Virologic Failures on Initial Boosted-PI Regimen Infrequently Possess Low-Level Variants with Major PI Resistance Mutations by Ultra-Deep Sequencing

Article · February 2012with55 Reads
DOI: 10.1371/journal.pone.0030118 · Source: PubMed
It is unknown whether HIV-positive patients experiencing virologic failure (VF) on boosted-PI (PI/r) regimens without drug resistant mutations (DRM) by standard genotyping harbor low-level PI resistant variants. CASTLE compared the efficacy of atazanavir/ritonavir (ATV/r) with lopinavir/ritonavir (LPV/r), each in combination with TVD in ARV-naïve subjects. To determine if VF on an initial PI/r-based regimen possess low-level resistant variants that may affect a subsequent PI-containing regimen. Patients experiencing VF on a Tenofovir/Emtricitabine+PI/r regimen were evaluated by ultra deep sequencing (UDS) for mutations classified/weighted by Stanford HIVdb. Samples were evaluated for variants to 0.4% levels. 36 VF subjects were evaluated by UDS; 24 had UDS for PI and RT DRMs. Of these 24, 19 (79.2%) had any DRM by UDS. The most common UDS-detected DRM were NRTI in 18 subjects: M184V/I (11), TAMs(7) & K65R(4); PI DRMs were detected in 9 subjects: M46I/V(5), F53L(2), I50V(1), D30N(1), and N88S(1). The remaining 12 subjects, all with VLs<10,000, had protease gene UDS, and 4 had low-level PI DRMs: F53L(2), L76V(1), I54S(1), G73S(1). Overall, 3/36(8.3%) subjects had DRMs identified with Stanford-HIVdb weights >12 for ATV or LPV: N88S (at 0.43% level-mutational load 1,828) in 1 subject on ATV; I50V (0.44%-mutational load 110) and L76V (0.52%-mutational load 20) in 1 subject each, both on LPV. All VF samples remained phenotypically susceptible to the treatment PI/r. Among persons experiencing VF without PI DRMs with standard genotyping on an initial PI/r regimen, low-level variants possessing major PI DRMs were present in a minority of cases, occurred in isolation, and did not result in phenotypic resistance. NRTI DRMs were detected in a high proportion of subjects. These data suggest that PIs may remain effective in subjects experiencing VF on a PI/r-based regimen when PI DRMs are not detected by standard or UDS genotyping.
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    • Ultra deep sequencing was performed as described in previous studies and the level of DS variant detection has been previously reported [27][28][29][30][31]. For the purpose of discussion in this paper, we reported a variant detection limit of >1% as resistant variants at this level have been shown to be clinically rele- vant [10,[32][33][34]. In addition, the level of >1% was chosen as the 454 platform used in this study has demonstrated high intralaboratory and interlaboratory consistency in the detection of mutants present at a frequency between 1 and 10% in a large multicentre evaluations [35– 37] The analysis for DRMs in this study was performed using Stanford HIV Drug-Resistance Database and all mutations with a resistance score of e 15 for any drug were considered (http://
    [Show abstract] [Hide abstract] ABSTRACT: Objective: Determine HIV drug resistance mutations (DRMs) prevalence at low and high levels in ART-experienced patients experiencing virologic failure (VF). Methods: 29 subjects from 18 counties in Hunan Province that experienced VF were evaluated for the prevalence of DRMs (Stanford DRMs with an algorithm value ≥15, include low-, intermediate and high-level resistance) by both Sanger sequencing (SS) and deep sequencing (DS) to 1% frequency levels. Results: DS was performed on samples from 29 ART-experienced subjects; the median viral load 4.95×104 c/ml; 82.76% subtype CRF01_AE. 58 DRMs were detected by DS. 18 DRMs were detected by SS. Of the 58 mutations detected by DS, 40 were at levels <20% frequency (26 NNRTI, 12 NRTI and 2 PI) and the majority of these 95.00% (38/40) were not detected by standard genotyping. Of these 40 low-level DRMs, 16 (40%) were detected at frequency levels of 1-4% and 24 (60%) at levels of 5-19%. SS detected 15 of 17 (88.24%) DRMs at levels ≥ 20% that were detected by DS. The only variable associated with the detection of DRMs by DS was ART adherence (missed doses in the prior 7 days); all patients that reported missing a dose in the last 7 days had DRMs detected by DS. Conclusions: DS of VF samples from treatment experienced subjects infected with primarily AE subtype frequently identified Stanford HIVdb NRTI and NNRTI resistance mutations with an algorithm value 15. Low frequency level resistant variants detected by DS were frequently missed by standard genotyping in VF specimens from antiretroviral-experienced subjects.
