GENERAL MICROBIOLOGY

SET 1:

1. Classify bacteria depending on their shapes

2. Structure of cell wall
3. Difference between cell walls of gram-positive and gram-negative bacteria
4. Antibiotics acting on it
5. Functions of various appendages
SET 2:

Identification Methods

SET 3:
Fluorescent microscopy
SET 4:
Immunofluorescence
SET5:
Physiology of bacteria
SET6:

Gene transfer

SET7:
Mechanisms of drug resistance

SET 8:
Sterilization and disinfection

SET 9:
1. Louis Pasteur (3)
2. Robert Koch
3. Lord lister
SET 10:
Bacterial capsule
SET 11:
L-forms of bacteria
SET 12:
1. Enriched / enrichment media
2. Selective media
3. Differential media
4. Transport media
5. Anaerobic media
SET 13:
Anaerobic culture methods
SET 14:
Antibiotic sensitivity test
SET15:
Transposable genetic elements
SET 16:
Nucleic acid probes

SET 17:

Bacterial spores
SET 18:
Bacterial virulence
SET 19:
1. Mutations
2. Phenotypic variations of bacteria
SET 20:
1. Plasmids
2. Extrachromosomal genetic elements
 SET 1

1. CLASSIFICATION OF BACTERIA
⮚ Bacteria is a prokaryotic cell that does not contain chlorophyll except blue green algae.
● Unicellular.
● Doesn’t show true branching
● Divide by binary fission.
⮚ Bacteria can be classified depending on their shape into:
● Coccus - Spherical
● Bacillus - Rod- shaped
● Coccobacillus - Oval and similar to coccus
● Vibrio - Curved or comma-shaped rod
● Spirillum - Thick and rigid spiral
● Spirochete - Thin and flexible spiral
● Actinomycetes - Branched filamentous
⮚ Bacteria can also be classified depending on their arrangement: In turn it depends on the plane
of division.
● Paired: Diplo
● Grape-like clusters: Staphylo
● Chain: Strepto

CLASSIFICATION BASED ON SHAPE

Reference: Essentials of Medical Microbiology, Apurba S Sastry E/3

2. STRUCTURE OF BACTERIA
⮚ All bacteria consist of:
● Rigid cell wall
● Plasma membrane
● Cytoplasmic structures- Internal structures
● External structures – Appendages

BACTERIAL CELL WALL:

⮚ Tough and rigid structure surrounding bacteria like a shell.

⮚ Thickness:10 – 25 nm ;Weight: - 20 – 25% of dry weight of cell.
⮚ It cannot be seen by direct light microscopy and does not stain simple stains.

FUNCTIONS:

⮚ Protection to cells against osmotic lysis.

⮚ Maintains shape
⮚ Confers rigidity and ductility.
⮚ Provides a rigid platform for surface appendages – fimbria, pili and flagella
⮚ Role in cell division.
⮚ Protection from toxic substances
⮚ Site of action for several antibiotics
⮚ Contains virulence factors like endotoxins contributing to pathogenicity
⮚ Possess target site for lysozyme and bacteriophage
⮚ Contain immunogenic antigens against which immunity can be raised
STRUCTURE OF CELL WALL:

⮚ Bacteria are divided into gram positive and gram negative based on gram staining
⮚ Cells of both types of bacteria differ widely

GRAM POSITIVE BACTERIAL CELL WALL:

⮚ Their cell wall contain:

● Thick homogenous peptidoglycan layer.
● Teichoic and lipoteichoic acid- interwoven.
● Cell wall outer surface studded with surface proteins.

PEPTIDOGLYCAN:

⮚ It is a polymer of peptidoglycan monomer (mucopeptide scaffolding).

⮚ It is called murein.
⮚ Vast polymer consisting of an interlocking chain of identical peptidoglycan monomer.
⮚ Peptidoglycan monomer: N-Acetyl Glucosamine (NAG) cross linked with N-Acetyl Muramic
Acid (NAM) by Tetrapeptide side chain and pentaglycine bridges with a pentapeptide
coming off from NAM.
⮚ Tetrapeptide side chain Composition: L alanine+ D glutamine +L lysine + D alanine.

Penta peptide bridges

TEICHOIC ACID:

⮚ Present in a significant amount.

⮚ Major surface antigen.
⮚ Water soluble polymer.
⮚ Composition: Polymer of Glycerol or Ribitol joined by Phosphate group.

Types:
FUNCTION OF GRAM-POSITIVE CELL WALL:

⮚ Surface protein serves as enzymes, adhesions, invasins

Reference: Essentials of Medical Microbiology, Apurba S Sastry E/3

GRAM NEGATIVE CELL WALL:

⮚ Complex structure
⮚ From outside to inside it has:
● Outer membrane
● Thin peptidoglycan layer
● Cytoplasmic membrane

OUTER MEMBRANE:
⮚ Phospholipid layer lying outside thin peptidoglycan layer.
⮚ It is firmly attached to peptidoglycan by covalent linkage of membrane protein (Braun’s
Lipoprotein)
⮚ It is made up of lipopolysaccharide
⮚ FUNCTIONS:
● Serves as a protective barrier to cells.
● Outer Membrane Proteins or Porins:
Specialized Proteins

3 porin molecules cluster together

Span the outer membrane

 Form a narrow channel

Molecules less than 600 D can pass through.

Larger molecules (vitamin B) transported by specific Carrier.

o Functions of outer membrane protein:

▪ Prevents loss of constituents such as periplasmic enzymes.
▪ Target site for phages, antibiotics and bacteriocin.

LIPOPOLYSACCHARIDES:

⮚ Present only in gram negative bacteria.

⮚ Consists of 3 parts:

Lipid A or Endotoxin- Region 3:

⮚ Composition: 2 glucosamine sugar derivatives each with 3 fatty acids and PO4 attached.
⮚ Buried in the outer membrane, the remainder of LPS molecules project from the surface.
⮚ Has endotoxic activities :
● Pyrogenicity
● Lethal effect
● Tissue Necrosis
● Anti complementary activity
● B cell mitogenicity
● Antitumor activity.

Core polysaccharide-Region 2:
⮚ Projected from Lipid A region.
⮚ Composition: 10- 12 sugar moieties.

O side chain or O antigen- Region 1:

⮚ Polysaccharide chain extending outwards from core polysaccharide.

⮚ Made of several sugar moieties.
⮚ Major surface antigen (Somatic Antigen) – induces Ab formation.
⮚ Used for serotyping.
PERIPLASMIC SPACE:

⮚ Space between inner cell membrane (with peptidoglycan layer in between) and outer membrane.
⮚ Contains various binding proteins for specific substrates.

FUNCTION OF GRAM NEGATIVE CELL WALL:

⮚ The outer membrane has the following roles:

● Many small molecules can pass through porins.
● Some toxic substances such as penicillin G and lysozyme from entering.

DEMONSTRATION OF CELL WALL:

⮚ It is not stained by simple dyes and can’t be seen by light microscope.

⮚ It is demonstrated by:
● Plasmolysis
● Microdissection
● Differential staining
● Reaction with specific antibody
● Electron microscope

3. DIFFERENCE BETWEEN CELL WALLS OF GRAM POSITIVE

AND GRAM NEGATIVE BACTERIA

Gram positive bacteria Gram negative bacteria

Thickness Thicker Thinner
Periplasmic Absent Present
Space
Outer Absent Present
membrane
Lipid Absent / Little Present
Teichoic Acid Present Absent
Variety of Few Several
Amino acid
AA & sulphur Absent Present
AA
Peptidoglycan 16 to 80 nm 2nm

4. ANTIBIOTICS ACTING ON CELL WALL

Antibiotics Resistance by
● Penicillin ● By synthesis of enzymes to inactivate the drug.
● Cephalosporin ● By altering the antibiotic binding site.
● Glycopeptides ● By producing an efflux pump.
● Fosfomycin
● Bacitracin

5. VARIOUS APPENDAGES OF BACTERIA

⮚ Bacterial cell wall appendages include:
● Capsule or slime layer
● Flagella
● Pili or fimbriae

CAPSULE / SLIME LAYER: (Refer SET 10)

FLAGELLA:
⮚ Thread like long sinuous cytoplasmic appendage
⮚ Protrude from cell wall
⮚ Confers motility to bacteria
⮚ Composed of Protein called flagellin.
⮚ All motile bacteria except spirochaetes possess one or more flagella.
ARRANGEMENT:

⮚ Flagella is arranged differently in different bacteria with respect to its surface:

● Monotrichous:
▪ Single polar flagellum – polar flagella may be single.
▪ Eg: Vibrio cholerae, Pseudomonas and Campylobacter.
● Lophotrichous:
▪ Tuft of flagella at one end or both ends.
▪ Eg: Spirilla
● Peritrichous:
▪ Flagella is distributed over the entire cell surface.
▪ Eg: Salmonella typhi, Escherichia coli.
● Amphitrichous:
▪ Single flagellum at both ends.
▪ Eg: Alcaligenes faecalis.

Reference: Essentials of Medical Microbiology, Apurba S Sastry E/3

ULTRASTRUCTURE OF FLAGELLA

⮚ A bacterial flagellum has 3 parts:

● Filament
● Basal body
● Hook
Filament:

⮚ Longest portion and extends from surface to tip.

⮚ Hollow rigid cylinder made up of single protein flagellin.
⮚ Flagellar antigens induce specific antibodies in high titre which are useful in serodiagnosis

Basal body:

⮚ Embedded in a cell and contains 2 – 4 rings connected to the central rod.

⮚ In gram negative bacteria:
● 4 rings named L, P, S, M.
● Outer L and P rings associated with Lipopolysaccharides and Peptidoglycan layer
respectively.
● Inner S ring in periplasmic space
● M ring which is in contact plasma membrane.
⮚ In gram positive bacteria:
● Only 2 rings, of which inner ring is connected to plasma membrane and outer ring
attached to peptidoglycan.

Hook:

⮚ Short curved, flexible segment– links filament to its basal body.

Reference: Essentials of Medical Microbiology, Apurba S Sastry E/3

DETECTION OF FLAGELLA:

⮚ Flagella - 0.02µm in thickness

Direct Demonstration of Flagella:

▪ Tannic acid staining (Leifson’s method and Ryu’s method)

▪ Electron microscope.

Indirect Demonstration of Flagella:

⮚ Craige tube method - Best method

⮚ Swarming growth of bacteria
⮚ Hanging drop - Most common
⮚ Semisolid medium
⮚ Dark ground or phase contrast microscopy

BACTERIAL MOTILITY:
Type of motility Example
Tumbling Listeria

Gliding Mycoplasma

Stately Clostridium

Darting Vibrio cholerae

Swarming Proteus, Clostridium tetani

Corkscrew, lashing Spirochete

FIMBRIAE / PILI:
⮚ Many gram negative and some gram positive bacteria possess short, fine hair like appendage-
thinner than flagella and not involved in motility called fimbriae / pili
⮚ Made up of protein called pillin
⮚ Antigenic antibodies against fimbrial antigens aren’t protective.
⮚ A bacterium can have as much as 1000 pilli.
⮚ Present in both motile and non motile cells.
FUNCTIONS:

⮚ Organ of adhesion – enhances virulence of bacteria

⮚ Certain fimbriae – sex pili - helps in bacterial gene transfer.

Types of pili Function

1.Common pili: ● They are small measuring 0.5 µm long and 10 nm
● Help in bacterial adhesion to thick
epithelial surface and colonization. ● Present in gram-positive and gram-negative
bacteria
● There are 6 types depending on morphology,
number per cell, adhesive property and antigenic
nature.

2.Sex pilli: ● Long thick tubular structures

● Help in bacterial conjugation by ● Special type of large fimbriae 1 – 10 per cell helps
forming conjugation tube from male in bacterial conjugation.
to female bacteria through which ● Present only in gram-negative bacteria.
bacterial gene transfer take place
3.Col1 (Colicin) pilli ● Large self transmissible plasmids

DETECTION OF FIMBRIAE:

Direct Demonstration:

⮚ Electron microscope

Indirect Demonstration:

⮚ Haemagglutination
⮚ Surface pellicles
 SET 2

METHODS IN LABORATORY DIAGNOSIS OF BACTERIAL

INFECTION

LABORATORY DIAGNOSIS:

⮚ Laboratory diagnosis of bacteria is useful for various purposes like

● Identification
● Treatment
● Surveillance purpose
● For outbreak investigation
● To start PEP
● To initiate appropriate infection control measures.
⮚ Steps in laboratory diagnosis:
● Specimen collection
● Direct detection:
o Microscopy
o Antigen detection
o Molecular methods
● Culture:
o Culture media
o Culture methods
o Culture morphology, smear and motility testing
● Identification:
o Biochemical identification
o Automated identification methods
● Antimicrobial susceptibility testing
● Serology
● Molecular methods
● Typing methods
SPECIMEN COLLECTION:

⮚ Specimen collection depends upon the type of underlying infection.

⮚ Proper specimen collection is very important for isolation of bacteria in culture.
⮚ General principles in specimen collection:
● Standard precautions
● Prior antibiotic administration
● Avoiding contamination
● Appropriate sterile containers
● Labelling
● Rejection of contaminated or improperly labelled specimen
● No use of formaldehyde to collect specimens.

SPECIMEN TRANSPORT:

⮚ Specimens should reach the lab as soon as possible.

⮚ Suitable transport medium is used if needed.
⮚ Specimens that require immediate transport: CSF and body fluids, ocular specimen, tissue
specimen,suprapubic aspirate and bone specimen.
⮚ Urine: 2 hours but with preservatives 24 hours
⮚ Stool culture: 1 hour but with transport medium 24 hours.
⮚ Rectal swab: 24 hours
⮚ For anaerobic culture: Put in Robertson’s cooked meat broth and transported immediately.

SPECIMEN STORAGE:

⮚ Most specimens are stored at room temperature upto 24 hours.

⮚ Blood cultures and sterile body fluids- 37º C.
⮚ Corneal scraping immediately placed at the bedside.
⮚ Stool culture - 72 hours at 4º C.
⮚ Urine, lower respiratory tract specimen and gastric biopsy - 24 hours at 4º C.
DIRECT DETECTION:

⮚ Important for early institution of antimicrobial therapy

STAINING TECHNIQUES:

⮚ Staining methods produce color contrast and increase the visibility of bacteria.
⮚ Before staining, the smears are fixed so that they will not be displaced during the staining
process.
⮚ FIXATION – done by 2 methods:
1. Heat fixation – by gently flame heating an air-dried film, used for bacterial smears
2. Methanol fixation – used for blood smear

COMMON STAINING TECHNIQUES:

1. Simple stain:
● Basic dyes, such as methylene blue or basic fuchsin are used as simple stains.
● They provide the color contrast, but impart same color to all the bacteria in the smear
2. Negative staining:
● A drop of bacterial suspension is mixed with dyes, such as Indian ink or nigrosine
● The background gets stained black whereas unstained bacterial / yeast capsules stand out in
contrast.
● Used in demonstration of yeast or capsules which do not take up simple stains.
3. Impregnation methods:
● Bacterial cells and structures that are too thin to be seen under the light microscope, are
thickened by impregnation of silver salts on their surface to make them visible.
● For demonstration of bacterial flagella and spirochetes
4. Differential stain:
● Here,two stains are used which impart different colors to different bacteria
● Help in differentiating bacteria

Commonly used differential stains:

1. Gram stain (differentiate bacteria into gram +ve and gram -ve groups)

2.Acid fast stain (into acid fast and non acid fast)
 3.Albert stain (differentiates bacteria having meta granules from other bacteria)

GRAM STAIN:

⮚ Introduced by Hans Christian Gram

PROCEDURE:

Fixation: The smear made on a slide from bacterial specimen is air dried & then heat fixed

STEPS:

● Primary stain Smear is stained with crystal violet/methyl violet/gentian violet

(pararosaniline dyes) for 1 min and then rinsed with water.
● Mordant Pour gram’s iodine (dilute iodine solution) for 1 min and rinse with water. It binds
to the dye to form bigger dye-iodine complexes in the cytoplasm.
● Decolorization Pour acetone (for 1-2 sec) / alcohol (20-30 sec) or acetone alcohol (10 sec)
and rinse immediately. It removes the primary stain from gram- negative bacteria while
gram-positive bacteria retains it.
● Counterstain Safranin or dilute carbol fuchsin is added for 30 sec (neutral red for gonococci).
The slide is rinsed, dried and viewed under oil immersion objective.

Reference: Essentials of Medical Microbiology, Apurba S Sastry E/3 Page No. 27 Fig. 3.3.1
INTERPRETATION:

Gram positive bacteria Gram negative bacteria

Resist decolorization Decolorized

Retain primary stain i.e.violet Take counterstain and appear pink

PRINCIPLE:

pH theory:
Gram positive cytoplasm

More acidic

Binds basic stain Primary stain retained better

Cell wall theory:

1) Gram positive cell: Retains primary stain
● Peptidoglycan wall is thicker - acts as permeability barrier and prevents loss of the stain
● Alcohol decreases the pore size-dye-iodine complex cannot penetrate.
2) Gram negative cell wall:
● Thin peptidoglycan wall
● more lipids- get dissolved by alcohol/acetone (decolourizer) - leads to pore formation-
dye-iodine complex escapes.

MODIFICATIONS OF GRAM STAINING:

⮚ Kopeloff and Beerman’s Modification: Primary stain-methyl violet and counterstain - basic
fuchsin.
⮚ Jensen’s Modification: Decolourizer - absolute alcohol and counter stain- neutral red-for
meningococci and gonococci.
⮚ Brown and Brenn Modification: For actinomycetes.
ACID FAST STAIN:

● 1st introduced by Paul Ehrlich.

● Modified by Ziehl neelsen ZN stain.
● To identify acid-fast organisms which is due to the presence of mycolic acid in the cell wall.

SMEAR PREPARATION:

● Prepared from the yellow purulent portion of the sputum on a clean slide near a flame (within 6
inches) to coagulate aerosols

HEAT FIXATION:

● The smear is dried for 15-30 min and heat fixed by passing 3-5 times over flame for 3-4 seconds.
● For the coagulation of proteinaceous material in order to fix the smear.

PROCEDURE:

1. Primary stain:
● Carbol fuchsin is poured for 5 minutes.
● Intermittent heating is done until vapour rises.
● Slide is rinsed with tap water-side appears red

2. Decolorization:
● 25% sulfuric acid is poured.
● It is allowed to stand for 2-4 mins.
● Rinsed with tap water and drained- slide appears light pink.
● Back of the slide is wiped clean with a swab dipped in sulfuric acid.
3. Counter staining:
● 0.1% methylene blue is poured and left for 30 seconds.
● Rinsed with tap water and dried.
● Examined using 40X lens to select suitable area and viewed under oil immersion
INTERPRETATION:

Acid fast structures (Mycobacterium tuberculosis) Non acid fast organisms

Retains carbol fuchsin (appear red) Take up counterstain & appear blue

MODIFICATIONS:

⮚ Cold Method (Kinyoun’s method): No intermittent heating is required.

ALBERT STAIN
⮚ To demonstrate metachromatic granules of Corynebacterium diphtheria

PROCEDURE:

1. The smear is heat fixed.

2. Covered with Albert I for 5 mins and excess is drained out

3. Albert II is added for 1 min

4. Slide is washed,blotted dry and examined under oil immersion

COMPOSITION:

● Albert I: Toluidine blue, malachite green, glacial acetic acid, alcohol (95% ethanol) & distilled
water.
● Albert II: Iodine in potassium iodide.

INTERPRETATION:

● Corynebacterium diphtheria appears as green bacilli arranged in cuneiform pattern, with bluish
black metachromatic granules at polar ends
OTHER TECHNIQUES:

⮚ Dark ground and phase contrast microscope

⮚ Hanging drop preparation

ANTIGEN DETECTION:

⮚ Various immunological methods such as latex agglutination, immunochromatographic test help

to detect antigen in specimens.
● CSF- Capsular antigen of Pneumococci, Meningococci and H. influenza
● Urinary antigen detection - Pneumococci and legionella.
● Tissue specimen and exudates - T.pallidum

MOLECULAR METHODS:

⮚ Bacterial DNA and RNA can be detected in clinical specimens by PCR.

CULTURE IDENTIFICATION:

⮚ Most common diagnostic method for detection of bacterial infections.

⮚ Steps:
● Inoculation on culture media
● Incubation
● Identification
● Antibiotic susceptibility testing

CULTURE MEDIA: Refer SET 12

CULTURE METHODS:

⮚ It involves inoculating the specimen on appropriate culture media, followed by incubation of the
culture plates in appropriate conditions.
⮚ Entire process should be carried out in a biological safety cabinet with appropriate PPE.

SELECTION OF MEDIA:

⮚ Depends on the type of specimen.

⮚ Commonly used – Combination of blood agar and MacConkey agar.

INOCULATION OF SPECIMEN:

⮚ Done with bacteriological loops made up of platinum or nichrome wire.

⮚ First heated in a Bunsen burner until red hot and cooled for 10 sec.

INOCULATION METHODS:

⮚ Types:
● For inoculating clinical specimens onto the culture media.
● For inoculating colonies on to various media for further processing.

STREAK CULTURE:

⮚ Most common inoculation method used for inoculation of specimens on solid media.
⮚ Also for obtaining individual isolated colonies from a mixed culture.
⮚ It involves:
● Streaking: A loopful of specimens smeared onto the solid media to form primary
inoculums and then spread over by streaking parallel lines to form secondary and tertiary
inoculums and a feathery tail.
● Intermittent heating: The loop is flamed and cooled in between the different sets of
streaks to obtain isolated colonies on the final streak.

LIQUID CULTURE:

⮚ For culturing the specimens which are directly inoculated by adding specimens into the liquid
medium.
⮚ Bacterial growth is detected by observing the turbidity
 ⮚ Uses:
● Blood or body fluid cultures
● MGIT
● Water analysis
⮚ Advantages: For culture of specimen
● Containing small quantity of bacteria
● Specimens containing antibiotics and other antibacterial substances.
● For large bacterial yield
⮚ Disadvantages:
● Do not provide pure culture with mixed inoculums
● No visible colonies- so no clue about the type of bacteria

LAWN OR CARPET CULTURE:

⮚ To carry out antimicrobial susceptibility testing by disk diffusion method.

⮚ Uniform lawn obtained by swabbing or flooding the culture plate with bacterial broth.

POUR PLATE CULTURE:

⮚ For quantifying bacterial load present in blood and urine

⮚ Serial dilutions of specimens are added on to molten agar.
⮚ After being cooled and solidified, petri dishes are incubated and the colony count is estimated.

STROKE CULTURE:

⮚ Carried out on agar slopes or slants by streaking the straight wire in zig zag fashion.
⮚ Used in biochemical tests.

STAB CULTURE:

⮚ Made by stabbing the semi-solid agar butt by a straight wire.

⮚ Used for motility testing by mannitol motility medium and triple sugar iron test.
INCUBATION:

⮚ Incubatory conditions vary depending on the bacteria isolated.

⮚ As most pathogenic bacteria grow best at 37º C.
⮚ The culture plates, biochemical test or AST are incubated at 37ºC aerobically (as they are mostly
aerobes or facultative anaerobes) overnight in an incubator.
⮚ For capnophilic bacteria: Candle jar is used- Candle is lighted and the jar is sealed- Provides 3 to
5% CO2 .
⮚ Microaerophilic bacteria: 5% oxygen for optimum growth.
⮚ Obligate anaerobes: Anaerobic culture methods (Refer SET 13).

COLONY MORPHOLOGY
⮚ After overnight incubation, the culture media are removed from the incubator and are examined
under bright illumination.
⮚ The appearance of bacterial colonies on culture medium is characteristic for many organisms;
which helps in their preliminary identification.
⮚ Following features of the colony are studied
● Size: In mm; e.g. pinhead size is a characteristic of staphylococcal colony while pinpoint
size is a characteristic of streptococcal colony.
● Shape: Circular or irregular.
● Consistency: Dry, moist or mucoid.
● Density: Opaque, translucent or translucent.
● Hemolysis on blood agar: Certain bacteria produce hemolysin enzymes that lyse the red
blood cells surrounding the colonies on blood agar, forming a zone of hemolysis.
❖ Partial or α hemolysis: Partial clearing of blood ( with RBC membrane intact) around
the colonies with green discoloration. Eg: Pneumococci, viridans streptococci)
❖ Complete or β hemolysis: Zone of complete clearing of blood around the colonies (
complete lysis of RBC). Eg: Staphylococcus aureus and Staphylococcus pyogenes
❖ No hemolysis or γ hemolysis: No colour change surrounding the colonies. Eg:
Enterococcus.
● Color of the colony: Colonies may be colored due to certain properties of the media or
organisms e.g. pink colonies produced by lactose fermenters on MacConkey agar.
● Pigment production:
 ❖ Diffusible pigments: Pigments diffuse into the surrounding medium.
Eg: Pseudomonas aeruginosa producing blue-green pigments.
❖ Non-diffusible pigments: Do not diffuse into surrounding medium-only colonies are
colors. Eg: S.aureus producing golden-yellow colonies.

CULTURE SMEAR AND MOTILITY:

⮚ Colonies grown on the culture media are subjected to Gram-staining and motility testing by
hanging drop method.

HANGING DROP PREPARATION:

⮚ Most common and easiest method to demonstrate bacterial motility.

⮚ A drop of bacterial broth is prepared on a coverslip and kept over a cavity slide.
⮚ Edge of the drop is focused under microscopy for demonstration of bacterial motility.
⮚ For identification of bacteria especially gram negative bacillus

CULTURE IDENTIFICATION

⮚ Identification of bacteria from culture is made either by conventional biochemical tests or

automated identification systems.

BIOCHEMICAL METHODS:

⮚ Based on the type of colony morphology and gram staining appearance observed in culture
smear, the appropriate biochemical tests are employed
1. Initially, Catalase and oxidase tests are done on all types of colonies grown on the media
2. For gram-negative bacilli: Indole test, citrate utilization test, urea hydrolysis test and triple
sugar iron test
3. For gram-positive cocci:
⮚ Coagulase test (for Staphylococcus aureus)
⮚ CAMP test/Christie – Atkins- Munch-Petersen Test (for group B Streptococcus)
 ⮚ Bile esculin hydrolysis test (for Enterococcus)
⮚ Heat tolerance test (for Enterococcus)
⮚ Inulin fermentation test (for Pneumococcus)
⮚ Bile solubility test (for Pneumococcus)
⮚ Antimicrobial susceptibility tests done for bacterial identification are as follows:
● Optochin susceptibility test – to differentiate pneumococcus (sensitive) from
viridans streptococci (resistant)
● Bacitracin susceptibility test – to differentiate group A (sensitive) from group B
(resistant) Streptococcus

CATALASE TEST

⮚ When a colony of any catalase producing bacteria is mixed with a drop of 3% H2O2 placed on a side,
effervescence or bubbles appear due to breakdown of H2O2 by catalase to produce oxygen
⮚ Primarily used to differentiate between Staphylococcus (catalase positive) and Streptococcus
(catalase negative)

OXIDASE TEST

⮚ Detects the presence of cytochrome oxidase enzyme in bacteria, which catalyzes the oxidation of
reduced cytochrome by atmospheric oxygen
⮚ Oxidase positive (deep purple): Pseudomonas, Bacillus, Haemophilus etc..
⮚ Oxidase negative (no colour change): Family of Enterobacteriaceae, Acinetobacter

INDOLE TEST

⮚ Detects the ability of certain bacteria to produce an enzyme tryptophanase that breaks down amino
acid tryptophan present in the medium into indole.
⮚ Kovac’s reagent is used which complexes with indole to produce cherry red coloured ring
⮚ Indole positive ( red coloured ring near the surface): E.coli, Vibrio cholera.
⮚ Indole negative (yellow coloured ring near the surface) : Klebsiella pneumoniae, Salmonella,
Pseudomonas, etc
CITRATE UTILIZATION TEST

⮚ Detects the ability of a few bacteria to utilize citrate as the sole source of carbon for their growth,
with production of alkaline metabolic products.
⮚ Test is performed on Simmon’s citrate medium.
⮚ Positive (green colour changes to blue) for Klebsiella pneumoniae, Citrobacter.
⮚ Negative (Original green colour remains) for E.coli, Shigella

UREA HYDROLYSIS TEST

⮚ Urease producing bacteria can split urea present in the medium to produce ammonia that makes the
medium alkaline.
⮚ Test done on Christensen’s urea medium which contains phenol red as indicator.
⮚ Positive for ( pink colour): Klebsiella pneumoniae, Helicobacter pylori.
⮚ Negative (no colour change): E.coli, Salmonella

TRIPLE SUGAR IRON (TSI) AGAR TEST

⮚ Important medium for the identification of gram-negative bacteria.

⮚ TSI medium contains three sugars- glucose., sucrose and lactose in the ratio of 1:10:10.
⮚ Uninoculated TSI medium is red in colour and has a slant and a butt.
⮚ After inoculation the medium is incubated at 37º C for 18-24 hours.

INTERPRETATION:

⮚ TSI detects three properties of bacteria, which include fermentation of sugars to produce acid
and / or gas and production of H2S.
⮚ Acid Production:
● If acid is produced the medium is turned yellow from red.
● Accordingly organisms are classified as:
❖ Nonfermenters: They do not ferment any sugar and the medium remains red
–Alkaline slant/alkaline butt (K/K). Eg: Pseudomonas and Acinetobacter.
 ❖ Glucose only fermenters: They ferment only glucose and produce little acid only
at the butt. So it results in alkaline slant/ acid butt.
Eg: Salmonella and Shigella.
❖ ≥2 Fermenters: They ferment glucose and also lactose and/or sucrose to produce
large amounts of acid so that the medium fully turns to yellow.-Acid slant/acid
butt (A/A). Eg: E.coli and Klebsiella.
⮚ Gas production: If gas is produced the medium is lifted up or broken with cracks. Eg: E.coli and
Klebsiella.
⮚ H2S Production: If H2S is produced it changes the medium black.Eg: Salmonella typhi, Proteus
vulgaris.

AUTOMATED SYSTEM FOR BACTERIAL IDENTIFICATION

⮚ They are revolutionary and have several advantages like:

● Produce faster results
● Identifies wider range of organisms with accuracy
⮚ Some of the automated systems are:
● MALDI-TOF
● VITEK 2
● Phoenix
● MicroScanWalkAway System

ANTIBIOTIC SUSCEPTIBILITY TESTING (Refer SET 14)

SEROLOGY:

⮚ These include detection of either antigen or antibody in the patient’s serum.

⮚ It involves various immunological assays like precipitation, agglutination, ELISA and rapid test.
⮚ Some important serological test include:
● Widal test for enteric fever.
● Standard agglutination test for brucellosis.
 ● Microscopic agglutination test and rapid diagnostic test for leptospirosis.
● Weil-Felix test for rickettsial infection.
● VDRL and RPR tests for syphilis.
● ELISA for various bacterial infections.

MOLECULAR METHODS:

⮚ Molecular methods are broadly grouped into amplification based and non – amplification based
methods.
⮚ Nucleic acid amplification techniques (NAATs) have been increasingly used in diagnostic
microbiology.
⮚ Various NAATs used are:
● Polymerase chain reaction (PCR)
● Real-time polymerase chain reaction (rt-PCR)
● Loop mediated isothermal amplification (LAMP)
● Automated PCR such as biofire filmarray.
● Automated real-time PCR such as cartridge based nucleic acid amplification test (CBNAAT)
⮚ Non amplification methods include DNA hybridization method e.g. line probe assay

POLYMERASE CHAIN REACTION (PCR):

⮚ Developed by Kary B Mullis.

⮚ Used to amplify a single or few copies of a piece of DNA to generate millions of copies of DNA

PRINCIPLE:

STEPS:

1. DNA extraction from the organism: Lysis of the organism and release of the DNA which may be
done by boiling, adding enzymes etc.
2. Amplification of extracted DNA: Carried out in a thermocycler. The extracted DNA is subjected to
repeated cycles (30-35) of amplification which takes about 3-4 hrs.

3. Each amplification cycle has 3 steps:

❖ DENATURATION (95°C) – Separation of dsDNA into two separate single strands
❖ ANNEALING (55°C) – Primer is a short oligonucleotide complementary to a small sequence
of target DNA. It anneals to the complementary site on the target ssDNA.
❖ EXTENSION (72 °C): Taq polymerase ( from Thermus aquaticus) enzyme keeps on adding the
free nucleotides to the growing end of the primer

4. Gel electrophoresis of amplified product: Amplified DNA is electrophoretically migrated according

to their molecular size by performing agarose gel electrophoresis. The amplified DNA forms a clear
band, which can be visualized under UV light.

APPLICATIONS:

❖ To amplify the DNA of an organism: Either directly from a sample or to confirm

organism growth in culture
❖ Detects genetic diseases such as sickle cell anemia, phenylketonuria
❖ To detect the genes in the organisms responsible for drug resistance (e.g. mec A gene
detection in staphylococcus aureus)

ADVANTAGES:

❖ More sensitive - It can amplify very few copies of specific DNA.

❖ More specific - Due to use of primers targeting specific DNA primer
 ❖ Also detect the organisms that are highly fastidious or non cultivable by conventional
culture methods

DISADVANTAGES:

❖ Conventional PCR detects only DNA and not RNA.

❖ Qualitative, not quantitative – conventional PCR cannot quantitate the amount of DNA present in
the sample. This is possible with real time PCR.
❖ Viability – cannot differentiate between viable or nonviable organisms.
❖ False positive amplification – due to contamination with environmental DNA.
❖ False negative – PCR inhibitors present in blood, feces etc. may inhibit amplification of target DNA

MODIFICATIONS:

⮚ Reverse Transcriptase PCR (Rt-PCR) – Conventional PCR amplifies only the DNA. For
amplifying RNA, RT-PCR is done
⮚ After RNA extraction, the first step is addition of reverse transcriptase enzyme that
converts RNA into DNA
⮚ Then, the amplification of DNA and gel documentation steps are similar to that of
conventional PCR
⮚ It is extremely useful for detection of RNA viruses or 16s rRNA genes of the organisms

⮚ Nested PCR– Two rounds of PCR amplification are carried out by using two primers that are
targeted against two different DNA sequences of same organism
⮚ The amplified products of the first round PCR is subjected to another round of
amplification using a second primer which targets the same organism but a different DNA
sequence
⮚ More sensitive and specific and yields a high quantity of DNA.
⮚ Used for detection of Mycobacterium tuberculosis in samples
⮚ More chances of contamination of the PCR tubes, which may lead to false positive results
⮚ Multiplex PCR:
⮚ Uses more than one primer which can detect many DNA sequences of several organisms
in one reaction
⮚ Useful for diagnosis of infectious diseases that are caused by more than one
organism-Syndromic approach.
⮚ There is a high risk of the reaction tubes to be contaminated with environmental DNA.

BIOFIRE FILM ASSAY:

⮚ It is a completely automated multiplex nested PCR system where all the steps from sample
preparation to amplification, detection and analysis are performed automatically by the system;
giving the result in about 1 hour.
⮚ Four panels are available such as respiratory, GI, meningitis-encephalitis and blood culture
identification panels; each panel comprises primers targeting 20-25 common pathogens infecting the
respective systems.
⮚ It has excellent specificity and sensitivity.
⮚ But expensive.

REAL TIME PCR (RT-PCR):

⮚ It is based on PCR technology, used to amplify and simultaneously detect or quantify a targeted
DNA molecule on a real time basis.
⮚ Reverse transcriptase real-time PCR formats can detect and quantify RNA molecules of the test
organism in the test sample.
⮚ Uses a different thermocycler than the conventional PCR, very expensive.
⮚ Advantages:
o Quantitative and takes less time.
o Contamination rate is extremely low.
o More sensitive and specific.
⮚ The detection of amplified nucleic acid in a real-time PCR reaction is carried out by using a variety
of fluorogenic molecules which may be either nonspecific or specific
LOOP MEDIATED ISOTHERMAL AMPLIFICATION:

⮚ Isothermal nucleic acid amplification technique.

⮚ Amplification is carried out at a constant temperature of 60–65° (in contrast to alternating
temperature cycles in PCR).
⮚ The isothermal nature of LAMP assay is due to the use of specific DNA polymerase enzymes which
have additional strand displacement capacity, e.g. polymerase derived from Geobacillus
stearothermophilus
⮚ It has been approved for tuberculosis.
⮚ Advantages:
● Cheaper and easy to perform
● More sensitive
● Amplicons can be directly detected by naked eyes by turbidity and visual fluorescence
detection.
⮚ Disadvantage: High false positive results due to cross contamination between reaction tubes.

NUCLEIC ACID PROBES: (Refer SET 16)

MICROBIAL TYPING

⮚ Refers to characterization of an organism beyond its species level.

⮚ It is important for hospital microbiologists and epidemiologists to determine the relatedness
between different strains of the same species.
⮚ This helps to:
● Investigate outbreaks
● Determine the source and route of infection
● Trace cross-connection
● Differentiate between avirulent and virulent strains
● Differentiate between recurrence and infection with new strain.
● Evaluate effectiveness of control measures.
CHARACTERISTICS OF TYPING METHODS:

⮚ A good typing method has the following properties:

● Typeability
● Reproducibility
● Discriminative power
● Practicality

CLASSIFICATION:

⮚ Typing methods are broadly classified as:

● Genotypic methods
● Phenotypic methods

PHENOTYPIC METHODS:

⮚ It includes:
● Bacteriophage typing
● Bacteriocin typing
● Biotyping
● Antibiogram typing
● Serotyping

GENOTYPIC METHODS:

⮚ These are more reliable and have better discriminative power and reproducibility than
phenotypic methods.
⮚ But these are expensive.
⮚ It includes:
● Restricted Fragment Length Polymorphism (RFLP)
● Ribotyping
● Pulse Field Gel Electrophoresis (PFGE)
● Amplified Fragment Length Polymorphism (AFLP)
● Sequencing based methods
SET 3

FLUORESCENCE MICROSCOPE
⮚ Fluorescence property is used to generate an image

PRINCIPLE:

⮚ When fluorescent dye is exposed to UV light rays (invisible, short wavelength), they are excited
and are said to fluoresce (becomes visible light of longer wavelength).
APPLICATION:

EPIFLUORESCENCE MICROSCOPE:

⮚ Simplest form of microscope

● AUTOFLUORESCENCE: Certain microbes directly fluoresce when placed under UV
lamps. Eg:Cyclospora ( a protozoan parasite).
● MICROBES COATED WITH FLUORESCENT DYE: Certain microbes fluoresce when
stained non-specifically by fluorochrome dyes such as auramine O and rhodamine.
➢ QBC (Quantitative buffy coat): Detection of parasites such as Plasmodium and
Filarial nematodes using Acridine orange dye.
➢ Detection of tubercle bacilli using Auramine phenol.
● IMMUNOFLUORESCENCE: Detection of cell surface antigen or antibody bound to
cell surface antigen using fluorescent dye tagged immunoglobulins.

CONFOCAL MICROSCOPE:
● Advanced fluorescence microscope.
● Uses point illumination and pinhole in an optically conjugate plane to eliminate
out-of-focus signal and to get a better resolution of the fluorescent image.

Reference: Essential of Medical Microbiology, Apurba S Sastry E/3 Page no. 9 Fig. 2.4A
SET 4

IMMUNOFLUORESCENCE
⮚ It is a technique used to detect cell surface antigens or antibodies bound to cell surface antigen.
⮚ It is commonly employed for detection of microbial antigens in body fluids , in tissues and also
for detection of autoantibodies in autoimmune diseases.

PRINCIPLE:

⮚ Shorter wavelength (UV rays) Longer wavelength (Visible light rays)

Absorbs Emits

⮚ Fluorescence: Fluorescent dye conjugates antibodies and such antibodies can be used to
detect antigens or antigen-antibody complexes on the cell surface.
⮚ Fluorescence dyes :
1. FITC ( Fluorescein Isothiocyanate) - blue green fluorescence
2. Lissamine rhodamine – orange red fluorescence.
⮚ Fluorescent microscope uses fluorescence properties and generates an image.

Reference: Essentials of Medical Microbiology, Apurba S Sastry E/3 Page No. 9 Fig. 2.4 A & B
TYPES OF IMMUNOFLUORESCENCE ASSAY:

⮚ Direct immunofluorescence assay

⮚ Indirect immunofluorescence assay

DIRECT IMMUNOFLUORESCENCE ASSAY:

⮚ Detects unknown antigen in a specimen.

⮚ Example: Diagnosis of rabies by detection of rabies virus antigen in brain smear.
⮚ Disadvantage: separate fluorescent labeled antibodies has to be prepared against each antigen
to be tested.

Reference: Essentials of Medical Microbiology, Apurba S Sastry E/3 Page No. 160, Fig. 12.10A
INDIRECT IMMUNOFLUORESCENCE ASSAY:

⮚ Detects antibodies in serum or other body fluids.

⮚ Disadvantage of direct immunofluorescence is overcome by indirect immunofluorescence.
⮚ Here , a single Anti Human Antibody conjugated with fluorescent dye can be used for detecting
antibodies to any antigen.
Reference: Essentials of Medical Microbiology, Apurba S Sastry E/3 Page No. 160 Fig 12.10B
 SET 5

PHYSIOLOGY OF BACTERIA

MULTIPLICATION OF BACTERIA:

BACTERIAL DIVISION:

⮚ Binary fission: Simple form of cell division where nuclear division precedes cytoplasmic
division.
⮚ Nuclear division: Two DNA strands separate and replicate to form 2 identical molecules of ds
DNA.
⮚ Cytoplasmic division:
● Transverse septum grows across the cell from the cell membrane followed by deposition
of cell wall materials.
● Then 2 daughter cells get separated.
● When the daughter cells remain partially attached, they are arranged in pair or chains
(streptococci) or in clusters (staphylococci)
BACTERIAL GROWTH:

⮚ Batch culture:
● Grown in liquid medium.
● Multiplication is arrested after a few cell divisions due to nutrient depletion or
accumulation of toxic products.
⮚ Continuous culture:
● Grown in chemostat or turbidostat.
● Continuous culture of bacteria maintained through replenishment of nutrients
⮚ Colony:
● Clone of cells derived from a single parent cell.
● Solid medium = Colony formation.
● Liquid medium = diffused growth

RATE OF MULTIPLICATION IN BACTERIA:

Generation Time or Population Doubling Time:

⮚ Time required for a bacterium to give rise to 2 daughter cells under optimum conditions.
⮚ Eg: E.coli – 20 mins, Mycobacterium tuberculosis – 10-15 hrs, Mycobacterium leprae – 12-13
days.

BACTERIAL COUNT:

⮚ Expressed in terms of total count and viable count.

⮚ Total Count:
● Indicates total number of bacteria in the specimen irrespective of whether they are live
or not.
● Counted under microscope using counting chamber or electronic device using Counting
chambers
⮚ Viable Count:
● Measures number of living cells in the specimen.
● By pour plate method or dilution method
● Dilution Method: suspension is diluted to a point beyond which unit quantities do not
yield growth when inoculated into a suitable liquid media (extinction)
▪ Used widely for ‘presumptive coliform count’ in drinking water
 ▪ No accurate values
● Plating Method: Appropriate dilutions are inoculated on solid media, gives estimation
of viable count

BACTERIAL GROWTH CURVE:

⮚ When a bacterium is inoculated into a suitable liquid culture medium and incubated, its growth
follows a definite course.
⮚ If bacterial counts are made at regular intervals after inoculation and plotted in relation to time, a
bacterial growth curve is obtained.

Bacterial Growth curve: The viable count shows the lag, log, stationary and decline phases; in the total count, the
phase of decline is not evident

Reference: Essentials of Medical Microbiology, Apurba S Sastry E/3 Page No. 23 Fig. 3.2.14

1) LAG PHASE:
● Period between inoculation and beginning of multiplication of bacteria.
● Do not start multiplying immediately as it takes time to build-up enzymes and
metabolites.-Adaptation time.
❖ Increase in size due to accumulation of enzymes and metabolites.
❖ Reaches maximum size at the end of phase.
 2) LOG PHASE:
● Period where bacteria divides exponentially.
● Growth curve takes a straight line.
❖ Decrease in size.
❖ Uniformly stained -Best stage to perform Gram stain.
❖ 🡩 Antibody susceptibility
❖ Biochemically active -Best stage to perform biochemical reaction.
3) STATIONARY PHASE:
● Cessation of bacterial growth due to exhaustion of nutrients and accumulation of toxic
products and autolytic enzymes.
● No. of progeny cells formed = no. of cells dying -replacement occurs
● No. of viable cells = same, Total count = increasing
❖ Bacteria become gram-variable.
❖ Sporulation.
❖ Formation of storage granules.
❖ Production of exotoxins, antibodies, bacteriocins.
4) DECLINE PHASE:
● Bacteria stop dividing completely.
● Cell death continues due to exhaustion of nutrients and accumulation of toxic products.
❖ Viable count but not in total count.
❖ Involution forms seen.
❖ Autolytic bacteria: Shows phase of decline of total count as well

NUTRITIONAL REQUIREMENTS OF BACTERIA

⮚ Minimum requirements essential for growth and multiplication of bacteria

● Water
● An energy source
● A carbon source
● An oxygen source
● A source of nitrogen and hydrogen
● Inorganic compounds –sulphur, phosphorus, potassium, sodium, etc
● Vitamin
ENERGY SOURCE:

⮚ Based on the source bacteria can be classified as:

● Phototrophs: Derive energy from sunlight.
● Chemotrophs: Derive energy from chemical reactions

CARBON SOURCE:

⮚ Based on the source bacteria can be classified as:

● Autotrophs: Synthesise their own organic material.
● Heterotrophs: Cannot synthesise their metabolites, utilise atmospheric carbon dioxide
and nitrogen.

OXYGEN REQUIREMENTS:

⮚ Based on the oxygen requirements bacteria can be classified as:

● AEROBIC BACTERIA: Requires oxygen to survive
▪ Obligate Aerobes:
o Grow only in the presence of oxygen.
o Eg: Pseudomonas,Nocardia, Mycobacterium tuberculosis,etc
▪ Facultative Anaerobes:
o Ordinarily aerobic but can also grow in the absence of oxygen.
o Eg: Escherichia coli, Staphylococcus aureus
● ANAEROBIC BACTERIA: Grow in the absence of oxygen (Clostridia)
▪ Obligate Anaerobes:
o Cannot survive in the presence of oxygen, as it is lethal to them.
o Eg: Clostridium tetani
▪ Facultative Aerobes:
o Anaerobes that can also grow aerobically.
o Eg: Lactobacillus
▪ Aerotolerant Anaerobes:
o They can tolerate oxygen for some time but do not use it.
o Eg: Clostridium histolyticum.
● MICROAEROPHILIC BACTERIA:
▪ Grow in presence of low oxygen tension -5%-10%
▪ Eg: Helicobacter and Campylobacter.
CARBON DIOXIDE:

⮚ Some bacteria require high amounts of carbon dioxide (5%-10%) to grow.

⮚ They are called capnophilic bacteria
⮚ Eg: Brucella abortus and Streptococcus pneumonia.

TEMPERATURE REQUIREMENTS:

⮚ Different bacteria have different optimal temperatures.

⮚ Most pathogenic bacteria grow optimally at 37°C.
⮚ Mesophilic Bacteria: They grow within 25-40 °C. Eg: Most of the pathogenic bacteria belong
to this category.
● Some have wider ranges - Pseudomonas (5-43°C).
● Some have restricted range - Gonococcus (30-39°C)
⮚ Psychrophilic Bacteria: They grow best in temperatures less than 20°C. Eg: Soil and water
saprophytes
⮚ Thermophilic Bacteria: They grow within the range 33-80°C. Eg: Bacillus stearothermophilus
⮚ Thermal death point: Lowest temperature that kills a bacterium under standard conditions in a
given time
o Under moist conditions: 50-65° (mesophilic bacteria), 100-120°C (spores)

BACTERIAL VITAMIN:

⮚ Some fastidious bacteria grow only in the presence of some organic compounds.
⮚ These are called ‘ Bacterial growth factors’ or ‘Bacterial vitamins’.
⮚ Thiamine, riboflavin, nicotinic acid, pyridoxine, folic acid and vitamin B12 are needed.

FACTORS AFFECTING GROWTH OF BACTERIA:

⮚ Several environmental factors affect the growth of bacteria. They are:

● Oxygen
● Carbon dioxide
● Temperature
● pH
 ● Light
● Osmotic effect
● Mechanical and sonic stress
● Moisture and desiccation

MOISTURE AND DESICCATION:

⮚ Moisture is important for bacterial growth because 80% of bacterial cells are made up of water.
⮚ Highly sensitive bacteria dry out easily. Eg:Treponema pallidum and Neisseria gonorrhoea.
⮚ Some withstand drying for months. Eg: Mycobacterium tuberculosis and Staphylococcus
aureus.
⮚ Lyophilisation / Freeze-drying / Cryodesiccation: Drying process in cold temperature and
vacuum used to preserve bacteria.

pH :

⮚ Most pathogenic bacteria grow between pH 7.2-7.6.

⮚ Some bacteria can grow in acidic medium < pH 4. Eg: Lactobacilli
⮚ Some can grow in alkaline medium, pH 8.2-8.9. Eg:Vibrio cholera

LIGHT:

⮚ Grow well in the dark except for phototrophs.

⮚ They are sensitive to UV rays and other radiations.
⮚ Photochromogenic mycobacteria produce pigments on exposure to light

OSMOTIC EFFECT:

⮚ Bacteria can withstand a wide range of external range of osmotic variation due to mechanical
strength of the cell wall.
⮚ But sudden exposure to
● Hypertonic solution- cell shrinkage- plasmolysis.
● Distilled water- cell swelling and rupture- plasmoptysis.
MECHANICAL AND SONIC STRESS:

⮚ Bacterial cell walls can be ruptured and disintegrated by vigorous shaking with glass beads or
exposure to ultraviolet rays.

BACTERIAL METABOLISM
⮚ It is the process by which a microbe obtains the energy and nutrients for its survival and
reproduction. It can be classified based on 3 principles:
1) Method of obtaining carbon for synthesising cell mass:
● Autotrophs: Synthesise organic compounds using atmospheric CO2.
● Heterotrophs: Use reduced, performed organic molecules as carbon sources.
2) Method of obtaining reducing equivalents (electrons):
● Lithotrophs: Obtain reducing equivalents from inorganic compounds.
● Organotrophs: Obtain reducing equivalents from organic compounds.
3) Method of obtaining energy:
● Chemotrophs: Obtain energy from chemical compounds.
● Phototrophs: Obtain energy from light.
⮚ Most of the pathogenic bacteria are Chemoorganoheterotrophs
 SET 6
GENE TRANSFER
⮚ Gene transfer is a process of transmitting genetic material from one bacterium to another.
⮚ Gene transfer in bacteria can be broadly divided into:

1. Vertical gene transfer:

❖ Transmission of genes from parent to offspring via replication and recombination
2. Horizontal Gene Transfer:
❖ Transmission of genes from bacterium to another neighbouring bacterium
● Transformation
● Transduction
● Conjugation
● Lysogenic conversion

VERTICAL GENE TRANSFER

REPLICATION
⮚ During replication of a bacterium genetic information is passed on to the progeny.

● Parent replicates into two double stranded DNA:

(1 TEMPLATE STRAND and 1 NEWLY SYNTHESISED STRAND) *2
● Small pieces of chromosomes are formed and moved to the two ends of the bacterial cell.
● Each of them are enclosed in plasma membrane and new cell wall develops
● Thus 2 daughter cells are formed.

RECOMBINATION
⮚ This is a technique whereby a piece of DNA that has been artificially created from two or
more sources is incorporated into a single bacterial molecule.
⮚ This results in the expression of the particular genetic information by the bacteria and its
progeny

DIFFERENT TYPES:

⮚ Homologous recombination:
 ❖ Between DNA molecules that have very similar sequences.
⮚ Non homologous recombination:
❖ Between pieces of DNA in which there is no large similarity in sequences.
⮚ Site - specific recombination:
❖ Between short sequences present in dissimilar molecules
⮚ Replicative recombination:
❖ Generates a new copy of a segment of the DNA.

HORIZONTAL GENE TRANSFER

TRANSFORMATION:
⮚ Transformation is a process of gene transfer by random uptake of free or naked DNA fragment
from the surrounding medium by a bacteria cell and incorporation of this DNA fragment into its
chromosome in a heritable form.
⮚ Studied in Streptococcus, Bacillus, Haemophilus, Acinetobacter and Pseudomonas.

Discovered by Griffith:
⮚ He demonstrated that injecting a mixture of live non-capsulated (Type II) S.Pneumoniae and
heat-killed capsulated S.Pneumoniae (type I) (neither of which individually fatal) could kill mice.
⮚ He stated that the live non-capsulated strains were transformed into the capsulated strains due to
transfer of the capsular genes released from the lysis of the killed capsulated strains,
⮚ Confirmed later by Avery, Macleod and McCarty in 1944.

Reference: Essentials of Medical Microbiology, Apurba S Sastry E/1 Page No. 71 Fig. 6.8
MECHANISM OF TRANSFORMATION

1) A long dsDNA fragment comes in contact with a competent bacterium and binds to
DNA-binding protein present on its surface and then it is nicked by a nuclease.
2) One strand is degraded by the recipient cell exonucleases.
3) The other strand associates with a competence specific protein and is internalized, which
requires energy expenditure.
4) The single strand enters into the cell and is integrated into the host chromosome in place of the
homologous region of the host DNA.

Reference: Essentials of Medical Microbiology, Apurba S Sastry E/1 Page No. 70 Fig. 6.7)

5) However, their uptake depends upon the competency of the bacteria present in the
surroundings (in case of natural transformation)

COMPETENCY OF THE BACTERIA:

⮚ Competent bacteria refers to the cells multiplying in the log phase of cell division and
expressing certain transformation promoting factors called competence factors.

● Bacteria expressing competence factors (E.g: S. pneumoniae) can uptake any DNA fragment
irrespective of source.
 ● But competence factors are not expressed by all bacteria that mediate transformation E.g:
Haemophilus influenzae. In such cases, the uptake of DNA occurs only from closely related
species.

TRANSDUCTION: (Also a SHORT NOTE)

⮚ The transfer of a portion of DNA from one bacterium to another by a bacteriophage.
(Viruses that infect bacteria: consists of nucleic acid and protein coat)
⮚ During the assembly of bacteriophage ‘packaging error’ might occur, which is that nucleic acid
of the host may get incorporated into the viral genome.
⮚ When this bacteriophage infects another bacteria the DNA taken from the previous cell is
transferred.
⮚ The recipient bacterium acquires new characters coded by the donor DNA.

ROLE OF TRANSDUCTION:
⮚ Transduction is not only confined to DNA it can also transduce episomes and plasmids.
⮚ Example: DRUG RESISTANCE: Penicillin resistance in staphylococci is due to plasmids
transferred from one bacterium to another.
⮚ Treatment: Transduction has also been proposed as a method of genetic engineering in the
treatment of some inborn metabolic defects.
⮚ Genetic mapping

TYPES OF TRANSDUCTION:

GENERALISED:
⮚ It involves transfer of any part of the donor bacterial genome at random into the recipient
bacteria.
⮚ Packaging errors may happen occasionally due to defective assembly of the daughter phages.
⮚ Instead of their own DNA, a part of the host DNA may accidentally get incorporated into the
daughter phage (Transducing phage).
⮚ The transducing phage injects its donor DNA into the recipient bacterial cell but it does not
initiate a lytic cycle as the original phage DNA is lost.

⮚ The donor DNA may have three fates:

 1. Abortive transduction:
● About 70-90% of the transferred DNA is not integrated with the recipient bacterial
chromosome, but often is able to survive and express itself.
2. Stable gene transfer:
● The donor DNA is integrated with the recipient bacterial chromosome.
3. Unstable gene transfer:
● In some cases, the donor DNA gets disintegrated by the host cell enzymes.

Reference: Essentials of Medical Microbiology, Apurba S Sastry E/1 Page No. 72 Fig. 6.9

RESTRICTED:
⮚ It involves transducing only a particular genetic segment of the bacterial chromosome present
adjacent to the phage DNA.
 ⮚ It occurs due to the defect in the disintegration of the lysogenic phage DNA from the bacterial
chromosome.
⮚ It has been studied in the ‘lambda’ stage of E.Coli.
⮚ The transfer of the donor DNA takes place in 2 ways :
1) Crossover between the donor DNA and a part of recipient DNA - integration of the
donor DNA into the recipient chromosome and a part of the recipient DNA into the phage
DNA.
2) The entire transducing genome acts as a prophage and gets integrated to the recipient
chromosome (only when the recipient is already affected by another bacteriophage).

Reference: Essentials of Medical Microbiology, Apurba S Sastry E/1 Page No. 72 Fig. 6.10
CONJUGATION (Also a Short note):
⮚ The transfer of genetic material from one bacterium (donor or male) to another bacterium
(recipient or female) by mating and forming a conjugation tube.
1) F+cell and F- cell Conjugation:
● F Plasmid (sex / Fertility Factor):
o It is conjugative plasmid which encodes for sex pilus (helps in forming
conjugation tube).
o Devoid of any other identifiable genetic markers such as drug resistance.
● Donor: Contain F Plasmid – F+ / Male.
● Recipient: Do not contain F Plasmid- F- / Female.
● The plasmid DNA replicates during conjugation , and is passed on to the recipient along
the conjugation tube. Thus, F- becomes F+
2) Hfr Cell:
● The F factor is sometimes an episome that exists in the ‘integrated state’ or inserted into
the host chromosome.
● Such cells can transfer chromosomal genes to recipients at high frequency and are
known as Hfr ( high frequency of recombination) cells.
● Only a few chromosomal genes along with a part of the F factor get transferred to the F-
cell as the connection between the cells breaks before the transfer of the whole genome.
● Thus the F- recipient does not become F+ cell after conjugation.

3) F’ prime Factor:
● F+ to Hfr is reversible, thus when the F factor reverts from being in the integrated state
to the free state it may carry some of the chromosomal genes from near its attachment,
such a factor is called F’ prime factor.
● When the F’ cell conjugates with the F- cell, it transfers the F’ factor and thus the
recipient also becomes an F' cell.
● This process is called sexduction.

4) Colicogenic (col) factor:

● Bacteriocins are the antibiotic-like substances produced by one bacterium that inhibit
other bacteria. Bacteriocin produced by coliform bacteria is called colicin.
● Other examples: pyocin by Pseudomonas. diptericin by Corynebacterium diphtheriae.
 ● The production of these bacteriocins are plasmid coded those plasmids are called col
factor, during conjugation this factor may get transferred

Resistance factor (R factor):

⮚ This plasmid leads to the spread of multiple drug resistance among bacteria.
⮚ This factor was reported when infection caused by Shigella strains resistant to sulfonamides ,
streptomycin, chloramphenicol and tetracycline was reported.
⮚ Also found out that the affected patients excreted E.coli strains in the feces that were resistant to the
same drugs.
❖ Which implied the transfer of multiple drug resistance between E.Coli and Shigella
strains. It was also demonstrated invivo and invitro

R Factor Plasmid :

⮚ Contains RTF and r determinants

1) RTF: Resistance Transfer Factor: It the plasmid responsible for conjugational transfer
● RTF may dissociate from r determinants and they can exist in separate plasmids in such
cases the resistance is not transferable
● RTF can have other determinants other than drug resistance attached to it.

● Eg: Enterotoxin and hemolysin production In enteropathogenic E.Coli

2) r determinant: R factor can have several r determinants and each r determinant coding for
resistance to one drug .
 A. F· X F-mating B. Hfr X F· mating C. F' X F- mating

Reference: Essentials of Medical Microbiology, Apurba S Sastry E/1 Page No. 74 Fig. 6.11

LYSOGENIC CONVERSION:
⮚ Bacteriophages have two types of life cycles in the bacterium:
● VIRULENT OR LYTIC CYCLE:
o Large number of progeny phages are formed and released causing death and
lysis of the host cell.
● TEMPERATE OR NON-LYTIC CYCLE:
o Here the host bacterium is unharmed, phage DNA remains integrated with the
chromosome as a prophage which multiplies with bacterial DNA.
o However, when the phage DNA tries to come out, it is disintegrated
from the host chromosome, comes out into the cytoplasm, and behaves as a lytic
phage.
 o The prophage acts as an additional chromosomal element which encodes for new
characters and is transferred to the daughter cells. This process is known as
lysogeny or lysogenic conversion.
o In lysogenic conversion, the phage DNA itself behaves as the new genetic element
in contrast to transduction.
o Eg: Diphtheria toxin is coded by a lysogenic phage DNA which is integrated with
the bacterial chromosome.
o Elimination of the phage from a toxigenic strain renders the bacterium
nontoxigenic.

FATE OF DONOR POST HORIZONTAL TRANSFER:

⮚ The donor DNA enters inside the recipient cell, and remains in the cytoplasm temporarily.
⮚ At this stage, the recipient cell is called MEROZYGOTE.

1) Recombination: The donor DNA integrates with the recipient chromosome either as a
replacement or additional element
2) Partially diploid cells: The donor DNA persists outside the host chromosome and the host cell
becomes partially diploid for a portion of the genome that is homologous to the donor DNA.
Such cells may or may not replicate to produce a clone of partially diploid cells.
3) Host restriction: The host cell nucleases may degrade the donor DNA if it is not homologous
to any part of the bacterial chromosome.
 SET 7

MECHANISM OF DRUG RESISTANCE

⮚ Development of resistance to an antimicrobial agent by a microorganism.
⮚ Emergence of resistance is a major problem worldwide in antimicrobial therapy.
⮚ Infections caused by resistant microorganisms often fail to respond to standard treatment,
resulting in prolonged illness, higher healthcare expenditures and a greater risk of health.
⮚ It is of two types
● ACQUIRED RESISTANCE
● INTRINSIC RESISTANCE

ACQUIRED RESISTANCE:
● Emergence of resistance in bacteria that are ordinarily susceptible to antimicrobial agents.
● By acquiring the genes coding for resistance.
● Most antimicrobial resistance belong to this category.
● Overuse and misuse of antimicrobial agents is the most important cause.
❖ Use of particular antibiotics poses selective pressure in a population which in turn
promotes resistant bacteria to thrive and susceptible bacteria to die.
❖ Resistant bacterial populations flourish in high antimicrobial use (selective advantage
over susceptible populations)
❖ The resistant strains then spread in the environment and transfer the genes coding for
resistance to other unrelated bacteria.
● Factors favouring spread of antimicrobial resistance are
➢ Poor infection control practices in hospitals.
➢ Inadequate sanitary conditions
➢ Inappropriate food-handling
➢ Irrational use of antibiotics by doctors.
➢ Uncontrolled sale of antibiotics over the counters without prescription.

INTRINSIC RESISTANCE:
● Innate ability of a bacteria to resist a class of antimicrobial agent.
● Due to inherent structural or functional characteristics.
● Has a defined pattern of resistance and is non-transferable.

ORGANISMS INTRINSIC RESISTANCE TO FOLLOWING DRUGS

Enterbacteriaceae To antimicrobial agents specific for gram positive organisms ;

clindamycin, daptomycin, fusidic acid, glycopeptides

(vancomycin), rifampin, macrolides (erythromycin, azithromycin)

Exceptions: Salmonella and Shigella are susceptible to azithromycin

Klebsiella pneumoniae Same as enterobacteriaceae plus ampicillin and

Ticarcillin

Citrobacter species Same as enterobacteriaceae plus ampicillin, 1st and 2nd

generation cephalosporins, cephamycins,

amoxicillin-clavulanate and amoxicillin-sulbactam

Enterobacter species Same as enterobacteriaceae plus ampicillin, 1st and 2nd

generation cephalosporins, cephamycins,

amoxicillin-clavulanate and amoxicillin-sulbactam

Proteeae tribe Same as enterobacteriaceae plus ampicillin, 1st and 2nd

generation cephalosporins, tetracyclines, tigecycline,

Nitrofurantoin and polymyxins.

Salmonella species Same as enterobacteriaceae plus aminoglycosides, 1st and 2nd

generation cephalosporins.

Shigella species Same as enterobacteriaceae plus aminoglycosides, 1st and 2nd

generation cephalosporins and cephamycins.

Serratia marcescens Same as enterobacteriaceae plus ampicillin, 1st and 2nd

generation cephalosporins, cephamycins,

amoxicillin-clavulanate and amoxicillin-sulbactam,

Nitrofurantoin and polymyxins.

Yersinia enterocolitica Same as enterobacteriaceae plus amoxicillin, ticarcillin,

1st generation cephalosporins and amoxicillin-clavulanate

Non-fermentative Penicillin (benzylpenicillin),1st and 2nd generation

Gram negative cephalosporins, cephamycins, clindamycin, daptomycin, fusidic

Bacteria [NF-GNB] acid, glycopeptides (vancomycin), rifampicin, linezolid, macrolides

Pseudomonas Same as NF-GNB plus ampicillin, amoxicillin,

aeruginosa amoxicillin-clavulanate, ampicillin-sulbactam, ertapenem,

Tetracyclines, chloramphenicol
Acinetobacter Same as NF-GNB plus ampicillin, amoxicillin,

baumannii Amoxicillin-clavulanate, ertapenem, chloramphenicol,

Aztreonam and fosfomycin

Stenotrophomonas Same as NF-GNB plus ampicillin, amoxicillin, cefotaxime

maltophilia Amoxicillin-clavulanate, ertapenem, chloramphenicol,

Aztreonam and fosfomycin, cefotaxime, polymyxins,

Aminoglycosides

Burkholderia Same as NF-GNB plus ampicillin, amoxicillin,

cepacia complex Amoxicillin-clavulanate, ertapenem, ampicillin-sulbactam

polymyxins, and fosfomycin

Gram-positive Aztreonam, polymyxin B / colistin and nalidixic acid

bacteria

S.aureus Same as other gram-positive bacteria

Enterococcus Same as other gram-positive bacteria plus cephalosporins,

Species aminoglycosides,clindamycin and clotrimazole

MUTATIONAL AND TRANSFERABLE RESISTANCE
⮚ In the presence of selective antibiotic pressure, bacteria acquire new resistance by two methods:
● MUTATIONAL RESISTANCE
● TRANSFERABLE RESISTANCE

MUTATIONAL RESISTANCE:
⮚ Can develop due to mutation in resident genes
● Typically seen in Mycobacterium tuberculosis developing resistance to anti-tubercular
drugs
● Usually low level resistance developed to one drug at a time, which can overcome by
using a combination of different classes of drugs
➔ Multi-drug therapy is used in tuberculosis using 4-5 different classes of drugs
[isoniazid, rifampicin, pyrazinamide, ethambutol and streptomycin]

TRANSFERABLE RESISTANCE:
⮚ Plasmid-coded
⮚ Usually transferred by conjugation or rarely by transduction or transformation.
⮚ The resistant coded plasmid (R plasmid) can carry multiple genes, each coding for resistance to
one class of antibiotics.
⮚ It results in a high degree of resistance which cannot be overcome by combination of drugs.

RESISTANCE TRANSFER FACTOR (RTF):

⮚ Is of great medical importance as it leads to spread of multiple drug resistance amongst bacteria.

⮚ First reported in 1959 when investigating the sudden increase in infections caused by Shigella

strains resistant simultaneously to sulphonamides, streptomycin, chloramphenicol and

tetracycline.
● Patients excreting such Shigella strains also shed their feces E.coli strains resistant to the
same drugs.
● Transfer of multiple drug resistance was demonstrated between E.coli and Shigella
strains both in vitro and in vivo

⮚ The resistance is plasmid-mediated and is transferred by conjugation

 ● This mechanism is known as transferable, episomal or infectious drug resistance.

⮚ Plasmid consists of two components:

● The transfer factor called the resistance transfer factor (RTF) is responsible for
conjugational transfer.
● Resistant determinant (r) is responsible for multiple drug resistance.

⮚ R factor can have several r determinants and resistance to as many as eight or more drugs can be

transferred simultaneously.

⮚ The RTF may dissociate from the r determinants, the two components existing as separate

plasmids.
● Host cell remains drug-resistant, but the resistance is not transferable.

⮚ Enterotoxin and hemolysin production in some enteropathogenic E.coli are transmitted by this

transfer factor.

Reference: Ananthanarayanan and Paniker's Textbook of Microbiology E/11 Fig 5.10

 MUTATIONAL DRUG RESISTANCE TRANSFERABLE DRUG RESISTANCE

1.Resistant to one drug at a time. 1.Multiple drug resistance at the same time

2.Low degree resistance 2.High degree resistance

3.Resistance can be overcome by combination 3.Resistance cannot be overcome by drug

of drugs. combinations.

4.Virulence of resistant mutants may be 4.Virulence is not decreased.

lowered.

5.Resistance is not transferable to other 5.Resistance is transferable to other organisms

organisms.

6.Resistance can spread to offspring (vertical 6.Resistance spreads by horizontal gene

gene transfer) transfer

MECHANISM OF ANTIMICROBIAL RESISTANCE

⮚ Bacteria develop antimicrobial resistance by the following mechanisms:
1. DECREASED PERMEABILITY ACROSS THE CELL WALL:
● Modify their cell membrane porin channels (either in frequency, size or selectivity) preventing
antimicrobials from entering into the cell.
● Observed in many gram negative bacteria such as Pseudomonas, Enterobacter and Klebsiella
species against drugs such as quinolones, imipenem and aminoglycosides.

2. EFFLUX PUMPS:
● Mediate expulsion of drugs from cell, soon after their entry (preventing intracellular
accumulation)
● Observed in
❖ E.coli and other Enterobacteriaceae against tetracyclines, chloramphenicol
❖ Staphylococci against macrolides and streptogramins
❖ Staphylococcus aureus and Streptococcus pneumoniae against fluoroquinolones.
 3. BY ENZYMATIC INACTIVATION:
● Bacteria may produce enzymes to inactivate the antimicrobial agents.
● Beta-lactamase enzyme: This enzyme (observed in both gram-positive and gram-negative
bacteria) breaks down the beta-lactam rings, thereby inactivating the beta-lactam antibiotics.
● Aminoglycoside modifying enzymes: They (acetyltransferases, adenylyltransferase and
phosphotransferases produced by both gram-negative and gram-positive bacteria) destroy the
structure of aminoglycosides.
● Chloramphenicol acetyltransferase: It is produced by members of Enterobacteriaceae which
destroys the structure of chloramphenicol.

4. BY MODIFYING TARGET SITES:

● Some bacteria modify the target site of the antimicrobial agent and thus prevent their action.
● MRSA (Methicillin-resistant Staphylococcus aureus): Target site of penicillin (penicillin binding
protein [PBP]) gets altered to PBP-2a. The altered PBP do not bind to beta-lactam antibiotics
and therefore prevent them from inhibiting cell wall synthesis.
● Beta-lactam resistance in Pneumococci due to alteration of PBP to PBP2b.
● Rifampicin resistance in Mycobacterium tuberculosis due to mutation in RNA polymerase.
● Streptomycin resistance in Mycobacterium tuberculosis is due to modification of ribosomal
proteins or 16 S rRNA.
● Vancomycin-resistant enterococci (VRE).

CLASS/MECHANISM SPECTRUM OF ACTIVITY MECHANISM OF RESISTANCE

INHIBIT CELL WALL SYNTHESIS

1.Beta-lactam antibiotics:

(bactericidal; block peptidoglycan cross linking by inhibiting the transpeptidase enzyme i.e
penicillin-binding protein)

Penicillins
Penicillin Mostly gram-positive bacteria:

Streptococcus pyogenes

Pneumococcus

Corynebacterium diphtheriae

Clostridium tetani

Clostridium perfringens

Meningococcal infection

Treponema pallidum 1.Drug inactivation by producing

beta-lactamase enzyme ;

Penicillinase- Same as penicillin plus penicillinase 2.Alteration of target site-PBP;

Resistant penicillin producing Staphylococcus aureus 3.Decreased permeability.

Aminopenicillins Same as penicillin plus

(Extended spectrum) Enterococcus faecalis

Escherichia coli

Helicobacter pylori

Salmonella

Shigella

Antipseudomonal Same as penicillin plus

Penicillins Pseudomonas aeruginosa

Cephalosporins:

1st generation Staphylococcus aureus

Staphylococcus epidermidis

Some gram-negative bacteria like

E.coli and Klebsiella

2nd generation Same as 1st generation plus

Increased gram-negative activity

Increased anaerobic activity ESBL (extended-spectrum

Beta lactamase

3rd generation. Decreased activity against gram-positives

compared to 1st and 2nd generation;

Increased gram-negative activity

4th generation. Good activity against gram-positive

and gram-negative bacteria including

Pseudomonas

5th generation. Same as 4th generation and MRSA

Beta lactam + Same spectrum as respective beta lactam

Beta lactamase Drug plus active against beta lactamase

Inhibitors producing bacteria;

 Have excellent anaerobic coverage

Carbapenems Broadest range of activity against most bacteria;

No action on MRSA and Mycoplasma

Monobactam Gram-negative rods

Other cell wall inhibitors:

Glycopeptides Active against most gram-positive Alteration of target site

including MRSA

Fosfomycin Inactivate enzyme MurA; active Alteration of target site;

against urinary tract pathogens Producing enzymes that inactivate

against both gram-negative and fosfomycin

gram-negative bacteria

Bacitracin Topical gram-positive cocci Not defined

infections

PROTEIN SYNTHESIS INHIBITION

Anti-30s ribosomal subunit:

Aminoglycosides Aerobic gram-negative bacteria Drug inactivation by

aminoglycoside

(Bactericidal: Often used for empirical therapy Modifying enzyme;decreased

Irreversible in adjunct with 3rd generation Permeability; decreased influx of

Binding to cephalosporins in respiratory drug

30S) infections, meningitis and subacute

endocarditis.

Tetracyclines Rickettsiae, chlamydiae, mycoplasma, Decreased intracellular drug

(Bacteriostatic: Spirochetes, accumulation;

Bind to 30S Yersinia pestis, Brucella, Campylobacter, Ribosomal target site alteration

And block tRNA Vibrio cholerae

attachment )

Glycylglycine

(Same as Staphylococcus, Enterococcus, Active drug efflux pump

Tetracycline) Acinetobacter,E.coli

Anti-50s ribosomal subunit:

Chloramphenicol Haemophilus influenzae Drug inactivation by producing

( bacteriostatic; Pyogenic meningitis chloramphenicol acetyltransferase;

Binds to 50S and Brain abscess Altering membrane transport

interfere with Anaerobic infection

peptide bond

formation)
Macrolides

(Bacteriostatic; Streptococcus Altering ribosomal target;

binds to 50S and Haemophilus influenzae Active efflux of antibiotic

prevent translocation Mycoplasma pneumoniae

of elongated peptide)

Ketolide Community acquired Altered target;active drug efflux

(same as macrolide) pneumonia

Lincosamides S.aureus (CA-MRSA,MSSA) Altered target

(binds to 50s and

blocks peptide bond

formation )

NUCLEIC ACID SYNTHESIS INHIBITORS

DNA synthesis inhibitors:

Fluoroquinolones E.coli, Klebsiella, Enterobacter Alteration of target; poor transport

(inhibit DNA salmonella, shigella, neisseria, across cell membrane

synthesis) vibrio cholerae, Pseudomonas

Nitroimidazoles Anaerobic organisms, entamoeba, decreased drug intake; active efflux;

(Damage DNA) Giardia, Trichomonas decreased drug activation

Nitrofurantoin urinary tract infection altered drug activating system

(Damages bacterial (E.coli, Klebsiella)

DNA)

RNA synthesis inhibitors:

Rifamycins M.tuberculosis,M.leprae, Alteration of target

(Inhibits RNA Staphylococcus aureus

polymerase)
 SET 8

STERILIZATION AND DISINFECTION

STERILIZATION:

Sterilization is defined as the process by which an article, body surface or medium is freed of all living
microorganisms either in the vegetative or spore state.

DISINFECTION:

The reduction of pathogenic organisms to a level at which they no longer constitute a risk.

ASEPSIS:

It is a process where the chemical agents (called antiseptics) are applied on to the body surfaces (skin),
which kill or inhibit the microorganisms present on skin.

DECONTAMINATION (CLEANING):

It refers to the reduction of pathogenic microbial population to a level at which items are considered
as safe to handle without protective attire.

DIFFERENCES BETWEEN STERILIZATION AND DISINFECTION:

STERILIZATION DISINFECTION

● It results in reduction of at least 106 log ● It results in reduction of at least 103 log
colony-forming units of colony-forming units of
microorganisms and their spores. microorganism, but not spores.
● Destruction of all living organisms. ● Destruction of potential pathogens
● Physical or chemical agents, used on and substantial reduction of microbial
inanimate and animate objects. population.
● Physical and chemical agents - used on
inanimate objects only.
CLASSIFICATION:

⮚ GENERAL CLASSIFICATION: Based on the nature of the sterilant or disinfectant

⮚ SPAULDING'S CLASSIFICATION: Based on the degree of risk of infection involved in

the use of the patient care items and equipment

DISINFECTANTS: Depending on their efficacy

VARIOUS STERILANTS, DISINFECTANTS AND CLEANSING AGENTS

AGENTS PHYSICAL METHODS CHEMICAL METHODS

STERILANTS:
● Steam sterilizer ● Ethylene oxide (ETO) sterilizer
● Agents of sterilization (autoclave) ● Plasma sterilizer
● Dry heat sterilizer (hot
air oven)
● Filtration
● Radiation: ionizing and
non-ionizing (infrared)
● Others: Incineration,
microwave

DISINFECTANTS: No physical methods in this ● Aldehydes - glutaraldehyde,

category orthophthaldehyde, formaldehyde,
● High-level: Can kill all
Peracetic acid
microorganisms and
● Hydrogen peroxide
bacterial spores if used
in sufficient
concentration and
suitable conditions.
● Alcohols-ethyl alcohol and
● Heat-based methods:
isopropyl alcohol
Pasteurization, boiling
● Intermediate-level:
and steaming.
Can destroy all
● Ultraviolet ● Phenolics-phenol, cresol, Lysol
microorganisms but
(non-ionizing)
not bacterial spores.
radiation ● Halogens-iodine and chlorine
 ● Low-level: Destroy No physical methods in this ● Quaternary ammonium compound
vegetative bacteria and category (QAC)
enveloped viruses; ● Chlorhexidine
variable action on
non-enveloped viruses,
and fungi, but no
action on tubercle
bacilli and spores.

CLEANING: ● Automated washers ● Enzymatic solution

such as ultrasonic ● Detergent
● Agents of cleaning
washers, ● Soap (antimicrobial or plain soap)
washer-disinfector and
automated cart
washers

FACTORS INFLUENCING EFFICACY OF STERILANT OR DISINFECTANT:

⮚ The efficiency of a sterilant/disinfectant is affected by various factors.

❖ Organism load:
● As the bioburden increases, the contact time of the disinfectant also needs to be increased.
❖ Nature of organisms:
● Organisms vary greatly in their resistance to disinfectants and sterilant.
● The decreasing order of resistance of an organism to various sterilants and disinfectants is
as follows: Prions> bacterial spores >coccidian oocyst >mycobacteria non-enveloped virus>
fungi >vegetative bacteria >enveloped viruses.
❖ Concentration:
● Use of agent at optimal concentration is necessary for desired antimicrobial action.
● High concentration may corrode the material and low concentration might be less effective.
❖ Contact time:
 ● It is the period of time a disinfectant is in direct contact with the surface or item to be
disinfected
● Most crucial factor-framed by application to the surface until complete drying.
● Lower exposure time – less effective killing of microbes.
❖ Temperature:
● Activity of most agents increases with temperature.
● But inappropriately high temperatures degrade the agent.
❖ Stability:
● Some agents are unstable at in-use concentration, e.g. hypochlorite, and should be freshly
prepared each day.
❖ Local pH:
● An increase in pH improves the antimicrobial activity of some disinfectants Like
glutaraldehyde, quaternary ammonium compound or QAC
● But increase in Ph decreases the antimicrobial activity phenols, hypochlorite, and iodine.
❖ Relative humidity:
● Important factor influencing the activity of gaseous disinfectant such as ethylene oxide
(ETO)
❖ Presence of organic matter:
● Organic matter such as pus, serum, blood, and stool can interfere with the antimicrobial
activity of some disinfectants (e.g. hypochlorites and QAC)
● This can be overcome by: i) mechanically cleaning the instrument or surface/floor before it
is subjected for disinfection/sterilization and (ii) increase exposure time or concentration of
the agent
● However, few other disinfectants such as phenolics or glutaraldehyde retain their efficacy in
the presence of organic matter.
❖ Biofilm:
● Formation of biofilm is another mechanism which prevents the entry of disinfectant /
sterilant to act on the microorganisms which are embedded inside the biofilm.

PROPERTY OF AN IDEAL STERILANT/DISINFECTANT:

⮚ An ideal disinfectant / sterilant should have various properties such as:

● (i) broader microbicidal activity
 ● (ii) fast acting
● (ii) not affected by environmental factors such as organic matter
● (iv) nontoxic
● (v) compatible with surfaces/materials to which it is used
● (vi) odorless or pleasant odor
● (vii) economical and
● (viii) environmental friendly

GENERAL CLASSIFICATION:

PHYSICAL METHODS:

⮚ Different types of physical methods of sterilization are:

● Drying
● Heat
● Filtration
● Vibration
● Ultrasonic vibration

DRYING:

⮚ It removes the moisture required for bacterial growth.

⮚ But it does not affect many microbes and spores.

HEAT:

⮚ Heat is the most reliable and commonly employed method of sterilization or disinfection.
⮚ Thermal death time: This is the minimum time required to kill a suspension of organisms at a
predetermined temperature in a specific environment.
⮚ Factors influencing sterilization by heat:
● Nature of heat - dry or moist
● Temperature and time
● Number of organisms present
● Characteristics of the organism-species, strain, presence of spores
● Type of material from which the organism must be eradicated.
 ⮚ Types:
● Dry heat
● Moist heat

DRY HEAT:

⮚ It kills organisms by exposing them to very high temperatures.

⮚ Mechanism of Action: It kills the organisms by charring, denaturation of bacterial protein,
oxidative damage and by the toxic effect of elevated levels of electrolytes.

PROCESS TEMPERATURE MATERIALS THAT ARE OTHER FEATURES

AND DURATION STERILIZED

1.Flaming:

● Items held in the ● Longer ● Longer time: ● Infective

flame of Bunsen time-red Inoculating wires or materials can be
burner. hot. loops, tips of forceps dipped in
● Shorter and searing spatulas. disinfectant
time-not red ● Shorter time: Fragile before flaming
hot. items-mouth of test to prevent
tubes. splattering

2.Incineration: ● 870˚C-1200 ● Biomedical waste - ● Terminal

˚C anatomical and sterilization-dis
microbiological waste. posal

3.Hot –air oven: ● Sterilization

control:
● Electrically ● 160˚C for 2 ● Glassware like glass
heated and fitted hours syringes, petri dishes,
with a fan-even ● Cooling flasks, pipettes and test
Biological indicator:
distribution of time-2 hours tubes.
Checking for
hot air ● Surgical instruments
destruction of spores
● Fitted with like scalpel, forceps,
(106) of nontoxigenic
thermostat. etc.
strains of Clostridium
● Chemicals, such as
tetani or Bacillus
liquid paraffin, fats,
 glycerol, and glove subtilis subspecies
powder, etc. niger.
Precautions:

● No overloading
and free air Thermocouples:
circulation. Records temperature by
● Materials should potentiometer.
be dried and
paper wrapped.
● Cotton plugs Browne's tube: It

–close mouths of contains a heat

test tubes and sensitive red dye which

flasks. turns green after being

● No inflammable exposed to a certain

materials should temperature for a

be kept, except definite period of time.

silicon rubber. ● Advantages:

Non-toxic,low
operating costs,
penetrates well and
non-corrosive for
metals.

● Disadvantage:

High temperatures are

not suitable for most
materials.
 HOT –AIR OVEN

Reference: https://images.app.goo.gl/tjhPMLReqmrdRJQg7

MOIST HEAT:

⮚ It kills microorganisms at a lower temperature than dry heat.

⮚ Mechanism of action: Moist heat kills the microorganisms by denaturation and coagulation of
proteins.
⮚ Types:
● At temperature below 100˚C - Pasteurization, water bath and fractional sterilization
(inspissations).
● At 100˚C- Boiling, steaming and tyndallization (intermittent sterilization).
● At a temperature above 100˚C - Autoclave.

AT TEMPERATURE BELOW 100˚C:

PROCESS TEMPERATURE AND MATERIALS OTHER FEATURES

DURATION THAT ARE
STERILIZED

1.Pasteurization: ● Holder method: ● Beverages ● All non sporing

63˚C for 30 ● Beer pathogens are
minutes killed except
Coxiella
 ● Flash method: ● Dairy burnetii in
72˚C for 20 products-mil Holder method
seconds followed k
by rapid cooling
to 13˚C or below.

2.Water bath: ● Bacterial ● Serum

vaccines: 60˚C ● Body fluids
for 1 hour. ● Vaccines
● Serum and heat
labile body
fluids: 56˚C for 1
hour.

3.Inspissator: ● 80˚C-85˚C for 30 ● Egg based ● Substances that

minutes media-Lowe are destroyed at
● Heating an article for 3
nstein Jensen high
successive days.
medium temperatures
● Principle: In
● Serum based are sterilized by
inspissator, the first
media- this method.
exposure kills all the
Loeffler’s
vegetative forms, and
serum slope
in the intervals
between, the remaining
spores germinate into
vegetative forms,
which are then killed
on subsequent heating;

AT 100˚C:

PROCESS DURATION MATERIALS THAT OTHERS FEATURES

ARE STERILIZED
1.Boiling:

● Easily available ● 10-30 minutes ● Does not kill

but not preferred bacterial spores

2.Steaming at
Atmospheric Pressure:

● Koch’s or
Arnold’s steam
sterilizer ● 90 minutes ● Articles and ● Kills most of

● Articles on culture media vegetative forms

perforated tray that are except thermophiles

decomposed at and spores
high
temperatures of
autoclave.

3.Tyndallisation or
Intermittent
Sterilization:

● Same as
inspissator ● 20 minutes for ● Gelatin and egg, ● May not kill spores
3 consecutive serum or sugar of certain anaerobes
days containing and thermophiles
media, which
are damaged at
higher
temperatures of
autoclave.

ABOVE 100˚C:

AUTOCLAVE (STEAM STERILIZER):

⮚ Principle:
 ● Water boils when its vapor pressure equals that of the surrounding atmosphere. So, when
the atmospheric pressure is raised, the boiling temperature is also raised.
● At normal pressure, water boils at 100˚C. But when pressure inside a closed vessel
increases, the temperature at which water boils also increases.
⮚ Components:
● Steam sterilizer is a pressure chamber; it consists of a cylinder, a lid and an electrical
heater.
● Pressure chamber: It consists of:
o A large cylinder (vertical or horizontal) made up of gunmetal or stainless steel, in
which the materials to be sterilized are placed
o A steam jacket (water compartment).

● Lid: It bears the following:

o A discharge tap for the passage of air and steam
o A pressure gauge (sets the pressure at a particular level)
o A safety valve (to remove the excess steam).
● Electrical heater: It is attached to the jacket; that heats the water to produce steam.

⮚ Mechanism of Action:
● The cylinder is filled with sufficient water and the material to be sterilized is placed
inside the pressure chamber.
● The lid is closed and the electrical heater is put on.
● The safety valve is adjusted to the required pressure.
● The process follows three phases:

CONDITIONING PHASE:
● After the water boils, the steam and air mixture is allowed to escape through the discharge tap
till all the air has been displaced.
● This can be tested by passing the steam-air mixture liberated from the discharge tap into a pail
of water through a connecting rubber tube.
● When the air bubbles stop coming in the pail, it indicates that all the air has been displaced by
steam.
● The discharge tap is then closed.
 ● The steam pressure rises inside and when it reaches the desired set level [e.g. 15 pounds
(lbs) per square inch in most cases], the safety valve opens and excess steam escapes out.

EXPOSURE PHASE:
The holding period is counted from this point of time, which is about 15 minutes in most cases.

EXHAUST PHASE:
● After the holding period, the electrical heater is stopped and the autoclave is allowed to cool till
the pressure gauge indicates that the pressure inside is equal to the atmospheric pressure.
● The discharge tap is opened slowly and air is allowed to enter the autoclave.
● The lid is now opened and the sterilized materials are removed.

