20 weeks of gestation: Birth defects uncommon.
254 | Microbiology
Laboratory diagnosis:
• Isolation: Tissue culture, detection by immunofluorescence, immunoperoxidase
staining.
• Serology: IgM antibodies, ELISA, RIA.
Prophylaxis:
• Live attenuated vaccine, RA 27/3 strain.
Picornaviridae:
Icosahedral, 28-30 nm (small size), single stranded RNA, linear positive sense,
nonenveloped

Three genera containing viruses infecting humans:

Genus Species
Enterovirus Polio virus 1-3
Coxsackieviruses A1-24 except 23
Coxsackieviruses B1-6
Echoviruses 1-34 except 10 and 28
Enteroviruses 68-71
Rhinovirus Rhinoviruses 1-100
Hepatovirus Hepatitis A virus ( earlier called as enterovirus 72)

Differences Between Coxsackie A and B Viruses

A B
Pathological changes induced by Generalised myositis Patchy focal myositis
inoculation of suckling mice Flaccid paralysis Spastic paralysis
Death within one week Localized lesions in the liver,
pancreas, myocardium
Number of types 23 (1-24 except 23) 6 (1-6)

Enterovirus Serotypes and Manifestations:

Manifestation Serotype(s) of Indicated Virus
Coxsackie virus Echovirus (E) and Enterovirus
(Ent)
Acute hemorrhagic conjunctivitis A24 Ent70 (MC)
Aseptic meningitis A2, 4, 7, 9, 10 E4, 6, 7, 9, 11, 13, 16, 18, 19,
B1–5 30,33
Ent70, 71 (MC)
Encephalitis A9 E3, 4, 6, 7, 9, 11, 18, 25, 30
B1–5 Ent71 (MC)
Virology | 255

Exanthem A4, 5, 9, 10, 16 E4–7, 9, 11, 16–19, 25, 30

B1, 3–5 Ent71 (MC)
Generalized disease of the newborn B1–5 E4–6, 7, 9, 11, 14, 16, 18, 19
Hand-foot-and-mouth disease A5, 7, 9, 10, 16 (MC) Ent71
B1, 2, 5
Herpangina A1–10, 16, 22 (MC) E6, 9, 11, 16, 17, 25, 30
B1–5 Ent71
Myocarditis, pericarditis A4, 9, 16 E6, 9, 11, 22
B1–5
Paralysis A4, 7, 9; E2–4, 6, 7, 9, 11, 18, 30
B1–5 Ent70, 71
Pleurodynia A1, 2, 4, 6, 9, 10, 16 E1–3, 6, 7, 9, 11, 12, 14, 16, 19,
B1–6 (MC) 24, 25, 30
Pneumonia A9, 16 E6, 7, 9, 11, 12, 19, 20, 30
B1–5 Ent68, 71

Polioviruses:
• Consists of three types (1-3) based on neutralization tests.
• Type1: Epidemic.
• Type2: Endemic infections.
• Type3: Occasionally associated with epidemics.
• Natural infections occurs only in man.
• Spread by faeco-oral route and droplet infection.
• Pathogenesis:
Virus multiplies in the tonsils and payer’s patches → spread to regional lymph nodes
(cervical, mesenteric) → viraemia → disseminated throughout the body including spinal
cord and brain.
• Clinical features:
ƒ Inapparent infection: 90-95% of individuals.
ƒ Minor illness/abortive poliomyelitis: Influenza like illness, 4-8%.
ƒ Non-paralytic poliomyelitis: Headache, neck stiffness, back pain, complete
recovery; 1-2%.
ƒ Paralytic poliomyelitis: Flaccid paralysis, may be spinal, bulbar or bulbospinal;
0.1-2%.
Laboratory diagnosis:
• Isolation: Tissue culture, CPE: cell retraction, cytoplasmic granularity, nuclear
pyknosis.
Prophylaxis:
• Inactivated polio (Salk) vaccine.
• Live attenuated polio (Sabin) vaccine:
ƒ Currently bivalent (1,3) vaccine used in the programme.
256 | Microbiology
Rhinoviruses:
• Differentiated from the enteroviruses by their acid lability (therefore not able to infect
intestinal tract) and their optimal temperature for replication (33°C).
• Consist of more than 100 serotypes
• Most important cause of common cold (>50% of common colds):
ƒ Infection transmitted by droplet infection.
Clinical features:
• Incubation period: 2-4 days.
• Rhinorrhea, nasal obstruction, sneezing, sore throat, cough, headache, malaise, mild
fever.
• Symptoms subside in about a week.
Laboratory diagnosis:
• Isolation: Tissue culture, incubation at 33°C.
• Antigen detection: ELISA.
• Nucleic acid detection: PCR.
Rhabdoviridae:
• Bullet shaped, 75 x 180nm, single stranded RNA, linear, non-segmented, negative-
sense, enveloped.
• Two genera:
ƒ Vesiculovirus: infections in animals.
ƒ Lyssavirus: rabies virus.
Rabies Virus:
• Rabies is a natural infection of dogs, foxes, wolves etc.
Animal Susceptibility to Rabies
Very High High Moderate Low
Foxes Hamsters Dogs Opossums
Coyotes Shanks Sheep
Jackals Raccoons Goats
Wolves Cats Horses
Cotton rats Bats Nonhuman primates
Rabbits
Cattle
• Man acquires infection by the bite of the rabid dog or other animals.
• Rarely, infection can occur following licks on abraded skin and intact mucosa.
• Bite of the animal results in deposition of rabies infected saliva in the muscle → virus
replicates at the local site → infects peripheral nerves → within the nerve fibres it
travels along the axon towards the central nervous system (speed of 3mm/hour) →
in central nervous system it multiplies and produces encephalitis → virus then spreads
outwards along the nerve trunks to various parts of the body including salivary glands
→ shed in saliva.
Virology | 257
• The presence of the virus in the saliva and irritability brought on by encephalitis
ensure the transmission and survival of the virus in the nature.
• Man is not highly susceptible, the incidence of human rabies after bites by known
rabid dogs is about 15%.
Clinical features:
• Incubation period: 1-2 months, shorter in children and than in adults, shorter in
persons bitten on the face or head than those bitten on the legs (related to the
distance virus has to travel to reach brain).
• Malaise, headache, fever, paraesthesia at the site of bite followed by anxiety,
hyperactivity, aggression, convulsions, hydrophobia. Finally patient develops coma
and death.
• Disease once developed is always fatal in about 4-14 days.
• Virus is secreted in saliva, urine and other secretions; human to human transmission
rare.
Immune response.
• Rabies virus ascends to the brain along the nerves and does not come in contact with
immune system. It is only after virus spreads form the CNS to different parts of the
body that antibodies are formed.
• By this time it is too late as irreversible damage of the neurons have already occurred.
• Antibody is protective if it is present before exposure (pre-exposure vaccination)
or after exposure (post-exposure vaccination or passive immunization) thereby
preventing the binding of the virus to the nerve fibres at the site of inoculation.

Fig.6.7: Rabies virus

Laboratory diagnosis:
• Demonstration of Negri bodies (intracytoplasmic, round, oval, eosinophilic with
basophilic inner granules) by Seller’s stain. Mainly found in pyramidal cells of ammon’s
horn, purkinje cells of hippocampus, brain stem, cerebellum.
• Demonstration of antigen by direct immunofluorescence.
258 | Microbiology
• Antemortem: Salivary, corneal smears, skin biopsy from nape of the neck (most
sensitive site).
• Postmortem: Impression smears of cut surface of salivary glands, hippocampus,
brain stem, cerebellum.
• Detection of genomic RNA or viral mRNA: PCR, DNA probes.
• Virus isolation:
ƒ Mouse inoculation: Suckling mouse.
ƒ Cell culture: No CPE, detection by fluorescent antibody staining.
• Serology: In CSF and serum by CFT, ELISA.

Fig. 6.8: Negri bodies

Rabies vaccines:
Neural vaccines:
• Pasteur vaccine.
• Fermi vaccine.
• Semple vaccine.
• Beta-propiolactone vaccine.
• Suckling mouse brain vaccine.
Non-neural vaccine:
• Duck egg vaccine.
• Cell culture vaccine.
ƒ First generation:
f Human diploid cell vaccine (HDCV)
ƒ Second generation vaccine:
f Purified chick embryo cell (PCEC) vaccine.
f Purified vero cell rabies vaccine (PVRV).
Dose 1 2 3 4 5
Essen regimen (IM) 0 3 7 14 28
Modified Thai regimen (ID) (2 doses) 0 3 7 0 28
Virology | 259
Arboviruses: Togaviridae, Flaviviridae, Bunyaviridae,Togaviridae:
Spherical, 70nm diameter, positive sense, single stranded RNA, enveloped.
Two genera:
Alphavirus: Arthropod borne viruses.
Rubivirus: Rubella virus, not arthropod borne.
Human Diseases Caused by Alphaviruses
Virus Vertebrate Mosquito vector Disease
reservoir
Eastern equine encephalitis Wild birds Culiseta, Aedes, Encephalitis
Culex
Western equine encephalitis Wild birds Culex Encephalitis
Venezuelan equine Horses Aedes, Culex Encephalitis
encephalitis
Chikungunya Monkeys, humans Aedes Fever, rash, arthralgia, myalgia
Onyong-nyong Humans Anopheles Fever, rash, arthralgia, myalgia
Mayaro Monkeys Haemagogus Fever, rash, arthralgia, myalgia
Ross river Marsupials, rodents Aedes, culex Fever, rash, arthralgia, myalgia
Sindbis Wild birds Culex, Culiseta, Fever, rash, arthralgia, myalgia
Aedes

Laboratory diagnosis:
• Intracerebral inoculation of suckling mice.
• Isolation: Tissue culture (vero, C6/36 mosquito cell line), detection of growth by
immunofluorescence.
• Serology: ELISA, HAI, CFT, NT.
Flaviviridae:
• Spherical, 40-50nm, single-stranded, positive sense RNA.
• Two genera of medical importance:
ƒ Flavivirus: Arthropod borne viruses.
ƒ Hepacivirus: Hepatitis C virus, non-arthropod borne.
Human Diseases Caused by Flaviviruses
Syndrome Vector Virus
Encephalitis Mosquito Japanese encephalitis
Murray valley encephalitis
St. Louise encephalitis
West Nile virus
Tick Louping ill
Powassan
Russian spring summer encephalitis
260 | Microbiology

Yellow fever Mosquito Yellow fever

(Aedes aegypti)
Dengue Mosquito Dengue 1,2,3,4
(Aedes aegypti)
Haemorrhagic fever Mosquito Dengue 1,2,3,4
(Aedes aegypti)
Tick Omsk hemorrhagic fever
Kyasanur forest disease

Laboratory diagnosis:
• Intracerebral inoculation of suckling mice.
• Isolation: Tissue culture (vero, C6/36 mosquito cell line), detection of growth by
immunofluorescence.
• Serology: ELISA, HAI, CFT, NT.
Bunyaviridae:
• Spherical, 80-120nm diameter, triple segmented, negative sense or ambisense, single
stranded RNA, enveloped.
• Four important genera:
ƒ Bunyavirus: Predominantly transmitted by mosquitoes.
ƒ Nairovirus: Predominantly transmitted by ticks.
ƒ Phlebovirus: Predominantly transmitted by sandflies.
ƒ Hantavirus: Rodent borne, not transmitted by arthropods.
Human Diseases Caused by Members of the Family Bunyaviridae

Genus Virus Vector Human disease

Bunyavirus La Crosse Mosquito Encephalitis

Oropouche Mosquito Fever, arthralgia, myalgia

Phlebovirus Rift valley fever Mosquito Fever, myalgia, retinitis, hemor-

rhagic fever

Sandfly fever Sandfly Fever, myalgia, conjunctivitis

Nairovirus Crimean-Congo Tick Hemorrhagic fever

hemorrhagic fever

Hantavirus Hantaan - Hemorrhagic fever, nephropathy

Belgrade - Hemorrhagic fever, nephropathy

Seoul - Nephropathy

Puumala - Nephropathy
Virology | 261
Laboratory diagnosis:
• Intracerebral inoculation of suckling mice.
• Isolation: Tissue culture (vero, C6/36 mosquito cell line), detection of growth by
immunofluorescence.
• Serology: ELISA, HAI, CFT, Nt.
Retroviridae:
• Spherical, 80-100nm, cylindric core, single stranded RNA, linear, positive sense,
diploid, enveloped. Reverse transcriptase enzyme makes DNA copy from genomic
RNA.
Human Retroviruses
Subfamily Genus Virus Disease
Oncovirinae Retrovirus HTLV1 Adult T cell leukemia
HTLV2 Infection in intravenous drug users,
not associated with any disease
Lentivirinae Lentivirus HIV1 AIDS
HIV2 AIDS
Spumavirinae Spumavirus Human foamy virus Not associated with any disease

Human Immunodeficiency Viruses:

• Two antigenic types HIV 1 and HIV2.
Differences Between Hiv1 And Hiv2
HIV1 HIV2
Related to Chimpanzee simian immunodefi- Sooty mangabay simian
ciency virus immunodeficiency virus
Geographical distribution World wide West Africa
Virulence More virulent Less virulent
Envelope antigens
Spike antigen gp120 gp140
Transmembrane pedicle antigen gp 41 gp 36
Matrix protein antigen p17 p16
Capsid protein antigen p24 p26
Groups and subtypes Divided into three groups based 5 subtypes (A-F)
on evn gene sequences
M (major), N (new), O (outlier)
M comprises of 8 subtypes or
clades (A,B,C,D,F,H,J, K)
O comprises of 9 subtypes
India commonest: C
Western countries: B
262 | Microbiology
The genome of HIV contains:
• Three structural genes:
Gag (group-specific antigen):
• Core protein : p24.
• Matrix protein: p17.
Pol (polymerase):
• Reverse transcriptase: p51.
• Protease: p11.
• Integrase: p32.
Env (envelope):
• Outer envelope glycoprorein: gp120.
• Transmembrane envelope glycoprotein: gp41.
• Six non-structural genes:
ƒ Vif, vpr, vpu (vpx instead of vpu in HIV2), rev, tat and nef.