    Full-text · Article · Feb 2016
    • According to our data, a minimum input of 20,000 copies/ml and an extraction volume of 500 μl plasma should be considered for UDS to guarantee successful amplification of all six amplicons, confirming the 454 recommendation of using the equivalent of at least 2,000 copies per 10 μl RNA [11, 12]. The sensitivity of UDS depends on the mutation-specific error rate, the read coverage at the drug resistance position, and the input viral load of the sample [14, 15,171819 33]. A coverage of at least 50 mutant reads in a total of 5000 reads at the position of interest is recommended to reliably detect minorities at 1% [34].
    [Show abstract] [Hide abstract] ABSTRACT: Pregnant HIV-infected women were screened for the development of HIV-1 drug resistance after implementation of a triple-antiretroviral transmission prophylaxis as recommended by the WHO in 2006. The study offered the opportunity to compare amplicon-based 454 ultra-deep sequencing (UDS) and allele-specific real-time PCR (ASPCR) for the detection of drug-resistant minor variants in the HIV-1 reverse transcriptase (RT).Plasma samples from 34 Tanzanian women were previously analysed by ASPCR for key resistance mutations in the viral RT selected by AZT, 3TC, and NVP (K70R, K103N, Y181C, M184V, T215Y/F). In this study, the RT region of the same samples was investigated by amplicon-based UDS for resistance mutations using the 454 GS FLX System.Drug-resistant HIV-variants were identified in 69% (20/29) of women by UDS and in 45% (13/29) by ASPCR. The absolute number of resistance mutations identified by UDS was twice that identified by ASPCR (45 vs 24). By UDS 14 of 24 ASPCR-detected resistance mutations were identified at the same position. The overall concordance between UDS and ASPCR was 61.0% (25/41). The proportions of variants quantified by UDS were approximately 2-3 times lower than by ASPCR. Amplicon generation from samples with viral loads below 20,000 copies/ml failed more frequently by UDS compared to ASPCR (limit of detection = 650 copies/ml), resulting in missing or insufficient sequence coverage.Both methods can provide useful information about drug-resistant minor HIV-1 variants. ASPCR has a higher sensitivity than UDS, but is restricted to single resistance mutations. In contrast, UDS is limited by its requirement for high viral loads to achieve sufficient sequence coverage, but the sequence information reveals the complete resistance patterns within the genomic region analysed. Improvements to the UDS limit of detection are in progress, and UDS could then facilitate monitoring of drug-resistant minor variants in the HIV-1 quasispecies.
    Full-text · Article · Oct 2015
    Claudia Kücherer+1 more author...
    • Our study shows that ultrasensitive genotyping is helpful to identify in which late presenters NNRTIbased ART is unlikely to fail. The lack of association of DRMVs with virological outcomes of PI/r-including ART is consistent with the previous studies [12,13,222324. Data on the clinical value of DRMVs on first-line PI/r ART is scarce, and definitions of protease inhibitor resistance used in previous studies often included naturally occurring polymorphisms, which have little or no effect on protease inhibitor susceptibility in the absence of major mutations.