Schematic representation of horizontal autoclave

Reference: Essentials of Medical Microbiology, Apurba S Sastry E/1 Page.No. 34 Fig. 3.4

⮚ USES:
● Autoclave is particularly useful for media containing water that cannot be sterilized by
dry heat.
● It is the method of choice for sterilizing the following:
a. Surgical instruments
b. Culture media
c. Autoclavable plastic containers
d. Plastic tubes and pipette tips
e. Solutions and water
f. Biohazardous waste
g. Glassware (autoclave resistible)
⮚ PRECAUTIONS:
● Autoclave should not be used for sterilizing waterproof materials, such as oil and
grease or dry materials, such as glove powder.
● Materials are loaded in, such a way that it allows efficient steam penetration. The
chamber should not be overfilled
● Material should not touch the sides or top of the chamber.
● The clean items and the wastes should be autoclaved separately.
● Polyethylene trays should not be used as they may melt and cause damage to the
autoclave.
⮚ ADVANTAGES:
● It is low cost than ETO and plasma sterilizers
● Sterilization cycles are fast compared to ETO sterilizers
● It is nontoxic and leaves no by-product behind (unlike ETO).
⮚ DISADVANTAGES:
● Heat can damage acrylics and styrene, PVC materials and corrode some metals.
● High temperature for a prolonged time can harm or shorten the life of some instruments.
● Moisture also can adversely affect the electronics and can cloud sensitive materials and
leave watermark stains on them.
⮚ STERILIZATION CONTROL:
The effectiveness of the sterilization done by autoclave can be monitored by:
● Biological indicator:
o Spores of Geobacillus stearothermophilus are the best indicator, because they are
resistant to steaming.
o Their spores are killed in 12 minutes at 121˚C.
● Chemical indicators:
o External pack control, e.g. autoclave tape
o Bowie-Dick test
o Internal pack control.
● Physical indicators: For example, digital displays on the equipment displaying temperature,
time and pressure
FLASH STERILIZATION:

⮚ Flash sterilization is a modification of conventional steam sterilization, designed to be used at

emergency or during unplanned procedures.
⮚ It involves fast sterilization (134°C for 3-10 minutes) of surgical instruments in an unwrapped
condition in steam sterilizers located close to the operation theatre .
⮚ This practice should only be restricted for emergency situations, e.g. instruments have been
contaminated and need to be replaced in the surgical field immediately.
⮚ It is not suitable for porous and cannulated instruments, implants and suction tubing
⮚ As the instruments are not packed, they remain wet following sterilization; therefore, there is a
high-risk.

FILTRATION:

⮚ Filtration is an excellent way to remove the microbial population in solutions of heat-labile

materials
⮚ Materials sterilized: Vaccine, antibiotics, toxin, serum and sugar solution as well as for purification
of air.
⮚ Types of Filters:
o Depth filters
o Membrane filters.

DEPTH FILTERS:

⮚ Mechanism of Action: They are porous filters that retain particles throughout the depth of
the filter, and are composed of random mats of metallic, polymeric, or inorganic materials.
⮚ These fillers rely on the density and thickness of the filler to trap particles rather than the pore
size.
⮚ Commonly used when the fluid to be filtered contains a high load of particles but are not
suitable for filtration of solution containing bacteria.
MEMBRANE FILTERS:

⮚ Mechanism of Action: They are porous and retain all the particles on the surface that are
larger than their pore size
⮚ Membrane fillers are made up of cellulose acetate, cellulose nitrate, polycarbonate,
polyvinylidene fluoride, or other synthetic materials.
⮚ Pore size: Average pore diameter of
o 0.22 µm - removes most of die bacteria but not viruses
o 0.45 µm - retain coliform bacteria in water microbiology
o 0.8 µm filters remove airborne microorganisms
⮚ Materials sterilized:
o Liquids: Sera, sugar and antibiotic solutions, separation of toxins and bacteriophages
from bacteria, to obtain bacteria free filtrates of clinical samples for virus isolation,
purification of water.
o Air:

⮚ Air filtration:
o Surgical masks: Filters microbes
o Air filter used in biological safety cabinets and laminar airflow systems
▪ HEPA fillers (High-efficiency particulate air filters): Removes 99.97% of
particles that have a size of 03 µm or more.
▪ ULPA filters (Ultra-low particulate/ penetration air): Can remove from the air
at least 99.999% of dust, pollen, mold, bacteria and any airborne particles with a
size of 0.12µm or larger.
⮚ Sterilization control: For membrane filters -Brevundimonas diminuta and Serratia
marcescens.
 RADIATION:

TYPE MECHANISM OF MATERIALS OTHER FEATURES

ACTION STERILIZED

1.Ionizing
Radiation:

● X-rays,
● Breakage of DNA ● Disposable plastic ● Advantages : high
gamma
supplies, such as penetrating power,
rays (from
disposable rubber or rapidity of action,
Cobalt 60
plastic syringes, and temperature is
source),
infusion sets and not raised
and cosmic
cadieiers. ● High penetrability
rays.
● Catgut sutures, bone ● Not effective against
and tissue grafts and viruses.
adhesive dressings as ● Sterilization
well as antibiotics control: Efficacy
and hormones. tested by using
● Irradiation of food Bacillus pumilus.
(permitted in some
countries)

2.Non-ionizing
Radiation:

● Infrared
● Causes ● Clean surfaces in ● Bacteria and viruses
and
destruction of operation theaters, are more easily
ultraviolet
nucleic acid laminar flow hoods killed.
radiations.
through induction as well as for water ● Not useful in killing
of thymine treatment spores.
dimers. ● Quite lethal but does
not penetrate glass,
● Recommended
dirt films, water.
dose is 250-300
● UV radiation burns
skin (erythema) and
 nm wavelength, damages eyes
for 30 minutes (keratoconjunctivitis)

3.Microwave: ● Microwaves are ● Small size:Soft ● Must only be used

radio-frequency contact lenses, dental with products that are
waves, which are instruments, compatible.
usually used at a dentures, and urinary
frequency of 2450 catheters (for
MHz. intermittent
● They produce self-catheterization)
friction of water ● Large size: Disposal
molecules which of biomedical waste
generates heat. (for plastic infectious
waste).

CHEMICAL METHODS:

⮚ Chemical agents used for sterilization are also known as disinfectants and the process is known as
disinfection.
⮚ Different types of chemical methods are:
● Alcohol: Ethyl alcohol, isopropyl alcohol
● Aldehydes: Formaldehyde, glutaraldehyde, Ortho-phthalaldehyde
● Phenolic compounds: Cresol, lysol chlorhexidine, chloroxylenol, hexachlorophene.
● Halogens: Chlorine, Iodine, iodophors
● Oxidizing agents: Hydrogen peroxide, per acetic acid
● Salts: Mercuric chloride, copper salts
● Surface active agents: Quaternary ammonium compounds and soaps
● Chlorhexidine gluconate:
● Dyes: Aniline dyes and acridine dyes
● Gas sterilization:
o Low temperature steam formaldehyde
o Ethylene oxide (ETO)
o Betapropiolactone (BPL)
o Plasma sterilization
 ALCOHOLS:

⮚ Most widely used disinfectants and antiseptics.

⮚ Mechanism of action: They act by denaturing proteins and possibly by dissolving membrane
lipids. But no action on spores
⮚ Uses:
● Methyl alcohol: Cleaning cabinets and incubators. Effective against fungal spores but
vapour is toxic and inflammable.
● Ethyl alcohol: Surgical spirit (70%) in hand rubs as antiseptics.
● Isopropyl alcohol: Clinical thermometers and small instruments are disinfected by
soaking in it for 10- 15 minutes.
⮚ In 70-80 % concentration - bactericidal and fungicidal but not sporicidal; some enveloped
viruses (HIV) are also destroyed.

ALDEHYDES:

⮚ Mechanism of action: They combine with nucleic acids, proteins and inactivate them,
probably by cross linking and alkylating the molecules.
⮚ Uses:

ALDEHYDE MATERIALS STERILIZED OTHER FEATURES

1.Formaldehyde:

● Formalin or ● Preservation of anatomical ● Toxic and irritant when

formol-40% specimen inhaled
formaldehyde ● Formaldehyde gas is used for ● Corrosive to the metals.
● Active against fumigation of closed areas, such
amino as operation theatres
group-bactericidal, ● Heat-sensitive catheters
sporicidal and ● Preparation of toxoid from
virucidal. toxin

2.Gluteraldehyde:
 ● 2% concentration ● Endoscopes, bronchoscopes, ● Active in the presence of
(2% Cidex) rubber anesthetic organic matter
● Effective against tubes,polythene tubing, plastic ● Disinfects objects within 20
tubercle bacilli, endotracheal tubes, minutes but may require as
fungi and viruses. cystoscopes. long as 10-14 hours to kill
spores.
● Fogging and cleaning of floor
● Available in inactive form;
and surfaces of critical areas
has to be activated by
such as operation theatre (e.g.
alkalinization before use.
Bacillocid Extra).
● Once activated, it remains
active only for 14 days.

3.Ortho-phthalaldehyde:

● 0.55% solution. ● Endoscopes and cystoscopes. ● Does not require activation,

● Low vapour property,
● Better odor
● More stable during storage
● More mycobactericidal
activity.

PHENOLIC COMPOUNDS:

⮚ The phenol and its derivatives (called phenolics) are produced by distillation of coal tar between
temperatures of l 70' C and 270'C.
⮚ Mechanism of action: Act by denaturing proteins and disrupting cell membranes, inactivation
of membrane-bound oxidases and dehydrogenases leading to lysis and death of organisms.
⮚ Used as disinfectants:
● Cresol, xylenol, Lysol and ortho-phenylphenol
● Retain activity in presence of organic manure.
● Toxic and irritant to skin
⮚ Used as antiseptics:
● More active against gram-positive than gram-negative bacteria.
● Chlorhexidine: Active ingredient of savlon (chlorhexidine and cetrimide).
 ● Chloroxylenol: Active ingredient of denol.
● Hexachlorophene: Cause brain damage-so use as antiseptic is restricted. Indicated only in
response to a staphylococcal outbreak.

HALOGENS:

⮚ Iodine and chlorine have antimicrobial activity.

⮚ Exist in a free state, and form salt with sodium and most other metals.

IODINE:

⮚ Bactericidal, with moderate action against spores. Active against the tubercle bacteria and
viruses.
⮚ Mechanism of action: Oxidizing cell constituents and iodinating cell proteins. At higher
concentrations, it may even kill some spores.
⮚ Tincture of iodine: Effective antiseptic, but can cause skin allergy and a yellow stain is left.
⮚ Iodophor: Preoperative antiseptics as well as disinfectants in laboratories.

CHLORINE:

⮚ Various preparations: chlorine gas, sodium hypochlorite (household bleach), calcium

hypochlorite (bleaching powder).
⮚ Mechanism of action: All preparations yield hypochlorous acid (HCIO), which causes
oxidation of cellular materials and destruction of vegetative bacteria and fungi, but not spores.
⮚ Uses:
● Disinfecting municipal water supplies and swimming pools
● In the dairy and food industries
● As laboratory disinfectant
● As bleaching agent, to remove stain from clothes
⮚ Disadvantages:
● Not active against Giardia and Cryptosporidium
● Corrosive
● Organic matter interferes with its action
● Form carcinogenic trihalomethanes with organic compounds
 ● Daily preparation required as sodium hypochlorite is unstable and disintegrates

OXIDIZING AGENTS:

OXIDIZING AGENT MECHANISM OF MATERIALS OTHER FEATURES

ACTION STERILIZED

1.Hydrogen peroxide:

● 3-6%-effective
against most
● It breaks off ● Disinfect ● Bactericidal and
organisms
H2O2 and ventilator, soft active against
● Catalase
liberates toxic contact lenses, and biofilms and
producing
free hydroxyl tonometer microorganisms
organisms and
radicals - biprisms. within it
spores - (10%) of
active ● Vaporized form ● Advantages: Does
H2O2
ingredients - used in plasma not coagulate
attack sterilization blood, does not fix
membrane, tissues to surfaces,
lipid, DNA, enhances removal
and other of organic matter
cellular from equipment, is
components. less toxic
● Inhibits DNA environmentally
synthesis, safe, neither
protein carcinogenic or
synthesis mutagenic.

● Disadvantages:
Expensive,
incompatible with
some
materials,produces
 eye irritation and
corneal damage.

2.Peracetic acid:

● Concentrations ● It denatures ● Disinfect ● Particularly against

less than 1 % are proteins, hemodialyzers antibiotic-resistant
sporicidal even at disrupts cell ● Used in plasma bacteria such as
low temperature. wall sterilization methicillin-resistant
permeability ● Sterilizing Staphylococcus
and oxidizes endoscope aureus,
sulfhydryl and vancomycin-resista
sulfur bonds in nt Enterococcus
proteins, and Clostridium
enzymes, and difficile.
other ● Corrode steel, iron,
metabolites. copper, brass and
bronze.

HEAVY METAL SALTS:

⮚ Salts of heavy metals, such as mercury, silver, arsenic, zinc and copper.
⮚ Mechanism of action: Heavy metals combine with bacterial cell proteins, often with their sulfhydryl
groups, and inactivate them.
⮚ They are more bacteriostatic than bactericidal
⮚ Uses:
● Silver sulfadiazine - On burns surfaces
● Si1ver nitrate (1%) solution - Eyes of infants to prevent ophthalmia neonatorum. Replaced by
erythromycin.
● Copper sulphate - effective fungicide (algicide) in lakes and swimming pools.
● Thiomersal (merthiolate) - preservative in vaccines, sera and other immunoglobulin
preparations.
SURFACE ACTIVE AGENTS:

⮚ These are substances that alter the energy relationship at interfaces, producing a reduction in
surface tension.
⮚ They are widely used as wetting agents, detergents and emulsifiers.
⮚ Mechanism of action: These act on the phosphate groups of the cell membrane and also enter the
cell. The membrane loses its semi-permeability and the cell proteins are denatured.
⮚ They act on bacteria, but not on spores, tubercle bacilli and most viruses.
⮚ They are classified into:
● Anionic surfactants:
o Common soaps - Strong detergent but weak antimicrobial properties.
o These agents are most active with acidic pH.
● Cationic surfactants:
o Quaternary ammonium compounds like Alkyl trimethyl ammonium salts, Acetyl
trimethyl ammonium bromide
o Disrupt microbial membranes and may also denature proteins.
o Kill most bacteria (gram-positives are better killed than gram-negatives) but not
M. tuberculosis or spores.
o Soaps prepared from saturated fatty acids-more effective against Gram-negative
bacilli and those prepared from unsaturated fatty acids (oleic acid) have greater
action against Gram-positive and the Neisseria group of organisms.
o They are stable, and nontoxic to skin,
o Inactivated by acidic pH, organic matter, hard water and soap.
● Non-ionic surfactants:
● Amphoteric surfactants:
o Tego compounds
o Possess detergent properties of anionic compounds and antimicrobial activity of
cationic compounds.
o Active over a wide range of pH but their activity is reduced in presence of organic
matter
o Antiseptics in dental practice - known to cause allergic reactions.
 CHLORHEXIDINE GLUCONATE:

⮚ Mechanism of action: CHG is a biguanide disinfectant, acts by disruption of cytoplasmic

membrane.
⮚ Uses: in antiseptic products, at various concentrations
● Hand hygiene product: Hand rub (0.5%), hand wash (4%) (e.g. Microshield, a commercial
product)
● Mouthwash (0.1-0.2%)
● Body wash solutions (used before surgery)
● Skin disinfectant before surgery (2%)
● Antiseptic: Wound cleaning - commercially available as savlon-CHG 0.3%,Cetrimide and
isopropyl alcohol.
⮚ Advantages: Residual activity and less irritant
⮚ Disadvantages: Slow activity, pH dependent activity, reduced activity in presence of organic
matter, dermatitis in case of prolonged use.

DYES:

DYE MECHANISM OF MATERIALS TO BE OTHER FEATURES

ACTION STERILIZED

1.Aniline dyes:

● Crystal violet, ● Interfere with the ● In laboratory as ● They are

gentian violet, synthesis of selective agents in non-toxic and
brilliant green peptidoglycan culture media (e.g. non-irritant to the
and malachite component of the malachite green in tissues.
green. cell wall. Lowenstein Jensen ● Their activity is
● More active medium, which is a reduced in
against selective medium presence of
gram-positive used for isolation of organic material,
bacteria than Mycobacterium. such as pus.
gram-negative Tuberculosis)
bacteria and
have no
 activity against
M.
tuberculosis.

2.Acriline dyes:

● Acriflavine, ● Interfere with the ● Skin and wound ● Not affected

euflavine, synthesis of antiseptics. presence of
proflavine and nucleic acids and organic matter
aminacrine. proteins in ● They are also
bacterial cells. more active
against
gram-positive
bacteria than
gram-negative
bacteria

GASEOUS STERILIZATION:

LOW TEMPERATURE STEAM FORMALDEHYDE:

⮚ Use: It was widely used for fumigation of operation theaters, wards and laboratories.
⮚ No longer preferred - irritant and toxic when inhaled.

BETAPROPIOLACTONE (BPL):

⮚ BPL gas (0.2%) is active against all microorganisms including spores

⮚ Use: Inactivation of vaccines.
⮚ Low penetrating power and is carcinogenic, hence not used for fumigation.

ETHYLENE OXIDE:

⮚ This is a colourless liquid with a boiling point of 10.7˚C and highly penetrating at normal
temperature pressure.
 ⮚ Mechanism of action:
● It acts by alkylating the amino, carboxyl, hydroxyl and sulfhydryl groups in protein
molecules within the microbes and spores.
● It also reacts with DNA and RNA (rendering them virucidal).
⮚ Use: Sterilization of many heat sensitive items, such as disposable plastic petri dishes and syringes,
heart-lung machine components, sutures, catheters, respirators and dental equipment.
⮚ Disadvantages: Potentially toxic to human beings, causing mutagenicity and carcinogenicity,
unsuitable for fumigating rooms because of its explosive property
⮚ Sterilization condition: Three factors influencing the rate of sterilization
● EtO concentration
● Humidity
● Temperature
⮚ Sterilization control: Bacillus globigii is used as a biological indicator to check the effectiveness
of sterilization.

PLASMA STERILIZATION:

⮚ Plasma sterilization is a recently introduced sterilization method; increasingly used nowadays .

⮚ Principle:
● Plasma refers to a gaseous state consisting of ions. photons and free electrons and
neutral uncharged particles (such as O and OH).
● These active agents present in plasma such as photons of ultraviolet rays and radicals (e.g.
O and OH) are capable of killing microorganisms and spores efficiently.
⮚ Plasma sterilizers:
● It is a special device used to create the plasma state (commercial brands, such as Sterrad and
Plazlyte).
● It has the following steps:
● Vacuum:
o Chamber is evacuated and a uniform vacuum is maintained inside the chamber.
● Chemical sterilants:
o Next step is injection of chemical hydrogen peroxide (H2O2) solution from a
cassette, which gets vaporized in the sterilization chamber to a concentration of 6
mg/L .
 o The H2O2 vapor diffuses through the chamber (50 minutes), phase exposes all
surfaces of the load to the sterilant.
o Low temperature is maintained 37-44°C throughout the cycle to maintain the
integrity of heat labile items.
● Gas plasma:
o In the next step, an electrical field is applied to the chamber to create a gas plasma.
o H2O2 breaks into radicals such as hydroxyl (OH-) and hydroperoxyl (HO2) which
initiate microbicidal action, which subsequently interact with essential cell
components (e.g.enzymes, nucleic acids)
● Final step:
o The excess the cycle (e.g. water vapor, oxygen) are nontoxic and therefore, there is
no need of an additional aeration step
⮚ Cycle duration: It has a cycle time of 75 min. The newer versions have shorter cycles of 52 min
and 24 min
⮚ Uses: Sterilization of surgical instruments, arthroscopes, ureteroscopes, micro and vascular scopes,
spine sets and laparoscopy.
⮚ Sterilization control: Tested by using Bacillus stearothermophilus, Bacillus subtilis . Physical and
chemical indicators are the same as discussed for the autoclave.

SPAULDING'S CLASSIFICATION OF MEDICAL DEVICES:

⮚ Earle H. Spaulding devised a rational approach to classify the patient-care items and equipment of a
hospital into four categories according to the degree of risk for infection involved in use of the
items.

MEDICAL DEFINITION EXAMPLES RECOMMENDED

DEVICE STERILIZATION OR
DISINFECTION

1.Critical devices Enter a normally Surgical instruments,cardiac and Heat based sterilization,
sterile site urinary catheters, implants, eye and chemical sterilant or
dental instruments high level disinfectant
2.Semi-critical Comes in Respiratory therapy equipments, High-level disinfectant
devices contact with anesthesia equipments, endoscopes,
mucus laryngoscope,
membranes or rectal/vaginal/esophageal probes
minor skin
breaches

3.Non-critical Comes in BP cuff, ECG electrodes, bedpans, Intermediate-level or

devices contact with crutches, stethoscope. thermometer low-level disinfectant
intact skin

4.Non-critical Less direct Surfaces of medical equipments, Low-level disinfectant

environmental contact with examination table, computers
surfaces patients

DISINFECTION OF OPERATION THEATRE:

⮚ Environmental cleaning in operation theatres minimizes patient’s HCW’s exposure to potentially
infectious microorganisms.
⮚ Surface Disinfection:
● Cleaning should be performed first with a cleansing agent.
● Followed by disinfection using an aldehyde-based disinfectant
⮚ Disinfection of OT is carried out in the following situations:
● First cleaning of the day (before cases begin)
● In between cases (cleaning 3-4 feet perimeter around the OT table
● Terminal cleaning of OT after the last case.
● Detailed wash-down of the OT complex once a week.
● During renovation or construction of OT or nearby places.
⮚ Fogging:
● Aerial disinfection-spraying of disinfectant.
● Fogger machine is used to spray glutaraldehyde, H2O2 or QAC based product
● The procedure takes 1-2 hours. The OT should be closed and the personnel should be
vacated.
 ● Indication: Routine periodic fogging is not recommended, but is indicated only when
any outbreak of infection is suspected or any change in infection control practice
implemented or during renovation or construction of OT or nearby places.

TESTING OF DISINFECTANT:

PHENOL COEFFICIENT (RIDEAL WALKER) TEST:

⮚ Phenol coefficient is determined by the dilution of the disinfectant in question which

sterilizes/disinfects the suspension of Salmonella Typhi in a given time divided by the dilution of
phenol which sterilizes/ disinfects the suspension in the same time
⮚ If the phenol coefficient is more than 1, the test disinfectant is said to be more effective than
phenol.
⮚ The drawbacks of Rideal Walker test are:
● Only the phenolic compounds can be assessed
● It does not assess the ability of the disinfectant to act in presence of organic matter.

CHICK MARTIN TEST:

⮚ It is a modification of Rideal and walker test

⮚ Disinfectants act in the presence of organic matter (e.g. dried yeast, feces, etc.) to simulate the
natural conditions.

CAPACITY (KELSEY-SYKES) TEST:

⮚ It tests the capacity of a disinfect to retain its activity when repeatedly used microbiologically
(i.e. when the microbiological load keeps increasing).

IN-USE (KELSEY AND MAURER) TEST :

⮚ It determines whether the chosen disinfectant is effective for actual use in hospital practice.
 ⮚ The efficiency of a new disinfectant is determined by its ability to inactivate a known number of
standard strain pathogenic Staphylococcus on a given surface within a certain time.

TEST FOR STERILANTS (INDICATORS):

⮚ Terminal cleaning of OT after the last case The efficacy of sterilizers can be assessed by using
physical, chemical and biological indicators.

PHYSICAL INDICATOR:

⮚ These are the digital displays of the sterilizer equipment showing parameters such as
temperature, pressure, time etc

CHEMICAL INDICATOR:

⮚ They use heat or chemical indicators that undergo a color change if the sterilization parameter
(eg: time, steam quality and temperature) for which it is issued is achieved.Common types used
are:
● Class I: Also called as exposure indicator or external pack control. They are used on the
external surface of each pack, to indicate that the pack has been directly exposed to the
sterilant. However, it does not assure sterility
● Class II: It is called as Bowie- Dick test or as equipment control; i.e. it checks the
efficacy of air removal, air leaks and steam penetration and ensures that the steam
sterilizer is functioning well.
● Class III and IV: Also called as internal pack control indicator. It is placed inside the
pack and therefore ensures whether the critical parameters such as time, steam quality
and temperature are attained inside the pack or not.

BIOLOGICAL INDICATORS:

⮚ It is the most reliable indicator as it uses the bacterial spores to check the effectiveness of the
sterilization.
⮚ The spores are highly resistant and only destroyed when the effective condition is achieved.
 ● Geobacillus stearothermophilus for steam sterilizer and gas plasma (hydrogen peroxide)
and liquid acetic acid sterilizer.
● Bacillus atrophaeus for ethylene oxide sterilizer and dry heat sterilizer (hot air oven).
⮚ Spores containing vials are incubated. Depending upon the incubators used, the result is
obtained in 24 min to 48 hours.
 SET 9

LOUIS PASTUER
● Father of microbiology
● Disproved the ‘Theory of spontaneous generation’ and proved ‘Theory of biogenesis’ by using
swan necked flask.
● Proposed principles of fermentation for preservation of food, and said that undesirable
microorganisms caused spoilage of food.
● Introduced methods of pasteurization of milk.
● Introduced sterilization techniques and developed steam steriliser, hot air oven and autoclave.
● Stated the importance of cotton wool plugs for protection of culture media from aerial
contamination.
● He differentiated between aerobic and anaerobic bacteria and coined the term ‘Anaerobic’.
● He worked on ‘Pebrine’, a silk-worm disease caused by a protozoan, and showed that infection
can be controlled by choosing worms free from the parasite for breeding.
● He gave the term ‘Vaccine’ (vacca=cow), in honour of Edward Jenner’s cowpox vaccine, to
various materials used to induce active immunity.
● Developed process of attenuation during his work on ‘Chicken cholera’ in fowls.
● He showed that anthrax disease in cattle and sheep is caused by a bacteria and discovered a ‘live
attenuated’ vaccine for anthrax by incubating the bacteria at 40-42°C.
● He also developed vaccines for cholera in fowls and rabies.
● Postulated Germ theory of disease and stated that disease cannot be caused by bad air or vapor,
but produced by microorganisms present in air.
● Introduced liquid media concept and used nutrient broth to grow microorganisms.
● He was the founder of Pasteur Institute, Paris-for mass anti-rabies treatment.
ROBERT KOCH
● Father of practical bacteriology
● Introduced solid media and used agar as solidifying agent for culture of bacteria
● Introduced methods for isolation of bacteria in pure culture
● Discovered bacteria such as anthrax bacilli, tubercle bacilli (Koch’s bacilli), cholera bacilli
● Described hanging drop method for testing motility
● Introduced staining technique by using aniline dye
● Koch’s phenomenon: Guinea pigs already infected with tubercle bacillus developed
hypersensitivity when injected with tubercle bacilli or it’s protein.

HENLE-KOCH’S POSTULATES:

Microorganisms can be accepted as the causative agent of an infectious disease only if the
following four criteria are fulfilled:
● The microorganism should be constantly associated with the lesions of the disease
● It should be possible to isolate the organism in pure culture from the lesion of the
disease.
● The same disease must result when the isolated microorganism is inoculated into a
suitable laboratory animal
● It should be possible to re-isolate the organism in pure culture from the lesion produced
in experimental animals.
● Other criteria introduced was that antibody to the causative organism should be
demonstrable in the patient’s serum.

Exception to Koch postulates:

Some bacteria do not satisfy one or more of the four criteria of Koch’s postulates. They are:
● Mycobacterium leprae and Treponema pallidum. They cannot be grown in vitro but can be
maintained in experimental animals.
● Neisseria gonorrhoea can be grown in vitro but there is no animal
model.
JOSEPH LISTER:
● Father of antiseptic surgery
● Concluded that sepsis may be due to microbial growth derived from the atmosphere
● Prevented postoperative sepsis by introducing antiseptic techniques
● Used disinfectants such as diluted carbolic acid during surgery to sterilize the instruments and
to clean the wounds.
● It saved millions of lives from death due to wound infections
 SET 10

CAPSULE
⮚ Amorphous viscid material outside cell wall – glycocalyx

● Most of the bacterial capsules are polysaccharide in nature, except in Bacillus anthracis where ít
is polypeptide in nature.

FUNCTIONS:

❖ Bacterial Virulence: Contributes to virulence by the following properties

o Protects from phagocytosis
o Prevents complement mediated cell lysis
o Prevents dying out (Desiccation)
o Protects from action of lysozyme and bacteriophages
o Capsules of some organisms are toxic causes abscess e.g. Bacteroides fragilis
o Help in biofilm formation (living ecosystems made of millions of adherent bacterial
cells within self-produced matrices) and adhesion
 ❖ As Vaccines:
o Some contain antigenic and anticapsular antibodies, hence capsular antigens are used
as potential vaccine candidates
o Capsular vaccines are available for bacteria, such as pneumococcus, meningococcus
and Haemophilus influenzae serotype-b

DEMONSTRATION OF CAPSULE:
❖ Negative Staining:
➢ By Indian ink and Nigrosin ink.
➢ Capsule appears as a clear refractile halo around the bacteria; where as both the
bacteria and the background appear black

❖ M’Faydean capsule stain:

➢ Staining with polychrome methylene blue stain for demonstration of Bacillus
anthracis capsule.
❖ Serological Test:
➢ Since capsule are antigenic demonstrated by specific anticapsular serum
➢ Quellung reaction:
➔ Capsular serotypes of Streptococcus pneumoniae can be detected by
adding antisera mixed with methylene blue.
➔ Capsule becomes swollen, refractile and delineated.

➢ Capsular antigen:

➔ It can be detected in the sample (e.g. CSF) by latex agglutination test by

using specific anticapsular antibodies coated on latex particles.
➔ This is available for pneumococcus, Cryptococcus, Haemophilus
influenzae and meningococcus.
 SET 11
L-FORMS OF BACTERIA

⮚ Cell wall deficient bacterial forms.

⮚ Discovered by E klieneberger while studying Streptobacillus moniliformis.
⮚ When bacteria lose their cell wall they become spherical, irrespective of shape.
⮚ It could happen spontaneously, or after exposure to penicillin or lysosome (agents that hinder
cell wall synthesis).
⮚ Play a major role in persistence of pyelonephritis and other chronic infections.

TYPES:
1. UNSTABLE FORMS:
● These are species that lose their cell wall in presence of penicillin or other inducing agents.
● Mechanism of resistance against penicillin.
● Can revert back to original morphology, once penicillin is removed.
I. Protoplasts:
✔ Gram positive bacteria
✔ Cell wall completely removed

II. Spheroplasts:
✔ Gram negative bacteria
✔ Cell wall partially removed

2. STABLE FORMS:
● These are species where the aberrant form (cell wall deficient forms)becomes permanent
feature of the strain and is retained in subcultures
● Example: Mycoplasmas lack cell walls permanently.
● It is hinted that mycoplasmas can be stable forms of L-forms.
● However, genetic, antigenic factors are not in favor of this hypothesis.
 SET 12
CULTURE MEDIA
ENRICHED MEDIA:

❖ Basal medium added with additional nutrients like blood, serum, egg it is called enriched media.
❖ Support growth of both fastidious and non - fastidious organisms / bacteria.

BLOOD AGAR:
● 5-10% sheep blood is added to molten nutrient agar (Peptone water + meat extract + 2%
agar) at 45o C.
● Supports the growth of both aerobic bacteria, anaerobic bacteria and fungi.
● Vitamin K, hemin and cysteine supplements enhance the growth of anaerobic bacteria.
● Tests the hemolytic property of bacteria - Partial/ α-hemolysis, Complete/ β-hemolysis
and γ-no hemolysis.
● Most widely used medium.

Reference: Complete Microbiology for MBBS by CP Baveja E/7 Page No. 739 Fig 49.1.7

CHOCOLATE AGAR:
● It is the heated blood agar, by adding 5-10% sheep blood to molten nutrient agar at 70o C
so that RBC content will be lysed and released which changes the colour of the medium
to brown.
● More nutritious than blood agar and even supports growth of Haemophilus influenzae,
Neisseria gonorrhoea, N. Meningitidis and Pneumococcus.
LOEFFLER’S SERUM SLOPE:
● Contain serum used for identifying Corynebacterium diphtheriae.

BLOOD CULTURE MEDIA:

● Used for isolating microorganisms from blood.
● Conventional or Automated blood culture media.

ENRICHMENT BROTH:

❖ Liquid media with inhibitory agents, which selectively allows growth of an organism while
inhibiting others, that is, commensals usually.
❖ Important in isolating pathogens from normal flora containing specimens -Stool and Sputum.
❖ Examples:
● Gram-negative broth - Shigella
● Selenite F broth- Shigella
● Alkaline peptone water (AWP)- Vibrio cholerae
● Tetrathionate broth-Salmonella typhi –inhibits coliforms.

SELECTIVE MEDIA:

❖ Solid media- contain inhibitory substances which inhibit normal flora and allow pathogens to
grow and form colonies in a specimen.
● Lowenstein-Jensen(LJ) medium: Mycobacterium tuberculosis isolation
o Composition: Mineral salts, asparagine, glycerol, malachite green and hen’s egg.
o Malachite green prevents the growth of other bacteria.
o It is sterilised by inspissation.
● Deoxycholate citrate agar (DCA): Salmonella & Shigella (enteric pathogens) from
stool.
● Xylose lysine deoxycholate(XLD): Salmonella & Shigella from stool.
● Thiosulfate citrate bile salt sucrose agar (TCBS): Vibrio species.
o Inhibits gut flora growth as it is alkaline.
 o Also acts as an indicator medium- Sucrose fermenters appear yellow and
non-sucrose fermenters appear green.
● Potassium tellurite agar (PTA)-McLeod’s medium: Corynebacterium diphtheriae.
● Thayer-Martin Medium: N. Gonorrhoeae
o It has antibiotics (vancomycin 3.0 mg, colistin 7.5 mg and nystatin 12.5 units per
mL of agar) in chocolate agar.
o To isolate N. Gonorrhoea from urethral and endocervical swabs by inhibiting the
growth of commensals.
o Incubated in the atmosphere with 3-10% CO2.

Reference: Complete Microbiology for MBBS, CP Baveja E/7 Page No. 32 Fig. 2.4.4, 2.4.5

DIFFERENTIAL MEDIA:

❖ This media differentiate two groups of bacteria by using an indicator, which changes the colour
of a particular group of colonies but not the other.
MACCONKEY AGAR:
● Enteric gram-negative bacteria isolation.
● Differential & low selective medium.
● Differentiates into lactose fermenters - LF (Escherichia coli & Klebsiella)- pink
colonies & non lactose fermenters - (NLF) (Shigella & Salmonella)- colourless
 colonies.
● Composed of peptone, lactose, agar and neutral red (indicator) & taurocholate.
● Combination of blood agar and MacConkey agar used too.

CYSTEINE LACTOSE ELECTROLYTE DEFICIENT (CLED) AGAR:

● Used to differentiate between LF & NLF.
● For processing urine specimens.
● Alternative to blood agar & MacConkey agar combination

Reference: Ananthanarayan and Paniker's Textbook of Microbiology E/11 Page No. 40 Fig. 4.5,4.6,
Page No. 41 Fig. 4.8

WILSON- BLAIR MEDIUM:

● Used to isolate lactose fermenting salmonellae.

● Sulphite is the indicator which is reduced by Salmonella typhi to sulphide to give a black
metallic sheen on the colony.
TRANSPORT MEDIA:

❖ Used for transport of clinical specimen when:

● It contains delicate organism
● Delay in transporting specimens to the laboratory

❖ Bacteria do not multiply in this media but only remain viable.

ORGANISM TRANSPORT

Neisseria ● Amies medium & Stuart’s medium

(reducing agent – thioglycolate, and
charcoal)

Vibrio cholerae ● VR (Venkataram-Ramakrishnan)

medium
● Autoclaved sea water
● Cary Blair medium

Shigella, Salmonella ● Buffered glycerol saline

● Cary Blair medium

ANAEROBIC MEDIA:

❖ Contain reducing substances - create lower redox potential

❖ Facilitate growth of obligate anaerobes- Clostridium
ROBERTSON’S COOKED MEAT BROTH:
➔ Contain chopped meat particles (beef heart) and, thioglycolate which provide
Glutathione (reducing agent) and, amino acids & Unsaturated fatty acids, respectively.
➔ It also contains dextrose, vitamin K, yeast extract and hemin.
➔ Supports the growth of both spore-forming and non-spore forming obligate anaerobes.
➔ Use: Maintenance of shock cultures.

OTHER ANAEROBIC MEDIA:

➔ Thioglycollate broth:
o Contains yeast extract, casitone, sodium chloride, L-cystine, thioglycolic acid,
 agar, methylene blue and deionised water at final pH of 7.2
o Supports the growth of anaerobes, aerobes, microaerophiles and facultative
anaerobes
➔ Anaerobic blood agar
➔ Neomycin blood agar
➔ Egg yolk agar
➔ Phenyl ethyl agar
➔ Bacteroides bile esculin (BEE) agar
➔ Brain heart infusion (BHIS) agar with Vitamin K and hemin supplements.

BLOOD CULTURE MEDIA:

❖ These are a type of enriched media used to isolate fastidious blood pathogens which are
usually in lesser quantity.
❖ There are two types of Blood culture media:
● Conventional blood culture media
● Automated blood culture media

CONVENTIONAL BLOOD CULTURE MEDIA:

● Monophasic medium: Brain-heart infusion broth
o Highly nutritious, buffered fluid culture prepared by non-enzymatic infusion
from calf brain and cow heart, often with added peptone and dextrose.
● Biphasic medium: Liquid phase containing BHI broth and a solid agar slope made of
BHI agar.
● Recovery of organisms is done by mixing blood with broth.
● Subcultures made from Monophasic BHI broth - have higher risk of contamination due
to opening of bottle cap when making subcultures every time.
● Subcultures made from Biphasic BHI broth - have lower risk of contamination.

AUTOMATED BLOOD CULTURE MEDIA:

● Continuous automated monitoring: Incubated bottles tilted every 10 minutes and
monitored for microbial growth every 10 minutes by the instrument which gives a signal
(beep or colour change) confirming it.
● Composition: Tryptic soy broth/ BHI with Polymeric resin beads.
 ● Specimens: Blood, bone marrow, and sterile body fluids like CSF, Peritoneal fluid,
Pleural fluid & Synovial fluid.
● More sensitive media
● Rapid method & Less labour intensive

Reference: Essentials of Medical Microbiology, Apurba S Sastry E/3 Page No. 33 Fig. 3.8

DISADVANTAGES OF AUTOMATED SYSTEMS:

● High cost of instrument
● Inability to observe colony morphology as liquid medium is used.

COMMERCIALLY AVAILABLE AUTOMATED SYSTEMS:

● BacT/ALERT 3D
● BacT/ALERT VIRTUO
● BACTEC
● MGIT: For detecting Mycobacterium tuberculosis
 SET 13

ANAEROBIC CULTURE METHODS

⮚ It is used to cultivate anaerobic organisms in oxygen free conditions.
⮚ The media used in this contains reducing agents
⮚ Commonly used anaerobic media are Robertson’s cooked meat medium and thioglycollate
medium.

BY DISPLACEMENT AND COMBUSTION OF OXYGEN

EVACUATION AND REPLACEMENT

⮚ It involves the evacuation of air from the jar and replacement by an inert gas like hydrogen
followed by removal of residual oxygen by use of a catalyst.
⮚ Types:
● Manual method: McIntosh and Fildes Anaerobic jar.
● Automated system: Anoxomat

MCINTOSH AND FILDES ANAEROBIC JAR:

● It was the most reliable and widely used anaerobic method.

● Not in use nowadays.
● Structure:
1. It consist of a stout glass or metal jar with a metal lid that is clamped airtight with a screw
2. The lid has two tubes with a tap, one acting as the gas inlet and the other as the outlet.
3. The lid has two terminals which can be connected to an electrical supply.
● Leading from the terminals and suspended by stout wires on the underside of the lid is a small,
grooved, porcelain spool around which is wrapped a layer of palladinised asbestos.
● Inoculated culture plates are placed in the jar and lid is clamped
● Alumina pellet coated with palladium and kept dry in a sachet within the jar ,acts as a catalyst at
room temperature
● Reduced methylene blue is used as an indicator and it remains colorless anaerobically but turns blue
on exposure to oxygen
 Reference: https://www.microrao.com/micronotes/mcintosh.pdf

ANOXOMAT:

● Automated,microprocessor- controlled system for the cultivation of anaerobic, microaerophilic and

capnophilic bacteria.
● It automatically evacuates air and replaces it with hydrogen from a cylinder.
● Sachet containing aluminium pellets coated with palladium is used to remove residual oxygen.
● This method is the most efficient means of bacterial cultivation.

INDICATORS OF ANAEROBIOSIS:
● Chemical indicator: Methylene blue-remains colourless in anaerobic conditions but turns blue
on exposure to oxygen.
● Biological indicator: Absence of growth of obligate anaerobes like Pseudomonas.

ABSORPTION OF OXYGEN BY CHEMICAL OR BIOLOGICAL METHOD:

GASPAK SYSTEM:

● It is commercially available as a disposable envelope, containing sodium bicarbonate and sodium

borohydride, which generate hydrogen and carbon dioxide in addition to water.
 ● The inoculated plates are kept in the jar and gaspak envelope is added with water, then the lid is
screwed tightly.
● Hydrogen and carbon dioxide are liberated and the traces of oxygen are removed by the catalyst-
aluminium pellets coated with palladium.
● This results in a combination of hydrogen and oxygen to produce an anaerobic environment.

● GENbag (bioMerieux): It is similar to the gaspak system and has an airtight transparent bag with
generator bag, which rapidly produces carbon dioxide and creates an anaerobic environment.

PRE-REDUCED ANAEROBIC SYSTEM (PRAS):

● It is prepared under oxygen- free conditions from initial sterilization process to packing in foil
packets.
● It is used for fastidious anaerobic cultures.

ANAEROBIC WORK STATION (‘ANAEROBIC GLOVE BOX’):

● It is an airtight, glass-fronted cabinet filled with inert gas, with an entry lock for the introduction
and removal of materials, and gloves for the hands.

● It provides facilities for easy processing, incubation, and examination of specimens without
exposure to oxygen.

REDUCING AGENTS:

● To achieve reduction of oxygen in the medium.

● Addition of a small quantity of agar enhances the anaerobic capacity of the medium by slowing
the diffusion of oxygen in it.
● Examples: 1% glucose, 0.1% thioglycolate, 0.1% ascorbic acid and 0.05% cysteine.

ROBERTSON’S COOKED MEAT (RCM) MEDIUM:

● Widely used fluid medium for the culture of anaerobes.

● It consists of fat-free, minced cooked meat in broth.
● It permits the growth of even strict anaerobes and indicates their saccharolytic or proteolytic
activities by the meat being turned red or black.

THIOGLYCOLATE BROTH:

● It contains hemin and vitamin K and serves as an enriched liquid medium for culturing
anaerobic and microaerophilic bacteria.

ANAEROBIC BROTH:

● Easily prepared medium

● Example: Smith-Noguchi medium, containing fresh animal tissue like rabbit
kidney,spleen,testes or heart, to cultivate Reiter’s strain of Treponema.
 SET 14

ANTIBIOTIC SENSITIVITY (OR SUSCEPTIBILITY) TEST

⮚ Antibiotic sensitivity test is done to help the clinicians to choose the right antibiotics for the
pathogenic bacteria to treat, that is, to tailor the empirical antibiotic therapy to pathogen-directed
therapy.
⮚ AST is performed only for pathogenic bacteria isolated from the specimen and not for
commensals.

DISK DIFFUSION TESTS:

● Most widely used method

● Uses filter paper discs charged with appropriate concentration of drugs(antibiotics)
● Test bacterium is inoculated on the medium and antibiotics discs are applied
● Sensitivity to the drugs is determined from the inhibition of bacterial growth around the disc
● Unsuitable for slow growing microbes
.

COLONY DISK DIFFUSION

A) STOKES DISC DIFFUSION METHOD:

● In Mueller Hinton(MH) agar plate ,test bacterium is inoculated on the central one third and
control on upper and lower thirds of plate
● Uninoculated gap of 2-3 mm wide should be kept between standard and test inocula where
antibiotic discs are applied
● Comparison of zones of inhibition between the standard and test bacteria indicates sensitivity/
resistance of the latter
o SUSCEPTIBLE: when it is inhibited by the concentration of the drug usually
achieved in the blood following dosage
o INTERMEDIATELY SUSCEPTIBLE: susceptible to higher than normal dosage
or when drug has efficacy when drugs are concentrated physiologically at the
body site
o RESISTANT: to the drug when its not inhibited
● In modified Stokes disc diffusion method, test bacterium is inoculated over the upper and
lower thirds and control on central one third of plate
● Plates are incubated at 370C for 16-18 hrs

● CHOICE OF ANTIBIOTIC DISC: only clinically relevant antibiotics are tested.

 Reference: https://images.app.goo.gl/4AsBJd8JgWaacQWy7

Reference: Essential of Medical Microbiology, Apurba S Sastry E/2 Page No. 91 Fig. 7.5

Report: By comparing the zones of inhibition of control and test bacterium

B) KIRBY-BAURER DISC DIFFUSION METHOD:

● Most widely used disk diffusion test

● Use a cotton swab to inoculate the CAMHA (Cation-adjusted Mueller- Hinton agar) plate by
streaking
● Allow 30 mins for drying
● 6 Antibiotic discs are placed 24mm space with one at center and others at periphery
o Diameter of zone of inhibition depends on:
Diffusibility of drug, disc concentration, composition of medium and
thickness,,pH and time of incubation
● Plates are incubated at 37C for 16-18 hrs

Report: by measure the zone of inhibition around each disc

SOME STANDARD ANTIBIOTICS INTERPRETATION CHART OF CLSI GUIDELINES

Antibiotics Disc Diameter of Zone of inhibition (in mm)

Concentration
in µg
Resistant Intermediate Sensitive
sensitive
Gentamicin 10 ≤12 13-14 ≥15

Amikacin 30 ≤14 15-16 ≥17

Erythromycin 15 ≤13 14-22 ≥23

Tetracycline 30 ≤14 15-18 ≥19

Ampicillin 10 ≤16 - ≥17

Vancomycin 30 ≤14 15-16 ≥17

Reference: Essential of Medical Microbiology, Apurba S Sastry E/2 Page No. 91 Table 7.6

DIRECT DISK DIFFUSION METHOD

C) PRIMARY DISC DIFFUSION METHOD:

● It is performed ,when results are required urgently and single pathogenic bacterium is
suspected in specimen
● It is no use when mixed growth of different bacterium is suspected
● results of the direct-DD test should always be verified by performing AST from the colony
subsequently

DILUTION TESTS

● Antibiotics are diluted and dilution is tested with the test organism
● Minimum inhibitory concentration (MIC): is the lowest concentration of an antimicrobial
agent that will inhibit the visible growth of a microorganism after overnight incubation
● Minimum bactericidal concentration (MBC) are determined
A) BROTH DILUTION METHOD:
● 2 types
✔ Macro broth dilution (performed in tubes)
✔ Microbroth dilution (in microtiter plate)
● Serial dilution of drug in MH broth are in tubes and standardised suspension of test bacterium
inoculated
● Incubate at 37C for 16-18 hrs
● MIC :
✔ Lowest concentration of drug at which there is no visible growth
✔ MIC inhibits the bacterial growth
✔ Uses :
i. for confirmation of results of antimicrobial susceptibility test from disc diffusion
tests
ii. for testing slow growing bacteria such as tubercle bacilli
● MBC:
✔ Determined by subculturing from each tube showing no growth on nutrient agar plate
without any antimicrobial agent
✔ MBC kills the bacterium
 Reference: Essential of Medical Microbiology, Apurba S Sastry E/3 Page No. 43 Fig. 3.3.26

B) AGAR DILUTION METHOD:

● Serial dilutions of drug are prepared in molten agar and poured into plates
● Many strains can be inoculated on each plate containing an antibiotic dilution
● Test strains are spot inoculated
EPSILOMETER TEST (E-TEST ):

This is a quantitative method of detecting MIC by using the principles of both dilution and diffusion of
antibiotics into the medium.

● Quantitative Test to detect Minimum inhibitory concentration (MIC) of antibiotic

● Recent modification of agar diffusion test
● It uses an absorbent strip with a known gradient of antibiotic concentration along its length
● Strip is placed on the agar plate inoculated with test organism
● Antibiotic diffuses into medium
● MIC is recorded as lowest concentration of gradient which inhibits growth of organism

Reference: Complete Microbiology for MBBS by Baveja E/1 Page No. 104 Fig. .5.2.3
AUTOMATED METHODS
● Rapid methods
● Based on microbroth dilution
● These are ,
✔ VITEK system (biomerieux):
o can perform AST of bacteria and yeasts
o It works on the principle of microbroth dilution
o It uses a reagent card containing 64 wells, which contain doubling dilution of
antimicrobial agents. The organism suspension is added to the Wells
o Incubated in the system at 35.5 ±1°C.
o Reading is taken once in every 15 minutes: measures the presence of any turbidity
(by nephelometry) which indicates the organism has grown in that antibiotic well
o Results are available within
8-10 hours - gram negative bacilli
16-18 hours - gram-positive cocci
✔ Phoenix system (Becton Dickinson)

✔ Microscan walkaway system

INTERPRETATION OF AST:
✔ The result of AST (whether disk diffusion or MIC based methods) is expressed :
o Susceptible (S): Indicates that the antibiotic is clinically effective when used in
standard therapeutic dose
o Intermediate (I): Indicates that the antibiotic is not clinically effective when used
in standard dose; but may be active when used in increased dose.
o Susceptible dose dependent (SDD): Indicates that the antibiotic will be clinically
active only if given in increased dose. This category is available only for few
agents such as cefepime for Enterobacteriaceae
o Resistant (R): Indicates that the antibiotic is NOT clinically effective when used in
either standard dose or increased dose; and therefore should not be included in the
treatment regimen.
MOLECULAR METHODS:

Polymerase Chain reaction (PCR) mostly used molecular method

● For MRSA (Methicillin resistant S.aureus) detection , mecA gene can be amplified and
identified
● Van gene can be amplified for vancomycin resistant S.aureus (VRSA) and vancomycin
resistant Enterococcus (VRE)

● GeneXpert for detection of rifampicin resistance (in M. tuberculosis) and line probe assay for
detection of resistance to many anti-tubercular drugs
 SET 15

TRANSPOSABLE GENETIC ELEMENTS

❖ Structurally and genetically discrete bacterial genes that are capable of intracellular transfer
between chromosomes, plasmids and chromosomes to plasmids are called transposons.
❖ These are also called jumping genes or mobile genetic elements.
❖ May sometimes confer survival advantages under appropriate environmental conditions.
❖ The process of intracellular transfer of transposons is called transposition.
❖ Transposons are:
● Segment of DNA with one or more in the Centre, and the two ends carrying inverted
repeat sequences of nucleotides (Nucleotide sequences complementary to each other but
in the reverse order).
● Has transposase gene
● Non self replicating
● Dependent on DNA for replication.

TYPES OF TRANSPOSONS:

INSERTION SEQUENCE TRANSPOSON:

● Simplest form
● Length:1-2 kilo Base pairs
● Because of the inverted repeats,each strand of the transposon can form a single
stranded loop carrying the transposase gene.
● There is a double stranded stem formed by hydrogen bonding between the terminal
inverted repeat sequences.
 Reference: Essentials of Medical Microbiology, Apurba S Sastry E/3 Page No. 59 Fig. 3.4 A, B

COMPOSITE TRANSPOSON:
● Larger transposons carrying additional genes for antibiotic resistance or toxin production.
● Has insertional sequences at the ends that are identical or similar.

Reference: Essentials of Medical Microbiology, Apurba S Sastry E/3 Page No. 59 Fig. 3.4
 SET 16
NUCLEIC ACID PROBES

❖ Nucleic acid probes are radiolabeled or fluorescent labelled, biotinylated copies of

cloned SS DNA or RNA.
❖ They are 20 – 25 nucleotides long and contain a unique nucleotide sequence which can
be used for detection of homogenous nucleic acid by Hybridization.
❖ Hybridization is a non amplification based molecular technique in which two single
strands of nucleic acid come together to form a double stable molecule.
❖ There are two types of Nucleic acid probes namely, DNA Probe (hybridizes DNA) and
RNA Probe (hybridizes RNA)
❖ Probes containing sequences unique to the microbe (strain, species or group) to be
detected can be added to the microbial cultures, body fluids or other clinical materials to
contain the microbe or it’s DNA
❖ Probe hybridizes with complementary specific sequences on the microbe's nucleic acid
❖ Done after enzymatic digestion of extracted nucleic acid so that it detects only specific
DNA fragment from the mixture ( Eg : Southern blot)

USES:

● Nucleic acid probe is used to detect the specific nucleic acid from
1) Clinical samples directly
2) Following amplification of small quantity of nucleic acid
from clinical sample (Eg: by Real time PCR)
3) From culture isolates

● DNA proves are used in the diagnosis of Infectious diseases.

ADVANTAGES:

● High degree specificity

 ● Ability to detect minute quantities of complementary DNA even in the presence of other
microbes
● Capacity to recognise microbes that are either difficult or impossible to culture.

DISADVANTAGES:

● For detection of nucleic acid from clinical specimens , probe - based methods have
lower sensitivity than amplification based methods.

❖ Line probe assay is a classical example of molecular test that uses nucleic acid probe
technology & is used for diagnosis of Tuberculosis.
 SET 17

BACTERIAL SPORES
⮚ Spores are a highly resistant resting (or dormant) stage of the bacteria formed in
unfavourable environmental conditions as a result of the depletion of exogenous
nutrients.

STRUCTURE:

⮚ Bacterial spore comprises several layers.

⮚ From innermost towards the outermost, the layers are: core → cortex → coat →
exosporium..
⮚ The core is the innermost part containing the DNA material and is separated from the
cortex by an inner membrane and the germ cell wall (delicate membrane from which the
cell wall of the future vegetative bacteria will develop).
⮚ Cortex and the coat layers (multilayered) lie external to the core, and are separated from
each other by an outer membrane
⮚ The outermost layer is called the exosporium which may have distinct ridges and
grooves.

Reference: Essentials of Medical Microbiology, Apurba S Sastry E/3

SPORULATION:

⮚ Sporulation (or sporogenesis) is the process of formation of spores from the vegetative
stage of bacteria.
⮚ Sporulation is initiated by the appearance of a clear area, usually near one end of the cell,
which gradually becomes more opaque to form the ‘forespore’.
⮚ ENDOSPORES: As bacterial spores are formed within the parent cells these are called
endospores.

⮚ It is not a method of reproduction because the bacteria do not divide during sporulation.

⮚ Sporulation occurs when growth stops due to lack of nutrients.

⮚ The mature spore formed is extremely resistant to heat and disinfectant; which is due to
the deposition of calcium and dipicolinic acid into the spore cortex.

GERMINATION:

⮚ Transformation of dormant spores into active vegetative cells when grown in a

nutrient-rich medium.

⮚ The spores lose their refractivity and swell.

⮚ The spore wall is shed and the germ cell appears by rupturing the spore coat and
elongates to form the vegetative bacteria.

SHAPE AND POSITION OF SPORES:

⮚ Position: Spores may be central, subterminal or terminal

⮚ Shape: They may be oval or spherical in shape
⮚ Width:
o The diameter of spore may be same or less than the width of bacteria
(non-bulging spore-Eg as in Bacillus)
 o May be wider than the bacillary body producing a distension or bulge in the cell
(bulging spore, e.g. as in Clostridium).

SPORICIDAL CHEMICAL:

⮚ Spores are resistant to most routinely used disinfectants.

⮚ So some sterilants and high-level disinfectants only can be used.

STERILANTS:

⮚ Ethylene oxide (ETO) steriliser

⮚ Autoclave
⮚ Plasma sterilization
⮚ Dry heat sterilizer (hot air oven)

HIGH LEVEL DISINFECTANT:

⮚ Capable of killing bacterial spores when used in sufficient concentration under suitable
conditions.
⮚ Glutaraldehyde used at 2% or 2.4% concentration kills spores in 10-14hours
⮚ Hydrogen peroxide is sporicidal at >4-5%

DEMONSTRATION:

⮚ In unstained preparations as refractile bodies.

⮚ Forespore stains intensely but once the spore envelope is formed, it doesn’t stain
readily.
⮚ Spores appear as unstained areas in Gram-stained preparations.
⮚ They are acid-fast and are stained by modification of the Ziehl-Neelsen technique.
 ⮚ Involution forms may develop due to ageing cultures and autolytic enzymes.

APPLICATION OF SPORES:

⮚ Spores of certain bacteria are indicators of proper sterilization.

⮚ Absence of the spores (inability to grow) after autoclaving or processing in hot air oven
indicates proper sterilization.
● Spores of Geobacillus stearothermophilus are used as sterilization control for
autoclave and plasma sterilizer.
● Spores of Bacillus atrophaeus are used as sterilization control for hot air oven and
ethylene oxide sterilizers.
⮚ Spores have also been used as agents of bioterrorism, e.g., endospores of Bacillus
anthracis were used in the 2001 anthrax bioterrorism attack

NON-SPORING ANAEROBES:

⮚ Peptostreptococcus
⮚ Bacteroides
⮚ Prevotella
⮚ Porphyromonas
 SET 18

BACTERIAL VIRULENCE
⮚ Pathogenicity: It is the ability of a microorganism to produce disease.
⮚ Virulence: It is the degree of the disease- producing property of the microorganism.
⮚ Different strains of the same species may exhibit varying degrees of virulence.
⮚ Example: The pathogenic species of M. Tuberculosis and polio virus contain strains of
varying degrees of virulence including those which are avirulent such as vaccine strain
(BCG).
⮚ The virulence of a strain may undergo spontaneous or induced variation.
⮚ Exaltation :
● Enhancement of virulence
● Induced experimentally by serial passage in susceptible hosts.
⮚ Attenuation :
● Reduction of virulence.
● Can be achieved by
o Passage through an unfavourable host.
o Repeated culture in Artificial media.
o Growth in high temperature.
o Prolonged storage in culture.
o Growth in the presence of weak antiseptics.

DETERMINANTS OF FEATURES
VIRULENCE

1.Adhesion ● Initial event of pathogenesis.

● Mediated by specialized molecules called Adhesins, that
bind to specific host cell receptors.
 ● Prevent the bacteria from being flushed away in
secretions.
● Adhesins are fimbriae, pili
● Non-pilus adhesions:
o Lipopolysaccharide, flagella, outer membrane
protein and glycocalyx.
o Biofilm formation: Biofilm is a group of bacterial
cells that attach to a surface, living or nonliving, by
excreting a sticky substance that encompasses the
bacteria in a matrix.
o Colonization factors.
● Loss of adhesin often renders the strain avirulent.
● Adhesins are made of proteins and antigenic in nature.
● Specific immunization with adhesins has been attempted
as a method of prophylaxis. Eg : Against Gonorrhea in
human beings.

2.Invasiveness ● Entry of bacteria into host cells, leading to spread within

the host tissues.
● Highly Invasive pathogen: Produce generalized lesion
(Eg : Streptococcal infection)
● Less invasive pathogen : Cause localized lesion
(Eg : Staphylococcal Infections) .
● Pathogen which lack invasion remain confined to site of
entry and produce disease ( Eg : Clostridium tetani)

3.Toxigenicity Bacteria produce 2 types of toxin.

● Endotoxins:
o Lipopolysaccharides, part of the cell wall of
gram negative bacteria.
o Heat stable
 o Poorly antigenic and can’t be toxoided.
o Non-specific action , not enzymatic and has low
potency
● Exotoxins:
o Proteins, secreted by both gram positive and
negative bacteria.
o Heat labile.
o Highly antigenic and can be converted into
toxoid by formaldehyde.
o Highly specific pharmacologic action on specific
sites, enzymic in action and has high potency
o Toxoid forms are used as vaccine (Eg: tetanus
toxoid)
4.Antiphagocytic factors ● Capsules:
o Capsules are well-organized amorphous
gelatinous layers around the bacterial cell wall
that oppose phagocytosis.
o Capsulated bacteria include Streptococcus
pneumonia, Klebsiella pneumonia, pneumococci,
Haemophilus influenzae
● Bacterial Surface Antigens:
o Vi antigen of S.Typhi and K antigens of E. coli
help them to withstand phagocytosis and lytic
activity of the complement system.
● Streptococcal M Protein:

o Present on group A streptococci which binds to

both fibrinogen and fibrin to the bacterial cell
wall
o Thus masks the bacterial receptors from
complement proteins.
 ● Cytotoxins:

o They either interfere with chemotaxis or kill

the phagocytes.
o Eg: Staphylococcus aureus produces haemolysin
(both RBC and WBC) and leucocidin (only
WBC).
5. Survival within phagocytes ● Escape from phagosomes:
o Some microbes escape into the cytoplasm of
the host cell before the fusion of the phagosome
with the lysosome.
o Eg: Rickettsiae
● Prevention of fusion and degranulation:
o Cells of some microbes modify the phagosomal
membrane in such a manner that fusion of
lysosome with phagosome is prevented.
o Eg: Chlamydia
● Resistance to lysosomal enzymes:
o Due to the presence of capsular polysaccharide
in Mycobacterium lepraemurium and mycoside
in M. tuberculosis.
● Interference with oxidative burst:
o Bacteria produce enzymes to decrease
production of H2O2 and superoxide.
o Staph. aureus produces catalase which breaks
down H2O2.
o Listeria monocytogenes produces superoxide
dismutase to neutralize oxygen radicals.
6.Genetic material: Plasmids ● Gene coding for some virulence characteristics are
plasmid borne.
 ● Can be transmitted by bacteriophages or by
conjugation.
● Phage directed virulence is seen in Diphtheria.
● These can code for surface antigen which help in
colonization (in E. coli) or enterotoxin (produced by
E.coli and S. aureus).
● The transferred plasmid can also be multiple drug
resistance plasmids (R plasmid), which increase the
severity of disease by their resistance to antibiotic
therapy.

7.Enzymes ● Some bacterial products, though devoid of intrinsic

toxicity, may contribute to virulence by inhibiting the
mechanism of host resistance or directly damage the
host tissue.
● Protease: Cleaves immunoglobulin A that protects
mucosal surface.
● Coagulase: Prevents phagocytosis by forming a fibrin
barrier around the bacteria. Eg: Staphylococcus aureus.
● Hyaluronidases: Split hyaluronic acid which is a
component of intercellular connective tissue and
facilitates spread of infection in tissue spaces.
● Fibrinolysin: Breaks the fibrin barrier and helps in
invasion.
● Collagenase: Breaks down collagen in connective tissue
and contributes to invasion. Eg: Clostridium perfringens
8.Infective dose ● Minimum Infecting Dose (MID): Minimum number of
bacteria required to produce clinical evidence of
infection in a susceptible host under standard conditions.
 ● Minimum Lethal Dose (MLD): Minimum number of
bacteria required to cause death of a susceptible host
under standard conditions.
● It depends on the virulence of the bacteria, that is,
higher the virulence, lower is the infecting dose.

9.Route of Infection ● Some bacteria can produce infection whatever may be

the mode of entry. Eg: Staphylococcus aureus.
● Some can produce infection only when they enter
through their optimal route. Eg: Vibrio cholera is
infective orally but is unable to cause infection when
introduced subcutaneously.
10.Siderophores and iron ● Some bacteria produce low molecular weight
acquisition compounds called siderophores which acquire iron
from host’s iron binding proteins, for their metabolism.
● It enhances their virulence.
11.Communicability ● The ability of a parasite to spread from one host to
another is called communicability.
● It does not influence the production of disease in an
individual host but determines the survival and
distribution of a parasite in a community.
● There is no correlation between virulence and
communicability.
 SET 19
1. GENOTYPIC VARIATION
⮚ Genetic variations occur in the genome and are stable and heritable.
⮚ They may occur by:
● Mutations: Result in vertical gene transfer.
● Gene transfer: Transformation, lysogenic conversion or transduction and
conjugation.-Result in horizontal gene transfer.

MUTATIONS:
⮚ Mutation is a random, undirected heritable variation caused by change in the nucleotide
sequence of the genome of the cell.
⮚ Mutation can involve any of the numerous genes present in bacterial chromosomes or
rarely plasmid.
⮚ The frequency of mutation ranges from 10-2 to 10-10 per bacterium per division.
⮚ It can be beneficial or harmful to the cell.
⮚ Mutations occur in one of the two ways:
● Spontaneous mutations: Mutations that occur naturally in any dividing cells that
arise occasionally without adding any mutagen.
● Induced mutations: These mutations on the other hand, are as a result of
exposure of the organism to a mutagen, an agent capable of inducing
mutagenesis. Examples of mutagens include
❖ Physical agents: E.g: Ultraviolet (UV) radiation- cytosine and thymine
are more vulnerable to UV rays, Ionising radiation-X rays, visible light,
heat.
❖ Chemical agents: E.g: Alkylating agents, 5-bromouracil, nitrous acid and
acridine dyes.
⮚ Mutation is best appreciated when it involves a function, which can be readily observed
by experimental methods.
⮚ For example, an E.coli mutant that loses its ability to ferment lactose can be readily
detected on MacConkey agar.
 ⮚ Mutation can affect any gene and hence may modify any characteristic of the
bacterium, for example:
● Sensitivity to bacteriophages.
● Loss of ability to produce capsules or flagella.
● Loss of virulence.
● Alteration in colony morphology.
● Alteration in pigment production.
● Drug susceptibility.
● Biochemical reactions.
● Antigenic structure

CLINICAL APPLICATION:
⮚ The practical importance of bacterial mutation is mainly in the field of drug resistance
and the development of live vaccines.

CLASSIFICATION OF MUTATION TYPES:

⮚ Mutations may occur in two ways:
● Small-scale mutations: They are more commonly seen in bacteria. Examples
include
❖ Point mutations - occur at a single nucleotide,
❖ Addition or deletion of single nucleotide pair
● Large-scale mutations: Occur in chromosomal structure
❖ These include deletion or addition of several nucleotide base pairs or
gene duplications.

FORWARD MUTATIONS

Substitution at single nucleotide base pair

At DNA level:

● It is a point mutation that changes a purine nucleotide to

1.Transition
another purine (A↔G) or a pyrimidine nucleotide to
another pyrimidine(C↔T)

● It refers to the substitution of a pyrimidine or vice versa in

2.Transversion
DNA (C/T↔ A/D)

At Codon Level:

● The new codons codes for the same amino acid, eg.
1.Silent mutation
AGG↔ CGG, both code for arginine

● The new codon codes forms different but functionally

2.Neutral mutation
equivalent amino acid: AAA(lysine) AGA(arginine)

● The new codon codes for a different amino acid

3.Missense mutation

● The new codon is a stop codon which causes termination eg.

4.Nonsense mutation
CAG↔ UAG

Addition or deletion at many nucleotide base-pairs

● Any addition or deletion of base pairs that is not a multiple

1.Frame - shift mutation
of three results in a shift in the normal reading frame of the
coded message forming a new set of triplet codons.
● They are usually very deleterious and may lead to synthesis
of non-functional proteins

REVERSE MUTATIONS
It is a second mutation that nullifies the effect of the first mutation and results In gaining back the function
of the wild phenotype.

● A true reverse mutation converts the mutant nucleotide

1.True reversion
sequence back to the wild type sequence.
● AAA (Lysine) forward mutation GAA(Glutamine) reverse
mutation AAA(Lysine) (wild type) →(mutant)→ (wild
type).

● Second mutation produces a different codon which codes for

2.Equivalent reversion
the same amino acid of wild type sequence.
● UCC (Serine) forward mutation GAA (Cystine} reverse
mutation AAA (Serine} (wild type)→ (mutant)→ (wild
type).

● It is a second mutation in a different gene that reverts the

3.Suppressor mutation
phenotypic effects of an already existing mutation.

DETECTION AND ISOLATION OF MUTATION:

⮚ It can be recognized both by genetic and phenotypic methods.
⮚ GENETIC METHODS:
● Gene probes
● Gene sequencing
⮚ PHENOTYPIC METHODS:
● Fluctuation Test: It demonstrates spontaneous mutations in bacteria.
● Replica Plating Method: It demonstrates auxotrophic mutants (mutants that
don’t grow in the absence of a particular nutrient).
● Ames Test: It is used to test the carcinogenicity of a mutagen.
2. PHENOTYPIC VARIATIONS OF BACTERIA
⮚ Phenotype: Physical expression of genotype in a given environment.
⮚ A cell may exhibit different phenotypic variations in different situations.
⮚ Phenotypic variation is determined by the environment and can be reversed by
changing the environment.
⮚ Examples:
● Salmonella typhi
➔ Normally flagellated
➔ When grown in phenol agar it becomes non- flagellated
➔ The condition is reversible when the subcultured from phenol agar to
broth
● Escherichia coli
➔ Lactose fermentation occurs in the medium containing lactose using
enzyme beta-galactosidase
➔ When grown in a medium containing only glucose, the enzyme will not be
produced
 SET 20
EXTRACHROMOSOMAL GENETIC ELEMENTS

TYPES:
➢ Plasmids
➢ Episomes

EPISOMES:
⮚ Extrachromosomal genetic elements which integrate with chromosomal DNA and
replicate.

PLASMIDS:

⮚ Extrachromosomal double stranded circular DNA molecules existing in free state in

cytoplasm of bacteria and some yeasts.

● They are not essential for the life of bacteria.

● They can be present singly or in multiple numbers – upto 40 or more per cell.
● They behave like Replicons, that is, have an Origin of replication & other genes
involved in replication.
● Capable of replicating independently.
● They can also be Episomes, that is, sometimes, plasmids integrate and replicate with
bacterial chromosomes.
Reference: Essentials of Medical Microbiology, Apurba S Sastry E/3 Page No. 52 Fig. 3.4.2

CLASSIFICATION OF PLASMIDS:

⮚ BASED ON ABILITY TO PERFORM CONJUGATION:

● Conjugative Plasmids: Self-transmissible to other bacteria by conjugation.
● Non-Conjugative Plasmids: Non-transmissible to other bacteria by themselves.

⮚ BASED ON COMPATIBILITY BETWEEN PLASMIDS:

● Compatible Plasmids: Two or more different plasmids can exist in one bacterial
cell if they are compatible.
● Incompatible Plasmids: One or the other plasmid will be rapidly lost from the cell,
as they might have the same replication or partition mechanism and thus compete
with each other.

⮚ BASED ON FUNCTION:
● Resistance Plasmids: Contain genes coding for resistance to antibiotics.
● Col Plasmids: Contain genes coding for bacteriocins (antibiotic-like protein
substances, produced by bacteria,that can kill other bacteria).
● Fertility Plasmids/ F-Plasmids: Contain tra-genes - code for sex pili which help in
conjugation by forming conjugation tube.
 ● Virulence Plasmids: Code for virulence factors and toxins. Examples include:
➔ Heat labile and heat stable toxin of E.coli
➔ Siderophore production
➔ Adherence Antigens (K88 Antigen in E.coli)

● Metabolic Plasmids: Help the host in various metabolic activities

➔ Unusual substance digestion- toluene, salicylate, camphor
➔ Urease synthesis
➔ Nitrogen fixation

PLASMID AS VECTOR:

⮚ Plasmids contain sites where genes can be artificially inserted by recombinant DNA
technology.

⮚ Due to their ability to transfer DNA from one cell to another, they can be used as vectors.

⮚ Hence, they are important vectors in protein production, gene therapy, etc.

CURING:
⮚ Process of eliminating plasmids from a bacteria.
⮚ May occur spontaneously or due to treatment of host cells with substances that inhibit
plasmid replication and growth at higher temperatures, without affecting host cells.
⮚ The substances or methods used include acridine, radiations, thymine starvation.
REFERENCES
Ananthanarayan and Paniker’s Textbook of Microbiology
▪ Tenth Edition
▪ Eleventh Edition

Essentials of Medical Microbiology, Apurba Sastry

▪ First Edition
▪ Second Edition
▪ Third Edition

Review of Microbiology and Immunology, Apurba Sastry, Sixth Edition

Complete Microbiology for MBBS, CP Baveja Seventh Edition

ESSAYS:
1. Innate Immunity.
2. Humoral Immunity.
3. Active and Passive Immunity.
● Acquired Immunity.
4. Human Leucocyte Antigen.
● Major Histocompatibility Complex.
● HLA typing.
5. Immunoglobulins.
● Ig G
● Ig M.
● Ig E.
● Ig A, (Secretary IgA).
● Abnormal Immunoglobulins.
● Ig M Detection in Infectious Diseases.
● Immuno Fluorescence Methods
6. B cells and T cells
● Cells involving immunity and their functions.
● Structure and Functions of B cells.
● Difference between the B & T cells, Development of T cells.
● T Cells.
● CD8 + T Cells.
● NK Cells.
● Cell-Mediated Immunity.
● Antigen Presenting Cells.
● B – Lymphocyte.
● T – lymphocyte.
● T Cell Subsets
7. Hypersensitivity
● Enumerate Hypersensitivity Reactions.
● Discuss in detail Type IV Hypersensitivity Reactions and a note on Schwartzman
reaction.
● Describe the Mechanism of Anaphylaxis.
● Define and Classify Hypersensitivity Reactions. Describe Type I Hypersensitivity.
● Type – IV Hypersensitivity.
● Type – II Hypersensitivity.
● Type – IV (delayed) Hypersensitivity.
● Type – III Hypersensitivity.
● Anaphylaxis
8. Define and Classify Antigen-Antibody reactions, Discuss the agglutination test with
clinical examples.
● Passive Agglutination.
● Define Agglutination and Precipitation.
9. Autoimmunity.
● Schwartzman’s Reaction.
● Autoimmunity.
● Mechanism of Autoimmunization (Auto Antibody Formation Theories).
● Systemic Autoimmune Diseases
10. Define Complement, write about Alternative C Pathway and add a note on the Biological
effects of Complement and Genetic deficiency of Complement components.
● Complement.
● Alternative Pathway of Complement.
● The biological function of Complement.
● Complement Deficiency Diseases.
11. Herd Immunity.
12. Cytokines.
● Lymphokines.
13. Infection
● Methods of Transmission of Infection.
● Source of Human Infection.
SHORT NOTES:
1. Heterophile Antigen.
2. Complement Fixation Test.
3. ELISA.
● Western Blot technique.
● Applications of ELISA.
● Coombs test.
4. Fluorescent Antibody techniques.
5. Elek’s Gel precipitation test.
6. Immunological Tolerance.
7. Theories of Immune Response.
8. Adjuvants.
9. Principles of Monoclonal Antibody Production.
● Monoclonal Antibody.
10. Serum Sickness.
11. P.K (Prausnitz – Kustner)Reactions.
12. Tumour Antigens.
13. Immuno Surveillance Possible Mechanisms.
14. Allograft Rejection.
● GVH Reaction.
15. Carriers.
16. Difference between the Exotoxin and Endotoxin.
● Exotoxins.
17. Protocols for safe Blood Transfusion.
ESSAY
 1.INNATE IMMUNITY

● The first line of defence – acts in minutes

● No prior microbial exposure
● Limited diversity
● Nonspecific
● No memory

MECHANISMS
RECEPTOR INTERACTION
● Attachment – binding of surface molecules of microbes 🡪 Innate immune cell
receptors

MICROBIAL SURFACE MOLECULES

● Conserved repeating molecular markers
● MAMPs – microbes associated molecular patterns e.g.: LPS, teichoic acid of
bacteria

PATTERN RECOGNITION RECEPTORS (PRRS)

● Receptors on immune cells recognize – MAMPs (coded by germline genes-
conserved)
● Toll-like receptor – main, named after fruit fly(Drosophila)

COMPONENTS
a)

b) PHAGOCYTOSIS

(Neutrophils, Monocytes, Macrophage 🡪 Recruited at Site

c) NATURAL KILLER CELLS – a kind of Lymphocyte

d) OTHER RARE LYMPHOCYTES :

● ɣƍ T cells - Intraepithelial Lymphocytes – Skin and Mucosa

● NK Tcells - Epthelium + Lymphoid Organ
● B-1 cells - Peritoneal cavity + Mucosal tissues
● Marginal zone B cells - edges lymphoid follicle in the spleen

e) MAST CELLS

● Respiratory and other mucos (IgE dep. Activation) / (MAMPs + TLRs)

f ) DENDRITIC CELLS:

● bridge b/w Innate and acquired immunity

g) COMPLEMENT ACTIVATION :

h)INFLAMMATORY RESPONSE :
i) NORMAL FLORA :

● Competes for resource

● Production of antimicrobial substances

j) CYTOKINES :

● Mediates cellular reactions (immune)

● TNF-α
● IL – 1, 6, 8, 12, 16
● INF – α, β
● TGF –β

k) ACUTE PHASE REACTANTS(APRS) :

Role: Wide

● Antimicrobial
● Anti-inflammatory
 ● Chelates Metals – makes – unavailable for Microbial growth

Production :

● Liver (MAIN)
● Endothelial cells
● Fibroblasts
● Monocytes
● Adipocytes

Essential of Medical Microbiology by apurba S Sastry - 3/E pg no.204 - fig.17.1

Ref: Essential of Medical Microbiology by apurba S Sastry - 3/E
 2.HUMORAL IMMUNITY
● It is the Primary defence against most Extracellular Bacterial Pathogens.
● Helps in defence against Viruses that infect through the Respiratory (or) Intestinal tract.
● Prevents recurrence of Virus infections and participates in the pathogenesis of Immediate
Hypersensitivity and certain Autoimmune Diseases.

STEP OF PRODUCTION: -
1. AFFERENT LIMB:
The entry of antigen, its distribution, and fate in the tissues and its contact with
appropriate immunocompetent cells.

2. CENTRAL FUNCTIONS: -
The processing of antigen by cells and the control of the antibody-forming process.

3. EFFERENT LIMB: -
The Secretion of Antibody, its Distribution in Tissues and Body fluid and the
Manifestation of its effects.

PHASES: -
1. Lag phase
2. Log phase
3. Plateau/Steady state
4. Phase of Decline

1° RESPONSE: -
● The antibody response to the initial antigenic stimulus is called 1° response.
● The 1° response is slow, sluggish, and short-lived.
A – An antigenic stimulus.
1 – Latent Period.
2 – Rise in titer of Serum Antibody.
3 – Steady-state of Antibody titer.
4 – Decline of Antibody titer.

(Ref: Ananthanarayan and Paniker's Textbook of Microbiology – Pg.139 – Fig 16.1)

2° RESPONSE: -
● The antibody response to subsequent stimuli with the same antigen produces a 2°
response.
● 2° response is prompt powerful, prolonged, and the antibody formed is IgG.
A, B, C – Repeated antigenic stimuli.
1 – 1° Immune Response.
2 – 2° Immune Response.
3 – Negative Phase.
4 – High Level of Antibody following Booster Injection.

(Ref: Ananthanarayan and Paniker's Textbook of Microbiology – Pg.139 – Fig 16.2)

NEGATIVE PHASE: A Temporary fall in the level of Circulating Antibody occurring due to the
Combination of Antigen with Antibody.

THE FATE OF ANTIGEN IN TISSUES: -

1. PARTICULATE ANTIGEN:
2 Phases

Non-Immune Immune
 The antigen is engulfed by Phagocytic After Antigen-Antibody complexes are
cells, broken down, and eliminated. formed and rapidly Phagocytosed.

2. SOLUBLE ANTIGEN:
3 Phases

Equilibration Metabolism Immune Elimination

Diffusion of Antigen to Catabolic Elimination by Antigen-Antibody

extravascular space decay complex formation

SPEED OF ELIMINATION: - Protein (Faster) > Polysaccharides

PRODUCTION OF ANTIBODIES: -

1. Capture and Processing of Antigen by APC

2. Presentation to appropriate MHC

3. T-cell dependent antigen 3. T-cell independent antigen

E.g., Proteins and linked to a T-cell receptor E.g., Polysaccharide

For CD4 Cells (TH Cells) ➜ Antigen must be presented by MHC class II
For CD8 Cells (TC Cells) ➜ Antigen must be presented by MHC class I
 4. Activation of TH Cells: -
Signals required for TH Cell activation

Binding of TCR to MHC-Antigen Complex Interleukin I produced by APC

5. Activated TH Cells produce IL-2 and cytokines for B-Cell Stimulation.

6. Activation of Tc Cells combined with MHC-Antigen complex.
7. TC Cells release cytotoxins and destroy the target (Virus/Tumor Cell).

MONOCLONAL ANTIBODIES: -
● Antibody produced by a Single Antibody Forming cell/clone against a Single Antigen
(or) Antigenic Determinant only.
● Are useful tools in Diagnostic and Research techniques.

FACTORS INFLUENCING ANTIBODY PRODUCTION: -

1. Genetic factors.
2. Age.
3. Nutritional Status.
4. Route of Administration.
5. Size and no. of dose.
6. Multiple antigens.
7. Adjuvants.
8. Immunosuppressive Agents.
9. Effect of Antibody.

GENETIC FACTORS:
Immune response genes control the individual’s capacity to respond or not to a particular
antigen.

AGE: -
● Antibody production starts at 3 – 6 months of age.
● Full Competence ➜ 5 – 7 years for IgG.
 ➜ 10 – 15 years for IgA.

NUTRITIONAL STATUS (VITAMINS & AMINO ACIDS):

Malnutrition Affects both Humoral and Cell-Mediated Immunity.

ROUTE OF ADMINISTRATION:
The humoral immune response is better following parenteral administration rather than the
oral/nasal route.

SIZE AND NO. OF DOSE:

The antigen is Effective only above a minimum critical dose, further increase in dose enhances
the intensity up to a certain limit.

ANAMNESTIC REACTION: -
Production of antibody in response to an antigenic stimulus of a heterologous but related
antibody that the host had earlier produced.

ADJUVANTS: -
Any substance that enhances the immunogenicity of an antigen.
 3.ACTIVE AND PASSIVE IMMUNITY

ACTIVE IMMUNITY: -
1. Active immunity is the resistance developed by an individual as a result of an
antigenic stimulus.
2. This involves the active functioning of the host’s immune apparatus leading to the
synthesis of antibodies and production of immunologically active cells.
3. There is a negative phase during which the level of measurable immunity lowers
than it was before the antigenic stimulus.
4. It sets in only after a latent period which is required for the immunological
machinery to be set in motion.
5. Active immunity is long-lasting. An individual who has been actively immunized
against antigen experiences the same antigen subsequently, the immune response
occurs more quickly and abundantly than during the first encounter. This is
known as Secondary response.
6. It develops the humoral and cellular immunity associated with immunological
memory.
7. Active immunization is more effective and confers better protection than passive
immunization.

8. PREMUNITION: A special type of Active immunity in which the immunity to

reinfection lasts only as long as the original infection remains active. E.g.
Syphilis.

NATURAL ACTIVE IMMUNITY: The immunity following bacterial infection is generally less
permanent than the following viral infection.

E.g.,
1. Life-Long Immunity ➜ Chickenpox, Measles.
2. Short-lived Immunity ➜ Influenza, Common Cold.

ARTIFICIAL ACTIVE IMMUNITY: -

Bacterial Vaccines Viral Vaccines

Live – BCG vaccine. Live – oral polio vaccine.

 Killed – Cholera vaccine. Killed – Injectable Polio vaccine.

Bacterial Products – Tetanus Toxoid. Subunit – Hepatitis B vaccine

PASSIVE IMMUNITY: -
1. The resistance that is transmitted passively to a recipient in a readymade form is known
as passive immunity.
2. There is no antigenic stimulus, instead preformed antibodies are administered.
3. There is no latent period in passive immunity, so protection is effective immediately.
4. There is no negative phase.
5. No secondary response occurs, as the foreign antibody administered second time is
eliminated more rapidly.
6. Passive immunity acts immediately and instantly.

NATURAL PASSIVE IMMUNITY: -

● Resistance passively transferred from mother to baby.

● The transport of antibodies across the placenta is an active process.
● The concentration of antibodies in fetal blood may sometimes be higher than that seen in
the mother.

ARTIFICIAL PASSIVE IMMUNITY: -

● The resistance is passively transferred to a recipient by the administration of antibodies.
● The agents used are hyperimmune sera of animal (or) human origin, convalescent sera,
and pooled human gamma globulin.
● These are used for prophylaxis and therapy.
● Disadvantages: Hypersensitivity and immune elimination.
● Convalescent sera: High levels of specific antibody.
● Pooled human gamma globulin contains antibodies against all common pathogens.

COMBINED IMMUNISATION:
● A combination of active and passive methods of immunization.
ADAPTIVE IMMUNITY: -
● It is the injection of immunologically competent lymphocytes.
● Used in the treatment of diseases like lepromatous leprosy.

ACQUIRED IMMUNITY: -
● The resistance that an individual acquires during his life is known as acquired immunity.

2 types

Active Passive

(As a result of antigenic stimuli) (Resistance transferred to the

recipient in readymade form)

Natural Artificial Natural Artificial

● It is mediated by antibodies and T-Cells.

● Component of 2nd line of defence.

CHARACTERISTICS: -
1. Specific.
2. Capable of self-recognition.
3. Immunological memory.
4. Diversity.
 ACTIVE IMMUNITY PASSIVE IMMUNITY

● Produced by the host immune system. ● Received.

● Induced by infection (or) by ● Readymade antibody

immunogens. transferred.

● Durable, effective protection. ● Transient, less effective.

● Immunological memory present. ● No memory.

● Booster effect on the subsequent dose. ● Subsequent doses are less

effective.

● Negative phases may occur. ● No negative phase.

 4. HUMAN LEUKOCYTE ANTIGEN

MAJOR HISTOCOMPATIBILITY COMPLEX

● It is a group of genes coding for a set of host cell-surface molecules
➔ These molecules bind peptide fragments from pathogens and display them on the
host cell for recognition by appropriate T cells
● Located in the short arm of chromosome 6

MHC MOLECULES:
● MHC coded proteins are also known as human leukocyte antigens (HLA) or
histocompatibility antigens
● Unique identification markers for every individual
● The genetic sequence of MHC genes is different for every individual
● Determines the compatibility between the graft and host tissues

STRUCTURE OF HLA COMPLEX

● It extends covering more than >100 genes
● Genes are clustered into three regions
➔ MHC region 1
➔ MHC region 2
➔ MHC region 3
(Ref - Essentials of medical microbiology by Apurba S Sastry - 3/E - pg. 179 - fig. 14.12)

MHC REGION 1
● Has HLA-A, HLA-B, HLA-C genes codes for HLA-A, HLA-B, HLA-C proteins
respectively
● Forms alpha chain of MHC class 1 molecule
● MHC- 1 Proteins are located on the surface of all nucleated cells (except sperm) and
platelets
➔ They present the peptide antigen to CD8 t-cells

MHC REGION 2
● Has DP, DQ, DR genes encoding for DP, DQ, DR proteins respectively
● Forms alpha and beta chains of MHC class II molecules
● MHC 2 proteins are located on the surface of antigen-presenting cells
➔ They present the peptide antigen of CD4 t-cells

MHC REGION 3
● Is not involved in antigen presentation
● It has genes coding for
➔ Complement factors
➔ Heat shock proteins
➔ Tumor necrosis factor
➔ Steroid 21 hydroxylase
STRUCTURE OF MHC MOLECULES

(Ref - Essentials of medical microbiology by Apurba S Sastry - 3/E - pg. 179 - fig. 14.13)

MHC class I MHC class II

Composed of an alpha chain (glycoprotein) Composed of one alpha chain and one beta
and beta 2 microglobulin chain

Has alpha 1, 2, 3 domains Has alpha 1 and 2, beta 1 and 2 domains

Peptide binding site - alpha1/alpha2 groove Peptide binding site alpha 1/ beta 1 groove

Alpha 3 binds to CD8 molecules Beta 2 binds to CD4 molecules

REGULATION OF MHC EXPRESSION

● Transcription factors on binding to promoter genes for MHC increase their transcription
● Cytokines influence MHC expression
● Corticosteroids and prostaglandin decrease the expression of MHC II molecules
● In many viral infections, MHC 1 expression is suppressed
HISTOCOMPATIBILITY
● Histocompatibility between the graft and recipient would decide whether the graft is
going to be accepted or rejected.
● Histocompatible - usually autografts and isografts
● Histoincompatible - usually allografts and xenografts
● MHC molecules are the most important transplant antigens which the recipient would
mount an immune response
● HLA typing is a laboratory test to determine histocompatibility before transplantation

HLA TYPING
● In this test, the donor's antigen expressed on the surface of leukocytes or their gene to that
of the recipient is matched.

METHODS OF HLA TYPING

● Phenotypic Method
➔ Microcytotoxicity
➔ Mixed lymphocyte reaction
● Genotypic methods
➔ PCR detecting HLA genes
➔ PCR - RFLP (Restriction Fragment Length Polymorphism)
➔ PCR - DNA sequencing
➔ PCR - SSP (PCR- Sequence-Specific Primer)
➔ PCR - SSOP (PCR - Sequence-Specific Oligonucleotide Probing)
● PCR-SSP, PCR-SSOP, PCR-DNA are the most reliable methods currently in use.
● It has high-resolution matching

(Ref - Essentials of medical microbiology by Apurba S Sastry - 3/E)

 5.IMMUNOGLOBULINS

Antibody or immunoglobulin - specialized glycoprotein, - produced from activated B cells

(plasma cells) – in response to antigen, - capable of combining with an antigen that triggered its
production.

CONSTITUENTS:- 20-25% of total serum proteins.

