This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Elie, C. M.
Right arrow Articles by Morrison, C. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Elie, C. M.
Right arrow Articles by Morrison, C. J.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, November 1998, p. 3260-3265, Vol. 36, No. 11
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Rapid Identification of Candida Species with Species-Specific DNA Probes

Cheryl M. Elie, Timothy J. Lott, Errol Reiss, and Christine J. Morrison*

Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia

Received 30 March 1998/Returned for modification 26 May 1998/Accepted 7 August 1998

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Rapid identification of Candida species has become more important because of an increase in infections caused by species other than Candida albicans, including species innately resistant to azole antifungal drugs. We previously developed a PCR assay with an enzyme immunoassay (EIA) format to detect amplicons from the five most common Candida species by using universal fungal primers and species-specific probes directed to the ITS2 region of the gene for rRNA. We designed probes to detect seven additional Candida species (C. guilliermondii, C. kefyr, C. lambica, C. lusitaniae, C. pelliculosa, C. rugosa, and C. zeylanoides) included in the API 20C sugar assimilation panel, five probes for species not identified by API 20C (C. haemulonii, C. norvegica, C. norvegensis, C. utilis, and C. viswanathii), and a probe for the newly described species C. dubliniensis, creating a panel of 18 Candida species probes. The PCR-EIA correctly identified multiple strains of each species tested, including five identified as C. albicans by the currently available API 20C database but determined to be C. dubliniensis by genotypic and nonroutine phenotypic characteristics. Species identification time was reduced from a mean of 3.5 days by conventional identification methods to 7 h by the PCR-EIA. This method is simple, rapid, and feasible for identifying Candida species in clinical laboratories that utilize molecular identification techniques and provides a novel method to differentiate the new species, C. dubliniensis, from C. albicans.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Rapid identification of Candida isolates to the species level in the clinical laboratory has become more important because the incidence of candidiasis continues to rise in proportion to a growing number of patients at risk for infection with Candida albicans and, recently, with innately azole-resistant non-albicans Candida species (5, 7, 29). This patient population has increased as a result of more intensive regimens of cancer therapy, complications of abdominal or cardiothoracic surgery, organ transplantations, burns, and trauma. Affected patients may be immunocompromised or not, and common risk factors include prolonged broad-spectrum antibiotic therapy, invasive devices such as indwelling Hickman catheters, and/or prolonged hospital stays (5, 7, 26). Under these conditions, an antibiotic-resistant replacement flora, including Candida species, can proliferate in the gut and invade deep tissues from mucosal foci. This is especially the case when mucosal integrity has been disrupted as a result of chemotherapy or surgery. In addition, as the number of risk factors increases, the odds of developing candidiasis multiply (26). Some Candida species, including C. glabrata and C. krusei, are emerging, possibly because they are innately less susceptible to azole drugs (16, 18, 29). In the case of C. parapsilosis, its ability to survive in the hospital environment, i.e., on the hands of healthcare workers, on intravenous devices, and in solutions, increases the possibility of its nosocomial transmission (5, 16). Consequently, rapid identification to the species level is necessary for more timely, targeted, and effective antifungal therapy and to facilitate hospital infection control measures.

Identification of Candida species by conventional morphology and assimilation tests can require 3 to 5 days or even longer for more difficult or unusual species (25). We previously employed universal fungal primers, multicopy gene targets, and species-specific probes directed to the ITS2 region of the rRNA-encoding gene (rDNA) to develop a rapid (1-day) PCR assay to detect candidemia (6, 20). Amplicons were detected in an enzyme immunoassay (EIA) format, and the method was referred to as PCR-EIA. Since the API 20C carbohydrate assimilation panel is limited to the identification of only certain species, DNA probes were designed to detect a total of 18 Candida species. Of these, the following 12 species can be identified by the current API 20C panel: C. albicans, C. glabrata, C. guilliermondii, C. kefyr, C. krusei, C. lambica, C. lusitaniae, C. parapsilosis, C. pelliculosa, C. rugosa, C. tropicalis, and C. zeylanoides. Five other, newly emerging Candida species, not identified by API 20C but readily identified by molecular probes, are C. haemulonii, C. norvegensis, C. norvegica, C. utilis, and C. viswanathii. A species-specific probe for the newly described Candida species, C. dubliniensis, was also designed. The resulting PCR-EIA identification matrix is simple, rapid, sensitive, and feasible for identifying Candida species in clinical laboratories.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Microorganisms. Clinical isolates or cultures obtained from the American Type Culture Collection (ATCC) were used in this study (see Tables 2 and 5). Isolates of Candida spp., Cryptococcus humicolus, Stephanoascus ciferrii, and Trichosporon cutaneum were grown in 50-ml Erlenmeyer flasks by seeding one 10-µl loopful of growth from an agar slant into 10 ml of YPD broth (1% yeast extract, 2% Bacto Peptone, 2% dextrose; Difco Laboratories, Detroit, Mich.). Cryptococcus neoformans serotypes A, B, C, and D were grown similarly; however, YPD broth was supplemented with 2.9% NaCl to reduce capsule formation. All broth cultures were grown at 35°C for 18 h in a rotary shaker set at 150 rpm prior to DNA extraction for prototype testing.

DNA isolation. DNA was extracted from all yeast species by using the Puregene DNA Isolation Kit (Gentra Systems Inc., Minneapolis, Minn.). This kit facilitates the rapid recovery of sufficient DNA for PCR amplification and allows multiple samples to be extracted in parallel. For example, multiple yeast isolates could be extracted at the same time so that a large number of samples could be processed quickly and efficiently on a given day. DNAs from filamentous and dimorphic fungi were obtained as previously described (6) or were a gift from Liliana de Aguirre, Instituto Investigaciones Veterinarias, Maracay, Venezuela. Quantification of DNA was performed by using a fluorometer and Hoechst 33258 Dye (Dyna Quant 200; Pharmacia Biotech, Piscataway, N.J.). DNA was diluted in TE buffer (10 mM Tris, 1 mM EDTA [pH 8.0]) so that a total of 1 ng of template DNA was added to each PCR vial.

