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Journal of Clinical Microbiology, February 2008, p. 443-446, Vol. 46, No. 2
0095-1137/08/$08.00+0     doi:10.1128/JCM.01986-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Candida bracarensis Detected among Isolates of Candida glabrata by Peptide Nucleic Acid Fluorescence In Situ Hybridization: Susceptibility Data and Documentation of Presumed Infection

Justin A. Bishop,¹ Nancy Chase,^1, Shelley S. Magill,^1,2, Cletus P. Kurtzman,³ Mark J. Fiandaca,⁴ and William G. Merz^1*

Departments of Pathology,¹ Medicine, The Johns Hopkins Medical Institutions, Baltimore, Maryland,² Microbial Genomics and Bioprocessing Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, Peoria, Illinois,³ AdvanDx, Inc., Woburn, Massachusetts⁴

Received 9 October 2007/ Returned for modification 1 November 2007/ Accepted 28 November 2007

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Molecular taxonomic studies have revealed new Candida species among phenotypically delineated species, the best example being Candida dubliniensis. This study was designed to determine the occurrence of two new molecularly defined species, Candida bracarensis and Candida nivariensis, which are closely related to and identified as Candida glabrata by phenotypic assays. A total of 137 recent clinical isolates of C. glabrata identified by phenotypic characteristics was tested with C. bracarensis and C. nivariensis species-specific peptide nucleic acid fluorescence in situ hybridization probes. Three of 137 (2.2%) isolates were positive with the C. bracarensis probe, whereas the control strain, but none of the clinical isolates, was positive with the C. nivariensis probe. D1/D2 sequencing confirmed the identification of the three isolates as representing C. bracarensis. Clinically, one C. bracarensis isolate was recovered from a presumed infection, a polymicrobial pelvic abscess in a patient with perforated diverticulitis. The other two isolates were recovered from two adult oncology patients who were only colonized. C. bracarensis was white on CHROMagar Candida, had variable API-20C patterns that overlapped with C. nivariensis and some C. glabrata isolates, and had variable results with a rapid trehalose assay. Interestingly, an isolate from one of the colonized oncology patients was resistant to fluconazole, itraconazole, voriconazole, and posaconazole in vitro. In summary, C. bracarensis was detected among clinical isolates of C. glabrata, while C. nivariensis was not. One C. bracarensis isolate causing a presumed deep infection was recovered, and another isolate was azole resistant. Whether clinical laboratories should identify C. bracarensis will require more data.

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Although Candida albicans remains the Candida species most commonly recovered from clinical specimens including blood, non-albicans Candida species have become increasingly prevalent in multiple centers and geographic regions over the last two decades (11, 13). Indeed, at our institution (The Johns Hopkins Hospital), non-albicans Candida species accounted for 54% and 64% of yeast recovered from blood in 2005 and 2006, respectively (our unpublished data). Additionally, it is vital to identify these non-albicans Candida species correctly, as they are often less susceptible or resistant to antifungal agents used to treat Candida infections (2, 11).

Molecular diagnostic methods have the potential to provide rapid, accurate identification. In addition, molecular assays, particularly rRNA gene sequencing, have identified new Candida species that share significant phenotypic characteristics with frequently encountered Candida spp. (1, 3, 4, 7, 14). Perhaps the best example was the discovery of Candida dubliniensis in 1995, a yeast that shares many phenotypic characteristics with C. albicans (12). Candida bracarensis and Candida nivariensis are two recently described species that are phenotypically identified as Candida glabrata species (1, 3, 4). However, little is known about the prevalence, antifungal susceptibility patterns, and clinical significances of these two newly discovered species of the C. glabrata clade. Therefore, the goals of this study were to explore the prevalences, antifungal susceptibility patterns, and clinical significances of C. bracarensis and C. nivariensis. We utilized novel, species-specific, peptide nucleic acid fluorescence in situ hybridization (PNA FISH) probes to identify these new species among clinical isolates identified phenotypically as representing C. glabrata.

