SARS-CoV-2 Variants Detection Strategies in Wastewater Samples Collected in the Bangkok Metropolitan Region
Abstract
:1. Introduction
2. Materials and Methods
2.1. Wastewater Sample Collection
2.2. Wastewater Enrichment and RNA Extraction
2.3. SARS-CoV-2 Real-Time PCR Detection
2.4. Evaluation of the Wastewater RNA Extraction Protocol
2.5. SARS-CoV-2 Variants of Concern (VOC) Detection by Multiplex PCR MassARRAY (PMA)
2.6. Whole-Genome Sequencing by Next-Generation Sequencing (NGS)
2.7. Data Analysis
3. Results
3.1. Country’s Reported Case Number
3.2. Evaluation of RNA Extraction Protocol
3.3. Limit of Detection of the SARS-CoV-2 RNA Assay from a Wastewater Sample
3.4. SARS-CoV-2 PCR Results in Wastewater Samples from the Community
3.5. SARS-CoV-2 PCR Results in Wastewater Samples from the Hospital
3.6. Screening of SARS-CoV-2 Variants in Wastewater
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Huang, C.; Wang, Y.; Li, X.; Ren, L.; Zhao, J.; Hu, Y.; Zhang, L.; Fan, G.; Xu, J.; Gu, X.; et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet 2020, 395, 497–506. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Situation of COVID-19 Cases Updated Weekly. Available online: https://covid19.ddc.moph.go.th (accessed on 20 March 2023).
- Yorsaeng, R.; Suntronwong, N.; Thongpan, I.; Chuchaona, W.; Lestari, F.B.; Pasittungkul, S.; Puenpa, J.; Atsawawaranunt, K.; Sharma, C.; Sudhinaraset, N.; et al. The impact of COVID-19 and control measures on public health in Thailand, 2020. PeerJ 2022, 10, e12960. [Google Scholar] [CrossRef] [PubMed]
- Guidelines for Integrated Management of State Quarantine Areas. (Thai) Ver. 1.09., 3 June 2020. Available online: https://ddc.moph.go.th/viralpneumonia/file/g_quarantine/g_quarantine_state190663.pdf (accessed on 2 February 2023).
- Wölfel, R.; Corman, V.M.; Guggemos, W.; Seilmaier, M.; Zange, S.; Müller, M.A.; Niemeyer, D.; Jones, T.C.; Vollmar, P.; Rothe, C.; et al. Virological assessment of hospitalized patients with COVID-2019. Nature 2020, 581, 465–469. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Bai, Y.; Yao, L.; Wei, T.; Tian, F.; Jin, D.Y.; Chen, L.; Wang, M. Presumed Asymptomatic Carrier Transmission of COVID-19. JAMA 2020, 323, 1406–1407. [Google Scholar] [CrossRef] [Green Version]
- Rimoldi, S.G.; Stefani, F.; Gigantiello, A.; Polesello, S.; Comandatore, F.; Mileto, D.; Maresca, M.; Longobardi, C.; Mancon, A.; Romeri, F.; et al. Presence and infectivity of SARS-CoV-2 virus in wastewaters and rivers. Sci. Total Environ. 2020, 744, 140911. [Google Scholar] [CrossRef]
- Randazzo, W.; Truchado, P.; Cuevas-Ferrando, E.; Simón, P.; Allende, A.; Sánchez, G. SARS-CoV-2 RNA in wastewater anticipated COVID-19 occurrence in a low prevalence area. Water Res. 2020, 181, 115942. [Google Scholar] [CrossRef]
- Ahmed, W.; Angel, N.; Edson, J.; Bibby, K.; Bivins, A.; O’Brien, J.W.; Choi, P.M.; Kitajima, M.; Simpson, S.L.; Li, J.; et al. First confirmed detection of SARS-CoV-2 in untreated wastewater in Australia: A proof of concept for the wastewater surveillance of COVID-19 in the community. Sci. Total Environ. 2020, 728, 138764. [Google Scholar] [CrossRef]
