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MICROBIOLOGY TEST - COMPLETE ANSWERS

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ESSAYS

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1. RABIES

Classification & Structure

• Family: Rhabdoviridae; Genus: Lyssavirus
• Bullet-shaped enveloped virus, 180 x 75 nm
• Single-stranded negative-sense RNA genome
• Nucleocapsid (N), Matrix (M), G glycoprotein (surface spike), RNA polymerase (L), Phosphoprotein (P)
• G glycoprotein: key antigen - induces virus-neutralizing antibodies; binds acetylcholine receptor (AchR) and neural cell adhesion molecule (NCAM) on host cells

Pathogenesis

1. Entry: Virus introduced via bite/wound/mucous membrane contamination from saliva of infected animal
2. Local replication: Virus replicates in muscle cells at site of inoculation
3. Peripheral nervous system invasion: Virus binds acetylcholine receptors at neuromuscular junctions and enters peripheral nerves - travels retrograde (centripetally) via axoplasmic flow
4. CNS invasion: Virus ascends to spinal cord and brain - preferentially infects neurons; replicates in hippocampus, basal ganglia, brainstem, cerebellum
5. Centrifugal spread: From CNS, virus spreads outward along peripheral nerves to salivary glands, skin, cornea, and other organs
6. Negri bodies: Eosinophilic intracytoplasmic inclusion bodies form in infected neurons (Purkinje cells of cerebellum, pyramidal cells of hippocampus - Sommer's sector) - pathognomonic
7. Incubation period: 10 days to 1 year (average 1-3 months); shorter for bites on face/head, longer for leg bites

Clinical Manifestations

1. Incubation (10 days - 1 year): No symptoms
2. Prodrome (2-10 days): Fever, headache, malaise, anorexia, nausea, pain/paresthesia at bite site (characteristic early sign)
3. Acute Neurologic Stage (Encephalitic phase):
   • Furious (encephalitic) type (80%): Hydrophobia (laryngeal spasm on attempting to swallow water), aerophobia, hypersalivation, hyperthermia, agitation, hallucinations, autonomic instability
   • Dumb (paralytic) type (20%): Ascending flaccid paralysis (Guillain-Barre-like), less dramatic; often misdiagnosed
4. Coma and Death: Follows within days of neurologic stage; almost uniformly fatal once symptoms appear

Laboratory Diagnosis

• Brain biopsy: DFA (gold standard), RT-PCR, histopathology showing Negri bodies (H&E or Seller's stain)
• Mouse inoculation: intracerebral inoculation of brain suspension
• Cell culture (Murine neuroblastoma cells)

Treatment & Prevention

• No specific antiviral treatment for established rabies - supportive care only
• Post-exposure prophylaxis (PEP):
  1. Thorough wound washing with soap and water, antiseptic
  2. Rabies Immune Globulin (RIG): half around wound, half IM - provides passive immunity immediately
  3. Inactivated rabies vaccine (HDCV or PCEC): Days 0, 3, 7, 14 (4 doses IM)
• Pre-exposure prophylaxis: 3 doses of vaccine on days 0, 7, 21-28 (for vets, lab workers, travelers)

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2. ORGANISMS CAUSING GENITAL ULCER + SYPHILIS

Organisms Causing Genital Ulcer (Mnemonic: SHTCH)

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SYPHILIS - Detailed

• Slim spirochete, 5-15 μm long, corkscrew shape with regular spirals
• Not culturable in vitro
• Stained by immunofluorescence, darkfield microscopy, silver impregnation (Warthin-Starry, Levaditi)
• No LPS; minimum surface proteins (immune evasion)
• Transmitted by sexual contact, vertical (congenital), rarely by blood transfusion

Clinical Manifestations

• Chancre: Single (usually), painless, indurated ulcer with clean base at site of inoculation (genitalia, lips, rectum)
• Non-tender regional lymphadenopathy (rubbery, discrete)
• Chancre heals spontaneously in 3-8 weeks

• Symmetric maculopapular rash - trunk, extremities, PALMS AND SOLES (distinctive)
• Generalized non-tender lymphadenopathy
• Condylomata lata: flat warty papules in moist areas (genitals, perianal) - highly infectious
• Mucous patches in mouth
• Fever, malaise, headache
• Alopecia ("moth-eaten" pattern)
• All lesions teeming with spirochetes

