Received 26 July 2006/Returned for modification 10 October 2006/Accepted 16 October 2006

JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 2007, p. 70 75 Vol. 45, No. 1 0095-1137/07/$08.00 0 doi:10.1128/jcm.01551-06 Copyright 2007, American Society for Microbiology. All Rights Reserved. Use of Fluconazole as a Surrogate Marker To Predict Susceptibility and Resistance to Voriconazole among 13,338 Clinical Isolates of Candida spp. Tested by Clinical and Laboratory Standards Institute-Recommended Broth Microdilution Methods M. A. Pfaller, 1,2 * S. A. Messer, 1 L. Boyken, 1 C. Rice, 1 S. Tendolkar, 1 R. J. Hollis, 1 and D. J. Diekema 1,3 Departments of Pathology 1 and Medicine, 3 Roy J. and Lucille A. Carver College of Medicine, and Department of Epidemiology, College of Public Health, University of Iowa, Iowa City, Iowa 52242 2 Received 26 July 2006/Returned for modification 10 October 2006/Accepted 16 October 2006 Clinical laboratories frequently face the problem of delayed availability of commercially prepared approved reagents for performing susceptibility testing of new antimicrobials. Although this problem is encountered more often with antibacterial agents, it is also an issue with antifungal agents. A current example is voriconazole, a new triazole antifungal with an expanded spectrum and potency against Candida spp., Aspergillus spp., and other opportunistic fungal pathogens. The present study addresses the use of fluconazole as a surrogate marker to predict the susceptibility of Candida spp. to voriconazole. Reference broth microdilution MIC results for 13,338 strains of Candida spp. isolated from more than 200 medical centers worldwide were used. Voriconazole MICs and interpretive categories (susceptible, <1 g/ml; susceptible dose dependent, 2 g/ml; resistant, >4 g/ml) were compared with those of fluconazole by regression statistics and error rate bounding analyses. For all 13,338 isolates, the absolute categorical agreement was 91.6% (false susceptible or very major error [VME], 0.0%). Since voriconazole is 16- to 32-fold more potent than fluconazole, the performance of fluconazole as a surrogate marker for voriconazole susceptibility was improved by designating those isolates with fluconazole MICs of <32 g/ml as being susceptible to voriconazole, resulting in a categorical agreement of 97% with 0.1% VME. Clinical laboratories performing antifungal susceptibility testing of fluconazole against Candida spp. can reliably use these results as surrogate markers until commercial FDA-approved voriconazole susceptibility tests become available. Downloaded from http://jcm.asm.org/ Voriconazole is a new triazole antifungal agent with broadspectrum activity against Candida spp., Cryptococcus neoformans, Aspergillus spp., and other opportunistic fungal pathogens (2, 9, 12, 21, 22, 25, 27). The in vitro activity of voriconazole against Candida spp. has been documented by broth dilution, agar disk diffusion, and Etest methods (24, 25, 27, 28, 30), and most recently clinically relevant interpretive breakpoints for MIC and disk diffusion testing of Candida spp. have been established by the Clinical and Laboratory Standards Institute (CLSI) (formerly NCCLS) (30). The U.S. Food and Drug Administration (FDA) has approved voriconazole for treatment of invasive candidiasis (including candidemia) in nonneutropenic patients (6, 12). Voriconazole has also been FDA approved for treatment of esophageal candidiasis, invasive aspergillosis, and serious infections caused by Fusarium spp. and by Scedosporium apiospermum in patients who are intolerant or refractory to other antifungal agents (9, 19, 21, 22). Similar indications for the use of voriconazole have been approved in Europe, including the use of this agent for the treatment of fluconazole-resistant serious invasive Candida infections (6, 12). * Corresponding author. Mailing address: Medical Microbiology Division, C606 GH, Department of Pathology, University