Detection of Dengue virus RNA in Wastewater during a Local Epidemic in Central Italy (August-October 2024)

Wastewater-based epidemiology (WBE) is a well-established tool for monitoring enteric and respiratory viruses. However, its potential application to vector-borne diseases, including dengue virus (DENV), remains largely underexplored. This study investigated the feasibility of using WBE to detect DENV serotype 2 RNA during the largest dengue outbreak recorded to date in Italy (216 confirmed and probable cases), that occurred in the Marche Region (central Italy) from August to October 2024. From 1 to 30 October, during the decline of the epidemic (30 reported cases), 27 wastewater samples were collected from wastewater treatment plants in the affected municipality of Fano and in the nearby urban center of Pesaro. Four viral RNA concentration methods were tested: polyethylene glycol/sodium chloride precipitation, Nanotrap^® Magnetic Virus Particles, electropositive membrane filtration, and solid fraction analysis. Only solid fraction analysis detected DENV-2 RNA in 9 samples, with digital RT-PCR showing greater sensitivity than Real-time RT-PCR. Virus concentrations ranged from 6.1 × 10¹ to 7.9 × 10² genome copies per gram of solid material. This study highlights the feasibility of WBE as a complementary surveillance tool for vector-borne diseases. Future efforts should focus on refining detection methods and in exploring surveillance strategies for timely wastewater sampling to improve early warning and/or outbreak management.