Low-Abundance Drug-Resistant Viral Variants in Chronically HIV-Infected, Antiretroviral Treatment-Naive Patients Significantly Impact Treatment Outcomes

454 Life Sciences, a Roche Company, Branford, Connecticut, USA.
The Journal of Infectious Diseases (Impact Factor: 6). 03/2009; 199(5):693-701. DOI: 10.1086/596736
Source: PubMed


Minor (i.e., <20% prevalence) drug-resistant human immunodeficiency virus (HIV) variants may go undetected, yet be clinically important.
To compare the prevalence of drug-resistant variants detected with standard and ultra-deep sequencing (detection down to 1% prevalence) and to determine the impact of minor resistant variants on virologic failure (VF).
The Flexible Initial Retrovirus Suppressive Therapies (FIRST) Study (N = 1397) compared 3 initial antiretroviral therapy (ART) strategies. A random subset (n = 491) had baseline testing for drug-resistance mutations performed by use of standard sequencing methods. Ultra-deep sequencing was performed on samples that had sufficient viral content (N = 264). Proportional hazards models were used to compare rates of VF for those who did and did not have mutations identified.
Mutations were detected by standard and ultra-deep sequencing (in 14% and 28% of participants, respectively; P < .001). Among individuals who initiated treatment with an ART regimen that combined nucleoside and nonnucleoside reverse-transcriptase inhibitors (hereafter, "NNRTI strategy"), all individuals who had an NNRTI-resistance mutation identified by ultra-deep sequencing experienced VF. When these individuals were compared with individuals who initiated treatment with the NNRTI strategy but who had no NNRTI-resistance mutations, the risk of VF was higher for those who had an NNRTI-resistance mutation detected by both methods (hazard ratio [HR], 12.40 [95% confidence interval {CI}, 3.41-45.10]) and those who had mutation(s) detected only with ultra-deep sequencing (HR, 2.50 [95% CI, 1.17-5.36]).
Ultra-deep sequencing identified a significantly larger proportion of HIV-infected, treatment-naive persons as harboring drug-resistant viral variants. Among participants who initiated treatment with the NNRTI strategy, the risk of VF was significantly greater for participants who had low- and high-prevalence NNRTI-resistant variants.

