Plasma biomarkers for bone infection

Abstract

The purpose is to provide plasma biomarkers for bone infections in humans. [Solution] The plasma biomarker for bone infection consists of one or more substances selected from the group consisting of sphingosine-1-phosphate, taurocholic acid, glycololic acid, 11a,b-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-dihydroxy-5a-pregnan-20-one-2, aldosterone, bisacrone-2, naringenin, tauro-a-muricholic acid-6, 1-deoxysphingosine, 12(S)-HETE-2, 2-arachidonoylglycerol, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3b-hydroxy-5-cholestenoic acid, AEA(22:4)-1, AEA(22:4)-2, bilverdin, sphingosine, malic acid, and succinic acid.

Description

[Technical Field]

[0001] The present invention relates to a plasma biomarker for bone infection, a method for determining bone infection using the plasma biomarker, and a kit for measuring the concentration of the plasma biomarker in human plasma. [Background technology]

[0002] As we enter a super-aging society, orthopedic surgery using biomaterials such as artificial joint replacement, spinal implant surgery, and artificial bones due to degenerative diseases and trauma has become widespread. However, bone infections are often associated with open bone and soft tissue injuries and implant surgery during treatment. Furthermore, elderly people who are susceptible to infection can develop hematogenous bone infections such as pyogenic spondylitis and pyogenic arthritis even without surgery or trauma, and the incidence of these infections is steadily increasing. The therapeutic effect of antibiotics on bone infections is poor compared to infections in other sites, as the drug is less easily transferred from the blood to tissues, and treatment is often prolonged. Therefore, early diagnosis is important, but diagnosis is often difficult because the early stage of infection only produces a mild inflammatory response and there are no specific infectious disease markers in blood tests. In particular, in the case of surgical site infections, it is difficult to distinguish between inflammation due to surgical invasion and inflammation due to infection, making early diagnosis of infection difficult, and delays in initiating treatment often lead to intractable disease.

[0003] Much research has been done on infectious disease biomarkers, but the majority of studies have focused on assessing the severity of sepsis in intensive care units and other settings that result in poor overall health. Given the underlying disease context, these markers often focus on inflammatory cytokines. However, bone infections are unique in that they do not result in a significant increase in inflammatory cytokines and poor overall health; rather, while overall health is maintained, they have a very high recurrence rate and are prone to developing into chronic infections. There have been no reports of specific biomarkers for bone infections, and there are very few reports focusing on metabolic products, even when including infections in other sites. Even for procalcitonin, one of the representative biomarkers, it has been reported that its sensitivity and specificity for bone infections is extremely low at around 40% (Non-patent document 1), and there is no doubt that biomarkers need to be explored from a new perspective.

[0004] We conducted basic research into bone infections and successfully generated a bacterial strain that stably emits light in vivo and in vitro. Using this bacterium, we successfully created a reproducible osteomyelitis model in mice (Funao H. et al., Infect Immun, 2011, Non-Patent Document 2) and a soft tissue infection model using the superficial gluteus maximus muscle (Yoshioka K. et al., PLOS ONE, 2014, Non-Patent Document 3). Furthermore, we conducted comprehensive metabolomic analysis of metabolites using the blood of mice with femoral osteomyelitis and reported that an increase in the sphingolipids sphingosine and sphinganine from the early stages of infection and a decrease in α-ketoglutarate and succinate in the TCA cycle could serve as specific biomarkers for early infection (Isogai N et al., Scientific Reports, 2020, Patent Document 1). The problem here is that the blood of human bone infection patients is similar to that of mice. The point is that it is unclear whether biomarkers are useful. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Republished Patent No. 2018-147472 [Non-patent literature]

[0006] [Non-Patent Document 1] Eur.J.Cli.Microbiol.InfectDis.32(6)807(2013) [Non-patent document 2] Funao H. et al. Infect Immun, 2011 [Non-patent document 3] Yoshioka K. et al. PLOS ONE, 2014 [Non-patent document 4] Anal.Chem.,2009,81,6165-61740010 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention has been made in consideration of the above-mentioned problems, and aims to provide plasma biomarkers for bone infections in humans, a method for determining bone infections using plasma biomarkers, and a kit for measuring plasma biomarker concentrations in human plasma. [Means for solving the problem]

