www.elsevier.com/locate/diagmicrobio

Diagnostic Microbiology and In

Antifungal susceptibility of 1000 Candida bloodstream isolates to

5 antifungal drugs: results of a multicenter study conducted in

Sao Paulo, Brazil, 1995–2003

Daniel Archimedes da Mattaa, Leila Paula de Almeidaa, Antonia Maria Machadoa,

Ana Carolina Azevedoa, Elisa Junko Ura Kusanob, Norma Fracalanza Travassosc,

Reinaldo Salomaoa,d, Arnaldo Lopes Colomboa,4aDivision of Infectious Diseases, Universidade Federal de Sao Paulo, SP 04023-062, Brazil

bMicrobiology Section, Hospital do Servidor Publico Estadual de Sao Paulo, SP 04038-034, BrazilcMicrobiology Section, Hospital Beneficencia Portuguesa, SP 01323-000, Brazil

dDivision of Infectious Diseases, Hospital e Maternidade Santa Marcelina, SP 08270-070, Brazil

Received 12 July 2006; accepted 21 October 2006

Abstract

We evaluated all Candida sp. bloodstream isolates obtained from patients admitted to 4 tertiary care hospitals between 1995 and 2003 in

the city of Sao Paulo, Brazil. Susceptibility to amphotericin B, 5-fluorocytosine, fluconazole (FCZ), itraconazole (ITZ), and voriconazole

(VCZ) was determined using the Clinical Laboratory Standards Institute broth microdilution method. We tested a total of 1000 strains,

including 400 strains of Candida albicans (40%), 243 of Candida tropicalis (24.3%), 238 of Candida parapsilosis (23.8%), 44 of C.

glabrata (4.4%), 30 of Candida guilliermondii (3%), and 25 of Candida rugosa (2.5%). Only 1.9% of the strains tested were susceptible in a

dose-dependent manner, and 0.2% of them were resistant to FCZ. Almost 100% of the strains were susceptible to VCZ. Despite that azole

resistance was a rare finding, a trend toward increased resistance among C. rugosa strains to FCZ and ITZ was noted.

D 2007 Elsevier Inc. All rights reserved.

Keywords: Candida spp.; Candidemia; Antifungal susceptibility testing; Resistance

1. Introduction

A progressive increase in the frequency of candidemia

has been observed worldwide, particularly among patients

receiving antibiotics, immunosuppressive therapy, or paren-

teral nutrition, as well as among patients exposed to invasive

medical procedures (Verduyn Lunel et al., 1999). Studies

conducted in tertiary care hospitals in different countries of

Europe and in the United States have shown incidence rates

of candidemia ranging from 0.17 to 0.76 and 0.28 to 0.96

per 1000 admissions, respectively (Kao et al., 1999;

Marchetti et al., 2004; Diekema et al., 2002; Richet et al.,

2002). It is worth mentioning that candidemia leads to an

0732-8893/$ – see front matter D 2007 Elsevier Inc. All rights reserved.

doi:10.1016/j.diagmicrobio.2006.10.011

4 Corresponding author. Tel.: +55-11-5081-3240; fax: +55-11-5083-

0806.

E-mail address: colomboal@terra.com.br (A.L. Colombo).

increased time of hospitalization and has a mortality rate of

about 50% (Gudlaugsson et al., 2003).

Antimicrobial resistance is a concern regarding human

pathogens including fungi. Antifungal resistance surveil-

lance programs have been conducted recently by different

groups, and there is mounting evidence suggesting that the

emergence of invasive infections due to Candida non-

albicans (CNA) spp. resistant to fluconazole (FCZ) may be

an increasing problem in several medical centers in the

world. The resistance rates of Candida glabrata blood-

stream isolates to FCZ range from 7% to 14% in American

hospitals and from 3.7% to 40% in European hospitals

(Chryssanthou, 2001; Diekema et al., 2002; Ostrosky-

Zeichner et al., 2003; Hajjeh et al., 2004; Cuenca-Estrella

et al., 2005; Tortorano et al., 2006).

