Mapping and characterization of vicriviroc resistance mutations from HIV-1 isolated from treatment-experienced subjects enrolled in a phase II study (VICTOR-E1).

OBJECTIVES: In the phase 2 VICTOR-E1 study, treatment-experienced subjects receiving 20 mg or 30 mg of the CCR5 antagonist vicriviroc (VCV), with a boosted protease containing optimized background regimen, experienced significantly greater reductions in HIV-1 viral load compared with control subjects. Among the 79 VCV-treated subjects, 15 experienced virologic failure, and of these 5 had VCV-resistant virus. This study investigated the molecular basis for the changes in susceptibility to VCV in these subjects. METHODS: Sequence analysis and phenotypic susceptibility testing was performed on envelope clones from VCV-resistant virus. For select clones, an exchange of mutations in the V3 loop was performed between phenotypically resistant clones and the corresponding susceptible clones. RESULTS: Phenotypic resistance was manifest by reductions in the maximum percent inhibition. Clonal analysis of envelopes from the 5 subjects identified multiple amino acid changes in gp160 that were exclusive to the resistant clones, however, none of the changes were conserved between subjects. Introduction of V3 loop substitutions from the resistant clones into the matched susceptible clones was not sufficient to reproduce the resistant phenotype. Likewise, changing the substitutions in the V3 loops from resistant clones to match susceptible clones only restored susceptibility in 1 clone. CONCLUSIONS: There were no clearly conserved patterns of mutations in gp160 associated with phenotypic resistance to VCV and mutations both within and outside of the V3 loop contributed to the resistance phenotype. These data suggest that genotypic tests for VCV susceptibility may require larger training sets and additional information beyond V3 sequences.

Impact of antifungal prophylaxis on colonization and azole susceptibility of Candida species.

Two large studies compared posaconazole and fluconazole or itraconazole for prophylaxis in subjects undergoing allogeneic hematopoietic stem cell transplantation or subjects with acute myelogenous leukemia. To assess the impact of prophylaxis on colonization and the development of resistance in Saccharomyces yeasts, identification and susceptibility testing were performed with yeasts cultured at regular intervals from mouth, throat, and stool samples. Prior to therapy, 34 to 50% of the subjects were colonized with yeasts. For all three drugs, the number of positive Candida albicans cultures decreased during drug therapy. In contrast, the proportion of subjects with positive C. glabrata cultures increased by two- and fourfold in the posaconazole and itraconazole arms, respectively. Likewise, in the fluconazole arm the proportion of subjects with positive C. krusei cultures increased twofold. C. glabrata was the species that most frequently exhibited decreases in susceptibility, and this trend did not differ significantly between the prophylactic regimens. For the subset of subjects from whom colonizing C. glabrata isolates were recovered at the baseline and the end of treatment, approximately 40% of the isolates exhibited more than fourfold increases in MICs during therapy. Molecular typing of the C. albicans and C. glabrata isolates confirmed that the majority of the baseline and end-of-treatment isolates were closely related, suggesting that they were persistent colonizers and not newly acquired. Overall breakthrough infections by Candida species were very rare (approximately 1%), and C. glabrata was the colonizing species that was the most frequently associated with breakthrough infections.

Changes in susceptibility to posaconazole in clinical isolates of Candida albicans.

OBJECTIVES: To characterize the molecular mechanisms responsible for reduced susceptibility to azoles in Candida albicans clinical isolates. MATERIALS AND METHODS: Seven sequential C. albicans isolates were cultured from an AIDS patient treated with posaconazole for refractory oropharyngeal candidiasis. Expression levels of the CDR1, CDR2 and MDR1 genes, encoding efflux pumps previously implicated in azole resistance, and ERG11, encoding the azole target site, were monitored using northern blot and real-time PCR. The ERG11 genes from all seven isolates were sequenced. RESULTS: The seven closely related isolates exhibited significant decreases in susceptibility to fluconazole (MIC >or= 32 mg/L) and voriconazole (MIC >or= 2 mg/L) and progressive decreases in susceptibility to both posaconazole (isolates 1-4 MIC 0.25 mg/L, isolates 5-7 MIC 2 mg/L) and itraconazole (isolates 1-4 MIC 1 mg/L, isolates 5-7 MIC > 8 mg/L). None of the isolates exhibited any significant changes in the expression levels of ERG11 or the efflux pump genes. All seven isolates had multiple mutations in ERG11; isolates one through four each had five missense mutations; four of the resultant amino acid changes were previously associated with azole resistance. The fifth isolate had an additional novel mutation in one copy of ERG11, resulting in a Pro-230 to Leu substitution. This mutation was present in both ERG11 genes in the last two isolates. Select ERG11 genes were expressed in Saccharomyces cerevisiae, the ERG11 allele with all six mutations conferred the highest level of posaconazole resistance. CONCLUSIONS: Multiple mutations in ERG11 are required to confer decreased susceptibility to posaconazole.