Establishing norms on the Japanese version of the Eyberg Child Behavior Inventory.

BACKGROUND: The Eyberg Child Behavior Inventory (ECBI) is one of the standardized parent rating scales used to identify disruptive behavior problems in children in Western countries. This study aimed to determine norms for the Japanese version of the ECBI, including clinical cutoff scores among the general population in Japan. METHODS: This study established norms for the Japanese version of the ECBI using a sample of 1,992 parents of children aged 2-7, living in Japan. The research evaluates the validity and the reliability of the ECBI scores for the Intensity Scale and the Problem Scale. After validation, a clinical cutoff value of the ECBI scores was calculated, setting the cutoff to above the +1 standard deviation (SD) level based on the population distribution. RESULTS: The means of the Intensity and Problem Scale scores were 100.07 and 6.57, respectively. Cronbach's α for both the Intensity and the Problem scores was 0.91. At this point, we propose cutoff scores of 125 for the Intensity Scale and 14 for the Problem Scale. CONCLUSIONS: Our results suggest that the Japanese version of the ECBI is highly reliable and may be useful as a tool for assessing behavior problems in children.

Adrenocortical Scintigraphy With SPECT/CT for the Diagnosis of Hepatic Adrenal Rest.

Hepatic adrenal rest include ectopic adrenal tissue in the liver and adrenohepatic fusion. Long-lasting hyperstimulation of that tissue induces hyperplasia sometimes simulating true tumors. True autonomic tumors originating from ectopic adrenal tissue with or without hyperfunction are rare. Hepatic adrenal rest sometimes has similar imaging findings to lipid-rich hepatocellular carcinoma or angiomyolipoma, containing obvious lipid component. But in our cases, tumors showed hypervascular patterns similar to hepatocellular adenoma or carcinoma. Therefore, adrenocortical scintigraphy with SPECT/CT should be taken into consideration to diagnose tumoral lesions in the posterior segment of the right hepatic lobe with or without an obvious lipid component.

New strain typing method with Sporothrix schenckii using mitochondrial DNA and polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) technique.

The complete sequences of mitochondrial DNA (mtDNA) from two strains of different genotypes, American Type Culture Collection 10268 of mtDNA type 1 and KMU2025 of mtDNA type 4, were determined. These are circular molecules, 27 125 and 26 095 bp in length, respectively. The greatest difference between the two strains was found in the region encompassed by atp9 and cox2 genes, which was amplified with polymerase chain reaction (PCR) and used for preliminary restriction fragment length polymorphism (RFLP) analysis. Eight isolates of five mtDNA types were used and RFLP patterns obtained with the restriction enzyme AseI showed that this method seems to have greater discrimination power than the other PCR-RFLP typing method using internal transcribed spacer regions of nuclear DNA.

Extra-scalp black dot ringworm caused by Trichophyton tonsurans among contact sports players.

We describe here two patients with tinea corporis exhibiting black dot ringworm (BDR). A cluster of black dots was observed on the extensor surfaces of the extremities of two rather hairy male patients, a 15-year-old judo practitioner and a 26-year-old combined martial arts fighter, during treatment of tinea corporis with topical antimycotics. Direct KOH examination showed that the black dots were composed of degenerated hair with numerous arthroconidia and were indistinguishable from BDR of tinea capitis. Trichophyton tonsurans was isolated from the dots of both patients. Although they were diagnosed with tinea corporis, they required 2-3 months of treatment with oral terbinafine. Dermatologists should be aware that BDR can appear on areas of the skin other than the scalp.

Mating genes of the Trichophyton mentagrophytes complex.

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.

Verification of a taxonomy of dermatophytes based on mating results and phylogenetic analyses.

A newly proposed taxonomy of Trichophyton mentagrophytes and related species was introduced and verified with the grounds for the new classification, phylogenetic analysis, and Templeton's cohesive species concept. So-called asexual species were shown to retain sexual ability and different host preferences were shown not to be comparable to different ecological niches. We showed that genealogical concordance phylogenetic species recognition (GCPSR) can be applied to so-called asexual Trichophyton species. The results of GCPSR analysis and mating experiments demonstrated that Arthroderma simii, A. vanbreuseghemii, T. mentagrophytes var. interdigitale, and T. tonsurans may be considered as single phylogenetic species, and that A. benhamiae, T. concentricum, T. verrucosum, and T. mentagrophytes var. erinacei may also be considered as single phylogenetic species. In addition, the conspecificity of three Arthroderma species may also be demonstrated in future. In conclusion, we should not hastily revise the nomenclature based on the phylogenetic tree from only one gene, because different genes can yield different phylogenetic trees. Therefore, we recommend retaining the previous taxonomy until an acceptable taxonomy is proposed.

Genetic relationship between Trichophyton mentagrophytes var. interdigitale and Arthroderma vanbreuseghemii.