STRUCTURE OF IMMUNOGLOBULIN:
● Y shaped heterodimer
● Composed of 4 polypeptide chains: 2 identical light chains (MW 25,000 Da each) and
2 identical heavy chains (MW 50,000 Da each).

Fig 11.2
Reference: Apurba S Sastry 3rd edition
CLASSES OF IMMUNOGLOBULIN
H chain type

L chain Type

HEAVY AND LIGHT CHAIN DOMAIN:

● Light chain – one variable domain (VL) and one constant domain (CL)
● Heavy chain – one variable domain (VH) and 3 to 4 constant domain (CH)

Heavy chain Number of constant domains

γ, α, δ 3 (CH1, CH2, CH3)

μ, ε 4 (CH1, CH2, CH3, CH4)

HINGE REGION: Sensitive to enzymatic digestions

● In γ, α, δ heavy chains - between CH1 and CH2 domain
● In μ, ε heavy chains – the absence of hinge region, instead their constant region has an
additional domain (CH4)
ENZYMATIC DEGRADATION:

Enzyme Fragments

Papain – above disulfide bridge of hinge region 2 Fab and Fc fragment

Pepsin- below disulfide bridge of hinge region F(ab’)2 fragments i.e. 2 Fab subunits bound
together and many smaller fragments

Mercaptoethanol – cleaves only disulfide bond 2 H chains and 2 L chains

reduction of Ig molecules sparing peptide bonds

IMMUNOGLOBULIN CLASS:
IgG:
● Secreted by plasma cells
● Most abundant
● Monomer
● Constitutes 65 – 70% of total Ig in the body.
● Has 4 subclasses IgG1, IgG2, IgG3, and IgG4 - that differ in the amino acid sequence of
the constant region of γ heavy chain.

FUNCTIONS OF IgG:
● Can cross placenta provide passive immunity to the fetus and newborn.
● Activates Classical complement pathway when IgG binds to an antigen.
● Free antigen in ECF when bound to IgG causes phagocytosis.
● Mediates Precipitation and neutralization reactions
● Produced in abundant amount in the secondary immune response.
 ● Mediate ag reaction by binding to Protein A of Staphylococcus aureus.

IgM:
● Secreted by plasma cells
● Has higher molecular weight
● Present only in the intravascular compartment
● Exists in both Pentameric and Monomeric form
● Pentamer – Monomeric units are joined together by the J chain and have 10
antigen-binding sites.

FUNCTIONS OF IgM :
● The first antibody to be produced in acute infection – primary immune response.
● Activated classical complement pathway through its pentameric form.
● Fuse with B cell surface in monomeric form and serves as B cell receptor for antigen
binding.
● Acts as opsonin
● The first antibody to be synthesized in fetal life (20 weeks) provides fetal immunity.
● Protects against intravascular microorganisms.
● Mediates agglutination in case of mismatched blood transfusion (Type II
Hypersensitivity).

IgM DETECTION IN INFECTIOUS DISEASES:

● If serum demonstration shows High IgM levels it indicates acute infection
● Detection is useful in the diagnosis of congenital infections, syphilis, rubella, HIV
infection, and toxoplasmosis.

IgA:
● The most abundant class is next to IgG.
● Constitutes 10 – 15 % of total Ig
● Exists in both monomeric and dimeric form

SERUM IgA:
● Predominantly monomeric form
● Interacts with Fc receptors - initiate antibody-dependent cell-mediated cytotoxicity
(ADCC), degranulation of immune cells, etc...
SECRETORY IgA:
● Dimeric in nature
● Two IgA monomer units linked by J chain, adding to that there is another joining segment
called Secretory component
● Dimers are synthesized by plasma cells near the mucosal epithelium
● The secretory component is synthesized by mucosal epithelial cells

LOCATION: In body secretions like milk, saliva, intestinal and respiratory tract mucosal
secretions.

FUNCTION:

● Mediates local or mucosal immunity

● Provides protection against pathogens by cross-linking bigger antigens with multiple
epitopes and preventing their entry through the mucosal surface
● Breast milk rich in secretory IgA provides protection to immunologically immature
infant’s gut.

SUBCLASSES OF IgA:

● IgA1: Dominant in serum

● IgA2: Higher concentrations in secretion.

IgE:
● Secreted by plasma cells
● Monomers
● Lowest serum concentration
● Extravascular in distribution
● Heat labile (inactivated at 56 degrees C)
● Affinity for the surface of mast cells.

FUNCTIONS OF IgE:
● Mediate Type I Hypersensitivity
● Seen in response to various allergic conditions such as asthma, anaphylaxis, hay fever.
● Elevated in helminthic infections by ADCC
IgD:
● Secreted by plasma cells
● Monomer
● Found on the surface of B cell – act as B cell receptor along with IgM

ABNORMAL Ig:
BENCE JONES PROTEINS:
● Produced in the neoplastic condition of plasma cells called multiple myeloma
● Also called light chain disease as cancerous plasma cells produce excess light chains
● It is accumulated in the patient’s serum and excreted in the urine.
● They get coagulated at 50-degree C and redissolving at 70-degree C.

WALDENSTROM’S MACROGLOBULINEMIA:
● B cell lymphoma producing excess IgM

HEAVY CHAIN DISEASE:

● Excessive production of heavy chains that are short and truncated.

CRYOGLOBULINEMIA:

● Seen in multiple myeloma and Hepatitis C infection.

IMMUNOFLUORESCENCE METHOD:
Fluorescence – absorbing high energy shorter wavelength UV light rays by fluorescent
compounds which in turn emit visible light rays with a low energy longer wavelength.

● Fluorescent dye is used to conjugate the antibody

● This labeled antibody can be used to detect antigen or antigen-antibody complex on cell
surfaces.
● The fluorescent compound commonly used is Fluorescein isothiocyanate (FITC)
TYPES:
DIRECT IMMUNOFLUORESCENCE ASSAY:

Sample containing cells with surface antigen is smeared on a slide

A primary antibody specific to the antigen, tagged with a fluorescent dye is added

The slide is washed to remove unbound antibodies

It is then viewed under a fluorescence microscope.

INDIRECT IMMUNOFLUORESCENCE ASSAY: (detects antibodies in serum)

Test serum-containing primary antibody is added to the slide

The slide is washed to remove unbound antibodies

The secondary antibody is added

Slide is washed

Viewed under microscope

APPLICATIONS:
⮚ Detection of Autoantibodies; E.g. Antinuclear antibodies in autoimmune diseases.
⮚ Detection of microbial agents; E.g. Rabies antigen in corneal smear
⮚ Detection of viral antigens in cell lines inoculated with specimens
Reference: Apurba S Sastry 3rd edition
 6. CELLS INVOLVED IN IMMUNITY AND THEIR
FUNCTIONS:

CYTOKINES – are soluble products secreted by cells of the immune system. E.g., interferons,
interleukins, tumor necrosis factor (TNF), etc ...,

LYMPHOID CELLS:
● They form the major component of cells of the immune system.

● The development of lymphoid cells occurs in lymphoid organs such as bone marrow
and thymus (primary/central lymphoid organs).

● Progenitor T-cells and B-cells originate in the bone marrow. B-cells develop in the
bone marrow itself but progenitor T-cells migrate to the thymus for further
development.

● CD/CLUSTER OF DIFFERENTIATION MOLECULES:

Surface markers are used to identify cells of the immune system.
 FUNCTIONS:
1. Act as surface receptors
2. Cell adhesion
Eg. CD4- helper T-cells
CD8 - cytotoxic T-cells

● NAIVE LYMPHOCYTES:
○ Resting lymphoid cells that do not interact with antigens.
○ Small in size
○ Life span- 1 to 3 months

● LYMPHOBLASTS:
○ Naive cells interact with an antigen and get activated in presence of certain
cytokines to become lymphoblast.

DIFFERENTIATES INTO:

Naive cell Effector cell Memory cell

Location Secondary lymphoid Inflamed tissues Both the locations of naive

(present organs, e.g., lymph nodes, and mucosal and effector cell
mostly in) spleen surfaces

Cell cycle Dormant (G0 phase) Active Dormant (G0 phase)

Morphology Small lymphocyte Large Small lymphocyte
lymphocyte

Life span Short Short Long

Function Transforms to effector cell Eliminate antigen Transforms to effector cell

on primary exposure to on secondary exposure to
antigen, occurs slowly due antigen, occurs fast without
to lag period a lag period

T- LYMPHOCYTES:
● Constitutes 70-80% of blood lymphocytes.
● Bears special surface receptor called T-cell receptor (TCR).
● TCR recognizes antigens presented by antigen-presenting cells (APC).

● ACTIVATION OF TCR:
➔ TCR has 2 polypeptides: alpha and beta chains
➔ Each polypeptide has 3 domains – extracellular, transmembrane, and cytoplasmic
tail.
➔ TCR is active when both alpha and beta chains form a complex with CD3
molecules.
➔ A CD3 molecule has 3 pairs of polypeptides:
1. zeta homodimer
2. Delta-epsilon heterodimer
3. Gamma-epsilon heterodimer

Antigen binds to alpha and beta chains of TCR, a signal is generated which is transmitted
through the CD3 complex leading to activation of T-cells.
● DEVELOPMENT OF T-LYMPHOCYTES:
➔ Occurs in the thymus under the influence of thymic hormones and lymphopoietic
growth factor IL-7.
➔ Characteristic changes occur in cell surface markers during their development.

● STEPS OF T-CELL DEVELOPMENT:

● The fate of Double positive T-cell:

 1. POSITIVE SELECTION: The DPT-cells that can recognize MHC complex are
positively selected. Hence, they are called MHC-restricted cells.
2. DEATH BY NEGLECT: 95% of DPT -cells fail +ve selection because they do not
recognize MHC complex.
3. NEGATIVE SELECTION: Some of the MHC-restricted T-cells react with
self-antigens. They are killed by apoptosis and removed.
4. MATURATION:
➔ The remaining DPT-cells shut off the expression of either CD4 or CD8.
Some express only CD4 and some express only CD8. Hence, they become
Single positive mature T-cells.
● E.g., T-H cells express only CD4 (i.e., CD4+ CD8-)
● T-C cells express only CD8 (i.e., CD4- CD8+)
➔ They acquire thymus-specific antigen, move to lymphoid organs, and
respond to antigenic stimuli.

● TYPES:
HELPER T-CELLS:
● Has CD4 surface receptors.
● Recognize antigenic peptides presented with MHC-II molecules.
● Has three types:

CYTOTOXIC T-CELLS:

● Has CD8 surface receptor.

● Recognize intracellular antigens presented with MHC-I molecules.
● Involved in the destruction of virus-infected cells and tumor cells.

REGULATORY T-CELLS:

● Regulates immune system and provides peripheral tolerance.

● Surface receptors: CD4, CD25, Foxp3.
● Deficiency of Foxp3 causes IPEX-Syndrome (Immune dysregulation,
Polyendocrinopathy, Enteropathy X-linked syndrome).

GAMMA-DELTA T-CELLS:
 ● TCR of these cells has gamma and delta peptides instead of alpha and beta peptides
hence lack both CD4 and CD8 surface receptors.
● They do not require antigen processing and MHC presentation.
● Found in gut mucosa within intraepithelial lymphocytes (IELs).
● They probably encounter the lipid antigens that enter through the intestinal mucosa.
● They are part of innate immunity.

B-LYMPHOCYTES:

● Constitutes 10 to 15% of blood lymphocytes.

PLASMA CELLS:

● They are antibody-secreting cells.

● Oval-shaped with oval-shaped nucleus(has cartwheel appearance).

● Short life span- 2 to 3 days

DIFFERENCE BETWEEN T-CELLS AND B-CELLS

Property T-cell B-cell

Origin Bone marrow Bone marrow

Maturation Thymus Bone marrow

Peripheral blood 70-80% of total lymphocytes 10-15% of total

Lymphocytes

Antigen recognition T-cell receptors complexed B-cell receptor -surface IgM or IgD
Receptors With CD3 complexed with Ig alpha/Ig beta

CD markers CD3,4,8 CD 19,21,24

Thymus specific antigen Present Absent

Microvilli on the surface Absent Present

NATURAL KILLER CELLS (NK CELLS):

● Large granular lymphocytes.

● They are cytotoxic but antigen nonspecific. Act against virus-infected cells and tumor
cells.
● Act as the first line of defense in innate immunity and do not require prior contact with
antigens. They don’t differentiate into memory cells.
● Cell markers: CD16 and CD56.
● MECHANISM OF NK CELL-MEDIATED CYTOTOXICITY:

➔ NK cells can distinguish between normal cells and altered cells because of the
presence of two types of receptors (theory of opposing-signals model).
1. Activation receptors:
● Engagement of these receptors with ligands present on target cells
activates NK cells.
2. Inhibitory receptors:
● Recognizes and binds to MHC-I molecules present in all normal
nucleated cells. Sends inhibitory signals to suppress NK cells even
if bound to activation receptors because the inhibitory signal is
dominant.
● MHC-I expression in virus-infected cells and tumor cells is
reduced hence no inhibitory signal.

● TARGET CELL DESTRUCTION:

Secretion of perforins

Formation of pores

on target cells

entry of granzymes
 lysis of target cells

MACROPHAGES:

● When monocytes migrate to tissues they become macrophages.

● If they are motile called free or wandering macrophages and if they reside in a
particular tissue and non-motile then called fixed macrophages.
● Two important functions are phagocytosis and antigen presentation.

● TYPES OF MACROPHAGES

Body sites Macrophage designation

Peripheral blood Monocytes

Tissues Macrophages

1. Liver Kupffer cells

2. Brain Microglial cells

3. Kidney Mesangial cells

4. Lungs Alveolar macrophages

5. Bone Osteoclasts

6. Inflammation site Langhans giant cells

7. Connective tissues Histiocytes

8. Placenta Hufbauer cells

9. Lymphoid follicle Tingible body macrophage

DENDRITIC CELLS:
● Possess long membranous cytoplasmic extensions.
● Their function is to capture, process, and present the antigenic peptides on their cell
surface to helper T-cells.

ANTIGEN-PRESENTING CELLS (APCS):

○ T-cells cannot recognize antigens by themselves hence APCs present antigenic
peptides to helper T cells and cytotoxic T cells by completing with MHC-II &
MHC-I respectively.
○ E.g., Dendritic cells, macrophages, and B-cells.
ANTIGEN-PRESENTING PATHWAYS:
i. CYTOSOLIC PATHWAY -endogenous antigens processed and presented along with
MHC-I to CD8 T-cells.
ii. ENDOCYTIC PATHWAY – exogenous antigens are processed and complexed with
MHC-II to CD4 T-cells.

References: Essentials of microbiology 3rd edition – Apurba Sastry

 7.HYPERSENSITIVITY

Hypersensitivity refers to the injurious consequences in the sensitized host, following subsequent
contact with specific antigens.

● Results due to abnormality of either humoral or cell-mediated immunity

● 4 types of Hypersensitivity reactions

Immediate Hypersensitivity reactions – Occur immediately (minutes – hours)

● Due to exaggerated humoral response (antibody-mediated)

TYPE 1: – IgE mediated

● Causes mast cell degranulation after contact with soluble antigen

TYPE 2: – IgG (or rarely IgM) mediated

● Causes complement activation or antibody-dependent cellular cytotoxicity (ADCC)
● In response to cell surface-bound antigen

TYPE 3:– Immune-complex mediated

● Due to interaction of soluble antigen + antibody = abnormal inflammation

DELAYED HYPERSENSITIVITY REACTION (TYPE4) – occurs 48-72 hrs after

antigen exposure

● Delayed hypersensitivity T cells mediated (T DTH cell) – sensitized T cells

● Delayed Hypersensitivity cannot be passively transferred by serum but can be transferred
by lymphocytes or the transfer factor.
 Ananthanarayan and Paniker’s Textbook of Microbiology – 10/E- Pg. 158 - Fig 14.5 (a) and (b)
Mechanism of cell-mediated hypersensitivity (delayed-type)

TYPES OF TYPE 4 HYPERSENSITIVITY REACTIONS:

1. TUBERCULIN INFECTION TYPE:

● Tuberculin test: Prototype of delayed hypersensitivity

● Sensitized individuals + Preparation of tuberculin antigen (glycerol extract of the tubercle
bacillus) intradermal injection 🡪 local reaction develops after 48-72 hours (induration
surrounded by erythema)
● Unsensitised individuals 🡪 No response
● Useful indication of the state of delayed hypersensitivity to the bacilli
● Develops in: acute/chronic bacterial, fungal, viral, parasitic infections, allograft reactions,
and in many autoimmune diseases

2. CONTACT DERMATITIS:

● Many antigens such as nickel, poison oak act by producing DTH response
● Substances (haptens) + Skin proteins (carrier) 🡪 Immunogenic Hapten complex
 ● Hapten-skin protein complex is internalized by skin APCs, then present to TH cells to
induce a TDH reaction
● Activated macrophages release lytic enzymes 🡪 skin lesions

ROLE OF DTH:

● PROTECTIVE RESPONSE: Pathogens are cleared with little tissue damage

● TISSUE DAMAGE RESPONSE: When intracellular microbes escape the macrophages,
enhanced phagocytic activity and release of various lytic enzymes by activated
macrophages leads to nonspecific tissue destruction

PATHOLOGY OF DTH REACTION (Granuloma formation)

Continuous DTH reaction for killing the intracellular microbes 🡪 Formation of granuloma

● Initial TH cell infiltrate 🡪 replaced by macrophages in 2-3 weeks

● Macrophages transform into 2 types of cells:
➔ Epithelioid cells - Become large, flat,
eosinophilic
➔ Epithelioid cells occasionally fuse (induced by
IFN-γ) to form multinucleated giant cells

Granuloma consists of an inner zone of epithelioid cells,

typically surrounded by a collar of lymphocytes and a
peripheral rim of fibroblasts and connective tissues

SCHWARTZMAN REACTION

● Intravenous injection of a substance followed by intradermal injection of the same agent

evokes reaction at the site of the second inoculation
 ● Leading to disseminated intravascular coagulation + thrombohemorrhagic reaction at the
local site

CLINICAL IMPLICATIONS:

● Waterhouse - Friderichsen syndrome

● Disseminated intravascular coagulation (DIC)

Features of various types of Hypersensitivity reactions

Type I Type II Type III Type IV

Immune response Humoral Humoral Humoral Cell mediated

altered

Immediate or Immediate Immediate Immediate Delayed

delayed

The duration 2-30 minutes 5-8 hours 2-8 hours 24-72 hours
between the
appearance of
symptoms and
antigen contact

Antigen Soluble Cell Soluble Soluble or

surface-bound bound

Mediator IgE IgG Ag-Ab complex TDTH cell

Effector Mast cell ADCC Complement Macrophage

mechanism degranulation Complement– activation and activation leads
mediated inflammatory to phagocytosis
cytolysis response or cell
cytotoxicity

Desensitization to Easy, but Easy, but Easy, but Difficult, but

the allergen short-lasting short-lasting short-lasting sustained
Typical Anaphylaxis Transfusion Arthus reaction Tuberculin test
manifestations Asthma reactions Serum sickness Granuloma
Atopic Rh Glomerulonephritis formation in
dermatitis compatibility Rheumatoid arthritis tuberculosis,
Hemolytic leprosy, etc
anemia Contact
dermatitis

ANAPHYLAXIS

● classical immediate hypersensitivity that occurs within seconds of the antigen-binding

with IgE bound to mast cells, basophils, and eosinophils
● Prior exposure to the allergen with an interval of at least 2-3 weeks before the sensitizing
dose and the shocking dose
● SHOCKING DOSE : IV > intraperitoneally and subcutaneously > intradermally
● Type 1 Hypersensitivity reactions (immediate anaphylactic hypersensitivity) are of 2
types:
➔ Systemic anaphylaxis: Acute, potentially systemic
➔ Localized anaphylaxis (atopy): Recurrent, non-fatal, typically localized

1. SYSTEMIC ANAPHYLAXIS

● It is an acute medical emergency condition, characterized by severe dyspnoea,

hypotension, and vascular collapse leading to death at times.
● Occurs within minutes of exposure to the allergen
● Unless treated, leads to death
● Drug of choice: Epinephrine
 2. LOCALISED ANAPHYLAXIS (ATOPY)

● The reaction is limited to a specific target tissue or organ and almost always inherited.
● ALLERGIC RHINITIS (Hay fever): Most common
➔ Results from exposure to airborne allergens with the conjunctiva and nasal
mucosa
➔ Symptoms: 🡩 watery secretions of the conjunctiva, nasal mucosa, and upper
respiratory tract sneezing, coughing
● ASTHMA: 2nd most common
➔ Involvement of lower respiratory mucosa 🡪 Contraction of bronchial smooth
muscles, airway edema, 🡩 mucus secretion 🡪 Bronchoconstriction & dyspnea
➔ ALLERGIC ASTHMA: induced by air-borne or blood-borne allergens (pollen,
dust, fumes, insect products, viral antigen)
➔ INTRINSIC ASTHMA: independent of allergen stimulation, induced by cold or
exercise
● FOOD ALLERGY: localized anaphylaxis
➔ Food allergens stimulate the mast cells lining gut mucosa to cause GI symptoms
(diarrhea, vomiting)
● Atopic urticaria (hives) – allergen is deposited on skin, causes local wheal and flare

● ATOPIC DERMATITIS (allergic eczema): inflammatory disease of the skin

➔ Developed during infancy
➔ Manifestation: Erythematous skin eruptions with pus
➔ Skin lesions have increased the response of TH2 cells and eosinophils

DRUG ALLERGY: Can be local or systemic anaphylaxis

MECHANISM OF ANAPHYLAXIS
HYPERSENSITIVITY

● Hypersensitivity refers to the injurious consequences in the sensitized host, following

subsequent contact with specific antigens.

CLASSIFICATION OF HYPERSENSITIVITY REACTIONS

● DURATION
➔ Immediate reactions (Type I, II, III)
➔ Delayed reaction (Type IV)
● MECHANISM – COOMBS & GELL CLASSIFICATION
➔ Immediate Hypersensitivity
■ Type I (Immediate anaphylactic hypersensitivity)
■ Type II (Cytolytic /cytotoxic hypersensitivity)
■ Type III (Immune complex-mediated hypersensitivity)
➔ Delayed Hypersensitivity
■ Type IV (Delayed T-cell mediated hypersensitivity)

TYPE I HYPERSENSITIVITY REACTION

● Altered immediate humoral response in response to soluble antigen, mediated by IgE

● EFFECTOR MECHANISM: Mast cell degranulation
● FACTORS AFFECTING HYPERSENSITIVITY: Genetic makeup, Allergic dose, TH1
vs TH2 response
● DETECTION OF TYPE 1 HYPERSENSITIVITY: Skin prick test, Total serum IgE
antibody, Allergen-specific IgE
● TREATMENT: Avoidance of contact with known allergens, hyposensitization,
monoclonal anti-IgE, drugs (antihistamines, epinephrine, cortisone)
MECHANISM OF HYPERSENSITIVITY TYPE 1 REACTION

(Read mechanism of anaphylaxis)

MANIFESTATION

● IMMEDIATE: (Read Mechanism of Anaphylaxis essay Qn)

● LATE: Mediators, eosinophilic influx, neutrophilic infiltration (4-6 hrs later)

MEDIATORS: released in the acute phase with cytokines, ECF, NCF, and various inflammatory
cells

EOSINOPHILIC INFLUX: Favoured by ECF (eosinophilic chemotactic factor), IL-5, GM-CSF

● Eosinophils (Fc receptors) bind directly to antibody-coated allergens 🡪 release toxic

granules from eosinophils 🡪 chronic inflammation of bronchial mucosa (persistent
asthma)

NEUTROPHILIC INFILTRATION: Induces by NCF (Neutrophil chemotactic factor) &

cytokines

● Activated neutrophils release mediators 🡪 inflammatory tissue damage & thickening of

basement membrane

HYPERSENSITIVITY TYPE II REACTION

● Mediator: Antibodies (IgG or rarely IgM)

● After Ag-Ab binding occurs, the Fc region of the antibody initiates the type II reactions
by the following 3 mechanisms:

COMPLEMENT-DEPENDENT REACTIONS

Fc region of the antibody (bound with antigen) can activate the classical pathway of the
complement system which leads to host injury mediated by:
1. COMPLEMENT-DEPENDENT CYTOLYSIS: Membrane attack complex (C5-C9)
formed 🡪 produce pores 🡪 lysis of target cells
2. COMPLEMENT–DEPENDENT INFLAMMATION: C3a and C5a (chemoattractants)
induce inflammatory response 🡪 tissue injury
3. OPSONIZATION: C3b and C4b (opsonins) deposit on target cells. Phagocytes engulf the
coated target cells via complement receptors

● Antibody-dependent cellular cytotoxicity (ADCC)

1.

● IgG antibodies coat target cells (Fab region). IgE (Fc region) binds to NK cells (Fc
receptors) 🡪 destruction of target cells
 ● Destruction of target cells that are too large to be phagocytized (Parasites, tumors, graft
rejection)
● IgE used sometimes (eosinophil-mediated killing of parasites)

AUTOANTIBODY MEDIATED (ANTIBODY-DEPENDENT CELLULAR

DYSFUNCTION OR ADCD)

● Host produces autoantibodies which bind and disturb the normal human self-antigens

Anti-receptor Ab: Ab directed against human receptors 🡪 inhibition/ excessive activation of

receptors 🡪 host injury

a. ACTIVATION OF THE RECEPTOR (Graves’ disease): Autoantibodies (LATS –

long-acting thyroid stimulators) 🡪 stimulate the thyroid cells to upregulate the production
of thyroid hormones
b. INHIBITION OF RECEPTORS (Myasthenia gravis): Ach receptor antibodies produced
🡪 Block Ach receptors 🡪 profound muscular weakness
c. Other eg: Goodpasture syndrome (Ab against Type IV collagen), Pernicious anemia (Ab
against intrinsic factor), Rheumatic fever (Ab against streptococcal antigens
cross-reacting with heart), Myocarditis in Chagas disease

HYPERSENSITIVITY TYPE III REACTION

● Develop as a result of the excess formation of immune complexes which initiate an

inflammatory response through activation of complement system leading to tissue injury
● Antigen involved: Exogenous antigens such as bacteria or virus or endogenous antigen
such as DNA

MECHANISM OF TISSUE INJURY

1. CLASSICAL COMPLEMENT ACTIVATION

 ● ANAPHYLATOXIN: Complement by-products C3a and C5a (anaphylatoxins) 🡪
localized mast cell degranulation & 🡩 in vascular permeability
● CHEMOATTRACTANT: C3a and C5a (chemoattractants) 🡪 recruitment of
neutrophils to the site of immune complex deposition
● ROLE OF NEUTROPHILS: Attempt to phagocytose the large immune
complexes and release a large number of lytic enzymes from the secretory
granules (frustrated phagocytosis) 🡪 tissue damage
2. PLATELET ACTIVATION
● Immune complexes bind to Fc receptors on platelets 🡪 Platelet activation 🡪
Platelet aggregation (microthrombi formation) 🡪 vasoactive amines released 🡪
tissue ischemia
3. ACTIVATION OF HAGEMAN FACTOR 🡪 leads to activation of kinin 🡪 causes
vasodilation and edema
TYPES OF TYPE III HYPERSENSITIVITY REACTION

● Arthus reaction (local manifestation of generalized hypersensitivity)

A localized area of tissue necrosis due to vasculitis resulting from acute immune complex
deposition at the site of inoculation of antigen

● Produced experimentally – injecting an antigen into the skin of a previously

immunized animal

Circulating antibodies bind with the antigen in the dermis and form immune complexes (fix the
complement) 🡪 localized immune complex-mediated inflammatory response (Arthus reaction).
In humans,

1. IN SKIN: Insect bites, allergic desensitization treatment (repeated injections of the same
antigen for long periods of time)

2. IN LUNGS: Inhalation of bacteria, fungi, spores, or proteins = intrapulmonary lesions

● Farmer’s lung –Inhalation of actinomycetes (Saccharopolyspora species) from moldy hay

● Bird-Fancier’s disease: Inhalation of serum proteins in dust from dried pigeon’s feces

GENERALIZED / SYSTEMIC TYPE III REACTIONS

● Formation of small-sized soluble Ag-Ab complexes

● Induces inflammatory reaction: deposition of immune complexes in various tissues
(vasculitis, glomerulonephritis, arthritis)
● Serum sickness (Historical example)
➔ Horse serum protein (foreign) – induces antibody formation in host 🡪 generation
of a large number of immune complexes
➔ After 7-8 days, manifestations arise 🡪 serum sickness (fever, weakness, vasculitis,
edema, erythema, lymphadenopathy, glomerulonephritis)
➔ Subsides gradually as immune complexes are cleared and free antibodies
accumulate
 8.ANTIGEN-ANTIBODY REACTION

● The antigen and antibody reaction is a bimolecular association where the antigen and
antibody combine with each other specifically, but it does not lead to an irreversible
alternation in either antibody or in antigen.

PROPERTIES OF ANTIGEN AND ANTIBODY REACTION:

1. SPECIFIC: Ag- Ab reaction involves specific interaction between the epitope of
the antigen with the corresponding paratope of the homologous antibody except in
Cross reaction due to sharing of epitopes among different antigens.

2. NONCOVALENT INTERACTIONS: the union of antigen and antibody requires

the formation of a large number of noncovalent interactions between them such as
hydrogen bonds, electrostatic interactions, hydrophobic interactions, and Vander
Wal forces.

3. STRENGTH: The strength or the firmness of the association is influenced by

affinity and avidity.
● Affinity = sum of noncovalent interactions between a single epitope of an
antigen with its corresponding paratope present on the antibody.
● Avidity = term used to describe the affinities of all the binding sites when
multivalent antibody reacts with a complex antigen carrying multiple
epitopes. It increases with time (though IgG has a low avidity initially,
later part of infection will have stronger avidity). Avidity will be much
higher than the individual affinity at each binding site, but lower than the
sum of all affinities. This is because the geometry of the multivalent
antibody gets stretched when it reacts with a complex antigen, thus
resulting in less optimal binding interactions.
Antigen and antibody reactions in vitro are known as serological reactions. Parameters used are:
● Sensitivity: the ability of a test to detect even a small quantity of antigen or
antibody. This property reduces false negatives.
● Specificity: the ability of the test to detect reactions between homologous
antigens and antibodies only, and with no other. This reduces false positives.
● Qualitative assays: refer to the mere detection of the presence of an antigen and
antibody.
● Quantitative assays: refer to the estimation of the quantity of an antigen or
antibody.

USES:
● In the body, they form the basis of antibody-mediated immunity in infectious diseases or
autoimmune diseases.
● In the laboratory, they help in the diagnosis of infections by detection and quantitation of
either antigens and antibodies.
 CLASSIFICATION:

CONVENTIONAL IMMUNOASSAY:
1. PRECIPITATION REACTION:
(Refer to Short notes Below)

2. AGGLUTINATION REACTION:
(Refer to Short notes Below)

3. COMPLEMENT FIXATION TEST:

● To detect the antibodies in the patient’s serum that are capable of fixing with
complements.
● It was widely used for the detection of complement-fixing antibodies in
Rickettsia, chlamydia, mycoplasma infections, and some arboviral infections.

4. NEUTRALIZATION TEST: (less commonly used)

● VIRAL NEUTRALIZATION TEST: detects the presence of neutralizing
antibodies in the patient’s serum.
 ● Serum+ live viral suspension-----> poured into a cell line------->specific serum
antibody neutralizes the surface antigen, making the virus unable to infect a cell
line.
● TOXIN-ANTITOXIN NEUTRALIZATION TEST: eg: Schick test (Diptheria
toxin-antitoxin neutralization test)
Naglers reaction (detection of alpha-toxin of clostridium perfringens).

NEWER TECHNIQUES:
1.ELISA (ENZYME-LINKED IMMUNOSORBENT ASSAY):
● It is an immunoassay that detects either antigen or antibodies in the
specimen, by using an enzyme-substrate chromogen system for detection.
● It has 2 components: Immunosorbent (absorbs the antigen and antibody
present in the serum)and enzyme (used to label antigen and antibody). A
substrate chromogen system is added at the final step.
(Ag- Ab complex)-enzyme + substrate -->activates the chromogen---> color change
detected by spectrophotometry.

STEPS:
● Addition of a reagent
● Incubation
● Washing the wells in the microtiter.
TYPES:
● DIRECT: for detection of antigen in test serum.
well+Ag (test serum) +primary Ab enzyme+ substrate-chromogen----->color
change
 ● INDIRECT: for detection of antibody in serum
Wells coated with Ag+primary Ab (test serum) +secondary Ab
enzyme+substrate- chromogen----->development of color

● SANDWICH: detection of antigen.

Wells coated with capture Ab+Ag (test serum) +primary Ab
enzyme+substrate chromogen----->color development.
 ● IgM ANTIBODY CAPTURE: this is known as amplified sandwich
ELISA.Used for dengue, Japanese encephalitis, leptospirosis, etc.
Wells coated with capture anti IgM Ab+IgM Ab (test serum)+recombinant
antigen+secondaryAbbiotin+avidin-enzyme+substrate-chromogen------>color
.
 ● COMPETITIVE: detection of antigen. In this, the antigen in the test serum
competes with another antigen of the same type coated on the well to bind
to the primary antibody.

Ag- Ab mixture+ well precoated with the same type of antigen---->free

antibodies bind to Ag------>washing secondary enzyme-conjugated
Ab------>washing+ substrate-chromogen------>color.

ADVANTAGES:
High sensitivity
More specific
DISADVANTAGES:
Time-consuming
Expensive
APPLICATION:
For detection of antigen(hepatitis B surface antigen and precore antigen) and
antibody (hepatitis B, HIV, dengue)
 2.ENZYME-LINKED FLUORESCENT ASSAY:
● It is a modification of ELISA. It differs from ELISA being an automated
system and the Ag-Ab-Enzyme complex is detected by the fluorometric
method. Commercially available systems are VIDAS and mini VIDAS.
● Procedure: same as ELISA

Solid phase receptacle+Ag (test serum) +primary Ab-enzyme+substrate

chromogen---->color change
Components used are different:
*Enzyme used is alkaline phosphate
*Substrate used is 4-methyl umbelliferyl phosphate
● ADVANTAGES: automated system
User friendly
More sensitive, specific
Less contamination
● DISADVANTAGES: expensive
Can run only 12-24 tests at a time
● APPLICATIONS:
*Detects infectious diseases like markers of hepatitis virus and HIV, varicella,
and rotavirus.
*Used as biomarkers, tumor markers, cardiac markers, and screening for
allergy.

3.IMMUNOFLUORESCENCE ASSAY:
● It is similar to ELISA but differs by using fluorescent dye for labeling of
antibodies. The fluorescent dye is used to conjugate the antibody and such
labeled antibody can be used to detect the antigens or antigen-antibody
complexes on the cell surface.
● TYPES:
➔ DIRECT: sample on slide primary Ab tagged with fluorescent
dye----->washed----->slide viewed under a fluorescence microscope.
 ➔ INDIRECT: serum-containing primary Ab is added on
slide--->washed+ secondary Ab----->washed----->viewed under a
fluorescence microscope.
● APPLICATIONS:

Detection of antinuclear antibody in autoimmune diseases, microbial antigens,

and viral antigens.

4.CHEMILUMINESCENCE LINKED IMMUNOASSAY:

● It is similar to ELISA; however, the chromogenic substance is replaced by
chemiluminescent compounds like luminol and acridinium ester that
generate light during a chemical reaction (luxogenic). The light can be
detected by a photomultiplier called a luminometer.
(Ag-Ab complex)-enzyme+chemiluminescent substrate------> product+light------>detected
by luminometer.
● ADVANTAGES:
10 more times more sensitive than ELISA.
Tests multiple samples at a time
● APPLICATIONS:
 Detection of antigen and antibodies against viral infections like hepatitis viruses,
HIV, TORCH infections, and biomarkers such as procalcitonin.

5.WESTERN BLOT:
● Detects specific proteins in a sample containing a mixture of antibodies
each targeted against different antigens of the same microbe.
● COMPONENTS:
*SDS PAGE: This is a method that separates complex protein antigen mixture into
individual fragments by treating with sodium dodecyl sulfate and subjected
polyacrylamide gel electrophoresis according to molecular weight.
*NCM blotting: fragments in the gel are transferred to a nitrocellulose membrane sheet.
*Enzyme immunoassay: NCM strip is then treated with patient’s samples containing
antibodies. Individual antibodies bind to respective antigen fragments present on NCM,
which can be subsequently detected by adding enzyme-linked anti-human antibodies.
● APPLICATIONS:
This has excellent specificity. Hence, it is used as a supplementary test to confirm the
results of ELISA or other higher sensitivity tests.
It is used to detect antibodies in various diseases such as HIV, Lyme's disease, herpes
simplex virus infection, and toxoplasmosis.
6.IMMUNOHISTOCHEMISTRY:
● It refers to the process of detecting antigens in cells of a tissue section by
exploiting the principle of using labeled antibodies binding specifically to
the antigens in biological tissues.
● It can be based on principles of ELISA or IFA
● The antibody is conjugated to an enzyme, such as peroxidase, that can
catalyze a color-producing reaction.
● Used in diagnosis of abnormal cells.

7.RAPID TESTS:
● These tests are also called point of care tests because unlike ELISA and
other immunoassays, the POC tests can be performed independently of
laboratory equipment and deliver instant results. Two principles of rapid
tests are available - lateral flow assay and flow-through assay. used for the
diagnosis of various diseases such as malaria, hepatitis B, hepatitis C,
HIV, leptospirosis, syphilis, etc.
 8.IMMUNOCHROMATOGRAPHIC TEST (LATERAL FLOW ASSAY):
● It is based on lateral flow technique
● Can be used for both antigen and antibody
● Antigen detection: The test system consists of an NCM and an absorbent pad.
● NCM has coated two places = test line, coated with monoclonal antibody
targeted against the test antigen, and a control line, coated with anti-species
immunoglobulin. Serum+well+antibody labeled with a chromogenic
marker (colloidal gold or silver) ------>Ag specific Ab colloidal gold
complex and Free colloidal gold-labeled
Ab move laterally along NCM-------->
● At the test line, Ag labeled Ab complex binds to monoclonal Ab to form a
colored band.
● At the control band, Free colloidal gold-labeled Ab binds to anti-human Ig to
form a colored band. (if not formed, then the test is invalid irrespective of test
band)

9. FLOW-THROUGH ASSAY:
● differs from ICT in 2 ways:
● Protein A is used for labeling antibodies instead of gold conjugate
● The sample flows vertically through NCM
● Used for antigen and antibody detection
● HIV TRIDOT test is a classical example. It detects antibodies to HIV1 and
2 separately in patients’ serum.
 Reference: (for diagrams and notes) Essentials of Medical Microbiology by Apurva Sastry,3rd
edition.

AGGLUTINATION REACTION:
● When an insoluble antigen is mixed with its antibody, in the presence of electrolytes at a
suitable temperature and pH, the particles are agglutinated or clumped.
● It is more sensitive than the precipitation test.
● Types:
● Direct
● Indirect (Passive)
● Reverse Passive

● Two main problems pertaining to agglutination are prozone phenomena (excess

antibodies fail to agglutinate with antigen)and blocking antibodies.

DIRECT AGGLUTINATION TEST:

● SLIDE AGGLUTINATION:
● TUBE AGGLUTINATION:
Slide agglutination Tube agglutination Microscopic agglutination

Done to confirm the The test is done for estimating Performed on a microtiter
identification and serotyping antibodies in serum. plate and the result is read
of bacterial colonies grown in under a microscope.
culture.

Used for blood grouping and Used for diagnosis of Done for leptospirosis
Cross-matching. ● Typhoid fever (Widal test):
detects antibodies against
both H (flagellar appears as
loose fluffy clumps) and O
(somatic-appears as chalky
white granular dense
deposits)
● Coombs antiglobulin test
● Acute brucellosis (standard
agglutination test)

The bacterial colony is mixed A fixed volume of a particulate

with a drop of saline on a antigen suspension is added to an
slide to form a uniform equal amount of serial dilutions of
smooth milky white serum sample containing antibodies
suspension. in a test tube

A drop of antiserum (serum

with appropriate antibody)
and shaken thoroughly

*Positive= clumping with *Positive=indicates agglutination

clearing suspension. along with supernatant
*Negative= suspension *Negative= no agglutination.
remains unchanged
INDIRECT OR PASSIVE AGGLUTINATION TEST:(for antibody detection)
● The soluble antigen is coated on a Carrier molecule (RBC, latex) so that the antibody
binds to the coated antigen, and agglutination takes place on the surface of the carrier
molecule.
● Eg: Indirect hemagglutination test: RBC is used as a carrier molecule.
● LATEX AGGLUTINATION TEST (for detection of antistreptolysin antibody):
polystyrene Latex is used as a carrier molecule.It o one of the most widely used tests
because it is simple and rapid.
● REVERSE PASSIVE AGGLUTINATION TEST:(for antigen detection) In this test, the
antibody is coated on a carrier molecule which detects antigen in the patient’s serum.
E.g.:

● REVERSE PASSIVE AGGLUTINATION ASSAY: RBC is used as a carrier molecule.

Earlier, it is used for the detection of hepatitis B surface antigen.
● LATEX AGGLUTINATION TEST: used for the detection of C reactive protein,
rheumatoid arthritis.
● COAGGLUTINATION TEST: staphylococcus aureus (protein A) acts as a carrier
molecule. Earlier, it was used to detect antigens from clinical specimens.
● HEMAGGLUTINATION TEST: It refers to the tests that use RBCs as a source of
antigen.
➔ It is of two types- direct and indirect.
➔ Direct agglutination test: serum antibodies directly agglutinate with
surface antigens of RBCs to produce a matt.
 ➔ PAUL BUNNELL TEST:(using sheep RBCs): for detection of Epstein
Barr virus antibodies in serum.

BLOOD GROUPING
● COLD AGGLUTINATION TEST (using humans RBCs): to detect mycoplasma
antibodies in serum.

PRECIPITATION REACTION:
● When a soluble antigen reacts with its antibody in the presence of optimal pH,
temperature, and electrolytes, it leads to the formation of antigen and antibody
complex in the form of
● Insoluble precipitation band: when solid medium is used{immunodiffusion}
● Insoluble floccules: when liquid medium is used {flocculation test}

CLINICAL APPLICATIONS:
● SLIDE FLOCCULATION TEST (for syphilis, caused by Treponema pallidum):

A drop of antigen + drop of patient’s serum containing antibody in a slide

the precipitate is formed as floccules.

 ● ELEKS GEL PRECIPITATION TEST (for Diptheria, caused by Corynebacterium
diphtheriae):
The strain isolated streaked onto a medium containing a filter paper soaker with
diphtheria antitoxin.
Toxin + antitoxin--------> arrow shaped precipitation band.
 9.AUTOIMMUNITY

SCHWARTZMAN’S REACTION

● The Shwartzman phenomenon is a rare reaction of a body to particular types of toxins, called
endotoxins, which cause thrombosis in the affected tissue.
● Ninety years ago, Gregory Shwartzman first reported an unusual discovery following the
intradermal injection of sterile culture filtrates from principally Gram-negative strains
from bacteria into normal rabbits.
● If this priming dose was followed in 24 h by a second intravenous challenge (the
provocative dose) from the same culture filtrate, dermal necrosis at the first injection site
would regularly occur.
● The occasional occurrence of typical pathological features of the generalized Shwartzman
reaction limited to a single organ is notable in many well-known clinical events (e.g.,
hyper-acute kidney transplant rejection, fulminant hepatic necrosis, or adrenal
apoplexy in Waterhouse-Fredrickson syndrome).

TYPES:
● Local Schwartzman reaction
● Systemic Schwartzman reaction

LOCAL SCHWARTZMAN REACTION:

● An intravenous preparatory injection of endotoxin followed by intradermal injection of
endotoxin 24 hours later elicits a thrombohaemorrhagic lesion only at the site of
intradermal injection of endotoxin in the local Shwartzman reaction.

SYSTEMIC SCHWARTZMAN REACTION

● Two intravenous injections of endotoxin spaced 24 hours apart induced a systemic
generalized Shwartzman reaction characterized by coagulopathy, petechial hemorrhages,
microthrombi, and decreased circulating platelets similar to DIC (disseminated
intravascular coagulation)

● Prior to the endotoxin injection, biopsies of the skin show normal vessels without
microthrombi or significant inflammation.
 ● Since endothelial cells line the small vessels in the dermis, where a Shwartzman reaction
appears to be initiated, it is likely that endothelial cells are important for initiating a
local Shwartzman reaction.

● IL-1 and TNF can substitute for the intradermal injection of endotoxin in the local
Shwartzman reaction, induce endothelial cells to become thrombogenic, and can induce
the expression of cell adhesion molecules on endothelial cells making endothelial cells
more sticky for leukocytes.

MECHANISM OF THE SCHWARTZMAN PHENOMENON:

● priming of resident macrophages by the first dose of LPS

● IL-12 mediated activation of lymphocytes which then produce large amounts of IFNℽ
that primes/Further activates the macrophages.
● The subsequent dose of LPS produces massive production of TNF⍺, IL-1, and other
inflammatory cytokines and procoagulants that induce local thrombosis and necrosis

( LPS-lipopolysaccharide; IL-interleukin; INF-interferon; TNF-tumor necrosis factor )

AUTOIMMUNITY:
● It is a condition in which the body’s own immunologically competent cells or
antibodies act against its self-antigens resulting in structural and functional damage.
● Normally immune system does not react to its own antigens due to a protective
mechanism called tolerance.
● Any breach in tolerance mechanisms predisposes to several autoimmune diseases.

TOLERANCE:
● It is a state of unresponsiveness of the immune system towards his/her own tissue
antigens.
● It is mediated by two broad mechanisms
➔ Central Tolerance:
➔ Peripheral Tolerance:

● CENTRAL TOLERANCE:
This refers to the deletion of self-reactive T and B lymphocytes during their
maturation in central lymphoid organs (i.e. in the thymus for T cells and in the bone
marrow for B cells )

● In thymus:
Any developing T cell that expresses a receptor for a self-antigen (presented by a
thymic APC in association with self - MHC) is negatively selected. i.e. deleted by
apoptosis.
● In bone marrow:
When a developing B cell in bone marrow encounter a self-antigen during their
development, the tolerance is developed by
i. Receptor editing:
 The process by which B cells reactivate the machinery of antigen receptor
gene rearrangement, so that a different (edited) B cell receptor will be
produced that no longer recognizes the self-antigen.
ii. Negative selection:
After receptor editing, if the B cells again recognize a self-antigen, then they
are destroyed by apoptosis.

However many self-reactive B and T cells escape into the circulation.

● PERIPHERAL TOLERANCE:
It is the collection of mechanisms that occur in the peripheral tissues to counteract
the self-reactive B and T cells that escape central tolerance.
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:
It is defined as unresponsiveness to antigenic stimulus.
If self-reactive T cells recognize APC with a self-antigen, that does not bear the
co-stimulators, a negative signal is derived and the cell becomes anergic.
3) PHENOTYPIC SKEWING:
The self-reactive T cells after being activated might secrete nonpathogenic
cytokines and chemokine receptors profile and hence fail to produce an
autoimmune response.
4) APOPTOSIS BY AICD:
The activation of self-reactive T cells induces upregulation of the Fas ligand which
interacts with the death receptor Fas leading to apoptosis.
This mechanism is known as activation-induced cell death.

5) REGULATORY T CELLS:
 These cells can downregulate the self-reactive T cells through secreting certain
cytokines (IL-10 and transforming growth factor-beta(TGF beta) or killing by direct
contact)
6) DENDRITIC CELLS(DCS):
Some immature and tolerogenic DCs downregulate the expression of costimulatory
ligands like CD40 and B7 or induce T regulatory cells.
7) SEQUESTRATION OF SELF-ANTIGEN:
Certain self-antigens can evade immune recognition by sequestration in
immunologically privileged sites e.g. corneal proteins, testicular antigens, and
antigens from the brain.
● B cells also exhibit peripheral tolerance. About 10% of the escaped self-reactive
B cells are destroyed in the spleen by several mechanisms such as
downregulation of a B cell growth factor called B cell-activating factor (BAFF).
Autoimmunity results due to failure of one or more of the Mechanisms of Immunological
Tolerance.
MECHANISM OF AUTO IMMUNIZATION (AUTOANTIBODY FORMATION
THEORIES)

a) BREAKDOWN OF T CELL ANERGY:

● Normal cells that do not usually express costimulatory molecules (B7) can be induced
to do so.
 ● Such induction may occur in presence of necrosis and local inflammation.
● This mechanism has been postulated for multiple sclerosis, rheumatoid arthritis, and
psoriasis.
b) FAILURE OF AICD:
● Failure of autoreactive T cells to undergo activation-induced cell death.
● It is observed in systemic lupus erythematous.
c) LOSS OF REGULATORY T CELLS:
● Autoimmunity can result from the loss of regulatory T cell-mediated suppression of
self-reactive lymphocytes.
d) PROVIDE T CELL HELP TO STIMULATE SELF REACTING B CELLS:
● Antibody response to self-antigens occurs only when potentially self-reactive B cells
receive help from T cells.
● e.g. In autoimmune hemolytic anemia, administration of certain drugs may induce
RBCs to create antigens recognizable by helper T cells.
e) RELEASE OF SEQUESTRATED ANTIGENS:
● Injury to certain organs may lead to the release of these antigens.
● These are seen as foreign by the immune system since they are never been exposed to
the tolerance mechanisms during the development of immune cells.
● e.g. Spermatazoa and ocular antigens released after trauma or surgery can cause
post-vasectomy orchitis and post-traumatic uveitis.
f) MOLECULAR MIMICRY:
● Some microbes share antigenic determinants (epitopes) with self-antigens and an
immune response against such microbes may produce antibodies that can cross-react
with self-antigens.
● e.g. Acute rheumatic fever results due to antibodies formed against streptococcal
antigens (M protein), cross-react with cardiac antigens (glycoproteins).
g) POLYCLONAL LYMPHOCYTE ACTIVATION:
● Several microbes and their products are capable of causing polyclonal (i.e. antigen
nonspecific) activation of B and T cells.
 ● Polyclonal T cell activation is induced by staphylococcus aureus and Polyclonal B
cell activation is induced by HIV.
h) EXPOSURE TO CRYPTIC SELF EPITOPES:
● During development, some nondominant cryptic epitopes remain sequestrated. Hence
T cell clones against such epitopes are not deleted.
● Such cryptic self-antigens can be released secondary to inflammation, can cause
an immune response.
i) EPITOPE SPREADING:
▪ The self-peptides released due to persistent inflammation induce tissue damage and
are processed and presented by APCs along with microbial peptides.
▪ It is possible that there may occur a shift or spread of T cell recognition to self
epitopes.
j) BYSTANDER ACTIVATION:
▪ It is the nonspecific activation of bystander self-reactive T helper 1 cells.
▪ Activation of these cells leads to cytokine influx which causes an increased
infiltration of various nonspecific T cells at the site of infection.
SYSTEMIC AUTOIMMUNE DISORDERS:

The autoimmune diseases that involve many organs and cause systemic manifestations are
called systemic autoimmune diseases.

SYSTEMIC LUPUS ERYTHEMATOSUS:

● Autoantibodies and self antigens:
 ➔ Autoantibodies are produced against various tissue antigens such as DNA, a
nuclear protein, RBC, and platelet membranes.
● Immune response and important features:
➔ Women are commonly affected
➔ Female: male ratio is 10:1
➔ Immune complexes are formed, which are deposited in various organs.
➔ Major symptoms are Fever, butterfly rash over cheeks, arthritis, pleurisy, and renal
dysfunction.

RHEUMATOID ARTHRITIS:
● Autoantibodies and self antigens:
➔ A group of antibodies against host IgG antibodies is called the RA factor.
➔ It is an IgM antibody directed against the Fc region of IgG
➔ Anticitrullinated peptide antibodies are also produced.
● Immune response and important features:
➔ Women of 40-60 years are commonly affected.
➔ Autoantibodies bind to circulatory IgG, which are deposited in the joints and can
activate the complement cascade.
➔ The main features are Arthritis ( chronic inflammation of the joints, begins at
synovium; most commonly affected are small joints of hands, feet, and cervical spine.
➔ Other features include hematologic, cardiovascular, and respiratory systems are
frequently affected.

SYSTEMIC SCLEROSIS / SCLERODERMA:

● Autoantibodies and self antigens:
➔ Nuclear antigens such as DNA topoisomerase and centromere are present in the
heart, lung. kidney, GIT, etc.
● Immune response and important features:
➔ T Helper cell and autoantibody-mediated
➔ Excessive fibrosis of the skin, throughout the body
 ➔ Two types:
DIFFUSE SCLERODERMA: Autoantibodies against DNA topoisomerase is
increased.
LIMITED SCLERODERMA: Autoantibody against centromere is increasingly
characterized by CREST syndrome – Calcinosis, Raynaud phenomenon, Esophageal
dysmotility, Sclerodactyly, Telangiectasia.

SERONEGATIVE SPONDYLOARTHROPATHIES:
● Self-antigens are present on:
➔ Sacroiliac joints and other vertebrae
● Common characteristics:
Present as rheumatoid arthritis-like features, but differ by:
➔ Association with HLA-B27
➔ Pathologic changes begin in the ligamentous attachments and not in the synovium
➔ Absence of RA (hence, the name seronegative)
➔ Involvement of sacroiliac joints

SJORGEN SYNDROME:
● Self-antigens are present on
➔ Ribonucleotide (RNP) antigens
➔ SS-A (Ro) and SS-B (La) are present on salivary glands, lacrimal glands, liver,
kidneys, thyroid.
● Important features:
➔ Leads to immune-mediated destruction of the lacrimal and salivary glands resulting in
dry eyes (keratoconjunctivitis sicca) and dry mouth (xerostomia).

REFERENCES:

● Essentials of medical microbiology – Apurba Sastry Third edition

 10.COMPLEMENT

● Complement (C) is defined as the system of soluble and cell-bound proteins that occur
normally in serum and are activated by antigen-antibody interactions.

COMPLEMENT PATHWAY:
● The C cascade is triggered by three parallel but independent mechanism or pathway,
which differs in the initial step of activation.
1.Classical C pathway
2.Alternative or Properdin Pathway
3. Lectin Pathway

ALTERNATIVE PATHWAY:
● This is an Antibody-Independent pathway
● The activation of C3 without the prior participation of C4b2a is known as the Alternative
Pathway.

ACTIVATORS: -
i. Bacterial endotoxins and teichoic acid of gram-positive bacteria
ii. IgA and D
iii. Cobra venom factor and the nephritic factor
iv. Zymosan
v. Some virus and virus-infected cell
vi. Parasites eg, trypanosomes

STEPS OF ALTERNATIVE PATHWAY:

● Binding of C3b to an activator.C3b is continuously generated in circulation but in a free
state, it is rapidly inactivated by serum protein factors H and I.
● Bound C3b interacts with serum protein called factor B (C3 pro activator) to form an
Mg-dependent complex ‘C3b, B’
● This complex is cleaved by a serum protein factor D (C3 pro activator convertase), into
two fragments, Ba and Bb.
● Fragment Ba is released into the medium and fragment Bb remains bound to C3b,
forming the esterase C3bBb complex, which is the alternative pathway C3 convertase.
● This is extremely liable. The function of properdin (factor P) is to stabilize the C3
convertase, which hydrolyses C3, leading to further steps in the cascade, as in the
classical pathway

Textbook Of Microbiology –Ananthanarayan and Paniker 11th Edition

BIOLOGICAL EFFECTS OF COMPLEMENT ACTION
PHAGOCYTOSIS
● The complement pathway facilitates the uptake and destruction of pathogens of the
phagocytic cell.
● This opsonic effect is based on the presence of phagocytic cells on the surface of
complement receptors.

INFLAMMATORY RESPONSE
● C fragments released during the cascade reaction helps in amplifying the inflammatory
response.
● C2 kinins are vasoactive amines and increase capillary permeability.
● C3a and C5a are anaphylatoxic and chemotactic.

HYPERSENSITIVITY REACTIONS
● C participates in cytotoxic(type II)and immune complex (type III) hypersensitivity
reactions
● The destruction of erythrocytes, following incompatible transfusion and
thrombocytopenia in sedormid purpura are type II reactions.
● C contributes to the pathogenesis of nephrotoxic nephritis and is required for the
production of immune complex diseases such as serum sickness and Arthus reaction.

AUTOIMMUNE DISEASE
● Serum C components are decreased in many autoimmune diseases such as systemic
lupus erythematosus and rheumatoid arthritis
● It also plays an important role in autoimmune hemolytic anemia.

ENDOTOXIC SHOCK
● Endotoxin activates the alternative C pathway.
● During endotoxic shock, there is massive C3 fixation and platelet adherence.
 ● Large-scale platelet lysis and the release of large amounts of platelet factors lead to
disseminated intravascular coagulation and thrombocytopenia.

IMMUNE ADHERENCE
● C bound to antigen-antibody complexes adheres to erythrocytes or to non-primates
platelets.
● C3 and C4 are necessary for immune adherence.

GENETIC DEFICIENCY OF COMPLEMENT SYSTEM

Group Deficiency Syndrome

I C1 inhibitor: autocatalytic Hereditary angioneurotic edema (episodic angioedema of

activation of C1 and the subcutaneous tissues or of the mucosa of the respiratory or
unrestrained breakdown of alimentary tracts)
C4 and C2

Il Early components of SLE and other collagen vascular diseases

classical pathway C1, C2.C4

III C3 and its regulatory protein Severe recurrent pyogenic infections

C3b inactivator

IV C5 to C8 Bacteremia, mainly with gram-negative diplococci,

toxoplasmosis

V C9 No particular disease
 11.HERD IMMUNITY

● The overall immunity of a community/ herd toward a pathogen

● Herd immunity plays a vital role in preventing epidemic disease
● When herd immunity is good, a large percentage of population immunity is present
towards the pathogen
● As a result, epidemics are less likely to occur and can be eradicated easily
● Elements contributing to strong herd immunity
○ Clinical and subclinical cases occurrence in a herd
○ Ongoing immunization process
○ Herd structure – a type of population
○ Type of pathogen
● Herd immunity following vaccination for diseases like
○ Diphtheria and Pertussis vaccine
○ Measles, Mumps, Rubella vaccine
○ Polio
○ Smallpox vaccine
 12.CYTOKINES

DEFINITION:
● Cytokines are a chemical substance which serves as messengers, mediating
interactions and communicators between various immune systems.

MAJOR CLASSES:

● Lymphokines produced by lymphocytes

● Monokines produced by macrophages/macrocytes

● Interleukins produced by WBCs

● Chemokines involved in chemotaxis and other WBCs behavior.

PROPERTIES:
● Produced only after activation of their cells of origin
● Unlike hormones, it has a broad range of effects
○ autocrine effects – acts on the same cell
○ paracrine effects – acts on adjacent cells
○ endocrine effects – acts on distant cells
● Cytokines work together and there are various types of interactions
○ PLEIOTROPY – the same cytokine having different actions on different cells
○ REDUNDANCY – different cytokines having the same effect on the same cells
○ SYNERGY – two cytokines augments each other’s actions
○ ANTAGONISM – may oppose each other actions
○ CASCADE – series of effects mediated by different cytokines
 Ref: Apurba S Sastry, 3rd edition, Chapter 14, Pg. 181.

STRUCTURE:
Glycoproteins weighing less than 30kDa and characterized into four groups

● Hematopoietin family
● Interferon family
● Chemokine family
● Tumor necrosis factor family

FUNCTIONS:
Cytokines are majorly produced by TH cells and macrophages and they produce
overlapping functions

● Promote the development of cellular and humoral immune responses

● Interferon-γ
● IL-2, IL-4, IL-5
 ● Promote various responses of innate immunity
● Induction of inflammatory responses – IL-1, IL-8, TNF-α
● Regulation pf hematopoiesis – IL-2, IL-3, IL-9, IL-11
● Antiviral activity – interferon α and β
● Antitumor activity – TNF α and β
● Pyrogenic activity – TNF- α, IL-1, and IL-6

CYTOKINES AND DISEASES:

Pathogenesis of several diseases characterized by increased expression of cytokines or
their receptors

Disease Pathogens Cytokines Produced by

Septic shock E. coli, Neisseria IL-1, TNF α Macrophages

meningitidis

Toxic shock syndrome Staphylococcus IL-1, TNF α activates T-cells that in turn activates
aureus macrophages

Cancers ed IL-6 TH2 cells, macrophages

Chaga’s disease Trypanosoma cruzi blocking IL-2 action by blocking IL-2 action inhibits TH1
activity leads to immunosuppression

Cytokine storm: Cytokines produced in excess leading to hypercytokinemia cause damage to tissues and
organs seen in graft versus host disease, acute respiratory distress syndrome, COVID-19, sepsis, etc.

CYTOKINES USED IN THERAPY:

● Used as Drugs

▪ Interferon – α for hepatitis B, hepatitis hairy cell leukemia, multiple myeloma

▪ Interferon – β for multiple sclerosis

▪ Interferon – γ For chronic granulomatous disease

● Cytokine toxin conjugates

Used to destroy target cells, here cytokines help binding to target cells so that toxins act
on them

Ref: Apurba S Sastry, 3rd edition, Chapter 14, Pg. 180 – 183
 13.INFECTION
METHODS OF TRANSMISSION OF INFECTION
● DIRECT TRANSMISSION: Occurs when infectious agents are transferred from one
person to another without a contaminated intermediate object. E.g.: Direct contact with body
fluids or bare contaminated hands.

● INDIRECT TRANSMISSION: Transfer through contaminated intermediate objects.

E.g.: Clothes, Fomite.

● AGENTS TRANSMITTED THROUGH CONTACT: Methicillin-Resistant S.

aureus (MRSA), Carbapenem-Resistant Enterobacteriaceae (CRE), Vancomycin-Resistant
Enterococci (VRE), Hepatitis A and E virus.

● AGENTS TRANSMITTED THROUGH DROPLET: Diphtheria, Haemophilus

influenzae type B, N. meningitides, B. pertussis, SARS-CoV2, Mycoplasma pneumonia,
Influenza viruses

● AGENTS TRANSMITTED THROUGH AEROSOL: Mycobacterium

tuberculosis, Measles virus, Varicella (chickenpox and zoster), Smallpox
Route Description

Contact transmission

Direct contact Skin to skin contact and thereby physical transfer of

microorganisms between a susceptible host and an
infected or colonized person (usually healthcare workers,
rarely other patients)
This is the most important and frequent mode of
transmission.

Indirect contact This involves contact of a susceptible host with

contaminated inanimate objects such as:
● Dressings, or gloves, instruments (e.g.
stethoscope)
● Parenteral transmission through Needle or sharp
prick injury, splashes of blood or body fluids or
excretions, contaminated saline flush, syringes,
vials, and bags

Inhalational mode

Droplet transmission Droplets of >5 um size can travel for a shorter distance
(<3 feet).
● Droplets generated from the infected person while
coughing, sneezing, and talking are propelled for
a short distance through the air and deposited on
the host's body.
● This is an important mode of transmission of
agents causing bacterial meningitis, diphtheria,
and RSV, etc.

Airborne transmission This refers to the airborne droplet nuclei (s 5 um size) or

dust particles that remain suspended in the air for a long
time and can travel a longer distance.
● This is a more efficient mode than droplet
transmission.
● Microorganisms transmitted by airborne
transmission include Legionella, Mycobacterium
tuberculosis, measles, and varicella-zoster
viruses.

Vector-borne transmission
 ● Via vectors such as mosquitoes, flies, etc carrying
the microorganisms.
● This is a rare mode of transmission in hospitals.

Common vehicle transmission

Such as food, water, devices, and equipment

SOURCES OF INFECTION:
● ENDOGENOUS SOURCE: Involve patient's microbial flora which may invade die
patient's body during some surgical or instrumental manipulations.
● EXOGENOUS SOURCE: From hospital environment, staff, or patients.
1.Environmental sources include inanimate objects, air, water, and food in the hospital.
● Inanimate objects 🡪 Medical equipment (endoscopes, catheters, etc.), bedpans, surfaces
contaminated by patients' excretions, blood, and body fluid.
2.Healthcare workers 🡪potential carriers, harboring many organisms, e.g. nasal carriers of
Methicillin-resistant Staphylococcus aureus (MRSA).
3. Other patients of the hospital may also be the source of infection.
SHORT NOTES
 1.HETEROPHILE ANTIGEN

HETEROPHILE ANTIGEN:
1. Antigens belonging to two different species are called heteroantigens. like plants,
animals, microorganisms.
2. They are heteroantigens that are present in two different species, but they share epitopes
with each other.
3. Antibodies formed against an antigen of one species cross-react with the other and vice
versa.

DIAGNOSIS
● Used in various serological tests.
●
NAME OF WEIL-FELIX PAUL-BUNNE COLD STREPTOCOCCUS
TEST REACTION L TEST AGLUTINATION MG TEST
TEST

DONE FOR Typhus Infectious Primary atypical Primary atypical

mononucleosis pneumonia pneumonia

CAUSATIVE Rickettsia Epstein-Barr Mycoplasma Mycoplasma

ORGANISM
virus pneumonia pneumonia

AG1 Rickettsial antigen Epstein Barr Mycoplasma Mycoplasma

antigen pneumonia antigen pneumonia antigen

AG2 Proteus Sheep red blood Human O RBCs Streptococcus MG

cell antigen

Reference: Essentials Of Medical Microbiology By Apurba S Sastry-3/E

 2.COMPLEMENT FIXATION TEST

● The term complement refers to a group of proteins normally found in the serum in an
inactive form, but when activated they augment the immune responses. They constitute
about 5% of normal serum proteins and their level does not increase following either
infection or vaccination.
● Complement fixation test (CFT) detects the antibodies in the patient’s serum that are
capable of fixing with complements. It was once very popular, now is almost obsolete.

APPLICATIONS:
● CFT was widely used for the detection of complement-fixing antibodies in Rickettsia,
Chlamydia, Mycoplasma infections, and some viral infections such as an arboviral virus.
● Complements are also used for various other serological tests such as the Treponema
pallidum immobilization test for syphilis and the Sabin-Feldman dye test for Toxoplasma.
 3.ELISA

● Enzyme-Linked Immunosorbent Assay is a technique for the detection of a variety of

antibodies or antigens or haptens in the specimen

GENERAL FEATURES :
● Tests for specific immunoglobulin classes
● Used for detection of antigens and antibodies
● Substrates are specific for each enzyme

Enzyme Substrate

Horseradish peroxidase Hydrogen peroxide

Alkaline phosphate p-nitrophenyl phosphate

PRINCIPLE :
TYPES OF ELISA:

ELISA Type Used for detection of The enzyme is labeled with

Direct ELISA Antigen Primary antibody

Indirect ELISA Antigen or Antibody Secondary antibody

Sandwich ELISA Antigen Primary antibody – direct sandwich ELISA

Secondary antibody – indirect sandwich
ELISA

Competitive Antigen or Antibody Secondary antibody

ELISA

ELISPOT Cells producing antibody Primary antibody

or cytokine

DIRECT ELISA:
 Essentials of medical microbiology by Apurba S Sastry E/2 Pg.no.136 Fig.no. 12.14

INDIRECT ELISA :

For Antibody detection For Antigen Detection

Antigen coated well Antigen (test serum or sample)

+ +
Serum (antibody to be detected ) Primary antibody
+ +
Secondary antibody labeled with an enzyme Secondary antibody labeled with an enzyme
+ +
Substrate Substrate

Colour detection Colour detection

 Essentials of medical microbiology by Apurba S Sastry E/2 Pg.no.136 Fig.no. 12.15

A - Antibody detection , B - Antigen detection

SANDWICH ELISA :

Direct Sandwich ELISA Indirect Sandwich ELISA

Antibody coated well Antibody coated well

+ +
Serum (Antigen) Serum (Antigen)
+ +
Primary antibody (conjugated) Primary antibody
+ +
Substrate Secondary Antibody (conjugated)
+
Colour detection Substrate

Colour detection
 Essentials of medical microbiology by Apurba S Sastry E/2 Pg.no.136 Fig.no. 12.16

A – Direct Sandwich B – Indirect Sandwich

COMPETITIVE ELISA:

For Antibody detection For Antigen detection

Antigen coated well Primary antibody

+ +
Serum (Non labeled Antibody) Serum (antigen)
+
Enzyme labeled Antibody Mixture
+
Substrate Antigen coated well

Free Primary antibody

If non labeled If non labeled (More antigen in serum -less free primary antibody
antibody present antibody absent present)
+
No color detection Colour detection Secondary Antibody (Conjugated)
+
 Substrate

Colour detection

ELISPOT (ENZYME-LINKED IMMUNOSORBENT SPOT) :

Essentials of medical microbiology by Apurba S Sastry E/2 Pg.no.137 Fig.no. 12.18

WESTERN BOLT
● Technique for detecting specific proteins separated by electrophoresis and by use of
labeled antibodies

PROCEDURE :
APPLICATIONS :
● High specificity
● Supplementary test to confirm the result of ELISA or other immunoassays
● Antibody detection in,
✔ HIV
✔ Lyme’s disease
✔ Herpes simplex virus infection
✔ Toxoplasmosis

APPLICATIONS OF ELISA
ELISA can be used both for antigen and antibody detection

Antigen detection Antibody detection

✔ Hepatitis B surface antigen (HBsAg) ✔ Hepatitis B

✔ NS1 antigen for dengue ✔ Hepatitis C
✔ Rotavirus ✔ HIV
✔ Dengue
✔ Toxoplasma
✔ Leishmaniasis

COOMB’S TEST
● It is an Antiglobulin test
● For detection of incomplete anti-Rh antibodies
PRINCIPLE:

Complete Microbiology for MBBS by Baveja E/1 Pg.no.151 Fig.no.6.5.5

TYPES:

USES:
● For detection of anti-Rh antibodies
● For a demonstration of any type of incomplete antibody
✔ Eg. Brucellosis
 4.FLUORESCENT ANTIBODY TECHNIQUE

● IMMUNOFLUORESCENCE ASSAY (IFA) is a technique similar to ELISA, but differs

by some important features:
○ Fluorescent dye is used instead of enzyme for labeling of antibody
○ It detects cell surface antigens. It is also used to detect antibodies bound to cell
surface antigens, unlike ELISA which detects free antigen or antibody.

PRINCIPLE
● Fluorescence refers to absorbing high energy-shorter wavelength ultraviolet light rays by
the fluorescent compounds and in turn, emitting visible light rays with a low
energy-longer wavelength.
● The Fluorescent dye is used to conjugate the antibody and such labeled antibody can be
used to detect the antigens or antigen-antibody complexes on the cell surface
● The Fluorescent compounds commonly used is fluorescein isothiocyanate

TYPES:
● Direct immunofluorescence assay
● Indirect immunofluorescence assay
● Flow cytometry

1. DIRECT IMMUNOFLUORESCENCE ASSAY

o Step 1: Sample containing cells carry specific antigens smeared on a slide
o Step 2: Primary antibody specific to antigen tagged with a fluorescent dye added
o Step 3: Slide washed to remove unbound antibodies and viewed under a
fluorescence microscope

2. INDIRECT FLUORESCENCE ASSAY

o Step 1: Test serum-containing primary antibody added to the slide
o Step 2: Slide washed to remove unbound antibodies and then secondary antibody
added
o Step 3: Slide washed and then viewed under a fluorescence microscope

Applications include
▪ detection of autoantibodies in autoimmune diseases
▪ detection of microbial antigens
▪ detection of viral antigens

3. FLOW CYTOMETRY
o Laser-based technology, quantitatively analyze and separate cells as they pass
through a laser beam
o Used to analyze multiple parameters of cells such as cell counting, sorting,
analysis of size, shape, granularity, DNA or RNA

Applications include
▪ CD4 T cell count in HIV infected patients
▪ Detection of leukocytes with specific markers for diagnosis of various
lymphomas
 5.ELEK’S GEL PRECIPITATION TEST

● In vitro test.
● As described by Elek, 1949.
● Type of immuno-diffusion in a gel.
● Media contains a filter paper soaked with antitoxin; the isolated strain is streaked onto it.
➔ If the stain is a toxin, it liberates the toxin.
➔ Toxin diffuses into the agar.
➔ Meets the antitoxin.
➔ Produce an arrow-shaped precipitation band.
● This test is used to find the relatedness between the strains found during outbreaks
● 3 Patterns are observed when precipitate bands of outbreak isolates (streaked adjacent)
meet with each other.
➔ Cross over with each other - unrelated strain.
➔ Spur formation - partially related strain.
➔ Fused - identical strain.

➔ Isolates 1 to 4: toxic strain - toxin released from the strain and antitoxin from the filter
paper has formed a band.
➔ Isolates 1 and 2: unrelated strains - precipitation bands crossed over.
➔ Isolates 2 and 3: partially related - spur formation (partial fusion of precipitation bands).
➔ Isolates 3 and 4: identical strains - the fusion of precipitation bands.
➔ Isolate 5: non-toxigenic - no precipitation band formed.
 6.IMMUNOLOGICAL TOLERANCE
(Essential of Medical Microbiology by apurba S Sastry - 3/E)

State –Individual –Capable of Developing Immune Response Against its own Antigen

CENTRAL TOLERANCE
● Self-reactive lymphocytes – killed at central lymphoid organs
●
● In Thymus:
Self-antigens presented by APCs with self MHC to T lymphocytes
– if found reactive 🡪apoptosis (NEGATIVE SELECTION)

● In Bone marrow:
Developing B cells- tolerance is attained by 🡪
○ Receptor editing: self-reactive B cells – antigen receptor gene rearrangement
○ Negative selection

● Central tolerance is not fully effective

PERIPHERAL TOLERANCE (a backup mechanism)

● IGNORANCE: never recognize self-antigen

● ANERGY: unresponsiveness to antigenic stimulus

○ T cell need MHC + TCR & B7(APC) + CD28(T) co-stimulus
🡪 Sensitization
○ It gets MHC + TCR & B7(APC) + CTLA-4(T) co-stimulus
🡪 Negative stimulus 🡪T cell becomes anergic

● PHENOTYPIC SKEWING:
 ○ T cells undergo activation 🡪but, nonpathogenic CYTOKINES are secreted

● ACTIVATION-INDUCED CELL DEATH (AICD) :

○ Activated cell 🡪 increased Fas 🡪 interaction with FasLingand 🡪Apoptosis

● Treg :
○ Downregulates using IL10, TGF-β, etc.
○ Death by direct contact

● DENDRITIC CELLS:
○ Immature DCs + tolerogenic DCs 🡪 processes Self-antigen
○ Downregulates expression of CD40, B7 🡨 DCs 🡪induction of regulatory T cells

● Sequestration of self-antigen at immune-privileged sites like cornea, testis, brain, etc.

 7.THEORIES OF IMMUNE RESPONSE

● INSTRUCTIVE THEORIES: Postulate that an immunocompetent cell is capable of

synthesizing antibodies of any specificity. The antigen encounters an immunocompetent
cell and instructs it to produce complementary antibody
● SELECTIVE THEORIES: Postulate that immunocompetent cells have a restricted
immunological range. The antigen exerts only a selective influence by stimulating the
appropriate immunocompetent cell to synthesis an antibody

CLONAL SELECTION THEORY:

● Burnet proposed the theory in 1957
● The theory emphasizes the immunological specificity to the cellular level
● According to this theory cells capable of reacting with different antigens were said to be
formed by a process of somatic mutation
● Clones of cells that had immunological reactivity with self-antigens were said to be
eliminated and are called forbidden clones
● The result of the contact with the specific antigen was cellular proliferation to form
clones synthesizing antibody

ABANDONED THEORIES:
SIDECHAIN THEORY:
● Proposed by Ehrlich
● The theory postulated that the cell receptors anchor nutrients to cells before
assimilation and that when foreign antigens are introduced, they combine with cell
receptors that have a complementary fit (side chain)
● This was said to inactivate the receptors and interfere with nutrient absorption
● As a compensatory mechanism, there was said to be an overproduction of the same type
of receptor which then circulates in the blood as antibodies

DIRECT TEMPLATE THEORIES:

● According to these theories, the antigen enters antibody-forming cells and serves as a
‘template’ against which antibodies are synthesized
● Thus produced antibodies have combing sites complementary to the antigenic
determinant
INDIRECT TEMPLATE THEORY:
● The entry of antigenic determinants into the antibody-producing cell induced in it a
heritable change
● A ‘genocopy’ of the antigenic determinant was said to be incorporated in the genome of
the cell and transmitted to the progeny cells (indirect template)

NATURAL SELECTION THEORY :

● According to this theory about a million globulin (antibody), molecules were formed in
embryonic life, which cover the full range of antigenic specificities. These globulins were
said to be ‘natural antibodies’
● When an antigen is introduced, it combines selectively with the globulin that had the
nearest complementary ‘fit’
● This complex was then said ‘home’ in on the antibody-forming cells and stimulate them
to produce the same kind of antibody.
● However, it didn’t explain the fact the immunological memory resides in cells and not in
serum
 8.ADJUVANTS

● A substance that enhances the immunogenicity of an antigen

● Usually added to vaccines to increase the immunogenicity of vaccine antigen

EXAMPLES OF ADJUVANT ACTIVITY:

● Alum
● Mineral oil
● Freund’s incomplete adjuvant – water in oil immersion containing a protein antigen in the
aqueous phase
● Freund’s complete adjuvant – a mixture of Freund’s incomplete adjuvant & suspension of
killed tubercle bacilli in the oil phase
● Lipopolysaccharide (LPS) fraction of gram-negative bacilli e.g. LPS of Bordetella
pertussis – excellent adjuvant for diphtheria & tetanus toxoids
● Other bacteria or their products – Mycobacterium Bovis - Toxoid (diphtheria toxoid &
tetanus toxoids – adjuvant for Haemophilus influenza – type b vaccine)
● Nonbacterial products – silica particles, beryllium, sulfate

MECHANISM OF ADJUVANT ACTION:

3 STEPS
1. Delaying the release of antigen – It precipitates the antigen which is released slowly, thus
prolonging the antigenic exposure
2. By activating phagocytosis – adjuvant – antigen precipitate is large, thus increases the
phagocytosis. The muramyl dipeptide of tubercle bacilli can activate the macrophages
directly
3. By activating helper T cells – activated macrophages release interleukin 11(IL 11) &
express a higher level of MCH-II; thus promote activation of helper T cells which
activates B cells to produce specific antibodies.
 9.MONOCLONAL ANTIBODIES
● Monoclonal antibodies are antibodies that are derived from a single clone of plasma cells
and are produced against a single antigenic determinant (epitope)of an antigen.

PRINCIPLE:
● Antibodies that are usually produced in response to a single antigen (multiple epitopes)
are heterogeneous as they are synthesized by several different clones of cells i.e.,
polyclonal.
● A single antibody-forming cell or clone produces antibodies directed against a single
antigen or antigenic determinant (epitope)only are monoclonal antibodies

TECHNIQUE:
● Antibody-forming spleen cells are fused with myeloma cells to produce hybrid cells
(hybridomas). the resultant hybridoma retains the ability of myeloma cells to multiply
indefinitely.
1. Animal (mice) is injected with an antigen and B lymphocytes are harvested from the
spleen.
2. Spleen cells (B lymphocytes) are then fused with mouse myeloma cells, grown in
culture, which are deficient in the enzyme Hypoxanthine Guanine Phosphoribosyl
Transferase (HGPRT). Fusion is done by incubating these cells in the presence of
Polyethylene Glycol(PEG).
3. The fused cells (hybrid cells) are grown in a basal culture medium containing
Hypoxanthine, Aminopterin, And Thymidine (HAT medium)
4. Only hybrid cells having properties of both the splenic lymphocytes (HGPRT+)and
myeloma cells (HGPRT-) can grow in culture. The enzyme HGPRT is necessary for
Nucleic Acid Synthesis and is provided by the Splenic Lymphocytes in Hybrid Cells.
Splenic Lymphocytes alone cannot replicate indefinitely while unfused myeloma cells
are killed by aminopterin in HAT medium.
5. Clones that secrete the desired antibody are selected for continuous cultivation. These
hybridomas can be maintained indefinitely and will continue to form monoclonal
antibodies.
APPLICATIONS OF MONOCLONAL ANTIBODIES:
● Therapeutic ---for treatment of lymphomas, rheumatoid arthritis, etc.
● Diagnostic imaging ---radiolabeled monoclonal antibodies are used to detect tumor
antigens.
● Diagnostic reagent---diagnosing infections (immunoassays) and measuring blood
levels of various drugs
● Research -in vitro production of immunoglobulins
 10.SERUM SICKNESS
● It is an example of a Type III hypersensitivity Reaction.
● It appears 7-12 days after injecting a high concentration of foreign serum (Heterologous
serum) such as Diphtheria Antitoxin, Horse tetanus antiserum (that was given for
protection).

CLINICAL FEATURES:
● Fever
● Lymphadenopathy
● Splenomegaly
● Arthritis
● Glomerulonephritis
● Endocarditis
● Vasculitis
● Urticarial rashes
● Abdominal pain
● Nausea and
● Vomiting.

PATHOGENESIS:
1. Formation of Immune complexes: it occurs following the entry of a large dose of antigen
into the body.
2. Deposition of immune complexes in the endothelial lining of blood vessels
3. Inflammatory infiltration: blood vessels (vasculitis), glomerular basement membrane
(glomerulonephritis), and synovial membrane (arthritis).

● It differs from other hypersensitivity reactions, in a way that a single injection can serve
both as the sensitizing and the shocking dose.
● It subsides gradually once the immune complexes are cleared and free antibodies
accumulate.

Fig: Type III Hypersensitivity Reaction

Ref: Robbins & Cotran Pathological Basis of Disease 10th ed Page 211 Fig.6.17
 (Ref: Essentials of Medical Microbiology by Apurba Sastry 3rd ed, page 200
Ananthnarayan and Paniker's Textbook of Microbiology 10th ed, page 168-169)
 11.PRAUSNITZ - KUSTNER REACTION ( P-K REACTION)
● It is an experiment to demonstrate Type 1 Hypersensitivity reactions
● Prausnitz and Kustner first demonstrated the antibody in the serum responsible for the
allergy (named as PK antibody or reaginic antibody)
● which is later known as IgE

EXPERIMENT
● Serum from an allergic person is injected intradermally into the nonallergic person
● Later when the appropriate allergen is injected at the same site a wheal and flare reactions
is developed at the site

WHEAL AND FLARE RESPONSE

● THREE STAGES
➔ The appearance of an erythematous area at the site of injury
➔ Development of a flare (erythema) surrounding the site
➔ Wheal (swelling and congestion) forms at the site as fluid under the skin from the
surrounding capillaries.

(Ref - Essentials of medical microbiology by Apurba S Sastry - 3/E)

 12.TUMOR ANTIGENS

● When a cell undergoes malignant transformation, it acquires new surface antigens. It may
also lose some normal antigens.
● This makes a tumor antigenically different from the normal tissues of the host and in turn
elicits an immune response.
● The tumor antigens are of two types
➔ TUMOR-SPECIFIC ANTIGENS
➔ TUMOR-ASSOCIATED ANTIGENS

TUMOR-SPECIFIC ANTIGENS:
● These antigens are present in malignant cells but absent in the corresponding normal cells
of the host.
● They induce an immune response when the tumor is transplanted in syngeneic animals.
● Such antigens, which induce tumor transplants rejection in immunized hosts are termed
tumor-specific transplantation antigens (TSTA) or tumor-associated transplantation
antigens (TATA).
● In chemically induced tumors, TSTA is tumor-specific.
● Different tumors possess different TSTA even though they are induced by the same
carcinogens.
● TSTA of virus-induced tumors is virus-specific.
● All tumors produced by one virus possess the same antigen ( even when the tumors occur
in different animal strains or species.
TUMOR-ASSOCIATED ANTIGENS:
This type of antigen is found in some tumors and may also be in few normal cells. They
are:

ONCOFETAL ANTIGENS:
● They are fetal antigens
● Found in embryonic and malignant cells but not in adult normal cells
● Synthesis represents the de-differentiation of malignant cells into more primitive
forms.
● EG: alpha-fetoproteins in hepatomas

CARCINOEMBRYONIC ANTIGEN
● It is a glycoprotein
● Detected in many patients with carcinoma of the colon (particularly in metastases)
● Also appears in the case of alcoholic cirrhosis.
● Little diagnostic value

DIFFERENTIATION ANTIGEN
● Prostate-specific antigen (PSA)- high levels are used as a diagnostic indicator in
prostate cancer
● Carbohydrate antigen 125 (CA125)- diagnostic and prognostic marker for ovarian
cancer.
 13.IMMUNOSURVEILLANCE

● Malignant cell arises by mutation of somatic cell frequently.

● Postulated that immune system keeps constant vigilance of this mutation and keep them
on spot.
● This is called immunosurveillance
● The primary function of cell-mediated immunity is to ‘seek and destroy’ malignant cell
that arises by somatic mutation.
● Increased incidence of cancer due to inefficiency of immunosurveillance that results due
to
○ Aging
○ Congenital immunodeficiency
○ Acquired immunodeficiency
● The development of the tumor represents the escape from this surveillance.
● The mechanism of such escape is not clear but various possibilities have been suggested.

MECHANISM OF ESCAPE
● Certain tumor cells may shed or stop expressing the surface antigen thus making the
tumor cell immunologically invisible Modulation of Surface Antigen
○ Some cancer produces a mucoprotein called sialomucin. It binds to the surface of
the tumor cell. Since sialomucin is a normal component, the tumor cell is not
recognized by the immune system Masking Tumor Antigens

● Certain tumor cell evokes immune system to produce blocking antibodies, which cannot
fix and activate complement, resulting in prevention of tumor cell lysis. Production Of
Blocking Antibody
 ○ Due to the fast rate of proliferation of malignant cells tumors may be able to
sneak through before the development of an effective immune response and once
they reach a certain mass, the tumor may be too great for the host immune system
to control the Fast Rate Of Proliferate Of Malignant Cell

● Some tumors may form cytokine-like Transforming Growth Factor β (TGF-β) which
suppressed CMI Suppression Of Cell-Mediated Immunity (CMI)

Reference: Textbook of Microbiology, Ananthanarayan and Paniker’s, 8th Edition

 14.ALLOGRAFT REJECTION

ALLOGRAFT
● It is a tissue transferred between genetically non-identical individuals of the same
species (e.g.; kidney, heart transplant).
● Allografts are the most commonly used graft in transplant centers.
● Allografts are usually histocompatible (antigenically dissimilar).
● Antigens of allografts against which the recipient mount in the an immune response
-TRANSPLANTATION ANTIGEN.
● E.g.; MHC molecules (major histocompatibility antigen), ABO and RH blood group,
Minor histocompatibility antigen (MHA).
● Immune responses against HLA molecules are weaker; hence rejection is less frequent
than MHC molecules.

EICHWALD -SILMSER EFFECT:

● It is unilateral sex-linked histocompatibility.
● The rejection of grafts when transferred from a male donor to a female recipient is more
as the graft tissue of a male donor will have male-specific MHA on the Y chromosome
which is absent in the female recipient.

FACTORS INFLUENCING ALLOGRAFT REJECTION

1. TISSUE INVOLVED: Skin grafts - rejected faster than other tissues.

2. GENETIC DISTANCE: between the donor and recipient.

More distance, more rejection.

3. IMMUNOLOGICAL MEMORY: Rejection is faster when another graft is received from

the same donor due to memory cells against the first graft.
AUTOGRAFT ACCEPTANCE: Skin graft transplanted to the same individual at a
different place.
● Revascularization --- 3-7 days
● Healing—7-10days
● Resolution and acceptance – 12-14 days

FIRST SET ACCEPTANCE: This is a type of primary graft rejection that develops when
allograft is placed for the first time to the recipient from a donor.
● Revascularisation—3-7 days
● Cellular infiltration—10 days
● Necrosis—10 days
● complete rejection—12-14 days

SECOND-SET REJECTION: Another graft from the same donor is transplanted, after
the first set response, it will be rejected in an accelerated fashion.
● Vascularisation – starts but interrupted by inflammation
● Necrosis –sets early and graft sloughs off on the 6th day.
MECHANISM OF GRAFT REJECTION:
● T cell-mediated ( against the MHC molecules)
1. SENSITIZATION PHASE

2. EFFECTOR PHASE
SENSITIZATION PHASE

EFFECTOR PHASE
PREVENTION OF GRAFT REJECTION
Before transplantation, histocompatibility tests should be done.

A. ABO blood group compatibility testing.

Donors Ag on leukocyte

B. HLA typing: Matched with the recipient

Gene

PHENOTYPIC GENOTYPIC

SEROLOGY: PCR detecting HLA genes

Microcytotoxicity PCR -RFLP
PCR-SSP
 TISSUE TYPING: Mixed PCR-DNA sequencing
lymphocyte reaction PCR-SSOP
Confirmatory analysis

TREATMENT :
● HYPERACUTE REJECTION: Immediate removal
● ACUTE REJECTION: Therapeutic regimens ( immunosuppressive drugs).
● CHRONIC REJECTION: Retransplant.

