Department of Microbiology and Infectious Diseases, Hôpital Maisonneuve-Rosemont, 5415 Boulevard LAssomption, Montréal, Québec, Canada H1T 2M4
Sir,
The activity of antifungal agents in vitro may vary according to procedural variations such as culture media, addition of supplements, incubation duration and endpoint determinations. The appropriate methods to assess the activity in vitro of the new candin antifungals have not been clearly defined. We assessed the influence of the addition of supplemental 2% glucose (2%G) to the culture medium on the activity of caspofungin in vitro against clinically significant Candida isolates.
A total of 50 isolates of Candida species recovered between 1996 and 2000 from blood cultures of cancer patients treated at Hôpital Maisonneuve-Rosemont, Montréal, Québec, Canada (HMR) were selected for testing. Antifungal susceptibility testing was carried out by a broth microdilution method according to the procedures described by the NCCLS.1 Standard caspofungin antifungal powder was supplied by Merck & Co. (Whitehouse Station, NJ, USA). Stock solutions were prepared in water. Serial two-fold dilutions were made in 2%G supplemented and non-supplemented RPMI 1640 medium (Gibco-BRL) buffered to pH 7.0 with 0.165 M MOPS buffer (Sigma) and 2%G supplemented and non-supplemented antibiotic medium 3 (AM3) (Difco Laboratories, Detroit, MI, USA). The final concentration of the solvent did not exceed 1% in any of the wells, with the final concentrations of caspofungin ranging from 0.006 to 64 mg/L. Drug-free and yeast-free controls were included. The trays were incubated in air at 35°C and MIC endpoints were read after 48 h of incubation. Following incubation, the trays were visually examined and the growth in each well was compared with that of the control (drug-free) well. The MICs were defined as the lowest concentration that resulted in a prominent decrease in turbidity compared with that of growth-control wells, using the turbidity numerical score proposed by the NCCLS.1 Candida parapsilosis ATCC 22019, Candida krusei ATCC 6258 and a fluconazole-resistant Candida albicans were used for quality control.
The effect of supplemental 2%G on RPMI-MOPS and AM3 after various incubation periods is shown in Table 1. Addition of 2%G to either RPMI-MOPS or AM3 had little effect on the observed MICs. Similarly, the length of incubation did not significantly influence the geometric mean MICs. After 48 h of incubation, MIC values were one- to two-fold higher than the 24 h values. Caspofungin, however, appears to be more potent in AM3, with geometric mean MICs four- to seven-fold lower in AM3 than in RPMI-MOPS. Test conditions have been found to have significant effects on the activity in vitro of azole and triazole antifungals.2 However, with the candin antifungals, the impact of test conditions is unclear. Krishnarao & Galgiani3 recently analysed the test conditions on the activities in vitro of caspofungin and LY303366 against yeast isolates and failed to identify any significant impact. In contrast, several authors showed that echinocandins LY303366 and cilofungin appear to be considerably more potent against yeasts when tested in AM3 compared with RPMI-MOPS.4,5 In the present study, we have made similar observations, with caspofungin showing an increased activity in AM3 when compared with RPMI-MOPS. However, the discrepancies between study results might have been caused by the testing systems used in the different studies; like Pfaller et al.,4 we used a broth microdilution system, whereas in their study, Krishnarao & Galgiani3 used a broth macrodilution system.
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In conclusion, AM3 appears to increase the activity in vitro of caspofungin against clinical Candida isolates. Addition of 2%G to either RPMI-MOPS or AM3 had little impact on the observed MICs, and in general the MICs observed after 48 h of incubation did not vary from those obtained after 24 h of incubation.
Acknowledgements
We thank Merck and Co. for providing the standard antifungal powder used in this study.
Footnotes
* Corresponding author. Tel: +1-514-252-3817; Fax: +1-514-252-3898; E-mail: laverdim{at}courrier.umontreal.ca
References
1 . National Committee for Clinical Laboratory Standards. (1997). Reference Method for Broth Dilution Antifungal Susceptibility of Yeasts: Approved Standard M27-A. NCCLS, Wayne, PA, USA.
2 . Galgiani, J. N. & Lewis, M. L. (1997). In vitro studies of the activities of the antifungal triazoles SCH56592 and itraconazole against Candida albicans, Cryptococcus neoformans and other pathogenic yeasts. Antimicrobial Agents and Chemotherapy 41, 1803.[Abstract]
3 . Krishnarao, T. & Galgiani, J. N. (1997). Comparison of the in vitro activities of the echinocandin LY303366, the pneumocandin MK-0991, and fluconazole against Candida species and Cryptococcus neoformans. Antimicrobial Agents and Chemotherapy 41, 195760.[Abstract]
4 . Pfaller, M. A., Messer, S. A. & Coffman, S. (1997). In vitro susceptibilities of clinical yeast isolates to a new echinocandin derivative LY30336, and other antifungal agents. Antimicrobial Agents and Chemotherapy 41, 7636.[Abstract]
5 . Gordee, R. S., Zeckner, D. J., Ellis, L. F., Thakker, L. & Howard, L. C. (1984). In vitro and in vivo anti-Candida activity and toxicology of LY121019. Journal of Antibiotics 37, 105465.[ISI][Medline]
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Cuenca-Estrella, M., Diaz-Guerra, T. M., Mellado, E. & Rodriguez-Tudela, J. L. (2001). Influence of glucose supplementation and inoculum size on growth kinetics and antifungal susceptibility testing of Candida species. Journal of Clinical Microbiology 39, 52532.