Zika Virus
Testing Indications
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 (PHAC), is available within the document Zika virus: For Health Professionals.
As of March 25, 2020, CATMAT has modified the recommendations for screening and management of Zika virus (ZIKV) advising that Viral RNA testing in Canada is only recommended for symptomatic individuals or those that fulfill certain testing criteria. Serology is no longer routinely recommended due to a lack of specificity. Testing is available for symptomatic individuals, pregnant women, and neonates/ infants born to confirmed or suspected mothers or with congenital Zika syndrome (CZS). Testing of asymptomatic individuals (men or non-pregnant women) is not recommended.
Testing Indications Table for Zika Virus:
| Clinical Features | ZIKA PCR1 | ZIKA Serology2,3 | Comments |
|---|---|---|---|
|
Asymptomatic men/non-pregnant women who have never experienced Zika-like illness or have recovered from Zika-like illness |
Not recommended |
Not recommended |
Order other testing as clinically indicated. |
|
Symptomatic individuals or those that fulfill other routine testing criteria (symptoms compatible with Zika virus infection, and onset within 14 days of last potential exposure) |
Recommended |
Not recommended |
Specimens may be submitted for PCR testing if collected within 14 days of symptom onset. |
|
Asymptomatic pregnant women and no concern about possible congenital infection (i.e. pregnancy is progressing normally) |
Not recommended |
Not recommended |
Given the low risk of ZIKV infection with associated low specificity and risk of false positive Zika virus serology, CATMAT recommends against testing of asymptomatic pregnant women. |
|
Symptomatic pregnant women within 12 weeks of symptom onset4 |
Recommended |
Not recommended |
Given the low risk of ZIKV infection with associated low specificity and risk of false positive Zika virus serology, CATMAT recommends against routine serology testing of symptomatic pregnant women. |
|
Pregnant women who are greater than 12 weeks of exposure or onset of symptoms |
Not recommended |
Not recommended |
Given the low risk of ZIKV infection with associated low specificity and risk of false positive Zika virus serology, CATMAT recommends against routine serology testing of symptomatic pregnant women. ZIKV PCR is unlikely to remain positive beyond 12 weeks following symptom onset or Zika virus exposure. ZIKV PCR and serology should be performed if congenital infection is clinically suspected (e.g. ultrasound findings suggestive of congenital Zika syndrome) – see below |
|
Suspected or confirmed Zika virus infection in pregnancy and fetal anomaly on antenatal ultrasound (e.g., microcephaly, CNS calcifications, arthrogryposis). |
Recommended |
Recommended |
ZIKV PCR and serology testing should be performed on maternal blood; ZIKV PCR should be performed on maternal urine; amniotic fluid PCR testing should be considered. |
|
Infant born to a woman with confirmed or suspected Zika virus infection during pregnancy, or with suspected congenital Zika syndrome (e.g. microcephaly, CNS calcification, arthrogryposis) |
Recommended |
Recommended |
Neonatal specimens should be collected within 2 days of birth if possible. Zika virus PCR should be performed on:
Zika virus serology should be performed on:
|
|
Patients with an acute neurological syndrome possibly linked with Zika virus infection (e.g., Guillain-Barré syndrome) and risk factors for Zika virus infection. |
Recommended |
Recommended |
Zika virus PCR should be performed on:
Zika virus serology should be performed on:
|
|
Patients with confirmed Zika positive partners |
See comment |
Not recommended |
ZIKV PCR testing should only be performed if the exposed partner develops ZIKV symptoms. It should not be performed on asymptomatic exposed patients unless congenital infection is suspected (see above). |
Notes:
- Serum as well as urine should be submitted for PCR testing within 14 days of symptom onset. Urine should be submitted for all patients undergoing PCR testing due to its higher sensitivity than serum. ZIKV screening serology (IgM and IgG) is performed at PHO’s laboratory. ZIKV confirmatory serology plaque reduction neutralization test (PRNT) will be performed at NML on Zika virus IgM and/ or IgG reactive or indeterminate specimens.
- In the rare circumstance that ZIKV serology is performed, a convalescent specimen may be collected 2-3 weeks after the acute specimen.
- Zika virus screening serology (IgM and IgG) is performed at PHO’s laboratory. Zika virus confirmatory serology (PRNT) is performed at NML on Zika virus screening serology reactive or indeterminate specimens.
- Chikungunya and dengue virus serology and PCR testing will be routinely performed on all symptomatic pregnant patients undergoing Zika virus PCR testing.
