Highly efficient and sensitive membrane-based concentration process allows quantification, surveillance, and sequencing of viruses in large volumes of wastewater
Authors: El soufi G. , Di Jorio L. , Gerber Z. , Cluzel N. , Van Assche J. , Delafoy D. , Olaso R. , Daviaud C. , Loustau T. , Schwartz C. , Trebouet D. , Hernalsteens O. , Marechal V. , Raffestin S. , Rousset D. , Van Lint C. , Deleuze JF. , Boni M. ,   OBEPINE consortium, Rohr O. , Villain-Gambier M. , Wallet C. 
Published: September 2023

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.

Graphical Abstract