In Situ Hybridization Detection of Dematiaceous Fungi in Formalin-Fixed, Paraffin-Embedded Sinonasal Specimens Using a Wide-Spectrum Oligonucleotide Probe.
Kathleen T Montone, Virginia A LiVolsi, Michael D Feldman, Irving Nachamkin. University of Pennsylvania, Philadelphia
Background: Dematiaceous fungi are a diverse group of “darkly” pigmented fungi which contain melanin in their cell walls and are commonly found in soil worldwide. While morphology and histochemical stains may aid identification in tissue sections, these means for species identification are not specific. In situ hybridization (ISH) for abundant fungal rRNA sequences may provide a means for detecting dematiaceous fungi and differentiating them from other clinically important filamentous fungi such as Aspergillus sp., Paecilomyces sp, and Fusarium sp. In this study, we report a rapid ISH assay for detecting dematiaceous fungi in formalin-fixed, paraffin-embedded tissue specimens.
Design: 29 patients with culture proven sinonasal dematiaceous fungal infections were utilized. These included Alternaria sp. (10), Bipolaris sp. (5), Curvularia sp. (10), Cladosporium sp. (1); Scopulariopsis sp. (1), Scedosporium prolificans and dematiaceous species, not otherwise specified (1). Rapid (<2.5 hour) ISH was performed using a 24 base oligonucleotide biotin-labeled DNA probe targeting the fungal rRNA gene sequences of a variety of dematiaceous fungi. By GenBank BLAST analysis, this sequence was shown to have 100% homology to nucleic acid sequences of a variety of fungi in the Phylum Ascomycota including Curvularia sp. and Bipolaris sp. and as well as uncultured soil fungi.
Results: ISH revealed positivity in 24 of 29 culture proven cases of dematiaceous fungal infection. The five negative cases included 2 Bipolaris sp, 2 Curvularia sp. and 1 case of S. prolificans. Two of the negative specimens had undergone decalcification in HCl-based decalcification solution prior to tissue processing. The dematiaceous fungal specific probe did not hybridize to culture positive examples of Rhizopus sp., Aspergillus sp, Fusarium sp., Paecilomyces sp., H. capsulatum, Candida sp., and B. dermatitidis.
Conclusions: ISH can rapidly detect several human disease-related dematiaeous fungi and differentiate them from other medically important filamentous fungi such as Zygomyces, Aspergillus, Fusarium and Paecilomyces which are often clinically treated differently. This method can be performed rapidly and is useful for fungal identification in routinely processed tissue specimens. ISH using probes targeting a variety of fungal pathogens will be useful for triaging tissue based filamentous fungal infections in which fungal cultures are not available.
Monday, February 28, 2011 1:00 PM
Poster Session II # 256, Monday Afternoon