No abstract

Both intrinsic and acquired resistance to amphotericin B have been documented for Candida lusitaniae. Amphotericin B remains the drug of choice for many critical fungal infections, and the detection of resistance is essential to monitor treatment effectively. The limitations of the National Committee for Clinical Laboratory Standards (NCCLS) reference methodology for detection of amphotericin B resistance are well documented, and several alternative methods have been proposed. Etest assays with RPMI and antibiotic medium 3 (AM3) agar were compared to the NCCLS M27-A broth macrodilution method using AM3 for amphotericin B resistance testing with 49 clinical isolates of C. lusitaniae. The panel included nine isolates with known or presumed resistance to amphotericin B on the basis of in vivo and/or in vitro data. The distribution of amphotericin B MICs by Etest with RPMI ranged from 0.032 to 16 g/ml and was bimodal. All of the putatively resistant isolates were inhibited by amphotericin B at >0.38 g/ml and could be categorized as resistant using this breakpoint. Etest with AM3 yielded a broader amphotericin B MIC range (0.047 to 32 g/ml), and there were six putatively resistant isolates for which MICs were >1 g/ml. The separation of putatively susceptible and resistant isolates was less obvious. Broth macrodilution with AM3 generated a unimodal distribution of MICs (ranging from 0.032 to 2 g/ml) and failed to discriminate most of the putatively resistant isolates at both 24 and 48 h. Etest using RPMI and, to a lesser extent, using AM3 provided better discrimination between amphotericin B-resistant and -susceptible isolates of C. lusitaniae.Amphotericin B is the drug of choice for many systemic fungal infections (7). Although rare, treatment failures associated with resistance to amphotericin B or resistance to amphotericin B and azoles in both immunocompromised and immunocompetent patients have been documented (3,13,18). Among the non-Candida albicans species, Candida lusitaniae is of special interest, owing to its uncommon susceptibility pattern (1, 5, 9). Rapidly acquired resistance to amphotericin B has been described or suspected, and some strains of C. lusitaniae may be intrinsically resistant (6, 15). The detection of resistance to amphotericin B is essential for treatment of C. lusitaniae-associated infections. In the past 10 years, there have been major advances in antifungal susceptibility testing, as illustrated by several methodology documents published by the National Committee for Clinical Laboratory Standards (NCCLS) (11). Although standardized NCCLS methods and MIC interpretive breakpoints are now available for azole and flucytosine susceptibility testing of yeasts, amphotericin B testing is still under investigation (4, 11). The methodology initially proposed by the NCCLS, i.e., a broth macrodilution procedure using RPMI 1640 medium, did not consistently detect amphotericin B-resistant isolates, nor does the microdilution format (17). Proposed alternative media and methods include the use of antibi...

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.

This study is registered on ClinicalTrials.gov, number NCT01613365.

The sterol and fatty acid compositions of four amphotericin B-resistant and of two amphotericin Bsusceptible Candida lusitaniae clinical isolates were determined. A flow cytofluorometric susceptibility test (FCST) with a membrane potential-sensitive cationic dye was used as a complement to the conventional method for selecting the isolates. Compared to susceptible isolates, resistant ones showed a greatly reduced ergosterol content and changes in sterol composition, consistent with a defect in ⌬837 isomerase. Within each group, no correlation between the sterol or fatty acid pattern or composition and both the degree of in vitro susceptibility and FCST MIC was found.