WO2018147472A1

WO2018147472A1 - Blood biomarker

Info

Publication number: WO2018147472A1
Application number: PCT/JP2018/005603
Authority: WO
Inventors: 賢石井; 宜広磯貝
Original assignee: 学校法人慶應義塾
Priority date: 2017-02-09
Filing date: 2018-02-09
Publication date: 2018-08-16
Also published as: JPWO2018147472A1

Abstract

The present invention relates to a blood biomarker of microorganisms, and a method for determining infection with microorganisms using the blood biomarker. This blood biomarker is characterized by comprising one or more substances in the TCA cycle selected from α-ketoglutaric acid, succinyl-CoA, succinic acid, fumaric acid and malic acid.

Description

Blood biomarker

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. However, 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. In general, 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. The reason is that in existing blood tests, the effects of surgical invasion remain for several days to a week, and cannot be distinguished from infection. In other words, since postoperative infections increase blood inflammatory cytokines due to the invasion of surgery itself, it is difficult to distinguish whether postoperative inflammation is due to surgical invasion or the onset of postoperative infection .

In addition to after surgery, infections may develop after trauma or in susceptible hosts. For example, infectious diseases may develop in patients with chronic inflammatory diseases such as autoimmune diseases, but early diagnosis thereof is difficult.

Pierrakos C, Vincent JL. Sepsis biomarkers: a review. Crit Care. 2010; 14 (1): R15. Reports 178 substances as biomarkers for all infectious diseases. However, none of them has been reported with high specificity in infectious diseases. Procalcitonin, which has been reported with high sensitivity specificity as a biomarker for infectious diseases, is also available from Shen CJ, Wu MS, Lin KH, Lin WL, Chen HC, Wu JY, et al. The use of procalcinin in the diagnosis of bone and joint effect: a systemic review and meta-analysis. Eur J Clin Microbiol Infect Dis. 2013 Jun; 32 (6): 807-14 (for example, p.813, lines 18-22), its usefulness has not been established in all infectious diseases.

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.

As described above, 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.

Japanese Patent Application No. 2011-95052

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 (α-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.

Although not necessarily limited, the present invention includes the following aspects.

[Aspect 1]
A microbial blood biomarker comprising one or more substances selected from the group consisting of α-ketoglutaric acid, succinyl-CoA, succinic acid, fumaric acid and malic acid in the TCA cycle.

[Aspect 2]
The blood biomarker according to aspect 1, wherein the presence or absence of infection by microorganisms is determined.

[Aspect 3]
3. The blood biomarker according to

aspect

1 or 2 for determining microbial infection in surgery.

[Aspect 4]
The blood concentration of one or more substances selected from the group consisting of α-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 α-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.

[Aspect 6]
The blood concentration of one or more substances selected from the group consisting of α-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 α-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.

[Aspect 8]
The blood concentration of one or more substances selected from the group consisting of α-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.

[Aspect 9]
The blood concentration of one or more substances selected from the group consisting of α-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.

[Aspect 10]
The blood concentration of one or more substances selected from the group consisting of α-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.

[Aspect 11]
The blood biomarker according to any one of aspects 1 to 10, wherein the substance in the TCA cycle is one or more substances selected from the group consisting of α-ketoglutaric acid, succinic acid, and fumaric acid.

[Aspect 12]
The blood concentration of one or more substances selected from the group consisting of α-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 biomarker according to the aspect 1 or 11, which is judged to be infected with a microorganism when it is reduced to 30% or less of the medium concentration.

[Aspect 13]
The blood concentration of one or more substances selected from the group consisting of α-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 biomarker according to the aspect 1 or 11, which is judged to be microbially infected when it is reduced to 10% or less of the medium concentration.

[Aspect 14]
The blood biomarker according to any one of aspects 1 to 13, wherein the microorganism is selected from the group consisting of bacteria, fungi and viruses.

[Aspect 15]
The blood biomarker according to any one of aspects 1-14, wherein the microorganism is a bacterium.

[Aspect 16]
The blood biomarker according to aspect 15, wherein the bacterium is a Gram-positive bacterium.

[Aspect 17]
The blood biomarker according to aspect 15 or 16, wherein the bacterium is selected from the group consisting of Staphylococcus aureus, Staphylococcus epidermidis, Acne and Enterococcus.

