key: cord-0717429-9gkoh0xc authors: Hillary, Luke S.; Farkas, Kata; Maher, Kathryn H.; Lucaci, Anita; Thorpe, Jamie; Distaso, Marco A.; Gaze, William H.; Paterson, Steve; Burke, Terry; Connor, Thomas R.; McDonald, James E.; Malham, Shelagh K.; Jones, David L. title: Monitoring SARS-CoV-2 in municipal wastewater to evaluate the success of lockdown measures for controlling COVID-19 in the UK date: 2021-05-08 journal: Water Res DOI: 10.1016/j.watres.2021.117214 sha: 737f51ca472fd82845bf9b50582ef480540d1b35 doc_id: 717429 cord_uid: 9gkoh0xc 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 sequencing 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. The emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), and the resulting global Coronavirus disease 2019 pandemic has had disastrous socioeconomic and political consequences worldwide (Chakraborty and Maity, 2020) . This led to the World Health Organisation (WHO) declaring the COVID-19 pandemic a global health emergency (WHO, 2020) . In response to this, many countries implemented a range of mitigation strategies to reduce the spread of disease, including social distancing, restricted movement, use of personal protection equipment, contact tracing, shielding of vulnerable populations, local or national lockdowns, and community mass testing (Cirrincione et al., 2020; Iacobucci, 2020) . These measures are of particular importance in urbanised areas where the spread of disease is most likely (Zhang and Schwartz, 2020) . These measures proved to be largely effective at reducing the first wave of COVID-19, albeit not completely eliminating infections (Goscé et al., 2020; Jarvis et al., 2020) . The occurrence of subsequent waves of COVID-19 is of significantconcern, as countries seek to learn from the effectiveness of the mitigation measures used during the first wave of infection (Aleta et al., 2020) . A large proportion of SARS-CoV-2 infections are asymptomatic or result in only a mild infection (Nishiura et al., 2020) . When symptoms do become apparent, this typically occurs 3-7 days after infection (Arons et al., 2020) and severity can vary widely across different sectors of society, disproportionately affecting the elderly . Evidence points towards the fact that individuals can transmit the virus unknowingly prior to developing symptoms. Furthermore, a-and pre-symptomatic individuals pose challenges to surveillance efforts to accurately estimate the presence and extent of infection in the community. In a more practical sense, both asymptomatic and pre-symptomatic individuals also pose a major threat to public health as they can unknowingly spread the virus to more vulnerable groups . Although mass community testing has been instigated in many countries to estimate the prevalence of COVID-19 in the population, this is costly and the demand for tests frequently exceeds the capacity of testing facilities (Barasa et al., 2020) . Focussing testing solely on symptomatic cases may also fail to capture asymptomatic and pre-symptomatic infections, and may focus on populations such as those who are hospitalised, meaning that surveillance is unavailable for the wider community. In some cases, it can also be difficult to obtain nasopharyngeal swabs from high-risk parts of the community due to a range of physical, logistical or cultural issues. Wastewater-based epidemiology (WBE) detects genome fragments of SARS-CoV-2 shed in faeces and urine, and represents an alternative strategy to monitor the levels of virus circulating at population-level scales Kitajima et al., 2020; Polo et al., 2020) . WBE approaches have previously been successful in evaluating the prevalence of other viral diseases (e.g. polio-, norovirus) and also for tracking the use of illicit substances, pharmaceuticals and exposure to xenobiotics (Castiglioni et al., 2014; Ozawa et al., 2019; Zuccato et al., 2008) . Monitoring viruses in wastewater also allows an evaluation of the potential risk posed by the discharge of treated and untreated wastewater into the wider environment. Overall, WBE may represent a cost-effective method for determining viral prevalence at the population-level, and has been used to monitor of SARS-CoV-2 in a range of countries (Supplementary Table 1 ). Despite the simplicity of the approach, the quantitative recovery of viruses and viral nucleic acids from wastewater is notoriously difficult (Farkas et al., 2018a) . For example, virus concentrations in wastewater can be heavily influenced by (i) dilution by rainfall and industrial inputs, (ii) the presence of compounds that may degrade the virus (e.g. detergents, pH, salt), (iii) the presence of substances that physically protect the virus (e.g. faecal matter), (iv) loss of