• No clinical signs; only serologic evidence
• Early latent (<1 year): infectious relapses possible
• Late latent (>1 year): not infectious except via blood/congenital

• Gummatous syphilis: Granulomatous lesions (gummata) in skin, bone, liver, testis - destructive but not infectious
• Cardiovascular syphilis: Aortitis (ascending aorta), aortic regurgitation, saccular aortic aneurysm (syphilitic "tree-bark" intima)
• Neurosyphilis:
  • Meningitis, meningovascular disease
  • Tabes dorsalis: demyelination of posterior columns/dorsal roots - ataxia, wide-based gait, foot slap, Charcot's joints, Argyll Robertson pupil (accommodates but doesn't react to light)
  • General paresis (GPI): dementia, psychosis
  • PARESIS mnemonic: Personality, Affect, Reflexes (hyperreflexia), Eye (Argyll Robertson), Sensory, Intellect, Speech

• Early (<2 years): Snuffles (mucopurulent rhinitis), maculopapular rash, hepatosplenomegaly, periostitis, condylomata lata
• Late (>2 years): Interstitial keratitis, Hutchinson's teeth (notched upper incisors), Clutton's joints, saddle-nose deformity, saber shins (Hutchinson's triad)

Laboratory Diagnosis

• Darkfield microscopy: Exudate from chancre/condylomata - demonstrates motile spirochetes (corkscrew motility with right-angle flexions); gold standard for primary syphilis
• Direct fluorescent antibody (DFA-TP): Smear from lesion; uses fluorescent anti-T. pallidum antibody; can be used on fixed material
• PCR: Highly sensitive and specific; useful when darkfield unavailable

• VDRL (Venereal Disease Research Laboratory): flocculation test; cardiolipin-lecithin-cholesterol antigen; cheap, simple, titers reflect disease activity (used for monitoring treatment response)
• RPR (Rapid Plasma Reagin): Similar to VDRL; uses charcoal particles; can be done on unheated serum
• Become positive 1-4 weeks after chancre
• False positives: SLE, antiphospholipid syndrome, malaria, pregnancy, TB, leprosy, viral infections

• FTA-ABS (Fluorescent Treponemal Antibody Absorption): Most sensitive; first to become positive; reference standard
• TPHA/TPPA (T. pallidum Hemagglutination/Particle Agglutination): Specific, simple
• MHA-TP: Microhemagglutination
• ELISA/CIA: Automated; used in modern labs
• Treponemal tests remain positive even after treatment (scar serology)

Treatment

• Primary, Secondary, Early Latent (<1 year): Benzathine Penicillin G 2.4 million units IM, single dose
• Late Latent, Tertiary (non-neuro): Benzathine Penicillin G 2.4 MU IM weekly x 3 weeks
• Neurosyphilis: Aqueous crystalline Penicillin G 18-24 million units/day IV x 10-14 days
• Penicillin allergy: Doxycycline 100 mg BD x 14 days (not in pregnancy); Ceftriaxone
• Congenital Syphilis: Aqueous Penicillin G IV x 10 days
• Jarisch-Herxheimer Reaction: Fever, rigors, headache 2-8 hrs after first dose of penicillin (due to cytokine release from dying spirochetes) - treat with antipyretics

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3. INFLUENZA VIRUS - Pathogenesis, Antigenic Variations, Complications, Lab Diagnosis

Classification & Structure

• Family: Orthomyxoviridae
• Types: A, B, C (Type A causes pandemics, B causes epidemics, C causes mild illness)
• Enveloped, segmented negative-sense RNA (8 segments in A & B, 7 in C)
• Key surface antigens:
  • Hemagglutinin (HA): 18 subtypes; mediates attachment to sialic acid receptors; target for neutralizing antibodies; HA1 - receptor binding, HA2 - membrane fusion
  • Neuraminidase (NA): 11 subtypes; cleaves sialic acid to release new virions; anti-NA antibodies limit spread
• NP (nucleoprotein) and M (matrix) proteins: type-specific antigens (differentiate A, B, C)
• M2 protein: ion channel - target of amantadine

