Detection of tropical fungi in formalin-fixed, paraffin-embedded tissue: still an indication for microscopy in times of sequence-based diagnosis?

Abstract

Introduction: The aim of the study was the evaluation of panfungal PCR protocols with subsequent sequence analysis for the diagnostic identification of invasive mycoses in formalin-fixed, paraffin-embedded tissue samples with rare tropical mycoses.

Materials and methods: Five different previously described panfungal PCR/sequencing protocols targeting 18S and 28S ribosomal RNA gene fragments as well as internal transcribed spacer 1 and 2 fragments were evaluated with a collection of 17 formalin-fixed, paraffin-embedded tissue samples of patients with rare and/or tropical invasive mycoses, comprising chromoblastomycosis, coccidioidomycosis, cryptococcosis, histoplasmosis, mucormycosis, mycetoma/maduromycosis, and rhinosporidiosis, in a proof-of-principle analysis.

Results: The primers of the panfungal PCRs readily and predominantly reacted with contaminating environmental fungi that had deposited on the paraffin blocks. Altogether three sequence results of histoplasmosis and mycetoma samples that matched the histological assessment were associated with sample age <10 years and virtually without PCR inhibition.

Conclusions: The high risk of amplifying environmental contaminants severely reduces the usefulness of the assessed panfungal PCR/sequencing protocols for the identification of rare and/or tropical mycoses in stored formalin-fixed, paraffin-embedded tissues. Histological assessment remains valuable for such indications if cultural differentiation is impossible from inactivated sample material.

Figure 1

Photographs of representative fungi-containing samples analyzed in this study. (a) Case 1. Chromoblastomycosis of the skin. Fungal elements can be recognized in HE-stained sections as brownish-yellow pigmented bodies (arrows). A separation of the thick-walled fungus is also visible (multiform body). (b) Case 9. Skin lesion in rhinosporidiosis. Numerous spherical structures varying in diameter in an age-dependent manner. Immature forms (trophocytes) contain nucleus (arrow) and cytoplasm. Mature forms (sporangia) contain numerous endospores (asterisk). The rupture of this form is also visible. HE staining. (c) Case 10. Mycetoma. Lobulated grain with light colored center (brown) in lung. Grocott stain. (d) Case 11. Histoplasma infection. The periodic acid-Schiff reaction reveals many ellipsoidal yeast cells (red) inside macrophages and a giant cell. The arrow shows a budding.