Eastern Equine 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 for the Eastern Equine Encephalitis virus (EEEV) at Public Health Ontario (PHO). The EEEV is a single-stranded RNA virus in the Togaviridae family (genus Alphavirus) that can cause severe neuroinvasive disease. The virus is transmitted primarily through the bite of infected mosquitoes in endemic areas (e.g. North America)1,2.
Testing Indications
Testing for EEEV infection may be considered when an individual displays clinically compatible signs/symptoms of infection and has relevant exposures (e.g. mosquito bites, travel to endemic areas, outdoor activities, among others)1-3. Many individuals exposed to EEEV remain asymptomatic, while others may develop a mild febrile illness that can progress to neuroinvasive disease1-3.
Serology is the preferred method to detect an EEEV infection1,4-5. Testing for EEEV by PCR is not routinely recommended and should only be considered for individuals that are immune compromised1. Testing of asymptomatic individuals is not recommended.
Due to the overlap in geographic distribution of the corresponding mosquito vectors, infection with West Nile Virus should also be considered if suspecting EEEV infection.
Acceptance/Rejection Criteria
Specimens received without the appropriate forms, relevant clinical information or approvals (See: Submission and Collection Notes) may not be tested.
Specimen Requirements
| Test Requested | Required Requisition(s) | Specimen Type | Minimum Volume | Collection Kit |
Eastern Equine Encephalitis serology |
Serum |
5.0 mL blood or 1.0 mL serum |
Clotted blood – Serum Separator tubes (SST) for serum |
|
Eastern Equine Encephalitis PCR |
Serum3 |
1.0 mL |
Clotted blood-Serum Separator tubes (SST) for serum |
|
Eastern Equine Encephalitis PCR |
Other specimens (e.g. CSF, plasma)3 |
400 µl |
Sterile container OR Plasma collected in non-heparin anticoagulant |
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 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 (NML) for relevant testing performed there.
Clinical information, including symptom onset date, any risk factors such as exposure to mosquitoes, and any recent travel, must be provided.
Molecular testing (EEEV PCR) is not performed routinely and must be pre-approved by a PHO Microbiologist. Contact PHO’s Laboratory customer service at 416-235-6556 or 1-877-604-4567 to request approval.
Timing of Specimen Collection
Serology:
Specimens for serology testing (acute and convalescent) should be collected 2-3 weeks apart from patients with clinical illness.
Molecular (PCR):
Specimens submitted for molecular testing (PCR) should be collected ASAP after the onset of symptoms, unless otherwise indicated via discussion with a PHO Microbiologist.
Limitations
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 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 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, an additional Arbovirus (Non-Zika) Testing Intake Form is not required. Specimens submitted without this mandatory information will not be tested until that information is provided.
Test Frequency and Turnaround Time (TAT)
Serology:
Serology screening for EEEV antibodies is performed once per week at PHO. The turnaround time (TAT) is up to 8 days from receipt at PHO. Reactive specimens are referred to the NML for additional confirmatory testing. The TAT for confirmatory testing is up to 14 calendar days after the completion of HI testing.
PCR:
Molecular testing (PCR) is performed at the NML if the request is approved by a PHO Microbiologist. This is not a routine test and TAT will be determined in consultation with the NML at the time of submission.
Serology:
EEEV serology screening is performed using Hemagglutination Inhibition (HI) Assay. Confirmatory testing is performed at the NML using a plaque reduction neutralization test (PRNT).
PCR:
Molecular testing is performed at the NML using a reverse transcriptase polymerase chain reaction (RT-PCR)
Algorithm
Serology:
Serum is first screened for EEEV antibodies (IgM/IgG) by HI. Specimens that are HI reactive (e.g. reactive- may be resulted as positive if tested at NML) will be further analyzed for the presence of neutralizing antibodies by PRNT. No further testing will be performed on specimens that are HI non-reactive.
PCR:
PCR testing by RT-PCR will be considered on a case-by-case basis as it is not a sensitive test and is not performed routinely. If CSF PCR testing is approved, submission of a paired serum for serology testing is recommended.
Interpretation
All results should be interpreted in the context of the specific clinical scenario. Given the overlap in the distribution of disease vectors, testing for other potential co-pathogens should be considered where applicable.
Serology:
| HI Result |
Interpretation |
Comments |
|---|---|---|
|
Non- Reactive <1:10 |
No serological evidence of infection |
Submit a second specimen for serologic testing in 2 to 3 weeks if clinically indicated. |
|
Reactive ≥1:10 |
May indicate infection |
Cross reactions may occur with other Flaviviruses such as Dengue virus, West Nile virus, Japanese encephalitis virus, Results should be interpreted in the context of the clinical and travel history, signs and symptoms of the patient. |
The HI assay detects total antibodies to EEEV (IgM/IgG). EEEV HI testing of a single serum specimen is insufficient to establish the diagnosis of an EEEV infection if a reactive result is obtained5. Submission of both acute and convalescent specimens is recommended to assist with interpretation and confirmation. Confirmatory testing of HI reactive specimens (those with HI titres ≥1:10) by PRNT is required to verify the presence of antibodies to EEEV. A ≥ 4-fold change in antibody titre between acute and convalescent specimens is indicative of an acute or recent infection.
Low HI reactive specimens (titre of 1:10) may be due to the persistence of antibodies to EEEV from a previous infection, cross-reactivity with antibodies to other arboviruses (e.g. Dengue virus, West Nile virus, Japanese Encephalitis virus or Yellow Fever virus), among other causes.
PCR:
A positive PCR result (POS) indicates that EEEV nucleic acids were detected in the specimen and indicates an acute/recent infection.
A negative PCR result (NEG) indicates that EEEV nucleic acids were not detected in the specimen. This does not exclude EEEV infection.
Reporting
Results are reported to the physician, authorized health care provider (General O. Reg 45/22, s.18) or submitter 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.
References
- Centers for Disease Control and Prevention. Eastern Equine Encephalitis Virus. 2024. Available from: https://www.cdc.gov/eastern-equine-encephalitis/site.html.
- Government of Canada. Mosquito-borne disease surveillance: seasonal update. 2024. Available from: https://health-infobase.canada.ca/zoonoses/mosquito/
- Government of Canada. Eastern and Western Equine Encephalitis virus: Infectious substances pathogen safety data sheet. 2024. Available from: https://www.canada.ca/en/public-health/services/laboratory-biosafety-biosecurity/pathogen-safety-data-sheets-risk-assessment/eastern-equine-encephalitis.html
- National Microbiology Laboratory. Arbovirus, Rabies, Rickettsia and Related Zoonotic Diseases. Available from: https://cnphi.canada.ca/gts/laboratory/1020
- Vetter SM. Eastern Equine Encephalitis Virus. In: Leber AL and Burnham CAD. Clinical Microbiology Procedures Handbook (5th ed). 2023. ASM Press. Washington DC, USA. 18.13.
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