Background:  Real-time polymerase chain reaction (RT-PCR) -based nucleic acid amplification and quantification of HIV-1 is a valuable, albeit expensive, clinical tool for early HIV-1 diagnosis and for evaluation of the efficacy of ART. Dried blood spots (DBS) on filter paper is a dependable and practical method to obtain, store, and transport blood samples. Rapid, cost-effective, and sensitive methods using DBS are needed for diagnosis and viral load quantification in resource-limited regions of the world.

Methods:  Whole blood samples were drawn from 27 HIV-1⁺ U.S. and non-U.S. patients, and from 34 HIV-1^– individuals. DBS were prepared with 50 mL of whole blood spotted onto SS903 and stored at –20^° C. HIV-1 RNA extracted from DBS was RT-PCR amplified in a 1-step single-tube LightCycler closed system using primers specific for LTR region of all HIV-1 clades, and quantified using fluorescent SyBrGreen dye. For quantification, cycle thresholds corresponding to serial dilutions of a known copy of HIV-1 molecular clone was used to plot a standard curve. Results were statistically analyzed to determine linear dynamic range, inter-assay precision, detection sensitivity, and specificity of the assay. The real-time LightCycler (rtLC) DBS assay was tested for genotype inclusivity using an international panel of HIV-1 isolates representing globally prevalent strains.

Results:  RtLC DBS assay has a dynamic range of 5 log₁₀, percentage coefficient of variation <5% up to a 4 log₁₀ dilution, and good correlation with branched DNA (r = 0.85) and Roche Ultrasensitive RNA (r = 0.82) commercial assays. Probit analysis determined 95% detection level at 28 copies/reaction sample. The sensitivity and specificity of the assay were 92% and 100%, respectively. The assay successfully detected HIV-1 RNA in a reference panel representing viral clades and circulating recombinant forms. The rtLC DBS assay cost $5/test, which is 40-fold less compared to commercial viral load assays, and needed minimal infrastructure.

Conclusions:  We have developed a rapid and sensitive RT-PCR assay to detect and quantify viral loads across different HIV-1 clades utilizing DBS. The results were equivalent to, and showed good correlation with, established gold standard viral load assays. The low cost of this assay makes it a promising test in resource-limited settings for early diagnosis of HIV-1 infection and for monitoring disease progression.