GENERAL MICROBIOLOGY

LOUIS PASTEUR
➢ Louis Pasteur is also known as the father of microbiology. He has many
contributions to microbiology:
1. He has proposed the principles of fermentation for preservation of food
2. He introduced sterilization techniques and developed a steam sterilizer,
hot air oven and autoclave.
3. He described the method of pasteurization of milk
4. He had also contributed for designing the vaccines against several
diseases such as anthrax, fowl cholera and rabies
5. He disproved the theory of spontaneous generation of disease and
postulated the ‘germ theory of disease’. He stated that disease cannot
be caused by bad air or vapor, but it is produced by the microorganisms
present in air.
6. Liquid media concept- He used nutrient broth to grow microorganisms.
7. He was the founder of the Pasteur Institute, Paris.

ROBERT KOCH
➢ Robert Koch provided remarkable contributions to the field of microbiology:
1. He used solid media for culture of bacteria-Eilshemius Hesse, the wife of
Walther Hesse, one of Koch’s assistants, had suggested the use of agar as
a solidifying agent.
2. He also introduced methods for isolation of bacteria in pure culture.
3. Described hanging drop method for testing motility.
4. Discovered bacteria such as the anthrax bacilli, tubercle bacilli and
cholera bacilli.
5. Introduced staining techniques by using aniline dye.
6. Koch’s phenomenon: Robert Koch observed that guinea pigs already
infected with tubercle bacillus developed a hypersensitivity reaction when
injected with tubercle bacilli or its protein. This reaction is called Koch’s
phenomenon.
7. Koch’s Postulates:

➢ According to Koch’s postulates, a microorganism can be accepted as the

causative agent of an infectious disease only if the following conditions are
fulfilled:
1. The microorganism should be constantly associated with the lesions of the
disease.
2. It should be possible to isolate the organism in pure culture from the
lesions of the disease.
3. The same disease must result when the isolated microorganism is
inoculated into a suitable laboratory animal.
4. It should be possible to re-isolate the organism in pure culture from the
lesions produced in the experimental animals.
➢ An additional fifth criterion was introduced subsequently which states that
antibody to the causative organism should be demonstrable in the patient’s
serum.
➢ Exceptions to Koch’s postulates: It is observed that it is not always possible

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 to apply these postulates to study all the human diseases. There are some
bacteria that do not satisfy all the four criteria of Koch’s postulates. Those
organisms are:
● Mycobacterium leprae and Treponema pallidum: They cannot be grown
in vitro; however can be maintained in animals.
● Neisseria gonorrhoeae: There is no animal model; however, bacteria can
be grown in vitro.
➢ Molecular Koch’s postulates: It was a modification of Koch’s postulates
(by Stanley Falkow). He stated that gene (coding for virulence) of a
microorganism should satisfy all the criteria of Koch’s postulates rather than the
microorganism itself.

MICROSCOPES
Types of Microscopes Description

Compound Microscope ➢ Parts

● Condensing lens, which focused
the light onto the stage
● Objective lens (10x 4Ox , 100x),
which is responsible for
magnification and the
Magnification power can be
changed, since they are mounted
on a revolving nosepiece.
1. 10x: Low dry
2. 40x: High dry
3. 100x: Oil immersion
4. Ocular/ Eyepiece Iens:
Magnification is always
fixed i.e., 10x
➢ It is the transmitted light that reaches
the observer's eyex
➢ Resolution of the compound microscope:
0.2 to 0.3 μ
➢ Magnification: 1000x

Dark field Microscope/ Dark ground ➢ There is a special condenser with a

microscope central opaque area (Central stop).
➢ This illuminates the object on the stage
with a hollow cone of light.
➢ It is used for seeing very slender
bacteria, typically the 'spirochetes'.
(Leptospira and Treponema).

Phase Contrast Microscope ➢ It is used to observe 'unstained live

cells'.
➢ It has a special component 'phase plate'.
➢ As light passes through internal
structures with different refractive
indices, different phases are formed.

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 This difference in the amplitude of light
are converted into different intensities
with the help of the phase plate.

Fluorescent Microscope ➢ It is used when 'fluorescent dyes' are

used to stain the cells.
➢ The property of fluorescent dyes is that
they absorb light Of shorter
wavelengths.
➢ Fluorochromes like auramine, rhodamine,
acridine orange,etc., have this special
property.
➢ The important components of the
fluorescent microscope are,
● Dichroic mirror,
● Excitation filter, and
● Emission filter.

Electron Microscope ➢ It was first introduced by 'Ernst Ruska'

and 'Max Knoll'.
➢ Here, instead of light, electrons are
used, which are generated by a special
cathode.
➢ Instead of lenses, to focus the objects
on the stage, electromagnets are used.
➢ The scattered electrons are received on
a fluorescent screen, on which the image
of the internal structures can be seen.
➢ Resolution of the electron microscope:
0.2 to 0.5 nm, which is 1/1000th of that
of a light microscope.
➢ 'Phosphotungstic acid' is a special type
of negative staining to see selected
structures.
➢ Electron microscopes are of two types:
Scanning and Transmission.
● Scanning electron microscope is
used to look for ' surface
morphology'
● Transmission electron microscope
is used to study the internal
structures of the cell.

IMPORTANT STAINS IN MICROBIOLOGY

Gram Stain ➢ Gram ss taining is used to demonstrate almost all bacteria
into gram-positive and gram-negative.
➢ Some exceptions to this are,
1. Mycoplasma (too small to be gram stained; 0.2 to 0.3 μ
in size).
2. Spirochetes (too slender to be gram stained).

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 3. Rickettsia and chlamydia (minute intracellular
bacteria) and
4. Mycobacteria (they have a complex cell wall covered
with lipids, which prevent the entry of gram staining
agents into the cytoplasm).
➢ But depending upon the structure of the cell wall, they can
be classified into gram-positive and gram-negative.
➢ Mycobacteria is 'gram positive', since its cell wall has several
layers of peptidoglycans.
➢ Spirochetes are 'gram negative', since they have the typical
outer membrane and periplasmic space.
➢ Poorly gram stained bacteria:
● They are larger enough to be gram stained, but they
do not take up the counter stain 'safranin' properly.
Examples are, Legionella and Haemophilus.
● So instead of safranin, 'basic Fuchsin' stain is used
which stain the cytoplasm pink in colour.

Silver stains ➢ Used in certain specific species.

➢ Dieterle silver stain: Legionella and Mycoplasma in tissue
sections.
➢ Warthin starry silver stain: aged for, Helicobacter and
Bartonella.
➢ Levadite and Fontana silver stain: Used for 'spirochetes'.
● For films, 'Fontana silver stain' is used.
● For tissue sections, 'Levadite silver s bain' is axed.
➢ Methenamine silver stain: Used for 'fungi'
● Examples are, Gomori's, Gregory's, Grocott's
methenamine silver stain.

Negative staining ➢ Used to stain the capsule of the bacteria, especially to

demonstrate cryptococcus (encapsulated fungus) in CSF,
blood.
➢ Examples are, Indiaink, Nigrosin.

special stains 1. Albert, Neisser Ponder stain:

● Used to stain the 'meta chromatic granules'.
● Classical example is 'Corynebacterium diphtheriae'.
2. Sudan bIack B:
● Used to demonstrate lipid granules in the cytoplasm
of the bacteria.
3. Iodine:
● Used to demonstrate polysaccharide granules in the
cytoplasm of the bacteria.

Acid fast stain ➢ Types include,

● Ziehl- Neelcon Stain (ZN stain) and modified IN Stain
(kinyoun stain).
● They are used for demonstrating bacteria such as,
Mycobacteria
➢ Certain partially acid fast stain s include,

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 ● Nocandia (0.5 to 1% H2S04)
● Legionella mcdade (0.5 to 1% H2S04) and
● Oocytes of cyclospora and cryptosporidium (5%
H2SO4).

SELECTIVE MEDIA
TYPES OF SELECTIVE MEDIA DESCRIPTION

Thayer Martin Medium ➢ Selective medium for ‘Neisseria’.

➢ this contains,
● Vancomycin
● Colistin, and
● Nystatin which inhibit the
growth of unwanted bacteria.
➢ There is also a modified Thayer
Martin Medium

Skirrow-Butzler Medium ➢ Selective media for ‘Campylobacter’.

➢ It is also used for ‘Helicobacter’.

Tinsdale and Macleod Medium ➢ Both these media contain ‘potassium

tellurite’, which acts as the
selective agent for
‘Corynebacterium’.
➢ Tinsdale is also called ‘Cysteine
tellurite blood agar’.

Salt agar ➢ All staphylococci can grow in the

presence of 7 to 10% of salt.
➢ Hence, salt agar is a selective
medium for ‘staphylococcus’.

Buffered Charcoal Yeast Extract (BCYE) ➢ Selective medium for ‘Legionella’.

agar

Cetrimide agar ➢ Selective medium for ‘Pseudomonas

aeruginosa’, which is resistant to
several chemical agents (cetrimide
is a chemical disinfecrant).

Cefsulodin-Irgasan-Novobiocin (CIN) ➢ Selective medium for ‘Yersinia’.

agar

Ashdown medium ➢ Selective medium for ‘Burkholderia

pseudomallei’.
➢ It is the causative agent of
‘melioidosis’.

Wilson and Blair Bismuth Sulphate ➢ Selective medium for only

(WBBS) medium ‘Salmonella’, not for Shigella.

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Polymyxin Lysozyme EDTA (Ethylene ➢ Selective medium for ‘Bacilus
Diamine Tetracetic Acid) Thalous anthracis’.
Acetate (PLET) medium

Mannitol Egg Yolk Phenol Red Polymyxin ➢ Selective medium for ‘Bacilius
Agar (MYPA) agar cereus’.

Thiosulphate Citrate Bile salts Sucrose ➢ Selective medium and differential

(TCBS) medium medium for ‘Vibrio’.
➢ Sucrose fermenters produce ‘yellow
coloured colonies’.
➢ Non-Sucrose fermenters produce
‘pale coloured colonies’.

Xylose Lysine Deoxycholate (XLD) agar ➢ Selective medium for ‘Salmonella’

and ‘Shigella’.

Differential media ➢ It helps to differentiate the groups

of bacterial based on the cultural
characteristics.
➢ Classical example is ‘blood agar’,
which helps us to differentiate α
and β hemolytic bacteria from γ
hemolytic bacteria.

Mac Conkey medium ➢ It helps us to differentiate

between Lactose fermenting from
Non-lactose fermenting gram
negative bacteria.
➢ Lactose fermenters produce ‘bright
pink colonies/ Magenta coloured
colonies’ on Mac Conkey agar.
➢ Non-Lactose fermentoters produce
‘pale coloured colonies’.

Mannitol salt agar ➢ It is a differential medium for

‘Staphylococcus’.
➢ It helps us to differentiate
mannitol fermenting Staphylococcus
(eg. Staphylococcus aureus) which
produce ‘yellow coloured colonies’ by
fermentingmannitol.
➢ Rest of the Staphylococcus which
are Non-mannitol fermenters
produce ‘pale coloured colonies’ in
this medium.

Hugh-Leifson Oxidative-Fermentative ➢ This medium helps us to

medium differentiate between oxidative and
fermentative utilisation of sugars.
➢ It is usually used as a pair of

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 culture media, where there is a pair
of test tubes containing glucose.
➢ The bacteria are inoculated into
both the test tubes and in one of
the test tubes, petroleum jellyis
added to create anaerobic condition.

➢ Bacteria are able to utilise sugars only in the presence of aerobic conditions and
hence the acid is produced only in the aerobic test tubes, not in the anaerobic
test tubes. This is called ‘oxidative utilisation of sugars’.
➢ If a bacterium is utilising glucose in both the test tubes, the test tubes will
change colours, into yellow colour, this bacterium is called as ‘fermentative
bacterium’.
➢ In case, if no colour change is prioduced or no acid is formed, it is called a
‘asacrolytic bacterium’. It is unable to use the simplest sugars (i.e. glucose) for its
metabolism.
➢ If a bacterium is aerobic, it only grows under aerobic conditions and if it utilises
glucose, it is called ‘oxidative bacterium’.
➢ If a bacterium is facultative anaerobe, it is able to utilise glucose both
aerobically and anaerobically and this is called ‘fermentative utilisation of sugars’.

ANTIBIOTIC SENSITIVITY TESTING

➢ Certain dilution methods are considered as reference methods for susceptibility
testing, as they help uis to estimate the exact minimum inhibitory concentration
(MIC) of that bacterium

Dilution method ➢ It can be donein broth media.

➢ If itis done in microtitre pl ater, it is called
'microbroth dilution' andis if it's done in test
tubes, it is called'macro broth dilution'.
➢ If a solid medium ins used, it is called 'agar
dilution'.
➢ Using these dilution methods, the exact MIC can
be calculated.
➢ Since, these dilution methods are time
consuming, they are only done in reference
laboratories.
➢ Recomended culture medium for susceptibility
testing: Cation Adjusted Mueller Hinton Agar
(CA-MHA)/ Mueller Hinton Broth (MHB).
➢ If required, 5% blood may be added for more
fastidious bacteria which requires extra
nutrition.

Disc diffusion methods ➢ very easy to use methods. Most commonly used
method worldwide is,
1. Kirby Bauer Disc diffusion method:
● The samples are integrated along
with single standardised
concentration of antibiotics in the

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 disc and the zone of inhibition
around the antibiotic discs is
measured after overnight
inoculation.
● The sensitivity of the bacterium
to a particular antibiotic is given
by the reference tables which
tells us whether the bacterium is
sensitive or resistant to a
particular antibiotic, according to
the diameter of the Zone of
inhibition around that particular
antibiotic.
2. Stokes disc diffusion methods:
● In the centre, the test sample is
Laun cultured in the centre of the
petri dish and on either side, We
have control strains of the
bacteria.
● The zone of inhibition is compared
with the test and the control and
tells us about sensitivity of the
bacteria for a particular
antibiotic.
● There is no need to refer the
standard tables.

Epsiloneter test ➢ A plastic strip is impregnated with a greater

concentration of antibiotic and the zone of
inhibition formed after overnight inoculation.
➢ It is also called 'E test'.
➢ The bacterium iss laun cultured in the petri dish
instead of (serial dilutions and the exact MIC
can be measured.
➢ The zone of inhibition will intersect the plastic
strip and that value will give the exact MIC
value.
➢ It is a combination of disc diffusion and dilution
method.

Standard Inoculum ➢ Itis a special suspension of the test bacterium

which is having the turbidity of 0.5 MCF
(McFarland).
➢ Before we do the sensitivity testing, this special
inoculum is prepared in the sterile peptone
water or nutrient broth with turbidity 0. S MCF
and this is used for susceptibility testing.

Quantitative tests of anti ➢ It can give us the estimate of the Minimum

biotic sensitivity Inhibitory Concentration (MIC).
➢ They are, Dilution test and the E tesst..

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 ➢ Disc Diffusion method is a qualitative test,
which tells us only about the sensitivity of the
bacteria.

Automated test ➢ These are based on broth dilution methods.

Different names are given by different
companies such as, Vitek, Micro scan, and
Phoenix.

CHROM agar ➢ A special chromogenic substrate is added into

the medium which will change colour if a
particular bacteria is present or growing in that
media.
➢ It helps us to identify special resistant bacteria
such as:
● Methicillin Resistant Staphylococcus
aureus (MRSA),
● Vancomycin Resistant Enterococcus
(VRE),
● Extended Spectrum Beta Lactamase
(ESBL) producing Escherichia coli and
Klebsiella pneumoniae.

BACTERIAL GROWTH CURVE

➢ This is done in the batch culture.
➢ A fresh sterile medium is taken and few colonies of bacteria are added into this
medium. At regular intervals without adding fresh nutrients, the number of live
bacteria (viable count) is counted and is plotted against the time in a graph called
'bacterial growth curve', which has 4 phases.

Lag phase ➢ It is the stage of 'adaptation'.

➢ The bacteria is adapting to the nutrients provided in the culture
medium and is variable.
➢ For some bacteria, it may be 30 minutes and for other bacteria,
it takes 1 hour.
➢ No replication is occurring in this stage.
➢ Therefore, the total count, i.e., number of live and dead bacteria
and the viable count remains constant.
➢ Although there is no replication, the bacteria is metabolically
active, because it is quickly synthesising all the enzymes that are
required for binary fission.
➢ Just at the end of the lag phase, the bacterial size is maximum.

Log phase ➢ It is the stage of 'active replication'.

➢ There is an exponential increase in the number of bacteria.
➢ The total count and the viable count starts increasing.
➢ This is the stage where the 'generation time' can be calculated.
Generation time is the time required for a bacterium to undergo 1
binary fission (doubling time).
➢ Since the bacteria is quickly replicating, the bacteria is 'small in

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 size'.
➢ 'Maximum effect of antibiotic' that has been added in the
culture medium is seen in the log phase.

Stationary ➢ There is gradual depletion in the nutrients.

phase ➢ Hence, some bacteria will start to die out.
➢ The number of Bacteria replicating is almost equal to the number
of bacteria dying.
➢ Hence, the total count is increasing, but the viable count remains
constant.
➢ 'Sporulation' occurs during this phase.
➢ The exotoxins and the antibiotic producing bacteria form their
products during this phase.

Death phase ➢ In this stage, all the nutrients are depleted and there are a lot of
toxic metabolites.
➢ No replication occurs in this phase and most of the bacteria are
dying out.
➢ The total count becomes constant and the viable count is reduced.
➢ Involution form: Sometimes the bacteria instead of dying out,
become metabolically inactive and it occurs during the death
phase.

STRUCTURES PRESENT IN BACTERIA

Glycocalyx ➢ It is the outermost structure present in the bacterium.
➢ If it is loose and I'll define it, it is called 'slime'.
➢ When the slime of a group of bacteria collects together, it is called
'biofilm'.
➢ Biofilm helps the bacteria to survive by Preventing Phagocytosis and
Reduces the entry of Antibiotics.
➢ It is a polysaccharide layer around the bacterial colony.
➢ Important bacterias that forms biofilm are:
● Streptococcus mutans (most common cause of dental caries).
● Staphylococcus epidermidis ( ost common cause of
bacteremia following intravenous catheterisation) and
● Pseudomonas aeruginosa

Capaule ➢ If the glycocalyx is well-defined and well demonstrated, it is called

'capsule'.
➢ Capsule is polysaccharide in nature.
➢ Since the capsule has no net charge, it doesn't take up the gram
stain.It can be demonstrated by 'negative staining' using India ink
or Nigrosin (the capsule is outlined against a dark background).
➢ Quellung reaction:
● Also called 'Neufeld reaction'.
● It is the swelling of the capsule in the presence of its
antiserum.
● It is positive for any capsulated bacteria such as:
1. Yersinia pestis,
2. Streptococcus pneumoniae,

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 3. Staphylococcus aureus,
4. Bacteroides fragilis,
5. Bordetella pertussis,
6. Haemophilus influenzae,
7. Vibrio parahaemolyticus,
8. Klebsiella pneumoniae,
9. Bacillus anthracis,
10. Meningococcus,
11. Clostridium perfringens and
12. Cryptococcus neoformans.
➢ Special cases:
● Polypeptide capsules: It is a polymer of D glutamic acid.
Present in Bacillus anthracis, Yersinia pestis.
● Non-antigenic capsules: Present in Bordetella pertussis and
Streptococcus pyogenes.
● Zwitter ion capsules: There are a lot of positive and negative
ions in the capsule which give them the special ability to
form abscess. Present in Bacteroides fragilis and
Staphylococcus aureus.

CELL WALL
Gram Positive Bacteria Gram Negative Bacteria

● Has several layers of ● It is a complex structure.

peptidoglycans, which are marine ● It has an outer membrane of
monsters. phospholipid bilateral, in which two
● Alternating molecules of types of proteins are embedded:
N-acetylglucosamine and N-acetyl Structural proteins and porin
muramic acid. proteins.
● 50 to 100 layers of peptidoglycan. ● Porins are responsible for
absorption of nutrients.

● Presence of teichoic acid (both ● Just beneath 5he outer membrane

lipoteichoic acid and cell wall lies the periplasmic space, in which
teichoic acid)helps in adhesion. 2 layers of marine monomers
● They extend throughout the cell (peptidoglycans) are present.
wall.
● They are polymers of ribitol
phosphate and glycerol phosphate.
● They are unique to the gram
positive cell wall.

● Thickness is approximately 80 nm. ● Lipopolysaccharide (or) endotoxin

are embedded in the outer
membrane.

● Cell membrane consists of ● Cell membrane consists of

phospholipid bilayer. phospholipid bilayer.

● Cell wall synthesising enzymes like

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 carboxypeptidases and
transpeptidases are present in the
cell membrane.

➢ Lipopolysaccharides (or) endotoxins:

● They are unique to the gram negative bacterial cell wall.
● They are made up of 3 parts:
1. Outermost part: 'O' antigen, which is polysaccharide in nature.
2. Middle part: Core oligosaccharide, which contains a short chain of
6 to 10 amino acids.
3. Innermost part: Lipid A molecule, which is embedded in the outer
membrane of the gram negative bacteria. This lipid A molecule is
responsible for endotoxic property.

ENDOTOXIN V/S EXOTOXIN

Endotoxin Exotoxin

➢ Embedded in the outer membrane ➢ Actively secreted by either gram

of the gram negative bacteria and is positive or gram negative bacteria.
released only on lysis of the ➢ Example: Diphtheria toxin, Cholera
bacteria. toxin, etc.,
➢ It is unique in gram negative ➢ Exception is 'botulinum toxin' which
bacteria. is released on lysis of the bacteria.
➢ Exception is Listeria, which is a
gram positive rod producing
endotoxin.

➢ Heat stable (relatively) ➢ Heat labile, except Staphylococcus

aureus enterotoxin, which is
responsible for the short incubation
period food poisoning.

➢ Very low antigenicity ➢ Highly antigenic

➢ Act by inducing the release of ➢ Polypeptide or complex proteins.

cytokines such as:
● Interleukin-1 (IL-1),
● Interleukin-6 (IL-6),
● Tumor Necrosis Factor
(TNF) alpha, mainly from the
innate immune cells like
macrophages, monocytesand
dendritic cells.

➢ It also activates alternate ➢ Highly variable actions like increase

complement pathways. cAMP, Inhibition of protein
synthesis, Necrotic effect, etc.,

PILI V/S FLAGELLA

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Pili/Fimbriae Flagella

➢ Slender in structure present on the ➢ Much longer than pili and they help
surface of the bacteria. in locomotion.

➢ Made up of protein called 'Pilin'. ➢ Made up of protein 'Flagellin'.

➢ It is of 2 types: ➢ Not all bacteria are motile. Hence

1. Common Pili: Help in flagella can be present or absent
adhesion of bacteria and (atrichous) in a given bacteria.
mainly present on surface of ➢ Most pathogenic cocci such as
gram negative bacteria. Staphylococcus, Streptococcus,
2. Sex Pili: Help in Neisseria are non-motile/ atrocious.
'conjugation'.
Produced by both gram
positive and gram negative
bacteria, if they have the 'F
plasmid' or sex plasmid
(fertility factor).

➢ Depending Upon the distribution of

flagellum, the bacteria are
classified into:
● Monotrichous: Single
flagellum at one of the poles.
● Amphitrichous: One or more
flagella at both poles.
● Lophotrichous: Tuft of
capillaries at one end.
● Peritrichous: Flagella are
present all around the
bacteria.

➢ Testing of motility:
● 'Motility test agar' is used for this purpose.
● The colonies of bacteria are stab inoculated into this soft agar
(semi-solid) medium, which contains 0.2 to 0.5% of agar (lesser than usual
of 1 to 2%).
● After a few hours of inoculation, if the bacteria is growing just along the
inoculate, then it is non-motile bacteria.
● If the bacteria can move through the semi-solid medium and turn the
medium turbid/ translucent gradually, then it is a motile bacteria.
● Sometimes, a chemical agent may also be added to enhance the Motility of
the bacteria and the chemical turns red in the presence of bacterial
metabolites.

SPORES
➢ Certain bacteria under unfavorable conditions such as, nutrient depletion have
the ability to form spores.
➢ It is not a method for reproduction, but a method of survival under unfavorable

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 conditions.
➢ It is a metabolically inactive stage and are formed in the Stationary phase.
➢ These are resistant to heat (due to the presence of the chemical dipicolinic acid
in the core of the spore) and chemicals (due to the presence of keratin coat
present on the surface of the spores).
➢ Important pathogenic bacteria which forms spores are:

Bacillus Clostridium

➢ Non-bulging (as broad as the ➢ Bulging (broader than the bacterial

bacillary body). body).

➢ Only in soil and culture. ➢ Form spores in soil, culture and

body, except clostridium
perfringens which will not perform
spores in the body.
➢ Clostridium perfringens form spores
in a special medium called 'Eliner's
medium'.

➢ High heat resistance. ➢ Relatively low resistance to heat.

➢ This is the reason, Bacillus ➢ Clostridium tetani and many other
stearothermophilus, Bacillus spores can easily be killed within 5
subtitles are being used for minutes at 100⁰C.
efficacy testing of autoclaves and ➢ Exception: Clostridium perfringens
hot air ovens. type A and Care highly heat
resistant (survive heat for 2 to 3
hours) and have the ability to
produce clostridial food poisoning.

GENE TRANSFER IN BACTERIA

❖ Transformation
➢ Uptake up soluble DNA fragments directly through the cell wall.
➢ Contain bacteria, which are called 'competent bacteria', have special
enzymes to take up the bacterial fragments from the environment directly
through the cell wall, incorporating them in their genes.
➢ It was demonstrated by Griffith' on Streptococcus pneumoniae.

Gene transfer via bacteriophages:

★ Transduction
➢ Bacteriophage is acting as a vehicle for transfer of bacterial fragments
from one bacterium (donor bacterium) to the recipient bacterium.
➢ It is of two types:
1. Generalized transduction:
● Bacteriophages follow 2 cycles in the bacterium.
● In lytic cycle, it uses the bacteria to synthesize its own
components and then induces the lysis of the bacteria, once
the daughter phages have been assembled.
● Generalized transduction occurs in a lytic cycle.

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 ● During the synthesis of the phage components in the
bacterial xytoplasm, there is fragmentation of the bacterial
DNA. The phage has taken over the bacterial metabolic
machinery as a result of which the bacterial chromosomes
get fragmented.
● During the assembly of the daughter phages, some bacterial
DNA fragments get incorporated into the phage heads.
These mispackaged phages are released and infects the new
bacterium, thereby transferring the DNA fragments from
the dead DNA (donor DNA) to the new recipient bacterium.
● This is called generalized, as any bacterial DNA fragments
can be incorporated into the new bacterium and this occurs
due to mispackaging during the assembly of daughter phages.
2. Specialized transduction:
● It occurs after a lysogenic cycle.
● When a bacteriophage infects the bacteria, instead of
undergoing the lytic cycle, the phage integrates with the
bacterial chromosomes. Such phages are called 'prophages'.
● After a few cycles of replication, when the prophage keeps
on passing to the future generation, there is induction of the
prophage. The prophage separates away from the bacterial
chromosome and during this disintegration, it takes away
some of the bacterial genes which are next to the site of
integration.
● Now this bacteria enters the lytic cycle and the
bacteriophage with newly acquired DNA fragments from the
donor bacterium is released and infect the new bacterium,
thereby transferring the specific DNA fragments from the
donor bacterium to the recipient bacterium.
● It occurs due to defective excision of the disintegrating
prophage.

★ Lysogenic Conversion:
➢ It occurs because of bacteriophages.
➢ During the lysogenic cycle, the prophage is integrated with the bacterial
chromosome.
➢ Generally The prophage is totally dormant in the lysogenic cycle.
Sometimes, genes of the prophage are expressed by the bacterium, by
which the bacterium acquires new property. It may express new antigen on
the surface or may produce new toxins.
➢ This is called 'lysogenic conversion', where the prophage genes are
expressed by the lysogenized bacterium.
➢ Classical examples are: Production of phage mediated toxins such as
diphtheria toxin, cholera toxin, verocytotoxin, botulinum toxin C and D,
pyrogenic toxins A and C of the Streptococcus pyogenes, etc.,
➢ Another example is Salmonella having the ability to express different
types of antigens on its surface when new types of phages infect it and
get integrated into its chromosome.

❖ Conjugation:
➢ It occurs due to the presence of the special plasmid called 'F

15
 plasmid'/Sex plasmid'/'Fertility factor'.
➢ This encodes the sex pilus, through which one strand of F plasmid is
transferred to another bacterium (F-bacterium) from the F+ bacterium.
➢ Then they act as the template for the synthesis of complementary strands
and the end result is the formation of two F+ bacterium.
➢ If the F plasmid contain antibiotic resistant genes, then it is called 'R
plasmid'.
➢ Most common mechanism of gene transfer in bacteria: Transduction.
➢ Most common mode of bacteria acquiring antibiotic resistance genes:
Conjugation.
➢ Transfer of general genes most commonly occurs through transduction,
but antibiotic resistance is via 'conjugation' (I.e.) R-plasmid is getting
transferred from one bacteria to the other.
➢ Most common mode by which Pneumococcus acquires antibiotic resistance
genes: Transformation.
➢ Transformation is the Uptake of soluble DNA fragments directly through
the cell wall.
➢ Most common mode by which Staphylococcus aureus acquires antibiotic
resistance genes: Transduction.

STERILIZATION AND DISINFECTION

Physical Methods Of Sterilization/ Disinfection

★ Heat Sterilization/Disinfection:
➔ Mechanism of Action:
➢ Dry heat kills the organisms by Charring, Oxidative damage,
Denaturation of bacterial protein and Elevated levels of
electrolytes (CODE).
➢ Moist heat kills the microorganisms by denaturation and coagulation
of proteins.
1. Dry Heat (Hot air oven):
➢ Holding temperature required: 160°C for 2 hours •
➢ Materials sterilized: Hot air oven is best method for
sterilization of:
● Glassware like glass syringes, petri dishes,
flasks, pipettes and test tubes.
● Surgical instruments like scalpels, scissors,
forceps, etc.
● Chemicals such as liquid paraffin, fats,
glycerol, and glove dust powder, etc. •
➢ Sterilization control:
● Spores (106) of nontoxigenic strains of
Clostridium tetani or Bacillus subtilis subsp.
niger
● Thermocouples and Browne’s tube.
2. Moist Heat at a Temperature below 100ºC:
➢ Pasteurization: Used for perishable beverages like
fruit and vegetable juices, beer, and dairy products
such as milk.

16
 ● Two methods: Holder method (63ºC for 30
min) and Flash method (72ºC for 20 sec
followed by cooling to 13ºC).
● All non-sporing pathogens are killed except
Coxiella burnetii which may survive holder's
method.
➢ Water bath: Used for disinfection of serum, body fluids,
and vaccines (60°C for one hour)
➢ Inspissation (Fractional sterilization):
● It is a process of heating an article on 3 successive
days at 80–85ºC for 30 min
● Used for sterilization of egg based (LJ and Dorset’s
egg medium) and serum based media (Loeffler’s serum
slope).
3. Moist Heat at a Temperature of 100ºC
➢ Boiling: Boiling of the items in water for 15 minutes
may kill most of the vegetative forms but not the
spores.
➢ Steaming: Koch’s or Arnold’s steam sterilizers are
used to provide a temperature of 100°C for 90
minutes. It is useful for those media which are
decomposed at high temperatures of autoclave. It
kills most of the vegetative forms but not the spores.
➢ Tyndallization or intermittent sterilization: Involves
steaming at 100°C for 20 min for 3 consecutive
days. It is used for sterilization of gelatin and egg,
serum or sugar containing media. It kills most of the
vegetative forms including spores.
4. Moist Heat at a Temperature above 100ºC (Autoclave)
➢ Principle: Autoclave functions similar to a pressure
cooker. At normal pressure, water boils at 100°C
but when pressure inside a closed vessel increases,
the temperature at which water boils also increases.
➢ Sterilization conditions: 121°C for 15 min at pressure
of 15 psi (most commonly used).
➢ Uses of autoclave: Autoclave is useful for surgical
instruments and culture media and those materials
which cannot withstand the higher temperature of
hot air oven or media containing water that cannot be
sterilized by dry heat.
➢ Sterilization control:
● Biological indicator-Spores of Geobacillus
stearothermophilus (best indicator)
● Thermocouple and indicators like Browne’s
tube, Autoclave tapes:
5. Filtration
➢ Filtration is an excellent way to remove the
microbial population in solutions of heat-labile
materials like vaccine, antibiotics, toxin, serum
and sugar solution as well as for purification of

17
 air in laminar air flow systems.
➢ There are two types of filters; depth and membrane
filters.
a. Depth filters: They are porous filters that
retain particles throughout the depth of
the filter, rather than just on the surface.
They are used for industrial applications
such as filtration of food and beverage, and
chemicals, but are not to filter bacteria.
Examples include:
● Candle filters made up of
diatomaceous earth (Berkefeld
filters), unglazed porcelain
(Chamberland filters)
● Asbestos filters (Seitz and Sterimat
filters)
● Sintered glass filters
b. Membrane filters: They are widely used
filters for bacterial filtration. They are
porous; retain all the particles on the surface
that are smaller than their pore size.
● Made up of cellulose acetate, cellulose
nitrate, polycarbonate, polyvinylidene
fluoride
● Pore size: Membrane filters have an
average pore diameter of 0.22 µm (MC
used)
● Filtration of air: Air filters are
membrane filters used to deliver
bacteria-free air. Examples:
❖ Surgical masks (that let air in
but keep microorganisms out).
❖ In biological safety cabinets and
laminar airflow systems; two
filters are used
● HEPA filters
(High-efficiency
particulate air
filters): HEPA filter
removes 99.97% of
particles of size ≥
0.3 µm.
● ULPA filters
(Ultra-low
particulate/penetrat
ion air filters):
Removes from the
air 99.999% of
particles of size ≥
0.12 µm.

18
 ● Sterilization control includes
Brevundimonas diminuta and Serratia
marcescens.
6. Radiation
Ionising radiations:
➢ Examples include, X-rays, gamma rays (from Cobalt
60 source), and cosmic rays.
➢ Mechanism: It causes breakage of DNA without
temperature rise (hence called cold sterilization).
➢ It destroys spores and vegetative cells, but is not
effective against viruses. It is used for: Disposable
plastics, e.g. rubber or plastic syringes, infusion sets
and catheters.
➢ Catgut sutures, bone and tissue grafts and adhesive
dressings, antibiotics and hormones.
➢ Advantages of Ionizing radiation:
● High penetrating power, Rapidity of action and
Temperature is not raised
➢ Sterilization control: Efficacy of ionising radiation is
tested by using Bacillus pumilus. Non-ionizing
radiation:
➢ Examples of non-ionizing radiation include infrared
and ultraviolet radiations.
➢ They are quite lethal but do not penetrate glass, dirt
films and water.
➢ Dose: 250–300 nm wavelength for 30 min
➢ Used for sterilization of clean surfaces in
operation theatres, laminar flow hoods as well as
for water treatment.

Chemical Agents Of Sterilization/ Disinfection

★ Alcohols
➢ They act on bacteria, fungi, some enveloped viru (e.g. HIV); but not spores.
Vegetative bacteria
➢ They act by denaturing proteins and possibly by dissolving membrane
lipids.
➢ Ethyl alcohol is used as surgical spirit (70%) in hand rubs as antiseptics.
➢ Isopropyl alcohol: Used for clinical thermometers.

★ Aldehydes
➢ They combine with nucleic acids and proteins and inactivate them, probably
by crosslinking and alkylating the molecules. They are sporicidal and can be
used as chemical sterilants.
1. Formaldehyde: it is best used for:
● Preservation of anatomical specimen.
● Formaldehyde gas is used for fumigation of closed areas
such as operation theatres.
● Preparation of toxoids from toxins. It is toxic, irritant and
corrosive to metals.
2. Glutaraldehyde is less toxic, less irritant and less corrosive, hence

19
 is best used to sterilize endoscopes and cystoscopes:
● It is used as 2% concentration (2% cidex) for 20 minutes.
● It has to be activated by alkalinization before use. Once
activated, it remains active only for 14 days.
3. Ortho-Phthalaldehyde (0.55%): It can also be used for
sterilization of endoscopes and cystoscopes and has many
advantages over glutaraldehyde:
● It does not require activation.
● Low vapor property.
● Better odor.
● More stable during storage.
● Increased mycobactericidal activity.

