Characterization of minority HIV-1 drug resistant variants in the United Kingdom following the verification of a deep sequencing-based HIV-1 genotyping and tropism assay

Abstract

BackgroundThe widespread global access to antiretroviral drugs has led to considerable reductions in morbidity and mortality but, unfortunately, the risk of virologic failure increases with the emergence, and potential transmission, of drug resistant viruses. Detecting and quantifying HIV-1 drug resistance has therefore become the standard of care when designing new antiretroviral regimens. The sensitivity of Sanger sequencing-based HIV-1 genotypic assays is limited by its inability to identify minority members of the quasispecies, i.e., it only detects variants present above ~ 20% of the viral population, thus, failing to detect minority variants below this threshold. It is clear that deep sequencing-based HIV-1 genotyping assays are an important step change towards accurately monitoring HIV-infected individuals.MethodsWe implemented and verified a clinically validated HIV-1 genotyping assay based on deep sequencing (DEEPGEN™) in two clinical laboratories in the United Kingdom: St. George’s University Hospitals Healthcare NHS Foundation Trust (London) and at NHS Lothian (Edinburgh), to characterize minority HIV-1 variants in 109 plasma samples from ART-naïve or -experienced individuals.ResultsAlthough subtype B HIV-1 strains were highly prevalent (44%, 48/109), most individuals were infected with non-B subtype viruses (i.e., A1, A2, C, D, F1, G, CRF02_AG, and CRF01_AE). DEEPGEN™ was able to accurately detect drug resistance-associated mutations not identified using standard Sanger sequencing-based tests, which correlated significantly with patient’s antiretroviral treatment histories. A higher proportion of minority PI-, NRTI-, and NNRTI-resistance mutations was detected in NHS Lothian patients compared to individuals from St. George’s, mainly M46I/L and I50 V (associated with PIs), D67 N, K65R, L74I, M184 V/I, and K219Q (NRTIs), and L100I (NNRTIs). Interestingly, we observed an inverse correlation between intra-patient HIV-1 diversity and CD4+ T cell counts in the NHS Lothian patients.ConclusionsThis is the first study evaluating the transition, training, and implementation of DEEPGEN™ between three clinical laboratories in two different countries. More importantly, we were able to characterize the HIV-1 drug resistance profile (including minority variants), coreceptor tropism, subtyping, and intra-patient viral diversity in patients from the United Kingdom, providing a rigorous foundation for basing clinical decisions on highly sensitive and cost-effective deep sequencing-based HIV-1 genotyping assays in the country.Electronic supplementary materialThe online version of this article (10.1186/s12981-018-0206-y) contains supplementary material, which is available to authorized users.

Pre-existing resistance in the latent reservoir can compromise VRC01 therapy during chronic HIV-1 infection

“…Testing our hypothesis experimentally would require detecting minority variants ‘within’ latently infected cells, highlighting the challenge involved. Current assays can rarely detect variants below a frequency of ~1% [ 34 ], implying that, given the prevalent estimates of the latent reservoir size of 10 5 –10 8 cells [ 4 , 35 ], variants present in as many as 10 3 latently infected cells may go undetected and be responsible for therapy failure. Indeed, in many individuals who failed rapidly in the trials above [ 17 ], viral genome sequencing could not detect VRC01 resistance pre-treatment [ 17 , 36 ].…”

Next-Generation Sequencing for HIV Drug Resistance Testing: Laboratory, Clinical, and Implementation Considerations

“…Each of these steps introduces variation and possible bias that affects the NGS output representativeness of the viral quasispecies, HIV drug resistance mutation (DRM) detection sensitivity and accuracy, and subsequently HIVDR interpretation, which will be discussed in the following sections ( Figure 1 ). Several commercial options for HIVDR testing using NGS are already available and have obtained regulatory approval as in vitro diagnostic (IVD) products by reference agencies ( Table 1 ) [ 13 , 14 , 15 ]. However, due to cost and flexibility issues, many laboratories have opted for developing and validating in house protocols.…”

“… a Validated test to be used in a CAP/CLIA-certified laboratories [ 13 , 14 ]. Abbreviations: ABL: Advanced biological Laboratories; CWRU: Case Western Reserve University; PR: protease; RT: Reverse transcriptase; IN: Integrase; PGM: Personal Genome Machine; CE-IVD: European CE Marking for In Vitro Diagnostic (IVD) devices; RUO: Research use only; TGA: Therapeutic Goods Administration in Australia; CAP/CLIA: College of American Pathologists/Clinical Laboratory Improvement Amendments; FDA: US Food and Drug Administration; HSA: Singapore Health Science Authority.…”

HIV-1 Drug Resistance Genotyping in Resource Limited Settings: Current and Future Perspectives in Sequencing Technologies

“…This, however, means that Sanger sequencing does not reliably detect mutations that are less represented within the viral pool, also known as low-abundance drug-resistant variants (LA-DRVs) [8,9]. Despite that, HIVDR mutations detected by Sanger sequencing have been shown to predict treatment response, making it a reliable method for use in making clinical decisions [10]. On the other hand, NGS is more sensitive, having the ability to detect LA-DRVs (i.e., viral variants at <20%) [11], and enabling quantitative detection of HIVDR mutations [12].…”