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Journal of Clinical Microbiology, April 2009, p. 1063-1073, Vol. 47, No. 4
0095-1137/09/$08.00+0     doi:10.1128/JCM.01558-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Species-Specific Identification of a Wide Range of Clinically Relevant Fungal Pathogens by Use of Luminex xMAP Technology ^,

C. Landlinger,^1,2 S. Preuner,¹ B. Willinger,³ B. Haberpursch,⁴ Z. Racil,⁵ J. Mayer,⁵ and T. Lion^1,2*

Division of Molecular Microbiology and Development of Genetic Diagnostics, Children's Cancer Research Institute,¹ LabDia Labordiagnostik GmbH, Vienna, Austria,² Division of Clinical Microbiology, Institute of Hygiene and Medical Microbiology, Medical University of Vienna, Vienna, Austria,³ Multimetrix GmbH, Heidelberg, Germany,⁴ Department of Internal Medicine Hemato-Oncology, University Hospital Brno and Masaryk University Brno, Brno, Czech Republic⁵

Received 12 August 2008/ Returned for modification 27 September 2008/ Accepted 18 February 2009

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In immunocompromised patients suffering from invasive fungal infection, rapid identification of the fungal species is a prerequisite for selection of the most appropriate antifungal treatment. We present an assay permitting reliable identification of a wide range of clinically relevant fungal pathogens based on the high-throughput Luminex microbead hybridization technology. The internal transcribed spacer (ITS2) region, which is highly variable among genomes of individual fungal species, was used to generate oligonucleotide hybridization probes for specific identification. The spectrum of pathogenic fungi covered by the assay includes the most commonly occurring species of the genera Aspergillus and Candida and a number of important emerging fungi, such as Cryptococcus, Fusarium, Trichosporon, Mucor, Rhizopus, Penicillium, Absidia, and Acremonium. Up to three different probes are employed for the detection of each fungal species. The redundancy in the design of the assay should ensure unambiguous fungus identification even in the presence of mutations in individual target regions. The current set of hybridization oligonucleotides includes 75 species- and genus-specific probes which had been carefully tested for specificity by repeated analysis of multiple reference strains. To provide adequate sensitivity for clinical application, the assay includes amplification of the ITS2 region by a seminested PCR approach prior to hybridization of the amplicons to the probe panel using the Luminex technology. A variety of fungal pathogens were successfully identified in clinical specimens that included peripheral blood, samples from biopsies of pulmonary infiltrations, and bronchotracheal secretions derived from patients with documented invasive fungal infections. Our observations demonstrate that the Luminex-based technology presented permits rapid and reliable identification of fungal species and may therefore be instrumental in routine clinical diagnostics.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Although the vast majority of invasive fungal infections are still caused by Aspergillus or Candida species, changes in epidemiology have become evident over the last years (11, 15, 33, 39). In view of the different drug resistance profiles of many clinically relevant fungal pathogens (34), the development of rapid methods for species-specific identification of clinically important fungi is desirable in order to permit selection of the most appropriate antifungal treatment. Traditional diagnostic approaches to the identification of fungal species are mainly based on phenotype analysis of fungal cultures. However, these approaches are time-consuming and show limited applicability for the detection of molds (29). Over the last years, a variety of molecular methods have been established for rapid and sensitive detection of fungal pathogens. Many of these assays are real-time quantitative PCR tests, mostly targeting the ribosomal multicopy gene (rDNA gene) (1, 3, 12-14, 16, 21, 30, 31, 35, 40). With these techniques, the fungal sequences of interest can be amplified by universal primers targeting highly conserved regions within the rDNA gene, and identification of individual species can either be performed by specific hybridization probes directed against variable sequences within the amplicons or by specific melting curve profiles. Many of these assays, however, only permit the detection of a limited number of fungal species.

Other approaches to the identification of fungal pathogens have been introduced, including fragment length analysis of the internal transcribed spacer 2 (ITS2) region by capillary electrophoresis (5, 7, 37), hybridization assays recognizing unique portions of the ITS2 sequence (6, 9, 42), microarray-based detection (4, 36), or DNA sequencing of the ITS regions (2, 28).

