amphotericin-b and 3-3--dipentyl-2-2--oxacarbocyanine

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.

Topics: Amphotericin B; Antifungal Agents; Candida; Carbocyanines; Drug Resistance, Microbial; Flow Cytometry; Fluorescent Dyes; Microbial Sensitivity Tests; Regression Analysis

Flow cytometry was used for measuring the effects of amphotericin B on the membrane of Leishmania infantum strains. The technique was adapted from the rapid flow cytometric membrane potential assay developed by Ordonez and Wehman (Cytometry 22:154-157, 1995) for evaluating antibiotic-susceptibility of Candida species. The study consisted of measuring membrane potential changes induced by amphotericin B in 3 initial strains and 12 laboratory-generated variants adapted to grow with amphotericin B. Results showed that, after 3 h of incubation, amphotericin B induced a dose-related decrease of membrane potential that reached its maximal level at the same concentrations that inhibited parasite growth. These results suggest that the flow cytometric membrane potential assay could be used to assess the susceptibility of Leishmania promastigotes to amphotericin B.

Topics: Amphotericin B; Animals; Antiprotozoal Agents; Carbocyanines; Dose-Response Relationship, Drug; Flow Cytometry; Fluorescent Dyes; Leishmania infantum; Membrane Potentials; Time Factors

The capacity of flow-cytometric techniques to detect drug-specific biochemical targets and side effects in Leishmania infantum promastigotes was estimated by assessing the effects of three antileishmanial drugs (pentamidine, allopurinol, and amphotericin B) on parasite metabolism. Cell cycle and total protein content were estimated by staining cells with propidium iodide and fluorescein isothiocyanate, nonprotein thiols were stained by mercury orange, and membrane potential was measured by the accumulation of 3,3'-dipenthyloxacarbocyanine iodide inside the cell. Results showed that dynamic studies in parasites treated with subtoxic concentrations of drugs allowed the detection of drug-specific targets: pentamidine primarily affected nonprotein thiol contents and DNA synthesis, allopurinol primarily affected intracellular protein contents, and amphotericin B primarily affected membrane potential. Moreover, the assessment of cellular functions in parasites treated with increasing concentrations of drugs certified the capacity of these techniques to establish dose-response curves and to permit the detection of side effects.

Topics: Allopurinol; Amphotericin B; Animals; Antiprotozoal Agents; Carbocyanines; Cell Cycle; Coloring Agents; DNA, Protozoan; Dose-Response Relationship, Drug; Flow Cytometry; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Leishmania infantum; Membrane Potentials; Pentamidine; Phenylmercury Compounds; Propidium; Protozoan Proteins; Sulfhydryl Compounds; Sulfhydryl Reagents