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MARY ANN JABRA-RIZK,1* A. A. M. A. BAQUI,1 JACQUELINE I. KELLEY,1. WILLIAM ..... Elie, C. M., B. A. Lasker, L. W. Mayer, W. R. Pruitt, G. Smith, D. Rimland,.
JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1999, p. 321–326 0095-1137/99/$04.0010 Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Vol. 37, No. 2

Identification of Candida dubliniensis in a Prospective Study of Patients in the United States MARY ANN JABRA-RIZK,1* A. A. M. A. BAQUI,1 JACQUELINE I. KELLEY,1 WILLIAM A. FALKLER, JR.,2 WILLIAM G. MERZ,3 1 AND TIMOTHY F. MEILLER Department of Oral Medicine1 and Department of OCBS,2 Dental School, University of Maryland, and Department of Pathology, The Johns Hopkins University,3 Baltimore, Maryland Received 23 July 1998/Returned for modification 9 October 1998/Accepted 31 October 1998

Although Candida albicans remains the fungal species most frequently isolated as an opportunistic oral pathogen, other yeast species are often identified in human immunodeficiency virus (HIV)-seropositive patients. Candida dubliniensis phenotypically resembles C. albicans in many respects, yet it can be identified and differentiated as a unique Candida species by its phenotypic and genetic profiles. The purpose of the present study was to prospectively test for the presence of C. dubliniensis among clinical isolates and to determine the clinical and demographic characteristics of patients harboring C. dubliniensis. Over a 90-day period, isolates from 724 patients that were presumptively identified as C. albicans were screened for C. dubliniensis by use of tests for germ tube and chlamydospore production, by detection of an inability to grow at 45°C, by colony color on CHROMagar Candida medium, and by the results of a sugar assimilation test with the API 20C AUX yeast identification system. Among 699 isolates retrieved from those specimens evaluated, 5 from 25 HIV-seropositive patients and 1 isolate from a patient whose HIV status was unknown were shown to be consistent by phenotyping and by electrophoretic karyotyping with the European reference strain of C. dubliniensis. One of the C. dubliniensis isolates had dose-dependent susceptibility to fluconazole (MIC, 16 mg/ml). These results confirm the presence of this interesting species in the United States and support the need for further investigations into the prevalence and pathogenesis of C. dubliniensis. Currently, methods for rapid identification and determination of the specific species of Candida, including C. dubliniensis and atypical Candida species that cause infections, are under study in various laboratories (3, 22). Numerous researchers are attempting to develop molecular probes (3) and to identify more detailed protein patterns for C. dubliniensis in order to further delineate the epidemiology and pathogenesis of this organism in HIV-infected patients. Although the extent of its contribution to the opportunistic infection of the oropharyngeal complex is not yet known, concerns over the occurrence of fluconazole resistance in clinical isolates have been raised. The observation of readily inducible stable fluconazole resistance in vitro has been made (11). If, indeed, C. dubliniensis represents a species that can rapidly develop resistance to antifungal therapy, then patients who have received multiple treatments for fungal infections throughout the course of their AIDS disease may be at increased risk for harboring C. dubliniensis as the predominant species in their oral cavities. The following are the results of a prospective study designed to recover and identify C. dubliniensis and presumptive clinical C. albicans isolates from the oral cavities of HIV-seropositive individuals.

