Technical validation of a multiplex real-time PCR for combined detection of Rift Valley fever, chikungunya, Zika and dengue viruses.

Journal: Journal of virological methods
Published Date: May 7, 2025

Abstract

Several arthropod-borne (arbo)-viruses have overlapping symptoms, insect vectors and geographical occurrence. With little known about the importance of arboviruses as cause of acute undifferentiated fever (AUF) in East and Central Africa (ECA), there is a clear need for a multiplex-PCR allowing for multi-pathogen surveillance. A multiplex real-time RT-PCR (RDCZ-multiplex) was developed and validated for the simultaneous detection of Rift Valley fever virus (RVFV), dengue virus 1-4 (DENV), chikungunya virus (CHIKV) and Zika virus (ZIKV). Phocine distemper virus (PDV) was added to the PCR as sample extraction control. Validation was conducted following the MIQE-guidelines using a panel of retrospective clinical samples and Quality Control for Molecular Diagnostics (QCMD, https://www.qcmd.org/en/) samples with the simplex-PCR as reference. These included samples from RVFV in animals (n=19), DENV (n=15), CHIKV (n=11), ZIKV (n=2) and YFV (n=1, QCMD), and 14 negative endemic controls. Extractions and PCRs were done with commercially available kits. Some loss of sensitivity was observed at low target concentrations for RVFV, DENV1 and DENV4, when comparing the standard curves of simplex-PCRs with the multiplex-PCR. The limit of detection of the multiplex-PCR was 2,064 copies/mL for CHIKV, 3,587 copies/mL for DENV1, 30,249 copies/mL for ZIKV and 73 PFU/mL for RVFV. Specificity of the multiplex-PCRs was 100%. For 12 out of 48 positive samples with high Cq values, RVFV (n=7), CHIKV (n=2), DENV1 (n=2), YFV (n=1), the multiplex-PCRs were negative. Although PCR sensitivity of the RDCZ-multiplex is slightly lower with low target concentrations, it offers a useful tool for molecular surveillance and clinical diagnosis for arboviruses for the ECA-region.

Authors

  • Anne Hauner
    Department of Biomedical Sciences, Unit of Virology, Institute of Tropical Medicine, Antwerp, Belgium. Electronic address: ahauner@itg.be.
  • Stijn Rogé
    DRC office, Institute of Tropical Medicine, Antwerp.
  • Veerle Vanlerberghe
    Department of Public Health, Unit of Emerging Infectious Diseases, Institute of Tropical Medicine, Antwerp, Belgium.
  • Luciana Lepore
    Department of Public Health, Unit of Emerging Infectious Diseases, Institute of Tropical Medicine, Antwerp, Belgium.
  • Fabrice Ndayisenga
    Rwanda Agriculture and Animal Resources Development Board Kigali, Rwanda.
  • Anselme Shyaka
    Center for One Health, University of Global Health Equity, Butaro, Rwanda.
  • Marjan Van Esbroeck
    Department of Clinical Science, Institute of Tropical Medicine, Antwerp, Belgium.
  • Silvia Situma
    Washington State University Global Health-Kenya, Nairobi, Kenya.
  • Carolyne Nasimiyu
    Washington State University Global Health-Kenya, Nairobi, Kenya.
  • Steve Ahuka-Mundeke
    National Institute for Biomedical Research INRB, Kinshasa, Democratic Republic of the Congo.
  • M Kariuki Njenga
    Washington State University, Pullman, USA.
  • Robert F Breiman
    Emory University, Atlanta, USA.
  • Justin Masumu
    National Institute for Biomedical Research INRB, Kinshasa, Democratic Republic of the Congo.
  • Daniel Mukadi-Bamuleka
    Rodolphe Mérieux INRB-Goma Laboratory, Goma, North Kivu, Democratic Republic of the Congo; Service of Microbiology, Department of Medical Biology, Faculty of Medicine, University of Kinshasa, Kinshasa, Democratic Republic of the Congo; Department of Virology, Institut National de Recherche Biomédicale, INRB, Kinshasa, Democratic Republic of the Congo.
  • Kevin K Ariën
    Department of Biomedical Sciences, Unit of Virology, Institute of Tropical Medicine, Antwerp, Belgium; Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium. Electronic address: karien@itg.be.

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