Wastewater-based epidemiology (WBE) has been applied for public health surveillance for several decades but gained unprecedented global prominence during the COVID-19 pandemic, leading to its broader adoption for multi-pathogen surveillance. However, the impact of heterogeneous urban wastewater infrastructure on the detection and interpretation of pathogen signals remains inadequately characterised, particularly in settings such as Mumbai, with fragmented and partially connected sewer networks. This study systematically compared multi-pathogen wastewater surveillance from centralised sewage treatment plants (STPs) and decentralised open drains (ODs), which represent aggregated and localised population catchments, respectively. Wastewater was collected during two monitoring periods (2023 and 2024) and analysed by RT-qPCR for SARS-CoV-2, influenza viruses, and respiratory syncytial virus (RSV). Comparative analyses included detection frequency, temporal dynamics, and patterns of viral co-occurrence between node types. Viral detection rates were highest in STP influent, consistent with signal integration over larger populations and reduced short-term variability. Conversely, open drains exhibited greater temporal fluctuation, which may be consistent with localised, short-term changes but with reduced signal persistence. In the absence of concurrent clinical data, these node-level differences are interpreted as patterns in the wastewater signal rather than demonstrated gains in epidemiological sensitivity or earlier detection. SARS-CoV-2 was consistently detected across both node types, while influenza virus detection was more sporadic and exhibited marked variability, suggesting seasonal transmission dynamics. Co-detection of influenza A and B was observed in 2023, and influenza A subtyping revealed limited within-type diversity; source attribution for the detected subtypes could not be determined. Collectively, these findings indicate that wastewater signal profiles reflect an interplay between pathogen-specific characteristics and the structural configuration of the sampling network. Overall, the findings support integrated surveillance frameworks that leverage both centralised and decentralised nodes, while highlighting the need for extended temporal monitoring and independent clinical validation to strengthen epidemiological inference.
Environmental surveillance of viral contaminants in water systems is an essential tool for assessing exposure risk and supporting early-warning frameworks for emerging infectious diseases. In this study, a comprehensive one-year surveillance of SARS-CoV-2 and six enteric viruses was conducted across diverse freshwater systems in Tehran, Iran — including rivers, wells, and traditional qanat (underground aqueduct) infrastructures. Water quality parameters (turbidity, pH, Total Dissolved Solids, nitrate) and fecal bacterial indicators (total coliforms, fecal coliforms, and E. coli) were also measured to contextualize viral contamination within a broader environmental risk framework. Between September 2020 and August 2021, a total of 118 water samples were collected and analyzed using quantitative molecular detection methods. Overall, 61 of 118 samples (51.7
Extreme flood events can overwhelm sanitation infrastructure and drive the widespread dispersal of viral pathogens into aquatic environments. In May 2024, Porto Alegre (southern Brazil) experienced an unprecedented flood that caused record-breaking overflow of the Guaíba hydrological system and prolonged urban inundation. Here we report a one-year longitudinal environmental viral surveillance study, comprising seven campaigns between May 2024 and April 2025. Surface waters were analyzed by RT-qPCR for SARS-CoV-2, norovirus genogroups I and II, hepatitis A virus (HAV) and hepatitis E virus (HEV). All five targets were detected simultaneously during the flood peak, reflecting widespread contamination possibly driven by sewage overflow and multi-site infrastructure failure. Thereafter, the number of targets detected per campaign was lower and fluctuating (two to four), tracing a transition from an initial multi-pathogen contamination pulse observed during the flood peak to persistent detection of viral genomic material in surface waters. SARS-CoV-2 was detected in every campaign, in a minority of sites in all campaigns except the last (C7, 14/22, 63.6
Long-term genomic surveillance of SARS-CoV-2 remains important as routine clinical testing and sequencing decline over time after the pandemic. We describe the temporal dynamics, methodological adaptation, and operational continuity of Italy’s national wastewater variant surveillance from October 2021 to December 2025. Across 51 monthly surveys, 6,278 wastewater samples collected from 167 wastewater treatment plants in 18 Regions and 2 Autonomous Provinces were analysed by RT-qPCR and targeted spike sequencing using short- and long-amplicon Sanger and Oxford Nanopore Technology approaches. Overall, 1,186 samples yielded a successful long amplicon and an interpretable Sanger sequence, while 248 regional pools were characterized by ONT during the funded surveillance phase from October 2021 to March 2023. The data documented the replacement of Delta by Omicron, followed by the circulation of BA.1*, BA.2* and BA.4/BA.5* and the subsequent emergence of BQ*, XBB*, EG.5*, and JN.1* lineages, with periods of persistence and co-circulation. The analytical workflow was adapted as lineage-defining mutations changed. Since March 2023, surveillance continued voluntarily through December 2025 using only long-amplicon Sanger sequencing. A qualitative comparison with national periodic flash surveys to estimate the prevalence of SARS-CoV-2 variants on biological samples showed that the two systems broadly captured the same succession of major lineage transitions, although the variant prevalence estimates and wastewater lineage proportions were not directly comparable. These findings support the feasibility of a targeted and adaptable strategy for sustaining nationwide wastewater genomic surveillance across emergency and post-emergency phases and for complementing clinical surveillance when routine testing and sequencing are reduced.
Human noroviruses and persistent bacterial pathogens remain major food-safety concerns. This screening study compared 40
