Hsiu‐Jung Lo , PhD
Source: Infection Control and Hospital Epidemiology, Vol. 25, No. 1 (January 2004), pp. 60-64
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A
MPHOTERICIN
B
AND
F
LUCONAZOLE
: T
HE
E
MERGENCE OF
F
LUCONAZOLE
R
ESISTANCE IN
C
ANDIDA TROPICALIS
Yun-Liang Yang, PhD; Yong-An Ho, BSc; Hsiao-Hsu Cheng, BSc; Monto Ho, MD; Hsiu-Jung Lo, PhD
In the past decade, nosocomial infections caused by yeast have increased significantly. In the United States, yeast infections rank as the fourth most common cause of nosocomial bloodstream infection.1,2 The prevalence of nosocomial candidemia increased 27-fold from 1981 through 1993 at a hospital in Taiwan.3,4 The dramatic increases in the prevalence of fungal infections are proba-bly the result of alterations in immune status associated with the acquired immunodeficiency syndrome epidemic, cancer chemotherapy, organ and bone marrow transplan-tation,5 frequent broad-spectrum antibacterial therapy, and invasive hospital procedures.
Currently available antifungal drugs can have trou-blesome side effects, are ineffective against some fungi, and lead to the development of resistance. Antifungal drug resistance has become an important issue for a vari-ety of fungal infections. Oropharyngeal candidiasis due to drug-resistant fungi is a major problem for patients
infect-ed with human immunodeficiency virus.6 One-third of patients with late-stage acquired immunodeficiency syn-drome had drug-resistant strains of Candida albicans in their mouth in one report.7 The rate of invasive fungal infection among liver transplant patients in another study was 11% and 40% of the patients with systemic fungal infec-tion died. Seventy-two percent of these deaths were attrib-uted to the fungal infection.8
Candidaspecies have varying degrees of susceptibil-ity to common antifungal agents. C. lusitaniae is relatively resistant to amphotericin B.9C. kruseiand C. glabrata are less susceptible to fluconazole than are other Candida species.10-12Although C. tropicalis is less commonly isolat-ed from clinical specimens than is C. albicans, it is one of the most common non-albicans Candida species13,14and it is always associated with diseases.15,16Among non-albicans
Candida species, C. tropicalis is considerably clinically important because it develops fluconazole resistance
rapid-Dr. Yang is from the Department of Biological Science and Technology, National Chiao Tung University, Hsinchu; and Drs. Monto Ho and Lo, Mr. Yong-An Ho, and Mr. Cheng are from the Division of Clinical Research, National Health Research Institutes, Taipei, Taiwan, Republic of China.
Address reprint requests to Hsiu-Jung Lo, PhD, Division of Clinical Research, National Health Research Institutes, 128, Yen-Chiu-Yuan Road, Section 2, Taipei, 11529, Taiwan, Republic of China.
The authors thank Dr. Y.-C. Chen for her helpful suggestions, and Bristol Myers Squibb and Pfizer for supplying the amphotericin B and flu-conazole, respectively. They also thank the following 22 participating hospitals for providing the isolates and information related to the isolates: Chang Gung Memorial Hospital at Linkou, Chang Gung Memorial Hospital at Keelung, Hsin-Chu Hospital, Lo-Tung Poh Ai Hospital, St. Mary Hospital, Taipei Municipal Yang-Ming Hospital, Taipei Municipal Zen Ai Hospital, Tao-Yuan General Hospital, Taiwan Adventist Hospital, Koo Foundation Sun Yat-sen Cancer Center, Tri Service General Hospital, Kuan-Tien General Hospital, Veterans General Hospital-Taichung, Zen Ai General Hospital, Chi Mei Hospital, Kaohsiung Medical College Chung-Ho Memorial Hospital, Kaohsiung Military Hospital, Tainan Municipal Hospital, Veterans General Hospital-Kaohsiung, Buddhist Tzu-Chi General Hospital, Hua-Lien Hospital, and Mackay Memorial Hospital Taitung Branch.
OBJECTIVE: To determine the susceptibilities of
Candidaspecies isolated from Taiwan to amphotericin B and flu-conazole.
