Testing clinical samples with the swab–to–RT-LAMP assay On the basis of these preliminary experiments, we decided to use swab samples either directly without any treatment (direct swab–to–RT-LAMP assay) or after heat treatment for 5 min at 95°C (hot swab–to–RT-LAMP assay). As an additional precaution, we kept the samples in the cold (using an ice-cold metal block) whenever possible. For testing large numbers of clinical samples, we performed the RT-LAMP assay using several 96-well plates. In total, we tested 209 different samples using the hot swab–to–RT-LAMP assay, and of these, 131 samples also were tested by the direct swab–to–RT-LAMP assay. Many samples were tested twice but using aliquots withdrawn at different time points (usually within 24 hours) from the swab samples stored at 4°C. This resulted in 235 direct swab–to–RT-LAMP assay measurements and 343 hot swab–to–RT-LAMP assay measurements (Fig. 5A). The hot swab–to–RT-LAMP assay detected a color change in the majority of samples with a CT < 30 with high sensitivity, whereas the direct swab–to–RT-LAMP assay only exhibited a high sensitivity for samples with a CT < 25 (Fig. 5 and Table 3). The heat treatment rendered the RT-LAMP assay more stringent as it reduced false positives and more sensitive for samples with a CT of 25 to 30. We found that some positive samples did not induce a color change but did so when assayed a second time. We therefore would recommend running this assay using technical duplicates. Fig. 5 Swab–to–RT-LAMP assay of clinical pharyngeal swab samples. (A) Skipping a prior RNA isolation step, pharyngeal swab samples were subjected to the RT-LAMP assay either directly (left) or after 5 min of heat treatment at 95°C (right). For each sample, scatter plots are used to compare the swab–to–RT-LAMP assay results (ΔOD values) with the results of RT-qPCR (CT values). The measurement time point was 30 min after the start of the 65°C incubation. (B) Shown is the sensitivity (right) and specificity (left) of the swab–to–RT-LAMP assay [derived from the data in (A)] using the decision threshold indicated by the horizontal gray line in (A). Specificity and sensitivity values (thick lines) are shown with their 95% confidence intervals (boxes) as in Fig. 3, with blue indicating the direct swab–to–RT-LAMP assay and red indicating the hot swab–to–RT-LAMP assay. (Also see table S3). Table 3 Shown is RT-qPCR and RT-LAMP testing of 592 clinical samples stratified into CT value bins (see Fig. 5A). Fig. 5A and table S3 show specificity and sensitivity values calculated from these numbers. Hot swab–to–RT-LAMP RT-LAMP CT Pos Neg Sum RT-qPCR Pos 0–25 38 4 42 25–30 17 5 22 30–35 5 23 28 35–40 0 36 36 Neg Neg 1 214 215 Sum 61 282 343 Direct swab–to–RT-LAMP RT-LAMP CT Pos Neg Sum RT-qPCR Pos 0–25 15 1 16 25–30 6 11 17 30–35 2 21 23 35–40 3 23 26 Neg Neg 9 144 153 Sum 35 200 235