RAM KRISHNA MEDICAL COLLEGE - MICROBIOLOGY 2nd SESSIONAL EXAM ANSWERS

Q.2 Long Essay (12 marks) - Hepatitis B

1. Most Probable Etiological Diagnosis (1 mark)

2. Laboratory Tests to Confirm Diagnosis (8 marks)

• HBV DNA (PCR/quantitative): Gold standard to assess viral load, guide antiviral therapy, and monitor treatment response. Values expressed as IU/mL.
• HBV genotyping: (A-H) guides treatment response prediction

• Serum bilirubin (elevated - direct + indirect)
• AST, ALT (elevated, ALT > AST in viral hepatitis)
• Alkaline phosphatase
• Serum albumin, prothrombin time (assess synthetic function)

• Complete blood count (CBC)
• Ultrasound abdomen - hepatomegaly, echogenicity
• Liver biopsy - histopathology for staging fibrosis (Knodell/METAVIR score); shows "ground-glass hepatocytes" (HBsAg accumulation in ER), Councilman bodies, piecemeal necrosis

• AFP (alpha-fetoprotein) - screen for hepatocellular carcinoma (HCC)
• Anti-HDV - screen for hepatitis D co-infection (requires HBsAg)

3. Preventive Measures (3 marks)

• Hepatitis B vaccine (recombinant HBsAg): 3-dose schedule - 0, 1, 6 months (IM deltoid). Produces Anti-HBs >10 mIU/mL = protective
• Universal vaccination: All newborns (birth dose within 24 hours), healthcare workers, high-risk groups

• Hepatitis B Immunoglobulin (HBIG): For post-exposure prophylaxis (needle-stick, neonates of HBsAg+ mothers). Given with vaccine at different sites.

• Screening blood/blood products before transfusion (ELISA for HBsAg)
• Use of disposable syringes and needles
• Universal precautions in healthcare settings
• Safe sex practices (condom use)
• Screening pregnant women for HBsAg
• Avoidance of sharing razors, toothbrushes, needles

Q.3 Reasoning Questions (5 x 3 = 15 marks)

A. Morphological Forms of Entamoeba histolytica - Why All Forms Are NOT Infective

1. Trophozoite (vegetative form): Active, motile, feeds by pseudopodia; contains ingested RBCs (hematophagous - pathognomonic). NOT infective when ingested - destroyed by gastric acid; cannot survive outside host.
2. Pre-cyst: Transitional stage, round, non-motile, glycogen mass present. Not infective.
3. Cyst (quadrinucleate - mature): The ONLY INFECTIVE FORM. Has a resistant chitinous wall that protects it from gastric acid and environmental conditions. Survives in soil and water for weeks. Can pass safely through stomach, excysts in small intestine.

B. Stain Used to Diagnose Herpes Infection and Why

C. Ways Mycoplasma is Similar to and Different from Viruses

• Passes through bacteriological filters (0.22 µm) - hence originally thought to be a virus
• Cannot be seen by light microscopy (too small, 0.2-0.8 µm)
• Causes atypical pneumonia (like some viruses)
• Cell culture is required for isolation
• Penicillin is ineffective (no cell wall - similar to viruses having no cell wall target)

• Contains BOTH DNA and RNA (viruses have only one type)
• Has ribosomes and can synthesize proteins independently
• Binary fission (not viral replication/assembly)
• Sensitive to antibiotics that target protein synthesis (macrolides, tetracyclines, fluoroquinolones)
• Can grow on cell-free artificial media (PPLO - Pleuropneumonia-Like Organism agar) - forms "fried egg" colonies
• Has a plasma membrane with sterols (unique among bacteria)