In view of the increasing need for clinically applicable technical approaches to rapid identification of fungal species, we have established a high-throughput assay facilitating reliable recognition of a broad range of pathogenic fungi that are relevant in the context of invasive infections. Our approach is based on the Luminex xMAP hybridization technology, which permits the analysis of up to 100 different target sequences in a single reaction vessel (10). Microbeads, uniquely defined by their specific spectral addresses, are covalently bound by fungus-specific hybridization probes. Biotinylated PCR-amplified target DNA is hybridized to microbead sets bearing oligonucleotide capture probes of interest. By adding a streptavidin-phycoerythrin reporter, all hybridized amplicons captured by their complementary nucleotide sequence on the microbeads are recognized and the median fluorescence intensity (MFI) is subsequently measured by flow cytometry. Recently, this technology has been successfully used for the detection of individual pathogenic fungi, such as Trichosporon spp. (9), Fusarium spp. (25), and several Candida species (6, 26, 27). The assay presented herein permits the rapid identification of a broad spectrum of fungal pathogens, including 10 fungal genera and 29 fungal species, covering both commonly occurring and emerging fungi, and the current panel can be readily extended to cover any other species of interest.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Fungal strains. Reference fungal strains of all species of interest were obtained from different institutions, including the American Type Culture Collection (ATCC, Rockville, MD), the German Collection of Microorganisms (DSM, Braunschweig, Germany), and the Institute of Hygiene and Medical Microbiology of the Medical University of Vienna (IHMM, Austria). Strains are listed in the table in the supplemental material.

Clinical materials. Specimens from patients with fungal infections, including samples from biopsies of pulmonary infiltrations, bronchoalveolar lavage, bronchotracheal secretions, and peripheral blood, were obtained after informed consent. The clinical samples and blood cultures were provided by the St. Anna Children's Hospital, Vienna, Austria; the Institute of Hygiene and Medical Microbiology, Medical University of Vienna, Austria; and the Department of Internal Medicine-Hematooncology, University Hospital Brno, Czech Republic. Peripheral blood specimens from healthy volunteer donors were used to test for cross-reactivity with human DNA.

DNA extraction. All steps were performed in a laminar flow hood using one-way sterile utensils. Reagents used for extraction were filtered through 0.2-µm sterile filters (Corning; Corning, Incorporated, Germany). (i) For fungal strains, a loopful from individual colonies of each fungus culture was homogenized in 500 µl of lyticase lysis buffer (LLB; 50 mM Tris [pH 7.6], 1 mM EDTA [pH 8.0], 0.2% 2-mercaptoethanol, and 1 U/100 µl recombinant lyticase [Sigma, Steinheim, Germany]) and incubated at 37°C for 1 h. After incubation, acid-washed glass beads 710 to 1,180 µm in diameter (Sigma) were added and the solution was vortexed thoroughly for 2 min. Amounts of 400 µl of the supernatant were used for DNA extraction on a MagNA Pure compact instrument using a MagNA Pure compact nucleic acid isolation kit I (Roche Diagnostics, Penzberg, Germany), as described by the manufacturer. DNA concentrations were determined by using a PicoGreen double-stranded DNA quantification kit (Molecular Probes, Inc., Eugene, OR) on an F-2500 fluorescence spectrophotometer (Hitachi, Japan). (ii) For peripheral blood specimens, after hypotonic lysis of the erythrocytes using red blood cell lysis buffer (10 mM Tris [pH 7.6], 5 mM MgCl₂, 10 mM NaCl [all from Sigma]), as described previously (19), the leukocytes were pelleted and resuspended in 470 µl LLB. The subsequent steps were identical to the extraction protocol described above. (iii) For blood culture specimens, 200-µl aliquots derived from blood cultures previously shown to be fungus positive were transferred to Falcon tubes and red blood cell lysis buffer was added. The subsequent procedure was as described above. (iv) For plasma containing white blood cells, peripheral blood specimens anticoagulated with EDTA were kept at 4°C for at least 4 h to sediment the red blood cells. The entire supernatant, i.e., plasma containing white blood cells, was used for DNA extraction. The samples were centrifuged at 15,000 x g for 10 min. Most of the plasma was removed, leaving a residual volume of 100 µl, and 430 µl of LLB was added. The DNA extraction was performed as described above. (v) For specimens from respiratory tract and lung biopsies, solid material was cut into small pieces and homogenized in 430 µl of LLB. The ensuing steps were as described above.

