A16 Next-generation sequencing to detect transmitted drug resistance mutations in Romanian people who inject drugs

Romania has faced an HIV outbreak among people who inject drugs (PWID) since 2011. The introduction of so-called ‘legal highs’ (amphetamine-type stimulants) on the drug market a few years prior contributed substantially to this outbreak. Next-generation sequencing (NGS) provides the possibility to detect drug resistance mutations with higher sensitivity than Sanger sequencing. The aim of this study was to search for transmitted drug resistance (TDR) mutations in strains from PWID recently diagnosed with HIV infection by parallel use of Sanger sequencing and NGS. The study was conducted on strains from 34 PWID diagnosed with HIV infection between 2016 and 2017. Sequencing was performed for the pol (PR, RT, and INT) and env (V2-V3 loop) regions. Sanger sequencing was performed with the commercial ViroseqTMHIV-1 Genotyping system (Abbott Laboratories) and with an in-house protocol for the env gene. NGS was performed in the same genomic regions using Nextera DNA Library Preparation Kit (Illumina) and the Miseq instrument (Illumina). NGS data were processed for error correction, read mapping, and detection of drug resistance mutations with HIV-1 Deepchek analysis software. Geno2pheno algorithm was used for viral tropism prediction and the WHO 2009 list for TDRM analysis. By using NGS, we detected seven cases (20.6%) of TDR in PWID and only two cases (5.8%) with standard sequencing. The TDR mutations detected by NGS were K103N, K101EN, Y181C, T215S in RT gene, I54V and M46L in PR, and none in INT. Two NNRTI mutations (K103N and K101EN) were detected in the same sample. Most of the TDR identified were present in the minority population (between 1% and 2% of the total reads) explaining the higher sensitivity of NGS method compared with standard sequencing. No significant differences were observed between these two methods when tropism prediction was analyzed. The majority of the viruses circulating in this group were R5-tropic. All strains showed more resistance mutations when analyzed by deep sequencing than by Sanger sequencing and more than previously observed in other risk groups. NGS proved to be a sensitive tool to detect TDR in newly infected PWID.