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

Abstract

A method for determining the degree of sensitivity of a strain of fungus to an antifungal agent by using the possible change in a chitin level in a population of cells of a strain of fungus to an antifungal agent. The change is determined compared to the chitin level of a population of cells of said strain of fungus in the absence of antifungal agent.

Claims

1. 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.

2. The method according to claim 1, further comprising determining a possible change in a length of vegetative germination hypha in said population of cells of said strain of fungus to determine the degree of sensitivity of said strain of fungus to the antifungal agent, said change in the length of the vegetative germination hypha being determined by comparing a length of vegetative germination hypha in said population of cells of said strain of fungus in the presence of the antifungal agent to a length of vegetative germination hypha of the population of cells of said strain of fungus in the absence of the antifungal agent, and wherein said fungus is a multicellular fungus.

3. The method according to claim 1, said fungus being an unicellular fungus and wherein the resistant phenotype of said strain of fungus with respect to said antifungal agent is determined by an increase in the chitin level of less than 10%, or a decrease in the chitin level, or an unchanged chitin level compared to the chitin level of a population of cells of said strain of fungus in the absence of antifungal agent, and wherein the minimum threshold of cells in said population of cells of said strain of fungus demonstrating an increase in the chitin level is at least 10%, or wherein the intermediate phenotype of said strain of fungus with respect to said antifungal agent is determined either by an increase in the chitin level of from 10% to a value less than 20% compared to the chitin level of a population of cells of said strain of fungus in the absence of antifungal agent, or by an increase in the chitin level greater than or equal to 20% compared to the chitin level of a population of cells of said strain of fungus in the absence of antifungal agent, and by a decrease in the number of cells of less than 0.3 log or an unchanged number of cells compared to the number of cells of a population of cells of said strain of fungus in the absence of antifungal agent, and wherein the minimum threshold of cells in said population of cells of said strain of fungus demonstrating an increase in the chitin level is at least 10%, or wherein the sensitive phenotype of said strain of fungus with respect to said antifungal agent is determined by an increase in the chitin level greater than or equal to 20% compared to the chitin level of a population of cells of said strain of fungus in the absence of antifungal agent, and by a decrease in the number of cells of at least 0.3 log compared to the number of cells of a population of cells of said strain of fungus in the absence of antifungal agent, and wherein the minimum threshold of cells in said population of cells of said strain of fungus demonstrating an increase in the chitin level is at least 10%, and wherein the minimum level of a decrease in the number of cells in said population of cells of said strain of fungus is at least 0.3 log.

4. The method according to claim 1, wherein the antifungal agent is selected from the group consisting of antifungal agents free from polyenes and antifungal agents that inhibit the synthesis of ergosterol.

5. The method according to claim 1, wherein the fungus is a yeast selected from the group consisting of the genera Candida, Cryptococcus, Saccharomyces, Trichosporon, Rhodotorula, and Malassezia.

6. The method according to claim 1, wherein said fungus is a unicellular fungus.

7. The method according to claim 1, wherein said degree of sensitivity corresponds to the resistant phenotype when the change in the chitin level in the population of cells of said strain of fungus in the presence of the antifungal agent is an increase in the chitin level of less than 10%, or a decrease in the chitin level, or an unchanged chitin level compared to the chitin level of a population of cells of said strain of fungus in the absence of antifungal agent, and wherein the minimum threshold of cells in said population of cells of said strain of fungus demonstrating an increase in the chitin level is at least 10%; wherein said degree of sensitivity corresponds to the intermediate phenotype when the change in the chitin level in the population of cells of said strain of fungus in the presence of the antifungal agent is either (1) an increase in the chitin level of from 10% to a value less than 20% compared to the chitin level of a population of cells of said strain of fungus in the absence of the antifungal agent, or (2) an increase in the chitin level greater than or equal to 20% compared to the chitin level of a population of cells of said strain of fungus in the absence of the antifungal agent and the change in the number of cells of said strain of fungus in the presence of the antifungal agent is a decrease of less than 0.3 log or an unchanged number of cells compared to the number of cells in a population of cells of said strain of fungus in the absence of the antifungal agent, and for both (1) and (2) wherein the minimum threshold of cells in said population of cells of said strain of fungus demonstrating an increase in the chitin level is at least 10%; and wherein said degree of sensitivity corresponds to the sensitive phenotype when the change in the chitin level in the population of cells of said strain of fungus in the presence of antifungal agent is an increase in the chitin level greater than or equal to 20% compared to the chitin level of a population of cells of said strain of fungus in the absence of the antifungal agent, and the change in the number of cells in said population of cells of said strain of fungus in the presence of the antifungal agent is a decrease in the number of cells of at least 0.3 log compared to the number of cells of a population of cells of said strain of fungus in the absence of the antifungal agent, and the minimum threshold of cells in said population of cells of said strain of fungus demonstrating an increase in the chitin level is at least 10%.

8. The method according to claim 4, wherein said fungus is a multicellular fungus, further comprising a step of determining a possible change in a length of vegetative germination hypha in said population of fungal cells in the presence of said antifungal agent, said possible change in the length of the vegetative germination hypha being determined compared to a length of vegetative germination hypha of the population of cells of said strain of fungus in the absence of said antifungal agent.

9. The method of claim 1, wherein a treatment of an infection of the strain of fungus is adapted based on whether it is concluded that the strain of fungus is the resistant phenotype, the intermediate phenotype, or the sensitive phenotype.

Description

FIGURES

(1) FIG. 1: Shows the effect of fluconazole on cells of the strain SC5314 of C. albicans. The solid curve shows the change in the chitin level in the wall of the cells as a function of the concentration of fluconazole (logarithmic scale). The dashed curve shows the change in the number of cells in the medium as a function of the concentration of fluconazole (logarithmic scale).

(2) FIG. 2: Shows the effect of voriconazole on cells of the strain SC5314 of C. albicans. The solid curve shows the change in the chitin level in the wall of the cells as a function of the concentration of voriconazole (logarithmic scale). The dashed curve shows the change in the number of cells in the medium as a function of the concentration of voriconazole (logarithmic scale).

(3) FIG. 3: Shows the effect of fluconazole on cells of the strain DSY296 of C. albicans. The solid curve shows the change in the chitin level in the wall of the cells as a function of the concentration of fluconazole (logarithmic scale). The dashed curve shows the change in the number of cells in the medium as a function of the concentration of fluconazole (logarithmic scale).

(4) FIG. 4: Shows the effect of voriconazole on cells of the strain DSY296 of C. albicans. The solid curve shows the change in the chitin level in the wall of the cells as a function of the concentration of voriconazole (logarithmic scale). The dashed curve shows the change in the number of cells in the medium as a function of the concentration of voriconazole (logarithmic scale).

(5) FIG. 5: Shows the effect of micafungin on cells of the strain TOP of C. albicans. The solid curve shows the change in the chitin level in the wall of the cells as a function of the concentration of micafungin (logarithmic scale). The dashed curve shows the change in the number of cells in the medium as a function of the concentration of micafungin (logarithmic scale).

(6) FIG. 6: Shows the effect of fluconazole on cells of the strain Tg5 of C. glabrata. The solid curve shows the change in the chitin level in the wall of the cells as a function of the concentration of fluconazole (logarithmic scale). The dashed curve shows the change in the number of cells in the medium as a function of the concentration of fluconazole (logarithmic scale).

(7) FIG. 7: Shows the effect of voriconazole on cells of the strain Tg5 of C. glabrata. The solid curve shows the change in the chitin level in the wall of the cells as a function of the concentration of voriconazole (logarithmic scale). The dashed curve shows the change in the number of cells in the medium as a function of the concentration of voriconazole (logarithmic scale).

(8) FIG. 8: Shows the effect of fluconazole on cells of the strain ATCC®7349 of C. tropicalis. The solid curve shows the change in the chitin level in the wall of the cells as a function of the concentration of fluconazole (logarithmic scale). The dashed curve shows the change in the number of cells in the medium as a function of the concentration of fluconazole (logarithmic scale).

(9) FIG. 9: Shows the effect of voriconazole on cells of the strain ATCC®7349 of C. tropicalis. The solid curve shows the change in the chitin level in the wall of the cells as a function of the concentration of voriconazole (logarithmic scale). The dashed curve shows the change in the number of cells in the medium as a function of the concentration of voriconazole (logarithmic scale).

(10) FIG. 10: Shows the effect of micafungin on cells of the strain ATCC®7349 of C. tropicalis. The solid curve shows the change in the chitin level in the wall of the cells as a function of the concentration of micafungin (logarithmic scale). The dashed curve shows the change in the number of cells in the medium as a function of the concentration of micafungin (logarithmic scale).

(11) FIG. 11: Shows the effect of micafungin on cells of the strain 13/5 of C. tropicalis. The solid curve shows the change in the chitin level in the wall of the cells as a function of the concentration of micafungin (logarithmic scale). The dashed curve shows the change in the number of cells in the medium as a function of the concentration of micafungin (logarithmic scale).

(12) FIG. 12: Shows the effect of fluconazole on cells of the strain ATCC®22019 of C. parapsilosis. The solid curve shows the change in the chitin level in the wall of the cells as a function of the concentration of fluconazole (logarithmic scale). The dashed curve shows the change in the number of cells in the medium as a function of the concentration of fluconazole (logarithmic scale).

(13) FIG. 13: Shows the effect of voriconazole on cells of the strain ATCC®22019 of C. parapsilosis. The solid curve shows the change in the chitin level in the wall of the cells as a function of the concentration of voriconazole (logarithmic scale). The dashed curve shows the change in the number of cells in the medium as a function of the concentration of voriconazole (logarithmic scale).

(14) FIG. 14: Shows the effect of micafungin on cells of the strain ATCC®22019 of C. parapsilosis. The solid curve shows the change in the chitin level in the wall of the cells as a function of the concentration of micafungin (logarithmic scale). The dashed curve shows the change in the number of cells in the medium as a function of the concentration of micafungin (logarithmic scale).

(15) FIG. 15: Shows the effect of fluconazole on cells of the strain 8/21 of C. parapsilosis. The solid curve shows the change in the chitin level in the wall of the cells as a function of the concentration of fluconazole (logarithmic scale). The dashed curve shows the change in the number of cells in the medium as a function of the concentration of fluconazole (logarithmic scale).

