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

Gilberto A Santiago,Gilberto A Santiago,Rebecca Roulo,Jorge L Muñoz-Jordan,Lauren E Andersen,Jesús Vázquez,Jesús Vázquez,Sean Courtney,Sean Courtney,Katia Y Matías,John Bowzard,Candimar Colón,Angela E Butler,Lauren E Andersen,Katia Y Matías,Candimar Colón,Julie M Villanueva,Julie M Villanueva,Rebecca Roulo,Angela E Butler

doi:10.1038/s41467-018-03772-1

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 103 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.

Highlights

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
In response to the diagnostic challenges presented by the ZIKV epidemic, the Centers for Disease Control and Prevention (CDC) developed the Trioplex real-time reverse transcriptasepolymerase chain reaction assay (Trioplex assay) for the concurrent detection of ZIKV, DENV, and CHIKV RNA in human serum, whole blood in ethylenediaminetetraacetic acid (EDTA) and cerebrospinal fluid (CSF) or the sole detection of ZIKV in human urine or amniotic fluid[27]
The Trioplex assay was designed for the detection of ZIKV, DENV, and CHIKV RNA in human diagnostic specimens

Summary

Introduction

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. In response to the diagnostic challenges presented by the ZIKV epidemic, the Centers for Disease Control and Prevention (CDC) developed the Trioplex real-time reverse transcriptasepolymerase chain reaction (real-time RT-PCR) assay (Trioplex assay) for the concurrent detection of ZIKV, DENV, and CHIKV RNA in human serum, whole blood in ethylenediaminetetraacetic acid (EDTA) and cerebrospinal fluid (CSF) or the sole detection of ZIKV in human urine or amniotic fluid[27]. The performance of the Trioplex assay including a selection of RNA extraction methods, PCR chemistries, and real-time PCR instruments was evaluated to facilitate implementation and standardization across public health laboratories globally[27]. We present the analytical and clinical performance evaluations of the Trioplex assay, and document its limit of detection (LoD) and utility as a flexible test in available diagnostic devices, allowing most public health laboratories to detect Zika, dengue, and chikungunya cases globally

Methods

Results

Conclusion