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Anti-Epileptic Drugs (AEDs)

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Overview and Treatment Goals

• ~50% respond to the first drug as monotherapy
• ~15% respond to a second drug as monotherapy
• Remaining cases are considered treatment-resistant

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Classification: 3 Generations of AEDs

First-Generation (Older) Drugs

Second-Generation (Newer) Drugs

Third-Generation (Newest) Drugs

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Mechanisms of Action

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Drug of Choice by Seizure Type

Caution: Carbamazepine and phenytoin can worsen absence, myoclonic, and atonic seizures - avoid in generalized epilepsies.

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Key Adverse Effects

Phenytoin

• Nystagmus, ataxia, diplopia (dose-related)
• Gingival hyperplasia, hirsutism, coarsening of facial features (cosmetic - especially in young women)
• Megaloblastic anemia (folate interference)
• Teratogenic (fetal hydantoin syndrome, cleft palate)
• Zero-order (saturable) kinetics - small dose increases cause disproportionate toxicity

Carbamazepine

• Diplopia, ataxia, drowsiness
• Hyponatremia (SIADH-like effect)
• Aplastic anemia, agranulocytosis (rare but serious - check CBC)
• Stevens-Johnson syndrome - associated with HLA-B*1502 in Asian populations
• Enzyme inducer (CYP3A4) - reduces OCP efficacy, warfarin, other AEDs

Valproate

• Hepatotoxicity (especially in children <2 years on polytherapy)
• Teratogenicity - neural tube defects (spina bifida), contraindicated in pregnancy if possible
• Weight gain, hair loss, tremor
• Thrombocytopenia
• Pancreatitis

Phenobarbital

• Sedation, cognitive impairment
• Dependence; abrupt withdrawal causes seizures
• Enzyme inducer
• Paradoxical hyperactivity in children

Lamotrigine

• Stevens-Johnson syndrome / toxic epidermal necrolysis - especially with rapid titration or when combined with valproate (which doubles lamotrigine levels)
• Dizziness, diplopia, headache
• Relatively safe in pregnancy compared to valproate

Levetiracetam

• Behavioral disturbance: irritability, aggression, mood changes
• Renally excreted - dose adjust in renal failure
• Minimal drug interactions (not a CYP inducer/inhibitor)
• Available IV - useful in acute settings

Ethosuximide

• GI upset (nausea, vomiting)
• Drowsiness, headache
• Only used for absence seizures - does NOT protect against GTC

Topiramate

• Cognitive slowing ("Dopamax"), word-finding difficulty
• Kidney stones (carbonic anhydrase inhibition)
• Weight loss (also used as migraine prophylaxis)
• Hyperthermia, metabolic acidosis

Gabapentin / Pregabalin

• Sedation, dizziness, weight gain
• No hepatic metabolism, no drug interactions
• Renally excreted - dose adjust in renal failure
• Pregabalin also approved for neuropathic pain and anxiety

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Key Drug Interactions

• Valproate + lamotrigine: Valproate inhibits lamotrigine glucuronidation - lamotrigine levels double; start at lower dose to avoid SJS
• Valproate + phenobarbital: Valproate displaces phenobarbital from protein; excess sedation
• Enzyme inducers (phenytoin, carbamazepine, phenobarbital, primidone): Reduce levels of warfarin, oral contraceptives, cyclosporine, and many other drugs
• Antacids: Reduce phenytoin absorption
• Erythromycin: Increases carbamazepine levels (toxic)
• Chloramphenicol: Increases phenytoin and phenobarbital levels

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Renal & Hepatic Dosing Considerations

• Renally excreted (dose-reduce in CKD): Levetiracetam, gabapentin, pregabalin, topiramate, ethosuximide - and these are also dialyzed out (supplement after dialysis)
• Hepatically metabolized: Most older AEDs - monitor more closely in liver failure; check free drug levels in hypoalbuminemia
• In uremia, free phenytoin levels should be targeted (1-2 µg/mL free) as total levels are unreliable

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Skin Reactions

• Maculopapular rash (most common, first month)
• Erythema multiforme
• Stevens-Johnson syndrome (SJS) and Toxic Epidermal Necrolysis (TEN)

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AEDs in Pregnancy

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Recent evidence note: A 2024 systematic review (PMID 39299778) examined paternal exposure to antiseizure medications and offspring outcomes - an emerging area of concern beyond maternal exposure. A 2025 network meta-analysis (PMID 39948216) assessed adjunctive treatment options for pediatric focal epilepsy and may update some of the pediatric drug choices above.

