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Review Article| Volume 42, ISSUE 2, P161-191, June 2022

An Overview of SARS-CoV-2 Molecular Diagnostics in Europe

Published:March 08, 2022DOI:https://doi.org/10.1016/j.cll.2022.02.005
An Overview of SARS-CoV-2 Molecular Diagnostics in Europe
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      Keywords

      Key points

      • •
        The COVID-19 pandemic has led not only to an influx of new molecular diagnostics but also a drive to modify existing technologies to allow the testing of thousands of patients daily over a variety of settings.
      • •
        The need for rapid turn-around times for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) testing for public health actions and patient care has led to the necessity for synchronously using multiple assays and platforms.
      • •
        Testing solutions exist for any scale of SARS-CoV-2 testing strategy.
      • •
        Overall SARS-CoV-2 molecular diagnostics seem to perform well; however, market saturation has left peer-reviewed real-world data lacking.
      • •
        With these new developments, diagnostic testing regulations for SARS-CoV-2 are paramount to aid manufacturers in achieving assay performance and for laboratories to use as a tool alongside local verification to determine the suitability of assays and platforms for use in future epidemics.

      Introduction

      An emerging viral pneumonia of unknown etiology was detected in patients from several health care facilities in the city of Wuhan in China on 30 December 2019.

      ProMED-mail. Published Date : 2020-01-05 18 : 15 : 37 Subject : PRO/AH/EDR > Undiagnosed Pneumonia - China ( HU ) ( 03 ): Updates , SARS , MERS Ruled out , WHO , RFI Archive Number : 20200105 . 6872267.; 2020.

      A novel coronavirus was identified initially termed “2019-nCoV” and designated as severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) with the clinical disease termed “coronavirus infectious disease-19” (COVID-19).

      Zhang Y-Z. Initial genome release of novel coronavirus. Virological. Available at: https://virological.org/t/novel-2019-coronavirus-genome/319. 2020. Accessed June 23, 2021.

      , 

      World Health Organization. Novel coronavirus (2019-NCoV) situation reports. Available at: https://www.who.int/docs/default-source/coronaviruse/situation-reports/20200121-sitrep-1-2019-ncov.pdf?sfvrsn=20a99c10_4. 2020. Accessed June 23, 2021.

      , 
      Coronaviridae Study Group of the International Committee on Taxonomy of Viruses
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      World Health Organization. Naming the coronavirus disease (COVID-19) and the virus that causes it. Available at: https://www.who.int/emergencies/diseases/novel-coronavirus-2019/technical-guidance/naming-the-coronavirus-disease-(covid-2019)-and-the-virus-that-causes-it. 2020. Accessed June 23, 2021.

      It has overwhelmed health care systems globally due to rapid asymptomatic spread and lethality leading the World Health Organization (WHO) to declare a COVID-19 pandemic on 11 March 2020.
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      Classification of severe acute respiratory syndrome coronavirus-2, virion, and genome

      SARS-CoV-2 is a betacoronavirus and one of the seven known members of the Coronaviridae family.
      Coronaviridae Study Group of the International Committee on Taxonomy of Viruses
      The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2.
      Nat Microbiol. 2020; 5: 536-544
      ,
      • Huang Y.
      • Yang C.
      • Xu X.
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      It is an enveloped positive-strand RNA virus (single linear RNA segment) with a genome length of 29,881 bp (GenBank no. MN908947). Its genome has 14 open reading frames (ORFs), which encode for 28 different proteins—4 structural proteins such as the S (spike), E (envelope), M (membrane), and N (nucleocapsid) proteins; 16 nonstructural proteins (NSP 1–16); and 8 accessory proteins as shown in Table 1.
      • Wu A.
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      Genome composition and divergence of the novel Coronavirus (2019-nCoV) originating in China.
      Cell Host Microbe. 2020; 27: 325-328
      Table 1Table showing SARS-CoV-2 structural and nonstructural proteins and their respective functions
      (Adapted from Suryawanshi and colleagues
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      • Agelidis A.
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      Dysregulation of cell signaling by SARS-CoV-2.
      Trends Microbiol. 2021; 29: 224-237
      (2021) and Wang and colleagues
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      SARS-CoV-2: structure, biology, and structure-based therapeutics development.
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      (2020)).
      GeneProteinFunctionReferences
      Structural protein
       Spike (S)SBinds to Angiotensin-Converting Enzyme 2 (ACE2) receptor and heparan sulfate for viral entry
      • Kakhki R.K.
      • Kakhki M.K.
      • Neshani A.
      COVID-19 target: a specific target for novel coronavirus detection.
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       Envelope (E)EVirion structure
      • Wu D.
      • Koganti R.
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      • et al.
      Vaccines and therapies in development for SARS-CoV-2 infections.
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       Membrane (M)MVirion structure
      • Wu D.
      • Koganti R.
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      Vaccines and therapies in development for SARS-CoV-2 infections.
      J Clin Med. 2020; 9: 1885
       Nucleocapsid (N)NContains genome; interferes with translation and cell cycle of the host cell.
      • Kim C.-H.
      SARS-CoV-2 evolutionary adaptation toward host entry and recognition of receptor O-acetyl sialylation in virus–host interaction.
      Int J Mol Sci. 2020; 21: 4549
      Nonstructural protein (NSP)
       ORF1aORF1bNSP-1RNA processing and replication
      • Huang C.
      • Lokugamage K.G.
      • Rozovics J.M.
      • et al.
      SARS coronavirus nsp1 protein induces template-dependent endonucleolytic cleavage of mRNAs: viral mRNAs are resistant to nsp1-induced RNA cleavage.
      PLOS Pathog. 2011; 7: e1002433
      NSP-2Modulation of survival signaling pathway of host cell
      • Cornillez-Ty C.T.
      • Liao L.
      • Yates 3rd, J.R.
      • et al.
      Severe acute respiratory syndrome coronavirus nonstructural protein 2 interacts with a host protein complex involved in mitochondrial biogenesis and intracellular signaling.
      J Virol. 2009; 83: 10314-10318
      NSP-3Possibly separates translated protein
      • Korber B.
      • Fischer W.M.
      • Gnanakaran S.
      • et al.
      Tracking changes in SARS-CoV-2 spike: evidence that D614G increases infectivity of the COVID-19 virus.
      Cell. 2020; 182: 812-827.e19
      NSP-4Contains transmembrane domain 2 (TM2) and modifies ER membranes
      • Oostra M.
      • Te Lintelo E.G.
      • Deijs M.
      • et al.
      Localization and membrane topology of coronavirus nonstructural protein 4: involvement of the early secretory pathway in replication.
      J Virol. 2007; 81: 12323-12336
      NSP-5Polyprotein replication
      • Chan J.F.-W.
      • Kok K.-H.
      • Zhu Z.
      • et al.
      Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan.
      Emerg Microbes Infect. 2020; 9: 221-236
      NSP-6Presumptive transmembrane domain
      • Benvenuto D.
      • Angeletti S.
      • Giovanetti M.
      • et al.
      Evolutionary analysis of SARS-CoV-2: how mutation of Non-Structural Protein 6 (NSP6) could affect viral autophagy.
      J Infect. 2020; 81: e24-e27
      NSP- 7 and NSP-8Increases the combination of NSP-12 and template-primer RNA
      • Cottam E.M.
      • Whelband M.C.
      • Wileman T.
      Coronavirus NSP6 restricts autophagosome expansion.
      Autophagy. 2014; 10: 1426-1441
      NSP-9ssRNA-binding protein
      • Cottam E.M.
      • Whelband M.C.
      • Wileman T.
      Coronavirus NSP6 restricts autophagosome expansion.
      Autophagy. 2014; 10: 1426-1441
      NSP-10Cap methylation of viral mRNAs
      • Wang Y.
      • Sun Y.
      • Wu A.
      • et al.
      Coronavirus nsp10/nsp16 methyltransferase can Be targeted by nsp10-derived peptide in vitro and in vivo to reduce replication and pathogenesis.
      J Virol. 2015; 89: 8416-8427
      NSP-11Unknown
      • Suryawanshi R.K.
      • Koganti R.
      • Agelidis A.
      • et al.
      Dysregulation of cell signaling by SARS-CoV-2.
      Trends Microbiol. 2021; 29: 224-237
      NSP-12RNA-dependent RNA polymerase (RdRp)
      • Wu F.
      • Zhao S.
      • Yu B.
      • et al.
      A new coronavirus associated with human respiratory disease in China.
      Nature. 2020; 579: 265-269
      NSP-13Binds with ATP and the zinc-binding domain - required for replication and transcription
      • Seybert A.
      • Hegyi A.
      • Siddell S.G.
      • et al.
      The human coronavirus 229E superfamily 1 helicase has RNA and DNA duplex-unwinding activities with 5'-to-3' polarity.
      Rna. 2000; 6: 1056-1068
      NSP-14Proofreading exoribonuclease domain
      • Chang C-k
      • Hou M.-H.
      • Chang C.-F.
      • et al.
      The SARS coronavirus nucleocapsid protein – forms and functions.
      Antiviral Res. 2014; 103: 39-50
      NSP-15Mn(2+)-dependent endoribonuclease activity
      • Chen Y.
      • Cai H.
      • Pan J.
      • et al.
      Functional screen reveals SARS coronavirus nonstructural protein nsp14 as a novel cap N7 methyltransferase.
      Proc Natl Acad Sci. 2009; 106: 3484
      NSP-162′-O-ribose methyltransferase
      • Naqvi A.A.T.
      • Fatima K.
      • Mohammad T.
      • et al.
      Insights into SARS-CoV-2 genome, structure, evolution, pathogenesis and therapies: structural genomics approach.
      Biochim Biophys Acta Mol Basis Dis. 2020; 1866: 165878
      ORF 3aIon channel protein—affected cytokine response
      • Castaño-Rodriguez C.
      • Honrubia J.M.
      • Gutiérrez-Álvarez J.
      • et al.
      Role of severe acute respiratory syndrome coronavirus viroporins E, 3a, and 8a in replication and pathogenesis.
      mBio. 2018; 9: e02325-17
      ORF 6Inhibits antiviral interferon response
      • Yount B.
      • Roberts R.S.
      • Sims A.C.
      • et al.
      Severe acute respiratory syndrome coronavirus group-specific open reading frames encode nonessential functions for replication in cell cultures and mice.
      J Virol. 2005; 79: 14909-14922
      ORF 7aInhibits antiviral interferon response and STAT1 phosphorylation
      • Xia H.
      • Cao Z.
      • Xie X.
      • et al.
      Evasion of type I interferon by SARS-CoV-2.
      Cell Rep. 2020; 33: 108234
      ORF 7bInhibits antiviral interferon response, STAT1, and STAT2 phosphorylation
      • Wang Y.
      • Sun Y.
      • Wu A.
      • et al.
      Coronavirus nsp10/nsp16 methyltransferase can Be targeted by nsp10-derived peptide in vitro and in vivo to reduce replication and pathogenesis.
      J Virol. 2015; 89: 8416-8427
      ORF 8Inhibits antiviral interferon response
      • Chen S.
      • Zheng X.
      • Zhu J.
      • et al.
      Extended ORF8 gene region is valuable in the epidemiological investigation of severe acute respiratory syndrome–similar coronavirus.
      J Infect Dis. 2020; 222: 223-233
      This table also breaks down the components of orf1ab complex.
      The genome commences with a 5′ untranslated region (UTR), then the replication complex (ORF1a and ORF1b) followed by the four structural proteins and 3′ UTR, ending with nonstructural ORFs and a poly(A) tail.
      • Wu A.
      • Peng Y.
      • Huang B.
      • et al.
      Genome composition and divergence of the novel Coronavirus (2019-nCoV) originating in China.
      Cell Host Microbe. 2020; 27: 325-328
      ,
      • Kim D.
      • Lee J.-Y.
      • Yang J.-S.
      • et al.
      The architecture of SARS-CoV-2 transcriptome.
      Cell. 2020; 181: 914-921.e10
      ORF1a contains 10 NSPs, while ORF1b contains 16 NSPs. The combination of ORF1a and ORF1b codes for polyproteins pp1a and pp1b that form the viral replication complex.
      • Wu A.
      • Peng Y.
      • Huang B.
      • et al.
      Genome composition and divergence of the novel Coronavirus (2019-nCoV) originating in China.
      Cell Host Microbe. 2020; 27: 325-328
      ,
      • Kim D.
      • Lee J.-Y.
      • Yang J.-S.
      • et al.
      The architecture of SARS-CoV-2 transcriptome.
      Cell. 2020; 181: 914-921.e10
      Structurally, the RNA genome is bound by the N protein, while the S, E, and M proteins together create the double-layered lipid viral envelope. The principle genes of diagnostic significance are the RdRp (NSP-12), various ORF1ab regions, and the viral structural proteins (S, E, and N).
      • Wu A.
      • Peng Y.
      • Huang B.
      • et al.
      Genome composition and divergence of the novel Coronavirus (2019-nCoV) originating in China.
      Cell Host Microbe. 2020; 27: 325-328

      History of severe acute respiratory syndrome coronavirus-2 molecular diagnostics

      The early sequencing of the SARS-CoV-2 genome and subsequent distribution of the genome sequence via Global Initiative on Sharing Avian Influenza Data (GISAID) enabled the development of nucleic acid amplification tests (NAATs), which became the cornerstone for the diagnosis of SARS-CoV-2. Although that is not the only molecular diagnostic technique, real-time polymerase chain reaction (RT-PCR) has become the mainstay across Europe with only limited use of other molecular techniques such as transcription-mediated amplification (TMA) or CRISPR.
      • Datta M.
      • Singh D.D.
      • Naqvi A.R.
      Molecular diagnostic tools for the detection of SARS-CoV-2.
      Int Rev Immunol. 2021; 40: 143-156
      ,
      • Gorzalski A.J.
      • Tian H.
      • Laverdure C.
      • et al.
      High-Throughput Transcription-mediated amplification on the Hologic Panther is a highly sensitive method of detection for SARS-CoV-2.
      J Clin Virol. 2020; 129: 104501
      One of the first published RT-PCR assays originated from Europe in January 2020 with primer probe sets targeting the E, N, and RdRp genes.
      • Corman V.M.
      • Landt O.
      • Kaiser M.
      • et al.
      Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR.
      Euro Surveill. 2020; 25: 2000045
      The RdRp assay included a Pan Sarbecco probe that detected SARS-CoV-1, SARS-CoV-2, and Bat-SARS-related-CoV with a second probe specific to SARS-CoV-2 leading to the recommendation of using the E gene assay as the first-line screening tool, followed by confirmatory testing with the RdRp gene assay.
      • Corman V.M.
      • Landt O.
      • Kaiser M.
      • et al.
      Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR.
      Euro Surveill. 2020; 25: 2000045
      A further assay was quickly developed by the Centers for Disease Control and Prevention (CDC) targeting multiple regions of the N gene, which has become the baseline assay for several commercially available molecular diagnostic tests.

      Centers for Disease Control and Prevention, CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel. Available at: https://www.fda.gov/media/134922/download. 2020. Accessed June 23, 2021

      , 

      CerTest BioTec. SARS-CoV-2 (N1 + N2) for BD MAXTM System Instructions for Use. Available at: https://www.certest.es/products/sars-cov-2-n1-n2-bd-maxtm-system/. 2021. Accessed June 23, 2021.

