O R I G I N A L A R T I C L E
Distribution and drug susceptibilities of
Candida species causing
candidemia from a medical center in central Taiwan
Te-Pin Chang•Mao-Wang Ho •Yun-Liang Yang•Pai-Chang Lo•Pei-Sheng Lin •An-Huei Wang• Hsiu-Jung Lo
Received: 15 January 2013 / Accepted: 17 May 2013 / Published online: 4 June 2013
Ó Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases 2013
Abstract Invasive fungal infections have increased sig-nificantly in the past few decades because of the increase in high-risk populations. To investigate the distribution and drug susceptibilities of such infections, we analyzed all 152 Candida isolates causing candidemia from 2004 to 2006 at the China Medical University Hospital, a medical center in central Taiwan. Candida albicans was the most common species, accounting for 52.6 % of the isolates, followed by C. tropicalis (19.7 %), C. parapsilosis (14.5 %), C. glab-rata (8.6 %), C. guilliermondii (3.9 %), and C. pelliculosa (0.7 %). All isolates were susceptible to amphotericin B, anidulafungin, micafungin, and voriconazole according to minimum inhibitory concentrations (MICs) after a 24-h incubation; 0.7 %, 6.6 %, and 7.9 % of isolates were resistant to amphotericin B, fluconazole, and voriconazole, respectively, after 48-h incubation. Both C. albicans and C. parapsilosis had high degrees of agreement for azoles between 24- and 48-h incubation periods, whereas C.
glabrata (38.5–46.2 %) and C. tropicalis (56.7–63.3 %) did not. The majority of the isolates with high azole MICs displayed a trailing growth phenotype. Hence, the MICs of different drugs after 24-h incubation may be considered for prognosis of candidemia.
Keywords Amphotericin B Azoles Candida species Candidemia Drug susceptibility Echinocandins
Introduction
The epidemiology of invasive fungal infections has become important in the past few decades because of the increased number of immunocompromised patients, the augmentation of invasive medical devices, and the exten-sive use of broad-spectrum antibiotics [1–3]. Candida species are the most frequently isolated fungal pathogens,
T.-P. Chang and M.-W. Ho contributed equally to this article. T.-P. Chang P.-C. Lo A.-H. Wang H.-J. Lo (&) National Institutes of Infectious Diseases and Vaccinology, National Health Research Institutes, 35 Keyan Road, Zhunan Town, Miaoli 350, Taiwan
e-mail: [email protected] Present Address: T.-P. Chang
Botanical Garden Division, Taiwan Forestry Research Institute, Taipei, Taiwan
M.-W. Ho
Section of Infectious Diseases, Department of Internal Medicine, China Medical University Hospital, Taichung, Taiwan Y.-L. Yang
Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan
Y.-L. Yang
Institute of Molecular Medicine and Bioengineering, National Chiao Tung University, Hsinchu, Taiwan
P.-S. Lin
Division of Biostatistics and Bioinformatics, IPHS, National Health Research Institutes, Miaoli, Taiwan
H.-J. Lo
School of Dentistry, China Medical University, Taichung, Taiwan
responsible for significant morbidity and mortality [4–9], and the leading causes of bloodstream infections globally [2, 8–14]. According to the results of hospital-based sur-veillance, European countries reported 0.5–0.7 cases per 10,000 patient-days in association with invasive candidia-sis [15,16], and in the United States, it was 1.5 cases per 10,000 patient-days. Globally, Brazil has the highest reported incidence of 3.7 cases per 10,000 patient-days [11,
17]. On average, it is about 1.5 cases per 10,000 patient-days in Taiwan [18–20].
Hundreds of species of Candida have been described, but only 30–40 species among these have been reported to cause diseases in humans [21, 22]. Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsi-losis, and Candida krusei are the five most common species causing candidemia, in combination accounting for more than 95 % of the cases [2,9,11,15].
Azole (fluconazole and voriconazole), echinocandin (anidulafungin, caspofungin, and micafungin), and polyene (amphotericin B) are the three classes of drugs most com-monly prescribed for treating systemic fungal infections. The emergence of fungal pathogens resistant to these drugs is a growing concern [3,23,24]. Hence, we have conducted studies on 152 Candida isolates causing candidemia in central Taiwan, collected from 2004 to 2006, to determine the species distribution and their susceptibility profiles to five antifungal agents (amphotericin B, anidulafungin, fluconazole, micafungin, and voriconazole). We also com-pared the categorical agreement of minimum inhibitory concentrations (MICs) of amphotericin B, fluconazole, and voriconazole after 24- and 48-h incubation.
