Evaluation of two RT-PCR screening assays for identifying SARS-CoV-2 variants

Migueres, Marion; Lhomme, Sébastien; Trémeaux, Pauline; Dimeglio, Chloé; Ranger, Noémie; Latour, Justine; Dubois, Maxime; Nicot, Florence; Miédougé, Marcel; Mansuy, Jean‐Michel; Izopet, Jacques

doi:10.1016/j.jcv.2021.104969

Cited by 31 publications

(33 citation statements)

References 18 publications

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“…There is a link between the nasopharyngeal (NP) SARS-CoV-2 load and infectivity [4]. Previous studies, including ours, have described greater viral loads in patients infected with the Alpha variant than in other SARS-CoV-2 lineages, which could explain its greater transmissibility [5]. In contrast, few studies have compared the NP SARS-CoV-2 RNA loads of patients infected with Delta and Alpha variants or whether the NP viral load differs with the host vaccination status.…”

Section: Introductionmentioning

confidence: 57%

Influence of the Delta Variant and Vaccination on the SARS-CoV-2 Viral Load

Migueres

Trémeaux

Raymond

et al. 2022

Viruses

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Studies comparing SARS-CoV-2 nasopharyngeal (NP) viral load (VL) according to virus variant and host vaccination status have yielded inconsistent results. We conducted a single center prospective study between July and September 2021 at the drive-through testing center of the Toulouse University Hospital. We compared the NP VL of 3775 patients infected by the Delta (n = 3637) and Alpha (n = 138) variants, respectively. Patient’s symptoms and vaccination status (2619 unvaccinated, 636 one dose and 520 two doses) were recorded. SARS-CoV-2 RNA testing and variant screening were assessed by using Thermo Fisher® TaqPath™ COVID-19 and ID solutions® ID™ SARS-CoV-2/VOC evolution Pentaplex assays. Delta SARS-CoV-2 infections were associated with higher VL than Alpha (coef = 0.68; p ≤ 0.01) independently of patient’s vaccination status, symptoms, age and sex. This difference was higher for patients diagnosed late after symptom onset (coef = 0.88; p = 0.01) than for those diagnosed early (coef = 0.43; p = 0.03). Infections in vaccinated patients were associated with lower VL (coef = −0.18; p ≤ 0.01) independently of virus variant, symptom, age and sex. Our results suggest that Delta infections could lead to higher VL and for a longer period compared to Alpha infections. By effectively reducing the NP VL, vaccination could allow for limiting viral spread, even with the Delta variant.

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Section: Introductionmentioning

confidence: 57%

Influence of the Delta Variant and Vaccination on the SARS-CoV-2 Viral Load

Migueres

Trémeaux

Raymond

et al. 2022

Viruses

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“…It is known that a low viral load is the main factor of discrepant results between sequencing and screening tests for SARS-CoV-2 variants [21]. Indeed, in this study, all samples tested had a high viral load (mean Ct value of 23.6 ± 3.8) in order to guarantee that the diagnostic evaluation of the assays was performed on good-quality samples.…”

Section: Discussionmentioning

confidence: 93%

“…By allowing a rapid and accurate detection of mutations, these studies have highlighted the importance of focusing resources of genome sequencing on the research of newly emerging variants[17][18][19][20]. To date, no data on the diagnostic performance of the other assays analyzed in the present study are available.It is known that a low viral load is the main factor of discrepant results between sequencing and screening tests for SARS-CoV-2 variants[21]. Indeed, in this study, all samples tested had a high viral load (mean Ct value of 23.6 ± 3.8) in order to guarantee that the diagnostic evaluation of the assays was performed on good-quality samples.…”

