Unveiling the enterovirus diversity in Barcelona, Spain (2020-2024) through wastewater and clinical surveillance

Enterovirus (EV) infections are common and often mild or asymptomatic but can occasionally cause serious illness in children. Routine clinical surveillance typically underestimates the full spectrum of circulating EVs, whereas wastewater-based epidemiology captures a broader viral diversity and may serve as an early warning tool. We conducted a 5-year surveillance (2020-2024) in Barcelona, Spain, combining biweekly wastewater sampling with clinical EV reporting during the SARS-CoV-2 pandemic and subsequent relaxation of restrictions. We quantified EV genome copies in sewage by RT-qPCR and performed partial VP1 sequencing on wastewater concentrates and on clinical specimens. Over the study period, wastewater EV loads broadly paralleled reported case trends, with good correlations over the years except in 2022. A monotonic relationship was observed for certain EV type trends, including CVA6, CVB2, CVB3, E11, and EV-D68, but there was low robustness in their predictive capacity for clinical cases. Overall, deep sequencing revealed 122 distinct EV types in wastewater versus 92 types in clinical samples; 66 types were found in both sources, whereas 56 types appeared only in sewage. Sequences from wastewater and clinical sources showed high genetic similarity. Dominant EV types shifted over time highlighting the virus dynamics. These findings demonstrate that integrating WS with clinical surveillance yields a more comprehensive picture of EV circulation, uncovering hidden viral diversity and enabling early detection of emerging public health threats.