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LONG ANSWER QUESTIONS (15 × 3 = 45 Marks)

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Q1. (15 Marks)

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a) Provisional Diagnosis

• Classic constitutional symptoms (low-grade fever, weight loss, loss of appetite = "B symptoms" of TB)
• Chronic productive cough >3 weeks is the hallmark of PTB
• Age group and duration strongly suggest post-primary (reactivation) TB

• Lung abscess / bronchiectasis
• Carcinoma of bronchus
• Chronic fungal infections (histoplasmosis, aspergillosis)

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b) Laboratory Diagnosis of Pulmonary Tuberculosis

1. Specimen Collection

• Early morning sputum (3 consecutive samples on 3 separate days)
• BAL (bronchoalveolar lavage) if sputum not adequate
• Gastric lavage in children

2. Direct Microscopy (Most rapid, widely used)

• Smear fixed on slide, stained with carbol fuchsin (hot stain)
• Decolorized with 20% H2SO4 (acid-fast decolorizer)
• Counterstained with methylene blue
• AFB appear red rods on blue background
• Sensitivity: 5,000-10,000 bacilli/mL needed for positive smear

• More sensitive than ZN
• Bacilli fluoresce yellow-orange on dark background
• Preferred for high-volume screening
• Positive fluorochrome smears must be confirmed by ZN

3. Culture (Gold Standard)

• Lowenstein-Jensen (LJ) medium - egg-based, results in 4-8 weeks
• Colonies: rough, dry, cream-colored, "cauliflower" or "breadcrumb" appearance
• Growth slower at 37°C, best at 35-37°C
• Middlebrook 7H10/7H11 - agar-based, transparent, slightly faster

• BACTEC 460 (radiometric), MGIT 960 (fluorometric)
• Detects growth in 1-3 weeks (much faster)
• MGIT 960 = most widely used liquid system today
• Method of choice for drug susceptibility testing

4. Identification of M. tuberculosis

• Niacin test: Positive (M. tb accumulates niacin)
• Nitrate reductase test: Positive
• Catalase test: Heat-labile (inactivated at 68°C) - distinguishes from NTM
• Pyrazinamidase: Positive
• Tween 80 hydrolysis: Negative

5. Molecular/Nucleic Acid Amplification Tests (NAAT)

• Xpert MTB/RIF (GeneXpert): WHO-endorsed, detects MTB AND rifampicin resistance simultaneously within 2 hours; sensitivity ~89%, specificity ~99%
• Line Probe Assay (LPA / GenoType MTBDRplus): Detects mutations in rpoB (RIF resistance) and katG/inhA (INH resistance) directly from smear-positive sputum
• TrueNAT MTB: Point-of-care chip-based PCR, approved by WHO

6. Immunological Tests (Indirect diagnosis)

• Tuberculin Skin Test (Mantoux test): Intradermal injection of 5 TU PPD (purified protein derivative); induration ≥10 mm at 48-72 hrs = positive (cut-off varies with population)
• IGRA (Interferon-Gamma Release Assay): QuantiFERON-TB Gold In-Tube, T-SPOT.TB; detects IFN-γ release by sensitized T cells in response to ESAT-6/CFP-10 antigens; more specific than Mantoux, unaffected by BCG

7. Histopathology (for extrapulmonary TB)

• Caseating granuloma with central caseous necrosis
• Surrounded by epithelioid cells, Langhans giant cells, lymphocytes

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c) Drug Susceptibility Testing (DST) and Drug Resistance Patterns

Drug Susceptibility Testing Methods

• Proportion method on LJ medium or Middlebrook 7H10 (takes 4-8 weeks)
• Drug incorporated at critical concentration; resistant if growth >1% of control
• Gold standard for DST but slow

• Faster (7-14 days)
• Approved for first-line drugs (INH, RIF, EMB, PZA, SM)

• Xpert MTB/RIF: Detects rpoB mutations → rifampicin resistance (proxy for MDR-TB)
• Line Probe Assay (LPA):
  • MTBDRplus: Detects INH and RIF resistance (first-line)
  • MTBDRsl: Detects fluoroquinolone and aminoglycoside resistance (second-line)
• Results in hours-to-days

