Kayoko Takizawa

Identification of pathogenic dematiaceous fungi and related taxa based on large subunit ribosomal DNA D1/D2 domain sequence analysis

The nucleotide sequences of the D1/D2 domains of large subunit (26S) ribosomal DNA for 76 strains of 46 species of pathogenic dematiaceous fungi and related taxa were determined. Intra-species sequence diversity of medically important dematiaceous fungi including Phialophora verrucosa, Fonsecaea pedrosoi, Fonsecaea compacta, Cladophialophora carrionii, Cladophialophora bantiana, Exophiala dermatitidis, Exophiala jeanselmei, Exophiala spinifera, Exophiala moniliae, and Hortaea werneckii were extremely small; as few as 0 changes were detected in C. bantiana, Fonsecaea and Exophiala species, 1 bp in C. carrionii and H. werneckii, and 2 bp in P. verrucosa. Inter-species nucleotide diversity between most species was higher. These data suggested that the D1/D2 domain is sufficiently variable for identification of pathogenic dematiaceous fungi and relevant species. The phylogenetic trees constructed from the sequence data revealed that most human pathogenic species formed a single cluster and that Cladosporium and Phialophora species were distributed polyphyletically into several clusters.

Molecular diversity of Fonsecaea (Chaetothyriales) causing chromoblastomycosis in southern China

Chromoblastomycosis is a chronic, cutaneous and subcutaneous infection caused by members of the order Chaetothyriales and commonly found in China. Among the etiologic agents, members of the genus Cladophialophora are predominant in northern China. Alternatively, Fonsecaea spp. are particularly common in southern China. However, the identification of Fonsecaea isolates recovered in China is difficult due to the fact that different species lack distinctive morphological characters. Therefore, the identification of 24 Fonsecaea isolates from symptomatic patients were re-evaluated by using morphology, ITS rDNA sequence diversity and partly through the use of randomly amplified polymorphic DNA (RAPD) typing. Twenty strains, including a morphological mutant were found to be Fonsecaea monophora, while four strains corresponded to F. pedrosoi. We have demonstrated that Fonsecaea monophora is the predominant etiologic agent of chromoblastomycosis in southern China and populations showed marked geographic structuring.

Rapid Identification of the Genus Fonsecaea by PCR with Specific Oligonucleotide Primers

ABSTRACT An oligonucleotide primer set based on internal transcribed spacer regions of ribosomal DNA for PCR which gives the amplicon for only the DNA from Fonsecaea species was designed. This set yielded an amplicon with 333 bp for all strains of Fonsecaea pedrosoi and Fonsecaea compacta examined but no amplicons for related dematiaceous fungi and pathogenic yeasts. PCR using this primer set was considered to be a useful method for the rapid identification of the genus Fonsecaea.

Specific Oligonucleotide Primers for Identification of Cladophialophora carrionii, a Causative Agent of Chromoblastomycosis

ABSTRACT Cladophialophora carrionii is one of the relatively common causative agents of chromoblastomycosis. We have developed the specific oligonucleotide primer set based on the internal transcribed spacer regions of ribosomal DNA for the rapid identification of this pathogen. PCR with this primer set amplified a 362-bp amplicon from C. carrionii strains. From other relevant dematiaceous species, including medically important dematiaceous fungi, such as Fonsecaea pedrosoi, Phialophora verrucosa, and Exophiala dermatitidis, and eight species of medically important yeasts, such as Candida albicans and Cryptococcus neoformans var. neoformans, the primer set did not produce any amplicon. PCR with this primer set may be a useful tool for the identification of C. carrionii.

First Case of Arthrographis kalrae Ethmoid Sinusitis and Ophthalmitis in the People's Republic of China

ABSTRACT We present here the first case in the Peoples Republic of China of human disease caused by the fungus Arthrographis kalrae. The male patient had fungal panophthalmitis and invasive sinusitis involving the maxillary and ethmoid sinuses. He was an apparently healthy man before receiving trauma to his left eye. He complained of pain and loss of visual acuity in the injured eye, which displayed redness and edema and eventually discharged pus. His symptoms became more severe after he was treated with steroids and several antibacterial agents. A computed tomography scan of the left eye revealed that the maxillary and ethmoid sinuses were involved. A smear of purulent material from the left eye orbit revealed fungal elements, and cultures of the material grew a fungus. The isolate was identified as A. kalrae based on gross and microscopic morphologies, biochemical assays, and DNA sequence analysis. The patient received amphotericin B intravenously, itraconazole orally, and atomized allitridum by nebulizing allitridum therapy. The patients wound healed following surgical intervention, but the patient lost the use of his left eye. This case should remind ophthalmologists and other clinicians to consider the possibility of infections being fungal when antibacterial agents have no effect and the patients condition worsens.

