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MICROBIOLOGY - PAPER I (RS-4 & RS-5)

Long Essay Answers (10 Marks Each)

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QUESTION 1 — Visceral Leishmaniasis (Kala-azar)

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a) Etiological Agent and Clinical Diagnosis

• Amastigote (Leishman-Donovan body / LD body): Oval, 2-3 μm, intracellular form found inside macrophages; has a nucleus and a rod-like kinetoplast appearing as a "dot and dash" on Giemsa stain
• Promastigote: Elongated, flagellated, found in sandfly gut and culture media

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b) Life Cycle and Pathogenesis of Leishmania donovani

Life Cycle Flowchart:

SANDFLY (Phlebotomus) — Female sandfly bites infected host
        │
        ▼
Ingests amastigotes from blood/macrophages
        │
        ▼
SANDFLY MIDGUT: Amastigotes transform into → PROMASTIGOTES (flagellated)
        │
        ▼
Promastigotes multiply by binary fission, migrate to proboscis
        │
        ▼
Sandfly bites new host — injects PROMASTIGOTES into skin
        │
        ▼
HUMAN HOST: Promastigotes phagocytosed by neutrophils/macrophages
        │
        ▼
Inside phagolysosome: Promastigotes → transform to AMASTIGOTES
        │
        ▼
Amastigotes multiply within macrophages (resist lysosomal killing)
        │
        ▼
Infected macrophages burst → amastigotes released
        │
        ▼
Taken up by other macrophages → spread via lymphatics/bloodstream
        │
        ▼
Reticuloendothelial system (RES): Spleen, Liver (Kupffer cells),
Bone marrow, Lymph nodes → massive colonization
        │
        ▼
KALA-AZAR: Hepatosplenomegaly, Pancytopenia, Fever, Weight loss

Pathogenesis:

1. Entry: Promastigotes injected by sandfly are phagocytosed by skin macrophages
2. Intracellular survival: L. donovani inhibits phagolysosome fusion and resists oxidative killing - the parasite lives and multiplies as amastigotes in macrophage cytoplasm
3. Dissemination: Infected macrophages carry the parasite via lymphatics to the liver, spleen, bone marrow, and lymph nodes (RES organs)
4. Hyperplasia of spleen: Marked splenomegaly due to RES proliferation
5. Bone marrow suppression: Amastigotes fill bone marrow macrophages → pancytopenia (anaemia, leukopenia, thrombocytopenia)
6. Immune dysregulation: Defective cell-mediated immunity (CMI), polyclonal B-cell activation → hypergammaglobulinemia
7. Fever: Irregular, hectic fever due to cytokine release (IL-1, TNF)
8. Post-kala-azar dermal leishmaniasis (PKDL): Occurs 1-2 years after treatment in Indian kala-azar - cutaneous vesicles with abundant parasites

Clinical Features Summary:

• Prolonged irregular fever (>2 weeks)
• Progressive weight loss and cachexia
• Massive splenomegaly (most prominent sign) + hepatomegaly
• Pancytopenia → anaemia, bleeding tendency, susceptibility to infections
• Hyperpigmentation of skin (hence "Kala-azar" = black fever in Hindi)
• Hypoalbuminemia and hypergammaglobulinemia

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c) Laboratory Diagnosis of Visceral Leishmaniasis

Flowchart - Laboratory Diagnosis:

SUSPECTED KALA-AZAR
         │
    ┌────┴────────────────────────────────────┐
    │                                         │
PARASITOLOGICAL                         SEROLOGICAL /
METHODS                                 IMMUNOLOGICAL
    │                                         │
    ├─ Bone Marrow Aspirate (GOLD            ├─ rK39 ICT strip test
    │  STANDARD): Giemsa stain               │  (Rapid, field diagnosis)
    │  → LD bodies in macrophages             │
    │                                         ├─ DAT (Direct Agglutination Test)
    ├─ Splenic Aspirate:                      │
    │  Most sensitive (>95%)                  ├─ ELISA (anti-Leishmania Ab)
    │  but risk of bleeding                   │
    │                                         ├─ CFT (Complement Fixation Test)
    ├─ Liver Biopsy:                          │  - Chopra's aldehyde test
    │  Less sensitive than spleen             │    (turbidity in hypergammaglobu-
    │                                         │    linaemia - non-specific)
    ├─ Lymph node aspirate                    │
    │                                         └─ Leishmanin (Montenegro)
    ├─ Peripheral blood smear (buffy coat)      skin test: NEGATIVE in
    │  - less sensitive                         active disease (CMI depressed)
    │
    ├─ NNN CULTURE MEDIUM: Promastigotes
    │  visible in 1-4 weeks
    │
    └─ PCR: Sensitive and specific,
       detects Leishmania DNA in blood/
       tissue. Used for species ID
         │
    MOLECULAR
    METHODS
         │
         └─ PCR on blood, bone marrow, or
            splenic aspirate

