enteric and respiratory viruses. However, its application to vector-borne diseases, including dengue virus (DENV), remains largely unexplored. This study aims to evaluate the feasibility of detecting DENV RNA in wastewater during a dengue outbreak in Central Italy, providing insights into the potential role of WBE as a complementary surveillance tool for arboviral infections. Methods: Between August and October 2024, a dengue outbreak in the municipality of Fano (Marche region) resulted in 218 confirmed autochthonous cases—the largest reported in Italy to date. Wastewater sampling was initiated in October 2024, during the decline of the epidemic curve. A total of 27 samples were collected from wastewater treatment plants in Fano and the nearby city of Pesaro. Four concentration methods were tested to optimise viral RNA recovery: a) polyethylene glycol/sodium chloride precipitation, b) Nanotrap® Magnetic Virus Particles, (c) electropositive membrane filtration, and (d) analysis of the solid fraction. Detection was performed using both real-time and digital PCR, targeting DENV serotype 2 RNA. Results and Conclusions: Among the concentration methods tested, only the solid fraction analysis successfully detected DENV-2 RNA in 9 of the 27 samples. Digital PCR showed greater sensitivity than real-time PCR, with viral concentrations ranging from 6.1 × 10¹ to 7.9 × 10² genome copies per gram of solid material. At the time of sampling, only six (confirmed and probable) cases had been reported, suggesting that WBE may detect viral RNA even in the presence of limited clinical cases. This study provides the first evidence of dengue virus RNA detection in wastewater in Italy during a local outbreak, demonstrating the potential of WBE as a complementary tool for the surveillance of vector-borne diseases. Although the delayed start of sampling limited real-time correlation with case data, the findings highlight the sensitivity of WBE in identifying low-level community transmission. Future work should focus on improving sampling timeliness, refining concentration protocols, and integrating WBE into public health preparedness strategies for emerging arboviral threats.
Detection of dengue virus rna in wastewater during an autochthonous outbreak in central italy: a pilot study on environmental surveillance of vector-borne diseases / Veneri, C.; Mancini, P.; Bonanno Ferraro, G.; Franco, A.; Iaconelli, M.; Brandtner, D.; Lucentini, L.; Venturi, G.; Marsili, G.; Del Manso, M.; Sacco, C.; Bella, A.; Riccardo, F.; Pezzotti, P.; Bolognini, L.; Magi, S.; Ballarini, E.; Grucci, A.; Suffredini, E.; La Rosa, G.. - (2025). ( 9th National Congress of the Italian Society for Virology Torino ).
Detection of dengue virus rna in wastewater during an autochthonous outbreak in central italy: a pilot study on environmental surveillance of vector-borne diseases
C. VeneriPrimo
;P. Mancini;A. Franco;F. Riccardo;E. Ballarini;
2025
Abstract
enteric and respiratory viruses. However, its application to vector-borne diseases, including dengue virus (DENV), remains largely unexplored. This study aims to evaluate the feasibility of detecting DENV RNA in wastewater during a dengue outbreak in Central Italy, providing insights into the potential role of WBE as a complementary surveillance tool for arboviral infections. Methods: Between August and October 2024, a dengue outbreak in the municipality of Fano (Marche region) resulted in 218 confirmed autochthonous cases—the largest reported in Italy to date. Wastewater sampling was initiated in October 2024, during the decline of the epidemic curve. A total of 27 samples were collected from wastewater treatment plants in Fano and the nearby city of Pesaro. Four concentration methods were tested to optimise viral RNA recovery: a) polyethylene glycol/sodium chloride precipitation, b) Nanotrap® Magnetic Virus Particles, (c) electropositive membrane filtration, and (d) analysis of the solid fraction. Detection was performed using both real-time and digital PCR, targeting DENV serotype 2 RNA. Results and Conclusions: Among the concentration methods tested, only the solid fraction analysis successfully detected DENV-2 RNA in 9 of the 27 samples. Digital PCR showed greater sensitivity than real-time PCR, with viral concentrations ranging from 6.1 × 10¹ to 7.9 × 10² genome copies per gram of solid material. At the time of sampling, only six (confirmed and probable) cases had been reported, suggesting that WBE may detect viral RNA even in the presence of limited clinical cases. This study provides the first evidence of dengue virus RNA detection in wastewater in Italy during a local outbreak, demonstrating the potential of WBE as a complementary tool for the surveillance of vector-borne diseases. Although the delayed start of sampling limited real-time correlation with case data, the findings highlight the sensitivity of WBE in identifying low-level community transmission. Future work should focus on improving sampling timeliness, refining concentration protocols, and integrating WBE into public health preparedness strategies for emerging arboviral threats.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