Modes of transmission:
Route Efficacy
Blood transfusion >90%
Perinatal 13-40%
Sexual intercourse:
Anal 1% per episode
Vaginal 0.1% per episode
Intravenous drug use 0.5-1%
Needle stick injury 0.3%

• In India and worldwide, commonest mode of transmission is heterosexual

route.
Pathogenesis:
• Receptor for HIV: CD4.
• Co-receptors: CXCR4, present on lymphocytes; CCR5, present on macrophages;
coreceptors are required for the fusion of viral envelope with cell membrane.
• AIDS is a unique sexually transmitted disease without local genital manifestations at
any time during the infection but with grave systemic manifestations.
• Infection of resident macrophages and submucosal lymphocytes in the genital tract or
rectum → virus transported to draining lymph nodes → replication → after 2-3 weeks
of viraemia → fall in CD4+ T cells, glandular like fever → within one month, viraemia
declines and illness subsides → followed by long asymptomatic period, average 10
years → low titres present in blood, but high level in lymph nodes → slow destruction
of CD4+ Tcells → when count falls below 200/µl, person becomes susceptible to
opportunistic infections and cancers → death due to opportunistic infections and
malignancy.
Virology | 263

Fig.6.9: Structure of HIV

OPPORTUNISTIC INFECTIONS and MALIGNANCIES

ASSOCIATED WITH HIV INFECTION
Bacterial:
M. tuberculosis (disseminated/ extrapulmonary)
M. avium complex
Salmonella (recurrent septicaemia)
Viral:
Cytomegalovirus.
Herpes simplex virus.
Varicella-zoster virus.
Epstein-Barr virus.
Human herpes virus 6.
Human herpes virus 8.
Fungal:
Candidiasis.
Cryptococcosis.
Aspergillosis.
Pneumocystis carinii pneumonia.
Parasitic:
Toxoplasmosis.
Cryptosporidiosis.
264 | Microbiology

Isosporiasis.
Microsporidiosis.
Generalized strongyloidiasis.
Malignancies:
Kaposi’s sarcoma.
B cell lymphoma.
Non-Hodgkin’s lymphoma.

Laboratory diagnosis (As per NACO):

Screening (Rapid) tests:
• Rapid tests: Time less than 30 minutes,.
• Do not require expensive equipment:
ƒ HIV spot and comb test.
Supplemental tests: (Not in NACO programme):
• Western blot assay.
• Immunofluorescence test.
Confirmatory tests:
• Virus isolation.
• Detecion of p24 antigen.
• Detection of viral nucleic acid: in situ hybridization, PCR.
Strategies for HIV testing:
• Strategy I: The serum is tested with one rapid test and if reactive, sample is
considered positive and if nonreactive it ia considered negative. Used for testing
blood for transfusion.
• Strategy II: The serum reactive with one rapid test is retested with a second rapid
test based on different antigen or different test principle. If found reactive on second
test it is reported as positive, otherwise negative. Used for HIV surveillance.
• Strategy III: The serum reactive with one rapid test is retested with another two
rapid tests. These tests should be of different antigen system of different principle.
A serum reactive in all three tests is considered as positive while serum sample
nonreactive in third rapid test is considered equivocal/ boderline. Such individuals
should be retested after three weeks. Used for diagnosis of HIV infection in an
individual case.
• CD4 count is not a criteria for initiation of ART now.

Postexposure Prophylaxis as per Naco Guidelines:

TLE Regimen:
• FDC containing Tenofovir (TDF) 300 mg plus Lamivudine (3TC) 300 mg plus Efavirenz
(EFV) 600 mg once daily for 28 days.
• It ideal to start the therapy within 2 hours and should not exceed 72 hours of exposure.
• Indicated to HCW exposed to Source positive for HIV, Any kind of exposure or Source
unknown.
Virology | 265
Concept 6.4: Important virusese
Learning Objectives
• Hepatitis
• Filoviridae
• Reoviridae
• Oncogenic viruses
• CoronaVirus

Time Needed
1 reading
st
90 mins
2nd look 30 mins

Hepatitis Viruses:
• Cause systemic disease primarily involving liver.
• Caused by:
ƒ Hepatitis A virus.
ƒ Hepatitis B virus.
ƒ Hepatitis C virus.
ƒ Hepatitis D virus.
ƒ Hepatitis E virus.
• Other viruses that can cause sporadic hepatitis are:
ƒ Yellow fever virus.
ƒ Cytomegalovirus.
ƒ Epstein-Barr virus.
ƒ Herpes simplex virus.
ƒ Enteroviruses.

Hepatitis A Virus:
• Member of picornaviridae.
• Previously classified as enterovirus 72.
• Now assigned to new genus Hepatovirus.
• 27nm, icoshedral, linear, single stranded RNA, linear, positive polarity, nonenveloped.
• Only one serotype exists.
• One of the most stable viruses infecting humans.
• Only one of the human hepatitis viruses that can be cultivated in vitro.
Pathogenesis:
• Virus is shed in the stools of infected persons.
• Infection is transmitted by faeco-oral route.
• First multiplies in the intestinal epithelial cells→ spreads to the liver via blood.
• Accounts for 25% of acute hepatitis cases world wide.
266 | Microbiology
Clinical features:
• Acute self limiting disease, I.P.: 2-6 weeks.
• Abrupt onset with fever, malaise, anorexia, nausea, lethargy followed by jaunduce
and hepatomegaly.
• Less than 0.5% cases develop fulminant hepatitis.
• Complete recovery in 8-12 weeks.
• Severity of the disease varies with the age.
• 5% children < 3 years develop jaundice.
• >50% of the adults develop jaundice.
• No extra hepatic manifestation, no carrier state.
• Not associated with cirrhosis/ heptaocellular carcinoma.
Laboratory diagnosis:
• Raised AST and ALT.
• Demonstration of virus particles: Immunoelectron microscopy.
• Antigen detection: ELISA.
• Serology: IgM antibodies, ELISA.
• Isolation: Tissue culture.
• Nucleic acid detection: PCR.
Prophylaxis: Killed vaccine available.

Hepatitis B Virus:
• Family: Hepadnaviridae.
• Consists of Hepatitis viruses of humans (HBV) and animals.
Morphology:
• Hepatitis B virus/ Dane particle: 42 nm, envelope contains hepatitis B suface antigen
(HBsAg). It encloses an inner icosahedral 27nm nucleocapsid. It contains Hepatitis
B core antigen. Inside the core is the genome of HBV and DNA-dependant DNA-
polymerase.
• HBV genome: 3.2 Kbp molecule of circular dsDNA. The plus strand is incomplete
leaving 15-50% of the molecule single stranded. The minus strand is complete and
contains four overlapping open reading frames.
P gene: Codes for DNA polymerase. It has three distinct enzymatic activities:
• DNA polymerase.
• Reverse transcriptase.
• RNaseH.
S gene: codes for protein which occurs in three forms
• Large protein: Translated from pre-S1, pre-S2 and S region; present in the infectious
virion.
• Middle sized protein: Translated from pre-S2 and S region.
• Small sized protein: Translated from S region (most commonly found). Basic
constituent of non-infectious HBsAg particles.
C gene: Has two initiation sites that divide it into a pre-C and C region producing two
distinct proteins HBe Ag (secreted from the virus) and HbcAg (not secreted) respectively.
Virology | 267

Fig.6.10: HBV

Morphological forms:
• Mature infectious virion/ Dane particle: 47nm diameter.
• Spherical particle: 22nm diameter, composed of HBsAg, non-infectious.
• Elongated tubules: 22nm diameter, length variable, composed of HBsAg, non-
infectious.
Antigenic structure:
• Group specific antigen ‘a’.
• Two type specific antigens: d or y/ w or r.
• Four antigenic types: adw, adr, ayw, ayr.
• Commonest subtype in India: ayw; Western countries: adw.
Cultivation:
• HBV has not been cultivated in the laboratory.
Pathogenesis:
Three main modes of transmission:
Parenteral:
• Accidental inoculation of body fluids during medical, surgical, dental procedures,
intravenous drug abusers, blood transfusion.
• Natal:
ƒ Prenatal: Transplacental.
ƒ Perinatal: Contamination of mucous membranes of the baby with maternal blood.
ƒ Post natal: Breast feeding.
ƒ 90% of the babies infected at birth become chronic carriers as compared to 10%
of those infected after the age of 6 years.
• Sexual.
Clinical features:
• Prodromal (preicteric phase); I.P.: 6 weeks to 6 months. Malaise, anorexia, nausea,
vomiting.
• Icteric phase: 2 days to 2 weeks after prodromal phase. Jaundice, pale stools, dark
urine.
• Convalescent phase: Malaise and fatigue lasting for several weeks.
268 | Microbiology
Outcome of infection:
• <1% of icteric cases develop fulminant hepatitis.
• 90-95% recover without sequelae.
• 5-10% develop chronic hepatitis which can lead to development of cirrhosis and
hepaocellular carcinoma.
Carriers:
• Persistence of HBsAg in the serum for more than six months.
Chronic Active Hepatitis Chronic Persitent Hepatitis
Replicative phase Non-replicatove phase
HbeAg+ HbeAg-
Anti-HbeAg- AntiHBeAg-
DNA exists in the free form in the hepatocyte DNA integrated into host genome
Intact virions present in the circulation No intact virions present in the circulation
More infectious Less infectious

Laboratory diagnosis:
• Elevated AST, ALT.
• Raised serum bilirubin.
Serological Markers of Hepatitis B Infection
HBsAg Anti- HBeAg Anti- Anti-HBcAg Interpretation
HBsAg HbeAg IgM IgG
+ - + - - - Incubation period
+ - + - + - Acute hepatitis
+ - + - - + Chronic active hepatitis
+ - - + - + Chronic persistent hepatitis
- + - - - + Past infection
- + - - - - Vaccination
• Epidemiological marker of HBV infection: IgG-Anti-HbcAg.
• Surrogate marker of HBV replication: HbeAg.
• Marker for immunity: Anti-HBs.
• First Serological Marker to be positive in Acute HBV: HBsAg.
Prophylaxis:
• Passive immunization: HBIG.
• Active immunization:
ƒ Plasma derived hepatitis B vaccine.
ƒ Recombinant yeast derived hepatitis B vaccine.
ƒ Recombinant Chinese hamster ovary cell hepatitis vaccine.
Virology | 269
Hepatitis C Virus:
• Belongs to family Flaviviridae.
• Placed in separate genus: Hepacivirus.
• 30-60nm, spherical, single standed RNA, positive sense, positive sense, enveloped.
• Classified into 11 genotypes based on heterogeneity of nucleotide sequence.
Pathogenesis:
• Three main modes of transmission.
• Parenteral.
ƒ Accidental inoculation of body fluids during medical, surgical, dental procedures,
intravenous drug abusers, blood transfusion
• Natal:
ƒ Prenatal: Transplacental.
ƒ Perinatal: Contamination of mucous membranes of the baby with maternal blood.
ƒ Post natal: Breast feeding.
• Sexual.
Clinical features:
• Incubation period: 6-8 weeks.
• About 75% infections sub-clinical.
• 50% patients develop chronic infection.
• Acute infection as compared to HBV infection is less severe, shorter duration of
prodromal phase, milder symptoms. >85% cases of acute infection develop chronic
disease.
• Fulminant infection: 0.1%.
• Patients with chronic disease may later on develop cirrhosis and hepatocellular
carcinoma.
Prophylaxis: No vaccine available currently.
Hepatitis D Virus:
• Defective virus requiring help from HBV (HBV necessary for the production of HDV
virions).
• Spherical, 36-38nm, HBsAg coat, HDAg nucleocapsid, single, circular RNA, minus
strand.
• Genus: Delta virus.
Pathogenesis:
• Parenteral.
• Perinatal.
• Sexual.
Two types of infection:
• Coinfection: Simultaneous infection with HBV, HDV. Most commonly results from par-
enteral transmission. Infection more severe than HBV alone. IgM-Anti-HBcAg+.
• Super infection: Infection of HBV carrier with HDV. Commoner and more serious than
coinfection (as liver function already compromised by HBV infection). Develops into
fulminant infection. IgG-Anti-HBcAg+.
270 | Microbiology
Laboratory diagnosis:
• HDAg detection: ELISA.
• HDV RNA detection: PCR.
• IgM Anti-HDV detection: ELISA.
Prophylaxis:
• Prevention of infection with HBV.

Hepatitis E Virus:
Belongs to family Caliciviridae:
• Spherical, 27-38nm, single stranded, positive sense RNA.
• Pathogenesis:
Ingestion of contaminated drinking water.
• Virus first identified in New Delhi, India in 1955.
Clinical features:
• I.P.: 2-8 weeks.
• Disease resembles that produced by Hepatitis A virus.
• Fulminant infection: 1-2% in general population; 10-20% in pregnant women.
• Does not progress to chronic infection, cirrhosis or hepatocellular carcinoma.
Laboratory diagnosis:
• Demonstration of virus particles: Immunoelectron microscopy.
• Antigen detection: ELISA.
• Serology: IgM antibodies, ELISA.
• Nucleic acid detection: PCR.
Filoviridae:

Fig.6.11: Ebola virus

• Long filamentous virus 80nm x 1000nm, enveloped, single stranded, negative sense
RNA.
• Marburg virus.
• Ebola virus.
Virology | 271
Pathogenesis:
• Highly virulent infection, invariably fatal.
• Transmitted to man from primates by aerosols, direct contact with blood, body fluids
etc.
Clinical features:
• I.P.: 3-15 days, patient develops headache, fever, myalgia, severe haemorrhages,
hypotension.
Laboratory diagnosis:
• Demonstration of virus: Electron microscopy.
• Isolation.
• Serology: Immunofluorescence.
Arenaviridae:
• Sandy appearance on electron microscopy (Arena means sand), due to the incorpo-
ration of the host cell ribosomes during assembly.
• 110-130nm, pleomorphic, single strand RNA, negative sense, two segments,
enveloped.
• Natural hosts are the rodents in which they cause persistent infection with viraemia.
Virus is shed in the urine.
Diseases Cause by Viruses in the Family Arenaviridae
Virus Geographical distribution Human disease
Lymphocytic choriomeningitis Worldwide Influenza-like illness, aseptic
meningitis, rarely encephalomyelitis
Lassa West Africa Hemorrhagic fever
Junin Argentina Argentine hemorrhagic fever
Machupo Bolivia Bolivian hemorrhagic fever
Guanarito Venezuela Venezuelan haemorrhagic fever

Reoviridae:
• Spherical virions, nonenveloped, segmented (10-12 segments), ds DNA.
• Medically important genera:
ƒ Orthoreovirus: 10 segments.
ƒ Orbivirus: 10 segments.
ƒ Rotavirus: 11 segments.
ƒ Coltivirus: 12 segments.
Coltivirus:
• 2 serotypes.
• Causes Colorado tick fever.
• Transmitted by ticks.
• I.P.: 3-6 days; myalgia, headache, leucopenia, rash.
• Complications: Meningoencephalitis, hemorrhagic fever.
272 | Microbiology
Orbivirus:
• 100 serotypes.
• Infections in animals; mild fevers in humans.
Orthreovirus:
• 3 serotypes.
• May be associated with enteritis in infants and children; upper respiratory tract
infections, neonatal biliary atresia.
Rotavirus:

Fig.6.12: Rotavirus

• Divided into six groups (A-F) and 14 serotypes (9 human, 5 animal).

