Monitoring the propagation of SARS CoV2 variants by tracking identified mutation in wastewater using specific RT-qPCR

Wurtzer, Sébastien; Waldman, Prunelle; Levert, Morgane; Mouchel, JM; Gorgé, Olivier; Boni, Mickaël; Maday, Yvon; Maréchal, Vincent; Moulin, Laurent

doi:10.1101/2021.03.10.21253291

Cited by 20 publications

(24 citation statements)

References 30 publications

(13 reference statements)

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“…Besides testing the untreated wastewater, we also quantified SARS-CoV-2 RNA in 8 samples of treated wastewater (four sampling weeks from both WWTP), which gave negative results for both plants (data not shown). This is in contrast with some recent studies that reported the detection of SARS-CoV-2 RNA by RT-qPCR in treated wastewaters and even in rivers (Nasseri et al, 2021;Rimoldi et al, 2020;Wurtzer et al, 2021Wurtzer et al, , 2020, although near the quantification limits. These findings could be important to indicate if the wastewater treatment technologies are being efficient in the removal of SARS-CoV-2 and could provide a comprehensive view on the fate of SARS-CoV-2 along the entire chain of WWTPs.…”

Section: Sars-cov-2 Detection In Liquid and Solid Wastewater Fractionscontrasting

confidence: 99%

Continuous monitoring of SARS-CoV-2 RNA in urban wastewater from Porto, Portugal: sampling and analysis protocols

Tomasino

Semedo

Vieira

et al. 2021

Preprint

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Research on the emerging COVID-19 pandemic is demonstrating that wastewater infrastructures can be used as public health observatories of virus circulation in human communities. Important efforts are being organized worldwide to implement sewage-based surveillance of SARS-CoV-2 that can be used for preventive or early warning purposes, informing preparedness and response measures. However, its successful implementation requires important and iterative methodological improvements, as well as the establishment of standardized methods. The aim of this study was to develop a continuous monitoring protocol for SARS-CoV-2 in wastewater, that could be used to model virus circulation within the communities, complementing the current clinical surveillance. Specific objectives included (1) optimization and validation of a sensitive method for virus quantification; (2) monitoring the time-evolution of SARS-CoV-2 in wastewater from two wastewater treatment plants (WWTPs) in the city of Porto, Portugal. Untreated wastewater samples were collected weekly from the two WWTPs between May 2020 and March 2021, encompassing two COVID-19 incidence peaks in the region (mid-November 2020 and mid-January 2021). In the first stage of this study, we compared, optimized and selected a sampling and analysis protocol that included RNA virus concentration through centrifugation, RNA extraction from both liquid and solid fractions and quantification by reverse transcription quantitative PCR (RT-qPCR). In the second stage, we used the selected methodology to track SARS-CoV-2 in the collected wastewater over time. SARS-CoV-2 RNA was detected in 39 and 37 out of 48 liquid and solid fraction samples of untreated wastewater, respectively. The copy numbers varied throughout the study between 0 and 0.15 copies/ng RNA and a good fit was observed between the SARS-CoV-2 RNA concentration in the untreated wastewater and the COVID-19 temporal trends in the study region. In agreement with the recent literature, the results from this study support the use of wastewater-based surveillance to complement clinical testing and evaluate temporal and spatial trends of the current pandemic.

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Section: Sars-cov-2 Detection In Liquid and Solid Wastewater Fractionscontrasting

confidence: 99%

Continuous monitoring of SARS-CoV-2 RNA in urban wastewater from Porto, Portugal: sampling and analysis protocols

Tomasino

Semedo

Vieira

et al. 2021

Preprint

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“…Most of these groups developed assays targeting only a single site for detection of each VOC lineage. They reported substantially lower sensitivity for their assays in comparison to the US CDC N1 and N2 assays and had poor amplification efficiencies (Graber et al, 2021, Wurtzer et al, 2021, Yaniv et al, 2021. A number of biomedical companies have also released products for the detection of SARS-CoV-2 VOCs in wastewater, but have not divulged additional details on their methods.…”

Section: Discussionmentioning

confidence: 99%

Quantitative detection of SARS-CoV-2 B.1.1.7 variant in wastewater by allele-specific RT-qPCR

