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MUHS Microbiology 2 - MBBS Exam Notes

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1. SALMONELLA / ENTERIC FEVER **

Biology

• Gram-negative, non-sporing, flagellated rods (motile, peritrichous)
• Antigens: H (flagellar), O (somatic/LPS), Vi (virulence capsular antigen - only Typhi)
• Facultative anaerobes; ferment glucose with gas (Typhi - no gas); oxidase negative

Pathogenesis - Enteric Fever (Typhoid)

• Ingestion of contaminated food/water -> Peyer's patches (terminal ileum) -> M cells invaded -> macrophages carry organisms -> mesenteric lymph nodes -> Primary bacteremia (1st week) -> liver, spleen, bone marrow -> multiplication -> Secondary bacteremia (2nd week) -> systemic disease
• Endotoxin causes fever; re-infection of Peyer's patches causes ulceration

Clinical Stages

Laboratory Diagnosis of Enteric Fever by Week

Widal Test (IMPORTANT - frequently asked)

• Principle: Tube agglutination test - detects antibodies (agglutinins) against O and H antigens of S. Typhi and S. Paratyphi
• Antigens used: TO (S. Typhi O), TH (S. Typhi H), AH (S. Paratyphi A H), BH (S. Paratyphi B H)
• Method: Serial dilutions of patient serum + antigen suspension -> incubate 37°C overnight -> read agglutination
• Interpretation:
  • Single titre: O ≥ 1:80 (non-endemic), O ≥ 1:160 (endemic) = significant
  • Paired sera (2-week interval): fourfold rise in titre = diagnostic
  • H agglutination (fluffy): past infection or vaccination
  • O agglutination (granular): current/recent infection (clinically more significant)
• Ideal time: 2nd week of fever (when antibodies appear)
• Limitations:
  • False positive: Brucellosis, malaria, liver disease, other Salmonella infections, previous vaccination
  • False negative: early disease (1st week), antibiotic treatment, immunocompromised
  • Does not distinguish current from past infection without paired sera
  • Cross-reactions with non-typhoidal Salmonella

First Week Diagnosis of Enteric Fever

• Blood culture - positive in 80-90% in 1st week
• Clot culture - buffy coat (contains more organisms)
• Bone marrow culture - remains positive even after antibiotics started
• Methods: Standard aerobic blood culture bottles; bile broth (enrichment); Castaneda's biphasic medium

Co-Agglutination Test (CoA test)

• Uses protein A of Staphylococcus aureus (Cowan I strain) coated with antibody to S. Typhi Vi antigen
• Detects Vi antigen in urine, serum, blood - identifies carriers
• Rapid, sensitive - positive in 1st week before Widal becomes positive
• Role: Early diagnosis, carrier detection

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2. SPIROCHETES ***

Leptospira - Laboratory Diagnosis

• MAT (Microscopic Agglutination Test): Gold standard; titre ≥ 1:100 (non-endemic), ≥ 1:400 (endemic) = significant; fourfold rise diagnostic
• ELISA: IgM ELISA (Leptocheck) - rapid, detects early; good for endemic areas
• Dark field microscopy: Motile spirochetes seen but unreliable (false positives)

Syphilis - Serological Diagnosis

• VDRL (Venereal Disease Research Laboratory)
• RPR (Rapid Plasma Reagin)

• TPHA (Treponema pallidum Haemagglutination Assay)
• FTA-ABS (Fluorescent Treponemal Antibody Absorbed) - most sensitive
• TPPA (Treponema pallidum Particle Agglutination)

VDRL - Principle, Applications, Advantages, Limitations

• Detects reagin (non-specific IgG and IgM antibodies) against cardiolipin-lecithin-cholesterol antigen
• Flocculation test - antigen forms visible floccules with patient antibody
• Performed on heated (inactivated) serum at 56°C for 30 min
• Read microscopically (vs RPR = macroscopic reading)