• Commonest cause of gastroenteritis in children below 2 years of age.
• Infection is transmitted by contaminated water and food.
• I.P.: 1-2days, vomiting, diarrhoea, dehydration.
• Laboratory diagnosis:
ƒ Antigen detection: Latex agglutination, passive hemagglutination, ELISA.
ƒ Detection of virus: Electron microscopy, immunoelectron microscopy.
• Vaccination: Two live attenuated vaccines available, given orally.
Viruses Associated with Acute Gasteroenteritis in Hiumans
Rotavirus
Group A Single most important cause (viral or bacterial) of endemic
severe diarrheal illness in infants and young children
worldwide.
Group B Outbreaks of diarrheal illness in adults and children in China.
Outbreaks of diarrheal illness in children.
Group C
Enteric adenoviruses Second most important viral agent of endemic diarrheal
illness in infants and young children worldwide.
Noroviruses Most common cause of epidemics of viral diarrhea in adults.
27nm, single stranded RNA, positive sense,
non enveloped
Astroviruses Sporadic cases and outbreaks of diarrheal illness in infants,
30nm, nonenvelped, RNA viruses young children and adults.
Virology | 273
Oncogenic Viruses:
Oncogenic Viruses Associated with Human Neoplasia
DNA viruses:
Papovaviridae Human papilloma virus Genital tumors, sqamous cell
carcinoma

Herpesviridae Epstein-Barr virus Nasopharyngeal carcinoma

Burkitt’s lymphoma
B cell lymphoma
Hodgkin’s disease

Herpes Simplex Virus 1 and 2 ? cervical cancer

Cytomegalovirus ? cervical cancer

Human Herpes Virus 8 Kaposi’s sarcoma

Hepadnaviridae HBV Hepatocellular carcinoma

Poxviridae Molluscipoxvirus Molluscum contagiosum

RNA viruses:
Retroviridae HTLV1 Adult T cell leukemia

Flaviviridae HCV Hepatocellular carcinoma

Coronavirus:
• RNA virus.
• ssRNA, Positive sense, Enveloped.
• Types of Corona viruses
ƒ Human coronavirus 229E (HCoV-229E)
ƒ Human coronavirus OC43 (HCoV-OC43)
ƒ SARS-CoV
ƒ Human coronavirus NL63 (HCoV-NL63)
ƒ Human coronavirus HKU1
ƒ Middle East respiratory syndrome coronavirus (MERS-CoV)
ƒ SARS CoV 2

Fig.6.13: Corona virus

274 | Microbiology
COVID 19
• IP – 3 – 13 days
• Transmission by droplets, Contaminated surfaces.

Fig.6.14: Covid 19 Clinical Course

• Associated with SARS (Severe Acute Respiratory Syndrome).

Fig.6.15: Covid 19 Symptoms

Virology | 275
Lab diagnosis
Viral culture
RT PCR
• S gene, N gene , E gene
• Antigen detection
• Antibody detection (not useful for acute clinical management)
Vaccines
• Killed / Inactivated vaccine (Covaxin)
• Vector based recombinant vaccine (Covishield/ Oxford)
• mRNA vaccine (Moderna)

Prions:
• Proteinaceous infectious particles.
• Highly resistant to proteases, heat, radiation.
• More difficult to inactivate as compared to bacterial spores.
• Can be inactivated by NaOH, Sodium hypochlorite, stronger method of autoclaving
at 134°C for 18 mins.
Diagnosis: Biopsy of brain tissue: spongiform encephalopathy.
Diseases of humans:
• Kuru.
• Creutzfeldt-Jacob disease.
• Gerstmann-Straussler-Scheinker syndrome.
• Familial fatal insomnia.
Diseases of animals:
• Scrapie of sheep and goats.
• Bovine spongiform encephalopathy of cattle.
• Mink and feline encephalopathy.
• Wasting disease of deer.
276 | Microbiology

Worksheet
• MCQ OF “VIROLOGY” FROM DQB

• EXTRA POINTS FROM DQB

1.

2.

3.

4.

5.

6.

7.

8.

9.

10
Virology | 277
Active Recall from Tables
Name of the inclusion bodies Examplse

Segmented RNA Viruses

Adenoviral diseases Serotypes

278 | Microbiology

Hepatitis B - stages Serological markers

Oncogenic viruses Associated tumours

7 Mycology

CONCEPTS
 Concept 7.1 Classification
 Concept 7.2 Superficial & Cutaneous mycoses
 Concept 7.3 Sub cutaneous mycoses
 Concept 7.4 Systemic Mycoses
 Concept 7.5 Opportunistic Mycoses
280 | Microbiology
Concept 7.1: Classification of Fungus
Learning Objectives
• Morphology of Fungus
• Different classification systems
• Sexual & Asexual spores
• Anti-fungal drugs

Time Needed
1st reading 30 mins
2 look
nd
10 mins

Introduction:
• Father of mycology – Raymond Jacques Adrien Sabouraud.

Characteristics of Fungi:
• Eukaryotic unicellular or multicellular organisms.
• Exists as saprophytes, commensals or parasites (MC saprophytes).
• Either pathogenic or non pathogenic.
• Cannot photosynthesize due to lack chlorophyll (heterotrophic).
• They absorb food (osmotrophic).
• Woronin bodies: Prevents loss of cytoplasm after hyphal injury in molds (not seen
in true yeasts).

Difference between Fungi and Bacteria:

Properties Fungi Bacteria
1. Cell structure Eukaryote. Prokaryote.
2. Cell Wall Composition Multilayered; Chitin, Mannan Peptidoglycan, LPS, Teichoic
and glucans. acid.
3. Cytoplasmic Membrane Sterols present except P.carinii. Sterols absent except
Mycoplasma.
4. Cytoplasmic Contents Membrane bound organelles like Membrane bound organelles
Mitochondria, golgi apparatus, absent.
ER present.
5. Nucleus True with nuclear membrane; No nucleus or nuclear membrane
paired chromosome. Single circular dsDNA
chromosome.
6. Diameter 5 – 10 µ; may be > 100µ < 2µ
7. Morphology Yeast: ovoid, round Cocci, bacilli, Some branching
Moulds: Filamentous
8. Spores Both asexual and sexual Asexual (Endospores)
Function-reproduction. Function-survival
Mycology | 281

9. Stain H+E, PAS, Meth. Ag Gm+ (few) Gram, ZN, Albert

Calcoflour white
10. Mode of reproduction Sexual / Asexual (Spores) Binary fission

Fungal Morphology:
1. Yeast.
Unicellular and reproduce by asexual process known as budding.
Exception:
i. The dimorphic fungi Penicillium marneffei reproduce asexually by binary fission
i.e. transverse septum formation.
ii. Cryptococcus neoformans can also reproduce by sexual process also showing
atelomorph state i.e. Filobasidiella neoformans.
Yeast may produce chains of elongated cells known as pseudohyphae.
Pseudohyphae True hyphae
Mechanism of formation Failure of separation of daughter cells during Apical elongation
budding process
Origin Constriction No constriction
Width More than half of parent yeast Less than half of parent
yeast
2. Mold
These are composed of branching hyphae growing by apical elongation after
germinating from spores.

Hyphae

Septum
Pigment

Septate Aseptate
Dematiaceous
Hyaline

Mycelium – Bundle of Hyphae

Aerial Vegetative

Fig.7.1: Fungal Hyphae

Fungal stains:
• Best stain to for demonstration of fungal cell wall is Methanamine silver.
• Lacto phenol cotton blue (LPCB) is used for culture identification and it is not for direct
specimen.
282 | Microbiology
• Nigrosin, India ink and Mucicarmine are for Cryptococcus.
• PAS is a used for demonstration of fungi in tissue sections.

Classification:

Systemic
Clinical
Morphological

Fig.7.2: Fungal Classification

Systemic:
On the basis of formation of sexual spores (Teleomorph) divided into 4 classes.
Sexual Spore Description Example
Zygomycetes Zygospores Non septate hyphae. Mucor, Rhizopus
Asexual spores are called Sporangiospores.
Ascomycetes Ascospores Ascospores are formed in a sac called Penicillium, Aspergillus,
ascus. Piedra hortae
Basidiomycetes Basidiospores Basidiospores are formed externally on Filobasidiella,
the tip of a pedestal called a basidium. Trichosporon species
Deuteromycetes/ - Fungi that lack a known sexual state. Coccidioides.
Fung imperfecti

Asexual Spores:
• Conidia (asexual spores) types:
ƒ Arthrospores - Arise by fragmentation of the ends of hyphae Eg. C.immitis.
ƒ Chlamydospores – Rounded, thick-walled Eg. spores of C. albicans.
ƒ Sporangiospores – Formed within a sac (sporangium) on a stalk by molds. Eg.
Rhizopus and Mucor.
ƒ Conidiospores - Conidia arise directly from conidiophore Eg. Aspergillus.
ƒ Blastospores – Formed by the budding process e.g. C. albicans.
Morphological classification:
• Yeasts:
ƒ Oval, round elongated unicellular fungi.
ƒ Reproduce by asexual process called budding.
ƒ E.g. Saccharomyces – non pathogenic yeast, Cryptococcus neoformans –
pathogenic yeast.
• Yeast like:
ƒ Yeast with Pseudohyphae formation. e.g. Candida.
• Molds:
ƒ Spores germinate to produce branching filaments called hyphae which forms
tangled mass of growth called mycelium.
Mycology | 283
ƒ May be septate or nonseptate (coenocytic).
ƒ E.g. Penicillium, Mucor, Rhizopus etc.
• Dimorphic fungi: Have yeast form in host at 37°C and hyphae (mycelial) form in vitro
(25°C) e.g.
ƒ Histoplasma capsulatum.
ƒ Coccidioides immitis.
ƒ Paracoccidioides brasiliensis.
ƒ Blastomyces dermatitidis.
ƒ Sporothrix schenkii.
ƒ Penicillium marneffei.
Clinical Classification:
• Superficial mycosis.
• Cutaneous Mycosis.
• Subcutaneous Mycosis.
• Systemic Mycosis.
• Opportunistic Mycosis.

Fig. 7.3: Asexual Fungal Spores

284 | Microbiology
Antifungal agents:
Group of compound Antifungal Agents Mechanism of action
Polyenes Amphotericin-B, Nystatin Bind to ergosterol
Azole derivatives Miconazole, Ketoconazole, Fluco- Inhibit cytochrome P-450 dependent
nazole, Itraconazole enzymes
Nucleoside analogues 5-flouro-cytosine Inhibits DNA and RNA synthesis
Grisans Griseofulvin Inhibits microtubular function
Allylamines Naftifine , Terbinafine Squalene epoxidase inhibitors
Thiocarbamates Tolnaftate , Toliciclate Squalene epoxidase inhibitors
Morpholines Amorolfine Inhibits ergosterol biosynthesis
Echinocandins Caspofungin, Anidulafungin, Mi- Cell wall inhibitor (inhibits cross linking of
cafungin b 1, 3 glucans)
Nikkomycin Nikkomycin X and Z Inhibits chitin

Fig.7.4: MoA of Antifungal Drugs

Antifungal susceptibility testing:

As per Clinical Laboratory Standard Institute (CLSI) guidelines:
1. Broth dilution method for yeast (M27-A2) and molds (M38-A).
2. Disk diffusion method (M44-A).
Other methods:
1. Etest. 2. Fungitest.
3. Spectrophotometric methods. 4. Flowcytometry.
Mycology | 285

Worksheet
• MCQ OF “MYCOLOGY” FROM DQB

• EXTRA POINTS FROM DQB

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10
286 | Microbiology
Active Recall from Tables
Antifungal agents Mode of Action

Sexual spores Asexual spores

Mycology | 287
Concept 7.2: Superficial & Cutaneous Mycosis
Learning Objectives:
• Tinea versicolor
• Dermatophytes

Time Needed
1 reading
st
45 mins
2 look
nd
15 mins

Superficial Mycosis:
• Limited to outermost layers of skin and its appendages. Immune response is rarely
induced

1 Pityriasis versicolor:
• Synonyms- Tinea versicolor, Tinea Flava, Dermatomycosis, Liver Spots,
Furfuracea.
• Superficial infection of horny layer(stratum corneum) of skin.
• Causative agent- Malassezia furfur (earlier Pityrosporum orbiculare/ovale) a lipophilic
yeast is normal skin flora (endogenous infection).
• Sites – Any part of body but MC neck, upper trunk, face and upper arms.
Clinical Presentation:
• Hypopigmented, hyperpigmented, leukodermal, erythematous or dark brown lesions
(versi means several).
• Sharp margins.
• Covered with scales.
• Non inflammatory.
• No itching.
• Cosmetic problem.
• Risk factor for invasive malassezia-TPN therapy.
Diagnosis:
• Wood’s lamp-golden yellow fluorescence.
• KOH wet mount-spaghetti and meat ball or banana and grapes.

Fig.7.5: M.furfur – Spaghetti – Meat ball appearence

288 | Microbiology
• Culture:
ƒ Sabourads dextrose agar overlaid by olive oil/oleic acid/glycerol.
ƒ Dixon’s agar.
ƒ GYP-S agar.
Treatment: Thorough scrubbing, Topical sodium thiosulphate, selenium sulphide
shampoo.
Severe cases – Topical / oral ketoconazole or Itraconoazole.

2. Tinea Nigra:
• Localized to stratum corneum.
• Palms and sole.
• Eitiological agent- Cladosporium werneckii or Exophilla werneckii (Phaeoid/
Dematiaceous/Pigmented fungi).
Clinical presentation:
• Brown to black pigmented macular patches.
• Non scaling.
• Sharply marginated.
• Usually asymptomatic.
• No inflammation.
Diagnosis:
Microscopy - 20% KOH
Culture-SDA with actidione at 25-30°C “drop of oil” appearance

3. Piedra:
Black piedra:
• Caused by ascomyceteous fungus – Piedraia hortae.
• Involves mainly scalp.
• Hard (discrete gritty) brown-black nodule in distal hair shaft (no alopecia).
• Itching absent.
• M/E – Round to Oval asci containing curved fusiform aseptate ascopores (sexual
sopres) that bear whip like appendages at both ends.
White piedra:
• Caused by Trichosporon beigelii.
• Involves axillary hair, beard, moustache and pubic hair (scalp less commonly
involved).
• Soft, greyish-white nodules in distal hair shaft (no alopecia).
• Itching present.
• Wood’s lamp-No fluorescence.
• M/E - arthroconidia are seen (Rectangular, elongated asexual spores).
• Culture-SDA without actidione at 37°C.
Mycology | 289
4. Dermatophytosis (Tinea or ring worm):
• Most common type of superficial mycosis seen in human beings.
• Caused by keratinophilic fungi known as dermatophytes.
• Infects keratinized tissue of skin, hair and nail (Dermatomycosis – skin infection
caused by any fungi like Candida, Fusarium etc).

Dermatophytosis caused by:

Genus Site Macroconidia Microconidia
Trichophyton Skin, hair and Pencil shaped, elongated, thin walled smooth. Plenty
nails Rarely seen.
Microsporum Skin, hair Spindle shaped with tapering ends, thick, rough Rarely seen
walled.
Plenty in number.
Epidermophyton Skin, nail Club shaped (clavate), smooth walled in clusters Not seen
Plenty in number.

