Study of genotypic and phenotypic HIV-1 dynamics of integrase mutations during raltegravir treatment: a refined analysis by ultra-deep 454 pyrosequencing

Abstract

Background: The dynamics of raltegravir-resistant variants and their impact on virologic response in 23 HIV-1-infected patients, who started a salvage raltegravir-containing regimen, were investigated.

Methods: Integrase population sequencing and Ultra-Deep-454 Pyrosequencing (UDPS) were performed on plasma samples at baseline and at raltegravir failure. All integrase mutations detected at a frequency ≥1% were considered to be reliable for the UDPS analyses. Phylogenetic and phenotypic resistance analyses were also performed.

Results: At baseline, primary resistance mutations were not detected by both population and UDPS genotypic assays; few secondary mutations (T97A-V151I-G163R) were rarely detected and did not show any statistically association either with virologic response at 24-weeks or with the development of resistant variants at failure. At UDPS, not all resistant variants appearing early during treatment evolved as major populations during failure; only specific resistance pathways (Y143R-Q148H/R-N155H) associated with an increased rate of fitness and phenotypic resistance were selected.

Conclusions: Resistance to raltegravir in integrase strand transfer inhibitor-naive patients remains today a rare event, which might be changed by future extensive use of such drugs. In our study, pathways of resistance at failure were not predicted by baseline mutations, suggesting that evolution plus stochastic selection plays a major role in the appearance of integrase-resistance mutations, whereas fitness and resistance are dominant factors acting for the late selection of resistant quasispecies.

Figure 1.

Evolution of viral haplotypes during raltegravir failure. Maximum likelihood trees inferred for the haplotypes (90–163 integrase positions) from patients ID12 (A), ID69 (B), and ID84 (C). Dots indicate major variants (prevalence, >50%) detected by Ultra-Deep-454 Pyrosequencing, and squares indicate specimens by population sequencing. T₀ indicates raltegravir baseline; T₁−T₁₂ , time (months) of sample collection during raltegravir treatment.