Wastewater-based epidemiology revealed in advance the increase of enterovirus circulation during the Covid-19 pandemic.

Wastewater-based epidemiology (WBE) was conducted to track Enteroviruses (EVs) circulation in the Milan metropolitan area (Northern Italy) during Covid-19 pandemic (March 2020-December 2022). 202 composite 24-hour wastewater samples (WWSs) were collected weekly from March 24, 2020, to December 29, 2022 at the inlet of two wastewater treatment plants (WWTP) in Milan (1.5 million inhabitants). EV-RNA was quantified and molecular characterization of non-polio EVs (NPEV) was performed by Sanger sequence analysis. Data from WWS were matched with virological data collected in the framework of Influenza-Like Illness (ILI) surveillance in the same place and time. EV-RNA was identified in 88.2 % of WWSs. The peak in EVs circulation was observed in late August 2020 (upon conclusion of the first national lockdown), in late August 2021, and in mid-April 2022. EV-RNA concentration in WWS (normalized as copies/d/1000 people) at peak of circulation presented a yearly increase (2020: 2.47 × 1010; 2021: 6.81 × 1010; 2022: 2.14 × 1011). This trend overlapped with trend in EV-positivity rate in ILI cases, expanded from 21.7 % in 2021 to 55.6 % in 2022. EV trends in WWS preceded clinical sample detections in 2021 and 2022 by eight and five weeks, respectively, acting as an early warning of outbreak. Although sequencing of EV-positive WWSs revealed the presence of multiple EV strains, typing remained inconclusive. Molecular characterization of EVs in clinical samples revealed the co-circulation of several genotypes: EV-A accounted for 60 % of EVs, EV-B for 16.7 %, EV-D68 for 23.3 %. EVs were circulating in Milan metropolitan area between March 2020 and December 2022. The epidemiological trends unfolded the progressive accumulation of EV transmission in the population after removal of Covid-19 restrictions. The increased circulation of EVs in 2021-2022 was identified at least 35 days in advance compared to the analysis of clinical data. The inconclusive results of Sanger sequencing lookout for improvement and innovative molecular approaches to deepen track EVs.