Survey of rapid development of environmental surveillance methods for SARS-CoV-2 detection in wastewater.
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ABSTRACT:
SUBMITTER: Zhou NA
PROVIDER: S-EPMC7808727 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Wastewater1 surveillance of SARS-CoV-2 may be a useful supplement to clinical surveillance as it is shed in feces, there are many asymptomatic cases, and diagnostic testing can have capacity limitations and extended time to results. Although numerous studies have utilized wastewater surveillance for SARS-CoV-2, the methods used were developed and/or standardized for other pathogens. This study evaluates multiple methods for concentration and recovery of SARS-CoV-2 and seeded human coronavirus OC43 from municipal primary wastewater and/or sludge from the Greater Seattle Area (March-July 2020). Methods evaluated include the bag-mediated filtration system (BMFS), with and without Vertrel™ extraction, skimmed milk flocculation, with and without Vertrel™ extraction, polyethylene glycol (PEG) precipitation, ultrafiltration, and sludge extraction. Total RNA was extracted from wastewater concentrates and analyzed for SARS-CoV-2 and OC43 with RT-qPCR. Skimmed milk flocculation without Vertrel™ extraction performed consistently over time and between treatment plants in Seattle-area wastewater with the lowest average OC43 Cq value and smallest variability (24.3; 95% CI: 23.8-24.9), most frequent SARS-CoV-2 detection (48.8% of sampling events), and highest average OC43 percent recovery (9.1%; 95% CI: 6.2-11.9%). Skimmed milk flocculation is also beneficial because it is feasible in low-resource settings. While the BMFS had the highest average volume assayed of 11.9 mL (95% CI: 10.7-13.1 mL), the average OC43 percent recovery was low (0.7%; 95% CI: 0.4-1.0%). Ultrafiltration and PEG precipitation had low average OC43 percent recoveries of 1.0% (95% CI: 0.5-1.6%) and 3.2% (95% CI: 1.3-5.1%), respectively. The slopes and efficiency for the SARS-CoV-2 standard curves were not consistent over time, confirming the need to include a standard curve each run rather than using a single curve for multiple plates. Results suggest that the concentration and detection methods used must be validated for the specific water matrix using a recovery control to assess performance over time.
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Project description:There have been over 507 million cases of COVID-19, the disease caused by the severe
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Project description:Wastewater-based epidemiology for SARS-CoV-2 RNA detection in wastewater is desirable for understanding COVID-19 in settings where financial resources and diagnostic facilities for mass individual testing are severely limited. We conducted a rapid review to map research evidence on the utilization of SARS-CoV-2 wastewater surveillance in Africa. We searched PubMed, Google Scholar, and the World Health Organization library databases for relevant reports, reviews, and primary observational studies. Eight studies met the inclusion criteria. Narrative synthesis of the findings from included primary studies revealed the testing methodologies utilized and that detected amount of SARS-CoV-2 viral RNA correlated with the number of new cases in the studied areas. The included reviews revealed the epidemiological significance and environmental risks of SARS-CoV-2 wastewater. Wastewater surveillance data at the community level can be leveraged for the rapid assessment of emerging threats and aid pandemic preparedness. Our rapid review revealed a glaring gap in the primary literature on SARS-CoV-2 wastewater surveillance on the continent, and accelerated and adequate investment into research is urgently needed to address this gap.
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Project description:In this study, 14 virus concentration protocols based on centrifugation, filtration, polyethylene glycol (PEG) precipitation and ultrafiltration were tested for their efficacy for the quantification of SARS-CoV-2 in wastewater samples. These protocols were paired with four RNA extraction procedures resulting in a combination of 50 unique approaches. Bovine respiratory syncytial virus (BRSV) was used as a process control and seeded in each wastewater sample subjected to all 50 protocols. The recovery of BRSV obtained through the application of 50 unique approaches ranged from <0.03 to 64.7% (±1.6%). Combination of centrifugation as the solid removal step, ultrafiltration (Amicon-UF-15; 100 kDa cut-off; protocol 9) as the primary virus concentration method, and Zymo Quick-RNA extraction kit provided the highest BRSV recovery (64.7 ± 1.6%). To determine the impact of prolonged storage of large wastewater sample volume (900 mL) at -20 °C on enveloped virus decay, the BRSV seeded wastewaters samples were stored at -20 °C up to 110 days and analyzed using the most efficient concentration (protocol 9) and extraction (Zymo Quick-RNA kit) methods. BRSV RNA followed a first-order decay rate (k = 0.04/h with r2 = 0.99) in wastewater. Finally, 21 wastewater influent samples from five wastewater treatment plants (WWTPs) in southern Maryland, USA were analyzed between May to August 2020 to determine SARS-CoV-2 RNA concentrations. SARS-CoV-2 RNA was quantifiable in 17/21 (81%) of the influent wastewater samples with concentration ranging from 1.10 (±0.10) × 104 to 2.38 (±0.16) × 106 gene copies/L. Among the RT-qPCR assays tested, US CDC N1 assay was the most sensitive followed by US CDC N2, E_Sarbeco, and RdRp assays. Data presented in this study may enhance our understanding on the effective concentration and extraction of SARS-CoV-2 from wastewater.