Seminested PCR amplification. Consensus primers for the ITS2 target region were used to minimize the number of reactions required for subsequent fungus identification. For the first round of amplification, the described universal ITS4 reverse primer (41) (5'-TCC TCC GCT TAT TGA TAT GCT-3') and a newly designed forward primer (5'-TTT CAA CAA YGG ATC TCT TGG-3'), designated ITS1A, were used to amplify a sequence covering the complete ITS1, 5.8S, and ITS2 regions, as well as portions of the 18S and 28S regions of the rDNA gene. Amplicons containing the entire ITS2 region were generated by a second round of amplification using the 5'-end biotinylated reverse primer ITS4 (sequence given above) and two 5'-end biotin-labeled forward primers, ITS86-I (5'-TGA ATC ATC GAR TCT TTG AAC G-3') and ITS86-II (5'-TGA ATC ATC GAG TTC TTG AAC G-3'), which hybridize to the 5.8S region of the rDNA gene. The second forward primer, ITS86-II, was necessary for adequate amplification of Candida krusei and Absidia corymbifera. The PCRs were set up in a total volume of 25 µl containing GeneAmp 1x PCR buffer II (Applied Biosystems [AB], Branchburg, NJ), 2.5 mM MgCl₂ (AB), 5 mM deoxynucleotide triphosphate, dATP, dCTP, dGTP, and a 1:8 ratio of dUTP to dTTP (Invitrogen, Lofer, Austria), 400 nM of each primer, 0.25 U heat-labile uracyl-DNA glycosylase (UDG; Roche) (for the first round of amplification only) (20), 2.5 U AmpliTaq DNA polymerase (AB), and molecular biology-grade water (Eppendorf, Hamburg, Germany). Amounts of 5 µl of template DNA were used for the first round of amplification, and 3 µl of the first-round PCR product served as template for the second round of amplification. The PCR was performed according to the following protocol: 10 min at 37°C (UDG activation); 95°C for 10 min (polymerase activation); 35 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 10 min, followed by cooling at 4°C. The amplification conditions for the second round of amplification were identical, omission of the initial UDG activation step being the only difference.

Probe design. The selection of hybridization probes was based on comprehensive ITS2 sequence alignments of all fungal strains listed in Table 1, employing a multiple sequence alignment program. We have screened and aligned the appropriate sequences of multiple clinical isolates registered in the GenBank database and performed meticulous sequence comparison of the available entries (up to 65 different entries per species). Due to the significant error rate within the GenBank entries (8, 24), selection of species-specific hybridization probes was based on the ITS2 consensus sequence of a broad spectrum of different isolates of individual fungal species of interest (i.e., intraspecies consensus). The second important criterion for the selection of species-specific probes was the greatest possible divergence to other fungal species of the same genus (i.e., interspecies diversity).

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TABLE 1. Species-specific and genus-specific hybridization probes

Additionally, probes with broader specificity covering selected fungal genera (genus-specific detection probes; Table 1) were designed to facilitate more-economical prescreening of clinical specimens. All probes were designed to display an optimal length of 21 nucleotides (range, 20 to 22) and a similar G+C content to facilitate multiplex hybridization under uniform conditions. The consensus hybridization temperature of 54°C was determined by using the software program Primer3. The absence of prospective cross-reactivity of the hybridization probes was determined by careful analysis of sequences across a large panel of organisms using the BLAST software. All probes were tagged with the 5'-end amino modifier C12, a reactive primary amino group that facilitated the coupling to the carboxyl group on the xMAP beads (Luminex), and a poly(T) linker (7 T) between the reacting amine and the hybridizing sequence.

