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Microbiology Assignment - IInd Year MBBS

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Question 1

Clinical Diagnosis

Etiological Agent

• Neonates: Streptococcus agalactiae (Group B Strep), E. coli, Listeria monocytogenes
• Infants/Children (1 month - 5 years): Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae type b
• School-age & adults: N. meningitidis, S. pneumoniae

Differential Diagnosis

Specimen Collection & Transport

• Specimen: Cerebrospinal fluid (CSF) - gold standard
• Collection: Lumbar puncture (L3-L4 or L4-L5 interspace), aseptic technique, collect 3 tubes:
  • Tube 1: Biochemistry (protein, glucose)
  • Tube 2: Microbiology (culture, Gram stain)
  • Tube 3: Cell count/cytology
• Also collect: Blood cultures (2 sets), nasopharyngeal swab
• Blood: For simultaneous blood culture before antibiotics
• Transport:
  • CSF should be transported immediately to the lab (within 30 minutes) at body temperature (35-37°C) - never refrigerate
  • Meningococci are fastidious and die rapidly at low temperatures
  • Use sterile, leak-proof containers

Laboratory Diagnosis

• Normal: crystal clear; Bacterial meningitis: turbid/purulent; TB meningitis: cobweb clot

1. Gram stain - Gram-negative diplococci (N. meningitidis), Gram-positive diplococci (S. pneumoniae), Gram-negative coccobacilli (H. influenzae)
2. Culture - Blood agar, Chocolate agar (incubated at 37°C, 5% CO₂); Sensitivity ~80%
3. Latex agglutination test - Rapid antigen detection (N. meningitidis, S. pneumoniae, H. influenzae, Group B Strep)
4. India ink preparation - For Cryptococcus neoformans (in immunocompromised)
5. PCR - Most sensitive, detects bacterial DNA even after antibiotics started
6. Counter immunoelectrophoresis (CIE) - Antigen detection

Treatment

• Add Dexamethasone 0.15 mg/kg IV 6-hourly x 4 days (started before or with first dose of antibiotic) - reduces mortality and complications especially in H. influenzae and pneumococcal meningitis

Prevention

• Meningococcal vaccines: MenACWY (quadrivalent), MenB vaccine
• Hib vaccine: Part of EPI schedule (DPT-Hib pentavalent vaccine)
• Pneumococcal vaccine (PCV13): Routine childhood immunization
• Chemoprophylaxis: Rifampicin (or ciprofloxacin/ceftriaxone) for close contacts of meningococcal meningitis

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Question 2

Clinical Condition

Etiological Agent

• Acid-fast bacillus (AFB), non-motile, non-sporing, aerobic
• Slow-growing (18-24 hours doubling time), takes 4-8 weeks to grow on culture
• Cell wall rich in mycolic acids - responsible for acid-fastness

Recent Advanced Techniques in Laboratory Diagnosis

• Sputum smear microscopy (ZN stain): Cheap, rapid, but low sensitivity (~60%). Requires 5,000-10,000 bacilli/mL
• Auramine-rhodamine fluorescence microscopy: More sensitive than ZN stain
• Culture: Gold standard - Lowenstein-Jensen (LJ) medium (egg-based, results in 4-8 weeks); BACTEC MGIT 960 liquid culture (results in 10-14 days)

Treatment (RNTCP/NTEP Guidelines, India)

• Intensive Phase (2 months): HRZE - Isoniazid (H) + Rifampicin (R) + Pyrazinamide (Z) + Ethambutol (E) daily
• Continuation Phase (4 months): HR - Isoniazid + Rifampicin daily
• Total duration: 6 months

• Pyridoxine (Vit B6): Given with isoniazid to prevent peripheral neuropathy
• Directly Observed Treatment (DOT) under NTEP is mandatory in India

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Question 3

Clinical Presentation

• Classic features: "Stepladder" pattern fever, relative bradycardia (Faget's sign - pulse rate lower than expected for the degree of fever), abdominal pain/distension, rose spots (2%), hepatosplenomegaly, constipation or diarrhea

Causative Agent

• Gram-negative rod, non-sporing, non-capsulated (except Vi antigen), peritrichous flagella
• Belongs to Enterobacteriaceae family
• S. paratyphi A, B, C - cause paratyphoid fever (milder)

Laboratory Procedures

• Bile broth or tryptose broth; positive in ~80% in 1st week
• Sensitivity decreases after 1st week

• Tube agglutination test
• Tests for O antigen (somatic) and H antigen (flagellar) antibodies
• Titre ≥1:160 for O agglutinins significant (in non-endemic areas)
• Significant rise in titre in paired sera (2 weeks apart) is more reliable
• Limitations: False positives in malaria, liver disease, immunization; False negatives in early disease

• Positive in 3rd week onward
• MacConkey agar - colorless (non-lactose fermenting) colonies