Culture grows at 25° C

Geophilic Saprophytes in soil (e.g. Microsporum gypseum, T.ajelloi, M.nanum)
Zoophilic Primarily animal pathogen (e.g. T. verucosum, M. canis)
Anthropophilic Highly infectious, affecting only human and are transmitted indirectly via fallen hairs
etc. (e.g. T. rubrum, M. audouinii, E. floccosum)

Fig.7.6: Epidermophyton floccosum – Macroconidia

Dermatophytid or ‘id’ reaction:

• Hypersensitivity to fungus antigens may lead to secondary eruption because of
circulation of allergenic products in sensitized patients or following commencement
of oral antifungal therapy.
290 | Microbiology
• Two types:
1. Lichen scrofulosorum-like.
2. Pompholyx-like (type III HTN).
Clinical Manifestations:
• Tinea capitis:
ƒ Endothrix – arthrospore formations occurs within the hair completely filling hair
shaft (Adamson’s fringe) caused by.
▫ T.tonsurans.
▫ T.violaceum.
▫ T.scrofulacium.

Fig.7.7: Endothrix

ƒ Ectothrix – arthrospore on the surface of hair shaft caused by:

▫ M.audounii.
▫ M.canis.
▫ T.mentagrophytes.

Fig.7.8: Ectothrix
Mycology | 291
ƒ Kerion – Painful inflammatory reaction producing boggy lesions on scalp:
▫ T.verrucosum.
▫ T.mentagrophytes.
ƒ Favus- air spaces within the hair shafts – cup like crust (scutula) forms around the
infected hair follicle minimal hair shaft involvement:
▫ T.schoenleinii.
ƒ Black dot – attack hair shaft by endothrix type invasion with abundant sporulation:
▫ T.tonsurans.
• Tinea Corporis: MC type of dermatophytosis in India caused by T rubrum:
▫ Majocchi’s granuloma.
• Tinea Imbricatacaused by T. concentricum.
• Tinea Gladiatorum in wrestlers caused by T tonsurans.
• Tinea Incognito – Clinical appearance modified due to application of topical
corticosteroids.
• Tinea Barbae – Barber’s itch.
• Tinea Cruris – Jock itch.
• Tinea Manuum – Hyperkeratosis of palms and fingers.
• Tinea Pedis – Athlete’s foot/Moccasin or Sandal ringworm.
• Tinea Unguium– Affects nail.

Fluorescence under Wood’s lamp - positive (UV light with peak of 365nm):
Bright green M. audouinii, M. canis
Blue green M. ferrugineum, M. distortum
Dull yellow M. gypseum
Dull green T. schoenleinii
Golden yellow Malassezia furfur
Coral red Corynebacterium minutissimum

Test to differentiate T. mentagrophytes from T. rubrum:

a. T mentagrophytes urease +ve.
b. T rubrum produce deep red pigment on cornmeal agar.
c. In vitro hair perforation test positive with T. mentagrophytes.
d. Spiral and rat tail hyphae seen in T. mentagrophytes.
e. Microconidia grape like in T.mentagrophytes and tear drop in T.rubrum.
292 | Microbiology

Worksheet
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• EXTRA POINTS FROM DQB

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5.

6.

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10
Mycology | 293
Active Recall from Tables
Dermatophyte Microconidia Macroconidia
294 | Microbiology
Concept 7.3: Subcutaneous Mycoses
Learning Objectives
• Mycetoma
• Sporotrichosis
• Chromoblastomycosis

Time Needed
1 reading
st
30 mins
2 look
nd
10 mins

Subcutaneous Mycoses:
1. Mycetoma – Slowly progressive,
chronic localized granulomatous
infection of skin, subcutaneous
tissues, fascia and bones, usually
post traumatic.
Mostly it affects foot and hand. It
is defined by a triad.
i. Subcutaneous swelling.
ii. Draining sinus tract.
iii. Grains / Granules.

Fig.7.9: Mycetoma
Types:
Eumycetoma Actinomycetoma(MC) Botryomycosis
(True fungal mycetoma) (Bacterial mycetoma)

Black grain: Actinomadura madurae Staphylococcus aureus

Madurella mycetomatis A pellitieri Pseudomonas aeruginosa
M. grisea Nocardia asteroides Coagulase negative Staphylococcus
Exophiala N. brasiliensis E. coli
Curvularia Streptomyces Proteus species
Streptococcus spp.
White grain:
Pseudallescheria boydii
Aspergillus nidulans
Acremonium falciforme
Fusarium
Mycology | 295

Clinical features Actinomycetoma Eumycetoma

Causative agent Bacteria. Fungi

Tumor mass Multiple, diffuse with ill-defined margins. Usually single with well-defined margins.

Sinuses Appear early and more in no. Appear late and less in no.

Opening of sinuses Raised, inflamed and flared up. Flat opening and not flared up.

Flap of opening Easily removed. Not easily removed.

Discharge Usually purulent. Serous or sero-sanguineous.

Grains White, very thin filaments (0.5µm - Black or white.

1µm in diameter).

Extent of involvement More extensive with punched out Less extensive with only osteosclerotic
osteolytic lesions. lesions.

ƒ Actinomycetomas may respond to Penicillin and sulphonamides and other

antibiotics.
ƒ Eumycetomas – are resistant and require amputation.
2. Sporotrichosis
ƒ Causative agent in Sporothrix
schenkii – Dimorphic fungus.
ƒ Rose gardener’s disease.
ƒ Chronic pyogenic granulomatous
infection of skin and subcutaneous
tissues may become disseminated
by lymphatic spread (series of
ulcers).
ƒ Risk factor-thorn prick in gardeners.
ƒ Epidemiology – Central and S.
America (Mexico, Brazil), Parts of
USA, S. Africa.
ƒ In India – Sub Himalayan areas
ranging from H. Pradesh to Assam.

Fig.7.10: Sporotrichosis
296 | Microbiology
Diagnosis:

Fig.7.11: Sporothrix schenkii – flower pot apperaence

• Cigar shaped yeast like cells (PAS or methanamine silver stain).

• Splendore Hoeplii phenomenon due to immune complex deposition.
• Asteroid phenomenon positive in HandE stain.
• Culture – mycelial phase at 25°C on S.D.A (thin hyphae with flower like conidia)
and yeast phase on media such as BHI or PINE’S media at 37°C and by animal
inoculation.

Fig.7.12: Sporotrichosis - Asteroid bodies

Treatment:
• Saturated solution of potassium iodide for cutaneous form. (obsolete now).
• IV Amphotericin- B for lymphcutaneousleisions.
• Can also use KNZ / Itraconazole orally.
Mycology | 297
3. Chromoblastomycosis:
ƒ Chronic localized infection of skin and subcutaneous tissue, most often involving
limb with brown walled, globose bodies 5-13µm in size, called sclerotic bodies
or muriform cells/ Medlar body copper-penny cells.
ƒ These bodies divide by separation along interface of double septa.
ƒ Stain with H and E. In PAS they stain dark red.
ƒ Lesion – Verrucous, crusted and raised, initially solitary but slow localized spread
occurs.

Fig.7.13: Copper penny bodies – Chromoblastomycosis

Causative Agents are phaeoid or dematiceous fungi (pigmented):

• Fonsecacea.
• Phialophora.
• Cladosporium.
• Rhinocladiella.
Treatment – Solitary lesion of short duration – surgical excision.
Medical treatment - Flucytosine with Itraconazole.
4. Phaeohyphomycosis:
ƒ Nonspecific solitary subcutaneous lesion.
ƒ Opportunistic deep seated fungus infections (brain abscess) lung infection.
ƒ Caused by dark pigmented fungus.
ƒ Diagnosis - Made at the time of surgery.
ƒ Microscopy.
▫ Distorted hyphal strands seen (pigmented).
▫ Sclerotic cells not seen (unlike chromoblastomycosis).
e.g. Acrophialophora, Alternaria, Athrinium, Cladophialophora, Cephaliophora, Cladorrhinum
etc.
298 | Microbiology

Worksheet
• MCQ OF “MYCOLOGY” FROM DQB

• EXTRA POINTS FROM DQB

1.

2.

3.

4.

5.

6.

7.

8.

9.

10
Mycology | 299
Active Recall from Tables
Actinomycotic mycetoma Eumycotic mycetoma
300 | Microbiology
Concept 7.4: Systemic Mycoses
Learning Objectives
• Histoplasmosis
• Blastomycosis
• Coccidioidomycosis
• Paracoccidioidomycosis

Time Needed
1st reading 45 mins
2 look
nd
20 mins

Systemic Mycosis:
All dimorphic – 25°C –mycelial and 37°C–yeast form.

1. Histoplasma capsulatum:
• Histoplasmosis / Darlings disease / Ohio Valley disease.
• Intracellular mycoses of Reticuloendothelial system.
• Histoplasma capsulatum var capsulatum (Not capsulated).
• Reported from India, Common in central + S.E. USA
ƒ African Histoplasmosis: Caused by Histoplasma capsulatum var doboisii. Not
reported from India.
ƒ Common endemic mycosis in pts with AIDS.
ƒ Environmental isolations - made from soil enriched with excreta from chicken,
starlings and bats.
ƒ No man to man / animal to man transmission.

Fig.7.14: Histoplasma capsulatum

Clinical features:
• Pulmonary – Acute, Chronic (Histoplasmoma).
• Cutaneous, Subcutaneous, Mucocutaneous.
• Disseminated.
Mycology | 301
• Primary lung infection - 95% of cases of histoplasmosis are inapparent, subclinical
or benign.
• 7% cases are mucocutaneous – MC form in INDIA.
• Reactivation - Disseminated infection, with involvement of the RES.
• Fever, weight loss, hepatosplenomegaly and lymphadenopathy are the common
clinical features.
• All stages of this disease may mimic tuberculosis.
• Chest X Ray shows calcification.
ƒ In tissues H. capsulatum is present inside phagocytic cells in yeast phase. Oval
yeast like cell 2 – 4 µm in diameter. They fill the cytoplasm of macrophages,
monocytes and PMN leucocytes.
ƒ SDA – 25 - 300C forms septate branching hyphae with conidia.
• Diagnostic form: Large tuberculate macroconidia.
• Diagnosis may be made by microscopic examination of stained smears of sputum,
bone marrow, blood and scrapings.
• Ag detection in serum and urine- helpful in early diagnosis (false positives may occur).
• Immunodiffusion for the detection of antibody is useful in the diagnosis however,
detection of ANTIBODIES in immunosuppressed patients is difficult, with 20-50% of
patients testing negative.
• Skin test – Histoplasmin skin test.
• Treatment – Amphotericin B.

2. Blastomyces dermatitidis:
• Blastomycosis, North American blastomycosis, Gilchrist disease.
• 2- 3 cases have been reported from India.
• Suppurative and granulomatus cutaneous lesions.
• Commonly causes self limited or localized pulmonary lesions.
• Chronic disseminated disease in immunocompromised – lungs, Other tissues ( skin
andbone).
• Chronic infection of lungs which may spread to other tissues particularly to skin, bone
and genitourinary tract. (Slowly progressive).
• Infection occurs by inhalation of conidia growing as saprophytes in the soil.

Fig.7.15: Figure of 8 – Blastomycosis

302 | Microbiology
Laboratory Diagnosis:
• Samples -Sputum, BAL, Lung Biopsy, Skin Biopsy.
• Direct microscopy -KOH / calcofluor white, PAS digest, Grocott’s methenamine silver
(GMS) or Gram stain.
• Tissue: Thick walled yeast cells producing buds on a broad base (figure of
eight).
• Culture: Culture is confirmed by conversion of yeast form to Mycelial form at 37°C on
Brain heart infusion agar/blood glucose cysteine agar/Kelley’s agar.
• Fungus is also seen surrounded by a refractile, eosinophilic halo called
splendore – Hoeppli or asteroid body due to immune complex deposition
around organism.
• For skin test → Ag derived from mycelial phase.
• For serology → Ag derived from yeast phase.
• Treatment – Itraconazole.

3. Coccidioides immitis:
• Coccidiomycosis, San Joaquin Valley fever, Desert rheumatism, Posada’s disease.
• MC deep mycosis in USA.
• Primarily an infection of lungs caused by Coccidioides immitis.
• Endemic in South, North and Central USA and Mexico. High endemicity in California,
Arizona.
• Not reported from India.
• Present in soil in form of arthroconidia. Which when inhaled by man cause
infection.
• Mainly self-limiting. In rare cases, progressive disease develops. Is not transmitted
from man to man.

Fig.7.16: Arthroconidia – Coccidioidomycosis

Laboratory Diagnosis:
• Tissue phase: At 37°C Spherule with a thick doubly refractile wall filled with
endospores.
• Mycelial phase: In soil and in culture (at 25°C) grows as mould with barrel shaped
arthroconidia. No sexual stage.
• Treatment: Itraconazole.
Mycology | 303
4. Paracoccidiomycosis (dimorphic) (Lutz mycosis):
• Chronic granulomatus infection of lungs, mucosa, skin and lymphatic system.
• Formerly known as South American blastomycosis (Central America to Argentina).
• Caused by: Paracoccidioides brasiliensis.
• Mode of infection: Inhalation.
• At 37°C → Multipolar budding (mickey mouse appearance):
ƒ Characterized by primary pulmonary infection that may spread by hematogenous
routes systemically.
ƒ Microscopical examination of tissues, sputum, biopsies show numerous yeast+
cells with multiple bud.

Fig.7.17: Captain Wheel – Paracoccidioidomycosis

C/F- Mucocut, Lymphatic (Bull neck). Visceral (pul).

Treatment – KNZ (DOC) But Itraconazole preferred of no. interference with liver and
Bone marrow function.
Sulfonamides are also effective.
304 | Microbiology

Worksheet
• MCQ OF “MYCOLOGY” FROM DQB

• EXTRA POINTS FROM DQB

1.

2.

3.

4.

5.

6.

7.

8.

9.

10
Mycology | 305
Active Recall from Tables
Name of the fungus Yeast form Mold form
306 | Microbiology
Concept 7.5: Opportunistic Mycoses
Learning Objectives
• Candidiasis
• Cryptococcsis
• Aspergillosis
• Zygomycosis
• Pneumocystosis
• Penicillosis

Time Needed
1 reading
st
60 mins
2 look
nd
15 mins

Opportunistic Mycoses:
1. Candidiasis:
• Commonest mycoses involving skin and its appendages, mucosa and internal organs.
• Caused by yeast like fungus Candida albicans (70 – 80%) and occasionally by C.
tropicalis, C. parapsilosis, C. krusei, C. guillermondi etc.
• Yeast like fungi with pseudohyphae (Pseudomycelium).
• They occur as normal flora of skin, mucosa and gastrointestinal tract.
Risk factors for candidiasis:
• CMI. • Pregnancy.
• DM (MC predisposing factor). • O.C.P
• AIDS. • Malignancy.
• I/V catheters. • Chemotherapy.
• Neutropenia. • Broad spectrum antibiotics and Steroids.

Infections:
• Mucocutaneous lesions - Oral thrush, Esophageal candidiasis, Vulvovaginitis.
• Skin and nail infections –Diaper rash, Balanitis, Intertrigo, Paronychia, Onychomycosis.
• Systemic candidiasis – Brain (MC), Heart, Kidney.

