The availability and recommendations for Zika testing may change as the outbreak evolves. Please return to this page for the latest information for testing for Zika virus in Ontario. This page was last updated The availability and recommendations for Zika testing may change as the outbreak evolves. Please return to this page for the latest information for testing for Zika virus in Ontario. This page was last updated July 27, 2016.
All samples submitted for testing must be accompanied by a separate Public Health Ontario Laboratory General Test Requisition for each sample type collected. All fields on each requisition must be completed. In addition, fill in the Mandatory Information Intake Form for Zika Virus Testing which requests the following mandatory information:
a. Order relevant Zika virus testing as directed in the Testing Guidance Table
b. Country (or countries) visited
c. Dates of travel (arrival to and departure from affected country)
d. Symptoms compatible with Zika virus infection
1. Currently symptomatic/recovered/never had symptoms
2. List all relevant symptoms
e. Date of symptom onset (omit if never had symptoms)
f. Date of sample collection
g. History of receiving any flavivirus vaccine (e.g., Japanese encephalitis vaccine, yellow fever vaccine) or previous flavivirus infection (e.g., West Nile virus, dengue virus)
h. Pregnancy status (Y/ N/ Not applicable)
1. If fetal or neonatal ultrasound performed, describe findings (normal, fetal microcephaly, CNS calcification, other).
i. Sexual contact with a confirmed case (Y/N)
j. Male who is part of a couple trying to get pregnant for medical reasons within 6 months of his departure from a Zika endemic area (Y/N).#
1. Provide relevant medical reason to justify testing in this scenario (this information is mandatory).
Altenatively, the mandatory information can be documented on the General Test Requisition.
Please see below for full details about testing indications, specimen requirements, handling, testing algorithm, result interpretation, turnaround time, and reporting.
The incubation period for Zika virus infection is approximately 3 to 12 days.
Patients with current symptoms compatible with Zika virus infection should be tested by PCR if:
a. they are within 14 days of symptom onset (see Timing of Specimen Collection below for important information about serum and urine collection times for molecular testing) , AND
b. onset was while in an endemic area, or within 2 weeks of departure from an endemic area OR
c. onset was within 2 weeks of sexual contact with a confirmed case of Zika virus disease
• In these cases, serology will also be performed if PCR is negative or if they are pregnant, neonates, or have atypical clinical presentations irrespective of PCR results.
Asymptomatic pregnant women with potential Zika virus exposure may be tested for Zika virus; serology will be performed in this situation.
• The National Microbiology Laboratory (NML) suggests waiting one month after departure from a Zika virus endemic area before performing Zika virus serology on asymptomatic pregnant patients. Serology tests may cross react with antibodies to other flaviviruses (secondary to concurrent or previous infection or vaccination). Molecular testing is not recommended on asymptomatic pregnant women after potential exposure as viral RNA is unlikely to be present in these patients. Health care providers and their patients should be aware that the diagnostic tests for Zika virus were primarily developed for use on patients who have recovered from, or are acutely unwell with, symptomatic Zika infection. The performance of these assays (sensitivity, specificity, positive and negative predictive values) when used in asymptomatic people are not known at this time. If making use of laboratory testing in asymptomatic pregnant patients, results should be interpreted with caution, and in the context of other available clinical and epidemiological information.
Testing is not indicated in non-pregnant patients after recovery from a self-limiting illness.#
Current US Centres for Disease Control (CDC) and Public Health Agency of Canada (PHAC) guidelines do not recommend testing non-pregnant people who have never experienced symptoms compatible with Zika virus infection.#
Serology +/- molecular testing from blood, serum or CSF: two tubes (when possible), each containing 2 to 5 ml blood in serum separator tubes (SST) or 1.0 ml serum; or 400 µl of CSF. Although PCR testing can be performed on EDTA blood, NML has identified serum as the preferred specimen type for both PCR and serology testing.
Additional molecular testing (Real time PCR): 5ml of urine; 400 µl of amniotic fluid or CSF; tissue. Any of these specimens must be submitted in a tightly sealed sterile container. (Leaking specimens will be rejected.)
1. Blood or serum must be submitted on all patients investigated for Zika virus infection regardless of other specimen types collected (e.g., CSF, tissue, urine, amniotic fluid.)