DESIGN: Prospective sur veillance study.
METHODS: Each hospital was asked to submit up to 10
C. albicansand 40 non-albicans Candida species during the col-lection period, from April 15 to June 15, 1999. One isolate was accepted from each episode of infection. The broth microdilution method was used to determine susceptibilities to amphotericin B and fluconazole.
RESULTS: Only 3 of 632 isolates, one each of C. famata,
C. krusei, and C. tropicalis, were resistant to amphotericin B. A total of 53 (8.4%) of 632 clinical yeast isolates, consisting of 4% C.
albicans, 8% C. glabrata, 15% C. tropicalis, and 70% C. krusei, were resistant to fluconazole. In contrast, no C. parapsilosis isolate was
resistant to fluconazole. Isolates from tertiary-care medical cen-ters had higher rates of resistance to fluconazole than did those from regional and local hospitals (11.4% vs 6.6%). Isolates from different sources showed different levels of susceptibility to flu-conazole. All of the isolates with the exception of C. tropicalis and
C. kruseiisolated from blood were susceptible to fluconazole. A pattern of co-resistance to both amphotericin B and fluconazole was observed.
CONCLUSIONS: Non-albicans Candida species had
higher rates of resistance to fluconazole than did C. albicans (44 of 395 [11.2%] vs 9 of 237 [3.8%]; P = .002). The increasing rate of fluconazole resistance in C. tropicalis (15%) is important because
C. tropicalisis one of the most commonly isolated non-albicans
Candidaspecies (Infect Control Hosp Epidemiol 2004;25:60-64).
ly17and the rate of resistance to fluconazole of clinical C.
tropicalisisolates is increasing.18-21
We previously reported a national sur vey in Taiwan in which 22 hospitals contributed 660 clinical yeast iso-lates.22The current study showed that the levels of sus-ceptibility to fluconazole of Candida species were differ-ent among differdiffer-ent species and also among the same species from different sources. Our data suggested that, in addition to C. krusei and C. glabrata, C. tropicalis should be on the list of yeast for which fluconazole resis-tance is common.
METHODS
Organisms and Medium
Yeast isolates were collected from 22 hospitals par-ticipating in Taiwan Sur veillance of Antimicrobial Resistance of Yeasts (TSARY). Six were tertiar y-care medical centers, 14 were regional hospitals, and 2 were local hospitals. Each hospital was asked to submit up to 10 C. albicans and 40 non-albicans Candida species
dur-ing the collection period, from April 15 to June 15, 1999. One isolate was accepted from each episode of infection. Isolates were stored frozen at -70°C in bead-containing Microbank cr yovials (PRO-LAB Diagnostics, Austin, TX). At the end of the collection period, isolates were kept frozen and transported to the National Health Research Institutes laborator y within 24 hours. The iso-lates were first subcultured on Sabouraud dextrose agar (BBL Becton Dickinson, Cockeysville, MD) to check for purity and identification. Pure isolates were labeled and stored in vials containing 50% glycerol at -70°C for fur-ther analysis.
Antifungal Susceptibility Testing
The minimum inhibitory concentration (MIC) to amphotericin B or fluconazole of each yeast was deter-mined by in vitro antifungal susceptibility testing accord-ing to guidelines published by the National Committee for Clinical Laboratory Standards.23 The powder of RPMI medium 1640 was provided by Gibco BRL (Cat.#
31800-TABLE 1
SUSCEPTIBILITIES TOAMPHOTERICINB
MIC (µg/mL) C. albicans C. tropicalis C. glabrata C. parapsilosis C. krusei Others Total
0.06 1 0 0 0 0 1 2 0.125 3 0 0 1 0 0 4 0.25 44 13 9 13 0 4 83 0.5 149 95 101 20 3 5 373 1 40 54 46 17 6 4 167 2 0 1 0 0 1 1 3 Total 237 163 156 51 10 15 632 MIC50 0.5 0.5 0.5 0.5 1 0.5 0.5 MIC90 1 1 1 1 2 1 1
MIC = minimum inhibitory concentration.
