Background:  More sensitive means of detecting resistance mutations and their linkage are needed to define HIV-1 diversity in treatment-experienced patients. To investigate the extent to which drug-resistance mutations are missed by standard genotyping methods, we performed single-genome sequencing and standard genotyping on the same plasma samples from patients with multidrug resistant HIV-1.

Methods:  Plasma samples were obtained from 28 patients, either drug naïve, on antiretroviral therapy, or known to be infected with multidrug resistant HIV-1. Standard genotypes were obtained by RT-PCR and sequencing of bulk PCR product.  For single-genome RT-PCR sequencing, cDNA derived from plasma RNA was serially diluted to a single copy, and a region encompassing p6, protease, and RT was amplified and sequenced. Sequences from 11 to 50 single viral genomes were obtained from each sample. Mutations included in the analysis were those defined as conferring resistance by the Stanford database.

Results:  All plasma samples tested contained between one and 10 drug-resistance mutations that were identified by single-genome sequencing but were not detected by standard genotype analysis. Drug-resistance mutations present in fewer than 10% of genomes were not detected by standard genotyping, whereas mutations present in 10 to 35% of single genomes were only present 25% of the time in a standard genotype. For example, in one patient, 10 mutations identified by single-genome sequencing and conferring resistance to PI, NRTI, and NNRTI escaped detection by standard genotype of the same plasma sample.  Each of these mutations was present in 5 to 20% of the 20 genomes analyzed; 15% of the genomes in this sample contained linked PI mutations (L10V, M46I, I84V, L90M, I93L), none of which was detected in the standard genotype. In another patient sample, 33% of genomes contained 5 linked RTI resistance mutations (A98S, K101E, Y181C, G190A, T215Y), none of which were detected by standard genotype.

Conclusions:  These results illustrate the inadequacy of standard genotype for detecting drug-resistance mutations present in less than 35% of the plasma virus population. Mutations present in <10% of the virus population were almost never detected by standard genotype and those present in 10 to 35% were usually not detected. In addition to its greater sensitivity, single-genome sequencing identifies linked mutations that confer high-level drug resistance. Such linkage cannot be detected in standard genotypes.

Keywords: Single Genome Sequencing; HIV Genotyping; Detection of Drug Resistance mutations