Scottish SARS-CoV-2 Wastewater Surveillance Program

In a recent study published on medRxiv* preprint server, researchers reported on the use of the wastewater-based epidemiological approach (WBE) to assess ribonucleic acid (RNA) levels of severe acute respiratory syndrome coronavirus 2 (SARS-CoV -2) being poured into Scottish sewage systems, during the coronavirus disease 2019 (COVID-19) pandemic, based on a national program dataset.

Study: SARS-CoV-2 RNA levels in Scotland’s sewage. Image Credit: Daniel Jedzura/Shutterstock

SARS-CoV-2 testing is important for monitoring and controlling COVID outbreaks, as it allows health authorities to appropriately manage people infected with SARS-CoV-2 based on their test trace results. In addition, viruses released into sewer systems by the general population can be detected and quantified at specific times.

The presence of SARS-CoV-2 particles in the feces of people infected with the virus correlates with the amount detected during community COVID-19 testing. Therefore, wastewater surveillance can monitor the extent of SARS-CoV-2 transmission in the community and has the potential to detect SARS-CoV-2 outbreaks at an early stage.

About the study

In the present study, the researchers described the monitoring of SARS-CoV-2 RNA levels by the WBE approach based on the near real-time dataset of a national program led by Scottish Water and the Scottish Environmental Protection Agency (SEPA) between May 2020 and February 2022.

Overview of the methodological workflow of the Scottish SARS-CoV-2 sewage surveillance programme. Incoming wastewater samples are collected by Scottish Water (1) and transferred to SEPA laboratories where the samples are stored and processed (2). SARS-CoV-2 viral levels are then quantified using RT-qPCR (3). Finally, the data obtained is shared on the public SEPA dashboard and with local authorities such as the Scottish government. Additionally, the detailed methodology and datasets are shared on open online platforms and repositories such as protocols.io, Zenodo, and GitHub 19–21,26,27. This figure was created with BioRender.com.

Wastewater samples were collected by the Scottish Water (SW) team of operators from 122 sites in 14 National Health Service (NHS) Board of Health areas in Scotland and transferred to SEPA Laboratories for storage and further processing. SARS-CoV-2 RNA was extracted from samples and quantified using quantitative reverse transcription polymerase chain reaction (RT-qPCR) assays of the SARS-CoV nucleocapsid 1 (N1) gene -2.

Samples were taken once or twice a week, up to four times a week, using a 24-hour autosampler or a grab sample from maintenance holes. Prior to treatment, wastewater samples were spiked with a known non-target Porcine Reproductive and Respiratory Syndrome Virus (PRRS) RNA target as a quality control measure. Cycle threshold values ​​(Ct) and gene copies per liter (gc/L) values ​​were calculated for each sample.

The resulting data was shared on public SEPA dashboards and with the Scottish Government. Additionally, datasets were shared on open online platforms and repositories such as GitHub, protocols.io, and Zenodo. Data were normalized for the final analysis, based on information such as site population, wastewater inflow values ​​and ammonia content, and expressed as millions of gene copies per person per person. day. [Mgc/pD]. Statistical analysis was carried out using Biomathematics and Statistics of Scotland (BioSS) and the data was presented in Scottish Government reports, published weekly.

Based on the number of N1 gene replicates per litre, wastewater samples were categorized as negative, weak positive, detected positive, non-quantifiable (DNQ positive), or positive. If ≥ 2 replicates did not produce a Ct, the sample was flagged as negative. If two replicates of three replicates or one replicate of two replicates showed positive signals, but the signals were below the limit of detection (LoD), the sample was reported as weakly positive. If the mean replicate values ​​were between the LoD (1,316 gc/L) and the limit of quantification (LoQ, 11,386 gc/L) but could not be the amount was not statistically significant, the sample d wastewater was declared DNQ positive. If the mean values ​​of all replicates exceeded the LoQ value, the sample was declared positive.

Results

In the analysis, 9.3%, 7.3%, 14% and 63.4% of the samples were reported as negative, weak positive, DNQ positive and positive, respectively. However, six percent of the study samples could not be classified. Data normalization showed that data obtained by the WBE approach correlated well with data published by local health authorities on SARS-CoV-2 pandemic waves.

Map of Scotland showing the sites from which sewage samples were taken for SARS-CoV-2 analysis. Colored circles show normalized levels of the SARS-CoV-2 virus in the last week of July 2021 in millions of gene copies per person per day ([Mgc/pD]; truncated at 90). Gray circles represent sites with no measurements that week. See Methods – Data Visualization for more details.

A nationwide increase in COVID-19 cases was reported between December 2020 and February 2021, which was followed by a decrease in the number of COVID-19 cases between February 2021 and mid-June 2021, after which COVID-19 cases have started to rise again. Similar COVID-19 trends were also observed after assessing SARS-CoV-2 RNA levels in wastewater by the WBE approach.

Similarly, COVID-19 cases peaked in September 2021, after which cases dropped and rebounded again with another peak between late December 2021 and February 2022, which was correlated with a directly proportional increase and decrease in SARS-CoV-2 RNA levels in sewage in Scotland, United Kingdom (UK). Additionally, in quality control experiments, a PRRS-causing viral pathogen was successfully detected, indicating that the study methodology was accurate and also that a negative test was most likely due to the absence of the SARS-CoV-2 N1 gene in the wastewater samples and not due to methodological failures.

Overall, the results showed that mathematical modeling of wastewater data could be used to estimate the daily number of COVID-19 cases and the national prevalence of SARS-CoV-2 infections. The WBE approach may be a useful aid in detecting predictive outbreaks of SARS-CoV-2 infections or similar viral infections in the future.

*Important Notice

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