[Aspect 18]
The blood biomarker according to any one of aspects 15 to 17, wherein the bacterium is Staphylococcus aureus or Staphylococcus epidermidis.

[Aspect 19]
The blood biomarker according to any one of aspects 15 to 18, wherein the bacterium is Staphylococcus aureus.

[Aspect 20]
The blood biomarker according to aspect 15, wherein the bacterium is a gram-negative bacterium.

[Aspect 21]
A method for determining microbial infection in surgery,
(1) Measure blood concentration of one or more substances selected from the group consisting of α-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.

[Aspect 22]
In the step (2), 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 determination method according to aspect 21, wherein it is determined that the microorganism is infected when it is reduced to 60% or less.

[Aspect 23]
In the step (2), 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 determination method according to

aspect

21 or 22, wherein when the concentration is reduced to 60% or less, it is determined that the microorganism is infected.

[Aspect 24]
The determination method according to any one of aspects 21 to 23, wherein the substance in the TCA circuit is one or more substances selected from the group consisting of α-ketoglutaric acid, succinic acid, and fumaric acid.

[Aspect 25]
In the step (2), 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. The determination method according to aspect 24, in which it is determined that the microorganism is infected when the value is also reduced to 30% or less.

[Aspect 26]
The determination method according to any one of aspects 21 to 25, wherein the microorganism is selected from the group consisting of bacteria, fungi and viruses.

[Aspect 27]
27. The determination method according to any one of aspects 21 to 26, wherein the microorganism is a bacterium.

[Aspect 28]
The determination method according to aspect 26 or 27, wherein the bacterium is selected from the group consisting of Staphylococcus aureus, Staphylococcus epidermidis, Acne and enterococci.

[Aspect 29]
The determination method according to any one of aspects 26 to 28, wherein the bacterium is Staphylococcus aureus or Staphylococcus epidermidis.

[Aspect 30]
30. The determination method according to any one of aspects 26 to 29, wherein the bacterium is Staphylococcus aureus.

[Aspect 31]
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 α-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.

[Aspect 32]
32. The determination method according to aspect 31, wherein the microorganism is selected from the group consisting of bacteria, fungi and viruses.

[Aspect 33]
The determination method according to aspect 31 or 32, wherein the microorganism is a bacterium.

[Aspect 34]
34. The determination method according to aspect 32 or 33, wherein the bacterium is selected from the group consisting of S. aureus, S. epidermidis, acne and enterococci.

[Aspect 35]
The determination method according to any one of aspects 32-34, wherein the bacterium is Staphylococcus aureus or Staphylococcus epidermidis.

[Aspect 36]
36. The determination method according to any one of aspects 32-35, wherein the bacterium is Staphylococcus aureus.

[Aspect 37]
34. The determination method according to any one of aspects 29 to 33, wherein “below the detection limit” is 0.03 μM or less.

[Aspect 38]
The determination method according to any one of aspects 31 to 37, wherein the subject is a mammal from one day after surgery.

[Aspect 39]
The determination method according to any one of aspects 31 to 38, wherein the subject is a mammal from 1 to 30 days after surgery.

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. In addition, detection and determination can be performed at an early stage of infection, which is impossible to detect with a conventional marker. Furthermore, since 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. Moreover, since it emits light only in the presence of oxygen and ATP, only living bacteria emit light, and no light is emitted when the bacteria die. 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) and the amount of luminescence (× 10 ⁶ photons / second / cm ² ) show a strong positive correlation. That is, the amount of bacteria can be calculated from the amount of luminescence. 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. In total, 297 substances were identified, and 66 kinds of substances having a high concentration in the infected group, of which 12 kinds were statistically significant. There were 195 low-concentration substances and 48 statistically significant substances in the infected group. 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. For each substance, the results of “control group”, “sham group”, and “infection group” are shown in order from the top of the graph. Three substances of succinic acid, α-ketoglutaric acid (2-oxoglutaric acid, 2-OG), and fumaric acid decreased to below the detection limit in the “infection group”. FIG. 9 is a schematic diagram of β-oxidation. In the examples, NAD + (nicotinamide adenine dinucleotide) was not detectable only in the “infected group” (depletion). This was thought to be because β-oxidation was enhanced by interpreting infection as a kind of starvation. 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 α-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. In the examples, five substances from α-ketoglutarate to malate in the TCA cycle were almost depleted only in the infected group. This is probably because NAD + necessary in the reaction in which α-ketoglutarate is generated from isocitrate was depleted due to the enhancement of β-oxidation in the infected group, so that no substance after α-ketoglutarate was produced. In the present invention, five substances of α-ketoglutarate, succinyl-CoA, succinic acid, fumaric acid and malic acid in the TCA cycle are used as blood biomarkers for early detection of infection. FIG. 13 shows the results of examining the presence of citric acid and succinic acid in blood (plasma) for three patients suspected of postoperative infection in orthopedics. Citric acid is a UV absorption measurement kit (F-kit citric acid, manufactured by JK International), and 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. Among them, substances involved in the TCA cycle are almost depleted only in the infected group from isocitrate, and α-ketoglutarate (2-oxoglutarate, 2-OG), succinyl-CoA, succinate, fumar The effects were found on five substances, acid and malic acid. The present invention utilizes these five substances as blood biomarkers of microbial infection.