[0008] The present inventors have conducted extensive research to solve the above problems and have found that 12 specific substances (sphingosine-1-phosphate, taurocholic acid, glycololic acid, 11a,b-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-dihydroxy-5a-pregnan-20-one-2, aldosterone, bisacrone-2, naringenin, tauro-α-muricholic acid-1, and tauro-α-muricholic acid-6) are involved in bone infection in humans. The present inventors discovered that the blood concentrations of these substances are increased in patients with the disease compared to those without the disease, and that the blood concentrations of 12 specific substances (1-deoxysphingosine, 12(S)-HETE-2, 2-arachidonoylglycerol, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3b-hydroxy-5-cholestenoic acid, AEA(22:4)-1, AEA(22:4)-2, bilverdin, sphingosine, malic acid, and succinic acid) are decreased in patients with the disease compared to those without the disease, and thus arrived at the present invention. The present invention includes, but is not limited to, the following aspects.

[0009] [1] A plasma biomarker for bone infection consisting of one or more substances selected from the group consisting of sphingosine-1-phosphate, taurocholic acid, glycololic acid, 11a,b-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-dihydroxy-5a-pregnan-20-one-2, aldosterone, bisacron-2, naringenin, tauro-α-muricholic acid-6, 1-deoxysphingosine, 12(S)-HETE-2, 2-arachidonoylglycerol, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3b-hydroxy-5-cholestenoic acid, AEA(22:4)-1, AEA(22:4)-2, bilverdin, sphingosine, malic acid, and succinic acid. [2] The plasma biomarker described in [1] for determining whether or not the bone infection has occurred. [3] The plasma biomarker described in [1] or [2] for determining the onset of bone infection during surgery. [4] Plasma biomarkers that indicate bone infection when the blood concentration of one or more substances selected from the group consisting of sphingosine-1-phosphate, taurocholic acid, glycocholate, 11a,b-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-dihydroxy-5a-pregnan-20-one-2, aldosterone, bisacron-2, naringenin, and tauro-α-muricholic acid-6 increases to 200% or more of the normal value three days or more after surgery. [5] Plasma biomarkers that indicate bone infection when the blood concentration of one or more substances selected from the group consisting of 1-deoxysphingosine, 12(S)-HETE-2, 2-arachidonoylglycerol, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3b-hydroxy-5-cholestenoic acid, AEA(22:4)-1, AEA(22:4)-2, bilverdin, sphingosine, malic acid, and succinic acid decreases to 60% or less of the normal value three days or more after surgery. [6] A method for determining the onset of bone infection during surgery, comprising: In the subject 3 days or later after surgery, measuring the blood concentration of one or more substances selected from the group consisting of sphingosine-1-phosphate, taurocholic acid, glycocholate, 11a,b-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-dihydroxy-5a-pregnan-20-one-2, aldosterone, bisacron-2, naringenin, and tauro-α-muricholic acid-6; If the blood levels of the above substances are higher than normal, A method for determining whether a bone infection has developed. [7] The method of claim 6, wherein if the blood concentration of the substance is increased to 200% or more of the normal value, it is determined that a bone infection has developed. [8] A method for determining the onset of bone infection during surgery, comprising: In the subject on or after 3 days after surgery, the blood concentration of one or more substances selected from the group consisting of 1-deoxysphingosine, 12(S)-HETE-2, 2-arachidonoylglycerol, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3b-hydroxy-5-cholestenoic acid, AEA(22:4)-1, AEA(22:4)-2, bilverdin, sphingosine, malic acid, and succinic acid is measured; If the blood levels of the above substances are lower than normal, A method for determining whether a bone infection has developed. [9] The method of determining whether a bone infection has developed when the blood concentration of the above substance is reduced to 60% or less of the normal value, as described in [8].

[10] A method for determining the onset of a bone infection in a subject, comprising: In subjects with elevated CRP levels, measuring the blood concentration of one or more substances selected from the group consisting of sphingosine-1-phosphate, taurocholic acid, glycololic acid, 11a,b-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-dihydroxy-5a-pregnan-20-one-2, aldosterone, bisacron-2, naringenin, and tauro-α-muricholic acid-6; If the blood concentration of the above substance is increased to 200% or more of the normal value, A method for determining whether a bone infection has developed.

[11] A method for determining the onset of a bone infection in a subject, comprising: In subjects with elevated CRP levels, blood concentrations of one or more substances selected from the group consisting of 1-deoxysphingosine, 12(S)-HETE-2, 2-arachidonoylglycerol, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3b-hydroxy-5-cholestenoic acid, AEA(22:4)-1, AEA(22:4)-2, bilverdin, sphingosine, malic acid, and succinic acid are measured; If the blood concentration of the above substance is reduced to 60% or less of the normal value, A method for determining whether a bone infection has developed.