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Control strains and clinical isolates tested. One hundred fifty recently collected clinical isolates that were previously identified as being C. glabrata isolates were selected for this study (Table 1). Criteria for inclusion were as follows: there was only one isolate per patient, and there was an attempt to represent consecutive isolates. These yeasts were identified by the staff of the Microbiology Laboratory at The Johns Hopkins Hospital using standard laboratory methods. Characteristics used for the identification of isolates as being C. glabrata isolates included negative germ tube test, small yeasts with no hypha production and no melanin production on cornmeal agar with caffeic acid, and positive results for glucose and trehalose fermentation. When identification remained uncertain, carbohydrate assimilation patterns were determined using the API-20C yeast identification system (BioMerieux, Hazelwood, MO). Purity and viability were ascertained before testing by subculturing on CHROMagar Candida (BD Diagnostics, Sparks, MD). The results for the 137 isolates on which all tests were performed are reported: 13 of the original 150 isolates failed to grow when subcultured.

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TABLE 1. Isolates of yeasts phenotypically identified as being C. glabrata and their clinical sources

Control strains included C. bracarensis type strain (TS) NRRL Y-48270 (CBS 10154), reported previously by Correia et al. (3); C. bracarensis HOP-15 (NRRL Y-27794), described previously by Page et al. (9); Candida nivariensis type strain NRRL Y-48269 (CBS 9983); described previously by Alcoba-Florez et al. (1); Nakaseomyces (Kluyveromyces) delphensis type strain NRRL Y-2379 (CBS 2170 or ATCC 24205); and C. glabrata CLC (a well-characterized control strain used for quality control of internal tests at The Johns Hopkins Clinical Mycology Laboratory).

PNA FISH. Species-specific probes for C. bracarensis and C. nivariensis (kindly provided by AdvanDx, Inc., Woburn, MA) were used according to the manufacturer's protocol for positive blood cultures (8). Briefly, an inoculum of a yeast culture phenotypically identified as being C. glabrata was suspended in saline at a concentration of at least a 0.5 McFarland standard; a drop of the suspension was placed onto Teflon-coated microscope slides, air dried, and fixed with methanol. One drop of hybridization solution was added, a coverslip was applied, and the slides were incubated on a slide warmer at 55°C for 90 min. The slides were then submerged for 30 min in a stringent wash solution (55°C) in a water bath, during which time the coverslips were removed. Slides were mounted with mounting fluid, and coverslips were again applied before examination with a fluorescence microscope (oil objective, 100x) equipped with a fluorescein isothiocyanate-Texas Red dual-band filter (filter no. AC003; AdvanDx). Two readers independently examined each slide. Distinct green fluorescence in multiple fields was considered to be a positive result (8).

D1/D2 LSU rRNA gene sequencing. Isolates that were reactive with the C. bracarensis or C. nivariensis probes and all control strains were sequenced using methods previously described by Kurtzman and Robnett (5, 6) for domains 1 and 2 (D1/D2) of the large-subunit (LSU) rRNA gene.

Patient information. Following institutional review board approval, the records for individuals with a positive culture for C. bracarensis were reviewed. Data collected included patient demographics, underlying illness, length of hospitalization, antifungal usage, signs and symptoms of infection, and other positive cultures for C. glabrata.

Mycologic studies. For control strains and C. bracarensis and C. nivariensis probe-positive isolates, colony color was assessed on CHROMagar Candida (Becton Dickinson Diagnostics, Sparks, MD) after incubation at 37°C for 24 and 48 h. Carbohydrate assimilation patterns were compared using API-20C (BioMerieux, Hazelwood, MO) according to the manufacturer's directions. Rapid trehalose assimilation was performed using rapid trehalose assimilation broth (Remel, Lenexa, KS) according to the manufacturer's directions.

In vitro susceptibility testing. In vitro susceptibility testing was performed on all control strains and C. bracarensis or C. nivariensis probe-positive isolates. The Sensititre YeastOne system (Trek Diagnostic Systems, Inc., Cleveland, OH) was used according to the manufacturer's directions. Antifungal drugs tested on the Sensititre panel were fluconazole (FLC), itraconazole (ITC), voriconazole (VRC), posaconazole, caspofungin (CASP), and flucytosine (5FC). Amphotericin B (AMB) was tested using E-strips (AB BIODISK, Solna, Sweden); the MIC was determined in accordance with the manufacturer's instructions.

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PNA FISH results for detection of C. bracarensis and C. nivariensis. Of the 137 C. glabrata isolates screened with the species-specific C. bracarensis PNA FISH probe, three isolates (Cagl-78, Cagl-112, and Cagl-121) were reactive, with fluorescent yeast cells detected. Two C. bracarensis control strains (C. bracarensis TS and C. bracarensis HOP-15) were also PNA FISH probe positive. On the other hand, none the 137 isolates showed any fluorescence with the C. nivariensis probe, whereas the control strain was positive. There was no noticeable cross-reactivity with the C. bracarensis or C. nivariensis probes.

