Mycology
Susceptibilities to amphotericin B and fluconazole of Candida species in
Taiwan Surveillance of Antimicrobial Resistance of Yeasts 2006
Yun-Liang Yang
a, An-Huei Wang
b, Chih-Wei Wang
b, Wei-Ting Cheng
b,
Shu-Ying Li
c, Hsiu-Jung Lo
b,⁎
TSARY Hospitals
aDepartment of Biological Science and Technology, National Chiao Tung University, Hsinchu 300, Taiwan, ROC bDivision of Clinical Research, National Health Research Institutes, Zhunan Town, Miaoli County 350, Taiwan, ROC
c
Division of Laboratory Research and Development, Center for Disease Control, Taipei 115, Taiwan, ROC Received 27 October 2007; accepted 16 January 2008
Abstract
Susceptibilities to amphotericin B and fluconazole of 964 Candida isolates collected in Taiwan Surveillance of Antimicrobial Resistance of Yeasts in 2006 were determined. There were 419 (43.5%) Candida albicans, 246 (25.5%) Candida tropicalis, 211 (21.9%) Candida glabrata, 62 (6.4%) Candida parapsilosis, 14 (1.5%) Candida krusei, and 12 (1.2%) others. Interestingly, 16 of the 17 amphotericin B-resistant isolates were non-albicans Candida species. The resistant rate to amphotericin B has decreased from 2.5% in 2002 to 1.8% in 2006. On the other hand, there were 132 C. tropicalis, 14 C. krusei, 10 C. albicans, and 9 C. glabrata isolates that had MICs to fluconazole ≥64 μg/mL. The prevalence of isolates with such high MICs was significantly higher than that in 2002 (17.1% versus 1.9%). Our results further indicate that most of the isolates with MICs to fluconazole≥64 μg/mL exhibited the “trailing” phenomenon.
© 2008 Elsevier Inc. All rights reserved.
Keywords: Candida; Susceptibility; Resistance
1. Introduction
Because of alterations in immune status and invasive hospital procedures (White et al., 1998; Yang and Lo, 2001), infections caused by opportunistic pathogens, such as yeasts, are becoming important causes of morbidity and mortality in immunocompromised patients. In the past 2 decades, nosocomial yeast infections have increased significantly worldwide. For example, the prevalence of nosocomial candidemia increased 27-fold from 1981 through 1993 at a major hospital in Taiwan (Chen et al., 1997; Hung et al., 1996). In the United States, yeast infection also ranks as the 4th most common cause of nosocomial bloodstream infection (Wisplinghoff et al., 2004). Several antifungal drugs have been applied to render the situation, and as a
result of broad prophylactic usages and long-term treatments with those drugs, the prevalence of drug resistance has become an important issue in various yeast infections, which have profound effects on human health (Marr et al., 2001; Pfaller et al., 2003; Yang et al., 2004b).
Candida species have various degrees of susceptibility to frequently used antifungal drugs. For instance, Candida lusitaniae is relatively resistant to amphotericin B (Hadfield et al., 1987). Candida krusei is intrinsically resistant to fluconazole, and Candida glabrata is less susceptible or has a higher MICs to it than other Candida species (Akova et al.,
1991; Orozco et al., 1998; Yang et al., 2004b). This
phenomenon illustrates the importance of identification and surveillance of Candida species in the clinical settings.
In 1999 and again in 2002, 2 national surveys in Taiwan Surveillance of Antimicrobial Resistance of Yeasts (TSARY) have been conducted. The drug susceptibilities of the 632 and 909 isolates collected in 1999 and 2002, respectively, have been determined (Yang et al., 2004b, 2005b). Among them, 0.5% (1999) and 2.5% (2002) were resistant to
Diagnostic Microbiology and Infectious Disease 61 (2008) 175–180
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⁎ Corresponding author. Tel.: 246-166x35516; fax: +886-37-586-457.
