Internal Transcribed Spacer Region Typing of Trichophyton interdigitale Isolated from Japanese Patients.

Trichophyton interdigitale, an anthropophilic species, is one of the main causative agents of tinea unguium and tinea pedis. T. interdigitale and the zoophilic species T. mentagrophytes are morphologically and physiologically very similar. Isolates of the T. interdigitale/T. mentagrophytes complex from around the world have been classified into more than 10 internal transcribed spacer (ITS) genotypes. In this study, we isolated T. interdigitale from Japanese patients and investigated which ITS type was more common. The ITS regions of 29 clinical isolates of T. interdigitale and one clinical isolate of T. mentagrophytes were sequenced. The phylogenetic analysis of the ITS region sequences revealed that the 29 isolates of T. interdigitale belong to ITS type II of T. interdigitale. The one clinical isolate of T. mentagrophytes was in the same cluster with ITS type II* of T. mentagrophytes. One terbinafine-resistant strain of T. interdigitale also belonged to ITS type II of T. interdigitale.

Clinical Isolate of a Multi-Antifungal-Resistant Trichophyton rubrum.

The multi-antifungal drug-resistant strain (NUBS21012) of Trichophyton rubrum was isolated from a patient with recurrent tinea corporis. The resistant strain encoded Phe at codon 393 instead of Leu (L393F) in the squalene epoxidase (SQLE) gene. The expression of genes encoding ATP-binding cassette transporter proteins increased in the strain compared to that of other strains. This result provides evidence that ATP-binding cassette transporter proteins are closely associated with azole resistance.

A deletion mutation in the amino acid sequence of squalene epoxidase in terbinafine-resistant Trichophytonrubrum.

INTRODUCTION: Trichophyton rubrum is an anthropophilic dermatophyte that is most frequently isolated from onychomycosis (tinea unguium) worldwide. T. rubrum strains showing resistance to the anti-fungal drug terbinafine (TRF) have also been isolated from human patients worldwide. METHODS: In this study, we isolated a TRF-resistant strain (N99-2) of T. rubrum from a patient with recurrent tinea unguium. In vitro susceptibility of the clinical isolate to TRF, itraconazole (ITZ), ravuconazole (RVZ), and luliconazole (LCZ) was investigated using the Clinical & Laboratory Standards Institute M38-A2 test. To identify mutations, we compared the gene sequence of N99-2 to that of a TRF-susceptible strain of T. rubrum. Results; In N99-2, the minimum inhibitory concentrations were 32 mg/L for TRF, <0.03 mg/L for ITZ, <0.03 mg/L for RVZ, and <0.03 mg/L for LCZ. The squalene epoxidase (SQLE) gene sequence in N99-2 was determined to be 1467 bp in length, and it encoded a protein of 488 amino acids, beginning with a putative initiating methionine (ATG). The following mutations were identified from the SQLE of N99-2: L393F and Y394del. CONCLUSIONS: This is the first report of the detection of a deletion mutation in SQLE in a TRF-resistant strain. The protein of SQLE is the target of TRF, and it is essential for cell membrane synthesis in dermatophytes. However, dermatophyte cells were found to undergo gene mutations to escape the effects of antifungal agents.

Mating Type Gene (MAT) and Itraconazole Susceptibility of Trichophyton tonsurans Strains Isolated in Japan.

Infection by Trichophyton tonsurans is an emerging fungal epidemic in Japan. Itraconazole (ITZ) and terbinafine have been used for the treatment of this infection for 15 years. However, patients with T. tonsurans infections have been shown to remain uncured or to become reinfected, suggesting that subclinical infection or polyphyletic strains and/or antifungal drug-resistant strains might be occurring in Japan. In this study, PCR analysis was performed to confirm the presence of the mating type locus MAT in genomic DNA from 60 Japanese clinical isolates of T. tonsurans, and to assess the previously postulated clonal origin of clinical isolates of this species. Antifungal susceptibility testing on isolates also was performed to confirm the absence of strains resistant to ITZ. PCR analysis proved that all 60 strains contained the MAT1-1 allele, while none contained the MAT1-2 allele. As determined by E-test, the mean MIC of ITZ in the 60 strains was 0.023 mg/L (range 0.002-0.125 mg/L). All strains of T. tonsurans isolated in Japan were clonal and were not resistant to ITZ. Therefore, dermatophytosis due to T. tonsurans is expected to respond to ITZ, since clinical isolates of T. tonsurans tested to date have been susceptible to this antifungal. This infection is proliferating as a subclinical infection in Japan.

