Mycology
Distribution and antifungal susceptibility of Candida species causing
candidemia from 1996 to 1999
Ming-Fang Cheng
a,b,c,e, Kwok-Woon Yu
c, Ran-Bin Tang
d, Yu-Hua Fan
f, Yun-Liang Yang
g,
Kai-Sheng Hsieh
a, Monto Ho
b, Hsiu-Jung Lo
b,*
aDepartment of Pediatrics, Veterans General Hospital, Kaohsiung, Taiwan bDivision of Clinical Research, National Health Research Institutes, Taipei, Taiwan
cDepartment of Microbiology, Veterans General Hospital, Kaohsiung,, Taiwan
dSection of Infection Disease, Department of Pediatrics, Veterans General Hospital, Kaohsiung,, Taiwan eNational Yang Ming University, Taipei, Taiwan
fKoahsiung Medical University, Kaohsiung, Kaohsiung, Taiwan
gDepartment of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan Received 11 June 2003; received in revised form 6 August 2003
Abstract
Susceptibilities to amphotericin B and fluconazole of 383 Candida species isolated from blood were determined. Candida albicans was the most common species (55.6%), followed by Candida parapsilosis (17.5%), Candida tropicalis (16.5%), Candida glabrata (5.2%),
Candida guilliermondii (2.3%), and others (2.9%). All but three isolates, Candida ciferrii, C. tropicalis, and C. glabrata, one each, were
susceptible to amphotericin B. A total of 367 (95.8%) and 15 (4.2%) isolates were susceptible and susceptible-dose dependent to fluconazole, respectively. Only one isolate, a C. glabrata, was resistant to fluconazole. Few patients (13%) having prior fluconazole treatments may explain the low rate of resistance to fluconazole in this study. © 2004 Elsevier Inc. All rights reserved.
Keywords: Candida species; Candidemia; Antifungal susceptibility; Resistance
1. Introduction
Candida species now rank as the fourth most common
cause of nosocomial bloodstream infections in the United States (Jarvis, 1995). Non-albicans Candida species, includ-ing uncommon Candida krusei, Candida guilliermondii, and Candida ciferrii, have played an important role in candidemia in the past decade (de Gentile et al., 1991; Furman and Ahearn, 1983; Pfaller et al., 2003). The in-crease in the proportion of bloodstream infections by non-albicans Candida species is likely to associate with follow-ing causes: improvement in diagnostic procedures; increasing number of critically ill patients; surgical proce-dures; cytotoxic therapy with prolonged neutropenia; other immunosuppressive therapies; use of broad-spectrum
anti-biotics and indwelling invasive devices; and intensive care support (Yang and Lo, 2001; Verduyn Lunel et al., 1999).
Due to differences in antifungal practices and infection control strategies, there are some variations in the distribu-tion of Candida species and their susceptibility to flucon-azole among isolates from different institutions, localities, and countries (Sanglard and Odds, 2002; St. Germain et al., 2001). Totally, 6% of Candida species were fluconazole resistant in one of teaching hospitals in Taiwan and in India (Hsueh et al., 2002; Chakrabarti et al., 2002). Among 88 non-albicans Candida species causing candidemia, 17% were fluconazole resistant in the Slovak Republic (Kovaci-cova et al., 2000). In order to understand the spectrum of
Candida species involved and the emergence of antifungal
resistance, we retrospectively surveyed the clinical Candida species isolated from blood over a 4-year period (1996 – 1999) at Veterans General Hospital-Taipei (VGH-TPE), Taiwan.
* Corresponding author. Tel.:⫹886-2-2652-4095; fax: ⫹886-2-2789-0254.