[12] The method of determining the presence or absence of a pathogenic variant of the present invention described in

[10] or

[11] , wherein the subject is a mammal.

[13] A test kit for bone infections configured to measure the concentration of the plasma marker described in [1] in human blood.

[14] The test kit according to

[13] , wherein the measurement is performed by at least one of dye colorimetry, ion chromatography, GC-MS, HPLC, immunochromatography, enzyme-linked immunosorbent assay (ELISA), chemiluminescence enzyme immunoassay (CLEIA), chemiluminescence immunoassay (CLIA), fluorescent enzyme immunoassay, aptamer method, immunoturbidimetry, immunonephelometry, latex agglutination, Jaffe method, and colloidal gold colorimetry. [Effects of the Invention]

[0010] The specific substances listed above show significant differences in their blood levels, either significantly increased or significantly decreased, in bone infection patients, making them highly effective as plasma biomarkers for bone infection. Furthermore, they enable detection or diagnosis at a very early stage of the onset of bone infection, an early stage that cannot be detected using conventional markers. Furthermore, because the plasma biomarkers of the present invention are based on the blood concentrations of these substances, measurement is simple, rapid, and inexpensive. [Brief explanation of the drawings]

[0011] [Figure 1] Figure 1 is a graph comparing the concentrations of four of the 12 substances that showed high levels in the group that developed bone infection between the control group and the infected group. [Figure 2] Figure 2 is a graph comparing the concentrations of four of the 12 substances that showed high levels in the group that developed bone infection between the control group and the infected group. [Figure 3] Figure 3 is a graph comparing the concentrations of four of the 12 substances that showed high levels in the group that developed bone infection between the control group and the infected group. [Figure 4] Figure 4 is a graph comparing the concentrations of four of the 12 substances that showed lower values in the group that developed bone infection between the control group and the infected group. [Figure 5] Figure 5 is a graph comparing the concentrations of four of the 12 substances that showed lower values in the group that developed bone infection between the control group and the infected group. [Figure 6] Figure 6 is a graph comparing the concentrations of four of the 12 substances that showed lower values in the group that developed bone infection between the control group and the infected group. DETAILED DESCRIPTION OF THE INVENTION

[0012] The inventors collected blood samples from human patients with bone infections and control patients who had difficulty in clinical differentiation, and performed metabolomic analysis on the samples matched with background data. As a result, 331 candidate compounds were identified. All of these are novel plasma biomarker candidates that have not been previously reported.

[0013] After orthopedic bone and joint surgery, C-reacted protein (CRP), an inflammatory response, transiently increases and then decreases. This study focused on patients who experienced a re-elevation of CRP during the decrease phase. Blood samples taken at the time of CRP re-elevation were compared using metabolomic analysis from three patients (control group) who spontaneously recovered without antibiotic administration and three patients (infection group) whose condition eventually worsened and were diagnosed with bone infection. As a result, 331 samples were detected as biomarker candidates, and the following 24 metabolites were identified as specific biomarkers for bone infection in humans based on reproducibility between samples. This invention utilizes these 24 substances as plasma biomarkers for bone infection in humans.

[0014] The present invention includes, but is not limited to, the following aspects. 1. Plasma biomarkers for bone infection The present invention relates to plasma biomarkers for bone infection.

[0015] (1) Plasma biomarker substances The blood biomarkers of the present invention consist of one or more substances selected from the group consisting of sphingosine-1-phosphate, taurocholic acid, glycololic acid, 11a,b-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-dihydroxy-5a-pregnan-20-one-2, aldosterone, bisacrone-2, naringenin, tauro-α-muricholic acid-1, tauro-α-muricholic acid-6, 1-deoxysphingosine, 12(S)-HETE-2, 2-arachidonoylglycerol, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3b-hydroxy-5-cholestenoic acid, AEA(22:4)-1, AEA(22:4)-2, bilverdin, sphingosine, malic acid, and succinic acid.

