No evidence for cross-contamination of dried blood spots excised using an office hole-punch for HIV-1 drug resistance genotyping

Sir, In a recent report, we described a laboratory procedure to perform HIV-1 genotypic drug resistance testing from dried blood spots (DBS). The methodology as a whole was subjected to rigorous validation. However, it has since been pointed out to us that we had not demonstrated that the process for cleaning the hole-punch used to excise DBS from filter paper was sufficient to prevent cross-contamination between samples. Our DBS excision technique involves the use of a hand-held 6 mm diameter office hole-punch (catalogue number 272575—Staples, Doncaster, UK). This is used to punch a pair of DBS per specimen from a filter paper collection card. It is cleaned by punching five filter paper spots from a fresh, unused card, before being used again to excise from a DBS specimen. In order to address these concerns, studies were performed to investigate the possibility that this procedure could generate false positives due to contamination of the hole-punch. Such methodological concerns are important considering the use of DBS as a widespread surveillance method. Ten DBS per specimen were prepared from three venous whole blood specimens collected with EDTA anticoagulant from HIV-infected individuals with known high viral load (.1 10 copies/mL). HIV viral load confirmation was performed using the COBAS Ampliprep HIV Taqman Assay (Roche, Lewes, UK). A further 30 DBS were prepared from normal human whole blood collected with citrate anticoagulant. DBS were prepared, stored and tested as per our earlier report. Two punches from each DBS were used for nucleic acid extraction. Alternate HIV and normal whole blood DBS were excised and tested consecutively. For each DBS replicate, PCR reactions to amplify fragments of the human b-globin gene, and two HIV subgenomic regions [protease spanning nucleotide positions 2057– 2979, reverse transcriptase (RT) 2813–3618 of the HIV genome, numbering as HXB2 reference strain, K03455], were carried out. For each normal whole blood replicate DBS, only the human b-globin gene fragment was successfully amplified. HIV nucleic acids were not detected by nested PCR in any normal whole blood replicate, in either the protease or RT regions. For each HIV whole blood DBS replicate, human b-globin and HIV protease and RT genes were detected by PCR. The outcome of these studies is summarized in Table 1, along with HIV viral load data for each patient specimen. Several studies have reported different methods to excise HIV DBS for drug resistance genotyping. The studies of Ziemniak et al. reported the excision of DBS using a sterile disposable razor blade. Bertagnolio et al. reported the use of disposable sterile scissors to excise DBS specimens. Others have reported the use of scissors and forceps sterilized between uses by spraying with a solution of 70% ethanol, before being completely dried. These methods for the excision of DBS from filter paper collection cards are cumbersome, timing-consuming, expensive and—in the case of razor blade use—potentially hazardous. A recent study by Driver et al. reported the use of a manual hole-punch to excise DBS for detection of HIV DNA by PCR, for use in the diagnosis of mother-to-child transmissions. Extensive replicate testing revealed no false positives regardless of whether the hole-punch was cleaned between DBS or not. Taken together with our study, the use of the manual hole-punch is a cost-effective, labour-saving method for DBS excision for HIV drug resistance genotyping, for which specimen crosscontamination is unlikely.