Zika Virus Specimen Submission Forms and Guidelines*

*Providers who have questions about Zika virus testing available through the MDHHS Bureau of Laboratories should contact their local health department. Contact information for Michigan local health departments is available on the Directory of Michigan Health Departments


Testing at the Michigan Department of Health and Human Services- Bureau of Labs (BOL)


Only specimens collected from patients meeting CDC’s clinical and epidemiological criteria should be tested for Zika virus, RNA or IgM. Top priority for public health’s response to Zika infection is to protect pregnant women and their unborn babies. Therefore testing is focused on pregnant women with partners who live in or traveled to an area with Zika. Pregnant women at risk and symptomatic individuals (men and women) are screened for Zika if the timing falls within the CDC guidelines found below and in Figures 1-3. Areas with active Zika transmission can be found at the CDC website: Areas with Risk of Zika.

Couples planning to conceive should consider avoiding travel to areas with local Zika virus transmission. Women who are exposed to the virus through travel or unprotected sex with a partner who has traveled to an area of risk should consult with their healthcare providers. Current recommendations from the CDC are that they should wait at least 8 weeks from symptom onset or last possible exposure. Men should avoid attempting conception for at least 6 months from symptom onset or last possible exposure. The BOL cannot test women or men for purposes of family planning or to see if an individual is infectious. This should be discussed with their primary healthcare provider.

Serum and urine are the primary diagnostic specimens for Zika virus screening and diagnosis. Other specimen types such as plasma, whole blood, cerebrospinal fluid (CSF), and amniotic fluid are authorized for use with some Zika RT-PCR RNA tests that have received a Food and Drug Administration (FDA) Emergency Use Authorization (EUA). All specimens must be accompanied by an additional “paired-serum” sample (an extra tube of serum drawn at the same collection time). This extra serum sample is necessary for reflex IgM testing.

NOTE: The Michigan BOL cannot accept plasma or whole blood at this time. We request two vials of serum and one vial of urine. If CSF or amniotic fluid is submitted, please submit 1 mL aliquots.

IMPORTANT: Please be especially careful when labeling specimen vials for submission as “urine” or “serum”. These two specimens can look identical.

Screening for Zika virus infection is outlined as described below. Algorithms for these testing recommendations based upon the most recent CDC Guidelines are found in Figures 1-3.

  • For SYMPTOMATIC patients with exposure to Zika virus, RNA nucleic acid testing (NAT or PCR) of serum and urine is recommended up to 2 weeks after symptom onset. If negative, serology is performed. An additional specimen collected 2-12 weeks after symptom onset should be tested by serology if initial specimen is negative, especially if patient is pregnant.(See Figure 1)

  • RNA testing of serum and urine is recommended for ASYMPTOMATIC PREGNANT women living in areas without active Zika virus transmission

a) if the collect date is < 2 weeks after the last possible exposure

b) for those who are evaluated 2-12 weeks after exposure and have been found to be Zikavirus IgM-positive.

  • ASYMPTOMATIC PREGNANT women with exposure to Zika may be offered screening with serologic testing within 2-12 weeks after the last date of possible exposure.
  • ASYMPTOMATIC PREGNANT women who live in areas with active Zika virus transmission should have Zika virus IgM testing as part of routine obstetric care during the 1st and 2nd trimesters, with immediate RNA testing of women who are IgM-positive; a positive RNA NAT test provides a definitive diagnosis of Zika virus infection

  • Testing NEWBORNS within 48 hours of birth by RT-PCR of serum and urine, plus IgM capture for Zika infection is recommended for

(a) infants born to mothers with laboratory evidence* of possible Zika virus infection, or for

(b) infants with signs of congenital Zika** syndrome at birth, or

(c) infants with a mother at risk from living in or traveling to an area with active Zika transmission, and/or had sex without a barrier method to prevent infection with a partner who lived in or traveled to an area with active Zika transmission.

NOTE: In each case testing the placenta by RT-PCR should be considered.

