Japanese Encephalitis - Laboratory Diagnosis

1. Specimen Collection

2. CSF Findings (Lumbar Puncture)

• WBC: 10-500 cells/μL (rarely up to 1,000); early PMN predominance, shifting to lymphocytic predominance later
• Protein: Elevated, 50-100 mg/dL
• Glucose: Normal
• CSF findings are non-specific but consistent with viral/aseptic meningoencephalitis

3. Serological Tests (PRIMARY DIAGNOSIS)

A. IgM Antibody Capture ELISA (MAC-ELISA) - First-Line Test

• Recommended by WHO as the first-line rapid diagnostic assay
• Detects JEV-specific IgM in CSF or serum
• IgM appears in CSF by ~4 days after symptom onset; in serum by ~7 days after onset; some sources state as early as 3 days post-symptom onset
• Sensitivity in CSF: 65-70% (commercial assays)
• Specificity: 89-100% in CSF
• Available at commercial reference laboratories; confirmatory testing at CDC
• Limitation: Cross-reacts with other flaviviruses (dengue, West Nile, Zika, yellow fever) - confirmatory testing required

B. Plaque Reduction Neutralization Test (PRNT) - Gold Standard for Confirmation

• Gold standard for flavivirus diagnosis
• Distinguishes JEV-specific antibodies from cross-reacting flaviviral antibodies
• Used when MAC-ELISA is positive or equivocal, or where dengue co-circulation is a concern
• Not widely available; typically performed at reference/CDC laboratories

C. IgG Antibody (Convalescent Serology)

• A 4-fold rise in IgG titer between acute and convalescent serum samples is confirmatory
• Used when PRNT is unavailable
• Requires paired samples (acute + 2-4 weeks later), limiting early diagnosis

4. Molecular Tests

RT-PCR

• Detects JEV RNA in blood or CSF during early/acute phase (first few days of illness)
• Virus is replaced by antibody response rapidly, making PCR insensitive once neurological symptoms are established
• Low viral load means NAAT/RT-PCR should NOT be used to rule out infection in immune-competent patients
• Virus isolation from blood is infrequent; however, JEV can be isolated from CSF in up to 1/3 of patients with severe disease

5. Summary of Diagnostic Methods

6. Neuroimaging (Supportive Diagnosis)

• T2/FLAIR hyperintense signal bilaterally in thalamus (most characteristic)
• Also: midbrain, basal ganglia, cerebellum
• Represents subcortical encephalitis

7. Differential Diagnosis Considerations

• Dengue virus
• West Nile virus
• Zika virus
• Yellow fever virus
• St. Louis encephalitis virus

Key Points

1. Viremia resolves early - do not rely on culture or PCR in established disease
2. IgM ELISA of CSF/serum is the standard first-line test (WHO recommendation)
3. PRNT is the gold standard for confirmation and flavivirus differentiation
4. CSF typically shows lymphocytic pleocytosis with normal glucose
5. MRI thalamic T2 hyperintensity is the characteristic imaging finding
6. JEV can occasionally be isolated from CSF in up to 1/3 of severe cases

Pathogenesis of Tetanus

1. The Organism

2. Entry and Germination

1. Spores are introduced into a wound contaminated with soil, foreign bodies, or feces (puncture wounds, lacerations, contaminated heroin, umbilical stump in neonates, surgical instruments, etc.)
2. Germination occurs when local tissue conditions reduce oxygen tension - devitalized/crushed tissue, foreign bodies, or co-infection create the anaerobic microenvironment needed
3. Vegetative cells proliferate locally. Importantly, C. tetani is non-invasive - it does not spread beyond the wound. There may be minimal or no visible wound inflammation
4. Toxin production occurs during the stationary phase of growth; the toxin is released when the cell lyses

3. The Two Toxins

4. Tetanospasmin - Structure

• Synthesized as a single 150-kDa polypeptide
• Cleaved by an endogenous bacterial protease into:
  • Light chain (A chain, ~50 kDa): toxic subunit - zinc endopeptidase activity
  • Heavy chain (B chain, ~100 kDa): binding and translocation subunit
• The two chains are held together by a disulfide bond + non-covalent forces

