key: cord-1005782-46r46ja3 authors: Chavarria-Miro, G.; Anfruns-Estrada, E.; Guix, S.; Paraira, M.; Galofre, B.; Saanchez, G.; Pinto, R.; Bosch, A. title: Sentinel surveillance of SARS-CoV-2 in wastewater anticipates the occurrence of COVID-19 cases date: 2020-06-13 journal: nan DOI: 10.1101/2020.06.13.20129627 sha: 6dd99293252ddfadd778f6b769ec0004683f4ba4 doc_id: 1005782 cord_uid: 46r46ja3 SARS-CoV-2 was detected in Barcelona sewage long before the declaration of the first COVID-19 case, indicating that the infection was present in the population before the first imported case was reported. Sentinel surveillance of SARS-CoV-2 in wastewater would enable adoption of immediate measures in the event of future COVID-19 waves. In early December 2019, COVID-19 originated in Wuhan, China, and reached thereafter many parts of the world, including Europe, where the first case was reported in France in late-January 2020 (1) . However, evidence points to the occurrence of cases in France already in late 2019. Despite COVID-19 is a respiratory disease, large amounts of coronavirus genomes are shed in the feces (2, 3) and ultimately reach wastewater (4, 5) . SARS-CoV-2 surveillance in sewage may be considered a sensitive tool to monitor the spread of the virus among the population. However, there is no epidemiological evidence that sewage could be a transmission route for SARS-CoV-2, through contamination of bathing areas or irrigation waters, because very few studies report culture of infectious virus from stool (6) . Even in respiratory samples, infectivity is only associated with very high genome copy numbers (7, 8) that very unlikely are found in wastewater. To date, Spain ranks in fourth place in absolute number of cases, and almost topping the list in number of cases and deaths per 1 million inhabitants, being Barcelona the second most affected area. The first case in Barcelona was reported in February 25, 2020 and the total number of cases at the end of May 2020 was over 17,500 (https://salutweb.gencat.cat/ca/inici/notapremsa/index.html?id=385948#googtrans(ca|en)). In order to elucidate the evolution of COVID-19 in Barcelona, 24-h composite raw sewage samples from two large wastewater treatment plants (WWTP1 and WWTP2) were weekly analyzed for the presence of SARS-CoV-2 from April 13, in the peak of the epidemics, to May 25. In addition, for WWTP2, frozen archival samples All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 13, 2020. . https://doi.org/10.1101/2020.06.13.20129627 doi: medRxiv preprint 3 from 2018 (January-March), 2019 (January, March, September-December) and 2020 (January-March) were also assayed. Eight hundred-milliliter samples of wastewater were concentrated through precipitation with 20% polyethylene-glycol 6000 and resuspended in 3 mL of PBS, pH 7.4 (9) . Nucleic acid extraction was performed from 1mL of the concentrate and eluted in 50 µL using the NucliSENS® miniMAG® extraction system (bioMérieux). For WWTP2, samples from December 2019 to May 2020 were available, which opened the possibility to better analyze the dynamics of genome copy numbers in sewage. These numbers, determined with IP2 and IP4 targets, followed a curve peaking between March 4 and May 4 ( Figure 1, panel D) . In the middle of the peak, an abrupt fall in genome copies was observed on April 27, likely due to a heavy rainfall event occurring during April 19-21, with daily pluviosity averages of 65 mm. These All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 13, 2020. . https://doi.org/10.1101/2020.06.13.20129627 doi: medRxiv preprint rainwaters are collected in storm water reservoirs of 375,500 m 3 that gradually drain into sewers, adding a considerable dilution factor to viruses in wastewater. This dilution effect was not observed in WWTP1 samples, since no large storm water reservoirs affect its sewerage network. A progressive decline in genome copy numbers in WWTP2 parallels the diminution in the number of estimated virus shedders in the community carriers may have been misclassified as influenza diagnoses in primary care, boosting community transmission before public health measures were taken (11) . Additionally, there is a significant proportion of asymptomatic carriers that shed SARS-CoV-2 and contribute to the virus spread (12) . Despite the apparent disappearance of SARS-CoV-2 in WWTP1 and WWTP2 around May 18-25, the analysis of grab samples, collected 8-9 AM, from four urban sewers revealed the occurrence of virus genomes (Figure 1 , panels G-J). Sewer1 drains into WWTP1, while sewer2, sewer3 and sewer4 drain into WWTP2. A higher dilution factor applies in the WWTPs than in the sewers, which together with differences applying between grab and composite samples, and bowel habits (13) , could explain why samples from the former came out to be negative for the virus, while genome copies could still be detected in the sewer samples. Sewer analysis may provide most relevant information on the localization of areas where COVID-19 cases reappear, enabling immediate response to prevent spread of the outbreak. Nevertheless, it represents a more laborious and costly approach that surveillance through WWTP monitoring. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 13, 2020. First cases of coronavirus disease 2019 (COVID-19) in the WHO European Region Viral load of SARS-CoV-2 in clinical samples Fecal specimen diagnosis 2019 novel coronavirusinfected pneumonia Presence of SARS-Coronavirus-2 in sewage. medRxiv SARS-CoV-2 in wastewater: potential health risk, but also data source Infectious SARS-CoV-2 in Feces of Patient with Severe COVID-19 Clinical presentation and virological assessment of hospitalized cases of coronavirus disease 2019 in a travel-associated transmission cluster Predicting infectious SARS-CoV-2 from diagnostic samples Glass wool concentration optimization for the detection of enveloped and nonenveloped waterborne viruses Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR Excess cases of influenza suggest an earlier start to the coronavirus epidemic in Spain than official figures tell us: an analysis of primary care electronic medical records from over 6 million people from Catalonia Asymptomatic cases with SARS-CoV-2 infection Defecation frequency and timing, and stool form in the general population: a prospective study Epidemiologic Features and Clinical Course of Patients Infected With SARS-CoV-2 in Singapore SARS-CoV-2 in Barcelona sewage and its association with COVID-19 cases. Panels A-F: SARS-CoV-2 genome copy levels in two WWTP, detected with targets IP2 and IP4 Each point represents the mean and standard deviation of 2-4 replicas of RT-qPCR assays. Absence of values at a given date means genome copy numbers below the theoretical limit of detection (dashed line for IP2, IP4, E and N2, and dotted line for N1), with the exception of N2 target on April 27 and May 4 for which aliquots to assay were no longer available. Some samples showed positivity below the theoretical limit of detection (WWTP1: N1 at WWTP2. A. SARS-CoV-2 genome detection with targets IP2 and IP4, E and N1 and N2. B. Real-time RT-PCR amplification plots of IP2 and This research was supported in part by the REVEAL project, funded by SUEZ Spain.We thank our colleagues from Aigües de Barcelona (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted June 13, 2020. . https://doi.org/10.1101/2020.06.13.20129627 doi: medRxiv preprint All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted June 13, 2020. . https://doi.org/10.1101/2020.06.13.20129627 doi: medRxiv preprint 7 All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted June 13, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted June 13, 2020. . https://doi.org/10.1101/2020.06.13.20129627 doi: medRxiv preprint All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted June 13, 2020. . https://doi.org/10.1101/2020.06.13.20129627 doi: medRxiv preprint All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted June 13, 2020. . https://doi.org/10.1101/2020.06.13.20129627 doi: medRxiv preprint