of Iowa College of Medicine, Iowa City, IA 52242. Phone: (319) 384-9566. Fax: (319) 356-4916. E-mail: michael-pfaller@uiowa.edu. Published ahead of print on 1 November 2006. Voriconazole has been shown to be efficacious in both primary (12) and salvage (19, 22) therapy of invasive candidiasis. However, several investigators have recently urged caution regarding its use in heavily azole-exposed patients due to the potential for cross-resistance, especially with fluconazole-resistant strains of Candida glabrata (1, 6, 8, 13, 20). Previously, we have shown that in vitro cross-resistance between fluconazole and voriconazole does exist among clinical isolates of Candida spp. (25, 27, 30). The clinical relevance of this cross-resistance has been documented in case series from three different institutions (1, 13, 20), where clinically significant microbial resistance to voriconazole has been reported among immunocompromised patients (e.g., intensive care and stem cell transplant patients) with a high level of azole (i.e., fluconazole before voriconazole) exposure. Although most of the resistant isolates in these series were C. glabrata, a number of C. albicans, C. tropicalis, and C. parapsilosis isolates were also found to have reduced susceptibility or resistance to both fluconazole and voriconazole (1, 13, 20). Thus, decreased susceptibility to fluconazole may precede, or even predict, decreased susceptibility to voriconazole, and diligent monitoring of candidal infections in highly compromised patients must be continued despite voriconazole coverage, particularly when voriconazole use is preceded by prolonged fluconazole exposure (1, 13, 20). These concerns have led Magill et al. (13) to institute reflexive fluconazole susceptibility testing of patients initial blood isolates as a means of on October 30, 2018 by guest 70

VOL. 45, 2007 SUSCEPTIBILITY TESTING OF VORICONAZOLE 71 Downloaded from http://jcm.asm.org/ FIG. 1. Scattergram comparing fluconazole and voriconazole MICs for 13,338 strains of Candida spp. An excellent correlation was observed (r 0.93; y 4.5 1.1x). The horizontal and vertical lines designate the respective breakpoints for fluconazole and voriconazole. (Adapted from reference 30.) identifying those that may not respond optimally to either fluconazole or voriconazole therapy. Despite the fact that interpretive breakpoints for voriconazole and Candida have been established by CLSI (30), the manufacture of commercially available antifungal susceptibility test systems that incorporate voriconazole is still under development, and none are FDA approved (3, 24). Currently, the Sensititre YeastOne colorimetric antifungal plate (TREK Diagnostic Systems, Cleveland, OH) is FDA approved for testing Candida spp. against fluconazole, itraconazole, and flucytosine (3). Previously, we have shown that fluconazole may serve as a surrogate marker for evaluating the susceptibility of Candida spp. to ravuconazole, an investigative triazole (29). This same approach should be applicable to voriconazole, allowing laboratories to use an FDAapproved commercial MIC panel containing fluconazole to predict the susceptibility of Candida isolates to voriconazole as a temporary measure until FDA approval for voriconazole testing has been granted. In the present study we utilized a large database of susceptibility test results for fluconazole and voriconazole, all determined by CLSI broth microdilution (BMD) methods, to first provide a very robust analysis of cross-resistance between the two agents and additionally to examine the usefulness of fluconazole as a surrogate marker for evaluating voriconazole susceptibility and resistance among Candida spp. MATERIALS AND METHODS Organisms. A total of 13,338 clinical isolates of Candida spp. obtained from more than 200 medical centers worldwide were tested (23). The collection included the following species and numbers of isolates: 7,725 isolates of C. albicans, 