a Dipartimento di Biotecnologie Cellulari ed Ematologia, University La Sapienza, Via Benevento 6, 00161 Rome; b Laboratorio di Batteriologia e Micologia Medica, Istituto Superiore di Sanita, Rome, Italy
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Abstract |
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Introduction |
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Materials and methods |
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C. albicans isolates from patients with haematological malignancies undergoing remission induction chemotherapy or autologous bone marrow transplantation were enrolled in this study. Until 1990, most patients received antifungal prophylaxis with oral amphotericin B suspension (500 mg every 6 h). Between 1990 and 1993 fluconazole was the main agent used in prophylaxis. Based on the evidence that fluconazole, at a dose of 150 mg/day, was as effective as oral amphotericin B in the prevention of fungal infections in leukaemic patients,10 administration of oral amphotericin B prophylaxis has become standard practice in our institution since 1994.
Candida sp. isolates
A total of 83 C. albicans isolates, from 80 patients, were used throughout this study. Twenty and 42 strains were isolated from mucosal surveillance cultures in 1987 and in 19971998, respectively, and 21 strains were isolated from blood during the period from 1991 to February 1998. Two reference strains (C. albicans ATCC 24433 and C. albicans ATCC 76615 ) were included in each set of experiments.
Antifungal agents
Fluconazole (2 mg/mL) and voriconazole (10 mg/mL) were prepared as stock solutions diluted in water. Both drugs were provided by Pfizer Central Research (Sandwich, UK).
NCCLS microdilution reference method
Antifungal agents were diluted with RPMI 1640 medium (Sigma Chemical Co., St Louis, MO) buffered to pH 7.0 with 0.165 M morpholinepropanesulphonic acid (MOPS) buffer (Sigma). Aliquots of 0.1 mL of the drug solutions were dispensed into each well of 96-well microtitre plates. The final concentrations of the antifungal agents ranged from 0.03 to 16 mg/L for voriconazole and 0.125 to 64 mg/L for fluconazole. C. albicans inocula, adjusted to a concentration of 1.0 x 1035.0 x 103 cfu/mL (twice the final inoculum) were added to each well of the microdilution plate. The MICs were determined after 48 h incubation at 35°C. The endpoint definition suggested by the NCCLS document M27-A4 for the microdilution method is "a prominent reduction in turbidity". However, as well as for the macrodilution method, the MIC was defined more precisely as the lowest drug concentration that produced an 80% reduction of growth compared with that of the drug-free growth control.
Modified microdilution method with RPMI2% glucose and MIC endpoint at 50% inhibition
Susceptibility testing was performed in RPMI 1640glucose (final concentration of glucose 20 g/L) medium buffered to pH 7.0 with 0.165 M MOPS buffer. The final concentrations of the antifungal agents ranged from 0.0078 to 4 mg/L for voriconazole and 0.125 to 64 mg/L for fluconazole. The MICs were determined after 24 h incubation at 35°C. Microtitre plates were read after shaking for 3 min at 70 rpm. The MIC was defined as the lowest drug concentration that produced a 50% reduction of growth.
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Results |
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The susceptibilities obtained by the NCCLS method for the 21 C. albicans strains isolated from blood are detailed in the Figure. Fluconazole MIC50 and MIC90 results were both 1 mg/L (range 0.25>64 mg/mL); only isolate 148/1995 was resistant to the drug (MIC > 64 mg/L), and five (24%) strains isolated since 1994 showed trailing growth close to 80% inhibition. Voriconazole was highly active (MIC50 0.03 mg/L; MIC90 0.125 mg/L; range
0.038) against all but the fluconazole-resistant isolate 148/1995 (MIC = 8 mg/L). However, in two (10%) of the 21 bloodstream isolates, the presence of a trailing endpoint close to 80% inhibition was observed. When tested by the modified method, fluconazole MIC50 and MIC90 results were 0.25 mg/L and 0.5 mg/L, respectively (range
0.1252 mg/L), and voriconazole MIC50 and MIC90 results were 0.015 mg/L and 0.03 mg/L, respectively (range
0.00780.03 mg/L). No trailing growth close to 50% inhibition was observed.