Supporting Hematological Findings:

Sources: Medical Microbiology 9e (Murray); Jawetz Melnick & Adelberg's Medical Microbiology 28e

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QUESTION 2 — Bacillary Dysentery (Shigellosis)

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a) Etiological Agent

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b) Pathogenesis and Complications

Pathogenesis Flowchart:

INGESTION of ≥10-100 Shigella organisms (fecal-oral route)
         │
         ▼
Reach SMALL INTESTINE → Initial colonization
         │
         ▼
Produce ENTEROTOXIN → early WATERY DIARRHEA
(first 12-24 hours)
         │
         ▼
Bacteria reach COLON (large intestine)
         │
         ▼
Invade M-cells (Microfold cells) overlying Peyer's patches
via TYPE III SECRETION SYSTEM (T3SS)
         │
         ▼
T3SS injects IpaA, IpaB, IpaC, IpaD proteins into epithelial cells
         │
         ▼
Membrane RUFFLING → Macropinocytosis → Bacterial engulfment
         │
         ▼
Shigella LYSE the phagosome → escape into CYTOPLASM
(unlike Salmonella which stays in vacuole)
         │
         ▼
ACTIN POLYMERIZATION propels bacteria cell-to-cell
(protected from immune clearance)
         │
         ▼
Induce APOPTOSIS of macrophages → release IL-1β
         │
         ▼
IL-1β attracts NEUTROPHILS (PMNs) → mucosal inflammation
(pus cells in stool!)
         │
         ▼
Disruption of INTESTINAL WALL INTEGRITY
         │
         ▼
BLOODY DYSENTERY: Blood, mucus, pus in stool
Severe cramps + Tenesmus (painful urge to defecate)
         │
         ▼
S. dysenteriae: SHIGA TOXIN (AB5 toxin)
B subunits → bind Gb3 receptor on endothelial cells
A subunit → cleaves 28S rRNA → BLOCKS PROTEIN SYNTHESIS
         │
         ▼
Intestinal epithelial damage + (in small subset) Glomerular
endothelial damage → HEMOLYTIC UREMIC SYNDROME (HUS)

Shiga Toxin Mechanism:

• Structure: 1 A subunit + 5 B subunits (AB5 toxin)
• Receptor: B subunits bind glycolipid Gb3 on host cells
• Action: A subunit cleaves 28S rRNA of 60S ribosomal subunit → blocks aminoacyl-tRNA binding → inhibits protein synthesis → cell death

Complications:

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c) Laboratory Diagnosis

Flowchart - Laboratory Diagnosis of Shigellosis:

CLINICAL SUSPICION (fever + tenesmus + bloody-mucoid stools)
              │
    ┌─────────┴────────────────────────┐
    │                                  │
MICROSCOPY                        CULTURE
(Immediate)                       (Definitive)
    │                                  │
    ├─ Stool wet mount:            Collect FRESH stool
    │  • Abundant pus cells (PMNs)     │
    │  • RBCs                          ├─ MacConkey agar:
    │  • Mucus                         │  PALE/COLORLESS colonies
    │  • NO cysts/ova                  │  (non-lactose fermenter)
    │    (distinguishes from           │
    │     parasitic dysentery)         ├─ XLD (Xylose Lysine
    │                                  │  Deoxycholate) agar:
    ├─ Giemsa stain (PMNs,             │  Pink/red colonies
    │  intracellular bacteria          │  (no H2S - no black center)
    │  in severe cases)                │
    │                                  ├─ Hektoen Enteric (HE) agar:
    │                                  │  Green colonies
    │                                  │
    │                                  ├─ Selenite F broth: enrichment
    │                                  │
    │                                  └─ BIOCHEMICAL ID:
    │                                     • Glucose fermented (acid, no gas)
    │                                     • Lactose: NOT fermented
    │                                     • H2S: Negative
    │                                     • Urease: Negative
    │                                     • Motility: Non-motile
    │
SEROLOGY                          MOLECULAR
    │                                  │
    ├─ Widal-type slide                └─ Multiplex PCR / NAATs
    │  agglutination with               (GOLD STANDARD - enteric
    │  Shigella antisera                multiplex panels)
    │  (confirm O-antigen               Detects virulence genes
    │   group A/B/C/D)                  (ipaH, Shiga toxin genes)
    │
    └─ Not routinely done
       (culture preferred)

Culture Characteristics on MacConkey Agar:

Additional Identification Steps:

