Abstract
The performance of Etest in fluconazole and voriconazole testing of 279 isolates of uncommon Candida spp. was assessed in comparison with the National Committee for Clinical Laboratory Standards (NCCLS)-approved standard broth microdilution (BMD) method. The NCCLS method employed RPMI 1640 broth medium, and MICs were read after incubation for 48 h at 35°C. Etest MICs were determined with RPMI agar containing 2% glucose and were read after incubation for 48 h at 35°C. The isolates include Candida krusei, C. lusitaniae, C. guilliermondii, C. kefyr, C. rugosa, C. lipolytica, C. pelliculosa, C. dubliniensis, C. famata, C. zeylanoides, C. inconspicua, and C. norvegensis. Overall agreement between Etest and BMD MICs was 96% for fluconazole and 95% for voriconazole. Where a discrepancy was observed between Etest and the reference method, the Etest tended to give lower values with both fluconazole and voriconazole. The Etest method using RPMI agar appears to be a useful method for determining fluconazole and voriconazole susceptibilities of uncommon species of Candida.
The Etest stable agar gradient MIC method (AB BIODISK, Solna, Sweden) has been shown to be useful in testing Candida spp. against a variety of antifungal agents, including fluconazole and voriconazole (1, 3, 9, 11, 12, 14, 16, 17, 22, 24). The species of Candida tested in these studies generally represent those most commonly isolated from clinical sources and are dominated by Candida albicans, C. glabrata, C. parapsilosis, and C. tropicalis, which account for 95 to 97% of all clinical isolates of Candida spp. (8, 13, 15, 18). Thus, although Etest has been validated for the four most common species of Candida, the evidence supporting its use in testing the less common species is lacking.
Among the approximately 17 species of Candida reported to cause bloodstream infections (BSI) (8), 12 or 13 of these species account for less than 5% of all Candida BSI (19). These rare species include among others, C. krusei, C. lusitaniae, C. guilliermondii, C. kefyr, C. rugosa, and C. dubliniensis, several of which may pose problems with antifungal resistance and nosocomial spread (4, 5, 7, 23, 26). Although less common than C. albicans, C. glabrata, C. parapsilosis, and C. tropicalis, these species may pose difficult management problems for individual patients, which may benefit from the application of antifungal susceptibility testing (21). Given the use of Etest for antifungal susceptibility testing of the common Candida spp. causing BSI, it is reasonable to validate its use for testing systemically active agents, such as fluconazole and voriconazole, against these less common species as well.
The purpose of the present study is to expand the Etest database for fluconazole and voriconazole by testing an international collection of 279 clinical BSI isolates of 12 uncommon species of Candida obtained from 68 different locations in 26 nations. The fluconazole and voriconazole MICs determined by Etest are compared to MICs determined by the National Committee for Clinical Laboratory Standards (NCCLS) reference broth microdilution (BMD) method, NCCLS M27-A (10).
MATERIALS AND METHODS
Organisms.
A total of 279 clinical isolates of Candida spp. obtained from 68 medical centers in North America (39 centers), Latin America (6 centers), Europe (15 centers), and the Asia-Pacific region (8 centers) were tested. The collection included the following numbers of isolates: 118, C. krusei; 56, C. lusitaniae; 53, C. guilliermondii; 11, C. kefyr; 10, C. rugosa; 8, C. lipolytica; 8, C. pelliculosa; 7, C. dubliniensis; 3, C. famata; 3, C. zeylanoides; 1, C. inconspicua; and 1, C. norvegensis. All were incident isolates obtained from blood cultures of 279 different patients with candidemia. Isolates were identified by using Vitek and API yeast identification systems (bioMerieux, Inc., Hazelwood, Mo.) and were supplemented by conventional methods as needed (25). Isolates were stored as water suspensions until used. Prior to testing, each isolate was passaged at least twice on potato dextrose agar (Remel, Lenexa, Kans.) to ensure purity and viability.
Susceptibility testing.
Reference antifungal susceptibility testing of Candida spp. was performed by BMD, as described by the NCCLS (10). Reference powders of fluconazole and voriconazole were obtained from Pfizer Pharmaceuticals (Groton, Conn.).