GVH REACTION ( GRAFT VERSUS HOST REACTION)

GRAFT IMMUNE RESPONSE AGAINST HOST

This is contrary to the usual situation of host rejections.

GVH OCCURS UNDER 3 CONDITIONS:

1. The graft should contain Immunocompetent T cells.
2. The recipient should contain transplantation antigens & is absent in graft.
3. The recipient may be immunosuppressant, so cannot mount an immune response against
the host.
 ACUTE OR FULMINANT GVH CHRONIC GVH DISEASE
DISEASE.
o Occurs after 100 days of
o Occurs 100 days transplantation.
post-transplantation.
o Occurs in bone marrow o Less severe form.
transplant.

CLINICAL MANISFESTATION:
CLINICAL MANIFESTATIONS:
Acute GVH manifestation
1. Hepatomegaly
+
2. Skin rash Damage to connective tissues,
exocrine glands.
3. Mucosal damage
4. Diarrhea

Experimentally GVH reaction can be

produced in MICE RUNT DISEASE

TREATMENT
GLUCOCORTICOIDS (I.V) for both acute and chronic GVH.
 15.CARRIERS

● The carriers are the APC’s and Plasma cells

ANTIGEN-PRESENTING CELLS:
● Although antigen presentation refers to the presentation of the antigenic peptide to both
TH and TC by complexing with MHC – II and I respectively
● However, APC’s in a strict sense implies to those cells that present antigenic peptides
along with MHC class II to TH cells
● Cells presenting antigenic peptides along with MHC class I to TH cells are not included
under APCs.
● These cells are usually virus-infected cells or tumor cells.
● They are often referred to as target cells as the activated Tc cells causes lysis of these cells

Eg: Dendritic cells, macrophages, and B cells are the major APCs, and Professional
APCs

● There are some other nonprofessional APC that occasionally present antigen to helper T
cells.

(Fibroblasts, Thymic epithelial cells, pancreatic beta cells, Vascular endothelial cells, Glial cells,
Thyroid epithelial cells)

PLASMA CELL:
● They are oval, large with an eccentrically oval nucleus containing large blocks of
peripheral chromatin (cartwheel appearance), and cytoplasm containing abundant
organelles. Their life span is 2-3 days.
 16. DIFFERENCE BETWEEN ENDOTOXIN AND EXOTOXIN

FEATURES ENDOTOXIN EXOTOXIN

Nature Lipopolysaccharides Proteins

Source Part of the cell wall of Secreted both by gram-positive

gram-negative bacteria and negative bacteria diffuse into
surrounding medium

Released by Cell lysis, not by secretion Actively secreted by bacteria

Heat stability Highly stable Heat labile, destroyed at 60°C

Mode of action ↑IL-1 and TNF - α Mostly enzyme-like action

Effect Non-specific Specific action on the particular

tissue

Tissue affinity No Specific affinity for tissues

Fatal dose Only large doses are fatal More potent, even in small doses

Antigenicity Poorly antigenic Highly antigenic

Neutralization by antibodies Ineffective Neutralized by specific

antibodies

Used for vaccine No effective vaccine is available Toxoid forms are used as a
using endotoxin vaccine
E.g.: tetanus toxoid

EXOTOXINS
● Heat labile proteins
● Secreted by both gram-positive and gram-negative bacteria
● Diffuse readily into surrounding medium
● Highly potent even in minute amounts
➔ Botulinum toxin is the most potent
➔ 39.2g of botulinum toxin is sufficient to eradicate the entire mankind
● Can be converted into vaccines (toxoid) after treatment using formaldehyde
● Toxoid lack toxigenicity but retains antigenicity and hence produce immunity
● Highly specific for a particular tissue
➔ Tetanus toxin for CNS
ORGANISM TOXIN (EXOTOXIN) MECHANISM

Staphylococcus aureus Enterotoxin, toxic shock Act as a superantigen, stimulate T cell

syndrome toxin nonspecifically, to

Streptococcus Pyogenes Streptococcal pyrogenic release large amounts of cytokine

exotoxin

Corynebacterium Diphtheria toxin Inhibits protein synthesis (by inhibiting

Diphtheriae elongation factor - 2)

Bacillus anthracis Anthrax toxin ↑cAMP in target cells, edema

Clostridium perfringens Α toxin, other major and Lecithinase and phospholipase activity
minor toxins →causes myonecrosis

Clostridium tetani Tetanus toxin Decrease in neurotransmitter (GABA and

(tetanospasmin) glycine) release from the inhibitory
neuron → spastic paralysis

Clostridium botulinum Botulinum toxin (BT) Decrease in neurotransmitter

(acetylcholine) release from the neuron →
flaccid paralysis

Escherichia coli Heat labile toxin (LT) Activation of adenylate cyclase → ↑cAMP
(diarrheagenic) in target cell → secretory diarrhea

Heat stable toxin (ST) ↑cGMP in target cell → secretory diarrhea

verocytotoxin Inhibit protein synthesis

Shigella dysenteriae type 1 Shiga toxin (By inhibiting ribosome)

Vibrio cholerae Cholera toxin (CT) Activation of adenylate cyclase → ↑cAMP

in target cell → secretory diarrhea

pseudomonas Exotoxin - A Inhibit protein synthesis

(By inhibiting elongation factor-2)
 17. PROTOCOLS FOR BLOOD TRANSFUSION

● DONOR ELIGIBILITY: Selection of donors should be done based on adequate history,

physical examination, encouraging known donors (e.g., family members).
● PROCESSING, HANDLING, AND STORAGE: Adequate skin disinfection and
diverting initial
30 mL of blood can reduce bacterial contamination effectively
● SCREENING FOR TTI’s: Screening for TTIs (Transfusion- Transmitted Infection). In
India, screening is done for HIV, HBV, HCV, syphilis, and malaria.
● SCREENING TESTS: They should satisfy the following properties
○ Need to be highly sensitive, detect early in the phase of infection and rapid
○ The gradual shift from tests based on antigens/antibodies to highly sensitive tests
based on nucleic acid amplification (NAAT) helps in detecting infections in the
window period (the period of early infectivity)
● STORAGE: Optimum storage conditions (e.g., temperature) should be maintained.
● PATHOGEN INACTIVATION: Effective pathogen inactivation procedures without
compromising the integrity of blood/blood products.
● QUALITY CONTROL: Quality audits and assessments should be carried out to ensure
the highest safety standards.

Ref: Apurba S Sastry, 3rd edition, Annexure 8, Pg. 827

ESSAY

1. Organisms causing meningitis it's pathogenicity, lab diagnosis of meningococcal

meningitis, the role of BACTEC in rapid diagnosis of causative agents in bacterial
meningitis
2. Anthracis
3. Clostridia:
4. Causative agents of enteric fever, diagnosis of typhoid fever.

5. Classify mycobacterium, details about pulmonary tuberculosis?

6. Dysentery, causative agents, details about bacillary dysentery.
7. Describe Corynebacterium diphtheriae. add a note on its prophylaxis

8. Name 3 coccobacillus : Bordetella pertussis

Haemophilus influenzae

Chlamydia trachomatis

Discuss in detail about etiology, pathogenesis, and lab diagnosis of Bordetella pertussis

9. Leptospirosis
10. Syphilis
11. Rickettsiaceae
12. Laboratory diagnosis of respiratory tuberculosis

SHORT NOTES
1. el tor vibrio
2. Gonorrhea
3. Non gonococcal urethritis (NGU)
4. x and v factors
5. What is the bile solubility test? Describe its principle
6. Zoonotic infections
7. BCG
8. Food poisoning
9. Unique characteristics of enterococcus
10. Nagler’s reaction
11. What is the disease caused by mycoplasma?
12. Llab diagnosis of typhoid fever:
13. Actinomycosis
14. Nonsporing anaerobic infections
15. Weil’s disease: (hepato- renal hemorrhagic syndrome)
16. Causative agent for relapsing fever:
17. Various mechanisms by which escherichia coli produce diarrhea
18. Mantoux test
19. Name 4 pigments produced by bacteria
20. Milk ring test and its uses
21. Neil mooser reaction
22. Write a note on satellitism
23. Clostridium botulinum
24. Quellung reaction
ESSAY
 1. MENINGITIS
● Meningitis is an inflammation of leptomeninges surrounding the brain and spinal cord with
involvement of the subarachnoid space
● TYPES OF MENINGITIS
○ Acute meningitis
○ Chronic meningitis
● Organisms causing bacterial meningitis
○ ACUTE OR PYOGENIC MENINGITIS
■ Streptococcus pneumonia is the most common cause of pyogenic meningitis
■ Other agents include meningococcus, Streptococcus agalactiae, Listeria, and
Haemophilus influenzae.
■ Neonates-streptococcus agalactiae, gram-negative bacilli such as Escherichia
coli and Klebsiella
■ Elderly - Streptococcus agalactiae and Listeria monocytogenes
○ CHRONIC BACTERIAL MENINGITIS
■ Mycobacterium tuberculosis
■ Borrelia birgdoferi
■ Treponema pallidum
■ Rate bacterial agents such as Nocardia, actinomyces, Tropheryma whipplei,
Leptospira, and brucella
● PATHOGENESIS - bacteria transmitted from person to person through droplets of
respiratory secretion from cases or nasopharyngeal carriers
○ Routes of infection
■ Hematogenous spread - most common route but entry into the subarachnoid
space is gained through the choroid plexus or through other blood vessels of
the brain.
■ Direct spread from an infected site present close to the meninges-otitis
media, mastoiditis, sinusitis, etc.
 ■ Anatomical defects in the central nervous system may occur as a result of
surgery, trauma, congenital defects, which can allow organisms for ready and
easy access to CNS
● CLINICAL MANIFESTATION
○ Important symptoms include fever vomiting intense headache altered consciousness
and occasionally photophobia
○ Signs of meningism
■ Nuchal rigidity
■ Kernig's sign
■ Brudzinski's sign
■ Organism specific finding (eg) purpuric rash as seen in meningococcal
meningitis
● LABORATORY DIAGNOSIS OF MENINGOCOCCAL MENINGITIS
○ Specimen collection and transport
■ First suspected meningococcal meningitis the specimens are nasopharyngeal
swabs pus or scrapings from rashes; should be carried in a transport medium
such as Stuart's medium.
○ Cytological and biochemical analysis

○ Gram staining
■ Gram-negative diplococci capsulated with adjacent sides flattened - Neisseria
meningitides which causes Meningococcal meningitis
● ROLE OF BACTEC IN RAPID DIAGNOSIS OF CAUSATIVE
AGENTS IN BACTERIAL MENINGITIS
○ BACTEC principle is based on fluorometric detection of growth; use an
oxygen-sensitive fluorescent dye present in the medium
○ Mycobacteria Growth Indicator Tube(MGIT) is an automated culture system
available for laboratory diagnosis of Chronic bacterial meningitis caused by
Mycobacterium tuberculosis.
○ MGIT works on the fluorometric principle of detection similar to BACTEC
 2. ANTHRACIS
● Bacillus anthracis is gram-positive, aerobic, non-motile, sporulation bacteria

SPORES:
● Formed under unfavorable conditions
● Highly resistant to physical and chemical agents
● Are formed in soil/culture, not in the animal body

VIRULENCE FACTORS:
(1)CAPSULE-

● made of polyglutamate
● Inhibits phagocytosis
● It is plasmid-borne

(2)TOXIN HAS THREE FRACTIONS

● Edema factor-active fragment

Acts as adenylyl cyclase and increases cAMP level
Cause edema
● Protective factor-binding fragment
Binds to host cell receptors and facilitates entry of other fragments into
host cells
● Lethal factor-causes cell death

CLINICAL MANIFESTATIONS:
ANIMAL ANTHRAX

● Anthrax is a zoonotic disease.

● Herbivores are more commonly affected than carnivores
● Ingestion of spores in the soil causes the disease
● Anthrax presents as fatal septicemia
● Infected animals discharge many bacilli from the mouth, nose, and rectum which
sporulate in soil and may act as a source of infection for man

HUMAN ANTHRAX

● Humans acquire disease via abraded skin

● Inhalation of spores
 ● Ingestion of anthrax infected animal carcasses

Types:

● Cutaneous anthrax-(hide porter’s disease)-malignant pustule is seen.

● Pulmonary anthrax-(wool sorter’s disease)-causes bioterrorism outbreaks commonly
Hemorrhagic pneumonia is seen
● Intestinal anthrax-when undercooked/uncooked meat is consumed

EPIDEMIOLOGY:
● High incidence in Africa, Central, and southern Asia
● Human anthrax
Non-industrial-agricultural exposure to animals
Industrial-from hides, hair, bristles, wools

LABORATORY DIAGNOSIS:
● Specimen collection-
● pus, swab, or tissue from the pustule
● Sputum in pulmonary anthrax
● Blood in septicemia
● CSF in hemorrhagic meningitis
● Gastric aspirate, feces, food in intestinal anthrax
● Ear lobes for dead animals
● Direct demonstration-
● Gram staining
● Mc Fadyean's reaction-Capsule appears as amorphous purple around bacilli when
reacted with Gurr’s polychrome methylene blue for 30sec
● Direct immunofluorescence test
● Ascoli’s thermo precipitation test
● Culture-
● B. anthracis is non-fastidious
● Nutrient agar-Medusa head appearance, irregular, opaque, grayish-white with
frosted glass appearance
● Blood agar-dry wrinkled, non-hemolytic colonies
● Gelatin stab agar-inverted fir tree appearance
● Selective media-Solid medium with penicillin-bacilli in a string of pearl appearance
● PLET medium
Reference: Ananthanarayan and Paniker’s Textbook of Microbiology-11/E-pg no.235-fig 24.4

● Culture smear-
● Gram-staining-bamboo stick appearance of bacilli with non-bulging spores
● Spores-demonstrated by Ashby’s method or acid-fast staining

Reference: Ananthanarayan and Paniker’s Textbook of Microbiology-11/E-pg no.232-fig 24.1

● Molecular diagnosis-PCR with specific primers

PREVENTION:
● Animals died of anthrax should be burnt or buried deep in lime pits
● Decontamination(by autoclaving) of animal products
● Protective clothing and gloves when handling infectious materials
● Immunization-Pasteur’s anthrax vaccine
● Sterne vaccine-live attenuated non-capsulated
● Mazzucchi vaccine

TREATMENT:
● Post-exposure prophylaxis-Adsorbed toxoid vaccine (BioThrax) along with
antimicrobial therapy
 3. CLOSTRIDIA
● Clostridia are obligate anaerobic gram-positive bacilli with bulging spores.
● Infection causing clostridia are
1. C. perfringens
2. C. tetani
3. C. botulinum
4. C. difficile

Clostridium tetani

MORPHOLOGY:
● Obligate anaerobic, gram-positive bacillus with terminal round spore (drumstick
appearance)
● Non-capsulated
● Has peritrichous flagella and exhibits swarming motility

PATHOGENESIS:

● C. Tetani produces two exotoxins – tetanolysin and tetanospasmin

● Tetanolysin is a hemolysin, not involved in the pathogenesis
● Tetanospasmin (tetanus toxin) is a neurotoxin and is responsible for disease
manifestations. It gets toxoided spontaneously or by formaldehyde.
● Mechanism of action:
Tetanus Toxin binds to receptors present on the motor nerve terminal

Causes presynaptic inhibition of glycine and GABA (inhibitory neurotransmitter)

Spastic muscle contraction

MODE OF TRANSMISSION:

● It is non-infectious (no person to person spread)

● Injury (superficial abrasions, puncture wounds, road traffic accidents)
● Surgery is done without proper asepsis
● In neonates following delivery due to unhygienic practices
● Otitis media

CLINICAL MANIFESTATIONS:

● The incubation period is about 6-10 days. Muscles of the face and jaw are
affected 1st(shorter distance for a toxin to reach presynaptic terminals)
● First symptom: increase in masseter tone leading to trismus or lockjaw,
followed by muscle pain and stiffness, back pain, and difficulty in swallowing
● As the disease progresses, painful spasm develops, it can be:
Localized: involves affected limb
Generalized: painful muscle spasm leading to descending spastic paralysis
● Deep tendon reflexes are exaggerated
● The autonomic disturbance is maximal during the 2nd week of severe tetanus,
characterized by:
Low or high BP
Tachycardia
Intestinal stasis
Sweating
Increased tracheal secretions
Acute renal failure
● In neonates, difficulty in feeding is the initial presentation

COMPLICATIONS:

● Risus sardonicus: characterized by abnormal, sustained spasms of facial

muscles that appear to produce grinning
● Opisthotonos position: the abnormal posture of the body occurring due to
generalized spastic contraction of the extensor muscle
LAB DIAGNOSIS:

Specimen:

● Necrotic tissue from the site of injury

Microscopy:

● Gram staining reveals drumstick appearance

● Unreliable (cannot distinguish from other clostridia)

Culture:

● More reliable
● Robertson cooked meat broth: C. tetani being proteolytic turns meat
particles black and produces foul order
● Blood agar with polymyxin B: C. tetani produces swarming growth

Toxigenicity test:

● Performed in vivo mouse inoculation test on specimens like serum or

urine.

PREVENTION:
Active immunization
● Monovalent vaccine: Tetanus Toxoid
● Combined vaccine: DPT (diphtheria pertussis and tetanus), Td (tetanus
and diphtheria), pentavalent vaccine (DPT, hepatitis B and Hib)
TREATMENT:

Passive immunization

● 250 IU of HTIG (Human Tetanus immunoglobulin) or 1500 IU of ATS

(Anti tetanus serum) given as a single IM dose.
● Effect of HTIG last for 30 days and ATS last for 7-10days

Combined immunization:

● Both active and passive immunization

 ● In a non-vaccinated person, 1st dose of TT is given in one arm along with
HTIG or ATS in another arm followed by a complete course of TT vaccine

Antibiotics:

● They play a minor role as they cannot neutralize toxins that are already
released
● They are useful in early infection, before the expression of toxin(<6hrs)
● They prevent further release of toxin
● Metronidazole is a drug of choice given for 7-10 days

Other measures:

● Endotracheal intubation and early tracheostomy are useful to protect the

airway
● Anti-spasmodic to eliminate reflex spasm
● Beta-blockers to control sympathetic hyperactivity
● Entry wound should be cleaned and debrided of necrotic material, to
remove anaerobic foci of infection
● The patient should be isolated in a separate room as any noxious stimulus
can aggravate the spasm

PREVENTION OF TETANUS AFTER INJURY:

Complete course of Not tetanus prone Tetanus prone

vaccination wound wound

Taken within 5 years nothing required Nothing required

Taken within >5to<10 Td 1 dose Td 1 dose

year

Taken >10 years back Td 1 dose Td 1 dose + HTIG

Unknown/incomplete Td complete dose Td complete dose

immunization +HTIG
 4. CAUSATIVE AGENTS OF ENTERIC FEVER

Salmonella is a gram-negative bacterium, belonging to the family Enterobacteriaceae.

TYPHOIDAL SALMONELLA: Includes

1. Salmonella typhi
2. Salmonella paratyphi A, B, C

● Enteric fever includes both typhoid fever and paratyphoid fever.

PATHOGENESIS:

● Infection is acquired by ingestion of contaminated food or water.

● Infective dose : 103 to 106 Bacilli
●
CLINICAL MANIFESTATIONS OF ENTERIC FEVER:

● The incubation period is 7-14 days

● Step ladder pyrexia
● Headache, Malaise, Anorexia
● Abdominal discomfort
● Rashes (Rose spots)
● Complications like intestinal bleeding, intestinal perforation, Meningitis,
Hepatosplenomegaly.

LABORATORY DIAGNOSIS OF TYPHOID FEVER:

Isolation of bacilli from a patient, Demonstration of Antibody in Patient serum.

1. Culture Isolation
● Blood Culture
85-90 % positive in the first week of illness
70% positive in the third or fourth week of illness
percentage positivity decreases thereafter
● Stool and Urine Culture
2. Culture Smear and Motility: Gram-stained smear reveals gram –ve bacteria. They are motile
with peritrichous flagella.
3. Biochemical identification
4. Slide agglutination test
5. Serum Antibody Detection (WIDAL TEST)
6. Antigen Detection: ELISA
7. Molecular Methods: Nested PCR
8. Non Specific Findings: WBC count – Neutropenia is seen in 15-25% of cases.
9. Antimicrobial Susceptibility Testing: Disk diffusion method on Mueller-Hinton Agar or MIC
based method (VITEK)
10. Detection of Carriers:
● Culture: By stool, urine, or bile culture.
● Detection of Vi Antibodies: here even a titer of 1:10 is considered a significant
OTHER ANTIBODY DETECTION TESTS:

● Typhidot test: It uses a dot ELISA format to detect both IgM and IgG separately after 2-3
days of infection.

1. CULTURE ISOLATION :

A)Blood Culture: Ideal Method

i) Inoculation of blood in the medium at dilution of 1:5 = Antibacterial components will

get diluted.
ii) Incubation @ 37℃ . Salmonella grows within 24 hrs.
iii) Subcultures are made and inoculated on blood agar and Mac Conkey agar.
iv) Colony Appearance on Blood Agar: Nonhemolytic moist colonies
v) Colony appearance on Mac Conkey agar: Non-Lactose Fermenting(NLF).
● Bone Marrow specimens: 55-90% sensitive, it is used when blood culture is
negative as the patient is on antibiotics.
● Duodenal Aspirate: if blood and bone marrow culture is –ve.
● Other specimens like rose spots, pus from suppurative lesions, CSF, Sputum,
Autopsy Specimen of the gallbladder, mesenteric lymph nodes where salmonella
can be isolated.
C)Urine Culture: Salmonella are shed in urine irregularly, infrequently. Urine samples are
centrifuged and deposits are inoculated in enrichment /selective media.

BIOCHEMICAL IDENTIFICATION :

S. Typhi is Anaerogenic

i. Indole test –ve

ii. Citrate test –ve
iii. Urease test –ve
iv. Triple Sugar Test: No gas production, production of small speck H2S.

SLIDE AGGLUTINATION TEST : It is done with polyvalent O and Polyvalent H antisera.

They react with isolated bacteria on the slide.

i) +ve agglutination implies isolation belongs to the genus Salmonella.

ii) Serotypes can be identified by specific O antisera
S.typhi agglutinates with O9 antisera
S.Paratyphi A agglutinates with O2 antisera
S.Paratyphi B agglutinates with O4 antisera.

WIDAL TEST: Discovered by Fernand Widal.

PRINCIPLE: Agglutination test, Detects H and O Antibodies in patients of enteric/

typhoid fever.
 Kauffmann-White Antigenic Classification

Salmonella Somatic O antigen Capsulated Vi Flagellar H

antigen antigen

S. Typhi 9,12 +(capsulated) d

S. Paratyphi A 1, 2 ,12 -(non capsulated) a/1,5

S.Paratyphi B 1, 4 ,5, 12 -(noncapsulated) b/1,2

APPARATUS: Two types of tubes

i. Felix Tube –A short round bottom tube for O agglutination.

ii. Dreyer’s Agglutination tube – A narrow tube with a conical bottom for H
agglutination.

ANTIGENS USED :

i) O antigen of S. Typhi (TO)

ii) H antigen of S.Typhi(TH)
iii) H antigen of S.Paratyphi A (AH)
iv) H antigen of S.Paratyphi B (BH).
The Paratyphi O is not employed as it cross-reacts with typhoid O antigen due to their
sharing factor 12.

PROCEDURE :

i. Patient serum is serially diluted from 1/10 to 1/640 in 4 sets of tubes.

ii. To each set of diluted sera four antigens, ( TO, TH, TA, TB) suspension are added.
iii. Incubated in a water bath @ 37℃ overnight.
RESULT:

O antibodies +O antigens = granular / H antibodies + H antigen = Fluffy/ Cotton

Chalk Agglutination./ disc shaped Wooly Agglutination.

● If Agglutination occurs, there is clear supernatant fluid.

● If Agglutination does not occur, Hazy supernatant fluid and button formation due to
deposition of antigen.

INTERPRETATION: The highest dilution of sera at which agglutination occurs in antibody

titer.

i) Significant Titre: Higher titer are significant. In India, a significant titer is

● H agglutination titre more than 200
● O agglutination titre more than 100.

In the above diagram, the TO titer is 1:160 implies it is a significant titer.

The titer is 1: 320 implies it is a significant titer.

● Demonstration of rising in titer by testing sera at a 1-week interval is more meaningful

than a single high titer.
 ● A rise in titer in anamnestic response is transient, usually falling after a week. In true
infection antibody titer increases fourfold after 1 week.

Widal test Result Suggestive of

Rise of TO and TH antibody Enteric fever due to S.typhi

Rise of TO and AH antibody Enteric fever due to S.Paratyphi A

Rise of TO and BH antibody Enteric fever due to S. Paratyphi B

Rise of TO antibody only Recent infection: due to S.Typhi or

Paratyphi A,B.

Rise of TH antibody only Convalescent stage/Anamnestic

response

Rise of all TH, AH, BH Post TAB vaccination.

False +ve result in:

i. Anamnestic response
ii. Bacteria fimbriae in Bacterial Antigen suspension.
iii. Prior Immunization ( TAB vaccine)

False –ve result in Early-stage ( 1st week of illness), Patient on Antibiotics.

TREATMENT :

● DOC : Ceftriaxone for empirical treatment.

● Alternative drug: Azithromycin, Ciprofloxacin
 5. MYCOBACTERIUM
✧ CLASSIFICATION OF MYCOBACTERIUM:

MYCOBACTERIA

M.tuberculosis complex. M.leprae. Non-tuberculous bacteria

(Further classified by Runyon)
❖ Photochromgens
❖ Scotochromgens
❖ Nonphotochromogens
❖ Rapid growers

PULMONARY TUBERCULOSIS :

● It is caused by the bacteria- Mycobacterium tuberculosis complex

M.tuberculosis complex includes
1. M.tuberculosis
2. M.bovis
3. Other members like M. caprae, M. africanum ,M. microti, M.
pinnipedii

MORPHOLOGY:
M.tuberculosis has an antigenic structure.
1. CELL WALL(INSOLUBLE)ANTIGENS:
The cell wall consists of several distinct layers
● Peptidoglycan layer: It maintains the shape and rigidity of the cell
● Arabinogalactan layer: It facilitates the survival of
M.tuberculosis within the macrophages
● Mycolic acid layer: It is the principal constituent, made up of
long-chain fatty acids attached to arabinogalactan. It confers very
low permeability to the cell wall and is responsible for acid fastness
and also reduces the entry of most antibiotics
● Outermost layer: It consists of lipids (mycocerosates and
acylglycerols), glycolipids, and mycosides (phenolic glycolipids)
 ● Proteins (e.g. porins, transport proteins): They are found
throughout the various layers
● Plasma membrane: This layer is present beneath the cell wall, into
which various proteins, phosphatidylinositol mannose, and
lipoarabinomannan {LAM) are inserted. LAM is an important
antigen, helps in attachment to host cells, and is also a target
antigen used for diagnosis.

2. CYTOPLASMIC(SOLUBLE)ANTIGENS:
These include antigen 5, antigen 6, antigen 60; and are used in serodiagnosis of
tuberculosis.

(fig. 63.1) Pg:624 (ref. Essential of Medical Microbiology by Apurva. S Sastry-3/E)

PATHOGENESIS:

❖ Source of infection: (1) human (e.g. cases of pulmonary tuberculosis), (2) bovine source
(e.g. consumption of unpasteurized infected milk).

❖ Mode of Transmission: M. tuberculosis is mainly transmitted by inhalation of droplet

nuclei, generated while coughing, sneezing, or speaking of infected patients.
Other modes are Inoculation and ingestion
 ❖ Risk factors: include low cell-mediated immunity, age(late adulthood and early
adulthood periods are prone), sex(Women at 25-35age, men at an older age are prone)
CLINICAL MANIFESTATIONS
Tuberculosis (TB) is classified as pulmonary and extra-pulmonary forms.
Pulmonary Tuberculosis (PTB)
Pulmonary tuberculosis (PTB) accounts for 80% of all cases of tuberculosis (TB).
It can be further categorized into primary or postprimary (secondary) types

(Table 63. 1) pg 626(ref. Essential of Medical Microbiology by Apurva. S Sastry-3/E)

LABORATORY DIAGNOSIS : Mycobacterium tuberculosis :

DIAGNOSIS OF ACTIVE TUBERCULOSIS
SPECIMEN COLLECTION:
• In pulmonary TB: Sputum (2 specimens-spot and early morning), gastric
aspirate (in children)
• In EPTB: Specimens vary depending on the site involved.
Digestion, decontamination, and concentration of specimen:
• Modified Petroff's method (4% NaOH) or NALC (N-acetyl-L-cysteine) + 2%
NaOH.
 DIRECT MICROSCOPY BY ACID-FAST STAINING:
• Ziehl-Neelsen (ZN) technique-long slender, beaded, less uniformly stained red
color acid-fast bacilli
• Kinyoun's cold acid-fast staining
Fluorescent (auramine) staining-it is more sensitive and Conventional culture
media- take 6-8 weeks
• Solid media, e.g. Lowenstein Jensen (LJ) medium- shows
rough, tough, and buff-colored colonies in 6-8 weeks
• Liquid media- Kirchner's medium and Middlebrook 7H9
medium.
AUTOMATED CULTURE METHODS:-(take 3-4 weeks)
• MGIT system: Detects growth and resistance to antitubercular
drugs (ATDs); with a turnaround time of 2-3 weeks
• BacT/ALERT: Detects growth
• Versa Tek System: Detects growth and resistance to ATDs.
CULTURE IDENTIFICATION:
• MPT 64 antigen detection-
• Biochemical identification- Niacin test (obsolete).
MOLECULAR METHODS:
• PCR detecting IS6110 gene
• CBNAAT (GeneXpert)- for identification and detection of resistance to
rifampicin; has a turnaround time 2 hour
• Line probe assay (e.g. Genotype TB)-for identification and detection of
resistance to 1st and 2nd line ATDs; has a turnaround time 2- 3 days.
DIAGNOSIS OF LATENT TUBERCULOSIS:
•By tuberculin test (e.g. Mantoux test) and interferon-gamma release assay (IGRA).
DRUG SUSCEPTIBILITY TESTING:
PHENOTYPIC METHOD:
❖ MGIT (used for 1st and 2nd line drugs): Resistance is determined by the
growth of TB bacilli in the drug-containing tube as compared to control tube
(drug-free) within 4-21days
❖ Proportion method (used for 1st and 2nd line drugs): An isolate is considered
resistant to a given drug when the growth of 1% or more is observed in the
drug-containing LJ medium compared to the control LJ medium without drug
after 42 days of incubation.

GENOTYPIC METHODS:
❖ GeneXpert (used only for rifampicin): Five probes targeting different wild
sequences of the rpoB gene are used. Turnaround time is <2 hours
❖ Line probe assay: Detects resistance to both 1st and 2nd line drugs in 2- 3 days
of turnaround time.
TREATMENT:
Anti-tubercular drugs (ATDs) can be classified into:
✔ First-line drugs
✔ Second-line drugs
FIRST-LINE DRUGS:
⮚ isoniazid (H)
⮚ Rifampin (R)
⮚ Pyrazinamide (Z)
⮚ Ethambutol (E)
⮚ Streptomycin (S)
SECOND-LINE DRUGS:
⮚ Ethionamide and prothionamide
⮚ Quinolones: levofloxacin, moxifloxacin and ofloxacin .
⮚ Aminoglycosides: kanamycin, capreomycin, and amikacin
⮚ Cycloserine and para-aminosalicylic acid
⮚ Macrolides: clarithromycin
 ⮚ Bedaquiline (approved in 2015)

Pg:632 Treatment regime based on drugs susceptibility testing (ref. Essential of

Medical Microbiology by Apurva. S Sastry-3/E)

RESISTANCE TO ANTITUBERCULAR DRUGS (ATDS)

MONO RESISTANCE
Defined as resistant to one first-line ATD only.
POLY RESISTANCE
Defined as resistant to > 1 first-line ATD except other than both INH and
rifampicin resistance to Antitubercular Drugs(ATDs)
RIFAMPICIN RESISTANCE (RR)
 Defined as rifampicin resistance with or without resistance to other ATDs
(excluding isoniazid).
MULTIDRUG-RESISTANT TUBERCULOSIS (MDR-TB)
MDR-TB is defined as resistance to isoniazid and rifampicin with or without
resistance to another first-line anti-tubercular drug
EXTENSIVELY DRUG-RESISTANT TUBERCULOSIS (XDR-TB)
They are MDR-TB cases which are also resistant to Fluoroquinolones
(ofloxacin/levofloxacin) and At least one injectable aminoglycosides (kanamycin,
amikacin or capreomycin).

DRUG RESISTANCE GENES FOR M.TUBERCLOSIS:

(table 63. 70 Pg: 634 (ref. Essential of Medical Microbiology by Apurva. S

Sastry-3/E)

BACILLUS CALMETTE GUERIN VACCINE (BCG): Vaccine for the prevention of

tuberculosis
⮚ BCG strain: Live-attenuated M. Bovis was the strain
⮚ Types of vaccine: BCG is available in two forms
• Liquid (fresh) form
 • Lyophilized from (freeze-dried) form
⮚ Administration of BCG
• Dose and strength: 0.1 mL containing 0.1 mg TU
• Alcohol should not be used to wipe the skin
• Site: It is given above the insertion of the left deltoid
• Route: It is administered by intradermal route by using a 26 gauge tuberculin
syringe
⮚ Indications of BCG:
• Direct BCG: BCG is directly given to newborns soon after birth. This strategy
is followed by most of the developing countries including India. If not given at
birth it can be given later, maximum up to 2 years
• Indirect BCG: BCG is given after performing the tuberculin test.
⮚ Contraindications to BCG include:
• HIV-positive child
• Child borne to AFB positive mother
• Child with low immunity
• Generalized eczema
• Pregnancy.
 6.BACILLARY DYSENTERY
Dysentery is characterized by diarrhea with increased blood and mucus, often associated with
fever, abdominal pains, and tenesmus.

CAUSATIVE AGENTS:

● Shigella species
● Campylobacter jejuni
● Enterohemorrhagic E. coli
● Enteroinvasive E. coli
● Vibrio parahaemolyticus

BACILLARY DYSENTERY

● Bacillary Dysentery is characterized by the passage of loose stool mixed with blood and
mucus.
● Causative agent is – Shigella.
● It is classified into four species and based on O antigen they are further classified into
several serotypes.
● Shigella dysenteriae has 15 serotypes.

PATHOGENESIS

● Mode of transmission:

Infection occurs by ingestion through contaminated fingers (most common), food, water and
rarely flies.

● Minimum infective dose:

As low as 10-100 bacilli can initiate the disease, probably because of the ability to survive in
gastric acidity.
 ● Entry via M cell:

● Exotoxins

Some serotypes of Shigella produce enterotoxins:

Shigella enterotoxin and Shiga toxin

● Endotoxins

Induces intestinal inflammation and ulceration.

CLINICAL MANIFESTATIONS

5 phases:

▪ INCUBATION PERIOD
Lasts for 1-4 days
▪ INITIAL PHASE
Watery diarrhea with fever, malaise, anorexia, and vomiting
▪ PHASE OF DYSENTERY
Frequent passage of bloody mucopurulent stools with increased tenesmus and abdominal
cramps.
▪ PHASE OF COMPLICATION
Seen in children less than 5 years of age
⮚ Intestinal complications- toxic megacolon, perforations, and rectal prolapse
⮚ Metabolic complications- hypoglycemia, hyponatremia, and dehydration
⮚ Ekiri Syndrome or toxic encephalopathy- altered consciousness, seizures,
delirium, abnormal posturing, and cerebral edema
 ▪ Postinfectious phase
After months, autoimmune reactions are developed

EPIDEMIOLOGY

▪ Risk factors include overcrowding, poor hygiene, and children less than 5 years
▪ It tends to occur as an epidemic in developing countries such as the Indian Subcontinent
and sub-Saharan Africa
▪ Humans are the natural host and cases are the only source of infection.
▪ Children less than 5 years account for nearly 69% of cases
▪ With improved sanitation, the incidence is decreasing and the drug resistance among
Shigella strains is increasing

LABORATORY DIAGNOSIS

The fresh stool is the ideal specimen.

PATHOLOGY:

▪ Shallow ulcer
▪ Inflamed intervening mucosa

STOOL MACROSCOPIC FEATURE:

▪ Number of motion: >10/day

▪ Amount: small quantity
▪ Colour: bright red
▪ Odorless
▪ Alkaline in nature
▪ Adherent to the container

MICROSCOPIC FEATURE:

▪ Presence of RBCs – discrete or rouleaux

▪ Pus cells- numerous
▪ Macrophages- numerous
Stools can be used to:

● Wet mount preparation

● Culture
● Selective media such as MacConkey Agar
● Culture smear and motility testing
● Serotyping
● Colicin typing

TREATMENT

● Ciprofloxacin is the drug of choice

● Alternative drugs include ceftriaxone, azithromycin, and fifth-generation quinolones
● Oral Rehydration Solution (ORS) should be started for correction of dehydration and
nutrition

PREVENTION

● Handwashing after handling children’s feces and before handling of food is highly
recommended
● Stool decontamination with Sodium hypochlorite
● No vaccine is available

REFERENCE:

Essentials of Medical Microbiology by Apurba S Sastry and Sandhya Bhat – Third Edition
 7. CORYNEBACTERIUM DIPHTHERIAE

Corynebacterium diphtheriae in methylene blue-stained smear

Ref: fig 24.1A pg no 261 essentials of medical microbiology by apurba Sastry 1st edition

● The causative agent of diphtheria infecting the throat if not treated properly, it may be
life-threatening

MORPHOLOGY:

● Gram-positive but tend to decolorize easily

● Encapsulated, nonsporing, nonmotile bacillus
● Frequently shows club-shaped swellings
● Has several species called diphtheroids
● Two characteristic features of Corynebacterium diphtheriae
● Chinese letter arrangement: appear as V or L shaped because the bacterial
cells divide and the daughter cells tend to be in acute angle to each other
● Metachromatic granules: when taking up the Loeffler’s methylene stain they
appear as bluish granules at the end of the poles of the bacilli. they are also
called volutin or babes-Ernst granules

Virulence factors(Diphtheriae toxin): Primary virulence factor responsible for the disease
STRUCTURE OF TOXIN:

MECHANISM OF DIPHTHERIA TOXIN:

FACTORS REGULATING TOXIN PRODUCTION:

● Remains toxigenic as long as the phage are present inside the bacilli
● Toxin production depends on iron concentration
● Diphtheria toxin is also produced by C. ulcerus and C. pseudotuberculosis

PATHOGENICITY:

● Incubation period : 3 to 4 days

● Diphtheria is toxemia but never bacteremia
● Bacilli are noninvasive but the toxin produced by them enters the bloodstream causing
the disease

RESPIRATORY DIPHTHERIA:

● The most common form of diphtheria

● Affected areas: nose and larynx
 ● Facial diphtheria:

Pseudomembrane coat is classically seen in diphtheria

Ref: pg no 263 fig.no 24.2A Essentials of medical microbiology by apurba Sastry 1st edition

Bull neck appearance

Ref: pg no 263 fig.no 24.2B essentials of medical microbiology by apurba Sastry 1st edition

● Elicits an inflammatory response

● This leads to the formation of mucosal ulcers lined by leathery greyish white
pseudomembranous coat
● Pseudomembrane extends in severe cases leads to airway obstruction
● Massive tonsillar swelling and neck edema bull neck appearance
SYSTEMIC COMPLICATIONS:

NEUROLOGIC MANIFESTATIONS :

● Polyneuropathy
● Peripheral neuropathy
● Ciliary paralysis

CARDIOLOGICAL MANIFESTATIONS:

● Myocarditis
● Arrhythmias
● Dilated cardiomyopathy

Polyneuropathy and myocarditis are the late toxic manifestations of diphtheria

LABORATORY DIAGNOSIS:

Specimen: throat swab and a portion of pseudomembrane

Direct smear:

Ref: fig 24.1B pg no 261 essentials of medical microbiology by apurba Sastry 1st edition

● Gram stain: club-shaped gram-positive bacilli with Chinese letter arrangement

Ref: fig 24.1C pg no 261 essentials of medical microbiology by apurba Sastry 1st edition

● Albert stain: green bacilli with bluish-black metachromatic granule

Culture media:

Ref: fig24.4A pg no 264 essentials of medical microbiology by apurba Sastry 1st edition

● Enriched medium: blood agar, chocolate agar, Loeffler’s serum slope

Ref: fig24.4B pg no264 essentials of medical microbiology by apurba Sastry 1st edition

● Selective medium: potassium tellurite agar produce black colonies

IDENTIFICATION:

● Biochemical tests such as hiss serum sugar fermentation test

● Automated identified systems such as MALDI-TOF or VITEK
● Diphtheria toxin identification:
● In vivo: guinea pig inoculation in subcutaneous and intracutaneous region
● In vitro:
 Elek’s gel precipitation test

Ref :fig.no24.5 pg no265 essentials of medical microbiology by apurba Sastry 1st edition

Elek's gel precipitation test

Detection of tox gene by PCR
Cytotoxicity on cell lines
Detection of toxin by ELISA or ICT

EPIDEMIOLOGY:

● Source of infection: by carriers

● Carrier: maybe nasal or throat. Nasal carriers are more dangerous than throat due to
frequent shedding
● Transmissions: by droplets
● Reservoir: humans
● Age: common among children of age 1 to 5. But due to immunization, it has been
changed to school children
● The situation in the world: due to widespread immunization, the cases are drastically
declined. but in recent years around 16648 cases are recorded
● The situation in India: India still has the highest cases of diphtheria ( around 8788) in
2018
PROPHYLAXIS:

Infection control measure: patient kept in an isolated room to prevent droplet infection

Post-exposure prophylaxis: booster dose of diphtheria vaccine + penicillin G +erythromycin (7

to 10 days) is given

VACCINATION:

● Induces antitoxin production in the body

● Can’t prevent the cutaneous diphtheria
● Can’t eliminate the carrier state

TYPES OF VACCINE:

● Single vaccine: Diphtheriae toxoid = diphtheriae toxin + formalin +alum (adjuvant)

● Combined vaccine:

DPT = DT( diphtheria toxoid)+ Pertussis+TT(tetanus toxoid)

DaPT = DT+PT+acellular pertussis(aP)

ADMINISTRATION OF DIPHTHERIA VACCINATION :

Schedule: by national immunization schedule of India 2020

Children:

DOSE TYPE OF DOSE GIVEN AT THE AGE OF

First dose Pentavalent vaccine 6th week
Second dose Pentavalent vaccine 10 th week
Third dose Pentavalent vaccine 14 th week
Fourth dose Booster dose 16 – 24 months
Fifth dose Booster dose 5 yrs
Sixth dose Booster dose 10 yrs
Seventh dose Booster dose 16 yrs
Pregnant women: two doses of Td at the one-month interval

Site: IM at the lateral aspect of the thigh

Storage: kept at 2-8*C

Adult immunization: recommended as cases are increasing across the world

● Adults who have completed primary vaccination: Td booster dose is indicated every 10
years till 65 years
● Adults who have not completed primary vaccination: 3 doses of Td given at 0, 1 month,
and 1 year

ADVERSE REACTION OF DPT ADMINISTRATION:

Mild: fever and local reaction

Severe: associated with neurological complications

Absolute contraindication:

● Hypersensitivity to the previous dose

● Progressive neurological disorder
 8. BORDETELLA PERTUSSIS

ETIOLOGY

● Bordetella is highly fastidious, very small, gram-negative coccobacilli, non-fermenter,

family Alcaligenaceae
● Bordetella pertussis: cause whooping cough in children, a highly contagious
vaccine-preventable bacterial disease
o characterized by a paroxysmal cough ending in a high-pitched inspiratory sound
described as "whoop".
o B. pertussis causes a violent paroxysmal productive cough in children called whooping
cough or pertussis.

PATHOGENESIS

▪ B. pertussis produces a wide array of toxins and biologically active products

▪ Pertussis Toxin (PT) most important virulence factor

▪ It causes ADP ribosylation of G protein, which activates adenylyl cyclase, leading to

increased concentrations of cAMP; responsible for producing a variety of biological
effects, such as:
✔ T cell mitogenicity
✔ Hemagglutination
✔ Adhesion to respiratory ciliated cells
✔ Inhibition of neutrophil oxidative burst monocyte migration, histamine release from mast
cells
✔ Induction of leukocytosis
✔ Enhancement of insulin secretion leading to hypoglycemia

▪ Tracheal cytotoxin

It is a part of cell wall peptidoglycan, which causes damage to the cilia of respiratory
epithelial cells by producing interleukin-1 and nitric oxide intracellularly.

▪ Adenylate cyclase toxin: It activates cyclic AMP, which impairs the host immune
function.

▪ Dermonecrotic toxin: contribute to respiratory mucosal damage.

▪ Endotoxin: has properties similar to those of other gram-negative bacterial LPS.

▪ Adhesins: role in bacterial attachment.

Examples include:

✔ Filamentous hemagglutinin (FHA)

✔ Pertactin, an outer-membrane protein
✔ Fimbriae or pili or agglutinogens
✔ BrkA (Bordetella resistance to killing) protein: mediates the serum resistance and
adhesion.

LAB DIAGNOSIS

• Specimen collection

Nasopharyngeal secretions are the best specimens which may be obtained by-

➔ Nasopharyngeal aspiration (best method)

➔ Perinasal swab
● For culture, alginate swabs are the best followed by dacron swabs.
● Cotton swabs are not satisfactory as fatty acids present in cotton may inhibit the growth
of the bacilli. However charcoal-impregnated cotton swabs (Stuart’s) may be useful.
● It is recommended to collect six swabs, at 1- 2 days intervals to achieve maximum yield.
 ● Transport: Specimens- processed immediately. If the delay is expected, then suitable
charcoal-based medium (Amies) can be used.

DIRECT DETECTION:

● B. Pertussis may be directly detected from nasopharyngeal secretions by direct

immunofluorescence test using fluorescein labeled polyclonal or monoclonal
antibodies.Because of poor sensitivity and specificity, it is not widely used.
● Culture: Nasopharyngeal Culture remains the gold standard method of diagnosis.
● B. pertussis is a strict aerobe, grows best at 35-37°C.
● It is fastidious, requires special complex media for primary isolation, such as-
● Charcoal agar supplemented with 10% horse blood and cephalexin (Regan and Lowe
medium). It is currently the medium of choice.
● Bordet Gengou glycerine-potato-blood agar was a traditional medium used before.
● Colonies are greyish while, convex with a shiny surface appear after 3- 5 days, described
as mercury drops or bisected pearls appearance

Reference: Essentials of medical microbiology by apurba Sastry –2/E–pg 368- fig 3.4
CULTURE SMEAR: Gram-staining of culture reveals small, ovoid coccobacilli (0.5 µm), tend
to arrange in loose clumps, with clear spaces in between giving a thumbprint appearance

Reference: Essentials of medical microbiology by apurba Sastry-2/E-pg:368-fig:3.4c

DETECTION SERUM ANTIBODIES

● Enzyme immunoassays (EIA~) using purified antigens of B. pertussis, such as PT, FHA,
and pertactin are the methods of choice.

MOLECULAR METHODS:

● PCR is being increasingly used in many laboratories replacing the culture, because of
increased sensitivity, specificity, and quicker results.
● The most common targeted genes are 1S481 and the PT promoter region genes.
TYPING OF B. PERTUSSIS

● It is important during outbreak investigation to find out the epidemiological link between
the isolates.
● Serotyping: It is based on two fimbrial antigens ( type 2 and 3) and one
lipooligosaccharide antigen (type l ) of Bordetella pertussis.
● Genotyping: It can be carried out by gene sequencing, and pulsed-field gel
electrophoresis~ (PFGE).
● Others: Lymphocytosis is common among young children but not among adolescents.
 9. LEPTOSPIROSIS

INTRODUCTION:
● Leptospirosis is a Spirochetal infection caused by Leptospira
● Leptospira is a thin, flexible, coiled helical bacteria.
● They have many tightly coiled spirals, with hooked ends.
● 6- 20 μm in length.

CLASSIFICATION:

● Leptospira is broadly classified into:

● L. interrogans
● L. biflexa
● These are further classified into various subgroups based on Liposaccharide antigens.

L. interrogans 26 Serogroups

L. biflexa 32 Serogroups

● SEROGROUPS OF LEPTOSPIRA INTERROGANS:

Australis Grippotyphosa Sarmin

Autumnalis Hebdomadis Sejroe

Ballum Icterohaemorrhagiae Semaranga

Bataviae Javanica Tarassovi

Canicola Leptonema Hurstbridge

Celledoni Lyme Ranarum

Cynopteri Mini Turneria

Djasiman Pomona Manhao

Pyogenes
PATHOGENESIS:

● There are two stages of pathogenesis:

FIRST PHASE (Septicemic phase)

Leptospira enter through the mucosa (Conjunctival /oral)

or abraded skin

L.interrogans enter the bloodstream and spread to various

organs including brain, liver, lung, heart & kidney.

● Vascular damage occurs Spirochetes seen in walls of capillaries, Small & Large
blood vessels.
● Penetration and invasion of tissues due to Active motility and Hyaluronidase
release.

SECOND PHASE: (Immune phase)

Antibodies develop and form

Antigen-Antibody complexes deposited in various organs

Renal colonization occurs where bacilli are adherent to the proximal tubular brush border

Excreted in urine
CLINICAL MANIFESTATION:
● INCUBATION PERIOD: 1 to 30 days.
● MILD ANICTERIC FEBRILE ILLNESS: Occurs in 90 % of patients.
● A biphasic course involving the Septicemic phase and
● Presents with flu-like illness with:
➔ Fever
➔ Chills
➔ Headache
➔ Nausea & Vomiting
➔ Conjunctival suffusion
➔ Myalgia
➔ Abdominal pain
 10. SYPHILIS

● Syphilis is a sexually transmitted disease caused by Treponema pallidum

● These bacteria come under the genus Spirochetes
● Trep- turn (helical); nema- thread-like ; pallidum- pale staining

PATHOGENESIS OF SYPHILIS

1) MODE OF TRANSMISSION
2) SPREAD

3) IP- INCUBATION PERIOD

● 9 to 90 days
● Note- blood is infectious even during incubation and early stages

CLINICAL MANIFESTATIONS

STAGES- 4
1) PRIMARY SYPHILIS
▪ 2 characteristic features are hard chancre and regional lymphadenopathy

NOTE:

If transmitted by

▪ Direct contact 🡪 primary chancre is extragenital, usually on fingers

▪ Blood transfusion 🡪 no chancre

2) SECONDARY SYPHILIS
● Develops after 6-12 weeks after healing of the primary lesion
● Skin and mucous membrane commonly affected
● Generalized lymphadenopathy
Note: condyloma lata vs accuminatum

3) LATENT SYPHILIS
● No lesions but the patients are serologically positive ie) Antigens and Antibodies present in
serum
● So the patient can transmit the infection via blood or in utero
4) LATE/TERTIARY SYPHILIS
● Gumma – destructive granulomatous lesions of skin, bones
● Neurosyphilis- chronic meningitis, general paralysis of insane and tabes dorsalis
● Cardiovascular syphilis- aneurysm of ascending aorta and aortic regurgitation
Note:

- latent + late/tertiary syphilis = Quaternary syphilis

CONGENITAL SYPHILIS

● vertical transmission from mother to baby

● consists of a triad of symptoms

DIAGNOSIS:

● laboratory diagnosis of treponema consists of demonstration, detection of antibodies, PCR

A) MICROSCOPY (common for all spirochetes)

1) DARK GROUND MICROSCOPE:

● Since Treponemes cannot be visualized by light microscope, the dark ground microscope is
used
● Advantages- no stain used so organisms are live and motile
● Motility- i) Flexion- extension motility
ii) Cock screw motility (similar to H. pylori)

Dark background, light spirochetes

Reference- Essentials of Microbiology Apurba Sastry 3/E pg no 759, fig 77.3A

2) DFA- TP (Direct Fluorescent Antibody Staining for
T. pallidum)
● Stain🡪 fluorescent- labeled monoclonal antibody targetted
against T. pallidum surface antigens
● Dark background, apple green fluorescent colored bacilli

Reference- Essentials of Microbiology Apurba Sastry 3/E pg no 759, fig 77.3B

3) BRIGHT-FIELD MICROSCOPY
● Since spirochetes are thin, silver impregnation stain is used
● 2 types of stain i) FONTANA stain- yellow background and brown spirochetes
ii) Levaditi stain
Reference- Essentials of Microbiology Apurba Sastry 3/E pg no 759, fig 77.3C

B) IMMUNOLOGICAL TEST/ SEROLOGY

● As microscopy is difficult and culture methods are not available, antibody detection methods
are of paramount importance in the diagnosis of syphilis

I) Non-Treponemal or Standard Test for Syphilis(STS)

● Here nonspecific antibodies (Reagin antibody) are detected
● These antibodies are secreted against Cardiolipin antigen present in Oxheart
● Type- Ig G or rarely Ig M
VDRL test

● This test was named after Venereal Disease Research Laboratory (VDRL), New York, where
the test was developed
● Principle🡪 Slide flocculation
● Procedure
Reference- Essentials of Microbiology Apurba Sastry 3/E pg no 760, fig 77.4
Uses- cheaper, preferred as a screening test (high sample load), detect neurosyphilis

RPR test

● Similar to VDRL with the following differences

● Prolonged shelf life, so used for the individual test (low sample load)
● No need for a microscope, results can be read with naked eyes
● More expensive

PROS AND CONS OF NON SPECIFIC OR NON TREPONEMAL TESTS

PROS

● To monitor prognosis
● IOC- investigation of choice
● VLDR- to detect neurosyphilis (CSF antibody)
● 98- 99 % specificity

CONS

● BFP- biologically false-positive reactions 🡪 positive results in nontreponemal tests, with

negative results in treponemal tests, in the absence of syphilis and not caused by technical
faults
● Prozone phenomena
● Low sensitivity

II) TREPONEMAL OR SPECIFIC TESTS:

● Here specific treponemal antibodies are detected in patient serum
● Extract of live or killed T. pallidum is used
● Advantage- High specificity and High sensitivity,
● Used as Confirmatory test
C) CULTURE
● Pathogenic Treponema are NONcultivable so Animal Inoculation is used
● Eg; Rabbits testes 🡪 Nichols Strain

D) PCR/ MOLECULAR METHODS

● To amplify and detect T. pallidum specific genes
● PCR is of paramount importance in the diagnosis of congenital and neurosyphilis

SYPHILIS AND HIV

● HIV 🡪 increases the risk of syphilis because Genital syphilis facilitates the transmission of
HIV through the abraded mucosa
● HIV patients if gets syphilis will rapidly progress to late stages of syphilis and also gets
neurosyphilis
● Also, diagnosis of syphilis in HIV patients is difficult
TREATMENT

● DOC of all stages of syphilis 🡪 Penicillin

● But for patients with penicillin allergy, tetracycline is used
PREVENTION:

● Treatment of cases and contacts (sexual partners)

● Education about safe sex practices
● Prophylactic use of barrier contraceptive methods.

Reference:

Essentials of Microbiology Apurba Sastry 3/E

 11. RICKETTSIACEAE
GENERAL FEATURES
● They are obligate intracellular organisms.
● Cannot grow on artificial media, but can grow on cell line /mice inoculation (except
bartonella).
● Transmitted by arthropod vectors (except Coxiella by inhalation mode.
● Final target site: vascular endothelial cells.

CLASSIFICATION
Order Rickettsiales

Family Rickettsiaceae Family anplasmataceae

Rickettsia Ehrlichia
Orientia Anaplasma
Wolbachia
Neorickettsia

FAMILY RICKETTSIACE
RICKETTSIAL INFECTIONS
1. Typus group.
2. Spotted fever group.

ANTIGENIC STRUCTURE
● OMP antigens – for serodiagnosis .
● LPS antigens - serves basis of Weil Felix test .

PATHOGENESIS

● Transmission: through arthropod vectors.

● Tick and mite through a bite can act as reservoirs to maintain the organism by
transovarial transmission.
● Louse and flea through rubbing or scratching the vector on abraded skin or mucosa.
● Spread: lymphatics then lymph nodes then bloodstream.
 ● Target sites: endothelial cells.
● Intracellular survival: though they get phagocyted they resist lysosomal killing and
inhibit phagolysosomal fusion.
● Multiplies by binary fission.
● Cell to cell spread by actin polymerization.

FEATURES

Epidemic typhus (louse-borne)

● Vector: louse.
● Species:R.prowazekki .
● Clinical manifestations: acute febrile disease accompanied by incubation period 1_2
weeks.
1. Headache
2. Eye discharge
3. Rashes: begins on the trunk then spreads to extremities except for palms and soles
4. Myalgia
5. CNS involvement: confusion, coma
● Risk factors: outbreaks occur in unhygienic conditions.
● Brill Zinsser disease: recrudescent illness and the R.prowazekii remains latent for years.

ENDEMIC TYPHUS (FLEA-BORNE)

● Vector: flea.
● Species: R.typhi
● Reservoir: rodents (Rattus rattus )
● Clinical manifestations: incubation period 1_2 weeks
 1. Similar to epidemic typhus but it is milder and rarely fatal.
2. Rashes on skin involving the trunk more often than the extremities.
● Geographical distribution: worldwide.

ROCKY MOUNTAIN SPOTTED FEVER (TICK-BORNE)

● Vector: Tick.
● Species: R.rickettsi
● Most severe form: associated with high mortality rate.
● Reservoir: ticks as both vector and reservoir.
● Clinical manifestations: incubation period 4_ 14 days.
1. RMS fever _ fatal
2. Rashes on palm and soles
3. Maculopapular and becomes hemorrhagic
4. CNS involvement
● Geographical distribution: America.

INDIAN TICK TYPHUS (TICK-BORNE)

● Clinical manifestations: Eschar at the site of tick bite is more severe in diabetes, alcoholic
patients.
● Vector: Tick.
● Species: R.conori
Geographical distribution: Europe, Asia.

RICKETTSIAL POX (MITE BORNE)

● Vector: mite.
● Species: R.akari
● Clinical manifestations:
1. Vesicular rashes
2. Eschar _ painless black lesions
3. Regional lymphadenopathy
● Geographical distribution: America and Ukraine.

LABORATORY DIAGNOSIS
WEIL FELIX TEST
● It is a heterophile agglutination test; where rickettsial antibodies are detected by using
certain Proteus strains (OX19, OX2, OXK strains) due to the cross-reactivity of alkali
stable LPS antigen.
 ● Procedure: It is a tube agglutination test; serial dilutions of patient serum are treated with
motile strains of P.vulgaris OX19 and OX2 and P.mirabilis OXK.
● Results:
1. In epidemic and endemic typhus _sera agglutinate mainly with OX19 and OX2 are
elevated
2. In tick-borne spotted fever _antibodies to OXK were raised.
3. The test is negative in rickettsial pox Qfever ehrlichiosis and bartonellosis .

False-positive titer
● It May be seen in presence of underlying Proteus infection .hence a fourfold rise of
antibody titer in paired Sera is more meaningful than a single titer.
It may occur due to excess antibodies in patients Sera (prozone phenomenon).this can be
obviated by testing with serial dilutions of the patient's sera.
● Weil Felix test being a non-specific test should always be confirmed by specific tests.

SPECIFIC ANTIBODY DETECTION TESTS

● Indirect immunofluorescence assay
● CFT
● Ig M capture Elisa
● Latex agglutination test

Other methods:
● Cutaneous biopsy – histological examination of sample from lesion.
● Isolation can be done by cell lines since they can’t grow in artificial media
● Neil Mosser reaction: specimens are inoculated in guinea pigs
1. R.rickettsiae: produces scrotal necrosis
2. R.prowazekki: produce only fever without any testicular inflammation
3. R.conori,R.akari:produce positive tunica reaction
● PCR.

TREATMENT OF RICKETTSIOSIS
● Doxycycline is the drug of choice for the most rickettsial illness.
● Chloramphenicol as an alternative.
SCRUB TYPHUS
● Agent :Orientia tsutsugamushi. It differs from rickettsia by both genetically as well as
lacking in LPS in the cell walls.
● Vector: mites the larval stage called chiggers stage of mite are the only stage that feeds on
humans hence scrub typhus is also known as chiggerosis
● Clinical manifestations: triad
1. Eschar
2. Lymphadenopathy
3. Maculopapular rash
● Antigenic diversity: three major types
1. Karo
2. Gillian
3. Kato
● Zoonotic tetrad: mites, rats, shrews, scrub vegetations, wet season.
● Indian scrub typhus is more common in India.
● Diagnosed by Weil Felix test (OXK raised).

EHRLICHIOSIS
● Pathogenic species: Ehrlichia chaffeensis ,Ehrlichia ewingii,Anaplasma
phagocytophilum,Neorickettsia sennestu
● Transmission: ticks except Neorickettsia sennestu_by fish carrying flukes.
● Epidemiology: USA and Asia.
● Clinical manifestations: headache, myalgia, lymphadenopathy, vomiting, diarrhea,
change in mental status.
● Inclusions: morula, elementary and initial bodies.
● Treatment: Doxycycline

Q FEVER
● Species: Coxiella burnetti
● Transmission: contaminated urine feces and milk of sheep and cattle.
● Geographical distribution: India, most part of the world except cold region.
● Clinical manifestations
1. Acute fever: pneumonia, hepatitis, pericarditis
2. Chronic fever: endocarditis
3. No rashes
● Lab diagnosis: isolation followed by IFA, PCR can also be done
● Treatment
1. Acute fever _ Doxycycline and quinolones
2. Chronic fever _ hydroxychloroquine
 ● Prevention _Vaccination, good disposal of cattle wastes, good pasteurization of milk.

BARTONELLOSIS
● B.hensele :
1. Transmission: cats
2. Cat scratch disease: causes regional lymphadenopathy and painless pustule.
● Bacillary angiomatosis: neovascular lesions in HIV infected persons.
● Bacillary peliosis: Angio proliferative disorder involving liver and spleen.
● B.quintana : Causes TRENCH FEVER.
● B.bacilliformis : Oroya fever or carissons disease, Verruca peruana.
Laboratory diagnosis
● Antibody detection_ IFA, PCR.
Treatment
● Cat scratch disease _azithromycin.
● Bacillary angiomatosis _ erythromycin or Doxycycline.
 12. LABORATORY DIAGNOSIS OF RESPIRATORY
TUBERCULOSIS

1. SPECIMEN COLLECTION:
Spot sample(on the same day under supervision)
2 sputum samples
early morning sample(on next day)
gastric aspirate – children(tend to swallow sputum)
- ICU patients (aspiration)
▪ alternatively, 2 spot samples at least one hour apart can be collected.
▪ Early morning sputum specimen: collected on an empty stomach, after rinsing the
mouth
▪ Deep inhalation and cough out from chest during exhalation and spit the sputum
in container
▪ Sputum – 3-5 ml, thick and purulent.

2. DIGESTION, DECONTAMINATION, AND CONCENTRATION:

Sputum and specimens are

i. Digested – to liquefy thick pus cells and homogenization.

ii. Decontaminated – to inhibit normal flora
iii. Concentrated – to increase yield.

METHODS:

A. Modified Petroff’s method- 4% NaOH ; recommended for LJ culture

B. NALC( N-acetyl -L-cysteine) + 2% NaOH: recommended for automated culture

systems.
 3. DIRECT MICROSCOPY BY ACID-FAST STAINING:

A. ZEIHL-NEELSEN (ZN) TECHNIQUE ( HOT METHOD )

Smears are prepared from the thick part of sputum

Smear is stained by acid-fast stain

M.tuberculosis appears slender, beaded, less uniformly stained red color acid-fast bacillus.

PRESUMPTIVE DIAGNOSIS: “acid-fast bacillus resembling M. tuberculosis are seen bt smear

microscopy by ZN stain “.

ADVANTAGES: rapid, easy to perform, cheaper

DISADVANTAGES: less sensitive than culture, cannot determine the viability.

B. KINYOUN’S COLD ACID – FAST STAINING

C. FLUORESCENCE STAINING:

- Primary stain- auramine -phenol (7-10 min )

- Decolourizer – 0.5 % acid alcohol ( 2min )
- Counterstain – 0.1% potassium permanganate ( 30 secs )

Examine under a fluorescent LED microscope

Bacilli – brilliant yellow against a dark background

⮚ Smears are screened faster – The screening method

⮚ More sensitive than ZN staining
A: ZN staining of sputum smear showing long, slender, and beaded red-colored acid-fast bacilli.

B. auramine phenol staining sputum smear – tubercle bacilli appear bright brilliant green against
a dark background.

REFERENCE: Essentials of medical microbiology by apurba Sastry- 3/E-pg no.628-fig 63.3A

and B.

4 . CULTURE METHODS:

A gold standard method of diagnosis of TB.

a. Conventional solid media ( Lowenstein – Jensen medium ):

Inoculated for 6- 8 weeks (long generation time of 10-15 hours )
Colonies: M. tuberculosis produces typical rough, tough, and buff-colored colonies.
b. Conventional liquid media: Kirchner’s medium and Middle brook 7H9 -used.
c. Automated liquid culture:
- Faster turnaround than compared to conventional culture methods
- Middle brook 7H9 medium + growth media + antibiotic mixture ----- used.
BACTEC MGIT ( mycobacteria growth indicator tube )-
- Detects growth of mycobacteria
- Performs drug susceptibility testing against all first-line and second-line
antitubercular drugs.
 CULTURE IDENTIFICATION :

Colonies of LJ media and broth from automated culture bottle

First subjected to acid-fast stain

If stain is positive, further tests are done

i) MPT 64 ANTIGEN – detection by rapid immunochromatographic

test(ICT),antigen-specific for M.tuberculosis complex and negative for
Nontuberculous mycobacteria.
ii) AUTOMATED IDENTIFICATION SYSTEM: such as MALDI-TOF
iii) BIOCHEMICAL TESTS: niacin test, rabbit pathogenicity test.

5. MOLECULAR METHODS:

i) PCR – Nested PCR – detects IS6110 gene.

- Automated Real-time PCR –

1. Catridge -based nucleic acid amplification test (CBNAAT) – eg;

GeneXpert-for identification and detection of rifampicin resistance-95%sensitive and
98%specific-turnaround time 2 hours.

2. Chip-based real-time PCR: eg; Truenat.

ii) Line Probe Assay (LPA):

⮚ involves probe-based detection of amplification DNA in specimen

⮚ used for identification and detection of resistance to 1st and 2nd line ATDs
⮚ Turnaround time 2-3 days.
SHORT NOTES
 1. EL TOR VIBRIO
⮚ It caused the 7th pandemic – originated In Indonesia in 1962
⮚ It produced milder cholera but was associated with more carrier rate than
classical.
⮚ O139 (BENGAL STRAIN )- isolated from Chennai and appears as derivative of
EL Tor, but has a distinct LPS and is capsulated.

PATHOGENESIS:

✔ TOXIN- MEDIATED (endotoxin ).

✔ Transmitted by – contaminated food and water
✔ In the small intestine, vibrio penetrate the mucous layer and reach
epithelial cells
✔ Adheres to the epithelium by type IV fimbria – toxin coregulated pilus.
✔ Gene for cholera toxin – phage coded.

CLINICAL MANIFESTATIONS

Incubation period – 24 to 48 hours

● Watery diarrhea
● Rice water stool
● Vomiting
● Muscle cramps

LABORATORY DIAGNOSIS:

1. SPECIMEN: freshly collected watery stool- for acute cases

Rectal swab – for convalescent patients or carrires.

1. TRANSPORT/HOLDING MEDIA: in 10-20 ml of :

● Venkatraman-ramakrishnan (VR) medium
● Alkaline salt transport medium
● Cary-Blair medium: for salmonella and shigella
● Autoclaved seawater.
 2. DIRECT MICROSCOPY :

Gram staining – of mucus flakes of feces – curved comma-shaped gram-negative rods,

arranged in rows- fish in stream appearance.

Motility testing by hanging method – darting motility.

3. CULTURE: aerobic and grows well in original media – nutrient agar.

Enrichment broth – alkaline peptone water(APW)

Monsur’s taurocholate tellurite peptone water

Selective media :

- TCBS Agar: yellow colonies- sucrose fermenters

- Alkaline bile salt agar (BSA): translucent oil drop colonies
- Monsur’s gelatin taurocholate trypticase tellurite agar (GTTTA): translucent
colonies with a black center and a turbid halo
- Mac Conkey agar: translucent and pale -pink on prolonged incubation – Late
lactose fermenters.

5. CULTURE SMEAR AND MOTILITY TESTING:

Culture smear – short curved gram-negative bacilli

Hanging drop – darting motility.

6 . IDENTIFICATION :

Automated systems – MALDI-TOF or VITEK

Conventional biochemical tests-

a. Catalase and oxidase +

b. ICUT test:

● indole test +
● Citrate test- variable
● Urease test – negative
 ● TSI ( triple sugar iron agar test ) – sucrose fermenter – shows acid
– gas absent – H2S absent.
c . Hemodigestion: on blood agar – greenish clearing around the main inoculum.
d.String test: vibrio mixed with 0.5% sodium deoxycholate on the slide, suspension loses
its turbidity and becomes mucoid – when lifted with string forms string.

7. BIOTYPING: classical and El tor biotypes are differentiated by:

Biotypes of V. cholerae 01 Classical biotype El Tor biotype

β-hemolysis on sheep blood agar Negative Positive
Polymyxin B Susceptible Resistant
Group IV phage susceptibility Susceptible Resistant
El Tor phage V susceptibility Resistant Susceptible
VP(Voges- Proskauer ) test Negative Positive

8. SEROGROUPING: species confirmed by agglutination test on the slide with V. cholerae

polyvalent O antisera.

First tested with O1 antisera

If found negative if agglutinated, then simultaneous serotyping with Ogawa

and Inaba antisera

Then test with O139 antisera

9. ANTIGEN DETECTION: by cholera dipstick assay

10. MOLECULAR METHOD: BioFire FilmArray –(automated multiplexed PCR).

11. ANTIMICROBIAL SUSCEPTIBILITY TESTING (AST): on Mueller Hilton agar by disc

diffusion method.

PREVENTION

GENERAL MEASURES :

● Provision of safe water

● Improved sanitary disposal of feces.
● Proper food sanitation
● Prompt outbreak investigation and taking necessary steps to reduce transmission

CHEMOPROPHYLAXIS:

Tetracycline – drug of choice

VACCINES:

Oral cholera vaccines

a. Killed whole-cell vaccine: whole-cell vaccine

Whole-cell recombinant B subunit vaccine
b. Oral live attenuated vaccines (OCV)
 2. GONORRHEA
It is a sexually transmitted disease.
CAUSATIVE AGENT – Neisseria gonorrhoeae- capsulated, gram-negative, kidney-shaped
diplococcus manifests as cervicitis, urethritis, conjunctivitis
VIRULENCE FACTORS:
1. Pili or fimbriae- helps in adhesion
2. Outer membrane proteins: protein I and protein II

CLINICAL MANIFESTATIONS:

IN MALES –

- Purulent urethral discharge

- Complications - Epididymitis, prostatitis, balanitis
- Water – can perineum – infection spreads to periurethral tissues causing abscess
with sinus formation.

IN FEMALES-

- Mucopurulent cervicitis
- Vulvovaginitis
- Spreads to the endometrium and fallopian tube
- Fitz-Hugh – Curtis syndrome - a complication of peritonitis and perihepatic
inflammation.

IN PREGNANT –

- Premature delivery
- Chorioamnionitis
- Sepsis in infant

LABORATORY DIAGNOSIS:

● SPECIMEN – urethral swab – men, vaginal swab – women

● TRANSPORT MEDIA: Stuart‘s medium
● MICROSCOPY: gram staining – gram-negative intracellular kidney-shaped diplococci
 ● CULTURE: Thayer martin medium
● IDENTIFICATION: gonococci are catalase and oxidase positive. Ferment only glucose.
● MOLECULAR METHOD: PCR
● TREATMENT: drug if choice – 3 rd generation cephalosporins.

PROPHYLAXIS: No vaccine.
 3. NON GONOCOCCAL URETHRITIS (NGU)
● It is a condition of chronic urethritis where gonococci can’t be demonstrated. The cause
could be gonococci but may not be detectable because they persist as L forms or it could
be caused by other microorganisms
● Sometimes: urethritis + conjunctivitis +arthritis 🡪 REITER’S SYNDROME

ONSET: take more than a week

URETHRAL DISCHARGE: mucous to mucopurulent

CAUSE DIAGNOSTIC METHODS TREATMENT

1) Bacterial Chlamydia trachomatis 🡪 culture on McCoy and Doxycycline,

HeLa cell lines AZITHROMY
CIN
Ureaplasma AZITHROMY
urealyticum CIN

Mycoplasma hominis

Gardenerella vaginalis

Acinetobacter lwoffi
2) Viral Herpes

Cytomegalovirus

3) Fungal Candida albicans detection of budding yeast Clotrimazole

in discharge

4) Protozoal Trichomonas vaginalis detection of trophozoite Metronidazole

 4. X and V Factors

● Haemophilus influenzae require accessory growth factors present in blood :

● FACTOR X: could be hemin/protoporphyrin IX / other iron-containing porphyrins
(group of heat-stable compounds)
● required for the synthesis of cytochrome, catalase, and peroxidase (involved in aerobic
respiration)
● This factor is required only during aerobic respiration
● FACTOR V: heat-labile factor present in RBCs, could be NAD or NADP. This is
synthesized by fungi bacteria (by staphylococcus aureus) plant and some animal cells.
● Haemophilus grows well on blood agar with factor V. Thus staph. aureus can also be
added to provide a source
● Ordinary blood agar is not suitable for its growth as V Factor is not freely available More
so, sheep blood contains NADase that destroys factor V.
● However, it is freely available in chocolate agar since at 750C NADase gets inactivated
and RBCs are lysed releasing Factor V.
 5. BILE SOLUBILITY TEST
● A bile solubility test is done to differentiate pneumococcus from other streptococci.
● Pneumococci are soluble in bile.

2 METHODS

TUBE METHOD PLATE METHOD

10% sodium deoxycholate solution + 1ml Loopful of 10% deoxycholate +

Broth culture (neutral Ph) S.Pneumoniae colony on blood agar

Culture clear due to lysis Colony lyses within few minutes

PRINCIPLE:

Based on the presence of autolytic enzyme amidase present in the pneumococcus.

 6. ZOONOTIC INFECTIONS
● A zoonosis is any disease or infection that is naturally transmissible from vertebrates to
humans

● Zoonotic infections associated with bacteria :

❖ TULARAEMIA
⮚ FRANCISELLA TULARENSIS is the causative agent of the infection
⮚ It is primarily a plague-like disease of rodents and other small animals
⮚ Results from
✔ Interactions with biting or blood-sucking insects (ticks and tabanid
flies)
✔ Ingestion of contaminated water or food
✔ Inhalation of infective aerosols
⮚ Clinical manifestations :
✔ Ulceroglandular tularemia: ulcerative lesions at the site of
inoculation with regional lymphadenopathy
✔ Pulmonary, oropharyngeal, oculoglandular, and typhoid like illness
✔ Complication: suppurated lymph node, acute kidney injury,
hepatitis, rhabdomyolysis
✔ Category A agent of bioterrorism
⮚ Lab diagnosis
✔ Culture: needs special media such as BCG agar
✔ Specimen: ulcer scrapings and lymph nodes biopsy
✔ Species identification is done by conventional biochemical tests or
by automated identification systems such as VITEK
⮚ Treatment
✔ Gentamicin is considered the drug of choice
✔ Doxycycline or ciprofloxacin can be given as alternatives

❖ PASTEURELLA INFECTION
⮚ Pasturella species are harbored as normal flora in the oral cavity of dogs
and cats
⮚ Clinical features
✔ The affected area becomes red, swollen, and painful with regional
lymphadenopathy
✔ In serious cases, bacteremia can result, causing osteomyelitis or
endocarditis, or meningitis
⮚ Lab diagnosis
✔ P.multocida is a gram-negative coccobacillus that grows in culture
media
✔ Identification is done by MALDI-TOF or VITEK
 ⮚ TREATMENT
✔ Penicillin G or amoxicillin-clavulanate

❖ CAPNOCYTOPHAGA INFECTION
⮚ Certain species are commensals in the mouth of a dog
⮚ C.canimorsus can cause fulminant septicemia following a dog bite
⮚ Associated risk factors
✔ Patients with anatomic or functional asplenia
✔ Heavy alcohol intake
✔ Liver cirrhosis
⮚ Lab diagnosis
✔ Fusiform or filamentous gram-negative coccobacilli
✔ Capnophilic
✔ Produce orange-colored colonies, take up to 14 days to grow
✔ Identification through MALDI-TOF and VITEK
⮚ Treatment
✔ Β lactam / β lactam inhibitor combinations such as ampicillin
sulbactam

❖ RAT BITE FEVER

⮚ Characterized by septic fever, petechial rashes, and painful polyarthritis
⮚ Caused by streptobacillus moniliformis and spirillum minus
⮚ Transmission
✔ Contact with rodents carrying these bacteria
✔ Consumption of food contaminated with urine and droppings of
rodents carrying bacteria
⮚ Treatment
✔ Penicillin

Reference :
Apurba s Sastry ; 3rd edition
 7. BCG
LIVE ATTENUATED VACCINE (BACTERIAL)
● The live attenuated vaccines lose their ability to induce disease but retain
immunogenicity.
● Attenuation is achieved by passing the organism into a foreign host.

ADVANTAGES
● More potent immunizing agent.
● Multiply inside the host hence antigenic dose would be larger than administered
● Capable of inducing the production of Ig A antibody production.

PRECAUTIONS
● Contraindications:
● should not be administered in immunodeficiency patients such as leukemia, lymphoma.
● Pregnancy.
● When two live vaccines are given, there should be an interval of at least 4 weeks.
● Dosage: given in single dose.
● The risk of gaining the virulence: should be given in an effective because there is always
a risk of gaining the virulence.
● Storage: must be cautiously retained.
REFERENCE
● Essentials of Apurba Shastry 3rd edition.
● Table 31.1 pg no:311; Essentials of Apurba Shastry 3rd edition.
 8. FOOD POISONING

❖ Food poisoning refers to the illness acquired through consumption of food or drinks
contaminated either with microorganisms or their toxins

BACTERIA CAUSING FOOD POISONING:

INCUBATION PERIOD 1 – 6 hrs

● Here the toxins are already present in the contaminated food (preformed toxins) that’s why
the incubation period is less

1) Staph aureus

TOXIN- Enterotoxin (preformed toxin)

● Heat stable toxin, resistant to gastric juice.

● Site of the action 🡪 vagus nerve, vomiting center, intestine
● Serotypes producing enterotoxin 🡪 Serotype A to E, G to P (15)
● Serotype F is responsible for Toxic shock syndrome and doesn’t cause food poisoning
● Detection of enterotoxin: ELISA, PCR, Latex agglutination test

SYMPTOMS

● Nausea, Vomiting
● Occasional diarrhea
● Hypotension
● Dehydration
● However, there is NO FEVER

TREATMENT: Supportive, correcting the electrolyte balance

2) Bacillus cereus
Reference- Essentials of Microbiology Apurba Sastry 3/E pg no 395

INCUBATION PERIOD 8- 16 hrs

● Here, diarrhea and abdominal cramps are present

1) Bacillus cereus (diarrheal type)

2) Clostridium perfringens
● After ingestion of contaminated food, the toxin causes severe abdominal cramps and
diarrhea

INCUBATION PERIOD >16 hrs

Vibrio cholera

Symptoms begin 1- 4 days after exposure, characterized by

● Watery diarrhea
● Abdominal cramps
● Fever, chills
● Nausea, vomiting

REFERENCE- Essentials of Microbiology Apurba Sastry 3/E

 9. UNIQUE CHARACTERISTICS OF ENTEROCOCCUS
● Enterococci show the following characteristics that help in their identification:
➔ They are gram-positive oval cocci arranged -in pairs; cocci in a pair are arranged
at an angle to each other (spectacle eyed appearance)
➔ Non-motile cocci (except gallinarum and casseliflavus).
➔ Blood agar: It produces non-hemolytic translucent colonies.
➔ MacConkey agar: It produces minute magenta pink colonies.
➔ Nutrient agar: It grows poorly.
➔ Bile aesculin hydrolysis test is positive
➔ PYR (Pyrrolidonyl-beta-naphthylamide) test is positive.
➔ they can grow in presence of extremes of conditions such as 6.5% NaCl, 40%
bile, pH 9.6, 45 degrees C, and 10 degrees C.
➔ Heat tolerance test: they are relatively heat resistant, can survive 60°C for 30
minutes.

Reference: Essentials of medical microbiology by apurba sastry-2/Epg.no 237.

 10. NAGLER’S REACTION

● Nagler’s or lecithinase reaction is used to detect Clostridium perfringes.

● C. perfringes produces α-toxin which has lecithinase activity.
● C. perfringes is grown on egg yolk or serum agar with antitoxin spread on one half of the
culture plate and the other half without antitoxin.

Reference:jaypeedigital.com

THE HALF OF THE PLATE WHICH HAS NO ANTITOXIN :

Lecithinase which is a phospholipase in the presence of Ca++ and Mg++ splits lecithin in the
medium into phosphorylcholine and triglyceride which is seen as a zone of opacity around
colonies.

THE HALF OF THE PLATE WHICH CONTAIN ANTITOXIN :

This opacity isn’t seen around colonies in antitoxin containing half of the plate, due to
toxin-antitoxin neutralization reaction,i.e., antitoxin neutralizes α toxin and hence it’s lecithinase
activity is lost and hence no opacity.

● This test is also positive for C.bifermentans, C.baratti, C.sordellii.

Reference: Ananthanarayan and Paniker’s Textbook of Microbiology – 11/E-pg no. 245,248 and
Essentials of Medical Microbiology 3/E-pg no. 539,540,541,542.
 11. MYCOPLASMA
● Smallest microbes. Pleuropneumonia-like organisms. (PPLO)
● Lack rigid cell wall
● Highly Pleomorphic.
● Better stained by Giemsa stain.
● Mycoplasma colonies – Fried egg appearance.

DISEASE CAUSED BY MYCOPLASMA :

● Mycoplasma Pneumoniae are strict human pathogen, transmitted by respiratory droplets.

● M.Pneumoniae produces various infections.
They are :
i. UPPER RESPIRATORY TRACT INFECTIONS: Pharyngitis,
Tracheobronchitis, Otitis media.
ii. PNEUMONIA: causes atypical community-acquired interstitial pneumonia.
♦ Also referred to as Eaton’s agent Pneumonia or Primary atypical pneumonia
or Walking Pneumonia.
♦ Onset is gradual with fever, malaise, headache, sore throat.
♦ Characterized by wheezing, dry cough, Peribronchial Pneumonia, Streaks of
interstitial infiltration on chest X-RAY.
iii. EXTRAPULMONARY MANIFESTATION :
♦ Neurologic: Meningoencephalitis, encephalitis.
♦ Dermatologic: Skin rashes, Steven Johnson Syndrome.
♦ Cardiac: Myocarditis, Pericarditis.
♦ Hematologic: Anemia.
● Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma urealyticum cause urogenital
infections. Non-gonococcal urethritis caused by Ureaplasma urealyticum.
✔ Transmitted by sexual contact.
✔ Causes urethritis, proctitis in males; cervicitis, vaginitis in females
 12. LAB DIAGNOSIS OF TYPHOID FEVER

Laboratory tests Diagnosis

Culture isolation: Blood and Bone marrow ✔ Bacterial colonies on broth.

culture on BHI broth/agar or on BACTEC in
1st week of illness. Incubated @37℃.

Stool culture: in 3-4 weeks of illness.

✔ Enrichment broth such as Selenite F

broth, tetrathionate broth.
✔ Low selective medium: MacConkey
agar. ⮚ Translucent NLF colonies
✔ Highly selective media :
● DCA(deoxycholate citrate ⮚ NLF colonies with black center
agar)
● XLD(Xylose lysine
deoxycholate) ⮚ Pink colonies with a black center.
● Wilson Blair green bismuth
sulfite. ⮚ Jet black colonies with metallic
sheen due to H2S production.

Culture smear and motility Gram-Negative, Motile

Biochemical Identification of S.Typhi

● Indole test o –ve

● Citrate test o –ve
● Urease test o –ve
 ● Triple sugar test o Small speck H2S production.

Widal test: 2-3 weeks of illness. Antibodies O antibodies produce granular chalky
are detected against TO, TH, BH antigens. clumps when O ag. H antibodies produce
cotton wools clumps with H antigen.

▪ InS. Typhi infection: TO and TH

antibodies increase.

Antigen detection (Serum and Urine) By ELISA

Molecular Method PCR detecting flagellin gene

Nonspecific findings Neutropenia, leukocytosis.

 13. ACTINOMYCOSIS:

It is a clinical condition caused by Actinomyces

Actinomyces are

▪ Gram –ve rods.

▪ Non- acid-fast
▪ Normal flora in the oral cavity, GIT, Vagina.

A.israelii is the most common species infecting man.

PATHOGENESIS: Actinomycosis is a chronic suppurative & granulomatous infection

characterized by multiple abscesses, formation of sinuses, discharge containing sulfur granules,
indurated swellings.
Reference : Essentials of Medical microbiology Apurba Sastry 3rd ed pg : 543.fig: 55.4

LAB DIAGNOSIS: The specimen to be collected include pus with sulfur granules, bronchial
washing, cervicovaginal secretion.

TREATMENT :

✔ Less duration of antibiotics usage may cause relapse.

✔ I.V Penicillin G for 4-6 weeks
✔ Followed by Oral Penicillin V for 6-12 months.
✔ Surgical Removal of affected tissues for the extensive lesion.
✔ Penicillin allergy patients are treated with Doxycycline, Ceftriaxone, Clindamycin.

REFERENCE: ESSENTIALS OF MEDICAL MICROBIOLOGY- APURBA SASTRY

3RD ED.
 14. NONSPORING ANAEROBIC INFECTIONS

CLINICAL PRESENTATIONS
i. ANAEROBIC COCCI
● Peptococcus and peptoStreptococcus - normal flora of skin
mouth intestine and vagina. However are recovered from various
clinical infections such as puerperal sepsis, skin and soft skin
infections, and brain abscess
● Veillonella - usually non-pathogenic
ii. GRAM-POSITIVE NONSPORING ANAEROBES
● Bifidobacterium species - beneficial normal flora in the mouth
and gut
● Eubacterium species - commensals in mouth and intestine
● Propionibacterium species - cause acne vulgaris and folliculitis.
Also, cause prosthetic hip joint and prosthetic heart valve
infections
● Lactobacillus species - normal flora of mouth gut and vagina
iii. GRAM-NEGATIVE NON - SPORING AND ANAEROBIC BACILLI
● Bacteroides fragilis
a. Virulence factor include
i. Capsular polysaccharide
ii. Lipopolysaccharide
 iii. Enterotoxin
b. It causes peritonitis and intraabdominal abscess following
bowel injury and pelvic inflammatory disease
c. Antral toxigenic strains can cause diarrhea
● Prevotella
a. Pigmented (eg) P.melaninogenica
i. Has been isolated from lung or liver abscess
mastoiditis pelvic and genitourinary infection and
lesions of intestine and mouth
b. Non-Pigmented (eg) P.buccalid
i. Cause periodontal disease and aspiration
pneumonitis
● Porphyromonas
a. P.gingivalis - periodontal disease
b. P.endodontalis - dental root canal infections
● Fusobacterium
a. F.nucleatum - Normal inhabitant of mouth but can cause
oral infection pleuropulmonary sepsis CNS infection and
septic arthritis
b. F.necrophorum - is an agent of Lumiere’s syndrome
● Leptotrichia buccalis
a. Part of normal oral flora
b. Are implicated in acute necrotizing gingivostomatitis
known as Vincent's angina.
Reference: Essentials of medical microbiology Apurba Sastry 3rd edition
 15. WEIL’S DISEASE
(Hepato- renal hemorrhagic syndrome)

➔ Severe form of icteric illness.

➔ Occurs in 10% of patients.
➔ Biphasic course may not be present.

CLINICAL FINDINGS INCLUDE:

➔ High grade fever

➔ Liver jaundice and raises liver enzymes
➔ Hemorrhages: Pulmonary hemorrhage, Gastrointestinal hemorrhage,
Conjunctival hemorrhage
➔ Raised Serum Urea and Creatinine with Renal failure.

LABORATORY DIAGNOSIS OF WEIL’S DISEASE:

LABORATORY FIRST STAGE SECOND STAGE

DIAGNOSIS (Septicemic phase) (Immune phase)

Specimens Blood and CSF Urine

Serology for Antibody Serum IgM absent Serum IgM present

detection

Antibiotic treatment Susceptible to Refractory to

antibiotics antibiotic treatment

LABORATORY DIAGNOSIS OF LEPTOSPIROSIS:

● Specimens used:
● First Stage: Blood & CSF
● Second Stage: Urine
1. MICROSCOPY:
● Wet Films: Observed under dark ground / Phase contrast microscope. Leptospira
exhibits spinning & translational movements. They are highly motile. Leptospira
interrogans- spirally coiled with hooked ends visualized.

