Clinical UM Guideline

This document addresses testing for Zika virus (ZIKV), a mosquito-borne flavivirus. Such testing includes RNA real time reverse transcription-polymerase chain reaction (RT-PCR), immunoglobulin M (IgM), and the plaque reduction neutralization test (PRNT).

Medically Necessary:

Zika virus testing is considered medically necessary for individuals in accordance with the recommendations of the U.S. Centers for Disease Control and Prevention (CDC).

When the CDC recommendations are updated, the updated guidance regarding testing for Zika virus disease is considered medically necessary as of the effective date of the updated recommendations.

The following tests for Zika virus disease are considered medically necessary, in accordance with the latest CDC recommendations:

• Zika RNA Real-Time RT-PCR (for serum, urine, combined serum and urine, cerebrospinal fluid, amniotic fluid and saliva);
• Zika Virus IgM and Neutralizing Antibody Titers (PRNT) (for serum testing).

Not Medically Necessary:

Zika virus testing is considered not medically necessary for individuals, when not in accordance with the most recent recommendations of the U.S. Centers for Disease Control and Prevention (CDC).

Notes: Probable Zika virus exposure is considered when an individual:

1. Resides in an area with ongoing Zika (ZIKV) transmission; or
2. Travel to a country or region with known ZIKV transmission; or
3. Has direct epidemiologic linkage to a person with laboratory evidence of recent ZIKV infection (for example, sexual contact, in utero or perinatal transmission, blood transfusion, organ transplantation); or
4. Association in time and place with a confirmed or probable case.

Clinical Criteria (signs and symptoms of Zika infection):
A person with one or more of the following:

• Acute onset of fever (measured or reported);
• Maculopapular rash;
• Arthralgia;
• Conjunctivitis;
• Guillain-Barré syndrome not known to be associated with another diagnosed etiology
• Complication of pregnancy:

Fetal loss in a mother with compatible illness and/or epidemiological risk factors; or
In utero findings of microcephaly and/or intracranial calcifications with maternal risk factors.

The following codes for treatments and procedures applicable to this document are included below for informational purposes. Inclusion or exclusion of a procedure, diagnosis or device code(s) does not constitute or imply member coverage or provider reimbursement policy. Please refer to the member's contract benefits in effect at the time of service to determine coverage or non-coverage of these services as it applies to an individual member.

When services may be Medically Necessary when criteria are met:

When services are Not Medically Necessary:
For the procedure codes listed above when criteria are not met or for situations designated in the Clinical Indications section as not medically necessary.

Summary

ZIKV is a mosquito-borne, single-stranded RNA flavivirus primarily transmitted through the bite of infected Aedes species mosquitoes. According to the CDC, ZIKV can also be spread via sexual contact, blood transfusion, and from a pregnant individual to their fetus. Most infected individuals experience mild or no symptoms; when present, symptoms may include fever, rash, arthralgia, and conjunctivitis lasting several days to a week. The virus is causally associated with congenital Zika syndrome, particularly microcephaly and other severe fetal brain anomalies when contracted during pregnancy. ZIKV infection has also been linked to Guillain-Barré syndrome, neuropathy, and myelitis in adults and children (CDC, 2025).

In 2016, the World Health Organization (WHO) declared Zika-related microcephaly a Public Health Emergency of International Concern (PHEIC), and later declared the end of the PHEIC in November of the same year. Although cases of Zika virus disease declined from 2017 onwards globally, transmission persists at low levels in several countries in the Americas and other endemic regions (WHO, 2022). Infants with possible congenital ZIKV infection should undergo RT-PCR and IgM testing of serum, urine, and cerebrospinal fluid when available.

The CDC advises against routine testing of asymptomatic pregnant persons without current or ongoing exposure due to declining transmission and limitations in interpreting serologic test results.

There is no vaccine to prevent or medicine to treat Zika virus disease. The CDC emphasizes prevention tactics, such as avoiding mosquito bites, using condoms to prevent sexual transmission, and taking travel precautions in areas with active Zika virus transmission. (CDC, 2025).

Discussion

ZIKV was originally isolated in 1947 from a sentinel primate in Uganda. According to the U.S. CDC, local mosquito-borne transmission of ZIKV in U.S. territories has been reported in the Commonwealth of Puerto Rico, the U.S. Virgin Islands, and American Samoa. Although most cases in residents of U.S. states were travel-associated, local transmission has been reported. Following the introduction and spread of ZIKV in the Americas in 2015, the number of travel-associated cases in U.S. states increased, with 5168 confirmed or probable cases of non-congenital ZIKV reported in 2016. The first autochthonous, mosquito-borne cases in the continental U.S. occurred in Florida in June 2016; local transmission peaked in August and then sharply declined. Although cases were reported from 49 states and the District of Columbia, approximately half (48%) were reported from three states (Florida 21%; New York 19%; and California 8%) (Hall, 2018). ZIKV is closely related to dengue, West Nile, Japanese encephalitis, and yellow fever viruses. Among flaviviruses, ZIKV and dengue virus share similar symptoms of infection, transmission cycles, and geographic distribution. Diagnostic testing for ZIKV infection can be accomplished using both molecular and serologic methods.

According to the initial U.S. CDC Morbidity and Mortality Weekly Report (MMWR), which was issued on May 10, 2016 and updated May 13, 2016, the following guidance was initially provided:

Criteria for ZIKV testing included persons who experienced two or more of the following symptoms: rash, fever, arthralgia or conjunctivitis during or within 2 weeks of return from an area with ZIKV activity, or who had an epidemiologic link to a ZIKV-infected traveler (sexual partner, household member, etc.). RT-PCR was routinely performed on urine, serum, or saliva specimens collected within 21 days of symptom onset. Clinicians were informed that only the serum RT-PCR and antibody tests were to be used for diagnostic purposes. Urine and saliva RT-PCR tests were only used for surveillance purposes. Serologic testing was performed on all serum specimens included in this analysis. The probable case definition criteria for ZIKV disease, based on serology, required ZIKV-specific immunoglobulin M (IgM) antibodies and no dengue virus-specific IgM antibodies detected in serum or cerebrospinal fluid.

