Background:  The role of minor drug-resistant variants in antiretroviral therapy failure had been recently demonstrated for non-nucleoside reverse transcriptase inhibitors (NNRTI). Genotypic profiles linked to nelfinavir (NFV) failure are mainly associated to the selection of either the D30N or the L90M mutation in the protease gene. It has been suggested that these two mutations are mutually exclusive during the evolutionary process. We investigated the role of minority populations on the evolution of genotypic and phenotypic resistance to a subsequent boosted protease inhibitor (PI) regimen after a first failure to nelfinavir.

Methods:  Standard HIV-1-resistance genotyping was performed by automated population-based full-sequence analysis. In order to determine the diversity of viral quasi-species, the protease genotype of individual molecular clones derived from plasma HIV-1 RNA was determined. Viral phenotype was determined by ViroLogic Inc.

Results:  Two resistance pathways were observed:  the addition of mutations to a currently dominant genotype when minority variants are not evidenced or the emergence of a minority variant as a dominant strain. When apparently only D30N is present on standard genotype, minor species harboring only L90M at very low level (2 per 100 clones) can be detected. In this case, after a subsequent boosted PI regimen used in the context of salvage to NFV, these L90M minor species can emerge as a dominant strain and the D30N species completely disappear under the selective pressure of the new boosted PI-based regimen. In contrast, when only D30N species can be detected by clonal analysis, the evolution of resistance pathways occurred adding other primary PI resistance mutations, such as L90M, in addition to the D30N mutation background. Interestingly, identical genotypic profile (D30N + N88D) on standard genotype displayed different PI susceptibility with the monoclonal viral isolate showing reduced susceptibility to NFV alone (> 10-fold), whereas oligoclonal viral isolate showed a >100-fold reduced susceptibility to NFV and reduced susceptibility to other PI.

Conclusions:  This suggests that a pre-existing minority population can drive the mutational resistance pathway observed after boosted PI failure and that minority viral populations not detected by standard genotype, here representing 2 % of the viral population, may influence the results of phenotypic tests.

Keywords: protease inhibitor; quasispecies; evolution