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Ravuconazole (RVCZ) is a new human anti-fungal azole drug available in Japan since 2018 and is a broad-spectrum agent that exhibits excellent activity against dermatophytes. In the present study, the in vitro RVCZ susceptibility of clinical isolates of anthropophilic dermatophytes, including Trichophyton interdigitale strains with either low susceptibility to itraconazole (ITCZ) or resistance to terbinafine (TEBR), was investigated using the Clinical & Laboratory Standards Institute M38-A2 test. The MICs of RCVZ for 20 clinical isolates of T. interdigitale were < 0.03125-0.125 mg/L; for 4 clinical isolates of T. rubrum, < 0.03125-0.0625 mg/L; and for 20 clinical isolates of T. tonsurans, < 0.03125 mg/L. Similarly, the MICs of RCVZ for the T. interdigitale strains with either low susceptibility to ITCZ or resistance to TEBR were also < 0.03125 mg/L. To our knowledge, this is first study to investigate the in vitro RVCZ susceptibility of T. interdigitale strains with either low susceptibility to ITCZ or resistance to TEBR. Our results indicated that RVCZ was the most effective drug against these strains.

Background: The genus Malassezia is comprised of a group of lipophilic yeasts that have evolved as skin commensals and opportunistic cutaneous pathogens of a variety of mammals and birds., Objectives: The objective of this document is to provide the veterinary community and other interested parties with current information on the ecology, pathophysiology, diagnosis, treatment and prevention of skin diseases associated with Malassezia yeasts in dogs and cats., Methods and Material: The authors served as a Guideline Panel (GP) and reviewed the literature available prior to October 2018. The GP prepared a detailed literature review and made recommendations on selected topics. The World Association of Veterinary Dermatology (WAVD) Clinical Consensus Guideline committee provided guidance and oversight for this process. The document was presented at two international meetings of veterinary dermatology societies and one international mycology workshop; it was made available for comment on the WAVD website for a period of six months. Comments were shared with the GP electronically and responses incorporated into the final document., Conclusions and Clinical Importance: There has been a remarkable expansion of knowledge on Malassezia yeasts and their role in animal disease, particularly since the early 1990's. Malassezia dermatitis in dogs and cats has evolved from a disease of obscurity and controversy on its existence, to now being a routine diagnosis in general veterinary practice. Clinical signs are well recognised and diagnostic approaches are well developed. A range of topical and systemic therapies is known to be effective, especially when predisposing factors are identified and corrected., (© 2019 ESVD and ACVD.)

Malassezia pachydermatis, a lipophilic and aerobic yeast, is a causative agent of Malassezia dermatitis, a common skin mycosis in dogs and cats. This fungus is also responsible for zoonotic fungal infections in human neonates. Ravuconazole (RVZ) is an antifungal azole compound and the active metabolite of fosravuconazole, which was approved for use in humans in Japan in 2018. In the present study, in vitro RVZ susceptibility and multi-azole resistance of 13 clinical M. pachydermatis strains was investigated using the modified Clinical and Laboratory Standards Institute M27-A3 test. The minimum inhibitory concentrations (MICs) for the 13 isolates ranged from 0.094 to >32 mg/L for itraconazole (ITZ) and from 0.5 to >32 mg/l for RVZ. Similarly, MICs for ITZ- or RVZ-resistant strains (MICs >32 mg/l) were also >32 mg/l for clotrimazole (CTZ), >32 mg/l for miconazole (MCZ), and 0.25 to >32 mg/L for voriconazole (VRZ). BLAST analysis using the NCBI database showed that ERG11 cDNA of the RVZ-resistant strain encoded Gly at codon 461 and Asp in cytochrome p450 encoded by M. pachydermatis ERG11 mRNA. This work is the first report to describe that an RVZ-resistant M. pachydermatis strain contains ERG11 mutations. The affinity of the protein encoded by ERG11 for RVZ may differ from that of ITZ. Therefore, RVZ has considerable therapeutic potential for treating ITZ-resistant canine Malassezia dermatitis. However, RVZ-resistant strains already exist in canine Malassezia dermatitis in Japan., (© The Author(s) 2019. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

