Method for determining the degree of sensitivity of a strain of fungus to an antifungal agent

A method for determining a degree of sensitivity of a population of cells of a strain of fungus capable of being stained by Calcofluor White to an antifungal agent,

• wherein the degree of sensitivity is determined by determining a possible change in a chitin level in cell walls of the population of cells of the strain of fungus, said possible change being determined by comparing a chitin level in cell walls of the population of cells in the presence of the antifungal agent to a chitin level in cells walls of the population of the cells in the absence of the antifungal agent,
• wherein the antifungal agent is capable of provoking a parietal stress that leads to an accumulation of the chitin in the cell walls of the population of cells to compensate for damage induced by the antifungal agent, and
• wherein the degree of sensitivity corresponds to a sensitive phenotype, or a resistant phenotype, or an intermediate phenotype of said strain of fungus with respect to the antifungal agent,
• the method comprising:
• (a) a step of contacting the population of cells of said strain of fungus with a gradient of concentrations of the antifungal agent varying from 0.0009 to 130 ug/ml, at a temperature of from 30 to 35 degrees C., for a period of time less than or equal to 48 h, so as to obtain a mixture of cells of said strain of fungus and antifungal agent;
• (b) a step of adding the fluorescent marker Calcofluor White to the mixture of cells of said strain of fungus and antifungal agent obtained previously, so as to obtain a mixture of cells of said strain of fungus, labelled by Calcofluor White, and antifungal agent;
• (c) a step of quantifying the chitin level, by high-content analysis fluorescence microscopy, of the labelled cells of said strain of fungus in the mixture obtained in the previous step;
• (d) a step of determining the possible change in the chitin level in the mixture of cells of said strain of fungus, labelled by Calcofluor White, and antifungal agent as a function of the concentrations of antifungal agent in the population of cells of said strain of fungus compared to the chitin level of a population of cells of said strain of fungus in the absence of antifungal agent;
• and
• in the case in which said possible change in the chitin level is an increase in the chitin level greater than or equal to 20%, said step d is followed by
• (e) a step of counting the cells in the mixture of cells of said strain of fungus, labelled by Calcofluor White, and antifungal agent;
• then by
• (f) a step of determining the possible change in the number of cells as a function of the concentration of antifungal agent in the population of cells of said strain of fungus compared to the number of cells in a population of cells of said strain of fungus in the absence of antifungal agent;
• when said change in the chitin level of said step d is an increase in the chitin level of less than 10% or a decrease in the chitin level, or the level is unchanged, it is concluded that said strain of fungus has a resistant phenotype;
• when said change in the chitin level of said step d is an increase of from 10% to a value less than 20%, it is concluded that said strain of fungus has an intermediate phenotype;
• when said change in the chitin level of said step d is an increase by a value greater than or equal to 20%, and when said change in the number of cells of said step f is a decrease of less than 0.3 log or an unchanged number of cells, it is concluded that said strain of fungus has an intermediate phenotype;
• and
• when said change in the chitin level of said step d is an increase by a value greater than or equal to 20%, and when said change in the number of cells is a decrease of at least 0.3 log, it is concluded that said strain of fungus has a sensitive phenotype.