*If maternal samples are collected around the time of delivery, which is >12 weeks after symptom onset or exposure, infant samples should be collected. If maternal IgM is negative, infant testing should be considered, because a negative IgM result does not rule out recent maternal Zika virus infection. If maternal IgM is positive or equivocal, infant testing should be based on maternal PRNT results (if neutralizing antibodies to Zika are detected, infant testing should be pursued).

**Microcephaly at birth is not a necessary feature of congenital Zika syndrome. Infants with a head circumference at birth in the normal range can have brain abnormalities consistent with congenital Zika syndrome. In addition, microcephaly from congenital infection can develop after birth.


It is important to note that Zika virus infection can cause signs and symptoms similar to those seen in patients with other arthropod-borne virus (arbovirus) infections, including dengue virus, a related flavivirus, and chikungunya (CHIK) virus, an unrelated alphavirus. It is also important to note that a positive result for one of these viruses does not preclude infection with the others. Co-infection with Zika virus and dengue or CHIK virus is rare, but we have seen it here in Michigan. And many infections with dengue virus have been detected in the BOL Zika laboratory. For this reason, the BOL tests for all three arbovirus infections, Zika, dengue and chikungunya, by both PCR and IgM capture ELISA. Remember that chikungunya is a BSL-3 agent, and the viral loads for CHIK in positive specimens are extremely high. Therefore, based upon a complete risk assessment, Zika virus serology testing at the BOL is performed in a BSL-2 laboratory with BSL-3 practices. (Pregnancy should be considered a significant factor in risk assessment for individuals working with Zika virus, and the involvement of pregnant workers working with Zika virus should be minimized.)


The first marker of Zika infection is RNA, detectable PCR, i.e. RT-PCR, NAT. PCR can be positive in the serum 24 hours (or less) after the onset of symptoms and usually remains detectable for 7-10 days. Urine (and saliva) can also be RNA-positive shortly after the onset of symptoms, but can remain positive for a longer period of time – up to 2 weeks. In pregnant women viremia can last longer with reports of detectable RNA in serum for 107 days after symptoms, presumably from the infected fetus. Recently whole blood has been cited as the most sensitive samples for RT-PCR, but not all laboratories have the automation required in the CDC’s standard operating procedure for whole blood extraction under the Emergency Use Authorization (EUA). Make sure the lab you are using can perform testing on the sample type you submit. (The BOL does NOT accept whole blood, plasma, semen, or saliva routinely at this time.) Cerebrospinal fluid (CSF), amniotic fluid and placenta also can be submitted for Zika RNA.  (The placenta will be forwarded to the CDC.)  The BOL performs the CDC Trioplex RT-PCR, a multiplex assay that detects RNA from Zika, dengue and/or chikungunya viruses.  The limits of detection in serum and urine for Zika virus are approximately 2.4 x 103 and 4.6 x 103 genome equivalents per mL, respectively.  The limits of detection for chikungunya and dengue viruses are approximately 1.3 x 105 and 4 x 104, genome copies per mL respectively.  Importantly, the Trioplex RT-PCR is approved only for serum specimens for chikungunya and dengue.

All positive PCR results are final and do not need to be confirmed. Negative results are reflexed to serology. For this reason ALL specimens submitted for Zika testing must be accompanied by serum.

Multiple nucleic acid tests have received EUA from FDA. FDA maintains a list on its website of all Zika virus EUAs. Please refer to the FDA website for a current list of available assays and associated letters of authorization, fact sheets and product labeling. Additional assay-specific information (e.g., performance characteristics) is included in the labeling. Information about molecular tests that have been cleared by FDA for detection of arboviruses other than Zika virus can be found in the FDA’s searchable database


Cerebrospinal Fluid: Cerebrospinal fluid is not a primary diagnostic specimen for Zika virus testing. However, if CSF is obtained during evaluation for other reasons, the specimen may be tested for the presence of anti-Zika IgM antibodies by ELISA and for the presence of Zika virus RNA by the Trioplex and possibly other molecular methods. CSF, along with a paired serum specimen, should be tested by Zika RNA NAT if collected <14 days following onset of symptoms. CSF and serum should be tested by antibody detection methods if collected >14 days after symptom onset, or if PCR is negative in samples collected <14 days after onset of symptoms.

