Multiplexed RT-qPCR Coupled with Whole-Genome Sequencing to Monitor a SARS-CoV-2 Omicron Variant of Concern in a Hospital Laboratory Setting in Latvia
Abstract
:1. Introduction
2. Materials and Methods
2.1. Setting
2.2. Detection of SARS-CoV-2 RNA
2.3. Detection of SARS-CoV-2 Variants by Multiplexed RT-qPCR
2.4. Whole-Genome Sequencing
2.5. Statistical Analysis
3. Results
3.1. Detection of SARS-CoV-2 Variants with Multiplexed RT-qPCR
3.2. Whole-Genome Sequencing
3.3. Validation of the Detection of the Three Targeted Omicron VOC Mutations ΔH69/V70, E484A, and N501Y) by Multiplexed RT-qPCR against WGS
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Carabelli, A.M.; Peacock, T.P.; Thorne, L.G.; Harvey, W.T.; Hughes, J.; de Silva, T.I.; Peacock, S.J.; Barclay, W.S.; de Silva, T.I.; Towers, G.J.; et al. SARS-CoV-2 Variant Biology: Immune Escape, Transmission and Fitness. Nat. Rev. Microbiol. 2023, 21, 162–177. [Google Scholar] [CrossRef] [PubMed]
- Brown, K.A.; Gubbay, J.; Hopkins, J.; Patel, S.; Buchan, S.A.; Daneman, N.; Goneau, L.W. S-Gene Target Failure as a Marker of Variant B.1.1.7 Among SARS-CoV-2 Isolates in the Greater Toronto Area, December 2020 to March 2021. JAMA 2021, 325, 2115–2116. [Google Scholar] [CrossRef]
- Volz, E.; Mishra, S.; Chand, M.; Barrett, J.C.; Johnson, R.; Geidelberg, L.; Hinsley, W.R.; Laydon, D.J.; Dabrera, G.; O’Toole, Á.; et al. Assessing Transmissibility of SARS-CoV-2 Lineage B.1.1.7 in England. Nature 2021, 593, 266–269. [Google Scholar] [CrossRef] [PubMed]
- Nörz, D.; Grunwald, M.; Tang, H.T.; Olearo, F.; Günther, T.; Robitaille, A.; Fischer, N.; Grundhoff, A.; Aepfelbacher, M.; Pfefferle, S.; et al. Rapid Automated Screening for SARS-CoV-2 B.1.617 Lineage Variants (Delta/Kappa) through a Versatile Toolset of qPCR-Based SNP Detection. Diagnostics 2021, 11, 1818. [Google Scholar] [CrossRef]
- Lai, E.; Kennedy, E.B.; Lozach, J.; Hayashibara, K.; Davis-Turak, J.; Becker, D.; Brzoska, P.; Cassens, T.; Diamond, E.; Gandhi, M.; et al. A Method for Variant Agnostic Detection of SARS-CoV-2, Rapid Monitoring of Circulating Variants, and Early Detection of Emergent Variants Such as Omicron. J. Clin. Microbiol. 2022, 60, e00342-22. [Google Scholar] [CrossRef]
- TaqMan SARS-CoV-2 Mutation Research Panel-LV. Available online: https://www.thermofisher.com/tr/en/home/clinical/clinical-genomics/pathogen-detection-solutions/real-time-pcr-research-solutions-sars-cov-2/mutation-panel.html (accessed on 20 February 2023).
- Seegene. More Advanced Solution for COVID-19 and Variants. Available online: https://www.seegene.com/advantages/complete_solution_for_the_covid_19_response (accessed on 1 June 2023).
