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Open accessJournal ArticleDOI: 10.1016/J.JES.2021.01.029

Comparison of approaches to quantify SARS-CoV-2 in wastewater using RT-qPCR: Results and implications from a collaborative inter-laboratory study in Canada

04 Mar 2021-Journal of Environmental Sciences-china (Elsevier)-Vol. 107, pp 218-229
Abstract: Detection of SARS-CoV-2 RNA in wastewater is a promising tool for informing public health decisions during the COVID-19 pandemic. However, approaches for its analysis by use of reverse transcription quantitative polymerase chain reaction (RT-qPCR) are still far from standardized globally. To characterize inter- and intra-laboratory variability among results when using various methods deployed across Canada, aliquots from a real wastewater sample were spiked with surrogates of SARS-CoV-2 (gamma-radiation inactivated SARS-CoV-2 and human coronavirus strain 229E [HCoV-229E]) at low and high levels then provided “blind” to eight laboratories. Concentration estimates reported by individual laboratories were consistently within a 1.0-log10 range for aliquots of the same spiked condition. All laboratories distinguished between low- and high-spikes for both surrogates. As expected, greater variability was observed in the results amongst laboratories than within individual laboratories, but SARS-CoV-2 RNA concentration estimates for each spiked condition remained mostly within 1.0-log10 ranges. The no-spike wastewater aliquots provided yielded non-detects or trace levels (

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29 results found


Open accessJournal ArticleDOI: 10.1016/J.WATRES.2021.117252
17 May 2021-Water Research
Abstract: Wastewater-based epidemiology (WBE) has been shown to coincide with, or anticipate, confirmed COVID-19 case numbers. During periods with high test positivity rates, however, case numbers may be underreported, whereas wastewater does not suffer from this limitation. Here we investigated how the dynamics of new COVID-19 infections estimated based on wastewater monitoring or confirmed cases compare to true COVID-19 incidence dynamics. We focused on the first pandemic wave in Switzerland (February to April, 2020), when test positivity ranged up to 26%. SARS-CoV-2 RNA loads were determined 2-4 times per week in three Swiss wastewater treatment plants (Lugano, Lausanne and Zurich). Wastewater and case data were combined with a shedding load distribution and an infection-to-case confirmation delay distribution, respectively, to estimate infection incidence dynamics. Finally, the estimates were compared to reference incidence dynamics determined by a validated compartmental model. Incidence dynamics estimated based on wastewater data were found to better track the timing and shape of the reference infection peak compared to estimates based on confirmed cases. In contrast, case confirmations provided a better estimate of the subsequent decline in infections. Under a regime of high-test positivity rates, WBE thus provides critical information that is complementary to clinical data to monitor the pandemic trajectory.

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23 Citations


Open accessJournal ArticleDOI: 10.1016/J.WATRES.2021.117214
Luke S. Hillary1, Kata Farkas1, Kathryn H. Maher2, Anita Lucaci3  +9 moreInstitutions (6)
15 Jul 2021-Water Research
Abstract: 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.

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Topics: Population (52%)

16 Citations


Open accessJournal ArticleDOI: 10.1016/J.SCITOTENV.2021.147183
Abstract: This study is the first focused on the presence of SARS-CoV-2 in different freshwater environments in an urban setting. Groundwater and surface water reservoirs for drinking water as well as water from receiving rivers of the Monterrey Metropolitan Area were sampled repeatedly during a SARS-CoV-2 peak phase between October 2020 and January 2021, and viral RNA was measured by quantitative reverse transcription polymerase chain reaction. Forty-four percent of the groundwater samples had detectable viral loads between 2.6 and 38.3 copies/ml. A significant correlation between viral load and sucralose concentration in groundwater reaffirmed the hypothesis of leaching and infiltrating effluent from surface and/or failing sewage pipes and emphasized the importance of water disinfection. Twelve percent of the surface water dam samples tested positive for viral RNA, with values varying between 3.3 and 3.8 copies/ml. Finally, 13% of the river samples were positive for viral RNA, with concentrations ranging from 2.5 to 7.0 copies/ml. Untreated wastewater samples taken in the same period showed viral loads of up to 3535 copies/ml, demonstrating a dilution effect and/or wastewater facilities efficiency of three orders of magnitude. Variations in the viral loads in the groundwater and surface water over time and at the submetropolitan level generally reflected the reported trends in infection cases for Monterrey. The viral loads in the freshwater environments of Monterrey represent a low risk for recreational activities according to a preliminary risk assessment model. However, this result should not be taken lightly due to uncertainty regarding data and model constraints and the possibility of situations where the infection risk may increase considerably.

