A. Hasegawa

Case report. First report on human ringworm caused by Arthroderma benhamiae in Japan transmitted from a rabbit

Summary. Two human cases of tinea corporis due to Arthroderma benhamiae (teleomorph of Trichophyton mentagrophytes) were described. They acquired the infection from their cross‐bred rabbit. The three clinical isolates from a human couple and a pet rabbit had been identified as A. benhamiae by chitin synthase 1 (CHS1) gene analysis as well as by mating experiments. There was no previous isolate of A. benhamiae from humans in Japan, although we had reported the first isolate of A. benhamiae from a rabbit in 1998. Therefore, this is the first report on human ringworm cases caused by A. benhamiae in Japan. It is anticipated that the human and animal cases of A. benhamiae infection could rise in number.

Clinically significant micafungin resistance in Candida albicans involves modification of a glucan synthase catalytic subunit GSC1 (FKS1) allele followed by loss of heterozygosity

OBJECTIVES To determine the mechanism of intermediate- and high-level echinocandin resistance, resulting from heterozygous and homozygous mutations in GSC1 (FKS1), in both laboratory-generated and clinical isolates of Candida albicans. METHODS The DNA sequences of the entire open reading frames of GSC1, GSL1 (FKS3) and RHO1, which may contribute to the beta-1,3-glucan synthase of a micafungin-susceptible strain and a resistant clinical isolate, were compared. A spontaneous heterozygous mutant isolated by selection for micafungin resistance, and a panel of laboratory-generated homozygous and heterozygous mutants that possessed combinations of the echinocandin-susceptible and -resistant alleles, or mutants with individual GSC1 alleles deleted, were used to compare levels of echinocandin resistance and inhibition of glucan synthase activity. RESULTS DNA sequence analysis identified a mutation, S645P, in both alleles of GSC1 from the clinical isolate. GSL1 had two homozygous amino acid changes and five non-synonymous nucleotide polymorphisms due to allelic variation. The predicted amino acid sequence of Rho1p was conserved between strains. Reconstruction of the heterozygous (S645/S645F) and homozygous (S645F/S645F) mutation showed that the homozygous mutation conferred a higher level of micafungin resistance (4 mg/L) than the heterozygous mutation (1 mg/L). Exposure of the heterozygous mutant to micafungin resulted in a loss of heterozygosity. Kinetic analysis of beta-1,3-glucan synthase activity showed that the homozygous and heterozygous mutations gave echinocandin susceptibility profiles that correlated with their MIC values. CONCLUSIONS A homozygous hot-spot mutation in GSC1, caused by mutation in one allele and then loss of heterozygosity, is required for high-level echinocandin resistance in C. albicans. Both alleles of GSC1 contribute equally and independently to beta-1,3-glucan synthase activity.

Effects of staphylococci on cytokine production from human keratinocytes

Background  Staphylococcus skin infection is characterized by the infiltration of numerous neutrophils within the epidermis; however, the precise mechanism of epidermal infiltration of neutrophils during skin infection with staphylococci is not well understood and the factors regulating the neutrophil recruitment are yet to be determined.

Identification of Sporothrix schenckii based on sequences of the chitin synthase 1 gene

Summary. Sporothrix schenckii is pathogenic to human and animals. To detect S. schenckii in the tissue, we designed specific oligonucleotide primers based on the chitin synthase 1 gene. Amplification products were selectively obtained from S. schenckii DNA. Polymerase chain reaction analysis with the primer pair S2–R2 was able to detect 10 pg genomic DNA of S. schenckii with ethidium bromide staining. This detection system will be useful as a microbiological tool for the diagnosis of human and animal sporotrichosis.

A new genotype of Arthroderma benhamiae

Nucleotide sequence analysis of chitin synthase 1 (CHS1) indicated 90% sequence similarities among human and animal isolates of Arthroderma benhamiae. In particular, greater than 99% similarity was noted in the nucleotide sequence among Americano-European race isolates, African race isolates, and five isolates from four-toed hedgehogs (Atelerix albiventris). The phylogenetic analysis indicated that the five isolates from hedgehogs were included in the same cluster and distinct from the Americano-European and the African race standard strains of A. benhamiae. These results were confirmed by ITS analysis. Therefore, it was proposed that genotypes could be established for the isolates of A. benhamiae in association with the mating behavior.

Plasma thrombin- antithrombin complex concentrations in dogs with malignant tumours

