Amphotericin B susceptibility testing of Candida lusitaniae isolates by flow cytofluorometry: comparison with the Etest and the NCCLS broth macrodilution method

A. Favela,*, F. Peyrona, M. De Méob, A. Michel-Nguyenc, J. Carrièred, C. Chastina and P. Reglia

a Laboratoire de Botanique, Cryptogamie et Biologie cellulaire (EA 864) Université de la Méditerranée, Faculté de Pharmacie, 13 385 Marseille Cedex 5 b Laboratoire de Biogénotoxicologie et Mutagénèse environnementale (EA 1784), Université de la Méditerranée, Faculté de Pharmacie, 13 385 Marseille Cedex 5; c Laboratoire de Bactériologie et Parasitologie, CHU Nord, 13 015 Marseille; d Service de Parasitologie-Mycologie, CHU Pitié-Salpêtrière, 75 651 Paris, Cedex 13, France


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A flow cytofluorometric susceptibility test (FCST) was used for rapid determination of the susceptibility of Candida lusitaniae isolates to amphotericin B. The test is based on the decrease in fluorescence intensity of cells stained with 3,3'-dipentyloxacarbocyanine iodide (DiOC5(3)), a membrane potential-sensitive cationic dye, after drug treatment. A total of 58 C. lusitaniae clinical isolates including strains known to be amphotericin B-resistant on the basis of in-vivo and/or in-vitro data were tested. MICs were determined concurrently by the NCCLS broth macrodilution method and the Etest, both with antibiotic medium 3. Regression analysis demonstrated that the data from the FCST and the Etest were better correlated (r = 0.93, n = 59, P < 0.001) than those from the FCST and the NCCLS method (r = 0.63, n = 59, P < 0.001). The FCST readily identified a series of putatively susceptible and resistant isolates. Our study points out the advantages of the flow cytometry approach in antifungal susceptibility testing of yeasts, since speed remains a major problem in conventional tests.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Candida lusitaniae is an emerging opportunistic pathogen; its incidence in nosocomial candidaemia is about 1%. 1 It often causes fatal diseases in severely immunocompromised patients. 2 C. lusitaniae is characterized by its ability to develop resistance to amphotericin B. 3,4 About 25% of the isolates are resistant to flucytosine. 4,5 Since the management of C. lusitaniae infections is difficult, and since amphotericin B remains the drug of choice, a rapid and reliable susceptibility test would be especially useful to monitor antifungal treatment effectively. Antifungal susceptibility testing is a rapidly developing field of research. The standardized method proposed in 1992 by the NCCLS 6 was updated in 1997. 7 Fluconazole and itraconazole interpretative breakpoints for MICs determined by this method have been established. 8 However, amphotericin B susceptibility testing remains problematic. Since the medium proposed initially, namely RPMI, was not reliable in discriminating between amphotericin B-susceptible and -resistant Candida sp. isolates, the antibiotic medium 3 (AM3) was proposed for investigation. 9,10 The Etest, which readily identifies amphotericin B-resistant Candida isolates, has also been proposed as an alternative to the NCCLS reference method. 11 These conventional methods have one major drawback: they are based on visual assessment of yeast growth and results are thus available at best after 24 h incubation.

Flow cytometry, which allows for high-speed, multiparametric analysis of individual cells in suspension, could overcome this drawback. This technique has a broad range of applications, and is currently used in haematology and cancer cytology, for example. Continuing improvements in sensitivity and specificity of instrumentation and techniques and the development of new dyes have led to a wide range of microbiological applications. 12 Flow cytometry for evaluating the activity of antifungal agents was initiated in 1990 by Pore, 13 yet few studies have addressed this approach. 14,15 Flow cytofluorometric susceptibility tests (FCSTs) require specific fluorescent probes suitable for monitoring the interactions between the antifungal agent and a target site of the fungal cell. Recently, our laboratory developed a rapid and accurate FCST using 3,3'-dipentyloxacarbocyanine iodide (DiOC5(3)), a cationic membrane potential-sensitive fluorescent dye, to assess the amphotericin B susceptibility of yeast isolates. 16

The aim of this study was to evaluate this flow cytofluorometric method with a series of C. lusitaniae isolates including some that were known to be amphotericin B-resistant on the basis of in-vivo and/or in-vitro data. 10 The results were compared with those obtained by the NCCLS broth macrodilution method and the Etest method, both with AM3.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Test organisms