1. Serotyping: Slide agglutination with polyvalent antisera (Groups A, B, C, D)

• Group A = S. dysenteriae (12 serotypes)
• Group B = S. flexneri (6 serotypes)
• Group C = S. boydii (18 serotypes)
• Group D = S. sonnei (1 serotype)

1. Antibiotic Sensitivity Testing: Required due to multidrug resistance; fluoroquinolones or azithromycin are standard empirical therapy
2. NAATs / Multiplex PCR: Now considered the gold standard for diagnosis - detects ipaH gene (invasion plasmid) and Shiga toxin genes

Sources: Medical Microbiology 9e (Murray/Rosenthal); Red Book 2021; Rosen's Emergency Medicine

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Quick Summary Table:

MICROBIOLOGY – Long Essay Answers (10 Marks Each)

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Q1 – Visceral Leishmaniasis (Kala-azar)

a) Etiological Agent & Clinical Diagnosis

• Agent: Leishmania donovani
• Diagnosis: Visceral Leishmaniasis (Kala-azar)
• Vector: Female Phlebotomus sandfly
• Key finding: Amastigotes (LD bodies) inside macrophages on Giemsa stain — oval, 2–3 µm, with nucleus + rod-like kinetoplast ("dot and dash")

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b) Life Cycle & Pathogenesis

Sandfly bites → injects PROMASTIGOTES into skin
        ↓
Phagocytosed by macrophages
        ↓
Transform into AMASTIGOTES (LD bodies) inside phagosome
        ↓
Resist lysosomal killing → multiply → macrophage bursts
        ↓
Spread via lymphatics/blood to RES organs
(Spleen, Liver, Bone Marrow, Lymph nodes)
        ↓
Massive splenomegaly + hepatomegaly
Bone marrow infiltration → Pancytopenia
Irregular fever, weight loss, anaemia
        ↓
Untreated → FATAL
        ↓ (post-treatment in India)
PKDL (Post Kala-azar Dermal Leishmaniasis)

• Inhibits phagolysosome fusion → survives intracellularly
• Depresses cell-mediated immunity (CMI)
• Polyclonal B-cell activation → hypergammaglobulinaemia
• Leishmanin skin test negative in active disease

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c) Laboratory Diagnosis

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Q2 – Bacillary Dysentery (Shigellosis)

a) Etiological Agent

• Organism: Shigella spp. (most likely S. flexneri in developing countries)
• Gram-negative, non-motile rod; non-lactose fermenter; oxidase-negative
• Infective dose: as low as 10–100 organisms
• 4 species: S. dysenteriae (most severe), S. flexneri, S. boydii, S. sonnei

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b) Pathogenesis & Complications

Ingestion of Shigella (fecal-oral route)
        ↓
Small intestine → ENTEROTOXIN → watery diarrhea (early)
        ↓
Reach COLON → invade M-cells via Type III Secretion System (T3SS)
        ↓
T3SS injects Ipa proteins (IpaA/B/C/D) → membrane ruffling
        ↓
Bacteria engulfed → LYSE phagosome → replicate in cytoplasm
        ↓
Actin polymerization → spread cell-to-cell
(protected from immune clearance)
        ↓
Induce APOPTOSIS in macrophages → IL-1β release
        ↓
PMN infiltration → mucosal destruction
        ↓
BLOODY DYSENTERY: blood + mucus + pus in stool + tenesmus
        ↓ (S. dysenteriae only)
SHIGA TOXIN (AB5): B subunits bind Gb3 receptor
A subunit cleaves 28S rRNA → blocks protein synthesis
        ↓
Intestinal damage ± Glomerular endothelial damage → HUS

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c) Laboratory Diagnosis

Fresh stool specimen collected
        ↓
┌─────────────────────┬──────────────────────────────┐
│   MICROSCOPY        │         CULTURE               │
│                     │                               │
│ Wet mount:          │ MacConkey agar:               │
│ • Abundant pus cells│ PALE/COLORLESS colonies       │
│ • RBCs + mucus      │ (non-lactose fermenter)       │
│ • NO ova/cysts      │                               │
│                     │ XLD / Hektoen agar:           │
│                     │ confirm non-H2S colonies      │
│                     │                               │
│                     │ Selenite F broth: enrichment  │
└─────────────────────┴──────────────────────────────┘
        ↓
Biochemical ID:
Lactose (-), H2S (-), Urease (-), Non-motile
        ↓
SEROTYPING:
Slide agglutination with antisera
Group A = S. dysenteriae
Group B = S. flexneri
Group C = S. boydii
Group D = S. sonnei
        ↓
PCR / NAATs (ipaH gene, Shiga toxin genes)
→ Gold standard, most sensitive

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At-a-Glance Comparison