      , 

      Becton Dickinson & Company. SARS-CoV-2 Reagents for BD MAX System Instructions for Use. Available at: https://www.fda.gov/media/136816/download. 2020. Accessed June 23, 2021.

      , 
      • Freire-Paspuel B.
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      • et al.
      Analytical and clinical comparison of Viasure (CerTest Biotec) and 2019-nCoV CDC (IDT) RT-qPCR kits for SARS-CoV2 diagnosis.
      Virology. 2021; 553: 154-156

      Diagnostic testing regulations in Europe

      At the start of the COVID pandemic, in vitro medical devices (IVD), including NAAT-based systems and assays, needed to comply with European Union Directive 98/79/EC In Vitro Diagnostic Directive (IVDD) and bear a Conformitè Europëenne (CE) symbol as proof, to be marketed in European Union (EU) and European Free Trade Association countries and Turkey and the United Kingdom.

      European Commission. Directive 98/79/EC of the European Parliament and of the Council of 27 October 1998 on in Vitro Diagnostic Medical Devices. Vol 75. Available at: https://www.legislation.gov.uk/eudr/1998/79/pdfs/eudr_19980079_1998-12-07_en.pdf. 2003. Accessed June 23, 2021.

      ,

      Stralin M. In which countries is CE marking required? Clever Compliance. Available at: https://support.ce-check.eu/hc/en-us/articles/360014076911-In-which-countries-is-CE-marking-required. 2020. Accessed June 2, 2021

      CE marking required the manufacturer to have verified compliance with legal requirements and prepared an EC declaration of conformity containing the device performance and safety data.

      European Commission. Q&A on in vitro diagnostic medical device conformity assessment and performance in the context of COVID-19. Available at: https://ec.europa.eu/health/system/files/2021-06/covid-19_ivd-qa_en_0.pdf. 2021. Accessed June 23, 2021

      This allowed the device to be CE marked if it was intended for use by health care professionals although specific national requirements may also have been required.

      European Commission. Directive 98/79/EC of the European Parliament and of the Council of 27 October 1998 on in Vitro Diagnostic Medical Devices. Vol 75. Available at: https://www.legislation.gov.uk/eudr/1998/79/pdfs/eudr_19980079_1998-12-07_en.pdf. 2003. Accessed June 23, 2021.

      Although the United Kingdom left the EU in 2020, it will still accept CE-marked kits until 2023 when the UK Conformity Assessed mark will be required to market IVDs in the United Kingdom.
      Medicines and healthcare products regulatory agency. Regulating medical devices in the UK.
      https://www.gov.uk/guidance/regulating-medical-devices-in-the-uk
      Date: 2020
      Under Directive 98/79/EC, devices could also be granted emergency market access in the interest of health protection, such as in the COVID-19 pandemic; this required a derogation to be issued by the competent authority of a country allowing temporary marketing of a device without a full declaration of conformity, which was valid only for that nation.

      European Commission. Directive 98/79/EC of the European Parliament and of the Council of 27 October 1998 on in Vitro Diagnostic Medical Devices. Vol 75. Available at: https://www.legislation.gov.uk/eudr/1998/79/pdfs/eudr_19980079_1998-12-07_en.pdf. 2003. Accessed June 23, 2021.

      ,

      European Commission. Q&A on in vitro diagnostic medical device conformity assessment and performance in the context of COVID-19. Available at: https://ec.europa.eu/health/system/files/2021-06/covid-19_ivd-qa_en_0.pdf. 2021. Accessed June 23, 2021

      As of May 2021, Directive 98/79/EC was replaced in the EU by Regulation (EU) 2017/746, which expands the risk-based device classification system alongside a requirement for device assessment by independent third parties and confirmation of test performance by EU reference laboratories before a CE mark is awarded.
      European Commission
      Regulation (EU) 2017/746 of the European parliament and of the council on in vitro diagnostic medical devices.
      Off J Eur Union. 2017; 5: 117-176
      All products currently on the market that comply with the old legislation will have to recertify according to the new regulations.
      European Commission
      Regulation (EU) 2017/746 of the European parliament and of the council on in vitro diagnostic medical devices.
      Off J Eur Union. 2017; 5: 117-176
      ,

      MedTech Europe. Is the IVD Regulation Framework Ready for Class D Devices? Available at: https://www.medtecheurope.org/wp-content/uploads/2020/10/medtech-europe-reflection-paper-class-d-infrastructure-under-ivdr-transition-october-2020-1.pdf. 2020. Accessed June 23, 2021

      Regulation (EU) 2017/746 still allows the national emergency market access of IVDs in the interest of protection of health if the derogation is issued by the country’s competent authority.
      European Commission
      Regulation (EU) 2017/746 of the European parliament and of the council on in vitro diagnostic medical devices.
      Off J Eur Union. 2017; 5: 117-176
      This change in regulation brings CE marking more in line with the more stringent Food and Drug Administration (FDA) approval process, which requires devices to be tested by clinical trial and licensed only for use in specific circumstances.
      • Mishra S.
      FDA, CE mark or something else?—thinking fast and slow.
      Indian Heart J. 2017; 69: 1-5
      On 17 June 2021, the UK government announced the intention to introduce a mandatory validation scheme initially for COVID-19 diagnostics to expand to cover all devices sold in the United Kingdom. This process would require manufacturers to provide a minimum set of standard performance data, which would undergo independent verification by specially commissioned laboratories. If successfully introduced, it would be a criminal offense to market devices that have failed or not undergone this mandatory validation in the United Kingdom under the Medicines and Medical Devices Act 2021.
      Department of Health and Social care
      Private COVID-19 testing validation.
      https://www.gov.uk/government/consultations/private-coronavirus-covid-19-testing-validation/private-covid-19-testing-validation
      Date: 2021
      The above pieces of legislation along with the European Commission’s guidelines for the Current Performance of COVID-19 Test Methods and Devices and Proposed Performance Criteria state the performance characteristics for IVDs, which includes but is not limited to analytical and diagnostic sensitivity and specificity, limits of detection (LODs), and expected values in normal and affected populations.

      European Commission. Directive 98/79/EC of the European Parliament and of the Council of 27 October 1998 on in Vitro Diagnostic Medical Devices. Vol 75. Available at: https://www.legislation.gov.uk/eudr/1998/79/pdfs/eudr_19980079_1998-12-07_en.pdf. 2003. Accessed June 23, 2021.

      ,
      European Commission
      Regulation (EU) 2017/746 of the European parliament and of the council on in vitro diagnostic medical devices.
      Off J Eur Union. 2017; 5: 117-176
      ,

      European Commission. Current performance of COVID-19 test methods and devices and proposed performance criteria. Available at: https://ec.europa.eu/docsroom/documents/40805. 2020. Accessed June 23, 2021

      No required values for these characteristics are published in these documents although common specifications are planned.

      MedTech Europe. Is the IVD Regulation Framework Ready for Class D Devices? Available at: https://www.medtecheurope.org/wp-content/uploads/2020/10/medtech-europe-reflection-paper-class-d-infrastructure-under-ivdr-transition-october-2020-1.pdf. 2020. Accessed June 23, 2021

      A list of CE-marked COVID-19 IVDs is maintained at the European commission’s Joint Research Centre In Vitro Diagnostic Devices and Test Methods Database.
      European Commission
      European commission COVID-19 in vitro diagnostic devices and test methods database.
      https://covid-19-diagnostics.jrc.ec.europa.eu
      Date: 2020
      As of 08/06/2021 325 CE-marked NAATs exist in this database originating from 240 unique manufacturers with 31 countries of origin. This database lacks key performance criteria for a significant number of entries including 120 tests with no stated LOD, 226 with no analytical sensitivity, 209 with no analytical specificity, and 200 with no clinical accuracy data. The entrance of many nontraditional manufacturers to the market has fueled a lack of peer-reviewed publications that make assessment of real-world performance difficult. An improved and standardized approach to market regulations would be welcomed as at present local validations/verifications of diagnostics are hugely important in ensuring the suitability of test selection for the intended purpose.
      In addition to CE marking, the WHO and national bodies such as the UK Medicines and Healthcare products Regulatory Agency (MHRA) have published target product profiles (TPPs) that outline performance characteristics that a test must meet to be considered successful for its intended use.
      World Health Organization
      COVID-19 target product profiles for priority diagnostics to support response to the COVID-19 pandemic v.1.0.
      World Health Organization, 2020
      , 
      Medicines and Healthcare products Regulatory Agency
      Target product profile: point of care SARS-CoV-2 detection tests.
      2020https://doi.org/10.1093/med/9780199609147.003.0077
      , 
      Medicines and Healthcare products Regulatory Agency
      Target product profile: laboratory-based SARS-CoV-2 viral detection tests.
      2020https://doi.org/10.1093/med/9780199609147.003.0077
      , 

      Medicines and Healthcare products Regulatory Agency. Guidance For industry and manufacturers: COVID-19 tests and testing kits. Available at: https://www.gov.uk/government/publications/how-tests-and-testing-kits-for-coronavirus-covid-19-work/for-industry-and-manufactures-covid-19-tests-and-testing-kits. 2021. Accessed June 23, 2021

      WHO and MHRA TPPs outline “acceptable” and “desirable” characteristics including ranges for parameters such as analytical sensitivity/LOD and clinical sensitivity.
      World Health Organization
      COVID-19 target product profiles for priority diagnostics to support response to the COVID-19 pandemic v.1.0.
      World Health Organization, 2020
      , 
      Medicines and Healthcare products Regulatory Agency
      Target product profile: point of care SARS-CoV-2 detection tests.
      2020https://doi.org/10.1093/med/9780199609147.003.0077
      , 
      Medicines and Healthcare products Regulatory Agency
      Target product profile: laboratory-based SARS-CoV-2 viral detection tests.
      2020https://doi.org/10.1093/med/9780199609147.003.0077
      These documents are not legally binding but were developed to aid manufacturers in achieving assay performance that would be desired for use in the field. Equally these documents can be used by laboratories as a tool alongside local verification to determine the suitability of an assay for use. A selection of characteristics for NAAT-based tests is listed in Tables 2 and 3 with the MHRA TPP showing much stricter acceptable criteria than the WHO criteria recommended for adoption by European Centre for Disease Prevention and Control (ECDC).
      World Health Organization
      COVID-19 target product profiles for priority diagnostics to support response to the COVID-19 pandemic v.1.0.
      World Health Organization, 2020
      , 
      Medicines and Healthcare products Regulatory Agency
      Target product profile: point of care SARS-CoV-2 detection tests.
      2020https://doi.org/10.1093/med/9780199609147.003.0077
      , 
      Medicines and Healthcare products Regulatory Agency
      Target product profile: laboratory-based SARS-CoV-2 viral detection tests.
      2020https://doi.org/10.1093/med/9780199609147.003.0077
      ,

      European Centre for Disease Prevention and Control. COVID-19 testing strategies and objectives key messages. Available at: https://www.ecdc.europa.eu/sites/default/files/documents/TestingStrategy_Objective-Sept-2020.pdf. 2020. Accessed June 23, 2021

      Table 2Selected target product profile characteristics for point-of-care SAR-CoV-2 detection tests
      World Health OrganizationMedicines and Healthcare Products Regulatory Agency
      ScopeDesiredAcceptableDesiredAcceptable
      Intended useIn areas with confirmed SAR-CoV-2 community-wide transmission. In suspected outbreak situations and to monitor trends in disease incidence.Aid in the triage of current SARS-CoV-2 infection during active infection.Aid in the triage of current SARS-CoV-2 infection during the acute phase of infection.
      Target populationPatients with acute or subacute respiratory symptoms; suspicious symptoms and contact with confirmed or probable case/living in the area of cluster/community transmission.People with/without SARS-CoV-2 clinical signs and symptoms if testing appropriate.People with clinical signs and symptoms associated with SAR-CoV-2 infection.
      Target user/settingsTrained staff in health care facilities or community level or self-administrated.Trained staff in health care facilities.Trained health care professional (governed by professional standards authority). In primary/secondary/community health care settings and nonhealth care settings.
      Target analyteSARS-CoV-2 only biomarker, for example, RNA, protein/antigen.SARS-CoV-2 only biomarker. Assumption SARS-CoV-1 not circulatingDual (or more) SARS-CoV-2 RNA or antigen targets.Single (or more) SARS-CoV-2 RNA or antigen target.
      Target typeAnterior nares, saliva/oral fluid, sputumNP or OP or nasal swab, nasal wash, sputumSputum, saliva, or other method not using invasive swabNP or OP, lower respiratory tract aspirate, BAL, nasopharyngeal wash/aspirate or nasal aspirate
      Clinical sensitivity≥90%≥80%>97% within confidence intervals of 93–100%
      Determined using at least 150 positive clinical samples covering a clinically meaningful range of viral loads.
      		>80% within 95% confidence intervals of 93–100%
      Determined using at least 150 positive clinical samples covering a clinically meaningful range of viral loads.
      Clinical specificity≥99%≥97%>99% within confidence intervals of 97–100%
      Determined using at least 250 negative clinical samples.
      		>95% within 95% confidence intervals of 90–100%b
      Analytical sensitivity (LOD)1 × 104 copies per ml or Ct≈>301 × 106 copies per ml or Ct ≈ 25–30<100 SARS-CoV-2 copies/ml<1000 SARS-CoV-2 copies/ml
      Technical Failure rate≤ 0.5%< 2%< 1%< 5%
      Turnaround time≤ 20 min≤ 40 min< 30 min< 2 h
      Throughput≥ 10/h per operator≥ 5/h per operator> 100 tests per unit per 12 h> 6 tests per unit per 12 h
      Abbreviations: BAL, bronchoalveolar; LOD, limit of detection; NP, nasopharyngeal swab; OP, oropharyngeal swab; Ct, Cycle threshold.
      a Determined using at least 150 positive clinical samples covering a clinically meaningful range of viral loads.
      b Determined using at least 250 negative clinical samples.
      Table 3Selected target product profile characteristics for high- and low-throughput diagnostic SAR-CoV-2 detection testing
      World Health OrganizationMedicines and Healthcare Products Regulatory Agency
      ScopeDesiredAcceptableDesiredAcceptable
      Intended useTo detect the presence of virus components to diagnose or confirm acute and subacute SARS-CoV-2 infection.Multiplex—determining current infection by detecting SARS-CoV-2 virus, differentiate other respiratory infections.Determining current infection by detecting SARS-CoV-2 virus.
      Target populationPatients with acute or subacute respiratory symptoms; suspicious symptoms and contact with confirmed or probable case/living in the area of cluster/community transmission.People with/without clinical signs associated with SARS-CoV-2 infection.People with clinical signs associated with SAR-CoV-2 infection.
      Target settings/usersHigh volume: reference laboratories/district hospitals/mobile laboratories. Laboratory technicians. Low volume: outpatient clinics, point of care or near-patient settings. Laboratory technicians/health care workers.Health care and medical laboratories. Trained health care professional (governed by professional standards authority) and suitably trained and assessed lab technician or scientist.
      Target analyteMust have at least one target specific for SARS-CoV-2 RNA or protein/antigen.Dual (or more) SARS-CoV-2 RNA. Multiplex panel for a range of infectious respiratory viruses.Single SARS-CoV-2 RNA.
      Target typeSamples amenable to self-collection: saliva/oral fluid, stool; inactivated samples.NP or OP or nasal swab. Washes-nasal, oropharyngeal, BAL. SputumOral fluidNP or OP, lower respiratory tract aspirate, BAL, nasopharyngeal wash/aspirate, or nasal aspirate.
      Clinical sensitivity≥98%≥95%>99%. 95% two-sided confidence interval > 97%
      Determined using at least 150 positive clinical samples covering a clinically meaningful range of viral loads.
      		>95%. 95% two-sided confidence interval > 90%
      Determined using at least 150 positive clinical samples covering a clinically meaningful range of viral loads.
      Clinical specificity≥99%≥99%>99%. 95% two-sided confidence interval > 97%
      Determined using at least 250 negative clinical samples.
      		>95%. 95% two-sided confidence interval > 90%
      Determined using at least 250 negative clinical samples.
      Analytical sensitivity (LOD)1 × 102 copies per ml in upper/lower respiratory tract specimens, stool1 × 103 copies per ml in any respiratory tract specimen.≤100 SARS-CoV-2 copies/ml≤1000 SARS-CoV-2 copies/ml
      Technical failure rateNANA<0.2%<1%
      Turnaround time< 45 min< 4 h< 90 min< 5 h
      ThroughputHigh volume: 200–500 tests in 4 h. Low Volume: 6 patients in 45 minHigh volume: 50–150 tests in 4 h. Low volume: 1–4 patients per 45 min> 200 tests in unit per 4 h> 50 tests in unit per 4 h
      Abbreviations: BAL, bronchoalveolar; LOD, limit of detection; NP, nasopharyngeal swab; OP, oropharyngeal swab.
      a Determined using at least 150 positive clinical samples covering a clinically meaningful range of viral loads.
      b Determined using at least 250 negative clinical samples.