Oligonucleotide synthesis of primers and probes. Oligodeoxyribonucleotide primers and probes were synthesized as described previously (6). Universal fungal primers ITS3 and ITS4 (28) were used to amplify the ITS2 region. Oligonucleotide probes were designed from sequence data for the ITS2 region of the Candida sp. rDNA (13, 14).

PCR amplification. The reaction mixture (100 µl) contained 10 µl of 10× PCR buffer (100 mM Tris-HCl, 500 mM KCl [pH 8.3]; Boehringer Mannheim, Indianapolis, Ind.), 6 µl of 25 mM MgCl2, 8 µl of a deoxynucleotide triphosphate mixture (1.25 mM each dATP, dCTP, dGTP, and dTTP), 1 µl of each primer (20 µM), 2.5 U of Taq DNA polymerase (TaKaRa Shuzo Co., Ltd., Shiga, Japan), 2 µl of template DNA (0.5 ng/µl), and sterile distilled water to bring the total volume to 100 µl. Vials were placed in the heating block of a model 9600 thermal cycler (Perkin-Elmer, Emeryville, Calif.) equilibrated at 95°C. PCR amplification conditions were 5 min of denaturation at 95°C, followed by 30 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 1 min. A final extension step of 72°C for 5 min was then conducted. Appropriate positive and negative controls were included, and PCR contamination precautions were followed (6, 9).

Agarose gel electrophoresis. Electrophoresis was conducted in TBE (0.1 M Tris, 0.09 M boric acid, 0.001 M EDTA [pH 8.4]) buffer at 76 V for approximately 1 h in gels composed of 1% (wt/vol) agarose (Boehringer Mannheim) and 1% (wt/vol) NuSieve (FMC Bioproducts, Rockland, Maine). Gels were stained with 0.5 µg of ethidium bromide per ml of deionized water for 30 min, followed by a 30-min wash in deionized water. DNA bands confirming a positive PCR were visualized with a UV transilluminator and photographed.

PCR-EIA. PCR-amplified DNA was hybridized to species-specific digoxigenin-labeled probes and to a generic biotinylated probe, and then the complex was added to streptavidin-coated microtitration plates and captured as previously described (6, 20). A colorimetric EIA was then conducted to detect captured DNA by using horseradish peroxidase-conjugated anti-digoxigenin antibodies (6, 20) in a manner very similar to that of other EIAs performed routinely in many clinical microbiology laboratories. All probes were tested in a matrix format against DNA from other Candida species, as well as against DNAs from other fungi (see Table 3). In this manner, all probes were tested against all of the target DNAs so that fungi could be identified by a discrete pattern of reactivity. When a probe cross-reacted with heterologous DNA, probes specific to the heterologous DNA were designed. Therefore, use of both probes as part of the matrix allows species-specific identification by a process of elimination and does not require additional steps or retesting of samples because all probes and all targets are included in the complete matrix from the beginning.

Statistical analyses. Student's t test was used to determine significant differences between mean absorbance values of homologous and nonhomologous probe reactions. Differences were considered significant when the value of P was less than or equal to 0.05.

Nucleotide sequence accession numbers. The GenBank accession numbers for C. dubliniensis and C. pelliculosa sequences are U96719 and U96720, respectively. The accession numbers for the DNA sequences of the other Candida species used in this study are published in references 13 and 14.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Specificity of digoxigenin-labeled probes. Eighteen Candida species probes (Table 1) were designed and tested in the PCR-EIA against the DNAs from the fungi listed in Table 2. The absorbance value obtained for each probe tested against its homologous DNA was significantly greater than that obtained when probes were tested against nonhomologous DNA (P <=  0.05), with two exceptions (Table 3). In the first instance, the probe for C. guilliermondii (GU) cross-reacted with DNA from C. zeylanoides (CZ). However, the probe for C. zeylanoides (CZ) did not cross-react with DNA from C. guilliermondii (GU). Therefore, by using both probes as part of the complete matrix, species-specific identification could be achieved by a process of elimination (Table 3). In the second instance, as previously described (6), the probe for C. glabrata, CG, cross-reacted with Saccharomyces cerevisiae DNA (mean absorbance, 0.882). In the present study, the CG probe also cross-reacted with C. pelliculosa and C. utilis DNAs, which had not been tested previously (mean absorbance ± standard deviation [SD], 0.810 ± 0.197 and 0.800 ± 0.648, respectively). Therefore, the CG probe was redesigned, resulting in the elimination of cross-reactions with all of the species tested except S. cerevisiae while retaining positive reactivity with homologous C. glabrata DNA (new CG probe named CGE, Table 3). Preliminary testing of an S. cerevisiae probe indicates that it does not cross-react with C. glabrata DNA (data not shown), allowing the differentiation of C. glabrata DNA from S. cerevisiae DNA.