D. Differentiation of Pneumococcal and Meningococcal Meningitis Based on CSF Microscopy

E. Why Screening for UTI Before Urine Culture is Useful

1. Cost-effectiveness: Urine culture is expensive and time-consuming (18-48 hours). Negative dipstick has high NPV (~95%), so culture can be avoided in clearly negative cases.
2. Leukocyte esterase detects pyuria (>10 WBC/HPF), indicating inflammation
3. Nitrite test detects Enterobacteriaceae (reduce nitrate to nitrite)
4. Reduces unnecessary cultures: Only samples with positive screening are sent for culture, reducing lab workload
5. Guides empirical treatment: Positive screen helps clinician start empirical antibiotics while awaiting culture
6. Avoids contamination confusion: Pyuria without bacteriuria (sterile pyuria) suggests TB, chlamydia, or other causes

Q.4 Short Essay Questions (3 x 6 = 18 marks)

A. Patient with Symmetrical Skin Lesions, Multiple Nodules, Plaques, Xanthoma-like Papules, Leonine Facies (1+3+2 = 6 marks)

1. Slit-skin smear (SSS): Smears taken from ear lobes, forehead, chin, affected skin. Stained with Ziehl-Neelsen (ZN) stain. In LL: Numerous acid-fast bacilli (AFB) arranged in bundles called "globi" within foamy macrophages (Virchow cells). Bacteriological Index (BI) 4+ to 6+.
2. Histopathology (skin biopsy):

• Lepromatous: Foamy (Virchow) macrophages packed with AFB; no granuloma; subepidermal "clear zone" (Grenz zone); lymphocytes sparse
• Tuberculoid: Well-formed epithelioid granulomas with Langhans giant cells; few/no AFB

1. Lepromin test (Mitsuda test): NEGATIVE in lepromatous leprosy (due to absent cell-mediated immunity). Measures host immunity, not diagnosis per se.
2. PCR: Highly sensitive; detects M. leprae DNA even in paucibacillary cases.
3. Mouse footpad inoculation: Gold standard for viable bacilli (research only).
4. Serological tests: Anti-PGL-1 (phenolic glycolipid-1) antibody - elevated in LL.

• Rifampicin 600 mg once monthly (supervised)
• Clofazimine 300 mg once monthly (supervised) + 50 mg daily (self-administered)
• Dapsone 100 mg daily (self-administered)

B. Laboratory Diagnosis of Pulmonary Tuberculosis (6 marks)

• ZN (Ziehl-Neelsen) stain: AFB appear bright red against blue background. Grading: 1+ to 3+ or scanty
• Auramine-Rhodamine fluorescence stain: More sensitive; AFB fluoresce yellow-orange. Preferred for screening large volumes

• Lowenstein-Jensen (LJ) medium: Solid egg-based medium. Growth in 4-8 weeks. Colonies: buff-colored, dry, rough, warty ("cauliflower-like")
• BACTEC MGIT 960 (Mycobacterium Growth Indicator Tube): Liquid broth, fluorescence-based. Results in 1-3 weeks. Preferred currently
• Followed by drug susceptibility testing (DST)

• GeneXpert MTB/RIF (Xpert MTB/RIF): WHO-endorsed; cartridge-based PCR detects MTB DNA AND rifampicin resistance (rpoB gene) within 2 hours. High sensitivity and specificity
• Line Probe Assay (LPA): Detects INH and RIF resistance genes (katG, inhA, rpoB)
• NAAT (Nucleic Acid Amplification Tests)

• Mantoux/Tuberculin Skin Test (TST): Intradermal PPD (5 TU); read at 48-72 hrs; induration ≥10 mm = positive (≥5 mm in immunocompromised)
• IGRA (Interferon-Gamma Release Assay) - QuantiFERON-TB Gold: Measures IFN-γ release by sensitized T-cells to ESAT-6 and CFP-10 antigens; more specific than TST (no BCG cross-reactivity)

