INTRODUCTION

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The fungi belonging to the genus Fusarium are well-known plant pathogens and food contaminants that also cause superficial and subcutaneous infections, such as onychomycosis and keratomycosis, in humans (9). They have recently emerged as major opportunistic agents in immunocompromised hosts, especially in patients with hemopathy (2, 7). They are now considered the third most common fungal genus (after Candida and Aspergillus) isolated from systemic infections in bone marrow transplantation patients (8). Four species account for more than 95% of human infections: Fusarium solani, Fusarium moniliforme (Fusarium verticilloides), and Fusarium oxysporum are each responsible for about 30% of the cases, whereas Fusarium dimerum is involved in 5% of the cases (6, 7). Diagnosis of Fusarium at the species level is based on conventional methods, which include the description of colonies on appropriate media (texture, color, and pigment etc.) and microscopic description of conidiogenous cells and conidia. This can be best observed after 2 weeks of incubation, lengthening the time for a definitive diagnosis. Because of important variations of characters, such as pigmentation and growth rate, that are often seen within a given species, only well-trained mycologists are able to ensure the diagnosis (6). The results are thus frequently inconclusive, as seen in various reports where one-third to one-half of the isolates are not identified at the species level (2, 7). Identification at the species level is important for epidemiological purposes and may become absolutely necessary because some new antifungal agents exhibit variable activity against Fusarium depending on the species (1, 13). In this work, we report a rapid method for the identification of significant Fusarium species involved in human infections by using rRNA gene (rDNA) sequencing.

	MATERIALS AND METHODS

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Strains. The fungal isolates used in this work are listed in Table 1. A total of 33 isolates belonging to six different Fusarium species were tested, including 18 reference strains and 15 clinical isolates collected from superficial and deep infections. Three clinical isolates and one reference strain belonging to the genus Acremonium and two Cylindrocarpon tonkinense reference strains were also studied due to the difficulties in distinguishing between these genera and Fusarium (6). In addition, nine control isolates from unrelated fungi were included to test the specificity of the designed primers (see below). The identification of Fusarium and Acremonium isolates was performed according to the colonial aspects and microscopic morphology after 7 and 14 days of culture at 25°C on potato dextrose agar (Difco Laboratories, Detroit, Mich.) plates in daylight. Examination of micro-, meso-, and macroconidia, chlamydospores, and phiallides allowed differentiation between the Fusarium species (4, 6). All of the isolates were stored in 10% glycerol at 80°C until tested.

DNA extraction. After a 3-day incubation at 35°C on Sabouraud agar (Sanofi-Diagnostic Pasteur, Marnes la Coquette, France) plates, cultures were discharged in 300 µl of distilled water in a microcentrifuge tube. A volume of 100 µl of Chelex solution (10% [wt/vol] Chelex-100 [Bio-Rad, Hercules, Calif.] in an aqueous solution of 0.1% [wt/vol] sodium dodecyl sulfate sodium salt [Sigma, St. Louis, Mo.], 1% [vol/vol] Nonidet P-40 [Sigma], and 1% [vol/vol] Tween 80 [Sigma]) was added. The tubes were incubated at 95°C for 30 min and then on ice for 5 min. DNA was removed from the supernatant after 5 min of centrifugation (10,000 × g) and stored at 80°C until used.

Amplification and sequencing. Two oligonucleotides (Fus1 [5'-TGAAATCTGGCTCTCGGG] and Fus2 [5'-CATGCGCGAACCTCAGTC]) were designed after comparison of Fusarium 28S rDNA sequences in the GenBank database. Amplification was performed in a volume of 50 µl with 10 mM Tris-HCl, 50 mM KCl, 2.5 mM MgCl₂, 0.01% (wt/vol) gelatin, 5 µl of DNA, a 0.25 µM concentration of each oligonucleotide primer, 0.2 mM deoxynucleoside triphosphates (Pharmacia, Uppsala, Sweden), 0.3 U of Taq Gold (PE Applied Biosystems, Foster City, Calif.), and 5 µl of DNA. Thermal cycling parameters were as follows: 10 min at 94°C, followed by 35 cycles of 30 s at 94°C, 90 s at 64°C, and 90 s at 72°C and a final extension of 10 min at 72°C. The PCR products were separated by electrophoresis and visualized under UV. Amplification products were purified by using microspin S-400 HR purification columns (Pharmacia). The sequence reactions were made with 5 µl of purified amplified DNA, 4 pmol of primers, and 4 µl of Big Dye Terminator (PE Applied Biosystems) according to the manufacturer's instructions. The extension products were then precipitated, washed, and resuspended in Template Suppression Reagent (PE Applied Biosystems). After denaturation at 95°C for 2 min, the samples were loaded onto POP6 capillary columns in a ABI Prism 310 Genetic Analyzer (PE Applied Biosystems). Sequences of both strands were determined.