                              
View this table:
[in this window]
[in a new window]
  		TABLE 1.   Synthetic oligonucleotides used in PCR and hybridization analyses

                              
View this table:
[in this window]
[in a new window]
  		TABLE 2.   Microorganisms tested against all probes

                              
View this table:
[in this window]
[in a new window]
  		TABLE 3.   Specificities of DNA probes

All of the negative controls tested, except S. cerevisiae and C. zeylanoides, as mentioned above, gave mean optical density OD values (± SD) ranging from 0.001 ± 0.001 to 0.009 ± 0.025 (Table 3). Minor cross-reaction was observed with the VS probe against C. tropicalis DNA (0.175 ± 0.093); however, the CT probe did not cross-react with C. viswanathii DNA (0.001 ± 0.001). Similarly, minor cross-reactions were observed when the CT and CU2 probes were used versus Aspergillus terreus (0.114 ± 0.052 and 0.147 ± 0.035, respectively), but an A. terreus-specific probe has been developed and has been reported separately (3). Finally, the CK probe demonstrated a minor cross-reaction with C. lambica DNA (0.114 ± 0.042), but the LA probe gave no cross-reaction with C. krusei DNA (0.002 ± 0.003). Therefore, all species could be differentiated by a process of elimination. In addition, compared to the absorbance values for their respective positive controls, the significantly weaker cross-reactions of the negative controls could be easily discriminated visually.

In addition, the ITS2 regions from seven strains of Candida famata were sequenced. Although all of the strains tested were obtained from the ATCC as C. famata or its teleomorph Debaryomyces hansenii, each strain showed sequence heterogeneity in this region (data not shown), suggesting that this species is a taxonomic complex of more than one species. An ITS2 probe designed for specificity to one of these strains was found to hybridize only with its own DNA and not to the DNA from any of the other four C. famata strains tested. None of the other non-C. famata Candida sp. probes reacted with DNA from the type culture of C. famata (ATCC 36239), indicating that C. famata (type culture) would not be misidentified as another Candida sp. by using these probes.

Multiple strains of several Candida species were tested, and some inherent variability in probe hybridization for strains of the same species was apparent in the range of standard deviations of the absorbance values observed (Table 4). However, all strains gave absorbance signals of sufficient strength to allow differentiation of truly positive from truly negative samples, with two exceptions. The CP probe tested against C. parapsilosis Lehmann group III DNAs (12) and the CH probe for C. haemulonii tested against C. haemulonii group 2 (strain 90.00.3593) DNA were significantly less reactive than with the respective positive control strains. These discrepant cases may indicate a finer taxonomic discrimination of isolates by genotypic than phenotypic methods (10-12, 30). Alternatively, combinations of probes could be designed for detection of all groups of C. parapsilosis and all groups of C. haemulonii in a clinical setting.

                              
View this table:
[in this window]
[in a new window]
  		TABLE 4.   Consistency of A650 values for isolates within a given species

Species-specific probes were also designed to discriminate between two species that have a phenotype in common. A new species, C. dubliniensis, first described by Sullivan et al. (23), is typically identified as C. albicans by routine phenotypic methods. The probes designed in this study readily discriminated C. albicans from C. dubliniensis (Table 5). The CA probe which detected C. albicans DNA did not react with DNA from any C. dubliniensis strain tested, and the DB probe for C. dubliniensis identification did not hybridize with DNA from any C. albicans strain tested. The CA probe also detected both C. stellatoidea type I and II DNAs and differentiated C. stellatoidea DNA from C. dubliniensis DNA (Table 5).

                              
View this table:
[in this window]
[in a new window]
  		TABLE 5.   Differentiation of C. albicans from C. dubliniensis by species-specific probes

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Previous research in this laboratory demonstrated that five Candida species-specific probes could be designed and adapted to a simple PCR-EIA format to detect Candida species DNA (6, 20). This report extends the range of probes to include a test matrix of 18 Candida species that is capable of complementing species identification by the API 20C carbohydrate assimilation system. Sixteen of the probes are totally specific and can be used to identify their respective Candida species, including C. dubliniensis. In addition, C. stellatoidea types I and II (10) can be differentiated from C. dubliniensis by these probes. The C. guilliermondii probe (GU) cross-reacted with C. zeylanoides DNA, but the C. zeylanoides probe (CZ) did not cross-react with C. guilliermondii DNA, allowing species-specific identification by a process of elimination. Future studies will attempt to design a species-specific probe for each. However, at present, positive identification can still be achieved by a process of elimination by using both probes in the matrix configuration. As designed, all of the probes can be used in the matrix at the same time so that all possible combinations of probes and target DNAs can be tested in a single run. As more probes are added to the matrix, automation and chip array technologies become attractive ways to identify large numbers of different organisms simply and rapidly. The previously published probe for C. glabrata, CG, was found to cross-react with C. pelliculosa and C. utilis DNAs in this study. Therefore, a new probe, CGE, was designed which eliminated all cross-reactions except for that with S. cerevisiae DNA. Such cross-reactivity should have little impact on clinical diagnosis, however, since it is unlikely, although not impossible, that S. cerevisiae would be found in blood cultures or other normally sterile deep tissue sites. Also, since clinical isolates of both C. glabrata and S. cerevisiae are innately fluconazole resistant (21, 24, 31), clinical treatment decisions would most likely not be negatively impacted by the inability to discriminate C. glabrata from S. cerevisiae. An S. cerevisiae probe has been developed (data not shown), and preliminary testing indicates that it is specific for S. cerevisiae detection. Therefore, this probe could be used in conjunction with the CGE probe to differentiate the two species by a process of elimination.

Standardization of DNA extraction for all Candida species is facilitated by using a broth culture method and a commercially available extraction kit. To facilitate prototype testing, isolates were grown overnight to obtain sufficiently large quantities of DNA for repeated analyses and probe development. In the clinical laboratory setting, sufficient DNA for routine testing, derived from primary cultures without subculturing, would further shorten the time required for species identification. Indeed, even species contained in mixed yeast cultures (C. albicans and C. glabrata) have been correctly identified from primary cultures in our laboratory by using species-specific probes (20).

