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Journal of Clinical Microbiology, June 2007, p. 1874-1883, Vol. 45, No. 6
0095-1137/07/$08.00+0     doi:10.1128/JCM.00223-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Identification of Genotypically Diverse Cryptococcus neoformans and Cryptococcus gattii Isolates by Luminex xMAP Technology

M. Bovers,¹ M. R. Diaz,² F. Hagen,¹ L. Spanjaard,³ B. Duim,⁴ C. E. Visser,⁴ H. L. Hoogveld,⁵ J. Scharringa,⁶ I. M. Hoepelman,⁶ J. W. Fell,² and T. Boekhout^1,6*

CBS—Fungal Biodiversity Centre, Utrecht, The Netherlands,¹ University of Miami, Rosenstiel School of Marine and Atmospheric Science (RSMAS), Key Biscayne, Florida,² The Netherlands Reference Laboratory for Bacterial Meningitis (AMC/RIVM), Department of Medical Microbiology, Academic Medical Center, Amsterdam, The Netherlands,³ Department of Medical Microbiology, Academic Medical Center, Amsterdam, The Netherlands,⁴ Netherlands Institute of Ecology (NIOO-KNAW), Centre for Limnology, Nieuwersluis, The Netherlands,⁵ Department of Internal Medicine and Infectious Diseases, University Medical Centre Utrecht, Utrecht, The Netherlands⁶

Received 29 January 2007/ Returned for modification 5 March 2007/ Accepted 11 April 2007

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A Luminex suspension array, which had been developed for identification of Cryptococcus neoformans and Cryptococcus gattii isolates, was tested by genotyping a set of 58 mostly clinical isolates. All genotypes of C. neoformans and C. gattii were included. In addition, cerebrospinal fluid (CSF) obtained from patients with cryptococcal meningitis was used to investigate the feasibility of the technique for identification of the infecting strain. The suspension array correctly identified haploid isolates in all cases. Furthermore, hybrid isolates possessing two alleles of the Luminex probe region could be identified as hybrids. In CSF specimens, the genotype of the cryptococcal strains responsible for infection could be identified after optimization of the PCR conditions. However, further optimization of the DNA extraction protocol is needed to enhance the usability of the method in clinical practice.

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Cryptococcus neoformans and Cryptococcus gattii are closely related pathogenic yeasts, as indicated by the previous description of C. gattii as a variety of C. neoformans (16). Recently, C. gattii has been described as a separate species because of differences in ecology, biochemical, and molecular characteristics (17, 18). C. neoformans and C. gattii both may cause meningoencephalitis, which is fatal unless treated. C. neoformans occurs globally and is found primarily in immunocompromised individuals, e.g., human immunodeficiency virus (HIV)-infected patients. Although the incidence of cryptococcosis in AIDS patients has decreased because of the introduction of the highly active antiretroviral treatment, cryptococcosis remains a serious disease, with a mortality rate of 10 to 30% in regions where access to treatment is limited (3, 22), and it continues to be the most important cause of fungal meningitis in immunocompromised patients. In contrast to C. neoformans, C. gattii mainly infects immunocompetent individuals and was thought to occur only in (sub)tropical areas. However, one of the genotypic groups of C. gattii is causing an ongoing outbreak on Vancouver Island (14, 15, 27), which indicates that C. gattii may also occur in more temperate areas.

C. neoformans and C. gattii differ not only in host range and geographic distribution, but they also differ in clinical manifestation. Although both species infect the central nervous system, C. gattii appears to invade the brain parenchyma more commonly than C. neoformans. Furthermore, in C. gattii-infected patients, pulmonary infections are more likely and pulmonary mass-like lesions occur more commonly than in C. neoformans-infected patients (23, 26). Patients infected with C. gattii seem to have had their symptoms longer before presentation and therapy is often needed for a longer period of time (23, 26). Because of the differences in clinical manifestations and the outcomes of disease, it is important to accurately identify the species responsible for the infection.

