Eastern Equine Encephalitis (EEE) 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.

Specimen Collection and Handling

Specimen Requirements

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

Eastern Equine Encephalitis

Blood or Serum

5.0 ml blood or 1.0 ml serum

Clotted blood - vacutainer tubes (SST) for serum4

Eastern Equine Encephalitis

Plasma (or serum) in EDTA

1.0 ml plasma or serum

Plasma with non-heparin anticoagulant (or serum), CSF

Eastern Equine Encephalitis

CSF (must be preapproved by PHOL microbiologist)

400 µl

Sterile container

Submission and Collection Notes


Effective October 3, 2022, the Arbovirus (Non-Zika) Testing Intake Form  is a mandatory requirement for Eastern equine encephalitis virus 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 exposure to mosquitoes, and any recent travel, must be provided.


If submitting CSF, a paired serum specimens must also be submitted.


Molecular testing is not a routine test, and must be preapproved by the PHO laboratory microbiologist. Contact PHO’s laboratory Customer Service at 416-235-6556 or 1-877-604-4567 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.

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.

Ship refrigerated specimens on ice packs and frozen specimens 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)

Serology is performed once a week at PHO’s laboratory using an Hemagglutination Inhibition (HI) Assay for the detection of antibodies to Eastern Equine Encephalitis virus. Turnaround time is up to 8 days from receipt at PHO’s laboratory.

Plaque Reduction Neutralization Test (PRNT) is performed at the National Microbiology Laboratory (NML). TAT is up to 14 Calendar days after the completion of HI testing.

Molecular testing by RT-PCR is performed at NML after approval by the PHO’s microbiologist. As this is not a routine test, NML will determine TAT after approval of testing.

Test Methods

Screening test: specimens for Eastern equine encephalitis virus serology are performed using Hemagglutination Inhibition (HI) Assay.

Confirmatory test: Plaque Reduction Neutralization Test (PRNT).

Optional test: molecular detection by RT-PCR.


Submitted serum specimens will first be analyzed by the Hemagglutination Inhibition (HI) Assay to determine if the patient has developed antibodies.

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

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

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


Results are reported to the ordering physician or health care provider as indicated on the requisition.

Positive results from patients with encephalitis are also reported to the Medical Officer of Health as per Health Protection and Promotion Act.

Additional Information

All molecular testing must be preapproved by the PHOL microbiologist. Contact PHOL Customer Service at 416-235-6556 or 1877-604-4567 to request approval.

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Laboratory Services


Updated 3 Oct 2022