Tick-borne encephalitis (TBE) Serology and PCR – Referred Out

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Background
This page provides information on the testing available through Public Health Ontario (PHO) for the tick-borne encephalitis virus (TBEV).

TBEV is a tick-borne RNA virus that is a member of the Flaviviridae family and is known to cause neuroinvasive disease in humans.
The primary route of TBEV transmission is through a tick bite, but other routes are possible (e.g. ingestion of contaminated unpasteurized milk products). TBEV is endemic to Europe and parts of Asia1.
Testing for TBEV infection involves serology and/or PCR depending on the specific clinical scenario.

This is a Referred Out Test. Specimens are sent to the National Microbiology Laboratory (NML) in Winnipeg. Testing is not performed at PHO.

Updates
Effective July 2, 2025, submission of the new Vector-borne and Zoonotic Virus Testing Intake Form is mandatory, along with the General Test Requisition when requesting specific vector-borne or zoonotic virus tests. The new intake form replaces both the Arbovirus (Non-Zika) Testing Intake Form and the Mandatory Intake Form for Zika Virus Testing.

* Copie d'impression non contrôlée. Valide uniquement le jour de l'impression : 05 juill. 2025.

Testing Indications

Testing for TBEV is indicated for individuals with:

compatible clinical symptoms and
appropriate exposures (e.g., mosquito bites, travel to or residence in a TBEV endemic area or an area with ongoing TBEV transmission, among others) and
no recent or prior history of TBEV vaccination

Most individuals infected with TBEV remain asymptomatic. Symptomatic individuals may present initially with an acute febrile illness up to 28 days after exposure (fever, fatigue, headache, muscular ache, and nausea)². This may progress to neuroinvasive disease with symptoms of meningitis and/or encephalitis in severe cases.

Serology testing is the preferred method to detect infections caused by TBEV. Molecular testing for TBEV is not routinely recommended but may be used to assist with the identification of acute infection in some individuals (e.g. immune compromised). Testing of asymptomatic individuals is not recommended.

All specimens for TBEV testing require shipment to PHO under the Emergency Response Assistance Plan (ERAP)³ regardless of patient’s clinical status and reason for testing. Contact PHO’s Laboratory Customer Service Centre at 416-235-6556/1-877-604-4567 or the After-Hours Emergency Duty Officer at 416-605-3113 prior to sample collection and submission. Submitters should ensure that a Transportation of Dangerous Goods (TDG) certified individual is available to ship the specimens. PHO’s laboratory will assist in facilitation of the ERAP process during the shipment.

Acceptance/Rejection Criteria

Specimens received without the appropriate forms (See: Submission and Collection Notes) are subject to cancellation.

Specimen Collection and Handling

Specimen Requirements

Test Requested	Required Requisition(s)	Specimen Type	Minimum Volume	Collection Kit
Tick-borne encephalitis virus serology	General Test Requisition Vector-borne and Zoonotic Virus Testing Intake Form	Serum³	0.5 mL	Red top or Serum separator tubes (SST)
Tick-borne encephalitis virus PCR	General Test Requisition Vector-borne and Zoonotic Virus Testing Intake Form	Serum³	5 ml blood or 1 ml serum	Red top or Serum separator tubes (SST)
Tick-borne encephalitis virus PCR	General Test Requisition Vector-borne and Zoonotic Virus Testing Intake Form	Serum³	0.5 mL	Sterile tube/container
Tick-borne encephalitis virus PCR	General Test Requisition Vector-borne and Zoonotic Virus Testing Intake Form	Serum³	-	Sterile container

Submission and Collection Notes

Label the specimen container(s) with the patient’s first and last name, date of collection, and one other unique identifier such as the patient’s date of birth or Health Card Number. For additional information see: Criteria for Acceptance of Patient Specimens. Failure to provide this information may result in rejection or testing delay

Each specimen submitted for testing must be accompanied by a separate PHO General Test Requisition with all fields completed.

It is MANDATORY to provide the clinical information, relevant travel(s), and relevant exposures for Vector-borne viruses requested on the Vector-borne and Zoonotic Virus Testing Intake Form. Test requests that are submitted without the appropriate mandatory information are subject to cancellation.