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 α-ketoglutaric acid, succinyl-CoA, succinic acid, fumaric acid and malic acid in the TCA cycle.

(1) Substances in the TCA cycle The “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 β-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 α-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 α-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 α-ketoglutaric acid, succinyl-CoA, succinic acid, fumaric acid and malic acid. It consists of the above or five substances. In the present invention, among the five substances, in particular, three substances of α-ketoglutaric acid, succinic acid and fumaric acid are clearly depleted in the microbial infection group (blood concentration is below detection limit). became. Therefore, preferably, the blood biomarker of the present invention is one or more substances selected from the group consisting of α-ketoglutaric acid, succinic acid and fumaric acid. Preferably, 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 α-ketoglutaric acid, succinic acid and fumaric acid.

(2) Determination target microorganism of blood biomarker of the present invention The blood biomarker of the present invention is a biomarker capable of determining the infection of a target microorganism. The term “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. In addition to bacteria (eubacteria) and archaea, fungi (fungi), eukaryotes such as protists, slime molds and algae, and small animals such as rotifers are also included. 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.

The “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” (Eubacteria) 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

In one embodiment of the present invention, 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). Monocytogenes, etc.), Streptococcus (Streptococcus, Green Streptococcus, β-hemolytic Streptococcus, etc.), Clostridium (Botulinum, Tetanus, etc.), Bacillus (Bacillus) (Anthrax) , Bacillus cereus, Bacillus subtilis, etc.).

The genus Staphylococcus is a Gram-positive cocci with a diameter of about 1 μm, and is a facultative and anaerobic organic vegetative bacterium with an irregular arrangement of grape tufts. As of 2016, 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. “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. Because of its strong adherence to plastic surfaces and the like, and because it is a resident microorganism of the epidermis, it may adhere to medical instruments such as catheters and heart valves during surgery and may enter the body. In particular, it may cause deep suppuration by adhering to and growing on medical devices of the type that are placed in the body. In addition to methicillin-sensitive Staphylococcus epidermidis (MSSE) that is resistant to the antibiotic methicillin, drug-resistant bacteria such as methicillin-resistant Staphylococcus epidermidis (MRSE) are also included.

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.

“Enterococcus” 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 . As the main species, E. coli of the genus Enterococcus. faecalis, E .; faecium, E.M. avium, E.I. caseliflavus, E.I. gallinarum, E.M. flavescens. The pathogenicity is weak and usually harmless, but there are known examples of opportunistic infections for patients who have decreased resistance to bacterial infection such as immunodeficiency after surgery, and may cause sepsis in some cases. In particular, vancomycin-resistant enterococci (VRE) are a problem.

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).

In one embodiment of the present invention, 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. 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 α-Proteobacteria such as Wolbachia It is. Other representative groups of Gram-negative bacteria include Pseudomonas aeruginosa, Acinetobacter, cyanobacteria, spirochetes, green sulfur bacteria, and Bacteroides.

In one embodiment of the present invention, 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. 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. Among Escherichia coli strains, those showing particularly strong pathogenicity are called pathogenic Escherichia coli. Also called pathogenic E. coli in the field of food hygiene. However, 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.

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. 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. Examples of 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.

(3) 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 α-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. Preferred are 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 α-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.

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. It has been found in the present invention that three substances, α-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.

