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Trichphyton indotineae, a species newly designated in 2020 independent of T. interdigitale, comprises highly terbinafine (TRF)-resistant dermatophytosis that is epidemic in North India and spreding to worldwide. Some clinical isolates of T. indotineae have been resistance both TRF and azoles that might be caused the treatment failure. To detect the azole resistance strains, we developed a long amplification PCR (LA-PCR) detection method for the tandem repeat of the CYP51B (encoding sterol 14a-demethylase gene) in T. indotineae. Contrasting the drug susceptibility test results with the LA-PCR results confirmed a trend toward low susceptibility to azole antifungal agents in strains with amplifications of 9.5 kbp or greater (3 or more copies of CYP51B). Our results suggest that the method could be detected rapidly of low-susceptibility strains to azole antifungal agents., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

In this study, we induced terbinafine (TRF) resistance in a T. rubrum strain in vitro for 18 months then compared the genomes of the TRF-resistant (N42-3) and TRF-susceptible wild-type (N42WT) strains to identify mutations. In the SQLE gene, N42WT had no mutation while N42-3 had a F397L mutation. We sequenced approximately 22.53 Mb of the genomes of the N43WT and N42-3 strains. Other than the F397L mutation in SQLE, there were three other genetic mutations in three different genes that were found in N42-3, but not in N43WT; however, these three mutations were not detected in other TRF-resistant T. rubrum strains. From this genome sequencing analysis, the only variation that was confirmed to be associated with drug resistance in the genome of the TRF-resistant T. rubrum was a hotspot mutation in SQLE., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

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., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Trichophyton equinum is a zoophilic dermatophyte that is frequently isolated from horse dermatophytosis and rare infections in humans. In the present study, molecular and physiological testing were performed on T. equinum isolates from dermatophytoses of Japanese racehorses to assess genotype and phenotype patterns of these strains. Comparative nucleotide sequence analysis showed that internal transcribed spacer (ITS) region sequences amplified from all Japanese isolates were 99.5% identical to T. equinum reference strains. ITS sequences amplified among the isolates were 100% (BT2) showed that isolates were 100% identical and harbored a "T" single nucleotide polymorphism at position 18. The sequences of β-tubulin (BT2) showed that isolates were 100% identical to T. equinum reference strains. The MAT1-2 allele was detected by PCR in all seven isolates, whereas none of the isolates contained the MAT1-1 allele. All isolates grew only on Trichophyton Agar 5 and did not grow on Trichophyton Agar 1 and 4, indicating nicotinic acid requirement. These results suggest that Japanese T. equinum isolates are derived from a clonal population.

Protothecosis refers to disease of humans and animals caused by infection with fungus-like, colourless microalgae of the genus Prototheca. Although protothecosis remains an uncommon infection, increasing numbers of human and animal cases are being diagnosed worldwide. This review summarises major new findings in basic science (sequencing analyses of sterol 14α-demethylase (CYP51/ERG11) genes and organelle genomes of Prototheca wickerhamii) to elucidate taxonomic features of this pathogen. Furthermore, this review updates and summarises the clinical features, diagnosis and treatment of protothecosis in dogs and cats. This content of this review is based on information presented at the medical phycology symposium held in the 20th Congress of the International Society for Human and Animal Mycology ( https://www.isham.org/ ).

In this report, we describe the first isolation of two highly terbinafine (TRF)-resistant Trichophyton interdigitale-like strains from a Nepali patient and an Indian patient with tinea corporis in Japan. These strains (designated NUBS19006 and NUBS19007) exhibited a TRF minimal inhibitory concentration (MIC) of > 32 mg/L and contained a missense mutation (Phe397Leu) in squalene epoxidase (SQLE) gene. The internal transcribed spacer (ITS) region sequences amplified from the isolates (NUBS19006 and NUBS19007) were 99.5% identical to Japanese isolates of T. interdigitale and T. interdigitale strain CBS 428.63. The homology of region sequences were also 97.6% identical to T. mentagrophytes strain CBS 318.56. Moreover, the ITS sequences amplified from the isolates were 100% identical to highly TRF-resistant strains of T. interdigitale, which were isolated in Delhi, India, and harbored mutations in SQLE. The urease test on Christensen's urease agar was positive for T. mentagrophytes and T. interdigitale after 7 days of incubation. On the other hand, the type strain of T. rubrum CBS 100081  T and highly TRF-resistant strains (NUBS19006 and NUBS19007) were negative on Christensen urease agar after 7 and 14 days of incubation. Moreover, NUBS19006 and NUBS19007 were also negative reaction on the hair perforation test. To avoid confusion in the taxonomy of the T. mentagrophytes/T. interdigitale complex, we suggest that the highly TRF-resistant Indian strains be considered a new species independent of T. interdigitale, according to clinical and mycological features.

