Comparative Investigation and Trends of Respiratory Viruses Using Wastewater-Based Epidemiological Surveillance in Patras, Greece

The COVID-19 pandemic has underscored the importance of alternative epidemiological tools capable of providing real-time, population-level insights into infectious disease dynamics. Wastewater-based epidemiology (WBE) has emerged as a powerful, non-invasive method for tracking viral transmission within communities. While initially focused on SARS-CoV-2, WBE now offers the potential for multiplex surveillance of a broader range of respiratory pathogens. This study applied WBE to monitor the circulation of six major respiratory viruses such as Human adenovirus (HAdV), SARS-CoV-2, Influenza A and B, Respiratory Syncytial Virus (RSV) A/B, and Rhinoviruses, over a six-month period (ISO weeks 2022-40 to 2023-13) in the city of Patras, Greece. Weekly composite samples from a central wastewater treatment plant were analyzed via quantitative PCR, and viral genome concentrations were normalized per 100,000 inhabitants. The results revealed distinct circulation patterns: HAdV and SARS-CoV-2 were detected consistently throughout the period, while Influenza A peaked during the winter months, followed by Influenza B. RSV, however, appeared earlier in the season (first detectable from ISO week 2022-46) and overlapped with Influenza. Rhinoviruses displayed intermittent peaks, indicating multiple waves or persistent low-level transmission. Correlation analyses showed strong positive associations between influenza viruses, RSV, and SARS-CoV-2, suggesting synchronized seasonal trends. Hierarchical cluster analysis classified the viruses into three distinct groups: (1) an epidemic cluster including Influenza A/B, RSV, and Rhinoviruses; (2) a persistently present cluster represented by HAdV; and (3) a separate episodic cluster characterized by SARS-CoV-2. These groupings reflect differences in viral epidemiology and shedding behaviour. This study confirms the effectiveness of WBE in tracking the temporal dynamics of multiple respiratory viruses and provides evidence of its utility in supplementing traditional clinical surveillance systems. The consistent detection of underreported pathogens such as HAdV underscores the added value of environmental monitoring for public health preparedness and early warning applications. These findings advocate for the integration of WBE into routine respiratory virus surveillance frameworks.