In this study, we used a nucleic acid based real-time PCR assay to detect and quantify plasma HIV-1 copy numbers on samples from 56 HIV-1 infected patients utilizing the DBS format. The assay results were comparable and correlated well with commercially available viral load assays (Siemens bDNA, Spearman r
0.89 and Roche Ultrasensitive Amplicor, Spearman r
0.91). In the patient cohort analyzed, the assay successfully detected all positive samples. The calculated specificity using known negative samples was 100%. The estimated 95% detection threshold was 136 copies and the dynamic range of the assay was 5 log10
. Finally, the assay successfully detected four major subtypes and 2 CRF of HIV-1.
A little over a decade ago, we demonstrated the utility of early combination antiretroviral therapy (ARV) in infants.
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.
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.
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.
Hence access to nucleic acid testing in these settings is currently very limited.
Studies to detect and quantify HIV-1 have traditionally involved two-step, nested or probe based PCR 
; more recently, real time PCR has been used for HIV detection and quantification.
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.
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.
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.
Comparable efficacy using multiple extractions and runs of our customized standards support the reliability of the DBS format and the rtLC DBS assay.
The rtLC DBS assay described herein is a one-step, walk away technique. Automation of this assay provides potential for high throughput with very small sample volumes, which makes the assay suitable for use in infants and children from whom one often has access to only small blood volumes. It is an assay system which will be cost-effective and easily adaptable to limited-resource settings, where the majority of new HIV-1 infections are seen today. The detection of PCR products by SYBR Green ensures good sensitivity. SYBR Green is relatively inexpensive compared to probe-based detection, and in general, SYBR Green detection is one cycle threshold or so more sensitive than probe-based assays.
Several previous studies have utilized SYBR Green for HIV-1 diagnosis and are reviewed by Espy et al.
The efficiency of this dye-based assay is also comparable with the currently available diagnostic assays on HIV. Aside from use in HIV diagnosis and quantification, SYBR Green has been widely used to detect and quantify diverse human pathogens.
The data in these reports strongly support the utility and reliability of SYBR Green for detection of specific PCR amplicons above the background. The use of degenerate LTR primers in the rtLC DBS assay allows for a wide range of genotype inclusivity.
Compared to commercial assays, the rtLC DBS assay is rapid and cost-effective. The equipment used in the rtLC assay is self-contained, occupies minimal bench-space and doesn't require accessory equipments (such as a plate washer, optical density reader, and incubator; ). Aside from the initial cost of obtaining the LightCycler instrument, a comparative analysis of assay costs and technician time reveals a 40-fold decrease in cost as well as 2.5 fold decrease in technician time (4.5 hours) associated with the rtLC DBS when compared to commercial kit based PCR assays ().
Infrastructure requirements of commercial versus rtLC DBS assays for viral load determination.
Cost and time comparisons of commercial verses rtLC DBS assays for viral load determination.
The reduced equipment requirements, personnel hours, and costs compared to the commercial ‘gold’ standard assays make the rtLC DBS assay attractive for transfer to and use in resource-limited settings. The success of the pilot field study on DBS from Congo emphasizes the utility and applicability of our assay although further studies with larger sample sizes are definitely warranted.
In summary, we have utilized a real-time LightCycler based PCR assay on small volumes of whole blood dried on filter paper to successfully detect and quantify viral loads across different HIV-1 clades. The use of dried blood spots provides a simple and inexpensive means for obtaining blood samples for analysis that minimizes the risk for contamination while maximizing the ability to obtain timely results. A major advantage of the rtLC DBS assay is that the amplification, real-time detection and quantification, and confirmation of amplicon-specificity by melting curve analysis are performed in a one-step, closed-tube format. In addition, viral load tests by rtLC DBS assay are substantially less expensive and logistically less intensive than commercial assays. Preliminary data suggest the adaptability of the assay into other real-time systems. Validation of these results with larger field studies would constitute a more robust evaluation and will have major implications for early diagnosis, disease management, and epidemiological- or resistance- surveillance studies in limited resource settings.