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RT-PCR genotyping assays to identify SARS-CoV-2 variants in England in 2021: a design and retrospective evaluation study

Publication date: 

17 Jan 2024


RT-PCR genotyping assays to identify SARS-CoV-2 variants in England in 2021: a design and retrospective evaluation study Bray, Neil et al. The Lancet Microbe, Volume 5, Issue 2, e173 - e180


Bray, N., Sopwith, W., Edmunds, M., Vansteenhouse, H., Feenstra, J.D.M., Jacobs, P., Rajput, K., O'Connell, A.M., Smith, M.L., Blomquist, P., Hatziioanou, D., Elson, R., Vivancos, R., Gallagher, E., Wigglesworth, M.J., Dominiczak, A., Hopkins, S., Lake, I.R.

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Background Whole-genome sequencing (WGS) is the gold standard diagnostic tool to identify and genetically characterise emerging pathogen mutations (variants), but cost, capacity, and timeliness limit its use when large populations need rapidly assessing. We assessed the potential of genotyping assays to provide accurate and timely variant information at scale by retrospectively examining surveillance for SARS-CoV-2 variants in England between March and September, 2021, when genotyping assays were used widely for variant detection. Methods We chose a panel of four RT-PCR genotyping assays to detect circulating variants of SARS-COV-2 in England and developed a decision algorithm to assign a probable SARS-CoV-2 variant to samples using the assay results. We extracted surveillance data from the UK Health Security Agency databases for 115 934 SARS-CoV-2-positive samples (March 1–Sept 6, 2021) when variant information was available from both genotyping and WGS. By comparing the genotyping and WGS variant result, we calculated accuracy metrics (ie, sensitivity, specificity, and positive predictive value [PPV]) and the time difference between the sample collection date and the availability of variant information. We assessed the number of samples with a variant assigned from genotyping or WGS, or both, over time. Findings Genotyping and an initial decision algorithm (April 10–May 11, 2021 data) were accurate for key variant assignment: sensitivities and PPVs were 0·99 (95% CI 0·99–0·99) for the alpha, 1·00 (1·00–1·00) for the beta, and 0·91 (0·80–1·00) for the gamma variants; specificities were 0·97 (0·96–0·98), 1·00 (1·00–1·00), and 1·00 (1·00–1·00), respectively. A subsequent decision algorithm over a longer time period (May 27–Sept 6, 2021 data) remained accurate for key variant assignment: sensitivities were 0·91 (95% CI 0·74–1·00) for the beta, 0·98 (0·98–0·99) for the delta, and 0·93 (0·81–1·00) for the gamma variants; specificities were 1·00 (1·00–1·00), 0·96 (0·96–0·97), and 1·00 (1·00–1·00), respectively; and PPVs were 0·83 (0·62–1·00), 1·00 (1·00–1·00), and 0·78 (0·59–0·97), respectively. Genotyping produced variant information a median of 3 days (IQR 2–4) after the sample collection date, which was faster than with WGS (9 days [8–11]). The flexibility of genotyping enabled a nine-times increase in the quantity of samples tested for variants by this method (from 5000 to 45 000). Interpretation RT-PCR genotyping assays are suitable for high-throughput variant surveillance and could complement WGS, enabling larger scale testing for known variants and timelier results, with important implications for effective public health responses and disease control globally, especially in settings with low WGS capacity. However, the choice of panels of RT-PCR assays is highly dependent on database information on circulating variants generated by WGS, which could limit the use of genotyping assays when new variants are emerging and spreading rapidly.