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Project description:Wastewater surveillance and quantitative analysis of SARS-CoV-2 RNA are increasingly used to monitor the spread of COVID-19 in the community. We studied the feasibility of applying the surveillance data for early detection of local outbreaks. A Monte Carlo simulation model was constructed, applying data on reported variation in RNA gene copy concentration in faeces and faecal masses shed. It showed that, even with a constant number of SARS-CoV-2 RNA shedders, the variation in concentrations found in wastewater samples will be large, and that it will be challenging to translate viral concentrations into incidence estimates, especially when the number of shedders is low. Potential signals for early detection of hypothetical outbreaks were analysed for their performance in terms of sensitivity and specificity of the signals. The results suggest that a sudden increase in incidence is not easily identified on the basis of wastewater surveillance data, especially in small sampling areas and in low-incidence situations. However, with a high number of shedders and when combining data from multiple consecutive tests, the performance of wastewater sampling is expected to improve considerably. The developed modelling approach can increase our understanding of the results from wastewater surveillance of SARS-CoV-2.
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Project description:The continuing emergence of SARS-CoV-2 variants of concern and variants of interest emphasizes the need for early detection and epidemiological surveillance of novel variants. We used genomic sequencing of 122 wastewater samples from three locations in Switzerland to monitor the local spread of B.1.1.7 (Alpha), B.1.351 (Beta) and P.1 (Gamma) variants of SARS-CoV-2 at a population level. We devised a bioinformatics method named COJAC (Co-Occurrence adJusted Analysis and Calling) that uses read pairs carrying multiple variant-specific signature mutations as a robust indicator of low-frequency variants. Application of COJAC revealed that a local outbreak of the Alpha variant in two Swiss cities was observable in wastewater up to 13 d before being first reported in clinical samples. We further confirmed the ability of COJAC to detect emerging variants early for the Delta variant by analysing an additional 1,339 wastewater samples. While sequencing data of single wastewater samples provide limited precision for the quantification of relative prevalence of a variant, we show that replicate and close-meshed longitudinal sequencing allow for robust estimation not only of the local prevalence but also of the transmission fitness advantage of any variant. We conclude that genomic sequencing and our computational analysis can provide population-level estimates of prevalence and fitness of emerging variants from wastewater samples earlier and on the basis of substantially fewer samples than from clinical samples. Our framework is being routinely used in large national projects in Switzerland and the UK.
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Project description:The effective application of wastewater surveillance is dependent on testing capacity and sensitivity to obtain high spatial resolution testing results for a timely targeted public health response. To achieve this purpose, the development of rapid, high-throughput, and sensitive virus concentration methods is urgently needed. Various protocols have been developed and implemented in wastewater surveillance networks so far, however, most of them lack the ability to scale up testing capacity or cannot achieve sufficient sensitivity for detecting SARS-CoV-2 RNA at low prevalence. In the present study, using positive raw wastewater in Hong Kong, a PEG precipitation-based three-step centrifugation method was developed, including low-speed centrifugation for large particles removal and the recovery of viral nucleic acid, and medium-speed centrifugation for the concentration of viral nucleic acid. This method could process over 100 samples by two persons per day to reach the process limit of detection (PLoD) of 3286 copies/L wastewater. Additionally, it was found that the testing capacity could be further increased by decreasing incubation and centrifugation time without significantly influencing the method sensitivity. The entire procedure uses ubiquitous reagents and instruments found in most laboratories to obtain robust testing results. This high-throughput, cost-effective, and sensitive tool will promote the establishment of nearly real-time wastewater surveillance networks for valuable public health information.
