Search

Trichosporon species are some of the most common pathogenic yeasts in Asia, and many are resistant to echinocandin antifungal drugs. Effective treatment of fungal infections requires the selection of appropriate antifungals and the accurate identification of the causal organism. However, in histopathological specimens Trichosporon spp. are often misidentified as Candida species due to morphological similarities. In situ hybridization (ISH) is a useful technique for identifying fungal species in formalin-fixed and paraffin-embedded (FFPE) tissue sections. Although many novel probes for ISH are available, the practical use of ISH for identification of fungi remains limited, in part due to the lack of adequate verifications. We conducted a two-center retrospective observational study in which the ISH technique was used to differentiate Trichosporon spp. and C. albicans in FFPE tissue from autopsy specimens. The study included 88 cases with blood stream yeast infection without Cryptococci extracted from 459 autopsy files of cases with proven invasive fungal infection (IFI). Positive signals for the Trichosporon spp. protein nucleic acid (PNA) probe and C. albicans PNA probe were seen for 7 and 35 cases, respectively, whereas the remaining 46 were negative for both. For the Trichosporon spp.- positive specimens, 5/7 were reported as candidiasis in autopsy records. Our results suggested that accurate histological identification of fungal infections remains challenging, but ISH may be a suitable approach to support histological findings. In addition, this retrospective study suggested that trichosporonosis may have high prevalence among cases of bloodstream yeast infections in Japan., (© The Author(s) 2019. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Although histopathology is required for definitive diagnosis of fungal infections, conclusive identification and discrimination of fungi in tissue sections and cytological preparations remain technically difficult. Therefore, new diagnostic tools are needed for the routine diagnosis of pathogenic fungi. In situ hybridization (ISH) is a non-culture based procedure that has many advantages over traditional diagnostics for identification of pathogenic fungi in histological specimens. This review highlights the basic ISH technique, with particular emphasis on using pretreatment of tissue sections prior to hybridization to solve problems associated with formalin fixation. With this modification, ISH has become a valuable tool that complements conventional histopathological diagnoses in formalin-fixed and paraffinembedded (FFPE) tissues. However, understanding the limitations imposed by formalin fixation is essential in developing suitable ISH protocols for fungal identification.

Although accurate and rapid diagnosis of fusariosis is now required, morphological similarities among molds make it difficult to histologically differentiate Fusarium spp. from other molds. In this chapter, we present our in situ hybridization (ISH) technique as a valuable tool to identify Fusarium spp. and emphasize the usefulness of the technique.

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.

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.

Notwithstanding the advantages of providing definitive diagnoses, the identification of fungi based on histopathological determination can be difficult and may lead to a pit of diagnoses. Therefore, the establishment of an auxiliary diagnostic method for use in routine pathological laboratories is desirable and should improve the above situation. Our previous studies have shown the superiority of in situ hybridization (ISH) for the detection of pathogenic fungi in histological specimens. This review focuses on the usefulness of ISH in the detection and identification of pathogenic fungi from formalin-fixed and paraffin-embedded (FFPE) tissue sections, and provides an overview of ISH for the diagnosis of fungal infection and retrospective autopsy analysis using molecular procedures. Based on the above, peptide nucleic acid (PNA) probes were shown to be superior in terms of the detection of target fungi and useful since histopathological diagnosis has the potential danger of being incorrect in the identification of fungi. In conclusion, we wish to emphasize that histopathological diagnosis in combination with molecular methods such as ISH and polymerase chain reaction (PCR) of FFPE samples should enhance the accuracy of identification in relation to fungi.

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.

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.

Invasive fungal infection (IFI) has a high mortality rate in patients who undergo hematopoietic stem cell transplantation, and it is often confirmed by postmortem dissection. When IFI is initially confirmed after an autopsy, the tissue culture and frozen section are challenging to secure, and in many cases, formalin-fixed, paraffin-embedded (FFPE) samples represent the only modality for identifying fungi. Histopathological diagnosis is a useful method in combination with molecular biological methods that can achieve more precise identification with reproducibility. Meanwhile, polymerase chain reaction (PCR) using fungal-specific primers helps identify fungi from FFPE tissues. Autopsy FFPE specimens have a disadvantage regarding the quality of DNA extracted compared with that of specimens obtained via biopsy or surgery. In the case of mucormycosis diagnosed postmortem histologically, we examined currently available molecular biological methods such as PCR, immunohistochemistry (IHC), and in situ hybridization (ISH) to identify fungi. It is reasonable that PCR with some modification is valuable for identifying fungi in autopsy FFPE specimens. However, PCR does not always correctly identify fungi in autopsy FFPE tissues, and other approaches such as ISH or IHC are worth considering for clarifying the broad classification (such as the genus- or species-level classification). [ABSTRACT FROM AUTHOR]

Background: Angiotensin-converting enzyme (ACE)2 forms angiotensin-1-7 which may protect kidney in a counterregulatory manner to angiotensin II. Recent studies revealed increased ACE and decreased ACE2 expression in kidneys of patients with diabetic nephropathy. However, these changes may not be specific for diabetic nephropathy. We studied ACE and ACE2 expression in patients with IgA nephropathy. Methods: Renal ACE and ACE2 expression was assessed by immunohistochemistry and in situ hybridization in 30 patients with IgA nephropathy and 21 healthy controls. Correlation between ACE and ACE2 expression and levels of various biochemical parameters was also assessed. Gene expression was also assessed in minimal change nephrotic syndrome (MCNS) and membranous nephropathy (MN) as disease controls. Results: Reduced ACE2 expression (p < 0.01) and increased ACE expression in glomeruli (p < 0.001), and reduced ACE2 expression in tubulointerstitium (p < 0.001) were observed in patients with IgA nephropathy compared to healthy controls, although the changes in ACE2 mRNA were not statistically significant. Reduced renal ACE2 expression was also found in MN but not in MCNS. Correlation between renal ACE and ACE2 expression and proteinuria was not observed in IgA nephropathy. Conclusion: IgA nephropathy is associated with increased ACE and decreased ACE2 expression in kidneys, as in diabetic nephropathy. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

In recent years, incidence of invasive fungal infection has been increasing, mostly due to advances in + medicine that may produce immunocompromised individuals. Candidial infection in the central nervous system (CNS) is one of the most serious forms of blood stream infection of Candida sp. and mortality is known to be more than 50%. In this research, we employed 27 autopsies with confirmed in vasive CNS yeast infection which were confirmed. In addition to detailed morphological analysis of yeast cells in lesions, in situ hybridization was carried out with an originally designed Candida -specific peptide nucleic acid (PNA) probe to identify the candidial infection of each patient. This was followed by histopathological investigation:invasiveness, shape, and distribution of yeast or yeasts with pseudohyphal growth, and a study regarding the correlation between histological characteristics and number of leukocytes in the peripheral blood just before death. Resalts showed that the, supratentorial region was the most common area of disseminated candidial infection in CNS, and that density was highest in the cerebral gray matter followed by the white matter and basal ganglia. On the other hand, regarding the lesions developed in the cortical area, the average distance from the brain surface was 4.026 mm. This area corresponding to the deeper cortex has a characteristic arterial structure that refers hairpin curving reverse. The structure may contribute to the high incidence of candidial foci in the deeper cortex, because of the increase in shear stress. [ABSTRACT FROM AUTHOR]