Six haploid genotypic groups within C. neoformans and C. gattii can be distinguished by several different molecular methods, e.g., by amplified fragment length polymorphism (AFLP) analysis (4), PCR fingerprinting (21), and intergenic spacer (IGS) genotyping (10). The haploid groups within C. neoformans correspond to the two varieties C. neoformans var. grubii and C. neoformans var. neoformans, while C. gattii can be divided into four genotypic groups. Besides these haploid groups, hybrids have been described as well. Hybrids between the two varieties of C. neoformans exist, these are the so-called AD hybrids (4, 6, 20, 28), and hybrids between C. neoformans var. neoformans and C. gattii have recently been described (5). The different genotypic groups and the relationship between variety, serotype, and the different genotyping methods are shown in Table 1.

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TABLE 1. Overview of the varieties, serotypes, and genotypes within C. neoformans and C. gattii.

Unfortunately, the diagnostic methods which are currently used do not discriminate between all genotypic groups. As a consequence, the differences in hosts and symptoms between the genotypic groups are not known, which is especially true for the genotypic groups within C. gattii. It is likely that more differences in host range and symptoms will be found when the exact genotype of the infecting cryptococcal strain is determined. Another disadvantage of the current diagnostic methods is that they take a considerable amount of time to complete (e.g., culturing) or they can only be used for a limited number of species (e.g., antigen detection). Recently, Luminex xMAP technology has been adapted for the detection of the genotypes within C. neoformans and C. gattii (9). The xMAP technology is based on uniquely color-coded microspheres, which allows as many as one hundred different species to be detected in a single reaction. This technology has been used for the detection of several species of bacteria and fungi (7, 8, 9, 11, 13, 24, 31). Recently, xMAP technology has been used in several diagnostic kits for the detection of bacterial and viral pathogens.

In our study, we used a set of 48 haploid and 10 hybrid isolates to test a Luminex suspension array, which had been developed for identification of C. neoformans and C. gattii strains (9). Our set contained isolates obtained from Dutch cryptococcosis patients in the period between 1977 and 2001, as well as C. gattii isolates from our own collection. In addition, cerebrospinal fluid (CSF) specimens obtained from patients diagnosed with cryptococcal meningitis were used to investigate the feasibility of this Luminex suspension array for the identification of cryptococci in clinical specimens.

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Genotyping of cryptococcal isolates. Thirty-four isolates obtained from Dutch patients and maintained in the cryptococcal collection of The Netherlands Reference Laboratory for Bacterial Meningitis (Academic Medical Center, Amsterdam, The Netherlands) were used for the genotyping assay. Because almost all of these isolates were C. neoformans, we included twenty additional C. gattii isolates and four additional hybrid isolates from our own collection. The origins of the haploid and hybrid isolates are shown in Tables 2 and 3, respectively.

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TABLE 2. Origins of haploid isolates and overview of results obtained by AFLP analysis, mating-serotype-specific PCRs, and Luminex suspension array

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TABLE 3. Origin of hybrid isolates and overview of results obtained by AFLP analysis, sequence analysis, mating-serotype-specific PCRs, and Luminex suspension arraya

DNA was isolated from cultures as described by Bovers et al. (5). Isolates that had not been genotyped before were analyzed by AFLP (4) and C. neoformans mating- and serotype-specific PCRs. PCR amplifications were performed in 20-µl volumes containing 1x PCR buffer (10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl₂, 0.01% gelatin, 0.1% Triton X-100, pH 8.3), 0.1 mM deoxynucleoside triphosphates, 0.5 U of Taq DNA polymerase (Gentaur, Bruxelles, Belgium), 2 to 3 µl of template DNA, and 0.1 µM of both primers. Amplification conditions were as follows: for serotype AD-MAT-specific primer pair JOHE1671/1672 (20), 96°C for 5 min, followed by 25 cycles of 96°C for 30 s, 66°C for 30 s, and 72°C for 30 s, and a final extension step of 72°C for 5 min; for serotype A-specific primer pair JOHE3241/JOHE2596 (20) and serotype D-specific primer pair JOHE3240/JOHE2596 (20), 96°C for 5 min, followed by 25 cycles of 96°C for 30 s, 55°C for 30 s, and 72°C for 45 s, and a final extension step of 72°C for 5 min. PCR conditions for the first serotype A-MATa-specific primer pair JOHE5169/JOHE5170 (20), the second serotype A-MATa-specific primer pair JOHE7270/JOHE7272 (1), the serotype A-MAT-specific primer pair JOHE7264/JOHE7265 (1), the serotype D-MATa-specific primer pair JOHE7273/JOHE7275 (1), and the serotype D-MAT-specific primer pair JOHE7267/JOHE7268 (1) were as follows: 96°C for 5 min, followed by 30 cycles of 96°C for 15 s, 66°C for 15 s, and 72°C for 1 min, and a final extension step of 72°C for 5 min.