Coupling of capture probes to xMAP Luminex beads. Amounts of 100 µl or 1.25 million of the respective Luminex beads, uniquely defined by different red to infrared fluorescent color tones, were used for coupling. The coupling efficiency was enhanced by bovine serum albumin coating. The 100-µl bead solutions were supplemented with 0.002% Tween 20, briefly vortexed, and centrifuged for 3 min at 11,000 x g. The beads were washed once with 500 µl activation buffer (100 mM NaH₂PO₄ · 2 H₂O [pH 6.1]) and resuspended in 192 µl activation buffer by brief vortexing and sonication. Amounts of 24 µl of fresh N-hydroxysulfosuccinimide sodium salt (NHS; Sigma) solution (50 mg/ml in activation buffer) and 24 µl of fresh N-(3-dimethylaminodipropyl)-N'-ethylcarbodiimide (EDC; Sigma) solution (50 mg/ml in activation buffer) were added, and the vials were incubated in the dark for 20 min in the ultrasonic bath. The beads were washed once with 500 µl 50 mM MES buffer (2-[morpholino]ethanesulfonic acid [Roth], 0.002% Tween 20 [pH 5.0]) and resuspended in 250 µl 50 mM MES buffer without Tween. Amounts of 250 µl of bovine serum albumin solution (0.5 mg/ml in MES buffer) were added, and the vials were incubated in the dark for 2 h under permanent agitation. Then the samples were supplemented with 0.002% Tween 20, washed twice with 500 µl 0.1 M MES (pH 5.0)-0.002% Tween 20, and resuspended in 30 µl 0.1 M MES. The beads were coupled in the dark with 10 µl of 100 pmol/µl capture probes in the presence of 10 µl of EDC (100 µg/µl in H₂O) for 1 h under agitation. After 30 min, 10-µl amounts of a freshly prepared EDC solution were added again. The beads were washed with 1 ml of 0.02% Tween 20 and subsequently with 1 ml of 0.1% sodium dodecyl sulfate. Finally, the beads were resuspended in 100 µl TE (10 mM Tris-HCl, 1 mM EDTA [pH 8.0]) buffer and stored in the dark at 4°C.

Hybridization assay and Luminex measurement. A number of parameters were carefully optimized to establish adequate conditions for hybridization and measurement by multiplex Luminex assays. The appropriate oligonucleotide-coupled xMAP beads were selected, resuspended by vortexing and sonication, and diluted 1:10 in TE buffer. The hybridization reaction mixture contained 0.5 µl of each type of coupled beads (approximately 5,000 beads per reaction), 33 µl of 1x tetramethyl ammonium chloride (TMAC; 3 M), 0.1% sarcosyl, 50 mM Tris-HCl (pH 8.0), 4 mM EDTA (pH 8.0), 5 µl of biotinylated PCR amplicons and was filled up with TE buffer to a total volume of 50 µl. The reaction mixture was incubated for 5 min at 95°C in a PCR thermocycler, followed by 15 min of incubation at 55°C. The use of high concentrations of TMAC, an ammonium salt agent that increases the stringency conditions of hybridization, was crucial for the discrimination between fungal target sequences differing by only 1 nucleotide. This compound renders the efficiency of oligonucleotide probe hybridization dependent on the length of the probe rather than on the base composition. After hybridization, the beads were pelleted for 3 min at 1,800 x g and washed once with 100 µl of 1x TMAC. Next, the beads were incubated with 100 µl conjugation buffer (80 mM Na₂HPO₄, 18 mM KH₂PO₄, 30 mM NaCl), including 200 ng streptavidin-R-phycoerythrin (Sigma), for 5 min in the dark. The washing buffer, the number of wash cycles, the concentration of streptavidin-R-phycoerythrin, and the incubation time for staining were optimized for multiplex, single-well Luminex assays containing pools of differentially coupled beads.

The subsequent detection of the hybridized probes was performed on the Luminex 100 apparatus by analyzing 50 beads of each set. The MFI values were generated with the Luminex software. The MFI values presented were subtracted from the background fluorescence determined by the parallel analysis of control samples containing all components except the PCR amplicons. When analyzing patient specimens, signals were regarded as positive if the fluorescence intensity was at least two times higher than the background noise.

Multiple positive and negative controls were included in each run to assess the amplification efficiency of the preceding seminested PCR amplification of the target sequences and to exclude the occurrence of contamination during analysis.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We have established a high-throughput multiplexed hybridization assay permitting rapid identification of a large spectrum of pathogenic fungi, including the most important Aspergillus (n = 7) and Candida species (n = 9), as well as emerging fungal genera, such as Cryptococcus, Trichosporon, Fusarium, Penicillium, Acremonium, Mucor, Rhizopus, and Absidia. The present detection panel includes 69 species-specific hybridization probes facilitating the identification of 29 different pathogenic fungi and 6 genus-specific probes (panprobes) for the identification of the genera Aspergillus, Penicillium, Candida, Trichosporon, and Fusarium (including two alternative pan-Aspergillus/Penicillium and pan-Trichosporon probes) (Table 1).

