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WO2018147472A1 - Biomarqueur sanguin - Google Patents

Biomarqueur sanguin Download PDF

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WO2018147472A1
WO2018147472A1 PCT/JP2018/005603 JP2018005603W WO2018147472A1 WO 2018147472 A1 WO2018147472 A1 WO 2018147472A1 JP 2018005603 W JP2018005603 W JP 2018005603W WO 2018147472 A1 WO2018147472 A1 WO 2018147472A1
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blood
acid
concentration
group
microorganism
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Japanese (ja)
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賢 石井
宜広 磯貝
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Keio University
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Keio University
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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor

Definitions

  • the present invention relates to a microbial blood biomarker and a method for determining microbial infection using the blood biomarker.
  • infectious diseases is a general term for unwanted reactions (diseases) that occur in a host due to infection with pathogens such as bacteria, fungi, viruses, parasites, and abnormal prions. Many infections do not show symptoms even after infection, and many infections develop symptoms after a certain period of time. Even today, infectious diseases dealing with diagnosis, treatment, and prevention of infectious diseases are developing against the background of advances in microbiology, immunology, pharmacology, internal medicine, external science, public health, etc. Overall, infections still account for the highest proportion of death. Diagnosing infections at an early stage and performing necessary treatment at an early stage is extremely important in terms of preventing the onset and seriousness of infections.
  • Infectious diseases of microorganisms such as bacteria may occur after surgery, for example.
  • the progress of surgical therapy is remarkable in various fields such as the orthopedic field, and it contributes to recovery of functional aspects such as exercise, pain, and viscera of many patients and prolongation of life prognosis.
  • post-operative infection is a complication that greatly reduces the outcome of treatment, and treatment by early detection is very important to minimize the effect.
  • postoperative infections are comprehensive due to worsening of pain at the surgical site, clinical symptoms such as discharge of pus, fever, and imaging findings, as well as inflammation findings of blood tests using leukocytes and C-reactive protein (CRP). Diagnosis is made. However, at present, it takes 1-2 weeks or more after the operation until both clinical symptoms and blood tests become apparent.
  • infections may develop after trauma or in susceptible hosts.
  • infectious diseases may develop in patients with chronic inflammatory diseases such as autoimmune diseases, but early diagnosis thereof is difficult.
  • JP 2011-95052 describes a disease marker for infectious diseases comprising ventraxin 3 and azulogisin, and a method for detecting the presence or absence of an infectious disease using the disease marker. Specifically disclosed is a method for detecting a complex of ventaxine 3 and azulodicin using an anti-ventlaxin 3 antibody, which involves a complicated process depending on the specificity of the antibody. Also, the sensitivity is not good.
  • biomarkers specific for diagnosing microbial infectious diseases have not yet been provided with biomarkers that are practical and capable of identifying infectious diseases at an early stage, despite their great clinical significance.
  • the present inventors As a result of diligent research to solve the above problems, the present inventors, as a result of infecting microorganisms, began to detect five specific substances ( ⁇ -ketoglutaric acid) in the TCA cycle (citric acid cycle) from the early stage of infection. , Succinyl-CoA, succinic acid, fumaric acid, and malic acid) were found to have a reduced blood concentration compared to the case of no infection, resulting in the present invention.
  • TCA cycle citric acid cycle
  • the present invention includes the following aspects.
  • a microbial blood biomarker comprising one or more substances selected from the group consisting of ⁇ -ketoglutaric acid, succinyl-CoA, succinic acid, fumaric acid and malic acid in the TCA cycle.
  • the blood concentration of one or more substances selected from the group consisting of ⁇ -ketoglutaric acid, succinyl-CoA, succinic acid, fumaric acid and malic acid in the TCA cycle in the blood after one day after the operation is The blood biomarker according to any one of aspects 1-3, wherein the blood biomarker according to any one of aspects 1-3 is determined to be infected with a microorganism when the concentration is reduced to 60% or less of the blood concentration of the previous corresponding substance.
  • Aspect 5 The blood concentration of one or more substances selected from the group consisting of ⁇ -ketoglutaric acid, succinyl-CoA, succinic acid, fumaric acid and malic acid in the TCA cycle in the blood 1-30 days after surgery,
  • the blood biomarker according to any one of aspects 1-3 wherein the blood biomarker according to any one of aspects 1-3 is determined to be infected with a microorganism when the concentration of the corresponding substance before surgery is reduced to 60% or less.
  • the blood concentration of one or more substances selected from the group consisting of ⁇ -ketoglutaric acid, succinyl-CoA, succinic acid, fumaric acid and malic acid in the TCA cycle in blood 1-14 days after surgery is The blood biomarker according to any one of aspects 1-3, wherein the blood biomarker according to any one of aspects 1-3 is determined to be infected with a microorganism when the concentration of the corresponding substance before surgery is reduced to 60% or less.