• Most sensitive (80-95%) even after antibiotics - gold standard

• O antibody (somatic) rises first, indicates active infection
• H antibody (flagellar) rises later, persists longer, may indicate past infection or vaccination

Drug of Choice (DOC)

• Fluoroquinolones (Ciprofloxacin 500mg BD x 10-14 days) - DOC for susceptible strains
• For NDTS (Nalidixic acid-resistant): Azithromycin (1g loading, then 500mg OD x 5 days) or Ceftriaxone
• For MDR typhoid: Ceftriaxone 2g IV daily x 10-14 days or Azithromycin
• For XDR typhoid (also resistant to fluoroquinolones + 3rd gen cephalosporins): Azithromycin or Meropenem

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Question 4

Probable Diagnosis

• If flank pain, fever, chills are present: Acute Pyelonephritis (upper UTI)

Etiological Agents

Specimen Collection

1. Patient cleans the periurethral area with antiseptic
2. First portion of urine discarded
3. Midstream urine collected in sterile wide-mouth container (60-100 mL)
4. Avoid contamination from vaginal secretions
5. Catheter specimen (CSU) or suprapubic aspirate if patient cannot provide CMSU (most sterile)
6. Transport within 2 hours to lab or refrigerate at 4°C (up to 24 hours)

Laboratory Diagnosis

• Turbid, foul-smelling urine suggestive

• Pus cells (WBC >5/hpf) - pyuria
• RBC, bacteria, casts (in pyelonephritis)

• Nitrite test - positive if Gram-negative bacteria (reduce nitrate to nitrite)
• Leukocyte esterase - indicates pyuria
• Both positive together: ~90% predictive for UTI

• Inoculate on CLED (Cysteine Lactose Electrolyte Deficient) medium or MacConkey agar
• Count colonies: ≥10⁵ CFU/mL (100,000 CFU/mL) in CMSU = significant bacteriuria (Kass criterion)
• Lower counts significant in symptomatic patients (≥10² in CMSU with symptoms)
• Identify organism and perform antibiotic sensitivity testing (AST)

• E. coli: Pink lactose-fermenting colonies on MacConkey; metallic sheen on EMB agar
• Klebsiella: Mucoid pink colonies on MacConkey
• Proteus: Swarming on blood agar; H₂S production

• Uncomplicated cystitis: Nitrofurantoin 100mg BD x 5 days or Trimethoprim-sulfamethoxazole or Fosfomycin 3g single dose
• Pyelonephritis: Fluoroquinolone (ciprofloxacin) or ceftriaxone IV for severe cases

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Question 5

Clinical Presentation & Causative Organism

• Causative organism: Corynebacterium diphtheriae
  • Gram-positive rod, club-shaped, arranged in "Chinese letter" or "V/L" patterns (snapping division)
  • Non-motile, non-sporing, non-capsulated
  • Metachromatic granules (Babes-Ernst/volutin granules) - stained with Albert's or Ponder's stain as bluish-black granules in yellowish-green background
  • 3 biotypes: Gravis, Intermedius, Mitis (Gravis most virulent)
  • Toxin-producing strains carry bacteriophage β (tox gene)

• Faucial diphtheria (most common): Gray-white pseudomembrane on tonsils/pharynx
• Membrane is tough, leathery, bleeds on removal
• "Bull neck" appearance (cervical lymphadenopathy + soft tissue edema)
• Toxin causes: Myocarditis (cardiac arrhythmia), neuropathy (palatal palsy, CN VI, VII), airway obstruction

Ideal Specimen

Collection & Transport

1. Use sterile swab on a wooden stick
2. Rub firmly at the edge of the membrane (not the center)
3. Two swabs: one for direct smear, one for culture
4. Transport in Loeffler's serum medium (enrichment transport medium) or Amies transport medium
5. Process within 2-4 hours; if delayed, inoculate into Loeffler's serum and refrigerate

Gold Standard for Laboratory Diagnosis

• Filter paper strip soaked in diphtheria antitoxin placed on agar plate
• Suspect colonies streaked at right angles to filter paper
• Toxin + antitoxin produce precipitin bands ("lines of identity") within 24-48 hours

1. Direct smear: Albert's stain - shows metachromatic granules
2. Culture media:
   • Loeffler's serum slope - C. diphtheriae grows readily; shows metachromatic granules
   • Tellurite medium (Hoyle's/Tinsdale): Selective; C. diphtheriae produces black/brown colonies (tellurite reduced to tellurium)
3. PCR - Detects tox gene rapidly (modern gold standard in labs)

Treatment

1. Diphtheria Antitoxin (DAT): Most important, given immediately (neutralizes circulating toxin)
   • Dose: 20,000-1,00,000 units depending on severity
   • Test for horse serum sensitivity first (desensitization if needed)
2. Antibiotics (to eliminate organism and prevent transmission):
   • Penicillin G (procaine penicillin) or Erythromycin x 14 days
3. Supportive care: Airway management, cardiac monitoring

