Successful Treatment of Sino-Orbital Aspergillus udagawae Infection Using Oral Posaconazole in a Cat.

A 10 yr old spayed female ragdoll cat presented with sudden onset of sneezing, nasal discharge, and stertor. There was no improvement in clinical signs despite treatment with antibiotics, feline interferon, and nebulization. A computed tomography (CT) scan revealed findings consistent with chronic rhinitis, and a tissue biopsy obtained by rhinoscopy led to a histopathologic diagnosis of sinonasal aspergillosis. Polymerase chain reaction amplification identified the causative agent as Aspergillus udagawae. Oral itraconazole therapy was initiated. However, the cat's clinical signs progressed to include left exophthalmos, nictitating membrane protrusion, and lacrimation. A second CT scan revealed a soft-tissue attenuating structure extending into the left retrobulbar space, confirming progression to sino-orbital aspergillosis (SOA). The oral medication was changed to posaconazole and continued for 5 mo, resulting in resolution of the clinical signs. The cat has remained asymptomatic over 24 mo since initial diagnosis. This case represents the first successful treatment of feline SOA caused by A udagawae infection with posaconazole. A udagawae is the second most common cause of SOA and is known to be intractable because of its low susceptibility to antifungal agents and poor response to topical clotrimazole. Posaconazole may be a valuable treatment option for SOA caused by A udagawae.

Viability of pathogenic dermatophytes during a 4-week treatment with 1% topical luliconazole for tinea pedis.

The viability of pathogenic fungi in the scale was investigated during topical administration of 1% luliconazole (LLCZ). Thirteen tinea pedis patients found to be positive on KOH examination were assessed by mycological examinations and quantitative real-time polymerase chain reaction (PCR) targeted internal transcribed spacer (ITS) in ribosomal RNA gene at the initial visit and after 2 and 4 weeks of treatment. Assays showed that the average copy number of ITS DNA had significantly decreased to 22.9% at 2 weeks and 4.8% at 4 weeks compared with the initial visit. LLCZ topical treatment could defeat almost pathogenic dermatophytes in the scales within 4 weeks.

Variations in ribosomal DNA copy numbers in a genome of Trichophyton interdigitale.

BACKGROUND: Ribosomal DNA (rDNA) reportedly has multiple copies in the fungal genome. The internal transcribed spacer (ITS) region in rDNA is useful for investigating relationships between close taxonomic relatives. Thus, ITS has been widely used as a target gene in medical mycology for the detection of pathogenic fungi and identification of fungal species. However, the rDNA copy number in a genome of Trichophyton interdigitale, the pathogen causing dermatophytosis, currently remains unknown. OBJECTIVE: Clarification of the rDNA copy number in a genome of T. interdigitale. METHODS: rDNA copy numbers in 64 clinical isolates of T. interdigitale were examined using quantitative real-time PCR (qPCR) with the absolute quantitative method targeting TruMDR2, a single-copy control gene and the ITS region in rDNA. RESULTS: The copy numbers of the rDNA subunit varied among the 64 strains tested, from 24 to 116 copies per genome. The average rDNA copy number ± standard deviation was 59 ± 16. No correlations were observed between the rDNA copy number and colony colour, colony morphology or molecular type of the non-transcribed spacer region in rDNA. Experiments on rDNA copy numbers obtained from independent colonies of each strain in single-spore cultures revealed that the copy number was homogeneous within each strain. CONCLUSION: This is the first study to estimate copy numbers of the rDNA subunit in a genome of T. interdigitale. The rDNA copy number of T. interdigitale varied among the strains tested and was homogeneous within each strain.

A Case of Cutaneous Mycosis Caused by Scedosporium dehoogii on an Immunocompromised Patient.

This report describes a 77-year-old man with cutaneous mycosis caused by Scedosporium dehoogii while taking oral betamethasone and tacrolimus for the treatment of rheumatoid arthritis. At examination in our clinic, the patient had multiple cystic lesions and nodules with slight tenderness, varying in size up to 4 cm, on his left knee and shin. He had not noticed any traumatic injury at the site of the lesions. Fungal cultures of samples taken from the abscesses, scales, and crusts of the lesions yielded white, later grayish brown, fluffy surfaced colonies. Partial sequencing of the ß-tubulin gene confirmed the species of the isolate. The patient was initially treated with oral voriconazole and local hyperthermia, but experienced hepatic injury 2 weeks later. His treatment was changed to itraconazole (ITC) and local hyperthermia, followed by a combination of ITC and terbinafine. The patient recovered completely during the 12-month course of treatment.

