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Diagnostic Approaches for Aspergillus Infections

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Front. Microbiol. | doi: 10.3389/fmicb.2018.00555

Comparison of two molecular assays for detection and characterization of Aspergillus fumigatus triazole resistance and Cyp51A mutations in clinical isolates and primary clinical samples of immunocompromised patients

Patricia Postina1, Julian Skladny1, Tobias Boch1, Oliver A. Cornely2,  Axel Hamprecht3, Peter-Michael Rath4, Jörg Steinmann5,  Oliver Bader6, Thomas Miethke7, Anne Dietz7, Natalia Merker1, Wolf-Karsten Hofmann1,  Dieter Buchheidt1* and Birgit Spiess1
  • 1Hematology and Oncology, Universitätsmedizin Mannheim (UMM), Germany
  • 2Department I of Internal Medicine, Universitätsklinikum köln, Germany
  • 3Institute of Medical Microbiology, Immunology and Hygiene, Universitätsklinikum köln, Germany
  • 4Institute of Medical Microbiology, Universitätsklinikum Essen, Germany
  • 5Institute of Clinical Hygiene, Medical Microbiology and Clinical Infectiology, Paracelsus Medizinischen Privatuniversität, Germany
  • 6Institute for Medical Microbiology, Universitätsmedizin Göttingen, Germany
  • 7Institute of Medical Microbiology and Hygiene, Universitätsmedizin Mannheim (UMM), Germany

In hematological patients, the incidence of invasive aspergillosis (IA) caused by azole resistant As-pergillus fumigatus (ARAf) is rising. As the diagnosis of IA is rarely based on positive culture in this group of patients, molecular detection of resistance mutations directly from clinical samples is crucial.
In addition to the in-house azole resistance ARAf polymerase chain reaction (PCR) assays detecting the frequent mutation combinations TR34/L98H, TR46/Y121F/T289A and M220 in the Aspergillus fumigatus (A. fumigatus) Cyp51A gene by subsequent DNA sequence analysis, we investigated in parallel the commercially available AsperGenius® real time PCR system in detecting the Cyp51A alterations TR34/L98H and Y121F/T289A directly from 52 clinical samples (15 biopsies, 22 bronchoalveolar lavage (BAL), 15 cerebrospinal fluid (CSF) samples) and ARAf isolates (n=3) of im-munocompromised patients. We analyzed DNA aliquots and compared both methods concerning amplification and detection of Aspergillus DNA and Cyp51A alterations.
As positive control for the feasibility of our novel Y121F and T289A PCR assays, we used two A. fumigatus isolates with the TR46/Y121F/T289A mutation combination isolated from hematological patients with known Cyp51A alterations and a lung biopsy sample of a patient with acute myeloid leukemia (AML).
The rate of positive ARAf PCR results plus successful sequencing using the ARAf PCR assays was 61 % in biopsies, 29 % in CSF, 67 % in BAL samples and 100 % in isolates. In comparison the amount of positive PCRs using the AsperGenius® assays was 47 % in biopsies, 42 % in CSF, 59 % in BAL samples and 100 % in isolates. Altogether 17 Cyp51A alterations were detected using our ARAf PCRs plus DNA sequencing and therefrom 10 alterations also by the AsperGenius® system.
The comparative evaluation of our data revealed that our conventional PCR assays are more sensitive in detecting ARAf in BAL and biopsy samples, whereby differences were not significant. The advantage of the AsperGenius® system is the time saving aspect. We consider non-culture based molecular detection of Aspergillus triazole resistance to be of high epidemiological and clinical relevance in patients with hematological malignancies.

Keywords: invasive aspergillosis, Triazole resistance, PCR, Clinical samples, Molecular assays

Received: 08 Feb 2018; Accepted: 12 Mar 2018.

Edited by:

Helmut J. Salzer, Forschungszentrum Borstel (LG), Germany

Reviewed by:

Masoomeh Shams-Ghahfarokhi, Tarbiat Modares University, Iran
Walter Buzina, Medizinische Universität Graz, Austria  

Copyright: © 2018 Postina, Skladny, Boch, Cornely, Hamprecht, Rath, Steinmann, Bader, Miethke, Dietz, Merker, Hofmann, Buchheidt and Spiess. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Prof. Dieter Buchheidt, Universitätsmedizin Mannheim (UMM), Hematology and Oncology, Mannheim, Germany,