(16) FIG. 16: Shows the effect of voriconazole on cells of the strain 8/21 of C. parapsilosis. The solid curve shows the change in the chitin level in the wall of the cells as a function of the concentration of voriconazole (logarithmic scale). The dashed curve shows the change in the number of cells in the medium as a function of the concentration of voriconazole (logarithmic scale).

(17) FIG. 17: Shows the effect of fluconazole on cells of the strain Tg5 of ATCC®6258 of C. krusei. The solid curve shows the change in the chitin level in the wall of the cells as a function of the concentration of fluconazole (logarithmic scale). The dashed curve shows the change in the number of cells in the medium as a function of the concentration of fluconazole (logarithmic scale).

(18) FIG. 18: Shows the effect of voriconazole on cells of the strain Tg5 of ATCC®6258 of C. krusei. The solid curve shows the change in the chitin level in the wall of the cells as a function of the concentration of voriconazole (logarithmic scale). The dashed curve shows the change in the number of cells in the medium as a function of the concentration of voriconazole (logarithmic scale).

(19) FIG. 19: Shows the effect of fluconazole on cells of the strain GRE32 of C. krusei. The solid curve shows the change in the chitin level in the wall of the cells as a function of the concentration of fluconazole (logarithmic scale). The dashed curve shows the change in the number of cells in the medium as a function of the concentration of fluconazole (logarithmic scale).

(20) FIG. 20: Shows the effect of voriconazole on cells of the strain GRE32 of C. krusei. The solid curve shows the change in the chitin level in the wall of the cells as a function of the concentration of voriconazole (logarithmic scale). The dashed curve shows the change in the number of cells in the medium as a function of the concentration of voriconazole (logarithmic scale).

EXAMPLES

(21) A—Examples of Unicellular Fungi

(22) I—Culture of Fungus in the Presence of an Antifungal Agent

(23) The strains of Candida were incubated beforehand at 30° C. overnight in a yeast extract medium—peptone dextrose (YPD) (1% bacto peptone, 0.5% yeast extract, 2% glucose, 1.5% agar).

(24) Yeast colonies were then removed from YPD medium plates and suspended in a 0.9% NaCl saline solution, in which the cell concentration was estimated by optical microscopy using Kova counting slides.

(25) A dilution was performed to obtain 15 ml of an inoculum at 3×10.sup.6 CFU/ml, in a synthetic complete (SC) medium at pH 7 (2% glucose, 0.5% ammonium sulfate, 0.17% nitrogen-containing yeast base, 0.2% synthetic complete mixture, and 10% HEPES 1.5M pH 7.2) for C. albicans, C. parapsilosis, C. tropicalis and C. krusei, and in a solution of RPMI 1640 pH 7.3 (10% HEPES 1M pH 7.2) for C. glabrata, so as to reduce the formation of septa and clusters.

(26) 1 ml of inoculum was then added to 2 ml of antifungal solution prepared in an SC or RPMI medium depending on the species of fungus, so as to obtain a final concentration of yeast of 10.sup.6 CFU/ml and the desired concentration of antifungal agent according to table 1.

(27) TABLE-US-00001 TABLE 1 Gradients of concentration of fluconazole, voriconazole and micafungin used for the yeast species Candida albicans, C. tropicalis, C. parapsilosis, C. glabrata and C. krusei. Antifungal agent Species Concentration (μg/ml) Fluconazole C. albicans, 16 8 4 2 1 0.5 0.25 0.125 0.062 0.031 0.015 C. tropicalis, C. parapsilosis C. glabrata, 128 64 32 16 8 4 2 1 0.5 0.25 0.125 C. krusei Voriconazole C. albicans, 5 2.5 1.25 0.625 0.312 0.156 0.078 0.039 0.019 0.009 0.004 C. tropicalis, C. parapsilosis C. glabrata, 8 4 2 1 0.5 0.25 0.125 0.062 0.031 0.015 0.007 C. krusei Micafungin C. albicans, 1 0.5 0.25 0.125 0.062 0.031 0.015 0.007 0.004 0.002 0.001 C. glabrata, C. tropicalis, C. parapsilosis, C. krusei

(28) After homogenisation, the cultures were placed at 30° C. at 200 rpm, with the exception of cultures of C. glabrata, which were placed at 35° C. at 200 rpm, and incubated for a period of 6.5 h or 24 h.

(29) II—Examination by High-Content Analysis (HCA) Microscopy

(30) After incubation, 100 μl of the culture of fungi obtained above were transferred in triplicate to 96-well plates, and 2.5 μl of Calcofluor White (CFW) were added to each well so as to mark the chitin of the cell walls of the yeasts.

(31) A step of acquiring images by automated fluorescence microscopy (ScanR screening station, Olympus), using a 40× lens and a CFW filter, made it possible to obtain 30 images per well so as to capture enough cells to provide statistically significant data. These images were analysed by software (ScanR analysis software, Olympus). Firstly, the background noise was processed so as to improve the contrast between the fluorescence of the yeasts and background noise. A pixel threshold was then defined. Segmentation of the fluorescent elements present in each of the acquired images was then performed using an algorithm predefined in the software, making it possible to determine the limit of each element on the basis of the fluorescence intensity of said element. A size parameter was then applied to select the yeasts and eliminate the aggregates and the fluorescent debris.

(32) Each element then corresponding to a yeast, it was thus possible, on the basis of these elements predefined beforehand, to define the fluorescence intensity for each of the yeasts, this intensity being directly correlated with the chitin content of their wall.

(33) On the basis of said defined elements, it was also possible to automatically count the yeasts,

(34) This data was then processed by a software (GraphPad Prism) so as to provide a representation in the form of a graph showing the change in the chitin level of the cell wall and the change in the number of cells in CFU/ml (colony-forming unit per ml) as a function of the concentration of antifungal agent, for each strain of yeast-antifungal agent pairing.

(35) When an increase in the chitin level having a value less than 10% or a decrease in the chitin level of less than 20% or an unchanged chitin level are observed compared to the chitin level of a population of cells of said fungus in the absence of antifungal agent, the strain is assumed to have the resistant phenotype.

(36) When an increase in the chitin level having a value of from 10% to a value less than 20% is observed compared to the chitin level of a population of cells of said fungus in the absence of antifungal agent, the strain is assumed to have the intermediate phenotype.

(37) When an increase in the chitin level greater than or equal to 20% is observed compared to the chitin level of a population of cells of said fungus in the absence of antifungal agent, as well as a decrease in the number of cells of less than 0.3 log or an unchanged number of cells compared to the number of cells in a population of cells of said fungus in the absence of antifungal agent, the strain is assumed to have the intermediate phenotype.

(38) When an increase in the chitin level greater than or equal to 20% is observed compared to the chitin level of a population of cells of said fungus in the absence of antifungal agent, as well as a decrease in the number of cells of at least 0.3 log compared to the number of cells in a population of cells of said fungus in the absence of antifungal agent, the strain is assumed to have the sensitive phenotype.

(39) It should be noted that all of the examples of the present application comprise graphs showing both the curve for the change in chitin level and the curve for the change in the number of cells, however the parameter of the change in the number of cells is not used in some cases for determination of the sensitivity of the strain of fungus.

(40) In fact, the generation of the two curves is inherent to the software used for the execution of the present examples.

(41) The consideration of a parameter other than the cell count for determining the sensitivity of a strain of fungus constitutes the uniqueness of this invention compared to existing tests based on a measurement of yeast growth, these being subject to the subjectivity of the reader.

(42) The aim of the present application is thus to find another criterion for determining the sensitivity of a strain of fungus, the cell count being included as supplementary piece of information in the majority of cases. This parameter, however, is necessary to distinguish between an intermediate phenotype and a sensitive phenotype when the change in chitin level is greater than or equal to 20%.

(43) III—Analysis by the Comparative Etest® Method, Marketed by Biomérieux

(44) The Etest® method is a commercial test for determining the sensitivity of a strain, most frequently used routinely in clinical mycology laboratories.

(45) The results obtained by HCA were compared with this obtained with this susceptibility test.

(46) The Etest® tests were performed according to the manufacturer's instructions. After distributing a standardised Mac Farland 0.5 inoculum (equivalent of 10.sup.8 CFU/ml) over plates of RPMI medium, said plates were incubated for 24 h at 35° C., with the exception of C. glabrata in which case the incubation was extended to 48 h so as to be able to determine the minimum inhibitory concentration (MIC).

(47) The results of the MICs obtained were interpreted in accordance with the tables of the CLSI (Clinical and Laboratory Standards Institute) and/or of EUCAST (European Committee on Antimicrobial Susceptibility Testing) (Arendrup M C, Cuenca-Estrella M, Lass-Flörl C, Hope W W (2014) Breakpoints for antifungal agents: an update from EUCAST focussing on echinocandins against Candida spp and triazoles against Aspergillus spp. Drug Resist Updat 16(6):81-95. doi:10.1016/j.drup.2014.01.001) (cf. Table 2 taken from Maubon D, Garnaud C, Calandra T, et al. (2014) Resistance of Candida spp. to antifungal drugs in the ICU: where are we now? Intensive Care Med. doi: 10.1007/s00134-014-3404-7) which list the clinical thresholds (CBPs) for the most common species of yeast of the genus Candida as a function of different antifungal agents, or according to the table of epidemiological thresholds (ECVs).

(48) The MIC thus makes it possible to determine the sensitivity of a strain to an antifungal agent. The CBPs or clinical thresholds make it possible to interpret this MIC and to predict a failure of therapy in the patient. The CBPs make it possible to establish the following categories: sensitive, intermediate, and resistant.

(49) If the value of the MIC is in the sensitive category, the likelihood of failure of the treatment is low. This likelihood increases in the intermediate and resistant categories.

(50) These tables make it possible to detect the acquired resistances which result primarily from the selection of mutants subjected to the pressure of pharmaceutical products in patients. They are thus specific to strains and should not be confused with intrinsic resistances, which are specific to species.