The genus Prototheca consists of achlorophyllic algae that are ubiquitous in the environment and animal intestines. However, this organism has forfeited its photosynthetic ability and switched to parasitism. In 1894, Krüger described two microorganisms isolated in Germany from mucous flux of Tilia and Ulmus spp., namely Prototheca moriformis and P. zopfii. Based on their yeast-like colony morphology, Krüger classified these organisms as fungi. The genus is now included within the class Trebouxiophyceae, order Chlorellales, and family Chlorellaceae. Historically, protothecosis and infections caused by green algae have been studied in the field of medical mycology. Prototheca spp. have been found to colonize human skin, fingernails, the respiratory tract, and digestive system. Although human infection by Prototheca is considered rare, an increase in infections has been noted among immunosuppressed patients, those on corticosteroid treatment, or both. Moreover, the first human outbreak of protothecal algaemia and sepsis was recently reported in a tertiary care chemotherapy oncology unit in 2018. Prototheca is also a causative pathogen of bovine disease. Prototheca zopfii and P. blaschkeae are associated with bovine mastitis, which causes a reduction in milk production and secretion of thin, watery milk containing white flakes. Economic losses are incurred either directly via reduced milk production and premature culling of affected animals or indirectly as a result of treatment and veterinary care expenses. Thus, knowledge of this fungal-like pathogen is essential in human and veterinary medicine. In this mini-review, I briefly introduce human and animal protothecoses.

Aspergillus caninus (synonym: Phialosimplex caninus) is an anamorphic fungus species associated with systemic infections in dogs that has been transferred from the genus Phialosimplex to Aspergillus. Here, we report the first case of canine A. caninus infection in Japan. A castrated Japanese Shiba Inu (6 years old; weight, 12.5 kg) was referred to the Yamaguchi University Animal Medical Center, Yamaguchi, Japan, in June 2017 showing vitality loss and depression. Computed tomography revealed iliac and splenic hilum lymphopathies, and histologic examination of an iliac lymph node by biopsy revealed granulomatous lesions with numerous oval to round yeast-like fungal cells. Aspergillus caninus was isolated from the biopsy samples, and in vitro susceptibility tests of the isolate to the antifungal drugs amphotericin B (AMB), fluconazole (FLZ), itraconazole (ITZ), voriconazole (VRZ), and micafungin (MCF) were performed by the E-test method. The isolate from this dog exhibited a minimal inhibitory concentration of < 0.002 µg/ml to AMB, > 256 µg/ml to FLZ, < 0.002 µg/ml to ITZ, < 0.002 µg/ml to VRZ, and < 0.002 µg/ml to MCF, indicating that the isolate was not susceptible to FLZ and susceptible to AMB, ITZ, VRZ, and MCF. Since the response of the patient dog to ITZ and VRZ treatments was poor, more aggressive management using combination therapies of ITZ with other antifungals may be necessary for treating canine A. caninus infection in dogs.