Specimens reported as positive had cycle threshold (Ct) values ≤ 38 for at least one of the replicates in both the primary and secondary RT-PCR assays. Specimens reported as equivocal had a Ct value ≤ 38 in the primary assay, but not the secondary assay. For the purpose of this analysis, equivocal specimens were considered as negative. Specimens reported as negative had Ct values > 38 in the primary assay and were not tested further. ZIKV and dengue virus IgM antibody testing was performed at BPHL (Florida Department of Health Bureau of Public Health Laboratories) using a laboratory-developed IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) based on a CDC flavivirus MAC-ELISA protocol. In March 2016, BPHL transitioned to the Food and Drug Administration’s Emergency Use Authorization (EUA) ZIKV MAC-ELISA developed by CDC. ZIKV antigen and positive control material were provided by CDC. A positive/negative (P/N) ratio was calculated from results of the MAC-ELISA for each specimen tested and was interpreted as the following: P/N ratios < 2 were reported as negative, P/N ratios 2 - < 3 were reported as equivocal, and P/N ratios ≥ 3 were reported as presumptive positive, as defined in the EUA (Bingham; MMWR, May 13, 2016).

Additional information was provided by the CDC MMWR issued on June 3, 2016, as follows:

For persons with suspected ZIKV disease, a positive RT-PCR result confirms ZIKV infection, but a negative result does not exclude infection. In these cases, antibody testing can identify additional recent ZIKV infections. If IgM test results are positive, equivocal, or inconclusive, performing a plaque reduction neutralization test (PRNT) is needed to confirm the diagnosis. However, recent evidence suggests that a 4-fold higher titer by PRNT might not discriminate between anti-ZIKV antibodies and cross-reacting antibodies in all persons who have been previously infected with, or vaccinated against, a related flavivirus. Thus, a more conservative approach to interpreting PRNT results is now recommended to reduce the possibility of missing the diagnosis of either ZIKV or dengue virus infection.

On the basis of the available data, the CDC recommended that ZIKV RT-PCR be performed on urine collected < 14 days after onset of symptoms in individuals with suspected ZIKV disease. ZIKV RT-PCR testing of urine should be performed in conjunction with serum testing if using specimens collected < 7 days after symptom onset. A positive result in either specimen type provides evidence of ZIKV infection (Bingham MMWR; Erratum, May 13, 2016).

On July 28, 2017, the CDC MMWR issued updated interim guidance for U.S. health care providers caring for pregnant women with possible ZIKV exposure that revised the routine testing procedures for asymptomatic women who may have traveled to a region where ZIKV is circulating. This interim guidance was released in response to the declining prevalence of ZIKV in the continental U.S. and emerging evidence indicating prolonged detection of ZIKV immunoglobulin (IgM) antibodies. According to the CDC interim guidance:

As the prevalence of ZIKV declines, the likelihood of false-positive test results increases. Further, emerging data indicate that ZIKV IgM antibodies can persist beyond 12 weeks after infection, and, therefore, cannot always reliably distinguish between an infection that occurred during the current pregnancy and one that occurred before the current pregnancy.

The CDC affirms that the new guidance should NOT be seen as a sign that ZIKV infections are any less dangerous for pregnant women. Instead, these recommendations reflect the limitations of the most commonly used blood test for the virus.

In February 2025, the CDC testing guidance was updated as follows:

Asymptomatic pregnant patient

Lived in or traveled to the United States and its territories during pregnancy

• Because no confirmed cases of Zika virus disease have been detected in the United States and its territories since 2018, routine Zika virus testing is not recommended.

Traveled to an area with an active CDC Zika Travel Health Notice during pregnancy

• NAAT testing may be considered up to 12 weeks after travel.

Traveled to an area with current or past Zika virus transmission outside the United States and its territories during pregnancy

• Routine testing is not recommended.
• If the decision is made to test, NAAT testing can be done up to 12 weeks after travel.

Symptomatic pregnant patient

Lived in or traveled to an area with an active CDC Zika Travel Health Notice during pregnancy
Or had sex during pregnancy with someone living in or with recent travel to an area with an active CDC Zika Travel Health Notice

• Specimens should be collected as soon as possible after onset of symptoms up to 12 weeks after symptom onset.
• Perform dengue and Zika virus NAAT and IgM testing on a serum specimen and Zika virus NAAT on a urine specimen.
• If Zika NAAT is positive and the Zika IgM is negative, repeat NAAT test on newly extracted RNA from same specimen to rule out false-positive results.
• If both dengue and Zika virus NAATs are negative but either IgM antibody test is positive, confirmatory PRNTs should be performed against dengue, Zika, and other flaviviruses endemic to the region where exposure occurred.

Lived in or traveled to an area with current or past Zika virus transmission during pregnancy

• Specimens should be collected as soon as possible after onset of symptoms up to 12 weeks after symptom onset.
• Perform dengue and Zika virus NAAT on a serum specimen and Zika virus NAAT on a urine specimen.
• If Zika NAAT is positive, repeat test on newly extracted RNA from same specimen to rule out false-positive results.
• Perform IgM testing for dengue only.
• If dengue NAAT or IgM test is positive, this provides adequate evidence of dengue infection, and no further testing is indicated.

Had sex during pregnancy with someone living in or with recent travel to an area with current or past Zika virus transmission

• Specimens should be collected as soon as possible after onset of symptoms up to 12 weeks after symptom onset.
• Only Zika NAAT should be performed.
• If Zika NAAT is positive, repeat test on newly extracted RNA from same specimen to rule out false-positive results.