Drug Resistance Definitions

Current Resistance Patterns and Treatment

• Preferred regimen: BPaL (Bedaquiline + Pretomanid + Linezolid) - 6-month "BPaLM" regimen now preferred
• Longer regimen (18-20 months): Bedaquiline + Moxifloxacin + Linezolid + Clofazimine
• Key new drugs: Bedaquiline (ATP synthase inhibitor), Delamanid (nitroimidazole), Pretomanid

• BPaL or BPaLM regimen
• Individualized therapy based on full DST

• WHO estimates ~450,000 new MDR/RIF-TB cases annually
• South-East Asia (India, China) and Eastern Europe have highest MDR-TB rates
• Treatment success for MDR-TB ~60%, for XDR-TB ~40%

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d) Pathogenesis of Tuberculosis

Step 1: Inhalation and Initial Infection

• Droplet nuclei (1-5 μm) containing 1-3 bacilli reach the alveoli and are ingested by alveolar macrophages
• M. tuberculosis survives within macrophages by inhibiting phagosome-lysosome fusion (via lipoarabinomannan - LAM)
• Initial multiplication is unchecked for 2-4 weeks (pre-allergy phase)

Step 2: Formation of Primary Complex (Ghon's Complex)

• Bacilli multiply locally → Ghon's focus (subpleural area, usually upper portion of lower lobe or lower portion of upper lobe)
• Spread via lymphatics to hilar lymph nodes → Ghon's complex = Ghon focus + hilar lymphadenopathy
• Bacilli disseminate hematogenously in this early phase (seeding of apices, meninges, kidneys, bones)

Step 3: Development of Immunity (Cell-Mediated Immunity)

• T cells (CD4+ Th1 cells) recognize mycobacterial antigens presented by macrophages
• Th1 cells release IFN-γ and TNF-α
• IFN-γ activates macrophages → enhanced killing via reactive oxygen/nitrogen species
• Tuberculin hypersensitivity develops at 4-8 weeks (Type IV / delayed hypersensitivity)

Step 4: Granuloma Formation

• Activated macrophages transform into epithelioid cells and fuse to form Langhans giant cells
• Granuloma = central caseous necrosis + epithelioid cells + Langhans giant cells + lymphocytes
• Caseous necrosis: unique to TB - hypoxic, low pH, fatty acid-rich environment; bacteriostatic but bacilli can survive
• In immunocompetent hosts: granuloma contains infection → latent TB

Step 5: Outcomes

• Due to waning immunity (HIV, malnutrition, steroid use, diabetes, aging)
• Apical segments preferred (high pO2, poor lymphatic drainage)
• Caseous material liquefies → cavity formation
• Liquefied caseum discharged into bronchus → cavitation, open TB, highly infectious sputum

• Cord factor (trehalose dimycolate): Toxic to mitochondria, inhibits leucocyte migration, causes serpentine growth
• Sulfatides: Inhibit phagosome-lysosome fusion
• LAM (Lipoarabinomannan): Scavenges reactive oxygen intermediates
• Wax D: Adjuvant property, stimulates cell-mediated immunity
• Cell wall mycolic acids: Acid-fastness, resistance to desiccation, drying

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Q2. (15 Marks)

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Bacterial Agents Causing Genital Ulcer Disease

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Pathogenesis of Primary Syphilis

Organism

Mechanism of Pathogenesis

• Transmission through sexual contact (risk ~30% per exposure)
• Treponemes enter through microscopic abrasions in mucosa or intact squamous mucosa of genitalia, rectum, mouth
• Attach to host cells via fibronectin-binding adhesins and outer membrane proteins

• Treponemes multiply at entry site; incubation period = 10-90 days (average 21 days)
• Host immune response activates macrophages and T cells
• Endarteritis obliterans: Treponemes surround and invade blood vessel walls → inflammatory infiltrate (plasma cells + lymphocytes) → endothelial swelling → obliterative endarteritis with reduced blood supply

