Discrimination of aspergillosis, mucormycosis, fusariosis and scedosporiosis in formalin-fixed paraffin-embedded tissue specimens using a real-time quantitative PCR assay

Elham Salehi, Willem Melchers, Jan Zoll, Mohammad T. Hedayati, Haleh Rafati, Henrich van der Lee, Paul E. Verweij, Seyedmojtaba (Amir) Seyedmousavi

Abstract: 

Background: The frequency of invasive fungal infections (IFIs) has increased significantly over the
past decades, associated with excessive morbidity and mortality. Early and reliable detection of a
fungal pathogen is crucial to guide the appropriate and successful treatment of IFIs. The purpose of
this study was to evaluate the ability of a rapid molecular assay to accurately detect and identify
causative agents of IFIs in formalin-fixed paraffin-embedded (FFPE) tissues.
Material/methods: In a retrospective multicenter study, tissue samples from 102 FFPE specimens
with histopathology results were tested. Two 4-5 μm FFPE tissue section from each specimen was
digested with proteinase K followed by automated nucleic acid extraction. A specific quantitative realtime
PCR assay, using fluorescently labeled primers, was performed to identify clinically important
genus and species of Aspergillus (A. fumigatus, A. flavus, A. terreus, A. nidulans and A. niger),
Fusarium (F. oxysporum, F. subglutinans, F. solani and F. dimerum), Scedosporium (S. apiospermum,
S. aurantiacum and S. prolificans) and the Zygomycetes (Rhizopus oryzae, Rhizopus microsporus,
Cunninghamella bertholletiae, Mucor spp., Lichtheimia spp., Syncephalastrum spp. and Rhizomucor
spp.)
Results: Overall, mold hyphae were detected histopathologically in 102 of the tissue specimens
corresponded to aspergillosis (59), mucormycosis (29), concomitant aspergillosis and mucormycosis
(4) or could not be further specified (10).
The qPCR assay used was highly sensitive and a range of fungal genera/species were identified,
including: A. fumigatus, A. flavus, A. terreus, F. oxysporum, F. solani, S. apiospermum, Rhizopus
oryzae, Mucor spp. and Syncephalastrum spp.
Fusarium oxysporum and F. solani DNA were amplified from three specimens initially diagnosed as
aspergillosis (by histopathology), respectively.
Aspergillus flavus, S. apiospermum and Syncephalastrum were detected from mucormycosis samples.
In addition, examination of four suspected samples as concomitant aspergillosis and mucormycosis
infection resulted to one R. oryzae and three A. flavus. The qPCR assay used failed to detect fungal
DNA in 9 samples, possibly as a result of the destruction of DNA before paraffin wax embedding.
Conclusions: Our results indicated that histopathological features of molds can easily be confused in
tissue sections. The quantitative PCR assays used in this study is a reliable tool, for rapid and
accurate identification to the genus and species levels of fungal pathogens directly from FFPE tissues.
This assay a rapid promising alternative/adjunctive tool that can be easily incorporated into clinical
mycology laboratories.

2016

Poster: 

AttachmentSize
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abstract No: 

#5401

Full conference title: 

26th European Congress of Clinical Microbiology and Infectious Diseases
    • ECCMID 26th (2016)