Background:  Dried blood spots (DBS) are an attractive alternative to plasma for HIV-1 drug-resistance testing in resource-limited settings. We recently showed that resistance genotypes generated from DBS and plasma are highly concordant, and that HIV-1 can be efficiently genotyped from DBS stored at -20ºC for prolonged periods (6 months to 4 years). However, storage at –20ºC may not always be possible in resource-limited areas where 4ºC or room temperature may represent a more feasible alternative. Here we evaluated the efficiency of genotyping from DBS stored at 4ºC for 1 year.

Methods:  A total of 40 DBS were prepared from residual diagnostic specimens collected from HIV subtype B-infected persons. The median plasma virus load was 13,680 RNA copies/mL (range 518 to 676,694). DBS were prepared by pipetting 50 µL of whole blood onto 903 filter paper cards. Cards were dried overnight at room temperature, placed in a gas impermeable, sealable plastic bag containing a silica gel desiccant, and stored at 4ºC. Total nucleic acids were extracted from one spot after 1 year using a modification of the Nuclisens assay. Resistance testing was done using the ViroSeq HIV-1 Genotyping System (1.8 kb) and an in-housereal-time polymerase chain reaction (RT-PCR) method that amplifies a smaller pol fragment (1 kb) and uses a nested PCR step.

Results: Using the ViroSeq assay, only 23 of the 40 (57.5%) DBS specimens were successfully genotyped; most of these specimens had plasma viremias >10,000 RNA copies/mL. An additional 5 specimens were amplifiable but failed to generate full-length RT and protease sequences. When the specimens were tested using an in house RT-nested PCR assay, 38 of the 40 DBS (95%) were successfully genotyped. Overall, resistance genotypes generated from the DBS and plasma were highly concordant.

Conclusions:  We show that drug-resistance genotyping from DBS stored for 1 year at 4ºC is highly efficient but requires the amplification of smaller pol fragments and the use of an in-house nested PCR protocol with quality controlled reagents to overcome possible losses in HIV-1 RNA integrity. These findings suggest that storage at 4ºC may be a suitable alternative to -20ºC for long-term storage of DBS. Our findings further highlight the promise of DBS as a convenient specimen type for drug resistance surveillance and monitoring in resource-limited settings where the use of antiretroviral drugs continues to increase.