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Topics: Viral load (54%), Surface water (52%)

11 Citations


Open accessPosted ContentDOI: 10.1101/2021.03.16.21253652
Aaron Bivins1, Devin North1, Zhenyu Wu1, Marlee Shaffer1  +2 moreInstitutions (2)
24 Mar 2021-medRxiv
Abstract: Wastewater surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA is being used to monitor Coronavirus Disease 2019 (COVID-19) trends in communities; however, within-day variation in primary influent concentrations of SARS-CoV-2 RNA remain largely uncharacterized. In the current study, grab sampling of primary influent was performed every 2 hours over two different 24-hour periods at two wastewater treatment plants (WWTPs) in northern Indiana, USA. In primary influent, uncorrected, recovery-corrected, and pepper mild mottle virus (PMMoV)-normalized SARS-CoV-2 RNA concentrations demonstrated ordinal agreement with increasing clinical COVID-19 positivity, but not COVID-19 cases. Primary influent SARS-CoV-2 RNA concentrations exhibited greater variation than PMMoV RNA concentrations as expected for lower shedding prevalence. The bovine respiratory syncytial virus (BRSV) process control recovery efficiency was low (mean: 0.91%) and highly variable (coefficient of variation: 51% - 206%) over the four sampling events with significant differences between the two WWTPs (p <0.0001). The process control recovery was similar to the independently assessed SARS-CoV-2 RNA recovery efficiency, which was also significantly different between the two WWTPs (p <0.0001). Recovery-corrected SARS-CoV-2 RNA concentrations better reflected within-day changes in primary influent flow rate and fecal content, as indicated by PMMoV concentrations. These observations highlight the importance of assessing the process recovery efficiency, which is highly variable, using an appropriate process control. Despite large variations, both recovery-corrected and PMMoV-normalized SARS-CoV-2 RNA concentrations in primary influent demonstrate potential for monitoring COVID-19 positivity trends in WWTPs serving peri-urban and rural areas.

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9 Citations


Open accessPosted ContentDOI: 10.1101/2021.03.25.21254344
26 Mar 2021-medRxiv
Abstract: Wastewater-based epidemiology (WBE) has been shown to coincide with, or anticipate, confirmed COVID-19 case numbers. During periods with high test positivity rates, however, case numbers may be underreported, whereas wastewater does not suffer from this limitation. Here we investigated how the dynamics of new COVID-19 infections estimated based on wastewater monitoring or confirmed cases compare to true COVID-19 incidence dynamics. We focused on the first pandemic wave in Switzerland (February to April, 2020), when test positivity ranged up to 26%. SARS-CoV-2 RNA loads were determined 2-4 times per week in three Swiss wastewater treatment plants (Lugano, Lausanne and Zurich). Wastewater and case data were combined with a shedding load distribution and an infection-to-case confirmation delay distribution, respectively, to estimate incidence dynamics. Finally, the estimates were compared to reference incidence dynamics determined by a validated compartmental model. Incidence dynamics estimated based on wastewater data were found to better track the timing and shape of the reference infection peak compared to estimates based on confirmed cases. In contrast, case confirmations provided a better estimate of the subsequent decline in infections. Under a regime of high-test positivity rates, WBE thus provides critical information that is complementary to clinical data to monitor the pandemic trajectory.

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8 Citations


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48 results found


Open accessJournal ArticleDOI: 10.1373/CLINCHEM.2008.112797
01 Apr 2009-Clinical Chemistry
Abstract: Background: Currently, a lack of consensus exists on how best to perform and interpret quantitative real-time PCR (qPCR) experiments. The problem is exacerbated by a lack of sufficient experimental detail in many publications, which impedes a reader’s ability to evaluate critically the quality of the results presented or to repeat the experiments. Content: The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines target the reliability of results to help ensure the integrity of the scientific literature, promote consistency between laboratories, and increase experimental transparency. MIQE is a set of guidelines that describe the minimum information necessary for evaluating qPCR experiments. Included is a checklist to accompany the initial submission of a manuscript to the publisher. By providing all relevant experimental conditions and assay characteristics, reviewers can assess the validity of the protocols used. Full disclosure of all reagents, sequences, and analysis methods is necessary to enable other investigators to reproduce results. MIQE details should be published either in abbreviated form or as an online supplement. Summary: Following these guidelines will encourage better experimental practice, allowing more reliable and unequivocal interpretation of qPCR results.