- Wurtzer, S.; Marechal, V.; Mouchel, J.M.; Maday, Y.; Teyssou, R.; Richard, E.; Almayrac, J.L.; Moulin, L. Evaluation of lockdown effect on SARS-CoV-2 dynamics through viral genome quantification in waste water, Greater Paris, France, 5 March to 23 April 2020. Eurosurveillance 2020, 25, 2000776. [Google Scholar] [CrossRef]
- Arora, P.; Kempf, A.; Nehlmeier, I.; Schulz, S.R.; Jäck, H.M.; Pöhlmann, S.; Hoffmann, M. Omicron sublineage BQ.1.1 resistance to monoclonal antibodies. Lancet Infect. Dis. 2022, 23, 22–23. [Google Scholar] [CrossRef]
- Wolfe, M.; Hughes, B.; Duong, D.; Chan-Herur, V.; Wigginton, K.R.; White, B.J.; Boehm, A.B. Detection of SARS-CoV-2 Variants Mu, Beta, Gamma, Lambda, Delta, Alpha, and Omicron in Wastewater Settled Solids Using Mutation-Specific Assays Is Associated with Regional Detection of Variants in Clinical Samples. Appl. Environ. Microbiol. 2022, 88, e0004522. [Google Scholar] [CrossRef]
- Agrawal, S.; Orschler, L.; Schubert, S.; Zachmann, K.; Heijnen, L.; Tavazzi, S.; Gawlik, B.M.; de Graaf, M.; Medema, G.; Lackner, S. Prevalence and circulation patterns of SARS-CoV-2 variants in European sewage mirror clinical data of 54 European cities. Water Res. 2022, 214, 118162. [Google Scholar] [CrossRef] [PubMed]
- Yu, A.T.; Hughes, B.; Wolfe, M.K.; Leon, T.; Duong, D.; Rabe, A.; Kennedy, L.C.; Ravuri, S.; White, B.J.; Wigginton, K.R.; et al. Estimating Relative Abundance of 2 SARS-CoV-2 Variants through Wastewater Surveillance at 2 Large Metropolitan Sites, United States. Emerg. Infect. Dis. 2022, 28, 940–947. [Google Scholar] [CrossRef] [PubMed]
- Wurtzer, S.; Waldman, P.; Ferrier-Rembert, A.; Frenois-Veyrat, G.; Mouchel, J.M.; Boni, M.; Maday, Y.; OBEPINE consortium; Marechal, V.; Moulin, L. Several forms of SARS-CoV-2 RNA can be detected in wastewaters: Implication for wastewater-based epidemiology and risk assessment. Water Res. 2021, 198, 117183. [Google Scholar] [CrossRef] [PubMed]
- Langenfeld, K.; Chin, K.; Roy, A.; Wigginton, K.; Duhaime, M.B. Comparison of ultrafiltration and iron chloride flocculation in the preparation of aquatic viromes from contrasting sample types. PeerJ 2021, 9, e11111. [Google Scholar] [CrossRef]
- Heijnen, L.; Elsinga, G.; de Graaf, M.; Molenkamp, R.; Koopmans, M.P.G.; Medema, G. Droplet digital RT-PCR to detect SARS-CoV-2 signature mutations of variants of concern in wastewater. Sci. Total Environ. 2021, 799, 149456. [Google Scholar] [CrossRef]
- Wacharapluesadee, S.; Hirunpatrawong, P.; Petcharat, S.; Torvorapanit, P.; Jitsatja, A.; Thippamom, N.; Ninwattana, N.; Phanlop, C.; Buathong, R.; Tangwangvivat, R.; et al. Simultaneous Detection of Omicron and Other SARS-CoV-2 Variants by Multiplex PCR MassARRAY Technology. Sci. Rep. 2023, 13, 2089. [Google Scholar] [CrossRef]
- Farr, B.; Rajan, D.; Betteridge, E.; Shirley, L.; Quail, M.; Park, N.; Redshaw, N.; Bronner, I.F.; Aigrain, L.; Goodwin, S.; et al. DNA Pipelines R&D Protocol Citation: DNA Pipelines R&D. 2020. Available online: https://www.protocols.io/view/covid-19-artic-v3-illumina-library-construction-an-j8nlk4b36g5r/v1 (accessed on 2 November 2020).
