In vitro evaluation of various antifungal agents alone and in combination by using an automatic turbidimetric system combined with viable count determinations

Abstract

A new method combining automatic turbidimetry and sequential viable count determinations was developed to evaluate the in vitro activity of various antifungal agents alone and in combination against three clinical isolates of Candida spp. (two Candida albicans and one C. tropicalis) at two inocula (105 and 106 CFU/ml). Specific parameters were derived from the time-kill curves: the maximal rate of killing, the lowest biomass, and the overnight biomass. Their intra-assay and between-assay reproducibilities were high, with respective standard deviations of 0.4 and 0.25 to 1.4 log CFU/ml. Amphotericin B alone showed a linear relationship between rate of killing or lowest biomass and the log of concentration from 0.03 to 4 mg/liter that was similar for the three strains tested. 5-Fluorocytosine (flucytosine) alone showed a dose-related reduction of overnight biomass for concentrations up to 8 mg/liter with no further increase at higher concentrations for one strain of C. albicans and a paradoxical decrease for one strain of C. tropicalis. Ketoconazole alone was found to be only fungistatic with no increased activity at concentrations up to 16 mg/liter. Amphotericin B plus flucytosine interacted synergistically in 46 to 60% of the combinations tested against C. tropicalis depending on the initial inoculum. Indifference was observed for the two strains of C. albicans. Amphotericin B or flucytosine plus ketoconazole was usually indifferent against the three tested strains.

Discovery of a Novel Dibromoquinoline Compound Exhibiting Potent Antifungal and Antivirulence Activity That Targets Metal Ion Homeostasis

“…Compound 4b exhibited a fungistatic effect against C. albicans , as no reduction in fungal CFU was observed over a 24 hour timeframe. This matched the behavior observed with 5-Fluorocytosine, a known fungistatic antifungal drug, against C. albicans 24 . At 10 × MIC, 4b and 5-Fluorocytosine did not generate a three-log 10 reduction in fungal CFU, indicating that compound 4b is a fungistatic agent (particularly against C. albicans ).…”

New in vitro assay based on glucose consumption for determining intraconazole and amphotericin B activities against Aspergillus fumigatus

“…Another method is determination of intracellular ATP dosage (13), which allows fungal biomass quantification. A combination of different techniques is also very efficient; drug evaluation is possible not only by measuring MICs but also by associating turbidimetric measurements with the number of living cells after treatment (39). Intracellular organelle metabolism can also be used as an indirect method of drug evaluation, and tetrazolium salt reduction by mitochondria allows for a delicate analysis (32).…”

“…A range of dilutions between 462 and 2.31 pug/ml was prepared from this stock solution. All dilutions were filtered on 0.22-pum-poresize Millex-GV filters (Millipore, Molshein, France) and were conserved at -20°C for no longer than 3 months before use as described previously (23,26,39). …”

Antifungal Susceptibility Testing

“…Third, the relevance of MLCs rather than MICs will need to be evaluated, and there will likely be many technical issues that must be resolved before a reproducible method for MLC determination is available. Fourth, increasingly detailed studies of the mechanisms of antifungal resistance of selected isolates have become available (65,76,77,89,147), and such isolates should prove to be useful for further validating any MIC methodology. Finally, all of these studies may benefit from the application of molecular genetic analysis.…”

“…Such studies can be implemented as kinetic turbidimetric and time-kill curves or as determinations of minimum lethal concentrations (MLCs) (147,149). A few reports have suggested that MLCs may be more important than MICs in predicting in vivo resistance (24,83,149).…”