title: Incidence of Cyp51 A key mutations in Aspergillus fumigatus-a study on primary clinical samples of immunocompromised patients in the period of 1995-2013.
abstract: 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, we screened our Aspergillus DNA sample collection for the occurrence of azole resistance mediating cyp51 A key mutations. Using two established, a modified and a novel polymerase chain reaction (PCR) assays followed by DNA sequence analysis to detect the most frequent mutations in the A. fumigatus cyp51 A gene conferring azole resistance (TR34 (tandem repeat), L98H and M220 alterations). We analyzed two itraconazole and voriconazole and two multi-azole resistant clinical isolates and screened 181 DNA aliquots derived from clinical samples (blood, bronchoalveolar lavage (BAL), biopsies, cerebrospinal fluid (CSF)) of 155 immunocompromised patients of our Aspergillus DNA sample collection, previously tested positive for Aspergillus DNA and collected between 1995 and 2013. Using a novel PCR assay for the detection of the cyp51 A 46 bp tandem repeat (TR46) directly from clinical samples, we found the alteration in a TR46/Y121F/T289A positive clinical isolate. Fifty stored DNA aliquots from clinical samples were TR46 negative. DNA sequence analysis revealed a single L98H mutation in 2010, two times the L98H alteration combined with TR34 in 2011 and 2012 and a so far unknown N90K mutation in 1998. In addition, four clinical isolates were tested positive for the TR34/L98H combination in the year 2012. We consider our assay of epidemiological relevance to detect A. fumigatus azole resistance in culture-negative clinical samples of immunocompromised patients; a prospective study is ongoing.
authors: Department of Hematology and Oncology, University Hospital Mannheim, University of Heidelberg, Mannheim, Germany.
authors: Birgit
authors: B
authors: Spiess
authors: Department of Hematology and Oncology, University Hospital Mannheim, University of Heidelberg, Mannheim, Germany.
authors: Patricia
authors: P
authors: Postina
authors: Department of Hematology and Oncology, University Hospital Mannheim, University of Heidelberg, Mannheim, Germany.
authors: Mark
authors: M
authors: Reinwald
authors: Department I of Internal Medicine, University Hospital of Cologne, Cologne; Clinical Trials Centre Cologne, ZKS Köln, BMBF 01KN1106, and Centre for Integrated Oncology CIO Köln Bonn, and Cologne Excellence Cluster on Cellular Stress Response in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany.
authors: Oliver A
authors: OA
authors: Cornely
authors: Institute for Medical Microbiology, Immunology and Hygiene, University Hospital of Cologne, Cologne, Germany.
authors: Axel
authors: A
authors: Hamprecht
authors: Section of Infectious Diseases and Tropical Medicine & Department of Pulmonology, University Hospital of Internal Medicine, Graz Medical University, Graz, Austria.
authors: Martin
authors: M
authors: Hoenigl
authors: Section of Hygiene and Medical Microbiology, Medical University of Innsbruck, Innsbruck, Austria.
authors: Cornelia
authors: C
authors: Lass-Flörl
authors: Institute of Medical Microbiology, University Hospital of Essen, Essen, Germany.
authors: Peter-Michael
authors: PM
authors: Rath
authors: Institute of Medical Microbiology, University Hospital of Essen, Essen, Germany.
authors: Jörg
authors: J
authors: Steinmann
authors: Institute of Medical Microbiology and Hygiene, University Hospital Mannheim, Mannheim, Germany.
authors: Thomas
authors: T
authors: Miethke
authors: Pediatric Hematology and Oncology, University Hospital Schleswig-Holstein, Campus Lübeck, Lübeck, Germany.
authors: Melchior
authors: M
authors: Lauten
authors: Department of Hematology and Oncology, University Hospital Mannheim, University of Heidelberg, Mannheim, Germany.
authors: Silke
authors: S
authors: Will
authors: Department of Hematology and Oncology, University Hospital Mannheim, University of Heidelberg, Mannheim, Germany.
authors: Natalia
authors: N
authors: Merker
authors: Department of Hematology and Oncology, University Hospital Mannheim, University of Heidelberg, Mannheim, Germany.
authors: Wolf-Karsten
authors: WK
authors: Hofmann
authors: Department of Hematology and Oncology, University Hospital Mannheim, University of Heidelberg, Mannheim, Germany.
authors: Dieter
authors: D
authors: Buchheidt
mesh_terms: D000935:Antifungal Agents / Q000494:pharmacology; D001228:Aspergillosis / Q000175:diagnosis / Q000209:etiology / Q000382:microbiology*; D001232:Aspergillus fumigatus / Q000187:drug effects / Q000235:genetics* / Q000276:immunology; D001393:Azoles / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D025141:Drug Resistance, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics*; D006801:Humans; D016867:Immunocompromised Host; D008826:Microbial Sensitivity Tests; D009154:Mutation; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
chemical_list: D000935:Antifungal Agents; D001393:Azoles; D005656:Fungal Proteins; D003577:Cytochrome P-450 Enzyme System; C465526:cytochrome P-450 CYP51A, Aspergillus