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MICROBIOLOGY - Complete Syllabus Answers

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SECTION I: GENERAL MICROBIOLOGY (CORE CONCEPTS)

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1. Differences Between Prokaryotes and Eukaryotes

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2. Louis Pasteur; Robert Koch's Postulates; Edward Jenner

• Disproved spontaneous generation (swan-neck flask experiment)
• Developed germ theory of disease
• Invented pasteurization
• Developed vaccines for chicken cholera, anthrax, and rabies
• Developed concept of attenuation (weakening) of pathogens for vaccines

1. The organism must be found in all cases of the disease
2. The organism must be isolated from the diseased host and grown in pure culture
3. The pure culture must cause disease when introduced into a healthy susceptible host
4. The organism must be re-isolated from the experimentally diseased host and shown to be identical to the original

• Cannot be applied to organisms that cannot be cultured (e.g., Treponema pallidum in artificial media, Mycobacterium leprae)
• Carrier state - healthy people carry pathogens without disease (e.g., Vibrio cholerae carriers)
• Multiple organisms cause same disease (e.g., pneumonia)
• Ethical constraints for human experimentation

• Observed that milkmaids who had cowpox did not get smallpox
• 1796: Inoculated James Phipps with cowpox (vaccinia) - first vaccination
• Led to development of smallpox vaccine and eventual eradication of smallpox (1980 - WHO)
• Father of immunology/vaccinology

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3. Bacterial Cell Wall; Bacterial Capsule and Flagella

• Thick peptidoglycan layer (20-80 nm)
• Teichoic acids (wall teichoic acids and lipoteichoic acids)
• No outer membrane
• Stains purple with Gram stain
• Examples: Staphylococcus, Streptococcus, Bacillus, Clostridium

• Thin peptidoglycan layer (2-7 nm)
• Outer membrane containing Lipopolysaccharide (LPS) = endotoxin
• Periplasmic space between inner and outer membranes
• Porins - channels for small molecules
• Stains pink/red with Gram stain
• Examples: E. coli, Salmonella, Pseudomonas, Neisseria

• Backbone: alternating N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG)
• Cross-linked by peptide bridges
• Target of penicillin (inhibits transpeptidase/PBP), lysozyme (cleaves NAM-NAG bonds)

• Mycoplasma - no cell wall, hence resistant to penicillin

• Polysaccharide layer outside cell wall (exception: Bacillus anthracis - poly-D-glutamic acid capsule)
• Functions:
  • Anti-phagocytic (main virulence factor) - inhibits opsonization
  • Protects against desiccation
  • Adhesion to surfaces
• Detected by: Quellung reaction (capsular swelling), India ink staining (negative staining - capsule appears as clear halo)
• Encapsulated bacteria ("Some Killers Have Pretty Nice Capsules"):
  • Streptococcus pneumoniae, Klebsiella pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Neisseria meningitidis, Cryptococcus neoformans

• Protein filaments used for motility
• Composed of flagellin protein
• Types based on arrangement:
  • Monotrichous - single polar flagellum (Vibrio cholerae)
  • Lophotrichous - tuft at one pole (Helicobacter pylori)
  • Amphitrichous - flagella at both poles
  • Peritrichous - flagella all around (Salmonella, E. coli)
  • Atrichous - no flagella (Klebsiella, Shigella)
• H antigen = flagellar antigen (used in serotyping Salmonella - Kauffmann-White scheme)
• Detected by: Leifson's stain, electron microscopy, hanging drop preparation (motility)

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4. Bacterial Growth Curve and Its Phases

• No increase in cell numbers
• Bacteria adjust to new environment, synthesize enzymes, RNA, proteins
• Duration depends on age of inoculum and nutrients available
• Metabolically very active

• Rapid doubling at a constant rate (generation time)
• Generation time varies: E. coli = 20 minutes, Mycobacterium tuberculosis = 12-24 hours
• Most susceptible to antibiotics (especially cell-wall active agents)
• Bacteria most uniform in size, morphology, staining

• Rate of growth = rate of death
• Nutrient depletion and toxic metabolite accumulation
• Spore formation begins in this phase
• Secondary metabolites (toxins, antibiotics) produced

• Death rate exceeds growth rate
• Exponential die-off
• Autolysis of cells

• Formula: G = t / n, where t = time, n = number of generations

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5. Bacterial Sporulation and Significance

• Bacillus species (aerobic): B. anthracis, B. cereus, B. subtilis
• Clostridium species (anaerobic): C. tetani, C. botulinum, C. perfringens, C. difficile

• Central: B. anthracis, C. perfringens
• Subterminal: B. cereus, C. botulinum
• Terminal: C. tetani (drumstick appearance), C. tetanomorphum

• Core (DNA + ribosomes)
• Inner membrane
• Cortex (peptidoglycan)
• Spore coat (keratin-like protein)
• Exosporium
• Dipicolinic acid (DPA) - unique to spores, responsible for heat resistance

• Extreme resistance to heat (withstand 100°C boiling for hours), chemicals, UV radiation, desiccation
• Autoclave (121°C, 15 psi, 15 min) required to kill spores
• Dry heat: 160°C for 2 hours
• Hospital infections (C. difficile spores persist on surfaces)
• Bioterrorism (B. anthracis spores)
• Food spoilage and food-borne illness (B. cereus, C. botulinum)

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6. Culture Media: Classification with Examples

• Liquid (broth): Nutrient broth, peptone water, thioglycollate broth
• Semi-solid: Used for motility testing (0.5% agar)
• Solid: Nutrient agar (1.5-2% agar)

• Simple/Basal: Nutrient agar, peptone water (supports most non-fastidious organisms)
• Enriched: Blood agar, Chocolate agar (5% lysed blood - for Haemophilus, Neisseria), Loeffler's serum slope (Corynebacterium)
• Selective: Inhibits unwanted organisms, selects one
  • MacConkey agar (Gram-negatives only; bile salts + crystal violet inhibit Gram-positives)
  • TCBS (Thiosulfate Citrate Bile Salts Sucrose) - for Vibrio
  • Mannitol Salt Agar - for Staphylococcus
  • XLD (Xylose Lysine Deoxycholate) - for Salmonella/Shigella
  • Tellurite media (Modified McLeod's) - for Corynebacterium diphtheriae
• Differential: Distinguishes between organisms by color/morphology
  • MacConkey (lactose fermenters = pink/red; non-fermenters = colorless)
  • CLED (Cystine Lactose Electrolyte Deficient) - for urine cultures
• Selective + Differential: MacConkey, TCBS
• Transport media: Preserve organisms during transport (don't support growth)
  • Cary-Blair (feces - for Vibrio, Salmonella, Shigella)
  • Stuart's (gonorrhea, general)
  • Amies (general)
  • Venkatraman-Ramakrishnan (VR) media (Vibrio cholerae)
• Enrichment media: Liquid media that selects for specific organisms
  • Selenite F broth (Salmonella/Shigella)
  • Alkaline peptone water (Vibrio cholerae - pH 8.6)
  • Robertson's cooked meat (Clostridia)
• Special media:
  • Lowenstein-Jensen (LJ) - for Mycobacterium tuberculosis
  • Sabouraud's Dextrose Agar (SDA) - for fungi
  • BCYE (Buffered Charcoal Yeast Extract) - for Legionella
  • Bordet-Gengou agar - for Bordetella pertussis (whooping cough)
  • Thayer-Martin - for Neisseria gonorrhoeae

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7. Gram Staining; Ziehl-Neelsen Acid Fast Staining

1. Flood with Crystal violet (primary stain) - 1 minute → all bacteria stain purple
2. Flood with Gram's iodine (mordant) - 1 minute → CV-I complex formed, larger molecule
3. Decolorize with acetone-alcohol - 30 seconds → Gram-positive retain stain (thick PG), Gram-negative lose stain (thin PG + LPS dissolves)
4. Counterstain with safranin/neutral red - 30 seconds → Gram-negative stain pink/red

• Gram-positive: Purple/violet
• Gram-negative: Pink/red

• Cocci: Staphylococcus (clusters), Streptococcus (chains), Enterococcus
• Rods: Bacillus, Clostridium, Lactobacillus, Corynebacterium (Chinese letter pattern), Listeria

• Cocci: Neisseria (diplococci), Moraxella
• Rods (Enterobacteriaceae): E. coli, Salmonella, Shigella, Klebsiella, Proteus
• Other rods: Pseudomonas, Haemophilus, Vibrio, Campylobacter, Helicobacter

• Mycobacteria (waxy cell wall)
• Mycoplasma (no cell wall)
• Treponema, Leptospira (too thin)
• Chlamydia, Rickettsia (intracellular)
• Legionella (stains poorly)

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1. Flood with Carbol-fuchsin (primary stain, red) - heat until steaming (Hot ZN) or prolonged application (Cold = Kinyoun method) → all bacteria stain red
2. Decolorize with 20% H₂SO₄ (acid-alcohol = 3% HCl in 95% alcohol) → acid-fast organisms retain red stain, others lose stain
3. Counterstain with Loeffler's methylene blue - 30 seconds → non-acid-fast stain blue

• Acid-fast (AFB): Bright red rods (beaded) on blue background
• Non-acid-fast: Blue

• Mycobacterium tuberculosis (and all Mycobacteria)
• Mycobacterium leprae
• Nocardia (weakly acid-fast - decolorize with 1% H₂SO₄)
• Cryptosporidium oocysts (modified ZN)
• Cystoisospora (Isospora) oocysts
• Cyclospora oocysts (variably acid-fast)

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8. Anaerobic Culture Methods

