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Antimicrob Agents Chemother. 2016 October; 60(10): 6173–6178.
Published online 2016 September 23. Prepublished online 2016 August 1. doi:  10.1128/AAC.01027-16
PMCID: PMC5038286

Intra- and Interlaboratory Agreement in Assessing the In Vitro Activity of Micafungin against Common and Rare Candida Species with the EUCAST, CLSI, and Etest Methods

Abstract

The emergence of resistant strains among common and rare Candida species necessitates continuous monitoring of the in vitro susceptibilities of those isolates. We therefore assessed the in vitro activities of micafungin against 1,099 molecularly identified isolates belonging to 5 common and 20 rare Candida species by the EUCAST, CLSI, and Etest methods, assessing both the intralaboratory agreement and the interlaboratory agreement for two centers. The median micafungin EUCAST MICs were as follows, from the lowest to the highest: for Candida albicans, 0.004 mg/liter; for C. glabrata, 0.016 mg/liter; for C. tropicalis, 0.031 mg/liter; for C. krusei, 0.125 mg/liter; for C. parapsilosis, 2 mg/liter. Among rare Candida species, high MICs were found for C. guilliermondii, C. lipolytica, C. orthopsilosis, C. metapsilosis, and C. fermentati. No resistant isolates were found by the CLSI method, whereas resistance rates of 1 to 2% were found by the EUCAST method. Overall, the EUCAST method resulted in MICs 1 to 2 dilutions higher than those found by the CLSI and Etest methods. The intra- and interlaboratory agreement between methods was >92%, except for the interlaboratory agreement between the EUCAST and CLSI methods (81%), where 17 to 31% of the differences were >2 2-fold dilutions for C. albicans, C. glabrata, C. tropicalis, and other rare Candida species and <6% for C. parapsilosis and C. krusei. For the other interlaboratory comparisons, the EUCAST method resulted in higher MICs than the Etest method for all species, but <7% of these differences were >2 2-fold dilutions. Overall, the CLSI method resulted in lower MICs than the Etest method, with 11% of all isolates demonstrating >2 2-fold-dilution differences (6 to 20% for C. albicans, C. tropicalis, and rare Candida species; <5% for C. glabrata, C. krusei, and C. parapsilosis) and smaller differences found after 24 h. Despite these differences, categorical agreement was excellent (>97%), with only 1 to 2% very major errors between the EUCAST method and the other two methods.

INTRODUCTION

Micafungin is an echinocandin used for the treatment and prophylaxis of Candida infections in both neutropenic and nonneutropenic patients (1, 2). Although micafungin has a broad spectrum of activity against different Candida species, including azole-resistant isolates, the emergence of resistance in common and rare Candida species necessitates in vitro susceptibility testing of those isolates (3). Methods for the determination of MICs have recently been published by the Clinical and Laboratory Standards Institute (CLSI) (4) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (5). Both of these methods are broth microdilution methods; they differ in glucose concentration, inoculum size, the shape of microplates, and the mode of reading. In addition, the Etest is often used to determine susceptibility in routine laboratories (6).

Although all these methods are reproducible and reliable in testing the in vitro susceptibility of Candida isolates, there are few comparative studies exploring the differences between them (6, 7). Most of those studies utilized small collections of isolates for common species tested in a single lab. In addition, in light of species-specific breakpoints, correct species identification is of paramount importance both for assessing in vitro susceptibility and for comparing methods. Correct identification is particularly challenging for rare species, where phenotypic tests have usually failed (8). We therefore assess the in vitro activities of micafungin against 1,099 molecularly identified isolates belonging to 5 common and 20 rare Candida species with the EUCAST, CLSI, and Etest methods, measuring both intralaboratory agreement (in one lab) and interlaboratory agreement (between two labs).

MATERIALS AND METHODS

Isolates.

