Mass spectrometric diagnosis of septicemia
View Patent ↗The invention mainly relates to the mass spectrometric identification of pathogens in blood cultures from bloodstream infections (septicemia). The invention provides a method with which microbial pathogens can be separated in purified form from blood after a relatively brief cultivation in a blood culture flask, without any interfering human proteins or any residual fractions of blood particles such as erythrocytes and leukocytes, and can be directly identified by mass spectrometric measurement of their protein profiles. The method is based on the use of relatively strong tensides to destroy the blood particles by dissolving the weak cell membranes and most of the internal structures of the blood particles; in spite of the fact that tensides are regarded as strong ionization inhibitors in MALDI and other ionization processes required for mass spectrometric measurements. This method allows unknown pathogens to be obtained in their pure form by centrifuging or filtration and to be identified on the taxonomic level of species or subspecies. Problems with DNA from high levels of leukocytes can be resolved by special measures. After sufficient cultivation, the identification in a mass spectrometric laboratory takes only half an hour.
1. A method for the mass spectrometric identification of microbes contained in blood, comprising the steps:
providing a blood sample that is suspected of containing microbes,
cultivating the blood sample in order to produce a number of microbes in a blood culture sample sufficient for mass spectrometric detection,
destroying blood particles of the blood culture sample while not destroying microbial cell structures by mixing the blood culture sample with an amphiphile, surface active substance,
separating the microbes from the destroyed blood particles by pelleting or sedimenting the microbes from the blood culture sample-substance mixture by centrifugation or filtration, respectively, wherein the steps of destroying blood particles and of separating the microbes are executed such that the pelleted or sedimented microbes appear white,
preparing a measurement sample from the pelleted or sedimented microbes for a subsequent mass spectrometric measurement,
acquiring a mass spectrum from the prepared measurement sample, and
identifying the microbes in the prepared measurement sample down to the species or subspecies level by the mass spectrum, wherein the microbes are identified using protein profiles in the mass spectrum that represent the masses and abundances of different soluble microbial proteins by means of a similarity analysis with reference spectra from a spectrum library,
where the method does not include any microbe reproduction step on a flat nutrient medium.
2. The method according to claim 1 , further comprising cultivating the blood sample before the mixing in the presence of anticoagulants and nutrients.
3. The method according to claim 1 , wherein the step of preparing the measurement sample further comprises washing the pelleted or sedimented microbes to remove remnants of the amphiphile, surface-active substance and traces of human proteins before the mass spectrum is acquired.
4. The method according to claim 3 , wherein distilled water is used as washing liquid.
5. The method according to claim 1 , wherein the amphiphile, surface-active substance is anionic.
6. The method according to claim 5 , wherein sodium dodecyl sulfate (SDS) is used as the anionic amphiphile, surface-active substance.
7. The method according to claim 1 , wherein the amphiphile, surface-active substance is provided as a solution.
8. The method according to claim 7 , wherein the solution contains a defoamer.
9. The method according to claim 1 , further comprising centrifuging the blood culture sample and removing the resultant supernatant before the mixing in order to remove liquid blood culture sample fractions that do not contain any analytical information of interest.
10. The method according to claim 9 , wherein the steps of centrifuging the blood culture sample, removing the supernatant and mixing with the amphiphile, surface-active substance is repeated in order to remove the last residues of blood particles.
11. The method according to claim 1 , wherein the step of preparing the measurement sample further comprises disintegrating the pelleted or sedimented microbes by physico-chemical means before the mass spectrum is acquired.
12. The method according to claim 11 , wherein the at least one of pelleted or sedimented microbes are disintegrated one of (i) physically by sonication or mechanical treatment and (ii) chemically by solutions containing acids like formic acid or trifluoro ethanoic acid, and organic solvents like acetonitrile.
13. The method according to claim 1 , wherein the step of preparing the measurement sample further comprises its preparation on a mass spectrometric sample support plate using a matrix substance in whose crystals the soluble proteins of the at least one of pelleted or sedimented microbes are embedded before the mass spectrum is acquired.
14. The method according to claim 1 , wherein the step of preparing the measurement sample further comprises its preparation by transferring some microbes of the at least one of pellet or sediment onto a mass spectrometric sample support where a matrix solution is added before the mass spectrum is acquired.
15. The method according to claim 1 , wherein the mass spectrum is acquired with a time-of-flight mass spectrometer using one of (i) ionization by matrix assisted laser desorption (MALDI), (ii) electrospray ionization (ESI), and (iii) atmospheric pressure chemical ionization (APCI).
16. The method according to claim 1 , wherein mass signals in a mass range between 3,000 and 15,000 daltons are evaluated for the identification.
17. The method according to claim 1 , wherein the microbes comprise at least one of (i) bacteria, (ii) single-cell fungi, (iii) yeast, (iv) mold, and (v) protozoa.
18. The method according to claim 1 , further comprising agitating the blood culture sample-substance mixture in order to promote thorough mixing before the microbes are pelleted or sedimented.
19. The method according to claim 1 , wherein the microbes are sedimented by micro-filtration.
20. The method according to claim 1 , wherein the amphiphile, surface-active substance is a denaturizing agent.