Interlaboratory comparison using inactivated SARS-CoV-2 variants as a feasible tool for quality control in COVID-19 wastewater monitoring.

Wastewater-based SARS-CoV-2 epidemiology (WBE) has proven as an excellent tool to monitor pandemic dynamics supporting individual testing strategies. WBE can also be used as an early warning system for monitoring the emergence of novel pathogens or viral variants. However, for a timely transmission of results, sophisticated sample logistics and analytics performed in decentralized laboratories close to the sampling sites are required. Since multiple decentralized laboratories commonly use custom in-house workflows for sample purification and PCR-analysis, comparative quality control of the analytical procedures is essential to report reliable and comparable results. In this study, we performed an interlaboratory comparison at laboratories specialized for PCR and high-throughput-sequencing (HTS)-based WBE analysis. Frozen reserve samples from low COVID-19 incidence periods were spiked with different inactivated authentic SARS-CoV-2 variants in graduated concentrations and ratios. Samples were sent to the participating laboratories for analysis using laboratory specific methods and the reported viral genome copy numbers and the detection of viral variants were compared with the expected values. All PCR-laboratories reported SARS-CoV-2 genome copy equivalents (GCE) for all spiked samples with a mean intra- and inter-laboratory variability of 19 % and 104 %, respectively, largely reproducing the spike-in scheme. PCR-based genotyping was, in dependence of the underlying PCR-assay performance, able to predict the relative amount of variant specific substitutions even in samples with low spike-in amount. The identification of variants by HTS, however, required >100 copies/ml wastewater and had limited predictive value when analyzing at a genome coverage below 60 %. This interlaboratory test demonstrates that despite highly heterogeneous isolation and analysis procedures, overall SARS-CoV-2 GCE and mutations were determined accurately. Hence, decentralized SARS-CoV-2 wastewater monitoring is feasible to generate comparable analysis results. However, since not all assays detected the correct variant, prior evaluation of PCR and sequencing workflows as well as sustained quality control such as interlaboratory comparisons are mandatory for correct variant detection.

SARS-CoV-2 wastewater surveillance in Germany: Long-term RT-digital droplet PCR monitoring, suitability of primer/probe combinations and biomarker stability.

In recent months, wastewater-based epidemiology (WBE) has been shown to be an important tool for early detection of SARS-CoV-2 circulation in the population. In this study, a detection methodology for SARS-CoV-2 RNA (wildtype and variants of concern) in wastewater was developed based on the detection of different target genes (E and ORF1ab) by polyethylene glycol (PEG) precipitation and digital droplet PCR. This methodology was used to determine the SARS-CoV-2 concentration and the proportion of N501Y mutation in raw sewage of the wastewater treatment plant of the city of Karlsruhe in south-western Germany over a period of 1 year (June 2020 to July 2021). Comparison of SARS-CoV-2 concentrations with reported COVID-19 cases in the catchment area showed a significant correlation. As the clinical SARS-CoV-2 official case report chain takes time, viral RNA titre trends appeared more than 12 days earlier than clinical data, demonstrating the potential of wastewater-based epidemiology as an early warning system. Parallel PCR analysis using seven primer and probe systems revealed similar gene copy numbers with E, ORF, RdRP2 and NSP9 assays. RdPP1 and NSP3 generally resulted in lower copy numbers, and in particular for N1 there was low correlation with the other assays. The occurrence of the N501Y mutation in the wastewater of Karlsruhe was consistent with the occurrence of the alpha-variant (B.1.1.7) in the corresponding individual clinical tests. In batch experiments SARS-CoV-2 RNA was stable for several days under anaerobic conditions, but the copy numbers decreased rapidly in the presence of dissolved oxygen. Overall, this study shows that wastewater-based epidemiology is a sensitive and robust approach to detect trends in the spread of SARS-CoV-2 at an early stage, contributing to successful pandemic management.

Wastewater-Based Epidemiology for SARS-CoV-2 Biomarkers: Evaluation of Normalization Methods in Small and Large Communities in Southern Germany.

