Mass spectrometric diagnosis of sepsis without blood culture
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.
1. A method for the identification of microbes from a bodily fluid, comprising:
(a) destroying human cells in the bodily fluid;
(b) separating the microbes from the bodily fluid;
(c) cultivating the microbes using a nutrient broth which does not contain antimicrobial components of the bodily fluid;
(d) separating the microbes from the nutrient broth; and
(e) identifying the microbes by mass spectrometry.
2. The method of claim 1 , wherein the bodily fluid is blood and the human cells are blood particles of the blood.
3. The method of claim 2 , wherein the blood particles are destroyed by adding distilled water to the bodily fluid.
4. The method of claim 2 , wherein a sample of the blood is centrifuged and the supernatant blood plasma is removed prior to the step destroying the blood particles.
5. The method according to claim 2 , wherein the step of destroying the blood particles and the subsequent separating of the microbes from the blood is done immediately after a sample of the blood is taken from a subject.
6. 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.
7. The method of claim 1 , wherein the cultivation of the microbes takes place in less than one milliliter of the nutrient broth.
8. The method of claim 1 , during the cultivation monitoring quantitative growth of the microbes.
9. The method of claim 8 , wherein the nutrient broth is optically clear and the quantitative growth of the microbes is monitored optically.
10. The method of claim 9 wherein the monitoring of the quantitative growth of the microbes is carried out by measuring fluorescence.
11. The method of claim 10 , 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.
12. The method of claim 9 , wherein the monitoring of the quantitative growth of the microbes comprises measuring scattered light.
13. The method of claim 8 , wherein the cultivation is carried out only until the monitoring of the quantitative growth of the microbes indicates sufficient microbes for the identification by mass spectrometry.
14. The method of claim 1 , wherein, after separating the microbes from the nutrient broth, the microbes are cleansed of foreign proteins by an aqueous solution of SDS (sodium dodecyl sulfate).
15. The method of claim 1 , wherein, after separating the microbes from the nutrient broth, a MALDI mass spectrum is acquired for the microbes and used in the identifying step.
16. The method of claim 1 , wherein a portion of the microbes growing in the nutrient broth culture is used for tests of their resistance to antibiotics.
17. The method according to claim 16 , wherein the nutrient broth is divided after a predetermined time into portions for cultures, wherein one culture is used for the identification of the microbes by mass spectrometry and the other cultures are used to determine the resistance of the microbes to antibiotics by cultivation in the presence of the antibiotics.
18. The method of claim 1 , wherein, during separating the microbes from the bodily fluid, the microbes are separated by centrifugation.
19. The method of claim 1 , wherein, during separating the microbes from the bodily fluid, the microbes are separated by filtration.