Article
Low prevalence of cyp51A key mutations in Aspergillus fumigatus in Germany in primary clinical samples (blood, BAL, biopsies, CSF) and isolates of immunocompromised patients
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Authors
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B. Spiess
- Department of Hematology and Oncology, University Hospital Mannheim, University of Heidelberg, Mannheim, Germany
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P. Postina
- Department of Hematology and Oncology, University Hospital Mannheim, University of Heidelberg, Mannheim, Germany
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M. Reinwald
- Department of Hematology and Oncology, University Hospital Mannheim, University of Heidelberg, Mannheim, Germany
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W. Seifarth
- Department of Hematology and Oncology, University Hospital Mannheim, University of Heidelberg, Mannheim, Germany
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S. Will
- Department of Hematology and Oncology, University Hospital Mannheim, University of Heidelberg, Mannheim, Germany
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O.A. Cornely
- 1st Department of Internal Medicine, Cologne University Hospital, Cologne, Germany
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M.J.G.T. Vehreschild
- 1st Department of Internal Medicine, Cologne University Hospital, Cologne, Germany
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P.-M. Rath
- Institute of Medical Microbiology, Essen University Hospital, Essen, Germany
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M. Lauten
- Pediatric Hematology and Oncology, University Hospital Schleswig-Holstein, Campus Lübeck, Lübeck, Germany
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W.-K. Hofmann
- Department of Hematology and Oncology, University Hospital Mannheim, University of Heidelberg, Mannheim, Germany
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D. Buchheidt
- Department of Hematology and Oncology, University Hospital Mannheim, University of Heidelberg, Mannheim, Germany
| Published: | June 18, 2013 |
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Outline
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Objectives: As the incidence of azole resistance in Aspergillus fumigatus is rising and the diagnosis of invasive aspergillosis (IA) in immunocompromised patients is rarely based on positive culture yield in this group of patients, we established the molecular detection of azole resistance directly from clinical samples (blood, bronchoalveolar lavage (BAL), cerebrospinal fluid (CSF), tissue biopsies) and screened our Aspergillus DNA sample collection for the occurrence of azole resistance mediating cyp51A key mutations.
Methods: Using the 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 134 clinical samples (94 BAL, 14 blood and 13 CSF 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 primarily this most sensitive assay, 79 of 134 samples yielded a positive signal, 55 samples, including all blood samples, were found to be PCR-negative. The positive-tested samples were further submitted to the TR and M220 PCR assays. Investigating the CSF samples, two samples were positive for L98H and TR PCR, four samples for the L98H PCR. Six samples were PCR-negative. In CSF samples no mutations could be detected.
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: In order to detect azole resistance mediating mutations of the A. fumigatus cyp51A gene directly from clinical samples, we optimised our published PCR assays. Positive PCR signals show the feasibility of the approach also for investigation of CSF samples. We consider our assay of epidemiological and clinical relevance to detect azole resistance.