2. Urine was found to be positive by PCR for a longer duration than serum (often up to 2 weeks), and recent data (see final reference below) suggests that it is also significantly more sensitive than serum for detection of Zika virus RNA during the early stages of acute infection (including during the first 5 days of illness). Thus it is recommended to collect urine for PCR testing on all patients being tested for Zika virus infection within 14 days of symptom onset; serum will also be tested by PCR if specimens are collected within 10 days of symptom onset.
3. Testing of amniotic fluid, CSF or tissue must be pre-approved by PHO microbiologist. Please contact PHO Laboratory Customer Service Centre at 416-235-6556 or 1-877-604-4567 before submission.
To order collection kits or other PHOL supplies complete the Requisition for Containers and Supplies. The form should be faxed to the Public Health Ontario Laboratory, Toronto at 416-235-5753 or your local PHOL.
Timing of specimen collection:Serology: Acute serology should be collected on symptomatic patients at the time of initial presentation. IgM antibody develops at ≥4 days after symptom onset, and usually persists for 2 to 12 weeks. Convalescent serology should be collected at least 2 – 3 weeks after the initial (acute) serology specimen is collected.*
The National Microbiology Laboratory (NML) suggests waiting one month after departure from a Zika virus endemic area before performing Zika virus serology on asymptomatic pregnant patients in order to ensure the highest sensitivity of this test.
Molecular (real-time PCR):
Serum specimens for PCR testing should be collected as soon as possible after symptom onset, but no later than 10 days following onset of illness; urine should be collected no later than 14 days after onset. In most cases, Zika virus is detected by PCR in serum up to 7 days, and in urine up to 10 days , following symptom onset; on some occasions, virus has persisted for several days longer. PCR sensitivity will be maximized if specimens are collected earlier in the course of illness.
Blood or serum: serum separator tubes (SST)
CSF, tissue, urine, amniotic fluid: sterile container
To order collection kits or other PHOL supplies complete the Requisition for Containers and Supplies
. The form should be faxed to the Public Health Ontario Laboratory, Toronto, at 416-235-5753
or your local PHOL.
Serology +/- molecular testing from blood, serum or CSF: two tubes (when possible), each containing 2 to 5 ml blood in serum separator tubes (SST) or 1.0 ml serum; or 1ml of CSF
All samples submitted for testing must be accompanied by a separate Public Health Ontario Laboratory General Test Requisition for each sample type collected. All fields on each requisition must be completed. In addition, it is MANDATORY to provide all information requested on the Mandatory Information Intake Form for Zika Virus Testing (see 'Important Notice' above). Alternatively, this mandatory information may be included on the PHOL requisition, but ALL information must be included.
Hemolysed, icteric, lipemic or microbially contaminated sera or plasma are not recommended for testing.
· 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.
· Blood and urine specimens should be stored at 2-8°C following collection and shipped to PHOL on ice packs.
· For any other specimen types for molecular testing, specimens may be stored at 2-8°C following collection and shipped to PHOL on ice packs, but should be frozen and shipped on dry ice if delivery to PHOL will take more than 72 hours.
Instructions for using SST tubes are found in the document titled: LAB-SD-008, Blood Collection Using Serum Separator Tubes.
Testing methods for Zika virus include molecular testing and serology. As of March 14, 2016, molecular (PCR) testing is offered at the Public Health Ontario (PHO) Laboratory for samples meeting criteria for Zika virus PCR testing.¶ Specimens meeting criteria for Zika virus serology testing are shipped from PHO Laboratory to the National Microbiology Laboratory (NML) in Winnipeg for testing.
Molecular testing is performed using RT-PCR, and serology testing performed with an in-house IgM ELISA assay developed by CDC; Zika IgM positive tests will be confirmed by Zika Virus plaque reduction neutralization test (PRNT).
Other potentially clinically relevant tests (e.g., chikungunya serology, dengue serology) will also be conducted if specifically requested on the PHOL General Test Requisition.
