Mycopathologia (2014) 178:243250

DOI 10.1007/s11046-014-9791-z

Nosocomial Bloodstream Candida Infections in a TertiaryCare Hospital in South Brazil: A 4-Year Survey
Viviane Gevezier da Costa Regina Mariuza Borsato Quesada Aline Tancler Stipp Abe
Luciana Furlaneto-Maia Marcia Cristina Furlaneto

Received: 14 September 2013 / Accepted: 28 July 2014 / Published online: 8 August 2014
Springer Science+Business Media Dordrecht 2014

Abstract The aims of this study were to evaluate the

epidemiology of nosocomial candidemia in a tertiary
hospital in South Brazil and the in vitro antifungal
susceptibility of isolates. Blood strains from 108
patients were identified by PCR-based method. Some
30.5 % of candidemia were caused by Candida
tropicalis, 28.7 % were due to Candida albicans,
24.1 % with Candida parapsilosis sensu stricto, 8.3 %
with Candida glabrata sensu lato, 1.8 % involved
Candida krusei and 6.6 % with other species. Candidemia was more common in intensive care unit
settings (66 %). In vitro susceptibility to antifungal
drugs was determined by a microdilution method; and
new species-specific clinical breakpoints for fluconazole and voriconazole were applied. Overall susceptibility rates were 100 % for itraconazole, 91 % for
fluconazole, 98 % for voriconazole and 99 % for
amphotericin B. Fluconazole resistance was mostly
among C. parapsilosis sensu stricto isolates (26.9 %).
V. G. da Costa  A. T. S. Abe  M. C. Furlaneto (&)
Department of Microbiology, Centre of Biological
Sciences, Parana State University, C.P. 6001, Londrina,
PR CEP: 86051990, Brazil
e-mail: furlaneto@uel.br
R. M. B. Quesada  A. T. S. Abe
Department of Clinical Analyses, Parana State University,
Londrina, Brazil
L. Furlaneto-Maia
Technological Federal University of Parana, Parana,
Brazil

Most of the findings reported here agreed with

epidemiological features common to other tertiary
hospitals in Brazil; but also revealed some peculiarities, such as a high frequency of C. tropicalis
associated with candidemia. Besides, high rate of
fluconazole resistance among C. parapsilosis stricto
sensu isolates was obtained when applying the new
species-specific clinical breakpoints.
Keywords Candida spp.  Candidemia 
Epidemiology  Antifungal susceptibility testing
Introduction
Bloodstream infections (BSIs) caused by Candida
species, i.e., candidemia, have been considerably
rising in the last decades and remain an important
comorbid condition in hospitals [1, 2]. The epidemiology of candidemia is generally characterized by
geographical and temporal variability. Historically,
Candida albicans has been the most common isolate
from candidemia; however, there has been a reported
increase in the incidence of nonCandida albicans
Candida (NCAC) species, some of which are associated with antifungal susceptibility patterns other than
that of C. albicans [3, 4].
A prospective nationwide surveillance in Brazilian
hospitals showed that Candida spp. accounted for 6 %
of all nosocomial BSIs where NCAC species
accounted for 65.7 % of the isolates [5]. The largest
candidemia multicentric study conducted in Latin

123

244

America reveals that among NCAC species, Candida

parapsilosis and Candida tropicalis were the most
frequent species in Brazilian tertiary-care hospitals [6].
The epidemiology of candidemia varies depending on
the geographic region and hospital characteristics. In this
regard, little is known about the epidemiology of
candidemia in South Brazil. Currently, there are three
previous local studies in this region of the country [79].
Since the outcomes of patients with candidemia are poor
and Candida spp. with increased resistance to antifungal
therapy may be associated with these results, the
knowledge of local epidemiologic trends and antifungal
susceptibility of etiological agents is critical. The objective of this study was to evaluate the incidence and
susceptibility profile of Candida spp. causative agents of
candidemia in tertiary-care hospital in South Brazil.
In line with results of other Brazilian studies, we
documented that a considerable proportion of Candida
BSIs was due to NCAC species; besides in our
institution, C. tropicalis outranked C. albicans.