Identification and genetic homogeneity of Trichophyton tonsurans isolated from several regions by random amplified polymorphic DNA

Trichophyton tonsurans is an anthropophilic dermatophyte mostly causing tinea capitis and tinea corporis. This study was carried out to identify T. tonsurans and to clarify whether it has any genetic differences depending on the phenotype or region of isolation by random amplified polymorphic DNA (RAPD) analysis with three random primers. The assay was performed in 11 Korean, 2 Japanese, 2 Taiwanese, 5 Brazilian and 1 American isolates of T. tonsurans together with the other 10 anamorphic species of dermatophytes and 3 Arthroderma spp. All tested species of dermatophytes showed distinct bands and T. tonsurans was differentiated from other dermatophytes. It was most clearly ifferentiated from T. mentagrophytes by using primer 5‘-GAAGGCTCCC-3’ (OPAO-15). No difference was found in RAPD band patterns in all strains of T. tonsurans with these random primers. It was considered that T. tonsurans is a genetically homogeneous species regardless of its isolation regions, morphologic or physiologic characteristics.

Identification of the first isolates of Trichosporon asahii var. asahii from disseminated trichosporonosis in China

Infection with Trichosporon asahii is a major cause of deep-seated and disseminated trichosporonosis, which is associated with a high mortality rate. Disseminated trichosporonosis in individuals with no underlying disease has not been reported. In this study, we report the identification of the first isolate of Trichosporon asahii var. asahii in China. Two isolates were obtained from the liver and skin of a patient with disseminated trichosporonosis who displayed no evidence of underlying disease. The morphologic and physiologic characteristics of the two isolates differed slightly from those of usual strains of T. asahii var. asahii, including the type strain CBS 2479. Ubiquinone-9 was identified as the major ubiquinone in both isolates. Sequence analysis of the LSUrDNA D1/D2, ITS, and IGS1 regions from the two isolates showed them to be T. asahii var. asahii, and random amplified polymorphic DNA analysis strongly suggested that they were the same strain.

A new indoloditerpene derivative, penijanthine A, isolated from Penicillium janthinellum

In a screen searching for new bioactive agents, a new indoloditerpene, penijanthine A (1), was isolated from Penicillium janthinellum IFM 55557. The structure of 1 was established on the basis of spectroscopic and chemical investigation, as well as detailed comparison with the spectroscopic and physico-chemical data of paxilline (2), which was isolated along with 1.

Three pyrrolyloctatetraenyl-α-pyrones from Auxarthron conjugatum

The red pigments, auxarconjugatins A, B and C, were isolated from mycelia of Auxarthron conjugatum, an ascomycetous fungus belonging to the Onygenaceae, in which the causative fungi of severe mycoses also belong. The structures of auxarconjugatins A, B and C, including the stereochemistry of the conjugated tetraene, were established by spectroscopic analyses. These compounds were in equilibrium with a few double bond stereoisomers of the double bonds when dissolved in MeOH or MeCN.

Intraspecific diversity of species of the Pseudallescheria boydii complex

In order to establish intraspecific diversity of Pseudallescheria boydii and Scedosporium apiospermum, and to develop tools for their identification, variability within P. boydii and related species was investigated at different levels of diversity. Sequences of the D1/D2 region of large subunit (LSU) and of the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) gene were analyzed for a set of 57 strains, as well as partial sequences of the elongation factor 1-alpha (EF 1-alpha). Incongruence among 3 locus lineages was detected by partition homogeneity test (PHT). The maximum parsimony (MP) tree of the combined sequence data set, with the exception of strain CBS 499.90, formed 3 clades with high bootstrap support, corresponding to previously described nuclear DNA (nDNA)/DNA reassociation groups. These groups are known to differ slightly in predilection and temperature relations. Using Structure software, population genetic analysis revealed 3 clusters within the complex on the basis of multi-locus genotype data. Strain distribution in the clusters was concordant with that in the 3 clades of combined multi-locus MP tree. Recombination among individuals of a clade in evolutional history was found in 2 of the 3 clades. There was population differentiation among the 3 clades. Restriction fragment length polymorphism (RFLP) analysis of the intergenic spacer (IGS) region of rDNA gene was added to further characterize subspecific entities. When the IGS regions of 22 strains were digested with the restriction endonucleases Hae III and Mbo I, seven and five distinct patterns were detected, respectively. This subtyping did not reveal any correspondence with geographic origin or clinical appearance. Though we need more evidence to locate the 3 clades of the P. boydii complex at species or population level, the sequence of the D1/D2 region is sufficiently variable for identification of taxa belonging to the P. boydii complex.