viral RNA during long transit times through the wastewater network due to decay and sorption, (v) variable shedding rates in the community, and (vi) inhibitory substances in the wastewater that may interfere with quantitative (reverse transcription)-PCR (q(RT-)PCR ) reactions (Polo et al., 2020) . In addition to these factors, the protocols used to concentrate and purify viral nucleic acids from wastewater samples can have substantial impacts on recovery, leading to underestimation of the quantities of the virus present in the wastewater system. Consequently, there is a need to better understand the factors that influence observable levels of SARS-CoV-2 in wastewater to allow validation of the approach for surveillance purposes. Largescale efforts to monitor changes in the SARS-CoV-2 genome and track its circulation at national and global scales have largely relied on the analysis of high-throughput sequencing of the SARS-CoV-2 genome in symptomatic individuals (Islam et al., 2020; Meredith et al., 2020; Plessis et al., 2021) . As retrospective screening of respiratory samples has detected asymptomatic cases of COVID-19 (Meredith et al., 2020) it suggests that lineages may appear in wastewater samples prior to observation in clinical cases. Because wastewater aggregates samples from across a community/area, sequencing of SARS-CoV-2 RNA recovered from wastewater is likely to contain multiple lineages and so analysis of this data also has the potential to assess the proportions of different lineages circulating in the wider population. This potentially enables the identification of lineages that are known to be present and early warning of new lineages not previously observed in a catchment. Here, we present a 3.5-month longitudinal analysis of SARS-CoV-2 RNA prevalence and genetic diversity across six different urban centres during the imposition and gradual lifting of the first national lockdown period in the UK (March-July 2020). The aims of this study were to (i) investigate the use of WBE for tracking SARS-CoV-2 after the implementation of national lockdown measures at six urban centres of varying size within the UK, (ii) determine the influence of environmental factors (e.g. flow) on levels of SARS-CoV-2 RNA and a human faecal marker virus (crAssphage) in wastewater, (iii) investigate the impact of wastewater treatment on the removal of SARS-CoV-2 RNA from wastewater, and (iv) assess the utility of WBE in understanding SARS-CoV-2 genetic variation through high-throughput sequencing. All laboratory procedures were carried out in line with Public Health England/ Public Health Wales advice on the handling of samples suspected of containing SARS-CoV-2. Untreated influent and treated effluent wastewater were collected from six wastewater treatment plants (WWTPs) located in Wales and Northwest England. The WWTPs served urban areas in the local authority areas of Gwynedd, Cardiff, Liverpool, Manchester, the Wirral and Wrexham, with a total combined population equivalent of ~3 million people (Suplementary Fig. 1 ). Untreated wastewater influent from the six WWTPs was sampled on a weekly basis between March and July 2020. Samples were collected in polypropylene bottles as single grab samples with the exception of the Wirral site, which was collected as a 24-hour composite sample using an autosampler. Grab samples were collected on weekdays between 08.00 and 09.00 h to ensure temporal comparability, and treated effluent also collected periodically at the same time as influent. Samples were transported on either the same day, or overnight on ice, to the laboratory, stored at 4 °C and processed within 24 h of receipt. Aliquots of wastewater samples (1.5 ml) were also frozen in polypropylene vials at -80 °C for subsequent physico-chemical analyses and extraction of pre-concentration viral nucleic acids. Wastewater samples were pasteurised before physicochemical analysis by heating to 60 C for 90 min. Wastewater ammonium concentrations were determined colorimetrically using the salicylic acid procedure of Mulvaney (1996) . Nitrate was determined colorimetrically using the vanadate procedure of Miranda et al. (2001) while molybdate-reactive phosphate (MRP) was determined according to Murphy and Riley (1962) . All analysis was performed in a 96-well plate format using a PowerWave XS Microplate Spectrophotometer (BioTek Instruments Inc., Winooski, VT). Wastewater electrical conductivity (EC) was measured using a Jenway 4520 conductivity meter and pH with a Hanna 209 pH meter (Hanna Instruments Ltd., Leighton Buzzard, UK). Duplicate samples of 50-100 mL of unpastuerised wastewater influent underwent centrifugation (10,000 g, 30 min, 4°C) and the supernatant and pellet