A total of 58 C. lusitaniae isolates were tested. Fifty-five isolates were from patients in oncology or intensive care units. They were recovered from upper respiratory tract (28 isolates), mouth (six isolates), stool (seven isolates), urine (five isolates), skin (one isolate), vagina (one isolate) and blood (seven isolates) cultures. They were identified by standard methods 17 and the ID 32C system (BioMérieux, Marcy l'Etoile, France). The 55 isolates included two recent isolates (94-6856 and 6-103) that were putatively resistant to amphotericin B on the basis of clinical data. Three C. lusitaniae isolates were kindly provided by J. Rex, University of Texas Medical School, Houston, TX, USA. One of these (CL 2887) was proven to be resistant to amphotericin B in an animal model of disseminated candidiasis, and the other two (Y533 and Y534) were presumed resistant after in-vitro testing. 10 Candida parapsilosis CL 524, previously identified as being susceptible to amphotericin B in an animal model, was also studied. 10 Isolates were maintained on Sabouraud glucose agar (BioMérieux) slants and stored at 4°C. Before being tested, they were subcultured on Sabouraud glucose agar (BioMérieux) and incubated at 35°C for 24 h. C. parapsilosis ATCC 22019 (MIC = 0.25–1 mg/L) and Candida tropicalis IP 1275-81 (MIC >= 2 mg/L) were included in each series of experiments as internal controls.

Antifungal agent

Amphotericin B (Sigma, St Louis, MO, USA) was dissolved in reagent-grade dimethyl sulphoxide (DMSO; Sigma) at a starting concentration of 12,800 mg/L. The stock solution was stored at -70°C until use. Amphotericin B Etest strips (AB Biodisk, Solna, Sweden) were kept at -20°C until use.

Standard antifungal susceptibility testing

The broth macrodilution susceptibility test was done according to the NCCLS M27-A method 7 with AM3 broth buffered to pH 7.0 (BioMérieux) and supplemented with glucose (2%). 10 The incubation period was 48 h at 35°C. The MIC was defined according to the NCCLS criteria (the MIC endpoint was read as the lowest amphotericin B concentration preventing any discernible growth).

Etest

The Etest was done according to the manufacturer's recommendations with solidified (2% agar) AM3 buffered to pH 7.0 (BioMérieux) and supplemented with glucose (2%). 11 The inoculum suspensions were adjusted spectrophotometrically at 530 nm to match the turbidity of a 0.5 McFarland standard. Agar plates were inoculated with a cotton swab, and they were allowed to dry for 15 min before the Etest strips were applied. The plates were incubated at 35°C and read at 24 and 48 h. The Etest MIC endpoint was the drug concentration at which the inhibition ellipse intercepted the scale on the antifungal strip.

FCST conditions

The conditions for the FCST were as previously described. 16 Briefly, the assay medium was RPMI 1640 with L-glutamine (Sigma) buffered to pH 7.0 with 0.165 M morpholinepropanesulphonic acid (MOPS). Drug dilutions were prepared to ten times the strength of the final concentration with medium as the diluent (e.g. 0.3–160 mg/L). Stock inoculum suspensions were made in sterile 0.85% saline and matched with a 0.5 McFarland standard. The adjusted yeast suspensions were diluted 1:10 with the medium to achieve a final concentration of 4–5 x 10 5 cfu/mL. The final inoculum was added in 0.9 mL volumes to glass tubes containing 0.1 mL of drug dilutions, except the growth control. Final concentrations of amphotericin B ranged from 0.03 to 16 mg/L. Tubes were incubated at 27°C for 30 min. DiOC5(3) (Molecular Probes, Eugene, OR, USA) was then added to all tubes at a final concentration of 0.5 mM. Flow cytofluorometric analysis was initiated 5 min after the dye had been added to the first tube.

Flow cytometry

Samples were run on a FACSort cytometer (Becton Dickinson, Sunnyvale, CA, USA) with a 15 mW, 488 nm argon ion laser, with a Consort 32 workstation combined with Lysis II software. Instrumental parameters were as follows: forward light scatter (FSC, log), sideways light scatter (SSC, log, 304 PMT voltage, 488/10 filter), and green fluorescence (FL1, log, 443 PMT voltage, 530/30 filter). An untreated control of each isolate was sampled first. A gate that excluded cell clusters and debris was adjusted from a cytogram obtained by plotting FSC against SSC. Five thousand cells per sample were analysed, and data were recorded as density plots of DiOC5(3) fluorescence (FL1) versus FSC and as histograms of fluorescence. The geometric mean of fluorescence for the defined population was calculated using Becton Dickinson software.