D1/D2 sequence results. Sequencing of the LSU D1/D2 regions confirmed the species identity of control strains and the three clinical isolates as being C. bracarensis isolates. The three clinical isolates differed from the type strain by 2 nucleotides, with a CT inversion near the middle of the sequence. The earlier clinical C. bracarensis control strain from our institution (9), Cagl-HOP-15, did not have this inversion.

Patient information. Pertinent clinical and demographic information for the three patients from whom the isolates were recovered is summarized in Table 2. One of the C. bracarensis strains (Cagl-121) was recovered from throat and another (Cagl-78) was recovered from stool from surveillance cultures of two oncology patients undergoing treatment for hematological malignancies. Both patients had received antifungal agents (both with AMB and VRC and one also with CASP) prior to the isolation of C. bracarensis. One of these patients had multiple previous surveillance cultures that were positive for C. glabrata, and both patients had subsequent positive C. glabrata cultures from their gastrointestinal tracts. These isolates were not available for testing with the C. bracarensis probe.

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TABLE 2. Demographic and clinical characteristics of patients from whom C. bracarensis was isolated

The third clinical C. bracarensis isolate (Cagl-112) was recovered from a specimen collected surgically in a case of pelvic abscess in a woman with a history of perforated diverticulitis but no underlying immunosuppressive conditions. In contrast to the other two cases, the patient had clinical signs and symptoms in addition to radiographic evidence of infection. This was a polymicrobial infection; Enterococcus faecium and coagulase-negative Staphylococcus were also recovered from the wound. Following percutaneous abscess drainage, the patient underwent a hemicolectomy and diverting ileostomy. She never received antifungal therapy, and no prior or subsequent cultures grew yeast.

Mycologic studies. The phenotypic characteristics of the three newly identified C. bracarensis isolates as well as those of all of the control isolates are summarized in Table 3. All of the organisms in question shared phenotypic morphological properties with no germ tube formation, no hypha production on cornmeal agar with caffeic acid, no phenoloxidase activity, and no urease activity. The recently collected clinical isolates of C. bracarensis as well as the C. bracarensis controls showed similar but not identical carbohydrate assimilation profiles; the isolates differed in their abilities to assimilate trehalose. Our control culture of C. nivariensis had phenotypic properties similar to those of C. glabrata and C. bracarensis and was trehalose assimilation negative.

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TABLE 3. Comparison of characteristics of C. bracarensis with C. glabrata, C. nivariensis, and N. delphensisa

In the original and subsequent descriptions of C. nivariensis, it was noted that this species produced white colonies, not the pink-purple colonies typically produced by C. glabrata, on CHROMagar Candida (1, 4). All of the three newly discovered clinical isolates and all control strains of C. bracarensis, C. nivariensis, and N. delphensis produced white colonies. The C. glabrata control strain produced pink colonies.

In vitro susceptibility studies. In vitro susceptibility testing of the clinical isolates and controls is presented in Table 4. The C. bracarensis isolate recovered from throat (Cagl-121) was resistant to all triazole agents tested, and the control strain of N. delphensis was resistant to FLC (128 µg/ml), ITC (8 µg/ml), and VRC (4 µg/ml). In contrast, the other two C. bracarensis isolates as well as the control strain of C. nivariensis displayed susceptibilities similar to those of our C. glabrata control strain. All isolates tested had low MICs of CASP, 5FC, and AMB.

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TABLE 4. Comparison of in vitro antifungal susceptibility of C. bracarensis with those of C. glabrata, C. nivariensis, and N. delphensis

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We identified three isolates of C. bracarensis among 137 yeast isolates identified phenotypically as representing C. glabrata, for a frequency of 2.2%. This equates to approximately 1 patient with a C. bracarensis isolate for every 50 patients with positive cultures for yeasts phenotypically identified as C. glabrata. D1/D2 sequencing confirmed the identification of the three recently discovered isolates. These findings confirm the original description by Correia and colleagues that C. bracarensis is a mimicker of C. glabrata and also suggest that it may have a wide geographic distribution, since it has been recovered from clinical specimens from Portugal, the United Kingdom, and the United States (3, 9; this study). Although C. bracarensis is a newly described species, we hypothesize that this species would be found among C. glabrata strains from well-maintained stock culture isolates collected decades ago. It should be emphasized that our study examined a limited number of isolates from one tertiary care medical center. Further studies from multiple geographical locations are needed to elucidate the epidemiology of infection, colonization, and antifungal resistance of C. bracarensis.