      Severe acute respiratory syndrome coronavirus-2 molecular diagnostics

      The scale of testing required to manage the SARS-COV-2 pandemic has been unprecedented with extensive yet flexible testing strategies being key to protecting public health through prompt isolation of cases.

      European Centre for Disease Prevention and Control. COVID-19 testing strategies and objectives key messages. Available at: https://www.ecdc.europa.eu/sites/default/files/documents/TestingStrategy_Objective-Sept-2020.pdf. 2020. Accessed June 23, 2021

      ,

      Royal College of Pathologists, COVID-19 testing, 2020, A National Strategy. Available at: https://www.rcpath.org/profession/on-the-agenda/covid-19-testing-a-national-strategy.html. 2020. Accessed June 23, 2021.

      The United Kingdom has undertaken a dual-arm approach to testing with twice weekly at home rapid antigen tests being freely available and actively encouraged in the asymptomatic general population and in laboratory NAAT being used for more sensitive screening of all hospital admissions including day case and those with symptoms consistent with COVID-19.
      • Torjesen I.
      Covid-19: how the UK is using lateral flow tests in the pandemic.
      BMJ. 2021; 372: 1-3
      ,
      UK Parliament
      Mass asymptomatic Covid-19 testing: strategy and accuracy.
      https://commonslibrary.parliament.uk/research-briefings/cbp-9223/
      Date: 2021
      The ECDC not only recommends the use of NAAT for all symptomatic cases but also acknowledges the role for rapid antigen tests in population screening.

      European Centre for Disease Prevention and Control. COVID-19 testing strategies and objectives key messages. Available at: https://www.ecdc.europa.eu/sites/default/files/documents/TestingStrategy_Objective-Sept-2020.pdf. 2020. Accessed June 23, 2021

      ,

      Royal College of Pathologists, COVID-19 testing, 2020, A National Strategy. Available at: https://www.rcpath.org/profession/on-the-agenda/covid-19-testing-a-national-strategy.html. 2020. Accessed June 23, 2021.

      The use of sensitive molecular diagnostic assays is important to the control of transmission. If SARS-CoV-2 infection is allowed to spread unchecked, the emergence of novel variants is likely to be enhanced as mutations in key genes continue to accumulate as part of the natural error-prone replication of RNA viruses. As mutations accumulate, it is not only possible that they can lead to increased pathogenicity or vaccine escape, but that they may also lead to detection failures in well-established diagnostic assays. It is now recommended that the presence of SARS-CoV-2 in clinical samples is determined through the detection of at least two distinct targets to mitigate this risk. The observation of the ThermoFisher S gene PCR assay failure in the United Kingdom for the B.1.1.7 Alpha variant, which would have led to significant numbers of false-negative tests being reported if this was being used as a single target assay, highlights the importance of a multi-target approach.

      Public Health England. Investigation of novel SARS-COV-2 variant Variant of Concern 202012/01 Detection of an epidemiological cluster associated with a new variant of concern Nomenclature of variants in the UK Current epidemiological findings. 2020;(December):1-11.

      To achieve testing on such an immense scale testing, a diverse approach has been required with laboratories often using multiple assays and platforms in unison. The following is by no means an extensive review of all diagnostic assays used in Europe but aims to provide an overview of some of the most common. Rapid antigen near patient point of care and isothermal amplification techniques are outside the scope of this review but will be covered elsewhere in this Clinics edition.

      Rapid molecular diagnostics

      Rapid, commercial, cartridge-based sample-to-answer molecular diagnostic platforms for the detection of SARS-CoV-2 have fulfilled an important niche in point-of-care settings and clinical laboratories. They are simple to use, provide accurate results within 1–2 h, have minimal hands-on time, and permit on-demand testing of urgent specimens.
      An overview of the main sample-to-answer platforms is presented in Table 4. These single-use tests often automate nucleic acid extraction, purification, amplification, detection, and interpretation of results. All the platforms presented are internally controlled yet only three use an endogenous sample control, which monitors for an adequately taken sample and sample degradation. Independent studies evaluating the performance of rapid RT-PCR tests have varied with few head-to-head comparisons although evaluations of these platforms are more extensively published due to their widespread use in non-specialist laboratories.
      Table 4An overview of rapid, cartridge-based, sample to answer SARS-CoV-2 molecular tests
      Test NameManufacturerTarget 1Target 2Internal ControlPlatformMaximum

      Sample Capacity      		Platform Run Time (min)Sample Input Volume (uL)
      Xpert Xpress SARS-CoV-2

      Xpert Xpress SARS-CoV-2/Flu/RSV      		CepheidN2EManufacturer SPCGeneXpert Dx and GeneXpert Infinity2–16 (Dx) or Up to 80 (Infinity)45300
      BioFire® Respiratory Panel 2.1 plus (RP2.1 plus)BioMerieuxSM

      Schizosaccharomyces pombe      		FilmArray 2.0 and FilmArray Torch2–1245300
      Cobas Liat SARS-CoV-2 and Influenza A/BRocheORF1 a/bNManufacturer SPCCobas Liat120200
      Novodiag COVID-19MobiDiagORF1 a/bNRNAse P and Manufacturer SPCNovodiag4–1660500
      VitaPCR SARS-CoV-2

      VitaPCR SARS-CoV-2/Flu AB      		Credo Diagnostics Biomedical PteNNβ-globinVitaPCR12030
      30 uL lysate (lysis buffer containing sample).
      Aries SARS-CoV-2LuminexORF1a/bNRNAse PAries12120200
      GenomEra SARS-CoV-2

      GenomEra SARS-CoV-2, Flu A/B+ RSV      		Abacus DiagnosticaRdRPE
      GenomEra SARS-CoV-2 contains E gene. GenomEra SARS-CoV-2, Flu A/B+ RSV contains only RdRP.
      		MS2GenomEra CDX47035
      50 uL of sample is heated and mixed with 1 mL of lysis buffer, after which 35 uL of processed sample is loaded onto the test chip.
      QIAstat-Dx Respiratory SARS-CoV-2 PanelQiagenORF1 a/b (RdRp)EMS2QIAstat Dx Analyzer170300
      GenMark ePlex SARS-CoV-2

      GenMark ePLex Respiratory Pathogen Panel 2 (RP2)      		GenMark DxNNManufacturer SPCePlex3 (ePlex NP) to 24 (ePlex 4 Tower)90200
      Abbreviations: N, nucleocapsid; E, envelope protein; S, spike glycoprotein; M, membrane protein; ORF1 a/b, open reading frame 1 a/b; RdRP, RNA-dependent RNA polymerase; SPC, sample process control.
      a 30 uL lysate (lysis buffer containing sample).
      b GenomEra SARS-CoV-2 contains E gene. GenomEra SARS-CoV-2, Flu A/B+ RSV contains only RdRP.
      c 50 uL of sample is heated and mixed with 1 mL of lysis buffer, after which 35 uL of processed sample is loaded onto the test chip.
      Unlike other applications, the rapid testing platforms exhibit significant variation in the technologies used. Cepheid Xpert Xpress, QiaStatDx, and VitaPCR SARS-CoV-2 rely on classic multiplex RT-PCR. Novodiag COVID-19

      MobiDiag. Complete solution for rapid molecular diagnostics of coronavirus infection. Available at: https://mobidiag.com/products/coronavirus/#Novodiag-COVID-19. 2021. Accessed June 14, 2021.

      is unique in its use of qPCR and microarray technology for the detection of SARS-CoV-2. GenomEra SARS-CoV-2

      Abacus Diagnostica. GenomEra SARS-CoV-2 Assay Kit Package Insert. Available at: https://www.abacusdiagnostica.com/products/sars-cov-2-2-0/. 2020. Accessed June 23, 2021.

      and GenomEra SARS-CoV-2 with Flu A/B+ RSV

      Abacus Diagnostica. GenomEra SARS-CoV-2 , Flu A/B + RSV Assay Kit Package Insert. Version 1.0. Available at: https://www.abacusdiagnostica.com/products/sars-cov-2-flu-a-b-rsv/. 2020. Accessed June 23, 2021

      use multiplex RT-PCR performed on chips. BioFire Respiratory Panel 2.1 plus (RP2.1plus)

      BioFire respiratory Panel 2.1 plus (RP2.1plus) Instructions for use. Available at: https://www.biomerieux-diagnostics.com/filmarrayr-respiratory-panel. 2020. Accessed June 23, 2021.

      achieves extensive multiplexing through an initial RT-PCR step before target amplification using numerous monoplex PCR reactions, which are detected using endpoint melt curve analysis. GenMark ePlex SARS-CoV-2

      GenMark Dx. EPlex SARS-CoV-2 Test Assay Manual. Available at: https://www.fda.gov/media/136282/download. 2020. Accessed June 28, 2021.

      and GenMark ePlex Respiratory Pathogen Panel 2 (RP2)

      GenMark Dx. EPlex Respiratory Pathogen Panel 2 Package Insert. Available at: https://www.fda.gov/media/142905/download. 2020. Accessed June 28, 2021.

      use RT-PCR in combination with electrowetting and GenMark’s eSensor technology involving electrochemical detection rather than optical detection of fluorescence.
      Aside from the variation in technologies, the rapid testing platforms also offer detection of the widest range of pathogens. With the exception of Luminex Aries, SARS-CoV-2 can be detected in isolation or in combination with influenza as a minimum.

      Cepheid. Xpert Xpress SARS-CoV-2/Flu/RSV Instructions For Use. Available at: https://www.cepheid.com/en/package-inserts/1913. 2021. Accessed June 28, 2021.

      ,
      • Fournier P.E.
      • Zandotti C.
      • Ninove L.
      • et al.
      Contribution of VitaPCR SARS-CoV-2 to the emergency diagnosis of COVID-19.
      J Clin Virol. 2020; 133: 337-339
      BioFire RP2.1plus

      BioFire respiratory Panel 2.1 plus (RP2.1plus) Instructions for use. Available at: https://www.biomerieux-diagnostics.com/filmarrayr-respiratory-panel. 2020. Accessed June 23, 2021.

      detects 23 respiratory pathogens, GenMark ePlex RP2

      GenMark Dx. EPlex Respiratory Pathogen Panel 2 Package Insert. Available at: https://www.fda.gov/media/142905/download. 2020. Accessed June 28, 2021.

      detects 25 respiratory pathogens, and the QIAstat-Dx Respiratory SARS-CoV-2 Panel

      Qiagen. QIAstat-Dx Respiratory Panel. Instructions for Use. Available at: https://www.qiagen.com/us/resources/download.aspx?id=e3f2dc10-c712-4bcf-9acd-211bd35df944&lang=en. 2020. Accessed June 28, 2021.

      detects 22 respiratory pathogens.
      Xpert Xpress SARS-CoV-2

      Cepheid. Xpert Xpress SARS-CoV-2 Instructions For Use. Available at: https://www.cepheid.com/en/package-inserts/1615. 2020. Accessed June 28, 2021.