Occurrence of Arthroderma benhamiae genotype in Japan.

In this study, we epidemiologically investigated on clinical isolates of Arthroderma benhamiae from humans and animals in Japan by internal transcribed spacer (ITS) region sequence analysis and mating type (MAT)-specific PCR. Seven of 8 A. benhamiae isolates from a human, rabbits and guinea pigs were identified as group I (white phenotype) by morphological characters and ITS region sequence analysis. One strain isolated from a degus (Octodon degus) produced colonies with few irregular folds and yellow velvety mycelium without macro- and microconidia. This strain resembled to group II (yellow phenotype) strain. ITS sequence analysis was also 100% identical to that of group II. MAT-specific PCR indicated that 6 of these 7 isolates of group I contained an alpha-box gene and that one strain contained high-mobility-group (HMG) gene. One strain of group II was revealed to have an alpha-box gene and no HMG gene. To our knowledge, it is the first A. benhamiae isolate of group II found in Japan. The A. benhamiae may be more widespread in worldwide than our surpassing what is common or usual or expected.

The MAT1-1:MAT1-2 ratio of Sporothrix globosa isolates in Japan.

In order to understand the reproductive biology of pathogenic species in the Sporothrix schenckii complex, we characterized the partial mating type (MAT1-1) loci of Sporothrix schenckii, as well as the S. globosa MAT1-1-1 gene, which encoded 262 amino acid sequences. The data confirmed that the MAT1-1 locus of S. globosa was divergent from the MAT1-2 locus of the opposite mating type, suggesting that the fungus is heterothallic. To determine the mating type ratio of 20 isolates from Japanese patients, we analyzed the MAT loci by specific PCR amplification of MAT1-1-1 and MAT1-2-1 genes. The MAT1-1-1 was detected in 5 isolates but not in the other 15 isolates with the presence of MAT1-2-1. The MAT1-1:1-2 ratio of S. globosa isolates in Japan was estimated to be 1:3. Phylogenetic analysis indicated that the sequences of the MAT1-1-1 were identical among S. globosa isolates but different from S. schenckii and Ophiostoma montium.

Genotype analyses of human commensal scalp fungi, Malassezia globosa, and Malassezia restricta on the scalps of patients with dandruff and healthy subjects.

Dandruff and seborrheic dermatitis are common afflictions of the human scalp caused by commensal scalp fungi belonging to the genus Malassezia. Malassezia globosa and Malassezia restricta are the predominant species found on the scalp. The intergenic spacer regions of these species' rRNA genes contain short sequence repeats (SSR): (GT)n and (CT)n in M. globosa and (CT)n and (AT)n in M. restricta. In the present study, we compared the genotypes (SSR) of M. globosa and M. restricta colonizing the scalps of patients with dandruff and healthy individuals. For M. globosa, the genotype (GT)10:(CT)8 (40.3 %, 25/62) was predominant followed by (GT)9:(CT)8 (14.5 %, 9/62) and (GT)11:(CT)8 (14.5 %, 9/62) in patients with dandruff, whereas the genotypes in healthy subjects were diverse. For M. restricta, the genotype (CT)6:(AT)6 (59.7 %, 37/62) was predominant followed by (CT)6:(AT)8 (24.2 %, 15/62) in patients with dandruff, while four genotypes, (CT)6:(AT)6 (10.5 %, 6/57), (CT)6:(AT)7 (22.8 %, 13/57), (CT)6:(AT)8 (17.5 %, 10/57), and (CT)6:(AT)10 (21.1 %, 12/57), accounted for 71.9 % of all combinations in healthy subjects. The results of this study suggest that the M. globosa genotype (GT)10:(CT)8 and the M. restricta genotype (CT)6:(AT)6 may be involved in the development of dandruff.

Isolation of dermatophytes and related species from domestic fowl (Gallus gallus domesticus).