Amniotic Fluid: If indicated, amniotic fluid may be tested by Trioplex and possibly some other EUAs, alongside paired serum and urine specimens. Consideration of amniocentesis should be individualized, because data regarding sensitivity and specificity of Zika virus testing at different time points during pregnancy to diagnose congenital Zika virus infection are limited. The presence of Zika virus RNA in the amniotic fluid might indicate fetal infection; however, a negative result does not exclude congenital Zika virus infection. Please see Oduyebo et al, 2016, for additional information regarding testing of amniotic fluid.

Tissue Specimens: There are currently no FDA authorized tests for Zika virus testing of tissue specimens, however, Zika, dengue, and chikungunya virus testing on fixed and frozen tissue at CDC may be considered on a case-by-case basis. Fixed tissues are preferred. Requests for testing should be coordinated through the BOL or local/state health departments; pre-approval is required before submission to CDC. Additional information about specimen collection and submission procedures is available on CDC’s website.


(Fact Sheet for Patients: Understanding Results from the Zika MAC-ELISA)

IgM rises after RNA, generally at 4-5 days and lasts for 12 weeks in most individuals, ranging from 8-12 weeks. It may be missed in specimens drawn <4 days from the onset of symptoms. Therefore, if the IgM is drawn early and yields a negative result in a pregnant patient (or her partner who exhibits the symptoms of Zika infection), an additional serum specimen should be drawn and submitted at a later date. (Note on the tube that this is a pregnancy follow-up serology for Zika.) Some laboratories use alternate EUA IgM test kits for detecting Zika IgM. Check to see if serum or plasma is preferred. The BOL requires serum. For all tests performed, a confirmation plaque-reduction, neutralization assay (PRNT) is required if the result is positive, equivocal or inconclusive for Zika IgM.  Sera are sent either to the CDC or to the BOL (Michigan residents) for PRNT confirmation.  A negative Zika IgM is final, unless the patient is pregnant and has not had a Zika PCR performed.  (Note when a follow-up specimen is recommended.)


On August 17, 2016, FDA issued an EUA for emergency use of InBios International, Inc.’s ZIKV Detect™ IgM Capture ELISA for the presumptive detection of Zika virus IgM antibodies in human sera. This is the first commercially available serological test for Zika available under EUA (the first serological test, the CDC Zika MAC-ELISA, was initially authorized for use in February 2016). This test is intended for use with human sera collected from individuals meeting CDC Zika virus clinical criteria (e.g., a history of clinical signs and symptoms associated with Zika virus infection) and/or CDC Zika virus epidemiological criteria (e.g., history of residence in or travel to a geographic region with active Zika transmission at the time of travel, or other epidemiological criteria for which Zika virus testing may be indicated).

Where there are presumptive Zika positive, possible Zika positive, or presumptive other flavivirus positive results from the ZIKV Detect™ IgM Capture ELISA, confirmation of the presence of anti-Zika IgM antibodies or other flavivirus IgM antibodies requires confirmation at the BOL (See the authorized Instructions for Use document.), using the latest CDC guideline for the diagnosis of Zika virus infection. Please note that the various ZIKA IgM antibody capture enzyme-linked immunosorbent assays (MAC-ELISAs): the CDC test, used by most public health laboratories and some reference laboratories, and InBios commercial kit assay, can result in discrepant results.


