key: cord-0814135-i924o2la authors: Randazzo, W.; Truchado, P.; Ferrando, E. C.; Simon, P.; Allende, A.; Sanchez, G. title: SARS-CoV-2 RNA titers in wastewater anticipated COVID-19 occurrence in a low prevalence area date: 2020-04-25 journal: nan DOI: 10.1101/2020.04.22.20075200 sha: 6c6d9e9ca234804ddd3efa58b1b65862e06d2c43 doc_id: 814135 cord_uid: i924o2la Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused more than 200,000 reported COVID-19 cases in Spain resulting in more than 20,800 deaths as of April 21, 2020. Faecal shedding of SARS-CoV-2 RNA from COVID-19 patients has extensively been reported. Therefore, we investigated the occurrence of SARS-CoV-2 RNA in six wastewater treatments plants (WWTPs) serving the major municipalities within the Region of Murcia (Spain), a low prevalence area. Firstly, an aluminum hydroxide adsorption-precipitation concentration method was tested using a porcine coronavirus (Porcine Epidemic Diarrhea Virus, PEDV) and mengovirus (MgV). The procedure resulted in average recoveries of 10.90% and 10.85% in influent water and 3.29% and 6.19% in effluent water samples for PEDV and MgV, respectively. Then, the method was used to monitor the occurrence of SARS-CoV-2 from March 12 to April 14, 2020 in influent, secondary and tertiary effluent water samples. By using the real-time RT-PCR (RT-qPCR) Diagnostic Panel validated by US CDC that targets three regions of the virus nucleocapsid (N) gene, we estimated quantification of SARS-CoV-2 RNA titers in untreated wastewater waters of 5.29 log genomic copies/L on average. Moreover, we tested as negative all secondary and tertiary treated water samples, highlighting that current water disinfection treatments applied in the analyzed WWTP are able to remove SARS-CoV-2 RNA. This environmental surveillance data were compared to declared COVID-19 cases at municipality level, revealing that SARS-CoV-2 was circulating among the population even before the first cases were reported by local or national authorities in many of the cities where wastewaters have been sampled. The detection of SARS-CoV-2 in wastewater in early stages of the spread of COVID-19 highlights the relevance of this strategy as an early indicator of the infection within a specific population. At this point, this environmental surveillance could be implemented by municipalities right away as a tool, designed to help authorities to coordinate the exit strategy to gradually lift its coronavirus lockdown. Coronaviruses (CoVs) are a family of viruses pathogenic for humans and animals 52 associated to respiratory and gastro-intestinal infections. CoVs used to be considered as 53 minor pathogens for humans as they were responsible of common cold or mild respiratory 54 infections in immunocompetent people. Nonetheless, the emergence of novel and highly 55 pathogenic zoonotic diseases caused by CoVs such as Severe Acute Respiratory (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 April 25, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 100 The porcine epidemic diarrhea virus (PEDV) strain CV777 and the mengovirus (MgV) 101 vMC0 (CECT 100000) were preliminary used to evaluate the aluminum hydroxide 102 adsorption-precipitation method previously described for concentrating enteric viruses 103 from wastewater and effluent water (30, 31). In brief, 200 mL of biobanked influent (n=2) 104 and effluent water samples (n=2) were artificially inoculated with PEDV and MgV. Then 105 pH was adjusted to 6.0 and Al(OH)3 precipitate formed by adding 1 part 0.9N AlCl3 106 (Acros organics, Geel, Belgium) solution to 100 parts of sample. The pH was readjusted 107 to 6.0 and sample mixed using an orbital shaker at 150 rpm for 15 min at room 108 temperature. Then, viruses were concentrated by centrifugation at 1,700 × g for 20 min. The pellet was resuspended in 10 mL of 3% beef extract pH 7.4, and samples were shaken 110 for 10 min at 150 rpm. Concentrate was recovered by centrifugation at 1,900 × g for 30 111 min and pellet resuspended in 1 mL of PBS. All wastewater and effluent water samples included in this study were processed as 113 described and MgV (5 log PCRU) was spiked as process control. 114 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 April 25, 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 April 25, 2020. Moreover, filtration through 10 kDa Centricon ® Plus-70 centrifugal device successfully 163 recovered SARS-CoV-2 in wastewater with recovery efficiencies of F-specific RNA 164 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 April 25, 2020. On average, SARS-CoV-2 RNA titers of 5.15 ± 0.25, 5.53 ± 0.24, and 5.49 ± 0.27 log 187 gc/L were quantified in wastewater by using N1, N2 and N3 primer/probe mixes, 188 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 April 25, 2020. . https://doi.org/10.1101/2020.04.22.20075200 doi: medRxiv preprint respectively. Titer of 4 and 5 to more than 6 log gc/L have been reported in Massachusetts 189 and France, respectively (23, 25). 190 We observed discrepancies among RT-qPCR N1, N2 and N3 assays for several water 191 samples in agreement to a previous report (22). This could be due to the different (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (Table 3 ) and plotted to the SARS-CoV-2 RNA mean loads as detected by three RT-217 qPCR assays (Figure 2) . On the other hand, we believe that this environmental surveillance could be used as an 234 instrument to drive the right decisions to reduce the risk of lifting restrictions too early. In fact, a very important question is to determine what needs to be implemented to have 236 reliable data to reduce the risk of a "second wave". Massive population tests are the first 237 choice, but in their absence, wastewater monitorization of SARS-CoV-2 RNA can give a 238 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 April 25, 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 authors acknowledge the "Entidad Regional de Saneamiento y Depuración de Aguas (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (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 April 25, 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 April 25, 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 April 25, 2020. Analytical sensitivity and 404 efficiency comparisons of SARS-COV-2 qRT-PCR assays 39. CDC. CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR 407 Diagnostic Panel Water, sanitation, hygiene and waste management for Water Transmission and COVID-19: Questions and Answers Stability of SARS-CoV-2 in different environmental 413 conditions. The Lancet Microbe Coronavirus and Risk Analysis 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 April 25, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (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 April 25, 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 April 25, 2020. . https://doi.org/10.1101/2020.04.22.20075200 doi: medRxiv preprint