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

Abstract

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 C_T 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.

Figure 1

Time to positivity (Tp) in minutes plotted against RT-qPCR C_T for each combination of method and sample type. Samples that were negative by RT-qPCR are not shown. Samples that were negative by reverse-transcription loop-mediated isothermal amplification (RT-LAMP) are shown with 0 time to positivity. Results of linear ordinary least squared regression are shown for samples that were RT-LAMP positive with the regression line and corresponding 95% CI represented by the blue lines and light blue shaded regions, respectively.

Figure 2

Performance of the RNA reverse-transcription loop-mediated isothermal amplification (RT-LAMP) and direct RT-LAMP assays on both saliva and swab samples, according to viral load groupings.

Figure 3

Forest plots for direct reverse-transcription loop-mediated isothermal amplification (RT-LAMP) per sample type, showing site heterogeneity in sensitivity and specificity, with overall estimates and the resulting expected sensitivity and specificity retrieved from each respective bivariate random effects model. FN, false negative; FP, false positive; NHS, National Health Service; TN, true negative; TP, true positive.

Figure 4

Forest plots for RNA reverse-transcription loop-mediated isothermal amplification (RT-LAMP) per sample type, showing site heterogeneity in sensitivity and specificity, with overall estimates and the resulting expected sensitivity and specificity retrieved from each respective bivariate random effects model. FN, false negative; FP, false positive; NHS, National Health Service; TN, true negative; TP, true positive.