In the case presented here, we describe the isolation of an azole-resistant strain of M. pachydermatis from a canine Malassezia dermatitis. The isolate (NUBS18001) from this case exhibited a minimum inhibitory concentration (MIC) of 320 μg/ml to itraconazole (ITZ) by broth microdilution (BM) assay, >32 μg/ml to ITZ by E-test, and >32 μg/ml to KTZ by E-test. Synergistic effects between FK506 and ITZ in the azole-resistant strain was evaluated using the microdilution checker-board method. The ITZ-resistant strain exhibited MICs of 320 μg/mL of ITZ alone and 5 μg/ml of FK506 alone; the addition of FK506 attenuated the ITZ MIC to 2.5 μg/ml, yielding an ITZ FICI value of 0.507. This result suggested that the combination of ITZ and FK506 exerted an additive effect against the ITZ-resistant strain. To understand the other mechanism inferred to be present in our multi-azole-resistant strain, we sequenced the ERG11 gene from this isolate, and detected missense mutations (A412G and C905T) in the sequence of the ERG11 open reading frame (ORF). To the best of our knowledge, this work is the first report that a multi-azole-resistant M. pacydermatis strain contains mutations in ERG11., (© The Author(s) 2018. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

A 2-year-old, exotic shorthair cat presented with baldness and mild scaling on trunk that was confirmed as Microsporum canis (M. canis) infection by the following methods. Wood's lamp and trichogram were used to demonstrate fungal elements suggestive of dermatophytosis consistent with M. canis. Dermatophyte test medium (DTM) and polymerase chain reaction (PCR) were used for identification. E-test and broth microdilution test were then utilized to estimate antifungal minimal inhibitory concentrations (MICs) towards ITZ and TRF respectively. The strain was isolated from the patient and revealed TRF MIC >32 µg/ml and ITZ MIC 0.023 µg/ml. Patient was cured of dermatophytosis with systemic ITZ.

A 73-year-old male who had been receiving immunosuppressive drugs for 15 years developed a nodule on the left buttock region. The nodule slowly grew into a 15-cm fluctuant multilocular subcutaneous cyst. Serum beta-D-glucan levels were high, and the yellow purulent fluid obtained from the cyst was positive for Trichophyton rubrum. Granuloma formation in the cyst wall and large abscesses in the central cystic area were found, and septated hyphae were observed in both tissues. The cyst was surgically removed, and followed by itraconazole treatment. Notably, the clinical manifestations closely resembled those of a huge atheroma.

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.

Topical or oral azole antifungals are commonly used in canine atopic dermatitis (AD), as the lipophilic yeast Malassezia pachydermatis exacerbates canine AD. To examine whether canine AD lesions harbor azole-resistant M. pachydermatis isolates in East Asia, we investigated the in vitro susceptibility of M. pachydermatis isolates to ketoconazole (KTZ) and itraconazole (ITZ) obtained from AD lesions of canines in Japan, Korea and Taiwan. The minimum inhibitory concentrations (MICs) of KTZ and ITZ were measured by the E-test using Sabouraud dextrose agar with 0.5% Tween 40. The MICs of KTZ and ITZ for isolates from canines with AD were significantly higher than the MICs for isolates from healthy canines. Our findings suggested that the clinical isolates from canine AD skin lesions were less susceptible to azoles than those from normal canine skin in East Asia.

Isolates of the yeast Malassezia pachydermatis obtained from skin samples of healthy dogs and of dogs with atopic dermatitis in Japan, Taiwan and Korea were molecularly characterized using intergenic pacer 1 (IGS1) region analysis. The percentage of IGS1 subtype isolates detected in healthy skin was as follows: 1A (6%), 1B (27%), 1C (11%), 2A (6%), 2B (6%), 3A (11%), 3B (6%), 3C (3%) and 3D (24%). In contrast, the most prevalent isolates detected in skin lesions of atopic dermatitis were subtype 3D in Japan and Taiwan and subtype 3C in Korea. All subtype isolates grew well on acidic medium (pH 6). However, subtype 3C and 3D isolates grew better than the other subtype isolates on medium at pH 8.