C. Rabies - Laboratory Diagnosis and Vaccination (6 marks)

1. Seller's stain (brain tissue - Ammon's horn/hippocampus): Demonstrates Negri bodies (eosinophilic intracytoplasmic inclusions, oval/round) - pathognomonic but sensitivity only 70-80%
2. Fluorescent Antibody Test (FAT/DFA): Gold standard. Detects viral antigen in brain impression smears using fluorescent-labeled anti-rabies antibody. WHO recommended. Rapid (hours), highly sensitive and specific
3. Mouse Inoculation Test: Intracerebral injection of brain tissue into mice; mice develop flaccid paralysis; confirmatory
4. Histopathology: H&E stain shows Negri bodies in Purkinje cells (cerebellum) and pyramidal cells (hippocampus)

1. Skin biopsy (nape of neck): DFA on hair follicle nerve endings - detects antigen
2. Corneal impression smear: DFA - viral antigen in corneal cells
3. Saliva/CSF/Tears: RT-PCR for rabies virus RNA - most sensitive ante-mortem test
4. Serum/CSF: Virus neutralization test (RFFIT) - detects antibodies (useful in vaccinated patients or late stages)

• Indication: Veterinarians, lab workers, wildlife handlers
• Cell culture vaccine (HDCV, PCECV, VERO cell): 3 doses on Days 0, 7, 21 (or 28) IM
• Booster every 2-3 years

• WHO category 3 bites (deep, multiple, mucosa): Immediate wound washing + RIG + vaccine
• Rabies Immunoglobulin (RIG):
• Human RIG (HRIG): 20 IU/kg - infiltrated around wound + remaining IM
• Equine RIG (ERIG): 40 IU/kg (cheaper, used in developing countries)
• Vaccine schedule: 5 doses - Days 0, 3, 7, 14, 28 (Essen regimen) OR 4 doses - Days 0 (x2 sites), 7, 21 (Zagreb regimen)
• Previously vaccinated: Only 2 vaccine doses (Days 0 and 3); NO RIG needed

Q.5 Short Answer Questions (5 x 5 = 25 marks)

A. Pathogenesis and Lab Diagnosis of Hydatid Cyst (5 marks)

1. Humans ingest eggs from contaminated food/water/dog feces
2. Eggs hatch in duodenum → oncospheres penetrate intestinal wall
3. Enter portal circulation → lodge in liver (most common - 75%) or lungs, brain, bone
4. Oncosphere develops into hydatid cyst over months-years
5. Cyst has 3 layers: Pericyst (host-derived), Ectocyst (laminated outer), Endocyst (germinal/inner layer - produces protoscolices and brood capsules)
6. Cyst grows slowly; rupture can cause anaphylaxis and secondary dissemination (daughter cysts)

1. Imaging: Ultrasound (WHO-IWGE classification, CE1-CE5), CT scan - cystic lesion with internal daughter cysts ("honeycomb" appearance), pathognomonic "water lily sign" (collapsed membranes)
2. Casoni intradermal test: Inject hydatid fluid intradermally; wheal >5 mm = positive (historical, low specificity)
3. Serology (main diagnostic tool):

• ELISA for anti-Echinococcus IgG (most used)
• Indirect Hemagglutination (IHA)
• Arc 5 precipitin line (immunoelectrophoresis) - highly specific
• Western blot for confirmation

1. Eosinophilia: Peripheral blood eosinophilia (present if cyst has leaked)
2. Aspiration (PAIR - Puncture, Aspiration, Injection, Re-aspiration): Hydatid fluid shows "hydatid sand" - scolices, hooks (double row of hooklets), calcareous corpuscles on microscopy. Blind aspiration is CONTRAINDICATED due to anaphylaxis risk; only done as therapeutic PAIR with precautions.

