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Journal of Clinical Microbiology, July 2005, p. 3324-3333, Vol. 43, No. 7
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.7.3324-3333.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Multilocus DNA Sequence Comparisons Rapidly Identify Pathogenic Molds

Departments of Laboratory Medicine,¹ Microbiology,University of Washington, Seattle, Washington,³ Clarity BioSciences, Inc., Carlsbad, California²

Received 10 December 2004/ Returned for modification 26 January 2005/ Accepted 24 March 2005

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion References

 
The increasing incidence of opportunistic fungal infections necessitates rapid and accurate identification of the associated fungi to facilitate optimal patient treatment. Traditional phenotype-based identification methods utilized in clinical laboratories rely on the production and recognition of reproductive structures, making identification difficult or impossible when these structures are not observed. We hypothesized that DNA sequence analysis of multiple loci is useful for rapidly identifying medically important molds. Our study included the analysis of the D1/D2 hypervariable region of the 28S ribosomal gene and the internal transcribed spacer (ITS) regions 1 and 2 of the rRNA operon. Two hundred one strains, including 143 clinical isolates and 58 reference and type strains, representing 43 recognized species and one possible new species, were examined. We generated a phenotypically validated database of 118 diagnostic alleles. DNA length polymorphisms detected among ITS1 and ITS2 PCR products can differentiate 20 of 33 species of molds tested, and ITS DNA sequence analysis permits identification of all species tested. For 42 of 44 species tested, conspecific strains displayed >99% sequence identity at ITS1 and ITS2; sequevars were detected in two species. For all 44 species, identifications by genotypic and traditional phenotypic methods were 100% concordant. Because dendrograms based on ITS sequence analysis are similar in topology to 28S-based trees, we conclude that ITS sequences provide phylogenetically valid information and can be utilized to identify clinically important molds. Additionally, this phenotypically validated database of ITS sequences will be useful for identifying new species of pathogenic molds.

	INTRODUCTION

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Fungal infections have been increasing in prevalence among hospitalized patients, particularly immunocompromised individuals. Invasive mold infections have very high mortality rates; successful treatment requires rapid and accurate identification of the pathogen. Aspergillus is the most prevalent infectious mold in immunocompromised patients; however, other molds, such as Fusarium spp. and Zygomycetes, are increasingly frequent causes of infection (2, 18, 30).

The phenotype-based identification methods routinely used in clinical laboratories require expertise and can be time-consuming and laborious. Isolates are identified by recognition of colonial morphology and microscopic reproductive structures; few biochemical tests are available to aid in identification. Phenotypic variants may not be identifiable or may be misidentified. Typically in the clinical laboratory, isolates that cannot be identified by reproductive structures are described as Mycelia sterilia, a name indicating a filamentous fungus that displays no distinguishing phenotypes recognized by routine clinical laboratory analyses.

Phenotypic methods can, in some cases, take weeks, a time frame that is not clinically useful. Rapid identification of molds causing invasive disease could facilitate the timely administration of effective therapy. For example, Aspergillus flavus and Aspergillus terreus have been reported to be resistant to amphotericin B (39, 45), and Aspergillus fumigatus can become resistant to itraconazole (9).

Molecular methods for identification of pathogenic fungi have been validated for use in clinical settings (6, 7, 10, 14, 20-22, 32, 38, 48). rRNA genes, including the 28S gene (26S gene in all yeasts), are conserved, accrue single nucleotide changes at a relatively low rate, and provide useful phylogenetic information (51). In eukaryotes, the rRNA operon includes internal transcribed spacer regions 1 and 2 (ITS1 and ITS2), which do not encode functional rRNAs or proteins. These loci have increased levels of DNA sequence diversity compared to other loci within the operon, including the 28S gene (19, 51). Thus, ITS sequences may provide accurate identification of closely related isolates and species which cannot readily be distinguished using 26S or 28S rRNA gene sequences. This hypothesis has been confirmed for yeasts isolated in clinical laboratories (6-8, 14, 43, 48). For example, ITS sequence analysis provides a more accurate taxonomic placement for a number of yeasts, including Cryptococcus humicolus, Pichia veronae, and P. fabianii (6), which were not resolved by analysis of 26S sequences. Henry et al. (22) demonstrated sufficient sequence diversity at ITS1 and ITS2 to distinguish six medically important Aspergillus species from one another. Turenne et al. (48) and De Baere et al. (8) used capillary electrophoresis to examine the lengths of ITS2 PCR products for identifying fungi. This method is rapid but does not provide species-specific identification for all organisms tested, demonstrating a need for additional analyses.

