Correction: Farkas et al. Concentration and Quantification of SARS-CoV-2 RNA in Wastewater Using Polyethylene Glycol-Based Concentration and qRT-PCR. Methods Protoc. 2021, 4, 17.

There was an error in the original article [...].

Concentration and Quantification of SARS-CoV-2 RNA in Wastewater Using Polyethylene Glycol-Based Concentration and qRT-PCR.

Wastewater-based epidemiology has become an important tool for the surveillance of SARS-CoV-2 outbreaks. However, the detection of viruses in sewage is challenging and to date there is no standard method available which has been validated for the sensitive detection of SARS-CoV-2. In this paper, we describe a simple concentration method based on polyethylene glycol (PEG) precipitation, followed by RNA extraction and a one-step quantitative reverse transcription PCR (qRT-PCR) for viral detection in wastewater. PEG-based concentration of viruses is a simple procedure which is not limited by the availability of expensive equipment and has reduced risk of disruption to consumable supply chains. The concentration and RNA extraction steps enable 900-1500× concentration of wastewater samples and sufficiently eliminates the majority of organic matter, which could inhibit the subsequent qRT-PCR assay. Due to the high variation in the physico-chemical properties of wastewater samples, we recommend the use of process control viruses to determine the efficiency of each step. This procedure enables the concentration and the extraction the DNA/RNA of different viruses and hence can be used for the surveillance of different viral targets for the comprehensive assessment of viral diseases in a community.

Monitoring SARS-CoV-2 in municipal wastewater to evaluate the success of lockdown measures for controlling COVID-19 in the UK.

SARS-CoV-2 and the resulting COVID-19 pandemic represents one of the greatest recent threats to human health, wellbeing and economic growth. Wastewater-based epidemiology (WBE) of human viruses can be a useful tool for population-scale monitoring of SARS-CoV-2 prevalence and epidemiology to help prevent further spread of the disease, particularly within urban centres. Here, we present a longitudinal analysis (March-July 2020) of SARS-CoV-2 RNA prevalence in sewage across six major urban centres in the UK (total population equivalent 3 million) by q(RT-)PCR and viral genome sequencing. Our results demonstrate that levels of SARS-CoV-2 RNA generally correlated with the abundance of clinical cases recorded within the community in large urban centres, with a marked decline in SARS-CoV-2 RNA abundance following the implementation of lockdown measures. The strength of this association was weaker in areas with lower confirmed COVID-19 case numbers. Further, sequence analysis of SARS-CoV-2 from wastewater suggested that multiple genetically distinct clusters were co-circulating in the local populations covered by our sample sites, and that the genetic variants observed in wastewater reflected similar SNPs observed in contemporaneous samples from cases tested in clinical diagnostic laboratories. We demonstrate how WBE can be used for both community-level detection and tracking of SARS-CoV-2 and other virus' prevalence, and can inform public health policy decisions. Although, greater understanding of the factors that affect SARS-CoV-2 RNA concentration in wastewater are needed for the full integration of WBE data into outbreak surveillance. In conclusion, our results lend support to the use of routine WBE for monitoring of SARS-CoV-2 and other human pathogenic viruses circulating in the population and assessment of the effectiveness of disease control measures.

Shedding of SARS-CoV-2 in feces and urine and its potential role in person-to-person transmission and the environment-based spread of COVID-19.

The recent detection of SARS-CoV-2 RNA in feces has led to speculation that it can be transmitted via the fecal-oral/ocular route. This review aims to critically evaluate the incidence of gastrointestinal (GI) symptoms, the quantity and infectivity of SARS-CoV-2 in feces and urine, and whether these pose an infection risk in sanitary settings, sewage networks, wastewater treatment plants, and the wider environment (e.g. rivers, lakes and marine waters). A review of 48 independent studies revealed that severe GI dysfunction is only evident in a small number of COVID-19 cases, with 11 ± 2% exhibiting diarrhea and 12 ± 3% exhibiting vomiting and nausea. In addition to these cases, SARS-CoV-2 RNA can be detected in feces from some asymptomatic, mildly- and pre-symptomatic individuals. Fecal shedding of the virus peaks in the symptomatic period and can persist for several weeks, but with declining abundances in the post-symptomatic phase. SARS-CoV-2 RNA is occasionally detected in urine, but reports in fecal samples are more frequent. The abundance of the virus genetic material in both urine (ca. 102-105 gc/ml) and feces (ca. 102-107 gc/ml) is much lower than in nasopharyngeal fluids (ca. 105-1011 gc/ml). There is strong evidence of multiplication of SARS-CoV-2 in the gut and infectious virus has occasionally been recovered from both urine and stool samples. The level and infectious capability of SARS-CoV-2 in vomit remain unknown. In comparison to enteric viruses transmitted via the fecal-oral route (e.g. norovirus, adenovirus), the likelihood of SARS-CoV-2 being transmitted via feces or urine appears much lower due to the lower relative amounts of virus present in feces/urine. The biggest risk of transmission will occur in clinical and care home settings where secondary handling of people and urine/fecal matter occurs. In addition, while SARS-CoV-2 RNA genetic material can be detected by in wastewater, this signal is greatly reduced by conventional treatment. Our analysis also suggests the likelihood of infection due to contact with sewage-contaminated water (e.g. swimming, surfing, angling) or food (e.g. salads, shellfish) is extremely low or negligible based on very low predicted abundances and limited environmental survival of SARS-CoV-2. These conclusions are corroborated by the fact that tens of million cases of COVID-19 have occurred globally, but exposure to feces or wastewater has never been implicated as a transmission vector.

Wastewater and public health: the potential of wastewater surveillance for monitoring COVID-19.

Pathogenic viruses represent one of the greatest threats to human well-being. As evidenced by the COVID-19 global pandemic, however, halting the spread of highly contagious diseases is notoriously difficult. Successful control strategies therefore have to rely on effective surveillance. Here, we describe how monitoring wastewater from urban areas can be used to detect the arrival and subsequent decline of pathogens, such as SARS-CoV-2. As the amount of virus shed in faeces and urine varies largely from person to person, it is very difficult to quantitatively determine the number of people who are infected in the population. More research on the surveillance of viruses in wastewater using accurate and validated methods, as well as subsequent risk analysis and modelling is paramount in understanding the dynamics of viral outbreaks.