(51) TABLE-US-00002 TABLE 2 Clinical thresholds of the tables of the CLSI (Clinical and Laboratory Standards Institute) and/or of EUCAST (European Committee on Antimicrobial Susceptibility Testing) for species of Candida (Source: Maubon D, Garnaud C, Calandra T, et al. (2014) Resistance of Candida spp. to antifungal drugs in the ICU: where are we now? Intensive Care Med. doi: 10.1007/s00134-014-3404-7). MIC (mg/L) minimum inhibitory concentration Non-species related C. albicans C. glabrata C. krusei C. parapsilosis C. tropicalis C. guilliermondii breakpoints.sup.1 Antifungal agent S≤ R> S≤ R> S≤ R> S≤ R> S≤ R> S≤ R> S≤ R> Amphotericin B EUCAST 1 1 1 1 1 1 1 1 1 1 IE IE IE IE CLSI ND ND ND ND ND ND ND ND ND ND ND ND 1 Fluconazole EUCAST 2 4 0.002 32 — — 2 4 2 4 IE.sup.2 IE.sup.2 2 4 CLSI 2 4 0.002 32 — — 2 4 2 4 ND ND ND ND Voriconazole EUCAST 0.12.sup.5 0.12.sup.5 IE.sup.2 IE.sup.2 IE.sup.2 IE.sup.2 0.12.sup.5 0.12.sup.5 0.12.sup.5 0.12.sup.5 IE.sup.2 IE.sup.2 IE IE CLSI 0.12 0.5 — — 0.5 1 0.12 0.5 0.12 0.5 ND ND ND ND Posaconazole EUCAST 0.06 0.06 IE2 IE2 IE2 IE2 0.06 0.06 0.06 0.06 IE.sup.2 IE.sup.2 IE IE CLSI ND ND ND ND ND ND ND ND ND ND ND ND ND ND Itraconazole EUCAST IP IP IP IP IP IP IP IP IP IP IP IP IP IP CLSI ND ND ND ND ND ND ND ND ND ND ND ND 0.125 0.5 Anidulafungin EUCAST 0.03 0.03 0.06 0.06 0.06 0.06 0.002 4 0.06 0.06 IE.sup.2 IE.sup.2 IE IE CLSI 0.25 0.5 0.12 0.25 0.25 0.5 2 4 0.25 0.5 2 4 ND ND Micafungin EUCAST 0.016 0.016 0.03 0.03 IE.sup.4 IE.sup.4 0.002 2 IE.sup.4 IE.sup.4 IE.sup.4 IE.sup.4 IE IE CLSI 0.25 0.5 0.06 0.125 0.25 0.5 2 4 0.25 0.5 2 4 ND ND Caspofungin EUCAST Note.sup.3 Note.sup.3 Note.sup.3 Note.sup.3 Note.sup.3 Note.sup.3 Note.sup.3 Note.sup.3 Note.sup.3 Note.sup.3 IE.sup.2 IE.sup.2 Note.sup.3 Note.sup.3 CLSI 0.25 0.5 0.12 0.25 0.25 0.5 2 4 0.25 0.5 2 4 ND ND Flucytosine EUCAST ND ND ND ND ND ND ND ND ND ND ND ND ND ND CLSI ND ND ND ND ND ND ND ND ND ND ND ND 4 16 ND: no data, DP: data in preparation, DI: data insufficient. .sup.1Thresholds determined primarily on the basis of pharmacokinetic data, independent of species. Should be used only for organisms that have no specific thresholds. .sup.2The thresholds for this species are generally higher than for C. albicans. .sup.3Due to a significant inter-laboratory variation for the MICs of caspofungin, the EUCAST thresholds were established for this agent. .sup.4The MICs for C. tropicalis are 1 to 2 dilutions higher than for C. albicans and C. glabrata. In the clinical study, the rate of therapeutic success was slightly lower for C. tropicalis than for C. albicans at the two doses (100 and 150 mg per day). However, the difference was not significant and the clinical relevance of this difference is unknown. The MICs for C. krusei are 3 dilutions higher than for C. albicans and, similarly, those of C. guilliermondii are 8 dilutions higher. In addition, only a small number of cases involve these species in the clinical tests. The data is insufficient to conclude that the wild population of these species can be considered as sensitive to micafungin. .sup.5The strains with MIC values above the S/I threshold are rare, or else not reported at all. The tests for identifying sensitivity performed on these isolates must be repeated, and if the result is confirmed the isolate must be sent to a reference laboratory. Until there is proof of the clinical response for the confirmed isolates with MIC above the current resistant clinical threshold (in italics), these isolates must be marked as resistant.

Example 1: Test of Sensitivity of the Strain SC5314 of C. albicans to Fluconazole

(52) The strain SC5314 of C. albicans was cultivated as described in paragraph I of the Examples section so as to obtain an inoculum at 3×10.sup.6 CFU/ml in 15 ml of SC medium.

(53) 2 ml of fluconazole solution prepared in the SC medium were then added to 1 ml of this inoculum so as to obtain a final concentration of yeast of 10.sup.6 CFU/ml and of 16 μg/ml in fluconazole.

(54) The inoculum was treated in the same way so as to obtain cultures of 10.sup.6 CFU/ml and the following concentrations of fluconazole: 0.015 μg/ml, 0.031 μg/ml, 0.062 μg/ml, 0.125 μg/ml, 0.25 μg/ml, 0.5 μg/ml, 1 μg/ml, 2 μg/ml, 4 μg/ml, 8 μg/ml, 16 μg/ml.

(55) A control without fluconazole was prepared by adding 2 ml of SC medium.

(56) After incubation for 6.5 h at 30° C. with stirring at 200 rpm, 100 μl of each culture were removed and transferred to 96-well plates. This procedure was performed in triplicate, and 2.5 μl of CFW were then added to each well.

(57) The chitin level in the cell wall of the yeasts was then measured by high-contact analysis (HCA) microscopy, as described in paragraph II of the Examples section, in triplicate for each condition of concentration of fluconazole and without fluconazole.

(58) The data obtained was then averaged for each different tested condition and was presented in the form of a graph, shown in FIG. 1, where the solid curve shows the change in chitin level as a function of the concentration of fluconazole.

(59) An increase having a value greater than or equal to 20% of the chitin level in the wall of the cells of the strain SC5314 of C. albicans in the presence of fluconazole was observed compared to the chitin level of a population of cells of the strain SC5314 of C. albicans in the absence of fluconazole.

(60) The number of cells for each triplicate of each condition of concentration of fluconazole and without fluconazole was then determined by high-content analysis (HCA) microscopy, as described in paragraph II of the Examples section.

(61) The data obtained was then averaged for each different tested condition and was presented in the form of a graph, shown in FIG. 1, where the dashed curve shows the change in the number of cells as a function of the concentration of fluconazole.

(62) A decrease of at least 0.3 log of the number of cells of the strain SC5314 of C. albicans as a function of the concentration of fluconazole in the medium was observed compared to the number of cells in a population of cells of the strain SC5314 of C. albicans in the absence of fluconazole.

(63) These results confirm the sensitive phenotype of the strain SC5314 of C. albicans with respect to fluconazole.

(64) This result was confirmed by that obtained with the Etest method. In fact, with this method an MIC of 0.125 μg/ml was obtained, and the interpretation by the EUCAST and CLSI tables made it possible to determine that, with an MIC lower than 2 μg/ml for fluconazole, this strain is sensitive to this antifungal agent.

Example 2: Test of Sensitivity of the Strain SC5314 of C. albicans to Voriconazole

(65) The strain SC5314 of C. albicans was cultivated as described in paragraph I of the Examples section so as to obtain an inoculum at 3×10.sup.6 CFU/ml in 15 ml of SC medium.

(66) 2 ml of voriconazole solution prepared in the SC medium were then added to 1 ml of this inoculum so as to obtain a final concentration of yeast of 10.sup.6 CFU/ml and of 5 μg/ml in voriconazole.

(67) The inoculum was treated in the same way so as to obtain cultures of 10.sup.6 CFU/ml and the following concentrations of voriconazole: 0.004 μg/ml, 0.009 μg/ml, 0.019 μg/ml, 0.039 μg/ml, 0.078 μg/ml, 0.156 μg/ml, 0.312 μg/ml, 0.625 μg/ml, 1.25 μg/ml, 2.5 μg/ml, 5 μg/ml.

(68) A control without voriconazole was prepared by adding 2 ml of SC medium.

(69) After incubation for 6.5 h at 30° C. with stirring at 200 rpm, 100 μl of each culture were removed and transferred to 96-well plates. This procedure was performed in triplicate, and 2.5 μl of CFW were then added to each well.

(70) The chitin level in the cell wall of the yeasts was then measured by high-contact analysis (HCA) microscopy, as described in paragraph II of the Examples section, in triplicate for each condition of concentration of antifungal agent and without antifungal agent.

(71) The data obtained was then averaged for each different tested condition and was presented in the form of a graph, shown in FIG. 2, where the solid curve shows the change in chitin level as a function of the concentration of voriconazole.

(72) An increase having a value greater than or equal to 20% of the chitin level in the wall of the cells of the strain SC5314 of C. albicans in the presence of voriconazole was observed compared to the chitin level of a population of cells of the strain SC5314 of C. albicans in the absence of voriconazole.

(73) The number of cells for each triplicate of each condition of concentration of voriconazole and without voriconazole was then determined by high-content analysis (HCA) microscopy, as described in paragraph II of the Examples section.

(74) The data obtained was then averaged for each different tested condition and was presented in the form of a graph, shown in FIG. 2, where the dashed curve shows the change in the number of cells as a function of the concentration of voriconazole.

(75) A decrease of at least 0.3 log of the number of cells of the strain SC5314 of C. albicans as a function of the concentration of voriconazole in the medium was observed compared to the number of cells in a population of cells of the strain SC5314 of C. albicans in the absence of voriconazole.

(76) These results confirm the sensitive phenotype of the strain SC5314 of C. albicans with respect to voriconazole.

(77) This result was confirmed by that obtained with the Etest method. In fact, with this method an MIC of 0.012 μg/ml was obtained, and the interpretation by the EUCAST and CLSI tables made it possible to determine that, with an MIC lower than 0.12 μg/ml for voriconazole, this strain is sensitive to this antifungal agent.

Example 3: Test of Sensitivity of the Strain DSY296 of C. albicans to Fluconazole

(78) The strain DSY296 of C. albicans was cultivated as described in paragraph I of the Examples section so as to obtain an inoculum at 3×10.sup.6 CFU/ml in 15 ml of SC medium.

(79) 2 ml of fluconazole solution prepared in the SC medium were then added to 1 ml of this inoculum so as to obtain a final concentration of yeast of 10.sup.6 CFU/ml and of 16 μg/ml in fluconazole.

(80) The inoculum was treated in the same way so as to obtain cultures of 10.sup.6 CFU/ml and the following concentrations of fluconazole: 0.015 μg/ml, 0.031 μg/ml, 0.062 μg/ml, 0.125 μg/ml, 0.25 μg/ml, 0.5 μg/ml, 1 μg/ml, 2 μg/ml, 4 μg/ml, 8 μg/ml, 16 μg/ml.