• Hallmark of primary syphilis
• Begins as a papule at entry site → ulcerates
• Hunterian chancre: Single, round/oval, painless (due to perineural invasion causing hypoesthesia), indurated ("cartilaginous" feel), clean base, raised edges, serous exudate
• Most common sites: glans penis, labia, cervix, perianal, oral
• Regional lymphadenopathy: painless, non-tender, firm, rubbery (reactive lymphadenitis)
• Chancre heals spontaneously in 3-8 weeks even without treatment

• Within hours of infection, treponemes disseminate via lymphatics and bloodstream
• Seeds multiple organs: skin, liver, CNS, eyes, bones
• Allows secondary syphilis to develop 4-10 weeks later

• Very low number of outer membrane proteins (only ~1% of E. coli)
• Rapid antigenic variation of surface proteins
• Low organism burden in each tissue - below "critical antigenic mass"
• These factors allow persistence despite host antibody and T-cell responses

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Laboratory Diagnosis of Primary Syphilis

1. Direct Detection (Demonstration of the organism)

• Exudate from the base of the ulcer collected on a glass slide
• Examined immediately under dark-field microscope
• T. pallidum appears as bright, white, corkscrew spirochetes against dark background with characteristic motility
• Limitation: Cannot distinguish from commensal oral treponemes; do NOT use for oral lesions

• Smear from ulcer, stained with fluorescein-labelled anti-T. pallidum antibody
• More specific than darkfield; can be used on fixed material
• No live organism needed

• Most sensitive and specific for direct detection
• Can be used on ulcer swab, tissue biopsy, CSF
• Differentiates T. pallidum from other treponemes

• Used on tissue sections (biopsy)
• Treponemes appear as black spirochetes against yellow background

2. Serological Tests (Indirect - detect host antibody response)

• Both detect reagin antibodies (IgG and IgM against cardiolipin released from damaged host cells)
• Results reported as titres (e.g., 1:8, 1:32)
• Prozone phenomenon: False-negative at high antibody titres; dilute specimen to detect
• False positives in: SLE, pregnancy, malaria, viral infections, TB, IV drug use
• Used to monitor treatment response (titre should fall 4-fold after treatment)

• Highly sensitive and specific for treponemal infection
• Remain positive for life (even after treatment) - cannot be used to monitor treatment
• FTA-ABS IgM: Positive earliest; important in congenital syphilis

3. Histopathology

• Biopsy of chancre: plasma cell infiltrate + obliterative endarteritis (diagnostic appearance)

Summary: Algorithm for Lab Diagnosis of Primary Syphilis

Genital ulcer
    ↓
Dark-field microscopy (immediate)
    ↓
Serology: RPR / VDRL (screening)
    ↓ if reactive
Confirm with TPHA / FTA-ABS
    ↓
PCR on swab (if dark-field equivocal)

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Q3. (15 Marks)

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Morphology

• Gram-positive, non-sporing, non-motile, non-capsulate rods
• Slender, slightly curved rods, club-shaped or tapered ends (Greek: koryne = club)
• Size: 1-8 μm × 0.3-0.8 μm
• Arranged in palisade formation (like a picket fence / "V" and "L" shapes), sometimes in clusters like "Chinese letters"
• Metachromatic granules (Babes-Ernst granules / volutin granules):
  • Composed of polymetaphosphate
  • Stain metachromatically with Albert's stain (toluidine blue / malachite green) - granules appear bluish-black against green-blue organism body
  • Demonstrated also with Loeffler's methylene blue (granules appear dark blue against light blue background)
  • Reflect club-shaped ends and are a hallmark feature

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Cultural Characteristics

• Aerobe/facultative anaerobe
• Growth at 37°C, pH 7.2-7.4
• Requires L-cystine and L-methionine for growth

• Primary isolation medium
• Coagulated horse/ox serum + glucose broth
• Colonies grow in 12-18 hours (much faster than other organisms)
• Metachromatic granules prominent on this medium
• Appearance: cream/grey, glistening

• Hoyle's medium (blood agar + potassium tellurite): Colonies appear grey-black due to reduction of potassium tellurite to tellurium
• Tinsdale's medium: Black colonies with brown halo (due to H2S production) - distinguishes C. diphtheriae from other corynebacteria
• McLeod's medium (blood agar + potassium tellurite): Similar

• Grey, non-haemolytic colonies (C. diphtheriae is usually non-haemolytic)

