Evaluation of the Enterovirus serotype monitoring approach for wastewater surveillance of hand foot and mouth disease using secondary epidemiological surveillance data

The serotypes of Enterovirus A (EVA) causing hand, foot, and mouth disease (HFMD) exhibit cyclical dominance, with the dominant serotypes being EVA71, Coxsackievirus A type 6 (CVA6), CVA10, and CVA16. In this study, a quadruplex digital polymerase chain reaction (dPCR) assay was developed for serotype-level monitoring in wastewater surveillance for HFMD. The quadruplex dPCR assay performance was comparable to that of the singleplex assays. Grab influent samples (n = 122) were collected weekly from a wastewater treatment plant in Yamanashi Prefecture, Japan, from March 2022 to July 2024. The dPCR results revealed that CVA6 had a lower prevalence in 2023 than in 2022 and 2024, while the detection ratio of EVA71 was significantly higher in 2023 than in 2022 (Chi-square test, P < 0.05). Triangulation of these results with a clinical report indicated the dominance of CVA6 in 2022 and 2024, but not in 2023, while EVA71 was found to be dominant in 2023. Secondary data revealed significantly higher HFMD cases in the catchment in the years when CVA6 was dominant and significantly lower in the years when EVA71 was dominant. The comparative analysis revealed a stronger correlation between CVA6 RNA concentrations and HFMD cases (r = 0.83) than that between a broad range of Enteroviruses, Pan-Enterovirus RNA concentrations (r = 0.47). Thus, serotype-level monitoring, particularly through quadruplex dPCR, provides valuable insights into EVA serotype dynamics, accurately reflects HFMD trends, and supports the monitoring of the cyclical patterns of EVA serotypes.