Flow cytometry and sequencing of hybrid isolates. The diploid nature of all hybrid isolates was confirmed by flow cytometry according to the method of Bovers et al. (5). Furthermore, partial sequences of the IGS1 region of the ribosomal DNA and laccase (CNLAC1) gene were determined for all hybrid isolates. The primer sequences were those used by Diaz et al. (12) and Xu et al. (32). The amplicons were cloned into Escherichia coli DH5 cells with a TA cloning kit (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Clones were picked randomly, amplified, and purified with a GFX PCR DNA and gel band purification kit (Amersham Biosciences, Piscataway, NY). A BigDye v3.1 Chemistry kit (Applied Biosystems, Foster City, CA) was used for sequencing, and the amplicons were analyzed on an ABI 3700XL DNA analyzer (Applied Biosystems).

Clinical specimens. Clinical specimens were obtained from The Netherlands Reference Laboratory for Bacterial Meningitis in Amsterdam, the University Medical Centre in Utrecht, and the Erasmus Medisch Centrum in Rotterdam, all in The Netherlands, and the University Hospital Gasthuisberg in Leuven, Belgium. CSF from patients with culture-proven cryptococcal meningitis had been stored for up to five years at –80°C. The origins and volumes of the CSF specimens are described in Table 4. After thawing the CSF samples, they were centrifuged for 10 min at 16,000 x g and the supernatant was removed. Five hundred microliters of distilled water was added, and the pellet was resuspended to remove human cells that might be present in the CSF. The samples were centrifuged for 10 min at 16,000 x g and the supernatant was removed. One milliliter of Novozym 234 (1 mg/ml) (Novo Industri, Bagsvaerd, Denmark) suspended in sorbitol buffer (1 M sorbitol, 0.1 M sodiumcitrate; pH 5.5) was added to the samples. The samples were incubated for one hour at 37°C to generate protoplasts, after which the samples were centrifuged for 5 min at 4,600 x g and the supernatant was removed. The tissue protocol of the QIAamp DNA Micro kit (QIAGEN, Venlo, The Netherlands) was used for DNA isolation. The DNA was eluted with 35 µl of AE buffer from the kit.

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TABLE 4. Origin and volume of CSF for which amplicons could be obtained and the results of Luminex identification

Luminex suspension array. The Luminex suspension array, which detects 5' biotin-labeled PCR amplicons hybridized to specific capture probes, was performed as described by Diaz and Fell (9). Specific oligonucleotide probes for each of the six haploid genotypic groups within the C. neoformans species complex as well as oligonucleotide probes targeting either C. neoformans or C. gattii were used. All probes had been designed based on the IGS1 region of the ribosomal DNA (9). An overview of the targets of each probe is given in Table 1. The probes were synthesized with a 5'-end Amino C-12 modification (Integrated DNA Technologies, Coralville, IA) and covalently coupled to different sets of 5.6-µm polystyrene carboxylated microspheres using a slightly modified carbodiimide method (8). Each microsphere set (MiraiBio, Alameda, CA) contains a unique spectral address by combining different ratios of red and infrared fluorochromes. In a typical reaction, 5 x 10⁶ microspheres were resuspended in 25 µl 0.1 M MES (2[N-morpholino]ethanesulfonic acid), pH 4.5, with a determined amount of probe (0.2 to 0.5 nmol). Probe coupling was performed as described by Diaz and Fell (8), and the microspheres were subsequently resuspended in 100 µl of TE buffer (10 mM Tris-HCl, 1 mM EDTA; pH 8). A microsphere mixture was made by adding approximately 5,000 microspheres for each of the eight probes to a 1.5x TMAC (3 M tetramethyl ammonium chloride, 50 mM Tris [pH 8], 4 mM EDTA [pH 8], 0.1% Sarkosyl) solution.

