Reverse-Transcription Loop-Mediated Isothermal Amplification Has High Accuracy for Detecting Severe Acute Respiratory Syndrome Coronavirus 2 in Saliva and Nasopharyngeal/Oropharyngeal Swabs from Asymptomatic and Symptomatic Individuals.

Previous studies have described reverse-transcription loop-mediated isothermal amplification (RT-LAMP) for the rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal/oropharyngeal swab and saliva samples. This multisite clinical evaluation describes the validation of an improved sample preparation method for extraction-free RT-LAMP and reports clinical performance of four RT-LAMP assay formats for SARS-CoV-2 detection. 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 samples from asymptomatic and symptomatic individuals across health care and community settings. For direct RT-LAMP, overall diagnostic sensitivity (DSe) was 70.35% (95% CI, 63.48%-76.60%) on swabs and 84.62% (95% CI, 79.50%-88.88%) on saliva, with diagnostic specificity of 100% (95% CI, 98.98%-100.00%) on swabs and 100% (95% CI, 99.72%-100.00%) on saliva, compared with quantitative RT-PCR (RT-qPCR); analyzing samples with RT-qPCR ORF1ab CT values of ≤25 and ≤33, DSe values were 100% (95% CI, 96.34%-100%) and 77.78% (95% CI, 70.99%-83.62%) for swabs, and 99.01% (95% CI, 94.61%-99.97%) and 87.61% (95% CI, 82.69%-91.54%) for saliva, respectively. For RNA RT-LAMP, overall DSe and diagnostic specificity were 96.06% (95% CI, 92.88%-98.12%) and 99.99% (95% CI, 99.95%-100%) for swabs, and 80.65% (95% CI, 73.54%-86.54%) and 99.99% (95% CI, 99.95%-100%) for saliva, respectively. These findings demonstrate that RT-LAMP is applicable to a variety of use cases, including frequent, interval-based direct RT-LAMP of saliva from asymptomatic individuals who may otherwise be missed using symptomatic testing alone.

Kidd SP ，Burns D ，Armson B ，Beggs AD ，Howson ELA ，Williams A ，Snell G ，Wise EL ，Goring A ，Vincent-Mistiaen Z ，Grippon S ，Sawyer J ，Cassar C ，Cross D ，Lewis T ，Reid SM ，Rivers S ，James J ，Skinner P ，Banyard A ，Davies K ，Ptasinska A ，Whalley C ，Ferguson J ，Bryer C ，Poxon C ，Bosworth A ，Kidd M ，Richter A ，Burton J ，Love H ，Fouch S ，Tillyer C ，Sowood A ，Patrick H ，Moore N ，Andreou M ，Morant N ，Houghton R ，Parker J ，Slater-Jefferies J ，Brown I ，Gretton C ，Deans Z ，Porter D ，Cortes NJ ，Douglas A ，Hill SL ，Godfrey KM ，Fowler VL ... -  《-》

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.

Rapid, 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. In 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. In 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%. LamPORE 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.

Ptasinska A ，Whalley C ，Bosworth A ，Poxon C ，Bryer C ，Machin N ，Grippon S ，Wise EL ，Armson B ，Howson ELA ，Goring A ，Snell G ，Forster J ，Mattocks C ，Frampton S ，Anderson R ，Cleary D ，Parker J ，Boukas K ，Graham N ，Cellura D ，Garratt E ，Skilton R ，Sheldon H ，Collins A ，Ahmad N ，Friar S ，Burns D ，Williams T ，Godfrey KM ，Deans Z ，Douglas A ，Hill S ，Kidd M ，Porter D ，Kidd SP ，Cortes NJ ，Fowler V ，Williams T ，Richter A ，Beggs AD ... -  《-》

Multisite Clinical Validation of Isothermal Amplification-Based SARS-CoV-2 Detection Assays Using Different Sampling Strategies.

