DEVELOPMENT AND VALIDATION OF A HIGH RESOLUTION MELTING ASSAY TO DETECT AZOLE RESISTANCE IN ASPERGILLUS FUMIGATUS

L Bernal-Martínez, H Gil, O Rivero-Menéndez, E Mellado, M Cuenca-Estrella, S Gago, A Alastruey-Izquierdo

Abstract: 

Purpose:
In the last years, there has been an emergence of azole-resistant Aspergillus fumigatus strains reported worldwide, representing a growing public health concern. The prompt identification of these resistances for the selection of the proper antifungal treatment is critical for the management of the A. fumigatus infected patients. A rapid molecular –based assay for the detection of the most frequent mutations related to azole resistance in A. fumigatus was designed and validated in this study.
Methods:
Five PCR reactions targeting the G54, L98, M220, G448 point mutations in cyp51A and, the three tandem repeats described in the promoter region of this gene (TR34, TR46 and TR53) were designed with similar cycling conditions to run simultaneously in a LC 480 Roche thermocycler. After amplification, a High Resolution Melting (HRM) assay for the identification of the amplicons according to the temperature of the melting curves was performed. For the standardization of this technique, 33 A. fumigatus strains with previously known mutations (4 susceptible and 29 resistant) were used. In addition, 80 A. fumigatus strains, including susceptible and azole resistance strains were blind tested for validation.
Results:
The method was able to specifically differentiate the mutations M220V, M220I, M220T, L98H, G448S and the three tandem repeats. The mutations G54E, G54W, G54V and G54R could not be distinguished, although they were clearly differentiated from the wild type and therefore detected as resistant. Regarding the validation, the success rate of this HRM blind study was 98.75%. Only one strain containing the mutation M220K was not properly identified.
Conclusions:
i) A single HRM assay to detect the most frequent mutations related to azole resistance in A. fumigatus was designed, standardized and validated.
ii) All resistant strains were clearly differentiated from the wild type by HRM, except one strain with the mutation M220K.
iii) This methodology is a reliable diagnostic tool for the rapid identification of azole resistant in A. fumigatus strains.

2016

abstract No: 

105

Full conference title: 

7th Advances Against Aspergillosis Conference