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Journal of Clinical Microbiology, April 2009, p. 1216-1217, Vol. 47, No. 4
0095-1137/09/$08.00+0     doi:10.1128/JCM.02315-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Identification of Candida nivariensis and Candida bracarensis in a Large Global Collection of Candida glabrata Isolates: Comparison to the Literature

Shawn R. Lockhart,^1* Shawn A. Messer,¹ Michael Gherna,² Justin A. Bishop,² William G. Merz,² Michael A. Pfaller,¹ and Daniel J. Diekema¹

University of Iowa, Carver College of Medicine, Iowa City, Iowa 52242,¹ Johns Hopkins Medical Institute, Baltimore, Maryland²

Received 2 December 2008/ Returned for modification 20 January 2009/ Accepted 21 January 2009

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We analyzed 1,598 Candida glabrata isolates for the presence of the cryptic species Candida nivariensis and Candida bracarensis. Both species were very rare in this collection (0.2% prevalence), despite the number of isolates analyzed and the global distribution of the isolates. We saw no associated antifungal resistance in C. nivariensis.

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As our methods of species identification have shifted from more traditional phenotypic methodologies to nucleic acid-based techniques, previously unrecognized "cryptic" species have been added to the list of human pathogens. Recent reports have added the Candida glabrata phenotypic mimics Candida nivariensis and Candida bracarensis to this list (2, 5). Following a report on 16 isolates from the United Kingdom which indicated that C. nivariensis was an emerging pathogen with multidrug resistance (4, 6), we sought to use our unique global collection of Candida isolates to determine what percentage of C. glabrata isolates were C. nivariensis and C. bracarensis and whether these isolates indeed had high rates of resistance to antifungal drugs.

All isolates were collected as part of the ARTEMIS antifungal surveillance program between 2001 and 2006 and identified as C. glabrata by the Vitek yeast identification system (BioMérieux, Durham, NC). A total of 1,598 isolates phenotypically identified as C. glabrata were included in this study. These isolates were distributed globally. Isolates were received from 98 medical centers in 28 countries on six continents (Table 1). All isolates were plated on BBL CHROMagar, and their color was scored as either mauve or white. All isolates were then analyzed by C. glabrata-specific PCR with the primers CGL1 (5'-TTATCACACGACTCGACACT-3') and CGL2 (5'-CCCACATACTGATATGGCCTACAA-3') as described previously (1, 7).

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TABLE 1. Numbers and origins of isolates used in this study

When the 1,598 presumed C. glabrata isolates were plated on CHROMagar, 1,584 were mauve and 14 were white. Out of the 1,598 tested by C. glabrata-specific PCR, 1,595 were positive, including 11 of the 14 that were white on CHROMagar. All 14 of the isolates that were white on CHROMagar were analyzed by peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) first with a probe that recognized C. glabrata and the two closely related species and then with probes specific for C. nivariensis or C. bracarensis (3, 9). The 11 isolates white on CHROMagar that were also C. glabrata PCR positive were also confirmed to be C. glabrata by PNA-FISH. Of the three isolates that were white on CHROMagar and negative by C. glabrata PCR, one was determined to be C. nivariensis and two were determined to be C. bracarensis by PNA-FISH. C. nivariensis and C. bracarensis isolates represented only 0.2% of the total C. glabrata complex isolates. The two C. bracarensis isolates originated in the United States at a single site in Wilmington, DE, but one was isolated from sputum in 2002 and the other was isolated from a bloodstream infection in 2004. The C. nivariensis isolate was from the pleural fluid of a patient in Westmead, Australia.

