West Nile Virus – Serology and PCR

Testing Indications

West Nile virus disease should be considered in any patient with:

  • febrile or acute neurological illness


  • recent exposure to: mosquitoes, blood transfusion , organ transplantation

This is especially true during the summer months.

The diagnosis should also be considered in any infant born to a mother infected with the virus during pregnancy or while breastfeeding.

West Nile virus should be considered in the differential diagnosis when the following diseases are suspected:

  • encephalitis and aseptic meningitis, such as: herpes simplex virus, enteroviruses
  • other arboviruses, such as: West Nile virus, Eastern equine encephalitis, Powassan virus

Specimen Collection and Handling

Specimen Requirements

Test Requested Required Requisition(s) Specimen Type Minimum Volume Collection Kit

West Nile Virus Serology

Blood or serum

5.0 ml blood or 1.0 ml serum

Vacutainer tubes (SST) 

West Nile Virus Serology


400 µl

Sterile container

West Nile Virus PCR1,4

EDTA blood, plasma (or serum)2

5.0 ml blood or 1.0 ml plasma or serum

Non-heparin anticoagulant/Lavender top tube

Submission and Collection Notes


Effective October 3, 2022, the Arbovirus (Non-Zika) Testing Intake Form  is a mandatory requirement for West Nile virus PCR testing. PHO’s laboratory 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 National Microbiology Laboratory for relevant testing performed there.


Clinical information, including any risk factors such as mosquito exposure, and any recent travel, must be provided.


CSF Serology is the preferred method of testing. If submitting CSF, a paired serum specimen must also be submitted.


Molecular testing must must be preapproved by the PHO laboratory microbiologist. Contact PHO laboratory Customer Service at 416-235-6556 or 1877-604-4567 to request approval.

Timing of Specimen Collection

Serum specimen (acute and convalescent) collected 2-3 weeks apart from patients with clinical illness.


Haemolysed, icteric, lipemic or microbial contaminated sera or plasma are not recommended for testing.

PCR is not recommended for West Nile viruses as it is not a sensitive test for West Nile viruses, unless collected within 5 days of neurological onset during active mosquito season. National Microbiology Laboratory (NML) will determine if molecular testing is feasible after serological testing is complete.

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 the PHO’s laboratory on ice packs.

Specimens for molecular testing should be frozen and shipped on dry ice.

Special Instructions

All specimens submitted for testing must be accompanied by a separate General Test Requisition for each specimen type collected. All fields on each requisition must be completed.

It is also MANDATORY to provide all information requested on the Arbovirus (Non-Zika) Information Intake Form. The Arbovirus (Non-Zika) Information Intake Form may be exempted if all mandatory information is available on the PHO’s General Test Requisition. Specimens submitted with missing mandatory information will not be tested until that information is provided.

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

West Nile virus IgG and IgM  Enzyme Linked Immunosorbent Assay (ELISA) testing is performed daily Monday to Friday during peak season (summer months) and up to twice per week at other times.

Turnaround time (TAT) for non-reactive ELISA IgG and IgM is up to 2 days during peak season and up to 5 days otherwise.

TAT for a confirmed reactive ELISA IgM is up to 5 days from receipt at PHO’s laboratory.

TAT for results on specimens tested by Plaque Reduction Neutralization Test (PRNT) is up to 10 days from receipt at PHO’s laboratory.

Test Methods

Screening test: specimens for West Nile virus-(WNV) IgG and IgM serology are performed using Enzyme Linked Immunosorbent Assay (ELISA).

Confirmatory test : Plaque Reduction Neutralization Test (PRNT)

Optional test: molecular detection by PCR


Submitted specimens will first be analyzed by the IgG and IgM ELISA to determine if the patient has developed antibodies.

Specimens that are ELISA reactive will be further analyzed for the presence of neutralizing antibodies by PRNT.

Specimens that are ELISA non-reactive will not be tested by PRNT.

PCR testing will be considered on a case-by-case basis.

West Nile avidity testing may be considered in order to differentiate recent (last few months) from remote infections. This should only be considered in patients with severe illness (e.g. hospitalized). Contact PHO Customer Service at 416-235-6556 or 1-877-604-4567 to request approval.

For further testing information refer to labstract titled: LAB-SD-011, West Nile Virus Update - Interpretation of serology results and investigation of cases with neurological involvement.


Reports are sent back to PHO’s laboratory from NML and are then reported and forwarded to the ordering physician or Health care Provider as indicated on the requisition.

Specimens that are positive for West Nile virus are reported to the Medical Officer of Health as per the Health Protection and Promotion Act.

Data and Analysis

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Laboratory Services


Updated 5 Oct 2022