WO2020202113A1 - Procédés de spectrométrie de masse et compositions pour l'identification de microbes à partir de sang par réduction de protéines parasites - Google Patents

Procédés de spectrométrie de masse et compositions pour l'identification de microbes à partir de sang par réduction de protéines parasites Download PDF

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Publication number
WO2020202113A1
WO2020202113A1 PCT/IB2020/053234 IB2020053234W WO2020202113A1 WO 2020202113 A1 WO2020202113 A1 WO 2020202113A1 IB 2020053234 W IB2020053234 W IB 2020053234W WO 2020202113 A1 WO2020202113 A1 WO 2020202113A1
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instances
protein analysis
microorganism
mass spectrometry
blood culture
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PCT/IB2020/053234
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English (en)
Inventor
Karita HAAPASALO-TUOMAINEN
Hanne AMDAHL
Anssi RANTAKARI
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Thermo Fisher Scientific Oy
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses

Definitions

  • Microbial blood infections such as sepsis
  • Microbial blood infections are a major and increasing cause of in-hospital morbidity and mortality.
  • the mortality rate associated with septic shock, patients with sepsis complicated by strongly reduced blood pressure, is as high as 45%. Therefore there is an urgent need to improve diagnosis and therapy planning for microbial blood infections such as sepsis.
  • Sepsis caused by gram-negative pathogens is considered as the most frequent, with the majority of gram-negative sepsis caused by Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa.
  • Gram-positive pathogens such as Staphylococci and
  • Streptococci are the second major cause of sepsis, but their incidence has risen over the last decade.
  • a third major group includes fungi, with fungal infections causing a relatively small percentage of sepsis cases, but with a relatively high mortality rate.
  • Blood cultures are typically required to detect the presence of microorganisms in the blood of sepsis patients, to identify the microorganism present and guide treatment.
  • a blood culture involves collecting patient blood in a dedicated blood culture bottle and incubating the blood culture bottle for hours or days. If a microorganism is present, it can proliferate in the bottle during the incubation. Automated systems can detect microorganism growth in the blood culture bottle. Microorganism isolates from positive blood culture bottles undergo further analysis allowing for an identification of the microorganism to be made.
  • Mass spectrometry has been applied to the identification of microorganisms found in a positive blood culture.
  • Mass spectrometric analysis provides a spectrum of proteins present in the microorganism isolated from the blood culture. The identity of the microorganism from the blood culture is determined by comparing its protein spectrum to a reference database.
  • a critical component of mass spectrometric analysis is sample preparation. This is especially important when the analyte of interest is a minor component of the sample milieu; such as in the instance of blood culture wherein a microorganism is a minor component. Blood proteins confound the mass spectrometric identification of a
  • microorganism isolated from a blood culture is isolated from a blood culture.
  • the instant disclosure provides unique methodologies and compositions for mitigating the confounding effects of blood proteins, particularly hemoglobin, in proteomic analyses such as mass spectrometry.
  • Blood culture is the most widely used diagnostic tool for the detection of bacteremia and fungemia. It is the gateway for diagnosing the etiology of bloodstream infections.
  • blood is taken from a patient and is used to inoculate a blood culture bottle. Ideally 20-30 ml of a patient's blood is drawn and introduced into a culture bottle containing microbial growth nutrients. These bottles are then incubated to allow for microorganism growth.
  • Human blood contains 4-6 x 10 6 red blood cells/ml, while in contrast the number of microorganisms in a positive blood culture, one in which microbial growth is identified, maybe only a few hundred to no more than a few thousand microorganisms per milliliter. This vast excess of red blood cells and associated proteins can suppress detection of microorganism proteins.
  • Hemoglobin accounts for 95% of the total red blood cell protein content.
  • Several approaches for the removal of hemoglobin have been reported. These approaches include the application of strong cation exchange, gel filtration, clear native electrophoresis, and solution phase isoelectric focusing. Hemoglobin has been removed from red blood cell lysates by taking advantage of its affinity for nickel, use of the polyelectrolyte
  • HemogloBindTM HemogloBindTM, recombinant antibodies, and ProteoMinerTM.
  • the instant disclosure provides a wholly unique approach in overcoming the confounding effects of blood proteins, such as hemoglobin on protein based, and especially mass spectrometric analyses, of microorganisms from blood culture.
  • the method disclosed entails in some embodiments contacting a sample taken from a blood culture bottle with a preparation composition, the preparation composition encompassing a protease. After contact with the preparation composition, a protein analysis is conducted to detect the presence of a microorganism. In some embodiments, in which a microorganism is present, the microorganism is identified to the genus level. In some embodiments, the protein analysis is mass spectrometry.
  • a microorganism protein mass spectrometry preparation vessel encompasses a container in which a preparation composition has been introduced.
  • the preparation composition encompasses a protease and a detergent.
  • the preparation composition is dried in the container before use.
  • Figure 1 Comparison of residual hemoglobin after treatment with various proteases. Absorbance of a remaining supernatant solution at 395 nm was measured after treating Group A Streptococcus bacteria grown in a blood culture with various proteases.
  • FIG. 1 Heat map showing proteoforms analyzed from protease treated samples by Liquid Chromatography - Mass Spectrometry (LC-MS).
  • the proteoforms from each sample (x-axis) obtained in the LC-MS analysis were clustered according to their mass as visualized in a dendrogram.
  • the dendrogram above shows distances between each samples according to their unique proteoform spectra.
  • the relative abundance of each proteoform is indicated in the heat map.
  • Pepsin and Papain reduced proteoforms obtained from hemoglobin but also assigned multiple residual peaks that were not found in trypsin or proteinase K samples. Samples treated with only saponin served as a negative enzyme control.
  • FIG. 3 Hemoglobin a/b elution profiles. Comparison of hemoglobin a/b liquid chromatography elution profiles showed that the hemoglobin b eluted later, both in trypsin and proteinase K samples while the elution of the fragment in pepsin sand papain eluted at the same retention time as most of the bacterial proteoforms. Samples with only saponin showed a broad retention time for hemoglobin that would cover most of the proteoforms obtained from bacterial samples. Pa - papain, Pe - pepsin, PK - proteinase K, S - saponin alone, T - trypsin.
  • FIG. 4 A schematic representation of a workflow used to reduce hemoglobin and mammalian cells from an aliquot taken from a microbe positive blood culture. From left to right, an aliquot of a blood culture is introduced into a preparation vessel, in the depicted scheme a microorganism protein mass spectrometry preparation vessel, containing trypsin and saponin. The vessel is subjected to sonication, followed by centrifugation and washing twice of the resulting microbe enriched pellet. A lysis solution is then added to the vessel in order to lyse microbes. To this, a storage solution can be added.
  • microbe debris is pellet by centrifugation and the microbe protein containing supernatant is removed for analysis.
  • the microbe proteins in the depicted scheme, are subjected to liquid chromatography followed by introduction into a mass spectrometer. The microbe from which the proteins are derived is then identified, in some instances to the strain level.
  • Proteins of interest to biological researchers are generally present in a complex mixture of other proteins, which presents two significant problems.
  • the dynamic range of protein concentrations in biological mixtures can exceed 10 orders of magnitude.
  • the more abundant species have a tendency to "drown out" the detection from less-abundant ones.
  • the second problem is that a complex mixture is very difficult to fully analyze simply because of the overwhelming number of components.
  • bacteria can bind host proteins.
  • Staphylococcus aureus binds hemoglobin, utilizing it as a source of iron.
  • Staphylococcus aureus is not alone as a microorganism that binds hemoglobin or other patient proteins. Because of all these factors, relative paucity of microorganism proteins, presence in a complex confounding environment of patient proteins, with some of these patient proteins binding to the microorganism, an overall methodology to identify a microorganism by protein analysis from a patient sample has remained elusive until the instant disclosure.
  • a "patient sample” refers to a biological fluid or a biological tissue taken from a human donor.
  • biological fluids include amniotic fluid, blood, cerebral spinal fluid, mucus, plasma, serum, saliva, semen, stool, sputum, tears and urine.
  • Biological tissues are aggregates of cells, usually of a particular kind. Examples of biological tissues include organs and tumors.
  • the methods disclosed herein utilize a patient sample.
  • the patient sample is contacted with a preparation composition forming a contacted patient sample.
  • the contacted patient sample subsequently undergoes a protein analysis to detect the presence of a microorganism.
  • veterinary sample refers to a biological fluid or a biological tissue taken from a non-human animal donor.
  • the veterinary sample is contacted with a preparation composition forming a contacted veterinary sample.
  • the contacted veterinary sample subsequently undergoes a protein analysis to detect the presence of a microorganism.
  • the patient or veterinary sample can be introduced to a culture medium.
  • the culture medium can be a liquid, semi-solid, or solid.
  • the combination of culture medium and patient or veterinary sample can be incubated for minutes, hours, days or weeks, to promote or allow for microorganism growth.
  • an aliquot of a liquid culture medium can be removed and contacted with a preparation composition. This contacted aliquot of a liquid culture medium subsequently undergoes protein analysis.
  • a colony can be chosen from the semi-solid or solid medium and contacted with a preparation composition. This contacted colony from a semi-solid or solid medium subsequently undergoes protein analysis.