All of the currently available commercial tests for species identification, such as the API 20C system, RapID, etc., require subculturing from clinical specimens to obtain pure cultures before inoculation of the test panels. Therefore, even if an overnight culture were required prior to PCR-EIA testing, the time to species identification after obtaining a pure culture is still reduced to 7 h rather than a mean of 3.5 days by conventional phenotypic identification methods. Also, species identification of unusual species such as C. norvegensis or C. utilis by conventional methods may require up to 4 or 5 weeks (25), whereas the PCR-EIA can identify these species in a single day. Since some species are innately resistant to certain drugs, e.g., C. krusei to fluconazole (16, 18, 29), accurate and timely species identification is important for selection of appropriately targeted therapy.

The recently described species C. dubliniensis was discovered when DNA from phenotypically identified "C. albicans" strains did not react with the C. albicans-specific mid-repetitive element Ca3 (2, 23). Molecular differences between C. albicans and C. dubliniensis were confirmed in the current research in that sufficiently significant sequence differences occurred in the ITS2 region to facilitate the development of species-specific probes. Although C. dubliniensis is not currently listed as one of the yeasts identified in the API 20C database, differences between C. albicans and C. dubliniensis in the assimilation of xylose and alpha -methyl-D-glucoside may prove useful for the phenotypic differentiation of these species (19). In addition, other physical (growth temperature) or chemical (beta -glucosidase activity) tests may also help discriminate C. albicans from C. dubliniensis (17, 22).

The C. dubliniensis strains listed in Table 5 were originally obtained in Europe and Australia. C. dubliniensis has also been isolated in North America, as reported by Kleinegger et al. (8) and Boucher et al. (1) in 1996. We confirmed the presence of C. dubliniensis in North America by using our probes to test DNA obtained from oropharyngeal isolates from a population of human immunodeficiency virus-positive persons in the Atlanta, Ga. area (4). To our knowledge, these are the first isolates of C. dubliniensis recovered from a human immunodeficiency virus-positive population in the United States (4, 19).

This genotype-based identification method has revealed the need for further taxonomic resolution of some species, for example, C. famata (teleomorph form, D. hansenii). Although the identities of seven C. famata strains tested in this study were confirmed by conventional phenotypic methods (data not shown), the ITS2 probe designed for one of these strains was found to hybridize only with its own DNA and not to the DNA of any of the other four C. famata strains tested. When the sequences of the ITS2 regions of these seven strains were determined and compared, greater differences were observed among these strains than among strains of other Candida species tested. Therefore, the complexity of this taxon is apparent and agrees with the DNA-DNA hybridization studies of Nishikawa et al. (15), who described a low percentage of hybridization between some DNAs from several C. famata strains. Because C. famata appears to be a taxonomic complex, further studies are needed to determine sequences which will allow the detection of clinically encountered members of this complex either by redefinition of these strains into subspecies or by the use of combinations of probes to simultaneously detect members of the complex. Additional molecular characterization is needed to clarify the taxonomy and identification of strains which appear to be C. famata by phenotypic criteria but differ by genotypic criteria.

Lower absorbance values were obtained for some strains of C. parapsilosis (group III) and C. haemulonii (group 2) than for their positive control strains. However, these values were still significantly greater than those for their respective negative controls. Because of the controversy surrounding C. parapsilosis group III and C. haemulonii group 2, the designation of a truly positive cutoff value for these groups awaits taxonomic resolution. The clinical significance of C. parapsilosis group III and C. haemulonii group 2 is not known, nor are the true incidence and prevalence of these groups. However, the CP probe correctly identified all clinical blood isolates of C. parapsilosis in a previous study (20). Therefore, it is likely that the CP probe, at least, can identify clinically relevant strains. The same is yet to be determined for the CH probe. In addition, combinations of probes, used in one reaction mixture, to identify all groups of C. parapsilosis and all groups of C. haemulonii could be designed if such a need were identified in the clinical setting.

The present method of sample preparation and PCR-EIA is amenable to automation, and the entire panel of 18 different probes can be tested against an unknown yeast in a simple microtitration plate format. Greater numbers of isolates will be tested in prospective clinical studies to validate the specificity of each probe. These probes were designed to detect 1 ng of target DNA recovered from Candida sp. cultures, and the limit of their sensitivity has not yet been determined in clinical samples. Previous studies using positive blood culture bottles suggest that these probes may be useful for the direct identification of Candida species from primary cultures (20). Minor probe cross-reactions should not be problematic, since a process of elimination with specific, non-cross-reacting, paired probes allows specific differentiation (e.g., the CK probe and C. lambica DNA). When sensitivity limits become an issue, such as in clinical samples containing unknown quantities of DNA, then a matrix of probes will differentiate truly positive samples from truly negative samples. Ultimately, once a sufficient panel of medically important Candida and/or yeast species has been constructed, the ideal assay will consist of probes attached to a nylon membrane or to wells of a microtiter plate, a single universal PCR using premixed PCR reagents, and the colorimetric development of the matrix array in a single assay. Commercially prepared, premixed PCR reagents are available, and prototype assays with such configurations have already been examined in our laboratory (27) and continue to be improved to optimize the speed and simplicity of species-specific yeast identification.

    ACKNOWLEDGMENTS

This research was supported in part through an Emerging Infectious Diseases Training Fellowship from the Association of State and Territorial Public Health Laboratory Directors for C.M.E. and also by an appointment to the Research Participation Program at the CDC, National Center for Infectious Diseases, Division of Bacterial and Mycotic Diseases, administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and CDC.

