Alu-HIV PCR was performed as described earlier (15 (link), 17 (link)) by use of 42 replicate reactions of a mix with gag-reverse HIV primer and Alu-forward primer as well as 42 replicates by use of only primers specific to HIV gag (HIV-only PCR). Samples were diluted to 2 or 10 μg DNA/mL and distributed in replicate PCR reactions containing 25 μL sample combined with 25 μL master mix, resulting in an equivalent of approximately 7500 cells (1 ng/μL PCR mix) or approximately 37 500 cells (5 ng/μL PCR mix) per 50 μL PCR replicate. All patient samples were run at 7500 cells/replicate PCR. We conducted the final nested qPCR in 20 μL with 10 μL of the first PCR product. This qPCR was optimized to enable robust amplification in a 1:1 dilution of PCR without PCR inhibition from pyrophosphates. We used master mixes and cycling conditions as previously described (15 (link)), and primer pairs are depicted in Supplemental Table 1, which accompanies the online version of this article at http://www.clinchem.org/content/vol60/issue6. Degenerate probes are no longer used for this assay, since they diminish robustness (unpublished data). PCR cycling was performed on an Applied Biosystems 7500 Real-Time PCR System, and Cq values were obtained by fit point analysis. Normalization to cell numbers was performed with a β-globin assay as described before (15 (link)).