Zika Virus - Serology and PCR

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This page provides information on the testing available for Zika virus (also known as Zika or Zika Fever) or ZIKV at Public Health Ontario’s (PHO) laboratory. The ZIKV is a single-stranded RNA virus that is a member of the Flaviviridae family. Testing for ZIKV at PHO’s laboratory involves PCR and/or serology depending on the specific clinical scenario.

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Testing Indications

Zika virus testing is limited to symptomatic individuals, pregnant women, and neonates/infants born to confirmed or suspected mothers or those with symptoms of congenital Zika syndrome (CZS). Refer to the Canadian Zika Virus Prevention and Treatment Guidelines and the Zika Virus: For Health Professionals resources developed by the Committee to Advise on Tropical Medicine and Travel (CATMAT) and the Public Health Agency of Canada (PHAC), respectively, for the most recent Canadian guidance on ZIKV infection prevention and disease management, including laboratory testing.

Molecular testing to detect ZIKV nucleic acids is the preferred testing modality when performed within 14 days of symptom onset.

Serology is not routinely recommended due to its lack of specificity for diagnosing recent infection.

For information on current areas of risk, refer to the CDC Zika Travel Information page.

Testing Indications Table for Zika Virus (ZIKV):

Patient category	ZIKV PCR¹	ZIKV Serology	Comments
Asymptomatic men/non-pregnant women with no ZIKV-like illness or have recovered from ZIKV-like illness²	Not recommended	Not recommended	Order other infectious disease testing as clinically indicated .
Symptomatic individuals with relevant travel, exposures or symptoms	Recommended	Not recommended	ZIKV PCR should be requested on serum and urine collected within 14 days of symptom onset or most recent exposure.
Asymptomatic pregnant women without any concern about possible congenital infection (i.e. pregnancy is progressing normally)	Not recommended	Not recommended	Not recommended due to a low risk of ZIKV infection and a risk of false positive ZIKV serology. Testing by PCR may be considered upon consultation with an Infectious Diseases specialist.
Symptomatic pregnant women within 12 weeks of symptom onset	Recommended	Not recommended	ZIKV PCR should be requested on maternal serum and urine collected within 12 weeks of symptom onset or most recent exposure.
Pregnant women who are greater than 12 weeks of exposure or onset of symptoms	Not recommended	Not recommended	Serologic testing is not recommended due to a low risk of ZIKV infection and a potential false positive ZIKV serology. Testing should only be considered if congenital infection is clinically suspected (e.g. ultrasound findings suggestive of CZS) – see below
Suspected or confirmed ZIKV infection in pregnancy and fetal anomaly on antenatal ultrasound (e.g., microcephaly, CNS calcifications, and arthrogryposis).	Recommended	Recommended	ZIKV PCR testing should be requested on maternal urine, while both ZIKV PCR and serology should be requested on maternal serum. Consider ZIKV PCR testing of amniotic fluid.
Infant born to a woman with confirmed or suspected ZIKV infection during pregnancy, or with suspected congenital infection	Recommended	Recommended	ZIKV PCR should be requested on: neonatal/infant blood and urine placenta and umbilical cord tissue. amniotic fluid (if collected during delivery) CSF (if lumbar puncture was performed). ZIKV serology should be performed on: blood CSF (if lumbar puncture was performed).
Acute ZIKV-related neurological syndrome (e.g., Guillain-Barré syndrome) and risk factors for ZIKV infection.	Recommended	Recommended	ZIKV PCR should be requested on: blood and urine CSF (if lumbar puncture was performed). ZIKV serology should be requested on: blood CSF (if lumbar puncture was performed).

Notes:

The ZIKV PCR also includes targets for Dengue and Chikungunya viruses. PCR testing for all three viruses will be performed at PHOL.
PCR should only be requested on patients with confirmed ZIKV positive partners if the exposed partner develops ZIKV-like symptoms or if congenital infection is suspected in an asymptomatic exposed patient.

Acceptance/Rejection Criteria

Donor testing for Zika virus is not available at PHO’s laboratory. Specimens from patients being screened as potential donors (e.g. organ, tissue, cells, fertility etc.) should be referred to a laboratory that performs donor screening assays. Specimens received for donor screening at PHO’s laboratory will be rejected .

