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Journal of Clinical Microbiology, January 2004, p. 404-407, Vol. 42, No. 1
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.1.404-407.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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

Paride Abliz, Kazutaka Fukushima,^* Kayoko Takizawa, and Kazuko Nishimura

Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, Chiba, Japan

Received 29 May 2003/ Returned for modification 28 July 2003/ Accepted 11 October 2003

Top Abstract Introduction Nucleotide sequence accession... References

 
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.

Top Abstract Introduction Nucleotide sequence accession... References

 
Cladophialophora carrionii (synonym, Cladophialophora ajelloi) is one of the relatively common causative agents of chromoblastomycosis, a chronic mycosis of skin and subcutaneous tissue. The most common appearance of such lesions is as verrucous plaques or nodules (13). This fungus is found primarily in arid areas of South America, Africa, and Australia (14) and was first described by Trejos in 1954 under the name Cladosporium carrionii (22). Recent taxonomic studies have moved pathogenic species of the genus Cladosporium, including C. carrionii, to the genus Cladophialophora (7). C. carrionii is considered the most important pathogenic species in this genus due to the many cases of illness caused by this fungus worldwide (4, 10, 12, 16, 19, 20, 25). Bronchitis in response to the allergenic mechanism of this fungus has also been reported (5). Infections caused by C. carrionii are chronic and appear as spreading mycoses of the skin, subcutaneous tissues, and nail (4, 13).

Morphological and physiological characteristics have been used predominantly for the identification of this fungus until recently. However, the similar morphological features of Cladophialophora species have sometimes caused misidentification of the fungus and also indicate the difficulties of differentiating species on the basis of these characteristics. In addition, cultivation for 2 or 3 weeks is needed to get a suitable amount of conidia production. The physiological profiles of pathogenic dematiaceous fungi were considered to have limited diagnostic value for characterizing these species (23).

In recent years, PCR-based methods have been used to identify pathogenic fungi, including medically important dematiaceous fungi (1, 2, 15, 18, 21). In the present study, we developed a rapid PCR-based approach for the detection of the medically important dematiaceous species C. carrionii.

Eighty-two strains of 54 species (Table 1), including medically relevant dematiaceous species and pathogenic yeasts, were examined. DNAs were prepared according to the method of Makimura et al. (17). Briefly, approximately 50 mg of fungal elements was suspended in 600 µl of extraction buffer (200 mM Tris-HCl [pH 7.5], 25 mM EDTA, 0.5% [wt/vol] sodium dodecyl sulfate, 250 mM NaCl) and then incubated at 100°C for 15 min. The solution was extracted with phenol-chloroform-isoamyl alcohol (25:24:1; vol/vol/vol). DNA was precipitated with cold isopropanol (-20°C). The internal transcribed spacer (ITS) regions were amplified with the following universal primers: ITS5, 5'-GGAAGTAAAAGTCGTAACAAGG-3', and ITS4, 5'-TCCTCCGCTTATTGATATGC-3' (24). Direct sequencing of PCR products corresponding to the ITS1-5.8S-ITS2 region of the ribosomal DNA (rDNA) was done with an ABI PRISM 3100 sequencer after labeling with BigDye Terminator Cycle Sequencing Ready Reaction (Applied Biosystems, Foster City, Calif.). Two external primers, ITS5 and ITS4, and two internal primers, ITS2 (5'-GCTGCGTTCTTCATCGATGC-3') and ITS3 (5'-GCATCGATGAAGAACGCAGC-3') (24), were used for sequencing. Fourteen strains of C. carrionii, three strains of Fonsecaea pedrosoi, a strain of Fonsecaea compacta, and strains of Cladophialophora arxii, Cladophialophora bantiana, Cladophialophora boppi, Cladophialophora emmonsii, Cladophialophora devriesii, Cladophialophora minourae, Phialophora verrucosa, Exophiala dermatitidis, Hortaea werneckii, Rhinocladiella aquaspersa, and Rhinocladiella atrovirens were sequenced. The data obtained from the sequencing are shown in Table 1. In addition to our data, the sequences of ITS regions from other dematiaceous fungal species and medically important yeasts were obtained from GenBank and aligned with CLUSTAL W (version 1.6) for primer design (11). We designed a primer set, 5'-TAAACCTCAT GTTGCTTCG-3' (Car-F) and 5'-TCGAGAM(A/C)CACTCGACCAA-3' (Car-R), based on the sequence of the ITS regions to amplify DNAs from C. carrionii. The primers were synthesized by and purchased from Sigma Genosys Japan (Tokyo, Japan). Each PCR (25 µl) consisted of 2.5 µl of template DNA, 2.5 µl (2 pmol) of each primer, 2 µl (2.5 mM) of dNTP mixture (Nippon Gene, Tokyo, Japan), 0.125 µl (5 U/µl) of Taq polymerase (Nippon Gene), and 2.5 µl of 10x reaction buffer (Nippon Gene). Amplification was performed with a PCR Thermal Cycler MP (TaKaRa Shuzo Co., Ltd., Tokyo, Japan) under the following conditions: 1 cycle at 95°C for 4 min, followed by 30 cycles at 94°C for 1 min, at 62°C for 1.5 min, and at 72°C for 1.5 min, with a final extension at 72°C for 10 min. After thermal cycling, 2 µl of each amplified product was separated by electrophoresis on a 1.5% agarose gel, stained with ethidium bromide, and visualized with UV light.

