1. Diagnostic Testing for Zika Virus Frederick S. Nolte, PhD, D(ABMM), F(AAM) Professor and Vice-Chair for Laboratory Medicine Department of Pathology and Laboratory Medicine

2. Zika Virus Mosquito-borne SS RNA virus in the genus Flavivirus › Closely related to dengue, West Nile, Japanese encephalitis and yellow fever viruses Zika, dengue, and chikungunya viruses share similar symptoms of infection, transmission cycles, and geographic distribution Diagnostic testing for Zika virus infection can be accomplished using both nucleic acid amplification and serologic tests 3D image of virus produced by cryo-electron microscopy Devika Sirohi et al. Science 2016;352:467-470 Aedes aegypti

3. Nucleic Acid Amplification Tests for Zika Virus Real time RT-PCR used to amplify and detect specific Zika virus gene sequences directly in serum, CSF, urine and amniotic fluid (5’ nuclease technology) Specific for Zika virus (no cross- reactions with other arboviruses) Zika virus present in serum for ≤7 days and in urine for ≤14 days after onset of symptoms Positive test confirms infection however a negative test does not rule out infection

4. Serological Tests IgM antibodies to Zika virus develop within the first 4 days of illness and persist for about 12 weeks following infection A Zika virus MAC-ELISA is used to screen for presence of antibody However, there are extensive cross reactions with other related Flaviviruses (dengue and West Nile) All positive and equivocal MAC ELISA tests must be confirmed with plaque reduction neutralization tests (PRNT) A negative MAC-ELISA rules out recent infection

5. Zika Virus Tests with FDA Emergency Use Authorization for Diagnosis An EUA is a tool that FDA can use to allow the use of certain medical products for emergencies based on scientific data › No FDA-cleared tests Real-time RT-PCR tests › RealStar® Zika Virus RT-PCR Kit U.S. (altona Diagnostics) › Serum and urine › Zika Virus RNA Qualitative Real-Time RT-PCR (Focus Diagnostics/Quest) › Serum › Trioplex Real-time RT-PCR Assay (CDC) › Detects and differentiates Zika, dengue, and chikungunya virus › Serum, CSF, urine and amniotic fluid Serological test › Zika Immunoglobulin M Antibody Capture Enzyme-Linked Immunosorbent Assay (Zika MAC-ELISA) (CDC) http://www.fda.gov/MedicalDevices/Safety/EmergencySituations/ucm161496.htm

6. Zika Virus Blood Donor Screening To date, there have been no confirmed blood transfusion-transmission cases in the United States. There is evidences that Zika virus can be spread through blood transfusions. Zika virus currently poses a low risk to the blood supply in the continental US, but this could change depending on how many people become infected with the virus. In areas of active transmission, FDA recommends that blood either be screened by laboratory testing, subjected to pathogen reduction technology (PRT), or outsourced from other areas. Although there is no FDA licensed test for Zika virus, on April 3, 2016, testing for Zika became available through an Investigational New Drug application for blood collected in Puerto Rico. › Roche Cobas Zika test http://www.cdc.gov/zika/transmission/blood-transfusion.html

7. Zika Virus Testing Logistics Symptomatic travelers, including pregnant women, who have visited areas with active Zika virus transmission and had illness onset during travel or within 2 weeks from returning are recommended for testing. › Asymptomatic pregnant women who have traveled to areas with active Zika virus transmission within the past 2 to 12 weeks or who had sexual contact with a male who traveled to an area of active Zika transmission and had clinical illness consistent with Zika virus can be offered testing. Report all suspected cases of Zika virus disease by calling the regional health department in your area. After calling the regional health department, regional staff will collect patient data using a standardized data collection tool and approve testing if needed. › Local laboratory completes DHEC BOL form

8. Specimen Collection Diagnosis › Serum (≥1 ml) › ≤7 days after onset of symptoms for real-time RT-PCR › ≥4 days after onset of symptoms for MAC-ELISA › Urine (0.5 to 1 ml) ≤14 days after onset of symptoms › CSF (≥1 ml) › Amniotic fluid (0.5 to 1 ml) › Semen (0.5 to 1 ml) Live birth with evidence of maternal or fetal infection › Placenta › Umbilical cord › Umbilical cord blood http://www.cdc.gov/zika/hc-providers/diagnostic.html

9. Zika Virus Testing Logistics Zika MAC-ELISA, and Trioplex Real-time RT-PCR Assay performed at DHEC BOL in Columbia › Negative MAC-ELISA results available ≤4 days › RT-PCR results available same day of receipt Confirmatory PRNT performed at CDC › Results available ≤3 weeks Surge capacity of DHEC BOL is 400 specimens/week To date MUSC Health has submitted samples from 15 patients who met CDC criteria for testing and none have been positive for Zika virus › As of June 3, 2016 there is one confirmed travel-associated case of Zika virus in South Carolina We have no plans to implement local testing at MUSC unless we become an area with active transmission Christy Jeffcoat, DHEC Virology Laboratory, Personal Communication

10. Challenges for Zika Virus Test Development Current screening and confirmatory serological tests are cumbersome and suffer from cross reactions with other Flaviviruses. › Zika MAC-ELISA and PRNT are beyond the capabilities of most clinical laboratories. › Cross reactions are problematic in Latin America where multiple Flavivirus infections are common. Development of recombinant antigens that will react with virus-specific antibodies is needed. Real-time PCR assays are more specific and easier to perform but only useful during a short window of acute infection. Lack of access to samples positive for Zika virus has hampered test development. › HHS recently funded a program to collect samples and make them available to diagnostic companies to validate new tests.