Currently, within the United States (but not Puerto Rico), when ELISA IgM antibody testing indicates the presence of anti-Zika IgM antibodies (positive, equivocal, presumptive or possible Zika virus positive result), PRNT, which measures virus-specific, neutralizing antibodies to Zika virus and other endemic flaviviruses, is required for diagnosis. PRNT must be conducted by CDC or a laboratory qualified by CDC. (PRNT is available at the BOL.  We have agreements with several reference laboratories to receive all specimens from Michigan residents who require Zika IgM confirmation testing.) If ELISA testing indicates a positive or equivocal result for dengue infection, confirmatory testing should be performed as indicated in the IgM assay labeling.  Given the high degree of antibody cross-reactivity observed with Zika and dengue infections, results of Zika/dengue PRNT testing should be interpreted alongside initial IgM assay results to assess the status and timing of infection.  The CDC Interim Guidance for Interpretation of Zika Virus Antibody Results (Rabe et al., 2016), contains specific information that guides the overall interpretation of combined results from Zika virus and dengue virus ELISA and PRNT.

Unfortunately PRNT is not always able to provide a definitive determination of the specific flavivirus causing a recent infection, particularly in persons with a prior history of flavivirus infection. For this reason, PRNT confirmation is not currently routinely recommended in Puerto Rico, where dengue virus is endemic and cross-reactivity is likely to occur in most cases.


It is important to note that serum is required for all diagnostic algorithms and thus a paired serum specimen must be submitted alongside all other sample types

Note to Healthcare Providers: To determine which specimen types can be tested and for specific specimen collection, handling and storage requirements, please consult the testing laboratory or the labeling information for current tests with Emergency Use Authorization.

Note to Healthcare Providers: Please do not submit urine in urine collection cups for Zika virus testing. Urine should be transferred to a clean vial with screw cap and O-ring to prevent leakage in transport.

Note to Healthcare Providers: Prior approval from your local health department or state epidemiologist is NO LONGER required. A new Michigan Zika Supplemental Questionnaire is required (See BOX B below). It must be filled out completely and sent with the patient specimen (s) to the BOL. Only pregnant or symptomatic non-pregnant patients visiting effected areas falling within the timeline recommended by the CDC Guidelines will be tested.

Testing Infants: If the infant’s initial serum sample is negative for RNA but is IgM-positive, then PRNT should be performed on the infant’s initial sample if it was not performed on the mother. However, PRNT cannot distinguish between maternal and infant antibodies at birth. For infants with an initial sample that was negative for Zika virus RNA, serologic testing (IgM followed by PRNT if indicated) at ≥18 months of life, when maternal antibody has waned can assist with diagnosis for congenital Zika virus infection.



2 mL serum for RNA + 2 mL serum for IgM + 2 mL urine for RNA

  • Always include a urine and two tubes of sera just in case the sample is reflexed for additional tests.
  • Serum MUST be submitted regardless of other sample types submitted.
  • Ship specimens on a frozen ice pack using screw-capped tubes with O-rings.
  • These shippers are available from the BOL by contacting Josh Hall at 517-335-9040 or by email at mdhhslab@michigan.gov.



  1. Patient History Form (Michigan Zika Supplemental Form) COMPLETELY Filled out.

  2. BOL Requisition Form 1 for each specimen source:

Laboratory Test Requisition


Testing algorithms (Figures 1, 2 and 3) were designed to accommodate the temporal nature of the appearance and disappearance of markers of Zika virus infection and to optimize testing for pregnant women. Figure 1: For symptomatic patients with specimens collected at <14 days post-onset of symptoms or post-exposure, test serum and urine with a Zika virus RNA NAT. (A RNA-positive Zika virus NAT result in any specimen is sufficient to diagnose Zika virus infection.) If Zika virus RNA NAT results are negative, serum should be tested for the presence of anti-Zika IgM. A reactive (Equivocal, Presumptive Positive, Inconclusive or Possible Zika Positive) anti-Zika IgM result is followed by PRNT to confirm the diagnosis. Figure 2: If specimens collected at > 14 days are Zika IgM equivocal or positive, and the patient is pregnant, perform RT-PCR prior to PRNT confirmation. Figure 3 If specimens collected at <14 days post-exposure are IgM negative by anti-Zika IgM and the patient is pregnant, recollect serum at a later date (2-12 weeks post exposure) and perform PCR on all IgM-positive or equivocal specimens prior to PRNT confirmation. (In this last case, BOL performs serology on PCR-negative specimens collected at <14 days, AND requests another specimen collected at 2-12 weeks, be submitted for repeat serology. The rational for this is that IgM antibodies CAN appear as early as 4 days after symptom onset/exposure, but false negative IgM tests are more likely for specimens collected at <14 days.)


