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In response to: Multiple assays in a real-time RT-PCR severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) panel can mitigate the risk of loss of sensitivity by new genomic variants during the COVID-19 outbreak

2021; Elsevier BV; Volume: 105; Linguagem: Inglês

10.1016/j.ijid.2021.01.049

1878-3511

Luis Peñarrubia, María Remedios Fernández Ruiz, Roberto Porco, Sonia N. Rao, Stephen A. Vella, Martí Juanola‐Falgarona, Davide Manissero, Marta López-Fontanals, Josep Pareja,

Viral gastroenteritis research and epidemiology

As an RNA virus, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) lacks a robust proofreading mechanism during replication, increasing the likelihood of evolutionary mutants (van Dorp et al., 2020van Dorp L. Richard D. Tan C.C.S. Shaw L.P. Acman M. Balloux F. et al.No evidence for increased transmissibility from recurrent mutations in SARS-CoV-2.Nat Commun. 2020; 11: 5986Crossref PubMed Scopus (169) Google Scholar). This is exemplified by the recent emergence of new SARS-CoV-2 variants associated with increased viral load (Kidd et al., 2020Kidd M. Richter A. Best A. Mirza J. Percival B. Mayhew M. et al."S-variant SARS-CoV-2 is associated with significantly higher viral loads in samples tested by ThermoFisher TaqPath RT-QPCR.medRxiv. 2020; https://doi.org/10.1101/2020.12.24.20248834Crossref Scopus (0) Google Scholar), most notably, the VOC-202012/01 (UK) and 501Y.V2 (South Africa) variants. Genomic analysis of these variants shows the highest mutation frequency occurring predominantly in the spike gene (Kupferschmidt, 2020Kupferschmidt K. Mutant coronavirus in the United Kingdom sets off alarms, but its importance remains unclear.Science. 2020; 20 (December)Google Scholar, Rambaut et al., 2020Rambaut A. Loman N. Pybus O. Barclay W. Barrett J. Carabelli A. et al.Preliminary genomic characterisation of an emergent SARS-CoV-2 lineage in the UK defined by a novel set of spike mutations. COVID-19 Genomics Consortium UK, 2020Google Scholar). Whether the expansion of these mutations is the result of selection or genetic drift is still under investigation (Tegally et al., 2020Tegally H. Wilkinson E. Giovanetti M. Iranzadeh A. Fonseca V. Giandhari J. et al.Emergence and rapid spread of a new severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) lineage with multiple spike mutations in South Africa.medRxiv. 2020; https://doi.org/10.1101/2020.12.21.20248640Crossref Scopus (0) Google Scholar, Kupferschmidt, 2021Kupferschmidt K. Fast-spreading UK virus variant raises alarms.Science. 2021; 371: 9-10Crossref PubMed Scopus (47) Google Scholar). However, the rapid spread of these variants has evoked public concern regarding the reliability of diagnostic reverse transcription polymerase chain reaction (RT-PCR) assays. We previously assessed the risk of loss of sensitivity of different published assays by the emergence of genetic variability through week 21 2020 (Penarrubia et al., 2020Penarrubia L. Ruiz M. Porco R Rao SN Juanola-Falgarona M Manissero D. et al.Multiple assays in a real-time RT-PCR SARS-CoV-2 panel can mitigate the risk of loss of sensitivity by new genomic variants during the COVID-19 outbreak.Int J Infect Dis. 2020; 97: 225-229Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). We concluded that targeting more than one genomic region mitigates the risk of loss of sensitivity by the accumulation of mutations in the primer binding regions. We also demonstrated that the QIAstat-Dx Respiratory SARS-CoV-2 panel (Qiagen, Hilden, Germany) successfully identified >99% of SARS-CoV-2 genomes with 100% oligonucleotide coverage. The remaining <1% of genomes exhibited a single variation in the annealing region of one of the oligonucleotides. From these, 0.02% of sequences showed a mismatch in a critical position. In-vitro testing showed no effect on the limit of detection of the panel, highlighting the robustness of its multi-target approach to maintain nominal sensitivity (Penarrubia et al., 2020Penarrubia L. Ruiz M. Porco R Rao SN Juanola-Falgarona M Manissero D. et al.Multiple assays in a real-time RT-PCR SARS-CoV-2 panel can mitigate the risk of loss of sensitivity by new genomic variants during the COVID-19 outbreak.Int J Infect Dis. 2020; 97: 225-229Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Following our previous methodology (Penarrubia et al., 2020Penarrubia L. Ruiz