Wastewater-based epidemiology (WBE) is currently used to monitor not only the spread of the viral SARS-CoV-2 pandemic but also that of other viruses in endemic conditions, particularly in the absence of syndromic surveillance. The continuous monitoring of sewage requires high expenditure and significant time investments, highlighting the need for standardized methods and structured monitoring strategies. In this context, we conducted weekly wastewater monitoring in northwestern Tuscany (Italy) and targeted human adenovirus (HAdV), norovirus genogroup II (NoVggII), enterovirus (EV), and SARS-CoV-2. Samples were collected at the entrances of treatment plants and concentrated using PEG/NaCl precipitation, and viral nucleic acids were extracted and detected through real-time reverse transcription qPCR. NoVggII was the most identified target (84.4%), followed by HAdV, SARS-CoV-2, and EV. Only HAdV and EV exhibited seasonal peaks in spring and summer. Compared with data that were previously collected in the same study area (from February 2021 to September 2021), the results for SARS-CoV-2 revealed a shift from an epidemic to an endemic pattern, at least in the region under investigation, which was likely due to viral mutations that led to the spreading of new variants with increased resistance to summer environmental conditions. In conclusion, using standardized methods and an efficient monitoring strategy, WBE proves valuable for viral surveillance in pandemic and epidemic scenarios, enabling the identification of temporal–local distribution patterns that are useful for making informed public health decisions.

Reusing reclaimed water is of paramount importance to achieve the 2030 Agenda for Sustainable Development Goals 6 and 13. In Europe, a recent Regulation set minimum requirements for water reuse in agriculture. However, some challenges remain considering microbial risks and their prevention. In this study, two urban wastewater treatment plants (WWTPs) were investigated from the perspective of reuse. A five-year investigation was performed on routine monitoring parameters collected under different weather conditions (wet/dry) and treatments (chlorination/non-chlorination) in inlet and outlet samples. Moreover, a three-month investigation focused on microbial parameters, including indicators, index pathogens (Human Adenovirus—HAdV, Salmonella spp.), and other viral pathogens (norovirus, enterovirus, and SARS-CoV-2). The long-term study revealed the compliance of both WWTPs for chemical parameters (organic substances and solids) in more than 90% of samples, whereas for Escherichia coli, the compliance ranged from 96.1% with chlorination under dry weather to 16.7% without chlorination in wet days. E. coli was positively associated with chemical oxygen demand (COD), which could be a promising and online measurable proxy of E. coli. The study on microbial performance demonstrated sound reliability in detecting E. coli as a suitable surrogate for Salmonella in chlorinated effluents, but neither bacterial nor viral indicators are able to represent HAdV. Although chlorination was able to remove most of the pathogens considered, the compliance with microbial indicators seems insufficient to represent viral water safety.

The current pandemic has provided an opportunity to test wastewater-based epidemiology (WBE) as a complementary method to SARS-CoV-2 monitoring in the community. However, WBE infection estimates can be affected by uncertainty factors, such as heterogeneity in analytical procedure, wastewater volume, and population size. In this paper, raw sewage SARS-CoV-2 samples were collected from four wastewater treatment plants (WWTPs) in Tuscany (Northwest Italy) between February and December 2021. During the surveillance period, viral concentration was based on polyethylene glycol (PEG), but its precipitation method was modified from biphasic separation to centrifugation. Therefore, in parallel, the recovery efficiency of each method was evaluated at lab-scale, using two spiking viruses (human coronavirus 229E and mengovirus vMC₀). SARS-CoV-2 genome was found in 80 (46.5%) of the 172 examined samples. Lab-scale experiments revealed that PEG precipitation using centrifugation had the best recovery efficiency (up to 30%). Viral SARS-CoV-2 load obtained from sewage data, adjusted by analytical method and normalized by population of each WWTP, showed a good association with the clinical data in the study area. This study highlights that environmental surveillance data need to be carefully analyzed before their use in the WBE, also considering the sensibility of the analytical methods.

During the COVID-19 pandemic, wastewater-based surveillance (WBS) showed great potential as an early warning system and could complement human clinical surveillance. This study aimed to highlight the added value of WBS for respiratory infections alongside different clinical surveillance systems. Sewage collected at the entry of four Wastewater Treatment Plants in Northern Tuscany (Italy) were analyzed for SARS-CoV-2, Human Adenovirus (HAdV), Respiratory Syncytial Virus (RSV) and Influenza Virus (IV), over two years. Clinical data for COVID-19 were available for the study area, while data for other viruses came from national virological surveillance. For SARS-CoV-2, the correlation was highly significant between clinical and hospitalization data (ρ = 0.8460), but not significant between wastewater and clinical or hospitalization data (ρ = 0.1682 and ρ = 0.0569, respectively). SARS-CoV-2 RNA was found in wastewater even in period when clinical cases were not reported, indicating a continuous community circulation. HAdVs were detected in 74.3 % of samples, but most of the sequences identified belonged to enteric species (HAdV-F41), indicating the need of distinguishing the species causing respiratory diseases for the surveillance. RSV were found only in winter 2022-2023, while IV had not been detected in wastewater, probably due to poor test sensitivity. In conclusion, although there may be various challenges in testing different targets, WBS can provide pathogen-specific situational assessment which complements existing surveillance systems.

Wastewater-Based Epidemiology (WBE) is currently used to monitor not only the spread of the pandemic virus SARS-CoV-2 but also for other viruses in endemic conditions, particularly in the absence of syndromic surveillance. Continuous monitoring of sewage requires high costs and significant time investment, highlighting the need for standardized methods and structured monitoring strategies. In this context, we conducted weekly wastewater monitoring in northwestern Italy, targeting Human Adenovirus (HAdV), Norovirus Genogroup II (NoVggII), Enterovirus (EV), and SARS-CoV-2. Samples collected at the entrance of treatment plants were concentrated using PEG/NaCl precipitation and viral nucleic acids were extracted and detected by real-time (RT-)qPCR. NoVggII resulted the most identified target (84.4%), followed by HAdV, SARS-CoV-2, and EV. Only HAdV and EV exhibited seasonal peaks in spring and summer. SARS-CoV-2 results compared with data previously collected in the same study area (February 2021 to September 2021) revealed a shift from an epidemic to a pandemic status, likely due to the evolution of variants. In conclusion, WBE, using standardized methods and an efficient monitoring strategy, proves valuable for virus surveillance in pandemic and epidemic scenarios, enabling the identification of temporal-local distribution patterns that are useful for making informed public health decision.