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.
A rapid and simple bead-bashing-based method for genomic DNA extraction from mammalian tissue
