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 November 2 , 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. Order
relevant Zika virus testing as directed in the Testing Guidance Table. In addition, fill in the Mandatory Information Intake Form for Zika Virus Testing which requests the following mandatory information:
a. Country (or countries) visited
b. Dates of travel (arrival to and departure from endemic or currently affected area*)
c. Symptoms compatible with Zika virus infection
1. Currently symptomatic/recovered/never had symptoms (NOTE: testing of non-pregnant patients who recovered or never had symptoms is not recommended; see Testing Indications section below for more information)
2. List all relevant symptoms
d. Date of symptom onset (omit if never had symptoms)
e. Date of sample collection
f. 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)
g. Pregnancy status (Y/ N/ Not applicable).
1. If yes,
i. Symptomatic: onset ≤14 days of specimen collection (Y/N)
ii. Asymptomatic: potential Zika exposure‡ ≤ 14 days prior to specimen collection.
(Y/N/collection date unknown)
2. If fetal or neonatal ultrasound performed, describe findings (normal, fetal microcephaly, CNS calcifications, other)
h. A symptomatic patient who had unprotected sexual contact within 14 days of symptom onset with a partner who, in the last 6 months, lived in or traveled to a Zika endemic or currently affected area (Y/N)
i. Male who is part of a couple trying to get pregnant for medical reasons within 6 months of his departure from a Zika endemic or currently affected 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 days to 2 weeks.
Patients with current symptoms compatible with Zika virus infection should be tested by PCR if:
a. they are within 14 days of symptom onset, AND
b. onset was within 14 days of last potential exposure‡
- 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.
As of August 22, 2016 asymptomatic pregnant women with potential Zika virus exposure‡ in the 14 days prior to sample collection will be tested for Zika virus by PCR and IgM.
Pregnant women (symptomatic or asymptomatic) who are between 2-12 weeks after exposure‡ should be tested by IgM serology. If Zika virus IgM is found to be positive or equivocal, a Zika PCR test will then be performed. Samples collected >12 weeks after exposure will be tested by Zika PRNT regardless of IgM results.
- A negative Zika IgM at 2 to 12 weeks following the last potential exposure indicates that infection is unlikely, though does not exclude it. Women who are initially tested within 14 days of last potential Zika virus exposure and are Zika PCR and IgM negative should have serology repeated 2 to 3 weeks later as IgM antibodies may not have developed at the time of initial testing.
- Serology tests may cross react with antibodies to other flaviviruses (secondary to concurrent or previous infection or vaccination). 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. When 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.
Current US Centers for Disease Control (CDC) and Public Health Agency of Canada (PHAC) guidelines do not recommend testing men and non-pregnant women who have never experienced symptoms or have had an uneventful recovery from an illness compatible with Zika virus infection without evidence of complications.#
Serology +/- molecular testing from 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. Clotted 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, 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). It is therefore recommended to collect urine in addition to clotted blood/serum for PCR testing on all patients being tested for Zika virus infection within 14 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: Initial/acute serology should be collected on all symptomatic patients and asymptomatic pregnant women at the time of first presentation. IgM antibody develops at ≥4 days after symptom onset, and usually persists for 2 to 12 weeks. Follow-up/convalescent serology should be collected at least 2 – 3 weeks after the initial serology specimen is collected.
Molecular (real-time PCR):
Clotted blood/serum and urine specimens for PCR testing should be collected as soon as possible after symptom onset, but no later than 14 days following onset of illness. Asymptomatic pregnant women should have clotted blood/serum and urine collected for PCR within 14 days of last potential exposure‡ to Zika virus.*
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.
- Clotted blood/serum 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 (at -80°C if possible) 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.
Molecular testing using RT-PCR assay is offered at the Public Health Ontario Laboratory (PHOL) since March 14, 2016 for specimens meeting testing criteria for Zika virus PCR.¶
Serology testing is performed at the National Microbiology Laboratory (NML) in Winnipeg using an in-house IgM ELISA assay developed by US CDC. Specimens meeting testing criteria for Zika virus serology are shipped from PHO Laboratory to the NML for testing. Zika IgM positive specimens will be confirmed by Zika virus plaque reduction neutralization test (PRNT).
