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      Performance of the Trioplex real-time RT-PCR assay for detection of Zika, dengue, and chikungunya viruses

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          Abstract

          The emergence and spread of Zika virus (ZIKV) presented a challenge to the diagnosis of ZIKV infections in areas with transmission of dengue (DENV) and chikungunya (CHIKV) viruses. To facilitate detection of ZIKV infections, and differentiate these infections from DENV and CHIKV, we developed the Trioplex real-time RT-PCR assay (Trioplex assay). Here, we describe the optimization of multiplex and singleplex formats of the assay for a variety of chemistries and instruments to facilitate global standardization and implementation. We evaluated the analytical performance of all Trioplex modalities for detection of these three pathogens in serum and whole blood, and for ZIKV in urine. The limit of detection for the three viruses and in different RNA-extraction modalities is near 10 3 genome copy equivalents per milliliter (GCE/mL). Simultaneous testing of more than one specimen type from each patient provides a 6.4% additional diagnostic sensitivity. Overall, the high sensitivity of the Trioplex assay demonstrates the utility of this assay ascertaining Zika cases.

          Abstract

          The Trioplex real-time RT-PCR assay was developed for detection of Zika virus infections in areas with dengue and chikungunya transmission. Here, Santiago et al. describe the optimization and clinical performance of the assay, showing high sensitivity for detection and differentiation of the three viruses.

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          Serotype-specific detection of dengue viruses in a fourplex real-time reverse transcriptase PCR assay.

          Barbara Johnson, Brandy J Russell, Robert Lanciotti (2005)
          The dengue (DEN) viruses are positive-strand RNA viruses in the genus Flavivirus. Dengue fever and dengue hemorrhagic fever/dengue shock syndrome are important human arboviral diseases caused by infection with one of four closely related but serologically distinct DEN viruses, designated DEN-1, DEN-2, DEN-3, and DEN-4 viruses. All four DEN serotypes are currently co-circulating throughout the subtropics and tropics, and genotypic variation occurs among isolates within a serotype. A real-time quantitative nucleic acid amplification assay has been developed to detect viral RNA of a single DEN virus serotype. Each primer-probe set is DEN serotype specific, yet detects all genotypes in a panel of 7 to 10 representative isolates of a serotype. In single reactions and in fourplex reactions (containing four primer-probe sets in a single reaction mixture), standard dilutions of virus equivalent to 0.002 PFU of DEN-2, DEN-3, and DEN-4 viruses were detected; the limit of detection of DEN-1 virus was 0.5 equivalent PFU. Singleplex and fourplex reactions were evaluated in a panel of 40 viremic serum specimens with 10 specimens per serotype, containing 0.002 to 6,000 equivalent PFU/reaction (0.4 to 1.2 x 10(6) PFU/ml). Viral RNA was detected in all viremic serum specimens in singleplex and fourplex reactions. Thus, this serotype-specific, fourplex real-time reverse transcriptase PCR nucleic acid detection assay can be used as a method for differential diagnosis of a specific DEN serotype in viremic dengue patients and as a tool for rapid identification and serotyping of DEN virus isolates.
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            Vital Signs: Update on Zika Virus–Associated Birth Defects and Evaluation of All U.S. Infants with Congenital Zika Virus Exposure — U.S. Zika Pregnancy Registry, 2016