Prevention

• DPT (DTP) vaccine - primary immunization at 6, 10, 14 weeks (EPI schedule)
• Booster at 18 months and 5 years (DPT booster)
• Td booster for adults every 10 years
• Schick test - obsolete test for immunity to diphtheria

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Question 6

Etiological Agent

• Gram-negative, comma-shaped rod (vibrio)
• Single polar flagellum (darting motility - "shooting star" in dark-field microscopy)
• Oxidase positive
• Produces cholera toxin (CT): B subunit binds to GM1 ganglioside → A subunit activates adenylate cyclase → ↑cAMP → hypersecretion of Cl⁻ and water → watery diarrhea ("rice-water stools")
• O1 biotypes: Classical and El Tor; serotypes: Inaba, Ogawa, Hikojima

How to Determine Serogroup

1. Slide agglutination test with polyvalent O1 antiserum, then with Inaba and Ogawa antisera
2. O139 serogroup has a polysaccharide capsule; agglutinates with O139 antiserum
3. Widal-type tube agglutination
4. PCR - most specific; detects ctxA/B gene (cholera toxin gene)

Transport of Specimen

• Specimen: Stool (watery - "rice-water"), rectal swab
• Transport medium: Venkatraman-Ramakrishnan (VR) transport medium (alkaline peptone water, pH 8.4-9.0) - best
• Alternatively: Cary-Blair transport medium or Alkaline peptone water (APW) for enrichment
• Process within 2 hours; if delayed, use transport medium and refrigerate

Laboratory Diagnosis

1. Dark-field microscopy: Darting motility of vibrios ("shooting star")
2. Enrichment culture: Alkaline peptone water (APW) pH 8.6, 37°C, 6-8 hours
3. Culture media:
   • TCBS (Thiosulfate Citrate Bile Salt Sucrose) agar - selective; V. cholerae produces yellow colonies (sucrose fermenter)
   • Blood agar: flat, rough colonies
4. Oxidase test: Positive (distinguishes from Enterobacteriaceae)
5. Hanging drop preparation: Darting/shooting star motility
6. Slide agglutination: With O1 and O139 antisera
7. String test: Positive (cholera vibrios lyse in 0.5% sodium deoxycholate)
8. Biotyping: El Tor biotype - Voges-Proskauer +ve, haemolysin, phage group IV resistance

Treatment

1. Oral Rehydration Therapy (ORT) - cornerstone of treatment; ORS (WHO formula)
2. IV Fluids (Ringer's lactate) for severe dehydration
3. Antibiotics (reduce duration and stool volume):
   • Tetracycline 500mg QID x 3 days (DOC in adults, resistant strains emerging)
   • Doxycycline 300mg single dose
   • Azithromycin (for children and pregnant women)
   • Ciprofloxacin - alternative
4. Zinc supplementation in children

Prevention

• Safe drinking water, sanitation, hygiene (WASH)
• Oral cholera vaccine (OCV): Shanchol or mORC-VAX (killed bivalent, 2 doses); WHO prequalified
• Killed oral whole-cell cholera vaccine (WC-rBS)
• Notification of cholera is mandatory (International Health Regulations)

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Question 7

Clinical Diagnosis

Etiological Agent

• Family: Flaviviridae, Genus: Flavivirus
• Single-stranded positive-sense RNA virus
• 4 serotypes: DENV-1, DENV-2, DENV-3, DENV-4
• Vector: Aedes aegypti (primary), Aedes albopictus (secondary) - daytime biting mosquitoes
• Second infection with different serotype → Dengue Hemorrhagic Fever (DHF) via "antibody-dependent enhancement (ADE)"

Laboratory Diagnosis

1. NS1 Antigen Test:
   • Detects non-structural protein 1 in blood
   • Positive from Day 1-5 (febrile phase)
   • Sensitivity ~90% in early infection
   • Rapid card test available
2. IgM ELISA (MAC-ELISA):
   • IgM appears from Day 5 onward
   • Indicates primary or recent infection
   • Gold standard for diagnosis after Day 5
   • IgM persists for 2-3 months
3. IgG ELISA:
   • Secondary infection: IgG rises rapidly (anamnestic response)
   • IgG:IgM ratio >1.2 suggests secondary infection
4. Dengue PCR (RT-PCR):
   • Most sensitive and specific
   • Detects and serotypes virus
   • Positive from Day 1-5
5. Dengue Viral Culture:
   • Research purpose only; C6/36 mosquito cell line
   • Not for routine diagnosis
6. Complete Blood Count:
   • Thrombocytopenia (<1,00,000/mm³) - hallmark
   • Leukopenia
   • Rising hematocrit (plasma leakage in DHF)
7. Tourniquet test (Rumpel-Leede test): >10 petechiae in a 2.5 cm² area - positive