Selection and design of hybridization probes. The variable ITS2 sequence within the multicopy rDNA gene cluster was chosen as the target region for the identification of a wide range of fungal pathogens. The ITS2 sequence is flanked by highly conserved nucleotide stretches and can therefore be easily amplified by a universal primer pair. A panel of genus- and species-specific probes hybridizing within the ITS2 region has been carefully designed to facilitate multiplex analysis of fungal pathogens using the Luminex assay. All probes were conceived to display a length of 20 to 22 nucleotides, a G+C content ranging from 40 to 80%, and a calculated hybridization temperature of 54°C in order to permit hybridization under uniform conditions (Table 1). Predicted formation of hairpin or other secondary structures or stretches of more than four G-C bases within the hybridizing sequence were exclusion criteria for the design and selection of oligonucleotide probes. For reliable identification of fungal species with highly conserved ITS2 sequences differing by a single base pair, optimal specificity of detection was achieved by placing the discriminating nucleotide in the center of the probe. Whenever possible, two or three probes targeting different portions of the ITS2 region within each fungal species of interest have been designed, in order to permit unambiguous identification even in the presence of mutations within individual target sequences. For most fungal species, three different probes were available for specific detection. In some instances, however, the generation of multiple probes displaying adequate specificity was not possible owing to the presence of very high G+C content within the discriminating sequences or of stretches of high homology to other species. Due to these limitations, only one species-specific probe could be designed for A. candidus, A. clavatus, Penicillium chrysogenum, Penicillium marneffei, and Penicillium simplicissimum.

In addition to probes facilitating the identification of individual fungal species, capture probes for genus-specific classification were generated by targeting regions of the ITS2 sequence that are conserved among species of individual fungal genera. The current panel includes two pan-Aspergillus/Penicillium probes (Pan-A/P1 and Pan-A/P2), two pan-Trichosporon probes (Pan-Tri1 and Pan-Tri2), one pan-Fusarium probe (Pan-Fus), and one pan-Candida (Pan-Can) probe (Table 1), facilitating economical prescreening of clinical specimens at the level of fungal genera. The two genus-specific probes Pan-A/P1 and Pan-A/P2 detect the entire range of Aspergillus and Penicillium species presented in Table 1. The Pan-Tri1 and Pan-Tri2 probes facilitate the detection of at least four clinically relevant Trichosporon species (Tables 1 and 2). The genus-specific Fusarium probe (Pan-Fus) permits the detection of the Fusarium species indicated (Tables 1 and 2). For reliable detection of Fusarium solani, however, more than 1 pg of template DNA is required (Table 2). Three species-specific probes (F.sol1, F.sol2, and F.sol3) were therefore designed to facilitate the detection and identification of Fusarium solani at lower concentrations also, down to 10 fg (Table 2). Based on sequence alignments, the spectrum of Candida species recognized by the genus-specific probe is significantly greater than the number of species tested (Table 1), but certain Candida species, including C. lipolytica, C. kefyr, C. inconspicua, and C. valida, are not reliably detected by this probe in clinical specimens containing very small amounts of fungal DNA. However, C. lipolytica, which is certainly the most common and clinically relevant pathogen of the Candida species not detected by the Pan-Can probe, can be reliably detected and identified by three species-specific probes included in the panel (Table 1).

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TABLE 2. Identification of zygomycetes, Trichosporon, Fusarium, and other emerging fungal species by species-specific and genus-specific hybridization probes

Assessment of specificity and reproducibility. The eligibility of probes for use in clinical testing was based on comprehensive sequence alignments of numerous clinical isolates of the species of interest registered in the GenBank database. For some fungal strains, we found isolates displaying minor intraspecies variations within the targeted ITS2 regions. However, all isolates showed 100% homology with at least one species-specific hybridization probe presented in Table 1. In addition to extensive sequence analyses, 82 different fungal strains/isolates of the fungal species covered by the assay were tested experimentally (see the table in the supplemental material). These strains were derived from reference collections, such as ATCC and DSM, or from the central diagnostic facility of the Medical University of Vienna, IHMM (see Materials and Methods). All fungal isolates used had been controlled by culture and sequence analysis. The specific hybridization probes showed adequate MFI values, permitting clear and reliable species identification in all cases (data not shown).

