Zika Virus - Serology and PCR

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

ZIKV is a mosquito-borne RNA virus that is a member of the Flaviviridae family.
The virus is transmitted primarily through the bite of an infected mosquito. Ongoing or previous transmission of ZIKV has been noted in countries in the Americas¹, Central Africa, Southeast Asia and the Pacific Islands².

Testing for ZIKV at PHO 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 : 12 juill. 2025.

Testing Indications

Note: A clinical risk assessment for ZIKV infection is recommended prior to submitting a test request, particularly for: individuals that are pregnant, infants born to women with suspected or confirmed ZIKV infection during pregnancy or those with symptoms of congenital Zika syndrome.

The most recent guidance on ZIKV infection prevention and disease management, including laboratory testing, has been published in the Canadian Zika Virus Prevention and Treatment Guidelines and the Zika Virus: For Health Professionals resources developed by the Committee to Advise on Tropical Medicine and Travel (CATMAT) and the Public Health Agency of Canada (PHAC), respectively.

Testing for ZIKV may be considered in individuals with^3,4:

Compatible signs/symptoms of infection and
Relevant exposure history (e.g. travel to or residence in areas with ongoing or previous ZIKV transmission, mosquito bites, sexual partner(s) with travel history to a country reporting ongoing transmission of ZIKV, among others) and/or
Pregnancy-associated clinical evidence suggestive of ZIKV infection (neonates born to women with suspected or confirmed ZIKV infection during pregnancy and/or suspicion of congenital Zika syndrome)

Molecular testing by PCR is the preferred method to detect infections caused by ZIKV. Testing for ZIKV by serology is not routinely recommended due to its lack of specificity. Testing of asymptomatic individuals, regardless of pregnancy status, is not recommended.

The table below summarizes Canadian ZIKV testing recommentations^3,4:

Table 1. Canadian testing recommendations for ZIKV from the Public Health Agency of Canada

Risk Population	ZIKV Testing Method Recommended PCR	ZIKV Testing Method Recommended Serology	Testing Comments
Asymptomatic male or non-pregnant individual - No ZIKV-like illness OR - No relevant exposures OR - Recovery from ZIKV-like illness	No	No	- ZIKV testing not recommended - Order other non-ZIKV infectious disease testing if clinically indicated.
Asymptomatic pregnant individual - No ZIKV-like illness - No relevant exposures	No	No	- ZIKV testing not recommended - Order other non-ZIKV infectious disease testing if clinically indicated.
Asymptomatic pregnant individual - No ZIKV-like illness - Relevant exposures in the past 12 weeks (e.g. travel to, residence in or a sexual partner with travel to a location with mosquito-borne transmission of ZIKV)	Possible	No	- ZIKV PCR may be considered up to 12 weeks after the last possible exposure - Consult an Infectious Diseases specialist to determine the appropriateness of testing.
Asymptomatic pregnant individual - No ZIKV-like illness - Relevant exposures occurred greater than 12 weeks ago (e.g. travel to, residence in or a sexual partner with travel to a location with mosquito-borne transmission of ZIKV)	No	No	- ZIKV testing not recommended - Order other non-ZIKV infectious disease testing if clinically indicated. - Testing should only be considered ≥12 weeks after the last possible exposure if there is a high suspicion of congenital infection (e.g., ultrasound findings or other indications)
Symptomatic male or non-pregnant individual - Acute ZIKV-like symptoms - Relevant exposures (e.g. travel to, residence in or a sexual partner with travel to a location with mosquito-borne transmission of ZIKV)	Yes	No	- ZIKV PCR can be performed on serum and urine collected within 14 days of symptom onset
Symptomatic pregnant individual - Acute ZIKV-like symptoms OR - Recovery from ZIKV-like illness - Relevant exposures (e.g. travel to, residence in or a sexual partner with travel to a location with mosquito-borne transmission of ZIKV)	Yes	No	- ZIKV PCR can be performed on maternal serum and urine collected up to 12 weeks after symptom onset
Pregnant individual with ultrasound findings consistent with congenital ZIKV infection^5,6 - Relevant exposures (e.g. travel to, residence in or a sexual partner with travel to a location with mosquito-borne transmission of ZIKV)	Yes	No	ZIKV PCR may be performed on: - neonate/infant serum and urine or CSF (if lumbar puncture was performed) - placenta and fetal tissue - amniotic fluid (if collected during delivery)
Infant born to mother with suspected or confirmed ZIKV infection during birth^5,6 - Relevant exposures (e.g. travel to, residence in or a sexual partner with travel to a location with mosquito-borne transmission of ZIKV)	Yes	Yes	ZIKV PCR may be performed on: - Neonate/infant serum and urine or CSF (if lumbar puncture performed) ZIKV IgM serology may be performed on: - serum and CSF (if lumbar puncture performed)

Acceptance/Rejection Criteria

Donor testing for Zika virus is not available at PHO’s laboratory. Specimens from patients being screened as potential donors (e.g. organ, tissue, cells, fertility etc.) should be referred to a laboratory that performs donor screening assays. Specimens received for donor screening at PHO’s laboratory will be rejected.