Testing for TBEV must be pre-approved by a PHO Microbiologist. Contact PHO’s Laboratory Customer Service Centre at 416-235-6556/1-877-604-4567 or the After-Hours Emergency Duty Officer at 416-605-3113 prior to sample collection and submission. Submission of the mandatory Vector-borne and Zoonotic Virus Testing Intake Form will initiate the review process at PHO provided the form contains all necessary information. Requests received without the form, forms submitted with insufficient information or insufficient justification for testing are subject to cancellation.

Molecular testing is not a routine test. The NML will determine if molecular testing is appropriate after serological testing is complete.

Centrifugation of serum tubes (e.g. SST) is not required and should be avoided.

For tissue specimens, refer to the NML’s guidance on collection method for TBEV testing³.

Timing of Specimen Collection

Serology:
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):
Specimens for molecular testing (RT-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 CSF, sera or plasma are not recommended for testing.

Storage and Transport

Requests for TBEV testing require prior approval by PHO. Contact PHO’s laboratory at 416-235-6556 or 1-877-604-4567 before collecting and sending the specimens.

Specimens should be transported according to TDG guidelines for Category A pathogens (UN2814 packaging) and require initiation of ERAP. Contact PHO’s Laboratory Customer Service Centre to request assistance in shipping these types of samples.

All clinical specimens must be shipped in accordance with the Transportation of Dangerous Goods Act/Regulations and Shipping Infectious Substance Bulletin

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.
Clotted blood/serum specimens should be stored at 2-8°C following collection and shipped to PHO on ice packs.
Ship refrigerated specimens (e.g., clotted blood, serum, CSF) on ice packs, and frozen specimens (e.g., serum, CSF, tissues) on dry ice. Do not ship clotted blood, EDTA/ heparin/citrated whole blood on dry ice.

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

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

The turnaround time for all TBE testing is 14 calendar days from receipt of specimens at NML.

Test Methods

TBEV serology (IgG and IgM) is performed at the NML using an Enzyme Linked Immunosorbent Assay (ELISA). The NML indicates that this test is performed for investigational purposes only⁴.

Refer to NML- Guide to Services for further information.

Molecular detection of TBEV is performed by RT-PCR at the NML

Interpretation

All results should be interpreted in the context of the specific clinical scenario. Given the overlap in distribution of disease vectors, testing for other potential pathogens should be considered, where applicable.

Serology:
A ≥4-fold increase in neutralizing antibody titre between acute and convalescent sera (collected 2 to 3 weeks apart) tested by PRNT is considered indicative of seropositivity.
Additional notes on TBEV serology:

Specific TBEV IgM antibodies can persist for up to 10 months.
Cross-reactivity (a reactive test result) may occur with antibodies for other Flaviviridae such as Dengue virus and West Nile virus; therefore, submitting both acute and convalescent sera is recommended.
Testing for other vector-borne viruses may be added by the NML to assist with interpretation.

Molecular (Real-Time PCR):
A positive PCR result indicates that TBEV nucleic acids were detected in the specimen and an acute/recent infection.

A negative PCR result indicates that TBEV nucleic acids were not detected in the specimen. This does not exclude TBEV 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 specimens are reported to the Medical Officer of Health as per the Ontario Health Protection and Promotion Act.

References

European Centre for Disease Prevention and Control. 2025. Tick-borne encephalitis. Available from: https://www.ecdc.europa.eu/en/tick-borne-encephalitis.
Public Health Agency of Canada. 2025. Tick-borne encephalitis (TBE). Available from: https://www.canada.ca/en/public-health/services/diseases/tick-borne-encephalitis.html
National Microbiology Laboratory. 2025. Molecular Detection of Tick-borne encephalitis virus. Available from: https://cnphi.canada.ca/gts/reference-diagnostic-test/5410?alphaReturn=pathogenByLetter&alphaChar=T
National Microbiology Laboratory. 2025. Detection of antibodies against Tick-borne encephalitis virus by ELISA. Available from: https://cnphi.canada.ca/gts/reference-diagnostic-test/11260?alphaReturn=pathogenByLetter&alphaChar=T