Fig.7.18: C. albicans – Germ tube test

Mycology | 307
C. albicans:
• Produces pseudohyphae both in culture and in tissues.
ƒ Presence of pseudohyphae indicate colonization.
• Candida species grow well on SDA and on blood agar also at 25°C– 37°C.
• C.albicans can be differentiated from other species by:
ƒ Germ tube Test.
ƒ Blastoconidia (spore formed during budding) on corn meal agar.
ƒ Chlamydospore production (corn meal agar).
ƒ Fermentation and Assimilation of sugars.
ƒ N2 utilization.
• GERM TUBE TEST (Reynolds Braude Phenomenon):
ƒ Formation of Germ Tube within 2hrs when incubated in rabbit serum at 37°C.
ƒ Positive: C.albicans, C.tropicalis, C.dubliniensis.
ƒ C. albicans will grow at 42°C where others fail to grow.
• C.albicans is responsible for about half of all cases of candidemia in hospitalized
patients and is most common cause of mucosal candidiasis.
C. dublinensis:
• New Candida species.
• Seen in HIV patients.
• Resistance to fluconazole.
• Dark green on chrom agar.
• Germ tube positive.
• Chlamydospores on special media.

Treatment:
Cutaneous candidiasis GV paint locally/Topical azoles (Clotrimazole)/ Nystatin

Mucocutaneous Oral Fluconazole

Systemic Amphotericin B/ Ketoconazole/ Fluconazole / Itraconazole

Oral thrush Clotrimazole troches

2. Cryptococcus:
• European blastomycosis / Torulopsis.
• Encapsulated yeast.
Serotypes:
• Four types (A, B, C, D).
Biotypes:
• Cryptococcus neoformans var neoformans (A, D).
• Cryptococcus neoformans var gattii (B, C).
• Cryptococcus neoformans var grubii (A) (Most infections are caused by serotype A).
308 | Microbiology
Reservoirs:
• C. neoformans reservoirs is pigeons droppings and Nitrogen rich soil.
• C. gattii reservoirs is Flowering Eucalyptus tree (red gum).
• In AIDS patients, C. neoformans is common.
• In nature yeast cells are minimally encapsulated and easily aerosolized.
• Cryptococcosis is presenting manifestation of AIDS in about 1/3rd of AIDS pts and
generally occurs in patients with CD4 counts of < 100/µl.
• MC cause of fungal meningitis in AIDS pt.
Pathogenesis:
• Mode of infection: Inhalation (MC) / Skin / Mucosa.
• Clinical presentation:
ƒ Pulmonary infection: Mild, Self-limiting, No calcification. Primary site.
• CNS Disease: Most Frequent presentation.
• Cryptococcal osteomyelitis, Cryptococcoma (Disseminated).
• Least common- Kidney.
Virulence factors:
a. Polysaccharide capsule – Not immunogenic, No anticapsular ab formed.
b. Melanin: Phenyloxidase enzyme responsible for production of melanin when grown
on substrate like Niger seed agar or L – dopa agar. In brain, dopamine, norepinephrine
are most effective substrates for enzyme.
c. Ability to grow at 37°C.
d. Urease positive.
e. Phospholipase.
f. Mannitol production.
g. Superoxide dismutase secretions.
C. neoformans:
a. Grows at 37°C.
b. Hydrolyzes urea.
c. Produce brown colonies on Niger seed agar / Bird seed agar.
d. Produce disease in mice (Animal pathogenicity test).

Fig.7.19: Cryptococcus – Nigrosin stain

Mycology | 309
Laboratory Diagnosis:
Clinical specimens:
• Cerebrospinal fluid (CSF).
• Biopsy tissue (brain, liver).
• Sputum, bronchial washings.
• Pus, blood and urine can be collected depending on the site of infection or the organ
system involved.
Direct Microscopy:
• CSF and Body fluids:
ƒ Unstained, wet preparation of CSF with India ink or Nigrosin – Capsule.
• Tissue sections:
ƒ PAS.
ƒ Mucicarmine stain for capsule.
ƒ Capsulated, budding yeast cells (4 -20µm) surrounded by Mucopolysacchride
capsule.
• Culture:
ƒ SDA (without cycloheximide)– smooth, mucoid, cream coloured colonies.
ƒ Growth at 37°C.
ƒ Niger seed agar/ Bird seed agar - brown colonies.
• C. neoformans: Lack of fermentative ability.
• Urea hydrolysis Positive.
• Capsular polysaccharide antigen can be detected in serum and CSF by latex
agglutination and ELISA (Highly sensitive and Specific).
Treatment – Amphotericin B/ Flucytosine / Fluconazole.
Fluconazole preferred over itraconazole because of better penetration of BBB.

3. Aspergillosis:
Septate hyaline hyphae with acute angle (45°) branching and fruiting body with condia.
a. A. fumigatus (MC).
b. A. flavus.
c. A. niger.
d. A. terreus.
e. A. nidulans.
• Ubiquitous in the environment, growing on dead leaves, stored grain, compost piles,
hay, and other decaying vegetation.
• In immunosupressive therapy and in AIDS patients, severe form of aspergillosis
(invasive disease) occurs.
• Malignancy (especially hematological malignancy) and Neutropenia.
• Renal transplants.
• Steroid therapy.
A. fumigatus - Uniserrate.
A. niger and A. flavus- Biserrate.
310 | Microbiology

Fig.7.21: Aspergillus fumigatus

Fig.7.20: Aspergillus flavus

Fig.7.22: Aspergillus niger

Clinical features:
a. ABPA (allergic bronchopulmonary aspergillosis) Type I and III HS reaction. Diagnostic
criteria: Coughing out of mucous plugs.
b. Fungus ball – Usually develops in preexisting cavities such as tuberculosis
• It is only colonization without invasion.
c. Invasive aspergillosis: Disseminated disease in severely immunocompromised,
diabetics, neutropenic patients, pulmonary nodules, endocarditis in
immunocompromised patients, patients undergoing open heart surgery.
d. Paranasal granuloma:
• MC form of human infections by Aspergillus- Otomycosis.
• MC cause of fungal corneal ulcer – Aspergillus/Candida / Fusarium.
• MC cause of paranasal sinus mycoses- Aspergillus.
Mycology | 311
Laboratory Diagnosis:
• Clinical material:
ƒ Sputum, bronchial washings and tracheal aspirates.
ƒ Tissue biopsies; Blood.
• Direct Microscopy:
• Sputum, washings and aspirates- 10% KOH and/or Gram stained smears are prepared.
• Tissue sections or sputum smears are stained with H and E, GMS and PAS digest.
• HYALINE, ACUTE ANGLE DICHOTOMOUSLY BRANCHED SEPTATE HYPHAE (“V form”/
Antler horn appearance).
• Biopsy and EVIDENCE OF TISSUE INVASION is of particular importance.
• Serology:
ƒ Galactomannan antigen detection – Important role in the early diagnosis of
invasive aspergillosis. (False positive galactomannan assay in case of ticarcillin/
piperacillin therapy).
ƒ Ag detection helps to define etiology in patients with negative culture, multiple
etiology and misdiagnosis.
ƒ Disappearance of Ag correlates with good clinical outcome.
ƒ Ab detection tests - Role in the diagnosis of allergic, aspergilloma, and invasive
aspergillosis.
• Treatment: IV Amphotericin B/ Voriconazole/ Itraconazole/ Posaconazole
(prophylactic).

4. Zygomycosis (Mucormycosis + Entomophthoramycosis):

• Broad aseptate hyaline hyphae with right / obtuse angle (90°) branching.
• Basidiobolus, Conidiobolus.
• Pathologically mucormycosis is characterized by vascular invasion because they
have predilection for elastic lamina of large and small arteries thus causing thrombosis,
hemorrhage, infarction and necrosis of tissue.
• All patients have serious underlying condition such as diabetes, immunosuppression,
burns and trauma.
• Etiology of Mucormycosis:
ƒ Rhizopus.
ƒ Mucor.
ƒ Absidia.
ƒ Rhizomucor.
ƒ Cunninghmella.
ƒ Absidia – Has rhizoids and sporangiophores that arise from the aerial mycelium in
between the rhizoids.
ƒ Rhizopus – Rhizoids and sporangiophores that arise in groups directly above
rhizoids.
ƒ Mucor – Does not possess rhizoids shows branching.
312 | Microbiology

Fig.7.23: Rhizopus Fig.7.24: Mucor

Zygomycosis may lead to:

• Rhinocerebral zygomycosis (In debilitated patients, Diabetic Ketoacidosis) (bread
mould).
• Pulmonary zygomycosis.
• Gastrointestinal zygomycosis.
• Cutaneous (Burns).
• Disseminated.
• Predisposing factors for mucormycosis.
• Uncontrolled DM.
• Organ transplant.
• Hematological malignancy.
• Patients on desferoxamine therapy.
• Treatment: IV Amphotericin B.

5. Pneumocytis jerovecii:
• Pneumocystis carinii is not a pathogen in healthy humans. In people with a weak
immune system, it can cause Pneumocystis carinii pneumonia (PCP) and in some
cases extrapulmonary spread.
• Subjects with CD4 counts below 200/microlitre and who are not receiving preventive
therapy are nine times more likely to develop PCP.
• Taxonomical classification into fungus due to:
ƒ RNA, mitrochondrial protein and major enzyme.
ƒ Presence of 1,3 glucan in cell wall.
ƒ Virulence factor – 95 -140 k Da major surface glycoprotein (MSG).
Mycology | 313
• Highly immunogenic.
• Undergoes antigenic variation and thus the organism evades host defenses.
• On a chest examination:
ƒ Crackles/crepts.
ƒ Signs of focal lung consolidation.
ƒ Acute bronchospasm.
ƒ Pneumatoceles and Pneumothorax rarely.
• On Chest X Ray:
ƒ Diffuse alveolar or interstitial pulmonary infiltrates are the classic findings.
ƒ Occasionally patchy asymmetric infiltrates are seen.
ƒ No abnormalities can also be detected.
Lab diagnosis:
• Clinical specimens:
ƒ Induced sputum (using 3% hypertonic saline) [55-95%]
ƒ Bronchioalveolar lavage fluid [79- 98%].
ƒ Bronchial or lung biopsy [94-100%].
ƒ BAL fluids are considered better than induced sputum samples, but since the load
is higher in HIV patients induced sputum samples give comparable results.
• Direct microscopy:
ƒ Direct Fluorescent Antibody (DFA) test.
ƒ Giemsa staining.
ƒ Gomori Methenamine Silver Staining (GMS).
ƒ Toludine blue O.

Fig. 7.25: Pneumocystis jirovecii

• Definitive diagnosis of PCP is established by demonstration of P. Jerovecii in the

suspected sample.
• P. jerovecii infection occurs in HIV infected patient at CD4 count < 200 /µl.
• Common in HIV patients.
314 | Microbiology
• Causes interstitial plasma cell pneumonia.
ƒ Life cycle: Trophozoite, sporozoite and cyst which contains 8 sporozoites. Typical
granular, foamy, honeycombed material seen by H and E stain of tissue.
• Can’t be grown on fungal culture media.
• Treatment: DOC is Trimethoprim + Sulfamethoxazole.
• Alternative: – TMP + Dapsone / Clindamycin + Primaquine; Atovaquone.
6. Penicillium marneffi:
• Opportunistic in HIV patients.
• Umbilicated molluscum contagiosum like papular lesions.
• Brick red pigment on SDA.
• Isolated from Bamboo rat.
• Shows thermal dimorphism.
• Endemicity in S.E. Asia (In India-Manipur).
• Infects RES, Lung: systemic mycosis.
• Morphology: Yeast phase -Sausage shaped cells with transverse septa which divide
by binary fission.
• Mycelial phase-Broomstick/paint brush appearance.
Important points to remember:
• Spherule: Coccidiomycosis.
• Sclerotic body: Chromoblastomycosis
• Splender Hoeppli / Asteroid phenomenon – Sporothrix, Blastomycosis.
• Non cultivable fungi: Loboa loboi, Pneumocystis carinii.
• Carminiphilic fungi – Cryptococcus Rhinosporiduim.
• Otomycosis – Aspergillus, Penicillium, Candida.
• Keratomycosis: Aspergillus, Fusarium, Curvularia, Alternaria, Candida etc.
• Cryptococcus – Pigeon dropping.
• Histoplasma – Chicken,Bat droppings.
• Farmer’s lung- Stored hay (Aspergillus, Penicillium).
• Bagassosis – Sugarcane dust inhalation (Thermoactinomyces).
• Byssinosis – Dusty cotton.
• Immunity after exposure to fungi confers partial protection against re-infection.
• CMI is of paramount importance in – Cryptococcosis, Histoplasmosis, Coccidioidomycosis.
• Fluconazole is ineffective in Aspergillosis and Mucormycosis
• Fluconazole is less effective than itraconazole in Blastomycosis, Histoplasmosis and
Sporotrichiosis.
• Cycloheximide: Suppresses saprophytic molds so you can isolate the slower growing
systemic molds.
• But it also suppresses important fungi like:
ƒ Trichosporon beigelii.
ƒ Candida tropicalis.
ƒ Cryptococcus neoformans.
ƒ Yeast of Blastomyces.
ƒ Yeast of Histoplasma.
Mycology | 315

Worksheet
• MCQ OF “MYCOLOGY” FROM DQB

• EXTRA POINTS FROM DQB

1.

2.

3.

4.

5.

6.

7.

8.

9.

10
316 | Microbiology
Active Recall from Tables
Aspergillus species Colony morphology Microscopy

Zygomycetes - Genus Morphology

8 Parasitology

CONCEPTS
 Concept 8.1 Amoeba
 Concept 8.2 Flagellates
 Concept 8.3 Sporozoa
 Concept 8.4 Cestodes
 Concept 8.5 Trematodes
 Concept 8.6 Nematodes
 Concept 8.7 Important Points
318 | Microbiology
Concept 8.1: Amoeba
Learning Objectives
• Introduction to parasitology
• Entamoeba histolytica
• Free living amoeba

Time Needed
1 reading
st
30 mins
2 look
nd
10 mins

ENDOPARASITE- Lives within host e.g. Leishmania (Infection).

Obligate endoparasite – Can’t exist without parasitic life e.g. Toxoplasma gondii
Facultative endoparasite – Can live either parasitic or free-living e.g. Naeglaria
Accidental endoparasite – Attacks unusual host eg. Echinococcus
Aberrant endoparasite – Parasite during its migration (in host) reaches a site where it can’t live/ develop
further e.g. Toxocara canis

ECTOPARASITE: Lives on outer surface or in superficial tissue e.g. lice (Infestation).

HOST – Org. which harbors parasite, provides nutrition, shelter
• Definitive – Harbors – Adult parasite
or
• Harbors most highly developed form
or
• Where sexual replic occurs
Intermediate – Harbors Asexual form.
RESERVOIR HOST : Harbors parasite. Important source of infection to another host.
PARATENIC HOST : Harbors parasite without any further development.
PROTOZOA : Unicellular, Eukaryotes, Single cell performs all functions -- Flagella /
Pseudopodia / Cilia are the organs of locomotion and may provide immunity to re-
infection.