• Gas-generating sachet (hydrogen + CO₂) + palladium catalyst
• Hydrogen reacts with O₂ in presence of catalyst: 2H₂ + O₂ → 2H₂O
• Indicator: methylene blue strip (colorless = anaerobic)

• Thioglycollate broth (contains sodium thioglycollate - absorbs O₂)
• Robertson's cooked meat (unsaturated fatty acids absorb O₂)
• Brewer's thioglycollate medium

• Pyrogallol + NaOH absorbs O₂ (laboratory only)

• Blood agar (enriched, plain, supplemented)
• Neomycin blood agar (selective)
• CCFA (Cycloserine Cefoxitin Fructose Agar) - for C. difficile

• Gram-positive: Clostridium tetani, C. botulinum, C. perfringens, C. difficile, Actinomyces
• Gram-negative: Bacteroides fragilis (most common anaerobic infection), Prevotella, Fusobacterium, Veillonella

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9. Methods of Sterilization: Physical and Chemical

• Dry heat:
  • Red heat (incineration): inoculating loops, platinum wire - 160°C until glowing
  • Flaming: forceps, spatulas, mouths of tubes
  • Hot air oven: 160°C/2 hr or 170°C/1 hr or 180°C/30 min - for glassware, powders, oils
  • Incineration: biohazard waste
• Moist heat:
  • Boiling at 100°C: kills vegetative forms in 10 min; NOT spores
  • Autoclave (steam under pressure): 121°C/15 psi/15 min - kills spores; gold standard for most materials
  • Tyndallization (fractional sterilization): 100°C on 3 consecutive days - kills spores via germination-then-kill cycles
  • Pasteurization: NOT sterilization - reduces pathogen load
    • HTST: 72°C for 15 seconds (High-Temperature Short-Time)
    • LTH: 63°C for 30 minutes (Low-Temperature Holding)
    • UHT: 135°C for 2 seconds (Ultra-High Temperature) - sterile milk

• Ionizing radiation (gamma rays, X-rays): Damages DNA; used for heat-sensitive items (syringes, catheters, sutures, prosthetic heart valves); cobalt-60 source; cold sterilization
• Non-ionizing (UV radiation): Damages DNA (thymine dimers); used for air/surface sterilization in OTs, laminar flow hoods; cannot penetrate solids or glass

• Removes organisms by physical sieving - does NOT kill them
• For heat-sensitive liquids (serums, vitamins, antibiotics, vaccines)
• Membrane filters: 0.22 µm pore size (removes bacteria + fungi)
• 0.01 µm filters: remove viruses
• Types: Seitz filter (asbestos), Berkefeld filter (diatomaceous earth), Millipore/Nuclepore (membrane)
• Laminar flow hoods also use HEPA filters (High-Efficiency Particulate Air) for air

• Glutaraldehyde (2%): "Cold sterilization" for heat-sensitive equipment (endoscopes, rubber items); 10 hrs for full sterilization, 20-30 min for high-level disinfection; alkaline glutaraldehyde (cidex) more active
• Formaldehyde: Gas (formalin fumigation of rooms/cabinets) and liquid; carcinogenic
• Ethylene oxide (ETO): Gas sterilant; used for electronics, plastics, heart-lung machines; penetrates packaging; leaves toxic residues - need aeration; 12% ETO + 88% Freon
• Hydrogen peroxide (6-30%): Plasma sterilization
• Peracetic acid: High-level disinfectant/sterilant for endoscopes

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10. Pasteurization and Filtration Techniques

• Named after Louis Pasteur
• Purpose: Reduce pathogen load in food/beverages - does NOT achieve sterility
• Kills: Brucella (most heat-resistant pathogen in milk), Mycobacterium bovis, Salmonella, Campylobacter, Listeria, Coxiella burnetii (most heat-resistant organism - if killed, all others are too - the pasteurization target)
• Methods (see above in moist heat)

• Asbestos (Seitz) filter: Depth filter; used in labs
• Berkefeld (kieselguhr/diatomaceous earth) filter: Depth filter; W (Wenig/fine), N (Normal/medium), V (Viel/coarse) grades
• Chamberland porcelain filter: Used by Pasteur and Koch; V, L, L₁, L₂, L₃ grades
• Millipore/Cellulose acetate membrane filter: Most commonly used today; gradocol membranes available in precise pore sizes; used to determine size of viruses
• Membrane filters also used in water quality testing (pour filter on agar, count colonies)

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11. Disinfection and Antiseptics: Hospital Application

• Sterilization: Kills all organisms including spores
• Disinfection: Kills or removes most pathogens (not necessarily spores) from inanimate surfaces
• Antisepsis: Killing/inhibiting organisms on living tissue
• Bactericidal: Kills bacteria
• Bacteriostatic: Inhibits bacterial growth (reversible)
• Sanitization: Reduces organisms to safe public health levels

• Phenol (carbolic acid): First antiseptic (Lister); 1-2% for surfaces; Cresol (Lysol) more active; mechanism = protein denaturation
• Chlorine compounds: Household bleach (sodium hypochlorite); drinking water (0.2-0.5 ppm); swimming pools; kills most organisms; inactivated by organic matter
• Iodine: Tincture of iodine (2% I₂ in 70% alcohol) for skin; Povidone iodine (Betadine) - less irritating, slow release; kills all bacteria, spores, fungi, viruses
• Alcohols: Ethanol (70%) and Isopropanol (70%); protein denaturation; rapid action; for skin and surfaces; not effective against spores or naked viruses; 70% more effective than 100% (requires water for activity)
• Quaternary ammonium compounds (QACs/Quats): Benzalkonium chloride (Zephiran); cationic surfactant; membrane disruption; ineffective against TB, spores, Pseudomonas; used for skin and instruments
• Chlorhexidine (Hibitane): Bisbiguanide; membrane disruption; bactericidal + some fungicidal; retained on skin (residual activity); used for surgical scrubs, wound care, catheter care; less effective against Gram-negatives
• Hydrogen peroxide (3%): Wound antiseptic; 6% = disinfectant
• Formaldehyde: Formalin (10% in water); room fumigation; tissue fixation; carcinogen

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12. Bacterial Genetics: Gene Transfer - Transformation, Transduction, Conjugation

• Uptake of naked DNA from the environment by a competent bacterium
• Natural transformation: Streptococcus pneumoniae (Griffith's experiment - rough to smooth), Haemophilus influenzae, Bacillus, Neisseria
• Mechanism: Competent cells express surface proteins (competence factors) that bind and import DNA
• Artificially induced by: CaCl₂ treatment, electroporation (used in cloning)
• Griffith's experiment (1928): Dead smooth + live rough → live smooth (established transformation principle)
• Avery, MacLeod, McCarty (1944): Proved the transforming principle is DNA

• Transfer of bacterial DNA via bacteriophage (virus)
• Generalized transduction:
  • Any piece of bacterial DNA is accidentally packaged into phage head
  • Lytic cycle phage (e.g., P1 phage in E. coli)
  • Any gene can be transferred
  • Example: Staphylococcus aureus resistance genes
• Specialized (Restricted) transduction:
  • Only specific genes adjacent to phage integration site are transferred
  • Lysogenic cycle phage (temperate phage)
  • Lambda phage: transfers genes between gal and bio operons
  • Phage converts bacteria (lysogenic conversion):
    • Corynebacterium diphtheriae toxin (tox gene from beta phage)
    • Clostridium botulinum toxin (types C, D from phage)
    • Streptococcus pyogenes erythrogenic toxin (scarlet fever)
    • Vibrio cholerae CTX phage (cholera toxin)
    • E. coli O157:H7 Shiga toxin (from phage)

• Direct cell-to-cell contact via sex pilus (F pilus)
• Requires F+ (donor) and F- (recipient) cells
• F factor (fertility factor): 100 kb plasmid encoding conjugation proteins
• F+ × F- → F+ (F factor transferred; chromosomal genes not transferred)
• Hfr (High frequency recombination): F factor integrated into chromosome → chromosomal genes transferred at high frequency; rarely makes recipient Hfr
• F' (F prime): F factor excises carrying chromosomal genes → sexduction/F-duction
• Most clinically important: transfer of resistance (R) plasmids - multidrugresistance spread
• Key difference: Only method that requires direct cell contact

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13. Plasmids and Transposons

• Extrachromosomal, circular, double-stranded DNA
• Self-replicating, independent of chromosome
• 1 kb to 1000 kb in size
• Not essential for normal growth (curable - eliminated by acridine orange, elevated temperature)
• Multiple copies per cell (copy number varies)

• F (Fertility) plasmid: Conjugation, gene transfer
• R (Resistance) plasmid: Carries antibiotic resistance genes; most clinically important
• Col (Colicin) plasmids: Produce bacteriocins (kill competing bacteria)
• Virulence plasmids: Toxin genes
  • E. coli heat-labile (LT) and heat-stable (ST) toxins on plasmids (ETEC)
  • Staphylococcal exfoliative toxin, TSST-1 on plasmids
  • Yersinia pestis virulence plasmids
• Metabolic plasmids: Degrade unusual compounds (e.g., camphor in Pseudomonas)
• Ti plasmid: In Agrobacterium tumefaciens (used in plant genetic engineering)

• Mobile genetic elements that can move (transpose) from one DNA location to another
• Can move between chromosome, plasmids, or between bacteria
• Contain inverted repeat sequences at ends
• Require enzyme transposase
• Types:
  • Insertion sequences (IS elements): Simplest; only transposase gene + inverted repeats
  • Composite transposons (Class I): Two IS elements flanking antibiotic resistance genes (e.g., Tn10 - tetracycline resistance)
  • Complex transposons (Class II): Single-unit; encode transposase + other genes (e.g., Tn3 - ampicillin resistance)