In contrast, C. nivariensis was not found among the 137 isolates of C. glabrata in the present study. Although the incidence of C. nivariensis remains unknown, our data suggest that this species is less common than C. bracarensis. Currently, to the best of our knowledge, this species has been recovered in Spain, Japan, and Indonesia (1, 4). Studies in multiple laboratories and geographic locations will be needed to address this question.

Evaluation of the clinical characteristics at the time of specimen collection for the patients from whom C. bracarensis was recovered yielded interesting findings. Specifically, there were additional "C. glabrata"-positive cultures recovered from the two oncology patients; it is likely that they were C. bracarensis cultures. If confirmed, these findings plus one reported previously in the literature (9) suggest that persistence of gastrointestinal tract colonization may occur with this species.

The other significant finding was that one of the three patients from whom we recovered C. bracarensis had evidence of a presumed deep infection; the type strain was the causal agent of vaginitis. This is the first report of recovery of C. bracarensis from a deep anatomic site and supports a presumption of infection since the organism was collected directly from the abscess and not from drainage tubes. The presence of this organism in a pelvic abscess also implies that it was probably a component of this individual's gastrointestinal tract. Preabscess cultures would have provided important data on whether this patient was colonized.

Our evaluation of common mycological characteristics of the three new C. bracarensis strains, as well as the control strains for the members of the C. glabrata clade, yielded no phenotypic test(s) that could distinguish these closely related species. It is interesting that all the members of the C. glabrata clade produced white colonies on CHROMagar Candida; these data suggest the potential utility for this medium as a screening tool for members of the C. glabrata clade.

The antifungal susceptibility data were particularly noteworthy. The C. bracarensis isolate recovered from the throat of one of the oncology patients (Cagl-121) was resistant to all triazoles tested. This patient had the longest hospital stay (95 days) of the three patients studied and had received prior AMB, VRC, and CASP treatment. Additionally, the control strain of N. delphensis was found to be resistant to FLC, ITC, and VRC. Finally, although the type strain that we used as our C. nivariensis control was susceptible to all agents tested, Fujita and colleagues previously reported pan-azole resistance in an isolate of C. nivariensis recovered from blood (4). These findings are clinically relevant because selection of antifungal therapy for candidiasis is often species based, and azoles are considered by many to be first-line antifungal drugs for many types of infections (2, 10). More studies will be needed to determine the extent of azole resistance in the newly described members of this clade.

In conclusion, three isolates of C. bracarensis, a member of the C. glabrata clade, were identified among isolates of C. glabrata. One patient in the present study had evidence of an infection, whereas the other two patients were colonized. One of the colonized patients' isolates was pan-azole resistant. Whether it is important to identify the individual species within the C. glabrata clade is unclear. Further data are needed to determine whether these species have clinically significant differences in antifungal susceptibilities and responses to antifungal therapy compared to C. glabrata.

 
We thank the staff of the Mycology Laboratory for help with the culture collection; AdvanDx Inc, Woburn, MA, for the species-specific probes; Eleanor Basehoar-Powers for D1/D2 sequences; and Tracey Ross for her technical efforts.

 
* Corresponding author. Mailing address: Department of Pathology, The Johns Hopkins Medical Institutions, Meyer B1-193, 600 N. Wolfe St., Baltimore, MD 21112. Phone: (410) 955-5077. Fax: (410) 614-8087. E-mail: wmerz{at}jhmi.edu

Published ahead of print on 12 December 2007.

Present address: Walter Reed Army Medical Center, 6900 Georgia Ave. NW, Washington, DC 20307.

Present address: Mycotic Diseases Branch, Division of Foodborne, Bacterial, and Mycotic Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road, Mailstop C-09, Atlanta, GA 30333.

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Journal of Clinical Microbiology, February 2008, p. 443-446, Vol. 46, No. 2
0095-1137/08/$08.00+0     doi:10.1128/JCM.01986-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bishop, J. A. Articles by Merz, W. G. Search for Related Content PubMed PubMed Citation Articles by Bishop, J. A. Articles by Merz, W. G.