● Staining: Stained by Silver impregnation stain - Fontana stain and modified

Steiner technique.
● Disadvantages: Require technical expertise and be less sensitive. Serum proteins
in blood resemble Leptospires.

2. CULTURE ISOLATION:
● Culture Condition: Incubated at 30o for 4 to 6 weeks. Culture fluid should be
examined under a dark ground microscope for leptospires’ presence.
● Culture Media: EMJH (Ellinghausen, McCullough, Johnson, Harris) medium,
Korthof’s and Fletcher’s medium.

3. SEROLOGY FOR ANTIBODY DETECTION:

Antibody detection tests are classified into:

Genus specific test:

Uses broadly reactive genus-specific antigen derived from a non-pathogenic L.biflexa
Patoc 1 strain.
● ELISA: detects IgM and IgG separately.
● Lepto dipstick array test: detects IgM antibodies.
● Immunochromatographic test: detects IgM and IgG separately.

Serovar-specific test:
● Microscopic agglutination test: Gold standard method and reference test for
Leptospirosis diagnosis. Used to detect the presence of antibodies.
 Patient’s serum mixed with live antigen suspension of leptospirosis endemic in the
locality, in a microtitre plate

Incubated for: 2-4 hrs at 30o

Examine under dark ground microscopy

Check for the presence of Agglutination.

Cross agglutination and absorption test:

● To detect relatedness between strains.

4. MOLECULAR METHODS:
● Polymerase chain reaction: Genes like 32 kDa lipoprotein gene, 16s/23s rRNA,
IS1533 insertion sequence targeted.
● PCR: Not antigen-specific
.
5. NON SPECIFIC FINDINGS:
● Elevated Blood Urea Nitrogen & Serum Creatinine
● Elevated Bilirubin and Liver enzymes in serum.

TREATMENT:
● Mild Leptospirosis:
● Oral doxycycline- 100 mg twice a day for 7 days
● Amoxicillin
● Severe Leptospirosis:
● Penicillin is the drug of choice- 1.5 million units IV, 4 times/day for 7 days.
● Cefotaxime
● Ceftriaxone
 16. CAUSATIVE AGENT FOR RELAPSING FEVER:
● Epidemic relapsing fever is caused by various species of Borrelia, a spirochete.
● Relapsing fever: Recurrent episodes of fever with nonspecific symptoms following
exposure to insect vectors.
● Two types:
Caused by: Vector

EPIDEMIC RF B.recurrentis Louse

ENDEMIC RF B.duttonii, B.hermsii, Lice

B.turicatae

● PATHOGENESIS:
Mode of transmission

EPIDEMIC RF Crushing of louse by scratching.

ENDEMIC RF Bite of infected lice.

Borrelial surface antigen undergoes repeated antigenic variation, hence evading the
immune host system.

● CLINICAL MANIFESTATIONS:
Recurrent febrile episodes - 3 to 5 days with intervening afebrile periods of 4-14
days.
Hemorrhages most likely in Epidemic RF (Epistaxis & blood-tinged sputum)
Neurologic features- seizures, meningitis & psychosis may occur in 10-30% of
cases, these are common in Epidemic RF.

● LABORATORY DIAGNOSIS:
● Specimen: Blood
● Microscopy:
➔ Peripheral thick/thin smear- stained by Wright or Giemsa stain
➔ Dark ground or Phase contrast microscopy- detect motile spirochetes
● Culture:
 ➔ Confirmation is done by isolation of Borrelia from blood in the afebrile
period
● Serology: To detect antibodies
➔ ELISA & IFA
➔ GlpQ assay
● Molecular method: PCR
● TREATMENT:
● Doxycycline and Erythromycin- drug of choice
● Single-dose for Epidemic RF & 7-10 days for Endemic RF.

Reference: Essentials of Medical Microbiology by Apurba S Sastry - 3/E

Reference: Complete Microbiology for MBBS by CP Baveja - 7th edition –pg 739 - Fig 49.1.7
17. VARIOUS MECHANISMS BY WHICH ESCHERICHIA COLI
PRODUCE DIARRHEA.
● E. coli causes diarrhea mainly through Enterotoxins. The various enterotoxins are,

1. LT (HEAT - LIABLE TOXIN ):-

● Mechanism of action: It has 2 peptide fragments A and B.

● Fragment B – It is a binding fragment; binds to GM1 ganglioside receptors present on the
intestinal epithelium following which A fragment is internalized.
● Fragment A – It is the active fragment, causes ADP ribosylation of G protein 🡪 activates
adenylate cyclase 🡪 results in the intracellular accumulation of cyclic AMP 🡪 leads to
increased outflow of water and electrolytes into the gut lumen, with consequent diarrhea.

2. ST (HEAT – STABLE TOXIN ):-

● Mechanism of action: It binds to the guanylate cyclase C 🡪 increased production of

cyclic GMP 🡪 fluid accumulation in the gut lumen 🡪 diarrhea.

3. VEROCYTOTOXIN OR SHIGA TOXIN:-

● Mechanism of action : It has 2 fragments A and B

● Fragment B binds to the globotriosyl ceramide ( gb3) receptor on intestinal epithelium.
● Fragment A is the active fragment . It inhibits protein synthesis by inhibiting 60S
ribosomal subunit.

There are 6 pathotypes of diarrheagenic Escherichia coli,

1. Enteropathogenic E. coli (EPEC)

2. Enterotoxigenic E. coli (ETEC)
3. Enteroinvasive E. coli (EIEC)
4. Enterohemorrhagic E. coli (EHEC)
5. Enteroaggregative E. coli (EACE)
6. Diffusely adherent E. coli (DAEC)
 ⮚ Enteropathogenic E. coli – causes infantile diarrhea.
Mech. Of action :
● Adhesion of intestinal mucosa, mediated by plasmid coated bundle forming pili,
which form cup-like projections called pedestals.
● A/E lesions ( attaching and effacing lesions ) are typical lesions produced on the
intestinal epithelium ; which leads to disruption of brush border epithelium causing
increased secretion and watery diarrhea.

⮚ Enteroinvasive E. coli – The epithelial cell invasion is mediated by

plasmid – coded antigen called Virulence marker antigen ( VMA ).

⮚ Enterotoxigenic E. coli – causes Traveler’s diarrhea.

Mech. of action :
● Attachment to intestinal mucosa is mediated by fimbrial protein called CFA (
colonization factor antigen )
● Toxin production – Heat liable toxin (acts by inc. cAMP) and heat stable toxin ( acts
by inc. cGMP).

⮚ Enterohemorrhagic E.coli – acts by secreting shiga toxin ( by inhibiting protein

synthesis by inhibiting 60S ribosome ).
⮚ Enteroaggregative E. coli – Intestinal colonization is mediated by aggregative
adhesion fimbriae I ( regulated by aggR gene ). It also produces EAST 1 toxin
(enteroaggregative heat stable enterotoxin 1 ).

REFERENCE:

Essentials of Medical Microbiology by Apurba S Sastry and Sandhya Bhat – Third Edition
 19. MANTOUX TEST

⮚ Also known as Tuberculin test/Pirquet test

⮚ Antigens used in tuberculin test:
• OT (Old tuberculin antigen): It is a crude preparation of tubercle bacilli, now
rarely used
• PPD (Purified protein derivative antigen): It is a purified preparation of the
active tuberculoprotein.
⮚ DOSAGE: It is expressed in the tuberculin unit (TU). One TU is equal to 0.01
mL of OT or 0.00002 mg of PPD
⮚ PROCEDURE:
• Mantoux test: It is the most commonly employed method. 0.1 mL of PPD
containing 1 TU is injected intradermally into the flexor surface of the forearm
⮚ Reading: It is taken after 48-72 hours. At the site of inoculation, an induration
surrounded by erythema is produced. If the width of the induration is:
• 2:10 mm: Positive (tuberculin reactors)
• 6-9 mm: Equivocal/doubtful reaction
• <5 mm: Negative reaction.
⮚ INTERPRETATION OF RESULT:
Adults: Positive tuberculin test in adults-only indicates present or past exposure
with tubercle bacilli but does not confirm the presence of active stage of the
disease.
Children: In children, a positive test indicates active infection and is used as a
diagnostic marker
⮚ False-positive: The test becomes positive after
• BCG vaccination (after 8-14 weeks)
• Nontuberculous mycobacteria infection.
⮚ False-negative: The test may become negative in various conditions such as early
or advanced TB, miliary TB, decreased immunity (HIV-infected people)
 19. NAME 4 PIGMENTS PRODUCED BY BACTERIA
Pigments Bacteria Color
Staphyloxanthin Staphylococcus aureus Golden yellow

Zeaxanthin Bacillus Brown

Pyocyanin Pseudomonas aeruginosa Blue-green

Anthraquinone Penicillium oxalium Red

20. MILK RING TEST AND ITS USES

● It is done to detect Brucella antibodies in milk
● Procedure: Sample of whole milk mixed well with a drop of stained Brucella antigen
(Concentrated suspension of killed Bacillus abortus/B.melitensis stained with
hematoxylin)
● Incubation in water bath at 70°C for 40-50 mins
● If antibodies present in milk bacilli agglutinated and rise with cream to form blue ring at
top leaving milk unstained
● Positive : Blue ring at top leaving milk unstained
● Negative: No ring .Milk remains uniformly blue
● Uses: For diagnosis of brucellosis in animals
● Screening test to detect presence of Brucella antibodies in infected cattle

21. NEIL MOOSER REACTION

● Animal pathogenicity testing done in guinea pigs to speciate rickettsial species
● R.typhi and R.prowazekii are similar but can be differentiated by which R.typhi transmit
endemic typhus while later does not
● When male guinea pigs inoculated intraperitoneally with blood from a case of endemic
typhus / culture of R.typhi
● They develop fever & characterised scrotal inflammation
● This is called Neil Mooser / Tunica reaction
● This reaction is negative with R.prowazekii
 22. VIRULENCE FACTORS OF STAPHYLOCOCCUS AUREUS
CELLWALL CELL SURFACE PROTEINS EXTRACELLULAR
ASSOCIATED ENZYMES
Peptidoglycan layer: Clumping factor/Bound coagulase: Coagulase:
● Thicker ● It is a fibrinogen binding adhesion ● Acts with
● Induce ● Responsible for slide coagulase test coagulase reacting
inflammatory ● Fibronectin binding adhesion factor (CRF) in
response ● Collagen binding adhesin plasma
● Endotoxin like ● To bind with &
activities activate
prothrombin
● Forming fibrin
clots that protect
bacteria from
phagocytosis
● This serves as the
basis for the tube
coagulase test
Teichoic acid: Protein A: ● Heat stable
● Made of ribitol ● 42k Da polypeptide encoded by spa thermonuclear
phosphate gene and DNase
polymers ● Anticomplementary,antiphagocytic, ● Staphylokinase/
● Helps in ○ Chemotactic,mitogenic fibrinolysin
adhesion of ● Induce platelet damage ● Breaks fibrin clot,
cocci to ● Inhibit opsonization spread infection
mucosal ● Mediates staphylococcal ● Hyaluronidase
surfaces co-agglutination reaction: ● Breaks connective
● Inhibit ● Protein A bind to the Fc terminal of IgG tissue
complement-m ● Leaving free Fab region that binds with ● Lipase and
ediated its specific antigen phospholipase
opsonization ● Microcapsule: ● Breakdown of
● Has polysaccharide lipids
● Inhibits phagocytosis by neutrophils
●
TOXINS OF STAPHYLOCOCCUS AUREUS
CYTOLYTIC TOXINS ENTEROTOXIN EPIDERMOLYSIS/
EXFOLIATIVE
Hemolysins: ● Responsible for ● Responsible for
● Alpha hemolysin- Staphylococcal food Staphylococcal Scalded
leucocidal, cytotoxic, poisoning Skin Syndrome (SSSS)
dermonecrotic ● A preformed toxin, ● Has 2 proteins ET-A &
● Lyse rabbit cells incubation period 1-6 hrs ET-B
● Beta hemolysins – is a ● Has 15 serotypes ● Localized tender
sphingomyelinase (A-E,G-P) blisters, bullae
● Lyse sheep cells ● Antigenic & is neutralized formation to exfoliation
● Gamma hemolysin by specific antibodies ● Separation of outer
● Delta hemolysin epidermal layer leaving
denuded underlying
skin- Nikolsky's skin
● Often occurs in
newborns (Ritters
disease)& children
● Milder forms-
pemphigus neonatorum,
bullous

Panton Valentine leucocidin Toxic Shock Syndrome Toxin

(PVL) ● TSST-1 & TSST-2
● Has bicomponent S ● Act as superantigen
and F ● Shows fever, hypotension,
● Gamma hemolysin and myalgia, diarrhea,
PVL are called ● Erythematous rash,
synergohymenotropic
toxins
● Leucocidal &
hemolytic
● PV toxin expressed on
MRSA

Ref: Ananthanarayan and Paniker’sTextbook of Microbiology Ed: 11

Essentials of Medical Microbiology by Apurba S Sastry Ed:3
 22. SATELLITISM:
Satellitism of H. influenzae (schematic diagram)

Ref: fig.no33.1 pg no 364 essentials of medical microbiology by apurba Sastry 1st edition

● Seen in the Haemophilus influenzae

● H. influenzae is highly fastidious require factor X and V for their growth
● H. influenzae grows well on blood agar and chocolate agar
● Colonies of H. influenzae can grow adjacent to S. aureus- satellitism

Satellitism of H. influenzae around S.aureus

Ref: fig.no33.2A pg no 364 essentials of medical microbiology by apurba Sastry 1st edition

● When S.aureus is streaked across blood agar plate perpendicular H. influenzae streak
line factor V is released
 ● Thus, it forms larger colonies across the S. aureus streak line which gradually decreases
away from the line
● This property is routinely employed for the isolation of H. influenzae

REFERENCE:

● Essentials of medical microbiology by apurba Sastry and Sandhya Bhat 3rd edition
● Ananth Narayanan and paniker’s textbook of microbiology seventh edition
● Images reference: essentials of medical microbiology by Apurba Sastry and Sandhya
Bhat First edition
 23. CLOSTRIDIUM BOTULINUM

MORPHOLOGY:

● Gram-positive spore forming (oval and subterminal) obligate anaerobes

● Non-capsulated
● Has peritrichous flagella
● Produces powerful toxin called botulinum which causes botulism

PATHOGENESIS:

C.botulinum is non-invasive. The pathogenesis is due to botulinum toxin

SEROTYPE:

● Has 8 serotypes—A, B, C1, C2, D, E, F&G

● A, B, E causes human diseases; A most severe
● All serotypes produce neurotoxin except C2 which produces an enterotoxin

TOXINS:

● BT differs from other toxins as it is produced intracellularly and appears outside

after autolysis of bacterial cell
● Nontoxic protoxin active neurotoxin
Trypsin or
Proteolytic enzymes

MECHANISM OF ACTION OF BT:

● After entry, BT is transported via the blood to peripheral cholinergic nerve

terminals
● The most common nerve terminals are NMJ, postganglionic parasympathetic
nerve endings, and peripheral ganglia. It does not affect CNS
● BT blocks the release of Ach, leading to paralysis
RECOVERY:

● Blocking of Ach release is permanent but action is short-lasting and recovery

occurs in 2-4 months, once new terminal axons sprout

THERAPEUTIC USES:

● BT can be used in the treatment of spasmodic conditions like strabismus,

blepharospasm, and myoclonus

CLINICAL MANIFESTATIONS:

Manifestations appear after an incubation time of 18-36 hrs which include:

● Diplopia, dysphasia, dysarthria

● Flaccid paralysis of voluntary muscles
● Decreased deep tendon reflexes
● Constipation
● Respiratory muscle paralysis may lead to death

TYPES OF BOTULISM:

FOODBORNE BOTULISM:

● Due to consumption of food contaminated with preformed BT

● Sources: homemade improperly canned food or fermented food and beverages
● Most cases are sporadic; outbreaks are rare

WOUND BOTULISM:

● Due to contamination of wounds with C. Botulinum spores

● Presents like foodborne botulism except for the absence of GIT features
● Can be recurrent in injection drug users
INFANT BOTULISM:

● Most common type (75% of total cases)

● Due to ingestion of contaminated food with spores of C. Botulinum
● Floppy child syndrome is seen which includes an inability to suck, swallow,
weakened voice, ptosis, floppy neck, and extreme weakness.
● Managed by supportive care and assisted feeding
● Rarely progresses to generalized flaccidity, respiratory failure, and sudden death

ADULT INTESTINAL BOTULISM:

● Rarely, in patients with suppressed normal flora, the colonized clostridial

spores may germinate producing toxin: as in infant botulism

IATROGENIC BOTULISM:

● Due to injection of an overdose of toxin while used for therapeutic

purpose

INHALATION BOTULISM:

● Aerosolization of spores may occur as in act of bioterrorism

LAB DIAGNOSIS:

ISOLATION OF THE BACILLI:

● Done on blood agar or Robertson’s cooked meat (RCM) broth

● In RCM broth they can be either proteolytic and saccharolytic turning the
meat particles black and pink respectively
● Growth on culture media can be confirmed by gram staining and
biochemical tests or by automated methods such as MALDI-TOF
● Serotyping is done with type-specific antisera
TOXIN DEMONSTRATION (MOUSE BIOASSAY):

● Specimens are injected into mice; dial develops paralysis in 48 hours; which can be
inhibited by prior administration of specific antitoxin.
● The sensitivity of the mouse bioassay varies inversely with the time elapsed between the
onset of symptoms and sample collection.

TREATMENT:

● Meticulous intensive care support is needed (such as mechanical ventilation if respiratory

paralysis develops).
● Botulinum antitoxin: It should be administered immediately on clinical suspicion,
without waiting for laboratory confirmation. Earlier the administration better is the cure
rate because antitoxin can neutralize the unbound free toxin molecules. However, once
toxin binds to nerve ending antitoxin has no role.
● In wound botulism, suspected wounds and abscesses should be cleaned, debrided, and
drained promptly Antibiotics such as penicillin or metronidazole may be useful.

CLOSTRIDIUM CAUSING GAS GANGRENE:

Established agents: C. peirfringens (most common, 60% of the total cases) and C. novyi and C.
septicum, (20- 40%).

• Probable agents: there are less commonly implicated; e.g.- C. histolyticum., C. sporogenes, C.
fallax, C. bifermentans, C. sordellii C. aerofoetidum C. tertium.
 24. QUELLUNG REACTION:
● Also known as Neufeld reaction
● This test was routinely done in the past at the bedside, directly from sputum
samples from pneumonia cases. When the specimen is treated with type-specific
antiserum, along with methylene blue dye: the capsule becomes swollen, sharply
delineated, and refractile
● Currently, serotyping is done by a latex agglutination test using type-specific
antisera.
Essays
 ESSAYS:
1. Hepatitis Viruses. Enumerate viruses causing Post – Transfusion hepatitis. Discuss in
detail about the morphology, pathogenesis, laboratory diagnosis and prophylaxis of
Hepatitis B virus (8)

2. Classify rhabdoviruses, details about rabies virus (6)

● SN covered in this: Rabies prophylaxis (5)

3. Classify arbovirus, details about Japanese encephalitis (5)

● SN covered in this: Japanese B encephalitis virus (6)

4. Describe in detail the pathogenesis, laboratory diagnosis, treatment, prevention and

control of dengue fever (5)
● SN covered in this:
i. Dengue fever (3)
ii. Dengue virus (2)

5. DNA viruses. Classify herpes viridae, details about herpes simplex virus, and detail about
Varicella Zoster Virus.
● SN covered in this:
i. Varicella zoster (2)
ii. Human herpes virus

6. Details about EBV (3)

● SN covered in this: EBV (3)

7. Classify picornaviruses. Describe the pathogenesis, clinical features and laboratory

diagnosis of poliovirus. Add a note on prophylaxis against Poliomyelitis. Viruses causing
aseptic meningitis (4)
● SN covered in this:
i. Immune prophylaxis of polio (2)
ii. Lab diagnosis of Poliomyelitis
iii. Aseptic meningitis
8. Describe the morphology of HIV. Describe the pathogenesis and Laboratory diagnosis of
HIV infection. Add a note on pre exposure prophylaxis (2)
● SN covered in this: Laboratory diagnosis of AIDS

9. Discuss briefly on Immunoprophylaxis of viral diseases. Discuss briefly on viral

vaccines.
● SN covered in this:
i. Oral / Sabin polio vaccine (2)
ii. Rabies vaccine (2)

10. Discuss various methods for isolation of viruses in the laboratory.

● SN covered in this:
i. Tissue culture for viruses (4)
ii. Viral inclusion bodies (4)
iii. Cytopathic effects (2)
iv. Viral cell culture
v. Egg culture

11. Describe the morphology, pathogenesis and laboratory diagnosis of Influenza virus.
● SN covered in this:
i. Diagnosis and prophylaxis of H1N1 infection
ii. Recent Swine flu pandemic
iii. Prophylaxis of influenza
iv. Influenza viruses

12. Discuss the pathogenesis, clinical features, complications, laboratory diagnosis of

Rubella virus. Add a note on its prevention.
● SN covered in this: MMR Vaccine (3)

13. Discuss the pathogenesis, clinical features, complications, laboratory diagnosis of

Measles virus. Add a note on its prevention.
● SN covered in this:
i. MMR Vaccine. (3)
ii. Warthin Finkeldey giant cells.(2)
iii. Measles virus (2)
14. Classify Coronaviruses. Discuss the morphology, pathogenesis, clinical features,
laboratory diagnosis, treatment & prevention of COVID-19. Explain in detail the recent
COVID-19 pandemic and other previous outbreaks.

15. Define & Classify Slow virus diseases. Discuss the mechanism, clinical manifestations,
laboratory diagnosis & treatment of Prion diseases. How will you sterilize the Prion
contaminated materials?
● SN covered in this: Slow viruses / slow viral disease of man (2)

16. Classify Paramyxoviruses. Write the morphology, pathogenesis, clinical Features,

laboratory diagnosis, treatment & prevention of Mumps. SN covered in it: Difference
between the orthomyxovirus and paramyxovirus.
 ESSAY 1
Hepatitis Viruses. Enumerate viruses causing Post – Transfusion hepatitis. Discuss in detail
about the morphology, pathogenesis, laboratory diagnosis and prophylaxis of Hepatitis B
virus (8)

GENERAL FEATURES OF HEPATITIS VIRUSES

 Reference: Essentials of Medical Microbiology, Apurba Sastry E/1: Page No. 527 & 528 Table
no. 50.1

Hepatitis B viruses are:

▪ Most widespread, produces acute self-limiting hepatitis which is subclinical or

symptomatic
▪ Only DNA virus among hepatitis viruses
▪ Belongs to Hepadnaviridae, genus Orthohepadnavirus

MORPHOLOGY:

Three morphologic forms:

1. Spherical forms: Numerous, small (22nm in diameter), made of HBsAg antigen.

2. Tubular or filamentous forms: 22nm diameter, 200 nm long, made of HBsAg antigen

3. Complete form or Dane particles: Large, less frequently observed, 42nm size spherical
virions

Reference: Essentials of Medical Microbiology, Apurba Sastry E/1: Page No. 530 Fig 50.2
PATHOGENESIS:

Mode of replication of HBV

Reference: Essentials of Medical Microbiology, Apurba Sastry E/1: Page No. 532 Fig no. 50.4

CLINICAL MANIFESTATIONS:
 Reference: Essentials of Medical Microbiology, Apurba Sastry E/1: Page No. 533 Fig no. 50.5

LABORATORY DIAGNOSIS:

- Definitive diagnosis based on serological demonstration of viral markers

Methods available:

▪ ELISA

▪ Chemiluminescence
▪ ICT
▪ Detection by PCR and quantified by RT PCR

Serological markers of Hepatitis B:

Reference: Essentials of Medical Microbiology, Apurba Sastry E/1: Page No. 534 Fig 50.6
PROPHYLAXIS:

ACTIVE IMMUNIZATION (HEPATITIS B VACCINE):

▪ Method of preparation: HBsAg antigen is used in baker’s yeast by recombinant

technology.
▪ Route of administration: Intramuscular route over deltoid region.
▪ Dosage: 10-20 microgram /dose

30 doses at 0, 3, 6 months

PASSIVE IMMUNIZATION (HEPATITIS B IMMUNOGLOBULIN / HBIG):

▪ Indications: Used immediate protection is required

▪ Timings: HBIG started immediately after accidental exposure.
▪ Recommended dose: 0.06ml / kg single dose by IM
▪ Combined immunization:

Vaccine + HBIG for neonates born to HBV infected mother

GENERAL PROPHYLACTIC MEASURES:

▪ Screening
▪ Safe sex
▪ Safe aseptic surgical practices
▪ Safe injection practices
▪ Health education
 ESSAY 2

CLASSIFY RHABDOVIRUSES,
DETAILS ABOUT RABIES VIRUS (6)
SHORT NOTE: RABIES PROPHYLAXIS (5)

RHABDOVIRUSES
▪ Bullet – shaped, enveloped viruses with a single – stranded RNA genome

CLASSIFICATION (Infecting humans)

⮚ Vesiculovirus
⮚ Lyssavirus

Reference: Ananthanarayan and Paniker’s Textbook of Microbiology E/10, Page no. 535, Fig no:
57.1

RABIES VIRUS:

MORPHOLOGY:

● Bullet shaped
● 180 x 75 nm in size
● One end rounded or conical and the other planar or concave.
 ● Lipoprotein envelope carries knob-like spikes, composed of glycoprotein G. Spikes
may be released by treating with lipid solvents or detergents.
● Beneath the envelope is the membrane or matrix (M) protein layer.
● The core of the virion consists of helically arranged ribonucleoprotein
● The genome is an unsegmented, linear, negative sense RNA.
● RNA-dependent RNA transcriptase and some structural proteins are present in
nucleocapsids.

RESISTANCE:
The virus is sensitive to:
● Ethanol
● Iodine preparations
● Quaternary ammonium compounds
● Soap and detergents
● Lipid solvents (ether, chloroform and acetone)
It is inactivated by:
● Phenol
● Formalin
● beta propionolactone
● Ultraviolet irradiation
● Sunlight
- Thermal inactivation occurs in one hour at 50°C and five minutes at 60°C.
- It dies at room temperature but can survive when stabilised by 50% glycerol for
weeks. It survives at 4°C for weeks.
- It can be preserved at -70°C or by lyophilisation.
- For storage in dry ice, the virus has to be sealed in vials as it is inactivated on
exposure to CO2.

ANTIGENIC PROPERTIES:
GLYCOPROTEIN: The surface spikes are composed of glycoprotein G, important for
pathogenesis, virulence and immunity.
Important properties are:
● It mediates the binding of the virus to acetylcholine receptors in neural tissues.
● It induces hemagglutination inhibiting (HI) antibodies.
● It induces neutralizing antibodies (protective antibodies)
● It stimulates cytotoxic T cell immunity.
● It is a serotype-specific antigen.
● Purified glycoprotein may act as a safe and effective subunit vaccine.

NUCLEOPROTEIN:
Properties of nucleocapsid protein are:
● It induces complement-fixing antibodies.
● The antibodies are not protective.
● The antigen is group specific and cross-reactions are seen with some rabies-related
viruses.
● The antiserum prepared against the nucleocapsid antigen is used in
immunofluorescence tests for diagnostic purpose
● Other antigens identified include two membrane proteins, glycolipid and
RNA-dependent RNA polymerase.

HOST RANGE AND CULTIVATION:

Animals:
● All mammals are susceptible to rabies infection, differences exist in susceptibility
between species.
● Cattle, cats and foxes are highly susceptible
● Skunks, opossums and fowl are relatively resistant.
● Humans and dogs occupy an intermediate position. Pups are more susceptible than adult
dogs.
● Experimental infection can be produced in any laboratory animal
● Mice are the animals of choice. They can be infected by any route. After intracerebral
Inoculation, they develop encephalitis and die within 5-30 days.
Street virus:
● The rabies virus isolated from natural human or animal infection is termed the street
virus.
● Inoculation through any route can cause fatal encephalitis in laboratory animals after a
long and variable incubation period of about 1-12 weeks.
● Intracytoplasmic inclusion bodies (Negri bodies) can be demonstrated in the brain of
animals dying of street virus infection.
● Negri bodies are composed of a finely fibrillar matrix and rabies virus particles and are
most abundant in the cerebellum and hippocampus.
Fixed virus:
● After several serial intracerebral passages in rabbits, the virus undergoes certain changes
and becomes what is called the fixed virus.
● The fixed virus is more neurotropic, though it is much less infective by other routes.
● After intracerebral inoculation, it produces fatal encephalitis after a short and fixed
incubation period of 6-7 days.
● Negri bodies are usually not demonstrable in the brain of animals dying of fixed virus
infection.
● The fixed virus is used for vaccine production.
Chick embryos:
● The rabies virus can be grown in chick embryos.
● The usual mode of inoculation is into the yolk sac.
● Serial propagation in chick embryos has led to the development of attenuated vaccine
strains like Flury and Kelev.
● Strains adapted to duck eggs which give high yields of the virus have been used for
the preparation of inactivated vaccines.
Tissue culture:
● The rabies virus can grow in primary and continuous cell cultures such as chick
embryo fibroblast, and porcine or hamster kidney but cytopathic effects are not
apparent and the yield of virus is low.
● The fixed virus strains adapted for growth in human diploid cell, chick embryo and
Vero cell cultures are used for the production of vaccines.
RABIES
● Rabies has been recognized since ancient times as a disease transmitted to humans and
animals by the bite of 'mad dogs'.
● The disease in human beings is called hydrophobia because the patient exhibits fear of
water, being incapable of drinking though subject to intolerable thirst.
● The causative agent has been associated with the saliva of rabid dogs.
● In a series of studies from 1881, Pasteur established that the rabies virus was present in
the brain of infected animals. By serial intracerebral passage in rabbits, he obtained the
fixed virus and demonstrated that dogs could be rendered immune by a series of
injections of fixed virus of graded infectivity.
● This vaccine was prepared by drying, for various periods, pieces of spinal cord from
rabbits infected with the fixed virus.
● In July 1885, Joseph Meister, a nine-year-old boy, severely bitten by a rabid dog and was
risk of developing rabies, was given a course of 13 inoculations of the infected cord
vaccine by Pasteur.
● The boy survived, this event was a milestone in the development of medicine.

PATHOGENICITY:
 Reference: Ananthanarayan and Paniker’s Textbook of Microbiology E/10, Page No. 536; Fig
57.2

CLINICAL STAGES:
STAGES OF DISEASE IN HUMANS:

1. Prodrome: (Early stage of this disease)

● The symptoms are fever, headache, malaise, fatigue, and anorexia.
● An early symptom is often a neuritic type of pain or paresthesia and fasciculation at the
site of virus entry.
 ● Apprehension, anxiety, agitation, irritability, nervousness, insomnia and depression
characterize the prodromal phase, which usually lasts 2-4 days.
2. Acute encephalitic phase: (Acute neurological phase)
● Begins with hyperactivity characteristically intermittent, with bizarre behavior, agitation
or seizures appearing between apparently normal periods
● The pathognomonic feature is difficulty in drinking with intense thirst.
● Patients may be able to swallow dry solids but not liquids.
● Attempts to drink bring on such painful spasms of the pharynx and larynx, producing
choking or gagging.
● The patients may fear even the sight or sound of water (hydrophobia).
● Generalized convulsions follow
3. Coma
4. Death occurs within 1-6 days due to respiratory arrest during convulsions.

STAGES OF DISEASE IN DOGS:

● In dogs, the incubation period is usually 3 – 6 weeks but may range from 10 days to a
year.
● The initial signs are troubled air and a change in disposition with restlessness, snapping at
imaginary objects, licking at the site of the bite.
● After 2-3 days of this prodromal stage, the disease develops into either the furious or
dumb type of rabies.
1. Furious rabies, which is more common, the dog runs amok, biting without
provocation and indiscriminately.
▪ The lower jaw droops and saliva drools from the mouth. Paralysis,
convulsions and death follow.
2. Dumb rabies is the paralytic form in which the animal lies huddled, unable to
feed.
▪ The dog may not bite but attempts to feed it are dangerous.
▪ The dumb form is as infectious as the furious type.
▪ Rabid dogs usually die in 3 – 5 days.
LABORATORY DIAGNOSIS:

i. SPECIMEN: Corneal smears and skin biopsy (from face or neck) or saliva antemortem,
and brain postmortem.
ii. DIRECT MICROSCOPY:
● Antemortem: Commonly used for diagnosis is the demonstration of rabies virus antigens by
immunofluorescence.
▪ Direct immunofluorescence is done using monoclonal antibodies tagged with
fluorescein isothiocyanate.
▪ Immunoperoxidase staining can be used in to antigen in tissues.
● Postmortem: Diagnosis may be made postmortem by demonstration of Negri bodies in the
brain, but they may be absent in about 20 % of cases.

ISOLATION:
i. Animal inoculation: Isolation of the virus by intracerebral inoculation in mice can be
attempted from the brain, CSF, saliva and urine (biological test).
● Chances of isolation are greater early in the disease
● A few days after onset, neutralizing antibodies appear and the virus can then be isolated
only occasionally
ii. Tissue culture: A more rapid and sensitive method is isolation of the virus in tissue
culture cell lines (WI 38, BHK 21, CER).
● CPE is minimal and so virus isolation is identified by immunofluorescence.
● A positive IF test can be obtained as early as 2 – 4 days after inoculation.
● The identity of the isolate can be established by the neutralization test with specific anti
rabies antibody.
iii. Antibody demonstration: High titre antibodies are present in the CSF in rabies but not
after immunisation.
iv. Molecular methods: Detection of rabies virus RNA by reverse transcription PCR is a
sensitive method.
RABIES PROPHYLAXIS
PRE – EXPOSURE:
● In animals, this is imperative but human pre – exposure immunization was only used in
persons at high risk, such as veterinarians and dog handlers because neural vaccines
carry some risk of serious complications .
● The introduction of cell culture vaccines, which are free from serious complications,
has made pre – exposure immunization in humans safe and feasible.

POST EXPOSURE:
● Specific prophylaxis is generally used after exposure to infection and is therefore called
antirabic treatment.
This consists of
1. Local treatment
2. Active immunization with antirabic vaccines
3. Passive immunization with antirabies serum.

Local treatment: Animal bites deposit the virus in the wound. Prompt cauterisation of the
wound therefore helps destroy the virus.
● The wound should be scrubbed well immediately with soap and water. This is a very
important step in the prevention of rabies as soap destroys the virus effectively.
● After washing the soap away completely, the wound is treated with quaternary
ammonium compounds (such as cetavlon), tincture or aqueous solution of iodine, or
alcohol (40-70%).
● Antirabic serum may be applied topically and infiltrated around the wound.

Antirabic vaccines:
These fall into two main categories:
1. Neural (associated with serious risk of neurological complications and are no longer
used)
2. Non – neural
1. Neural vaccines:
Semple vaccine: This vaccine developed by Semple (1911) at the Central Research Institute,
Kasauli (India) was the most widely used vaccine.

● It is a 5% suspension of sheep brain infected with fixed virus and inactivated with phenol
at 37°C, leaving no residual live virus.
Beta propionolactone (BPL) vaccine: Modified form of the Semple vaccine, in which beta
propiolactone is used as the inactivating agent instead of phenol.
● It is believed to be more antigenic, so smaller doses are considered adequate.
● The major antirabic vaccine producing laboratories in India manufacture BPL vaccine.
Suckling mouse brain vaccines: The encephalitogenic factor in brain tissue is a basic protein
associated with myelin.
● It is scanty or absent in the non – myelinated neural tissue of newborn animals.
● So vaccines were developed using infant mouse, rat or rabbit brains.
● It is impractical in India as very large quantities are required.

2. Non – neural vaccines:

Egg vaccines:
Duck egg vaccine
● Prepared from a fixed virus adapted for growth in duck eggs and inactivated with beta
propiolactone, but was discontinued due to poor immunogenicity.
● A purified, more potent duck egg vaccine was developed, but was supplanted by tissue
culture vaccines which became available then.
Live attenuated chick embryo vaccines: (not in use now)
Two types of vaccines were developed with the Flury strain:
A. Low Egg Passage (LEP) vaccine at 40-50 egg passage level for immunization of dogs.
B. High Egg Passage (HEP) vaccine at 180 passage level for cattle and cats.

TISSUE CULTURE VACCINES:

● Human diploid cell (HDC) vaccine:
▪ The first cell culture vaccine
 ▪ Developed by Koprowsky, Wiktor and Plotkin.
▪ It is a purified and concentrated preparation of fixed rabies virus (Pitman- Moore
strain)
▪ Grown on human diploid cells (WI 38 or MRC5) and inactivated with beta
propiolactone or tri-n-butyl phosphate
▪ highly antigenic
▪ Free from serious side effects.
⮚ Disadvantage - its high cost.
● Other equally effective and more economical vaccines have been developed ,they are
1. Primary cell culture vaccines grown on chick embryos.
2. Continuous cell culture vaccines grown on the Vero cell line of the monkey kidney
● Cell culture vaccines are available in India are:
1. Human diploid cell (HDC) vaccine
2. Purified chick embryo cell (PCEC) vaccine
3. Purified Vero cell (PVC) vaccine
● All three of them are equally safe and effective. These are currently used for
immunization.
 ESSAY 3
CLASSIFY ARBOVIRUS, DETAILS ABOUT JAPANESE ENCEPHALITIS (5)
SHORT NOTE: JAPANESE B ENCEPHALITIS VIRUS (6)

ARBOVIRUSES
▪ Arthropod - borne viruses
▪ RNA viruses
▪ Transmitted by blood sucking arthropods from one vertebrae host to another
▪ They multiply inside the insects

CLASSIFICATION:
● Has 5 different families
▪ Togaviridae
▪ Flaviviridae
▪ Bunyaviridae
▪ Reoviridae
▪ Rhabdoviridae
Some examples of Arbovirus:

Virus Manifestation Distribution Vector Reservoir

Family: Togaviridae

Chikungunya Fever, arthritis Asia, Africa Aedes aegypti Monkeys

virus

Eastern equine Encephalitis East part of Aedes, Culex Birds

encephalitis North America
virus

Family: Flaviviridae

Japanese B Encephalitis South East Asia Culex Pigs, birds

encephalitis tritaeniorhynchu
s
 Dengue virus Hemorrhagic India Aedes aegypti -
fever

Yellow fever Hemorrhagic West Africa Aedes aegypti Monkeys

virus fever

Kyasanur forest Hemorrhagic Russia Tick Monkeys and rat

disease virus fever

Zika virus Fever and Brazil Aedes aegypti Monkeys

arthritis

Family: Bunyaviridae

California Encephalitis USA Aedes triseriatus Rodents

encephalitis
virus

Family: Reoviridae

Colorado tick Fever, rarely America Tick Rodents

fever virus encephalitis

Family: Rhabdoviridae

Chandipura virus Encephalitis India Sand fly -

JAPANESE ENCEPHALITIS
● Belongs to family Flaviviridae
● Enveloped virus
● Contain ss RNA

EPIDEMIOLOGY:
● Vector
▪ Transmitted by bite of Culex mosquito (C. tritaeniorhynchus)
● Transmission cycle
▪ Infects several non human hosts e.g. animals and birds
▪ Animal hosts – pigs, cattle, horses and humans
▪ Bird hosts – Ardeid birds
 ▪ Humans are considered as dead end
(Pigs → Culex → pigs)
(Ardeid birds → Culex → Ardeid birds)
● Age
▪ Most cases are common in children below 15 years (but infants are not affected)

CLINICAL MANIFESTATIONS:
● Incubation period: 5-15 days
● Subclinical infection is common - It shows the iceberg phenomenon.
● Clinical course is divided into three stages:
1. Prodromal stage: Febrile illness; the onset of which maybe either abrupt, acute
or subacute (commonly)
2. Acute encephalitis stage: Characterized by acute onset of fever, mental
confusion, disorientation, delirium, seizures, or coma
3. Late stage and sequelae: Patient may be fully recovered or retain some
neurological deficits permanently (Case fatality 20-40%)

LABORATORY DIAGNOSIS:
▪ IgM capture antibody ELISA
▪ RT PCR

TREATMENT:
- Only supportive measures and no specific antiviral drugs are available.

PREVENTION:
● Children residing in rural areas and individuals living in endemic areas are recommended
to take vaccines.
● Vaccines licensed in India are:
▪ Live attenuated SA 14-14-2 vaccine
▪ Inactivated JE vaccine
▪ Catch up vaccination
▪ Combined vaccine
 ESSAY 4
DESCRIBE IN DETAIL THE PATHOGENESIS, LABORATORY DIAGNOSIS,
TREATMENT, PREVENTION AND CONTROL OF DENGUE FEVER (5)

SHORT NOTE: DENGUE VIRUS

DENGUE VIRUS
▪ Most common arbovirus found in India.
▪ Has four serotypes (DEN-1 to DEN-4). Recently, the fifth serotype (DEN-5) was
discovered in 2013 from Bangkok. It is also called break-bone fever.
▪ Dengue virus can be detected in blood from 1 day before the onset of symptoms up to 5
days thereafter.

VECTOR:
Aedes aegypti is the principal vector followed by Aedes albopictus. They bite during the day
time.

● Aegypti is a nervous feeder (so, it bites repeatedly to more than one person to complete a
blood meal) and resides in domestic places, hence is the most efficient vector.
● Aedes albopictus is found in peripheral urban areas. It is an aggressive and concordant
feeder i.e. can complete its blood meal in one go; hence is less efficient in transmission.
● Aedes becomes infective only when it feeds on viremic patients (generally from a day
before to the end of the febrile period i.e. 5 days.)
● Extrinsic incubation period of 8-10 days is needed before Aedes to become infective.
However, 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.
● Infection with dengue virus induces the production of neutralizing and non-neutralizing
antibodies.
● The neutralizing antibodies are protective in nature. Such antibodies are produced
against the infective serotype (which lasts lifelong) as well as against other serotypes
(which last for some time). Hence, protection to infective serotypes stays lifelong but
cross protection to other serotypes diminishes over a few months.
● The non-neutralizing antibodies last lifelong and are heterotypic in nature; i.e they are
produced against other serotypes but not against the infective serotype. Such antibodies
produced following the first serotype infection, can bind to a second serotype; but instead
of neutralizing the second serotype, it protects it from the host immune system by
inhibiting the bystander B cell activation against the second serotype. This is called
antibody dependent enhancement (ADE) which explains the reason behind the severity
of secondary dengue infection. Among all the serotype combinations, ADE is remarkably
observed when serotype 1 infection is followed by serotype 2, which also claims to be the
most severe form of dengue infection.

LABORATORY DIAGNOSIS:
1. NS1 Antigen Detection:
ELISA and ICT formats are available for detecting NS1 antigen in serum. They gained
recent popularity because of the early detection of the infection.
● 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.
2. Antibody Detection:
● In primary infection: Antibody response is slow and of low titer. IgM appears
first after 5 days of fever and disappears within 90 days. IgG is detectable at low
titer in 14-21 days of illness, and then it slowly increases.
● In secondary infection: IgG antibody titers raise rapidly. IgG is often cross
reactive with many Flaviviruses and may give false positive result after recent
infection or vaccination with yellow fever virus or JE. In contrast, IgM titer is
significantly low and may be undetectable.
● In past infection: Low levels of IgG remain detectable for over 60 years and, in
the absence of symptoms, is a useful indicator of past infection.
 ● MAC-ELISA is the most recommended tool available currently for dengue
infection. It has a sensitivity and specificity of approximately 90% and 98%
respectively.
● Formats are available for detection of both IgM and IgG separately.
● Rapid tests such as dipstick assays are also available.
● Other antibody detection assays used previously are:
a) HAJ (Hemagglutination inhibition test)
b) CFT (Complement fixation test)
c) Neutralization tests such as plaque reduction test, neutralization and micro
neutralization tests.
3. Virus Detection
Dengue virus can be detected in blood from 1 day before the onset of symptoms upto 5
days thereafter.
● 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. It is the most
sensitive and specific assay, can be used for detection of serotypes and
quantification of viral load in blood.

DENGUE FEVER
▪ Acute febrile illness with two or more of the following manifestations: Headache,
retro-orbital pain, myalgia, arthralgia, rash, rubelliform exanthema, hemorrhagic
manifestations.
▪ Dengue manifests after an incubation period of 3-14 days. This acute febrile phase
usually lasts 2 – 7 days.

It is characterized by:

● Abrupt onset of high fever (also called biphasic fever, break bone fever or saddle back
fever).
● Skin erythema and pain in the back and limbs (break-bone fever)
● Maculopapular rashes over the chest and upper limbs.
● Severe frontal headache.
● Muscle and Joint pains
● Lymphadenopathy
● Retro orbital pain
● Loss of appetite, nausea and vomiting
● Photophobia, accompanied by facial flushing
Dengue hemorrhagic fever (DHF): These patients become worse around the time of
defervescence, when the temperature drops to 37.5-38°C or less and remains below this level,
usually on days 3-8 of illness. It is characterised by clinical criteria of dengue fever plus
hemorrhagic tendencies evidenced by one or more of the following:

● Positive tourniquet test (>20 petechial spots per square inch area in cubital fossa.
● High grade continuous fever
● Petechiae, ecchymoses or purpura
● Bleeding from mucosa, gastrointestinal tract. injection sites or other sites
● Raised hematocrit (packed cell volume) by 20%
● Thrombocytopenia (platelet count < l Lakh/mm3)
● Hepatomegaly
● Signs of plasma leakage (pleural effusion, ascites, hypoproteinemia)
Dengue shock syndrome (DSS): All the above criteria for DHF with evidence of circulatory
failure manifested by rapid and weak pulse and narrow pulse pressure (< 20 mm Hg) or
hypotension for age, cold and clammy skin and restlessness.
PREVENTION AND CONTROL:
Vaccine: While no licensed dengue vaccine is available, several vaccine trials are currently being
evaluated.

● Live-attenuated tetravalent vaccine based on chimeric yellow fever-dengue virus

(CYD-TDV) has been developed by Sanofi Pasteur Company.
● Mosquito control measures.

TREATMENT:
▪ There is no specific treatment for dengue.
▪ Supportive management, with cold tepid sponging, paracetamol for fever (Aspirin/
NSAIDS like Ibuprofen, etc., should be avoided since it may cause gastritis, vomiting,
acidosis, platelet dysfunction and severe bleeding); fluid and electrolyte replacement and
platelet infusion when counts are 10,000 and less, should be done.
▪ Dengue shock is treated with whole blood transfusion and management of shock.
 ESSAY 5
DNA VIRUSES. CLASSIFY HERPES VIRIDAE, DETAILS ABOUT HERPES SIMPLEX
VIRUS, DETAIL ABOUT VARICELLA ZOSTER VIRUS.
SHORT NOTE: VARICELLA ZOSTER (2)

HUMAN HERPES VIRUS

DNA viruses include:
▪ Herpes viruses
▪ Pox viruses
▪ Parvoviruses
▪ Human papillomaviruses
Herpes viridae consists of a group of viruses which establish latent or persistent infections in
their hosts and later on undergo periodic reactivation.

CLASSIFICATION:
Family- "Herpesviridae"

SUBFAMILY GENUS SPECIES

("herpesvirinae")
Official name Common name

Alpha Simplex virus Human herpesvirus 1 Herpes simplex virus

type 1

Human herpesvirus 2 Herpes simplex virus

type 2

Varicellovirus Human herpesvirus 3 Varicella-zoster virus

Beta Cytomegalovirus Human herpesvirus 4 Cytomegalovirus

Roseolovirus Human herpesvirus 5 Human herpesvirus 6

Human herpesvirus 6 Human herpesvirus 7

Gamma Lymphocryptovirus Human herpesvirus 7 Epstein-barr virus

 Rhadinovirus Human herpesvirus 8 Kaposi's sarcoma
associated
herpesvirus

MORPHOLOGY:
▪ Large, spherical in shape
▪ Linear ds DNA
▪ Icosahedral symmetry
▪ Nucleocapsid is surrounded by lipid envelope inserted with glycoprotein spikes
▪ Tegument – between capsid and envelope

Reference: Essentials of Medical Microbiology, Apurba Sastry E/3 Page No. 550, Fig. 56.1

HERPES SIMPLEX VIRUS

▪ Belong to alpha subfamily of herpes viridae
▪ Replicate fast, spread fast
▪ Cytolytic
▪ Undergo latency in nerve cells and reactivate later causing recurrent lesions
▪ They are of two types HSV-1 and HSV-2
PATHOGENESIS:

HERPES SIMPLEX VIRUS 1 HERPES SIMPLEX VIRUS 2

Transmission occurs through Transmission occurs through

● Oropharyngeal contact with infected ● Sexual contact
saliva ● Vertical mode rarely (from mother to
● Direct skin contact foetus)

Latency in trigeminal ganglia Latency in sacral ganglia

Young children are affected Young adults are affected

Skin lesions – above the waist Skin lesions – below the waist
(Most common site of infection – around (Most common site of infection – genital
mouth) area)

RECURRENT INFECTIONS:
Provocative stimuli (fever, axonal injury, UV rays, physical or emotional stress)
⬇️
Reactivation of the latent virus
⬇️
Via axonal spread
⬇️
Back to peripheral site and further replicates in skin and mucosa producing secondary lesions
Recurrent infections are less extensive and severe

CLINICAL MANIFESTATIONS:
Incubation period: 1 to 26 days
❏ Oral- facial mucosal lesions
➔ Gingivostomatitis and pharyngitis
➔ Herpes labialis (painful vesicles near lips)
➔ Tonsillitis and vesicular lesions on the eyelids
❏ Cutaneous lesions
➔ Herpetic Whitlow
 ➔ Febrile blisters
➔ Eczema herpeticum
➔ Erythema multiforme
➔ Herpes gladiatorial
❏ Genital lesions
➔ Bilateral, painful, multiple, tiny vesicular ulcers
❏ CNS infections
➔ Encephalitis, meningitis, Bell's palsy
❏ Ocular manifestations
➔ Keratoconjunctivitis, corneal ulcer and blindness
❏ Visceral and disseminated herpes
➔ Pneumonitis, tracheobronchitis and hepatitis
❏ Neonatal herpes
➔ Transmitted more commonly during birth than in utero. Always symptomatic.

EPIDEMIOLOGICAL PATTERN:
HSV-1:
● Primary infection occurs early in life and is a either asymptomatic or remains confined to
oropharyngeal disease
● Antibodies develop but they fail to eliminate the virus from the body
HSV-2:
● Primary infection occurs in adult life.
● Antibodies develop only in less number of people

LABORATORY DIAGNOSIS:
▪ Cytopathology (Tzanck preparation)
▪ Viral antigen detection in specimen by direct IF
▪ HSV DNA detection by PCR
▪ Antibody detection by ELISA
TREATMENT:
▪ Acyclovir is the drug of choice.
▪ It acts by inhibiting viral DNA polymerase.

PREVENTION:
▪ Use of condom to prevent genital herpes
▪ Neonatal herpes can be prevented by prior administration of acyclovir to the mother
during third trimester of pregnancy or delivery by elective Caesarean section

VARICELLA – ZOSTER VIRUS INFECTIONS

- Varicella – zoster virus produces vesicular eruptions (rashes) on the skin and the mucous
membrane in the form of:
● Chicken pox
● Zoster or shingles

CHICKEN POX
- Generalized diffuse bilateral vesicular rashes which occur following primary infection
usually affecting children.

PATHOGENESIS:
Virus enters through the upper respiratory mucosa or the conjunctiva by
● Aerosol ( most common)
● Contact transmission
Infected mononuclear cells in blood carried from local site to the target sites like
● Skin (rashes)
● Respiratory tract (shed in respiratory secretions)
● Neurones (undergoes latency)
CLINICAL MANIFESTATIONS:
● Incubation period- 10-21 days
● Rashes
▪ Rashes are vesicular
▪ Centripetal, bilateral and diffuse in distribution
▪ Rashes appear in multiple crops
▪ Fever appears with each crop of rashes
● Chickenpox in adults causes more severe Bulbous and hemorrhagic rashes leaving behind
pitted scars on skin after recovery.

COMPLICATIONS:
Seen more common in adults and in immunocompromised individuals
▪ Secondary bacterial infections of the skin
▪ Cerebellar ataxia, encephalitis in children
▪ Varicella pneumonia (common in adults)
▪ Nephritis, arthritis and myocarditis.
Chickenpox in pregnancy can affect both mother and the foetus
● Mothers are at high risk of developing Varicella pneumonia
● Foetus is at higher risk of developing congenital Varicella syndrome

ZOSTER OR SHINGLES
Occurs due to reactivation of latent Varicella zoster virus
● In old age (above 60 years)
● In immunocompromised individual
● Occasional in healthy adults

CLINICAL MANIFESTATIONS:
▪ Severe pain in area of skin or mucosa supplied by ganglia
▪ Rashes of unilateral, segmental confined to the area of skin supplied by affected nerve
▪ Ophthalmic branch of trigeminal nerve is the most common nerve involved
COMPLICATIONS OF ZOSTER:
▪ Zoster ophthalmicus (unilateral painful crops of skin rashes around eyes)
▪ Post herpetic neuralgia (pain at local site lasting for months)
▪ Ramsay hunt syndrome (when geniculate ganglion of facial nerve is affected)
▪ Pneumonia

LABORATORY DIAGNOSIS:
▪ Cytopathology
▪ Specimen collection from lesions
▪ Virus isolation
▪ Varicella zoster specific gene detection by PCR
▪ Specific IgM and IgG antibody detection by ELISA
▪ Specific antigen detection by direct immunofluorescence staining

TREATMENT:
● Acyclovir, Famciclovir or Valacyclovir are the drugs of choice.

PREVENTION:
▪ Live attenuated vaccine using Oka strain of Varicella zoster is given to children after one
year of age.
▪ Varicella-Zoster immunoglobulin is useful for post exposure prophylaxis within 96 hours
of exposure in
● Adults at higher risk of Varicella related death
● Neonates born to mother suffering from chickenpox
▪ Isolation of patients infected with Varicella zoster virus.
 ESSAY 6
DETAILS ABOUT EBV
SHORT NOTE: EBV

EPSTEIN – BARR VIRUS

▪ Member of the gamma sub-family of Herpesviridae that causes infectious mononucleosis.
▪ Also associated with several human tumors, including nasopharyngeal carcinoma,
Burkitt's lymphoma, Hodgkin's disease and B cell lymphoma.

ANTIGENS OF EBV:
Divided into three classes:

• Latent phase antigens: They are synthesized during the period of latency, e.g., EBV nuclear
antigen (EBNA): II has six subtypes, Latent membrane protein (LMP): II has two subtypes.

• Early antigens: They are non-structural proteins which help in viral replication.

• Late antigens: they are the structural proteins that form viral capsid and envelope.

PATHOGENESIS:
Primary Infection

EBV is transmitted by oropharyngeal contact through infected salivary secretions.

▪ EBV receptors: EBV binds to specific receptors present on B cell (CD21 or CR2) which
are also receptors for C3b component of complement. Such receptors are also present on
pharyngeal epithelial cells.
▪ Primary infection occurs in the oropharynx. EBV replicates in epithelial cells surface B
lymphocytes of the pharynx and salivary glands.
▪ Following entry into the B cells, EBV directly enters into the latent phase without
completing the viral replication.
▪ Though, majority of the infected cells are eliminated, a small number of infected cells
(one in 10"- 10" B cells) may persist for lifetime. Virus spreads from the oropharynx to
other sites of the body and is capable of undergoing reactivation later.
▪ Viral shedding continues in oropharyngeal secretions at low levels for weeks to months
and serves as a source of infection.
 ▪ In children, most primary infections are subclinical, but young adults often develop a
condition called acute infectious mononucleosis. Infected B cells become immortalized
by the virus and synthesize large number of variety of immunoglobulins (polyclonal),
many of those are autoantibodies (e.g., heterophile antibody to sheep RBC antigen) In
response to this, the bystander CD8 T Lymphocytes are stimulated and appear atypical.

CLINICAL MANIFESTATIONS:
1. Infectious Mononucleosis

2. EBV-associated Malignancies

3. Lymphoproliferative disorder (seen among immunodeficient patients, e.g. Duncan

syndrome which is an X-linked recessive disease affecting young boys.)

4. Hairy cell leukoplakia (Wart-like growth of epithelial cells of die tongue developed in
some HIV-infected patients and transplant recipients )

5. Chronic fatigue syndrome

INFECTIOUS MONONUCLEOSIS (KISSING DISEASE):

▪ Age: Young adults of developed countries are usually affected.

▪ It is characterized by:

● Headache, fever, malaise

● Pharyngitis

● Cervical lymphadenopathy

● Hepatosplenomegaly

● Rashes following ampicillin therapy

● Atypical lymphocytosis (CD8 T cells)

● Autoantibodies reactive to sheep RBC antigen (detected by Paul Bunnell test)

EBV-ASSOCIATED MALIGNANCIES:
 1. Burkitt's lymphoma (tumor of the jaw seen in children and young adults): EBV is
associated with 90% of African and 20% of non-African cases of Burkitt's lymphoma.

▪ Most of the cases have pre-existing mutation {t(8;14)}

▪ Falciparum malaria may impair host CMI and stimulate the EBV-infected B cells.

2. Nasopharyngeal Carcinoma: It is seen among Chinese people who have a history of

intake of salted fish (nitrosamine) and herbal snuff (phorbol ester).
3. Hodgkin’s lymphoma: (especially the mixed – cellularity type): EBV DNA is found in
Reed-Sternberg cells, in atleast50% of cases of Hodgkin’s lymphoma.
4. NHL (Non-Hodgkin's lymphoma): All central nervous system non-Hodgkin's
lymphomas and 50% of systemic non-Hodgkin's lymphomas are EBV positive.

EPIDEMIOLOGY:
▪ Worldwide in distribution:
▪ Age: EBV infections are most common in early childhood, with a second peak during
late adolescence. However, infectious mononucleosis is common among young adults of
developed countries.
▪ Prevalence: EBV is common in all parts of the world, with > 90% of adults being
seropositive and develops antibodies to EBV.
▪ Transmission: Intimate and prolonged oral contact is required for effective transmission.
- EBV is spread by direct contact with oral secretions, e.g. salivary contact during kissing
- Other modes are blood transfusion and following bone marrow transplantation.
- Source: Asymptomatic seropositive individuals shed the virus in oropharyngeal
secretions. Shedding is more in immuno-compromised patients

LABORATORY DIAGNOSIS:
Antibody Detection: Heterophile Agglutination Test

▪ Paul-Bunnell test: It is a tube agglutination test that uses sheep RBCs to detect
heterophile antibodies in a patient's serum.
▪ Procedure: Serial dilutions of inactivated (56°C for30 minutes) patient's serum are mixed
with equal volumes of 1 % sheep RBC's and then the tubes are incubated at 37°C for four
hours.
▪ Result: Agglutination titre of > 256 is considered as significant.
 ▪ False positive: Heterophile antibodies are non-specific, may also be present following
serum therapy or even in some normal individuals; hence confirmation is must.
▪ Differential absorption is done for confirmation.
▪ Patient's serum is first made to react with guinea pig kidney cells and ox red cells,
following which the Paul-Bunnell test is repeated.
▪ Monospot test is modified heterophile agglutination test is available commercially.

● It is a simple slide agglutination test that uses horse RBCs instead of sheep
RBCs.

● Test serum is priorly treated with guinea pig kidney and ox red cells.

● It has largely replaced the differential absorption test, and has excellent
sensitivity (75%) and specificity (90%).

▪ Heterophile antibodies appear early (40% in the first week and 80- 90% in the third week
of illness), then disappear within 3 months.
▪ Heterophile antibodies are not detectable in children < 5 years, in elderly or in patients
with atypical symptoms.

PREVENTION:
The isolation of patients with infectious mononucleosis is not needed as temporary contact does
not transmit the infection. No vaccine is currently available. A vaccine trial using EBV
glycoprotein was found to be ineffective.
 ESSAY 7
CLASSIFY PICORNAVIRUSES. DESCRIBE THE PATHOGENESIS, CLINICAL FEATURES
AND LAB DIAGNOSIS OF POLIOVIRUS. ADD A NOTE ON PROPHYLAXIS AGAINST
POLIOMYELITIS. VIRUSES CAUSING ASEPTIC MENINGITIS.
SHORT NOTES:
i. IMMUNE PROPHYLAXIS OF POLIO
ii. LAB DIAGNOSIS OF POLIOMYELITIS
iii. ASEPTIC MENINGITIS

PICORNAVIRUSES
MAJOR GROUPS:
● Enteroviruses
● Rhinoviruses
Enteroviruses:
▪ Transmission- Feco oral route
▪ Doesn't cause intestinal manifestation
▪ > 115 human serotype

CLINICAL MANIFESTATIONS OF VARIOUS VIRUSES:

● Polio viruses – meningitis
● Coxsackie viruses( B,A7,A9) – aseptic meningitis
● Echo viruses – aseptic meningitis
● Parechoviruses – aseptic meningitis
● Enteroviruses 71 – aseptic meningitis
● Enterovirus 68 – pneumonia
● Enterovirus 70 – acute hemorrhagic conjunctivitis
● Enterovirus 72 – reclassified into hepatitis A virus

POLIOVIRUS
- Causes a highly infectious childhood disease called Polio / poliomyelitis causing acute
flaccid paralysis due to involvement of the nervous system.
MORPHOLOGY:
▪ Type of Enterovirus (Family: Picornaviridae)
▪ Size: 28-30nm
▪ Shape: Spherical
▪ Symmetry: Icosahedral
▪ Envelope: non enveloped
▪ Capsid: Composed of 60 subunits (capsomeres)
o Each capsomeres has 4 viral proteins (VP1-VP4)
o Each parechoviruses - 3 proteins
▪ Nucleic acid: single stranded +ve sense linear RNA

ANTIGENIC TYPES:
● Wild polioviruses (WPV) – Cause natural disease
● Vaccine derived polioviruses (VDPV) - These are vaccine strains which regained
neurovirulence and produce diseases in man

SITE OF ACTION:
- Motor nerve ending (i.e. anterior horn cells of spinal cord) and leads to muscle weakness
and flaccid paralysis

NEURON DEGENERATION:
Virus infected neurons undergo degeneration.

Earliest change degeneration of Nissle's body- aggregated ribosomes in cytoplasm of neuron.

CLINICAL MANIFESTATION:

Inapparent infection 91-96% of cases Asymptomatic

Absorptive infection 5% Minor symptoms: fever,

malaise, anorexia, sore
throat, myalgia, headache
Non paralytic poliomyelitis 1% Aseptic meningitis

Paralytic poliomyelitis < 1% Descending asymmetric

acute flaccid paralysis

● Proximal muscles are affected earlier than distal muscles

● Paralysis starts at hip forwarded towards extremities

WILD POLIO VIRUS (WPV):

⮚ Strains: Wild poliovirus type 1 (WPV1), Wild poliovirus type 2 (WPV2), Wild
poliovirus type 3 (WPV3)
⮚ Strains are identical, produce similar manifestations, distinct genetically and
immunologically
⮚ WPV2 + WPV3 were eradicated in 1999 & 2019 respectively. Currently all cases are
caused by WPV1

VACCINE DERIVED POLIOVIRUSES (VDPVS):

● Produce poliomyelitis
● Clinically indistinguishable
● 3 types:
▪ circulating VDPVs (cVDPVs)
▪ immunodeficiency associated VDPVs (iVDPVs)
▪ ambiguous VDPVs (aVDPVs)

cVDPVs:
▪ Circulate in community
▪ Spread person to person by Feco oral transmission
▪ Occurrence is more common

PATHOGENESIS:
1. Transmission:
▪ Feco oral route
▪ Respiratory droplets (via inhalation)
▪ Conjunctival contact
 2. Multiply locally in intestinal epithelial cells, submucosal lymphoid tissue,tonsil,Peyer
patches
● Hematogenous spread:
1. Regional lymph nodes

2. Spill into blood stream (primary viremia)

3. Further multiplication in reticuloendothelial system

4. Again enter blood stream (secondary viremia)

5. Spinal cord & Brain

● Neural spread:
1. Following tonsillectomy

2. Spread via glossopharyngeal nerve

LABORATORY DIAGNOSIS:
1. Viral isolation-

● Specimen: Throat swab (upto 3 weeks of illness), Rectal swab (upto 12 weeks)
Viral isolation from CSF (blood is very rare)

● Transportation: Kept in frozen state

● Cell line: Primary monkey kidney cell – most recommended cell line
● Cell growth is identified by:
▪ Cytopathogenic effects
▪ Antigen detection
▪ PCR (targets VPI region of poliovirus)

2. Antibody detection :

● A rise in antibody titre in paired Sera collected at 1-2 weeks interval - suggestive of
poliomyelitis
● Most recommended method: Neutralisation test
● Only 1st infection with poliovirus produces strictly type specific responses
● Subsequent infections induce antibodies against group specific antigen common to all three
serotypes.
PROPHYLAXIS
Vaccine
Both inactivated and live polio virus vaccines are available

● Injectable polio vaccine

● Oral polio vaccine
▪ Injectable polio vaccine (Salk) - Virus is grown in monkey kidney cell line
1.5 ml of vaccine contains
● 40 units of type 1
● 8 units of type 2
● 32 units of type 3
▪ Oral polio vaccine (Sabin)
1.5 ml of vaccine contains
● 3 lakhs of type 1 virus
● 1 lakhs of type 2 virus
● 3 lakhs units of type 3 virus

RHINOVIRUSES:
● Respiratory route
 ● Cause Common cold
● >100 antigenic types

VIRUSES CAUSING ASEPTIC MENINGITIS:

● Group A and B Coxsackieviruses – Most commonly by A7 and A9 serotypes
● They are one of the type of Enterovirus
● They are classified into group A and B
● And each group is further classified as different serotypes

CLINICAL MANIFESTATIONS:

LABORATORY DIAGNOSIS:
● Specimen collected – Stools, Swabs, CSF
● Isolation of virus – Suckling mouse intracerebral inoculation
● PCR vp1 gene is amplified
 ESSAY 8
DESCRIBE THE MORPHOLOGY OF HIV, DESCRIBE THE PATHOGENESIS AND
LAB DIAGNOSIS OF HIV INFECTION. ADD A NOTE ON PRE EXPOSURE
PROPHYLAXIS
SHORT NOTE: LABORATORY DIAGNOSIS OF AIDS

HUMAN IMMUNODEFICIENCY VIRUS

- Belongs to Retroviridae family and genus Lentivirus

MORPHOLOGY:
▪ Spherical and 80-110 nm in size
▪ HIV is an enveloped virus
▪ ENVELOPE:
a) Lipid part – derived from the host cell membrane
b) Protein part – has 2 components:
1) Glycoprotein 120 (gp 120) – it is projected as knob like spikes on the
surface.
2) Glycoprotein 41 (gp41) – forms anchoring transmembrane pedicles
▪ NUCLEOCAPSID:
1. Capsid is isohedral in symmetry

2. Made of core protein.

3. Has a dense cylindrical inner core which has

- 2 identical copies of single stranded positive sense linear RNA

- Viral enzymes such as reverse transcriptase, integrate, proteases

HIV genes:

▪ Three structural genes – gag, pol, env

▪ Six non structural or regulatory genes

PATHOGENESIS:
Mode of transmission:
a) Sexual mode – most common method, accounts for 75% of total cases
b) Blood transfusion is the least common mode of transmission (5%) and risk of
transmission is maximum (90-95%)

Receptor attachment:

• CD 4 receptor on host cell surface is the main receptor and binds to gp 120
• Chemokine receptors act as second co- receptors and bind to gp120.
Ex: CXCR4 present on T lymphocytes.
CCR5 on cells of macrophage lineage.
• DC-SIGN facilitates transport of HIV by dendritic cells to lymphoid
organs.
REPLICATION:
1. After attachment of receptor to gp120, fusion of HIV to host cell by fusion protein gp 41
2. Nucleocapsid enter into host cell cytoplasm
3. Uncoating and release of 2 copies of ssRNA ,viral enzymes
4. Reverse transcriptase transcribes ssRNA to ssDNA
5. Endonuclease degrades ssRNA and ssDNA replicates to form dsDNA
6. The viral dsDNA gets integrated into host cell chromosome and forms pro virus with help
of integrase enzyme
7. HIV exhibits a latency period and it is able to replicate even in that state

DISEASE PROGRESSION:
Progressors Develops into AIDS % of PLHA
Typical progressor Within 10 years 80-90%
Rapid progressor Within 2-3 years 5-10%
Long term Non- progressor After long time (10-30 years) without A 5%
shows < 5000 HIV RNA copies /ml
Elite controller After long time (10-30 years) without A < 1%
shows < 50 RNA copies /ml

LABORATORY DIAGNOSIS:
SCREENING TESTS (Detection of antibody):

1. ELISA (Enzyme Linked ImmunoSorbent Assay ):

▪ Most commonly performed screening tests. Easy to perform, sensitive, specific,

cost effective
▪ Types of ELISA-
● Indirect ELISA
● Competitive ELISA
● Sandwich ELISA
▪ Types of ELISA kits:
▪ Third generation ELISA uses recombinant and synthetic peptides as
antigen to detect HIV antibodies
 ▪ Fourth generation ELISA detects both HIV antibodies and p24 antigen
2. RAPID /SIMPLE TESTS:
● Works on various principles like: Immunochromatography, Dipstick / Comb
tests, particle agglutination assays
● Requires less than 30minutes to perform and does not require special
equipment.

SUPPLEMENTARY TESTS:
WESTERN BLOT ASSAY:

▪ Detects individual antibodies in serum separately against various antigenic fragments.

▪ Works on the principle of immunoblotting technique.
▪ Antigen antibody complexes appear as distinct bands on nitrocellulose strips.

CONFIRMATORY TESTS:
DETECTION OF p24 CORE ANTIGEN:

▪ P24 antigen is detectable after 12-26 days of infection, lasts for 3-6 weeks
▪ Detected by 4th generation ELISA
▪ Less sensitive as antibody formed binds to p24 antigen and the antigen antibody complex is
eliminated from blood

VIRAL RNA DETECTION :

▪ GOLD STANDARD method for confirmatory diagnosis

▪ Most sensitive, specific method
▪ Best tool for diagnosis of HIV during window period

DNA PCR:

▪ Extremely useful for diagnosis of pediatric HIV

▪ Differentiates latent HIV infection from active viral transcription
NON SPECIFIC IMMUNOLOGICAL TESTS:
CD4 CELL COUNT:

▪ By flow cytometry method

▪ Useful for assessing the risk of infections and monitoring the response to antiretroviral
therapy

POST EXPOSURE PROPHYLAXIS:

▪ Required to reduce the risk of transmission after occupational exposure

▪ Duration: Started within 2h of exposure and to be continued for 28 days

▪ Indication: If source is unknown / positive for HIV
▪ TL+LR Regimen: consists of Tenofovir – Lamivudine plus Lopinavir – Ritonavir
 ESSAY 9
DISCUSS BRIEFLY ON IMMUNOPROPHYLAXIS OF VIRAL DISEASES. DISCUSS
BRIEFLY ON VIRAL VACCINES.
SHORT NOTES:
i. ORAL / SABIN POLIO VACCINE
ii. RABIES VACCINE

IMMUNOPROPHYLAXIS OF VIRAL DISEASE

ACTIVE IMMUNIZATION PASSIVE IMMUNIZATION

● (Pre-exposure prophylaxis) (Post-exposure prophylaxis)

● VIRAL VACCINES IMMUNOGLOBULINS
IMMUNOGLOBULINS:
● Used when an individual is immunodeficient
● No memory cells involved
● No role in prevention of subsequent infections
● Human Igs are used nowadays
● Ex: Human Igs are available for Mumps, Measles, Rabies

COMBINED IMMUNIZATION
Simultaneous administration of vaccines and immunoglobulins in post exposure prophylaxis is
extremely useful.

Ex: Rabies

LIVE ATTENUATED VACCINES

● Preparation: Attenuated by serial passages (except: smallpox)
● Advantages: Gives strong and long-lasting immunity, single dose is sufficient (except:
OPV)
● Disadvantages: Less stable, Risk in immunodeficient and pregnant people.

Ex: MUMPS, MEASLES, RUBELLA, CHICKEN POX, SMALLPOX

KILLED VACCINES
● Preparation: Inactivating viruses with formalin, phenol and not with UV since it has a
risk of multiplicity reactivation
● Advantages: Stable, safe for immunodeficient and pregnant people
● Disadvantage: Adverse side effects due to reactogenicity which can be reduced by
purification of viruses.
Ex: Rabies (Neural and Non-neural)

SUBUNIT VACCINE
● Preparation: rDNA technology
● Advantage: No side effects
● Disadvantage: Costly

ORAL POLIO VACCINE/ SABIN VACCINE (SHORT NOTE):

▪ Live attenuated vaccine
▪ Formulation: Trivalent (1,2,3 serotypes)
o Bivalent (1,3 serotypes)
o Monovalent (any one serotype)
▪ National immunization schedule: 5 DOSES (2 dose of Bivalent)
o zero dose – birth
o 1,2,3 – 6th 10th 14th week
o Booster – 1 – 24 months
▪ Efficacy: 90 – 100% (1/2 doses of OPV)

ADVANTAGES:
● HERD IMMUNITY:
OPV shorts sheds in feces
Spread in community
Induce herd immunity
● LOCAL IMMUNITY
OPV induces mucosal IgA production
● Cheap
DISADVANTAGES:
● Unstable – Require stringent conditions

Storage at – 20 degree Celsius, Stabled in MgCl2,

Maintained in pH less than 7

● Risk to give to immunodeficient and pregnant peoples

● Efficacy decreases when breastfeeding, diarrhea, interference by enterovirus
● OPV can cause VAPP and VDPV
VAPP-Vaccine Associated Paralytic Poliomyelitis
VDPV- Vaccine derived Polio virus

RABIES VACCINE (SHORT NOTE)

 Reference: Essentials of Medical Microbiology, Apurba Sastry E/3: Page No. 722

TYPES:
● Purified Chick Embryo Cell (PCEC)
● Purified Vero Cell (PVC)
● Human Diploid Cell (HDC)

ROUTES: Intradermal or Intramuscular

SITE: Deltoid for adults, Anterolateral area of thigh for children (age below 2)

DOSE: One ID dose- 0.5 ml, One IM dose – entire vial

SCHEDULE OF PEP REGIMEN: (PEP- Pre exposure prophylaxis)

ID regimens are cost effective, dose sparing, time sparing. So ID is preferred over IM

ID PEP regimen (2-2-2): 2-site on 0,3,7 days

IM PEP regimen (4 doses)

1. Site on 0,3,7 and 4th dose between 14 to 28 days

2. Site on day 0 and 1 – site IM on days 7 and 21

INTERCHANGE:
Changes in vaccine product routes (IM or ID) during the same PEP course are acceptable if
unavoidable.

IF DELAYED:
If vaccine dose is delayed for any reason PEP regimen should be resumed.

PEP FOR INDIVIDUALS PREVIOUSLY VACCINATED:

● RIG is not necessary regardless of exposure category.
 ● They need local wound care and an accelerated vaccine regimen consisting of any
of the following schedules
▪ Site ID vaccine given on days 0 and 3 or
▪ Site IM vaccine given on days 0 and 3 or
▪ Site ID vaccine given on day 0 only (left and right deltoids, thigh)

NOTE: If repeat exposure occurs within 3 months of receiving PEP only local wound treatment
is required.

PEP IS RECOMMENDED IN TWO CONDITIONS:

● For individuals at higher occupational risk
● For subpopulation in remote endemic areas

PEP REGIMEN (FOR ALL AGES):

Schedule:

▪ Site ID vaccine given on days 0 and 7

▪ Site IM vaccine given on days 0 and 7

Booster:

No need to take PEP booster periodically only if continued high risk of rabies exposure remains
a routine PEP booster is recommended.

Following Exposures:

● An accelerated vaccine regimen is indicated

● Rabies Immunoglobulin (RIG) is not necessary.
 ESSAY 10
DISCUSS VARIOUS METHODS FOR ISOLATION OF VIRUSES IN THE
LABORATORY.
SHORT NOTES:
i. TISSUE CULTURE FOR VIRUSES
ii. VIRAL INCLUSION BODIES
iii. CYTOPATHIC EFFECTS
iv. VIRAL CELL CULTURE
v. EGG CULTURE

LABORATORY ISOLATION OF VIRUSES :

- Viruses cannot be grown on artificial culture due to

▪ Lack of individual metabolic machinery
▪ Total dependence on host for replication
- Specimens collected should be transported immediately to the laboratory along with
refrigeration in liquid N2 at -196℃, as most viruses are heat labile.

ANIMAL INOCULATION:
- Due to ethical issues surrounding the use of animals in scientific study, animal
inoculation is restricted to the study of viral pathogenesis and viral vaccination trails.

Egg culture:

Embryonated hen’s egg is used for inoculation.

Sites for inoculation in embryonated egg:

1. Yolk sac: Arbovirus inoculation

2. Amniotic sac: Isolation of influenza virus
3. Allantoic sac: As it is a larger sac, it is used for better yield of viral vaccines for influenza
virus and yellow fever
4. Chorioallantoic Membrane: Isolation of poxvirus
This method was largely used in the past but is limited in present times.

Tissue culture:

Poliovirus – first cultivated through tissue culture

Three types:

1. Organ culture: Used for certain viruses which have affinity towards specific organs.

Ex: Tracheal ring culture for coronavirus

2. Explant culture: Minced tissue is grown as explants

3. Cell line culture: Cell lines are prepared and used for isolation.

Preparation of cell lines:

Tissue

Individual cells

Suspend the cells in viral growth medium containing vitamins, minerals, growth supplements,
pH buffer, phenol red as pH indicator

Viral growth medium is suspended in tissue culture flask

Monolayer is formed on glass surface after incubation

TYPES OF CELL LINES:
1. Primary cell lines:
● Normal tissue
● Freshly taken from organs
● Limited divisions of up to 5-10 times
● Diploid karyotype
● Used for primary isolation and vaccine production
● Ex: Human amnion cell line
2. Secondary cell lines
● Taken from normal tissue
● 10-50 divisions
● Diploid karyotype
● Ex- Human fibroblast cell line
3. Continuous cell lines:
● Derived from cancer cells
● Capable of indefinite divisions
● Altered haploid karyotype
● Ex- HeLa cell line
DETECTION OF VIRAL GROWTH IN CULTURE

CYTOPATHIC EFFECT:

- Morphological changes produced by a host cell shown by a virus in a cell line as seen
through a light microscope is called cytopathic effect.
▪ CPE occurs when an infected cell is lysed by a reproducing virus.
▪ Morphological changes:
1. Rounding of cell
2. Syncytia formation
3. Appearance of cytoplasmic inclusion bodies
▪ Not all viruses produce CPE
▪ Type of CPE is different for different viruses, and is used for their identification

Shell vial technique:

▪ Used for viruses taking long time to show CPE

▪ Method:
Sample centrifugation

Enhancing cell contact and viral replication

Detection of viral antigen in host cell by direct fluorescence method

Other methods:

1. Detection of viral genes by PCR

2. Electron microscopy
 ESSAY 11

DESCRIBE THE MORPHOLOGY, PATHOGENESIS AND LAB DIAGNOSIS

INFLUENZA VIRUS.
SHORT NOTES:
i. DIAGNOSIS AND PROPHYLAXIS OF H1N1 INFECTION.
ii. RECENT SWINE FLU PANDEMIC.
iii. PROPHYLAXIS OF INFLUENZA.
iv. INFLUENZA VIRUSES.

INFLUENZA VIRUS
Family – Orthomyxoviridae

Four genera – Influenza A, B, C, D

MORPHOLOGY
● Shape- spherical
● Size- 80-120 nm
● Enveloped virus with two peplomers – Haemagglutinin and neuraminidase
● Helical nucleocapsid
● Negative single stranded segmented RNA genome (Influenza A &B - 8 segments of RNA
and Influenza C&D - 7 segments of RNA)
● Replication occurs in nucleus (* only RNA virus replicate in nucleus)
● Viral proteins- 8 structural {PB1, PB2, PA, NP, HA, NA, M1, M2} and 2 non-structural
proteins {NS1, NS2}
 Reference: Essentials of Medical Microbiology, Apurba Sastry E/3: Page No. 648 Fig. 66.1

Gene Gene product Function

segment

1 PB1 Polymerase

2 PB2 Polymerase

3 PA Polymerase

4 HA Haemagglutinin – binds to receptor on RBC forms

hemagglutination

5 NP Nucleocapsid protein, encloses genome

6 NA Neuraminidase – replaces HA from RBC and cause

elution (reversal of hemagglutination)

7 M1 Matrix protein – forms shell underneath envelope

M2 Ion channels – transport molecules across envelope

8 NS1 Interferon antagonist, inhibits pre-mRNA splicing

NS2 Transport of molecules across nucleus

TYPES OF INFLUENZA VIRUS

Based on ribonucleoprotein (RNP) and matrix protein (M protein) influenza viruses
is divided into 4 genera: A, B, C and D

INFLUENZA A VIRUSES
● Genome segments - 8
● Host range – animals, birds & humans
● Epidemiology – epidemic & pandemic
● Subtypes – based on HA & NA .... HA subtypes: 18 & NA subtypes: 11
● Human subtypes: 6 HA (H1, H2, H3, HT, H7 & H9) & 2 NA (N1 & N2)

INFLUENZA B VIRUSES
● Genome segments – 8
 ● Host range – humans
● Subtypes – no subtypes
● But diverged into lineages
● Either belong to either B/Yamagata or B/Victoria lineage

INFLUENZA C VIRUSES
● Genome segments – 7
● Host range – man, pigs
● Less frequently detected, cause mild infections

INFLUENZA D VIRUSES
● Genome segments – 7
● Host range – animals
● Not pathogenic to humans

PATHOGENESIS
Inhalation of respiratory droplets, via contact with contaminated surfaces or fomites

HA attaches to specific sialic acid receptors on respiratory mucosa

Virus enters the cell

Virus replicate in the infected cells

Infectious daughter virions spread to adjacent respiratory epithelial cells

 Virus spread to lower respiratory tract or spills over bloodstream

Cause cellular destruction and desquamation of mucosa of respiratory tract

Predisposes to secondary bacterial infection

RECENT SWINE FLU PANDEMIC:

● Virus – Influenza A (H1N1)
● Emerged in California

EPIDEMIOLOGY:
● Origin: Genetic reassortment of 4 strains – 1 human strain + 2 swine strains + 1 avian
strain – mixing occurred in pigs
● Transmission: Human to human
● Less virulent (as it lacks PB1 F2 protein) so, it has caused more morbidity but less
mortality

CLINICAL FEATURES:
● Uncomplicated influenza: Mild upper respiratory tract illness and diarrhoea
● Complicated influenza: Secondary bacterial pneumonia, dehydration, CNS involvement
and multiorgan failure.
 Reference: Essentials of Medical Microbiology, Apurba Sastry E/3: Page No. 652 Fig. 66.2

CATEGORISATION OF SEASONAL INFLUENZA A/H1N1:

Category Definition Laboratory testing, treatment, isolation

Category A Influenza like illness (ILI) Laboratory tests - not required

Mild fever, cough/sore throat, headache Treatment - only symptomatic, antiviral

diarrhoea and vomiting drugs not required

Isolation- confine at home, avoid contact

with public and family members

Category B Category A plus any one: Laboratory tests- not required

a) High- grade fever, sore throat Treatment - symptomatic requires

treatment, antiviral drug require(
b) Presence of risk factors like Diabetes oseltamivir or Zanamivir)
mellitus, obesity, AIDS, chronic pulmonary,
cardiac and renal disorders Isolation- confine at home, avoid contact
with public and family members

Category C Severe acute respiratory syndrome (SARI) Laboratory tests- required

Category B plus any one Immediate hospitalization- required

a) breathlessness, chest pain, fall in BP, sputum Treatment - start antiviral drugs
+ blood, bluish discoloration of nails immediately without delay

b) children having ILI manifest with red flag Isolation - droplet precaution to be
signs - inability to feed well, difficulty in followed
breathing

c) worsening of underlying chronic causes

Reference: Essentials of Medical Microbiology, Apurba Sastry E/3: Page No. 652 Fig. 66.2
LABORATORY DIAGNOSIS
1. SPECIMEN COLLECTION:
▪ Nasopharyngeal swab, lavage fluid, nasal aspirate
▪ Transport – swab is kept inside viral transport media at 4°C

2. ISOLATION OF VIRUS:

▪ Inoculation in embryonated eggs and primary monkey kidney cell lines

▪ Growth is detected by hemadsorption, haemagglutination test
▪ As it is difficult to isolate, it is not routinely used

3. DIRECT IMMUNOFLUORESCENCE TEST:

▪ Viral antigens coated on epithelial cells can be detected in nasal aspirates by

using fluorescent tagged antibodies
▪ Rapid but less sensitive

4. MOLECULAR METHODS:

A) RT-PCR:

▪ More sensitive, specific and rapid

▪ Turnaround time < 1day
▪ Also detect specific type and subtype of influenza virus

B) Real - time RT-PCR:

▪ Gold standard method – quantitative, highly sensitivity and specificity

than RT-PCR
▪ Turnaround time 2-3 hours
▪ Simultaneously detect three common seasonal flu strains - A/H1N1,
A/H3N2 and type B

Real-time RT-PCR for seasonal influenza types

C) Bio Fire Film Array Respiratory Panel (RP):

▪ Detect 20 respiratory pathogens simultaneously like Influenza A, Influenza A/H1,

Influenza A/H3, Influenza A/H1-2009 and Influenza B

5. ANTIBODY DETECTION (SEROLOGY):

▪ Detect subtype specific serum antibodies using specific antigens

▪ Useful for sero – epidemiology purpose, not for clinical diagnosis
▪ Available tests – ELISA, neutralization test and haemagglutination inhibition test
(HAI)

TREATMENT:
● Specific antiviral therapy
● Start within 48hrs of onset of symptoms
● Neuraminidase inhibitors
▪ Administered for influenza A and Influenza B infections
▪ Drug of choice for A/H1N1 2009 flu, A/H5N1 avian flu and Influenza B
▪ Dosage –
✔ Oseltamivir (Tamiflu 75mg tablets)
✔ Zanamivir (10mg, inhalation form)
▪ Schedule – For treatment – given twice a day for 5 days
▪ For chemoprophylaxis – given once daily
Duration depends on clinical setting; usually 7 days
● Matrix protein M2 inhibitor
▪ Amantadine and rimantadine - given for influenza A virus infection
▪ Other strains like A/H1N1 2009 FLU and A/H5N1 avian flu and Influenza B
virus had developed resistance
 ESSAY 12
DISCUSS THE PATHOGENESIS, CLINICAL FEATURES, COMPLICATIONS,
LABORATORY DIAGNOSIS OF RUBELLA VIRUS. ADD A NOTE ON ITS
PREVENTION.
SHORT NOTE: MMR VACCINE

RUBELLA VIRUS (GERMAN MEASLES)

● Mild exanthematous fever
● With Transient fever, lymphadenopathy
● Pregnant women – causes congenital malformations
● Trivial (MMR)

PROPERTIES:
● Pleomorphic, spherical
● 50-70 nm in diameter
● Single stranded RNA genome
● Surrounded by hemagglutinin peplomers
● Resembles Toga virus
● Family: Togaviridae
● Genus: Rubivirus
● Accumulates human RBC at 4°C

CLINICAL FEATURES:
● Infection by Inhalation
● Incubation period: 2-3 weeks
● Rash on face, neck, trunk, palms and soles
● Mainly in children

COMPLICATIONS:
● Arthralgia, arthritis
● Common in women
 ● Causes chromosomal breakages, inhibition of mitoses
● Virus in throat disappear in 7 days
● Viremia at 7th day before rash
● Infection to fetus by maternal blood

CONGENITAL RUBELLA:
Fetal damage by stage of pregnancy

1. Very early pregnancy: Ends in abortion

2. First trimester: 90% of congenital
3. Later on in pregnancy: Developmental retardation in child

CONGENITAL RUBELLA SYNDROME:

● Causes are cardiac defects, cataract, deafness
● Expanded Rubella Syndrome:
Hepatosplenomegaly

Thrombocytopenic purpura

Myocarditis

Bone lesions

● Present in all excretions

● Present in tissues as cataractous lenses for several years

LABORATORY DIAGNOSIS:
● No routine diagnosis needed
● Diagnosis important in suspected pregnancy

DIAGNOSIS IN PREGNANCY:
1. SEROLOGY:
▪ Mainstay of diagnosis
▪ ELISA – to detect IgM, IgG
▪ IgM without IgG – Current acute Infection
 ▪ IgG without IgM – Past infection / vaccination / Infection
▪ Screening Test – TORCH Panel (In pregnant women)
2. CULTIVATION:
▪ Grown in 1° cell cultures, continuous cell lines
▪ Virus isolation is not commonly used for diagnosis due to delay
▪ Isolated from blood, throat swabs in rabbit
▪ Virus grows better in 33 – 35 ° C

DIAGNOSIS OF CONGENITAL RUBELLA:

1. SEROLOGY:
▪ IgM in newborn indicates intrauterine infection
▪ IgM not crosses placenta indicates response of fetus to infection
▪ IgG present in newborn after 6 months in congenital infection
2. ISOLATION:
▪ From urine, throat swabs, leucocytes bone marrow, CSF

PROPHYLAXIS:
● Confers lasting immunity as it has 1 antigenic type
● Live attenuated infection developed in tissue culture
● RA 27/3 strain is the vaccine used today
● Given by subcutaneous injection alone / combination with MMR vaccine
● Lymphadenopathy, rash, arthralgia may occur sometimes
● Not prescribed for immunodeficient patients
● Pregnancy should be avoided for 3 months after vaccination
● Not teratogenic

EPIDEMIOLOGY:
▪ Worldwide in distribution
▪ 80 – 90 % are immune by 15 years
▪ 10 – 20 % of mothers are non-immune, vulnerable
 ESSAY 13
DISCUSS THE PATHOGENESIS, CLINICAL FEATURES, COMPLICATIONS,
LABORATORY DIAGNOSIS OF MEASLES VIRUS. ADD A NOTE ON ITS
PREVENTION.
SHORT NOTES:
1. MMR VACCINE
2. WARTHIN FINKELDEY GIANT CELLS
3. MEASLES VIRUS

MEASLES

- Childhood disease (highly contagious)

- Characterized by – fever and respiratory symptoms, followed by typical maculopapular
rash

PATHOGENESIS:
● Transmission: Occurs predominantly via the respiratory route either by Droplet inhalation
or Small-aerosol particles
● Spread: The virus multiplies locally in the respiratory tract; then spreads to the regional
lymph nodes --- enters into the bloodstream and infect monocytes (primary viremia)
----further multiplies in reticuloendothelial system ---- spills over into blood (secondary
viremia) --- disseminates to various sites.
● Target sites: The virus is predominantly seeded in the epithelial surfaces of the body,
including the skin, respiratory tract, and conjunctiva.

