Zika Virus

Testing Indications

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:

  • they are within 14 days of symptom onset, AND 
  • onset was within 14 days of last potential exposure1 
    • 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 exposure1 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 exposure1 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. 
  • Pregnant women with repeated potential exposure after initial negative Zika testing should be retested to evaluate for subsequent infection. 

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 complications4.

Important notice

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 February 13, 2018.

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:

  • country (or countries) visited
  • dates of travel (arrival to and departure from endemic or currently affected area2)
  • symptoms compatible with Zika virus infection 
    • 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)
    • list all relevant symptoms
  • date of symptom onset (omit if never had symptoms)
  • date of sample collection
  • 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)
  • pregnancy status (Y/ N/ Not applicable).
    • if yes, indicate date of last menstrual period (LMP) or estimated date of confinement (EDC), plus one of:
      • i. symptomatic: onset ≤14 days of specimen collection (Y/N)
      • ii. asymptomatic: potential Zika exposure1 ≤ 14 days prior to specimen collection. (Y/N/collection date unknown)
    • if fetal or neonatal ultrasound performed, describe findings (normal, fetal microcephaly, CNS calcifications, other)
  • 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)
  • female or male who is part of a couple trying to get pregnant within 2 or 6 months, respectively, of departure from an area with Zika transmission. (Y/N)4
    • a relevant medical reason must be provided to justify testing instead of deferring conception attempt.

Alternatively, 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.

Specimen Collection and Handling

Specimen Requirements

Test Requested Required Requisition(s) Specimen Type Minimum Volume Collection Kit

Zika Virus Serology

Blood / serum

2 x 2.0 ml blood or 2x 1.0 ml serum

Vaccutainer or serum separator tubes (SST)

Zika Virus Serology

CSF

400 µl

Sterile container

Zika Virus PCR (Molecular Testing)

Urine, or amniotic fluid, or CSF, or tissue

5.0 ml (Urine)

400 µl (CSF, amniotic fluid)

1.0 gram (Tissue)

Sterile container

Submission and Collection Notes

1

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. Serum is the preferred specimen type for both PCR and serology testing.

2

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.

3

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.)

4

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.

5

Urine should be collected on all neonates investigated for Zika virus within 48 hours postpartum.

6

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.

7

Instructions for using SST tubes are found in the document titled: LAB-SD-008, Blood Collection using Serum Separator Tubes

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 exposure1 to Zika virus2.

Limitations

Hemolysed, icteric, lipemic or microbially contaminated sera or plasma are not recommended for testing.

Preparation Prior to Transport

Label the specimen container with the patient’s full name, date of collection and one other unique identifier such as the patient’s date of birth or Health Card Number. Failure to provide this information may result in rejection or testing delay.

Special Instructions

  • 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

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

Serology is performed at the National Microbiology Laboratoroy (NML). Turnaround time is one month from receipt by PHO laboratory.

Molecular testing is performed by PHOL and by NML.

PHO laboratory turnaround time for molecular testing is up to 5 days from receipt at PHO laboratory and 10 days if tested at NML.

Turnaround time may be longer if supplementary testing/ gene sequencing is required.

Reporting

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.

Test Methods

Testing methods for Zika virus include molecular testing and serology. Molecular testing using RT-PCR assay is offered at the Public Health Ontario laboratory since March 14, 2016 for specimens meeting testing criteria for Zika virus PCR3.

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 PHO laboratory General Test Requisition. 

 

Algorithm

See Testing Guidance Table

Specimens submitted for Zika virus testing will undergo the following investigations, where indicated:

  1. Zika virus real-time PCR at PHO laboratory3, 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.  
  2. 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.
  3. 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 exposure1 will also be tested by PCR. 
  4. 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 exposure1, and serology testing if collected beyond that period, as described above. 
  5. 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. 
  6. Specimens submitted from non-pregnant patients who never exhibited symptoms or have recovered from their illness without evidence of complications will not be routinely accepted for testing4.

Interpretation

Zika virus infection is laboratory-confirmed by either one or a combination of the following:

  1. Detection of Zika virus by RT-PCR
  2. A positive Zika virus IgM (or dengue IgM) with Zika virus PRNT confirmation (as outlined below)
  3. 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 seropositivity5.

A negative serological or molecular (RT-PCR) result does not rule out Zika virus infection.

Footnotes

1 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: CDC Areas with Risk of Zika page.

2 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). 

3 PHO laboratory 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.

4 The Canadian Recommendations on the Prevention of Zika Virus, revised January 16, 2017, 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.
  • Serological testing of male individuals with a history of travel to an area with Zika virus transmission but no history of related symptoms will be considered if their partners plan on becoming pregnant within 6 months of travel to an affected area.
  • Samples should be collected at least two weeks after return and follow up serology several weeks later is advised due to possible variations in immune response to viral infection.
  • Additionally, pre-conceptual testing of male or female individuals with a history of travel to an area with ZIKV transmission but no history of related symptoms will be considered on a case-by-case basis by the NML if conception cannot be delayed for medical reasons. In this circumstance, it is recommended that test results be used as described above. It may be necessary to discuss these cases with your local or provincial laboratory before ordering the test.”

5 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.



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Updated 4 Jan 2019