(81) A control without fluconazole was prepared by adding 2 ml of SC medium.

(82) After incubation for 6.5 h at 30° C. with stirring at 200 rpm, 100 μl of each culture were removed and transferred to 96-well plates. This procedure was performed in triplicate, and 2.5 μl of CFW were then added to each well.

(83) The chitin level in the cell wall of the yeasts was then measured by high-contact analysis (HCA) microscopy, as described in paragraph II of the Examples section, in triplicate for each condition of concentration of fluconazole and without fluconazole.

(84) The data obtained was then averaged for each different tested condition and was presented in the form of a graph, shown in FIG. 3, where the solid curve shows the change in chitin level as a function of the concentration of fluconazole.

(85) A decrease in the chitin level of less than 20% in the wall of the cells of the strain DSY296 of C. albicans in the presence of fluconazole was observed compared to the chitin level of cells of the strain DSY296 of C. albicans in the absence of fluconazole.

(86) This result confirms the resistant phenotype of the strain DSY296 of C. albicans with respect to fluconazole.

(87) This result was confirmed by that obtained with the Etest method. In fact, with this method an MIC of 96 μg/ml was obtained, and the interpretation by the EUCAST and CLSI tables made it possible to determine that, with an MIC greater than 4 μg/ml for fluconazole, this strain is resistant to this antifungal agent.

Example 4: Test of Sensitivity of the Strain DSY296 of C. albicans to Voriconazole

(88) The strain DSY296 of C. albicans was cultivated as described in paragraph I of the Examples section so as to obtain an inoculum at 3×10.sup.6 CFU/ml in 15 ml of SC medium.

(89) 2 ml of voriconazole solution prepared in the SC medium were then added to 1 ml of this inoculum so as to obtain a final concentration of yeast of 10.sup.6 CFU/ml and of 5 μg/ml in voriconazole.

(90) The inoculum was treated in the same way so as to obtain cultures of 10.sup.6 CFU/ml and the following concentrations of voriconazole: 0.004 μg/ml, 0.009 μg/ml, 0.019 μg/ml, 0.039 μg/ml, 0.078 μg/ml, 0.156 μg/ml, 0.312 μg/ml, 0.625 μg/ml, 1.25 μg/ml, 2.5 μg/ml, 5 μg/ml.

(91) A control without voriconazole was prepared by adding 2 ml of SC medium.

(92) After incubation for 6.5 h at 30° C. with stirring at 200 rpm, 100 μl of each culture were removed and transferred to 96-well plates. This procedure was performed in triplicate, and 2.5 μl of CFW were then added to each well.

(93) The chitin level in the cell wall of the yeasts was then measured by high-contact analysis (HCA) microscopy, as described in paragraph II of the Examples section, in triplicate for each condition of concentration of voriconazole and without voriconazole.

(94) The data obtained was then averaged for each different tested condition and was presented in the form of a graph, shown in FIG. 4, where the solid curve shows the change in chitin level as a function of the concentration of voriconazole.

(95) A decrease in the chitin level of less than 20% in the wall of the cells of the strain DSY296 of C. albicans in the presence of voriconazole was observed compared to the chitin level of cells of the strain DSY296 of C. albicans in the absence of voriconazole.

(96) This result confirms the resistant phenotype of the strain DSY296 of C. albicans with respect to voriconazole.

(97) This result was confirmed by that obtained with the Etest method. In fact, with this method an MIC of 1 μg/ml was obtained, and the interpretation by the EUCAST and CLSI tables made it possible to determine that, with an MIC greater than 0.12 μg/ml and greater than 0.5 μg/ml respectively for voriconazole, this strain is resistant to this antifungal agent.

Example 5: Test of Sensitivity of the Strain TOP of C. albicans to Micafungin

(98) The strain TOP of C. albicans was cultivated as described in paragraph I of the Examples section so as to obtain an inoculum at 3×10.sup.6 CFU/ml in 15 ml of SC medium.

(99) 2 ml of micafungin solution prepared in the SC medium were then added to 1 ml of this inoculum so as to obtain a final concentration of yeast of 10.sup.6 CFU/ml and of 1 μg/ml in micafungin.

(100) The inoculum was treated in the same way so as to obtain cultures of 10.sup.6 CFU/ml and the following concentrations of fluconazole: 0.0009 μg/ml, 0.0019 μg/ml, 0.039 μg/ml, 0.007 μg/ml, 0.015 μg/ml, 0.031 μg/ml, 0.062 μg/ml, 0.125 μg/ml, 0.25 μg/ml, 0.5 μg/ml, 1 μg/ml.

(101) A control without voriconazole was prepared by adding 2 ml of SC medium.

(102) After incubation for 6.5 h at 30° C. with stirring at 200 rpm, 100 μl of each culture were removed and transferred to 96-well plates. This procedure was performed in triplicate, and 2.5 μl of CFW were then added to each well.

(103) The chitin level in the cell wall of the yeasts was then measured by high-contact analysis (HCA) microscopy, as described in paragraph II of the Examples section, in triplicate for each condition of concentration of micafungin and without micafungin.

(104) The data obtained was then averaged for each different tested condition and was presented in the form of a graph, shown in FIG. 5, where the solid curve shows the change in chitin level as a function of the concentration of micafungin.

(105) An increase in the chitin level of less than 10% in the wall of the cells of the strain TOP of C. albicans in the presence of micafungin was observed compared to the chitin level of cells of the strain TOP of C. albicans in the absence of micafungin.

(106) This result confirms the resistant phenotype of the strain TOP of C. albicans with respect to micafungin.

(107) This result was confirmed by that obtained with the Etest method. In fact, with this method an MIC of 1 μg/ml was obtained, and the interpretation by the EUCAST and CLSI tables made it possible to determine that, with an MIC greater than 0.016 μg/ml and greater than 0.5 μg/ml respectively for micafungin, this strain is resistant to this antifungal agent.

Example 6: Test of Sensitivity of the Strain Tg5 of C. glabrata to Fluconazole

(108) The strain Tg5 of C. glabrata was cultivated as described in paragraph I of the Examples section so as to obtain an inoculum at 3×10.sup.6 CFU/ml in 15 ml of RPMI medium.

(109) 2 ml of fluconazole solution prepared in the RPMI medium were then added to 1 ml of this inoculum so as to obtain a final concentration of yeast of 10.sup.6 CFU/ml and of 128 μg/ml in fluconazole.

(110) The inoculum was treated in the same way so as to obtain cultures of 10.sup.6 CFU/ml and the following concentrations of fluconazole: 0.125 μg/ml, 0.25 μg/ml, 0.5 μg/ml, 1 μg/ml, 2 μg/ml, 4 μg/ml, 8 μg/ml, 16 μg/ml, 32 μg/ml, 64 μg/ml, 128 μg/ml.

(111) A control without fluconazole was prepared by adding 2 ml of RPMI medium.

(112) After incubation for 6.5 h at 35° C. with stirring at 200 rpm, 100 μl of each culture were removed and transferred to 96-well plates. This procedure was performed in triplicate, and 2.5 μl of CFW were then added to each well.

(113) The chitin level in the cell wall of the yeasts was then measured by high-contact analysis (HCA) microscopy, as described in paragraph II of the Examples section, in triplicate for each condition of concentration of fluconazole and without fluconazole.

(114) The data obtained was then averaged for each different tested condition and was presented in the form of a graph, shown in FIG. 6, where the solid curve shows the change in chitin level as a function of the concentration of fluconazole.

(115) A decrease in the chitin level of less than 20% in the wall of the cells of the strain Tg5 of C. glabrata in the presence of fluconazole was observed compared to the chitin level of cells of the strain Tg5 of C. glabrata in the absence of fluconazole.

(116) This result confirms the resistant phenotype of the strain Tg5 of C. glabrata with respect to fluconazole.

(117) This result was confirmed by that obtained with the Etest method. In fact, with this method an MIC greater than 256 μg/ml was obtained, and the interpretation by the EUCAST and CLSI tables made it possible to determine that, with an MIC greater than 32 μg/ml for fluconazole, this strain is resistant to this antifungal agent.

Example 7: Test of Sensitivity of the Strain Tg5 of C. glabrata to Voriconazole

(118) The strain Tg5 of C. glabrata was cultivated as described in paragraph I of the Examples section so as to obtain an inoculum at 3×10.sup.6 CFU/ml in 15 ml of RPMI medium.

(119) 2 ml of voriconazole solution prepared in the RPMI medium were then added to 1 ml of this inoculum so as to obtain a final concentration of yeast of 10.sup.6 CFU/ml and of 8 μg/ml in voriconazole.

(120) The inoculum was treated in the same way so as to obtain cultures of 10.sup.6 CFU/ml and the following concentrations of voriconazole: 0.007 μg/ml, 0.015 μg/ml, 0.031 μg/ml, 0.062 μg/ml, 0.125 μg/ml, 0.25 μg/ml, 0.5 μg/ml, 1 μg/ml, 2 μg/ml, 4 μg/ml, 8 μg/ml.

(121) A control without voriconazole was also prepared by adding 2 ml of RPMI medium.

(122) After incubation for 6.5 h at 35° C. with stirring at 200 rpm, 100 μl of each culture were removed and transferred to 96-well plates. This procedure was performed in triplicate, and 2.5 μl of CFW were then added to each well.

(123) The chitin level in the cell wall of the yeasts was then measured by high-contact analysis (HCA) microscopy, as described in paragraph II of the Examples section, in triplicate for each condition of concentration of voriconazole and without voriconazole.

(124) The data obtained was then averaged for each different tested condition and was presented in the form of a graph, shown in FIG. 7, where the solid curve shows the change in chitin level as a function of the concentration of voriconazole.

(125) A decrease in the chitin level of less than 20% in the wall of the cells of the strain Tg5 of C. glabrata in the presence of voriconazole was observed compared to the chitin level of cells of the strain Tg5 of C. glabrata in the absence of voriconazole.

(126) This result confirms the resistant phenotype of the strain Tg5 of C. glabrata with respect to voriconazole.

(127) This result was confirmed by that obtained with the Etest method. In fact, with this method an MIC of 8 μg/ml was obtained, and the interpretation by the EUCAST table made it possible to determine that, with an MIC greater than 0.5 μg/ml for voriconazole, this strain is resistant to this antifungal agent.

