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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 ofAspergillus ,Fusarium ,Scedosporium , and theMucormycetes . 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, includingAspergillus fumigatus ,Aspergillus flavus ,Aspergillus terreus ,Aspergillus niger ,Fusarium oxysporum ,Fusarium solani ,Scedosporium apiospermum ,Rhizopus oryzae ,Rhizopus microsporus ,Mucor spp., andSyncephalastrum .Fusarium oxysporum andF. solani DNA was amplified from five specimens from patients initially diagnosed by histopathology as having aspergillosis.Aspergillus flavus ,S. apiospermum , andSyncephalastrum 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 twoA. flavus isolates, oneMucor isolate, and only one sample having bothR. oryzae andA. 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.

journal of clinical microbiology

Discrimination of Aspergillosis, Mucormycosis, Fusariosis, and Scedosporiosis in Formalin-Fixed Paraffin-Embedded Tissue Specimens by Use of Multiple Real-Time Quantitative PCR Assays