The surveillance programs focused on candidemia in

South American hospitals remain limited. In addition, there

are few studies extensive enough to provide temporal and

fectious Disease 57 (2007) 399–404

D.A. da Matta et al. / Diagnostic Microbiology and Infectious Disease 57 (2007) 399–404400

geographic trends in terms of the emergence of antifungal

resistance in our region. In the study described here, we

provided data on the trend of species distribution and azole

resistance among 1000 Candida bloodstream isolates

obtained from patients admitted to 4 medical centers from

1995 to 2003.

Table 1

Species distribution of 1000 Candida bloodstream isolates stratified in 2

periods: 1995 to 1999 and 2000 to 2003

Candida spp. Period 1

(1995–1999),

n (%)

Period 2

(2000–2003),

n (%)

Total,

n (%)

P

C. albicans 129 (43) 271 (38.6) 400 (40) .18

C. tropicalis 74 (24.7) 169 (24.1) 243 (24.3) .82

C. parapsilosis 58 (19.5) 180 (25.6) 238 (23.8) .03

C. glabrata 16 (5.5) 28 (4) 44 (4.4) .33

C. guilliermondii 12 (4) 18 (2.7) 30 (3) .22

C. rugosa 6 (2) 19 (2.7) 25 (2.5) .51

C. krusei 1 (0.3) 5 (0.7) 6 (0.6) .47

Other Candida spp. 3a (1) 11b (1.6) 14 (1.4) .48

Total 299 701 1000

a C. pelliculosa (2) and C. zeylanoides (1).b C. lusitaniae (5); C. pelliculosa (4); C. famata (1), and

C. lipolytica (1).

2. Materials and methods

2.1. Samples

We evaluated all Candida sp. bloodstream isolates

obtained from patients admitted to 4 tertiary care hospitals

between January 1995 and December 2003 in the city of

Sao Paulo, Brazil: Hospital Sao Paulo, Hospital do Servidor

Publico Estadual, Casa de Saude Santa Marcelina, and

Hospital da Beneficencia Portuguesa. The isolates of

Candida spp. recovered from blood cultures from each

center were sent to the Special Mycology Laboratory, at the

Universidade Federal de Sao Paulo, for further species

identification and antifungal susceptibility testing. Isolates

were stored on YPD glycerol (1% yeast extract, 2%

peptone, 2% dextrose, and 3% glycerol) at �70 8C for

different periods before they were tested.

2.2. Yeast identification procedures

The isolates were plated onto CHROMagar Candida

(CHROMagar Microbiology, Paris, France) to check the

purity and viability of all original yeast cultures. C. albicans

isolates were identified if they exhibited green colonies on

CHROMagar Candida and formed chlamydospores on corn

meal-Tween 80 agar. CNA species isolates were identified

on the basis of their micromorphology on corn meal-Tween

80 agar and biochemical tests performed with the commer-

cial system ID 32C (bioMerieux Marcy l’Etoile, France)

(Warren and Hazen, 1999).

2.3. In vitro susceptibility testing

Antifungal susceptibility testing was performed using the

Clinical Laboratory Standards Institute (CLSI, 2002,

formerly National Committee for Clinical Laboratory

Standards) broth microdilution assay method. The follow-

ing antifungal drugs, supplied by the manufacturers as pure

standard compounds, were tested: amphotericin B (AMB)

and 5-fluorocytosine (5FC) (Sigma Chemical, St. Louis,

MO), FCZ and voriconazole (VCZ) (Pfizer, New York,

NY), and itraconazole (ITZ) (Janssen Pharmaceutica, Titus-

ville, NJ). Briefly, the medium used was RPMI-1640

(Sigma Chemical), with l-glutamine, without bicarbonate,

and buffered at pH 7.0 with 3-(N-morpholino)propanesul-

fonic acid (0.165 mol/L, Sigma Chemical). The yeast

inoculum suspension was prepared by using a spectropho-

tometer to obtain a final yeast concentration containing 0.5

to 2.5 � 103 cells/mL. The plates were incubated at 35 8Cfor 48 h. Quality control was ensured by testing the CLSI-

recommended strains, Candida krusei ATCC 6258 and

C. glabrata ATCC 22019. The MIC for AMB was

considered the lowest tested concentration able to prevent

any visible growth. The MICs for each azole and 5FC were

defined as the lowest concentration resulting in a

prominent inhibition of growth (~50%) compared with the

control growth (CLSI, 2002).

