Comparison of illumina and 454 deep sequencing in participants failing raltegravir-based antiretroviral therapy.

BACKGROUND: The impact of raltegravir-resistant HIV-1 minority variants (MVs) on raltegravir treatment failure is unknown. Illumina sequencing offers greater throughput than 454, but sequence analysis tools for viral sequencing are needed. We evaluated Illumina and 454 for the detection of HIV-1 raltegravir-resistant MVs. METHODS: A5262 was a single-arm study of raltegravir and darunavir/ritonavir in treatment-naïve patients. Pre-treatment plasma was obtained from 5 participants with raltegravir resistance at the time of virologic failure. A control library was created by pooling integrase clones at predefined proportions. Multiplexed sequencing was performed with Illumina and 454 platforms at comparable costs. Illumina sequence analysis was performed with the novel snp-assess tool and 454 sequencing was analyzed with V-Phaser. RESULTS: Illumina sequencing resulted in significantly higher sequence coverage and a 0.095% limit of detection. Illumina accurately detected all MVs in the control library at ≥0.5% and 7/10 MVs expected at 0.1%. 454 sequencing failed to detect any MVs at 0.1% with 5 false positive calls. For MVs detected in the patient samples by both 454 and Illumina, the correlation in the detected variant frequencies was high (R2 = 0.92, P<0.001). Illumina sequencing detected 2.4-fold greater nucleotide MVs and 2.9-fold greater amino acid MVs compared to 454. The only raltegravir-resistant MV detected was an E138K mutation in one participant by Illumina sequencing, but not by 454. CONCLUSIONS: In participants of A5262 with raltegravir resistance at virologic failure, baseline raltegravir-resistant MVs were rarely detected. At comparable costs to 454 sequencing, Illumina demonstrated greater depth of coverage, increased sensitivity for detecting HIV MVs, and fewer false positive variant calls.

Associations of T cell activation and inflammatory biomarkers with virological response to darunavir/ritonavir plus raltegravir therapy.

OBJECTIVES: One of the goals of antiretroviral therapy (ART) is to attenuate HIV-induced systemic immune activation and inflammation. We determined the dynamics of biomarkers of immune activation, microbial translocation and inflammation during initial ART with a nucleos(t)ide-sparing regimen of darunavir/ritonavir plus raltegravir. We also evaluated associations between these biomarkers and the virological response to the regimen. METHODS: We determined baseline and week 24 and 48 levels of CD4+ and CD8+ T cell activation (% HLA-DR+/CD38+), interleukin-6 (IL-6), interferon-γ-inducible protein-10 (IP-10), soluble CD14 (sCD14), D-dimer and lipopolysaccharide. Associations between the biomarkers at baseline were assessed using Spearman's rank correlation. The Wilcoxon signed rank test analysed changes from baseline. Comparisons between groups were made using the Wilcoxon rank sum test, and Cox proportional hazards models assessed predictors of virological failure (VF). RESULTS: Assays were completed on 107 of 112 subjects after excluding five subjects who had only baseline samples. The subjects included were 94 (88%) men with a median age of 37 years, a median baseline CD4 count of 261.5 cells/mm(3) and a median baseline viral load (VL) of 75 876 copies/mL. Subjects with a baseline VL >100 000 copies/mL had higher baseline T cell activation, IL-6, IP-10, sCD14 and D-dimer. These biomarkers declined during treatment (P < 0.05). Although subjects who experienced VF had higher baseline CD4+ T cell activation (P = 0.035), only baseline VL independently predicted VF (hazard ratio for >100 000 versus ≤100 000 copies/mL was 4.5-5.6, P ≤ 0.002). CONCLUSIONS: Darunavir/ritonavir plus raltegravir attenuated immune activation, inflammation and microbial translocation. T cell activation remained higher in subjects with VF than those without. Baseline VL >100 000 copies/mL remained the primary driver of VF.

Efficacy of a nucleoside-sparing regimen of darunavir/ritonavir plus raltegravir in treatment-naive HIV-1-infected patients (ACTG A5262).

OBJECTIVE: To explore darunavir/ritonavir (DRV/r) plus raltegravir (RAL) combination therapy in antiretroviral-naive patients. DESIGN: Phase IIb, single-arm, open-label, multicenter study. METHODS: One hundred and twelve antiretroviral-naive, HIV-1-infected patients received DRV/r 800/100 mg once daily and RAL 400 mg twice daily. Primary endpoint was virologic failure by week 24. Virologic failure was defined as confirmed viral load of 1000 copies/ml or more at week 12, or an increase of more than 0.5 log(10) copies/ml in viral load from week 4 to 12, or a confirmed viral load of more than 50 copies/ml at or after week 24. Protease and integrase genes were sequenced in patients experiencing virologic failure. RESULTS: Virologic failure rate was 16% [95% confidence interval (CI) 10-24] by week 24 and 26% (95% CI 19-36) by week 48 in an intent-to-treat analysis. Viral load at virologic failure was 51-200 copies/ml in 17/28 failures. Adjusting for age and sex, virologic failure was associated with baseline viral load of more than 100,000 copies/ml [hazard ratio 3.76, 95% CI (1.52-9.31), P = 0.004] and lower CD4 cell count [0.77 per 100 cells/µl increase (95% CI 0.61-0.98), P = 0.037]. When trough RAL concentrations were included as a time-varying covariate in the analysis, virologic failure remained associated with baseline viral load more than 100,000 copies/ml [hazard ratio = 4.67 (95% CI 1.93-11.25), P < 0.001], whereas RAL level below detection limit in plasma at one or more previous visits was associated with increased hazard [hazard ratio = 3.42 (95% CI 1.41-8.26), P = 0.006]. All five participants with integrase mutations during virologic failure had baseline viral load more than 100,000 copies/ml. CONCLUSION: DRV/r plus RAL was effective and well tolerated in most patients, but virologic failure and integrase resistance were common, particularly in patients with baseline viral load more than 100,000 copies/ml.