Example 8: Test of Sensitivity of the Strain ATCC®7349 of C. tropicalis to Fluconazole

(128) The strain ATCC®7349 of C. tropicalis was cultivated as described in paragraph I of the Examples section so as to obtain an inoculum at 3×10.sup.6 CFU/ml in 15 ml of SC medium.

(129) 2 ml of fluconazole solution prepared in the SC medium were then added to 1 ml of this inoculum so as to obtain a final concentration of yeast of 10.sup.6 CFU/ml and of 16 μg/ml in fluconazole.

(130) The inoculum was treated in the same way so as to obtain cultures of 10.sup.6 CFU/ml and the following concentrations of fluconazole: 0.015 μg/ml, 0.031 μg/ml, 0.062 μg/ml, 0.125 μg/ml, 0.25 μg/ml, 0.5 μg/ml, 1 μg/ml, 2 μg/ml, 4 μg/ml, 8 μg/ml, 16 μg/ml.

(131) A control without fluconazole was prepared by adding 2 ml of SC medium.

(132) After incubation for 24 h at 30° C. with stirring at 200 rpm, 100 μl of each culture were removed and transferred to 96-well plates. This procedure was performed in triplicate, and 2.5 μl of CFW were then added to each well.

(133) The chitin level in the cell wall of the yeasts was then measured by high-contact analysis (HCA) microscopy, as described in paragraph II of the Examples section, in triplicate for each condition of concentration of fluconazole and without fluconazole.

(134) The data obtained was then averaged for each different tested condition and was presented in the form of a graph, shown in FIG. 8, where the solid curve shows the change in chitin level as a function of the concentration of fluconazole.

(135) An increase having a value greater than or equal to 20% of the chitin level in the wall of the cells of the strain ATCC®7349 of C. tropicalis in the presence of fluconazole was observed compared to the chitin level of a population of cells of the strain ATCC®7349 of C. tropicalis in the absence of fluconazole.

(136) The number of cells for each triplicate of each condition of concentration of fluconazole and without fluconazole was then determined by high-content analysis (HCA) microscopy, as described in paragraph II of the Examples section.

(137) The data obtained was then averaged for each different tested condition and was presented in the form of a graph, shown in FIG. 8, where the dashed curve shows the change in the number of cells as a function of the concentration of fluconazole.

(138) A decrease of at least 0.3 log of the number of cells of the strain ATCC®7349 of C. tropicalis as a function of the concentration of fluconazole in the medium was observed compared to the number of cells of the strain ATCC®7349 of C. tropicalis in a population of cells in the absence of fluconazole.

(139) These results confirm the sensitive phenotype of the strain ATCC®7349 of C. tropicalis with respect to fluconazole.

(140) This result was confirmed by that obtained with the Etest method. In fact, with this method an MIC of 0.38 μg/ml was obtained, and the interpretation by the EUCAST and CLSI tables made it possible to determine that, with an MIC lower than 2 μg/ml for fluconazole, this strain is sensitive to this antifungal agent.

Example 9: Test of Sensitivity of the Strain ATCC®7349 of C. tropicalis to Voriconazole

(141) The strain ATCC®7349 of C. tropicalis was cultivated as described in paragraph I of the Examples section so as to obtain an inoculum at 3×10.sup.6 CFU/ml in 15 ml of SC medium.

(142) 2 ml of voriconazole solution prepared in the SC medium were then added to 1 ml of this inoculum so as to obtain a final concentration of yeast of 10.sup.6 CFU/ml and of 5 μg/ml in voriconazole.

(143) The inoculum was treated in the same way so as to obtain cultures of 10.sup.6 CFU/ml and the following concentrations of voriconazole: 0.004 μg/ml, 0.009 μg/ml, 0.019 μg/ml, 0.039 μg/ml, 0.078 μg/ml, 0.156 μg/ml, 0.312 μg/ml, 0.625 μg/ml, 1.25 μg/ml, 2.5 μg/ml, 5 μg/ml.

(144) A control without voriconazole was prepared by adding 2 ml of SC medium.

(145) After incubation for 6.5 h at 30° C. with stirring at 200 rpm, 100 μl of each culture were removed and transferred to 96-well plates. This procedure was performed in triplicate, and 2.5 μl of CFW were then added to each well.

(146) The chitin level in the cell wall of the yeasts was then measured by high-contact analysis (HCA) microscopy, as described in paragraph II of the Examples section, in triplicate for each condition of concentration of voriconazole and without voriconazole.

(147) The data obtained was then averaged for each tested condition and was presented in the form of a graph, shown in FIG. 9, where the solid curve shows the change in chitin level as a function of the concentration of voriconazole.

(148) An increase having a value greater than or equal to 20% of the chitin level in the wall of the cells of the strain ATCC®7349 of C. tropicalis in the presence of voriconazole was observed compared to the chitin level of a population of cells of the strain ATCC®7349 of C. tropicalis in the absence of voriconazole.

(149) The number of cells for each triplicate of each condition of concentration of antifungal agent and without antifungal agent was then determined by high-content analysis (HCA) microscopy, as described in paragraph II of the Examples section.

(150) The data obtained was then averaged for each tested condition and was presented in the form of a graph, shown in FIG. 9, where the dashed curve shows the change in the number of cells as a function of the concentration of voriconazole.

(151) A decrease of at least 0.3 log of the number of cells of the strain ATCC®7349 of C. tropicalis as a function of the concentration of voriconazole in the medium was observed compared to the number of cells of the strain ATCC®7349 of C. tropicalis in a population of cells in the absence of voriconazole.

(152) These results confirm the sensitive phenotype of the strain ATCC®7349 of C. tropicalis with respect to voriconazole.

(153) This result was confirmed by that obtained with the Etest method. In fact, with this method an MIC of 0.023 μg/ml was obtained, and the interpretation by the EUCAST and CLSI tables made it possible to determine that, with an MIC lower than 0.12 μg/ml for voriconazole, this strain is sensitive to this antifungal agent.

Example 10: Test of Sensitivity of the Strain ATCC®7349 of C. tropicalis to Micafungin

(154) The strain ATCC®7349 of C. tropicalis was cultivated as described in paragraph I of the Examples section so as to obtain an inoculum at 3×10.sup.6 CFU/ml in 15 ml of SC medium.

(155) 2 ml of micafungin solution prepared in the SC medium were then added to 1 ml of this inoculum so as to obtain a final concentration of yeast of 10.sup.6 CFU/ml and of 1 μg/ml in micafungin.

(156) The inoculum was treated in the same way so as to obtain cultures of 10.sup.6 CFU/ml and the following concentrations of fluconazole: 0.0009 μg/ml, 0.019 μg/ml, 0.039 μg/ml, 0.007 μg/ml, 0.015 μg/ml, 0.031 μg/ml, 0.062 μg/ml, 0.125 μg/ml, 0.25 μg/ml, 0.5 μg/ml, 1 μg/ml.

(157) A control without voriconazole was prepared by adding 2 ml of SC medium.

(158) After incubation for 6.5 h at 30° C. with stirring at 200 rpm, 100 μl of each culture were removed and transferred to 96-well plates. This procedure was performed in triplicate, and 2.5 μl of CFW were then added to each well.

(159) The chitin level in the cell wall of the yeasts was then measured by high-contact analysis (HCA) microscopy, as described in paragraph II of the Examples section, in triplicate for each condition of concentration of micafungin and without micafungin.

(160) The data obtained was then averaged for each different tested condition and was presented in the form of a graph, shown in FIG. 10, where the solid curve shows the change in chitin level as a function of the concentration of micafungin.

(161) An increase having a value greater than or equal to 20% of the chitin level in the wall of the cells of the strain ATCC®7349 of C. tropicalis in the presence of micafungin was observed compared to that of cells in the absence of micafungin.

(162) The number of cells for each triplicate of each condition of concentration of micafungin and without micafungin was then determined by high-content analysis (HCA) microscopy, as described in paragraph II of the Examples section.

(163) The data obtained was then averaged for each different tested condition and was presented in the form of a graph, shown in FIG. 10, where the dashed curve shows the change in the number of cells as a function of the concentration of micafungin.

(164) A decrease of at least 0.3 log of the number of cells of the strain ATCC®7349 of C. tropicalis as a function of the concentration of micafungin in the medium was observed compared to the number of cells in a population of cells in the absence of micafungin.

(165) These results confirm the sensitive phenotype of the strain ATCC®7349 of C. tropicalis with respect to micafungin.

(166) This result was confirmed by that obtained with the Etest method. In fact, with this method an MIC of 0.016 μg/ml was obtained, and the interpretation by the CLSI table made it possible to determine that, with an MIC lower than 0.25 μg/ml for micafungin, this strain is sensitive to this antifungal agent.

Example 11: Test of Sensitivity of the Strain 13/5 of C. tropicalis to Micafungin

(167) The strain 13/5 of C. tropicalis was cultivated as described in paragraph I of the Examples section so as to obtain an inoculum at 3×10.sup.6 CFU/ml in 15 ml of SC medium.

(168) 2 ml of micafungin solution prepared in the SC medium were then added to 1 ml of this inoculum so as to obtain a final concentration of yeast of 10.sup.6 CFU/ml and of 1 μg/ml in micafungin.

(169) The inoculum was treated in the same way so as to obtain cultures of 10.sup.6 CFU/ml and the following concentrations of fluconazole: 0.0009 μg/ml, 0.019 μg/ml, 0.039 μg/ml, 0.007 μg/ml, 0.015 μg/ml, 0.031 μg/ml, 0.062 μg/ml, 0.125 μg/ml, 0.25 μg/ml, 0.5 μg/ml, 1 μg/ml.

(170) A control without voriconazole was prepared by adding 2 ml of SC medium.

(171) After incubation for 6.5 h at 30° C. with stirring at 200 rpm, 100 μl of each culture were removed and transferred to 96-well plates. This procedure was performed in triplicate, and 2.5 μl of CFW were then added to each well.

(172) The chitin level in the cell wall of the yeasts was then measured by high-contact analysis (HCA) microscopy, as described in paragraph II of the Examples section, in triplicate for each condition of concentration of micafungin and without micafungin.

(173) The data obtained was then averaged for each different tested condition and was presented in the form of a graph, shown in FIG. 11, where the solid curve shows the change in chitin level as a function of the concentration of micafungin.

(174) An increase in the chitin level of less than 10% in the wall of the cells of the strain 13/5 of C. tropicalis in the presence of micafungin was observed compared to the chitin level of cells of the strain 13/5 of C. tropicalis in the absence of micafungin.