The breakpoints for azoles and 5FC MICs were those

suggested by the CLSI (2002) M27-A2 document and Pfaller

et al. (2006a). Because of the lack of consensus about the

definition of MIC breakpoints for AMB, arbitrary values

suggested by a previous study were used by Nguyen et al.

(1998). Isolates with MICs V1 Ag/mL for AMB, V4 Ag/mL

for 5FC, V8 Ag/mL for FCZ, V0.125 Ag/mL for ITZ, and

V1 Ag/mL for VCZ were considered susceptible. Isolates

with MICs from 8 to 16 Ag/mL were classified as inter-

mediate to 5FC. Isolates with MICs from 16 to 32 Ag/mL for

FCZ, 0.25 to 0.5 Ag/mL for ITZ, and 2 Ag/mL for VCZ were

considered as susceptible in a dose-dependent manner

(SDD). MICs z2 Ag/mL for AMB, z32 Ag/mL for 5FC,

z64 Ag/mL for FCZ, z1 Ag/mL for ITZ, and z4 Ag/mL for

VCZ were considered resistant.

Analysis of the temporal influence on species distribution

and antifungal susceptibility pattern was arbitrarily per-

formed considering 2 different intervals: 1995 to 1999

(period 1) and 2000 to 2003 (period 2). Statistical analysis

was performed by using v2 and Kruskal–Wallis test when

appropriated with the software SPSS 11.0.1 software (SPSS,

Chicago, IL).

3. Results

3.1. Distribution of Candida spp.

Table 1 summarizes the distribution of 1000 Candida sp.

bloodstream isolates. Only a single candidemic episode for

each patient was considered for the study. Four hundred of

them were C. albicans (40%), 243 Candida tropicalis

(24.3%), 238 C. parapsilosis (23.8%), 44 C. glabrata

Table 2

In vitro susceptibilities of 1000 Candida bloodstream isolates tested against FCZ, ITZ, and VCZ

Candida spp. (n) FCZ ITZ VCZ

Ag/mL % Ag/mL % Ag/mL %

MIC50/MIC90 Sa/SDDb/Rc MIC50/MIC90 S/SDD/R MIC50/MIC90 S/SDD/R

C. albicans (400) 0.25/0.5 100/0/0 0.03/0.06 99.5/0.5/0 0.03/0.03 100/0/0

C. tropicalis (243) 0.5/1 100/0/0 0.03/0.06 98.4/1.6/0 0.03/0.06 100/0/0

C. parapsilosis (238) 0.5/2 100/0/0 0.03/0.06 98.8/0.8/0.4 0.03/0.03 100/0/0

C. glabrata (44) 4/8 93.2/2.3/4.5 0.25/1 34.1/50/15.9 0.125/0.25 93.2/2.3/4.5

C. guilliermondii (30) 2/4 96.7/3.3/0 0.25/0.25 43.4/50/6.6 0.06/0.125 100/0/0

C. rugosa (25) 2/32 56/44/0 0.03/0.5 68/32/0 0.25/0.5 100/0/0

C. krusei (6)d 16/– 0/100/0 0.25/– 33.5/66.5/0 0.25/– 100/0/0

Othere (14) 2/2 100/0/0 0.03/0.125 92.9/7.1/0 0.03/0.06 100/0/0

Total (1000) 0.5/2 97.9/1.9/0.2 0.03/0.125 93.2/5.8/1 0.03/0.06 99.7/0.1/0.2

a Percentage of isolates susceptible to azoles by using CLSI (2002).b Percentage of isolates susceptible-dose dependent to azoles by using CLSI (2002).c Percentage of isolates resistant to azoles by using CLSI (2002).d Isolates of C. krusei should be always assumed to be intrinsically resistant to fluconazole (CLSI, 2002).e C. pelliculosa (6), C. lusitaniae (5), C. famata (1), C. lipolytica (1), and C. zeylanoides (1).