B. Laboratory Diagnosis of Mycetoma (5 marks)

• Eumycetoma: True fungi (e.g., Madurella mycetomatis, Scedosporium apiospermum)
• Actinomycetoma: Aerobic actinomycetes (e.g., Nocardia brasiliensis, Actinomadura madurae)

1. Macroscopic examination of grains (MOST IMPORTANT initial step):

• Color and texture of grains help predict organism:
• Black grains → Madurella spp. (eumycetoma)
• White/yellow grains → Actinomadura or Nocardia (actinomycetoma)
• Red grains → Actinomadura pelletieri

1. Gram stain/ZN stain of grains:

• Actinomycetoma: Gram-positive, branching filaments; partially acid-fast (Nocardia)
• Eumycetoma: Broad, non-branching hyphae with chlamydospores

1. KOH preparation: Grains in 10-20% KOH - shows fungal hyphae (eumycetoma)
2. Culture:

• Sabouraud's Dextrose Agar (SDA) at 25-30°C - for fungi
• Blood agar/nutrient agar aerobically - for actinomycetes
• Growth is slow (weeks)

1. Histopathology (skin/tissue biopsy):

• Grains surrounded by neutrophilic abscess and fibrosis
• Grain morphology distinguishes eumycetoma vs actinomycetoma

1. Molecular: PCR/sequencing for species identification
2. X-ray/MRI: Bone involvement (periostitis, "soap bubble" lytic lesions)

C. Clinical and Laboratory Approaches to Diagnose Etiological Agents of Vulvovaginitis (5 marks)

• Wet mount (saline): Budding yeast cells and pseudohyphae
• KOH preparation (10%): Dissolves epithelial cells, shows hyphae/pseudohyphae clearly - best rapid test
• Gram stain: Gram-positive budding yeast
• Culture: Sabouraud's agar - cream-colored colonies; germ tube test (for C. albicans)

• Wet mount (saline - gold standard for direct diagnosis): Motile, pear-shaped flagellated protozoa with jerky/tumbling motility. Sensitivity ~60-70%
• NAAT (PCR): Most sensitive and specific - recommended by CDC
• Culture: In Diamond's or InPouch TV medium (most sensitive non-molecular)
• Pap smear: May show TV incidentally
• Rapid antigen tests (OSOM TV test)

• Amsel's criteria (3 of 4 required):

1. Homogeneous grey discharge
2. pH >4.5
3. Positive "whiff test" (amine odor with 10% KOH)
4. Clue cells on wet mount (epithelial cells covered by bacteria - stippled/granular appearance)

• Nugent score: Gram stain of vaginal smear; score 7-10 = BV (large Gram-positive rods decrease, Gram-variable coccobacilli increase)
• Culture: HVS on blood agar - Gardnerella shows beta-hemolysis

D. Pathogenesis and Laboratory Diagnosis of Diphtheria (5 marks)

1. Droplet infection → colonizes pharynx/larynx/nasopharynx
2. Only toxigenic strains (infected by bacteriophage carrying tox gene) cause disease
3. Diphtheria exotoxin (A-B type):

• B fragment: Binds to host cell receptors (heparin-binding EGF)
• A fragment (active): Catalyzes ADP-ribosylation of EF-2 (Elongation Factor-2) → irreversibly inhibits protein synthesis → cell death

1. Local: Pseudomembrane formation in throat (fibrin + necrotic cells + bacteria + neutrophils) - tough, grayish-white, bleeds on removal
2. Systemic toxin effects:

• Myocarditis (most common cause of death) - heart block, arrhythmia
• Demyelinating neuropathy - palatal palsy (nasal regurgitation), oculomotor palsy, peripheral neuritis
• Adrenal necrosis

1. Specimens: Throat/nasal swabs (from edge of membrane), membrane fragment
2. Direct Microscopy:

• Albert's stain: Blue-green bacilli with dark-blue/black metachromatic granules (volutin/Babes-Ernst granules) at poles - "Chinese letter" arrangement
• Loeffler's Methylene Blue: Shows granules well