We thank J. H. Shin, Chonnam University Medical School, Kwangju, Korea, for blood culture isolates of C. pelliculosa and C. guilliermondii; R. Cherniak, Georgia State University, Atlanta, for isolates of C. neoformans serotypes A, B, C, and D; P. F. Lehmann, Medical College of Ohio, Toledo, for isolates of C. parapsilosis groups I, II, and III; D. Ahearn and S. Meyer, Georgia State University, Atlanta, for isolates of C. haemulonii and C. dubliniensis; S. Lockhart, University of Iowa, Iowa City, for isolates of C. dubliniensis; L. de Aguirre, Instituto Investigaciones Veterinarias, Maracay, Venezuela, for DNA extracted from filamentous fungi; and B. A. Lasker, CDC, Atlanta, Ga., for DNA extracted from dimorphic fungi and S. cerevisiae.

    FOOTNOTES

* Corresponding author. Mailing address: Mailstop G-11, Centers for Disease Control and Prevention, Atlanta, GA 30333. Phone: (404) 639-3128. Fax: (404) 639-3546. E-mail: cjm3{at}cdc.gov.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1. Boucher, H., S. Mercure, S. Montplaisir, and G. Lemay. 1996. A novel group I intron in Candida dubliniensis is homologous to a Candida albicans intron. Gene 180:189-196[Medline].
2. Coleman, D. C., D. J. Sullivan, D. E. Bennett, G. P. Moran, H. J. Barry, and D. B. Shanley. 1997. Candidiasis: the emergence of a novel species, Candida dubliniensis. AIDS 11:557-567[Medline].
3. de Aguirre, L. A., H. Vaishnav, J. M. Westerman, E. Reiss, and C. J. Morrison. 1997. Rapid differentiation of Aspergillus species from other filamentous fungi and yeasts using species-specific DNA probes, abstr. C234, p. 161. In Abstracts of the 97th General Meeting of the American Society for Microbiology 1997. American Society for Microbiology, Washington, D.C.
4. Elie, C. M., B. A. Lasker, L. W. Mayer, W. R. Pruitt, G. Smith, D. Rimland, L. Gallagher, E. Reiss, C. J. Morrison, and M. M. McNeil. 1998. Rapid differentiation of Candida dubliniensis from atypical C. albicans isolates using species-specific DNA probes, abstr. C290, p. 179. In Abstracts of the 98th General Meeting of the American Society for Microbiology 1998. American Society for Microbiology, Washington, D.C.
5. Fridkin, S. K., and W. R. Jarvis. 1996. Epidemiology of nosocomial fungal infections. Clin. Microbiol. Rev. 9:499-511[Abstract].
6. Fujita, S.-I., B. A. Lasker, T. J. Lott, E. Reiss, and C. J. Morrison. 1995. Microtitration plate enzyme immunoassay to detect PCR-amplified DNA from Candida species in blood. J. Clin. Microbiol. 33:962-967[Abstract].
7. Jarvis, W. R. 1995. Epidemiology of nosocomial fungal infections, with emphasis on Candida species. Clin. Infect. Dis. 20:1526-1530[Medline].
8. Kleinegger, C. L., S. R. Lockhart, K. Vargas, and D. R. Soll. 1996. Frequency, intensity, species, and strains of oral Candida vary as a function of host age. J. Clin. Microbiol. 34:2246-2254[Abstract].
9. Kwok, S., and R. Higuchi. 1989. Avoiding false positives with PCR. Nature (London) 339:237-238[Medline].
10. Kwon-Chung, K. J., W. S. Riggsby, R. A. Uphoff, J. B. Hicks, W. L. Whelan, E. Reiss, B. B. Magee, and B. L. Wickes. 1989. Genetic differences between type I and type II Candida stellatoidea. Infect. Immun. 57:527-532[Abstract/Free Full Text].
11. Lehmann, P. F., L.-C. Wu, W. R. Pruitt, S. A. Meyer, and D. G. Ahearn. 1993. Unrelatedness of groups of yeasts within the Candida haemulonii complex. J. Clin. Microbiol. 31:1683-1687[Abstract/Free Full Text].
12. Lin, D., L.-C. Wu, M. G. Rinaldi, and P. F. Lehmann. 1995. Three distinct genotypes within Candida parapsilosis from clinical sources. J. Clin. Microbiol. 33:1815-1821[Abstract].
13. Lott, T. J., B. M. Burns, R. Zancope-Oliveira, C. M. Elie, and E. Reiss. 1998. Sequence analysis of the internal transcribed spacer 2 (ITS2) from yeast species within the genus Candida. Curr. Microbiol. 36:63-69[Medline].