D.A. da Matta et al. / Diagnostic Microbiology and Infectious Disease 57 (2007) 399–404 401

(4.4%), 30Candida guilliermondii (3%), 25Candida rugosa

(2.5%), 6 C. krusei and Candida pelliculosa (0.6%), and

5 Candida lusitaniae (0.5%), and there was 1 isolate of each

species of Candida famata, Candida lipolytica, and Candida

zeylanoides (0.1%). The species distribution throughout the

9-year period was stratified by 2 different intervals: 1995 to

1999 (period 1) and 2000 to 2003 (period 2). The rank order

of Candida sp. distribution was relatively stable over the

9-year period of study, except for the increased number of

C. parapsilosis strains isolated in the 2nd period (19.5 �25.6, P = .03). Except for the 25 strains of C. rugosa that

were isolated from patients admitted in a single institution,

the rank order of Candida sp. distribution was basically the

same within the 4 medical centers.

3.2. Antifungal susceptibility testing

MIC values of 1000 strains of Candida spp. ranged

from 0.06 to 1 Ag/mL for AMB, 0.125 to z64 Ag/mL for

5FC and FCZ, 0.03 to 8 Ag/mL for ITZ, and 0.03 to 4 Ag/mL for VCZ. Table 2 summarizes the MICs at which 50%

(MIC50) and 90% (MIC90) of the isolates tested were

inhibited for each drug and the susceptibility categories to

FCZ, ITZ, and VCZ according to CLSI (2002) and Pfaller

et al. (2006a).

Table 3

Susceptibility profile of 1000 Candida bloodstream isolates tested against 5 antif

Candida spp. (n1/n2) % Susceptible isolates tested during time

AMB 5-Fluorocytosine

C. albicans (129/271) 100/100 96.9/98.9

C. tropicalis (74/169) 100/100 97.3/91.1

C. parapsilosis (58/180) 100/100 98.3/97.2

C. glabrata (16/28) 100/100 100/100

C. guilliermondii (12/18) 100/100 100/100

C. rugosa (6/19) 100/100 100/100

Total (299/701) 100/100 97.4/96.3

a Diferences were not statistically significant to fluconazole ( P = .55) and ib Diferences were statistically significant to fluconazole ( P = .01) and itraco

3.3. Amphotericin B

None of the strains showed MIC values z2 Ag/mL for

AMB, including the 5 strains of C. lusitaniae.

3.4. 5-Fluorocytosine

Twenty-five isolates (2.5%) were resistant to 5FC,

including 13 strains of C. tropicalis, 7 of C. albicans, 4

of C. parapsilosis, and 1 of C. krusei.

3.5. Fluconazole

All strains of C. albicans, C. tropicalis, C. parapsilosis,

C. pelliculosa , C. lusitaniae , C. zeylanoides , and

C. lipolytica strains had MIC values V8 Ag/mL and were

classified as susceptible to FCZ. A total of 19 (1.9%) isolates

were considered SDD to FCZ, including 11 strains of

C. rugosa, 6 of C. krusei, 1 of C. guilliermondii, and 1 of

C. glabrata. Only 2 (0.2%) C. glabrata isolates were

resistant to this antifungal agent (MIC z64 Ag/mL).

3.6. Itraconazole

A total of 58 (5.8%) isolates were SDD to ITZ, including

22 strains of C. glabrata, 15 of C. guilliermondii, 8 of

C. rugosa strains, 4 of C. krusei, 4 of C. tropicalis, 2 of

ungal drugs

intervals: 1995–1999/2000–2003

FCZ ITZ VCZ

100/100 99.2/99.6 100/100

100/100 97.3/98.8 100/100

100/100 96.5/99.5 100/100

100/89.3a 50/28.6a 100/89.3

91.7/100 50/59 100/100

100/42b 100/57.9b 100/100

99.3/97.3 93/93.3 100/99.6

traconazole ( P = .16).

nazole ( P = .06).

D.A. da Matta et al. / Diagnostic Microbiology and Infectious Disease 57 (2007) 399–404402

C. parapsilosis, 2 of C. albicans, and 1 of C. pelliculosa.

Seven strains of C. glabrata, 2 of C. guilliermondii, and 1 of

C. parapsilosis were resistant to this antifungal drug.

3.7. Voriconazole

All the isolates that were susceptible in vitro to FCZ and

ITZ were also susceptible to VCZ. Among the 19 isolates

classified as SDD to FCZ, only 1 of them was SDD to VCZ.