We have developed a rapid molecular method for identifying pathogenic molds based on the lengths and sequences of ITS1 and ITS2. The combination of ITS1 and ITS2 length polymorphisms identifies 20 of 33 species of molds tested. Analysis of ITS1 and ITS2 DNA sequences unambiguously identified all molds tested to the species level; 44 species were represented in the analysis. Identification by ITS DNA sequence analyses was concordant with, but more specific than, identification by 28S DNA sequence analysis and phenotypic analyses. ITS-based phylogenetic trees are similar in overall topology to those constructed with 28S sequences, demonstrating that ITS loci are taxonomically informative. Therefore, phylograms constructed with ITS sequences will allow accurate taxonomic assignment of previously unidentified or uncharacterized molds. We present here a database of ITS DNA sequences, validated with 28S DNA sequence and phenotypic analyses, that will allow rapid identification of molds isolated in the clinical laboratory by ITS DNA sequence comparisons.

	MATERIALS AND METHODS

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Strains. Two hundred one strains of molds, representing 44 species, were characterized in this study. Our phenotypically validated database includes information from type strains obtained from either the American Type Culture Collection (ATCC) (n = 10) or the Centraalbureau voor Schimmelcultures (CBS) (n = 17). Clinical strains (University of Washington Fungal Project [UWFP]) (n = 143) were isolated in the mycology laboratory at the University of Washington Medical Center. ITS1+2 (n = 13), 28S (n = 15), or ITS1+2 and 28S (n = 3) DNA sequence information from 31 reference strains (including 22 type strains) available in public databases was included in the sequence analyses. (The ITS region referred to here as ITS1+2 is amplified using primer binding sites in the 3' end of the 18S rRNA gene and the 5' end of the 28S rRNA gene: the resulting PCR product includes the entirety of ITS1, the 5.8S rRNA gene, and ITS2.). Strains are listed in Table 2. An additional 42 clinical isolates (Aspergillus flavus [n = 1], A. fumigatus [n = 24], A. niger [n = 9], and Trichophyton rubrum [n = 8]) were analyzed by phenotypic methods and ITS1 and ITS2 length polymorphism analysis only (Table 1).

DNA extraction. Mold isolates grown on SDA plates at 30°C for 72 h were resuspended in 1 ml lysis buffer (100 mM Tris, 0.5% [wt/vol] sodium dodecyl sulfate, 3 mM EDTA, pH 7.5) containing approximately one-third volume 0.5-mm glass beads in 2-ml Bead-Beater tubes (BioSpec Products, Inc., Bartlesville, OK). The vials were placed into a Mini-BeadBeater-8 (BioSpec Products, Inc., Bartlesville, OK) at high speed (3,200 rpm) for 3 min and then placed on ice. After centrifugation at 20,000 x g for 5 min, 800 µl of supernatant was transferred to a new tube. Twenty microliters of proteinase K (20 mg/ml) was added, the tubes were incubated for 1 h at 37°C, and the proteinase K was inactivated by incubation at 65°C for 20 min. The DNA was extracted with 700 µl Tris-saturated phenol chloroform (1:1) two times followed by a chloroform extraction and ethanol precipitation. The DNA pellet was dried and resuspended in 150 µl of sterile nuclease-free water and treated with 12 µl of RNase (10 mg/ml) for 1 h at 37°C.

PCR and DNA sequencing. The ITS1+2 regions were PCR amplified using primers ITS1 and ITS4 (6, 7), and both strands of the PCR products were directly sequenced (7). In separate experiments, the ITS1 locus was amplified using primers ITS1 and ITS2 (6), and similarly, the ITS2 locus was amplified using primers ITS3 and ITS4 (7). The lengths of the resultant amplicons were determined with single-nucleotide precision by automated capillary electrophoresis under denaturing conditions using an ABI310 genetic analyzer and GeneScan software (PE Applied Biosystems, Foster City, CA) as described previously (7). PCR amplification and sequencing of the D1/D2 hypervariable region of the 28S rRNA gene was performed using primers NL-1 and NL-4 as described previously (28).

Sequence and phylogenetic analysis. DNA sequences were aligned, edited, and analyzed as described previously (7); in addition, GeneStream align (35; http://xylian.igh.cnrs.fr/) was used. Manual editing was performed in Jalview version 1.3b (M. Clamp, European Bioinformatics Institute [http://circinus.ebi.ac.uk:6543/jalview/]) and with standard word processing software. CLUSTAL_X version 1.81 (47) was used for phylogenetic analysis. Dendrograms constructed with the neighbor-joining treeing algorithm were evaluated with 1,000 bootstrap analyses using CLUSTAL_X and visualized in Treeview version 1.5.3 (34).

