HIV-1 drug resistance genotyping from dried blood spots stored for 1 year at 4°C

Abstract

Background: Dried blood spots (DBSs) are an attractive alternative to plasma for HIV-1 drug resistance testing in resource-limited settings. We recently showed that HIV-1 can be efficiently genotyped from DBSs stored at -20°C for prolonged periods (0.5-4 years). Here, we evaluated the efficiency of genotyping from DBSs stored at 4°C for 1 year. Methods: A total of 40 DBSs were prepared from residual diagnostic specimens collected from HIV subtype B-infected persons and were stored with desiccant at 4°C. Total nucleic acids were extracted after 1 year using a modification of the Nuclisens assay. Resistance testing was performed using the ViroSeq HIV-1 assay and an in-house nested RT-PCR method validated for HIV-1 subtype B that amplifies a smaller (1 kb) pol fragment. Results: Using the ViroSeq assay, only 23 of the 40 (57.5%) DBS specimens were successfully genotyped; 22 of these specimens had plasma viraemia >10 000 RNA copies/mL. When the specimens were tested using the in-house assay, 38 of the 40 DBSs (95%) were successfully genotyped. Overall, resistance genotypes generated from the DBSs and plasma were highly concordant. Conclusions: We show that drug resistance genotyping from DBSs stored at 4°C with desiccant is highly efficient but requires the amplification of small pol fragments and the use of an in-house nested PCR protocol with quality-controlled reagents. These findings suggest that 4°C may represent a suitable temperature for long-term storage of DBSs. © The Author 2008. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.