To amplify the IGS1 region, forward primer IG1F (5'-CAG ACG ACT TGA ATG GGA ACG-3') and reverse primer IG2R (5'-ATG CAT AGA AAG CTG TTG G-3') were used (12). The reverse primer was biotinylated at the 5' end. The 1x HotStarTaq MasterMix (QIAGEN, Valencia, CA) containing 1.5 mM MgCl₂, 0.2 mM deoxynucleoside triphosphates, and 2.5 units of HotStarTaq polymerase was used for all PCRs. DNA had been extracted from the cryptococcal isolates prior to amplification, although Diaz and Fell (9) directly used yeast cells for PCR amplification. PCRs were carried out in a total volume of 25 µl. Primers IG1F and IG2R (0.6 µM) and 1.5 µl of template DNA were added to the MasterMix. Amplification conditions were as follows: 95°C for 15 min, followed by 35 cycles of 95°C for 30 s, 50°C for 30 s, and 72°C for 30 s, and a final extension step of 72°C for 7 min.

PCR amplification of the first two clinical samples was carried out using the HotStarTaq MasterMix in a 50-µl total volume containing 3 µl of DNA and 0.4 µM of both primer IG1F and primer IG2R. Amplification conditions were as described above.

Optimization of the PCR conditions resulted in the following reaction conditions, which were used for the remaining clinical samples. PCR amplification was carried out with the HotStarTaq MasterMix in a 50-µl total volume containing 0.2% bovine serum albumin, 8 µl of DNA, and 0.6 µM of both primer IG1F and primer IG2R. Amplification conditions were as follows: 95°C for 15 min, followed by 40 cycles of 95°C for 30 s, 50°C for 30 s, and 69°C for 30 s, and a final extension step of 69°C for 9 min. Amplicons were cleaned with a QIAGEN purification kit (QIAGEN, Valencia, CA) and eluted with elution buffer (10 mM Tris-HCl [pH 8.5]).

To genotype the cryptococcal isolates, 5 µl of biotinylated amplicon was diluted with 12 µl of TE buffer (pH 8), and to genotype the clinical samples, 15 µl of biotinylated amplicon was diluted with 2 µl of TE buffer (pH 8). Thirty-three microliters of the microsphere mixture was added. Each amplicon was tested in duplicate with the Luminex suspension array. The hybridization reaction was performed as described by Diaz and Fell (9).

The hybridized samples were analyzed on the Luminex 100 analyzer (Luminex Corporation, Austin, TX). One hundred microspheres of each set were analyzed, which represents a hundred replicate measurements. Median fluorescence intensity (MFI) values were calculated with a digital signal processor and Luminex 1.7 proprietary software. A positive signal was defined as a signal that is at least twice the background level after subtraction of the background.

Nucleotide sequence accession numbers. All sequences were deposited at GenBank under the accession numbers DQ286656 to DQ286661, DQ286665 to DQ286670, and EF100569 to EF100594.

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AFLP analysis and mating-serotype-specific PCRs performed on the isolates that had not been genotyped before showed that twenty-one isolates belonged to C. neoformans var. grubii MAT serotype A (AFLP1), four isolates were C. neoformans var. neoformans MAT serotype D (AFLP2), and two isolates were C. neoformans var. neoformans MATa serotype D (AFLP2). Finally, one isolate belonged to the C. gattii AFLP4 genotype. Some hybrid isolates between the two varieties of C. neoformans were detected: two MATa serotype A-MAT serotype D (AFLP3) and two MATa serotype D-MAT serotype A (AFLP3) isolates. Results of the AFLP analysis and the mating-serotype-specific PCRs are presented in Tables 2 and 3.

