Detection Patterns and Temporal Dynamics of Multiple Pathogens in Wastewater Using a TaqMan Array Card

Abstract

Background: Wastewater-based surveillance (WBS) complements clinical surveillance by capturing community-level circulation of infectious diseases, including asymptomatic infections.However, most multipathogen WBS studies have focused on pathogen detection and detection frequency, with limited evaluation of temporal changes using semi-quantitative Ct values.Objectives: This study aimed to characterize multipathogen detection patterns in wastewater using a customdesigned TaqMan Array Card (TAC) and explore temporal changes using semi-quantitative Ct values.Methods: Weekly influent samples (500 mL) were collected from two wastewater treatment plants in Cheonan and Asan during weeks 21~52 of 2025 (64 total samples).The TAC targeted 31 pathogen analysis groups encompassing respiratory, enteric, and selected high-concern pathogens, along with seven antimicrobial resistance genes (ARGs).Pathogen occurrence was evaluated based on week-based detection status and detection frequency, while Ct values were interpreted as a semi-quantitative indicator for exploratory assessment of temporal changes.Samples failing the predefined quality control criteria were re-extracted and reanalyzed, and all samples were ultimately included in the final analysis.Results: Ten pathogens, including SARS-CoV-2, norovirus, adenovirus type F, and human rhinovirus, were detected in 100% of the study weeks.Influenza A virus and respiratory syncytial virus (RSV) showed increased detection during winter, with influenza A/H3 and RSV type B predominating among their respective subtypes.Semi-quantitative Ct values showed no consistent increasing or decreasing trend for SARS-CoV-2, but they declined during winter for norovirus and influenza A. In contrast, RSV showed increased winter detection without a corresponding decrease in Ct values.bla_KPC, bla_NDM, and vanA/B/ C were detected in all study weeks, and mecA and bla_OXA also showed high detection frequencies.Conclusions: TAC-based WBS enabled simultaneous characterization of multipathogen detection patterns, antimicrobial resistance gene detection, and exploratory assessment of temporal changes using semiquantitative Ct values.These findings support TAC-based WBS as an integrated tool for community-level infectious disease surveillance.