Presence of SARS-Coronavirus-2 in sewage

Gertjan Medema; Leo Heijnen; Goffe Elsinga; Ronald Italiaander; Anke Brouwer

To get an indication of the sensitivity of the monitoring of sewage, a proxy for the prevalence of COVID-19 in the cities served by the WWTP sampled was created using: 1. the number of COVID-19 cases on March 5 or 16, as reported for the cities that are served by each of the WWTP as numerator, and 2. the number of people served by each of the WWTP as denominator. The latter was estimated from the capacity (in inhabitant equivalents) of each of the WWTP. The prevalence at the airport could not be estimated, as the number of COVID-19 cases were not reported for this denominator. This yielded a rough estimate of the prevalence, since the service areas of WWTP do not precisely overlap with the city boundaries. Also, there was some delay in notification of cases by the local health authorities to the national surveillance system which could mean that the actual prevalence was slightly higher than reported. Moreover, the reported prevalence are the data from cases with COVID-19 as confirmed by laboratory diagnosis; a significant proportion of COVID-19 goes undetected, since people with mild symptoms are not tested. A study among healthcare workers in two hospitals in the Netherlands indicated that SARS-CoV-2 was already circulating undetected in the community prior to February 27, when the first COVID-19 case was reported, suggesting that there is a high prevalence of mild COVID-19 in the community. 22 The detection of N1 in WWTP Amersfoort on March 5, when no cases were reported in Amersfoort, also suggests virus circulation in the population before COVID-19 cases are reported through the health surveillance system. Comparing Tables 3 and 4 shows that the N1 primer/probe set started to produce a signal in sewage samples when the observed COVID-19 prevalence was around or even below 1.0 case in 100,000 people and the N3 and E set started to yield positive signals when the observed prevalence was 3.5 case per 100,000 people or more, although not consistently, since sewage from WWTP Amersfoort did not yield positive results with set N3 and E. Given the roughness of the prevalence estimates, these numbers are indicative, but do indicate that sewage surveillance with the method used in this study is sensitive. However, reliable quantification of SARS-CoV-2 with RT-qPCR in sewage will be required to make reliable surveillance feasible. Therefore, the development of controls to consistently monitor coronavirus recovery and to measure viral RNA yield and check for RT-PCR inhibition is of great importance. The analyses of an added quantified suspension of another human coronavirus (such as 229E) 23 to the sewage samples can potentially be used as an easy control to make reliable quantification possible. Also, digital droplet PCR could aid in the quantification of SARS-CoV-2 in water, as shown for other RNA viruses. 24 The detection of the virus in sewage, even when the COVID-19 prevalence is low, indicates that sewage surveillance could be used to monitor the circulation of the virus in the population and as early warning tool for increased circulation in the coming winter or unaffected populations. Amsterdam 0,0 0,3 5.5