Received 31 March 2009/Returned for modification 26 May 2009/Accepted 22 June 2009

JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 2009, p. 2766 2771 Vol. 47, No. 9 0095-1137/09/$08.00 0 doi:10.1128/jcm.00654-09 Copyright 2009, American Society for Microbiology. All Rights Reserved. Comparison of 24-Hour and 48-Hour Voriconazole MICs as Determined by the Clinical and Laboratory Standards Institute Broth Microdilution Method (M27-A3 Document) in Three Laboratories: Results Obtained with 2,162 Clinical Isolates of Candida spp. and Other Yeasts Ana Espinel-Ingroff, 1 * E. Canton, 2 J. Peman, 2 M. G. Rinaldi, 3 and A. W. Fothergill 3 VCU Medical Center, Richmond, Virginia 1 ; Hospital Universitario La Fe, Valencia, Spain 2 ; and University of Texas Health Science Center, San Antonio, Texas 3 Received 31 March 2009/Returned for modification 26 May 2009/Accepted 22 June 2009 We evaluated the performance of the 24-h broth microdilution voriconazole MIC by obtaining MICs for 2,162 clinical isolates of Candida spp. and other yeasts; the 24-h results were compared to 48-h reference MICs to assess essential, as well as categorical, agreement. Although the overall essential agreement was 88.6%, it ranged from 96.4 to 100% for 6 of the 11 species or groups of yeasts tested. The overall categorical agreement was 93.2%, and it was above 90% for eight species. However, unacceptable percentages of very major errors (false susceptibility) were observed for Candida albicans (2.7%), C. glabrata (4.1%), C. tropicalis (9.7%), and other less common yeast species (9.8%). Since it is essential to identify potentially resistant isolates and breakpoints are based on 48-h MICs, it appears that the 24-h MIC is not as clinically useful as the 48-h reference MIC. However, further characterization of these falsely susceptible MICs for three of the four common Candida spp. is needed to understand whether these errors are due to trailing misinterpretation or if the 48-h incubation is required to detect voriconazole resistance. Either in vivo versus in vitro correlations or the determination of resistance mechanisms should be investigated. Candida spp. and Aspergillus spp. are responsible for the majority (80 to 90%) of fungal infections. During the last several years, new antifungal agents (echinocandins and triazoles) have been licensed for the systemic treatment of fungal infections. Among the triazoles, voriconazole is available for the oral or intravenous treatment of mold and yeast infections (e.g., primary treatment of invasive candidiasis, including candidemia, in neutropenic and nonneutropenic patients). These events have underscored the need for testing the antifungal susceptibilities of fungal pathogens to these agents. The Clinical and Laboratory Standards Institute (CLSI) has developed reference methods (CLSI M27-A3 and M44-A documents) for antifungal susceptibility testing of Candida spp. and Cryptococcus neoformans (3, 5, 6). In addition to the guidelines for testing voriconazole, the CLSI has established interpretive breakpoints for this agent versus Candida spp. (5, 6, 19). These microdilution MIC breakpoints were based on the determination of voriconazole MICs after 48 h of incubation. However, most common Candida spp. have suitable growth for MIC determination at 24 h; a shorter incubation time is more efficient and practical for use in the clinical laboratory and is currently used to determine endpoints for the echinocandins, amphotericin B, and fluconazole. The interlaboratory reproducibility of the 24-h voriconazole result, as well as the categorical agreement between 24- and 48-h MICs were previously addressed in a collaborative study for a small number of Candida isolates (8). The purpose of the present study was to * Corresponding author. Mailing address: 3804 Dover Rd., Richmond, VA 23221. Phone: (804) 358-5895. Fax: (804) 828-3097. E-mail: avingrof@vcu.edu. Published ahead of print on 1 July 2009. further evaluate the suitability of CLSI 24 h voriconazole MIC results with 2,162 clinical isolates of Candida and other yeast species. The evaluation involved (i) the compatibility or essential agreement between 24- and 48-h voriconazole MICs (within 2 log 2 dilutions of the reference 48 h