1,966 isolates of C. glabrata, 1,623 isolates of C. parapsilosis, 1,253 isolates of C. tropicalis, 312 isolates of C. krusei, 134 isolates of C. lusitaniae, 103 isolates of C. dubliniensis, 92 isolates of C. guilliermondii, 34 isolates of C. pelliculosa, 33 isolates of C. kefyr, 19 isolates of C. famata, 19 isolates of C. rugosa, 7 isolates of C. lipolytica, 5 isolates of C. zeylanoides, 3 isolates of C. inconspicua, 2 isolates of C. lambica, 2 isolates of C. sake, 1 isolate of C. norvegensis, and 5 isolates of Candida spp. not otherwise identified. All were incident isolates from individual patients and were obtained from blood or other normally sterile body fluids (23, 25 27). Isolates were identified by using Vitek and API yeast identification systems (biomerieux, Inc, Hazelwood, MO) supplemented by conventional methods as needed (7). The C. dubliniensis isolates were from mucosal infections and were identified by specific probe hybridization (10). Isolates were stored as water suspensions until used. Prior to testing, each isolate was passaged at least twice on potato dextrose agar (Remel, Lenexa, KS) and CHROMagar (Hardy Laboratories, Santa Maria, CA) to ensure purity and viability. on October 30, 2018 by guest

72 PFALLER ET AL. J. CLIN. MICROBIOL. TABLE 1. Fluconazole and voriconazole MIC distribution profiles for 13,338 clinical isolates of Candida spp. tested by Clinical and Laboratory Standards Institute-recommended broth microdilution methods a Species (no. of isolates tested) Antifungal agent No. of isolates at MIC ( g/ml) of: 0.007 0.015 0.03 0.06 0.12 0.25 0.5 1 2 4 8 16 32 64 C. albicans (7,725) Fluconazole 2,121 4,387 751 210 104 33 32 31 7 49 Voriconazole 5,624 1,417 372 181 39 38 17 12 10 1 14 C. glabrata (1,966) Fluconazole 1 4 6 18 164 477 547 491 77 181 Voriconazole 3 5 29 237 584 584 223 139 89 50 21 2 C. parapsilosis (1,623) Fluconazole 4 163 636 479 213 55 27 30 8 8 Voriconazole 265 589 409 219 70 47 12 7 4 1 C. tropicalis (1,253) Fluconazole 21 240 390 322 202 36 14 7 2 19 Voriconazole 120 203 382 386 100 27 8 6 3 4 9 5 C. krusei (312) Fluconazole 2 7 31 146 126 Voriconazole 1 28 137 126 18 1 1 C. lusitaniae (134) Fluconazole 5 44 43 22 11 3 2 1 2 1 Voriconazole 77 36 8 7 2 2 1 1 C. dubliniensis (103) Fluconazole 46 39 4 1 4 2 4 3 Voriconazole 60 24 8 1 2 4 2 2 C. guilliermondii (92) Fluconazole 1 1 3 32 31 15 5 1 3 Voriconazole 2 3 14 36 18 9 7 2 1 C. pelliculosa (34) Fluconazole 6 21 7 Voriconazole 1 20 11 2 C. kefyr (33) Fluconazole 4 16 11 2 Voriconazole 18 12 3 C. famata (19) Fluconazole 1 3 3 1 4 2 5 Voriconazole 2 3 2 2 2 5 3 C. rugosa (19) Fluconazole 13 2 4 Voriconazole 10 1 5 1 2 Candida spp. b (25) Fluconazole 1 1 3 2 2 7 2 1 6 Voriconazole 3 2 4 2 7 1 3 1 1 1 Total (13,338) Fluconazole 2,203 4,896 1,849 1,064 740 674 661 607 248 396 Voriconazole 6,174 2,300 1,232 1,079 874 869 404 183 110 57 31 25 a Broth microdilution MICs determined in accordance with CLSI M27-A2. b Includes C. lipolytica (7 isolates), C. zeylanoides (5 isolates), C. inconspicua (3 isolates), C. lambica (2 isolates), C. sake (2 isolates), C. norvegensis (1 isolate), and Candida spp. not otherwise identified (5 isolates). Susceptibility testing. Reference antifungal susceptibility testing of all isolates was performed by BMD as described by the CLSI (15). Reference powders of fluconazole and voriconazole were obtained from Pfizer Inc. (Groton, CT). MIC interpretive criteria for fluconazole were those published by Rex et al. (32, 33) and recently confirmed by Pfaller et al. (31). Breakpoints were as follows: susceptible (S), 8 g/ml; susceptible dose dependent (SDD), 16 to 32 g/ml; and resistant (R), 64 g/ml. The interpretive breakpoints for voriconazole were those proposed by the CLSI and published by Pfaller et al. (30): S, 1 g/ml; SDD, 2 g/ml; and R, 4 g/ml. Analysis of results. All MIC results (in g/ml) for fluconazole were directly compared with those for voriconazole by regression statistics