Ultra-deep sequencing improves the detection of drug resistance in cellular DNA from HIV-infected patients on ART with suppressed viraemia.

BACKGROUND

Standard genotypic tests performed on HIV DNA from patients on suppressive ART, with previous resistance-associated mutations (RAMs) detected in their plasma, underestimate resistance. We thus compared ultra-deep sequencing (UDS) with bulk sequencing of DNA to detect RAMs previously identified in plasma.

METHODS

We sequenced the DNA of 169 highly treatment experienced patients with suppressed viraemia (ANRS 138-EASIER trial). Protease (PR) and reverse transcriptase (RT) genes from HIV DNA were sequenced by bulk sequencing and UDS, comparing 1% and 20% as thresholds of detection for UDS.

RESULTS

Patients were highly treatment experienced (13.6 years). UDS of DNA was successful for the RT and PR genes in 133 (79%) and 137 (81%) patients, respectively. The detection of RAMs was similar by bulk sequencing and UDS with a 20% cut-off. However, the detection of RAMs by UDS with a 1% cut-off was significantly higher than that of bulk sequencing for RT codons D67N (65.4% versus 52.3%), M184V (66.2% versus 52.3%), L210W (48.9% versus 36.4%) and T215Y (57.9% versus 42.1%) and PR codons M46I (46% versus 26%), I54L (12.4% versus 3.9%), V82A (44.5% versus 29.9%) and L90M (57.7% versus 42.5%).

CONCLUSIONS

Genotypic resistance testing of cellular HIV DNA of well-controlled patients should use UDS technology with a sensitivity threshold of 1% to improve the detection of the resistant reservoir.