• Spread antibiotic resistance between plasmids and chromosomes
• Create new resistance gene combinations
• Tn1546 - vancomycin resistance in Enterococcus (VRE)

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14. Mutations and Antibiotic Resistance

• Point mutation: Single base change
  • Silent (synonymous): No amino acid change
  • Missense: Different amino acid substituted
  • Nonsense: Stop codon created → truncated protein
• Frameshift mutation: Insertion or deletion of bases (not in multiples of 3) → reading frame shifts completely
• Insertion/deletion: Can be in-frame or frameshift
• Transition: Purine → purine or pyrimidine → pyrimidine
• Transversion: Purine → pyrimidine or vice versa

• Physical: UV (thymine dimers), ionizing radiation
• Chemical: Base analogs (5-BrU), alkylating agents (mustard gas), deaminating agents (nitrous acid), intercalating agents (acridine orange - frameshift)

• Beta-lactamases: Destroy beta-lactam ring of penicillin/cephalosporins
  • ESBL (Extended Spectrum Beta-Lactamases): Klebsiella, E. coli - hydrolyze 3rd/4th gen cephalosporins + aztreonam; treat with carbapenems
  • Carbapenemases: Klebsiella pneumoniae Carbapenemase (KPC), NDM-1 (New Delhi Metallo-beta-lactamase), OXA-48 - hydrolyze even carbapenems; treatment nightmare
• Aminoglycoside-modifying enzymes: acetyltransferases, phosphotransferases, adenyltransferases
• Chloramphenicol acetyltransferase (CAT)

• PBP (Penicillin-Binding Protein) alteration: MRSA - mecA gene on SCCmec element → PBP2a with low affinity for all beta-lactams
• Ribosomal methylation: Erythromycin resistance in Streptococcus (erm genes)
• DNA gyrase mutation: Fluoroquinolone resistance
• Altered cell wall precursors (D-Ala-D-Lac instead of D-Ala-D-Ala): VRE vancomycin resistance (van genes)

• Porin mutations: Decreased outer membrane permeability (Pseudomonas, Klebsiella)
• Reduces entry of beta-lactams, carbapenems, aminoglycosides

• Active expulsion of antibiotic from cell
• MDR efflux pumps: Pseudomonas (MexAB-OprM), Staphylococcus (NorA)
• Confers resistance to fluoroquinolones, tetracyclines, macrolides

• Trimethoprim resistance: Overproduction of DHFR or alternative DHFR with low binding
• Sulfonamide resistance: Alternative PABA pathway or DHPS alteration

• mecA gene → PBP2a → low affinity for all beta-lactams
• Treatment: Vancomycin (drug of choice), Linezolid, Daptomycin, Tigecycline

• Van genes → D-Ala-D-Lac substitution → vancomycin cannot bind
• VanA: Resistant to vancomycin + teicoplanin (high-level)
• VanB: Resistant to vancomycin only
• Treatment: Linezolid, Quinupristin-dalfopristin (not E. faecalis), Daptomycin

• TEM, SHV, CTX-M type enzymes
• Hydrolyze all penicillins + cephalosporins (1st-4th gen) + aztreonam
• Treatment: Carbapenems (imipenem, meropenem) = drug of choice
• Screen by: Cephalosporin + clavulanate - zone of inhibition increases (confirmatory test)

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15. Normal Flora of Human Body and Its Significance

• Colonization resistance: Prevents pathogen colonization by competition (space + nutrients) and production of bacteriocins
• Stimulates development of immune system (GALT - Gut-Associated Lymphoid Tissue)
• Vitamin synthesis: Vitamin K and B12 by gut bacteria
• Breakdown of complex carbohydrates and bile salts
• Maintains mucosal integrity

• Opportunistic infections when host immunity fails
• Post-surgical infections if normal flora enters sterile sites
• Antibiotic-associated diarrhea: Disruption of normal flora allows C. difficile overgrowth (pseudomembranous colitis)
• Endogenous infections in immunocompromised patients

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16. Pathogenicity and Virulence Factors

• Pili/fimbriae: E. coli (ETEC - CFA I/II; UPEC - type 1 fimbriae for bladder epithelium; P-fimbriae for kidney)
• Surface adhesins: FimH, intimin (EPEC)
• Biofilm formation: S. epidermidis (medical devices)

• Hyaluronidase: Spreads through tissue ("spreading factor") - Streptococcus, Staphylococcus, Clostridium
• Collagenase: C. perfringens (gas gangrene - collagen destruction)
• Coagulase: S. aureus - fibrin clot formation (protects from phagocytosis)
• Kinases (streptokinase, staphylokinase): Fibrinolysis
• DNase, Lipase: Tissue destruction

• Exotoxins: Secreted proteins; heat-labile (mostly); specific receptors; highly potent; stimulate antitoxin (immunogenic)
  • Neurotoxins: Tetanus toxin (C. tetani - blocks inhibitory interneurons), Botulinum toxin (C. botulinum - blocks ACh release at NMJ)
  • Enterotoxins: Cholera toxin (V. cholerae - ADP-ribosylates Gs → permanent activation → ↑cAMP → Cl⁻/water secretion), E. coli LT (similar), E. coli ST (activates guanylate cyclase → ↑cGMP)
  • Cytotoxins: Shiga toxin (Shigella/STEC - inhibits 60S ribosome → cell death → HUS), Diphtheria toxin (inhibits EF-2 → halts protein synthesis)
• Endotoxin (LPS): Lipopolysaccharide from Gram-negative outer membrane
  • Lipid A = active toxic component
  • Released on bacterial death
  • Heat-stable, not destroyed by autoclaving
  • Effects: Fever (IL-1, IL-6, TNF-α), hypotension, DIC, complement activation, endotoxic shock/septic shock
  • Detected by: Limulus amebocyte lysate (LAL) test

• Capsule: Primary anti-phagocytic factor (S. pneumoniae, Klebsiella, H. influenzae, N. meningitidis)
• Protein A (S. aureus): Binds Fc region of IgG → blocks opsonization
• M protein (S. pyogenes): Anti-phagocytic, binds factor H
• Leukocidins (PVL - Panton-Valentine Leukocidin): Kills neutrophils and macrophages (S. aureus)
• IgA protease: Cleaves secretory IgA (N. gonorrhoeae, S. pneumoniae, H. influenzae)

• Inhibits phagolysosome fusion: M. tuberculosis, Legionella
• Escapes into cytoplasm: Listeria (actin rocket motility), Shigella
• Survives in phagolysosome: Leishmania, Coxiella burnetii

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17. Quorum Sensing and Biofilms

• Cell-to-cell communication system in bacteria using chemical signals (autoinducers)
• When population density reaches threshold, autoinducer level triggers gene expression changes
• Named because bacteria "sense" when a quorum (sufficient number) is present

1. Bacteria produce and secrete autoinducers (AHL - acyl homoserine lactones in Gram-negative; oligopeptides in Gram-positive)
2. As population grows, autoinducer accumulates
3. At threshold concentration, autoinducer binds receptor → gene expression changes
4. Coordinate behaviors: Biofilm formation, toxin production, virulence factor expression, sporulation, bioluminescence

• Pseudomonas aeruginosa: Las-R/Las-I and Rhl-R/Rhl-I systems control virulence (biofilm, elastase, pyocyanin)
• Staphylococcus aureus: Agr (accessory gene regulator) system controls toxin production and biofilm dispersal
• Vibrio fischeri: Bioluminescence in deep-sea fish (model organism for QS)
• Streptococcus mutans: Dental caries formation

• Structured communities of bacteria embedded in extracellular polysaccharide (EPS) matrix attached to surfaces
• Typically 100-1000x more resistant to antibiotics than planktonic cells
• EPS matrix = "slime layer" (alginate in Pseudomonas)

1. Reversible attachment (initial adhesion)
2. Irreversible attachment (pili, adhesins)
3. EPS matrix production and microcolony formation
4. Maturation (3D architecture, water channels)
5. Dispersal (bacteria detach for new sites)

• Indwelling medical device infections: Urinary catheters (CAUTI), IV lines (CLABSI), prosthetic valves, joint prostheses, pacemakers
• Main organisms: S. epidermidis, S. aureus, Pseudomonas aeruginosa, Candida
• Chronic infections: Pseudomonas in cystic fibrosis lungs
• Dental plaque (S. mutans)
• Resistance mechanisms: EPS barrier, slow growth, persister cells, phenotypic variation

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18. Methods of Microbial Identification (Staining, Culture, Serology)

• Gram stain, ZN stain (covered above)
• Albert's stain: Metachromatic granules (volutin/Babes-Ernst granules) of Corynebacterium diphtheriae - blue-black granules on green background (safety pin appearance, clubbing)
• Wayson's stain: Bipolar staining of Yersinia pestis (safety pin appearance)
• Giemsa stain: Blood parasites (Plasmodium, Leishmania), Chlamydia (LGV), Rickettsia
• PAS stain: Fungi (cell wall carbohydrates), Whipple's disease (Tropheryma whipplei)
• India ink stain: Capsule of Cryptococcus neoformans (negative stain - clear halo around yeast)
• Fluorescent stains: Auramine-rhodamine (for TB screening - AFB appear golden)
• Calcofluor white: Fungi (cell wall chitin fluoresces)