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
Zika virus serology	General Test Requisition Vector-borne and Zoonotic Virus Testing Intake Form	Serum	2 tubes (if possible) of 2-5 ml each (Minimum for neonates is 1 tube of 1.5 ml)	Red top or Serum separator tubes (SST)
Zika virus PCR	General Test Requisition Vector-borne and Zoonotic Virus Testing Intake Form	Serum	2 tubes (if possible) of 2-5 ml each (Minimum for neonates is 1 tube of 1.5 ml)	Red top or Serum separator tubes (SST)
Zika virus PCR	General Test Requisition Vector-borne and Zoonotic Virus Testing Intake Form	Urine	5 ml	Sterile container
Zika virus PCR	General Test Requisition Vector-borne and Zoonotic Virus Testing Intake Form	Plasma	2 - 5 ml	EDTA tube

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.

Serum should be submitted for all individuals under investigation for ZIKV. Collect serum and urine for all patients with suspected ZIKV infection, including neonates. Viral RNA can be detected for prolonged periods in urine, and it is more sensitive than serum during the early stages of acute infection (including during the first 5 days of illness).

PCR testing is not validated for specimen types not listed in the table above (e.g. amniotic fluid, CSF, tissue). 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

Molecular (real-time RT-PCR):
Specimens submitted for molecular testing (PCR) should be collected as soon as possible after the onset of symptoms (within 14 days³, unless the individual is pregnant), unless otherwise indicated via discussion with a PHO Microbiologist.

For pregnant individuals, serum and urine may be collected for ZIKV PCR testing for up to 12 weeks following symptom onset (if symptomatic) or the last possible exposure to ZIKV (if asymptomatic). Collect neonatal specimens for ZIKV PCR within 2 days of birth, if possible.

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. Collect neonatal specimens for ZIKV serology within 2 days of birth, if possible.

Limitations

Hemolysed, icteric, lipemic or microbial contaminated sera or plasma are not acceptable for testing.

Cord blood is not acceptable for testing due to potential difficulties in differentiating fetal and maternal blood sources when sampling the umbilical cord.

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, CSF and urine specimens should be stored at 2-8°C following collection and shipped to PHO’s laboratory on ice packs.

All other 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)

Molecular (real-time RT-PCR):
ZIKV PCR is performed twice per week at PHO. The TAT for ZIKV PCR testing at PHO is up to 5 business days from receipt at the laboratory. The TAT may be longer if supplementary testing/gene sequencing is required.

Serology:
The TAT for ZIKV serology testing of serum at PHO is up to 5 business days from receipt at the laboratory. Upon special request, CSF specimens are referred to the National Microbiology Laboratory (NML) for ZIKV serology. The TAT for ZIKV CSF serology is up to 21 days from receipt at PHO’s laboratory.

Confirmatory ZIKV serology is performed at the NML. The TAT for confirmatory ZIKV serology results by PRNT is 4 to 6 weeks from receipt at PHO.

Test Methods

Molecular (real-time RT-PCR):
PHO uses a laboratory-developed multiplex real-time polymerase chain reaction (RT-PCR) assay to detect Zika, Dengue and Chikungunya virus nucleic acids in parallel⁷.

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.

Serology:
Serum specimens submitted for ZIKV serology are screened for IgM and IgG antibodies at PHO using commercial Enzyme-Linked Immunosorbent Assays (ELISA). Confirmatory ZIKV serology testing is performed using a plaque reduction neutralization test (PRNT) at the NML.

Note: CSF specimens are referred to the NML for testing upon special request using a laboratory developed IgM assay.

Algorithm

Molecular (real-time RT-PCR):
Specimens submitted for ZIKV PCR will be tested by PHO’s laboratory developed test (LDT) RT-PCR assay for Dengue virus, Chikungunya virus and Zika virus.

Serology:
Serum is first screened for ZIKV antibodies (IgM and IgG) by ELISA. Specimens that are IgM and/or IgG reactive in PHO will be sent to the NML for PRNT confirmation. The NML may perform PRNT for other related Flaviviruses (e.g. Dengue) to assist with result interpretation, as appropriate8.

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.

Molecular (real-time RT-PCR):
A positive PCR result indicates that ZIKV nucleic acids were detected in the specimen and are suggestive of an acute/recent ZIKV infection.