In one aspect, the blood biomarker of the present invention determines the infection of microorganisms in surgery. Hereinafter, 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.

In one embodiment of the present invention, α-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. Preferably, after 2 days after surgery, after 3 days. Preferably, it is within 30 days, within 14 days, within 7 days, and within 5 days after surgery. Preferably, 1-30 days after surgery, 1-14 days after surgery, and 1-7 days after surgery. More preferably, it is 1-6 days, 2-5 days, 3-4 days.

As a result of comparing the blood concentration after surgery with the concentration of the above-mentioned substance before surgery, if the blood concentration of one or more substances is reduced compared to the blood concentration of the corresponding substance before surgery, microorganisms It is determined to be infected. “When it is decreased” means, for example, but not limited to, as a result of comparing the blood concentration after surgery with the concentration of the above-mentioned substance before surgery, the blood concentration of one or more substances corresponds to that before surgery. When it is reduced to 60% or less compared to the blood concentration of the substance to be treated, it is more preferably 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less. In one embodiment, the blood concentration after surgery is below the detection limit. For example, the detection limit or less is 0.05 μM or less, 0.03 μM or less, or 0.01 μM or less. In particular, the three substances α-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 α-ketoglutaric acid, succinyl-CoA, succinic acid, fumaric acid and malic acid in the TCA cycle in the blood after 1 day after surgery, A blood biomarker that is judged to be infected with a microorganism when it is reduced to 60% or less of the blood concentration of the corresponding substance before surgery.
The blood concentration of one or more substances selected from the group consisting of α-ketoglutaric acid, succinyl-CoA, succinic acid, fumaric acid and malic acid in the TCA cycle in blood 1-30 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 α-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 α-ketoglutaric acid, succinyl-CoA, succinic acid, fumaric acid and malic acid in the TCA cycle in blood 1-7 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 α-ketoglutaric acid, succinyl-CoA, succinic acid, fumaric acid and malic acid in the TCA cycle in the blood after 1 day after surgery, A blood biomarker that is judged to be infected with a microorganism when it is reduced to 50% or less of the blood concentration of the corresponding substance before surgery.
The blood concentration of one or more substances selected from the group consisting of α-ketoglutaric acid, succinyl-CoA, succinic acid, fumaric acid and malic acid in the TCA cycle in the blood after 1 day after surgery, A blood biomarker that is judged to be infected with a microorganism when it is reduced to 30% or less of the blood concentration of the corresponding substance before surgery.
The blood concentration of one or more substances selected from the group consisting of α-ketoglutaric acid, succinyl-CoA, succinic acid, fumaric acid and malic acid in the TCA cycle in the blood after 1 day after surgery, A blood biomarker that is judged to be infected with a microorganism when it is reduced to 10% or less of the blood concentration of the corresponding substance before surgery.
The blood concentration of one or more substances selected from the group consisting of α-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 30% or less of the blood concentration.
The blood concentration of one or more substances selected from the group consisting of α-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.

2. 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 α-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.

Definitions of “microorganism”, “one or more substances selected from the group consisting of α-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”.

Regarding “(2) Measuring blood concentration of substance measured in step (1) in the subject after surgery”, 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.
-In the above step (2), 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 determination method of determining that it is infected with microorganisms when it has decreased to 60% or less of the concentration.
-In the above step (2), 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.
-In the step (2), 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 determination method of determining that it is infected with microorganisms when it has decreased to 30% or less.

3. 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 α-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.

It has been found in the present invention that three substances, α-ketoglutaric acid, succinic acid and fumaric acid, have a blood concentration below the detection limit due to microbial infection. Therefore, even if normal values are not measured for these three substances, it can be determined that the microorganism is infected when the blood concentration is below the detection limit.

Definition and explanation of “microorganism”, “one or more substances selected from the group consisting of α-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.
-Determination method in which the subject is a mammal from 1 day after surgery.
-A determination method, wherein the subject is a mammal 1-30 days after surgery.

Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited to these examples. Those skilled in the art can easily modify and change the present invention based on the description of the present specification, and these are included in the technical scope of the present invention.

Example 1 Search for New Biomarker Using Osteomyelitis Model Mouse In this example, 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.

Materials and Methods (1) 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.

