Sensitivity of Candida albicans isolates to caspofungin – comparison of microdilution method and E-test procedure

Introduction
The rate of serious fungal infections caused by Candida species, i.e. candidaemia and candidiasis, has soared lately as a consequence of the increasing number of patients whose immunity is compromised. This problem affects HIV/AIDS people, patients undergoing aggressive therapies for cancer and autoimmune diseases, organ or tissues recipients, and patients in neonatal intensive care units. Although yeasts belonging to non-albicans species have emerged recently as frequent aetiological agents of fungal infections, the main pathogen remains Candida albicans; it can cause either mucosal or systemic diseases [1-3].
Caspofungin is an echinocandin with a novel mode of action – inhibition of (1,3)-b-glucan synthesis. This drug is indicated for treatment of invasive aspergillosis and invasive candidiasis in cases of peritonitis, candidaemia in neutropenic patients, intra-abdominal ulcer, infection of the pleural cavity, oesophageal candidiasis, as well as empirical treatment of patients with fever and neutropenia if fungus infection (Aspergillus or Candida) is highly suspected [1].
Caspofungin exhibits concentration-dependent fungicidal or fungistatic activity against Candida spp. [4]. The broth microdilution method recommended by CLSI (Clinical Laboratory Standards Institute) for assessing in vitro activity of antifungals is the one widely used in laboratories for estimating the potency of caspofungin. However, this procedure is laborious and time-consuming, so an alternative, more practical but reliable method is in demand, e.g. E-test [5-7].
The aim of our study was to (i) estimate in vitro activity of caspofungin against yeasts of C. albicans, (ii) evaluate the agreement between minimal inhibitory concentrations (MICs) of caspofungin obtained by the CLSI reference broth microdilution method and the E-test procedure.

Material and methods
Microorganisms
A collection of 96 C. albicans isolates was tested. They were obtained from the nasopharynx of hospitalised and ambulatory adults and children between 2004 and 2006. The number of patients participating in the study was 565, including 188 adults and 447 children. The throat and nasal swabs were streaked onto Sabouraud dextrose agar with or without chloramphenicol in order to isolate the yeast-like fungi. Identification was carried out using microscopic methods (Gram stain), biochemical microtests API 20C Aux (bioMerieux) and by testing the formation of chlamydospores. All yeast isolates were stored on Sabouraud dextrose agar until the study was performed. Before the experiment, each isolate was passaged onto fresh agar to ensure purity and optimal growth characteristics.
Caspofungin
Standard antifungal powder of caspofungin acetate was examined (Merck & Co., Inc., USA). Stock solution containing 16 mg/ml was prepared in sterile distilled water and stored frozen at –20°C until use. E-test strips with caspofungin were purchased from AB Biodisk, Sweden. Susceptibility testing in vitro.
Broth microdilution method
Broth microdilution testing was performed according to CLSI directions [6]. Serial two-fold dilutions of caspofungin were made in RPMI 1640 medium (Sigma) buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS) buffer (Sigma). Final concentrations of caspofungin ranged from 0.03 to 32 mg/l. Stock inoculum suspensions of yeasts were adjusted to optical density corresponding with 0.5 Mc Farland standard, i.e. 1 to 5 × 106 cells/ml in sterile 0.85% NaCl, and then suitably diluted at first 1 : 100 and then 1 : 20 with RPMI 1640 medium, according to the procedures accepted by CLSI. After 48 h of incubation at 35°C, the MICs were visually evaluated and defined as the lowest drug concentration that showed complete growth inhibition. Drug-free and yeast-free controls were included. All experiments were done in triplicate. Representative data are presented.
E-test procedure
The E-test method was performed according to the manufacturer’s guidelines (AB Biodisk). The strips of caspofungin possess continuous concentration gradients of 0.002 to 32 mg/l. The yeast inocula were prepared in sterile 0.85% NaCl to match the turbidity of 0.5 Mc Farland standard, i.e. 1 to 5 × 106 cells/ml. 90 mm-diameter plates containing RPMI 1640 agar medium supplemented with 2% glucose and MOPS were used. The RPMI agar was inoculated by using a sterile swab dipped in a yeast suspension and the plates were left at room temperature for about 15 min in order to allow moisture to be absorbed completely. Then, an E-test strip was applied onto each plate. The plates were incubated at 35°C for 48 h. Minimal inhibitory concentrations values were read at the point of intersection between inhibition ellipse edge and the scale on the E-test strip. Micro-organisms inside the inhibition zone were not taken into account. All experiments were done in triplicate. Representative data are presented.
Minimal fungicidal concentration
In order to determine the minimal fungicidal concentration (MFC) of caspofungin, 10 µl from each tube from the broth microdilution method that showed thorough growth inhibition, from the last positive one and from the growth control, was streaked onto Sabouraud dextrose agar plates. After 48 h of incubation at 35°C, the MFCs were assessed visually as the lowest drug concentration at which there was no growth. All experiments were done in triplicate. Representative data are presented.
Correlation coefficient
The Kappa test was used to calculate the degree of agreement. We converted our results for statistical analysis requirements and treated the MIC values obtained by both methods within two dilutions as compatible. Kappa coefficients greater than 0.75 represent excellent agreement, coefficients below 0.4 represent poor agreement and coefficients between 0.4 and 0.75 represent fair to good agreement [8].
Quality control
Quality control was performed for broth microdilution and E-test methods by using the CLSI reference strain C. parapsilosis ATCC 22019. The MICs of caspofungin obtained for the quality control isolate (C. parapsilosis ATCC 22019) were 1 mg/l evaluated by the microdilution method and 0.5 mg/l assessed by the E-test strips, i.e. within the control range (0.5-2 mg/l) established by Barry et al. [9].

