Rapid Assessment of SARS-CoV-2 Variant-Associated Mutations in Wastewater Using Real-Time RT-PCR

Kata Farkas; Cameron Pellett; Rachel Williams; Natasha Alex-Sanders; Irene Bassano; Mathew R. Brown; Hubert Denise; Jasmine M.S. Grimsley; Jessica L. Kevill; Mohammad S. Khalifa; Igor Pântea; Rich Story; Matthew J. Wade; Nick Woodhall; Davey L. Jones

doi:10.1128/spectrum.03177-22

Rapid Assessment of SARS-CoV-2 Variant-Associated Mutations in Wastewater Using Real-Time RT-PCR

Kata Farkas^*, Cameron Pellett, Rachel Williams, Natasha Alex-Sanders, Irene Bassano, Mathew R. Brown, Hubert Denise, Jasmine M.S. Grimsley, Jessica L. Kevill, Mohammad S. Khalifa, Igor Pântea, Rich Story, Matthew J. Wade, Nick Woodhall, Davey L. Jones

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

6 Citations (Scopus)

Abstract

Within months of the COVID-19 pandemic being declared on March 20, 2020, novel, more infectious variants of SARS-CoV-2 began to be detected in geospatially distinct regions of the world. With international travel being a lead cause of spread of the disease, the importance of rapidly identifying variants entering a country is critical. In this study, we utilized wastewater-based epidemiology (WBE) to monitor the presence of variants in wastewater generated in managed COVID-19 quarantine facilities for international air passengers entering the United Kingdom. Specifically, we developed multiplex reverse transcription quantitative PCR (RT-qPCR) assays for the identification of defining mutations associated with Beta (K417N), Gamma (K417T), Delta (156/157DEL), and Kappa (E154K) variants which were globally prevalent at the time of sampling (April to July 2021). The assays sporadically detected mutations associated with the Beta, Gamma, and Kappa variants in 0.7%, 2.3%, and 0.4% of all samples, respectively. The Delta variant was identified in 13.3% of samples, with peak detection rates and concentrations observed in May 2021 (24%), concurrent with its emergence in the United Kingdom. The RT-qPCR results correlated well with those from sequencing, suggesting that PCR-based detection is a good predictor for variant presence; although, inadequate probe binding may lead to false positive or negative results. Our findings suggest that WBE coupled with RT-qPCR may be used as a rapid, initial assessment to identify emerging variants at international borders and mass quarantining facilities. IMPORTANCE With the global spread of COVID-19, it is essential to identify emerging variants which may be more harmful or able to escape vaccines rapidly. To date, the gold standard to assess variants circulating in communities has been the sequencing of the S gene or the whole genome of SARS-CoV-2; however, that approach is time-consuming and expensive. In this study, we developed two duplex RT-qPCR assays to detect and quantify defining mutations associated with the Beta, Gamma, Delta, and Kappa variants. The assays were validated using RNA extracts derived from wastewater samples taken at quarantine facilities. The results showed good correlation with the results of sequencing and demonstrated the emergence of the Delta variant in the United Kingdom in May 2021. The assays developed here enable the assessment of variant-specific mutations within 2 h after the RNA extract was generated which is essential for outbreak rapid response.

Original language	English
Journal	Microbiology Spectrum
Volume	11
Issue number	1
DOIs	https://doi.org/10.1128/spectrum.03177-22
Publication status	Published - Jan 2023

Bibliographical note

Funding Information:
The work was supported by the Environmental Monitoring for Health Protection Program using funds provided by the United Kingdom Department of Health and Social Care. The funder was involved in sampling design and organized sampling through external partners. We thank Daphne Beniston and Andrew Singer (Accelerated Capability Environment, Homeland Security, UK) for their help contribution organizing the project. We also thank Richard Stanton (Cardiff University, UK) for providing the inactivated SARS-CoV-2 virus stocks. We declare no conflict of interest.

Funding Information:
The work was supported by the Environmental Monitoring for Health Protection Program using funds provided by the United Kingdom Department of Health and Social Care. The funder was involved in sampling design and organized sampling through external partners. We thank Daphne Beniston and Andrew Singer (Accelerated Capability Environment, Homeland Security, UK) for their help contribution organizing the project. We also thank Richard Stanton (Cardiff University, UK) for providing the inactivated SARSCoV-2 virus stocks. We declare no conflict of interest. Kata Farkas: Conceptualization, Methodology, Investigation, Formal analysis, Writing & editing, Funding acquisition, Supervision. Cameron Pellett: Formal analysis, Writing & editing. Rachel Williams: Conceptualization, Methodology, Formal analysis, Writing & editing, Supervision. Natasha Alex-Sanders: Investigation, Writing & editing. Irene Bassano: Methodology, Investigation, Formal analysis, Writing & editing. Mathew Brown: Methodology, Investigation, Formal analysis, Writing & editing. Hubert Denise: Formal analysis, Writing & editing. Jasmine M.S. Grimsley: Conceptualization, Writing & editing, Funding acquisition. Jessica Kevill: Conceptualization, Methodology, Writing & editing. Mohammad Khalifa: Formal analysis, Writing & editing. Igor Pântea: Methodology, Writing & editing. Rich Story: Conceptualization, Writing & editing. Matthew J. Wade: Conceptualization, Writing & editing, Funding acquisition. Nick Woodhall: Methodology, Investigation, Writing & editing. Davey L. Jones: Conceptualization,Methodology,Writing & editing, Funding acquisition, Supervision.