★ Phenolic compounds
➢ Phenolics as disinfectants: Cresol, xylenol, Lysol and ortho-phenylphenol
are used as disinfectants in laboratories and hospitals.
● All have the ability to retain activity in the presence of organic
matter.
● They are toxic and irritant to skin, hence used as disinfectants but
not as antiseptics.
➢ Phenolics as antiseptics: Certain phenolics are less irritant to skin,
persist in skin for longer period and are widely used as antiseptics. In
general they are more active against gram positive than gram-negative
bacteria.
● Chlorhexidine: It is an active ingredient of savlon (chlorhexidine
and cetrimide)
● Chloroxylenol: It is an active ingredient of dettol.
● Hexachlorophene: As it can cause brain damage, hence its use as
antiseptic is restricted only to a staphylococcal outbreak.

★ Halogens
➢ Iodine: It is used as a skin antiseptic and kills microorganisms by
oxidizing cell constituents and iodinating cell proteins, e.g. Tincture of
iodine (2%) and Iodophor (iodine complexed with an organic carrier) e.g.
Betadine.
➢ Chlorine: It is the most commonly used disinfectant:
● For municipal water supplies and swimming pools and is also
employed in the dairy and food industries
● As laboratory disinfectant
● As bleaching agent: to remove the stain from clothes. •
● Preparations: It may be available as: (i) chlorine gas, (ii) sodium
hypochlorite (household bleach, 5.25%), or (iii) calcium hypochlorite
(bleaching powder)
● Mechanisms: All preparations yield hypochlorous acid (HClO)
which causes oxidative destruction of vegetative bacteria and
fungi, but not spores.
● Disadvantages:
➔ Carcinogenic
➔ Organic matter interferes with its action, hence excess
chlorine always is added to water to ensure microbial
destruction

20
 ➔ Need daily preparation
➔ They are not active against Giardia and Cryptosporidium,
➔ Sodium hypochlorite is corrosive and should be handled
cautiously.

★ Oxidising Agents
➔ Hydrogen Peroxide (H2O2)
➢ Mode of action: It is a chemical sterilant, acts by liberating
toxic free hydroxyl radicals which attack membrane, lipid, DNA,
and other cellular components.
➢ Concentration of (H2O2 3–6% is ideal, except for catalase
producing organisms and spores which require 10% of H2O2 .
➢ Used to disinfect ventilators, soft contact lenses, and tonometer
biprisms. Vaporized H2O2 is used for plasma sterilization.
➢ Advantage: 1. It acts perfect even in the presence of organic
matter 2. Low toxicity 3. Environmentally safe.
➔ Peracetic acid
➢ It is a chemical sterilant, often used in conjunction with H2O2 ,to
disinfect hemodialyzer and in plasma sterilization. It is also used
for sterilizing endoscopes. However, it may corrode steel, iron,
copper, brass and bronze.
➔ Plasma Sterilization
➢ This was recently introduced sterilization device (e.g. Sterrad and
Plazlyte) used for creating plasma state, so as to maintain a uniform
vacuum inside the chamber.
● Chemical sterilizers such as H2O2 alone or a mixture of H2O2
and peracetic acid
● Active agent is Ultraviolet (UV) photons and radicals (e.g. O
and OH): kill microorganisms and spores.
● Low temperature is maintained (<50°C , so best for heat
labile surgical instruments
● Sterilization control: Geobacillus stearothermophilus,
Bacillus subtilis subsp. niger.

★ Heavy Metal Salts

➢ Heavy metallic salts are of limited use in certain areas:
● Silver sulfadiazine is used on burned surfaces.
● Silver nitrate (1%) solution used for eyes of infants to prevent
ophthalmia neonatorum.
● Copper sulfate is an effective fungicide (algicide) in lakes and
swimming pools.
● Mercury salts such as mercuric chloride, thimerosal and
mercurochrome were known antiseptics in the past. Thiomersal
(merthiolate) is used as a preservative in vaccines and sera.
● Mechanism of action: Heavy metals combine with bacterial cell
proteins, often with their sulfhydryl groups, and inactivate them.
They may also precipitate cell proteins. Many heavy metals are
more bacteriostatic than bactericidal.

★ Surface Active Agents

➢ They lower the surface tension between two liquids or between a liquid and

21
 a solid. Surfactants may act as detergents, wetting agents, and
emulsifiers because they have both polar hydrophilic and nonpolar
hydrophobic ends.
1. Cationic surfactants (Quaternary ammonium compounds):
● They disrupt microbial membranes and may also denature
proteins.
● They kill most bacteria (gram-positives are better killed
than gram-negatives) but not M. tuberculosis or spores.
● Nontoxic but are inactivated by acidic pH, organic matter,
hard water and soap.
● Cationic detergents are often used as disinfectants for
food utensils and small instruments and as skin antiseptics.
● Examples include:
➔ Acetyl trimethyl ammonium bromide (cetavlon or
cetrimide)
➔ Alkyltrimethylammonium salts
➔ Benzalkonium chloride and Cetylpyridinium chloride
2. Anionic surfactants, e.g. soaps, have strong detergent but weak
antimicrobial properties. They are active at acidic pH.
3. The amphoteric surfactants: They have both detergent and
antimicrobial activity.
● They are active over a wide range of pH, but was reduced
in presence of organic matter.
● E.g. ‘Tego compounds’: Used as antiseptics in dental
practice, but cause allergic reactions.

★ Dyes
➢ Aniline and acridine dyes have been used extensively as skin and wound
antiseptics.
1. Aniline dyes: E.g. crystal violet, gentian violet, brilliant green and
malachite green:
● They are more active against gram-positive bacteria than
gram-negative and have no activity against M. tuberculosis.
● They are non-toxic and non-irritant to the tissues.
● Their activity is reduced in presence of organic material
such as pus.
● They interfere with the synthesis of peptidoglycan
components of the cell wall.
● These dyes are used in the laboratory as selective agents in
culture media (e.g. malachite green in LJ medium)
2. Acridine dyes: These include acriflavine, euflavine, proflavine and
aminacrine:
● They are affected very little by the presence of organic
material.
● More active against gram-positive bacteria but are not as
selective as the aniline dyes.
● They interfere with the synthesis of nucleic acids and
proteins in bacterial cells.

★ Gaseous Sterilization
➔ Ethylene Oxide (ETO)

22
 ➢ Ethylene oxide sterilizer is one of the widely used gaseous
chemical sterilants in present days.
● It has high penetration power, has both microbicidal and
sporicidal activity; acts by combining with cell proteins. •
● However, it is highly inflammable, irritant, explosive and
carcinogenic. Hence it is usually supplied in a 10 to 20%
concentration mixed with inert gases.
● Sterilization condition: 5 to 8 hours at 38°C or 3 to 4 hours
at 54°C.
● Sterilization control: Bacillus globigii.
● Use: For sterilization of many heat sensitive items such as
disposable plastic petri dishes and syringes, heart-lung
machine, sutures, catheters, respirators and dental
equipment.
➢ The decreasing order of resistance of microorganisms to
disinfectant or sterilizing agents is as follows:
● Prions (highest resistance) > Cryptosporidium oocysts >
Coccidian cyst > Bacterial spores > Mycobacteria > Other
parasite cysts (Giardia) > Small non-enveloped viruses >
Protozoan Trophozoites > Gram-negative bacteria > Fungi
> Large non-enveloped viruses > Gram-positive bacteria >
Enveloped viruses.
➢ Sporicidal agents include:
● EFGH: Ethylene oxide, Formaldehyde, Glutaraldehyde,
Hydrogen peroxide.
● 3P: Peracetic acid, O-Phthalic acid and Plasma sterilization .
● Autoclave and Hot air oven.

BIOCHEMICAL TESTS IN MICROBIOLOGY

★ Catalase test:
➢ All bacteria are catalase positive except:
● All anaerobes (Actinomyces, Bacteroides, Clostridium and
Lactobacillus).
● Streptococcaceae members.
● Shigella, dysenteriae type 1.

★ Oxidase test:
➢ All bacteria are oxidase positive except:
● Corynebacterium.
● Enterobacteriaceae.
● Staphylococci.
● Streptococcaceae.

★ Urease test:
➢ Urease positive organisms are:
● Proteus.
● Ureaplasma.
● Nocardia.
● Cryptococcus.
● Helicobacter.

23
 ● Monganella.
● Staphylococcus aureus, S. saprophyticus.
● Klebsiella.
● Brucellosis.

➢ Utilisation of sugars by bacterium: Medium used is Hugh Leifison Oxidative

fermentative medium.
➢ If it is saccharolytic, it will wither be
● Oxidative: Strict aerobic like Pseudomonas, Neisseria, Brucellosis,
Bordetella. Or
● Fermentative: Facultative anaerobes like Corynebacterium, Hemophilus,
Enterobacteriaceae, Staphylococcus, Streptococcus in both aerobic and
anaerobic conditions.
● Assacharolytic: Moraxella, Acinetobacter, Compylobacter, Helicobacter.

IMMUNOLOGY

24
IMMUNITY
Immunity is of two types:

Innate Immunity ➢ It has an immediate response and is first to encounter the

pathogen.
➢ The receptors recognize broad molecular patterns shared
by several pathogens. EgLipopolysaccharide (LPS)- Toll-like
receptors of innate immunity recognize molecular patterns
in LPS shared by several pathogens (all gram-negative
bacteria).
➢ Toll like receptor 5- Recognizes flagellin which is shared by
all flagellated bacteria. Such patterns are called Pathogen
Associated Molecular Patterns (PAMPS).
➢ The receptors of innate immunity are called Pattern
Recognizing Receptors (PRRs). They have broad specificity
and limited diversity.
➢ These receptors are encoded in the Germaine DNA.
Examples: Toll like receptors, C-reactive protein, Mannose
binding lettin, etc.,

Adaptive Immunity ➢ It is activated only on exposure to the pathogenand hence

there is a lag time of response.
➢ The highly specific receptor of adaptive immunity
recognizes organism specific antigens.
➢ The receptors of adaptive immunity are T-cel. Receptors
and B-cell receptors.
➢ They are organism specific and has narrow specificity.
There is unlimited diversity of these receptors.
➢ The receptors of adaptive immunity are generated by a
phenomenon called somatic recombination or rearrangement
which occur during the maturation of the T-cell in the
thymus and B-cell in the bone marrow.
➢ Memory response is present in adaptive immunity.

TOLL LIKE RECEPTORS

TLR Ligands (PAMP) Microbes

TLR 1(Cytosolic) Tracy lipopeptides Mycobacteria and

Gram-Negative bacteria

TLR 2 (Cytosolic) Peptidoglycans Gram-Positive bacteria

Zymosan Fungi

Lippmann, Lipoproteins Mycobacteria and other

bacteria

TLR 3 (Endosomal) Double stranded RNA Viruses

25
TLR 4 (Cytosolic) Lipopolysaccharide Gram-Negative bacteria

F protein Respiratory Syncytial Virus

(RSV)

Mannans Fungi

TLR 5 (Cytosolic) Flagellin Bacteria

TLR 6 (Cytosolic) Diacyl lipopeptides Mycobacteria and other

bacteria

Zymosan Fungi

TLR 7 & 8 (Endosomal) Single stranded RNA Viruses

TLR 10 Profilin Toxoplasma

HAPTENS
➢ Haptens are low molecular weight molecules that:
● Lack immunogenicity (cannot induce immune response), but:
● Retain antigenicity or immunological reactivity (i.e. can bind to their
specific antibody or T-cell receptor).
➢ However, Haptens can become immunogenic when combined with a larger
protein molecule called ‘carrier’.
➢ It is observed that hapten-carrier conjugate induce antibodies specific for:
● Epitopes of hapten
● Unaltered epitopes on the carrier protein and
● New epitopes formed by combined parts of both the hapten and carrier
➢ Haptens may be classified as complex or simple:
● Complex haptens contain two or more epitopes; they can react with
specific antibodies and the hapten-antibody complex can be visualized
by various methods such as precipitation reaction.
● Simple haptens usually contain only one epitope (univalent). Such
haptens can bind to the antibodies but the hapten antibody complex
cannot be not visualized as it is believed that precipitation to
happen, it requires the antigen to have at least two or more
epitopes.

IMMUNOGLOBULINS
★ Structure of Immunoglobulins
➢ Antibody or immunoglobulin is a ‘Y- shaped’ heterodimer;composed of
four polypeptide chains: two light (L) chains and two heavy (H) chains:
● All four H and L chains are bound to each other by disulfide
bonds, and by noncovalent interactions such as salt linkages,
hydrogen bonds, and hydrophobic bonds.
● Chains have two ends: An amino terminal end (NH3) and a
carboxyl terminal end (COOH).
● There are five classes of H chains (γ, α,µ,δ and ε) and two

26
 classes of light chains (κ and λ).
● Any antibody contains only one type of light chain and one type of
heavy chain.
● Based on the constant region of the heavy chains (γ, α,µ,δ
and ε); Ig has been classified into five types; lgG, IgA, IgM, IgD
and IgE respectively.
● Each H and L chain comprises of two regions: Variable region and
constant region
● Within the variable region, there are some zones or hot
spots called hypervariable regions or complementarity
determining regions that show higher variability. There are
three hot spots in the L and four in the H chain.
● Paratope is the site on the hypervariable regions that make
actual contact with the epitope of an antigen.
● Hinge region:
➔ It is the junction formed between the constant region of
heavy chains of IgG, IgA and IgD. It is absent in IgE and
IgM.
➔ This region is rich in proline and cysteine. The hinge region is
quite flexible, thus helps the antibody in reaching towards
the antigen.
● Enzymatic digestion:
➔ Papain digestion: generates Two Fab and one Fc fragment
➔ Pepsin digestion: generates One F(ab')2 and Many smaller
fragments
➔ Mercaptoethanol digestion: generates four fragments (two
H and 2 L chains)
● Immunoglobulin chains coded by different chromosomes:
➔ Heavy chains is coded by chromosome-14
➔ Light chain kappa is coded by chromosome-2
➔ Light chain lambda is coded by chromosome-22

★ Functions of Immunoglobulins
➢ Antigen binding (by Fab region)
➢ Effector functions (by Fc region)
● Fixation of complement: Antibody coating the target cell binds to
complement through its Fc receptor which leads to complement
mediated target cell lysis
● Binding to various cell types: Phagocytic cells, lymphocytes,
platelets, mast cells, NK cell, eosinophils and basophils bear Fc
receptors (FcR) that bind to the Fc region of immunoglobulins.
★ Properties of Various Immunoglobulin
➢ IgG Antibody:
● IgG is highest for DHS (decreasing order for DHS
is—GAMDE i.e. highest is IgG and lowest IgE): ○ Daily
production, ○ Half life (23 days), ○ Serum
concentration
● Four subtypes: IgG1-4 (Decreasing order for DHS is IgG 1 > IgG 2 >
IgG 3 > IgG 4)
➔ IgG is Responsible for:

27
 ➔ Precipitation,
➔ Neutralization,
➔ NK cell binding (to perform ADCC)
➔ Classical complement binding (IgM > IgG 3 > IgG 1 > IgG2)
(IgG 4 does not fix complement)
➔ Coagglutination by binding to S.aureus protein A
(Except IgG 3) ○Opsonization
● IgG appears late, so indicates past/chronic infection
● IgG avidity increases with time – So, detection of less avidity IgG
indicates relatively recent infection
● Secreted in placenta (Maximum placental transfer IgG1, minimum
IgG2)
● Secreted in breast milk
● Helps in phagocytosis by binding to FcR on phagocytes (Except
IgG2)
➢ Ig E Antibody:
● It is the only Heat labile antibody
● Lowest for DHS
● Responsible for- Type I hypersensitivity reaction
● Homocytotropic (Species specific) antibody
● Also called as Reagin antibody
● Raised in helminthic infections
➢ IgA Antibody:
IgA is the second most abundant antibody (2nd highest for DHS). It is of
two types:
● Serum IgA: Predominantly in monomeric form.
● Secretory IgA (SIgA): It is dimeric (valency four); both are joined
by J chain. In addition, there is another joining segment called
secretory component (synthesized by mucosal epithelium).
● Secretory IgA is responsible for Mucosal/local immunity. IgA also
exist in two subclasses/isotypes: IgA is mainly found in serum. IgA2
predominates in secretions.
➢ Ig D Antibody:
● Surface immunoglobulin on the surface of B-cells
● Possess highest carbohydrate content
➢ IgM Antibody:
➔ IgM is highest for MIS:
➔ Molecular weight (900,000),
➔ Intravascular distribution (blood Antibody) (80%),
➔ Sedimentation coefficient (19),
● Pentameric in nature with 10 valency
➔ IgM (and IgD) act as surface immunoglobulin on the surface of
B-cells
➔ IgM is the first antibody to appear following infection, indicates
recent infection
➔ IgM is the first antibody to appear in intrauterine life also (20
weeks): Indicates congenital infection
➔ IgM is responsible for (or mediates):
➔ Agglutination,
➔ Haemolysis,

28
 ➔ Opsonization,
➔ Classical complement pathway binding
● Example:
➔ Antibody in typhoid,
➔ Reagin Antibody (syphilis)
➔ Natural antibody of ABO, Rh system.

★ Abnormal Immunoglobulin
1. Bence Jones Proteins: They are produced in multiple myeloma (light chain
disease).
● The cancerous plasma cells produce excess light chains (Bence
Jones proteins) which are accumulated in the patient's serum and
excreted in urine.
● Such proteins have a unique property of getting coagulated at 50°C
and redissolving again at 70°C.
2. Waldenstrom’s Macroglobulinemia: It is lymphoma affecting B-cells
producing excess IgM. It has been seen in multiple myeloma. Somatic
mutations in MYD 88 gene occur in over 90% of patients.
3. Heavy Chain Disease: It is characterized by an excessive production
of heavy chains that are short and truncated. Four types of heavy
chain disease have been recognized based on H chain involved—alpha
(Seligmann’s disease), gamma (Franklin’s disease), mu and delta chain
disease.
4. Cryoglobulinemia: It is a condition where the blood contains
cryoglobulins; a type of Ig that becomes insoluble (precipitate) at
low temperatures but redissolves again if the blood is heated:
● Cryoglobulins usually consist of IgM directed against the Fc region
of IgG.
● They have been associated with multiple myeloma and hepatitis C
infection.

★ Ig Specificity or Antigenic Determinants of Ig:

➢ Epitope: Antigenic determinant of Ag against which Ab is raised.
➢ Paratope: Specific site of Ab that reacts with the corresponding epitope.
➢ Idiotope: Antigenic determinant of Ab against which Ab is raised, vary in
variable regions of the H & L chain.
➢ Isotope: Classes and subclasses of Ab, which vary in constant regions of
the H chain.

ANTIGEN-ANTIBODY REACTIONS
Immunoassay method Molecules used for labeling Types of visible effect

Enzyme Linked Enzyme Color change is detected by

Immunosorbent Assay spectrophotometer
(ELISA)

Immunofluorescence Fluorescent dye Emits light, detected by

Assay (IFA) fluorescence microscope

Radioimmunoassay (RIA) Radioactive isotope Emits β and γ radiations,

29
 detected by β and γ
counters

Chemiluminescence-linked Emits light, detected by Emits light, detected by

Immunoassay (CLIA) luminometer luminometer

Immunohistochemistry Enzyme or Fluorescent dye Color change (naked eye) or

(IHC) Fluorescence microscope

Western Blot (WB) Enzyme Color band (naked eye)

Immunochromatographic Colloidal gold or silver Color band, (naked eye)

test (RAPID TEST)

Flow through assay Protein A conjugate Color band, (naked eye)

(RAPID TEST)

Immunoferritin Electron Electron dense molecules Appears as black dot under

Microscopy (IEM) (e.g ferritin) electron microscope

COMPLEMENT AND ITS PATHWAYS

➢ Complement are a group of proteins normally found in serum in inactive
form, but when activated they augment the immune responses:
● Constitute about 5% of normal serum proteins
● Antigen nonspecific: Their level does not increase following either
infection or vaccination.
● Species nonspecific
● Heat labile: 56°C for 30 minutes
● Binds to Fc region of antibody: IgM (binds strongly) followed by IgG3 → 1 →
2
● Site of Synthesis of Complements: Mainly by liver, also by GIT,
macrophage and spleen.

★ Complement Pathways
➢ There are three pathways of complement activation:
1. Classical pathway: This is an Ab dependent pathway, triggered by
the Ag-Ab complex.
2. Alternative pathway: This is an Ab independent pathway,
triggered by the antigen directly.
3. Lectin pathway resembles classical pathway, but it is Ab
independent.
★ Stages of Complement Activation
➢ There are four main stages in the activation of any of the complement
pathways:
1. Initiation of the pathway
2. Formation of C3 convertase
3. Formation of C5 convertase
4. Formation of membrane attack complex (MAC)
➢ All the three pathways differ from each other in their initiation till
formation of C3 convertase. Then, the remaining stages are identical in all

30
 the pathways.

★ Biological Role of Complement

➢ Target cell lysis: MAC makes pores or channels in the target cell
membrane
➢ Inflammatory response: Complement by-products such as C3a, C4a and
C5a act as anaphylatoxins and chemotactic.
➢ Opsonization: C3b and C4b act as major opsonins
➢ Mediate hypersensitivity reaction II and III
➢ Removes the immune complexes from blood to spleen- by C3b
➢ Immune adherence: CR2 acts as EBV receptors
➢ Kinin like activity (↑vascular permeability): C2b
➢ Viral neutralization

Difference between three complement pathways

Classical Alternate Lectin pathway

Activator (initiator) Antigen antibody Endotoxin IgA, Carbohydrate

complex IgD, Cobra venom residue of bacterial
Nephritic factor cell wall (mannose
binding protein) that
binds to host lectin
antigen.

1st complement C1 C3b C4

activated

C3 convertase C14b2a C3bBb MBL/MASP-C4b2a

C5 convertase (C3 C14b2a3b C3bBb3b MBL/MASP-C4b2a3

convertase + 3b) b

Complement level in All C1-C9: Low C1,C4,C2- Normal C1- Normal Others-
the serum Others- Low Low

Immunity Acquired Innate Innate

Complement Deficiency Diseases

I. Complement Pathway(s) involved Disease/pathology

protein
deficiencies

C1, C2, C3, C4 C1, C2,C4-Classical SLE, glomerulonephritis and pyogenic

pathway C3- Common infections
deficiency

Properdin, Factor D Alternative pathway Neisseria and pyogenic infection

Membrane attack Common deficiency Disseminated Neisseria infection

complex (C5-C9)

31
II. Complement regulatory protein deficiencies

C1 esterase Overactive classical Hereditary angioneurotic edema

inhibitor pathway

DAF (Decay Deregulated C3 PNH (Paroxysmal nocturnal

accelerating factor) convertase Increased hemoglobinurea)
and CD59 RBC lysis

Factor I Deregulated classical Immune complex disease; recurrent

pathway with over pyogenic infections
consumption of C3

Factor H Deregulated Immune complex disease; pyogenic

alternative pathway infection
with increased C3
convertase activity

MACROPHAGES
➢ Monocytes/macrophages originate from bone marrow, from a separate
granulocyte monocyte progenitor cell. Monocytes are the largest blood cells

32
 present in blood. They do not divide and within 8 hours they migrate to tissues.
➢ Macrophages differ from monocytes in the following:
● 5–10 folds larger than monocytes
● Contain more lysozymes, organelles, enzymes and cytokines
● Possess greater phagocytic activity and have a longer life in tissues
(months to years)
➢ Two major functions of macrophage are Phagocytosis and Antigen presentation.

Body sites Macrophage designation

Peripheral blood Monocytes

Tissues Macrophages

Liver Kupffer cells

Brain Microglial cells

Kidney Mesangial cells

Lungs Alveolar macrophages

Bone Osteoclasts

Inflammation site Epithelioid cells Multinucleated cell (Langhans

giant-cells)

Connective tissues Histiocytes

Placenta Hofbauer cell

Lymphoid follicle Tingible body macrophage

MAJOR HISTOCOMPATIBILITY COMPLEX

➢ MHC molecules or human leukocyte antigens (HLA) serve as a unique
identification marker for every individual as the genetic sequence of MHC
genes is different for every individual.
➢ They also determine the histocompatibility between the donor graft and the
recipient.
➢ In humans, HLA complex coding for MHC proteins are located in the short
arm of chromosome-6.
➢ The genes are clustered in three regions named as MHC region-I, II and III
➢ MHC I and II help in antigen presentation to T-cells:
● MHC I presents intracellular antigen on viral/tumor cells to cytotoxic
T-cells
● MHC II presents extracellular antigen on APCs to helper T-cells
➢ MHC III does not help in Ag presentation, but code for various proteins
such as complement factors (C2, C4, C3 convertase, factor B and properdin),
heat shock protein, TNF-α and β and steroid 21-hydroxylases.

Differences between MHC class I and MHC class II molecules

33
 MHC class I MHC class II

Present on All nucleated cells (except Antigen presenting cells

sperms) and platelets (APCs)

Peptide antigen is presented to CD8 T-cells presented to CD4 T-cells

Nature of peptide antigen Endogenous or intracellular Exogenous

(viral / tumor Ag)

Peptide antigen (size) 8–10 amino acid long 13–18 amino acid long

Antigen presentation By Cytosolic pathway By Endocytic pathway

Peptide-binding site α1/α2 groove α1/β1 groove

CD4 or CD8 binding site α3 binds to CD8 molecules β2 binds to CD4 on TH

on TC cells cells

CYTOKINES
Cytokine Cytokine Secreting Cells Targe T-cells and functions
s

Interleukins (IL)

IL-1 Produced by all nucleated 1. TH cells - IL-1 produced by APCs

cells (Mainly by APCs such stimulates TH cells activation and
as Macrophages, monocytes proliferation:
dendritic cell, B-cells and ● Promotes IL-2 secretion by TH
endothelial cell) cells.
● Induces IL-2 receptor
expression on TH cells.
● Induces ↑MHC-II expression on
APCs.
2. B-cell: Promotes B-cell development
and maturation.
3. Liver: Induces synthesis of acute
phase reactant proteins.
4. Hypothalamus: induction of fever.
5. Macrophage and neutrophil activation:
↑ expression of ICAM.

IL-2 TH 1 cells Induces proliferation of activated TH cells,

TC cells and some NK cells (Previously called
T-cell growth factor).

IL-3 TH cell, NK cell, Mast cell 1. Stimulates hematopoiesis (acts as

multi-CSF).
2. Mast cell degranulation-↑ histamine
secretion.

34
IL-4 TH 2 cells 1. TH cells Promote TH 2 cell activity and
inhibit TH 1 cell.
2. B-cell: Promote B-cells activation and
proliferation and induce B-cell class
switch over to produce IgE, IgG4,
IgG1; previously called B-cell growth
factor.
3. Macrophage and APCs: Induce ↑MHC-II
expression.

IL-5 TH 2 cells Promote eosinophil growth and

differentiation.

IL-6 TH 2 cells, macrophages IL-1 and TNF like effects (synergistic effect)
Promotes B cell proliferation and antibody
production.

IL-7 Bone marrow/thymic Serves as a growth factor for T-cell and

stromal cells B-cell precursors.

IL-8 Macrophages, endothelial Attracts neutrophils, NK cells, eosinophils and

cells basophils.

IL-9 TH cells Hematopoietic and thymopoietic effects.

IL-10 TH 2 cells Reduces cytokine production by TH 1 cell.

IL-11 Bone marrow stromal cells Hematopoietic effect (B-cell and platelet
development) Liver: Induce synthesis of acute
phase reactant protein.

IL-12 Macrophages Promote TH 1 cell induction and inhibit TH 2

activity; promotes CMI responses, NK cell
stimulatory factor.

IL-13 TH 2 cells Mimic IL-4 function

IL-17 CD4+ activated memory TH Initiates and maintains inflammation

cell

Interferons (IFN)

IFN-α Leukocytes Antiviral activity

IFN-β Fibroblasts Antiviral activity

IFN-γ TH and TC cells, NK cells 1. Macrophage: Activates the resting

macrophages into activated
macrophage
2. B-cells: Activate B-cells to produce
IgG
3. Promotes inflammation of delayed type
of hypersensitivity (along with

35
 TNF-β)
4. TH 2 cell: Inhibits TH 2 cell
proliferation

Tumor Necrosis Factor (TNF)

TNF-α Macrophage 1. IL-1 like effect

2. Tumor cells: Promote vascular
thrombosis and tumor necrosis
3. Inflammatory cells: Induce cytokine
secretion
4. Induces lipolysis, causes extensive
weight loss associated with chronic
inflammation

TNF- β TH 1 cell and TC cell ● Tumor cells: Similar effect like TNF-α
● Macrophage: Enhance phagocytic
activity

Colony Stimulating Factors (CSF)

GM-CSF Fibroblasts, endothelium, Macrophage and granulocyte growth

T-cells, macrophages stimulation

G-CSF Bone marrow stromal cells, Granulocyte growth stimulation

macrophages

M-CSF Fibroblasts, endothelium Macrophage growth stimulation

Others

TGF- β Macrophages, masT-cells T 1. Inhibit T and B cell proliferation and

and B-cells, platelet hematopoiesis
2. Promote wound healing
3. Promotes class switching of B-cells to
the IgA class

CELL-MEDIATED IMMUNE RESPONSES

➢ CMI provides immunity against: (i) microbes residing in intracellular milieu (both
obligate and facultative) (ii) tumor cells, (iii) Mediate delayed/type IV
hypersensitivity (iv) plays key role in transplantation immunity and
graft-versus- host reaction.
➢ Effector cells of CMI include:
● Antigen specific cells: (i) Cytotoxic T-cells (principal mediator of CMI)
● Antigen nonspecific cells: (ii) NK cells, (iii) Cells performing ADCC
(NK cells, macrophages, neutrophil and eosinophils)

★ Cytotoxic T Lymphocytes
➢ CD8TC cells are the principal effector cells of CMI. Activation of Naive
TC cells requires these specific signals.
1. Antigen-specific signal: TCR of naive TC cells binds to MHC

36
 I-peptide complex of target cells.
2. CD8 of TC cells also interacts with a domain of MHC-I.
3. Costimulatory signal: CD28 of naive TC cells interacts with B7
molecule on target cells.
4. Cytokine signal: IL-2 (secreted by TH 1 cell) acts on TC cells
➢ The activated TC cells produce two types of lethal enzymes; called (i)
perforins (for pores on the target cells) and (ii) granzymes (destroy the
target cells).

★ Natural Killer Cells

➢ NK cells are large granular lymphocytes that constitute 10–15% of
peripheral blood lymphocytes:
● They are derived from a separate lymphoid lineage. NK cells are
cytotoxic but antigen nonspecific.
● They are part of innate immunity, act as first line of
defense and do not require prior contact with the antigen.
● Mechanism of NK cell mediated cytotoxicity:
➔ Receptor interaction: When activation receptors (e.g.
NKR-P1, CD16) present on NK cells are engaged with ligands
present on the target cells; NK cells become activated.
➔ Target cell destruction: is similar to that of TC cells, i.e.
via secreting perforins and granzymes. However, the NK
cells enzymes are constitutively expressed (i.e. they are
cytotoxic all the time, even without exposure to the
antigen).

★ Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC )

➢ A number of nonspecific cytotoxic cells express receptors (FcR) on
their surface that can bind to the Fc region of any Ig:
● These cells can bind to Fc portion of the antibody coated on the
target cells, and subsequently cause lysis of the target cell by
releasing various cytotoxic factors such as:
➔ NK cells secrete perforins, and granzymes
➔ Neutrophils releases lytic enzymes
➔ Eosinophils can release lytic enzymes and perforins; protects
against helminths
➔ Macrophages produce lytic enzymes and TNF
● Although these cytotoxic cells are nonspecific for the antigen,
the specificity of the antibody directs them towards the
specific target cells. This type of cytotoxicity is referred to
as antibody-dependent cell-mediated cytotoxicity (ADCC).

★ Assessment/Detection of CMI
I. Mixed-lymphocyte reaction (MLR),
II. Cell-mediated lympholysis (CML),
III. The graft versus host reaction (GVH) in experimental animals.

TYPE I HYPERSENSITIVITY REACTION

★ Mechanism
❖ Type-I Hypersensitivity reaction occurs through two phases:

37
 ➢ Sensitization phase: Priming dose of antigen (allergen)→ processed by
APC → antigenic peptide presented to T-cell → TH 2 activated → secretes
IL4 → Acts on B-cell → IgE produced → Mast cells coated with IgE (Fc) at
mucosal sites.
➢ Effector phase: Shocking (subsequent) dose of allergen → IgE (Fab)
binds to Antigen → Mast cell degranulation to release mediators which can
be primary mediators (preformed, released immediately) and secondary
mediators (released after synthesis). The mediators have various
pharmacological actions.

Primary mediators Action

Histamine, Heparin and Serotonin ↑Vascular permeability, ↑Smooth-muscle

contraction

Eosinophil chemotactic factor (ECF-A) Eosinophil chemotaxis

Neutrophil chemotactic factor (NCF-A) Neutrophil chemotaxis

Proteases Bronchial mucus secretion; Degradation of

blood-vessel basement membrane

Secondary mediators Action

Platelet-activating factor Platelet aggregation and degranulation;

Contraction of pulmonary smooth muscles

Leukotrienes (slow reactive substance of ↑ Vascular permeability; Contraction of

anaphylaxis, SRS-A) pulmonary smooth muscles

Prostaglandins ↑Vasodilation; Contraction of pulmonary

smooth muscles; Platelet aggregation

Bradykinin ↑Vascular permeability; Smooth-muscle

contraction

Cytokines (IL-1 and TNF-α) Systemic anaphylaxis; ↑ Expression of cell

adhesion molecules (CAMs) on venular
endothelial cells

Common allergens associated with Type 1 Hypersensitivity reaction

Food Nuts, egg, peas, sea food, beans, milk

Plants and pollens Rye grass, rag weed, timothy grass

Proteins Foreign serum vaccines

Drugs Penicillin, sulfonamides, local anesthetics

and salicylates

Insect bite products Venom of bee, wasp, ant, cockroach calyx

and dust mites

38
Others Mold spores, animal hair and dander

★ Examples of Type I Hypersensitivity Reaction

➢ Experiments to demonstrate type I hypersensitivity reaction: P-K
reaction, Schultz Dale phenomenon and Theobald smith phenomenon
➢ Systemic anaphylaxis
➢ Localized anaphylaxis (atopy) such as:
● Allergic rhinitis (or hay fever)
● Asthma
● Food allergy
● Atopic urticarial
● Atopic dermatitis (allergic eczema)
● Drug allergy
● Wheal and flare reaction.
➢ Parasitic diseases/tests:
● Casoni test (hydatid disease)
● Tropical Pulmonary Eosinophilia (TPE)
● Loeffler’s pneumonia (Ascaris)
● Ground itch (Hookworm)
● Leakage of hydatid fluid
● Cercarial dermatitis/swimmer’s itch (Schistosoma).

★ Detection of Type I Hypersensitivity

➢ Skin prick test
➢ Radioimmunosorbent test (RIST): It quantitatively detects the total
serum IgE
➢ Radioallergosorbent test (RAST): It quantifies the serum level of
allergen specific IgE.

★ Treatment of Type I Hypersensitivity Reaction

➢ Avoidance of contact with known allergens
➢ Hyposensitization: Repeated exposure to increased subcutaneous doses of
allergens can reduce or eliminate the allergic response to the same
allergen.
➢ Humanized Monoclonal anti-IgE-It can bind and block IgE.
➢ Drugs: Several drugs are useful in suppressing type-1 response such as
antihistamines, adrenaline, cortisone, theophylline and cromolyn sodium.

