JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 2010, p. 4677–4679
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 48, No. 12
Candida bracarensis Bloodstream Infection in an
Thomas A. Warren,1Lisa McTaggart,2Susan E. Richardson,1,2,3and Sean X. Zhang1,2*
Department of Laboratory Medicine and Pathobiology, University of Toronto,1Public Health Laboratories, Ontario Agency for
Health Protection and Promotion,2and Division of Microbiology, Hospital for Sick Children,3Toronto, Ontario, Canada
Received 15 July 2010/Returned for modification 30 August 2010/Accepted 21 September 2010
Candida bracarensis is a recently described Candida species which is phenotypically similar to Candida
glabrata. A case of C. bracarensis bloodstream infection in a bone marrow transplant patient is described and
confirms this organism as an opportunistic human pathogen. The organism can be distinguished from C.
glabrata by its white color on CHROMagar and by DNA sequence analysis using D1/D2 and internal tran-
scribed spacer (ITS) primers.
A 50-year-old male underwent a matched related-donor
bone marrow transplant after a diagnosis of chronic lympho-
cytic leukemia made 7 years previously. The patient’s post-
transplant course was complicated by significant graft-versus-
host disease (GVHD) of the skin, liver, and bowel. Four
months after transplant, the GVHD necessitated admission to
hospital and he was treated with cyclosporine and corticoste-
roids. In hospital, he had multiple infectious complications,
including herpes simplex virus mucositis and cytomegalovirus
viremia, treated with antiviral therapy. Seven weeks after ad-
mission to hospital, the patient developed respiratory failure
and sepsis, which necessitated transfer to the intensive care
unit (ICU). Klebsiella pneumoniae was cultured from both the
urine and the blood, and Staphylococcus aureus was cultured
from bronchoalveolar lavage fluid. He was treated with appro-
priate antibiotic therapy.
Three weeks after admission to the ICU, two blood cultures
became positive, and yeast cells were seen on the Gram stain.
After 24 h of growth, small white colonies were seen on blood
and chocolate agar media. A wet prep showed budding yeast
cells, and a germ tube test was negative. On Saboraud’s agar,
colonies appeared white and creamy. On cornmeal agar, hy-
phae or pseudohyphae were not observed microscopically.
Both isolates produced white colonies on BBL CHROMagar
(BD diagnostics, Maryland). Biochemically, the isolates were
positive for the rapid trehalose assay (Remel, Lenexa, KS) and
positive for assimilation of lysine and glucose (Table 1). Iden-
tification using the API 20C AUX system (bioMe ´rieux, Inc.,
NC) showed a low-percentage identity match with C. glabrata
(?50%). Thus, molecular analysis was applied to further char-
acterize these strains.
Both isolates were negative as determined by a C. glabrata-
specific PCR, with gel electrophoresis endpoint detection using
primers targeting the internal transcribed spacer (ITS) region
and PCR conditions described previously (8) (Table 1). The
ITS1-5.8S-ITS2 region and the D1/D2 region of 26S ribosomal
DNA (nucleotides 63 to 642) were amplified and sequenced.
PCRs were conducted using the Phire Hot Start DNA poly-
merase kit (New England Biolabs, Massachusetts) according to
the manufacturer’s instructions and using the following ampli-
fication conditions: 98°C for 30 s, followed by 35 cycles of 98°C
for 5 s, annealing temperature (see below) for 5 s, and 72°C for
20 s, followed by a final extension of 72°C for 1 min. The ITS
region was amplified using the primers ITS-1 (5?-TCCGTAG
GTGAACCTGCGG-3?) and ITS-4 (5?-TCCTCCGCTTATTG
ATATGC-3?) (12) and an annealing temperature of 56°C,
while amplification of D1/D2 required the primers NL-1 (5?-
GCATATCAATAAGCGGAGGAAAAG-3?) and NL-4 (5?-G
GTTCCGTGTTTCAAGACGG-3?) (9) and an annealing tem-
perature of 60°C. PCR amplicons were sequenced using the
ABI 3130xl genetic analyzer (Life Technologies, Carlsbad,
CA) according to the manufacturer’s instructions. Sequence
alignment and cluster analysis of the ITS and D1/D2 regions of
the clinical isolates along with the type strains C. nivariensis
NRRL Y-48269T, C. bracarensis NRRL Y-48270T, C. glabrata
NRRL Y-65T, and C. albicans NRRL Y-12983Twere per-
formed by the neighbor-joining (NJ) method using the Bio-
Numerics v.6.0 software program (Applied Maths, Inc., Austin,
TX). The ITS and D1/D2 sequences of the clinical isolates
were identical to each other and showed 98.7% and 99.7%
similarities, respectively, to the type strain of C. bracarensis.
Furthermore, NJ phylogenetic analysis of both D1/D2 (Fig. 1)
and ITS (data not shown) demonstrated the clinical isolates
clustering more closely with C. bracarensis than any of the
other type strains. Based on the results from phenotypic and
molecular analysis (Table 1 and Fig. 1), we identified the iso-
lates as C. bracarensis.
Antifungal drug susceptibilities of the two isolates were
tested using the Yeast YO9 panel (Trek Diagnostic, Cleveland,
OH). Both isolates were susceptible to all the drugs tested
except for itraconazole, to which a category of “susceptible
dose dependent” was assigned (Table 2). The patient was
treated with a 2-week course of caspofungin. Subsequent blood
* Corresponding author. Present address: Division of Medical Mi-
crobiology, the Johns Hopkins University School of Medicine, 600
North Wolfe St., Meyer B1-193, Baltimore, MD 21287. Phone: (410)
955-5077. Fax: (410) 614-8087. E-mail: email@example.com.