Study design and statistical analysis

A total of 60 Candida spp. isolates were tested once by the three methods. The same commercial lot of AM3 was used in the Etest and in the NCCLS broth macrodilution method. A set of putatively amphotericin B-resistant and -susceptible isolates was tested in a second experiment to assess the reproducibility of the results. Regarding the FCST, dose– response relationships were determined with a non-linear model: F = ab/(b + C) where F is the mean fluorescence, a and b are coefficients calculated by non-linear regression analysis, and C is the amphotericin B concentration (mg/L). For each isolate, the drug concentration producing an 80% decrease in fluorescence mean relative to the fluorescence mean of the drug-free control (IC80 = 4b) was calculated. The correlation between the IC80 and the M27-A MIC, and that between the IC80 and the Etest MIC, were determined. For each linear model, analysis of variance was performed. All calculations were performed with `Table Curve 2D' software (Jandel Scientific, version 3.0).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Fifty-eight C. lusitaniae clinical isolates and the putatively amphotericin B-susceptible C. parapsilosis 524 were tested for susceptibility to amphotericin B by the flow cytofluorometric method with DiOC5(3). Cell membrane depolarization in amphotericin B-treated cells was evidenced by a decrease in fluorochrome uptake which was dose-dependent. Figure 1shows representative density plots (FL1/FSC) for C. parapsilosis 524 (Figure 1a)and C. lusitaniae 2887 (Figure 1b), two isolates characterized on the basis of in-vivo testing as susceptible and resistant, respectively, to amphotericin B. The effects of two significant concentrations of amphotericin B (0.12 and 8 mg/L), near the respective MICs, are depicted. The MIC ranges and the MICs required to inhibit 50% and 90% of the isolates (MIC50s and MIC90s respectively) by the three test methods are summarized in Table I. For the control organism, C. parapsilosis ATCC 22019, a tight distribution of the MICs, within the expected range, was observed whatever the test method. Resistance to amphotericin B for the control organism C. tropicalis IP 1275-81 was detected with the three test methods, and extremely high Etest and NCCLS MIC values were obtained. This isolate was also consistently identified with the M27-A method using RPMI medium (MIC = 4 mg/L, data not shown). For the clinical isolates, the Etest and the FCST provided ranges of MICs broader than those obtained with the NCCLS broth macrodilution method. The MIC50 and MIC90 were similar for all the test methods. The majority of the isolates were inhibited by amphotericin B at concentrations <=1 mg/L. The regression analysis showed that the correlation between the FCST MICs and the 48 h Etest MICs (r = 0.93, n = 59, P < 0.001; Figure 2) was better than that between FCST MICs and the NCCLS MICs (r = 0.63, n = 59, P < 0.001; data not shown). Identical results were obtained when FCST MICs were plotted against 24 h Etest MICs (r = 0.93, n = 59, P < 0.001; data not shown). As shown in Figure 2 with the Etest and FCST, the resistant isolates were clearly distinct from the other clinical isolates. Table II details results from two separate experiments with the set of putatively amphotericin B-resistant and -susceptible isolates, along with some clinical data. C. lusitaniae 2-367, an isolate whose status was unknown and that exhibited high MICs, is also included in this table. Whatever the test method, the MICs were within one two-fold dilution between the two experiments and they were in agreement with clinical data. For the putatively resistant isolates, the Etest and the FCST provided MICs higher than those obtained by the broth macrodilution method. C. lusitaniae Y534 exhibited the lowest 48 h MICs of the resistant set; the highest MIC value was obtained with the FCST. For the susceptible C. parapsilosis 524, the results of each experiment were highly consistent with the three methods (MICs within 0.12 mg/L ± one dilution) as they were between the two separate experiments.



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Figure 1. Effect of amphotericin B on DiOC 5(3) staining of (a) C. parapsilosis 524 and (b) C. lusitaniae 2887. Fluorescence (FL1) versus forward scatter (FSC) density plot profiles of cells exposed to characteristic amphotericin B concentrations (0.12 mg/L and 8 mg/L) for 30 min.

 

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Table I. Amphotericin B MICs determined by three test methods for 59 clinical isolates of C. lusitaniae and the control organisms
 


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Figure 2. Regression analysis correlating FCST MICs with Etest MICs. (a) All the points. (b) Set of points ranging from 0 to 2 mg/L in the Etest (x axis). The regression statistic is y = 0.65x +0.59, r= 0.93.