E-mail address: [email protected] (H.-J. Lo).
www.elsevier.com/locate/diagmicrobio 48 (2004) 33–37
0732-8893/04/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2003.08.002
2. Materials and methods
2.1. Strains and media
Strains (one strain per species per patient) of Candida species isolated from blood were collected from April 1996 to December 1999 at VGH-TPE, a teaching hospital in Taiwan with 2800 beds. This hospital provides both primary and tertiary medical care, admitting an average of 69,000 patients each year. The isolates were first subcultured on Sabouraud dextrose agar (SDA, BBL, Becton Dickinson and Company, Cockeysville, MD) plates to check for pu-rity. Pure isolates were labeled and stored at ⫺70°C for further analysis. Brain heart infusion agar or broth (BHI, DIFCO, Becton Dickinson and Company, Sparks, MD), SDA plates, and RPMI 1640 (GibcoBRL, Inchinnan, Pais-ley, UK) were used routinely for inoculation.
Organisms were identified to species level by germ tube test, morphology evaluation on cornmeal-Tween 80 agar, carbohydrate assimilation tests with API -32C strips (bio-Me´rieux Vitek, Inc., Hazelwood, Mo.), and Yeast Biochem-ical Card (YBC, bioMe´rieux Vitek, Inc., Hazelwood, Mo.) in the laboratory at VGH-TPE and/or at National Health Research Institutes (NHRI), Taipei, Taiwan.
Of 415 Candida isolates, 30 isolates were not recovered from ⫺70°C and two failed to grow in the RPMI 1640 medium. A total of 383 Candida species were enrolled into this study.
2.2. Antifungal susceptibility testing
Susceptibility to antifungal drugs was determined in the laboratory at NHRI. Minimum inhibitory concentrations (MIC) to amphotericin B and fluconazole were determined by a broth microdilution method according to the recom-mendations of the National Committee for Clinical Labo-ratory Standards (NCCLS), approved standard M27-A (Na-tional Committee for Clinical Laboratory Standards, 1997).
Candida albicans ATCC 14053, C. krusei ATCC 6258, Candida parapsilosis ATCC 22019, and Candida glabrata
ATCC 9003 were quality control strains in each batch of clinical isolates.
The MIC to amphotericin B was defined as minimum inhibitory concentration of amphotericin B reducing the turbidity to greater than 95%, after 48 h of incubation at 35°C. The MIC to fluconazole was defined at 50% reduction of turbidity. The interpretative breakpoints for both ampho-tericin B and fluconazole were determined according to the NCCLS guidelines. Isolates with resistance to amphotericin B were defined as MIC ⭌2g/ml. Resistant, susceptible-dose dependent, and susceptible to fluconazole were defined as MICs⭌64g/ml, 16–32 g/ml, and ⬉8 g/ml, respec-tively. The MIC of 50% of all isolates was defined as MIC50.
2.3. Clinical data collection
Since there were isolates of C. albicans, C. ciferrii C.
glabrata, C. guilliermondii, and C. tropicalis having
flucon-azole MICs ⭌16 g/ml, all 92 patients infected with C.
glabrata, C. guilliermondii, or C. tropicalis and two patients
infected with either C. albicans or C. ciferrii who showed fluconazole MICs⭌16g/ml were reviewed. The clinical data were collected retrospectively and inclusion criteria for patients were based on the presence of at least one positive blood culture with Candida species from April 1996 to December 1999 at VGH-TPE. The predisposing events within 30 days before developing candidemia were evalu-ated and the laboratory data within seven days that the first positive blood cultures were analyzed.
3. Results
3.1. Distribution of species
A total of 436 fungal pathogens causing fungemia were collected from April 1996 to December 1999 in the micro-biology laboratory at VGH-TPE. Candida species were the most common pathogens, accounting for 415 isolates (95.3%), followed by 11 (2.5%) Trichosporon species, 8 (1.8%) Cryptococcus neoformans, 1 (0.2%) Sporothrix
schenckii, and 1 (0.2%) Zygomycetes. On average, there
were 1.3 fungal infections per 1000 discharges during the study period.
Of 383 Candida species tested for susceptibility to anti-fungal agents, C. albicans was the most common species (213, 55.6%), followed by C. parapsilosis (67, 17.5%), C.
tropicalis (63, 16.5%), C. glabrata (20, 5.2%), C. guillier-mondii (9, 2.3%), C. peniculosa (7, 1.8%), C. famata (3,
0.8%), and C. ciferrii (1, 0.3%) (Table 1).