1. Culture:

• Loeffler's Serum Slope: Rapid growth (6-8 hrs); shows granules clearly
• Tellurite Blood Agar (Hoyle's/McLeod's): Selective medium; C. diphtheriae reduces tellurite → black colonies (gray-black). Tellurite inhibits other organisms
• Colony types: Gravis (large, gray), Mitis (small, black), Intermedius

1. Toxigenicity Testing (ESSENTIAL - not all strains are toxigenic):

• Elek's gel precipitation test (immunoprecipitation): Filter paper strip soaked in antitoxin placed on agar; organism streaked perpendicular; toxin + antitoxin form precipitin lines at 45° = toxigenic
• PCR for tox gene
• Vero cell cytotoxicity assay

1. Schick test (historical): Tests susceptibility to diphtheria toxin in a population

E. Laboratory Diagnosis of Gas Gangrene (5 marks)

1. Specimen: Wound exudate, tissue biopsy, blood culture
2. Gram Stain (rapid, very helpful):

• Large Gram-positive rods, often "box-car" shaped
• Notably ABSENT or very few neutrophils (toxin kills WBCs)
• Spores (oval, central/subterminal) may be seen
• This pattern (large GPR + no PMNs) is highly suggestive of gas gangrene

1. Culture (anaerobic - essential):

• Robertson's Cooked Meat (RCM) medium: Enrichment; blackening + rancid odor = C. perfringens
• Blood agar (anaerobic): Double zone of hemolysis (C. perfringens) - inner zone beta, outer zone alpha
• Nagler's reaction (lecithinase test): On egg yolk agar; C. perfringens produces alpha-toxin (lecithinase) → opalescent halo around colony; inhibited by specific antitoxin on one half = positive Nagler reaction
• Stormy fermentation in litmus milk medium

1. X-ray/CT: Gas in tissues (feathery pattern along fascial planes) - clinical/radiological diagnosis is made first
2. Toxin detection: ELISA or PCR for C. perfringens alpha toxin
3. Blood culture: For bacteremia/sepsis

Q.6 Short Answer Questions (5 x 4 = 20 marks)

A. Non-Suppurative Complications of Streptococcus pyogenes (Group A Strep) (4 marks)

• Follows pharyngitis (not skin infection)
• Molecular mimicry: Anti-streptococcal M protein antibodies cross-react with cardiac antigens
• Jones Criteria: Major (carditis, polyarthritis, chorea, erythema marginatum, subcutaneous nodules) + Minor (fever, elevated ESR/CRP, prolonged PR interval) + evidence of preceding GAS infection
• Can cause rheumatic heart disease (mitral stenosis most common sequela)

• Follows pharyngitis OR skin infection (impetigo) - nephritogenic strains (M types 1, 4, 12, 49)
• Immune complex (type III hypersensitivity) deposition in glomeruli → complement activation → glomerular damage
• Features: Hematuria ("cola-colored" urine), proteinuria, hypertension, periorbital edema, reduced GFR
• Typically self-limiting; anti-DNase B titers elevated (better marker than ASO for skin strains)

B. Free-Living Amoebae (4 marks)

• Found in warm freshwater (lakes, hot springs, swimming pools)
• Entry: Nasal route while swimming → olfactory nerve → brain (cribriform plate)
• PAM: Acute, rapidly fatal (>97% mortality), fulminant hemorrhagic meningoencephalitis
• CSF: Like bacterial meningitis (PMNs, no organism on Gram stain); motile amoebae on wet mount of fresh CSF
• Treatment: Amphotericin B (intrathecal) + miltefosine (poor prognosis)

• Causes Granulomatous Amoebic Encephalitis (GAE): Subacute, in immunocompromised (AIDS, transplant); hematogenous spread from lungs/skin
• Acanthamoeba Keratitis (AK): Contact lens wearers; painful corneal ulcer with ring infiltrate; can cause blindness
• Lab: Corneal scrapings - trophozoites/cysts on Giemsa; culture on non-nutrient agar with E. coli overlay (amoeba eats bacteria - tracks visible)

