Drug resistance testing is an important tool in the management of HIV-1 infection. As access to genotypic resistance assays is limited in low- and middle-income settings, alternatives are warranted. The aim of the study was to adapt a phenotypic drug susceptibility assay, ExaVir Drug, for detection of resistance to the second generation non-nucleoside reverse transcriptase inhibitor (NNRTI) etravirine (ETR). Five NNRTI resistant mutant forms of RT were produced (L100I, K103N, L100I/K103N, Y181C, V179D) in order to validate the assay for ETR. Furthermore, HIV-1 RT was purified from plasma samples (n = 28) obtained from treatment naïve and experienced HIV-1 infected patients, and ETR drug susceptibility (IC(50)) was estimated. The direct sequencing of the pol gene was performed. The recombinant RT mutants had the expected changes in drug sensitivity patterns. The RTs isolated from plasma of therapy naïve individuals showed low IC(50) for ETR. In the plasma virus from treatment experienced patients with Y181C, A98G, V108I, and/or K101E mutations in the pol gene, higher IC(50) values were found in line with reduced susceptibility data for ETR. This study demonstrates that ExaVir® Drug, a simple enzymatic phenotypic assay, can be used for detection of ETR resistance, including cross-resistance to other NNRTIs, in clinical samples. A further evaluation is needed to define clinical cut-offs; however the assay is an alternative to more costly HIV drug resistance tests, especially in low-income countries.
BackgroundA sensitive, phenotypic reverse transcriptase (RT)-based drug susceptibility assay for the detection of etravirine (ETR) resistance in patient isolates was developed and compared with the results from direct sequencing and ultra-deep pyrosequencing (UDPS).MethodsSamples were obtained from 15 patients with antiretroviral therapy (ART) failure and from five non-nucleoside reverse transcriptase inhibitor (NNRTI)-naïve patients of whom four were infected by an NNRTI-resistant strain (transmitted drug resistance, TDR). In five patients, two consecutive samples (a and b) were taken for follow up of the virological response. HIV-1 RT was purified and drug susceptibility (IC50) to ETR was estimated. Direct sequencing was performed in all samples and UDPS in samples from nine patients.ResultsIncreased IC50 to ETR was found in samples from 13 patients where direct sequencing predicted resistance in only four. UDPS identified additional (N = 11) NNRTI resistance associated mutations (RAMs) in six of nine tested patients. During early failure, IC50 increases were observed in three of six patients without any ETR-RAMs detected by direct sequencing. In further two patients, who stopped NNRTI before sampling, increased IC50 values were found shortly after, despite absence of ETR-RAMs. In two patients who had stopped NNRTI for >1 year, a concordance between phenotype and genotypes was found. Two patients with TDR had increased IC50 despite no ETR-RAMs were detected by direct sequencing. UDPS revealed additional ETR-RAMs in four patients with a discrepancy between phenotype and direct sequencing.ConclusionsThe RT-based phenotypic assay showed decreased ETR susceptibility in patients where direct sequencing predicted ETR-sensitive virus. This increased phenotypic sensitivity was to a large extent supported by UDPS and treatment history. Our method could be valuable for further studies on the phenotypic kinetics of NNRTI resistance. The clinical relevance remains to be studied in larger patient-populations.
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