key: cord-0787718-kllgnncl authors: Agrawal, S.; Orschler, L.; Lackner, S. title: Long-term monitoring of SARS-CoV-2 in wastewater of the Frankfurt metropolitan area in Southern Germany date: 2020-10-27 journal: nan DOI: 10.1101/2020.10.26.20215020 sha: 0c3bb5e598a5415ea64c1f7a4c998887e636725a doc_id: 787718 cord_uid: kllgnncl Wastewater-based epidemiology (WBE) is a great approach that enables us to comprehensively monitor the community to determine the scale and dynamics of infections in a city, particularly in metropolitan cities with a high population density. Therefore, we monitored the time course of the SARS-CoV-2 RNA concentration in raw sewage in the Frankfurt metropolitan area, the European financial center. To determine the SARS-CoV-2 concentration in sewage, we continuously collected samples from two wastewater treatment plant (WWTP) influents (Niederrad and Sindlingen) serving the Frankfurt metropolitan area and performed RT-qPCR analysis targeting three genes (N gene, S gene, and ORF1ab gene). In August, a resurgence in the SARS-CoV-2 RNA load was observed, reaching 3 x 10^13 copies/day, which represents similar levels compared to April with approx. 2 x 10^14 copies/day. This corresponds to an also continuous increase again in COVID-19 cases in Frankfurt since August, with an average of 28.6 incidences, compared to 28.7 incidences in April. Different temporal dynamics were observed between different sampling points, indicating local dynamics in COVID-19 cases within the Frankfurt metropolitan area. The SARS-CoV-2 load to the WWTP Niederrad ranged from approx. 4 x 10^11 to 1 x 10^15 copies/day, the load to the WWTP Sindlingen from approx. 1 x 10^11 to 2 x 10^14 copies/day, which resulted in a preceding increase in these loading in July ahead of the weekly averaged incidences. The study shows that WBE has the potential as early warning system for SARS-CoV-2 infections and as monitoring system to identify global hotspots of COVID 19. The ongoing pandemic of the coronavirus disease 2019 (COVID-19) is a public health 37 emergency of global concern and is expressed by symptoms like fever, myalgia, 38 fatigue, dry cough. The disease, caused by severe acute respiratory syndrome 39 PCR efficiencies, threshold and baseline setting are provided in the supplementary 112 information. 113 114 We investigated the largest metropolitan region in southern Germany (Frankfurt am 117 Main) using three separate sampling points in the catchment of the two large WWTPs 118 (Table 1 ) and this data was compared to the COVID-19 cases in the area. 119 Epidemiological data on COVID-19 in the studied area was retrieved from the publicly 120 available repository of the Robert Koch Institute (https://survstat.rki.de/Default.aspx). 121 of August compared to April, whereas the overall trend for Germany only saw a slight 133 increase in cases during that time. Additionally, it is necessary to mention, that during 134 July and August the German government started free-of-cost testing for people 135 returning from other countries as well as risk regions to control returnees from holiday. 136 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted October 27, 2020. ; This may have also led to an increase in the reported positive COVID-19 case in the 137 summer months. 138 139 The highest numbers of COVID-19 cases in the city of Frankfurt am Main occurred in 141 April and August 2020, with 28 cases per 100.000 people as three-week average. In 142 contrast, the trend throughout Germany showed a much higher first peak in April with 143 44 cases per 100.000 people at the maximum and only 10 in August. Therefore, we 144 used the COVID-19 cases and the corresponding SARS-CoV-2 loads to the WWTPs 145 both in April and August to compare both time points of the pandemic (Figure 2A) . 146 Our results revealed, that the load of SARS-CoV-2 virus in the wastewater samples 147 was only slightly higher in April with a maximum of 1.4 x 10 15 copies/d compared to a 148 maximum of 5.37 x 10 14 copies/d in August for individual influent wastewater samples. 149 Additionally, we evaluated the correlation between the incidences reported against the 150 sum of the SARS-CoV-2 load measured in all the samples ( Figure 2B ). It is clearly 151 visible that with the increase in incidences, a corresponding increase in the viral load 152 was observed as also shown in the Spearman's rank correlation coefficient rs of 0.7464 153 (p (2-tailed) = 0.00217). However, there is a certain scattering in the data, which might 154 be due to the variance in the measured viral load for different sampling points, as 155 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted October 27, 2020. ; https://doi.org/10.1101/2020.10.26.20215020 doi: medRxiv preprint ranged between 1.29 x 10 12 copies/d on the 06.07.2020 as lowest and 1.63 x 10 15 163 copies/d on the 21.04.2020 as highest. In June and July, the incidences were less than 164 ten COVID-cases per 100.000 persons per week and the load ranged between 165 1.29 x 10 12 and 1.91 x 10 13 copies/d. The comparison with the COVID-19 cases 166 revealed that the increase in the SARS-CoV-2 load clearly preceded the reported 167 cases with the first step-increase in the middle of July 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted October 27, 2020. The WWTP Niederrad also receives sewage from Griesheim, a part of the Frankfurt 199 metropolitan area, through a separate canal where sampling was possible. Therefore, 200 to generate more localized information, we separately monitored sewage from 201 Griesheim as well. Interestingly, we did not detect any SARS-CoV-2 in a few of the 202 samples from Griesheim during the study period. From the 14 sampling days, six 203 samples showed results of SARS-CoV-2 below the limit of detection (LOD), especially 204 at the beginning of the study in June and July 2020, which corresponds well with the 205 low number of COVID-19 cases reported during this time. Like other sampling points, 206 a moderate increase of virus load in the wastewater from Griesheim was observed, 207 with a specific peak in August when the load was as high as 6.48 x 10 13 copies/day 208 (see Figure 5) . is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted October 27, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted October 27, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted October 27, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted October 27, 2020. ; 360 361 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted October 27, 2020. ; https://doi.org/10.1101/2020.10.26.20215020 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted October 27, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted October 27, 2020. ; https://doi.org/10.1101/2020.10.26.20215020 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted October 27, 2020. In case of the positive control, we included for each qPCR run triplicates of the four 430 different concentration (i.e. 1x10 1 , 2x10 1 , 2x10 2 , 2x10 3 copies per reaction) of the 431 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted October 27, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted October 27, 2020. ; https://doi.org/10.1101/2020.10.26.20215020 doi: medRxiv preprint COVID-19) and the Virus That Causes It Concerns and 274 Strategies for Wastewater Treatment during COVID-19 Pandemic to Stop Plausible 275 Transmission. Resources, Conservation and Recycling The Presence of SARS-CoV-2 RNA in the Feces 279 of COVID-19 Patients Urine from an Asymptomatic Patient with Novel Coronavirus 283 2019 Infection: A Case Report SARS-CoV-2 from Faeces to Wastewater Treatment: What Do We Know? A 287 Review Step in Implementing the Wastewater Based Epidemiology for the COVID-19