A total of 1,099 Candida isolates were collected from 871 patients with no prior echinocandin exposure who entered into clinical trials of invasive and esophageal candidiasis from 2002 to 2004. The isolates were all identified using molecular techniques, including amplified fragment length polymorphism (AFLP) and internal transcribed spacer (ITS) analysis, as described elsewhere (9). They included 584 Candida albicans, 180 C. tropicalis, 122 C. parapsilosis, 86 C. glabrata, and 30 C. krusei isolates, as well as 97 isolates belonging to other Candida species (20 C. guilliermondii, 15 C. orthopsilosis, 11 C. lusitaniae, 8 C. dubliniensis, 7 C. rugosa, 7 C. kefyr, 6 C. pelliculosa, 5 C. fabianii, 3 C. lipolytica, 3 C. metapsilosis, 2 C. utilis, 2 C. fermentati, 2 C. intermedia, 1 C. inconspicua, 1 C. pararugosa, 1 C. famata, 1 C. palmioleophila, 1 C. xestobii, and 1 C. viswanathii isolate). The quality control (QC) strains C. parapsilosis ATCC 22019 and C. krusei ATCC 6258 were used in each experiment.

Susceptibility testing.

In vitro testing of susceptibility to the antifungal micafungin was performed according to the EUCAST method (EDef 7.2) and the CLSI M27-A3 method (4, 10). The Etest (bioMérieux, Solna, Sweden) was performed according to the manufacturer's instructions and was read after 24 h and 48 h.

Analysis.

Micafungin MICs were determined by each method in two different laboratories for all 1,099 isolates. The intralaboratory agreement among the three methods was calculated for a selection of 200 isolates (50 C. albicans, 40 C. parapsilosis, 40 C. glabrata, 40 C. tropicalis, and 30 C. krusei isolates), which were tested in parallel by each method. The median MIC and the range of MICs were calculated for each species separately and for all isolates together in both the intralaboratory and the interlaboratory comparison. The percentages of susceptible (S), intermediate (I), and resistant (R) isolates were calculated for each method and for the five common species C. albicans, C. parapsilosis, C. glabrata, C. tropicalis, and C. krusei using the EUCAST and CLSI breakpoints. For C. tropicalis and C. krusei, for which no EUCAST breakpoints have been determined, the epidemiological cutoff values of 0.064 and 0.25 mg/liter, respectively, were used. For the Etest, given the lack of method-specific breakpoints, the susceptibility categories were determined using either CLSI or EUCAST breakpoints solely for comparison, and the essential agreement rates between the Etest and each of the two reference methods were also included in the analysis.

To compare the methods, MIC values for each strain were transformed by taking the log2. For a proper comparison, Etest values were rounded to the next highest value in a 2-fold dilution range. The differences were assessed statistically with a paired t test adjusted for multiple comparisons using Bonferroni's adjustment. The t test assessed whether the differences observed between MICs were systematic or were due to random error. In order to assess the microbiological significance of these differences, the percentage of isolates with >2 2-fold differences was calculated. Finally, in order to assess the potential clinical significance of these differences, the percentages of minor, major, and very major errors were calculated as the percentage of isolates classified either S or R with one method and I with the reference comparator method (or vice versa), R with one method and S with the reference comparator method, and S with one method and R with the reference comparator method, respectively. The reference method compared to the Etest method was either the EUCAST or the CLSI method; the reference method compared to the EUCAST method was the CLSI method. For the Etest method, analysis was performed for 24-h and 48-h results.

RESULTS

Tables 1 and and22 show the micafungin MIC data for Candida species for each method and the intra- and interlaboratory agreement, respectively. The median micafungin MICs were ranked from low to high as follows: C. albicans, C. glabrata, C. tropicalis, C. krusei, C. parapsilosis. This order was more pronounced with EUCAST (0.004, 0.016, 0.031, 0.125, and 2 mg/liter, respectively) than with the CLSI and Etest methods. The median MICs for the other Candida species were close to the C. krusei MICs. Table 3 shows the MICs of rare Candida species, with the highest MICs (EUCAST MICs, >0.25 mg/liter) observed for C. guilliermondii, C. lipolytica, C. orthopsilosis, C. metapsilosis, C. fermentati, and C. xestobii, followed by C. lusitaniae, C. rugosa, and C. pararugosa (EUCAST MICs, 0.125 to 0.25 mg/liter), whereas the other Candida species had lower MICs (EUCAST MICs, <0.125 mg/liter). Similar differences were found with the CLSI and Etest methods.

TABLE 1
Intralaboratory agreement between test methodsa
TABLE 2
Interlaboratory agreement between test methodsa
TABLE 3
In vitro susceptibilities of rare Candida species to micafungin

Intralaboratory agreement.