Sequence alignments and phylogenetic trees. Sequences were edited with the Sequence Navigator software (PE Applied Biosystems). Multiple sequence alignments were performed with the Clustal X version 1.64 software (Higgins, Heidelberg, Germany). Trees and matrix distances were constructed with the Phylip package 3.572 by using the neighbor-joining method. The degree of confidence in phylogenetic branching was assessed by using 1,000 bootstrap resamplings.

Nucleotide sequence accession numbers. The nucleotide sequences of the strains and isolates tested in this work appear in the GenBank nucleotide sequence database under the accession numbers shown in Table 1.

	RESULTS AND DISCUSSION

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In this work, we compared the conventional methods for identification of Fusarium species with a 28S rDNA sequence method. Using the newly designed primers Fus1 and Fus2, we were able to amplify a 480-bp fragment from 33 Fusarium strains and isolates and from strains and isolates from the closely related genera Acremonium and Cylindrocarpon. In contrast, we failed to amplify the DNAs from nine reference strains belonging to the unrelated species listed in Table 1. Sequencing of the PCR products led to 10 sequence classes for Fusarium strains and isolates, each containing 1 to 10 isolates. Comparison with Fusarium sequences from the GenBank database demonstrates complete interspecies identity with those of Fusarium semitectum (accession no. X80813), F. oxysporum (U34537 and U34542), and F. moniliforme (U34528). The sequence of F. dimerum (U88105) exhibits close similarity with that of F. dimerum sequence class 2, with a difference of only 6 nucleotides (98.6% homology). Comparison of sequences for F. solani is possible only with sequences obtained from F. solani forma speciales phaseoli isolates (L36629, L36630, L36632, and L36634), which are involved in the soybean sudden death syndrome. They exhibit a closer homology with C. tonkinense sequence class 2 (98.16%) than with F. solani sequence class 1 (96.74%). Multiple alignment of our sequence classes was carried out by using Clustal X software (15). These data were used to construct an unrooted phylogenetic tree by the neighbor-joining method (12). As illustrated in Fig. 1, each Fusarium species as determined by conventional methods formed a distinct clade, with a minimum bootstrap confidence value of 71% for F. solani (Fig. 1). Interspecies homologies for Fusarium never reached higher than 99.01% (i.e., for Fusarium chlamydosporum and F. semitectum) (Fig. 2). In contrast, with the exception of F. dimerum (discussed below), intraspecies homologies ranged from 99.09% for F. solani to 100% for F. oxysporum. An isolate thus can be readily assigned to one species when its homology with one of the sequence classes is higher than 99.1%.

View larger version (14K): [in this window] [in a new window]   FIG. 1.   Unrooted phylogenetic tree derived from 28S rDNA sequences of Fusarium spp., C. tonkinense, and Acremonium spp. The tree was constructed by using the neighbor-joining method. Bar, 1% estimated sequence divergence. cl, class.

View larger version (27K): [in this window] [in a new window]   FIG. 2.   Homology between Fusarium spp., Acremonium spp., and C. tonkinense, derived from 28S rDNA sequences. The sequence classes (cl) correspond to those in Table 1.

The isolate IHEM 5322, considered F. dimerum, exhibited a unique sequence, with 8.25% divergence from the other sequence class containing three strains of F. dimerum. Interestingly, this isolate had deletions at positions 398 and 403, a characteristic shared only with the other sequence class corresponding to F. dimerum. We have no explanation for this discrepancy. (It should be mentioned that this strain had been collected not from a patient but from the recycled water of a spray humidifier.) Such a discrepancy between a molecular method (in this case using species-specific probes) and the conventional method has already been described for a Fusarium reference strain (3), suggesting that conventional methods may not be accurate for some strains with atypical phenotypic characteristics.

While the aim of this work was not a phylogenic reevaluation, we noted that there are closer relationships between some genera tested in this study than between some species within the Fusarium genus. This is illustrated by the grouping of F. solani, Acremonium spp., and C. tonkinense (intersequence class divergences lower than 6.24%), whereas F. moniliforme and F. oxysporum exhibited mean divergences from F. solani between 6.98 and 7.42%, respectively. Interestingly, F. solani, Acremonium spp., and Cylindrocarpon are characterized by teleomorphs belonging to the genus Nectria, whereas F. moniliforme and F. oxysporum have teleomorphs belonging to the genus Giberella, reflecting the heterogeneity of the genus Fusarium (5, 10, 11).

For diagnosis purposes, the sequencing method showed its objective value by easily and correctly identifying a nonpigmented F. solani isolate (N96144236), independent of the mycologist's experience. Also, isolates with uncertain or wrong conventional identifications were identified in agreement with the Institute of Hygiene and Epidemiology Mycology reference center identification. Finally, the sequencing method allows identification within 48 to 72 h following a 3-day culture of the isolate, as opposed to the time-consuming conventional methods. Recently, an exoantigen test was proposed for the species identification of Fusarium, which requires the production of antiserum against specific antigens (6 weeks of incubation) and the extraction of exoantigens from 10-day-old cultures (14). Our results demonstrate that 28S rDNA sequencing is a valuable method for the identification of significant Fusarium species involved in human infections.