Pregnant patient having a fetus with prenatal ultrasound findings consistent with congenital Zika virus infection

Lived in or traveled during pregnancy to an area with an active CDC Zika Travel Health Notice or current or past Zika virus transmission
OR had sex during pregnancy with someone living in or with recent travel to an area with an active CDC Zika Travel Health Notice or current or past Zika virus transmission

• Zika virus NAAT and IgM testing should be performed on serum and NAAT on urine.
• If the Zika virus NAATs are negative and the IgM is positive, confirmatory PRNTs should be performed against Zika and dengue.
• If amniocentesis is being performed as part of clinical care, Zika virus NAAT of amniocentesis specimens should also be performed and results interpreted within the context of the limitations of amniotic fluid testing (CDC, 2025).
• Testing of placental and fetal tissues may also be considered.

Symptomatic non-pregnant patient

Living in or with recent travel to the United States and its territories

• Because no confirmed cases of Zika virus disease have been detected in the United States and its territories since 2018, routine Zika virus testing is not recommended.

Living in or with recent travel to an area with an active CDC Zika Travel Health Notice or current or past Zika virus transmission outside the United States and its territories

• Dengue and Zika virus NAATs should be performed on serum collected ≤ 7 days after symptom onset. A positive NAAT result typically provides evidence of acute infection.
• Perform dengue and Zika virus IgM antibody testing on NAAT-negative serum specimens and serum collected >7 days after onset of symptoms.
• If either dengue or Zika virus IgM antibody testing is positive, and definitive diagnosis is needed for clinical or epidemiologic purposes, confirmatory PRNTs should be performed against dengue, Zika, and other flaviviruses endemic to the region where exposure occurred.

Asymptomatic non-pregnant patients

• Testing for dengue or Zika viruses is not recommended for this group.

Infant with possible congenital Zika virus infection
With a mother with possible Zika virus exposure during pregnancy

• Collect specimens as soon as possible after birth.
• Zika virus NAAT and IgM testing should be performed on infant serum and NAAT on infant urine.
• If cerebrospinal fluid (CSF) is obtained for other purposes, NAAT and IgM antibody testing should be performed on CSF.
• If the infant's serum is IgM non-negative and NAAT negative, but PRNT was not performed on the mother's serum, PRNT for Zika and dengue viruses should be performed on the infant serum.
• Perform PRNT on a sample collected from an infant aged 18 months or older whose initial sample collected at birth was IgM non-negative and neutralizing antibodies were detected by PRNT in either the infant's or mother's sample.

Nucleic acid amplification test, or NAAT, is a generic term referring to all molecular tests used to detect viral genomic material. For additional information about tests for ZIKV, see: https://www.cdc.gov/zika/hcp/diagnosis-testing/. Updated February 12, 2025. Accessed on May 22, 2025.

Regarding sexual transmission and prevention, the CDC provides the following guidance (May 21, 2019):

• ZIKV can be passed through sex from a person with ZIKV to his or her partners.
• Sex includes vaginal, anal, and oral sex and the sharing of sex toys.
• ZIKV can be passed through sex even in a committed relationship.
• The timeframes that people can pass ZIKV through sex are different because ZIKV virus can stay in semen longer than in other body fluids.
• There is no available test to know if you have ZIKV in your semen or how likely you are to pass ZIKV through sex. ZIKV can stay in your semen and may be passed to your partner (and the fetus) for months after infection, even if you have no symptoms.

Current data do not definitively establish the duration of infectious Zika virus presence in semen. Although Zika virus RNA has been frequently detected in semen, its presence does not necessarily indicate infectious virus or correlate with sexual transmission risk. The largest cohort study to date found that Zika virus RNA shedding in semen declined significantly within 3 months of symptom onset, with fewer than 7% of participants showing detectable RNA beyond 90 days. The estimated mean time to RNA clearance from semen was 54 days, consistent with findings from smaller studies (CDC, 2025).

NAT is a ZIKV RNA nucleic acid test (cobas Zika, Roche Molecular Systems, Inc., Pleasanton, CA) which was authorized by the FDA under an investigational new drug application (IND) (March 30, 2016). RT-PCR is an example of an NAT test. For symptomatic pregnant women with possible exposure to ZIKV, NAT should be performed concurrently with IgM serology.

On September 29, 2016, the CDC updated its guideline recommendations for infants and children as follows:

• Infants and children can acquire ZIKV congenitally and postnatally. Guidance is available on testing, clinical management and prevention of ZIKV for children under the age of 18;
• Testing of infants with possible congenital ZIKV infection should be guided by:
  • Whether the infant has abnormalities consistent with congenital ZIKV syndrome (e.g., microcephaly, intracranial calcifications, or other brain or eye abnormalities);
  • The mother’s ZIKV testing results.
• Congenital ZIKV infection can be diagnosed by RT-PCR and through serologic testing.
• Postnatal ZIKV disease should be suspected in an infant or child < 18 years old who:
  • Has traveled to or lived in an area with active ZIKV transmission in the last 2 weeks;
  • Has two or more symptoms of ZIKV: fever, rash, conjunctivitis, or arthralgia;
  • Has another possible exposure to ZIKV (sexual contact with a person who lives in or traveled to an area with ZIKV or an association in time and place with a confirmed or probable case).
• Perinatal ZIKV disease should also be suspected in an infant in the first 2 weeks of life if:
  • The mother traveled to or resided in an affected area within 2 weeks of delivery;
  • The infant has two or more of the following manifestations: fever, rash, conjunctivitis, or arthralgia.
• For diagnosing postnatal ZIKV infection, RT-PCR is recommended during the first two weeks after symptom onset, serologic testing is recommended 2-12 weeks after symptom onset (CDC, September 29, 2016.  https://www.cdc.gov/zika/hcp/diagnosis-testing/. Accessed on May 22, 2025.

On August 30-31, 2017 the CDC, in collaboration with the American Academy of Pediatrics (AAP) and the American College of Obstetricians and Gynecologists (ACOG), convened the Forum on the Diagnosis, Evaluation, and Management of ZIKV infection among infants, “With the goal of obtaining individual expert opinion to inform development of updated guidance for diagnosing, evaluating, and managing infants with possible congenital ZIKV and to identify strategies to enhance communication and coordination of care of mothers and infants affected by ZIKV.” Additional detailed information is available at:https://www.cdc.gov/mmwr/volumes/66/wr/mm6641a1.htm#suggestedcitation. Accessed on May 20, 2025.