Concurrent Double Fungal Infections of the Skin Caused by Phialemoniopsis endophytica and Exophiala jeanselmei in a Patient with Microscopic Polyangiitis.

is missing (Short communication).

High prevalence of superficial white onychomycosis by Trichophyton interdigitale in a Japanese nursing home with a geriatric hospital.

A mycological survey on feet was performed in a nursing home with a geriatric hospital to ascertain the prevalence of tinea lesions. Of 100 subjects, comprising 62 in the nursing home and 38 in the geriatric wing, 70 were diagnosed with tinea pedis, tinea unguium (onychomycosis) or both of which 54 had onychomycosis alone, nine tinea pedis alone and seven had co-existing onychomycosis and tinea pedis. The most common clinical type of onychomycosis was distal lateral subungual onychomycosis (DLSO) at 30 cases, followed by superficial white onychomycosis (SWO) at 23 cases. Fifteen strains of Trichophyton (T.) interdigitale isolated from 23 SWO patients comprised six molecular types (D2II, nine cases; C2II, two cases; four other types, one case of each), based on the non-transcribed spacer region (NTS) of the ribosomal DNA. The pathogen of three other SWO cases was identified as T. rubrum. Direct physical contact between the subjects was unlikely because they were bedridden most of the time. Nine T. interdigitale strains were isolated from a bathtub used by patients on the floor with a high incidence of SWO alone, and all nine strains were D2II type, which suggests nosocomial infection. Consequently, the hospital infection control policy committee was consulted, bathing arrangements were changed, and nursing staff were educated about onychomycosis.

Molecular Markers Useful for Intraspecies Subtyping and Strain Differentiation of Dermatophytes.

Dermatophytosis is a very common skin disorder and the most frequent infection encountered by practicing dermatologists. The identification, pathogenicity, biology, and epidemiology of dermatophytes, the causative agents of dermatophytosis, are of interest for both dermatologists and medical mycologists. Recent advances in molecular methods have provided new techniques for identifying dermatophytes, including intraspecies variations. Intraspecies subtyping and strain differentiation have made possible the tracking of infections, the identification of common sources of infections, recurrence or reinfection after treatment, and analysis of strain virulence and drug resistance. This review describes molecular methods of intraspecies subtyping and strain differentiation, including analyses of mitochondrial DNA and non-transcribed spacer regions of ribosomal RNA genes, random amplification of polymorphic DNA, and microsatellite markers, along with their advantages and limitations.

Pathogenic Dermatophytes Survive in Nail Lesions During Oral Terbinafine Treatment for Tinea Unguium.

Tinea unguium caused by dermatophyte species are usually treated with oral antimycotic, terbinafine (TBF). To understand the mechanisms of improvement and recalcitrance of tinea unguium by oral TBF treatment, a method of quantifying dermatophyte viability in the nail was developed, and the viability of dermatophytes was analyzed in toenail lesions of 14 patients with KOH-positive tinea unguium treated with oral TBF 125 mg/day for up to 16 weeks. Mycological tests, including KOH examination and fungal culture, and targeted quantitative real-time PCR for internal transcribed spacer (ITS) region, including rRNA, were demonstrated at the initial visit and after 8 and 16 weeks of treatment. Assays in eight patients showed that average ITS DNA amount significantly decreased, to 44% at 8 weeks and 36% at 16 weeks compared with 100% at initial visit. No significant difference was observed between at 8 and 16 weeks, despite the TBF concentration in the nail supposedly more than 10-fold higher than the minimum fungicidal concentration for dermatophytes. This finding suggests the pathogenic dermatophytes in nail lesions could survive in a dormant form, such as arthroconidia, during oral TBF treatment. Both antimycotic activity and nail growth are important factors in treatment of tinea unguium.

Glycyrrhetinic acid inhibits contact hypersensitivity induced by trichophytin via dectin-1.