      is the most widely evaluated rapid test with a recent systematic review and meta-analysis encompassing 1734 subjects determining a pooled sensitivity of 99% (97–99, 95% CI) and a specificity of 97% (95–98, 95% CI).
      • Lee J.
      • Song J.U.
      Diagnostic accuracy of the Cepheid Xpert Xpress and the Abbott ID NOW assay for rapid detection of SARS-CoV-2: a systematic review and meta-analysis.
      J Med Virol. 2021; : 1-9
      Reported sensitivities for other platforms range from 90 to 100% with particular issues noted for samples with high cycle threshold (Ct) values in some studies.
      • Fournier P.E.
      • Zandotti C.
      • Ninove L.
      • et al.
      Contribution of VitaPCR SARS-CoV-2 to the emergency diagnosis of COVID-19.
      J Clin Virol. 2020; 133: 337-339
      ,
      • Fitoussi F.
      • Dupont R.
      • Tonen-Wolyec S.
      • et al.
      Performances of the VitaPCRTM SARS-CoV-2 Assay during the second wave of the COVID-19 epidemic in France.
      J Med Virol. 2021; : 1-7
      , 
      • Eckbo E.J.
      • Locher K.
      • Caza M.
      • et al.
      Evaluation of the BioFire COVID-19 test and Respiratory Panel 2.1 for rapid identification of SARS-CoV-2 in nasopharyngeal swab samples.
      Diagn Microbiol Infect Dis. 2021; 99: 115260
      , 
      • Creager H.M.
      • Cabrera B.
      • Schnaubelt A.
      • et al.
      Clinical evaluation of the BioFire® Respiratory Panel 2.1 and detection of SARS-CoV-2.
      J Clin Virol. 2020 Aug; 129: 104538https://doi.org/10.1016/j.jcv.2020.104538
      , 
      • Visseaux B.
      • Le Hingrat Q.
      • Collin G.
      • et al.
      Evaluation of the QIAstat-dx respiratory SARS-CoV-2 Panel, the first rapid multiplex PCR commercial assay for SARS-CoV- 2 detection.
      J Clin Microbiol. 2020; 58: 1-5
      Fitoussi and colleagues (2021)
      • Fitoussi F.
      • Dupont R.
      • Tonen-Wolyec S.
      • et al.
      Performances of the VitaPCRTM SARS-CoV-2 Assay during the second wave of the COVID-19 epidemic in France.
      J Med Virol. 2021; : 1-7
      found a VitaPCR SARS-CoV-2 sensitivity of 60% for samples that were positive at Ct > 33 using a comparator N gene assay; however, VitaPCR involves no formal RNA extraction and purification that may account for this poor performance.
      • Fitoussi F.
      • Dupont R.
      • Tonen-Wolyec S.
      • et al.
      Performances of the VitaPCRTM SARS-CoV-2 Assay during the second wave of the COVID-19 epidemic in France.
      J Med Virol. 2021; : 1-7
      All tests in Table 4 were shown to be near 100% specific except for the VitaPCR SARS-CoV-2 and QIAstat-Dx.
      • Fournier P.E.
      • Zandotti C.
      • Ninove L.
      • et al.
      Contribution of VitaPCR SARS-CoV-2 to the emergency diagnosis of COVID-19.
      J Clin Virol. 2020; 133: 337-339
      ,
      • Visseaux B.
      • Le Hingrat Q.
      • Collin G.
      • et al.
      Evaluation of the QIAstat-dx respiratory SARS-CoV-2 Panel, the first rapid multiplex PCR commercial assay for SARS-CoV- 2 detection.
      J Clin Microbiol. 2020; 58: 1-5
      The VitaPCR gave a specificity of 94.7% in one study due to its increased sensitivity over the comparator assay, and a second study showed an improved sensitivity of 99%.
      • Fournier P.E.
      • Zandotti C.
      • Ninove L.
      • et al.
      Contribution of VitaPCR SARS-CoV-2 to the emergency diagnosis of COVID-19.
      J Clin Virol. 2020; 133: 337-339
      ,
      • Fitoussi F.
      • Dupont R.
      • Tonen-Wolyec S.
      • et al.
      Performances of the VitaPCRTM SARS-CoV-2 Assay during the second wave of the COVID-19 epidemic in France.
      J Med Virol. 2021; : 1-7
      The QIAStat-Dx gave a specificity of 93% compared with a WHO-recommended RT-PCR.
      • Visseaux B.
      • Le Hingrat Q.
      • Collin G.
      • et al.
      Evaluation of the QIAstat-dx respiratory SARS-CoV-2 Panel, the first rapid multiplex PCR commercial assay for SARS-CoV- 2 detection.
      J Clin Microbiol. 2020; 58: 1-5
      Evaluations often used small sample sets, due to a limited availability of reagents and used various SARS-CoV-2 reference controls, making LOD comparisons difficult. Reported LODs varied from 100 copies/ml for Xpert Xpress SARS-CoV-2 to 3000 genome copy equivalents for the Aries SARS-CoV-2.

      Luminex. ARIES SARS-CoV-2 Assay Package Insert. Available at: https://www.fda.gov/media/136693/download. 2020. Accessed June 28, 2021.

      Several platforms fail to achieve the MHRA TPP “acceptable” LOD criteria of 1000 copies/ml; GenomEra SARS-CoV-2, Flu A/B+ RSV at 2857 copies/mL,

      Abacus Diagnostica. GenomEra SARS-CoV-2 , Flu A/B + RSV Assay Kit Package Insert. Version 1.0. Available at: https://www.abacusdiagnostica.com/products/sars-cov-2-flu-a-b-rsv/. 2020. Accessed June 23, 2021

      Novodiag COVID-19

      MobiDiag. Complete solution for rapid molecular diagnostics of coronavirus infection. Available at: https://mobidiag.com/products/coronavirus/#Novodiag-COVID-19. 2021. Accessed June 14, 2021.

      at 1815 copies/mL when using collection devices other than the provided medium nucleic acid amplification test;

      MobiDiag. Novodiag COVID-19 Instructions For Use. Document Version 6-0. 2020.

      and both the GenMark ePlex SARS-CoV-2

      GenMark Dx. EPlex SARS-CoV-2 Test Assay Manual. Available at: https://www.fda.gov/media/136282/download. 2020. Accessed June 28, 2021.

      and the QIAstat-Dx Respiratory SARS-CoV-2 Panel

      Qiagen. QIAstat-Dx Respiratory Panel. Instructions for Use. Available at: https://www.qiagen.com/us/resources/download.aspx?id=e3f2dc10-c712-4bcf-9acd-211bd35df944&lang=en. 2020. Accessed June 28, 2021.

      at 1000 copies/ml.
      The main limitations of the rapid sample-to-answer platforms include their high cost per test and low sample throughput. Moreover, despite their low complexity, rapid platforms are not infallible, and they are sensitive molecular tests that can be compromised without meticulous sample processing and good laboratory practice. Notably, BioFire and ePlex platforms do not output Ct values, meaning there is no indication of SARS-CoV-2 viral burden that can be of interest to the clinician as higher viral loads have been associated with increased SARS-CoV-2 mortality.
      • Magleby R.
      • Westblade L.F.
      • Trzebucki A.
      • et al.
      Impact of severe acute respiratory syndrome Coronavirus 2 Viral load on risk of intubation and mortality among hospitalized patients with coronavirus disease 2019.
      Clin Infect Dis. 2020;

      Stand-alone real-time polymerase chain reaction kits

      One of the biggest barriers to the implementation of SARS-CoV-2 testing in non-specialist laboratories early in the pandemic was the availability of the correct equipment to enable the rapid introduction of testing. The solution to this problem for many manufacturers was the rapid introduction to the market of stand-alone assays encompassing kits, which include the reagents necessary for reverse-transcription PCR, including controls, but that are not tied to a specific extraction or PCR platform. They offer flexibility over more “closed” systems as they can potentially be run on existing instrumentation, precluding the requirement for purchasing new and often expensive equipment. Use of such reagents requires more extensive validation than end-to-end systems, and the onus on providing this validation, including sample preparation and the compatibility of any instrumentation with a particular kit, will fall on the individual laboratory. Some suppliers provide details of compatible platforms, but many do not, and it is this lack of data that have allowed many substandard kits to enter the market. Over 200 CE-marked manual RT-PCR kits are listed on the COVID-19 In Vitro Diagnostic Medical Devices database,
      European Commission
      European commission COVID-19 in vitro diagnostic devices and test methods database.
      https://covid-19-diagnostics.jrc.ec.europa.eu
      Date: 2020
      a selection of which are shown in Table 5 along with some of their main attributes.
      • Freire-Paspuel B.
      • Vega-Mariño P.
      • Velez A.
      • et al.
      Analytical and clinical comparison of Viasure (CerTest Biotec) and 2019-nCoV CDC (IDT) RT-qPCR kits for SARS-CoV2 diagnosis.
      Virology. 2021; 553: 154-156
      ,
      • Visseaux B.
      • Le Q.
      • Collin G.
      • et al.
      Evaluation of the RealStar® SARS-CoV-2 RT-PCR kit RUO performances and limit of detection.
      J Clin Virol. 2020; 129: 104520
      , 

      Public Health England. Rapid Assessment of the genetic signatures EasyScreen SARS -CoV-2 detection kit. Available at: https://www.gov.uk/government/publications/covid-19-phe-laboratory-assessments-of-molecular-tests. 2020. Accessed June 23, 2021.

      , 
      • Freire-Paspuel B.
      • Garcia-Bereguiain M.A.
      Analytical sensitivity and clinical performance of a triplex RT-qPCR assay using CDC N1, N2, and RP targets for SARS-CoV-2 diagnosis.
      Int J Infect Dis. 2021; 102: 14-16
      , 
      • Kenyeres B.
      • Anosi N.
      • Banyai K.
      • et al.
      Comparison of four PCR and two point of care assays used in the laboratory detection of SARS-CoV-2.
      J Virol Methods. 2021; : 293
      , 
      • Görzer I.
      • Buchta C.
      • Chiba P.
      • et al.
      First results of a national external quality assessment scheme for the detection of SARS-CoV-2 genome sequences.
      J Clin Virol. 2020; 129https://doi.org/10.1016/j.jcv.2020.104537
      , 
      • Labbé A.C.
      • Benoit P.
      • Gobeille Paré S.
      • et al.
      Comparison of saliva with oral and nasopharyngeal swabs for SARS-CoV-2 detection on various commercial and laboratory-developed assays.
      J Med Virol. 2021; : 1-6https://doi.org/10.1002/jmv.27026
      , 
      • Garg A.
      • Ghoshal U.
      • Patel S.S.
      • et al.
      Evaluation of seven commercial RT-PCR kits for COVID-19 testing in pooled clinical specimens.
      J Med Virol. 2021; 93: 2281-2286
      , 
      • Price T.K.
      • Bowland B.C.
      • Chandrasekaran S.
      • et al.
      Performance characteristics of severe acute respiratory syndrome coronavirus 2 RT-PCR tests in a single health system.
      J Mol Diagn. 2021; 23: 159-163
      , 
      • Cuong H.Q.
      • Hai N.D.
      • Linh H.T.
      • et al.
      Comparison of primer-probe sets among different master mixes for laboratory screening of severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2).
      Biomed Res Int. 2020; : 2020https://doi.org/10.1155/2020/7610678
      , 
      • Procop G.W.
      • Brock J.E.
      • Reineks E.Z.
      • et al.
      A comparison of five SARS-CoV-2 molecular assays with clinical correlations.
      Am J Clin Pathol. 2021; 155: 69-78
      , 
      • Wirden M.
      • Feghoul L.
      • Bertine M.
      • et al.
      Multicenter comparison of the Cobas 6800 system with the RealStar RT-PCR kit for the detection of SARS-CoV-2.
      J Clin Virol. 2020; : 130
      , 
      • Kohmer N.
      • Rabenau H.F.
      • Hoehl S.
      • et al.
      Comparative analysis of point-of-care, high-throughput and laboratory-developed SARS-CoV-2 nucleic acid amplification tests (NATs).
      J Virol Methods. 2021; : 291https://doi.org/10.1016/j.jviromet.2021.114102
      , 
      • Lee C.K.
      • Tham J.W.M.
      • Png S.
      • et al.
      Clinical performance of Roche xobas 6800, Luminex ARIES, MiRXES fortitude kit 2.1, altona RealStar, and applied Biosystems TaqPath for SARS-CoV-2 detection in nasopharyngeal swabs.
      J Med Virol. 2021; : 1-5https://doi.org/10.1002/jmv.26940
      , 
      • van Kasteren P.B.
      • van der Veer B.
      • van den Brink S.
      • et al.
      Comparison of seven commercial RT-PCR diagnostic kits for COVID-19.
      J Clin Virol. 2020; : 128
      , 
      • Merindol N.
      • Pépin G.
      • Marchand C.
      • et al.
      SARS-CoV-2 detection by direct rRT-PCR without RNA extraction.
      J Clin Virol. 2020; 128: 3-6
      , 
      • Schnuriger A.
      • Perrier M.
      • Marinho V.
      • et al.
      Caution in interpretation of SARS-CoV-2 quantification based on RT-PCR cycle threshold value.
      Diagn Microbiol Infect Dis. 2021; 100: 2-5https://doi.org/10.1016/j.diagmicrobio.2021.115366
      , 

      Public Health England. Rapid assessment of biomerieux real-time detection kit. Available at: https://www.gov.uk/government/publications/covid-19-phe-laboratory-assessments-of-molecular-tests. 2020. Accessed June 23, 2021.

      , 
      • Tastanova A.
      • Stoffel C.I.
      • Dzung A.
      • et al.
      A comparative study of real-time RT-PCR–based SARS-CoV-2 detection methods and its application to human-derived and surface swabbed material.
      J Mol Diagn. 2021; 23: 796-804
      Table 5An overview of stand-alone RT-PCR suppliers and kits available in the EU. Details are taken from company websites and/or accompanying literature
      SupplierKit NameTarget 1Target 2Target 3Internal ControlNo’ of Tests/KitCompatible PlatformsAnalytical SensitivityReferences
      AltonaRealStar® SARS-CoV-2 Virus RT-PCR Kit 1.0ESManufacturer SPC384/4800Bio-Rad CFX96,

      Bio-Rad CFX96 deep-well,

      ABI QuantStudio,

      ABI 7500,

      Roche LightCyler 480, Qiagen Rotor-Gene Q      		E = 0.025 pfu/mL

      S = 0.014 pfu/mL
      • Visseaux B.
      • Le Q.
      • Collin G.
      • et al.
      Evaluation of the RealStar® SARS-CoV-2 RT-PCR kit RUO performances and limit of detection.
      J Clin Virol. 2020; 129: 104520


      • Wirden M.
      • Feghoul L.
      • Bertine M.
      • et al.
      Multicenter comparison of the Cobas 6800 system with the RealStar RT-PCR kit for the detection of SARS-CoV-2.
      J Clin Virol. 2020; : 130


      • Kohmer N.
      • Rabenau H.F.
      • Hoehl S.
      • et al.
      Comparative analysis of point-of-care, high-throughput and laboratory-developed SARS-CoV-2 nucleic acid amplification tests (NATs).
      J Virol Methods. 2021; : 291https://doi.org/10.1016/j.jviromet.2021.114102


      • Lee C.K.
      • Tham J.W.M.
      • Png S.
      • et al.
      Clinical performance of Roche xobas 6800, Luminex ARIES, MiRXES fortitude kit 2.1, altona RealStar, and applied Biosystems TaqPath for SARS-CoV-2 detection in nasopharyngeal swabs.
      J Med Virol. 2021; : 1-5https://doi.org/10.1002/jmv.26940


      • van Kasteren P.B.
      • van der Veer B.
      • van den Brink S.
      • et al.
      Comparison of seven commercial RT-PCR diagnostic kits for COVID-19.
      J Clin Virol. 2020; : 128


      • Merindol N.
      • Pépin G.
      • Marchand C.
      • et al.
      SARS-CoV-2 detection by direct rRT-PCR without RNA extraction.
      J Clin Virol. 2020; 128: 3-6
      Anatolia Geneworks

      /Launch      		Bosphore Novel Coronavirus (2019-nCoV) Detection Kit v4Orf1abNERNAse P50/100Not statedorf1ab = 0.86 copies/ul

      N = 0.82 copies/ul

      E = 1.02 copies/ul
      • Schnuriger A.
      • Perrier M.
      • Marinho V.
      • et al.
      Caution in interpretation of SARS-CoV-2 quantification based on RT-PCR cycle threshold value.
      Diagn Microbiol Infect Dis. 2021; 100: 2-5https://doi.org/10.1016/j.diagmicrobio.2021.115366
      BiomaximaSARS-CoV-2 Real-Time PCR LAB-KITTMOrf1abNManufacturer SPC96 (12 × 8 well strips)"Open PCR systems"10 copies/reactionNo literature found
      Indicates that using the kit name in combination with either "COVID-19″ or "SARS CoV-2″ as the search term in PubMed and Google Scholar yielded no significant results.
      BioMerieuxArgene SARS Cov-2 R-GeneNRdRpEEndogenous (HPRT1) and Manufacturer SPC120ABI 7500,

      ABI QuantStudio5,

      Roche LightCycler 480, Bio-Rad CFX96,

      Qiagen Rotor-Gene Q      		0.43 TCID50/mL (equivalent to 380 copies/mL).