MIC) regardless of breakpoint agreement and (ii) the categorical agreement and error rates according to CLSI interpretive breakpoints for voriconazole. Since fluconazole has been proposed as a surrogate marker to predict resistance to voriconazole, fluconazole MICs were determined for 731 of the 2,162 isolates included here. MATERIALS AND METHODS Study design. We compared the MICs obtained in three independent laboratories by the CLSI reference M27-A3 broth microdilution method (5) to those obtained at 24 h by the same method. A panel of 2,162 isolates of Candida and other yeasts recovered from clinical specimens (blood and other normally sterile sites) in three medical centers were included in the study; the two quality control (QC) isolates (Candida parapsilosis ATCC 22019 and C. krusei ATCC 6258) were also included. In addition, fluconazole MICs were determined for 731 of the 2,162 isolates, including isolates categorized as either susceptible, susceptible dose dependent or resistant to voriconazole. Each first day voriconazole MIC was compared to the reference second day result for the evaluations of both essential and categorical agreement; voriconazole 24 h MICs were also compared to fluconazole endpoints for the selected set of 731 isolates. Clinical isolates. A total of 2,162 isolates from three culture collections (VCU Medical Center, Richmond, VA; the Fungus Testing Laboratory at the University of Texas Health Science Center, San Antonio, TX; and the Hospital Universitario La Fe, Valencia, Spain) included 681 C. albicans, 12C. dubliniensis, 11 C. famata, 535 C. glabrata,42c. guilliermondii, 106 C. krusei,74c. lusitaniae, 424 C. parapsilosis, 238 C. tropicalis, 8 isolates of less common Candida spp., and 31 isolates of other yeasts or yeastlike organisms (listed as other yeasts in Tables 1 and 2). The set included 150 resistant and 72 susceptible dose-dependent isolates (at 48 h) to voriconazole; some of these isolates were recovered from patients enrolled in the phase III voriconazole clinical trials (13), as well as from other patients. The CLSI QC isolates C. parapsilosis ATCC 22019 and C. krusei ATCC 2766

VOL. 47, 2009 COMPARISON OF 24- AND 48-H VORICONAZOLE MICs FOR YEASTS 2767 TABLE 1. Susceptibilities of 2,162 isolates of Candida spp. and other yeastlike organisms to voriconazole as determined by the CLSI broth microdilution method (M27-A3) and read after 24 and 48 h of incubation Species No. of values a Incubation period (h) MIC ( g/ml) b Range MIC 50 MIC 90 R (Pearson) Essential MIC agreement (%) c C. albicans 671 24 0.008 8 0.03 0.5 681 48 0.008 8 0.06 1 0.822 96.4 C. dubliniensis 11 24 0.008 0.03 0.008 0.016 12 48 0.008 0.06 0.016 0.016 0.795 100 C. famata 11 24 0.008 8 0.008 2 11 48 0.008 8 0.016 4 0.97 100 C. glabrata 531 24 0.008 8 0.06 1 535 48 0.008 8 0.25 2 0.842 73.3 C. guilliermondii 41 24 0.008 2 0.06 0.5 42 48 0.008 2 0.12 1 0.904 87.8 C. krusei 106 24 0.008 2 0.25 0.5 106 48 0.008 8 0.5 1 0.774 97.2 C. lusitaniae 74 24 0.008 1 0.016 0.06 74 48 0.008 8 0.016 0.12 0.922 97.2 C. parapsilosis 420 24 0.008 4 0.016 0.03 424 48 0.008 4 0.03 0.12 0.832 98.6 C. tropicalis 237 24 0.008 8 0.03 0.5 238 48 0.008 8 0.06 8 0.56 77.2 Candida spp. d 8 24 0.008 0.25 0.03 0.25 8 48 0.008 1 0.12 1 0.895 75 Other yeasts e 31 24 0.008 4 0.03 0.25 31 48 0.008 4 0.06 4 0.4 83.9 Total 2,141 24 0.008 8 0.03 0.5 2,162 48 0.008 8 0.06 2 0.8 88.6 a The MICs were not determined at 24 h for 21 isolates among some species due to either no or insufficient growth. b MIC 50 and MIC 90 were the MICs encompassing 50% and 90% of the isolates tested, respectively. c Agreement between 24 and 48 h or between 48 and 72 h (Cryptococcus neoformans) broth microdilution MICs. d Including C. ciferii (one isolate), C. haemulonii (three isolates), C. inconspicua (one isolate), C. lambica (one isolate), and C. lipolytica (two isolates). e Including Blastoschizomyces capitatus (two isolates), C. neoformans (four isolates), Geotrichum spp. (two isolates), Pichia spp. (one isolate), Rhodotorula mucilaginosa (three isolates), R. minuta (two isolates), Saccharomyces cerevisiae (three isolates), Trichosporon asahii (twelve isolates), T. cutaneum (one isolate), and Yarrowia lipolytica (one isolate). Downloaded from