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Journal of Clinical Microbiology, September 1998, p. 2742-2744, Vol. 36, No. 9
Department of Microbiology, Meiji College of
Pharmacy, Tanashi, Tokyo, Japan
Received 22 December 1997/Returned for modification 29 April
1998/Accepted 8 June 1998
Trichosporon asahii is a major causative agent of
deep-seated trichosporonosis, which has a high mortality rate. To
detect T. asahii, we have developed specific
oligonucleotide primers based on the internal transcribed spacer
regions of this organism's genome. Amplification products were
selectively obtained from only T. asahii DNA; the DNAs
of other Trichosporon species, as well as those of
medically relevant yeasts such as Candida albicans, Cryptococcus neoformans, and Malassezia furfur,
were not amplified. This detection system will be useful as a
microbiological tool for the diagnosis of trichosporonosis.
Trichosporon
Behrend is a medically important genus that includes the causative
agents of deep-seated, mucosa-associated, and superficial
infections, including white piedra. Recently, the taxonomy of the
genus Trichosporon was significantly revised on
the basis of partial sequences of large-subunit (LSU) rRNA and DNA
relatedness (3, 18, 22). The causative agent of trichosporonosis was previously believed to be
Trichosporon cutaneum, but on the basis of the new
taxonomy, it has been demonstrated that six
Trichosporon species, T. asahii,
T. asteroides, T. cutaneum, T. inkin, T. mucoides, and
T. ovoides, are all associated with this infection
(2, 5, 19, 20). It was also shown that the major causative
agents of trichosporonosis differ according to the site of
infection. T. asahii and T. mucoides
are involved in deep-seated infections. T. asteroides
and T. cutaneum are associated with superficial
infections. T. ovoides is involved in capital white
piedra, and T. inkin is associated with white piedra of the genital area. Since the first report of a brain abscess due to
Trichosporon infection, there have been an
increasing number of reports concerning this infectious agent (13,
26, 28). The majority of those patients with fatal disseminated
fungemia were afflicted with leukemia or lymphoma and were in a
profound neutropenic state when the
Trichosporon infection developed. Deep-seated trichosporonosis is especially life
threatening, with a high mortality rate; the prognosis for
patients is very poor. In taxonomic investigations, most of the
isolates obtained from patients with deep-seated
trichosporonosis were identified as T. asahii (2, 5, 19, 20). Early diagnosis and treatment
are therefore of paramount importance to
trichosporonosis patients. Since we had already
developed genus-specific primers for Trichosporon
species, including nonpathogenic species (23), in the
present study we developed a rapid PCR-based approach to the detection
of a major causative agent of trichosporonosis, T. asahii.
The 78 strains used in this study, including 14 strains of
T. asahii, are listed in Table
1. They include all
species (17 species and 5 varieties) of the genus
Trichosporon, as well as related medically relevant
yeasts. Trichosporon clinical isolates have been
identified on the basis of a nuclear DNA-DNA hybridization method
(19, 20). For basidiomycetous yeasts, DNA was extracted by
the method of Makimura et al. (9). For ascomycetous
yeasts, a DNA extraction kit (Nucleon MiY; Amersham International plc, Buckinghamshire, United Kingdom) was used according to the
manufacturer's instructions. The internal transcribed spacer
(ITS) regions were amplified with primers pITS1
(5'-TCCGTAGGTGAACCTGCGG-3') and pITS4 (5'-TCCTCCGCTTATTGATATG-3'), which were derived from
conserved regions of the small-subunit (SSU) and LSU rRNA genes,
respectively. Direct sequencing of the PCR-amplified ITS1, ITS2, and
5.8S rRNA gene was performed with an ABI PRISM cycle sequencing kit
(Applied Biosystems, Foster City, Calif.). To determine the
sequences, two external primers, pITS1 and pITS4, were used.
T. asahii var. asahii CBS 2479, T. asahii var. coremiformis CBS 2482, T. asahii var. faecalis CBS 4828, and
T. asteroides CBS 2481 were sequenced. They are all
type strains. To select primers that would specifically amplify only
T. asahii, the sequences of the ITSs of potentially pathogenic yeasts, obtained from DNA sequence libraries, were aligned.
The primers, chosen to align with regions which were not conserved
in other medically relevant yeasts, were TAAF (forward; 5'-GGATCATTAGTGATTGCCTTTATA-3') and pITS4 (reverse;
5'-TCCTCCGCTTATTGATATG-3'). The oligonucleotide primers were
obtained from Greiner Japan (Tokyo, Japan). Amplification reactions
were performed in PCR buffer containing the following: 50 mM KCl; 10 mM
Tris-HCl (pH 8.3); 1.0 mM MgCl2; 25 mM each dATP, dCTP,
dGTP, and dTTP; 2 mM each oligonucleotide primers; and 0.5 U of
Taq DNA polymerase (Takara, Shiga, Japan). The reaction
mixtures were amplified in a Perkin-Elmer 9700 thermal cycler, using
the following program: 94°C for 3 min, followed by 30 cycles
consisting of 94°C for 30 s, 58°C for 30 s, and 72°C for 40 min, with a final extension period at 72°C for 10 min. After thermal cycling, 3 µl of the amplified product was run on a 1.5% (wt/vol) agarose gel, stained with ethidium bromide, and visualized under a UV light.
