Affiliation:
1. Mycology Reference Laboratory, National Centre for Microbiology, Instituto de Salud Carlos III, Madrid, Spain
2. Spanish Network for Research in Infectious Diseases, Instituto de Salud Carlos III, Madrid, Spain
3. European Program for Public Health Microbiology Training, European Centre for Disease Prevention and Control, Stockholm, Sweden
Abstract
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.
Funder
MINECO | Instituto de Salud Carlos III
Publisher
American Society for Microbiology
Subject
Infectious Diseases,Pharmacology (medical),Pharmacology
Reference55 articles.
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