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10,655 Citations


Open accessJournal ArticleDOI: 10.1038/SJ.GENE.6364190
Jim F. Huggett1, Keertan Dheda1, Keertan Dheda2, Stephen A. Bustin3  +1 moreInstitutions (3)
01 Jun 2005-Genes and Immunity
Abstract: Real-time RT-PCR has become a common technique, no longer limited to specialist core facilities. It is in many cases the only method for measuring mRNA levels of vivo low copy number targets of interest for which alternative assays either do not exist or lack the required sensitivity. Benefits of this procedure over conventional methods for measuring RNA include its sensitivity, large dynamic range, the potential for high throughout as well as accurate quantification. To achieve this, however, appropriate normalisation strategies are required to control for experimental error introduced during the multistage process required to extract and process the RNA. There are many strategies that can be chosen; these include normalisation to sample size, total RNA and the popular practice of measuring an internal reference or housekeeping gene. However, these methods are frequently applied without appropriate validation. In this review we discuss the relative merits of different normalisation strategies and suggest a method of validation that will enable the measurement of biologically meaningful results.

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1,665 Citations


Open accessJournal ArticleDOI: 10.1111/J.1365-2672.2012.05384.X
Abstract: The polymerase chain reaction (PCR) is increasingly used as the standard method for detection and characterization of microorganisms and genetic markers in a variety of sample types. However, the method is prone to inhibiting substances, which may be present in the analysed sample and which may affect the sensitivity of the assay or even lead to false-negative results. The PCR inhibitors represent a diverse group of substances with different properties and mechanisms of action. Some of them are predominantly found in specific types of samples thus necessitating matrix-specific protocols for preparation of nucleic acids before PCR. A variety of protocols have been developed to remove the PCR inhibitors. This review focuses on the general properties of PCR inhibitors and their occurrence in specific matrices. Strategies for their removal from the sample and for quality control by assessing their influence on the individual PCR test are presented and discussed.

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1,079 Citations


Open accessJournal ArticleDOI: 10.1016/J.SCITOTENV.2020.138764
Warish Ahmed1, Nicola Angel2, Janette Edson2, Kyle Bibby3  +14 moreInstitutions (4)
Abstract: Infection with SARS-CoV-2, the etiologic agent of the ongoing COVID-19 pandemic, is accompanied by the shedding of the virus in stool. Therefore, the quantification of SARS-CoV-2 in wastewater affords the ability to monitor the prevalence of infections among the population via wastewater-based epidemiology (WBE). In the current work, SARS-CoV-2 RNA was concentrated from wastewater in a catchment in Australia and viral RNA copies were enumerated using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) resulting in two positive detections within a six day period from the same wastewater treatment plant (WWTP). The estimated viral RNA copy numbers observed in the wastewater were then used to estimate the number of infected individuals in the catchment via Monte Carlo simulation. Given the uncertainty and variation in the input parameters, the model estimated a median range of 171 to 1,090 infected persons in the catchment, which is in reasonable agreement with clinical observations. This work highlights the viability of WBE for monitoring infectious diseases, such as COVID-19, in communities. The work also draws attention to the need for further methodological and molecular assay validation for enveloped viruses in wastewater.

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Topics: Population (52%), Wastewater (51%)

735 Citations


Open accessJournal ArticleDOI: 10.1016/J.IJANTIMICAG.2020.105951
Heng Li1, Shang-Ming Liu1, Xiao-Hua Yu2, Shi-Lin Tang1  +1 moreInstitutions (2)
Abstract: Coronavirus disease 2019 (COVID-19) originated in the city of Wuhan, Hubei Province, Central China, and has spread quickly to 72 countries to date. COVID-19 is caused by a novel coronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [previously provisionally known as 2019 novel coronavirus (2019-nCoV)]. At present, the newly identified SARS-CoV-2 has caused a large number of deaths with tens of thousands of confirmed cases worldwide, posing a serious threat to public health. However, there are no clinically approved vaccines or specific therapeutic drugs available for COVID-19. Intensive research on the newly emerged SARS-CoV-2 is urgently needed to elucidate the pathogenic mechanisms and epidemiological characteristics and to identify potential drug targets, which will contribute to the development of effective prevention and treatment strategies. Hence, this review will focus on recent progress regarding the structure of SARS-CoV-2 and the characteristics of COVID-19, such as the aetiology, pathogenesis and epidemiological characteristics.

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Topics: Coronavirus (57%)

513 Citations