3.2. Susceptibility to amphotericin B and fluconazole
The MICs of the Candida species to amphotericin B and fluconazole are listed in Table 1. The range of MICs of amphotericin B was 0.25 to 2g/ml, and the MIC50was 0.5
g/ml. Three isolates, one of each C. ciferrii, C. glabrata, and C. tropicalis had MICs of amphotericin B equal to 2 g/ml.
The range of MICs to fluconazole was from 0.125 to 128
g/ml, and the MIC50was 0.5g/ml. A total of 15 (4.2%)
isolates were susceptible-dose dependent. They included nine fatality cases (one C. tropicalis, two C. guilliermondii, and six C. glabrata) and six cured cases (one C. ciferrii, one
C. albicans, one C. glabrata, and three C. guilliermondii).
Only one isolate, a C. glabrata, was resistant to fluconazole with its MIC equal to 128g/ml (Table 2).
3.3. Clinical data
All 16 patients infected by Candida species having flu-conazole MICsⱖ16 g/ml are listed in Table 2. Candida
glabrata and C. ciferrii had higher amphotericin B MICs
than other Candida species. Of these 16 patients, 13 had catheters, and only 3 patients were previously treated with antifungal drugs.
4. Discussion
It has been reported that beside C. parapsilosis, non-albicans Candida species including C. krusei (75%), C.
glabrata (35%), C. tropicalis (10 –25%), and Candida lus-itaniae (10 –25%) causing candidemia having had higher
resistance rates to fluconazole than C. albicans (Krcmery and Barnes, 2002; Pfaller et al., 2003). Of 383 isolates collected from the VGH-TPE, only one was resistant to
fluconazole. Of 92 reviewed patients infected with C.
gla-brata, C. guilliermondii, or C. tropicalis, only 13% of the
patients (12/92) had prior fluconazole treatments. This fact may explain the low rate of resistance to fluconazole in this study. This is consistent with the idea that continuous ex-posure to azoles seems to have an essential role in devel-oping resistance to fluconazole in Candida species (Kon-toyiannis, 2002).
Although C. albicans remains the most common species causing candidemia, the prevalence of non-albicans
Can-dida species has increased over the last decade (Kao et al.,
1999; Pfaller et al., 1999; Sanglard and Odds, 2002). The prevalence of C. glabrata candidemia has been increasing due to its high MIC to fluconazole. It is the most common
non-albicans Candida species in some other studies
(San-glard and Odds, 2002; Pfaller et al., 1999). However, C.
glabrata caused only 5.2% of candidemia in this study,
which is relatively low compared to recent studies (Sanglard and Odds, 2002). There was no C. krusei isolated in this
Table 1
In vitro susceptibilities of Candida species to amphotericin B and fluconazole
Species Amphotericin B Fluconazole
No. of isolates
MIC (g/ml) No. (%) of which
MICsⱖ 2g/ml
MIC (g/ml) No. (%) of which
MICsⱖ 16g/ml
Range (mean) MIC50 Range (mean) MIC50
C. albicans 213 0.25–1 (0.5) 0.5 0 0.125–32 (0.6) 0.25 1 (0.5) C. parapsilosis 67 0.5–1 (0.7) 0.5 0 0.125–2 (1.1) 1 0 C. tropicalis 63 0.25–2 (0.7) 0.5 1 (1.6) 0.125–8 (1.6) 11 1 (1.6) C. glabrata 20 0.5–2 (1.0) 1 1 (5) 2–128 (16.5) 8 8 (40) C. guilliermondii 9 0.25–0.5 (0.4) 0.25 0 2–32 (12.2) 16 5 (55.5) C. peniculosa 7 0.25–0.5 (0.4) 0.25 0 1–4 (3.3) 4 0 C. famata 3 0.25–1 (0.6) 0.5 0 0.255–8 (4.1) 4 0 C. ciferrii 1 2 1 (100) 32 1 (100) Total 383 0.25–2 (0.6) 0.5 3 (0.8) 0.125–128 (2.1) 0.5 16 (4.2)
MIC, minimum inhibitory concentrations.