Classification of Amoeba:
Intestinal species Extra intestinal species
• E. histolytica • E. gingivalis
• E. hartmanni • Acanthamoeba spp.
• E. coli • Naegleria fowleri
• E. polecki
• Endolimax nana
Parasitology | 319
Entamoeba histolytica:
• Third leading parasitic cause of death after Schistosoma and Malaria.
• Man is the commonest source of infection.
• Large intestine is affected, flask shaped ulcer, MC- site- Ilecocaecal junction.
Trophozoite- Invasive form:

Fig.8.1: E.histolytica – Quadrinucleate cyst

• Size: 8 – 30 µm.
• Nucleus: Single and spherical.
• Central karyosome (eccentric in E. coli).
• Cytoplasm: Clear ectoplasm and granular endoplasm [Diff. not marked in E. coli].
• RBC s found in endoplasm (negative in E. coli).
• Actively motile with help of pseudopodia.
Cyst - Infective stage:
• 10 to 16 µm.
• Mature cyst is Quadrinucleate.
• Immature cyst- Cytoplasm contains chromatid bars and glycogen mass.
• Nucleus is lined with chromatin granules.

Precyst:
Stage between trophozoite and cyst.
Pathogenesis:
• E. histolytica is resistant to complement mediated lysis (due to cysteine proteinase
which is an important virulence factor).
• Amoebic dysentery – flask shaped ulcers.
• Most common extraintestinal manifestation - Amoebic Liver abscess.
• Lab diagnosis – Intestinal- Stool – Ova / Cyst, sigmoidoscopy.
320 | Microbiology
• Liver abscess – Trophozoites, EIA, PCR, ICT, DNA probes.
ƒ Anchovy sauce pus.
In > 90% of patients with colitis / amoeboma / liver abscess → ELISA and agar gel
diffusion assays for antibody are positive. Positive titres revert to negative within 6 – 12
months of treatment.
IHA:
• More difficult than ELISA.
• Titres may remain upto 10 years.
ƒ Tretment of amoebic colitis and liver abscess: Metronidazole, tinidazole, secnidazole
and cornidazole.
ƒ Luminal agents: Iodoquinol, diloxanide furoate or paromomycin should follow.
Pathogenic free living amoeba
1. Naeglaria fowleri – Primary amoebic meningoencephalitis (PAM) – Fulminant,
rapidly fatal (Mainly in children and young adults):
ƒ 11 cases in India (Rural 140 cases).
ƒ Moist soil, fresh water (swimming) [cyst, troph. (both infective)].
Life cycle:

Free living amoeba: Trophozoite, Cyst in water

Death
Inhalation / Aspiration of organism
PAM
By human

Brain

Olfactory Penetration of nasal mucosa

• Morphological Forms: Trophozoite/Amoebae (flagellated), Cyst.

• Cyst – Thick, double layered, usually absent in clinical samples (Brain etc.).
• Infective form – Trophozoite and flagellate form both.
• Definitive diagnosis – Demonstration of MOTILE trophozoites in CSF / Bx.
• Treatment – Amphotericin B intrathecal / IV.
• Amphotericin B + Miconazole, RF, CM.
2. Acanthmoeba spp. (A.castelli, A.culbertsoni):
• Healthy host → Amoebic keratitis.
• Immunodeficient host → Granulomatous amoebic meningoencephalitis (Subacute or
chronic disease with focal granulomatous lesions in brain).
Parasitology | 321
• Dust, Aerosol → Infections of lung, skin → Haematogenous spread – Brain. Indolent
course of encephalitis. Examination of CSF for trophozoites is diagnostic but LP is
contraindicated because of ↑ICT. Infection is almost uniformly fatal.
ƒ Trophozoite – Spiny projection, No flagellate form.
ƒ Cyst (is present in clinical sample).
• No flagellate form. Both cyst and trophozoite are infective.
• Transmission: Dust, aerosol → Lens solution → Keratitis or direct eye exposure. Treat-
ment – Polyhexamethylene biguamide, Chlorhexidine.
• Direct invasion through broken skin can also occur. Cutaneous ulcer / hard nodules
with amoeba are frequently seen in AIDS.
• Lung → CNS.
3. Balmuthia mandrillaria → Relatively uncommon. No flagellate stage. Causes me-
ningoencephalitis in immunocompetent host and has branching pseudopodia.
322 | Microbiology
Concept 8.2: Flagellates
Learning Objectives:
• Classification
• Giardia
• Trichomonas
• Trypanosoma
• Leishmania

Time Needed
1 reading
st
30 mins
2nd look 10 mins

Flagellates:
1. Giardia intestinalis:
• Falling leaf motility/gliding.
• Small intestine affected – duodenum and upper jejunum.
• Trophozoite Cyst
• Pear shaped Infective stage (oval and Quadrinucleate)
• B/L symmetrial Four nuclei, central axostyle
• 2 nuclei, 2 axostyles.
• 4 pairs of flagella.
• No haematogenous dissemination- never invasive.
• Can cause fat / carbohydrate malabsorption in children.
• MC parasitic cause of traveller’s diarrhea
• Steatorrhoea.

Fig.8.2: Giardia intestinalis- trophozoite

Parasitology | 323
Diagnosis:
• Identification of cysts or trophozoites in stool sample.
• A duodenal aspirate/biopsy may also be useful to detect trophozoite presence.
• Entero test/string test.
• An Enzyme-Linked Immunosorbent Assay (ELISA) to detect Giardia antigen in the
stool.
Treatment: Metronidazole, Tinidazole.
2. Trichomonas Vaginalis – Twitching motility.
ƒ Only Trophozoites – Pear shaped, 7-23 µ long and 5-15 µ wide.
ƒ 1 flagellum posterior and 3-5 flagella anterior.
ƒ No cystic stage.
• Trichomoniasis – most common STD (Parasitic).
• Trichomonal vaginitis- foul smelling discharge, strawberry appearance.
• Diagnosis:
ƒ Microscopic examination: Vaginal discharge, uretheral discharge, PAP smear;
demonstration of trophozoites.
ƒ Whiff test.
ƒ pH of vagina > 4.5.
ƒ Culture: Anaerobically- Diamond's, Hollanders media, Plastic envelope medium.
ƒ Polymerase chain reaction (PCR).

Fig.8.3: Trichomonas hominis – trophozoite

3. BLOOD and TISSUE FLAGELLATE

A. Leishmania:
• Old World:
ƒ Visceral.
ƒ Cutaneous.
Vector – Phlebotomus
• New World:
ƒ Mucocutaneous.
ƒ Cutaneous.
324 | Microbiology
Vector – Lutzomyia (sandfly)

1. OLD WORLD – India, Africa

Important species – L. donovani, L.major, L.tropica, L.aethopica, L.infantum
2. NEW WORLD - Species which are of medical importance in New world Leishmaniasis
(Central America, Mexico). Important species – L.chagasi, L.amazonensis, L.mexicana,
L. brasiliensis

Clinical manifestations:
Visceral leishmaniasis (VL) or Kala-azar:
L. donovani, L. infantum, L.chagasi, L.tropica.

The cutaneous leishmaniasis (CL) or oriental sore:

L.major, L. tropica, L.aethiopica,( old world),
L. mexicana, L. amazonensis. (new world),
Chiklero ulcer (hand and head)

Leishmaniasis recidivans (LR) -unusual sequel of oriental sore by.

L.tropica, associated with strong cell mediated immune response.

Diffuse cutaneous leishmaniasis (DCL) -anergic variant of localized CL,lesions are disseminated.
L.mexicana, L.amazonensis and L. aethiopica.

Mucocutaneous leishmaniasis (MCL), or espundia - extensive and disfiguring destruction of mucous

membranes.
L.braziliensis and L.guyanensis.

Two morphological forms:

• Amastigotes - Ovoid (3-5µm), non-motile, intracellular stage found in the mammalian
hosts.
• Promastigotes – Elongated (8-15µm), motile, extracellular stage, found in sandfly
hosts.
• Amastigotes – in RE cells of man
ƒ Spleen → 98% positivity.
ƒ Bone marrow – 54 – 86% - preferred method.
ƒ Liver 70%.
ƒ LN – 64%.
STAIN – Giemsa / Leishman.
Lab diagnosis:
• Amastigotes – smear.
• Culture NNN, Schniders liquid medium
• Serology - ↑ gamma globulin → Aldehyde, Antimony test, CFT – WKK ag (M. tb ag).
• Specific – IM, ELISA or rapid tests using rk39 antigen.
Parasitology | 325
Leishmanin skin test:
(Montenegro Test):
• Negative in acute case of Kala azar.
• Treatment – Amphotericin B (liposomal preparation).
B Trypanosoma:
• Trypanosoma cruzi→ Chaga’s disease (S. American Trypanosomiasis)
VECTOR: Reduvid bug.
Cycle:
Amastigote, trypomastigote → infective form (metacyclic) → released with faeces while
insect is taking blood meal and then faeces is rubbed into the bite wound site.
ƒ ACUTE- Local swelling (a chagoma) where the parasite entered the body.
ƒ Romaña’s sign: Includes swelling of the eyelids near the bite wound or accidentally
rubbed into the eye.
ƒ CHRONIC: Two thirds patients: cardiomyopathy, cardiomegaly.
ƒ About one third of patients go on to develop digestive system (megacolon and
megaesophagus).
• Trypanosoma brucei→ African sleeping sickness, Vector, Tsetse fly.
• African trypanosomiasis:
ƒ T. gambiense (W. Africa).
ƒ T. rhodisense (E. Africa).
• Stage 1 (early or hemolymphatic, stage) -Generalized or regional lymphadenopathy
(Posterior cervical lymphadenopathy [Winterbottom sign].
• Stage 2 (late or CNS, stage) - Daytime somnolence followed by night time insomnia.
326 | Microbiology
Concept 8.3: Sporozoa
Learning Objectives:
• Classification
• Plasmodium
• Toxoplasmaosis
• Coccidian parasites

Time Needed
1st reading 60 mins
2 look
nd
20 mins

SPOROZOA:
• Blood spp: Plasmodium spp.
• Tissue spp: Toxoplasma spp.
• Intestinal spp: CystiCystoisospora spp, Cryptosporidium opp.

Life cycle of malarial parasite:

• Infective form for man- sporozoite.
• Infective form for mosquito- gametocyte.

Immunity:
• Species specific, stage specific and strain specific.
• Lasts only till malarial parasite infection remains active: Premunition immunity.
• Innate:
ƒ Red cell polymorphisms associated with some protection.
▫ Hemoglobin S sickle cell trait or disease.
▫ Hemoglobin C and hemoglobin E.
▫ Thalessemia – α and β.
▫ Glucose – 6 – phosphate dehydrogenase deficiency (G6PD).
ƒ Red cell membrane changes.
ƒ Absence of certain Duffy coat antigens improves resistance to P.vivax
Parasitology | 327

Fig.8.4: Life cycle of Malarial parasite

P. vivax – Most common in India.

P. ovale – Not found in India.
P. falciparum P. vivax P. malariae P. ovale
Incubation Period 10-14 days 10-14 days 18 days – 6 wks 10-14 days
Asexual Phase 48 hours 48 hours 72 hours 48 hours
Form in P/S Rings, Gametocyte √ Rings, √ √
Gametocyte
Schizont
Ring stage Small, delicate Xle √ Large, prominent √ √
ring, for accole single ring
Late trophozoite Compact Large Band like Compact
(growing form)
Schizont Small compact Large Small Medium
(-) in P/S (+) in P/S (+) in P/S (+) in P/S
328 | Microbiology

Merozoite Grape like cluster Grape like cluster Rosette Irregular

Microgametocyte Kidney shaped √ spherical √ √
Macrogamete Crescent, blue √ spherical √ √
cytoplasm
Pigments Maurer’s (Black) Schuffner (yellow Ziemann (Brown James (Dark
brown) Black) Brown)
Host cell Normal size Enlarged (immature Not enlarged Slightly enlarged
blast)

Lab Diagnosis:
• Microscopy: This depends on the demonstration of parasites in the peripheral smear
after staining.
• GIEMSA STAIN: Most common (gold standerd).
• Thick smear: Quantization of the parasitaemia increases concentration, more
sensitivity.
• Thin smear: For species identification.
• Quantitative Buffy Coat/QBC: Quantitative assay.
• Immunochromatographic tests: Very good quick method, good sensitivity,
rapid.
• Histidine rich protein-II(HRP-II) (P. falciparum).
• Lactate dehydrogenase (LDH) (P.falciparum and P. vivax) -Optimal.
• Culture: Not for diagnosis but for epidemiological studies and drug resistance.
• RPMI 1640 MEDIUM containing human O group RBC’s (Roswell Park Memorial
Institute).
Drug Resistance in Malaria:
• Chloroquine resistance P. falciparum → Columbia, Thailand / late 1900 s.
• WHO def. → Ability of parasite to multiply or survive in presence of concentration of
drug that normally destroys parasite of same spp.
• WHO class → On the basis of counting number of trophozoites in peripheral smear
upto 7 days after treatment

Fig. 8.5: P. falciparum - Gametocyte

Parasitology | 329
• R I → Following treatment, parasitemia clears for at least 2 days but recrudes (+).
• R II → Following treatment, there is less than 25% reduction but not clearance of
parasite.
• R III → Following treatment, there is no reduction of parasite.

Fig. 8.6: P.falciparum – Accole

Malarial Vaccine:
• The immunity to malaria infection is incomplete, so there is no model for what
constitutes effective immunity.
• Plasmodium can vary some of its critical antigenic structures, plus the different stages
of the protozoa have different antigens.
• Antigens currently being studied as candidates for a vaccine include: circumsporo-
zoite protein (CSP), merozoite surface protein 1 (MSP-1), erythrocyte-binding anti-
gen 175 (EBA 175), apical-merozoite antigen (APA-1), gametocyte antigens (Pfs25),
and pre-erythrocyte liver-stage antigen 3 (LSA-3).
• Field trials of malaria vaccines to date have only had limited success, so currently
there is no good vaccine.
Babesia:
• Pigment (–), multiple stages + in single RBC (maltese cross).
• Organism shows slow antigenic variation.
ƒ Vector is tick.
ƒ Gametocyte (–).
ƒ E/C trophozoite (in heavy infestation) clinically – no cerebral manifestation.
• Severe infection in splenectomised patient.
• Treatment:
ƒ Supportive care.
ƒ Clindamycin + quinine.
ƒ Azithromycin + quinine (I /C pt).
330 | Microbiology
COCCIDIA:
1. Toxoplasma gondii:
Intracelluar
All stages (tachyzoite, tissue cyst, sporulated oocyst, bradyzoite) are infective.
CAT – reservoir
1. Cat faeces – infected food (oocyst).
2. Undercooked meat (tissue cyst).
▫ Most common manifestation of congenital toxoplasmosis: Chorioretinitis.
▫ Most common manifestation of toxoplasmosis in HIV patients: Encephalitis.
▫ Most common manifestation of acquired toxoplasmosis: Asymptomatic and if
symptomatic Cervical lymphadenopathy.
Life Cycle of Toxoplasma:

Mammals / birds (Trophozite, cyst in meat)

Trophozoite in meat
Humans (Intermediate Host) Oocyst in faeces

CAT
CAT Oocyst

• Trophozoites (Tachyzoite): Crescent shaped (2-3 µm x 4 -8 µ long).