Etest strips for fluconazole and voriconazole were provided by AB BIODISK. MICs using Etest were determined as described previously (11, 14) by using 150-mm-diameter plates containing RPMI agar with 2% glucose (RPG; Remel), an inoculum suspension adjusted to the turbidity of a 0.5 McFarland standard (106 cells/ml), and incubation at 35°C for 48 h. Both fluconazole and voriconazole strips were placed on the same plate. The MICs of both fluconazole and voriconazole were read at the lowest concentration at which the border of the elliptical inhibition zone intercepted the scale on the strip. Any growth, such as microcolonies, throughout a discernible inhibition ellipse was ignored.
MIC interpretive criteria for fluconazole were those published by Rex et al. (20) and the NCCLS (10). Breakpoints were as follows: susceptible (S), ≤ 8 μg/ml; susceptible-dose dependent, 16 to 32 μg/ml; and resistant (R), ≥ 64 μg/ml. Interpretive breakpoints have not yet been established for voriconazole.
QC.
Quality control (QC) was performed for BMD and Etest in accordance with NCCLS document M27-A by using C. krusei ATCC 6258 and C. parapsilosis ATCC 22019 (2, 10). QC determinations made on each day of testing were within the control limits for fluconazole and voriconazole described by Barry et al. (2, 3).
Analysis of results.
Etest MICs for fluconazole and voriconazole read at 48 h were compared to reference BMD MICs read at 48 h. The reference microdilution MICs and Etest MICs were determined in two different time periods and were read independently by two different individuals; i.e., the testing was blinded. Etest MICs were rounded to the next even log2 concentration in order to simplify analysis (3, 11, 14). Discrepancies of no more than 2 dilutions were used to calculate the percent agreement.
The interpretive breakpoints described by NCCLS (10) were utilized to determine the categorical agreement between the Etest and BMD results for fluconazole. Major errors were identified as a classification of R by Etest and S by BMD, very major errors were when the Etest results was S and BMD was R, and minor errors were when one of the test results was S or R and the other was susceptible-dose dependent.
RESULTS AND DISCUSSION
Tables 1 and 2 summarize the in vitro susceptibilities of 279 BSI isolates of uncommon Candida spp. to fluconazole and voriconazole, respectively, as determined by the reference BMD and Etest methods. The data are presented in a continuous fashion as cumulative percentages of organisms susceptible at each dilution throughout the dilution series. The fluconazole MICs obtained by both BMD and Etest demonstrate excellent activity (≥ 95% S) against C. lusitaniae, C. kefyr, C. pelliculosa, C. dubliniensis, C. zeylanoides, and C. inconspicua and relatively poor activity against C. krusei, C. guilliermondii, C. rugosa, C. lipolytica, and C. norvegensis (Table 1).
