Zika Virus

Testing Indications

Important notice:

The availability and recommendations for Zika testing may change as knowledge about Zika virus, its epidemiology, and associated guidelines evolve. Please refer to the Canadian Zika Virus Prevention and Treatment Guidelines, developed by the Committee to Advise on Tropical Medicine and Travel (CATMAT), for the most recent Canadian guidance for health care professionals on Zika virus infection prevention and patient disease management, including laboratory testing. Additional information on laboratory testing, provided by the Public Health Agency of Canada, is available within the document Zika virus: For Health Professionals. Please return to this webpage for the latest information on testing for Zika virus in Ontario, which is primarily based on the current Canadian guidelines referred to above. This page was last updated April 29, 2019.

All specimens submitted for testing must be accompanied by a separate Public Health Ontario Laboratories General Test Requisition for each sample type collected. Order relevant Zika virus testing as directed in the Testing Guidance Table. 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:

  • Countries visited
  • Dates of travel (arrival to and departure from an area of risk)
  • Indicate whether the patient is currently symptomatic, recovered, or 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)
  • If the patient is symptomatic, or recovered, list all relevant symptoms 
  • Date of symptom onset (omit if never had symptoms)
  • Indicate if the patient is a newborn or infant that was potentially exposed during pregnancy, regardless of 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 the patient is pregnant, indicate the date of last menstrual period (LMP) or estimated date of confinement (EDC), plus one of:
      • Pregnant Symptomatic: onset ≤12 weeks of specimen collection (Y/N)
      • Pregnant Asymptomatic: potential Zika exposure ≤ 12 weeks prior to specimen collection; (Y/N/collection date unknown). 
    • If fetal or neonatal ultrasound performed, describe findings (normal, fetal microcephaly, CNS calcifications, other)
  • Symptomatic patient who had unprotected sexual contact within 14 days of symptom onset with a partner who, in the last 2 months (if partner is female) or 3 months (if partner is male), lived in or traveled to an area of risk (Y/N)
  • Female or male who is part of a couple trying to get pregnant within 2 or 3 months, respectively, of departure from an area of risk‡, and pregnancy cannot be delayed for medical reasons. (Y/N)#
    • The relevant medical reason must be provided to justify testing instead of deferring conception attempt.

Alternatively, the above mandatory information can be documented on the General Test Requisition.

See below for full details about testing indications, specimen requirements and handling, testing algorithm, result interpretation, turnaround time, and reporting.

Testing Indications for Zika Virus:

The incubation period for Zika virus infection is approximately 3 to 14 days.

In Canada, Zika virus testing (RNA detection and serology) is only routinely recommended and available for symptomatic individuals and pregnant women. Testing of asymptomatic individuals (men or non-pregnant women) is not routinely offered #.

Nonpregnant patients with current symptoms compatible with Zika virus infection, and onset within 14 days of last potential exposure will be tested for Zika virus. Testing includes:

  • Zika virus PCR as well as IgM and IgG screening serology if they are within 14 days of symptom onset, OR
  • IgM and IgG screening serology if symptoms persist beyond 14 days.

Pregnant women (symptomatic or asymptomatic) who are within 12 weeks of exposure or symptom onset will be tested by PCR as well as IgM and IgG screening serology. If tested >12 weeks after symptom onset or most recent exposure, only serology will be conducted.

Note: The Canadian Zika Virus Prevention and Treatment Guidelines state, “Screening of asymptomatic pregnant women with possible exposure during pregnancy or during the peri-conception period should be discussed on a case-by-case basis between the woman and her health care provider. The declining incidence of Zika transmission in most areas of the world means that infection rates in this population will be very low, and the risk of false positive laboratory results, particularly for serology, correspondingly elevated. False positive diagnoses would have important implications for adverse events related to unwarranted additional testing and anxiety, as well as resource utilization. The decision whether to screen should take into account intensity of the potential exposure, the use of prevention measures, and the transmission trends at the location of potential exposure. In most cases, screening is not recommended.”….”The decision to test should include consideration of how the results of the screening tests would be used to inform subsequent decisions.”

