Flow cytometry data processing for antimicrobial agent sensibility prediction

Patent metadata

Field	Value
Publication number	US-11649476-B2
Application number	US-201716316294-A
Country	US
Kind code	B2
Filing date	Jul 6, 2017
Priority date	Jul 8, 2016
Publication date	May 16, 2023
Grant date	May 16, 2023

Abstract

A method for quantifying the sensibility of a test microorganism to a concentration of an antimicrobial agent includes: preparing two liquid samples including the microorganism, one having the antimicrobial agent and one without; for each sample acquiring, by a flow cytometer, a digital set values including a fluorescence, forward, or side scatter distribution, and computing: a first coordinate value corresponding to the acquired distribution main mode and an acquired distribution first area for values greater than the first coordinate value, and a second coordinate value, greater than the first, for which an acquired distribution second area between the values equals a first area predefined percentage over 50%; computing a ratio according to:
Q = QT ⁡ ( ATB ) - Mode ⁡ ( ATB ) QT ⁡ ( no ⁢ ⁢ ATB ) - Mode ⁡ ( no ⁢ ⁢ ATB )
where Mode(ATB) and QT(ATB) are the first and second coordinate values with the antimicrobial agent concentration, and Mode(no ATB) and QT(no ATB) are respectively the first and second coordinate values without the antimicrobial agent concentration.

First claim

The invention claimed is:

1. A method for quantifying sensibility of a test microorganism to a concentration of an antimicrobial agent, comprising:
preparing at least two liquid samples comprising a population of the test microorganism and a viability fluorescence marker targeting the test microorganism in which at least one of the liquid samples comprises the antimicrobial agent at the concentration and another of the liquid samples does not comprise the antimicrobial agent;
performing flow cytometry on the liquid samples with a flow cytometer so as to acquire a digital set of values comprising a fluorescence distribution and/or a forward scatter distribution and/or a side scatter distribution of the population of test microorganism in each of the liquid samples, wherein:
the flow cytometer comprises a fluidic system, at least one light source, an optical system comprising excitation optics and collection optics, and an electronic system; and
an acquired distribution of each sample is subject to computation to calculate:
a first coordinate value corresponding to the main mode of the acquired distribution;
a first area of the acquired distribution for values greater than the first coordinate value;
a second coordinate value that is greater than the first coordinate value;
a second area of the acquired distribution between the first and second coordinate values that equals a predefined percentage of the first area over 50%; and
a ratio according to the relation:

Q
=

QT
⁡

(
ATB
)

-

Mode
⁡

(
ATB
)

QT
⁡

(

no
⁢

⁢
ATB

)

-

Mode
⁡

(

no
⁢

⁢
ATB

)

where QT is a notation for quantile and ATB is a notation for the antimicrobial agent, Mode (ATB) and QT(ATB) are respectively the first and second coordinate values with the concentration of the antimicrobial agent, and Mode(no ATB) and QT(no ATB) are respectively the first and second coordinate values without any concentration of the antimicrobial agent.

2. The method according to claim 1 , wherein the acquired distribution comprises the fluorescence distribution and the coordinate values are fluorescence values.

3. The method according to claim 2 , wherein:
a system comprises the flow cytometer and a computer unit connected to the flow cytometer; and
the system is installed in a clinical laboratory so that the method is performed in the clinical laboratory.

4. The method according to claim 2 , wherein the predefined percentage of the first area is over 70%.

5. The method according to claim 2 , wherein the antimicrobial agent is an antibiotic and the population of test microorganism are bacteria.

6. A method for providing a prediction model of sensibility phenotype to an antimicrobial agent amongst susceptible, intermediate, and resistant phenotypes, comprising a learning stage that comprises:
choosing a set of microorganisms comprising susceptible, intermediate, and resistant phenotype microorganisms in which the phenotypes are determined based on susceptible and resistant breakpoint concentrations of the antimicrobial agent so as to generate a digital set of sensibility phenotypes of the set of microorganisms;
preparing liquid samples for each microorganism of the set of microorganisms comprising a population of the microorganism, a viability fluorescence marker targeting the microorganism, and the antimicrobial agent in which the liquid samples comprises at least two different concentrations of the antimicrobial agent;
performing flow cytometry on the liquid samples with a flow cytometer so as to acquire a digital set of values comprising a fluorescence distribution and/or a forward scatter distribution and/or a side scatter distribution of the population of microorganism in each of the liquid samples; and
generating a feature vector based on the sets of values acquired for each microorganism of the set of microorganisms via a computer unit, wherein:
the flow cytometer comprises a fluidic system, at least one light source, an optical system comprising excitation optics and collection optics, and an electronic system; and
generation of the feature vector for each of the different concentrations of the antimicrobial agent comprises computing:
a first coordinate value corresponding to the main mode of an acquired distribution;
a first area of the acquired distribution for values greater than the first coordinate value;
a second coordinate value that is greater than the first coordinate value;
a second area of the acquired distribution between the first and second coordinate values that equals a predefined percentage of the first area over 50%; and
a ratio according to the relation:

Q
=

QT
⁡

(
ATB
)

-

Mode
⁡

(
ATB
)

QT
⁡

(

no
⁢

⁢
ATB

)

-

Mode
⁡

(

no
⁢

⁢
ATB

)

where QT is a notation for quantile and ATB is a notation for the antimicrobial agent, Mode(ATB) and QT(ATB) are respectively the first and second coordinate values with the

Assignees

• Biomerieux Sa

Inventors

• Ramjeet Mahendrasingh
• Mahe Pierre
• Kaneko Gaël
• Chapel Margaux

Classifications

• G16B40/20

  Supervised data analysis · CPC title

• G01N2021/6439

  with indicators, stains, dyes, tags, labels, marks · CPC title

• G16B40/10

  Signal processing, e.g. from mass spectrometry [MS] or from PCR · CPC title

• G01N21/6428

  Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53) · CPC title

• G06N20/10

  using kernel methods, e.g. support vector machines [SVM] · CPC title