Prevalence of low-level HIV-1 variants with reverse transcriptase mutation K65R and the effect of antiretroviral drug exposure on variant levels

Background: It has been reported that treatment-naive individuals infected with HIV-1 subtype C may be more likely to harbour viral variants possessing a K65R reverse transcriptase gene mutation. The objectives of this study were to determine the prevalence of low-level K65R variants within different HIV-1 subtypes and to assess the effects of antiretroviral exposure on K65R variant levels. Methods: Treatment-naive individuals infected with different HIV-1 subtypes were genotyped by ultra-deep sequencing. Samples were evaluated for low-level variants to 0.4% or 1% levels depending upon viral load. Estimated mutational load was calculated by multiplying the percentage of the variant by the plasma viral load. Results: A total of 411 treatment-naive individuals were evaluated by ultra-deep sequencing to 1% levels; 4 subjects (0.97%) had K65R variants at >= 1% or had a very high mutation load. All four subjects had variants with linked drug resistance mutations suggesting transmitted resistant variants. 147 ARV-naive subjects were sequenced to 0.4% levels; 8.8% (13/147) had K65R low-level variants identified: 2.2% (2/92) in subtype B, 35.7% (10/28) in subtype C (P<0.001 for B versus C) and 3.7% (1/27) in non-B/C subtypes. The 13 ARV-naive subjects with K65R variants at <1% received tenofovir plus emtricitabine plus a ritonavir-boosted protease inhibitor (TDF+FTC+PI/r) and 5 subsequently experienced virological failure. There was no enhancement in K65R levels by percentage or mutational load compared to pre-therapy levels. Conclusions: Low-level K65R variants were more frequently identified in subtype C. K65R variants at >1% levels likely represent transmitted resistant variants. The clinical implication of low-level K65R variants below 1% in treatment-naive subjects who receive TDF+FTC+PI/r remains to be determined as the majority are very low-level and did not increase after antiretroviral exposure.