- Li, H.; Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 2009, 25, 1754–1760. [Google Scholar] [CrossRef] [Green Version]
- Khare, S.; Gurry, C.; Freitas, L.; Schultz, M.B.; Bach, G.; Diallo, A.; Akite, N.; Ho, J.; Lee, R.T.; Yeo, W.; et al. GISAID’s Role in Pandemic Response. China CDC Wkly. 2021, 3, 1049–1051. [Google Scholar] [CrossRef]
- Okonechnikov, K.; Golosova, O.; Fursov, M.; UGENE team. Unipro UGENE: A unified bioinformatics toolkit. Bioinformatics 2012, 28, 1166–1167. [Google Scholar] [CrossRef] [Green Version]
- Tamáš, M.; Potocarova, A.; Konecna, B.; Klucar, Ľ.; Mackulak, T. Wastewater Sequencing-An Innovative Method for Variant Monitoring of SARS-CoV-2 in Populations. Int. J. Environ. Res. Public Health 2022, 19, 9749. [Google Scholar] [CrossRef]
- Expert Consultation on Public Health Needs Related to Surveillance of SARS-CoV-2 in Wastewater: Summary Report: Virtual Meeting, Copenhagen: WHO Regional Office for Europe. 2020. Available online: https://apps.who.int/iris/handle/10665/339487 (accessed on 30 November 2020).
- Wastewater Surveillance for COVID-19. Michigan: State of Michigan. Available online: www.michigan.gov/coronavirus/0,9753,7-406-98163_98173-545439--,00.html (accessed on 11 March 2022).
- Hart, O.E.; Halden, R.U. Computational analysis of SARSCoV-2/COVID-19 surveillance by wastewater-based epidemiology locally and globally: Feasibility, economy, opportunities and challenges. Sci. Total Environ. 2020, 730, 138875. [Google Scholar] [CrossRef] [PubMed]
- Zheng, X.; Deng, Y.; Xu, X.; Li, S.; Zhang, Y.; Ding, J.; On, H.Y.; Lai, J.C.C.; In Yau, C.; Chin, A.W.H.; et al. Comparison of virus concentration methods and RNA extraction methods for SARS-CoV-2 wastewater surveillance. Sci. Total Environ. 2022, 824, 153687. [Google Scholar] [CrossRef] [PubMed]
- Thongpradit, S.; Prasongtanakij, S.; Srisala, S.; Kumsang, Y.; Chanprasertyothin, S.; Boonkongchuen, P.; Pitidhammabhorn, D.; Manomaipiboon, P.; Somchaiyanon, P.; Chandanachulaka, S.; et al. A Simple Method to Detect SARS-CoV-2 in Wastewater at Low Virus Concentration. J. Environ. Public Health 2022, 2022, 4867626. [Google Scholar] [CrossRef] [PubMed]
- Banko, A.; Petrovic, G.; Miljanovic, D.; Loncar, A.; Vukcevic, M.; Despot, D.; Cirkovic, A. Comparison and Sensitivity Evaluation of Three Different Commercial Real-Time Quantitative PCR Kits for SARS-CoV-2 Detection. Viruses 2021, 13, 1321. [Google Scholar] [CrossRef] [PubMed]
- Jones, D.L.; Rhymes, J.M.; Wade, M.J.; Kevill, J.L.; Malham, S.K.; Grimsley, J.M.S.; Rimmer, C.; Weightman, A.J.; Farkas, K. Suitability of aircraft wastewater for pathogen detection and public health surveillance. Sci. Total Environ. 2023, 856, 159162. [Google Scholar] [CrossRef]
- Castro-Gutierrez, V.; Hassard, F.; Vu, M.; Leitao, R.; Burczynska, B.; Wildeboer, D.; Stanton, I.; Rahimzadeh, S.; Baio, G.; Garelick, H.; et al. Monitoring occurrence of SARS-CoV-2 in school populations: A wastewater-based approach. PLoS ONE 2022, 17, e0270168. [Google Scholar] [CrossRef]
- Lee, W.L.; Imakaev, M.; Armas, F.; McElroy, K.A.; Gu, X.; Duvallet, C.; Chandra, F.; Chen, H.; Leifels, M.; Mendola, S.; et al. Quantitative SARS-CoV-2 Alpha Variant, B.1.1.7 Tracking in Wastewater by Allele-Specific RT-qPCR. Environ. Sci. Technol. Lett. 2021, 8, 675–682. [Google Scholar] [CrossRef]
