DNA Extraction. One spot each of DBS filter paper was processed to extract DNA using the previously described resinbased Chelex (Bio-Rad, Hercules, CA) method. [26] Briefly, the paper punched from one entire DBS was immersed in 1 mL of whole blood specimen wash solution (Amplicor HIV-1 Monitor test v1.5, Roche Molecular Systems) and rocked for 2 hours at room temperature. After a quick spin for 5 minutes at high speed, the red-tinged buffer was removed and discarded. The DBS paper was washed once, and then immersed in 250ul of Chelex resin resuspended in DNA dilution buffer (10% v/v in 10 mM Tris buffer, pH 8.3 containing 50 mM KCl). DNA kit Internal Control (3.3ul) was added to the DBS sample, mixed frequently to keep the resin in suspension, and was heated to 100uC for 1 hour, with a 10 second vortex after the first 30 minutes of heating. After a spin for 3 minutes to pellet the resin, the supernatant containing the extracted DNA was removed and stored at 280uC until HIV-reactivity of the extract was determined using Roche Amplicor HIV-1 DNA Amplicor 1.5 v kit according to a previously published protocol. [26] The cellular equivalents of DNA in each test sample were determined in a real-time Taqman PCR assay by probing for CCR5 copy number using forward primer 59-GCTGTGTT-TGCGTCTCTCCCAGGA-39 and reverse primer 59-CTC-ACAGCCCTGTGCCTCTTCTTC-39, and the corresponding fluorogenic probe 59FAM-AGCAGCGGCAGGACCAGCCC-CAAG-TAMRA 39. Known copies of plasmid carrying the CCR5 gene were used to generate the standard curve, from which the number of CCR5 copies and cellular equivalents were determined for each sample. Real-time PCR analyses on RNA extracted from infant DBS was performed as described below.