• Congenital infection – Severe if mother acquires the infection during 1st / 2nd
trimester. However transmission rate is higher with progress of pregnancy. (Max. in
3rd trimester).
• Infants at birth / later – Retinochoroiditis, cerebral.
• Calcification and occasional hydrocephalus or microcephaly, convulsions.
• Diagnosis: Tachyzoites – LN, bone marrow, brain (smears) (I/P inoculation in mice).
• Serology – Sabinfeldmen (obsolete), EIA, DOT blot assay.
• Others: PCR, Tissue culture, animal inoculation, (I/P in mice).
Toxoplasmosis: Test of choice for diagnosis:
• Pregnant female- IgM ELISA, IgG Avidity test , B1 antigen detection.
• Congenital infection – IgM in fetal blood, IgA can also be used (Experimental but
better sensitivity).
• Immunocompromised – IgM and IgG detection.
Parasitology | 331
Toxoplasmosis Treatment Varies:
• IMMUNOCOMPETENT HOSTS:
ƒ No treatment is typically indicated.
• CONGENITAL TOXOPLASMOSIS:
ƒ Combination of pyrimethamine and sulfadiazine (or clindamycin if sulfadiazine not tolerated).
ƒ Prolonged therapy (often 1 year).
• TOXOPLASMOSIS DURING PREGNANCY:
ƒ Spiramycin is typically used (not FDA approved).
• PROPHYLAXIS IN HIV PATIENTS:
ƒ Trimethoprim-sulfamethoxazole.
ƒ Atovaquone useful in encephalitis.
2. Cryptosporidium:
• C. parvum – pathogenic to man.
• Habitat – Intestinal tract. Man is the reservoir of infection. Cyst released in stool is
immediately infective.
Life cycle:
Cow / Man / Cat

Excystation
Excretion in faeces

Sporozoite
Thick (infection to
susceptible host
Sporulated oocyst Trophozoite
Thin (Auto infection)

Meronts (1,2nd)
Unsporulated oocyst

Macro gametocyte + Micro gametocycte

Fig.8.7: a. Cryptosporidium b. Cyclospora c. Cystoisospora – Oocysts

332 | Microbiology
• Infection in immunocompetent – Mild watery diarrhea.
• Infection in immunocompromised – Severe profuse diarrhea (Cholera like) can affect
gall bladder, pancreas etc.
• Lab diagnosis → oocyst (acid fast) in Stool – Modified ZN stain, DFA.
• Serodiagnosis – No effective antimicrobial agent has been found. Spiramycin, Par-
amomycin has been tried in few cases. Nitazoxamide used in USA for treatment in
children.
Others – Cyclospora, Microspora, Cystoisospora.
Cystoisospora:
• Cyst released in stool is not immediately infective.
• Treatment: Trimethoprim – Sulphamethoxazole
• Pyrimethamine / ciprofloxacin can also be used.
Microsporidia (currently it belongs to Fungi):
• Obligate I/C spore forming protozoa.
• Small gram positive organisms with mature spores – 0.5 – 2 µm, 1 – 4 µm. It causes
intestinal infection, keratoconjunctivitis, sinusitis, disseminated infection (In AIDS
patients). Treat with Albendazole.
Sarcocystis:
• Muscular sarcocystosis- S.lindemanni (man-I.D.).
• Intestinal sarcocystosis.
• S.hominis (Man-DH, Cattle-ID).
• S.suihominis (Man-DH, Pigs- ID).

Balantidium Coli
Balantidium coli – only spp. pathogenic to man:
• Largest protozoa.
• Habitat – large intstine of man + PIGS.
• Reservoir – PIG.
• Morphology – Trophozoite and cyst.
• Troph. – Revolving motility.
ƒ Two nuclei → Macronucleus
ƒ Micronucleus
ƒ Cyst - Infective form.
ƒ 50 – 60 µm in size
ƒ Two nuclei +
• Diagnosis – Dysentery
• In large intestine → ulcers mimic amoebic ulcers but never invade muscular layer.
• Treatment – Tetracycline – DOC
ƒ Others – MNZ
Parasitology | 333
Concept 8.4 . Cestodes
Learning Objectives:
• Classification of helminths
• D latum
• T. saginata
• T. solium
• E. granulosus
• H. nana

Time Needed
1 reading
st
45 mins
2 look
nd
15 mins

HELMINTHS:
Cestodes Trematodes Nematodes

• Segmented (tape like). • Unsegmented (Leaf like). • Unsegmented.

• Hermaphrodite. • Gen. Hermaphrodite. (Elongate, cylindrical).

• Gut absent. • Except - schistosomes. • Separate sexes (dioecious).

• Infection by ingestion • Incomplete . Gut, anus absent. • Gut present and complete anus
of encysted larvae. • Mainly by larval stage by skin present.

• No body cavity. penetration or ingestion. • Infection by ingestion / Skin pen-

• No body cavity. etration.

• Body cavity present.

• Nematodes : Rounds worms.

• Platyhelminths : Tapeworms (cestodes) and Flukes (Trematodes).
Cestodes:
Taenia Taenia solium Diphyllobothrium Echinococcus Echinococcus Hymenolepis 334
|
saginata latum granulosus multilocularis nana
Stage
Adult

Definitive host Humans Humans Humans, Cats, Dog Dogs, Wolves Foxes Humans, Rodents

Location Gut lumen Gut lumen Gut lumen Gut lumen Gut lumen Gut lumen
Microbiology

Length (m) 4-6 2-4 3-10 0.005 0.005 0.02-0.04

Attachment Disks Disks, Hooklets Grooves Disks, hooklets Disks, hooklets Disks, hooklets
device

Mature segment Elongated Elongated Broad Elongated Elongated Broad

Egg

Maturation status Embryonated Embryonated Nonembryonated Embryonated Embryonated Embryonated

Distinguishing Radial striations Radial striations Operculated Radial striations Radial striations Polar filaments
characteristic

Larval No Yes No Yes Yes Yes

development in
humans

Larva

Intermediate host Cattle Swine, humans Copepods, Fishes Herbivores, Humans Field mice, Humans Humans, Rodents

Location Tissue Tissue Tissue Tissue Tissue Gut mucosa

From Cysticercus Cysticercus Procercoid (copepod) Hydatid cyst Hydatid cyst Cysticercoid

Plerocercoid (fish)
Parasitology | 335
D. latum:
• Largest helminth.
• Fish tapeworm.
• Definitive host – Man.
• Intermediate host:
ƒ Ist – Cyclops (procercoid).
ƒ 2nd – fish (plerocercoid).
ƒ Infection form – 3rd stage larva (pleurocercoid).

Fig.8.8: Life cycle of D. latum

Clinical features – Abdominal pain, diarrhea, Megaloblastic Anaemia Taenia

Fig.8.9: Life cycle of Taenia

336 | Microbiology
T. saginata:
• Cysticercus bovis (Larva).
• Cow: Intermediate host.
• Beef tapeworm, Unarmed tapeworm.
T. solium:
• Cysticercus cellulosae (larva).
• Pig (intermediate host).
• Pork tapeworm or armed tapeworm
• Neurocysticercosis – T. solium → Man eats eggs and becomes intermediate–host
(larval form development)
Echinococcus granulosus:
• Dog tapeworm.
• Definitive host: Dog.
• Intermediate Host: Man.

Fig.8.10: Life cycle of Echinococcus granulosus

Hydatid cyst → Unilocular, Casoni test – Immediate hypersensitivity reaction – Injection

of 2 ml filtered (Sterile) hydatid fluid I/D → Read in ½ hour → ≥ 5 cm wheal ≥ (+).
Ectocyst (outer).
Endocyst (inner germinal layer).
a. Gives rise to brood capsules with scolices.
b. Secretes hydatid fluid which is highly antigenic and toxic . It is:
ƒ Acidic (6.7).
ƒ Electrolyte rich.
ƒ Low specific gravity.
Parasitology | 337
Echinococcus multilocularis: Multilocular hydatid disease in man- mistaken as malignant
tumor. Has ability to metastasize.
H. nana
• Dwarf tapeworm.
• Egg is infective to man.
• Only one host involved.

Fig.8.11: Life cycle of H.nana

H. dimmunita → Rat tapeworm.

Fig.8.12: Tapeworm – Hexacanth egg

338 | Microbiology
Concept 8.5: Trematodes
Learning Objectives:
• Blood flukes
• Liver flukes
• Liver flukes
• Lung flukes

Time Needed
1st reading 45 mins
2 look
nd
10 mins

Blood Flukes:
Bilharziasis:
1. Schistosoma haematobium – Vesical and pelvic venous plexus
ƒ Associated with bladder cancer.
ƒ Egg has terminal spine.
• Mode of transmission – Bathing in contaminated water where cercaria larva penetrates
skin.
• Intermediate host- Snail( infected by miracidium larva).
• Sign and symptoms – hematuria, dysurea, urgency and Squamus cell carcinoma.
2. S. mansoni (dysentery) – Sigmoidorectal plexus( inferior mesenteric vein).
ƒ Egg has lateral spine.
• Mode of transmission and intermediate host - Same as S. haematobium.
3. S. japonicum – (Katayama disease) -ilio caecal plexus(Eosinophilic diarrhoea).
ƒ Egg has central spine.
• Mode of transmission and intermediate host - Same as S. haematobium.
Infective form – Cercaria (larva) released from intermediate host (snail)
↓
Penetrates skin of man → egg (Non operculated)
Treatment – Praziquantel.
Liver flukes:
Sheep Liver fluke – Fasciola hepatica. Sheep is definitive host and snail and water cress
(1st + 2nd) are intermediate hosts. Mode of transmission - Man gets accidental infection by
ingestion of aquatic vegetation contaminated with encysted metacercaria.
IH:
• 1st: Snail.
• 2nd: Crab / Creyfish.
Intestinal fluke → Fasciolopsis buski, Largest fluke
Parasitology | 339
IH:
• 1st: Snail.
• 2nd: Fish.
Lung fluke – Paragonimus westermani, Definitive host man; Intermediate host:
• 1st: Snail.
• 2nd: Crab.
Mode of transmission- Ingestion of raw, undercookes crab/ crayfish
Causes endemic hemoptysis
Oriental / Chinese liver fluke – Clonorchis sinensis. Definitive host Man; intermediate host:
• 1st: Snail.
• 2nd: Fish.
ƒ Mode of transmission- ingestion of raw , undercooked freshwater fish.
ƒ Clinical features - Cholangitis, cholangiocarcinoma, biliary obstruction.
ƒ Lab diagnosis- Stool examination, entero test.
Scientific and Epidemiology Disease- How Major Disease Manifestations,
Common Producing form Infection Diagnostic Stage, and
Name and its Location Occurs Specimen of Choice
in Host

Fasciolopsis Fast East. Adults live in Ingestion Edema, Eosinophilia, Diarrhea,

buski (large small intestine. of encysted Malabsorption, and even death
intestinal fluke) metacercariae in heavy infection; diagnosis:
on raw Egg in feces.
vegetation.

Fasciola Worldwide Adults live in bile Ingestion Traumatic tissue damage and
hepatica (sheep (in sheep- ducts. of encysted irritation to the liver and bile
liver fluke) raising and metacercariae ducts, jaundice and eosinophilia
(zoonosis) cattle-raising on raw can occur; diagnosis: eggs in
areas), humans vegetation. feces.
(accidental
host), sheep
(natural host).

Clonorchis Far East. Adults live in bile Ingestion Jaundice and eosinophilia in
sinensis ducts. of encysted acute phase, long-term heavy
(Oriental or metacercariae infections lead to functional
Chinese liver in un-cooked impairment of liver; diagnosis:
fluke) fish. egg in feces.

Paragonimus Far East, Adults live Ingestion Chronic fibrotic disease

westermani India, and encysted in lung. of encysted resembling tuberculosis (cough
(oriental lung parts of Africa. metacercariae with blood- tinged sputum);
fluke) in un- cooked diagnosis: egg in feces.
crab or
crayfish.
340 | Microbiology

Heterophyes Fast East. Adults live in Ingestion No intestinal symptoms unless

heterophyes; small intestine. of encysted very heavy infection; diagnosis:
Metagonumus metacercariae egg in feces.
yokogawai (the in uncooked
heterophyids) fish.

Schistosoma Arica, Adult in Vernules Fork-tailed Granuloma formation around

mansoni Middle East, of the colon (eggs cercariae eggs ( i.e., in liver, intestine,
(manson’s and South trapped in liver burrow into and bladder), toxic and allergic
blood fluke, America. and other tissues). the capillary reactions: nephrotic syndrome;
bilharziasis, bed of feet, diagnosis: eggs in feces and
swamp fever) legs, or arms. rectal biopsy, rarely in urine.

Schistosoma Far East. As for As for As for schistosoma mansoni,

japonicum schistosoma schistosoma but symptoms are more
(oriental blood mansoni. mansoni. severe because of greater egg
fluke) production; diagnosis: eggs in
feces and rectal biopsy, rarely
in urine.

Schistosoma Africa, Middle Adults in venules As for Bladder coloc with blood
Haematobium East, and of bladder and schistosoma and pus, nephrotic syndrome,
(bladder fluke) Portugal. rectum, eggs mansoni. symptomatic symptoms are
caught in tissues. mild, pulmonary involvement
from eggs in lungs, has been
associated with cancer of the
bladder; diagnosis: eggs in
urine, rarely in feces.

Swimmer’s itch Worldwide. Cercariae of Fork-tailed

(zoonosis) schistosomes that cercariae
usually parasitize burrow into
mammals and skin of human
birds enter human in water.
sking.