	RESULTS

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ITS DNA sequence polymorphisms identify pathogenic molds to the species level. To test the hypothesis that genetic information in the ITS1 and ITS2 loci of pathogenic molds is diagnostically useful, we analyzed ITS length (Table 1) and sequence polymorphisms (Table 2). One hundred sixty-six strains representing 33 species of molds were analyzed to determine whether ITS1 and ITS2 PCR product length polymorphisms (Table 1) are sufficient to identify clinically relevant molds to the species level. All isolates were identified by traditional colonial and morphological analyses and appropriate biochemical tests (see Materials and Methods). Single PCR products amplified from purified mold genomic DNA were analyzed by capillary electrophoresis under denaturing conditions, which allows rapid determination of DNA fragment length with single-base-pair accuracy (7). ITS1 (3' end of the 18S rRNA gene, all of ITS1, and the 5' end of the 5.8S gene) and ITS2 (the 3' end of the 5.8S gene, all of ITS2, and the 5' end of the 28S gene) PCR product length polymorphisms distinguish 20 of 33 mold species tested (Table 1). Intraspecies ITS1 and ITS2 length variations obscure the distinction of Penicillium sumatrense from Emericella quadralineata and Penicillium commune from Penicillium oxalicum (Table 1). In contrast, isolates of Aspergillus fumigatus and Pseudallescheria boydii also displayed intraspecies variation in ITS PCR product lengths. However, they are identified by characteristic length polymorphisms because the distribution of ITS lengths for each group is sufficiently different to distinguish them from other molds. All PCR product length measurements were confirmed by direct sequencing (see below), and a close correlation between the electrophoresis results (Table 1) and the actual number of nucleotides that comprise the ITS1 and ITS2 amplicons was observed.

The entire ITS locus (ITS1+2 from the 3' end of the 18S gene to the 5' end of the 28S gene (ITS1 plus the entire 5.8S gene plus ITS2), and the D1/D2 hypervariable region of the 28S rRNA gene were analyzed by direct sequencing of both the forward and reverse strands of the respective PCR products (Table 2). Forty-four species were represented in the analysis, and type strain data for all 43 recognized species was included and validated by phenotypic analyses; one probable new species was also identified (see below). One hundred forty-three clinical isolates and 27 type strains were analyzed phenotypically and genotypically in our laboratory. Inclusion of sequence information for 31 reference strains from GenBank was justified by identity to DNA sequences from fungal isolates phenotypically and genotypically identified in our laboratory (Table 2). ITS1 and ITS2 DNA sequence analyses specifically identify all 44 species tested. Conspecific strains (including type strains) demonstrate >99% sequence identity at the ITS1 and ITS2 loci for all but two species tested (Cladosporium cladosporioides and Mucor racemosus) (Table 2 and below). Further, phenotypic designations were 100% concordant with ITS1, ITS2, and 28S DNA sequence-based identifications.

ITS sequence analyses refine identification: sequevars and probable new species defined. Our data and those of others have demonstrated that conspecific fungi are >99% identical at the 28S/26S and ITS loci (6, 7, 28, 43). Genetic variants among isolates of a species are designated sequevars (7) when an isolate is (i) phenotypically consistent with the type strain, (ii) genotypically 99% identical to the type strain at multiple loci, and (iii) <99% identical to the type strain at one diagnostic locus (6). We identified isolates of several species of molds displaying increased sequence diversity (<99% identity) at one of the ITS loci, together with high levels of conservation (99% identity) at the 28S gene and the other ITS locus and phenotypic concordance (Table 3). These data (i) indicate that sequence diversity independently accumulates in each locus (ITS1, ITS2, and 28S) and (ii) are consistent with previous observations of clinically important yeasts (6). On this basis, we submit that the analyses summarized in Table 2 represent multilocus sequence-based identifications.

Similarly, traditional phenotypic analyses in the clinical laboratory readily identify Mucor spp. to the genus level, while genetic analyses can differentiate Mucor racemosus from other Mucor species. A group of five clinical isolates (UWFP-788, -790, -827, -959, and -1088) that exhibit phenotypes consistent with Mucor spp. are 100% identical to each other at the 28S, ITS1, and ITS2 loci and show >99% identity to the M. racemosus type strain (CBS 260.68) at 28S and ITS1 (Table 3). However, identity between the clinical isolates and the type strain at ITS2 is 98.6%. Therefore, the clinical isolates are designated M. racemosus sequevar 1.