According to the October 7, 2016 MMWR on Zika testing, the following is provided:

Persons with possible ZIKV exposure who have symptoms of ZIKV disease should receive testing in accordance with CDC interim guidance: “Algorithm for U.S. Testing of Symptomatic Individuals.” CDC does not recommend ZIKV testing of nonpregnant persons with possible ZIKV exposure who do not have symptoms of ZIKV disease, including persons who are planning to attempt conception, or to assess the risk for sexual transmission of ZIKV. ZIKV testing for this purpose remains of uncertain value, because current understanding of the duration and pattern of shedding of ZIKV in reproductive tissues is limited. Information on the performance of serologic ZIKV testing remains limited, with falsely positive tests resulting in avoidable stress and expense and falsely negative tests providing false reassurance and possibly leading to inadvertent fetal exposure to ZIKV (Petersen; MMWR, October 7, 2016).

According to the World Health Organization (WHO, 2019), laboratory confirmation of ZIKV is considered the following:

1. Presence of ZIKV RNA or antigen in serum or other samples (e.g., saliva, tissues, urine, whole blood); or
2. IgM antibody against ZIKV positive and PRNT₉₀ for ZIKV with titre ≥ 20 and ZIKV PRNT₉₀ titre ratio ≥ 4 compared to other flaviviruses; and exclusion of other flaviviruses.

Regarding interpretation of ZIKV test results, the CDC provides the following:

For persons with suspected ZIKV disease, a positive RT-PCR result confirms ZIKV infection, and no antibody testing is indicated. However, because of the decline in the level of viremia over time and possible inaccuracy in reporting of dates of illness onset, a negative RT-PCR result does not exclude ZIKV infection. Therefore, serum IgM antibody testing for ZIKV and dengue virus infections should be performed if RT-PCR is negative. For serum specimens collected < 7 days after onset of symptoms, the combination of a negative RT-PCR result and negative IgM antibody testing suggests that there was no recent infection. However, a negative IgM antibody test, in the absence of RT-PCR testing, might reflect specimen collection before development of detectable antibodies and does not rule out infection with the viruses for which testing was performed. For specimens collected from 7 days to 12 weeks after onset of symptoms, a negative IgM antibody result to both ZIKV and dengue viruses rules out recent infection with either virus.

If either the ZIKV or dengue virus IgM antibody testing yields positive, equivocal, or inconclusive results, PRNTs against ZIKV and dengue viruses (or other flaviviruses endemic to the region where exposure occurred) should be performed. A PRNT using a 90% cutoff value with a titer ≥ 10 (the typical starting serum dilution used to establish the presence of virus-specific neutralizing antibodies) against ZIKV, together with negative PRNTs (< 10) against other flaviviruses is confirmatory for recent infection with ZIKV. A PRNT titer ≥ 10 for both ZIKV and dengue virus (or another flavivirus) provides evidence of a recent infection with a flavivirus but precludes identification of the specific infecting virus. A negative PRNT against ZIKV in a specimen that is collected > 7 days after illness onset rules out ZIKV infection. For specimens collected < 7 days after onset of symptoms, the combination of a negative RT-PCR and a PRNT titer < 10 suggests that there was no infection with ZIKV. However, in the absence of RT-PCR testing, a PRNT titer < 10 might reflect specimen collection before development of detectable neutralizing antibodies and does not rule out infection with the viruses for which testing was conducted. Without confirmatory PRNTs, it is not possible to determine whether a presumptive positive IgM antibody result against ZIKV reflects recent flavivirus infection or a false-positive result.

Regarding the adverse effects of ZIKV on the unborn fetus of infected pregnant women, Rasmussen and colleagues reviewed the available evidence in 2016 and concluded that, “Sufficient evidence has accumulated that infers a causal relationship between prenatal ZIKV and microcephaly and other severe brain anomalies” (Rasmussen, 2016). Other investigators studied the 2013-2014 ZIKV outbreak in French Polynesia where the risk of microcephaly due to ZIKV in the first trimester of pregnancy was 0.95% (95% confidence interval [CI]; 0.34 to 1.91), on the basis of 8 microcephaly cases identified retrospectively in a population of approximately 270,000 people with an estimated rate of ZIKV of 66%. Additional data was analyzed from Bahia, Yap Island, the Federated States of Micronesia and French Polynesia and assessed for the association of ZIKV risk with microcephaly cases, reported in the Brazilian Live Births Information System between July 2015 and February 2016. The investigators reported a strong association between the risk of microcephaly and ZIKV risk in the first trimester and a negligible association in the second and third trimesters. The estimated baseline risk for microcephaly was low, approximately 2 per 10,000 births, but the estimated risk due to ZIKV in the first trimester ranged from 0.88% (95% CI; 0.80 to 0.97) when an 80% overall ZIKV rate was assumed and 100% over-reporting of microcephaly cases, to 13.2% (95% CI; 12.0 to 14.4) when a 10% ZIKV rate was assumed and no over-reporting. The authors noted the uncertainties and limitations with all current estimates of microcephaly risk associated with ZIKV. Additional recent studies have revealed associations between symptomatic ZIKV during all trimesters and adverse pregnancy outcomes with potential peak risk during gestational weeks 14 to 17. Microcephaly is only one possible adverse outcome among a spectrum of conditions that may be part of congenital Zika syndrome. More data are needed to refine gestational age-specific risk estimates for microcephaly and other outcomes related to ZIKV, especially in relation to symptomatic and asymptomatic infection (Johansson, 2016). Recently ZIKV has also been associated with cases of Guillain-Barré syndrome in the infected individual.