TYPE II HYPERSENSITIVITY REACTION

➢ In type-II reactions, the host injury is mediated by antibodies (IgG or
rarely IgM) which interact with various types of antigens such as:
● Host cell surface antigens (e.g. RBC membrane antigens like blood
group and Rh antigens)
● Extracellular matrix antigens or
● Exogenous antigens absorbed on host cells (e.g. a drug coating on RBC
membrane).
➢ After Ag-Ab binding occurs, the Fc region of antibody initiates the
type-II reactions by the following three broad mechanisms:

★ Antibody (Fc) Activating Complement System

39
 ➢ By complement dependent cytolysis (due to MAC), inflammation (by
C5a, C3a), opsonization (by C3b and C4b)
➢ It is seen in following conditions:
● Transfusion reaction (ABO incompatibility)
● Erythroblastosis fetalis
● Autoimmune hemolytic anemia, agranulocytosis, or
thrombocytopenia
● Drug induced hemolytic anemia
● Pemphigus vulgaris
● Hyper acute graft rejection.

★ Antibody (FC Portion) Interacting with Fc Receptors on Target Cells

➢ Antibody dependent cellular cytotoxicity (ADCC)
➢ Opsonization.

★ Antibody Dependent Cellular Dysfunction or ADCD

➢ Autoantibody Mediated:
● Activation of receptor, e.g. Grave’s disease
● Inhibition of receptor, e.g. Myasthenia gravis
● Other examples of ADCD:
➔ Good pasture syndrome (antibody produced against type IV
collagen)
➔ Pernicious anemia (antibody directed against intrinsic
factor)
➔ Rheumatic fever (antibody against streptococcal antigens
cross reacting with heart)
➔ Myocarditis in Chagas disease.
TYPE III HYPERSENSITIVITY REACTION
➢ Type-III hypersensitivity reactions are as a result of excess formation of
immune complexes (Ag-Ab complexes) which initiate an inflammatory response
through activation of complement system leading to tissue injury:
● Localized or Arthus reaction
➔ In skin: (i) following insect bites or (ii) during allergic
desensitization
➔ In lungs (i) Farmer’s lung (Saccharopolyspora species), (ii)
Bird-Fancier’s disease
● Generalized or Systemic type III Reactions, e.g. Serum sickness.

Diseases associated with generalized type III hypersensitivity reactions:

❖ Connective tissue disorders: Due to autoantibodies forming immunocomplexes

with self-antigens
➢ SLE (Systemic lupus erythematosus): Anti-DNA Ab
➢ Rheumatoid arthritis: Ab against human immunoglobulin
➢ PAN (Polyarteritis nodosa)

❖ Parasitic diseases: Resulting from immunocomplex deposition

➢ Nephrotic syndrome in Plasmodium malariae
➢ Katayama fever in schistosomiasis
➢ African trypanosomiasis

40
 ❖ Bacterial diseases resulting from immunocomplex deposition
➢ Streptococcus pyogenes: Poststreptococcal glomerulonephritis
➢ Mycobacterium leprae (Lepra reaction type 2)

❖ Viral diseases with immunocomplex deposition

➢ Hepatitis B (arthritis)
➢ Hepatitis C (arthritis)
➢ Infectious mononucleosis (Epstein Barr Virus)
➢ Dengue (arthritis)

❖ Others:
➢ Subacute bacterial endocarditis
➢ Serum sickness

TYPE IV HYPERSENSITIVITY REACTION

➢ Type-IV hypersensitivity reactions differ from other types in various ways:
● It is delayed type (occurs after 48–72 hours of antigen exposure)
● Cell mediated: Characteristic cells called TDTH cells are the principal
mediators
● Tissue injury occurs predominantly due to activated macrophages.

❖ Mechanism of Type-IV Reactions

➢ Sensitization phase (occurs 1–2 weeks following Ag exposure):
APCs process and present the antigenic peptides to TH cells. TH cells
are differentiated to TH 1 cells which further differentiates to form TDTH
cells
➢ Effector phase: The TDTH cells, on subsequent contact with the antigen,
secrete variety of cytokines such as:
➔ Interferon γ
➔ IL-2
➔ MCAF (Monocyte chemotactic and activating factor)
➔ TNF-β
➔ MIF (migration inhibitory factor)
➔ IL-3
➔ GM-CSF (granulocyte-monocyte colony stimulating factor).
➢ Cytokines in turn perform various functions which may be either
protective type or tissue damage type.

Infections/conditions associated with type IV hypersensitivity reactions:

➢ Intracellular ➢ Intracellular fungi: ➢ Noninfectious

bacteria: ● Pneumocystis conditions:
● Mycobacterium jirovecii ● Diabetes
leprae ● Candida mellitus type
● M.tuberculosis albicans 1
● Listeria ● Histoplasma ● Multiple
monocytogenes capsulatum sclerosis
● Brucella ● Cryptococcus ● Peripheral
abortus neoformans neuropathies
● Hashimoto’s

41
 thyroiditis
● Crohn’s
disease
● Chronic
transplant
rejection
● Graft-versu
host disease

➢ Intracellular viruses: ➢ Skin tests to ➢ Granuloma

● Herpes simplex demonstrate DTH formation seen in:
virus ● Tuberculin Tuberculosis,
● Variola test sarcoidosis,
(smallpox) (Mantoux schistosomiasis and
● Measles virus test) other trematode
● Lepromin infections
test ➢ Other example:
● Montenegro Lepra reaction type
test I
(leishmaniasi
s)
● Frie test –
done in LGV

➢ Contact dermatitis: Following exposure to contact

antigens:
● Nickel, poison ivy, poison oak, picryl chloride

IMMUNOLOGICAL TOLERANCE
➢ Immunological tolerance is a state in which an individual is incapable of
developing an immune response against his own tissue antigens. It is mediated by
two broad mechanisms— central tolerance and peripheral tolerance.

★ Central Tolerance
➢ This refers to the deletion of self-reactive T and B lymphocytes
during their maturation in central lymphoid organs
● In thymus: Removes the self reacting T-cells by negative selection
● In bone marrow for B-cells: Removes the self reacting B-cells by
negative selection and receptor editing

★ Peripheral Tolerance
➢ This refers to several back-up mechanisms that occur in the peripheral
tissues to counteract the self-reactive T-cells that escape central
tolerance. It is provided by several mechanisms:
1. Ignorance: The self-reactive T-cells might never encounter the
self-antigen which they recognize and therefore remain in a state
of ignorance.
2. Anergy: By blocking co-stimulatory signal by binding of B7
molecules on APC to CTLA-4 molecules on T-cells
3. Phenotypic skewing: Self-reactive T-cells stimulated by

42
 self-antigens secrete nonpathogenic cytokines
4. Apoptosis of Self-reactive T-cells by activation-induced cell
death (AICD)
5. Regulatory T-cells (Treg cells) can down regulate the self-reactive
T-cells
6. Dendritic cells (DCs): When certain dendritic cells, such as
immature DCs and tolerogenic DCs capture the self-antigen for
processing, they down regulate the expression of molecules of
costimulatory ligands, such as CD40 and B7 molecules or act
indirectly by induction of regulatory T-cells.
7. Sequestration of self-antigen in immunologically privileged
sites, e.g. corneal proteins, testicular and brain antigens.

MECHANISM OF AUTOIMMUNITY
➢ Breakdown of CTLA-4 mediated T-Cell Anergy: Seen in multiple sclerosis,
rheumatoid arthritis and psoriasis
➢ Failure of AICD (activation-induced cell death): Seen in SLE
➢ Loss of Treg cells
➢ Providing T-cell help to stimulate self-reacting B-cells
➢ Release of Sequestered Antigens (spermatozoa and ocular antigens) due to injury
to organs
➢ Molecular Mimicry, e.g. in post streptococcal acute rheumatic fever and
glomerulonephritis
➢ Polyclonal Lymphocyte Activation: Mediated by Superantigens, EBV and HIV
➢ Exposure of cryptic self-epitopes
➢ Epitope spreading
➢ Bystander activation.

CLASSIFICATION OF IMMUNOLOGICAL DIORDERS

➢ Immunodeficiency is a state where the defense mechanisms of the body are
impaired, leading to enhanced susceptibility to microbial infections as well as to
certain forms of cancer. It is broadly classified as primary or secondary.
● Primary immunodeficiency diseases result from inherited defects
affecting immune system development.
● Secondary immunodeficiency diseases are secondary to some other disease
process that interferes with the proper functioning of the immune system
(e.g. infection, malnutrition, aging, immunosuppression, autoimmunity, or
chemotherapy). They are more common than primary immunodeficiency
diseases.

Primary immunodeficiency diseases:

I. Humoral immunodeficiency (B-cell defects)

1. Bruton disease (X-linked agammaglobulinemia)
2. Common variable immunodeficiency
3. Isolated IgA deficiency
4. Hyper-IgM syndrome
5. Transient hypogammaglobulinemia of infancy

43
 II. Cellular immunodeficiencies (T–cell defects)
1. DiGeorge syndrome (Thymic hypoplasia)
2. Chronic mucocutaneous candidiasis
3. Purine nucleoside phosphorylase (PNP) deficiency

III. Combined immunodeficiencies (B and T-cell defects)

1. Severe combined immunodeficiencies
2. Wiskott-Aldrich syndrome
3. Ataxia telangiectasia
4. Nezelof syndrome

IV. Disorders of phagocytosis

1. Chronic granulomatous disease
2. Myeloperoxidase deficiency
3. Chediak-Higashi syndrome
4. Leukocyte adhesion deficiency
5. Lazy leukocyte syndrome
6. Job’s syndrome or Hyper-IgE syndrome
7. Tuftsin deficiency
8. Shwachman’s disease

V. Disorders of complement
1. Complement component deficiencies 2Complement regulatory protein defi
2. Complement regulatory protein deficiencies

Risk of secondary infections in various immunodeficiencies

Pathogen T-cell defect B-cell defect Granulocyte Complement

defect defect

Bacteria Bacterial sepsis Streptococci, Staphylococci, Neisseria,

Staphylococci, Pseudomonas Toxoplasma
Haemophilus Nocardia Other pyogenic
influenzae Infections

Viruses CMV, EBV, Enterovirus -

Severe encephalitis
varicella,
Chronic
infections with
respiratory and
intestinal
viruses

Fungi Candida, - Candida,

Pneumocystis Aspergillus
jirovecii

Parasites - Giardiasis -

Special Aggressive Recurrent -

features disease with sinopulmonary

44
 opportunistic infections,
pathogens, Sepsis, chronic
failure to clear meningitis
infections

SYSTEMIC BACTERIOLOGY

STAPHYLOCOCCUS AUREUS
➢ Staphylococcus aureus is catalase positive, coagulase positive, facultative
anaerobe, non-motile, non-sporing and occasionally capsulated.

● In Greek, Staphyle means bunch of grapes.

● Staphylococcus was discovered by Sir Alexander Ogston and
S.aureus was named by Rosenbach.

Virulence factors

Cell wall factors Activity

Peptidoglycan More thicker,

Confers cell rigidity and induces inflammatory
response

Techoic acid Helps in adhesion to mucosal surfaces and

prevent opsonisation

Clumping factor/Bound coagulase Responsible for slide coagulase reaction

Protein A Antiphagocytic, anticomplementary, chemotactic

Binds to Fc region of IgG leaving Fab region
free to bind to an Antigen - Basis of
Coagglutination reaction

45
Toxins Activity

Membrane active toxins

A. Hemolysins

α Hemolysin Inactivated at 70°C; reactivated paradoxically

at 100°C (due to denaturation of a heat labile
inactivator at 100°C) Leucocidal, Cytotoxic,
dermonecrotic, lethal

β Hemolysin Sphingomyelinase
Lyses sheep RBC, but not human or rabbit RBC
Exhibits hot-cold phenomenon

γ Hemolysin Bicomponent protein,

Lyses rabbit sheep and human RBCs

δ Hemolysin Detergent like, Lyses rabbit, sheep and human

RBCs

B. Leucocidins/ Panton Two components F and S

valentine (PV) toxins Damage PMN and macrophages
Associated with Community acquired MRSA
Synergohymenotropic toxins: Bicomponent
toxins such as γ and PV toxin act synergistically
and are called as Synergohymenotropic toxins

C. Other toxins

Epidermolytic toxin (Exfoliative Mainly belong to phage group II

toxin) Scalded skin syndrome (Nikolsky’s sign-
epidermal layer separated)
Severe- Ritter disease (newborn),
toxic epidermal necrolysis (TEN) (adult)
Milder- Pemphigus neonatorum, bullous impetigo

Enterotoxins Produced by 50% of clinical isolates

Cause food poisoning
Incubatory Period: 1-6 hr due to preformed
toxin
Site of action: The toxin stimulates the vagus
nerve and vomiting center of the brain.
It also stimulates the intestinal peristaltic
activity.
Heat stable (not destroyed after heating food)
Most common food items involved are milk
products, bakery food, custards, potato salad,
or processed meats.
Multiple antigenic type (A–E, G–I, R-T and V)
(MC- type A)

46
Toxic shock syndrome toxin Most strains belong to phage group I
Enterotoxins F (pyrogenic exotoxin C) is the
most common TSST, followed by Enterotoxin
B,C
Risk factor: Use of vaginal tampon by
menstruating females (however, males and
nonmenstruating females also get effected
rarely)
Anti TSST1 Antibody is protective
Manifestations: Rash, fever, hypotension and
Multi organ failure
Diagnosis: Detection of TSST by latex
agglutination test and enzyme immunoassay.
Detection of TSST genes 1 and 2 by PCR
Treatment: Clindamycin (reduces toxin
synthesis)

➢ Extracellular enzymes:
● Specific to S.aureus: Coagulase, heat stable thermonuclease, DNase,
phosphatase
● Present in most staphylococci: Protease, lipase, staphylokinase
(fibrinolysin), hyaluronidase

★ Pathogenesis
➢ Staphylococcus aureus is the MC agent of the following conditions:
● Skin and soft tissue infections
● Botryomycosis (mycetoma-like condition)
● Tropical pyomyositis – S. aureus, (acute bacterial myositis –
Group A Streptococcus) (Overall - S.aureus)
● Osteomyelitis and septic arthritis (MC site- knee)
● Postoperative parotitis
● Paronychia
● Pyomyositis (skeletal muscle infection): In tropics and HIV
infected people (Overall MC agent - S.aureus, except in acute
bacterial myositis – Group A Streptococcus is the MC agent)
● Pneumatocele-Shaggy, thin-walled cavities in lungs) in neonates
● Abscess: Psoas abscess and epidural abscess
● Surgical wound infection
● Folliculitis, furuncle, carbuncle and Hidradenitis suppurativa
● Mastitis and breast abscess (in nursing mothers)
● Toxin-mediated diseases: Toxic shock syndrome, food
poisoning, scalded-skin syndrome

➢ Infections associated with Community: Associated Methicillin Resistant S.

aureus (CA-MRSA) While skin and soft tissues are the most common sites for
CA-MRSA strains; 5–10% of strains are invasive and can cause various invasive
infections, such as:
● Necrotizing pneumonia
● Sepsis with Waterhouse: Friderichsen syndrome or purpura fulminans
(S. aureus is rare cause; most commonly caused by meningococci).
● Necrotizing fasciitis (S. aureus is a rare cause, Streptococcus

47
 pyogenes is the most common cause)

★ Endocarditis:
➢ MC cause of Native valve endocarditis: Overall or Hospital
acquired-S.aureus, Community acquired- S.viridans
➢ MC cause of Prosthetic valve endocarditis
● Early prosthetic valve endocarditis (<12 months):
Staphylococcus epidermidis
● Late prosthetic valve endocarditis (>12 months): Viridans
streptococci
● Overall MC cause of prosthetic valve endocarditis,
Staphylococcus epidermidis
➢ MC cause of Endocarditis in IV drug users:
● Rt sided – Staphylococcus aureus,
● Lt sided – Enterococcus >Staphylococcus aureus
● Over all – Staphylococcus aureus
➢ MC cause of Subacute endocarditis – Viridans streptococci

Laboratory Diagnosis

Direct smear microscopy Pus cells with gram positive cocci in cluster

Culture

Nutrient agar Golden yellow pigmented colonies (pigments

made up of beta carotene)

Blood agar Colonies with narrow zone of β-hemolysis

Selective media ● Mannitol salt agar (yellow colonies due to

mannitol fermentation)
● Salt milk agar
● Ludlam’s medium

Culture smear microscopy Gram-positive cocci in clusters

Biochemical identification

Catalase test Positive

Of test shows fermentative pattern

Coagulase test (slide and tube) Positive

Heat stable thermo nuclease Positive

test

DNase test Positive

Phosphatase test Positive

Mannitol sugar Fermented

48
potassium tellurite agar Black colored colonies

Gelatin liquefaction Positive

Protein A Detection

MC method for typing of Phage typing (pattern method)

S.aureus

National reference centre for in Maulana Azad Medical College, Delhi.

phage typing

Epidemic strain of S. aureus Phage type 80/81

It causes outbreaks in hospitals

Treatment of Staphylococcus aureus Infections

Since S. aureus rapidly develops drug resistance, antibiotics should be cautiously
chosen

Parenteral therapy for serious infections:

Sensitive to penicillin DOC: Penicillin G

Sensitive to methicillin DOC: Nafcillin or oxacillin

Resistant to methicillin (MRSA) DOC: Vancomycin (15–20 mg/kg bd)

Alternate drugs: See text below

Empirical therapy (if MRSA Vancomycin with or without an aminoglycoside

status not yet known) Vancomycin is indicated only if MRSA risk is
high or condition is serious, e.g. cardiac Oral
therapy for skin and soft tissue infections
Sensitive to methicillin implant

Oral therapy for skin and soft tissue infections

Sensitive to methicillin Dicloxacillin, cephalexin

Resistant to methicillin (MRSA) Clindamycin

Alternate drugs: Cotrimoxazole, doxycycline,
linezolid

★ Drug Resistance in S. aureus

➢ Resistance in S. aureus to β lactam antibiotics

➢ S. aureus shows resistance to β lactam antibiotics in various ways:
1. Production of β lactamase enzyme: β lactamase or penicillinse
enzymes cleave the β lactam ring:
➢ This resistance is plasmid coded, can be transferred between
S.aureus strains by transduction.
➢ It is produced by > 90% of strains of S.aureus.
➢ This resistance can be overcome by addition of β lactamase
inhibitors such as clavulanic acid or sulbactam.

49
2. By alterations of PBP: It is shown by MRSA strains
➢ Methicillin Resistant Staphylococcus aureus (MRSA)
MRSA is mediated by mecA gene; which is a chromosomally
coded. It alters penicillin binding protein (PBP) present on
S.aureus cell membrane to PBP-2a:
● PBP is an essential protein needed for cell wall synthesis of
bacteria. β lactam drugs bind and inhibit this protein,
there by inhibiting cell wall synthesis.
● The altered PBP2a of MRSA strains has less affinity for β
lactam antibiotics; hence MRSA strains are resistant to all
β lactam antibiotics.
● BORSA strains (Borderline Oxacillin resistant S.aureus):
Occasionally a non-mecA gene mediated low level resistance to
oxacillin is observed in some strains of S.aureus, which is due
to hyper production of β lactamase.
● There is an increasing trend of MRSA rate over last few
decades. Though it varies from place to place, overall
about 30–40% strains of S. aureus are MRSA.
● MRSA rate in India is 30-40%. It is lowest in Scandinavian
countries
➢ Detection of MRSA
● Antimicrobial susceptibility test: Disk diffusion test can be
done by using cefoxitin or oxacillin disks.
➔ Cefoxitin is the recommended disk to be used.
➔ If oxacillin disk is used, then certain conditions to be
maintained such as—using media containing 2–4% NaCl,
incubation at 30 °C for 24 hours.
● Oxacillin screen agar: Adding oxacillin 6 µg/ml and NaCl (2–4%)
to the medium.
● PCR detecting mecA gene • Latex agglutination test detecting
PBP-2a
➢ Treatment of MRSA
● Vancomycin is the drug of choice for MRSA.
● Alternate drugs include:
➔ Teicoplanin, linezolid, quinupristin-dalfopristin,
tigecycline, oritavancin
➔ Daptomycin (for endocarditis and complicated skin
infections),
➔ Mupirocin 2% ointment (for nasal carriers of MRSA)
● However, even simple orally effective drugs such as
tetracycline, erythromycin or cotrimoxazole may also be
effective. These can be indicated in non-serious conditions,
caused by CA-MRSA strains if found to be susceptible
based on antimicrobial susceptibility report.
● All β lactam drugs should be avoided. However, 5th
generation cephalosporins, such as Ceftobiprole, ceftaroline,
ceftolozane have shown some activity against MRSA.

★ Resistance to Vancomycin (VRSA and VISA)

➢ Erroneous and overuse of vancomycin may lead to emergence of

50
 resistance to vancomycin, which may be of two types:
● VRSA (Vancomycin Resistant S. aureus): High grade
resistance with MIC ≥ 16 µg/ml
● VISA (Vancomycin Intermediate S. aureus): Low grade
resistance with MIC 4-8 µg/ml
● Epidemiology: VRSA is very rare. In India, it is reported
from few places such as Hyderabad, Kolkata and Lucknow.
However, VISA is more frequently reported. Mechanisms:
➔ VRSA is mediated by van gene. A The van A gene is
believed to be acquired from a vancomycin-resistant
strain of Enterococcus fecalis by horizontal conjugal
transfer.
➔ VISA is due to increase in cell wall thickness of S.
aureus.
● Treatment of VRSA/VISA: Linezolid, telavancin,
daptomycin and quinupristin/ dalfopristin are the effective
drugs. Vancomycin and Teicoplanin are not effective.

➢ S. aureus carriers
● About 25–50% of healthy population are carriers of S.aureus.
● MC site of colonization: Anterior nares and Skin, (perineum, axilla,
groins)
● MC way of spread of infection in hospital- through the hands of
hospital staff
● Most effective way to prevent the hospital infection – handwashing
● DOC for nasal carriers of MRSA: Mupirocin 2% ointment.

★ COAGULASE NEGATIVE STAPHYLOCOCCUS (CoNS)

They are mostly the normal flora of skin.
➢ Staphylococcus Epidermidis
● MC CoNS—Accounts for 60–70% of CoNS
● Produces polysaccharide glycocalyx (slime) (Biofilm production)
● Adhere to any implanted foreign bodies like valvular shunts,
prosthetic devices
● Infections:
➔ Endocarditis with insertion of valvular prosthesis
➔ Ventricular shunt infections and Stitch abscess.
➢ Staphylococcus Saprophyticus
● It causes UTI in young sexually active females.
● It is resistant to novobiocin.
STREPTOCOCCUS PYOGENES (GROUP A STREPTOCOCCUS)
★ Classification
➢ On the basis of hemolysis, streptococci can be divided into 3 groups
1. α Hemolytic: (Partial or green hemolysis), e.g. Streptococcus
Viridans, Streptococcus pneumoniae
2. β Hemolytic: (Complete or yellowish hemolysis), e.g. β
haemolytic Streptococcus
3. γ Hemolytic: (no hemolysis is seen), e.g. Enterococci
➢ Lancefield’s grouping (for β haemolytic streptococci)
● Based on carbohydrate antigen in cell wall, the β haemolytic

51
 streptococci are further divided into 20 serogroups: Group A
to V except I and J.
● Carbohydrate antigen extracted by HCl (Lancefield’s
method), Formamide (Fuller’s method), Enzymatic (Maxted’s)
or autoclaving.
➢ Streptococcus group A (S. pyogenes) is further subdivided based on
➔ Griffith typing: Based on M protein (> 100 M serotypes) or
➔ emm typing: Based on gene coding for M protein, > 124 emm
genotypes identified.

★ Virulence factors

Cell wall antigens ● Inner thick peptidoglycan layer (confers cell wall
rigidity, induces inflammatory response and has
thrombolytic activity)
● C-carbohydrate antigen: Present as middle layer
and is group specific
● Outer layer of protein (M, T, R) and lipoteichoic
acid (helps in adhesion)
● M protein:
➔ Mediates adherence to epithelial cells,
inhibits phagocytosis
➔ Binds to fibrinogen and neutrophils leadings
to release of inflammatory mediators that
induce vascular leakage (streptococcal
toxic shock).
➔ M protein is further divided into Class I
and Class II. Antibodies to class IM
protein are responsible for pathogenesis of
rheumatic fever.

Capsule ● Expressed by mucoid strains, made-up of

hyaluronic acid.
● Capsule is anti-phagocytic, helps in adhesion; but
it is not antigenic.

SPE (Streptococcal ● 3 Types (SPE A, B and C): Type A and C are, e.g. o
pyrogenic exotoxin) or Superantigens
Erythrogenic toxin ● Type A and C bacteriophage coded, B toxin
chromosomal mediated
● Pathogenic role: Associated with the
pathogenesis of scarlet fever, necrotizing
fasciitis and streptococcal toxic shock syndrome.
● Dick test: Intradermal injection of SPE produces
erythema only in those children who are
susceptible to develop scarlet fever.
● Schultz Charlton reaction (blanching of rash
after injection of anti SPE antibodies): Used for
diagnosing scarlet fever in past

Hemolysins Streptolysin O and Streptolysin S (see table below)

52
Streptokinase ● Fibrinolysin (activates plasminogen)
● Rapid spread: By preventing the formation of
fibrin barrier.
● Therapeutically used in treatment of coronary
thrombosis.

DNase ➢ Also called Deoxyribonuclease or Streptodornanse

(4 types: A, B,C,D)
● Diagnostic use: Anti-DNase B > 300–350 U
is useful for the retrospective diagnosis of
skin infections (pyoderma) and acute
glomerulonephritis where ASO titer is low
● Therapeutic use: Preparation containing
streptodornase and streptokinase can be
used to liquefy the thick exudates in
empyema cases.

Other enzymes ● Hyaluronidase (spreading factor): Expressed by

noncapsulated strains, such as M type 4 and 22. It
breaks down the hyaluronic acid of the tissues,
thus helps in the spread of infection along the
intercellular space
● Serum opacity factor: Lipoproteinase enzyme in
nature
● NADase, C5a peptidase and SpyCEP (inactivates
IL- 8)

Manifestations
Streptococcus pyogenes causes both suppurative and nonsuppurative
manifestations.

❖ Suppurative Manifestations
➢ Respiratory infections: • Pharyngitis/sore throat (MC cause, 20–40%
of all cases) • Pneumonia and empyema
➢ Scarlet fever (MC cause ): Now rare, characterized by: • Pharyngitis
and Sandpaper rashes, strawberry tongue • Pastia’s lines- prominent
rashes in skin folds • Pathogenesis is due to SPE toxin (Dick test +ve)
➢ Skin and soft tissue infections:
● Impetigo (pyoderma): (MC cause)
➔ Seen in children, poor hygiene, warm climate
➔ Characterized by pustular lesions that
becomehoneycomb like crusts, no fever, painless.
➔ Associated with higher M types, and nephritogenic
strains.
● Cellulitis and erysipelas (MC cause):
➔ Tender, bright red, swollen and indurated peaud’orange
texture of skin (due to involvement of the superficial
lymphatics) along with fever and chills.
➔ MC site- malar area of the face, seen in older people.
➢ Deep soft tissue infections:
● Necrotizing fasciitis or streptococcal hemolytic gangrene- S.

53
 pyogenes is MC cause (60%), it is rapidly spreading, hence S.
pyogenes is also called flesh eating bacteria
● Toxic shock syndrome (staphylococcal TSS is MC, but
bacteremia is MC in streptococcal TSS)
● Streptococcal myositis (S. aureus is MC cause of myositis)
➢ Complications:
● Puerperal sepsis (Group B Streptococcus is MC cause),
● Others: Otitis media, Quinsy, Ludwig’s angina, pneumonia (post
viral), osteomyelitis, meningitis

❖ Nonsuppurative Complications
➢ Streptococcal antigens show molecular mimicry with human antigens.
Due to antigenic cross reactivity, antibodies produced against
previous streptococcal infections cross react with human tissues to
produce lesions. This accounts for a number of nonsuppurative
complications such as:
● Acute rheumatic fever
● Poststreptococcal glomerulonephritis (PSGN)
● Guttate psoriasis
● Reactive arthritis
● Pediatric Autoimmune Neuropsychiatric Disorders Associated
with Streptococcus pyogenes (PANDAS)

★ Laboratory Diagnosis

Transport medium Pike’s medium

Direct smear Pus cells with gram-positive cocci in short chains

microscopy

Culture ● Blood agar: Pinpoint colony with a wide zone of

β-hemolysis
● Selective media: Crystal violet blood agar and
PNF (polymyxin B, neomycin, fusidic acid) media
● Liquid media: Granular turbidity with powdery
deposit

Biochemical ● Catalase negative,

identification: ● Bacitracin sensitive and
● Pyrrolidonyl Arylamidase (PYR) test is positive

Typing ● Lancefield grouping: Shows group A

Streptococcus
● Typing of group A Streptococcus: Griffith
typing and emm typing

Serology ➢ ASO antibodies and Anti-DNase B antibodies

● ASO antibodies titer is elevated > 200
Todd unit/ml in most streptococcal
infections except in pyoderma and PSGN.
● Anti-DNase-B Ab – Titer > 300–350
units/ml is diagnostic of PSGN and

54
 pyoderma.
● Other antibodies elevated are
Antihyaluronidase and antistreptokinase
antibodies.

★ Treatment of streptococcal infection

Penicillin is the drug of choice for all type of streptococcal infections

Condition Treatment recommended

Pharyngitis Benzathine penicillin G, IM single dose or oral penicillin V

for 10 days

Erysipelas/Cellulitis ● Mild- Procaine penicillin

● Severe- Penicillin G

Necrotizing fasciitis Surgical debridement (most crucial) + Penicillin G +

Clindamycin

Pneumonia and Penicillin G + drainage of empyema

empyema

Streptococcal TSS Penicillin G + Clindamycin + immunoglobulin (to SPE)

Rheumatic fever ➢ Benzathine penicillin G, IM single dose; or oral

Penicillin V for 10 days
➢ Long-term maintenance therapy with penicillin G
monthly:
● For 5 yrs or until 21 yrs of age, (without
carditis) • For 10 yrs (with carditis)
● up to 40 yrs of age/lifelong (with residual
heart disease)

PSGN Benzathine penicillin G, IM single dose; or oral penicillin V

for 10 days

Treatment of asymptomatic carriers

Pharyngeal carrier Penicillin V + rifampicin

Rectal carriers Vancomycin + rifampicin

➢ Prophylaxis
● Penicillin V + rifampicin Vancomycin + rifampicin Long-term
maintenance therapy with penicillin (alternative-sulfadiazine or
erythromycin in penicillin allergy) is required for children who develop
early signs of rheumatic fever. This prevents streptococcal
reinfection and further damage to heart.

GROUP B STREPTOCOCCUS (S. AGALACTIAE)

★ Pathogenesis:

55
 ➢ Approximately 30% of women are vaginal or rectal carriers of group B
Streptococcus. Hence, the infection is common in neonates and in
pregnancy. It is a major cause of:
● Neonatal sepsis and meningitis: Neonatal sepsis can be of two
types-early onset and late onset type
● Puerperal sepsis and peripartum fever
● Infections in elderly people with underlying illness, such as
diabetes mellitus or malignancy: Cellulitis and soft tissue
infections, UTI, pneumonia, and endocarditis.

★ Laboratory Diagnosis: It can be differentiated from Group A Streptococcus

by following biochemical tests
● CAMP positive, Hippurate hydrolysis test positive, Bacitracin resistant
and PYR test is negative
● Orange pigment production- enhanced in Islam’s medium.
● β hemolytic colonies, which are mucoid and slightly larger (2 mm).
● It has a capsular polysaccharide, which can be typed into nine
serotypes.

Characters S. pyogenes S. agalactiae

Lancefield group Group A Group B

Bacitracin sensitivity test Sensitive Resistant

PYR test Positive Negative

Hippurate hydrolysis test Negative Positive

CAMP test Negative Positive

β hemolytic colonies 0.5–1 mm, pin point Mucoid, larger (2 mm)

Early and late onset Group B Streptococcus disease in neonates

Characteristics Early-onset disease Late-onset disease

Age of onset 0–6 days of birth 7–90 days of birth

Increased risk following Prematurity and prolonged Not associated

obstetric complications labor

Mode of transmission to During or before birth Contact with a colonized

the baby from the colonized mother and nursing
maternal genital tract personnel

Common clinical Pneumonia and/or Bacteremia and meningitis

manifestations respiratory distress (most common)
syndrome followed by
meningitis

Common serotypes Ia, III, V, II, Ib III predominates

56
Case fatality rate 4.7% 2.8%

ENTEROCOCCUS
➢ The enterococci were initially grouped under group-D Streptococcus, but
later, it has been reclassified as a separate genus Enterococcus under family
Enterococcaceae.
● Enterococci are the part of normal flora of human GIT. At the
same time, they are also increasingly important agents of human
disease especially in hospitals mainly because of their resistance to
antibiotics.
● E. faecalis is the most common species found in clinical specimens;
whereas E. faecium is more drug resistant than E. faecalis.

➢ Various Clinical Manifestations Include

● Urinary tract infections (cystitis, urethritis, pyelonephritis and
prostatitis)
● Bacteremia and mitral valve endocarditis (in IV drug abusers)
● Intra-abdominal, pelvic, and soft tissue Infection
● Late-onset neonatal sepsis and meningitis
● Infection on burn surface.

❖ Laboratory Diagnosis
➢ Enterococci show the following characteristics that help in the
identification:
● They are gram-positive oval cocci arranged in pairs (spectacle
eyed appearance)
● Nonmotile cocci (except E. gallinarum and E. casseliflavus)
● Blood agar: It produces nonhemolytic, translucent colonies
(rarely produces α or β hemolysis)
● MacConkey agar: It produces minute magenta pink colonies.
● Bile aesculin hydrolysis test is positive
● PYR test is positive
● Growth occurs in presence of:
➔ 6.5% NaCl, 40% bile and pH 9.6
➔ Heat tolerance test: They are relatively heat resistant,
can survive 60°C for 30 minutes.

❖ Treatment
➢ Most strains of enterococci are resistant to penicillins,
aminoglycosides and sulfonamides. They show intrinsic resistance to
cephalosporins and cotrimoxazole.
● Resistance is overcome by combination therapy with penicillin
and aminoglycoside (due to synergistic effect) and is the
standard therapy for life-threatening enterococcal infections;
however in UTI, monotherapy with ampicillin or
nitrofurantoin is sufficient. Resistance to this combination
therapy may also develop.
● Vancomycin is usually indicated in resistant cases but resistance
to vancomycin has also been reported.

57
 ★ Vancomycin Resistant Enterococci (VRE)
➢ Vancomycin resistance in enterococci has been increasingly reported
nowadays.
● VRE is mediated by Van gene, which codes for altered target
site for vancomycin in the cell wall (i.e. D-alanyl-D-alanine side
chain of peptidoglycan layer is altered to D-alanylD-serine or
D-alanyl-D-lactate) and this altered side chains have less
affinity for binding to vancomycin.
● Van gene has 9 genotypes. Important ones are: VanA to VanE.
➔ Strains with VanA gene show high level resistance to
both glycopeptides- vancomycin and teicoplanin.
➔ Strains with VanB gene show low level resistance to
vancomycin, but sensitive to teicoplanin.
➔ E. gallinarum and E. casseliflavus possess VanC genes
which is chromosomal coded (other genotypes transposon
coded), and they show intrinsic resistance to both
glycopeptides.
● Screening of patients for VRE is carried out by: Rectal swab
culturing on Bile esculin azide agar with 6 μg/ml vancomycin

VIRIDANS STREPTOCOCCI
➢ Viridans streptococci are α hemolytic, commensal of mouth
● S. mutans: Causes dental caries and plaques
● S. sanguis: Causes subacute bacterial endocarditis
● S. milleri group: Produces suppurative infections, differ in
hemolytic pattern (may be α, β or γ hemolytic).