      Public Health England. Rapid assessment of biomerieux real-time detection kit. Available at: https://www.gov.uk/government/publications/covid-19-phe-laboratory-assessments-of-molecular-tests. 2020. Accessed June 23, 2021.

      Bio-RadReliance SARS-CoV-2 RT-PCR Assay KitN1N2RNAse P200Bio-Rad CFX96,

      ABI 7500      		125–250 copies/mlNo literature found
      Indicates that using the kit name in combination with either "COVID-19″ or "SARS CoV-2″ as the search term in PubMed and Google Scholar yielded no significant results.
      ClonitQuanty COVID-19 v2 (quantitative)N1N2RNAse P96ABI 7500,

      Qiagen Rotor Gene Q,

      Bio-Rad CFX96      		Not statedNo literature found
      Indicates that using the kit name in combination with either "COVID-19″ or "SARS CoV-2″ as the search term in PubMed and Google Scholar yielded no significant results.
      ClonitCOVID 19 HT Screen (qualitative)N1N2Manufacturer SPC96ABI 7500,

      Qiagen Rotor Gene Q,

      Bio-Rad CFX96      		Not statedNo literature found
      Indicates that using the kit name in combination with either "COVID-19″ or "SARS CoV-2″ as the search term in PubMed and Google Scholar yielded no significant results.
      EuroimmunEuroRealTime SARS-CoV-2Orf1abNManufacturer SPC25–1000Roche LightCycler 480,

      ABI 7500,

      Bio-Rad CFX 96,

      Qiagen Rotor-Gene Q, qTower 3      		1 copy/ul
      • Tastanova A.
      • Stoffel C.I.
      • Dzung A.
      • et al.
      A comparative study of real-time RT-PCR–based SARS-CoV-2 detection methods and its application to human-derived and surface swabbed material.
      J Mol Diagn. 2021; 23: 796-804
      Genetic SignaturesEasyScreen SARS-CoV-2 Detection KitNEManufacturer SPC96ABI Quantstudio 5Not stated

      Public Health England. Rapid Assessment of the genetic signatures EasyScreen SARS -CoV-2 detection kit. Available at: https://www.gov.uk/government/publications/covid-19-phe-laboratory-assessments-of-molecular-tests. 2020. Accessed June 23, 2021.

      IDT2019-nCov CDC AssayN1N2RNAse P96ABI 75001–3 copies/ul
      • Freire-Paspuel B.
      • Vega-Mariño P.
      • Velez A.
      • et al.
      Analytical and clinical comparison of Viasure (CerTest Biotec) and 2019-nCoV CDC (IDT) RT-qPCR kits for SARS-CoV2 diagnosis.
      Virology. 2021; 553: 154-156


      • Freire-Paspuel B.
      • Garcia-Bereguiain M.A.
      Analytical sensitivity and clinical performance of a triplex RT-qPCR assay using CDC N1, N2, and RP targets for SARS-CoV-2 diagnosis.
      Int J Infect Dis. 2021; 102: 14-16
      MenariniCorona MELTOrf1abOrf1abHuman GADPH100Most commercial Real Time PCR instruments20 copies/reactionNo literature found
      Indicates that using the kit name in combination with either "COVID-19″ or "SARS CoV-2″ as the search term in PubMed and Google Scholar yielded no significant results.
      Perkin ElmerSARS-CoV-2 Real-time RT-PCR AssayOrf1abNMS248Bio-Rad CFX96/385,

      ABI 7500,

      ABI QuantStudio, qTower 3      		20 copies/mlNo literature found
      Indicates that using the kit name in combination with either "COVID-19″ or "SARS CoV-2″ as the search term in PubMed and Google Scholar yielded no significant results.
      Primerdesigngenesig® COVID-19 2G Real-Time PCR assayOrf1abSManufacturer SPC96ABI 7500,

      Bio-Rad CFX Connect,

      Roche LightCycler 480, genesig® q32      		0.4 copies/ul
      • van Kasteren P.B.
      • van der Veer B.
      • van den Brink S.
      • et al.
      Comparison of seven commercial RT-PCR diagnostic kits for COVID-19.
      J Clin Virol. 2020; : 128


      • Kenyeres B.
      • Anosi N.
      • Banyai K.
      • et al.
      Comparison of four PCR and two point of care assays used in the laboratory detection of SARS-CoV-2.
      J Virol Methods. 2021; : 293


      • Görzer I.
      • Buchta C.
      • Chiba P.
      • et al.
      First results of a national external quality assessment scheme for the detection of SARS-CoV-2 genome sequences.
      J Clin Virol. 2020; 129https://doi.org/10.1016/j.jcv.2020.104537
      RIDA®GENESARS-CoV-2EManufacturer SPC100/200RIDA CYCLER,

      Roche LightCycler 480, Mx3005P,

      ABI 7500,

      Bio-Rad CFX96,

      Qiagen Rotor-Gene Q      		50 copies/reaction
      • Labbé A.C.
      • Benoit P.
      • Gobeille Paré S.
      • et al.
      Comparison of saliva with oral and nasopharyngeal swabs for SARS-CoV-2 detection on various commercial and laboratory-developed assays.
      J Med Virol. 2021; : 1-6https://doi.org/10.1002/jmv.27026
      SeegeneAllplex 2019-nCOVRdRpNEManufacturer SPC50/100Roche LightCycler 480 (minimum)1–4 copies/ul
      • Kohmer N.
      • Rabenau H.F.
      • Hoehl S.
      • et al.
      Comparative analysis of point-of-care, high-throughput and laboratory-developed SARS-CoV-2 nucleic acid amplification tests (NATs).
      J Virol Methods. 2021; : 291https://doi.org/10.1016/j.jviromet.2021.114102


      • van Kasteren P.B.
      • van der Veer B.
      • van den Brink S.
      • et al.
      Comparison of seven commercial RT-PCR diagnostic kits for COVID-19.
      J Clin Virol. 2020; : 128


      • Merindol N.
      • Pépin G.
      • Marchand C.
      • et al.
      SARS-CoV-2 detection by direct rRT-PCR without RNA extraction.
      J Clin Virol. 2020; 128: 3-6


      • Garg A.
      • Ghoshal U.
      • Patel S.S.
      • et al.
      Evaluation of seven commercial RT-PCR kits for COVID-19 testing in pooled clinical specimens.
      J Med Virol. 2021; 93: 2281-2286
      SerosepRespibio SARS-CoV-2Not statedNot stated96Roche LightCycler 480,

      ABI 7500      		Not statedNo literature found
      Indicates that using the kit name in combination with either "COVID-19″ or "SARS CoV-2″ as the search term in PubMed and Google Scholar yielded no significant results.
      ThermofisherTaqPath COVID-19 CE-IVD RT-PCR Kit,SNorf1abMS2Up to 1000 (96- and 384-well format)ABI 7500,

      ABI Quantstudio 5      		10 genome copy equivalents/reaction
      • Lee C.K.
      • Tham J.W.M.
      • Png S.
      • et al.
      Clinical performance of Roche xobas 6800, Luminex ARIES, MiRXES fortitude kit 2.1, altona RealStar, and applied Biosystems TaqPath for SARS-CoV-2 detection in nasopharyngeal swabs.
      J Med Virol. 2021; : 1-5https://doi.org/10.1002/jmv.26940


      • Garg A.
      • Ghoshal U.
      • Patel S.S.
      • et al.
      Evaluation of seven commercial RT-PCR kits for COVID-19 testing in pooled clinical specimens.
      J Med Virol. 2021; 93: 2281-2286


      • Price T.K.
      • Bowland B.C.
      • Chandrasekaran S.
      • et al.
      Performance characteristics of severe acute respiratory syndrome coronavirus 2 RT-PCR tests in a single health system.
      J Mol Diagn. 2021; 23: 159-163
      TIBMOL BIOLDual Target SARSNEUBC Human mRNA96Roche LightCycler 480Not stated
      • Cuong H.Q.
      • Hai N.D.
      • Linh H.T.
      • et al.
      Comparison of primer-probe sets among different master mixes for laboratory screening of severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2).
      Biomed Res Int. 2020; : 2020https://doi.org/10.1155/2020/7610678


      • Procop G.W.
      • Brock J.E.
      • Reineks E.Z.
      • et al.
      A comparison of five SARS-CoV-2 molecular assays with clinical correlations.
      Am J Clin Pathol. 2021; 155: 69-78
      ViaSure (CerTest Biotech)SARS-CoV-2 Real Time PCROrf1abNNot stated96"Most open PCR systems"1–10 copies/reaction
      • Freire-Paspuel B.
      • Vega-Mariño P.
      • Velez A.
      • et al.
      Analytical and clinical comparison of Viasure (CerTest Biotec) and 2019-nCoV CDC (IDT) RT-qPCR kits for SARS-CoV2 diagnosis.
      Virology. 2021; 553: 154-156
      VirCellSARS-CoV-2 Real Time PCR KitNERNAse P48"Most open PCR systems"3–5 copies/reactionNo literature found
      Indicates that using the kit name in combination with either "COVID-19″ or "SARS CoV-2″ as the search term in PubMed and Google Scholar yielded no significant results.
      Abbreviations: N, nucleocapsid; E, envelope protein; S, spike glycoprotein; ORF1 a/b, open reading frame 1 a/b; RdRP, RNA-dependent RNA polymerase; SPC, sample process control.
      a Indicates that using the kit name in combination with either "COVID-19″ or "SARS CoV-2″ as the search term in PubMed and Google Scholar yielded no significant results.
      Kit formats are broadly similar and include minimal necessary reagents (primer/probe mixes, controls). Reagents may be provided either lyophilized or “wet” most commonly in tubes but also as eight-well strips. Although earlier kits relied on a single viral gene target, these have now been largely superseded by dual or triple target assays that focus on some combination of the E, N, S, and Orf1a genes. Although this has made the assays more robust in dealing with the emergence of novel SARS-CoV-2 variants, it has also complicated the interpretation of results when some gene targets fail to amplify. Furthermore, most kits supply an internal control (IC), which may be either endogenous (eg RNase P)
      • Freire-Paspuel B.
      • Vega-Mariño P.
      • Velez A.
      • et al.
      Analytical and clinical comparison of Viasure (CerTest Biotec) and 2019-nCoV CDC (IDT) RT-qPCR kits for SARS-CoV2 diagnosis.
      Virology. 2021; 553: 154-156
      ,
      • Freire-Paspuel B.
      • Garcia-Bereguiain M.A.
      Analytical sensitivity and clinical performance of a triplex RT-qPCR assay using CDC N1, N2, and RP targets for SARS-CoV-2 diagnosis.
      Int J Infect Dis. 2021; 102: 14-16
      ,
      • Cuong H.Q.
      • Hai N.D.
      • Linh H.T.
      • et al.
      Comparison of primer-probe sets among different master mixes for laboratory screening of severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2).
      Biomed Res Int. 2020; : 2020https://doi.org/10.1155/2020/7610678
      ,
      • Procop G.W.
      • Brock J.E.
      • Reineks E.Z.
      • et al.
      A comparison of five SARS-CoV-2 molecular assays with clinical correlations.
      Am J Clin Pathol. 2021; 155: 69-78
      ,
      • Schnuriger A.
      • Perrier M.
      • Marinho V.
      • et al.
      Caution in interpretation of SARS-CoV-2 quantification based on RT-PCR cycle threshold value.
      Diagn Microbiol Infect Dis. 2021; 100: 2-5https://doi.org/10.1016/j.diagmicrobio.2021.115366
      or exogenous (eg MS2),
      • Garg A.
      • Ghoshal U.
      • Patel S.S.
      • et al.
      Evaluation of seven commercial RT-PCR kits for COVID-19 testing in pooled clinical specimens.
      J Med Virol. 2021; 93: 2281-2286
      ,
      • Price T.K.
      • Bowland B.C.
      • Chandrasekaran S.
      • et al.
      Performance characteristics of severe acute respiratory syndrome coronavirus 2 RT-PCR tests in a single health system.
      J Mol Diagn. 2021; 23: 159-163
      ,
      • Lee C.K.
      • Tham J.W.M.
      • Png S.
      • et al.
      Clinical performance of Roche xobas 6800, Luminex ARIES, MiRXES fortitude kit 2.1, altona RealStar, and applied Biosystems TaqPath for SARS-CoV-2 detection in nasopharyngeal swabs.
      J Med Virol. 2021; : 1-5https://doi.org/10.1002/jmv.26940
      which can be used either as full process controls or solely as PCR controls. Some kits include both endogenous and exogenous ICs

      Public Health England. Rapid assessment of biomerieux real-time detection kit. Available at: https://www.gov.uk/government/publications/covid-19-phe-laboratory-assessments-of-molecular-tests. 2020. Accessed June 23, 2021.

      although some fail to disclose the IC origin.
      • Visseaux B.
      • Le Hingrat Q.
      • Collin G.
      • et al.
      Evaluation of the QIAstat-dx respiratory SARS-CoV-2 Panel, the first rapid multiplex PCR commercial assay for SARS-CoV- 2 detection.
      J Clin Microbiol. 2020; 58: 1-5
      ,
      • Visseaux B.
      • Le Q.
      • Collin G.
      • et al.
      Evaluation of the RealStar® SARS-CoV-2 RT-PCR kit RUO performances and limit of detection.
      J Clin Virol. 2020; 129: 104520
      ,

      Public Health England. Rapid Assessment of the genetic signatures EasyScreen SARS -CoV-2 detection kit. Available at: https://www.gov.uk/government/publications/covid-19-phe-laboratory-assessments-of-molecular-tests. 2020. Accessed June 23, 2021.