14. Lott, T. J., R. Kuykendall, and E. Reiss. 1993. Nucleotide sequence analysis of the 5.8S rDNA and adjacent ITS2 region of Candida albicans and related species. Yeast 2:1199-1206.
15. Nishikawa, H., H. Tomomatsu, T. Sugita, R. Ikeda, and T. Shinoda. 1996. Taxonomic position of clinical isolates of Candida famata. J. Med. Vet. Mycol. 34:411-419[Medline].
16. Pfaller, M. A. 1995. Nosocomial fungal infections: epidemiology of candidiasis. J. Hosp. Infect. 30(Suppl.):329-338.
17. Pinjon, E., D. Sullivan, I. Salkin, D. Shanley, and D. Coleman. 1998. Simple, inexpensive, reliable method for differentiation of Candida dubliniensis from Candida albicans. J. Clin. Microbiol. 36:2093-2095[Abstract/Free Full Text].
18. Rex, J. H., M. A. Pfaller, A. L. Barry, P. W. Nelson, and C. D. Webb for the NIAID Mycoses Study Group, and The Candidemia Study Group. 1995. Antifungal susceptibility testing of isolates from a randomized multicenter trial of fluconazole versus amphotericin B as treatment of nonneutropenic patients with candidemia. Antimicrob. Agents Chemother. 39:40-44[Abstract].
19. Salkin, I. F., W. R. Pruitt, A. A. Padhye, D. Sullivan, D. Coleman, and D. H. Pincus. 1998. Distinctive carbohydrate assimilation profiles used to identify the first clinical isolates of Candida dubliniensis recovered in the United States. J. Clin. Microbiol. 36:1467[Free Full Text]. (Letter.)
20. Shin, J. H., F. S. Nolte, and C. J. Morrison. 1997. Rapid identification of Candida species in blood culture by a clinically useful polymerase chain reaction method. J. Clin. Microbiol. 35:1454-1459[Abstract].
21. Sobel, J. D., J. Vazquez, M. Lynch, C. Meriwether, and M. J. Zervos. 1993. Vaginitis due to Saccharomyces cerevisiae: epidemiology, clinical aspects, and therapy. Clin. Infect. Dis. 16:93-99[Medline].
22. Sullivan, D., and D. Coleman. 1998. Candida dubliniensis: characteristics and identification. J. Clin. Microbiol. 36:329-334[Free Full Text].
23. Sullivan, D. J., T. J. Westerneng, K. A. Haynes, D. E. Bennett, and D. C. Coleman. 1995. Candida dubliniensis sp. nov.: phenotypic and molecular characterization of a novel species associated with oral candidosis in HIV-infected individuals. Microbiology 141:1507-1521[Abstract/Free Full Text].
24. Tiballi, R. N., J. E. Spiegel, L. T. Zarins, and C. A. Kauffman. 1995. Saccharomyces cerevisiae infections and antifungal susceptibility studies by colorimetric and broth macrodilution methods. Diagn. Microbiol. Infect. Dis. 23:135-140[Medline].
25. Warren, N. G., and K. C. Hazen. 1995. Candida, Cryptococcus, and other yeasts of medical importance, p. 723-737. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.), Manual of clinical microbiology, 6th ed. American Society for Microbiology, Washington, D.C.
26. Wenzel, R. P. 1995. Nosocomial candidemia: risk factors and attributable mortality. Clin. Infect. Dis. 20:1531-1534[Medline].
27. Westerman, J. M., C. M. Elie, and C. J. Morrison. 1998. Improved identification of Candida species using biotinylated species-specific DNA probes, abstr. J107, p. 481. In Abstracts of the 38th Interscience Conference on Antimicrobial Agents and Chemotherapy. American Society for Microbiology, Washington, D.C.
28. White, T. J., T. D. Burns, S. B. Lee, and J. W. Taylor. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, p. 315-322. In M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White (ed.), PCR protocols. Academic Press, San Diego, Calif.
29. Wingard, J. R. 1995. Importance of Candida species other than C. albicans as pathogens in oncology patients. Clin. Infect. Dis. 20:115-125[Medline].
30. Zeng, S., L.-C. Wu, and P. F. Lehmann. 1996. Random amplified polymorphic DNA analysis of culture collection strains of Candida species. J. Med. Vet. Mycol. 34:293-297[Medline].
31. Zerva, L., R. J. Hollis, and M. A. Pfaller. 1996. In vitro susceptibility testing and DNA typing of Saccharomyces cerevisiae clinical isolates. J. Clin. Microbiol. 34:3031-3034[Abstract].

