A Prospective Diagnostic Study to Measure the Accuracy of Detection of SARS-CoV-2 Variants Of Concern (VOC) Utilising a Novel RT-PCR GENotyping Algorithm in an <i>in silico</i> Evaluation (VOC-GENIE)

Abstract

Background: SARS-CoV-2 variants of concern (VOCs) have been associated with higher rate of transmission, and evasion of immunisation and antibody therapeutics. Variant sequencing is widely utilized in the UK. However, only 0.5% (~650k) of the 133 million cumulative positive cases worldwide were sequenced (in GISAID) on 08 April 2021 with 97% from Europe and North America and only ~0.25% (~320k) were variant sequences. This may be due to the lack of availability, high cost, infrastructure and expert staff required for sequencing. Public health decisions based on a non-randomised sample of 0.5% of the population may be insufficiently powered, and subject to sampling bias and systematic error. In addition, sequencing is rarely available in situ in a clinically relevant timeframe and thus, is not currently compatible with diagnosis and treatment patient care pathways. Therefore, we investigated an alternative approach using polymerase chain reaction (PCR) genotyping to detect the key single nucleotide polymorphisms (SNPs) associated with increased transmission and immune evasion in SARS-CoV-2 variants.Methods: We investigated the utility of SARS-CoV-2 SNP detection with a panel of PCR-genotyping assays in a large data set of 640,482 SARS-CoV-2 high quality, full length sequences using a prospective in silico trial design and explored the potential impact of rapid in situ variant testing on the COVID-19 diagnosis and treatment patient pathway. Results: Five SNPs were selected by screening the published literature for a reported association with increased transmission and / or immune evasion. 344881 sequences contained one or more of the five SNPs. This algorithm of SNPs was found to be able to identify the four variants of concern (VOCs) and sequences containing the E484K and L452R escape mutations.Interpretation: The in silico analysis suggest that the key mutations and variants of SARS-CoV-2 may be reliably detected using a focused algorithm of biologically relevant SNPs. This highlights the potential for rapid in situ PCR genotyping to compliment or replace sequencing or to be utilized instead of sequences in settings where sequencing is not feasible, accessible or affordable. Rapid detection of variants with in situ PCR genotyping may facilitate a more effective COVID-19 diagnosis and treatment patient pathway. Funding: The study was funded by Primer Design (UK), with kind contributions from all academic partners.Declaration of Interests: Stephen Kidd, Nick Cortes, Nathan Moore, Kate Templeton, Alex Richter and Alice Goring have no conflicting interests. R.A Trevor, Daryl Borley, Paul Oladimeji, Prachi Teltumbde, Seden Grippon, and Aida Sanchez-Bretano are employees of Novacyt group, which is a medical diagnostics company operating in the COVID-19 variant testing field. R.A Trevor has no additional direct conflicts but is a shareholder in a number of un-related private and public companies that do not operate in the COVID-19 or diagnostics field. Joanne Martin has no direct conflicts of interest. She is a principal investigator of a care home trial using Novacyt rapid testing and National Specialty Advisor for Pathology for NHS England and Improvement. She is a director and shareholder of Biomoti a drug delivery company and has a shareholding in Glyconics, a diagnostics company.