Development of a peptide nucleic acid probe to Trichosporon species and identification of trichosporonosis by use of in situ hybridization in formalin-fixed and paraffin-embedded (FFPE) sections

Abstract

In order to identify Trichosporon species in formalin-fixed and paraffin-embedded sections from which visual discrimination of non-glabrata Candida species is mostly ineffective but critical for the choice of antifungals, we tested the usefulness of a newly designed peptide nucleic acid probe (PNA) for in situ hybridization (ISH). Results confirmed the usefulness of ISH with our PNA probe in identifying Trichosporon species from Candida albicans.

Fig 1

Specificity verification of the Trichosporon spp. PNA probe and assessments of rRNA retention and its hybridizability in experimentally infected mice. (A) ISH using the Trichosporon spp. PNA probe in renal tissue from mice infected with T. asahii. Strong positive signals against 28S rRNA of Trichosporon spp. were observed in the specimen. (B) ISH using the Trichosporon spp. PNA probe in renal tissue from mice infected with C. albicans. Positive signals were not observed in the specimen. (C) ISH result with the panfungal PNA probe in renal tissue from mice infected with T. asahii. Strong positive signals were observed in the specimen. (D) ISH result with the panfungal PNA probe in renal tissue from mice infected with C. albicans. Strong positive signals were observed in the specimen. Magnification, ×400.

Fig 2

Results of ISH with a pulmonary lesion of disseminated trichosporonosis confirmed by DNA sequence analysis. (A) Pathological findings with hematoxylin and eosin stain. Histological examination revealed foci consisting of yeast formations of organisms. (B) Findings with Grocott's stain. Grocott's stain showed oval or square yeast-like elements within foci of infection. (C) Result of ISH with the Trichosporon spp. PNA probe. The PNA probe against Trichosporon spp. was strongly reactive with the yeast-like elements of Trichosporon spp. (D) ISH result with the C. albicans PNA probe. The PNA probe against C. albicans was not reactive with any Trichosporon spp. organisms.

Fig 3

Results of ISH for pulmonary lesions in the case of culture-proven C. albicans. (A) Pathological findings with hematoxylin and eosin stain. Histological examination revealed foci consisting of pseudohyphal and yeast formations of organisms. (B) Results with Grocott's stain. Grocott's stain showed oval yeast-like and pseudohyphal elements within the foci of infection. (C) ISH result with the Trichosporon spp. PNA probe. The PNA probe against Trichosporon spp. was not reactive with any organisms of C. albicans. (D) ISH result with the C. albicans PNA probe. The PNA probe against C. albicans was strongly reactive with pseudohyphal and yeast-like elements of C. albicans.