  • liquid blood culture refers to the instance in which blood drawn from a patient or non-human animal donor is introduced into a liquid culture medium.
  • liquid culture medium examples of commercially available brands of liquid culture medium used in a liquid blood culture sample are Thermo ScientificTM VersaTREKTM, BDTM BactecTM, and bioMerieux BACT/ALERTTM. This mixture of blood and liquid culture medium is often incubated for minutes, hours, days or weeks, to promote or allow for microorganism growth.
  • an aliquot from a liquid blood culture is contacted with a preparation composition forming a contacted liquid blood culture.
  • the contacted liquid blood culture subsequently undergoes a protein analysis to detect the presence of a microorganism.
  • the methods disclose herein utilize a colony from a plate blood culture.
  • a "plate blood culture” refers to the instance in which blood drawn from a patient or animal is introduced onto or into a semi-solid or solid culture medium. Examples of semi solid or solid culture medium include blood agar and chocolate agar. This mixture of blood and semi-solid or solid culture medium is often incubated for hours, days or weeks, to promote microorganism growth.
  • a colony from the plate blood culture is contacted with a preparation composition forming a contacted colony. The contacted colony subsequently undergoes protein analysis.
  • Microorganism refers generally to unicellular organisms. Examples of unicellular organisms include bacteria, yeasts and molds. Bacteria are often divided into two broad categories based on their Gram stain characteristics; Gram-positive and Gram-negative. Examples of Gram-positive bacteria include bacteria of the following genera: Enterococcus, Streptococcus, Staphylococcus, Bacillus, Paenibacillus, Lactobacillus, Listeria,
  • Gram-negative bacteria include bacteria of the following genera: Pseudomonas, Escherichia, Salmonella, Shigella, Enterobacter, Klebsiella, Serratia, Proteus, Campylobacter,
  • yeasts and molds include those of the following genera: Candida, Cryptococcus, Nocardia, Penicillium, Alternaria, Rhodotorula, Aspergillus, Fusarium, Saccharomyces and Trichosporon.
  • the methods disclosed herein utilize a patient sample or veterinary sample.
  • the patient or veterinary sample is contacted with a preparation composition forming a contacted patient or veterinary sample.
  • the contacted patient or veterinary sample subsequently undergoes a protein analysis to detect the presence of a microorganism.
  • the microorganism is a Gram-positive bacteria.
  • the microorganism is a Gram-negative bacteria.
  • the microorganism is a yeast. While in still further instances, the microorganism is a mold.
  • the patient or veterinary sample can be introduced to a culture medium.
  • the culture medium can be a liquid, semi-solid, or solid.
  • the combination of culture medium and patient or veterinary sample can be incubated for minutes, hours, days or weeks, to promote or allow for microorganism growth.
  • the microorganism is a Gram positive bacteria.
  • the microorganism is a Gram-negative bacteria.
  • the microorganism is a yeast.
  • the microorganism is a mold.
  • a colony can be chosen from the semi-solid or solid medium and contacted with a preparation composition.
  • the colony can be, in some instances, a Gram-positive bacteria. In other instances, the colony is a Gram negative bacteria. In still other instances, the colony is a yeast. In still further instances, the colony is a mold. This contacted colony from a semi-solid or solid medium subsequently undergoes protein analysis.
  • an aliquot from a liquid blood culture is contacted with a preparation composition forming a contacted liquid blood culture.
  • the contacted liquid blood culture subsequently undergoes a protein analysis to detect the presence of a microorganism.
  • the microorganism is a Gram-positive bacteria.
  • the microorganism is a Gram-negative bacteria.
  • the microorganism is a yeast. While in still further instances, the microorganism is a mold.
  • the microorganism from the contacted liquid blood culture that undergoes a protein analysis is a Gram-positive bacteria but not a Gram-negative bacteria, a yeast or a mold.
  • the Gram-positive bacteria are of the genus Staphylococcus.
  • the microorganism from the contacted liquid blood culture that undergoes a protein analysis is a Gram-negative bacteria but not a Gram-positive bacteria, a yeast or a mold.
  • the microorganism from the contacted liquid blood culture that undergoes a protein analysis is a yeast but not a Gram-negative bacteria, a Gram-positive bacteria, or a mold.
  • the microorganism from the contacted liquid blood culture that undergoes a protein analysis is a mold but not a Gram negative bacteria, a Gram-positive bacteria, or a yeast.
  • the methods disclose herein utilize a colony from a plate blood culture.
  • a colony from the plate blood culture is contacted with a preparation composition forming a contacted colony.
  • the colony can be, in some instances, a Gram-positive bacteria.
  • the colony is a Gram-negative bacteria.
  • the colony is a yeast.
  • the colony is a mold. This contacted colony from a semi-solid or solid medium subsequently undergoes protein analysis.
  • Protein analysis is applied in the methods disclosed herein.
  • Protein analysis refers to techniques capable of detecting proteins. Examples of protein analysis techniques include polyacrylamide gel electrophoresis (PAGE) (one and two-dimensional), Western blot, enzyme-linked immunosorbent assay (ELISA), immuno-precipitation, liquid chromatography (including high performance liquid chromatography), and mass
  • Protein analysis techniques are capable of being combined.
  • a non-limiting example of a combined protein analysis technique is the combination of liquid
  • Mass spectrometry is an analytical technique involving a process in which a sample is turned into ions and the ions are sorted based on their mass to charge ratio.
  • ionization techniques electrospray (ESI) and matrix assisted laser desorption/ionization (MALDI).
  • ESI electrospray
  • MALDI matrix assisted laser desorption/ionization
  • ions are generated at atmospheric pressure by passing a fluid with the analyte through a capillary. The capillary is electrically charged. As the fluid emerges from the capillary, an electrostatic spray of fluid containing analyte is generated. As the charged droplets move from the capillary, ions become free of the solvent that surrounds them, and these ions make their way into the mass analyzer of the
  • ions in MALDI are produced in discrete events by pulsed-laser irradiation of the analyte.
  • the analyte is co-crystallized with a solid matrix that can absorb the wavelength of light emitted by the laser. The solid matrix is then struck by laser light, causing ions to be emitted. The mechanism underlying how the ions are formed is not understood. The ions then enter the mass analyzer.
  • protein analysis is conducted. For instance a patient sample or veterinary sample is contacted with a preparation composition forming a contacted patient or veterinary sample. The contacted patient or veterinary sample subsequently undergoes a protein analysis to detect the presence of a microorganism.
  • the microorganism is a Gram-positive bacteria.
  • the microorganism is a Gram-negative bacteria.
  • the microorganism is a yeast. While in still further instances, the microorganism is a mold.
  • the protein analysis is by PAGE.
  • the protein analysis is by Western blot.
  • the protein analysis is by ELISA.
  • the protein analysis is by immuno-precipitation.
  • the protein analysis is by liquid
  • the protein analysis is by mass spectrometry. In still other instances, the protein analysis is a combination of two or three or four protein analysis techniques.
  • the mass spectrometry is ESI. In other instances, the mass spectrometry is MALDI.
  • the patient or veterinary sample can be introduced to a culture medium.
  • the culture medium can be a liquid, semi-solid, or solid.
  • the combination of culture medium and patient or veterinary sample can be incubated for minutes, hours, days or weeks, to promote or allow for microorganism growth.
  • the microorganism is a Gram positive bacteria.
  • the microorganism is a Gram-negative bacteria.
  • the microorganism is a yeast.
  • the microorganism is a mold.
  • the protein analysis is by PAGE. In other instances, the protein analysis is by Western blot. In some instances, the protein analysis is by ELISA. In some instances, the protein analysis is by immuno-precipitation. In some instances, the protein analysis is by liquid chromatography. In some instances, the protein analysis is by mass spectrometry. In still other instances, the protein analysis is a combination of two or three or four protein analysis techniques.
  • the mass spectrometry is ESI. In other instances, the mass spectrometry is MALDI.
  • a colony can be chosen from the semi-solid or solid medium and contacted with a preparation composition.
  • the colony can be, in some instances, a Gram-positive bacteria. In other instances, the colony is a Gram negative bacteria. In still other instances, the colony is a yeast. In still further instances, the colony is a mold.
  • This contacted colony from a semi-solid or solid medium subsequently undergoes protein analysis.
  • the protein analysis is by PAGE.
  • the protein analysis is by Western blot.
  • the protein analysis is by ELISA.
  • the protein analysis is by immuno-precipitation.
  • the protein analysis is by liquid chromatography. In some instances, the protein analysis is by mass spectrometry. In still other instances, the protein analysis is a combination of two or three or four protein analysis techniques.
  • the mass spectrometry is ESI. In other instances, the mass spectrometry is MALDI.
  • an aliquot from a liquid blood culture is contacted with a preparation composition forming a contacted liquid blood culture.
  • the contacted liquid blood culture subsequently undergoes a protein analysis to detect the presence of a microorganism.
  • the microorganism is a Gram-positive bacteria.
  • the microorganism is a Gram-negative bacteria.
  • the microorganism is a yeast. While in still further instances, the microorganism is a mold.
  • the protein analysis is by PAGE.
  • the protein analysis is by Western blot.
  • the protein analysis is by ELISA.
  • the protein analysis is by immuno-precipitation.
  • the protein analysis is by liquid chromatography.
  • the protein analysis is by mass spectrometry.