• Identification by colony morphology, color, smell, pigment
• Biochemical tests:
  • Catalase test: Staph (positive - bubbles) vs Strep (negative)
  • Coagulase test: S. aureus (positive) vs S. epidermidis (negative)
  • Oxidase test: Neisseria, Pseudomonas, Vibrio, Campylobacter (positive); Enterobacteriaceae (negative)
  • Indole test (from tryptophan): E. coli (positive), Proteus vulgaris (positive); Salmonella, Klebsiella (negative)
  • IMViC (E. coli): ++ -- (Indole, Methyl red, VP, Citrate)
  • Urease test: H. pylori, Proteus, Klebsiella, Brucella
  • API strips, VITEK system (automated biochemical identification)

• Detection of antibodies or antigens in serum
• ELISA, Western blot, agglutination tests (covered in Immunology section)
• Widal test: Tube agglutination for typhoid (O + H antigens)
• ASO (Antistreptolysin O) titre: Past Group A Strep infection
• VDRL/RPR: Syphilis screening (non-treponemal tests)
• TPHA, FTA-ABS: Confirmatory treponemal tests for syphilis

• PCR (Polymerase Chain Reaction): Highly sensitive, specific; diagnose TB, HIV, HPV, C. difficile
• 16S rRNA sequencing: Universal bacterial identification
• MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight): Rapid protein fingerprinting for species identification - now standard in most labs
• PFGE (Pulsed Field Gel Electrophoresis): Strain typing for outbreak investigation
• WGS (Whole Genome Sequencing): Gold standard for epidemiology

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19. Antimicrobial Susceptibility Testing (Kirby-Bauer, MIC)

• Also called Disc diffusion test
• Principle: Antibiotic-impregnated discs placed on Mueller-Hinton agar (standard medium) inoculated with test organism; antibiotic diffuses creating gradient; bacteria grow where concentration is below MIC
• Method:
  1. Prepare 0.5 McFarland turbidity suspension of organism
  2. Spread lawn on Mueller-Hinton agar
  3. Apply antibiotic discs (standard potency)
  4. Incubate 35-37°C for 16-18 hours
  5. Measure zone of inhibition diameter in mm
• Interpretation: Compare to CLSI (Clinical and Laboratory Standards Institute) / EUCAST breakpoints:
  • Sensitive (S): Zone ≥ standard threshold → treat with standard dose
  • Intermediate (I): Between S and R → treat with high dose or use when drug concentrates at site
  • Resistant (R): Zone ≤ threshold → drug ineffective
• Advantages: Simple, inexpensive, flexible
• Disadvantages: Qualitative only (not quantitative); doesn't give MIC; not suitable for slow-growing organisms or anaerobes (E-test better)

• Lowest concentration of antibiotic that inhibits visible growth after overnight incubation
• Broth dilution method: Serial 2-fold dilutions of antibiotic in broth tubes; inoculate each with standard bacterial suspension; incubate; lowest clear tube = MIC
  • Macrobroth dilution (original)
  • Microbroth dilution (96-well plate) - most common now
• MBC (Minimum Bactericidal Concentration): Lowest concentration that kills ≥99.9% of organisms (subcultured from clear MIC tubes onto antibiotic-free medium)
• E-test (Epsilometer test): Plastic strip with continuous gradient of antibiotic concentrations; place on seeded agar; elliptical zone of inhibition forms; MIC read at intersection of growth with strip - combines ease of disc diffusion with quantitative MIC

• PK/PD parameters guide dosing:
  • Time-dependent killing (beta-lactams, carbapenems): Keep concentration above MIC for >50% of dosing interval → more frequent dosing
  • Concentration-dependent killing (aminoglycosides, fluoroquinolones): Maximize peak concentration → once-daily dosing

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20. Mechanisms of Antimicrobial Drug Resistance in Bacteria (ESBL, MRSA, VRE)

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21. Nosocomial Infections: Causes, Prevention

• Infections acquired in healthcare settings, not present/incubating at admission
• Onset: ≥48 hours after admission

1. CAUTI (Catheter-Associated Urinary Tract Infection) - most common (40% of HAIs)
2. VAP (Ventilator-Associated Pneumonia) - highest mortality
3. CLABSI (Central Line-Associated Bloodstream Infection) - highest cost
4. SSI (Surgical Site Infection)
5. CDAD (Clostridioides difficile-Associated Disease)

• Gram-negative: E. coli, Klebsiella (ESBL), Pseudomonas aeruginosa, Acinetobacter baumannii
• Gram-positive: MRSA, S. epidermidis (biofilm on devices), Enterococcus (VRE)
• Fungi: Candida species (esp. ICU patients)
• Spores: C. difficile (CDI)

• Hand hygiene (most important single measure): WHO 5 moments (before patient contact, before aseptic procedure, after body fluid exposure, after patient contact, after contact with patient surroundings)
• Alcohol-based hand rubs for routine decontamination; soap and water for C. difficile (spores)
• Standard precautions: For all patients regardless of diagnosis
• Transmission-based precautions:
  • Contact precautions: MRSA, VRE, C. difficile, multi-drug resistant organisms
  • Droplet precautions: Influenza, Meningococcus, Mumps
  • Airborne precautions: TB, Chickenpox, Measles (negative pressure rooms, N95 respirators)
• Device bundles:
  • Central line bundle (CLABSI prevention): Hand hygiene, maximal barrier precautions, chlorhexidine skin antisepsis, optimal catheter site selection, daily review
  • Ventilator bundle (VAP prevention): HOB elevation 30-45°, oral chlorhexidine, sedation vacation, subglottic suctioning
  • Urinary catheter bundle: Insert only when indicated, remove as soon as possible
• Antibiotic stewardship: Prevent resistance development
• Proper instrument sterilization, disinfection
• Surveillance and reporting

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22. Biomedical Waste Management: Color Coding, Disposal; Biosafety Levels and Universal Precautions

• Yellow bag: Incineration (anatomical waste); chemical treatment + shredding (cytotoxic)
• Red bag: Autoclave (steam sterilization) → shredding → to authorized recycler
• White puncture-proof container: Encapsulation/plasma pyrolysis/incineration
• Liquid waste: ETP (Effluent Treatment Plant) before discharge

• Treat all blood and body fluids as potentially infectious, regardless of known status
• Components:
  1. Hand hygiene before and after patient contact
  2. PPE (gloves, mask, gown, eye protection) as appropriate
  3. Safe handling and disposal of sharps - NEVER recap needles with two hands
  4. Safe disposal of biohazardous waste
  5. Respiratory hygiene/cough etiquette
  6. Safe injection practices
  7. Proper handling of patient care equipment

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SECTION II: IMMUNOLOGY

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1. Innate vs Adaptive Immunity; Natural vs Artificial; Passive and Herd Immunity

• Present from birth; non-specific; no memory
• Rapid response (minutes to hours)
• Components:
  • Physical/anatomical barriers: Skin, mucous membranes, cilia, gastric acid, lysozyme in tears/saliva
  • Cellular: Neutrophils (first responders), macrophages, NK cells, mast cells, basophils, eosinophils, dendritic cells
  • Soluble: Complement, acute phase proteins (CRP, fibrinogen, serum amyloid A), interferons (type I - IFN-α, IFN-β), lysozyme, defensins
  • Pattern Recognition Receptors (PRRs) recognize PAMPs (Pathogen-Associated Molecular Patterns):
    • Toll-Like Receptors (TLRs): TLR4-LPS, TLR3-dsRNA, TLR9-unmethylated CpG DNA
    • NOD-like receptors (NLRs), RIG-I (RNA helicase), Mannose receptor
  • DAMPs (Damage-Associated Molecular Patterns): Released by dying host cells; activate sterile inflammation

• Specific, has memory, slower (days to weeks for primary response)
• Components:
  • T cells (cell-mediated): CD4+ helper, CD8+ cytotoxic, Treg
  • B cells/Plasma cells (humoral): Antibody production
• Features: Specificity, Diversity (10^11 different antibodies), Memory (faster/stronger secondary response), Self-tolerance

• Active immunity: Immunological memory is generated; long-lasting
• Passive immunity: No memory; temporary (weeks to months); provides immediate protection

• Antitoxins: Tetanus antitoxin (TAT), diphtheria antitoxin
• Anti-venom serum
• HBIG (Hepatitis B Immunoglobulin) post-exposure prophylaxis
• Anti-D immunoglobulin (Rh prophylaxis)
• IVIG (Intravenous Immunoglobulin) for immunodeficient patients

• When sufficient proportion of a population is immune → indirect protection to non-immune individuals
• Threshold (Herd Immunity Threshold, HIT) = 1 - 1/R₀
• Where R₀ = Basic Reproduction Number (average infections caused by one case in fully susceptible population)

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2. Antigen, Epitope, Hapten: Definitions and Examples

• Any substance that can bind to an antibody or T-cell receptor
• Immunogen: Antigen that can trigger an immune response
• Properties of good immunogen: Foreign, high molecular weight (>10,000 Da), chemical complexity, accessibility of epitopes, appropriate route of entry
• Complete antigen: Both immunogenic AND antigenic (e.g., proteins, large polysaccharides)
• Incomplete antigen (Hapten): Antigenic but NOT immunogenic alone

• The specific molecular site on an antigen that binds to an antibody or TCR
• A single antigen may have multiple epitopes (polyvalent)
• B cell epitopes: Usually conformational (3D structure); can be linear
• T cell epitopes: Must be linear peptides presented by MHC; processed by APCs
• Haptens as epitopes: A hapten acts as an epitope when conjugated to a carrier protein

