Characterization of clinical isolates of pathogenic <Emphasis Type="Italic">Nocardia</Emphasis> strains and related actinomycetes in Thailand from 1996 to 2003

In Thailand from 1996 to 2003, 171 strains of pathogenic aerobic actinomycetes from clinical specimens were isolated. Of those strains, 134 were mycolic acid containing actinomycetes, including 96 strains of Nocardia species. Others included 10 strains of Gordonia, 14 strains of Rhodococcus, and 22 strains of Mycobacterium. One strain each of the genera Tsukamurella and Corynebacterium were also isolated. Also identified were 27 strains of non-mycolic acid containing actinomycetes. Our identification studies of 96 strains of Nocardia species showed that significant pathogens in Thailand were N. beijingensis (18 strains), N. cyriacigeorgica (13 strains), and N. farcinica (34 strains); the most prevalent species was N. farcinica (35.4%). We also isolated four strains of N. asiatica, five strains of N. asteroides sensu stricto, four strains of N. nova, seven strains of N. otitidiscaviarum, eight strains of N. transvalensis, and two strains of N. pseudobrasiliensis.     

Random amplified polymorphic DNA (RAPD) analysis ofPenicillium marneffei strains isolated from AIDS patients in Thailand

Results of random amplified polymorphic DNA (RAPD) analysis using three different primers showed that 16 strains ofPenicillium marneffei isolated from AIDS patients in Thailand belonged to a genetically homogenous group, but different slightly from an isolate from bamboo rat in China. Six PCR fragments (from about, 200 to 600 bp) that were commonly observed in the RAPD fingerprint of all strains were extracted and sequented. Usefulness of this sequence information for identification ofP. marneffei is discussed.     

Genotyping and Antifungal Drug Susceptibility of the Pathogenic Yeast <Emphasis Type="Italic">Trichosporon asahii</Emphasis> Isolated from Thai Patients

Trichosporonosis due to Trichosporon asahii is a life-threatening infection with a very poor prognosis. We analyzed the genotype of intergenic transcribed spacer (IGS) region 1 of the rRNA gene and determined the drug susceptibility of 101 T. asahii isolates obtained from Thai patients to collect basic information on trichosporonosis in Thailand. Of the five genotypes in the IGS region identified in this study, types 1 and 3 were predominant in Thailand. The distribution in Thailand differs from that in other countries, suggesting that there is a geographic substructure among T. asahii clinical isolates. Voriconazole appeared to be the most active drug.     

Real-time PCR assay to detect DNA in sera for the diagnosis of deep-seated trichosporonosis

Deep-seated trichosporonosis due to Trichosporon asahii is life-threatening and has high mortality. A real-time PCR assay to detect T. asahii DNA in sera for diagnosis of this fungal infection was developed. The assay showed a higher sensitivity than polysaccharide antigen detection method. Our new real-time PCR assay may be used for diagnosing deep-seated trichosporonosis due to T. asahii.     

The first isolation of ustilaginomycetous anamorphic yeasts,Pseudozyma species,from patients' blood and a description of two new species: P. parantarctica and P. thailandica

The genus Pseudozyma is ustilaginomycetous anamorphic yeasts, and are mainly isolated from plants. We isolated three Pseudozyma strains from the blood of patients in Thailand. While one isolate was identified as P. antarctica by rDNA sequence analysis, the other two were considered to be new species and were named P. parantarctica and P. thailandica. The three isolates proved to be resistant to 5-flucytosine, and P. thailandica was also resistant to fluconazole and itraconazole. As far as we know, this is the first isolation of Pseudozyma strains from humans. The two new species are described.     

The intergenic spacer region of the ribosomal RNA gene of Penicillium marneffei shows almost no DNA sequence diversity

The fungus Penicillium marneffei causes fatal systemic infections and is endemic in many parts of South-East Asia, especially Thailand. The intergenic spacer (IGS) region, the most variable region of rRNA genes, was found to be highly conserved among 58 P. marneffei strains. IGS analysis might not be suitable for molecular epidemiological analysis of P. marneffei infections.     