To evaluate the specificity and reproducibility of the entire panel of 69 species-specific and 6 genus-specific detection probes, each individual fungal target detection test was assessed by a minimum of three independent analyses of a well-defined reference strain. In each analysis, 1 pg of genomic DNA derived from a defined fungal strain was amplified by seminested PCR and hybridized to specific probes coupled to uniquely defined, color-coded microbeads. The subsequent measurements of fluorescence signals were performed in triplicates. The mean MFI values indicating the binding efficacy of individual species-specific and genus-specific hybridization probes were calculated and are presented in Tables 2, 3, and 4. The signal intensities exhibited by individual probes upon PCR product analysis of 1 pg genomic template ranged from >100 to >4,000 MFI above the background levels. The variability of signal intensities was dependent on the G+C content, the self-complementarity, and the poly(A/T/C/G) content of the hybridization probes (Tables 2, 3, and 4). Assessment of the intra-assay variability revealed standard deviations of MFI values below 3%, and the interassay variability was below 10% in most instances. A small number of probes, however, revealed greater fluctuation of MFI values (data not shown). The Luminex technology is therefore only adequate for semiquantitative analysis of the fungal targets.

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TABLE 3. Identification of Aspergillus and Penicillium species by species-specific and genus-specific hybridization probes

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TABLE 4. Identification of Candida species by species-specific and genus-specific hybridization probes

An important requirement for the specificity of all genus-specific and species-specific hybridization probes was the absence of relevant cross-reactivity with incompletely homologous fungal target sequences. In rare instances, however, cross-reactivities not interfering with unequivocal fungal species or genus identification were observed (Tables 2, 3, and 4). Four probes showed cross-reactivities revealing MFI values similar to the specific signals: A.fum2 with Penicillium chrysogenum, A.fla3 with A. terreus, A.nid1 with A. versicolor, and F.oxy2 with Fusarium verticilloides (Table 3). Minor cross-reactivities were observed with five probes: A.fum2 with A. flavus, C.lus3 with C. parapsilosis, C.par1 with C. albicans, A.fla3 with A. glaucus, and Pan-Tri2 with C. albicans (Tables 2, 3, and 4). Despite the cross-reactivity of some probes with certain fungal species, unambiguous species identification was not compromised. This was attributable to the fact that the cross-reactivities did not occur in both directions (e.g., A. terreus showed some cross-reactivity with one of the probes for A. flavus (A.fla3), but A. flavus did not cross-react with the probes for A. terreus (Table 3). Moreover, additional probes lacking any cross-reactivity are available for most fungal species (Tables 2, 3, and 4).

Detection limit. A panfungal seminested PCR protocol was established for efficient amplification of the ITS2 region of all fungal species tested to ensure high sensitivity of the Luminex hybridization assay. To assess the detection limit of individual hybridization assays, fungal DNA from the strains of interest (Tables 2, 3, and 4) was serially diluted across a range of 10 fg to 10 pg, amplified by seminested PCR, and subjected to Luminex analysis. For most hybridization probes, good fluorescence signals were obtained with template concentrations down to 10 fg, which corresponds to a fraction of a single fungal genome. However, a subset of probes, including A.can1, A.ter1, A.fla2, Ac.str1, Ac.stri2, Ac.str3, Ab.cor1, Ab.cor2, Ab.cor3, C.gla1, Cr.neo1, Cr.neo2, F.oxy1, P.mar1, Pan-Tri1, Pan-Tri-2, and R.ory1, revealed better reproducibility at 100 fg template DNA. To assess the detection limits of the Luminex assay for clinical specimens, 1-ml aliquots of peripheral blood from healthy volunteer donors were spiked with 10-fold serial dilutions covering a range of 1 to 10⁵ conidia from A. fumigatus and A. flavus and cells of C. albicans as representatives of molds and yeasts, respectively. Upon DNA extraction and amplification of the entire ITS2 region, species-specific Luminex detection assays were performed as described in Materials and Methods. Fluorescence signals were obtained down to concentrations of a single organism per ml peripheral blood, but 10 organisms per ml provided better reproducibility of detection (data not shown). Peripheral blood from healthy volunteer donors that was not spiked with any fungal organism remained negative (data not shown).