• Small molecule (< 1000 Da) that binds specifically to an antibody but CANNOT by itself stimulate antibody production
• Requires coupling to a larger carrier protein to become immunogenic (Hapten-Carrier concept, Landsteiner)
• When hapten-carrier conjugate is injected:
  • B cells recognize the hapten
  • T cells recognize the carrier
  • T-B cooperation leads to anti-hapten antibodies
• Examples:
  • Penicillin: Hapten - binds to serum proteins → penicilloyl-protein conjugate → IgE antibodies → anaphylaxis
  • DNCB (Dinitrochlorobenzene): Contact hypersensitivity testing hapten
  • Poison ivy (urushiol): Hapten causing contact dermatitis
  • Dinitrophenol (DNP): Classic experimental hapten
  • Drugs: Many drugs (aspirin, sulfonamides, quinidine) are haptens

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3. Immunoglobulins - Classes, Structure and Function (IgG, IgA, IgM, IgE, IgD)

• 4-chain structure: 2 identical heavy chains (H) + 2 identical light chains (L)
• Linked by disulfide bonds
• Light chains: Kappa (κ) or Lambda (λ)
• Heavy chains determine class: γ(IgG), α(IgA), µ(IgM), ε(IgE), δ(IgD)

• Fab (Fragment antigen binding): Variable regions (VH + VL) + constant region CH1 + CL
• Fc (Fragment crystallizable): CH2 + CH3 of heavy chains
• Hinge region: Between Fab and Fc; flexible; contains disulfide bonds; susceptible to papain/pepsin cleavage
• Papain cleavage: 2 Fab + 1 Fc
• Pepsin cleavage: F(ab')₂ + pFc' (destroyed)
• CDR (Complementarity-Determining Regions): Hypervariable regions within V domains; actual antigen contact

• Most abundant (75-80% of total serum Ig)
• 4 subclasses: IgG1, IgG2, IgG3, IgG4
• ONLY Ig that crosses placenta (via FcRn receptor) → neonatal protection (maternal IgG lasts 3-6 months)
• Secondary (anamnestic) immune response - predominant Ig
• Functions: Neutralization, opsonization, ADCC, complement activation (IgG1 > IgG2 > IgG3; IgG4 does NOT activate complement via classical pathway)
• Monovalent in solution; can activate complement

• Largest Ig (pentameric - 5 units joined by J chain)
• 10% of serum Ig; MW ~900 kDa
• First antibody produced in primary immune response (acute infection marker)
• Does NOT cross placenta - IgM in newborn = congenital infection (TORCH)
• Most efficient complement activator (classical pathway)
• Best agglutinating antibody (10 antigen-binding sites)
• ABO blood group antibodies are IgM
• Natural antibodies (not requiring prior exposure) are IgM

• Second most abundant in serum (15%)
• Predominant Ig in secretions (secretory IgA = sIgA): Saliva, tears, breast milk, GI tract secretions, respiratory secretions
• Serum IgA: Monomer; 2 subclasses (IgA1, IgA2)
• Secretory IgA: Dimer + J chain + secretory component (SC) - SC protects against proteolysis
• Secretory component produced by epithelial cells
• First line of mucosal defense
• Does NOT activate complement (classical pathway); may activate alternative pathway
• Breast milk sIgA protects neonates from GI infections
• IgA deficiency: Most common primary Ig deficiency; recurrent sinopulmonary infections; risk of anaphylaxis to blood products

• Lowest serum concentration (<0.001%)
• Heavy chain = epsilon (ε)
• Binds with high affinity to FcεRI on mast cells and basophils
• Cross-linking by antigen triggers mast cell degranulation → immediate hypersensitivity (Type I) / allergic reactions / anaphylaxis
• Elevated in: Allergies, asthma, eczema, parasitic infections (helminth defense)
• Half-life: 2.5 days (serum); weeks (bound to mast cells)
• Role in ADCC against helminths (parasitic worms) - eosinophil-mediated

• <1% of serum Ig
• Primary role: B cell antigen receptor (BCR) on naive B cells (co-expressed with IgM as BCR)
• Signaling during B cell activation and differentiation
• No well-defined effector function in serum
• Elevated in IgD myeloma and in some autoimmune conditions

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4. Humoral vs Cell-Mediated Immunity

• Mediated by antibodies produced by B cells (plasma cells)
• Effective against: Extracellular bacteria, toxins, viruses (before cell entry), parasites
• Process:
  1. Antigen recognized by BCR (surface Ig) on B cell
  2. T-dependent antigens (proteins): Require CD4+ T helper cells; produce memory; class switching (IgG, IgA, IgE)
  3. T-independent antigens (LPS, polysaccharides): No T cell help needed; no memory; primarily IgM
  4. B cell differentiates into plasma cells (antibody factories) + memory B cells
  5. Antibody effector functions: Neutralization, opsonization, complement activation, ADCC

• Mediated by T cells; no antibody involvement
• Effective against: Intracellular pathogens (viruses, TB, Listeria, Leishmania), fungi, transplant rejection, tumor cells
• Key T cell subtypes:
  • CD4+ Th1 (Helper 1): Activate macrophages (via IFN-γ) → killing of intracellular organisms; delayed hypersensitivity reactions; IL-2, IFN-γ
  • CD4+ Th2: Help B cells; promote IgE (allergy, antiparasite); IL-4, IL-5, IL-13
  • CD4+ Th17: Neutrophil recruitment; mucosal immunity; IL-17, IL-21; autoimmunity
  • CD8+ CTL (Cytotoxic T Lymphocytes): Kill infected cells (via perforin/granzymes), tumor cells, transplant rejection; recognize antigen on MHC class I
  • Treg (Regulatory T cells): Suppress immune responses; prevent autoimmunity; IL-10, TGF-β, CTLA-4

• Mantoux test (PPD/tuberculin): Type IV hypersensitivity reaction; tests prior TB exposure
• Contact sensitivity: DNCB test

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5. Antigen-Antibody Reactions - Types, Principle, Clinical Applications (Agglutination, Precipitation)

• Ag-Ab reactions are reversible, non-covalent
• Forces: Hydrogen bonds, electrostatic, Van der Waals, hydrophobic interactions
• Law of mass action applies
• Affinity: Strength of single Ag-Ab bond (intrinsic)
• Avidity: Overall binding strength of multiple bonds (multivalent interactions - IgM very high avidity)
• Specificity: Each antibody binds its specific epitope (cross-reactivity can occur with structurally similar epitopes)

• Clumping of particulate antigens (bacteria, RBCs, latex particles) by antibodies
• Requires at least divalent antibody (IgM is best - pentavalent)
• Direct agglutination: Antibody directly agglutinates bacteria/RBCs
  • ABO blood typing: Reagent antibodies agglutinate RBCs
  • Widal test: Patient serum agglutinates killed Salmonella typhi (O and H antigens)
  • Brucella agglutination test (Wrights)
• Passive (Indirect) agglutination: Soluble antigen coated onto inert carrier (latex, bentonite, RBCs) then agglutinated by antibody
  • Latex agglutination for CRP, cryptococcal antigen, H. influenzae, Group B Strep
  • RPHA (Reverse Passive Hemagglutination): Antibody-coated RBCs agglutinated by antigen
• Prozone phenomenon: False negative agglutination due to antibody excess - each antigen molecule fully saturated by antibodies, no lattice formation; solution = dilute the serum
• Coombs test (Antiglobulin test):
  • Direct Coombs: Detect antibody/complement already on RBCs (hemolytic disease of newborn, autoimmune hemolytic anemia)
  • Indirect Coombs: Detect antibodies in serum that would coat RBCs (pre-transfusion crossmatch)

• Antigen is soluble (not particulate); antibody-antigen complexes precipitate
• Requires optimal antigen-antibody ratio (equivalence zone)
• Prozone (antibody excess): No precipitation; false negative
• Postzone (antigen excess): No precipitation; false negative
• Precipitation techniques:
  • Ouchterlony double diffusion (gel diffusion): Antigen and antibody diffuse toward each other in agar; precipitin line forms at equivalence
    • Line of identity (same epitopes), partial identity, non-identity
    • Used for: Aspergillus precipitins, hepatitis antigens
  • Radial immunodiffusion (Mancini): Antigen placed in wells, antibody in agar; ring diameter² proportional to Ag concentration; quantitative
  • Immunoelectrophoresis: Separate proteins by electrophoresis then overlay antiserum; for identifying paraprotein bands in myeloma
  • Immunofixation: More sensitive variant; diagnose specific monoclonal proteins

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6. Immunofluorescence; Prozone Phenomenon

• Antibodies labeled with fluorescent dye (fluorochrome) used to detect antigens in tissue or antibodies in serum
• Fluorochromes: Fluorescein isothiocyanate (FITC - green), Rhodamine (red), Phycoerythrin (PE)
• Detected by fluorescence microscope (UV light source)

• Direct IF (DIF): Fluorescein-labeled antibody applied directly to specimen
  • Rapid, one-step, but requires labeled antibody for each antigen
  • Used for: Diagnosis of HSV, rabies (antigen detection), pemphigus/pemphigoid (skin biopsy showing IgG/C3 deposits)
  • Pneumocystis jirovecii: Monoclonal antibody DIF for BAL diagnosis
• Indirect IF (IIF/IDAT): Two-step method
  1. Primary unlabeled antibody binds antigen in tissue
  2. Fluorescent anti-immunoglobulin (secondary antibody) binds primary antibody
  • More sensitive (signal amplification), flexible
  • Used for: ANA (antinuclear antibodies) in SLE, ANCA (antineutrophil cytoplasmic antibodies) in vasculitis, anti-dsDNA, anti-thyroid antibodies