Materials and methods
Clinical isolates and patient age groups
All isolates causing candidemia were collected from October 2004 to December 2006 in central Taiwan at the China Medical University Hospital (CMUH), a medical center with approximately 2,000 beds. A total of 152 Candida isolates were collected and characterized in the present study. In addition to the sources of isolates, the patient age data were also recorded, and the patients were divided into three groups by age:\18 years, 19–65 years, and[65 years [25,26].
Identification of isolates
All Candida isolates were first identified by the CMUH and reassessed in the laboratory at National Health Research Institutes (NHRI). The identification procedure at NHRI for the candidemia isolates was based on our previous
reports [27–29]. All isolates were tested with the VITEK Yeast Biochemical Card (YBC) (bioMe´rieux, Marcy l’Etoile, France) for species identification. When the YBC identification probability was less than 90 % or when the identification of an organism was inconsistent between the hospital and the NHRI laboratories, sequences of the internal transcribed spacer (ITS) region and/or the D1/D2 region of ribosomal DNA were used for species identifi-cation. The ITS region was amplified with the primers ITS1, 50-TCCGTAGGTGAACCTGCGG-3, and ITS4 50 -TCCTCCGCTTATTGATATGC-30, and the D1/D2 region was amplified with the primers NL1 50-GCATATCAAT AAGCGGAGGAAAAG-30 and NL4 50-GGTCCGTG TTTCAAGACGG-30[30,31].
Antifungal susceptibility testing
The MICs of the five agents were determined by the same in vitro antifungal susceptibility testing established in our laboratory [32], according to the guidelines of M27-A3 recommended by the Clinical and Laboratory Standards Institute (CLSI) [33]. RPMI medium 1640 (31800-022; Gibco BRL, Gaithersburg, MD, USA) was used for growth and dilution of the yeasts. Strains from the American Type Culture Collection (ATCC), including C. albicans (ATCC 90028), C. krusei (ATCC 6258), and C. parapsilosis (ATCC 22019), were used as the standard controls. Growth of each isolate was measured by the Biotrak II plate spectrophotometric reader (Amersham Biosciences, Bio-chrom, Cambridge, England) after incubation at 35°C for 24 h for all five drugs and 48 h for amphotericin B, fluconazole, and voriconazole.
Standard powders of amphotericin B kindly provided by Bristol Myers Squibb, anidulafungin, fluconazole, and vo-riconazole by Pfizer, and micafungin by Astellas Pharms were dissolved in dimethyl sulfoxide (DMSO). The final concentrations of anidulafungin, micafungin, and vorico-nazole were 0.0156–8 mg/l, amphotericin B, 0.0313–16
mg/l, and fluconazole, 0.125–64 mg/l.
The newly defined species-specific breakpoints after 24-h incubation for the five common Candida species C. albicans, C. krusei, C. glabrata, C. parapsilosis, and C. tropicalis were applied in the present study [34]. For the species for which clinical breakpoints have not been established, we applied epidemiological cutoff values instead [34].
The MICs were defined as the concentration of drugs capable of reducing the turbidity of cells to greater than 50 % for anidulafungin, fluconazole, micafungin, and vo-riconazole and completely inhibiting cell growth for amphotericin B. The MICs of 50 % and 90 % of the total population were defined as MIC50and MIC90, respectively. The epidemiological cutoff value of amphotericin B was 2 mg/l. For fluconazole, with C. albicans, C. tropicalis, and
C. parapsilosis after 24-h incubation, MICsB2 mg/l were considered to be susceptible,C8 mg/l resistant, and 4 mg/l susceptible-dose dependent (S-DD); for C. glabrata, MICs B32 mg/l were susceptible and C64 mg/l resistant after 24 h. The epidemiological cutoff values for Candida guil-liermondii and Candida pelliculosa were 8 and 4 mg/l, respectively. When the incubations were extended to 48 h, then MICs B8 mg/l were susceptible, C64 mg/l resistant, and 16–32 mg/l S-DD for all species.
For voriconazole, with C. albicans, C. tropicalis, and C. parapsilosis, MICs B0.125 mg/l were susceptible, C1 mg/l resistant, and 0.25–0.5 mg/l intermediate after 24-h incubation. The epidemiological cutoff value for C. guilliermondii and C. pelliculosa was the same, 0.25 mg/l. MICs\1 mg/l were susceptible, 2 mg/l intermediate, and C4 mg/l resistant after 48 h.