and by scattergram (Fig. 1). The error rate bounding method to minimize intermethod interpretive error was also applied using the interpretive breakpoints described above. Acceptable error limits used in this comparison were those cited by the CLSI (8) and by other authors (4, 11). The definitions of errors used in this analysis were as follows: a very major error (VME), or a false-susceptible error, was an S result for the surrogate marker fluconazole and an R result for voriconazole; a major error (ME), or a false-resistant error, was an R result for fluconazole and an S result for voriconazole; and minor errors occurred when the result for one of the agents was S or R and that for the other agent was SDD. In general, for an agent to be considered a reliable surrogate, the VME rate should be 1.5% of all results, and the absolute categorical agreement between methods should be 90% (4, 11, 14). RESULTS AND DISCUSSION Table 1 depicts the MIC distribution profiles for fluconazole and voriconazole determined for 13,338 strains of Candida spp. by using CLSI (15)-validated BMD methods. Overall, 12,087 isolates (90.6%) were S, 855 (6.4%) were SDD, and 396 (3.0%) were categorized as R to fluconazole. Conversely, 13,115 (98.3%) were S, 110 (0.8%) were SDD, and 113 (0.9%) were R to voriconazole at MIC breakpoints of 1 g/ml, 2 g/ml, and 4 g/ml, respectively. The modal MIC for voriconazole was 0.007 g/ml, compared to 0.25 g/ml for fluconazole. De-

VOL. 45, 2007 SUSCEPTIBILITY TESTING OF VORICONAZOLE 73 TABLE 2. Use of fluconazole to predict susceptibility patterns of voriconazole, using 13,338 clinical isolates of Candida spp. from the Global Antifungal Surveillance Program TABLE 3. Absolute categorical agreement and error rates when the azole surrogate fluconazole result was used to predict voriconazole susceptibility of Candida spp. Species (no. of isolates tested) Fluconazole susceptibility category a No. (%) in voriconazole category b : S ( 1 g/ml) SDD (2 g/ml) R ( 4 g/ml) Organism(s) tested No. of isolates % a : Agreement VME ME Minor errors All Candida (13,338) S 12,087 (90.6) 0 (0.0) 0 (0.0) SDD 836 (6.3) 16 (0.1) 3 (0.1) R 192 (1.4) 94 (0.7) 110 (0.8) All Candida minus C. krusei (13,026) S 12,078 (92.7) 0 (0.0) 0 (0.0) SDD 659 (5.1) 16 (0.1) 3 (0.1) R 68 (0.5) 93 (0.7) 109 (0.8) C. albicans (7,725) S 7,638 (98.9) 0. (0.0) 0 (0.0) SDD 37 (0.5) 1 (0.01) 0 (0.0) R 25 (0.3) 9 (0.1) 15 (0.19) C. glabrata (1,966) S 1,217 (61.9) 0 (0.0) 0 (0.0) SDD 557 (28.3) 10 (0.5) 1 (0.1) R 30 (1.5) 79 (4.0) 72 (3.7) C. parapsilosis (1,623) S 1,577 (97.2) 0. (0.0) 0 (0.0) SDD 36 (2.2) 2 (0.1) 0 (0.0) R 5 (0.3) 2 (0.1) 1 (0.1) C. tropicalis (1,253) S 1,223 (97.6) 0 (0.0) 0 (0.0) SDD 5 (0.4) 2 (0.2) 2 (0.2) R 2 (0.2) 1 (0.1) 16 (1.3) C. krusei (312) S 9 (2.8) 0 (0.0) 0 (0.0) SDD 177 (56.7) 0 (0.0) 0 (0.0) R 124 (39.7) 1 (0.4) 1 (0.4) C. lusitaniae (134) S 130 (97.0) 0 (0.0) 0 (0.0) SDD 2 (1.4) 1 (0.8) 0 (0.0) R 1 (0.8) 0 (0.0) 0 (0.0) C. dubliniensis (103) S 94 (91.3) 0 (0.0) 0 (0.0) SDD 6 (5.8) 0 (0.0) 0 (0.0) R 1 (1.0) 0 (0.0) 2 (1.9) C. guilliermondii (92) S 83 (90.2) 0 (0.0) 0 (0.0) SDD 6 (6.5) 0 (0.0) 0 (0.0) R 0 (0.0) 2 (2.2) 1 (1.1) C. pellliculosa (34) S 34 (100) 0 (0.0) 0 (0.0) SDD 0 (0.0) 0 (0.0) 0 (0.0) C. kefyr (33) S 33 (100) 0 (0.0) 0 (0.0) SDD 0 (0.0) 0 (0.0) 0 (0.0) C. famata (19) S 14 (73.7) 0 (0.0) 0 (0.0) SDD 5 (26.3) 0 (0.0) 0 (0.0) C. rugosa (19) S 19 (100) 0 (0.0) 0 (0.0) SDD 0 (0.0) 0 (0.0) 0 (0.0) a S, MIC of 8 g/ml; SDD, MIC of 16 to 32 g/ml; R, MIC of 64 g/ml. b Voriconazole susceptibility categories are as described by Pfaller et al. (30). creased potencies of both fluconazole and voriconazole were observed among C. glabrata (modal MICs of 8 g/ml and 0.12 to 0.25 g/ml, respectively) and C. krusei (modal MICs of 32 g/ml and 0.25 g/ml, respectively), although 99% of C. krusei isolates were susceptible to voriconazole at the 1- g/ml breakpoint. The extent of cross-resistance between fluconazole