- Graber, T.E.; Mercier, É.; Bhatnagar, K.; Fuzzen, M.; D’Aoust, P.M.; Hoang, H.D.; Tian, X.; Towhid, S.T.; Plaza-Diaz, J.; Eid, W.; et al. Near real-time determination of B.1.1.7 in proportion to total SARS-CoV-2 viral load in wastewater using an allele-specific primer extension PCR strategy. Water Res. 2021, 205, 117681. [Google Scholar] [CrossRef]
- Yaniv, K.; Ozer, E.; Shagan, M.; Lakkakula, S.; Plotkin, N.; Bhandarkar, N.S.; Kushmaro, A. Direct RT-qPCR assay for SARS-CoV-2 variants of concern (Alpha, B.1.1.7 and Beta, B.1.351) detection and quantification in wastewater. Environ. Res. 2021, 201, 111653. [Google Scholar] [CrossRef]
- Zhao, F.; Lu, J.; Lu, B.; Qin, T.; Wang, X.; Hou, X.; Meng, F.; Xu, X.; Li, T.; Zhou, H.; et al. Novel Strategy for the Detection of SARS-CoV-2 Variants Based on Multiplex PCR-Mass Spectrometry Minisequencing Technology. Microbiol. Spectr. 2021, 9, e0126721. [Google Scholar] [CrossRef]
Site No. | Site Character | Samples Collection Period | Sampling Time (Frequency) [No. of Sample/Time] * | No. Tested Sample | No. Positive Sample | |
---|---|---|---|---|---|---|
Start Date | End Date | |||||
1 | Hotel A | 23 November 2020 | 22 March 2021 | 16 (weekly) [2] | 32 | 12 (1) |
2 | Hotel B | 19 January 2021 | 24 March 2021 | 10 (weekly) [3] | 30 | 11 (2) |
3 | Field hospital | 22 September 2021 | 27 October 2021 | 6 (weekly) [6] | 36 | 19 |
4 | Condominium | 12 July 2021 | 2 August 2021 | 4 (weekly) [4] | 16 | 16 |
5 | Market A | 12 July 2021 | 2 August 2021 | 4 (weekly) [1] | 4 | 4 |
6 | Market B | 14 March 2021 | 22 March 2021 | 2 (weekly) [5,6] | 11 | 6 |
7 | Factory A | 11 October 2021 | 9 November 2021 | 1 [1] | 7 | 1 |
8 | Factory B | 18 October 2021 | 18 October 2021 | 1 [1] | 1 | 0 |
9 | Factory C | 12 July 2021 | 2 August 2021 | 4 (weekly) [4] | 16 | 1 |
10 | Factory D | 12 July 2021 | 2 August 2021 | 4 (weekly) [3] | 12 | 9 |
11 | Construction Camp A | 25 November 2021 | 25 November 2021 | 1 [2] | 2 | 0 |
12 | Construction Camp B | 27 September 2021 | 27 September 2021 | 1 [2] | 2 | 2 |
13 | Hospital | 22 December 2021 | 23 February 2022 | 1–2, 14 sites [1] | 25 | 14 |
14 | Aircraft | 29 November 2021 | 21 December 2021 | 20 planes [1] | 20 | 7 |
TOTAL | 215 | 102 |
Virus Concentration | Direct Extraction PCR Ct Value * | Spike into Wastewater Sample PCR Ct Value * | ||||
---|---|---|---|---|---|---|
Copies/mL | E | ORF1ab | N | E | ORF1ab | N |
107 | 16.23 | 15.52 | 14.24 | 18.90 | 17.88 | 16.81 |
106 | 20.08 | 19.38 | 18.15 | 23.16 | 22.27 | 21.10 |
105 | 23.25 | 22.58 | 21.29 | 26.42 | 25.72 | 24.46 |
104 | 27.07 | 26.65 | 25.11 | 28.35 | 27.94 | 26.43 |
103 | 30.86 | 30.45 | 28.75 | 32.09 | 30.59 | 29.68 |
102 | 32.13 | 31.15 | 29.88 | - | - | - |
10 | - | - | - | - | - | - |
1 | - | - | - | - | - | - |
Total Viral Copies | LOD Copies/mL | PCR Ct Value * | |||
---|---|---|---|---|---|
E | ORF1ab | N | Internal Control | ||
2 × 106 | 4 × 104 | 18.90 | 17.88 | 16.81 | 18.53 |
2 × 105 | 4 × 103 | 23.16 | 22.27 | 21.10 | 18.73 |
2 × 104 | 4 × 102 | 26.42 | 25.72 | 24.46 | 18.64 |
2 × 103 | 40 | 28.35 | 27.94 | 26.43 | 18.56 |
2 × 102 | 4 | 32.09 | 30.59 | 29.68 | 18.89 |
20 | 0.4 | ND | ND | ND | 18.84 |
