COVID-19 and Wastewater-based Epidemiology: A flexible approach to monitoring SARS-CoV-2 and its variants in Trentino wastewater to support the Health Authorities

During the past three years, we assisted to the rise of a new pathogen that afflicted the world with a global pandemic. Working in an era of rapid change has posed important challenges and the focus of research has shifted more and more toward topics of greater social utility. However, this period has also brought a new role for wastewater highlighting how it can provide insight into the health of a community. This is the approach of Wastewater-based Epidemiology (WBE). The work presented here aimed to deepen this approach not only at the theoretical level but also contributing with an ongoing monitoring of about 30 months. The main objectives were (i) to collect information on the recently discovered SARS-CoV-2 virus, its biology, transmission mechanism, and role in wastewater treatment plants (WWTP); (ii) set up a surveillance system that would allow to monitor SARS-CoV-2 infections over time, obtaining early information on its spread among the population to support the Health Authority. Starting from a detailed study of the shedding mechanisms of SARS-CoV-2 in the feces of infected patients, we moved on to the evaluation of the viral concentrations in the sewage system and the wastewater entering the WWTP. The possibility of a faecal-oral transmission route of the virus was investigated by evaluating the data about viability and infectivity in wastewater. The natural processes of decay of the virus in wastewater and the reduction of its concentration in the different treatment stages of WWTPs were explored in literature and with experimental data. At the same time, we developed a SARS-CoV-2 surveillance system in wastewater by applying different detection methods. Some practical and scientific aspects of the analysis protocol have been studied in depth such as the choice of the type of sample, the storage temperatures, and the pre-heat treatments aimed at making the analysis safer for the operator. The choice of the concentration method was evaluated to comply with the low concentration of the viral titer and therefore the crucial importance of this phase of the protocol. During the monitoring campaign, we further investigated aspects related to data processing and developed normalization approaches. Samples from WWTPs in the province of Trento were analysed weekly and sampling frequencies and curve smoothing methods deriving from those data were evaluated. The trend curves thus obtained were compared with those deriving from clinical data provided by the local Health Authority and signals of early warnings of virus diffusion trends in the population were highlighted. With the alternation of the different variants of the virus and the evidence of their importance in the development of new waves of infection, a PCR based genotyping method has been devised to rapidly identify the already known variants. In conclusion, this research project addressed a broad spectrum of aspects related to the WBE approach in contrasting the COVID-19 emergency and confirmed that wastewater could be a valuable source of information and management support for this and other emerging pathogens or micropollutants.