Purpureocillium lilacinum is an opportunistic pathogenic fungus associated with endophthalmitis and keratitis. Previously, we isolated the strain P. lilacinum IFM 63780, notable for its high resistance to polyhexamethylene biguanide hydrochloride (PHMB), a common disinfectant and antiseptic used in dermatology and ophthalmology. In this study, we assessed the drug resistance profiles within the genus Purpureocillium. The minimum inhibitory concentration of PHMB for P. lilacinum and Purpureocillium takamizusanense was significantly higher compared to other species, while susceptibilities to other antibiotics and antifungal agents were consistent across all species. We developed species-specific primers for identifying P. lilacinum (PlF/R) and P. takamizusanense (PtF/R) by targeting the β-tubulin gene. These primers effectively amplified species-specific sequences, even when used in mixed primer pairs for the multiplex PCR, and did not cross-react with other species. This rapid and straightforward method provides a highly specific and reliable approach for detecting P. lilacinum and P. takamizusanense.

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Aspergillus fumigatus causes fatal infections in immunocompromised individuals, with the increasing number of azole-resistant strains leading to high mortality rates. This study aimed to develop a novel in vitro model using electrical impedance to continuously evaluate interactions between A. fumigatus and human cells and antifungal agent efficacy. A. fumigatus conidia and human cell lines (THP-1 macrophages and A549 alveolar epithelial cells) were cultured. Electrical impedance and fluorescence were observed using the xCELLigence RTCA E-Sight system. Conidia were seeded at various multiplicity of infection (MOI) values, and cell damage was assessed. In addition, the inhibition of cell damage by A. fumigatus in response to antifungal agents was evaluated. The time needed for electrical impedance to fall by half in macrophages was 31.5 hours for MOI 0.1 and 14.1 hours for MOI 8. Therefore, higher conidial concentrations led to faster decreases in electric impedance, indicating increased cytotoxicity. Macrophages showed a gradual decrease in electric impedance with mycelial growth, whereas A549 cells displayed a rapid electric impedance decline after mycelial growth. Azoles and amphotericin B suppressed the electric impedance decrease above their minimum inhibitory concentration, while echinocandins resulted in a continuous electric impedance decrease regardless of concentration. This study demonstrated that electrical impedance constitutes an objective method for continuously evaluating the cytotoxicity of A. fumigatus and antifungal efficacy. This novel in vitro model offers a new standard for studying interactions between filamentous fungi and human cells. Further validation using clinical isolates and other fungi is required.

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The rapid identification of microbes using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is gaining attention, particularly in clinical laboratories and hygiene management in food manufacturing. However, unlike bacteria, technical issues related to preprocessing and a lack of comprehensive reference libraries pose challenges in fungi. In this study, we constructed a new MALDI-TOF MS database, named EMALiMB, that expands the existing reference library to accurately identify a wider range of microbial species. The new reference library included 75 genera and 430 species of Ascomycota, 77 genera and 213 species of Basidiomycota, i.e. a total of 643 species of fungi, and six species belonging to the genus Prototheca. All strains were selected to complement taxa that were either not registered in the current library or were insufficiently represented, owing to a small number of strains. For example, 107 Candida species included pathogens, but also non-pathogenic species, inhabiting in the environment, and phylogenetically closely related with clinical relevants. Additionally, we improved the ionization of basidiomycetous yeasts and filamentous species, and Trichophyton, which had not been sufficiently ionized before, by incorporating bead-crushing in the pretreatment. The accuracy of this new reference library was evaluated using 384 clinical and environmental yeast isolates. A slight but remarkable increase in accuracy from 85.20% to 87.28% and in the mean score from 2.15 to 2.27 was obtained. The coverage rate for tested species improved significantly, from 80% to 88.57% for clinically relevant species, and from 52.38% to 76.19% for species isolated from environments.