All haploid isolates were identified by two Luminex suspension array probes (Fig. 1). Probes CNNb and CNN1b identified the AFLP1 isolates as C. neoformans var. grubii (AFLP1), and probes CNNb and CNN2d identified the AFLP2 isolates as C. neoformans var. neoformans (AFLP2). Probe CNG correctly identified all C. gattii isolates as C. gattii. In addition, probe CNG4c identified the AFLP4 isolates, probe CNG5b identified the AFLP5 isolates, probe CNG3 identified the AFLP6 isolates, and probe CNG6 identified the AFLP7 isolates. These results show that the Luminex suspension array correctly genotyped all haploid strains (Table 2).

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FIG. 1. Results obtained with the Luminex suspension array for all six haploid groups within C. neoformans and C. gattii. Examples of the results obtained with the AFLP1, -2, -4, -5, -6, and -7 genotypic groups are depicted in panels a, b, c, d, e, and f, respectively.

The Luminex suspension array was also used to genotype ten hybrid isolates. Five out of seven serotype AD (AFLP3) hybrid isolates, namely AMC881205I, CDC92-26, Kl#1, Kl#45, and ZG287, were identified as hybrids between the two varieties of C. neoformans by probes CNNb, CNN1b, and CNN2d (Fig. 2a). However, two serotype AD (AFLP3) hybrid isolates, namely AMC890351 and AMC891529, were identified as C. neoformans var. neoformans by probes CNNb and CNN2d (Fig. 2b). The three serotype BD (AFLP8) hybrid isolates were identified as hybrids between C. neoformans var. neoformans and C. gattii AFLP4 by probes CNG, CNG4c, CNNb, and CNN2d (Fig. 3). Interestingly, when the suspension array identified a hybrid isolate, low signal intensities, namely 16% to 53% of an average positive signal, were obtained for probes which identified one of the parental genotypes. The results of the suspension array correlated with the number of clones that were found for each allele. For example, more AFLP4 than AFLP2 IGS1 clones were obtained for the serotype BD hybrid isolates. The Luminex probes gave a similar outcome: the signals for probes CNG and CNG4c, which identify the C. gattii AFLP4 genotype, had normal intensities, i.e., 546 to 940 MFI. The probes which identify C. neoformans var. neoformans (AFLP2), namely CNNb and CNN2d, were positive with MFI values ranging from 119 to 311, but the MFI values that were obtained were only 16% to 19% of an average positive signal (Fig. 3).

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FIG. 2. Results obtained with the Luminex suspension array for all of the serotype AD (AFLP3) hybrids of C. neoformans. Panel a shows five serotype AD hybrids that were identified by three probes, and panel b shows two serotype AD hybrids that were identified by two probes.

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FIG. 3. Results obtained with the Luminex suspension array for the serotype BD (AFLP8) hybrid isolates.

Flow cytometry confirmed that all hybrid isolates were diploid or close to diploid (data not shown). In addition, when a part of the CNLAC1 region was cloned and sequenced, two alleles could be obtained for all hybrid isolates. However, when the IGS1 region was used for cloning and sequencing, two alleles were obtained for five serotype AD (AFLP3) hybrid isolates, namely AMC881205I, CDC92-26, Kl#1, Kl#45, and ZG287, but only one IGS1 allele was found in two serotype AD (AFLP3) hybrid isolates, namely AMC890351 and AMC891529, even though thirty clones were sequenced. All three serotype BD (AFLP8) hybrid isolates possessed two IGS1 alleles. Our results show that all hybrid isolates were diploid or close to diploid, they possessed two CNLAC1 alleles, and most hybrid isolates possessed two IGS1 alleles. All hybrid isolates that possessed two IGS1 alleles, the region on which the Luminex probes are based, could be identified as hybrids. In order to further improve the identification of cryptococcal hybrids, a probe derived from another gene could be included. In a multigene study of thirty-one serotype AD hybrid isolates (AFLP3), five isolates possessed two IGS1 alleles, but twenty-seven isolates had two TEF1 alleles, twenty-six isolates possessed two RPB1 alleles, and twenty-three isolates had two CNLAC1 alleles (M. Bovers, unpublished data). This indicates that CNLAC1, RPB1, and TEF1 are potential regions that could be used to improve the identification of cryptococcal hybrids.

