Clonal resistance analyses of HIV type-1 after failure of therapy with didanosine, lamivudine and tenofovir

Background The rapid failure of initial therapy with combinations of nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) that exclude zidovudine has not been fully explained by standard virus population analyses of HIV type-1 (HIV-1) drug resistance. We therefore investigated HIV-1 genotype and phenotype at the single genome level in samples from patients on a failing regimen of tenofovir (TNV), didanosine (ddI) and lamivudine (3TC). Methods Single genome sequencing was performed on 9 failure samples containing both K65R and M184V mutations by standard genotype, either as wild-type/mutant mixtures (6/9) or as mutant only (3/9). Recombinant clones with different combinations of observed mutations were generated and tested for NRTI susceptibility. Results Of the 204 single genome sequences analysed, 50% were K65R/M184V double mutants, 38% were M184V single mutants, 10% were M184I single mutants and only 1% (2 sequences) were K65R single mutants. Phenotypic testing of recombinant clones showed a significant increase in resistance for double mutants: mean fold resistance to abacavir, ddI and TNV was 6.5, 4.3 and 1.6 for K65R/M184V double mutants versus 2.5, 1.9 and 0.6 for M184V single mutants, respectively (P