DNA sequence analyses of multiple loci can also identify species that are not differentiated by phenotypic or single-locus sequence analyses. Two of the 143 clinical isolates we analyzed, strains UWFP-777 and UWFP-969, potentially represent a new species that is closely related to Mucor ramosissimus. The strains, isolated from two different patients, have identical ITS1, ITS2, and 28S DNA sequences and exhibit phenotypes consistent with Mucor spp. These strains display 99.2% identity to the M. ramosissimus type strain ATCC 28933 at the 28S locus but only 95.1% and 96.0% identity at ITS1 and ITS2, respectively (Table 3). Sequence diversity of this magnitude at two loci compared to the type strain sequences suggests that UWFP-777 and -969 most likely represent members of another species of Mucor closely related to M. ramosissimus. These data show that analysis of multiple loci extends the specificity of identification of Mucor species beyond the capabilities of either traditional methods routinely used in the clinical laboratory or 28S DNA sequence analysis alone.

Phylogenetic analysis. rRNA gene sequences, including 28S sequences, have been shown to provide taxonomically useful information (51). To determine whether ITS sequences could accurately identify relationships among diverse mold taxa, we compared phylogenetic trees constructed with ITS sequences to those constructed with 28S sequences. The topologies of the ITS and 28S trees are highly similar (Fig. 1). In addition, high bootstrap values are observed at the deeply branching nodes, and similarly high values are observed at branches separating more closely related genera. Thus, the ITS tree demonstrates that there are species-specific, phylogenetically informative, taxonomically useful sequences within the ITS loci of pathogenic molds. The high level of intraspecies sequence identity (Table 2) demonstrate the stability of the loci within species, and the high bootstrap values (Fig. 1) provide confidence in the species-specific designations obtained from our sequence analyses. The data presented in Fig. 1 and Table 2 indicate that previously uncharacterized molds could be identified in the clinical laboratory by placing their ITS sequences into an established tree constructed with DNA sequence information derived from phenotypically validated mold isolates.

View larger version (65K): [in this window] [in a new window]   FIG. 1. 28S and ITS1/ITS2 DNA sequence-based phylogenetic trees of clinically significant molds. Trees were drawn using the neighbor-joining method with 1,000 bootstrap analyses. Numbers at the nodes indicate the bootstrap values, and the bars indicate relative genetic distance. The source of the organism or sequence is indicated in Table 2. The zygomycetes Mucor spp. and Rhizopus spp. were used as outgroups in both trees. The trees include organisms for which DNA from the type strain was sequenced in our laboratory or for which type strain sequence data were verified by identity to a DNA sequence generated in our laboratory from a conspecific isolate. (A) ITS-based tree of 32 molds. Analysis included partial 18S and 28S sequences and complete ITS1, 5.8S, and ITS2 sequences. (B) 28S-based tree of 27 molds. Analysis included the D1/D2 hypervariable region of the 28S rRNA gene.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

The integration of molecular analyses with traditional phenotypic methods of fungal identification can significantly increase the specificity and decrease the turnaround time for the identification of clinically important molds. Production of reproductive structures or expression of specific biochemical phenotypes by fungi grown in culture is required for phenotypic analyses. Previously uncharacterized molds are difficult to identify by such methods, yet accurate taxonomic placement of the uncharacterized molds, and hence their identity, can be inferred from their phylogenetic relationships with well-characterized mold isolates. Molecular analyses of multiple DNA loci can provide a more rapid (24 h versus several weeks) and sometimes more specific identification of an organism than traditional culture-based methods: identification is refined by including ITS and 28S sequence analyses with phenotypic characterization. The increased level of sequence variation at ITS loci compared to the 28S locus will facilitate the future development of rapid, hybridization-based identification methods (38).

Using phenotypic methods alone, an isolate of Cladosporium spp. can be differentiated from Cladophialophora spp. in a time frame of 1 to 4 weeks. In addition to colonial and microscopic morphology, two tests are performed to identify the organism in our laboratory: gelatin liquefaction and temperature tolerance (growth at 37°C and 42°C) (29). Molecular analysis of the ITS1, ITS2, and 28S DNA sequences of the isolate can identify C. cladosporioides to the species and sequevar levels. This analysis can be completed in 24 h once the organism is isolated from a patient specimen.

