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

Lataillade, Max; Chiarella, Jennifer; Yang, Rong; DeGrosky, Michelle; Uy, Jonathan; Seekins, Daniel; Simen, Birgitte B.; John, Elizabeth St.; Moreno, Elizabeth A; Kozal, Michael J.

doi:10.1371/journal.pone.0030118

Cited by 33 publications

(28 citation statements)

References 10 publications

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“…This patient experienced viral failure on an FTC/TDF-ritonavirboosted FPV regimen but had no significant PI mutations detected by TG. Such a finding is in accord with previous reports of prevalence of PI minor mutations in patients who experienced virological failure on boosted-PI regimens but had no drug-resistant mutations detected by standard drug resistance genotyping (23,26).…”

Section: Discussionsupporting

confidence: 92%

Evaluation of a Benchtop HIV Ultradeep Pyrosequencing Drug Resistance Assay in the Clinical Laboratory

et al. 2013

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f Detection of low-abundance drug resistance mutations (DRMs) of HIV-1 is an evolving approach in clinical practice. Ultradeep pyrosequencing has shown to be effective in detecting such mutations. The lack of a standardized commercially based assay limits the wide use of this method in clinical settings. 454 Life Sciences (Roche) is developing an HIV ultradeep pyrosequencing assay for their benchtop sequencer. We assessed the prototype plate in the clinical laboratory. Plasma samples genotyped by the standardized TruGene kit were retrospectively tested by this assay. Drug-treated subjects failing therapy and drug-naive patients were included. DRM analysis was based on the International AIDS Society USA DRM list and the Stanford algorithm. The prototype assay detected all of the DRMs detected by TruGene and additional 50 low-abundance DRMs. Several patients had lowabundance D67N, K70R, and M184V reverse transcriptase inhibitor mutations that persisted long after discontinuation of the drug that elicited these mutations. Additional patient harbored low-abundance V32I major protease inhibitor mutation, which under darunavir selection evolved later to be detected by TruGene. Stanford analysis suggested that some of the low-abundance DRMs were likely to affect the resistance burden in these subjects. The prototype assay performs at least as well as TruGene and has the advantage of detecting low-abundance drug resistance mutations undetected by TruGene. Its ease of use and lab-scale platform will likely facilitate its use in the clinical laboratory. The extent to which the detection of low-abundance DRMs will affect patient management is still unknown, but it is hoped that use of such an assay in clinical practice will help resolve this important question.

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Section: Discussionsupporting

confidence: 92%

Evaluation of a Benchtop HIV Ultradeep Pyrosequencing Drug Resistance Assay in the Clinical Laboratory

et al. 2013

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“…Massively parallel sequencing technologies, such as 454 pyrosequencing (23), also can detect and quantify low-frequency drug resistance variants in HIV populations from individuals (24)(25)(26). In this study, we compared the detection of drug resistance mutations by the OLA versus 454 pyrosequencing, to evaluate the sensitivity of the OLA to detect and to quantify mutants in clinical specimens across different HIV subtypes and across HIV codons associated with NNRTI and lamivudine (2=,3=-dideoxy-3=-thiacytidine [3TC]) resistance.…”

mentioning

confidence: 99%

Validation of an Oligonucleotide Ligation Assay for Quantification of Human Immunodeficiency Virus Type 1 Drug-Resistant Mutants by Use of Massively Parallel Sequencing

Beck¹,

Deng

Payant³

et al. 2014

J Clin Microbiol

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Global HIV treatment programs need sensitive and affordable tests to monitor HIV drug resistance. We compared mutant detection by the oligonucleotide ligation assay (OLA), an economical and simple test, to massively parallel sequencing. Nonnucleoside reverse transcriptase inhibitor (K103N, V106M, Y181C, and G190A) and lamivudine (M184V) resistance mutations were quantified in blood-derived plasma RNA and cell DNA specimens by OLA and 454 pyrosequencing. A median of 1,000 HIV DNA or RNA templates (range, 163 to 1,874 templates) from blood specimens collected in Mozambique (n ‫؍‬ 60) and Kenya (n ‫؍‬ 51) were analyzed at 4 codons in each sample (n ‫؍‬ 441 codons assessed). Mutations were detected at 75 (17%) codons by OLA sensitive to 2.0%, at 71 codons (16%; P ‫؍‬ 0.78) by pyrosequencing using a cutoff value of >2.0%, and at 125 codons (28%; P < 0.0001) by pyrosequencing sensitive to 0.1%. Discrepancies between the assays included 15 codons with mutant concentrations of ϳ2%, one at 8.8% by pyrosequencing and not detected by OLA, and one at 69% by OLA and not detected by pyrosequencing. The latter two cases were associated with genetic polymorphisms in the regions critical for ligation of the OLA probes and pyrosequencing primers, respectively. Overall, mutant concentrations quantified by the two methods correlated well across the codons tested (R 2 > 0.8). Repeat pyrosequencing of 13 specimens showed reproducible detection of 5/24 mutations at <2% and 6/6 at >2%. In conclusion, the OLA and pyrosequencing performed similarly in the quantification of nonnucleoside reverse transcriptase inhibitor and lamivudine mutations present at >2% of the viral population in clinical specimens. While pyrosequencing was more sensitive, detection of mutants below 2% was not reproducible.

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“…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 antiretroviral therapy failure. To the best of our knowledge, no study has used deep sequencing to associate the effect of low-level INSTI-resistance variants prior the initiation of an INSTI-based antiretroviral regimen.…”

Section: Discussionmentioning

confidence: 93%

Contribution of Human Immunodeficiency Virus Type 1 Minority Variants to Reduced Drug Susceptibility in Patients on an Integrase Strand Transfer Inhibitor-Based Therapy

et al. 2014

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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.

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

Cited by 33 publications

References 10 publications

Evaluation of a Benchtop HIV Ultradeep Pyrosequencing Drug Resistance Assay in the Clinical Laboratory

Evaluation of a Benchtop HIV Ultradeep Pyrosequencing Drug Resistance Assay in the Clinical Laboratory

Validation of an Oligonucleotide Ligation Assay for Quantification of Human Immunodeficiency Virus Type 1 Drug-Resistant Mutants by Use of Massively Parallel Sequencing

Contribution of Human Immunodeficiency Virus Type 1 Minority Variants to Reduced Drug Susceptibility in Patients on an Integrase Strand Transfer Inhibitor-Based Therapy

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