Epidemiological surveillance of SARS-CoV-2 by genome quantification in wastewater applied to a city in the northeast of France: Comparison of ultrafiltration- and protein precipitation-based methods

Abstract

The aim of the present study was to develop a simple, sensitive, and specific approach to quantifying the SARS-CoV-2 genome in wastewater and to evaluate this approach as a means of epidemiological surveillance. Twelve wastewater samples were collected from a metropolitan area in north-eastern France during April and May 2020. In addition to the quantification of the SARS-CoV-2 genome, F-specific RNA phages of genogroup II (FRNAPH GGII), naturally present in wastewater, were used as an internal process control for the viral concentration and processing of RT-PCR inhibitors. A concentration method was required to allow the quantification of the SARS-CoV-2 genome over the longest possible period. A procedure combining ultrafiltration, phenol-chloroform-isoamyl alcohol purification, and the additional purification of the RNA extracts was chosen for the quantification of the SARS-CoV-2 genome in 100-mL wastewater samples. At the same time, the COVID-19 outbreak was evaluated through patients from the neighbouring University Hospital of Nancy, France. A regular decrease in the concentration of the SARS-CoV-2 genome from ~10⁴ gc/L to ~10² gc/L of wastewater was observed over the eight weeks of the study, during which the population was placed under lockdown. The SARS-CoV-2 genome was even undetectable during one week in the second half of May and present but non-quantifiable in the last sample (28 May). A concordant circulation in the human community was highlighted by virological diagnosis using respiratory samples, which showed a decrease in the number of COVID-19 cases from 677 to 52 per week over the same period. The environmental surveillance of COVID-19 using a reliable viral quantification procedure to test wastewater is a key approach. The real-time detection of viral genomes can allow us to predict and monitor the circulation of SARS-CoV-2 in clinical settings and survey the entire urban human population.

Keywords: Concentration methods; Prevalence; SARS-CoV-2; Wastewater.

Fig. 1

Quantitative time-course monitoring of SARS-CoV-2 and FRNAPH-GGII genomes in wastewater samples in the Nancy metropolitan area and evolution of the number of cases per week at the local University Hospital. Quantification in 100 mL of wastewater (ultrafiltration) of the SARS-CoV-2 genome targeting RdRp (full triangles) and E (full squares) genes and of the FRNAPH GGII genome (full diamonds). Quantification in 5 mL of wastewater: open forms. Negative samples of the SARS-CoV-2 genome are marked with an asterisk. The sample positive for the RdRp gene but below the LoQ is marked with a plus sign. The number of cases of COVID-19 detected at the local University Hospital is shown per week (bars). The limit of detection (LoD) for RdRp and E genes (2 × 10² gc/L) is represented by a white arrow on the left y-axis. The lines given for genome concentration values are only designed to provide a visual guide.