Zika Virus – PCR and Serology
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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 (Flaviviridae family) that is transmitted to humans through the bite of infected mosquitos.
- Transmission of ZIKV has occurred in the Americas1, Central Africa, Southeast Asia and the Pacific Islands2.
ZIKV testing may include PCR or serology depending on the clinical context. Molecular testing by PCR is the preferred method to detect acute ZIKV infection. Serologic testing for ZIKV by serology is not routinely recommended.
Asymptomatic individuals should not be tested.
Updates
- Effective May 4, 2026, ZIKV serology requests will be referred to the National Microbiology Laboratory. PHO continues to offer ZIKV PCR as the preferred diagnostic method for detecting ZIKV infection.
Testing Indications
A clinical risk assessment is recommended for ZIKV, with specific reference to travel and exposure history prior to symptom onset and possible alternative diagnoses.
Based on current Canadian ZIKV testing guidance, testing should be considered for individuals that meet the following criteria:
| Risk Group | ZIKV Testing Method PCR | ZIKV Testing Method Serology | Notes |
|---|---|---|---|
| Asymptomatic | |||
Asymptomatic male or non-pregnant individual with - No ZIKV-like illness OR |
No |
No |
- ZIKV testing not recommended |
Asymptomatic pregnant individual with - No ZIKV-like illness |
No |
No |
- ZIKV testing not recommended |
Asymptomatic pregnant individual with - No ZIKV-like illness |
Possible |
No |
- ZIKV PCR from maternal serum may be considered up to 12 weeks after the last possible exposure |
Asymptomatic pregnant individual with - No ZIKV-like illness |
No |
No |
- ZIKV testing not recommended |
| Symptomatic | |||
Symptomatic male or non-pregnant individual with - Acute ZIKV-like symptoms |
Yes |
No |
- Submit serum and urine collected within 14 days of symptom onset for ZIKV PCR |
Symptomatic pregnant individual with - Acute ZIKV-like symptoms OR |
Yes |
No |
- Submit maternal serum and urine collected within 12 weeks after symptom onset for ZIKV PCR |
| Other Recommendations5,6 | |||
Pregnant individual with prenatal ultrasound findings consistent with congenital ZIKV infection with - Relevant exposures (e.g. travel to, residence in or a sexual partner with travel to a location with mosquito-borne transmission of ZIKV) |
Yes |
Possible |
- Submit maternal serum and urine for ZIKV PCR |
Infant born to mother with suspected or confirmed ZIKV infection during birth with - 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 |
- Submit neonate/infant serum, urine or CSF (only if lumbar puncture was performed) for ZIKV PCR |
Acceptance/Rejection Criteria
ZIKV PCR will be performed for specimens that meet the eligibility criteria listed above.
Serologic testing for ZIKV will be limited to only the following groups:
- Pregnant individuals with suspected or confirmed ZIKV infection during pregnancy and evidence of compatible fetal abnormalities on a prenatal ultrasound; and
- Infants born to individuals with suspected or confirmed ZIKV infection during pregnancy, where congenital ZIKV infection is suspected.
ZIKV donor testing is not offered by PHO. Specimens collected for donor screening purposes (e.g. organ, tissue, cellular, reproductive, etc.) should be directed to laboratories that perform donor screening assays. Any specimens submitted to PHO for donor screening will be rejected.
Specimens received without the appropriate forms (See: Submission and Collection Notes) or those that do not meet the criteria above are subject to cancellation.
Specimen Requirements
| Test Requested | Required Requisition(s) | Specimen Type | Minimum Volume | Collection Kit |
Zika virus PCR |
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 |
Urine |
5 ml |
Sterile container |
|
Zika virus PCR |
Plasma |
2 - 5 ml |
EDTA tube |
|
Zika virus serology |
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) |
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.
Clinical information and relevant travel and exposure histories for vector-borne viruses must be provided on the Vector-borne and Zoonotic Virus Testing Intake Form. This information will be reviewed by a PHO Microbiologist to determine eligibility. Failure to provide this information may result in rejection or testing delays.
