We previously reported that the Malassezia microbiota of the external auditory canal were composed principally of Malassezia slooffiae and Malassezia restricta. In the present study, the fungal microbiota of the external auditory canal were comprehensively analyzed by pyrosequencing on an FLX-454 platform. Of 17 samples tested, the fungal microbiota of 14 samples were composed of > 90% Malassezia species; either M. slooffiae or M. restricta was abundant in individual samples. The external auditory canal exhibits a lower fungal diversity than do other bodily sites.
In Japan, Fusarium species are known etiological agents of human fungal infection; however, there has been no report of a large-scale epidemiological study on the etiological agents of fusariosis. A total of 73 Fusarium isolates from patients with invasive fusariosis (IF, n= 36) or superficial fusariosis (SF, n= 37), which were obtained at hospitals located in 28 prefectures in Japan between 1998 and 2015, were used for this study. Fusarium isolates were identified using Fusarium- and Fusarium solani species complex (FSSC) -specific real-time PCR and partial DNA sequences of the elongation factor-1 alpha (EF-1α) gene and the nuclear ribosomal internal transcribed spacer (ITS) region. FSSC was predominately isolated from both patients with IF and SF (IF, 77.8% and SF, 67.6%). Distribution of the phylogenetic species of FSSC isolates from patients with IF and SF exhibited different spectra; specifically, F. keratoplasticum (FSSC 2) (25.0%) was the most frequent isolate from patients with IF, whereas F. falciforme (FSSC 3+4) (32.4%) was the most frequent isolate from patients with SF. Fusarium sp. (FSSC 5) was the second most frequent isolate from both patients with IF and SF (IF, 22.2% and SF, 24.3%). Notably, F. petroliphilum (FSSC 1) was isolated only from patients with IF. Each species was isolated from a broad geographic area, and an epidemic was not observed. This is the first epidemiological study of Fusarium species causing IF and SF in Japan.
The binding of Candida albicans cells to chitin was examined in a cell-binding assay. Microscopic observations indicated that both living and heat-killed Candida cells bound to chitin-coated substrates. C. albicans preferentially bound to chitin-coated plastic plates over chitosan-coated and uncoated plates. We prepared 125I-labeled Candida cells for quantitative analysis of their binding to chitin. Heat-killed 125I-labeled Candida cells bound to chitin-coated plates in a time-dependent manner until 1.5 hours after start of incubation at 4℃. The binding of 125I-labeled Candida cells to chitin-coated plates was inhibited by adding unlabeled living or unlabeled heat-killed Candida cells. The binding of Candida to chitin was also reduced by addition of 25 mg/ml chitin or chitosan up to 10%. N-acetylglucosamine (GlcNAc), which is a constituent of chitin, inhibited binding of Candida to chitin in a dose-dependent manner between 12.5 and 200 mM. Glucosamine, which is a constituent of chitosan, showed no such inhibitory effect. These findings suggest that the binding of Candida to chitin may be mediated by recognition of GlcNAc.
The mechanisms of azole resistance in Trichosporon asahii have not yet been fully clarified. We previously showed that T. asahii has the ERG11 gene, coding lanosterol 14-α-demethylase (Erg11 protein; Erg11p), which is the primary target of azoles. A single amino acid substitution at G453R in Erg11p was found to induce changes in the affinity of this enzyme for azoles, especially fluconazole, in vitro. In the present study, we investigated the DNA sequences of the ERG11 gene using six different strains of clinically isolated T. asahii that were highly resistant to multi-azoles, including fluconazole, itraconazole, and voriconazole. All of the T. asahii strains had a point mutation (G448A) that caused a single amino acid substitution at G150S in Erg11p. This amino acid is highly conserved among major fungal pathogens. We identified a new point mutation in the ERG11 gene that is common to clinically isolated azole-resistant T. asahii strains, suggesting that this mutation is associated with the multi-azole resistance of T. asahii.
A 62-year-old male with numerous subcutaneous nodules in the lower extremities was referred to The University of Tokyo Hospital. The patient suffered from systemic lupus erythematosus (SLE), diabetes mellitus, and persisting hepatic dysfunction, and had been treated for SLE with oral prednisolone 20 mg/day and oral cyclosporine 3 mg/kg/day. The culture of scales collected from the patient’s skin surface on Sabouraud’s dextrose agar medium showed features of Trichophyton rubrum. Topically applied bifonazole cream was effective for tinea corporis, but oral griseofulvin 500 mg/day was discontinued after 2-month administration because of deteriorated liver function. All the nodules were resected surgically. Histologically, resected granulomas showed dermal abscesses that were tightly encapsulated by fibrous capsules. Grocott staining revealed numerous fungal elements within abscesses. The patient’s condition indicated the need to perform histopathological examination of granuloma trichophyticum in order to determine whether it is tightly encapsulated. Namely, the presence of cystic granuloma trichophyticum with abscesses encapsulated by fibrous capsules suggested that the patient should be treated by surgical resection of the lesions.
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.
Aspergillus species are ubiquitous in the environment and Aspergillus fumigatus can cause life-threatening infections in immunocompromised patients. β-1,3-/1,6-glucan is a major fungal cell wall polysaccharide that has various biological effects on the infected host, but little is known about the influence of β-glucan on the fungus itself. In a previous study, we demonstrated that the cell wall β-glucan content could be increased in Aspergillus spp. by addition of β-glucan to the culture medium. In this study, we investigated the influence of β-glucan on the susceptibility of A. fumigatus to antifungal agents. A. fumigatus was cultured in the presence or absence of β-glucan for antifungal susceptibility testing based on changes of the growth rate and morphology. Susceptibility to micafungin, a β-glucan synthase inhibitor, was about 10-fold lower when β-glucan was added to the culture medium. On the other hand, susceptibility to amphotericin B and voriconazole was similar in either the presence or absence of β-glucan. These results strongly suggest that β-glucan has an important physiological role in Aspergillus spp.