HIV-1 minority resistance mutations detected with Next Generation Sequencing in treatment naïve patients are not explained by past transmission events.. Evaluation of the potential of next generation sequencing to monitor transmitted HIV-1 drug resistance mutations in newly diagnosed therapy naïve patients.

Background: Sanger based sequencing (SBS) is the gold standard method for detection of HIV-1 transmitted drug resistance mutations (TDRM). SBS allows for detection of TDRM present in 20% of the viral population. Next generation sequencing (NGS) methods can detect minority variants (MV) at significantly lower occurrences than SBS and might therefore not only improve TDRM-surveillance but also information about suitable HIV-therapeutics at treatment-baseline. Objectives: To evaluate the potential advantages of NGS as compared to the golden-standard SBS analysis in assessment of TDRM prevalence among HIV-1-treatment naïve patients at different clinical stages of presentation (late presenters (LP) versus non-late presenters (NLP)). Study design: Samples from 93 patients (53 LP and 40 non-late presenters) diagnosed during the period of 2001-2016 were used to generate duplicated RT-PCR products of the pol gene. PCR products were subsequently analyzed by NGS on the Illumina MiSeq platform. TDRM were identified from phylogenetic cluster analysis of possible matching sequences. Results: A total of 70 drug resistance mutations (DRM) and 8 TDRM were detected by NGS at the 1% cut-off level as compared with 18 DRM detected by SBS. However, all eight identified TDRM were also detectable by SBS. Significantly more DRM were detected by NGS in HIV-specimens from LP, which also harbored more diverse and less identical sequences. Conclusion: This study using NGS rejects the hypothesis that the true TDRM prevalence is under-reported due to limitations in the current ability to detect minority TDRM by SBS. However, NGS improved detection of DRM among LP.