retained. Supernatants were concentrated to 500 µL using Centriprep 50 kDa MWCO centrifugal concentrators (Merck KGaA, Germany). For wastewater effluent samples (see Supplementary Table 5 ), 1-2 L of each effluent was initially concentrated using tangential flow ultrafiltration with a 100 kDa PES membrane (Spectrumlabs, USA) as previously described (Farkas et al., 2018c) , followed by secondary concentration using Centriprep concentrators as described above. Selected wastewater concentrates, centrifugation pellets and unconcentrated wastewater samples were spiked with approximately 4×10 5 genome copies (gc) of murine norovirus (MNV) as a viral RNA extraction control. Positive and negative nucleic acid control extractions of nuclease-free water with or without the same quantity of MNV spike-in were used to quantify MNV recovery by q(RT-)PCR and to check for cross-contamination during the nucleic acid extraction process or q(RT-)PCR assay setup (described in section 2.4). The supplemented with 2% foetal bovine serum (FBS) at 37 °C in 5% CO 2 for two days. Viruses were harvested by three cycles of freeze-thawing (-20°C/+37°C) followed by centrifugation and 100× dilution of the supernatant in phosphate-buffered saline pH 7.4. Aliquots of MNV stock were stored at -80°C until use. The MNV and BV2 tissue stocks were kindly provided by Prof Ian Goodfellow (University of Cambridge, UK). Nucleic acids were extracted using the NucliSENS MiniMag Nucleic Acid Purification System (BioMérieux SA, Marcy-l'Étoile, France) according to the manufacturer's protocol as described elsewhere (Farkas et al., 2021) in a final volume of 50 (last week of March 2020) or 100 µL (April-July 2020) of elution buffer. Extracted nucleic acids were stored at -80 °C prior to q(RT-)PCR quantification. The nucleic acid extractions and q(RT-)PCR assay setups were carried out in separate laboratories inside class II microbiological safety cabinets to minimise the risk of contamination. The q(RT-)PCR assays were carried out in a QuantStudio ® Flex 6 Real-Time PCR System (Applied Biosystems, USA) using primers, probes and reaction conditions described in Supplementary Table 2 . SARS-CoV-2 N1 and MNV RNA were quantified using a duplex q(RT-)PCR assay or in triplex with SARS-CoV-2 E gene, as described in Farkas et al. (2021) . The 25 μL reaction mix contained 1×RNA Ultrasense Reaction Mix with 1 µL RNA Ultrasense Enzyme Mix (Invitrogen, USA), 12.5 pmol of the forward and the reverse primers, 6.25 pmol of the probe/probes, 0.1× ROX reference dye, 1.25 µg bovine serum albumin (BSA) and 2-5 μL of the extracted wastewater RNA, molecular grade water as a negative control or virus standards. Initially, 5 µL of extracted RNA was tested for wastewater samples. If the MNV recovery was lower than 1%, samples were retested with 2 µL sample/reaction to assess inhibition of the q(RT-)PCR assay, however this was found to be detrimental to assay sensitivity. All data-points used in the analysis came from assays of 5 L of extracted nucleic acids. CrAssphage was used as a marker of human faecal abundance/loading in the wastewater (Farkas et al., 2019; Stachler et al., 2018) . CrAssphage DNA was quantified using a singleplex qPCR as described previously (Farkas et al., 2019) . The 20 µL reaction mix contained 1× KAPA Probe Force qPCR mix (KAPA Biosystems, USA) with 10 pmol of the forward, 10 pmol of the reverse primers, 5 pmol of the probe, 1 µg bovine serum albumin, and 2 µL and 4 µL of the concentrated and original wastewater nucleic acid extracts or controls. A serial dilution of DNA standards within the range of 10 5 -10 0 genome copies (gc) µL -1 was used for quantification. For SARS-CoV-2, commercially available circular plasmids carrying the N gene or E gene were used (Integrated DNA Technologies Inc., Coralville, IA). Plasmid DNA concentrations were halved when setting up serial dilutions to account for ssRNA producing half the fluorescence signal of dsDNA at the same concentration. For MNV and crAssphage, custom-made, single-stranded oligo DNA sequences carrying the target region were used (Life Technologies, USA). Negative controls (molecular grade water) were included in each run. All samples, standards and controls were run in duplicate and the mean value for each extraction replicate used for further analysis. The limit of detection (LoD) and limit of quantification (LoQ) of the triplex q(RT-)PCR assays were determined previously (Farkas et al., 2021) by running wastewater samples spiked with low concentrations of SARS-CoV-2 (1-150 gc µL -1 N1 CDC and 1-200 gc µL -1 E Sarbeco) and MNV RNA (1-80 gc µL -1 ) in ten replicates. The q(RT-PCR) assay LoD (the lowest concentration where all replicates were positive) were 1.7, 