Publisher Copyright:
© 2023 Farkas et al.

Keywords

Digital PCR
airport sewage surveillance
human health risk
quarantine hotel monitoring
single nucleotide polymorphism
variant of concern

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

Access to Document

10.1128/spectrum.03177-22

Cite this

Farkas, K., Pellett, C., Williams, R., Alex-Sanders, N., Bassano, I., Brown, M. R., Denise, H., Grimsley, J. M. S., Kevill, J. L., Khalifa, M. S., Pântea, I., Story, R., Wade, M. J., Woodhall, N., & Jones, D. L. (2023). Rapid Assessment of SARS-CoV-2 Variant-Associated Mutations in Wastewater Using Real-Time RT-PCR. Microbiology Spectrum, 11(1). https://doi.org/10.1128/spectrum.03177-22

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title = "Rapid Assessment of SARS-CoV-2 Variant-Associated Mutations in Wastewater Using Real-Time RT-PCR",

abstract = "Within months of the COVID-19 pandemic being declared on March 20, 2020, novel, more infectious variants of SARS-CoV-2 began to be detected in geospatially distinct regions of the world. With international travel being a lead cause of spread of the disease, the importance of rapidly identifying variants entering a country is critical. In this study, we utilized wastewater-based epidemiology (WBE) to monitor the presence of variants in wastewater generated in managed COVID-19 quarantine facilities for international air passengers entering the United Kingdom. Specifically, we developed multiplex reverse transcription quantitative PCR (RT-qPCR) assays for the identification of defining mutations associated with Beta (K417N), Gamma (K417T), Delta (156/157DEL), and Kappa (E154K) variants which were globally prevalent at the time of sampling (April to July 2021). The assays sporadically detected mutations associated with the Beta, Gamma, and Kappa variants in 0.7%, 2.3%, and 0.4% of all samples, respectively. The Delta variant was identified in 13.3% of samples, with peak detection rates and concentrations observed in May 2021 (24%), concurrent with its emergence in the United Kingdom. The RT-qPCR results correlated well with those from sequencing, suggesting that PCR-based detection is a good predictor for variant presence; although, inadequate probe binding may lead to false positive or negative results. Our findings suggest that WBE coupled with RT-qPCR may be used as a rapid, initial assessment to identify emerging variants at international borders and mass quarantining facilities. IMPORTANCE With the global spread of COVID-19, it is essential to identify emerging variants which may be more harmful or able to escape vaccines rapidly. To date, the gold standard to assess variants circulating in communities has been the sequencing of the S gene or the whole genome of SARS-CoV-2; however, that approach is time-consuming and expensive. In this study, we developed two duplex RT-qPCR assays to detect and quantify defining mutations associated with the Beta, Gamma, Delta, and Kappa variants. The assays were validated using RNA extracts derived from wastewater samples taken at quarantine facilities. The results showed good correlation with the results of sequencing and demonstrated the emergence of the Delta variant in the United Kingdom in May 2021. The assays developed here enable the assessment of variant-specific mutations within 2 h after the RNA extract was generated which is essential for outbreak rapid response.",

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note = "Funding Information: The work was supported by the Environmental Monitoring for Health Protection Program using funds provided by the United Kingdom Department of Health and Social Care. The funder was involved in sampling design and organized sampling through external partners. We thank Daphne Beniston and Andrew Singer (Accelerated Capability Environment, Homeland Security, UK) for their help contribution organizing the project. We also thank Richard Stanton (Cardiff University, UK) for providing the inactivated SARS-CoV-2 virus stocks. We declare no conflict of interest. Funding Information: The work was supported by the Environmental Monitoring for Health Protection Program using funds provided by the United Kingdom Department of Health and Social Care. The funder was involved in sampling design and organized sampling through external partners. We thank Daphne Beniston and Andrew Singer (Accelerated Capability Environment, Homeland Security, UK) for their help contribution organizing the project. We also thank Richard Stanton (Cardiff University, UK) for providing the inactivated SARSCoV-2 virus stocks. We declare no conflict of interest. Kata Farkas: Conceptualization, Methodology, Investigation, Formal analysis, Writing & editing, Funding acquisition, Supervision. Cameron Pellett: Formal analysis, Writing & editing. Rachel Williams: Conceptualization, Methodology, Formal analysis, Writing & editing, Supervision. Natasha Alex-Sanders: Investigation, Writing & editing. Irene Bassano: Methodology, Investigation, Formal analysis, Writing & editing. Mathew Brown: Methodology, Investigation, Formal analysis, Writing & editing. Hubert Denise: Formal analysis, Writing & editing. Jasmine M.S. Grimsley: Conceptualization, Writing & editing, Funding acquisition. Jessica Kevill: Conceptualization, Methodology, Writing & editing. Mohammad Khalifa: Formal analysis, Writing & editing. Igor P{\^a}ntea: Methodology, Writing & editing. Rich Story: Conceptualization, Writing & editing. Matthew J. Wade: Conceptualization, Writing & editing, Funding acquisition. Nick Woodhall: Methodology, Investigation, Writing & editing. Davey L. Jones: Conceptualization,Methodology,Writing & editing, Funding acquisition, Supervision. Publisher Copyright: {\textcopyright} 2023 Farkas et al.",

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AU - Brown, Mathew R.

