Ultra-Deep Sequencing of HIV-1 Reverse Transcriptase Before Start of an NNRTI-based Regimen in Treatment-naive Patients

Abstract

There are conflicting data on the impact of low frequency HIV-1 drug-resistant mutants on the response of first-line highly active antiretroviral therapy (HAART), more specifically containing a NNRTI. As population sequencing does not detect resistant viruses representing less than 15-25% of the viral population, more sensitive techniques have been developed but still need clinical validation. We evaluated ultra-deep sequencing (UDPS), recently more available and affordable, as a tool for the detection of HIV-1 minority species carrying drug resistant mutation (DRM) in a clinical setting. A retrospective analysis of the reverse transcriptase (RT) gene of plasma HIV-1 from 70 patients starting a NNRTI based regimen was performed. Minority populations were defined as representing > 1% and < 20% of the total viral population. Using UDPS, we could not confirm an association between the presence of low minority variants harbouring RT mutations at the start of therapy and primary or secondary therapeutic failure.

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.…”

“…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.…”

Sanger and Next Generation Sequencing Approaches to Evaluate HIV-1 Virus in Blood Compartments

“…Nevertheless, low-frequency mutations may emerge in patients with inadequate treatment management [49,50,51]. Recently, NGS sequencing has been proposed as an alternative to sequencing-based methods to identify mutations associated with resistance to antiretrovirals [52,53]. The NGS-based HIV assay is a new platform that allows the monitoring of low abundance viral variants.…”

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

“…Furthermore, it was also demonstrated that these low-abundance mutations can have a significant impact on virological failure, mostly for non-nucleoside analog RT inhibitor (NNRTI) mutations (6,(15)(16)(17)(18). However, despite its high accuracy and effectiveness, the GS FLX platform exceeds the capacity of smalland medium-scale projects, limiting its application for routine testing.…”