  • Aspect 7 The blood concentration of one or more substances selected from the group consisting of ⁇ -ketoglutaric acid, succinyl-CoA, succinic acid, fumaric acid and malic acid in the TCA cycle in the blood 1-7 days after the operation,
  • the blood biomarker according to any one of aspects 1-3 wherein the blood biomarker according to any one of aspects 1-3 is determined to be infected with a microorganism when the concentration of the corresponding substance before surgery is reduced to 60% or less.
  • the blood concentration of one or more substances selected from the group consisting of ⁇ -ketoglutaric acid, succinyl-CoA, succinic acid, fumaric acid and malic acid in the TCA cycle in the blood after one day after the operation is The blood biomarker according to any one of aspects 1 to 7, wherein the blood biomarker is determined to be infected with a microorganism when the concentration is reduced to 50% or less of the previous corresponding substance in blood.
  • the blood concentration of one or more substances selected from the group consisting of ⁇ -ketoglutaric acid, succinyl-CoA, succinic acid, fumaric acid and malic acid in the TCA cycle in the blood after one day after the operation is The blood biomarker according to any one of aspects 1 to 7, wherein the blood biomarker according to any one of aspects 1 to 7 is judged to be infected with a microorganism when the blood concentration of the previous corresponding substance is reduced to 30% or less.
  • the blood concentration of one or more substances selected from the group consisting of ⁇ -ketoglutaric acid, succinyl-CoA, succinic acid, fumaric acid and malic acid in the TCA cycle in the blood after one day after the operation is The blood biomarker according to any one of aspects 1 to 7, wherein the blood biomarker according to any one of aspects 1 to 7 is judged to be infected with a microorganism when the blood concentration of the corresponding substance is decreased to 10% or less.
  • the blood concentration of one or more substances selected from the group consisting of ⁇ -ketoglutaric acid, succinic acid, and fumaric acid in the TCA circuit in the blood after one day after surgery is the blood of the corresponding substance before surgery.
  • the blood concentration of one or more substances selected from the group consisting of ⁇ -ketoglutaric acid, succinic acid, and fumaric acid in the TCA circuit in the blood after one day after surgery is the blood of the corresponding substance before surgery.
  • a method for determining microbial infection in surgery (1) Measure blood concentration of one or more substances selected from the group consisting of ⁇ -ketoglutaric acid, succinyl-CoA, succinic acid, fumaric acid, and malic acid in the TCA circuit in the subject before surgery. , (2) In the subject after surgery, measure the blood concentration of the substance measured in step (1), (3) If the concentration measured in (2) is lower than the concentration measured in (1), it is determined that the organism is infected. The determination method.
  • the blood concentration of the substance measured in the step (1) is measured in the subject after the first day after the operation, and the blood concentration measured in (2) is the blood concentration measured in (1).
  • the blood concentration of the substance measured in the step (1) is measured in a subject 1 to 30 days after the operation, and the blood concentration measured in (2) is the blood concentration measured in (1).
  • the blood concentration of the substance measured in the step (1) is measured in a subject after 1 day after the operation, and the blood concentration measured in (2) is more than the blood concentration measured in (1). 25.
  • a method for determining microbial infection in a subject comprising: Measuring the blood concentration of one or more substances selected from the group consisting of ⁇ -ketoglutarate, succinate and fumaric acid in the TCA cycle; The said determination method of determining that it is infected with microorganisms, when the blood concentration of the said substance is below a detection limit.
  • the present invention is based on the discovery that in the analysis of metabolites, substances of the TCA cycle that have not been focused on until now have changed significantly.
  • the particular substance of the TCA cycle is very effective as a biomarker of microbial infection because of the marked difference that it is significantly reduced or hardly detectable.
  • detection and determination can be performed at an early stage of infection, which is impossible to detect with a conventional marker.
  • the biomarker of the present invention is based on the blood concentration of these substances, the measurement is simple, rapid and inexpensive.
  • FIG. 1A shows an instrument (product name: IVIS-Lumina LT) (manufactured by Summit Pharmaceuticals International Co., Ltd.) that can capture weak light in the living body using the ultra-sensitive CCD camera used in the optical imaging of the example. It is a photograph.
  • FIG. 1B is a photographic diagram showing the degree of bacterial infection by optical imaging in a femur osteomyelitis model mouse infused with luminescent bacteria.
  • the ultra-sensitive CCD camera detects the faint light emitted by the luminescent bacteria from the body surface of the animal, and the site where the bacteria are present is rendered as a light image with a pseudo color.
  • the red part has more bacteria and the part has less bacteria.
