A little over a decade ago, we demonstrated the utility of early combination antiretroviral therapy (ARV) in infants. [28, 29] HIV infection is associated with particularly high morbidity and mortality in limited-resource settings and a recent randomized trial conducted in South Africa demonstrated reduced morbidity and mortality in infants treated with early combination ARV. [30] In a recent WHO consultants' meeting, revision of current guidelines was recommended to include routine early diagnosis and treatment of HIV positive infants under 1 year of age. [31] Nucleic acid based testing is the gold standard for early diagnosis. Commercially available assays (bDNA, Roche Amplicor Ultrasensitive, and Cobas) are relatively expensive and require significant infrastructure and technical expertise to allow transfer of technology to resource-limited settings. [2, 8] Hence access to nucleic acid testing in these settings is currently very limited. [6, 17, 18, 32, 33] Studies to detect and quantify HIV-1 have traditionally involved two-step, nested or probe based PCR [34] [35] [36] [37] ; more recently, real time PCR has been used for HIV detection and quantification. [6, 22, 24, 34, 38, 39] Multiple investigators have documented the utility of DBS sample collection for early HIV-1 detection in infants, viral load monitoring, and surveillance of seropositivity and drug resistance in laboratories and clinics that lack facilities for refrigeration or sample processing. [8, 17, 20, [22] [23] [24] 38, [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] The DBS format greatly facilitates the logistics of sample collection, processing, and shipping for limited resource settings. Whole blood saved as DBS can be transported or mailed to reference laboratories without refrigeration and has low biohazard risk. Optimal storage conditions for DBS and long term stability of DNA and RNA from DBS under different storage conditions have been extensively documented. [23, 44, 47, 51, 53] Our pilot studies with a small cohort of patient samples to assess the effect of temperature and time, albeit one week, on the stability of RNA demonstrated no difference, and were in agreement with previous larger studies. [39, [54] [55] [56] Comparable efficacy using multiple extractions and runs of our customized standards support the reliability of the DBS format and the rtLC DBS assay.