Rapid detection of cyp51A 98 mutations in triazole-resistant environmental and clinical Aspergillus fumigatus isolates by multiplex allele-specific PCR

Ref ID: 19332

Author:

Suhail Ahmad, Ziauddin Khan, Jacques Meis, Ajmal Theyyathe

Author address:

Department of Microbiology, Faculty of Medicine, Kuwait University, P. O. Box 24923, Safat 13001,
Kuwait

Full conference title:

Asian Mycological Congress 2013 and the 13th International Marine and Freshwater Mycology Symposium

Date: 19 August 2014

Abstract:

Invasive aspergillosis (IA) caused principally by Aspergillus fumigatus is associated with high
mortality rates in immunocompromised patients. New triazoles (itraconazole and voriconazole) are
primary therapies for IA. 14- -sterol demethylase (cyp51A) codon 98 (cyp51A98) mutations confer
resistance to triazoles in A. fumigatus isolates. This study evaluated a multiplex allele-specific PCR
(MAS-PCR) assay targeting cyp51A98 for rapid detection of triazole resistance in environmental and
clinical A. fumigatus isolates for proper patient management. Reference A. fumigatus strains carrying
wild-type and L98H mutation at cyp51A98 were used. MAS-PCR used three primers designed to detect
presence of CTC (wild-type) or CAC (mutant) allele at cyp51A98. Amplicons were detected by agarose
gel electrophoresis. Environmental (n=131) and clinical (n=44) A. fumigatus isolates were used. These
included triazole-susceptible (n=125) and -resistant (n=50) strains as determined by Etest. Results of
MAS-PCR were confirmed by direct DNA sequencing of cyp51A98 region from selected isolates.
MAS-PCR with reference A. fumigatus strains yielded expected results. The 125 triazole-susceptible and
46 of 50 triazole-resistant A. fumigatus isolates were correctly identified by MAS-PCR. Of the remaining 4
triazole-resistant isolates, two each contained a mutation at cyp51A codons 54 and 220 which are
outside the region of analysis by MAS-PCR. The MAS-PCR assay rapidly detected L98H mutation in
cyp51A gene that confers resistance to triazoles in 92% triazole-resistant A. fumigatus strains and thus,
may help in early recognition of triazole-resistant A. fumigatus strains. The cyp51A98 mutation was
detected in environmental but not in clinical A. fumigatus isolates in Kuwait. Supported by KURS grant
MI01/09.

Abstract Number: P-47

Conference Year: 2013

Link to conference website: NULL

New link: NULL

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