3.8 and 3.1 gc µL -1 for the N gene, E gene and MNV, respectively. The LoQ (the lowest concentration where the coefficient of variance was below 0.25) were 11.8, 25.1 and 32.1 gc µL -1 for the N gene, E gene and MNV, respectively. Data were analysed using QuantStudio™ Real-Time PCR The relationship between SNP and indel site frequency and the proportion of the genome with coverage at greater than 50× coverage and the log 10 gc µL -1 were examined with We Results for SARS-CoV-2 RNA concentrations from q(RT-)PCR quantification are displayed as unadjusted mean genome copies/ 100 ml of wastewater rather than normalised by crAssphage concentrations as factors such as extraction efficiency can vary depending on the virus used (Medema et al., 2020) . Although studies suggest that 24-hour composite sampling is more representative than grab sampling, it has been shown that grab samples are accurate to within an order of magnitude , Ahmed et al., 2021 . Further, our previous work has shown limited diurnal variability, particularly in large wastewater catchments where transit times can be up to 24 hours and where large amounts of mixing occurs in the network (Farkas et al., 2018b) . Transit times may also influence observable virus quantities due to degradation of viral nucleic acids as they pass through the sewage system; however, SARS-CoV-2 RNA has been shown to be relatively stable in wastewater under environmental conditions, with a T 90 of 24 or 28 days at 15 or 4 C (Ahmed et al., 2020b) . We compared mean SARS-CoV-2 RNA concentrations to daily flow and influent wastewater chemistry but found no statistically significant correlations (see supplementary table 4 ). The highly abundant bacteriophage crAssphage was used as a human faecal marker. No correlation was found between crAssphage and SARS-CoV-2 nucleic acid concentrations (Spearman, p = 0.8341). No effect on crAssphage concentration was observable from sampling week (Kruskal-Wallis, p = 0.9042), but a significant effect was found between crAssphage concentration and WWTP site (Kruskal-Wallis, p = 0.01751). These data indicate that faecal loading was constant throughout the study period and that different WWTPs have different balances of human waste and industrial/ other domestic wastewater sources. late June. These can occur due to surge testing following local workplace-related outbreaks and changes in testing eligibility during the study, and highlight the inherent difficulties in comparing wastewater loads to positive tests when testing is both limited and non-random. SARS-CoV-2 RNA concentration and daily positive tests ( Fig. 1b and Supplementary Fig. 3) . Negative correlations were also observed between viral concentrations in all sites and time Further exploration of site-specific factors and improved access to higher resolution spatial distributions of positive test locations is required to improve the accuracy of WBE in predicting COVID-19 prevalence amongst local populations as part of national monitoring programmes. Previous studies have corrected SARS-CoV-2 RNA concentration for WWTP flow , and adjusted cases or positive tests for differences between local authority populations and WWTP catchment areas (Medema et al., 2020) . Statistically, we found no benefit of correcting for these factors on Spearman correlation coefficients between WWTP SARS-CoV-2 RNA concentration and positive tests/ COVID-19 related deaths (see Supplemental Fig. 3) , however due to differences between WWTP sites and sewersheds, we would caution against making extensive quantitative comparisons between sites. Our data confirm that SARS-CoV-2 RNA is readily detectable in wastewater influent across a range of concentrations from <1.2  10 3 ( 0.05; Fig. 4a and b) , but a reduced model suggested that there was heterogeneity among sites (X 2 = 11.57, df = 5, p = 0.041; Fig. 4c ). The index of SNP plus indel frequency was not related to log population equivalent served by each wastewater treatment plant (Spearman's  = 0.251, p = 0.251; Fig. 4d ). This is explained by the presence of multiple viral lineages present within the sample, corresponding to the diverse infections in the population represented in the wastewater sample. A substantial fraction of the detected SNPs has previously been identified in clinical samples across the UK, and has the potential to be informative for distinguishing viral lineages (Supplementary Table 6 ). Multiple SARS-CoV-2 lineages can be present within a single wastewater sample. Samples have the potential to contain viruses from both symptomatic and asymptomatic individuals within the community, as SARS-CoV-2 has been detected in the faeces of both asymptomatic and symptomatic individuals Tang et al., 