Transmitted HIV-1 Drug Resistance in a Large International Cohort Using Next-Generation Sequencing

Results From the Strategic Timing of Antiretroviral Treatment (START) Study

JD Baxter; D Dunn; A Tostevin; RL Marvig; M Bennedbæk; A Cozzi-Lepri; S Sharma; MJ Kozal; M Gompels; AN Pinto; J Lundgren


HIV Medicine. 2021;22(5):360-371. 

In This Article

Abstract and Introduction


Objectives: The aim of this analysis was to characterize transmitted drug resistance (TDR) in Strategic Timing of Antiretroviral Treatment (START) study participants by next-generation sequencing (NGS), a sensitive assay capable of detecting low-frequency variants.

Methods: Stored plasma from participants with entry HIV RNA > 1000 copies/mL were analysed by NGS (Illumina MiSeq). TDR was based on the WHO 2009 surveillance definition with the addition of reverse transcriptase (RT) mutations T215N and E138K, and integrase strand transfer inhibitor (INSTI) surveillance mutations (Stanford HIVdb). Drug resistance mutations (DRMs) detected at three thresholds are reported: > 2%, 5% and 20% of the viral population.

Results: Between 2009 and 2013, START enrolled 4684 antiretroviral therapy (ART)-naïve individuals in 35 countries. Baseline NGS data at study entry were available for 2902 participants. Overall prevalence rates of TDR using a detection threshold of 2%/5%/20% were 9.2%/5.6%/3.2% for nucleoside reverse transcriptase inhibitors (NRTIs), 9.2%/6.6%/4.9% for non-NRTIs, 11.4%/5.5%/2.4% for protease inhibitors (PIs) and 3.5%/1.6%/0.1% for INSTI DRMs and varied by geographic region. Using the 2% detection threshold, individual DRMs with the highest prevalence were: PI M46IL (5.5%), RT K103NS (3.5%), RT G190ASE (3.1%), T215ISCDVEN (2.5%), RT M41L (2.2%), RT K219QENR (1.7%) and PI D30N (1.6%). INSTI DRMs were detected almost exclusively below the 20% detection threshold, most commonly Y143H (0.4%), Q148R (0.4%) and T66I (0.4%).

Conclusions: Use of NGS in this study population resulted in the detection of a large proportion of low-level variants which would not have been detected by traditional Sanger sequencing. Global surveillance studies utilizing NGS should provide a more comprehensive assessment of TDR prevalence in different regions of the world.


Transmitted drug resistance (TDR) refers to the presence of one or more HIV-1 drug resistance mutations (DRMs) in individuals with no prior history of antiretroviral drug exposure. TDR has been associated with reduced susceptibility to antiretroviral agents and increases the risk of a suboptimal virological response to initial antiretroviral therapy (ART).[1,2] Multiple studies from the US and Europe have described the prevalence of TDR in treatment-naïve individuals, which typically ranges from 5% to 15%.[3–8] Available data from resource limited countries have shown that TDR is becoming an emerging health issue, with increasing prevalence rates being reported, particularly for nonnucleoside reverse transcriptase inhibitors (NNRTIs).[3,9]

Global surveillance studies of TDR have primarily used standard Sanger sequencing looking for reverse transcriptase (RT) and protease inhibitor (PI) DRMs, with prevalence rates varying by geographic region.[3,9] There are limited data on global TDR using next-generation sequencing (NGS), which is a highly sensitive assay capable of detecting low-frequency (minor) variants.[10–12] NGS can identify the prevalence of DRMs associated with TDR in a population of individuals while allowing for the detection of low-frequency drug-resistant related variants which may have reduced viral fitness.

The Strategic Timing of Antiretroviral Treatment (START) study is an international trial of immediate vs. deferred ART initiation among treatment-naïve HIV-positive individuals with CD4 counts > 500 cells/μL. Participants were enrolled from 215 sites in 35 countries, representing both resource-rich and resource-limited regions of the world and included North America, Europe, Australia, Latin America, Africa and Asia. A study of TDR in a subset of START study participants was previously described using locally performed Sanger sequencing available almost exclusively in resource-rich regions.[13] In this analysis, we applied a more sensitive assay for detection of drug resistance using NGS to determine TDR prevalence rates from stored specimens for nucleoside reverse transcriptase inhibitor (NRTI), NNRTI, PI and integrase strand transfer inhibitor (INSTI) DRMs at study entry in the entire START study population.