[0016] The present inventors have found that patients with bone infection have higher levels of sphingosine-1-phosphate, taurocholic acid, glycocholate, 11a,b-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-dihydroxy-5a-pregnan-20-one-2, aldosterone, bisacron-2, naringenin, tauro-α-muricholic acid-1, and 11a,b-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-dihydroxy-5a-pregnan-20-one-2, aldosterone, bisacron-2, naringenin, and tauro-α-muricholic acid-1. The present inventors have found that tauro-α-muricholic acid-6 is increased, and 1-deoxysphingosine, 12(S)-HETE-2, 2-arachidonoylglycerol, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3b-hydroxy-5-cholestenoic acid, AEA(22:4)-1, AEA(22:4)-2, bilverdin, sphingosine, malic acid, and succinic acid are decreased, and have arrived at the present invention. The present invention relates to sphingosine-1-phosphate, taurocholic acid, glycololic acid, 11a,b-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-dihydroxy-5a-pregnan-20-one-2, aldosterone, bisacrone-2, naringenin, tauro-α-muricholic acid-6, 1-deoxysphingosine, 12(S)-HETE-2, 2-arachidonoylglycerol, 3 ...3-hydroxyprogesterone, 3-hydroxyprogesterone, 3-hydroxyprogesterone, 3-hydroxyprogesterone, 3-hydroxyprogesterone, 3-hydroxyprogesterone, 3-hydroxyprogesterone, 3-hydroxyprogesterone, 3-hydroxyprogesterone, 3-hydroxyprogesterone, 3-hydroxypro It is a plasma biomarker consisting of one or more substances selected from the group consisting of hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3b-hydroxy-5-cholestenoic acid, AEA(22:4)-1, AEA(22:4)-2, bilverdin, sphingosine, malic acid, and succinic acid. If the blood concentration of one or more of these substances is increased or decreased compared to normal values (before infection), it is determined that a bone infection has developed.

[0017] The blood biomarkers of the present invention include sphingosine-1-phosphate, taurocholic acid, glycorolic acid, 11a,b-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-dihydroxy-5a-pregnan-20-one-2, aldosterone, bisacrone-2, naringenin, tauro-α-muricholic acid-6, 1-deoxysphingosine, 12(S)-HETE-2, 2-arachidonoylglycerol, 3-hydroxydodecanoic acid, and 3-hydroxytetradecane. The composition of the present invention comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, nineteen or more, twenty or more, twenty-one or more, twenty-two or more, twenty-three or more, or twenty-four or more of 24 substances selected from the group consisting of benzoyl peroxidase (BPH), ...

[0018] In the present invention, it was revealed that of the 24 substances, 12 substances, namely, sphingosine-1-phosphate, taurocholic acid, glycololic acid, 11a,b-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-dihydroxy-5a-pregnan-20-one-2, aldosterone, bisacrone-2, naringenin, and tauro-α-muricholic acid-6, were increased in the group that developed bone infection.

[0019] In the present invention, it was revealed that of the 24 substances, 12 substances, namely 1-deoxysphingosine, 12(S)-HETE-2, 2-arachidonoylglycerol, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3b-hydroxy-5-cholestenoic acid, AEA(22:4)-1, AEA(22:4)-2, bilverdin, sphingosine, malic acid, and succinic acid, were decreased in the group that developed bone infection.

[0020] (2) Bone infection Most orthopedic diseases are benign, and treatments such as surgery aim to improve daily function. Unlike sepsis, which significantly increases inflammatory cytokines and leads to poor overall health, postoperative bone infections are unique infections characterized by a high recurrence rate and a tendency to develop into chronic infections, while maintaining overall health. Postoperative infections can significantly reduce treatment outcomes, and treatment often requires invasive reoperation to cleanse the infected area. The therapeutic effect of antibiotics on bone infections is poor compared to infections in other sites, as the drug is less easily transferred from the blood to tissues, and treatment is often prolonged. Therefore, early diagnosis is important, but diagnosis is often difficult because the early stage of infection only produces a mild inflammatory response and there are no specific infectious disease markers in blood tests. In particular, in the case of surgical site infections, it is difficult to distinguish between inflammation due to surgical invasion and inflammation due to infection, making early diagnosis of infection difficult, and delays in initiating treatment often lead to intractable disease. Furthermore, elderly people who are susceptible to infection can develop hematogenous bone infections such as pyogenic spondylitis and pyogenic arthritis even without surgery or trauma. Unprovoked bone infections in elderly people are often difficult to diagnose, especially when symptoms are minimal, and treatment is often delayed. If it becomes possible to detect bone infections early, treat them early, and prevent them from becoming chronic, it is expected that we can expect to see significant reductions in medical costs, as this will reduce medical expenses that would otherwise be incurred through prolonged treatment and ensure the ability to function daily, which would otherwise be impaired.