TABLE 1.
In vitro susceptibility of uncommon Candida spp. to fluconazole determined by BMD and Etest
| Species (no. of isolates tested) | Test method | Cumulative no. of isolates (%) susceptible at MIC (μg/ml):
|
% Agreementa | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 0.12 | 0.25 | 0.5 | 1 | 2 | 4 | 8 | 16 | 32 | 64 | |||
| C. krusei (118) | BMD | 0 | 0 | 0 | 0 | 0 | 0 | 2 (2) | 13 (11) | 59 (50) | 112 (95) | |
| Etest | 0 | 0 | 0 | 0 | 0 | 0 | 1 (1) | 3 (3) | 37 (31) | 81 (69) | 97 (114/118) | |
| C. lusitaniae (56) | BMD | 2 (4) | 18 (32) | 41 (73) | 48 (86) | 52 (93) | 52 (93) | 53 (95) | 54 (96) | 54 (96) | 56 (100) | |
| Etest | 5 (9) | 18 (32) | 36 (64) | 47 (84) | 52 (93) | 52 (93) | 53 (95) | 58 (95) | 55 (98) | 55 (98) | 100 (56/56) | |
| C. guilliermondii (53) | BMD | 0 | 0 | 1 (2) | 6 (11) | 16 (30) | 33 (62) | 44 (83) | 49 (92) | 49 (92) | 49 (92) | |
| Etest | 0 | 4 (8) | 8 (15) | 12 (23) | 27 (51) | 41 (77) | 50 (94) | 51 (96) | 52 (98) | 52 (98) | 91 (48/56) | |
| C. kefyr (11) | BMD | 0 | 6 (55) | 10 (91) | 11 (100) | |||||||
| Etest | 1 (9) | 7 (64) | 10 (91) | 11 (100) | 100 (11/11) | |||||||
| C. rugosa (10) | BMD | 0 | 0 | 0 | 1 (10) | 3 (30) | 6 (60) | 7 (70) | 9 (90) | 10 (100) | ||
| Etest | 0 | 0 | 0 | 1 (10) | 4 (40) | 6 (60) | 7 (70) | 7 (70) | 8 (80) | 10 (100) | 100 (10/10) | |
| C. lipolytica (8) | BMD | 0 | 0 | 0 | 0 | 0 | 5 (63) | 6 (75) | 6 (75) | 6 (75) | 7 (88) | |
| Etest | 0 | 0 | 0 | 0 | 1 (13) | 5 (63) | 6 (75) | 6 (75) | 7 (88) | 7 (88) | 100 (8/8) | |
| C. pelliculosa (8) | BMD | 0 | 0 | 0 | 0 | 0 | 6 (75) | 8 (100) | ||||
| Etest | 0 | 0 | 1 (13) | 1 (13) | 3 (38) | 4 (50) | 8 (100) | 100 (8/8) | ||||
| C. dubliniensis (7) | BMD | 1 (14) | 7 (100) | |||||||||
| Etest | 4 (57) | 6 (86) | 7 (100) | 100 (7/7) | ||||||||
| C. famata (3) | BMD | 0 | 1 (33) | 2 (67) | 2 (67) | 2 (67) | 2 (67) | 2 (67) | 3 (100) | |||
| Etest | 1 (33) | 1 (33) | 2 (67) | 2 (67) | 3 (100) | 67 (2/3) | ||||||
| C. zeylanoides (3) | BMD | 1 (33) | 1 (33) | 2 (67) | 3 (100) | |||||||
| Etest | 1 (33) | 1 (33) | 2 (67) | 3 (100) | 100 (3/3) | |||||||
| C. inconspicua (1) | BMD | 0 | 0 | 0 | 0 | 0 | 0 | 1 (100) | ||||
| Etest | 0 | 0 | 0 | 0 | 0 | 1 (100) | 100 (1/1) | |||||
| C. norvegensis (1) | BMD | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 (100) | |||
| Etest | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 (0/1) | |
| All species (279) | BMD | 4 (1) | 33 (12) | 63 (23) | 78 (28) | 94 (34) | 125 (45) | 144 (52) | 165 (59) | 212 (76) | 268 (96) | |
| Etest | 12 (4) | 37 (13) | 66 (24) | 84 (30) | 111 (40) | 133 (48) | 150 (54) | 153 (55) | 192 (69) | 238 (85) | 96 (268/279) | |
% agreement signifies the percentage of Etest MICs within 2 dilutions of the reference BMD MICs determined following 48 h of incubation.
TABLE 2.