Susceptibilities to echinocandins were suggested to be determined after 24-h incubation. For C. albicans and C. tropicalis, MICsB0.25 mg/l were susceptible, C1 mg/l resistant, and 0.5 mg/l intermediate. For C. parapsilosis and C. guilliermondii, the breakpoints were changed to\2 mg/l susceptible, 4 mg/l intermediate, and[8 mg/l resistant. For anidulafungin, with C. glabrata, MICsB0.125 mg/l were susceptible,C0.5 mg/l resistant, and 0.25 mg/l intermedi-ate. For micafungin, MICs B0.06 mg/l were susceptible, C0.25 mg/l resistant, and 0.125 mg/l intermediate.
Data analysis
Categorical agreement (CA) was assigned to compare the susceptibility testing results with the MIC values at 24 h and at 48 h for amphotericin B, fluconazole, and vorico-nazole that fell within the same interpretive categories. Agreement was defined as discrepancies between each MIC pair compared within ±1 twofold dilution. We developed the saw-like calculation to describe the CA by using R statistics software (R Foundation for Statistical Computing, Vienna, Austria). The statistical significance of the differences in frequencies and proportions was deter-mined by the chi-square test with Mantel–Haenszel correction.
Results
Species distribution
We recovered 15, 69, and 68 isolates in 2004, 2005, and 2006, respectively. A total of 9 isolates were speciated differently by the hospital and the NHRI laboratories: 3 C. tropicalis isolates were identified as Candida intermedia by CMUH, 2 C. glabrata, 1 C. guilliermondii, and 1 C. tropicalis as C. albicans, 1 C. parapsilosis as C. sake,
and 1 C. parapsilosis as C. tropicalis. VITEK failed to identify 14 isolates, including 8 C. parapsilosis, 5 C. guilliermondii, and 1 C. tropicalis. All these 23 isolates were speciated according to their rDNA sequences.
Of the 152 isolates, C. albicans was the most common species, accounting for 52.6 %, followed by C. tropicalis (19.7 %), C. parapsilosis (14.5 %), C. glabrata (8.6 %), C. guilliermondii (3.9 %), and C. pelliculosa (0.7 %) (Table1). Patient age ranged from 1 to 91 years (average age, 58± 24.7 years). Of the isolates, 11.2 %, 39.5 %, and 49.3 % were recovered from patients in the age groups of B18, 19–65, and C66 years, respectively (Table2). Among different species, the majority of the C. tropicalis isolates (18/30, 60 %; p = 0.04) were recovered from patients in the age group 19–65 years. More C. parapsi-losis were recovered from younger patients than from other age groups (10/17 vs. 12/135; p\ 0.001). Among the 17 isolates collected from patients aged B18 years, 10 (58.8 %) were C. parapsilosis.
Susceptibility to amphotericin B
The range of amphotericin B MICs of the 152 isolates was from 0.03 to 1 mg/l and 0.125 to 4 mg/l after 24- and 48-h incubations, respectively (Table1). The MIC50values for amphotericin B were 0.125 and 0.5 mg/l after 24- and 48-h incubations, respectively. Only 1 isolate of C. glabrata had an 4 mg/l amphotericin B MIC after 48-h incubation. Susceptibility to azole drugs
The range of fluconazole MICs of the 152 isolates was from 0.125 to 64 mg/l (Table1). The values of MIC50 for fluconazole were 0.125 and 0.25 mg/l after 24- and 48-h incubations, respectively. One C. parapsilosis iso-late (8 mg/l MIC) was resistant to fluconazole after 24-h incubation. Nevertheless, there were 10 isolates, includ-ing 7 C. tropicalis, and 1 each C. albicans, C. glabrata, and C. parapsilosis, with fluconazole MICs C64 mg/l after 48-h incubation. The range of voriconazole MICs of the 152 isolates was 0.03–0.5 mg/l after 24-h incu-bation and 0.03–16 mg/l after 48-h incuincu-bation (Table1). The values of MIC50 for voriconazole remained the same, staying at 0.03 mg/l, after 24- and 48-h incuba-tions. No isolate was resistant to voriconazole according to the MICs after 24-h incubation. After 48-h incubation, 10 C. tropicalis and 2 C. albicans had voriconazole MICs of 16 mg/l.
