Japanese Encephalitis Virus – Serology and PCR
Consistent with O. Reg. 671/92 of the French Language Services Act, laboratory testing information on this page is only available in English because it is scientific or technical in nature and is for use only by qualified health care providers and not by members of the public.
Background
This page provides Serology and PCR testing information available through Public Health Ontario (PHO) for the Japanese encephalitis virus (JEV). The JEV is an RNA virus that is a member of the Flaviviridae family and is genetically related to other common arboviruses, including Dengue virus and West Nile virus. The primary mechanism for JEV transmission is through the bite of an infected mosquito in an area that is considered endemic (e.g. South East Asia).
Testing Indications
This is a Referred Out Test. Specimens are sent to the National Microbiology Laboratory (NML) in Winnipeg. Testing is not performed at PHO’s laboratory.
Most individuals infected with JEV remain asymptomatic. Those who develop symptoms may experience an acute febrile illness within 14 days of exposure, which may progress to a severe neuroinvasive disease1. Testing is recommended for individuals with compatible clinical symptoms and appropriate exposures (e.g. mosquito bites, travel to or residence in a JEV endemic area or an area with ongoing JEV transmission, among others)1-5.
Serology testing is the preferred method to detect infections caused by JEV1-5. Molecular testing for JEV is not routinely recommended. It may be used to assist with the identification of an acute infection in some individuals (e.g. immunocompromised). Testing of asymptomatic individuals is not recommended.
Acceptance/Rejection Criteria
Specimens received without the appropriate forms, clinical information or approvals (See: Submission and Collection Notes) will not be tested.
Specimen Requirements
| Test Requested | Required Requisition(s) | Specimen Type | Minimum Volume | Collection Kit |
Japanese Encephalitis serology |
Serum |
5.0 ml Blood or 1.0 ml serum |
Clotted blood – Serum Separator Tubes (SST) for serum |
|
Japanese Encephalitis PCR |
Serum |
400 µl |
Clotted blood – Serum Separator Tubes (SST) for serum |
|
Japanese Encephalitis PCR |
Other specimen types (e.g. plasma, CSF, tissues) |
400 µl |
EDTA/lavender top tube (plasma) Sterile container (other specimens) |
Submission and Collection Notes
Effective October 3, 2022, the Arbovirus (Non-Zika) Testing Intake Form is a mandatory requirement for Japanese encephalitis virus testing. PHO utilizes the information on the requisition and the mandatory intake form to assess testing criteria, assign appropriate tests, and provide mandatory information required by the NML for relevant testing performed there. Clinical information, including any risk factors such as mosquito exposure, recent travel prior to symptom onset, and recent or previous vaccination to JEV must be provided.
If submitting CSF, a paired serum specimen must also be submitted.
Molecular testing is not performed routinely and must be pre-approved by the PHO laboratory microbiologist. Contact PHO laboratory customer service at 416-235-6556 or 1-877-604-4567 to request approval.
Timing of Specimen Collection
Serology:
Acute and convalescent clotted blood or serum specimens for serology should be collected 2-3 weeks apart from patients with clinical illness1,3-5.
PCR:
Specimens for JEV PCR testing should be collected ASAP after the onset of symptoms3,4 unless otherwise indicated after discussion with a PHO Microbiologist.
Limitations
Haemolysed, icteric, lipemic or microbial contaminated sera or plasma are not recommended for testing.
The NML indicates that their laboratory-developed test has not been fully validated/verified.
Storage and Transport
Centrifuge if using SST. Place specimen in biohazard bag and seal. Specimens should be stored at 2-8°C following collection and shipped to PHO’s laboratory on ice packs.
Specimens for molecular testing should be frozen and shipped on dry ice.
Ship refrigerated specimens (e.g., clotted blood, serum, CSF) on ice packs, and frozen specimens (e.g., serum, CSF) on dry ice. Do not ship clotted blood and/or EDTA blood in a frozen state.
All clinical specimens must be shipped in accordance to the Transportation of Dangerous Good Act.
Special Instructions
Each specimen submitted for testing must be accompanied by a separate PHO General Test Requisition. All fields on each requisition must be completed.