            Megan Reynolds, Abbey M. Jones, Emily Petersen … (2017)
            Background In collaboration with state, tribal, local, and territorial health departments, CDC established the U.S. Zika Pregnancy Registry (USZPR) in early 2016 to monitor pregnant women with laboratory evidence of possible recent Zika virus infection and their infants. Methods This report includes an analysis of completed pregnancies (which include live births and pregnancy losses, regardless of gestational age) in the 50 U.S. states and the District of Columbia (DC) with laboratory evidence of possible recent Zika virus infection reported to the USZPR from January 15 to December 27, 2016. Birth defects potentially associated with Zika virus infection during pregnancy include brain abnormalities and/or microcephaly, eye abnormalities, other consequences of central nervous system dysfunction, and neural tube defects and other early brain malformations. Results During the analysis period, 1,297 pregnant women in 44 states were reported to the USZPR. Zika virus–associated birth defects were reported for 51 (5%) of the 972 fetuses/infants from completed pregnancies with laboratory evidence of possible recent Zika virus infection (95% confidence interval [CI] = 4%–7%); the proportion was higher when restricted to pregnancies with laboratory-confirmed Zika virus infection (24/250 completed pregnancies [10%, 95% CI = 7%–14%]). Birth defects were reported in 15% (95% CI = 8%–26%) of fetuses/infants of completed pregnancies with confirmed Zika virus infection in the first trimester. Among 895 liveborn infants from pregnancies with possible recent Zika virus infection, postnatal neuroimaging was reported for 221 (25%), and Zika virus testing of at least one infant specimen was reported for 585 (65%). Conclusions and Implications for Public Health Practice These findings highlight why pregnant women should avoid Zika virus exposure. Because the full clinical spectrum of congenital Zika virus infection is not yet known, all infants born to women with laboratory evidence of possible recent Zika virus infection during pregnancy should receive postnatal neuroimaging and Zika virus testing in addition to a comprehensive newborn physical exam and hearing screen. Identification and follow-up care of infants born to women with laboratory evidence of possible recent Zika virus infection during pregnancy and infants with possible congenital Zika virus infection can ensure that appropriate clinical services are available.
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              Interim Guidance for Interpretation of Zika Virus Antibody Test Results.

              Ingrid B. Rabe, J. Staples, Julie Villanueva … (2016)
              Zika virus is a single-stranded RNA virus in the genus Flavivirus and is closely related to dengue, West Nile, Japanese encephalitis, and yellow fever viruses (1,2). Among flaviviruses, Zika and dengue virus share similar symptoms of infection, transmission cycles, and geographic distribution. Diagnostic testing for Zika virus infection can be accomplished using both molecular and serologic methods. For persons with suspected Zika virus disease, a positive real-time reverse transcription-polymerase chain reaction (rRT-PCR) result confirms Zika virus infection, but a negative rRT-PCR result does not exclude infection (3-7). In these cases, immunoglobulin (Ig) M and neutralizing antibody testing can identify additional recent Zika virus infections (6,7). However, Zika virus antibody test results can be difficult to interpret because of cross-reactivity with other flaviviruses, which can preclude identification of the specific infecting virus, especially when the person previously was infected with or vaccinated against a related flavivirus (8). This is important because the results of Zika and dengue virus testing will guide clinical management. Pregnant women with laboratory evidence of Zika virus infection should be evaluated and managed for possible adverse pregnancy outcomes and be reported to the U.S. Zika Pregnancy Registry or the Puerto Rico Zika Active Pregnancy Surveillance System for clinical follow-up (9,10). All patients with clinically suspected dengue should have proper management to reduce the risk for hemorrhage and shock (11). If serologic testing indicates recent flavivirus infection that could be caused by either Zika or dengue virus, patients should be clinically managed for both infections because they might have been infected with either virus.
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                Author and article information

                Contributors
                Jorge L. Muñoz-Jordan: JMunoz@cdc.gov
                Journal
                Journal ID (nlm-ta): Nat Commun
                Journal ID (iso-abbrev): Nat Commun
                Title: Nature Communications
                Publisher: Nature Publishing Group UK (London )
                ISSN (Electronic): 2041-1723
                Publication date (Electronic): 11 April 2018
                Publication date PMC-release: 11 April 2018
                Publication date Collection: 2018
                Volume: 9
                Electronic Location Identifier: 1391
                Affiliations
                [1 ]ISNI 0000 0001 2163 0069, GRID grid.416738.f, Division of Vector Borne Diseases, Dengue Branch, , Centers for Disease Control and Prevention, National Centers for Emerging and Zoonotic Infectious Diseases, ; San Juan, PR 00920 USA
                [2 ]ISNI 0000 0001 2163 0069, GRID grid.416738.f, Division of Preparedness and Emerging Infections, , Centers for Disease Control and Prevention, National Centers for Emerging and Zoonotic Infectious Diseases, ; Atlanta, GA 30333 USA
                Article
                Publisher ID: 3772
                DOI: 10.1038/s41467-018-03772-1
                PMC ID: 5895813
                PubMed ID: 29643334
                SO-VID: ade52121-7fa0-406a-91f0-a61e8ae0f6f6
                Copyright © © The Author(s) 2018
                License:

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                Date received : 1 November 2017
                Date accepted : 9 March 2018
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                Subject: Article
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                issue-copyright-statement © The Author(s) 2018

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