Highly Efficient and Sensitive Membrane-Based Concentration Process Allows Quantification, Surveillance, and Sequencing of Viruses in Large Volumes of Wastewater

Wastewater-based epidemiology is experiencing exponential development. Despite undeniable advantages compared to patient-centered approaches (cost, anonymity, survey of large populations without bias, detection of asymptomatic infected peoples…), major technical limitations persist. Among them is the low sensitivity of the current methods used for quantifying and sequencing viral genomes from wastewater. In situations of low viral circulation, during initial stages of viral emergences, or in countries experiencing heavy rains, the extremely low concentrations of viruses in wastewater may fall below the limit of detection of the current methods. The availability and cost of the commercial kits, as well as the requirement of expensive materials, can also present major blocks to the development of wastewater-based epidemiological survey, specifically in low-income countries. Thereby, highly sensitive, low cost and open-access methods are still needed to increase the predictability of the viral emergences, to survey low-circulating viruses and to allow wastewater-based surveillance. Here, we outline and characterize new protocols for concentrating, quantifying, monitoring, and sequencing SARS-CoV-2 from large volumes (500 mL-1L) of raw wastewater. Our nucleic acid extraction technique (the routine C: 5ml method) does not require sophisticated equipment such as automatons and is not reliant on commercial kits, making it readily available to a broader range of laboratories for routine epidemiological survey. Furthermore, we demonstrate the efficiency, the repeatability, and the high sensitivity of a new membrane-based concentration method (MBC: 500 mL method) for enveloped (SARS-CoV-2) and naked (FRNAPH GGII) viruses. We show that the MBC method allows the quantification and the monitoring of viruses in wastewater with a significantly improved sensitivity. In contexts of low viral circulation, we report quantifications of SARS-CoV-2 in wastewater at concentrations below 100 genome copies per liter and as low as 40 genome copies per liter. In highly diluted samples collected in wastewater treatment plants of French Guiana, we confirmed the accuracy of the MBC method compared to the estimations done with the C method. Finally, we demonstrate that both the C and MBC methods are compatible with SARS-CoV-2 sequencing. We show that the quality of the sequence is correlated with the concentration of the extracted viral genome. Of note, the quality of the sequences obtained with some MBC processed wastewater was improved by dilutions or enzyme substitutions suggesting the presence of specific enzyme inhibitors in some wastewater. To the best of our knowledge, our MBC method is the first efficient, sensitive, repeatable, and up-scalable method characterized for SARS-CoV-2 quantification and sequencing from large volumes of wastewater. ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was supported by the University of Strasbourg, the French scientific group of interest GIS-OBEPINE, The Agence de l Eau Rhin Meuse, by the Grand Est region, the French national agencies for research ANR (COVIDEU project) and ANRS-MIE (EmerEaUde project). Work in C.V.L. lab was supported by the Belgian National Fund for Scientific Research (F.R.S.-FNRS, Belgium), the ULB-COVID-Fund and the Les Amis des Instituts Pasteur a Bruxelles, asbl. The laboratory of C.V.L. is part of the ULB-Cancer Research Center (U-CRC) (Faculty of Medicine, ULB). C.V.L. is Directrice de Recherches of the Belgian National Fund for Scientific Research (F.R.S.-FNRS, Belgium). O.H. was a fellow of the Belgian Fonds pour la formation a la Recherche dans l Industrie et dans l Agriculture (FRIA) du F.R.S.-FNRS (Contract F 3/5/5 - FRIA/FC - 6035) and is a fellow of Les Amis des Instituts Pasteur a Bruxelles, asbl. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present work are contained in the manuscript [1]: pending:yes