Thirty-nine Arthroderma vanbreuseghemii isolates identified by mating behavior were assayed to determine their ribosomal RNA gene internal transcribed spacer (ITS) region genotypes. Twenty-two isolates of mating type (+) and 4 of (-) showed the genotype of Trichophyton mentagrophytes var. interdigitale, 9 isolates of mating type (+) and 4 of (-) showed the genotype of A. vanbreuseghemii. Nine of 14 isolates with granular or asteroid colony morphology showed the genotype of T. mentagrophytes var. interdigitale. There was no relation between ITS genotype and morphology or mating type of the isolates. Isolates of T. mentagrophytes var. interdigitale genotype were shown to include non-sexually degenerate zoophilic strains with granular colony morphology as well as sexually and morphologically degenerate anthropophilic strains and may be just one genotype of a heterogeneous species, A. vanbreuseghemii.

Multiple gene analyses are necessary to understand accurate phylogenetic relationships among Trichophyton species.

Phylogenetic relationships among 34 isolates from 11 Trichophyton and 3 Arthroderma species were investigated using the nucleotide sequences from 4 DNA regions: internal transcribed spacers (ITS) 1 and 2 including the 5.8S rRNA gene, and the actin (ACT), DNA topoisomerase (TOP) 2 and glyceraldehyde-3-phosphate dehydrogenase (GPD) genes. All four phylogenetic trees showed that the 34 isolates can be divided into 3 clades, the Arthroderma simii, A. benhamiae and Trichophyton rubrum clades. The Shimodaira-Hasegawa test (SH test) revealed significant topological incongruities within the A. benhamiae and A. simii clades. Although branching patterns of the 3 clades were inconsistent among the four trees, the SH test did not support these differences except that the best tree topology according to ACT sequences was significantly rejected by the TOP data set. These results show that multiple gene analyses are necessary to more precisely understand the phylogenetic relationships among these fungi.

Arthroderma benhamiae (the teleomorph of Trichophyton mentagrophytes) mating type-specific genes.

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.

Matings among three teleomorphs of Trichophyton mentagrophytes.

Three genetically hybrid F1 progenies produced between a clinical isolate of Arthroderma simii (KMU4810) and a tester strain of A. vanbreuseghemii (RV27961) were crossed with two tester strains of A. vanbreuseghemii (RV27961 and RV27960) and a tester strain of A. benhamiae (RV30001), respectively. Three crossings yielded hybrid second progenies. Another interspecies crossing between A. simii (KMU4810) and a tester strain of A. benhamiae (RV26680) yielded one hybrid F1 progeny (Asb57). The second crossings of F1 progeny (Asb57) with A. vanbreuseghemii (RV27961) and A. benhamiae (RV30001) yielded many hybrid second progenies. Some hybrid second progenies produced between F1 progeny and A. vanbreuseghemii were confirmed to have genes from three species. The gene exchangeability among three Arthroderma species was shown and the meaning of these events discussed.

Molecular typing of Trichophyton mentagrophytes var. interdigitale isolated in a university hospital in Japan based on the non-transcribed spacer region of the ribosomal RNA gene.

Detecting intraspecies polymorphisms in fungi causing dermatophytoses is important in elucidating routes of infection and determining whether Tinea recurrence is caused by exacerbation or re-infection. In fungi, the non-transcribed spacer region (NTS) of the ribosomal RNA gene shows the greatest accumulation of base sequence mutations. We therefore assessed NTS sequences in 64 clinical isolates of Trichophyton mentagrophytes var. interdigitale, the second most common species of dermatophytes in Japan. These isolates were among the clinical isolates of dermatophytes in the Department of Dermatology, Kanazawa Medical University Hospital in 2006 and were obtained by morphological and molecular biological identification methods. DNA was extracted from each isolate, as well as from one isolate maintained in our department, to detect length polymorphisms at each of three variable loci, TmiS0, TmiS1 and TmiS2, of the NTS for subtyping. We observed seven patterns for TmiS0, six patterns for TmiS1 and three patterns for TmiS2. The combinations of these patterns enabled us to classify the 65 isolates into 15 types. The most prevalent, constituted 46% (30/65) of all isolates. Eleven types were new combinations, whereas the other four were previously described. These results suggest that this method may be used to determine the molecular epidemiology of T. mentagrophytes var. interdigitale in Japan, because it generated results rapidly and in a sensitive manner.

Successful mating of Trichophyton rubrum with Arthroderma simii.

Crossing of Trichophyton rubrum with Arthroderma simii yielded many ascomata around the fluffy T. rubrum colonies. One of the 35 supposed ascospores isolated from matured ascoma was shown to be a hybrid of the two species. Hybrids were observed within the genotypes of four different genes, i.e., rRNA, actin, DNA topoisomerase II and cytochrome b. These results strongly suggest that T. rubrum is not an asexual or clonal species.

Successful mating of a human isolate of Arthroderma simii with a tester strain of A. vanbreuseghemii.