Among the many opportunistic infections observed in human immunodeficiency virus (HIV)-infected patients, oral candidiasis ranks high in terms of incidence. The yeast Candida albicans has long been considered the predominant etiologic agent of candidiasis. Over the last decade, however, there has been an increase in the incidence in immunocompromised individuals of candidiasis caused by other Candida species, such as C. tropicalis, C. krusei, C. glabrata, and C. lusitaniae (10, 24, 26, 27). In 1995, a new species of Candida which had phenotypic characteristics similar to those of C. albicans was characterized and was named C. dubliniensis (19, 20). The clinical significance of any new species, including C. dubliniensis, is primarily based on the ability to determine if the pathogenesis or management of the infection is different from that of an infection caused by other members of the genus, especially C. albicans. For C. dubliniensis, the clinical significance seems to be its association with HIV-seropositive individuals (1, 2, 8, 11, 20– 23). Furthermore, within the HIV-seropositive population, intravenous drug abuse has been reported as a risk factor in those patients found to be harboring C. dubliniensis (1, 21). To date, virulence factors exclusive to C. dubliniensis have not been elucidated but are under investigation (8). Standard clinical laboratory procedure designates yeast cultures that form germ tubes and chlamydospores as members of the species C. albicans (12). Since C. dubliniensis strains share these characteristics, it is likely that some C. dubliniensis strains have been and will continue to be identified in the clinical laboratory as C. albicans.

MATERIALS AND METHODS Presumptive C. albicans isolates. Isolates (n 5 699) collected between January and March 1998 were identified, by using standard criteria (25), as C. albicans by the clinical laboratories at the University of Maryland and The Johns Hopkins Medical Institutions. They were reevaluated by differential growth on Sabouraud dextrose agar (SDA; Difco Laboratories, Detroit, Mich.) at 45°C as the first step in identifying any isolate that may have been C. dubliniensis. Isolates from HIV-seropositive patients. Twenty-five additional isolates recovered from HIV-seropositive individuals managed at the University of Maryland Dental School were also included for a final sample population of 724. Of those HIV-infected patients sampled, 26% were females and 74% were males, with an age range of 28 to 46 years. Oral samples were obtained from the middorsum of the tongue with a sterile swab, which was immediately used to inoculate and

* Corresponding author. Mailing address: Department of Oral Medicine, Dental School, UMAB, 666 W. Baltimore St., Baltimore, MD 21201. Phone: (410) 708-7628. Fax: (410) 706-0519. E-mail: mrizk @umaryland.edu. 321

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J. CLIN. MICROBIOL. al. (6). Briefly, yeast isolates were incubated overnight in yeast extract-peptonedextrose broth medium. The cells were packed by centrifugation, washed twice, and suspended in sodium EDTA. Spheroblasts were produced by treating the yeast cells with lyticase (Sigma Chemical Co., St. Louis, Mo.) at 37°C for 20 min. Following incubation, the yeast suspension was then mixed with low-meltingpoint agarose, and aliquots of the yeast-agarose suspension were placed in individual molds to form agarose plugs. The hardened agarose plugs were then incubated at 50°C for 15 h in buffer containing proteinase K. Agarose plugs in which yeast chromosomal DNA was embedded were loaded into the wells of a 1.0% agarose gel in 0.53 Tris-borate-EDTA (TBE). Electrophoresis was carried out in 0.53 TBE at 4°C with a contour-clamped homogeneous field electrophoresis system (CHEF-DR II; Bio-Rad, Hercules, Calif.), with 120-s pulses of 120 V for 24 h followed by 350-s alternating pulses at 150 V for 48 h. The gels were stained with ethidium bromide. The bands were visualized with UV light (354 nm), photographed, and scanned to develop a permanent record. All isolates meeting the phenotypic criteria of C. dubliniensis, a C. albicans control, and C. dubliniensis CD36 were karyotyped. Electrophoretic karyotyping was repeated four times for each of the isolates, reference strains, and controls. In order to begin to investigate strain stability, two samples were taken from a single patient 5 weeks apart and were used in the karyotyping experiments as two separate samples, resulting in the evaluation of a total of seven isolates. Fluconazole susceptibility testing. A broth macrodilution susceptibility assay was carried out by the method outlined in the report describing the National Committee for Clinical Laboratory Standards M27-A reference method (12). Briefly, drug screens were prepared in the following way: Twofold dilutions of fluconazole were made in sterile distilled water to final concentrations ranging from 64.0 to 0.125 mg/ml. The MIC at 48 h was used to determine resistance or susceptibility. The MIC was recorded as the highest concentration of drug demonstrating at least an 80% reduction in turbidity compared to that for the positive control tube. Interpretation of the results was performed according to the guidelines of Rex et al. (16), as follows: an MIC at 48 h of #8 mg/ml, susceptible, an MIC of 16 to 32 mg/ml, dose-dependent susceptible; and an MIC of $64 mg/ml, resistant (4, 12, 16). For quality control of susceptibility testing, purity plates were checked at 24 or 48 h for organism purity, and the numbers of CFU per milliliter were determined to ensure proper dilution.