  • FIG. 2 is a diagram showing the correlation between the amount of bacteria and the amount of luminescence.
  • the amount of bacteria ⁇ 10 7 CFU
  • the amount of luminescence ⁇ 10 6 photons / second / cm 2
  • FIG. 3 is a schematic diagram showing a process of producing a femur osteomyelitis model mouse in the Example and confirming infection by optical imaging.
  • FIG. 4 is a table in which “infectious group”, “sham group”, and “control group” in the examples are arranged with or without surgery for drilling the femur and with or without injection of luminous bacteria.
  • FIG. 5 is a diagram showing conditions for selecting blood biomarkers for determining the infection of microorganisms based on the results of metabolomic analysis. A substance that is not different between the control group and the sham group and that has a high (or low) value only in the infected group was selected because it is important as a blood biomarker that can determine infection after surgery early.
  • FIG. 6 is a diagram showing the results of metabolomic analysis. Red (+1 to +3) indicates a high value, and green (-1 to -3) indicates a low value.
  • FIG. 7 is a graph showing the relative values of the sham group and the infected group when the control is set to 1.0 for 12 substances having high values in the infected group. For each substance, the results of “control group”, “sham group”, and “infection group” are shown in order from the top of the graph.
  • FIG. 8 is a graph showing the relative values of the sham group and the infected group when the control is 1.0 for 48 substances that have a low value in the infected group.
  • FIG. 9 is a schematic diagram of ⁇ -oxidation.
  • NAD + nicotinamide adenine dinucleotide
  • FIG. 10 is a schematic diagram of a TCA circuit.
  • FIG. 11 shows how the substances in the TCA circuit differ between the “control group”, “sham group”, and “infection group” as a result of the metabolome analysis, and the results of FIGS. 7 and 8 are shown in the TCA circuit diagram. It is displayed. In the graph regarding each substance, blue (left), green (center), and red (right) indicate the results of “control group”, “sham group”, and “infection group”, respectively. It is understood that substances downstream of ⁇ -ketoglutaric acid (2-OG) in the TCA cycle are depleted only in the “infected group”, while being maintained in the “control group” and the “sham group”.
  • FIG. 12 is a schematic diagram for understanding the technical idea of the present invention.
  • Citric acid is a UV absorption measurement kit (F-kit citric acid, manufactured by JK International)
  • succinic acid is a colorimetric measurement kit (Succinate Colorometric Assay Kit), manufactured by BioVision ).
  • the inventors of the present invention were 12 osteomyelitis model mice (infection group) that underwent surgery to inject bacteria into the femur in 12-week-old mice, and 12 mice (sham group) that were subjected to the same operation but did not inject bacteria. ), 12 animals (control group) that did not perform surgery. On the third day after surgery, blood was collected from the abdominal aorta, and metabolomic analysis was performed to detect about 1200 metabolites. As a result, 12 types of substances having specifically high values and 48 types of substances having specifically low values were identified in osteomyelitis model mice.
  • the present invention includes, but is not limited to, the following aspects. 1. TECHNICAL FIELD
  • the present invention relates to a blood biomarker in a microorganism.
  • the blood biomarker of the present invention comprises one or more substances selected from the group consisting of ⁇ -ketoglutaric acid, succinyl-CoA, succinic acid, fumaric acid and malic acid in the TCA cycle.
  • TCA cycle is the most important biochemical reaction circuit related to aerobic metabolism, and is found in all living organisms that perform enzyme respiration. Acetyl-CoA produced by glycolysis and ⁇ -oxidation of fatty acids is incorporated into this circuit and oxidized to produce ATP, NADH used in the electron transfer system, and the like, enabling efficient energy production (Fig. 10). It also supplies biosynthetic precursors such as amino acids.
  • a TCA circuit may also be referred to as a TCA cycle, a tricarboxylic acid circuit, a citric acid circuit, a Krebs circuit, and the like.
  • the present inventors have found that ⁇ -ketoglutaric acid, succinyl-CoA, succinic acid, fumaric acid and malic acid in the TCA cycle are decreased in microbially infected mice compared to uninfected mice.
  • the inventor came up with the present invention.
  • the present invention is a blood biomarker composed of one or more substances selected from the group consisting of ⁇ -ketoglutarate, succinyl-CoA, succinic acid, fumaric acid and malic acid, and one or more of these substances More blood levels are reduced compared to normal values (before infection), or one or more of these substances are depleted, ie blood levels are below the detection limit If so, determine that the person is infected with a microorganism.
  • the blood biomarker of the present invention is one or more, two or more, three or more, four or more among five substances selected from the group consisting of ⁇ -ketoglutaric acid, succinyl-CoA, succinic acid, fumaric acid and malic acid. It consists of the above or five substances.