M. Porco R Rao SN Juanola-Falgarona M Manissero D. et al.Multiple assays in a real-time RT-PCR SARS-CoV-2 panel can mitigate the risk of loss of sensitivity by new genomic variants during the COVID-19 outbreak.Int J Infect Dis. 2020; 97: 225-229Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), we re-evaluated the impact on sensitivity of the mutations of SARS-CoV-2 variants under investigation according to the European Centre for Disease Prevention and Control, 2021European Centre for Disease Prevention and Control Risk related to spread of new SARS-CoV-2 variants of concern in the EU/EEA, first update – 21 January 2021. ECDC, Stockholm2021Google Scholar (Table 1) for both the QIAstat-Dx Respiratory SARS-CoV-2 panel and the NeuMoDx SARS-CoV-2 assay (Qiagen; https://www.neumodx.com/sars-cov-2/). Both of these assays use a dual target approach; the QIAstat-Dx-Respiratory panel targets the RdRp gene and the E gene, and the NeuMoDx SARS-CoV-2 assay targets the Nsp2 gene and the N gene. The analysis found that the mutations present in the listed viral strains are not expected to affect the sensitivity and/or inclusivity of the QIAstat-Dx Respiratory SARS-CoV-2 panel or the NeuMoDx SARS-CoV-2 assay.Table 1XXX.SARS-CoV-2 variantMutation (amino acid change)Reported public health impactGeographical regionImpact on assay sensitivityQIAstat-Dx Respiratory SARS-CoV-2 panel (RdRp and E assays)NeuMoDx SARS-CoV-2 panel (Nsp2 and N assays)VOC 202012/01Deletion (spike): Δ69-70, Δ144 Substitution (spike): N501Y, A570D, P681H, T716I, S982A, D1118HReport of increased transmissibility from the UKPrevalent in parts of the UK, cases detected increasingly in other countriesNo impactNo impact501.V2Substitution (spike): D80A, D215 G, E484 K, N501Y and A701VReport of increased transmissibility from South AfricaDominant in South Africa, two cases recently detected in the UKNo impactNo impactDanish mink variantDeletion (spike):Δ69-70 Substitution (spike):Y453FTransmission from mink to humans and community spread confirmed. No changes in transmissibility reportedPrevalent in Denmark. Not detected elsewhereNo impactNo impactDanish mink cluster 5Deletion (spike):Δ69-70 Substitution (spike):Y453 F, I692 V, M1229IPreliminary report of moderate reduction of neutralization by convalescent seraDenmark, not observed since September 2020No impactNo impactVarious variants with spike amino acid change N439KDeletion (spike):often Δ69-70Reports of minor reduction of neutralization by convalescent seraCommon in Czechia, Denmark and Ireland. Found in lower proportions in many countriesNo impactNo impactNext strain cluster 20A.EU1Substitution (spike): A222VRapid increase in Spain and then the rest of the EU/EEA at the start of the second wave, probably due to random events and travel patternsFirst observed in Spain. Most common variant in the EU/EEANo impactNo impactNext strain cluster 20A.EU2Substitution (spike): S477N (nucleocapsid): A376TRapid increase in France at the start of the second wave, probably due to founder effectsFirst observed in France, and also prevalent in Belgium, Czechia, Denmark, Hungary, the Netherlands and SwitzerlandNo impactNo impactD614GSubstitution (spike): D614GRapid increase during the early stages of the pandemic in the EU/EEA and then worldwide, probably due to a mix of founder effects and increased transmissibilityWorldwide. All other variants described here have descended from this oneNo impactNo impactEU/EEA, European Union/European Economic Area.Table adapted from: https://www.ecdc.europa.eu/sites/default/files/documents/COVID-19-risk-related-to-spread-of-new-SARS-CoV-2-variants-EU-EEA.pdf. Open table in a new tab EU/EEA, European Union/European Economic Area. Table adapted from: https://www.ecdc.europa.eu/sites/default/files/documents/COVID-19-risk-related-to-spread-of-new-SARS-CoV-2-variants-EU-EEA.pdf. The SARS-CoV-2 mutation rate will continue to increase the number of variants. Although reports of increased infectivity have appeared, no effect on the virulence of COVID-19 has been reported to date. Here, our results confirm the findings of previous work showing that multi-target real-time RT-PCR SARS-CoV-2 detection can mitigate the risk of loss of sensitivity. In this regard, continuous monitoring of genomic variants by assay manufacturers is essential to provide a rapid response in case the need arises for assay redesign. All authors are employees of QIAGEN. None.