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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.

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.

Clinical diagnosis of invasive fungal infections (IFIs) is sometimes difficult, and obtaining an accurate assessment of trends concerning the prevalence of IFIs is a challenge. The aim of this study was to determine trends in the prevalence of IFIs from an autopsy survey. The retrospective review of autopsy records stored in Toho University was performed on all documented cases with fungal infection from 1955 to 2006. A total of 411 cases of IFIs were detected among 10 297 autopsies. The prevalence of candidiasis decreased from 3.6% (1981-93) to 2.0% (1994-2006), and that of aspergillosis increased throughout the 52-year period and reached 2.0% (1994-2006). The prevalence of IFIs in the patient group comprising haematological disorders was significantly higher (19.9%) than in other patient groups (2.9%), of which the odds ratio was 18.4 for mucormycosis and 10.0 for aspergillosis. The lung was the most common organ involved irrespective of major fungal species, and most cases with candidiasis showed multiple-organ infection. Results confirmed the increasing prevalence of aspergillosis and high risk of IFIs in the patient group with haematological disorders. IFIs were also detected in an immunocompromised state caused not only by primary disease but also by treatment with anti-tumour drugs and corticosteroids., (© 2012 Blackwell Verlag GmbH.)

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.

Our analysis reported here, is the first one in the world to make a nationwide-level estimate on numbers of visceral mycoses in case of leukemia and myelodysplastic syndrome (MDS)in Japan. The data on visceral mycoses in cases reported in the" Annual of the Pathological Autopsy Cases in Japan" published by the Japanese society of pathology in 2002 and 2006, and the data in the vital statics in Japan published by Ministry of health, labour and welfare were analyzed epidemiologically. The estimated numbers of visceral mycoses were 2,250 out of the estimated total death 8,976 in 2001, and 2290 out of 9,805 in 2005, respectively, in cases of leukemia and MDS in Japan. Furthermore, the estimated severe cases that we thought direct cause of death were 1,454 in 2001, and 1,464 in 2005, respectively. In the severe cases, the most causative agents were Aspergillus. The estimated numbers of Candida and Zygomycetes were interestingly almost same in the severe cases, but the lethal rate in Zygomycetes was about 60-80 % , almost twice as that in Candida. We think it is imperative to continuously survey and watch these rates towards the future.

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.

One year after the release of the Japanese "Guidelines for the Diagnosis and Treatment of Deep-Seated Mycosis" we conducted a survey of clinicians to determine the extent to which the new guidelines had penetrated clinical practice, and compared these results with those of a previous survey. The responses to the current survey regarding the diagnosis and treatment had changed from those of the previous survey to reflect the new recommendations, showing that the guidelines have had an effect on clinical practice. However, the current survey highlights a need to provide more practical information in the guidelines for use in the clinical settings. In particular, physicians expect the guidelines to proactively provide more information about the features of both current and new drugs. In addition, an effective drug against the genus Aspergillus is eagerly awaited. However, because it is difficult to differentiate among filamentous fungi, there is a need for a drug with broad-spectrum coverage against filamentous fungi. Investigation of combination therapy consequently becomes necessary. Definitive diagnoses of invasive fungal infection are too scarce at the national level. The cooperation of clinicians for organizing more definitive diagnoses would be appreciated when the guidelines are revised.

Background Mucormycosis is a potentially fatal fungal infection, and there is limited information on its precise epidemiology and treatment practices, including the optimal dosage of liposomal amphotericin B. Methods A retrospective, multicenter, nationwide analysis of 82 proven and probable cases of mucormycosis was performed. Cases between 2015 and 2022 were collected from 51 hospitals in Japan by hematologists and infectious disease specialists. The study included the epidemiology, treatment details, and association between the dose of liposomal amphotericin B and the outcome. Results The lungs were the most commonly involved organ (70.7% of cases), and 35.4% of patients had disseminated disease. Rhizopus spp. Cunninghamella spp. and Mucor spp. were the most common organisms. Mortality at 4 weeks was 41.5%. The survivors had a shorter duration of neutropenia (P =.006) and less persistent hyperglycemia (P =.023). The site of infection and species of Mucorales had no detectable effect on survival. Survival did not differ between patients receiving liposomal amphotericin B at 5 mg/kg/d relative to those receiving >5 mg/kg/d (P =.625). Using Cox proportional hazards models and adjusting for confounders, the hazard ratio for the influence of >5 mg/kg/d liposomal amphotericin B on 4-week survival was 0.86 (95% CI, 0.28–2.68; P =.796) compared with 5 mg/kg/d. Conclusions This study provides important insights into the precise epidemiology and treatment practices of mucormycosis. Treatment with liposomal amphotericin B at doses higher than 5 mg/kg/d did not improve outcomes relative to 5 mg/kg/d. [ABSTRACT FROM AUTHOR]

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]

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]

Blastomycosis is a fungal infectious disease that can occur in both immunocompromised and immunocompetent populations endemic in North America, with no previous reports in Japan. A 26-year-old Japanese female patient with no relevant medical history presented intermittent left back pain and an abnormal shadow in the left upper lung field eight months ago at a local clinic. She was referred to our hospital for further evaluation and treatment. The patient currently lives in Japan, but until two years ago had spent several years in New York, Vermont and California. Chest computed tomography revealed a 30 mm mass with a cavity in the left pulmonary apex. The specimens obtained by transbronchial biopsy showed periodic acid-Schiff stain (PAS)-positive and Grocott-positive yeast-like fungi scattered among the granulomas, with no malignant findings, and the initial pathology did not lead to a definitive diagnosis. She was empirically started on fluconazole because of onset of multiple subcutaneous abscesses and was referred to the Medical Mycology Research Center. Although antibody tests could not diagnose the disease, blastomycosis was suspected based on the pathology of the skin and lung tissue at the Medical Mycology Research Center, and Blastomyces dermatitidis was identified by ITS analysis of the rRNA region. Her symptoms and CT findings gradually improved with fluconazole. We reported the first Japanese case of blastomycosis with pulmonary and cutaneous involvement in Japan. As the number of overseas travelers is expected to continue increasing, we would like to emphasize the importance of travel history interviews and information of blastomycosis. [ABSTRACT FROM AUTHOR]