A consortium led by the FiW and the ISA, together with the Goethe University Frankfurt, has succeeded in publishing the first study in Germany on the detection of SARS-CoV-2 viruses in wastewater for COVID-19 monitoring of the population and assessment of potential infection risks. In cooperation with six water boards in NRW, a screening of influent and selected effluent samples from nine wastewater treatment plants was conducted during the first pandemic wave. The SARS-CoV-2 genes detected in the wastewater were found to be non-infectious in in vitro cell tests. The results of the study were picked up by numerous press articles (SPIEGEL, BILD, WELT, Rheinischer Post and others).
SARS-CoV-2 is an enveloped RNA virus of approx. 100 nm in diameter. In addition to laboratory diagnostic detection of the virus in sputum, SARS-CoV-2 RNA can also be detected in the stool with up to 108 gene copies/ml in up to 67 % of patients, sometimes even in asymptomatic cases. Since the beginning of the pandemic, research groups have therefore been working on methods to use wastewater samples to determine the infection figures of all inhabitants connected to a sewage treatment plant. If the sensitivity and selectivity of the method are sufficient, such analyses could serve authorities, for example, as an integral "all-warning system" as the measures ordered lead to decreasing case numbers in the total population in sewage treatment plant catchment areas. In the Netherlands, such an information system is already established.
Method development and validation
Virus detection is performed by quantitative RT-qPCR for several CoV-2 genes. Using older reserve samples from 2017 and 2018, i.e. before the pandemic outbreak, a methodology was developed and validated in which two gene primers (M gene and RdRP gene) are used in combination. This was done in order to achieve sufficient sensitivity of the analytics on the one hand, and on the other hand to selectively quantify only SARS-CoV-2, excluding other non-disease-causing coronaviruses in wastewater circulating in the population. The plausibility of the results was then critically reviewed and confirmed via Sanger sequencing.
Screening in North Rhine-Westphalia
Our analyses revealed 3 to 20 gene copies per millilitre of raw sewage in all nine sewage treatment plants sampled during the first pandemic wave in April 2020. This is a concentration level that was also measured in studies in the Netherlands and the USA. In addition to the aqueous phase, SARS-CoV-2 can also be detected in the solid phase, sometimes at higher concentrations. As expected, the retention of genetic material is incomplete in conventional wastewater treatment plants. However, after ozonation at the Aachen-Soers wastewater treatment plant, the lowest gene concentrations of all samples examined were measured.
The measured viral load of a wastewater treatment plant was correlated with the number of COVID-19 infected persons in the catchment area of the wastewater treatment plant reported to the public health authorities. In the largest wastewater treatment plant, with an estimated virus load of 6 trillion (6 x 1012) gene equivalents per day, 1037 acute cases were reported in the catchment area, whereas in smaller wastewater treatment plants, with a virus load two orders of magnitude lower, 36 cases were reported.
The sensitivity is sufficient to indicate as an early warning system when the 7-day incidence falls below 50 incidences per 100,000 inhabitants. For reliable use in wastewater-based epidemiology, further method improvements are possible, including the detection of genetic material bound to solid phases and the use of bioindicators. In the second pandemic wave, the data density is to be increased by measuring long-term hydrographs of individual wastewater treatment plants.
Replication tests and infectivity
While our work based on small-volume laboratory studies with approx. 1 to 10 gene copies indicates that the detected RNA fragments are not infectious, load calculations suggest that approx. 6*1010 to 6*1012 SARS-CoV-2 gene equivalents are emitted into the receiving waters per day in individual sewage treatment plants in NRW. Because of the high loads and the low retention capacity of conventional sewage treatment plants, the behaviour of SARS-CoV-2 in the water cycle must be investigated in more detail. An application has been submitted to the MULNV NRW for this purpose.
Research Institute for Water and
Waste Management at RWTH Aachen e. V.
Dr. sc. Dipl.-Ing. Frank-Andreas Weber
+49 241 80 2 68 25 / E-Mail ›
Institute for Urban Water Management at RWTH Aachen University
Univ.-Prof. Dr.-Ing. habil. Thomas Wintgens
+49 241 80 2 52 07 / E-Mail ›
apl. Prof. Dr. Volker Linnemann
+49 241 80 9 15 23 / E-Mail ›
The study was carried out on the initiative of the FiW and the ISA without third-party funding. FiW e. V. gratefully acknowledges support from the Wilo Foundation.
Institute for Medical Virology of the University Hospital Frankfurt (KGU); Goethe University Frankfurt (GUF)
Sandra Westhaus, Frank-Andreas Weber, Sabrina Schiwy, Volker Linnemann, Markus Brinkmann, Marek Widera, Carola Greve, Axel Janke, Henner Hollert, Thomas Wintgens, Sandra Ciesek.