RESULTS FIG. 1. Identification of germ tube- and chlamydospore-positive yeast-like colonies after 48 h of incubation at 37°C. a, observed during initial growth and may wane upon subculturing; b, in the bioMerieux Vitek API 20C AUX database, there is no current assimilation profile which will identify C. dubliniensis. streak an SDA plate for organism isolation. Figure 1 illustrates the entire diagnostic protocol used to eliminate detailed tests for other Candida species and, ultimately, to identify those isolates with characteristics consistent with those of C. dubliniensis. These primary isolates were incubated at 37°C for 48 to 72 h and evaluated daily for growth of yeast colonies. All plates demonstrating growth were evaluated for germ tube formation and chlamydospore production (see the method described below). Isolates that were found to be positive for germ tubes and chlamydospores were then subcultured on SDA and were incubated at 45°C for 24 h. All germ tube- and chlamydospore-positive isolates that failed to grow or that grew poorly at 45°C were considered suspicious and were further evaluated as described below. Yeast identification. Briefly, to determine germ tube and chlamydospore production, Tween 80–oxgall–caffeic acid (TOC) agar (Remel, Lenexa, Kans.) plates were streaked and stabbed with a 48-h-old yeast colony, covered with a sterile coverslip, incubated at 37°C for 3 h, and subsequently observed for germ tube production. TOC agar plates were then incubated at room temperature for 2 to 3 days in the dark to promote the production of chlamydospores, hyphae, and pseudohyphae. Alternatively, cornmeal agar (BBL, Cockeysville, Md.) was used in selected confirmational studies in our laboratory. Germ tube production and chlamydospore production were observed by phase-contrast light microscopy. Isolates that met these initial criteria were then subcultured along with C. albicans ATCC 18804 and the type strain C. dubliniensis CD36 (courtesy of Derek Sullivan, lodged with the British National Collection of Pathogenic Fungi under, accession no. NCPF 3949) on CHROMagar Candida medium (CHROMagar, Paris, France), a relatively new differential and isolation medium for Candida species (14). The plates were incubated at 37°C for 48 h. CHROMagar Candida medium comprises (per liter) peptone (10 g), glucose (20 g), agar (15 g), chloramphenicol (0.5 g), and a proprietary “chromogenic” mixture (2 g). The isolates were further characterized for substrate assimilation profiles by using the API 20C AUX system (bioMerieux Vitek, Inc., Hazelwood, Mo.). The API 20C AUX system uses a micromethod and consists of a series of cupules containing dehydrated substrates for assimilation reactions. These assimilation tests allow the identification of the most clinically significant yeasts after 72 h of incubation at 30°C. Key sugar metabolism profiles allow the differentiation of Candida species. Preparation of yeast DNA for electrophoretic karyotyping. The preparation of intact DNA in agarose plugs was performed by the method described by King et