The 2 C. glabrata strains resistant to FCZ were classified as

SDD and resistant to VCZ.

3.8. Analysis of the temporal influence on the azole

antifungal susceptibility

Table 3 summarizes the susceptibilities in vitro of

5 antifungal agents against 1000 Candida sp. strains tested

throughout the 2 intervals: 1995 to 1999 (period 1) and 2000

to 2003 (period 2). Isolates remained highly susceptible to

AMB (100%), 5FC (~97%), FCZ (99–97%), ITZ (~93%),

and VCZ (100–99.7%) over the entire 9-year period. Only

isolates of C. rugosa and C. glabrata spp. showed a trend to

increased resistance to azoles in the 2nd period. The rates of

susceptibility of C. rugosa strains decrease from 100% to

42% to FCZ (P = .01) and 100% to 58.1% to ITZ (P = .06)

throughout the period. Despite that some differences were

noted on the susceptibility of C. glabrata strains throughout

the period of the study, the values were not statistically

significant.

4. Discussion

This study represents the largest series published on the

microbiologic profile of candidemia episodes in Brazilian

hospitals. Our data confirm the results previously published

on the high prevalence of C. tropicalis and C. parapsilosis

among CNA species associated with candidemia in Brazil

(Colombo et al., 1999; Costa et al., 2000; Goldani and

Mario, 2003; Antunes et al., 2004; Colombo et al., 2006).

On the other hand, our results point to a trend of decreased

susceptibility to FCZ in C. rugosa strains isolated in a

single institution.

AlthoughC. albicans remains the most frequently isolated

species in this series, CNA isolates accounted for 60% of all

candidemic episodes. Unlike the epidemiology of North

Hemisphere countries, in Brazil, there is still a predominance

of CNA species susceptible to FCZ represented by

C. parapsilosis and C. tropicalis strains (Colombo et al.,

1999; Antunes et al., 2004; Aquino et al., 2005; Colombo et

al., 2006). According to our findings, candidemia due to

C. glabrata persists at a low frequency in Brazilian hospitals

accounting for less than 5% of the candidemic episodes. By

analyzing the data obtained throughout the 9-year period,

excluding the rising incidence of C. parapsilosis strains from

1995 to 2003 (19% versus 25%, P = .03), there were no

changes in distribution pattern of Candida spp.

It is worth mentioning that the 25 episodes of fungemia

due to C. rugosa were documented in a single institution.

This species of Candida is considered rare among infections

in humans. However, Colombo et al. (2003) documented an

outbreak of fungemia caused by this species in the same

hospital. Having in mind that new isolations occurred after

the outbreak and throughout the year, it is clear that this

pathogenic agent was able to adapt itself to the hospital

environment, causing new episodes of candidemia. Actual-

ly, C. rugosa is a fungus that appears to be emerging as a

cause of invasive infection in Latin America as suggested by

Pfaller et al. (2006b).

In accordance with other series published in different

countries, isolates from bloodstream infections caused by

C. albicans, C. parapsilosis , and C. tropicalis spp.

presented high susceptibility to all antifungal agents tested.

Resistance to azoles in C. albicans isolates associated with

fungemia remains low and is mostly limited to immunosup-

pressed patients with hematologic disorders receiving

prophylaxis with high doses of FCZ (Marr et al., 2000;

Ostrosky-Zeichner et al., 2003; Hajjeh et al., 2004).

All Candida isolates in our investigation had MIC values

V1 Ag/mL for AMB. Higher AMB MICs were not seen with

our C. lusitaniae strains, a species often considered as less

susceptible to AMB. This finding is in accordance with data

published by Ostrosky-Zeichner et al. (2003). In fact,

resistance to AMB in C. lusitaniae is not still well

understood, although there are data supporting mutations

in sterol pathways and other data supporting phenotypic

switching (Miller et al., 2006).

Although the CLSI method has proven to be reliable and

reproducible, it generates a restricted range of AMB MICs,

precluding discrimination between susceptible and resistant

isolates of Candida spp. (Park et al., 2006). Some studies

have emphasized that the E-test has significant value for the

determination of AMB MICs and represents one of the more

reliable ways to identify resistant isolates (Rex et al., 2001;

McClenny et al., 2002). However, interpretive breakpoints

for AMB susceptibility testing have remained controversial,

and therefore, routine AMB testing for Candida spp.

isolated from the bloodstream is probably not indicated

(Park et al., 2006).