Molecular Epidemiology Reveals Genetic Diversity amongst Isolates of the Cryptococcus neoformans/C. gattii Species Complex in Thailand

To gain a more detailed picture of cryptococcosis in Thailand, a retrospective study of 498 C. neoformans and C. gattii isolates has been conducted. Among these, 386, 83 and 29 strains were from clinical, environmental and veterinary sources, respectively. A total of 485 C. neoformans and 13 C. gattii strains were studied. The majority of the strains (68.9%) were isolated from males (mean age of 37.97 years), 88.5% of C. neoformans and only 37.5% of C. gattii strains were from HIV patients. URA5-RFLP and/or M13 PCR-fingerprinting analysis revealed that the majority of the isolates were C. neoformans molecular type VNI regardless of their sources (94.8%; 94.6% of the clinical, 98.8% of the environmental and 86.2% of the veterinary isolates). In addition, the molecular types VNII (2.4%; 66.7% of the clinical and 33.3% of the veterinary isolates), VNIV (0.2%; 100% environmental isolate), VGI (0.2%; 100% clinical isolate) and VGII (2.4%; 100% clinical isolates) were found less frequently. Multilocus Sequence Type (MLST) analysis using the ISHAM consensus MLST scheme for the C. neoformans/C. gattii species complex identified a total of 20 sequence types (ST) in Thailand combining current and previous data. The Thai isolates are an integrated part of the global cryptococcal population genetic structure, with ST30 for C. gattii and ST82, ST83, ST137, ST141, ST172 and ST173 for C. neoformans being unique to Thailand. Most of the C. gattii isolates were ST7 = VGIIb, which is identical to the less virulent minor Vancouver island outbreak genotype, indicating Thailand as a stepping stone in the global spread of this outbreak strain. The current study revealed a greater genetic diversity and a wider range of major molecular types being present amongst Thai cryptococcal isolates than previously reported.     

Nocardia beijingensis,is a Pathogenic Bacterium to Humans: The First Infectious Cases in Thailand and Japan

Nocardia beijingensis, a recently established new species, is an isolate from soil in China. During our taxonomic studies on 450 nocardial clinical isolates in Thailand and Japan, 17 strains from Thailand and 1 strain from Japan were found to have a similar physiological characteristic to those of N. beijingensis, such as a drug susceptibility pattern to three antimicrobial agents. Our phylogenetic studies on these 18 strains by 16S rRNA gene sequence analysis confirmed that these strains belong to N. beijingensis species. Phylogenetically, these newly isolated N. beijingensis strains were found to be classified into two distinct clades: one is a Japanese clade and other is a Chinese clade, including a reference strain and 17 Thai strains. This is the first report of human infection due to N. beijingensis strains, and we propose that the bacterium be categorized as an opportunistic infectious group regardless of its original isolation from soil.     

Phylogeography of the fungal pathogen Histoplasma capsulatum

Until recently, Histoplasma capsulatum was believed to harbour three varieties, var. capsulatum (chiefly a New World human pathogen), var. duboisii (an African human pathogen) and var. farciminosum (an Old World horse pathogen), which varied in clinical manifestations and geographical distribution. We analysed the phylogenetic relationships of 137 individuals representing the three varieties from six continents using DNA sequence variation in four independent protein‐coding genes. At least eight clades were idengified: (i) North American class 1 clade; (ii) North American class 2 clade; (iii) Latin American group A clade; (iv) Latin American group B clade; (v) Australian clade; (vi) Netherlands (Indonesian?) clade; (vii) Eurasian clade and (viii) African clade. Seven of eight clades represented genetically isolated groups that may be recognized as phylogenetic species. The sole exception was the Eurasian clade which originated from within the Latin American group A clade. The phylogenetic relationships among the clades made a star phylogeny. Histoplasma capsulatum var. capsulatum individuals were found in all eight clades. The African clade included all of the H. capsulatum var. duboisii individuals as well as individuals of the other two varieties. The 13 individuals of var. farciminosum were distributed among three phylogenetic species. These findings suggest that the three varieties of Histoplasma are phylogenetically meaningless. Instead we have to recognize the existence of genetically distinct geographical populations or phylogenetic species. Combining DNA substitution rates of protein‐coding genes with the phylogeny suggests that the radiation of Histoplasma started between 3 and 13 million years ago in Latin America.     

Comparative evaluation of Trichosporon asahii susceptibility using ASTY colorimetric microdilution and CLSI M27‐A2 broth microdilution reference methods

The in vitro activity of AMPH‐B, 5‐FC, FLCZ, MCZ, ITCZ, and VRCZ against 50 isolates of T. asahii was determined using CLSI M27‐A2 microdilution and ASTY colorimetric methods. Observed agreement ranged from 96 to 100% according to the drug. Overall, the agreement between two methods was 97.7%. The ASTY colorimetric method was thus determined to be comparable to the CLSI reference method when testing the susceptibility of T. asahii to a variety of antifungal agents.