If these are used, it is possible to confirm whether or not the luminescent bacteria are alive in the living body by optical imaging (FIGS. 1A and 1B). It has been observed that this detectable intensity of luminescence and the amount of bacteria are positively correlated (FIG. 2).

(2) Femur osteomyelitis model mouse 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 μl, concentration 1.0 × 10 ⁸ CFU) were injected as an infected group.

This is a model that can create femur osteomyelitis (infection state) very stably. In this example, 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 μl). The sample was centrifuged and 300 μl of plasma component was collected and used for analysis.

(3) 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.

(2) About 1,200 kinds of molecular data such as sugars, amino acids, lipids, steroid derivatives, etc. were comprehensively measured from the blood collected in the “femur osteomyelitis model mouse” and subjected to metabolomic analysis. For the metabolomic analysis of this example, Dual Scan (CE-TOFMS and LC-TOFMS) manufactured by HumanMetabolome was used.

In the comparison between the infected group and the sham group, the difference due to the presence or absence of bacterial infection was determined, and in the comparison between the sham group and the control group, the difference due to the presence or absence of surgery was determined. In this analysis, a substance that has no difference between the control group and the sham group and that has a high value (or low value) only in the infected group is considered to be important as a blood biomarker that can identify infection at an early stage without being affected by surgery. (Figure 5).

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 β-oxidation and TCA cycle were very characteristic.

As shown in FIG. 8, the three substances α-ketoglutaric acid, succinic acid and fumaric acid were below the detection limit (not detected). Malic acid was reduced to about 30% compared to the control group (70% decrease).

(2) β-oxidation (Figure 9)
β-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. In this example, NAD + (nicotinamide adenine dinucleotide) necessary for this β-oxidation reaction was not detectable only in the infected group and was specifically depleted. This was thought to be because β-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"

(3) 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). As a result of metabolome analysis, it was found that in the infected group, substances from α-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 α-ketoglutaric acid from isocitrate is inhibited. NAD + is required in the reaction to produce α-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 β-oxidation (FIG. 12). Therefore, substances from α-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 In this example, the presence of succinic acid in human blood (in the plasma) and the presence or absence of infection were verified. Specifically, 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.

All patients had continuation of slight fever and swelling of the wound in about one week after the operation, and postoperative infection could not be ruled out. For blood collection, a 1 ml vacuum blood collection tube (including EDTA2K / manufactured by TERUMO) was used, immediately centrifuged at 4 ° C., 1200 rpm / min × 10 minutes, and stored frozen at −80 ° C. The sample was naturally thawed at room temperature, and citric acid was measured using an enzymatic UV absorption measurement kit (F-kit citric acid, manufactured by JK International Co.), and succinic acid was measured using a colorimetric method (succinic acid). Each measurement was performed with an acid measurement kit (Succinate Colorimetric Assay Kit, manufactured by BioVision).

As shown in FIG. 13, succinic acid was detected in patient 1 and below the detection limit (40 μM), 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.

From this example, it was shown that the absence (non-detection) of succinic acid in human blood (plasma) is associated with infection, which proved that succinic acid is useful as a biomarker for infection.

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. In addition, detection and determination can be performed at an early stage of infection, which is impossible to detect with a conventional marker. Furthermore, since 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. Furthermore, it is not only useful as a blood biomarker for infection of microorganisms, but also contributes to the elucidation of the reaction mechanism itself against microorganisms in vivo, particularly bacterial infections. The present invention provides innovative results in the field of infectious disease research.