  • the protein analysis is a combination of two or three or four protein analysis techniques.
  • the mass spectrometry is ESI. In other instances, the mass spectrometry is MALDI.
  • the microorganism from the contacted liquid blood culture that undergoes a protein analysis is a Gram-positive bacteria but not a Gram-negative bacteria, a yeast or a mold. In some instances, the Gram-positive bacteria are of the genus Staphylococcus. In some instances, the microorganism from the contacted liquid blood culture that undergoes a protein analysis is a Gram-negative bacteria but not a Gram-positive bacteria, a yeast or a mold.
  • the microorganism from the contacted liquid blood culture that undergoes a protein analysis is a yeast but not a Gram-negative bacteria, a Gram-positive bacteria, or a mold. In some instances, the microorganism from the contacted liquid blood culture that undergoes a protein analysis is a mold but not a Gram negative bacteria, a Gram-positive bacteria, or a yeast. In some instances, the protein analysis is by PAGE. In other instances, the protein analysis is by Western blot. In some instances, the protein analysis is by ELISA. In some instances, the protein analysis is by immuno-precipitation. In some instances, the protein analysis is by liquid chromatography.
  • the protein analysis is by mass spectrometry. In still other instances, the protein analysis is a combination of two or three or four protein analysis techniques.
  • the mass spectrometry is ESI. In other instances, the mass spectrometry is MALDI.
  • the methods disclose herein utilize a colony from a plate blood culture.
  • a colony from the plate blood culture is contacted with a preparation composition forming a contacted colony.
  • the colony can be, in some instances, a Gram-positive bacteria.
  • the colony is a Gram-negative bacteria.
  • the colony is a yeast.
  • the colony is a mold. This contacted colony from a semi-solid or solid medium subsequently undergoes protein analysis.
  • the protein analysis is by PAGE.
  • the protein analysis is by Western blot.
  • the protein analysis is by ELISA.
  • the protein analysis is by immuno-precipitation.
  • the protein analysis is by liquid chromatography.
  • the protein analysis is by mass spectrometry.
  • the protein analysis is a combination of two or three or four protein analysis techniques.
  • the mass spectrometry is ESI. In other instances, the mass spectrometry is MALDI. Liquid chromatography - Mass spectrometry (LC-MS)
  • Liquid chromatography is a technique used to separate an individual compound from a mixture. This separation occurs based on the interactions of the compound with the mobile and stationary phases.
  • Liquid-solid column chromatography the most commonly used type of liquid chromatography, involves a liquid mobile phase which filters through a solid stationary phase bringing the separated compounds with it. As the mobile passes through the solid stationary phase held in a column, the compounds separate into bands. As each compound is eluted from the column, each can be collected and analyzed separately.
  • Solid phase extraction is another term used to describe liquid
  • SPE can be divided into three general types: normal phase, reverse phase and ion exchange.
  • Normal phase SPE typically involves a polar analyte, a mid- to nonpolar matrix and a polar stationary phase. Retention of an analyte under normal phase conditions is primarily due to interactions between polar functional groups of the analyte and the polar groups of the solid phase. Reverse phase separations involve a polar or moderately polar mobile phase and a nonpolar stationary phase. The analyte of interest is typically mid- to nonpolar.
  • Ion exchange SPE is used for compounds that are charged when in solution.
  • the methods disclosed herein utilize a protein analysis, wherein the protein analysis is a combination of liquid chromatography and mass spectrometry.
  • a patient sample or veterinary sample is contacted with a preparation composition forming a contacted patient or veterinary sample.
  • the contacted patient or veterinary sample subsequently undergoes a protein analysis to detect the presence of a microorganism.
  • the microorganism is a Gram-positive bacteria.
  • the microorganism is a Gram-negative bacteria.
  • the microorganism is a yeast.
  • the microorganism is a mold.
  • the protein analysis is by liquid chromatography combined with mass spectrometry.
  • the protein analysis is by liquid chromatography combined with mass spectrometry, wherein the mass spectrometer is ESI.
  • the patient or veterinary sample can be introduced to a culture medium.
  • the culture medium can be a liquid, semi-solid, or solid.
  • the combination of culture medium and patient or veterinary sample can be incubated for minutes, hours, days or weeks, to promote or allow for microorganism growth.
  • the microorganism is a Gram-positive bacteria.
  • the microorganism is a Gram-negative bacteria.
  • the microorganism is a yeast.
  • the microorganism is a mold.
  • an aliquot of a liquid culture medium can be removed and contacted with a preparation composition. This contacted aliquot of a liquid culture medium subsequently undergoes protein analysis.
  • the protein analysis is by liquid chromatography combined with mass spectrometry.
  • the protein analysis is by liquid chromatography combined with mass spectrometry, wherein the mass spectrometer is ESI.
  • a colony can be chosen from the semi-solid or solid medium and contacted with a preparation composition.
  • the colony can be, in some instances, a Gram-positive bacteria. In other instances, the colony is a Gram negative bacteria. In still other instances, the colony is a yeast. In still further instances, the colony is a mold. This contacted colony from a semi-solid or solid medium subsequently undergoes protein analysis. In some instances, the protein analysis is by liquid
  • the protein analysis is by liquid chromatography combined with mass spectrometry, wherein the mass spectrometer is ESI.
  • an aliquot from a liquid blood culture is contacted with a preparation composition forming a contacted liquid blood culture.
  • the contacted liquid blood culture subsequently undergoes a protein analysis to detect the presence of a microorganism.
  • the microorganism is a Gram-positive bacteria.
  • the microorganism is a Gram-negative bacteria.
  • the microorganism is a yeast.
  • the microorganism is a mold.
  • the protein analysis is by liquid chromatography combined with mass spectrometry.
  • the protein analysis is by liquid chromatography combined with mass spectrometry, wherein the mass spectrometer is ESI.
  • the microorganism from the contacted liquid blood culture that undergoes a protein analysis is a Gram-positive bacteria but not a Gram-negative bacteria, a yeast or a mold.
  • the Gram-positive bacteria are of the genus Staphylococcus.
  • the microorganism from the contacted liquid blood culture that undergoes a protein analysis is a Gram-negative bacteria but not a Gram-positive bacteria, a yeast or a mold.
  • the microorganism from the contacted liquid blood culture that undergoes a protein analysis is a yeast but not a Gram-negative bacteria, a Gram-positive bacteria, or a mold.
  • the microorganism from the contacted liquid blood culture that undergoes a protein analysis is a mold but not a Gram negative bacteria, a Gram-positive bacteria, or a yeast.
  • the protein analysis is by liquid chromatography combined with mass spectrometry.
  • the protein analysis is by liquid chromatography combined with mass spectrometry, wherein the mass spectrometer is ESI.
  • the methods disclose herein utilize a colony from a plate blood culture.
  • a colony from the plate blood culture is contacted with a preparation composition forming a contacted colony.
  • the colony can be, in some instances, a Gram-positive bacteria.
  • the colony is a Gram-negative bacteria.
  • the colony is a yeast.
  • the colony is a mold. This contacted colony from a semi-solid or solid medium subsequently undergoes protein analysis.
  • the protein analysis is by liquid chromatography combined with mass spectrometry.
  • the protein analysis is by liquid chromatography combined with mass spectrometry, wherein the mass spectrometer is ESI.
  • enrichment protocol refers to a process meant to result in an increase in microorganisms after the process relative to some starting mixture; for instance, starting with a patient or veterinary sample, a culture medium, a liquid culture medium, a blood culture, a blood culture medium or a liquid blood culture medium.
  • processes used within the enrichment protocol include filtration, capture and centrifugation.
  • capture include the employing of affinity reagents such as antibodies, lectins, for instance, mannan- binding lectin, and apolipoprotein H.
  • Centrifugation includes application of sufficient centrifugal force to deposit microorganisms on the bottom of a vessel or vessel side, as exemplified by a pellet. Centrifugation also includes a multi-speed centrifugation, also referred to as differential centrifugation, in which a relatively low centrifugal force is applied, resulting in the deposition of non-microorganisms to the bottom of a vessel or vessel side, while a proportion of microorganisms are not deposited. This relatively low centrifugal force is followed by the application of a relatively higher centrifugal force, this higher force causing microorganisms to be deposited on the bottom of a vessel or vessel side.
  • Centrifugation also includes density gradient centrifugation.
  • density gradient centrifugation a solution with a density differing from microorganisms is added to the vessel. The density of the added solution can be higher or lower than that of microorganisms.
  • Application of centrifugal force results in microorganisms being sequestered above or below or within the solution based upon density.
  • One, two, or three of these processes can be incorporated into an enrichment protocol.
  • an enrichment protocol is utilized. For instance a patient sample or veterinary sample is contacted with a preparation composition forming a contacted patient or veterinary sample. The contacted patient or veterinary sample subsequently undergoes an enrichment protocol followed by a protein analysis to detect the presence of a microorganism.
  • the enrichment protocol encompasses filtration.
  • the enrichment protocol encompasses capture.
  • the enrichment protocol encompasses centrifugation.
  • the microorganism is a Gram-positive bacteria.
  • the microorganism is a Gram negative bacteria.
  • the microorganism is a yeast. While in still further instances, the microorganism is a mold.
