Rapid In Vitro Evaluation of Antiretroviral Barrier to Resistance at Therapeutic Drug Levels.

Failure of combination antiretroviral (ARV) therapy in HIV-infected patients is often associated with the emergence of drug resistance-associated mutations (RAMs). To facilitate analysis of the barrier to resistance at therapeutically relevant ARV concentrations, we performed fixed-dose in vitro HIV-1 drug resistance selection assays using the immortalized MT-2 T-cell line and primary human CD4+ T cells with a panel of FDA-approved ARVs, each at their respective cell culture equivalent clinical trough concentration (CCE Cmin). At high multiples of its CCE Cmin, emtricitabine (FTC) selected for the rapid emergence of M184I/V, a result consistent with resistance emergence in vivo. While the rate of viral breakthrough in the presence of rilpivirine or efavirenz was delayed relative to FTC, both inhibitors selected for virus with known clinically relevant RAMs. No viral breakthrough was observed for the protease inhibitor atazanavir even at subtherapeutic drug concentrations, which is consistent with its previously characterized high in vivo barrier to resistance. Depending on assay conditions, treatment with integrase inhibitors elvitegravir and raltegravir resulted in breakthrough of both resistant and wild-type virus. The RAMs observed in drug selections were not detected above a 2% threshold by deep sequencing in the in vitro virus inoculum, and only rarely in isolates from treatment-naive HIV+ patients. These new viral breakthrough assays facilitate the analysis of multiple experimental replicates and conditions in parallel and provide a rapid quantitative means to evaluate drug resistance emergence at therapeutically relevant drug concentrations, which should facilitate the identification of new ARVs with a high barrier to resistance.

Parameters Influencing Baseline HIV-1 Genotypic Tropism Testing Related to Clinical Outcome in Patients on Maraviroc.

OBJECTIVES: We analysed the impact of different parameters on genotypic tropism testing related to clinical outcome prediction in 108 patients on maraviroc (MVC) treatment. METHODS: 87 RNA and 60 DNA samples were used. The viral tropism was predicted using the geno2pheno[coreceptor] and T-CUP tools with FPR cut-offs ranging from 1%-20%. Additionally, 27 RNA and 28 DNA samples were analysed in triplicate, 43 samples with the ESTA assay and 45 with next-generation sequencing. The influence of the genotypic susceptibility score (GSS) and 16 MVC-resistance mutations on clinical outcome was also studied. RESULTS: Concordance between single-amplification testing compared to ESTA and to NGS was in the order of 80%. Concordance with NGS was higher at lower FPR cut-offs. Detection of baseline R5 viruses in RNA and DNA samples by all methods significantly correlated with treatment success, even with FPR cut-offs of 3.75%-7.5%. Triple amplification did not improve the prediction value but reduced the number of patients eligible for MVC. No influence of the GSS or MVC-resistance mutations but adherence to treatment, on the clinical outcome was detected. CONCLUSIONS: Proviral DNA is valid to select candidates for MVC treatment. FPR cut-offs of 5%-7.5% and single amplification from RNA or DNA would assure a safe administration of MVC without excluding many patients who could benefit from this drug. In addition, the new prediction system T-CUP produced reliable results.