Using wastewater to monitor COVID-19

Tracking of wastewater has historically played an important role in the development of early warning systems (EWS) for various pathogens. Monitoring wastewater involves the collection and analysis of a pooled community sample and can provide information on community-level health risks. Unlike individual testing, wastewater surveillance is independent of health-seeking behaviour. To date, wastewater surveillance is recognised as an important complementary public health tool which can support clinical testing.

Since March 2020, over 50 countries have successfully applied wastewater-based epidemiology (WBE) to track the spread of COVID-19. In many cases, SARS-CoV-2 wastewater surveillance programmes have also been used to monitor variants, including known variants of interest or concern, and in certain settings new and emerging variants. Sequencing the genomes of viruses is crucial to understand the evolution of several pathogens in relation to public health interventions. Scientists can now successfully study the evolution of the SARS-CoV-2 genome by continuously sequencing the order of nucleotides that make up its genetic code, not only within humans but also from an environmental source like wastewater.

Two main methods are used for tracking variants in wastewater: reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and next-generation sequencing (NGS), both have advantages and limitations. Advanced NGS technologies have enabled scientists to sequence long- and short-read SARS-CoV-2 fragments. Prior to sequencing the 30 Kb genome of the virus in the wastewater sample, scientists extract total RNA, run RT-PCR testing with SARS-CoV-2 primers and perform quantity and quality checks (Figure 1).

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Figure 1 – Sample collection, RNA extraction, RT-PCR testing and whole genome sequencing of SARS-CoV-2 from wastewater.

Sequencing the whole genome of SARS-CoV-2 enables scientists to closely monitor the changing order of nucleotides through base mutations that constitute the characteristics seen in variants of concern (VOCs). Phenotypic mutations may lead to aberrant changes in virus biology, which include transmissibility, infectivity, pathogenicity and antigenicity. Sequencing of viral genomes is thus a crucial aspect in the epidemiology and aetiology of SARS-CoV-2 VOCs survival. Once samples have been sequenced, the data needs to be processed, assessed for quality, and analysed to identify VOCs.

It should be noted that compared to clinical samples, the identification of SARS-CoV-2 VOCs through wastewater sequencing has its limitations. The most challenging is that the amplicons generated during sequencing are from viruses shed from thousands of individuals and it is therefore unlikely that mutations in different amplicons can reliably be associated with a single genome. One can however still detect signature mutations associated with specific VOCs. The schematic representation shown in Figure 2 gives an overview of a standard data analysis pipeline with examples of some of the frequently used tools.

Quality control = FastQC, Trimmomatic, bbduk (short reads) or FastQC, BoardION, NanoFilt (ONT reads) > Alignment (mapping) against reference = BWA, bowtie, STARaligner (short reads) or Medeka, BWA, minimap2 (ONT reads) > Coverage check (filtering) = SAMtools, BEDtools > Variant calling = SAMtools, GATK (short reads) or Medaka, SAMtools, GATK (ONT reads) > Consensus fasta > SARS-CoV-2 lineage alignment = Pangolin, custom designed pipelines”>

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Figure 2 – Schematic representation of the data analysis and variant identification pipeline showing the most frequently used tools.

Further reading

Wastewater sequencing – an innovative method for variant monitoring of SARS-CoV-2 in populations

Wastewater surveillance of pathogens can inform public health responses

Pathogen surveillance through monitoring of sewer systems