Monitoring SARS-CoV-2 circulation and diversity through community wastewater sequencing

Abstract

The current SARS-CoV-2 pandemic has rapidly become a major global health problem for which public health surveillance is crucial to monitor virus spread. Given the presence of viral RNA in feces in around 40% of infected persons, wastewater-based epidemiology has been proposed as an addition to disease-based surveillance to assess the spread of the virus at the community level. Here we have explored the possibility of using next-generation sequencing (NGS) of sewage samples to evaluate the diversity of SARS-CoV-2 at the community level from routine wastewater testing, and compared these results with the virus diversity in patients from the Netherlands and Belgium. Phylogenetic analysis revealed the presence of viruses belonging to the most prevalent clades (19A, 20A and 20B) in both countries. Clades 19B and 20C were not identified, while they were present in clinical samples during the same period. Low frequency variant (LFV) analysis showed that some known LFVs can be associated with particular clusters within a clade, different to those of their consensus sequences, suggesting the presence of at least 2 clades within a single sewage sample. Additionally, combining genome consensus and LFV analyses we found a total of 57 unique mutations in the SARS-CoV-2 genome which have not been described before. In conclusion, this work illustrates how NGS analysis of wastewater can be used to approximate the diversity of SARS-CoV-2 viruses circulating in a community.

SARS-CoV-2 detection and genomic sequencing from hospital surface samples collected at UC Davis

“…(33)). Furthemore, environmental samples in particular may have been degraded or diluted, affecting the genomic RNA available for reverse transcription, as observed in multiple studies of environmental samples ((34–36).…”

Droplet Digital RT-PCR to detect SARS-CoV-2 variants of concern in wastewater

“…Whole genome sequencing of SARS-CoV-2 from clinical samples from infected individuals is commonly used to identify VoC’s (Tegally, Wilkinson et al 2021). Although whole genome sequencing has shown to be feasible to give insight in the sequence variations of SARS-CoV-2 genomes in sewage (Izquierdo-Lara, Elsinga et al 2020) and has been used to detect mutations associated with variants of concern in wastewater(Jahn, Dreifuss et al 2021), this requires deep sequencing and bioinformatics to determine the presence of mutations associated with VoC’s in wastewater, and is provides less quantitative results than ddPCR. An advantage of whole genome sequencing of wastewater samples is that it can provide more extensive information about the co-occurrence of the range of mutations and deletions associated with VoC.…”

“…However, there are currently no methods available to quantify the presence of different VoC’s in sewage. Sequence analysis of RNA isolated from sewage can specifically detect and identify SARS-CoV-2 RNA sequence variation (Izquierdo-Lara, Elsinga et al 2020) and sequence analysis has the potential to identify VoC in sewage. However, it is expected that this needs deep sequencing efforts and bioinformatics to find rare mutations in sewage samples which likely contain mixtures of SARS-CoV-2 lineages.…”

Detection and surveillance of SARS-CoV-2 genomic variants in wastewater

“…Viral RNA of SARS-CoV-2 infected persons can be found in faeces (Gupta et al 2020) and accordingly, at the community level, in the wastewater collected in wastewater treatment plants (WWTPs) (Kitajima et al 2020; Medema et al 2020; Gonzalez et al 2020). In principle, wastewater samples can provide a snapshot of the circulating viral lineages and their diversity in the community (Izquierdo Lara et al 2020; Nemudryi et al 2020; Martin et al 2020; Crits-Christoph et al 2020) and serve as an efficient and complementary approach to genomic epidemiology based on individual patient samples (Nadeau et al 2020).…”