Lee

McElroy²,

Armas

et al. 2021

Preprint

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Wastewater-based epidemiology (WBE) has emerged as a critical public health tool in tracking the SARS-CoV-2 epidemic. Monitoring SARS-CoV-2 variants of concern in wastewater has to-date relied on genomic sequencing, which lacks sensitivity necessary to detect low variant abundances in diluted and mixed wastewater samples. Here, we develop and present an open-source method based on allele specific RT-qPCR (AS RT-qPCR) that detects and quantifies the B.1.1.7 variant, targeting spike protein mutations at three independent genomic loci highly predictive of B.1.1.7 (HV69/70del, Y144del, and A570D). Our assays can reliably detect and quantify low levels of B.1.1.7 with low cross-reactivity, and at variant proportions between 0.1% and 1% in a background of mixed SARS-CoV-2. Applying our method to wastewater samples from the United States, we track B.1.1.7 occurrence over time in 19 communities. AS RT-qPCR results align with clinical trends, and summation of B.1.1.7 and wild-type sequences quantified by our assays strongly correlate with SARS-CoV-2 levels indicated by the US CDC N1/N2 assay. This work paves the path for rapid inexpensive surveillance of B.1.1.7 and other SARS-CoV-2 variants in wastewater.

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“…This method, constrained by the same bottlenecks of cost and infrastructure requirement as clinical sequencing, is further hampered by challenges in detection of low-frequency variants and is poorly quantitative due to the lack of robust modelling to quantify variant titers (Fuqua et al, 2021;Van Poelvoorde et al, 2021) . RT-qPCR-based methods have been developed for variant identification in clinical samples (Clark et al, 2021;Vogels et al, 2021;Wang et al, 2021) Yaniv et al, 2021aYaniv et al, , 2021bWurtzer et al, 2021 ). While RT-qPCR methods cannot discover new variants or identify variants beyond what they are designed for, they are more sensitive and quantitative, providing a readout from sample to data, in just hours.…”

Section: Introductionmentioning

confidence: 99%

“…RT-qPCR-based methods have been developed for variant identification in clinical samples (Clark et al, 2021;Vogels et al, 2021;Wang et al, 2021) but are not yet validated for quantification of variant mixtures, which are expected in wastewater samples. This motivates the development of specialized methods for quantification of variant mixtures in environmental samples such as wastewater (Graber et al, 2021;Yaniv et al, 2021aYaniv et al, , 2021bWurtzer et al, 2021 ). While RT-qPCR methods cannot discover new variants or identify variants beyond what they are designed for, they are more sensitive and quantitative, providing a readout from sample to data, in just hours.…”

Section: Introductionmentioning

confidence: 99%

Quantitative SARS-CoV-2 tracking of variants Delta, Delta plus, Kappa and Beta in wastewater by allele-specific RT-qPCR

Lee

Armas

et al. 2021

Preprint

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The Delta (B.1.617.2) variant has caused major devastation in India and other countries around the world. First detected in October 2020, it has now spread to more than 100 countries, prompting WHO to declare it as a global variant of concern (VOC). The Delta (B.1.617.2), Delta plus (B.1.617.2.1) and Kappa (B.1.617.1) variants are all sub-lineages of the original B.1.617 variant. Prior to the inception of B.1.617, vaccine rollout, safe-distancing and timely lockdowns greatly reduced COVID-19 hospitalizations and deaths. However, the Delta variant, allegedly more infectious and for which existing vaccines seemed less effective, has catalyzed the resurgence of cases. Therefore, there is an imperative need for increased surveillance of the B.1.617 variants. While the Beta variant is increasingly outpaced by the Delta variant, the spread of the Beta variant remains of concern due to its vaccine resistance. Efforts have been made to utilize wastewater-based surveillance for community-based tracking of SARS-CoV-2 variants, however wastewater with its low SARS-CoV-2 viral titers and mixtures of viral variants, requires assays to be variant-specific yet accurately quantitative for meaningful interpretation. Following on the design principles of our previous assays for the Alpha variant, here we report allele-specific and multiplex-compatible RT-qPCR assays targeting mutations T19R, D80A, K417N, T478K and E484Q, for quantitative detection and discrimination of the Delta, Delta plus, Kappa and Beta variants in wastewater. This method is open-sourced and can be implemented using commercially available RT-qPCR protocols, and would be an important tool for tracking the spread of B.1.617 and the Beta variants in communities.

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Monitoring the propagation of SARS CoV2 variants by tracking identified mutation in wastewater using specific RT-qPCR

Cited by 20 publications

References 30 publications

Continuous monitoring of SARS-CoV-2 RNA in urban wastewater from Porto, Portugal: sampling and analysis protocols

Continuous monitoring of SARS-CoV-2 RNA in urban wastewater from Porto, Portugal: sampling and analysis protocols

Quantitative detection of SARS-CoV-2 B.1.1.7 variant in wastewater by allele-specific RT-qPCR

Quantitative SARS-CoV-2 tracking of variants Delta, Delta plus, Kappa and Beta in wastewater by allele-specific RT-qPCR

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