1. Screening for syphilis in blood donors, antenatal screening
2. Neurosyphilis diagnosis (CSF VDRL - most specific test for neurosyphilis)
3. Monitoring treatment response (titres fall with treatment; 4-fold fall = adequate response)
4. Screening STI clinics

• Cheap, simple, rapid
• Quantitative - useful for monitoring treatment
• Suitable for mass screening

• Biological false positives (BFP):
  • Acute BFP (<6 months): Viral infections (EBV, hepatitis, measles, varicella), malaria, mycoplasma pneumonia
  • Chronic BFP (>6 months): Autoimmune diseases (SLE - most common), elderly, IV drug users, leprosy, malaria
• Does not detect early primary syphilis (before reagin develops)
• Cannot confirm treponemal infection (non-specific)
• Prozone phenomenon (very high titres may give false negative - excess antibody)
• CSF may be blood-contaminated giving false positive

• Very early primary syphilis
• Prozone phenomenon (see above)
• Late tertiary syphilis (low/absent reagin)

Leptospirosis - Pathogenesis

• Entry via skin abrasions, mucous membranes, conjunctiva - occupational exposure (farmers, vets, sewage workers)
• Leptospira survive in kidneys of reservoir hosts (rats, dogs, cattle) -> shed in urine -> contaminates water/soil
• Leptospiraemia (1st week): fever, myalgia, conjunctival suffusion
• Immune phase (2nd week): antibody formation, organ damage - Weil's disease (jaundice, AKI, haemorrhage), uveitis, aseptic meningitis
• Weil's disease = severe leptospirosis: jaundice + renal failure + haemorrhage (triad)
• Pathology: vasculitis of capillaries -> multi-organ involvement

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3. HIV **

Definition - Window Period

• Antibody test window: ~23-90 days (3rd generation ELISA)
• 4th generation combo (Ag/Ab) test window: 18-45 days
• NAAT/RNA PCR window: 10-33 days (shortest)
• Patient is infectious during window period despite negative test

Etiology

• Family: Retroviridae, Genus: Lentivirus
• RNA virus (ssRNA, positive-sense) - 2 copies
• Species: HIV-1 (pandemic, more virulent) and HIV-2 (West Africa, less transmissible)

Structure of HIV (Diagram Points)

• Outer envelope (lipid bilayer) with gp120 (surface glycoprotein) and gp41 (transmembrane)
• gp120 binds CD4 receptor; gp41 mediates fusion
• Matrix protein p17
• Core: p24 capsid (diagnostic marker - detected in Ag/Ab combo test)
• Inside: 2 copies ssRNA, reverse transcriptase (p51/p66), integrase (p32), protease (p10)
• Genes: gag (structural - p24, p17, p9, p7), pol (enzymes - RT, integrase, protease), env (envelope - gp120, gp41), plus regulatory genes (tat, rev, nef, vif, vpr, vpu)

Pathogenesis

1. Entry: gp120 binds CD4 (primary receptor) + co-receptor CCR5 (macrophages/monocytes - early infection) or CXCR4 (T cells - late infection) -> gp41 mediates fusion
2. Reverse transcription: ssRNA -> dsDNA (by reverse transcriptase)
3. Integration: dsDNA -> proviral DNA integrated into host genome (by integrase) - latent reservoir
4. Replication: Provirus activated -> new virions budded from cell (protease cleaves polyproteins)
5. CD4 T cell depletion: Direct cytopathic effect, immune activation, apoptosis -> progressive immunodeficiency
6. Normal CD4: 500-1500 cells/μL; AIDS defined as CD4 <200 cells/μL or AIDS-defining illness

Two Opportunistic Infections in HIV *

1. Pneumocystis jirovecii Pneumonia (PCP) - most common OI; CD4 <200; interstitial pneumonia
2. Cryptococcal meningitis - CD4 <100; headache, fever, raised ICP; India ink positive