Specimen Collection and Handling

Specimen Requirements

Test Requested	Required Requisition(s)	Specimen Type	Minimum Volume	Collection Kit
Zika virus PCR and/or Zika virus serology testing	General Test Requisition Mandatory Information Intake Form for Zika Virus Testing	Serum	2 tubes (if possible) of 2-5 ml each (Minimum for neonates is 1 tube of 1.5 ml)	Serum separator tubes (SST) or red top tube
Zika virus PCR testing	General Test Requisition Mandatory Information Intake Form for Zika Virus Testing	Urine	5 ml	Sterile container
Zika virus PCR testing	General Test Requisition Mandatory Information Intake Form for Zika Virus Testing	Plasma²	2 - 5 ml	EDTA tube
Zika virus PCR testing	General Test Requisition Mandatory Information Intake Form for Zika Virus Testing	CSF (Testing must be pre-approved by PHOL Microbiologist prior to receipt of specimen)	400 µl	Sterile container
Zika virus PCR testing	General Test Requisition Mandatory Information Intake Form for Zika Virus Testing	Amniotic fluid (Testing must be pre-approved by PHOL Microbiologist prior to receipt of specimen)	400 µl	Sterile container
Zika virus PCR testing	General Test Requisition Mandatory Information Intake Form for Zika Virus Testing	Tissue (Testing must be pre-approved by PHOL Microbiologist prior to receipt of specimen)		Sterile container

Submission and Collection Notes

Serum is the preferred blood specimen type for both PCR and serology. Submit serum for all patients being investigated for ZIKV infection irrespective of other specimen types collected (e.g. CSF, tissue, urine, amniotic fluid).

Collect urine and serum for PCR of all patients with suspected ZIKV infection, including neonates (for neonates collect sample within 48 hours postpartum). Viral RNA can be detected for prolonged periods in urine and it is more sensitive than serum during the early stages of acute infection (including during the first 5 days of illness).

Testing of amniotic fluid, CSF or tissue must be pre-approved by a PHO Microbiologist. Please contact PHO’s laboratory Customer Service Centre at 416-235-6556 or 1-877-604-4567 before submission.

Each specimen type submitted for testing must be accompanied by a separate PHO laboratory General Test Requisition (e.g. serum, CSF). Order the relevant ZIKV testing as indicated above. In addition, the Mandatory Information Intake Form for Zika Virus Testing MUST be completed. All fields must be completed on each requisition and the intake form.

Timing of Specimen Collection

Molecular (real-time RT-PCR):
Serum and urine should be submitted for ZIKV PCR testing if collected within 14 days of symptom onset (within 7 days is preferred), unless the patient is pregnant.

For pregnant women, serum and urine should be collected for ZIKV PCR testing as soon as possible following symptom onset or the last potential exposure (if asymptomatic). As these patients may have a prolonged viremia, specimens can be collected for ZIKV PCR testing for up to 12 weeks following symptom onset or last potential exposure to ZIKV (if asymptomatic).

Collect neonatal specimens for ZIKV PCR within 2 days of birth, if possible.

Serology:
Acute and convalescent sera should be collected for serologic testing, where applicable. The convalescent serum specimen should be collected at least 2 to 3 weeks after the initial acute specimen.

Collect neonatal specimens for ZIKV serology within 2 days of birth, if possible.

Limitations

Hemolysed, icteric, lipemic or microbial contaminated sera or plasma are not acceptable for testing.

Cord blood is not acceptable for testing due to potential difficulties in differentiating fetal and maternal blood sources when sampling the umbilical cord.

Storage and Transport

All clinical specimens must be shipped in accordance with the Transportation of Dangerous Goods Act/Regulations.

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.

All other specimens submitted for molecular testing should be stored at 2-8°C following collection and shipped to PHO’s laboratory on ice packs. If a delay in transport to PHO’s laboratory is anticipated (more than 72 hours), specimens should be frozen (at -80°C if possible) and shipped on dry ice.

Special Instructions

Each specimen submitted for testing must be accompanied by a separate PHO General Test Requisition form. All fields on each requisition must be completed.

It is MANDATORY to submit the Mandatory Information Intake Form for Zika Testing(MIIF) where specified, with all fields completed. If all of the requested information from the Intake Form has been provided on the PHO General Test Requisition, we do not require an additional Mandatory information Intake Form (MIIF) for Zika Virus testing. Specimens submitted with this mandatory information missing will not be tested until that information is provided.