Resistance to 5FC was documented in 2.5% of all the

isolates tested. This finding is in accordance with the data

generated by Pfaller et al. (2002) where only 3% of 8000

isolates tested were resistant to 5FC.

In our study, bloodstream isolates of C. tropicalis proved

to be susceptible to azoles. In other studies, C. tropicalis

strains related to candidemic episodes have shown resis-

tance rates ranging from 0% to 12% (Diekema et al., 2002;

Godoy et al., 2003; Ostrosky-Zeichner et al., 2003; Cheng

et al., 2004; Hajjeh et al., 2004; Colombo et al., 2006). Such

significant variation in resistance rates of C. tropicalis

strains may be in part related to the in vitro phenomenon

known as trailing growth. It is well known that C. tropicalis

and C. albicans strains can exhibit heavy trailing in the

broth microdilution method after 48 h of incubation

(Arthington-Skaggs et al., 2000). It is our belief that only

D.A. da Matta et al. / Diagnostic Microbiology and Infectious Disease 57 (2007) 399–404 403

a limited number of C. tropicalis bloodstream isolates are

able to express the mechanisms of azole resistance already

described by Vandeputte et al. (2005).

We obtained only 2 strains of C. glabrata resistant to

FCZ, representing less than 5% of the C. glabrata strains

tested. This finding suggests that resistance to FCZ against

Candida spp., including C. glabrata strains, is still rare in

clinical isolates from fungemia in Brazil. Recently, Colombo

et al. (2006) reported antifungal susceptibility data of 712

candidemic episodes documented in 11 medical centers from

9 Brazilian cities. The resistance rate to FCZ among C.

glabrata strains in that study was 5.7%. In contrast to our

findings, the resistance rate to FCZ among C. glabrata

isolates in bloodstream infections is higher in the United

States (7–14%) and Europe (4–40%) (Chryssanthou, 2001;

Diekema et al., 2002; Ostrosky-Zeichner et al., 2003;

Tortorano et al., 2006; Hajjeh et al., 2004; Cuenca-Estrella

et al., 2005). These differences may be related to the low use

of FCZ in prophylaxis and empirical therapy regimens in

Brazilian public hospitals as compared with American and

European medical centers (Colombo et al., 1999).

In spite of the low occurrence of isolates less susceptible

to FCZ in this series, it should be pointed out that there

was an increase in the isolation of C. rugosa strains

with reduced susceptibility to this drug during the study

(100–42%, P = .01). The trend toward increased resistance

among C. rugosa isolates to azoles is interesting and will

require further investigation to determine the factors behind

these observations.

In this study, VCZwas the triazole with the highest in vitro

antifungal activity against all Candida strains with 99.7% of

all the isolates tested susceptible to this antifungal agent. Of

note, 2 C. glabrata isolates resistant to FCZ also exhibited

lower susceptibility or resistance to VCZ. Although VCZ

displayed improved potency compared with FCZ, it is

apparent that cross-resistance to these agents may be

observed among the rare bloodstream infection isolates of

Candida spp. that are resistant to FCZ (Pfaller et al., 2001).

In conclusion, we were able to demonstrate that in

Brazilian hospitals, most CNA species are represented by

C. tropicalis and C. parapsilosis isolates still highly

susceptible to antifungal agents. Otherwise, we noted a

trend toward increased resistance among C. rugosa strains

to azoles in a single institution. Our data highlight the

relevance to continue surveillance programs to evaluate

trends of species distribution and resistance of Candida

isolates to antifungal drugs. This aspect becomes even more

important when we consider that new drugs have been

included in recent years in the therapeutic arsenal of

antifungal agents, and we must monitor their impact in the

epidemiology of candidemia.

Acknowledgments

The authors thank Dr. Beth Arthington-Skaggs for

critical reading of the manuscript.

This study was supported by CAPES (Coordenacao de

Aperfeicoamento de Pessoal de Nıvel Superior) and

FAPESP (Fundacao de Amparo a Pesquisa do Estado de

Sao Paulo), Brazil.

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