    [Show abstract] [Hide abstract] ABSTRACT: This article aims to investigate if the detection of preexisting drug-resistant minority variant (DRMV) and/or X4 HIV-1 variants could improve the efficacy of first-line combined antiretroviral therapy (ART) in late presenters. Post-hoc, combined analysis of two open-label, prospective, randomized clinical trials comparing first-line ART with efavirenz (EFV) vs. ritonavir-boosted protease inhibitor (PI/r)-based regimens in ART-naive, HIV-1-infected patients, with CD4 T-cell counts less than 100 cells/μl and wild-type HIV-1 by bulk sequencing. Pre-ART samples were reanalyzed for the presence of DRMVs and X4 HIV-1 using 454 sequencing. Kaplan-Meier curves and Cox regression were used to evaluate the association between X4 HIV and DRMVs and risk of virological failure. From 141 evaluable patients, 57 received EFV, and 84 received PI/r, including first-line ART. Median pre-ART CD4 T-cell counts and HIV-1 RNA levels were 39 cells/μl and 257 424 copies/ml, respectively; 35.5% of patients had X4 HIV variants. Detection of DRMVs leading to an ART-specific cumulative HIVdb score of at least 10 increased the risk of virological failure in patients initiating EFV [log-rank P = 0.048, hazard ratio = 4.3 (95% confidence interval: 0.8, 25.0), P = 0.074], but not in those starting PI/r. Presence of X4 HIV did not affect virological outcomes, but was associated with impaired CD4 T-cell count recovery over 2 years (214 vs. 315 cells/μl with X4 vs. R5 HIV-1 tropism, respectively, P = 0.017). Accounting for preexisting DRMVs may improve the outcomes of first-line nonnucleoside reverse transcriptase inhibitor-based ART in late presenters with advanced immune suppression. Presence of X4 HIV-1 at diagnosis predicts impaired immune restoration under ART.
    Full-text · Article · Jul 2015
    • Today, deep sequencing methodologies are capable of generating an extraordinary number of sequences (reads), which make them the ideal tool to study intra-and inter-host viral diversity, virus transmission and adaptation dynamics, and disease progression (Fig. 3). Recent strategies to study HIV variability have involved deep sequencing of nearly complete viral genomes949596 or specific genomic regions [26,979899100101102103104105106107. Other studies have focused on the analysis of HIV intra-patient evolution, such as the characterization of transmitted HIV and persistence of minority variants [108], estimation of primary infection dates [109] , description of intrahost evolution dynamics during the course of infection with or without antiretroviral treatment [95,98,103,107,110,111]. Others have analyzed hypermutation patterns [112], viral evolution in different host compartments [113] or in response to the host immune system114115116, and simultaneous assessment of replication fitness of different drug resistant variants [117].
    [Show abstract] [Hide abstract] ABSTRACT: Population (Sanger) sequencing has been the standard method in basic and clinical DNA sequencing for almost 40 years; however, next-generation (deep) sequencing methodologies are now revolutionizing the field of genomics, and clinical virology is no exception. Deep sequencing is highly efficient, producing an enormous amount of information at low cost in a relatively short period of time. High-throughput sequencing techniques have enabled significant contributions to multiples areas in virology, including virus discovery and metagenomics (viromes), molecular epidemiology, pathogenesis, and studies of how viruses to escape the host immune system and antiviral pressures. In addition, new and more affordable deep sequencing-based assays are now being implemented in clinical laboratories. Here, we review the use of the current deep sequencing platforms in virology, focusing on three of the most studied viruses: human immunodeficiency virus (HIV), hepatitis C virus (HCV), and influenza virus.
    Full-text · Article · Sep 2014
    • Detection of low-abundance NNRTI-or NRTI-resistant HIV-1 variants prior the initiation of antiretroviral therapy seems to correlate with a higher risk of virologic failure [32,44,45,65,70]. On the other hand, similar studies have not been able to associate the presence at baseline of low-frequency HIV-1 variants resistant to PI [67], NRTI [68,69], or NNRTI [66] with antiretroviralFigure 6. HIV-1 coreceptor tropism determination using deep sequencing (DEEPGEN [43]) and a phenotypic assay (VERITROP [54]). (A) Hierarchical clustering analysis was used to group the two HIV-1 coreceptor tropism determinations by similarity.