• Homogeneous: Anti-dsDNA, anti-histone → SLE
• Speckled: Anti-Sm, anti-RNP, anti-SSA/SSB
• Nucleolar: Anti-RNA polymerase → Scleroderma
• Centromere: Anti-CENP-B → Limited systemic sclerosis (CREST)
• Cytoplasmic: Anti-Jo-1 → Polymyositis

• Paradoxical failure to detect an antigen-antibody reaction despite both being present, due to EXCESS ANTIBODY
• At high antibody concentrations, each antigen is saturated by antibodies from all sides → no cross-linking → no lattice/precipitate/agglutination
• False negative result
• Solution: Dilute the serum
• Classic example: Prozone in syphilis VDRL test (high-titer patients may show false negative if serum not diluted)
• Also seen in Brucella agglutination, Widal test
• Note: POST-zone = antigen excess → also no lattice/false negative

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7. Antigen-Presenting Cells and MHC I/II (Macrophages, Phagocytes; HLA; T and B cells; NK cells, IL)

• Express MHC molecules and present peptide antigens to T cells
• Professional APCs (express MHC II constitutively):
  • Dendritic cells (DCs): Most potent APCs; "sentinels" of innate immunity; capture antigen in periphery → migrate to lymph nodes → activate naive T cells; Langerhans cells (skin DCs); follicular DCs
  • Macrophages: Phagocytose and process antigens; also effector cells; secrete cytokines (IL-1, IL-6, TNF-α, IL-12); activated by IFN-γ
  • B cells: Present antigens to CD4+ T cells; BCR-mediated endocytosis of antigen

• Also called HLA (Human Leukocyte Antigens) system in humans
• Located on chromosome 6 (short arm)
• Highly polymorphic (hundreds of alleles at each locus)

• Expressed on ALL nucleated cells
• Presents endogenous antigens (intracellular, e.g., viral proteins, tumor antigens)
• Pathway: Proteasome → TAP (Transporter Associated with Antigen Processing) → ER → peptide loaded onto MHC I → Golgi → cell surface
• Recognized by CD8+ cytotoxic T cells (CTLs)
• Loci: HLA-A, HLA-B, HLA-C
• Structure: α chain (polymorphic) + β₂-microglobulin (non-polymorphic, chromosome 15)

• Expressed only on professional APCs (dendritic cells, macrophages, B cells) + thymic epithelium
• Presents exogenous antigens (phagocytosed/endocytosed)
• Pathway: Endosome → lysosome (protease digestion) → Ii chain (invariant chain, CLIP) displaced by HLa-DM → peptide loaded → cell surface
• Recognized by CD4+ helper T cells
• Loci: HLA-DR (most polymorphic, most important clinically), HLA-DQ, HLA-DP
• Structure: α chain + β chain (both polymorphic)

• Innate lymphoid cells; large granular lymphocytes
• Do NOT require prior sensitization or MHC-restricted recognition
• Kill virus-infected cells and tumor cells that DOWNREGULATE MHC I (missing self - NK cells attack)
• Regulated by:
  • Activating receptors: NKp30, NKp44, NKG2D (recognize stress ligands on infected/tumor cells)
  • Inhibitory receptors: KIR (Killer Immunoglobulin-like Receptors), NKG2A - recognize self MHC I → inhibition
• Kill via: Perforin-granzyme pathway, FasL-Fas pathway, ADCC (CD16/FcγRIII on NK cells)
• Produce: IFN-γ (anti-viral, macrophage activation), TNF-α

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8. Cytokines and Their Immunological Role

• Type I IFNs (IFN-α, IFN-β): Innate antiviral response; induced by viral dsRNA (TLR3); upregulate MHC I, activate NK cells; used therapeutically (IFN-α for HCV, HBV, melanoma)
• Type II IFN (IFN-γ): Produced by T and NK cells; activates macrophages to kill intracellular organisms; key for granuloma formation (TB); upregulates MHC I and II

• CC chemokines (CCL): CCL2 (MCP-1, monocytes), CCL5 (RANTES, T cells)
• CXC chemokines (CXCL): CXCL8 (IL-8, neutrophils), CXCL10 (IP-10, T cells)

• Etanercept, Infliximab, Adalimumab: Anti-TNF-α (RA, IBD, psoriasis)
• Tocilizumab: Anti-IL-6R (RA, cytokine storm in COVID-19)
• Dupilumab: Anti-IL-4Rα (atopic dermatitis, asthma)

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9. Complement Pathways (Classical, Alternative, Lectin)

• Triggered by: Antigen-antibody complexes (IgG or IgM bound to antigen); IgM is 1000x more efficient than IgG per molecule
• Activation sequence: C1q → C1r → C1s → C4 → C2 → C3 convertase (C4b2a) → C3 cleavage → C5 convertase (C4b2a3b) → C5 cleavage → C5b-9 (MAC)
• C1q binds Fc of antibody → conformational change → activates C1r → C1s → cleavage of C4 and C2

• Triggered by: Mannose-Binding Lectin (MBL) binding to mannose residues on pathogen surfaces; also ficolins
• MBL → MASP-1/MASP-2 (serine proteases similar to C1r/C1s) → cleavage of C4 and C2 (same as classical from here)
• Innate immunity; does not require antibody

• Triggered by: Direct spontaneous hydrolysis of C3 (C3 tickover) on pathogen surfaces lacking regulatory proteins
• C3 → C3(H₂O) + Factor B → Factor D cleaves B → C3bBb = C3 convertase → amplification loop
• Stabilized by Properdin (Factor P) on pathogen surface; inhibited by Factor H and Factor I on host cells
• Most ancient pathway; continuous low-level activation; amplification loop greatly amplifies classical and lectin

• C5 → C5a + C5b
• C5b + C6 + C7 + C8 + multiple C9 = MAC (Membrane Attack Complex)
• MAC inserts into bacterial membrane → osmotic lysis
• Critical for killing: Neisseria meningitidis and Neisseria gonorrhoeae (terminal complement deficiency → recurrent Neisseria infections)

• DAF (Decay Accelerating Factor = CD55): Prevents complement on host cells - accelerates decay of C3 convertase
• CD59 (Protectin): Inhibits MAC formation on host cells
• Deficiency of DAF and CD59 → PNH (Paroxysmal Nocturnal Hemoglobinuria): Episodes of intravascular hemolysis

• C1q, C2, C4 deficiency → SLE-like disease (impaired immune complex clearance)
• C3 deficiency → recurrent encapsulated bacterial infections (most severe)
• Terminal deficiency (C5-C9) → recurrent Neisseria infections
• Properdin deficiency → susceptibility to Neisseria
• C1-inhibitor deficiency → Hereditary Angioedema (HAE): excessive C2/C4 activation → bradykinin; treat with C1-INH concentrate, icatibant, ecallantide

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10. Hypersensitivity Reactions (Type I to IV) with Examples

• Mediator: IgE
• Mechanism: First exposure → sensitization (antigen-specific IgE produced → binds to FcεRI on mast cells/basophils); Re-exposure → antigen cross-links IgE → mast cell degranulation → histamine, leukotrienes (LTC4, LTD4), prostaglandins, tryptase release
• Time: Immediate (seconds to minutes; late phase reaction 4-6 hrs)
• Examples: Anaphylaxis, allergic asthma, allergic rhinitis (hay fever), urticaria, angioedema, food allergies (nuts, shellfish), penicillin allergy, bee sting allergy, atopic dermatitis
• Treatment: Epinephrine (anaphylaxis), antihistamines, corticosteroids, desensitization

• Mediator: IgG or IgM against cell surface / tissue antigens
• Mechanism: Antibody binds → complement activation (lysis) OR phagocytosis (opsonization) OR ADCC via NK cells/macrophages
• Time: Minutes to hours
• Examples:
  • Autoimmune hemolytic anemia (anti-RBC antibodies)
  • Transfusion reactions (incompatible blood group)
  • Hemolytic disease of newborn (Rh incompatibility - anti-D IgG crosses placenta)
  • ITP (Immune Thrombocytopenic Purpura - anti-platelet antibodies)
  • Goodpasture syndrome (anti-GBM antibodies → glomerulonephritis + pulmonary hemorrhage)
  • Myasthenia gravis (anti-AChR antibodies → block neuromuscular junction → muscle weakness)
  • Graves' disease (anti-TSH receptor antibodies → stimulatory → hyperthyroidism - Type II stimulatory variant)
  • Pemphigus vulgaris (anti-desmoglein antibodies → acantholysis)

• Mediator: IgG or IgM + antigen = immune complexes deposited in tissues
• Mechanism: Immune complexes deposit in vessel walls/basement membranes → complement activation (C3a, C5a) → neutrophil recruitment → lysosomal enzyme release → tissue damage
• Time: 6-12 hours (Arthus); 1-3 weeks (serum sickness)
• Examples:
  • Serum sickness: After antitoxin (horse serum) administration; fever, rash, arthralgias, proteinuria 1-3 weeks later
  • SLE (anti-dsDNA + dsDNA immune complexes in glomeruli, skin, joints)
  • Post-streptococcal glomerulonephritis: Anti-streptococcal antibodies + streptococcal antigens → IC in glomeruli (lumpy bumpy deposits on IF)
  • Rheumatoid arthritis (RF IgM + IgG IC in joints)
  • Arthus reaction (local, experimental): Intradermal antigen in pre-sensitized individual → local IC deposition → necrosis
  • Farmer's lung (hypersensitivity pneumonitis): Thermophilic actinomycetes antigens + IgG