  • the blood biomarker of the present invention is one or more substances selected from the group consisting of ⁇ -ketoglutaric acid, succinic acid and fumaric acid.
  • the blood biomarker of the present invention consists of one or more, two or more or three substances among three substances selected from the group consisting of ⁇ -ketoglutaric acid, succinic acid and fumaric acid.
  • the blood biomarker of the present invention is a biomarker capable of determining the infection of a target microorganism.
  • microorganism is a generic term that refers to a microscopic organism having a size that cannot be distinguished with the naked eye and that can be observed with a microscope or the like.
  • bacteria eubacteria
  • archaea fungi
  • eukaryotes such as protists, slime molds and algae
  • small animals such as rotifers
  • the “microorganism” that can be determined using the blood biomarker of the present invention is not limited to a living organism in a narrow sense, and may be a minute substance that can infect an object (living body) and can cause some kind of change. Therefore, a substance capable of infecting a living body such as “virus” is also included in the “microorganism” in the present invention and the present specification.
  • microorganism in the present invention is preferably selected from the group consisting of bacteria, fungi and viruses. More preferably, it is a bacterium. Bacteria are one aspect of microorganisms that are most concerned about infection (to avoid infection) in surgical procedures.
  • Bacteria “Bacteria” is a prokaryote having a cell membrane composed of fatty acid ester of sn-glycerol triphosphate, and is a group of organisms including Escherichia coli, Bacillus subtilis, cyanobacteria, etc. Causes infectious diseases such as animals.
  • Archaea which has a cell membrane composed of an isobrenoid ether of sn-glycerol monophosphate, belong to a different strain from bacteria (eubacteria).
  • Bacteria are classified by Gram staining (difference between cell positive and negative bacteria due to cell wall difference), structural or anatomical properties (direct observation), chemical properties (lipid structure, etc.) The
  • the bacterium is a Gram-positive bacterium.
  • Gram-positive bacteria is a general term for bacteria that are stained dark blue or purple by Gram staining, and generally has a feature that there is a thick peptide glucan layer having no outer membrane. Many Gram-positive bacteria are classified as Firmictes and Radiomycota. The genus of bacteria belonging to Gram-positive bacteria includes Staphylococcus, Propionibacterium (Acne, etc.), Enterococcus (Enterococcus), Listeria (Listeria).
  • Streptococcus Streptococcus, Green Streptococcus, ⁇ -hemolytic Streptococcus, etc.
  • Clostridium Botulinum, Tetanus, etc.
  • Bacillus Bacillus cereus, Bacillus subtilis, etc.
  • the genus Staphylococcus is a Gram-positive cocci with a diameter of about 1 ⁇ m, and is a facultative and anaerobic organic vegetative bacterium with an irregular arrangement of grape tufts.
  • bacteria belonging to the genus Staphylococcus are classified into 35 types, and are roughly divided into three species: “Staphylococcus aureus”, “Staphylococcus epidermidis”, and “Rotogenic staphylococci”.
  • Staphylococcus aureus is a permanent bacterium in the skin and digestive tract of humans and animals, and various epidermic infections such as abscesses and food poisoning, as well as severe and fatal diseases such as pneumonia, meningitis and sepsis It is also the cause of common infections. Infection with Staphylococcus aureus is one of the most susceptible to infections in surgical procedures (to avoid infection), which is often found in sites confined to the epidermis and tissues just below it.
  • Staphylococcus aureus includes drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin Staphylococcus aureus (VRSA) in addition to methicillin-sensitive Staphylococcus aureus (MSSA) that is resistant to the antibiotic methicillin.
  • MRSA methicillin-resistant Staphylococcus aureus
  • VRSA vancomycin Staphylococcus aureus
  • MSSA methicillin-sensitive Staphylococcus aureus
  • Staphylococcus epidermidis is mainly present in the nasal cavity and epidermis. It is usually non-pathogenic and is a barrier that protects the epidermis from other pathogenic bacteria and plays a role in keeping the epidermis healthy, but it may cause pathogenicity when it enters the body.
  • MSSE methicillin-sensitive Staphylococcus epidermidis
  • MRSE methicillin-resistant Staphylococcus epidermidis
  • Propionibacterium genus is an spore-free anaerobic bacterium that is a resident bacterium on the skin and mucous membranes and produces propionic acid from sugar.
  • Representative myomas include acne (Propionibacterium acnes), which is said to be the cause of acne.
  • Enterococci is a general term for about 20 species belonging to the genus Enterococcus rather than a specific bacterial species.
  • Enterococci are a group of resident bacteria mainly present in the intestinal tract of mammals including humans and take the form of cocci. This is a facultative anaerobic and Gram-positive streptococci that is generally detected in the intestines of healthy humans and that breaks down glucose, maltose, lactose, and sucrose and withstands heating at 60 ° C for 30 minutes .