A case of tinea corporis caused by Microsporum gallinae was found in 2011 in Okinawa, located in the southern part of Japan. The patient was a 96-year-old, otherwise healthy, Japanese man, who had been working as a breeder of fighting cocks for more than 70 years. He was bitten on his right forearm by one of the cocks and a few weeks later, two erythematous macules appeared on the right forearm, accompanied by a slight itchy sensation. While the first isolate of this dermatophyte was recovered from the region by Miyasato et al. in 2011, it was not obtained from the same fighting cock owned by the patient. However, frequent exchanges of fighting cocks and special domestic breeds of chickens related to fighting, mating, and/or bird fairs are common among the fans and breeders. We investigated 238 chickens and 71 fighting cocks in Okinawa and in the suburbs of Tokyo (Chiba, Tokyo, Ibaraki, and Sizuoka). One isolate of M. gallinae from a fighting cock in Chiba Prefecture in the Tokyo metropolitan area exhibited a different genotype, with a single base difference from the patient isolate based on the internal transcribed spacer 1-5.8s-ITS2 regions (ITS1-5.8S-ITS2) of the ribosomal RNA gene sequence. The isolation of M. gallinae from a fighting cock on the mainland of Japan is the first such finding in animals in our country.

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.

Human cases of dermatophytoses are occasionally transmitted from animals, and suffered from tinea corporis and sometimes Kerion celsi. The most frequent causative agent of these diseases is Microsporum canis. The other dermatophyte, Arthroderma benhamiae is now prevailing in rabbits, rodents and hedgehogs that are popular household pets in Japan. Therefore, some human cases of A. benhamiae infection were reported and the transmission of this infection from rabbits and rodents was confirmed.Cryptococcosis is regarded as dangerous zoonosis, but its transmission from animal to peoples has not been documented in Japan. Animal cases of cryptococcosis are possible to increase in number by developing immunosuppressive animals as well as by spreading of newly introduced C. gattii to Japan.Animal cases of sporotrichosis are rarely reported in Japan. However, feline sporotrichosis should be prevented and promptly treated since it easily transmitted to people from cat lesions and the exudates where copious numbers of organisms are found in tissues.

The present study describes the isolation of Fusarium sporotrichioides from a canine cutaneous ulceration. A 2-year-old male Beagle dog weighing 8.6 kg, with a history of immune-mediated hemolytic anemia (IMHA), had been treated with prednisone for 9 months. Physical examination revealed cutaneous ulceration on the left foreleg. Histopathological examination of skin samples from the ulcerative area revealed many branching hyphae surrounding neutrophils. Since itraconazole (ITZ) is recommended for miscellaneous fungal infections, the dog was treated with ITZ. However, the ulcerative lesions did not improve and after 3 weeks of treatment the dog died due to renal failure. No autopsy was performed. Since the isolate recovered from the biopsy specimen was identified as Fusarium species by morphological characteristics, the animal was diagnosed as having had an infection caused by this mould. The dog's prior prednisone treatment may have played a role in establishing the fungal infection. Comparative sequence analyses of the ITS regions of the clinical isolate with those in GenBank showed that it was 100% identical to F. sporotrichioides and less than 96% similar to ITS of other Fusarium species. Based on these findings, F. sporotrichioides was established as the etiologic agent of the canine infection, a situation that has not been previously reported in dogs, as well as humans.

The characteristic pathological feature of dermatomycosis is numerous neutrophilic infiltrates within the epidermis. However, the precise mechanism of this infiltration remains unknown. In this study, interleukins 1 beta, 6, and 8, monocyte chemotactic protein-1 (MCP-1), and tumor necrosis factor (TNF)-alpha levels in the medium where keratinocytes were co-cultured with Candida albicans, Malassezia and Trichophyton mentagrophytes, were determined by enzyme-linked immunosorbent assays (ELISAs) in order to estimate the effect of these fungi on the cytokine production from human keratinocytes. The IL-8 level in the supernatants increased with 1 to 14 hours of co-culture in response to live C. albicans, but the other cytokines were undetectable. Furthermore, the mRNA of IL- 8 in keratinocytes was also confirmed to increased. This data suggested that C. albicans directly induce interleukin 8 production from human keratinocytes without activated macrophages. The IL-6, IL-8, and TNF-alpha levels in the culture supernatants increased with 1 to 24 hours of co-culture with keratinocytes and Malassezia species but the MCP-1 level was undetectable. The IL-8 and TNF-alpha levels in the culture supernatants increased with 1 to 24 hours of co-culture with keratinocytes and Trichophyton mentagrophytes but the other cytokine levels were undetectable. The ELISA analysis of cytokine production by human keratinocytes will provide useful information in understanding the pathogenesis of dermatomycosis.