• Mediator: T cells (CD4+ Th1 primarily; CD8+ CTLs)
• No antibody involvement
• Mechanism: Sensitization by previous exposure; re-exposure → CD4+ T cells recognize MHC II + antigen → secrete IFN-γ, TNF-β → macrophage activation → tissue damage; peak response 48-72 hours
• Time: 24-72 hours (delayed)
• Examples:
  • Tuberculin/Mantoux test (PPD): Gold standard test for CMI; positive = ≥10 mm induration at 48-72 hrs (5 mm for HIV/immunocompromised, 15 mm for low-risk)
  • Contact dermatitis: Poison ivy, DNCB, nickel (hapten + carrier protein → sensitization → Type IV)
  • Graft rejection: Chronic rejection and GVHD are Type IV
  • Granuloma formation: TB, Leprosy, Sarcoidosis, Schistosomiasis (CD4+ T cells + macrophages forming epithelioid granulomas with Langhans giant cells)

• A - Anaphylactic (Type I) - IgE
• C - Cytotoxic (Type II) - IgG/IgM + cell surface Ag
• I - Immune Complex (Type III) - Ag+Ab IC
• D - Delayed (Type IV) - T cell mediated

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11. Autoimmune Diseases and Immunological Basis/Mechanisms of Autoimmunity

• Central tolerance:
  • T cells: Thymic selection - positive selection (recognize self MHC) → negative selection (high affinity for self antigen → apoptosis - clonal deletion); AIRE (Autoimmune Regulator) gene expresses peripheral antigens in thymus
  • B cells: Bone marrow - receptor editing or clonal deletion of self-reactive B cells
• Peripheral tolerance:
  • Anergy: T cell recognizes antigen without co-stimulation → anergy (functional unresponsiveness)
  • Treg cells: Suppress self-reactive T cells; FOXP3 mutation → IPEX syndrome (autoimmune polyendocrinopathy)
  • Fas-FasL (apoptosis of activated T cells - AICD)
  • Ignorance: Immune cells ignore antigens in immune-privileged sites (eye, testis, brain)

1. Molecular mimicry: Pathogen antigen resembles self-antigen → anti-pathogen antibodies cross-react with self
   • GAS M protein → cross-reacts with cardiac myosin → Rheumatic fever (anti-heart antibodies)
   • Campylobacter jejuni LPS → cross-reacts with gangliosides → Guillain-Barre syndrome
2. Bystander activation: Non-specific inflammation activates self-reactive T cells
3. Release of sequestered antigens: Trauma exposes immune-privileged antigens (sympathetic ophthalmia - eye trauma exposes lens protein)
4. Loss of Treg function: FOXP3 mutation, deficiency
5. Excessive T cell help / co-stimulation
6. Polyclonal B cell activation: EBV activates B cells non-specifically
7. Hormonal factors: Female hormones (estrogen) promote autoimmunity (SLE: F:M = 9:1)
8. Genetic susceptibility: HLA associations (see above)

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12. Immunodeficiency Diseases: Congenital and Acquired (e.g., HIV)

• X-linked agammaglobulinemia (Bruton's): Mutation in Btk (Bruton tyrosine kinase) → no B cell maturation beyond pro-B cell; boys only; recurrent bacterial infections after 6 months (when maternal IgG wanes); no tonsils/lymph nodes; treat with IVIG
• Selective IgA deficiency: Most common primary ID; recurrent sinopulmonary infections; risk of anaphylaxis to blood products (have anti-IgA antibodies); generally mild
• Common Variable Immunodeficiency (CVID): Late onset (20-30s); low Ig; recurrent infections; increased lymphoma risk; B cells present but don't differentiate
• Transient hypogammaglobulinemia of infancy: Physiological dip extends; resolves by 2-4 years

• DiGeorge syndrome (22q11.2 deletion): Thymic aplasia → no T cells; also hypoparathyroidism (hypocalcemia), conotruncal cardiac defects; recurrent viral/fungal infections; CATCH-22 (Cardiac defects, Abnormal facies, Thymic hypoplasia, Cleft palate, Hypocalcemia)
• Wiskott-Aldrich syndrome (WAS): X-linked; WASp gene mutation; triad: thrombocytopenia + eczema + combined immunodeficiency; small platelets; increased lymphoma risk

• SCID (Severe Combined Immunodeficiency): No T or B cells; earliest onset; die in first year without treatment; causes:
  • X-linked SCID (most common): IL-2Rγ chain (common gamma chain) mutation → failure of cytokine signaling (affects IL-2, IL-4, IL-7, IL-9, IL-15, IL-21)
  • Adenosine deaminase (ADA) deficiency: dATP accumulates → T/B cell toxicity (first gene therapy success)
• Ataxia-telangiectasia (AT): ATM gene (DNA repair) mutation; cerebellar ataxia, telangiectasias, combined ID, elevated AFP, increased malignancy (leukemia/lymphoma)
• Hyper-IgM syndrome: CD40L (CD154) deficiency on T cells → cannot signal B cells to class switch → high IgM, absent IgG/IgA/IgE; opportunistic infections (PCP)

• Chronic Granulomatous Disease (CGD): NADPH oxidase deficiency → inability to produce reactive oxygen species → cannot kill catalase-positive organisms (S. aureus, Klebsiella, Aspergillus, Pseudomonas); granuloma formation; NBT (Nitroblue tetrazolium) test = negative (no color change)
• Leukocyte Adhesion Deficiency (LAD): CD18 (β2 integrin) mutation → neutrophils can't adhere/migrate to sites of infection; omphalitis (delayed cord separation), no pus formation, high peripheral neutrophil count
• Chédiak-Higashi syndrome: LYST gene mutation → giant granules in neutrophils (partial albinism, nystagmus, recurrent pyogenic infections, peripheral neuropathy)

1. gp120 binds CD4 on T helper cells → conformational change → co-receptor binding:
   • CCR5 (M-tropic/R5 strains): Early infection; macrophages and T cells; CCR5Δ32 mutation → near complete resistance to HIV
   • CXCR4 (T-tropic/X4 strains): Late infection; T cells
2. gp41 mediates fusion → viral RNA enters cell
3. Reverse transcriptase: RNA → DNA (error-prone; generates diversity/resistance)
4. Integrase: Integrates viral DNA into host chromosome (provirus)
5. Latency: Reservoir in resting memory CD4+ T cells (cannot be eliminated)
6. Replication: 10^9-10^10 virions/day
7. CD4+ T cell depletion → immunosuppression

1. Acute HIV (Acute retroviral syndrome): 2-4 weeks after exposure; mononucleosis-like illness (fever, lymphadenopathy, pharyngitis, rash, myalgia); very high viral load
2. Clinical latency: Asymptomatic; 2-10 years; CD4+ T cells slowly declining; viral load detectable
3. AIDS: CD4 < 200 cells/µL OR AIDS-defining condition

• CD4 < 200: PCP (Pneumocystis jirovecii pneumonia - most common AIDS-defining illness), Toxoplasmosis of brain
• CD4 < 100: Cryptosporidiosis, Histoplasmosis, Microsporidiosis, Cryptococcal meningitis
• CD4 < 50: CMV retinitis (blindness), MAC (Mycobacterium avium complex) disseminated infection
• Any CD4: Kaposi sarcoma (HHV-8), NHL (EBV), Wasting syndrome, AIDS dementia complex, Invasive cervical cancer, Esophageal candidiasis, Recurrent bacterial pneumonia

• ELISA (4th generation p24 Ag + anti-HIV Ab) → screen
• Western blot: Confirmatory (requires 2 of 3 bands: p24, gp41, gp120/160)
• HIV RNA PCR (viral load): Monitoring treatment; acute infection diagnosis (window period)
• CD4 count: Staging and OI prophylaxis threshold

• NRTI (Nucleoside Reverse Transcriptase Inhibitors): Zidovudine (AZT), Tenofovir (TDF), Emtricitabine (FTC), Lamivudine (3TC), Abacavir
• NNRTI (Non-nucleoside RTI): Efavirenz, Nevirapine, Rilpivirine
• PI (Protease Inhibitors): Lopinavir/ritonavir, Atazanavir, Darunavir
• INSTI (Integrase Strand Transfer Inhibitors): Raltegravir, Dolutegravir, Bictegravir
• Fusion inhibitors: Enfuvirtide (T-20)
• CCR5 antagonist: Maraviroc

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13. ELISA, RIA, Western Blot: Principles and Uses

• Antigen coated on plate → enzyme-labeled antibody binds → add substrate → color change (proportional to antigen)

• Antigen coated on plate → patient's serum antibody binds → enzyme-labeled anti-human Ig binds → substrate → color
• Used for: Anti-HIV antibodies, ANA, anti-HCV, anti-dsDNA

• Capture antibody coated on plate → antigen from sample binds → detection antibody binds → enzyme-labeled secondary antibody → substrate → color
• High specificity (requires two antibodies)
• Used for: HIV p24 antigen, TB antigen, cytokine measurement (ELISA-based assays)

• Unknown antigen competes with labeled antigen for antibody binding
• Higher unknown antigen = lower signal
• Used for: Haptens, small molecules

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• First immunoassay; developed by Berson and Yalow (Nobel Prize 1977 - Yalow)
• Principle: Competition between labeled (radioactive ¹²⁵I) antigen and unlabeled patient antigen for a fixed amount of specific antibody
• More labeled antigen displaced = more unknown antigen in sample (inverse relationship)
• Measurement: Gamma counter (radioactivity)
• Advantages: Extremely sensitive (detects picomolar concentrations), precise
• Disadvantages: Radioactivity hazard, short shelf life of labeled antigen, requires special facilities
• Uses: Thyroid hormones (T3, T4), insulin, growth hormone, HBsAg (original test), cortisol, LH/FSH
• Largely replaced by ELISA and chemiluminescence assays in routine labs