➢ Treatment: Usually sensitive to penicillin (except neutropenic patients with

bacteremia; where vancomycin is given).

PNEUMOCOCCUS V/S VIRIDANS STREPTOCOCCI

➢ S. pneumoniae can be differentiated from Viridans streptococci by various
features:

Properties Pneumococcus Viridans streptococci

Morphology Lanceolate or flame shaped Round/oval

Arrangement Gram-positive cocci in pairs Gram-positive cocci in long

chains

Capsule Present Absent

On blood agar Draughtsman or carom coin colony Convex shaped colony

Liquid medium Uniform turbidity Granular turbidity

Bile solubility Soluble in bile Insoluble in bile

Inulin fermentation Fermenter Nonfermenter

58
Optochin Sensitive Resistant

Mice Pathogenicity Pathogenic Nonpathogenic

NEISSERIA MENINGITIDIS (MENINGOCOCCUS)

➢ Virulence Factors
● Capsular polysaccharide: It prevents the bacteria from phagocytosis,
can be typed into 13 serogroups:
➔ Only 5 serogroups account for the majority of cases: A, B, C, Y,
and W135;
➔ Other capsular serotypes and noncapsulated strains (16% of
isolates are not capsulated) colonize the nasopharynx of
asymptomatic carriers.
● Outermembrane proteins: OMP is used to classify serogroups to
serotypes.
● Lipopolysaccharide and endotoxin: Causes endothelial injury leads to:
➔ Increased vascular permeability leading to loss of fluid and
shock
➔ Intravascular thrombosis leading to disseminated intravascular
coagulation (DIC)
➔ Waterhouse-Friderichsen syndrome
➔ Myocardial dysfunction
● IgA proteases—Cleave mucosal IgA
● Transferrin binding protein.
● Adhesins: Mediated by OPA protein and pili.

❖ Epidemiology
Worldwide, nearly 5 lakh cases of meningococcal disease occur each
year, and 10% of those die.
➢ Disease pattern: There are several patterns of the disease noted:
● Epidemics occurs mainly in sub-Saharan Africa—Due to group A
(mainly) and W135.
● Outbreaks—mainly due to serogroup C (in semi-closed
communities such as schools, military camps , etc.).
● Hyperendemic disease (> 10 cases per 100,000 population) Due
to serogroup B.
● Sporadic cases (0.3–5 cases per 100,000 population) can occur
due to all, i.e. A, B, C, Y, and W135.
➢ High prevalence area is sub-Saharan belt of Africa (from Ethiopia to
Senegal)
➢ Seasonal variation is seen commonly in winter and spring (cold and dry
climate)
➢ Age: Meningitis is common in early childhood (3 months to 5 years).
➢ Risk factors that promote colonization include:
● Overcrowding and semi-closed communities such as schools,
military and refugee
● Travellers (Hajj pilgrims) and smoking
● Viral and Mycoplasma infection of the respiratory tract
➢ Risk factors that promotes disease include:
● Deficiency of terminal complement components (C5–C9)

59
 ● Hypogammaglobulinemia
● Hyposplenism.

❖ Pathogenesis
➢ Source: MC source of infection is human nasopharyngeal carriers
(mainly children).
➢ Carrier rate may vary from 5–10% (during inter epidemic period) up
to 70–80% (during epidemic).
➢ Mode of transmission is by droplet inhalation
➢ Spread of infection: From nasopharynx, meningococci reach the
meninges either by:
● Hematogenous route (most common) or
● By direct olfactory nerve spread through cribriform plate or
● Rarely through conjunctiva.
➢ Case fatality ratio is 80% (falls to 10% if early treatment is started).

❖ Clinical Manifestations
➢ Asymptomatic colonization is the most common presentation. Various
manifestations include
● Rashes: A nonblanching rash (petechial or purpuric) develops in >
80% of cases.
● Septicemia: It is attributed to endotoxin induced endothelial
injury
● Waterhouse-Friderichsen syndrome: It is a severe form of
fulminant meningococcemia, characterized by large purpuric
rashes (purpura fulminans), shock, DIC, bilateral adrenal
hemorrhage and multi-organ failure.
● Pyogenic meningitis: Commonly affects young children (3–5
years of age).
● Chronic meningococcemia: Occurs rarely and characterized by
petechial rash, fever, arthritis, and splenomegaly.
● Postmeningococcal reactive disease: Immune complexes
develop 4–10 days later, lead to manifestations like arthritis,
rash, iritis, pericarditis, polyserositis and fever.

❖ Laboratory Diagnosis
➢ Specimen:
● For cases: Blood and CSF
● For carriers: Nasopharyngeal swab
➢ CSF examination:
● First portion is centrifuged and used for:
➔ Capsular antigen detection
➔ Biochemical analysis: ↑CSF pressure, ↑protein and ↓glucose
in CSF
➔ Gram staining: Pus cells with gram-negative
diplococci, lens-shaped ○ Second portion: For
culture on blood agar, chocolate agar ○ Third
portion is enriched in BHI broth and incubated
for 7 days
➢ Nasopharyngeal swab culture: On Thayer Martin medium
➢ Biochemical tests:

60
 ● Oxidase and catalase positive
● Ferment glucose and maltose but not sucros
➢ Serogrouping: by latex agglutination test:
➢ Serology: Antibodies to capsular Ag (ELISA), Useful in retrospective
diagnosis of disease
➢ Molecular diagnosis: By multiplex PCR.

❖ Treatment
➢ DOC for treatment → Ceftriaxone and cefotaxime
➢ DOC for carriers and prophylaxis→ Ceftriaxone (DOC), others:
Rifampicin and ciprof loxacin.

❖ Vaccine
➢ Polyvalent vaccine containing → Four groups A,C,Y, W135
● No vaccine for Group B:
➔ As Group B capsule is made up Sialic acid residue which is
encephalitogenic and poorly immunogenic
➔ However, Outer membrane vesicles (OMVs) based
vaccines trails are going on.

NEISSERIA GONORRHOEAE (GONOCOCCUS)

N.gonorrhoeae is noncapsulated, Gram negative kidney shaped diplococci.
❖ Virulence Factors
➢ Pili or fimbriae: Principal virulence factor, helps in adhesion and inhibit
phagocytosis.
➢ Outer membrane proteins:
● Porin (protein I): They form membrane channels (pores) There
are two major serotypes: Por B.1A and PorB.1B serotypes.
➔ PorB.1A strains associated with local and
disseminated gonococcal infections (DGI)
➔ PorB.1B strains usually cause local genital infections.
● Opacity-associated protein (Protein II): It helps in adhesion,
and invasion
➢ Transferrin-binding and lactoferrin binding protein: It is required for
uptake of iron
➢ IgA1 protease: It degrades mucosal IgA antibody
➢ Lipooligosaccharide (LOS): It differs from LPS of
Enterobacteriaceae by lacking the repetitive O side chain.

❖ Typing of Gonococci
➢ Serotyping is based on protein-I (porin).
➢ Auxotyping: Typing is based on nutritional requirements of the
strains, e.g. AHU auxotype needs arginine, hypoxanthine and uracil as
growth factors.

❖ Clinical Manifestations
➢ Gonorrhea is a venereal disease reported since ancient time.
1. In males:
➢ Acute urethritis is the most common manifestation.
Purulent urethral discharge (the word ‘gonorrhea’ is

61
 derived from flow of seed resembling semen)
➢ The usual incubation period is 2–7 days.
➢ Complications: Epididymitis, prostatitis, edema of the
penis, and balanitis.
➢ Infection may spread to periurethral tissues causing
abscess with sinus formation (known as water-can
perineum).
2. In females:
➢ Gonococcal infection is less severe in females with more
asymptomatic carriage:
● Mucopurulent cervicitis is the most common
presentation.
● Vulvovaginiti seen in prepubertal girls and
postmenopausal women, but not in adult females
as the adult vagina is resistant to gonococcal
infection (due to its low pH and thick stratified
squamous epithelium).
● Salpingitis and pelvic inflammatory disease may
lead to sterility.
● Fitz-Hugh-Curtis syndrome: It is a rare,
characterised by peritonitis and associated
perihepatitis.
3. In both the sexes:
➢ Anorectal gonorrhea (as acute proctitis): Rectal
isolates are usually drug resistant.
➢ Pharyngeal gonorrhea (spread by orogenital sex)
➢ Ocular gonorrhea.
4. In neonates (Ophthalmia neonatorum):
➢ Characterized by purulent eye discharge, occurs within
2–5 days of birth.
➢ Transmission occurs during birth from colonized maternal
genital flora.
➢ Treatment: Silver nitrate solution into the eyes of
newborn (Crede’s method).
5. Disseminated gonococcal infection (DGI):
➢ Occurs in 0.5–3% of untreated persons:
● DGI is characterized by polyarthritis and rarely
dermatitis and endocarditis.
● It is most commonly associated with PorB.1A
serotypes and AHU auxotypes.
● Menstruation and complement (C5–C9) deficiency
are risk factors for DGI
6. In HIV-infected persons: Enhances the transmission of HIV.

❖ Laboratory Diagnosis
➢ Sample
● Males: Urethral discharge
● Females: Endocervical swab (high vaginal swab not
recommended).
● For DGI: Blood culture and synovial fluid culture.

62
 ➢ Transport media
● Charcoal impregnated swabs/medium (Stuart/Amies media)
● For longer holding period: CO2 generating system (JEMBEC
system)
➢ Gram staining
● For males: Gram staining of urethral discharge is more sensitive
(90%): Based on which treatment can be started
● For females: Gram staining is less sensitive (50–60%) due to
presence of commensal Neisseria spp. in genital tract. So,
Endocervical culture is recommended.
Endocervical culture: Gonococci are difficult to grow than
meningococci:
● Culture media in acute gonorrhoeae: Chocolate agar and
Mueller-Hinton agar
● Selective media are useful in chronic cases:
➔ Thayer: Martin media
➔ Modified New York city medium
➔ Martin: Lewis Media
➢ Treatment • DOC: Single-dose regimen of Ceftriaxone and cefotaxime
• Treat both the partners, regimen should also include azithromycin for
Chlamydia • Most strains are resistant to penicillin due to penicillinase
production. Such strains are called as PPNG strains (Penicillinase
producing strains of N. gonorrhoeae)

NON-GONOCOCCAL URETHRITIS (NGU)

Agent ➢ Bacterial:
● Chlamydia trachomatis- MC cause of NGU
● Ureaplasma urealyticum
● Mycoplasma hominis
● Some cases may be due to gonococcal infection,
the cocci persisting as L forms and hence
undetectable by routine tests
➢ Viruses: Herpesvirus and cytomegalovirus
➢ Fungi: Candida albicans
➢ Parasites: Trichomonas vaginalis

Onset Longer (> 1 week)

Urethral discharge Mucous to mucopurulent

Complication Reiter’s syndrome: Characterized by conjunctivitis,

urethritis, arthritis and mucosal lesions

Other Complications ➢ Males: Epididymitis, prostatitis, seminal vesiculitis and

balanitis
➢ Females: Salpingitis and pelvic inflammatory disease
and Fitz-Hugh-Curtis syndrome

Diagnosis ➢ For Chlamydia: Culture on McCoy and HeLa cell lines

➢ Trichomonas: Detection of trophozoite

63
 ➢ Candida: Detection of budding yeast cells in discharge
➢ PCR: Can be done for HSV or Chlamydia

Treatment ➢ For Chlamydia: Doxycycline

➢ For Trichomonas: Metronidazole
➢ For Candida: Clotrimazole (as vaginal cream or tablet)

CORYNEBACTERIUM DIPTHERIAE
❖ Morphology: Corynebacterium are club-shaped gram-positive, noncapsulated,
non-sporing, non-motile rods.

❖ Pathogenicity
➢ Diphtheria is toxemia but never a bacteremia:
● Bacilli are noninvasive, present only at local site (pharynx),
secrete the toxins which spread by bloodstream to various
organs.
● It is the toxin which is responsible for all types of
manifestations including local (respiratory) and systemic
complications (except the skin lesions which may be caused
due to the organism).

❖ Laboratory Diagnosis:
➢ Laboratory diagnosis of diphtheria is necessary only for:
● Confirmation of clinical diagnosis
● Initiating the control measures
● Epidemiological purposes (Not to start treatment)
➢ Culture media:
● Enriched medium: Such as Loeffler’s serum slope
➔ Detects growth early (6–8 hrs)
➔ Best medium for metachromatic granules production
● Selective medium: Such as PTA and Tinsdale medium
➔ Best media for isolation
➔ Black colonies appear only after 48 hours
➢ Toxin detection:
● In vivo tests (Guinea pigs inoculation)
● In vitro tests
➔ Elek's gel precipitation test
➔ Detection of tox gene- by PCR
➔ Detection of diphtheria toxin by ELISA
➔ Cytotoxicity produced on cell lines

❖ Prophylaxis (Vaccination)
➢ Active immunization is done by diphtheria toxoid that induces
antitoxin production in the body.
➢ Protective titer of antitoxin is > 0.01 Unit/ml.
➢ Herd immunity of > 70% is required to prevent epidemic spread of
diphtheria.
➢ However, vaccine is not effective for:
● Prevention of cutaneous diphtheria
● Elimination of carrier stage

64
 ❖ Types of vaccine:
➢ Single vaccine: Diphtheria toxoid (alum or formal precipitated)
➢ Combined vaccine: Nondiphtheriae Corynebacterium species
● DPT: Contains DT (diphtheria toxoid), Pertussis (whole cell)
and TT (tetanus toxoid)
● DaPT: Contains DT, TT and acellular pertussis (aP)
● DT: Contains DT and TT
● dT: Contains adult dose diphtheria toxoid (d) and TT.

ANTHRAX
Cutaneous anthrax Pulmonary anthrax

Also called Hide porter’s disease (as it commonly Wool sorter’s disease (as it
occurs in dock workers carrying loads is seen in workers of wool
of hides and skins on their bare backs) factory, due to inhalation
of dust from infected
wool)

Transmission Cutaneous exposure to spores (enter Inhalation of spores

through abraded skin)

Characterized ➢ Malignant pustule: ➢ Hemorrhagic

by ● Painless coal-black, pneumonia: Bacilli
necrotic eschar spread by
surrounded by lymphatics or blood,
non-pitting indurated leading to:
edema. ● Bacteremia
● The name anthrax, which ● Hemorrhagic
means coal, comes from mediastinitis
the black color of the ● Hemorrhagic
eschar. meningitis
● However, it is a
nonmalignant condition.

Occupations Dock worker, butcher, abattoir and Wool factory

farmer

Occurrence Most Common (95%) Rare

Prognosis Self-limiting, rarely becomes fatal if Fatal

untreated

Bioterrorism Rarely associated with bioterrorism Most common form to be

associated with
bioterrorism

❖ Laboratory Diagnosis (Anthrax vs Anthracoid Bacilli)

➢ B. anthracis can be differentiated from other Bacillus species
(Anthracoid Bacilli) by the presence of following properties:
● B. anthracis is Nonmotile and Capsulated.
● MC Fadyean reaction: Polypeptide capsule is seen as

65
 amorphous purple material surrounding blue bacilli when stained
with polychrome methylene blue.
● Gram staining: chain of bacilli arranged in bamboo stick
appearance.
● On Agar plate: Medusa head appearance colony (under low
power microscope)
● Gelatin stab: Appear as inverted fir tree appearance
● Solid media with penicillin: String of pearl appearance in
culture smear
● Blood agar: nonhemolytic colonies
● Selective medium: PLET media
● DFA (Direct fluorescent antibody test): Detects capsular
antigen. It is used for confirmation of diagnosis during
bioterrorism outbreaks.
● Ascoli’s thermo precipitin test: It is a ring precipitation test,
detects anthrax antigens.
● Spores can be demonstrated by phase contrast microscope or
use of special stains such as hot malachite green (Ashby’s
method) or 0.25% sulfuric acid (spores are acid fast).
● Lipid granules can be demonstrated by Sudan black B (Burdon’s
method).

GAS GANGRENE
➢ Gas gangrene (by (Oakley) is defined as a rapidly spreading, edematous
myonecrosis, occurring in association with severely crushed wounds
contaminated with pathogenic clostridia, particularly with C. perfringens. It
is always polymicrobial:
● Established agents: C. perfringens (MC, 60%) and C. novyi and C.
septicum (20–40%).
● Probable agents: C. histolyticum, C. sporogenes, C. fallax, C.
bifermentans, C. sordellii, C. aero foetidum and C. tertium.

❖ Pathogenesis of Gas Gangrene

➢ The development of gas gangrene requires:
● Anaerobic environment: Crushing injuries of muscles (road
traffic accidents) or foreign bodies (bullet injuries)
● Contamination of wound with clostridial spores present in
the soil (during war or road traffic accident) or clothes.
● Rarely, spontaneous non-traumatic gas gangrene occurs via
invasion of bowel clostridia; it occurs in people with
gastrointestinal pathologies (e.g. colonic malignancy).

❖ Incubation Period of Gas Gangrene

➢ The incubation period is variable, depending upon the nature of
injury, infective dose and clostridial species involved:
● 10–48 hrs for C. perfringens, 2–3 days for C. septicum and 5–6
days for C. novyi

➢ Gas Gangrene is Clinically Characterized By

● Sudden onset of excruciating pain at the affected site

66
 ● Rapid development of a foul-smelling thin serosanguineous discharge
● Gas bubbles (crepitus) in the muscle planes and brawny edema and
induration
● Shock and organ failure develop later
● Associated with higher mortality rate (50%).

❖ Laboratory Diagnosis
➢ Specimen: Necrotic tissues, muscle fragments and exudates from
deeper wound.
➢ Direct microscopy:
● Thick, stubby, boxcar-shaped gram-positive bacilli without
spore – suggestive of C. perfringens
● Gram-positive bacilli with spore- suggestive of other
Clostridium species
➢ Culture Media: Robertson cooked meat broth (RCM), egg yolk agar, etc.
➢ Identification of C. perfringens:
● Target hemolysis (double zone hemolysis)
● Nagler’s reaction: Opalescence surrounding the streak line
on egg yolk agar, inhibited by adding anti-alpha toxin.
● Reverse CAMP test: Positive.

❖ Treatment
➢ Early surgical debridement is the most crucial step in the management
of gas gangrene.
➢ Antibiotics: Penicillin + clindamycin are recommended for 10–14 days.
➢ Hyperbaric oxygen: It may kill the obligate anaerobic clostridia, such
as C. perfringens
➢ Passive immunization with anti α toxin antiserum.

TETANUS
❖ Mode of Transmission
➢ Tetanus bacilli enter through:
● Injury like road traffic accidents, unsterile
surgery/abortion/delivery, otitis media (otogenic tetanus)
● It is noninfectious: There is no person to person spread

❖ Clinical Manifestations
➢ Incubation period is about 6–10 days. Shorter the IP, graver is the
prognosis.
➢ Muscles of the face and jaw are often affected first (due to
shorter distances for the toxin to reach the nerve terminals).
➢ 1st symptom: Increase in the masseter tone leading to trismus or
lock jaw, followed by muscle pain and stiffness, back pain, and
difficulty in swallowing.
➢ In neonates, difficulty in feeding is the usual presentation
➢ As the disease progresses, painful muscle spasm develops which may
be:
● Localized: involves the affected limb
● Generalized painful muscle spasm: leads to descending spastic
paralysis.

67
 ➢ Hands, feet are spared and mentation is unimpaired
➢ Deep tendon reflexes are exaggerated
➢ Autonomic disturbance is maximal during the second week of
severe tetanuscharacterized by low or high blood pressure,
tachycardia, intestinal stasis, sweating, increased tracheal secretions
and acute renal failure.

❖ Complications
➢ Risus sardonicus: Characteristic, abnormal, sustained spasm of the
facial muscles that appears to produce grinning.
➢ Opisthotonos position of the body occurs due to generalized
spastic contraction of extensor muscles.
➢ Respiratory muscles spasm-may cause airway obstruction. Warm
climate, rural area with fertile soil is associated with increased risk.

❖ Laboratory Diagnosis
➢ Treatment should be started immediately based on clinical diagnosis.
Laboratory diagnosis helps only in confirmation.
● Specimen: Excised tissue bits from the necrotic depths of
wounds.
● Gram staining:
➔ Reveal gram-positive bacilli with terminal and round
spores (drumstick appearance)
➔ However microscopy alone is unreliable as it cannot
distinguish C. tetani from morphologically similar
clostridia like C. tetanomorphum and C. sphenoides.
● Culture: Culture is more reliable than microscopy:
➔ In RCM broth: C. tetani, being proteolytic turns the
meat black and produces foul odor.
➔ Blood agar: C. tetani produces characteristic swarming
growth.
● Toxigenicity Test: For demonstration of toxin production
➔ In vitro hemolysis inhibition test: detects tetanolysin
➔ In vivo mouse inoculation test: detects tetanospasmin

❖ Immunoprophylaxis:
➢ Tetanus toxoid (TT) is used for active immunization. It is
available either as (i) Monovalent vaccine as TT and (ii) Combined
vaccine as DPT
● Primary immunization of children: Under national
immunization schedule of India, total seven doses are given;
three doses of DPT at 6, 10 and 14 weeks of birth
followed by two booster doses of DPT at 16–24 weeks and
5 years followed by two additional doses of TT at 10 yrs and
16 yrs.
● Adult immunization:
➔ It is indicated if primary immunization is not
administered in childhood. Four doses of TT is given; 2
doses of TT at 1 month interval followed by 2
booster doses at 1 yr and 6 yrs.
● Site: TT is given deep IM at anterolateral aspect of thigh

68
 (children) and in deltoid (adults).
● Protective titer of tetanus antitoxin is ≥ 0.01
unit/ml.

BOTULINUM TOXIN
➢ C. botulinum is noninvasive and the pathogenesis is due to production of
powerful neurotoxin ‘botulinum toxin’(BT), probably the most toxic substance
known to be lethal to mankind:
● Serotype: Based on light chain, there are eight serotypes-A, B, C1, C2,
D, E, F and G:
➔ Serotypes A, B, E commonly cause human disease; most severe
being serotype A.
➔ All serotypes produce neurotoxin; except C2 which produces an
enterotoxin
➔ Botulinum toxin Type C and D are bacteriophage coded
● BT is produced intracellularly, not secreted and appears outside
only after autolysis of bacterial cell.
● BT is synthesized as protoxin, converted into active form by
proteolytic enzymes.
● Mechanism: BT blocks the release of acetylcholine in
neuromuscular junction, which leads to flaccid paralysis.
● Therapeutic uses: As BT produces flaccid paralysis it can be
used therapeutically for the treatment of spasmodic conditions, such
as strabismus, blepharospasm and myoclonus.
● BT is also produced by other clostridia, such as C. butyricum,C. baratti
and C. argentinense.
● Recovery: Blocking of acetylcholine release is permanent, but the
action is short lasting as the recovery occurs in 2–4 months, once
the new terminal axons sprout.

CLOSTRIDIUM DIFFICILE
➢ Clostridium difficile is the agent of ‘pseudomembranous colitis’which occurs
almost exclusively in association with prolonged antimicrobial use. It was
so named due to unusual difficulties involved in the isolation of C. difficile.

❖ Pathogenesis
➢ Clostridium difficile infection is associated with the following risk
factors:
● Prolonged hospital stay: Spores from hospital environment
gets colonized in colon of patients.
● Prolonged antimicrobial use can result in disruption of the
normal colonic flora:
➔ Cephalosporins (e.g. ceftriaxone) are frequently
responsible for this condition.
➔ Other antibiotics, such as clindamycin, ampicillin and
fluoroquinolones (ciprofloxacin)
➔ However, all antibiotics, including vancomycin and
metronidazole (which are the DOC in C. difficile

69
 infection) have been found to carry a risk of
infection, if given for prolonged duration.
● Toxin production: Pathogenesis is toxin mediated. C.
difficile (nontoxigenic strains) may be a part of normal
intestinal flora, however only the toxigenic strains can
cause pseudomembranous colitis:
➔ It produces two toxins: Toxin A (enterotoxin) and
Toxin B (cytotoxin). Both are important for
pathogenesis.
➔ Infants do not develop symptoms because they lack
suitable mucosal toxin receptors.
● Other risk factors include older age, underlying illness,
intestinal surgery, use of electronic rectal thermometers and
antacid treatment.

❖ Clinical Manifestations
➢ Diarrhea is the most common manifestation caused by C. difficile.
Other manifestations include fever, abdominal pain and leukocytosis.
Blood in stool is uncommon.
➢ Pseudomembrane formation over colonic mucosa with a relapse seen in
15–30% of cases.

❖ Laboratory Diagnosis
➢ Laboratory diagnosis of C. difficile infection depends on isolation of
the bacilli followed by toxigenicity testing:
➢ Stool culture on selective media, such as CCFA (cefoxitin cycloserine
fructose agar) or CCYA (cefoxitin cycloserine egg yolk agar). Stool
culture is highly sensitive but not specific. Toxin demonstration is
more meaningful.
➢ Toxin demonstration: Toxins can be detected by various methods:
● Cell culture cytotoxin test: It is highly specific but not as
sensitive as stool culture; it is time consuming.
● Enzyme immunoassay for toxin A and/toxins B in stool is rapid,
but not sensitive.
● PCR for C. difficile toxin B gene in stool it is highly specific and
sensitive.
➢ Colonoscopy is highly specific if pseudomembranes are seen, but
sensitivity is low.
➢ Histopathology of colonic pseudomembrane is also highly specific.

❖ Treatment
➢ Initial episode, mild to moderate cases: Oral metronidazole is the
drug of choice
➢ Recurrent episodes or severe cases: Vancomycin is the drug of choice
➢ Severe complicated or fulminant infection: Vancomycin plus IV
metronidazole

NON-SPORING ANAEROBES
❖ Common disease
➢ Mobilincus: Bacterial vaginosis (also caused by Gardnerella)

70
 ➢ Leptotrichia or Fusobacterium fusiformis: Agent of Vincent’s angina
(also caused by Borrelia vincentii)
➢ Anaerobic cocci: Puerperal sepsis and other female genital tract
infection (also by B. fragilis and Prevotella) Skin and soft tissue
infections.
➢ Bacteroides fragilis:
● Non-sporing anaerobe, capsulated gram-negative bacilli
● MC commensal in human intestine
● MC anaerobe to cause infection, causes abdominal infection
● Endotoxin is less toxic than that of aerobic gram-negative bacilli
➢ Fusobacterium necrophorum: Agent of Lemierre’s syndrome (is a form
of thrombophlebitis)

ESCHERICHIA COLI
❖ Clinical Manifestations of E.coli Infection
1. Urinary tract infection (UTI): Caused by uropathogenic E.coli
(UPEC) (described later)
2. Diarrhoea: Caused by six types diarrheagenic E.coli
3. Other syndromes:
➢ Abdominal infections: E.coli is the most common cause of both
primary bacterial peritonitis (occurs spontaneously) and
secondary bacterial peritonitis (occurs secondary to intestinal
perforation. It also causes visceral abscesses, such as hepatic
abscess.
➢ Pneumonia (especially in hospitalized patients): ventilator
associated pneumonia)
➢ Meningitis (especially neonatal meningitis)
➢ Wound and soft tissue infection.
➢ Osteomyelitis
➢ Endovascular infection and bacteremia.

❖ Diarrheagenic E. coli
1. Enteropathogenic E. coli (EPEC)
➢ EPEC frequently cause infantile diarrhea (outbreaks) and
rarely sporadic diarrhea in adults.
● It is nontoxigenic and noninvasive
● Mechanism of diarrhea:
➔ Adhesion to intestinal mucosa, mediated by
plasmid coded bundle-forming pili
➔ A/E lesions (attaching and effacing lesions) on
the intestinal epithelium.
2. Enterotoxigenic E. coli (ETEC)
➢ ETEC is the most common cause of traveler’s diarrhea causing
25–75% of cases:
● It causes acute watery diarrhea in infants and adults.
● Common serotypes associated are: O6, O8, O15, O25,
O27, O153, O159, etc.
● It is toxigenic but not invasive
● Pathogenesis:

71
 ➔ Attachment to intestinal mucosa is mediated by
CFA (Colonization Factor Ag)
➔ Toxins: (i) heat labile toxin or LT (↑cAMP), (ii) heat
stable toxin or ST (↑cGMP)
➔ Diagnosis is done by detection of toxins by in
vitro and in vivo methods.
3. Enteroinvasive E. coli (EIEC)
➢ Common serotypes associated with EIEC are O28, O112, O114,
O124, O136, O152, etc.
● Pathogenesis: EIEC is not toxigenic but invasive. The
epithelial cell invasion is mediated by a plasmid coded
antigen called virulence marker antigen (VMA).
● EIEC is biochemically, genetically and pathogenically
closely related to Shigella.
● Manifestations include ulceration of bowel, dysentery
(resembling shigellosis).
● Diagnosis:
➔ Detection of VMA by ELISA
➔ HeLa cell invasion assay
➔ Sereny test (inoculation into guinea pig eyes
produces conjunctivitis)
➔ EIEC are biochemically atypical being nonmotile
and lactose nonfermenters.

SHIGELLOSIS
❖ Pathogenicity
➢ Transmission: (i) ingestion through contaminated fingers (MC),
food, and water or flies and (ii) rarely by homosexuals
➢ Infective dose:
● Shigella has a low Infective dose (10 to 100 bacilli). Others with
low infective dose include EHEC, Entamoeba histolytica and
Giardia.
● Salmonella Typhi: 103–106 bacilli
● Vibrio cholerae: 106–108 bacilli
➢ Bacilli enter the mucosa via M cells.
➢ Invasion: Mediated by a large virulence plasmid
➢ Direct cell to cell spread: This occurs by inducing actin polymerization
of host cells.
➢ Exotoxins:
● Shigella enterotoxin (ShET 1 and 2)- found essentially in S.
flexneri
● Shiga toxin is a cytotoxin, produced by S.dysenteriae type1.
➢ Endotoxin induces intestinal inflammation and ulcerations.

❖ Complications:
➢ Intestinal complications, such as toxic megacolon, perforations and
rectal prolapse.
➢ Metabolic compilations, such as hypoglycemia, hyponatremia, and
dehydration.

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 ➢ Ekiri syndrome or toxic encephalopathy is a metabolic complication of
shigellosis.
➢ Postinfectious phase: Patients expressing HLA-B27 develop
autoimmune reactions, such as reactive arthritis, ocular
inflammation, and urethritis months after S. flexneri infection (3%
of cases).
➢ HUS and HC is produced by S. dysenteriae Type 1 (due to producing
Shiga toxin, hence called Shiga bacillus)
➢ Rarely, it causes bacteremia, meningitis, pneumonia, vaginitis and
keratoconjunctivitis.

❖ Laboratory Diagnosis:
➢ Specimen: mucus flakes of stool
➢ Culture Media: (Common media for both Shigella and Salmonella):
● Transport media: Sach’s buffered glycerol saline
● Selective media: DCA (deoxycholate citrate agar), XLD, SS
Agar
● Enrichment Broth: Gram-Negative broth, selenite F broth,
tetrathionate broth.
➢ Important biochemical properties:
● Nonmotile, Nonlactose fermenter except: S. sonnei (Late
lactose fermenter)
● Catalase +ve except: S. dysenteriae type 1
● Mannitol fermenting except: S. dysenteriae
➢ Typing of Shigella:
● Serotyping S. dysenteriae: 15, S. flexneri 6 , S. boydii 19
serotypes, S. sonnei 1
● Colicin typing (Bacteriocin typing) done for S. sonnei (has 26
colicin types).

SALMONELLA
➢ Gram negative bacteria.
➢ Mostly motile.
➢ S. gallinarum and S. pullorum are non motile.
➢ Kaufmann and White Scheme is used to classify Salmonella.

❖ Salmonella typhi
➢ Causes Typhoid fever
➢ Enteric fever is caused by Salmonella typhi and Salmonella paratyphi A,
B & C.
➢ Transmitted by Faeco-oral contamination.
➢ Step ladder pattern fever.
➢ Rose spots seen.
➢ Longitudinal ulcers seen.
➢ Per Soup Diarrhea seen.

❖ Laboratory Diagnosis
➢ Blood culture: 1st week
➢ Agglutination test: 2nd week
➢ Stool Culture: 3rd week

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 ➢ Urine culture: 4th week

➢ Blood Culture
● Overall best
● Blood:Culture fluid :: 1:10
➔ Bile broth or glucose broth can be used.
● Add Sodium Polyanethol Sulfonate (SPS) to remove antibiotic effect.

❖ Widal test
➢ Highly sensitive but poorly specific.
● Also positive in (Biologically False Positive):
➔ Infectious Mono Nucleosis.
➔ Malaria.
➔ SLE (Autoimmune Diseases)
➢ Antibody titre against
● O antigen should be 1:100 to be Positive.
● H antigen should be 1:200 to be Positive.
➔ Flagellar antigen is more immunogenic.
➢ Paired testing should be done, after two weeks of initial testing
● In 2nd test, 4 fold increase in titre should be present to say it
as Positive.
➢ Antibody titre values are not fixed, they change according to locality
➢ Types:
● Slide Widal test
● Tube Widal test
➔ Serial dilution with normal saline
➔ Prozone phenomenon is avoided.

❖ Stool and Urine Culture

➢ Enrichment media: Seminole F Broth is used.
➢ Selective media:
● DeoxyCholate Agar (DCA)
● Xylose Lysine Deoxycholate (XLD)
● Salmonella Shigella Agar (SSA)
● Hektoen Enteric Agar (HEA)

❖ Vi Antigen
➢ Vi mostly covering up "O".
➢ Vi phage typing can be used for epidemiological studies.

❖ Drug of Choice (DOC): 3rd Generation Cephalosporins.

❖ Vaccines for Typhoid Fever

➢ Parenteral TAB Vaccine
● It is heat-killed whole cell typhoid/paratyphoid A and B; no
longer in use due to its side effects.
➢ Parenteral Vi Polysaccharide Vaccine
● It is composed of Vi capsular polysaccharide antigen derived
from S.Typhi strain Ty2.
➔ Dosage: Single dose containing 25 μg of Vi antigen is
given IM or subcutaneously.

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 ➔ Vaccine confers protection for 2 years.
➔ VI antigen elicits T independent IgG antibody
response, booster given @ 2 years.
➔ Age: It is given only after 2 years of age.

VIBRIO CHOLERAE
➢ On the basis of O1 Antigen Vibrio classified into:

Classical Eltor

Polymyxin B Sensitivity Sensitive Resistant

Mukherjee phage IV Susceptibile Resistant

Susceptibility

Voges Proskauer (VP) Negative Positive

CAMP Negative Positive

Chick Erythrocyte Negative Positive

Agglutination test

❖ Sub-divisions:
➢ Classical:Ogava, Inaba, Hikojima
➢ Eltor: Ogava, Inaba, Hikojima
❖ Non O1
➢ O2- O139
➢ NAG: Non Agglutinable Vibrios

❖ Cholera Toxin
➢ A: ADP Ribosylation of GTP -> Increase in Adenylate Cyclase activity. ->
Increase in CAMP.
➢ B: Binds to GM1 Ganglioside receptor.
➢ Intracellular water comes to lumen resulting in massivediarrhea.
➢ Massive Diarrhea:
● Non-inflammatory diarrhea.
● Rice watery stools.
● Resembles Arsenic Metal poisoning.