• Others: CMV retinitis (CD4 <50), Toxoplasmosis (CD4 <100), MAC (CD4 <50), Candidiasis (oral/esophageal)

Laboratory Diagnosis of HIV

1. Screening: 4th generation ELISA (HIV Ag/Ab combo) - detects p24 antigen + IgG/IgM antibodies
2. Confirmatory: Western Blot (detects antibodies to specific bands - gp120, gp41, p24, p31 = positive if ≥2 bands)
3. NAAT/RNA PCR: Detects viral RNA - used in window period, infants <18 months, acute HIV

HIV Testing in India - Strategies

• ICTC (Integrated Counselling and Testing Centre) - free, confidential testing
• 4th generation testing now recommended as first-line in India
• NAAT for infants born to HIV+ mothers (at 6 weeks)
• Dried Blood Spot (DBS) used for early infant diagnosis

Organisms Causing STDs *

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4. INFLUENZA VIRUS & HIV - Diagram

Influenza Virus Structure

• Family: Orthomyxoviridae
• Segmented ssRNA (negative-sense), 8 segments (allows reassortment)
• Envelope with spikes: Haemagglutinin (HA) - 16 subtypes - mediates attachment and fusion; Neuraminidase (NA) - 9 subtypes - releases new virions from cell
• Matrix protein M2 (ion channel - target of amantadine)
• Types A, B, C based on NP and M1 antigens
• Influenza A: most important; infects humans, birds, pigs; undergoes antigenic shift + drift
• Antigenic drift = minor changes by point mutations (seasonal flu)
• Antigenic shift = major changes by reassortment (pandemic flu)

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5. RHABDOVIRUSES ** (Rabies)

Immunoprophylaxis **

• For high-risk groups: veterinarians, lab workers, spelunkers, travelers to endemic areas
• Schedule: Days 0, 7, 21 (or 28) - 3 doses IM deltoid
• Check neutralizing antibody titre at 1-2 years; booster if titre <0.5 IU/mL

• Wound care: Immediate thorough washing with soap & water (15 min), povidone-iodine
• Rabies Immunoglobulin (RIG):
  • Human RIG (HRIG): 20 IU/kg; or Equine RIG (ERIG): 40 IU/kg
  • Inject as much as possible into wound, remainder IM
  • Only for Category III exposures and immunocompromised
  • Given on Day 0 only (passive immunity during window)
• Rabies vaccine (active immunization):

Dosage Schedule

Non-Neural Vaccines and Schedule (important)

• Semple vaccine (from sheep brain) - can cause neuroparalytic accidents (ADEM), no longer recommended

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6. MALARIA - Laboratory Diagnosis

Methods:

1. Peripheral Blood Smear (PBS) - Gold standard:
   • Thick smear: More sensitive (concentrates RBCs) - for detection
   • Thin smear: Species identification (morphology preserved)
   • Stain: Leishman or Giemsa stain
   • Best time to collect: During fever spike or 1 hour before expected spike
   • Examine: 100-200 fields before calling negative
2. Rapid Diagnostic Tests (RDTs):
   • Detect parasite antigens (HRP-2 for P. falciparum; pLDH for all species)
   • Quick (15 min), no microscope needed; used in field conditions
3. QBC (Quantitative Buffy Coat): Fluorescence microscopy with acridine orange; quick but requires centrifuge
4. PCR: Most sensitive and specific; differentiates species; used in mixed infections, reference labs
5. Serology (IFAT/ELISA): For epidemiological surveys, not acute diagnosis

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7. Plasmodium falciparum * - Complications and Lab Diagnosis

Complications *

1. Cerebral malaria - most serious; ring hemorrhages (Dürck's granulomas); coma, seizures
2. Blackwater fever - massive intravascular hemolysis -> haemoglobinuria (black urine) -> AKI
3. Algid malaria - septicaemic shock picture; cold clammy skin, hypotension
4. Pulmonary edema / ARDS
5. Hypoglycemia (esp. in pregnancy and children)
6. Severe anemia (Hb <7g/dL)
7. AKI (acute kidney injury)
8. Hyperparasitemia (>5% RBCs parasitized)
9. Splenic rupture (rare)