Trichophyton infection is highly prevalent and tends to be recurrent. Therefore, it is important to develop new therapeutic agents. Previously, we established a mouse model of Trichophyton-induced contact hypersensitivity (CHS) and demonstrated that dectin-1 was involved in inflammation induced by trichophytin, the Trichophyton antigen. Here, we used that model to investigate glycyrrhetinic acid (GA) from plants of the genus Glycyrrhiza as a potential anti-inflammatory agent against superficial mycoses. GA suppressed swelling and the expression of inflammatory cytokines, including macrophage inflammatory protein (MIP)-2, interleukin (IL)-6, tumor necrosis factor (TNF)-α and interferon (IFN)-γ mRNA. Anti-MIP-2 antibody suppressed trichophytin-induced inflammation, and antidectin-1 antibody suppressed zymosan-induced MIP-2 production in keratinocyte cells. These results suggest that MIP-2 is produced by dectin-1 activation and is involved in inflammation associated with CHS to trichophytin. GA also suppressed zymosan-induced MIP-2 and interleukin (IL)-8, production in mouse and human macrophages and keratinocytes. Furthermore, GA suppressed the phosphorylation of spleen tyrosine kinase (Syk) and inhibitor of nuclear factor-kappa B (IκBα) and the degradation of IκBα in zymosan-simulated RAW264.7 cells. The results of this study suggest that GA suppresses inflammation induced by trichophytin, partly by the downregulation of Syk phosphorylation.

A critical role of Dectin-1 in hypersensitivity pneumonitis.

OBJECTIVES AND DESIGN: Hypersensitivity pneumonitis (HP) is a pulmonary disease caused by repeated exposure to various aspiration antigens, including bacteria and fungi. Although TLRs are known to be required for the generation of HP triggered by bacteria, the significance of fungal receptors remains unclear. The present study aimed to investigate whether Dectin-1 and Dectin-2 contribute to the development of experimental HP triggered by the fungus Trichosporon asahii (T. asahii) that causes summer-type HP. MATERIALS AND METHODS: We investigated the binding between Dectin-Fc protein and T. asahii by a dot blot assay. We performed the histological and flow cytometric analysis in the HP model using Dectin-1-deficient (Dectin-1(-/-)) and Dectin-2(-/-) mice. We also investigated Th17/Th1 responses in lung cells, and measured an IL-17-promoting cytokine IL-23 from bone marrow-derived dendritic cells (BMDCs) by ELISA. RESULTS: Dectin-1 bound more strongly to T. asahii than Dectin-2. Dectin-1(-/-) mice barely developed HP, whereas both wild-type mice and Dectin-2(-/-) mice developed similar lung diseases. Dectin-1 deficiency decreased the infiltration of neutrophils and monocyte-derived macrophages and repressed the expansion of lung CD4(+)IL-17A(+) cells. The production of IL-23 p19 was reduced in Dectin-1(-/-) BMDCs. CONCLUSIONS: These data suggested Dectin-1 plays a critical role in the development of fungus-induced HP.

Case of dermatophyte abscess caused by Trichophyton rubrum: a case report and review of the literature.

A 54-year-old Japanese man without apparent immunosuppression presented with nodules with purulent drainage on the right lower leg. He had ringworm of the right leg and tinea unguium. A biopsy specimen of the nodule showed intradermal abscesses with fungal elements, and Trichophyton rubrum was cultured from both the pus and the biopsy specimen. Treatment with oral terbinafine resolved the nodules. Dermatophyte abscess is a rare, deep and invasive dermatophytosis, which is often associated with immunocompromised conditions. We provide a review of the literature including Japanese cases.

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.

Quantification of dermatophyte viability for evaluation of antifungal effect by quantitative PCR.

Dermatophytosis is a common disease caused by dermatophyte fungi such as Trichophyton rubrum and Trichophyton mentagrophytes. A method of quantifying fungal viability in the lesions of dermatophytosis is indispensable for understanding the therapeutic process and outcome; however, no such method has yet been developed. The aim of this study was to develop a method for quantifying dermatophyte viability by quantitative polymerase chain reaction (qPCR). The internal transcribed spacer (ITS) and D1/D2 regions, including each of rRNA and rDNA, were chosen as the targets, and dermatophyte-specific primer pairs were designed corresponding to ITS and D1/D2 regions. The amounts of target RNA and DNA after heat or antifungal treatment were measured by qPCR and compared with colony-forming unit (CFU) counts. RNA and DNA could extract from dermatophytes by mechanical pulverization of conidia using a Multi-Beads Shocker cell disruptor. Our method was sufficiently sensitive to detect 10 copies by qPCR using both ITS and D1/D2 primer pairs. The most sensitive target was ITS-cDNA after heat or antifungal treatment, and essentially consistent with CFU counts. On the other hands, ITS-DNA and D1/D2-DNA were not decreased soon after heat or antifungal treatment, but those were decreased significantly and reflected the CFU counts after 48 h of antifungal treatment. We conclude that ITS-cDNA is useful mainly for quantifying dermatophyte viability at early responses, but ITS-DNA and D1/D2-DNA are also available for evaluation, which does not need an early response.

