Molecular tools for the detection of azole antifungal drug resistant phenotypes in aspergillus species

Abstract

Azole drugs are the first-line treatment option for invasive aspergillosis. Over the past few years, azole secondary resistance incidence in Aspergillus fumigatus strains is increasing. Similarly, the isolation of naturally azole resistant cryptic species of Aspergillus section Fumigati is starting to emerge. These issues became an important clinical concern considering that invasive aspergillosis treatment due to azole-susceptible strains are already difficult. Recently, an expert panel concluded that antifungal susceptibility testing of isolates should be performed if azole treatment is indicated. However, in vitro susceptibility testing by using commercially available or standard procedures takes at least 48 hours. Azole resistance in Aspergillus fumigatus is mainly linked with amino acid substitutions at Cyp51Ap or a combination of mutations and overexpression of the CYP51A gene. There are at least 15 resistance alleles described (seven of them molecularly confirmed by gene replacements and/or knockouts experiments) but few are prevalent. Whether or not the mutations at CYP51A are confirmed or prevalent it is clear that the nature and position of the substitution influence the pattern of azole resistance. For example, it is now clear that itraconazole/posaconazole resistance and cross-azole resistance are linked with substitutions at Cyp51A glycine 54 (G54) and subtition at leucine 98 combined with a higher CYP51A expression due to a tandem repetition of a 34-pb sequence in the CYP51A gene promoter, respectively. To circumvent the delays of the available susceptibility testing procedures and taking advantage of the correlation between CYP51A mutations and A. fumigatus susceptibility patterns, several molecular tools for the detection of azole resistance were developed. The aims of this presentation are: (i) to review the published diagnostic tools describing its advantages and disadvantages, (ii) to discuss the usefulness of these tools as a non-culture based diagnostic methods and (iii) to show our experience in this subject.