CLINICAL MANIFESTATIONS:
 - Incubation period is about 10 days which may be shorter in infants and longer (up 3
weeks) in adults. Disease can be divided into three stages.
i) Prodromal Stage:
▪ This stage lasts for 4 days (i.e., from 10th to 14th day of Infection) and is characterized
by manifestations such as
- Fever occurs on day 1 (i.e., on 10th day of infection).
- Koplik's spots are pathognomonic of measles, appear after two days following fever
(i.e., on 12th day of infection) and are characterized by White to bluish spot (1mm size)
surrounded by an erythema. Appear first on buccal mucosa near second lower molars.
Rapidly spread to involve the entire buccal mucosa and then fade with the onset of rash.
- Non-specific symptoms may be present such as cough, nasal discharge, and redness of
eye, diarrhea or vomiting.
ii) Eruptive Stage:
▪ Maculopapular dusky red rashes appear after 4 days of fever (i.e., on 14th day of
infection).
- Rashes typically appear first behind the ears -- then
- Spread to face, arm, trunk and legs -- then fade in the same order after 4 days of onset.
- Rashes are typically absent in HIV infected people.

Fever (10th day) - > Koplik's spot (12th day) - >rash (14th day)

iii) Post Measles Stage:

- It is characterized by weight loss and weakness. There may be failure to recover and
gradual deterioration into chronic illness.

COMPLICATIONS:
1. Secondary Bacterial Infections:

▪ Following measles, there is profound immune suppression and fall of cell mediated
immunity which in turn predisposes to various secondary bacterial infections.
▪ Otitis media and bronchopneumonia are most common.
 ▪ Recurrence of fever or failure of fever to subside with the rash.
▪ Worsening of underlying tuberculosis with a false positive Mantoux test.

2. Complications Due to Measles Virus Itself:

▪ Giant-cell pneumonitis (Hecht's pneumonia) in immunocompromised children,

and HIV infected people.
▪ Acute laryngotracheobronchitis (croup)
▪ Diarrhea leads to malnutrition including vitamin A deficiency.
3. Central Nervous System Complications:
- CNS complications are rare, but most severe.
▪ Post-measles encephalomyelitis: It develops within 2 weeks of onset of rash. It represents
an autoimmune response against the myelin basic protein.
▪ Measles inclusion body encephalitis occurs months after rashes.
▪ Subacute sclerosing pan encephalitis (SSPE): It is a slowly progressive disease
characterized by seizures and progressive deterioration of cognitive and motor functions.
SSPE belongs to group C slow virus infection, caused by a defective measles virus.
▪ Age: SSPE typically develops if the primary measles virus infection occurs in children
less than 2 years of age. SSPE usually develops after 7- 13 years after primary measles
infection. It is fatal within 1- 3 years of onset. High titre antibody to measles virus in CSF
is diagnostic.

LABORATORY DIAGNOSIS:
1. SPECIMEN: Nasopharyngeal swab

2. ANTIGEN DETECTION: By using anti-nucleoprotein antibodies in virus infected cells.

3. VIRUS ISOLATION: Monkey or human kidney cells / Vero cell line produces cytopathic
effect as multinucleated giant cells (Warthin Finkeldey cells).
Reference: Essentials of Medical Microbiology, Apurba Sastry E/2: Page No. 486 Fig. 44.7 C

WARTHIN FINKELDEY CELLS:

- They are the multinucleated giant cells containing both intranuclear and intracytoplasmic
antibodies. It is a cytopathic effect observed after 7 to 10 days of inoculation into cell
lines.
- Shell vial culture is recommended for early detection in 2- 3 days.

4. ANTIBODY DETECTION:

▪ Detection of measles-specific IgM antibody in serum or oral fluid or four-fold rise of IgG
antibody titre between acute and convalescent-phase sera is taken as significant.
▪ Demonstration of high titer measles antibody in the CSF is diagnostic of SSPE.
▪ ELISA is the most recommended test that uses recombinant measles nucleoprotein (NP)
antigen.

5. REVERSE TRANSCRIPTASE PCR (RT-PCR): Specific for measles RNA detection is

available.

▪ It is extremely sensitive and specific; it may also permit characterization of measles virus
genotypes for molecular epidemiologic studies.
▪ It can distinguish wild-type from vaccine virus strains.
▪ RNA can be detected in specimens up to 10- 14 days post rashes, in contrast to virus
isolation, which often becomes negative after 3 days of rash.
PREVENTION:
- By live Attenuated Measles Vaccine
▪ Strains: Most attenuated strains in use currently are derived from the original Edmonston
strain.
▪ Vaccine is prepared in the chick embryo cell line.
▪ Reconstitution: Vaccine is available in lyophilized form and it has to be reconstituted with
distilled water and then should be used within 4 hours.
▪ Vaccine is thermo labile, hence it must be stored at - 20°C.
▪ One dose (0.5 mL) containing more than 1000 infective viral units is administered
subcutaneously.
▪ Indication: Under the national immunization schedule of India, measles vaccine is given at 9
months (because maternal antibody disappears by this time) along with vitamin-A
supplements.
▪ However, it can be given at 6 months during a measles outbreak, in that case a second dose
should be given at 9 months.
▪ Combined vaccines: Measles vaccine is available in combined form with mumps and
rubella vaccine (MMR vaccine) and with varicella (MMR-V vaccine).
▪ Side effects: Mild measles like illness may develop in 15-20% of vaccines. There is no
spread of the vaccine virus in the community. Toxic shock syndrome (due to contamination
of vial with Staphylococcus aureus toxins).
▪ Contacts: Susceptible contacts over 9-12 months may be protected against measles if the
measles vaccine is given within 3 days of exposure. This is because the incubation period of
measles induced by the vaccine strain is about 7 days, compared to 10 days for the naturally
occurring measles. Measles immunoglobulin (Ig) can also be given within 3 days, at a WHO
recommended dose of 0.25 mg/ kg of bodyweight. However, both vaccines and Ig should not
be given together. At least 8- 12 weeks of gap must be maintained.
 ESSAY 14
CLASSIFY CORONAVIRUSES. DISCUSS THE MORPHOLOGY, PATHOGENESIS,
CLINICAL FEATURES, LABORATORY DIAGNOSIS, TREATMENT & PREVENTION
OF COVID-19. EXPLAIN IN DETAIL THE RECENT COVID-19 PANDEMIC AND
OTHER PREVIOUS OUTBREAKS.

COVID – 19
CLASSIFICATION OF CORONAVIRUS:
Family: Coronaviridae

Subfamily: Coronavirinae and Torovirinae

Genera:

Coronaviridae:

● Alphacoronavirus
● Beta coronavirus
● Gamma coronavirus
● Delta coronavirus
Alphacoronavirus:

● Human coronavirus 229E

● Human coronavirus NL63
Beta coronavirus:

● Human coronavirus HKU1

● Human coronavirus OC43
● SARS – CoV
● Mers – CoV
● SARS-CoV-2 (COVID – 19)
MORPHOLOGY:

Reference: Essentials of Medical Microbiology, Apurba Sastry E/3: Page No. 663 Fig. 67.3

● Enveloped, petal shaped peplomers

● Pleomorphic
● Helical symmetry
● Unsegmented +ve sense RNA genome
● Consists of 4 structural and16 non structural proteins
● Structural proteins
- Nucleocapsid – Consists of positive sense single stranded RNA

- Spike protein – Helps in attachment

It consists of two subunits:

▪ S1 subunit- posses receptor binding domain binds to ACE2 receptor

▪ S2 subunit-facilitates viral cell membrane fusion

- Membrane glycoprotein- gives shape to virus

- Envelope protein- transmembrane protein with ion channel activity

● Non-structural proteins – help in replication of virus

Ex: RNA dependent RNA polymerase

Helicase
PATHOGENESIS:
● Mode of transmission – droplet transmission, airborne transmission
● Incubation period – 5 to 6 days as long as 14 days
● Colonize in upper respiratory tract and reach the alveoli

VIRAL ENTRY:
 Reference: Essentials of Medical Microbiology, Apurba Sastry E/3

VIRAL REPLICATION:

Reference: Essentials of Medical Microbiology, Apurba Sastry E/3: Page No. 652 Fig. 66.2

HOST RESPONSE:
● Damage to pneumocytes – release of cytokines – activate pulmonary macrophages –
release IL-1, IL-6, TNF
● These cytokines – cause contraction of endothelial cells and increase vascular
permeability – accumulation of fluid in the alveoli
● Compression of alveoli – decrease surfactant – collapse of alveoli
● Neutrophils also migrate across the vascular wall and release protease and reactive
oxygen species
● They cause injury to damage to nearby type1 pneumocyte – cause difficult to exchange –
hypoxemia
 ● Alveolar macrophage activates T cell – release interferons and also CD8 T cell kill the
virus infected cells
● In later stage – recruitment of fibroblasts causes lung fibrosis – respiratory failure

Reference: Essentials of Medical Microbiology, Apurba Sastry E/3

- Hypoxemia stimulate sympathetic system – Increased heart rate and increase respiratory
rate
- As inflammatory response increases, it increases capillary permeability in other organs
- It decreases blood volume that leads to hypotension and result in decreased organ
perfusion
- As a result – multiple organ failure

CLINICAL FEATURES:
● Breathlessness
● Cough with expectoration
● Fever
● Sore throat
 ● Headache
● Loss of taste or smell
● Repeated shaking with chills
● Muscle or body aches

LABORATORY DIAGNOSIS:
● Sample collection – nasopharyngeal swab, oropharyngeal swab
● Swab used – dacron / rayon / polyester swab
● Transport medium – viral transport medium

1. NUCLEIC ACID AMPLIFICATION TEST -RT PCR

▪ Viral RNA is converted into complementary DNA using reverse transcriptase
enzyme

Reference: Essentials of Medical Microbiology, Apurba Sastry E/2: Page No. 65

▪ Gold standard test for diagnosis of COVID – 19

▪ Average time taken – 4 to 5 hours
▪ Gene targets –
- For screening – spike protein, envelope protein, membrane protein, nucleocapsid protein
- For confirmation – RNA dependent RNA polymerase, open reading frames (ORF1a/b)

● It is detected by quantitative PCR

Principle – Target gene is first amplified

● When the amplicon binds to probe the fluorescence is emitted

● No of virions inversely proportion of cycle time
● Sample is positive- cycle time greater than 40 cycles
 2. ANTIGEN DETECTION TEST

▪ To detect nucleocapsid and spike protein antigen using immunochromatographic test

Procedure:

- Nasopharyngeal or oropharyngeal swab used – added to viral extraction buffer – mixed

and add 2 to 3 drops to a well of strip – wait for 15 minutes
- Positive – control and test band
- Negative – control band (if negative, confirm by RTPCR)

Reference: Essentials of Medical Microbiology, Apurba Sastry E/2: Page No. 142 Fig. 12.24

▪ ELISA and Chemi Immunofluorescence assay also used

3. ANTIBODY DETECTION TEST

● IgM: It appears at the end of first week and disappears in 3-4 weeks
● IgG: At the end of Second week
● Detected by immunochromatographic test

INTERPRETATION:

● IgM – Infection occurred in last one week

● IgG and IgM infection occurred in last 14 days
 ● IgG – Infection occurred in 3-4 weeks back
● Only control band – not an infection or early infection
● It is useful in:
- Sero surveillance purpose
- The survey in high risk (health workers, frontline workers)

4. PROGNOSTIC MARKERS

● Elevated lL-6 – Indicates cytokine storm

● Elevated d-dimer – Indicates fibrin degradation product underlying coagulopathy
● Elevated serum ferritin – Indicate inflammation
● Severe lymphopenia
● Elevated C – reactive protein – Indicates acute inflammation

5. CT SCAN – Ground glass appearance

TREATMENT:
Symptomatic management:

- Supplemental oxygen therapy given immediately

- High flow nasal cannula oxygenation
- Non invasive mechanical ventilation
- Investigational therapy

Drugs:

● Imedinivimab, Casirivimab – Inhibit binding to ace-2

● Favipiravir, Remedesivir – Drugs which inhibit RNA polymerase
● Lopinavir, Ritovir – protease inhibitors
● Tocilizumab – Inhibit IL- 6
● Drugs which reduce mortality
- Steroids - reduce cytokine production
- Anticoagulants- low molecular weight heparin and unfractionated heparin

CHEMOPROPHYLAXIS – Hydroxychloroquine

● Indication – all asymptomatic health care workers, asymptomatic frontline workers

 ● Contraindication – retinopathy, Hypersensitivity to HCQ, pre-existing cardiomyopathy,
pregnancy and lactation
● ECG to be done before prescribing and during the course to check for prolongation of
QT Interval

COVID -19 VACCINE:

▪ Covaxin – Inactivated vaccine contains spike protein
(2 doses 4 weeks apart- IM route)

▪ Covishield – Modified chimpanzee adenovirus expressing spike protein

(2 doses 4weeks apart- IM route)

PREVENTION:
For health care workers

● Hand hygiene
● Personal protective equipment
● Environmental cleaning

For general public:

● Hand wash
● Social distancing
● Environmental cleaning
● Wearing cloth mask

COVID- 19 PANDEMIC:
● SARS-CoV- 2 originated in Wuhan, China on Dec 2019
● On 30th Jan 2020, India reported the first case
● On 11th Feb 2020, new coronavirus called COVID 19
● On 11 March 2020, WHO officially declared COVID 19 as Pandemic
● Covid 19 spread started on 13 March 2020
● During the later phase there is emergence of new variants – UK variant, South African
variant
OTHER RECENT OUTBREAKS:
SARS-CoV – first pandemic of 21st century

● Originated in china on 2002- 2003

● From civet cat, raccoon dogs
● Clinical manifestations- sore throat, fever, muscle pain, headache
MERS- CoV

● Originated in Saudi Arabia- 2012

● From dromedary camels
● Clinical manifestation- fever, cough, shortness of breath

Reference: Essentials of Medical Microbiology, Apurba Sastry E/3: Page No. 661 Fig. 67.2
 ESSAY 15
DEFINE & CLASSIFY SLOW VIRUS DISEASES. DISCUSS THE MECHANISM,
CLINICAL MANIFESTATIONS, LABORATORY DIAGNOSIS & TREATMENT OF
PRION DISEASES. HOW WILL YOU STERILIZE THE PRION CONTAMINATED
MATERIALS?

SHORT NOTE: SLOW VIRUSES / SLOW VIRAL DISEASE OF MAN

SLOW VIRUS DISEASES

- Group of infections in animals and human beings with –
1. Very long incubation periods ranging from months to years

2. Slow, relentless course of illness lasting for months or years, with remissions
and exacerbations

3. Predilection for involvement of the central nervous system

4. Absence of immune response or an immune response that does not arrest the
disease, but may actually contribute to pathogenesis

5. Genetic predisposition

6. Invariable fatal termination

CLASSIFICATION:
a) Group A: Caused by serologically related, non-oncogenic retroviruses called Lentiviruses

1. Sheep –Visna (demyelinating disease), Maedi (progressive pneumonia)

2. Humans – HIV (AIDS)
b) Group B: Prion diseases of the CNS

Collectively known as the subacute spongiform viral encephalopathies

1. Human disease: Kuru, Creutzfeldt-Jakob disease

2. Animal disease: Scrapie, Mink encephalopathy, Mad cow disease
 c) Group C: 2 unrelated CNS diseases of human beings

1. Subacute sclerosing panencephalitis (SSPE)

2. Progressive multifocal leukoencephalopathy (PML)

GROUP B – PRION DISEASES

- Transmissible spongiform encephalopathies (subacute spongiform viral
encephalopathies)
- Chronic progressive degenerative diseases of the CNS

PRIONS:
i. Infectious agents
ii. Proteinaceous in nature
iii. Devoid of DNA and RNA
iv. Unusually resistant to physical and chemical agents such as heat, irradiation and
formalin
v. Can be transmitted to experimental animals by parenteral and oral challenge

MECHANISM OF PRION DISEASES:

1. Once infected, the prion proteins (PrPsc) are carried to the neurons
2. They bind to the normal PrPc on the cell surface
3. This causes release of PrPc from cell surface
4. Followed by post translational modification– elongated polypeptide PrPc becomes
globular polypeptide PrPsc
5. The cell synthesizes new PrPc and the cycle is repeated
6. As a result, large amount of PrPsc is formed
7. PrPsc are aggregated as amyloid-like plaques in the brain
8. As these plaques consist of host proteins, there is lack of an immune response or
inflammation
9. PrPsc are internalized by neurons and get accumulated inside the cytoplasmic vacuoles
giving the cell a spongiform appearance
10. PrPsc are transmissible – infectious, inheritable
PATHOLOGY:
Spongiform encephalopathy – main pathology seen in CNS:

1. Vacuolation of the neurons

2. Formation of amyloid-containing plaques and fibrils
3. Proliferation and hypertrophy of astrocytes
4. Fusion of neurons and adjacent glial cells

CLINICAL MANIFESTATIONS:
Incubation period – varies from months to years (longest being30 years). But once disease
sets in, progression is fast

1. Prodromal phase lasts for 3- 5 months, followed by appearance of manifestations such as

loss of muscle control, shivering, myoclonic jerks, tremors, loss of coordination and
rapidly progressive dementia
2. Death occurs within 1 year of onset of disease

HUMAN PRION DISEASES:

CREUTZFELDT-JAKOB DISEASE (CJD):

1. Classical form – most common (85% cases)

2. A subacute, presenile encephalopathy
3. Progressive incoordination and dementia
4. Ending fatally in about a year
Other Forms:

a) Sporadic CJD: Due to somatic mutation or spontaneous conversion of PrPc into

PrPsc
b) Iatrogenic CJD (50-75 yrs age): Transmitted by direct contact during some
medical or surgical procedures contaminated with prion tissues
i. Corneal transplantation
ii. Injection of the pituitary growth hormone
iii. Dura mater graft implantation
iv. EEG electrode implantation
c) Variant CJD (Below 30 yrs age): Believed to be transmitted through consumption
of contaminated beef with BSE prions
 d) Familial CJD: Due to inheritance of mutation of the PrP gene
i. Gerstmann-Straussler-Scheinker (GSS) syndrome
ii. Fatal familial insomnia

KURU:

1. Was seen only in the Fore tribe inhabiting the eastern highlands of New Guinea.
2. The disease had an incubation period of 5-10 years
3. Progressive cerebellar ataxia and tremors
4. Ending fatally in 3-6 months
5. Believed to have been introduced through cannibalism and maintained by the tribal
custom of eating the dead bodies of relatives after death as a part of a ritual
6. The disease has disappeared following the abolition of cannibalism

ANIMAL PRION DISEASES:

1. Scrapie: Prototype prion disease, affects sheep
2. Mink encephalopathy: Scrapie-like disease of mink
3. Bovine spongiform encephalopathy (BSE): Mad cow disease

LABORATORY DIAGNOSIS:
1. Measurement of PrPsc by conformation dependent immunoassay – most definitive
diagnostic tool for prion diseases
2. Neuropathological diagnosis in brain biopsies: The pathological hallmarks of prion
diseases seen under light microscopy, are spongiform degeneration and astrocytic gliosis
with lack of inflammatory response
3. Sequencing the PRNP gene to identify the mutation - important in familial forms of
prion diseases
4. Abnormal EEG: In late stage of the disease, high-voltage, triphasic sharp discharges are
observed

TREATMENT:
No known effective therapy for preventing or treating prion diseases
DECONTAMINATION:
- Prions are extremely resistant to most of the common sterilization procedures.
Recommended methods for sterilization of material contaminated with prion proteins are:
1. Autoclaving at 134°C for 1- 1.5 hour
2. Treatment with 1 N NaOH for 1 hour
3. Treatment with 0.5% sodium hypochlorite for 2 hours
- Prions if bound to the stainless steel should be treated with an acidic detergent solution
prior to autoclaving; rendering them susceptible to inactivation
 ESSAY 16
CLASSIFY PARAMYXOVIRUSES. WRITE THE MORPHOLOGY, PATHOGENESIS,
CLINICAL FEATURES, LABORATORY DIAGNOSIS, TREATMENT & PREVENTION
OF MUMPS.
SHORT NOTE: DIFFERENCE BETWEEN ORTHOMYXOVIRUS AND
PARAMYXOVIRUS.

MYXOVIRUSES
- Myxo means mucin
- Myxoviruses binds to the mucoprotein receptor on nasopharyngeal
mucosa and in RBC and cause haemagglutination

FAMILY OF MYXOVIRUSES:

PARAMYXOVIRIDAE:
These group of viruses are transmitted via respiratory route and causes
● Localized respiratory infection (influenza virus)
● Spread throughout body causing infection (mumps, measles)

CLASSIFICATION IN PARAMYXOVIRIDAE FAMILY:

SUBFAMILY GENERA MEMBERS LARGE gp F p Hemolysin

 PARAMYXOVIRI RESPIROVIRUS PARAINFLUENZA 1,3 HN type + +
NAE RUBULAVIRUS MUMPS, HN type + +
PARAINFLUENZA 2, 4a,4b
MORBILLIVIRUS MEASLES H type + +
HENIPAVIRUS *HENDRA/NIPAH G type + Not identified
PNEUMOVIRINA PNEUMOVIRUS RESPIRATORY G type + -
E
SYNCYTIAL VIRUS
METAPNEUMO HUMAN G type + -
VIRUS METAPNEUMOVIRUS

Abbreviations:
HN – have both hemagglutinin and neuraminidase activities;
H – have only neuraminidase activity;
G – do not have both hemagglutinin and neuraminidase activity;
gp – glycoprotein;
f p – fusion protein
*Only the GENERA OF ZOONOTIC PARAMYXO VIRUSES including nipah and hendra viruses infect
mostly bats, occasionally infecting humans. All other genera are pathogenic to humans

DIFFERENTIATION BETWEEN PARAMYXOVIRUSES AND ORTHOMYXOVIRUSES

PROPERTIES ORTHOMYXOVIRIDAE PARAMYXOVIRIDAE

Size 80-120nm 100-300nm
Shape Spherical, Rarely Pleomorphic
Filamentous
Nucleic Acid Negative Sense Ssrna, Segmented, Negative Sense Ssrna,
Eight Pieces Unsegmented, Single Piece

Genetic Stability Variable Stable

Genetic Recombination Seen Not Seen
Site For Rna Replication Nucleus Cytoplasm
Antigenic Variation Seen Not Seen

Important Human Influenza Virus Parainfluenza Virus, Mumps,

Pathogens Rsv, Measles,
Metapneumovirus
Antigens (Ha/Na) Both Ha And Na Spikes Are Ha Spike Present In- Parainfluenza,
Present, Hemagglutination Is Measles, Mumps
Reversible (Elution Is Seen) Na Spike Present In- Parainfluenza,
Mumps Inclusion Body-
Intracytoplasmic (Exception
Measles It Is
Both Intracytoplasmic And
Intranuclear)

MUMPS
MORPHOLOGY:

Reference: Essentials of Medical Microbiology, Apurba S Sastry E/2 Page No.

501 Fig. 44.5

Shape: Pleomorphic
Size: 100-300nm
RNA:
● Non – segmented
● Negative sense ssRNA
● Helical symmetry and surrounded with nucleocapsid

VIRAL PROTEINS:
6 Structural proteins
1. FUSION PROTEIN- Have the role of syncytium formation and hemolysin activity
2. HN GLYCOPROTEIN - Has both activity of
● Hemagglutinin (binds to sialic acid receptors on epithelial cells and cause viral entry)
● Neuraminidase (degrades sialic acid receptors on host cells and helps release of viral
particle form infected cells)

3. MATRIX PROTEIN-
● Protein shell layer for protection
● Ion channels for transport

ENVELOPE: Made of lipid, has H and N glycoprotein embedded in it

PATHOGENESIS:

CLINICAL FEATURES:
● INCUBATION PERIOD – 19days (Range 7-23)
TREATMENT:

▪ Treatment is mostly symptomatic

▪ Mumps’ immunoglobulin is available, but is not effective, hence not recommended.

PREVENTION:

VACCINES:
● Live attenuated vaccine is followed worldwide
● Trivalent MMR: Live attenuated measles-mumps-rubella
● Quadrivalent MMR: Additional varicella vaccine
● Monovalent mumps vaccines are not commonly used
● SCHEDULES: 2 Doses subcutaneously at 1yr and4-6yrs
● EFFICACY: About 88% after 2 nd dose. Long term immunity is unknown
Short Notes and Sets
of Short Notes
Short Notes:
1. Viral haemorrhagic fever (6)
2. Interferon (6)
3. Antigenic drift and shift (5)
4. Chikungunya fever (3)
5. Bacteriophage (2)
6. Kyasanur forest disease (KFD) (2)
7. Coxsackie viruses. (2)
8. Suckling mice – Definition and uses in Virology
9. Rhinovirus infection
10. Hemagglutination inhibition test
11. Yellow fever
12. Specimen collection, transport and lab
13. Antiviral agents
14. Cutaneous and genital warts

Sets of Short notes:

● SSN 1:
1) Viral diarrhoea (4)
2) Rota Virus (3)
3) Viral gastroenteritis
● SSN 2:
1) Viral replication
2) Viral multiplication
Both 1 & 2 have similar answers.
3) Viral hemagglutinin
● SSN 3:
1) Epidemic keratoconjunctivitis

● SSN 4:
1) Hepatitis E
2) Type C hepatitis
● SSN 5:
1) Mechanism of viral oncogenesis
2) Oncogenes
3) APUD cell tumors
● SSN 6:
1) Latent viral infections
2) Congenital viral infection
Short Notes
 SN1: VIRAL HAEMORRHAGIC FEVER
- Haemorrhagic manifestations may occur in patients suffering from several virus
infections.
1. Exanthematous fevers
▪ Smallpox
▪ Chicken pox
▪ Measles
2. Mosquito borne diseases
▪ Yellow fever
▪ Dengue
▪ Chikungunya
3. Tick borne fevers
▪ Kyasanur forest disease (KFD)
▪ Omsk hemorrhagic fever
▪ Crimean – Congo haemorrhagic fever
4. Arenaviruses
▪ Lassa fever
▪ South American haemorrhagic fever
5. Filoviruses
▪ Marburg virus
▪ Ebola virus
6. Hantaviruses
▪ Hantaan virus
▪ Belgrade virus
▪ Seoul
 SN2: INTERFERONS
● Proteins of cytokine family
● Produced within hours in response to viral infection
● Role in innate antiviral immune response
● Modulate humoral and cellular immunity
● Have broad cell growth regulatory activities

ANTIVIRAL EFFECTS
● Non – specific defence of host against viral infection
● Interferon itself is not the antiviral agent, it moves to other cells, induces an antiviral
agent
● Does not protect virus infected cells
● Always host species specific in function / not specific for a given virus
● Inhibits replication of wide variety of DNA and RNA virus
● Extremely potent. Very small amount fewer than 50 molecules of interferons per cell are
sufficient to induce antiviral state
If IFNs are added to cells before infection, there is marked inhibition of viral replication
 SN3: ANTIGENIC DRIFT AND SHIFT

Antigenic drift Antigenic shift

Minor antigenic changes in either Major antigenic changes in HA or NA, resulting
haemagglutination (HA) or neuraminidase in emergence of new subtype unrelated
(NA) or both. antigenically to predecessor strains.
Gradual sequential change occurring regularly Abrupt, drastic, discontinuous variation in
at frequent intervals. antigenic structure.
Change results due to mutation and selection. Change results from gene reassortment
(recombination) in doubly infected cells.
Accounts for periodic episodes of influenza. Widely spread causing major epidemics or
pandemics.
New antigens, though different from previous Antibodies to the predecessor virus cannot
antigens, are still related to them, so that neutralize the new variants.
antisera to the predecessor virus strains react
with mutant to a certain extent.
 SN4: CHIKUNGUNYA FEVER
Re-emerging disease characterized by fever with arthralgia.

VECTOR: Aedes aegypti

SYMPTOMS:
● Sudden onset of fever – Typically biphasic with a period of remission after 1-6 days
● Crippling joint pain
● Lymphadenopathy
● Conjunctivitis
● Maculopapular rash – Common
● Hemorrhagic manifestation – Rare
● Chikungunya cannot be differentiated from uncomplicated dengue

REASONS OF RE-EMERGENCE:
● New mutation (E1-Alanine 226Valine) Alanine in 226th position of E1 glycoprotein
gene is replaced by Valine
● New vector – Aedes albopictus

DIAGNOSIS:
i) Detection of IgM or IgG in paired serum sample – ELISA
ii) To detect viral RNA – RT PCR

TREATMENT: No vaccine is available.

SN5: BACTERIOPHAGE
Bacteriophages: Viruses that attack bacteria

MORPHOLOGY:
▪ 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 TYPES:

1. LYTIC PHASE: Phage replicates in cytoplasm and lyses host bacteria to come out.
It resembles the replication of other DNA viruses; except:
● In penetration step: Phages are attached to bacterial cell walls 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.
2. 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:
1. Phage typing: Phage typing is employed for typing the following bacteria:
Ex. Vi antigen typing of Salmonella typhi
2. Phage assay: To estimate the no. of viable phages in preparations.
3. 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.
4. Used in diagnosis: Mycobacteriophages are used for the identification of Mycobacterium
tuberculosis.
5. Used as a cloning vector.
6. Transduction: In Staphylococcus aureus, the plasmids coding for β-lactamases are transferred
between the strains by transduction.
SN6: KYASANUR FOREST DISEASE (KFD)
● Identified in 1957 from monkeys from the Kyasanur Forest.

EPIDEMIOLOGY:
• Vector: Hard ticks (Haemaphysalis spinigera) are the vectors of KFD virus and once
infected, they remain infected for life.

• Hosts: Monkeys, rodents and squirrels are common hosts which maintain the virus through
animal-tick cycles. Reservoirs are the rats and squirrels.

Amplifier hosts are the monkeys, where the virus multiplies exponentially. Man is an incidental
host and considered as a dead end.

• In monkeys: KFD virus has been a cause of epizootics with high fatality in primates especially
in monkeys, hence known as Monkey's disease.

• Situated in India: KFD is currently endemic in five districts of Karnataka.

There is a declining trend of incidence after the initiation of vaccines.

CLINICAL MANIFESTATION IN HUMANS:

• Incubation period varies from 3-8 days.

• First stage (haemorrhagic fever): It starts as acute high fever with malaise and frontal
headaches, followed by haemorrhagic symptoms, such as bleeding from the nasal cavity, throat,
and gums, as well as gastrointestinal bleeding.

• Second stage in the form of meningoencephalitis may occur 7-21 days after the first stage.

LABORATORY DIAGNOSIS:
Diagnosis is made by virus isolation from blood or by IgM antibody Detection by ELISA.
• Recently, nested RT-PCR and real time RT-PCR have been developed detecting viral RNA
(NS-5 non coding region) in serum samples and can provide early, rapid and accurate diagnosis
of die infection.

• Non-specific findings such as leukopenia, thrombocytopenia and decreased haematocrit,

albuminuria and abnormal CSF are found in the second stage.

KILLED KFD VACCINE:

A formalin-inactivated chick embryo vaccine has been developed for KFD.

• Schedule: two doses at intervals of 2 months, followed by booster doses at 6-9 months and
then every 5 years.
SN7: COXSACKIE VIRUSES
Family: Picornaviridae
Sub family: Enterovirus
● ssRNA, non - enveloped virus
● Infects only suckling mice and not adults
● Incubation period 2-9 days in humans
● Two groups – based on pathological changes in suckling mice & neutralization tests
1. A group –has 24 serotypes
2. B group– has 6 serotypes
● B groups share common complement fixing antigen

A GROUP.
▪ Inoculated in suckling mice
▪ Generalised myositis
▪ Flaccid paralysis
▪ Leads to death within a week
B GROUP
▪ Inoculated in suckling mice
▪ Patchy focal myositis
▪ Spastic paralysis
▪ Necrosis of brown fat
▪ Often results in hepatitis, pancreatitis, myocarditis, encephalitis

HOST RANGE AND CULTIVATION:

● Isolation of the virus and inoculation into suckling mice by intracerebral or subcutaneous
or intraperitoneal routes
● All coxsackie B groups grow well in monkey kidney cell culture while only A7 and A9
of group A virus grows in it

CLINICAL FEATURES OF GROUP A VIRUS:

● Herpangina (vesicular pharyngitis)
● Common in children
● Caused by A2, A6, A8 & A10 virus
● Severe febrile pharyngitis with headache, vomiting, pain in abdomen
● Characteristic lesions: Small vesicles on fauces and on posterior pharyngeal wall, which
breaks to form ulcers
 ● Aseptic meningitis
● Mostly caused by group A and all group B viruses
● Characterised by presence of maculopapular rash
● HAND FOOT AND MOUTH DISEASE (HFMD)
● Exanthematous fever
● Affects mainly young children
● Characterised by clusters of papulovesicular lesions in skin and oral mucosa
● Mainly caused by coxsackie A16, A9 and B1-3 viruses
● Benign illness resolving in 1-2 weeks
● Minor respiratory infections
● Similar to common cold
● Caused by A10, A21, A24 &B3 viruses
● Acute hemorrhagic conjunctivitis
● Caused by A 24 virus
● Self-limiting subconjunctival haemorrhage
● Incubation period – 1 day / complete recovery within 8-10 days
● Has explosive epidemics among adults in 1969-71 in Africa, South East Asia

CLINICAL FEATURES OF GROUP B VIRUS

● Bornholme disease (epidemic myalgia/pleurodynia)
● Febrile disease with sharp piercing pain in chest and abdomen
● Myocarditis & pericarditis
● Most common in newborn – high fatality
● Sometimes in older children and adult
● Juvenile diabetes
● Most commonly caused by B4 virus
● Orchitis – inflammation of testis
● Transplacental & neonatal transmission
● Resulting in serious disseminated diseases including
● Hepatitis, meningoencephalitis, adrenocortical involvement
● Post viral fatigue syndrome
● Generalised diseases of infants – affects multiple organs

LABORATORY DIAGNOSIS:
1. ANIMAL INOCULATION:
▪ Virus isolated from lesions / fauces
▪ By inoculating into suckling mice intracerebrally
▪ Group A virus produce flaccid paralysis
▪ Group B virus produce spastic paralysis
2. TISSUE CULTURE:
 - All serotypes do not grow in cell lines hence tissue culture is not useful
3. SERODIAGNOSIS:
- As there are existence of several antigenic types, this will also not be useful
4. SEROLOGY: Performed to detect neutralizing antibodies
5. PCR TARGETING SPECIFIC GENES is highly useful as it is rapid, more sensitive,
serotype specific.

EPIDEMIOLOGY:
▪ Primarily coxsackie viruses inhabits alimentary canal
▪ Spreads by feco – oral route
▪ Coxsackie B virus shows epidemics in every 2-5 years
 SN8: SUCKLING MICE – DEFINITION AND USES IN
VIROLOGY
- Very susceptible to coxsackie and arboviruses, many of which do not grow in any other
system.
- Method for cultivation of viruses via animal inoculation.
- Earliest methods – Using human volunteers
Now – Using lab animals (mice)

ROUTES OF INOCULATION:
1. Intracerebral
2. Subcutaneous
3. Intraperitoneal
4. Intranasal.

OTHER ANIMALS USED: Guinea pigs, rabbits and ferrets.

INDICATIONS OF GROWTH OF VIRUS:

1. Death
2. Disease
3. Visible lesions

DISADVANTAGES:
1. Immunity may interfere with viral growth
2. Animals often harbour latent viruses.

OTHER USES:
Study of -
1. Pathogenesis,
2. Immune response,
3. Epidemiology

PATTERNS SEEN IN DIFFERENT VIRUSES:

1. Coxsackieviruses:
a) Group A viruses:
Produce generalized myositis and flaccid paralysis, leading to death within a week.
b) Group B viruses:
 Produce patchy focal myositis, spastic paralysis, necrosis of brown fat and, often,
pancreatitis, hepatitis, myocarditis and encephalitis.

2. Arboviruses (e.g., Togaviridae, Flaviviridae, etc):

Upon intracerebral inoculation, the animals develop fatal encephalitis. Though serial blind
passages may be necessary in some cases.
 SN9: RHINOVIRUS INFECTION
- Also known as Common cold

RHINOVIRUSES:
▪ Human rhinoviruses consist of 3 species (A, B and C)
▪ More than 150 serotypes are found
▪ Use host ICAM-I as receptor
▪ They belong to enteroviruses except

CLINICAL FEATURES:
● Incubation period is about 2-4 days
● Sneezing, nasal discharge, nasal obstruction, sore throat and no fever
● Primary disease presents as RHINOSINUSITIS
Secondary bacterial infection in children may cause otitis media, sinusitis, bronchitis or
pneumonitis

LABORATORY DIAGNOSIS:
▪ Viruses can be grown in WI-38 and MRC-5 cell lines
▪ Organ culture of ferret and human tracheal epithelium may be necessary in fastidious
strains

TREATMENT: Symptomatic treatment

SN10: HAEMAGGLUTINATION INHIBITION TEST

- Type of neutralization test

- Once used for the diagnosis of viral diseases like influenza

PRINCIPLE:
Anti-H antibodies present in the patient’s sera agglutinates the haemagglutinin
antigen present in viruses.

+ +

PROCEDURE:
▪ In this procedure the flu infected person’s serum containing the anti-HA antibodies
are made to react with the flu virus containing HA surface antigens.
▪ The flu virus has the ability to cause agglutination of human RBCs. Because the
virus has been already attacked by the antibody of the patient’s serum, the virus will
not be present to clump the RBCs, and hence the RBCs will settle at the bottom in
the form of a button.
▪ By calculating the amount of serum required to prevent agglutination of RBC we can
quantify the severity of infection that is expressed in terms of HI titres.
 SN11: YELLOW FEVER
➔ It is an acute, febrile illness caused by yellow fever virus.
➔ In severe cases it is characterized by liver dysfunction which leads to jaundice, renal
dysfunction, haemorrhage and mortality.
➔ It is endemic to West Africa and Central South America.

YELLOW FEVER VIRUS

▪ Arbovirus
▪ Belongs to family Flaviviridae
▪ Enveloped virus, containing single stranded RNA (ssRNA).

TRANSMISSION:
➔ Vector for infection in humans is by the bite of Aedes aegypti or the tiger mosquito.
➔ Transmission cycle:
▪ Jungle cycle: It occurs between monkeys and forest mosquitoes
▪ Urban cycle: it occurs between humans and Aedes aegypti.

CLINICAL MANIFESTATIONS:
▪ Incubation period - 3-6 days
▪ In early stage of disease
➔ Presence of fever, chills, headache, dizziness, myalgia and backache followed by
nausea, vomiting and bradycardia.
➔ Infected person is viremic in this stage.
▪ In severe cases
➔ Haemorrhage
➔ Platelet dysfunction
➔ Renal dysfunction
➔ Hepatitis- Torres bodies (intranuclear inclusions inside hepatocytes), jaundice
seen
 ➔ Mortality rate is high (mainly in children and elderly)

LABORATORY DIAGNOSIS:
➔ Serology: IgM ELISA
➔ Molecular method: RT-PCR (more confirmatory)

EPIDEMIOLOGY:
▪ Majority of outbreaks occur in Africa
▪ All age groups are susceptible
▪ In India strict guidelines for vigilance and quarantine of travellers in the international
airports is the reason for absence of yellow fever

YELLOW FEVER 17D VACCINE

▪ Live attenuated vaccine
▪ It is prepared in allantoic cavity of chick embryo
▪ Dosage: single dose, given subcutaneously
▪ Vaccine is effective within 7 days of administration
▪ Efficacy lasts for up to 35 years
Contraindications of yellow fever vaccine:
➔ Children < 9 months
➔ Pregnancy (except during outbreak)
➔ HIV infected people
➔ People with allergy to egg
 SN12: VIRAL SPECIMEN COLLECTION, TRANSPORT AND
LABORATORY DIAGNOSIS

SPECIMEN COLLECTION:
○ Specimens can be collected from the patient in the forms of swabs, sputum, urine,
aspirates, tissue specimens, body fluids, scrapings (like corneal scrapings) and by
stool collection from the respected infected areas.
○ These specimens are transported in viral transport media.
○ Viral transport media prevents drying of the specimen, maintains
viability of the viruses and prevents overgrowth by
contaminating flora.
○ The specimen should be held at 4°C during transport for most viral specimens.
○ These specimens should not be transported in FORMALIN as they cut and
destroy the DNA into small pieces.

SPECIMEN TRANSPORT:
○ Most of the specimens should be transported within 2 hours.
○ In cases of CSF, Body Fluids - Immediate transport is required.
○ In Urine, Rectal swabs - If with added preservatives like . .
boric acid transport duration is . . acceptable
up to 24 hours.
○ In cases of stool culture - Without medium - 2 hours
With medium - 24 hours is . .
acceptable (Cary-Blair medium)

LABORATORY DIAGNOSIS:
○ After receiving the specimen, tests are proceeded as fast as possible to achieve
rapid diagnosis.
○ The tests can be in the form of microscopical, staining, immunological,
serological or by molecular methods.
○ The results of the tests must be conveyed to the treating physician who has
requested the investigation and must be conveyed in standard reporting formats
in such a way that the physician or patient is able to get accurate and reliable
results which are clear and easy to understand.
○ A wrong report or an incomplete one might put the patient in danger of wrong
treatment or inadequate management.
SN 13. ANTIVIRAL AGENTS

Reference: Baveja Textbook of Microbiology- Fifth Edition Page No. 432

SN 14: CUTANEOUS AND GENITAL WARTS
➔ Caused by human papillomavirus (HPV)
▪ DNA virus
▪ Non – enveloped, have icosahedral capsids and contain circular double stranded
DNA
▪ Belongs to papillomavirus family
▪ Has selective tropism for Epithelium of skin and mucous membrane
▪ Has more than 100 serotypes
▪ They can produce infections ranging from benign warts to malignant neoplasia of
the cervix.

CUTANEOUS WARTS:
➔ Small, hard, rough growth on skin
➔ It is of following types
● Common skin warts (verruca vulgaris) - common in children
● Flat warts (verruca plana)- common in children
● Plantar warts (verruca plantaris)- common in adolescents

ANOGENITAL WARTS (CONDYLOMA ACUMINATUM):

➔ Caused by serotypes types 6 and 11 of human papillomavirus
● Have low malignant potential
➔ Found in genital area such as
● Penile shaft
● Scrotum
● Labia majora of the vagina
● Anal area
➔ Generally pink in colour
➔ Project out from surface of the skin
 ➔ Size may vary (from small to large masses)

LABORATORY DIAGNOSIS:
▪ Most lesions are visible to naked eye. 5% acetic acid solution is applied to
improve visibility
▪ Molecular methods: PCR

TREATMENT:
▪ Removal of the lesion by
● Cryosurgery
● Electrodesiccation
● Surgical excision
● Laser therapy
▪ Topical preparations of
● Interferon, podophyllum used for genital warts.

PREVENTION:
● HPV vaccine
● Barrier method of contraception (prevention of anogenital warts)
Sets of Short notes
 SSN 1
1) VIRAL DIARRHOEA
2) ROTA VIRUS

▪ Rotaviruses are the most common cause of Diarrheal illness in children.

▪ Rotaviruses have icosahedral symmetry.
▪ Surrounded by a triple layered capsid.
▪ Possess segmented dsRNA (11 segments)
▪ Proteins: There are six structural viral proteins (VP1 to VP7 except VP5) and six
non-structural proteins (NSPI 6).

PATHOGENESIS:
▪ Rotaviruses infect and ultimately destroy the mature enterocytes in the villi of the
proximal small intestine; however, the gastric and colonic mucosa are spared.
▪ They multiply in the cytoplasm of enterocytes and damage their transport mechanisms
resulting in secretory diarrhoea.
▪ The non-structural protein-NSP4, acts as enterotoxin and induces secretion by altering
epithelial cell function and permeability.

CLINICAL MANIFESTATIONS:
The incubation period is about 1- 3 days. It has an abrupt onset, characterized by vomiting
followed by watery diarrhoea, fever and abdominal pain.

LABORATORY DIAGNOSIS:
• Direct detection of virus: Faeces collected early in the illness is the most ideal specimen.
Rotaviruses can be demonstrated in stool by lmmuno Electron microscopy (lEM). Rotaviruses
have a sharp edged triple shelled capsid; look like the spokes grouped around the hub of a wheel.
 Reference: Essential of Medical Microbiology, Apurba Sastry E/2 Page No. 547 Fig. 49.3

• Detection of viral antigen in stool by ELISA and latex agglutination-based methods.

• RT-PCR is the most sensitive detection method for detection of rotavirus from stool.

VACCINE: Rotavac and Rotarix are available.

3. VIRAL GASTROENTERITIS
Viral etiology accounts for the most of the acute infectious gastroenteritis worldwide.

Viral gastroenteritis most commonly occurs among children.

Persons of all ages can be affected. Several enteric viruses can cause acute gastroenteritis in
humans, most common being rotavirus.
Reference: Essentials of Medical Microbiology, Apurba Sastry E/2 Page No. 546 Table 49.
 SSN 2
1. VIRAL REPLICATION
2. VIRAL MULTIPLICATION
Viruses do not undergo binary fission (seen in bacteria), but undergo complex ways of cell
division.
Replication of viruses passes through six sequential steps:
i) Adsorption/attachment:
▪ First and most specific step of viral replication.
▪ Involves receptor interactions between virus and host.
ii) Penetration
After attachment, virus particles penetrate into host cells either by:
▪ Phagocytosis (Viropexis) – through receptor-mediated endocytosis
▪ Membrane fusion – seen in HIV
▪ Injection of nucleic acid – seen in bacteriophages.
iii) Uncoating
Capsid is lysed (due to host lysozymes) and nucleus acid is released – this is absent for
bacteriophages.
iv) Biosynthesis of various viral components:
⮚ Nucleic acid
⮚ Capsid protein
⮚ Enzymes
⮚ Other regulatory proteins
Site of nucleic acid replication:
▪ DNA viruses – DNA replication occurs in nucleus (except in Poxviruses)
▪ RNA viruses – RNA replication occurs in cytoplasm (except in Retroviruses and
Orthomyxoviruses)
v) Maturation - Takes place in the nucleus or cytoplasm.
vi) Release
Release of daughter visions occur by:
▪ Lysis of host cells – seen in non-enveloped viruses mad bacteriophages
▪ Budding through host cell membrane – seen in enveloped viruses

ECLIPSE PHASE:
● Interval between entry of virus into host cell till appearance of first infectious virus particle
● During this period, virus cannot be demonstrated inside the host cell.
● Duration:
▪ Bacteriophages – 15 – 30 minutes
▪ Most of the animal viruses – 15 – 30 hours

3. VIRAL HAEMAGGLUTINATION
● Large number of viruses contain haemagglutinin spikes (peplomers) on the capsid or
envelope which can agglutinate erythrocytes of different species.
● Viral haemagglutinin (glycoprotein) has special affinity for different glycoprotein located
in receptor areas on the surface of erythrocyte.

PROCEDURE
▪ This test provides a simple and rapid method for detection of viruses in egg or tissue
culture fluid.
▪ When erythrocytes are added to serial dilutions of viral suspension, virus and
erythrocytes collide in the suspension and adhere to each other resulting in
hemagglutination.
▪ Highest dilutions that provide hemagglutination provides the titer.
▪ Erythrocytes which are not agglutinated settle at the bottom in the form of ‘button’,
while agglutinated erythrocytes are seen spread into shield-like pattern.
● Hemagglutination reaction is specifically inhibited by antibody to the virus.
● Hemagglutination inhibition test (HI) – Routinely used for detecting antiviral antibody in
diagnosis and research.
● Some viruses, particularly influenza and parainfluenza viruses also carry on their surface
another peplomer, the enzyme neuraminidase which acts on receptors on erythrocytes and
destroys them – Receptor Destroying Enzyme (RDE).
● Destruction of surface receptors results in reversal of hemagglutination and release of
viruses from the surface of erythrocytes – Elution.
● After elution, receptors are irreversibly damaged and erythrocytes are no longer
agglutinable by that particular virus. The free viruses are unharmed.

Reference: Baveja Textbook of Microbiology – Fifth edition Page No.414

 SSN 3:
1. EPIDEMIC KERATOCONJUNCTIVITIS
● Disease is produced by Adenovirus –non enveloped DNA virus, space vehicle-shaped.
● Serious condition that appears as an epidemic.
● Usually caused by type 8 and less often by types 19 and 37.
 SSN 4
1. HEPATITIS E

- Caused by hepatitis virus E.

- Enterically transmitted hepatitis affects young adults
Family: Heperviridae
Genus: Hepe virus

MORPHOLOGY:
▪ Small, non-enveloped, icosahedral
▪ +ve sense SSRNA, specific Ag

TRANSMISSION: Contamination of food and water intake

CLINICAL MANIFESTATIONS:
● Incubation period 14 – 60 days
● Self-recovering acute hepatitis
● Pregnant women are more prone
● No chronic or carrier state
HEV HAV

Secondary attacks are less Secondary attacks are more

Affects young adults Affects children

LABORATORY DIAGNOSIS:
● RT PCR – HEV RNA in stools
● ELECTRON MICROSCOPY - HEV RNA in stools
● ELISA – Serum IgM and IgG anti HEV are seen

TREATMENT: No specific antiviral drugs

PREVENTION: HEV 239 vaccine

 2. TYPE C HEPATITIS
- Most common post transfusion hepatitis
- Caused by Hepatitis C virus
Family: Flaviviridae
Genus: Hepacivirus

MORPHOLOGY:
● Spherical, enveloped
● +ve ss RNA virus
● Proteins
▪ Core proteins, E1, E2
▪ NS2, NS3, NS4A, NS4B, NS5A, NS5B
▪ P7 membrane proteins

TRANSMISSION:
● Mostly via contaminated blood, needle stick injury
● From infected Drug addicts who share needles
● Vertical transmission also can occur and not transmitted through lactation

CLINICAL MANIFESTATIONS:
● Incubation period 15-160 days
● Acute hepatitis that spontaneously clears within 12weeks
● Sometimes may become chronic hepatitis, cirrhosis, hepatocellular carcinoma
● Some extrahepatic manifestation
○ Mixed cryoglobulinemia
○ Glomerulonephritis
○ Arthritis and joint pain

LABORATORY DIAGNOSIS:
● ELISA:
- HCV Ab 3rd generation via antigens NS5 with core protein
- HCV core antigen is tested
● MOLECULAR METHODS
- HCV RNA is detected through real time RT PCR
● Gold standard is liver biopsy
TESTING SEQUENCES:
1. Anti HCV Ab test:
● If +ve, HCV RNA tested for active infection
● If -ve, no action needed
2.Hepatitis C screening:
It is done for
● >18 years, HIV patients
● Pregnant, IV drug addicts

TREATMENT:
● Pegylated interferon + Ribavirin
● Direct acting antivirals
○ NS3/4A inhibitors
▪ Grazoprevir
▪ Paritaprevir
○ NS5B inhibitors
▪ Dasabuvir
○ NS5A inhibitors
▪ Daclatasvir
Both are used as combined regimen for 12- 24 weeks
 SSN5
1.MECHANISM OF VIRAL ONCOGENESIS
- Viral oncogenesis is a complex multistep process that takes place over a long period of
time. Oncoviruses are usually not cytolytic; they transduce or activate oncogenes.
Oncogenic viruses transforming host cells can be classified under two types:

ONCOGENIC DNA VIRUSES

● Under the influence of the virus, the host cell undergoes malignant transformation similar
to lysogenic conversion in bacteria.
● The cell is not destroyed nor is a virus produced but there is altered expression of
pre-existing cellular genes (which regulate host cell growth) such as Proto-oncogenes,
Tumor suppressor gene, Apoptosis regulatory genes and DNA repair genes that interfere
with cell cycle causing uncontrolled growth and transformation.
● Two events are necessary for viral transformation: a persistent association of viral
genes with the cell, and the expression of certain viral “transforming” proteins.
● Examples of oncogenic DNA viruses:
a) Human herpesviruses (EBV, HHV-8 or KSHV)
b) Human hepatitis B virus
c) Human Papillomavirus and Polyomavirus

ONCOGENIC RNA VIRUSES

● First, most retroviruses do not kill the host cell, but rather set up a permanent infection
with continual virus production. Second, a DNA copy of the RNA genome is integrated
into the host cell DNA by a virally encoded integrase.
● Retroviruses are known to transform cells by two different mechanisms:
i. ACUTE TRANSFORMING OR DIRECT ACTING RETROVIRUSES:
▪ They are certain animal retroviruses (e.g., Rous sarcoma virus) that carry viral
oncogene which they directly insert into the host chromosome.
▪ Oncogenes encode a protein that interferes with cell signalling that results in
uncontrolled cell proliferation and cell transformation.
 ii. SLOW TRANSFORMING OR INDIRECT ACTING RETROVIRUSES:
▪ Viral genome can insert anywhere in the host chromosomes randomly and not
necessarily adjacent to proto-oncogenes.
▪ They do not have viral oncogenes, but possess an additional regulatory gene
(e.g., taxgene for HTLV-1 and tatgene for HIV). These genes induce or alter
the expression of pre-existing genes.
▪ E.g., Transactivating factor (Tax) turns on cellular genes, causing cell
proliferation.
● Examples of oncogenic RNA viruses:
a) Retroviruses
b) Human T-lymphotropic viruses I and II (HTLV-I and -II)
c) Hepatitis C virus

2. ONCOGENES
● Oncogenes or cancer genes are genes which encode proteins that trigger the
transformation of normal cells into cancer cells.
● Oncogenes present on the viral genome are called viral oncogenes(V-onc). These
genes are expressed by recombination between retroviral and cellular genes. More than
30 oncogenes have now been found since the original oncogene was identified in Rous
sarcoma virus (called v-src, where the v stands for viral).
● Oncogenes isolated from cancerous cells are called cellular oncogenes (C-onc). These
genes contain introns characteristic of eukaryotic genes.
● The cellular counterpart of oncogenes present in normal cells (not of viral origin)
are called proto-oncogenes. These genes have some essential functions in normal cells
such as coding for proteins involved in regulating cell growth and differentiation. When
mutated they form oncogenes.
● Transfection is the preferred method of study of oncogenes.
● Examples of few oncogenes:
Viral oncogene Origin Neutral Human gene tumour
V-src Chicken Sarcoma C-src
V-ras Rat Sarcoma C-ras
V-myc Chicken Leukemia C-myc
V-fes Cat Sarcoma C-fes
V-sis Monkey Sarcoma C-sis
V-mos Mouse Sarcoma C-mos

3. APUD CELL TUMOURS

⮚ Amine Precursor Uptake Decarboxylation (APUD) cells are cells with special
characteristics such as:
● High amine content
● Capacity of amine precursor uptake
● Property of decarboxylation
⮚ Tumours arising from these cells are called Apudomas. Since APUD cells have peptide
synthesizing properties, common presentations of these tumours are due to increased
neuro-endocrine secretions. Hence, they are also called Neuro-endocrine Tumours
(NETs)
⮚ NETs can arise in many different areas of the body, and are most often located in
the intestine (called carcinoid tumours), pancreas or the lungs.
⮚ Some tumours under NETs include:
▪ Neuroendocrine tumor of the anterior pituitary
▪ Medullary carcinoma
▪ Pulmonary neuroendocrine tumors (e.g., SLC)
▪ Gastroenteropancreatic neuroendocrine tumors (GEP-NET)
▪ Merkel Cell Carcinoma
▪ Inherited conditions such as (MEN- 1 and 2, Von-Hippel Lindau disease)
MERKEL CELL CARCINOMA
● It is also known as cutaneous Apudoma.
● It can be primary neuroendocrine carcinoma of the skin, primary small cell carcinoma
of the skin, or trabecular carcinoma of the skin
● Merkel-cell carcinoma usually arises on the head, neck, and extremities, as well as in
the perianal region and on the eyelid.
● About 80% of MCC tumor patients are infected with Merkel Cell Virus.
● Merkel Cell virus is a double stranded DNA Oncogenic virus under the class
Polyomaviruses.
● The virus is integrated into the host genome in a monoclonal pattern and the viral
replication takes place in the nucleus of the infected cells.
● Transcription of early genes is performed by host RNA polymerase, which leads to
synthesis of early proteins. The early proteins regulate viral transcription, DNA
replication, cell division, and transformation.
● Merkel Cell Virus is usually inactive in infected patients and the patient only becomes
symptomatic defective immune functions such as malignancy, HIV infection, and
organ transplant patients. Conversely, patients with brisk immune responses have
been shown to have improved prognosis.
 SSN6
1) LATENT VIRAL INFECTIONS
- In latent infections, the disease is not produced but the virus is not eradicated from the
host.
- The equilibrium between the host and the parasite is achieved in various ways by
different parasites and hosts.
- A latent viral infection does not cause any noticeable symptoms and can last a long time
before becoming active and cause symptoms.
- The virus may exist in a truly latent non-infectious occult form, possibly as an integrated
genome or an episomal agent, or as an infectious and continuously replicating agent,
termed a persistent viral infection.
● Recurrent herpes simplex and herpes zoster are the examples of latent viral infections
where clinical manifestations appear after prolonged periods of quiescence during which
the virus remains hidden in the nerve root ganglia.
● Persistent tolerant infection occurs when the virus is readily demonstrable in the tissues
of the host but neither the disease nor immune response develops. The host is
immunologically tolerant to the virus as a result of congenital or neonatal infection. Disease
sets in when the tolerance is interrupted.
Example: lymphocytic choriomeningitis of mice.
● The virus may exist as an infectious and continuously replicating agent, termed a
persistent viral infection.
In chronic persistent infections the viruses evade the immune response by several
mechanisms. They are:
a) Generation of cells that escape a cell-mediated immune response.
b) Down regulation of MHC production in infected cells so that they are not recognized and
destroyed by T cells.
c) Infection of cells in immunoprivileged sites such as the brain.

VIRAL LATENCY:
▪ Ability of a pathogenic virus to lie dormant within a cell, denoted as
the lysogenic part of the viral life cycle.
▪ Latent viral infection - type of persistent viral infection which is distinguished from
a chronic viral infection
▪ Latency is the phase in certain viruses' life cycles in which, after initial infection,
proliferation of virus particles ceases. However, the viral genome is not eradicated.
▪ The virus can reactivate and begin producing large amounts of viral progeny without the
host becoming reinfected by new outside virus, and stays within the host indefinitely.
 ▪ Virus latency is not to be confused with clinical latency during the incubation
period when a virus is not dormant.

▪ The mechanisms of viral latency are:

- Episomal latency
- Proviral latency
- Maintaining latency

EPISOMAL LATENCY
Episomal latency refers to the use of genetic episomes during latency. In this latency
type, viral genes are stabilized, floating in the cytoplasm or nucleus as distinct objects, either as
linear or lariat structures.
Example: Herpes virus family

PROVIRAL LATENCY
A provirus is a virus genome that is integrated into the DNA of a host cell. One of the
best-studied viruses that do this is HIV.
HIV uses reverse transcriptase to create a DNA copy of its RNA genome. HIV latency allows
the virus to largely avoid the immune system. Like other viruses that go latent, it does not
typically cause symptoms while latent. Unfortunately, HIV in proviral latency is nearly

MAINTAINING LATENCY
Both proviral and episomal latency may require maintenance for continued infection and fidelity
of viral genes. Latency is generally maintained by viral genes expressed primarily during latency.
Expression of these latency-associated genes may function to keep the viral genome from being
digested by cellular ribozymes or being found out by the immune system.
Example: latency associated transcripts (LAT) in herpes simplex virus
 2) CONGENITAL VIRAL INFECTIONS

- Congenital infections affect the unborn fetus or newborn infant. They are generally
caused by viruses that may be picked up by the baby at any time during the
pregnancy up through the time of delivery.
- The viruses initially infect the mother who subsequently may pass it to the baby either
directly through the placenta or at the time of delivery as the baby passes through the
birth canal.
- Mothers generally do not feel sick with the viruses. Sometimes they have flu-like
symptoms. Even if the mother is known to have a viral illness during her pregnancy, her
immune system may prevent the virus from infecting the fetus or newborn infant.
- Vertical transmission is the natural mode of spread of many tumor viruses. The avian
leukosis virus is transmitted in ovo and murine mammary virus through breast milk.
The more common viruses linked to congenital infections include the Cytomegalovirus (CMV),
Herpes, Rubella (German measles), Parvovirus, Varicella (chickenpox), and Enteroviruses.
Rubella and Cytomegalovirus produce maldevelopment or severe neonatal diseases.

PATHOPHYSIOLOGY:
▪ These infectious agents can cross the placental barrier and spread to the fetus in utero
which can cause fetal loss, the emergence of certain congenital
malformation, prematurity or chronic postnatal infection.
▪ The transplacental spread of these organisms to the fetus might be associated
with chronic infection because of the immaturity of the fetus’ immune system. These
organisms are usually not very virulent and the immune system of the developing fetus
can develop tolerance to them.
▪ If this happens, the fetal immune system will fail to eliminate the infecting organisms and
chronic infection might occur.

DIAGNOSIS:
▪ Diagnosis of congenital infections is difficult in early stages. Most congenital infections
in the fetus and newborn baby are totally silent and asymptomatic. But can be serious and
cause profound damage to the body resulting in birth defects or even death.
▪ It can quietly and slowly damage the body, causing medical and developmental problems
that only show up months or even years later.
▪ Diagnosis of a congenital infection can sometimes be made by the obstetrician or
pediatrician based upon the mother’s symptoms, the baby’s physical findings before (by
ultrasound) or after birth, as well as by blood tests on both mother and baby.
 ▪ Infants with silent congenital infections may not exhibit disabilities for months or years.
▪ Hence, it is important that all babies born with known or suspected congenital infections
be followed closely to detect signs of developmental problems at the earliest possible age.
▪ Close, early follow-up will permit the introduction of necessary interventional therapies
at the earliest time possible.

COMPLICATIONS DUE TO CONGENITAL INFECTION:

MEDICAL COMPLICATIONS:
- Calcifications in the brain associated with brain damage may be seen with CMV
infections. The brain grows poorly and the head subsequently appears small
(microcephaly).
- Hydrocephalus and groin hernias may also occur. Diabetes mellitus and heart
problems can be seen with congenital Rubella infections. Recurrent eye and skin
infections are typical for Herpes.

DEVELOPMENTAL COMPLICATIONS:
▪ Infants with congenital infections may suffer particular damage to the developing brain
and sensory organs.
▪ Subsequent effects of the infection are quite diverse, resulting in a broad range of
developmental outcomes.
▪ Hearing loss is the most common developmental disability, especially from CMV and
Rubella infections. It may be present at birth or develop later in childhood and be
progressive. Hearing loss may be difficult to detect in infancy.
▪ Visual impairments are common, especially with Herpes and Rubella infections. The
impairments result from the development of cataracts or from actual destruction of the
tissues of the eye.
▪ Mild to severe brain damage may occur, resulting in various degrees of mental
retardation, learning and behavioural disorders, and autism. Special education is
frequently required.
REFERENCES
Ananthanarayan and Paniker’s Textbook of Microbiology
▪ Tenth Edition
▪ Eleventh Edition

Essentials of Medical Microbiology, Apurba Sastry

▪ First Edition
▪ Second Edition
▪ Third Edition

Review of Microbiology and Immunology, Apurba Sastry, Sixth Edition

Sherris Medical Microbiology, Sixth Edition

Baveja Textbook of Microbiology, Fifth Edition

Egg innovations and strategies for improvements by Kateri Bertran, Patti J.

Miller, published in 2017
 MEDICAL MYCOLOGY

SET 1
Essay: Mycotic mycetoma [1]
SN: Mycetoma / Madura mycosis [7]
SN: Mycotic mycetoma [2]

SET 2
SN: Dermatophytes [13]

SET 3
SN: Tinea versicolor [1]
SN: Chlamydospores [1]

SET 4
SN: Sporotrichosis / Sporothrix schenckii [4]

SET 5
SN: Chromomycosis [1]
SN: Chromoblastomycosis [1]

SET 6
SN: Rhinosporidiosis / Rhinosporidium seeberi [2]

SET 7
SN: Histoplasma capsulatum / Histoplasmosis [3]
SN: Coccidioidomycosis [1]

SET 8
SN: Cryptococcus neoformans / Cryptococcosis [8]

SN: Lab diagnosis of Cryptococcal meningitis [1]

SET 9
SN: Aspergillosis [1]

SN: Aspergilloma [1]

SET 10
SN: Mycotic keratitis [1]
SN: Mycotoxins / Mycotoxicosis [5]
SN: Otomycosis [1]

SET 11
SN: Corn meal agar [1]
SN: Germ tube test [1]
SN: Sabouraud’s medium [1]

SET 12
SN: Candida
 SET 1

MYCETOMA (MADUROMYCOSIS / MADURA FOOT)

● Chronic, slowly progressive granulomatous infection of the skin and subcutaneous tissues.
● Clinical triad:
▪ Swelling
▪ Discharging sinuses
▪ Presence of granules in the discharge.

TYPES OF MYCETOMA AND CAUSATIVE AGENTS:

● Caused by
▪ Fungal agents (Eumycetoma)
▪ Bacterial agents (Actinomycetoma)
● Third category - Botryomycosis refers to a mycetoma – like condition caused by some
bacteria such as Staphylococcus aureus.
PATHOGENESIS:
● Mycetoma is produced by the introduction of microorganisms (bacteria or fungi) via
localized trauma to the skin with thorns, wood splinters, or implantation with solid objects.
● Then the disease evolves slowly; initially micro abscesses are formed by die polymorphs,
replaced later by chronic granulomatous tissue in skin and subcutaneous tissues.
● Clinically, the disease begins as small, firm nodules that can persist (mini-mycetoma) or
evolve to form extensive suppurative lesions that in some cases can reach more than 20 cm in
diameter.
● Eumycetoma tends to be more localized than actinomycetoma.
● Human-to-human or animal-to-human transmission has not been described for eumycetoma,
but nosocomial transmission of Nocardia farcinica, one of the agents of actinomycetoma in
postoperative surgical site infections, has been reported.

CLINICAL MANIFESTATIONS:
● Hallmark of mycetoma is presence of clinical triad consisting of:
▪ Tumour – like swelling, i.e., tumefaction
▪ Discharging sinuses
▪ Discharge oozing from sinuses containing granules.
● Most common site affected: Feet
● May involve underlying fasciae and bones, producing osteolytic or osteosclerotic bony
lesions. Lesions are usually painless.
LABORATORY DIAGNOSIS:
i) SPECIMEN COLLECTION:
- The lesions should be cleaned with antiseptics and the grains should be collected
on sterile gauze by pressing die sinuses from periphery or by using a loop.
ii) DIRECT EXAMINATION
● Granules are thoroughly washed in sterile saline; crushed between the slides and
examined.
● Macroscopic appearance of granules such as colour, size, shape, texture may
provide important clues to identify the etiological agent.
● If eumycetoma is suspected: Grains are subjected to KOH mount, which reveals
hyphae of 2-6 μm width along with chlamydospores at margin.
iii) CULTURE
● Granules obtained from deep biopsies are the best specimen for culture as it
contains live organisms. Both fungal (e.g., SDA) and bacteriological media (such
as Lowenstein Jensen media) should be included in the panel.
● Identification of die eumycetoma agents is usually carried out by observation of
the growth rate, colony morphology, production of conidia and their sugar
assimilation patterns.
● Agents of actinomycetoma can be identified by their growth rate, colony
morphology, urease test, acid fastness and decomposition of media containing
casein, tyrosine, xanthine.

TREATMENT:
● The management of eumycetoma is difficult, involving surgical debridement or excision
and chemotherapy.
● Chemotherapeutic agents must be given for long periods to adequately penetrate these
lesions.
● Treatment of mycetoma consists of surgical removal of the lesion followed by use of:
▪ Antifungal agents for eumycetoma (Itraconazole, Ketoconazole or Amphotericin
B for 8- 24 months) or
 ▪ Antibiotics for actinomycetoma such as Welsh regimen (Amikacin plus
Cotrimoxazole).

CONTROL:
- Properly cleaning wounds and wearing shoes are reasonable control measures.

DIFFERENTIAL DIAGNOSIS:
● Mycetoma due to actinomycetes should be differentiated from actinomycosis, which is an
endogenous suppurative infection caused by Actinomyces israelii, other species
of Actinomyces, or related bacteria, typically affecting the cervicofacial, thoracic, and
pelvic sites (the latter is usually associated with the use of intrauterine devices).
● The branching bacteria that cause actinomycosis are non–acid-fast anaerobic or
microaerophilic bacteria.
● These bacteria are smaller than 1 µm in diameter, smaller than eumycotic agents.
● Alternatively, the agents that cause actinomycetoma are always aerobic and are
sometimes weakly acid-fast.
 SET 2

DERMATOPHYTES
● Dermatophytosis (tinea / ringworm) - most common superficial mycoses affecting skin,
hair and nail; caused by a group of related fungi called dermatophytes, that are capable of
infecting keratinized tissues.
● These include:
⮚ Trichophyton species: Infect skin, hair and nail
⮚ Microsporum species: Infect skin and hair
⮚ Epidermophyton species: Infect skin and nail.
- Depending on the usual habitat, dermatophytes are classified as;

PATHOGENESIS:

Dermatophyte infection is acquired by direct contact with soil, animals or humans infected with
fungal spores.

● Skin: Dermatophytes grow in a centrifugal pattern in the stratum corneum; leading to

formation of characteristic well-demarcated annular- or ring-shaped pruritic scaly skin
lesions with central clearing and raised edges.
● Nails: They invade the nails through the lateral or superficial nail plates and then spread
throughout the nails.
● Hair shafts: Dermatophytes can invade within the hair shaft or may be found surrounding
it. Hairs become brittle and areas of alopecia may appear. A deep and persistent
suppurative folliculitis may be produced; called Majocchi granuloma.
 ● Lesions are not produced by the tissue invasion by the fungi; but in response to the host's
inflammatory reaction elicited by fungal antigens
● Males are more commonly infected than females as progesterone is inhibitory to
dermatophyte growth
● Severity depends on the infecting fungi, immune status of the host and the site of lesion

CLINICAL TYPES:

- Depending on the site of involvement, various clinical types of dermatophytic or tinea or

ringworm infections are produced. These include:
● Tinea capitis (infection of the scalp)
▪ Kerion
▪ Favus
▪ Ectothrix
▪ Endothrix
● Tinea corporis
● Tinea pedis (athlete foot)
● Tinea cruris (or jock itch)
● Tinea barbae
● Tinea faciei
● Tinea imbricata
● Tinea unguium (nail plate infection)
● Tinea manuum

DERMATOPHYTID OR ID REACTION

Occasionally, hypersensitivity to dermatophyte antigens may occur, which leads to appearance of

secondary eruption in sensitised patients because of circulation of allergenic products. However,
these lesions are distinct from the primary ringworm lesions as they occur distal to the primary
site and fungal culture often turns negative.
LABORATORY DIAGNOSIS:
i) SPECIMEN COLLECTION:

Skin scrapings, hair plucks (broken or scaly ones) and nail clippings are obtained
from the active margin of the lesions and are kept in folded black paper. Hairs
should be plucked, but not cut.

ii) DIRECT EXAMINATION:

Specimen is mounted in KOH (10%for skin scrapings or hair, 20-40% for nail
clippings) or calcofluor white stain and is examined for the presence of thin
septate hyaline hyphae with arthroconidia. When hair is involved, the
arthroconidia may be found on the surface of the hair shaft (ectothrix) or within
the shaft (endothrix)

iii) CULTURE:
- Specimens should be inoculated onto SDA containing cycloheximide and
incubated at 26-28°C for 4 weeks. Potato dextrose agar is used to stimulate the
sporulation.
- Identification is made by:
● Macroscopic appearance of the colonies such as-rate of growth, texture,
pigmentations, colony topography
● Microscopic appearance: The colonies are teased and LPCB mount is
made to demonstrate the hyphae and spores (or conidia):
▪ Conidia: Two types of spores or conidia are observed such
as small unicellular microconidia, and large septate
macroconidia; both are used for identification of species.
 ▪ Special hyphae: Dermatophytes possess thin septate hyaline
hyphae; some species have specialized hyphae such as
spiral hyphae, racquet hyphae and favic chandeliers.

iv) WOOD'S LAMP EXAMINATION:

Certain dermatophytes fluoresce when the infected lesions are viewed

under Wood's lamp. It is usually positive for various Microsporum species
and Trichophyton schoenleinii.

v) HAIR PERFORATION TEST:

- It is positive for Trichophyton mentagrophytes and Microsporum canis.
vi) UREASE TEST:
- Trichophyton mentagrophytes is urease positive
vii) DERMATOPHYTE TEST MEDIUM & DERMATOPHYTE
IDENTIFICATION MEDIUM:
- They are used for presumptive identification. These tests are based on color
change in the medium due to production of alkali metabolites
viii) MOLECULAR METHODS:
- PCR can be used to detect species specific genes (e.g., chitin synthase gene)
 ix) SKIN TEST:
- It is done for detecting hypersensitivity to dermatophyte antigen (trichophyton).

TREATMENT:

● Oral terbinafine or itraconazole are the drugs of choice for treatment of

dermatophytosis.
● Alternate: Oral griseofulvin and ketoconazole may be given
● Topical lotion such as Whitfield ointment or tolnaftate can be applied.
 Set 3

TINEA VERSICOLOR (PITYRIASIS VERSICOLOR)

● Chronic recurrent condition affecting the superficial layer (Stratum corneum) of skin by
lipophilic fungus Malassezia furfur.

CLINICAL MANIFESTATIONS:

● Flat round scaly patches of hypo to hyperpigmentation of skin

● Seborrheic dermatitis - dandruff (Erythematous pruritic scaly lesions) in adults
● Cradle cap in babies
● Folliculitis (hair follicle infection)
● Atopic dermatitis
● Disseminated infection occurs rarely.

FEATURES OF THE LESION:

● Lesions: Non - Inflammatory & non-pruritic

● Disease is more common in areas rich in sebaceous glands - neck, chest, upper arms.
● Lesions can be mistaken for vitiligo, but later is not scaly.

LABORATORY DIAGNOSIS:

i) Direct microscopy:
- Spaghetti & meatballs appearance -Budding yeasts & short septate hyphae are
seen by performing skin scrapings after treating with 10% KOH.
ii) Wood’s Lamp examination: Golden yellow fluorescence.
iii) Urease test: Positive.
 iv) Culture: Fried egg colonies in Sabouraud's Dextrose Agar (SDA) medium & after
incubating for 5-7 days.

TREATMENT:

Topical lotions:

● Ketoconazole shampoo / cream

● Selenium sulfide shampoo for two weeks
● Terbinafine cream

CHLAMYDOSPORE

● Thick-walled asexual vegetative resting spores developed by rounding up and

thickening of hyphal segments.
● Has 2 layers:
▪ outer thick layer - exospore
▪ inner delicate & smooth layer - endosperm
● After dispersal, chlamydospores fall on soil & lead a saprophytic life.
● On germination basidiospores are capable of infecting the organism.
● Candida albicans produce thick-walled chlamydospores on Cornmeal agar culture at 20
degree Celsius (Dalmau plate culture).
 Set 4

SPOROTRICHOSIS

● Caused by a dimorphic fungus Sporothrix schenckii.

PATHOGENESIS:

● Inoculated by minor trauma or thorn pick

● Its proteinases help local invasions and spreads through lymphatics.

CLINICAL MANIFESTATIONS:

● Incubation period: Three weeks

● Seen as chronic subcutaneous pyogranulomatous disease
● Most common type:
▪ Lymphocutaneous type
- Lymph nodes are enlarged, suppurative and cord like feeling on Palpation
- Sporotrichoid pattern of spread is seen. i.e., 1st the superficial cutaneous
lesion that progress along the dermal lymphatics and sub cutaneous
lymphatics
▪ Other types include osteoarticular, pulmonary type, disseminated sporotrichosis,
fixed cutaneous type

LABORATORY DIAGNOSIS:

i) DIRECT MICROSCOPY:
- Swabs from KOH mount or calcofluor staining elongated yeast cells
ii) HISTOPATHOLOGICAL EXAMINATION:
- Cigar shaped asteroid body
 - Asteroid body - central basophilic yeast cell surrounded by radiating
extension of eosinophilic mass

iii) CULTURE:
- Cultured on SDA and duplicated with blood agar incubated at
▪ 27°C flower like pattern is seen mycelial form
▪ 37°C yeast form is seen
iv) SEROLOGY:
- Latex agglutination test for serum antibodies
v) SKIN TEST:
- With sporotrichin antigen hypersensitivity reaction is seen

TREATMENT:

- Itraconazole is effective in over 90% of patients with lymphocutaneous infection;

Fluconazole and Terbinafine can also be administered.
- For disseminated infection, Amphotericin B is the DOC.
 Set 5

CHROMOMYCOSIS
● Group of clinical manifestations caused by various dematiaceous fungi. These include:
▪ Chromoblastomycosis
▪ Sporotrichosis
▪ Rhinosporidiosis
▪ Subcutaneous zygomycosis

CHROMOBLASTOMYCOSIS

● Slow growing chronic subcutaneous lesions caused by dematiaceous or phaeoid fungi

● Produce characteristic morphology called sclerotic body
● Agents include:
▪ Fonsecaea compacta
▪ Phialophora verrucose
▪ Cladosporium carrionii
▪ Rhinocladiella aquaspersa
● Features of lesions:
▪ Slow growing
▪ Polymorphic
▪ Verrucose / crusted / ulcerative / nodular / tumor - like
● Most commonly seen in:
▪ Tropical
▪ Subtropical areas
▪ Rural areas
● Sclerotic bodies:
▪ Medlar bodies / Muriform cells / golden brown septate “copper pennies”
 Set 6

RHINOSPORIDIOSIS / RHINOSPORIDIUM SEEBERI:

● Chronic granulomatous disease
● Characterized by large friable polyps in the nose (most common site), conjunctiva and
occasionally in ears, larynx, bronchus and genitalia.

DISTRIBUTION:
- Common in tropical countries, especially in Sri Lanka and India (Tamil Nadu,
Kerala, Odisha and Andhra Pradesh)

CAUSATIVE AGENT: Rhinosporidium seeberi

● Taxonomic classification: Previously classified under fungi, now it is considered to be an
aquatic protistan parasite.

DIAGNOSIS:
● Histopathology of the polyps demonstrates spherules (large sporangia that contain
numerous endospores).
● Stained better with mucicarmine stain.

TREATMENT:
● Radical surgery with cauterization.
● Dapsone has been found to be effective.
● Recurrence is common.
Rhinosporidium seeberi spherules containing sporangia filled with endospores (H and E stain).
 Set 7

HISTOPLASMOSIS / HISTOPLASMA CAPSULATUM

(DARLING’S DISEASE)

● Systemic granulomatous disease caused by dimorphic fungi (non capsulated)

VARIANTS:

▪ H. capsulatum var. capsulatum - causes classical histoplasmosis (most common)

▪ H. capsulatum var. duboisii - causes African histoplasmosis
▪ H. capsulatum var. farciminosum- causes epizootic histoplasmosis

PATHOGENESIS:

Transmission:

- By inhalation of spores (microconidia) usually circulate in air after the

contaminated soil is disturbed

● Infected people show strong CMI response in 2 weeks, granulomas formed later healed
with fibrosis and calcification
● Disseminated infections are seen in patients with impaired CMI response
CLINICAL MANIFESTATIONS:

● PULMONARY HISTOPLASMOSIS – Most common

● ACUTE FORM - Mild flu - like illness, pulmonary infiltrates in chest x-ray with hilar or
mediastinal lymphadenopathy
● CHRONIC FORM - Seen in smokers with underlying structural lung disease
● MUCOCUTANEOUS HISTOPLASMOSIS:
- Skin and oral mucosal lesions are developed (oral lesions are particularly seen in
India)

● DISSEMINATED HISTOPLASMOSIS:
- Develops in people if CMI is very low (eg: HIV)
- Common sites affected are spleen, bone marrow, liver, eyes

LABORATORY DIAGNOSIS:

i) DIRECT MICROSCOPY:

- Histopathological staining of specimens (sputum, blood) reveals tiny oval yeast

cells with narrow based budding within the macrophages.
 ii) SEROLOGY:
- Antibody in serum can be detected by CFT and immunodiffusion test

NOTE: Antibody appears after one month of infection so, more useful in chronic stage (often
negative in in early course and in disseminated stage)

- False positive may occur due to past infection or cross infection with Blastomyces
iii) SKIN TEST:
- Delayed type hypersensitivity response
iv) MOLECULAR TEST:
- PCR targeting specific ITS D1/D2 gene

TREATMENT:

● Liposomal amphotericin B – Acute and disseminated

● Itraconazole - Chronic
 COCCIDIOIDOMYCOSIS (VALLEY FEVER / DESERT
RHEUMATISM)
● Systemic fungal disease caused by a dimorphic fungus called Coccidioides

CAUSATIVE AGENTS:

▪ Coccidioides immitis
▪ Coccidioides posadasii

PATHOGENESIS:

TRANSMISSION: Inhalation of Arthroconidia

CLINICAL MANIFESTATIONS:

● 60% of patients are asymptomatic

● In remaining patients,
- PULMONARY COCCIDIOIDOMYCOSIS is most common form present as
pneumonia, cavities, pleural effusions or nodule formations
- Secondary to pulmonary coccidioidomycosis- skin lesions (rashes/erythema
nodosum/ arthritis)
 - DISSEMINATED FORM- males and persons with low CMI (HIV) are at high
risk, sites - joints, meninges, skin, bone.

LABORATORY DIAGNOSIS:

i) DIRECT MICROSCOPY:
- Histopathological staining of specimens (sputum, or tissue biopsy) reveals
spherules

ii) SEROLOGY:
- Antibodies are detected by immunodiffusion test and CFT
iii) SKIN TEST:
- Done by fungal extracts (coccidioidin or spherulin), delayed hypersensitivity
reaction indicates past infection.

TREATMENT:

● Itraconazole
● Amphotericin B for diffuse pneumonia with pulmonary sequelae.
 SET 8

CRYPTOCOCCUS NEOFORMANS / CRYPTOCOCCOSIS

● Cryptococcus neoformans – yeast (basidiomycete)
● Sexual forms / Teleomorphs are Filobasidiella
● Causes fatal diseases like
▪ Cutaneous Cryptococcosis
▪ Pulmonary Cryptococcosis
▪ Cryptococcal meningitis

SPECIES AND SEROTYPES:

PATHOGENESIS:

● Candida neoformans causes infections mostly in immunocompromised individuals like

AIDS patients.
● Candida gattii causes infections in immunocompetent individuals.
● Infection is acquired by
▪ Inhalation
▪ Skin / mucosa
● Cutaneous Cryptococcosis varies from small ulcers to large granulomata
 ● In immunocompromised individuals, lungs are not efficient in exhibiting defense
mechanism, so pulmonary infection arises
● Cryptococcus either directly migrates across blood-brain barrier or carried inside
macrophages as Trojan horse
● Cryptococcal meningitis is potentially fatal meningitis often seen associated with AIDS
patients

VIRULENCE FACTORS:

● Polysaccharide capsule- antiphagocytic and inhibits host immune response

● Melanin producing ability by phenyl oxidase enzyme
● Production of phospholipase and Urease

RISK FACTORS:

● AIDS patients with T-cell counts <200/μL

● Patients with hematologic malignancies
● Transplant recipients
● Patients on immunosuppressive or steroid therapy

CLINICAL MANIFESTATIONS:

● Pulmonary Cryptococcosis - First and common manifestation

● Cryptococcal meningitis:
▪ chronic meningitis
▪ Headache
▪ Fever
▪ Sensory and memory loss
▪ Cranial nerve paresis
▪ Loss of vision
● Cutaneous Cryptococcosis-Skin lesions
● Bones, joints may be involved
TREATMENT:

● Cryptococcosis without CNS involvement-Fluconazole

● AIDS patients with CNS involvement-Amphotericin B ±flucytosine for 2 weeks followed
by oral fluconazole therapy until T cell count raises > 200/μL for 6 months

LAB DIAGNOSIS OF CRYPTOCOCCAL MENINGITIS

i) SPECIMEN COLLECTION:
▪ CSF
ii) DIRECT COLLECTION METHODS:
▪ Negative staining: Modified India ink stain and Nigrosin stain is used
▪ Capsule appears thick, refractile delineated clear space surrounding round
budding yeast cells against black background

▪ Gram staining: Gram-positive round budding yeast cells are seen

▪ Mucicarmine stain: Carminophilic cell wall of Candida neoformans is stained
▪ Masson-Fontana stain: Melanin production is demonstrated
▪ Alcian blue stain: Capsule is demonstrated
▪ Latex agglutination test: Capsular antigens are detected
▪ Rapid, sensitive, specific method
iii) CULTURE:
▪ Medium - Sabouraud’s dextrose agar
▪ Conditions-37oC temperature
 ▪ Time-2-3 weeks
▪ Identification - Mucoid, creamy white colored colonies

iv) CONFIRMATION:
▪ Niger seed agar and Bird seed agar - Melanin production is demonstrated
▪ Urease test positive
▪ Assimilation of inositol and nitrate
▪ Growth at 37oC
▪ Mouse pathogenicity test
▪ MALDI-TOF
 SET 9

ASPERGILLOSIS
● Aspergillosis – invasive & allergic disease

CAUSATIVE AGENT: Aspergillus

● Hyaline mould
● Important species of aspergillus are:
▪ Aspergillus fumigatus
▪ Aspergillus flavus
▪ Aspergillus niger

PATHOGENESIS:

● Aspergillus species are widely distributed in nature

● Most commonly growing on decaying plants, producing chains of conidia.
● Transmission occurs by inhalation of airborne conidia.

RISK FACTORS:
● Glucocorticoid use (the most important risk factor)
● Profound neutropenia
● Neutrophil dysfunction
● Underlying pneumonia, COPD, TB.
● Anti-tumor necrosis factor therapy.

CLINICAL MANIFESTATIONS:

Incubation period: 2 to 90 days (varies depending on the site of involvement).

PULMONARY ASPERGILLOSIS :

- Most common form.

Manifestations :
 ▪ Allergic bronchopulmonary aspergillosis (ABPA)
▪ Severe bronchial asthma
▪ Aspergilloma (fungal ball)
▪ Chronic cavitary pulmonary aspergillosis
▪ Acute angioinvasive pulmonary aspergillosis

OTHER FORMS OF ASPERGILLOSIS :

Manifestations :

▪ ● Invasive sinusitis – Acute, Chronic

● Chronic granulomatous sinusitis
● Maxillary fungal ball
● Allergic sinusitis
▪ Cardiac aspergillosis – Endocarditis , Pericarditis
▪ Cerebral aspergillosis – Brain abcess , Meningitis
▪ Ocular aspergillosis – Keratitis , Endophthalmitis
▪ Ear infecion – Otitis externa
▪ Nail bed infecion – Onychomycosis
▪ Mycotoxicosis
● Aspergillus flavus produces aflatoxin – causes liver carcinoma
 ASPERGILLOMA
● Fungal ball grows within and is usually restricted to an existing lung cavity, for example,
due to tuberculosis or bronchiectasis.
● In this type of colonizing aspergillosis, surgical removal becomes necessary because the
disease commonly causes massive hemoptysis.