http://jcm.asm.org/ 6258 were tested each time a set of clinical isolates was evaluated in each laboratory; voriconazole and fluconazole MIC results were within the established QC MIC limits (5, 6). Each isolate represented a unique strain from a single patient with the exception of 20 isolates (serial isolates from four patients); isolates were maintained at 70 C and subcultured onto antimicrobial-free medium to ensure viability and purity prior to testing. Antifungal susceptibility testing. Visual MICs were determined for the whole set of 2,162 clinical isolates with voriconazole at both 24 and 48 h and for the selected set of 731 isolates with fluconazole. Broth microdilution MICs. Reference microdilution trays, containing serial drug dilutions of either voriconazole or fluconazole (Pfizer Central Research, New York, NY), were prepared by following the CLSI M27-A3 guidelines (5). Voriconazole drug concentrations ranged from 0.008 to 16 g/ml and fluconazole from 0.12 to 64 g/ml. The microdilution wells containing 100 l ofthe twofold serial dilutions of the antifungal drugs in standard RPMI 1640 medium (0.2% glucose) were inoculated with 100 l of inoculum containing between 1.0 10 3 and 5 10 3 CFU/ml. After inoculation of the microdilution trays, the plates were incubated at 35 C in a non-co 2 incubator. Voriconazole MICs were visually determined after 24 and 48 h and fluconazole at 48 h. MICs corresponded to the lowest drug dilution that showed prominent growth inhibition (50% or more) compared to the growth control (5). Statistical analysis. Both on-scale (e.g., 0.08 and 16 g/ml) and off-scale (e.g., 0.08 and 16 g/ml) voriconazole MICs were included in the analysis. For the comparison between the two incubation times, each voriconazole 24 h MIC was compared to the 48 h result using the latter result as the reference test. Voriconazole MICs were considered in essential agreement when the discrepancies between the two incubation times were no more than 2 log 2 dilutions (two wells). In addition, the interpretive CLSI criteria (M27-A3 document) for voriconazole were used to evaluate the categorical agreement between 24-h and reference 48-h MIC results (5, 6, 19). Errors were calculated as follows: (i) very major errors, when the reference MIC indicated resistance while the 24-h result indicated susceptible; (ii) major errors, when the 24-h result categorized the isolate as resistant and the reference as susceptible; and (iii) minor errors, when there was a single categorical shift between the two results (e.g., susceptible to susceptible dose dependent). For the correlation between voriconazole 24- and 48-h MICs, a linear regression analysis using the least-square method (Pearson correlation coefficient; MS Excel software) was performed by plotting 24 h MICs against their respective reference 48-h MIC endpoints for 2,141 of the 2,162 isolates for which it was possible to determine MICs at both incubation times (17). Using fluconazole values as surrogate predictors of voriconazole resistance (22), each fluconazole MIC for the set of 731 isolates was compared to their corresponding 24 h and reference 48 h voriconazole MIC results. RESULTS AND DISCUSSION Although several studies have compared fluconazole 24- and 48-h MICs as determined by the CLSI broth microdilution method (2, 15, 16, 17, 23, 24), only one study has recently compared voriconazole 24 and 48 h MICs (8). One of the purposes of these studies was to evaluate the possibility of providing earlier results since prompt treatment leads to a better response to antifungal therapy (12). The other concern has been the difficulty in determining 48 h fluconazole MICs, where trailing growth is more prominent and could lead to false fluconazole resistance (2, 15, 23, 24). Since breakpoints have been available for most antifungal agents for testing Candida spp., the more recent evaluations have demonstrated that the 24-h fluconazole endpoint predicted clinical outcome to fluconazole therapy as well as the reference 48-h result (16) and that the categorical agreement was quite suitable ( 94%) for most common species (8, 17). The exceptions in these on October 6, 2018 by guest