To clarify the terbinafine (TRF) resistance mechanism in a TRF-resistant strain of Microsporum canis, the expression of the pleiotropic drug resistance (PDR1), multidrug resistance (MDR1), MDR2 and MDR4 genes were investigated by real-time quantitative PCR (RT-qPCR) analysis, given the known interaction of the corresponding proteins with antifungals and with the efflux blocker FK506. The expression of the PDR1, MDR1, MDR2 and MDR4 genes was 2-4 times higher in the TRF-resistant strain grown in the presence of 0.14 µg/mL of TRF than in TRF-susceptible strains cultured in the absence of TRF. The TRF-resistant strain exhibited MICs of > 32 µg/mL for TRF alone; this resistance was attenuated to an MIC of 8 µg/mL in the presence of FK506, indicating that the TRF inhibitory concentration index value was < 0.75. The additive effect of the efflux blocker FK506 on TRF resistance was detected in the TRF-resistant strain. These results indicated that the TRF resistance in this strain reflects overexpression of genes encoding ABC transporter proteins.

In the initial online publication, the name of author Hock Siew Han was given incorrectly as Han Hock Siew. The original article has been corrected.

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.

We report here, to the best of our knowledge, the first description of an in vitro fluconazole (FLZ)-resistant Cryptococcus neoformans var. grubii from a case of feline cryptococcosis. In vitro testing demonstrated that this isolate was resistant to FLZ (minimum inhibitory concentration, MIC, of 128 μg/ml) but remained susceptible to amphotericin B (0.064 µg/ml), itraconazole (0.38 µg/ml), voriconazole (0.023 µg/ml), and posaconazole (0.125 µg/ml). The predicted amino acid sequence of the lanosterol 14-α demethylase (ERG11) protein in the isolate was identical to that of the C. neoformans var. grubii reference strain, indicating that resistance was not mediated by mutation of the target gene's open reading frame. The RT-qPCR analysis for ERG11 and ATP-binding cassette (ABC) transporter-encoding gene (AFR1) indicated that the isolate increased transcription factor function of ERG11 and AFR1 than that of FLZ-susceptive strains. This observation, in combination with the lack of resistance to other azoles (that is, lack of crossresistance), suggests that resistance in our isolate was the result of overexpression of the endogenous ERG11 and ABC transporter.

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.

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.

Trichophyton verrucosum is a zoophilic dermatophyte species that is the most frequent etiologic agent of bovine dermatophytosis throughout the world. Since no teleomorph of T. verrucosum has been found, polymerase chain reaction (PCR) analysis on the genome of T. verrucosum isolated from the Czech Republic and Japan was performed to confirm the presence of a mating type locus in the genome of the fungus and to clarify its classification and ecological characteristics. The mating type gene (MAT1-2) allele was detected by PCR analysis in all 22 isolates (four isolates from the Czech Republic and 18 isolates from Japan). The nucleotide sequence of the region exhibited 99-100 % identity among all isolates, including the reference strain of T. verrucosum. Phylogenetic analysis revealed that the sequences of the internal transcribed spacer region at the MAT1-2 locus clustered together in the isolates examined, forming a branch distinct from that of the other dermatophyte species. These results suggest that T. verrucosum is a clonal offshoot that has drifted away from Arthroderma benhamiae.

Systemic mycoses in killer whales (Orcinus orca) are rare diseases, but have been reported. Two killer whales died by fungal infections at the Port of Nagoya Public Aquarium in Japan. In this study, the fungal flora of the pool environment at the aquarium was characterized. Alternaria spp., Aspergillus spp. (A. fumigatus, A. niger, A. versicolor), Fusarium spp. and Penicillium spp. were isolated from the air and the pool surroundings. The other isolates were identified as fungal species non-pathogenic for mammals. However, the species of fungi isolated from the environmental samples in this study were not the same as those isolated from the cases of disease in killer whales previously reported.

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.

