Diagnostic accuracy of loop-mediated isothermal amplification coupled to nanopore sequencing (LamPORE) for the detection of SARS-CoV-2 infection at scale in symptomatic and asymptomatic populations

Anetta Ptasińska ,Seden Grippon,Alex Richter,Andrew Bosworth,Konstantinos Boukas,Emma L Wise,Nusreen Ahmad,Jade Forster,Charlotte Poxon,Emma Garratt,Tim Williams,Gemma Snell,Tony Williams,Emma L.A Howson,Daniel Burns,Keith M Godfrey,Angela Douglas,Rebecca Anderson,Nicholas Machin,Joe Parker,Nichola Graham,Doriana Cellura,Simon Friar,Alla Collins,Deborah Porter,Bryony Armson,Stephen P Kidd,Alice Goring,Claire Bryer,Hana Sheldon,Michael Kidd,Sue Hill,Sarah E Frampton ,Celina M Whalley ,Christopher Mattocks ,Veronica L Fowler ,Zandra C Deans ,Nicholas Cortes ,David W Cleary ,Rachel J Skilton ,Andrew D Beggs

doi:10.1016/j.cmi.2021.04.008

Abstract

ObjectivesRapid, high throughput diagnostics are a valuable tool, allowing the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in populations so as to identify and isolate people with asymptomatic and symptomatic infections. Reagent shortages and restricted access to high throughput testing solutions have limited the effectiveness of conventional assays such as quantitative RT-PCR (RT-qPCR), particularly throughout the first months of the coronavirus disease 2019 pandemic. We investigated the use of LamPORE, where loop-mediated isothermal amplification (LAMP) is coupled to nanopore sequencing technology, for the detection of SARS-CoV-2 in symptomatic and asymptomatic populations. MethodsIn an asymptomatic prospective cohort, for 3 weeks in September 2020, health-care workers across four sites (Birmingham, Southampton, Basingstoke and Manchester) self-swabbed with nasopharyngeal swabs weekly and supplied a saliva specimen daily. These samples were tested for SARS-CoV-2 RNA using the Oxford Nanopore LamPORE system and a reference RT-qPCR assay on extracted sample RNA. A second retrospective cohort of 848 patients with influenza-like illness from March 2020 to June 2020 were similarly tested from nasopharyngeal swabs. ResultsIn the asymptomatic cohort a total of 1200 participants supplied 23 427 samples (3966 swab, 19 461 saliva) over a 3-week period. The incidence of SARS-CoV-2 detection using LamPORE was 0.95%. Diagnostic sensitivity and specificity of LamPORE was >99.5% (decreasing to approximately 98% when clustered estimation was used) in both swab and saliva asymptomatic samples when compared with the reference RT-qPCR test. In the retrospective symptomatic cohort, the incidence was 13.4% and the sensitivity and specificity were 100%. ConclusionsLamPORE is a highly accurate methodology for the detection of SARS-CoV-2 in both symptomatic and asymptomatic population settings and can be used as an alternative to RT-qPCR.

Highlights

Coronavirus disease 2019, caused by an emergent novel betacoronavirus known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); represents a public health emergency [1].Rapid detection of infected cases in order to limit transmission, remains challenging as most validated methods use quantitative RT-PCR (RT-qPCR) [2]
SARS-CoV-2 reads were detected in the 0.2 copies/mL sample but this was below the threshold for calling a positive sample in LamPORE and were not detected with RT-qPCR (Table 1, Fig. 3)
We found that LamPORE has high sensitivity and specificity (>99%) in both the asymptomatic and symptomatic populations, directly comparable to RT-qPCR, and has comparable predictive ability across a range of uses in varying levels of population prevalence

Summary

Introduction

Coronavirus disease 2019, caused by an emergent novel betacoronavirus known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); represents a public health emergency [1].Rapid detection of infected cases in order to limit transmission, remains challenging as most validated methods use quantitative RT-PCR (RT-qPCR) [2]. Considered the reference standard for diagnosis, RT-qPCR is laborious and can be difficult to scale up for mass-testing; in addition, competition for reagents/equipment from many laboratories may lead to widespread reagent shortages. Laboratories throughout the UK used primers that were designed to target sequences within the RNAdependent polymerase gene (RdRp) [3]; these lacked sensitivity [4]. RT-qPCR primer sets were introduced that targeted the envelope (E), nucleocapsid (N) and ORF1ab genes, which provided the necessary increased sensitivity [5,6]. Loop-mediated isothermal amplification (LAMP) offers an alternative to RT-qPCR [7]. This reaction typically takes 20e30 minutes, which is considerably quicker than PCR [8]

Objectives

Results

Conclusion