[0021] (3) Blood concentrations of plasma biomarkers The present invention relates to a method for determining whether or not a bone infection has developed by using one or more substances selected from the group consisting of sphingosine-1-phosphate, taurocholic acid, glycololic acid, 11a,b-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-dihydroxy-5a-pregnan-20-one-2, aldosterone, bisacrone-2, naringenin, tauro-α-muricholic acid-6, 1-deoxysphingosine, 12(S)-HETE-2, 2-arachidonoylglycerol, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3b-hydroxy-5-cholestenoic acid, AEA(22:4)-1, AEA(22:4)-2, bilverdin, sphingosine, malic acid, and succinic acid as plasma biomarkers (indicators). The development of bone infections includes osteomyelitis, pyogenic spondylitis, pyogenic arthritis, bone and joint disease, and postoperative infections.

[0022] The "subject" for which the plasma biomarker of the present invention can be used is not particularly limited, as long as it is a living organism that may develop a bone infection. Preferred are mammals such as humans, monkeys, gorillas, orangutans, cows, horses, dogs, cats, mice, rats, rabbits, sheep, goats, and pigs. Primates such as humans and monkeys are more preferred. Humans are most preferred.

[0023] The present inventors have investigated the effects of sphingosine-1-phosphate, taurocholic acid, glycololic acid, 11a,b-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-dihydroxy-5a-pregnan-20-one-2, aldosterone, bisacrone-2, naringenin, tauro-α-muricholic acid-6, 1-deoxysphingosine, 12(S)-HETE-2, 2-arachidonoyl The present inventors have discovered that the blood concentrations of one or more substances selected from the group consisting of glycerol, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3b-hydroxy-5-cholestenoic acid, AEA(22:4)-1, AEA(22:4)-2, bilverdin, sphingosine, malic acid, and succinic acid increase or decrease with the onset of bone infection compared to normal levels (in cases without bone infection). Therefore, a bone infection is determined to be present when the levels of these substances are increased or decreased compared to normal levels (in cases without bone infection).

[0024] The blood concentrations of these substances can be measured, for example, as follows. For example, in the case of a human, 3 ml of blood is collected. For example, the blood concentrations can be measured using an enzymatic method, such as the F-Kit series (manufactured by Roche Diagnostics). For example, succinic acid can be measured using a succinate colorimetric assay kit (manufactured by BioVision). Alternatively, each substance can be measured by the method described in Anal. Chem., 2009, 81, 6165-6174 (Non-Patent Document 4).

[0025] By measuring the blood concentrations (normal values) of each substance in patients who have not developed bone infections in advance, it is possible to compare the blood concentrations after events that may lead to bone infections, such as surgery, trauma, contact with an infected person, and local redness, swelling, or heat.

[0026] In one embodiment, the plasma biomarker for bone infection of the present invention can be used to determine the presence or absence of the onset of a bone infection. Microbial infection can occur due to surgery (during or after surgery), treatment (injection, etc.), trauma, or when the host is susceptible to infection (for example, due to autoimmune diseases such as rheumatoid arthritis, liver dysfunction, renal dysfunction, immunodeficiency, etc.).

[0027] In one embodiment, the plasma biomarkers of the present invention are used to determine microbial infection during surgery. Hereinafter, the present invention will be described in a non-limiting manner, taking "surgery" as an example of a situation in which the possibility of infection is suspected.

[0028] In one embodiment of the present invention, for example, sphingosine-1-phosphate, taurocholic acid, glycocholate, 11a,b-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-dihydroxy-5a-pregnan-20-one-2, aldosterone, bisacron-2, naringenin, tauro-α-muricholic acid-6, 1-deoxysphingosine-1-phosphate ...15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 15(S The blood concentrations of one or more substances selected from the group consisting of mannose, 12(S)-HETE-2, 2-arachidonoylglycerol, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3b-hydroxy-5-cholestenoic acid, AEA(22:4)-1, AEA(22:4)-2, bilverdin, sphingosine, malic acid, and succinic acid are compared with the blood concentrations (normal values) of each substance in patients without bone infection. Preferably, the blood concentration is measured 1 day or later, 2 days or later, or 3 days or later after surgery. Alternatively, the blood concentration is measured within 30 days, 20 days, 10 days, or 5 days after surgery.