  • the protein analysis is by PAGE. In other instances, the protein analysis is by Western blot. In some instances, the protein analysis is by ELISA. In some instances, the protein analysis is by immuno-precipitation. In some instances, the protein analysis is by liquid chromatography. In some instances, the protein analysis is by mass spectrometry. In still other instances, the protein analysis is a combination of two or three or four protein analysis techniques.
  • the mass spectrometry is ESI. In other instances, the mass spectrometry is MALDI. In some instances, the protein analysis is by liquid chromatography combined with mass spectrometry. In some instances, the protein analysis is by liquid chromatography combined with mass spectrometry, wherein the mass spectrometer is ESI.
  • the patient or veterinary sample can be introduced to a culture medium.
  • the culture medium can be a liquid, semi-solid, or solid.
  • the combination of culture medium and patient or veterinary sample can be incubated for minutes, hours, days or weeks, to promote or allow for microorganism growth.
  • the microorganism is a Gram positive bacteria.
  • the microorganism is a Gram-negative bacteria.
  • the microorganism is a yeast. While in still further instances, the microorganism is a mold.
  • an aliquot of a liquid culture medium can be removed and contacted with a preparation composition and subsequently undergoes an enrichment protocol followed by a protein analysis to detect the presence of a microorganism.
  • the enrichment protocol encompasses filtration.
  • the enrichment protocol encompasses capture.
  • the enrichment protocol encompasses centrifugation.
  • the protein analysis is by PAGE.
  • the protein analysis is by Western blot.
  • the protein analysis is by ELISA.
  • the protein analysis is by immuno-precipitation.
  • the protein analysis is by liquid chromatography.
  • the protein analysis is by mass spectrometry. In still other instances, the protein analysis is a combination of two or three or four protein analysis techniques.
  • the mass spectrometry is ESI. In other instances, the mass spectrometry is MALDI. In some instances, the protein analysis is by liquid chromatography combined with mass spectrometry. In some instances, the protein analysis is by liquid chromatography combined with mass spectrometry, wherein the mass spectrometer is ESI.
  • a colony can be chosen from the semi-solid or solid medium and contacted with a preparation composition.
  • the colony can be, in some instances, a Gram-positive bacteria. In other instances, the colony is a Gram negative bacteria. In still other instances, the colony is a yeast. In still further instances, the colony is a mold.
  • This contacted colony from a semi-solid or solid medium subsequently undergoes an enrichment protocol followed by a protein analysis to detect the presence of a microorganism. In some instances the enrichment protocol encompasses filtration. In some instances the enrichment protocol encompasses capture. In some instances the enrichment protocol encompasses centrifugation.
  • the protein analysis is by PAGE. In other instances, the protein analysis is by Western blot. In some instances, the protein analysis is by ELISA. In some instances, the protein analysis is by immuno-precipitation. In some instances, the protein analysis is by liquid chromatography. In some instances, the protein analysis is by mass spectrometry. In still other instances, the protein analysis is a combination of two or three or four protein analysis techniques. [0065] In some instances, the mass spectrometry is ESI. In other instances, the mass spectrometry is MALDI. In some instances, the protein analysis is by liquid chromatography combined with mass spectrometry. In some instances, the protein analysis is by liquid chromatography combined with mass spectrometry, wherein the mass spectrometer is ESI.
  • an aliquot from a liquid blood culture is contacted with a preparation composition forming a contacted liquid blood culture.
  • the contacted liquid blood culture subsequently undergoes an enrichment protocol followed by a protein analysis to detect the presence of a microorganism.
  • the enrichment protocol encompasses filtration.
  • the enrichment protocol encompasses capture.
  • the enrichment protocol encompasses centrifugation.
  • the microorganism is a Gram-positive bacteria.
  • the microorganism is a Gram negative bacteria.
  • the microorganism is a yeast. While in still further instances, the microorganism is a mold.
  • the protein analysis is by PAGE.
  • the protein analysis is by Western blot. In some instances, the protein analysis is by ELISA. In some instances, the protein analysis is by immuno-precipitation. In some instances, the protein analysis is by liquid chromatography. In some instances, the protein analysis is by mass spectrometry. In still other instances, the protein analysis is a combination of two or three or four protein analysis techniques.
  • the mass spectrometry is ESI. In other instances, the mass spectrometry is MALDI. In some instances, the protein analysis is by liquid chromatography combined with mass spectrometry. In some instances, the protein analysis is by liquid chromatography combined with mass spectrometry, wherein the mass spectrometer is ESI.
  • the microorganism from the contacted liquid blood culture undergoes an enrichment protocol followed by a protein analysis is a Gram-negative bacteria but not a Gram-positive bacteria, a yeast or a mold.
  • the microorganism from the contacted liquid blood culture undergoes an enrichment protocol followed by a protein analysis is a Gram-positive bacteria, but not a Gram-negative bacteria, yeast or a mold.
  • the microorganism from the contacted liquid blood culture undergoes an enrichment protocol followed by a protein analysis is a yeast but not a Gram negative bacteria, a Gram-positive bacteria, or a mold.
  • the microorganism from the contacted liquid blood culture undergoes an enrichment protocol followed by a protein analysis is a yeast but not a Gram negative bacteria, a Gram-positive bacteria, or a mold.
  • the a yeast but not a Gram negative bacteria, a Gram-positive bacteria, or a mold.
  • the microorganism from the contacted liquid blood culture that undergoes an enrichment protocol followed by is a mold but not a Gram-negative bacteria, a Gram-positive bacteria, or a yeast.
  • the enrichment protocol encompasses filtration.
  • the enrichment protocol encompasses capture.
  • the enrichment protocol encompasses centrifugation.
  • the protein analysis is by PAGE.
  • the protein analysis is by Western blot.
  • the protein analysis is by ELISA.
  • the protein analysis is by immuno-precipitation.
  • the protein analysis is by liquid chromatography.
  • the protein analysis is by mass spectrometry.
  • the protein analysis is a combination of two or three or four protein analysis techniques.
  • the mass spectrometry is ESI. In other instances, the mass spectrometry is MALDI. In some instances, the protein analysis is by liquid chromatography combined with mass spectrometry. In some instances, the protein analysis is by liquid chromatography combined with mass spectrometry, wherein the mass spectrometer is ESI.
  • the methods disclose herein utilize a colony from a plate blood culture.
  • a colony from the plate blood culture is contacted with a preparation composition forming a contacted colony.
  • the colony can be, in some instances, a Gram-positive bacteria.
  • the colony is a Gram-negative bacteria.
  • the colony is a yeast.
  • the colony is a mold. This contacted colony from a semi-solid or solid medium subsequently undergoes an enrichment protocol followed by a protein analysis to detect the presence of a microorganism.
  • the enrichment protocol encompasses filtration.
  • the enrichment protocol encompasses capture.
  • the enrichment protocol encompasses centrifugation.
  • the protein analysis is by PAGE.
  • the protein analysis is by Western blot.
  • the protein analysis is by ELISA.
  • the protein analysis is by immuno-precipitation.
  • the protein analysis is by liquid chromatography.
  • the protein analysis is by mass spectrometry.
  • the protein analysis is a combination of two or three or four protein analysis techniques.
  • the mass spectrometry is ESI. In other instances, the mass spectrometry is MALDI. In some instances, the protein analysis is by liquid chromatography combined with mass spectrometry. In some instances, the protein analysis is by liquid chromatography combined with mass spectrometry, wherein the mass spectrometer is ESI. Lysis protocol
  • the methods disclosed herein include a lysis protocol.
  • "Lysis protocol” refers to a process meant to result in the disruption of a microorganism's cell membrane and cell wall, if the microorganism has a cell wall. Often the lysis protocol results in the cellular contents of the microorganism coming in contact with, or leaking into, the extracellular environment.
  • the process can be mediated by mechanical forces or chemicals. Examples of mechanical forces include sonication, freeze-thaw cycles and application of pressure, for instance, with a French press.
  • a chemical process is often associated with a composition encompassing an enzyme such as lysozyme, ostensibly to disrupt cell walls, and detergents. Sometimes these chemical compositions include a base such as sodium hydroxide.
  • a lysis protocol is utilized. For instance a patient sample or veterinary sample is contacted with a preparation composition forming a contacted patient or veterinary sample. The contacted patient or veterinary sample subsequently undergoes an enrichment protocol followed by a lysis protocol then a protein analysis to detect the presence of a microorganism.
  • the enrichment protocol encompasses filtration. In some instances the enrichment protocol encompasses capture. In some instances the enrichment protocol encompasses centrifugation.
  • the lysis protocol is mediated by mechanical force. In other instances, the lysis protocol is by applying a chemical composition. In still other instances, the lysis protocol is mediated by applying mechanical force and a chemical composition.
  • the microorganism is a Gram-positive bacteria. In other instances, the microorganism is a Gram negative bacteria. In still other instances, the microorganism is a yeast. While in still further instances, the microorganism is a mold.
  • the protein analysis is by PAGE. In other instances, the protein analysis is by Western blot. In some instances, the protein analysis is by ELISA. In some instances, the protein analysis is by immuno-precipitation. In some instances, the protein analysis is by liquid chromatography. In some instances, the protein analysis is by mass spectrometry. In still other instances, the protein analysis is a combination of two or three or four protein analysis techniques.