❖ Culture media:
➢ Transport media: Maintains viability, no multiplication.
● Venkataraman Ramakrishna Medium
● Cary Blair Media
➢ Enrichment media: Alkaline Peptone Warer
➢ Selective media:
● Thiosulphate Bile Salt Sucrose (TCBS)
➔ Green -> Yellow (dlt sucrose lysis)
➔ Best Selective media

❖ Vibrio cholerae
➢ Catalase positive

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 ➢ Oxidase positive
➢ Indole positive
➢ Nitrates to Nitrates Reduction test Positive
➢ Sucrose lysis
➢ String test Positive

➢ On Hanging drop, Darting motility is seen

❖ Vaccine
➢ Injectable killed vaccines: They are no longer in use. As they provide
little protection, produces adverse effects.
➢ Oral cholera vaccines (OCV) are currently in practice. Two types of
oral vaccines.
1. Killed Whole-Cell Vaccine
● Two types: Whole cell (WC) vaccine and Whole cell
recombinant B subunit cholera vaccine (WC/rBS)
(Dukoral)
● Schedule: Two oral doses are given at 7 days gap. C/I to
children < 2 years.
● Protection is short lived. (At 6 months, 58% for WC
vaccine and 85% for WC/rBS vaccine)
● Children are better protected than adults.
● WHO recommends for using vaccine during epidemics in
the community but not during inter epidemic period.
2. Oral live attenuated vaccines:
● CVD 103-HgR, Peru-15 and V. cholerae 638 for
classical and/or El Tor biotypes of V. cholerae O1.
● CVD-112 and Bengal-15 vaccine trials are ongoing for V.
cholerae O139.

❖ Halophilic Vibrios
➢ Vibrio Parahemolyticus: (7-8% halophilic)
● Causes sea food poisoning.
● Kanagawa phenomenon on Wagatsuma Agar (enhanced β
hemolysis)
➢ Vibrio vulnificus: (8% halophilic)
➢ Vibrio alginolyticus: (10% halophilic)

HELICOBACTER PYLORI
★ Laboratory Diagnosis

❖ Invasive Test
➢ Endoscopy guided multiple biopsies can be taken from gastric mucosa
(antrum and corpus) and are subjected to:
● Histopathology with Warthin starry silver staining
● Microbiological methods:
➔ Gram staining: Curved gram-negative bacilli with seagull
shaped morphology
➔ Culture media for H.pylori: Culture is the most
specific test, however, it is not sensitive.

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 ● Media for Campylobacter can be used, such as
Skirrow’s media
● Chocolate agar can be used
● Plates are incubated at 37°C under microaerophilic
condition
➔ Biochemical tests: Oxidase, catalase and urease tests
are positive.
● Biopsy urease test (rapid urease test): Detects urease activity
in gastric biopsies. It is rapid, sensitive, and cheap.

❖ Noninvasive Test
➢ Urea breath test: It is very popular now a days as it is noninvasive and
is:
➔ Most consistent and accurate test
➔ Most sensitive, quick and simple
➔ Used for monitoring of treatment (becomes negative after
improvement) • Stool antigen (coproantigen) assay: Used for (i)
Monitoring of treatment, (ii) Screening of children.
➢ Antibody (IgG) detection by ELISA: Used for (i) Screening before
endoscopy, (ii) Seroepidemiological study

PSEUDOMONAS
❖ Clinical Manifestations
➢ Most of the infections are encountered in hospitalized patients.
● Pneumonia: (VAP or Ventilator-Associated Pneumonia).
● Chronic respiratory tract infections: Occurs in patients with
cystic fibrosis (in Caucasian populations), bronchiectasis or
chronic panbronchiolitis (in Japan):
➔ The mucoid strains (possessing alginate layer) of
Pseudomonas commonly cause such infections.
➔ Structural abnormalities of the airways result in mucus
stasis.
● Ear infections: Swimmer’s ear (among children) and malignant
otitis externa (in elderly diabetic patients).
● Eyeinfections such as corneal ulcers (in contact lens wearers)
and endophthalmitis
● Shanghai fever: A mild febrile illness resembling typhoid fever.
● Skin and soft tissue infections:
➔ Burns patients: Pseudomonas is the most common
organism to infect the burn wounds.
➔ Ecthyma gangrenosum is an acute necrotizing
condition results from bacteremia), occurs more
commonly in patients with febrile neutropenia and AIDS.
➔ Pseudomonas dermatitis: Cause outbreaks in spas, and
swimming pools
➔ Toe-web infections (in the tropics)
➔ Green nail syndrome: It is a ‘paronychia’ results from
prolonged submersion of the hands in water.
● Other infections:

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 ➔ Cellulitis (characterized by blue green pus)
➔ Bone and joint infections such as osteomyelitis and septic
arthritis
➔ Meningitis (in postoperative or post-traumatic patients)
➔ UTI (urinary tract infection) in catheterized patients.

❖ Laboratory Diagnosis
➢ Pseudomonas is nonfastidious, obligate aerobe and is motile with single
polar flagellum:
● It produces large, opaque, irregular colonies with a metallic
sheen (iridescence)
● Diffusible pigments: Blue green (pyocyanin) or yellow green
(pyoverdin) pigmentation
● Pigment production can be enhanced in special media such as
King’s media
● Most colonies have a characteristic sweet ether or alcohol-like
fruity odor
● Blood agar: It produces β hemolytic colonies on blood agar
● MacConkey agar: Produce pale nonlactose fermenting colonies
● Selective media-cetrimide agar
● Oxidase and catalase positive
● Nonfermenter: It does not ferment any sugars, but utilizes
sugars oxidatively.
● OF test (Hugh and Leifson oxidative fermentative test) shows
oxidative pattern.

LABORATORY DIAGNOSIS IN PRAGUE

❖ Laboratory Diagnosis
➢ Specimen: Pus aspirated from bubo, Sputum and blood.
➢ Direct Microscopy:
● Gram staining: Reveals pus cells and gram-negative oval
coccobacilli with rounded ends surrounded by capsule.
● Wayson stain (or methylene blue staining): Demonstrates bipolar
appearance.
➢ Culture:
● Y. pestis grows best at 27°C but the capsule develops best at
37°C.
● Blood agar: Colonies are nonhemolytic and dark brown pigmented
● MacConkey agar: NLF colonies
● Stalactite growth seen on nutrient broth with oil or ghee
floated on top.
➢ F1 Antigen detection by direct immunofluorescence test, ELISA
➢ Antibodies to F-1 antigen may be detected by CFT, ELISA
➢ PCR can be done targeting gene coding F1 antigen, pesticin gene.

HEMOPHILUS INFLUENZAE
❖ Infections caused:
➢ Central Nervous system infections:

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 ● Pyogenic meningitis in < 2 years of age
● Subdural effusion, MC CNS complication
➢ Epiglotitis
➢ Lobar pneumonia
➢ Less common invasive conditions seen in children include:
● Nonmotile and Oxidase negative
● Inability to grow on MacConkey agar
● Inoculation into Guinea pigs can cause testicular swelling
(Strauss reaction).

HEMOPHILUS DUCREYI
❖ H. Ducreyi
➢ Causes Chancroid/softsore: Characterized by painful lymph node,
tender non-indurated and bleeding genital ulcer
➢ Chancroid increases both transmission and the degree of susceptibility
to HIV infection
➢ Indirectsmear: Pleomorphic gram-negative coccobacilli that:
● Show bipolar staining
● Occurs in parallel chains called in ‘School of fish’ or ‘rail road
track’ appearance
➢ Antigenically homogenous
➢ Culture Medium used:
● Rabbit blood agar or Chocolate agar with 1% isovitalex,
Vancomycin
● Chorioallantoic membrane (CAM)
➢ Drug of choice: Azithromycin (1 g oral; single dose), treatment of all
sexual partners.

BORDETELLA
❖ Virulence Factors
➢ Toxins:
● Pertussis toxin (PT) expressed only by B.pertussis, similar to
cholera toxin in its structure and function (↑cAMP)
● Other toxins: Tracheal cytotoxin, adenylate cyclase toxin,
dermonecrotic toxin and Endotoxin
● Adhesins: They play a role in bacterial attachment:
➔ Filamentous hemagglutinin (FHA)
➔ Pertactin, an outer-membrane protein
➔ Fimbriae or pili or agglutinogens.

BRUCELLOSIS
➢ Brucellosisis primarily a zoonotic disease acquired from animals such as
sheep, goat, or cattle.

❖ Nomen species classification is based on:

➢ Preference of animal host
➢ CO2 requirement

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 ➢ H2S production
➢ Genetic composition
➢ Bacteriophage susceptibility
➢ Tolerance to bacteriostatic dyes
➢ Agglutination with monospecific antisera
❖ Preference of animal host:
➢ B. melitensis: Sheep, goat and camel
➢ B.abortus: Cattle and buffalo
➢ B. suis: Pigs
➢ B. canis: Dogs
➢ B. ovis: Sheep
➢ B. neotomae: Desert rodents

❖ Clinical Manifestations
➢ Classictriad: Fever with night sweats; arthralgia/arthritis and
hepatosplenomegaly
➢ Typhoid-like illness: Overall, brucellosis resembles typhoid like
illness except that it is less acute, less severe with undulating
pattern of fever (or Malta fever or Mediterranean fever) and
more musculoskeletal symptoms.

❖ Laboratory Diagnosis
1. Culture and Identification
➢ Sample: Blood, bone marrow, CSF, joint fluid or other tissues.
➢ Cultural media: Biphasic blood culture bottles media
(Castaneda’s) made up of Brain heart infusion (BHI)
broth/agar
➢ Erythritol: Improves growth
➢ Automated techniques such as BACTEC and BacT/Alert systems.
2. Antibody Detection by Standard Agglutination Test (SAT)
➢ It remains the gold standard test serological test:
● It is a tube agglutination test detecting antibodies in
serum by using standard strain of B.abortus:
● SAT detects IgM antibodies against antigens of
smooth LPS: Hence useful for acute brucellosis
● False negative SAT may occur due to:
➔ Prozone phenomenon (due to excess of antibodies
in patient’s sera)
➔ Presence of blocking’ or non-agglutinating IgG or
IgA antibodies
● False positive SAT may occur due to-antigenic cross
reacting gram-negative bacteria such as E.coli.
● CFT and ELISA: Are preferred in chronic brucellosis
for Ab detection.
3. Other Tests
➢ PCR using primers for rrs-rrl gene, Omp2 gene
➢ Brucellin skin test
➢ Guinea pig inoculation
➢ Tbilisi phage typing is done
➢ Diagnosis of Brucellosis in Animals: Antibody detection in milk
by (i) Milk ring test, (ii) Rose Bengal card test and (iii) Whey

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 agglutination test

❖ Treatment
➢ Gold standard regimen in adults: Streptomycin plus doxycycline
➢ WHO regimen in adults: Rifampin plus doxycycline
➢ Relapse or treatment failure occurs in 5–10% of cases.
➢ For CNS involvement: Ceftriaxone is added to the regimen and
treatment is prolonged for 3–6 months.

MYCOBACTERIUM TUBERCULOSIS
➢ Acid fast
● Ability to resist decolourization,
● Depends on Mycolic acid content
➢ Virulence Factors
● Cord factor
● Lipo arabinomannan
➢ In children, lower lobes are involved
● Ghon's focus + Hilar lymphadenopathy -> Ghon's Complex
● Calcified Hilar lymphadenopathy -> Ranke's Complex
➢ Post primary Tuberculosis, upper lobes are involved
● Subpleural focus -> Simon's focus
● B/L infraclavicular lesions -> Assman's focus
➢ Skin TB
● Most Common Primary lesion-> Lupus Vulgaris (Apple Jelly nodules)
● 2nd most common lesion -> Scrofuloderma
➢ GI TB
● Most Common site -> Ileo Ceacal Junction
➢ Bone TB -> Pott's Spine

❖ Laboratory Diagnosis
1. Sputum examination
➢ 2 Spectrum samples taken
1. Onset sample
2. Early morning sample
➢ Concentration method -> Petroff's method
➢ Thick Spectrum + NaOH/ HCl -> Liquified Spectrum
a. In staining
➢ Sputum smear -> Carbol fuschin -> Intermittent
heating -> 20% H2SO4 (Decolouriser) -> Methylene
Blue -> Blue background; pinkish to rec coloured
bacilli.
b. Lowen Stein Jensen media (LJ media)
➢ Eggs are used for solidification
➢ Selective media agent: Malachite green
➢ Green coloured media
➢ Rough, tough, buff colonies
● Rough : wrinkled appearance
● Tough : difficult to remove
● Buff : yellowish brown
2. MTB PCR methods

81
 3. Gene Xpert r/f (detection of Rifampicin resistance)
4. Cartridge Based Nucleic Wcid Amplification Test (CBNAAT)
5. Line Probe Assays
6. MTB PCR + Multidrug Resistance detection
➢ Can give the results within 2 hours.
➢ Drawback of PCR : Does not differentiate between active and
latent TB
7. Quantiferon TB Gold Assay (Interferon Gamma Release Assay- IGRA)
➢ Mycobacterial antigens -> stimulates Blood sensitized T
Lymphocytes -> Measure Interferon γ activity (activity of
sensitized T Lymphocytes) -> Prior exposure will be known.
➢ Drawback : Does not differentiate between active and latent
TB.
8. Auramine-Rhodamine Staining
➢ Flourescent staining
➢ Done in cases of more load
➢ Drawback: False Positive rates are high.
9. Tuberculin test/ Montoux test
➢ 0.1 mL Purified Protein Derivative (PPD)
➢ Into flexor aspect of forearm.
➢ After 3 days, measure induration (Hardness Diameter)
● <5 mm : negative
● 5-10 mm : Equivocal
● >10 mm : positive
➢ False Positive
● Recent BCG Vaccination
● Atypical Mycobacteria infection
➢ False negative
● HIV +ve (Advanced stage)
● Miliary TB
● Malignancy
● Immunosuppression
➢ Delayed Hypersensitivity reaction

❖ Bacillus Calamete Guerin (BCG) Vaccine

➢ Derived from Danish 1331 strain of Mycobacterium bovis
➢ Intradermal
➢ Diluent : Normal saline
➢ Efficacy : 0-80 %

ATYPICAL MYCOBACTERIA
❖ Runyon's Classification
➢ Photo chromogens
● Grow in light
➔ Mycobacterium marine
➔ Mycobacterium asiatucum
➔ Mycobacterium simiae
➔ Mycobacterium kansasii
➢ Scoto chromogens

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 ●Grow in darkness
➔ Mycobacterium szulgai
➔ Mycobacterium scrofulaceum
➔ Mycobacterium gordanae
➢ Non chromogens
● Neither light nor darkness required
➔ Mycobacterium avium complex/ Mycobacterium
intracellulare a.k.a Battey's bacillus
➔ Mycobacterium xenopi
➔ Mycobacterium ulcerans
➢ Rapid growers
➔ Mycobacterium cheloni
➔ Mycobacterium fortuitum
➔ Mycobacterium phlei
➔ Mycobacterium smegmatis

❖ Diagnosis:
➢ Swimming pool granuloma : Mycobacterium marinum
➢ Buruli ulcer : Mycobacterium ulcerans
➢ Post injection abscess: Rapid growers
➢ Most atypical bacterial cases: Cutaneous infections and
lymphadenopathy
➢ Atypical Mycobacteria which mimics Mycobacterium tuberculosis in lung
involvement: Mycobacterium kansasii

MYCOBACTERIUM LEPRAE
➢ Causes Hansen's disease
➢ Not cultured in pure culture media (doesn't follow Koch’s postulates)
● Grows j foot pad of Armadillo (Nine banded)
● Grows in foot pad of mice.b

❖ Lepromin test
➢ Type IV Hypersensitivity Reaction
➢ Lepromin test is a prognostic test, not a diagnostic test.

Early Lepromin Reaction/ Fernandez Late Lepromin Reaction/ Misuda

Reaction Reaction

➢ Read after 3 days ➢ Reads after 3 weeks

➢ Measures induration diameter ➢ Measures Nodular Diameter

❖ Lepra reactions
➢ Can occur spontaneously or after the treatment
➢ Lepra reaction is a case of emergency
● DOC: Glucocorticoids

Type I Lepra Reaction Type II Lepra Reaction

➢ Down grading Reaction ➢ Erythema Nodosum

➢ Type IV Hypersensitivity Reaction ➢ Type III Hypersensitivity

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 ➢ Most common feature: edema reaction
➢ Most common nerve involved: ulnar ➢ Immune complex mediated
reaction
➢ Crops of Macclesfield and nodules
over the skin (TNF α)

❖ Laboratory Diagnosis
➢ Slit skin smear examination Taken
● Taken from
➔ B/L earlobes
➔ Forehead
➔ Chin
➔ Buttocks
➔ Nasal mucosal swabs
● In staining
➔ Add Carbol fuschin, do intermittent heating
➔ Add 5% H2SO4
➔ Add Methylene Blue
➔ Blue background
➔ Cigar shape/ Globi like arrangement of leprae seen

❖ Treatment:
1. Pauce bacillary
➢ Dapsone
➢ Rifampicin
For 6 months
2. Multi bacillary
➢ Dapsone
➢ Clofazimine
➢ Rifampicin
For 1 year
➢ For prognosis of leprosy
● Morphological index: better
➔ Measures percentage of solid stained bacilli (live bacilli)

SPIROCHETES
Pathogenic Spirochetes Disease Transmission

Treponema

T.pallidum Syphilis Sexual

T.pertenue Yaws Direct contact

(Non-veneral Treponema)
T.endemicum Endemic syphilis

T.carateum Pinta

Borrelia

B.recurrentis Relapsing fever (epidemic) Louse borne

84
B.duttonii, B.hermsii Relapsing fever (endemic) Tick borne

B.burgdorferi Lyme disease Tick borne

B.vincentii Vincent’s angina Direct contact

Leptospira

L.interrogans Leptospirosis (Weil’s Contact with rodent urine

disease)

TREPONEMA PALLIDUM
➢ Causes Syphilis
➢ Incubation period: 9-90 days.

❖ Stages

1. Primary ➢ Hard, painless chances seen

2. Secondary ➢ Rashes over the palms and soles

are present
➢ Condyloma Lata lesions are seen
Most infectious lesion

3. Latent ➢ Gummas (skin lesions) Seen

4. Tertiary ➢ Complications
● Neurosyphilis
➔ Presents with
General Paralysis of
Insane (GPI)
➔ Tabea dorsalis
● Cardiovascular syphilis
➔ Presents with
aneurysm of aorta

❖ Laboratory Diagnosis

1. Non Treponemal tests

Venereal Disease Research Lab Rapid Plasma Reagent (RPR)

(VDRL)

➢ Needs a microscope ➢ Better test

➢ Fluid is to be prepared and to be ● Card test
used in 24 hours ● No fluid is prepared
➢ Done with CSF (neurosyphilis) ➢ In CSF sample, RPR falls
➢ Best test to assess prognosis

2. Treponemal tests
➢ Flourescent treponemal antibody assay (FTA-ABS)
➢ Treponena pallidum Immobilization Assay (TPI)

85
 ➢ Treponema pallidum Haemagglutination Assay (TPHA)
➢ Treponema pallidum Particulate Agglutination Assay (TPPA)

➢ Most sensitive: FTA-ABS

➢ Most specific:
● TPI
➔ But routinely not done
➔ Involvement of live treponema
● TPPA
➔ 2nd most specific test
● TPHA

MYCOPLASMA
➢ Mycoplasma are the smallest free living organism known.

❖ General Properties
➢ Filterable (Hence known as Eaton’s agent)
➢ Formerly called PPLO- Pleuro pneumonia like organism.
➢ Lack rigid cell wall. Peptidoglycan layer is absent; replaced by
cholesterol.
➢ Hence they are resistance to cell wall active antibiotics like beta
lactams.

❖ Clinical Feature
➢ Incubation period is 2–4 weeks , person to person spread by respiratory
droplets.
➢ MC manifestation-upper respiratory illness
➢ MC cause of community acquired atypical pneumonia in adults.
➢ Pneumonia is called Primary atypical pneumonia (PAP) or ‘walking’
pneumonia or Eaton agent pneumonia
➢ Extrapulmonary Manifestations-neurologic, dermatologic, cardiac,
rheumatologic, and hematologic.
Note: Ureoplasma urealyticum cause NGU, epididymitis, vaginitis and cervicitis.

❖ Laboratory Diagnosis
➢ Poorly gram-negative, shows pleomorphism, resemble like L forms
➢ Staining with Dienes stain: Block of agar containing Mycoplasma
colony added to methylene blue is observed under microscope.
➢ Show gliding mobility ( however, they lack flagella, pili)
➢ Culture medium: PPLO broth and PPLO agar
➢ Produces Fried egg colonies.
➢ Antigen detection by Direct Immunofluorescence test
➢ PCR: More sensitive while culture is more specific.
➢ Detection of Antibody:
● Heterophile antibody:
➔ Cold agglutination test: Detects Mycoplasma
antibody by using human ‘O’ RBC antigen.
➔ Streptococcus MG test: Detects Mycoplasma
antibody by using Streptococcus MG antigen.
● Specific antibody:

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 ➔ CFT (complement fixation test)
➔ ELISA for IgM, IgG and IgA detection
➢ The combination of PCR for respiratory tract secretions and
serologic testing constitutes the most sensitive and rapid approach to
the diagnosis of M. pneumoniae infection.

❖ Treatment
➢ Mycoplasma pneumoniae and Ureoplasma urealyticum: DOC—
Azithromycin
➢ M. hominis: DOC—Doxycycline

ACTINOMYCETES AND NOCARDIA

➢ Actinomycetes are Gram-positive branching filamentous bacteria
➢ Human pathogenic actinomycetes: (1) Actinomyces, (2) Streptomyces, (3)
Nocardia, (4) Actinomadura
● Actinomyces is non-acid fast and anaerobic
● Nocardia: Aerobe and acid fast (1% sulfuric acid)
● Streptomyces and Actinomadura are aerobes and non-acid fast – Can
cause actinomycetoma

Actinomyces Nocardia

Acid-fastness Non-Acid fast Partially acid fast

O2 requirement A Anaerobe Obligate aerobe

Sugar Fermenter Utilizes sugar oxidatively

Habitat Found as oral flora Infections Usual habitat is soil

occur endogenously Infections occur
exogenously

Risk factors Disease occurs in Usually affects people with

immunocompetent host low immunity

Clinical forms Cervicofacial, abdominal and Pulmonary, CNS,

other forms Actinomycetoma

Granules Sulfur granules are hard and not Granules are soft and
emulsifiable, consist of branching lobulated. Commonly found
filamentous bacilli and in mycetoma, rare in other
surrounded by clubs (sunray conditions
appearance)

Culture Spidery molar teeth colony in Colonies are creamy,

solid media Fluffy ball at bottom wrinkled and orange to
of the liquid medium pink. Recovery done by:
➢ Selective media
➢ Paraffin bait
technique
➢ LJ medium

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Drug of choice Penicillin Sulfonamide or
Cotrimoxazole

RAT BITE FEVER

➢ Agents: Streptobacillus moniliformis and Spirillum minus

Streptobacillary rat bite fever Spirillary rat bite fever

Agent Streptobacillus moniliformis Spirillum minus

Disease also called Haverhill fever (USA), also called Sodoku (Japan)
Erythema arthriticum epidemicum

Incubation period 7–10 days 1–3 weeks

Clinical feature Septic fever, rashes, painful Similar features plus

polyarthritis with frequent lymph node enlargement
relapses

Laboratory ➢ Gram-negative, pleomorphic ➢ Gram-negative,

diagnosis bacilli in chain with beaded spirally coiled bacilli
or with fusiform swellings ➢ Motile
➢ Nonmotile (amphitrichous
➢ Readily developing into L flagella)
forms ➢ Not cultured on
➢ Culture: It grows on media artificial media
containing serum protein, ➢ Can be isolated
egg yolk, or starch. from guinea pigs or
mice

Artificial media Cultivable Noncultivable

Drug of choice Penicillin Penicillin

GARDNERELLA VAGINALIS
➢ It causes bacterial vaginosis.
➢ It is a gram variable (mostly gram-negative coccobacilli), possess
metachromatic granules
➢ Other organisms implicated in BV: Mycoplasma hominis, Mobilincus,
Prevotella, Ureaplasma and Peptostreptococcus
➢ Amsel’s criteria: Bacterial vaginosis is diagnosed if any 3 of the
following 4 findings are present:
● Profuse thin (low viscous), white homogeneous vaginal discharge
● pH of vaginal discharge > 4.5 (due to Decrease in the concentrations
of Lactobacilli)
● Fishy odor accentuated by vaginal secretions mixed with 10% solution
of KOH (Whiff test).
● Clue cells: Vaginal epithelial cells coated with coccobacilli.
➢ Nugent’s score is followed to count the no. of Gardnerella

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 vaginalis, Mobiluncus and Lactobacilli in the Gram stained
smear of vaginal discharge. A score of ≥ 7 is diagnostic.
➢ Treatment: DOC Metronidazole.

QUERY FEVER (Q FEVER)

➢ Caused by Coxiella burnetti.
➢ Contaminates milk
➢ Killed by Flash method of pasteurization.
➢ Method of Transmission: Aerosol transmission (no arthropod transmission.
➢ No rash occurs.
➢ Culture negative endocarditis.

CHLAMYDIA (ATP PARASITE)

❖ Forms:
➢ Extracellular/ Elementary body: Infectious
➢ Intracellular/ Reticulate body: Replicating

❖ C. trachomarosis
➢ Serogroups A, B, Ba, C : Causes Trachoma
➢ Serogroups D-K causes
● Inclusion conjunctivitis
● Infantile pneumonia
● Genital Chlamydiasis
➢ Serogroups L1, L2, L3 causes lymphogranuloma venerum (L2X1)

❖ C. psittaci
➢ Causes bird pneumonia
● Mainly to parrots (helps in aerosol transmission)

❖ C. pnemoniae
➢ Causes Adult pneumonia
➢ Along with atherosclerosis
➢ Along with asthma (to be confirmed)
➢ Inclusion bodies
● Trachoma: Halberdt Prowazek bodies (HP bodiez)
● Molluscum contagiousum: Henderson Peterson bodies (HP bodies)
● Psittacosis: Levinthaal Cole Lilee bodies
➢ Culture
● On McCoy cell line (Continous cell line)
● On yolk sac of eggs (Hen's)
➢ Investigation of Choice: Nucleic Acid Amplification Test (NAAT)

❖ LGV
➢ Painless ulcer
➢ Painful lymphnodes: Para rectal lymphnodes involved
➢ Frie's skin test is done
➢ Complications:
● Esthiomene: Rectal and Vulval structures

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VIROLOGY

VIRAL REPLICATION
➢ Viruses do not undergo binary fission (seen in bacteria), but undergo a
complex way of cell division. Replication of viruses passes through six
sequential steps:
1. Adsorption/ attachment is the first and the most specific step of
viral replication. It involves receptor interactions between virus and
host.
2. Penetration: After attachment, the virus particles penetrate into the
host cells either by

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 ● Phagocytosis (or viropexis)- Through receptor mediated
endocytosis
● Membrane fusion: seen in HIV ○ Injection of nucleic
acid: seen in bacteriophages
3. Uncoating: Capsid is lysed (due to host lysozymes) and the
nucleic acid is released. This step is absent for bacteriophages.
4. Biosynthesis of various viral components: i) nucleic acid, ii) capsid
protein, iii) enzymes iv) other regulatory proteins Site of Nucleic acid
replication
● In DNA viruses, the DNA replication occurs in the nucleus;
except in poxviruses (cytoplasm).
● In RNA viruses: The RNA replication occurs in cytoplasm;
except in retroviruses and orthomyxoviruses (nucleus).
5. Assembly: Viral nucleic acid and proteins are packaged together to
form progeny viruses (nucleocapsids).
● DNA viruses are assembled in the nucleus except
hepadnaviruses and poxviruses (in cytoplasm)
● RNA viruses are assembled in the cytoplasm.
6. Maturation: Take place either in the nucleus or cytoplasm or
membranes Release of daughter virions occur either by:
● Lysis of the host cells as shown by nonenveloped viruses and
bacteriophages.
● Budding through the host cell membrane as shown by enveloped
viruses.
7. Eclipse phase: It is the interval between entry of the virus into
host cell till the appearance of the first infectious virus particle.
● During this period, the virus cannot be demonstrated inside the
host cell.
● The duration of the eclipse phase is about 15 to 30 minutes
for bacteriophages and 15-30 hours for most of the animal
viruses.

VIRAL CULTIVATION
➢ Viruses cannot be grown on artificial cell free media (However, grow in
animals, eggs or tissue culture)
1. Animal Inoculation
● Suckling Mice is used for cultivation of certain viruses such as
Coxsackie and arboviruses.
➔ Coxsackie A-produces flaccid paralysis in mice
➔ Coxsackie B-produces spastic paralysis in mice
2. Embryonated Egg Inoculation
● Embryonated egg has four sites for cultivation of virus
1. Chorioallantoic membrane (CAM): Few viruses
produce lesions called pocks, e.g. Vaccinia, Variola, HSV
1 and 2
2. Yolk sac: Arboviruses (e.g. JEV, Saint Louis and West
Nile virus), Rickettsia, Chlamydia and Hemophilus ducreyi.
3. Amniotic membrane: Influenza culture (for diagnosis)

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 4. Allantoic cavity: Used for vaccine preparation for
Influenza, Yellow fever (17D), Rabies (Flury).
3. Tissue Culture
● Organ culture: Whole organ is used, tracheal ring used for
coronaviruses
● Explant culture: Minced organ is used, e.g. adenoid explant used
for Adenovirus
● Cell Line: Tissues are completely digested and the individual
cells are mixed with viral growth medium and dispensed in tissue
culture flask.
★ Types of Cell Lines
➢ Primary cell line
● Rhesus Kidney cell line
● Human amniotic cell line
● Chick embryo fibroblast
➢ Secondary cell line
● Human fibroblast cell line
● MRC-5 and WI-38 cell line
➢ Continuous cell line
● HeLa cell line
● HEp-2 cell line
● KB cell line
● McCoy cell line
● Vero cell line
● BHK cell line
● Detroit 6 cell line
● Chang C/I/L/K cell line

DETECTION OF GROWTH IN TISSUE CULTURE

➢ Cytopathic effect (CPE)
● It is defined as the morphological change produced by the virus in the
cell line detected by light microscope.
➢ Viral Interference: The growth of a non-CPE virus in cell culture can be
detected by the subsequent challenge to the cell line with a known CPE virus.
The growth of the first virus inhibit infection by the second virus. For
example, rubella is a non-CPE virus but prevents the replication of
enteroviruses which are known to produce CPE.
➢ To detect viral antigens in infected cell line- i) Direct IF assay, ii)
Immunoperoxidase staining, iii) Hemadsorption.
➢ Electron microscopy: to detect viral particles in infected cell lines
➢ Viral genes detection: by using PCR or nucleic acid probes.

STRUCTURE OF VIRUSES
➢ Viruses consist of nucleocapsid (nucleic acid and capsid), which is
further surrounded by envelope (in some viruses). Capsid is a protein layer; is
made up capsomer units.
Nucleic Acid: Viruses possess either DNA or RNA, but never both
● Genomic size:

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 ➔ Largest is retroviruses (7–11.5 kbp),
➔ Smallest Hepatitis D (1.7 kb) followed by Hepatitis B (3.2 kbp)
● All the DNA viruses are double stranded, except Parvoviruses (the only
SS DNA virus)
● All the RNA viruses have one copy of single stranded unsegmented
RNA except:
➔ Reo viruses (the only double stranded RNA virus)
➔ Retroviruses including HIV (possess 2 copies of SS RNA)
● Segmented RNA Viruses (code-BIRA)
➔ Bunya virus (3 segments)
➔ Influenza virus (8 segments),
➔ Rotavirus (11 segments),
➔ Arenavirus (2 segments) e.g. LCM i.e. lymphocytic
choriomeningitis virus
● Most RNA viruses possess positive sense RNA except: Myxoviruses,
Rabies, Filoviruses, Bunyaviruses and Arenaviruses (bear negative sense
SS RNA).

VIRAL INCLUSION BODIES

➢ They are the aggregates of virions or viral proteins and other products of
viral replication by which they can be demonstrated in virus infected cells
under the light microscope.

➢ Intracytoplasmic inclusion bodies ➢ Intranuclear inclusion bodies

● Negri bodies–seen in Rabies A. Cowdry type A inclusions
virus Paschen body–seen in ● Torres body–seen in
Variola virus Guarnieri Yellow fever
bodies–seen in Vaccinia ● Lipschultz
virus Bollinger bodies–seen body–seen in Herpes
in Fowl pox virus Molluscum simplex
bodies–seen in Molluscum B. Cowdry type B
contagiosum virus inclusions–seen in Poliovirus
● Perinuclear cytoplasmic (non specific, may be
body- seen in Reovirus illusory type)

➢ Intracytoplasmic and intranuclear inclusion bodies: Seen in Measles and

Cytomegalovirus (Owl’s eye appearance)

VIRAL INTERFERONS
➢ Interferons (IFNs) are the cytokines, produced by host cells on
induction by viral or nonviral inducers.
● Classification: Interferons are classified into three groups,
designated as IFN-α, β and γ.
● Mechanism of action: IFN has no direct action on viruses and it
does not protect the virus-infected cell that produces it. However,
it induces the other host cells to produce certain proteins called
translation inhibition proteins (TIPs), that inhibit viral protein
synthesis by selectively inhibiting the translation of viral mRNA,

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 without affecting cellular mRNA. • IFNs are host specific but not
virus specific
● Inducers: both viral and nonviral agents can induce IFN synthesis. In
general, RNA viruses and avirulent viruses are strong inducers.
Examples of potent inducers are:
➔ Viruses: Togaviruses, vesicular stomatitis virus, Sendai virus and
NDV (New Castle Disease virus)
➔ Nucleic acids (double-standard RNA)
➔ Synthetic polymers (e.g. Poly I:C)
➔ Bacterial endotoxin
● IFN induction is much quicker than the antibody response: IFN
synthesis begins within about an hour of induction and reaches high
levels in 6–12 hours
● Resistance: IFNs are proteins, hence inactivated by proteases, but not
by nucleases or lipases. They are heat stable and also stable to wide
ranges of pH (except IFN-γ).
● Interferon assay: is based on their biological activity. Being poorly
antigenic, they cannot be detected serologically.
● Application of IFN
➔ IFN-α is used in:
★ Topically: in rhinovirus infection, genital warts and
herpetic keratitis.
★ Systemically: in chronic hepatitis B, C and D infections,
hairy cell leukemia and Kaposi’s sarcoma
➔ IFN-β: It is used in multiple sclerosis
➔ IFN-γ: It is used in chronic granulomatous disease and
osteopetrosis.

Property IFN-α IFN-β IFN-γ

Formerly called as Leukocyte IFN Fibroblast IFN Immune IFN

Type of Type I Type I Type II

designation

Produced by host Most cell types Most cell Lymphocytes

cell (Mainly types-(mainly (mainly TH 1 cells,
macrophages) fibroblasts) rarely CD8 T cells,
NK cell)

Inducing agent Viruses; dsRNA Viruses; dsRNA Mitogens

Action Antiviral action Antiviral action 1. Immuno

↑MHC-I expression ↑MHC-I expression regulatory function
Activates NK cells Activates NK cells Stimulates
Antiproliferative Antiproliferative macrophages
function ↑MHC-I & II
expression
2. Anti-
proliferative
function

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Stability at pH Stable Stable Labile
2.0

Chromosomal 9 9 12
location of genes

IFN receptor IFN -α/β receptor IFN -α/β receptor IFN-γ receptor

IFN receptor 21 21 6
genes located on
chromosome no.

BACTERIOPHAGES
➢ Bacteriophages are viruses that attack bacteria
● Bacteriophages are typically tadpole-shaped possessing a hexagonal
head and a tail attached with tail fibers.
● Hexagonal head contains tightly coiled dsDNA, enclosed by capsid
(protein coat)
➢ Altered morphology may be seen in some phages:
● Shape: Spherical or filamentous instead of hexagonal.
● Nucleic acid: May contain ssDNA or RNA instead of dsDNA.

