Background:  Early diagnosis of HIV is vital to identify infected children for ART. The global pediatric epidemic is essentially located in low-resource settings where collection of dried blood spots (DBS) from infants for viral detection assays is more feasible than collection of liquid blood samples. Laboratory experience of processing DBS in high-throughput, routine molecular laboratories is lacking. We assessed the risk of contamination between samples during the excision of DBS for HIV-1 DNA polymerase chain reaction (PCR) testing using a manual punch and an automated punching system.

Methods:  For both the manual and automated punch, each spot from a known HIV^– patient was excised after a spot from a known HIV-infected patient. A hand-held punch was used to punch 3 to 4  6-mm discs (±50 mL) from 180 samples and 1 of 3 cleaning methods was applied between each sample:  1) punch swabbed with Virkon/ethanol, 2) punching a clean card, and 3) no cleaning (n = 60 for each method). For the automated punch 7  3.5-mm discs (±75 mL) were excised per spot from 202 samples and a clean card punched between each sample. Genomic DNA was extracted from the discs and proviral DNA amplified and detected using the Roche Amplicor™ HIV-1 DNA assay version 1.5.

Results:  The manual punching method produced no false positive HIV DNA PCR results. We obtained 1 and 4 equivocal results with cleaning interventions 1) and 2), respectively. This may represent a degree of contamination that was insufficient to affect the assay’s specificity. The automated punch yielded 2 equivocal and 2 false positive results (specificity 98%). Of the latter, 1 sample of DNA extracted from the same disc produced a negative HIV DNA PCR result confirming that contamination had arisen downstream from the excision step. The other sample for which a false positive result was obtained could not be retested (specificity 99%).

Conclusions:  Manual and automated punching methods for DBS present a low risk of cross-contamination. These data overestimate the contamination risk since the percentage of positive samples (50%) is double that encountered in the Johannesburg molecular laboratory. Although the automated punch is preferable for a high-throughput laboratory, it is expensive and requires a substantial amount of laboratory space. The prospect of cross contamination during excision of DBS should no longer be allowed to hamper implementation of widespread, routine DBS HIV-1 DNA PCR testing.