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

Abstract

h In a retrospective multicenter study, 102 formalin-fixed paraffin-embedded (FFPE) tissue specimens with histopathology results were tested. Two 4-to 5-m 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.T he frequency of invasive fungal diseases (IFDs) has increased significantly over the past 3 decades, and they are associated with excessive morbidity and mortality in immunocompromised hosts, patients hospitalized with severe underlying diseases (e.g., acute myelogenous leukemia), those requiring complex surgical procedures (e.g., trauma patients), and individuals who require support in intensive care units (1-5).The most well-known cause of opportunistic filamentous mycoses is Aspergillus fumigatus (6). However, the epidemiology of IFDs due to filamentous fungi has been expanded well beyond A. fumigatus, including non-fumigatus species of Aspergillus, the mucormycetes, Fusarium and Scedosporium species, and a wide variety of melanized fungi (4, 7).Given the complexity of the population of patients at risk and the diverse and increasing arrays of fungal pathogens, which show significantly different antifungal susceptibilities, (3) early and reliable detection of a causative fungal pathogen is crucial to guide the appropriate and successful treatment of IFDs (8, 9).Formalin-fixed paraffin-embedded (FFPE) tissues obtained from patients with prov...

Molecular Identification of Saprochaete capitata in Human Blood and Paraffinized Tissue Samples

“…2B, lanes 2 to 7). This finding can complement those of studies addressing the rapid identification of clinical relevant yeasts and filamentous fungi in paraffin-embedded tissues and blood samples by PCR-based molecular techniques (5)(6)(7)(8)(9)(10).…”

Invasive Pulmonary Aspergillosis

“…189,190 Detection of fungal DNA from formalin fixed and paraffin-embedded tissues can, however, be impaired by degradation of DNA and presence of PCR inhibitors in these samples. 191…”

Fungal Infections Complicating Lung Transplantation

“…26 Real-time PCR may be useful to discriminate species and genus among molds (e.g., Aspergillus, Mucor, Fusarium, and Scedosporium) in formalin-fixed paraffin embedded specimens. 82 A recent ISHLT guideline recommends against use of serum GM and BAL-PCR, while recommending that BAL-GM can be used for IA diagnosis. 26…”

“…114 Serological assay for Aspergillus GM and β-D-glucan are negative in mucormycosis. 116 Quantitative PCR in serum or tissue are available for some species (e.g., Mucor, Rhizopus, Lichtheimia, and Rhizomucor) 82,[206][207][208] and may be superior to culture. [209][210][211]…”