Pretreatment HIV drug resistance in the start study using next generation sequencing

Abstract

Background: In START, an international trial of immediate verses deferred ART initiation among ART-naive HIV-infected persons with CD4 counts >500 cells/muL, study entry HIV-1 was characterized by next generation sequencing (NGS); a sensitive assay capable of detecting low-frequency variants associated with pre-treatment HIV-1 drug resistance (PDR). Methods: Stored plasma specimens from participants with study entry HIV RNA >1,000 copies/ml were analyzed by NGS (Ilumina MiSeq). PDR was based on the WHO 2009 surveillance definition with the addition of reverse transcriptase (RT) mutations T215N and E138K, and integrase inhibitor (INI) mutations (Stanford HIVdb). Drug resistance mutations (DRMs) detected at two thresholds are reported, >=2% verses >20% of the viral population (the latter comparable to the detection threshold for Sanger sequencing). Results: Between 2009-2013, the START trial enrolled 4,684 ART-naive individuals in 35 countries. Baseline NGS data at study entry was available for 3,365 participants; median CD4 count 642 cells/muL, median HIV RNA 19,800 copies/mL, and median time since diagnosis was 0.9 years. The overall prevalence of PDR in START using a detection threshold of >=2%/>20% was 19.7/8.3% for RT-protease (PR) DRMs (6.1/2.7% for NRTIs, 7.0/4.3% NNRTIs, 9.0/2.1% PIs) and 0.9/<0.1% for INI DRMs. The prevalence of RT-PR PDR was highest in Australia and the USA, while lowest in Africa (figure). Prevalence of NNRTI PDR using the >=2%/>20% thresholds by region was: USA 11.7/9.4%, Latin America 7.7/5.8%, Europe 6.8/3.3%, Asia 5.7/2.2%, Australia 5.5/2.7%, and Africa 4.1/2.2%. Using the >=2% detection threshold, the six individual DRMs with the highest prevalence were: RT K103N 2.9/2.7%, M41L 1.6/1.2%, G190E 1.3/0.1%, PR M46I 3.1/0.4%, M46L 1.3/0.5%, and D30N 1.3/0.2% at the >=2%/>20% thresholds. RT M184I 0.4/0%, D67G 0.1/0%, L74V 0.1/0%, and Y188C 0.1/0% were only detected as minor variants. The most frequently detected INI DRMs were Y143H 0.2/0%, T66I 0.1/<0.1%, and Y143C 0.1/0%. Conclusion: The START trial represents one of the largest global cohorts with NGS characterization of PDR. Overall prevalence of PDR using the >=2% detection threshold was 19.7% for RT-PR DRMs, while only 8.3% using the 20% threshold and varied by region. INI DRMs were detected in 0.9% of the study cohort primarily as minor variants. NGS would be expected to detect a substantially higher prevalence of PDR than traditional Sanger sequencing, particularly for DRMs occurring predominately as minor variants. (Figure Presented).