[0029] When the blood concentrations after surgery are compared with the blood concentrations (normal values) of each substance in cases without bone infection, if the blood concentrations of one or more substances selected from the group consisting of sphingosine-1-phosphate, taurocholic acid, glycololic acid, 11a,b-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-dihydroxy-5a-pregnan-20-one-2, aldosterone, bisacron-2, naringenin, and tauro-α-muricholic acid-6 are increased compared to the blood concentrations (normal values) of each substance in cases without bone infection, it is determined that a bone infection has occurred.

[0030] "Increased" means, for example, when the blood concentration after surgery is compared with the blood concentration (normal value) of a case in which a bone infection has not developed, that the blood concentration of one or more substances has increased by, for example, 200% or more compared to the blood concentration (normal value) of a case in which a bone infection has not developed, more preferably 300% or more, even more preferably 500% or more, and particularly preferably 1000% or more.

[0031] When the blood concentrations after surgery are compared with the blood concentrations (normal values) of each substance in patients who have not developed a bone infection, if the blood concentrations of one or more substances selected from the group consisting of 1-deoxysphingosine, 12(S)-HETE-2, 2-arachidonoylglycerol, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3b-hydroxy-5-cholestenoic acid, AEA(22:4)-1, AEA(22:4)-2, bilverdin, sphingosine, malic acid, and succinic acid are decreased compared to the blood concentrations (normal values) of each substance in patients who have not developed a bone infection, it is determined that a bone infection has developed.

[0032] "When it is decreased" means, but is not limited to, for example, when the blood concentration after surgery is compared with the blood concentration (normal value) of a case in which a bone infection has not developed, and the blood concentration of one or more substances is found to be decreased to, for example, 60% or less compared to the blood concentration (normal value) of a case in which a bone infection has not developed, more preferably 40% or less, even more preferably 30% or less, and particularly preferably 20% or less.

[0033] The blood biomarkers of the present invention include, but are not limited to, the following embodiments. That is, these are plasma biomarkers that indicate the development of a bone infection when the blood concentration of one or more substances selected from the group consisting of sphingosine-1-phosphate, taurocholic acid, glycocholate, 11a,b-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-dihydroxy-5a-pregnan-20-one-2, aldosterone, bisacron-2, naringenin, and tauro-a-muricholic acid-6 in the blood increases to 200% or more of the blood concentration (normal value) of the corresponding substance before surgery on or after three days after surgery.

[0034] The blood biomarkers of the present invention include, but are not limited to, the following embodiments. That is, it is a plasma biomarker that indicates the development of a bone infection when the blood concentration of one or more substances selected from the group consisting of 1-deoxysphingosine, 12(S)-HETE-2, 2-arachidonoylglycerol, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3b-hydroxy-5-cholestenoic acid, AEA(22:4)-1, AEA(22:4)-2, bilverdin, sphingosine, malic acid, and succinic acid in the blood from three days after surgery is reduced to 60% or less of the blood concentration (normal value) of the corresponding substance before surgery.

[0035] 2. Method for determining the onset of bone infection during surgery The present invention also relates to a method for determining the onset of bone infection during surgery.

[0036] The determination method of the present invention comprises: measuring blood levels of one or more substances selected from the group consisting of sphingosine-1-phosphate, taurocholic acid, glycocholate, 11a,b-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-dihydroxy-5a-pregnan-20-one-2, aldosterone, bisacron-2, naringenin, and tauro-α-muricholic acid-6 in the subject after surgery; If the measured blood concentration is higher than normal, it is determined that a bone infection has developed. This process includes the following steps.

[0037] The determination method of the present invention comprises: measuring blood levels of one or more substances selected from the group consisting of 1-deoxysphingosine, 12(S)-HETE-2, 2-arachidonoylglycerol, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3b-hydroxy-5-cholestenoic acid, AEA(22:4)-1, AEA(22:4)-2, bilverdin, sphingosine, malic acid, and succinic acid in the subject after surgery; If the measured blood concentration is lower than normal, it is determined that a bone infection has developed. This process includes the following steps.

[0038] The definitions and explanations of "bone infection," "one or more substances selected from the group consisting of sphingosine-1-phosphate, taurocholic acid, glycololic acid, 11a,b-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-dihydroxy-5a-pregnan-20-one-2, aldosterone, bisacrone-2, naringenin, tauro-α-muricholic acid-6, 1-deoxysphingosine, 12(S)-HETE-2, 2-arachidonoylglycerol, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3b-hydroxy-5-cholestenoic acid, AEA(22:4)-1, AEA(22:4)-2, bilverdin, sphingosine, malic acid, and succinic acid," and "blood concentration" are as described in "1. Plasma markers for bone infection."