Molecular and serology tests for dengue and Chikungunya are available and will be conducted as described in the algorithm below. Other potentially clinically relevant tests 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:
- Zika virus real-time PCR at PHO Laboratory¶, with some PCR tests repeated at NML. PCR testing will only be performed on serum and urine specimens if collected from symptomatic patients or asymptomatic pregnant women within the period specified above.
- Zika virus serology (IgM ELISA) will be performed by NML on specimens collected from symptomatic patients between 2-12 weeks post onset. In addition serology will be performed on Zika virus PCR-positive specimens from patients who are pregnant, neonates, and those with atypical clinical presentations. Samples collected from pregnant woman >12 weeks post onset or exposure will be tested by Zika PRNT regardless of their Zika IgM ELISA result.
- Zika virus IgM ELISA reactive specimens, and dengue virus IgM ELISA reactive specimens from pregnant patients, will undergo Zika virus 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. Zika virus IgM reactive or equivocal specimens collected from pregnant women 2-12 weeks after symptom onset or potential Zika exposure‡ will also be tested by PCR.
- Specimens submitted from asymptomatic pregnant patients will undergo Zika virus PCR and Zika and dengue virus IgM serology if collected within 14 days of last potential exposure‡, and serology testing if collected beyond that period, as described above.
- Chikungunya and dengue virus PCR and serology testing will be routinely performed on symptomatic pregnant patients undergoing Zika virus PCR testing to rule out alternative or concurrent diagnoses in these instances. All dengue IgM positive specimens from pregnant women will also be sent for Zika PRNT due to cross reactivity among the flavivirus assays.
- Specimens submitted from non-pregnant patients who never exhibited symptoms or have recovered from their illness without evidence of complications will not be accepted for testing #.
Zika virus infection is laboratory-confirmed by either one or a combination of the following:
a. Detection of Zika virus by RT-PCR
b. A positive Zika virus IgM (or dengue IgM) with Zika virus PRNT confirmation (as outlined below)
c. Zika virus PRNT seroconversion (greater than 4 fold increase) between acute and convalescent specimens (with absence of cross reactivity to other flaviviruses)
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 greater than 4-fold that of other flaviviruses (e.g. dengue) will be considered confirmed seropositive for Zika virus. Those with titres 4 fold or less that of comparator flaviviruses will be considered inconclusive for Zika virus seropositivity.µ
A negative serological or molecular (RT-PCR) result does not rule out Zika virus infection.
‡ Zika virus exposure is defined as travel to a Zika endemic or currently affected area, or unprotected sexual contact with a partner who, in the last 6 months, lived in or travelled to a Zika endemic or currently affected area. For current information about areas with active Zika virus transmission see: http://www.cdc.gov/zika/geo/index.html
* In most cases, Zika virus is detected by PCR in serum up to 7 days, and in urine up to 14 days, following symptom onset. On some occasions, Zika virus viremia has persisted for several days longer, and in some cases has been shown to persist in the blood of pregnant women for more extended periods. PCR sensitivity will be maximized if specimens are collected earlier in the course of illness, and preliminary data suggests urine is more sensitive during all stages of acute illness so should be submitted on all patients undergoing PCR testing (see final reference below).
¶ 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 sensitivity and specificity. The commercial assay will allow PHOL to shorten the TAT for molecular testing, and will be used as PHOL’s principal assay going forward.
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 symptomatic pregnant women will continue to be sent to NML for replicate testing.
# 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.”
µ As of May 2016, to increase assay specificity, NML increased the PRNT cutoff titre for Zika serology to be interpreted as seropositive from ≥4 fold the titre of the comparator flavivirus (usually dengue) to >4 fold when both are tested in parallel .
For further information and reference documents 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.