The ViroSeq kit successfully genotyped approximately half of DBS specimens from patients with ART failure in rural Tanzania. Although efficient in patients with high viral loads, the ViroSeq kit failed to genotype most DBS specimens with viral loads of <10
000 copies/mL. In the specimens that yielded a genotype, however, there was high concordance with results from a plasma-based assay.
Three previous studies have assessed the ViroSeq kit in conjunction with DBS. Masciotra et al
successfully genotyped all DBS with viral loads of >2000 copies/mL, compared with 55% of those with viral loads of <2000 copies/mL. Youngpairoj et al
reported that only 8% of DBS could be genotyped when viral load was <10
000 copies/mL, compared with 81% when viral load was >10
000 copies/mL, similar to our results. Finally, in a recent study from Mexico, Lira et al
successfully genotyped 60% of DBS with viral loads of >14
000 copies/mL, but none of the DBS with viral loads below this level. All these studies found high concordance between nucleotide sequences derived from DBS and plasma, in line with our findings. Our study, however, is the first to assess the ViroSeq kit with DBS from patients infected with non-B subtypes. The ViroSeq kit was originally optimized for HIV-1 subtype B;11
hence, future DBS studies should aim to include various subtypes, including subtype A, which frequently failed amplification in our study, even in specimens with high viral loads.
Previously, we reported 94% amplification success from DBS collected in duplicate with DBS utilized in the present study and stored under less favourable conditions, using an in-house RT-nested PCR method.7
Youngpairoj et al
reported similar results; overall amplification success was 57.5% with the ViroSeq kit and 95% with an in-house nested assay. In-house assays typically reduce the costs by >50% compared with commercial kits.12
Furthermore, the use of a nested PCR protocol appears to increase sensitivity, particularly in samples with low-level viraemia, such as patients with early treatment failure. However, in resource-limited settings, where the selection of second-line antiretroviral drugs is scarce, the WHO recommends that first-line treatment be conserved as long as viral load does not exceed 10
Thus, the ViroSeq kit in conjunction with DBS could be an acceptable option for resistance testing under the WHO guidelines, given that genotyping is restricted to patients who reach the viral load threshold for regimen switch. Nonetheless, further refinement of the ViroSeq kit with DBS is warranted in order to increase sensitivity.
Our study was limited by a relatively small sample size. Moreover, although DBS and plasma were both genotyped using the ViroSeq kit, the use of different amplification methods might have contributed to discordances between the two specimen types. The main strength of our study was that we used samples obtained in rural Africa, and we believe our results reflect ‘real-life’ performance of DBS-based resistance monitoring in the field. However, temperature and humidity conditions differ from place to place, and the effect of various storage conditions should be studied in more detail.
In conclusion, we found that the ViroSeq kit performed well using DBS from patients with major virological failure, but failed to genotype most DBS with viral loads of <10
000 copies/mL. In DBS samples that yielded a genotype, there was high concordance between mutations found in DBS and plasma. Using the WHO guidelines for ART in resource-limited settings, recommending regimen switch only in patients with viral loads of >10
000 copies/mL, the ViroSeq kit in conjunction with DBS could be an acceptable option for drug resistance testing.