Powassan Virus – Serology and PCR

Testing Indications

Testing for Powassan virus infection is indicated when a patient displays clinically compatible signs/symptoms of infection and/or has a compatible exposure history (e.g. outdoor activity in an endemic area, a noted tick bite). Serology is the primary testing modality for Powassan virus infection.

Most Powassan virus infections are asymptomatic. Symptoms of Powassan virus infection can develop from 1 to 4 weeks following a tick bite.¹ Individuals may develop an initial prodrome of fever, chills, headache, gastrointestinal symptoms, myalgias, confusion, weakness, or nuchal rigidity, among others. Neuroinvasive disease may present as meningitis and/or encephalitis accompanied by neurological deterioration or other focal neurologic signs/symptoms. Testing should not be performed on asymptomatic individuals.

Acceptance/Rejection Criteria

• Specimens received without the appropriate forms (See: Submission and Collection Notes) will not be tested.

Timing of Specimen Collection

Serologic testing for Powassan virus:
Acute and convalescent sera should be collected for serologic testing, where applicable. The convalescent serum specimen should be collected at least 2 to 3 weeks after the initial acute specimen.

Molecular (real-time RT-PCR) testing for Powassan virus:
Collect specimens for molecular testing early in the course of illness.  See acceptance/rejection criteria above.

Limitations

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

Storage and Transport

All clinical specimens must be shipped in accordance with the Transportation of Dangerous Goods Act/Regulations.

• For serum separator tubes: centrifuge sample prior to placing in biohazard bag.
• Place each specimen type in an individual biohazard bag and seal. Insert the corresponding requisition in the pocket on the outside of each sealed biohazard bag.

All specimens submitted for molecular testing should be stored at 2-8°C following collection and shipped to PHO’s laboratory on ice packs. If a delay in transport to PHO’s laboratory is anticipated (more than 72 hours), specimens should be frozen (at -80°C if possible) and shipped on dry ice.

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) Testing 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.

Note: For arbovirus testing that will be referred to the NML (as indicated in the TIS), no additional NML requisition is required.

Test Frequency and Turnaround Time (TAT)

The TAT for Powassan virus serology screening (HI assay) at PHO’s laboratory is up to 8 days from receipt at the laboratory. Testing is performed once per week.

Specimens that are HI screen reactive at PHO’s laboratory are referred to the NML for confirmatory testing within 7 days of the reactive result. The TAT for confirmatory Powassan serology testing by PRNT is determined by the NML and may require up to an additional 21 calendar days following receipt of the specimen at the NML.

PHO’s laboratory uses a hemagglutination inhibition (HI) assay to screen for Powassan virus antibodies (IgM and IgG).

Confirmatory Powassan virus serologic testing is performed using a plaque reduction neutralization test (PRNT) at the NML. Specimens that are reactive in PHO’s laboratory will be sent to the NML for confirmation.

Molecular detection of Powassan virus targets is performed by RT-PCR at the NML. This is not a sensitive test for the detection of Powassan virus and is not routinely recommended.

Algorithm

Serum specimens submitted for serology will first be analyzed by the HI assay to test for the presence of Powassan antibodies.

Specimens that are HI reactive will be further analyzed for the presence of neutralizing antibodies by PRNT. HI non-reactive samples will not be tested by PRNT.

PCR testing is not performed routinely and will only be considered on a case-by-case basis, in consultation with the Microbiologist.

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 may be considered, where applicable.

Serologic testing for Powassan virus:

The presence of Powassan virus antibodies in serum (titres of ≥1:10) may indicate a recent or prior infection. Cross reactions (a reactive test result) may occur with other Flaviviruses such as St. Louis Encephalitis, Dengue, West Nile, Japanese B Encephalitis, and Yellow Fever.

A ≥4-fold increase in neutralizing antibody titre between acute and convalescent sera collected 2-3 weeks apart and tested by PRNT is considered indicative of seropositivity.

Molecular (real-time RT-PCR) testing for Powassan virus:

A positive PCR result indicates that Powassan virus nucleic acids were detected in the specimen and an acute/recent infection.

A negative PCR result indicates that Powassan virus nucleic acids were not detected in the specimen. This does not exclude Powassan virus infection.

Reporting

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.

References

1. Centers for Disease Control and Prevention. Antibody Testing [Internet]. Atlanta: Centers for Disease Control and Prevention; 2023 [cited 2023-07-18].