Background: Dried Blood Spots (DBS) are simpler to prepare, store, and transport than plasma and may represent a good alternative for drug resistance genotyping. However, the utility of DBS for drug resistance monitoring is not known. We investigated the efficiency of amplification and sequencing of HIV-1 protease/reverse transcriptase from DBS stored at different conditions, the nature of the amplified product (DNA/RNA), and the homology between plasma and DBS sequences.

Methods: Thirty seven plasma/DBS from untreated persons from Cameroon (9 subtype A, 1 subtype F, and 25 CRF02 based on gp41), and two matched plasma/DBS panels (A and B) generated using blood collected from 6 HIV-infected donors (Virology Quality Assessment (VQA)) were evaluated (903 paper). DBS from Cameroonian patients were stored at -20ºC for 2-3 years (median plasma VL = 23,715; Amplicor v1.5). The two VQA panels consisted of 3 samples each, with low, medium, or high RNA VL. Panel A was stored at -20ºC for 6 years and contained viruses with resistance mutations. Panel B contained wild type viruses and was stored for 5 years at both -70ºC and room temperature. Total nucleic acids were extracted from 50ul spots using the Nuclisens method. A 1.023 Kb PR/RT fragment was amplified by RT-nested PCR and sequenced. Amplifications were also done without RT to evaluate the contribution of proviral DNA.

Results: Amplification and sequencing were successful in the 3 DBS from panel A and in 2 of the 3 DBS from panel B stored at -70ºC. None of the DBS from panel B that were stored at room temperature were successfully amplified. Thirty four of the 37 (92%) DBS from Cameroonian patients were amplified, including 6/9 (67%) with VL<10,000 RNA copies/ml, 13/13 with VL between 10,000-50,000, and 15/15 with VL>50,000. Proviral DNA contributed significantly to DBS sequences in 4/5 spots from panels A and B, and in 21/37 (54%) specimens from Cameroon. The similarity between plasma and DBS PR/RT sequences in 23 Cameroonian specimens was 98.1%"1.6%. PR/RT resistance mutations from plasma and DBS from panel A were generally concordant although one sample showed 181C only in plasma.

Conclusions: Our results show the feasibility of using DBS for drug resistance surveillance. Long-term storage at -20ºC appears to preserve nucleic acids in DBS. The frequent contribution of proviral DNA to DBS sequences highlights the need for a wider evaluation of the concordance of resistance genotypes between plasma and DBS.