Treatment

• No specific antiviral therapy available
• Supportive management:
  • Paracetamol for fever and pain (avoid NSAIDs and aspirin - risk of bleeding)
  • Oral hydration; IV fluids (isotonic crystalloids) for dengue with warning signs
  • Monitor CBC, hematocrit, platelet count daily
  • Platelet transfusion only if <10,000/mm³ or active bleeding
• Warning Signs (require hospitalization): Abdominal pain, persistent vomiting, mucosal bleeding, rapid breathing, fluid accumulation, postural hypotension

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Question 8

Category of Exposure

• Single or multiple transdermal bites or scratches, contamination of mucous membrane with saliva (i.e., licks on broken skin) - especially on the face/head, neck, hands, genitals

Immediate Wound Management

1. Wash the wound immediately and vigorously for at least 15 minutes with soap and water, povidone-iodine, or detergent under running water
2. Apply antiseptic: Povidone-iodine (Betadine) solution
3. Do NOT suture the wound primarily (if suturing necessary, give RIG locally first)
4. Do NOT apply herbal remedies or bandage tightly
5. Wound washing is the single most important measure to prevent rabies - can reduce viral load by up to 90%
6. Check tetanus immunization status

Role of Rabies Immunoglobulin (RIG)

• Provides immediate passive immunity (passive immunization) until active immunity from vaccine develops
• Indicated for all Category III exposures in previously unvaccinated individuals
• Human Rabies Immunoglobulin (HRIG): 20 IU/kg body weight
• Equine Rabies Immunoglobulin (ERIG): 40 IU/kg body weight (cheaper, test for sensitivity)
• Administration: Maximum possible dose infiltrated into and around the wound; remainder given IM at a site distant from the vaccine
• Given on Day 0 only (single dose)
• Do NOT give in same syringe as vaccine; do NOT give in same anatomical site as vaccine

Vaccine Schedule

• Dose on Days: 0, 3, 7, 14, 28 (5 doses)

• Day 0: 2 doses (one in each deltoid), Day 7: 1 dose, Day 21: 1 dose
• Total: 4 doses (WHO accepted)

• Days 0, 3, 7, 28 - 0.1 mL intradermally at 2 sites
• More economical, WHO recommended for resource-limited settings

• Pre-exposure prophylaxis (PrEP): 3 doses on Days 0, 7, 21/28 - for veterinarians, lab workers
• Note: Once rabies symptoms appear, there is no effective treatment (case fatality ~100%)

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Question 9 (Q10 in PDF)

Suspected Diagnosis

Screening Test

• Detects both HIV-1 & HIV-2 antibodies + p24 antigen (combined assay)
• High sensitivity (>99.5%), used as screening test
• Reactive result must be confirmed

Confirmatory Strategy (NACO Guidelines, India)

• Test 1 (A1): ELISA - if reactive
• Test 2 (A2): Different ELISA principle - if reactive
• Test 3 (A3): Third ELISA - used to resolve discordant results
• Two reactive tests = HIV positive (no Western Blot needed in India's revised strategy)

• Positive: ≥2 of gp160/gp120, gp41, p24 bands present

Window Period

• Window period = time between HIV infection and detectable antibodies
• With 4th generation assay (detects p24 antigen + antibodies): ~11-14 days (reduced from earlier 21-22 days for antibody-only tests)
• With 3rd generation ELISA: ~21-22 days

First Marker to Appear After Infection

Common Opportunistic Infections (OIs) in AIDS (CD4 count determines risk)

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Question 10 (Q11 in PDF)

Three C's of Measles (Prodromal Phase)

1. Cough
2. Coryza (runny nose)
3. Conjunctivitis (red eyes, photophobia)

Koplik Spots

• Pathognomonic (diagnostic) enanthem of measles
• Appear 1-2 days before the rash (prodromal phase)
• Bluish-white or grayish-white spots with a red halo
• Located on the buccal mucosa opposite the lower molar teeth
• Described as "grains of salt on red background" or "table salt on a brick-red wall"
• Disappear shortly after the rash appears
• Caused by: Focal necrosis of mucosa with inflammation

Laboratory Tests for Confirmation

• Causative agent: Measles virus (Morbillivirus, family Paramyxoviridae) - single-stranded negative-sense RNA
• Rash: Maculopapular, starts behind ears → face → spreads downward (centrifugal spread)
• Complications: Pneumonia, encephalitis, SSPE (subacute sclerosing panencephalitis - late)
• Prevention: MMR vaccine (live attenuated)

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Question 11 (Q12 in PDF)