Journal of Clinical Microbiology, November 1998, p. 3260-3265, Vol. 36, No. 11
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Wang, Q.-M., Li, J., Wang, S.-A., Bai, F.-Y. (2008). Rapid Differentiation of Phenotypically Similar Yeast Species by Single-Strand Conformation Polymorphism Analysis of Ribosomal DNA. Appl. Environ. Microbiol. 74: 2604-2611 [Abstract] [Full Text]  
  • Zeng, X., Kong, F., Halliday, C., Chen, S., Lau, A., Playford, G., Sorrell, T. C. (2007). Reverse Line Blot Hybridization Assay for Identification of Medically Important Fungi from Culture and Clinical Specimens. J. Clin. Microbiol. 45: 2872-2880 [Abstract] [Full Text]  
  • Baskova, L., Landlinger, C., Preuner, S., Lion, T. (2007). The Pan-AC assay: a single-reaction real-time PCR test for quantitative detection of a broad range of Aspergillus and Candida species. J Med Microbiol 56: 1167-1173 [Abstract] [Full Text]  
  • Metwally, L., Hogg, G., Coyle, P. V., Hay, R. J., Hedderwick, S., McCloskey, B., O'Neill, H. J., Ong, G. M., Thompson, G., Webb, C. H., McMullan, R. (2007). Rapid differentiation between fluconazole-sensitive and -resistant species of Candida directly from positive blood-culture bottles by real-time PCR. J Med Microbiol 56: 964-970 [Abstract] [Full Text]  
  • Medeiros, E. A. S., Lott, T. J., Colombo, A. L., Godoy, P., Coutinho, A. P., Braga, M. S., Nucci, M., Brandt, M. E. (2007). Evidence for a Pseudo-Outbreak of Candida guilliermondii Fungemia in a University Hospital in Brazil. J. Clin. Microbiol. 45: 942-947 [Abstract] [Full Text]  
  • Leaw, S. N., Chang, H. C., Sun, H. F., Barton, R., Bouchara, J.-P., Chang, T. C. (2006). Identification of medically important yeast species by sequence analysis of the internal transcribed spacer regions.. J. Clin. Microbiol. 44: 693-699 [Abstract] [Full Text]  
  • Playford, E. G., Kong, F., Sun, Y., Wang, H., Halliday, C., Sorrell, T. C. (2006). Simultaneous detection and identification of Candida, Aspergillus, and cryptococcus species by reverse line blot hybridization.. J. Clin. Microbiol. 44: 876-880 [Abstract] [Full Text]  
  • Marot-Leblond, A., Beucher, B., David, S., Nail-Billaud, S., Robert, R. (2006). Development and Evaluation of a Rapid Latex Agglutination Test Using a Monoclonal Antibody To Identify Candida dubliniensis Colonies. J. Clin. Microbiol. 44: 138-142 [Abstract] [Full Text]  
  • Leinberger, D. M., Schumacher, U., Autenrieth, I. B., Bachmann, T. T. (2005). Development of a DNA Microarray for Detection and Identification of Fungal Pathogens Involved in Invasive Mycoses. J. Clin. Microbiol. 43: 4943-4953 [Abstract] [Full Text]  
  • Diaz, M. R., Fell, J. W. (2005). Use of a Suspension Array for Rapid Identification of the Varieties and Genotypes of the Cryptococcus neoformans Species Complex. J. Clin. Microbiol. 43: 3662-3672 [Abstract] [Full Text]  
  • Borst, A., Raimer, M. T., Warnock, D. W., Morrison, C. J., Arthington-Skaggs, B. A. (2005). Rapid Acquisition of Stable Azole Resistance by Candida glabrata Isolates Obtained before the Clinical Introduction of Fluconazole. Antimicrob. Agents Chemother. 49: 783-787 [Abstract] [Full Text]  
  • Gutzmer, R., Mommert, S., Kuttler, U., Werfel, T., Kapp, A. (2004). Rapid identification and differentiation of fungal DNA in dermatological specimens by LightCycler PCR. J Med Microbiol 53: 1207-1214 [Abstract] [Full Text]  
  • Marot-Leblond, A., Grimaud, L., David, S., Sullivan, D. J., Coleman, D. C., Ponton, J., Robert, R. (2004). Evaluation of a Rapid Immunochromatographic Assay for Identification of Candida albicans and Candida dubliniensis. J. Clin. Microbiol. 42: 4956-4960 [Abstract] [Full Text]  
  • Khan, Z. U., Ahmad, S., Mokaddas, E., Chandy, R. (2004). Tobacco Agar, a New Medium for Differentiating Candida dubliniensis from Candida albicans. J. Clin. Microbiol. 42: 4796-4798 [Abstract] [Full Text]  
  • de Aguirre, L., Hurst, S. F., Choi, J. S., Shin, J. H., Hinrikson, H. P., Morrison, C. J. (2004). Rapid Differentiation of Aspergillus Species from Other Medically Important Opportunistic Molds and Yeasts by PCR-Enzyme Immunoassay. J. Clin. Microbiol. 42: 3495-3504 [Abstract] [Full Text]  
  • Ahmad, S., Khan, Z., Mokaddas, E., Khan, Z. U. (2004). Isolation and molecular identification of Candida dubliniensis from non-human immunodeficiency virus-infected patients in Kuwait. J Med Microbiol 53: 633-637 [Abstract] [Full Text]  
  • Schurko, A. M., Mendoza, L., de Cock, A. W. A. M., Bedard, J. E. J., Klassen, G. R. (2004). Development of a Species-Specific Probe for Pythium insidiosum and the Diagnosis of Pythiosis. J. Clin. Microbiol. 42: 2411-2418 [Abstract] [Full Text]  
  • Hajjeh, R. A., Sofair, A. N., Harrison, L. H., Lyon, G. M., Arthington-Skaggs, B. A., Mirza, S. A., Phelan, M., Morgan, J., Lee-Yang, W., Ciblak, M. A., Benjamin, L. E., Thomson Sanza, L., Huie, S., Yeo, S. F., Brandt, M. E., Warnock, D. W. (2004). Incidence of Bloodstream Infections Due to Candida Species and In Vitro Susceptibilities of Isolates Collected from 1998 to 2000 in a Population-Based Active Surveillance Program. J. Clin. Microbiol. 42: 1519-1527 [Abstract] [Full Text]  
  • Johnson, M. D., MacDougall, C., Ostrosky-Zeichner, L., Perfect, J. R., Rex, J. H. (2004). Combination Antifungal Therapy. Antimicrob. Agents Chemother. 48: 693-715 [Full Text]  
  • Coignard, C., Hurst, S. F., Benjamin, L. E., Brandt, M. E., Warnock, D. W., Morrison, C. J. (2004). Resolution of Discrepant Results for Candida Species Identification by Using DNA Probes. J. Clin. Microbiol. 42: 858-861 [Abstract] [Full Text]  