• Ferments glucose and maltose (not sucrose or lactose)
• Reduces nitrate to nitrite
• Urease negative (distinguishes from C. ulcerans)
• Cystinase positive (shown on Tinsdale medium)

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Pathogenicity

Toxin Production

• Only lysogenic strains (carrying bacteriophage β/corynephage) produce diphtheria toxin
• The tox gene is encoded in the phage genome

Diphtheria Toxin - Mechanism of Action

1. Toxin = A-B toxin (two fragments)
2. Fragment B (binding): Binds to host cell receptor (heparin-binding EGF-like growth factor receptor - HB-EGF), present on heart, nerve, adrenal, kidney cells
3. Toxin enters cell by receptor-mediated endocytosis
4. In acidified endosome, Fragment A translocates into cytoplasm
5. Fragment A (active): Catalyzes ADP-ribosylation of Elongation Factor-2 (EF-2)
6. Inactivated EF-2 cannot function in protein synthesis → irreversible inhibition of protein synthesis → cell death
7. One molecule of toxin can kill a cell (extremely potent)
8. 0.1 μg/kg body weight = lethal dose in guinea pigs

Clinical Disease

• Proliferates on upper respiratory tract mucosa (pharynx, tonsils, larynx, trachea)
• Exotoxin causes necrosis of epithelium
• Fibrinopurulent exudate coagulates on necrotic surface → pseudomembrane (tough, grayish-white, firmly adherent - bleeds on removal)
• Neck edema + lymphadenopathy → "bull neck" appearance

• Myocarditis (2nd week): Arrhythmias, heart block, cardiac failure - most common cause of death
• Polyneuropathy:
  • Palatal palsy (early, 3rd week): Nasal voice, regurgitation
  • Oculomotor paralysis, facial palsy
  • Late peripheral motor neuropathy (6-8 weeks)
• Adrenal hemorrhagic necrosis
• Renal tubular necrosis

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Laboratory Diagnosis

1. Specimen

• Swab from throat and nasopharynx (from beneath the pseudomembrane edge)
• Both throat AND nose swabs should be taken
• If membrane present, piece of membrane also collected

2. Direct Microscopy

• Gram stain: Gram-positive rods in typical arrangements
• Albert's stain: Metachromatic granules (bluish-black) in green rods - characteristic appearance
• Loeffler's methylene blue: Dark blue granules

3. Culture

• Loeffler's serum slope (12-18 hrs): Creamy colonies with granules
• Tellurite blood agar (Hoyle's/Tinsdale): Black colonies with halo (selective + differential)
• Subculture to blood agar for biochemical identification

4. Identification

• Colonial morphology, Gram stain, Albert's stain
• Biochemical reactions (fermentation reactions, urease, nitrate, cystinase)
• MALDI-TOF MS (modern rapid identification)

5. Toxigenicity Testing (Most important step)

• Strip of filter paper soaked in antitoxin placed on tellurite blood agar plate
• Specimen streaked at right angles to the strip
• Both organism and known toxigenic control streak
• Toxin + antitoxin diffuse toward each other in agar
• Lines of precipitation (precipitin lines) at 45° to the strip = toxigenic
• Takes 24-48 hours

• Rapid, sensitive, detects tox gene directly
• Does NOT confirm toxin production (gene present but may not be expressed)

• Toxin kills VERO cells; neutralized by antitoxin

• Classic in vivo test; rarely used now

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Prevention

1. Active Immunization (Most important)

• Diphtheria Toxoid (formalin-treated toxin): Stimulates production of antitoxin
• Incorporated into:
  • DPT vaccine (Diphtheria-Pertussis-Tetanus): 6 weeks, 10 weeks, 14 weeks, booster at 18 months
  • DT (for children if pertussis contraindicated)
  • Td (adult-type, reduced antigen content): Booster every 10 years
  • Tdap: Tetanus, reduced diphtheria, acellular pertussis

2. Passive Immunization

• Diphtheria Antitoxin (DAT): 10,000-100,000 units IM/IV for clinical cases
• Must be given early - antitoxin cannot neutralize toxin already bound to tissues

3. Antibiotic Prophylaxis

• Erythromycin or benzylpenicillin for close contacts
• Eradicate carrier state

4. Schick Test (historical)

• Tests for susceptibility to diphtheria
• Intradermal injection of 1/50 MLD of diphtheria toxin
• Positive (susceptible): area of redness/induration at 24-48 hrs (no antitoxin present)
• Negative (immune): No reaction