      ,
      • Kenyeres B.
      • Anosi N.
      • Banyai K.
      • et al.
      Comparison of four PCR and two point of care assays used in the laboratory detection of SARS-CoV-2.
      J Virol Methods. 2021; : 293
      ,
      • Görzer I.
      • Buchta C.
      • Chiba P.
      • et al.
      First results of a national external quality assessment scheme for the detection of SARS-CoV-2 genome sequences.
      J Clin Virol. 2020; 129https://doi.org/10.1016/j.jcv.2020.104537
      ,
      • Kohmer N.
      • Rabenau H.F.
      • Hoehl S.
      • et al.
      Comparative analysis of point-of-care, high-throughput and laboratory-developed SARS-CoV-2 nucleic acid amplification tests (NATs).
      J Virol Methods. 2021; : 291https://doi.org/10.1016/j.jviromet.2021.114102
      , 
      • Lee C.K.
      • Tham J.W.M.
      • Png S.
      • et al.
      Clinical performance of Roche xobas 6800, Luminex ARIES, MiRXES fortitude kit 2.1, altona RealStar, and applied Biosystems TaqPath for SARS-CoV-2 detection in nasopharyngeal swabs.
      J Med Virol. 2021; : 1-5https://doi.org/10.1002/jmv.26940
      , 
      • van Kasteren P.B.
      • van der Veer B.
      • van den Brink S.
      • et al.
      Comparison of seven commercial RT-PCR diagnostic kits for COVID-19.
      J Clin Virol. 2020; : 128
      , 
      • Merindol N.
      • Pépin G.
      • Marchand C.
      • et al.
      SARS-CoV-2 detection by direct rRT-PCR without RNA extraction.
      J Clin Virol. 2020; 128: 3-6
      ,
      • Tastanova A.
      • Stoffel C.I.
      • Dzung A.
      • et al.
      A comparative study of real-time RT-PCR–based SARS-CoV-2 detection methods and its application to human-derived and surface swabbed material.
      J Mol Diagn. 2021; 23: 796-804
      The number of tests per kit ranges from 48 to 4800 allowing for a wide range of throughputs although this will also depend on the number of wells required per sample and whether they are being tested in 96- or 384-well format. Many assays exploiting RT-PCR can typically use up to four different fluorescent reporter dyes, including the IC, but others are not so comprehensively multiplexed and require two or even three wells for each sample. At least one kit (Menarini)

      A.Menarini Diagnostics. CORONAMELT; 2020. Available at: https://www.menarinidiagnostics.com/en-us/Home/Laboratory-products/COVID-19/Viral-RNA-Detection/CoronaMelt/Overview. Accessed June 28, 2021.

      uses melt curve analysis in preference to hydrolysis probes, negating the requirement for multiple fluorescent reporter dyes. Although not shown in Table 5, many SARS-CoV-2 kits are also formulated as multiplexes with other respiratory viruses, most commonly influenza and respiratory syncytial virus (RSV), for example, Altona,
      Altona Diagnostics. The altona Diagnostics product portfolio.
      https://altona-diagnostics.com/en/the-altona-product-lines.html
      Date: 2021
      Viasure,

      CerTest BioTec. VIASURE real time PCR detection kits SARS-CoV-2, FLU & RSV. Available at: https://www.certest.es/products/sars-cov-2-flu-rsv/, 2021. Accessed June 28, 2021.

      and ThermoFisher.
      Thermo Fisher scientific. TaqManTM SARS-CoV-2, Flu A/B, RSV RT-PCR assay kit.
      https://www.thermofisher.com/order/catalog/product/A47702#/A47702https://www.thermofisher.com/order/catalog/product/A47702#/A47702
      Date: 2021
      This will usually require the addition of an extra well for each sample and/or the use of a single dye for multiple gene targets of the same virus. The actual throughput for these assays will depend heavily on the extraction and PCR equipment chosen for use and the level of automation. Use of an automated end-to-end system like the Roche FLOW could produce in excess of 1000 results in a 24hr period from experience in our local laboratory.
      Owing to the pressure to manufacture diagnostic kits rapidly as the pandemic took hold, much of the technical and clinical validation data used minimal data sets. Unlike the rapid platforms that are in widespread use, peer-reviewed literature is sparse for many stand-alone kits and in some cases completely absent. For those referenced assays in Table 5, the LOD was most commonly in the range of 1–20 copies/reaction although this was liable to small variations depending on the extraction and eluate volume and the volume of eluate used in the PCR. When comparisons between kits using clinical samples or External Quality Assurance (EQA) samples were performed, most kits performed comparably with only small variations in results between the Altona,
      • Visseaux B.
      • Le Hingrat Q.
      • Collin G.
      • et al.
      Evaluation of the QIAstat-dx respiratory SARS-CoV-2 Panel, the first rapid multiplex PCR commercial assay for SARS-CoV- 2 detection.
      J Clin Microbiol. 2020; 58: 1-5
      ,
      • Visseaux B.
      • Le Q.
      • Collin G.
      • et al.
      Evaluation of the RealStar® SARS-CoV-2 RT-PCR kit RUO performances and limit of detection.
      J Clin Virol. 2020; 129: 104520
      ,
      • Wirden M.
      • Feghoul L.
      • Bertine M.
      • et al.
      Multicenter comparison of the Cobas 6800 system with the RealStar RT-PCR kit for the detection of SARS-CoV-2.
      J Clin Virol. 2020; : 130
      , 
      • Kohmer N.
      • Rabenau H.F.
      • Hoehl S.
      • et al.
      Comparative analysis of point-of-care, high-throughput and laboratory-developed SARS-CoV-2 nucleic acid amplification tests (NATs).
      J Virol Methods. 2021; : 291https://doi.org/10.1016/j.jviromet.2021.114102
      , 
      • Lee C.K.
      • Tham J.W.M.
      • Png S.
      • et al.
      Clinical performance of Roche xobas 6800, Luminex ARIES, MiRXES fortitude kit 2.1, altona RealStar, and applied Biosystems TaqPath for SARS-CoV-2 detection in nasopharyngeal swabs.
      J Med Virol. 2021; : 1-5https://doi.org/10.1002/jmv.26940
      , 
      • van Kasteren P.B.
      • van der Veer B.
      • van den Brink S.
      • et al.
      Comparison of seven commercial RT-PCR diagnostic kits for COVID-19.
      J Clin Virol. 2020; : 128
      , 
      • Merindol N.
      • Pépin G.
      • Marchand C.
      • et al.
      SARS-CoV-2 detection by direct rRT-PCR without RNA extraction.
      J Clin Virol. 2020; 128: 3-6
      Integrated DNA Technologies (IDT),
      • Freire-Paspuel B.
      • Vega-Mariño P.
      • Velez A.
      • et al.
      Analytical and clinical comparison of Viasure (CerTest Biotec) and 2019-nCoV CDC (IDT) RT-qPCR kits for SARS-CoV2 diagnosis.
      Virology. 2021; 553: 154-156
      ,
      • Freire-Paspuel B.
      • Garcia-Bereguiain M.A.
      Analytical sensitivity and clinical performance of a triplex RT-qPCR assay using CDC N1, N2, and RP targets for SARS-CoV-2 diagnosis.
      Int J Infect Dis. 2021; 102: 14-16
      Seegene,
      • Garg A.
      • Ghoshal U.
      • Patel S.S.
      • et al.
      Evaluation of seven commercial RT-PCR kits for COVID-19 testing in pooled clinical specimens.
      J Med Virol. 2021; 93: 2281-2286
      ,
      • Kohmer N.
      • Rabenau H.F.
      • Hoehl S.
      • et al.
      Comparative analysis of point-of-care, high-throughput and laboratory-developed SARS-CoV-2 nucleic acid amplification tests (NATs).
      J Virol Methods. 2021; : 291https://doi.org/10.1016/j.jviromet.2021.114102
      ,
      • van Kasteren P.B.
      • van der Veer B.
      • van den Brink S.
      • et al.
      Comparison of seven commercial RT-PCR diagnostic kits for COVID-19.
      J Clin Virol. 2020; : 128
      ,
      • Merindol N.
      • Pépin G.
      • Marchand C.
      • et al.
      SARS-CoV-2 detection by direct rRT-PCR without RNA extraction.
      J Clin Virol. 2020; 128: 3-6
      TaqPath,
      • Garg A.
      • Ghoshal U.
      • Patel S.S.
      • et al.
      Evaluation of seven commercial RT-PCR kits for COVID-19 testing in pooled clinical specimens.
      J Med Virol. 2021; 93: 2281-2286
      ,
      • Price T.K.
      • Bowland B.C.
      • Chandrasekaran S.
      • et al.
      Performance characteristics of severe acute respiratory syndrome coronavirus 2 RT-PCR tests in a single health system.
      J Mol Diagn. 2021; 23: 159-163
      ,
      • Lee C.K.
      • Tham J.W.M.
      • Png S.
      • et al.
      Clinical performance of Roche xobas 6800, Luminex ARIES, MiRXES fortitude kit 2.1, altona RealStar, and applied Biosystems TaqPath for SARS-CoV-2 detection in nasopharyngeal swabs.
      J Med Virol. 2021; : 1-5https://doi.org/10.1002/jmv.26940
      Viasure,
      • Freire-Paspuel B.
      • Vega-Mariño P.
      • Velez A.
      • et al.
      Analytical and clinical comparison of Viasure (CerTest Biotec) and 2019-nCoV CDC (IDT) RT-qPCR kits for SARS-CoV2 diagnosis.
      Virology. 2021; 553: 154-156
      and Tib MolBiol kits.
      • Cuong H.Q.
      • Hai N.D.
      • Linh H.T.
      • et al.
      Comparison of primer-probe sets among different master mixes for laboratory screening of severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2).
      Biomed Res Int. 2020; : 2020https://doi.org/10.1155/2020/7610678
      ,
      • Procop G.W.
      • Brock J.E.
      • Reineks E.Z.
      • et al.
      A comparison of five SARS-CoV-2 molecular assays with clinical correlations.
      Am J Clin Pathol. 2021; 155: 69-78
      Specificity was 100% in virtually all cases.
      Stand-alone kits offer a convenient alternative to more closed systems allowing rapid implementation on existing equipment. However, despite a broad agreement in the performance of these assays on clinical specimens, the sheer number of kits available means that in-house validation is essential before implementation as a clinical service.

      Low-throughput testing platforms

      The use of stand-alone PCR kits is not always an attractive option for laboratories, particularly if the existing molecular diagnostic infrastructure is not in place. Manufacturers identified a niche in the market for automated low-to-medium input end-to-end solutions, which could be easily introduced to laboratories with minimal molecular diagnostic experience. All platforms assessed here use multiplex RT-PCR with all assays containing an IC except the Virokey SARS-CoV-2, which contains neither an endogenous nor manufacturer-provided IC (Table 6).
      Vela diagnostics. ViroKey SARS-CoV-2 RT-PCR test v2.0 (CE-IVD).
      https://www.veladx.com/product/qpcr-respiratory-infections/virokey-sars-cov-2-rt-pcr-test-v20-ce-ivd.html
      Date: 2021
      False-negative results will not be identified by the failure to include an IC to demonstrate either sample adequacy or PCR failure. The Qiagen NeuMoDx has the best throughput of these systems at 435 samples in 24hr and also has the advantage of being a true random access platform with a quick time to result of only 1hr 25 min.
      • Lima A.
      • Healer V.
      • Vendrone E.
      • et al.
      Validation of a modified CDC assay and performance comparison with the NeuMoDxTM and DiaSorin® automated assays for rapid detection of SARS-CoV-2 in respiratory specimens.
      J Clin Virol. 2020 Dec; 133: 104688https://doi.org/10.1016/j.jcv.2020.104688
      Table 6An overview of low- to mid-throughput end-to-end testing platforms for SARS-CoV-2
      Supplier/PlatformAssayTarget 1Target 2Internal ControlAnalytical SensitivityBatch SizePlatform Run TimeThroughput 24hrReferences
      Mobidiag Amplidiag EasyAmplidiag COVID-19Orf1NRNAse P313 copies/ml483.5 h288
      MobiDiag. Coronavirus solutions.
      https://mobidiag.com/products/coronavirus/
      Date: 2021


      MobiDiag. Amplidiag COVID-19 Instructions for Use. 2020.



      • Jokela P.
      • Jääskeläinen A.E.
      • Jarva H.
      • et al.
      SARS-CoV-2 sample-to-answer nucleic acid testing in a tertiary care emergency department: evaluation and utility.
      J Clin Virol. 2020; 131https://doi.org/10.1016/j.jcv.2020.104614
      BD MAXBD SARS-CoV-2N1N2RNAse P640 genomic copy equivalents242.5 h216

      Becton Dickinson & Company. SARS-CoV-2 Reagents for BD MAX System Instructions for Use. Available at: https://www.fda.gov/media/136816/download. 2020. Accessed June 23, 2021.

      EliTech Elite InGeniusSARS-CoV-2 PLUS ELITe MGB KitOrf1abOrf8RNAse P111 genomic copy equivalents122.5 h108
      EliTech Group
      SARS-CoV-2 PLUS ELITe MGB kit.
      https://www.elitechgroup.com/product/sars-cov-2-plus-elite-mgb-kit
      Date: 2020
      ViaSure (CerTest Biotech)SARS-CoV-2 (N1 + N2) – BD MAXN1N2RNAse P≥ 5 genome copies per reaction242.5 h216

      CerTest BioTec. SARS-CoV-2 (N1 + N2) for BD MAXTM System Instructions for Use. Available at: https://www.certest.es/products/sars-cov-2-n1-n2-bd-maxtm-system/. 2021. Accessed June 23, 2021.

      Vela Diagnostics SentosaViroKey SARS-CoV-2 RT-PCR Test v2.0Orf1aNNone200 genome equivalents/ml464 h276
      Vela diagnostics. ViroKey SARS-CoV-2 RT-PCR test v2.0 (CE-IVD).
      https://www.veladx.com/product/qpcr-respiratory-infections/virokey-sars-cov-2-rt-pcr-test-v20-ce-ivd.html
      Date: 2021
      Aus Diagnostics HighPlex 24SARS-CoV-2 influenza and RSV 8-wellOrf1Orf8Endogenous and Manufacturer SPC2150–4325 copies/ml244.5 h120

      Aus Diagnostics. SARS-COV-2, INFLUENZA AND RSV 8-WELL Instructions for Use. 2021.



      Aus diagnostics. Highplex AllianceTM.
      https://www.ausdiagnostics.com/highplex-alliance
      Date: 2021
      NeuMoDx™NeuMoDx™ SARS-CoV-2 AssayNsp2NManufacturer SPC200
      • Kim D.
      • Lee J.-Y.
      • Yang J.-S.
      • et al.
      The architecture of SARS-CoV-2 transcriptome.
      Cell. 2020; 181: 914-921.e10
      		Random Access1 h 25 min435
      • Mostafa H.H.
      • Hardick J.
      • Morehead E.
      • et al.
      Comparison of the analytical sensitivity of seven commonly used commercial SARS-CoV-2 automated molecular assays.
      J Clin Virol. 2020; 130https://doi.org/10.1016/j.jcv.2020.104578
      Abbreviations: N, nucleocapsid; ORF1 a/b, open reading frame 1 a/b; Orf 8, open reading frame 8; SPC, sample process control.
      Peer-reviewed literature for these platforms is significantly lacking over all other investigated areas with most performance data presented here being sourced from the manufacturer’s literature. The BD MAX system can use a variety of kits from different manufacturers including SARS-CoV-2 in isolation or with other respiratory pathogens such as influenza. The BD MAX SARS-CoV-2 assays, including the ViaSure SARS-CoV-2 N1 + N2 assay, have repeatedly shown 100% sensitivity but the specificity of greater than 95% both in manufacturers post-market surveillance and in real-world data. Fears around the production of false-positive results led the FDA to release a product notice recommending confirmation of all positive results generated by the BD MAX; however, both of the assessed assays are based on the CDC N gene assay, which has been shown to be highly sensitive.

      CerTest BioTec. SARS-CoV-2 (N1 + N2) for BD MAXTM System Instructions for Use. Available at: https://www.certest.es/products/sars-cov-2-n1-n2-bd-maxtm-system/. 2021. Accessed June 23, 2021.

      ,

      Becton Dickinson & Company. SARS-CoV-2 Reagents for BD MAX System Instructions for Use. Available at: https://www.fda.gov/media/136816/download. 2020. Accessed June 23, 2021.