Materials and Methods

Study Setting and Population
We evaluated all Candida sp. bloodstream isolates
obtained from patients admitted at the Hospital
Universitario de Londrina (HU), a general tertiarycare hospital with 333 beds, including 43 intensive
care unit (ICU) beds, in the southern Brazil from
January 2006 to December 2007 and January 2010 to
December 2011. We included only the Candida sp.
isolate obtained from the first episode of nosocomial
candidemia of each patient. Nosocomial candidemia
was defined as the recovery of Candida spp. in culture
from a patients blood sample more than 48 h after
hospital admission. The study protocol was approved
by ethics committee: Comite de Etica em Pesquisas
em Seres Humanos da Universidade Estadual de
Londrina (CONEP 268, PF no 234/09). Samples were
inoculated on Sabouraud Dextrose Agar supplemented
with chloramphenicol (100 lg/mL). The plates were
incubated at 28 C for 48 h.
Clinical Data
Date of sex, age, hospital unit and associated diseases
were registered for each patient. At the collection date

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Mycopathologia (2014) 178:243250

of the first positive fungal blood culture, antimicrobial

therapy prescribed (prophylactic or therapeutic), previous immunosuppression (chemotherapy), corticotherapy, parenteral nutrition and mechanical
ventilation for more than 24 h before the episode of
fungemia were registered in a database. Underlying
diseases were also registered.
Yeast Identification Procedures
Species identification was based on colony morphology on chromogenic agar CHROMagar Candida
(CHROMagar, Paris, France). Definitive identification
of all clinical isolates assayed in this work belonging
to the genus Candida was performed using a PCRbased method. PCR identification was carried out
using species-specific forward primers (ITS1 and
ITS2) corresponding to intergenic spacer regions and
ITS4 as universal reverse primer located at the 26S
rDNA. Identity of species belong to C. parapsilosis
complex was obtained by the employment of primers
for URA3 gene (orotidine-50 -phosphate decarboxylase) (Table 1), which allowed us to distinguish C.
parapsilosis sensu stricto from the cryptospecies
belonging to the C. parapsilosis complex (Candida
orthopsilosis and Candida metapsilosis). PCR was
carried out as described by Furlaneto et al. [13].
Antifungal Susceptibility Testing
The thawed cultures were subcultured twice, and
then, antifungal susceptibility testing was performed
using the AFST-EUCAST Def. 7.1 broth microdilution assay method (AFST-EUCAST 2008) [14]. The
yeast isolates were grown on Sabouraud dextrose
agar for 24 h at 37 C. Suspensions were prepared in
sterile saline (0.85 %) and adjusted to 15 9 105
cells/mL. The following antifungal drugs were
tested: fluconazole, voriconazole, itraconazole and
amphotericin B (Sigma Chemical, St. Louis, MO,
USA). All antifungal drugs were supplied by the
manufacturers as pure standard compounds. Serial
dilutions of the drugs were performed in RPMI-1640
medium, supplemented with glucose 2 % (w/v) and
buffered with 0.165 M morpholinepropanesulfonic
acid (MOPSSigma-Aldrich). The final ranges of
the drugs dilutions tested were as follows: fluconazole (0.12564 mg/L), itraconazole (0.0158 lg/mL),
voriconazole (0.0158 lg/mL) and amphotericin B

Mycopathologia (2014) 178:243250

Table 1 Species-specific
primers employed for
identification of Candida
spp.

