The clinical sensitivity of the assay was determined using DBS specimens of 32 patients with known infection and viral loads above the detection threshold of the bDNA and Roche Ultrasensitive assays (Table 1 ). All 32 samples were positive using the rtLC DBS assay, yielding a clinical sensitivity of 100% for this cohort. An additional 5 DBS samples drawn from infants of indeterminate HIV-1 infection status tested positive on the rtLC DBS assay; all 5 were also positive on the commercial DNA assay. The clinical specificity of the rtLC DBS assay was determined using DBS specimens from 27 healthy HIV-1 negative adult donors and 17 HIV-1 negative infants born to HIV-1 positive women. All of these samples were negative for HIV-1 RNA, resulting in a clinical specificity of 100%. The information on the clinical specificity of rtLC DBS assay in HIV negative donors is provided in Table S1 in the supplementary section.