Detection of dengue virus and chikungunya virus in wastewater in Portugal: an exploratory surveillance study.

Background: The global distribution and prevalence of arboviral diseases have increased in recent years, driven by factors such as climate change, biodiversity loss, globalisation, and urbanisation. These diseases are often underestimated due to uneven surveillance and unreported asymptomatic cases. Current surveillance relies on vector and clinical surveillance. In this study, we aimed to explore wastewater-based surveillance (WBS) as an additional tool for dengue virus (DENV) and chikungunya virus (CHIKV) tracking.

Methods: In this exploratory surveillance study, WBS was done at eleven wastewater treatment plants in three regions in Portugal (North, Lisboa and Vale do Tejo, and south). Using quantitative RT-PCR, we quantified in raw wastewater the RNA concentrations of DENV and CHIKV (non-structural viral protein 1 [nsP1] and envelope protein [E1] genes) once every 2 weeks for a period of 11 months, between May 16, 2022, and April 19, 2023. Results were normalised with crAssphage (concentration of target viral RNA divided by the concentration of crAssphage DNA) and provided as median normalised viral load. Prevalence (proportion of positive samples) and viral quantities were summarised for the total sampling period, by calendar month, and by seasons.

Findings: 273 samples were collected from 11 wastewater treatment plants situated across the North (n=75 samples), Lisboa and Vale do Tejo (n=98), and south (n=100) regions of Portugal. DENV was detected in 68 (25%) of 273 samples, with a median viral load of 1·1 × 10^-4 (IQR 3·2 × 10^-5 to 8·0 × 10^-4). CHIKV was detected in 30 (11%) of 273 samples, with median viral loads of 3·1 × 10^-4 (1·6 × 10^-4 to 6·4 × 10^-4; nsP1 gene) and 7·8 × 10^-4 (4·2 × 10^-4 to 2·0 × 10^-³; E1 gene). The pattern of occurrence of CHIKV was similar between regions whereas slight differences were found for DENV. When combining results for the three studied regions, DENV prevalence and viral load had two seasonal peaks (summer and winter) and CHIKV prevalence and viral load had a single peak during March and April of 2023.

Interpretation: This study highlights the potential of WBS as a potent tool for gauging the epidemiological landscape of DENV and CHIKV in Portugal, where autochthonous cases have not yet been detected. WBS could serve as an additional element to conventional surveillance approaches, especially in areas where real-time clinical surveillance data are scarce or delayed.

Funding: EU Emergency Support Instrument and Fundação para a Ciencia e Tecnologia.