Wastewater Monitoring for Epidemiological Situation Assessment (AMELAG) − Completed projects
Date: 07/03/2025
Data Validation and Normalization – Research at the Environmental Protection Agency in the Field of Wastewater Technology Research, Wastewater Disposal
The concentration of SARS-CoV-2 in wastewater can be strongly influenced by changes in the wastewater composition, for example, due to rainfall events. This makes trend detection more difficult. The flow rate is a common parameter used to account for fluctuations in wastewater composition. However, there are also several alternative parameters and other possibilities. Therefore, the UBA (Environmental Protection Agency) is developing methods that allow an assessment of different validation and normalization approaches. The goal is to provide an objective, site-specific evaluation of the various parameters and how the use of the appropriate approach can improve trend detection.
Publications
Saravia, C. et al. “Wastewater-based epidemiology: deriving a SARS-CoV-2 data validation method to assess data quality and to improve trend recognition”, 2024, Frontiers in Public Health, 12/2024
https://www.frontiersin.org/journals/public-health/articles/10.3389/fpubh.2024.1497100/full
Trace Substance Analysis for Investigating the Suitability of Further Normalization Parameters – Research at the Environmental Protection Agency in the Field of Wastewater Analytics, Monitoring Procedures
When determining the viral load in wastewater, a "normalization" of the measured viral load must be carried out. "Normalization" means that efforts are made to balance fluctuations in wastewater volume and thus the composition of wastewater constituents. The better the normalization, the more accurately the viral load can be calculated in relation to the population share. Instead of the possibility of normalization through the average flow rate, quantification of surrogate viruses, or usual accompanying parameters like conductivity and ammonium, further possibilities and parameters for normalization at different wastewater treatment plant locations will be tested in cooperation with the Federal Institute for Materials Research and Testing (BAM) through trace substance analysis. Special, high-throughput test methods (ELISA) will be used for this purpose, modified for the highly sensitive measurement of pharmaceutical residues, ingredients, and metabolic products. The individual trace substances will be evaluated for their suitability as human fecal indicators at the respective locations.
Laboratory Harmonization / Standardization / Comparative Studies – Research at the Environmental Protection Agency in the Field of Wastewater Analytics, Monitoring Procedures
The current concentrations of SARS-CoV-2 in wastewater are measured by over 20 different laboratories as part of AMELAG. Different methods are used, including sample concentration, virus RNA extraction, gene sequences detected in PCR, and the PCR analysis used. In the framework of this research project, the UBA will identify and cluster laboratory methods and then aim for harmonization. Since it is assumed that various methods lead to comparable analytical results, a quality assessment of the data in relation to the respective analysis will be carried out. This will also aid in the preparation and design of a comparative test, which will also be conducted as part of the AMELAG project. This test will help determine detection limits, among other things. In addition, other pathogens relevant to public health, such as influenza and RSV, will be tested and established for routine laboratory testing.
Development of Methods for Detecting Pathogens and Antimicrobial Resistances (AMR) in Wastewater Samples – Research at the Environmental Protection Agency in the Field of Microbiological Risks
Concepts are being developed for methods to reliably detect relevant pathogens (including their antibiotic resistances) in wastewater samples. The focus is on enterobacteria with clinically important antibiotic resistances. To identify these in wastewater samples, a multi-stage screening process is being developed, which aims to detect both living bacteria directly and resistance genes and further sequence information. Mass spectrometry techniques, determination of minimum inhibitory concentrations for antibiotic substances, as well as molecular biological and sequencing-based methods are used in this process.
Method Development and Establishment of Detection Methods for Other Public Health-Relevant Viral Pathogens – Research at the Environmental Protection Agency in the Field of Microbiological Risks
Influenza A/B virus gene fragments are detected using molecular biological laboratory methods, and the results are evaluated. The methods are being prepared for possible implementation in routine wastewater surveillance. Furthermore, as part of AMELAG, new methods for detecting other respiratory and gastrointestinal pathogens in wastewater are being developed and tested using various preparation and extraction methods. Depending on the properties of the pathogens, different preparation methods are required for nucleic acid enrichment and extraction. Various concentration methods will be compared, experimental series will be designed using wastewater samples to which inactivated viruses or viral nucleic acids are added, and the methods will be validated. Additionally, experiments will be conducted to determine the detection limits. The goal is to develop quality-assured and valid laboratory methods that can be gradually applied through continuous optimization and harmonization in wastewater surveillance.
