Single-Reaction Multiplex Reverse Transcription PCR for Detection of Zika, Chikungunya, and Dengue Viruses

Clinical manifestations of Zika virus, chikungunya virus, and dengue virus infections can be similar. To improve virus detection, streamline molecular workflow, and decrease test costs, we developed and evaluated a multiplex real-time reverse transcription PCR for these viruses.

Z ika virus is a mosquitoborne flavivirus that, in 2015, spread throughout the tropical and subtropical regions of the Western Hemisphere. In January 2016, the first autochthonous cases of Zika fever were confirmed in Nicaragua (1). The diagnosis of human Zika virus infections is confounded by a nonspecific clinical presentation, which overlaps substantially with that of dengue virus (DENV) and chikungunya virus (CHIKV) (2,3) and by cross-reaction with DENV IgM and DENV nonstructural protein 1 in assays for Zika virus (4)(5)(6)(7).
Molecular assays can detect and differentiate these 3 pathogens during the acute phase of illness. Although a number of molecular tests have been published for detecting DENV and CHIKV, only 2 Zika virus real-time reverse transcription PCRs (rRT-PCRs) have been reported and were characterized by using human specimens (6)(7)(8). These assays are run as individual reactions, and molecular testing for all 3 viruses, using established protocols, requires multiple reactions for a single patient sample (6,9,10). We describe a Zika virus rRT-PCR that was designed to be run in multiplex with published assays for pan-DENV and CHIKV detection (11,12). We then evaluated the singlereaction multiplex rRT-PCR for Zika virus, CHIKV, and DENV (referred to as the ZCD assay) by testing clinical samples from persons with suspected cases in Nicaragua.

The Study
The Zika virus primers and probe (online Technical Appendix Table 1 (http://wwwnc.cdc.gov/EID/article/22/7/16-0326-Techapp1.pdf) were designed by using all complete or nearly complete (>10,000 kb) Zika virus genome sequences available in GenBank (n = 21) accessed March 28, 2014). Target sequences were subsequently confirmed to match strains from the Americas. All rRT-PCR reactions were performed on an ABI 7500 instrument (Applied Biosystems, Foster City, CA, USA) by using 25-mL reactions of the SuperScript III Platinum One-Step qRT-PCR kit (Life Technologies, Carlsbad, CA, USA) and 5 mL of RNA template. Cycling conditions for the ZCD assay were as follows: 52°C for 15 min; 94°C for 2 min; 45 cycles at 94°C for 15 sec, 55°C for 20 sec (acquisition), and 68°C for 20 sec. Each run included a no-template control and positive controls for Zika virus, CHIKV, and DENV.
De-identified serum samples, collected from Nicaraguan patients with suspected Zika virus, CHIKV, and/or DENV infections, were tested (online Technical Appendix). We tested 216 samples by using the ZCD assay and the pan-DENV-CHIKV rRT-PCR, which is a validated duplex assay containing the DENV and CHIKV primers and probes used in the ZCD assay (12). Both assays were performed on an ABI7500 (Applied Biosystems) ( Table  1) The first case of Zika virus infection in Nicaragua was detected with the ZCD assay during the assay comparison described above. After Zika virus identification, 133 consecutive samples were tested by using both the ZCD assay and a comparator Zika virus rRT-PCR targeting the capsid gene (6) ( Table 2). When the comparator rRT-PCR was analyzed according to the published validation (C t <38.5 defining a positive result), these assays demonstrated only moderate agreement (k = 0.47), and Zika virus was detected in significantly more samples by using the ZCD assay (p<0.001). Of the 31 samples positive only for Zika virus in the ZCD assay, 22 (71%) produced a C t (>38.5) that was reached later in the comparator Zika virus rRT-PCR. These

Conclusions
The ZCD assay improved detection of Zika virus relative to the comparator rRT-PCR, and 31 samples were positive only for Zika virus by the ZCD assay when the comparator was interpreted as published (6). Notably, 22 (71%) of these 31 samples produced a late signal in the comparator Zika virus rRT-PCR (C t >38.5), indicating that these most likely are true, late-positive results. Improved sensitivity for Zika virus is needed given the low viremia detected in clinical samples and the current lack of accurate alternative diagnostics, such as serology (6,7,15). Additionally, the ZCD assay identified 17 co-infections in Zika viruspositive patients. Although preliminary, these data provide evidence for the utility of a multiplex diagnostic test for these pathogens.
The performance of the ZCD assay for DENV detection was similar to that of the pan-DENV-CHIKV rRT-PCR, and the analytical sensitivity for CHIKV was similar in both assays (12). CHIKV detection in clinical samples in the ZCD assay and pan-DENV-CHIKV rRT-PCR demonstrated good agreement, although both assays contain the same CHIKV primers and probes. Discrepant samples all had C t of >37.36, which correspond to 10 copies/mL of eluate and fall below the 95% LLOD. Although ZCD assay results for these CHIKV-positive samples were reproducible, the clinical significance of such low-level viremia in patients with suspected chikungunya fever is unclear and warrants further study.
A limitation to our study is the use of a single comparator Zika virus rRT-PCR. This assay was 1 of 2 rRT-PCRs developed for the 2007 Yap Island Zika virus strain (6). The second rRT-PCR, targeting the membrane gene, was evaluated for the current study but proved consistently less analytically sensitive. Therefore, performance of this second Zika virus assay most likely would not have affected result interpretation.
In conclusion, the single-reaction multiplex ZCD assay detected and differentiated Zika virus, CHIKV, and DENV. This assay should streamline molecular workflow and decrease test costs while improving detection of these 3 human arboviruses.  Ministry of Health, and the Sustainable Sciences Institute in Managua, Nicaragua.
Patent applications or provisional patent applications that cover the ZCD multiplex assay and the primers and probes described in this report have been filed (J.J.W. and B.A.P.).

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