  • VRE vancomycin-resistant enterococci
  • the bacterium is preferably selected from the group consisting of Staphylococcus aureus, Staphylococcus epidermidis, acne and enterococci. More preferably, the bacterium is S. aureus or S. epidermidis. In one embodiment, S. aureus. In one embodiment, methicillin resistant Staphylococcus aureus (MRSA) or methicillin resistant Staphylococcus aureus (MRSE).
  • MRSA methicillin resistant Staphylococcus aureus
  • MRSE methicillin resistant Staphylococcus aureus
  • the bacterium is a “gram-negative bacterium”.
  • Gram-negative bacteria is a general term for bacteria that do not stain purple (crystal violet) in Gram staining and appear red or pink.
  • Gram-negative bacteria have characteristics such as a cytoplasmic membrane and a thin peptidoglycan layer (thicker layer in Gram-positive bacteria). Certain components of the cell wall are involved in the pathogenicity of Gram-negative bacteria.
  • the outer leaf of the membrane of Gram-negative bacteria is composed of complex lipopolysaccharide (LPS) whose lipid site functions as an endotoxin. When endotoxin enters the circulatory system, it causes fever, respiratory prompting and hypotension. Death may occur if endotoxin shock is caused.
  • LPS complex lipopolysaccharide
  • In humans LPS induces innate immune responses through cytokine production and activation of the immune system. Inflammation is a normal reaction due to cytokine production and can be harmful to the host.
  • Proteobacteria are a major group of Gram-negative bacteria, including E. coli, Salmonella, Helicobacter, Enterobacteriaceae, Pseudomonas, Moraxella, Stenotrophomona, Buderobibrio, Acetic acid bacteria, Legionella, and ⁇ -Proteobacteria such as Wolbachia It is.
  • Other representative groups of Gram-negative bacteria include Pseudomonas aeruginosa, Acinetobacter, cyanobacteria, spirochetes, green sulfur bacteria, and Bacteroides.
  • the bacterium is Escherichia coli, Salmonella, Helicobacter, Pseudomonas aeruginosa, or Acinetobacter.
  • Escherichia coli is a Gram-negative bacilli that belongs to facultative anaerobes and is one of the major species of bacteria present in the environment. Many strains of E. coli have been reported, and some have properties that can be harmful to animals. Most healthy adults have strains that cause diarrhea and do not show any symptoms, but those who are debilitated by infants or illness, or taking certain drugs In some people, special strains can cause illness and sometimes death. For example, in the case of a human, it becomes a pathogen when it enters the blood or urinary system (in case of ectopic infection), not in the large intestine. Because of the production of endotoxin (lipopolysaccharide), sepsis from E.
  • endotoxin lipopolysaccharide
  • Escherichia coli causes severe endotoxin shock.
  • Urinary tract infections are the most common cause of sepsis (if it becomes apparent), but Escherichia coli is the most common cause of urinary tract infections.
  • Escherichia coli strains those showing particularly strong pathogenicity are called pathogenic Escherichia coli.
  • pathogenic Escherichia coli Also called pathogenic E. coli in the field of food hygiene.
  • Shigella strains that cause dysentery among pathogenic Escherichia coli are called Shigella and are treated differently due to hygiene management problems.
  • Fungi Fungus
  • fungi is a general term for organisms generally called mushrooms, molds, and yeasts, and refers to organisms belonging to the fungal kingdom. It is a heterotrophic organism that uses external organic matter, secretes degrading enzymes, digests nutrients outside the cell, and ingests it from the cell surface.
  • fungi In the field of medicine and veterinary medicine, fungi are called fungi, and the study is called medical mycology. Infections caused by fungi are generally called mycosis, and superficial mycosis where the affected area stops in the keratin of the skin and does not reach the dermis, deep superficial mycosis that affects the subcutaneous tissue after the dermis, brain, It is roughly classified into deep mycosis (systemic mycosis, visceral mycosis) that extends to internal organs such as lungs and heart.
  • mycosis systemic mycosis, visceral mycosis
  • fections Diseases that infect humans and other animals with fungi (infections) include ringworms caused by ringworms, candidiasis caused by Candida, candidiasis caused by Candida, cryptococcosis caused by Cryptococcus, aspergillosis caused by Aspergillus, pneumocystis ( Pneumocystis pneumonia due to Pneumocystis) is a clinical problem.
  • Virus A “virus” is a microscopic structure that can replicate itself using cells of other organisms. It consists of a protein shell and a nucleic acid contained in the protein shell. Viruses do not have cells as constituent units, but they have genes and have the characteristics of organisms that can be propagated using cells of other organisms. Viruses can affect host homeostasis when infecting, and can act as pathogens. Microorganisms whose infection can be determined by the blood biomarker of the present invention include “viruses” that are minute infectious agents.
