title: In vitro acquisition of secondary azole resistance in Aspergillus fumigatus isolates after prolonged exposure to itraconazole: presence of heteroresistant populations.
abstract: Secondary resistance to azoles in Aspergillus fumigatus isolates from patients taking long-term itraconazole therapy has been described. We studied the acquisition of secondary azole resistance in 20 A. fumigatus isolates with no mutations at codon 54, 98, 138, 220, 432, or 448 in the cyp51A gene. Adjusted conidium inocula (3 × 10(7) CFU/ml) of each isolate were prepared and progressively or directly exposed to increasing itraconazole concentrations, ranging from 0.5 μg/ml to 16 μg/ml. Itraconazole, voriconazole, and posaconazole MICs were determined using the CLSI M38-A2 procedure before (MIC(initial)) and after (MIC(final)) exposure to itraconazole. In both procedures, the MIC(final) was significantly higher than the MIC(initial). However, after progressive exposure to itraconazole, the MICs of the three azoles were higher than after direct exposure. No mutations were found at codon 54, 98, 138, 220, 432, or 448 in the cyp51A gene of isolates growing at the highest concentration of itraconazole. More concentrated conidium inocula (2 × 10(9) CFU/ml) plated in itraconazole at 4 μg/ml revealed the presence of heteroresistant populations in two initially wild-type isolates. These isolates became resistant to itraconazole and posaconazole only after use of the concentrated inoculum. These heteroresistant isolates harbored a mutation at codon G54, and the MICs of itraconazole and posaconazole were >16 μg/ml. In all procedures, A. fumigatus short tandem repeat (STRAf) typing was used to demonstrate that the genotype did not change before or after exposure to itraconazole.
authors: Clinical Microbiology and Infectious Diseases Department, Hospital General Universitario Gregorio Marañón, Universidad Complutense de Madrid, Madrid, Spain.
authors: Pilar
authors: P
authors: Escribano
authors: Sandra
authors: S
authors: Recio
authors: Teresa
authors: T
authors: Peláez
authors: Milagros
authors: M
authors: González-Rivera
authors: Emilio
authors: E
authors: Bouza
authors: Jesús
authors: J
authors: Guinea
mesh_terms: D000935:Antifungal Agents / Q000008:administration & dosage*; D001228:Aspergillosis / Q000188:drug therapy* / Q000382:microbiology; D001232:Aspergillus fumigatus / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification; D003062:Codon; D003470:Culture Media; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D004252:DNA Mutational Analysis; D025141:Drug Resistance, Fungal / Q000187:drug effects*; D005656:Fungal Proteins / Q000235:genetics*; D006801:Humans; D017964:Itraconazole / Q000008:administration & dosage*; D008826:Microbial Sensitivity Tests; D009154:Mutation; D016533:Mycological Typing Techniques; D011743:Pyrimidines / Q000008:administration & dosage; D013997:Time Factors; D014230:Triazoles / Q000008:administration & dosage; D065819:Voriconazole
chemical_list: D000935:Antifungal Agents; D003062:Codon; D003470:Culture Media; D005656:Fungal Proteins; D011743:Pyrimidines; D014230:Triazoles; D017964:Itraconazole; C101425:posaconazole; D003577:Cytochrome P-450 Enzyme System; C465526:cytochrome P-450 CYP51A, Aspergillus; D065819:Voriconazole