LABORATORY DIAGNOSIS:

i) SPECIMEN:
▪ Sputum
▪ Tissue biopsy
ii) DIRECT EXAMINATION:
- KOH (10%) mount / Histopathological staining of specimens will relieve – a
characteristic narrow septate hyaline hyphae with acute angle branching.
iii) CULTURE:
- Specimens are inoculated onto SDA
- Incubated at 25° C
- Species identification – macroscopic and microscopic (LPCB mount) appearance
of colonies.
▪ Colonies consist of – hyaline septate hyphae from which conidiophores
arise, which end at the vesicle. [vesicle shape – either tubular/ globular]
 ▪ From the vesicle – finger-like projection of conidia producing cells arises
called phialides / sterigmata.
▪ Phialides arranged in either one / two rows, the first row is called
Metulae.
iv) ANTIGEN DETECTION:
- β -d- glucan antigen assay: - marker of invasive fungal infection
- Galactomannan antigen:
▪ Aspergillus specific antigen
▪ Can be detected by ELISA
▪ Helps in early diagnosis
v) ANTIBODY DETECTION:
- Detection of serum antibodies is very useful for chronic invasive aspergillosis &
aspergilloma, where the culture is usually negative.
- In allergic syndromes such as ABPA & severe asthma – specific serum IgE levels
are elevated.
vi) SKIN TEST:
- Positive skin test to various antigen extracts of aspergillus indicates
hypersensitivity response and is usually positive for various allergic types of
aspergillus.

TREATMENT:

TYPE DRUG OF CHOICE / INDICATIONS

For invasive aspergillosis Voriconazole
For ABPA Itraconazole
For single aspergilloma Surgery is indicated
For chronic pulmonary aspergillosis Itraconazole / voriconazole
For prophylaxis Posaconazole is indicated
 Set 10

MYCOTIC KERATITIS
● Invasive infection of cornea usually following corneal trauma.

OTHER NAMES:

▪ Oculomycosis
▪ Keratomycosis
▪ Fungal keratitis

CAUSATIVE AGENTS:

● Many saprophytic fungi cause ocular infection.

Ex:

Aspergillus niger, Aspergillus fumigatus, Aspergillus flavus

Candida albicans

Fusarium

PATHOGENESIS AND CLINICAL FEATURES:

● Predisposing factors – Corneal injury and bacterial infection.

● Widespread use of corticosteroids in ophthalmology has resulted in increased incidence
of keratomycosis.
● Fungal spores colonise the injured tissue and initiate an inflammatory reaction, leading to
hypopyon ulcer and endophthalmitis.

LABORATORY DIAGNOSIS:

i) Specimen:
- Corneal scrapings collected from slit lamp examination are used
for microscopy and culture.
TREATMENT:

Local application of

▪ Amphotericin B
▪ Nystatin
▪ Pimaricin (Natamycin)

MYCOTOXINS / MYCOTOXICOSES
● Mycotoxins – Natural products

– Produced by fungi found in some articles of food

● Mycotoxicosis – Ingestion of food contaminated with mycotoxins

AFLATOXIN:

● Best known mycotoxin.

● Aspergillus flavus – Aflatoxin B1.
● Aspergillus parasiticus – Aflatoxin B1 B2 G1 G2.
● Frequently present in mouldy foods, particularly in groundnuts, corn, peas.
● Highly toxic to animals, birds, and human beings.
● Known to cause hematoma in ducklings, rats and their possible carcinogenic effect in
human beings – caused a great concern.
 OTOMYCOSIS
● Fungal infection of external ear.

CAUSATIVE AGENTS:

▪ Aspergillus niger, Aspergillus fumigatus

▪ Penicillium

SYMPTOMS:

▪ Itching
▪ Pain
▪ Deafness
▪ Secondary bacterial infections (due to Pseudomonas and Proteus) cause suppuration.

LABORATORY DIAGNOSIS:

Demonstration of fungi in

i) SCRAPINGS:
- Potassium hydroxide (KOH) preparation is used.
ii) CULTURE: Done on Sabouraud Dextrose Agar (SDA)
iii) MICROSCOPY
- Performed from fungal colony (in teased mounts – prepared in
Lactophenol Cotton Blue) to study the morphology of hyphae,
spores and other structures
 Set 11
CORN MEAL AGAR

● Nutritionally deficient media developed by Hazen and Reed.

● Composed of cornmeal infusion, distilled water and agar.
● Used for stimulation of chlamydospore production and maintenance of fungal
culture.
● Candida albicans produces round thick walled chlamydospore.
● Addition of Tween 80 results in rapid and abundant chlamydospore formation.
● Sensitive to light and temperature, hence stored at 2 – 80C away from direct light.

GERM TUBE TEST

● Culture identification test and specific test for Candida albicans.

● Also known as REYNOLDS BRAUDE PHENOMENON.
● Candida albicans when grown in human or sheep serum at 370C for 2 – 3 hours
form Germ tubes.
● Germ tubes are long tube-like projections from yeast cells. ½ width and 3-4 times
length of yeast cell.
● Positive test: A short tube-like extension with no constriction at the point of
origin.
Ex: Candida albicans, Candida dubliniensis
● Negative test: No constriction or extension seen.

SABOURAUD’S DEXTROSE AGAR

● Commonly used culture media for isolation, cultivation, maintenance of

pathogenic and non-pathogenic species of fungi.
● Also used as selective media for isolation of dermatophytes.
● pH is acidic around 5 – 5.6.
● Composed of:
 ▪ Peptones (Act as nitrogen and vitamin source)
▪ Dextrose (Act as energy and carbon source)
▪ Agar
▪ Distilled water

● Antibiotics such as Gentamicin, Tetracycline, Chloramphenicol can be added as

selective agents to inhibit the growth of bacteria and facilitate growth of fungi.
● Morphology:

FUNGI COLONY MORPHOLOGY

Aspergillus flavus Yellow green powdery and pale yellow

on reverse

Aspergillus niger Initial white later black which gives

rise to salt and pepper appearance

Aspergillus fumigatus Blue green powdery and pale yellow on

reverse

Candida albicans Slightly dome smooth white colonies

 Set 12

CANDIDA
● Yeast – like fungus
● Produce pseudohyphae
● Candidiasis – most common fungal disease in human
Mycoses produced by Candida:
▪ Superficial (on skin, mucous membrane etc.)
▪ Systemic
▪ Opportunistic (in opportunities such as low immunity etc.)

SPECIES:
▪ Candida albicans- most common and most pathogenic Candida
▪ Candida glabrata
▪ Candida dubliniensis
▪ Candida tropicalis
▪ Candida krusei
▪ Candida kefyr
▪ Candida viswanathii
▪ Candida parapsilosis
▪ Candida guilliermondii

PATHOGENESIS:
1. Predisposing factors
▪ Age- infancy, old age
▪ Pregnancy
▪ Low immunity (like in people with HIV etc.)
▪ Broad spectrum antibiotics
▪ Febrile neutropenia
▪ Zn or Fe deficiency
 ▪ Diabetes mellitus
2. Virulence factors
● Adhesins: Helps to stick to skin and mucosa
● Enzymes: Tissue invasion by aspartyl proteases and serine proteinases
● Toxins: Glycoproteins in cell wall which are pyrogenic in nature
● Pseudohyphae: Phospholipase released from hyphal tip may help in invasion
● Phenotypic dimorphism / switching (In Candida albicans)
▪ transform between 3 phenotypic forms Yeast (blastospores), pseudohyphae, true
hyphae
▪ helps to adapt changing environment & to escape host immunity

CLINICAL MANIFESTATIONS:
MUCOSAL CANDIDIASIS
1. Oral thrush (Oropharyngeal Candidiasis)
White adherent painless patches in mouth
2. Candidal vulvovaginitis
Pruritus, pain & vaginal discharge (Whitish curd like in severe cases)
3. Balanitis & Balanoposthitis
In uncircumcised males
4. Esophageal candidiasis
5. Angular stomatitis & Denture stomatitis
6. Chronic mucocutaneous candidiasis
● It is seen in infants and children with deficient CMI (T cell defect)
 ● Lesions in hair, nail, skin & mucous membrane Which are usually resistant to
treatment.
● It is associated with other endocrine abnormalities

CUTANEOUS CANDIDIASIS
1. Intertrigo
Associated with tight fitting undergarment and sweating
Erythema, pustules in skin folds
2. Paronychia
Involve nail skin interface
3. Onychomycosis
Fungal infection of nail
4. Diaper candidiasis
Pustular rashes
5. Perianal candidiasis
6. Erosio interdigitalis blastomycetica
Infection of web spaces of hands or toes
7. Generalized disseminated cutaneous candidiasis in infants

INVASIVE CANDIDIASIS
- Result from hematogenous / local spread of fungi
1. Urinary tract infection
2. Pulmonary candidiasis
3. Septicemia - Mainly by Candida albicans & Candida glabrata
4. Arthritis & Osteomyelitis
5. Meningitis
6. Ocular- keratoconjunctivitis & endophthalmitis
7. Hepato Splenic candidiasis
8. Disseminated candidiasis
9. Nosocomial candidiasis - Mainly by Candida glabrata
ALLERGIC CANDIDIASIS
● Allergic reaction to metabolites of candida
● Vesicular lesions in web spaces of hands & other areas
● Similar to dermatophytid reaction
● Candid + dermatophytid reaction=> id reaction
OTHER ALLERGIC REACTION
● Gastritis
● Irritable bowel syndrome
● Eczema

LABORATORY DIAGNOSIS:
i) SPECIMEN COLLECTION
- Depends on site of infection
▪ Mucosal patch
▪ Skin scraping
▪ Nail scraping
▪ Sputum
▪ Urine
▪ Blood
ii) DIRECT MICROSCOPY
- Gram positive oval budding yeast cells with pseudohyphae
iii) CULTURE
- SDA (Sabouraud’s Dextrose Agar) & Blood agar
Produce Creamy white smooth pasty colony within 1-2 days
Incubated at 37° with antibiotics
Candida Glabrata alone does not show pseudohyphae
TESTS FOR SPECIES IDENTIFICATION:
1. Germ tube test [Reynolds Braude phenomenon]:
- Specific test for Candida albicans (may give positive for Candida dubliniensis)
▪ Colonies are mixed with human/sleep serum & incubated for 2
hours
▪ Wet mount preparation is examined under microscope
▪ Germ tubes are formed
2. Dalmau plate culture
- On corn meal agar
- Candida albicans produce thick walled Chlamydospores
3. CHROM agar
- Different species produce different colored colonies
4. Growth at 45°
- Candida albicans can grow
- Candida dubliniensis cannot grow
5. Sugar assimilation and sugar fermentation test
6. Molecular methods such as PCR
7. Immunodiagnosis:
▪ Antibody detection: Detect serum antibodies against cell wall mannan antigen
▪ Antigen detection: Detect cell wall mannan antigen, Cytoplasmic antigen
▪ Enzyme detection: Eg. Enolase
▪ Test for metabolites: Eg. mannitol, arabinitol
▪ G test: Detect alpha-1,3glucan

TREATMENT:
Candidiasis Drug of choice
Cutaneous candidiasis / oral thrush Topical azole

Esophageal and vulvovaginal candidiasis Oral fluconazole

Disseminated candidiasis Amphotericin B
REFERENCES:
● Essentials of Medical Microbiology – Apurba S Sastry – First Edition
● Essentials of Medical Microbiology – Apurba S Sastry – Third Edition
● Jawetz, Melnick & Adelberg’s Medical Microbiology – Twenty Eighth Edition
● Ananthanarayan and Paniker’s Textbook of Microbiology – Tenth Edition
● Ananthanarayan and Paniker’s Textbook of Microbiology – Eleventh Edition
● Baveja Textbook of Microbiology – Fifth Edition
● Review of Microbiology and Immunology – Apurba S Sastry – Sixth Edition
PARASITOLOGY
ESSAYS:
1. Important protozoan parasites of man. Details about malignant tertian malaria along
with its complications and lab diagnosis (1)

Hemoparasites, details about Plasmodium falciparum (4)

Classify Sporozoa, the life cycle of Plasmodium falciparum, differentiate it from

others, laboratory diagnosis of malignant tertian malaria (1)

Short notes:

1. Exoerythrocytic cycle.
2. Erythrocytic stage of Plasmodium vivax.
3. Plasmodium falciparum.
4. Black Water fever.
5. Gametocytes of Plasmodium Falciparum
6. Complications of Falciparum malaria.
7. The life cycle of Plasmodium vivax.
8. Exoerythrocytic schizogony.
9. Lab diagnosis of malaria.

Short notes under essay:

1. Plasmodium falciparum
2. Malignant tertian malaria complications and laboratory diagnosis

2. Parasitic zoonotic diseases, details about toxoplasmosis (1)

Short notes:

1. Congenital toxoplasmosis.
2. Toxoplasma gondii or toxoplasmosis (2)

Short notes under essay:

1. Parasitic zoonotic diseases

3. Hemoflagellates and their morphology. Details about Leishmania donovani or Kala-azar -

morphology, life cycle, clinical features, laboratory diagnosis (4)
 Short notes:

1. Laboratory diagnosis and morphology of KALA AZAR (3)

Short notes under essay:

1. Hemoflagellates and their morphology

4. List common blood & tissue flagellates causing human disease. Describe the pathogenesis,
clinical features, laboratory diagnosis & prophylaxis of Chagas disease (1)

5. Classify cestodes, details about taenia solium lab diagnosis of tapeworm infection -(3)

Short notes:

1. The life cycle of taenia solium

2. Larval form of cestodes
3. Neurocysticercosis
4. Cysticercus cellulosae (5)

6. Classify cestodes, details about Echinococcus granulosus (5)

Short notes:

1. Pathogenesis and laboratory diagnosis of hydatid diseases

2. Casoni test
3. Hydatid cyst

7. Intestinal parasites of man. Details about enterobiasis (1)

Short notes:

1. Thread Worm

8. Intestinal nematodes. Details about Ancylostoma duodenale, laboratory diagnosis of

hookworm infection, and enumerate parasites causing anemia (3)

Short notes:

1. Pernicious anemia
 2. Ancylostoma duodenale
3. The Life cycle of the hookworm
4. The life cycle of A. duodenale

9. Viviparous nematodes. Life Cycle, laboratory diagnosis of anyone, prevention, and control
of bancroftian filariasis (1)

Classify nematodes, details about filariasis (2)

Tissue or somatic nematodes. Details about Wuchereria bancrofti (3)

Short notes:

1. Microfilaria of Wuchereria Bancrofti.(9)

2. Lab diagnosis of filariasis.
3. Microfilaria.

Short notes under essay:

1. Viviparous nematodes
2. Tissue or somatic nematodes

10. Ascaris lumbricoides Life Cycle, pathogenesis, laboratory diagnosis, and classification of
nematodes (3)

Short notes:

1. Classification of nematodes according to the habitat of adult worms.

2. Life cycle Ascaris lumbricoides.

Short notes under essay:

1. Ascaris lumbricoides

11. Life Cycle of Dracunculus medinensis

Short notes under essay:

1. Dracunculus medinensis
SHORT NOTES:
1. 1) Life cycle of Entamoeba histolytica

2) Lab diagnosis Intestinal Amoebiasis

3) Extraintestinal amoebiasis and Amoebic liver abscess

4) Primary Amoebic Meningoencephalitis (6)

2. 1) Free-living amoeba (4)

3. 1) Trichomonas

2) Giardiasis

3) Trichomonas vaginalis (4)

4. 1) Morphology of pathogenic intestinal protozoans

5. 1) Pneumocystis jiroveci.

6. 1) Balantidium coli

7. 1) Redia and cercaria

8. 1) Fasciola hepatica.

9. 1) Lung fluke (2)

10. 1) Dwarf tapeworm

2) Larval forms of Diphyllobothrium latum

11. 1) Trichinella spiralis (2)

12. 1) Pathogenesis of Strongyloides stercoralis

13. 1) Onchocerca volvulus

14. 1) Role of cyclops in parasitic disease.

2) Cyclops (2)

15. 1) Visceral larva migrans

2) Cutaneous larva migrans

3) Larva migrans (5)

16. 1) Loa loa

17. 1) Cryptosporidium parvum

18. 1) Examination of feces for parasitic infections

2) Stool examination of parasitic infections

19.1) Viviparous parasites

20. 1) Bile stained eggs (2)

ESSAYS
 1.PROTOZOAN PARASITES OF MAN

● The parasite is a living organism which lives in or upon another organism ( host ) and
derives the nutrients directly from it, without giving any benefit to the host.
● Parasites are classified into endoparasites and ectoparasites.
● Endoparasites are further classified into Protozoa and Helminths.
● Protozoa are unicellular eukaryotic cells that perform all physiological functions.
● Helminths are elongated flat or round worm-like parasites measuring few millimeters
to as long as few meters.

Hemoparasites, as the name suggests, is an animal parasite living in the blood of a

vertebrate. And examples of hemiparasites include Babesia sp., Plasmodium sp.,
Trypanosomas, and Filarial nematodes.

IMPORTANT PROTOZOANS OF MAN:

The various important Protozoan parasites are classified into 3 categories.

Amoeba :

⮚ Entamoeba Histolytica
⮚ Free-living amoeba: Naegleria, Acanthamoeba, Balamuthia

Flagellates :

⮚ Intestinal flagellates : Giardia

⮚ Genital flagellates: Trichomonas
⮚ Hemoflagellates: Leishmania and Trypanosoma

Apicomplexa :

⮚ Malaria parasites and Babesia

⮚ Opportunistic Coccidian parasites: Toxoplasma, Cryptosporidium, Cyclospora,
Cytoisospora, and Sarcocystis

Miscellaneous Protozoa :

⮚ Balantidum Coli
⮚ Blastocystis
⮚ Microsporidia

⮚ Sporozoans are organisms characterized by being one-celled, non-motile, parasitic,

and spore-forming.
⮚ Most of them have an alteration of sexual and asexual stages in their life cycle.
⮚ Examples include Plasmodium, Piroplasms, Monocystis.
MALARIA:
Malaria is one of the oldest documented diseases of mankind.

The agent of Malaria is a protozoan parasite, the Plasmodium.

AGENT:

Although several species of plasmodium exist, only 5 of them are infective to humans

- P.Vivax, causing Benign Tertian malaria

- P.Falciparum, causing Malignant tertian malaria
- P.Malariae, causing benign quartan malaria
- P.Ovale, causing Ovale tertian malaria
- P.Knowlesi causes Quotidian/Simian malaria

LIFE CYCLE:

Plasmodium completes its life cycle in two hosts:

⮚ Definitive host: Female Anopheles Mosquito

⮚ Intermediate host: Man
HUMAN CYCLE:
The infective form of plasmodium is Sporozoites.

In humans, only the asexual cycle takes place, and It takes place in 3 stages

- Pre-erythrocytic Schizogony
- Erythrocytic Schizogony
- Gametogony

PRE-ERYTHROCYTIC SCHIZOGONY / HEPATIC STAGE /

EXOERYTHROCYTIC STAGE / TISSUE STAGE :

⮚ Occurs in hepatocytes
⮚ Occurs before the invasion of RBCs
⮚ Sporozoites enter into the Hepatocyte to form Trophozoite.
⮚ Trophozoites further develop to form Pre-erythrocytic Schizont
⮚ Pre-erythrocytic Schizont contains several merozoites, released on the rupture of
hepatocyte
⮚ Merozoites then attack RBCs to initiate the Erythrocytic stage
⮚ 1 sporozoite can give rise to 5000-30000 merozoites.
⮚ Duration: 5 – 15 days depending on the series.

ERYTHROCYTIC SCHIZOGONY
⮚ The hepatic merozoites after being released from Pre-erythrocytic schizont, attack
RBCs
⮚ Attachment is mediated by a specific surface Receptor – glycophorin receptor on the
RBC surface, enter by endocytosis, and are contained within a parasitophorous
vacuole inside the RBCs.
⮚ Hepatic merozoites transform into Trophozoites
⮚ Early trophozoites are called ring forms
⮚ Late trophozoites are irregular in shape
⮚ PLasmodiumfeeds on Hemoglobin, hence Malarian pigment Haemozoin is seen in the
late trophozoite stage
⮚ Late Trophozoite undergoes Schizogony to form 6-30 daughter merozoites to become
the Erythrocytic Schizont
⮚ Then the RBC ruptures, releasing the daughter merozoites.
⮚ Erythrocytic Schizogony in the case of Plasmodium Falciparum occurs only in the
visceral capillaries, hence only ring forms can be seen in the peripheral smear.
INCUBATION PERIOD :
⮚ One liver cycle + 2-3 cycles of RBC development

GAMETOGONY:

⮚ Gametocytes are the infective form of mosquitos

⮚ After a series of Erythrocytic cycles, some merozoites after entering into RBCs,
instead of developing into Trophozoites, they transform into sexual forms called
Gametocytes
⮚ Trophozoite gives rise to Macrogametocyte ( Female ) and Microgametocyte ( Male ).
⮚ At least 12 gametocytes per microlitre of blood must be present to transmit the
infection

SPOROGONY :

⮚ Mosquito Cycle
⮚ Gametocytes -🡪 Gametes -🡪 Zygote 🡪 Motile Ookinte 🡪 Oocyst 🡪 Sporozoites
 ⮚ Each Oocyst undergoes Sporogony ( Meiosis ) to produce four Spindle-shaped
Sporozoites.
⮚ Duration for Sporogony / Extrinsic incubation period of plasmodium is 10-14 days.

SECONDARY EXOERYTHROCYTIC SCHIZOGONY :

⮚ Reactivation of Hypnozoites ( latent forms in the hepatocytes )

⮚ Hypnozoites are seen in: P.Vivax and P.Ovale
⮚ Responsible for Relapse of Malaria

DIFFERENCES BETWEEN THE DIFFERENT PLASMODIUM.

Type P.Falciparum P.Vivax P.Ovale P.Malariae P.Knowlesi
Duration of 48 hours 48 hours 48 hours 72 hours 24 hours
Erythrocytic
Schizogony
RBC All cell ages Reticulocytes Reticulocytes Older cells All ages
Preference
Size of RBCs RBC not Enlarged Enlarged Not enlarged Not enlarged
enlarged
Clefts Maurer Schuffner Schuffner Zeimann’s

Pigment Black Yellow Brown Dark brown Brown-black Dark brown

PATHOGENESIS AND CLINICAL FEATURES :

⮚ Headache
⮚ Malaise
⮚ Fever + Chills + Rigor
⮚ Mild anemia
⮚ Febrile Paroxysm( cold stage, Hot stage, and Sweating stage )
⮚ Benign Malaria
⮚ Malignant Malaria

Benign Malaria: Triad of Febrile Paroxysm , Anemia and Splenomegaly

Malignant Malaria :

● The pathogenesis of Plasmodium Falciparum is different from other species.

● Sequestration is the special ability of Plasmodium Falciparum and it is mediated by
Cytoadherence, Rosetting, and Agglutination.
● Cytoadherence is mediated by Plasmodium Falciparum Erythrocyte membrane
Protein-1 ( PfEMP-1 ), which binds to specific receptors of vascular endothelium of
deep organs.
● PfEMP-1 also causes binding of non-infected RBC with each other and with infected
RBCs leading to Rosetting.
● Plasmodium Falciparum infection is more acute and severe with more complications
than Benign Malaria.

COMPLICATIONS OF MALIGNANT TERTIAN MALARIA :

● Cerebral Malaria – leading to vascular occlusion and cerebral anoxia

● Pernicious Anemia – Blackwater fever and Algid malaria and septicemic malaria
● Blackwater fever: Characterised by sudden intravascular hemolysis followed by
Fever, Hemoglobinuria, and Dark urine. It occurs following quinine treatment to
subjects previously infected with Plasmodium Falciparum.
● Pulmonary edema an Adult Respiratory Distress syndrome
● Renal failure
● Severe jaundice
● Acidosis

DIAGNOSIS OF MALARIA:
● MICROSCOPIC TESTS :
⮚ Peripheral blood smear – the gold standard

Thick smear 🡪 More sensitive

Thin smear 🡪 Speciation can be done using the particular features

⮚ FLUORESCENCE MICROSCOPY
⮚ Quantitative Buffy Coat examination: Parasitised RBCs appear as Brilliant Green
Dots
● NON-MICROSCOPIC TESTS
⮚ Antigen detection tests: Detect Pan malarial Ag ( LDH, Aldolase ) or Falciparum
specific ( HRP-II)
⮚ Culture: RPMI 1640 medium
⮚ Molecular diagnosis: PCR
 2. PARASITIC ZOONOTIC DISEASES
● Toxoplasma gondii is an obligate intracellular parasite affecting a wide range of
mammals and birds including humans.
● Though human infection is very common affecting nearly one­third of the world’s
population; clinical manifestations are relatively rare, mostly restricting to
opportunistic infections in immune-compromised persons and congenital infection in
fetuses.

MORPHOLOGY: It exists in three morphological forms—two asexual forms (tachyzoite

and tissue cyst) and asexual form (oocyst).

TACHYZOITE :
● It is an actively multiplying form (trophozoite), usually seen in acute infection.
● Crescent-shaped, having a pointed anterior end and a blunt posterior end
● It measures approximately 6 µm in length and 2 µm in breadth; contains several
dense granules and a round nucleus situated between the center and posterior end
● They can infect all mammalian (nucleated) cells except red blood cells (RBCs)
● At the anterior end, the tachyzoites contain special organelles like rhoptries and
micronemes which are crucial for the adhesion and invasion into the host cell
● Inside the host cell, tachyzoites are surrounded by a parasitophorous vacuole within
which they divide asexually by a process called internal budding or endodyogeny by
which daughter trophozoites are formed within the parent cell.
● They often form rosettes surrounding the host nucleus
● The host cell becomes distended by the proliferating tachyzoites and appears as a
pseudocyst.
● Later on, the host cell ruptures releasing the tachyzoites that infect other adjoining
cells. Tissue cyst It is the resting stage of the parasite, usually seen in chronic
infections.
● The parasite multiplies within the host cells and produces round to oval cyst
containing many crescent-shaped slowly multiplying trophozoites called bradyzoites,
surrounded by a cyst wall
● Tissue cysts vary in size. Younger ones that measure 2–5 µm in size and contain few
bradyzoites
● Older tissue cysts may reach more than100 µm size and contain several thousand
bradyzoites

BRADYZOITES:
● Measure 7 µm in length and 1.5 µm in breadth
● More slender, crescent-shaped with a nucleus situated posteriorly
● Contains several strongly periodic acid Schiff stain (PAS) positive amylopectin
granules
● Multiply slowly
 ● Seen in chronic infection
● More resistant to gastric juice
● The cyst wall of the tissue cyst is eosinophilic and weakly PAS-positive
● Conversion of the tachyzoites to bradyzoites can be triggered by many factors
like interferon­g (IFN­g), nitric oxide (NO), heat shock proteins, pH, and
temperature changes
● Most common site of the tissue cysts­muscles and brain (can be found in any
organ)
● They appear spherical in the brain and oval inside the muscle tissue

LIFE CYCLE
● Host: The life cycle involves two hosts:
● 1. Definitive hosts are cats and other felines; where the sexual cycle takes place
● 2. Intermediate hosts are a man and other mammals (goat, sheep, pig, cattle, and
certain birds); where the asexual cycle takes place.

PATHOGENICITY AND CLINICAL FEATURES :

❖ Toxoplasmosis is one of the most common parasitic zoonotic infections affecting
a wide range of mammals and birds.
❖ Its prevalence in humans varies from 5–75% and depends on various risk factors
like The geographical area (cold area, hot arid climatic conditions, high altitudes
are associated with a low prevalence)
❖ Age: It commonly affects older age and fetus
 ❖ Exposure to cat and cat’s feces
❖ Food habits: Ingestion of uncooked cat and other animal meat (seen in countries
like France)—at higher risk z Immune status
❖ Patients associated with HIV, malignancies, and other immunocompromised
conditions are at high risk
❖ Patients undergoing blood transfusion, and organ transplantations are at higher
risk.

CONGENITAL TOXOPLASMOSIS:
● Mother acquiring Toxoplasma infection in pregnancy is usually asymptomatic.
● However, she can transmit the infection to the fetus.
● Gestational age is the main factor influencing the fetal outcome. As the gestation
proceeds, the chance of transmission increases but the severity of the infection
declines
● If the mother becomes infected during the first trimester, the incidence of
transplacental infection is lowest (15%), but the disease in the neonate is most severe
● If maternal infection occurs during the third trimester, the incidence of transplacental
infection is maximum (65%), but the infant is usually asymptomatic at birth
● If the mother is infected before pregnancy, then the fetus is mostly uninfected except
when the mother is immunocompromised
● Initially though asymptomatic, but the persistence of infection in the newborn child
can result in severe disease
● Ocular involvement: Most frequently it causes chorioretinitis leading to profound
visual impairment. Other ocular manifestations include blurred vision, scotoma,
photophobia, strabismus, and glaucoma.

LABORATORY DIAGNOSIS:
● Direct microscopy: Detect tachyzoites in blood and tissue cyst in tissue biopsy
❖ Giemsa, PAS, silver stains, immunoperoxidase stain
❖ Direct fluorescent antibody test
● Antibody detection
❖ Sabin Feldman dye test
❖ Detection of IgG in serum—ELISA, IFA
❖ IgG avidity test
❖ Detection of IgM in serum—ELISA, IFA, and IgM-ISAGA
❖ Differential absorption test
❖ Detection of IgA—double sandwich ELISA
● Detection of Toxoplasma antigen—ELISA
● Molecular diagnosis—PCR
 ● Animal inoculation—intraperitoneal inoculation into mice
● Tissue culture—murine alveolar and peripheral macrophage cell line.
● Imaging methods—CT and MRI to detect TE

TREATMENT:
❖ Immunocompetent patients: Immunocompetent patients with the only
lymphadenopathy do not require specific therapy unless they have persistent, severe
symptoms.
❖ Patients with ocular toxoplasmosis are usually treated for 1 month with
pyrimethamine plus either sulfadiazine or clindamycin and sometimes with
prednisolone.
❖ Congenital toxoplasmosis: Neonates with congenital toxoplasmosis are treated with
daily oral pyrimethamine (1 mg/ kg) and sulfadiazine (100 mg/kg) with folinic acid
for 1 year.
❖ Immunocompromised patients: Toxoplasmosis is rapidly fatal in
immunocompromised patients. If not treated, it may progress to encephalitis. So
treatment is essential.
❖ (i) Primary prophylaxis Acquired immunodeficiency syndrome (AIDS) patients with
Toxoplasma infection, having CD4+ T lymphocyte count of less than 100/µL should
receive prophylaxis against TE.
❖ Trimethoprim-sulfamethoxazole (cotrimoxazole) is the drug of choice z
Dapsone-pyrimethamine, atovaquone with or without pyrimethamine can be given an
alternate z
❖ Prophylaxis can be discontinued in patients who have responded to antiretroviral
therapy (ART) and whose CD4+ T lymphocyte count has been more than 200/µL for
3 months.
❖ Secondary prophylaxis (Long-term maintenance therapy) is Required for
HIV-positive patients who are previously treated for toxoplasmosis.
❖ Should be started if the CD4+ T lymphocyte count decreases to less than 200/µL z
However, it can be discontinued if the patient is asymptomatic, and have a CD4+ T
lymphocyte count of more than 200/µL for at least 6 months.
 3.HEMOFLAGALLETE
● flagellated protozoa that are found in peripheral blood circulation
Examples - Leishmania and Trypanosoma - both are transmitted by the bite of the insect
Vector.

MORPHOLOGY

● In Leishmania - amastigote and promastigote forms are seen.

o Amastigotes - diagnostic form, found in man
o Promastigotes - infective stage to man, found in the insect vector

LEISHMANIASIS
● Caused by obligatory intracellular protozoa of the genus Leishmania - primarily affects
the reticuloendothelial system of the host.
● Vector-bite of the female sandfly vector.
● CLINICAL FORMS:
o Visceral leishmaniasis (VL)
o Post–kala-azar dermal leishmaniasis (PKDL) - occurs few months to years
following VL.
o Cutaneous forms - cutaneous leishmaniasis (CL), diffuse cutaneous
leishmaniasis (DCL), leishmaniasis recidivism (LR), and mucocutaneous
leishmaniasis (MCL).
VISCERAL LEISHMANIASIS
● Mainly caused by L. donovani and L. infantum, together known as L. donovani complex
and also, L. chagasi.
● L. donovani - most common species causing VL

LIFE CYCLE (Leishmania donovani)

PATHOGENICITY
● The phagocytosis of the promastigotes - facilitated by binding of its surface antigens -
gp-63 and LPG to specific receptors on macrophages.
● LPG - principle virulence factor - prevents phagosome maturation and protects the
parasite against hydrolytic enzymes secreted from the phagolysosome.
● GPI (glycosyl-phosphatidyl-inositol) - a major surface protein on amastigotes, helps in
protecting from phagolysosomal attack inside the macrophage.

HOST IMMUNE RESPONSE

● Depending on the host immune response (TH1 or TH2) - amastigotes are either killed or
allowed to multiply inside the macrophages.
CLINICAL FEATURES (VL)
● VL - also called kala-azar (a Hindi term meaning “black fever”).
● Incubation period - 2–6 months.
● Hallmark of VL - pentad of fever, progressive weight loss, hepatosplenomegaly,
pancytopenia, and hypergammaglobulinemia
● Fever
● Splenomegaly
● Hepatomegaly
● Lymphadenopathy
● Hyperpigmentation
● Pedal edema and ascites
● Hematological abnormalities – pancytopenia and hypergammaglobulinemia

POST-KALA-AZAR DERMAL LEISHMANIASIS (PKDL)

● Non-ulcerative lesion of skin following treatment with antimonials.
● Aggravated by exposure to sunlight.
● Develops as a hypopigmented macule near the mouth which spreads to the face, arms and
trunk and finally becomes nodules resembling leprosy.

LAB DIAGNOSIS
● DIAGNOSIS: Detection of amastigotes in the nodular lesions and by direct agglutination
test (DAT)
● OCULAR LESIONS - conjunctivitis and uveitis
● Sometimes, PKDL may directly occur in subclinical patients without a history of VL

LABORATORY DIAGNOSIS OF VL
● MICROSCOPY—Giemsa staining, detects LD bodies (i.e. macrophage filled with
amastigote forms)
o Splenic aspiration: Most sensitive
o Bone marrow aspiration: Most commonly preferred
o Lymph node aspiration
 o Liver biopsy
o Peripheral blood smear (in HIV-infected people)
o Biopsy of various organs (in HIV-infected people)
● CULTURE (detects promastigotes)—useful for species identification and drug sensitivity
testing
o NNN medium
o Schneider’s liquid medium
● ANTIBODY DETECTION in serum
o ELISA, IFA, direct agglutination test &ICT
● ANTIGEN DETECTION - carbohydrate antigen in the urine (latex agglutination test)
● Molecular method - PCR, real-time PCR detecting specific kinetoplast (mitochondrial)
DNA
● LEISHMANIA TEST (Montenegro tests) - indicates good CMI (DTH reaction);
positive in all stages, except active VL and diffuse CL
● ANIMAL INOCULATION—golden hamster
● NONSPECIFIC TESTS TO DETECT:
o Hypergammaglobulinemia - by Napier’s aldehyde test and Chopra’s antimony
test
o Pancytopenia - by complete blood count

TREATMENT
● The various drugs used in the treatment of VL are:
o (i) Pentavalent antimonials, Drug of choice
o (ii) Liposomal amphotericin B,
o (iii) Miltefosine,
o (iv) Paromomycin.
 4.BLOOD AND TISSUE FLAGELLATES

LEISHMANIA
▪ Leishmania donovani complex – Visceral leishmaniasis
▪ Leishmania braziliensis complex – Mucocutaneous leishmaniasis

TRYPANOSOMES
▪ Trypanosoma brucei – Sleeping sickness
▪ Trypanosoma cruzi – Chaga’s disease
Trypanosoma cruzi causes Chaga’s disease.

PATHOGENESIS:
● Incubation period: 1 – 2 weeks
● Depends on the intracellular multiplication of amastigotes and causes damage to
cells and tissues at various sites.
▪ Myocardium
▪ Skeletal muscles Commonly affected
▪ Neurological cells
▪ Reticulo-endothelial system

CLINICAL FEATURES:
Chaga’s disease
Acute Chronic
Common in children Common in adults
Fever Cardiac changes
Generalized edema Megaesophagus
Acute myocarditis Megacolon
Meningoencephalitis

▪ Subcutaneous inflammatory nodules develop at the site of entry of Trypanosoma

cruzi - Chagoma.
▪ Entry through the conjunctiva, the patient develops painless, unilateral edema,
conjunctivitis – Romana's sign.
 LAB DIAGNOSIS:
a) Specimens:
Blood
CSF
Tissue
b) Microscopy:
Trypomastigotes are found in peripheral blood by direct microscopy of
Giemsa stained blood films.
c) Culture: Novy, McNeal, and Nicolle (NNN) medium
d) Antigen detection: ELISA
e) PCR
f) Animal inoculation:
Blood or CSF is inoculated intraperitoneally into mice.
Trypomastigotes appear in the blood of animals in few days.
g) Xenodiagnosis:
A non-infected triatomine bug is allowed to feed in the patient, and its feces
are wheel examined 2 weeks later for the presence of the parasite.
h) Serology:
CFT Complement Fixation Test
IFA
IHA
ELISA
Card agglutination trypanosomiasis test
i) Intradermal test:
Extracts of T.cruzi culture (cruzin) are used for intradermal inoculation.
j) Biopsy of lymph nodes or tissue may reveal amastigote forms.

TREATMENT:
▪ Nifurtimox
▪ Benznidazole

PROPHYLAXIS:
▪ Control and elimination of vector and insecticides.
▪ Better housing provisions.

References: Medical Parasitology (Baveja) Fourth Edition

 5. CESTODES : TAENIA SOLIUM

● Cestodes are long, segmented, dorsoventrally flattened, tape like worms

also called tapeworms

CLASSIFICATION OF CESTODES (based on habitat):

1. INTESTINAL CESTODES:
-they are adult worms
Ex:
● Diphyllobothrium species
● Taenia solium and Taenia saginata
● Hymenolepis species
● Taenia Asiatica, Hymenolepis diminuta, Dipylidium caninum

2. SOMATIC/TISSUE CESTODES:
-larvae in human muscles or organs.
Ex:
● Taenia solium (Cysticercosis- CNS,muscle,eye)
● Echinococcus species (hydatid disease-liver)

TAENIA SOLIUM (ARMED TAPEWORM):

MORPHOLOGY:

ADULT WORM:
● Length - 2-4 meters
● Has three parts - Head(scolex),neck,strobila(body)
● Head - small, globular, four suckers present, bears rostellum with two rows of
hooklets
● Neck
 ● Strobila contains proglottids(segments)
1) Immature segments- undifferentiated male and female reproductive organs
2) Mature segment- both male and female organs in the same segment
(hermaphrodites)
3) Gravid segments- after fertilization uterus is filled with eggs

EGGS:
● Formed after fertilization and fill the gravid proglottids
● Released in feces
● Diagnostic form

LARVAE:

● Cysticercus cellulosae
● Location - pig's muscles and man's muscle, eye, and brain
● Infective form
DISEASES CAUSED BY TAENIA SOLIUM:

1. Intestinal taeniasis (intestine)

2. Cysticercosis (CNS, muscle, eye)

LIFE CYCLE:

Disease Intestinal taeniasis Cysticercosis

Host Definitive host - man

Intermediate host - pig intermediate host - man

Infective form Larva - cysticercus

Egg
cellulose

Diagnostic form Egg in feces Larva in tissue

Mode of transmission Ingestion of contaminated

Contaminated food and pork
water, auto-infection

Organ infected GIT CNS, muscle, eye

LIFE CYCLE IN INTESTINAL TAENIASIS:

LIFE CYCLE IN CYSTICERCOSIS:

1. Enters man through infection of contaminated food or water with eggs of T.solium (or)
auto-infection
2. Embryo released from eggs
3. Embryo penetrates intestine and enters the portal circulation
4. Reaches muscle, eye, brain, subcutaneous tissue ( where becomes larva in 7-9
weeks)
5. Deposited as cyst
6. 2-3 months - mature cyst
LARVAE OF CESTODES:

● Taenia - Cysticercus
● Hymenolepis - cysticercoid
● Echinococcus - hydatid cyst
● Diphyllobothrium
1) Coracidium
2) Procercoid
3) Plerocercoid

LAB DIAGNOSIS OF TAPEWORM INFECTION:

INTESTINAL TAENIASIS:

● STOOL EXAMINATION:

- Wet mount stool examination

- Multiple stool examination
- Anal swabs
- Eggs - non-acid fast
- Proglottids
- Scolex - armed with rostellum and hooks

● TAENIA SPECIFIC ANTIGEN DETECTION IN STOOL:

-ELISA to detect coproantigen in stool

● MOLECULAR METHODS:

-PCR targeting mitochondrial DNA

CYSTICERCOSIS:

● Radiodiagnosis - CT scan and MRI ( for detecting number, location, size of cysticerci,
and stage of disease)
● Antibody detection in serum or CSF
-ELISA (using crude extract of cysticerci)
-Western blot(using 13kDA LLGP Ag)
● Antigen detection in serum or CSF
● Lymphocyte transformation test
● Histopathology - to detect cysticerci
● FNAC of the cyst and then staining with Giemsa
● Fundoscopy - to detect larvae
● Modified Del Brutto diagnostic criteria

DIPYLIDIUM CANINUM INFECTION:

● Eggs in feces
- 25-40 micrometer size
- Egg packets group of 15
- Barrel-shaped proglottids with two germinal pores

DIPHYLLOBOTHRIASI

● Stool examination - eggs

● Molecular method - PCR
● Blood examination - reveals eosinophilia and megaloblastic anemia

NEUROCYSTICERCOSIS:

● Most common parasitic CNS infestation of man

● Age - 30-50 years
● Types:
 1. Parenchymal - in the brain parenchyma
2. Extraparenchymal - meninges, ventricles, spinal cord, subarachnoid space

● Clinical features:
-Seizure
-Hydrocephalus
-increased intracranial pressure
-hypertension- headache, vomiting, vertigo
-chronic meningitis
-focal neurological deficits
-psychological disorders and dementia
-cerebral arteritis
-basal and ventricular involvement

● Four morphological stages:

1)vesicular form
2)necrotic
3)nodular
4)calcified

● NCC and HIV coinfection

● Prevention:
-good personal hygiene
-effective fecal disposal
-vaccine to prevent porcine cysticercosis

CYSTICERCUS CELLULOSAE:
● 5mm long,8-10mm wide, spherical shape, yellowish-white
● Contains two chambers
-outer chamber - bladder-like sac filled with 0.5 ml vesicular fluid
-inner chamber - contains growing scolex
● Racemose cysticerci:
- When parasites are lodged in a spacious area, they grow into larger and
lobulated cysticerci(>20cm) with 60ml of vesicular fluid
- Do not contain scolices and resemble metastatic tumor
- Mainly in the brain and spinal cord
- Prognosis poor
- If increased intracranial pressure is present, surgery is required
 6. CESTODES: ECHINOCOCCUS GRANULOSUS
● Segmented tape like worms with variable sizes

CLASSIFICATION:
1. PSEUDOPHYLLIDEAN TAPEWORM

Diphyllobothrium latum - fish tapeworm

Sparganum mansoni - cause spargonosis

2. CYCLOPHYLLIDEAN TAPEWORM

- Genus taenia

T.saginata - beef tapeworm

T.solium - pork tapeworm

- Genus Echinococcus

E.granulosus - dog tapeworm causing hydatid disease

E.multilocularis - multilocular hydatid disease

- Genus hymenolepis

H.nana - dwarf tapeworm

- Genus diphlidium

G.canium - double pored dog tapeworm

- Genus multiceps

M.multiceps - cause coenurosis in man

ECHINOCOCCUS GRANULOSUS
● Has dog as the definitive host and sheep and humans as the principal intermediate
host. In humans unilocular Echinococcosis or hydatid disease.

MORPHOLOGY :

● Small tapeworm, measuring 3-6 mm in length

● Consist of the scolex, short neck, trunk
● Scolex is pyriform with 4 sucker and prominent rostellum bearing 2 booklets
 ● The trunk is composed of 3 proglottides, anterior immature, middle mature, posterior
gravid.
● Terminal proglottid is longer and wider and contains a branched uterus filled with
egg

LIFE CYCLE :

● The dog is the definitive host. Adult worm line in duodenum and jejunum of dog
with its scolex buried in the mucosa
● Eggs of these worms passed in dog faces. Sheep and cattle may ingest them
● Eggshell disintegrated in duodenum setting free the Embryo which penetrates the
intestinal wall and enters portal circulation.
● Liver act as the first filter, some embryo get arrested in sinusoidal capillaries. Escaped
Embryo enter pulmonary capillaries. Lung act as a second filter.
● A few enter systemic circulation, get lodged in organs and tissues like the spleen,
kidney, eye, brain, and bones
● At the site of deposition, the embryo develops into a cyst filled with fluid. this
becomes a hydatid cyst. It enlarges and reaches a diameter of 1 cm in about 6 months
● Growing cyst evokes host tissue reaction leading to deposition of a fibrous capsule
around it
● It has outer thick opaque cuticles and an inner thin germinal layer containing
nucleated cells
● The germinal cell is the site of asexual reproduction and also secrete hydatid fluid
which fills the cyst
● Hydatid fluid is clear, colorless, has a pH of 6.7 containing salt and protein. It is a
good antigen and is used in diagnostic test
● From the germinal layer, gemmules protrude into the cyst lumen. They enlarge,
become vacuolated, and filled with fluid. They are called brood capsules. They
initially attached to the germinal layer, later escape free into the cyst cavity
● From the inner wall of the brood capsule, protoscolices develop, complete to
invaginated scolex with sucker and hooklets.
● Several thousand protoscolices develop in mature hydatid cyst. These protoscolices
along with free brood capsules are called hydatid sand
● Mature hydatid cyst may produce daughter and granddaughter cyst. The cyst grows
slowly, often take 20 years to become big enough to cause clinical symptoms
● Sometimes, scolices may escape and get transported to other parts of the body
producing secondary hydatid cyst
● Some cyst are sterile, never produce brood capsules, while some brood capsules may
not produce scolices. These are called acephalocyst
● When hydatid cyst are formed inside bones, the outer cuticles is not well developed.
They migrate along the bony canals and crude bone tissue. This is osseous hydatid
 PATHOGENESIS :

● Infection occurs by ingestion of eggs passed by infected dogs. This may occur by
eating food contaminated with dog feces, contaminating hands while fondling pet
dog,s or kissing pet dogs
● At any time, but disease develops only after several years when a hydatid cyst had
grown enough to cause symptoms. Disease results from the pressure effect caused by
enlarging cyst
● Liver - hepatomegaly, pain, obstructive jaundice
● Lung - cough, haemoptysis, chest pain, dyspnoea
● Kidney - pain, haematuria
● The host is sensitized to antigen by minute amounts of hydatid fluid seeping out
through the capsule, hypersensitivity reactions occur
● If a cyst rupture spontaneously or during surgery, massive release of fluid may cause
severe, even fatal anaphylaxis

DIAGNOSIS:

1. IMMUNE TEST OR CASINO’S TEST:

● Antigen in hydatid fluid are collected and sterilized and injected intradermally
on one arm and an equal amount volume of saline is injected into another arm
● In positive cases, a large wheal with multiple pseudopodial projections
appears within 20 to 30 minutes at the test site and fades in an hour. A
secondary reaction consisting of edema and induration appear after 8 hrs
● The test is very sensitive but non-specific. A false-positive reaction may
appear in many conditions.
2. SEROLOGICAL TEST :
● The serological test used are CFT, latex agglutination, IFA,
immunoelectrophoresis, and ELISA.
● CFT - not sensitive and false-positive reaction is seen. It is useful after
surgical removal when a negative test has a better prognostic value
● Slide agglutination and IEP using hydatid fluid fraction 5 antigen are used.
3. OTHER TESTS
● Radiological examination and other imaging techniques such s
ultrasonography and CT scan revealed the diagnosis
● Exploratory puncture of cyst yields hydatid fluid and demonstrations of
scolices in hydatid sand provide a conclusive diagnosis. But the procedure is
very risky there is a chance of cyst rupture.

TREATMENT:
● Surgical removal of the cyst is the best mode of treatment. But recurrence is common.
● Drug treatment ( mebendazole, albendazole, praziquantel ) has limited application.
 7. INTESTINAL PARASITES OF MAN. DETAILS ABOUT
ENTEROBIASIS

The intestinal parasites that infect man are broadly classified into
1. Protozoan
2. Helminthes

INTESTINAL PROTOZOAN INFECTIONS:

● The protozoan parasites which cause intestinal infection include:
● Intestinal amoebae: Entamoeba histolytica
● Intestinal flagellate: Giardia lamblia Cause giardiasis
● Opportunistic intestinal coccidian parasites: Cryptosporidium, Cyclospora, and
Cystoisospora
● Others: Balantidium coli, Blastocystis hominis, Sarcocystis, and Microsporidia (now
considered as fungi).

INTESTINAL AMOEBA:
● Amoebae – single-celled protozoan parasites that constantly change their shape - due
to the presence of an organ of locomotion called “pseudopodium”.
● Based on the habitat they are of two types -
o Intestinal amoebae; Entamoeba histolytica
o Free-living amoebae; Naegleria, Acanthamoeba, Balamuthia.

OPPORTUNISTIC INTESTINAL COCCIDIAN PARASITIC INFECTIONS

● Cryptosporidium, Cyclospora, and Cystoisospora are the intestinal coccidian parasites
– cause opportunistic infections in HIV-infected patients producing profuse watery
diarrhea.

BALANTIDIASIS:
● Balantidiasis – an intestinal infection caused by a protozoan parasite Balantidium coli.
● Largest protozoan and the only ciliated parasite of humans.
BLASTOCYSTOSIS:
● Blastocystosis – infection caused by a protozoan parasite Blastocystis hominis.
● Considered – most common commensal protozoa of the intestine

CHAGAS’ DISEASE:
● In chronic Chagas’ disease, the causative agent, Trypanosoma cruzi multiplies in the
muscles of GIT, leading to megaesophagus (manifested as dysphagia, chest pain, and
regurgitation) and megacolon (manifested as abdominal pain and chronic
constipation)

MICROSPORIDIOSIS (FUNGI CAUSING INTESTINAL INFECTIONS)

● Microsporidia - lower eukaryotic, spore-forming obligate intracellular parasites, cause
opportunistic infection in HIV-infected patients.

INTESTINAL HELMINTHIC INFECTIONS:

INTESTINAL CESTODES:
● Diphyllobothrium, Taenia saginata, T. solium, Hymenolepis nana, and Dipylidium
caninum

INTESTINAL TREMATODE:
o Intestinal flukes, e.g. Fasciolopsis buski
o Blood flukes - Schistosoma mansoni and S. japonicum reside in the
mesenteric venous plexus of GIT and cause various GI symptoms including
dysentery.

INTESTINAL NEMATODE:
o Small intestinal nematodes - Ascaris, hookworm, and Strongyloides
o Large intestinal nematodes - Trichuris and Enterobius.
● Intestinal nematodes of lower animals rarely infect man. Examples - Toxocara,
Angiostrongylus, etc.

INTESTINAL CESTODE INFECTIONS:

● Cestodes - long, segmented, flattened dorsoventrally, tape-like worms - also called
tapeworms.
● Based on habitat, they are classified into two groups:
 o 1. Intestinal cestodes
o 2. Somatic/tissue cestodes

INTESTINAL TREMATODE INFECTIONS:

● Trematodes (or flukes) - unsegmented, leaf-like, and flatworms.
● Based on the habitat, trematodes are classified as follows:
o Intestinal flukes
o Blood flukes
o Hepatic flukes
o Lung flukes
o Intestinal flukes – e.g., Fasciolopsis buski.
o Blood flukes – e.g., Schistosoma - S. mansoni, S. japonicum, and S. haematobium.
o Hepatic flukes—e.g. Fasciola hepatica and F. gigantica in the liver, Clonorchis,
and Opisthorchis in the bile duct.
o Lung flukes – e.g., Paragonimus westermani

INTESTINAL NEMATODE INFECTIONS:

i) Classification based on Habitat
1. Intestinal Nematodes
o Small intestinal nematodes — Ascaris, hookworm, and
Strongyloides
o Large intestinal nematodes — Trichuris and Enterobius
2. Tissue or Somatic Nematodes
▪ Filarial nematodes:
o Wuchereria bancrofti and Brugia malayi cause lymphatic filariasis
o Loa loa, Onchocerca volvulus, and Mansonella cause cutaneous
filariasis
▪ Dracunculus medinensis: Causes Guinea-worm disease.
▪ Trichinella spiralis: Produces profuse watery diarrhea, followed by
myalgia due to deposition of encysted larvae in muscles

ii) Classification based on they Lay Egg or Larva

1. Oviparous:
● Examples – Hookworm, Ascaris, Trichuris, Enterobius, etc.
● Diagnosis is made by detection of eggs in feces
 2. Viviparous: Female worms directly give birth to larvae; there is no egg stage
● Examples – Filarial worms, Trichinella and Dracunculus
● Diagnosis is made by detection of larvae in tissues or blood
3. Ovoviviparous: Female worms lay eggs containing larvae; which
immediately hatch out
● Example – Strongyloides species
● Larvae – Diagnostic form detected in stool examination.

ENTEROBIASIS:
● Enterobiasis – common parasitic infection in children caused by the intestinal
nematode, Enterobius vermicularis.
● It is also called pinworm or threadworm infection (as the adult worm of Enterobius
is small, white, and thread-like).

LIFECYCLE:

Mode of transmission: Infection occurs via Autoinfection

Two types:
i) Exogenous autoinfection, by transferring eggs to the mouth with hands that have scratched
the perianal area,
ii) Endogenous autoinfection, by retrograde migration of the larva hatched from the eggs in
the perianal skin.
Through ingestion of eggs in the environment (e.g., surfaces, clothes, bed linens, etc.)
through contaminated fingers.
PATHOGENICITY & CLINICAL FEATURES:
● Most of the infections are asymptomatic. Few develop clinical diseases.
● Age and sex: Females, children, and young adults are often symptomatic than males
and older people
● Cardinal symptoms: Perianal pruritus is often worsened at night due to the nocturnal
migration of the female worm.
● Migration of the worm: Rarely, pinworms invade the female genital tract, causing
vulvovaginitis and pelvic or peritoneal granulomas.
● Eosinophilia is inconsistent.

LABORATORY DIAGNOSIS:
● Microscopy of perianal skin samples is the test of choice which detects
characteristic eggs. The specimen is collected by two methods.
o Cellophane tape
o NIH swab
● There are few other points to consider while specimen collection:
● Number of specimens: A series of 4–6 consecutive tapes - necessary as the female
worms migrate intermittently
● Timing: Samples should be collected when the chance of egg deposition is more -
late in the evening when the patient has been sleeping for several hours, or first thing
in the morning
● The female worms lay eggs in the perianal area; not in the rectum.
● Hence, eggs are rarely detected by stool examination; around 5% of cases
● The adult female worms may occasionally be found in the feces or crawling onto the
perianal skin.

TREATMENT:
● One of the following drugs can be given:
o Mebendazole (100 mg once) or
o Albendazole (400 mg once) or
o Pyrantel pamoate (11 mg/kg once; maximum, 1 g)
● The same treatment should be repeated after 2 weeks
● Treatment of household members is advocated to eliminate asymptomatic reservoirs
of potential reinfection.
PREVENTION:
● Improving personal hygiene
● Proper washing of bedclothes
● Keeping nails short and clean
● Frequent hand washing
 8. INTESTINAL NEMATODES

● Nematodes--widespread animal group—classified into intestinal and tissue/somatic

nematodes

● Intestinal nematodes include –

1. Small intestinal nematodes- Ascaris, hookworm, Strongyloides
2. Large intestinal nematodes- Trichuris and Enterobius

● Classification based on whether they lay eggs or larva

1. Oviparous: lay eggs

e.g.: most intestinal nematodes except Strongyloides

2. Viviparous: give birth to larvae

e.g: most somatic nematodes

3. Ovo-viviparous: lay eggs containing larvae that immediately hatch out

e.g: Strongyloides

LIFE CYCLE OF HOOKWORM/ANCYLOSTOMA DUODENALE

● 2nd most common helminthic infection in the world
● An important cause of iron deficiency anemia
● CLASSIFICATION :
1. Ancylostoma duodenale: Old world hookworm
2. Necator americanus: New world hookworm

● HOST: only humans

● HABITAT: Jejanum of the small intestine
● MODE OF INFECTION: skin penetration by Filariform larvae/ Stage 3 larvae/ L3
larvae
● EPIDEMIOLOGY:
1. Widespread infection
2. Males and young adults more commonly affected
3. Anemia caused as a result is more severe in children and pregnant women
4. Chandler’s index: used to estimate the morbidity and mortality in the community
due to hookworm infection

● MORPHOLOGY:
EGGS
1. Oval
2. 60*40 micrometers
 3. Surrounded by thin eggshell- clear space between shell and embryo
present
4. Not bile stained
5. Segmented ovum – 4-8 blastomeres present
6. Eggs of A. duodenale and N. americanus are morphologically
indistinguishable

RHABDITIFORM LARVAE
L1 larvae of A. duodenale and N. americanus are morphologically indistinguishable.

ADULTS
1. 7-13mm in size
2. Bent in the anterior end
3. Adults of A. duodenale and N. americanus are differentiated by buccal
capsule with teeth and cutting plates respectively
● LIFE CYCLE:

1. Filariform larvae penetrate the skin

2. Enters circulation
3. Reaches rt side of heart and lungs
4. Breaks through the alveolar walls
5. Climbs respiratory tract
6. Coughed up and swallowed
7. Reaches the small intestine and develops into an adult
 ● PATHOGENICITY:
Secrete chemicals which will expose capillaries below the mucosa and extract blood
using mouthparts – teeth/ cutting plates

● CLINICAL FEATURES:
Due to larva:
1. Ground itch- Rash/ Vesicular eruptions – at the site of entry
2. Transient pneumonitis – milder than in ascariasis- as the larvae pass through
the lungs
Due to adults: most cases are asymptomatic
1. Less worm load – early intestinal phase – inflammatory diarrhea, epigastric
pain, eosinophilia
2. Heavy worm load – late intestinal phase – blood loss causing
a. Iron deficiency anemia
b. Hypoproteinemia
c. Weakness
d. Shortness of breath
3. Wakanda syndrome – accidental ingestion of filariform larvae. Manifestations
a. Gastrointestinal and respiratory: nausea, vomiting, pharyngeal
irritation, cough, dyspnea, hoarseness

● LAB DIAGNOSIS

1. PCR – targeting mitochondrial cytochrome oxidase gene

2. The concentration of stool sample: eggs observed
a. In stool sample that is not fresh, eggs may hatch out -rhabditiform larvae
with rhabditiform esophagus
● TREATMENT

i. Anti parasitic – Albendazole – 400mg – once

ii. Symptomatic treatment
a. Iron deficiency anemia – oral iron and folic acid

PARASITES CAUSING ANEMIA:

Parasite Anaemia caused Mechanism
A.duodenale Iron deficiency anemia Blood ingestion
N. americanus
Trichuriasis Iron deficiency anemia Blood loss
Diphyllobothrium latum Megaloblastic anemia Dissociation of the Vit B12-
intrinsic factor complex
within the gut lumen
S. haemotobium Normochromic anemia Hemorrhage
P. falciparum - malaria Normochromic anemia Hemolysis, hypersplenism
L. donovani Normochromic anemia Spleen and bone marrow
involvement
PERNICIOUS ANEMIA
● Diphyllobothriasis is caused by an intestinal cestode- Diphyllobothrium latum / fish
tapeworm
● Largest parasite in the human intestine
● The adult worm causes dissociation of the Vit B12- intrinsic factor complex within
the gut lumen 🡪 decreased absorption of B12 in the ileum
● Vit B12 deficiency 🡪megaloblastic anemia
● Some ppl exhibit neurologic sequelae like paresthesia
● Anemia observed in 2% of the infected patients- especially in the elderly – attributed
to genetic predisposition
 9. VIVIPAROUS NEMATODES
● The viviparous nematode is a category of classification that deals with the nematodes
which produce larvae.
● Example: Trichinella, Wuchereria, Brugia, Dracunculus

LIFECYCLE OF BANCROFTIAN FILARIASIS

LABORATORY DIAGNOSIS

● Demonstration of microfilariae by thin or thick smear stained with Giemsa or by QBC

Examination. W.Bancrofti- tail tip pointed, free of nuclei, B.Malayai- tail tip blunt, nuclei
extended up to the tail tip.
● ANTIGEN DETECTION (ELISA, ICT)- detects antigen by using mAb to Og4C3 Ag and
AD12 Ag
● ANTIBODY DETECTION
● A flow-through assay using WbSXP-1 Ag
● Luciferase immunoprecipitation system using Wb 123 Ag
● Imaging methods- filarial dance sign in USG, indicates the serpentine movement of adult
worms in lymphatics.
● Molecular methods- real-time PCR Detecting genes such as Sspl repeat, pWb12 repeat,
pWb-35, etc
● Other methods- eosinophilia, elevated IgE.
PREVENTION AND CONTROL OF BANCROFTIAN FILARIASIS

ANTI-LARVAL MEASURE
Chemicals used- mosquito larvicidal oil, pyrethrum based oil, organo-phosphorus larvicide

ANTI-ADULT MEASURE
Pyrethrum spray can be used
DDT and HCH are not effective.

THE NATIONAL VECTOR-BORNE DISEASE CONTROL PROGRAM

The national filariasis control program in India is active since 1955.
Was integrated with NVBDCP in 2006.

CLASSIFICATION OF NEMATODES.

INTESTINAL NEMATODES TISSUE/ SOMATIC NEMATODES

A. SMALL INTESTINE A. LYMPHATIC TISSUE
1. Ascaris lumbricoides 1. Wucheria bancrofti
2. Ancylostoma duodenale 2. Brugia malayi
3. Necator americanus
4. Strongyloides stercoralis
5. Trichinella spiralis
6. Capillaria philippinensis
B. LARGE INTESTINE B. SUBCUTANEOUS TISSUE
1. Enterobius vermicularis 1. Loa loa
2. Trichuris 2. Onchocerca volvulus
C. Conjunctiva
1. Loa loa
D. BODY CAVITY
1. Mansonella perstans
2. M.ozzardi

FILARIASIS

● Filariasis (or philariasis) is a parasitic disease caused by an infection with roundworms of

the Filarioidea type.
● These are spread by blood-feeding black flies and mosquitoes.
● This disease belongs to the group of diseases called helminthiasis.
● Eight known filarial nematodes use humans as their definitive hosts
WUCHERIA BANCROFTI

● The primary causative agent of lymphatic filariasis

● Accounts for > 90% of cases of lymphatic filariasis

LIFE CYCLE:
● Host and vector: W.bancrofti completes its life cycle in two hosts:
● 1. Man ( definitive host)
● 2. Intermediate host ( mosquito)
● Infective form (L3) filariform larvae are the infective form, found in the proboscis of the
mosquito.

MICROFILARIA OF WUCHERERIA
● They are colorless and Transparent with blunt heads and pointed tails.
● Covered by a hyaline sheath
● Since the sheath is longer than the Embryo, the microfilaria can move forwards and
backward.
● The somatic cells or nuclei appear as granules in the central axis of the embryo and extend
from head to tail-end.
● However, the granules do not extend up to the tip and are a distinguishing feature of
Microfilaria bancrofti.

LABORATORY DIAGNOSIS OF FILARIASIS

1. Direct evidence
2. Indirect evidence

1. DIRECT EVIDENCE
● Appears in the large colony at night
● Membrane filtration technique and sedimentation technique
● Demonstrated via anticoagulated blood
● Acrid one orange microhematocrit tube technique
● Fluorescent microscopy

2. INDIRECT EVIDENCE
● Antigen detection
● DNA probes
● PCR

MICROFILARIA
● The microfilaria (plural microfilariae) is an early stage in the life cycle of certain
parasitic nematodes in the family Onchocercidae.
● In these species, the adults live in a tissue or the circulatory system of vertebrates (the
definitive host).
● They release microfilariae into the bloodstream of the vertebrate host. The
microfilariae are taken up by blood-feeding arthropod vectors (the "intermediate
host").
 ● In the intermediate host, the microfilariae develop into infective larvae that can be
transmitted to a new vertebrate host.
● The presence of microfilariae in the host bloodstream is called microfilaraemia

VIVIPAROUS NEMATODES
● female worms directly give birth to larvae
● There is no egg stage
● Example: Trichinella, Wuchereria, Brugia, Dracunculus

TISSUE / SOMATIC NEMATODES

● Nematodes which infect the different tissues are called tissue or somatic nematodes.
● The filarial nematodes are the major group of tissue-nematodes.
● The filarial nematodes belong to the superfamily Filarioidea; they are slender, thread-like
worms.
● They inhabit the circulatory and lymphatic channels, connective tissue, and serous cavities.
● The female worms are viviparous.
● Eight species of filarial worms infect humans, who are the definitive hosts.
 10. ASCARIS LUMBRICOIDES

● Ascariasis - infection of the small intestine caused by Ascaris lumbricoides.

● It is the largest nematode parasitizing the human intestine.
● Commonly called a roundworm.
● It is a soil-transmitted helminth
● Ascaris lumbricoides - cosmopolitan in distribution
● Clay soils - most favorable for the development of Ascaris eggs

MORPHOLOGY:

Ascaris exists in three forms: adult, larvae (four stages), and egg. The adult worm is
cylindrical

The female worms liberate two types of eggs

(1) fertilized eggs, and (2) unfertilized eggs.

LIFE CYCLE:

● Involves only one host (Man)

● Embryonated eggs containing the L2 larvae are the infective form.
● Mode of transmission: Ingestion of embryonated eggs from contaminated soil, food,
and water.
● Migratory phase: Following ingestion the eggs hatch out to liberate the L2 larvae,
which molt to form L3 which penetrates the intestine and reach the right side of the
heart and lungs via portal circulation and pulmonary capillaries where they molt to
form L4.L4 larvae in the lungs migrate to the pharynx and finally swallowed to reach
the intestine
● Intestinal phase: Adult worms are formed.Following fertilization female worms lay
fertilized eggs which are passed into feces
● Development in soil: Fertilized eggs molt twice become embryonated which is
infective to man.
 ● Embryonated eggs survive as far as 15 years as they are highly resistant due to thick
eggshell containing lipoprotein Ascaroside

PATHOGENESIS:
● Pathogenesis caused by Ascaris infection is attributed to:
(i) the host immune response,
(ii) migration of larva
(iii) mechanical obstruction by the adult worms
(iv) nutritional deficiencies due to the presence of adult worms.
The incubation period is about 60–70 days

PULMONARY PHASE:
● Results from migrating larvae in the lungs – provoke an immune-mediated
hypersensitivity response.
● Symptoms - observed in the second week after the ingestion of eggs; characterized by
non-productive cough, chest discomfort, and fever
● Eosinophilic pneumonia (Loeffler’s syndrome)
INTESTINAL PHASE:
● Results due to the effect of adult worms in the intestine.
● Asymptomatic
● Malnutrition and growth retardation
● Intestinal complications
● Extraintestinal complications
● Allergic manifestations like fever, urticaria, angioneurotic edema, and conjunctivitis

LABORATORY DIAGNOSIS:
Egg Detection (Stool Examination):
● Fertilized and unfertilized eggs- Saline and Iodine wet mount.
● Concentration techniques by sedimentation method.
● Floatation method for stool concentration not preferred

Adult worm detection:

● Occasionally detected in stool or sputum of patients by the naked eye
● Barium meal X-ray of the GIT
● Two worms lying parallel- trolley car lines appearance in X-ray
● Ultrasound or cholangiopancreatography - Extraintestinal sites.

Larva detection:
● During the early pulmonary migratory phase, larvae can be found in sputum or gastric
aspirates before the eggs appear in the stool.

Serology:
● Antibody detection (by ELISA and other formats)

MOLECULAR METHOD:
● PCR - developed targeting internal transcribed spacer region (ITS1) or cytochrome
oxidase-1 of Ascaris egg in the stool.
● Multiplex PCR
● Real-time PCR

OTHER METHOD:
● Eosinophilia is prominent during the early lung stage
● Presence of Charcot-Leyden crystals in sputum and stool

TREATMENT:
● Antiparasitic drugs:Albendazole or mebendazole
● Alternatively, drugs like ivermectin and nitazoxanide are also effective
● Pregnancy- Pyrantel pamoate
 ● Symptomatic treatment

NEMATODES
CLASSIFICATION BASED ON HABITAT
● Intestinal Nematodes
● Tissue or Somatic Nematodes

INTESTINAL NEMATODES
● Small intestinal nematodes—Ascaris, hookworm, and Strongyloides
● Large intestinal nematodes—Trichuris and Enterobius.

TISSUE OR SOMATIC NEMATODE:

● Filarial nematodes:
Wuchereria bancrofti and Brugia malayi - lymphatic filariasis
Loa loa, Onchocerca volvulus, and Mansonella -cutaneous filariasis
● Dracunculus medinensis- Guinea-worm disease.
● Trichinella spiralis: Watery diarrhea followed by myalgia

CLASSIFICATION BASED ON THE LAY EGG OR LARVA

1.OVIPAROUS: Following fertilization, the female worms produce eggs that take some time
to hatch out to form larvae in the environment
● Most intestinal nematodes are oviparous except Strongyloides.
● Examples - Hookworm, Ascaris, Trichuris, Enterobius
● Diagnosis: Detection of eggs in feces.
2.VIVIPAROUS: Female worms directly give birth to larvae; there is no egg stage
● Most somatic nematodes are viviparous.
● Examples - filarial worms, Trichinella and Dracunculus
● Diagnosis: Detection of larvae in tissues or blood.
3. OVOVIVIPAROUS: Female worms lay eggs containing larvae; which immediately hatch
out
● Example - Strongyloides species
● Larvae - diagnostic form detected in stool examination.
 11. THE LIFE CYCLE OF DRACUNCULUS MEDINENSIS
● Dracunculus Medinensis is the causative agent of Dracunculiasis.
● It is a somatic nematode and is also called a Guinea worm.
● It is characterized by Cutaneous blisters.
● It is a crippling parasitic disease on the verge of eradication, currently endemic to
only 3-4 countries, with only a total of 54 cases was reported in 2019 from Chad and
Ethiopia.

LIFE CYCLE :
● Definitive host: Man
● Intermediate host: Cyclops
● Mode of transmission: Man gets infected by drinking water from a stagnant pool
containing cyclops infected with L3 larva (an infective form of Dracunculus
Medinensis ).
● The Cyclops are then digested in the stomach releasing the L3 larvae.
● The larvae penetrate the small intestine and migrate to the thoracic muscle, develop to
from adult worms.
● The female worms migrate throughout the body and ultimately reach the skin,
particularly over the ankle, feet, and lower legs. When the skin comes in contact with
water, blisters are induced by the female worm, which eventually ruptures releasing
the L1 larvae into the water.
TREATMENT :
● No Antihelminthic drug is found
● Worm removal: Worms are slowly and gently extracted over a period of 15-20 days
using a small stick and wounding out daily with small traction.
● Use Antiseptics to prevent secondary bacterial infections
Short Notes
 1.1 LIFE CYCLE OF ENTAMOEBA HISTOLYTICA
● Host: Humans
● Habitat: Caecum and Colon
● Infective form: Quadrinucleate cyst
● Mode of infection: Feco-oral route
● Exist in 3 stages – Trophozoite, Pre-cyst, and Cyst
● Trophozoite form is invasive, feeding as well as replicating form
● A cyst is a diagnostic form

1.2 LABORATORY DIAGNOSIS OF INTESTINAL

AMOEBIASIS
SPECIMEN:
A stool sample is collected

Permanent stains used are Iron hematoxylin stain, Trichrome stain, Modified ZN stain
STOOL MICROSCOPY:
· Liquid stools: Trophozoites mainly seen

· Semi-solid stools: Cysts

· Has poor sensitivity

Nucleic Acid Amplification test (NAAT) – gold standard test

SEROLOGY: Antibodies appear in the 2nd-3rd week, but persist for years.

1.3 EXTRAINTESTINAL AMOEBIASIS AND AMOEBIC

LIVER ABSCESS
● 5-10% of invasive amebiasis
● Trophozoites reach the liver, most commonly the right lobe of the liver
● 10 times more common in men than in women.
● Rare in children
● Fever + Dull ache in hypochondrium/epigastrium
● Hepatomegaly with point tenderness just beneath the ribs

1.4 PRIMARY AMOEBIC MENINGOENCEPHALITIS

· Acute: Incubation period 2-5 days

· Almost resembles bacterial meningitis

· Present with change in taste/smell à nausea, vomiting, and headache

· Can be due to Naegleria fowleri and even Para VahlKampfia.

· Mortality: 98%

DIAGNOSIS: CSF specimen, Polymorphs increased, Protein raised, Pressure raised,

Glucose normal/Low.

CULTURE: Non-nutrient agar (NNA) with lawn culture of E. coli

TREATMENT: Amphotericin B
Gold standard Diagnostic test: Nucleic Acid amplification test
 2.1 FREE-LIVING AMOEBAE INFECTIONS
· Small, freely living, widely distributed in soil and water and can cause opportunistic
infections in humans.

· Naegleria fowleri is a causative agent of primary amoebic meningoencephalitis

(PAM).

· Acanthamoeba species causes granulomatous amoebic encephalitis (GAE) and

amoebic keratitis in contact lens wearers.

· Balamuthia mandrillaris causes GAE.

· Sappinia species: causes encephalitis.

· Naegleria is a free-living amoeba, typically found in warm freshwater, such as ponds,

lakes, rivers, and swimming pools.

· Exists in nature as cyst and trophozoite forms

LIFECYCLE AND PATHOGENESIS:

PRIMARY AMOEBIC MENINGOENCEPHALITIS:

· Acute suppurative fulminant infection of CNS known

· PAM: It is so named because to distinguish it from the secondary invasions of CNS

caused by E. histolytica

· Healthy children or young adults with a recent history of swimming in fresh hot water

· Incubation period: 1-2 days to 2 weeks after exposure.

 · The initial symptoms include changes in the taste and smell (due to olfactory nerve
involvement) followed by headache, anorexia, nausea, vomiting, high fever, and signs
of meningeal involvement like stiff neck and a positive Kernig's sign

· Secondary symptoms include confusion, hallucinations, lack of attention, ataxia, and

seizures

· The mortality rate is nearly 98%.

LABORATORY DIAGNOSIS:
· CSF analysis: CSF is thick purulent, elevated protein and reduced sugar level (mimic
bacterial meningitis)

· CSF microscopy: Detection of characteristic trophozoites in CSF confirms the

diagnosis

· Histopathology: Brain biopsied- trophozoites - sky blue cytoplasm with a pink

nucleus

· Culture: Non-nutrient agar (Page’s saline and 1.5% agar). Naegleria feeds on bacteria
and crawls over the lawn culture of E. coli to produce trails (Trail sign)

· Enflagellation test

· Isoenzyme analysis

· Molecular methods

· Imaging methods

TREATMENT;
· No effective treatment

· Amphotericin B, rifampicin, sulfisoxazole, and antifungals like miconazole,

fluconazole, and miltefosine are also found to be effective.

ACANTHAMOEBA INFECTIONS:
· Ubiquitous and present worldwide, isolated from soil, fresh and brackish waters.

· Important ones- A. astronyxis, A. castellanii (type species), A. culbertsoni, and A.

polyphagia

· Acanthamoeba may harbor bacterial pathogens such as Legionella

LIFE CYCLE AND PATHOGENESIS:

12

GRANULOMATOUS AMOEBIC ENCEPHALITIS

CSF microscopy: The presence of characteristic trophozoites (or occasionally cyst) confirms
the diagnosis.

Wet mount examination and phase-contrast microscopy are performed.

Permanent staining: Done for cytospin centrifuged CSF and brain biopsy (more reliable
than CSF) using hematoxylin and eosin stain or PAS stain

Calcofluor stain: Visualize the double-walled cyst

IFAT (indirect fluorescent antibody technique) with specific antisera can be used for
speciation of Acanthamoeba. A. culbertsoni and A. castellanii

Culture: Inoculated onto non-nutrient agar with a bacterial supplement, and incubated at
30°C.

Molecular methods

Imaging method

TREATMENT:
· There are no therapies with proven efficacy.

· Combination therapy - pentamidine, an azole, sulfonamide (e.g., cotrimoxazole), and

possibly flucytosine.
BALAMUTHIA MANDRILLARIS INFECTION

LIFECYCLE:
Similar to Acanthamoeba – has trophozoite and cyst form (no flagellated form)

· Trophozoite is – irregular with extensive branching, single nucleus, centrally located

karyosome with no peripheral chromatin

· Cyst – surrounded by a three-layered cyst wall, and an abnormally large, vesicular

nucleus

CLINICAL FEATURES:

· Enter the body - respiratory tract or open wounds.

· In CNS, it causes GAE.

· Also cause skin lesions.

· Unlike Acanthamoeba, Balamuthia encephalitis – found in immunocompetent

individuals

LABORATORY DIAGNOSIS:
Trophozoites & cysts - found in CSF and brain biopsy.
 · Balamuthia can be differentiated from Acanthamoeba species by:

o Cyst: - Tri-layered

o Indirect fluorescent antibody (IFA) test

o Culture on cell lines but not on an agar plate

o PCR targeting mitochondrial small subunit rRNA gene

TREATMENT:
· Multidrug therapy - combinations of pentamidine, azithromycin, fluconazole,
amphotericin, and miltefosine.
 3.1 TRICHOMONAS
- Trichomonas differ from other flagellates as they lack the cyst stage. They exist as only
trophozoites.

- Three species of Trichomonas infect humans.

1. Trichomonas vaginalis is the only pathogen residing in the genital tract

2. Pentatrichomonas hominis: Non-pathogen, resides in the large intestine

3. Trichomonas tenax: Nonpathogenic, resides in the mouth (teeth and gums)

TRICHOMONAS VAGINALIS:
· It is the most common parasitic cause of sexually transmitted diseases (STDs).

· Females are more commonly affected than males.

· Though it is a eukaryote, its metabolism is similar to a primitive anaerobic bacterium

where carbohydrate is utilized fermentatively. It is unable to synthesize fatty acids,
sterols, purines, and pyrimidines and hence depends on exogenous sources.

CLINICAL FEATURES:
Acute infection (vulvovaginitis):

· Females are commonly affected, characterized by profuse foul-smelling purulent

vaginal discharge. Discharge may be frothy (10% of cases) and yellowish-green color
mixed with a number of polymorphonuclear leukocytes.

· Strawberry appearance of vaginal mucosa (Colpitis macularis). It is characterized

by small punctate hemorrhagic spots on vaginal and cervical mucosa.

· Other features include dysuria and lower abdominal pain.

· In males, the common features are nongonococcal urethritis and rarely epididymitis,
prostatitis, and penile ulcerations.

Chronic infection: In the chronic stage, the disease is mild with pruritus and pain during
coitus. Vaginal discharge is scanty, mixed with mucus.

COMPLICATIONS: Rarely it is associated with complications like pyosalpinx,

endometritis, infertility, low birth weight, and cervical erosions.
PENTATRICHOMONAS HOMINIS:
· It is a harmless commensal present in the large intestine.

· Trophozoite is pyriform shaped, measuring 5–15 µm long and 7–10 µm wide, similar
to that of T. vaginalis, except that the undulating membrane is extended throughout the
body.

TRICHOMONAS TENAX:
· T. tenax is a harmless commensal in the mouth (gum and tartar of the teeth).

· However, few cases of respiratory infection and thoracic abscesses are reported from
Western Europe particularly in patients with cancer or other underlying lung diseases.

· The trophozoite is similar to P. hominis (the undulating membrane is extended

throughout the body), but it is smaller (5–12 µm long and 5–10 µm wide) and is more
slender.

3.2 GIARDIASIS
· Caused by Giardia lamblia residing in the duodenum and upper jejunum.

· Host: Giardia completes its life cycle in one host.

· Infective form: Mature cyst

· Mode of transmission: Man acquires infection by ingestion of food and water

contaminated with mature cysts or rarely by sexual route (mainly in homosexuals)

CLINICAL FEATURES:
The clinical course of giardiasis can be divided into three stages:

1. Asymptomatic carriers:

· Most infected persons are asymptomatic, harboring the cysts and spreading
the infection

2. Acute giardiasis:

· Incubation period varies from 1 week to 3 weeks (average 12–20 days).

· Symptoms may develop suddenly or gradually.

· Common symptoms include diarrhea, abdominal pain, bloating, belching,

flatus and vomiting.
 · Diarrhea is often foul-smelling with fat and mucus but no blood

· The acute stage lasts for 1 week but usually resolves spontaneously.

· Very rarely, in some children may last for months

3. Chronic giardiasis:

· It may present with or without a previous acute symptomatic episode

· Symptoms are intermittent and recurring

· Common symptoms include recurrent episodes of foul-smelling diarrhea, foul

flatus, sulfurous belching with rotten egg taste, and profound weight loss
leading to growth retardation

· Uncommon symptoms such as – fever, presence of blood and/or mucus in the

stools, and other signs and symptoms of colitis

· Extraintestinal manifestations have been described, such as urticaria, anterior

uveitis, salt, and pepper retinal changes, and arthritis.

LABORATORY DIAGNOSIS:
· Stool examination – Detects cysts and trophozoites

· Enterotest

· Antigen detection in the stool (copro-antigen) – ELISA, ICT

· Antibody detection in serum – ELISA, IFA

· Culture

· Molecular method – PCR

· Radiological findings – Barium meal, X-ray

 3.3 TRICHOMAS VAGINALIS
MORPHOLOGY:
· Trophozoites are the only stage, there is no cystic stage.

· Trophozoites: It is pear (pyriform) shaped, measures 7–23 µm and 5–15 µm

wide resides in the vagina and urethra of women and urethra, seminal vesicle
and prostate of men.

· It shows characteristic jerky or twitchy motility in saline mount preparation.

· It bears five flagella – four anterior flagella and one lateral flagellum called a
recurrent flagellum

LIFE CYCLE:
· Trophozoites are the infective stage as well as the diagnostic stage.

· Asymptomatic females are the reservoir of infection and transmit the disease by
sexual route

· Trophozoites divide by longitudinal binary fission giving rise to a number of

daughter trophozoites in the urogenital tract which can infect other individuals.

PATHOGENESIS AND CLINICAL FEATURES:

· Trichomoniasis is the most common parasitic cause of STDs.

· It is worldwide in distribution and accounts for 10% of cases of vulvovaginitis

· Incubation period is variable (4–28 days)

· Predisposing factors:

● Binding to the vaginal epithelium by various metabolic enzymes

secreted by the trophozoites like adhesins, proteolytic enzymes,
iron-regulated proteins, erythrocyte binding proteins, etc
● Vaginal pH of more than 4.5 facilitates infection
● Hormonal levels
● Coexisting vaginal flora
● Strain and relative concentration of the organisms present in the vagina
ASYMPTOMATIC INFECTION:

25–50% of individuals are asymptomatic, harboring the trophozoites, and can transmit the
infection

ACUTE INFECTION (VULVOVAGINITIS):

● Females are commonly affected and are presented as vulvovaginitis, characterized by

profuse foul-smelling purulent vaginal discharge.
● Discharge may be frothy (10% of cases) and yellowish-green color mixed with a
number of polymorphonuclear leukocytes
● Strawberry appearance of vaginal mucosa (Colpitis macularis) is observed in 2% of
patients. It is characterized by small punctate hemorrhagic spots on vaginal and
cervical mucosa
● Other features include dysuria and lower abdominal pain
● In males, the common features are nongonococcal urethritis and rarely epididymitis,
prostatitis, and penile ulcerations

Chronic infection: In the chronic stage, the disease is mild with pruritus and pain during
coitus. Vaginal discharge is scanty, mixed with mucus

COMPLICATIONS: Rarely it is associated with complications like pyosalpinx,

endometritis, infertility, low birth weight, and cervical erosion.

LABORATORY DIAGNOSIS:
· Direct microscopy

· Wet saline mounting

· Permanent stain

· Acridine orange fluorescent stain

· Direct fluorescent antibody test

· Culture – gold standard method

· Antigen detection in vaginal secretion – ELISA, ICT, etc

· Antibody detection – ELISA

· Molecular method – PCR

· Other supportive tests

· Raised vaginal pH

· Positive whiff test

 4.1. MORPHOLOGY OF PATHOGENIC INTESTINAL
PROTOZOA
Protozoan parasites causing intestinal infection include:
● Intestinal amoeba – Entamoeba histolytica
● Intestinal flagellate – Giardia lamblia
● Opportunistic intestinal coccidian parasite – Cryptosporidium, Cyclospora
● Others – Balantidium coli, Blastocystis hominis, Sarcocystis, Microsporidia

INTESTINAL AMOEBA - ENTAMOEBA HISTOLYTICA

Exists in three stages:

TROPHOZOITE:
● Invasive as well as feeding and replicating form.
● Present in feces of patients with active disease.
● 15- 20 micrometer in size.
● Contain a single nucleus with central karyosome and fine peripheral chromatin
granules lining the nucleus, in between them exist the spoke-like arrangement of
achromatic fibrils (cartwheel appearance).
● Possess pseudopodia (finger-like projection) that help in locomotion.
● Better appreciated in saline mount than iodine mount as iodine kills trophozoite.

PRE CYST: Intermediate stage

CYST:
● Diagnostic form found in feces of carriers as well as affected person.
● 12- 15 micrometer in size.
● Can be mature or immature. The immature cyst contains 1 or 2 nuclei.
● A mature cyst is quadrinucleated and is the infective form.
● The nucleus is the same as in trophozoite.
● Better appreciated in iodine mount than saline mount.

INTESTINAL FLAGELLATE
● Inhabited in the crypt of the duodenum and upper part of the jejunum.
● Occur in 2 forms.

PEAR-SHAPED TROPHOZOITE:
● Pathogenic and feeding form.
● 10 - 20 micrometre in length; 5- 15 micrometre in width.
● Front - pear-shaped; lateral - spoon or sickle-shaped.
● Has falling leaf-like motility.
 ● Have one pair of nuclei, four pairs of flagella.
● Better appreciated in saline mount & permanent staining.

TETRA NUCLEATED OVAL CYST:

● Infective & diagnostic form
● Oval-shaped & contain 4 nuclei.
● Present in the stool of both carriers and active diseased persons.

OPPORTUNISTIC INTESTINAL COCCIDIAN PARASITIC

INFECTION
- Their life cycle passes through various stages as oocyst, trophozoite, schizont,
merozoite, the zygote.

OOCYST:
● Infective and diagnostic form
● Round to oval-shaped, surrounded by cyst wall and bear sporozoites.
● Acid-fast in nature but does not stain by iodine.
● Extremely resistant to routine chlorination, heat, and other disinfectants.
Cryptosporidium – oocyst is round, 4- 6 micrometers in size, and contains 4
sporozoites.
Cyclospora – oocyst is round, 8- 10 micrometer in size, containing 2 sporocysts (each
has 2 sporozoites).
Cystoisospora – oocyst is oval and larger, 20- 33 micrometers in size, containing 2
sporocysts (each has 4 sporozoites).

OTHERS:
Balantidium coli:
● Exists in 2 forms:
1. Trophozoite – found in the stool of affected people.
2. Cyst – found in both carriers and chronic cases.
● Both are nucleated having a large macronucleus and a small micronucleus.

Blastocystis hominis:
● Occur in 6 forms.
▪ Vacuolar form
▪ Avacuolar form
▪ Multi vacuolar form
▪ Amoeboid form
▪ Granular form
▪ Cyst form
 5.1. PNEUMOCYSTIS JIROVECI / P. CARINII
● It was generally considered a sporozoan parasite. Analysis of its chromosomal and
mitochondrial genes indicates; it is more closely related to fungi than to protozoa.
● It lives within the alveoli of the lungs.
● Occurs in 2 forms.
1. Trophozoite
2. Cyst.

TROPHOZOITE:
● 1- 5 micrometer in size.
● Amoeboid in shape
● Has a central nucleus
● The cytoplasm contains mitochondria, ribosomes, ER & various granules.
● Divides by binary fission.
● Some become encysted & produce 8 daughter trophozoites within the cyst. Daughter
trophozoites are also called intracystic bodies or sporozoites.

CYST:
● Mature cyst in thick-walled
● Up to 10 micrometers in diameter
● When the cyst collapses, it releases trophozoite initiating another cycle of
multiplication either in the same host or in another host if spread by coughing.
● Collapsed cysts can be seen as irregular crescentic bodies.
 ● P. jiroveci is normally a commensal in the lung, spread by respiratory droplets.