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confidence: 93%

Comparative Analysis of Five Multiplex RT-PCR Assays in the Screening of SARS-CoV-2 Variants

et al. 2022

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The rapid and presumptive detection of SARS-CoV-2 variants may be performed using multiplex RT-PCR assays. The aim of this study was to evaluate the diagnostic performance of five qualitative RT-PCR tests as compared with next-generation sequencing (NGS). We retrospectively examined a multi-variant panel (n = 72) of SARS-CoV-2-positive nasopharyngeal swabs categorized as variants of concern (Alpha, Beta, Gamma and Delta), variants under monitoring (Iota and Kappa) and wild-type strains circulating in Liguria (Italy) from January to August 2021. First, NGS libraries of study samples were prepared and mapped to the reference genome. Then, specimens were screened for the detection of L452R, W152C, K417T, K417N, E484Q, E484K and N501Y mutations using the SARS-CoV-2 Variants II Assay Allplex, UltraGene Assay SARS-CoV-2 452R & 484K & 484Q Mutations V1, COVID-19 Ultra Variant Catcher, SARS-CoV-2 Extended ELITe MGB and Simplexa SARS-CoV-2 Variants Direct. The overall accuracy of these assays ranged from 96.9% to 100%. Specificity and sensitivity were 100% and 96–100%, respectively. We highly recommend the use of these assays as second-level tests in the routine workflow of SARS-CoV-2 laboratory diagnostics, as they are accurate, user friendly, low cost, may identify specific mutations in about 2 h–3 h and, therefore, optimize the surveillance of SARS-CoV-2 variants.

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“…Here, we describe a new assay that combines general detection of SC-2, together with specific classification of the two major variants Alpha and Delta. Previous reports of in-house selective assays, as well as commercial variant detection kits, rely mostly on the identification of common mutations that are not specific to a single variant (Migueres et al 2021, Lu et al 2021, Zelyas et al 2021. Other VOC detection approaches rely on multi-step expensive procedures (Williams et al 2021, Tierling et al 2021).…”

Section: Discussionmentioning

confidence: 99%

Specific Detection of Sars-Cov-2 Variants B.1.1.7 (Alpha) and B.1.617.2 (Delta) Using a One-Step Quantitative PCR Assay

Erster

Mendelson

Kabat

et al. 2021

Preprint

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In this report, we describe the development of an RT-qPCR assay, termed Alpha Delta assay, which can detect SARS-COV-2 (SC-2) and distinguish between the Alpha (B.1.1.7) and Delta (B.1.617.2) variants. The Alpha- and Delta-specific reactions in the assay target mutations that are strongly linked to the target variant. The Alpha reaction targets the D3L substitution in N gene, and the Delta reaction targets the spike gene 156-158 mutations. Additionally, we describe a second Delta-specific assay that we use as a confirmatory test for the Alpha Delta assay that targets the 119-120 deletion in the Orf8 gene. Both reactions have similar sensitivities of 15-25 copies per reaction, similar to the sensitivity of commercial SC-2 detection tests. The Alpha Delta assay and the Orf8-119del assay were successfully used to classify clinical samples that were subsequently analyzed by whole genome sequencing. Lastly, the capability of the Alpha Delta assay and Orf8-119del assay to identify correctly the presence of Delta RNA in wastewater samples was demonstrated. This study provides a rapid, sensitive and cost-effective tool for detecting and classifying two worldwide dominant SC-2 variants. It also highlights the importance of a timely diagnostic response to the emergence of new SC-2 variants with significant consequences on global health.

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Evaluation of two RT-PCR screening assays for identifying SARS-CoV-2 variants

Cited by 31 publications

References 18 publications

Influence of the Delta Variant and Vaccination on the SARS-CoV-2 Viral Load

Influence of the Delta Variant and Vaccination on the SARS-CoV-2 Viral Load

Comparative Analysis of Five Multiplex RT-PCR Assays in the Screening of SARS-CoV-2 Variants

Specific Detection of Sars-Cov-2 Variants B.1.1.7 (Alpha) and B.1.617.2 (Delta) Using a One-Step Quantitative PCR Assay

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