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)

BACKGROUND: 

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.

METHODS: 

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.

RESULTS: 

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.

CONCLUSION: 

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.

CLINICAL TRIAL: AUFI in Goma, DRC: Clinicaltrials.gov NCT05139524, registered on December 1 st, 2021.

SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12879-026-12570-w.