If Zika virus IgM and/or IgG screening serology is reactive or inconclusive, the specimen will be forwarded to The National Microbiology Laboratory for confirmatory serology by plaque reduction neutralization test (PRNT).

  • A negative Zika IgM AND IgG at 1 to 2 months following the last potential exposure indicates that infection is unlikely, though does not exclude it. Pregnant women who are initially tested within this time frame and are Zika PCR and serology negative should have serology repeated 2 to 3 weeks later as 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).Healthcare 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.

Neonates or infants with confirmed fetal or maternal Zika virus infection during pregnancy, or risk factors for maternal Zika virus infection and suspected fetal anomaly on antenatal ultrasound or on assessment at birth (e.g. microcephaly, CNS calcifications, arthrogryposis) should undergo Zika virus testing. Testing will include Zika virus serology and PCR.

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.#

Specimen Collection and Handling

Specimen Requirements

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

Zika serology and/or molecular testing

Blood

2 tubes (if possible) of 2-5 ml each

(Minimum for neonates is 1 tube of 1.5 ml)

Serum separator tubes (SST)

Zika serology and/or molecular testing

Serum

1.0 ml

Serum separator tubes (SST)

Additional molecular testing

CSF

400 µl

Sterile container

Additional molecular testing

Urine

5.0 ml

Sterile container

Additional molecular testing

Amniotic fluid

400 µl

Sterile container

Additional molecular testing

Tissue

Sterile container

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

Although PCR testing can be performed on EDTA blood, serum is the preferred blood specimen type for both PCR and serology testing.

3

Urine was found to be positive by PCR for a longer duration than serum, and data suggest 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)1. It is therefore recommended to collect urine in addition to clotted blood /serum on all patients, including neonates, undergoing Zika virus PCR testing.

4

Serum and urine should be collected on all neonates investigated for Zika virus within 48 hours postpartum.

5

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.

6

To order collection kits or other PHO Laboratory 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 PHO Laboratory.

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.

If testing asymptomatic persons to assist with pregnancy planning (when conception cannot be delayed for medical reasons), testing should be done at least 14 days after last exposure to allow time for antibodies to develop. 

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. Pregnant women should also have clotted blood/serum and urine collected for PCR as soon as possible following symptom onset or last potential exposure (if asymptomatic), but because viremia can persist longer in this patient group, these can be done up to 12 weeks following symptom onset or last potential exposure to Zika virus (if asymptomatic).*

 

Limitations

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

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 PHO Laboratory on ice packs.

For any other specimens submitted for molecular testing, specimens may be stored at 2-8°C following collection and shipped to PHO Laboratory on ice packs, but should be frozen (at -80°C if possible) and shipped on dry ice if delivery to PHO Laboratory 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.

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

IgM and IgG screening serology TAT is 5 days.

PRNT confirmatory serology TAT is one month (done at NML). 

Molecular testing TAT is up to 5 days for PHO Laboratory results; 10 days for NML results. TAT 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.

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 (PHO Labatory) since March 14, 2016 for specimens meeting testing criteria for Zika virus PCR.¶ 

IgM and IgG Zika ELISA screening serology is done at PHO Laboratory as of May 6, 2019, using the following kits:

  • InBiOS ZIKV Detect™ 2.0 IgM Capture ELISA (MAC-ELISA) – possible results include Zika virus reactive; Flavivirus (non-Zika) reactive, or Zika virus non-reactive. 
  • Euroimmun Anti-Zika Virus ELISA (IgG) – possible results include Zika virus IgG reactive, indeterminate (antibody status inconclusive) or non-reactive. 

Specimens in which IgM and/or IgG antibodies are detected (i.e. reactive), or are inconclusive, are forwarded to NML for confirmatory Zika virus PRNT serology. NML may also repeat the screening IgM serology, using an in-house IgM ELISA (MAC-ELISA) developed by US CDC, as well as the IgG serology (using the same Euroimmun assay used at PHO Laboratory)

In an evaluation by NML, the InBios ZIKV Detect IgM Capture ELISA (an earlier version of the assay PHO Laboratory is using) in combination with the Euroimmun Anti-Zika Virus ELISA (IgG) demonstrated sensitivity of 97.2%, and specificity of 96.0% when compared to PRNT. Cross-reactivities of InBios ZIKV Detect IgM Capture ELISA and Euroimmun Anti-Zika Virus ELISA (IgG) were 71.5% and 50.0%, respectively, with sera positive for dengue virus antibodies2. Based on their evaluation, NML has recommended that provincial laboratories use these two assays in combination to perform Zika virus screening serology. 