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SHORT NOTES (5 × 7 = 35 Marks)

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Q4. Laboratory Diagnosis of Enteric Fever (5 Marks)

Specimens and Timing

1. Blood Culture (Most important in early disease)

• Volume: 10-15 mL blood (80-90% sensitive)
• Media: Brain Heart Infusion broth, BACTEC
• Blood:broth ratio = 1:10 (dilutes inhibitory substances in blood)
• Subculture to MacConkey agar + blood agar
• S. Typhi colonies: pale/colourless on MacConkey (non-lactose fermenter), H2S on XLD agar

2. Bone Marrow Culture

• Most sensitive (~95%), gold standard
• Positive even if antibiotics started
• Invasive; reserved for difficult/treated cases

3. Stool Culture (Week 3 onwards)

• MacConkey agar: pale/colourless colonies
• XLD (Xylose Lysine Deoxycholate) agar: black-centred colonies with H2S
• Selenite F broth or tetrathionate broth used as enrichment

4. Urine Culture

• Positive in 3rd week
• Less commonly used

5. Widal Test (see Q5 below)

6. Newer Serological Tests

• Typhidot (dot EIA): Detects IgM and IgG against 50 kDa outer membrane protein of S. Typhi; positive from day 2-3 of fever
• Typhidot-M: IgM only; detects acute infection
• Tubex test: Semi-quantitative inhibition assay detecting anti-O9 IgM antibodies; result in 2 minutes

7. Molecular Tests

• PCR targeting the flagellin gene (fliC) or Vi antigen gene (viaB)
• High sensitivity and specificity; not yet widely available

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Q5. Widal Test and Its Interpretation (5 Marks)

Principle

• S. Typhi O (somatic/LPS antigen)
• S. Typhi H (flagellar antigen)
• S. Paratyphi A H, B H, C H

Method

Interpretation

O vs. H Agglutinins

Limitations and False Results

• Previous typhoid vaccination (high H titre)
• Other Salmonella infections (Weil-Felix cross-reactions)
• Other infections: malaria, brucellosis, liver disease, SBE
• Previous typhoid infection (anamnestic/Booster response)
• Technical errors (rough colonies used as antigen)

• Early infection (antibodies not yet risen)
• Early antibiotic treatment
• Immunosuppression
• Very virulent organism with rapid fatal course

Clinical significance rules:

1. Rising titre > single titre
2. O titre is more diagnostic than H titre
3. Both O AND H titres positive is more significant
4. H titre alone may indicate past infection or vaccination
5. Widal is not recommended as sole diagnostic test; must correlate with clinical picture

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Q6. Pathogenesis and Non-Suppurative Complications of Streptococcus pyogenes (5 Marks)

Pathogenesis

• Entry via throat (pharyngitis) or skin (impetigo, cellulitis)
• M protein enables binding to pharyngeal epithelium
• Spreading factors facilitate local tissue invasion
• Pyrogenic exotoxins trigger massive cytokine release

Non-Suppurative Complications

1. Acute Rheumatic Fever (ARF) - after throat infection only

• Antibodies against streptococcal M protein cross-react with cardiac myosin and sarcolemmal antigens
• CD4+ T cells also cross-react with cardiac antigens
• Inflammation of heart (carditis), joints (arthritis), and other tissues

• Major: Carditis, Polyarthritis, Chorea, Erythema marginatum, Subcutaneous nodules
• Minor: Fever, raised ESR/CRP, prolonged PR interval

2. Acute Post-Streptococcal Glomerulonephritis (APSGN) - after throat OR skin infection

• Streptococcal antigens + host antibodies → immune complexes
• Deposits in subepithelial space ("humps") → complement activation → glomerular injury
• "Lumpy-bumpy" deposits on immunofluorescence
• "Hump-shaped deposits" on electron microscopy

Key Differences

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Q7. Laboratory Diagnosis of Gas Gangrene (5 Marks)

Specimens

• Wound exudate, tissue biopsy, blood (for bacteremia)
• Collected anaerobically (use anaerobic transport media/swabs)

