Minor HIV-1 Variants with the K103N Resistance Mutation during Intermittent Efavirenz-Containing Antiretroviral Therapy and Virological Failure

The impact of minor drug-resistant variants of the type 1 immunodeficiency virus (HIV-1) on the failure of antiretroviral therapy remains unclear. We have evaluated the importance of detecting minor populations of viruses resistant to non-nucleoside reverse-transcriptase inhibitors (NNRTI) during intermittent antiretroviral therapy, a high-risk context for the emergence of drug-resistant HIV-1. We carried out a longitudinal study on plasma samples taken from 21 patients given efavirenz and enrolled in the intermittent arm of the ANRS 106 trial. Allele-specific real-time PCR was used to detect and quantify minor K103N mutants during off-therapy periods. The concordance with ultra-deep pyrosequencing was assessed for 11 patients. The pharmacokinetics of efavirenz was assayed to determine whether its variability could influence the emergence of K103N mutants. Allele-specific real-time PCR detected K103N mutants in 15 of the 19 analyzable patients at the end of an off-therapy period while direct sequencing detected mutants in only 6 patients. The frequency of K103N mutants was <0.1% in 7 patients by allele-specific real-time PCR without further selection, and >0.1% in 8. It was 0.1%–10% in 6 of these 8 patients. The mutated virus populations of 4 of these 6 patients underwent further selection and treatment failed for 2 of them. The K103N mutant frequency was >10% in the remaining 2, treatment failed for one. The copy numbers of K103N variants quantified by allele-specific real-time PCR and ultra-deep pyrosequencing agreed closely (ρ = 0.89 P<0.0001). The half-life of efavirenz was higher (50.5 hours) in the 8 patients in whom K103N emerged (>0.1%) than in the 11 patients in whom it did not (32 hours) (P = 0.04). Thus ultrasensitive methods could prove more useful than direct sequencing for predicting treatment failure in some patients. However the presence of minor NNRTI-resistant viruses need not always result in virological escape. TRIAL REGISTRATION: ClinicalTrials.gov NCT00122551