The mating type (-)-specific gene of the alpha-box and the mating type (+)-specific gene of the high-mobility group (HMG) DNA-binding domain were confirmed in zoophilic dematophytes of Arthroderma simii and A. vanbreuseghemii. The sequence of the alpha-box gene was 1,375 bp, containing 2 exons (from 172 to 463 bp and from 513 to 1,375 bp) in the A. simii (-) mating type strain and 1,380 bp, containing 2 exons (from 177 to 468 bp and from 518 to 1,380 bp) in the A. vanbreuseghemii (-) mating type strain. The sequence of the HMG gene was 1,871 bp, containing 2 exons (from 181 to 362 bp and from 426 to 1,440 bp, coding a protein of 398 amino acids) in the A. simii (+) mating type strain and 1,811 bp containing 2 exons (from 158 to 339 bp and from 403 to 1,381 bp, coding a protein of 386 amino acids) in the A. vanbreuseghemii (+) mating type strain. Of 15 animal isolates and 72 human isolates examined, the alpha-box gene was detected in five of the animal isolates and in none of the human isolates, while the HMG gene was detected in the other 10 of the animal isolates and in all of the human isolates. Phylogenetic analysis of the alpha-box and HMG genes of Trichophyton mentagrophytes complex strains and the Microsporum gypseum strain revealed that these strains were divided into 4 clusters; the first cluster consisting of A. vanbreuseghemii and the isolates from animals and humans, the second cluster consisting of A. simii, the third cluster consisting of A. benhamiae and the fourth cluster consisting of M. gypseum. These results indicate that anthropophilic T. mentagrophytes evolved from the A. vanbreuseghemii (+) mating strain.

This report describes the first documented case of subcutaneous infection due to Cryptococcus flavescens in a dog. The chief symptoms of the patient dog were abscessed lesions on the dorsal muzzle, right eyelid, and lower jaw. Biopsy specimens from the lesions on the dorsal muzzle and lower jaw showed pyogranulomatous inflammation with numerous yeast cells. The patient dog was diagnosed with a subcutaneous fungal infection and orally received 5 mg/kg itraconazole once a day for 2 months, the abscesses disappeared. After 1 month at the end of treatment, the skin lesions did not redevelop. Isolates from the biopsy specimens were identified as C. flavescens by molecular analysis as well as morphologic and biochemical examination, indicating that C. flavescens is a potential canine pathogen.

Marek's disease virus (MDV) is an oncogenic herpesvirus that causes malignant lymphomas in chickens. Recent field isolates of MDV have tended to exhibit increasing virulence, and MDV strains are currently classified into four categories based on their relative virulence. Meq, a putative MDV oncoprotein, resembles the Jun/Fos family of basic leucine zipper (bZIP) transcription factors and can regulate the expression of viral and cellular genes as a homodimer or as a heterodimer with a variety of bZIP family proteins. MDV isolates display distinct diversity and point mutations in Meq, which may contribute to changes in the transcriptional activities of Meq and subsequently, to observed increases in MDV oncogenicity. In this study, we introduced mutations into the meq gene and used dual luciferase reporter assays to analyze the transcriptional activities of the resulting Meq proteins to determine whether distinct mutations in Meq could be responsible for differences in transcriptional activity among MDV strains. A proline-to-alanine substitution at position 217, the second position of one of the proline direct repeats in the transactivation domain, enhanced the transactivation activity of Meq. In addition, we found that two substitutions at positions 283 and 320 affected transactivation activity. These results suggest that the distinct diversity of and point mutations in the Meq proteins are responsible for differences in transactivation activity among MDV strains.

This study first report to identify the mating type (-)-specific gene of alpha-box and the mating type (+)-specific gene of the high-mobility-group (HMG) DNA-binding domain in zoophilic dermatophytes of Arthroderma benhamiae in an effort to understand the epidemiological characteristics of Trichophyton mentagrophytes. The sequence of the alpha-box gene (1,387 bp) was found to contain two exons, from 184 to 475 bp and from 525 to 1,387 bp, coding a protein of 384 amino acids, beginning with a putative initiating methionine (ATG). The sequence of the HMG gene (1,910 bp) contained two exons, from 234 to 415 bp and from 479 to 1,457 bp, coding a protein of 386 amino acids, beginning with a putative initiating methionine (ATG). PCR analysis detected the alpha-box gene in A. benhamiae (-) mating type strains but not in (+) mating type strains. On the other hand, the HMG gene was detected in A. benhamiae (+) mating type strains but not in (-) mating type strains. These findings suggest that the HMG and alpha-box genes could be specific to the (+) and (-) mating types, respectively.

