Some pathogens are excreted in sufficient quantities by infected individuals through stool, urine, saliva, and other bodily fluids, to make them detectable in wastewater. This allows for a testing strategy-independent monitoring of pathogen circulation, capturing individuals who might not undergo diagnostic testing due to mild symptoms. Wastewater testing can provide trends in pathogen loads and circulating variants for specific geographic regions and populations. Results should always be evaluated alongside other data sources.

Date:  13/07/2023

Yes, it is possible to sequence the nucleic acids of pathogens in wastewater samples. For instance, variants of the SARS-CoV-2 virus can be identified in wastewater to understand their circulation in the population.

Date:  13/07/2023

Wastewater surveillance is an additional tool for assessing epidemiological situations. Especially if differences to other surveillance parameters are identified, the comparison between surveillance strategies might hint at possible causes, such as test utilization rates, reporting delays, or changes in virus shedding rates due to variant differences or immunity. Insights from infection dynamics can be used to adjust e.g. testing strategies.

Date:  13/07/2023

The ability to detect rising or falling SARS-CoV-2 infection dynamics earlier than other surveillance systems depends on factors such as the prevailing virus variant, testing strategy, and reporting delays. Literature shows varying lead times; some studies observed a lead time of up to three weeks, while others found none.

Date:  13/07/2023

Regular samples are collected from the influents of wastewater treatment plants. In specialized laboratories, these samples are prepared by concentrating pathogens and isolating their nucleic acids. Pathogens are detected through PCR when present in sufficient concentrations. For further semiquantitative evaluation, corrections are applied using viral fecal indicators or precipitation levels at the time of sampling.

Date:  13/07/2023

Within the AMELAG project, 168 wastewater treatment plants currently test for SARS-CoV-2, covering approximately 32% of the population. At least 80 plants test for Influenza A and B viruses, covering at least 15% of the population.

Date:  29/10/2024

Wastewater composition varies daily and weekly. During weekdays, most human wastewater is discharged in the mornings, while it is more evenly distributed throughout the day on weekends. To obtain representative samples, composite samples are collected. Over 24 hours, 0.5-liter samples are taken hourly, combined, homogenized, and prepared as a 1-liter analysis sample for the lab. Sampling is currently conducted in raw wastewater, immediately after preliminary cleaning.

Date:  13/07/2023

Samples prepared through appropriate concentration and extraction can be stored for PCR analysis for about 4–7 days at 4°C. Longer storage times, storage above 4°C, or freezing raw wastewater significantly reduce analysis results.

Date:  13/07/2023

Polymerase chain reaction (PCR) is used to detect pathogens in wastewater. PCR identifies genetic information (DNA or RNA) of a pathogen, enriching and amplifying specific genes or gene fragments before detection. PCR is a key molecular biology tool and is widely used as a routine procedure.

Date:  13/07/2023

Changes in lab methods, such as sample preparation or PCR analysis, can alter sensitivity, potentially causing abrupt shifts in data trends. These changes are marked with a vertical dashed line in weekly reports.

Date:  10/07/2024

Due to differences in sampling, lab techniques, normalization, and pathogen-specific factors, absolute virus loads should not be compared between sites or pathogens. However, trends and changes in virus loads can be compared.

Date:  31/10/2024

PCR analyses follow quality standards, including controls to ensure reliability. For SARS-CoV-2, two to three genes are amplified in a single PCR, providing redundancy to compensate for errors.

Date:  29/10/2024

The limit of detection is the minimum pathogen concentration detectable by PCR, while the limit of quantification is the minimum concentration measurable or quantifiable. Values below the detection limit are considered absent, whereas values below the quantification limit indicate the presence of pathogen components but are unreliable for quantification.

Date:  29/10/2024

Influent samples are collected with accompanying parameters (e.g., weather data, flow rate, pH, temperature). Samples are sent to laboratories for biomarker analysis. Data are checked for plausibility and quality at the Federal Environment Agency. For SARS-CoV-2, data are normalized to account for effects like rainfall. Influenza data are not normalized, as it offers no quality improvement.

Date:  29/10/2024

Gene concentrations are log-transformed. Aggregated values (e.g., weekly averages) are smoothed using LOESS regression, and confidence intervals are calculated based on the t-distribution. Trends are analyzed by calculating weekly percentage changes. 