For the direct detection of dermatophytes in skin scrapings and hairs from animals, a primer pair specific to the chitin synthase 1 (CHS1) gene of dermatophytes was constructed. By PCR analysis with the primer pair, dermatophyte DNA could be diagnosed directly and rapidly in clinical skin samples.

A nonsporulating isolate from a dog with dermatophytosis was identified as Trichophyton rubrum by molecular analysis. The nucleotide sequence analysis of the chitin synthase 1 (CHS1) gene from the isolate indicated more than 99% sequence similarity with other human and canine isolates of T rubrum. The molecular typing suggested that isolates of T. rubrum from human and canine sources were genetically identical.

The genus Malassezia has been revised to include six lipophilic species and one nonlipophilic species. These Malassezia species have been investigated to differentiate their morphological and physiological characteristics. However, assimilation of amino acids as a nitrogen source by these species was not well elucidated. In the present study, isolates of Malassezia species were examined with a glycine medium (containing 7-266 mmol glycine, 7.4 mmol KH(2)PO(4), 4.1 mmol MgSO(4)7H(2)O, 29.6 mmol thiamine, 0.5% Tween-80 and 2% agar) and a modified Dixon glycine medium (0.6% peptone, 3.6% malt extract, 2% ox-bile, 1% Tween-40, 0.2% glycerol, 0.2% oleic acid, 7 mmol glycine and 2% agar). All M. furfur isolates developed on the glycine medium, assimilating glycine at concentrations of at least 7 mmol l(-1). However, the other six Malassezia species were unable to grow on the glycine medium. Also, many colonies of M. furfur grew rapidly, within 2-3 days on the modified Dixon glycine medium, although the other six species showed slow and poor development. From these results, it was suggested that M. furfur might be able to utilize glycine as a single nitrogen source, which the other Malassezia species could not. Therefore, glycine medium was recommended for the differentiation of M. furfur from other species of Malassezia.

In Japan, several isolates of Arthroderma benhamiae, a teleomorphic member of Trichophyton mentagrophytes complex, which were not found by earlier mating studies, have recently been recovered from human and animal dermatophytoses. In the present study, intraspecies polymorphism of A. benhamiae isolated in Japan was investigated using restriction fragment length polymorphisms (RFLP) of the non-transcribed spacer (NTS) region of the ribosomal DNA (rDNA), a method introduced to detect intraspecies polymorphisms of other dermatophyte species, such as T. rubrum. Based on their restriction profiles, there were five DNA types out of eight strains of A. benhamiae isolated in Japan. None of the five DNA types were found among the registered tester strains of A. benhamiae. Therefore, several different strains of A. benhamiae may have been brought into Japan separately.

Molecular investigation of 110 clinical isolates of non-lipid-dependent Malassezia pachydermatis from dogs and cats was carried out by random amplification of polymorphic DNA (RAPD) and chitin synthase 2 (CHS2) gene sequence analyses. The RAPD analysis indicated that the clinical isolates of M. pachydermatis constituted four distinct genetic types (A, B, C and D). Moreover, the results from CHS2 gene analysis completely agreed with those from the RAPD analyses. The clinical isolates of M. pachydermatis were obtained from normal external ears, lesions of atopic dermatitis, flea allergic dermatitis, otitis externa, pyoderma and seborrheic dermatitidis in dogs and cats. Type A consisted of 93 clinical isolates as well as the ex-neotype strain of M. pachydermatis. The isolates of type A M. pachydermatis originated from lesions of all kinds of diseases. They were predominant on dog and cat skin. The other types, B, C, and D were isolated mainly from otitis externa.

The isolate from the rabbit with dermatophytosis which was transmitted to the owners was proved to be Arthroderma benhamiae (-) by mating experiments as well as by chitin synthase 1 (CHSI) gene analysis.