- Cord blood is not recommended for testing due to possible difficulties differentiating fetal and maternal source of blood when sampling the umbilical cord.
- CSF serology is performed at NML.
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: 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.
The incubation period for Zika virus infection is approximately 3 to 14 days.
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.
Specimen Requirements
| Test Requested | Required Requisition(s) | Specimen Type | Minimum Volume | Collection Kit |
Zika PCR and/or Zika serology testing |
Serum |
2 tubes (if possible) of 2-5 ml each |
Serum separator tubes (SST) or red top tube |
|
Additional Zika PCR testing |
Plasma2 |
2 - 5 ml |
EDTA tube |
|
Additional Zika PCR testing |
Urine |
5.0 ml |
Sterile container |
|
Additional Zika PCR testing |
CSF5 |
400 µl |
Sterile container |
|
Additional Zika PCR testing |
Amniotic fluid5 |
400 µl |
Sterile container |
|
Additional Zika PCR testing |
Tissue |
Sterile container |
Submission and Collection Notes
Serum must be submitted for all patients investigated for Zika virus infection regardless of other specimen types collected (e.g. CSF, tissue, urine, amniotic fluid).
Although PCR testing can be performed on EDTA blood, serum is the preferred blood specimen type for both PCR and serology testing.
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 serum on all patients, including neonates, undergoing Zika virus PCR testing.
Serum and urine should be collected on all neonates investigated for Zika virus within 48 hours postpartum.
Testing of amniotic fluid, CSF or tissue must be pre-approved by PHO microbiologist. Please contact PHO’s laboratory Customer Service Centre at 416-235-6556 or 1-877-604-4567 before submission.
All specimens submitted for testing must be accompanied by a separate PHO laboratory General Test Requisition for each specimen type collected (e.g. serum, CSF). Order relevant Zika virus testing as directed in the Testing Indications 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)
- 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 specimen 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
- 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)
- If the patient is pregnant, indicate the date of last menstrual period (LMP) or estimated date of confinement (EDC), plus one of:
- 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.
Timing of Specimen Collection
Molecular (real-time PCR):
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 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).
Serology (if requested):
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.
Limitations
Hemolysed, icteric, lipemic or microbial contaminated sera or plasma are not recommended for testing.
Storage and 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.
- Serum and urine specimens should be stored at 2-8°C following collection and shipped to PHO’s 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’s laboratory on ice packs, but should be frozen (at -80°C if possible) and shipped on dry ice if delivery to PHO’s laboratory will take more than 72 hours.
Special Instructions
All specimens submitted for testing must be accompanied by a separate PHO Laboratories General Test Requisition for each specimen type collected. All fields on each requisition must be completed.
It is MANDATORY to provide all information requested on the Mandatory Information Intake Form for Zika Testing (see “Important Notice” above). Alternatively, this mandatory information may be included on the PHO laboratory requisition, but ALL information must be included. Specimens submitted with missing mandatory information will not be tested until that information is provided.
Test Frequency and Turnaround Time (TAT)
PHO’s laboratory molecular testing TAT is up to 5 days. NML testing TAT is 10 days from receipt at NML. TAT may be longer if supplementary testing/gene sequencing is required.
IgM and IgG screening serology TAT is 5 days from receipt at PHO’s laboratory.
PRNT confirmatory serology is performed at NML and the TAT is one month from receipt at PHO’s laboratory.
Testing methods for Zika virus include molecular testing and serology.
Molecular testing using a Lab developed Arbovirus Real-Time polymerase chain reaction (PCR) panel which simultaneously tests for Zika, Chikungunya and Dengue virus targets.
A RT-PCR assay is offered at PHO’s laboratory for specimens meeting testing criteria for Zika virus PCR.
IgM and IgG Zika ELISA screening serology is done at PHO’s laboratory 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, and Euroimmun Anti-Zika Virus ELISA (IgG).
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.
Other potentially clinically relevant tests will also be conducted if specifically requested on the PHOL General Test Requisition.
Algorithm
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 >12 weeks after symptom onset will be prioritized for IgG serology, and will undergo IgM serology if sufficient volume remains.
PCR results reported by PHO’s laboratory are final results.
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 in the Testing Indications table.
- 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.
- 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.
Interpretation
Zika virus infection is laboratory-confirmed by either one or a combination of the following:
- Detection of Zika virus by RT-PCR
- A positive Zika virus serology (IgM and/or IgG; or dengue IgM) with Zika virus PRNT confirmation (as outlined below)
- 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.
References
- 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.
- 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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