The LOESS regression uses a locally weighted regression such that forecasts of a viral load incorporate a certain amount of all detected viral loads around the forecasted value. Influence on this forecast decreases with increasing distance from the forecasted value. The forecasted values form a graph which is estimated separately for each analysis. The Akaike-Criterium with correction factor (AICc) is used to determine the number of viral loads considered. This optimizes estimation quality. The result is a smooth curve and a forecast viral load for each point in time.

Newly added values might change the estimation of the curve – also of past values.

Date:  10/11/2023

Yes, wastewater samples are regularly tested for pathogens like SARS-CoV-2 and Influenza A and B in other countries. Wastewater surveillance is gaining importance globally and being expanded in many regions. 

Date:  29/10/2024

Normalization is based on dry weather inflow at treatment plants, adjusting for dilution effects like rainfall. The ratio of current flow to dry weather flow is multiplied by the geometric mean of PCR results.

For normalizing SARS-CoV-2 data, the dry-weather inflow of the treatment plant serves as a reference. Dry-weather inflow refers to the flow rate unaffected by precipitation or snowmelt.

The current normalization method involves calculating the ratio of inflow during the sampling period (Q_{KA, current}) to dry-weather inflow, then multiplying by the geometric mean of PCR values (Genes(average)). The dry-weather inflow is determined using the median of all previously reported inflow values (Q_{KA, median}).

The normalized value is calculated as follows:

Genes normalized = (Q_{KA, current} / Q_{KA, median}) * Genes (average)

where Q_{KA, current}: is the inflow rate during the sampling period

Q_{KA, median}: is the median inflow rate at the plant

Normalized PCR data are then used in statistical analyses. Other virus raw data, like influenza, are not normalized as normalization has not improved data quality.

Date:  29/10/2024

For SARS-CoV-2, at least two genomic regions are measured. If at least half of the values are below the limit of quantification, half of the limit of quantification is used as the value for calculations. 

For Influenza A and B viruses, typically one genomic region is measured. If the value is below the limit of quantification, half of the limit of quantification is used for calculations. 

The limits of quantification vary depending on the laboratory method, pathogen, and genomic region.

Date:  29/10/2024

Trend analyses are possible, but precise incidence or prevalence estimates are not feasible due to unknown factors, such as virus shedding rates and variability among variants and immunity levels. Varying environmental influences and properties of virus variants will probably require regular adaption of analysis and modelling.

Date:  13/07/2023

Wastewater-based surveillance operates independently of the testing strategy and behavior of the population. Different sampling strategies and analytical methods can address various questions. While individual testing provides actionable insights for specific persons, this is not possible with wastewater monitoring. However, wastewater-based surveillance can describe the trend of SARS-CoV-2 infection dynamics. The results of wastewater surveillance must be interpreted in the context of other surveillance systems to generate added value for situational management.

Date:  13/07/2023

Because wastewater testing cannot provide information on all aspects of public health, it serves as a complement to existing surveillance systems. The following aspects cannot be addressed through wastewater testing: the virulence and clinical relevance of detected pathogens or their variants, disease severity, healthcare burden, affected population groups (e.g., gender or age distribution), risk and protective factors, and vaccine effectiveness. Additionally, deriving individual infection control measures is not possible.

Date:  13/07/2023

Many pathogens can be detected in wastewater. Surveillance of poliovirus has been established in some countries for decades. For possible candidate pathogens, e.g. relevance for public health, possibility for public health interventions or risk of reintroduction are to consider. AMELAG is conducting surveillance for SARS-CoV-2 as well as Influenza virus. Research is ongoing for RSV and antibiotic-resistant bacteria. 

Date:  29/10/2024

Data are available through the RKI Open Data Policy on GitHub and Zenodo (https://github.com/robert-koch-institut/Abwassersurveillance_AMELAG & https://zenodo.org/records/10782702). A comprehensive description of epidemiological wastewater surveillance data is published weekly on the AMELAG homepage (www.rki.de/wastewater). Reports and analysis are included in RKI's weekly reports on respiratory diseases (https://influenza.rki.de/Wochenberichte.aspx) and the Pandemic Radar (Ministry of Health; https://infektionsradar.gesund.bund.de/de).

Date:  29/10/2024

In singular cases, infectious SARS-CoV-2 was detected in feces of infected individuals. Those could, in theory, reach a wastewater treatment plant. However, until now, no infectious SARS-CoV-2 could be detected in wastewater.

Date:  13/07/2023