Phenotypic characterization. Of the 699 yeast isolates initially identified as C. albicans, 8 were considered suspicious due to weak or no growth at 45°C (Table 1). The growth of these isolates on CHROMagar Candida medium and determination of their assimilation profiles with the API 20C AUX system identified seven of the eight isolates as C. albicans. The one remaining isolate was included in the electrophoretic karyotyping protocol. Of the 25 isolates from HIV-seropositive patients obtained by culture of clinical specimens, 5 of the isolates proved to be atypical in that they grew well at 37°C but failed to grow at 45°C on SDA. However, C. albicans ATCC 18804 consistently grew well at this temperature. These six suspicious isolates were reconfirmed to be germ tube positive and to produce abundant chlamydospores on TOC and cornmeal agars. Chlamydospores of these six isolates were often attached in the characteristic triplet or pair arrangement at the end of short, hyperbranching pseudohyphae (see Fig. 2 for typical patterns for our unknown isolates compared to those for C. albicans isolates). Specimens were viewed with and without selective staining with 1.0% (wt/vol) lactophenol cotton blue (25), which in some cases allowed the easier visualization of chlamydospores. The C. albicans reference strains also produced germ tubes and fewer chlamydospores on longer hyphae, and the chlamydospores were rarely present in pairs or triplets. The colors of the suspicious colonies on CHROMagar Candida medium were compared to the characteristic dark green of C. dubliniensis CD36. The colors of the colonies were also compared to the blue-green colonies formed by C. albicans ATCC 18804, which was used as a control in all of the experiments. The sugar assimilation profiles obtained for the six isolates with the API 20C AUX yeast identification systems gave “no identification.” The results of these tests were often ambiguous and difficult to interpret.

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C. glabrata

C. albicans

2

1

1a

Growth at 45°C

Dark green

Dark green

Pink or white

Pale halo green

Color on CHROMagar Candida medium

No IDb

Characteristic

Characteristic

Species

C. dubliniensis

2 No ID (6176130, 6176130, 6173330, 6172170, 2552130, 6176170, and 6176170)

1 Singlets

Short, lateral hyperbranching

Long, limited branching

Hyphae

,8 bands, usually .1 Mb

2

Doublets

Short, lateral hyperbranching

Chlamydospore pattern

.1 Mb

1

Doublets and triplets

Germ tube formation

.8 bands, 1 or more ,1 Mb

1

No. and size of bands obtained by electrophoretic karyotyping

.8 bands, 1 or more ,1 Mb

TABLE 1. Typical characteristics of Candida species

API 20C AUK profile identification

Usually positive growth at 45°C; weak or negative growth at 45°C has been observed in some strains. ID, identification. The profiles do not resemble those of any of the taxa stored in the database.

Unknowns (CD-012-1, CD-012-2, CD-014, CD-016, CD-017, CD-021, and CD-410)