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TABLE 1. Strains examined in this study and PCR results

Our ITS sequence data for 14 strains of C. carrionii revealed that there were five base pairs in intraspecies nucleotide substitutions. The reverse primer (Car-R) was constructed at the location that showed high variation in interspecies and a nucleotide substitution in intraspecies of C. carrionii. The specificity of our newly designed primer set is summarized in Table 1. This primer set amplified the expected 362-bp fragment only from DNAs extracted from the 20 strains of C. carrionii (Fig. 1). In order to verify the specificity of the primers, we performed PCR analysis of multiple strains of medically important dematiaceous species such as F. pedrosoi, P. verrucosa, E. dermatitidis, Exophiala jeanselmei, and C. bantiana. We also used this primer set for PCR analysis for species of the genera Cladophialophora, Fonsecaea, Phialophora, Cladosporium, Exophiala, Rhinocladiella, Alternaria, Aureobasidium, Hortaea, Hormoconis, Capronia, and eight species of medically important yeasts, including Candida albicans and Cryptococcus neoformans var. neoformans. None of these tested species yielded any amplification product (data not shown). PCR bands were obtained within 7 h from initiating the DNA extractions: 3 h for preparation of the template DNAs, 3 h for PCR, and 1 h for electrophoresis, staining, and detecting the band profiles.

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FIG. 1. Agarose gel electrophoresis of PCR products of C. carrionii. Lane 1, 100-bp ladder; lanes 2 to 21, C. carrionii.

Recent taxonomic studies have shown that the genus Cladophialophora consists of eight species: C. arxii, C. bantiana, C. boppi, C. carrionii, C. devriesi, C. emmonsii, C. minourae, and C. modesta (7, 8). Among these eight species, C. carrionii is the most common pathogen in chromoblastomycosis. C. carrionii has traditionally been identified according to morphological and physiological characteristics and by antigen detection methods (7, 9); however, these methods are time consuming and require technical experience. C. bantiana is also a common pathogenic species that has been isolated as an etiologic agent of brain abscesses and that has been shown to have neurotropic properties. Because C. carrionii and C. bantiana are morphologically very similar, differentiation according to morphological characteristics is difficult. Analysis of ribosomal DNAs by restriction fragment length polymorphisms revealed that C. carrionii and C. bantiana have DNA restriction patterns that are identical to those of most enzymes (6). Our PCR assay offers a reliable method to discriminate these species.

The rapid and accurate identification of causative agents is necessary in medicine. The ITS region located between the 18S and 28S ribosomal DNAs is subdivided into two spacers, ITS1 and ITS2, separated by the 5.8S conserved region. ITS regions are thought to have higher rates of mutation than those of 18S, 5.8S, and 28S rDNAs. Therefore, ITS regions are being used to design specific PCR primers for the rapid identification of human fungal pathogens, including dematiaceous fungi. The ITS sequences of 14 strains of C. carrionii revealed that they have intraspecies variation. However, the interspecies variation of C. carrionii with other dematiaceous fungi is more remarkable. Both of the primers Car-F and Car-R are constructed from the regions with high interspecies variation among all species examined. Furthermore, we designed the reverse primer (Car-R) to allow the possible substitution of nucleotide A or C at the position of a nucleotide substitution in the region originally used for the primer design. The design may have contributed to our getting the expected results in the present study. This suggests that primers designed with possible alternative nucleotides may be useful for enhancing the specificity as well as the efficiency of PCR assays. Indeed, the assay using our newly designed primer set was enhanced to yield the expected band for C. carrionii but not for any of the other species examined. The D1/D2 domain sequence analysis of rDNA has been useful for identifying pathogenic dematiaceous fungi at the species level (3). However, such analysis requires expensive equipment and reagents and takes longer than the PCR identification method; the PCR with our designed primer set gives a result within 7 h. As a matter of course, this assay is a more rapid and accurate method than morphological and physiological identification methods.

In conclusion, we developed a species-specific PCR strategy for identification of a dematiaceous pathogen, C. carrionii. The species specificity of our PCR assay was verified. A PCR analysis with this primer set may be a useful tool for the identification of C. carrionii, a causative agent of chromoblastomycosis.

Top Abstract Introduction Nucleotide sequence accession... References

 
The sequences determined as a result of the present study were registered in the DNA Data Bank of Japan under the accession numbers shown in Table 1.

 
This study was performed as part of the program "Frontier Studies and International Networking of Genetic Resources in Pathogenic Fungi and Actinomycetes (FN-GRPF)" through special coordination funds for promoting science and technology from the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government, 2003.

 
* Corresponding author. Mailing address: Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8673, Japan. Phone: 81-43-226-2797. Fax: 81-43-226-2797. E-mail: kfuky{at}faculty.chiba-u.jp.

Top Abstract Introduction Nucleotide sequence accession... References

 

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Journal of Clinical Microbiology, January 2004, p. 404-407, Vol. 42, No. 1
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.1.404-407.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Abliz, P. Articles by Nishimura, K. Search for Related Content PubMed PubMed Citation Articles by Abliz, P. Articles by Nishimura, K.