Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2025 Jun 24;20(6):e0309626.
doi: 10.1371/journal.pone.0309626. eCollection 2025.

Assessment of a multiplex arbovirus PCR Detection Test in an area endemic for Chikungunya, Zika, and Dengue viruses: An evaluation of kit performance characteristics in line with Clinical Laboratory Improvement Amendments (CLIA) Standards

Leonard Kingwara  1 , Lazarus Odeny  2 , Vera Onwonga  1 , Rukia Madada  1 , Marygorett Mbeneka  1 , Jully Okonji  1 , Victoria Mwende  1 , Charles Rombo  1 , Shalyn Akasa  1 , Millicent Ndia  1 , Fredrick Ouma  1 , Isabella Ayagah  1 , Abdi Roba  1 , Kamene Kimenye  1
Affiliations

Assessment of a multiplex arbovirus PCR Detection Test in an area endemic for Chikungunya, Zika, and Dengue viruses: An evaluation of kit performance characteristics in line with Clinical Laboratory Improvement Amendments (CLIA) Standards

Leonard Kingwara et al. PLoS One. .

Abstract

Introduction: Several multiplex and singleplex PCR systems exist for the detection of Dengue(DENV), Zika(ZIKV), and Chikungunya(CHIKV) viruses; however, their performance characteristics in East Africa remain unassessed. In this study, we investigated the TaqMan® Arbovirus Triplex Kit, which allows for the simultaneous identification of CHIKV, DENV, and ZIKV viruses in serum samples and singleplex assays, including the RealStar® Chikungunya RT-PCR Kit 2.0, RealStar® Dengue RT-PCR Kit 3.0, and RealStar® Zika Virus RT-PCR Kit 1.0.

Methods: We used Clinical Laboratory Improvement validation methods and residual specimens sourced from DENV, CHIKV, and ZIKV outbreaks, from Kenya, Brazil, and the Democratic Republic of Congo(DRC) in March 2024. Quality control was ensured through Quality Control for Medical diagnostics (QCMD) commercial positive panels. Gold standard results were established through consensus from multiple kits, including the TaqMan® Arbovirus Triplex Kit, Illumina viral surveillance panel (Illumina VSP), and Altona singleplex kit. Data were analyzed using R to determine specificity, sensitivity, negative predictive value (NPV), positive predictive value (PPV), and diagnostic odds ratio, with probit analysis employed to evaluate the limit of detection.

Results: The TaqMan® Arbovirus Kit showed sensitivity exceeding 95% for all three virus targets: 97.98% for DENV, and 100% for both CHIKV and ZIKV. Simillarly, the RealStar® RT-PCR Kits exhibited sensitivities: 93.94% for CHIKV, 90.91% for DENV, and 100% for ZIKV. All kits demonstrated specificity above 99%, with a positive predictive value (PPV) over 99% and a negative predictive value (NPV) exceeding 97%. Probit analysis revealed that the TaqMan® Arbovirus Kit also had a lower detection limit compared to the RealStar kit for all tested viruses.

Conclusion: The TaqMan® Arbovirus Kit provides multiplex capabilities and performance similar to the RealStar® CHIKV, DENV, and ZIKV RT-PCR Kits. Consequently, these kits can be used interchangeably for detecting DENV, CHIKV, and ZIKV viruses in outbreak situations.

PubMed Disclaimer

Conflict of interest statement

No authors have competing interests.

Figures

Fig 1
Fig 1. Sensitivity and specificity by platform and virus.
Fig 2
Fig 2. Releationship between input concentration and ct value.
Fig 3
Fig 3. Probit Curve analysis.
See this image and copyright information in PMC

References

    1. Pabbaraju K, Wong S, Gill K, Fonseca K, Tipples GA, Tellier R. Simultaneous detection of Zika, Chikungunya and Dengue viruses by a multiplex real-time RT-PCR assay. J Clin Virol. 2016;83:66–71. doi: 10.1016/j.jcv.2016.09.001 - DOI - PubMed
    1. Cantero C, Cardozo F, Waggoner JJ, Pinsky BA, Espínola A, Infanzón B, et al. Implementation of a Multiplex rRT-PCR for Zika, Chikungunya, and Dengue Viruses: Improving Arboviral Detection in an Endemic Region. Am J Trop Med Hyg. 2020;102(3):625–8. doi: 10.4269/ajtmh.19-0707 - DOI - PMC - PubMed
    1. Wu W, Wang J, Yu N, Yan J, Zhuo Z, Chen M. Development of multiplex real-time reverse-transcriptase polymerase chain reaction assay for simultaneous detection of Zika, dengue, yellow fever, and chikungunya viruses in a single tube. J Med Virol. 2018;90(11). - PubMed
    1. Lura T, Su T, Thieme J, Brown MQ. A validated triplex RT-qPCR protocol to simultaneously detect chikungunya, dengue and Zika viruses in mosquitoes. J Vector Borne Dis. 2022;59(3):198–205. doi: 10.4103/0972-9062.316275 - DOI - PubMed
    1. Mansuy J-M, Lhomme S, Cazabat M, Pasquier C, Martin-Blondel G, Izopet J. Detection of Zika, dengue and chikungunya viruses using single-reaction multiplex real-time RT-PCR. Diagn Microbiol Infect Dis. 2018;92(4):284–7. doi: 10.1016/j.diagmicrobio.2018.06.019 - DOI - PubMed

MeSH terms

Substances