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Antimicrobial Agents and Chemotherapy, August 2005, p. 3486-3488, Vol. 49, No. 8
0066-4804/05/$08.00+0 doi:10.1128/AAC.49.8.3486-3488.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Department of Medical Microbiology, University of Debrecen, Debrecen, Hungary,1 Faculty of Health Sciences, University of Debrecen, Debrecen, Hungary,2 Department of Genetics, University of Debrecen, Debrecen, Hungary3
Received 27 January 2005/ Returned for modification 27 March 2005/ Accepted 15 May 2005
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Caspofungin exhibits a rapid fungicidal effect against Candida species, which has been proven by flow cytometry (4). A standard method for the determination of the minimum fungicidal concentration (MFC) has not yet been proposed (10), although Cantón et al. (2) recently described a method which seems reliable to detect the 
99.9% killing rate caused by amphotericin B in the case of Candida species. Similar experiments determining caspofungin MFCs have not yet been performed (10).
C. inconspicua was the sixth most frequent Candida species in our laboratory in 2002 (L. Majoros, G. Kardos, C. Miszti, J. Szabó, and B. Szabó, Abstr. 23rd Int. Spec. Symp. Yeast, abstr. O-6-05, 2003), and due to its decreased susceptibility to fluconazole, it represents a therapeutic problem. The aim of this study was to examine the in vitro efficacy of caspofungin against fluconazole-resistant clinical isolates of C. inconspicua by using the standard BMD method (9) and Etest. In the BMD test, MICs were determined by using partial inhibition and total inhibition endpoint criteria after either 24 or 48 h incubation. We also determined MFCs to C. inconspicua clinical isolates by using the method described by Cantón et al. (2) with modifications (see below).
We used the same 48 clinical isolates of C. inconspicua as in our previous study (8). The identification of isolates was performed as described earlier (7). The testing of isolates was performed in duplicate, and all tests were repeated at least twice with each method. For interpretation, we used the breakpoint proposed by Stone et al. (12): isolates with MICs of >1 µg/ml were considered resistant, and isolates with lower MICs were regarded as susceptible.
BMD. The reference BMD method was performed according to the guidelines of CLSI (formerly NCCLS) (9). Caspofungin (Merck Research Laboratories) was dissolved in sterile distilled water. Stock solutions were diluted with RPMI 1640 medium (with l-glutamine but without bicarbonate) (Sigma), supplemented with glucose (2%), and buffered to pH 7.0 with 0.165 M MOPS (morpholinepropanesulfonic acid) (Sigma). The final concentration range of caspofungin was 0.03 to 8 µg/ml.
Test plates were incubated at 35°C and read visually after 24 and 48 h. We used two endpoint criteria for MIC determination: (i) total inhibition (MICTI), the lowest concentration of caspofungin that yielded no visible growth (a clear well); and (ii) partial inhibition (MICPI), the lowest concentration that produced a prominent decrease in turbidity compared to that of the drug-free control. MICPIs read after 24 h were used as reference MICs. Quality control strains of Candida parapsilosis (ATCC 22019) and C. krusei (ATCC 6258) were included in each test.
Etest. Caspofungin Etest strips (Merck Research Laboratories) were applied to RPMI 1640 agar with 2% glucose and buffered to pH 7.0 with MOPS. The plates were incubated at 35°C and read after 24 and 48 h.
MFC.
For MFC determination, we used the method described by Cantón et al. (2) with the following modifications. BMD was performed using approximately 105 CFU/ml yeast inoculum (3); otherwise, the method was according to the CLSI guidelines (9). The content of each well containing drug concentrations corresponding to and higher than the MICPI read after 24 h was transferred onto drug-free Sabouraud dextrose agar plates. Plates were incubated at 35°C for 48 h. The MFC was defined as the lowest caspofungin concentration that killed 99.9% of the final inoculum (3-log reduction). In a repeated experiment, MICPI determination and transfer to antifungal-free medium were performed after 48 h of incubation.