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Terbinafine (TBF) and azoles are commonly used to treat fungal infections such as tinea pedis and tinea unguium. TBF-resistant Trichophyton species have been increasingly reported globally; however, the research has primarily focused on Trichophyton rubrum. In other words, there are limited studies that exist on other causative Trichophyton species, such as Trichophyton interdigitale, Trichophyton mentagrophytes, and Trichophyton indotineae. This study aimed to determine the prevalence and resistance mechanisms of TBF-resistant Trichophyton isolates in Japan. Screening of 701 clinical isolates preserved at the Medical Mycology Research Center, Chiba University, Japan, identified 20 resistant strains (2.9% prevalence), including 16 T. rubrum, two T. interdigitale, one T. mentagrophytes, and one T. indotineae. Minimum inhibitory concentrations (MICs) to TBF ranged from 1 to ≥ 32 µg/mL. Additionally, strains showing TBF resistance and reduced susceptibility to azoles were identified in T. rubrum, T. mentagrophytes, and T. indotineae. The squalene epoxidase gene sequencing targeted by TBF revealed amino acid mutations, such as Leu393Ser, Leu393Phe, and Phe397Leu in T. rubrum and Ser392Ala and Leu419Phe in other species. Notably, the Phe397 mutation correlated with high MICs (≥ 32 μg/mL), indicating its significant role in TBF resistance. This study detected a novel isolate of T. mentagrophytes showing TBF resistance and reduced susceptibility to azoles. The study underscores the need for ongoing surveillance and monitoring of antifungal resistance patterns for TBF and azole antifungal agents, considering the increasing prevalence of resistant isolates.

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In recent years, increased azole-resistant Aspergillus fumigatus (ARAf) isolation has been reported worldwide, and it poses a major threat. The main cause of resistance originates from mutations in the cyp51A gene, which encodes 14α-demethylase. Among the clinical isolates we received before 2024, a designated point mutation in their Cyp51A protein was the main mechanism of resistance for ARAf. However, from 2024, ARAf with tandem repeats (TRs) of 34 or 46 base-pairs in the promoter region of the cyp51A gene and amino acid substitutions in the coding region (TR-ARAf), which have been widely reported overseas but seldom found in Japan, has become isolated more frequently. Among the 111 A. fumigatus strains referred to our institution in 2024, ten strains were ARAf, of which four were TR-ARAf. Our study indicates that the prevalence of TR-ARAf in Japan is potentially on the rise, possibly posing a serious threat to public health. This trend might have serious implications for the management of aspergillosis in the country in the near future.

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Although culture remains the gold standard for detecting etiologic fungal organisms, histopathology plays a crucial role in confirming fungal infections by demonstrating these organisms in tissue. The morphology of fungi in tissue can often be specific to their genus or order, aiding pathological diagnosis. However, the presence of atypical fungal forms can complicate identification. For example, Aspergillus hyphae, which are typically thin and exhibit dichotomous acute-angle branching, may display atypical features such as varicose changes, twisting, and hyperseptation. These morphological variations can hinder the accurate identification of Aspergillus in tissue. Similarly, characteristics of hyphae from hyaline fungi, including Fusarium, Scedosporium, Trichosporon, and Mucorales, must be carefully interpreted, considering the inherent limitations of morphological diagnosis. Tinctorial characteristics are often useful for identifying fungi in tissue. For instance, brown coloration in hematoxylin and eosin-stained tissue and Fontana-Masson stain positivity can help differentiate dematiaceous fungi from hyaline fungi. However, these staining characteristics have limitations and potential pitfalls. Pale, brown-colored dematiaceous fungi may occasionally appear, and hyaline fungi may sometimes exhibit Fontana-Masson stain positivity, leading to diagnostic challenges. Understanding these morphological and tinctorial variations is essential for accurate histopathological identification of fungal infections in tissue.

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In Japan, two topical drugs and three oral antifungal drugs are prescribed for onychomycosis. Fosravuconazole L-lysine ethanolate (F-RVCZ) is an azole antifungal drug that has been prescribed in Japan since 2018 for the oral treatment of onychomycosis. Herein, we review the real-world evidence of F-RVCZ treatment for cutaneous fungal infection in Japan. The number of prescriptions for F-RVCZ has been increasing. It is more effective than topical drugs and has a favorable safety profile. On the other hand, side effects include elevation of liver enzymes, such as γ-GTP, and renal dysfunction in aged patients. Real-world case reports suggest that F-RVCZ is effective in treating other forms of tinea such as tinea capitis and tinea corporis. The diagnosis of onychomycosis is typically based on KOH direct examination; however, the dermatophyte test strip is used in specific situations where microscopy is difficult to perform such as in house-call medical practice.

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This study evaluated a lateral flow dermatophyte antigen kit (Kit) for rapid diagnosis of feline dermatophytosis. The Kit, originally used for human onychomycosis, was tested on 20 healthy cats and six cats with dermatophytosis. Hair samples from healthy cats yielded negative Kit results, although fungal culture detected environmental fungi in 4 of 20 cases. In dermatophytosis cases, the Kit correctly identified all six infected cats, including two cats tested with a single infected hair. Cultures confirmed Microsporum canis in all positive cases. These findings suggest that the kit may be useful as new rapid screening test for animal dermatophytosis.

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