We investigated 793 bird combs [645 chickens and 148 fighting cocks (Shamo)] to determine the prevalence of dermatophytes and their related fungal species. The targeted fungal species were recovered from 195 of the 793 examined birds (24.6 %). Prevalence ratios were compared in temperate (the mainland) and subtropical (Nansei Islands) areas, genders, strains, breeding scale (individual and farm), and housing system (in cage and free ranging). The frequency of the fungal species in the mainland, males, fighting cocks, breeding scale by individual nursing, and free-range housing system exhibited significantly higher positive ratios than that in the other groups. A total of 224 dermatophytes and related species were isolated, including 101 Arthroderma (Ar.) multifidum, 83 Aphanoascus (Ap.) terreus, five Uncinocarpus queenslandicus, two U. reesii, two Ap. pinarensis, one Amauroascus kuehnii, one Ar. simii, one Gymnoascus petalosporus, one Microsporum gallinae, and 28 Chrysosporium-like (Chrysosporium spp.) isolates, which were identified using internal transcribed spacer regions of ribosomal RNA gene sequences. The predominant fungal species in the mainland was Ap. terreus and that in the Nansei Islands was Ar. multifidum. Pathogenic fungal species to humans and animals were limited to M. gallinae and Ar. simii, which corresponded to 0.025 % of the isolates in this study.

Intractable Nocardial mycetoma with possible colonisation by Candida species.

A Japanese male in his 30s with no underlying medical condition presented with painless nodules after being bitten by a dog during a stay in Bali, Indonesia, 7 years earlier. He was referred to our department with multiple ulcers, nodules, and masses on the right leg. The final diagnosis was mycetoma caused by Nocardia vulneris, which may have been exacerbated by colonization of Candida parapsilosis and C. tropicalis as these yeasts were isolated by culture from the tissue. Treatment with minocycline hydrochloride and sulfamethoxazole trimethoprim showed partial efficacy, but the addition of posaconazole achieved significant efficacy. This suggests that the surmised coexistence of pathogenic yeasts of lower virulency may have made mycetoma in this case intractable.

Mating type gene (MAT1-1) in Japanese isolates of Trichophyton rubrum.

Trichophyton rubrum is an anthropophilic species that is the most frequent etiologic agent of human dermatophytosis throughout the world. No teleomorph has been identified for T. rubrum strains. This study used PCR analysis to confirm the presence of a mating type locus in the genome of Japanese isolates of T. rubrum. To clarify the epidemiological and ecological characteristics of this fungus, mating type sequences were tested for correlation of MAT genotype to mating type. This study examined clinical isolates of T. rubrum that had been obtained from 206 human cases of tinea pedis and tinea unguium in Japan, including those from Fukuoka (29 strains), Gifu (23 strains), Kanazawa (63 strains), and Tokyo (91 strains), along with 10 isolates derived from 10 cases of canine dermatophytosis. PCR detected the presence of MAT1-1 in all of the human and animal isolates. Therefore, all isolates examined were expected to react as (-) type on the mating test and not as (+) type.

In vitro Azole Susceptibility Testing of Japanese Isolates of Terbinafine-Resistant Trichophyton indotineae and Trichophyton rubrum.

Multi-antifungal-resistant strains of Trichophyton indotineae and Trichophyton rubrum have been isolated in Japan. In the present study, we examined the in vitro susceptibility of terbinafine (TRBF) -resistant isolates of T. indotineae and T. rubrum to efinaconazole (EFCZ) and luliconazole (LUCZ). In all isolates, the minimum inhibitory concentrations were ≥ 32 mg/l for TRBF, < 0.03 to 16 mg/l for itraconazole, < 0.03 to 16 mg/l for ravuconazole, < 0.03 to 0.5 mg/l for LUCZ, and < 0.03 to 4 mg/l for EFCZ. Of note, T. rubrum NUBS21012 and T. indotineae NUBS 19006T showed resistance to LUCZ and/or EFCZ unlike the other isolates.

A Case of Kerion Celsi due to Trichophyton tonsurans.

The patient was a 13-year-old boy who was a member of the judo club at his junior high school. Approximately 1 week prior to his presentation, he developed multiple erythematous pilaris papules on his occipital area and was treated by a local doctor. The erythematous lesions expanded to 10 × 10 cm, showing granulation with drainage and strong spontaneous pain. At this point, he visited our hospital. He was diagnosed with kerion celsi due to Trichophyton tonsurans by fungal examination. The patient was treated with terbinafine (125 mg/day) for 6 weeks, and a brush test at 6 weeks was negative. All 18 members of the judo club, including this patient, were investigated; brush tests were positive in 4 cases, and one was positive for tinea corporis alone. The patient's family members parents were both negative. When an athlete is diagnosed with ringworm, T. tonsurans infection should be considered, and testing and treatment of family members and fellow athletes should be carried out to prevent the spread of infection.

Epidemiological study of antifungal-resistant dermatophytes isolated from Japanese patients.

