Wastewater monitoring for SARS-CoV-2 is a valuable tool for surveillance in public health. However, reliable analytical methods and appropriate approaches for the normalization of results are important requirements for implementing state-wide monitoring programs. In times of insufficient case reporting, the evaluation of wastewater data is challenging. Between December 2021 and July 2022, we analyzed 646 samples from 23 WWTPs in Thuringia, Germany. We investigated the performance of a direct capture-based method for RNA extraction (4S-method) and evaluated four normalization methods (NH₄-N, COD, N_tot, and PMMoV) in a pooled analysis using different epidemiological metrics. The performance requirements of the 4S method were well met. The method could be successfully applied to implement a state-wide wastewater monitoring program including a large number of medium and small wastewater treatment plants (<100,000 p.e) in high spatial density. Correlations between wastewater data and 7-day incidence or 7-day-hospitalization incidence were strong and independent from the normalization method. For the test positivity rate, PMMoV-normalized data showed a better correlation than data normalized with chemical markers. In times of low testing frequency and insufficient case reporting, 7-day-incidence data might become less reliable. Alternative epidemiological metrics like hospital admissions and test positivity data are increasingly important for evaluating wastewater monitoring data and normalization methods. Furthermore, future studies need to address the variance in biological replicates of wastewater.

This study addresses the need for reliable methods in wastewater-based antimicrobial resistance monitoring by comparing three sample preparation approaches for analyzing intracellular DNA (iDNA) and extracellular DNA (exDNA) in raw wastewater: a filtration-based method, a centrifugation-based method, and a combination of the two (combined). We evaluated method repeatability, operational time, usability, consumable costs, qualitative workflow characteristics, and descriptive recovery. Selected antibiotic resistance genes (ARGs) (sul1, bla_OXA48 group, bla_VIM1 group, vanA, tetW, QnrS1) and the mobile genetic element intI1 were quantified by digital PCR. Most target signals occurred in the iDNA-enriched fraction (>98%). Filtration and the combined method received higher usability scores than centrifugation, whereas the combined workflow required approximately 15% higher consumable costs. Storage effects were target-, fraction-, and temperature-dependent. No common holding time satisfied the complete two-sided operational acceptance criteria. Considering decreases in relative recovery alone, storage at 5 °C for up to six days did not reduce any mean recovery below its target- and fraction-specific lower limit. Storage at −20 °C produced decreases in iDNA recovery after 24 h. These findings offer practical guidance for PCR-based AMR monitoring in wastewater.

Since the COVID-19 pandemic, wastewater monitoring has become an additional tool in the surveillance of infectious diseases. Many EU countries put wastewater surveillance systems (WSS) in place to track SARS-CoV-2 and its variants and other pathogens, such as the influenza virus or Respiratory syncytial virus (RSV). In Germany, several research and pilot projects funded by the EU, the Federal Ministry of Education and Research, the Federal Ministry of Health, and projects at Federal State level have been launched in the last four years. In Germany, wastewater monitoring was not implemented as a public health tool before the COVID-19 pandemic, but in September 2022, it has been legally determined in the German infection protection act (Infektionsschutzgesetz, IfSG). As Germany is a federal state, competencies in epidemic management partly belong to the 16 federal states (“Länder”). In the federal states, the local health authorities at the county (“Kreise”) level also have specific risk management and communication competencies. Furthermore, WSS has been incorporated into the revised Urban Wastewater Treatment Directive (EU) 2024/3019. For this reason, the federal states and local health authorities play a pivotal role in successfully implementing wastewater monitoring as a supplementary component of disease surveillance in Germany. Between November 2021 and August 2022, the federal state of Thuringia, Germany, supported a pilot study to implement a surveillance system for SARS-CoV-2-RNA in wastewater of 23 wastewater treatment plants in 17 counties in Thuringia. Here, we describe the study design and the system behind the logistics and the planning, and we provide an overview of the options for involving the public health service. Furthermore, the possibilities for IT concepts and approaches to innovative AI solutions are shown. We also aim to explore the feasibility and potential barriers to further implementing wastewater surveillance as a supplementary public health tool in Thuringia.