Sample No. | Collected Date | Collection Site’s Name/Building Function | PCR Results | PCR Ct Value | ||
---|---|---|---|---|---|---|
E | N | ORF1ab | ||||
A211098 | 24 December 2021 | C1.1A/non covid IPD + OPD | Not detected | - | - | - |
A211099 | 24 December 2021 | C1.1B/non covid IPD + OPD | Not detected | - | - | - |
PO22001 | 20 January 2022 | C1.1A/non covid IPD + OPD | Not detected | - | - | - |
PO22002 | 20 January 2022 | C1.1B/non covid IPD + OPD | Not detected | - | - | - |
PO22023 | 25 January 2022 | C1.2/non covid IPD + OPD | Not detected | - | - | - |
PO22005 | 20 January 2022 | C1.3/OR | Not detected | - | - | - |
PO22035 | 7 February 2022 | C1.3/OR | Not detected | - | - | - |
PO22006 | 20 January 2022 | C1.4/OPD | Detected | 31.94 | 30.41 | 29.33 |
PO22039 | 7 February 2022 | C1.4/ OPD | Detected | 31.358 | 29.857 | 30.78 |
PO22019 | 24 January 2022 | C1.5/OPD | Detected | 32.625 | 31.508 | 31.346 |
PO22043 | 23 February 2022 | C1.5/OPD | Detected | 30.6 | 28.68 | 30.18 |
PO22021 | 25 January 2022 | C1.6/OPD | Not detected | - | - | - |
PO22037 | 7 February 2022 | C1.7/COVID ward | Detected | 26.994 | 26.023 | 26.247 |
A211094 | 22 December 2021 | C1.8A/main treatment tank | Detected | 27.076 | 24.261 | 26.962 |
A211095 | 22 December 2021 | C1.8B/ main treatment tank | Detected | 32.882 | 31.167 | 33.869 |
PO22003 | 20 January 2022 | C2.1A/office building | Detected | 31.25 | 29.3 | 28.76 |
PO22004 | 20 January 2022 | C2.1B/office building | Detected | 34.11 | 31.83 | 33.09 |
PO22009 | 24 January 2022 | C2.1A/office building | Detected | 34.139 | 31.057 | 33.58 |
PO22007 | 20 January 2022 | C2.2/canteen | Detected | 31.44 | 32.68 | 32.89 |
PO22025 | 25 January 2022 | C2.2/canteen | Detected | 31.035 | 30.125 | 30.736 |
PO22011 | 24 January 2022 | C3.1/staff dormitory | Detected | 30.489 | 27.536 | 29.185 |
PO22013 | 24 January 2022 | C3.2/staff dormitory | Detected | 34.042 | 30.549 | 30.994 |
PO22015 | 24 January 2022 | C3.3/staff dormitory | Not detected | - | - | - |
PO22017 | 24 January 2022 | C3.4/staff dormitory | Not detected | - | - | - |
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Tangwangvivat, R.; Wacharapluesadee, S.; Pinyopornpanish, P.; Petcharat, S.; Hearn, S.M.; Thippamom, N.; Phiancharoen, C.; Hirunpatrawong, P.; Duangkaewkart, P.; Supataragul, A.; et al. SARS-CoV-2 Variants Detection Strategies in Wastewater Samples Collected in the Bangkok Metropolitan Region. Viruses 2023, 15, 876. https://doi.org/10.3390/v15040876
Tangwangvivat R, Wacharapluesadee S, Pinyopornpanish P, Petcharat S, Hearn SM, Thippamom N, Phiancharoen C, Hirunpatrawong P, Duangkaewkart P, Supataragul A, et al. SARS-CoV-2 Variants Detection Strategies in Wastewater Samples Collected in the Bangkok Metropolitan Region. Viruses. 2023; 15(4):876. https://doi.org/10.3390/v15040876
Chicago/Turabian StyleTangwangvivat, Ratanaporn, Supaporn Wacharapluesadee, Papassorn Pinyopornpanish, Sininat Petcharat, Suthida Muangnoicharoen Hearn, Nattakarn Thippamom, Chadaporn Phiancharoen, Piyapha Hirunpatrawong, Phattra Duangkaewkart, Ananporn Supataragul, and et al. 2023. "SARS-CoV-2 Variants Detection Strategies in Wastewater Samples Collected in the Bangkok Metropolitan Region" Viruses 15, no. 4: 876. https://doi.org/10.3390/v15040876