  • Viruses are broadly classified into DNA viruses that have DNA as a major component and RNA viruses that have RNA as a component because of the difference in genetic material.
  • the DNA virus includes, but is not limited to, smallpox virus, herpes simplex virus, varicella-zoster virus, adenovirus, papilloma virus, hepatitis B virus, EB virus, parvovirus and the like.
  • RNA viruses include, but are not limited to, influenza virus, norovirus, SARS virus, rubella virus, poliovirus, Ebola virus, yellow fever virus, dengue virus, hepatitis C virus, human immunodeficiency virus, measles virus, mumps virus, etc. Is included.
  • Blood concentration of blood biomarker in the present invention, whether or not a microorganism such as a bacterium has infected a subject is determined based on whether ⁇ -ketoglutarate, succinyl-CoA, succinic acid, fumaric acid or apple in the TCA cycle.
  • One or more substances selected from the group consisting of acids are determined as blood biomarkers (indicators). Infection means that a pathogen such as a microorganism having a smaller volume infests or grows in the body or surface of an organism.
  • the “subject” to which the blood biomarker of the present invention can be used is not particularly limited as long as it is a living organism that can be infected by microorganisms and become a host.
  • mammals such as humans, monkeys, gorillas, orangutans, cows, horses, dogs, cats, mice, rats, rabbits, sheep, goats and pigs. More preferred are primates such as humans and monkeys. Most preferred is a human.
  • the inventors of the TCA circuit have a blood concentration of one or more substances selected from the group consisting of ⁇ -ketoglutaric acid, succinyl-CoA, succinic acid, fumaric acid, and malic acid due to infection by microorganisms.
  • the present invention was conceived by finding that it decreases compared to normal values (before infection), or that one or more of these substances are depleted. Therefore, these substances are reduced compared to normal values (before infection), or one or more of these substances are depleted, that is, the blood concentration is below the detection limit If so, determine that the person is infected with a microorganism.
  • the blood concentration of each of these substances can be measured, for example, as follows. Without limitation, for example, in the case of a human, 3 ml of blood is collected. For example, it can be measured by an enzymatic method, F-kit series (manufactured by Roche Diagnostics). For example, succinic acid can be measured using a succinic acid measurement kit (Succinate Colorimetric Assay Kit, manufactured by BioVision). Alternatively, for example, Anal. Chem. , 2009, 81, 6165-6174, each substance can be measured.
  • each substance that is not infected with microorganisms By measuring the blood concentration of each substance that is not infected with microorganisms in advance, it can be compared with the blood concentration after occurrence of a suspected possibility of infection.
  • the circumstances in which the possibility of infection is suspected include, for example, surgical operation, trauma, contact with an infected person, local redness, swelling, thermal sensation, and the like.
  • three substances, ⁇ -ketoglutaric acid, succinic acid and fumaric acid have blood concentrations below the detection limit due to microbial infection. Therefore, these three substances can be used as blood biomarkers even when normal values are not measured.
  • the blood biomarker of a microorganism of the present invention can be used for determining the presence or absence of a microorganism infection as one aspect.
  • Microbial infection can be caused by surgery (during surgery, after surgery), treatment (injection, etc.), trauma, etc., or when the host is an easily infected host (such as rheumatoid arthritis, autoimmune diseases, liver dysfunction, renal dysfunction, This may occur in the case of immunodeficiency.
  • the blood biomarker of the present invention determines the infection of microorganisms in surgery.
  • the aspect of the present invention will be described as an example of a situation in which “surgical operation” is suspected of the possibility of infection without limitation.
  • ⁇ -ketoglutaric acid, succinyl-CoA, succinic acid, fumaric acid, and malic acid in the TCA cycle in blood after 1 day after surgery (example of suspected infection)
  • the blood concentration of one or more substances selected from the group consisting of is compared with the blood concentration of the corresponding substance prior to surgery.
  • it is within 30 days, within 14 days, within 7 days, and within 5 days after surgery.
  • the blood concentration after surgery is below the detection limit.
  • the detection limit or less is 0.05 ⁇ M or less, 0.03 ⁇ M or less, or 0.01 ⁇ M or less.
  • the three substances ⁇ -ketoglutaric acid, succinic acid and fumaric acid are markedly reduced by infection.
  • the blood biomarker of the present invention includes, but is not limited to, for example, the following embodiments.
  • the blood concentration of one or more substances selected from the group consisting of ⁇ -ketoglutarate, succinyl-CoA, succinic acid, fumaric acid and malic acid in the TCA cycle in blood 1-14 days after surgery A blood biomarker that is determined to be infected with a microorganism when the blood concentration of the corresponding substance before surgery is reduced to 60% or less.