See Testing Guidance Table
Specimens submitted for Zika virus testing will undergo the following investigations, where indicated:
a. Zika virus real-time PCRs at PHO Laboratory¶, with some PCR tests repeated at NML. PCR testing will only be performed on serum and urine specimens if collected within the period specified above. Zika virus serology will be performed by NML in Zika virus PCR-positive patients who are pregnant, neonates, and those with atypical clinical presentations. Chikungunya and dengue PCR will also be performed at PHO on all specimens undergoing Zika virus PCR testing to rule out alternative or concurrent diagnoses in these instances.
b. Zika virus IgM ELISA. IgM reactive specimens will undergo neutralization (PRNT) assays at NML. Zika PRNT reactive specimens will also be tested against other relevant flaviviruses (e.g. dengue) due to possible cross reactivities among different flaviviruses.
c. Specimens submitted from asymptomatic pregnant patients will only undergo Zika virus serology testing, as described above.*
d. Specimens submitted from non-pregnant patients who never exhibited symptoms will not be accepted for testing.#
Detection of Zika virus by RT-PCR, a positive Zika virus IgM with PRNT confirmation (as outlined below), or Zika virus PRNT seroconversion (4 fold or greater increase) between acute and convalescent specimens (with absence of cross reactivity to other flaviviruses) is sufficient for laboratory confirmation of Zika virus infection.
Zika virus IgM reactive specimens are considered indicative of a recent flavivirus infection. IgM antibodies against Zika virus, dengue virus, and other flaviviruses including West Nile virus, have strong cross reactivity in serological assays; current assays cannot reliably distinguish between Zika, dengue virus and other flavivirus infections. These specimens will be further investigated by neutralization assays (PRNT).
Because PRNT can also cross react among different flaviviruses, this assay is run in parallel with other relevant flaviviruses to which the patient may have been exposed (e.g. dengue virus). Zika PRNT reactive specimens with a Zika titre ≥4-fold that of other flaviviruses (e.g. dengue) will be considered confirmed seropositive for Zika virus. Those with titres <4 fold that of comparator flaviviruses will be considered inconclusive for Zika virus seropositivity.
Detection of Zika virus by RT-PCR, a positive Zika virus IgM with PRNT confirmation (as outlined above), or Zika virus PRNT seroconversion (4 fold or greater increase) between acute and convalescent specimens (with absence of cross reactivity to other flaviviruses) is sufficient for laboratory confirmation of Zika virus infection.
A negative serological or molecular (RT-PCR) result does not rule out Zika virus infection.
¶ PHOL commenced Zika virus PCR testing and reporting on March 14, 2016 using a protocol developed at US CDC, which is also in use at NML. On July 27, 2016 PHOL implemented PCR testing by a commercial RT-PCR kit (RealStar® Zika Virus RT-PCR Kit, Altona, Hamburg). This test was verified against the US CDC’s PCR test and was found to be of similar or superior sensitivity and specificity. The commercial assay will allow PHOL to shorten the TAT for molecular testing.
As of May 18, 2016, PCR results reported by PHOL on blood and urine specimens are final results. Less commonly submitted specimens (e.g. CSF, tissue) will continue to be reported as provisional and will be sent to NML for repeat/parallel testing. Note: all specimens collected on pregnant women will continue to be sent to NML for replicate testing.
*NML suggests waiting one month after departure from a Zika virus endemic area before performing Zika virus serology on asymptomatic pregnant patients (to allow time for antibodies to develop).
# The Canadian Recommendations on the Prevention of Zika Virus, revised May 5, 2016, state: “Serologic testing may be considered for male returned travellers whose clinically compatible illness has resolved, and are at least two weeks post exposure, in order to assess for potential contagiousness to sexual partners. The same would theoretically apply to males who have travelled and remain asymptomatic but testing is not currently being offered to this group in Canada. Testing an asymptomatic individual simply out of curiosity concerning their serostatus would not be a prudent use of limited resources.”
For further information on Zika virus, see:
Serology TAT is one month.
Molecular testing TAT is up to 5 days for PHOL results; 10 days for NML results. TAT may be longer if supplementary testing/ gene sequencing is required.
STAT testing is not available.
Results are reported to the ordering physician or health care provider as indicated on the requisition.
Although Zika virus is not reportable in Ontario, positive results from patients with encephalitis are reported to the Medical Officer of Health as per Health Protection and Promotion Act.