IT has been suggested that coagulation abnormalities such as disseminated intravascular coagulation (DIC) might be caused by the excessive activation of coagulation in dogs with malignant tumours (O’Keefe and Couto 1988). Since excessive activation of coagulation leads to over-generation of thrombin, increased concentrations of thrombin measured in circulating blood would indicate an activation of coagulation. Thrombin-antithrombin complexes (TATs), which are formed rapidly after thrombin production, have been identified as a marker of coagulation activation (Pelzer and others 1988). The plasma TAT concentration has been reported to be useful for evaluating the activation of coagulation in dogs (Ravanat and others 1995), and is available as a marker of the hypercoagulable state in dogs with Cushing’s syndrome (Jacoby and others 2001). However, the plasma TAT concentrations in dogs with malignant tumours have not been reported. This short communication describes the plasma TAT concentrations in dogs with benign or malignant tumours and the incidence of a hypercoagulable state in dogs with malignant tumours. The plasma TAT concentrations of three groups of dogs were examined. Group 1 comprised 16 clinically healthy adult dogs; the dogs were considered clinically normal on the basis of physical examination, routine haematological examination and serum biochemical analysis. Group 2 comprised 11 dogs with benign tumours: five with an adenoma, three with a leiomyoma, two with a haemangioma and one with a lipoma. Group 3 consisted of 62 dogs with malignant tumours, this group was further divided into four subgroups: 27 dogs with epithelial tumours, 17 with mesenchymal tumours except haemangiosarcoma; 10 with haemangiosarcomas and eight with haematopoietic tumours. The dogs in groups 2 and 3 were referred to the Animal Medical Center of Nihon University and their tumours were diagnosed by histopathological examination. None of the dogs received any anticoagulants or blood products before blood sampling. Blood samples were collected into tubes containing 0·13M trisodium citrate (nine parts blood to one part anticoagulant) and centrifuged at 2000 g for 10 minutes, and the citrated plasma was frozen at –30°C until analysis. The plasma TAT concentrations were measured by enzyme immunoassay (TAT Test Kokusai-F; International Reagents Corporation). Mann-Whitney U tests were used to compare plasma TAT concentrations between the groups. For statistical analysis, plasma TAT concentrations undetectable by the assay method used (<0·4 ng/ml) were regarded as 0·4 ng/ml. To detect the incidence of a hypercoagulable state, a reference range of plasma TAT concentration was established as the mean (2sd) concentration obtained from group 1. Dogs with plasma TAT concentrations above the reference range were regarded as displaying a hypercoagulable state. The median (range) plasma TAT concentrations were 0·5 (<0·4 to 0·6) ng/ml in group 1, 0·4 (<0·4 to 6·3) ng/ml in group 2 and 1·3 (0·4 to 49·3) ng/ml in group 3; concentrations were significantly higher in group 3 than in group 1 (P<0·0001). However, there was no significant difference in the plasma TAT concentrations of groups 1 and 2 (P=0·8823) (Fig 1). Within group 3, the TAT concentrations were 0·8 (0·4 to 3·8) ng/ml for the dogs with epithelial tumours, 0·7 (0·4 to 49·3) ng/ml for those with mesenchymal tumours, 17·3 (4·4 to 42·5) ng/ml for those with haemangiosarcomas, and 8·2 (2·1 to 19·0) ng/ml for those with haematopoietic tumours. The plasma TAT concentrations were significantly elevated in all subgroups of group 3 (P<0·001, P<0·01, P<0·0001 and P<0·0001, respectively) compared with group 1 (Fig 2). The mean (2sd) plasma TAT concentration of dogs in group 1 was 0·49 (0·12) ng/ml, and thus dogs with plasma TAT concentrations in excess of 0·61 ng/ml were considered as disVeterinary Record (2005) 156, 839-840