1. Direct Microscopy (Rapid, highly informative)

• Gram stain of wound exudate:
  • Large, boxcar-shaped Gram-positive rods
  • Absence of leucocytes (pus cells) - alpha toxin destroys PMNs (this is pathognomonic)
  • Oval, subterminal spores (but spores often absent in tissue specimens as sporulation requires O2)
  • May see Gram-negative rods if mixed infection

2. Anaerobic Culture (Definitive)

• Inoculate onto:
  • Blood agar (anaerobic incubation): C. perfringens = double zone of haemolysis (inner zone complete haemolysis by theta toxin + outer zone incomplete by alpha toxin)
  • Egg yolk agar (EYA): Lecithinase (Nagler's reaction) - zone of opacity around colonies, inhibited by C. perfringens antitoxin on one half of plate (Nagler's reaction - specific for C. perfringens)
  • Robertson's cooked meat medium (RCMM): Enrichment; turns pink (not black = differentiates from C. sporogenes)
  • Neomycin blood agar: Selective (inhibits non-spore-formers and many other organisms)
• Incubate at 37°C under anaerobic conditions (anaerobic jar / MGPS / glove box)
• C. perfringens grows rapidly (4-6 hours), large irregular spreading colonies

3. Identification of C. perfringens

• Large Gram-positive rods, no motility (non-motile - distinguishes from other clostridia)
• Double zone haemolysis on blood agar
• Stormy clot/stormy fermentation in milk media: clot is torn by gas
• Lecithinase positive (Nagler reaction)
• Reverse CAMP test: Inhibition of S. aureus haemolysis at junction
• Biochemical: Ferments glucose, lactose, sucrose, maltose; H2S positive; nitrate positive

4. Molecular Tests

• PCR for alpha toxin gene (pfoA) and other toxin genes
• Useful for direct detection in tissue without culture

5. Imaging (Clinical)

• X-ray: Gas in tissues (radiolucent streaks along fascial planes)
• CT scan: More sensitive for gas localization

6. Toxin Detection

• ELISA for alpha toxin (lecithinase C)
• Rarely required for clinical diagnosis

Histopathology

• Tissue biopsy: myonecrosis with large Gram-positive rods, minimal inflammatory cells
• Gas bubbles within muscle fibers

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Q8. Morphology and Cultural Characteristics of Vibrio cholerae (5 Marks)

Morphology

• Curved, comma-shaped Gram-negative rod (like a comma or half-moon)
• Size: 1.5-3 μm × 0.2-0.4 μm
• Single polar flagellum (monotrichous) - responsible for darting/shooting-star motility
• In old cultures, can appear straight
• Non-capsulate (except V. cholerae O139)
• Non-sporing
• Hanging drop preparation: Darting/shooting-star motility - highly characteristic
• Motility inhibited by specific O1 antiserum → string test (immobilization)

Cultural Characteristics

Growth Requirements

• Aerobe/facultative anaerobe
• Halophilic (salt-loving) - grows best in 0.5-3% NaCl
• Temperature: 18-37°C (optimal 37°C)
• Alkaliphilic - grows at pH 8.5-9.0 (optimal), survives at pH 9-10 (most other organisms die)

Selective Media

• pH 8.6
• V. cholerae grows rapidly and forms a pellicle (surface film) at top of broth within 6-8 hours
• This pellicle is subcultured for further isolation
• Reason: Alkaline pH kills most enteric organisms, allowing selective growth

• Most widely used selective medium for Vibrio species
• V. cholerae = Yellow colonies (sucrose fermenter; lowers pH, turns pH indicator yellow)
• V. parahaemolyticus = Blue-green colonies (non-sucrose fermenter)
• Contains bile salts and citrate to inhibit other organisms

• V. cholerae = Translucent colonies with dark grey centre (tellurite reduction)

• V. cholerae = Pale/non-lactose fermenting colonies

Serogroups and Biotypes

• Based on O (somatic) antigen - >200 serogroups identified
• Only O1 and O139 cause epidemic cholera
• O1 serotypes: Inaba (A, C), Ogawa (A, B), Hikojima (A, B, C)
• O1 biotypes: Classical (historical), El Tor (current pandemic strain)