Pathogenesis

1. Transmission: Droplet inhalation (respiratory route)
2. Attachment: HA binds sialic acid residues on epithelial cells of upper and lower respiratory tract
3. Entry & Uncoating: Receptor-mediated endocytosis; low pH in endosome triggers conformational change in HA2 (fusion peptide exposed) - viral envelope fuses with endosomal membrane; M2 ion channel acidifies virion interior - RNA released into cytoplasm
4. Replication: Viral RNA transcribed in nucleus (unique for RNA virus); mRNA exported to cytoplasm for protein synthesis; progeny genomes assembled
5. Release: NA cleaves sialic acid from cell surface; new virions bud out
6. Tissue damage: Viral cytopathic effect on ciliated epithelium; loss of mucociliary clearance; cytokine storm (IFN-α, IFN-β, TNF-α, IL-6) causes systemic symptoms (fever, myalgia)
7. Innate immune evasion: NS1 protein inhibits interferon production

Antigenic Variations

• Continuous point mutations in HA and NA genes during replication (error-prone RNA polymerase has no proofreading)
• Results in amino acid changes at 5 exposed antigenic sites on HA
• Gradual accumulation of mutations; need 2+ mutations for epidemiological significance
• Existing antibodies partially protect - hence annual flu vaccine is reformulated
• Occurs in Influenza A AND B

• Sudden, drastic change in HA and/or NA surface proteins
• Mechanism: Genetic Reassortment - when a host (typically pig, "mixing vessel") is coinfected with human and avian influenza A viruses, their segmented RNA genomes can mix, producing a novel hybrid virus with entirely new HA/NA combination
• Results in a new subtype that the human population has NO prior immunity to
• Can cause global pandemics with very high mortality
• Occurs ONLY in Influenza A (because only Influenza A has animal reservoirs)

Complications

• Primary viral pneumonia: Most severe; bilateral interstitial pneumonitis; rare but high mortality
• Secondary bacterial pneumonia: Most common complication; due to loss of mucociliary clearance; organisms: S. aureus (most dangerous, produces protease cleaving HA), S. pneumoniae, H. influenzae
• Combined viral-bacterial pneumonia: 3x more common than primary viral pneumonia
• ARDS (Acute Respiratory Distress Syndrome)

• Reye Syndrome: Acute encephalopathy + fatty liver in children/adolescents (2-16 yrs); associated with salicylate use; mortality 10-40%; associated with Influenza B > A, and VZV
• Myocarditis, pericarditis
• Encephalitis
• Guillain-Barre Syndrome (rare)
• Myositis (particularly in children)
• Otitis media (in children)

Laboratory Diagnosis

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SHORT NOTES

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4. LABORATORY DIAGNOSIS OF MENINGITIS

Specimen: CSF (Lumbar puncture - L3/L4 or L4/L5)

1. Gram Stain:
   • N. meningitidis: Gram-negative diplococci (intracellular)
   • S. pneumoniae: Gram-positive diplococci (lancet-shaped)
   • H. influenzae: Gram-negative coccobacilli
   • L. monocytogenes: Gram-positive bacilli
   • E. coli: Gram-negative bacilli (neonates)
2. India Ink Preparation: Capsule of Cryptococcus neoformans appears as clear halo against black background
3. Ziehl-Neelsen (ZN) Stain / Fluorescent auramine stain: AFB for M. tuberculosis
4. Culture:
   • Blood agar, chocolate agar (CO2), MacConkey agar
   • Inoculate immediately; transport at 37°C
   • Sabouraud agar for fungi
5. Antigen Detection (latex agglutination/CIE):
   • Rapid; detects S. pneumoniae, N. meningitidis (A, C, Y, W135), H. influenzae type b, Group B strep, Cryptococcus
   • Cryptococcal antigen (CRAG): highly sensitive for cryptococcal meningitis
6. PCR / Multiplex PCR (BioFire FilmArray):
   • Detects bacterial, viral, fungal pathogens simultaneously
   • High sensitivity/specificity; results in hours
   • Especially useful when prior antibiotics given
7. Adenosine Deaminase (ADA): Elevated in TB meningitis (>10 U/L)
8. VDRL on CSF: For neurosyphilis diagnosis
9. Blood cultures: Positive in ~80% bacterial meningitis
10. Counterimmunoelectrophoresis (CIE): Antigen detection in CSF, blood, urine

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5. PRIMARY AMOEBIC MENINGOENCEPHALITIS (PAM)

Epidemiology

• Worldwide distribution; found in warm freshwater (lakes, rivers, geothermal springs, poorly chlorinated pools), soil
• Predominantly affects children and young adults; males > females (76%)
• Most cases in summer months; associated with swimming/diving in warm freshwater
• Rare but almost universally fatal (>97% mortality)
• Cases also linked to nasal irrigation with inadequately treated water

