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Multiple mutations in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) increase transmission, disease severity, and immune evasion and facilitate zoonotic or anthropozoonotic infections. Four such mutations, ΔH69/V70, L452R, E484K, and N501Y, occurred in the SARS-CoV-2 spike glycoprotein in combinations that allow the simultaneous detection of VOCs. Here, we present two flexible reverse transcription-quantitative PCR (RT-qPCR) platforms for small- and large-scale screening (also known as variant PCR) to detect these mutations and schemes for adapting the platforms to future mutations. The large-scale RT-qPCR platform was validated by pairwise matching of RT-qPCR results with whole-genome sequencing (WGS) consensus genomes, showing high specificity and sensitivity. Both platforms are valuable examples of complementing WGS to support the rapid detection of VOCs. Our mutational signature approach served as an important intervention measure for the Danish public health system to detect and delay the emergence of new VOCs. IMPORTANCE Denmark weathered the SARS-CoV-2 crisis with relatively low rates of infection and death. Intensive testing strategies with the aim of detecting SARS-CoV-2 in symptomatic and nonsymptomatic individuals were available by establishing a national test system called TestCenter Denmark. This testing regime included the detection of SARS-CoV-2 signature mutations, with referral to the national health system, thereby delaying outbreaks of variants of concern. Our study describes the design of the large-scale RT-qPCR platform established at TestCenter Denmark in conjunction with whole-genome sequencing to report mutations of concern to the national health system. Validation of the large-scale RT-qPCR platform using paired WGS consensus genomes showed high sensitivity and specificity. For smaller laboratories with limited infrastructure, we developed a flexible small-scale RT-qPCR platform to detect three signature mutations in a single run. The RT-qPCR platforms are important tools to support the control of the SARS-CoV-2 endemic in Denmark.

Fast surveillance strategies are needed to control the spread of new emerging SARS-CoV-2 variants and gain time for evaluation of their pathogenic potential. This was essential for the Omicron variant (B.1.1.529) that replaced the Delta variant (B.1.617.2) and is currently the dominant SARS-CoV-2 variant circulating worldwide. RT-qPCR strategies complement whole genome sequencing, especially in resource lean countries, but mutations in the targeting primer and probe sequences of new emerging variants can lead to a failure of the existing RT-qPCRs. Here, we introduced an RT-qPCR platform for detecting the Delta- and the Omicron variant simultaneously using a degenerate probe targeting the key ΔH69/V70 mutation in the spike protein. By inclusion of the L452R mutation into the RT-qPCR platform, we could detect not only the Delta and the Omicron variants, but also the Omicron sub-lineages BA.1, BA.2 and BA.4/BA.5. The RT-qPCR platform was validated in small- and large-scale. It can easily be incorporated for continued monitoring of Omicron sub-lineages, and offers a fast adaption strategy of existing RT-qPCRs to detect new emerging SARS-CoV-2 variants using degenerate probes., Competing Interests: The authors have declardd that no competing interest exist.

SARS coronavirus 2 (SARS-CoV-2) continues to evolve and new variants emerge. Using nationwide Danish data, we estimate the transmission dynamics of SARS-CoV-2 Omicron subvariants BA.1 and BA.2 within households. Among 22,678 primary cases, we identified 17,319 secondary infections among 50,588 household contacts during a 1-7 day follow-up. The secondary attack rate (SAR) was 29% and 39% in households infected with Omicron BA.1 and BA.2, respectively. BA.2 was associated with increased susceptibility of infection for unvaccinated household contacts (Odds Ratio (OR) 1.99; 95%-CI 1.72-2.31), fully vaccinated contacts (OR 2.26; 95%-CI 1.95-2.62) and booster-vaccinated contacts (OR 2.65; 95%-CI 2.29-3.08), compared to BA.1. We also found increased infectiousness from unvaccinated primary cases infected with BA.2 compared to BA.1 (OR 2.47; 95%-CI 2.15-2.84), but not for fully vaccinated (OR 0.66; 95%-CI 0.57-0.78) or booster-vaccinated primary cases (OR 0.69; 95%-CI 0.59-0.82). Omicron BA.2 is inherently more transmissible than BA.1. Its immune-evasive properties also reduce the protective effect of vaccination against infection, but do not increase infectiousness of breakthrough infections from vaccinated individuals., (© 2022. The Author(s).)

In late 2021, the Omicron SARS-CoV-2 variant overtook the previously dominant Delta variant, but the extent to which this transition was driven by immune evasion or a change in the inherent transmissibility is currently unclear. We estimate SARS-CoV-2 transmission within Danish households during December 2021. Among 26,675 households (8,568 with the Omicron VOC), we identified 14,140 secondary infections within a 1-7-day follow-up period. The secondary attack rate was 29% and 21% in households infected with Omicron and Delta, respectively. For Omicron, the odds of infection were 1.10 (95%-CI: 1.00-1.21) times higher for unvaccinated, 2.38 (95%-CI: 2.23-2.54) times higher for fully vaccinated and 3.20 (95%-CI: 2.67-3.83) times higher for booster-vaccinated contacts compared to Delta. We conclude that the transition from Delta to Omicron VOC was primarily driven by immune evasiveness and to a lesser extent an inherent increase in the basic transmissibility of the Omicron variant., (© 2022. The Author(s).)

Objectives: The aim of this study was to develop a RT-PCR assay for the specific detection of the SARS-CoV-2 Omicron Variant of Concern (VOC) as a rapid alternative to sequencing., Methods: A RT-PCR was designed in silico and then validated using characterised clinical samples containing Omicron (both BA.1 and BA.2 lineages) and the Omicron synthetic RNA genome. As negative controls, SARS-CoV-2 positive clinical samples collected in May 2020, and synthetic RNA genomes of the isolate Wuhan Hu-1 and of the Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Kappa (B.1.617.1), Iota (B.1.526), Epsilon (B.1.429) and Delta (B.1.617.2) SARS-CoV-2 VOC were used., Results: Experiments performed using as templates the synthetic RNA genomes demonstrate the high specificity of the PCR-method for the SARS-CoV-2 Omicron. Despite the synthetic RNAs were used at high copy numbers, specific signal was mainly detected with the Omicron synthetic genome. Only a non-specific late signal was detected using the Alpha variant genome, but these results were considered negligible as Alpha VOC has been replaced by the Delta and it is not circulating anymore in the world. Using our method, we confirmed the presence of Omicron on clinical samples containing this variant but not of other SARS-CoV-2 lineages. The method is highly sensitive and can detect up to 1 cp of the Omicron virus per µl., Conclusions: The method presented here, in combination with other methods in use for detection of SARS-CoV-2, can be used for an early identification of Omicron., (Copyright © 2022. Published by Elsevier B.V.)

By 9 December 2021, 785 SARS-CoV-2 Omicron variant cases have been identified in Denmark. Most cases were fully (76%) or booster-vaccinated (7.1%); 34 (4.3%) had a previous SARS-CoV-2 infection. The majority of cases with available information reported symptoms (509/666; 76%) and most were infected in Denmark (588/644; 91%). One in five cases cannot be linked to previous cases, indicating widespread community transmission. Nine cases have been hospitalised, one required intensive care and no deaths have been registered.