Application of In Situ Hybridization to Tissue Sections for Identification of Molds Causing Invasive Fungal Infection

Minoru Shinozaki; Yoichiro Okubo; Haruo Nakayama; Aki Mitsuda; Tadashi Ide; Somay Yamagata Murayama; Kazutoshi Shibuya

doi:10.3314/jjmm.50.075

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Application of In Situ Hybridization to Tissue Sections for Identification of Molds Causing Invasive Fungal Infection

Minoru Shinozaki, Yoichiro Okubo, Haruo Nakayama, Aki Mitsuda, Tadashi Ide, Somay Yamagata Murayama, Kazutoshi Shibuya

Author information

Minoru Shinozaki
Department of Surgical Pathology, Toho University School of Medicine,6-11-1 Omori-Nishi, Ota-Ku, Tokyo143-8541, Japan
Yoichiro Okubo
Department of Surgical Pathology, Toho University School of Medicine,6-11-1 Omori-Nishi, Ota-Ku, Tokyo143-8541, Japan
Haruo Nakayama
Department of Surgical Pathology, Toho University School of Medicine,6-11-1 Omori-Nishi, Ota-Ku, Tokyo143-8541, Japan
Aki Mitsuda
Department of Surgical Pathology, Toho University School of Medicine,6-11-1 Omori-Nishi, Ota-Ku, Tokyo143-8541, Japan
Tadashi Ide
Department of Surgical Pathology, Toho University School of Medicine,6-11-1 Omori-Nishi, Ota-Ku, Tokyo143-8541, Japan
Somay Yamagata Murayama
Laboratory of Molecular Epidemiology for Infectious Agents,Graduate School of Infection Control Sciences & Kitasato Institute for Life Sciences Kitasato University,5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
Kazutoshi Shibuya
Department of Surgical Pathology, Toho University School of Medicine,6-11-1 Omori-Nishi, Ota-Ku, Tokyo143-8541, Japan

Keywords: in situ hybridization, Aspergillus fumigatus, histopathological diagnosis, invasive fungal infection, polymerase chain reaction (PCR)

JOURNAL FREE ACCESS

2009 Volume 50 Issue 2 Pages 075-083

DOI https://doi.org/10.3314/jjmm.50.075

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Abstract

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.

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