The Eurofins Covid-19 SentinelTM program was developed to monitor the evolution of the pandemic and for early detection of outbreaks. The study objective was to develop a wastewater testing method to analyze SARS-CoV-2 as an indicator of community infection rate resurgence of COVID-19 or in well-defined sites such as production facilities or nursing homes. Eurofins performed >700 tests on 78 unique samples from 18 sites in Denmark, France and Belgium. Ten variant test protocols were trialed. Protocol variations trialed included centrifugation, precipitation of the SARS-CoV-2 RNA, agitation prior to precipitation, cooling, and pasteurization of the samples. A method was succesfully developed and reliability was supported by stability, reproducibility, and dilution & linearity studies. Results obtained showed a direct link to number of RNA copies in the sample using a calibration curve with synthetic SARS-CoV-2. Analysis was performed on both the liquid phase and solid phase of wastewater samples, with virus RNA detected in both phases but more frequently in the liquid phase. The virus was present in a sample from a Danish community wastewater treatment plant collected on February 24, 3 days before the first COVID-19 case was officially reported in the country. The greatest concentration of virus detected corresponded to when the COVID-19 crisis was at its peak in Denmark. Based on studies carried out in a Danish hospital, the wastewater testing method is expected to be able to detect a community COVID-19 prevalence rate as low as a 0,02%-0,1% (i.e. between 2 virus shedders per 10000 and 1 virus shedder per 1000). The wastewater testing method was used to monitor a Danish Community after a COVID-19 outbreak and it was shown that the method can be used as a semi-quantitative method to monitor the development of an outbreak.

Estimates of viral content can be used as an indicator of the community infection rate, and as an indicator of resurgence of COVID-19 in well-defined sites such as production facilities, hospitals, nursing homes and wastewater treatment plants. The Eurofins Covid-19 Sentinel™ program was developed to monitor the evolution of the Covid-19 pandemic, and for early detection of corona disease outbreaks in local communities. This paper provides an extension of the Eurofins Sentinel program, and demonstrates further protocol optimization, method validation and real life application. The validation of the Eurofins Covid-19 Sentinel™ protocol include analysis of optimal storage conditions, sample stability, estimates of reproducibility, sensitivity, precision, and precision in viral recovery upon dilution of wastewater samples. The Eurofins Covid-19 Sentinel™ wastewater method was later implemented in the national wastewater surveillance program in Denmark, as it provides a robust, sensitive and semi-quantitative method to monitor the spread and occurrence of SARS-CoV-2 virus within local populations.