Tenofovir (TDF)-Selected or Abacavir (ABC)-Selected Low-Frequency HIV Type 1 Subpopulations During Failure with Persistent Viremia as Detected by Ultradeep Pyrosequencing

Abstract

Detection of drug resistance is critical for determining antiretroviral treatment options. Ultradeep pyrosequencing (UDPS; 454 Life Sciences) is capable of detecting virus variant subpopulations with much greater sensitivity than population sequencing, which typically has a detection limit around 20%. UDPS of the HIV-1 reverse transcriptase (RT) (amino acids 56-120) was performed to detect the key mutations K65R and L74V associated with tenofovir and abacavir use. Plasma specimens from subjects with persistent rebound viremia following suppression on tenofovir (n = 8) or abacavir (n = 9)-based therapy were studied. Samples from a subject treated with zidovudine/lamivudine/efavirenz with a similar loss of virologic response served as a control. HIV-1 plasma RNA was ≥3.68 log(10) copies/ml at all time points sequenced. The median number of UDPS sequences analyzed/time point was 33,246. Among the eight tenofovir-treated subjects, three showed high-frequency (>20%) RT K65R at the time of failure, whereas one showed low-frequency (<20%) L74V; no low-frequency K65R was detected in these subjects. Among the nine abacavir-treated subjects, three showed low-frequency K65R; no L74V was detected in these patients. No K65R or L74V was detected in the samples from the control subject. At failure, other RT mutations were detected, including low-frequency NNRTI-resistant species detected at ≥1 time point in nine subjects; the key NNRTI mutation K103N, however, was always observed at >20% frequency. Although UDPS is useful in the detection of low-frequency subpopulations with transmitted resistance in antiviral-naive patients, it may have less utility in treatment-experienced patients with persistent viremia on therapy.

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

“…Nonetheless, using a deep sequencing-based HIV-1 genotyping assay (DEEPGEN) [43] we were able to identify low-level HIV-1 variants (at a frequency of <20% of the population) carrying 59 additional drug-resistance mutations in the protease, reverse transcriptase and integrase coding regions of viruses from these highly antiretroviral-experienced patients. Previous studies have described similar findings, although analyzing no more than one or two drug-targeted regions at a time, in antiretroviral-naïve [65], [66], [73], [74], [75], [76] or -experienced HIV-infected individuals [43], [68], [69], [77], [78], [79], [80], with the detection of minority mutations usually correlating with historical antiretroviral treatment. In the case of INSTI-resistance mutations, using deep sequencing we corroborated and quantified all the amino acid substitutions identified by Sanger sequencing, even in positions labeled as mixtures, e.g., E92E/Q (55.1% E92Q) or N155N/H (79.9% N155H) in patients 08–172 and 08–177, respectively.…”

“…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.…”

“…While a few studies have failed to establish an association between the presence of minority variants carrying drug resistance mutations and therapeutic failure [66], [67], [68], [69], many others have associated the identification of low-abundance drug-resistant viral variants as having an impact in treatment outcome [32], [35], [44], [45], [65], [70]. It is possible that the threshold for the identification of significant low-level variants may be mutation and/or antiretroviral drug dependent [31]; thus, it is clear that additional studies based on reliable ultrasensitive assays are needed to better understand the clinical relevance of these minority HIV-1 variants.…”

Dynamics of Envelope Evolution in Clade C SHIV-Infected Pig-Tailed Macaques during Disease Progression Analyzed by Ultra-Deep Pyrosequencing

“…The high sensitivity of this technology in detecting minor populations makes it an ideal approach for our study. UDPS has been used by several groups to study minor drug resistance mutations, cytotoxic T-lymphocytes (CTL) escape pathways and small envelope region such as V3 [17], [18], [19], [20], [21], [22]. To our knowledge, this is the first study to use UDPS to resolve HIV-1 clade C envelope evolution in infected pig-tailed macaques during disease progression which is marked by a consistent decline in CD4 + T-cell count and the present of high plasma viral load.…”

Genotypic resistance testing in routine clinical care

“…However, due to the innate error rate of reaction enzymes, it has been reported that that the sensitivity of UDS is limited to about 0.5% [56]. D'Aquila et al [57] found that UDS rarely detected K65R and L74V not already found by conventional sequencing among patients failing regimens containing TDF or ABC and concluded it had little utility in treatment-experienced patients.…”