and voriconazole may be seen more clearly in Table 2 and Fig. 1. All Candida 13,338 91.6 0.0 1.4 7.0 All Candida minus 13,026 93.6 (97.7) 0.0 (0.1) 0.5 (1.4) 5.9 (0.8) C. krusei C. albicans 7,725 99.1 0.0 0.3 0.6 C. glabrata 1,966 66.1 (93.9) 0.0 (0.1) 1.5 (1.5) 32.4 (4.5) C. parapsilosis 1,623 97.4 0.0 0.3 2.3 C. tropicalis 1,253 99.1 0.0 0.2 0.7 C. krusei 312 3.2 0.0 39.7 57.1 C. lusitaniae 134 97.8 0.0 0.8 1.4 C. dubliniensis 103 93.2 0.0 1.0 5.8 C. guilliermondii 92 91.3 0.0 0.0 8.7 C. pelliculosa 34 100 0.0 0.0 0.0 C. kefyr 33 100 0.0 0.0 0.0 C. famata 19 73.7 (100) 0.0 0.0 26.3 (0.0) C. rugosa 19 100 0.0 0.0 0.0 a Values in parentheses are categorical agreement and error rates obtained using the following categories for fluconazole: susceptible, MIC of 32 g/ml (S and SDD combined); resistant, MIC of 64 g/ml. Although the MICs for voriconazole were at least 16- to 32- fold lower than the corresponding fluconazole MICs for each isolate tested, there was a strong positive correlation (r 0.93) between voriconazole and fluconazole MICs (Fig. 1). All of the fluconazole-susceptible isolates were susceptible to voriconazole, as were 97.8% of the fluconazole-sdd isolates (Table 2). Among 396 fluconazole-resistant isolates, 192 (48%) were susceptible, 94 (24%) were SDD, and 110 (28%) were resistant to voriconazole. The CLSI does not recommend that laboratories test C. krusei against fluconazole, given its poor clinical response to this agent and the fact that the intrinsic resistance manifested by this species may be underrepresented by the in vitro results (5, 15, 26, 29, 31, 32). In contrast, voriconazole appears quite active against C. krusei (310 of 312 isolates [99%] were susceptible at an MIC of 1 g/ml [Table 2]), and the clinical activity looks promising as well (5, 12, 19, 22). When the C. krusei results are eliminated from the total, we again find that 100% of fluconazole-susceptible isolates and 97% of fluconazole-sdd isolates are susceptible to voriconazole but that only 25% of fluconazole-resistant isolates are susceptible to voriconazole (Table 2). Alternatively, if one considers all isolates identified as C. krusei to be resistant to fluconazole, then 378 of 582 fluconazole-resistant isolates (65%) would be susceptible to voriconazole (data not shown). When the fluconazole test result category (S, SDD, or R) was used to predict the voriconazole category, the absolute categorical agreement between the test results was 91.6%, with no VME (false-susceptible error), 1.4% ME (false-resistant error), and a 7.0% minor error rate (Table 3). Given the fact that the fluconazole results clearly do not predict the susceptibility of C. krusei to voriconazole (Tables 1 and 2), we have also factored the C. krusei results out of the analysis, with a resulting improvement in categorical agreement (93.6%) and a decrease in both ME (0.5%) and minor errors (5.9%) (Table 3). The absolute categorical agreements for 12 individual species of Candida are shown in Table 3. A categorical agreement

74 PFALLER ET AL. J. CLIN. MICROBIOL. of 91% or better (range, 91.3 to 100%) was observed for all species except C. glabrata, C. famata, and C. krusei. As seen with C. krusei, the fluconazole results also underestimated the activity of voriconazole against C. glabrata and C. famata (Tables 2 and 3). All fluconazole-susceptible isolates of these two species were also susceptible to voriconazole (Table 2). Likewise, 98% of C. glabrata isolates and all five C. famata isolates that were SDD to fluconazole were susceptible to voriconazole. In contrast, only 16.5% of fluconazole-resistant isolates of C. glabrata were susceptible to voriconazole, and 40% were resistant. Importantly, 72 of 73 voriconazole-resistant isolates (98.6%) were also resistant to fluconazole. Clearly, it is most important to detect those isolates of C. glabrata that may be resistant to voriconazole, and in that regard fluconazole performs quite well