a

b

Electrophoretic karyotype analysis. Eight to nine DNA bands ranging from ;1.9 to ,1 Mb were separated by pulsedfield gel electrophoresis with the seven suspected C. dubliniensis isolates and reference strain CD36. The electrophoretic karyotyping patterns for all seven isolates revealed the presence of a chromosome-sized band of ,1 Mb. This band is not seen in the seven bands (.1 Mb) observed in tests with C. albicans ATCC 18804 (Table 1 and Fig. 3). The variation in the mobilities of the DNA molecules yielded six electrophoretic karyotyping patterns with the seven isolates. However, identical electrophoretic karyotyping patterns (lanes 2 and 3) were found for the two isolates recovered on two separate occasions from the same patient, demonstrating strain stability. Fluconazole susceptibility testing. The results of fluconazole susceptibility testing for the seven isolates presumptively identified as C. dubliniensis are illustrated in Table 2. Six of the seven isolates were susceptible (MICs, #0.25 mg/ml). One isolate demonstrated dose-dependent susceptibility (MIC, 16 mg/ ml). The two isolates obtained from the same patient were susceptible (MICs, 0.25 mg/ml). Characteristics of the patients positive for C. dubliniensis. Five of the six colonized patients were HIV seropositive; the HIV status of the remaining patient was unknown. Two patients had a history of intravenous drug abuse, and two claimed to have been at risk for HIV transmission by way of homosexual activity. The source of HIV infection for the remaining patient was multifactorial. Five of the six patients were males. Additionally, five of the six patients had previously been treated with fluconazole for oropharyngeal candidiasis at some point in time, but not within the last 9 months. Two of these six patients harboring C. dubliniensis were also cultured positive for C. albicans. DISCUSSION In this prospective study, the presence of Candida species with characteristics consistent with those of C. dubliniensis was observed in five HIV-seropositive patients and one patient whose isolate was obtained from among 699 presumptive C. albicans isolates from patients whose HIV status was unknown. Strain stability was demonstrated by sampling one of the HIVseropositive patients on two different occasions 5 weeks apart. C. dubliniensis was identified by cultural methods, formation of chlamydospores and germ tubes, and the inability to grow at 45°C. Although all of our C. dubliniensis isolates displayed the typical abundant production of chlamydospores, some of the C. albicans isolates tested produced chlamydospores similar in number and arrangement to those produced by C. dubliniensis, as reported previously by other investigators (7, 22). Therefore, this morphological feature was not relied on for the initial identification of C. dubliniensis. Since the current API 20C AUX database does not include assimilation profiles for C. dubliniensis, none of the isolates was identified by this system. Salkin et al. (17) reported that C. dubliniensis could be differentiated from C. albicans on the basis of the specific biocodes produced by C. dubliniensis. Although several of the biocodes for our isolates matched the biocodes of Salkin et al. (17), differences in the biocodes of other isolates were observed (Table 1). This may have been due to the visual subjectivity and irreproducibility of the results obtained with some substrates (22). Finally, a small-molecular-weight chromosome, revealed by electrophoretic karyotyping, provided a profile consistent with that of the European reference strain of C. dubliniensis. The five isolates and the C. dubliniensis reference strain were susceptible to fluconazole when they were tested by the broth

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FIG. 2. Phase-contrast micrographs showing chlamydospore production on TOC agar plates incubated at 25°C for 48 h. (A) Chlamydospores and pseudohyphae produced by C. albicans ATCC 18804; (b) typical production of abundant chlamydospores by the C. dubliniensis isolates in our study; the terminal pairs arrangement is shown. Magnifications, 3320.

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FIG. 3. Electrophoretic karyotypes of Candida isolates. Lanes: 1, Succharomyces cerevisiae chromosomes, which were used as molecular mass standards (Bio-Rad); 2, C. dubliniensis CD-012-1; 3, C. dubliniensis CD-012-2; 4, C. dubliniensis CD-014; 5, C. dubliniensis CD-016; 6, C. dubliniensis CD-017; 7, C. dubliniensis CD-021; 8, C. dubliniensis CD-410; 9, C. albicans ATCC 18804; and 10, type strain C. dubliniensis CD36.

macrodilution method, while one isolate demonstrated dosedependent susceptibility (4, 12, 16). Although C. dubliniensis has been shown to be inherently susceptible to the same range of antifungal drugs as C. albicans, a study performed by Moran et al. (11) indicates that C. dubliniensis is capable of developing stable fluconazole resistance in vitro at a high frequency. It could therefore be postulated that C. dubliniensis may emerge initially as a resistant organism when C. albicans is treated successfully with fluconazole or other antifungal therapy. It would be interesting to determine whether patients who had received multiple treatments with antifungal therapy were more prone to the development of C. dubliniensis as their resident Candida species. In addition, McCullough et al. (8) have shown that C. dubliniensis isolates may be more virulent, since they have significantly higher levels of proteinase activity and greater levels of adherence to buccal epithelial cells than do typical C. albicans strains. Whether this equates to increased virulence needs to be determined. In our procedures for the identification of C. dubliniensis, growth at 45°C was used as an initial screen following determination of germ tube and chlamydospore production. Our results confirm that no C. dubliniensis strain will grow at 45°C, whereas C. albicans strains will often grow weakly at 45°C (7, 15). In these cases further testing by determination of the strains’ profiles with the API 20C AUX system and growth on CHROMagar Candida medium was used to distinguish C. albicans from C. dubliniensis. Therefore, growth or lack of growth TABLE 2. In vitro activity of fluconazole against C. dubliniensis Isolate

