In vitro activity of fluconazole and voriconazole against isolates of Candida albicans from patients with haematological malignancies

Corrado Girmeniaa,*, Clelia Tuccinardia, Stefania Santillia, Francesca Mondellob, Monica Monacoa, Antonio Cassoneb and Pietro Martinoa

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We evaluated the activity of fluconazole and voriconazole against 83 Candida albicans isolates from patients with haematological malignancies, comparing the NCCLS microdilution method (M27-A) with a modified method with RPMI–2% glucose and MIC endpoint at 50% inhibition. Both drugs were highly active regardless of the year, the site of isolation of the strain and the test method employed. In several strains isolated during the last few years, trailing growth leading to difficulty in interpretation of the endpoint of the test has been observed for both drugs by the NCCLS method, but not by the modified method. In our experience, azole resistance of C. albicans is still not a clinical problem, however, the emerging phenomenon of the ‘trailing effect’ by the NCCLS method, even though resolvable by technical modifications, seems at least to indicate a reduction in the inhibitory activity of the azoles.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The widespread use of fluconazole has been accompanied by a rising incidence of resistant Candida albicans isolates from late-stage AIDS patients.1 In contrast, the true incidence and clinical impact of fluconazole resistance in cancer patients are controversial issues.2,3 The difficulties in the assessment of the degree of resistance to fluconazole and the other azoles are related to technical problems in the determination of their in vitro activity. The antifungal susceptibility method recommended by the National Committee for Clinical Laboratory Standards (NCCLS) (the M27-A method) still has some limitations.4 When testing azoles, a trailing endpoint, caused by a gradual decrease in growth rather than a distinct endpoint resulting from complete inhibition of growth, can lead to known susceptible strains appearing resistant.5 In view of the above, modifications of the NCCLS method have been proposed.69 We evaluated the susceptibility to fluconazole and voriconazole of 83 C. albicans strains from patients with haematological malignancies, comparing the NCCLS reference microdilution method with a modified NCCLS microdilution method with RPMI–2% glucose, agitation and MIC endpoint at 50% inhibition.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients

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 1997–1998, 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 103–5.0 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 RPMI–2% glucose and MIC endpoint at 50% inhibition

Susceptibility testing was performed in RPMI 1640–glucose (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.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The Table summarizes the in vitro activity of fluconazole and voriconazole against 62 isolates of C. albicans from surveillance cultures determined by the two methods. The 20 strains isolated in 1987 were susceptible to both drugs when tested by the NCCLS method. The MIC at which 90% of the isolates were inhibited (MIC90) was 1 mg/L of fluconazole and 0.125 mg/L of voriconazole, and no trailing growth close to 80% inhibition was observed. For the 42 strains isolated in 1997–1998, the fluconazole MIC90 was 4 mg/L and the voriconazole MIC90 was 0.125 mg/L. A trailing growth close to 80% inhibition was observed in 33% of the isolates for fluconazole and 26% for voriconazole. When tested with the modified method, all the isolates regardless of the year of isolation were highly susceptible to fluconazole and voriconazole. The interpretation of the susceptibility data was easy, as distinct endpoints of growth inhibition were produced without trailing growth close to 50% inhibition.

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 <=0.03–8) 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 <=0.125–2 mg/L), and voriconazole MIC50 and MIC90 results were 0.015 mg/L and 0.03 mg/L, respectively (range <=0.0078–0.03 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 (400–800 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).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The so-called ‘trailing effect’ represents a crucial difficulty in determination of the MIC of azoles, which could lead to the interpretation of erroneously high MICs for susceptible strains. Therefore, modifications to the NCCLS method in terms of incubation time, buffer and glucose concentration, pH, inoculum size and endpoint determination criteria have been proposed.69 The use of RPMI–2% glucose and reading the endpoint at 80% reduction of growth, allowed for the rapid (24 h) and objective determination of the MICs compared with the standard NCCLS method.7 In a multicentre study, agitation before reading and the use of 50% reduction of growth as the endpoint criterion improved intra-laboratory and inter-laboratory agreement and increased the frequency of interpretable MICs.6 We have used a method that is based on RPMI–2% glucose, agitation and 50% growth inhibition as the endpoint criterion. By employing this method we have shown a slight reduction in the fluconazole and voriconazole MICs against most C. albicans isolates compared with those obtained by the standard NCCLS methodology. No trailing growth close to 50% inhibition was observed and results were therefore easy to interpret. Susceptibility of the C. albicans strains, as observed by the modified method, was confirmed in vivo by the efficacy of fluconazole therapy in 10 cases, including that of the bloodstream infection due to isolate 148/1995, which was resistant to fluconazole and voriconazole by the standard NCCLS method, but was susceptible to both drugs when the modified method was used. Our preliminary data support the evidence that modifications of the NCCLS method could reduce the bias of subjective interpretation and therefore lead to improvements in the reproducibility of antifungal susceptibility tests. However, our data seem to show that the ‘trailing effect’ is not only a technical problem related to the method employed. Unlike C. albicans strains isolated before or in the first years of the clinical use of fluconazole, several strains isolated in recent years were characterized by growth patterns that led to difficulty in interpretation of results.

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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Table. In vitro susceptibility of 62 Candida albicans isolates from surveillance cultures NCCLS method (endpoint at 80% inhibition) Modified method (endpoint at 50% inhibition)
 



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Figure. In vitro susceptibility to fluconazole and voriconazole of 21 Candida albicans isolates from blood as determined by the NCCLS reference method.

 

    Notes
 
* Corresponding author. Tel: +39-06-857951; Fax: +39-06-44241984; E-mail: girmenia{at}bce.med.uniroma1.it Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Rex, J. H., Rinaldi, M. G. & Pfaller, M. A. (1995). Resistance of Candida species to fluconazole. Antimicrobial Agents and Chemotherapy 39, 1–8.[Free Full Text]

2 . 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, 734–8.[Abstract/Free Full Text]

3 . 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, 1463–4.[Abstract/Free Full Text]

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, 367–81.[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, 2387–91.[Abstract]

7 . Rodriguez-Tudela, J. L., Berenguer, J., Martinez-Suarez, J. V. & Sanchez, R. (1996). Comparison of a spectrophotometric microdilution method with RPMI–2% 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, 1998–2003.

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, 1473–6.[Abstract]

9 . 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, 1383–6.[Abstract/Free Full Text]

10 . 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, 913–18.[Abstract/Free Full Text]

Received 8 October 1999; returned 25 January 2000; revised 21 February 2000; accepted 22 May 2000