ABSTRACT
Previous studies have described RT-LAMP methodology for the rapid detection of SARS-CoV-2 in nasopharyngeal/oropharyngeal swab and saliva samples. Here we describe the validation of an improved simple sample preparation method for Direct SARS-CoV-2 RT-LAMP, removing the need for RNA extraction, using 559 swabs and 86,760 saliva samples from asymptomatic and symptomatic individuals across multiple healthcare settings. Using this improved method we report a diagnostic sensitivity (DSe) of 70.35% (95% CI 63.48-76.60%) on swabs and 84.62% (79.50-88.88%) on saliva, with diagnostic specificity (DSp) 100% (98.98-100.00%) on swabs and 100% (99.72-100.00%) on saliva when compared to RT-qPCR. Analysing samples with RT-qPCR ORF1ab CT values of <25 and <33 (high and medium-high viral loads, respectively), we found DSe of 100% (96.34-100%) and 77.78% (70.99-83.62%) for swabs, and 99.01% (94.61-99.97%) and 87.32% (80.71-92.31%) for saliva. We also describe RNA RT-LAMP (on extracted RNA) performed on 12,619 swabs and 12,521 saliva samples to provide updated performance data with DSe and DSp of 95.98% (92.74-98.06%) and 99.99% (99.95-100%) for swabs, and 80.65% (73.54-86.54%) and 99.99% (99.95-100%) for saliva, respectively. We also report on daily samples collected from one individual from symptom onset where both Direct and RNA RT-LAMP detected SARS-CoV-2 in saliva collected on all six days where symptoms were recorded, with RNA RT-LAMP detecting SARS-CoV-2 for an additional further day. The findings from these studies demonstrate that RT-LAMP testing of swabs and saliva is potentially applicable to a variety of use-cases, including frequent, interval-based testing of saliva from asymptomatic individuals via Direct RT-LAMP that may be missed using symptomatic testing alone.
ABSTRACT
The COVID-19 pandemic has illustrated the importance of rapid, accurate diagnostic testing for the effective triaging and cohorting of patients and timely tracking and tracing of cases. However, a surge in diagnostic testing quickly resulted in worldwide competition for the same sample preparation and real-time RT-PCR diagnostic reagents (rRT-PCR). Consequently, Hampshire Hospitals NHS Foundation Trust, UK sought to diversify their diagnostic portfolio by exploring alternative amplification chemistries including those that permit direct testing without RNA extraction. This study describes the validation of a SARS-CoV-2 RT-LAMP assay, which is an isothermal, autocycling, strand displacement nucleic acid amplification technique which can be performed on extracted RNA (RNA RT-LAMP) or directly from swab (Direct RT-LAMP). Analytical specificity (ASp) of this new RT-LAMP assay was 100% and analytical sensitivity (ASe) was between 1x101 and 1x102 copies when using a synthetic DNA target. The overall diagnostic sensitivity (DSe) and specificity (DSp) of RNA RT LAMP was 97% and 99% respectively, relative to the standard of care (SoC) rRT-PCR. When a CT cut-off of 33 was employed, above which increasingly, evidence suggests there is a very low risk of patients shedding infectious virus, the diagnostic sensitivity was 100%. The DSe and DSp of Direct-RT LAMP was 67% and 97%, respectively. When setting CT cut-offs of [≤]33 and [≤]25, the DSe increased to 75% and 100%, respectively. Time from swab-to-result for a strong positive sample (CT < 25) was < 15 minutes. We propose that RNA RT-LAMP could replace rRT-PCR where there is a need for increase in throughput, whereas Direct RT-LAMP could be used as a screening tool for triaging patients into appropriate hospitals wards, at GP surgeries and in care homes, or for population screening to identify highly contagious individuals (super shedders). Direct RT-LAMP could also be used during times of high prevalence to save critical extraction and rRT-PCR reagents by screening out those strong positives from diagnostic pipelines.
Subject(s)
COVID-19ABSTRACT
We gratefully acknowledge the UK COVID-19 Genomics Consortium (COG UK) for funding, and Public Health Wales / Cardiff University and MRC-University of Glasgow Centre for Virus Research for making their COG-UK sequence data publicly available. COG-UK is supported by funding from the Medical Research Council (MRC) part of UK Research & Innovation (UKRI), the National Institute of Health Research (NIHR) and Genome Research Limited, operating as the Wellcome Sanger Institute. The research was supported by the Wellcome Trust Core Award Grant Number 203141/Z/16/Z with funding from the NIHR Oxford BRC. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. We are deeply grateful to Robert Esnouf and the BMRC Research Computing team for unfailing assistance with computational infrastructure. We also thank Benjamin Carpenter and James Docker for assistance in the laboratory, and Lorne Lonie, Maria Lopopolo, Chris Allen, John Broxholme and the WHG high-throughput genomics team for sequencing and quality control. The HIV clone p92BR025.8 was obtained through the Centre For AIDS Reagents from Drs Beatrice Hahn and Feng Gao, and the UNAIDS Virus Network (courtesy of the NIH AIDS Research and Reference Reagent Program). KAL is supported by The Wellcome Trust and The Royal Society (107652/Z/15/Z). MH, LF, MdC, GMC, NO, LAD, DB, CF and TG are supported by Li Ka Shing Foundation funding awarded to CF. PS is supported by a Wellcome Investigator Award (WT103767MA). SummarySARS-CoV-2, the causative agent of COVID-19, emerged in late 2019 causing a global pandemic, with the United Kingdom (UK) one of the hardest hit countries. Rapid sequencing and publication of consensus genomes have enabled phylogenetic analysis of the virus, demonstrating SARS-CoV-2 evolves relatively slowly1, but with multiple sites in the genome that appear inconsistent with the overall consensus phylogeny2. To understand these discrepancies, we used veSEQ3, a targeted RNA-seq approach, to quantify minor allele frequencies in 413 clinical samples from two UK locations. We show that SARS-CoV-2 infections are characterised by extensive within-host diversity, which is frequently shared among infected individuals with patterns consistent with geographical structure. These results were reproducible in data from two other sequencing locations in the UK, where we find evidence of mixed infection by major circulating lineages with patterns that cannot readily be explained by artefacts in the data. We conclude that SARS-CoV-2 diversity is transmissible, and propose that geographic patterns are generated by transient co-circulation of distinct viral populations. Co-transmission of mixed populations could open opportunities for resolving clusters of transmission and understanding pathogenesis.