F forward primer,
R reverse primer

bp Base pars

245

Species

Sequence (50 30 )a

Amplicon size
source (bp)b

C. albicans

TCAACTTGTCACACCAGATTATT

402

[10]

C. tropicalis

AAGAATTTAACGTGGAAACTTA

149

[10]

C. parapsilosis complex
C. glabrata sensu lato

GGCGGAGTATAAACTAATGGATAG
CACGATTCGACACTTTCTAATT

126
632

[10]
[10]

C. krusei

GATTTAGTACTACACTGCGTG

475

[10]

Universal reverse primer ITS4

TCCTCCGCTTATTGATATGC

[11]

C. parapsilosis sensu stricto F

AGACTTGGGTATTACGTTGT

727

[12]

C. parapsilosis sensu stricto R

CAGGAGTCATGATTACCC

(0.0316 lg/mL). C. krusei ATCC 6258 and C.

parapsilosis ATCC 22019 were included in all tests
for quality control. The MIC endpoint in the
amphotericin B tests was the lowest concentration
capable of preventing 90 % growth. The MICs for
each azole were defined as the lowest concentration
resulting in a 50 % inhibition of growth compared
with the growth of the control.
The drugs breakpoints were applied following the
AFST-EUCAST recommendations and the recently
published species-specific clinical breakpoints and
epidemiological cutoff values (ECVs) [3, 14]. Fluconazole MIC results of [4 lg/mL were considered
resistant for C. albicans, C. parapsilosis and C.
tropicalis. All isolates of C. krusei were classified as
resistant to fluconazole. Fluconazole MIC results of
[32 lg/mL were considered resistant for C. glabrata
[15]. The resistance breakpoint for voriconazole was
[0.12 lg/mL for C. albicans, C. parapsilosis and C.
tropicalis. Voriconazole MIC results of [1 lg/mL
were considered resistant for C. glabrata and C.
krusei. For amphotericin B, the values of MIC[1 lg/
mL were used to classify resistant strains. Itraconazole
MIC results of MIC [0.5 lg/mL were considered
resistant for C. albicans, C. parapsilosis and C.
tropicalis [16]. Itraconazole MIC results of [2 and
[1 lg/mL were considered resistant for C. glabrata
and C. krusei, respectively [16]. MICs values for
which 50 % (MIC 50) and 90 % (MIC 90) of the
growth of the isolates was inhibited were established
for each analyzed drug.
Statistical Analysis
The chi-square test or Fishers exact test was used to
compare categorical variables, when appropriate.
P \ 0.05 was considered to be significant.

[12]

Results
A total of 108 patients with the diagnosis of candidemia were enrolled in the study during the established
time period. The demographic and clinical characteristics of patients with candidemia are summarized in
Table 2. Ninety-seven cases were analyzed, and the
remaining seven records were excluded from the study
due to incomplete medical records. Four patients had
polymicrobial candidemia. Demographics at the time
of diagnosis were 45 females (46.4 %) and 52 males
(53.6 %). Candidemia occurred in neonates (22.7 %),
38.1 % in adults between 19 and 60 years old and
25.8 % in elderly patients. The study included 64
patients in ICU, of which 20.6 % were in the neonatal
ICU and 45.4 % were in the adult ICU.
Analysis of the potential predisposing factors
revealed that 97.9 % of the cases received broad
spectrum antibiotics, 72.1 % had undergone a surgical
intervention, 64.4 % required mechanical ventilation,
and 67 % had a urinary catheter. Other evaluated
factors included underlying diseases, such as respiratory tract disease (47.4 %), gastrointestinal disease
(28.9 %) and cardiovascular disease (27.8 %).
The distribution of isolated Candida species is
shown in Table 3. Of the isolates obtained from
candidemia, 71.3 % were NCAC species. C. tropicalis
(30.5 %) and C. albicans (28.7 %) were the two most
frequent species, followed by C. parapsilosis sensu
stricto (24.1 %), C. glabrata sensu lato (8.3 %), C.
krusei (1.8 %) and all other species (6.6 %). None of
the isolates of the complex C. parapsilosis were C.
orthopsilosis (formerly C. parapsilosis group II) or C.
metapsilosis (formerly C. parapsilosis group III). Four
patients presented more than one different Candida
spp: two patients presented with C. albicans, C.
parapsilosis and C. tropicalis; one patient presented

