Prevalence and circulation patterns of SARS-CoV-2 variants in European sewage mirror clinical data of 54 European cities

Abstract

For community-level monitoring, the European Commission under the EU Sewage Sentinel System recommends wastewater-based SARS-CoV-2 surveillance. Tracking SARS-CoV-2 variants in a community is pivotal for appropriate public health response. Genome sequencing of SARS-CoV-2 in wastewater samples for tracking variants is challenging, often resulting in low coverage genome sequences, thereby impeding the detection of the SARS-CoV-2 mutations. Therefore, we aimed at high-coverage SARS-CoV-2 genome sequences from sewage samples which we successfully accomplished. This first pan-European surveillance compared the mutation profiles associated with the variants of concerns: B.1.1.7, P.1, B.1.351 and B.1.617.2 across 20 European countries, including 54 municipalities. The results highlight that SARS-CoV-2 variants detected in the wastewater samples mirror the variants profiles reported in clinical data. This study demonstrated that >98% coverage of SARS-CoV-2 genomic sequences is possible and can be used to track SARS-CoV-2 mutations in wastewater to support identifying variants circulating in a city at the community level.

Keywords: European sewage; High-coverage sequencing; Metagenomics; SARS-CoV-2 RNA; Wastewater-based epidemiology.

Graphical abstract

Fig. 1

COVID-19 situation in 20 European countries from clinical sequencing data. (A) Maps showing the incidence rate of the COVID-19 positive cases reported in the countries, during weeks 10 to 13, from when the wastewater samples originated. (B) Relative abundance of all sequences for the countries from where wastewater samples were analyzed, available in GISAID (https://www.gisaid.org) on 31–05–2021. All the sequences except B.1.1.7, B.1.351, P.1, and B.1.617 were categorized as “Others”.

Fig. 2

Amino Acid Mutations detected in wastewater samples. (Left) Allele frequency of the mutations (relative to the reference genome Wuhan-Hu-1 [GenBank accession numbers NC_045512 and MN908947.3]), associated with the variant of concern (VOCs), in each wastewater sample. (Middle) Number of mutations (count) detected in each sample corresponding to each VOC. (Right) Heatmap showing the presence (green) / absence (white) of mutations having more than 50% allele frequency. AA mutation: Amino acid mutations; AF: Allele Frequency; S: spike Protein; ORF8: open reading frame 8; ORF3a: open reading frame 3a; and N: nucleocapsid protein.

Fig. 3

Abundance of the reads associated with Spike protein AA mutations used by the ECDC for the characterization of the VOCs.

Fig. 4

Heatmap showing the read abundance of the top 50 AA mutations found across all the samples.