Establishment of Methods for Detecting Viruses in Wastewater to Assess the Infection Situation in the Population (EViAb)
The Technical University of Dresden is analyzing the wastewater from eight wastewater treatment plants in the state of Saxony as part of this project. This will establish the foundations for creating a panel of viruses whose detection in wastewater will support the interpretation of the infection situation for pathogens beyond SARS-CoV-2. Viruses that also develop pandemic patterns and regularly exhibit seasonal incidence peaks were selected: the influenza virus and the Respiratory Syncytial Virus (RSV). In addition to optimizing the enrichment of these viruses from wastewater and quantitatively assessing the efficiency of the methods used (detection limit, recovery rate), inactivation tests will help estimate the effects of different durations between excretion and analysis on the measured virus concentration.
Development of a Nationwide Wastewater Surveillance System in Thuringia Using Mobility Data and Artificial Intelligence (Wastewater Surveillance TH)
As part of the project, up to 14 wastewater treatment plants in Thuringia will be sampled weekly and tested for SARS-CoV-2 and influenza. To describe the spread of specific pathogens, mobility data will be combined with the wastewater monitoring data. The aim is to uncover where infections are spreading more rapidly and to explore the relationships between local conditions and the nationwide development of the pandemic. This will help better understand the spread patterns during the pandemic.
Establishment of a Multiplex PCR from Wastewater for Detection and Characterization of RSV as Part of the SARS-CoV-2 Wastewater Monitoring
As part of the AMELAG project, the University Hospitals of Bonn and Düsseldorf are jointly researching the potential to detect and characterize additional pathogens via wastewater monitoring. In the pilot phase, a multiplex PCR is being introduced to detect SARS-CoV-2, the Respiratory Syncytial Virus (RSV), and the influenza virus simultaneously. At the same time, timely sequencing of RSV in wastewater samples is being carried out to identify circulating virus variants. Due to the proximity of the wastewater treatment plant to the research laboratories, the study will also examine how quickly wastewater testing can be performed when long transport routes are eliminated and what obstacles hinder real-time detection. The goal of these investigations is to further develop wastewater surveillance as an early warning system for various infectious events and to evaluate the utility of this data for public health services.
Sequencing of Wastewater Samples in Germany as a Surveillance System for Pathogens (TU Darmstadt)
There are various variants of a pathogen. These differ in their genetic information, which can be read using a technique called sequencing. Sequencing from wastewater is more challenging than from clinical samples because genes from many different people are present, and the genetic material in wastewater has broken into fragments. However, known gene sequences can be used to assemble these fragments and determine the likelihood of variants. TU Darmstadt, in a collaborative project with the RKI (Robert Koch Institute), carries out this process weekly at seven locations for SARS-CoV-2. The results are presented in the AMELAG weekly report. In this way, circulating variants can be identified, and changes in the composition of variants can be determined. The goal is also to determine the distribution of variants for the influenza pathogen. TU Darmstadt is conducting research to see how accurately this can be achieved.
National Pilot Project ESI-CorA
In the ESI-CorA pilot project, 20 wastewater treatment plant locations in Germany were selected to begin monitoring in February 2022. An expansion phase incorporated 28 additional sites into the pilot project, with sampling and SARS-CoV-2 analytics funded by the Federal Ministry of Education and Research or directly by a federal state. The implementation and operation of wastewater-based surveillance were scientifically supported by various institutions. A key goal was to harmonize technical procedures for wastewater monitoring across the different sites. Methods for normalizing raw data, PCR analysis used, and calculating trend dynamics were also investigated. The project was funded under the European Commission's Emergency Support Instrument (duration: November 2021 to March 2023).