    [Show abstract] [Hide abstract] ABSTRACT: The role of HIV-1 minority variants on transmission, pathogenesis, and virologic failure to antiretroviral regimens has been explored; however, most studies of low-level HIV-1 drug-resistant variants have focused in single target regions. Here we used a novel HIV-1 genotypic assay based on deep sequencing, DEEPGEN (Gibson et al 2014 Antimicrob Agents Chemother 58∶2167) to simultaneously analyze the presence of minority variants carrying mutations associated with reduced susceptibility to protease (PR), reverse transcriptase (RT), and integrase strand transfer integrase inhibitors (INSTIs), as well as HIV-1 coreceptor tropism. gag-p2/NCp7/p1/p6/pol-PR/RT/INT and env/C2V3 PCR products were obtained from twelve heavily treatment-experienced patients experiencing virologic failure while participating in a 48-week dose-ranging study of elvitegravir (GS-US-183-0105). Deep sequencing results were compared with (i) virological response to treatment, (ii) genotyping based on population sequencing, (iii) phenotyping data using PhenoSense and VIRALARTS, and (iv) HIV-1 coreceptor tropism based on the phenotypic test VERITROP. Most patients failed the antiretroviral regimen with numerous pre-existing mutations in the PR and RT, and additionally newly acquired INSTI-resistance mutations as determined by population sequencing (mean 9.4, 5.3, and 1.4 PI- RTI-, and INSTI-resistance mutations, respectively). Interestingly, since DEEPGEN allows the accurate detection of amino acid substitutions at frequencies as low as 1% of the population, a series of additional drug resistance mutations were detected by deep sequencing (mean 2.5, 1.5, and 0.9, respectively). The presence of these low-abundance HIV-1 variants was associated with drug susceptibility, replicative fitness, and coreceptor tropism determined using sensitive phenotypic assays, enhancing the overall burden of resistance to all four antiretroviral drug classes. Further longitudinal studies based on deep sequencing tests will help to clarify (i) the potential impact of minority HIV-1 drug resistant variants in response to antiretroviral therapy and (ii) the importance of the detection of HIV minority variants in the clinical practice.
    Full-text · Article · Aug 2014
    • In such a setting, bPI-containing regimen might be a better suited first-line ART option in RLS that might improve rates of viral suppression and reduce treatment-emergent drug resistance . In case of virologic failure with bPI, due to the absence of major protease inhibitor mutations and infrequent minority variant resistant mutations [35], bPI could possibly be reused with a different antiretroviral backbone including integrase inhibitors [36]. In the next years, availability of other classes for second-line regimens , decreased prices and development of new drug formulation, following WHO prequalification and FDA approval of atazanavir/ritonavir FDC in late 2011, should facilitate strategic decisions regarding future switch of first-line combination ART to bPI. and all the staff (doctors, nurses, social workers, data and administrative workers) of the five clinics of Lubumbashi.
    [Show abstract] [Hide abstract] ABSTRACT: Objective: To compare WHO first-line antiretroviral therapy (ART) with nonnucleoside reverse transcriptase inhibitors (NNRTI)-based regimen with a boosted protease inhibitor (bPI) regimen in a resource-limited setting regarding treatment outcome and emergence of drug resistance mutations (DRMs). Methods: Treatment-naive adults were randomized to nevirapine (NVP) or ritonavir-boosted lopinavir (LPV/r) regimens each in combination with tenofovir (TDF)/emtricitabine (FTC) or zidovudine (ZDV)/lamivudine (3TC). Primary endpoint was the incidence of therapeutical (clinical and/or virologic) failure at week 48 with follow-up till week 96. Results: Four hundred and twenty-five patients (120 men; 305 women) received at least one dose of the study drug. mITT analysis showed no difference in proportion of therapeutical failure between treatment arms [67/209 (32%) in NVP vs. 63/216 (29%) LPV/r at week 48 (P = 0.53); 88/209 (42%) in NVP vs. 83/216 (38%) in LPV/r at week 96 (P = 0.49)]. Per-protocol analysis demonstrated significantly more virologic failure with NVP than with LPV/r regimens [at week 48: 19/167 (11%) vs. 7/166 (4%), P = 0.014; at week 96: 27/158 (17%) vs. 13/159 (8%), P = 0.019)]. Drug resistance mutations to NNRTI were detected in 19 out of 22 (86.3%) and dual-class resistance to nucleoside reverse transcriptase inhibitor (NRTI) and NNRTI in 15 out of 27 (68.2%) of NVP failing patients. K65R mutation was present in seven out of 14 patients failing NVP-TDF/FTC regimen. No major protease inhibitor-DRM was detected among LPV/r failing patients. Discontinuation for adverse events was similar between treatment groups. Conclusion: In resource-limited settings, first-line NNRTI-NRTI regimen as compared with bPI-based regimen provides similar outcome but is associated with a significantly higher number of virologic failure and resistance mutations in both classes that jeopardize future options for second-line therapy.
    Full-text · Article · May 2014
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January 2011 · Antiviral therapy · Impact Factor: 3.02
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