as a surrogate marker; there were no false-susceptible results (VME) and only 1.5% false-resistant results (ME). Given that 98% of C. glabrata isolates that were SDD to fluconazole were susceptible to voriconazole, one may improve the ability of the fluconazole MIC test to predict susceptibility of C. glabrata to voriconazole by combining the fluconazole S and SDD categories and using fluconazole MICs of 32 g/ml to identify voriconazole-susceptible isolates and MICs of 64 g/ml to identify voriconazole resistance. Using this criterion, the categorical agreement for C. glabrata improves to 93.9%, with 0.1% VME, 1.5% ME, and 4.5% minor errors (Table 3). Similarly, the categorical agreement for C. famata improves from 73.7% to 100%. Applying this modified criterion to the entire collection of isolates (minus C. krusei) results in an overall categorical agreement of 97.7%, with 0.1% VME and 1.4% ME (Table 3). Previously, we conducted a similar analysis using fluconazole results to predict the susceptibility of Candida spp. to ravuconazole as a proof of concept regarding the use of surrogate markers or class representatives in antifungal susceptibility testing (29). This strategy has been used for decades in antibacterial susceptibility testing to develop practical alternatives for the microbiology laboratory when specific diagnostic susceptibility testing reagents are limited or unavailable (11, 14, 16 18). Given the lack of FDA-approved testing systems for voriconazole, the approach described in the present study provides a useful strategy for laboratories in the effort to optimize antifungal therapy of candidal infections. In addition to providing a strategy to predict voriconazole susceptibility and resistance among Candida spp., these results provide strong support for the concerns of several investigators regarding the issue of cross-resistance between fluconazole and voriconazole (1, 8, 13, 20). By using an extensive global collection of clinically important isolates encompassing more than 12 species, we validate concerns originating from single-center experiences and rightly focus attention on C. glabrata as the species most likely to demonstrate cross-resistance among the azoles. Fluconazole functioned well as a surrogate marker for voriconazole susceptibility when applied to this collection of clinically significant isolates of Candida spp. The absolute categorical agreement of 91.6% (93.6% without C. krusei), with no VME among more than 13,000 isolates tested, easily meets the recognized criteria for a reliable surrogate marker as applied to antibacterial susceptibility testing (4, 11, 14, 16 18). Given the greater potency of voriconazole over fluconazole, the use of fluconazole as a surrogate marker for voriconazole susceptibility was clearly improved by designating those isolates with fluconazole MICs of 32 g/ml (S and SDD categories combined) as being susceptible to voriconazole, with the resistant category staying the same at 64 g/ml. The resulting 97.7% categorical agreement and 0.1% VME rate is excellent for a surrogate marker test. In conclusion, we have demonstrated the existence of crossresistance between fluconazole and voriconazole, with the greatest emphasis on C. glabrata. Furthermore, we have shown that the availability of voriconazole susceptibility testing results for Candida spp., in any medical center currently performing antifungal susceptibility testing of fluconazole, can be accomplished by using the fluconazole result as a surrogate marker for voriconazole susceptibility and resistance. Specifically, fluconazole MICs of 32 g/ml predict susceptibility and MICs of 64 g/ml predict resistance to voriconazole. The occurrence of false-susceptible and false-resistant errors with this expanded application of the class representative concept to selected triazoles was very low and was acceptable for surrogate marker testing. 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