Test results generated for each specimen should be reported to clinicians as specified in the assay instructions for use. Pregnancy status should also be reported to allow health care providers to readily identify these women. Results generated by methods used under FDA EUA must be accompanied by the appropriate fact sheets when reported back to providers and patients.  Fact Sheets have been prepared for health care providers and patients to help interpret the results of testing. Authorized Fact Sheets for each assay under EUA are posted to the FDA website

Please note that Zika, dengue and chikungunya virus infections are all on the 2016 list of nationally notifiable conditions. Therefore, results of testing should be reported back to state or local health department staff to facilitate investigation and classification of the case and reporting to CDC.


Should you have questions about Zika testing, please call 517-335-5067 and ask for one of the following:

Janice Matthews-Greer, PhD, ABMM, Virology & Immunology Section Manager

Kris Smith, Immunology Unit Manager

Bruce Robeson, Virology Unit Manager


Ades AE, Newell ML, Peckham CS, et al. European Collaborative Study. Children born to women with HIV-1 infection: natural history and risk of transmission. The Lancet. 1991; 337(8736):253–260.

Bingham AM, Cone M, Mock V, 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 Morb Mortal Wkly Rep 2016; 65. DOI: http://dx.doi.org/10.15585/mmwr.mm6518e2.

Gourinat AC, O’Connor O, Calvez E, Goarant C, Dupont-Rouzeyrol M. Detection of Zika virus in urine. Emerg Infect Dis. 2015; 21(1):84–86.

Interim Guidance for Zika Virus Testing of Urine — United States, 2016. MMWR Morb Mortal Wkly Rep 2016; 65. DOI: http://dx.doi.org/10.15585/mmwr.mm6518e1.

Oduyebo T, Igbinosa, I, Petersen EF et al. Update: Interim Guidance for Health Care Providers Caring for Pregnant Women with Possible Zika Vi.us Exposure — United States, July 2016. MMWR Morb Mortal Wkly Rep 2016; 65(29);739–744. DOI: http://www.cdc.gov/mmwr/volumes/65/wr/mm6529e1.htm?s_cid=mm6529e1_e.

Rabe IB, Staples JE, Villanueva J, et al. Interim Guidance for Interpretation of Zika Virus Antibody Test Results. MMWR Morb Mortal Wkly Rep 2016;65. DOI: http://dx.doi.org/10.15585/mmwr.mm6521e1.

Lustig Y, Mendelson E, Paran N, Melamed S, Schwartz E. 2016. Detection of Zika virus RNA in whole blood of imported Zika virus disease cases up to 2 months after symptom onset, Israel, December 2015 to April 2016. Euro Surveill. 2016; 21(26).

Russell, K, Oliver S, Lewis, L et al. Update: Interim Guidance for the Evaluation and Management of Infants with Possible Congenital Zika Virus Infection — United States, August 2016. MMWR Morb Mortal Wkly Rep 2016;65 (33);870–878DOI: https://www.cdc.gov/mmwr/volumes/65/wr/mm6533e2.htm?s_cid=mm6533e2_w(https://www.cdc.gov/mmwr/volumes/65/wr/mm6533e2.htm)

Suy A, Rodo C, Vazquez E et al. Prolonged Zika virus Viremia during Pregnancy. New Engl J Med. Dec 7, 2016; online at NEJM.org.

For information on Zika symptoms, pregnancy and exposure risk, please refer to current CDC clinical guidance(https://www.cdc.gov/zika/hc-providers/index.html)

For clinical information about chikungunya virus infection, including clinical evaluation guidance, may be found on CDC’s website(https://www.cdc.gov/chikungunya/hc/index.html)

Added 12/15/2016