- Berno, G.; Fabeni, L.; Matusali, G.; Gruber, C.E.M.; Rueca, M.; Giombini, E.; Garbuglia, A.R. SARS-CoV-2 Variants Identification: Overview of Molecular Existing Methods. Pathogens 2022, 11, 1058. [Google Scholar] [CrossRef] [PubMed]
- Greninger, A.L.; Dien Bard, J.; Colgrove, R.C.; Graf, E.H.; Hanson, K.E.; Hayden, M.K.; Humphries, R.M.; Lowe, C.F.; Miller, M.B.; Pillai, D.R.; et al. Clinical and Infection Prevention Applications of Severe Acute Respiratory Syndrome Coronavirus 2 Genoty**: An Infectious Diseases Society of America/American Society for Microbiology Consensus Review Document. Clin. Infect. Dis. 2022, 74, 1496–1502. [Google Scholar] [CrossRef]
- Walker, A.S.; Vihta, K.-D.; Gethings, O.; Pritchard, E.; Jones, J.; House, T.; Bell, I.; Bell, J.I.; Newton, J.N.; Farrar, J.; et al. Tracking the Emergence of SARS-CoV-2 Alpha Variant in the United Kingdom. N. Engl. J. Med. 2021, 385, 2582–2585. [Google Scholar] [CrossRef]
- McMillen, T.; Jani, K.; Robilotti, E.V.; Kamboj, M.; Babady, N.E. The Spike Gene Target Failure (SGTF) Genomic Signature Is Highly Accurate for the Identification of Alpha and Omicron SARS-CoV-2 Variants. Sci. Rep. 2022, 12, 18968. [Google Scholar] [CrossRef]
- Caputo, V.; Calvino, G.; Strafella, C.; Termine, A.; Fabrizio, C.; Trastulli, G.; Ingrascì, A.; Peconi, C.; Bardini, S.; Rossini, A.; et al. Tracking the Initial Diffusion of SARS-CoV-2 Omicron Variant in Italy by RT-PCR and Comparison with Alpha and Delta Variants Spreading. Diagnostics 2022, 12, 467. [Google Scholar] [CrossRef]
- Zelyas, N.; Pabbaraju, K.; Croxen, M.A.; Lynch, T.; Buss, E.; Murphy, S.A.; Shokoples, S.; Wong, A.; Kanji, J.N.; Tipples, G. Precision Response to the Rise of the SARS-CoV-2 B.1.1.7 Variant of Concern by Combining Novel PCR Assays and Genome Sequencing for Rapid Variant Detection and Surveillance. Microbiol. Spectr. 2021, 9, e00315-21. [Google Scholar] [CrossRef] [PubMed]
- Fu, J.Y.L.; Chong, Y.M.; Sam, I.-C.; Chan, Y.F. SARS-CoV-2 Multiplex RT-PCR to Detect Variants of Concern (VOCs) in Malaysia, between January to May 2021. J. Virol. Methods 2022, 301, 114462. [Google Scholar] [CrossRef] [PubMed]
- Kami, W.; Kinjo, T.; Arakaki, W.; Oki, H.; Motooka, D.; Nakamura, S.; Fujita, J. Rapid and Simultaneous Identification of Three Mutations by the NovaplexTM SARS-CoV-2 Variants I Assay Kit. J. Clin. Virol. 2021, 141, 104877. [Google Scholar] [CrossRef]
- Carpenter, R.E.; Tamrakar, V.; Chahar, H.; Vine, T.; Sharma, R. Confirming Multiplex RT-qPCR Use in COVID-19 with Next-Generation Sequencing: Strategies for Epidemiological Advantage. Glob. Health Epidemiol. Genom. 2022, 2022, 2270965. [Google Scholar] [CrossRef] [PubMed]
- Nielsen, M.C.; Machado, R.R.G.; Mitchell, B.M.; McConnell, A.J.; Saada, N.I.; Weaver, S.C.; Ren, P. A Comparison of Seegene Technologies Novaplex SARS-CoV-2 Variants I, II, and IV Assays with Spike Gene Sequencing for Detection of Known Severe Acute Respiratory Syndrome Coronavirus 2 Variants. J. Mol. Diagn. 2022, 24, 455–461. [Google Scholar] [CrossRef] [PubMed]
- Neopane, P.; Nypaver, J.; Shrestha, R.; Beqaj, S.S. SARS-CoV-2 Variants Detection Using TaqMan SARS-CoV-2 Mutation Panel Molecular Genoty** Assays. Infect. Drug Resist. 2021, 14, 4471–4479. [Google Scholar] [CrossRef]
- Vogels, C.B.F.; Breban, M.I.; Ott, I.M.; Alpert, T.; Petrone, M.E.; Watkins, A.E.; Kalinich, C.C.; Earnest, R.; Rothman, J.E.; de Jesus, J.G.; et al. Multiplex qPCR Discriminates Variants of Concern to Enhance Global Surveillance of SARS-CoV-2. PLoS Biol. 2021, 19, e3001236. [Google Scholar] [CrossRef]
