HIV-1 specific amplicons were detected using SYBR Green technology according to manufacturer's instructions (Qiagen, Valencia, CA). The number of HIV-1 RNA copies in each test template was measured by its threshold cycle (C t ) and determined from the standard curve of serially diluted customized DBS standards using software for data analysis (Sequence Detector version 1.6, PE Applied Biosystems). For each experiment, a standard curve was generated from serial endpoint dilutions (586,000 to 37 copies) of the customized DBS standards. The threshold cycle values were plotted against copy numbers to construct the standard curve. Quantification of HIV-1 RNA in each test sample was back calculated and viral load was expressed as copies/ml. Using the protocol described above, DBS was extracted, run and analyzed in the rtLC DBS assay by personnel with molecular and virology expertise who were blind to the results.