Results
The MICs of caspofungin for C. albicans isolates evaluated by the microdilution method ranged from 0.06 to 4 mg/l and from 0.047 to 0.5 mg/l when the E-test procedure was applied. Caspofungin inhibited 50 and 90% of the isolates at 0.25 mg/l (MIC50) and 0.5 mg/l (MIC90), respectively, when the CLSI reference method was used. Both MIC50 and MIC90 assessed by the E-test strips were 0.12 mg/l. All examined strains were susceptible to caspofungin. Determining the MIC of caspofungin by means of the microdilution method, the increased turbidity of the fungal inoculum at the highest drug concentrations was recorded for 26 C. albicans strains (27.1% of all isolates tested). This “Eagle effect” in accordance with the literature data was neglected for the MIC readings [10].
The MFC of caspofungin for C. albicans isolates ranged from 0.06 to >16 mg/l with the MFC90 of 1 mg/l. In the course of MFCs determination the correlation between the turbidity at the highest drug concentrations during the MIC reading and the lack of fungicidal effect of caspofungin at these high concentrations was observed. This pheno-menon was ignored where MFCs were estimated. Activity of caspofungin for 2 isolates tested (2.1%) was fungistatic. For 53 C. albicans strains (55.2%) the MFC/MIC ratio was 1, for 30 strains (31.2%) it was 2, and for another 11 isolates (11.5%) MFC = 4 × MIC.
The E-test MIC values were compared with the MICs obtained by the microdilution method (Table I). As the scale of the E-test strip possesses a continuous gradient of the drug concentrations, the MICs in-between twofold dilutions were raised to the next twofold level in order to make them correspondent with the dilution scheme of the CLSI recommended procedure. The percentages of agreement within ±1 and ±2 dilutions between the broth microdilution MICs and MICs assessed by use of E-tests were 49 and 88%, respectively. All the MICs values were read after 48 h of incubation. Kappa coefficient calculated for the MICs outcomes provided by both techniques was 0.88.

Discussion
Candida albicans is still notorious for being a causative agent of fungal infections associated with high morbidity and mortality rates, especially in immunosuppressed patients. Although modern medicine employs a wide range of antifungal agents, mucosal and invasive candidiasis are not always treated successfully due to emerging resistance of the yeasts belonging to Candida spp. [1, 2, 4, 11]. It is known that the isolates of C. albicans obtained from carriers may be useful in predicting drug resistant patterns of invading isolates, since candidiases are usually endogenous in origin [12].
Caspofungin, a member of a novel echinocandin family, appears to be a potent fungicidal or rarely fungistatic antibiotic against all strains of C. albicans tested in our in vitro study. The MICs (MIC90 0.5 and 0.12 mg/l) and MFCs (MFC90 1 mg/l) obtained in this research were comparable to the values presented by other authors [10, 13-15]. For comparison, according to our published [16] and unpublished data, MICs90 of the routinely used antifungal reagents amphotericin B, flucytosine, and fluconazole, for C. albicans isolates were 1, 0.25-0.5, 2-3 mg/l, respectively. These values are in accordance with the outcomes achieved by Marco et al. [17]. A remarkably low MFC/MIC ratio Ł 2 and Ł 4 for successively 83 (86%) and 94 (98%) examined isolates confirms excellent anti-candidal in vitro activity of caspofungin. According to the literature, similar low MFC/MIC ratios were noted for other echinocandins – micafungin and anidulafungin [18, 19]. Taking into account that the mean trough plasma concentration of caspofungin when administered at clinical doses is greater than 1 mg/l [4], 99% of our isolates, i.e. 95 strains, were inhibited and 92%, i.e. 88 strains, were killed within this therapeutically attainable concentration. After all, the clinical correlation between the in vitro and in vivo responses cannot be unmistakably evaluated, because neither the echinocandin breakpoints nor the complete standardisation of the susceptibility testing procedure has been fully established yet [7, 20, 21].
The paradoxical growth of C. albicans isolates in broth at some supra-MIC and supra-MFC concentrations of caspofungin noted during our investigation is known in the literature as the “Eagle effect”. This phenomenon has not been reported for other echinocandins and according to the literature [18, 19] and our unpublished data, it is rare for Candida non-albicans species. Notably, the concentrations of caspofungin at which this paradoxical growth is observed in vitro are within the range achieved in human serum after the recommended dosage. The peak plasma concentration of caspofungin after a single intravenous infusion of 70 mg amounts even to 16 mg/l and the concentrations in some tissues appear to greatly exceed the serum values. Although the clinical relevance of the “Eagle effect” is unknown, a paradoxical effect was not reproducibly demonstrated in vivo in murine systemic models of candidiasis [22-25].
Comparing caspofungin MIC values for our isolates determined by the CLSI reference method with those obtained by E-tests a high similarity of the outcomes within 2 dilutions was noted. The correlation between readings provided by these two procedures was statistically significant and confirmed by Kappa coefficient of 0.88. In general, when a discrepancy was reported between both techniques the E-test MICs were the lower ones. Our data are consistent with the outcomes of other authors [5, 7, 19].
In conclusion, caspofungin seems to be a highly effective antifungal agent against C. albicans, with potent in vitro fungicidal activity towards the majority of the isolates. Therefore, it may constitute an interesting therapeutic option for the treatment of C. albicans infections. In addition, the E-test method proved to be a reliable technique for testing in vitro susceptibility of C. albicans to caspofungin.

Acknowledgments
This research was supported by the grant PW86/07 from Medical in Lublin.

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