  • the mass spectrometry is ESI. In other instances, the mass spectrometry is MALDI. In some instances, the protein analysis is by liquid chromatography combined with mass spectrometry. In some instances, the protein analysis is by liquid chromatography combined with mass spectrometry, wherein the mass spectrometer is ESI. [0075]
  • the patient or veterinary sample can be introduced to a culture medium.
  • the culture medium can be a liquid, semi-solid, or solid.
  • the combination of culture medium and patient or veterinary sample can be incubated for minutes, hours, days or weeks, to promote or allow for microorganism growth. In some instances, the microorganism is a Gram positive bacteria.
  • the microorganism is a Gram-negative bacteria. In still other instances, the microorganism is a yeast. While in still further instances, the microorganism is a mold.
  • an aliquot of a liquid culture medium can be removed and contacted with a preparation composition and subsequently undergoes an enrichment protocol, then a lysis protocol, followed by a protein analysis to detect the presence of a microorganism.
  • the enrichment protocol encompasses filtration.
  • the enrichment protocol encompasses capture.
  • the enrichment protocol encompasses centrifugation.
  • the lysis protocol is mediated by mechanical force. In other instances, the lysis protocol is by applying a chemical composition.
  • the lysis protocol is mediated by applying mechanical force and a chemical composition.
  • the protein analysis is by PAGE.
  • the protein analysis is by Western blot.
  • the protein analysis is by ELISA.
  • the protein analysis is by immuno-precipitation.
  • the protein analysis is by liquid
  • the protein analysis is by mass spectrometry. In still other instances, the protein analysis is a combination of two or three or four protein analysis techniques.
  • the mass spectrometry is ESI. In other instances, the mass spectrometry is MALDI. In some instances, the protein analysis is by liquid chromatography combined with mass spectrometry. In some instances, the protein analysis is by liquid chromatography combined with mass spectrometry, wherein the mass spectrometer is ESI.
  • a colony can be chosen from the semi-solid or solid medium and contacted with a preparation composition.
  • the colony can be, in some instances, a Gram-positive bacteria. In other instances, the colony is a Gram negative bacteria. In still other instances, the colony is a yeast. In still further instances, the colony is a mold.
  • This contacted colony from a semi-solid or solid medium subsequently undergoes an enrichment protocol followed by lysis protocol before protein analysis to detect the presence of a microorganism. In some instances the enrichment protocol encompasses filtration. In some instances the enrichment protocol encompasses capture. In some instances the enrichment protocol encompasses centrifugation.
  • the lysis protocol is mediated by mechanical force. In other instances, the lysis protocol is by applying a chemical composition. In still other instances, the lysis protocol is mediated by applying mechanical force and a chemical composition.
  • the protein analysis is by PAGE. In other instances, the protein analysis is by Western blot. In some instances, the protein analysis is by ELISA. In some instances, the protein analysis is by immuno-precipitation. In some instances, the protein analysis is by liquid chromatography. In some instances, the protein analysis is by mass spectrometry. In still other instances, the protein analysis is a combination of two or three or four protein analysis techniques.
  • the mass spectrometry is ESI. In other instances, the mass spectrometry is MALDI. In some instances, the protein analysis is by liquid chromatography combined with mass spectrometry. In some instances, the protein analysis is by liquid chromatography combined with mass spectrometry, wherein the mass spectrometer is ESI.
  • an aliquot from a liquid blood culture is contacted with a preparation composition forming a contacted liquid blood culture.
  • the contacted liquid blood culture subsequently undergoes an enrichment protocol followed by a lysis protocol then a protein analysis to detect the presence of a microorganism.
  • the enrichment protocol encompasses filtration.
  • the enrichment protocol encompasses capture.
  • the enrichment protocol encompasses
  • the lysis protocol is mediated by mechanical force. In other instances, the lysis protocol is by applying a chemical composition. In still other instances, the lysis protocol is mediated by applying mechanical force and a chemical composition.
  • the microorganism is a Gram-positive bacteria. In other instances, the microorganism is a Gram-negative bacteria. In still other instances, the microorganism is a yeast. While in still further instances, the microorganism is a mold.
  • the protein analysis is by PAGE. In other instances, the protein analysis is by Western blot. In some instances, the protein analysis is by ELISA. In some instances, the protein analysis is by immuno-precipitation. In some instances, the protein analysis is by liquid
  • the protein analysis is by mass spectrometry. In still other instances, the protein analysis is a combination of two or three or four protein analysis techniques.
  • the mass spectrometry is ESI. In other instances, the mass spectrometry is MALDI. In some instances, the protein analysis is by liquid chromatography combined with mass spectrometry. In some instances, the protein analysis is by liquid chromatography combined with mass spectrometry, wherein the mass spectrometer is ESI.
  • the microorganism from the contacted liquid blood culture undergoes an enrichment protocol followed by a lysis protocol then subject to protein analysis is a Gram-negative bacteria but not a Gram-positive bacteria, a yeast or a mold.
  • the microorganism from the contacted liquid blood culture undergoes an enrichment protocol followed by lysis protocol then subject to protein analysis is a Gram positive bacteria, but not a Gram-negative bacteria, yeast or a mold.
  • the microorganism from the contacted liquid blood culture undergoes an enrichment protocol followed by lysis protocol then subject to protein analysis is a yeast but not a Gram-negative bacteria, a Gram-positive bacteria, or a mold.
  • the microorganism from the contacted liquid blood culture that undergoes an enrichment protocol followed by a lysis protocol then subject to protein analysis is a mold but not a Gram-negative bacteria, a Gram positive bacteria, or a yeast.
  • the enrichment protocol encompasses filtration.
  • the enrichment protocol encompasses capture.
  • the enrichment protocol encompasses centrifugation.
  • the lysis protocol is mediated by mechanical force.
  • the lysis protocol is by applying a chemical composition.
  • the lysis protocol is mediated by applying mechanical force and a chemical composition.
  • the protein analysis is by PAGE.
  • the protein analysis is by Western blot.
  • the protein analysis is by ELISA.
  • the protein analysis is by immuno-precipitation.
  • the protein analysis is by liquid chromatography.
  • the protein analysis is by mass spectrometry. In still other instances, the protein analysis is a combination of two or three or four protein analysis techniques.
  • the mass spectrometry is ESI. In other instances, the mass spectrometry is MALDI. In some instances, the protein analysis is by liquid chromatography combined with mass spectrometry. In some instances, the protein analysis is by liquid chromatography combined with mass spectrometry, wherein the mass spectrometer is ESI.
  • the methods disclose herein utilize a colony from a plate blood culture.
  • a colony from the plate blood culture is contacted with a preparation composition forming a contacted colony.
  • the colony can be, in some instances, a Gram-positive bacteria.
  • the colony is a Gram-negative bacteria.
  • the colony is a yeast.
  • the colony is a mold. This contacted colony from a semi-solid or solid medium subsequently undergoes an enrichment protocol followed by a lysis protocol and then protein analysis to detect the presence of a microorganism.
  • the enrichment protocol encompasses filtration.
  • the enrichment protocol encompasses capture.
  • the enrichment protocol encompasses centrifugation.
  • the lysis protocol is mediated by mechanical force.
  • the lysis protocol is by applying a chemical composition.
  • the lysis protocol is mediated by applying mechanical force and a chemical composition.
  • the protein analysis is by PAGE.
  • the protein analysis is by Western blot.
  • the protein analysis is by ELISA.
  • the protein analysis is by immuno-precipitation.
  • the protein analysis is by liquid chromatography.
  • the protein analysis is by mass spectrometry.
  • the protein analysis is a combination of two or three or four protein analysis techniques.
  • the mass spectrometry is ESI. In other instances, the mass spectrometry is MALDI. In some instances, the protein analysis is by liquid chromatography combined with mass spectrometry. In some instances, the protein analysis is by liquid chromatography combined with mass spectrometry, wherein the mass spectrometer is ESI.
  • the microorganism when a microorganism is determined to be present, the microorganism is identified.
  • identify a microorganism or “identification of a microorganism” or the “the microorganism is identified” refers to determining the taxonomy of the microorganism.
  • the microorganism is identified to the family level.
  • the microorganism is identified to the genus level.
  • the microorganism is identified to the species level.
  • the microorganism is identified to the strain level.
  • the microorganism is identified. For instance a patient sample or veterinary sample is contacted with a preparation composition forming a contacted patient or veterinary sample. The contacted patient or veterinary sample subsequently undergoes an enrichment protocol followed by a lysis protocol then a protein analysis to detect the presence of a microorganism. In some instances, the detected microorganism is identified. In some instances, the microorganism is identified to the family level. In some instances, the microorganism is identified to the genus level. In some instances, the microorganism is identified to the species level. In some instances, the microorganism is identified to the strain level. In some instances the enrichment protocol encompasses filtration.
  • the enrichment protocol encompasses capture. In some instances the enrichment protocol encompasses centrifugation. In some instance, the lysis protocol is mediated by mechanical force. In other instances, the lysis protocol is by applying a chemical composition. In still other instances, the lysis protocol is mediated by applying mechanical force and a chemical composition. In some instances, the
  • microorganism is a Gram-positive bacteria. In other instances, the microorganism is a Gram negative bacteria. In still other instances, the microorganism is a yeast. While in still further instances, the microorganism is a mold.
