Could wastewater epidemiology help track the circulation of SARS-CoV-2 variants?

Broad concerns over severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), are raised in the wake of the high transmission and rapid mutation of the virus. In the recent past, using genomic epidemiology, scientists have detected some fast-spreading and highly virulent SARS-CoV-2 variants – underlining the importance of sequencing analyses. The patterns and abundance of these VOCs (variants of concern) are studied from the clinical samples from COVID-positive patients.

While wastewater-based epidemiology (WBE) has typically been focused on pharmaceutical drugs in domestic wastewater, it is now an emerging tool to monitor the circulation and dynamics of SARS-CoV-2 at the community level. WBE employs quantitative polymerase chain reaction (qPCR) to determine SARS-CoV-2 titers in sewage for correlation with the reported number of SARS-CoV-2 positive cases.

However, very few studies presently utilize the combined potential of genomic epidemiology and WBE to determine the SARS-CoV-2 genomic variants circulating in a specific region. Therefore, in a recent study, researchers assessed the next-generation sequencing (NGS) of wastewater samples to gather information about the diversity of SARS-CoV-2 variants and associated mutations at the community level.

In a new study, released as a preprint on the medRxiv* server, researchers from Germany, Italy and Netherlands demonstrated the utility of integrating genomic and wastewater based epidemiology (WBE) to provide unprecedented insight into the abundance of SARS-CoV-2 and the profile of the mutations associated with the VOCs (i.e., B.1.1.7, B.1.351, P.1, and B.1.617.2) in the wastewater samples.

We determined the relative abundance of the VOCs based on the abundance of reads associated with certain AA mutations,” write the researchers.

They conducted a pan-European study under the umbrella of the EU Sewage Sentinel System for SARS-CoV-2. The researchers say that this is a direct result of what is called “The HERA Incubator,” which assesses the information provided by the next-generation sequencing (NGS) of wastewater samples.

The researchers performed this research on wastewater influent 24h composite samples collected from wastewater treatment plants across 20 European countries, including 54 municipalities. They collected the samples from the 10^th to 30^th March, 2021.

The researchers presented a picture of COVID-19’s prevalence in the 20 European countries by using clinical sequencing data (in terms of positive cases and relative abundance of mutational sequences) and then related it to the data from their wastewater sampling.

Interestingly, the team identified mutations associated with VOCs (633 mutations) in the wastewater samples. For example, the W131C mutation was only detected in wastewater samples from Denmark; it is one of the important mutations in ORF3a, which is found to assist the ion channel formation and thereby supports the virus in its infectivity. Likewise, they reported the A220V mutation, only identified in samples from Lithuania, which corresponded with the high count of A220V reported in clinical patient samples.

Across the twenty European countries twenty-six ORF1ab mutations, fourteen spike protein, eight nucleocapsids (N) protein, six ORF8, and three ORF3 mutations were among the dominant mutations, exhibiting spatial and temporal variation,” they noted.

Mutations such as D614G mutation and P681H were dominant in both clinical and wastewater samples, demonstrating the pattern of genomic variants and the abundance of VOCs to be consistent between the clinical and the wastewater sequencing data.

Notably, the researchers observed that the wastewater sequencing data also revealed genomic variants which are not reported as dominant in clinical data. These findings support the notion that wastewater sequencing data can provide novel insights into the prevalence of mutations at the community level.

Across most of the wastewater samples, we detected a high occurrence of ORF8 mutations (i.e. Q27stop, R52I), which provides evidence for the circulation of SARS-COV-2 variants containing these mutations in the sampled regions”

While this study emphasizes that sequencing for SARS-CoV-2 in wastewater can provide additional information alongside clinical data about the prevalence of mutations, the researchers suggest that this sequencing surveillance should be considered complementary.

This study clearly shows that the surveillance of SARS-CoV-2 mutation profiles associated with VOCs in wastewater samples is possible using NGS, and also provides valuable insights augmenting the clinical epidemiological research. The data generated also presents the possibility to attain a sufficient coverage of the SARS-CoV-2 genome from the wastewater samples, the researchers write.

*Important notice

medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Written by

Dr. Ramya Dwivedi

Ramya has a Ph.D. in Biotechnology from the National Chemical Laboratories (CSIR-NCL), in Pune. Her work consisted of functionalizing nanoparticles with different molecules of biological interest, studying the reaction system and establishing useful applications.