E-mail address:hjlo@nhri.org.tw(H.-J. Lo).
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amphotericin B. There were 8.4% of the isolates in 1999 that had MICs to fluconazole≥64 μg/mL, whereas only 1.9% in 2002 (Yang et al., 2004b, 2005b). In 2006, a follow-up survey was taken place, and 964 Candida isolates were collected and analyzed. The article is to report the result of the susceptibilities to antifungal drugs of Candida species in TSARY 2006 and the trends of the resistance in Taiwan from 1999 to 2006.
2. Materials and methods 2.1. Organisms and media
Yeast isolates were collected according to previous studies (Lo et al., 2001; Yang et al., 2005b) from the 22 hospitals participating in TSARY from July to September in 2006. Each hospital was asked to submit all yeast pathogens from blood and the first 10 Candida albicans and 40 non-albicans Candida species isolates from nonsterile sites. In principle, only 1 isolate was accepted from each specimen. Nevertheless, when there were multiple species isolated from 1 specimen, 1 isolate from each species was analyzed. All the collected isolates were stored frozen at −70 °C in bead-containing Microbank cryovials (Pro-Lab Diagnostics, Austin, TX). After their arrival at the laboratory at National Health Research Institutes (NHRI), Taiwan, ROC, these isolates were 1st subcultured on Sabouraud dextrose agar (Becton Dickinson, Cockeysville, MD) to assess the purity and identification. Pure isolates were labeled and stored in vials containing 50% glycerol at −70 °C awaiting further analyses.
2.2. Identification
The identifications of the isolates were reassessed in the laboratory at the NHRI. The identification procedure for the yeast isolates was modified based on our previous report (Lo
et al., 2001). All isolates were subjected to API-32C
(bioMérieux, St. Louis, MO). The VITEK Yeast Biochemical Card (YBC, bioMérieux) would then be used for the identification of the isolates when the API-32C showed less than 90% confidence and for the isolates whose information was inconsistent to that provided by the hospitals. The sequence of internal transcribed spaces of ribosomal DNA (Leaw et al., 2006, 2007) was applied for further assessment when both API-32C and YBC failed.
2.3. Antifungal susceptibility testing
The MICs to amphotericin B and to fluconazole of each isolate were determined by the same in vitro antifungal susceptibility testing as those in TSARY 1999 and TSARY 2002 (Yang et al., 2004b, 2005b), according to the guidelines of M27A published in 1997 by the Clinical and Laboratory Standards Institute (CLSI,1997). The RPMI medium 1640 (31800-022) provided by Gibco BRL was used for the testing. Strains from American Type Culture Collection
including C. albicans (ATCC 90028), C. krusei (ATCC 6258), and Candida parapsilosis (ATCC 22019) were used as the standard controls. The final growth of each isolate was measured by Biotrak II plate reader (Amersham Biosciences, Cambridge, UK) after incubation at 35 °C for 48 h. The MICs of some isolates were also measured by Etest (AB BIODISK, Solna, Sweden) to assess the results of the broth microdilution method.
The MICs to amphotericin B and to fluconazole were defined as the MICs of drugs capable of reducing the turbidity of cells to greater than 95% and 50%, respectively. For susceptibility to amphotericin B, isolates with MIC ≥2 μg/mL were considered to be resistant, and those with MIC ≤1 μg/mL were susceptible. For susceptibility to fluconazole, isolates with MIC≥64 μg/mL were considered to be resistant, whereas those with MIC ≤8 μg/mL were susceptible. Isolates with MICs falling in between (16–32 μg/mL) were susceptible dose dependent. The MICs of 50% and 90% of the total population were defined as MIC50and
MIC90, respectively.
