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Previous studies have described RT-LAMP methodology for the rapid detection of SARS-CoV-2 in nasopharyngeal (NP) and oropharyngeal (OP) swab and saliva samples. This study describes the validation of an improved sample preparation method for extraction free RT-LAMP and defines the clinical performance of four different RT-LAMP assay formats for detection of SARS-CoV-2 within a multisite clinical evaluation. Direct RT-LAMP was performed on 559 swabs and 86,760 saliva samples and RNA RT-LAMP on extracted RNA from 12,619 swabs and 12,521 saliva from asymptomatic and symptomatic individuals across healthcare and community settings. For Direct RT-LAMP, overall diagnostic sensitivity (DSe) of 70.35% (95% CI 63.48-76.60%) on swabs and 84.62% (79.50-88.88%) on saliva was observed, with diagnostic specificity (DSp) of 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, DSe of 100% (96.34-100%) and 77.78% (70.99-83.62%) for swabs were observed, and 99.01% (94.61-99.97%) and 87.61% (82.69-91.54%) for saliva, respectively. For RNA RT-LAMP, overall DSe and DSp were 96.06% (92.88-98.12%) and 99.99% (99.95-100%) for swabs, and 80.65% (73.54-86.54%) and 99.99% (99.95-100%) for saliva, respectively. These findings demonstrate that RT-LAMP is applicable to a variety of use-cases, including frequent, interval-based testing of saliva with Direct RT-LAMP from asymptomatic individuals that may otherwise be missed using symptomatic testing alone.
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BackgroundSARS-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. MethodsWe 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. ResultsFive 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. InterpretationThe 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. FundingThe study was funded by Primer Design (UK), with kind contributions from all academic partners.
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IntroductionRapid, high throughput diagnostics are a valuable tool, allowing the detection of SARS-CoV-2 in populations, in order 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 reverse transcriptase quantitative PCR (RT-qPCR), particularly throughout the first months of the COVID-19 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 three 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 - June 2020, were similarly tested from nasopharyngeal swabs. ResultsIn the asymptomatic cohort a total of 1200 participants supplied 23,427 samples (3,966 swab, 19,461 saliva) over a three-week period. The incidence of SARS-CoV-2 detection using LamPORE was 0.95%. Diagnostic sensitivity and specificity of LamPORE was >99.5% in both swab and saliva asymptomatic samples when compared to 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.