Test Information Sheet

Testing Indications

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Special Indications

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Specimen Collection and Handling

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Specimen Requirements

Test Requested Required Requisition(s) Specimen Type Minimum Volume Collection Kit
Serology and/or PCR
Whole blood, serum (preferred) 2 tubes (when possible), each containing 2 to 5ml blood  Serum Separator Tube (SST) or red top vacutainer
Kit not provided by PHO
PCR
CSF, urine, tissue, amniotic fluid 1.0ml serum Sterile Container
Order #390087

Submission and Collection Notes

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
Urine should be collected on all neonates investigated for Zika virus within 48 hours postpartum.
4
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.

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.*

Limitations

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Preparation Prior to Transport

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.

Special Instructions

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

Test Frequency and Turnaround Time (TAT)

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.

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 (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.

Algorithm

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 routinely accepted for testing..

Interpretation

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.

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

Labstracts

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Data and Analysis

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: http://www.cdc.gov/zika/geo/index.html

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. 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.

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 .

Updated 8 July 2020