We performed a cluster analysis of human and animal pathogenic Microsporum species and their teleomorphic states, Arthroderma species, including A. otae-related species (M. canis, M. audouinii, M. distortum, M. equinum, M. langeronii, and M. ferrugineum) and M. gypseum complex (A. fulvum, A. gypseum, and A. incurvatum) using DNA sequences of nuclear ribosomal internal transcribed spacer 1 (ITS1). The dendrogram showed the members of A. otae-related species to be monophyletic and to construct an extremely closely related cluster with a long horizontal branch. This ITS1-homologous group of A. otae was organized in 6 unique genotypes, while sequences of the members of the ITS1-homologous group of M. gypseum complex are more diverse. This ITS1-based database of Microsporum species and their teleomorphic states will provide a useful and reliable species identification system: it is time-saving (takes two to three days), accurate and applicable even to strains with atypical morphological features or in a non-culturable state.

In the present study, to confirm Microsporum canis infection rapidly, we detected the chitin synthase gene 1 (CHS1) gene of M. canis in the hair and skin samples of four dogs with dermatophytosis. Amplification of the DNAs in the four samples with CHS1 primers yielded fragments of about 620-bp. Nucleotide sequence analysis of the CHS1 gene fragments from samples and a reference strain of M. canis gene showed more than 99% similarity. The method presented in this study can rapidly detect the DNA of M. canis in skin scrapings, and we anticipate that it will be a useful microbiological tool for the diagnosis of M. canis infections in animals and humans.

Molecular characterization of 18 species belonging to the genus Microsporum isolated from human and animal dermatophytosis was carried out by random amplification of polymorphic DNA (RAPD) and Southern hybridization analyses. All of these 18 Microsporum species showed different band patterns specific to each species in RAPD analysis. Southern hybridization analysis using C3 probe derived from a RAPD band from Arthroderma otae, teleomorph of M. canis, revealed distinct bands in 14 of the 18 Microsporum species but not in the other four species. The sizes of the hybridized bands of the 14 species were specific to each species, except three species (A. otae, M. equinum and M. ferrugineum) showing bands of the same size. These findings indicate that the RAPD and the Southern hybridization analyses could be effective to differentiate the species of Microsporum.

Clinical isolates of Microsporum canis and M. gypseum from humans, dogs and cats were examined by random amplification of polymorphic DNA (RAPD) and Southern hybridization analyses. The RAPD band patterns of six clinical isolates of M. canis were identical to those of standard strains of Arthroderma otae. Of nine clinical isolates of M. gypseum seven and two isolates showed RAPD patterns identical to those of standard strains of A. gypseum and A. incurvatum respectively. Southern blot analysis using a probe (C3) obtained from A. otae DNA revealed that six clinical isolates of M. canis showed specific bands identical to those detected in the standard strains of A. otae. Of nine clinical isolates of M. gypseum, seven and two isolates showed bands hybridized by the C3 probe identical to those detected in A. gypseum and A. incurvatum respectively. Furthermore, the results from mating experiments on these nine clinical isolates of M. gypseum showed complete agreement with the results from RAPD and Southern hybridization analyses. These findings clearly indicate that RAPD and Southern hybridization analyses are very useful in the identification of clinical isolates of M. canis and M. gypseum.

A clinical isolate of Trichophyton mentagrophytes from rabbit was examined by polymerase chain reaction (PCR) analysis and a mating experiment. The species-specific primers designed from the nucleotide sequences of the chitin synthase 1 (CHS1) gene in the teleomorph of Arthroderma benhamiae amplified a fragment from genomic DNA samples of A. benhamiae and the clinical isolate but not from those of A. simii and A. vanbreuseghemii. On the other hand, the species-specific primers of A. simii and A. vanbreuseghemii did not amplify any fragment from the genomic DNA of the clinical isolates. When the isolate was respectively crossed with (+) or (-) tester strains of A. benhamiae, A. simii and A. vanbreuseghemii, ascospores were produced in the crossing with the A. benhamiae (+) strain. Therefore, the isolate was identified to be A. benhamiae (-), confirming the result of molecular analysis. This is the first report on the isolation of A. benhamiae in Japan.