In vitro susceptibility of uncommon Candida spp. to voriconazole determined by BMD and Etest
| Species (no. of isolates tested) | Test method | Cumulative no. of isolates (%) susceptible at MIC (μg/ml):
|
% Agreementa | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 0.07 | 0.015 | 0.03 | 0.06 | 0.12 | 0.25 | 0.5 | 1 | 2 | 4 | 8 | |||
| C. krusei (118) | BMD | 0 | 0 | 0 | 1 (1) | 13 (11) | 62 (53) | 110 (93) | 116 (98) | 118 (100) | |||
| Etest | 0 | 0 | 0 | 4 (3) | 16 (14) | 84 (71) | 113 (96) | 117 (99) | 117 (99) | 118 (100) | 99 (117/118) | ||
| C. lusitaniae (56) | BMD | 38 (68) | 51 (91) | 53 (95) | 53 (95) | 53 (95) | 54 (96) | 56 (100) | |||||
| Etest | 37 (66) | 51 (91) | 52 (93) | 53 (95) | 53 (95) | 54 (96) | 56 (100) | 100 (56/56) | |||||
| C. guilliermondii (53) | BMD | 3 (6) | 5 (9) | 10 (19) | 27 (51) | 35 (66) | 43 (81) | 49 (92) | 49 (92) | 51 (96) | 51 (96) | 51 (96) | |
| Etest | 7 (13) | 12 (23) | 28 (53) | 46 (87) | 49 (92) | 51 (96) | 52 (98) | 53 (100) | 79 (42/53) | ||||
| C. kefyr (11) | BMD | 4 (36) | 8 (73) | 11 (100) | |||||||||
| Etest | 0 | 6 (55) | 11 (100) | 100 (11/11) | |||||||||
| C. rugosa (10) | BMD | 1 (10) | 2 (20) | 3 (30) | 6 (60) | 7 (70) | 10 (100) | ||||||
| Etest | 1 (10) | 4 (40) | 7 (70) | 7 (70) | 9 (90) | 10 (100) | 100 (10/10) | ||||||
| C. lipolytica (8) | BMD | 0 | 0 | 1 (13) | 3 (38) | 6 (75) | 6 (75) | 6 (75) | 7 (88) | 7 (88) | 8 (100) | ||
| Etest | 0 | 1 (13) | 4 (50) | 6 (75) | 6 (75) | 6 (75) | 7 (88) | 7 (88) | 7 (88) | 7 (88) | 7 (88) | 100 (8/8) | |
| C. pelliculosa (8) | BMD | 0 | 0 | 0 | 0 | 4 (50) | 8 (100) | ||||||
| Etest | 0 | 1 (13) | 4 (50) | 6 (75) | 6 (75) | 6 (75) | 7 (88) | 7 (88) | 7 (88) | 7 (88) | 7 (88) | 100 (8/8) | |
| C. pelliculosa (8) | BMD | 0 | 0 | 0 | 0 | 4 (50) | 8 (100) | ||||||
| Etest | 0 | 1 (13) | 2 (25) | 3 (38) | 5 (63) | 8 (100) | 88 (7/8) | ||||||
| C. dubliniensis (7) | BMD | 6 (86) | 7 (100) | ||||||||||
| Etest | 7 (100) | 100 (7/7) | |||||||||||
| C. famata (3) | BMD | 1 (33) | 1 (33) | 2 (67) | 2 (67) | 2 (67) | 3 (100) | ||||||
| Etest | 2 (67) | 2 (67) | 3 (100) | 67 (2/3) | |||||||||
| C. zeylanoides (3) | BMD | 2 (67) | 2 (67) | 2 (67) | 2 (67) | 3 (100) | |||||||
| Etest | 2 (67) | 2 (67) | 3 (100) | 100 (3/3) | |||||||||
| C. inconspicua (1) | BMD | 0 | 0 | 0 | 0 | 1 (100) | |||||||
| Etest | 0 | 0 | 1 (100) | 100 (1/1) | |||||||||
| C. norvegensis (1) | BMD | 0 | 0 | 0 | 0 | 1 (100) | |||||||
| Etest | 0 | 0 | 0 | 0 | 0 | 1 (100) | 100 (1/1) | ||||||
| All species (279) | BMD | 55 (20) | 76 (27) | 89 (32) | 112 (40) | 143 (51) | 209 (75) | 265 (95) | 272 (97) | 276 (99) | 277 (99) | 277 (99) | |
| Etest | 62 (22) | 91 (33) | 118 (42) | 144 (52) | 163 (58) | 239 (86) | 272 (97) | 277 (99) | 277 (99) | 278 (99) | 278 (99) | 95 (265/279) | |
% agreement signifies the percentage of Etest MICs within 2 dilutions of the reference BMD MICs determined following 48 h of incubation.