Multilocus analysis allows the separation of possible new species that are not differentiated by standard clinical laboratory phenotypic analyses or single-locus analyses alone. Voigt et al. (50) used 18S and 28S sequences to describe the phylogeny of medically important zygomycetes, including six species of Mucor. Mucor ramosissimus (NRRL 3042) and Mucor circinelloides (NRRL 3631) show 99.8% identity at the 18S locus and 99.2% identity at the 28S locus. The strain of M. ramosissimus used in the Voigt et al. study, NRRL 3042, shows 100% identity at the 28S locus to the M. ramosissimus type strain included in our study, ATCC 28933. Consistent with the apparent monophylogeny of these species based on 18S and 28S analysis alone, we identified two clinical isolates (UWFP-777 and -969) that display >99% 28S DNA sequence identity to the type strain of M. ramosissimus (ATCC 28933); however, these isolates show only 98.9% identity at the 28S locus to that of M. circinelloides NRRL 3631, demonstrating that UWFP-777 and -969 are not M. circinelloides. ITS DNA sequence analyses clearly differentiate UWFP-777 and -969 from the M. ramosissimus type strain because they show significant sequence divergence at ITS1 and ITS2 (95.1 and 96.0%, respectively) compared with ATCC 28933. These strains may represent another species closely related to M. ramosissimus that would not be recognized by 28S or phenotypic analysis alone.

The Trichophyton rubrum complex has recently been characterized at the molecular level (17). A group of 11 species and varieties were reclassified as T. rubrum based on analysis of ITS1, ITS2, and 5.8S DNA sequence analyses, PCR fingerprinting, and amplified fragment length polymorphism analysis. ITS-based phylograms of this group revealed a tree in which 11 species and varieties grouped together in two distinct clades. The ITS DNA sequences from different species within the former T. rubrum group are not identical to each other, as evidenced by the arrangement of the ITS-based phylogenetic tree (17). Our analysis supports the polyphyletic nature of the newly designated T. rubrum group. The eight clinical isolates that we analyzed are 100% identical to each other at ITS1, ITS2, and 28S and are 100% identical at ITS1 and ITS2 to the strain designated the neotype strain for T. rubrum (CBS 392.68). Included in the T. rubrum group by Graser et al. (17) is the strain originally designated the type strain for Trichophyton fluviomuniense, CBS 592.68, which shows only 98.3% identity to the UWFP clinical isolates and to the T. rubrum neotype strain (CBS 392.68) at ITS1 and ITS2. Demonstration of <99% identity at two diagnostic loci suggests to us that this expanded T. rubrum group comprises at least two species, T. rubrum and T. fluviomuniense.

The methods we describe are rapid. After a mold is cultured on agar medium (72 h), identification can be achieved within 24 h. The 43 recognized species that we analyzed represent 19 genera; 80.3% of all mold isolates (n = 1,164, isolated in a 12-month period) identified by phenotypic methods in our laboratory belong to the 19 genera tested in this study. These 1,164 strains include 146 Penicillium isolates (12.5% of the total) that were not identified to the species level. An additional 112 isolates of the 1,164 (9.62%) were identified phenotypically as a mold but were not identified to the genus level. In the first 12 months in which DNA sequence analysis was integrated into our clinical laboratory's identification algorithm, 89 molds were identified by DNA sequence analyses. Isolates were analyzed genotypically if they did not produce reproductive structures within 10 days of isolation or if structures were produced but if phenotypic evaluation did not result in definitive identification. Fifty-seven of the 89 isolates produced only sterile hyphae after 10 days and, without DNA sequence analyses, would have been reported as M. sterilia or, if possible, more specifically identified after further incubation. Of these 57, 16 were identified to the species level, including three atypical A. fumigatus isolates, and 16 were identified to the genus level. The remaining 25 isolates were assigned a taxonomic orientation based on the relationship of their DNA sequences to those of related organisms in the database. Thus, DNA sequence-based methods can identify isolates that otherwise defy identification by traditional phenotypic analyses. The integration of multilocus sequence analyses with phenotype-based identification algorithms in a clinical laboratory provides a rapid and definitive identification that, in some cases, surpasses the specificity of identification by 28S sequence analyses and phenotypic methods alone (Table 2). We conclude that the phenotypically validated ITS DNA sequence database will be useful for identification of routinely isolated molds, previously unidentified molds, and molds that do not show specific or expected morphological and biochemical phenotypes.

	ACKNOWLEDGMENTS

 
We thank Jennifer Prentice for her technical assistance and expertise and Elaine Brooks for assistance preparing the manuscript.

This publication was made possible in part by grant number 7R44GM57669-04 from the National Institute of General Medical Sciences to Clarity BioSciences, Inc.

The contents of this paper are solely the responsibility of the authors and do not necessarily represent the official views of the NIGMS.

	FOOTNOTES

 
* Corresponding author. Mailing address: Departments of Laboratory Medicine and Microbiology, University of Washington, Box 357110, Seattle, WA 98195. Phone: (206) 598-6131. Fax: (206) 598-6189. E-mail: cookson{at}u.washington.edu.

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