A23 Identification of HIV drug resistance mutation patterns using illumina MiSeq next generation sequencing in patients failing second-line boosted protease inhibitor therapy in Nigeria

Abstract

There is limited information of patterns of protease inhibitor(PI) resistance in adults failing second-line therapy. Next generation sequencing (NGS) detects drug resistance mutations aslow as 1%. However, the clinical implications of these minorityvariants to treatment outcomes are still in debate.Approximately 5% of antiretroviral treatment (ART) exposedpatients in our treatment program are on second-line boostedprotease inhibitor (PI). Population-based sequencing conductedon some of these patients revealed no major HIV drug resistance mutations (DRMs) to PIs. We compared population-basedsequencing and NGS results with the view of identifyingpatterns of drug resistance mutations and minority variants.Forty-eight plasma samples from 40 patients on second-lineNRTI/NNRTI and boosted PI regimens with evidence of virologicfailures (VL 1,000 copies/ml) were used in this study. Of these,eight were obtained at the time of first-line failure while theremaining 40 at the time of second-line failure. Ultra-deepsequencing sample preparation was achieved using IlluminaNextera XT protocol. This required that target amplicon be subjected to fragmentation, tagging, indexing, size exclusion beadpurification, normalization, and pooling. MiSeq data analysiswas performed using the Geneious software by applying 1% cutoff at major drug resistance sites. Electropherogram data weregenerated using ABI 3130 genetic analyzer and analysis performed using Stanford Genotyping Resistance InterpretationAlgorithm available at http://sierra2.stanford.edu/sierra/servlet/JSierra and IAS-USA 2015 Drug Resistance Interpretation list.MiSeq sequencing showed that 53% (n = 23) of the patientsdeveloped PI resistance, 93% (n = 40) had NRTI resistance, and70% (n = 30) had NNRTI resistance. Of the DRMs detected in protease, L90M mutation was the most common mutation (28%,n = 12) followed by L76V (21%, n = 9), then I47V (7%, n = 3) andI84V (7%, n = 3). Among the NRTI associated mutations L74Vwas the predominant mutation (77%, n = 33) followed by M184V/I (60%, n = 26) then TAMS (51%, n = 22). Of these, 33% of patients(n = 14) showed NRTI NNRTI mutations, 39% (n = 17) showedNRTI NNRTI PI mutations, 7% (n = 3) showed NRTI PI mutations, whereas 21% (n = 9) and 2.3% (n = 1) exclusively showedNRTI and NNRTIs mutations respectively. Twenty-eight samples that had both MiSeq and Sanger sequencing data wereavailable for a comparison of mutational patterns in the PIregion. Miseq sequencing revealed minority PI mutations in 10samples that were wild type by Sanger sequencing and onesample showed mutations in both Sanger and NGS. The tensamples revealing mutations based on MiSeq data comprised ofminority variants including L90M (50%, n = 5), L76V (20%, n = 2),I50V (10%, n = 1), and N88S (20%, n= 2). Our data suggest thateven in the absence of PI mutations based on Sanger data, thoseminority variants can be present. NGS revealed the presence ofPI resistance mutations in patients who had wild-type usingpopulation-based sequencing. Given that patient regimenrevealed that minority variants were unlikely selected by ARTpressure, our results suggest poor adherence as the likely contributor to second-line failure due to the high genetic barrier ofPIs. Since ART adherence in these patients was monitored usingclinico-immunological parameters and virological tests onlywhen treatment failure was suspected, our results suggest theneed for routine virological monitoring. This should provideearly opportunity for adherence intervention and therebyavoiding the need for switch to salvage or third-line treatmentoptions, which is more expensive and not readily available inour setting.