Unravelling the dynamics of selection of multiresistant variants to integrase inhibitors in an HIV-1-infected child using ultra-deep sequencing.

Background: Ultra-deep sequencing (UDS) allows detection of minority resistant variants (MRVs) with a threshold of 1% and could be useful to identify variants harbouring single or multiple drug-resistance mutations (DRMs).
Objectives: We analysed the integrase gene region longitudinally using UDS in an HIV-1-infected child rapidly failing a raltegravir-based regimen.
Methods: Longitudinal plasma samples at baseline and weeks 4, 8, 13, 17 and 39 were obtained, as well as the mother's baseline plasma sample. Sanger sequencing and UDS were performed on the integrase gene using Roche 454 GS-Junior. A bioinformatic workflow was developed to identify the major DRMs, accessory mutations and the linkage between mutations.
Results: In Sanger sequencing and UDS, no MRV in the integrase gene was detected at baseline in either the mother or the child. The major DRM N155H conferring resistance to raltegravir and elvitegravir was detected in 4% of the sequences by week 4 using UDS, whereas it was not detected by Sanger sequencing. The double mutant E92Q + N155H, conferring resistance to the entire integrase inhibitor class, including dolutegravir, emerged at week 8 (16%) and became rapidly dominant (57% by week 13). At the last timepoint under raltegravir (week 17), Y143R emerged, leading to different resistance mutation patterns: single mutants N155H (47%) and Y143R (24%) and double mutants E92Q + N155H (13%), Y143R + N155H (2%) and E92Q + Y143R (2%). The polymorphic substitution M50I was preferentially selected on resistant variants, especially on E92Q + N155H variants. Conclusions: This case study illustrates the usefulness of UDS in detecting early MRVs and determining the dynamics of selected HIV-1 variants in longitudinal analysis.