Mating type gene (MAT1-2) of Trichophyton verrucosum.

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.

Detection of varicella-zoster virus in two dermatomes of herpes zoster duplex bilateralis in an immunocompetent host.

An 85-year-old woman with no history of herpes zoster (HZ) presented with a primary lesion of erythema and blistering on her left thigh and a secondary similar lesion on her right chest which had appeared at 4 and 3 days before presentation, respectively. Tzanck smears for both lesions were positive, revealing multinucleated giant cells. Immunochromatography to detect varicella-zoster virus (VZV) antigen (DermaQuick®VZV) showed positive on the left thigh at initial onset but negative on the right chest at subsequent onset. The latter repeatedly tested negative for VZV by DermaQuick®VZV. A skin biopsy of the subsequent onset area revealed giant cells, and inclusion bodies were observed in the epidermis. Immunohistochemical staining with anti-VZV antibody and polymerase chain reaction to detect VZV DNA were positive. The patient was diagnosed with HZ duplex bilateralis and treated with acyclovir. The right thoracic region of the posterior part of the lesion became negative for DermaQuick®VZV. It is thought that expression of viral antigens was suppressed in the right thoracic region, i.e., the late-onset area.

Vasculitis-like herpes zoster in the course of treatment with tofacitinib in ulcerative colitis: An assessment of local viral distribution by RNA in situ hybridization.

A 67-year-old man had taken the janus kinase (JAK) inhibitor, tofacitinib, for ulcerative colitis. He was referred to our department for a refractory ulcer on his lower leg. We suspected vasculitis and performed skin biopsy. Histopathological examination showed multinucleated giant cells in the epidermis and fibrinoid degeneration of small vessels in the upper dermis. Varicella zoster virus (VZV) DNA was detected by polymerase chain reaction and we diagnosed the patient with atypical vasculitis-like herpes zoster. The patient was treated with oral valacyclovir, but the rash persisted and took 2 months to heal. Immunostaining using anti-VZV antibody was positive mainly in epidermal keratinocytes, but was also observed to be positive in cells in the dermis. We further performed RNA in situ hybridization using a VZV ORF9 mRNA probe and clearly showed that the distribution of VZV mRNA extended into the dermis, including the dermal vessel walls and the eccrine sweat glands as well as the epidermis. The internal administration of JAK inhibitors may induce regional widespread VZV infection including vessels and involved in the formation of prolonged vasculitis-like manifestation. RNA in situ hybridization can be a potent tool for detecting the spread of VZV infection in the skin.

Case of tinea corporis caused by a terbinafine-sensitive Trichophyton indotineae strain in a Vietnamese worker in Japan.

A 42-year-old Vietnamese egg factory worker in Ishikawa prefecture, Japan, presented with itchy concentric erythema on the trunk and left calf. The lesions tested positive by direct potassium hydroxide examination, and two fungal strains were isolated. The isolates produced conidia abundantly and were morphologically indistinguishable from Trichophyton mentagrophytes/interdigitale, but were identified as Trichophyton indotineae by internal transcribed spacer sequence of ribosomal DNA. The lesions were refractory to treatment with topical luliconazole (LLCZ) cream for 4 weeks but subsided with oral itraconazole (ITCZ) 100 mg/day for 4 weeks in combination with topical lanoconazole (LCZ) cream. The lesions recurred 6 weeks after discontinuation of oral ITCZ, and an additional isolate was cultured. The minimum inhibitory concentrations of antimycotics for the isolate cultured at the first visit were: terbinafine (TBF) 0.03 µg/mL, ITCZ 0.015 µg/mL, LLCZ 0.0005 µg/mL, and LCZ 0.002 µg/mL. No TBF-resistant mutation in the amino acid sequence of squalene epoxidase, i.e., Leu 393 Ser/Phe or Phe 397 Leu, was detected in the isolate. The reason for recalcitrance in this case, despite the isolate's sensitivity to antimycotics, was unclear. Possible factors include insufficient use of the antimycotics, incomplete removal of abundantly produced conidia from the lesions, the patient's environment, and a language gap between the patient and physician hindering communication.