Context Evidence regarding the impact of minority, or low frequency, HIV-1 drug-resistant variants on the effectiveness of first-line antiretroviral treatment (ART) is conflicting. Objective To evaluate the association of pre-existing HIV-1 minority drug-resistant variants with risk of first-line non-nucleoside reverse transcriptase inhibitor (NNRTI)-based antiretroviral virologic failure. Data Sources We searched published and unpublished studies in MEDLINE (1966 through December, 2010), EMBASE (1974 through December, 2010), conference abstracts, and article references. Authors of all studies were contacted for detailed laboratory, ART, and adherence data. Study Selection and Data Abstraction Studies involving ART-naive participants initiating NNRTI-based regimens were included. Participants were included if all drugs in their ART regimen were fully active by standard HIV population sequencing. Cox proportional hazard models using pooled patient-level data were used to estimate the risk of virologic failure based on a Prentice weighted case-cohort analysis stratified by study. Data Synthesis Individual data from 10 studies and 985 participants were available for the primary analysis. Minority HIV-1 drug resistance mutations were associated with an increased risk of virologic failure (HR 2.3 [95% CI, 1.7–3.3], P<0.001) after controlling for medication adherence, ethnicity, baseline CD4 cell count and plasma HIV-1 RNA levels. The increased risk of virologic failure was most strongly associated with minority variants resistant to NNRTIs (HR 2.6 [95% CI, 1.9–3.5], P<0.001). Among participants from the cohort studies, 35% of those with detectable minority variants experienced virologic failure as compared to 15% of those without minority variants. The presence of minority variants was associated with 2.5–3 times the risk of virologic failure at either ≥95% or <95% overall medication adherence. A dose-dependent increased risk of virologic failure was found in participants with a higher proportion or quantity of drug-resistant variants. Conclusion In this pooled analysis, minority HIV-1 resistance mutations, particularly involving NNRTI-resistance, were significantly associated with a dose-dependent increased risk of virologic failure with first-line ART.

Many viruses, including the clinically relevant RNA viruses HIV (human immunodeficiency virus) and HCV (hepatitis C virus), exist in large populations and display high genetic heterogeneity within and between infected hosts. Assessing intra-patient viral genetic diversity is essential for understanding the evolutionary dynamics of viruses, for designing effective vaccines, and for the success of antiviral therapy. Next-generation sequencing (NGS) technologies allow the rapid and cost-effective acquisition of thousands to millions of short DNA sequences from a single sample. However, this approach entails several challenges in experimental design and computational data analysis. Here, we review the entire process of inferring viral diversity from sample collection to computing measures of genetic diversity. We discuss sample preparation, including reverse transcription and amplification, and the effect of experimental conditions on diversity estimates due to in vitro base substitutions, insertions, deletions, and recombination. The use of different NGS platforms and their sequencing error profiles are compared in the context of various applications of diversity estimation, ranging from the detection of single nucleotide variants (SNVs) to the reconstruction of whole-genome haplotypes. We describe the statistical and computational challenges arising from these technical artifacts, and we review existing approaches, including available software, for their solution. Finally, we discuss open problems, and highlight successful biomedical applications and potential future clinical use of NGS to estimate viral diversity.

Minority quasispecies of drug-resistant viruses, detected at baseline, can rapidly outgrow and become the major virus population and subsequently lead to early therapy failure in treatment-naive patients who receive antiretroviral therapy regimens with a low genetic resistance barrier.

Next-generation sequencing (NGS) technologies enable new insights into the diversity of virus populations within their hosts. Diversity estimation is currently restricted to single-nucleotide variants or to local fragments of no more than a few hundred nucleotides defined by the length of sequence reads. To study complex heterogeneous virus populations comprehensively, novel methods are required that allow for complete reconstruction of the individual viral haplotypes. Here, we show that assembly of whole viral genomes of ∼8600 nucleotides length is feasible from mixtures of heterogeneous HIV-1 strains derived from defined combinations of cloned virus strains and from clinical samples of an HIV-1 superinfected individual. Haplotype reconstruction was achieved using optimized experimental protocols and computational methods for amplification, sequencing and assembly. We comparatively assessed the performance of the three NGS platforms 454 Life Sciences/Roche, Illumina and Pacific Biosciences for this task. Our results prove and delineate the feasibility of NGS-based full-length viral haplotype reconstruction and provide new tools for studying evolution and pathogenesis of viruses.