An epidemiological study of antifungal drug-resistant dermatophytes was conducted as a follow-up to our 2020 survey. Dermatophytes were isolated in 2022 from the same dermatology clinics as in the previous study. In total, 288 Trichophyton interdigitale and Trichophyton rubrum clinical isolates were obtained from 288 human cases of dermatophytosis in Tokyo, Saitama, Shizuoka, and Kumamoto, Japan. Four strains were found to be resistant to terbinafine (TRF) and susceptible to itraconazole (ITZ), luliconazole (LCZ), and ravuconazole (RVZ), and three other strains were found to be resistant to ITZ and susceptible to TRF, LCZ, and RVZ. We determined the sequences of the squalene epoxidase (SQLE)-encoding gene in the three TRF-resistant T. rubrum strains, and found that two strains harbored L393F missense mutations, and one strain harbored a F397L missense mutation. The results of the present study indicated that the prevalence of TRF-resistant dermatophytes has not increased since 2020. However, TRF-resistant T. interdigitale (L393F mutation) was isolated for the first time, indicating that attention should be paid to the presence of TRF-resistant T. interdigitale in the future. We also examined for the first time the epidemiology of ITZ-resistant T. rubrum in Japanese patients. Although the number of ITZ-resistant strains was not large, the results confirmed that ITZ-resistant T. rubrum strains do exist in Japanese patients.

In vitro Azole Susceptibility Testing of Japanese Isolates of Itraconazole-Resistant Dermatophytes.

We conducted antifungal susceptibility testing on itraconazole (ITCZ)-resistant isolates of Trichophyton interdigitale and Trichophyton rubrum collected from Japanese patients in 2021 and 2022. The aim of the present study was to determine the most effective drug against ITCZ-resistant strains of dermatophytes. In all isolates, the minimum inhibitory concentrations (MICs) were > 32 mg/l for ITCZ, < 0.03 to 0.5 mg/l for ravuconazole (RVCZ), and < 0.03 mg/l for efinaconazole (EFCZ), luliconazole (LUCZ), and terbinafine (TRBF). Thus, in tinea unguium cases with ITCZ-resistant strains, treatment should be switched to TRBF or other azoles with a stronger antifungal efficacy, such as EFCZ, LUCZ, or RVCZ, and treatment must continue until the infectious organisms are completely eliminated.

Antifungal Susceptibility Testing for Microsporum canis from Cats in Japan.

Control of infection caused by Microsporum canis in pet animals are important for prevention of zoonosis. Treatments for animal dermatophytosis have generally consisted of itraconazole (ITZ) and terbinafine (TRF); however, a TRF-resistant M. canis strain from a case of feline dermatophytosis has been reported. In the present study, we examined the in vitro susceptibility of clinical isolates of M. canis to new antifungal drugs, such as ravuconazole (RVZ) and luliconazole (LCZ). The results indicated that RVZ and LCZ are more effective than ITZ and TRF. Therefore, oral administration of RVZ or topical application of LCZ may serve as new treatment options.

Dermatophyte antigen kit: A useful diagnostic tool for onychomycosis.

A dermatophyte antigen kit (DQT) was released in Japan as an in vitro diagnostic tool to identify tinea unguium in June 2022. From July 2022 to February 2023, we examined 75 potassium hydroxide (KOH)-negative patients (male, n = 23; female, n = 52; mean ± SD age, 63.6 ± 13.9 years) and determined the accuracy in confirming the fungal element with ZoomBlue™ staining at 400× magnification. The DQT results were classified into three categories. DQT-positive onychomycosis was detected in 27 patients with tinea unguium and two with non-dermatophyte onychomycosis. Fungal cultures were positive in 14 (51.8%) patients (Trichophyton rubrum [n = 11], T. interdigitale [n = 1], Fusarium solani [n = 1], and Talaromyces muroii [n = 1]). DQT-negative onychomycosis included ten patients with cured tinea unguium and 3 with Candida onychomycosis. Twenty-three patients had DQT-negative mimics for onychomycosis (onychauxis [n = 11], traumatic onycholysis [n = 8], yellow nail syndrome [n = 5], pincer nail deformity [n = 3], brittle nail syndrome [n = 2], contact dermatitis [n = 2], lichen planus [n = 1] and psoriasis [n = 1]). Because sparse, atrophic and/or fragmented mycelia are invisible in direct microscopy with potassium hydroxide (KOH) at 100× magnification, DQT was beneficial for diagnosing onychomycosis.