Statistical Analyses. The customized DBS standards serial dilutions were tested in 25 separate runs to determine the threshold, inter-assay precision and linearity of the rtLC DBS assay. Trained statisticians performed data analyses. Probit analysis was used to determine 95% and 99% detection limits. The likelihood ratio test was used to evaluate the effect of extractions and runs; the fitness was measured by R-squared value. Spearman correlation coefficients were calculated to determine the relationship between the HIV-1 RNA levels quantified by the Roche Ultrasensitive and bDNA assays with the rtLC DBS technique. The sensitivity of the rtLC DBS assay was calculated as the number of positive results divided by the total number of samples from infected patients who had plasma viral load above the threshold level of detection of commercial assays (bDNA and Roche Ultrasensitive) expressed as a percentage. The specificity was calculated as the number of negative samples divided by the total number of known negative specimens obtained from normal healthy donors and uninfected infants expressed as a percentage. Paired Wilcoxon tests were used to determine the effect of temperature and time on RNA stability and assay performance.