Clinical Presentation

• Typical presentation: Facial swelling, periorbital edema, black eschar over nose/palate, necrosis of nasal turbinates, proptosis
• Can invade orbits (orbital cellulitis), cranial nerves, and brain
• Predisposing factors: Uncontrolled diabetes mellitus (most common - diabetic ketoacidosis), prolonged corticosteroid use, hematological malignancies, organ transplants (gained prominence during COVID-19 pandemic)

Etiological Agent

• Rhizopus arrhizus (R. oryzae) - most common
• Rhizopus microsporus
• Mucor spp.
• Cunninghamella spp.
• Lichtheimia (Absidia) corymbifera

• Broad, ribbon-like, aseptate (or sparsely septate) hyphae
• Right angle branching (vs. Aspergillus: 45° branching, septate hyphae)
• Sporangiophores with rhizoids (in Rhizopus) - distinguishes from Mucor (no rhizoids)

Laboratory Diagnosis

1. Direct microscopy (KOH preparation): Reveals broad, aseptate, ribbon-like hyphae - most rapid
2. Histopathology (PAS stain / GMS stain): Tissue biopsy from affected area; shows necrotic tissue with hyphae invading blood vessels (angioinvasion) → thrombosis → necrosis (black eschar)
3. Culture: Sabouraud's Dextrose Agar (SDA) - rapidly growing gray/white fluffy colonies; identified by slide culture
4. CT/MRI: Assesses extent of disease (orbit, sinuses, brain involvement)
5. Serum β-D-Glucan: Negative in Mucormycosis (differentiates from other fungal infections like Aspergillus, Candida where it's positive)
6. Serum Galactomannan: Negative (positive in Aspergillosis)
7. PCR: Not widely available but emerging

Treatment

1. Control underlying predisposing factor: Optimize glycemic control, stop corticosteroids
2. Antifungal therapy:
   • Liposomal Amphotericin B (LAmB): 5-10 mg/kg/day IV - drug of choice
   • Isavuconazole - alternative, oral bioavailability; FDA approved
   • Posaconazole (step-down oral therapy after initial AmB)
3. Surgical debridement: Aggressive surgical removal of all necrotic tissue - equally important as antifungal therapy
4. Adjunctive therapy: Hyperbaric oxygen (controversial), Iron chelators avoidance (deferoxamine worsens mucormycosis)
5. Mortality high (30-90%) if untreated

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Question 12 (Q13 in PDF)

Most Likely Diagnosis

Causative Organism

• Trichophyton rubrum - most common overall cause of tinea
• Trichophyton mentagrophytes
• Epidermophyton floccosum (no hair involvement)

• Trichophyton - infects skin, hair, nails
• Microsporum - infects skin and hair (not nails)
• Epidermophyton - infects skin and nails (not hair)

Specimen Collection

• Skin scrapings from the active edge (margin) of the lesion (raised scaly margin) - highest fungal load at the margin
• Collection method:
  1. Clean the area with 70% alcohol and allow to dry
  2. Scrape with a sterile blunt scalpel or edge of glass slide
  3. Collect scrapings from the advancing edge (not center)
  4. Collect sufficient material (multiple scrapings)
  5. Place in black paper (for visibility) or sterile container

How to Sample the Lesion

• The active margin (peripheral edge) should be sampled, not the central clearing
• For scalp: Pluck hair stubs/broken hairs and scrape the scalp at the lesion border
• Use a curette or blunt scalpel

Laboratory Tests

1. KOH (Potassium Hydroxide) Preparation - Direct Microscopy:
   • 10-20% KOH dissolves keratin, clears background
   • Reveals: Hyphae (branching septate hyphae) and arthrospores
   • Rapid (30 min), cheap, no equipment needed
   • Calcofluor white stain + fluorescence microscopy: More sensitive
2. Culture (Gold Standard):
   • Sabouraud's Dextrose Agar (SDA) with cycloheximide and chloramphenicol
   • Incubation: 25-30°C, 2-4 weeks
   • M. canis: Fluffy white/pale yellow colonies; reverse yellow/orange; macroconidia: spindle-shaped with ≥6 cells and echinulate surface
   • T. rubrum: White to cream colonies; cherry-red pigment on reverse; teardrop-shaped microconidia
3. Wood's Lamp Examination:
   • UV light 365 nm; M. canis and M. audouinii - bright greenish-yellow fluorescence on infected hairs (ectothrix)
   • Not all dermatophytes fluoresce
4. Histopathology: PAS stain on skin biopsy if needed

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Question 13 (Q14 in PDF)

Most Likely Diagnosis

Common Causative Organism

• HSV-1 can also cause genital herpes (~10-20%) and is increasingly common
• Family: Herpesviridae; double-stranded DNA virus; enveloped
• Primary infection: More severe
• Recurrent infection: Milder, shorter duration

Why is This Condition Recurrent?