The following is excerpted from the instructions for use of the Trioplex Real-time RT-PCR Assay (Trioplex rRT-PCR) which was developed by the CDC (2016) and has been authorized by the FDA under the Emergency Use Authorization (EUA) as follows:

The Trioplex Real-time RT-PCR assay is intended for the qualitative detection and differentiation of RNA from ZIKV, dengue virus, and chikungunya virus in human sera or cerebrospinal fluid (collected alongside a patient-matched serum specimen), and for the qualitative detection of ZIKV RNA in urine and amniotic fluid (each collected alongside a patient-matched serum specimen). The assay is intended for use with specimens collected from individuals meeting CDC ZIKV clinical criteria (e.g., clinical signs and symptoms associated with ZIKV infection) and/or CDC ZIKV epidemiological criteria (e.g., history of residence in or travel to a geographic region with active ZIKV transmission at the time of travel, or other epidemiologic criteria for which ZIKV testing may be indicated as part of a public health investigation). Testing is limited to qualified laboratories designated by the CDC. Assay results are for the identification of Zika, dengue, and chikungunya viral RNA. Viral RNA is generally detectable in serum during the acute phase of infection (approximately 7 days following onset of symptoms, if present). Positive results are indicative of current infection. Laboratories are required to report all results to the appropriate public health authorities. Within the U.S. and its territories results must be reported to CDC. Negative Trioplex rRT-PCR results do not rule out dengue, chikungunya and/or ZIKV and should not be used as the sole basis for patient management decisions. Negative results must be combined with clinical observations, patient history, and epidemiological information.

The Food and Drug Administration (FDA, 2016) issued an Emergency Use Authorization (EUA) for the CDC Zika IgM Antibody Capture Enzyme-Linked Immunosorbent Assay (Zika MAC-ELISA) for antibody testing as follows:

An enzyme-linked immunosorbent assay (ELISA) can be used to detect anti-ZIKV IgM antibodies in serum or cerebrospinal fluid; however, the ZIKV IgM ELISA can provide false-positive results because of cross-reacting IgM antibodies against related flaviviruses or nonspecific reactivity. This assay has been introduced and is being used in qualified public health and Department of Defense laboratories in the United States. The Zika MAC-ELISA is used for the qualitative detection of ZIKV IgM antibodies in serum or cerebrospinal fluid collected from persons meeting the clinical and epidemiologic criteria for suspected ZIKV disease. Results are reported as positive (termed “presumptive positive” to denote the need to perform a confirmatory PRNT), equivocal, negative, or inconclusive (i.e., results uninterpretable because of high background optical density). To resolve false-positive results that might be caused by cross-reactivity or nonspecific reactivity, presumptive positive results should be confirmed with PRNT against ZIKV, dengue, and other flaviviruses to which the person might have been exposed. In addition, equivocal and inconclusive results that are not resolved by retesting also should have PRNT performed to rule out a false-positive result…If serologic testing indicates recent flavivirus infection that could be caused by either ZIKV or dengue virus, individuals should be clinically managed for both infections because they might have been infected with either virus.

On May 23, 2019, the FDA authorized marketing of the ZIKV Detect^™ 2.0 IgM Capture ELISA (InBios International, Inc., Seattle, WA) to detect ZIKV IgM antibodies in human blood. The ZIKV Detect 2.0 IgM Capture ELISA is the first diagnostic test for ZIKV that the FDA has allowed to be marketed in the U.S. Prior to this time, no test for ZIKV had been approved or cleared by the FDA. This FDA approval was based on data review through the De Novo premarket review pathway which then serves as a resource for future FDA approvals.

Multiple additional tests have been authorized by the FDA under an EUA for ZIKV testing at FDA approved laboratories, including the cobas^® Zika test, (Roche Molecular Systems Inc., Pleasanton, CA) for screening individual blood donations. On August 15, 2018 according to the FDA, the Procleix^® Zika virus assay was approved for blood screening on the Procleix Panther^® system (Grifols Diagnostic Solutions, Inc., Barcelona, Spain). This assay is approved for detecting ZIKV in donated individual or pooled plasma specimens, making it useful for blood banks. It is also approved to test plasma or serum samples to screen other living or dead organ donors and human cells, tissues and cellular and tissue-based products. Detailed information for each authorized test is available at: http://www.fda.gov/MedicalDevices/Safety/EmergencySituations/ucm161496.htm#zika. Accessed on May 20, 2025.

On June 27, 2016, the FDA announced that Inovio Pharmaceuticals, Inc. (Plymouth, PA) and GeneOne Life Science, Inc. (Seoul, South Korea; VGXI, Inc., TX) have been given approval to commence a Phase 1 clinical trial to evaluate their ZIKV vaccine GLS-5700. This 40-subject trial is an open label, dose-ranging study which will evaluate the vaccine’s safety, tolerability and immunogenicity. The GLS-5700 assay is administered intradermally with the CELLECTRA^®, Inovio’s proprietary DNA delivery device. A preliminary report of interim analysis at 14 weeks, (following the third dose of vaccine), was published in 2017 indicating no serious adverse events. Local reactions at the vaccination site, (for example, injection-site pain, redness, swelling, and itching), occurred in approximately 50% of the participants. After the third dose of vaccine, binding antibodies, (as measured on enzyme-linked immunosorbent assay), were detected in all the participants, with geometric mean titers of 1642 and 2871 in recipients of 1 mg and 2 mg of vaccine, respectively, and neutralizing antibodies developed in 62% of the samples on Vero-cell assay. On neuronal-cell assay, there was a 90% inhibition of ZIKV infection in 70% of the serum samples and 50% inhibition in 95% of the samples reported. Although promising, the authors acknowledged that further study is warranted to determine the safety and efficacy of this vaccine (Tebas, 2017). As of August 1, 2019 this study (NCT02809443) was listed as completed.

An updated version of the study results was published in 2021 (Tebas, 2021). In addition to the previous findings, results were presented indicating protection of mice from a lethal dose of ZIKV by human postvaccination serum. IFNAR knockout mice (bred with deletion of genes encoding interferon-α and interferon-β receptors) have a defective immune system, and when infected with ZIKV normally die within 6-7 days. When these mice were first injected with human serum collected at week 14 (after the third dose of the vaccine) before being infected with ZIKV, 92% of the mice survived the infection. No mice receiving baseline (prevaccination) serum survived. These results suggest that the antibody response generated in humans by the vaccine was protective in this infection model. Despite the promising results, further studies involving larger randomized trials in a region where ZIKV is endemic will be required to address the efficacy of this ZIKV vaccine in humans.