Susceptibility to echinocandins
MICs of echinocandins were determined after 24-h incu-bation only [35]. The values of MIC50for echinocandin of
Table 1 Susceptibilities to various antifungal drugs of the 152 isolates causing candidemia Type of drug Drug Incubation
(h)
MIC (mg/l)
Range MIC50 MIC90 %R %CA
Total (n= 152) Polyene AMB 24 0.03–1 0.125 1 0 72.4 48 0.125–4 0.5 1 0.7 Azole FLU 24 0.125–32 0.125 2 1.3 88.8 48 0.125–64 0.25 8 6.6 VOR 24 0.03–0.5 0.03 0.03 0 82.2 48 0.03–16 0.03 0.5 7.9 Echinocandin ANI 24 0.016–2 0.016 0.5 0 94.7 MICA 24 0.016–1 0.016 0.25 0 95.4 Candida albicans (n= 80) Polyene AMB 24 0.03–1 0.125 0.5 0 76.3 48 0.25–1 0.5 1 0 Azole FLU 24 0.125–1 0.125 0.25 1.2 90.0 48 0.125–64 0.125 1 1.2 VOR 24 0.03 0.03 0.03 0 91.3 48 0.03–16 0.03 0.06 2.5 Echinocandin ANI 24 0.016–0.03 0.016 0.016 0 95.0 MICA 24 0.016 0.016 0.016 0 97.5 Candida tropicalis (n= 30) Polyene AMB 24 0.125–0.5 0.125 0.5 0 56.7 48 0.25–1 0.5 1 0 Azole FLU 24 0.125–0.25 0.125 0.25 0 63.3 48 0.125–64 0.5 64 23.3 VOR 24 0.03 0.03 0.03 0 56.7 48 0.03–16 0.03 16 33.3 Echinocandin ANI 24 0.016–0.06 0.03 0.03 0 93.3 MICA 24 0.016–0.03 0.016 0.016 0 93.3 Candida parapsilosis (n= 22) Polyene AMB 24 0.06–1 0.125 0.5 0 68.2 48 0.125–2 0.5 1 4.5 Azole FLU 24 0.125–8 0.5 2 4.5 95.5 48 0.25–64 0.5 2 4.5 VOR 24 0.03–0.125 0.03 0.03 0 95.5 48 0.03–0.5 0.03 0.125 0 Echinocandin ANI 24 0.06–2 0.5 1 0 86.4 MICA 24 0.016–1 25 1 0 72.7 Candida glabrata (n= 13) Polyene AMB 24 0.125–1 1 1 0 92.3 48 0.5–4 1 2 15.4 Azole FLU 24 1–32 2 4 0 46.2 48 1–64 8 16 7.7 VOR 24 0.03–0.5 0.03 0.06 0 38.5 48 0.03–2 0.25 0.5 0 Echinocandin ANI 24 0.03–0.06 0.06 0.06 0 100 MICA 24 0.016–0.03 0.016 0.016 0 100 Others (n= 7) Polyene AMB 24 0.06–0.25 0.125 0.25 0 ND
the 152 isolates ranged from 0.016 to 2 mg/l (Table1). The MIC50s of both anidulafungin and micafungin were the same at 0.016 mg/l. All isolates were susceptible to both anidulafungin and micafungin.
Categorical agreement between 24- and 48-h incubations
According to the guidelines, the interpretations of drug susceptibilities were not completely consistent after 24- and 48-h incubations. In the present study, we used categorical agreement to test the consistency between the MICs after 24- and 48-h incubations. Of the 152 isolates, consistency for azoles ranged from 82.2 % to 88.8 %, and for amphotericin B, it was 72.4 %. Even though the agreement was not perfect for amphotericin B, the majority of the 152 isolates were susceptible to amphotericin B after 24- and 48-h incubations. The agreement of azoles between the two incubation periods varied in different species. It showed good agreement for C. albicans (90–91.3 %), and C. parapsilosis (95.5 %). In contrast, agreement was poor for C. glabrata (38.5–46.2 %) and C. tropicalis (56.7–63.3 %). Discussion
In the present study, 95 % of the 152 isolates belonged to one of the four major species: C. albicans, C. glabrata,
C. parapsilosis, and C. tropicalis. Although the prevalence of candidemia caused by non-albicans Candida species has increased, C. albicans is still the most common species causing candidemia, which is consistent with most reports [2,8,9,36]. Among the phenomena associated with drug resistance, ‘trailing’ describes the reduced but persistent growth that some isolates exhibit at drug concentrations above the MIC in broth dilution tests [29,37]. When the MIC of an isolate after 48-h incubation is approximately fourfold higher than that at the 24-h point, the isolate is defined to have trailing growth [29,37]. In the present study, the trailing growth phenotype was more prevalent in C. tropicalis than in other species, which is consistent with our previous findings in a national surveillance, Taiwan Surveillance of Antimi-crobial Resistance of Yeasts (TSARY) [29]. In the present study, no C. albicans was detected to be azole resistant after 24-h incubation. Hence, the isolates with high MICs after 48-h incubation may be caused by trailing growth. Further-more, a ‘‘true’’ azole-resistant C. albicans isolated from blood was not detected in TSARYs, in 2006 and 2010 [29,38].