Life Cycle / Pathogenesis

1. Trophozoites (free-swimming form) or flagellate form present in warm freshwater
2. Enters through nasal mucosa during water contact (swimming, diving)
3. Penetrates nasal epithelium and traverses cribriform plate of ethmoid bone
4. Invades olfactory nerves and olfactory bulbs
5. Reaches CNS - produces severe hemorrhagic necrotizing meningoencephalitis
6. Amoeba phagocytizes neurons (direct tissue destruction by contact-mediated killing and secretion of phospholipases, proteases)
7. Intense neutrophilic inflammatory response
8. Death within 1-12 days of symptom onset

Clinical Features

• Incubation: 1-7 days
• Sudden onset: severe frontal headache, high fever, nausea, vomiting
• Changes in smell/taste (olfactory involvement)
• Rapid progression to neck stiffness, photophobia, altered consciousness, seizures, coma
• Death within 3-7 days

Laboratory Diagnosis

Treatment

• Very poor prognosis; no proven regimen
• Amphotericin B (IV + intrathecal): Drug of choice; liposomal form preferred
• Miltefosine (recently FDA-approved compassionate use) + Amphotericin B + Fluconazole
• Rifampicin, Azithromycin: Adjuncts
• Only a handful of survivors worldwide (most treated with combination regimens including miltefosine)

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6. ETIOLOGY AND LABORATORY DIAGNOSIS OF UTI

Etiology

• E. coli, Klebsiella, Proteus, Pseudomonas aeruginosa, Enterococcus, Staphylococcus aureus, Candida spp.
• Catheter-associated UTI (CAUTI): Pseudomonas, Candida, Enterococcus

• Staphylococcus aureus bacteriuria: suspect hematogenous seeding
• Candida: immunocompromised, catheterized patients
• Mycobacterium tuberculosis: sterile pyuria (no growth on routine culture)
• Schistosoma haematobium: hematuria (not UTI per se)

• Type 1 fimbriae (mannose-sensitive): bind uroepithelium
• P fimbriae (mannose-resistant, Pap): bind P blood group antigens; important in pyelonephritis
• Hemolysin: cytotoxic
• Aerobactin: iron acquisition

Laboratory Diagnosis

• Midstream clean-catch urine (MSCU) - most common
• Catheter specimen
• Suprapubic aspirate (gold standard; any growth significant)

• Nitrite test: Bacteria (Enterobacteriaceae) reduce nitrate to nitrite - positive = infection
• Leukocyte esterase: Pyuria marker
• Hematuria: Micro/macroscopic blood

• Pyuria: >10 WBC/mm3 in uncentrifuged urine (or >5 WBC/HPF centrifuged)
• Bacteriuria: >1 organism/HPF in uncentrifuged Gram-stained urine correlates with >10^5 CFU/mL
• Casts: WBC casts = pyelonephritis

• Culture on CLED agar (Cystine Lactose Electrolyte Deficient) - prevents swarming of Proteus; or Blood agar + MacConkey agar
• Significant bacteriuria: ≥10^5 CFU/mL (10^5/mL) - Kass criterion for asymptomatic women
• ≥10^3 CFU/mL in symptomatic women is clinically significant
• Any growth from suprapubic aspirate is significant
• Antibiotic sensitivity testing (Kirby-Bauer/MIC)

• Antibody-coated bacteria (ACB) test: Distinguishes upper (pyelonephritis - positive) from lower UTI
• Blood culture: In pyelonephritis/urosepsis
• Serum procalcitonin, CRP: In febrile UTI

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7. PATHOGENESIS OF DIPHTHERIA

• Gram-positive pleomorphic bacilli
• Arranged in V, L, or palisade (Chinese letter) pattern
• Volutin granules (metachromatic granules / Babes-Ernst granules): stain purple with toluidine blue (Albert's or Neisser's stain)
• Toxigenic strains carry beta-bacteriophage (corynephage) containing tox gene (lysogenic conversion)

Pathogenesis (Step by Step)

• C. diphtheriae colonizes pharynx, larynx, tonsils (less commonly nose, skin, wound, vagina)
• Respiratory droplets / direct contact transmission
• Bacteria do NOT invade tissues - remain localized at site of infection