The detection limit of the suspension array was calculated to vary from 4 x 10¹ to 2 x 10³ cells for the different probes (9). In clinical practice, the concentration of cryptococcal cells ranges from 1,000 to 10,000,000 cells per ml of CSF (25). Therefore, the Luminex technology could be a powerful tool for the detection and identification of cryptococcal cells in CSF. CSF specimens from various Dutch and Belgian hospitals that had been stored for up to five years at –80°C were used to test the Luminex suspension array on clinical specimens. Only nine out of twenty CSF specimens obtained from patients with culture-proven cryptococcal meningitis gave an amplicon of the targeted IGS locus. Unfortunately, we do not have additional information, i.e., about the number of (undamaged) cells or about the presence of interfering substances (30) in the samples, to explain why an amplicon could not be obtained. Amplicons of the PCR-positive samples were subsequently used for genotypic identification by the suspension array (Fig. 4), and the results are shown in Table 4. The first two samples that were analyzed, namely JS9901 and AMC2010576, had probe signals that were too low to be considered positive. Optimization of the PCR conditions, i.e., adding 0.2% bovine serum albumin and increasing the amount of DNA and primers in the reaction mix as well as decreasing the temperature of the elongation step to 69°C, improved detection. As a consequence, the infecting agent of the remaining seven samples could successfully be identified at species and genotypic levels, but because no CSF of JS9901 and AMC2010576 remained, the genotype of the infecting agent could not be determined for those two samples. Probes CNNb and CNN1b, with MFI signals ranging from 260 to 1,402, identified the infecting agent in L5c, AMC2040592, and AMC2031845 as C. neoformans var. grubii. Probes CNNb and CNN2d identified the source of infection of AMC2010488, L4, and AMC2031402 as C. neoformans var. neoformans, with MFI signals ranging from 112 to 1,780. Probes CNNb, CNN1b, and CNN2d, with MFI signals ranging from 199 to 1,446, identified the cryptococcal strain responsible for infection of the patient of specimen L5d as a serotype AD (AFLP3) strain of C. neoformans. Interestingly, CSF specimens L5c and L5d were obtained from the same patient, thus suggesting a dual infection by a haploid C. neoformans var. neoformans and a serotype AD (AFLP3) hybrid strain. Our results show that the suspension array is highly specific, as both varieties of C. neoformans (C. neoformans var. grubii and C. neoformans var. neoformans) and a hybrid could be identified in CSF specimens. The clinical applicability of the Luminex suspension array might be improved by optimization of the DNA isolation protocol, as this is one of the critical steps in any molecular detection system (2). In addition, to determine the robustness of the method, fresh clinical specimens for which the amount of cryptococcal cells and the antigen titers are known should be tested and follow-up studies should be performed on samples with false-negative results.

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FIG. 4. Luminex suspension array results for the amplicons obtained from CSF specimens.

In summary, the Luminex suspension array has the potential to become an efficient diagnostic method with high specificity that not only identifies cryptococcal isolates at the species and genotype levels but that also allows identification of hybrid isolates that possess two IGS1 alleles. Furthermore, our results show that the Luminex suspension array is able to identify cryptococci in CSF specimens. Identification in CSF occurs at the species, genotype, and hybrid levels, but optimization of DNA extraction methods is needed before the method is suited for routine use in clinical laboratories.

 
Our special gratitude goes to A. van Belkum (Erasmus Medical Center, Rotterdam, The Netherlands) and K. Lagrou (University Hospital Leuven, Leuven, Belgium) for sending us clinical specimens.

This research was partially funded by National Institutes of Health grant 1-UO1 AI53879-01, the ‘Odo van Vloten fonds’ and the ‘Netherlands-Florida Scholarship Foundation’.

 
* Corresponding author. Mailing address: CBS—Fungal Biodiversity Centre, Uppsalalaan 8, NL-3584CT Utrecht, The Netherlands. Phone: 31(0)30-2122671. Fax: 31(0)30-2512097. E-mail: boekhout{at}cbs.knaw.nl

Published ahead of print on 18 April 2007.

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Journal of Clinical Microbiology, June 2007, p. 1874-1883, Vol. 45, No. 6
0095-1137/07/$08.00+0     doi:10.1128/JCM.00223-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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