  • the protein analysis is by PAGE. In other instances, the protein analysis is by Western blot. In some instances, the protein analysis is by ELISA. In some instances, the protein analysis is by immuno-precipitation. In some instances, the protein analysis is by liquid chromatography. In some instances, the protein analysis is by mass spectrometry. In still other instances, the protein analysis is a combination of two or three or four protein analysis techniques. In some instances, the mass spectrometry is ESI. In other instances, the mass spectrometry is MALDI. In some instances, the protein analysis is by liquid chromatography combined with mass
  • the protein analysis is by liquid chromatography combined with mass spectrometry, wherein the mass spectrometer is ESI.
  • the patient or veterinary sample can be introduced to a culture medium.
  • the culture medium can be a liquid, semi-solid, or solid.
  • the combination of culture medium and patient or veterinary sample can be incubated for minutes, hours, days or weeks, to promote or allow for microorganism growth.
  • the microorganism is a Gram positive bacteria.
  • the microorganism is a Gram-negative bacteria.
  • the microorganism is a yeast. While in still further instances, the microorganism is a mold.
  • an aliquot of a liquid culture medium can be removed and contacted with a preparation composition and subsequently undergoes an enrichment protocol, then a lysis protocol, followed by a protein analysis to detect the presence of a microorganism.
  • the detected microorganism is identified.
  • the microorganism is identified to the family level.
  • the microorganism is identified to the genus level.
  • the microorganism is identified to the species level.
  • the microorganism is identified to the strain level.
  • the enrichment protocol encompasses filtration.
  • the enrichment protocol encompasses capture.
  • the enrichment protocol encompasses centrifugation.
  • the lysis protocol is mediated by mechanical force. In other instances, the lysis protocol is by applying a chemical composition. In still other instances, the lysis protocol is mediated by applying mechanical force and a chemical composition.
  • the protein analysis is by PAGE. In other instances, the protein analysis is by Western blot. In some instances, the protein analysis is by ELISA. In some instances, the protein analysis is by immuno-precipitation. In some instances, the protein analysis is by liquid chromatography.
  • the protein analysis is by mass spectrometry. In still other instances, the protein analysis is a combination of two or three or four protein analysis techniques. In some instances, the mass spectrometry is ESI. In other instances, the mass spectrometry is MALDI. In some instances, the protein analysis is by liquid chromatography combined with mass spectrometry. In some instances, the protein analysis is by liquid chromatography combined with mass spectrometry, wherein the mass spectrometer is ESI.
  • a colony can be chosen from the semi-solid or solid medium and contacted with a preparation composition.
  • the colony can be, in some instances, a Gram-positive bacteria. In other instances, the colony is a Gram negative bacteria. In still other instances, the colony is a yeast. In still further instances, the colony is a mold. This contacted colony from a semi-solid or solid medium subsequently undergoes an enrichment protocol followed by lysis protocol before protein analysis to detect the presence of a microorganism. In some instances, the detected microorganism is identified. In some instances, the microorganism is identified to the family level. In some instances, the microorganism is identified to the genus level.
  • the microorganism is identified to the species level. In some instances, the microorganism is identified to the strain level. In some instances the enrichment protocol encompasses filtration. In some instances the enrichment protocol encompasses capture. In some instances the enrichment protocol encompasses centrifugation. In some instance, the lysis protocol is mediated by mechanical force. In other instances, the lysis protocol is by applying a chemical composition. In still other instances, the lysis protocol is mediated by applying mechanical force and a chemical composition. In some instances, the protein analysis is by PAGE. In other instances, the protein analysis is by Western blot. In some instances, the protein analysis is by ELISA. In some instances, the protein analysis is by immuno-precipitation.
  • the protein analysis is by liquid chromatography. In some instances, the protein analysis is by mass spectrometry. In still other instances, the protein analysis is a combination of two or three or four protein analysis techniques. In some instances, the mass spectrometry is ESI. In other instances, the mass spectrometry is MALDI. In some instances, the protein analysis is by liquid chromatography combined with mass spectrometry. In some instances, the protein analysis is by liquid chromatography combined with mass spectrometry, wherein the mass spectrometer is ESI.
  • an aliquot from a liquid blood culture is contacted with a preparation composition forming a contacted liquid blood culture.
  • the contacted liquid blood culture subsequently undergoes an enrichment protocol followed by a lysis protocol then a protein analysis to detect the presence of a microorganism.
  • the detected microorganism is identified.
  • the microorganism is identified to the family level.
  • the microorganism is identified to the genus level.
  • the microorganism is identified to the species level.
  • the microorganism is identified to the strain level.
  • the enrichment protocol encompasses filtration. In some instances the enrichment protocol encompasses capture.
  • the enrichment protocol encompasses centrifugation.
  • the lysis protocol is mediated by mechanical force.
  • the lysis protocol is by applying a chemical composition.
  • the lysis protocol is mediated by applying mechanical force and a chemical composition.
  • the lysis protocol is mediated by applying mechanical force and a chemical composition.
  • microorganism is a Gram-positive bacteria. In other instances, the microorganism is a Gram negative bacteria. In still other instances, the microorganism is a yeast. While in still further instances, the microorganism is a mold.
  • the protein analysis is by PAGE. In other instances, the protein analysis is by Western blot. In some instances, the protein analysis is by ELISA. In some instances, the protein analysis is by immuno-precipitation. In some instances, the protein analysis is by liquid chromatography. In some instances, the protein analysis is by mass spectrometry. In still other instances, the protein analysis is a combination of two or three or four protein analysis techniques. In some instances, the mass spectrometry is ESI. In other instances, the mass spectrometry is MALDI. In some instances, the protein analysis is by liquid chromatography combined with mass
  • the protein analysis is by liquid chromatography combined with mass spectrometry, wherein the mass spectrometer is ESI.
  • the microorganism from the contacted liquid blood culture undergoes an enrichment protocol followed by a lysis protocol then subject to protein analysis is a Gram-negative bacteria but not a Gram-positive bacteria, a yeast or a mold.
  • the microorganism from the contacted liquid blood culture undergoes an enrichment protocol followed by lysis protocol then subject to protein analysis is a Gram positive bacteria, but not a Gram-negative bacteria, yeast or a mold.
  • the microorganism from the contacted liquid blood culture undergoes an enrichment protocol followed by lysis protocol then subject to protein analysis is a yeast but not a Gram-negative bacteria, a Gram-positive bacteria, or a mold.
  • the microorganism from the contacted liquid blood culture that undergoes an enrichment protocol followed by a lysis protocol then subject to protein analysis is a mold but not a Gram-negative bacteria, a Gram positive bacteria, or a yeast.
  • the detected microorganism is identified.
  • the microorganism is identified to the family level.
  • the microorganism is identified to the genus level.
  • the microorganism is identified to the species level.
  • the microorganism is identified to the strain level.
  • the enrichment protocol encompasses filtration.
  • the enrichment protocol encompasses capture.
  • the enrichment protocol encompasses centrifugation.
  • the lysis protocol is mediated by mechanical force. In other instances, the lysis protocol is by applying a chemical composition. In still other instances, the lysis protocol is mediated by applying mechanical force and a chemical composition.
  • the protein analysis is by PAGE. In other instances, the protein analysis is by Western blot. In some instances, the protein analysis is by ELISA. In some instances, the protein analysis is by immuno-precipitation. In some instances, the protein analysis is by liquid chromatography. In some instances, the protein analysis is by mass spectrometry. In still other instances, the protein analysis is a combination of two or three or four protein analysis techniques. In some instances, the mass spectrometry is ESI.
  • the mass spectrometry is MALDI.
  • the protein analysis is by liquid chromatography combined with mass spectrometry.
  • the protein analysis is by liquid chromatography combined with mass spectrometry, wherein the mass spectrometer is ESI.
  • the methods disclose herein utilize a colony from a plate blood culture.
  • a colony from the plate blood culture is contacted with a preparation composition forming a contacted colony.
  • the colony can be, in some instances, a Gram-positive bacteria.
  • the colony is a Gram-negative bacteria.
  • the colony is a yeast.
  • the colony is a mold. This contacted colony from a semi-solid or solid medium subsequently undergoes an enrichment protocol followed by a lysis protocol and then protein analysis to detect the presence of a microorganism.
  • the detected microorganism is identified.
  • the microorganism is identified to the family level.
  • the microorganism is identified to the genus level. In some instances, the microorganism is identified to the species level. In some instances, the microorganism is identified to the strain level. In some instances the enrichment protocol encompasses filtration. In some instances the enrichment protocol encompasses capture. In some instances the enrichment protocol encompasses centrifugation. In some instance, the lysis protocol is mediated by mechanical force. In other instances, the lysis protocol is by applying a chemical composition. In still other instances, the lysis protocol is mediated by applying mechanical force and a chemical composition. In some instances, the protein analysis is by PAGE. In other instances, the protein analysis is by Western blot. In some instances, the protein analysis is by ELISA.
  • the protein analysis is by immuno-precipitation. In some instances, the protein analysis is by liquid chromatography. In some instances, the protein analysis is by mass spectrometry. In still other instances, the protein analysis is a combination of two or three or four protein analysis techniques. In some instances, the mass spectrometry is ESI.
  • the mass spectrometry is MALDI.
  • the protein analysis is by liquid chromatography combined with mass spectrometry.