?Published ahead of print on 29 September 2010.
cultures performed while the patient was on caspofungin
therapy were negative. However, he ultimately succumbed
to his underlying disease (GVHD) 32 days after the positive
Candida culture. His death was felt not to be related to the
Candida species are the third-most-common cause of noso-
comial bloodstream infections in ICU patients (13). Although
C. albicans remains the predominant cause of candidemia,
recent epidemiological data show that C. glabrata has emerged
as the second-most-common cause of candidemia in the
United States (11). Recently, two new species phenotypically
similar to C. glabrata have been described, C. nivariensis (1)
and C. bracarensis (5). While C. nivariensis has been described
as a new multidrug-resistant pathogen (4), little is known about
C. bracarensis. To date, only seven human clinical isolates have
been described in the literature. One was isolated from a
vaginal exudate in a Portuguese hospital and the other from a
blood culture in a United Kingdom hospital (5). Subsequently,
three more isolates were identified by retesting 137 C. glabrata
clinical isolates using a C. bracarensis-specific probe in a ter-
tiary hospital in the United States (3). Of these, one was from
a pelvic abscess of a patient with perforated diverticulitis and
two were from the throat and stool of oncology patients with-
out evidence of infection. More recently, 2 more isolates were
identified from a large global collection of C. glabrata isolates
(1,598 isolates), of which 1 was isolated from sputum culture
and the other from a blood culture (7).
Similar to other Candida species, C. bracarensis has been
recovered from multiple body sites, especially mucosal surfaces
(Table 2), and is clearly associated with infection and coloni-
zation (3, 5, 7). Our patient presented with multiple risk factors
for developing invasive candidiasis. He was immunocompro-
mised by therapeutic immunosuppression (cyclosporine and
methylprednisolone) and by his underlying disease condition
(leukemia, bone marrow transplant, and GVHD). GVHD of
the skin, liver, and bowel may result in mucosal damage lead-
ing to increased fungal translocation of C. bracarensis into the
bloodstream (6). Our patient was in the ICU for 20 days prior
to the positive culture results. Other relevant risk factors in-
cluded central venous catheterization and broad-spectrum an-
C. bracarensis and C. glabrata are morphologically and bio-
chemically indistinguishable except for the color change on
CHROMagar and lysine assimilation (Table 1). C. bracarensis
and C. nivariensis both produced white colonies on CHROMagar,
but C. bracarensis was positive for the rapid trehalose test
whereas C. nivariensis was negative (Table 1). Unlike findings
in other studies, our two isolates were not initially identified as
C. glabrata by the API 20C system, suggesting that C. braca-
rensis can be either misidentified as C. glabrata or left uniden-
tified by the biochemical analysis. Based on data from our-
selves and others (1–3, 5, 7), C. bracarensis can be
presumptively identified based on the production of white col-
onies on CHROMagar agar, microscopic features of small
budding yeast cells without hyphae or pseudohyphae, and a
positive rapid trehalose test. Identification can be further con-
firmed by DNA sequence analysis targeting the ITS and/or
D1/D2 regions. Therefore, CHROMagar should be included in
the workup if small budding yeast cells are seen from a positive
The current prevalence of this organism is based on retesting
of C. glabrata isolates (3, 7). This approach may possibly un-
derestimate the current prevalence of C. bracarensis, since our
two C. bracarensis isolates were initially not identified as C.
glabrata. Accurate identification of Candida species can have
important implications for the treatment of invasive candidia-
sis (10). This species is an opportunistic fungal pathogen caus-
ing invasive candidiasis in immunocompromised patients. In-
cluding the case reported here, three of the patients from
whom C. bracarensis was isolated were patients with hemato-
logical malignancies (3). Moreover, it will be important to
determine the antifungal drug susceptibilities of future isolates
of this organism. Two of the eight C. bracarensis isolates tested
for antifungal drug susceptibilities were drug resistant, of
which one was resistant to amphotericin B and one was resis-
tant to all the azoles (Table 2). The true incidence of this
FIG. 1. Neighbor-joining tree based on nucleotide sequences of the
D1/D2 region of 26S rRNA of two clinical isolates and type strains of
C. bracarensis, C. nivariensis, C. glabrata, and C. albicans. Bootstrap
replication percentages (1,000 replicates) are indicated at the nodes.
Bar, 1% nucleotide sequence divergence.
TABLE 1. Characteristics of the two clinical isolates
Assay result Assimilation at 48 h
Clinical isolate 1
Clinical isolate 2
C. bracarensis type strain
C. nivariensis type strain
C. glabrata type strain
C. albicans type strain
4678CASE REPORTSJ. CLIN. MICROBIOL.
organism in cases of invasive candidiasis has yet to be deter-
We thank the staff of the Medical Mycology section at the Public
Health Laboratory, Ontario Agency for Health Protection and Pro-
motion, for help with culture identification and collection.
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TABLE 2. Antifungal drug susceptibility profiles of C. bracarensisa
MIC (?g/ml) of drug
5-FCAMB FLCITC VRCPSCCASP MICAANID
Clinical isolate 1 (this study)
Clinical isolate 2 (this study)
C. bracarensis TSa(3)
C. bracarensis no. 1 (7)
C. bracarensis no. 2 (7)
aC. bracarensis TS, C. bracarensis type strain NRRL Y-48270T (CBS10154); AMB, amphotericin B; FLC, fluconazole; ITC, itraconazole; VRC, voriconazole; PSC,
posaconazole; CASP, caspofungin; MIC, micafungin; ANID, anidulafungin.
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