Serum and urine specimens should be collected from all patients under investigation for ZIKV infection, including neonates. Because viral RNA may persist longer in urine, testing can be more sensitive than serum during the early phase of acute infection, including the first 5 days of illness.
PCR testing for specimen types not listed in the table above (e.g. amniotic fluid, CSF, tissue) has not been validated and requires review by a PHO Microbiologist. Submission of the mandatory Vector-borne and Zoonotic Virus Testing Intake Form, completed with all required information, will initiate the review. Requests submitted without this form, with incomplete information or without adequate justification for testing are subject to cancellation.
Serologic testing of CSF specimens is performed at the NML only upon special request.
Timing of Specimen Collection
Molecular (real-time RT-PCR):
Specimens for ZIKV PCR testing should be collected within 14 days of symptom onset3, unless the individual is pregnant. For pregnant individuals, serum and urine specimens may be collected up to 12 weeks after symptom onset (if symptomatic) or after the last possible exposure to ZIKV (if asymptomatic). Neonatal specimens should be collected within 2 days of birth, if possible.
Serology:
Submission of both an acute specimen (collected ≥7 days after symptom onset) and a convalescent specimen (collected 2-3 weeks after the acute specimen) is required to complete laboratory testing. 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.
Test Frequency and Turnaround Time (TAT)
Molecular (real-time RT-PCR):
PHO performs ZIKV PCR twice per week with a TAT of up to 5 business days from receipt at the laboratory.
Serology:
Eligible specimens will be forwarded to the NML for serologic testing. Testing is not performed routinely and a minimum of 21 calendar days may be required for screening results after referral by PHO. Confirmatory testing of reactive specimens by PRNT may require 4 to 6 weeks.
Molecular (real-time RT-PCR):
PHO tests for nucleic acids of ZIKV, Dengue and Chikungunya viruses in parallel using a laboratory-developed multiplex real-time polymerase chain reaction (RT-PCR) assay.7 Specimens submitted within 14 days of symptom onset will be tested by PCR.
Serology:
NML performs testing, in whole or in part, using a laboratory-developed test which has not been fully validated/verified due to lack of a well-characterized panel. Reactive results are confirmed by plaque reduction neutralization testing (PRNT).
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 ZIKV.7 Results for all three viruses will be tested and reported if a PCR request is received for any of these viral targets.
Serology:
ZIKV serology requests are reviewed by a PHO Microbiologist using the information provided on the Vector-borne and Zoonotic Virus Testing Intake Form. Eligible specimens are forwarded to the National Microbiology Laboratory (NML) for ZIKV serology testing.6
Interpretation
All ZIKV 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 ‘Detected’ result indicates that ZIKV nucleic acids were detected in the specimen, consistent with an acute or recent ZIKV infection.
A ‘Not Detected’ result indicates that ZIKV nucleic acids were not detected in the specimen. However, this does not exclude a ZIKV infection.
An “Indeterminate” result indicates the test was inconclusive, and ZIKV infection cannot be confirmed or excluded. An additional specimen should be submitted for testing if there is suspicion of ZIKV infection.
Serology:
Serologic testing is not routinely recommended and results should be interpreted with caution.
Additional notes on ZIKV serology:
- A reactive ZIKV serology result may indicate detection of antibodies to ZIKV. However, IgM reactivity may occur following recent, resolved or past infection with ZIKV or a related Flavivirus due to cross-reactivity (e.g. Dengue virus, West Nile virus or Yellow Fever virus) or following recent vaccination. Confirmatory testing by PRNT is required for reactive ZIKV serology results.
- Seroconversion is determined by a ≥4-fold increase in ZIKV neutralizing antibody titre by PRNT between acute and convalescent serum specimens collected 2 to 3 weeks apart.
- A non-reactive ZIKV IgM and/or IgG result suggests the absence of detectable ZIKV antibodies. However, this does not exclude an acute ZIKV infection if specimens were collected before sufficient time had elapsed for antibody development.
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
- 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.
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