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

If specimens of low volume are received for testing, priority of testing will be as follows:

  • Specimens received within 14 days of symptom onset (or last exposure) will be prioritized for PCR testing first, then IgM serology, followed by IgG serology
  • Specimens received within 2 to 12 weeks after onset of symptoms (or last exposure) will be prioritized for IgM serology, and will undergo IgG serology if sufficient specimen remains.
  • Specimens received >12 weeks after symptom onset will be prioritized for IgG serology, and will undergo IgM serology if sufficient volume remains. 
Specimens submitted for Zika virus testing will undergo the following investigations, where indicated:
  1. 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.  
  2. Zika virus serology (IgM and IgG ELISA) will be performed at PHOL on specimens collected from patients who meet testing criteria outlined above.
  3. Zika virus IgM and/or IgG ELISA reactive or inconclusive specimens 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 reactivity among different flaviviruses. 
  4. Specimens submitted from asymptomatic pregnant patients will undergo Zika virus PCR and Zika and dengue virus IgM and IgG serology if collected within 12 weeks of last potential exposure
  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 reactive 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 testing #.

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 serology (IgM and/or IgG; or dengue IgM) with Zika virus PRNT confirmation (as outlined below) 
  3. Zika virus PRNT seroconversion (greater than 4 fold increase) between initial/acute and convalescent/follow-up specimens (with absence of cross reactivity to other flaviviruses) 

Zika virus [or flavivirus (non-Zika)] IgM or IgG serology reactive or inconclusive specimens are considered indicative of a recent flavivirus infection.  IgM and IgG 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.

 

Footnotes

Zika virus exposure is defined as travel to an area of risk, or unprotected sexual contact with a partner who, in the last 2 months (if the partner is female) or 3 months (if the partner is male), lived in or travelled to an area of risk. For current information about areas with active Zika virus transmission see: or https://www.canada.ca/en/public-health/services/diseases/zika-virus/affected-countries-areas.html or https://wwwnc.cdc.gov/travel/page/zika-travel-information  

* 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 suggest that urine is more sensitive during all stages of acute illness so should be submitted on all patients undergoing PCR testing1

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 PHO Laboratory 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. 

As of May 18, 2016, PCR results reported by PHO Laboratory 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. 

As of May 6, 2019, IgM and IgG screening serology is conducted at PHO Laboratory. Specimens that are IgM and/or IgG reactive or inconclusive are forwarded to NML for confirmatory Zika virus PRNT. 

# See The Canadian Zika Virus Prevention and Treatment Recommendations, and Zika Virus: For Health Professionals, for more information on testing asymptomatic patients for Zika virus for the purpose of pregnancy planning. 

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.

References

  1. Comparison of Test Results for Zika Virus RNA in Urine, Serum, and Saliva Specimens from Persons with Travel-Associated Zika Virus Disease — Florida, 2016. Bingham AM1, Cone M, Mock V, Heberlein-Larson L, Stanek D, Blackmore C, Likos A. MMWR Morb Mortal Wkly Rep. 2016 May 13;65(18):475-8. doi: 10.15585/mmwr.mm6518e2.
  2. Establishment of a comprehensive and high throughput serological algorithm for Zika virus diagnostic testing. Diagn Microbiol Infect Dis. 2019 Jan 14. pii: S0732-8893(18)30423-1. doi: 10.1016/j.diagmicrobio.2019.01.004. [Epub ahead of print]
    Mendoza EJ, Makowski K, Barairo N, Holloway K, Dimitrova K, Sloan A, Vendramelli R, Ranadheera C, Safronetz D, Drebot MA, Wood H.
 


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Updated 6 May 2019