Statistical Analyses of Correlation between Fluconazole MICs for Candida spp. Assessed by Standard Methods Set Forth by the European Committee on Antimicrobial Susceptibility Testing (E.Dis. 7.1) and CLSI (M27-A2)

The European Committee on Antimicrobial Susceptibility Testing(EUCAST) Subcommittee on Antifungal Susceptibility Testing recentlypublished a standard for determining the susceptibility of fermentativeyeasts to antifungals. From the beginning, the EUCAST and itsNorth American counterpart, the CLSI, decided to work togetherin order to establish common standards. As part of this exercise,the susceptibility of a set of 475 yeast isolates was testedby both standards. The intraclass correlation coefficient andthe equations defining the linear regression between both methodswere estimated. Both methods produced very similar results,with an intraclass correlation coefficient of 0.954 (0.945 to0.962), although linear regression analysis shows that the EUCASTstandard resulted in slightly lower MICs. There were only eightisolates showing at least four twofold dilution MIC differencesbetween both standards. After 24 h of incubation, the MICs obtainedby the CLSI method were equivalent to those obtained by theEUCAST standard. In summary, both methods produce very similarMICs, indicating that methodology does not pose any obstacleto obtaining uniform standards for antifungal susceptibilitytesting of yeasts.

INTRODUCTION

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INTRODUCTION

The Subcommittee on Antifungal Susceptibility Testing (AFST)of the European Committee on Antimicrobial Susceptibility Testing(EUCAST) has published a standard for the determination of antifungalsusceptibility testing of fermentative yeasts (7). Hitherto,only the M27-A2 reference method for broth dilution antifungalsusceptibility testing of yeasts issued by the CLSI (formerlyNCCLS) was available (4). Besides being established to devisea standard European methodology for antifungal susceptibilitytesting, determine breakpoints for antifungal drugs, and establishexpert rules for interpreting these antimicrobial susceptibilitytests, the AFST also set out to produce results concordant withthose obtained using the CLSI method. Moreover, the EUCAST andCLSI opted to work together with the aim of establishing commonstandards.

The standards of the EUCAST AFST and CLSI for determining theMICs of yeasts to antifungal drugs differ in several importantrespects (Table 1) and might be expected to result in majordifferences between the MICs generated using each method forthe same strains. To investigate this, a series of Candida specieswere tested by both methods to determine the relationship betweenthe MICs obtained and to identify any discrepancies that mighthave occurred.

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TABLE 1. Differences between the EUCAST AFST and CLSI M27-A2 yeast standards

MATERIALS AND METHODS

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MATERIALS AND METHODS

Microorganisms. A set of 475 distinct clinical isolates containing 149 isolatesof Candida albicans, 86 of Candida glabrata, 73 of Candida krusei,87 of Candida parapsilosis, and 80 of Candida tropicalis wasselected.

Antifungal susceptibility testing. The methods described in CLSI M27-A2 (4) and EUCAST E.Dis. 7.1(7) were followed strictly for testing the susceptibility tofluconazole. Strains were tested once by both methods at thesame time. Candida krusei ATCC 6258 and C. parapsilosis ATCC22019 were included for quality control. MICs were read at 24h for the EUCAST AFST method and at 48 h for the CLSI M27-A2method.

Statistical analysis. Statistical analysis was done using SPSS, version 13.0 (SPSS,S.L. Madrid, Spain).

MIC values were transformed to log₂. Linear regression analysisfor both methods was done to test the linearity of the relationshipbetween the CLSI M27-A2 and EUCAST AFST MICs. A two-way randomeffect model was utilized to calculate the intraclass correlationcoefficient (ICC) with a confidence interval of 95% using thefollowing equation: ICC = (group mean square – error meansquare)/(group mean square + error mean square) (3). The ICChas a maximum value of 1 if there is a perfect correlation anda minimum value of –1 if there is a complete absence ofcorrelation.

A difference of at least four twofold dilutions was considereda major discrepancy between the EUCAST AFST and CLSI M27-A2methods.

RESULTS

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RESULTS

The set of 475 strains was used for defining the mathematicalequivalencies between the EUCAST and CLSI methods. The regressionline between MICs obtained by each method showed that the MICswere generally equivalent (Fig. 1). Mean MICs generated by bothmethods were equivalent up to a mean MIC of 2 mg/liter (Table2), but thereafter, MICs generated by the CLSI M27-A2 methodwere twofold higher than those generated by the EUCAST AFSTmethod.

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FIG. 1. Regression line of log₂ MICs obtained by the EUCAST E.Dis 7.1 and CLSI M27-A2 methods.

