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J Antimicrob Chemother. 2010 May;65(5):901-5. doi: 10.1093/jac/dkq041. Epub 2010 Feb 18.

Novel mixed-format real-time PCR assay to detect mutations conferring resistance to triazoles in Aspergillus fumigatus and prevalence of multi-triazole resistance among clinical isolates in the Netherlands.

Author information

1
Department of Medical Microbiology and Infectious Diseases, Canisius Wilhelmina Hospital, Nijmegen, The Netherlands. c.klaassen@cwz.nl

Abstract

OBJECTIVES:

The aim of this study was: (i) to study the prevalence of triazole-resistant Aspergillus fumigatus isolates in the Netherlands; and (ii) to design rapid real-time PCR methods to identify such isolates.

METHODS:

A novel mixed-format real-time PCR assay is described for the detection of mutations leading to triazole resistance in A. fumigatus. One set of PCR primers and a probe carrying a single fluorescent label in combination with a double-stranded DNA fluorescent dye allow simultaneous detection of (a) specific mutation(s) as well as of the amplified product that serves as an internal amplification control. The method was applied to a random collection of 209 clinical isolates from throughout the Netherlands and was compared with phenotypic susceptibility testing.

RESULTS:

A total of four triazole-resistant isolates were identified, resulting in a prevalence of resistant isolates of <2%. All four isolates contained an identical combination of mutations leading to multi-triazole resistance, as reported before by others. Molecular testing results were 100% concordant with phenotypic susceptibility testing.

CONCLUSIONS:

Although in specific patient populations the prevalence of resistance in A. fumigatus may be an emerging problem, in the general population it is still relatively low. The novel real-time PCR format allows rapid and reliable identification of such isolates.

PMID:
20167588
DOI:
10.1093/jac/dkq041
[Indexed for MEDLINE]

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