Why only P. falciparum causes severe disease:

• Invades ALL ages of RBCs (others: only young or old)
• Produces knobs on RBC surface -> cytoadherence to endothelium -> microvascular obstruction
• Rosette formation (infected RBCs cluster with uninfected)
• Sequestration in deep vessels (not cleared by spleen)

Lab Features of P. falciparum:

• Earliest ring forms (trophozoites) seen: Delicate, small "appliqué" or accole forms
• Multiple rings per RBC (common)
• RBC not enlarged; Maurer's clefts (vs Schüffner's dots in vivax/ovale)
• No visible schizonts/gametocytes in peripheral blood (except in severe disease)
• Gametocytes: Banana/crescent-shaped (pathognomonic)

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8. LD Bodies - Leishmania donovani

• Intracellular amastigote forms of Leishmania donovani
• Seen inside macrophages (monocytes) in tissues - liver, spleen, bone marrow, lymph nodes
• Shape: Small (2-4 μm), oval; nucleus + kinetoplast (rod-shaped DNA-containing organelle)
• Kinetoplast is distinctive - seen as a small red/purple rod next to nucleus
• Stain: Giemsa or Leishman stain
• Found in: splenic aspirate, bone marrow aspirate, liver biopsy, buffy coat smear
• Highest yield: Splenic aspirate (95%) - but risk of bleeding; bone marrow aspirate safer (70-80%)

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9. Leishmania donovani - LAQ

Life Cycle

1. Sandfly (vector + definitive host): Phlebotomus species (India)
   • Sandfly ingests blood with amastigotes -> transform to promastigotes in gut -> multiply -> migrate to proboscis
2. Humans (intermediate host):
   • Promastigotes injected during sandfly bite -> taken up by macrophages -> transform to amastigotes -> multiply -> macrophages rupture -> infect new macrophages

• Promastigote (flagellated, elongated): in sandfly
• Amastigote (LD body, non-flagellated): in humans (intracellular)

Kala Azar (Visceral Leishmaniasis) - Pathogenicity *

• Promastigotes resist complement lysis -> enter macrophages via receptor-mediated phagocytosis
• Survive inside phagolysosome by inhibiting lysosomal enzymes, scavenging reactive oxygen species
• Reticuloendothelial system (RES) hyperplasia -> massive splenomegaly, hepatomegaly
• Bone marrow infiltration -> pancytopenia
• Hypergammaglobulinaemia (polyclonal)
• Clinical: Prolonged fever, massive splenomegaly > hepatomegaly, wasting, darkening of skin ("Kala azar" = black fever in Hindi)
• Complications: Secondary infections (TB, pneumonia), haemorrhage, PKDL (Post Kala Azar Dermal Leishmaniasis) - occurs months-years after treatment; rash; source of reinfection

Laboratory Diagnosis *

1. Demonstration of LD bodies (parasitological - definitive):
   • Splenic aspirate (best yield, >95%) - contraindicated if PT/INR abnormal
   • Bone marrow aspirate (safe, ~70-80%)
   • Liver biopsy, lymph node biopsy, buffy coat
   • Stain: Giemsa/Leishman
2. Serological tests:
   • rK39 rapid test - most commonly used; highly sensitive/specific for visceral leishmaniasis; lateral flow assay
   • DAT (Direct Agglutination Test): titre ≥ 1:3200 significant
   • ELISA, IFAT
3. Formol gel test (Aldehyde test / Napier's test):
   • 1-2 drops of formalin added to 1 mL serum -> opaque white gel = positive
   • Indicates hypergammaglobulinaemia; non-specific (also positive in other chronic infections)
   • Positive from 3rd month of illness onwards
4. Montenegro (leishmanin) skin test:
   • Tests delayed hypersensitivity; NEGATIVE during active kala azar (cell-mediated immunity suppressed); positive after cure
5. PCR: Most sensitive; can be done on peripheral blood