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TABLE 2. Equivalencies between MICs obtained by the CLSI M27-A2 and EUCAST E.Dis 7.1 AFST methods

There were eight isolates that showed a major discrepancy (Table3). Four isolates of C. tropicalis and one isolate of C. albicansexhibited high MICs by the CLSI M27-A2 method and low MICs bythe EUCAST AFST method. However, the MICs obtained by the CLSIM27-A2 method at 24 h were within the range of those obtainedby the EUCAST M27-A2 method. The remaining three C. albicansisolates with discrepant MICs exhibited high MICs by the EUCASTAFST method and low MICs by the CLSI M27-A2 method.

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TABLE 3. Major MIC discrepancies between the EUCAST E.Dis 7.1 and CLSI M27-A2 methods

ICCs were above 0.83 in every case (Table 4). The overall ICCwas 0.954 (95% confidence interval, 0.945 to 0.962). The slopeand intercept values indicate that the CLSI M27-A2 method resultsin slightly higher MICs than the EUCAST AFST method.

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TABLE 4. ICCs and 95% confidence intervals for fluconazole MICs obtained by the EUCAST E.Dis 7.1 and CLSI M27-A2 methods

DISCUSSION

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DISCUSSION

The results of this study showed there were few major differencesbetween the MICs resulting from the CLSI M27-A2 and EUCAST AFSTmethods. Not only did the regression analysis indicate closeproximity of the MICs generated by each method but the ICCsconfirmed this. Moreover, the relationship held true for valuesof up to 2 mg/liter. Thereafter, mean MICs generated by theCLSI M27-A2 method were twice those resulting from the EUCASTAFST method. Major discrepancies were found only for 8 isolates(1.6%) of the 475 tested.

These results were not expected given the differences betweenthe two methods in terms of inoculum, incubation time, and concentrationof glucose in the medium. The simplest explanation may wellbe that the extra glucose and higher inoculum allow sufficientgrowth for reading the MICs after 24 h rather than having towait an extra 24 h (8). Besides assuming that a final densityof 10⁸ CFU/ml is needed for visible growth, that a lag periodof 4 h occurs in the medium, and that a generation time of 120min is achieved under these conditions, the EUCAST AFST methodwith the 100-fold-higher inoculum would patently reach thisthreshold well ahead of the CLSI M27-A2 method. It has previouslybeen documented that a higher inoculum size significantly shortenedthe length of the lag phase and that the supplementation ofglucose increased the growth of Candida spp. Thus, this medium-inoculumcombination shortened the lag phase and yielded elevated opticaldensities after 24 h (2). Whatever the explanation, these resultsare encouraging as they suggest that there is no major differencebetween the two methods except that the EUCAST AFST method isquicker. Moreover, it would appear that the MICs generated byeither method are equivalent up to 2 mg/liter, that is, withinthe susceptibility range for clinical success. That MICs of4 mg/liter and higher generated by the CLSI M27-A2 method tendto be twofold higher than those obtained with the EUCAST AFSTmethod may not pose any real difficulties since this differenceis within the acceptable range.

Of the eight isolates showing significantly discrepant MICs,five showed high MICs with the CLSI M27-A2 method and low MICswith the EUCAST AFST method. Fortunately, the results of MICsobtained after 24 h with the CLSI M27-A2 method were concordantwith those generated with the EUCAST AFST method. Two investigationsof a murine model of invasive candidosis of several isolateswith low MICs at 24 h and high MICs at 48 h (1, 6) concludedthat the MICs read at 24 h for a 50% reduction in growth correlatedwith the in vivo response, i.e., under conditions adopted bythe EUCAST AFST method (7). In addition, Revankar et al. showedthat oropharyngeal candidosis caused by strains susceptibleat 24 h and resistant at 48 h (significant trailing growth)responded to doses of fluconazole as low as 100 mg/day (5).

In summary, the CLSI M27-A2 and EUCAST AFST standards for yeastsresult in essentially equivalent MICs. This would suggest thatthe method for determining the MICs will not account for anydifferences that might be found between the breakpoints setby the CLSI and those eventually adopted by the EUCAST AFST.

FOOTNOTES

* Corresponding author. Mailing address: Servicio de Micología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Ctra. Majadahonda Pozuelo km 2, 28220 Majadahonda, Madrid, Spain. Phone: 34 91 822 3919. Fax: 34 91 509 7966. E-mail: juanl.rodriguez-tudela{at}isciii.es.

Published ahead of print on 8 November 2006.

REFERENCES

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