Antimicrobial susceptibility
OwnerCidalia AZEVEDO PINA VAZinventor
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Abstract
The invention relates to methods and kits for the detection and determination of the susceptibility of microorganisms to therapeutic agents. The invention relates to the rapid determination of bacterial susceptibility to antibiotics. The invention also extends to methods of sample preparation and microorganism extraction from a biological sample, and to methods of identifying microorganisms, and to kits and panels specifically designed to determine the susceptibility of extracted microorganisms to antimicrobial agents. The invention is especially useful in clinical diagnostic and veterinary medicine.
Claims39
A method for determining the susceptibility phenotype, to at least one therapeutic agent, of a microorganism present in a biological sample, the method comprising:
- (i) introducing a biological sample comprising a microorganism into each of:
- (a) one or more of a first, test reservoir,
- (b) one or more of a second, positive control reservoir, and
- (c) one or more of a third, negative control reservoir, wherein the negative control reservoir comprises non-viable microorganisms;
- (ii) contacting the biological sample in the one or more first, test reservoir with at least one therapeutic agent;
- (iii) contacting the biological sample in the one or more first, second and third reservoir with at least one fluorescent marker;
- and
- (iv) performing a fluorescence analysis in order to obtain one or more fluorescence parameters for the biological sample in each of the reservoirs,
- wherein the microorganism's susceptibility phenotype to the at least one therapeutic agent is obtained by comparing one or more fluorescence parameters between the reservoirs.
The method according to any preceding claim, wherein the one or more third negative control reservoir are rendered non-viable by exposure to a cell-killing agent, optionally wherein the one or more third negative control reservoir comprises the cell-killing agent, optionally wherein the one or more third negative control reservoir comprises at least 60%, 70%, or 80% non-viable cells.
The method according to any preceding claim, wherein the susceptibility phenotype is susceptible, intermediate or resistant.
The method according to any preceding claim, wherein the method further comprises introducing the biological sample in one or more of a fourth auto-fluorescence control reservoir, which does not comprise the therapeutic agent or the fluorescent marker.
The method according to any preceding claim, wherein the method further comprises a sample preparation step.
The method according to any preceding claim, wherein the biological sample is of a human, animal or environmental origin, preferably wherein the sample is of a human origin.
The method according to any preceding claim, wherein the microorganism is selected from the group consisting of:
- a bacterium, a virus, a fungus or a protozoan, preferably wherein the microorganism is a bacterium.
The method according to any preceding claim, wherein the therapeutic agent is selected from the group consisting of:
- an antibiotic, an antiviral, an antifungal;
- and an antiprotozoan agent, preferably wherein the therapeutic agent is an antibiotic.
The method according to any preceding claim, wherein the fluorescent analysis is a flow cytometry analysis or a laser scanning analysis, preferably wherein the fluorescent analysis is a flow cytometry analysis.
The method according to any preceding claim, wherein the one or more flow cytometric parameters comprise forward scatter and/or side scatter and/or fluorescence parameters.
The method according to any preceding claim, wherein the fluorescent marker is selected from a group consisting of:
- acridine dye;
- cyanine dye;
- fluorone dye;
- oxazin dye;
- phenanthridine dye;
- and a rhodamine dye.
The method according to any preceding claim, wherein the fluorescent marker is selected from the group consisting of:
- CTC (5-Cyano-2,3-ditolyl tetrazolium chloride), Calcein AM, Dihydrorhodamine 123, DIBAC4(3), DioC 2(3), Fluorescein Diacetate, 5CFDA, AM, CFDA-SE, Propidium Iodine, SYTO 16 Green Fluorescent, and Nucleic Acid Stain.
The method according to any preceding claim, wherein the reservoir is a container, a tube or a well, preferably wherein the reservoir is a well.
A method of purifying a microorganism from a biological sample comprising:
- (i) obtaining a biological sample comprising a microorganism;
- and
- (ii) contacting the sample with a density gradient solution, to thereby purify the microorganism.
Use of a density gradient solution, for purifying a microorganism from a biological sample.
Dependencies are inferred from the claim text.