Among the phenomena associated with resistance, “trailing” describes the reduced but persistent growth that some isolates exhibit at drug concentrations above the MIC in broth dilution tests with azole antifungal agents, such as fluconazole (Lee et al., 2004). When the MIC of an isolate measured after 48-h incubation is approximately 4-fold higher than that at the 24-h point (Arthington-Skaggs et al., 2002), the isolate is defined to have trailing growth. 2.4. Database and analysis
The database for this study contained the characteristic information of each submitted isolate: hospital origin, location and type of the hospital, and identification and source of the isolate. The statistic significance of the differences in frequencies and proportions was determined by theχ2test with Yates' correction.
3. Results and discussion
3.1. Distribution of Candida species
The distribution of Candida species in TSARY 2006 (Table 1) was similar to that of 2 previous surveys in 1999 and 2002 (Yang et al., 2004b, 2005a). C. albicans was the most frequently isolated species, accounting for 43.5% of the total isolates. Candida tropicalis (25.5%) and C. glabrata (21.9%) were the 2 most frequently isolated non-albicans Candida species, followed by C. parapsilosis (6.4%), C. krusei (1.5%), and others (1.2%). When classified according to the sources (Table 1), there were 406 (42.1%) isolates from urine, 158 (16.4%) from sputum, 145 (15%) from blood, 43 (4.5%) from wound, 40 (4.1%) from pus, 40 (4.1%) from catheter tip, 38 (4%) from ascites, and 94 (9.8%) from other 45 different sites.
Different Candida species had various prevalence in different body sites. Although C. albicans was still the most
common single species (56.6%) causing candidemia in our study as well as others' (Cheng et al., 2004; Peman et al.,
2005; Pfaller et al., 2007a; Yang et al., 2006), the
prevalence of non-albicans Candida species had increased. Though C. tropicalis was the most common non-albicans Candida species in our collection, C. glabrata was the most common one from urine (145/277, 52.3%). The majority of C. glabrata (68.7%) was isolated from urine samples, consistent with the previous reports that C. glabrata is 2nd only to C. albicans as a cause of candiduria (Pfaller et al., 1999; Yang et al., 2004b).
3.2. Susceptibilities to amphotericin B of Candida species The range of MICs to amphotericin B of the 964 isolates was from 0.125 to 2 μg/mL (Table 2). C. krusei was less susceptible to amphotericin B than any other species because the MIC50of this species was 1μg/mL. The overall resistant
rate to amphotericin B has decreased from 2.5% in TSARY 2002 (Yang et al., 2005b) to 1.8% in TSARY 2006. In recent years, fungal infections caused by non-albicans Candida species have increased dramatically (Abi-Said et al., 1997; Slavin et al., 1995; Walsh et al., 2004). The phenomenon was also reflected in the distribution of resistant isolates in others' (Slavin et al., 1995; Walsh et al., 1998) as well as our studies. All 3 amphotericin B-resistant isolates in TSARY 1999 were non-albicans Candida species (Yang et al., 2004b). Further-more, 20 of the 23 amphotericin B-resistant isolates in
TSARY 2002 were also non-albicans Candida species (Yang et al., 2005b). Of the 17 amphotericin B-resistant isolates in TSARY 2006, 16 were non-albicans Candida species. They consisted of 12 C. tropicalis, 2 C. krusei, and 1 each of C. glabrata and Candida curvatus.
The prevalence of amphotericin B resistance of C. krusei (14.3%) or C. tropicalis (4.9%) was significantly higher than that of C. glabrata (0.5%) or C. albicans (0.2%) (P b 0.05). Among the frequently isolated non-albicans Candida species, no amphotericin B-resistant isolate of C. parapsilosis was detected in this study (Table 2), as well as in the 2 previous TSARYs (Yang et al., 2004b, 2005b). A total of 2, 2, and 5 C. lusitaniae were collected in TSARY 1999, 2002, and 2006, respectively. Up-to-date amphoter-icin B resistance of C. lusitaniae is not an issue for concern since all 9 C. lusitaniae was susceptible to amphotericin B. The MIC50 and MIC90 of those isolates were 0.25 and
1 μg/mL, respectively.
