Low prevalence of Aspergillus fumigatus cyp51A key mutations in clinical samples and isolates of immunocompromised patients in Germany

Ref ID: 19204

Author:

B. Spiess*, P. Postina, M. Reinwald, W. Seifarth, S. Will, J.J. Vehreschild, M.J.G.T. Vehreschild, M. Lauten, P.-M. Rath, W.-K. Hofmann, D. Buchheidt

Author address:

Mannheim, Cologne, Lübeck, Essen, DE

Full conference title:

23rd European Congress of Clinical Microbiology and
Infectious Diseases

Date: 27 April 2014

Abstract:

Objectives. Given recent reports of azole resistance in Aspergillus fumigatus and the low diagnostic yield of culture based methods for detecting invasive aspergillosis (IA) in immunocompromised patients, we established molecular assays to detect azole resistance directly from primary clinical samples (blood, bronchoalveolar lavage (BAL), cerebrospinal fluid (CSF), tissue biopsies). Our Aspergillus DNA sample collection was screened for the occurrence of azole resistance mediating cyp51A key mutations.
Methods. Using established polymerase chain reaction (PCR) assays followed by DNA sequence analysis to detect the most frequent mutations in the A. fumigatus cyp51A gene conferring azole resistance (TR (tandem repeat) alteration in the promoter region, L98H and M220 alterations), we screened 117 clinical samples (14 blood, 13 CSF and 77 BAL samples, 13 tissue biopsies) from our Aspergillus DNA sample collection previously tested positive for Aspergillus DNA using our diagnostic nested PCR.
Results. The detection threshold for the L98H PCR assay was 200 fg of A. fumigatus DNA. Using this most sensitive assay, 62 of 117 samples yielded a positive signal, 55 samples, including all blood samples, were found to be PCR-negative. The PCR positive-tested samples were further submitted to the TR and M220 PCR assays.
Investigating the CSF samples, two samples were PCR-positive for L98H and TR PCR, four samples for the L98H PCR, but no mutations could be detected by subsequent DNA sequence analysis. Six samples were PCR-negative. DNA sequence analysis revealed a single L98H mutation in a lung tissue specimen of a steroid treated COPD patient and a L98H alteration in combination with the TR in a brain tissue sample of a patient with ALL and in both the BAL sample and the corresponding isolate of a patient with AML. In addition, an isolate of a lung tissue specimen was tested positive for the L98H/TR combination.
Conclusions. We optimized our PCR and DNA sequencing based assays in order to detect azole resistance mediating mutations of the A. fumigatus cyp51A gene directly from clinical samples. Positive findings show the feasibility of the approach. We consider our assay of high epidemiological and clinical relevance to detect azole resistance aiming to optimise antifungal therapy in patients with IA.
Supported by a grant of the Deutsche Krebshilfe / Dr. Mildred Scheel Stiftung für Krebsforschung (Project number 109 411).

Abstract Number: P993

Conference Year: 2013

Link to conference website: http://registration.akm.ch/einsicht.php?XNABSTRACT_ID=161523&XNSPRACHE_ID=2&XNKONGRESS_ID=180&XNMASK

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