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Journal of Clinical Microbiology, February 2003, p. 873-876, Vol. 41, No. 2
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.2.873-876.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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

Paride Abliz, Kazutaka Fukushima,^* Kayoko Takizawa, Norikazu Nieda, Makoto Miyaji, and Kazuko Nishimura

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

Received 26 February 2002/ Returned for modification 8 August 2002/ Accepted 15 November 2002

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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.

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The five etiologic agents of chromoblastomycosis that have been recognized worldwide are Fonsecaea pedrosoi, the principal agent, and Phialophora verrucosa, Cladophialophora carrionii, Fonsecaea compacta, and Rhinocladiella aquaspersa, in order of detection (15, 20). In recent years, increasing numbers of chromoblastomycosis cases and invasive infections caused by F. pedrosoi have been reported (1, 4, 10, 18, 19, 21). According to some investigators, infections by F. pedrosoi account for the overwhelming majority of cases (90 to 93%) (1, 4). F. compacta is a rare cause of chromoblastomycosis (8), with only a few cases reported (1, 3). Traditional methods of identification based on morphological characteristics and antigen detection have been used clinically (7, 13). However, these methods are time-consuming and possess low specificity.

In recent years, PCR with specific primers has been used for the detection and identification of the pathogenic fungi (11, 17, 24). However, a rapid detection system for the identification of Fonsecaea species has not yet been developed. In this paper, we report the development of a rapid and specific PCR method for the detection of Fonsecaea species.

Eighty-four strains of 51 species (Table 1), including medically relevant dematiaceous fungal species and medically important pathogenic yeasts, were examined. DNA was prepared according to the method of Makimura et al. (16). About 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). After being mixed by vortex for 15 s, the mixture was incubated at 100°C for 15 min, kept on ice for 60 min, and centrifuged at 14,000 x g for 15 min. The supernatant was transferred to new tubes, and the solution was extracted once with phenol-chloroform-isoamyl alcohol (25:24:1 [vol/vol]). DNA was precipitated with cold isopropanol (-20°C), dried, and resuspended in 100 µl of distilled water. The internal transcribed spacer (ITS) regions were amplified with universal primers ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') (25). Direct sequencing of the PCR amplicons corresponding to the ITS1-5.8S-ITS2 region of ribosomal DNA (rDNA) was performed with an ABI PRISM 3100 sequencer with an ABI PRISM BigDye terminator sequencing kit (Applied Biosystems, Foster City, Calif.). External primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 were used for sequencing. Six strains of F. pedrosoi, including type strain CBS 271.37, three strains of F. compacta, and strains of Cladophialophora carrionii, Cladophialophora bantiana, Cladophialophora devriesii, Cladophialophora minourae, Phialophora verrucosa, Exophiala dermatitidis, Hortaea werneckii, R. aquaspersa, and Rhinocladiella atrovirens were sequenced. The sequences of the ITS1-5.8S-ITS2 regions of other dematiaceous fungal species and medically important yeasts were obtained from GenBank and were aligned. On the basis of these sequences, a set of oligonucleotide primers, Fon-F (forward; 5'-TAATGCGGGTGTTGCCTCTG-3') and Fon-R (reverse; 5'-AGGGGTGGAAAGTGTGAACT-3') was designed and was obtained from Sigma Genosys Japan kk (Tokyo, Japan). The PCR mixture (25 µl) was composed of 2.5 µl of template DNA, 2.5 µl (2 pmol) of each primer, 2 µl of (2.5 mM) deoxynucleotide triphosphate 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). The PCR was performed with a PCR Thermal Cycler MP (TaKaRa, Tokyo, Japan) according to the following program sequence: 95°C for 4 min, followed by 30 cycles consisting of 94°C for 1 min, 64°C for 2.5 min, and 72°C for 2.5 min, with a final extension at 72°C for 10 min. After thermal cycling, 2 µl of the amplified product was run on a 1.5% agarose gel, stained with ethidium bromide, and visualized with UV light.

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TABLE 1. Strains examined and PCR results

The necessity of prompt and accurate identification of pathogenic fungi is increasing, since such information can directly influence therapy and prognosis. To aid in the rapid identification of Fonsecaea species, we designed a set of genus-specific PCR primers from the sequence of the rDNA ITS region. This set amplified DNA from only the two Fonsecaea species and yielded a 333-bp fragment for all strains of the two species, as shown in Fig. 1. We performed PCR amplification on multiple strains of medically important dematiaceous species, including C. carrionii, P. verrucosa, E. dermatitidis, and E. jeanselmei, and verified that none of these strains produced visible amplicons, nor did other relevant dematiaceous fungal species and medically important yeasts examined (Fig. 2).