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    • "The new technologies of next-generation sequencing can detect and quantify minority HIV-1 drug resistance mutations. Reports of feasibility and clinical relevance have been presented for HIV-1 drug resistance testing (Codoner et al., 2011; Hoffmann et al., 2007; Lataillade et al., 2010; Le et al., 2009; Simen et al., 2009; Wang et al., 2007) and reviewed in (Beerenwinkel et al., 2012) and for analyzing HIV-1 tropism and coreceptor usage (Abbate et al., 2011; D ¨ aumer et al., 2011; Swenson et al., 2011; Tsibris et al., 2009). Prior to incorporating next-generation sequencing into daily routine diagnoses, proper validation based on consensus criteria is a prerequisite. "
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    ABSTRACT: The detection of mutant spectra within the viral quasispecies is critical for therapeutic management of HIV-1 infections. Routine clinical application of ultrasensitive genotyping requires reproducibility and concordance within and between laboratories. The goal of the study was to evaluate a new protocol on HIV-1 drug resistance testing by 454 ultra-deep pyrosequencing (454-UDS) in an international multicenter study. Sixteen blinded HIV-1 subtype B-samples were provided for 454-UDS as both RNA and cDNA with viral titers of 88,600-573,000 HIV-1 RNA copies/ml. Eight overlapping amplicons spanning protease (PR) codons 10-99 and reverse transcriptase (RT) codons 1-251 were generated using molecular barcoded primers. 454-UDS were performed using the 454 Life Sciences/Roche GS FLX platform. PR and RT sequences were analyzed using 454 Life Sciences Amplicon Variant Analyzer (AVA) software. Quantified variation data were analyzed for intra-laboratory reproducibility and inter-laboratory concordance. Routine population sequencing was performed using the ViroSeq HIV-1 genotyping system. Eleven laboratories and the reference laboratory 454 Life Sciences sequenced the HIV-1 sample set. Data presented are derived from seven laboratories and the reference laboratory since severe study protocol execution errors occurred in four laboratories leading to exclusion. The median sequencing depth across all sites was 1,364 reads per position (IQR=809-2,065). 100% of the ViroSeq-reported mutations were also detected by 454-UDS. Minority HIV-1 drug resistance mutations, defined as HIV-1 drug resistance mutations identified at frequencies of 1-25%, were only detected by 454-UDS. Analysis of 10 preselected majority and minority mutations were consistently found across sites. The analysis of drug-resistance mutations detected between 1-10% demonstrated high intra- and inter-laboratory consistency in frequency estimates for both RNA and prepared cDNA samples, indicating robustness of the method. HIV-1 drug resistance testing using 454 ultra-deep pyrosequencing results in an accurate and highly reproducible, albeit complex, approach to the analysis of HIV-1 mutant spectra, even at frequencies well below those detected by routine population sequencing.
    Full-text · Article · Apr 2014 · Journal of virological methods
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    • "The impact of minor variants on disease progression and HIV evolution is controversial. In drug resistance studies, some have reported an association between minor variants and poor clinical outcome [35-40], while others have found no such association [41-44]. Another important application of MPS is for the detection of minor variants in acute HIV infection, and possibility of early evolution resulting from immune selection pressure as detectable viremia emerges [27]. "
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    ABSTRACT: 454 pyrosequencing, a massively parallel sequencing (MPS) technology, is often used to study HIV genetic variation. However, the substantial mismatch error rate of the PCR required to prepare HIV-containing samples for pyrosequencing has limited the detection of rare variants within viral populations to those present above ~1%. To improve detection of rare variants, we varied PCR enzymes and conditions to identify those that combined high sensitivity with a low error rate. Substitution errors were found to vary up to 3-fold between the different enzymes tested. The sensitivity of each enzyme, which impacts the number of templates amplified for pyrosequencing, was shown to vary, although not consistently across genes and different samples. We also describe an amplicon-based method to improve the consistency of read coverage over stretches of the HIV-1 genome. Twenty-two primers were designed to amplify 11 overlapping amplicons in the HIV-1 clade B gag-pol and env gp120 coding regions to encompass 4.7 kb of the viral genome per sample at sensitivities as low as 0.01-0.2%.
    Full-text · Article · Oct 2013 · PLoS ONE
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    • "Massively parallel sequencing (MPS) technologies, such as 454 pyrosequencing (Margulies et al., 2005), are becoming common to rapidly and cost-effectively detect and quantitate rare sequence variants. Pyrosequencing generates up to millions of reads that can include rare variants to detect low frequency drug resistance and immune escape variants in viral (Human immunodeficiency virus [HIV] and Simian immunodeficiency virus [SIV]) populations (Bimber et al., 2009, 2010; Burwitz et al., 2011; Fischer et al., 2010; Hedskog et al., 2010; Henn et al., 2012; Love et al., 2010; O&apos;Connor et al., 2012; Poon et al., 2010; Simen et al., 2009; Tsibris et al., 2009; Wang et al., 2007). However, the PCR required before pyrosequencing of HIV/SIV populations introduces misincorporation errors, and the pyrosequencing process introduces a significant number of indels and carryforward errors (Margulies et al., 2005). "
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    ABSTRACT: Pyrosequencing technology provides an important new approach to more extensively characterize diverse sequence populations and detect low frequency variants. However, the promise of this technology has been difficult to realize, as careful correction of sequencing errors is crucial to distinguish rare variants (∼1%) in an infected host with high sensitivity and specificity. We developed a new approach, referred to as Indel and Carryforward Correction, or ICC, to cluster sequences without substitutions and locally correct only indel and carryforward sequencing errors within clusters to ensure that no rare variants are lost. ICC performs sequence clustering in the order of (a) homopolymer indel patterns only, (b) indel patterns only, and (c) carryforward errors only, without the requirement of a distance cut-off value. Overall, ICC removed 93%-95% of sequencing errors found in control data sets. On pyrosequencing data from a PCR fragment derived from 15 HIV-1 plasmid clones mixed at various frequencies as low as 0.1%, ICC achieved the highest sensitivity and similar specificity compared to other commonly used error correction and variant calling algorithms.Availability and implementation: Source code is freely available for download at It is implemented in Perl and supported on Linux, Mac OS X and MS Windows. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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