Fig.8.13: Schistosoma hematobium – Egg

Parasitology | 341

Fig.8.14: Schistosoma mansoni – Egg

Fig.8.15: Trematode – Egg with operculum

342 | Microbiology
Concept 8.6: Nematodes
Learning Objectives:
• Intestinal nematodes
• Tissue nematodes

Time Needed
1 reading
st
45 mins
2 look
nd
15 mins

Intestinal Nematodes
Small Intestine
• Ascaris lumbricoides(Round worm)
• Ancylostoma duodenale (Hook worm)
• Strongyloides stercoralis

Large Intestine
• Enterobius vermicularis( Seat worm,Pin worm)
• Trichuris trichiura(Whip worm)

Tissue nematode
Lymphatics
• Wuchereria bancrofti
• Brugia malayi

Conjunctiva
• Loa loa (African eye worm)

Mesentery
• Mansonella perstans
• Mansonella ozzardi

Subcutaneous tissues
• Dracunculus medinensis ( Guinea worm )
• Loa loa (African eye worm)
• Onchocerca volvulus (Eye worm)

According to egg/ larva producing capacity

Name Birth to Example
Viviparous Larva • D.medinensis
• W.bancrofti
• B.malayi
• T.spiralis
Parasitology | 343

Oviparous Laying eggs • A.lumbricoides

• T.trichiuria
• A.duodenale
• N.americanus
• E.vermicularis
Ovo-viviparous Egg containing larva which • S.stercoralis
immediately hatchout

Mode of infection
Mode Forms Examples
Ingestion Eggs contaminated food A. lumbricoides,
E. vermicularis
Growing embryo in intermediate host T. trichiura
D. medinensis
Encysted embryo in flesh T. spiralis
Penetration through skin Filariform larva A. duodenale
N. americanus
S. stercoralis
Bite Blood sucking insects Filarioideae
Inhalation Dust containing eggs A. lumbricoides
E. vermicularis

Human Intestinal Parasitic Nematodes

Parasitic Nematode
Feature Ascaris Ancylostoma Strongyloides Trichuris Enterobius
lumbricoides duodenale stercoralis trichiura vermicularis
(Roundworm) (Hookworm) (Whipworm) (Pinworm)
Infective stage Egg Filariform larva Filariform larva Egg Egg
Mode of Oral Percutaneou Percutaneous or Oral Oral
transmission autoinfection
Habitat Jejunal lumen Jejunal mucosa Small-bowel Cecum, colonic Cecum,
mucosa mucosa appendix
Pulmonary Yes Yes Yes No No
passage of
larvae
Clinical feaures Rarely Iron-deficiency Gastrointestinal Gastrointestinal Perianal pruritus
gastrointestinal anemia in heavy symptoms; symptoms,
or biliary infection malabsorption anemia
obstruction or sepsis in
hyperinfection
344 | Microbiology

Diagnostic stage Eggs in stool Eggs in fresh Larvae in stool Eggs in stool Eggs from
stool, larvae in or duodenal perianal skin on
old stool aspirate; cellulose acetate
sputum in tape
hyperinfection
Treatment Mebendazole Mebenda Ivermectin Mebendazole Mebendazole
Albendazole zole Albendazole Albendazole

Trichuris trichiura- (whip worm)

• Habitat: Adult worm live in large intestine-caecum, appendix
• Eggs :
ƒ Brown, bile stained egg
ƒ Barrel shaped with a mucus plug at each pole.
ƒ Floats in saturated solution of common salt.
ƒ Embryonated egg is infectious.
Trichuriasis.
• Acute appendicitis.
• Abdominal pain, mucus diarrhea, blood streaked stool and loss of weight.
• Prolapse of rectum in children.
• Hemolytic Anaemia
Laboratory diagnosis-
• Stool examination- characteristic egg.
• Adult worm occasionally found in stool.

Fig.8.16: Trichuris trichiura – Egg

Treatment- Thiabendazole and mebendazole.

Ascaris lumbricoides-
• Largest intestinal nematode parasite of humans.
• Most infected individuals have low worm burdens and are asymptomatic
Life Cycle
• Adult worms live in the lumen of the small intestine
• Feco-oral route
Parasitology | 345
Clinical Features
• Nonproductive cough
• Loefflers pneumonia
ƒ Chest x-ray- evidence of eosinophilic pneumonitis -Löffler’s syndrome
ƒ In heavy infections- pain and small-bowel obstruction, perforation, intussusception,
or volvulus, biliary colic, cholecystitis, cholangitis, pancreatitis, intrahepatic
abscesses.
Laboratory Findings
• Microscopic detection of characteristic Ascaris eggs – stool / bile

Fig.8.17: Ascaris – Fertilized egg Fig.8.18: Ascaris – unfertilized egg

Treatment- Albendazole, mebendazole effective

Hookworm - A. duodenale and N. americanus
• Infective stage & mode of infection- Filariform larvae, penetrate the skin

Clinical Features
• Most hookworm infections are asymptomatic.
• Infective larvae may provoke pruritic maculopapular dermatitis (ground itch”) at
the site of skin penetration as well as serpiginous tracks of subcutaneous migration
in previously sensitized hosts
• Chronic hookworm infection leads- iron deficiency.
• Hypochromic microcytic anemia, occasionally with eosinophilia or hypoalbuminemia,
is characteristic of hookworm disease.
Laboratory diagnosis
• Oval non bile stained eggs, that floats in saturated salt solution- in the feces.
• Stool-concentration procedures - to detect light infections
• Eggs of the two species are indistinguishable by light microscopy.
• If stool sample that is not fresh, the eggs may have hatched to release rhabditiform
larvae, which need to be differentiated from those of S. stercoralis.
346 | Microbiology

Fig.8.19: Hookworm egg

Treatment- albendazole, mebendazole

Strongyloides stercoralis
• Dwarf thread worm- Smallest pathogenic nematode causing human infection
• Has both parasitic and free living stages
• Lifecycle –
ƒ Completed in a single host, man is the principle host.
ƒ Dog and other animals can act as reservoir hosts
ƒ Has potential for autoinfection and multiplication within infected host.

Clinical spectrum-
• Acute strongyloidosis – presents as acute watery or mucoid diarrhoea
• Chronic strongyloidosis – diffuse abdominal pain, nausea, vomiting, diarrhoea
• Hyper- infection syndrome – corticosteroides, tacrolimus, chemotherapeutic agents,
radiation therapy etc
• Laboratory diagnosis-
• Specimens to be collected –
ƒ Stool
ƒ Duodenal aspirate
ƒ Sputum
ƒ Urine, etc.
• Larval demonstration in stool.
• Serology- IgG ELISA – about 85 – 95% sensitiivity.
Treatment-
• Ivermectin is drug of choice or thiabendazole and albendazole.

Tissue Nematodes:
Trichinella spiralis - Larvae contained in pork (striated muscle) is infective to man.
Not IN INDIA.
• In int. of man, adult worm develop – relases larvae.
Parasitology | 347
• Larva enters circulation → striated muscle of man (dead end).
ƒ Pig – Reservoir host.
ƒ No intermediate host.
ƒ Definitive host –Pig, man, rat.
ƒ Skin test – Brachman`s test

Somatic Nematodes:
1. Wucheraria bancrofti – sheathed microfilaria
↓
Infective form: 3rd stage larva.
Microfilariae in blood.
• Hydrocoele.
• Elephantiasis.
• Chyluria.
ƒ Meyers-Kouwenaar syndrome → caused by 1st stage larva (microfilaria)

Fig.8.20: W. bancrofti – microfilariae

Occult Filariasis:
• MF (-) in P/S.
• Affects lungs, liver, spleen along with lymphatics.
• Responds to DEC. Tropical pulmonary Eosinophilia.
• AB++ (Weingarten syndrome).
Parasite Periodicity Mf. Morphology Habitat (adult Vector
and Mf)
1. Wucheraria bancrofti Nocturnal No nucleus in tail Lymph. System Culex, Aedes
(Adenolymphangitis, tip (Sheathed) (A) bld (Mf) Mansoni;
Elephantiasis) Anopheles
2. M.f mallayi Nocturnal 2 Nuclei (+) in Lymph. Syst (A) Mansonia,
(Adenolymphangitis, tail tip (Sheathed) Bld (MF) Anopheles
Elephantiasis) Aedes
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3. Loa loa→ Diurnal Nuclei, (+) at tail Connectiva Flies: Chrysops

CALABAR SWELLING (12.00 noon tip (Sheathed) tissue,
-2.00 pm) conjunctiva
(Adult), Blood
(Microfilariae)
4. Onchocerca volvulus - Nuclear (+) side Connective Black flies:
(Blindness S/C nodules) by side in tail tip tissue (A) similium
(Unsheathed) Skin (Mf)

Fig.8.21: Loa loa - Microfilariae

Microfilariae:
A. Sheathed Microfilariae found in Blood
ƒ Wuchereria bancrofti.
ƒ Brugia malayi.
ƒ Loa Loa.
B. Unsheathed Microfilariae found in Blood
ƒ Mansonella ozzardi.
ƒ Mansonella perstans.
C. Unsheathed Microfilariae found in skin.
ƒ Mansonella streptocerca.
ƒ Oncocerca volvulus.
D. Dracunculus medinensis: Guinea worm, Medina worm.
ƒ Av. 100cm.
ƒ Infective form: Third stage larva (control → Nylon mesh to filter Cyclops in H2O).
ƒ Water purification of well using Niridazole.
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Life cycle of Ascasis lumbricoides

Adult worm in intestine

Swallowed Eggs in faeces (fertilized + unfertilized)

Trachea Develop to infective stage in soil

Alveoli of lung (moulting) Egg ingested hatch in intestine

Heart

Larvae develop and penetrate intestine

Migrate via blood stream
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Concept 8.7: Important Points
Learning Objectives:
• Important points
• Two Intermediate Hosts

Time Needed
1 reading
st
30 mins
2 look
nd
15 mins

Important points and table for revision

Parasite Infective stage
E.histolytica Cyst
Giardia intestinalis Cyst
E.coli Cyst
Cystoisospora Cyst
Trichomonas vaginalis Trophozoite
Leishmania Promastigote
Trypanosoma Metacyclic trypomastigote
Plasmodium Sporozoite
Babesia Sporozoite
Toxoplasma All (bradycyst, Merozoite, Sporulated oocyst)
Cryptosporidium Thick walled cyst
Pneumocystis carinii Cyst
Acanthmoeba, Naeglaria Cyst, Trophozoite

• Unilocular H. cyst : E .granulosus

• Multilocular : E.multilocularis
• Coenurus : Taenia multiceps
• Polycystic : E.vogelii multiocular
• T.saginata : Cysticercus bovis
• T.solium : Cysticercus cellulose (pig)
Schistosoma haematobium:
• Terminal spine in egg.
• Infects vesical plexus.
• Non operculated.
Schistosoma mansoni:
• Egg has lateral spine.
• Sigmoidorectal plexus involved.
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Schistosoma japonicum – Egg has central spine (katayama syndrome). Ileocaecal plexus
involved.
• Oviparous –Egg laid – unsegmented ova –Ascaris ; Trichuris trichuria.
ƒ Segmented ovum – hookworm.
• Viviparous – larva laid – T.spiralis.
ƒ W.bancrofli.
ƒ B.malayi.
ƒ D. medinensis.
• Ovoviviparous – Egg containing larva are laid – S.stercoralis.
• Trichuris trichura – Whip worm.
• A. lumbricoides – Roundworm;
• Dwarf tapeworm – H. nana.
• D. latum – Fish tapeworm.
• E. vermicularis – Thread worm /pin worm / seat worm.
• S. stercoralis – Dwarf round worm.
• E. granulosus – Dog tapeworm.
Non bile stained eggs
• Hookworm: A duodenale, N. americanus
• H. nana
• E. vermicularis
• Eggs which do not float in saturated salt solution.
ƒ Unfertilized eggs of Ascaris.
ƒ Taenia.
ƒ Intestinal flukes.
• Largest protozoa – B.coli.
• Largest helminth- D.latum.
• Schistosomes – non operculated eggs.
• Fasicola, D. latum, Paragonimus  operculated eggs.
• Auto infection is seen in:
ƒ Cryptospora.
ƒ H nana.
ƒ Ecchinococus.
ƒ Taenia.
ƒ S. stercoralis.

No intermediate host:

PROTOZOA:
→ E. histolytica
→ Giardia lamblia
→ Chilomastix mesnili
→ Trichomonas vaginalis
→ Balantidium coli
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HELMINITHS:
→ Enterobius vermicularis
→ Trichuris trichura
→ Ascaris lumbricoides
→ Ancylostoma duodenale
→ Necator americanus
→ H. nana

Two Intermediate Hosts:

Intermediate hosts:
• Snail, crustacean (Crab).
• Cyclops, fish.
• Snail, fish.
• Snail, plant (acquatic vegetations).
Parasites:
• Paragonimus westermani.
• D.latum.
• Clonorchis sinensis.
• Fasciola spp.
Parasites entering through skin penetration:
• A. duodoenale.
• Necator americanus.
• Strongyloides stercoralis.
• Schistosoma mansoni.
• Schistosoma Japonicum.
• Schistosoma haematobium.
• A.caninum.
• A. braziliensis.
Parasites transmitted by sexual contact:
• Trichomonas vaginalis.
• Entamoeba histolytica.
• Giardia lamblia.
Parasites transmitted congenitally:
• Toxoplasma gondii.
• Plasmodium spp.
• Microsporidia.
• Trypanosoma cruzi.
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Parasites infecting different tissues:
A. Parasites infecting intestines:
ƒ Entamoeba hystolytica.
ƒ Giardia lamblia.
ƒ Balantidium coli.
ƒ Cryptosora.
ƒ Cystoisospora belli.
B. Parasites infecting Liver:
ƒ Entamoeba hystolytica.
ƒ Echinococcus granulosus.
ƒ Fasciola hepatica.
C. Parasites infecting brain:
ƒ Plasmodium falciparum.
ƒ Neglaria fowleri.
ƒ Acanthamoeba.
ƒ Trypanosoma spp.
ƒ Toxoplasma gondi.
ƒ Entamoeba hystolytica.
ƒ Echinococcus granulosus.
ƒ Taenia solium.
D. Parasites infecting lung:
ƒ Paragonimus westermani.
E. Parasites infecting Lymphatic System:
ƒ Wuchereria bancrofti.
ƒ Brugia malayi.
Parasites associated with anemia:
• Hookworms.
• A. duodenale: Iron deficiency anemia.
• N. americanus: Iron deficiency anemia.
• Diphyllobothrium latum → Megaloblastic anemia.
• Haemolytic anemia: Malaria.
• Trichuris trichiura: Prolonged massive infection leads to iron deficiency anemia.
• Leishmania donovani.
Neoplasia:
• C. sinensis → Cholangiocarcinoma.
• Opisthorchis viverrini → Cholangiocarcinoma.
• S. heamatobium → Vesical carcinoma.
Obligate intracellular parasite:
• Plasmodium spp.
• Babesia spp.
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• Leishmania spp.
• Toxoplasma spp.
• Trypanosoma cruzi.
• Microsporidia.
Premunition:
• In most of the parasitic infections, immunity lasts only till original infection remains
active → Premunition or infection immunity. (e g. malaria, syphilis).
• A possible exception → Cutaneous leishmaniasis, in which ulcer heals leaving behind
good protection against reinfection.
Skin Tests:
a. Immediate Hypersensitivity
ƒ Erythema and induration after 30 minutes of infection.
ƒ Seen in Hydatid disease.
▫ Filariasis.
▫ Schistosomiasis.
▫ Ascariasis.
▫ Strongyloidiasis.
b. Delayed hypersensitivity (48 – 72 hours.).
ƒ Leishmaniasis.
ƒ Trypanosomiasis.
ƒ Toxoplasmosis.
ƒ Amoebiasis.
Parasitology | 355

Worksheet
• MCQ OF “PARASITOLOGY” FROM DQB

• EXTRA POINTS FROM DQB

1.

2.

3.

4.

5.

6.

7.

8.

9.

10
356 | Microbiology
Active Recall from Tables
Clues Examples

Cutaneous larva migrans

Human as an intermediate host

Autoinfection causing parasites

Eggs floating in the saturated salt solution

Anaemia causing parasites

Parasites having two intermediate hosts