  • the blood concentration of one or more substances selected from the group consisting of ⁇ -ketoglutaric acid, succinic acid and fumaric acid in the TCA cycle in the blood after the first day after the operation, the blood concentration of the corresponding substance before the operation A blood biomarker that is judged to be microbially infected when it is reduced to 10% or less of the blood concentration.
  • the present invention also relates to a method for determining microbial infection in surgery.
  • the determination method of the present invention includes: (1) Measure blood concentration of one or more substances selected from the group consisting of ⁇ -ketoglutaric acid, succinyl-CoA, succinic acid, fumaric acid, and malic acid in the TCA circuit in the subject before surgery. , (2) In the subject after surgery, measure the blood concentration of the substance measured in step (1), (3) If the concentration measured in (2) is lower than the concentration measured in (1), it is determined that the organism is infected. Including the process.
  • microorganism one or more substances selected from the group consisting of ⁇ -ketoglutarate, succinyl-CoA, succinic acid, fumaric acid and malic acid in the TCA cycle”, “blood concentration”, etc. The explanation is as described in “1. Blood biomarkers in microorganisms”.
  • the concentration measured in (2) is ( The case where the concentration is lower than the concentration measured in 1) is the same as described in “1. Blood biomarker in microorganism”.
  • the determination method of the present invention includes, but is not limited to, for example, the following aspects.
  • the blood concentration of the substance measured in step (1) is measured in the subject after 1 day after the operation, and the blood concentration measured in (2) is the blood concentration measured in (1)
  • the blood concentration of the substance measured in step (1) was measured in the subject 1 day to 30 days after the operation, and the blood concentration measured in (2) was measured in (1) The determination method of determining that it is infected with microorganisms when it has decreased to 60% or less of the blood concentration.
  • the blood concentration of the substance measured in the step (1) is measured in the subject after 1 day after the operation, and the blood concentration measured in (2) is the blood concentration measured in (1).
  • the present invention also relates to a method for determining microbial infection in a subject.
  • the determination method of the present invention includes: Measuring the blood concentration of one or more substances selected from the group consisting of ⁇ -ketoglutarate, succinate and fumaric acid in the TCA cycle; If the blood concentration of the substance is below the detection limit, it is determined that the substance is infected with a microorganism.
  • microorganism “one or more substances selected from the group consisting of ⁇ -ketoglutaric acid, succinic acid, fumaric acid in TCA cycle”, “blood concentration”, “under detection limit”, etc. , “1. Blood biomarkers in microorganisms”.
  • the determination method of the present invention includes, but is not limited to, for example, the following aspects.
  • -The below detection limit is the determination method which is 0.03 micromol or less.
  • Example 1 Search for New Biomarker Using Osteomyelitis Model Mouse a search for a new biomarker using an osteomyelitis model mouse was performed. Specifically, twelve mice with osteomyelitis who underwent surgery to inject bacteria into the femur in 12-week-old mice (infected group), and 12 mice that performed only the same operation but did not inject bacteria (sham group) Twelve animals (control group) that did not perform surgery were used. On the third day after surgery, blood was collected from the abdominal aorta, and metabolomic analysis was performed to detect about 1200 metabolites.
  • Optical Imaging and Luminescent Bacteria In this example, optical imaging (IVIS imaging system) and luminescent bacteria (luciferase-expressing Staphylococcus aureus) were used.
  • Optical imaging is an instrument (product name: IVIS-Lumina LT) (manufactured by Summit Pharmaceuticals International) that can capture weak light in the living body using an ultra-sensitive CCD camera, and the luminescent bacteria are weak as long as they survive.
  • a bacterium that has been gene-transfected so as to emit light, and obtained is obtained by introducing Staphylococcus aureus (Xen-29) manufactured by Caliper LS and Photolabhabdus lumscens luxABCDE using Luria Bertani medium manufactured by Sigma-Aldrich.
  • Staphylococcus aureus Xen-29
  • Photolabhabdus lumscens luxABCDE manufactured by Sigma-Aldrich.
  • FIG. 3 shows a process for producing a femur osteomyelitis model mouse. Surgery was performed to drill a thighbone in a 12-week-old mouse, and luminescent bacteria (1 ⁇ l, concentration 1.0 ⁇ 10 8 CFU) were injected as an infected group.
  • the study uses 12 model mice (infected group), 12 mice that do only surgery and do not inject bacteria (sham group), and 12 mice that do not perform surgery (control group), and compare them. (FIG. 4).
  • the anesthesia for the operation was ether anesthesia that did not affect the blood collection results, and blood was collected from the abdominal aorta 3 days after the operation (each 500 ⁇ l). The sample was centrifuged and 300 ⁇ l of plasma component was collected and used for analysis.