2768 ESPINEL-INGROFF ET AL. J. CLIN. MICROBIOL. TABLE 2. Categorical agreement between 24 and 48 h CLSI broth microdilution voriconazole MIC pairs (2,141) of Candida spp. and other yeastlike organisms Species (no. of values) a Incubation period (h) b % of MICs by category c % Errors S S-DD R Minor Major Very major % Categorical agreement (no. discrepant results) d C. albicans (671)* 48 90.7 1.3 8 24 92.4 1.9 5.7 1.2 1 2.7 95.1 (33) C. dubliniensis (11)* 48 100 0 0 24 100 0 0 0 0 0 100 (0) C. famata (11) 48 81.8 0 18.2 24 82 9 9 9.1 0 0 91 (1) C. glabrata (531)* 48 83.2 8.1 8.7 24 94.2 2.4 3.4 7.7 0 4.1 87.9 (64) C. guilliermondii (41)* 48 90.2 9.8 0 24 97.6 2.4 0 7.3 0 0 92.7 (3) C. krusei (106) 48 95.3 3.8 0.9 24 99.1 0.9 0 4.7 0 0 95.3 (5) C. lusitaniae (74) 48 98.6 0 1.4 24 100 0 0 0 0 1.4 98.6 (1) C. parapsilosis (420)* 48 99.3 0.5 0.2 24 99.8 0 0.2 0.5 0 0 99.5 (2) C. tropicalis (237)* 48 80.2 4.2 15.6 24 92.4 2.1 5.5 3.8 0.4 9.7 86.7 (33) Candida spp. (8) 48 100 0 0 24 100 0 0 0 0 0 100 (0) Other yeasts (31) 48 87.1 0 12.9 24 93.6 3.2 3.2 3.2 3.2 9.8 83.8 (5) Values at 48 h (2,162)* 48 89.8 3.4 6.8 Values at 24 h (2,141)* 24 95 1.6 3.3 3.3 0.4 3.1 93.2 (147) a *, MICs were not determined at 24 h for 21 isolates among these species due to either no or insufficient growth. b 24- and 48-h MIC pairs are presented: the MICs visually determined at these incubation times by the CLSI broth microdilution method (M27-A3) are shown. c That is, the percentages of 24- and 48-h MICs that were within the CLSI breakpoint categorization for voriconazole (susceptible S, MICs 1 g/ml; susceptible dose dependent S-DD, MIC 2 g/ml; and resistant R, MICs 4 g/ml). d That is the percentages of MIC pairs (24-and 48-h reference) that were in agreement regarding the CLSI breakpoint categorization. The numbers of 24-h MICs per species that fell into a different CLSI breakpoint category are indicated in parentheses. Downloaded from http://jcm.asm.org/ fluconazole studies were C. krusei (57 to 63% categorical agreement) and C. glabrata (69% categorical agreement) due to large percentages of minor errors (33 to 43%) (8, 17). Based on those results, the last edition of the M27 document (5) states that the 24-h fluconazole reading is acceptable. However, the acceptable incubation time is 48 h for voriconazole since both fluconazole and voriconazole breakpoints have been established based on 48-h MIC data (5, 19). Because of that, our goal was to evaluate the performance of the 24-h voriconazole MIC result in the same manner that Pfaller et al. (17) evaluated the 24-h fluconazole endpoint. Our voriconazole in vitro data for the 2,162 yeast isolates have been gathered during the last 8 years in three reference laboratories. According to the CLSI guidelines, both 24- and 48-h voriconazole MICs were obtained for all of the isolates. Of the 2,162 isolates, 24-h MICs were not available for 21 isolates (1%) due to either no growth or insufficient growth. Therefore, most isolates had sufficient growth at 24 h for MIC determination as previously reported (7, 8, 17, 24). Table 1 summarizes voriconazole MICs for the 2,162 isolates stratified by species as determined by the CLSI microdilution method at both 24 and 48 h. With the exception of isolates of C. tropicalis and the 31 isolates grouped as other yeasts, MIC 90 results were within one or two dilutions at both incubation times (results being consistently higher at 48 h). Pfaller et al. (17) found similarities for the same species during their comparison of fluconazole 24-h and reference 48-h MICs. The susceptibility of our isolates to voriconazole was more similar to those obtained for the 1,763 yeasts recovered in the phase III voriconazole clinical trials (MIC 90 s for C. albicans and C. glabrata of 0.25 and 4 g/ml, respectively, versus the MIC 90 sof1and2 g/ml, respectively) (13) than those obtained in other studies (MIC 90 s below 1 g/ml for most of the species, except for the MIC 90 [1 g/ml] for C. glabrata) (19). Our set of organisms included 200 isolates from patients with noncandidemia infections enrolled in these clinical trials. Because 24-h MICs were not available for 21 of the 2,162 isolates, the evaluation of essential agreement between 24-h and reference 48-h MIC pairs regardless of categorical classification was performed with 2,141 MIC