ESTABLISHING AN EFFICIENT PATHOGEN MONITORING SERVICE: A COMPARATIVE STUDY OF VIRAL EXTRACTION METHODS FROM WASTEWATER

Wastewater-based Epidemiology (WBE) is an epidemiological tool used since the early 2000s to observe a community’s health status and habits by analysing targeted biomarkers in wastewater. This approach has proven successful during the COVID-19 pandemic, allowing rapid and preventive surveillance of the spread of the virus and its variants in the population. Subsequently, numerous wastewater monitoring networks were created at national and international levels, and this approach was included in European legislation to combat biological emergencies. At the end of the pandemic, this type of surveillance was directed at other viruses, such as influenza, West Nile, and monkeypox viruses, as well as at antimicrobial resistance, one of the most significant current global threats. However, challenges such as data quality and the standardisation of analysis methods remain, and addressing these is crucial for using this tool to develop data-driven healthcare decisions. We aim to establish a surveillance service for emerging pathogens and beyond, utilising metagenomics to promote preventive mitigation and countermeasures. In the project’s first phase, we focus on an efficient, reliable, and applicable method for extracting and purifying the entire nucleic acid material, both DNA and RNA, both viral and bacterial, from the wastewater sample, even in contexts lacking sophisticated equipment. We compared three methods of concentrating nucleic acids in environmental matrices: the PEG/NaCl precipitation protocol used by the Italian network for monitoring SARS-CoV-2 in wastewater, the filtration of the aqueous sample with 0.22 μm filters (commonly used for the detection of antibiotic resistance genes and bacteria), and engineered beads for the capture and concentration of microorganisms. The efficiency of the concentration methods was tested on composite samples of raw wastewater entering two plants in Trento and spiked with selected viruses (SARS-CoV-2, H1N1, and Murine Norovirus). Here, we present a comparison of the methods by evaluating the yields and quality of extracted DNA and RNA, their reproducibility, and the extent of virus material recovered. By identifying an optimised protocol for processing wastewater samples, these findings will contribute to the development of a robust and adaptable surveillance service for emerging pathogens and antimicrobial resistance.