key: cord-0793148-2144hj7d authors: Migueres, M; Lhomme, S; Trémeaux, P; Dimeglio, C; Ranger, N; Latour, J; Dubois, M; Nicot, F; Miedouge, M; Mansuy, JM; Izopet, J title: Evaluation of two RT-PCR screening assays for identifying SARS-CoV-2 variants date: 2021-09-02 journal: J Clin Virol DOI: 10.1016/j.jcv.2021.104969 sha: fd3f2a3c3ab65532774ad77ef7cd4b7995ca6d5f doc_id: 793148 cord_uid: 2144hj7d BACKGROUND: The recent emergence of new SARS CoV-2 variants (variants of concern, VOC) that spread rapidly and may lead to immune escape has emphasized the urgent need to monitor and control their spread. METHODS: We analyzed 2018 SARS-CoV-2 positive specimens collected between February 9 and March 22, 2021 using the Thermofisher® TaqPath(TM) COVID-19 CE-IVD RT-PCR kit (TaqPath) and the ID solutions® ID™ SARS-CoV-2/UK/SA Variant Triplex RT-PCR (ID triplex) assay to screen for VOCs. RESULTS: The ID triplex assay identified 62.8% of them as VOCs: 61.8% B.1.1.7 and 0.9% B.1.351/P.1. The agreement between the ID triplex results for B.1.1.7 and the TaqPath S gene target failure (SGTF)/ S gene target late detection (SGTL) profile for this variant agreed very well (k=0.86). A low virus load was the main cause of discrepancies. Sequencing discordant results with both assays indicated that the TaqPath assay detected the B.1.1.7 lineage slightly better. Both assays suggested that the virus loads of B.1.1.7 variants were significantly higher than those of non-B.1.1.7 strains. Only 10/20 B.1.351/P.1 strains detected with the ID triplex assay were confirmed by sequencing. CONCLUSIONS: We conclude that the SGTF/SGTL profiles identified using the TaqPath assay and ID triplex results are suitable for detecting the B.1.1.7 lineage. The ID triplex assay, which rapidly determines all three current VOCs simultaneously, could be a valuable tool for limiting virus spread by supporting contact-tracing and isolation. Several SARS-CoV-2 variants of concern (VOCs) have emerged in recent months. The alpha variant, also referred as B.1.1.7 lineage, appeared in December 2020, in the United Kingdom [1] [2] [3] [4] . Its increased transmissibility ensured that it quickly became the dominant strain in England and other countries [5] [6] [7] [8] [9] . The emergence of this VOC has been associated with a rapid increase in case numbers and hospitalisation rates in several countries 7, 10, 11 but there is some debate as to its link with disease severity and mortality [12] [13] [14] [17] [18] [19] . It is essential to monitor these variants because of their increased transmissibility and potential resistance to host immunity and vaccination. Many countries have consequently increased their sequencing capacity of the whole SARS-CoV-2 genome. Unfortunately, whole genome sequencing is slower than PCR testing and so is not suitable for contact tracing to limit virus spreading. Alternative methods for rapid VOC detection and contact tracing, such as RT-PCR-based screening assays have been developed; these generate results in just a few hours 6, [20] [21] [22] This study was done to evaluate the capacity of the ID triplex assay to detect VOCs and to determine its correlation with the TaqPath assay used as first line screening RT-PCR assay. We also assessed the virus loads of each variant. All nasopharyngeal specimens sent to the Toulouse university hospital for SARS-CoV-2 detection between February 9 and March 22 were tested within 24 hours of collection. Each positive specimen was then screened for the presence of VOCs. RNA was extracted on an MGI SP-960 instrument, a high-throughput fully automated workstation, using the MGIEasy Nucleic Acid Extraction kit (MGI TM ) and amplified on the The sequence data were processed using DRAGEN COVID Lineage (v3.5.1) (Illumina Inc.). Pangolin lineage. Samples for which genome coverage