A negative PCR result indicates that ZIKV nucleic acids were not detected. This does not exclude ZIKV infection.

Serology:
Consult the table below for interpretations of ZIKV serologic testing. Serology testing is not routinely recommended for ZIKV and results should be interpreted with caution.

Table 2. Interpretation of ZIKV Serologic 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 ZIKV nucleic acids were also not detected by PCR.
Non-reactive	Indeterminate	ZIKV antibody status inconclusive. Previous ZIKV 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 ZIKV IgG suggests a non-specific reaction.
Non-reactive	Reactive	Previous ZIKV 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	Indeterminate	ZIKV 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 ZIKV IgM or IgG antibodies suggest a non-specific reaction.
Indeterminate	Non-reactive	ZIKV antibody status inconclusive. Advise a follow-up specimen in 2 to 3 weeks to assist with interpretation. Persistent indeterminate results for ZIKV IgM suggests a non-specific reaction.
Indeterminate	Reactive	ZIKV antibody status inconclusive. Previous ZIKV 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 ZIKV IgM antibodies may suggest a non-specific reaction.
Reactive	Non-reactive	Acute or recent ZIKV infection possible. Advise a follow-up in 2 to 3 weeks to assist with interpretation. If this result was obtained on the follow-up serum specimen, a non-specific IgM reaction may be possible. Other etiologic agents may be investigated.
Reactive	Indeterminate	Acute or recent ZIKV infection possible. Advise a follow-up specimen in 2 to 3 weeks to assist with interpretation.
Reactive	Reactive	Acute or recent ZIKV infection possible. Advise a follow-up specimen in 2 to 3 weeks to assist with interpretation, if clinically indicated.

Additional notes on ZIKV serology:

Serologic cross-reactions in IgM or IgG may occur due to a recent or past infection with ZIKV or a related Flaviviruses (e.g. Dengue virus, West Nile virus). Reactive serology results may also occur following vaccination against a relevant Flavivirus.
A reactive ZIKV IgM and/or IgG result (indicating the present of anti-ZIKV antibodies) may indicate a recent or previous infection. A ≥-fold increase in ZIKV neutralizing antibody (PRNT) titres between acute and convalescent sera collected 2 to 3 weeks apart is considered indicative of seroconversion
IgM antibodies may persist for several months following infection. Serologic reactivity in this setting may reflect a recent or resolved infection.

A non-reactive ZIKV IgM and/or IgG result may indicate there was no ZIKV infection. However, it cannot exclude an acute ZIKV infection if the time elapsed between symptom onset and specimen collection was insufficient to develop antibodies. In this situation, this does not rule out ZIKV infection.

Reporting

Results are reported to the ordering physician or authorized health care provider (General O. Reg 45/22, s.18) as indicated on the requisition.

References

Pan American Health Organization. 2025. Cases of Zika Virus Disease. Available from: https://ais.paho.org/ha_viz/zika/zika_Weekly_Agg_tben.asp
World Health Organization (WHO). 2024. Countries and territories with current or previous Zika virus transmission. Available from: https://www.who.int/images/default-source/wpro/health-topic/zika/zika_2024.png?sfvrsn=d4d18799_2
Public Health Agency of Canada. 2024. Zika virus: for health professionals. Available from: https://www.canada.ca/en/public-health/services/diseases/zika-virus/health-professionals.html
Bingham AM, Cone M, Mock V, Heberlein-Larson L, Stanek D, Blackmore C, et al. Comparison of Test Results for Zika Virus RNA in Urine, Serum, and Saliva Specimens from Persons with Travel-Associated Zika Virus Disease — Florida, 2016. MMWR Morbidity and Mortality Weekly Report. 2016 May 13; 65(18):475–8.
Centers for Disease Prevention and Control. 2025. Clinical Testing and Diagnosis for Zika Virus Disease. Available from: https://www.cdc.gov/zika/hcp/diagnosis-testing/index.html
Committee to Advise on Tropical Medicine and Travel (CATMAT). 2020. Zika Virus Prevention and Treatment Recommendations. Available from: https://www.canada.ca/en/public-health/services/publications/diseases-conditions/zika-virus-prevention-treatment-recommendations.html
Mendoza EJ, Makowski K, Barairo N, Holloway K, Dimitrova K, Sloan A, et al. Establishment of a comprehensive and high throughput serological algorithm for Zika virus diagnostic testing. Diagnostic Microbiology and Infectious Disease. 2019 Jun; 94(2):140–6.
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.

* Copie d'impression non contrôlée. Valide uniquement le jour de l'impression : 12 juill. 2025.

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