Fig. 5. Experimental validation of simple and combinatorial pooling.

(A) Five pools (columns of matrix), each consisting of 24 nasopharyngeal swab samples (rows of matrix: 23 negative samples per pool and 1 positive, with viral load indicated on right) were tested by viral extraction and RT-qPCR. Pooled results indicated as negative (blue), inconclusive (yellow), or positive (red). (B) Six combinatorial pools (columns) of 48 samples (rows: 47 negative and 1 positive with viral load of 12,300) were tested as above. Pools 1, 2, and 4 tested positive. Arrows indicate two samples that were in pools 1, 2, and 4: sample 32 (negative) and sample 48 (positive). (C) Previously tested de-identified samples were pooled using a combinatorial design with 96 samples, 6 pools, and 2 pools per sample. Thirty positive samples were randomly distributed across 10 batches of the design. Viral RNA extraction and RT-qPCR were performed on each pool, with the results used to identify potentially positive samples. (D) Samples were pooled according to a simple design (48 pools with 48 samples per pool). Twenty-four positive samples were randomly distributed among the pools (establishing 1% prevalence). The pooled test results were used with an MLE procedure to estimate prevalence (0.87%), and bootstrapping was used to estimate a 95% confidence interval (0.52 to 1.37%).