pairs (Table 1). The overall essential agreement was 88.6%; however, essential agreement was excellent ( 96%) for 6 of the 11 species or groups of isolates tested. Our essential agreement for these six species was similar to that obtained in previous comparisons of 24-h versus reference 48-h voriconazole (8) and fluconazole (8, 17) MICs among the most common species with the exception of C. glabrata and C. tropicalis (73.3 and 77.2%, respectively) (Table 1). Although in the collaborative study (8) the agreement between the two incubation times was lower for these two species than for the other species with voriconazole, their agreement was higher (89 and 93%, respectively) than in the present study (73.3 and 77.2%, respectively). The acceptable percentage of essential agreement between two methods is usually 90% (4). During performance evaluations of 24-h broth dilution methods such as EUCAST, YeastOne, and Vitek-2, the essential agreement has been species and incuba- on October 6, 2018 by guest

VOL. 47, 2009 COMPARISON OF 24- AND 48-H VORICONAZOLE MICs FOR YEASTS 2769 FIG. 1. Comparison of voriconazole broth microdilution MICs at 24 and 48 h for 2,141 Candida spp. and other yeast isolates. Interpretive MIC breakpoints are indicated by the horizontal and vertical lines. tion time variable, but results also have been frequently below 90% for three of the four most common species of Candida (C. albicans, C. glabrata, and C. tropicalis) (1, 7, 10, 20, 21). Our and these prior results underscore once more the impact of the incubation time on MIC results. Unfortunately, it is among these species where high voriconazole MICs can be obtained, especially for C. glabrata, where the percentage of success to voriconazole therapy was the lowest (55%) during clinical trials (19). Figure 1 provides the correlation between 24-h and reference 48-h voriconazole MICs for the 2,141 pairs; the horizontal and vertical lines indicate the CLSI breakpoints for voriconazole. Suitable values (R 0.7 and R 2 0.5) were obtained for the overall comparison, as well as for most of the species tested. The exceptions were C. tropicalis (R, 0.56) and the 31 isolates listed as other yeasts (R, 0.4) (Table 1). Although the overall categorical agreement was 93.2%, this agreement was below 90% for C. glabrata, C. tropicalis, other less common Candida spp. and other yeasts. In addition, high percentages of very major errors (false susceptibility) were observed for the same two species (C. glabrata [4.1%] and C. tropicalis [9.7%]), as well as for the group listed as other yeasts (9.8%); the percentage of very major errors for C. albicans was also above the criteria of 1.5% very major categorical errors used by the U.S. Food and Drug Administration for clearance of susceptibility test methods (11). Our percentages of very major errors were also higher (Table 2) than those previously obtained (0 to 1.8% very major errors) during comparisons between either both incubation times or between M27 MICs versus M44 disk or Neo-Sensitabs tablet results for both fluconazole (8, 16 18) and voriconazole (9, 18, 19). Regarding minor errors, our results (7.7% minor errors) were lower than those reported for C. glabrata against fluconazole (43.1% minor errors) and voriconazole (13% minor errors) but higher for C. albicans and C. tropicalis (1.2 and 3.8% minor errors, respectively) than previous data (0 to 1% minor errors for both triazoles) (8, 17) (Table 2). The Food and Drug Administration target for major errors is 3% (false resistance); only the results for other yeasts were not suitable regarding this criterion (11). Among these isolates, major errors were observed for three of the five Rhodotorula spp. isolates included in the study (data not listed in Table 2); as indicated in Table 2, other major errors were found among C. albicans and C. tropicalis. Major errors are usually reported when two methodologies are compared including during the comparison of the 24-h versus the 48-h fluconazole results (17). The reason for major errors is not clearly understood, but it could be attributed to the poor growth of certain isolates at 24 h. Because of that, sufficient growth (heavy and confluent growth covering the bottom of the growth control well), as