TABLE 2
SUSCEPTIBILITIES TOFLUCONAZOLE
MIC (µg/mL) C. albicans C. tropicalis C. glabrata C. parapsilosis C. krusei Others Total
0.125 83 4 0 2 0 0 89 0.25 108 8 0 3 0 1 120 0.5 16 19 1 17 0 4 57 1 4 36 1 19 0 1 61 2 1 42 7 6 0 2 58 4 2 8 67 2 0 4 83 8 7 12 45 1 0 1 66 16 4 10 18 1 1 2 36 32 3 0 4 0 2 0 9 ⭓ 64 9 24 13 0 7 0 53 Total 237 163 156 51 10 15 632 MIC50 0.5 2 8 1 64 2 1 MIC90 4 64 32 2 64 16 16
022; Becton Dickinson, Sparks, MD). Several strains from the American Type Culture Collection were used as stan-dard controls. The final growth of each isolate was mea-sured by a Spectra MAX Plus (Molecular Devices Corp., Sunnyvale, CA) after 48 hours of incubation at 35°C. The MIC of each isolate was measured at least twice. When the results of two susceptibility tests differed by more than onefold dilution, they were retested. MICs of some isolates were measured by Etest (AB Biodisk, Solna, Sweden) to confirm our microdilution results.
The interpretation of MICs was according to the guidelines of the National Committee for Clinical Laboratory Standards. The MICs to amphotericin B and fluconazole were defined as those that could reduce the turbidity of cells by greater than 95% and 50%, respectively. Isolates with a MIC of 2 µg/mL or greater were considered to be resistant to amphotericin B. Isolates with a MIC of 1 µg/mL or less were considered to be susceptible. Isolates with a MIC of 64 µg/mL or more were considered to be resistant to fluconazole. Isolates with a MIC of 8 µg/mL or less were considered to be susceptible. Isolates with a MIC between 16 and 32 µg/mL were susceptible-dose depen-dent. The MICs of 50% and 90% of the total population were defined as MIC50and MIC90, respectively.
Database and Analysis
In addition to identifying isolates, contributing hos-pitals also provided the following information about each isolate: location and type of hospital, genus and species as identified by each hospital, source (urine, sputum, blood, wound, and 48 other sources), and procedures for identi-fication. An analysis was performed using Epi-Info soft-ware (version 6.04; Centers for Disease Control and Prevention, Atlanta, GA).24The significance of differences in proportions was determined by the chi-square test with Yate’s correction.
RESULTS
Candida Species From Different Sources
A total of 632 isolates were analyzed for their sus-ceptibilities to amphotericin B and fluconazole. C. albi-canswas the most common species (37.5% of all isolates). C. tropicalis(25.8%) and C. glabrata (24.7%) were the two most common non-albicans Candida species followed by C. parapsilosis(8.1%), C. krusei (1.6%), and others (2.3%). When classified according to the sources, 273 (43.2%) of the isolates were from urine, 152 (24.1%) were from spu-tum, 51 (8.1%) were from blood, 49 (7.8%) were from wounds, and 107 (16.8%) were from other sites.