In the context of the COVID-19 pandemic, wastewater-based epidemiology (WBE) emerged as a useful tool to account for the prevalence of SARS-CoV-2 infections on a population scale. In this study, we analyzed wastewater samples from three large (>300,000 people served) and four small (<25,000 people served) communities throughout southern Germany from August to December 2021, capturing the fourth infection wave in Germany dominated by the Delta variant (B.1.617.2). As dilution can skew the SARS-CoV-2 biomarker concentrations in wastewater, normalization to wastewater parameters can improve the relationship between SARS-CoV-2 biomarker data and clinical prevalence data. In this study, we investigated the suitability and performance of various normalization parameters. Influent flow data showed strong relationships to precipitation data; accordingly, flow-normalization reacted distinctly to precipitation events. Normalization by surrogate viruses CrAssphage and pepper mild mottle virus showed varying performance for different sampling sites. The best normalization performance was achieved with a mixed fecal indicator calculated from both surrogate viruses. Analyzing the temporal and spatial variation of normalization parameters proved to be useful to explain normalization performance. Overall, our findings indicate that the performance of surrogate viruses, flow, and hydro-chemical data is site-specific. We recommend testing the suitability of normalization parameters individually for specific sewage systems.

[Innovative SARS-CoV-2 crisis management in the public health sector: Corona dashboard and wastewater surveillance using the example of Berchtesgadener Land, Germany]. / Innovatives SARS-CoV-2-Krisenmanagement im öffentlichen Gesundheitswesen: Corona-Dashboard und Abwasserfrühwarnsystem am Beispiel Berchtesgadener Land.

BACKGROUND: The rise of an infectious disease crisis such as the SARS-CoV­2 pandemic posed significant challenges for the administrative structures of the public health service, which resulted in varying levels of efficiency in outbreak management as a function of staffing and digital resources. This substantially impeded the integration of innovative pandemic outbreak management tools. Innovative crisis management, such as cluster tracking, risk group testing, georeferencing, or the integration of wastewater surveillance recommended by the EU Commission, was made significantly more difficult. AIM: In this case study in Berchtesgadener Land, we present the integration of an area-wide georeferenced wastewater surveillance system that captured 95% of the entire population since November 2020. METHODOLOGY: Sampling occurred twice a week at nine municipal wastewater treatment plants and directly from the main sewer at three locations. Samples were pre-treated by centrifugation and subsequently analyzed by digital droplet polymerase chain reaction (PCR) targeting four specific genes of SARS-CoV­2. RESULTS: The integration of an area-wide georeferenced wastewater surveillance system was successful. Wastewater occurrences are plotted for each municipality against cumulative infections over seven days per 100,000 inhabitants. Changes in the infection pattern in individual communities are noticeable ten days ahead of the official case numbers with a sensitivity of approximately 20 in 100,000 inhabitants. DISCUSSION: The integration of this innovative approach to provide a comprehensive overview of the situation by employing a digital dashboard and the use of an early warning system via quantitative wastewater surveillance resulted in very efficient, proactive management, which might serve as a blueprint for other municipalities in Germany.

Dynamics of pathogens and fecal indicators during riverbank filtration in times of high and low river levels.

Riverbank filtration is an established and quantitatively important approach to mine high-quality raw water for drinking water production. Bacterial fecal indicators are routinely used to monitor hygienic raw water quality, however, their applicability in viral contamination has been questioned repeatedly. Additionally, there are concerns that the increasing frequency and intensity of meteorological and hydrological events, i.e., heavy precipitation and droughts leading to high and low river levels, may impair riverbank filtration performance. In this study, we explored the removal of adenovirus compared with several commonly used bacterial and viral water quality indicators during different river levels. In a seasonal study, water from the Rhine River, a series of groundwater monitoring wells, and a production well were regularly collected and analyzed for adenovirus, coliphages, E. coli, C. perfringens, coliform bacteria, the total number of prokaryotic cells (TCC), and the number of virus-like particles (TVPC) using molecular and cultivation-based assays. Additionally, basic physico-chemical parameters, including temperature, pH, dissolved organic carbon, and nutrients, were measured. The highest log10 reduction during the >72 m of riverbank filtration from the river channel to the production well was observed for coliforms (>3.7 log10), followed by E. coli (>3.4 log10), somatic coliphages (>3.1 log10), C. perfringens (>2.5 log10), and F+ coliphages (>2.1 log10) at high river levels. Adenovirus decreased by 1.6-3.1 log units in the first monitoring well (>32 m) and was not detected in further distant wells. The highest removal efficiency of adenovirus and most other viral and bacterial fecal indicators was achieved during high river levels, which were characterized by increased numbers of pathogens and indicators. During low river levels, coliforms and C. perfringens were occasionally present in raw water at the production well. Adenovirus, quantified via droplet digital PCR, correlated with E. coli, somatic coliphages, TCC, TVPC, pH, and DOC at high river levels. At low river levels, adenoviruses correlated with coliforms, TVPC, pH, and water travel time. We conclude that although standard fecal indicators are insufficient for assessing hygienic raw water quality, a combination of E. coli, coliforms and somatic coliphages can assess riverbank filtration performance in adenovirus removal. Furthermore, effects of extreme hydrological events should be studied on an event-to-event basis at high spatial and temporal resolutions. Finally, there is an urgent need for a lower limit of detection for pathogenic viruses in natural waters. Preconcentration of viral particles from larger water volumes (>100 L) constitutes a promising strategy.