The isolate from urine of a dog with cystitis was molecularly identified Candida tropicalis and its minimum inhibitory concentration (MIC) was determined by a microdilution method. The 25S ribosomal DNA sequence analysis indicated that the clinical isolate was essentially identical to that of C. tropicalis and distinct from other Candida species. The MIC(50) and the MIC(90) of fluconazole (FLZ) for the clinical isolate of C. tropicalis was 6.25 and 25 microg/ml, respectively, indicating that susceptibility of the clinical isolate of C. tropicalis to FLZ was less than for other strains of C. tropicalis as well as C. albicans. The molecular analysis as presented in this study assisted the diagnosis of candidiasis by identifying the yeasts in urine samples within 2 days. The patient dog, a 10-year-old male Shih Tzu dog (7.0 kg) referred for examination of cystitis was successfully treated with itraconazole.

Cryptococcus neoformans produces an extracellular polysaccharide capsule that is related to its virulence. The production of capsular components was reported to be accelerated when cultured on media with lower amount of glucose. In this study, relationship between capsule synthesis and expression of capsule-associated genes (CAP genes) was investigated by quantitative real-time PCR analysis. Normally encapsulated strains and a stable acapsular strain were cultured in 1% polypepton medium with 0.1% or 15% glucose. The results of assessment of the capsule size showed that the capsule of yeast cells cultured in the medium with low amount of glucose was thicker than that with high amount of glucose. The CAP gene expressions of normally encapsulated strains were higher in the medium with 0.1% glucose than in the medium with 15% glucose. Furthermore, CAP10, CAP59 and CAP60 genes were expressed very low in a stable acapsular strain, and CAP64 gene was not expressed. Results of assessment of capsule size and CAP gene expressions by quantitative real-time PCR analysis indicated that CAP gene expressions might be related to the production of capsule, and that glucose concentration in culture media might be related to the expression of CAP genes.

A 6-month-old male Japanese domestic cat with otitis externa due to Aspergillus fumigatus was treated with antifungal agents for 25 days and appeared to be cured. Many yeast colonies however developed from the ear canal samples on Sabouraud's dextrose agar at 27 degrees C for 5 days, instead of A. fumigatus. This yeast colony was cream-colored and slim in texture with smooth and highly glossy surface after 5-day incubation on Sabouraud's dextrose agar at 27 degrees C. The isolate was identified as Cryptococcus magnus by mycological analysis and 28S ribosomal analysis.

The Nucleotide sequence of the chitin synthase 1 (CHS1) gene of Epidermophyton floccosum, an anthrophophilic dermatophyte which is the type species of the genus Epidermophyton was analyzed to determine its phylogenetic relation to eight other dermatophyte species belonging to the genera Microsporum and Trichophyton, which were sequenced in our previous studies. A genomic DNA fragment about 620 bp in length of the CHS1 gene was amplified from E. floccosum by polymerase chain reaction (PCR) and was sequenced. The CHS1 nucleotide sequence showed more than 85% similarity with sequences derived from the other dermatophytes. Phylogenetic analyses of the sequences from E. floccosum revealed that the genus Epidermophyton may be genetically distinct from Microsporum and Trichophyton.

A clinical isolate from a black-dot ringworm lesion of a 28-year-old female Japanese was investigated by morphological and biochemical analyses as well as molecular analyses. The isolate grew well on thiamine enriched agar and did not produce violet pigment, macro-conidia or micro-conidia on Sabouraud's dextrose agar. Approximately 620-bp genomic DNA fragments of the CHS1 gene were amplified from Trichophyton mentagrophytes, T. rubrum, T. tonsurans and T. violaceum by polymerase chain reaction (PCR) and sequenced. The chitin synthase 1 (CHS1) nucleotide sequences of the clinical isolate showed more than 97% similarity to that of T. violaceum and less than 96% similarity to that of T. mentagrophytes, T. rubrum and T. tonsurans. The phylogenetic analysis of their sequences revealed that the clinical isolate was genetically close to T. violaceum and distinct from T. mentagrophytes, T. rubrum and T. tonsurans. Therefore, the isolate was confirmed as T. violaceum by mycological examination and molecular analyses.