Determinants of Azole Resistance in Aspergillus

Background: Azole resistance of Aspergillus fumigatus is an increasingly challenging problem for the treatment of patients with aspergillosis. Azole drugs, such as voriconazole, target the enzyme lanosterol 14-α-demethylase, which is encoded by the cyp51A gene. A. fumigatus strains with certain mutations in the promoter region and in parts of the coding region of cyp51A, such as such as TR34/L98H, were previously shown to be resistant to azole drugs.

Methods: Fungal isolates from City of Hope patients were screened for voriconazole resistance. Resistant strains with voriconazole minimum inhibitory concentrations (MIC)s > 2.0 mg/L were collected and analyzed for mutations in the cyp51A gene region by DNA sequencing. Isolates with amino acid sequences that corresponded to Aspergillus lentulus, were speciated by sequencing six gene regions: β –tubulin (BenA), rodletA (rodA), Salt-Responsive Gene (SRG), Internal Transcribed Spacer regions (ITS), mitochondrial cytochrome b (mtcytb), and calmodulin (Cam). Gene regions were analyzed for sequence homology by NCBI nucleotide BLAST.

Results: DNA sequences were obtained for the cyp51a gene of twenty voriconazole resistant isolates, with MIC > 2 mg/L, four susceptible strains with MIC values < 1, and four non-resistant control A. fumigatus strains. Ten of the resistant isolates had no mutations, and six had at least one mutation in the cyp51A gene and promoter region. Most of the mutations were previously reported in other azole resistant A. fumigatus strains. However, four isolates had multiple mutations that indicated the presence of A. lentulus sequences. To further investigate these discrepancies we speciated the isolates in question and determined that one isolate, with an MIC value of 8 mg/L, was Aspergillus novofumigatus, while three isolates with respective MICs of 4, 4, and 16 mg/L, were identified as A. lentulus.

Conclusion: The TR34/L98H mutation was absent in all of our voriconazole resistant isolates. Due to similarities in morphology, the rapid discrimination of both species remains a key challenge. Aspergillus species have different susceptibilities to specific antifungal agents; therefore, quick and accurate identification for appropriate treatment is a necessity.