 

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Table II. Detection of amphotericin B resistance by three susceptibility tests in two separate experiments
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
While there has been considerable progress in the development of standard, reproducible and clinically relevant methods for antifungal susceptibility testing of yeasts, amphotericin B susceptibility testing remains a poorly documented field. The apparently low incidence of amphotericin B-resistance among Candida spp. isolates has undoubtedly contributed to this situation. Renewed interest has come from the emergence of clinical failure associated with the development of resistance to amphotericin B during therapy, especially with C. lusitaniae, and the possibility of cross-resistance to amphotericin B in Candida albicans isolates resistant to fluconazole. 2,3,4,18 The fungicidal activity of amphotericin B is known to correlate with the physiochemical damage of the cell membrane. 19 A new approach to susceptibility testing consists in monitoring the drug-induced cellular effects by flow cytometry. The present study confirms and extends the results of our preliminary evaluation of the flow cytofluorometric method. 16 FCST with DiOC5(3) allowed rapid evaluation of amphotericin B susceptibility of C. lusitaniae isolates and clear discrimination between amphotericin B-susceptible and -resistant isolates. As previously reported, 11 the Etest using AM3 readily identified resistant isolates, generating MICs higher than those obtained by the broth macrodilution method. Likewise the FCST provided a wide distribution of MICs with high values for these isolates, and the correlation between the two methods was statistically significant. With regard to the conventional methods, our results for three of the putatively resistant isolates (C. lusitaniae 2887, Y533 and Y534) were slightly different from those of other authors. The Etest MICs obtained by Wanger et al. 11 were higher than ours as were, to a lesser extent, the NCCLS MICs obtained by Rex et al. 10 These differences could be explained by lot-to-lot variability of the non-standardized AM3. The impact of this parameter was recently pointed out by Lozano-Chiu et al. 20 For C. tropicalis IP 1275-81, AM3 excessively boosts the MICs and the best agreement was between FCST MICs and MICs obtained by the broth macrodilution method with RPMI medium. The short incubation time involved in the FCST minimizes the effect of the test medium, in addition to allowing early data acquisition and analysis. In summary, when the ability of the NCCLS M27-P method to detect amphotericin B resistance was called into question, it rapidly appeared that an adaptation of the test medium and/or the test format was required. Nevertheless, as mentioned in the NCCLS M27-A protocol, results obtained with AM3 must be carefully interpreted. On the other hand, although reliable Etest results can be obtained after 24 h of incubation, this method is still too time-consuming to monitor therapy effectively in the rare but often dramatic cases in which resistance to amphotericin B is suspected. The present study shows that the DiOC5(3) FCST provides rapid, reliable, and accurate results. Additional studies are needed to improve standardization of this method and to document intra- and inter-laboratory reproducibility.


    Acknowledgments
 
The authors are grateful to A. Datry and P. Bouchara for providing some C. lusitaniae isolates. They thank H. Guiraud-Dauriac for helpful comments and J. Piras for preparing the manuscript.


    Notes
 
* Laboratoire de Botanique, Cryptogamie et Biologie cellulaire, Faculté de Pharmacie, 27 Boulevard J. Moulin, 13 385 Marseille Cedex 5, France. Tel:+33-04-91-83-55-53; Fax:+33-04-91-80-26-12 Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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4 . Pfaller, M. A., Messer, S. A. & Hollis, R. J. (1994).Strain delineation and antifungal susceptibilities of epidemiologically related and unrelated isolates of Candida lusitaniae. Diagnostic Microbiology and Infectious Disease 20, 127–33.[ISI][Medline]

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6 . National Committee for Clinical Laboratory Standards. (1992). Reference Method for Broth Dilution Antifungal Susceptibility Testing for Yeasts: Proposed Standard M27-P. NCCLS, Villanova, PA.

7 . National Committee for Clinical Laboratory Standards. (1997). Reference Method for Broth Dilution Antifungal Susceptibility Testing for Yeasts: Approved Standard M27-A. NCCLS, Wayne, PA.

8 . Rex, J. H., Pfaller, M. A., Galgiani, J. N., Bartlett, M. S., Espinel-Ingroff, A., Ghannoum, M. A. et al. (1997).Development of interpretive breakpoints for antifungal susceptibility testing: conceptual framework and analysis of in vitroin vivo correlation data for fluconazole, itraconazole and Candida infections. Sub-committee for Clinical Laboratory Standards. Clinical Infectious Dieases 24, 235–47.

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20 . Lozano-Chiu, M., Nelson, P. W., Lancaster, M., Pfaller, M. A. & Rex, J. H. (1997).Lot-to-lot variability of antibiotic medium 3 used for testing susceptibility of Candida isolates to amphotericin B. Journal of Clinical Microbiology 35, 270–2.[Abstract]

Received 20 April 1998; returned 15 June 1998; revised 6 July 1998; accepted 17 September 1998