2020) . Previous studies have sequenced SARS-CoV-2 genomes from wastewater (Ahmed et al., 2020a; Izquierdo-Lara et al., n.d.; Martin et al., 2020; Nemudryi et al., 2020) . We have shown not only that viral genome sequences can be recovered from wastewater samples, but that they exhibit substantial diversity across dozens of samples. Sequencing the genomes therefore has the potential to assess the diversity of viral infections in the wastewater catchment population and to identify emerging genetic variants before they are seen in clinical samples. In support of this, preliminary analysis suggests that the detected SNPs were consistent with those detected previously in clinical samples (see Supplementary Table 6 ). However, because the SNPs from wastewater samples are not phased across the genome, and because the genome coverage is imperfect, assigning viral lineages to samples will require a bespoke statistical framework to be developed. Attempting to quantitatively link observed viral RNA concentrations to detectable cases is challenging (Medema et al., 2020) . Many assumptions need to be made regarding the persistence of SARS-CoV-2 in wastewater, quantities of the virus shed in faeces and the influence of water chemistry (Ahmed et al., 2020a) . Sample processing methodology can also be a substantial source of variability. Concentration method, qPCR assay design and inter-lab variation can create variation in detectable SARS-CoV-2 RNA quantities (Pecson et al., 2021; Westhaus et al., 2021) . Use of appropriate process controls is necessary to monitor the effects of these factors when making intra-and inter-laboratory comparisons. Choice of process control is complex as a closely related surrogate virus should be used where available and further global collaboration and coordination is required to widen access to WBE technologies (Polo et al., 2020) . In addition to this, the effects of SARS-CoV-2 on global supply chains and the need to perform WBE at scale create additional pressures where sub-optimal protocols may become necessary in the future to achieve testing scale desired for national monitoring programs. Despite the possible sources of variability mentioned above, we have demonstrated that WBE is suitable for quantitatively tracking the course of the early stages of the SARS-CoV-2 pandemic and the effects of public health interventions, even in the early stages of a novel outbreak, where lack of surge capacity prevents optimal sampling. We highlight how tiled primer array sequencing complements q(RT-)PCR based detection of SARS-CoV-2 and enhances the sensitivity and usefulness of WBE in detecting the presence of novel mutations in the SARS-CoV-2 genome. Early detection of viral pathogens by q(RT-)PCR requires a suitable assay and routine monitoring of WWTPs however alternative technologies such as viral metagenomics may be more suited to initial detection of emerging and unknown pathogens . Our results suggest that viral amplicon sequencing could be more sensitive than q(RT-)PCR for detection of known pathogens. In future, monitoring could be targeted towards ports of entry and major metropolitan centres to maximise the likelihood of detection (Medema et al., 2020) .  Our results demonstrate that levels of SARS-CoV-2 RNA in wastewater generally correlated well with the abundance of clinical COVID-19 cases recorded within the community in large urban centres.  At the population level, wastewater-based epidemiology was used to confirm the success of lockdown measures (i.e. restricted movement and human-to human contact) implemented at the national scale to control the transmission of SARS-CoV-2.  The genetic diversity of SARS-CoV-2 from wastewater suggests that multiple genetically distinct clusters were co-circulating present in the local populations, and that the genetic variants observed in wastewater reflect similar SNPs observed in samples from nasopharyngeal swabs taken contemporaneously at clinical testing centres.  A greater understanding of the factors that affect SARS-CoV-2 RNA quantification in wastewater is still required to enable the full integration of wastewater-based epidemiology data into wider outbreak surveillance programmes.  Our results lend support to the use of routine wastewater-based epidemiology to monitor SARS-CoV-2 and other human pathogenic viruses circulating in the population and to assess the effectiveness of disease control measures. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 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Environ. Health Perspect Response Programme (projects NE/V004883/1 and NE/V010441/1) and the Centre for Environmental Biotechnology Project funded though the European Regional Development