As of March 2017, 64 countries and territories had travel notices for active Zika virus transmissions. CDC developed the Trioplex rRT-PCR test to detect evidence of Zika virus infection and aid in differentiating this infection from dengue and chikungunya virus infections, all of which are spread by the same types of Aedes species mosquitoes and cause similar illness. The Trioplex Real-time RT-PCR assay is for qualitative detection and differentiation of RNA from dengue, chikungunya and Zika viruses in serum, whole blood, urine, amniotic fluid and cerebral spinal fluid, and for the qualitative detection of Zika virus RNA in urine and amniotic fluid. This assay protocol is designed to facilitate simultaneous testing for the three viruses using a single sample in the same plate well (multiplex). A singleplex reaction (measuring one analyte at a time) is also an option for chikungunya and dengue testing in serum, whole blood, and cerebrospinal fluid (other specimen types if one primer/probe set per well is preferred). The U.S. Food and Drug Administration (FDA) issued an Emergency Use Authorization (EUA) for the Trioplex assay on March 17, 2016.