• Local tissue necrosis at site of colonization
• Fibrinous exudate forms containing organisms, dead neutrophils, RBCs, and fibrin
• Forms characteristic grayish-white pseudomembrane (tightly adherent, bleeds on removal) - most commonly on tonsils/pharynx
• Extension to larynx, trachea, bronchi may cause obstruction (croup/"bull neck" = extensive cervical lymphadenopathy)

• Only toxigenic strains (those lysogenized by beta-phage carrying tox gene) produce the exotoxin
• Diphtheria toxin is a heat-labile protein consisting of:
  • Fragment B (binding fragment): Binds heparin-binding EGF (HB-EGF) receptor on susceptible cells (heart, nerve, kidney cells)
  • Fragment A (active fragment): Translocated into cytoplasm; enzymatically active
• Mechanism: Fragment A ADP-ribosylates EF-2 (Elongation Factor-2 = Translocation factor), irreversibly inactivating it
• Result: Inhibition of protein synthesis in host cells - cell death

• Myocarditis: Most common cause of death; occurs 1-2 weeks after onset; cardiac arrhythmias, heart block, CCF; fatty degeneration of myocardium
• Neuropathy (demyelinating):
  • Early (local): Palatal palsy (nasal voice/regurgitation) - days 1-2 weeks
  • Oculomotor paralysis (ciliary palsy, diplopia) - weeks 3-5
  • Late: Peripheral motor neuropathy, diaphragmatic palsy (respiratory failure)
• Renal tubular necrosis
• Adrenal hemorrhage

• Tox gene on phage: low iron conditions induce high toxin production (DtxR - iron-dependent repressor; iron depletion lifts repression of tox gene)
• Cord factor (trehalose dimycolate): Present in corynebacteria
• Surface proteins mediating adherence

Lab Diagnosis of Diphtheria (brief):

• Swab from throat/membrane - culture on Loeffler's serum slope, Tellurite blood agar (gray-black colonies)
• Albert's stain or Neisser's stain for metachromatic granules
• Toxigenicity testing: Elek plate test (immunodiffusion - precipitation line = toxin production) or PCR for tox gene

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8. NON-GONOCOCCAL URETHRITIS (NGU)

Definition

Etiology

Clinical Features

• Incubation: 7-21 days (vs 2-7 days for gonorrhea)
• Scant mucoid/mucopurulent urethral discharge (vs profuse purulent in gonorrhea)
• Mild dysuria; may be asymptomatic (30%)
• Meatal erythema

• Men: Epididymo-orchitis, prostatitis, Reiter's syndrome (urethritis + arthritis + conjunctivitis + uveitis)
• Women: Cervicitis, PID, ectopic pregnancy, infertility
• Neonates: Conjunctivitis, pneumonitis (from Chlamydia)

Laboratory Diagnosis

1. Urethral smear (Gram stain):

   • 5 PMN/oil immersion field (x1000) in urethral discharge

   • Absence of Gram-negative intracellular diplococci (distinguishes from gonorrhea)
   • First-catch urine: >10 WBC/HPF
2. Culture:
   • C. trachomatis: McCoy cells (cyclohexamide-treated); iodine or Giemsa stain for intracytoplasmic inclusions; technically difficult
   • Mycoplasma/Ureaplasma: Special broth (PPLO/Shepard's medium); A7 agar - tiny "fried-egg" colonies
3. Nucleic Acid Amplification Tests (NAATs) - Gold Standard:
   • PCR, TMA (transcription-mediated amplification), SDA (strand displacement amplification)
   • Detects C. trachomatis and N. gonorrhoeae simultaneously (dual NAAT)
   • Specimen: First-catch urine, urethral/vaginal swab, self-collected swab
   • Most sensitive and specific (>95%)
4. Direct Immunofluorescence (DIF):
   • Fluorescent antibody staining of urethral smear
   • Detects chlamydial elementary bodies
5. ELISA: Antigen detection (lower sensitivity than NAAT)
6. Serology: Not useful for diagnosis of acute urethritis

Treatment

• First-line (Chlamydia/NGU): Doxycycline 100 mg BD x 7 days OR Azithromycin 1g single oral dose
• If Mycoplasma genitalium suspected/treatment failure: Moxifloxacin 400 mg OD x 7 days
• If Trichomonas: Metronidazole 2g single dose
• Partner notification and treatment is essential

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