Implementation of a Multiplex rRT-PCR for Zika, Chikungunya, and Dengue Viruses: Improving Arboviral Detection in an Endemic Region

Abstract

Arboviral diagnosis has been complicated throughout the tropical and subtropical Americas by the recent co-circulation of Zika virus (ZIKV), chikungunya virus (CHIKV), and dengue virus (DENV). The aim of this study was to implement a multiplex real-time RT-PCR (rRT-PCR) for ZIKV, CHIKV, and DENV in Paraguay to test patients who were clinically suspected of having dengue. We tested 110 sera from patients who presented to the Hospital de Clínicas in 2016 and had testing for DENV nonstructural protein 1 (NS1; 40 positive and 70 negative). Using a composite reference standard, we confirmed 51 dengue cases (46.4%): 38/40 NS1 positive and 13/70 NS1 negative. Chikungunya virus and ZIKV were detected in one sample each, both were DENV NS1 negative. The NS1 test demonstrated good agreement with rRT-PCR for DENV. However, multiplex rRT-PCR identified a subset of dengue cases and additional arboviral infections that would not be detected if NS1 assays are relied upon for diagnosis.

Comparison of Anti-Dengue and Anti-Zika IgG on a Plasmonic Gold Platform with Neutralization Testing

“…Molecular testing was performed at IICS-UNA to 1) detect acute DENV, ZIKV, and chikungunya virus (CHIKV) infections, and 2) determine DENV serotype and quantify viral load using validated and published real-time reverse transcription (rRT)-PCRs, as described. [25][26][27][28] For the current study, sera were selected from 77 individuals who presented £ 8 days after symptom onset and tested positive (n = 38) or negative (n = 39) for DENV by rRT-PCR.…”

Characterization of Dengue Virus 4 Cases in Paraguay, 2019–2020

“…Serum was obtained at the enrollment visit, aliquoted, and stored at −80 °C. Participants were screened for DENV by testing for the non-structural protein 1 (NS1) antigen and/or DENV RNA in a multiplex rRT-PCR for Zika, chikungunya, and dengue (the ZCD assay) [ 33 , 39 , 40 ]. NS1 testing was performed at IICS-UNA using the Standard Q Dengue Duo rapid immunochromatographic test (SD Biosensor, Suwon, South Korea) according to manufacturer recommendations.…”

“…For automated extractions, nucleic acids were extracted from 200 µL of serum and eluted in 60 μL of buffer. A total of 5 μL of eluate was then used in ZCD and DMPT reactions, and both assays were performed and interpreted as previously described [ 39 , 40 , 41 ]. Serum viral load was quantified from 4-point standard curves prepared with synthesized DENV target sequences and included on dedicated DMPT runs.…”