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Background <p>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.</p> Methods <p>The ZYDC-PCR was validated following MIQE guidelines, using Quality Control for Molecular Diagnostics (QCMD) and retrospective clinical samples at laboratories in Belgium (<i>n</i> = 44) and Cuba (<i>n</i> = 97). These samples consisted of samples positive for ZIKV (<i>n</i> = 9), CHIKV (<i>n</i> = 11), DENV1 (<i>n</i> = 8), DENV2 (<i>n</i> = 5), DENV3 (<i>n</i> = 26) and DENV4 (<i>n</i> = 47), as well as 14 negative endemic controls from DRC, Oropouche virus (OROV) positive (<i>n</i> = 9) and samples negative for DENV and OROV (<i>n</i> = 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 (<i>n</i> = 621), in Antwerp (<i>n</i> = 104) and in both laboratories (<i>n</i> = 99). Extractions and PCRs were done with commercially available kits.</p> Results <p>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 &gt; 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.</p> Conclusion <p>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.</p> Clinical Trial <p>AUFI in Goma, DRC: Clinicaltrials.gov NCT05139524, registered on December 1<sup>st</sup>, 2021.</p>

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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)

  • 期刊论文

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  • 发表日期：

    2026年2月6日

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  • Anne Hauner,
  • Ana Julia Benítez,
  • Noella Mulopo-Mukanya,
  • Stijn Rogé,
  • Silvia Serrano,
  • Veerle Vanlerberghe,
  • Luciana Lepore,
  • Marjan Van Esbroeck,
  • Justin Masumu,
  • Carolyne Nasimiyu,
  • M. Kariuki Njenga,
  • Maria Eugenia Toledo Romani,
  • Yenisbel V. Portal,
  • Mayling Álvarez,
  • Rosa Ramírez-Bartutis,
  • María G. Guzmán,
  • Steve Ahuka-Mundeke,
  • Daniel Mukadi-Bamuleka,
  • Kevin K. Ariën

摘要

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 1st, 2021.