The MICs obtained by both BMD and Etest methods demonstrated that voriconazole was very active against all of the uncommon species of Candida (92 to 100% S if MIC ≤ 1 μg/ml) with the exception of C. lipolytica (88% S at MIC if 1 μg/ml) (Table 2). Overall, 97 and 99% of isolates were inhibited at ≤1μg/ml by BMD and Etest, respectively (Table 2).
As reported previously for the more common Candida spp. (3, 11, 14), the agreement between BMD and Etest for both fluconazole and voriconazole with the uncommon Candida spp. was excellent (Tables 1 and 2). The agreement was 96% for fluconazole and 95% for voriconazole.
The agreement between fluconazole Etest and BMD MICs was > 97% for all species, with the exception of C. guilliermondii (91%), C. famata (two of three, 67%), and C. norvegensis (zero of one, 0%) (Table 1). When discrepancies were observed between the results obtained by Etest and BMD for fluconazole, the Etest generally provided lower MICs, although Etest MICs tended to be higher than BMD MICs if C. krusei was being tested. The overall categorical agreement between Etest and BMD results for fluconazole was 81%, with 1.1% very major errors, 0% major errors, and 17.9% minor errors (data not shown). This agreement is lower than that reported by Barry et al. (3) when the more common species of Candida are tested, largely due to a greater number of minor errors attributed to C. krusei.
The agreement between voriconazole Etest and BMD MICs was > 99% for all species, with the exception of C. guilliermondii (79%), C. pelliculosa (seven of eight, 88%), and C. famata (two of three, 67%) (Table 2). As with fluconazole, when discrepancies occurred between the results obtained by Etest and BMD for voriconazole, the Etest provided lower MICs.
The results of this study confirm and extend those of previous reports regarding the ability of Etest to generate fluconazole and voriconazole MIC data for the less common species of Candida (1, 6, 9, 11, 14). Previously, agreement was demonstrated between Etest (by using RPG) and the reference BMD method of 94% for fluconazole and 98% for voriconazole in studies where the vast majority of isolates were the more common species of Candida (11, 14). Testing smaller numbers of C. krusei, C. lusitaniae, and C. guilliermondii isolates revealed agreements between Etest and BMD of 97 to 100% for fluconazole (11) and 100% for voriconazole (14). Favel et al. (6) reported an agreement between Etest and BMD of 92% when testing fluconazole against 35 isolates of C. lusitaniae.
In summary, we have provided documentation of the ability of Etest to generate fluconazole and voriconazole MIC data for uncommon species of Candida that are comparable to those obtained by the NCCLS BMD method. RPMI agar with 2% glucose may be used to determine reference quality MICs of fluconazole and voriconazole against these rare agents of candidemia as well as the more common species detected in the clinical laboratory. Although the species tested in this study are uncommon causes of BSI, the fact that several may exhibit innate or acquired resistance to both amphotericin B and fluconazole (4, 5, 7, 23, 26) emphasizes the importance of reference quality antifungal testing capabilities in aiding management decisions (21). Given these concerns, the potent in vitro activity of voriconazole against these species is notable (Table 2).
Acknowledgments
Linda Elliott provided excellent support in the preparation of the manuscript.
The International Fungal Surveillance Program was supported in part by research grants from Bristol-Myers Squibb (SENTRY Program), Pfizer (ARTEMIS Program), and Schering Plough (Global Surveillance Program).
We express our appreciation to all International Fungal Surveillance Program participants: L. Steele-Moore, Christiana Care Health Services, Wilmington, Del.; G. Denys, Clarion Health Methodist Hospital, Indianapolis, Ind.; C. Staley, Henry Ford Hospital, Detroit, Mich.; J. R. Dipersio, Summa Health System, Akron, Ohio; M. Saubolle, Good Samaritan Regional Medical Center, Phoenix, Ariz.; M. L. Wilson, Denver General Hospital, Denver, Colo.; G. D. Overturf, University of New Mexico Hospital, Albuquerque; L. R. Peterson, Northwestern Memorial Hospital, Chicago, Ill.; P. C. Schreckenberger, University of Illinois at Chicago, Chicago; G. V. Doern, University of Iowa Hospitals and Clinics, Iowa City; et al.
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