Biochemical Reactions

• Oxidase: Positive (key feature - distinguishes from Enterobacteriaceae)
• Catalase: Positive
• Indole: Positive
• VP (Voges-Proskauer): Positive (El Tor)
• Nitrate reduction: Positive
• String test (bile solubility test with 0.5% sodium deoxycholate): Positive (bacteria form string = positive)
• Ferments sucrose, mannose; does NOT ferment arabinose

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Q9. Atypical Mycobacteria (NTM) - Classification and Clinical Significance (5 Marks)

Introduction

Runyon Classification (Based on growth rate and pigmentation)

Clinical Diseases

• Resembles pulmonary TB clinically and radiologically
• Most common cause: M. avium complex (MAC) and M. kansasii
• Two patterns:
  • Fibrocavitary (upper lobe cavities, like TB) - older men, smokers
  • Nodular bronchiectatic (right middle lobe/lingula) - postmenopausal women ("Lady Windermere syndrome")
• Diagnosis: ≥2 positive sputum cultures OR 1 positive BAL culture OR positive tissue biopsy

• Occurs when CD4 count <50 cells/μL
• Fever, night sweats, drenching sweats, hepatosplenomegaly, anaemia
• Diagnosis: Blood culture (BACTEC); AFB stain and culture of bone marrow/liver biopsy

• M. scrofulaceum, M. avium - cervical lymphadenitis in children (ages 1-5)
• Unilateral, painless, no systemic illness; tuberculin test weakly positive
• Treatment: surgical excision (not antituberculous drugs)

• M. marinum: "Swimming pool granuloma" or "fish tank granuloma" - nodular lesion on hands/elbows after exposure to contaminated water/fish tanks
• M. ulcerans: "Buruli ulcer" - painless skin ulcer with undermined edges; toxin (mycolactone) causes extensive necrosis; commonest in West Africa
• M. fortuitum/chelonae: Post-injection/post-surgical abscesses, catheter site infections

• M. marinum, M. kansasii, M. avium can cause osteomyelitis and septic arthritis

Key Distinguishing Features from M. tuberculosis

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Q10. Anaerobic Culture Methods and Their Applications (5 Marks)

Why Anaerobic Culture?

Specimen Collection (Critical first step)

• Must be collected and transported in anaerobic conditions
• Acceptable specimens: pus (aspirated), tissue biopsy, blood, CSF, deep wound swabs (NOT surface swabs)
• Transport media: Thioglycollate broth, Port-A-Cul system, anaerobic swab transport system
• Process specimens as quickly as possible (<30 min)

Methods of Anaerobic Incubation

1. Anaerobic Jar (McIntosh-Fildes jar)

• Gas pak: Commercial sachet containing sodium borohydride + sodium bicarbonate + citric acid + water
• When water added → H2 + CO2 released
• Palladium catalyst converts H2 + residual O2 → H2O
• Final atmosphere: ~80% N2, 10% H2, 10% CO2, O2 < 1%
• Indicator strip (methylene blue): Colourless = anaerobic; Blue = O2 present
• Simple, widely used in most labs
• Limitation: Cannot open jar to add/remove plates during incubation

2. Anaerobic Cabinet / Glove Box (Gold standard)

• Large chamber maintained at <0.5 ppm O2
• Passes specimens through an airlock (exchange lock)
• All manipulations done through gloves fixed to front wall
• Best for strict anaerobes; allows continuous anaerobic processing
• Expensive; used in reference labs

3. Candle Jar

• NOT truly anaerobic - only reduces O2 to ~14% and adds 3-5% CO2
• Used for capnophilic organisms (Neisseria, Haemophilus)
• NOT suitable for obligate anaerobes

4. Robertson's Cooked Meat Medium (RCMM)

• Liquid enrichment medium: Minced cooked ox heart muscle in broth (pH 7.5)
• Meat particles absorb O2 and act as reducing agents
• Used for enrichment and maintenance of anaerobes
• C. perfringens: pinkish, gas production
• C. sporogenes: blackening, putrid odour

5. Thioglycollate Broth

• Sodium thioglycollate acts as reducing agent
• O2 diffuses into top layer; anaerobes grow at bottom
• Useful as transport medium and broth enrichment

6. GasPak Pouch / Anaero-Pak (Modern compact systems)

• Pre-filled pouches that generate anaerobic atmosphere
• Used for smaller labs; easier to handle

Media Used for Anaerobic Culture

Applications of Anaerobic Culture

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