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Mycopathologia (2014) 178:243250

Table 2 Demographic characteristics and predisposing factors

of patients with bloodstream yeast infections from 2006 to
2007, and 2010 to 2011 in a tertiary-care hospital

Table 3 Distribution of the five most frequently isolated

species of Candida (by study period) in 108 episodes of
candidemia

Characteristics

Patients

Species

20042007
N (%)

20102011
N (%)

Total
N (%)

52

53.6

C. tropicalis

12 (30)

21 (30.9)

33 (30.5)

C. albicans

12 (30)

19 (27.9)

31 (28.7)

C. parapsilosis sensu
stricto

11 (27.9)

15 (22.1)

26 (24.1)

Sex
Male
Age (years)
Neonates

22

B118

13

22.7
13.4

1935

13

13.4

3660

24

24.7

C61

25

25.8

ICU neonatal

20

20.6

ICU adult
Pediatrics

44
4

45.4
4.1

Others

29

29.9

Premature birth

19

19.6

Trauma

12

12.4

Burns

10

10.3

Gastrointestinal disease

28

28.9

Diabetes mellitus

14

14.4

C. glabrata sensu lato

2 (5)

7 (10.3)

9 (8.3)

C. krusei

2 (5)

2 (1.8)

Hospitalization unit

Underlying disease

Cardiovascular disease

27

27.8

Cancer

10

10.3

Leukemia

3.1

Neurological disease

13

13.4

Respiratory tract disease

46

47.4

95
35

97.9
36.1

Risk factor
Previous antibiotic therapy
Previous corticoid therapy
Mechanical ventilation

72

74.2

Urinary catheter

65

67

Previous abdominal surgery

33

34

Parenteral nutrition

52

53.6

Previous non abdominal surgery

37

38.1

Neutropenia

11

11.3

6.2

Chemotherapy

with C. albicans and C. tropicalis, and the other

patient presented with C. parapsilosis and C.
tropicalis.
Table 4 provides the major clinical characteristics
of patients sorted by the three most frequent Candida
species. Some 13 % of C. parapsilosis-infected
patients had gastrointestinal disease, and the percentage was significantly lower (P \ 0.05). About 3.8 %

123

of C. tropicalis-infected patients had cancer, and the

percentage was significantly lower (P \ 0.05). The
parenteral nutrition and abdominal surgery were not
significantly different among the three Candida species (P [ 0.05).
The antifungal susceptibility profile of the most
frequently isolated species is detailed in Table 5. The
rate of fluconazole-nonsusceptible was 3.7 % (1/27)
for C. albicans, 3.2 % (1/31) for C. tropicalis and
26.9 % (7/26) for C. parapsilosis sensu stricto
(Table 5). In this study, we observed cross-resistance
between fluconazole and voriconazole for two isolates
being one C. albicans and one C. tropicalis. MICs
below the susceptibility breakpoints were found for
100 % of the Candida isolates for itraconazole.
Resistance to voriconazole was detected in 1.98 %
of the isolates, including 1 isolate of C. albicans
(3.7 %) and one isolate of C. tropicalis (3.2 %),
indicating the presence of resistance mechanisms.
With regards to amphotericin B, one C. albicans
isolate was resistant. The MICs for the control strains
were within the expected range (Table 5).