Desai KT ，Alfaro K ，Mendoza L ，Faron M ，Mesich B ，Maza M ，Dominguez R ，Valenzuela A ，Acosta CD ，Martínez M ，Felix JC ，Masch R ，Smith JS ，Gabrilovich S ，Wu T ，Plump M ，Novetsky AP ，Einstein MH ，Douglas NC ，Cremer M ，Wentzensen N ... -  《Microbiology Spectrum》

Development and Clinical Application of a Rapid and Sensitive Loop-Mediated Isothermal Amplification Test for SARS-CoV-2 Infection.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak urgently necessitates sensitive and convenient COVID-19 diagnostics for the containment and timely treatment of patients. We aimed to develop and validate a novel reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay to detect SARS-CoV-2. Patients with suspected COVID-19 and close contacts were recruited from two hospitals between 26 January and 8 April 2020. Respiratory samples were collected and tested using RT-LAMP, and the results were compared with those obtained by reverse transcription-quantitative PCR (RT-qPCR). Samples yielding inconsistent results between these two methods were subjected to next-generation sequencing for confirmation. RT-LAMP was also applied to an asymptomatic COVID-19 carrier and patients with other respiratory viral infections. Samples were collected from a cohort of 129 cases (329 nasopharyngeal swabs) and an independent cohort of 76 patients (152 nasopharyngeal swabs and sputum samples). The RT-LAMP assay was validated to be accurate (overall sensitivity and specificity of 88.89% and 99.00%, respectively) and diagnostically useful (positive and negative likelihood ratios of 88.89 and 0.11, respectively). RT-LAMP showed increased sensitivity (88.89% versus 81.48%) and high consistency (kappa, 0.92) compared to those of RT-qPCR for SARS-CoV-2 screening while requiring only constant-temperature heating and visual inspection. The time required for RT-LAMP was less than 1 h from sample preparation to the result. In addition, RT-LAMP was feasible for use with asymptomatic patients and did not cross-react with other respiratory pathogens. The developed RT-LAMP assay offers rapid, sensitive, and straightforward detection of SARS-CoV-2 infection and may aid the expansion of COVID-19 testing in the public domain and hospitals.IMPORTANCE We developed a visual and rapid reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay targeting the S gene for SARS-CoV-2 infection. The strength of our study was that we validated the RT-LAMP assay using 481 clinical respiratory samples from two prospective cohorts of suspected COVID-19 patients and on the serial samples from an asymptomatic carrier. The developed RT-LAMP approach showed an increased sensitivity (88.89%) and high consistency (kappa, 0.92) compared with those of reverse transcription-quantitative PCR (RT-qPCR) for SARS-CoV-2 screening while requiring only constant-temperature heating and visual inspection, facilitating SARS-CoV-2 screening in well-equipped labs as well as in the field. The time required for RT-LAMP was less than 1 h from sample preparation to the result (more than 2 h for RT-qPCR). This study showed that the RT-LAMP assay was a simple, rapid, and sensitive approach for SARS-CoV-2 infection and can facilitate COVID-19 diagnosis, especially in resource-poor settings.

Hu X ，Deng Q ，Li J ，Chen J ，Wang Z ，Zhang X ，Fang Z ，Li H ，Zhao Y ，Yu P ，Li W ，Wang X ，Li S ，Zhang L ，Hou T ... -  《mSphere》

A highly effective reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid detection of SARS-CoV-2 infection.

The COVID-19 pandemic has illustrated the importance of simple, rapid and accurate diagnostic testing. This study describes the validation of a new rapid SARS-CoV-2 RT-LAMP assay for use on extracted RNA or directly from swab offering an alternative diagnostic pathway that does not rely on traditional reagents that are often in short supply during a pandemic. Analytical specificity (ASp) of this new RT-LAMP assay was 100% and analytical sensitivity (ASe) was between 1 × 101 and 1 × 102 copies per reaction 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 rRT-PCR. When a CT cut-off of 33 was employed, above which increasingly evidence suggests there is a low risk of patients shedding infectious virus, the diagnostic sensitivity was 100%. The DSe and DSp of Direct RT-LAMP (that does not require RNA extraction) 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, CT < 25, was < 15 min. We propose that RNA RT-LAMP could replace rRT-PCR where there is a need for increased sample throughput and Direct RT-LAMP as a near-patient screening tool to rapidly identify highly contagious individuals within emergency departments and care homes during times of increased disease prevalence ensuring negative results still get laboratory confirmation.

Fowler VL ，Armson B ，Gonzales JL ，Wise EL ，Howson ELA ，Vincent-Mistiaen Z ，Fouch S ，Maltby CJ ，Grippon S ，Munro S ，Jones L ，Holmes T ，Tillyer C ，Elwell J ，Sowood A ，de Peyer O ，Dixon S ，Hatcher T ，Patrick H ，Laxman S ，Walsh C ，Andreou M ，Morant N ，Clark D ，Moore N ，Houghton R ，Cortes NJ ，Kidd SP ... -  《-》