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10. Wuchereria bancrofti - Morphology & Lab Diagnosis

Morphology

• Adult worms: White, thread-like; male (4 cm), female (8-10 cm); live in lymphatics
• Microfilaria (diagnostic stage):
  • 244-296 μm long
  • Sheath present (pink/pale-staining with Giemsa)
  • No nuclei in tail tip (distinguishes from Brugia)
  • Nocturnal periodicity in India - microfilariae appear in peripheral blood between 10 PM - 4 AM (maximal at midnight) - related to sleeping habit
  • Exception: South Pacific (diurnal subperiodic) and Calcutta (non-periodic) strains

Laboratory Diagnosis

1. Blood examination for microfilariae:
   • Night blood smear (collect 10 PM - 2 AM)
   • Thick smear + Giemsa/Leishman stain
   • Knott's concentration technique (lysis with 2% formalin + centrifuge) - increases sensitivity
   • Membrane filtration technique (most sensitive)
2. DEC provocative test (daytime): Give 100 mg diethylcarbamazine (DEC) -> microfilariae appear in blood within 30-60 min; used when can't collect night blood
3. Antigen detection (immunochromatographic test - ICT): Detects circulating filarial antigen (CFA); highly sensitive/specific; can be done day or night; Og4C3 ELISA for quantification
4. Serology (ELISA/IFAT): Detects antibodies; less specific (cross-reactions)
5. Hydrocele/chylous fluid: May contain microfilariae
6. Imaging: Ultrasound of scrotal lymphatics - "filarial dance sign" (live adult worms moving in lymphatics)

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11. Histoplasma capsulatum - Morphology, Growth, Pathogenesis

Morphology

• Dimorphic fungus:
  • 25°C (room temp/mould phase): White/brown fluffy colony; macroconidia (tuberculate) + microconidia - macroconidia have finger-like projections (tuberculate macroconidia are pathognomonic)
  • 37°C (body temp/yeast phase - in tissues): Small (2-4 μm) oval yeast cells inside macrophages; budding yeasts with narrow-based buds
• Despite name, NO capsule (name is a misnomer - clear halo seen in H&E was artifact of shrinkage)
• Stain: GMS (Gomori Methenamine Silver) or PAS in tissue

Growth Characters

• Slow-growing (2-4 weeks at 25°C)
• Grows on SDA (Sabouraud's Dextrose Agar) with antibiotics and BHI blood agar at 37°C
• Colony: White/buff fluffy at 25°C -> turns brown with age
• Safety: Biosafety Level 3 (highly infectious spores)

Pathogenesis

• Conidia inhaled (from bat/bird droppings - caves, old buildings, Ohio/Mississippi river valleys)
• Microconidia reach alveoli -> engulfed by macrophages -> convert to yeast at 37°C -> intracellular survival (resist phagolysomal killing at low temp, early infection)
• Primary infection: Usually asymptomatic (immunocompetent) or flu-like illness
• Cell-mediated immunity (CMI) controls infection -> granuloma formation -> calcification (lung and liver calcifications)
• Immunocompromised (HIV, CD4 <150): Progressive disseminated histoplasmosis -> fever, hepatosplenomegaly, pancytopenia, mucosal ulcers, adrenal involvement (causes Addison's disease)

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12. Opportunistic Mycoses * - Short Note

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13. Candida albicans *

• Morphology: Gram-positive yeast (2-6 μm oval); produces pseudohyphae and true hyphae; blastoconidia (budding yeast cells), chlamydoconidia (thick-walled terminal spores on cornmeal agar)
• Germ tube test: Serum at 37°C for 2-3 hours -> short unbranched outgrowth (germ tube) = positive for C. albicans (and C. dubliniensis); this is the hallmark identifying test
• Colony: White/cream, pasty, "yeasty smell" on SDA; smooth
• Chrom agar: Green colonies (C. albicans), pink (C. tropicalis), blue/purple (C. glabrata)
• Infections: Oral thrush (pseudomembranes - white plaques on erythematous base), vaginal candidiasis, diaper rash, esophageal candidiasis (dysphagia - AIDS-defining), invasive/systemic candidiasis (candidemia)