➢ Life Cycle of Bacteriophage

Based on two types of life cycle bacteriophages are classified into:
Lytic and lysogenic or temperate phage
● Lytic phase: Phage replicates in the cytoplasm and lyses the host
bacterium to come out. It resembles the replication of other DNA
viruses; except that:
➔ In penetration step: Phages are attached to bacterial cell wall
as ‘ghosts’.
➔ There is no uncoating step as seen with other viruses.
➔ Release of the daughter phages occur by lysis of the host
bacterium.
➔ Duration of Eclipse phase is about 15 to 30 minutes; in contrast
to 15-30 hours for most of the animal viruses.
● Lysogenic or Temperate phage: Phage DNA is incorporated within
host DNA and remains dormant as prophage.
➢ Lysogenic to lytic interconversion: When the temperate phages want to
come out, they get excised from bacterial chromosome, then transform to
lytic phages, multiply in the cytoplasm and are released by lysis.

➢ Uses of Bacteriophage
● Phage typing: Phage typing is employed for typing the following
bacteria:
➔ Staphylococcus aureus
➔ Vi antigen typing of Salmonella Typhi,
➔ Vibrio cholerae (Basu Mukherjee phage typing)
➔ Brucella (Tbilisi phage typing)
➔ Corynebacterium diphtheriae
● Phage assay: To estimate the no. of viable phages in preparations.

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 ● Used in treatment (Phage therapy): Lytic phages can kill the
bacteria, hence may be used for treatment of bacterial infection, such
as post-burn and wound infections.
● Used in diagnosis: Mycobacteriophages are used for the
identification of M.tuberculosis.
● Used as a cloning vector.
● Transduction: In S.aureus, the plasmids coding for β-lactamases are
transferred between the strains by transduction.
● Codes for toxins: The phage genomes code for the following bacterial
toxins
➔ A, C of Streptococcal pyrogenic toxin
➔ Botulinum toxin C, D
➔ Cholera toxin
➔ Diphtheria toxin
➔ EHEC (Enterohemorrhagic E.coli) (Verocytotoin)
● Alter antigenic property of bacteria: e.g. in Salmonella.

MOLLUSCUM CONTAGIOSUM VIRUS

➢ Lesions: It produces pink pearly wart-like lesions with a characteristic
dimple at the center (umbilicated).
● Lack of associated inflammation and necrosis
● Lesion are found anywhere on the body except on the palms and soles.
Genital lesions are seen in adults.
➢ Transmission:
● Children (MC): Spread by direct and indirect contact.
● Rarely sexual transmission has been reported in young adults.
➢ Laboratory diagnosis:
● Molluscum bodies are the intracytoplasmic eosinophilic inclusions seen
in skin scrapings (histopathological stains)
● Not cultivable: It cannot be propagated in tissue culture, egg or in
animals.
➢ Treatment: Surgical removal of the lesions by ablation.
➢ Prognosis: Self-limiting, except in HIV.

HERPESVIRUS
➢ The members of Herpesviridae possess the following properties:
● Large (150–200 nm size), spherical in shape with icosahedral symmetry.
● Tegument: It is an amorphous, asymmetric structure present between
the capsid and envelope
● Establish latent or persistent infections in their hosts and undergo
periodic reactivation
● Possess dsDNA and replicates by rolling circle mechanism.

Subfamily Duration of Site of latency Species

replication
(Cytopathology)

Alpha Short (12-18 hours) Neurons Herpes simplex

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 Cytolytic virus type 1 and 2
Varicella-zoster
virus

Beta Long (> 24 hours), Glands, Kidneys Cytomegalovirus

Cytomegalic

Long (> 24 hours) T-cells Human herpesvirus

Lymphoproliferativ 6 and 7
e

Gamma Variable, Lympho B-Cells Epstein-Barr virus

proliferative and Human
herpesvirus 8

HERPES SIMPLEX VIRUS (HSV 1 AND 2)

➢ HSV 1 and 2 are Antigenically related to each other. (All other Herpes viruses
are not related to each antigenically).
➢ Primarily Infections occur via skin breaks or via Mucous membranes.
➢ These viruses become latent in the 'neurons' of the sensory ganglia (HSV 1
become latent in the Dorsal Root Ganglia (DRG) (since HSV 1 above the waist)
and the HSV 2 become latent in the 'sacral ganglia' (HSV 2 below the waist).
➢ Reactivation is very common, sometimes predisposed by any kind of physical or
emotional stress, fever, exposure to ultraviolet rays.
➢ Reactivation can be symptomatic or asymptomatic.
➢ Even in asymptomatic patients, the virus is present in the secretions

➢ Infections caused by HSV:

● Both HSV 1 and HSV 2 present with mucocutaneous lesions and extra
mucocutaneous lesions.
● Mucocutaneous lesions are characterized by painful vesicles followed
by pustules whose top break up to form erythematosus ulcer (which are
very painful) then they heal by crusting.
➔ Oropharyngeal disease: Gingivostomatitis, Herpes Pharyngitis
and Herpes labilis.
➔ Keratoconjunctivitis
➔ Herpes genitalis and Herpes proctitis (predominantly caused by
HSV 2).
➔ Herpetic Whitlow: Occupational disease seen mainly in medical
professionals while examining the patients with herpetic
lesions).
➔ Herpes gladiatorum: Seen in sports persons, due to lots of
physical contact.

➢ Diagnosis of mucocutaneous lesions:

● Tzanck smear:
➔ Scraping from the base of the ulcer is taken and stained with
'Giemsa/ Wright stain'.
➔ Multinucleated giant cells with ground glass chromatic and
faceted nuclei are seen.

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 ➔ It is much less sensitive than the direct flourescent antibody
testing in the specimen for detecting antigens.
➔ Gold standard investigation for diagnosis: 'Nucleic Acid
Amplification Test (NAAT) and is most sensitive.

➢ Treatment: Acyclovir and Valacyclovir

➢ Extra mucocutaneous infections:

● Encephalitis: HSV 1 >> 2 are implicated as the most common cause for
sporadic encephalitis in the world. 90 to 95% of these cases is because
of HSV 1.
● Mollaret meningitis: Also known as 'benign recurrent lymphocyte
meningitis'. More common cause is HSV 2 > 1.
● Other CNS lesions include 'Transverse myelitis' and 'Bell's Palsy'.
● Neonatal Herpes: A pregnant female with asymptomatic or symptomatic
carriage of HSV 1 or 2 in her genital tract can transmit the infection to
her neonates during delivery. The neonates can develop generalized
painful vesicular eruption all Over his/her body along with encephalitis
and pneumonia.

VARICELLA ZOSTER VIRUS

➢ VZV produces vesicular rashes on the skin and mucous membranes in two
forms:

1. Chickenpox
➢ Clinical Manifestations
● Incubation period is about 10–21 days (2–3 weeks).
● Typical description of chickenpox rashes
➔ Vesicular, bilateral, diffuse and centripetal (start on the
face and trunk, spread rapidly to involve flexor surfaces).
➔ Rashes appear in multiple crops, fever appears with each
crop of rashes
● Chickenpox is a disease of childhood.
● When occured in adults, it is more severe with bullous and
hemorrhagic rash.
➢ Complications
Complications are more common in adults and in immunocompromised
individuals.
● MC infectious complication is: Secondary bacterial infection of
the skin.
● MC extracutaneous complication: CNS involvement (cerebellar
ataxia, encephalitis and aseptic meningitis): Usually occurs in
children.
● Most serious complication is: Varicella pneumonia, develops
commonly in pregnant women.
● Reye’s syndrome: Fatty degeneration of liver following
salicylate (aspirin) intake. Chickenpox in pregnancy affects both
mother and the fetus.
● Mothers are at high-risk of developing varicella pneumonia
● Fetal or congenital varicella syndrome: VZV is highly

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 teratogenic.
➔ Risk is maximum when mother acquires a primary
infection during pregnancy.
➔ Late first trimester/early second trimester: Congenital
malformation in fetusis more frequent, characterized
by cicatricial skin lesions, limb hypoplasia and
microcephaly.
● Infection near delivery
➔ If mother gets infection > 5 days before delivery: Then
baby is mostly asymptomatic due to protective maternal
Ab
➔ If mother gets infection before 5 days to after 2 days
of delivery: Maternal Ab would not have produced in such
a short time. This leads to dissemination of virus in the
baby to cause neonatal varicella (a severe form of
chickenpox).
➢ Epidemiology
Chickenpox is a highly contagious disease.
● Period of infectivity: 2 days before the onset of rash to 5
days after thereafter, until the vesicles are crusted.
● One attack gives lifelong immunity
● Reservoir: Humans are the only known reservoir hosts.
● Source of infection: Patients are the only source, there are no
carriers.
● Secondary attack rate is about 70–90%.

2. Zoster or Shingles
➢ Zoster usually occurs due to reactivation of latent VZV in old age (> 60
year age), in ↓immunity or rarely in healthy adults.
➢ Rashes: They are unilateral and segmental, confined to the area
of skin supplied by the affected nerves.
➢ Complications
● Post-herpetic neuralgia (pain at local site): MC complication in
elderly patients.
● Zoster ophthalmicus: Unilateral painful crops of skin rashes
surrounding the eye.
● Ramsay Hunt syndrome develops when geniculate ganglion of
facial nerve is involved. It is characterized by tetrad of facial
nerve palsy plus vesicles on tympanic membrane, external
auditory meatus and the tongue.
➢ Vaccine
● Live attenuated vaccine using the Oka strain of VZV is available.
● It is given to children after 1 year of age; 2 doses, first
dose is given at 12–15 months and second dose at 4–6 yrs.
➢ Treatment
● Acyclovir is the drug of choice. It can prevent the
complications of chickenpox and can also halt the progression of
zoster in adults, but cannot prevent post-herpetic neuralgia.
● VZIG (Varicella zoster immunoglobulin)
➔ It is recommended for postexposure prophylaxis. It is

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 given within 96 hrs (preferably within 72 hrs) of
exposure.
➔ It is also indicated for neonates born to mothers
suffering from chickenpox if the onset of chickenpox in
the mother is between < 5 days before delivery till 48
hrs after delivery. VZIG is not indicated if the mother
has zoster.

CYTOMEGALOVIRUS (CMV)
➢ CMV is the largest virus in Herpesviridae family. It is so named because it
causes massive enlargement of infected host cells.
● Host specificity: CMV are strictly species-specific.
● Cell type specificity: CMV infects kidney and salivary glands.
● Cell–to-cell spread: CMV is almost always closely associated with the
cells and spread primarily cell-to-cell, so that very little virus may be
cell-free.

❖ Clinical Manifestations
➢ Congenital CMV Infection
● CMV is probably the MC intrauterine infection associated with
congenital defects, affecting near 1% of infants born.
● Cytomegalic inclusion disease develops in about 5% of the
infected fetus. The remaining are although asymptomatic at
birth, 5–25% of them may develop significant psychomotor,
hearing, ocular, or dental defects within 2 years.
● Congenital defects include:
➔ MC defects are petechiae, hepatosplenomegaly, and
jaundice.
➔ Less common: Microcephaly, cerebral calcifications,
IUGR, and prematurity.
● Risk is maximum if the infection occurs in early pregnancy and
if the mother is primarily infected during pregnancy.
➢ Perinatal CMV Infection
● Transmission to newborn occurs during: (i) Delivery, (ii)
Postnatal—through infected breast milk/secretion of mother.
● Mostly asymptomatic, but shed virus in urine up to several years.
● Few infants, especially premature babies develop interstitial
pneumonitis.
➢ Immunocompetent Adults • Mononucleosis like syndrome develops in
healthy adults following blood transfusion

Infectious mononucleosis Mononucleosis like

syndrome

Agent Epstein Barr Virus (EBV) CMV (20-50%)

HHV-6, Toxoplasma,
Ehrlichia, HIV

Atypical lymphocytosis Seen Seen

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Clinical symptoms Fever, myalgia, rashes Similar presentation,
Hepatosplenomegaly except that exudative
Exudative pharyngitis, pharyngitis, cervical
Cervical lymphadenopathy lymphadenopathy are
absent

Heterophile antibodies Elevated (Paul-Bunnell test) Negative

Specific antibodies Antibodies to specific EBV Antibodies to CMV or

antigens are elevated other agents may be
elevated.

➢ In the Immunocompromised Host

★ Negative Antibodies to CMV or other agents may be elevated. CMV
produces severe infection in immunosuppressed individuals; due to
reactivation of latent CMV viruses.
● In untreated AIDS patients with CD4 T-cell count < 50/µl—CMV
may cause Chorioretinitis (MC presentation), gastroenteritis and
dementia.
● Organ transplant recipients: CMV is probably the MC viral infection in
transplant recipients.
➔ CMV infection occurs usually between 1 to 4 months following
transplantation.
➔ Bilateral interstitial pneumonia in bone marrow transplant
recipients.

❖ Epidemiology
➢ Transmission: In contrast to HSV, CMV transmission requires close
contact
● Oral and respiratory contact is the predominant mode
● Others-Transplacental, blood transfusion (risk is 0.1–10%) and
sexual
➢ Reservoir: Humans are the only known host for CMV.
➢ Prevalence: In under-developed nations, 90% of people are being
seropositive in contrast to 40–70% in developed nations.

❖ Laboratory Diagnosis
➢ Detection of inclusion bodies in urine: CMV produces both
perinuclear cytoplasmic and intranuclear inclusions (describe as Owl’s
eye appearance)
➢ Virus Isolation: CMV can be isolated from throat washings and urine.
● Human fibroblasts are the most ideal cell lines, growth occurs
in 2–3 weeks.
● Shell vial technique: Used to detect growth in 4–5 days.
➢ Antibody detection:
● IgM antibodies are indicative of active infection.
● Four fold rise of IgG indicates recurrent infection.
● Antibodies are often undetectable in immunocompromised
patients.
➢ Antigen detection: CMV-specific pp65 antigens. It is highly
specific and reliable method

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 ➢ PCR detects specific CMV DNA in blood or body fluids such as CSF.

❖ Treatment
➢ CMV does not respond to acyclovir.
● Ganciclovir is the DOC for cytomegalic inclusion disease or
retinitis or transplant infections.
● Others: Valganciclovir (given orally), foscarnet (DOC in
ganciclovir-resistant cases), cidofovir and CMV Ig.

ADENOVIRUS
➢ It is non enveloped DNA virus, space vehicle shaped

➢ Disease produced by adenovirus ➢ Adenovirus serotype associated

● Hemorrhagic cystitis ● Adenovirus type 11 and 21
● Infant diarrhea (boys)
➢ Ocular Infections ● Adenovirus serotype 40, 41
● Epidemic ● Adenovirus type 8,19, 37
keratoconjunctivitis (Shepard eye, industrial
● Pharyngoconjunctival fever worker)
or Swimming pool ● Adenovirus type 3,7,14
conjunctivitis (follicular) (tends to occur in
outbreaks, at children’s
summer camps)

➢ Respiratory Diseases ● Serotypes 1, 2, 3 and 5

● Upper respiratory tract ● Serotypes 3, 7, and 21
infection ● In military recruits, associated
● Pneumonia with type 4 and 7
● Acute respiratory disease
syndrome

Transplant recipients ● Serotypes 34 and 35. ↑ risk to

develop pneumonia, hepatitis,
nephritis, colitis, encephalitis, and
hemorrhagic cystitis

PICORNAVIRUSES
➢ Picornaviruses are very small (28–30 nm size) and nonenveloped viruses;
divided into two major groups:
● Enteroviruses: Transmitted by fecal-oral route. However, they do not
cause intestinal symptoms, but are associated with systemic
manifestations. Examples:
➔ Polio (3 serotypes)
➔ Coxsackie A (1–24)
➔ Coxsackie B (1–6)
➔ Echovirus (1–33)
➔ Parechovirus (1–3)
➔ Enteroviruses (68–71)
➔ Enterovirus 72 is reclassified as Hepatitis A virus.

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 ● Rhinoviruses: Transmitted by respiratory mode. It is the MC cause of
common cold.

❖ Polio serotypes:
➢ Type 1
● MC serotype to cause epidemics.
● Only serotype that is endemic currently in the world.
➢ Type 2
● Most antigenic and hence easiest serotype to be eradicated.
● No natural case since 1999.
● MC serotype found among the VDPV strains.
➢ Type 3
● No natural case since 2013.
● MC serotype to cause VAPP

❖ Polio pathogenesis:
➢ MC Transmission: Feco-oral route
➢ Host cell Receptor: Via CD155 receptors
➢ Site of action: Motor nerve ending, i.e. anterior horn cells of the spinal
cord that leads to flaccid paralysis.
➢ Spread- Hematogenous (MC) > Direct Neural spread.

❖ Clinical Manifestations
➢ The incubation period is usually 7–14 days. It manifests in four forms:
● Inapparent infection: Following infection, the majority
(91–96%) of cases are asymptomatic.
● Abortive infection: 5% of patients develop minor illness (fever
and malaise).
● Nonparalytic Poliomyelitis: Seen in 1% of patients, presented
as aseptic meningitis.
● Paralytic Poliomyelitis is the least common form (< 1%):
➔ Characterized by: Descending asymmetric acute flaccid
paralysis (AFP)
➔ Proximal muscles are affected earlier than the distal muscles;
paralysis starts at hip → proceeds towards extremities—that
leads to the characteristic tripod sign (child sits with flexed
hip, both arms are extended towards the back).
➔ Site of involvement can be spinal, bulbospinal and bulbar.
➔ Cranial nerve involvement seen, but there is no sensory
loss.
➔ Risk factors: Paralytic disease is more common among:
★ Older children and adults, pregnant women and
heavy muscular exercise
★ Tonsillectomy predisposes to bulbar poliomyelitis
★ IM injections increase the risk of paralysis in the
involved limb.

❖ Laboratory Diagnosis
➢ Specimen: Blood (3–5 days), throat swab (up to 1 week), CSF, feces (up
to 6–8 week)
➢ Virus isolation: Primary monkey kidney cell line is used.

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 ● Virus growth can be identified by various methods.
➔ CPE: Described as crenation and degeneration of the
entire cell sheet.
➔ Antigen can be detected and serotyped by
neutralization with specific antiserum.
➔ Specific Gene detection by PCR
● Stool culture: Recommended for AFP surveillance and laboratory
confirmation
● Cultures of CSF, serum or throat swab are positive less
frequently, but indicative of disease.
➢ Antibody detection: Neutralizing Antibody and CFT Antibody

❖ Polio Vaccines
➢ Two types of polio vaccines are in use (OPV and IPV).

Polio Vaccine Salk (Injectable) Sabin (Oral)

Preparation ➢ Formalin killed preparation ➢ Each dose contains

in MKC (Monkey kidney cell TCID50 of:
line) ➢ Type 1: 3 lakh, Type 2:
➢ Total 80 units of D-antigen 1 lakh, Type 3: 3 lakh,
of: ➢ Markers of
● Type 1 (40 units), attenuation: Vaccine
● Type 2 (8 units), virus:
● Type 3 (32 units) ● Phenotypic
markers:
Vaccine virus will
not grow in
presence of low
levels
bicarbonate, at
40°C, MKC and is
inactivated by
specific antisera
● Genotypic
markers:
Detects specific
genes
ofattenuated
virus

Dose ➢ Four doses: ➢ Total five doses:

● 1st three doses: at ➢ Zero dose: Given at
1-2 months gap, birth
● 4th booster: after ➢ 1st/2nd /3rd: at
6-12 months gap 6/10/14 weeks of age,
Booster: 16-24 months

Safety Relatively safer than OPV Safe except in:

Immunocompromised patients,
pregnancy, old age

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Efficacy 80-90% by full course of IPV ➢ 90-100% efficacy is
Immune response is slower than achieved even by 1 or 2
OPV doses of OPV
➢ Efficacy decreases by:
● Interference by
other
enteroviruses
● Diarrheal
diseases
● Breastfeeding

Economy Relatively expensive Economical

Duration of Short, need booster doses Long lasting

Protection periodically

In epidemics Can precipitate paralysis Can be used safely

Herd immunity Not provided Provided due to feco-oral

spread of vaccine virus

Local immunity Weakly stimulated Strongly stimulated (due to

IgA antibody)

Can prevent Only paralysis Paralysis and intestinal

reinfection

Storage Relatively stable Does not require Should be stored at (–20°C)

condition stringent condition Stabilized in MgCl2 , pH<7

VAPP and VDPV Zero chance Relatively more chance

➢ VAPP and VDPV:

● VAPP strains are OPV-like isolates, which differ from OPV by < 1%
● Vaccine-derived polioviruses (VDPVs)
● VDPV differ from OPV by > 1% for Serotypes 1 and 3 and > 0.6% for
Sabin type 2.

➢ VDPVs can be categorized as:

● Circulating VDPVs (cVDPVs)
● Immunodeficiency-associated VDPVs (iVDPVs)
● Ambiguous VDPVs (aVDPVs).

➢ Clinical: subclinical ratio in Polio:

● For every clinical case, there may be 1000 children and 75 adults of
subclinical cases.

➢ Polio cases reported in the World:

● In 2016, only 35 wPV cases (71 cases in 2015) and 4 cVDPV cases (27
cases in 2015) have been reported.
● Pakistan accounted for maximum no. of natural cases.
● Lao People's Democratic Republic is the only non endemic country to

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 report cVDPV cases (3 no.).

➢ IPV and bOPV in India:

● IPV introduced in India: from November 2015
● Bivalent OPV will be introduced in India: from April 2016

➢ IPV in India:
● Govt. of India introduced IPV under universal immunization programme
● Withdrawal of Serotype-2: Trivalent OPV will be replaced by
Bivalent OPV (serotype 1 and 3) six months after starting IPV (i.e. mid
2016)
● From November 2015.
● One dose of IPV
● This IPV dose is extra dose; over and above the Trivalent OPV
● In a phased manner
● In the first phase, IPV has been introduced in six high risk states:
Assam, Gujarat, Punjab, Bihar, Madhya Pradesh, and Uttar Pradesh.

➢ Indian Govt. Action plan: Switch over of tOPV to bOPV and IPV
● From November 2015 to March 2016: Three doses of trivalent OPV
plus one dose of IPV along with the third dose of OPV at 14 weeks.
● From April 2016: Bivalent OPV three dose plus one dose of IPV along
with the third dose of bOPV at 14 weeks.

➢ Endgame Strategic Plan:

● Interruption of polio virus transmission
● Strengthening immunisation
● Implementing containment of polioviruses
● Legacy planning.

COXSACKIEVIRUSES
Group A Coxsackieviruses Group B Coxsackieviruses

Suckling mouse intracerebral inoculation:

➢ Flaccid paralysis in mice ➢ Spastic paralysis in mice

➢ Generalized myositis ➢ Focal myositis and Necrosis of
brown fat

Manifestations

1. Aseptic meningitis (A7, A9) More organ involvement:

2. Herpangina (vesicular Pharyngitis) 1. Aseptic meningitis (B1-6)
3. Hand foot and mouth disease (also 2. Pleurodynia (Epidemic myalgia or
by Enterovirus:71) Bornholm disease)
4. Acute hemorrhagic conjunctivitis- 3. Myocarditis, pericarditis
Caused by Coxsackie-A24 and 4. Hepatitis and Pneumonia
Enterovirus 70 5. Pancreatitis leading to Juvenile
Diabetes mellitus – Coxsackie B4
6. Generalized disease of infants

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RABIES VIRUS
❖ Morphology
➢ Bullet-shaped, enveloped virus. Envelope is embedded with glycoprotein
antigen spikes.
➢ Nucleocapsid (made up of nucleoprotein) has a helical symmetry and
comprises of negative sense ssRNA
➢ Antigens: Rabies has two major antigens; Glycoprotein G and
Nucleoprotein.

❖ Pathogenesis
➢ Transmission:
● Bite: Rabies virus is usually transmitted to humans by the bite
of an infected animal.
➔ Dog bite is the most common mode
➔ Other animal bites: monkey, sheep, goat, etc. (except rat
bite and human bite)
➔ Human-to-human transmission is theoretically possible
but is extremely rare.
● Non-bite exposures are rare such as:
➔ Lick on abrasion or mucosa
➔ Inhalation of infected bats aerosols.
➔ Corneal transplantation
➢ Route of spread:
● Viral replication in muscle → bind to nicotinic Ach receptors at
NM junctions → spreads centripetally along peripheral motor
nerves → dorsal route ganglia of spinal cord → infect brain
neurons (brainstem and mental system) → centrifugal spread via
sensory and autonomic nerves to cornea, salivary gland, skin and other
organs
➢ Speed of Rabies Virus progress in axon – 250 mm/day.

❖ Laboratory Diagnosis
1. Rabies antigen detection
➢ Direct IF test detecting rabies nucleoprotein antigens in
specimens.
➢ The best specimen is the hair follicle of the nape of the neck
(most sensitive).
➢ Corneal impression smear: Positive in late stage with a
sensitivity of 30%.
2. Viral Isolation
➢ Mouse inoculation: Intracerebral inoculation into suckling mice
➢ Cell lines: Mouse neuroblastoma cell lines and baby hamster
kidney (BHK) cell lines are the preferred.
3. Antibody detection:
➢ Detection of CSF antibodies is more significant than serum
antibodies as serum antibodies appear late and can also be
present after vaccination. Various antibody detection tests
include:
● Mouse neutralization test (MNT)

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 ● Rapid fluorescent focus inhibition test (RFFIT)
● Fluorescent antibody virus neutralization (FAVN)
● Indirect fluorescence assay (IFA)
● Hemagglutination inhibition test (HAI)
● Complement fixation test (CFT)
4. Viral RNA detection
➢ Reverse transcription-polymerase chain reaction (RT-PCR)
➢ Most sensitive and specific assay available at present for the
diagnosis of rabies.
5. Negri body detection
➢ Negri body detection is pathognomonic for post mortem
diagnosis of rabies. However, it may not be detected in 20%
of cases. Therefore, the absence of Negri bodies does not
rule out the diagnosis of rabies.
➢ They are intracytoplasmic eosinophilic inclusions with
characteristic basophilic inner granules.
➢ Sharply demarcated, spherical to oval, and about 2–10 µm in size.
➢ MC sites are neurons of cerebellum and hippocampus; however,
less frequently in cortical and brainstem neurons.
➢ Commonly used stains are: Histological stains such as H and E
and Sellers stains (basic fuchsin and methylene blue).

❖ Regimens for Rabies vaccine:

➢ Post Exposure prophylaxis: Five doses on days 0, 3, 7, 14 and 28 and
booster at 90 days
➢ Preexposure prophylaxis: Three doses on day 0, 7, 28 days and booster
every 2 years
➢ Postexposure prophylaxis to previously vaccinated people
● Severe bite or titer unknown: 3 doses of vaccine- 0,3,7 days
● Less severe bite or titer > 0.5 IU/ml: 2 doses of vaccine 0,3
days
CHIKUNGUNYA
Chikungunya fever is a re-emerging disease characterized by fever with arthralgia.
➢ Human Transmission: Aedes mosquito, primarily Aedes aegypti which bites
during day time, rarely by vertical transmission from mother to fetus, blood
transfusion
➢ Clinical Manifestations:
● Incubation period is about 5 days (3–7 days)
● Most common symptoms are fever and severe joint pain (due to
arthritis)
● Arthritis is polyarticular (migratory), affecting the small joints.
➢ Epidemiology:

● India: Reported during 1963–1973; e.g. Kolkata in 1963 and South India
in 1964. Since then, it was clinically quiescent in the world between
1973–2005.
● Re-emergence (Reunion Outbreak): In 2005, Chikungunya re-emerged in
Reunion Island of the Indian Ocean and then spread to India and other
countries.

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 ●India (at present): Chikungunya is endemic in several states.
➔ States: Karnataka, Tamil Nadu, Andhra Pradesh and West
Bengal have reported higher number of cases.
➔ Karnataka accounted for the maximum cases in the year 2013
and 2014.
➢ Genotypes: It has three genotypes: West African, East African and Asian
genotypes.
● Most Indian cases before 1973 were due to Asian genotypes.
● However, the Reunion outbreak was caused due to a mutated strain and
is responsible for most of the current outbreaks in India as well as in
other parts of the world.
➢ Reasons for re-emergence:
● New mutation (E1-A226V): Chikungunya virus underwent an important
mutation. Alanine in the 226 position of E1 glycoprotein gene is
replaced by valine.
● New vector (Aedes albopictus): Mutated virus was found to be 100
times more infective to A.albopictus than to A. aegypti.
➢ Laboratory diagnosis
● Viral isolation (in mosquito cell lines) and real time RT- PCR are
best for early diagnosis.
● Serum antibody detection: MAC ELISA is the best serology test.
● Biological markers like IL-1β, IL-6 are increased and RANTES
levels are decreased in chikungunya infection.

JAPANESE B ENCEPHALITIS (JE)

Japanese B encephalitis is the leading cause of viral encephalitis in Asia, including
India. It was first seen in Japan (1871); however, it is now uncommon in Japan.
➢ Vector: Culex mosquito:
● C. tritaeniorhynchus is the major vector worldwide including India.
● C. vishnui is the next common vector found in India.
➢ Transmission cycle: JE virus infects several animals and birds. Two
transmission cycles are predominant.
● Pigs → Culex → Pigs
● Ardeid birds → Culex → Ardeid birds
➢ Animal hosts:
● Pigs are considered as the amplifier host. JE virus multiplies
exponentially in pigs without causing any manifestation.
● Cattle and buffaloes may act as mosquito attractants.
● Horses are probably the only animal to be symptomatic and show
encephalitis.
● Humans are considered as dead end; there is no man → mosquito →
man cycle (unlike in dengue)
➢ Bird hosts: Ardeid (wading) birds such as herons, cattle egrets, and ducks
are the important reservoir.

❖ Epidemiology
➢ Geographical distribution: Currently, JE is endemic in the Southeast
Asian region.
● It is common in India, Nepal, Pakistan, Thailand, Vietnam and

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 Malaysia.
●Because of immunization, its incidence has been declining in
Japan and Korea.
● In India: JE has been reported since 1955. JE is endemic in 15
states; Uttar Pradesh (Gorakhpur district) accounting for the
largest burden followed by Assam, West Bengal, Bihar, Tamil
Nadu and Karnataka.
➢ Age: 85% of cases occur in children below 15 years (but infants are not
affected).
➢ Seasonal Variation: Common in the rainy season with (maximum
mosquito activity).

❖ Clinical Manifestations
JE is the most common cause of epidemic encephalitis:
➢ Incubation period: Varies from 5–15 days.
➢ Subclinical infection is common: JE typically shows iceberg
phenomena. Cases are much less compared to subclinical/inapparent
infection with a ratio of 1:300-1000.
➢ Even during an epidemic the number of cases are just 1–2 per village.
➢ Clinical course of the disease can be divided into three stages:
Prodromal stage, Acute encephalitis stage and Late stage with
sequelae of neurological deficits permanently.

❖ Vaccine Prophylaxis
1. Live attenuated SA 14-14-2 vaccine:
➢ It is prepared from SA 14-14-2 strain
➢ It is cell line derived; primary hamster kidney cells are
commonly used.
➢ Single dose is given subcutaneously, followed by a booster dose
after 1 year.
➢ It is manufactured in China, but now licensed in India.
➢ Under the Universal Immunization Programme, it is given to
children (1–15 years) targeting 83 endemic districts of four
states–UP, Karnataka, West Bengal and Assam.
2. Inactivated vaccine (Nakayama strain and Beijing strain):
➢ It is a mouse brain derived formalin inactivated vaccine.
➢ It is prepared in Central Research Institute, Kasauli (India).
3. Inactivated vaccine (Beijing P3 strain): It is a cell line derived
vaccine.

DENGUE VIRUSES
➢ Dengue virus is the most common arbovirus found in India. It has four
serotypes (DEN-1, to DEN-4). Recently, the fifth serotype (DEN-5) was
discovered in 2013 from Bangkok.

❖ Vector
Aedes aegypti is the principal vector (most efficient vector) followed by
Aedes albopictus. They bite during the day time.
➢ Aedes acquires infection by feeding on viremic patients (from a day
before to 5 days later, i.e. the end of the febrile period).

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 ➢ Extrinsic incubation period of 8–10 days is needed before Aedes
becomes infective.
➢ Once infected, it remains infectious for life.
➢ Aedes can pass the dengue virus to its offspring by transovarial
transmission.
➢ Transmission cycle: Man and Aedes are the principal reservoirs.
Transmission cycle does not involve other animals.

❖ Pathogenesis
➢ Primary dengue infection occurs when a person is infected with
dengue virus for the first time with any one serotype.
➢ Months to years later, a more severe form of dengue illness may appear
(called secondary dengue infection) due to infection with another
second serotype which is different from the first serotype causing
primary infection.
➢ The severity of secondary dengue infection occurs due to a unique
immunological phenomena called antibody dependent enhancement
(ADE), i.e. the non-neutralizing antibody produced against the first
serotype will combine, cover and protect the second serotype from
host immune response.
➢ ADE is remarkably observed when serotype 1 infection is followed by
serotype 2, which also claims to be the most severe form and prone to
develop into DHF and DSS.
➢ Serotype 2 is apparently more dangerous than other serotypes.

❖ Geographical Distribution
➢ Global Scenario: Tropical countries of Southeast Asia and Western
Pacific are at highest risk.
➢ Situation in India:
● Disease is prevalent in most of the urban cities/towns
affecting almost 31 states/ Union territories.
● Maximum cases have been reported from Kerala, Tamil Nadu,
Karnataka, Orissa, Delhi, Maharashtra and Gujarat.
● Maharashtra followed by Orissa accounted for maximum cases in
2014.
● All four dengue serotypes have been isolated from India. (DEN-1
and 2 are widespread).

❖ Laboratory Diagnosis
➢ NS1 antigen detection: ELISA and ICT are available for detecting
NS1 antigen in serum.
● Early detection: NS1 antigen becomes detectable from day-1 of
fever and remains positive up to 18 days.
● Highly specific: It differentiates between flaviviruses. It
can also be specific to different dengue serotypes.
➢ Antibody detection:
● In primary infection: IgM appears first after 5 days of
fever and disappears within 90 days, followed by IgG (14–21
days of illness).
● In secondary infection: Four fold rise of IgG antibody titers
occurs.

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 ●MAC: ELISA is the most recommended test with excellent
sensitivity and specificity. It can detect IgM and IgG
separately.
● Other antibody detection assays used previously are:
➢ HAI (Hemagglutination inhibition test)
➢ CFT (Complement fixation test)
➢ Neutralization tests such as plaque reduction test,
neutralization and microneutralization tests
➢ Virus detection: Dengue virus can be detected in blood from 1 day
before the onset of symptoms and 5 days thereafter. It is done by:
● Virus isolation can be done by inoculation into mosquito cell lines
or in mice
● Detection of specific genes of viral RNA by real time RT- PCR.

YELLOW FEVER VIRUS

Yellow fever is endemic in West Africa and Central South America. It is not found in
the rest of the World including India.
➢ Typing: At least seven genotypes of yellow fever virus have been
identified based on genomic sequence, five in Africa and two in South
America. There is only one stereotype.
➢ Vector: Humans get the infection by the bite of Aedes aegypti or the tiger
mosquito.
➢ Transmission cycle: Two major cycles of transmission have been recognized:
● Jungle cycle: Occurs between monkeys and forest mosquitoes.
● Urban cycle: Occurs between humans and urban mosquitoes (Aedes
aegypti)
➢ India: Yellow fever has not invaded India yet. Various reasons have been
hypothesized to explain the absence of yellow fever in India:
● Measures for the travelers taken at the international airports in India:
➔ Unvaccinated travelers coming from endemic zone to India will
be kept in quarantine for 6 days
➔ Breteau index or the Aedes aegypti index should be less than
one; surrounding 400 mt of airport.
● Cross reacting dengue antibodies provide protection against yellow
fever. However, yellow fever immunization does not protect from
dengue.
➢ Clinical manifestations: Incubation period is about: 3–6 days. Common
features include:
● Jaundice (hence the name yellow fever)
● Midzonal necrosis and presence of councilman bodies
● Intranuclear inclusions may be seen inside the hepatocytes called as
Torres bodies.