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Project description:The ongoing COVID-19 pandemic is caused by SARs-CoV-2. The virus is transmitted from person to person through droplet infections i.e. when infected person is in close contact with another person. In January 2020, first report of detection of SARS-CoV-2 in faeces, has made it clear that human wastewater might contain this virus. This may illustrate the probability of environmentally facilitated transmission, mainly the sewage, however, environmental conditions that could facilitate faecal oral transmission is not yet clear. We used existing Pakistan polio environment surveillance network to investigate presence of SARs-CoV-2 using three commercially available kits and E-Gene detection published assay for surety and confirmatory of positivity. A Two-phase separation method is used for sample clarification and concentration. An additional high-speed centrifugation (14000Xg for 30 min) step was introduced, prior RNA extraction, to increase viral RNA yield resulting a decrease in Cq value. A total of 78 wastewater samples collected from 38 districts across Pakistan, 74 wastewater samples from existing polio environment surveillance sites, 3 from drains of COVID-19 infected areas and 1 from COVID 19 quarantine center drainage, were tested for presence of SARs-CoV-2. 21 wastewater samples (27%) from 13 districts turned to be positive on RT-qPCR. SARs-COV-2 RNA positive samples from areas with COVID 19 patients and quarantine center strengthen the findings and use of wastewater surveillance in future. Furthermore, sequence data of partial ORF 1a generated from COVID 19 patient quarantine center drainage sample also reinforce our findings that SARs-CoV-2 can be detected in wastewater. This study finding indicates that SARs-CoV-2 detection through wastewater surveillance has an epidemiologic potential that can be used as supplementary system to monitor viral tracking and circulation in cities with lower COVID-19 testing capacity or heavily populated areas where door-to-door tracing may not be possible. However, attention is needed on virus concentration and detection assay to increase the sensitivity. Development of highly sensitive assay will be an indicator for virus monitoring and to provide early warning signs.
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Project description:In the global strategy for polio eradication, environmental surveillance (ES) has been established worldwide to monitor polioviruses. In addition, nonpolio enteroviruses are simultaneously isolated from wastewater under this ES program. Hence, ES can be used to monitor enteroviruses in sewage to supplement clinical surveillance. In response to the coronavirus disease 2019 (COVID-19) pandemic, we also monitored severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in sewage using the polio ES system in Japan. Enterovirus and SARS-CoV-2 were detected in sewage from January 2019 to December 2021 and from August 2020 to November 2021, respectively. Enterovirus species such as echoviruses and coxsackieviruses were frequently detected by ES in 2019, indicating the circulation of these viruses. After the onset of the COVID-19 pandemic, sewage enterovirus detection and related patient reports were notably reduced in 2020 to 2021, suggesting changes in the hygiene behaviors of the human population in response to the pandemic. Our comparative experiment with a total of 520 reverse transcription-quantitative PCR (RT-qPCR) assays for SARS-CoV-2 detection demonstrated that the solid-based method had a significantly higher detection rate than that of the liquid-based method (24.6% and 15.9%, respectively). Moreover, the resulting RNA concentrations were correlated with the number of new COVID-19 cases (Spearman's r = 0.61). These findings indicate that the existing polio ES system can be effectively used for enterovirus and SARS-CoV-2 sewage monitoring using different procedures such as virus isolation and molecular-based detection. IMPORTANCE Long-term efforts are required to implement surveillance programs for the ongoing COVID-19 pandemic, and they will be required even in the postpandemic era. We adopted the existing polio environmental surveillance (ES) system for SARS-CoV-2 sewage monitoring in Japan as a practical and cost-effective approach. Moreover, the ES system routinely detects enteroviruses from wastewater and, therefore, can be used for enterovirus monitoring. The liquid fraction of the sewage sample is used for poliovirus and enterovirus detection, and the solid fraction can be used for SARS-CoV-2 RNA detection. The present study demonstrates how the existing ES system can be used for monitoring enteroviruses and SARS-CoV-2 in sewage.
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Project description:The SARS-CoV-2 Omicron variant emerged in South Africa in November 2021, and has later been identified worldwide, raising serious concerns. A real-time RT-PCR assay was designed for the rapid screening of the Omicron variant, targeting characteristic mutations of the spike gene. The assay was used to test 737 sewage samples collected throughout Italy (19/21 Regions) between 11 November and 25 December 2021, with the aim of assessing the spread of the Omicron variant in the country. Positive samples were also tested with a real-time RT-PCR developed by the European Commission, Joint Research Centre (JRC), and through nested RT-PCR followed by Sanger sequencing. Overall, 115 samples tested positive for Omicron SARS-CoV-2 variant. The first occurrence was detected on 7 December, in Veneto, North Italy. Later on, the variant spread extremely fast in three weeks, with prevalence of positive wastewater samples rising from 1.0% (1/104 samples) in the week 5-11 December, to 17.5% (25/143 samples) in the week 12-18, to 65.9% (89/135 samples) in the week 19-25, in line with the increase in cases of infection with the Omicron variant observed during December in Italy. Similarly, the number of Regions/Autonomous Provinces in which the variant was detected increased from one in the first week, to 11 in the second, and to 17 in the last one. The presence of the Omicron variant was confirmed by the JRC real-time RT-PCR in 79.1% (91/115) of the positive samples, and by Sanger sequencing in 66% (64/97) of PCR amplicons. In conclusion, we designed an RT-qPCR assay capable to detect the Omicron variant, which can be successfully used for the purpose of wastewater-based epidemiology. We also described the history of the introduction and diffusion of the Omicron variant in the Italian population and territory, confirming the effectiveness of sewage monitoring as a powerful surveillance tool.
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