Development and Validation of a High-Resolution Melting Assay To Detect Azole Resistance in Aspergillus fumigatus

Abstract

The global emergence of azole-resistant Aspergillus fumigatus strains is a growing public health concern. Different patterns of azole resistance are linked to mutations in cyp51A. Therefore, accurate characterization of the mechanisms underlying azole resistance is critical to guide selection of the most appropriate antifungal agent for patients with aspergillosis. This study describes a new sequencing-free molecular screening tool for early detection of the most frequent mutations known to be associated with azole resistance in A. fumigatus. PCRs targeting cyp51A mutations at positions G54, Y121, G448, and M220 and targeting different tandem repeats (TRs) in the promoter region were designed. All PCRs were performed simultaneously, using the same cycling conditions. Amplicons were then distinguished using a high-resolution melting assay. For standardization, 30 well-characterized azole-resistant A. fumigatus strains were used, yielding melting curve clusters for different resistance mechanisms for each target and allowing detection of the most frequent azole resistance mutations, i.e., G54E, G54V, G54R, G54W, Y121F, M220V, M220I, M220T, M220K, and G448S, and the tandem repeats TR 34 , TR 46 , and TR 53 . Validation of the method was performed using a blind panel of 80 A. fumigatus azole-susceptible or azole-resistant strains. All strains included in the blind panel were properly classified as susceptible or resistant with the developed method. The implementation of this screening method can reduce the time needed for the detection of azole-resistant A. fumigatus isolates and therefore facilitate selection of the best antifungal therapy in patients with aspergillosis.

Method-Dependent Epidemiological Cutoff Values for Detection of Triazole Resistance in Candida and Aspergillus Species for the Sensititre YeastOne Colorimetric Broth and Etest Agar Diffusion Methods

“…These fungal infections may cause elevated levels of morbidity and mortality among immunocompromised patients (3)(4)(5). The impact of azole resistance and its prevalence has been widely recognized, and various mechanisms of mutational resistance have been elucidated in the four most common species of Candida, especially in Candida albicans, and in Aspergillus fumigatus (6)(7)(8)(9)(10). In most Candida isolates, azole resistance (or unusually high or increased MICs) are mostly associated with two main molecular mechanisms, among others: an increase (overexpression) of the azole target azole sterol demethylase or alterations (amino acid substitutions) in either the gene ERG11, as the enzyme is encoded during the fungal ergosterol biosynthesis pathway, or the MRR1 transcriptional regulator (6,8,9).…”

Rapid and accurate identification of black aspergilli from grapes using high‐resolution melting (HRM) analysis

“…A. fumigatus , A. lentulus ) and Versicolores (i.e. A. sydowii , A. versicolor , A. creber ), as well as A. flavus , A. niger and A. tubingensis , among others, or discriminate among A. fumigatus fungicide‐resistant mutants . These reports clearly depict the usefulness and applicability of HRM to identification of Aspergillus spp.…”

“…A. sydowii, A. versicolor, A. creber), as well as A. flavus, A. niger and A. tubingensis, among others, [36][37][38][39] or discriminate among A. fumigatus fungicide-resistant mutants. 40 These reports clearly depict the usefulness and applicability of HRM to identification of Aspergillus spp. and, with respect to A. niger and A. tubingensis species, their broader importance in health issues.…”

Detecting Azole-Antifungal Resistance in Aspergillus fumigatus by Pyrosequencing

“…Numerous in-house assays ranging from nested PCR, mixed-format and real-time PCR approaches have been developed and tested [103][104][105][106][107][108][109][110]. Other molecular approaches (reviewed in [105]) include the analysis of high resolution melt curves [111,112]. However, despite all this work, only three commercial kits are currently available (AsperGenius, PathoNostics, Maastricht, Netherlands, MycoGENIE, Ademtech, Pessac, France and Fungiplex ® Aspergillus Azole-R IVD PCR, Bruker Daltonik GmbH, Bremen, Germany), attesting to the difficulty of developing robust molecular assays [113].…”