1. After primary infection, HSV travels along sensory neurons via retrograde axonal transport to the dorsal root ganglia (sacral ganglia S2, S3, S4) for genital HSV-2
2. The virus establishes latent infection in the ganglia (circular episomal DNA, minimal gene expression - only Latency Associated Transcripts/LATs expressed)
3. Triggered by stimuli (stress, fever, immunosuppression, UV light, menstruation, trauma), virus reactivates, travels via anterograde axonal transport back to skin/mucosa
4. Results in recurrent eruptions, often at the same dermatome
5. Immune system cannot eliminate latent virus in neurons
6. Frequency decreases over years

Ideal Specimen for Laboratory Diagnosis

• Vesicle fluid from an early, unroofed vesicle (before ulceration) - best specimen
• Swab from the base of an ulcer (if vesicles already ruptured) - using viral transport medium (VTM)
• Blood for serology (IgM/IgG) in primary infection

Gold Standard Lab Test

• MRC-5 human diploid fibroblasts or Vero cells
• Cytopathic Effect (CPE): Rounded, balloon-like cells, polykaryocytes (multinucleated giant cells)
• Results in 1-5 days

• More sensitive than culture (~99% vs ~70-80%)
• Can distinguish HSV-1 vs HSV-2 (typing)
• Can be done on vesicle fluid, swab, CSF
• Rapid (~hours)
• Preferred in clinical practice today

1. Tzanck smear (Tzanck test): Scraping from base of vesicle stained with Giemsa - shows multinucleated giant cells (Tzanck cells); not specific (positive in all herpesviruses); quick bedside test
2. Direct Fluorescent Antibody (DFA): Detects viral antigens in smear; rapid
3. Serology (type-specific IgG ELISA): Useful for detecting past infection (HSV-1 vs HSV-2 glycoprotein G-based assay); not useful for diagnosing acute infection

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Question 14 (Q15 in PDF)

Most Likely Diagnosis

Etiological Agent

• Protozoan parasite (amoeba), phylum Sarcomastigophora
• Two forms: Trophozoite (invasive) and Cyst (infective form)
• Trophozoite: 20-40 µm; contains ingested RBCs (erythrophagocytosis) - pathognomonic
• Cyst: 10-16 µm; quadrinucleate (4 nuclei), chromatid bars with rounded ends
• Transmission: Feco-oral (ingestion of cysts in contaminated food/water)
• Mechanism: Trophozoites invade colon → portal blood → liver → abscess

Why is the Right Lobe More Commonly Involved?

• The right lobe of the liver receives the majority of portal blood flow from the superior mesenteric vein (draining the right colon/ileocecal region - most commonly affected by amoebae)
• Streamline flow in the portal vein: right branch carries blood from ileocecal region to right lobe
• Right lobe is also larger (comprises ~65-70% of liver volume)
• Hence, parasites from the right colon preferentially reach the right lobe via the right branch of the portal vein

Laboratory Investigations

Ideal Specimen for Microbiological Diagnosis

• "Anchovy sauce" / chocolate-brown pus (lysed blood + necrotic debris)
• Trophozoites found in the LAST portion of the aspirate (not the central pus) or from the abscess wall scraping
• Note: Central pus is mostly acellular; trophozoites at periphery

1. Amoebic serology (ELISA): Detects anti-amoeba antibodies; sensitivity >90% for ALA; remains positive years after infection
2. Indirect Hemagglutination Assay (IHA): Traditional serological test
3. Counter Immunoelectrophoresis (CIE)
4. Stool microscopy: Fresh stool within 30 minutes; look for trophozoites with ingested RBCs on warm stage microscopy
5. PCR: Most sensitive and specific; differentiates E. histolytica from E. dispar (non-pathogenic look-alike)

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Question 15 (Q16 in PDF)

First Step After Exposure

1. Immediately wash the injury site with soap and water for 15 minutes; squeeze to express blood
2. Apply antiseptic (povidone-iodine)
3. Report to occupational health/infection control immediately
4. Draw blood from the healthcare worker (baseline serology) and from the source patient (with consent)

Serological Tests to Perform

• HBsAg, Anti-HBs (antibody to surface antigen), Anti-HBc (IgM + Total)

• HBsAg, HBeAg, HBV DNA (viral load)

When is Hepatitis B Immunoglobulin (HBIG) Indicated?

• Unvaccinated HCW exposed to HBsAg-positive source: Give HBIG immediately (within 24 hours, ideally) + start HBV vaccine series
• Previously vaccinated but anti-HBs <10 mIU/mL (non-responder): Give HBIG + revaccinate
• Previously vaccinated and anti-HBs ≥10 mIU/mL (responder): No HBIG needed
• Dose: HBIG 0.06 mL/kg IM at a different site from vaccine

Interpretation of Serological Markers

What Indicates Post-Vaccination Immunity?

• Anti-HBs appears without Anti-HBc (no Anti-HBc in purely vaccinated individuals)

What Indicates High Infectivity?