Reynolds-Braude Phenomenon (Q14)

• Formation of germ tubes when C. albicans is incubated in human or animal serum at 37°C for 2-3 hours
• Germ tubes are true hyphae-like extensions from yeast cells WITHOUT a constriction at point of origin (distinguishes from pseudohyphae which have constrictions)
• Used to identify C. albicans and C. dubliniensis from other Candida species
• Significance: Simple, rapid screening test performed directly from positive blood culture or colonies

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15. Fungi in HIV - Opportunistic Infections; Candida Lab Diagnosis

Fungi causing Opportunistic Infections in HIV-infected individuals:

1. Candida albicans - oral/esophageal/vaginal/systemic
2. Cryptococcus neoformans - meningitis, cryptococcemia
3. Pneumocystis jirovecii - PCP (interstitial pneumonia)
4. Aspergillus fumigatus - invasive pulmonary aspergillosis
5. Histoplasma capsulatum - disseminated histoplasmosis
6. Coccidioides immitis - coccidioidomycosis (endemic areas)
7. Penicillium marneffei (Talaromyces marneffei) - Southeast Asia
8. Mucor/Rhizopus - mucormycosis

Candida albicans - Laboratory Diagnosis:

1. Direct microscopy: KOH mount or Gram stain - Gram-positive oval yeast + pseudohyphae
2. Germ tube test (Reynolds-Braude): Serum 37°C 2-3h - tubes = C. albicans
3. Culture: SDA + chloramphenicol (37°C and 25°C); creamy white colonies
4. Chlamydoconidia: On cornmeal Tween-80 agar - terminal thick-walled spores
5. CHROMagar Candida: Differential chromogenic medium - green colonies = C. albicans
6. VITEK 2 / API 20C: Automated biochemical identification
7. Serology: Beta-D-glucan assay (pan-fungal marker); Candida mannan antigen (invasive)
8. Blood cultures: BACTEC/BacT/ALERT for candidemia (bottles: continuous monitoring)

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LAQ Topics Summary

Malignant Tertian Malaria (P. falciparum) - Life Cycle, Complications, Lab Diagnosis

• Exo-erythrocytic (liver) stage: Sporozoites injected by Anopheles mosquito -> hepatocytes -> liver schizonts -> merozoites released (no hypnozoites in P. falciparum)
• Erythrocytic stage: Merozoites invade RBCs -> ring stage -> trophozoite -> schizont -> 8-32 merozoites per RBC -> rupture (every 48 hours = "malignant tertian") -> fever paroxysm
• Merozoites -> male/female gametocytes (crescent-shaped) -> ingested by mosquito
• Sexual cycle in mosquito: Gametocytes -> gametes -> zygote -> ookinete -> oocyst -> sporozoites -> salivary glands

P. vivax - Morphology, Life Cycle, Lab Diagnosis

• Morphology: Enlarged, pale RBC with Schüffner's dots; amoeboid trophozoite; schizont 12-24 merozoites; mature gametocytes (round)
• Hypnozoites in liver (cause relapses - can recur months-years later); no true relapses in falciparum
• Tertian fever = 48-hour cycle (benign tertian); less severe than falciparum
• Lab: Giemsa smear shows enlarged RBC + Schüffner's dots

4 Fungi causing Opportunistic Fungal Infections + Lab Diagnosis of Candidiasis:

1. Candida albicans 2. Cryptococcus neoformans 3. Aspergillus fumigatus 4. Pneumocystis jirovecii

• (See detailed sections above)

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Quick Mnemonics

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