  • the protein analysis is by liquid chromatography combined with mass spectrometry, wherein the mass spectrometer is ESI.
  • the preparation composition encompasses a proteolytic enzyme, also referred to as a protease, and in some instances a detergent. Aside from a proteolytic enzyme, and in some instances a detergent, the preparation composition encompasses other components, such as biological buffers, in which the integrity of microorganism cell walls or membranes are largely maintained. Examples of suitable biological buffers would be isotonic, or nearly so, and at neutral, or near neutral, pH. In some instances, the biological buffer would be a phosphate buffer, such as a sodium/potassium phosphate buffer. In other instances, the biological buffer would be a carbonate buffer.
  • proteases refers to an enzyme exhibiting the ability to hydrolyze peptides or substrates having peptide linkages.
  • proteases of mixed nucleophile, superfamily A PA clan. This clan contains both cysteine and serine proteases.
  • cysteine and serine proteases the chymotrypsin-like serine proteases are of particular interest to the compositions and methods disclosed herein.
  • trypsin is of interest.
  • serine protease proteinase K is also of interest.
  • the amount of protease present in the preparation composition can be from 0.1 - 100 pg, from 0.1 - 75 pg, 0.1 - 50 pg, 0.1 - 25 pg, 0.1 - 10 pg, or 0.1 - 5 pg. In some instances, the amount of protease present is 1 pg, 2 pg, 3 pg, 4 pg, 5 pg, 6 pg, 7 pg, 8 pg, 9 pg, 10 pg, 11 pg, or 12 pg.
  • the amount of protease present is 0.1 pg, 0.2 pg, 0.3 pg, 0.4 pg, 0.5 pg, 0.6 pg, 0.7 pg, 0.8 pg, or 0.9 pg. In still other instances, the amount of protease present is 0.6 pg or about 0.6 pg.
  • the preparation composition in addition to a protease, encompasses a detergent.
  • Detergents of particular interest in the compositions and methods disclosed herein are amphipathic glycosides, and in particular saponin.
  • the microorganism is identified. For instance a patient sample or veterinary sample is contacted with a preparation composition forming a contacted patient or veterinary sample. The contacted patient or veterinary sample subsequently undergoes an enrichment protocol followed by a lysis protocol then a protein analysis to detect the presence of a microorganism.
  • the preparation composition encompasses trypsin.
  • the preparation composition encompasses saponin.
  • the preparation composition encompasses trypsin and saponin.
  • the preparation composition encompasses proteinase K.
  • the preparation composition encompasses proteinase K and saponin.
  • the detected microorganism is identified.
  • the microorganism is identified to the family level. In some instances, the microorganism is identified to the genus level. In some instances, the microorganism is identified to the species level. In some instances, the microorganism is identified to the strain level. In some instances the enrichment protocol encompasses filtration. In some instances the enrichment protocol encompasses capture. In some instances the enrichment protocol encompasses centrifugation. In some instance, the lysis protocol is mediated by mechanical force. In other instances, the lysis protocol is by applying a chemical composition. In still other instances, the lysis protocol is mediated by applying mechanical force and a chemical composition. In some instances, the microorganism is a Gram-positive bacteria.
  • the microorganism is a Gram-negative bacteria. In still other instances, the microorganism is a yeast. While in still further instances, the microorganism is a mold.
  • the protein analysis is by PAGE. In other instances, the protein analysis is by Western blot. In some instances, the protein analysis is by ELISA. In some instances, the protein analysis is by immuno-precipitation. In some instances, the protein analysis is by liquid
  • the protein analysis is by mass spectrometry. In still other instances, the protein analysis is a combination of two or three or four protein analysis techniques. In some instances, the mass spectrometry is ESI. In other instances, the mass spectrometry is MALDI. In some instances, the protein analysis is by liquid chromatography combined with mass spectrometry. In some instances, the protein analysis is by liquid chromatography combined with mass spectrometry, wherein the mass spectrometer is ESI.
  • the patient or veterinary sample can be introduced to a culture medium.
  • the culture medium can be a liquid, semi-solid, or solid.
  • the combination of culture medium and patient or veterinary sample can be incubated for minutes, hours, days or weeks, to promote or allow for microorganism growth.
  • the microorganism is a Gram positive bacteria.
  • the microorganism is a Gram-negative bacteria.
  • the microorganism is a yeast. While in still further instances, the microorganism is a mold.
  • an aliquot of a liquid culture medium can be removed and contacted with a preparation composition and subsequently undergoes an enrichment protocol, then a lysis protocol, followed by a protein analysis to detect the presence of a microorganism.
  • the preparation composition encompasses trypsin.
  • the preparation composition encompasses saponin.
  • the preparation composition encompasses trypsin and saponin.
  • the preparation composition encompasses proteinase K.
  • the preparation composition encompasses proteinase K and saponin.
  • the detected microorganism is identified.
  • the microorganism is identified to the family level.
  • the microorganism is identified to the genus level. In some instances, the microorganism is identified to the species level. In some instances, the microorganism is identified to the strain level. In some instances the enrichment protocol encompasses filtration. In some instances the enrichment protocol encompasses capture. In some instances the enrichment protocol encompasses centrifugation. In some instance, the lysis protocol is mediated by mechanical force. In other instances, the lysis protocol is by applying a chemical composition. In still other instances, the lysis protocol is mediated by applying mechanical force and a chemical composition. In some instances, the protein analysis is by PAGE. In other instances, the protein analysis is by Western blot. In some instances, the protein analysis is by ELISA.
  • the protein analysis is by immuno-precipitation. In some instances, the protein analysis is by liquid chromatography. In some instances, the protein analysis is by mass spectrometry. In still other instances, the protein analysis is a combination of two or three or four protein analysis techniques. In some instances, the mass spectrometry is ESI. In other instances, the mass spectrometry is MALDI. In some instances, the protein analysis is by liquid chromatography combined with mass spectrometry. In some instances, the protein analysis is by liquid chromatography combined with mass spectrometry, wherein the mass spectrometer is ESI.
  • a colony can be chosen from the semi-solid or solid medium and contacted with a preparation composition.
  • the preparation composition encompasses trypsin.
  • the preparation composition encompasses saponin.
  • the preparation composition encompasses trypsin and saponin.
  • the colony can be, in some instances, a Gram-positive bacteria.
  • the colony is a Gram-negative bacteria.
  • the colony is a yeast.
  • the colony is a mold. This contacted colony from a semi-solid or solid medium subsequently undergoes an enrichment protocol followed by lysis protocol before protein analysis to detect the presence of a microorganism.
  • the detected microorganism is identified. In some instances, the microorganism is identified to the family level. In some instances, the microorganism is identified to the genus level. In some instances, the microorganism is identified to the species level. In some instances, the microorganism is identified to the strain level. In some instances the enrichment protocol encompasses filtration. In some instances the enrichment protocol encompasses capture. In some instances the enrichment protocol encompasses centrifugation. In some instance, the lysis protocol is mediated by mechanical force. In other instances, the lysis protocol is by applying a chemical composition. In still other instances, the lysis protocol is mediated by applying mechanical force and a chemical composition. In some instances, the protein analysis is by PAGE.
  • the protein analysis is by Western blot. In some instances, the protein analysis is by ELISA. In some instances, the protein analysis is by immuno-precipitation. In some instances, the protein analysis is by liquid chromatography. In some instances, the protein analysis is by mass spectrometry. In still other instances, the protein analysis is a combination of two or three or four protein analysis techniques. In some instances, the mass spectrometry is ESI.
  • the mass spectrometry is MALDI.
  • the protein analysis is by liquid chromatography combined with mass spectrometry.
  • the protein analysis is by liquid chromatography combined with mass spectrometry, wherein the mass spectrometer is ESI.
  • an aliquot from a liquid blood culture is contacted with a preparation composition forming a contacted liquid blood culture.
  • the preparation composition encompasses trypsin.
  • the preparation composition encompasses saponin.
  • the preparation composition encompasses trypsin and saponin.
  • the preparation composition encompasses proteinase K.
  • the preparation composition encompasses proteinase K and saponin.
  • the contacted liquid blood culture subsequently undergoes an enrichment protocol followed by a lysis protocol then a protein analysis to detect the presence of a microorganism.
  • the detected microorganism is identified.
  • the microorganism is identified to the family level.
  • the microorganism is identified to the genus level. In some instances, the microorganism is identified to the species level. In some instances, the microorganism is identified to the strain level. In some instances the enrichment protocol encompasses filtration. In some instances the enrichment protocol encompasses capture. In some instances the enrichment protocol encompasses centrifugation. In some instance, the lysis protocol is mediated by mechanical force. In other instances, the lysis protocol is by applying a chemical composition. In still other instances, the lysis protocol is mediated by applying mechanical force and a chemical composition. In some instances, the microorganism is a Gram-positive bacteria. In other instances, the microorganism is a Gram-negative bacteria. In still other instances, the microorganism is a yeast.
  • the microorganism is a mold.