Identification of fungal species in blood cultures and clinical specimens. The initial series of tests included the identification of fungal species in Candida-positive blood cultures obtained from 10 patients. Upon extraction of DNA from the respective blood cultures and seminested PCR amplification of the ITS2 target region, Luminex hybridization assays were performed. All specimens analyzed showed high fluorescence signals with the genus-specific Candida probe (Pan-Can), and the species-specific probes readily permitted the identification of the Candida species present, including C. albicans (n = 6), C. dubliniensis (n = 1), C. glabrata (n = 1), C. tropicalis (n = 1), and C. parapsilosis (n = 1) (Table 5). Comparison with the results obtained by conventional blood culture revealed identical results in 9 of 10 instances. In one specimen, culture indicated the presence of C. albicans, while the Luminex assay unambiguously identified the species as C. dubliniensis (Table 5). In a subsequent series of tests, additional clinical specimens derived from 10 cancer patients with proven (n = 3), probable (n = 2), and suspected (n = 5) invasive fungal infection, according to the EORTC (European Organization for Research and Treatment of Cancer) criteria, were investigated. The specimens, including two lung biopsy samples, two plasma specimens, two blood specimens, two bronchotracheal secretion samples, one bronchoalveolar lavage sample, and one paraffin-embedded lung tissue sample, were analyzed with the Luminex hybridization assay. All specimens revealed specific hybridization signals facilitating the identification of various fungal pathogens, including C. lipolytica, A. fumigatus, A. flavus, Penicillium chrysogenum, Rhizopus oryzae, and Trichosporon spp. (Table 6). A low-level of cross-reactivity of individual probes was observed, but it did not interfere with clear identification of the species present (Table 6). The results obtained with the Luminex detection assays were identical to those obtained by other methods, including microbiological culture and DNA sequencing (Table 6).

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TABLE 5. Analysis of blood culture-positive specimens

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TABLE 6. Analysis of clinical specimens with documented fungal infections

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We present a test system, based on the Luminex hybridization technology, for genus- and species-specific diagnosis of clinically important fungal pathogens. The intended application of the assay is rapid identification of invasive fungi in clinical specimens from immunosuppressed patients, in whom timely detection of the specific fungus type(s) present is critical for the optimal choice of treatment and its success. The method presented offers major advantages over traditional diagnostic approaches primarily based on microbiological culture. Fungus identification by blood culture is less sensitive and often fails to detect invasive infections caused by aspergilli or other molds (29). Moreover, considerable experience is necessary for correct phenotypic identification of the steadily expanding range of pathogenic and opportunistic fungi. The discrepant result of one clinical specimen identified as C. dubliniensis by the Luminex assay and C. albicans by blood culture (Table 5) could be attributable to the major phenotypic (22) but only limited genetic similarities between these two species which have likely led to incorrect typing by routine fungus culture. Unequivocal identification of C. albicans and C. dubliniensis, however, may be relevant for the most appropriate antifungal treatment. Although the majority of C. dubliniensis isolates are susceptible to currently used antifungal drugs, it has been shown that isolates of this species, unlike those of C. albicans, can rapidly develop stable resistance to fluconazole upon exposure in vitro (23).

The amount of fungal pathogens in clinical specimens, particularly in peripheral blood, is usually very low (18) but may nonetheless indicate a life-threatening situation in immunocompromised patients. High sensitivity of detection is therefore a prerequisite for clinical diagnosis of invasive fungal infections in this setting. To achieve adequate sensitivity of the Luminex hybridization technique presented, a two-step preamplification of fungal DNA was performed, using a newly designed and optimized forward primer (ITS1A) for the first round of amplification. The employment of this preamplification protocol permitted reproducible species identification with the Luminex hybridization assay down to 10 to 100 fg of fungal genomic DNA. This detection limit corresponds to 0.3 to 3 pathogens, based on an average fungal genome mass of approximately 35 fg.

The putative occurrence of intraspecies variations in the target regions of different fungal isolates could represent a possible impediment to the sensitivity and specificity of detection. In order to overcome this potential problem, three different hybridization probes were designed for the detection of each species, whenever possible, to permit unambiguous identification of the fungal pathogen even in the presence of mutations in one of the target regions. Comprehensive ITS2 sequence alignments, including sequences of up to 65 clinical isolates, of the individual fungal species of interest listed in the GenBank database revealed 100% homology with at least one hybridization probe presented in Table 1.

