Chikungunya Virus- Serology and PCR

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Background
This page provides information on the testing available for Chikungunya virus (CHIKV) at Public Health Ontario (PHO).

The CHIKV is a mosquito-borne, single-stranded, enveloped RNA virus that is a member of the Togaviridae family.
The virus is transmitted through the bite of an infected mosquito in specific geographic regions.¹ The CHIKV is endemic to Africa, Central and South America, Southeast Asia, and the Indian subcontinent.^1,2

Testing for CHIKV infection involves PCR and/or serology depending on the specific clinical scenario.

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 : 04 juill. 2025.

Testing Indications

Testing for CHIKV infection is indicated in patients with:

Compatible clinical signs/symptoms
Relevant exposures (e.g. travel to or residence in a CHIKV endemic area or area with ongoing CHIKV transmission, noted mosquito bites, among others)

Individuals that develop CHIKV disease may present with a mild febrile illness (e.g. an acute onset of fever, rash and/or polyarthralgia) up to 12 days after exposure. Other acute symptoms may include malaise, headache, photophobia, nausea, vomiting, and/or pharyngitis.

The detection of CHIKV is accomplished by testing serum or plasma for the presence of viral nucleic acid or virus-specific antibodies.³ Molecular testing by PCR is preferred if the specimen is collected within 14 days of symptom onset. Testing of asymptomatic individuals is not recommended.

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
Chikungunya virus serology	General Test Requisition	Serum	5 ml blood or 1 ml serum	Red top tube or Serum separator tubes (SST)
Chikungunya virus PCR	General Test Requisition Vector-borne and Zoonotic Virus Testing Intake Form	Serum or plasma	5 ml blood or 1 ml serum/plasma	Red top tube or Serum separator tubes (SST), EDTA or heparin tubes

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.

For Chikungunya virus PCR testing, 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.

PCR testing is not validated for specimen types not listed in the table above (e.g. urine, CSF). These requests must be approved by a PHO Microbiologist. 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.

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 PCR):
Clotted blood, serum or plasma specimens for PCR testing should be collected as soon as possible after symptom onset, but no later than 14 days following onset of illness.

Limitations

Hemolysed, icteric, lipemic or microbially 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.
Clotted blood/serum/plasma specimens should be stored at 2-8°C following collection and shipped to PHO on ice packs.

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)

Serology:
CHIKV serology testing is performed once per week at PHO.
Turnaround time (TAT) is up to 8 days from receipt at PHO.

Molecular (Real-Time PCR):
CHIKV PCR is performed twice per week at PHO.
TAT is up to 5 days from receipt at PHO.

Test Methods

Serology:
Specimens submitted for CHIKV serology are tested for IgM and IgG antibodies using a commercial Enzyme Linked Immunosorbent Assay (ELISA).

Molecular (Real-Time PCR):
PHO uses a laboratory-developed (LDT) multiplex real-time polymerase chain reaction (RT-PCR) assay to detect the Chikungunya, Dengue and Zika virus nucleic acids in parallel3. All three viruses will be tested and reported if a PCR request is received for any of these viral targets. Specimens submitted within 14 days of symptom onset will be tested by PCR.

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 pathogens should be considered, where applicable.

Serology:
Consult the table below for interpretations of CHIKV serologic testing.

Table 1. Interpretation of CHIKV Serology tests

IgM ELISA	IgG ELISA	Possible Interpretation and Recommendations
Non-reactive	Non-reactive	No serological evidence of infection. Advise a follow-up specimen in 2 to 3 weeks if clinically indicated, unless CHIKV nucleic acids were also not detected by PCR.
Non-reactive	Indeterminate	CHIKV antibody status inconclusive. Previous CHIKV or other arboviral infection with waning immunity, or prior arboviral vaccination possible. Advise a follow-up specimen in 2 to 3 weeks to assist with interpretation if an acute or recent infection is suspected. Persistent indeterminate results for CHIKV IgG antibodies suggest a non-specific reaction.
Non-reactive	Reactive	Previous CHIKV or other arboviral infection, or prior arboviral vaccination likely. Advise a follow-up specimen in 2 to 3 weeks to assist with interpretation. Consider investigating other possible etiologic agents if the individual continues to have symptoms.
Indeterminate	Non-reactive	CHIKV antibody status inconclusive. Advise a follow-up specimen in 2 to 3 weeks to assist with interpretation. Persistent indeterminate results for CHIKV IgM suggests a non-specific reaction.
Indeterminate	Indeterminate	CHIKV antibody status inconclusive. Advise a follow-up specimen in 2 to 3 weeks to assist with interpretation if an acute or recent infection is suspected. Persistent indeterminate results for CHIKV IgM or IgG antibodies suggest a non-specific reaction.
Indeterminate	Reactive	CHIKV antibody status inconclusive. Previous CHIKV or other arboviral infection with waning immunity, or prior arboviral vaccination possible. Advise a follow-up specimen in 2 to 3 weeks to assist with interpretation if an acute or recent infection is suspected. Persistent indeterminate results for CHIKV IgM or IgG antibodies suggest a non-specific reaction.
Reactive	Non-reactive	Acute or recent CHIKV infection possible. Advise a follow up specimen in 2 to 3 weeks to assist with interpretation. If this result was obtained on a second serum specimen, a non-specific IgM reaction may be possible. Other etiologic agents may be investigated.
Reactive	Indeterminate	Acute or recent CHIKV infection possible. Advise a follow-up specimen in 2 to 3 weeks to assist with interpretation.
Reactive	Reactive	Acute or recent CHIKV infection possible. Advise a follow up specimen in 2 to 3 weeks to assist with interpretation, if clinically indicated.

Additional notes on CHIKV serology:

A ≥ 4-fold increase in neutralizing antibody titre between acute and convalescent sera collected 2 to 3 weeks apart is considered indicative of an acute or recent infection.
A non-reactive result (IgM/IgG not detected) may not exclude an acute CHIKV infection if the time elapsed between symptom onset and specimen collection was insufficient to develop antibodies.

Molecular (real-time RT-PCR):
A positive PCR result (Chikungunya virus detected by RT-PCR) indicates that CHIKV nucleic acids were detected in the specimen and indicative of an acute/recent infection.

A negative PCR result (Chikungunya virus not detected by RT-PCR) indicates that CHIKV nucleic acids were not detected in the specimen. This does not exclude CHIKV 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.

References

Centers for Disease Control and Prevention. Chikungunya Virus [Internet]. Atlanta: Centers for Disease Control and Prevention; [cited 2024 Jan 22]. Available from: https://www.cdc.gov/chikungunya/index.html
Centers for Disease Control and Prevention. Chikungunya Virus - Healthcare Providers: Diagnostic Testing [Internet]. Atlanta: Centers for Disease Control and Prevention; [cited 2024 Jan 22]. Available from: https://www.cdc.gov/chikungunya/hcp/diagnosis-testing/?CDC_AAref_Val=https://www.cdc.gov/chikungunya/hc/diagnostic.html
Pabbaraju K, Wong S, Gill K, Fonseca K, Tipples GA, Tellier R. 2016. Simultaneous detection of Zika, Chikungunya and Dengue viruses by a multiplex real-time RT-PCR assay. J. Clin. Virol. 83:66-71.