To date, the total number of published C. bracarensis and C. nivariensis isolates is small. The first three C. nivariensis isolates were reported from the Canary Islands in 2005 from a pulmonary abscess, blood, and urine (1). Since then, single isolates have been reported from a catheter-associated bloodstream infection in Japan and as a cause of oropharyngeal candidiasis in an AIDS patient in Indonesia (6, 12). An additional 16 isolates were reported from the United Kingdom, and 9 of these were from sterile body sites, including blood, ascitic fluid, and peritoneal fluid (4). Another isolate was recently recovered from the blood of a patient in Atlanta, GA (M. Brandt, A. Balajee, and S. Lockhart, unpublished observations).

The number of C. bracarensis isolates is even lower. Following the publication of the first two isolates as a cause of vulvovaginal candidiasis in Portugal and from a blood culture in the United Kingdom in 2006 (5), only three more have been published, isolates from the throat and stool of two oncology patients and from a pelvic abscess, all from Baltimore, MD (3). One other isolate has been recovered from a recent study of C. glabrata bloodstream isolates from a 1992 surveillance in San Francisco, CA (J. Frade and S. Lockhart, unpublished observations).

Antifungal susceptibility testing of the C. nivariensis and C. bracarensis isolates was performed by broth microdilution with fluconazole, caspofungin, anidulafungin, and micafungin and by Etest for amphotericin B as outlined in Clinical and Laboratory Standards Institute document M27-A2 and as previously described (8, 10) All MICs were determined visually. Echinocandin MICs were determined at 24 h, while azole and amphotericin B values were determined at 48 h. Despite reports of almost uniform azole resistance in isolates of C. nivariensis (4, 6), our C. nivariensis isolate from Australia exhibited neither azole nor echinocandin resistance (Table 2). The same was reported for the isolate from Indonesia (12) However, one of the C. bracarensis isolates reported here had an Etest amphotericin B MIC of 8 µg/ml (Table 2).

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TABLE 2. MICs for the non-C. glabrata isolates from global surveillance

The study described in this report is the largest to date which has looked at the prevalence of C. nivariensis and C. bracarensis on a global scale. Despite the analysis of 1,598 C. glabrata isolates from 28 countries, our data indicate that C. nivariensis and C. bracarensis isolates make up only a very small percentage of the C. glabrata clinical isolates from global surveillance. Other published data indicate that they may be more locally or regionally prevalent (3, 4). Colony color on CHROMagar may be a very good way to rule C. nivariensis and C. bracarensis isolates in or rule out among C. glabrata clinical isolates. While not all of the Candida isolates that were white on CHROMagar were C. bracarensis or C. nivariensis, all of the C. nivariensis and C. bracarensis isolates have been white on CHROMagar. Because of a documented increase in their incidence, a documented rise in the MICs of many antifungal drugs toward C. glabrata (11), and the propensity of at least some isolates of C. nivariensis and C. bracarensis to exhibit antifungal resistance, it would be prudent to continue to monitor for these emerging pathogens.

 
This work was funded by a grant from Pfizer, Inc.

The findings and conclusions of this article are ours and do not necessarily represent the views of the Centers for Disease Control and Prevention.

 
* Corresponding author. Present address: Centers for Disease Control and Prevention, 1600 Clifton Rd., Mailstop G-11, Atlanta, GA 30333. Phone: (404) 639-2569. Fax: (404) 639-3546. E-mail: gyi2{at}cdc.gov

Published ahead of print on 4 February 2009.

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Journal of Clinical Microbiology, April 2009, p. 1216-1217, Vol. 47, No. 4
0095-1137/09/$08.00+0     doi:10.1128/JCM.02315-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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• Marklein, G., Josten, M., Klanke, U., Muller, E., Horre, R., Maier, T., Wenzel, T., Kostrzewa, M., Bierbaum, G., Hoerauf, A., Sahl, H.-G. (2009). Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Fast and Reliable Identification of Clinical Yeast Isolates. J. Clin. Microbiol. 47: 2912-2917 [Abstract] [Full Text]  

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 Lockhart, S. R. Articles by Diekema, D. J. Search for Related Content PubMed PubMed Citation Articles by Lockhart, S. R. Articles by Diekema, D. J.