Few patients had received fluconazole for prophylaxis or treatment and such exposure did not correlate with reduced susceptibility of subsequent isolates to the azole or difficulty in interpretation of trailing endpoints. Eleven of the 21 patients with candidaemia were treated with iv or oral fluconazole (400800 mg/day). Only one patient died due to disseminated candidosis 2 days after the start of antifungal therapy. The other 10 patients responded to treatment, including the patient who developed a skin infection and septicaemia due to strain 148/1995 at the onset of his acute myeloid leukaemia and before hospital admission. Despite in vitro resistance of the microorganism to fluconazole as determined by the NCCLS reference method (demonstrated retrospectively), he was cured rapidly with fluconazole therapy (800 mg/day, iv).
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Discussion |
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The true clinical impact of fluconazole resistance in cancer patients is not known. In a survey conducted at a large cancer centre,2 a 20% incidence of fluconazoleresistant C. albicans clinical isolates in recent years was associated with increasing fluconazole use, even if an individual association with previous use of the drug was not demonstrated. In contrast, in a recent study of yeast isolates causing bloodstream infections in neutropenic patients, fluconazole resistance was not identified in any of the C. albicans isolates tested.3
In our experience in patients with haematological malignancies, resistance of C. albicans is not a clinical problem. It could be hypothesized that the selective use of fluconazole at our centre could have a role in the low incidence of fluconazole resistance. On the other hand, the increasing problem of the trailing effect observed in susceptibility testing conducted by the NCCLS method, even though technically resolvable, seems at least to indicate a reduction in the in vitro inhibitory activity of fluconazole and to a lesser extent voriconazole. This phenomenon does not appear to be related to the individual use of fluconazole but it could be hypothesized that the general pool of organisms is becoming more fluconazole tolerant.
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Notes |
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References |
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2
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Boschman, C. R., Bodnar, U. R., Tornatore, M. A., Obias, A. A., Noskin, G. A., Englund, K. et al. (1998). Thirteen-year evolution of azole resistance in yeast isolates and prevalence of resistant strains carried by cancer patients at a large medical center. Antimicrobial Agents and Chemotherapy 42, 7348.
3
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Hoban, D. J., Zhanel, G. G. & Karlowsky, J. A. (1999). In vitro susceptibilities of Candida and Cryptococcus neoformans isolates from blood cultures of neutropenic patients. Antimicrobial Agents and Chemotherapy 43, 14634.
4 . National Committee for Clinical Laboratory Standards. (1997). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeast: Approved Standard M27-A. NCCLS, Wayne, PA.
5 . Rex, J. H., Pfaller, M. A., Rinaldi, M. G., Polak, A. & Galgiani, J. N. (1993). Antifungal susceptibility testing. Clinical Microbiology Reviews 6, 36781.[Abstract]
6 . Anaissie, E. J., Paetznick, V. L., Ensign, L. G., Espinel-Ingroff, A., Galgiani, J. N., Hitchcock, C. A. et al. (1996). Microdilution antifungal susceptibility testing of Candida albicans and Cryptococcus neoformans with and without agitation: an eight-centre collaborative study. Antimicrobial Agents and Chemotherapy 40, 238791.[Abstract]
7 . Rodriguez-Tudela, J. L., Berenguer, J., Martinez-Suarez, J. V. & Sanchez, R. (1996). Comparison of a spectrophotometric microdilution method with RPMI2% glucose with the National Committee for Clinical Laboratory Standards reference macrodilution method M27-P for in vitro susceptibility testing of amphotericin B, flucytosine, and fluconazole against Candida albicans. Antimicrobial Agents and Chemotherapy 40, 19982003.
8 . Tornatore, M. A., Noskin, G. A., Hacek, D. M., Obias, A. A. & Peterson, L. R. (1997). Effects of incubation time and buffer concentration on in vitro activities of antifungal agents against Candida albicans. Journal of Clinical Microbiology 35, 14736.[Abstract]
9
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Marr, K. A., Rustad, T. R., Rex, J. H. & White, T. C. (1999). The trailing end point phenotype in antifungal susceptibility testing is pH dependent. Antimicrobial Agents and Chemotherapy 43, 13836.
10
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Menichetti, F., Del Favero, A., Martino, P., Bucaneve, G., Micozzi, A., D'Antonio, D. et al. (1994). Preventing fungal infection in neutropenic patients with acute leukemia: fluconazole compared with oral amphotericin B. The GIMEMA Infection Program. Annals of Internal Medicine 120, 91318.
Received 8 October 1999; returned 25 January 2000; revised 21 February 2000; accepted 22 May 2000