To establish and evaluate a tiered molecular surveillance workflow integrating arbovirus detection, dengue virus serotyping, and genomic epidemiology across 4 regions of Senegal.
Approach:
Study Design and Population: This observational molecular surveillance study prospectively enrolled 367 individuals presenting with febrile or nonfebrile illness in Diourbel, Touba, Thiès, and Kédougou between September and December 2025.
Molecular Screening: Multiplex RT-qPCR was used to detect dengue virus (DENV), Zika virus (ZIKV), and chikungunya virus (CHIKV). Forty-three samples from Touba were tested individually, while the remaining samples were tested in 108 pools containing 3 individuals each.
Dengue Serotyping: DENV-positive samples were characterized using RT recombinase polymerase amplification (RT-RPA) assays targeting DENV serotypes 1 through 4. RT-RPA was also evaluated against RT-qPCR in a tested subset.
Genomic Analysis: RT-RPA-positive samples underwent unbiased metagenomic and targeted amplicon-based sequencing. Recovered DENV-2 genomes were analyzed phylogenetically to determine genotype, lineage, relatedness, and estimated emergence.
Serologic Testing: DENV-1-4 NS1-specific IgM was measured to provide additional evidence of recent dengue transmission.
Key Findings:
Among 43 individually tested samples from Touba, DENV was detected in 20.9% (9/43), ZIKV in 9.3% (4/43), and CHIKV in 11.6% (5/43).
Among 108 pooled tests across all sites, DENV was detected in 18.5% (20/108), ZIKV in 5.6% (6/108), and CHIKV in 10.2% (11/108). These pool-level percentages cannot be interpreted as individual prevalence.
DENV-1-4 NS1-specific IgM was detected in 15 of 367 participants (4.1%).
RT-RPA results were completely concordant with RT-qPCR within the tested subset, and all serotyped DENV-positive samples were identified as DENV-2; DENV-1, DENV-3, and DENV-4 were not detected.
Targeted amplicon sequencing recovered near-complete DENV-2 genomes from 14 of 18 RT-RPA-positive samples (77.8%), compared with sufficient genomic coverage in 8 of 18 samples (44.4%) using metagenomic sequencing.
All recovered study sequences clustered within the DENV-2 cosmopolitan genotype, genotype II, lineage II-F.1.1, and were closely related to sequences reported from West Africa and other regions.
Time-resolved phylogenetic reconstruction estimated that the most recent common ancestor of the study sequences existed between 2022 and 2023, consistent with recent emergence or introduction of the circulating lineage followed by rapid expansion.
Metagenomic sequencing identified Plasmodium falciparum reads in 3 samples despite negative routine malaria rapid tests, including 2 samples also positive for DENV-2.
Interpretation:
The findings provide evidence that DENV, ZIKV, and CHIKV were concurrently circulating among the sampled population in Senegal during the study period. The tiered workflow allowed broad molecular screening followed by rapid DENV serotyping and targeted sequencing of selected positive samples. Whole-genome results supported recent expansion of a DENV-2 lineage connected to regional and global transmission networks. However, pooled testing prevents estimation of individual-level prevalence, and exclusive detection of DENV-2 applies to the samples serotyped in this study rather than establishing the absence of other serotypes throughout Senegal.
Limitations:
Pooled testing was used for most participants, limiting individual-level prevalence estimates and potentially underestimating infection burden.
The surveillance period lasted only from September through December 2025 and may not represent seasonal variation in arbovirus transmission.
Individual RT-qPCR results were available only for 43 samples from Touba, whereas the remaining specimens were analyzed in pools.
Genomic analyses were restricted to DENV-2-positive samples and did not characterize the genetic diversity of the detected ZIKV and CHIKV.
The study was designed to characterize arboviral circulation rather than estimate population-level prevalence, and no formal sample-size calculation was performed.
The study did not systematically evaluate malaria diagnostic performance or quantify parasite burden, so the clinical significance of the metagenomic Plasmodium falciparum findings remains uncertain.
Conclusion:
The study demonstrated the application of a tiered surveillance framework combining individual or pooled multiplex RT-qPCR, RT-RPA dengue serotyping, and targeted genomic sequencing. The workflow documented concurrent DENV, ZIKV, and CHIKV circulation and identified recent expansion of DENV-2 genotype II, lineage II-F.1.1. The approach offers a potentially scalable model for linking molecular detection with genomic epidemiology in resource-limited settings, although its surveillance performance and applicability require evaluation across longer periods and broader populations.