      ,
      • Navarathna D.H.
      • Sharp S.
      • Lukey J.
      • et al.
      Understanding false positives and the detection of SARS-CoV-2 using the Cepheid Xpert Xpress SARS-CoV-2 and BD MAX SARS-CoV-2 assays.
      Diagn Microbiol Infect Dis. 2021; 100https://doi.org/10.1016/j.diagmicrobio.2021.115334
      The Amplidiag COVID-19 assay was highly sensitive showing greater than 98% agreement compared directly with Cobas 6800 SARS-CoV-2. All other assessed platforms as shown in Table 6 were also found to have acceptable sensitivity and specificity of greater than 96% based on manufacturer’s data only.
      Vela diagnostics. ViroKey SARS-CoV-2 RT-PCR test v2.0 (CE-IVD).
      https://www.veladx.com/product/qpcr-respiratory-infections/virokey-sars-cov-2-rt-pcr-test-v20-ce-ivd.html
      Date: 2021
      ,
      • Lima A.
      • Healer V.
      • Vendrone E.
      • et al.
      Validation of a modified CDC assay and performance comparison with the NeuMoDxTM and DiaSorin® automated assays for rapid detection of SARS-CoV-2 in respiratory specimens.
      J Clin Virol. 2020 Dec; 133: 104688https://doi.org/10.1016/j.jcv.2020.104688
      ,
      EliTech Group
      SARS-CoV-2 PLUS ELITe MGB kit.
      https://www.elitechgroup.com/product/sars-cov-2-plus-elite-mgb-kit
      Date: 2020
      , 

      Aus Diagnostics. SARS-COV-2, INFLUENZA AND RSV 8-WELL Instructions for Use. 2021.

      , 
      • Mannonen L.
      • Kallio-Kokko H.
      • Loginov R.
      • et al.
      Comparison of two commercial platforms and a laboratory-developed test for detection of severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) RNA.
      J Mol Diagn. 2021; 23: 407-416
      , 
      • Mostafa H.H.
      • Hardick J.
      • Morehead E.
      • et al.
      Comparison of the analytical sensitivity of seven commonly used commercial SARS-CoV-2 automated molecular assays.
      J Clin Virol. 2020; 130https://doi.org/10.1016/j.jcv.2020.104578
      All assessed platforms were shown to have good analytical sensitivity as outlined in Table 6 with the exception of Aus Diagnostics SARS-CoV-2, influenza, and RSV, which has an LOD on 2150 to 4325 copies/ml.

      Aus Diagnostics. SARS-COV-2, INFLUENZA AND RSV 8-WELL Instructions for Use. 2021.

      Real-world testing of the Amplidiag COVID-19 also highlighted a failure to detect an EQA sample at 3300 copies/ml suggesting the manufacturer published LOD of 313 copies/ml may not be reliable.
      • Mannonen L.
      • Kallio-Kokko H.
      • Loginov R.
      • et al.
      Comparison of two commercial platforms and a laboratory-developed test for detection of severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) RNA.
      J Mol Diagn. 2021; 23: 407-416
      Local verification of the manufacturer’s claims is important before the introduction of any test into routine use to ensure discrepancies such as this are detected.
      The expected 24hr throughput for these systems is modest, and these systems are likely to be sited in laboratories that do not undertake 24/7 working meaning their full potential cannot be met. Although this may be the case, these automated solutions can offer easy-to-use solutions for laboratories with limited molecular experience. This has been important in providing the ability to decrease time to result over sending samples to specialist reference laboratories for testing, which in turn can reduce transmission risk particularly in health care settings.

      High-throughput testing platforms

      Several high-throughput platforms have been introduced for the detection of SARS-CoV-2 RNA offering end-to-end automated testing of samples from nucleic acid extraction through to amplification and detection. The introduction of high-throughput screening platforms into laboratories can improve laboratory efficiency and turnaround times while reducing staff hands-on time
      • Aretzweiler G.
      • Leuchter S.
      • Simon C.O.
      • et al.
      Generating timely molecular diagnostic test results: workflow comparison of the cobas® 6800/8800 to Panther.
      Expert Rev Mol Diagn. 2019; 19: 951-957
      and facilitating a substantial increase in a testing capacity. The main high-throughput testing platforms and associated assays are listed in Table 7. All are RT-PCR-based assays except the Hologic Aptima SARS-CoV-2 assay that use TMA. All assays listed use a minimum of two different SARS-CoV-2 targets to reduce the risk of false negatives due to primer/probe mismatches caused by sequence variability.
      • Khan K.A.
      • Cheung P.
      Presence of mismatches between diagnostic PCR assays and coronavirus SARS-CoV-2 genome: sequence mismatches in SARS-CoV-2 PCR.
      R Soc Open Sci. 2020; 7: 200636
      Multiple comparisons between the high-throughput platforms and standard RT-PCR demonstrate a high level of diagnostic performance. The Panther Fusion had an overall agreement of 96.4% compared with the Roche Cobas 6800 SARS-CoV-2 assay
      • Craney A.R.
      • Velu P.D.
      • Satlin M.J.
      • et al.
      Comparison of two high-throughput reverse transcription-PCR systems for the detection of severe acute respiratory syndrome coronavirus 2.
      J Clin Microbiol. 2020; 58: 1-6
      with a similar finding in a separate study.
      • Lieberman J.
      • Pepper G.
      • Naccache S.
      • et al.
      Comparison of commercially available and laboratory developed assays for in vitro detection of SARS-CoV-2 in clinical laboratories.
      J Clin Microbiol. 2020; 58: e00821-20
      An agreement of 98.3% was found when comparing the Cobas to the Abbott Alinity M SARS-CoV-2 AMP,
      • Kogoj R.
      • Kmetič P.
      • Valenčak A.O.
      • et al.
      Real-life head-to-head comparison of performance of two high-throughput automated assays for detection of SARS-CoV-2 RNA in nasopharyngeal swabs: the Alinity m SARS-CoV-2 and cobas 6800 SARS-CoV-2 assays.
      J Mol Diagn. 2021; https://doi.org/10.1016/j.jmoldx.2021.05.003
      and in a three-way comparison between these platforms and the Panther Fusion, the overall agreement was 99.7%.
      • Perchetti G.A.
      • Pepper G.
      • Shrestha L.
      • et al.
      Performance characteristics of the Abbott Alinity m SARS-CoV-2 assay.
      J Clin Virol. 2021; 140: 104869
      When the TMA-based Aptima assay was compared with the Panther Fusion and rapid low-throughput BioFire Defense COVID-19 test, it produced a positive percent agreement of 98.7% compared with the consensus and a 100% agreement for negative results.
      • Smith E.
      • Zhen W.
      • Manji R.
      • et al.
      Analytical and clinical comparison of three nucleic acid amplification tests for SARS-CoV-2 detection.
      J Clin Microbiol. 2020; 58: e01134-20
      Table 7An overview of high-throughput molecular diagnostic platforms for SARS-CoV-2
      PlatformAssayTarget 1Target 2Target 3Internal ControlAnalytical Sensitivity SARS-CoV-2 RNA c/mlPlatform Run TimeThroughput 24hrLoadingReferences
      Abbott m2000Abbott RealTime SARS-CoV-2RdRpNManufacturer SPC53
      • Fung B.
      • Gopez A.
      • Servellita V.
      • et al.
      Direct comparison of SARS-CoV-2 analytical limits of detection across seven molecular assays.
      J Clin Microbiol. 2020; 58: e01535-20
      		4 h470Batch
      • Fung B.
      • Gopez A.
      • Servellita V.
      • et al.
      Direct comparison of SARS-CoV-2 analytical limits of detection across seven molecular assays.
      J Clin Microbiol. 2020; 58: e01535-20
      Abbott Alinity MSARS-CoV-2 AMP KitRdRpNManufacturer SPC (DNA)50
      • Perchetti G.A.
      • Pepper G.
      • Shrestha L.
      • et al.
      Performance characteristics of the Abbott Alinity m SARS-CoV-2 assay.
      J Clin Virol. 2021; 140: 104869
      		2 h 35 min to first results1080Random Access
      • Perchetti G.A.
      • Pepper G.
      • Shrestha L.
      • et al.
      Performance characteristics of the Abbott Alinity m SARS-CoV-2 assay.
      J Clin Virol. 2021; 140: 104869
      Hologic Panther®Aptima® SARS-CoV-2 AssayOrf1Ab Region 1Orf1ab Region 2Manufacturer SPC83–194
      • Pham J.
      • Meyer S.
      • Nguyen C.
      • et al.
      Performance characteristics of a high-throughput automated.
      J Clin Microbiol. 2020; : 1-6
      ,
      • Schneider M.
      • Iftner T.
      • Ganzenmueller T.
      Evaluation of the analytical performance and specificity of a SARS-CoV-2 transcription-mediated amplification assay.
      J Virol Methods. 2021; 294: 114182
      		3.5 h to the first result1150Batch
      • Pham J.
      • Meyer S.
      • Nguyen C.
      • et al.
      Performance characteristics of a high-throughput automated.
      J Clin Microbiol. 2020; : 1-6


      • Schneider M.
      • Iftner T.
      • Ganzenmueller T.
      Evaluation of the analytical performance and specificity of a SARS-CoV-2 transcription-mediated amplification assay.
      J Virol Methods. 2021; 294: 114182
      Hologic Panther Fusion®Panther Fusion® SARS-CoV-2 AssayOrf1Ab Region 1Orf1ab Region 2Manufacturer SPC74–100
      • Fung B.
      • Gopez A.
      • Servellita V.
      • et al.
      Direct comparison of SARS-CoV-2 analytical limits of detection across seven molecular assays.
      J Clin Microbiol. 2020; 58: e01535-20
      ,
      • Zhen W.
      • Manji R.
      • Smith E.
      • et al.
      Comparison of four molecular in vitro diagnostic assays for the detection of sars-cov-2 in nasopharyngeal specimens.
      J Clin Microbiol. 2020; 58
      ,
      • Wong R.C.W.
      • Wong A.H.
      • Ho Y.I.I.
      • et al.
      Performance evaluation of Panther Fusion SARS-CoV-2 assay for detection of SARS-CoV-2 from deep throat saliva, nasopharyngeal, and lower-respiratory-tract specimens.
      J Med Virol. 2021; 93: 1226-1228
      		2.4 h to first results1440Random Access
      • Fung B.
      • Gopez A.
      • Servellita V.
      • et al.
      Direct comparison of SARS-CoV-2 analytical limits of detection across seven molecular assays.
      J Clin Microbiol. 2020; 58: e01535-20


      • Zhen W.
      • Manji R.
      • Smith E.
      • et al.
      Comparison of four molecular in vitro diagnostic assays for the detection of sars-cov-2 in nasopharyngeal specimens.
      J Clin Microbiol. 2020; 58


      • Wong R.C.W.
      • Wong A.H.
      • Ho Y.I.I.
      • et al.
      Performance evaluation of Panther Fusion SARS-CoV-2 assay for detection of SARS-CoV-2 from deep throat saliva, nasopharyngeal, and lower-respiratory-tract specimens.
      J Med Virol. 2021; 93: 1226-1228
      Roche Cobas® 6800cobas® SARS-CoV-2Orf1abEManufacturer SPC<10–85
      • Fung B.
      • Gopez A.
      • Servellita V.
      • et al.
      Direct comparison of SARS-CoV-2 analytical limits of detection across seven molecular assays.
      J Clin Microbiol. 2020; 58: e01535-20
      ,
      • Dust K.
      • Hedley A.
      • Nichol K.
      • et al.
      Comparison of commercial assays and laboratory developed tests for detection of SARS-CoV-2.
      J Virol Methods. 2020; 285
      		3.4 h to first results1440Batch
      • Fung B.
      • Gopez A.
      • Servellita V.
      • et al.
      Direct comparison of SARS-CoV-2 analytical limits of detection across seven molecular assays.
      J Clin Microbiol. 2020; 58: e01535-20


      • Dust K.
      • Hedley A.
      • Nichol K.
      • et al.
      Comparison of commercial assays and laboratory developed tests for detection of SARS-CoV-2.
      J Virol Methods. 2020; 285
      Roche Cobas® 88004128Batch
      Cepheid InfinityXpert Xpress SARS-CoV-2NEManufacturer SPC10050 min per cartridgeUp to 1920Random Access
      Cepheid. GeneXpert-Infinity.
      https://www.cepheid.com/en_US/systems/GeneXpert-Family-of-Systems/GeneXpert-Infinity
      Date: 2021
      Thermofisher AmplitudeTaqPath COVID-19 HTS geneNOrf1abMS2N/A3 h 30 min to first result8000BatchNo literature found
      Comparing analytical sensitivity is difficult due to differences in methods between studies, but generally all have high analytical sensitivities with LODs of 200 copies/ml or below, as collated from several studies and listed in Table 1. The TMA-based Aptima assay was shown to have a lower LOD when compared with standard RT-PCR,
      • Gorzalski A.J.
      • Tian H.
      • Laverdure C.
      • et al.
      High-Throughput Transcription-mediated amplification on the Hologic Panther is a highly sensitive method of detection for SARS-CoV-2.
      J Clin Virol. 2020; 129: 104501
      although when compared directly against the Roche Cobas and Abbott m2000, the Cobas test had the lowest LOD,
      • Mostafa H.H.
      • Hardick J.
      • Morehead E.
      • et al.
      Comparison of the analytical sensitivity of seven commonly used commercial SARS-CoV-2 automated molecular assays.
      J Clin Virol. 2020; 130https://doi.org/10.1016/j.jcv.2020.104578
      a similar finding when the Cobas was directly compared with the Abbott m2000 and Panther Fusion.
      • Fung B.
      • Gopez A.
      • Servellita V.
      • et al.
      Direct comparison of SARS-CoV-2 analytical limits of detection across seven molecular assays.
      J Clin Microbiol. 2020; 58: e01535-20
      All systems offer a throughput of 1000 samples or more in a 24hr period. The highest throughput systems are the Roche Cobas 8800 system and the recently introduced Thermofisher Amplitude running the Taqpath COVID-19 assay, which claims a very high throughput of 8000 samples from a single platform over 24 hours. The Taqpath COVID-19 assay has been evaluated as a standard RT-PCR
      • Garg A.
      • Ghoshal U.
      • Patel S.S.
      • et al.
      Evaluation of seven commercial RT-PCR kits for COVID-19 testing in pooled clinical specimens.
      J Med Virol. 2021; 93: 2281-2286
      assay, but no published data exist for the diagnostic performance of the complete Amplitude system. Assays for these high-throughput platforms are being updated to include additional respiratory targets to meet the predicted increases in RSV and seasonal influenza infections once nonpharmaceutical interventions for COVID are removed.
      • Baker R.E.
      • Park S.W.
      • Yang W.
      • et al.
      The impact of COVID-19 nonpharmaceutical interventions on the future dynamics of endemic infections.
      Proc Natl Acad Sci U S A. 2020; 117: 30547-30553
      These include the Roche Cobas SARS-CoV-2 and Influenza A/B for the 6800/800 systems, the Aptima SARS-CoV-2/Flu Assay for the Hologic Panther system, and the m RESP-4-PLEX ASSAY for the Abbott Alinity system.
      • Cheng A.
      • Riedel S.
      • Arnaout R.
      • et al.
      Verification of the Abbott Alinity m Resp-4-Plex Assay for detection of SARS-CoV-2, influenza A/B, and respiratory syncytial virus.
      medRxiv. 2021;
      The Cepheid GeneXpert infinity platform can give users the option to run up to 80 Xpert Xpress SARS-CoV-2 cartridges simultaneously with no increase in run time over the smaller cepheid instruments making this a high-throughput low complexity solution for laboratory settings.
      Cepheid. GeneXpert-Infinity.
      https://www.cepheid.com/en_US/systems/GeneXpert-Family-of-Systems/GeneXpert-Infinity
      Date: 2021

