Genotyping of intraspecies polymorphisms of Sporothrix globosa using partial sequence of mitochondrial DNA.

Restriction fragment length polymorphism (RFLP) of mitochondrial DNA (mtDNA) had been used for molecular identification of Sporothrix spp., which is the causative fungi of sporotrichosis and the most prevalent deep-seated dermatomycosis. Also, mtDNA-RFLP had been used to investigate the molecular epidemiology of sporotrichosis. While the current standard for molecular diagnosis is performed by sequence analysis of the calmodulin gene (CAL), correspondence between the results from CAL and mtDNA is of diagnostic and epidemiological interest. Here, we investigated the correspondence between CAL and mtDNA used for molecular identification of Sporothrix globosa and S. schenckii, which are two major species. We also investigated and propose molecular markers suitable to describe the epidemiology of S. globosa, which is considered as a species with few intraspecific polymorphisms. Eighty-seven strains morphologically identified as S. schenckii sensu lato were investigated. They were identified as group A (17 types, 17 strains) or B (14 types, 70 strains) by mtDNA-RFLP. Partial sequences of CAL, internal transcribed spacer, and spacer between atp9 and cox2 genes of mtDNA of these strains were determined. All group A strains corresponded to S. schenckii, and group B to S. globosa. The sequences of the amplicons targeted on the spacer region in mtDNA of S. globosa ranged 510-515 bp in length and exhibited 10 molecular variations, whereas CAL indicated seven molecular variations. In conclusion, most of the S. schenckii sensu lato strains isolated from Japanese sporotrichosis patients were confirmed as S. globosa, because group B, which comprised the majority of strains, matched perfectly with S. globosa by the CAL sequencing study. We proposed sequence variations in the spacer between atp9 and cox2 genes of mtDNA as a suitable molecular epidemiological marker for S. globosa.

Polyclonality of Trichophyton rubrum Isolates in aDermatophytosis Patient with Multiple Lesions.

We cultured 15 isolates of Trichophyton rubrum and one isolate of Trichophyton mentagrophytes from an 82-year-old male tinea patient with multiple lesions. To determine whether feet lesions were the source of dermatophytes of other tinea lesions, we extracted total cellular DNA from the T. rubrum isolates（13 from feet, two from right waist and buttock）. PCR targeting the non-transcribed spacer（NTS）region of ribosomal RNA gene was performed. Molecular polymorphisms were detected by length variation of amplicons.Four molecular types were found among the 15 isolates. The predominant type, which we previously named Type III, comprised seven isolates cultured from both feet and from left waist and buttock. This was followed by Type VI, five isolates; Type V, two isolates; and Type IV, one isolate. Apart from type III, which was cultured from both feet, isolates were cultured from one foot only. The patient was successfully treated for all types with a six-month course of oral terbinafine and topical luliconazole. The molecular typing supported the notion that tinea pedis was the source of tinea corporis in the patient.