AU - Denise, Hubert

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N2 - Within months of the COVID-19 pandemic being declared on March 20, 2020, novel, more infectious variants of SARS-CoV-2 began to be detected in geospatially distinct regions of the world. With international travel being a lead cause of spread of the disease, the importance of rapidly identifying variants entering a country is critical. In this study, we utilized wastewater-based epidemiology (WBE) to monitor the presence of variants in wastewater generated in managed COVID-19 quarantine facilities for international air passengers entering the United Kingdom. Specifically, we developed multiplex reverse transcription quantitative PCR (RT-qPCR) assays for the identification of defining mutations associated with Beta (K417N), Gamma (K417T), Delta (156/157DEL), and Kappa (E154K) variants which were globally prevalent at the time of sampling (April to July 2021). The assays sporadically detected mutations associated with the Beta, Gamma, and Kappa variants in 0.7%, 2.3%, and 0.4% of all samples, respectively. The Delta variant was identified in 13.3% of samples, with peak detection rates and concentrations observed in May 2021 (24%), concurrent with its emergence in the United Kingdom. The RT-qPCR results correlated well with those from sequencing, suggesting that PCR-based detection is a good predictor for variant presence; although, inadequate probe binding may lead to false positive or negative results. Our findings suggest that WBE coupled with RT-qPCR may be used as a rapid, initial assessment to identify emerging variants at international borders and mass quarantining facilities. IMPORTANCE With the global spread of COVID-19, it is essential to identify emerging variants which may be more harmful or able to escape vaccines rapidly. To date, the gold standard to assess variants circulating in communities has been the sequencing of the S gene or the whole genome of SARS-CoV-2; however, that approach is time-consuming and expensive. In this study, we developed two duplex RT-qPCR assays to detect and quantify defining mutations associated with the Beta, Gamma, Delta, and Kappa variants. The assays were validated using RNA extracts derived from wastewater samples taken at quarantine facilities. The results showed good correlation with the results of sequencing and demonstrated the emergence of the Delta variant in the United Kingdom in May 2021. The assays developed here enable the assessment of variant-specific mutations within 2 h after the RNA extract was generated which is essential for outbreak rapid response.

AB - Within months of the COVID-19 pandemic being declared on March 20, 2020, novel, more infectious variants of SARS-CoV-2 began to be detected in geospatially distinct regions of the world. With international travel being a lead cause of spread of the disease, the importance of rapidly identifying variants entering a country is critical. In this study, we utilized wastewater-based epidemiology (WBE) to monitor the presence of variants in wastewater generated in managed COVID-19 quarantine facilities for international air passengers entering the United Kingdom. Specifically, we developed multiplex reverse transcription quantitative PCR (RT-qPCR) assays for the identification of defining mutations associated with Beta (K417N), Gamma (K417T), Delta (156/157DEL), and Kappa (E154K) variants which were globally prevalent at the time of sampling (April to July 2021). The assays sporadically detected mutations associated with the Beta, Gamma, and Kappa variants in 0.7%, 2.3%, and 0.4% of all samples, respectively. The Delta variant was identified in 13.3% of samples, with peak detection rates and concentrations observed in May 2021 (24%), concurrent with its emergence in the United Kingdom. The RT-qPCR results correlated well with those from sequencing, suggesting that PCR-based detection is a good predictor for variant presence; although, inadequate probe binding may lead to false positive or negative results. Our findings suggest that WBE coupled with RT-qPCR may be used as a rapid, initial assessment to identify emerging variants at international borders and mass quarantining facilities. IMPORTANCE With the global spread of COVID-19, it is essential to identify emerging variants which may be more harmful or able to escape vaccines rapidly. To date, the gold standard to assess variants circulating in communities has been the sequencing of the S gene or the whole genome of SARS-CoV-2; however, that approach is time-consuming and expensive. In this study, we developed two duplex RT-qPCR assays to detect and quantify defining mutations associated with the Beta, Gamma, Delta, and Kappa variants. The assays were validated using RNA extracts derived from wastewater samples taken at quarantine facilities. The results showed good correlation with the results of sequencing and demonstrated the emergence of the Delta variant in the United Kingdom in May 2021. The assays developed here enable the assessment of variant-specific mutations within 2 h after the RNA extract was generated which is essential for outbreak rapid response.

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KW - airport sewage surveillance

KW - human health risk

KW - quarantine hotel monitoring

KW - single nucleotide polymorphism

KW - variant of concern

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Rapid Assessment of SARS-CoV-2 Variant-Associated Mutations in Wastewater Using Real-Time RT-PCR

Abstract

Bibliographical note

Keywords

UN SDGs

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