described in recent CLSI documents, should be observed in order to report 24 MICs. Since we had 67 isolates for which voriconazole MICs switched from susceptible at 24 h to resistant at 48 h and fluconazole has been proposed as a surrogate marker for voriconazole resistance (22), we compared fluconazole to voriconazole MICs for 731 isolates; 34 of these 67 isolates were included as follows: 12 C. albicans, 6 C. glabrata, 13 C. tropicalis, and three Rhodotorula spp. All six C. glabrata and the three isolates of Rhodotorula spp. were resistant to fluconazole, which indicated that the 48-h results could be more useful in detecting potential voriconazole resistance for these species. This was confirmed by the examination of other voriconazole-

2770 ESPINEL-INGROFF ET AL. J. CLIN. MICROBIOL. resistant isolates of these species for which fluconazole MICs were determined. The fluconazole category as a marker of voriconazole resistance for C. albicans and especially for C. tropicalis did not yield results as clear as those for C. glabrata and Rhodotorula spp. Eight (67%) of the twelve C. albicans and six (46%) of the thirteen C. tropicalis isolates were either fluconazole resistant or susceptible dose dependent, but the other eleven isolates were fluconazole susceptible (very major error or false susceptibility). Similar patterns were observed for other resistant isolates for which fluconazole MICs were determined for these two species. Magill et al. (14) found that 32% of fluconazole-resistant isolates were resistant to voriconazole. Pfaller et al. (22) reported no very major errors among their 73 voriconazole-resistant C. glabrata isolates (72 were also resistant to fluconazole) or for any other species during their evaluation of the fluconazole category as predictor of voriconazole resistance. Their other voriconazole resistant species were either fluconazole resistant or susceptible dose dependent. It is preferable to err on the side of false resistance than false susceptibility in the selection of the therapeutic agent. Therefore, these results suggest that testing should be continued at both incubation times until this dilemma is resolved by either correlation studies with in vivo data (clinical or animal models) or a mechanism-of-resistance evaluation for these isolates. It could be that some of our voriconazole-resistant 48-h MICs for C. albicans and especially for C. tropicalis, which grow at a faster rate than other Candida spp., were the result of a misinterpretation of trailing growth, as has been previously demonstrated in vivo (patient and an animal model) or by sterol quantitation for fluconazole (2, 23, 24). In the case of fluconazole, adjustment of the medium ph has been shown to alleviate the trailing problem to some extent; a similar observation has been reported using the YeastOne colorimetric method (10, 15, 21). In conclusion, in contrast to the fluconazole data, our results indicated that the 24-h voriconazole MIC is not as useful for detecting resistance for three of the more common Candida spp. Because of this, voriconazole MICs should continue to be obtained either at both incubation times or at 48 h as stated in the CLSI M27-A3 document. However, further characterization of isolates where the categorical classification shifted from susceptible at 24 h to resistant at 48 h should determine whether this shift was the result of the misinterpretation of trailing growth or if the longer incubation is needed to determine voriconazole resistance. The latter has been confirmed for triazole resistance when testing Aspergillus spp. ACKNOWLEDGMENTS This study was supported in part by research grants from Pfizer Spain (to E.C. and J.P.). REFERENCES 1. Alexander, B. D., T. C. Byrne, K. L. Smith, K. E. Hanson, K. J. Anstrom, J. R. Perfect, and L. B. Reller. 2007. Comparative evaluation of Etest and Sensititre YeastOne panels against the Clinical and Laboratory Standards Institute M27 A2 reference broth microdilution method for testing Candida susceptibility to seven antifungal agents. J. Clin. Microbiol. 45:698 706. 2. Arthington-Skaggs, B. A., W. Lee-Yang, M. A. Ciblak, J. P. Frade, M. E. Brandt, R. A. Hajjeh, L. H. Harrison, A. 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