Molecular diagnosis of feline sporotrichosis

SPOROTRICHOSIS is a chronic infection caused by Sporothrix schenckii, and usually presents as a fixed cutaneous or lymphocutaneous infection in animals (Kwon-Chung and Benett 1992, Rosser and Dunstan 1998). Since a number of diseases can resemble sporotrichosis quite closely (KwonChung and Bennett 1992), it should be diagnosed definitively on the basis of histopathological and mycological findings. Direct microscopic examination is rapid, but can be quite insensitive and is reliant on the operator’s skills. Kano and others (2003) developed a PCR method to detect S schenckii directly in biopsy samples from human patients with sporotrichosis using specific oligonucleotide primers based on the chitin synthase 1 (CHS1) gene of S schenckii (Kano and others 2001). The primer pair (S2 and R2) did not amplify DNAs from other pathogenic fungi (Aspergillus fumigatus, Blastomyces dermatitidis, Candida species, Cryptococcus neoformans, dermatophytes, Malassezia species and Histoplasma capsulatum), bacteria (Staphylococcus aureus), normal human skin cells or normal feline skin cells (Kano and others 2001). The sensitivity of the PCR was equivalent to the detection of 10 pg of S schenckii genomic DNA (Kano and others 2001). This short communication describes a study to identify S schenckii DNA directly from biopsy specimens obtained from a case of feline sporotrichosis. A 23-month-old male crossbred cat, weighing 5·4 kg, was referred to the Gifu University Animal Medical Center with abscesses and a draining puncture wound on the left foreleg and right hindleg which had been present for seven months. The lesions had been drained surgically and treated with chitin and chitosan. A subcutaneous nodule, 18 x 6 x 15 mm in size, was found on the right hindleg. Histopathological analysis of a biopsy of the nodule revealed granulomatous inflammation and many globular and ovoid budding yeast cells (Fig 1), with no asteroid bodies. Therefore, molecular diagnosis of sporotrichosis was performed. DNA was extracted from the biopsy. The tissue sample (approximately 100 mg) was digested at 37°C for 16 hours with 100 μg of proteinase K in 400 μl of a lysis buffer containing 0·1mM Tris-HCl (pH 8·0) and 0·3 per cent 2-mercaptoethanol. The DNA was obtained by phenol and chloroform extraction and then dissolved in Tris-EDTA buffer (10mM Tris-HCl [pH 8·0] and 1mM EDTA) and used directly for PCR amplification. A sample (200 ng) of DNA was amplified by PCR in a 30 μl reaction mixture containing 10mM Tris-HCl (pH 8·3), 50mM potassium chloride, 1·5mM magnesium chloride, 0·001 per cent gelatin, 200μM of each deoxynucleoside triphosphate, 1·0 U Taq polymerase (Takara) and 0·5 μg of the primer pair. To detect S schenckii rapidly, species-specific primers for PCR based on its CHS1 gene sequence were used (Kano and others 2001, 2003). The sequence of the forward primer (S2) was 5′-TGGGCGTCTACCAAGAGGGTATTGC-3′ (nucleotides 173 to 197 of the S schenckii CHS1 gene) (GenBank accession number L24908) and the sequence of the reverse primer (R2) was 5′-GCACATGGGCTCAAGATCAAAGGCC-3′ (nucleotides 468 to 492 of the S schenckii CHS1 gene). With these primers, a 318 base pair (bp) fragment of the CHS1 gene was expected to be amplified. The PCR amplification was carried out for 40 cycles consisting of template denaturation (one minute at 94°C), primer annealing (two minutes at 68°C) and polymerisation (two minutes at 72°C). Then, 10 μl of PCR products were separated in a 2 per cent (w/v) agarose gel, stained with ethidium bromide and visualised with ultraviolet light. The S2-R2 primer pair amplified approximately 320 bp fragments of the DNA from the clinical specimen and a control sample of S schenckii DNA (Fig 2). The primer pair did not amplify 320 bp fragments from normal feline skin DNA (Fig 2). Therefore, the case was diagnosed molecularly as sporotrichosis. The biopsy material from the nodule on the right hindleg was cultured on sunflower seed agar at 27°C for one week and then examined morphologically. The clinical isolate developed a black colony with a grey aerial surface after incubation for two weeks on sunflower seed agar. Microscopic examination of the isolate revealed one-cell, dark brown, thick-walled, subglobular, elliptical to cylindrical, smooth and hyaline conidia, which were 2 to 3 x 3 to 6 μm in size, produced on hyphae and in a bouquet at the tip of conidiophores. The conidiophores were slender and tapered from 1 to 2 μm diameter at the base to 0·5 to 1 μm at the tip. Since these findings were consistent with the description of S schenckii by Kwon-Chung and Bennett (1992), the isolate was identified as S schenckii. The cat was treated with itraconazole (Itrizole; Janssen Pharmaceutical) administered orally at a dose of 15 mg/kg once a day. After two months of treatment, the nodule had healed and the itraconazole was discontinued. The cat was in Veterinary Record (2005) 156, 484-485

Chitin synthase 1 gene of Arthroderma benhamiae isolates in Japan

Summary.  In the present study, the chitin synthase 1 (CHS1) gene of eight clinical isolates of Arthroderma benhamiae in Japan was investigated. Nucleotide sequence analysis of the CHS1 gene fragments from clinical isolates of A. benhamiae and from standard strains of Americano‐European race and African race A. benhamiae indicated more than 90% similarities among these dermatophytes. An especially high degree of similarity was noted in nucleotide sequence CHS1 gene fragments, with more than 99% among eight clinical isolates and standard strains of the Americano‐European race of A. benhamiae. The phylogenetic analysis of their sequences revealed that the eight clinical isolates and the standard strains of the Americano‐European race of A. benhamiae were included in the same cluster, and that the African race of A. benhamiae formed a cluster that was distinct from the Americano‐European race of A. benhamiae, A. simii and A. vanbreuseghemii.

Susceptibility testing of Malassezia pachydermatis using the urea broth microdilution method

Summary.  The in vitro susceptibility of 24 isolates of Malassezia pachydermatis to four antifungal drugs in combination with lysozyme was determined using a urea broth microdilution method. The antifungal activities of each drug alone against 24 isolates of M. pachydermatis were determined as the mean minimal inhibitory concentrations (MICs). MICs of bifonazole, itraconazole, amorolfine and terbinafine were 3.2 µg ml−1, 1.6 µg ml−1, 25 µg ml−1 and 3.2 µg ml−1, respectively. Lysozyme alone inhibited the growth of M. pachydermatis in a dose‐dependent manner, although the lysozyme was unable to kill the cells of M. pachydermatis at the highest concentration of 20 µg ml−1. Furthermore, the mean MICs of bifonazole, itraconazole, amorolfine and terbinafine in combination with lysozyme were the same as the results for each drug alone. Although the activity of antifungal drugs in combination with lysozyme is enhanced for other fungi. These results suggested that M. pachydermatis might not be affected by the hosts natural defences.

Thiazole orange-positive platelets in healthy and thrombocytopenic dogs

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