Discussion
Considerable changes in the relative frequency of
candidemia due to various Candida species as well as
their in vitro susceptibility patterns have been noted in
several parts of the world. In this study, we aimed to
determine the epidemiology of candidemia and susceptibility profile of Candida spp. in a Brazilian
tertiary-care hospital located at South region of Brazil,
in which no epidemiological data are yet available.
Candida spp. represented the seventh cause of BSIs in

Mycopathologia (2014) 178:243250

Table 4 Distribution of the
most common Candida
species according to clinical
characteristics

247

C. albicans
(n = 26)

C. tropicalis
(n = 29)

C. parapsilosis sensu
stricto (n = 23)

Characteristics

All episodes
(n = 93a)

Neonates

22

9 (34.6)

4 (13.8)

8 (34.8)

ICU admission

64

18 (69.2)

19 (65.5)

16 (69.6)

Data are presented as the

number of patients with the
percentage given in
parenthesis

Burns
Gastrointestinal disease

10
25

2 (7.7)
10 (38.5)

2 (6.9)
9 (31)

4 (17.4)
3 (13)

Abdominal surgery

32

10 (38.5)

11 (37.9)

Cancer

10

1 (3.8)

4 (13.8)

Patients who had more

than one species of Candida
bloodstream were excluded
from analysis

6 (26.1)
3 (13)

Mechanical ventilation

69

19 (73.1)

21 (72.4)

16 (69.6)

Parenteral nutrition

50

16 (61.5)

16 (55.2)

13 (56.5)

our institution (data not shown), similarly to found in a

large database of nosocomial BSIs in Brazil [5].
Consistent with recent Brazilian series [1721], the
frequency of NCAC species herein observed was
greater than C. albicans, showing the importance of
NCAC species in Brazil. As shown in Table 3, in our
institution, C. tropicalis was the species most often
isolated from candidemia (30.5 %), which frequency
was higher than those observed in other Brazilian
medical centers (4.927.3 %) [7, 8, 17, 2123]. The
reasons why C. tropicalis outranks C. albicans are
unknown. Local epidemiological factors may make
important contributions to this finding, which may
include the absence of antifungal prophylaxis in our
institution. According to Chai et al. [24], there are data
supporting the potential for transmission of C. tropicalis within the hospital environment, including
cross-transmission between healthcare workers and
patients.
Although C. parapsilosis alone or in association
with C. tropicalis is the second most prevalent
Candida species after C. albicans in the majority of
Brazilian series, few studies evaluate the newly
described cryptospecies belonging to the C. parapsilosis complex. This is an important issue to address
since some studies have reported that the prevalence of
these species could vary according to the geographic
region [25]; besides, differences in antifungal susceptibility among cryptic species have been observed
[26].
Our data indicate that C. glabrata occurs at low
frequency (the fourth etiological agent) as observed in
previous studies at different regions of Brazil [8, 17,
20, 22, 27, 28]; however, a trend for an increase in the
incidence of C. glabrata candidemia in Brazilian

hospitals has been recently reported [21, 28]. A high

rate of C. glabrata candidemia (30 %) was found in
Brazilian hospitals with heavy use of fluconazole [29].
Demographic and clinical characteristics of
patients with candidemia are shown in Table 2. In
our institution, candidemia occurred more frequently
among males, which is in agreement with previously
Brazilian studies [8, 9]. The distribution of isolates
was more frequent in ICU patients than those on the
wards (Table 2). This finding is in accordance with
other studies in which candidemia has been frequently
found in ICU [22, 23]. Earlier studies consider
parenteral nutrition as important risk factor for candidemia due to C. parapsilosis [30]. In our study, the
frequency of candidemia due to C. parapsilosis did not
differ to that due to neither C. albicans (56.5 vs.
61.5 %; P = 0.72) nor C. tropicalis (56.5 vs. 55.2 %;
P = 0.92).
In general, hospital-acquired candidemia leads to
high mortality that is clearly associated with the
variation in different Candida species among susceptibility to antifungal agents and with delayed initiation
of effective antifungal therapy [31]. In the present
study, resistance to fluconazole was observed in 9
(8.9 %) isolates (C. albicans, 3.7 %; vs. nonalbicans
Candida species, 30.1 %; P \ 0.001); the largest
resistance to fluconazole was detected among C.
parapsilosis stricto sensu isolates (Table 5) when
applying the new species-specific clinical breakpoints.
This resistance rate was higher than that obtained
when the susceptibility was interpreted using previous
CLSI breakpoints for fluconazole (data not shown).
Similar data were observed in a recent multicentre
prospective survey conducted in Spain [32]. These
authors emphasize the repercussions of the recent

