Simple Wastewater Preparation Protocol Applied to Monitor the Emergence of the Omicron 21L/BA.2 Variant by Genome Sequencing

Abstract

Detecting and monitoring viruses in wastewater samples have been reported as useful ways of tracking SARS-CoV-2 epidemic trends. However, there is currently no unanimously recognised method of processing samples to identify and quantify SARS-CoV-2 variants in wastewater. We aimed to implement a method that was as simple as possible in order to be used universally. In a study performed between January 2022 and June 2022 in the city of Marseille, France, we first evaluated the impact of the sample preservation strategy. We then compared ultracentrifugation to ultrafiltration and several steps of filtration to determine the optimal approach for virus concentration. As a proof-of-concept, the definitive protocol was applied to next-generation sequencing of SARS-CoV-2 in wastewater to monitor the emergence of the Omicron variant in the city. For sewage water to be processed in the week following the sampling, storage at +4 °C is sufficient, with less than 1 Ct loss. Filtration with a 5 µm syringe filter, then with a 0.8 µm filtration unit, followed by ultrafiltration was the optimal protocol, leading to an average increase of 3.24 Ct when the starting Ct was on average 38 in the wastewater. This made it possible to observe the emergence of the Omicron 21L/BA.2 variant after Omicron 21K/BA.1 by genome sequencing over a period ranging from 20 February to 10 April 2022 in agreement with observations based on patient data. To conclude, by using a simple method requiring only basic filters and a centrifuge as equipment, it is possible to accurately track the relative incidence rates and the emergence of SARS-CoV-2 variants based on sewage samples.

Timely Monitoring of SARS-CoV-2 RNA Fragments in Wastewater Shows the Emergence of JN.1 (BA.2.86.1.1, Clade 23I) in Berlin, Germany

“…Our results support Recommendation (EU) 2021/472 [ 16 ], which includes the tracking of variants in wastewater as “a cost effective, rapid and reliable source of information on the spread of SARS-CoV-2 in the population and that it can form a valuable part of an increased genomic and epidemiological surveillance” [ 16 ]. The genomic surveillance of wastewater for SARS-CoV-2 has been implemented in other countries and regions [ 1 , 4 , 17 , 18 , 19 ], but information from other countries is often unpublished or challenging to find. Therefore, genomic wastewater surveillance results should be collated at the EU-level to complement genomic surveillance from clinical samples.…”