Claims

A microbial blood biomarker comprising one or more substances selected from the group consisting of α-ketoglutaric acid, succinyl-CoA, succinic acid, fumaric acid and malic acid in the TCA circuit.
The blood biomarker according to claim 1, wherein the presence or absence of infection by microorganisms is determined.
The blood biomarker according to claim 1 or 2, which is used to determine infection of microorganisms in surgery.
The blood concentration of one or more substances selected from the group consisting of α-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 claims 1 to 3, wherein the blood biomarker according to any one of claims 1 to 3 is judged to be infected with a microorganism when the blood concentration of the corresponding substance is decreased to 60% or less.
The blood concentration of one or more substances selected from the group consisting of α-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 claims 1 to 3, wherein the blood biomarker according to any one of claims 1 to 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 α-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 claims 1 to 3, wherein the blood biomarker according to any one of claims 1 to 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 α-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 claims 1 to 3, wherein the blood biomarker according to any one of claims 1 to 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 α-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 claims 1 to 7, wherein the blood biomarker according to any one of claims 1 to 7 is judged to be infected with a microorganism when the blood concentration of the previous corresponding substance is reduced to 50% or less.
The blood concentration of one or more substances selected from the group consisting of α-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 claims 1 to 7, wherein the blood biomarker according to any one of claims 1 to 7 is judged to be infected with a microorganism when the blood concentration of the corresponding substance is reduced to 30% or less.
The blood concentration of one or more substances selected from the group consisting of α-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 claims 1 to 7, wherein the blood biomarker according to any one of claims 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 biomarker according to any one of claims 1 to 10, wherein the substance in the TCA circuit is one or more substances selected from the group consisting of α-ketoglutaric acid, succinic acid, and fumaric acid.
The blood concentration of one or more substances selected from the group consisting of α-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 biomarker according to claim 1 or 11, wherein the blood biomarker is determined to be infected with a microorganism when the concentration is reduced to 30% or less of the medium concentration.
The blood concentration of one or more substances selected from the group consisting of α-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 biomarker according to claim 1 or 11, wherein the blood biomarker is determined to be infected with a microorganism when the concentration is reduced to 10% or less of the medium concentration.
The blood biomarker according to any one of claims 1 to 13, wherein the microorganism is selected from the group consisting of bacteria, fungi and viruses.
The blood biomarker according to any one of claims 1 to 14, wherein the microorganism is a bacterium.
The blood biomarker according to claim 15, wherein the bacterium is a Gram-positive bacterium.
The blood biomarker according to claim 15 or 16, wherein the bacterium is selected from the group consisting of Staphylococcus aureus, Staphylococcus epidermidis, Acne and enterococci.
The blood biomarker according to any one of claims 15 to 17, wherein the bacterium is Staphylococcus aureus or Staphylococcus epidermidis.
The blood biomarker according to any one of claims 15 to 18, wherein the bacterium is Staphylococcus aureus.
The blood biomarker according to claim 15, wherein the bacterium is a Gram-negative bacterium.
A method for determining microbial infection in surgery,
(1) Measure blood concentration of one or more substances selected from the group consisting of α-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.
In the step (2), 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 determination method according to claim 21, wherein it is determined that the microorganism is infected when it is reduced to 60% or less.
In the step (2), 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 determination method according to claim 21 or 22, wherein when the concentration is reduced to 60% or less, it is determined that the microorganism is infected.
The determination method according to any one of claims 21 to 23, wherein the substance in the TCA circuit is one or more substances selected from the group consisting of α-ketoglutaric acid, succinic acid, and fumaric acid.
In the step (2), 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. The determination method according to claim 24, wherein it is determined that the microorganism is infected when the value is also reduced to 30% or less.
The determination method according to any one of claims 21 to 25, wherein the microorganism is selected from the group consisting of bacteria, fungi and viruses.
The determination method according to any one of claims 21 to 26, wherein the microorganism is a bacterium.
The determination method according to claim 26 or 27, wherein the bacterium is selected from the group consisting of Staphylococcus aureus, Staphylococcus epidermidis, Acne and enterococci.
The determination method according to any one of claims 26 to 28, wherein the bacterium is Staphylococcus aureus or Staphylococcus epidermidis.
The determination method according to any one of claims 26 to 29, wherein the bacterium is Staphylococcus aureus.
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 α-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.
32. The determination method according to claim 31, wherein the microorganism is selected from the group consisting of bacteria, fungi and viruses.
The determination method according to claim 31 or 32, wherein the microorganism is a bacterium.
The determination method according to claim 32 or 33, wherein the bacterium is selected from the group consisting of Staphylococcus aureus, Staphylococcus epidermidis, Acne and enterococci.
35. The determination method according to any one of claims 32-34, wherein the bacterium is Staphylococcus aureus or Staphylococcus epidermidis.
The determination method according to any one of claims 32-35, wherein the bacterium is Staphylococcus aureus.
The determination method according to any one of claims 29 to 33, wherein "below the detection limit" is 0.03 µM or less.
The determination method according to any one of claims 31 to 37, wherein the subject is a mammal from one day after surgery.
The determination method according to any one of claims 31 to 38, wherein the subject is a mammal 1 to 30 days after surgery.