Background:  Serodiagnosis of HIV-infection in infants born to seropositive mothers is problematic due to the prolonged presence of maternal antibodies.  A commercial polymerase chain reaction (PCR)-based assay (Roche Amplicor DNA version 1.5) to detect HIV DNA sequences from dried blood spot (DBS) was found to accurately identify HIV-infected infants in resource-limited countries.  We developed and evaluated a real-time reverse transcription PCR assay using DBS collected from three African countries.

Methods:  A simple magnetic bead-based method is used to isolate total nucleic acid from a 6 mm DBS disc for HIV detection using a qualitative real-time, duplex RT PCR assay. This approach was evaluated with DBS specimens from HIV-1 exposed infants in Uganda (n=128), Cameroon (n=315) and Kenya (n=410).  In Uganda, the DBS-based real-time results were compared with plasma (200ul)-based real-time results (gold standard).  In Cameroon and Kenya, DBS-based real-time results were compared with DBS-based Roche assay results (gold standard).

Results:  We first examined the stability of HIV-1 LTR nucleic acid in the DBS and found that it was stable for more than 2 months if DBS was stored properly at room temperature in a humidity-free environment.  The concordance of the results of DBS-based real-time assay and the results by plasma samples in Uganda was 99.2%.  The concordance of our real-time assay and Roche assay in the DBS samples from Cameroon and Kenya was 99.7%.  One sample from Uganda with negative plasma HIV test and two samples from Cameroon with negative Amplicor DNA results were found to be positive by our assay.  These samples were found to be positive for HIV gag and integrase sequences.

Conclusions:  Because of the simplicity of specimen collection, nucleic acid extraction, detection, low cost and high sensitivity, the DBS-based HIV real-time assay could be useful for pediatric HIV diagnosis and care/treatment programs in resource-limited settings.