was at least 75% and spike coverage was at least 85% were further analyzed. The consensus sequences were aligned on the SARS-CoV-2 Wuhan-Hu-1 reference genome (NC_045512.2) and 109 GISAID sequences of different Pangolin lineages detected in France using MAFFT (v.7.475) 25 . We used phylogenetic analysis of this alignment to confirm Nextclade and Pangolin analyses. The ability of the two assays to detect B. We compared viral loads between variant by using Ct values as surrogates. Comparison of the Ct values for N and ORF1ab targets in SGTF/SGTL and non-SGTF-SGTL samples analysed 13 with the TaqPath assay showed that the Ct values for N and ORF1ab targets in SGTF/SGTL were significantly lower than those for non-SGTF/SGTL; median Ct value difference: 4.5 (p<0.0001) for the N gene and 3.4 (p=0.0009) for ORF1ab (Figure 2A, 2B) 30 ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Investigation of novel SARS-CoV-2 variants of concern Fast-spreading U.K. virus variant raises alarms Early transmissibility assessment of the N501Y mutant strains of SARS-CoV-2 in the United Kingdom Emergence of a new SARS-CoV-2 variant in the UK Emergence of SARS-CoV-2 B.1.1.7 Lineage -United States SARS-CoV-2 N501Y Introductions and Transmissions in Switzerland from Beginning of Diagnostic Screening and Whole Genome Sequencing Tracking SARS-CoV-2 lineage B.1.1.7 dissemination: insights from nationwide spike gene target failure (SGTF) and spike gene late detection (SGTL) data, Portugal, week 49 2020 to week 3 2021 Early assessment of diffusion and possible expansion of SARS-CoV-2 7, variant of concern 202012/01) in France Early introductions and transmission of SARS-CoV-2 variant B.1.1.7 in the United States SARS-CoV-2 -increased circulation of variants of concern and vaccine rollout in the EU/EEA -14th update Estimated transmissibility and impact of SARS-CoV-2 lineage B.1.1.7 in England Genomic characteristics and clinical effect of the emergent SARS UK: a whole-genome sequencing and hospital-based cohort study Increased mortality in community-tested cases of SARS-CoV-2 Changes in symptomatology, reinfection, and transmissibility associated with the SARS-CoV-2 variant B.1.1.7: an ecological study Genetic Variants of SARS-CoV-2 May Lead to False Negative Results with Molecular Tests for Detection of SARS-CoV-2 -Letter to Clinical Laboratory Staff and Health Care Providers S-variant SARS-CoV-2 lineage B1.1.7 is associated with significantly higher viral loads in samples tested by ThermoFisher TaqPath RT-qPCR Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7 Emerging SARS-CoV-2 variants reduce neutralization sensitivity to convalescent sera and monoclonal antibodies Resistance of SARS-CoV-2 variants to neutralization by monoclonal and serum-derived polyclonal antibodies Multiplex qPCR discriminates variants of concern to enhance global surveillance of SARS-CoV-2 Rapid and simultaneous identification of three mutations by the CoV-2 variants I assay kit RT-qPCR Assays for Rapid Detection of the N501Y, 69-70del, K417N, and E484K SARS-CoV-2 Mutations: A Screening Strategy to Identify Variants With Clinical Impact Detecting Rapid Spread of SARS-CoV-2 Variants High throughput detection and genetic epidemiology of SARS-CoV-2 using COVIDSeq next-generation sequencing MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform Detection of a SARS-CoV-2 variant of concern in South Africa Infection sustained by lineage B.1.1.7 of SARS-CoV-2 is characterised by longer persistence and higher viral RNA loads in nasopharyngeal swabs Increased transmissibility and global spread of SARS-CoV-2 variants of concern as at The SARS-CoV-2 B.1.351 lineage (VOC β) is outgrowing the lineage (VOC α) in some French regions in The English text was edited by Dr Owen Parkes. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.