Optimization of a 6-plex Crystal Digital PCR® assay and its application to simultaneous surveillance of enteric and respiratory viruses in wastewater

Multiplex digital PCR (dPCR) approaches are commonly employed in wastewater-based epidemiology (WBE) studies. However, optimizing the dPCR workflow is a critical step to ensure its reliability and accuracy before application. In this study, a 6-plex Crystal Digital PCR® (cdPCR) workflow was optimized for the simultaneous detection of six epidemiologically important pathogens, including three enteric viruses, noroviruses of genogroups I and II (NoV-GI and GII) and enteroviruses (EnV), and three respiratory viruses, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent of coronavirus disease 2019 (COVID-19), influenza A virus (InfA), and respiratory syncytial virus B (RSVB), in wastewater. Four cDNA input ratios (20 %-70 %) and two extraction kits were evaluated for optimization, with 30 % cDNA input and the AllPrep PowerViral DNA/RNA Kit (Qiagen) exhibiting optimal performance. The optimized 6-plex cdPCR assay was applied to a year-long wastewater surveillance study in Japan (n = 52), revealing distinct trends and prevalence ratios for enteric and respiratory viruses. NoV-GII was detected in 96 % of the samples with the highest mean concentration (6.1 ± 0.6 log₁₀ copies/L), while SARS-CoV-2 and InfA were detected in 60 % and 50 % of the samples, respectively, which reflected the circulation of these pathogens within the community. Notably, RSVB was detected less frequently (25 %), in line with the fewer cases of RSVB reported during the study period. The wastewater concentrations of EnV and InfA showed significant positive correlations with hand foot and mouth disease and herpangina and influenza cases, respectively. However, no positive correlations were observed for RSV and COVID-19, possibly due to the testing of RSVB while RSVA was more prevalent and also due to cluster outbreaks. These findings demonstrated the utility of the 6-plex cdPCR assay in detecting pathogens and provided insights into community disease trends, representing an advancement in WBE.