KYASANUR FOREST DISEASE VIRUS (KFD)

KFD virus was identified in 1957 from monkeys from the Kyasanur Forest in
Shimoga district of Karnataka, India.
➢ Vector: Hard ticks (Haemaphysalis spinigera)
➢ Hosts:

112
 ● Reservoirs are the rats and squirrels
● Amplifier hosts are the monkeys (KFD is known as Monkey’s disease).
● Man is an incidental host and considered as a dead end.
➢ Clinical Manifestation in humans: Incubation period varies from 3–8 days.
First stage (hemorrhagic fever) occurs followed by the second phase of
meningoencephalitis.
➢ Seasonality: KFD is increasingly reported in dry months (January-June) which
coincides with human activity in the forest.
➢ Situation in India:
● Endemic in 5 Districts of Karnataka, Shimoga, North Kannada, South
Kannada, Chikmagalur and Udupi
● Largest outbreak occurred in 1983–84. There is a declining trend
of incidence after the initiation of vaccine in 1999. Currently only focal
cases occur.
➢ Killed KFD vaccine: It is recommended in endemic areas of Karnataka
(all villages within 5 km of endemic foci).

INFLUENZA VIRUS
❖ General Properties
➢ Influenza virus is spherical and possesses helical symmetry
➢ Viral RNA comprises of eight segments of negative sense single
stranded RNA
➢ Site of RNA replication: In the nucleus (in contrast to
cytoplasm by most other RNA viruses)
➢ Viral proteins: Influenza virus contains eight structural proteins
(PB1, PB2, PA, NP, HA, NA, M1 and M2) and two nonstructural
proteins (NS1 and NS2).

➢ Antigens of Influenza:
● Hemagglutinin (HA): Cause hemagglutination
● Neuraminidase (NA): causes reversal of hemagglutination called Elution.

➢ Antigenic Variation
● Influenza virus type A and to less extent type B undergo
antigenic variation, which is of two types: antigenic shift and drift.
Type C influenza virus is stable.

Antigenic Shift Antigenic Drift

➢ Occur in Type A ➢ Occur in type A, to less extent

➢ Results in Pandemic and major type B
epidemic ➢ Results in periodic Epidemic and
➢ Due to genetic reassortment sporadic cases
➢ Occurs every 10–20 years ➢ Due to point mutation
➢ Occurs every 2–3 years

❖ Laboratory Diagnosis
➢ Specimen collection: Nasopharyngeal swab (polyester or Dacron
swabs).
➢ Transported in viral transport media, kept at 40°C up to 4 days,

113
 thereafter at 700°C.
➢ Isolation of virus: Embryonated eggs and primary monkey kidney cell
lines.
➢ Egg inoculation
● Amniotic cavity inoculation: It supports growth of Influenza A,
B, C
● Allantoic cavity inoculation: Supports growth of only Influenza
A
● Growth is detected by: Hemagglutination with Fowl and Guinea
pig RBC
● Type A: Agglutinates with Guinea pig RBC, Type C:
Agglutinates with fowl RBC at 4°C; and Type B: Agglutinates
with both.
➢ Antigen detection from nasopharyngeal cells by direct IF test.
➢ Antibody detection: Fourfold rise in the antibody titer is more
significant:
● Hemagglutination inhibition test (HAI)
● Neutralisation test: It is the most specific and the best
predictor of susceptibility to infection, but is time-consuming
and difficult to perform.
● ELISA is more sensitive than other assays.
➢ Molecular methods:
● Reverse transcriptase PCR (RT-PCR) is the most sensitive, 2
type specific.
● Real time RT: PCR can be used to quantitate the viral load in the
clinical sample.

MEASLES (RUBEOLA) VIRUS

➢ Measles is an acute, highly contagious childhood disease characterized
by fever and respiratory symptoms, and rash.
➢ Transmission occurs predominantly via the respiratory route.

❖ Clinical Manifestations
Incubation period is about 10 days which may be shorter in infants and
longer (up to 3 weeks) in adults
➢ Fever is the first manifestation, occurs on day-1 (i.e. on 10th day of
infection)
➢ Koplik’s spots are pathognomonic of measles, appear after two days
following fever and is characterized by:
● White to blush spot (1 mm size) surrounded by an erythema
● Appear first on buccal mucosa near second lower molars,
rapidly spread to entire buccal mucosa
➢ Rash: Maculopapular dusky red rashes appear after four days of
fever (i.e. at 14th day of infection).
● Rashes appear first behind the ears → then spread to face, arm,
trunk → then fade in the same order.
● Rashes are typically absent in HIV infected people.
Incubation period (10 days) → Fever (10th day)→ Koplik’s spot (12th day) → rash (14th day)

❖ Complications of Measles:

114
 ➢ Otitis media and bronchopneumonia are the most common
➢ Subacute sclerosing panencephalitis (SSPE) is the rarest but most
severe.

❖ Laboratory Diagnosis
➢ Cell lines: Monkey or human kidney cells or a lymphoblastoid cell line
(B95-a) are optimal cell lines used for isolation of measles.
Vero/hSLAM cell line is the CDC recommended cell line.
➢ Cytopathic effect: Multinucleated giant cells (Warthin-Finkeldey
cells) containing both intranuclear and intracytoplasmic inclusion
bodies.

RESPIRATORY SYNCYTIAL VIRUS

➢ Clinical Manifestations
● Infants: RSV is the most common cause of lower respiratory
tract infection below 1 year of age, causing bronchiolitis, pneumonia,
and tracheobronchitis.
● Adults: RSV produces influenza-like upper respiratory symptoms.
● RSV can cause exacerbation and worsening of asthma or COPD.
● Recurrent infection is common, but is much milder (common cold).
➢ Laboratory Diagnosis
● Virus isolation: HeLa and HEp-2 are the most sensitive cell lines for
virus isolation.
● A characteristic cytopathic effect, syncytium formation
(multinucleated giant cell) appears after 10 days, hence it is named as
syncytial virus.
➢ Epidemiology
● Seasonality: Rainfall, in winter and spring.
● Age: Infants between the ages of 6 weeks to 6 months of age.
● Subgroups: RSV can be typed into two subgroups; Subgroup A
infections appear to cause more severe illness.
➢ Treatment
● Ribavirin is the drug of choice. It is indicated for severe
infections in infants. However its beneficial effect to older
children and adults is doubtful. It is administered as aerosol for
3–6 days.

HEPATITIS VIRUSES
Properties HAV HBV HCV HDV HEV

Common Infectious Serum Non A non B Delta agent Non A non B

name hepatitis hepatitis or enteric
post-transf transmitted
usion hepatitis
hepatitis

Family Enterovirus- Hepadnaviri Flaviviridae Unclassified Unclassified

72 dae viroid-like Caliciviridae
(Picornavirid -like

115
 ae)

Virion 27 nm, 42 nm, 60 nm, 35 nm, 30–32 nm,

icosahedral spherical spherical spherical icosahedral

Envelope No Yes (HBsAg) Yes Yes (HBsAg) No

Genome ssRNA dsDNA ssRNA ssRNA ssRNA

Stability Heat and Acid-sensiti Ether-sensi Acid-sensiti Heat-stable

acid-stable ve tive, ve
acid-sensiti
ve

Onset Abrupt Insidious Insidious Insidious Abrupt

Age Children, Young Any age, but Any age Young

Young adults more (similar to adults
adults babies, common in HBV) (20–40
toddlers adults years)

Route Feco-oral Blood (MC) Blood (MC) Blood (MC) Feco-oral

Sexual, Sexual (+/-) Sexual(++)
Vertical Vertical Vertical(+)
(+/-)

Incubation 15–45 days 30–180 days 15–160 days 30–180 days 14–60 days
period (Average (Average (Average (Average (Average
30) 60–90) 50) 60–90) 40)

Fulminant Rare (0.1%) Rare Rare (0.1%) Frequent Usually rare

disease (0.1–1%) (5–20%) (1–2%)
Pregnancy-
20–40%

Carrier None Yes Yes Variable None

(0.1–30%) (1.5–3.2%)

Chronicity None Occasional Common Common None

(1–10%) (85%)

Oncogenic No Yes Yes +/ No

(neonate)

Prevalence High High Moderate Low, Regional

regional

Associated Secondary HCC, HCC, HCC, Secondary

other attack rate cirrhosis, cirrhosis, cirrhosis, attack rate
features 10–20% Autoimmune Autoimmune fulminant (1–2%)
disorder - AGN, hepatitis Rarely seen
like AGN, arthritis, in western
arthritis, cryoglobulin countries

116
 PAN emia

Prognosis Excellent Worse with Moderate Acute-good Good

age Chronic-
poor

Prophylaxis Immunoglob HBIG, None HBV vaccine Vaccine

ulin, Recombinan (no vaccine (HEV239)
Inactivated t vaccine for HBV (only in
vaccine carriers) China)

Therapy None Pegylated Pegylated Interferon None

interferon interferon ±
Lamivudine plus
ribavirin

HUMAN IMMUNODEFICIENCY VIRUS (HIV)

❖ Morphology
➢ Envelope: HIV and other lentiviruses are spherical and 80–110 nm in
size, possess an envelope; made-up of a lipid layer in which two
proteins are embedded:
● Glycoprotein 120 (gp 120) are projected as knob like spikes on
the surface
● Glycoprotein 41 (gp 41): They form anchoring transmembrane
pedicles.
➢ Nucleocapsid: Capsid is icosahedral in symmetry, made-up of core
protein. Inside, there is an inner core which encloses:
● RNA: Two identical copies of single-stranded positive sense
linear RNA
● Viral enzymes, such as reverse transcriptase, integrase and
proteases that are closely associated with HIV RNA.

❖ Antigenic Variation and Diversity of HIV

➢ HIV shows extensive antigenic diversity because of undergoing high
rates of mutation especially env gene and due to the error prone
nature of reverse transcriptase enzyme.

❖ Pathogenesis
➢ Mode of Transmission
● Most common mode of transmission in World: Sexual (75%)
(vaginal, 60% > anal, 15%) > Parent to child (10%) > Injection drug
abuse (10%) > Blood transfusion (5%) > Needle- stick exposure
(0.1%)
● Most common mode of transmission in India: Heterosexual
(87.4%) > Parent to child (5.4%) > Injection drug abuse (1.6%) >
Homosexual (1.5%) > Blood transfusion and Needlestick exposure
(together 1%).
● Risk of transmission: Blood transfusion (90-95%) > Parent to
child (20–40%) > Injection drug abuse (0.5–1.0%) > Needlestick
exposure (0.3%) > Sexual intercourse (Anal 0.0650.5% > vaginal

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 0.05–0.1% > oral 0.005–0.1%).
➢ Receptor Attachment and Fusion
● Main receptor: gp120 of HIV binds to the CD4 receptor on the
host cell surface. CD4 molecules are mainly expressed on helper
T-cells; but also on the surface of various other cells like
monocytes, macrophages, langerhans cells, astrocytes,
keratinocytes and glial cells.
● A second coreceptor is necessary for fusion of HIV by binding
to gp120 and to gain entry into the host cell, e.g.
➔ CXCR4 molecules present on T-lymphocytes
➔ CCR5 molecules present on cells of macrophage lineage
● DC-SIGN, a dendritic cell-specific lectin receptor can also bind
to HIV-1 but does not mediate cell entry. Rather, it may
facilitate transport of HIV by dendritic cells to lymphoid organs
where HIV replicates further in T-cells.
➢ Replication
● After fusion, HIV undergoes penetration & uncoating → HIV RNA
is converted to HIV DNA by RT enzyme→ Preintegration
complex is formed, comprises of linear dsDNA, gag matrix protein,
accessory Vpr protein and viral integrase which is transported into the
host cell nucleus.
● Integration: The viral dsDNA gets integrated into the host
cell chromosome; mediated by viral integrase. The integrated
virus is called provirus.
● Latency: In the integrated state, HIV establishes a latent
infection for a variable period. However, HIV is different
from other latent viruses as it is able to replicate even in
latent state and is infectious to the neighboring cells.

❖ Laboratory Diagnosis
Tests for detecting HIV infection

Specific tests for HIV infection Nonspecific/Immunological methods

➢ Screening tests (ERS) (IgG ➢ Low CD4 T-cell count

antibody detection, high
sensitivity):
● ELISA (takes 2–3 hours)
● Rapid/ Simple test (takes <
30 minutes)

➢ Supplemental tests (antibody ➢ Hypergammaglobulinemia due to

detection, high specificity): polyclonal B cell activation leading
● Western blot assay • to formation of abnormal Ig, such
Immunofluorescence assay as:
● Radioimmuno-precipitation ● Neopterin
assay (RIPA) ● β2-macroglobulin
● Line immunoassay (LIA)

➢ Confirmatory tests ➢ Altered CD4 : CD8 T-cell ratio

● p24 antigen detection

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 ● Viral culture by
Cocultivation technique
● HIV RNA (‘gold standard’
for confirmation of HIV
diagnosis)
➔ Reverse
transcriptase PCR
(RT-PCR)
➔ Branched DNA assay
➔ NASBA (Nucleic
acid sequence based
amplification)
➔ Real time RT-PCR
for estimating viral
load
● HIV DNA detection: Useful
for diagnosis of pediatric
HIV

❖ NACO Guidelines for Postexposure Prophylaxis (PEP)

➢ TLE Regimen
● Single tablet containing Tenofovir ( TDF) 300 mg plus
Lamivudine (3TC) 300 mg plus Efavirenz (EFV) 600 mg once daily
for 4 weeks.
● Ideally, therapy should be started within 2 hours and
definitely within 72 hours of exposure.
● Indicated to HCW exposed to
➔ Source positive for HIV (low risk/asymptomatic or high
risk/symptomatic) and
➔ Any kind of exposure (mild, moderate or severe
exposure).
● Source unknown

SLOW VIRAL DISEASES

➢ Slow virus diseases are:
● Long IP
● Predilection for CNS
● Strong genetic predisposition
● Lack in antigenicity -leads to
➔ Lack of immune response
➔ Lack of associated inflammation
● Does not produce cytopathologic effect in vitro

VIRUSES CAUSING GASTROENTERITIS

➢ Norwalk virus is a calicivirus in the family Caliciviridae. The caliciviruses are
small, nonenveloped, single-stranded RNA viruses.
➢ The caliciviruses although show many features similar to those of

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 picornaviruses, they differ from those by having a large genome and by having
distinct spikes on the surface.
➢ There are two important caliciviruses, which cause infection in humans:
Norwalk virus and Hepatitis E virus.

VIRAL HEMORRHAGIC FEVERS

➢ Most viral hemorrhagic fevers (VHF) are caused by 12 distinct enveloped RNA
viruses that belong to four families: Arenaviridae, Bunyaviridae, Filoviridae,
and Flaviviridae. The manifestations of the disease vary depending upon the
agent causing the disease. Circulatory dysfunction, increased vascu-lar
permeability, and diffuse hemorrhage, however, are the serious and terminal
manifestations of the disease. With recog-nition of outbreak of infection
caused by Ebola virus near the city of Kikwik, Zaire (Africa), the condition has
now received worldwide attention. Viral hemorrhagic fevers caused by
differ-ent viruses show following features:
● Viral agents are usually arthropod-borne. Mosquitoes are primarily
responsible for transmitting the disease.
● Person-to-person transmission may occur in many VHFs by direct
contact with infected patients, their blood, or their secretions and
excretions.
● Rats and mice are the usual animal reservoirs for many of the VHFs.
However, domestic livestock, monkeys, and other primates may also
serve as intermediate hosts.
● In this condition, hemorrhage is typically present in many organs, and
effusions are common in serous cavities. Widespread necrosis may be
present in any organ system, and varies from modest and focal to
mas-sive in extent. Liver and lymphoid systems are usually involved.
➢ Ebola and Marburg are two most important causative agents of VHFs with a
mortality of 25–100%. Both viruses are found in Africa and possibly in
Philippines. Zaire subtype of Ebola virus has been associated with a high rate
of infection, especially in Zaire, Africa. Ebola infection during pregnancy has
been con-sistently fatal. The vector responsible for transmission of Ebola
virus is not known, but infected primates appear to be respon-sible. Later
close contact among humans or primates appears to spread the infection.
Aerosol transmission is suggested to occur in monkeys.
➢ Incubation period of Ebola and Marburg infection varies from 2 to 14 days.
Symptoms are nonspecific. An insidious or sudden onset of fever, chills,
malaise, generalized myal-gias and arthralgias, headache, anorexia, and cough
are some of the common symptoms. The condition may also be associated with
sore throat, epigastric pain, vomiting, and diarrhea.
➢ No specific antiviral agents are available for the treatment for Ebola or
Marburg virus. Avoidance of insect bites from the vectors and that of
exposure to rodent sources of infection is the most important measure for
preventing the condition. Barrier nursing and needle sterilization in African
hospitals are important to eliminate epidemics of Ebola and Marburg diseases

CLINICAL SYNDROMES OF RUBELLA

❖ Rubella

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 ➢ Incubation period varies from 14 to 21 days. Rubella is a milder and
more subtle disease than measles. A three-day maculopap-ular or
macular rash, which starts on the face and progresses downward to
involve the extremities, is the characteristic presentation in
symptomatic cases. The rash typically lasts 3 days. Tender
lymphadenopathy that affects all the nodes but most commonly affects
suboccipital, postauricular, anterior, and posterior cervical nodes is the
hallmark of rubella.
➢ In adults, rubella produces a more severe disease with mani-festations
of arthralgia and polyarthritis, and rarely thrombo-cytopenia or post
infectious encephalopathy.

❖ Congenital rubella syndrome

➢ Congenital rubella syndrome is the most severe and important
complication of rubella, which occurs in the fetus of pregnant women
without immunity to the virus. The fetus is at major risk until fifth
month of pregnancy. Maternal immunity to the virus due to prior
exposure or vaccination prevents spread of the virus to the fetus. In
the first trimester, 80% of the infants would be affected, and severity
of the disease depends on how early the infection occurs. Cataract,
mental retardation, and deafness are the most common manifestations
of congenital rubella infection.
➢ The congenital rubella results in congenital anomalies or even death of
the fetus. In addition, the infants infected in utero continue to
excrete rubella virus for up to 1 year. These children constitute a
public health hazard because they are considered as an exposure
threat to nonimmune pregnant women. The virus can be transmitted to
pregnant women from children.

MYCOLOGY

DERMATOPHYTOSES
➢ Dermatophytosis (or tinea or ringworm) is the commonest superficial mycoses
infecting keratinized tissues:

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 1. Trichophyton species: Infect skin, hair and nail
2. Microsporum species: Infect skin and hair
3. Epidermophyton species: Infect skin and nail
➢ Depending on the usual habitat dermatophytes are classified as follows:
● Anthropophilic species (infect only humans): Most common type of
dermatophyte infection in man, produce mild and chronic lesions but
respond poorly to treatment.
● Geophilic (soil species) and zoophilic species (infects animals):
Produce more acute inflammatory response and severe infections; but
they tend to resolve more quickly.

➢ Dermatophytid or ID reaction
● Occasionally, hypersensitivity to dermatophyte antigens may occur
which leads to appearance of secondary eruption in sensitized patients
because of circulation of allergenic products. However, these lesions
are distinct from the primary ringworm lesions as they occur distal to
primary site and fungal culture often turns negative.

❖ Laboratory Diagnosis
➢ Wood's Lamp Examination:Certain dermatophytes fluoresce when the
infected lesions are viewed under Woods lamp.
● This is due to the presence of pteridine pigment in the cell wall.
● It is positive for various Microsporum species and Trichophyton
schoenleinii. Negative for other dermatophytes.
➢ Specimen Collection:Skin scrapings, hair plucks (broken or scaly ones)
and nail clippings are obtained from the active margin of the lesion and
are kept in folded black paper.
➢ Direct examination (10% KOH mount): Reveals thin septate hyaline
hyphae with arthroconidia.
➢ Culture on SDA followed by LPCB mount of the colonies: Reveals two
types of spores (macroconidia and microconidia), based on which
speciation is done.
❖ Other methods of diagnosis
➢ Apart from culture, there are several other methods for identification
of dermatophytes:
● Hair perforation test is positive for Trichophyton
mentagrophytes and Microsporum canis.
● Urease test: Positive for Trichophyton mentagrophytes
● Dermatophyte test medium and dermatophyte identification
medium
● PCR can be used to detect species specific genes (e.g. chitin
synthase gene)
● Skin test for detecting hypersensitivity to dermatophyte
antigen (Trichophyton).

❖ Treatment
➢ Oral terbinafine or itraconazole are the drugs of choice for treatment.
Duration of treatment is 1–2 weeks for skin lesions, 6 weeks for hair
infection, 3 months for onychomycosis.
➢ Alternate: Oral griseofulvin and ketoconazole may be given
➢ Topical lotion, such as whitfield ointment or tolnaftate can be applied.

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MYCETOMA
➢ Mycetoma is a chronic, slowly progressive granulomatous infection of the skin
and subcutaneous tissues.
● Clinically, it is manifested as a triad of swelling, discharging sinuses and
presence of granules in the discharge.
● Mycetoma (also known as Maduramycosis or Madura foot) is of two
types: eumycetoma and actinomycetoma.

❖ Epidemiology
➢ Mycetoma is endemic in Africa, India and the Central and South
Americas.
● Overall, actinomycetoma is more common (60%) than
eumycetoma (40%) globally.
● Eumycetoma is more common in Africa.
● In India, Rajasthan reports maximum cases of mycetoma per
year followed by Tamil Nadu and West Bengal.
Actinomycetoma predominates in India (65%) except in
Rajasthan where eumycetoma is more common.

❖ Laboratory Diagnosis
➢ Direct Examination
● Granules are thoroughly washed in sterile saline; crushed
between the slides and examined
1. Macroscopic appearance of granules, such as color,
size, shape, texture
2. If eumycetoma is suspected: Grains are subjected to
KOH mount which reveals hyphae of 2–6 µm
3. If actinomycetoma is suspected: Gram staining
which reveals filamentous Gram-positive bacilli (0.5–1
µm wide). Modified acid fast stain is performed if
Nocardia is suspected as it is partially acid fast.
4. Histopathological staining of the granules:
➔ Eumycetoma: Reveals granulomatous reaction with
palisade arrangement of hyphae in the cement
substance
➔ Actinomycetoma: Shows granulomatous reaction
with filamentous bacteria at the margin.
➢ Culture
● Granules obtained from deep biopsies are the best
specimen for culture. Both fungal (e.g. SDA) and Lowenstein
Jensen media (for Nocardia) should be used.

❖ Treatment
➢ Treatment of mycetoma consists of surgical removal of the lesion
followed by use of:
● Antifungal agents for eumycetoma (itraconazole or amphotericin
B for 8–24 months)
● Antibiotics for actinomycetoma, such as Welsh regimen
(amikacin plus cotrimoxazole).

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SPOROTRICHOSIS
➢ Sporotrichosis or Rose Gardener's disease is chronic subcutaneous
granulomatous disease; caused by a thermally dimorphic fungus Sporothrix
schenckii. Various clinical manifestations have been observed.
● Nodulo-ulcerative lesions (painless) spreading along the lymphatics
● Lymph nodes become enlarged, suppurative and indurated
● Other rare clinical types are osteoarticular type, pulmonary type,
disseminated sporotrichosis.

❖ Epidemiology
➢ Prevalent in tropical countries with high humidity (South Africa and
India).
➢ In India, sporotrichosis is prevalent in sub Himalayan hilly areas
(from Himachal Pradesh to Assam).
➢ Risk factors include people walking barefoot (such as farmers and
gardeners).

❖ Laboratory Diagnosis
➢ Direct Microscopy by H and E staining of tissue sections reveals
cigar shaped asteroid bodies. It is described as a central basophilic
yeast cell surrounded by eosinophilic mass, composed of
antigen-antibody complexes. Such an eosinophilic halo is described as
the Splendore-Hoeppli phenomenon.
➢ Culture: Specimens are inoculated on SDA and incubated at 25°C and
37°C
● At 25°C: It produces mycelial form, consisting of slender
hyphae with conidia arranged in flower like pattern
● At 37°C: It produces yeast form, characterized by moist
creamy white colonies
➢ Serology: Latex agglutination test detects serum antibodies in
patients with extracutaneous form
➢ Skin test may demonstrate delayed type of hypersensitivity reaction
against sporotrichosis antigen.

❖ Treatment
➢ Itraconazole is the DOC, except for disseminated infection
(amphotericin B is DOC).

RHINOSPORIDIOSIS
Rhinosporidiosis is a chronic granulomatous disease, characterized by large friable
polyps in the nose (MC site), conjunctiva and occasionally in ears, larynx, bronchus and
genitalia.
➢ Agent: It is caused by Rhinosporidium seeberi, recently reclassified as an
Aquatic Protist Parasite(Mesomycetozoea); previously classified as lower
aquatic fungi.
➢ Source: Stagnant water is the main source of infection. Fungal spores are
inhaled while taking bath in ponds and rivers.
➢ Distribution: Tropical countries especially in Sri Lanka and India

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 (Tamilnadu, Kerala, Orissa and Andhra Pradesh)
➢ Diagnosis:
● Histopathology of polyp reveals spherules (large sporangia containing
numerous endospores).
● R.seeberi has not been cultivated yet.
● It is stained better with mucicarmine stain.
➢ Treatment: Radical surgery with cauterization is the mainstay of treatment.
Dapsone has been found to be effective. Recurrence is common.

HISTOPLASMOSIS
❖ Laboratory Diagnosis
➢ Histopathological staining of specimens reveals tiny oval yeast cells
(2–4 µm size) with narrow based budding within the macrophages and
underlying granulomatous response
➢ Culture is the gold standard method of diagnosis: Histoplasma is a
dimorphic fungus, hence:
● At 25°C: Produces white mycelial colonies that consist of
two types of conidia: Thick tuberculate macroconidia
(characteristic) and thin microconidia
● At 37°C: It gets converted into yeast form (creamy white
colonies) in special Kelley’s media.
➢ Antibodies in serum can be detected by CFT and immunodiffusion test.
➢ Skin Test may be done to demonstrate delayed type hypersensitivity.

CRYPTOCOCCOSIS
Cryptococcus has two species, C. neoformans and C.gattii and four serotypes
A,B,C and D.
➢ C.neoformans occurs in two varieties - Cryptococcus neoformans var. grubii
and Cryptococcus neoformans var. neoformans; which correlate with
serotypes A and D, respectively.
➢ C.gattii is antigenically diverse, corresponding to the serotypes B and C.
➢ Most laboratories do not routinely distinguish between the types, and report
all isolates simply as C.neoformans.
➢ CNS spread: The unique feature of Cryptococcus is its ability to cross
the blood-brain barrier which occurs when yeast cells either migrate directly
across the endothelium or are carried inside the macrophages as ‘Trojan
horse’.
➢ Virulence Factors of Cryptococcus that favor invasion and spread of
infection include:
● Polysaccharide capsule.
● Ability to make melanin by producing phenyloxidase enzyme.
● Production of other enzymes, such as phospholipase and urease
➢ Risk factors: Individuals at high-risk for cryptococcosis include:
● Patients with advanced HIV infection with CD4 T-cell counts <
200/µl is the most important risk factor for C.neoformans. However,
C.gattii is not associated with HIV. It usually causes infection in
immunocompetent individuals.
● Patients with hematologic malignancies

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 ● Transplant recipients
● Patients on immunosuppressive or steroid therapy.

❖ Laboratory Diagnosis
➢ Direct detection methods:
● Negative staining by modified India ink stain and nigrosin stain
to demonstrate the capsule which appears as refractile
delineated clear space surrounding the round budding yeast cells
against black ground. Sensitivity is 60–70%.
● Gram staining may show Gram-positive round budding yeast cells
● Other stains: Mucicarmine stain Masson-Fontana stain, Alcian
blue stain
● Capsular antigen detection from CSF or serum by latex
agglutination test is a rapid and sensitive (95% ).
➢ Confirmation of Cryptococcus species is made by:
● Culture on SDA: Colonies appear as mucoid creamy white yeast
like colonies
● Niger seed agar and bird seed agar is used to demonstrate
melanin production
● Growth at 37°C, urease test positive
● Assimilation of inositol and nitrate and mouse pathogenicity test
positive.

OPPORTUNISTIC MYCOSES
Disease Causative fungus

Candidiasis Candida albicans, Candida tropicalis and

other

Aspergillosis Aspergillus fumigatus, Aspergillus flavus,

Aspergillus niger and other diseases

Zygomycosis Mucor, Rhizomes and Absidia speciss.

Pneumocystis carinii pneumonia (PCP) Pneumocystis jirovecii

Penicilliosis Penicillium marneffei

Pseudallescheria boydii infection Pseudallescheria boydii

Fusarium solani infection Fusarium solani

Meningitis Cryptococcus neoformans

CANDIDIASIS
➢ Candidiasis accounts for the most common fungal infection in humans both
in HIV and nonHIV infected people; caused by Candida, a yeast-like fungus

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 that produces pseudohyphae. Various species of Candida include:
● Candida albicans: The most common and most pathogenic species
● Other rare species are C. tropicalis, C. glabrata, C. krusei, C.
parapsilosis, C. dubliniensis, C. kefyr, and C. viswanathii.

❖ Clinical Manifestations
➢ Candida species are a part of normal flora of the skin and
mucosa including gut flora. In the presence of opportunistic
conditions, they can cause various infections.
1. Mucosal candidiasis:
➢ Oropharyngeal candidiasis (oral thrush) presents as
white, adherent, painless patches in the mouth
➢ Candidal vulvovaginitis thin whitish curd like vaginal
discharge
➢ Balanitis and balanoposthitis (occurring in uncircumcised
males)
➢ Esophageal candidiasis
➢ Angular stomatitis and denture stomatitis
➢ Chronic mucocutaneous candidiasis: Seen in infants
with deficient CMI, resistant to treatment.
2. Cutaneous candidiasis:
➢ Intertrigo, Paronychia and onychomycosis (fungal
infection of nail)
➢ Diaper candidiasis in infants, Perianal candidiasis
➢ Erosio interdigitalis blastomycetica, an infection
between the digits of the hands or toes
➢ Generalized disseminated cutaneous candidiasis, seen in
infants.
3. Invasive candidiasis: Results from hematogenous or local
spread of the fungi. Various forms are:
➢ UTI, Pulmonary candidiasis, meningitis, osteomyelitis
and Hepatosplenic and disseminated candidiasis
➢ Septicemia (C.albicans and C.glabrata).
➢ Ocular: Keratoconjunctivitis and endophthalmitis
➢ Nosocomial candidiasis (mainly by C. glabrata)
4. Allergic candidiasis Include:
➢ Candidid: Vesicular lesions in the web space of hands,
similar to that of dermatophytid reaction (both
conditions are together called ‘ID’ reaction)
➢ Other allergic reactions include: Gastritis, irritable
bowel syndrome and eczema.

❖ Laboratory Diagnosis
➢ Direct microscopy: Gram-positive oval budding yeast cells (4–6 µm
size) with pseudohyphae.
➢ Culture on SDA: Colonies are described as creamy white, smooth,
and pasty with typical yeasty odor.
➢ Tests For Species identification:
● Germ tube test: It is also called Reynolds Braude
phenomenon, specific test for C. albicans.
➔ It is differentiated from pseudohyphae as there is no

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 constriction at the origin
➔ Though the test is specific for C. albicans, it may
also be positive for C. dubliniensis.
➢ Dalmau plate culture on Cornmeal agar C. albicans produces thick
walled chlamydospores
● CHROM agar: Different Candida species produce different
colored colonies on CHROM agar.
● Growth at 45°C: It differentiates C. albicans (grows well)
from C.dubliniensis (does not grow at 45°C).
● Sugar fermentation test and sugar assimilation test.
➢ Immunodiagnosis:
● Antibody detection: Against cell wall mannan antigen.
● Antigen detection: Cell wall mannan and cytoplasmic antigens
can be detected by ELISA
● Enzyme detection: Specific for Candida such as enolase,
aspartate proteinase, etc.
● Test for metabolites specific for Candida such as mannitol,
arabinitol can be detected.
● G test is done for detection of α1-3 glucan.

ASPERGILLOSIS
➢ Aspergillus species are widely distributed on decaying plants, producing chains
of conidia.
➢ Risk Factors for invasive aspergillosis are:
● Glucocorticoid use (the most important risk factor)
● Profound neutropenia or Neutrophil dysfunction
● Underlying pneumonia or COPD, tuberculosis or sarcoidosis
● Antitumor necrosis factor therapy.

❖ Clinical Manifestations
➢ Clinical manifestations of aspergillosis depend on the site of
involvement. The incubation period varies from 2 to 90 days.
1. Pulmonary Aspergillosis (MC form): Various forms include
Allergic bronchopulmonary aspergillosis (ABPA), Asthma,
Extrinsic allergic alveolitis, Aspergilloma (fungal ball) and
Chronic cavitary pulmonary aspergillosis
2. Invasive sinusitis: Invasive sinusitis, chronic granulomatous
sinusitis, maxillary fungal ball and allergic fungal sinusitis
3. Ocular aspergillosis: Keratitis and endophthalmitis
4. Ear infection: Otitis externa
5. Others: Endocarditis, brain abscess, skin lesions and
onychomycosis.
➢ Clinical manifestations also depend on the species involved:
● A. fumigatus accounts for most of the cases of acute pulmonary
and allergic aspergillosis.
● A. flavus is more common in hospitals and causes more sinus,
skin and ocular infections than A. fumigatus.
● A. niger can cause invasive infection but more commonly
colonizes the respiratory tract and causes otitis externa.

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ZYGOMYCOSIS
➢ Zygomycosis or mucormycosis represents group of life-threatening
infections caused by aseptate fungi belonging to the phylum zygomycota.
Examples include: Rhizopus, Mucor and Absidia
➢ Predisposing factors: Agents of mucormycosis require iron as growth
factor. Hence conditions with increased iron load are at higher risk of
developing invasive mucormycosis, such as:
● Diabetic ketoacidosis (DKA) is the most important risk factor
● End stage renal disease
● Patients taking iron therapy or deferoxamine (iron chelator)
● Defects in phagocytic functions (e.g. neutropenia or steroid therapy).

❖ Clinical Manifestations
➢ Agents of mucormycosis are angioinvasive in nature. There are six
types of clinical presentations:
1. Rhinocerebral mucormycosis: MC form; presents as orbital
cellulitis, proptosis and vision loss
2. Pulmonary mucormycosis is the second MC form, occurs in
patients with leukemia.
3. Cutaneous mucormycosis
4. Gastrointestinal mucormycosis, such as necrotizing
enterocolitis; seen commonly in premature neonates
5. Disseminated mucormycosis: Brain is the most common site of
dissemination Miscellaneous forms may involve any body site,
including bones, trachea and kidneys, etc.

❖ Laboratory Diagnosis
➢ Histopathological staining of tissue biopsies shows broad aseptate
hyaline hyphae with wide angle branching
➢ Culture on SDA at 25°C reveals cottony woolly colonies which are
initially white, later become brown black due to sporulation giving rise
to salt and pepper appearance
➢ Microscopic appearance: LPCB mount of the colonies reveals broad
aseptate hyaline hyphae, from which sporangiophore arise which ends
at sporangium containing numerous sporangiospores
➢ Rhizoid: Some species bear a unique root like growth called
rhizoid which provides initial clue for identification of the fungus.
Species can be differentiated depending on the position of the
rhizoid with respect to sporangiophore:
● Rhizopus bears nodal rhizoid
● Absidia bears internodal rhizoid
● Mucor: rhizoid is absent.

❖ Treatment
➢ Amphotericin B deoxycholate remains the drug of choice for all forms
of mucormycosis except the mild localized skin lesions in
immunocompetent patients, which can be removed surgically.