It is MANDATORY to submit the Arbovirus (Non-Zika) Information Intake Form, where specified, with all fields completed. If all of the requested information from the Arbovirus (Non-Zika) Intake Form has been provided on the PHO General Test Requisition, we do not require an additional Arbovirus (Non-Zika) Testing Intake Form. Specimens submitted with this mandatory information missing will not be tested until that information is provided.
Test Frequency and Turnaround Time (TAT)
This is a Referred Out Test. Specimens are referred to the NML in Winnipeg.
Serology and molecular testing TAT is up to 28 days from receipt at PHO.
All JEV testing is performed at the NML.
Serology:
Screening testing for total antibodies to JEV (IgM/IgG) is performed using a Hemagglutination Inhibition Assay (HI). Confirmatory serology testing is performed by Plaque Reduction Neutralization Testing (PRNT)1.
PCR:
Molecular testing is performed by reverse transcriptase polymerase reaction (RT-PCR) on a case-by-case basis1 if approved by a PHO Microbiologist.
Algorithm
Serology:
Serum specimens submitted for JEV serology are first screened for antibodies to JEV by HI. Specimens that are negative by HI are not tested further. Specimens that are HI positive are reflexed to PRNT for confirmation.
Note: Due to the cross-reactivity of anti-Flaviviridae antibodies, PRNT for other arboviruses may also be performed by the NML on JEV HI positive specimens to assist with interpretation of a positive result.
PCR:
Molecular testing is performed on a case-by-case basis if approved by a PHO Microbiologist.
Interpretation
Serology:
An HI positive (POS) result from the NML indicates that anti-JEV antibodies (IgM/IgG) may be present in the specimen. This screening result may represent recent or prior exposure to JEV (including previous vaccination) or a related Flavivirus due to the cross-reactivity of anti-Flaviviridae antibodies and requires further confirmation1,5. A positive PRNT serology is indicative that the patient’s serum contains neutralizing antibodies to JEV. Collection of both acute and convalescent serum specimens is recommended if suspecting acute or recent infection.
An HI negative (NEG) result from the NML indicates that antibodies to JEV were not detected1. PRNT is not performed in these situations. Depending on the time elapsed between symptom onset and collection of the specimen, this may be due to the delayed development of an antibody response. Submit an additional convalescent serum specimen within 2 to 3 weeks if clinically indicated.
PCR:
A positive (POS) RT-PCR result from the NML indicates that JEV nucleic acids were detected in the specimen and is suggestive of an acute/recent infection. Additional confirmation may be required1.
A negative (NEG) RT-PCR result from the NML indicates that JEV nucleic acids were not detected in the specimen. This should not be used to rule out an acute infection due to the lack of sensitivity of this assay due to the characteristics of JEV viremia5.
Reporting
Reports are sent back to PHO’s laboratory from NML, and reports are forwarded to the ordering physician or authorized health care provider (General O. Reg 45/22, s.18) or submitter as indicated on the requisition.
Specimens that are positive for Japanese encephalitis virus are reported to the Medical Officer of Health as per the Health Protection and Promotion Act.
References
- National Microbiology Laboratory (NML). Arbovirus, Rabies, Rickettsia and Related Zoonotic Diseases. 2024. Available from: https://cnphi.canada.ca/gts/laboratory/1020
- Public Health Agency of Canada (PHAC). Japanese encephalitis vaccine: Canadian Immunization Guide for Health Professionals. 2024. Available from: https://www.canada.ca/en/public-health/services/publications/healthy-living/canadian-immunization-guide-part-4-active-vaccines/page-11-japanese-encephalitis-vaccine.html
- World Health Organization (WHO). Manual for Laboratory Diagnosis of Japanese Encephalitis Virus Infection. 2007. Available from: https://cdn.who.int/media/docs/default-source/immunization/vpd_surveillance/lab_networks/manual-lab-diagnosis-je.pdf?sfvrsn=e2b62a35_4
- Centers for Disease Control and Prevention (CDC). Japanese Encephalitis Virus. 2024. Available from: https://www.cdc.gov/japanese-encephalitis/index.html
- Hills S, Lindsey N, and Fischer M. Japanese Encephalitis. In CDC Yellow Book 2024. 2023. Available from: https://wwwnc.cdc.gov/travel/yellowbook/2024/infections-diseases/japanese-encephalitis#diagnosis
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