• High HBV DNA (viral load) also indicates high infectivity
• HBsAg alone indicates infection but HBeAg + high HBV DNA indicates maximum infectivity

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Question 16 (Partially on Page 2/3)

Most Likely Congenital Infection

• Classic triad: Cataracts + Sensorineural deafness + Congenital heart disease (PDA/pulmonary artery stenosis)
• Other features: Microcephaly, "blueberry muffin" rash (dermal erythropoiesis), hepatosplenomegaly, thrombocytopenic purpura, glaucoma, psychomotor retardation

Important Maternal History

• History of rubella-like rash (maculopapular, starting on face) during 1st trimester (highest risk)
• Rubella exposure in first 12 weeks: ~85% risk of CRS
• Vaccination history (rubella vaccine)
• Contact with a case of rubella during pregnancy
• Antenatal serology (rubella IgM/IgG)

Laboratory Test Confirming Congenital Infection

1. Rubella-specific IgM in newborn blood: Indicates intrauterine infection (IgM does not cross placenta)
2. Persistent rubella IgG beyond 6-12 months of age (maternally acquired IgG declines; if persists, indicates active infection)
3. RT-PCR on throat swab, urine, CSF: Detects rubella virus RNA
4. Virus isolation (research): From throat, urine, CSF - rubella can be shed for months in CRS infants
5. Prenatal: Amniocentesis + PCR for intrauterine diagnosis

Preventive Measures

• Rubella vaccine (live attenuated, MMR vaccine) - given at 9-12 months (India - MR vaccine), 15-18 months (MMR)
• Vaccination of adolescent girls and women of childbearing age (check seronegative status)
• Contraindication: Rubella vaccine should not be given to pregnant women; pregnancy should be avoided for 1 month after vaccination
• Antenatal rubella screening
• Contact tracing and isolation of cases

Why Early Diagnosis is Important

1. Prevents spread: CRS infants shed live virus and are infectious to contacts for months
2. Intervention: Early hearing aids, corrective surgery for cataracts (before 6 weeks - critical window for visual development), cardiac surgery for CHD
3. Developmental support: Early physiotherapy, speech therapy improves outcomes
4. Inform vaccination programs: Monitor CRS for rubella elimination program
5. Maternal counseling for future pregnancies

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Question 17 (Q19 in PDF)

Most Likely Diagnosis

• CSF profile: Clear/xanthochromic, lymphocytic pleocytosis (10-500 cells), high protein (100-500 mg/dL), very low glucose (<50% of blood glucose), cobweb clot on standing
• This is the classic "chronic meningitis" presentation

Microbiological Tests

1. Smear for AFB (ZN stain): Low sensitivity (~10-40%); requires 10,000 bacilli/mL
2. CSF culture on LJ medium: Gold standard but takes 4-8 weeks; sensitivity ~45-90% if large volume used
3. Adenosine Deaminase (ADA) in CSF: Elevated (>10 U/L) - supports TBM diagnosis; easy and cheap
4. MGIT liquid culture: Results in 10-14 days; better sensitivity than LJ
5. CSF: Fibrin/Cobweb clot formation on standing at 4°C for 30 min - supports TBM

Role of Molecular Testing

• Rapid diagnosis when culture takes weeks
• Detects rifampicin resistance for MDR-TBM
• Guides early treatment initiation, reducing morbidity

Why is Diagnosis Often Delayed?

1. Insidious onset (subacute/chronic, over weeks to months)
2. Non-specific early symptoms (headache, low-grade fever, malaise) mimicking viral illness
3. Low sensitivity of smear - easy to miss few bacilli
4. Culture takes 4-8 weeks - definitive diagnosis delayed
5. Lack of history of pulmonary TB in many cases (especially children)
6. CSF findings not always classic (may be atypical)
7. Limited availability of molecular tests in resource-limited settings
8. Differential diagnosis is broad (viral meningitis, cryptococcal, bacterial)

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Question 18 (Q20 in PDF)

Diagnosis

• Temporal lobe or cerebellar abscess (from chronic otitis media/mastoiditis)

Common Organisms Implicated

Why is Lumbar Puncture Contraindicated?