Some fungal genera display very limited variability within their ITS2 sequences, thereby preventing the design of multiple genus- or species-specific probes targeting different regions. This problem became apparent in the differentiation between the genera Aspergillus and Penicillium, where the very high level of homology within the ITS2 region precluded the design of genus-specific probes permitting reliable discrimination. However, the pan-Aspergillus/Penicillium probes were employed to facilitate economical prescreening of clinical specimens at the level of fungal genera. In specimens testing positive in the prescreening, subsequent identification at the level of individual species was based on the application of species-specific hybridization probes (Table 3). Although there are reports of the occurrence of invasive penicilliosis, particularly from Southeast Asia, which is mostly caused by Penicillium marneffei (32, 38), the presence of Penicillium spp. in clinical specimens from European and North American patients is generally regarded as environmental contamination. The inclusion of probes for specific identification of the most-common environmental Penicillium species may therefore help prevent false diagnosis of aspergillosis in the clinical setting.

Our current detection panel includes 69 species-specific and 6 genus-specific hybridization probes targeting the variable ITS2 region (Table 1). To render the detection system highly flexible, the probes were designed in a manner permitting any combination in individual multiplex tests. This permits the establishment of subpanels for the identification of individual fungal species and genera of interest, depending on the specific requirements of any particular application. For application in the identification of invasive fungal pathogens in the routine clinical setting, we have assembled the hybridization probes into two detection assays: a mold identification assay comprising all Aspergillus-, Penicillium-, Fusarium-, and Acremonium-specific probes, and a yeast/Zygomycetes identification assay comprising all Candida-, Trichosporon-, Cryptococcus-, Mucor-, Rhizopus-, and Absidia-specific probes. The Luminex hybridization assay presented was designed for specific identification of fungal genera and species and not for quantification of fungus load. Although at least semiquantitative analysis would be feasible upon the introduction of appropriate controls, we generally use a real-time panfungal PCR screening assay for detection of fungal infection and fungal load assessment prior to specific fungus typing by the Luminex assay. The panfungal real-time PCR test developed in our laboratory (L. Baskova and T. Lion, patent application 06817468.9, Europe) comprises two reactions, one covering a broad spectrum of molds and the other a broad spectrum of yeasts and Zygomycetes. For the analysis of clinical samples presented here, specimens that tested positive for mold DNA by real-time PCR were subjected to the Luminex mold assay to identify the pathogen(s) present, whereas yeast- or Zygomycetes-positive real-time PCR samples were further analyzed with the yeast/Zygomycetes Luminex assay.

An essential feature of the Luminex method described herein is the detection of potential coinfections with multiple fungal species in patients revealing positive findings in broad-spectrum real-time PCR screening tests. Concomitant infections with two or more different fungi are not uncommon in immunocompromised individuals, and their identification may be an important prerequisite for determining the most appropriate antifungal therapy.

The Luminex assay described facilitates high-throughput species-specific identification of a wide range of pathogenic fungi within a few hours. The detection spectrum of the Luminex assay comprises more than 30 fungal species but can be readily extended to meet general or local requirements of species identification. Upon confirmation of its utility in large-scale clinical studies, this method may contribute to improved management of invasive fungal infections.

 
This study was supported by grants from the Austrian Science Fund (FWF) (P16929-B13), the Austrian Center for Innovation and Technology (ZIT) (Calls Co-Operate Enlarged-Vienna 2005 and Life Sciences-Vienna 2006), and Fonds der Stadt Wien für innovative interdisziplinäre Krebsforschung.

 
* Corresponding author. Mailing address: CCRI, Kinderspitalgasse 6, A-1090 Vienna, Austria. Phone: 43-0-1-40470 489. Fax: 43-0-1-40470 437. E-mail: Thomas.Lion{at}ccri.at

Published ahead of print on 25 February 2009.

Supplemental material for this article may be found at http://jcm.asm.org/.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Journal of Clinical Microbiology, April 2009, p. 1063-1073, Vol. 47, No. 4
0095-1137/09/$08.00+0     doi:10.1128/JCM.01558-08
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