ABSTRACT
In situ hybridization (ISH) has been recognized as an important technique for identifying the causative fungi in the foci of infection observed in histopathological specimens which was processed from formalin-fixed and paraffin-embedded (FFPE) tissues. However, few basic studies have conducted an evaluation of the DNA preservation for use in ISH in comparison to polymerase chain reaction (PCR). The latter is a DNA amplification-based modality. In the present study, we analyzed 65 FFPE lung tissue specimens collected from autopsy cases for comparing the usefulness of ISH and PCR analysis. As a result, the positive identification rates for PCR were strikingly low; a majority of these results can be assumed to be false negative because the presence of fungi had been confirmed by histopathological analysis. In contrast, panfungal ISH targeting of the 28S rRNA showed a higher sensitivity than the 230-bp panfungal PCR primers did (80.0% versus 4.6%, respectively). Furthermore, over 60% of the samples we examined showed a favorable intensity of the ISH signal. Therefore, in conventional postmortem FFPE tissues, the state of DNA preservation may be more favorable for ISH than PCR analysis.
Subject(s)
Fungi/isolation & purification , In Situ Hybridization/methods , Mycology/methods , Mycoses/diagnosis , Pathology, Molecular/methods , Polymerase Chain Reaction/methods , Adolescent , Adult , Aged , Aged, 80 and over , Child , DNA, Fungal/genetics , Female , Fungi/genetics , Humans , Infant , Male , Middle Aged , Mycoses/microbiology , Sensitivity and Specificity , Young AdultABSTRACT
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.
Subject(s)
In Situ Hybridization/methods , Oligonucleotide Probes , Pathology, Molecular/methods , Peptide Nucleic Acids , Trichosporon/isolation & purification , Trichosporonosis/diagnosis , Candida albicans/genetics , Formaldehyde/metabolism , Humans , Oligonucleotide Probes/genetics , Paraffin Embedding , Peptide Nucleic Acids/genetics , Tissue Fixation , Trichosporon/geneticsABSTRACT
Fusarium has recently emerged as an opportunistic pathogen of humans, but the histological differentiation of Fusarium from Aspergillus and Scedosporium is particularly difficult because these fungi may induce similar clinical features and exhibit filamentous development in host tissues. Thus, there is a need to establish rapid and reliable methods that are applicable to pathological diagnoses. The aim of this study was to evaluate and establish in situ hybridization (ISH) using peptide nucleic acid (PNA) probes targeting the 28S rRNA to identify Fusarium species in tissue sections. This technique was validated using both formalin-fixed and paraffin-embedded pulmonary tissues from mice infected with seven different species of fungi and cell blocks from fungal cultures of 30 strains. As a result, strong positive signals were observed within fungal organisms present in tissues of the lung from mice infected with Fusarium solani. Furthermore, this probe reacted strongly with both F. solani and Fusarium oxysporum in sections from cell blocks. Although some cross-reactivity occurred with the Pseudallescheria boydii in sections from cell blocks, the signal intensity was low and most hyphae were not reactive. In conclusion, it was confirmed that ISH with PNA probes is accurate and is a valuable tool for identifying Fusarium spp. among organisms that have identical morphological features in formalin-fixed and paraffin-embedded sections.
Subject(s)
Fusarium/isolation & purification , In Situ Hybridization/methods , Mycoses/diagnosis , Oligonucleotide Probes , Pathology, Molecular/methods , Peptide Nucleic Acids , Animals , Base Sequence , DNA, Fungal/genetics , DNA, Ribosomal/genetics , Formaldehyde , Male , Mice , Mice, Inbred ICR , Molecular Sequence Data , Paraffin Embedding , RNA, Ribosomal, 28S/genetics , Sensitivity and Specificity , Sequence AlignmentABSTRACT
The present article describes our studies to know the usefulness of in situ hybridization (ISH) to identify various kinds of mold observed in tissue sections and / or cytological preparations from the lesions of patients with invasive fungal infection. To establish the precise procedure for ISH in formalin-fixed and paraffin-embedded sections, various pretreatments were attempted. The condition finally chosen is written here providing a favorable outcome regarding to both intensity and specificity of signals on outline of molds observed in the tissue sections when specimens were treated with both heat and proteinase K and, solutions were adjusted to higher pH value.Therefore, usefulness of promising probes, two each DNA and peptide nucleic acid (PNA) were verified with a favorable pretreatment condition, using lungs of mice experimentally infected and / or those obtained from autopsies with invasive mold infection. As the result, DNA probes targeting alkaline proteinase (ALP) gene and retrotransposon Afut-1 gene of Aspergillus fumigatus showed specific signal intensity for the Aspergillus species and A. fumigatus, respectively. PNA probes for Candida albicans and the Fusarium species also showed satisfactory specificity. We wish to emphasize that ISH can be a valuable tool to identify medically important molds in formalin-fixed and paraffin-embedded tissue sections or cytological preparations.