(175) This result confirms the resistant phenotype of the strain 13/5 of C. tropicalis with respect to micafungin.

(176) This result was confirmed by that obtained with the Etest method. In fact, with this method an MIC of 1.5 μg/ml was obtained, and the interpretation by the CLSI table made it possible to determine that, with an MIC greater than 0.5 μg/ml for micafungin, this strain is resistant to this antifungal agent.

Example 12: Test of Sensitivity of the Strain ATCC®22019 of C. parapsilosis to Fluconazole

(177) The strain ATCC®22019 of C. parapsilosis was cultivated as described in paragraph I of the Examples section so as to obtain an inoculum at 3×10.sup.6 CFU/ml in 15 ml of SC medium.

(178) 2 ml of fluconazole solution prepared in the SC medium were then added to 1 ml of this inoculum so as to obtain a final concentration of yeast of 10.sup.6 CFU/ml and of 16 μg/ml in fluconazole.

(179) The inoculum was treated in the same way so as to obtain cultures of 10.sup.6 CFU/ml and the following concentrations of fluconazole: 0.015 μg/ml, 0.031 μg/ml, 0.062 μg/ml, 0.125 μg/ml, 0.25 μg/ml, 0.5 μg/ml, 1 μg/ml, 2 μg/ml, 4 μg/ml, 8 μg/ml, 16 μg/ml.

(180) A control without fluconazole was prepared by adding 2 ml of SC medium.

(181) After incubation for 6.5 h at 30° C. with stirring at 200 rpm, 100 μl of each culture were removed and transferred to 96-well plates. This procedure was performed in triplicate, and 2.5 μl of CFW were then added to each well.

(182) The chitin level in the cell wall of the yeasts was then measured by high-contact analysis (HCA) microscopy, as described in paragraph II of the Examples section, in triplicate for each condition of concentration of fluconazole and without fluconazole.

(183) The data obtained was then averaged for each different tested condition and was presented in the form of a graph, shown in FIG. 12, where the solid curve shows the change in chitin level as a function of the concentration of fluconazole.

(184) An increase having a value greater than or equal to 20% of the chitin level in the wall of the cells of the strain ATCC®22019 of C. parapsilosis in the presence of fluconazole was observed compared to the chitin level of a population of cells of the strain ATCC®22019 of C. parapsilosis in the absence of fluconazole.

(185) The number of cells for each triplicate of each condition of concentration of fluconazole and without fluconazole was then determined by high-content analysis (HCA) microscopy, as described in paragraph II of the Examples section.

(186) The data obtained was then averaged for each different tested condition and was presented in the form of a graph, shown in FIG. 12, where the dashed curve shows the change in the number of cells as a function of the concentration of fluconazole.

(187) A decrease of at least 0.3 log of the number of cells of the strain ATCC®22019 of C. parapsilosis as a function of the concentration of fluconazole in the medium was observed compared to the number of cells in a population of cells in the absence of fluconazole.

(188) These results confirm the sensitive phenotype of the strain ATCC®22019 of C. parapsilosis with respect to fluconazole.

(189) This result was confirmed by that obtained with the Etest method. In fact, with this method an MIC of 1 μg/ml was obtained, and the interpretation by the EUCAST and CLSI tables made it possible to determine that, with an MIC lower than 2 μg/ml for fluconazole, this strain is sensitive to this antifungal agent.

Example 13: Test of Sensitivity of the Strain ATCC®22019 of C. parapsilosis to Voriconazole

(190) The strain ATCC®22019 of C. parapsilosis was cultivated as described in paragraph I of the Examples section so as to obtain an inoculum at 3×10.sup.6 CFU/ml in 15 ml of SC medium.

(191) 2 ml of voriconazole solution prepared in the SC medium were then added to 1 ml of this inoculum so as to obtain a final concentration of yeast of 10.sup.6 CFU/ml and of 5 μg/ml in voriconazole.

(192) The inoculum was treated in the same way so as to obtain cultures of 10.sup.6 CFU/ml and the following concentrations of voriconazole: 0.004 μg/ml, 0.009 μg/ml, 0.019 μg/ml, 0.039 μg/ml, 0.078 μg/ml, 0.156 μg/ml, 0.312 μg/ml, 0.625 μg/ml, 1.25 μg/ml, 2.5 μg/ml, 5 μg/ml.

(193) A control without voriconazole was prepared by adding 2 ml of SC medium.

(194) After incubation for 6.5 h at 30° C. with stirring at 200 rpm, 100 μl of each culture were removed and transferred to 96-well plates. This procedure was performed in triplicate, and 2.5 μl of CFW were then added to each well.

(195) The chitin level in the cell wall of the yeasts was then measured by high-contact analysis (HCA) microscopy, as described in paragraph II of the Examples section, in triplicate for each condition of concentration of voriconazole and without voriconazole.

(196) The data obtained was then averaged for each different tested condition and was presented in the form of a graph, shown in FIG. 13, where the solid curve shows the change in chitin level as a function of the concentration of voriconazole.

(197) An increase having a value greater than or equal to 20% of the chitin level in the wall of the cells of the strain ATCC®22019 of C. parapsilosis in the presence of voriconazole was observed compared to the chitin level of a population of cells of the strain ATCC®22019 of C. parapsilosis in the absence of voriconazole.

(198) The number of cells for each triplicate of each condition of concentration of voriconazole and without voriconazole was then determined by high-content analysis (HCA) microscopy, as described in paragraph II of the Examples section.

(199) The data obtained was then averaged for each different tested condition and was presented in the form of a graph, shown in FIG. 13, where the dashed curve shows the change in the number of cells as a function of the concentration of voriconazole.

(200) A decrease of at least 0.3 log of the number of cells of the strain ATCC®22019 of C. parapsilosis as a function of the concentration of antifungal agent in the medium was observed compared to the number of cells in a population of cells of the strain ATCC®22019 of C. parapsilosis in the absence of antifungal agent.

(201) These results confirm the sensitive phenotype of the strain ATCC®22019 of C. parapsilosis with respect to voriconazole.

(202) This result was confirmed by that obtained with the Etest method. In fact, with this method an MIC of 0.023 μg/ml was obtained, and the interpretation by the EUCAST and CLSI tables made it possible to determine that, with an MIC lower than 0.12 μg/ml for voriconazole, this strain is sensitive to this antifungal agent.

Example 14: Test of Sensitivity of the Strain ATCC®22019 of C. parapsilosis to Micafungin

(203) The strain ATCC®22019 of C. parapsilosis was cultivated as described in paragraph I of the Examples section so as to obtain an inoculum at 3×10.sup.6 CFU/ml in 15 ml of SC medium.

(204) 2 ml of micafungin solution prepared in the SC medium were then added to 1 ml of this inoculum so as to obtain a final concentration of yeast of 10.sup.6 CFU/ml and of 1 μg/ml in micafungin.

(205) The inoculum was treated in the same way so as to obtain cultures of 10.sup.6 CFU/ml and the following concentrations of fluconazole: 0.0009 μg/ml, 0.019 μg/ml, 0.039 μg/ml, 0.007 μg/ml, 0.015 μg/ml, 0.031 μg/ml, 0.062 μg/ml, 0.125 μg/ml, 0.25 μg/ml, 0.5 μg/ml, 1 μg/ml.

(206) A control without voriconazole was prepared by adding 2 ml of SC medium.

(207) After incubation for 24 h at 30° C. with stirring at 200 rpm, 100 μl of each culture were removed and transferred to 96-well plates. This procedure was performed in triplicate, and 2.5 μl of CFW were then added to each well.

(208) The chitin level in the cell wall of the yeasts was then measured by high-contact analysis (HCA) microscopy, as described in paragraph II of the Examples section, in triplicate for each condition of concentration of micafungin and without micafungin.

(209) The data obtained was then averaged for each different tested condition and was presented in the form of a graph, shown in FIG. 15, where the solid curve shows the change in chitin level as a function of the concentration of micafungin.

(210) An increase having a value greater than or equal to 20% of the chitin level in the wall of the cells of the strain ATCC®22019 of C. parapsilosis in the presence of micafungin was observed compared to the chitin level of cells of the strain ATCC®22019 of C. parapsilosis in the absence of micafungin.

(211) The number of cells for each triplicate of each condition of concentration of micafungin and without micafungin was then determined by high-content analysis (HCA) microscopy, as described in paragraph II of the Examples section.

(212) The data obtained was then averaged for each different tested condition and was presented in the form of a graph, shown in FIG. 14, where the dashed curve shows the change in the number of cells as a function of the concentration of micafungin.

(213) A decrease of at least 0.3 log of the number of cells of the strain ATCC®22019 of C. parapsilosis as a function of the concentration of micafungin in the medium was observed compared to the number of cells in a population of cells of the strain ATCC®22019 of C. parapsilosis in the absence of micafungin.

(214) These results confirm the sensitive phenotype of the strain ATCC®22019 of C. parapsilosis with respect to micafungin.

(215) This result was confirmed by that obtained with the Etest method. In fact, with this method an MIC of 0.19 μg/ml was obtained, and the interpretation by the CLSI table made it possible to determine that, with an MIC lower than 2 μg/ml for micafungin, this strain is sensitive to this antifungal agent.

Example 15: Test of Sensitivity of the Strain 8/21 of C. parapsilosis to Fluconazole

(216) The strain 8/21 of C. tropicalis was cultivated as described in paragraph I of the Examples section so as to obtain an inoculum at 3×10.sup.6 CFU/ml in 15 ml of SC medium.

(217) 2 ml of fluconazole solution prepared in the SC medium were then added to 1 ml of this inoculum so as to obtain a final concentration of yeast of 10.sup.6 CFU/ml and of 16 μg/ml in fluconazole.

(218) The inoculum was treated in the same way so as to obtain cultures of 10.sup.6 CFU/ml and the following concentrations of fluconazole: 0.015 μg/ml, 0.031 μg/ml, 0.062 μg/ml, 0.125 μg/ml, 0.25 μg/ml, 0.5 μg/ml, 1 μg/ml, 2 μg/ml, 4 μg/ml, 8 μg/ml, 16 μg/ml.

(219) A control without fluconazole was prepared by adding 2 ml of SC medium.

(220) After incubation for 6.5 h at 30° C. with stirring at 200 rpm, 100 μl of each culture were removed and transferred to 96-well plates. This procedure was performed in triplicate, and 2.5 μl of CFW were then added to each well.