      Severe acute respiratory syndrome coronavirus-2 genotyping

      All viruses mutate, particularly RNA viruses, and the infection rate of SARS-Cov-2 on a large susceptible population has greatly increased the opportunity for mutations to occur. These mutations have led to variants of concern (VOCs) emerging with the potential of enhanced fitness, specifically toward increased transmissibility
      • Shahhosseini N.
      • Babuadze G.
      • Wong G.
      • et al.
      Mutation signatures and in silico docking of novel sars-cov-2 variants of concern.
      Microorganisms. 2021; 9: 1-15https://doi.org/10.3390/microorganisms9050926
      ,
      • Davies N.G.
      • Abbott S.
      • Barnard R.
      • et al.
      Estimated transmissibility and impact of SARS-CoV-2 lineage B.1.1.7 in England.
      Science. 2021; 372
      and vaccine evasion.
      • Tegally H.
      • Wilkinson E.
      • Giovanetti M.
      • 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; 2https://doi.org/10.1101/2020.12.21.20248640
      , 
      • Andreano E.
      • Piccini G.
      • Licastro D.
      • et al.
      SARS-CoV-2 escape in vitro from a highly neutralizing COVID-19 convalescent plasma.
      bioRxiv. 2020; https://doi.org/10.1101/2020.12.28.424451
      , 
      • Greaney A.J.
      • Loes A.N.
      • Crawford K.H.D.
      • et al.
      Comprehensive mapping of mutations in the SARS-CoV-2 receptor-binding domain that affect recognition by polyclonal human plasma antibodies.
      Cell Host Microbe. 2021; 29: 463-476.e6
      , 
      • Ramanathan K.
      • Antognini D.
      • Combes A.
      • et al.
      Comprehensive mapping of mutations in the SARS CoV- 2 receptor-binding domain that affect recognition by polyclonal human plasma antibodies.
      Cell Host Microbe. 2020; : 19-21
      , 
      • Weisblum Y.
      • Schmidt F.
      • Zhang F.
      • et al.
      Escape from neutralizing antibodies 1 by SARS-CoV-2 spike protein variants.
      Elife. 2020; 9: 1
      The first VOC (B.1.1.7—Alpha) was detected in the south of England and sequenced in September 2020.

      Rambaut A, Loman N, Pybus O, et al. Preliminary genomic characterisation of an emergent SARS-CoV-2 lineage in the UK defined by a novel set of spike mutations. Available at: https://virological.org/t/preliminary-genomic-characterisation-of-an-emergent-sars-cov-2-lineage-in-the-uk-defined-by-a-novel-set-of-spike-mutations/563. 2021. Accessed June 23, 2021.

      Soon after, new VOCs were identified from various locations across the world, each VOC becoming a prominent strain within their area of origin.
      • Sabino E.C.
      • Buss L.F.
      • Carvalho M.P.S.
      • et al.
      Resurgence of COVID-19 in Manaus, Brazil, despite high seroprevalence.
      Lancet. 2021; 397: 452-455
      Genomic sequencing is an invaluable tool in managing the pandemic due to its ability to detect unknown variations, which may indicate the emergence of a new VOC and the need for the development of new diagnostic assays. The United Kingdom currently sequences all SARS-CoV-2-positive samples where it is technically achievable; however, it can be slow, technically demanding, and currently has limited global availability.

      Royal College of Pathologists, COVID-19 testing, 2020, A National Strategy. Available at: https://www.rcpath.org/profession/on-the-agenda/covid-19-testing-a-national-strategy.html. 2020. Accessed June 23, 2021.

      One solution to identifying known SARS-CoV-2 lineages without the need for genomic sequencing is the development of real-time genotyping PCR assays.
      Rapid real-time genotyping PCR assays usually target a single nucleotide polymorphism (SNP), with the most discriminatory targets often located within the S-gene. These types of mutations invariably lead to nonsynonymous amino acid substitutions. SNPs within this region can cause changes in the receptor-binding motif with successful variants retaining an increased affinity of the S-protein to the human angiotensin 2 receptor (ACE2).

      Rambaut A, Loman N, Pybus O, et al. Preliminary genomic characterisation of an emergent SARS-CoV-2 lineage in the UK defined by a novel set of spike mutations. Available at: https://virological.org/t/preliminary-genomic-characterisation-of-an-emergent-sars-cov-2-lineage-in-the-uk-defined-by-a-novel-set-of-spike-mutations/563. 2021. Accessed June 23, 2021.

      ,
      • Yi C.
      • Sun X.
      • Ye J.
      • et al.
      Key residues of the receptor binding motif in the spike protein of SARS-CoV-2 that interact with ACE2 and neutralizing antibodies.
      Cell Mol Immunol. 2020; 17: 621-630
      , 
      • Cagliani R.
      • Forni D.
      • Clerici M.
      • et al.
      Computational inference of selection underlying the evolution of the novel coronavirus, severe acute respiratory syndrome coronavirus 2.
      J Virol. 2020; 94: e00411-20
      , 
      • Ou J.
      • Zhou Z.
      • Dai R.
      • et al.
      Emergence of rbd mutations in circulating sars-cov-2 strains enhancing the structural stability and human ace2 receptor affinity of the spike protein.
      bioRxiv. 2020; : 1-30
      Identification of these distinct mutations can be used as markers to detect specific VOC lineages.
      It is often the case that one distinct mutation may be present in several VOCs. For example, the presence of the N501Y mutation alone can be distinctive of the B.1.1.7 lineage, but the N501Y is also present in the B.1.351 and P1 VOC alongside the E484 K and K417 N or K417 T mutations, respectively; although the E484 K mutation is also occasionally seen in the B.1.1.7 lineage. It is often necessary to assay multiple targets to reliably determine the likely SARS-CoV-2 lineage. The range of SNP assays used will need to be modified as the new VOC are identified through whole-genome sequencing strategies.
      Public Health England currently uses the Applied Biosystems (Waltham, Massachusetts, USA) RT-PCR genotyping assay for the rapid detection of variants. This genotyping assay has a sufficient repertoire of target mutations to reliably cover all the major VOC currently recognized by the WHO and most of the variants of interest.
      World Health Organization
      Tracking SARS-CoV-2 variants.
      https://www.who.int/en/activities/tracking-SARS-CoV-2-variants/
      Date: 2021
      ,
      Thermo Fisher scientific. TaqMan SARS-CoV-2 mutation Panel.
      https://www.thermofisher.com/uk/en/home/clinical/clinical-genomics/pathogen-detection-solutions/real-time-pcr-research-solutions-sars-cov-2/mutation-panel.html
      Date: 2021
      The current selection consists of 32 assays that can detect 30 SNPs and 2 deletions. Each assay is duplex in format detecting the mutant and the original SARS-CoV-2 reference/wild-type sequence on two different fluorescent dye layers. The high specificity of each assay target results in a significant reduction in the sensitivity, and it is advised by the manufacturer to only use extracted RNA from specimens with a CT of ≤30 where this information is available.
      Thermo Fisher scientific. TaqMan SARS-CoV-2 mutation Panel.
      https://www.thermofisher.com/uk/en/home/clinical/clinical-genomics/pathogen-detection-solutions/real-time-pcr-research-solutions-sars-cov-2/mutation-panel.html
      Date: 2021
      There are several VOC assays in development or in early stages of marketing as shown in Table 8, many of which exist in stand-alone format to allow a reactive and rapid introduction of new SNP assays to the market as dictated by circulating variants. Agena Bioscience has developed the MassARRAY SARS-CoV-2 Variant Panel capable of detecting 15 variants over 36 gene targets in a two-well multiplex end-point RT-PCR assay.
      Bioscience A. MassARRAY® SARS-CoV-2 variant Panel (RUO).
      https://www.agenabio.com/wp-content/uploads/2021/03/GEN0048-05-SC2-Variant-Product-Sheet.pdf
      Date: 2021
      Table 8A small selection of SNP PCR assays available in Europe for the detection of SARS-CoV-2 variants of concern
      ManufacturerAssayTargetsVariantReferences
      EliTechSARS-CoV-2 Variants ELITe MGB® Kit
      • •
        S gene, E484 K
      • •
        S gene, N501Y
      		Alpha
      EliTech Group
      SARS-CoV-2 variants ELITe MGB® kit.
      https://www.elitechgroup.com/product/sars-cov-2-variants-elite-mgb-kit
      ViaSure (CerTest Biotech)SARS-CoV-2 & UK Variant
      • •
        HV 69/70 s gene deletion
      		Alpha
      CerTest BioTec. SARS-CoV-2 & UK variant (S UK, ORF1ab and N genes).
      https://www.certest.es/products/sars-cov-2-uk-variant-s-uk-orf1ab-and-n-genes/
      Date: 2021
      Anatolia Geneworks/LaunchBosphore SARS-CoV-2 UK. Variant Detection Kit
      • •
        A570D
      • •
        P681H
      • •
        Y144del
      		Alpha
      Antolia geneworks. Bosphore SARS-CoV-2 variant detection kit v1.
      http://www.anatoliageneworks.com/en/kitler.asp?id=375&baslik=Bosphore SARS-CoV-2 Variant Detection Kit v1&bas=Bosphore SARS-CoV-2 Variant Detection Kit v1
      Date: 2021
      ThermofisherTaqMan Custom SNP Assays
      • Bottom of Form
      • •
        D215 G
      • •
        D614 G
      • •
        HV 69/70 s gene deletion
      • •
        Y144del
      • •
        E484 K
      • •
        E484Q
      • •
        F888 L
      • •
        K417 N
      • •
        K417 T
      • •
        L18 F
      • •
        L452 R
      • •
        N439 K
      • •
        N501Y
      • •
        P681H
      • •
        P681 R
      • •
        S13I
      • •
        S477 N
      • •
        T20 N
      • •
        V1176 F
      		Alpha

      Beta

      Gamma

      Delta

      Plus numerous variants of interest depending on combination used
      Thermo Fisher scientific. TaqMan SARS-CoV-2 mutation Panel.
      https://www.thermofisher.com/uk/en/home/clinical/clinical-genomics/pathogen-detection-solutions/real-time-pcr-research-solutions-sars-cov-2/mutation-panel.html
      Date: 2021


      World Health Organization
      Tracking SARS-CoV-2 variants.
      https://www.who.int/en/activities/tracking-SARS-CoV-2-variants/
      Date: 2021
      TIBMOL BIOLVirSNiP Assays
      • •
        H66D
      • •
        A67 V
      • •
        HV 69/70 s gene deletion
      • •
        D253 G
      • •
        K417 N
      • •
        K417 T
      • •
        L452 R
      • •
        Y453 F
      • •
        T478 K
      • •
        E484 K
      • •
        E484Q
      • •
        N501Y
      • •
        A570D
      • •
        P681H
      • •
        P681 R
      • •
        F888 L
      • •
        Q949 R
      • •
        V1176 F
      		Alpha

      Beta

      Gamma

      Delta

      Plus numerous variants of interest depending on combination used
      TIB MOLBIOL. SARS kits and VirSNiP assays.
      https://www.tib-molbiol.de/covid-19
      Date: 2021
      Agena BioscienceMassARRAY SARS-CoV-2 Variant Panel
      • •
        L452 R
      • •
        E484Q
      • •
        P681 R
      • •
        T478 K
      • •
        T19 R
      • •
        P681H
      • •
        N501Y
      • •
        A570D
      • •
        HV 69/70 s gene deletion
      • •
        S982 A
      • •
        T716I
      • •
        Y144del
      • •
        D80 A
      • •
        D215 G
      • •
        K417 N
      • •
        E484 K
      • •
        A701 V
      • •
        L18 F
      • •
        L242_L244del
      • •
        Q677H
      • •
        D253 G
      • •
        L5F
      • •
        T95I
      • •
        S477 N
      • •
        D80 G
      • •
        S13I
      • •
        W152 C
      • •
        N439 K
      • •
        K1191 N
      • •
        Q493 K
      • •
        I692 V
      • •
        Y453 F
      • •
        N501 T
      • •
        Q677P
      		15 variants of interest including:

      Alpha

      Beta

      Gamma

      Delta
      Bioscience A. MassARRAY® SARS-CoV-2 variant Panel (RUO).
      https://www.agenabio.com/wp-content/uploads/2021/03/GEN0048-05-SC2-Variant-Product-Sheet.pdf
      Date: 2021
      The use of SNP genotyping assays for the detection of SARS-CoV-2 VOC can be an effective early warning system for emerging VOC within a population, with quicker turnaround times compared with genomic sequencing. Data produced from this method can help scientists to quickly predict the prevalence of a VOC within a given population and may provide evidence toward vaccine effectiveness for new variants when collated with data regarding new infections or hospitalizations.

      Summary

      The COVID-19 pandemic will have a long-reaching impact on molecular diagnostic testing. The speed at which molecular diagnostics entered the market has been unrivaled with strategies suitable for all desired testing throughputs available within a few short months. The overall analytical and clinical accuracy data for solutions marketed within Europe have generally been found to be satisfactory although published LODs can be variable. At the outset of the pandemic manufacturers, claims were not required to be independently verified in Europe, and outside the most used rapid or high-throughput testing platforms, peer-reviewed real-world data are sparse. Welcome changes to regulations for devices in Europe are on the horizon, but local laboratory validations will still play a key role in the future. With the increasing prevalence of new SARS-CoV-2 VOC and the need for enhanced surveillance, there is still potential for new developments in SARS-CoV-2 molecular diagnostics.

      Clinics care points

      • •
        evere acute respiratory syndrome coronavirus-2 (SARS-CoV-2) required the rapid expansion of virological diagnostic techniques to ensure adequate testing capacity in the pandemic settings.
      • •
        Rapid, molecular diagnostic platforms fulfill an important niche in point-of-care settings and clinical laboratories. They provide quick accurate results require minimal hands-on time and permit on-demand testing of urgent specimens, which is pertinent for non-COVID patient care.
      • •
        High-throughput platforms improve laboratory efficiency and turnaround times while reducing staff hands-on time. This leads to an increase in the testing capacity of diagnostic laboratories to help meet the clinical demand throughout pandemics.
      • •
        The use of SNP genotyping assays for the detection of SARS-CoV-2 VOCs can be an effective early warning system for emerging VOCs within a population, with faster turnaround times compared with genomic sequencing. This can assist with public health surveillance and provide high-quality evidence toward vaccine effectiveness.

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      Publication history

      Published online: March 08, 2022

      Footnotes

      Funding: None declared.

      Conflict of interest: None declared.

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      DOI: https://doi.org/10.1016/j.cll.2022.02.005

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