Discrimination of Aspergillosis, Mucormycosis, Fusariosis, and Scedosporiosis in Formalin-Fixed Paraffin-Embedded Tissue Specimens by Use of Multiple Real-Time Quantitative PCR Assays.
In a retrospective multicenter study, 102 formalin-fixed paraffin-embedded (FFPE) tissue specimens with histopathology results were tested. Two 4- to 5-μm FFPE tissue sections from each specimen were digested with proteinase K, followed by automated nucleic acid extraction. Multiple real-time quantitative PCR (qPCR) assays targeting the internal transcribed spacer 2 (ITS2) region of ribosomal DNA, using fluorescently labeled primers, was performed to identify clinically important genera and species of Aspergillus, Fusarium, Scedosporium, and the Mucormycetes The molecular identification was correlated with results from histological examination. One of the main findings of our study was the high sensitivity of the automated DNA extraction method, which was estimated to be 94%. The qPCR procedure that was evaluated identified a range of fungal genera/species, including Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Aspergillus niger, Fusarium oxysporum, Fusarium solani, Scedosporium apiospermum, Rhizopus oryzae, Rhizopus microsporus, Mucor spp., and Syncephalastrum Fusarium oxysporum and F. solani DNA was amplified from five specimens from patients initially diagnosed by histopathology as having aspergillosis. Aspergillus flavus, S. apiospermum, and Syncephalastrum were detected from histopathological mucormycosis samples. In addition, examination of four samples from patients suspected of having concomitant aspergillosis and mucormycosis infections resulted in the identification of two A. flavus isolates, one Mucor isolate, and only one sample having both R. oryzae and A. flavus Our results indicate that histopathological features of molds may be easily confused in tissue sections. The qPCR assay used in this study is a reliable tool for the rapid and accurate identification of fungal pathogens to the genus and species levels directly from FFPE tissues.
Student Research Committee, Mazandaran University of Medical Sciences, Sari, Iran.|Department of Medical Mycology and Parasitology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran.
Elham
E
Salehi
Department of Medical Mycology and Parasitology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran.|Invasive Fungi Research Center, Mazandaran University of Medical Sciences, Sari, Iran.
Mohammad T
0000-0001-6415-4648
MT
Hedayati
Department of Medical Microbiology, Radboud University Medical Centre, and Centre of Expertise in Mycology Radboudumc/CWZ, Nijmegen, The Netherlands.
Jan
J
Zoll
Department of Biochemistry, Erasmus University Medical Center, Rotterdam, The Netherlands.
Haleh
H
Rafati
Department of Pathology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran.
Maryam
M
Ghasemi
Transplantation Research Center, National Research Institute for Tuberculosis and Lung Disease, Department of Pathology Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Atosa
A
Doroudinia
Department of Medical Mycology and Parasitology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran.|Invasive Fungi Research Center, Mazandaran University of Medical Sciences, Sari, Iran.
Mahdi
M
Abastabar
Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran.
Ali
A
Tolooe
Department of Medical Microbiology, Radboud University Medical Centre, and Centre of Expertise in Mycology Radboudumc/CWZ, Nijmegen, The Netherlands.
Eveline
E
Snelders
Department of Medical Microbiology, Radboud University Medical Centre, and Centre of Expertise in Mycology Radboudumc/CWZ, Nijmegen, The Netherlands.
Henrich A
HA
van der Lee
Department of Medical Microbiology, Radboud University Medical Centre, and Centre of Expertise in Mycology Radboudumc/CWZ, Nijmegen, The Netherlands.
Antonius J M M
AJ
Rijs
Department of Medical Microbiology, Radboud University Medical Centre, and Centre of Expertise in Mycology Radboudumc/CWZ, Nijmegen, The Netherlands.
Paul E
PE
Verweij
Invasive Fungi Research Center, Mazandaran University of Medical Sciences, Sari, Iran Seyedmousavi@nih.gov Willem.Melchers@radboudumc.nl.|Department of Medical Microbiology, Radboud University Medical Centre, and Centre of Expertise in Mycology Radboudumc/CWZ, Nijmegen, The Netherlands.
Seyedmojtaba
0000-0002-6194-7447
S
Seyedmousavi
Department of Medical Microbiology, Radboud University Medical Centre, and Centre of Expertise in Mycology Radboudumc/CWZ, Nijmegen, The Netherlands Seyedmousavi@nih.gov Willem.Melchers@radboudumc.nl.
Willem J G
WJ
Melchers
D001230:Aspergillus / Q000235:genetics / Q000302:isolation & purification*; D057205:Automation, Laboratory / Q000379:methods; D021903:DNA, Ribosomal Spacer / Q000737:chemistry / Q000235:genetics; D003937:Diagnosis, Differential; D004202:Disinfectants; D005404:Fixatives; D005557:Formaldehyde; D005670:Fusarium / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D009090:Mucorales / Q000235:genetics / Q000302:isolation & purification*; D009181:Mycoses / Q000175:diagnosis*; D010232:Paraffin; D057089:Pathology, Molecular / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D021681:Scedosporium / Q000235:genetics / Q000302:isolation & purification*; D013048:Specimen Handling / Q000379:methods; D016707:Tissue Fixation
D021903:DNA, Ribosomal Spacer; D004202:Disinfectants; D005404:Fixatives; D005557:Formaldehyde; D010232:Paraffin