Mass spectrometric diagnosis of sepsis without blood culture

Patent metadata

Field	Value
Publication number	US-10006076-B2
Application number	US-201114113354-A
Country	US
Kind code	B2
Filing date	Jul 29, 2011
Priority date	Aug 2, 2010
Publication date	Jun 26, 2018
Grant date	Jun 26, 2018

Abstract

The invention relates to methods and instruments for the rapid detection and rapid mass spectrometric identification of microbial infective agents in blood or other body fluids. The invention recognizes that blood is not a good environment for the cultivation of microbes and provides a method which (a) largely destroys or dissolves the human particles in body fluids, such as erythrocytes and leukocytes in blood, without impairing the ability of the microbes to reproduce, (b) separates the microbial pathogens from the fluid, (c) cultivates them in a nutrient broth which contains none of the antimicrobial components of the body fluids, (d) separates them from the nutrient broth, and (e) identifies the microbes by a mass spectrum of the microbial proteins. The dissolution of the human particles also releases the microbes nesting in macrophages. The cultivation in an optically clear nutrient broth with optimum composition not only accelerates the propagation of the microbes compared to all other cultivation methods, but also makes it possible to continuously measure their quantitative growth starting from a low microbe density. This firstly allows the mass spectrometric identification to be carried out at the earliest possible time, secondly provides a positive detection of microbes far ahead of their identification, which can be lifesaving for the patient; and thirdly makes it possible to start the determination of resistances early.

First claim

What is claimed is:

1. A method for the identification of microbes from a bodily fluid comprising:
a) dissolving the blood particles in the bodily fluid;
b) separating the microbes from the bodily fluid;
c) cultivating the microbes in a nutrient broth which does not contain the antimicrobial components of the bodily fluid;
d) separating the microbes from the nutrient broth; and
e) identifying the microbes by similarity analysis of a mass spectrum of the microbial proteins and proteins in reference spectra.

2. The method of claim 1 , wherein the bodily fluid is blood.

3. The method of claim 1 , wherein the blood particles are dissolved by adding a surfactant to the bodily fluid.

4. The method of claim 1 , wherein the blood particles are dissolved in an aqueous solution of a saponin with added foam inhibitor.

5. The method of claim 1 , wherein, after the step of separating the microbes from the bodily fluid, it is investigated whether microbes have been separated, and the steps of cultivating, separating the microbes from the nutrient broth and identifying the microbes by mass spectrometry are carried out only if microbes have been detected.

6. The method of claim 1 , wherein the cultivation in the nutrient broth takes place in less than one milliliter of nutrient broth.

7. The method of claim 1 , wherein the quantitative growth of the microbes is monitored during the cultivation in the nutrient broth.

8. The method of claim 7 , wherein the cultivation in the nutrient broth is carried out only until the monitoring indicates sufficient microbes for a mass spectrometric identification.

9. The method of claim 7 , wherein the monitoring of the quantitative growth of the microbes is earned out by measuring fluorescence.

10. The method of claim 9 , wherein for the monitoring of the quantitative growth of the microbes by fluorescence, a substance is added to the nutrient broth whose breakdown by the microbes or attachment to the microbes causes a measurable fluorescence.

11. The method of claim 1 , wherein, after separating the microbes separated from the nutrient broth, the microbes are cleansed of foreign proteins by washing with an aqueous solution of SDS (sodium dodecyl sulfate).

12. The method of claim 1 , wherein for the acquisition of the mass spectrum in the identification step, the microbe proteins are ionized by matrix-assisted laser desorption (MALDI).

13. The method of claim 1 , wherein a portion of the microbes growing in the broth culture is used for tests of their resistance to antibiotics.

14. The method of claim 1 , wherein, in separating the microbes from the bodily fluid, the microbes are separated by centrifugation.

15. The method of claim 1 , wherein, in separating the microbes from the bodily fluid, the microbes are separated by filtration.

16. The method of claim 2 , wherein a sample of the blood is centrifuged and the supernatant blood plasma is removed prior to dissolving the blood particles and wherein, after dissolving the blood particles, the resulting material comprising the dissolved blood particles and the microbes is added to distilled water or a surfactant.

17. The method according to claim 2 , wherein dissolving the blood particles and the subsequent separation of the microbes from the blood is done immediately after a sample of the blood is taken from a subject.

18. The method according to claim 2 , wherein the volume of the nutrient broth is smaller than that of the blood.

19. The method of claim 7 , wherein the nutrient broth is optically clear and the growth is monitored optically.

20. The method according to claim 13 , herein the nutrient broth is divided after a predetermined time into portions for cultures, wherein one culture is used for identification of the microbes and the other cultures are used to determine the resistance of the microbes to antibiotics cultivation in the presence of the antibiotics.

Assignees

• Franzen Jochen
• Kostrzewa Markus
• Maier Thomas
• Michelmann Karsten
• Pusch Wolfgang
• Bruker Daltonik Gmbh

Inventors

• Franzen Jochen
• Kostrzewa Markus
• Maier Thomas
• Michelmann Karsten
• Pusch Wolfgang

Classifications

• C12Q1/04 Primary

  Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor {(C12Q1/6897 takes precedence)} · CPC title

• G01N21/77

  by observing the effect on a chemical indicator · CPC title

• G01N2800/26

  Infectious diseases, e.g. generalised sepsis · CPC title

• G01N33/6851

  Methods of protein analysis involving laser desorption ionisation mass spectrometry · CPC title

• G01N2021/7786

  Fluorescence · CPC title