SARS-CoV-2 variant trends in Ireland: Wastewater-based epidemiology and clinical surveillance.

SARS-CoV-2 RNA quantification in wastewater is an important tool for monitoring the prevalence of COVID-19 disease on a community scale which complements case-based surveillance systems. As novel variants of concern (VOCs) emerge there is also a need to identify the primary circulating variants in a community, accomplished to date by sequencing clinical samples. Quantifying variants in wastewater offers a cost-effective means to augment these sequencing efforts. In this study, SARS-CoV-2 N1 RNA concentrations and daily loadings were determined and compared to case-based data collected as part of a national surveillance programme to determine the validity of wastewater surveillance to monitor infection spread in the greater Dublin area. Further, sequencing of clinical samples was conducted to determine the primary SARS-CoV-2 lineages circulating in Dublin. Finally, digital PCR was employed to determine whether SARS-CoV-2 VOCs, Alpha and Delta, were quantifiable from wastewater. No lead or lag time was observed between SARS-CoV-2 wastewater and case-based data and SARS-CoV-2 trends in Dublin wastewater significantly correlated with the notification of confirmed cases through case-based surveillance preceding collection with a 5-day average. This demonstrates that viral RNA in Dublin's wastewater mirrors the spread of infection in the community. Clinical sequence data demonstrated that increased COVID-19 cases during Ireland's third wave coincided with the introduction of the Alpha variant, while the fourth wave coincided with increased prevalence of the Delta variant. Interestingly, the Alpha variant was detected in Dublin wastewater prior to the first genome being sequenced from clinical samples, while the Delta variant was identified at the same time in clinical and wastewater samples. This work demonstrates the validity of wastewater surveillance for monitoring SARS-CoV-2 infections and also highlights its effectiveness in identifying circulating variants which may prove useful when sequencing capacity is limited.

Coprostanol as a Population Biomarker for SARS-CoV-2 Wastewater Surveillance Studies

Wastewater surveillance is a cost-effective tool for monitoring SARS-CoV-2 transmission in a community. However, challenges remain with regard to interpretating such studies, not least in how to compare SARS-CoV-2 levels between different-sized wastewater treatment plants. Viral faecal indicators, including crAssphage and pepper mild mottle virus, have been proposed as population biomarkers to normalise SARS-CoV-2 levels in wastewater. However, as these indicators exhibit variability between individuals and may not be excreted by everyone, their utility as population biomarkers may be limited. Coprostanol, meanwhile, is a bacterial metabolite of cholesterol which is excreted by all individuals. In this study, composite influent samples were collected from a large- and medium-sized wastewater treatment plant in Dublin, Ireland and SARS-CoV-2 N1, crAssphage, pepper mild mottle virus, HF183 and coprostanol levels were determined. SARS-CoV-2 N1 RNA was detected and quantified in all samples from both treatment plants. Regardless of treatment plant size, coprostanol levels exhibited the lowest variation in composite influent samples, while crAssphage exhibited the greatest variation. Moreover, the strongest correlations were observed between SARS-CoV-2 levels and national and Dublin COVID-19 cases when levels were normalised to coprostanol. This work demonstrates the usefulness of coprostanol as a population biomarker for wastewater surveillance studies.

Decay of infectious SARS-CoV-2 and surrogates in aquatic environments.

The introduction of SARS-CoV-2 containing human stool and sewage into water bodies may raise public health concerns. However, assessment of public health risks by faecally contaminated water is limited by a lack of knowledge regarding the persistence of infectious SARS-CoV-2 in water. In the present study the decay rates of viable infectious SARS-CoV-2 and SARS-CoV-2 RNA were determined in river and seawater at 4 and 20°C. These decay rates were compared to S. typhimurium bacteriophage MS2 and pepper mild mottle virus (PMMoV). Persistence of viable SARS-CoV-2 was temperature dependent, remaining infectious for significantly longer periods of time in both freshwater and seawater at 4°C than at 20°C. T90 for infectious SARS-CoV-2 in river water was 2.3 days and 3.8 days at 20°C and 4°C, respectively. The T90 values were 1.1 days and 2.2 days in seawater at 20°C and 4°C, respectively. In contrast to the rapid inactivation of infectious SARS-CoV-2 in river and sea water, viral RNA was relatively stable. The RNA decay rates were increased in non-sterilised river and seawater, presumably due to the presence of microbiota. The decay rates of infectious MS2, MS2 RNA and PMMoV RNA differed significantly from the decay rate of SARS-CoV-2 RNA, suggesting that their use as surrogate markers for the persistence of SARS-CoV-2 in the environment is limited.