Evaluation and application of molecular denitrification monitoring methods in the northern Lake Tai, China.

Worldwide, excessive reactive nitrogen in groundwater and surface waters is a growing problem, especially in areas that face rapid urbanization and industrialization. One example for environmental nitrogen pollution is the Lake Tai, China's third largest freshwater Lake, located in the Yangtze River basin. Due to the rapid development of the surrounding area, nitrogen compounds like nitrate are discharged into the Lake. Consequently, eutrophication and harmful algae blooms increased and led to the production of toxins directly affecting water consumers through the water supply chain. Denitrification is the main process that attenuates nitrate by converting it into atmospheric nitrogen and represents an intrinsic natural process to compensate the excess reactive nitrogen. In this study, the methodology to detect nitrate reducing bacteria on a functional gene and transcriptional level was optimized and verified in laboratory experiments with a pure culture of Pseudomonas veronii, isolated from Lake Tai. We demonstrated that transcripts analysis (mRNA) did correspond with nitrate reduction activity. Subsequently, the abundance and the activity of nitrate reducing bacteria in Lake Tai were assessed using the developed methods. We demonstrated that nitrate reducing bacteria can be found throughout all sediment and water samples taken from the northern Lake Tai in September 2017. Measurements of narG transcripts also indicated the activity of the membrane-bound nitrate reductase in the water samples. However, the bioinformatic analysis of narG sequences showed varying binding efficiency of primer and gene sites in dependence of phylogenetic groups, which may lead to an underestimation in the qPCR method. Thus, it is important to point out the precautions and limitations of primer systems to monitor nitrogen transformation by qPCR in the environment. Based on this study, mRNA detection methods are suitable for improved microbiological monitoring of denitrification, as an intrinsic process in Lake Tai to mitigate the inflowing reactive nitrogen compounds.

Heterogeneous asymmetric recombinase polymerase amplification (haRPA) for rapid hygiene control of large-volume water samples.

Hygiene of drinking water is periodically controlled by cultivation and enumeration of indicator bacteria. Rapid and comprehensive measurements of emerging pathogens are of increasing interest to improve drinking water safety. In this study, the feasibility to detect bacteriophage PhiX174 as a potential indicator for virus contamination in large volumes of water is demonstrated. Three consecutive concentration methods (continuous ultrafiltration, monolithic adsorption filtration, and centrifugal ultrafiltration) were combined to concentrate phages stepwise from 1250 L drinking water into 1 mL. Heterogeneous asymmetric recombinase polymerase amplification (haRPA) is applied as rapid detection method. Field measurements were conducted to test the developed system for hygiene online monitoring under realistic conditions. We could show that this system allows the detection of artificial contaminations of bacteriophage PhiX174 in drinking water pipelines.

Long amplicon (LA)-qPCR for the discrimination of infectious and noninfectious phix174 bacteriophages after UV inactivation.

Waterborne viruses are increasingly being considered in risk assessment schemes. In general, virus detection by culture methods is time consuming. In contrast, detection by quantitative polymerase chain reaction (qPCR) is more rapid and therefore, more suitable for monitoring. At present, qPCR lacks the essential ability for discriminating between infectious and non-infectious viruses, thus limiting its applicability for monitoring disinfection processes. In this study, a method was developed to quantify UV inactivation by long amplicon (LA)-qPCR. Bacteriophage phiX174 was used as a surrogate for human pathogenic viruses. A qPCR protocol was developed with new sets of primers, resulting in amplicon lengths of 108, 250, 456, 568, 955, 1063, 1544, and 1764 nucleotides. The log reduction of gene copies increased with increasing amplicon length. Additional treatment with the intercalating dye, PMA, had no effect, indicating that the bacteriophage capsids were not damaged by low pressure UV irradiation. A qPCR of nearly the complete genome (approx. 5000 nucleotides) showed similar results to the plaque assay. The log reduction in qPCR correlates with [specific amplicon length x UV dose]. The normalized DNA effect constant can be applied to calculate phiX174 inactivation based on qPCR detection.