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• Principle: Proteins separated by SDS-PAGE (by molecular weight) → transferred (blotted) to nitrocellulose membrane → blocked → probed with primary antibody → secondary antibody (enzyme-linked) → substrate → band at specific molecular weight
• Uses:
  • HIV confirmatory test (detect anti-HIV antibodies against specific viral proteins p24, gp41, gp120, p31/integrase) - positive: 2/3 specific bands
  • Lyme disease confirmation (anti-Borrelia antibodies)
  • Diagnosis of CJD (prion protein PrPsc)
  • Detection of specific proteins in research
• Features: Highly specific (identifies protein by size); slower than ELISA; not easily automated; qualitative/semi-quantitative

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14. Transplantation - Types, Reactions, HLA Typing

• Occurs within minutes to hours
• Pre-formed antibodies (anti-HLA or anti-ABO) in recipient react with graft vessels
• Complement activation → thrombosis → vascular occlusion → organ necrosis
• Prevention: ABO matching, crossmatch (test recipient serum + donor lymphocytes)
• Irreversible; graft must be removed

• Days to weeks after transplant
• T cell mediated (CD4+ and CD8+ T cells against donor MHC)
• Direct pathway: Host T cells recognize intact donor MHC (like alloreaction)
• Indirect pathway: Host T cells recognize donor MHC peptides presented by host APCs
• Features: Organ dysfunction, histology shows lymphocytic infiltration
• Treatable: High-dose corticosteroids, anti-thymocyte globulin (ATG)

• Months to years after transplant
• Combination of immune and non-immune factors
• Intimal proliferation of graft vessels → ischemia → fibrosis
• Not well understood; poor response to treatment
• Major cause of long-term graft loss

• Immunocompetent donor T cells attack host tissues
• Occurs in bone marrow/stem cell transplantation
• Acute GVHD: <100 days; skin rash, diarrhea, hepatitis
• Chronic GVHD: >100 days; scleroderma-like skin changes, sicca syndrome, bronchiolitis obliterans

• Essential for matching transplant donor-recipient pairs
• Methods:
  • Serological typing: Complement-dependent cytotoxicity (CDC) - patient lymphocytes + antisera + complement → cell death if HLA present
  • Molecular typing (DNA-based): PCR-SSP (Sequence-Specific Primers), PCR-SSO (Sequence-Specific Oligonucleotides), SBT (Sequence-Based Typing) - most accurate
  • Crossmatch: Mix recipient serum + donor lymphocytes + complement → positive (death) = contraindication
• Most critical loci to match: HLA-DR > HLA-B > HLA-A (for renal transplant)
• Perfect 6-antigen match (HLA-A, -B, -DR on each chromosome) = best outcome
• Living related donors have highest match probability

• Calcineurin inhibitors: Tacrolimus (FK506), Cyclosporine (binds cyclophilin → inhibits calcineurin → blocks IL-2 transcription)
• mTOR inhibitors: Sirolimus (Rapamycin), Everolimus
• Antimetabolites: Mycophenolate mofetil (MMF) - blocks purine synthesis in lymphocytes, Azathioprine
• Corticosteroids: Prednisolone
• Biologics: Basiliximab (anti-IL-2Rα/CD25), Belatacept (CTLA-4-Ig, blocks co-stimulation), ATG

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15. Vaccines: Live vs Killed, Examples, Contraindications, Cold Chain and Storage; Recombinant Vaccines

• Contain weakened (attenuated) live organisms
• Produce long-lasting, strong immunity (mimic natural infection)
• Both humoral + cell-mediated immunity
• Fewer doses needed (usually single dose for most)
• Examples:
  • Bacterial: BCG (Bacille Calmette-Guérin, M. bovis - TB), Ty21a (oral typhoid)
  • Viral: OPV (oral polio - Sabin), MMR (Measles-Mumps-Rubella), Varicella (VZV), Yellow fever, Rotavirus, Nasal influenza (FluMist), Dengue (Dengvaxia)
• Contraindications: Immunocompromised patients (HIV with CD4 <200, malignancy, on immunosuppressants), pregnant women (theoretical risk), recent immunoglobulin/blood products (delay 3 months)

• Contain dead organisms (killed by heat, formaldehyde, beta-propiolactone)
• Safer; cannot cause disease
• Weaker immune response; require multiple doses + boosters
• Require adjuvants (alum, AS04) to boost response
• Examples:
  • Bacterial: Whole-cell pertussis (wP), Cholera (oral), TAB (Typhoid-Paratyphoid A+B)
  • Viral: IPV (inactivated polio - Salk), Hepatitis A, Rabies, Influenza (injectable), Japanese encephalitis

• Only specific antigenic parts (proteins, polysaccharides)
• Very safe; cannot replicate
• Toxoid vaccines: Inactivated toxins (formaldehyde-treated): Tetanus toxoid (TT), Diphtheria toxoid (DT); excellent immunogenic; safe in pregnancy
• Polysaccharide vaccines: Pure capsular polysaccharide; T-independent; no memory; poor in infants <2 years
  • Pneumovax (PPSV23 - 23-valent pneumococcal polysaccharide)
  • Menomune (meningococcal)
• Conjugate vaccines: Polysaccharide coupled to carrier protein → T-dependent → memory + effective in infants
  • PCV13/PCV15/PCV20 (Prevnar - pneumococcal conjugate)
  • Hib (Haemophilus influenzae type b)
  • MCV4 (meningococcal conjugate)

• Antigen produced by genetic engineering
• Gene encoding protective antigen inserted into vector (yeast, bacteria, virus)
• Examples:
  • HBV vaccine: HBsAg expressed in Saccharomyces cerevisiae (baker's yeast) - safest, most widely used recombinant vaccine
  • HPV vaccines: Gardasil (quadrivalent HPV 6,11,16,18), Cervarix (bivalent HPV 16,18); virus-like particles (VLPs) - self-assembling HBsAg capsids with no genetic material
  • Typhoid Vi capsular polysaccharide vaccine: Semi-recombinant
  • COVID-19 vaccines: mRNA vaccines (Pfizer/BioNTech, Moderna) - mRNA encoding spike protein; adenoviral vector vaccines (AstraZeneca/Oxford, Janssen)

• Birth: BCG, OPV-0 (Hepatitis B₀)
• 6 weeks: DTwP₁ + IPV₁ + Hib₁ + HBV₁ + PCV₁ + Rotavirus₁ (Pentavalent)
• 10 weeks: DTwP₂ + IPV₂ + Hib₂ + HBV₂ + Rotavirus₂
• 14 weeks: DTwP₃ + IPV₃ + Hib₃ + Rotavirus₃ + PCV₂
• 9 months: MR vaccine + Vitamin A
• 16-24 months: DPT booster 1 + OPV booster + MMR + PCV booster + Vitamin A
• 5-6 years: DPT booster 2
• 10-16 years: TT
• Pregnant women: TT (TD) × 2 doses

• System of refrigerated transport and storage from manufacturer to recipient to maintain vaccine potency
• Most vaccines: 2-8°C (refrigerator)
• OPV: -20°C (freezer); can be kept at 2-8°C for short periods
• Vaccines sensitive to freezing: DPT, HBV, IPV, TT (freezing destroys adjuvant - alum - irreversible damage; shake test detects freezing damage)
• Vaccines sensitive to heat: OPV, MMR, BCG (live vaccines most heat-sensitive)
• Cold chain equipment: Cold room (≤-20°C or 2-8°C), ILR (Ice-Lined Refrigerator), ice pack, vaccine carriers, cold boxes

• Heat-sensitive label on vials that changes color with heat exposure
• Color change (dark) = vaccine exposed to excessive heat; discard
• Used to track cold chain breaks

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16. Immunodiagnostic Tools in Clinical Microbiology

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SECTION III: RESPIRATORY TRACT INFECTIONS

• Streptococcus pyogenes (Group A Strep): Pharyngitis ("strep throat"), tonsillitis; complications: Rheumatic fever, Post-streptococcal GN
• Corynebacterium diphtheriae: Diphtheria - pseudomembrane, bull-neck, myocarditis, neuropathy; exotoxin (beta-phage encoded) inhibits EF-2
• Bordetella pertussis: Whooping cough (pertussis) - catarrhal → paroxysmal (inspiratory whoop + post-tussive vomiting) → convalescent; lymphocytosis; pertussis toxin (ADP-ribosylates Gi → ↑cAMP); culture on Bordet-Gengou agar

• Streptococcus pneumoniae: Most common bacterial pneumonia; lobar pneumonia; rusty sputum; blood culture + sputum culture; treated with penicillin/amoxicillin
• Klebsiella pneumoniae: Alcoholics/debilitated patients; thick "currant jelly" sputum; upper lobe cavitation; hospital-acquired pneumonia
• Staphylococcus aureus: Post-influenza pneumonia; necrotizing; empyema, pneumatoceles; MRSA important
• Pseudomonas aeruginosa: Cystic fibrosis, hospital-acquired; mucoid strains; treat with antipseudomonal agents
• Mycobacterium tuberculosis: Primary (Ghon focus) and secondary (post-primary/reactivation); cavitation upper lobes; ZN stain; LJ culture; GeneXpert; RNTCP/NTEP; DOT therapy
• Atypical pneumonia organisms: Mycoplasma pneumoniae (walking pneumonia, cold agglutinins, IgM), Chlamydophila pneumoniae, Legionella pneumophila (Legionnaire's disease - BCYE culture, Legionella urinary antigen)
• Viral pneumonia: Influenza A/B, RSV (infants), Adenovirus, SARS-CoV-2

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