3.3. Susceptibilities to fluconazole of Candida species from different sources
For the susceptibility to fluconazole, a total of 756 (78.4%), 43 (4.5%), and 165 (17.1%) isolates had MICs≤8, 16 to 32, and ≥64 μg/mL, respectively. The MIC50 and
MIC90of these isolates were 1 and 64μg/mL, respectively.
Interestingly, among the isolates with MICs ≥64 μg/mL, those from sputum (27.2%) appear to have a higher ratio than
Table 2
The susceptibility to amphotericin B of Candida species
MICs (μg/mL) C. albicans C. tropicalis C. glabrata C. parapsilosis C. krusei Others Total
0.125 16 (3.8)a 0 4 (1.9) 0 0 1 (8.3) 21 (2.2) 0.25 213 (50.8) 45 (18.3) 49 (23.2) 23 (37.1) 0 6 (50) 336 (34.8) 0.5 134 (31.9) 126 (51.2) 113 (53.6) 29 (46.8) 5 (35.7) 2 (16.7) 409 (42.4) 1 55 (13.1) 63 (25.6) 44 (20.8) 10 (16.1) 7 (50) 2 (16.7) 181 (18.8) 2 1 (0.2) 12 (4.9) 1 (0.5) 0 2 (14.3) 1 (8.3) 17 (1.8) Total 419 246 211 62 14 12 964 MIC50 0.25 0.5 0.5 0.5 1 0.25 0.5 MIC90 1 1 1 1 2 1 1
aNumber of isolates (percentage).
Table 1
The distribution of Candida species from different sources
Sources C. albicans C. tropicalis C. glabrata C. parapsilosis C. krusei Others Total
Urine 129 109 145 13 5 5 406 (42.1)a Sputum 70 65 19 2 2 0 158 (16.4) Blood 82 32 20 9 1 1 145 (15.1) Wound 21 6 6 5 1 4 43 (4.5) Pus 20 9 2 6 2 1 40 (4.1) Tipb 20 13 3 4 0 0 40 (4.1) Ascitesc 24 7 4 2 1 0 38 (3.9) Others 53 5 12 21 2 1 94 (9.8) Total 419 (43.5) 246 (25.5) 211 (21.9) 62 (6.4) 14 (1.5) 12 (1.2) 964 a
Number of isolates (percentage).
bCatheter tips.
those from ascites (7.9%), wound (11.6%), blood (13.1%), and urine (17.7%) (P = 0.05) (Table 3).
When comparisons were made among species, there were distinct variations in the fluconazole susceptibility. Of the 5 common Candida species, all C. krusei had MICs≥64 μg/ mL. In contrast, none of the C. parapsilosis did. These results were consistent with previous reports (Akova et al., 1991; Pfaller et al., 2000, 2004; Yang et al., 2004a, 2005b). Furthermore, susceptibilities among different species from blood also varied. All C. albicans, C. glabrata, and C. parapsilosis were susceptible to fluconazole, whereas 18 of the 32 (56.3%) C. tropicalis isolates had MICs≥64 μg/mL (Table 3).
Interestingly, C. glabrata having MICs to fluconazole ≥64 μg/mL was lower than what has been reported in other geologic areas (Fleck et al., 2007; Pfaller et al., 2007b; St Germain et al., 2001). In the 3 TSARYs, the portions of isolates having MICs ≥64 μg/mL were 8.3%, 1.6%, and
4.3% isolates from 1999, 2002, and 2006, respectively. It has been reported that continuous exposure to azoles seems to have a major impact on developing resistance to fluconazole in Candida species, especially for C. glabrata ( Kontoyian-nis, 2002). Therefore, our previous observation that only 13% of the candidemia 92 patients had prior fluconazole treatments (Cheng et al., 2004) may explain the low fluconazole-resistant C. glabrata in Taiwan.