CD-012-1 CD-012-2 CD-014 CD-016 CD-017 CD-021 CD-410

MIC (mg/ml)

Sensitivity

0.25 0.25 0.25 0.125 0.5 1.0 16.0

Susceptible Susceptible Susceptible Susceptible Susceptible Susceptible Dose-dependent susceptible

325

at 45°C may still be a very reliable and discriminatory screen for C. dubliniensis (15), whereas it had previously been thought (21) that a lack of growth at 42°C may have been adequate. The reliability of the use of the CHROMagar Candida medium as a way of differentiating between C. albicans and C. dubliniensis isolates on the basis of colony color is debatable. According to Schoofs et al. (18) and Sullivan and Coleman (22), the dark green color seen with C. dubliniensis within 48 h of growth at 37°C is not retained in subculture. Our experience with the use of CHROMagar Candida medium, however, was similar to that of Odds et al. (14), who had no problem obtaining dark green colonies in subcultures when the plates were incubated in the dark at room temperature for more than 48 h. Many questions regarding the role that C. dubliniensis plays in oral diseases, the factors that encourage its growth, its occurrence in patient groups other than the HIV-positive population, and the relationship of its coexistence with C. albicans and other fungal species in the oral cavity remains to be answered. The data regarding our six patients and their previous histories clearly illustrate this coexistence; however, the statistical significance of these findings will be elucidated only as more patients are studied. Each of our patients had been infected with HIV for longer than 6 years and had subsequently received numerous treatments for oral or disseminated fungal infections. The effects of previous treatment for oral candidiasis presumably caused by C. albicans pose an interesting question for the mechanism of selection for C. dubliniensis. It could be postulated that C. dubliniensis may appear as a resistant organism when a C. albicans infection is successfully treated with fluconazole or some other antifungal therapy. Strain stability was demonstrated in one patient by using electrophoretic karyotyping and two isolates which were obtained from that patient 5 weeks apart. For both isolates from that patient, MICs were 0.25 mg/ml. The isolates were of the same type as defined by electrophoretic karyotyping, and isolates of that electrophoretic karyotype were not found in other patients. Therefore, the electrophoretic karyotyping method, when applied for strain delineation, as was done previously for other Candida species (5, 9, 10), provided confirmation, and it also had the discriminatory power to provide important future clinical data concerning the epidemiology, pathogenesis, and interrelationships of the medically important yeasts. The continued prospective investigation of the presence of C. dubliniensis in populations with fungal infections and its coexistence with other fungi is warranted. ACKNOWLEDGMENTS We thank Derek Sullivan for providing the reference strain used as a comparative control and for ongoing collaboration as we plan further characterization of our isolates. Also, the assistance of Robert Nauman with the microscopy, Susan Harrington with the electrophoretic karyotyping, and Mark Romagnoli with the fluconazole susceptibility testing is greatly appreciated. REFERENCES 1. Boerlin, P., F. Loerlin-Petzold, C. Durussel, M. Addo, J.-L. Pagani, J.-P. Chave, and J. Bille. 1995. Cluster of oral atypical Candida albicans isolates in a group of human immunodeficiency virus-positive drug users. J. Clin. Microbiol. 33:1129–1135. 2. Coleman, D. C., D. J. Sullivan, D. E. Bennett, G. P. Moran, H. J. Barry, and D. B. Shanky. 1997. Candidiasis: the emergence of a novel species, Candida dubliniensis. AIDS 11:557–567. 3. 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. C-290. p. 179. In Abstracts of the 98th General Meeting of the American Society for Microbiology 1998. American Society for Microbiology, Washington, D.C. 4. Isenberg, H. D. (ed.) 1992. In vitro antifungal susceptibility testing of yeasts,

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