In the intralaboratory comparison, there were statistically significant differences between the EUCAST and CLSI methods, which were most pronounced for C. albicans and C. parapsilosis, for which average EUCAST MICs were 1 2-fold dilution higher than average CLSI MICs, whereas for the other Candida species, the average differences were <0.5 2-fold dilution (Table 1). However, <4% of those differences were >2 2-fold dilutions. EUCAST MICs were ca. 1 dilution higher than Etest method MICs for the non-C. albicans species. In contrast, MICs for non-C. albicans species by the CLSI method were lower than those obtained by the Etest method after 24 h, whereas the opposite was observed for C. albicans. However, <6% of those differences were >2 2-fold dilutions.

Interlaboratory agreement.

The differences found in the intralaboratory comparison were consistent and enlarged in the interlaboratory comparison (Table 2). Overall, the average MICs were 1 to 2 2-fold dilutions higher for the EUCAST method than for the CLSI method, with 19% of the isolates demonstrating differences of >2 2-fold dilutions. This percentage ranged from 0% for C. krusei to 31% for C. tropicalis. With the exception of C. albicans, where similar MICs were found, the average EUCAST MICs were 0.9 to 1.7 dilutions higher than the Etest MICs, particularly after 24 h. However, <7% of those differences were >2 2-fold dilutions. Compared to the CLSI method, the Etest yielded higher MICs for C. albicans and C. tropicalis, with 8% and 11% of the isolates showing differences of >2 2-fold dilutions after 24 h and 48 h, respectively. Of note are the differences for C. tropicalis and rare Candida spp., where 15 to 20% of the differences were >2 2-fold dilutions.

Categorical agreement.

Overall, most isolates were susceptible (>98%) with each method and breakpoint, with the exception of C. parapsilosis and EUCAST, where most isolates were intermediate because of the wide range of the intermediate susceptibility breakpoint (0.004 to 2 mg/liter). More resistant isolates were detected with EUCAST breakpoints (1 to 2%) than with CLSI breakpoints (0%). The resistant isolates belonged to C. albicans, C. tropicalis, and C. parapsilosis. Table 4 shows the percentages of categorical agreement between the methods. Overall, the percentage of agreement between methods was >97%, with 1 to 2% very major errors between the EUCAST and CLSI methods. The same error rates were found with Etest when EUCAST breakpoints were applied. Between the CLSI and Etest methods, 1 to 2% minor errors and no very major errors were found.

TABLE 4
Categorical agreement between the different methods

DISCUSSION

Micafungin demonstrated potent in vitro activity against most Candida species except C. parapsilosis, followed by C. krusei, among the common Candida species and C. guilliermondii, C. lipolytica, C. orthopsilosis, C. metapsilosis, C. fermentati, and C. xestobii, followed by C. lusitaniae, C. rugosa, and C. pararugosa, among the rare Candida species. Overall, the EUCAST method resulted in MICs 1 to 2 dilutions higher than the CLSI and Etest MICs. These differences were more pronounced when tests were performed in different labs, with 19% of all isolates having MIC differences of >2 2-fold dilutions. The smallest differences were found for C. krusei and the highest with C. tropicalis. Although for C. albicans, micafungin MICs were similar between the EUCAST and the Etest methods, for most Candida species, the MICs with the EUCAST method were around 1 dilution higher than that with the Etest method. Overall, the CLSI method resulted in lower MICs than the Etest method, with 11% of all isolates demonstrating differences of >2 2-fold dilutions. These differences were pronounced after 48 h. Despite these differences, categorical agreement was excellent (>97%), with only 1 to 2% very major errors between EUCAST and the other two methods; EUCAST detected more resistant isolates.