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

The TAT for Zika virus PCR testing at PHO’s laboratory is up to 5 business days from receipt at the laboratory, but may be longer if supplementary testing/gene sequencing is required.

The TAT for Zika virus serology testing of serum (IgM and IgG) at PHO’s laboratory is up to 5 business days from receipt at the laboratory.

Zika virus serology testing of CSF specimens is performed at the National Microbiology Laboratory (NML) in Winnipeg. The TAT for Zika virus CSF serology is within 21 days from receipt at the laboratory.

Confirmatory Zika serologic testing by PRNT is performed at the NML. The TAT is 4 to 6 weeks from receipt at PHO’s laboratory.

Test Methods

PHO’s laboratory uses a laboratory-developed multiplex RT-PCR assay that simultaneously tests for targets in Zika, Dengue and Chikungunya viruses. PCR results reported by PHO’s laboratory are final results and results for all three targets will be released. Specimens submitted within 14 days of symptom onset will be tested by PCR.

Serum specimens submitted for ZIKV serology are screened for IgM and IgG antibodies at PHO’s laboratory using two commercial enzyme-linked immunosorbent assays (ELISA). A two-assay combination approach for ZIKV screening is recommended by the NML due to cross-reactivity between Zika and Dengue virus antibodies. PHO’s laboratory uses the InBiOS ZIKV Detect™ 2.0 IgM Capture ELISA (IgM antibodies) and the Euroimmun Anti-Zika Virus ELISA (IgG) assays for ZIKV serology testing.

Cerebrospinal fluid specimens submitted for ZIKV serology are referred to the NML for testing. The NML uses an IgM assay developed by the US CDC and a commercial IgG assay (Euroimmun Anti-Zika Virus ELISA IgG).

Confirmatory ZIKV serology testing is performed using a plaque reduction neutralization test (PRNT) at the NML. Specimens that are IgM and/or IgG reactive in PHO’s laboratory will be sent to the NML for confirmation. The NML ZIKV PRNT assay is run in parallel with PRNT for other Flaviviruses (e.g. Dengue virus), as cross-reactivity of IgM and IgG antibodies among different Flariviruses has been observed in serology screening assays.

Note: For pregnant females meeting the ZIKV serology testing criteria, Dengue and Chikungunya serology testing (IgM and IgG) will also be performed at PHOL.

Interpretation

Zika virus PCR:

A positive PCR result indicates that ZIKV nucleic acids were detected and an acute/recent ZIKV infection.
A negative PCR result indicates that ZIKV nucleic acids were not detected but does not rule out ZIKV infection.

Zika virus Serology:

Reactive or inconclusive ZIKV IgM and/or IgG serology results are indicative of a recent Flavivirus infection (ZIKV or a non-ZIKV Flavivirus). Distinguishing among Flaviviruses (e.g. ZIKV, Dengue virus, West Nile virus) by serology can prove difficult due to cross-reactivity among IgM and IgG antibodies raised against these pathogens.
Specimens that are reactive by ZIKV PRNT with a ZIKV titre greater than 4-fold that of other Flaviviruses will be considered ZIKV seropositive. Specimens with titres 4 fold or less than that of comparator Flaviviruses will be considered inconclusive for ZIKV seropositivity.
A negative serological result does not rule out ZIKV infection.

Reporting

Results are reported to the physician, authorized health care provider (General O. Reg 45/22, s.18) or submitter as indicated on the requisition.

References

Bingham AM, Cone M, Mock V, Heberlein-Larson L, Stanek D, Blackmore C, et al. Comparison of Test Results for Zika Virus RNA in Urine, Serum, and Saliva Specimens from Persons with Travel-Associated Zika Virus Disease — Florida, 2016. MMWR Morbidity and Mortality Weekly Report. 2016 May 13; 65(18):475–8.
Mendoza EJ, Makowski K, Barairo N, Holloway K, Dimitrova K, Sloan A, et al. Establishment of a comprehensive and high throughput serological algorithm for Zika virus diagnostic testing. Diagnostic Microbiology and Infectious Disease. 2019 Jun; 94(2):140–6.

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Mis à jour le 29 août 2023