PENICILLIOSIS

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 ➢ Penicillium species are usually found in environment and as laboratory
contaminants. Rarely infects humans.
● Common manifestations: endophthalmitis, otomycosis, onychomycosis
and allergic pneumonitis
● Microscopy: Reveal hyaline thin septate hyphae, vesicles are absent,
and conidia arranged as brush border appearance.

➢ Penicillium Marneffei
● Penicillium marneffei is a thermally dimorphic fungus that causes
opportunistic infection in HIV infected patients.
➔ Systemic infection mimicking that of disseminated
histoplasmosis
➔ Skin lesions: Warty lesions mimicking that of Molluscum
contagiosum are seen.
● It is endemic in SE Asian countries including Thailand, Vietnam and
India (Manipur).
● P. marneffei is found mostly in rural areas and bamboo rats are the
reservoirs of infection
● Lab Diagnosis:
➔ Direct Microscopy: Shows oval or elliptical yeast cells with
central septation, which indicates that these cells divide by
transverse fission rather than budding
➔ Culture: P. marneffei being dimorphic; produces yeast like
colonies at 37°C and mold form at 25°C. The mold form has a
characteristic brick red pigment.
● Treatment: AIDS patients with severe penicilliosis are treated with
amphotericin B till the condition improves followed by maintenance
therapy with itraconazole for 12 weeks. In mild penicilliosis,
itraconazole is recommended for 12 weeks.

CLINICAL MICROBIOLOGY

URINARY TRACT INFECTION

Bacterial agents: Other agents

➢ Gram-negative bacilli: ➢ Fungus:

● Escherichia coli- ● Candida albicans
Commonest agent of UTI
● Proteus mirabilis ➢ Parasites:
● Klebsiella pneumoniae ● Schistosoma haematobium
● Pseudomonas aeruginosa ● Trichomonas vaginalis
● Acinetobacter species
● Enterobacter species
● Serratia species

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 ➢ Gram-positive cocci: ➢ Viruses:
● Staphylococcus ● Herpes simplex virus
saprophyticus ● Adenovirus
● Staphylococcus aureus ● JC and BK virus
● Staphylococcus epidermidis ● Cytomegalovirus
● Enterococcus spp.

INFECTIOUS AGENTS CAUSING FOOD POISONING

Organisms Symptoms Common food sources

1–6 h incubation period

Staphylococcus aureus Nausea, vomiting, diarrhea Ham, poultry, potato or

egg salad, mayonnaise,
cream pastries

Bacillus cereus Nausea, vomiting, diarrhea Fried rice

Clostridium botulinum Nausea, vomiting, diarrhea Canned food

8–16 h incubation period

Clostridium perfringens Abdominal cramps, Beef, poultry, legumes,

diarrhea (vomiting rare) gravies

Bacillus cereus Abdominal cramps, Meats, vegetables, dried

diarrhea (vomiting rare) beans, cereals

> 16 h incubation period

Vibrio cholerae Watery diarrhea Shellfish, water

Enterotoxigenic Watery diarrhea Salads, cheese, meat,

Escherichia coli water

Enterohemorrhagic Bloody diarrhea Ground beef, roast beef,

Escherichia coli salami, raw milk, raw
vegetables, apple juice

Salmonella species Inflammatory diarrhea Beef, poultry, eggs, dairy

products

Campylobacter jejuni Inflammatory diarrhea Poultry, raw milk

Shigella species Dysentery Potato or egg salad,

lettuce, raw vegetables

Vibrio parahaemolyticus Dysentery Mollusks, crustaceans

MENINGITIS
Pyogenic meningitis Aseptic meningitis

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Neonates or infants of ➢ Escherichia coli Viruses
0–2 months ➢ Group B ➢ Enteroviruses
streptococcus (S. (Polioviruses,
agalactiae) echoviruses,
➢ Listeria Coxsackie viruses):
monocytogenes The most common
➢ Other agents Herpes
Gram-negative simplex virus 1 and
bacilli (like 2
Klebsiella ➢ Other Herpes
pneumoniae) group: Varicella
zoster, CMV, EBV
2–20 years ➢ Neisseria ➢ Myxoviruses:
meningitidis: Most Influenza A and B,
common agent parainfluenza virus,
➢ Haemophilus and mumps virus
influenzae ➢ Arboviruses, and
➢ Streptococcus adenoviruses,
pneumoniae ➢ Rubella viruses and
HIV

> 20 years (adults) Streptococcus pneumoniae: Bacteria: Treponema

Most common agent pallidum, and Leptospira
Haemophilus influenzae
Neisseria meningitidis Parasites- Naegleria
species, Acanthamoeba
species and Toxoplasma
gondii

Overall Most common agent is Fungi: Cryptococcus

Streptococcus pneumoniae neoformans

FEVER OF UNKNOWN ORIGIN

➢ Petersdorf and Beeson classification (1961) was traditionally used for defining
PUO.
● Temperatures of > 38.3°C (>101°F)
● For a duration of > 3 weeks; and
● Failure to reach a diagnosis despite 1 week of inpatient investigation.
➢ This classification has stood for more than 30 years, but later in 90s, it was
revised as Durack and Street classification. Thereafter it was further
modified in 2015. According to Harrison 19th edition, FUO is now defined as:
1. Fever > 38.3°C (101°F) on at least two occasions
2. Illness duration of > 3 weeks
3. No known immunocompromised state
4. Diagnosis that remains uncertain after a thorough history-taking,
physical examination and the obligatory investigations. Infections
(36%) accounts for majority of FUO cases followed by Neoplasms
(19%).

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METHODS OF WATER ANALYSIS
➢ Presumptive coliform count (Multiple tube method): This detects the
probable number of coliform bacilli in water.
● It is done by calculating as the most probable number (MPN) of
coliform organisms in 100 ml water
● Medium: MacConkey purple broth (double strength and single strength)
in is the standard medium of choice
● Detection of coliform bacteria does not always indicate fecal
contamination as some of them may be found in the environment.
Hence, it is further tested by differential coliform count to
detect the fecal E. coli.
➢ Eijkman test: It is done to confirm that the coliform bacilli detected
in the presumptive test are fecal thermo tolerant E.coli which grows at
44 °C with indole and gas production and lactose fermentation. Brilliant
green bile broth is used.
➢ Other methods
● Clostridium perfringens detection
● Enzyme detection: β galactosidase (coliform bacilli specific enzyme)
and β glucuronidase (fecal E.coli specific)
● Membrane filtration method
● Examination for specific water borne pathogens such as
Salmonella Typhi and Vibrio cholerae

MMR VACCINE
➢ Measles vaccine is usually given with the mumps and rubella vaccines in
children 12–15 months of age and older.
● Two doses of MMR vaccine are recommended for all children on or
after the first birthday, including those who previously received the
monovalent measles vaccine.
● The first dose is generally given at 12–15 months of age, and the
second dose is generally given at 4–6 years of age. There must be a
minimum of 4 weeks between the two doses.
● The second dose of MMR vaccine provides an added safeguard against
all three diseases, but is recommended primarily to prevent outbreaks
of measles
➢ Students who are exposed to an outbreak but have not already received two
doses of the vaccine and who do not have other proof of immunity may be
excluded from school for the entire duration of the outbreak or are required
to receive the measles vaccination. The second dose of the measles vaccine
series is effective when given as early as 1 month after the first dose, and
this schedule is used when protection is needed quickly. Ninety-five percent of
those who receive the MMR or monovalent measles vaccine at 12 months of
age or older become immune after the first dose. After the second dose,
99.7% of those immunized are protected. Immunity is lifelong.
➢ There are hypotheses that the MMR vaccine causes autism. However, the best
available science indicates that the develop-ment of autism is not related to
the use of the MMR or any other vaccine. One small study seemed to postulate
such a link, but has subsequently been disproved by many other larger studies.
Ten of the thirteen authors of that study later retracted from their

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 suggestion of a link between MMR vaccine and autism.

BIOMEDICAL WASTE MANAGEMENT RULE 2016

Categor Type of waste Type of bag/ Treatment/ Disposal
y container options

Yellow Human anatomical Yellow coloured non Incineration/ Plasma

waste chlorinated plastic pyrolysis/ deep burial
bags
Animal anatomical
waste

Soiled waste Incineration/ Plasma

Pyrolysis/ deep burial/
autoclaving or
hydroclaving +
shredding/mutilation

Expired/ discarded Yellow coloured Incineration (cytotoxic

medicines- containers/ non drugs at temperature >
pharmaceutical chlorinated plastic 1200°C)
waste, cytotoxic bags
drugs

Chemical waste Yellow coloured incineration or Plasma

containers/ non pyrolysis or Encapsulation
chlorinated plastic
bags

Chemical liquid waste Separate collection Pre-treated before

system leading to mixing with other
effluent treatment wastewater
system

Discarded linen Non-chlorinated yellow Non- chlorinated chemical

contaminated with plastic bags/ suitable disinfection followed by
blood/ body fluids packing material incineration/ plasma
pyrolysis

Microbiology, other Autoclave safe plastic Pre-treat to sterilize with

clinical lab waste, bag/ container non-chlorinated chemicals
blood bags, live/ onsite as per NACO/
attenuated vaccines WHO guidelines +
Incineration.

Red Contaminated Waste Red coloured Autoclaving/ microwaving/

(Recyclable) non-chlorinated plastic hydroclaving + shredding
bags or containers Mutilation/ sterilisation
shredding. Treated waste
sent to registered or
authorized recyclers or

134
 for energy recovery or
plastics to diesel or fuel
oil or for road making,

White Waste sharps Puncture proof, Leak Autoclaving/ dry heat

(Translu including metal sharp proof, tamper proof sterilization+ shredding/
cent) containers mutilation Encapsulation in
metal container or cement
concrete Sanitary
landfill/ designated
concrete waste sharp pit

Blue Glassware Cardboard boxes with Disinfection (by soaking

Metallic body blue colored marking the washed glass waste
implants after cleaning with
detergent and Sodium
Hypochlorite treatment)/
through autoclaving/
microwaving/ hydroclaving
+ recycling

PARASITOLOGY

135
INTERMEDIATE HOST
➢ Intermediate host: Asexual cycle takes place.
● Direct/Simple life cycle: Parasites that need only one host (man)

Protozoa Helminths

➢ Entamoeba histolytica ➢ Cestodes: Hymenolepis nana

➢ Free living ameba ➢ Nematodes:
➢ Giardia lamblia ● Ascaris lumbricoides
➢ Trichomonas vaginalis ● Hookworm
➢ Balantidium coli ● Enterobius vermicularis
➢ Cryptosporidium parvum ● Trichuris trichiura
➢ Cyclospora cayetanensis ● Strongyloides spp.
➢ Isospora belli

INTESTINAL AMEBAE : ENTAMOEBA HISTOLYTICA

➢ Habitat: Large intestine of man
➢ Morphology: 3 stages: Trophozoites, Pre-cyst and cyst.
➢ Life cycle
● Host: Humans are the only host
● Infective form: Mature quadrinucleate cyst
● Mode of transmission: Faeco-oral route and rarely sexual
transmission (20–30% in homosexuals)
● Excystation occurs in small intestine → eight small metacystic trophozoites are
released→ carried to large intestine → trophozoites colonize the GIT
mucosa → depending on nutritional status and host immunity, there may
have different courses (i) Asymptomatic cyst passers, (ii) Amebic dysentery, (iii)
Amebic liver abscess.
● Encystation: Occurs in the large intestine → first precyst → immature
cysts and→ later mature quadrinucleate cysts are released in the feces
(diagnostic form).

➢ Virulence Factors
● Amebic Lectin antigen: Helps in adhesion
● Cysteine proteinase Amoebapore, Hydrolytic enzymes, Neuraminidase
and metallic collagenase

❖ Intestinal Amebiasis
➢ Males and females are affected equally with a ratio of 1:1
➢ Amebic ulcer:
● Flask-shaped (broad base with a narrow neck)
● Most common site: Ileocecal region
➢ Complications of intestinal amebiasis:
● Fulminant amebic colitis
● Amebic appendicitis
● Intestinal perforation and amebic peritonitis
● Toxic megacolon and Intussusception
● Perianal skin ulcers
● Ameboma (Amebic granuloma): Diffuse pseudotumor like mass

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 found in rectosigmoid region, seen in chronic amebiasis.

❖ Amebic Liver Abscess

➢ MC site involved: Posterior superior surface of right lobe of liver
➢ Sex: Male:female ratio is 9:1
➢ Anchovy sauce pus: Liver abscess pus is thick chocolate brown colour
➢ Other organs affected: Lung, brain, skin.

❖ Laboratory Diagnosis
➢ Minimum of three stool samples on consecutive days: As amebae are
shed intermittently.
➢ Stool Microscopy: done to demonstrate:
● Trophozoites: Indicates active infection
● Quadrinucleate cysts: Indicates carrier state
➢ Cyst and trophozoites of E. histolytica should be differentiated
from E. coli which is a commensal in stool.
➢ Stool culture
➢ Polyxenic culture:
● Contains bacterial supplement providing nourishment to ameba.
● Used in chronic and asymptomatic carriers passing less number
of cysts.
● 50–70% sensitivity and 100% specificity (gold standard).
● Various culture media used are:
➔ National Institute of Health (NIH) media
➔ Boeck and Drbohlav egg serum medium containing Locke’s
solution
➔ Balamuth’s medium, Nelson’s medium and Robinson’s
medium.
➢ Axenic culture: It lacks bacterial supplement, e.g. Diamond’s medium.
Axenic culture is useful when the bacterial flora interferes with the
test results such as:
● Studying pathogenicity of ameba
● Testing antiemetic drug susceptibility
● Preparation of amebic antigen in mass for serological tests
● For harvesting the parasite to determine the zymodeme pattern.
➢ Stool antigen detection (copro-antigen): By ELISA or ICT detecting
lectin antigen
➢ Amebic antigen in serum indicates recent and active infection. ELISA
is done for lectin antigen
➢ Amebic antibody: Serum antibodies appear only in the later stages of
intestinal amebiasis, e.g. ELISA, IFA, indirect hemagglutination test
(IHA). • Zymodeme Analysis: Detecting isoenzyme markers like
malic enzyme, hexokinase, isomerase.
➢ Nested multiplex PCR
➢ Charcot Leyden crystal in stool and moderate leukocytosis in blood.

❖ Laboratory Diagnosis of Amebic Liver Abscess

➢ Microscopy and Histopathology of liver pus shows trophozoites but
never cyst. This is specific but of low sensitivity (< 25%)
➢ Stool microscopy and culture is not useful
➢ Lectin antigen is usually absent in stool but can be demonstrated in

137
 serum, liver pus and saliva
➢ Antibody detection: Antibodies are often elevated and can be
detected by ELISA and IHA. However, antibodies persist even after
the cure, so it cannot differentiate recent and old infection.
➢ PCR done on amebic liver pus
➢ USG of liver shows the site of the abscess and its extension.

NAEGLERIA FOWLERI
Character Naegleria Fowleri

Disease Primary amebic meningoencephalitis

Risk factor Swimming in contaminated water

Infective form Trophozoites

Transmission Respiratory mode

Clinical course Acute

Pathology Diffuse suppurative changes

Trophozoites ➢ Two forms, ameboid and flagellated form

➢ Blunt pseudopodium (lobopodia) 8–15 µm size

Cyst ➢ Not present in tissue or CSF

➢ Small (7–15 µm), thick smooth double wall

Spread Direct neural spread

CSF Leukocytes Neutrophils

Culture ➢ Require bacterial supplement

➢ Do not grow with > 0.4% NaCl

CT scan Unremarkable, no specific feature

Diagnostic form in CSF Trophozoites

Climate Temperate

GIARDIA LAMBLIA (INTESTINAL FLAGELLATES)

➢ Habitat: Mucosa of duodenum and upper ileum of man.
➢ Morphology: 2 forms
● Trophozoites
➔ Front view: Tear-drop shaped/racket shaped/piriform shaped,
Lateral view: sickle/ spoon shaped
➔ Shows falling leaf like motility, 15—20 µm size
➔ It has 2 nuclei, 4 pairs of flagella, 2 Axostyles, 2
parabasal body and two Ventral sucking disk

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 ● Cyst: Mature cyst is oval, 10–14 µm size, consists of 4 nuclei
and an axostyle. Cysts are passed in feces.

❖ Life Cycle
➢ Infective stage: Cyst, Infective dose: As few as 10 to 25 cysts
➢ Route of infection: Feco-oral route
➢ Cyst transforms to trophozoites which multiply in duodenum,
attach to GIT mucosa by adhesive disk and later on, shed in the
lumen, transforms to cysts which are passed in feces. They are the
diagnostic form of the parasite.
➢ Susceptibility to infection: Children, HIV and Individuals with
Achlorhydria and Hypochlorhydria.

❖ Pathogenesis
➢ It causes abnormalities of villous structure and causes
malabsorption (lipids and lipid soluble vitamins)
➢ Malabsorption: There could be various types which include:
● Malabsorption of fat (steatorrhea): Leads to foul smelling
profuse frothy diarrhea.
● Disaccharidase deficiencies (lactate, xylose): Leading to lactose
intolerance.
● Malabsorption of vitamin B12 and folic acid and protein loosing
enteropathy.
➢ Antigenic variation in ‘variant surface protein’ of Giardia: results
in chronic and persistent infection.

❖ Lab Diagnosis
➢ Samples collected: Stool sample and duodenal contents
➢ Microscopy: Demonstrates either trophozoites (active infection) or
cyst (carrier)
➢ String test/Entero test: Bile stained mucus is collected for
examination of Trophozoites
➢ Antigen detection in stool (coproantigen) by ELISA or ICT: indicates
active infection
➢ Serum Antibody detection by ELISA, IFA: indicates past infection

TRICHOMONAS VAGINALIS (GENITAL FLAGELLATES)

➢ MC parasitic cause of STD and NG (nongonococcal urethritis) Habitat:
Urethra, vagina and prostate.

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 ❖ Morphology
➢ Trophozoite is the only form. No cystic stage.
➢ Trophozoite: Shows twitching or jerky motility, pear-shaped
consists of 5 flagella (4 anterior + 1 recurrent flagella)
supported by undulating membrane and a fibrillary structure called
as costa.

❖ Life Cycle
➢ Mode of transmission: Sexual route
➢ Reservoir of infection: Woman
➢ Infective stage and diagnostic stage: Trophozoites
➢ Trophozoites divide by longitudinal binary fission.

❖ Clinical Disease
➢ Incubation period: 4–28 days
➢ In men: Asymptomatic or Urethritis, Prostatitis and Cystitis
➢ In women: Asymptomatic or vulvo-vaginitis—Characterized by:
● Profuse vaginal discharge with offensive smell, high pH (> 4.5)
● Smell gets accentuated by adding 10% KOH (whiff test)
● Strawberry appearance of vaginal mucosa (Colpitis
macularis)—seen in 2% of cases.

❖ Lab Diagnosis
➢ Sample collected: Vaginal, and urethral discharges, Prostatic
secretions or Urine sediment
➢ Microscopy of Vaginal, and urethral discharges: Actively motile
(Jerky motility) trophozoites
➢ Staining: Giemsa and papanicolaou staining or Direct fluorescent
antibody (DAF) test
➢ Culture: More sensitive, gold standard e.g. Lash’s cysteine hydrolysate
serum media
➢ Antigen detection in vaginal smear by ICT or ELISA: More
sensitive than microscopy and indicates recent infection.
➢ Serology by ELISA: Antibodies persist for long, so indicates past
infection • PCR.

❖ Treatment
➢ Drug of choice: Metronidazole or tinidazole (2 grams, single dose)
to both the sexual partners. Resistance is rare but has been
reported.

LEISHMANIA
➢ Infective form: Promastigote
➢ Mode of transmission: By bite of female sandfly during the late evening or
night time.
➢ Minimum 10–1000 promastigotes per infective bite are required to initiate the
infection.
➢ Promastiogotes enter into skin macrophages, transform into amastigotes
which are carried out in the circulation to various organs like liver, spleen, and
bone marrow

140
➢ Diagnostic from in humans Amastigote form inside the macrophages- k/a LD
body.

❖ Kala-Azar
➢ Bone marrow involvement leads to:
● Anaemia, leucopenia, thrombocytopenia
● Hypergammaglobulinemia
➢ Spleen ↑ , Liver ↑
➢ Fever and Hyperpigmentation (Indian cases)
➢ LN↑ (African but not in Indian cases)
➢ Kala-azar with HIV:
● Absence of hepatosplenomegaly
● GIT and resp. symptoms
● Mainly it is reported from Southern Europe (France, Italy, Spain
and Portugal)
● In India, it is reported from Bihar and sub-Himalayan
region and other North Indian states
● Diagnosis: Amastigote detected from BAL and buffy coat
region of blood. Antibodies are negative.

❖ PKDL (Post Kala-azar Dermal Leishmaniasis)

➢ Seen in 2–10 year after treatment of VL (< 6 months in Sudan)
➢ Incidence among patients with VL: 50%(Sudan), 2–20% (Indian)
➢ Nonulcerative hypopigmented macular lesions, becomes nodular later
➢ Persists for 20 years (few months for Sudan cases)
➢ Treatment: Longer courses of antimonials, resolve slowly.

❖ Virulence Factors
➢ Glycoprotein (gp-63), Lipophosphoglycan (LPG) and
Glycosylphosphatidylinositol.

❖ Host Immune Response

➢ T helper 1 (Th1) Response:
● Protective response
● Leishmanin skin test +ve
➢ T helper 2 (Th2) Response
➢ Indicates: Disease progression
➢ Leishmanin skin test -ve:

❖ Laboratory Diagnosis
➢ Sample:
● Spleen (most Sensitive)/Bone marrow aspiration (Commonly
preferred sample)
● Lymph Node aspirate (Not useful in Indian cases)
● Blood: Buffy coat region and BAL (for HIV infected)
➢ Microscopy: Stained peripheral blood smear examination
demonstrates LD bodies (amastigotes)
➢ Culture: NNN medium (McNeal, Novy and Nicolle) and Schneider’s
Liquid medium (Amastigotes transform to promastigotes)
➢ Serological tests:
● Hypergammaglobulinemia: Detected by Napier’s aldehyde and

141
 Chopra’s antimony test
●CFT: Using tubercle bacilli antigen like WKK antigen
●Immunochromatographic test (ICT): Antibodies to rK39
antigen
● Direct agglutination test (DAT), ELISA, IFA
➢ Leishmanin (Montenegro) skin test (indicates delayed
hypersensitivity reaction):
● It is positive in people with good cell mediated immunity,
Observed in Patients with CL, LR, Recovered from VL
● However, this test is negative in (when CMI is low):
Patients with active VL and DCL
➢ PCR: To detect Kinetoplast DNA
➢ Animal inoculation: Chinese and golden Hamsters.

❖ Treatment
➢ Pentavalent antimonial: DOC in most endemic regions of the world,
except in Bihar
● Sodium stibogluconate
● Meglumine antimoniate
➢ Amphotericin B: DOC in Bihar
➢ Paromomycin - DIffuse CL (L.ethiopia)
➢ Miltefosine.
➢ Pentamidine - DOC in CL due to L.guyanensis
➢ Prevention: Insect control. Sleeping at top floors also can prevent
transmission.

PLASMODIUM FALCIPARUM
❖ Life Cycle
➢ Definitive host: Female Anopheles mosquitoes
➢ Intermediate host: Man. 48 72 24
➢ Infective form to man: Sporozoites presesnt in the salivary gland of
mosquito
➢ Infective form to man when transmitted by blood transfusion or
vertical mode- trophozoite
➢ When transmitted by blood transfusion or vertical mode the
difference in the life cycle is:
● There is no liver stage, and infective form is trophozoite>
merozoite
● Hypnozoites are not produced
● Hence, there is no relapse
● So no need of primaquine
➢ Infective form to mosquito: Gametocyte
➢ To infect mosquitoes: the gametocytes should be mature, viable,
count > 12 per cubic mm of blood.
➢ Asexual cycle in man:
● After the mosquito bite: Sporozoites are discharged to blood,
carried to liver
● Liver cycle (pre or exoerythrocytic cycle): Sporozoites
transform to trophozoites → pre-erythrocytic schizont undergoes

142
 schizogony to produce → PE merozoites
● RBC cycle: PE merozoites infect RBCs → transform to early
trophozoites (ring form) → late trophozoites → erythrocytic
schizont → Undergoes schizogony to produce merozoites
● Release of merozoites leads to appearance of clinical
manifestations
● Merozoites either attack RBCs to repeat the cycle or
transform to gametocytes which infect mosquito
➢ Sexual cycle (Sporogony): Begins when gametocytes infect the female
anopheles mosquito and then transform to gametes → zygote → ookinete →
oocyst → sporozoites
➢ Recrudescence
● Seen in P. falciparum and P. malariae infections
➔ Falciparum malaria: Recrudescence is due to
persistence of drug resistant parasites and fever
reappears after 2–3 weeks of completion of treatment.
➔ In P. malariae infection, long term recrudescences are
seen for as long as for 60 years. This is due to
long term survival of erythrocytic stages at a low
undetectable level in blood.
➢ Relapse
● Due to hypnozoites (resting stage) which may reactivate after
2-3 years of malaria
● Seen in P. vivax and P. ovale
● Treatment of relapse: Primaquine

❖ Pathogenesis of P. falciparum
➢ Sequestration, i.e. holding back of the parasite in the blood vessels of
deep visceral organs like brain, kidney, etc. leads to vascular occlusion.
● Cytoadherence (binding of RBC to endothelium) – Pf EMP
● Rossetting- unparasitized RBC clump together with parasitized
RBC
➢ Cytokines released: d/t GPI-glucosylphosphatidyl Inositol
➢ Antigenic diversity of Pf EMP

❖ Clinical Features of Malaria

➢ Fever, Sweating
➢ Anemia
➢ Splenomegaly (enlarged spleen)
➢ Irritability
➢ Coma, Retinal Hemorrhages
➢ Algid Malaria (a shock like syndrome)
➢ Respiratory distress syndrome.
❖ Complications
➢ Cerebral malaria
➢ Black water fever
➢ Tropical splenomegaly syndrome
➢ Malarial hyperpyrexia
➢ Gastrointestinal disorders.
➢ Algid malaria
➢ Nephrotic syndrome: P. malariae infection

143
 ➢ Promotes Burkitt’s Lymphoma.

❖ Laboratory Diagnosis
➢ Microscopic Tests
● Examination of peripheral blood smears (thin and thick
blood films): Gold standard method:
➔ Thick smear: More sensitive (40 times), used for
quantification and malaria pigment detection
➔ Thin smears: Used for speciation
➔ Feathery tail end of the smear should be examined
➔ At least 200–300 oil immersion fields should be
examined before considered as negative
● Stains used:
➔ SB (Jaswant Singh and Bhattacharya) stain – Used in
malaria control programme in India
➔ Leishman’s, Giemsa, and Field’s, Wright’s
● Fluorescence microscopy (Kawamoto technique)- by Acridine
orange stain
● Quantitative Buffy coat examination (QBC) Rapid method for
detection of parasites:
➔ Blood is collecetd in a capillary tube coated with acridine
orange + centrifuge the capillary tube + Examine the
buffy coat region (junction of RBC & WBC) under UV.
➢ Non-microscopic Tests
● Antigen detection tests: Rapid diagnostic tests (RDTs) or
Immunochromatographic tests (ICTs)
➔ Rapid and simple but less sensitive, costly and may give
false +ve in RA factor +ve cases
➔ LDH and Aldolase: Common to all Plasmodium species
➔ HRP-2 Ag detection: Specific for P. falciparum
● Antibody detection methods:
➔ Epidemiological survey in malaria.
➔ Screening of blood bank: To identify the infected
donors
● Culture of malarial parasites: Tragger and Jensen method
using RPMI 1640 medium is used for research purpose
● Molecular methods: PCR using PBRK1 primer
➔ It is 100 times more sensitive than that of thick blood
smear.
➔ Speciation can be done.
➔ Drug resistance genes can be detected.

TOXOPLASMA GONDII
❖ Life Cycle
➢ Worldwide distribution, infects a wide range of animals.
➢ Three morphological forms: Sporulated oocyst, crescentic tachyzoites,
tissue cyst containing bradyzoites
➢ Infective form: All three morphological forms are infectious
Toxoplasma Gondii:

144
 ➢ MC mode: Ingestion of sporulated oocysts from contaminated soil,
food, or water
➢ Definitive hosts are cat and other felines
➢ Intermediate hosts are man and other mammals (goat, sheep)
➢ Transmission: ○ MC mode: Ingestion of sporulated oocysts
from contaminated soil, food, or water ○ Ingestion of
tissue cyst containing bradyzoites from undercooked meat
○ By blood transfusion or vertical transmission
tachyzoites are the infective form.
➢ Sporulated oocyst transforms into tachyzoites which multiply actively
in blood, then finally transforms into tissue cyst containing the
bradyzoites (resting stage) which get deposited in various organs.

❖ Clinical Features
➢ Congenital toxoplasmosis:
● 1st Trimester: More severe infection
● 3rd Trimester: More chance of transmission
● If Mother is previously infected: Fetus is asymptomatic.
● Incidence: Approximately 1 per 1000 live births.
● Featured by: 3C + 2M (chorioretinitis, cerebral calcification,
convulsion, microcephaly and mental retardation)
● Most common manifestation: Chorioretinitis
● Diagnosis:
➔ IgM detection in fetal blood, IgA can also be used
(experimental but better sensitivity)
➔ Toxoplasma Ag in amniotic fluid, PCR to detect
Toxoplasma genes.
➢ Adult: Mostly asymptomatic, most common manifestation – Cervical LN↑
➢ In HIV pt:
● Association rate 15–40%.
● MC manifestation encephalitis
● MC site involved: Brainstem ○ Occurs when CD4 < 100/µl
● Other manifestations: Pulmonary infections and chorioretinitis.

❖ Diagnosis
➢ Microscopy:
● Blood smear: Comma shaped Tachyzoites (indicates active
lesion)
● Smear from biopsy from organs: Tissue cyst with bradyzoites
(indicates chronic or past infection)
➢ Antibody detection:
● Sabin Feldman test:
➔ Gold standard method, highly sensitive & specific but
cannot differentiate recent and past infection
➔ Pt serum + live tachyzoites + complement + methylene
blue- Incubated
➔ Ab in pt’s serum binds to tachyzoites along with
complement that leads to tachyzoites becomes distorted
and colorless.
● Other- ELISA, IFA
➢ PCR

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 ➢ Animal Inoculation.

TAENIA SOLIUM AND TAENIA SAGINATA

Features Taenia solium Taenia saginata

Hosts in life cycle Two Two

Life Cycle

Habitat in humans Small intestine Small intestine

Brain
Muscles

Infection in Cysticerous cellulosae in pork Cysticerous bovis in beef

humans Eggs -> IMH

Pathogenicity Abdominal pain Abdominal pain

Nodules
Seizures

Diagnosis Non-operculated eggs with Non-operculated eggs with

oncosphere oncosphere

ECHINOCOCCUS GRANULOSUS
❖ Life Cycle
➢ Definitive host: Dog and wild carnivores.
➢ Intermediate hosts: Man and other herbivorous animals.
➢ Man is an accidental host (dead end).
➢ Eggs: Infective stage of the parasite.
➢ Eggs transform to larva (hydatid cyst) that penetrate GIT and
migrates to various organs like liver.

❖ Clinical Features

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 ➢ Hydatid disease: Hepatomegaly (60—70% of cases), then lungs
➢ E. multilocularis:
● Causes Alveolar Hydatid disease because cyst has multiple
locules but has no fluid/free brood capsule
● 90% liver involvement, rapidly metastasizes (mimic malignant
tumor)
➢ E. oligarthrus and E. vogeli: Causes Polycystic Hydatid disease.

❖ Diagnosis
➢ Hydatid fluid microscopy:
● Wet mount examination to demonstrates protoscolices and
brood capsule
● Acid fast staining of centrifuged deposit
● Histological examination
➢ Casoni’s skin test: Example of immediate hypersensitivity reaction
➢ Antibody: Indicates past infection, used for seroepidemiology:
● Screening: IHA, CIEP, ELISA
● Confirm: Western Blot (against antigen B fragment)
➢ Detection of antigen: Indicates Recent infection
➢ Imaging methods like USG, MRI and X-ray: Demonstrates
size, exact location and extension of the cysts
➢ Water lily sign in USG: Due to collapsed cyst (floating membrane)
floating in the abdomen
➢ Tests to monitor the response to treatment: Imaging methods
and Antigen detection methods.

❖ Treatment
➢ Treatment of choice: Surgery
➢ DOC: Albendazole and mebendazole
➢ Commonly preferred method: Percutaneous Aspiration Injection
Reaspiration (PAIR) of the cyst.

CYSTICEROUS CELLULOSAE
➢ Potentially dangerous systemic disease.
➢ Definitive host: Man, Intermediate host: Man
➢ Transmission: (i) Ingestion of food/water contaminated with eggs, (ii)
autoinfection
➢ Eggs develop to larva (Cysticercus cellulosae) in human intestine
➢ Larvae penetrate the intestine and get deposited in-MC sites- CNS
(60-90%) followed by Eye and muscle.

SCHISTOSOMA HEMATOBIUM – (BLOOD FLUKE)

➢ Resides in: Vesical and pelvic venous plexus
➢ Associated with:
● Hematuria
● Hydroureter and hydronephrosis
● Bladder Carcinoma: Sqamous cell Ca (in high worm burden) > transitional
cell Ca (low worm burden)

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 ➢ Egg has terminal spine
➢ Antibody detection:
● HAMA-FAST: ELISA (Falcon assay screening test ELISA) using S.
haematobium adult worm microsomal antigen (HAMA).
● HAMA Western blot: Specific
● Other methods: Cercarial Huller reaction, IFA, IHA
➢ Antigen detection:
● Circulating cathodic antigen (CCA) in urine and
circulating anodic antigen (CAA) in serum. ○ It indicates
recent infection and can be used for monitoring the
treatment.

NEMATODES
Feature Ascaris Hookworm Strongy Trichuris Enterobius
(Roundworm) loides (Whipworm) (Pin/thread
worm)

Infective Egg Filariform Filarifor Egg Egg

stage larva m larva

Route Oral Skin Skin or Oral Oral or auto

autoinfe infection
ction

Location Small intestine Small Small Large Large

intestine intestin intestine intestine
e (Cecum, (Cecum,
colon) appendix)

Lung stage Yes Yes Yes No No

Principal GIT symptoms Ground itch GIT Dysentery, Perianal

symptoms Malabsorption Serpiginous sympto Iron def. pruritus
Intussusception tracks Mild ms, anemia, worse at
Loeffler pneumonitis Malabso Rectal night
syndrome GIT rption, prolapse,
symptoms Hyperin Growth
Iron def. fection retardation
anemia syndrom
e.

Diagnostic UnEmbryonated Eggs in Rhabditi UnEmbryona embryonate

stage eggs in stool fresh stool, form ted eggs in d eggs from
(Fertilized and larvae in old UnEemb stool perianal skin
unfertilized) stool ryonate collected by
d larvae NIH swab
in stool or Cello
phane tape

Eggs Fertilised egg: Oval, Bile Rhabditi Barrel Bile non

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 Round to oval, non stained form shaped stained
bile stained, Segmented larva: Mucus plug Planoconvex
thick albumin ovum (Four Long at ends, bile eggs
coat, floats on blastomeres buccal stained containing
saturated salt ) cavity larva inside
Unfertilized Genital
eggs: primordi
Elongated, um: Less
rectangular, bile promine
stained thin nt, small
coat, does not
float on
saturated salt.

Fecundity 2.4 Lakh Ancylostoma 5000– 3000–7000 2000

(eggs/ : 10,000
day/worm) 10,00025,00
0 Necator:
400010,000

Incubation 60–75 days 40–100 days 17–28 70–90 days 35–45 days
period days

Longevity 1 year N. Decades 5 year 2 months

americanus: (owing
2–5 year A. to
duodenale: autoinfe
6–8 year ction)

Treatment Albendazole Albendazole Ivermec Albendazole Mebendazol

tin e

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