The above assay tests and additional tests and laboratories have been authorized by the FDA under the EUA to perform NAAT for the qualitative detection of RNA for the ZIKV in human specimens, (such as serum, urine, amniotic fluid, cerebrospinal fluid). These laboratories are certified under the Clinical Laboratory Improvement Amendments of 1988 (CLIA) to perform high complexity tests, or are certified by similarly qualified non-U.S. laboratories, subject to, “The duration of the declaration that circumstances exist justifying authorization of the emergency use of in-vitro diagnostic tests for the detection of, or diagnosis of, ZIKV” (FDA, 2017).

On May 2, 2018, the FDA issued revised guidance for establishments that make donor eligibility determinations for donors of human cells, tissues, and cellular and tissue-based products: Donor Screening Recommendations to Reduce the Risk of Transmission of Zika Virus by Human Cells, Tissues, and Cellular and Tissue-Based Products; Guidance for Industry. This update supports the continuation of recommendations to screen living donors for risks of infection with ZIKV based on geographic areas with risk. On July 6, 2018, the FDA announced the availability of a revised final guidance: Revised Recommendations for Reducing the Risk of Zika Virus Transmission by Blood and Blood Components. This revised guidance replaces the August 2016 guidance, which recommended universal nucleic acid testing for ZIKV of individual units of blood donated in the U.S. states and territories. The revised guidance explains that, in order to comply with applicable testing regulations, blood establishments must continue to test all donated whole blood and blood components for ZIKV using a nucleic acid test. The revised guidance explains the basis for the FDA’s determination that pooled testing of donations using a screening test licensed for such use by the FDA is a sufficient method for complying with these regulations and effectively reducing the risk of ZIKV transmission, unless there is an increased risk of local mosquito-borne transmission of ZIKV in a specific geographic area that would trigger individual donation testing in that location. Alternatively, blood establishments may use an FDA-approved pathogen-reduction device for plasma and certain platelet products (Federal Register notice, 2018).

 According to the CDC National Notifiable Diseases Surveillance System (NNDSS), the following is provided as a summary of criteria and classifications for ZIKV and ZIKV disease (June 2016):

Clinical Criteria:

A person with one or more of the following:

• Acute onset of fever (measured or reported);
• Maculopapular rash;
• Arthralgia;
• Conjunctivitis;
• Complication of pregnancy:
  • fetal loss in a mother with compatible illness and/or epidemiologic risk factors; OR
  • in utero findings of microcephaly and/or intracranial calcifications with maternal risk factors
• Guillain-Barré syndrome not known to be associated with another diagnosed etiology.

Epidemiologic Linkage:

• Travel to a country or region with known ZIKV transmission; OR
• Sexual contact with a laboratory confirmed case of ZIKV infection; OR
• Receipt of blood or blood products within 30 days of symptom onset; OR
• Organ transplant recipient within 30 days of symptom onset; OR
• Association in time and place with a confirmed or probable case.

Case Classification:

Probable ZIKV Disease meets clinical criteria AND:

• Resides in, or has recently traveled to, an area with ongoing ZIKV transmission, OR;
• Has direct epidemiologic linkage to a person with laboratory evidence of recent ZIKV infection (for example, sexual contact, in utero or perinatal transmission, blood transfusion, organ transplantation); OR
• Association in time and place with a confirmed or probable case.

AND meets the following laboratory criteria:

• Positive ZIKV-specific IgM antibodies in serum or cerebrospinal fluid (CSF); AND
• Negative dengue virus-specific immunoglobulin M (IgM) antibodies; AND
• No neutralizing antibody testing performed; OR
• Less than four-fold difference in neutralizing antibody titers between ZIKV and dengue or other flaviviruses endemic to the region where exposure occurred.

Confirmed ZIKV Infection meets clinical criteria AND has laboratory evidence of recent ZIKV infection by:

• Detection of ZIKV by culture, viral antigen or viral ribonucleic acid (RNA) in serum, CSF, tissue, or other specimen (for example, amniotic fluid, urine, semen, saliva); OR
• ZIKV IgM antibodies in serum or CSF with ZIKV neutralizing antibody titers 4-fold or greater than neutralizing antibody titers against dengue or other flaviviruses endemic to the region where exposure occurred.

Emergency Use Authorization (EUA): A temporary approval issued by the FDA which, for the ZIKV testing, is based on data submitted by CDC to FDA, and on the U.S. Secretary of Health and Human Services’ (HHS) declaration that circumstances exist to justify the emergency use of in vitro diagnostic tests for the detection of ZIKV and/or diagnosis of ZIKV infection. “This EUA will terminate when the HHS Secretary’s declaration terminates, unless FDA revokes it sooner” (FDA, 2016).

Immunoglobulin M (IgM): A test for quantitative immunoglobulins (or antibodies) which is used to confirm an infectious process, including a diagnosis of ZIKV.

Plaque Reduction Neutralization test (PRNT): A highly specialized test of serum used to confirm a diagnosis of ZIKV when prior IgM testing has yielded positive or inconclusive results.

Real Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR or rRT-PCR) testing: A molecular test for the presence of viral RNA which is used in the diagnostic workup of suspected ZIKV and other infectious conditions. A positive RT-PCR result for ZIKV is considered conclusive.