TABLE 3
SUSCEPTIBILITIES OFCANDIDASPECIESFROMDIFFERENTSOURCES TOFLUCONAZOLE
Source C. albicans C. tropicalis C. glabrata C. parapsilosis C. krusei Others Subtotal Blood S 100 (15)* 58.4 (7) 87.5 (7) 100 (13) 0 100 (2) 86.3 (44) SDD 0 8.3 (1) 12.5 (1) 0 0 0 3.9 (2) R 0 33.3 (4) 0 0 100 (1) 0 9.8 (5) Sputum S 90.2 (74) 81.4 (35) 55 (11) 100 (3) 0 66.7 (2) 82.2 (125) SDD 3.7 (3) 4.7 (2) 30 (6) 0 0 33.3 (1) 7.9 (12) R 6.1 (5) 13.9 (6) 15 (3) 0 100 (1) 0 9.9 (15) Urine S 94.9 (75) 75.3 (55) 80.2 (85) 85.7 (6) 0 100 (3) 82 (224) SDD 3.8 (3) 6.9 (5) 12.3 (13) 14.3 (1) 40 (2) 0 8.8 (24) R 1.3 (1) 17.8 (13) 7.5 (8) 0 60 (3) 0 9.2 (25) Wound S 90 (18) 92.3 (12) 100 (4) 100 (11) 0 0 91.8 (45) SDD 0 7.7 (1) 0 0 0 100 (1) 4.1 (2) R 10 (2) 0 0 0 0 0 4.1 (2) Others S 95.2 (39) 91 (20) 77.8 (14) 100 (17) 0 100 (6) 89.7 (96) SDD 2.4 (1) 4.5 (1) 11.1 (2) 0 33.3 (1) 0 4.7 (5) R 2.4 (1) 4.5 (1) 11.1 (2) 0 66.7 (2) 0 5.6 (6) Subtotal S 93.2 (221) 79.2 (129) 77.6 (121) 98 (50) 0 86.7 (13) 84.5 (534) SDD 3 (7) 6.1 (10) 14.1 (22) 2 (1) 30 (3) 13.3 (2) 7.1 (45) R 3.8 (9) 14.7 (24) 8.3 (13) 0 70 (7) 0 8.4 (53) Total 37.5 (237) 25.8 (163) 24.7 (156) 8.1 (51) 1.6 (10) 2.3 (15) 100 (632)
S = susceptible; SDD = susceptible-dose dependent; R = resistant. *Percentage (number of isolates).
Susceptibilities to Amphotericin B and Fluconazole
The susceptibilities to amphotericin B are listed in Table 1. Only 3 of 632 isolates, one each of C. famata, C. krusei, and C. tropicalis, were resistant to amphotericin B. The MICs ranged from 0.06 to 2 µg/mL. The MIC50of these isolates was 0.5 µg/mL and the MIC90was 1 µg/mL. C. krusei were less susceptible to amphotericin B than were the other species, with MICs ranging from 0.5 to 2 µg/mL, a MIC50of 1 µg/mL, and a MIC90of 2 µg/mL.
The susceptibilities to fluconazole are listed in Table 2. A total of 53 (8.4%) and 45 (7.1%) isolates were resistant and susceptible-dose dependent. The MIC50of these isolates was 1 µg/mL and the MIC90was 16 µg/mL. C. krusei (70%), C. tropicalis (15%), and C. glabrata (8%) isolates had higher rates of resistance to fluconazole than did C. albicans (4%) and other Candida species (0%). No C. krusei isolate was sus-ceptible to fluconazole and the MIC50of this species was 64 µg/mL. Approximately 78% and 79% of C. glabrata and C. tropicalisisolates, respectively, were susceptible to flucona-zole. No C. parapsilosis isolate was resistant to fluconaflucona-zole. Non-albicans Candida species had higher rates of resistance to fluconazole than did C. albicans (44 of 395 [11.2%] vs 9 of 237 [3.8%]; P = .002). Isolates from hospitals affiliated with tertiary-care medical centers had a higher rate of resistance to fluconazole than did those from regional and local hospi-tals (25 of 219 [11.4%] vs 27 of 410 [6.6%]; P = .05).
Species and Sources Accounting for Susceptibilities to Fluconazole
Candidaspecies isolated from different sources had different susceptibilities to fluconazole (Table 3). All isolates from blood, with the exception of 4 C. tropicalis isolates and 1 C. krusei isolate, were susceptible to fluconazole (Table 3). Candidaspecies isolated from different sources had differ-ent MICs (MIC50and MIC90) to fluconazole (Table 4). The MIC50of isolates from urine (4 µg/mL) was higher than that of isolates from other sources (0.5 to 1 µg/mL). The MIC90 of isolates from wounds (8 µg/mL) was lower than that of isolates from blood, sputum, and urine (16 to 32 µg/mL).