5. Step-by-Step Mechanism of Action

Step 1 - Binding at the Neuromuscular Junction

• The carboxyl-terminal domain of the heavy chain (B chain) binds with high affinity to polysialoganglioside receptors (sialic acid receptors) and adjacent glycoproteins on the presynaptic membranes of peripheral motor neurons

Step 2 - Internalization and Retrograde Axonal Transport

• The toxin-receptor complex is internalized into endosomal vesicles
• These vesicles undergo retrograde axonal transport along motor neuron axons - travelling from the peripheral nerve terminal back to the motor neuron soma in the anterior horn of the spinal cord (and brainstem for cranial nerves)
• The toxin can also spread hematogenously to peripheral nerves throughout the body

Step 3 - Transcytosis into Inhibitory Interneurons

• At the spinal cord, the toxin undergoes trans-synaptic spread - it crosses the synapse and enters inhibitory interneurons (glycinergic and GABAergic neurons)
• The endosome becomes acidified, causing a conformational change in the N-terminal domain of the heavy chain
• This causes the heavy chain to insert into the endosomal membrane, allowing the light chain to translocate into the cytosol

Step 4 - Cleavage of SNARE Protein (Synaptobrevin/VAMP)

• The light chain zinc endopeptidase cleaves synaptobrevin (also called VAMP - vesicle-associated membrane protein)
• Synaptobrevin is a SNARE protein required for docking of neurotransmitter vesicles onto the presynaptic membrane
• Without synaptobrevin, inhibitory neurotransmitter vesicles cannot fuse and release their contents

Step 5 - Block of Inhibitory Neurotransmission

• Release of glycine (from spinal inhibitory interneurons) and GABA (from brainstem interneurons) is blocked
• These inhibitory neurotransmitters normally suppress alpha-motor neuron firing between voluntary movements

Step 6 - Unopposed Motor Neuron Excitation → Spastic Paralysis

• Without inhibitory control, alpha-motor neurons undergo sustained, unregulated excitatory discharge
• Agonist and antagonist muscle groups contract simultaneously and continuously → spastic paralysis and tetanic muscle spasms
• This is the opposite of botulinum toxin (which blocks acetylcholine release at the NMJ → flaccid paralysis)

6. Sympathetic Involvement

• Tetanospasmin also affects preganglionic sympathetic neurons and parasympathetic centers
• Loss of inhibitory control of the autonomic nervous system leads to sympathetic overactivity:
  • Markedly elevated circulating catecholamines
  • Tachycardia, hypertension, hyperpyrexia
  • Cardiac dysrhythmias, diaphoresis
• Autonomic dysfunction is the major cause of death in patients who survive the initial acute phase

7. Why Toxin Binding is Irreversible

• Tetanospasmin binds irreversibly at the synapse
• Recovery depends entirely on the growth of new axonal terminals - this is why clinical tetanus can persist for weeks to months
• Antitoxin (TIG/HTIG) can only neutralize unbound, circulating toxin - it cannot reverse already-bound toxin

8. Summary Diagram of Pathogenesis

Wound contamination with C. tetani spores
        ↓
Germination in anaerobic devitalized tissue
        ↓
Vegetative cells produce Tetanospasmin (A-B toxin, MW 150 kDa)
        ↓
Toxin released on cell lysis
        ↓
Heavy chain binds polysialoganglioside receptors on motor nerve endings
        ↓
Retrograde axonal transport to spinal cord / brainstem
        ↓
Trans-synaptic spread to inhibitory interneurons (Glycinergic / GABAergic)
        ↓
Light chain cleaves Synaptobrevin (SNARE protein)
        ↓
Block of Glycine + GABA release
        ↓
Unopposed motor neuron discharge
        ↓
Spastic paralysis + Tetanic spasms
  +
Autonomic dysfunction (sympathetic storm)

9. Clinical Correlation to Pathogenesis