Peer Reviewed Publications:

1. Dang J, Tiwari SK, Lichinchi G, et al. Zika Virus depletes neural progenitors in human cerebral organoids through activation of the innate immune receptor TLR3. Cell Stem Cell. 2016; 19(2):258-265.
2. Driggers RW, Ho CY, Korhonen EM, et al. Zika virus infection with prolonged maternal viremia and fetal brain abnormalities. N Engl J Med. 2016; 374(22):2142-2151.
3. Faria NR, Azevedo RS, Kraemer MU, et al. Zika virus in the Americas: early epidemiological and genetic findings. Science. 2016; 352(6283):345-349.
4. Fitzgerald B, Boyle C, Honein MA. Birth defects potentially related to Zika virus infection during pregnancy in the United States. JAMA. 2018; 319(12):1195-1196.
5. Garcez PP, Loiola EC, Madeiro da Costa R, et al. Zika virus impairs growth in human neurospheres and brain organoids. Science. 2016; 352(6287):816-818.
6. Granger D, Hilgart H, Misner L, et al. Serologic testing for Zika virus: comparison of three Zika virus IGM-screening enzyme-linked immunosorbent assays and initial laboratory experiences. J Clin Microbiol. 2017; 55(7):2127-2136.
7. Johansson MA, Mier-y-Teran-Romero L, Reefhuis J, et al. Zika and the risk of microcephaly. N Engl J Med. 2016; 375(1):1-4.
8. Mead PS, Duggal NK, Hook SA, et al. Zika virus shedding in semen of symptomatic infected men. N Engl J Med. 2018; 378(15):1377-1385.
9. Mckenzie BA, Wilson AE, Zohdy S. Aedes albopictus is a competent vector of Zika virus: a meta-analysis. PLoS. 2019; 14(5):1-16.
10. Mlakar J, Korva M, Tul N, et al. Zika virus associated with microcephaly. N Engl J Med. 2016; 374(10):951-958.
11. (No authors provided). Zika virus infection: global update on epidemiology and potentially associated clinical manifestations. Wkly Epidemiol Rec. 2016; 91(7):73-81.
12. Paz-Bailey G, Rosenberg ES, Doyle K, et al. Persistence of Zika virus in body fluids-final report. N Engl J Med. 2018; 379:1234-1243.
13. Rasmussen SA, Jamieson DJ, Honein MA, et al. Zika virus and birth defects: reviewing the evidence for causality. N Engl J Med. 2016; 374(20):1981-1987.
14. Ravichandran S, Hahn M, Belaunzarán-Zamudio PF, et al. Differential human antibody repertoires following Zika infection and the implications for serodiagnostics and disease outcome. Nat Commun. 2019; 10(1):1943.
15. Rolfe AJ, Bosco DB, Wang J, et al. Bioinformatic analysis reveals the expression of unique transcriptomic signatures in Zika virus infected human neural stem cells. Cell Biosci. 2016; 6:42.
16. Saá P, Proctor M, Foster G, et al. Investigational testing for Zika virus among U.S. blood donors. N Engl J Med. 2018; 378(19):1778-1788.
17. Saiee F, Badawi A. Maternal infection with Zika virus and prevalence of congenital disorders in infants: systematic review and meta-analysis. Can J Public Health. 2019; 110(5):638-648.
18. Santiago GA, Vázquez J, Courtney S, et al. Performance of the Trioplex real-time RT-PCR assay for detection of Zika, dengue, and chikungunya viruses. Nat Commun. 2018; 9:1391.
19. Tang H, Hammack C, Ogden SC, et al. Zika virus infects human cortical neural progenitors and attenuates their growth. Cell Stem Cell. 2016; 18(5):587-590.
20. Tebas P, Roberts CC, Muthumani K, et al. Safety and immunogenicity of an anti-Zika virus DNA vaccine. N Engl J Med. 2021; 385(12):e35.

Government Agency, Medical Society, and Other Authoritative Publications:

