Mutations in the cyp51A gene and susceptibility to itraconazole in Aspergillus fumigatus serially isolated from a patient with lung aspergilloma.
OBJECTIVE
To monitor changes in itraconazole susceptibility of isolates from a patient undergoing treatment for pulmonary Aspergillus infection and relate these changes to genotypic/phenotypic alterations.


METHODS
Six Aspergillus fumigatus isolates were serially recovered from the patient. Itraconazole MICs were determined by Etest and NCCLS methodology. Growth characteristics and phenotype were monitored. Molecular analysis included random amplified polymorphic DNA (RAPD) assay and sequencing of the cyp51A gene.


RESULTS
The MIC of itraconazole against the first isolate before treatment was 0.25 mg/L; the MIC against the second isolate, recovered after 6 months of itraconazole therapy, was >16 mg/L; and that against the third isolate, obtained 2 months after discontinuation of the therapy, was 0.5 mg/L. The MIC against the last three isolates, acquired after restoration of itraconazole therapy for 4-7 months, was >16 mg/L. The six isolates shared identical band patterns of RAPD assay using four primers and the same sequence in intertranscribed spacers (ITS). Therefore, the six isolates were likely to be the same strain of A. fumigatus, and mutations involving itraconazole resistance possibly occurred in these isolates after prolonged itraconazole therapy. Sequencing of the cyp51A gene in the coding region revealed a mutation of M220I in cytochrome P450 sterol 14-alpha-demethylase in the second resistant isolate and a mutation of G54R in the last three resistant isolates. Expression changes of some pump genes, such as MDR3, may also, in part, be related to the resistance to itraconazole.


CONCLUSIONS
We conclude that resistance of A. fumigatus to itraconazole occurred in a patient treated with the drug, and the resistance may result from mutations in the cyp51A gene-the gene encoding the target enzyme for itraconazole.
Chen
Jian
J
Department of Dermatology, Peking University First Hospital, Research Center for Medical Mycology, Peking University, No. 8 Xishiku St, West District, Beijing, 100034, People's Republic of China.
Li
Houmin
H
Li
Ruoyu
R
Bu
Dingfang
D
Wan
Zhe
Z
D000935:Antifungal Agents / Q000008:administration & dosage / Q000494:pharmacology* / Q000627:therapeutic use; D001228:Aspergillosis / Q000188:drug therapy / Q000382:microbiology; D001232:Aspergillus fumigatus / Q000145:classification / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D004334:Drug Administration Schedule; D025141:Drug Resistance, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics*; D006801:Humans; D017964:Itraconazole / Q000008:administration & dosage / Q000494:pharmacology* / Q000627:therapeutic use; D008172:Lung Diseases, Fungal / Q000188:drug therapy / Q000382:microbiology; D008297:Male; D008826:Microbial Sensitivity Tests; D008875:Middle Aged; D009154:Mutation; D016533:Mycological Typing Techniques
D000935:Antifungal Agents; D005656:Fungal Proteins; D017964:Itraconazole; D003577:Cytochrome P-450 Enzyme System; C465526:cytochrome P-450 CYP51A, Aspergillus