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

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 2064 copies/ml for CHIKV, 3587 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.

Keywords: Arbovirus; Chikungunya virus; Dengue virus; Real-time multiplex RT-PCR; Rift valley fever virus; Validation; Zika virus.

Fig. 1.

Occurrence of arboviruses (RVFV, ZIKV, DENV, CHIKV) and the suitability for Aedes aegypti on the African continent. RVFV occurrence data are acute human cases reported during 1999–2021 (Gerken et al., 2022). The occurrence data for DENV, CHIKV and ZIKV spans the years of 1927 to March 2024, assembled by Lim et al. (Lim et al., 2025). For Aedes aegypti the mean weighted suitability ranging from 0 (blue) to 1 (red), see Longbottom et al. 2023 (Longbottom et al., 2023).

Fig. 2.

The limit of detection for the RDCZ-Multiplex and typical viral load ranges in patients for RVFV, ZIKV, DENV and CHIKV. The limit of detection for each pathogen was determined with Probit regression in PFU/ml for RVFV and in copies/ml for ZIKV, DENV and CHIKV. The blue symbol represents the 95 % limit of detection for the RDCZ-multiplex PCR. The viral load in patients was calculated as median from references. The lowest and highest value were the median of the lowest and highest value given in the references. For ZIKV 5 references were available (Waggoner et al., 2016; Medina et al., 2019; Santiago et al., 2019; Musso et al., 2017; Aubry et al., 2016), for CHIKV 7 references (Panning et al., 2008; Waggoner et al., 2016; Laurent et al., 2007; Raghavendhar et al., 2019; Weaver and Lecuit, 2015; Staikowsky et al., 2009; Thiberville et al., 2013), for DENV 7 references (Waggoner et al., 2016; Stittleburg et al., 2020; Guilarde et al., 2008; Laue et al., 1999; Pozo-Aguilar et al., 2014; Perdomo-Celis et al., 2017; Tricou et al., 2011) and for RVFV 3 references (Njenga et al., 2009; de St Maurice et al., 2018; van Jansen Vuren et al., 2015).