HIV-1 minority resistance mutations detected with Next Generation Sequencing in treatment naïve patients are not explained by past transmission events.
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.