3.4. Trailing growth
Among the phenomena associated with resistance, trailing describes the reduced but persistent growth that some isolates exhibit at drug concentrations above the MIC in broth dilution tests with azole antifungal agents, such as fluconazole (Lee et al., 2004). Trailing may interfere the observation of resistance level in vivo (Arthington-Skaggs et al.,2002). Thus, we have also determined whether those 165 isolates with MICs≥64 μg/mL to fluconazole exhibit
Table 3
The susceptibility to fluconazole of Candida species from different sources
MICs (μg/mL) C. albicans C. tropicalis C. glabrata C. parapsilosis C. krusei Others Total Urine ≦8 123 (95.3)a 53 (48.6) 117 (80.7) 13 (100) 0 5 (100) 311 (76.6) 16−32 0 1 (0.9) 22 (15.2) 0 0 0 23 (5.7) ≧64 6 (4.7) 55 (50.5) 6 (4.1) 0 5 (100) 0 72 (17.7) Subtotal 129 109 145 13 5 5 406 Sputum ≦8 69 (98.6) 25 (38.5) 13 (68.4) 2 (100) 0 0 109 (69) 16–32 1 (1.4) 0 5 (26.3) 0 0 0 6 (3.8) ≧64 0 40 (61.5) 1 (5.3) 0 2 (100) 0 43 (27.2) Subtotal 70 65 19 2 2 0 158 Blood ≦8 82 (100) 10 (31.2) 16 (80) 9 (100) 0 1 (100) 118 (81.4) 16–32 0 4 (12.5) 4 (20) 0 0 0 8 (5.5) ≧64 0 18 (56.3) 0 0 1 (100) 0 19 (13.1) Subtotal 82 32 20 9 1 1 145 Wound ≦8 21 (100) 2 (3.3) 5 (83.3) 5 (100) 0 3 (75) 36 (83.7) 16–32 0 0 1 (16.7) 0 0 1 (25) 2 (4.7) ≧64 0 4 (66.7) 0 0 1 (100) 0 5 (11.6) Subtotal 21 6 6 5 1 4 43 Pus ≦8 19 (95) 4 (44.4) 2 (100) 6 (100) 0 1 (100) 32 (80) 16–32 0 0 0 0 0 0 0 ≧64 1 (5) 5 (55.6) 0 0 2 (100) 0 8 (20) Subtotal 20 9 2 6 2 1 40 Tipb ≦8 18 (90) 7 (53.8) 2 (66.7) 4 (100) 0 0 31 (77.5) 16–32 0 1 (7.7) 0 0 0 0 1 (2.5) ≧64 2 (10) 5 (38.5) 1 (33.3) 0 0 0 8 (20) Subtotal 20 13 3 4 0 0 40 Ascitesc ≦8 24 (100) 5 (71.4) 4 (100) 2 (100) 0 0 35 (92.1) 16–32 0 0 0 0 0 0 0 ≧64 0 2 (28.6) 0 0 1 (100) 0 3 (7.9) Subtotal 24 7 4 2 1 0 38 Others ≦8 51 (96.2) 2 (40) 10 (83.4) 20 (95.2) 0.0 1 (100) 84 (89.4) 16–32 1 (1.9) 0.0 1 (8.3) 1 (4.8) 0.0 0.0 3 (3.2) ≧64 1 (1.9) 3 (30) 1 (8.3) 0 2 (100) 0 7 (7.4) Subtotal 53 5 12 21 2 1 94 All ≦8 407 (97.1) 108 (43.9) 169 (80.1) 61 (98.4) 0 11 (91.7) 756 (78.4) 16–32 2 (0.5) 6 (2.4) 33 (15.6) 1 (1.6) 0 1 (8.3) 43 (4.5) ≧64 10 (2.4) 132 (53.7) 9 (4.3) 0 14 (100) 0 165 (17.1) Total 419 246 211 62 14 12 964
aNumber of isolates (percentage). bCatheter tips.