  • Selvarangan, R., Bui, U., Limaye, A. P., Cookson, B. T. (2003). Rapid Identification of Commonly Encountered Candida Species Directly from Blood Culture Bottles. J. Clin. Microbiol. 41: 5660-5664 [Abstract] [Full Text]  
  • Hsu, M.-C., Chen, K.-W., Lo, H.-J., Chen, Y.-C., Liao, M.-H., Lin, Y.-H., Li, S.-Y. (2003). Species identification of medically important fungi by use of real-time LightCycler PCR. J Med Microbiol 52: 1071-1076 [Abstract] [Full Text]  
  • Fotedar, R., Al Hedaithy, S. S. A. (2003). Candida dubliniensis at a University Hospital in Saudi Arabia. J. Clin. Microbiol. 41: 1907-1911 [Abstract] [Full Text]  
  • Borst, A., Theelen, B., Reinders, E., Boekhout, T., Fluit, A. C., Savelkoul, P. H. M. (2003). Use of Amplified Fragment Length Polymorphism Analysis To Identify Medically Important Candida spp., Including C. dubliniensis. J. Clin. Microbiol. 41: 1357-1362 [Abstract] [Full Text]  
  • Bautista-Munoz, C., Boldo, X. M., Villa-Tanaca, L., Hernandez-Rodriguez, C. (2003). Identification of Candida spp. by Randomly Amplified Polymorphic DNA Analysis and Differentiation between Candida albicans and Candida dubliniensis by Direct PCR Methods. J. Clin. Microbiol. 41: 414-420 [Abstract] [Full Text]  
  • Xu, J, Millar, B C, Moore, J E, McClurg, R, Walker, M J, Evans, J, Hedderwick, S, McMullan, R (2002). Comparison of API20C with molecular identification of Candida spp isolated from bloodstream infections. J. Clin. Pathol. 55: 774-777 [Abstract] [Full Text]  
  • Luo, G., Mitchell, T. G. (2002). Rapid Identification of Pathogenic Fungi Directly from Cultures by Using Multiplex PCR. J. Clin. Microbiol. 40: 2860-2865 [Abstract] [Full Text]  
  • Yeo, S. F., Wong, B. (2002). Current Status of Nonculture Methods for Diagnosis of Invasive Fungal Infections. Clin. Microbiol. Rev. 15: 465-484 [Abstract] [Full Text]  
  • Rodero, L., Cuenca-Estrella, M., Cordoba, S., Cahn, P., Davel, G., Kaufman, S., Guelfand, L., Rodriguez-Tudela, J. L. (2002). Transient Fungemia Caused by an Amphotericin B-Resistant Isolate of Candida haemulonii. J. Clin. Microbiol. 40: 2266-2269 [Abstract] [Full Text]  
  • Gee, S. F., Joly, S., Soll, D. R., Meis, J. F. G. M., Verweij, P. E., Polacheck, I., Sullivan, D. J., Coleman, D. C. (2002). Identification of Four Distinct Genotypes of Candida dubliniensis and Detection of Microevolution In Vitro and In Vivo. J. Clin. Microbiol. 40: 556-574 [Abstract] [Full Text]  
  • Oliveira, K., Haase, G., Kurtzman, C., Hyldig-Nielsen, J. J., Stender, H. (2001). Differentiation of Candida albicans and Candida dubliniensis by Fluorescent In Situ Hybridization with Peptide Nucleic Acid Probes. J. Clin. Microbiol. 39: 4138-4141 [Abstract] [Full Text]  
  • Chang, H. C., Leaw, S. N., Huang, A. H., Wu, T. L., Chang, T. C. (2001). Rapid Identification of Yeasts in Positive Blood Cultures by a Multiplex PCR Method. J. Clin. Microbiol. 39: 3466-3471 [Abstract] [Full Text]  
  • Lindsley, M. D., Hurst, S. F., Iqbal, N. J., Morrison, C. J. (2001). Rapid Identification of Dimorphic and Yeast-Like Fungal Pathogens Using Specific DNA Probes. J. Clin. Microbiol. 39: 3505-3511 [Abstract] [Full Text]  
  • Guiver, M, Levi, K, Oppenheim, B A (2001). Rapid identification of candida species by TaqMan PCR. J. Clin. Pathol. 54: 362-366 [Abstract] [Full Text]  
  • Martin, C., Roberts, D., van der Weide, M., Rossau, R., Jannes, G., Smith, T., Maher, M. (2000). Development of a PCR-Based Line Probe Assay for Identification of Fungal Pathogens. J. Clin. Microbiol. 38: 3735-3742 [Abstract] [Full Text]  
  • Park, S., Wong, M., Marras, S. A. E., Cross, E. W., Kiehn, T. E., Chaturvedi, V., Tyagi, S., Perlin, D. S. (2000). Rapid Identification of Candida dubliniensis Using a Species-Specific Molecular Beacon. J. Clin. Microbiol. 38: 2829-2836 [Abstract] [Full Text]  
  • Chen, Y. C., Eisner, J. D., Kattar, M. M., Rassoulian-Barrett, S. L., LaFe, K., Yarfitz, S. L., Limaye, A. P., Cookson, B. T. (2000). Identification of Medically Important Yeasts Using PCR-Based Detection of DNA Sequence Polymorphisms in the Internal Transcribed Spacer 2 Region of the rRNA Genes. J. Clin. Microbiol. 38: 2302-2310 [Abstract] [Full Text]  
  • Posteraro, B., Sanguinetti, M., Masucci, L., Romano, L., Morace, G., Fadda, G. (2000). Reverse Cross Blot Hybridization Assay for Rapid Detection of PCR-Amplified DNA from Candida Species, Cryptococcus neoformans, and Saccharomyces cerevisiae in Clinical Samples. J. Clin. Microbiol. 38: 1609-1614 [Abstract] [Full Text]  
  • Polacheck, I., Strahilevitz, J., Sullivan, D., Donnelly, S., Salkin, I. F., Coleman, D. C. (2000). Recovery of Candida dubliniensis from Non-Human Immunodeficiency Virus-Infected Patients in Israel. J. Clin. Microbiol. 38: 170-174 [Abstract] [Full Text]  
  • Turenne, C. Y., Sanche, S. E., Hoban, D. J., Karlowsky, J. A., Kabani, A. M. (1999). Rapid Identification of Fungi by Using the ITS2 Genetic Region and an Automated Fluorescent Capillary Electrophoresis System. J. Clin. Microbiol. 37: 1846-1851 [Abstract] [Full Text]  
  • Shin, J. H., Nolte, F. S., Holloway, B. P., Morrison, C. J. (1999). Rapid Identification of up to Three Candida Species in a Single Reaction Tube by a 5' Exonuclease Assay Using Fluorescent DNA Probes. J. Clin. Microbiol. 37: 165-170 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Elie, C. M.
Right arrow Articles by Morrison, C. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Elie, C. M.
Right arrow Articles by Morrison, C. J.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»