(221) The chitin level in the cell wall of the yeasts was then measured by high-contact analysis (HCA) microscopy, as described in paragraph II of the Examples section, in triplicate for each condition of concentration of fluconazole and without fluconazole.

(222) The data obtained was then averaged for each different tested condition and was presented in the form of a graph, shown in FIG. 15, where the solid curve shows the change in chitin level as a function of the concentration of fluconazole.

(223) An increase in the chitin level of less than 10% in the wall of the cells of the strain 8/21 of C. parapsilosis in the presence of fluconazole was observed compared to the chitin level of cells of the strain 8/21 of C. parapsilosis in the absence of fluconazole.

(224) This result confirms the resistant phenotype of the strain 8/21 of C. parapsilosis with respect to fluconazole.

(225) This result was confirmed by that obtained with the Etest method. In fact, with this method an MIC of 8 μg/ml was obtained, and the interpretation by the EUCAST and CLSI tables made it possible to determine that, with an MIC greater than 4 μg/ml for fluconazole, this strain is resistant to this antifungal agent.

Example 16: Test of Sensitivity of the Strain 8/21 of C. parapsilosis to Voriconazole

(226) The strain 8/21 of C. tropicalis was cultivated as described in paragraph I of the Examples section so as to obtain an inoculum at 3×10.sup.6 CFU/ml in 15 ml of SC medium.

(227) 2 ml of voriconazole solution prepared in the SC medium were then added to 1 ml of this inoculum so as to obtain a final concentration of yeast of 10.sup.6 CFU/ml and of 5 μg/ml in voriconazole.

(228) The inoculum was treated in the same way so as to obtain cultures of 10.sup.6 CFU/ml and the following concentrations of voriconazole: 0.004 μg/ml, 0.009 μg/ml, 0.019 μg/ml, 0.039 μg/ml, 0.078 μg/ml, 0.156 μg/ml, 0.312 μg/ml, 0.625 μg/ml, 1.25 μg/ml, 2.5 μg/ml, 5 μg/ml.

(229) A control without voriconazole was prepared by adding 2 ml of SC medium.

(230) After incubation for 6.5 h at 30° C. with stirring at 200 rpm, 100 μl of each culture were removed and transferred to 96-well plates. This procedure was performed in triplicate, and 2.5 μl of CFW were then added to each well.

(231) The chitin level in the cell wall of the yeasts was then measured by high-contact analysis (HCA) microscopy, as described in paragraph II of the Examples section, in triplicate for each condition of concentration of voriconazole and without voriconazole.

(232) The data obtained was then averaged for each different tested condition and was presented in the form of a graph, shown in FIG. 16, where the solid curve shows the change in chitin level as a function of the concentration of voriconazole.

(233) An increase in the chitin level of less than 10% in the wall of the cells of the strain 8/21 of C. parapsilosis in the presence of voriconazole was observed compared to the chitin level of cells of the strain 8/21 of C. parapsilosis in the absence of voriconazole.

(234) This result confirms the resistant phenotype of the strain 8/21 of C. parapsilosis with respect to voriconazole.

(235) This result was confirmed by that obtained by means of the Etest method, and the interpretation by the EUCAST table made it possible to determine that, with an MIC of 4 μg/ml for voriconazole, this strain is resistant to this antifungal agent.

(236) This result was confirmed by that obtained with the Etest method. In fact, with this method an MIC of 4 μg/ml was obtained, and the interpretation by the EUCAST and CLSI tables made it possible to determine that, with an MIC greater than 0.12 μg/ml and greater than 0.5 μg/ml respectively for voriconazole, this strain is resistant to this antifungal agent.

Example 17: Test of Sensitivity of the Strain ATCC®6258 of C. krusei to Fluconazole

(237) The strain ATCC®6258 of C. krusei was cultivated as described in paragraph I of the Examples section so as to obtain an inoculum at 3×10.sup.6 CFU/ml in 15 ml of SC medium.

(238) 2 ml of fluconazole solution prepared in the SC medium were then added to 1 ml of this inoculum so as to obtain a final concentration of yeast of 10.sup.6 CFU/ml and of 128 μg/ml in fluconazole.

(239) The inoculum was treated in the same way so as to obtain cultures of 10.sup.6 CFU/ml and the following concentrations of fluconazole: 0.125 μg/ml, 0.25 μg/ml, 0.5 μg/ml, 1 μg/ml, 2 μg/ml, 4 μg/ml, 8 μg/ml, 16 μg/ml, 32 μg/ml, 64 μg/ml, 128 μg/ml.

(240) A control without fluconazole was prepared by adding 2 ml of SC medium.

(241) After incubation for 6.5 h at 30° C. with stirring at 200 rpm, 100 μl of each culture were removed and transferred to 96-well plates. This procedure was performed in triplicate, and 2.5 μl of CFW were then added to each well.

(242) The chitin level in the cell wall of the yeasts was then measured by high-contact analysis (HCA) microscopy, as described in paragraph II of the Examples section, in triplicate for each condition of concentration of fluconazole and without fluconazole.

(243) The data obtained was then averaged for each different tested condition and was presented in the form of a graph, shown in FIG. 17, where the solid curve shows the change in chitin level as a function of the concentration of fluconazole.

(244) An increase in the chitin level of less than 10% in the wall of the cells of the strain ATCC®6258 of C. krusei in the presence of fluconazole was observed compared to the chitin level of cells of the strain ATCC®6258 of C. krusei in the absence of fluconazole.

(245) This result confirms the resistant phenotype of the strain ATCC®6258 of C. krusei with respect to fluconazole.

(246) This result was confirmed by that obtained with the Etest method. In fact, with this method an MIC of 64 μg/ml was obtained, and the interpretation by the EUCAST table made it possible to determine that, with an MIC greater than 32 μg/ml for fluconazole, this strain is resistant to this antifungal agent. The interpretation was obtained in this case by extrapolation of the EUCAST and CLSI data for C. glabrata.

Example 18: Test of Sensitivity of the Strain ATCC®6258 of C. krusei to Voriconazole

(247) The strain ATCC®6258 of C. krusei was cultivated as described in paragraph I of the Examples section so as to obtain an inoculum at 3×10.sup.6 CFU/ml in 15 ml of RPMI medium.

(248) 2 ml of voriconazole solution prepared in the RPMI medium were then added to 1 ml of this inoculum so as to obtain a final concentration of yeast of 10.sup.6 CFU/ml and of 8 μg/ml in voriconazole.

(249) The inoculum was treated in the same way so as to obtain cultures of 10.sup.6 CFU/ml and the following concentrations of voriconazole: 0.007 μg/ml, 0.015 μg/ml, 0.031 μg/ml, 0.062 μg/ml, 0.125 μg/ml, 0.25 μg/ml, 0.5 μg/ml, 1 μg/ml, 2 μg/ml, 4 μg/ml, 8 μg/ml.

(250) A control without voriconazole was also prepared by adding 2 ml of RPMI medium.

(251) After incubation for 24 h at 30° C. with stirring at 200 rpm, 100 μl of each culture were removed and transferred to 96-well plates. This procedure was performed in triplicate, and 2.5 μl of CFW were then added to each well.

(252) The chitin level in the cell wall of the yeasts was then measured by high-contact analysis (HCA) microscopy, as described in paragraph II of the Examples section, in triplicate for each condition of concentration of voriconazole and without voriconazole.

(253) The data obtained was then averaged for each different tested condition and was presented in the form of a graph, shown in FIG. 18, where the solid curve shows the change in chitin level as a function of the concentration of voriconazole.

(254) An increase having a value greater than or equal to 20% of the chitin level in the wall of the cells of the strain ATCC®6258 of C. krusei in the presence of voriconazole was observed compared to the chitin level of a population of cells of the strain ATCC®6258 of C. krusei in the absence of voriconazole.

(255) The number of cells for each triplicate of each condition of concentration of voriconazole and without voriconazole was then determined by high-content analysis (HCA) microscopy, as described in paragraph II of the Examples section.

(256) The data obtained was then averaged for each different tested condition and was presented in the form of a graph, shown in FIG. 18, where the dashed curve shows the change in the number of cells as a function of the concentration of voriconazole.

(257) A decrease of less than 0.3 log of the number of cells of the strain ATCC®6258 of C. krusei as a function of the concentration of antifungal agent in the medium was observed compared to the number of cells in a population of cells of the strain ATCC®6258 of C. krusei in the absence of antifungal agent.

(258) This result confirms the intermediate phenotype of the strain ATCC®6258 of C. krusei with respect to voriconazole.

(259) This result was confirmed by that obtained with the Etest method. In fact, with this method an MIC of 1 μg/ml was obtained, and the interpretation by the CLSI table made it possible to determine that, with an MIC equal to 1 μg/ml for voriconazole, this strain has an intermediate phenotype with respect to this antifungal agent.

Example 19: Test of Sensitivity of the Strain GRE32 of C. krusei to Fluconazole

(260) The strain GRE32 of C. krusei was cultivated as described in paragraph I of the Examples section so as to obtain an inoculum at 3×10.sup.6 CFU/ml in 15 ml of SC medium.

(261) 2 ml of fluconazole solution prepared in the SC medium were then added to 1 ml of this inoculum so as to obtain a final concentration of yeast of 10.sup.6 CFU/ml and of 128 μg/ml in fluconazole.

(262) The inoculum was treated in the same way so as to obtain cultures of 10.sup.6 CFU/ml and the following concentrations of fluconazole: 0.125 μg/ml, 0.25 μg/ml, 0.5 μg/ml, 1 μg/ml, 2 μg/ml, 4 μg/ml, 8 μg/ml, 16 μg/ml, 32 μg/ml, 64 μg/ml, 128 μg/ml.

(263) A control without fluconazole was prepared by adding 2 ml of SC medium.

(264) After incubation for 6.5 h at 30° C. with stirring at 200 rpm, 100 μl of each culture were removed and transferred to 96-well plates. This procedure was performed in triplicate, and 2.5 μl of CFW were then added to each well.

(265) The chitin level in the cell wall of the yeasts was then measured by high-contact analysis (HCA) microscopy, as described in paragraph II of the Examples section, in triplicate for each condition of concentration of fluconazole and without fluconazole.

(266) The data obtained was then averaged for each different tested condition and was presented in the form of a graph, shown in FIG. 19, where the solid curve shows the change in chitin level as a function of the concentration of fluconazole.