• GAE in both immunocompetent and immunocompromised
• No reliable treatment

• CSF wet mount (motile trophozoites)
• Culture on non-nutrient agar (tracks of amoeba eating bacteria)
• Immunofluorescence, PCR
• Brain biopsy - cysts and trophozoites

C. Trichomoniasis (4 marks)

• Women: Yellow-green, frothy, malodorous vaginal discharge; vulvovaginal pruritus and burning; "strawberry cervix" (punctate hemorrhages on cervix) - pathognomonic but seen in only 2%
• Men: Usually asymptomatic; urethritis, balanitis

1. Wet mount (saline): Motile, pear-shaped protozoa with characteristic jerky/tumbling motility. Sensitivity ~50-70% in women, lower in men.
2. NAAT (PCR): Most sensitive and specific - gold standard currently. Detects TV DNA in vaginal/urethral swabs, urine.
3. Culture (Diamond's medium or InPouch TV): More sensitive than wet mount; 5-7 days.
4. Rapid antigen tests: OSOM Trichomonas Rapid Test - results in 10 min.
5. Pap smear: Incidental finding (low sensitivity).
6. Acridine orange stain: Fluorescent staining of trophozoites.

D. MRSA - Methicillin-Resistant Staphylococcus aureus (4 marks)

• mecA gene (on mobile genetic element SCCmec) encodes PBP2a (PBP2') - modified penicillin-binding protein with very low affinity for all beta-lactams (penicillins, cephalosporins, carbapenems) → all beta-lactams ineffective

• HA-MRSA (Healthcare-Associated): Hospitals; pneumonia, bacteremia, wound infections; multi-drug resistant; SCCmec types I, II, III
• CA-MRSA (Community-Associated): Skin and soft tissue infections (furuncles, abscesses); Panton-Valentine Leukocidin (PVL) positive; SCCmec type IV; USA300 strain common

1. Culture: Blood agar/mannitol salt agar; golden-yellow, beta-hemolytic colonies; catalase +, coagulase +
2. Cefoxitin disc diffusion (30 µg): Zone ≤21 mm = MRSA (surrogate for mecA). Preferred over oxacillin disk
3. MIC determination: Oxacillin MIC ≥4 µg/mL = MRSA
4. Chromogenic agar (MRSA Select/CHROMagar): Mauve/pink colonies on selective agar - rapid screening
5. PCR for mecA gene: Gold standard molecular method; rapid, highly specific
6. Latex agglutination (PBP2a detection): Detects PBP2a protein in bacterial extract

E. Legionnaires' Disease (4 marks)

• Legionnaires' disease: Severe pneumonia with extrapulmonary features - high fever, dry cough, confusion, diarrhea, hyponatremia (SIADH), elevated CK, deranged LFTs
• Pontiac fever: Self-limiting flu-like illness without pneumonia

1. Urinary Antigen Test (UAT): Detects L. pneumophila serogroup 1 antigen in urine. Rapid (minutes), specific, remains positive for weeks. Test of choice for rapid diagnosis. Sensitivity ~70-80%.
2. Culture (Gold Standard):

• BCYE (Buffered Charcoal Yeast Extract) agar supplemented with L-cysteine and iron - Legionella requires L-cysteine for growth (auxotrophic)
• Grows in 3-7 days; small, glistening colonies with "ground-glass" appearance
• Specimens: Sputum, BAL, bronchial washings

1. Direct Fluorescent Antibody (DFA): Detects antigen in respiratory secretions; rapid but less sensitive
2. Serology (IFA): 4-fold rise in antibody titer between acute and convalescent samples (2-6 weeks apart) = diagnostic. Retrospective diagnosis only.
3. PCR: Highly sensitive; detects all serogroups; used on respiratory specimens/urine
4. Gram stain: Poorly staining (faint) Gram-negative rods; silver stain (Dieterle/Warthin-Starry) for tissue sections