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FIG. 1. Agarose gel electrophoresis of PCR products of Fonsecaea species. Lane 1, 100-bp ladder; lanes 2 to 4, F. compacta; lanes 5 to 22, F. pedrosoi.

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FIG.2. Agarose gel electrophoresis of PCR products of Fonsecaea species, relevant dematiaceous fungi, and medically important yeasts. (A) Lane 1, 100-bp ladder; lane 2, F. compacta; lane 3, F. pedrosoi; lane 4, Phialophora verrucosa; lane 5, Phialophora alba; lane 6, Phialophora americana; lane 7, Phialophora atrovirens; lane 8, Phialophora bubakii; lane 9, Phialophora cinerescens; lane 10, Phialophora fastigiata; lane 11, Phialophora heteromorpha; lane 12, Phialophora lagerbergii; lane 13, Phialophora melinii; lane 14, Phialophora oxyspora; lane 15, Phialophora repens; lane 16, Phialophora richardsiae; lane17, Phaeoacremonium parasitica; lane 18, Lecythophora hoffmannii; lane 19, Lecythophora mutabilis. (B) Lane 1, 100-bp ladder; lane 2, F. compacta; lane 3, F. pedrosoi; lane 4, Cladophialophora carrionii; lane 5, Cladophialophora bantiana; lane 6, Cladophialophora devriesii; lane 7, Cladophialophora minourae; lane 8, Cladosporium cladosporioides; lane 9, Cladosporium colocasiae; lane 10, Cladosporium coralloides; lane 11, Cladosporium elatum; lane 12, Cladosporium fulvum; lane 13, Cladosporium herbarum; lane 14, Cladosporium minusculum; lane 15, Cladosporium resinae; lane 16, Cladosporium resinae f. sp. avellaneum; lane 17, Cladosporium sphaerospermum; lane 18, Cladosporium variabile. (C) Lane 1, 100-bp ladder; lane 2, F. compacta; lane 3, F. pedrosoi; lane 4, Exophiala alcalophila; lane 5, E. dermatitidis; lane 6, E. jeanselmei; lane 7, Exophiala moniliae; lane 8, Exophiala spinifera; lane 9, H. werneckii; lane 10, Alternaria alternata; lane 11, Aureobasidium pullulans; lane 12, R. aquaspersa; lane 13, R. atrovirens; lane 14, Candida albicans; lane 15, Candida dubliniensis; lane 16, Candida glabrata; lane 17, Candida parapsilosis; lane 18, Candida tropicalis; lane 19, Cryptococcus neoformans var. neoformans; lane 20, Malassezia furfur; lane 21, Trichosporon asahii var. asahii.

The ITS region, located between the 18S and 26S nuclear rDNA sequences, includes two spacers (ITS1 and ITS2) separated by a 5.8S conserved region. Interspecies sequence differences in the ITS1 and ITS2 regions have been used to detect and identify fungal species (9, 14, 23). The ITS region contains diagnostic sequences that distinguish interspecific-level divergence of organisms (12). From the alignment of the ITS sequence data in our study, two species of Fonsecaea were found to show minimal intraspecies variation, whereas there was high diversity among the dematiaceous species examined. F. compacta has been reported to be a dysplastic variety of F. pedrosoi (5). In addition, 18S and ITS restriction fragment length polymorphism patterns for this species identical to those for F. pedrosoi have been demonstrated by the use of several restriction enzymes (2, 6). However, the two species show distinct morphological characteristics; F. pedrosoi produces slender conidia generated by loosely branched conidiogenous cells, whereas F. compacta produces spherical conidia generated by densely clustered conidiogenous cells (8). This variability in morphology supports the concept that these two species are distinct from each other, and confirming or denying that concept may require looking at several other genetic markers.

Our primer set rapidly discriminated between F. pedrosoi and R. aquaspersa, which had previously been assigned to the genus Acrotheca (7, 22).

In conclusion, we designed Fonsecaea genus-specific primers based on the rDNA ITS region. These, combined with simple PCR, permitted the specific and rapid detection of Fonsecaea species, indicating their potential use in the identification of Fonsecaea species.

Nucleotide sequence accession numbers. Accession numbers for the ITS regions sequenced in this study are provided 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, the Japanese Government, 2002.

 
* Corresponding author. Mailing address: Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, 1-8-1 Inohana, Chuo-ku, 260-8673 Chiba, Japan. Phone: 81-43-226-2797. Fax: 81-43-226-2797 or 2486. E-mail: kfuky{at}myco.pf.chiba-u.ac.jp.

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Journal of Clinical Microbiology, February 2003, p. 873-876, Vol. 41, No. 2
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.2.873-876.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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