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

Nicholas Silver,Christine Sayir,Kate Templeton,Mary Paynter,Georgina Mcallister,Maureen Atchley,David Carrington,Kicki Bergefall,David J Alouani,Freddie H Sharkey,Miguel E Quiñones-Mateu,Dane Winner,John Short

doi:10.1186/s12981-018-0206-y

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.

Highlights

The widespread global access to antiretroviral drugs has led to considerable reductions in morbidity and mortality but, the risk of virologic failure increases with the emergence, and potential transmis‐ sion, of drug resistant viruses
At the time of the study, most patients on treatment had received a median of 2.3 PIs (IQR: 0-5), all were treated with nucleoside/tide reverse transcriptase (NRTI)
George’s and NHS Lothian patients, respectively), approximately half of the patients in each cohort were exposed to non-nucleoside reverse transcriptase (NNRTI) (24/45 and 12/22), and around one-third to INSTIs (17/45 and 6/22)

Summary

Introduction

The widespread global access to antiretroviral drugs has led to considerable reductions in morbidity and mortality but, the risk of virologic failure increases with the emergence, and potential transmis‐ sion, of drug resistant viruses. HIV-1 genotypes based on Sanger sequencing can only detect HIV-1 variants present at frequencies above approximately 20% of the viral quasispecies [14,15,16,17,18], failing to quantify low-levels of HIV-1 drug resistant variants [10, 19]. These variants, usually present as minority members of the virus population, can be selected and become predominant under the appropriate pressure by antiretroviral drugs [20,21,22]. The clinical significance of these minority drug resistant HIV-1 variants is still on discussion [29,30,31,32,33], numerous groups are using these assays to monitor HIV-1 drug resistance and to better understand the role of low-level HIV-1 variants on transmission, disease progression, and HIV-1 cure strategies [reviewed on [10, 11]]

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Results

Discussion

Conclusion