The observation in the present study that a high preva-lence of infections are caused by C. parapsilosis in younger patients is consistent with the previous finding that C. parapsilosis was the most common species causing inva-sive candidiasis in children [36].
The prevalence of non-albicans Candida species varies among different areas. In the present study, C. tropicalis was
Table 2 Distribution of Candida species according to the age of patients
Age (years) C. albicans C. tropicalis C. glabrata C. parapsilosis Others Total B18 5 (29.4)a 1 (5.9) 0 10 (58.8)* 1 (5.9) 17 19–65 30 (50) 18 (30)* 3 (5) 5 (8.3) 4 (6.7) 60 C66 45 (60) 11 (14.7) 10 (13.3) 7 (9.3) 2 (2.7) 75 Total 80 (52.6) 30 (19.7) 13 (8.6) 22 (14.5) 6 (3.9) 152
a Number of isolates (percentage in each age group)
*p-value less than 0.05, considered significant Table 1 continued
Type of drug Drug Incubation (h)
MIC (mg/l)
Range MIC50 MIC90 %R %CA
48 0.25–0.5 0.25 0.5 0 ND Azole FLU 24 1–8 2 8 0 ND 48 1–16 4 16 0 ND VOR 24 0.03–0.06 0.03 0.06 0 ND 48 0.03–0.25 0.06 0.25 0 ND Echinocandin ANI 24 0.016–2 0.25 2 0 ND MICA 24 0.016–0.25 0.125 0.25 0 ND
h hours, MIC minimum inhibitory concentration, n number of isolates, %R percent resistant, %CA percent categorical agreement, AMB amphotericin B, ANI anidulafungin, FLU fluconazole, MICA micafungin, VOR voriconazole
the most common non-albicans Candida species causing candidemia, which is consistent with previous reports in Taiwan [4,6,9], as well as in other areas, such as Brazil (21.7 %) [39]. In contrast, C. glabrata is the most common non-albicans Candida species causing candidemia in Wes-tern countries. For example, in the study conducted by Pfaller et al. [40], among the 4,067 isolates causing candi-demia, 26.7 % were C. glabrata, whereas only 8.7 % were C. tropicalis. In Taiwan, the low prevalence of candidemia was caused by C. glabrata. The majority of C. glabrata collected in TSARYs were from urine [28,29,40]. Thus, only 11.2 % (55/492) of cases of candidemia were caused by C. glabrata [9,39]. In the present study, only 8.6 % of the 152 isolates were C. glabrata.
No C. krusei was collected in the present study. The prevalence of candidemia caused by C. krusei in Taiwan was 0.8 % (4/492) in the previous three TSARYs [9,38], 0.5 % in a medical center in southern Taiwan [4], and none in northern Taiwan [6]. In the study of Pfaller et al. [40], among the 4,067 isolates causing candidemia, 3.4 % were C. krusei. Furthermore, the prevalence of candidemia caused by C. krusei was 3.5 % in another study conducted in Brazil from 2006 to 2010. Hence, the prevalence of candidemia caused by C. krusei in Taiwan is lower than that in other areas [8/1,615 (0.5 %) vs. 149/4,380 (3.4 %); p\ 0.001]. Azole prophylaxis is one risk factor for C. glabrata and C. krusei infections [41]. The low prevalence of C. glabrata and C. krusei infections in Taiwan may be a result of infrequent fluconazole prophylaxis [42].
Epidemiological cutoff values and species-specific clini-cal breakpoints for fluconazole [43], voriconazole [44], and the echinocandins [35] for some Candida species have been established to minimize the trailing and to support a shorter time for reporting MICs. In conclusion, our results suggest that the MICs of different drugs after 24-h incubation may be adequate for predicting the prognosis of candidemia.
Acknowledgments We thank Bristol Myers Squibb for providing amphotericin B, Pfizer for providing anidulafungin, fluconazole, and voriconazole, and Astellas Pharms for micafungin. This work was supported in part by grants 00A1-ID-PP-04-014 and 01A1-IV-PP-04-014 from National Health Research Institutes.
Conflict of interest The authors declare that they have no conflict of interest.
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