  • Metabolome analysis In addition to nucleic acids (DNA) and proteins, there are many small molecules such as sugars, organic acids, amino acids, lipids, and steroid derivatives in the living body, and there are thousands of types. Many of these are metabolites produced by metabolic activities such as enzymes. A collective catalog of all small molecules in a living body including metabolic intermediates, hormones, signal molecules, secondary metabolites, and the like contained in a single tissue is called a “metabolome”. The metabolome changes every hour. Metabolome analysis is based on bioinformatics methods to study the actual state of metabolism and the total diversity of metabolic pathways that are slightly different in each layer of cells, tissues, organs, individuals, and species. This is a technique that can comprehensively analyze the concentration at once.
  • Results and Discussion (1) The results of metabolome analysis are shown in FIG. A total of 297 substances were identified. Comparing the concentration of each substance within each group, 66 substances were high in the infected group, of which 12 were statistically significant (FIG. 7). In the infected group, there were 195 low-concentration substances and 48 statistically significant substances (FIG. 8). Among them, changes in the values of substances involved in metabolic pathways such as ⁇ -oxidation and TCA cycle were very characteristic.
  • ⁇ -oxidation is a reaction for producing acetyl CoA from fatty acid, and is a method for producing energy from fat in a starved state.
  • Infection is a kind of starvation for the body.
  • NAD + nicotinamide adenine dinucleotide
  • ⁇ -oxidation was enhanced by interpreting infection as a kind of starvation.
  • the depletion of NAD + due to this infection state (starvation state) is caused not only in the femur osteomyelitis model mouse of this example but also in mammals including humans, and not only S. aureus but also other bacteria, various microorganisms. If the living body becomes starved due to the "infection"
  • TCA circuit (FIGS. 10 to 12)
  • the TCA cycle is one of the most important metabolic pathways of living organisms that produce energy in vivo based on acetyl-CoA (FIG. 10).
  • acetyl-CoA As a result of metabolome analysis, it was found that in the infected group, substances from ⁇ -ketoglutarate to malate in the TCA cycle were almost depleted (FIG. 11). This is a result that can be explained if the process of producing ⁇ -ketoglutaric acid from isocitrate is inhibited.
  • NAD + is required in the reaction to produce ⁇ -ketoglutaric acid, and it is considered that this reaction was delayed in the infected group due to depletion of NAD + due to the above-described enhancement of ⁇ -oxidation (FIG. 12). Therefore, substances from ⁇ -ketoglutarate to malate in the TCA cycle are effective as blood biomarkers for early detection of infection.
  • Example 2 Verification of the presence of succinic acid in human blood and the presence or absence of infection
  • the presence of succinic acid in human blood (in the plasma) and the presence or absence of infection were verified.
  • blood was collected from cases (3 cases) suspected of postoperative infection for the purpose of early detection of postoperative infection in orthopedics.
  • Patient 1 45-year-old male Blood was collected on the 9th day after surgery because infection was suspected after surgery for multiple cavernous hemangioma.
  • Patient 2 A 50-year-old woman After the operation for lumbar degenerative spondylolisthesis, slight fever continued.
  • Patient 3 71-year-old man After surgery for schwannomas, slight wound healing was observed, so blood was collected on the 7th postoperative day.
  • succinic acid was detected in patient 1 and below the detection limit (40 ⁇ M), but succinic acid was detected in patients 2 and 3.
  • Citric acid was detected in all patients. As a result, it was found that patient 1 was infected, and treatment for infectious diseases such as administration of antibiotics was performed. Patients 2 and 3 were not infected and treatment of the infection was unnecessary.
  • the present invention is based on the discovery that in the analysis of metabolites, substances of the TCA cycle that have not been focused on until now have changed significantly.
  • the particular substance of the TCA cycle is very effective as a biomarker of microbial infection because of the marked difference that it is significantly reduced or hardly detectable.
  • detection and determination can be performed at an early stage of infection, which is impossible to detect with a conventional marker.
  • the blood biomarker of the present invention is based on the blood concentration of these substances, the measurement is simple, rapid and inexpensive. Therefore, as a blood biomarker and determination method for specifically determining the infection of microorganisms, it is of great clinical significance in terms of early diagnosis and early treatment.
  • the present invention provides innovative results in the field of infectious disease research.

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Abstract

L'invention concerne un biomarqueur sanguin de micro-organismes et un procédé de détermination d'une infection par des micro-organismes à l'aide du biomarqueur sanguin. Ce biomarqueur sanguin est caractérisé en ce qu'il comprend au moins une substance dans le cycle TCA choisie parmi l'acide α-cétoglutarique, la succinyl-CoA, l'acide succinique, l'acide fumarique et l'acide malique.
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