1. Adebanjo T, Godfred-Cato S, Viens L, et al. Update: interim guidance for the diagnosis, evaluation, and management of infants with possible congenital zika virus infection — United States, October 2017. MMWR Morb Mortal Wkly Rep. 2017; 66:1089-1099. Available at: https://www.cdc.gov/mmwr/volumes/66/wr/mm6641a1.htm#suggestedcitation. Accessed on May 22, 2025.
2. 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. Available at: http://www.cdc.gov/mmwr/volumes/65/wr/mm6518e2.htm. Accessed on May 22, 2025.
3. Centers for Disease Control (CDC). Dedicated web site for information about Zika Virus including approved laboratories designated for performing Zika testing by the CDC. Updated November 2, 2022. Available at: https://www.cdc.gov/zika/index.html. Accessed on May 22, 2025.
4. Centers for Disease Control (CDC). National Notifiable Diseases System (NNDSS). Zika Virus Disease and Zika Virus Infection 2016 Case Definition, Approved June 2016. Available at: https://ndc.services.cdc.gov/case-definitions/zika-virus-disease-and-zika-virus-infection-2016-06-01/. Page last reviewed: April 16, 2021. Accessed on May 22, 2025.
5. Centers for Disease Control (CDC). Zika Virus. About Zika. January 30, 2025. Available at: https://www.cdc.gov/zika/about/index.html. Accessed on May 22, 2025.
6. Centers for Disease Control (CDC). Zika Virus. Clinical Considerations for Pregnant Women with Possible Zika Virus Infection. Health Care Providers. January 31, 2025. Available at: https://www.cdc.gov/zika/hcp/clinical-pregnant/index.html. Accessed on May 30, 2025.
7. Centers for Disease Control (CDC). Zika Virus. Clinical Testing and Diagnosis for Zika Virus Disease. Last updated February 12, 2025. Available at: https://www.cdc.gov/zika/hcp/diagnosis-testing/?CDC_AAref_Val=https://www.cdc.gov/zika/hc-providers/testing-guidance.html. Accessed on May 22, 2025.
8. Centers for Disease Control (CDC). Zika Virus. Countries & Territories at Risk for Zika. Last updated February 25, 2025. Available at:  https://www.cdc.gov/zika/geo/index.html. Accessed on May 22, 2025.
9. Centers for Disease Control (CDC). Zika Virus. Recommendations for Travelers and People Living Abroad. Last updated February 25, 2025. Available at: https://www.cdc.gov/zika/travel/?CDC_AAref_Val=https://www.cdc.gov/zika/prevention/sexual-transmission-prevention.html. Accessed on May 21, 2025.
10. Centers for Disease Control (CDC). Testing Guidance. New zika and dengue testing guidance (Updated January 30, 2025). Available at: https://www.cdc.gov/zika/hcp/sexual-transmission/. Accessed on May 28, 2025.
11. Goodman AB, Dziuban EJ, Powell K, et al. Characteristics of Children Aged <18 Years with Zika Virus Disease Acquired Postnatally — U.S. States, January 2015-July 2016. MMWR Morb Mortal Wkly Rep. 2016; 65:1082-1085.
12. Hall V, Walker WL, Lindsey NP, et al. Update: Noncongenital Zika Virus Disease Cases — 50 U.S. States and the District of Columbia, 2016. MMWR Morb Mortal Wkly Rep. 2018; 67:265-269.
13. GeneOne Life Science, Inc., Inovio Pharmaceuticals. Study of GLS-5700 in healthy volunteers. NLM Identifier: NCT02809443. Last updated November 7, 2024. Available at: https://clinicaltrials.gov/ct2/show/NCT02809443. Accessed on May 21, 2025.
14. Oduyebo T, Igbinosa I, Petersen EE, et al. U.S. Morbidity and Mortality Weekly Report (MMWR). Update: Interim Guidance for Health Care Providers Caring for Pregnant Women with Possible Zika Virus Exposure — United States, July 2016. Morb Mortal Wkly Rep. 2016; 65:739-744.
15. Oduyebo T, Polen KD, Walke HT, et al. Update: Interim Guidance for Health Care Providers Caring for Pregnant Women with Possible Zika Virus Exposure — United States (Including U.S. Territories), July 2017. MMWR Morb Mortal Wkly Rep. 2017; 66:781-793.
16. Petersen EE, Meaney-Delman D, Neblett-Fanfair R, et al. Update: Interim Guidance for Preconception Counseling and Prevention of Sexual Transmission of Zika Virus for Persons with Possible Zika Virus Exposure — United States, September 2016. MMWR Morb Mortal Wkly Rep. 2016; 65:1077-1081.
17. Rabe IB, Staples JE, Villanueva J, et al. Interim Guidance for Interpretation of Zika Virus Antibody Test Results. U.S. Morbidity and Mortality Weekly Report (MMWR). Morb Mortal Wkly Rep. 2016; 65.
18. Sharp TM, Fischer M, Muñoz-Jordán JL, et al. Dengue and Zika virus diagnostic testing for patients with a clinically compatible illness and risk for infection with both viruses. MMWR Recomm Rep. 2019; 68(RR-1):1-10.
19. Staples JE, Dziuban EJ, Fischer M, et al. Interim guidelines for the evaluation and testing of infants with possible congenital zika virus infection — United States, 2016. MMWR Morb Mortal Wkly Rep. 2016; 65:63-67.
20. U.S. Food and Drug Administration (FDA). Emergency Use Authorizations (EUA) for an updated list of all diagnostic tests, serologic assays, vaccines under development and additional chronological information from the FDA. 2024. Available at: https://www.fda.gov/EmergencyPreparedness/Counterterrorism/MedicalCountermeasures/MCMIssues/ucm485199.htm. Accessed on May 22-8, 2025.
21. U.S. Food and Drug Administration (FDA) Dept of Health and Human Services (HHS). Revised recommendations for reducing the risk of zika virus transmission by blood and blood components; guidance for industry. July 9, 2018. Available at: https://www.federalregister.gov/documents/2018/07/09/2018-14537/revised-recommendations-for-reducing-the-risk-of-zika-virus-transmission-by-blood-and-blood. Accessed on May 22, 2025.
22. U.S. Morbidity and Mortality Weekly Report (MMWR). Interim guidance for Zika virus testing of urine. MMWR updated May 10, 2016. Morb Mortal Wkly Rep. 2016; 65:484.
23. U.S. Morbidity and Mortality Weekly Report (MMWR). Erratum: MMWR. Morb Mortal Wkly Rep. 2016; 65(18):484.
24. U.S. Morbidity and Mortality Weekly Report (MMWR). Transmission of Zika virus through sexual contact with travelers to areas of ongoing transmission — continental United States, 2016. Morb Mortal Wkly Rep. 2016; 65:215-216.
25. World Health Organization (WHO). Zika Virus Key Facts. December 8, 2022. Available at: https://www.who.int/news-room/fact-sheets/detail/zika-virus. Accessed on May 22, 2025.
26. World Health Organization (WHO). Zika Virus Outbreak Toolbox. Updated April 2024. Available at: https://www.who.int/emergencies/outbreak-toolkit/disease-outbreak-toolboxes/zika-outbreak-toolbox. Accessed on May 22, 2025.

ADVIA Centaur Zika IgM
Aptima^® Zika Virus Assay, Hologic Inc.
Baebies Seeker^™ Analyzer, Baebies, Inc.
DPP^® Zika IgM System
GLS-5700, Vaccine
IgM
LightMix^® Zika rRT-PCR Test, Roche Molecular Systems Inc.
NAT (cobas Zika nucleic acid test), Roche Molecular Systems, Inc.
PRNT, Testing
PROCLEIX, Hologic Inc., Grifols
RealStar^® Zika Virus RT-PCR Kit, Altona Diagnostics
rRT/PCR, Assay
Sentosa^® SA ZIKV RT-PCR, Vela Diagnostics USA, Inc.
Trioplex, rRT/PCR Test
VERSANT^® Zika RNA 1.0 Assay (kPCR), Siemens Healthcare Diagnostics, Inc.
ZIKV Detect^™ IgM Capture ELISA, InBios International, Inc.
Zika MAC-ELISA, CDC
Zika Virus Real-Time RT-PCR, Viracor-IBT Laboratories, Inc.
Zika Virus RNA Qualitative Real-Time RT-PCR Test, Focus Diagnostics
Zika Virus Detection by RT-PCR, ARUP Laboratories
ZIKV Detect^™ 2.0 IgM Capture ELISA

The use of specific product names is illustrative only. It is not intended to be a recommendation of one product over another, and is not intended to represent a complete listing of all products available. 

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