trailing, and the results are summarized in Table 4. High percentage (64.8%) of those isolates indeed exhibited the trailing phenomenon. When classified according to species, 72.7% (96/132) C. tropicalis, 70% (7/10) C. albicans, and 44.4% (4/9) C. glabrata isolates exhibited the trailing phenomenon, whereas none of the C. krusei isolates did. 3.5. Conclusion
In TSARY 1999, there were 14.7% C. tropicalis isolates having MICs≥64 μg/mL (Yang et al., 2004b). In contrast, none of the 244 C. tropicalis from TSARY 2002 had MICs ≥64 μg/mL (Yang et al., 2005b). Surprisingly, in TSARY 2006, among the 246 C. tropicalis isolates, 132 (53.7%) had MICs≥64 μg/mL. Recently, we have reported an associa-tion between fluconazole susceptibility and genetic related-ness among C. tropicalis isolates from TSARY 1999 (Chou
et al., 2007; Wang et al., 2007). The DST140 was a
predominant type of C. tropicalis isolates among those having MICs ≥64 μg/mL in TSARY 1999 (Chou et al., 2007). Furthermore, none of the 17 tested fluconazole-susceptible C. tropicalis isolates collected from TSARY 2002 were DST140 type (Chou et al., 2007). Interestingly, our preliminary result showed that DST140 was detected again among C. tropicalis isolates collected in TSARY 2006. This result may explain the dramatic fluctuation in fluconazole susceptibility among 3 TSARYs. Whether this is an endemic problem requires further investigation.
The increasing rate of reduced susceptibility to flucona-zole in C. tropicalis has considerable clinical importance, because this species is 1 of the most frequently isolated non-albicans Candida species (Cheng et al., 2004; Hung et al., 2005; Lo et al., 2005; Pfaller et al., 2000; Prasad et al., 1999). Furthermore, C. tropicalis develops drug resistance in the presence of fluconazole much more rapidly than C. albicans (Barchiesi et al., 2000; Calvet et al., 1997). In addition, C. krusei isolates with high MICs are also closely related (Chou et al., 2007; Wang et al., 2007). These findings may explain why, to fluconazole, C. tropicalis has a higher rate to have MICs ≥64 μg/mL than C. albicans. Here we have identified that 72.7% (96/132) C. tropicalis exhibited the
trailing phenomenon by a definition that the MIC of an isolate after 48-h incubation is 4-fold higher than that at the 24-h point. Nevertheless, whether the remaining 36 (14.6%) C. tropicalis isolates are “truly resistant” requires further investigation.
Acknowledgments
The authors would like to thank Bristol Myers Squibb (New Brunswick, NJ) and Pfizer (New York, NY) for supplying the amphotericin B and fluconazole, respectively. They would also like to acknowledge Dr. Y.C. Chen for her assistance and helpful suggestion. They also thank the 22 participating hospitals for providing clinical isolates and information regarding those isolates. They are Asia East Memorial Hospital, Buddhist Tzu-Chi General Hospital, Chang Gung Memorial Chiayi Christian Hospital, Hospital at Kaohsiung, Chang-Hwa Christian Hospital, Cheng Ching Hospital, Chung Shan Medical Dental College Hospital, Kaohsiung Military Hospital, Kaohsiung Medical College Chung-Ho Memorial Hospital, Kuan-Tien General Hospital, Lo-Hsu Foundation Lo-Tung Poh Ai Hospital, Miin Sheng General Hospital, National Cheng Kung University Hospital, Show Chwan Memorial Hospital, Sin-Lau Christian Hospital, St. Mary Hospital, Taipei Municipal Chung Hsiao Hospital, Taipei Municipal Hoping Hospital, Veterans General Hospital-Taichung, Veterans General Hospital-Kaohsiung, Zen Ai General Hospital, and Tungs' Taichung NetroHarbor Hospital, Sha Lu branch. We thank Dr. T.L. Lauderdale and Mr. S.H. Hsu for their technical assistance. This study was supported by the grant NHRI CL-096-PP07.
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