An isolate of Arthroderma simii was successfully mated with a tester strain of A. vanbreuseghemii cultured on the plate of simple agar with some hair on it at 27 degrees C. Confirmation of sexual reproduction was made by the detection of hybrids of two parental genotypes. The implications of this result are discussed from the viewpoint of a reevaluation of the species boundaries of dermatophytes.

Mitochondrial DNA analysis of Sporothrix schenckii in India, Thailand, Brazil, Colombia, Guatemala and Mexico.

Up to now, 30 mitochondrial DNA(mtDNA)and 4 rDNA types of Sporothrix schenckii strains have been identified. Here, seventy-six isolates of S. schenckii from Mexico, Guatemala, Brazil, Thailand and India were genotyped and studied epidemiologically by mtDNA restriction fragment length polymorphisms(RFLP)and internal transcribed spacer region(ITS)-RFLP analysis and two new mtDNA types, Type 31 and Type 32, were found. Type 30, previously reported by Mora-Cabrera et al. was confirmed to be Type 3 and designated as blank. Of 48 isolates from Mexico, 41 belonged to Group A wherein Type 2(13 isolates), Type 3(10)and Type 28(7)were dominant. All ten isolates from India and Thailand belonged to Group B. The 52 Group A and 24 Group B isolates corresponded to rDNA Type I and Type IV , respectively, reported by Watanabe et al.(Nippon Ishinkin Gakkai Zasshi 45: 165-175, 2004).

Different genes can result in different phylogenetic relationships in trichophyton species.

To determine the relationships among Trichophyton species we constructed three phylogenies, based on the nucleotide sequences of the actin, rRNA and DNA topoisomerase II genes. These phylogenies showed several conflicting branch points. For example, strains of T. verrucosum,T. concentricum and T. mentagrophytes var. erinacei were mingled with strains of Arthroderma benhamiae and could not be separated into their own phylogenic groups. In addition, strains of A. vanbreuseghemii, T. tonsurans, T. mentagrophytes var. interdigitale and T. mentagrophytes var. quinckeanum were mingled with strains of A. simii and could not be separated into their own phylogenic groups. T. rubrum and T. violaceum made up a clade, which was phylogenetically related to the A. benhamiae clade or A. simii clade, depending on the gene examined. These findings indicate the need to reevaluate the boundaries among Trichophyton species using an alternative to morphological or molecular biological methods.

Tinea corporis with acute inflammation caused by Trichophyton tonsurans.

A 13-year-old Japanese boy presented with acute skin inflammation on the extremities. He belonged to a judo club of a junior high school in which club tinea capitis and tinea corporis seemed to be prevalent. Vesicles and pustules appeared on his right forearm and right leg. They increased in numbers and formed annular lesions. Pruritic erythema appeared surrounding these lesions. Direct microscopic examination of the lesions detected hyphae, and culture for the fungi yielded yellowish colonies. The result of culture from pustules revealed Staphylococcus aureus. At first, a topical antifungal drug and systemic antibiotics seemed to cure annular lesions, but pustules arose again. A large surrounding erythema was cured by topical treatment with a steroid agent. A biopsy specimen from a pustule showed hyphae of fungi within a hair shaft and in the bulb. The restriction fragment length polymorphism in the internal transcribed spacer regions of the ribosomal gene (polymerase chain reaction restriction fragment length polymorphism) revealed a banding pattern compatible with Trichophyton tonsurans. Treatment with systemic itraconazole was begun and lesions disappeared immediately. Systemic antifungal therapy was needed in our case. Tinea corporis with inflammation necessitates systemic antifungal therapy.

Epidemiology of sporadic (non-epidemic) cases of Trichophyton tonsurans infection in Japan based on PCR-RFLP analysis of non-transcribed spacer region of ribosomal RNA gene.

A number of cases of Trichophyton tonsurans infection have been reported among sportsmen and women participating in wrestling, judo, and sumo wrestling in Japan, but there have also been sporadic reports of cases with no history of contact with these sports. A molecular method using restriction enzyme analysis of PCR-amplified fragments targeting the non-transcribed spacer region (NTS) of ribosomal RNA gene in fungal nuclei was applied to T. tonsurans strains isolated from sporadic cases in Japan. Five of 6 molecular types recorded in Japan, i.e., NTS types I, II, IV, V, and VI, and two new types, designated NTS VII and NTS VIII, were observed among 10 strains isolated from sporadic cases. The NTS IV strains, considered not to be related to the present epidemic, were found to be the most prevalent molecular type accounting for 4 of the 10 strains isolated. NTS I was the most prevalent type in the current epidemic in Japan, but it was cultured from only one patient who was later noted to be the daughter of a retired judo practitioner. Four subjects had histories of living abroad and were considered to have been infected outside Japan. The strains in these cases were NTS II, V, VI, and VII. The results of this study suggested that the NTS IV strains were originally present in Japan at a low incidence, but that there has been a recent influx of NTS I, II, V, VI, and VII from abroad, which has been accompanied by the secondary spread of strains from wrestlers and practitioners of martial arts to the general community.