P 1630 Discrimination of aspergillosis, mucormycosis, fusariosis and scedosporiosis in formalin-fixed paraffin-embedded tissue specimens using a real-time quantitative PCR assay

Elham Salehi, 1 Mohammad T. Hedayati, 2, 3* Jan Zoll, 4 Willem J.G. Melchers, 4Haleh Rafati,5 Maryam Ghasemi, 6 Atosa Doroudinia, 7 Ali Tolooe-Zarrin, 8Henrich A. van der Lee, 4 Antonius J. M. M. Rijs, 4 Paul E. Verweij, 4 Seyedmojtaba Seyedmousavi, 3, 4, 9*

1 Student Research Committee, Mazandaran UMS, Sari, Iran 2Department of Medical Mycology and Parasitology, School of Medicine, Mazandaran UMS Sari, Iran3Invasive Fungi Research Center, Mazandaran UMS, Sari, Iran4 Radboudumc, Nijmegen, The Netherlands 5ErasmusMC, Rotterdam, The Netherlands 6Department of Pathology, School of Medicine, Mazandaran UMS, Sari, Iran 7Transplantation Research Center, National Research Institute for Tuberculosis and Lung Disease, Department of Pathology Faculty of Medicine, Shahid Beheshti UMS, Tehran, Iran 8Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran. 9Department of Medical Microbiology and Infectious Diseases, Erasmus UMC, the Netherlands.

Introduction Methods (cont.) Histopathology qPCR assay

The frequency of invasive fungal infections (IFIs) has increased We further compared the results of histopathological analysis and qPCR A. fumigatus (9), A. flavus (20), A. terreus (1), significantly over the past 3 decades, associated with excessive morbidity assay with the panfungal PCR assay targeting the ITS rRNA gene. The Aspergillosis (59) A. niger (1), F. oxysporum (4), F. solani (1), R. oryzae (1) and mortality in immunocompromised hosts, patients hospitalized with PCR products were sequenced and compared with sequences in the R. oryzae (8), Mucor (3), R. microspores (1), severe underlying diseases (eg; acute myelogenous leukemia), those GenBank database to identify the causal pathogen. The molecular Mucormycosis (29) A. flavus + Mucor (2), A. flavus + R. oryzae (1), requiring complex surgical procedures (eg; trauma patients), and identification was correlated with results from histological examination. A. flavus (2) Syncephalastrum (1), individuals who require support in intensive care units . Early and reliable Scedosporium (1) detection of a fungal pathogen is crucial to guide the appropriate and Concomitant aspergillosis and A. flavus (2), Mucor (1), R. oryzae + A. flavus (1), Results and Discussion successful treatment of IFIs. The purpose of this study was to evaluate mucormycosis (4)

the ability of a rapid molecular assay to accurately detect and identify Overall, mold hyphae were detected histopathologically in 102 of the tissue Unknown (10) causative agents of IFIs in formalin-fixed paraffin-embedded (FFPE) specimens corresponded to aspergillosis (59), mucormycosis (29), Table 1. Results of histopathological analysis and qPCR assay in 102 biopsy tissues. concomitant aspergillosis and mucormycosis (4) or could not be further specimens obtained from patients who had suspected mold infections. specified (10). Methods The qPCR assay used was highly sensitive and a range of fungal genera/ species were identified, including: A. fumigatus, A. flavus, A. terreus, F. Conclusions: In a retrospective multicenter study, tissue samples from 102 FFPE oxysporum, F. solani, S. apiospermum, Rhizopus oryzae, Mucor spp. and • Our results indicated that histopathological features of specimens with histopathology results were tested. Syncephalastrum spp. In contrast, the positive identification rates for molds can easily be confused in tissue sections. Two 4-5 µm FFPE tissue section from each specimen was digested with panfungal PCR-sequencing assay were low (60%). • The quantitative PCR assays used in this study is a proteinase K followed by automated nucleic acid extraction. Fusarium oxysporum and F. solani DNA were amplified from five reliable tool, for rapid and accurate identification to the A specific quantitative real-time quantitative PCR (q-PCR) assay, using specimens initially diagnosed as aspergillosis (by histopathology), genus and species levels of fungal pathogens directly from fluorescently labeled primers, was performed to identify clinically respectively. Aspergillus flavus, S. apiospermum and Syncephalastrum FFPE tissues. important genus and species of Aspergillus (A. fumigatus, A. flavus, A. were detected from mucormycosis samples. • This assay a rapid promising alternative/adjunctive tool terreus, A. nidulans and A. niger), Fusarium (F. oxysporum, F. In addition, examination of four suspected samples as concomitant that can be easily incorporated into clinical mycology subglutinans, F. solani and F. dimerum), Scedosporium (S. apiospermum, aspergillosis and mucormycosis infection resulted to two A. flavus, one laboratories. S. aurantiacum and S. prolificans) and the Zygomycetes (Rhizopus Mucor, and only one sample having both R. oryzae and A. flavus. oryzae, Rhizopus microsporus, Cunninghamella bertholletiae, Mucor spp., The qPCR assay failed to detect fungal DNA in 9 samples, possibly as a Lichtheimia spp., Syncephalastrum spp. and Rhizomucor spp.). result of the destruction of DNA before paraffin wax embedding Key words

Real time qPCR, paraffin embedded tissue, mucormycosis, aspergillosis, fusariosis, scedosporiosis

Disclosure: This research was supported by a collaborative research from MazandaranUMS, Iran Department of Medical Microbiology and Infectious Diseases, ErasmusMC , Rotterdam, The Netherlands. and RadboudUMC, The Netherlands. Contact: Dr. Seyedmojtaba (Amir) Seyedmousavi [email protected]