Rapid detection of cyp51A codon 98 mutations in azole-resistant Aspergillus fumigatus isolates by multiplex allele-specific polymerase chain reaction (PCR) assay

Ref ID: 19198

Author:

Z. Khan*, S. Ahmad, J.F. Meis, A. Theyyathel

Author address:

Safat, KW; Nijmegen, NL

Full conference title:

23rd European Congress of Clinical Microbiology and
Infectious Diseases

Date: 27 April 2014

Abstract:

Objective: Invasive aspergillosis (IA) caused by Aspergillus fumigatus is associated with high rates of morbidity and mortality in immunocompromised hosts. Although the organism is intrinsically resistant to fluconazole, new triazoles (itraconazole and voriconazole) are active and are common choice for primary therapy of IA due to favorable pharmacokinetic and safety profiles. Mutations at codon 98 (L98H) of cyp51A (cyp51A98) encoding 14- alpha
-sterol demethylase have been identified as major resistance conferring mechanism in triazole-resistant clinical A. fumigatus isolates. This study developed a multiplex allele-specific PCR (MAS-PCR) assay targeting cyp51A98 for rapid detection of azole-resistant Aspergillus fumigatus isolates for proper patient management.
Methods: Reference A. fumigatus strains carrying wild-type and L98H mutation at cyp51A98 were used for establishing MAS-PCR. The MAS-PCR was performed by using three primers designed to detect the presence of CTC (wild-type) or CAC (mutant) allele at cyp51A98 and the amplicons were detected by agarose gel electrophoresis. Azole susceptible (n=63) and -resistant (n=44) A. fumigatus isolates were used to evaluate the performance of MAS-PCR. Drug susceptibility testing (DST) of A. fumigatus isolates to azoles and other antifungal drugs was determined by Etest. The results of MAS-PCR were confirmed by direct DNA sequencing of cyp51A98 region from selected isolates.
Results: The MAS-PCR performed with reference A. fumigatus strains carrying wild-type (CTC) and CTC to CAC (L98H) mutation at cyp51A98 yielded expected results. The 63 azole-susceptible and 39 of 44 azole-resistant A. fumigatus isolates were correctly identified by MAS-PCR. Of the remaining 5 azole-resistant isolates, two each contained a mutation at cyp51A codons 54 and 220 and one isolate contained hetero-resistance at cyp51A98.
Conclusions: A simple, MAS-PCR assay has been successfully developed to facilitate rapid detection of L98H mutation in cyp51A gene that confers resistance to azoles in A. fumigatus strains. The assay will help in early recognition of azole-resistant A. fumigatus strains for proper management of patients with IA.
Supported by KURA grant MI 01/09.

Abstract Number: P992

Conference Year: 2013

Link to conference website: http://registration.akm.ch/einsicht.php?XNABSTRACT_ID=163304&XNSPRACHE_ID=2&XNKONGRESS_ID=180&XNMASK

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