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.

Wastewater-based epidemiology (WBE) has been widely employed to track the spread of human pathogens; however, correlating wastewater data with clinical surveillance remains challenging due to population variability and environmental factors affecting wastewater composition. This study evaluated different SARS-CoV-2 normalization methods, comparing static population estimates with dynamic normalization based on common physicochemical parameters: chemical oxygen demand (COD), biochemical oxygen demand (BOD₅), and ammonia (NH₄-N). Wastewater samples were collected from four urban wastewater treatment plants (WWTPs) in northwestern Tuscany (Italy) from February 2021 to March 2023. The correlations between normalized viral loads and clinical COVID-19 cases were highest for static normalization (ρ = 0.405), followed closely by dynamic normalization using COD and BOD₅ (ρ = 0.378 each). Normalization based on NH₄-N was less effective. These findings suggest that chemical parameters, particularly COD and BOD₅, offer a valid alternative for viral normalization when population estimates or flow rate measurements are unavailable. These parameters provide a cost-effective and practical approach for improving WBE reliability, particularly in resource-limited settings. Our results reinforce the importance of normalization in WBE to enhance its representativeness and applicability for public health surveillance.

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.

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.

Groundwater is one of the major sources of water supply for human needs. But anthropic activities such as agriculture are causing significant volume depletion and quality deterioration, favoring microbial contamination that has a negative impact on human health. The geological characteristics of the ground can influence the transport of microorganisms, especially if made of permeable rock. Furthermore, irrigation with untreated or partially treated wastewater can represent an additional health risk due to the potential transmission of pathogens to food. The aim of our research is to provide an interdisciplinary perspective on this issue by integrating hygienic, geological, and agronomic skills. Water samplings are scheduled seasonally by four monitoring campaigns in five sampling points placed in two Southern Italy regions, Apulia (one point at the outlet and two wells near the wastewater plant at Carpignano Salentino, Lecce province, Italy) and Sicily (two wells at Scicli and Pozzallo, Ragusa province, Italy) Laboratory experiments of microorganism transport in permeable rocks will be carried out under saturated and unsaturated conditions. A mathematical model of transport through porous media will be implemented and validated with laboratory measurements. The model will be used to develop a monitoring tool to control sites in Apulia and Sicily where periodic cultural and molecular detection of pathogenic bacteria, viruses, and protozoa will also be taken. In addition, an analysis of the microbiological contamination of herbaceous crops due to the use of low-quality water will be conducted to assess the Quantitative Microbial Risk Assessment (QMRA). The project will provide methodological tools to evaluate anthropogenic pressures and their impact on environmental matrices. The results will allow these pressures to be modulated to minimize environmental and agri-food microbiological contamination and protect public health.