- Ayadi, W.; Taktak, A.; Gargouri, S.; Smaoui, F.; Chtourou, A.; Skouri-Gargouri, H.; Derbel, R.; Sassi, A.H.; Gargouri, A.; Hammami, A.; et al. Development of a Simple Genoty** Method Based on Indel Mutations to Rapidly Screen SARS-CoV-2 Circulating Variants: Delta, Omicron BA.1 and BA.2. J. Virol. Methods 2022, 307, 114570. [Google Scholar] [CrossRef]
- Wilhelm, A.; Widera, M.; Grikscheit, K.; Toptan, T.; Schenk, B.; Pallas, C.; Metzler, M.; Kohmer, N.; Hoehl, S.; Marschalek, R.; et al. Limited Neutralisation of the SARS-CoV-2 Omicron Subvariants BA.1 and BA.2 by Convalescent and Vaccine Serum and Monoclonal Antibodies. eBioMedicine 2022, 82, 104158. [Google Scholar] [CrossRef]
- Planas, D.; Saunders, N.; Maes, P.; Guivel-Benhassine, F.; Planchais, C.; Buchrieser, J.; Bolland, W.-H.; Porrot, F.; Staropoli, I.; Lemoine, F.; et al. Considerable Escape of SARS-CoV-2 Omicron to Antibody Neutralization. Nature 2022, 602, 671–675. [Google Scholar] [CrossRef]
- Cao, Y.; Wang, J.; Jian, F.; ** Reverse Transcriptase PCR for Population-Level Variant Screening and Epidemiologic Surveillance. J. Clin. Microbiol. 2021, 59, e0085921. [Google Scholar] [CrossRef]
n | Detection of Targets, Multiplexed RT-qPCR | Presence of Targets, WGS | n | ||
---|---|---|---|---|---|
2 | ΔH69/V70, E484A, N501Y | Omicron, BA.1 | - | Delta, B.1.617.2 Delta, AY.121 | 1 1 |
5 | E484A, N501Y | Omicron, BA.2 | ΔH69/V70, E484A, N501Y ΔH69/V70, E484A, N501Y ΔH69/V70, E484A, N501Y | Omicron, BA.1.17.2 Omicron, BA.1.1 Omicron, BA.2 * | 1 2 2 |
1 | ΔH69/V70, N501Y | Other | ΔH69/V70, E484A, N501Y | Omicron, BA.1.17.2 | 1 |
18 | - | Delta | ΔH69/V70, E484A, N501Y ΔH69/V70, E484A, N501Y ΔH69/V70, E484A, N501Y ΔH69/V70, E484A, N501Y ΔH69/V70, E484A, N501Y ΔH69/V70, E484A, N501Y ΔH69/V70, E484A, N501Y | Omicron, BA.1 Omicron, BA.1.1 Omicron, BA.1.10 Omicron, BA.1.15 Omicron, BA.1.15.1 Omicron, BA.1.17 Omicron, BA.1.17.2 | 6 4 1 1 1 1 4 |
Measures | Target | ||
---|---|---|---|
ΔH69/V70 | E484A | N501Y | |
True positives (TP) | 686 | 933 | 942 |
True negatives (TN) | 255 | 10 | 2 |
False positives (FP) | 21 | 19 | 18 |
False negatives (FN) | 2 | 2 | 2 |
Correct classification rate (CCR), % (95% CI) | 97.6 (96.7–98.6) | 97.8 (96.9–98.7) | 97.9 (97.0–98.8) |
Sensitivity, % (95% CI) | 97.0 (96.0–98.1) | 98.0 (97.1–98.9) | 98.1 (97.3–99.0) |
Specificity, % (95% CI) | 99.2 (98.7–99.8) | 83.3 (81.0–85.7) | 50.0 (46.8–53.2) |
Positive predictive value (PPV), % (95% CI) | 99.7 (99.4–100) | 99.8 (99.5–100) | 99.8 (99.5–100) |
Negative predictive value (NPV), % (95% CI) | 92.4 (90.7–94.1) | 34.5 (31.5–37.5) | 10.0 (8.1–11.9) |
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Niedre-Otomere, B.; Kampenusa, I.; Trofimova, J.; Bodrenko, J.; Vangravs, R.; Skenders, G.; Nikisins, S.; Savicka, O. Multiplexed RT-qPCR Coupled with Whole-Genome Sequencing to Monitor a SARS-CoV-2 Omicron Variant of Concern in a Hospital Laboratory Setting in Latvia. Diagnostics 2023, 13, 3467. https://doi.org/10.3390/diagnostics13223467
Niedre-Otomere B, Kampenusa I, Trofimova J, Bodrenko J, Vangravs R, Skenders G, Nikisins S, Savicka O. Multiplexed RT-qPCR Coupled with Whole-Genome Sequencing to Monitor a SARS-CoV-2 Omicron Variant of Concern in a Hospital Laboratory Setting in Latvia. Diagnostics. 2023; 13(22):3467. https://doi.org/10.3390/diagnostics13223467
Chicago/Turabian StyleNiedre-Otomere, Baiba, Inara Kampenusa, Julija Trofimova, Jevgenijs Bodrenko, Reinis Vangravs, Girts Skenders, Sergejs Nikisins, and Oksana Savicka. 2023. "Multiplexed RT-qPCR Coupled with Whole-Genome Sequencing to Monitor a SARS-CoV-2 Omicron Variant of Concern in a Hospital Laboratory Setting in Latvia" Diagnostics 13, no. 22: 3467. https://doi.org/10.3390/diagnostics13223467