Diagnosis

Diagnosis of JE involves

  • Collection of Specimens:

  • Detection and Isolation of Virus

Collection of Specimens:

  • CSF (Cerebro Spinal Fluid)

  • Blood

Collection of CSF:

  • CSF can be collected by Lumbar puncture from the suspected cases during the early acute phase.

  • Collected specimens must be stored at 4 C on dry ice and transposrted to laboratory

Collection of CSF from Dead Patients:

  • Smal brain tissues can be collected from different parts of the brain - cerebral crotex, cerebellum, basal nuclei and brain stem.

  • Small pieces of brain tissues can be obtained by biobsy, using a VIM-Silverman needle inserted via the nose through the cribriform pate of the ethmoid bone.

  • The brain tissues should be immersed in 2ml of 10% glecerol-saline (pH 7.4)

  • Specimens for virus isolation should be transported in sealed containers in dry ice or liquid nitrogen.

Collection of Blood

  • Blood specimens should be collected from suspected JE cases in the acute phase

  • A second, convalescent specimen should be collected after 2-4 weeks.

  • Venous blood is collected aseptically, kept at room temperature for 15 min, to clot, then kept at 4c for one hour.

  • The serum has to be seperated from the clot and stored in sterile container at 4o c

Detection and Isolation of Virus

  • Detection of Antigen

  • Detection of Antibody

Detection of Antigen from CSF

1. Immunofluorescent Assay (IFA)

  • For detection of JEV antigen in CSF as early as the second day of illness

  • JE Antigen can be detected using immunoperoxidase

  • When IgM antibodies are not detected in the CSF IFA is useful.

2. Reverse passive Haemagglutination (RPHA) test

  • RPHA is valuable if CSF samples are obtained within 10 days of onset of clinical symptoms.

  • Monoclonal Antibodies are used that are directed against envelope glycoprotein (V3) of JEV

  • RPHA is capable of detecting free antigen as well as antigen complexed with antibodies.

  • JE virus genome can be detected using the Polymerase Chain Reaction (PCR) for blood of CSF specimens collected from the acute stage.

  • Virus isolation can be achieved by inoculation of specimens into infant mice.

Detection of Antibody from Serum

  • Haemagglutination- Inhibition Test (HIT)

  • JComplement Fixation Test (CFT); Kinetic CFT

  • JNeutralization Test NT)

  • Immunodiffusion

All the above tests are based on the rise of IgG antibody titre in paired sera collected at an interval of 15-20 days.

Detection of Antibody from Serum and CSF

IgM captured Enzyme Linked Immunosorbant Assay (MAC ELISA):

  • gM antibodies to JEV in the CSF and Serum can be detected using conjugated anti IgM

  • IgM antibodies are present in all cases beyond the seventh day of illness.

  • MAC ELISA is advantageous in providing diagnosis with a single specimen of CSF.

  • The cross reactivity with other closely related flaviviruses is negligible.
Last Updated on:06/05/2014