PNEUMOCYSTIS:
- It is an opportunistic infection; a clinical disease is found only when the resistance is
very low as in premature & malnourished infants and in AIDS and other
immunodeficiencies.

HISTOPATHOLOGY:
Multiplication induces a hyaline or foamy alveolar exudate containing numerous
lymphocytes, macrophages, and plasma cells but no polymorph. The stained section shows a
characteristic honeycomb pattern.

SYMPTOMS: Nonproductive cough, breathlessness, and cyanosis.

DIAGNOSIS:
- Demonstrating the parasite in sputum, tracheobronchial lavage, or transbronchial
biopsy specimens.
- The cyst can be stained by Giemsa or methenamine silver techniques.
- Immunofluorescence has been used for demonstrating cysts.
- P. jiroveci antigen can be demonstrated by ELISA.

TREATMENT:
- Cotrimoxazole
- Pentamidine
 6.1. BALANTIDIUM COLI
● Largest protozoan and the only ciliated parasite of humans.

HABITAT: Resides in the large intestine of man, pig (main reservoir), and other animals.
MORPHOLOGY: It exists in two forms:
(1) Trophozoite (found in the dysenteric stool)
(2) Cyst (found in carriers and chronic cases).

Both the forms are binucleated, having a large macronucleus and a small micronucleus.
▪ Trophozoite: It is found in the active stage of the disease and is considered an invasive
form.
▪ Cyst: It is a round, thick, and transparent cyst wall. It also contains two nuclei
(macronucleus and micronucleus) and vacuoles.

LIFECYCLE:
Host: The life cycle is completed in a single host.
▪ Natural host: Pig
▪ Accidental host: Man
Infective form: Cyst
Mode of transmission: Man gets infection by ingestion of food and water
contaminated with cysts
LABORATORY DIAGNOSIS:
● Stool Examination:
- Repeated stool examination should be done as the parasite is excreted
intermittently.
- Trophozoites are detected in acute disease (dysenteric stool). Trophozoites are
easy to identify by their rotatory motility, large size kidney-shaped macronucleus,
and presence of cilia
- Cysts are seen in chronic cases or carriers, Cyst is round, measures 40–60 µm in
size, is surrounded by a cyst wall, and has two nuclei.
● Histopathological staining of the biopsy tissue or scrapping of the ulcers taken by
sigmoidoscopy reveals a cluster of trophozoites, cysts, and lymphocytic infiltration
found in the submucosa
 7.1 REDIA AND CERCARIA
- Larval forms of trematodes.

REDIA LARVA:

▪ Redia emerges from the Sporocyst by rupture of its body wall. Each Redia is an
elongated oval hollow body covered with a thin cuticle. At the anterior end, there is a
muscular collar. The mouth is at the anterior end which opens into the muscular
pharynx, which in its turn opens into a small intestine. Little behind the muscular
collar is the birth pore.

▪ The posterior part of the body has a pair of blunt conical processes called lappets.
Above each lappet lies the excretory pore. The Redia larva comprises germ cells
which by division produces Cercaria larvae in autumn. These come out through
birth-pore.

CERCARIA LARVA:

▪ Each Redia produces 14 to 20 Cercaria larvae. These larvae come out through the
birth pore of the Redia larva and enter into the snail’s digestive gland. A fully grown
Cercaria is a flattened, heart-shaped body with an exceedingly contractile tail, more
than double as long as the body proper.

▪ There is an oral sucker at the anterior end and a little behind there is a ventral sucker,
placed in the middle of the lower surface of the body. In the center of the oral sucker,
there is a mouth that leads into the pharynx. The pharynx finally opens into the bifid
 intestine. The body contains flame cells (Protonephridia), germ cells, and gland cells
(Cystogenous gland cells)

TYPES OF CERCARIA:
1.Furcocercus cercaria: Elongated body with forked tail (e.g., Schistosoma)
2.Microcercus cercaria: Oval body with short stumpy tail (e.g., Paragonimus)
3.Lophocercus cercaria: Cercaria is armed with spines and has a large fluted tail and
conspicuous eyespots, (e.g., Clonorchis, Metagonimus, and Heterophyes)
4.Pleurolophocercus cercaria: Cercaria is armed with spines and has pigmented
eyespots with a long-keeled tail (e.g., Opisthorchis)
 8.1 FASCIOLA HEPATICA

● A trematode - unsegmented, leaf like, flatworm

● Hepatic fluke/liver fluke

MORPHOLOGY: Three forms - adult worm, egg, larvae

ADULT WORM :

● Unsegmented and dorsoventrally flattened

● 1 mm to 60mm
● Has two suckers, an incomplete digestive system, nervous system, and excretory
system
● Hermaphrodites

EGGS: Operculated

LARVAE:

● Five larval forms

1) Miracidium
2) Sporocyst
3) Redia
4) Cercaria
5) Metacercaria
LIFE CYCLE :

Host Definitive host - man

Intermediate host
1) Snail
2) Aquatic plants

Mode of transmission Ingestion

Infective form Metacercaria larva

Diagnostic form Operculated eggs

Organ infected Liver

Disease caused: Fascioliasis
● Clinical features :
Hepatomegaly
Eosinophilia
Liver rot
- Bile duct obstruction
 - Biliary tract dilatation
- Biliary cirrhosis
- Houlzen or Marrara syndrome

● Lab diagnosis :

- Stool microscopy - characteristic operculated eggs

- Antibody detection - ELISA and Western blot
- Molecular methods - PCR
- Imaging methods - ultrasound, CT scan, MRI scan

● Prevention :

- Sanitary disposal of sewage and control of snail hosts

- Avoiding consumption of alfalfa juice, raw water plants and cleaning them
before use
 9.1. LUNG FLUKE

- Trematode residing in the lungs

- Eg: Paragonimus westermani (oriental lung fluke)

MORPHOLOGY:

Shape Leaf-like, unsegmented

Suckers Present
Head end No hooklets

Alimentary canal Present-Incomplete

Body cavity Absent

Sexes Monoecious

LIFECYCLE:

Host Definitive host – Man

Intermediate host:

1) Snail

2) Crab or crayfish

Mode of transmission Ingestion

Larvae Five larval stages – Cercaria,

metacercaria, redia, miracidium, sporocyst

Infective form Metacercaria larva

Diagnostic form Eggs

PATHOGENESIS:
Multiplication of adult worm
● In lung causes Pulmonary Paragonimiasis (Endemic Haemoptysis)

● In extrapulmonary sites causes Extrapulmonary Paragonimiasis

CLINICAL MANIFESTATIONS:
Pulmonary Paragonimiasis (Endemic Haemoptysis):

● Caused by Paragonimus westermani

● The adult worm causes eosinophilic granulomatous inflammation in the

lungs

● Subsequently forms cysts surrounding the worms commonly in the right lung

PRESENTING FEATURES:
- Cough with brownish blood-tinged rusty sputum

- Frank hemoptysis
 - In chronic cases, bronchitis, bronchiectasis, pneumonia, lung abscess

Extrapulmonary Paragonimiasis:

● Caused by Paragonimus species

● Due to the migration of worms from the ruptured cysts to the liver, spleen,
abdominal wall, and brain

● Eg: Cerebral Paragonimiasis, Cutaneous Paragonimiasis

LABORATORY DIAGNOSIS:
1. SPUTUM MICROSCOPY: Sample - early morning, deeply coughed sputum

- Multiple sputum examination

- Sputum concentration by formalin ether sedimentation

- Stool microscopy to detect eggs in children

2. SEROLOGY: During the early part of the disease

a) Antibody detection tests:

- DIGFA (Dot-Immunogold Filtration Assay) – For serum antibodies to P.
westermani

- ELISA - For parasite-specific IgG or IgE in pleural fluid

b) Antigen detection tests:

- Dot ELISA format – To detect species-specific and stage-specific antigens

3. OTHER TESTS:

- Peripheral blood eosinophilia

- Radiological tests like MRI, CT scan, chest x-ray

TREATMENT:
● Praziquantel
● Surgical management for pulmonary or cerebral lesions
PREVENTION:
● Sanitary disposal of sputum
● Control of snails
● Treatment of cases
● Health education
 10.1. DWARF TAPEWORM

● Hymenolepis nana

● Smallest cestode infecting man

● Causes Hymenolepiasis – the most common tapeworm infection throughout

the world, higher prevalence among children

LIFECYCLE:

Host Mode of Infective form Diagnostic Organs

transmission form infected
Definitive host Ingestion Embryonated Embryonated GIT
Man (only host) Auto-infection eggs eggs
CLINICAL MANIFESTATIONS:
- Asymptomatic (usually)

- Anorexia, abdominal pain, headache, dizziness, diarrhea with mucus (when

worm burden exceeds)

LABORATORY DIAGNOSIS:
● Stool microscopy – eggs

● Stool concentration – If the egg load is less

TREATMENT:

- Praziquantel against both adult worms and cysticercoid larvae

- Nitazoxanide or Niclosamide

PREVENTION:
● Good personal hygiene

● Improved sanitation

10.2. LARVAL FORMS OF DIPHYLLOBOTHRIUM LATUM

● Diphyllobothriasis – Intestinal parasitic infection, caused by a cestode
Diphyllobothrium latum, also known as fish tapeworm.
● The largest known parasite is found in the human intestine.
● D. latum has three morphological forms:
1. Adult worm
2. Operculated egg
3. Larvae: Three stages
The first stage (Coracidium)
Second stage (Procercoid larva)
Third stage (Plerocercoid larva).
● Definitive Host:
▪ Humans (Common)
▪ Dogs, cats, and foxes (Rare)
There are two intermediate hosts:
First intermediate hosts: Freshwater copepods mainly of the genera Cyclops and
Diaptomus
Second intermediate hosts: Freshwater fishes (pike, salmon, perch, and trout)
Infective form: Third stage plerocercoid larvae.

MODES OF TRANSMISSION: Humans get the infection by ingestion of

undercooked freshwater fish containing third-stage plerocercoid larva.
● Human GIT: Plerocercoid larva develops into an adult worm in the human small
intestine; which sexually matures and fertilization takes place to produce eggs that are
released in feces (Diagnostic form)
● In Cyclops: Eggs in water transform into L1 larva which infects Cyclops. In Cyclops,
L1 develops into L2 which infects Fish
● In Fish: L2 larva develops into form L3 which becomes the infective form
12.1 PATHOGENESIS OF STRONGYLOIDES STERCORALIS
● The infection caused by S. stercoralis is known as strongyloidiasis
● Generally, it is benign and asymptomatic
● In symptomatic cases, it can be classified as

1. Cutaneous

2. Pulmonary

3. Intestinal

Hyper infection: Severe disease seen in immunocompromised persons

Generalized strongyloidiasis may be found in AIDS

CUTANEOUS
There may be dermatitis with erythema and itching at the site of penetration of filariform
larvae.

Common symptoms of chronic strongyloidiasis: Pruritus &amp; Urticaria

Larva currens is a characteristic sign of strongyloidiasis

PULMONARY
The larvae of S. stercoralis migrate through the lungs and escape from the pulmonary

capillaries into the alveoli leading to hemorrhages in the lung alveoli.

Bronchopneumonia may be present, which may lead to chronic bronchitis or asthmatic

symptoms.

Rhabditiform larvae may be found in the sputum.

INTESTINAL
Patients develop intermittent abdominal pain, distension, and diarrhea alternating with

constipation.

The symptoms may resemble those of malabsorption or peptic ulcer

HYPERINFLATION:
S. stercoralis hyper infection is frequently seen in debilitated individuals particularly in those
with cellular immune defects. It may also be seen in patients with immunosuppressive
therapy and co-infection with HIV. Extensive internal reinfection takes place.
 13.1. ONCHOCERCA VOLVULUS
● The causative agent of “river blindness” in man.
● Endemic in West Africa and also in South and Central America.

LIFE CYCLE:
● Life cycle – similar to – W. bancrofti, except the vector is Simulium (black flies).
● Following the bite of black flies - L3 larvae - transmitted to man and transform into
adult worms - migrate to subcutaneous tissues and eyes
● Gravid female worms release microfilariae - migrate to the blood - infective to the
black flies.

CLINICAL FEATURES:
● Skin (Dermatitis)
▪ Leopard skin
▪ Sowda
● Onchocercoma
● Ocular lesions
● Lymphadenopathy

LABORATORY DIAGNOSIS:
DETECTION OF THE PARASITE:
● In a skin snip smear – a gold standard method for diagnosis of onchocerciasis.
● Microfilariae – found either in the skin (90%) or in the nodules (10%).
● Microfilariae: Measure 254 μm long, pointed tail tip without any nuclei – unsheathed
and nonperiodic.
● Adult worms - detected from the biopsy of the subcutaneous nodules - less sensitive

OTHER METHODS:
● Serology
● Molecular methods
● Mazzotti skin test
TREATMENT OF ONCHOCERCIASIS:
● Ivermectin – Drug of choice for onchocerciasis
● Given orally in a single dose of 150 μg/kg – yearly or biannually.
● Surgical excision – recommended when nodules are located on the head.
 14.1 & 14.2. CYCLOPS
Cyclops is one of the most common genera of freshwater copepods, comprising over
400 species. Together with other similar-sized non-copepod fresh-water crustaceans,
especially Cladocera, they are commonly called water fleas. It comes under the phylum
Arthropoda of the kingdom Animalia.

ROLE OF CYCLOPS IN PARASITIC INFECTION:

Cyclops are intermediate hosts for; 1. Dracunculus medinensis
2. Diphyllobothrium latum
3. Spirometra
4. Gnathostoma spinigerum

DRACUNCULUS INFECTION:
1. Caused by Dracunculus medinensis,
2. Cyclops is an intermediate host in which an embryo undergoes
developmental changes.
3. The mode of infection is by drinking unfiltered water.

DEVELOPMENT OF LARVA IN CYCLOPS

● Larva swims in water & survive for a week
● Larva are swallowed by freshwater Cyclops [intermediate host]
● Larva penetrate the gut wall of Cyclops & its body cavity, twice molts
● In 2-4 weeks develops into infective form L3
● The entire life cycle takes about a year, formation of blisters & other
clinical manifestation

PROPHYLAXIS:
● Filter water through clothes
● Chemical treatment using Abate [temephos]

DIPHYLLOBOTHRIASIS INFECTION:
1. Cyclops is the first intermediate host.
2. The coracidium larvae in water are ingested by Cyclops.
3. In the midgut of Cyclops, coracidium casts off its ciliated coat & by 6
hooklets penetrates unto hemocele.
4. In 3 weeks, transformation to procercoid larva occurs.
 5. Procercoid larva is ingested by fishes & changes into sparganum and
causes subsequent infection.

PROPHYLAXIS:
● Proper cooking of fish
● Boiling water

SPARGANOSIS:
1. Caused by Spirometra, Cyclops is the first intermediate host.
2. Adult worm in the intestine of dogs Eggs in feces Eggs
in freshwater coracidium release ingested by Cyclops
develops to Procercoid larvae ingested by fish & subsequent
infection.

PROPHYLAXIS:
● Proper cooking of fish
● Boiling water

GNATHOSTOMIASIS INFECTION:
1. Caused by Gnathostoma spinigerum, Cyclops is the first intermediate
host.
2. The L1 larva is ingested by Cyclops → second stage larva appears
ingestion → by fishes.

PROPHYLAXIS:
● Proper cooking of fish
● Boiling water
 15.1 VISCERAL LARVA MIGRANS
● Primarily caused by Toxocara -Toxocara canis (dog) ,Toxocara catis (cat)
● Other Causes:
1. Ascaris suum (pig)

● Ingestion of eggs of nonhuman roundworms🡪 Rhabditiform larva 🡪 Filariform larva

🡪 Pass-through blood and lymph 🡪 Reach right side of the heart and lungs 🡪 Unable
to pass through pulmonary capillaries 🡪 invade visceral organs and cause eosinophilic
granuloma.

● Any organ can be affected, mostly liver🡪 HEPATOSPLENOMEGALY, common.

● Younger children affected
● Other features: lymphadenopathy, lung involvement, urticaria, nodules, seizures from
cerebral involvement

15.2 CUTANEOUS LARVA MIGRANS

● Aka creeping eruptions
● Larva migration occurs in the skin and subcutaneous tissue
● Causes:
1. Ancylostoma braziliense (dog)
2. Ancylostoma caninum (dog)
3. Bunostomum
4. Uncinaria (dog)
 5. Gnathostoma Spinigerum

● Mode of infection: skin penetration by nonhuman hookworms

● Filariform larvae 🡪 Can’t cross the basement membrane of skin 🡪 Keep migrating in
the skin for weeks to months 🡪 Die in a few weeks in the skin
● Serpiginous eruptions are seen along migrating paths
● Migrate at approx. 11mm/hr ( slower than Strongyloides)
● Generally resolves spontaneously
● Treatment: Ivermectin

15.3 LARVA MIGRANS

● Larva migrans describe a parasitic disease involving the migration of immature
(larval) worms in various parts of the body.
● After accidental infection 🡪 Life cycle gets arrested 🡪 Wander aimlessly in the body
● Occurs in one of three forms.
● When the worms migrate through the skin of the host 🡪 cutaneous (skin) larva
migrans.
● If the worm larvae migrate through various internal organs of the host 🡪 visceral
larva migrans.
● Sometimes the worm larvae may invade the eye of the host 🡪 ocular (eye) larva
migrans.
● Humans and a wide range of animal species can be affected by this disease.
● OCULAR LARVA MIGRANS :
1. Most commonly caused by Toxocara larva.
2. Unilateral painless chorioretinal granuloma in the posterior pole – most
common presentation.
 16. LOA LOA
● Filarial nematode a.k.a African eye worm
● Cutaneous manifestations‐ subcutaneous tissue and eye
● LIFE CYCLE:

● CLINICAL FEATURES:
1. Calabar/fugitive swelling: subcutaneous swelling on extremities‐
2. Ocular manifestations‐ conjunctival granuloma, edema of eyelid
leading to proptosis
3. Meningoencephalitis‐ most severe complication
4. Nephropathy and cardiomyopathy – rare complications

● LAB DIAGNOSIS
1. Peripheral blood smear: microfilaria, sheathed, long, have a column of
nuclei, show diurnal periodicity
2. Biopsy of subcutaneous selling: adult worms
3. Nested‐PCR – detection of specific DNA
4. Antibody detection‐ detects antibodies to L1‐SXP‐1 antigen

● TREATMENT
1. Diethylcarbamazine‐ DOC
2. Glucocorticoids‐ reduce inflammation against microfilariae
3. Albendazole / ivermectin
4. Surgical removal (rare)
 17. CRYPTOSPORIDIUM PARVUM
● Intestinal coccidian parasite
● Causes opportunistic infections in HIV patients
● Cause watery diarrhea
● This species affects – mammals including humans
● Morphology of oocyst :
■ Can be thick/thin-walled
■ Round
■ Measure 4‐6 micrometers
■ Contains 4 sporozoites
● LIFE CYCLE:
● PATHOGENESIS
1. Excystation: in the small intestine and release of sporozoites
2. Attachment: pf sporozoites to the small intestinal epithelium facilitated by
CP47
3. Penetration: using discharges from the apicomplexan present in sporozoite‐
invasion
4. Vacuole: parasite forms a parasitophorous vacuole near the microvilli surface
5. Cytokine release: parasite activates host immune system
6. Increased intestinal secretion of water and decreased sodium
absorption
● CLINICAL FEATURES
1. Immunocompetent hosts
■ Asymptomatic‐ mostly
■ Symptomatic – symptoms develop after approx. a week
➔ Watery non‐bloody stools, abdominal pain, fever, anorexia
➔ Self‐limiting
2. Immunocompromised hosts
a. Chronic, persistent profuse diarrhea
b. Extraintestinal manifestations
● LABORATORY DIAGNOSIS
1. Direct fluorescent antibody staining: gold standard test
2. Acid‐fast staining: uniformly acid‐fast
3. Autofluorescence‐ no, but can be stained with fluorescent dye.
4. Antigen detection
a. ELISA
b. ICT
5. Antibody detection‐ ELISA
6. Real-time PCR
7. BioFire Film Array
8. Fecal leukocyte marker

● TREATMENT
1. Mild cases: self-limiting, ORS
2. Severe cases: Nitazoxanide – 3 days or Paromomycin
 18.1 EXAMINATION OF FECES FOR PARASITIC
INFECTIONS
Feces should be examined for their color, consistency, odor, presence of blood or mucus. In
some instances, parasites may be seen on gross inspection as in the case of roundworms,
pinworm, tapeworm proglottids.

1. MICROSCOPY:
The microscope should be equipped with a micrometer eyepiece as it is essential to measure
the size of the parasite

It should include contributory findings such as the presence of Charcot Leyden crystals and
cellular exudate

2. WET MOUNTS:
Made by emulsifying a small quantity of feces in a drop of saline placed on the slide and
applying a coverslip on top, avoiding air bubbles. If the feces contain mucus, it is advisable to
prepare film with mucus.

Eosin, 1% aqueous solution used for staining. Eosin stains everything except lining
protoplasm. Chromatid bodies and nuclei of amoebic cysts can be seen distinctly. It also
indicates the viability of cyst

3. THICK SMEAR:
About 50 mg feces is taken on the slide and covered with wettable cellophane coverslip
soaked in glycerine containing aqueous malachite green and left for an hour, in which
glycerine clear feces and helminths eggs can be seen distinctly.

Not useful in routine examination and also not useful for protozoa or helminth larvae.

4. PERMANENT STAINED SMEAR:

These are employed for the identification of protozoa in feces and also as permanent records.
There are 2 methods commonly used,

· Iron hematoxylin stain

· Wheatley's trichrome stain

5. CONCENTRATION METHODS:
Concentration may be done using fish or preserved feces. There are 2 commonly used
methods, floatation, and sedimentation
In the floatation method, feces is suspended in a high specific gravity solution, so that
parasitic eggs and cyst can float. This is useful for protozoa cyst and eggs of nematodes and
small tapeworms. But it doesn't detect unfertilized roundworm eggs, nematode eggs,
nematode larvae, and eggs of most trematodes and large tapeworms.

In the sedimentation method, feces are suspended in a low specific gravity solution, so that
eggs and cysts can get sedimented. This method is useful for all helminth eggs and protozoan
cysts.

6. EGG COUNTS:
A semiquantitative assessment of the worm can be made by estimating the number of eggs
passed in the stool. This is done by egg counts and by relating the counts to the number of
worms present by assuming the number of eggs passed per worm per day.

These are at best approximations and only a rough indication of worm burden can be
obtained. Egg count helps to classify helminth infections as heavy, moderate, or light.

7. FECAL CULTURE:
Fecal culture is not used for routine diagnosis, but species identification

Eg: In differentiation between Ancylostoma and Necator.

 19.1 VIVIPAROUS NEMATODES
· Female worms directly give birth to larvae

· There is no egg stage

· Examples: Trichinella, Wuchereria, Brugia, Dracunculus

TISSUE / SOMATIC NEMATODES

● Nematodes that infect the different tissues are called tissue or somatic
nematodes.
● The filarial nematodes are the major group of tissue-nematodes.
● The filarial nematodes belong to the superfamily Filarioidea; they are slender,
thread-like worms.
● They inhabit the circulatory and lymphatic channels, connective tissue, and
serous cavities.
● The female worms are viviparous.
● Eight species of filarial worms infect humans, who are the definitive hosts.
Wuchereria bancrofti:
1. Adult worms reside in the lymphatic system.
2. Undergo fertilization
3. Release L1 larvae – microfilariae
4. Males die after mating, females live up to 20 yrs
5. A gravid female worm can discharge 50,000
microfilariae/day.

Brugia malayi
1. Adult worms reside in the lymphatic system

Dracunculus medinensis
1. Adult female worm releases L1 larvae in water
2. L1 larvae molt into L3 larvae inside an intermediate host –
Cyclops
3. Enters humans when they ingest water contaminated with
Cyclops
4. Causes cutaneous blisters

Trichinella spiralis
 20.1 BILE STAINED EGGS
∙ Bile staining properties are well appreciated in saline mount.
∙ Bile stained eggs appear golden brown and non‐bile stained eggs appear
colorless ∙ Not well-appreciated in iodine stain

Include‐
∙ Diphyllobothrium latum‐ eggs do not float in saturated salt solution and are
operculated ∙ Taenia species
∙ Schistosoma species‐ non‐operculated eggs
∙ Fasciola hepatica – operculated eggs
∙ Fasciolopsis buski – operculated eggs
∙ Ascaris lumbricoides‐ fertilized eggs float and unfertilized eggs do not float in
saturated salt solution
∙ Trichuris trichiura – float in saturated salt solution
Eggs of most intestinal parasites when they pass through the intestine are stained by bile.
The exceptions are Enterobius, hookworm, and Hymenolepis nana; these eggs are
non‐bile stained.
 APPLIED MICROBIOLOGY

1. E – Hospital associated infections

2. E - Emerging vs non emerging infections
3. E – Pyrexia of Unknown Origin
4. E – Zoonosis
5. SN – Normal flora of human body
Prebiotics vs Probiotics
Harmful effects of normal flora
Bacteriology of milk
6. SN – Diarrhea vs dysentery
7. SN – Bacteraemia and septicaemia
8. SN – Food poisoning
9. SN – Antibiogram
10.SN – Coagulase
11.SN – Significant bacteriuria
12.SN – Laboratory diagnosis of water & water borne diseases
13.SN – National Immunisation schedule
Biomedical waste management
14.SN – Urinary tract infections
15.SN – Sexually transmitted diseases
16.SN – Congenital infections
17.SN – Vector borne diseases
18.SN – Bacteriology of milk
19.SN – Faecal examination concentration methods
PCR
20.SN – Recombinant technology and implications
 ESSAY 1

WHAT ARE THE MAJOR TYPES OF HOSPITAL INFECTIONS?

EXPLAIN THEM IN DETAIL. LIST THE UNIVERSAL PRECAUTIONS
TAKEN TO PREVENT HOSPITAL ASSOCIATED INFECTIONS.

HOSPITAL ACQUIRED INFECTIONS

Infections that occur after 48 hours of admission / procedure in the hospital and up to 30 days
after procedure / discharge from the hospital.

TYPES OF HOSPITAL ASSOCIATED INFECTIONS:

The common types of HAI are:
1. Catheter – Related Blood stream infection – CRBSI, CLABSI
2. Ventilator associated Pneumonia
3. Catheter – Associated Urinary Tract Infections
4. Surgical site infection
5. Post – antibiotic Diarrhea
6. Others – Hepatitis, Tetanus

1. CATHETER-RELATED BLOODSTREAM INFECTION:

- Commonly caused by infected intravenous cannula

RISK FACTORS:

Device related:

● Central or Peripheral lines

● Type of Catheter
● Number of Lumens

Patient Related:

● Immunocompromised
● Total parenteral nutrition
 ● Loss of Skin Integrity

Caregiver Related: Poor Hand Hygiene

COMMON ORGANISMS:
● Intrinsic Contamination: Klebsiella, Enterobacter, Pseudomonas
● Extrinsic Contamination: CoNS, S. aureus
PREVENTION:
● Hand Hygiene before and after insertion
● Use Maximum sterile PPE
● Care of IV lines
● Skin preparation by Antiseptics

2. VENTILATOR- ASSOCIATED PNEUMONIA

Defined as the Pneumonia that occurs 48-72 hours after Endotracheal Ventilation.

RISK FACTOR:
Device Related – Nasogastric Tube
Ventilation Duration
Intervention Related – Stress Ulcer prophylaxis
Tracheostomy

COMMON ORGANISMS:
Generally caused by Multi-drug resistant strains of
● Acinetobacter baumannii
● Pseudomonas
● Klebsiella

PREVENTION:
● Daily Oral care with Chlorhexidine 2% solution
● Adherence to Hand Hygiene
● Elevation of the head of bed to 30-450
3. CATHETER- ASSOCIATED URINARY TRACT INFECTION

● Most Common HAI

● Caused due to the Presence of an Indwelling Catheter

RISK FACTORS:
Device Related: Long-Term Catheterization
Latex type of Catheter
Patient Related: Female Gender
Older Age
Diabetes Mellitus
Incomplete emptying of bladder

COMMON ORGANISMS:
● E. coli
● Proteus
● Enterococcus

PREVENTION:
● Catheter care
● Appropriate type of Catheter for long- term use
● Using Closed drainage system
● Using Sterile Items for Catheter Insertion

4. SURGICAL SITE INFECTION

● Infections that develop at the Surgical Site within 30 days of Surgery

● Can cause Morbidity and Mortality if left untreated.

RISK FACTORS:
Patient Related: Age>60 years
Malnutrition, Diabetes
Higher Wound Class
 Immunosuppression
Procedure Related: Improper surgical scab
Inadequate skin antisepsis
Prolonged Operative Time

COMMON ORGANISM:
● S. pyogenes
● MRSA
● E. coli

PREVENTION:
● Preoperative Bathing
● SAP must be provided
● Surgical hand disinfection
● Surgical site preparation

5. OTHER INFECTIONS

NEEDLESTICK AND SHARPS INJURIES

● Carries risk of infection by bloodborne pathogens like Hepatitis – B, C, D and HIV
● They can arise due to:
1. Device with needles
2. Syringes with Exposed Needles
3. Butterfly needles

6. ANTIBIOTIC – ASSOCIATED DIARRHEA

● Occurs due to prolonged uses of Antibiotics

● The Normal Gut Flora gets suppressed due to antibiotics

COMMON ORGANISMS: Clostridium difficile

 Reference: Ananthanarayan and Paniker’s Textbook of Microbiology E/11 Page No. 602

STANDARD / UNIVERSAL PRECAUTIONS OF HAIS:

● They are indicated while handling all Patients, Specimens and Sharps.
● Components include:
1. Hand Hygiene
2. Personal Protective Equipment (PPE)
3. Biomedical Waste
4. Spillage Cleaning
5. Disinfection of Patient care items
6. Environmental Cleaning
7. Sharp
8. Respiratory hygiene and cough etiquette

HAND HYGIENE:

- The Most important measure to avoid transmission of harmful microbes

TYPES:
● Hand Rub
 ● Hand Wash

5 MOMENTS FOR HAND HYGIENE:

1. Before touching a patient
2. Before Cleaning / Aseptic procedures
3. After Body Fluid Exposure
4. After Touching a Patient
5. After Touching Patient’s Surroundings.

PERSONAL PROTECTIVE EQUIPMENT (PPE):

● Used to Protect the Skin and Mucous membranes of HCWS from exposure to
Body fluids and from the HCW to the Patient.

TYPES:
1. Gloves
2. Should be worn during Anticipation of Contact
1. Should not be Reused
2. Same glove Shouldn’t be used on more than one patient.
3. Mask
4. Plastic apron / Gown
⮚ Should be worn to Protect Skin and Clothing during procedures
where contact of Body Fluid is anticipated
o Goggles / Face Shield
⮚ Used in Procedures that are likely to generate Sprays of Body Fluids.
5. Shoe Cover
6. Head Cover

BIOMEDICAL WASTE:

● All Biomedical Waste should be Segregated and Disposed appropriately.

DISINFECTION OF PATIENT CARE ITEMS:

● Ensure that all Patient Care Items such as Instruments, Devices, and Linens are
disinfected before reuse.

SPILLAGE CLEANING:

● Spillage of body fluids should be considered infectious and to be Cleaned at the

earliest.
● The area is cleaned with Hypochlorite.

SHARP:

● Safe use and Disposal of Sharp

● Sharp are disposed in Sharp Box which is a puncture – proof container

ENVIRONMENTAL CLEANING:

● Sterilization and Disinfection of Surface and floors in a hospital is important in

preventing Transmission of HAIs.

ESSAY 2

EMERGING AND RE-EMERGING INFECTIONS

EMERGING INFECTIONS:

● Infectious diseases
● Infections in humans have increased in the past 2 decades or threatens to increase in
near future
These include:

1. New infections, as a result of evolution

2. Known infections, spreading to new geographical populations
3. Previously unidentified infections
 Causes:

● Ecological disruption and human intrusion into new ecological system

● Climate change
● Urbanization and Industrialization
● International trade of goods and services
● Increased number of immunocompromised hosts

Ex: COVID-19 pandemic

RE-EMERGING INFECTIONS:

● Old infections
● Clinically silent, or reduced in numbers, and have re-emerged in the community

This can occur as a result of:

1. Antimicrobial resistance in agents

2. Breakdown in public health measures

Ex: Chikungunya re-emergence in 2005

Drug resistance and re-emergence:

▪ Occurs as a result of development of antimicrobial resistance

▪ Ex:
● Multidrug resistant tuberculosis (MDR-TB)
● Extensively drug resistant tuberculosis (EDR-TB)
● Vancomycin resistant enterococci (VRE)

MANAGEMENT OF EMERGING INFECTIOUS DISEASE:

- A proactive and planned approach to ensure the appropriate prevention and control of
the spread of disease.

Strategic planning should include:

1. Phase I (non-alert) is a routine, preparatory state

 2. Phase II (alert) is the detection, confirmation and declaration of changes
identified during non-alert conditions
3. Phase III (response) includes the ongoing assessment of information and the
planning and implementation of an appropriate response, which includes the
coordination and mobilization of resources to support intervention activities
4. Phase IV (follow-up) activities include re-evaluation

RECOMMENDATION:

● Strengthening epidemiological surveillance & laboratory capabilities and services.

Establishment of a rapid response team.
● Monitoring antimicrobial resistance.
● Establishment of international disease surveillance, networking and advocacy.
● Screening on International travels and trades.
● Networks of laboratories that link countries and regions need to be established.
● Strong national and regional public health systems.

Essay 3
PYREXIA OF UNKNOWN ORIGIN

Fever persisting for more than 2 weeks, with no clear diagnosis despite intelligent and
intensive investigation.

● Pyrexia of unknown origin (PUO) was classically defined as a temperature above

38.0°C on multiple occasions for more than 3 weeks, without diagnosis, despite initial
investigation in hospital for 1 week.
● The definition has been relaxed to allow for investigation over 3 days of inpatient
care, three outpatient visits or 1 week of intensive ambulatory investigation.
● Subsets of PUO are described as HIV-1 related, immune-deficient or nosocomial.
● Up to one-third of cases of PUO remain undiagnosed.
ETIOLOGY:
CLINICAL ASSESSMENT:
● Rare causes, such as periodic fever syndromes, should be considered in those with a
family history.
● Children and younger adults are more likely to have infectious causes – in particular,
viral infections.
● Older adults are more likely to have certain infectious and non-infectious causes.
● Detailed history and examination should be repeated at regular intervals to detect
emerging features (e.g., rashes, signs of infective endocarditis or features of
vasculitis).
● In men, the prostate should be considered as a potential source of infection.
● Clinicians should be alert to the possibility of factitious fever, in which high
temperature recordings are engineered by the patient.
INVESTIGATIONS:
● If initial investigation of fever is negative, further microbiological and
non-microbiological investigations should be considered.
● As with initial investigation of fever described above, the selection and prioritisation
of tests will be influenced by the geographical location of potential exposure to
pathogens.
● These will usually include: induced sputum or other specimens for mycobacterial
stains and culture
• serological tests, including an HIV test, and ferritin estimation
• imaging of the abdomen by ultrasonography or computed tomography (CT)
• echocardiography.
● Lesions identified on imaging should usually be biopsied in order to seek evidence of
relevant pathogens by culture, histopathology or nucleic acid detection.
● Particularly in patients who have received prior antimicrobials, 16S rRNA analysis
may aid diagnosis if a microorganism is not cultured.
● The chance of a successful diagnosis is greatest if procedures for obtaining and
transporting the correct samples in the appropriate media are carefully planned
between the clinical team, the radiologist or surgeon performing the procedure, and
the local microbiologist and histopathologist.
● Positron emission tomography (PET) scans – for diagnosis of vasculitis or help
selection of biopsy sites.
● Liver biopsy – to identify idiopathic granulomatous hepatitis – if there are
biochemical or radiological abnormalities.
● Bone marrow biopsies reveal haematological malignancy, myelodysplasia or
tuberculosis and for identifying brucellosis, typhoid fever or visceral leishmaniasis.
● Bone marrow – for culture and microscopy.
● Laparoscopy is occasionally undertaken with biopsy of abnormal tissues.
● Splenic aspiration – diagnostic test for suspected visceral leishmaniasis.
● Temporal artery biopsy should be considered in patients over the age of 50 years,
even in the absence of physical signs or a raised ESR.
PROGNOSIS:

- No cause is found in approximately 10% of PUO cases, but as long as there is no

significant weight loss or signs of another disease, the long-term mortality is low.
 ESSAY 4
ZOONOSIS
Any disease or infection that is naturally transmissible from vertebrate animals to humans.
Zoonoses may be associated with bacterial, viral, or parasitic, or due to fungal agents.

CLASSIFICATION (in terms of their reservoir hosts):

* Anthropozoonoses: Infections transmitted from animals to man.
* Zooanthroponoses: From man to animals
* Amphixenoses: In both man and animals transmitted in either direction.

COMMON ZOONOTIC INFECTIONS:

1. PLAGUE (YERSINIA PESTIS)
The tribe Yersinieae – genus Yersinia – 11species (3 are important human pathogens)
1) Yersinia pestis: It is the agent of plague
2) Y. pseudotuberculosis and Y. enterocolitica – both cause yersiniosis, a
self-limiting gastrointestinal illness

EPIDEMIOLOGICAL FACTORS:
* Reservoir: Wild rodents, such as gerbils (Tatera indica), field mice and the
bandicoot
*Source of infection- infected wild rodents, rat fleas and cases of pneumonic plague
*Vector:
1) Rat flea (common); species – Xenopsylla cheopis (most efficient) and X.
astia and X. brasiliensis
2) Human flea (Pulex irritans) – rare*

MODE OF TRANSMISSION:
- Bite of an infected rat flea (most common), human flea
- Direct contact with tissues of infected animal (rodents)
- Droplet inhalation (man to man)
 *Blocked flea: In a blood meal, the fleas suck blood containing bacilli from infected
rodents. In the gut of the flea, the bacilli multiply enormously and may block the
proventriculus-called as blocked flea.
*Extrinsic incubation period – Interval between the flea acquiring the infection
through blood meal and becoming blocked flea (2 weeks for Xenopsylla cheopis)
*Cheopis index – Average number of X. cheopis per rat Plague outbreak in places
having cheopis index > 1

VIRULENCE FACTORS OF Y. PESTIS:

Fraction 1 (F1) antigen: It is a capsular protein antigen, encoded by a plasmid (pFra);
principal virulence factor; acts by inhibiting phagocytosis. It is a highly antigenic -
Immunodiagnostic marker of infection.
Other virulence factors – Phospholipase D, surface proteases, pH 6 antigen,
lipopolysaccharide, type III secretion system, adhesins and siderophore.

HUMAN PLAGUE: CLINICAL TYPES

(1) Bubonic, (case-fatality ratio is nearly 30-60%)
(2) Pneumonic – Rare but highly infectious (C: F=30-100%)
(3) Septicaemic

BUBONIC PLAGUE – Most common type

Transmission – By the bite of an infected rat flea.
Incubation period – About 2-7 days
Symptoms – Fever, malaise, headache, and painful lymphadenitis
Buboes: Regional lymph nodes appear as tense, tender swellings called buboes; site- inguinal
(common), crural, (axillary & cervical in children), or submaxillary.

PNEUMONIC PLAGUE -
Transmission – Inhalation of bacilli in droplets expelled from contaminated persons or
animals.
Incubation period – About 1-3 days
Symptoms – Fever, headache, and respiratory symptoms (productive cough or haemoptysis,
dyspnea, and chest pain)
Agent of bioterrorism-aerosolized Y. pestis is a possible source of bioterrorism attack,

SEPTICAEMIC PLAGUE (BLACK DEATH)

Primary septicaemic plague – rare; Secondary septicaemic plague – common
Incubation period – about 2-7 days
Symptoms – haemorrhages in skin and mucosa → gangrene of the affected site

LABORATORY DIAGNOSIS OF PLAGUE:

SPECIMEN COLLECTION
• Bubonic plague – pus or fluid aspirated from buboes
• Pneumonic plague – sputum and blood
• Septicaemic plague – blood and splenic aspirate (post-mortem)

TRANSPORT MEDIUM (e.g., Cary-Blair medium)

DIRECT MICROSCOPY-
*Gram staining
*Wayson stain or methylene blue staining

CULTURE (Y. pestis is aerobic and facultatively anaerobic)

*Blood agar
*MacConkey agar

CULTURE SMEAR
Gram staining of culture smear reveals pleomorphism.

MOTILITY TESTING
Y. pestis-nonmotile both at 25°C and 37°C; other Yersinia species-motile at 25°C and
non-motile at 37°C.
IDENTIFICATION

AUTOMATED IDENTIFICATION SYSTEMS

*MALDI-TOF – can be used to differentiate three biotypes of Y. pestis

CONVENTIONAL BIOCHEMICAL TESTS

*ICUT tests:
Indole test (negative), citrate test (negative), urease test (negative) and TSI (triple
sugar iron agar) test shows alkaline/acid.

TREATMENT: “antibiotics”
Streptomycin has been the choice of treatment for plague(past)-given for 10 days.
Gentamicin is superior to streptomycin and currently recommended.
Levofloxacin, doxycycline, and chloramphenicol are also effective.

PREVENTION:
*Control of cases by early diagnosis, Isolation precaution
*Control of fleas by use of effective insecticides, control of rodents.
*Vaccine:
• Formalin killed vaccine (Sokhey's modification of original Haffkine vaccine) given
subcutaneously, two doses 4 weeks apart and a booster after 6 months.
• Live attenuated vaccine based on strain EV76

TULARAEMIA
CAUSATIVE AGENT – FRANCISELLA TULARENSIS
Primarily a plague-like disease of rodents and other small animals
F. tularensis - An agent of bioterrorism.

PREVALENCE:
F. tularensis – Four subspecies: Tularensis (most common & most virulent), holarctica,
novicida and mediasiatica.
CLINICAL MANIFESTATIONS:
* Ulceroglandular tularemia: most common form (75-85% of total cases) - ulcerative lesion
at the site of inoculation, with regional lymphadenopathy
Other forms-Pulmonary, oropharyngeal, oculoglandular form and typhoid-like illness
* Complications -Suppurated lymph nodes, acute kidney injury, hepatitis, rhabdomyolysis,
empyema, pericarditis, meningitis, osteomyelitis, and endocarditis.

LABORATORY DIAGNOSIS:
• Culture: special media-BCG agar (blood cysteine glucose agar)
• Specimen: Ulcer scrapings, and lymph node biopsy
• Species identification from colonies –
-By conventional biochemical tests or by automated identification systems such as VITEK.
- Antibody detection is the mainstay of diagnosis-Agglutination tests, ELISA, PCR.

TREATMENT:
Gentamicin – the drug of choice; given for 7-10 days. Doxycycline or ciprofloxacin –
alternatives.

3. BITE WOUND INFECTIONS

Bites and scratches from animals and humans allow the inoculation of microorganisms that
are commonly found in the animal's oral cavity, nose, or nail by lodging on the wound
surface.
* Can breach skin barrier and penetrate deeper causing osteomyelitis and septic arthritis.
* Invasion of lymphatics and blood cause bacteraemia, meningitis, brain abscess, and
endocarditis.
• Rarely, infection of the cutaneous nerves carries organisms to CNS (e.g., rabies causing
encephalitis).

DOG BITES
Accounts for 80% of all animal-bite wounds; infection- 15-20%
* Age/gender: children>adults, and males>females
*Site: often upper extremity, except for children <4 years -in head and neck region
*Microbiology:
- Aerobes - B-haemolytic streptococci, Pasteurella species, Staphylococcus, Eikenella
corrodens, and Capnocytophaga canimorsus.
- Anaerobes -Actinomyces, Fusobacterium, Prevotella, and Porphyromonas species
- Organisms causing systemic diseases: Rabies and tetanus.

CAT BITES
Cat bites and scratches → infection (>50% of cases) - higher risk of causing penetrating
injury-septic arthritis and osteomyelitis
*Victims -women>men
*Microbiology: Pasteurella multocida, Bartonella henselae (Causes cat-scratch disease),
Tularemia (Francisella tularensis), S. aureus, Anaerobes. Organisms causing systemic
disease – rabies (rare) and tetanus.

HUMAN BITES
Human bites may take place during fights, domestic abuse, sexual activity, or healthcare
workers caring for patients.
Infection-less → (10-15% of the time)
*Types of human bites: two types
- Occlusional injuries: inflicted by actual biting
- Clenched-fist injuries (more common): Occurs during fights.
* Microbiology:
- Aerobes – viridans streptococci, S. aureus, Eikenella corrodens (common in clenched-fist
injury), and Haemophilus influenzae
- Anaerobes – Fusobacterium nucleatum and Prevotella, Porphyromonas, and Pepto
streptococcus species
- Hospitalized patients → Enterobacteriaceae, non-fermenters in addition

OTHER ANIMAL BITES

* Rat bite infections, Snakebites, Bites of Old-World monkeys (Macaca)&seals
LABORATORY DIAGNOSIS FOR BITE WOUND INFECTIONS:
Specimen:
- Purulent exudate aspirated from depth of wound.
- Wound swab (most common)

AGENTS CAUSING BITE-WOUND INFECTIONS

- Pasteurellosis: (P. multocida – common species)

CLINICAL FEATURES: In humans, P. multocida is the most common cause of wound

infections after dog or cat bites.

SYMPTOMS: Affected area > red, swollen and painful with regional lymphadenopathy and
bacteraemia (severe cases) > osteomyelitis or endocarditis or meningitis.

LABORATORY DIAGNOSIS: Automated methods – MALDI-TOF or VITEK

TREATMENT: Penicillin G or amoxicillin-clavulanate is the drug of choice.

Capnocytophaga Infection Species:

C. ochracea, C. gingivalis and C. sputigena – In human mouth, cause periodontal diseases,
and sepsis / meningitis in immunocompromised hosts.
C. canimorsus and C. cynodegmi – In dogs.

LABORATORY DIAGNOSIS: Appearance - fusiform or filamentous gram-ve coccobacilli

- Automated methods such as MALDI TOF or VITEK.

TREATMENT: As they produce B-lactamases, ß lactam inhibitor combo i.e.: ampicillin

sulbactam is used as the drug of choice.
 4. RAT-BITE FEVER

Characterized by septic fever, petechial rashes, and painful polyarthritis with frequent
relapses. This is known as Haverhill fever or epidemic arthritic erythema.
It is caused by:
(1) Streptobacillus moniliformis
(2) Spirillum minus.

TRANSMISSION:
- By contact with rodents carrying these bacteria and Consumption of contaminated food or
water.
Streptobacillus moniliformis:
Gram-negative, highly pleomorphic nonmotile bacilli, which is frequently arranged in chains
and tangled filaments with bulbous swellings.
It has a tendency to form an L-form.

Spirillum minus: (known as Sudoku) -They are rigid, spirally coiled motile bacilli It doesn't
grow in artificial media.

TREATMENT:
Penicillin is the treatment of choice.
IMPORTANT ZOONOTIC INFECTIONS AFFECTING HUMAN
BEINGS & THEIR SOURCES
 Essay 5

NORMAL MICROBIAL FLORA OF HUMAN BODY AND

BACTERIOLOGY OF WATER, AIR AND FOOD

A. Explain the normal flora of the human body and their role?
ROLE OF BACTERIA:

1. MOUTH
● Oral bacteria produce certain vitamin c and cofactor like vitamin K, Biotin,
Riboflavin
● Prevention of colonization by exogenous pathogens.
● Help in maturation of the host immune system.
● Digestible enzymes like salivary amylase.

2. NASOPHARYNX:
● It can be considered the natural habitat of the common pathogenic bacteria which
causes infection of the nose, throat and bronchi.
● After insulin therapy, they may be predominant flora.

3. GIT:
● Produce vit B12, Vit K
● Control growth of harmful bacteria.
● Breakdown poison in large intestine.
● Bacteria produces enzyme that digest carbohydrate in plant cell wall

4. FGT –VAGINA:
● Normal vaginal flora is predominantly aerobic.
● Normal vaginal flora is dominated by lactobacilli.
● Lactic acid helps to maintain a normal vaginal pH of 3.8 to 4.2
● Acidic environment and other host immune factors inhibit the overgrowth of bacteria
● Some lactobacilli also produce H2O2, a potent microbicide.

5. SKIN:
● Skin is the organ of the human body that protects from the pathogens from the
environment and retards the loss of excessive water.
● Its other functions are insulation, temperature regulation sensation and synthesis of
vitamin D
 B. What is the difference between prebiotics and probiotics?

PREBIOTICS PROBIOTICS

● Prebiotics are defined as a ● Probiotics are referred to as live active

non-digestible special form of fibre microorganisms (bacteria) that when
or carbohydrate. administered in adequate amount will
have beneficial effect to its host.

● The powder from prebiotics can ● Probiotics are more fragile. They are
survive heat, cold, acid and even vulnerable to heat and stomach acid.
time. They may also kill over time

● Prebiotics perform their role by ● Probiotics fight the harmful bacterial

nourishing the bacteria that lives in species present in the gut.
the intestines.

● Prebiotics have been argued to be ● There are various stains of probiotics

beneficial in irritable bowel bacteria which are proven to be effective
syndrome, inflammatory bowel in the relief of irritable bowel syndrome
disease, colon polyps, cancer and and bowel infections symptoms.
even leaky guts.

● Serve as food for friendly bacteria ● Bacteria or yeast

within the gut.
 ● Available as food supplements and ● Available as food supplements and in
naturally occurring in certain foods certain food containing live cultures
such as chicory root, Jerusalem such as yoghurt, coconut.
artichoke, onion, leek, garlic, carrots
and dandelion.

C. What are the harmful effects that can be brought in by the

normal flora of the human body?

- There exists a delicate balance between the human body and the microbial flora that
live symbiotically with the host or as commensals.
- Any factor affecting this balance results in adverse effects.

Major factor responsible for altering the balance and causing infections are:

● Prolonged antibiotics therapy, immunosuppressive agents and immunodeficiency

diseases.
● Alteration of pH (as in the genitourinary tract in the elderly and during
pregnancy)
● Increased virulence of the commensals, production of toxins and change in
microbial flora.

HARMFUL EFFECTS:

❖ May be agents of the disease:

● Members of normal flora may cause various endogenous diseases.
 ● When the host immunity is lowered, the transient flora may invade and
produce disease. e.g., gram negative organism (E. coli) colonizing the
respiratory tract can cause pneumonia.
● If they enter the wrong site or tissue (e.g., Blood, sterile body cavities)- then
even the resistant flora can produce disease. e.g., E. coli which is a resident
flora of the intestine may cause urinary tract infection if entered into the
urinary tract.
❖ Transfer to susceptible hosts:
● Some pathogens of humans that are members of the normal flora for one host
can produce disease if transferred to the other host.
● E.g., The pathogen that colonizes the upper respiratory tract (such as
meningococcus, pneumococcus etc.) can produce disease in the susceptible
hosts.
❖ Bacterial synergism:
● Bacterial vitamins and growth factors produced by members of the normal
flora may promote the growth of the potential pathogens.
❖ Contribute to the drug resistance of pathogens:
● Some members of normal flora produce enzyme such as beta lactamases
which destroy the beta lactam antibiotics
● Thus, indirectly contributing to the drug resistance of pathogens that are
otherwise susceptible to the drug.
❖ Competition for host nutrients:
● Bacteria in GIT absorb some of the host’s nutrients for their survival.

DISEASES PRODUCED BY NORMAL ANATOMICAL SITE FROM WHICH THE

FLORA FLORA IS TRANSFERRED

❖ Urogenital infections including UTI ❖ Intestinal flora such as Escherichia coli,

Klebsiella, Proteus.

❖ Endocarditis ❖ Oral flora (Viridans streptococci)

❖ Dental caries and periodontal disease ❖ Oral flora (Streptococcus mutans)

❖ Peritonitis, abdominal infection ❖ Intestinal flora

❖ Pneumonia ❖ Transient respiratory flora

❖ Septicaemia ❖ From any site

D. Explain the bacteriology and the bacteriological examination

of milk?

BACTERIOLOGY:

● It is the branch of biology that deals with the study of minute organisms
called bacteria (singular bacterium).
● A branch of microbiology dealing with the identification, study, cultivation of
bacteria and with their application in medicine, agriculture, industry and
biotechnology.

BACTERIA:

● It is a single celled, often parasite microorganism without distinct nuclei or organized

cell structures. Various species are responsible for decay, fermentation, nitrogen
fixation and many plant and animal diseases.

They are:

● Prokaryotes
● Single celled organisms
● Size microscopic
● E. coli is 1.3 µm wide x1.0 µm long. 625 E. coli to make 1 inch.
BACTERIOLOGICAL EXAMINATION OF MILK:

- Routine bacteriological examination of milk consists of the following

● Viable method
● Test for coliform bacilli
● Methylene blue reduction test
● Phosphatase test
● Turbidity test

VIABLE COUNT:

Method:

● This is estimated by performing plate counts with serial dilution of milk sample.
● Raw milk always contains bacteria, varying in number from about 500 to several
million per ml.

Significance:

● The plate count gives a rough and direct assessment of the viable bacteria in the
milk
● It is easily explainable to the producer and gives a fair idea of the improvement
or deterioration in the condition of production.

TEST FOR COLIFORM BACILLI:

Method:

● This is performed by inoculating varying dilutions of milk into MacConkey’s

fluid medium
● And nothing about the production of acid and gas after incubation.

Significance:

● Contamination with coliforms comes mainly from dust, dirty utensils and daily
workers.
 ● This method is a useful indicator of faecal contamination and also contamination
by dust or unclean utensils.

METHYLENE BLUE REDUCTION TEST:

Method:

● This is the simple substitute for the viable count.

● It depends on the reduction of methylene blue by bacteria in milk when
incubated at 37ºC in complete darkness.

Significance:

● The rate of reduction is related to the degree of bacterial contamination.

● Raw milk is considered satisfactory if it fails to reduce the dye in 30 mins.
● The resazurin test is similar, except that the dye resazurin, on reduction, passes
through a series of colour changes- from blue to pink to colourless. This colour
change is noted after incubation with the milk for 10 mins.

PHOSPHATASE TEST:

Method:

● This is a check whether milk has been pasteurised.

● The enzyme phosphatase, which is normally present in milk, is inactivated if
pasteurization has been performed properly.

Significance:

● Residual phosphatase activity indicates that pasteurization has been inadequate.

● This test, if positive after proper pasteurization of milk, shows contamination after
pasteurization.
TURBIDITY TEST:

● This is a check on the sterilization of milk.

● If milk has been boiled or heated to the temperature prescribed for sterilization, all
heat – coagulable protein are precipitated
● If ammonium sulphate is then added to the milk, filtered and boiled for 5 mins, no
turbidity results.
● This test distinguishes between pasteurized and sterilized milk.
 SHORT NOTES 6

DIFFERENTIATE DIARRHEA AND DYSENTERY

DIARRHEA DYSENTERY

● Diarrhea is presented as watery stool ● Dysentery is presented as a mucoid stool that

with no blood and mucus. may be accompanied by blood.

● The patient may or may not be ● The patient usually complains of cramps and
accompanied by cramps or pain pain in the lower abdominal area.

● Fever is less common in diarrhea ● Fever is more common in dysentery

● Diarrhea is a disease that affect the ● Dysentery is a disease that affect the colon.
small bowel.

● Diarrhea infection is located and ● Dysentery not only upper epithelial cells and
targets only intestinal lumen and upper targeted but colon ulceration also results.
epithelial cells.

● There is no cell death in diarrhea and ● When a person get dysentery, the upper
the infection is only caused because of epithelial cells are attacked and destroyed by
the release of some toxins by the the pathogen or disease-causing agent.
infecting agent.

● The antimicrobial that are used to treat ● Treatment for dysentery can eradicate the
diarrhea do not eradicate the toxin left pathogen that is causing the infection and stop
behind. the inflammation.
● The effects of diarrhea are not that ● Dysentery can cause a lot of complications. If
serious, apart from a risk of left untreated.
dehydration.

● Diarrhea is mostly viral. E. coli can ● Dysentery is mostly bacterial E. coli, Shigella
also cause watery diarrhea. and Salmonella are the most common causative
organisms.

● Diarrhea does not need antibiotics. ● Dysentery almost always requires antibiotic
Oral rehydration solution or treatment. Intravenous antibiotics may be
intravenous fluid therapy may be used. needed in severely ill children.
 SHORT NOTE 7:

BACTEREMIA AND SEPTICEMIA

BACTEREMIA:
● The transient presence of Bacteria in the Bloodstream without Multiplication.
TYPES:
o TRANSIENT BACTEREMIA:
- May occur Spontaneously or with Minor events
- May lead to Septicaemia
- Normally cured by the Host immune mechanisms

o CONTINUOUS BACTEREMIA:
- Microbes are released into blood at a Fairly Constant Rate.
- Occurs in
● Septic Shock
● Endocarditis
● Early stages of: Enteric Fever and Brucellosis

o INTERMITTENT BACTEREMIA:
- Microbes are released Intermittently into blood.
- Occurs in
● Sequestered focus of infection (e.g., Undrained Abscess)
● Early Course of - Meningitis, Pneumonia, Septic Arthritis.

SEPTICEMIA:
❖ Refers to the Presence of Microbial Antigens and Endo / Exotoxins in the
Bloodstream.
❖ Sepsis – Response the Host mounts against these products.
❖ There is Active Bacterial Multiplication.
ETIOLOGY:

GRAM – POSITIVE COCCI

Staphylococci

Beta-haemolytic streptococci

Enterococci

Pneumococci

GRAM – NEGATIVE COCCI

Meningococci

GRAM – POSITIVE BACILLI

Bacillus anthracis

Listeria

GRAM – NEGATIVE BACILLI

E. coli

Klebsiella

PATHOGENESIS:

Bacteria may enter bloodstream:

– From an infective focus with the help of phagocytic cells carrying microbes
into capillaries or the lymphatic system.
– From breakages of blood vessels adjacent to the skin or mucosal surfaces.
– By introduction of contaminated material directly into the vascular system
SIGNS AND SYMPTOMS:

● Hypothermia / Fever
● Chills
● Hyperventilation
● Subsequent Respiratory Alkalosis

COMPLICATIONS:

▪ Septic
▪ Endotoxic
▪ Septic shock
▪ Acute renal failure
▪ Shock may lead to multiple organ failure (e.g., heart, lungs, liver, kidneys)
 SHORT NOTE 8:
FOOD POISONING

Food poisoning refers to an illness acquired through consumption of food or drink

contaminated either with microorganisms or their toxins.

CAUSES OF FOOD POISONING:

1-6 HOURS INCUBATION PERIOD:

Organisms Symptoms Common food sources

Staphylococcus aureus Vomiting, diarrhoea, Ham, poultry, salad, dairy

abdominal cramps products, pastries
due to Enterotoxin -
preformed heat stable toxin

Bacillus cereus (emetic) Vomiting, diarrhoea, Chinese fried rice

abdominal cramps
due to heat stable preformed
emetic toxin

8 - 16 HOURS INCUBATION PERIOD:

Organisms Symptoms Common food sources

Clostridium perfringens Abdominal cramps, diarrhoea Beef, poultry, legumes,

gravies

Bacillus cereus (diarrheal) Abdominal cramps, diarrhoea Meats, vegetables, dried

beans, cereals

> 16 HOURS INCUBATION PERIOD:

Organisms Symptoms Common food sources

Vibrio cholerae Watery diarrhoea Shellfish, water

Enterotoxigenic E. coli Watery diarrhoea Salads, cheese, meat

Enterohemorrhagic E. coli Bloody diarrhoea Ground beef, milk

Non typhoidal salmonella Inflammatory diarrhoea Meat, eggs, milk, juice

Shigella species Dysentery Potato, egg, salad, lettuce

Vibrio parahaemolyticus Dysentery Raw or undercooked shellfish

particularly oysters

Campylobacter jejuni Inflammatory diarrhoea Poultry, raw milk

Clostridium botulinum Flaccid paralysis diplopia Homemade improperly

dysphagia canned foot and honey in
infants

Listeria monocytogenes Fever and myalgia in Soft cheeses, raw sprouts,

pregnant women meats

Norovirus (virus) Watery diarrhoea, vomiting, Salads, fresh fruits, shellfish

abdominal cramps

Cyclospora (parasite) Watery diarrhoea, abdominal Raw fruits or vegetables

cramps herbs

Mycotoxicosis (fungus) Depends on type of fungal Nuts, maize, wheat, cereals

toxin.
e.g.: aflatoxin causes
hepatoma

TREATMENT:

Type of food poisoning Treatment

Staphylococcal food poisoning Supportive by correcting fluid and electrolyte

 imbalance

Bacillus cereus food poisoning Clindamycin, erythromycin, vancomycin,

tetracycline (resistant to penicillin as it
produces beta lactamase)

Shigellosis Fluoroquinolone like ciprofloxacin

Cholera Azithromycin

Food Botulism Heptavalent botulism equine serum antitoxin,

antibiotics like penicillin or metronidazole
 SHORT NOTE 9:
ANTIBIOGRAM

Overall profile of antimicrobial susceptibility testing results of a specific microorganism to a

battery of antimicrobial agents

ANTIBIOGRAM TYPING:
It classifies the organism into different groups based on their resistance pattern to different
antimicrobials.

HOSPITAL ANTIBIOGRAM :
Periodic summary of antimicrobial susceptibilities of local bacterial isolates submitted to the
hospital's clinical microbiology laboratory.
● It is the responsibility of the department of Microbiology to construct a hospital
antibiogram and share it with clinicians.

WHY DO WE NEED ANTIBIOGRAM?

● Antimicrobial-resistant bacterial infections are a challenging problem in the hospital
setting.
● Infections caused by resistant- and multidrug-resistant (MDR) bacteria not only
increase morbidity and mortality, but also increase overall healthcare costs, primarily
by prolonging hospital length of stay.

PROCEDURE:

1→Sample collection (E.g. - Blood, Pus, Urine, Sputum)

2→Isolation & Identification of microbes

3→Sample cultured and antibiotic disks added

4→After 24-48hours, Zones of inhibition (Clearings) are measured

5→Tables framed-labelling of microbes as Susceptible, Dose-dependent, Intermediate or
Resistant.

USES:
● Guides the clinicians in selecting the best empirical antimicrobial treatment-as
Inappropriate antimicrobial selection also has the potential to increase the risk for
resistance development.
● Useful tool for detecting and monitoring trends in antimicrobial resistance within the
hospital.
● Used to compare susceptibility rates across institutions.
● Since antimicrobial susceptibility testing is routinely done in any hospital, this typing
system provides the first clue to a microbiologist about outbreaks occurring in a
hospital.
● Evaluate the efficacy of a new antibiotics.
● Study the epidemiology of resistance.
 SHORT NOTE 10:
COAGULASE TEST

Biochemical test used to differentiate Staphylococcus aureus (positive) which produce the
enzyme coagulase, from S. epidermidis and S. saprophyticus (negative) which do not
produce coagulase i.e., Coagulase Negative Staphylococcus (CONS).

TYPES OF COAGULASE TEST:

A. Tube / Free coagulase test
B. Slide / Bound coagulase test

A. TUBE OR FREE COAGULASE TEST

Tube coagulase test detects the free coagulase which is an extracellular product of
Staphylococcus aureus.
PROCEDURE:
● 0.1 ml of a young broth culture or agar culture suspension of the isolate is
added to 0.5 ml heparinized or oxalated human or rabbit plasma.
● The tubes are incubated in the water bath at 37 degree Celsius for 3 to 6 hours
to demonstrate clotting.
PRINCIPLE:
1. Activation of plasma coagulase reacting factor (CRF) found in rabbit
plasma.
2. CRF reacts with coagulase.
 3. Formation of coagulase CRF complex.
4. Complex reacts with fibrinogen and activates it.
5. Fibrin clot is produced which is a positive test.

B. SLIDE OR BOUND COAGULASE TEST:

Slide coagulase tests detect the bound coagulase called the clumping factor.
PROCEDURE:
A drop of human or rabbit plasma is added to an emulsion of an isolate in a drop of
saline and mixed.
PRINCIPLE:
1. Clumping factor is bound to the bacterial cell wall.
2. It reacts directly with fibrinogen of human or rabbit plasma.
3. It results in the alteration of fibrinogen.
4. Precipitates on the staphylococcal cell.
5. Clumping of cocci occurs which is a positive test.
 SHORT NOTES 11:
SIGNIFICANT BACTERIURIA

● Bacteriological diagnosis of UTI is done by demonstrating significant bacteriuria

using semi-quantitative cultures developed by Kass.

FACT:
Normal urine is sterile but during voiding may become contaminated with
genital commensals i.e., normal urethral flora. The counts in the contaminated
urine would be lower than that in the case of UTI.

COUNT:
● Greater than or equal to 10⁵ CFU/ml = SIGNIFICANT indicates infection.
● Between 10⁴ to 10⁵ CFU/ml = DOUBTFUL significance, must be clinically
correlated.
● Low count of less than 10⁴ CFU/ml = CONTAMINATION of urine during voiding
with commensal bacteria.

CONDITIONS WHERE LOW COUNTS CAN BE SIGNIFICANT:

● Patient on antibiotic or on diuretic treatment.
● Infection with some gram-positive organisms like Staphylococcus aureus.
● Pyelonephritis and acute urethral syndrome.
● Sample taken by suprapubic aspiration.
● In catheterized patients:
If the patient is symptomatic then a count of greater than or equal to 10³ CFU/ ml
is considered significant.
 SHORT NOTES 12:

A. LABORATORY ASSESSMENT INVOLVED IN THE

ESTIMATION OF PORTABILITY OF DRINKING
WATER

LABORATORY TESTING OF DRINKING WATER

MULTIPLE TUBE METHOD:

● Most common method

● Involves mixing specific amount of water to multiple tubes containing culture
medium

PROCEDURE:

● Most hospital water supplies are unpolluted

● Testing of unpolluted water is as follows
1. 50ml of water is added to one tube of 50ml of culture
2. 10ml of water is added to 5 tubes of 10ml of culture
3. After 24-48hrs of incubation,
I. Medium turns yellow to purple
i. Due to lactose fermentation
ii. Indicates presence of coliform bacteria
II. Medium becomes turbid
III. Gas is collected in Durham’s tube
4. Number of tubes turning purple is matched against McCardy
statistical table to determine MPN of coliform count per 100ml of
water
5. Depending upon MPN/100ml, the quality of water can be
interpreted
DIFFERENTIAL COLIFORM TEST (EIJKMAN TEST):

● Positive multiple tube test does not always indicate faecal contamination, as coliform
bacteria are naturally present
● This test is done to detect faecal E. coli
● Positive tubes from multiple tube test are sub cultured on lactose culture

RESULT:
1. Brilliant green broth is seen
2. Detection of lactose fermentation with acid and gas production at 44℃
3. Positive indole test at 44℃

MEMBRANE FILTRATION METHOD:

PROCEDURE:

1. Filtration of known amount of water through cellulose membrane of pore size

0.2µm-0.45µm
2. Bacteria retained on surface on membrane is cultured and incubated
3. Yellow colonies of coliform are obtained which can be counted to CFU/100ml of
water

ADVANTAGES:

1. Checking dialysis water

2. Testing a large amount of water

DISADVANTAGES:

1. Not suitable for turbid water

2. Expensive than multiple tube water
PRESENCE ABSENCE METHOD – MANJAS METHOD:

● Qualitative method
● Detects the presence or absence of an organism
● H2S coated strips used to detect Salmonella in water
ADVANTAGES:
1. Monitoring good quality drinking water
2. In outbreak situations where urgent reports are needed

B. WATER BORNE DISEASES

Hospital water may serve as a reservoir for waterborne pathogens.

Microbial contamination of water in hospital settings are of 2 types:

1. Enteric pathogens:
● Common in communities surrounding health care settings
● Due to faecal contamination of drinking water
● Transmitted by ingestion of contaminated water
● Cause diarrheal outbreaks and extraintestinal diseases

Ex:

i. Bacteria: E. coli, vibrio cholerae

ii. Viruses: Hepatitis A and E viruses, polioviruses
iii. Parasites: Entamoeba histolytica, Cyclospora

2. Common hospital pathogens:

● Includes multidrug resistant gram-negative bacilli, non-tuberculous
mycobacteria, etc.
● Commonly present in hospital environment, can contaminate hospital water
reservoirs such as potable water, dialysis water, ice and ice machines
● Transmitted by ingestion, aspiration, etc.
● Outbreaks caused by these microorganisms are a serious threat to high-risk
patients who are critically ill or immunocompromised.

- Several methods have been employed for detection and elimination of

microbial contamination of water.
 Short Notes 13:

A. What are routine and individual vaccines? Explain the

National Immunisation Schedule.

ROUTINE VACCINES
❖ They have been developed based on the prevalence of Infectious Diseases, their
Public Health Importance, availability, cost- benefit factors and Logistics.
❖ In India, the Expanded Programme on Immunisation (EPI) and the Universal
Immunisation Programme (UIP) provide Routine Vaccinations to provide protection
against VPDs for much of the target population.
❖ E.g.: BCG, OPV, Hep B birth dose given at Birth.

INDIVIDUAL VACCINES
❖ These are supplemented by Individual initiative.
❖ These vaccines may be omitted under National programmes due to Economic
Limitations.
❖ E.g.
● Varicella vaccine
● Typhoid vaccine

NATIONAL IMMUNISATION SCHEDULE

❖ Immunisation is one of the Most Logical and Cost-effective strategies of any
country for the Prevention of Childhood Sickness and Disabilities.
❖ The National Immunisation Schedule is recommended by the Ministry of Health,
Government of India.
❖ It includes those vaccines that are given free of cost to all children of our country.
Reference: Ananthanarayan and Paniker’s Textbook of Microbiology E/11
B. HOW DO WE SEGREGATE DIFFERENT BIOMEDICAL
WASTES?
 Short note 14:

URINARY TRACT INFECTION (UTI)

Disease caused by microbial invasion of the urinary tract that extends from the renal cortex of
the kidney to the urethral meatus.

CLASSIFICATION:
Two types – based on the anatomical sites involved

Two Types-based on the source of infection

- Health care associated example CAUTI (catheter associated UTI) – most common
- Community-acquired

PREDISPOSING FACTORS:
● Gender: Female > Male
● Age: Incidence of UTI increases with age
● Pregnancy: Anatomical and hormonal changes > asymptomatic bacteriuria.
● Structural and functional abnormality of urinary tract
1)Structural obstruction- urethral structure, renal and ureteric stones, prostatic
hypertrophy
2)Functional Obstruction-neurogenic bladder.
● Bacterial virulence – Pili, Fimbriae; Vesicoureteral reflux; Genetic factors.

ETIOLOGY:
Escherichia coli (uropathogenic coli) – common cause of UTI – 70 % of total cases.
PATHOGENESIS:
By two routes – ascending and descending routes.

ASCENDING ROUTE DESCENDING ROUTE

Most common route: Invasion of renal parenchyma through

Enteric endogenous bacteria enter the urinary hematogenous seeding of pathogens as the
tract by Intercourse or catheterization. spread of bacteraemia.

Gram negative bacilli – E. coli Gram positive bacilli – Mycobacterium

tuberculosis

Fungus – Candida albicans Salmonella

Gram positive cocci – Enterococcus species, Gram positive cocci - Staphylococcus aureus
staphylococcus saprophyticus

Parasites – Schistosoma haematobium, Leptospira

Trichomonas vaginalis

CLINICAL MANIFESTATIONS:
▪ Lower UTI: Asymptomatic bacteriuria, cystitis, urethritis, acute urethral syndrome
▪ Upper UTI: Pyelonephritis, ureteritis, perinephric abscess, renal abscess, renal
tuberculosis
▪ Immunological sequela: Post-streptococcal glomerulonephritis (PSGN)
 SHORT NOTES 15:

CAUSES AND PREVENTION OF SEXUALLY

TRANSMITTED DISEASES

Sexually transmitted diseases are a group of communicable diseases transmitted by sexual

contact.

CAUSES OF STDs:

1. Agents causing local manifestations: (genital tract infections)

● Lesions common in both sexes: Genital ulcers, urethritis
● Female genital tract infections: Vulvovaginitis, cervicitis
● Male genital tract infections: Prostatitis, Epididymis
2. Agents causing systemic manifestations: without producing local manifestations
● Do not primarily affect the genital system
● HIV, Hepatitis B

PREVENTION OF STDs:

▪ Education about safe sex practices

▪ Implementation of healthy sex practices
▪ Prophylactic use of barrier contraceptives
▪ Tracing and treatment of contacts
▪ Periodic screening of high-risk groups
 SHORT NOTES 16:

CONGENITAL INFECTIONS

→ Vertical transmission refers to the spread of infections from mother-to-baby.

→ Routes of infection:
● transplacental route (congenital infection)
● during delivery
● after delivery
→ A congenital infection is an infection that crosses the placenta to infect the foetus.
→ They often lead to defects in foetal development or even death.
→ TORCH is an acronym used for some common congenital infections.
● Toxoplasmosis
● Other infections
■ congenital syphilis
■ hepatitis B,
■ Coxsackie virus
■ Epstein-Barr virus
■ Varicella-Zoster virus
■ Plasmodium falciparum
■ Human Parvovirus
● Rubella
● Cytomegalovirus (CMV)
● Herpes simplex virus

PERINATAL INFECTIONS (DURING DELIVERY)

→ Infections occur while the baby moves through an infected birth canal.
→ Usually caused by the agents of STD s.
→ Also include the infections transmitted through contamination with faecal matter
during delivery.
→ Common examples of agents causing perinatal infections include:
● CMV
 ● Neisseria gonorrhoeae
● Chlamydia species
● Herpes simplex virus
● Human papillomavirus (genital warts)
● Group B streptococci

POSTNATAL INFECTIONS (AFTER DELIVERY)

● These infections spread from mother to baby following delivery, usually
during breastfeeding.
● Causative organisms
■ CMV
■ HIV
■ Group B streptococci.

CONGENITAL TOXOPLASMOSIS:
→ Toxoplasma is the most common parasite to be teratogenic.
→ Incidence – 1 per 1000 live births.
→ Causes encephalitis in HIV-infected individuals
→ Important cause of repeated abortion and infertility.

TRANSMISSION:
→ Mother acquiring Toxoplasma infection in pregnancy is usually asymptomatic.
→ Transplacental transmission of T. gondii from mother to-foetus can occur at any time
during the pregnancy.
→ Tachyzoites are the infective form

GESTATIONAL AGE:
→ The main factor that influences the foetal outcome
→ As the gestation proceeds, the chance of transmission of infection increases but the
severity of the infection declines
CLINICAL MANIFESTATIONS:
→ The classical triad:
● Chorioretinitis
● Hydrocephalus
● Intracranial calcifications

→ Other manifestations:
● Stillbirth
● Psychomotor disturbance
● Microcephaly

→ Ocular involvement:
● Eyes are involved later in life (2nd 3rd decade) when the cysts ruptures
● Causes bilateral chorioretinitis leading to profound visual impairment
● Other manifestations
▪ Blurred Vision
▪ Scotoma
▪ Photophobia
▪ Strabismus
▪ Glaucoma
● If ocular involvement occurs without a history of congenital infection, it is
mostly unilateral.

LABORATORY DIAGNOSIS:

ANTENATAL DIAGNOSIS:
→ Acute infection – Ultrasonography of foetus should be done at 20-24 weeks of
gestation and repeated every 2-4 weeks for detecting the lesions of congenital
infection

→ PCR and/or isolation: Amniotic fluid sample is collected, centrifuged and the pellet is
subjected to PCR and/or isolation in mouse or tissue culture If either or both found
positive, then antenatal T diagnosis is confirmed
 → If both negative: Warrant’s evaluation of the neonate to rule out any remote
possibility of infection.

POSTNATAL DIAGNOSIS:

▪ Isolation of the parasite at the time of delivery must be attempted from amniotic
fluid, placenta and cord leukocyte

▪ IgM and IgG: New-born and maternal sera are subjected to detect IgG
(Sabin-Feldman dye test, IFA or ELISA) and IgM (ELISA or IFA)

▪ IgG titre of 21,000 in neonate: Indicates possible T diagnosis which should be

confirmed by IgM testing
▪ IgM titre of neonate 21:4 after 2 weeks of age indicates probable diagnosis and guides
the clinicians to initiate the treatment to the neonate.
▪ Other tests IgA detection (neonatal and maternal blood)
o IgA appears to be more sensitive than IgM for the diagnosis. IgA antibodies
usually disappear within 10 days of birth, hence persistence of IgA beyond 10
days confirms the postnatal infection
o PCR in neonatal and maternal blood detecting specific genes of T. gondii also
confirms the diagnosis
o IgE detection (neonatal and maternal blood)
 Short Notes 17:
ROLE OF VECTORS IN TRANSMISSION OF INFECTIOUS
DISEASES

VECTOR-BORNE INFECTIONS:
- Vectors are living organisms that can transmit infectious pathogens
(parasites, viruses and bacteria) between humans, or from animals to
humans.

TYPES OF TRANSMISSION:
MECHANICAL TRANSMISSION:
The disease agent does not replicate or develop in/on the in vector; it is simply transported by
the vector (e.g., housefly) from one animal or environment to man.

BIOLOGICAL TRANSMISSION:

Steps:

1→Pathogen (through blood meal)

2→Enters vector

3→Replicates and /develops inside vector

4→Regurgitated /injected into susceptible animal

TYPES OF BIOLOGICAL TRANSMISSION:

TYPE DEVELOPMENT EXAMPLE

•Propagative Pathogen only multiplies Yersinia pestis in rat fleas

 inside the vector

• Cyclodevelopmental Pathogen develops into the Wuchereria bancrofti in

next stage without mosquitoes
multiplying

• Cyclopropagative Pathogen multiplies and also Plasmodium species in

enters to next developmental mosquito
stage

• Salivaria (anterior station) ● Pathogen multiplies Trypanosoma brucei in Tsetse

and reaches the fly
salivary glands of the
vector
● Infection by
regurgitating/ biting

• Stercoraria (posterior ● Pathogen multiplies Trypanosoma cruzi in

station) and reaches the Reduviid bug
rectum of the vector
● Infective forms are
released through
faeces

• Transovarian transmission Parent vector passes the Rickettsia rickettsii in ticks

pathogen to off springs and
the latter spreads infection to
Susceptible hosts
 Short Notes 18:
BACTERIOLOGY OF MILK
TYPES OF BACTERIA IN MILK

i) Acid-forming bacteria
i. Lactic streptococci
ii. S. lactis
iii. Enterococcus faecalis
iv. Lactobacilli
● Ferment the lactose in milk, producing acids, mainly lactic acids, which lead
to the formation of a smooth, gelatinous curd.

ii) Alkali-forming bacteria

i. Alcaligenes spp.,
ii. Aerobic spore bearers
iii. Achromobacter species.
● These render the milk alkaline.

iii) Gas-forming bacteria

i. Coliform bacilli
ii. C. perfringens
iii. C. butyricum.
● They produce acid and gas-a smooth, gelatinous curd riddled with gas bubbles
is formed.
● Coliform bacilli are responsible for the ropiness in milk.

iv) Proteolytic bacteria

i. Bacillus subtilis
ii. Bacillus cereus,
iii. Proteus vulgaris,
iv. Staphylococci
v. Micrococci
 v) Inert bacteria
● Produce no visible change in milk.
i. Cocci of the udder,
ii. Members of the Achromobacter group
iii. Pathogenic organisms in milk.

vi) Human milk

i. S. epidermidis
ii. S. mitis
iii. Gaffkya tetragena
iv. S. aureus

MILK BORNE DISEASES

Milk may be contaminated with

● Streptobacillus moniliformis from the nasal secretions of with rats
● Campylobacter jejuni from animal faeces.
● Yersinia enterocolitica

STERILISATION OF MILK

1. Pasteurisation
▪ In this method, all vegetative pathogens are killed by heating milk to 63-66°C
and maintaining it at this temperature for 30 minutes (the holder method) or by
heating milk to a temperature of 71°C and holding it at that temperature for at
least 15 seconds.
▪ The second step of this method is the rapid cooling of milk to 10°C or less.
▪ The phosphatase test is used to check the adequacy of pasteurisation.

2. Boiling
 ▪ The commonly-used household method of heating milk at or around boiling
point (which destroys all but the most resistant spores) is adequate for short
term purposes.
▪ The efficacy of such treatment is tested by the turbidity test.

3. Ultraheat treatment
▪ In this method, milk is heated to 132°C for one second under specified
conditions.

BACTERIOLOGICAL EXAMINATION OF MILK

1. Viable count
a. This is estimated by performing plate counts with serial dilutions of the milk
sample.
b. Raw milk always contains bacteria, varying in number from about 500 to
several million per ml.
c. The plate count gives a rough and direct assessment of the viable bacteria in a
sample of milk.
d. It is easily explainable to the producer and gives a fair idea of the
improvement or deterioration in the conditions of production.

2. Test for coliform bacilli

Indicator of
a. faecal contamination
b. contamination by dust
c. unclean utensils.

3. Methylene blue reduction test

a. Substitute for the viable count.

 b. It depends on the reduction of methylene blue by bacteria in milk when
incubated at 37°C in complete darkness.
c. The rate of reduction is related to the degree of bacterial contamination.
d. Raw milk is considered satisfactory if it fails to reduce the dye in 30 minutes.
e. The resazurin test is similar, except that the dye resazurin, on reduction, passes
through a series of colour changes from blue to pink to colourless.
f. The colour change is noted after incubation with the milk for 10 minutes.

4. Phosphatase test

a. This is a check on whether milk has been pasteurised.

b. The enzyme phosphatase, which is normally present in milk, is inactivated if
pasteurisation has been performed properly.

EXAMINATION FOR SPECIFIC PATHOGENS:

▪ Tubercle bacillus
o The milk is centrifuged at 3,000 rpm for 30 minutes and the sediment
inoculated into two guinea pigs.

▪ Brucella
o Isolation of brucella may be attempted by inoculating cream heavily on serum
dextrose agar or by injecting a centrifuged deposit of the milk sample
intramuscularly into guinea pigs.
o The animals are sacrificed after six weeks and the serum tested for agglutinins
and the spleen inoculated in culture media.
o Brucellosis in animals can also be detected by demonstrating the antibodies in
milk by the milk-ring or the whey agglutination tests.
 SHORT NOTES 19:
A. WHAT ARE THE CONCENTRATION METHODS FOR
FAECAL EXAMINATION?

Stool analysis:

It is a series of tests done on a stool (faeces) sample for the differential diagnosis of certain
diseases of the digestive system and include infection (such as from parasites, virus or
bacteria), poor nutrient absorption or cancer.

CLINICAL SIGNIFICANCE OF STOOL ANALYSIS:

● Diagnosis of digestive system infectious diseases: Bacteria, parasites, virus and

fungi.
● Diagnosis of pancreas disorder (inflammation); which is associated with
malabsorption of nutrients.
● Primary screening test for some type of digestive system malignancy such as:
Colon cancer.
● Primary screening test for peptic ulcer disease and some types of anaemia.

STOOL EXAMINATION DONE IN:

● Patients with abdominal pain

● Patients with diarrhoea
● Patient with anaemia
CONCENTRATION TECHNIQUES FOR FAECAL EXAMINATION:

CONCENTRATION TECHNIQUE:

● These methods are used when ova or parasites are not found in direct saline
preparation.
● But their presence is highly suspected or symptoms persist. ova of certain parasites
are scanty. E.g., Schistosoma, Taenia.
a) Flotation technique:
This method uses the high specific gravity of a solution to float the lighter ova and
cyst. They can be improved by centrifugation.
ADVANTAGE:
 ● Easy to perform
DISADVANTAGE:
● Delay in examination can result in distortion
● Larvae and some fluke eggs do not concentrate.
● Frequent checking of specific gravity.
b) Sedimentation technique:
● Use solution of lower specific gravity than the parasitic organisms (formalin
ethyl acetate technique)
● Recommended for general diagnostic laboratories due to easy to perform and
less prone to technical error.
E.g., Formalin ether sedimentation technique
● It is the recommended concentration procedures
● Most types of worm eggs (roundworm, tapeworms, schistosomes, fluke eggs),
larvae and protozoan cysts may be recovered by this method.
ADVANTAGES:
● Speed: One sample can be processed in 5 minutes.
● Broad spectrum: It will recover most ova, cyst, and larvae.
● The morphology of most parasites is retained for easy identification.

DISADVANTAGES:

● Requires several pieces of apparatus which does not make it an easy.

● The preparation contains some debris.
● Ether is flammable. Formalin is an irritant.
● Hymenolepis nana and Fasciola spp. Do not concentrate well.
 B. EXPLAIN THE LABORATORY USE OF PCR AND TYPES
IN MICROBIOLOGICAL DIAGNOSIS

- Technique in molecular biology used to amplify a single or few copies of a piece of DNA
to generate millions of copies of DNA. It was developed by Kary B Mullis.

It involves 3 basic steps:

1) DNA extraction from the organism.

2) Amplification of extracted DNA
● Denaturation at 95ºC
● Priming annealing (55ºC)
● Extension of the primer (72ºC)

3) Gel electrophoresis of amplified products.

LABORATORY APPLICATIONS OF PCR:

● It is an indispensable technique used in medical diagnostics and research

laboratories.
● Subcloning DNA targets using PCR
● PCR mediated in vitro mutagenesis
● Amplification of differentially expressed gene sequences
● Differential display reverse transcription PCR.

APPLICATIONS OF PCR:

● More sensitive: It can amplify very few copies of a specific DNA, so it is more
sensitive.
● More specific: Use of primers targeting specific DNA sequences of the organism
makes the PCR assays highly specific.
● PCR can be done to amplify the DNA of the organism
1) Either directly from the sample or
2) To confirm the organism grown in culture.
 ● PCR can also detect the organism that are highly fastidious or non-cultivated by
conventional culture methods
● PCR can be used to detect the genes in the organism responsible for drug resistance
(Eg mec A gene detection in Staphylococcus aureus)
● Detects genetic disease such as sickle cell anaemia, phenylketonuria, muscular
dystrophy.

DISADVANTAGES OF PCR:

● Qualitative, not quantitative

● Viability – cannot differentiate viable or non-viable.
● False positive amplification
● False negative

TYPES OF PCR:

● Long PCR
● Nested PCR
● Inverse PCR
● Quantitative PCR
● Hot start PCR

1) Long PCR:
It is used to amplify DNA over the entire length up to 25kb of genomic DNA
segments cloned.

2) Nested PCR:
Involve two consecutive PCR reactions of 25 cycles. The first PCR uses primers
external to the sequence of interest. The second PCR uses the product of the first PCR
in conjunction with one or more nested primers to amplify the sequence within the
region flanked by the initial set of primers.

3) Inverse PCR:
Used to amplify DNA of unknown sequence that is adjacent to known DNA
sequence.
4) Quantitative PCR:
Product amplification with respect to time, which is compared with standard DNA.
5) Hot start PCR:
Used to optimize the yield of the desired amplified product in PCR and
simultaneously to suppress nonspecific amplification.
 SHORT NOTE 20:
IMMUNOPROPHYLAXIS

▪ Vaccine is an immunobiological preparation that provides specific protection against a

given disease.
▪ Following vaccine administration, the immunogen (active ingredient of the vaccine)
stimulates the immune system of the body to produce active immunity in the form of
protective antibody and/or immunocompetent T cell response.

DNA VACCINE:

▪ DNA vaccines are experimental at present, have many advantages such as cost
effectiveness and mounting a stronger and wider range of immune response.
▪ The small pieces of DNA containing genes from the pathogenic microorganism are
injected into the host.
▪ The gene of interest gets integrated with the host cell genome and starts transcribing
the proteins against which the host mounts an immune response.
▪ Several vaccine trials are going on based on DNA vaccines.

EDIBLE VACCINE:

▪ New concept- introduced recently

▪ The gene encoding the orally active antigenic protein is isolated from the pathogen
and is transferred to suitable plant bacteria, which are then used to infect a
transgenic plant (e.g., banana, potato, etc.)
▪ The plants infected by the bacteria then start producing the antigen of interest on a
large scale.
▪ The appropriate plant parts having the antigen may be fed raw to animals or humans
to bring about immunization
▪ The advantages of the edible vaccines are:
● Low cost
 ● Ability to produce in large scale
● Administered orally
● Induce local immunity
● Heat stable

APPLICATIONS:

→ The edible vaccines are still under experimental stage; some formulations available
include
● Transgenic potatoes and tomatoes against diarrhoea
● Edible banana against Norwalk virus
REFERENCES

Ananthanarayan and Paniker’s Textbook of Microbiology

▪ Tenth Edition
▪ Eleventh Edition

Essentials of Medical Microbiology, Apurba Sastry

▪ First Edition
▪ Second Edition
▪ Third Edition

Review of Microbiology and Immunology, Apurba Sastry, Sixth Edition

Complete Microbiology for MBBS, CP Baveja Seventh Edition