To our knowledge, this is the largest comparative study of these three methods assessing both intra- and interlaboratory agreement. In a comparative study of the three methods for 133 Candida isolates, the essential agreement (differences within 2 2-fold dilutions) was >90%, with the EUCAST method identifying more resistant strains (6). This is in agreement with the present study, where the EUCAST method detected more resistant strains, and most (>92%) differences in the intralaboratory comparison were within 2 2-fold dilutions for the five common species. However, in the interlaboratory comparison, using a larger collection of isolates belonging to common and rare Candida species, the overall essential agreement dropped to 81% between the EUCAST and CLSI methods. Noticeable were the differences for C. tropicalis and rare Candida species, where 31% and 28% of the differences were >2 2-fold dilutions. The corresponding rates were 17% for C. albicans and 19% for C. glabrata, whereas for C. parapsilosis and C. krusei, these differences were <6%. In another comparative study with 357 Candida isolates, the modal MICs of the EUCAST method were 1 to 2 dilutions lower than the CLSI modal MICs for all five Candida common species, with >94% essential agreement and >91% categorical agreement (7). Unfortunately, a paired comparison was not made to assess whether the EUCAST method consistently resulted in lower micafungin MICs than the CLSI method (6). Finally, in a larger study with 584 Candida isolates belonging to common species, the essential/categorical agreement between the EUCAST and CLSI methods was 93%/>90% for micafungin, with the EUCAST method resulting in modal MICs for C. albicans (n = 251) and C. parapsilosis (n = 224)—but not C. tropicalis (n = 46), C. glabrata (n = 37), and C. krusei (n = 11)—that were 1 2-fold-dilution higher and detecting more resistant isolates than the CLSI method (2.7% versus 1.5%, respectively), in agreement with our study (11).

When the Etest method was compared with the EUCAST method for micafungin against 160 Candida isolates, the MIC90 with the EUCAST method was 2 dilutions higher than the MIC90 with the Etest method after 24 h, whereas the opposite was observed with the Etest after 48 h, in agreement with the present findings (12). However, most (>93%) differences between the EUCAST and Etest methods were within 2 2-fold dilutions in the present study. In another intralaboratory study, the Etest method gave MICs similar to those with the EUCAST and CLSI methods; the only remarkable difference was the 1-to-2 2-fold dilution-lower modal MICs for C. parapsilosis and C. guilliermondii with the Etest method than with the other methods (6). In the present study, the average Etest MIC for C. parapsilosis was lower than the EUCAST MICs after both 24 and 48 h, whereas compared to the CLSI MICs, lower Etest MICs were found after 24 h. No significant differences was found in the classification of isolates between the Etest and the two reference methods. Because of the lack of specific breakpoints for the Etest and the use of EUCAST and CLSI breakpoints, Etest classification may not reflect the accurate susceptibility for all isolates. The essential (within 2 dilutions) intra- and interlaboratory agreement between the Etest and the two reference methods was >92% and >89%, respectively, with most differences observed between the Etest read at 48 h and the CLSI method for C. tropicalis and other Candida species. A recently published multicenter study showed >90% essential and categorical agreement between Etest and EUCAST testing of the susceptibilities of 933 Candida isolates to micafungin (13).

Few studies have included such a large collection of isolates, including rare Candida species for which in vitro susceptibility data are limited. The higher MICs (EUCAST MICs, >0.25 mg/liter) for the C. parapsilosis complex and C. guilliermondii have been described previously (6, 11, 14). The present study shows also that C. lipolytica, C. fermentati, and C. xestobii also had similarly high MICs (15). Reduced susceptibility to micafungin was found for C. krusei, C. lusitaniae, C. rugosa, and C. pararugosa (EUCAST MICs, 0.125 to 0.25 mg/liter), whereas for all the other Candida species, lower MICs were found.

A limitation of the present study is the fact that most of the isolates were susceptible, since the collection of isolates came from the first clinical trials with micafungin, where the prevalence of echinocandin resistance was low. Therefore, no information is available for the performance of each test at detecting resistant mutants.

Thus, although the three methods tend to give similar results for common Candida species in intralaboratory comparisons, testing larger collections of isolates of common and rare species and in different centers may result in differences of >2 2-fold dilutions, as we found in the present study. In vitro conditions and interlaboratory experimental variation are well known to have a major impact on antifungal susceptibility testing (16). The error rate for the classification of isolates was very low, <2%, using either the EUCAST or the CLSI breakpoints. Low error rates were previously found between the three methods for common Candida species, except for C. glabrata, where 6 to 9% very major errors were found using the epidemiological cutoff values (6). Overall, the EUCAST method consistently gave MICs 1 to 2 2-fold dilutions higher, whereas the CLSI resulted in lower MICs, with the Etest MICs lying between the EUCAST and CLSI MICs. These differences had only a minor impact on the final classification of the isolates.

Funding Statement

Part of this study was sponsored by an unrestricted grant from Astellas Pharma Europe, Inc.

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