Technical validation and implementation of a multiplex real-time PCR for differential diagnostic detection of Zika virus, yellow fever virus, dengue virus and chikungunya virus (ZYDC-PCR)

Arthropod-borne (arbo)-viruses, especially dengue (DENV), Zika (ZIKV), chikungunya (CHIKV) and yellow fever virus (YFV) are a public health threat worldwide. Timely and reliable diagnostics are key for early case detection, proper patient management and targeted public health interventions. We developed, validated and implemented a real-time reverse transcription (RT)-PCR (ZYDC-PCR) for the simultaneous identification of these four arboviruses. The ZYDC-PCR was validated following MIQE guidelines, using Quality Control for Molecular Diagnostics (QCMD) and retrospective clinical samples at laboratories in Belgium (n = 44) and Cuba (n = 97). These samples consisted of samples positive for ZIKV (n = 9), CHIKV (n = 11), DENV1 (n = 8), DENV2 (n = 5), DENV3 (n = 26) and DENV4 (n = 47), as well as 14 negative endemic controls from DRC, Oropouche virus (OROV) positive (n = 9) and samples negative for DENV and OROV (n = 10). The ZYDC-PCR was implemented for an exploratory study in the Democratic Republic of the Congo (DRC), with 725 samples tested in total, in DRC (n = 621), in Antwerp (n = 104) and in both laboratories (n = 99). Extractions and PCRs were done with commercially available kits. The results for the technical validation were satisfactory. Detection limits were 11,760 copies (cp)/mL for ZIKV, 1510 cp/mL for CHIKV, 2330 cp/mL for DENV1, and 6800 cp/mL for YFV. For 15 samples (4 ZIKV, 1 CHIKV, 3 DENV1, 1 DENV2, 6 DENV3) with Cq values > 36.5 in the simplex-PCR, the multiplex ZYDC-PCR tested negative. Specificity for cross reactivity samples and retrospective samples tested in Belgium and Cuba was 100%. The sensitivity for the DENV positive samples in Cuba was 91.5%. All samples from DRC tested negative for the four arboviruses. This study’s strength lies in its successful implementation across diverse settings-three geographic (Belgium, DRC, Cuba) locations, multiple PCR machines and kits, and varying lab expertise levels. Despite a slight reduction of sensitivity with low viremia, the ZYDC-PCRs ability to detect multiple arboviruses simultaneously makes it a valuable tool for global molecular surveillance and clinical diagnostics. AUFI in Goma, DRC: Clinicaltrials.gov NCT05139524, registered on December 1st, 2021.