Co-resistance to Both Amphotericin B and Fluconazole
The trend of co-resistance to amphotericin B and
fluconazole is outlined in Table 5. Fewer isolates with MICs to amphotericin B of 0.5 µg/mL or less were resis-tant to fluconazole compared with isolates with higher MICs (1 µg/mL or greater) to amphotericin B (30 of 462 vs 23 of 170; P = .007). Isolates with lower MICs to ampho-tericin B (0.5 µg/mL or less) had lower MIC50and MIC90 to fluconazole than did isolates with higher MICs (1 µg/mL or greater) to amphotericin B (1 vs 2 µg/mL and 16 vs 64 µg/mL, respectively).
DISCUSSION
According to in vitro antifungal susceptibility test-ing, 0.5% and 8.4% of isolates were considered to be resis-tant to amphotericin B and fluconazole, respectively. The difference in rate of resistance between fluconazole and amphotericin B is a result of different mechanisms of anti-fungal activity,6,25 frequency of use, different molecular mechanisms of drug resistance,5,26 or all three. Two of three isolates resistant to amphotericin B (one C. krusei and one C. tropicalis) were also resistant to fluconazole. Although the rate of resistance to amphotericin B was low, there was a trend of co-resistance to amphotericin B and fluconazole.
Fungal infections caused by non-albicans Candida species have been increasing dramatically.27-29 C. krusei and C. glabrata have been considered intrinsically more resistant to fluconazole.10,11In this study, no C. krusei iso-late was susceptible to fluconazole, but C. glabrata was less resistant to fluconazole than in other studies (8% vs 13% or 45.4%).19,30Overall, non-albicans Candida species had higher rates of resistance to fluconazole than did C. albicans. Most of the Candida species isolates from blood
TABLE 4
SUSCEPTIBILITIES OFCANDIDASPECIESFROMDIFFERENTSOURCES TOFLUCONAZOLE
C. albicans C. tropicalis C. glabrata C. parapsilosis Source MICs (µg/mL) MIC50 MIC90 MICs MIC50 MIC90 MICs MIC50 MIC90 MICs MIC50 MIC90
Blood 0.125–0.50 0.25 0.5 0.5–64 5 64 4.0–16 4 16 0.125–2 0.5 1
Sputum 0.125–512 0.25 8 0.25–64 2 64 1.0–64 8 64 0.5–2 1 2
Urine 0.125–64 0.25 2 0.125–64 2 64 0.5–128 4 24 0.5–16 1 8
Wound 0.125–64 0.25 1 0.25–16 2 2 4.0–8 4 8 0.5–4 1 2
MIC = minimum inhibitory concentration.
TABLE 5
SUSCEPTIBILITIES TOBOTHAMPHOTERICINB ANDFLUCONAZOLE
Amphotericin B Fluconazole
MIC (µg/mL) MIC50 MIC90 MIC ⭓ 64 MIC ⭐ 32
⭐ 0.5 1 2 30 (6.5%) 432 (93.5%) ⭓ 1 16 64 23 (13.5%) 147 (86.5%)
C. tropicalisisolates from blood were resistant to flucona-zole. The increasing rate of fluconazole resistance in C. tropicalis(15%) is important because C. tropicalis is one of the most commonly isolated non-albicans Candida species.13,14C. tropicalisdevelops drug resistance in the presence of fluconazole much more rapidly than does C. albicans.17,31These findings may explain why C. tropicalis (15%) had a higher rate of resistance to fluconazole than did C. albicans (4%) and C. glabrata (8%).
Isolates of the same species from different sources showed different susceptibilities to fluconazole. For example, C. glabrata isolates from sputum had a higher rate of resistance (15%) to fluconazole than did those from blood (0%) or urine (7.5%), whereas C. tropicalis isolates from blood had a higher rate of resistance (33.3%) to flu-conazole than did those from sputum (13.9%) or urine (17.8%). The fact that isolates from tertiary-care medical centers had higher rates of resistance to fluconazole than did those from regional and local hospitals needs to be investigated further.
Different levels of susceptibility to fluconazole in the same species from different sources complicates diagnosis and treatment in the clinical setting. Hence, accurate identification to the species level and susceptibil-ity testing are crucial for clinical management, especially for the emerging non-albicans Candida species that are resistant to fluconazole.
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