Table 2
Information of patients infected by Candida species having fluconazole MICsⱖ 16g/ml
patient year species MIC (Flu)* MIC (AmB) Outcome treatment Immunosupression Gender Catheters prior treatment
1 1997 cal 32 0.5 Cured Flu No Female Yes No
2 1996 cci 32 2 Cured no No Male Yes No
3 1997 cgl 16 1 Dead Flu No Male Yes No
4 1997 cgl 128 1 Cured Flu No Female No No
5 1997 cgl 16 2 Dead no Yes Male Yes Itr
6 1998 cgl 16 1 Dead AmB No Male Yes No
7 1998 cgl 16 1 Dead AmB No Female Yes No
8 1998 cgl 16 1 Dead Flu No Female Yes No
9 1998 cgl 16 1 Cured no No Male Yes No
10 1999 cgl 16 1 Dead Flu No Male No No
11 1997 cgu 32 0.25 Cured AmB No Female No No
12 1997 cgu 16 0.5 Dead Flu No Male Yes No
13 1997 cgu 16 0.5 Cured AmB No Female Yes No
14 1999 cgu 16 0.5 Dead Flu No Male Yes Flu and AMB
15 1999 cgu 16 0.25 Cured no No Male Yes No
16 1996 ctr 16 0.5 Dead Flu Yes Female Yes Flu
study. This indicates that infection of C. krusei has re-mained a rare cause even though it is intrinsically resistant to fluconazole (Colombo et al., 1999; Kao et al., 1999; Sandven et al., 1998). Azole prophylaxis is one risk factor for C. glabrata and C. krusei infections (Krcmery and Barnes, 2002). Low prevalence of C. glabrata and C. krusei infections in this study may be a result of infrequent flu-conazole prophylaxis during the study period.
Mardani et al. reported nine candidemia caused by C.
guilliermondii from 1988 to 1998 in cancer patients. Five of
those candidemia (55.6%) occurred as a breakthrough in-fection while patients were on antifungal prophylaxis with agents such as fluconazole, itraconazole, or amphotericin B (Mardani et al., 2000). Poor responses to antifungal therapy in vivo and emergence of resistance to amphotericin B during therapy had also been described by other studies (Tietz et al., 1999; Bulmer et al., 1999; Karlowsky et al., 1997). In this study, C. guilliermondii had the highest sus-ceptible-dose dependent rate to fluconazole, which is con-sistent with a recent report concerning one hospital in Tai-wan (Lee et al., 2002).
It has been suggested that C. ciferrii was an etiologic agent of superficial yeast infections in humans even though this species was thought to be a saprophyte previously (Furman and Ahearn, 1983; de Gentile et al., 1995). This species has been reported causing invasive fungal infections in humans in Australia and being a fluconazole-resistant fungus (Gunsilius et al., 2001). In this study, only one C.
ciferrii was isolated and it was resistant to amphotericin B
and susceptible-dose dependent to fluconazole.
In addition to the common non-albicans Candida spe-cies, including C. glabrata, C. parapsilosis, and C.
tropi-calis, the increasing trend of unusual Candida species, such
as C. lusitaniae, C. guilliermondii and C. ciferrii should be noted (Krcmery and Barnes, 2002). Previous study also showed that these rare Candida species appear to be more resistant to currently antifungal agents (Pfaller et al., 2003). Following works would be to determine the risk factors for non-albicans Candida species infections.
Acknowledgments
We would like to thank Mrs. Ing-Ming Liu at VGH-TPE for her help in collecting the isolates. We are also indebted to Mr. Hsiao-Hsu Cheng and Dr. Feng-Jui Chen at the National Health Research Institutes, Taipei, Taiwan, for their help in data processing. We would also like to thank Drs. Calvin M. Kunin and Lawrence C. McDonald for their helpful discussion and suggestions.
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