We summarize our results in Table 1. Caspofungin demonstrated excellent activity (48/48 susceptible) against clinical isolates of C. inconspicua when we used the partial inhibition criterion read at 24 and 48 h (MIC range, 0.06 to 0.25 and 0.12 to 0.5 µg/ml, respectively). MICTIs obtained after both 24- and 48-h incubation tended to be higher (generally with two dilutions) than the MICPI read at 24 h. Our findings are concordant with the results published by Pfaller et al. (11), who demonstrated one- to two-dilutions-higher caspofungin MICs by using the total inhibition endpoint criterion than MICs obtained using partial inhibition in the case of Candida albicans.
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Comparing the MICs obtained with the methods mentioned above to our reference MICs (partial inhibition and evaluation at 24 h), good agreement was observed only with the MICPIs read at 48 h and with the Etest results read at 24 h (Table 1). Comparing MICs obtained with total inhibition endpoint criterion read at 48 h, Etest MICs read at 24 and 48 h showed only 39.6 and 60.4% agreement, respectively. Similarly, Laverdiere et al. (6) and Chryssanthou and Cuenca-Estrella (3) obtained good agreement between the standard BMD method (total inhibition and 48 h incubation) and Etest only when they defined agreement less stringently at ±2 dilutions.
After 24- and 48-h incubations using high inoculum size (105 CFU/ml) and partial inhibition, MICs were the same or one dilution higher than the MICPIs read at 24 h, in accordance with previously described findings (3).
The MFC range of caspofungin after 24 h incubation was 0.12 to 1 µg/ml (90% MFC [MFC90], 0.5 µg/ml). The MFC was equal to 1x or 2x MICPI in the cases of 83.3% (40/48) of the isolates and never exceeded 4x MIC.
After a 48-h incubation, the obtained MFCs were lower (0.12 to 0.25 µg/ml; MFC90, 0.25 µg/ml), apparently due to the longer exposure. MFCs were equal to the MICPIs read at 24 h, with the exception of five isolates, for which MFCs were 2x MICPI.
Agreement between the MICTI at 48 h and MFC at 24 h within ±1 dilution was only 45.8%. MFCs, with the exception of two isolates, did not reach the MICTI read at 48 h. The correlation between the MICTI at 48 h and the MFC at 48 h was similarly poor. These findings suggest that after 24-h incubation, virtually all cells are unviable in the wells showing partial inhibition.
This assumption is supported by the findings of Klepser et al. (5), who demonstrated by using scanning electron microscopy that yeast cells exposed to a concentration greater than the MIC80 of another echinocandin, LY303366, exhibited substantial ultrastructure abnormalities and lack of viability signs. Similarly, we proved by using transmission electron microscopy that in the case of the total inhibition endpoint criterion, the falsely elevated MICs (MIC90, 1 µg/ml) were caused by unviable cells and cell debris (data not shown). Our results, together with the findings mentioned above (5), indicate that differences between the reference MICs and MICTIs read at 48 h are most probably due to dead yeast cells and cell debris rather than living cells.
The good agreement between MICPI read at 24 h and MFCs after 24 and 48 h incubation time and the inconsistency of MFCs and MICTIs suggest that the MICTI read at either 24 h or 48 h overestimates the MIC considerably and can frequently lead to falsely elevated MICs, at least in the case of C. inconspicua. However, Pfaller et al. (11) and Bartizal and Odds (1) similarly reported lower MICPIs than MICTIs in cases of other species, including C. albicans, Candida glabrata, and C. krusei, suggesting that our findings are likely to be applicable to other Candida species as well. To confirm this assumption, further studies are currently going on in our laboratory.
Summarizing our results, we demonstrated good in vitro efficacy of caspofungin against the fluconazole-resistant C. inconspicua strains by using MICPI after a 24-h incubation. Our results also showed that the caspofungin Etest read at 24 h may be a reliable substitute for the BMD, at least in the case of C. inconspicua. We found that MFCs of caspofungin correspond well to MICPIs read after 24-h incubation but not to MICTIs read at either 24 or 48 h. This difference was caused by trailing wells containing mainly unviable cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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