(267) A decrease in the chitin level of less than 20% in the wall of the cells of the strain GRE32 of C. krusei in the presence of fluconazole was observed compared to the chitin level of cells of the strain GRE32 of C. krusei in the absence of fluconazole.

(268) This result confirms the resistant phenotype of the strain GRE32 of C. krusei with respect to fluconazole.

(269) This result was confirmed by that obtained with the Etest method. In fact, with this method an MIC of 64 μg/ml was obtained, and the interpretation by the EUCAST table made it possible to determine that, with an MIC greater than 32 μg/ml for fluconazole, this strain is resistant to this antifungal agent. The interpretation was obtained in this case by extrapolation of the EUCAST and CLSI data for C. glabrata.

Example 20: Test of Sensitivity of the Strain GRE32 of C. krusei to Voriconazole

(270) The strain GRE32 of C. krusei was cultivated as described in paragraph I of the Examples section so as to obtain an inoculum at 3×10.sup.6 CFU/ml in 15 ml of SC medium.

(271) 2 ml of voriconazole solution prepared in the RPMI medium were then added to 1 ml of this inoculum so as to obtain a final concentration of yeast of 10.sup.6 CFU/ml and of 8 μg/ml in voriconazole.

(272) The inoculum was treated in the same way so as to obtain cultures of 10.sup.6 CFU/ml and the following concentrations of voriconazole: 0.007 μg/ml, 0.015 μg/ml, 0.031 μg/ml, 0.062 μg/ml, 0.125 μg/ml, 0.25 μg/ml, 0.5 μg/ml, 1 μg/ml, 2 μg/ml, 4 μg/ml, 8 μg/ml.

(273) A control without voriconazole was also prepared by adding 2 ml of RPMI medium.

(274) After incubation for 24 h at 30° C. with stirring at 200 rpm, 100 μl of each culture were removed and transferred to 96-well plates. This procedure was performed in triplicate, and 2.5 μl of CFW were then added to each well.

(275) The chitin level in the cell wall of the yeasts was then measured by high-contact analysis (HCA) microscopy, as described in paragraph II of the Examples section, in triplicate for each condition of concentration of voriconazole and without voriconazole.

(276) The data obtained was then averaged for each different tested condition and was presented in the form of a graph, shown in FIG. 20, where the solid curve shows the change in chitin level as a function of the concentration of voriconazole.

(277) An increase in the chitin level greater than or equal to 20% in the wall of the cells of the strain GRE32 of C. krusei in the presence of voriconazole was observed compared to the chitin level of cells of the strain GRE32 of C. krusei in the absence of voriconazole.

(278) The number of cells for each triplicate of each condition of concentration of voriconazole and without voriconazole was then determined by high-content analysis (HCA) microscopy, as described in paragraph II of the Examples section.

(279) The data obtained was then averaged for each different tested condition and was presented in the form of a graph, shown in FIG. 20, where the dashed curve shows the change in the number of cells as a function of the concentration of voriconazole.

(280) A decrease of less than 0.3 log of the number of cells of the strain GRE32 of C. krusei as a function of the concentration of voriconazole in the medium was observed compared to the number of cells in a population of cells of the strain GRE32 of C. krusei in the absence of voriconazole.

(281) These results confirm the intermediate phenotype of the strain GRE32 of C. krusei with respect to voriconazole.

(282) This result was confirmed by that obtained with the Etest method. In fact, with this method an MIC of 1 μg/ml was obtained, and the interpretation by the CLSI table made it possible to determine that, with an MIC equal to 1 μg/ml for voriconazole, this strain has an intermediate phenotype with respect to this antifungal agent.

(283) B—Examples of Multicellular Fungi

(284) I—Culture of Multicellular Fungus in the Presence of an Antifungal Agent

(285) The strains of multicellular fungus were incubated beforehand at 30° C. overnight in a yeast extract medium—peptone dextrose (YPD) (1% bacto peptone, 0.5% yeast extract, 2% glucose, 1.5% agar).

(286) Multicellular fungus conidia were then removed from YPD medium plates and suspended in a 0.9% NaCl saline solution, in which the cell concentration was estimated by optical microscopy using Kova counting slides.

(287) A dilution was performed to obtain a final inoculum at 10.sup.6 CFU/ml, in a synthetic complete (SC) medium at pH 7 (2% glucose, 0.5% ammonium sulfate, 0.17% nitrogen-containing yeast base, 0.2% synthetic complete mixture, and 10% HEPES 1.5M pH 7.2) a or an RPMI medium depending on the fungus, from the genera Aspergillus, Fusarium, Scedosporium, Lichteimia, Rhizopus, Rhizomucor, Mucor, Paecylomyces, and the species Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Fusarium solani, Fusarium oxysporum, Scedosporium apiospermum, Scedosporium prolificans, Mucor racemosa, Lichteimia corymbifera, Rhizopus oryzae, Rhizomucor pusillus, so as to obtain a final yeast concentration of 10.sup.6 CFU/ml and the desired concentration of antifungal agent according to table 3.

(288) TABLE-US-00003 TABLE 3 Gradients of concentration (μg/ml) of fluconazole, posaconazole, voriconazole, itraconazole, isavuconazole for the class of azoles and micafungin, anidulafungin and caspofungin for the class of class of echinocandins, and amphotericin B and nystatin for the class of polyenes, for the species of multicellular fungus. Antifungal agent Concentration (μg/ml) fluconazole 8 4 2 1 0.5 0.25 0.125 0.062 0.031 0.015 0.007 posaconazole voriconazole isavuconazole amphotericin B nystatin micafungin 2 1 0.5 0.25 0.125 0.062 0.031 0.015 0.007 0.003 0.0017 anidulafungin caspofungin

(289) After homogenisation, the cultures were placed at 30° C. at 200 rpm and incubated for a period of 4 h, 6 h or 24 h.

(290) II—Examination by High-Content Analysis (HCA) Microscopy

(291) After incubation, the culture of fungi obtained above were transferred in triplicate to 96-well plates, and 2.5 μl of Calcofluor White (CFW) were added to each well so as to mark the chitin of the cell walls of the conidia and vegetative germination hyphae.

(292) A step of acquiring images by automated fluorescence microscopy (ScanR screening station, Olympus), using a 40× lens and a CFW filter, make it possible to obtain 30 images per well so as to capture enough conidia and vegetative germination hyphae to provide measurements of the length of the germinative hyphae and the proportion of conidia yielding germinative hyphae. These images were analysed by ScanR analysis software (Olympus). Firstly, the background noise was processed so as to improve the contrast between the fluorescence of the conidia and germinative hyphae and background noise. A pixel threshold was then defined. Segmentation of the fluorescent elements present in each of the acquired images was then performed using an algorithm predefined in the software, making it possible to determine the limit of this element on the basis of the fluorescence intensity of each element. A parameter of size was then applied so as to select the conidia and the germinative hyphae and measure the length of these germinative hyphae.

(293) For each element then corresponding to a conidia or a germinative hyphae, it was thus possible, on the basis of these elements predefined beforehand, to define the fluorescence intensity for each of these elements, this intensity being directly correlated with the chitin content of their wall.

(294) This data was then processed by a software (GraphPad Prism) so as to provide a representation in the form of a graph showing the change in the chitin level of the wall of the conidia and vegetative hyphae and the change in the length of the vegetative hyphae as a function of the concentration of antifungal agent, for each strain of multicellular fungus-antifungal agent pairing.

(295) When an increase in the chitin level having a value less than 10% or a decrease in the chitin level of less than 20% or an unchanged chitin level are observed compared to the chitin level of a population of cells of said fungus in the absence of antifungal agent, the strain is assumed to have the resistant phenotype.

(296) When an increase in the chitin level having a value of from 10% to a value less than 20% is observed compared to the chitin level of a population of cells of said fungus in the absence of antifungal agent, the strain is assumed to have the intermediate phenotype.

(297) When an increase in the chitin level greater than or equal to 20% is observed compared to the chitin level of a population of cells of said fungus in the absence of antifungal agent, as well as a decrease in the length of the germinative hyphae of less than 10% or an unchanged length of the germinative hyphae compared to the length of germinative hyphae in a population of cells of said fungus in the absence of antifungal agent, the strain is assumed to have the intermediate phenotype.

(298) When an increase in the chitin level greater than or equal to 20% is observed compared to the chitin level of a population of cells of said fungus in the absence of antifungal agent, as well as a decrease in the length of germinative hyphae of at least 10% compared to the number of cells in a population of cells of said fungus in the absence of antifungal agent, the strain is assumed to have the sensitive phenotype.

(299) The consideration of a parameter other than the cell count for determining the sensitivity of a strain of fungus constitutes the uniqueness of this invention compared to existing tests based on a measurement of fungus growth, these being subject to the subjectivity of the reader.

(300) III—Analysis by the Comparative Etest® Method, Marketed by Biomérieux

(301) The Etest® method is a commercial test for determining the sensitivity of a strain, most frequently used routinely in clinical mycology laboratories.

(302) The results obtained by HCA were compared with those obtained with this susceptibility test.

(303) The Etest® tests were performed according to the manufacturer's instructions. After distributing a standardised Mac Farland 0.5 inoculum (equivalent of 10.sup.8 CFU/ml) over plates of RPMI medium, said plates were incubated for 24 h at 35° C. so as to be able to determine the minimum inhibitory concentration (MIC).

(304) The results of the MICs obtained were interpreted according to the tables of EUCAST (European Committee on Antimicrobial Susceptibility Testing www.eucast.org) which list the clinical thresholds (CBPs) for the most common species of multicellular fungi of the genus Aspergillus as a function of different antifungal agents. There are currently no interpretation criteria for the other genera of multicellular fungi including Fusarium, Scedosporium, Lichteimia, Rhizopus, Rhizomucor, Mucor, Paecylomyces.

(305) The MIC thus makes it possible to determine the sensitivity of a strain to an antifungal agent. The CBPs or clinical thresholds make it possible to interpret this MIC and to predict a failure of therapy in the patient. The CBPs make it possible to establish the following categories: sensitive, intermediate, and resistant.

(306) If the value of the MIC is in the sensitive category, the likelihood of failure of the treatment is low. This likelihood increases in the intermediate and resistant categories.

(307) These tables make it possible to detect the acquired resistances which result primarily from the selection of mutants subjected to the pressure of pharmaceutical products in patients. They are thus specific to strains and should not be confused with intrinsic resistances, which are specific to species.