• Risk of cerebral herniation (coning)
• Brain abscess raises intracranial pressure (ICP). LP suddenly reduces pressure in the spinal compartment below the foramen magnum, creating a pressure gradient that forces the brain (especially the uncus of temporal lobe or cerebellar tonsils) through the foramen magnum → Transtentorial or tonsillar herniation → Death
• LP is absolutely contraindicated when CT shows:
  • Mass lesion (abscess, tumor)
  • Midline shift
  • Loss of cisterns
  • Papilledema

Specimen for Culture

1. Aspirated pus from abscess (stereotactic CT-guided aspiration) - best specimen; direct culture on blood agar, chocolate agar, anaerobic media
2. Both aerobic AND anaerobic cultures essential (brain abscesses often polymicrobial with anaerobes)
3. Blood cultures (2 sets) - positive in ~10-15% of cases

Treatment Planning

1. Medical: Empirical antibiotics based on likely source:
   • Ceftriaxone (broad-spectrum Gram-positive + Gram-negative) + Metronidazole (anaerobic coverage) x 6-8 weeks
   • If post-traumatic/post-op: Add Vancomycin (for MRSA)
   • If immunocompromised: Add antifungal (voriconazole for Aspergillus) or anti-Toxoplasma therapy
2. Surgical:
   • Aspiration (stereotactic): For abscesses >2.5 cm, or in accessible areas, or progressive despite antibiotics
   • Excision: For multiloculated, traumatic, or fungal abscesses
   • Drainage of source (mastoidectomy for otogenic abscess)
3. Monitoring: Repeat CT/MRI at 2-4 weeks; treat seizures with antiepileptics; manage ICP

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Question 19 (Q20 cont. in PDF)

Diagnosis

Causative Organism

• Encapsulated yeast; sexual form: Filobasidiella neoformans (Basidiomycete)
• Serotypes A, B, C, D; serotype A (C. neoformans var. grubii) most common in HIV patients
• C. gattii - more virulent, affects immunocompetent; endemic in certain areas
• Capsule: Polysaccharide capsule (glucuronoxylomannan) - major virulence factor; inhibits phagocytosis, impairs immune response, responsible for high ICP by impeding CSF absorption
• Melanin production (laccase) - helps evade immunity
• Urease positive

Rapid Bedside Lab Test

• CSF mixed with India ink on a slide
• Capsule appears as a clear halo (unstained capsule) against black background
• Budding yeast cells with "lollipop" appearance
• Sensitivity: ~50-80% in HIV patients (high fungal burden)
• Rapid, cheap, available at bedside

• Cryptococcal Antigen (CrAg) test (Lateral Flow Assay): Most sensitive rapid test; detects capsular polysaccharide antigen; sensitivity >99% in HIV; can be done on blood or CSF; now WHO-recommended for screening at CD4 <200

Why is Intracranial Pressure Monitoring Important?

1. Cryptococcal meningitis causes markedly elevated ICP (CSF opening pressure often >250 mm H₂O due to obstruction of CSF absorption by capsular polysaccharide)
2. Elevated ICP is the main determinant of early mortality in cryptococcal meningitis
3. Therapeutic LPs: Serial lumbar punctures to drain CSF and reduce ICP improve outcomes significantly
4. Target: Reduce opening pressure by 50% or to <200 mm H₂O
5. Elevated ICP causes: Blindness (optic nerve damage), deafness, cranial nerve palsies, herniation, death
6. ICP monitoring guides frequency of therapeutic LPs and decision for CSF shunting if needed

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Question 20

Likely Diagnosis

Differentiation: Invasive vs Non-Invasive Diarrhea

Common Organisms

• Shigella dysenteriae (type 1 - most severe, Shiga toxin)
• Shigella flexneri, S. sonnei, S. boydii
• Campylobacter jejuni
• Salmonella enteritidis/typhimurium
• EIEC (E. coli Enteroinvasive)
• Entamoeba histolytica (amoebic dysentery - subacute)
• Yersinia enterocolitica

Stool Findings Expected

• 10 WBCs/hpf (high power field) strongly suggests bacterial invasion

• Trophozoites with ingested RBCs = E. histolytica

Laboratory Tests to Confirm Diagnosis

1. Stool microscopy: WBCs, RBCs, mucus; trophozoites if amoebic
2. Stool culture (Gold Standard):
   • MacConkey agar: Non-lactose fermenting colonies (Shigella, Salmonella)
   • Selective media: XLD (Xylose Lysine Deoxycholate) agar, SS (Salmonella-Shigella) agar, Hektoen enteric agar
   • Shigella: Non-motile, non-H₂S-producing, non-lactose fermenter; identified by biochemical tests and agglutination with polyvalent antisera
3. Serotyping: With specific antisera (Group A = S. dysenteriae, B = S. flexneri, C = S. boydii, D = S. sonnei)
4. PCR (Multiplex GI Panel): Detects multiple pathogens simultaneously; very sensitive
5. Enzyme immunoassay (EIA): For C. difficile (if hospital-acquired)
6. Blood culture: For systemic spread (especially in immunocompromised)
7. Sigmoidoscopy/Biopsy: In chronic cases; shows mucosal inflammation, flask-shaped ulcers (amoeba)

• Ciprofloxacin 500 mg BD x 3-5 days (DOC for adults)
• Azithromycin for children and in resistant cases
• Oral rehydration; avoid anti-motility agents (may worsen toxin-mediated disease)

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