  • the protein analysis is by PAGE. In other instances, the protein analysis is by Western blot. In some instances, the protein analysis is by ELISA. In some instances, the protein analysis is by immuno-precipitation. In some instances, the protein analysis is by liquid chromatography. In some instances, the protein analysis is by mass spectrometry. In still other instances, the protein analysis is a combination of two or three or four protein analysis techniques. In some instances, the mass spectrometry is ESI. In other instances, the mass spectrometry is MALDI. In some instances, the protein analysis is by liquid chromatography combined with mass spectrometry. In some instances, the protein analysis is by liquid chromatography combined with mass spectrometry, wherein the mass spectrometer is ESI.
  • the microorganism from the contacted liquid blood culture undergoes an enrichment protocol followed by a lysis protocol then subject to protein analysis is a Gram-negative bacteria but not a Gram-positive bacteria, a yeast or a mold.
  • the microorganism from the contacted liquid blood culture undergoes an enrichment protocol followed by lysis protocol then subject to protein analysis is a Gram positive bacteria, but not a Gram-negative bacteria, yeast or a mold.
  • the microorganism from the contacted liquid blood culture undergoes an enrichment protocol followed by lysis protocol then subject to protein analysis is a yeast but not a Gram-negative bacteria, a Gram-positive bacteria, or a mold.
  • the microorganism from the contacted liquid blood culture that undergoes an enrichment protocol followed by a lysis protocol then subject to protein analysis is a mold but not a Gram-negative bacteria, a Gram positive bacteria, or a yeast.
  • the detected microorganism is identified.
  • the microorganism is identified to the family level.
  • the microorganism is identified to the genus level.
  • the microorganism is identified to the species level.
  • the microorganism is identified to the strain level.
  • the enrichment protocol encompasses filtration.
  • the enrichment protocol encompasses capture.
  • the enrichment protocol encompasses centrifugation.
  • the lysis protocol is mediated by mechanical force. In other instances, the lysis protocol is by applying a chemical composition. In still other instances, the lysis protocol is mediated by applying mechanical force and a chemical composition.
  • the protein analysis is by PAGE. In other instances, the protein analysis is by Western blot. In some instances, the protein analysis is by ELISA. In some instances, the protein analysis is by immuno-precipitation. In some instances, the protein analysis is by liquid chromatography. In some instances, the protein analysis is by mass spectrometry. In still other instances, the protein analysis is a combination of two or three or four protein analysis techniques. In some instances, the mass spectrometry is ESI.
  • the mass spectrometry is MALDI.
  • the protein analysis is by liquid chromatography combined with mass spectrometry.
  • the protein analysis is by liquid chromatography combined with mass spectrometry, wherein the mass spectrometer is ESI.
  • the methods disclose herein utilize a colony from a plate blood culture.
  • a colony from the plate blood culture is contacted with a preparation composition forming a contacted colony.
  • the preparation composition encompasses trypsin.
  • the preparation composition encompasses saponin.
  • the preparation composition encompasses trypsin and saponin.
  • the preparation composition encompasses proteinase K.
  • the preparation composition encompasses proteinase K and saponin.
  • the colony can be, in some instances, a Gram-positive bacteria. In other instances, the colony is a Gram-negative bacteria. In still other instances, the colony is a yeast. In still further instances, the colony is a mold. This contacted colony from a semi-solid or solid medium subsequently undergoes an enrichment protocol followed by a lysis protocol and then protein analysis to detect the presence of a microorganism. In some instances, the detected microorganism is identified. In some instances, the microorganism is identified to the family level. In some instances, the microorganism is identified to the genus level. In some instances, the microorganism is identified to the species level.
  • the microorganism is identified to the strain level.
  • the enrichment protocol encompasses filtration. In some instances the enrichment protocol encompasses capture. In some instances the enrichment protocol encompasses centrifugation.
  • the lysis protocol is mediated by mechanical force. In other instances, the lysis protocol is by applying a chemical composition. In still other instances, the lysis protocol is mediated by applying mechanical force and a chemical composition.
  • the protein analysis is by PAGE. In other instances, the protein analysis is by Western blot. In some instances, the protein analysis is by ELISA. In some instances, the protein analysis is by immuno-precipitation. In some instances, the protein analysis is by liquid chromatography.
  • the protein analysis is by mass spectrometry. In still other instances, the protein analysis is a combination of two or three or four protein analysis techniques. In some instances, the mass spectrometry is ESI. In other instances, the mass spectrometry is MALDI. In some instances, the protein analysis is by liquid chromatography combined with mass spectrometry. In some instances, the protein analysis is by liquid chromatography combined with mass spectrometry, wherein the mass spectrometer is ESI.
  • Group A Streptococcus (strain PI 25) was introduced into a culture bottle containing blood. After overnight incubation to allow for bacterial growth, 150 m ⁇ was removed from the blood culture bottle and added to a tube containing one of four proteases (trypsin, pepsin, papain or proteinase K) and 5% saponin. After this, the absorbance of the solution was measured at 395 nm. Figure 1 shows a profound reduction in hemoglobin after trypsin treatment.
  • preparation of vessels for subsequent use are produced by first dissolving trypsin powder in a solution containing saponin. After 30 minutes incubation, the trypsin/saponin mixture is filtered, using a filter with a 0.2 pm mesh. 3 pi of the
  • trypsin/saponin mixture 200 mg/ml trypsin and 50 mg/ml saponin
  • trypsin/saponin mixture 200 mg/ml trypsin and 50 mg/ml saponin
  • a blood culture sample (150 pi) is removed from a blood culture bottle and added to a treatment vessel containing the dried trypsin/saponin mixture.
  • the treatment vessel, with the added blood culture sample is then subjected to sonication.
  • the sonication is thought to act, in part, to mix the contents of the treatment vessel.
  • the treatment vessel is subjected to centrifugation (2 minutes, 12,000 g).
  • the resulting supernatant is discarded, and a 10 mM Na/K phosphate wash buffer is added to the treatment vessel.
  • the vessel is once again subjected to sonication; 12 seconds of sonication, then 5 seconds of no sonication, followed by 12 seconds of sonication.
  • the treatment vessel is once again subjected to centrifugation (45 seconds, 12,000g) with the resulting supernatant discarded. After discarding the supernatant, a lysis solution (50% Formic acid, 25% Acetonitrile, 25% H2O) is added to the treatment vessel. The treatment vessel is then subjected to 45 seconds of sonication, after which a storage solution containing 50% Acetonitrile, 50 % H2O is added and the treatment vessel is subjected to 10 seconds of sonication.
  • a lysis solution 50% Formic acid, 25% Acetonitrile, 25% H2O
  • the treatment vessel In preparation for liquid chromatography-mass spectrometric analysis, the treatment vessel, following the 10 seconds of sonication, is subjected to centrifugation (5 minutes, 12,000g). The resulting supernatant is removed and applied to a liquid chromatography column for downstream protein analysis.

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Abstract

L'identification directe de micro-organismes à partir de bouillons de culture de sang positifs par analyse de protéines reste un défi. Bien que les bouillons de culture de sang positifs soient généralement mono-bactériens ou mono-fongiques, la présence de protéines provenant de globules rouges, de leucocytes et du sérum parasite l'analyse. L'invention concerne des compositions uniques et des procédés pour surmonter les défis présentés par les protéines parasites dans des cultures de sang positives. Les compositions et les procédés de l'invention sont notamment applicables à des analyses par spectrométrie de masse pour l'identification de micro-organismes dans une culture de sang.
PCT/IB2020/053234 2019-04-05 2020-04-03 Procédés de spectrométrie de masse et compositions pour l'identification de microbes à partir de sang par réduction de protéines parasites WO2020202113A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1877571A2 (fr) * 2005-05-03 2008-01-16 Acolyte Biomedica Limited Distinction de cellules dans un echantillon par inactivation d'enzyme extracellulaire avant liberation d'une enzyme intracellulaire
US20140186832A1 (en) * 2011-05-20 2014-07-03 Martin Fuchs Selective ultrasonic lysis of blood and other biological fluids and tissues
US20140221223A1 (en) * 2012-12-10 2014-08-07 Advandx, Inc. Use of Probes for Mass Spectrometric Identification and Resistance Determination of Microorganisms or Cells
WO2018037089A1 (fr) * 2016-08-25 2018-03-01 Thermo Fisher Scientific Oy Procédés et ensembles de spectrométrie de masse pour identifier un micro-organisme

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1877571A2 (fr) * 2005-05-03 2008-01-16 Acolyte Biomedica Limited Distinction de cellules dans un echantillon par inactivation d'enzyme extracellulaire avant liberation d'une enzyme intracellulaire
US20140186832A1 (en) * 2011-05-20 2014-07-03 Martin Fuchs Selective ultrasonic lysis of blood and other biological fluids and tissues
US20140221223A1 (en) * 2012-12-10 2014-08-07 Advandx, Inc. Use of Probes for Mass Spectrometric Identification and Resistance Determination of Microorganisms or Cells
WO2018037089A1 (fr) * 2016-08-25 2018-03-01 Thermo Fisher Scientific Oy Procédés et ensembles de spectrométrie de masse pour identifier un micro-organisme

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MAGDALENA KLIEM ET AL: "The essence on mass spectrometry based microbial diagnostics", CURRENT OPINION IN MICROBIOLOGY, vol. 15, no. 3, 28 February 2012 (2012-02-28), pages 397 - 402, XP028449300, ISSN: 1369-5274, [retrieved on 20120228], DOI: 10.1016/J.MIB.2012.02.006 *

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