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Mycopathologia (2014) 178:243250

Table 5 Antifungal in vitro susceptibility of Candida isolates

to fluconazole, voriconazole, itraconazole, voriconazole and
amphotericin B
Species (n)

C. albicans (27)

C. tropicalis (31)

C. parapsilosis
sensu stricto
(26)

C. glabrata sensu
lato (9)

C. krusei (2)

All organisms
(101)

Antifungals

MIC range
(lg/mL)

No. of R
isolates
(%)

Fluconazole

0.1264

1 (3.7)

Itraconazole
Voriconazole

0.010.25
0.0150.5

0
1 (3.7)

Amphotericin B

0.122

1 (3.7)

Fluconazole

0.1232

1 (3.2)

Itraconazole

0.030.25

Voriconazole

0.0150.25

1 (3.2)

Amphotericin B

0.121

Fluconazole

0.516

7 (26.9)

Itraconazole

0.010.5

Voriconazole

0.0150.12

Amphotericin B

0.121

Fluconazole

1632

Itraconazole

0.251

Voriconazole

0.251

Amphotericin B

0.251

Fluconazole

6464

nt

Itraconazole
Voriconazole

0.120.25
0.251

0
0

Amphotericin B

11

Fluconazole

0.1264

9 (8.91)

Itraconazole

0.011

Voriconazole

0.0151

2 (1.98)

Amphotericin B

0.122

1 (0.99)

Fluconazole

Itraconazole

0.03

Voriconazole

0.03

Amphotericin B
Fluconazole

0.5
16

Itraconazole

0.06

Voriconazole

0.12

Amphotericin B

Quality control
strains
C. parapsilosis
ATCC 22019

C. krusei ATCC
6258

species-specific clinical breakpoints on resistance

rates. Resistance to fluconazole was not observed in
C. glabrata isolates, although high MIC values
(1632 lg/mL) were observed (Table 5) similarly to
previous described in another Brazilian medical center
[20]. Here, one isolate of C. albicans was resistant to
amphotericin B, although most studies report a lack of

123

amphotericin B resistance in Candida species [33, 34],

while other Brazilian studies also found Candida
species resistant to this drug [17, 19]. Concerning
therapeutic results against the resistant Candida spp.,
in our study, three patients did not receive antifungal
therapy due to death soon after diagnostic confirmation. Three patients received first-line fluconazole
therapy and had persistent positive blood cultures for
in vitro fluconazole-resistant C. parapsilosis sensu
stricto. These patients at high risk for candidemia were
exposure to fluconazole previous to diagnostic.
Cross-resistance between fluconazole and voriconazole was observed in one isolate each of C. abicans
and C. tropicalis, similarly to found by other authors
[4, 20, 34]. Susceptibility to echinocandins was not
determined in this study because in our institution,
patients were not treated with these drugs at the
beginning of the survey period. However, given the
poor prognosis of candidemia in Brazil, it has recently
been stated that echinocandins are the best option for
initial treatment of this infection, despite the high MIC
values obtained for C. parapsilosis [35].
In conclusion, our study revealed that a large
proportion of candidemia is due to NCAC, with C.
tropicalis outranking C. albicans. Thus, performing
local epidemiological surveillance studies is important
to evaluate potential changes in species distribution
and antimicrobial susceptibility and also to guide
therapeutic choices.
Acknowledgments This work was supported by Fundacao
Araucaria/Governo do ParanaBrazil and PROPPG/UELBrazil. V.G.C. was fellowship-holder of Coordenacao de
Aperfeicoamento de Pessoal de Nvel Superior (CAPES)
Brazil.
Conflict of interest The authors report no conflicts of interest.

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