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

Patent metadata

Field	Value
Publication number	US-11415513-B2
Application number	US-201615744682-A
Country	US
Kind code	B2
Filing date	Jul 15, 2016
Priority date	Jul 15, 2015
Publication date	Aug 16, 2022
Grant date	Aug 16, 2022

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.

First claim

The invention claimed is:

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 le

Assignees

• Univ Grenoble Alpes
• Centre Hospitalier Univ De Grenoble
• Centre Hospitalier Regional De Grenoble

Inventors

• Morin-Aldebert Delphine Paule Renee
• Cornet-Gigou Murielle Monique Jacqueline

Classifications

• C12Q1/18 Primary

  Testing for antimicrobial activity of a material · CPC title

• G01N2333/37

  from fungi · CPC title

• C12Q2304/00

  Chemical means of detecting microorganisms · CPC title

• C12Q1/02

  involving viable microorganisms · CPC title

• G01N2400/28

  Chitin, chitosan · CPC title