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Identification of Trichosporon asahii by PCR Based on
Sequences of the Internal Transcribed Spacer Regions
and
ABSTRACT
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TABLE 1.
Specificity of the primers for T. asahii
and related species
Table 1 shows the specificity of the oligonucleotide primers that we designed against the medically relevant yeasts. The primers amplified the DNAs of only T. asahii, including all three varieties, and produced approximately 500-bp fragments (Fig. 1). We used 14 strains of T. asahii, including clinical isolates obtained from blood, feces, sputum, lung tissue, and urine. All of the strains produced the specific DNA fragment. DNAs of the other Trichosporon species, as well as those of medically relevant yeasts such as Candida albicans, Cryptococcus neoformans, and Malassezia furfur, were not amplified by the detection system. Our data show that of the DNAs of the pathogenic yeasts, these primers selectively amplify only that of T. asahii.
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To design specific oligonucleotide primers that would amplify only T. asahii sequences, we sequenced the ITS regions, including the end of the SSU rRNA, of three varieties of T. asahii and the closest phylogenetically related species, T. asteroides. Our primers amplified all three varieties of T. asahii. Their ITS1 region sequences were almost identical, and differential sequences were not found. Molecular phylogenetic analysis based on the sequences of SSU and/or LSU rRNA genes demonstrated that the three varieties of T. asahii are located in the same cluster (1, 3, 16, 17). Moreover, they showed intermediate levels of nuclear DNA relatedness (40 to 56%) to each other in a DNA-DNA hybridization experiment (18). Taxonomic investigations showed that most of the clinical isolates obtained from deep-seated trichosporonosis patients were T. asahii (2, 5, 19, 20). In addition, of the three varieties of T. asahii, the primary causative agent is the variety asahii. T. asahii var. coremiformis and faecalis are also occasionally isolated from clinical specimens (19, 20). Detection of all three varieties of T. asahii at once would be clinically significant. To detect these pathogenic fungi, primers for PCR have been designed on the basis of the sequences of SSU or LSU rRNA genes (4, 25). However, the relatively high level of sequence similarity observed between some species appears to limit the value of SSU rRNA for differentiating phylogenetically closely related species.
The ITS region is located between the SSU and LSU rRNA genes. The ITS region is subdivided into the ITS1 region, which separates the SSU and 5.8S rRNA genes, and the ITS2 region, which is found between the 5.8S and LSU rRNA genes. It is generally thought that the ITS regions have higher rates of divergence than SSU, 5.8S, or LSU rRNA genes. Therefore, on the basis of the sequences of the ITS regions, highly specific primers for PCR can be designed. By analyzing the sequences of the ITS regions, all Trichosporon isolates may be easily identified to the species level.
Several techniques for identification of Trichosporon species have been previously reported. Guého et al. (2) mentioned that six pathogenic species were clearly differentiated by several key characteristics: a combination of assimilation of carbon compounds, cycloheximide resistance, and ability to grow at 37°C. Immunohistochemical identification techniques were also reported by some researchers (8, 12). However, the cell wall antigens of Trichosporon species cross-react with the capsular polysaccharide of Cryptococcus neoformans (10, 11, 15). Shinoda and coworkers prepared specific-factor sera for Trichosporon species, and their studies indicated that Trichosporon species have at least four different serotypes: I, II, III, and I-III (7, 14). In addition, the serotypes correlated well with the molecular phylogenetic tree based on LSU rRNA sequences (16). Pathogenic Trichosporon species have serotype I (T. cutaneum and T. mucoides) or serotype II (T. asahii, T. asteroides, T. inkin, and T. ovoides), and serotype III and I-III species, such as T. gracile, are not responsible for infection. Serotyping provides useful information for tentative identification (7). While these physiological and biochemical identification techniques are easy and convenient, the pathogen is not rapidly detected. Deep-seated trichosporonosis is life threatening, with a high mortality rate, and the prognosis for the patient is very poor. Some authors have reported that the mortality rate (despite antifungal therapy, including amphotericin B) is 64 to 88% (6, 24). Our primers amplified T. asahii DNA from clinical specimen, although a significant number of specimens have not yet been studied (unpublished data). A rapid identification method such as PCR is a useful microbiological tool for the diagnosis of trichosporonosis.
In conclusion, we successfully developed species-specific primers for T. asahii based on the sequences of the ITS regions. We expect this detection system to be applicable to the clinical diagnosis of trichosporonosis.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology, Meiji College of Pharmacy, 1-22-1 Yato-cho, Tanashi, Tokyo 188-0001, Japan. Phone: 81-424-21-0339. Fax: 81-424-21-1489. E-mail: shinoda{at}my-pharm.ac.jp.
Present address: Department of Immunobiology, Meiji College of
Pharmacy, Tanashi, Tokyo, Japan.
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Journal of Clinical Microbiology, September 1998, p. 2742-2744, Vol. 36, No. 9
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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