[0039] Regarding "measuring the blood concentration of one or more substances selected from the group consisting of sphingosine-1-phosphate, taurocholic acid, glycololic acid, 11a,b-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-dihydroxy-5a-pregnan-20-one-2, aldosterone, bisacron-2, naringenin, and tauro-α-muricholic acid-6 in postoperative subjects," the number of days after surgery on which to measure and "if the measured concentration is increased above normal" are the same as those described in "1. Plasma biomarkers for bone infections."

[0040] The determination method of the present invention includes, but is not limited to, the following aspects. That is, this is a method for determining whether a subject has developed a bone infection by measuring the blood concentration of one or more substances selected from the group consisting of sphingosine-1-phosphate, taurocholic acid, glycololic acid, 11a,b-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-dihydroxy-5a-pregnan-20-one-2, aldosterone, bisacron-2, naringenin, and tauro-a-muricholic acid-6 in a subject three days or more after surgery, and determining that the subject has developed a bone infection if the measured blood concentration increases to 200% or more of the normal value.

[0041] Regarding "measuring the blood concentration of one or more substances selected from the group consisting of 1-deoxysphingosine, 12(S)-HETE-2, 2-arachidonoylglycerol, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3b-hydroxy-5-cholestenoic acid, AEA(22:4)-1, AEA(22:4)-2, bilverdin, sphingosine, malic acid, and succinic acid in postoperative subjects," the number of days after surgery on which to measure the concentration and "if the measured concentration is lower than the normal value" are the same as those described in "1. Plasma biomarkers for bone infections."

[0042] The determination method of the present invention includes, but is not limited to, the following aspects. That is, this is a method for determining whether a subject has developed a bone infection if the blood concentration of one or more substances selected from the group consisting of 1-deoxysphingosine, 12(S)-HETE-2, 2-arachidonoylglycerol, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3b-hydroxy-5-cholestenoic acid, AEA(22:4)-1, AEA(22:4)-2, bilverdin, sphingosine, malic acid, and succinic acid decreases to 60% or less of the normal value in the subject three days or more after surgery.

[0043] 3. Method for determining the onset of bone infection in a subject The present invention also relates to a method for determining the onset of a bone infection in a subject.

[0044] The determination method of the present invention comprises: In subjects with elevated CRP levels, blood levels of one or more substances selected from the group consisting of sphingosine-1-phosphate, taurocholic acid, glycocholate, 11a,b-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-dihydroxy-5a-pregnan-20-one-2, aldosterone, bisacron-2, naringenin, and tauro-α-muricholic acid-6 are measured; If the measured concentration is higher than normal, it is determined that a bone infection has developed. This process includes the following steps.

[0045] The determination method of the present invention comprises: In subjects with elevated CRP levels, blood concentrations of one or more substances selected from the group consisting of 1-deoxysphingosine, 12(S)-HETE-2, 2-arachidonoylglycerol, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3b-hydroxy-5-cholestenoic acid, AEA(22:4)-1, AEA(22:4)-2, bilverdin, sphingosine, malic acid, and succinic acid are measured; If the measured concentration is lower than normal, it is determined that a bone infection has developed. This process includes the following steps.

[0046] The definitions and explanations of "bone infection," "one or more substances selected from the group consisting of sphingosine-1-phosphate, taurocholic acid, glycololic acid, 11a,b-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-dihydroxy-5a-pregnan-20-one-2, aldosterone, bisacrone-2, naringenin, tauro-α-muricholic acid-6, 1-deoxysphingosine, 12(S)-HETE-2, 2-arachidonoylglycerol, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3b-hydroxy-5-cholestenoic acid, AEA(22:4)-1, AEA(22:4)-2, bilverdin, sphingosine, malic acid, and succinic acid," and "blood concentration" are as described in "1. Plasma markers for bone infection."

[0047] Regarding "measuring the blood concentration of one or more substances selected from the group consisting of sphingosine-1-phosphate, taurocholic acid, glycololic acid, 11a,b-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-dihydroxy-5a-pregnan-20-one-2, aldosterone, bisacron-2, naringenin, and tauro-α-muricholic acid-6 in subjects with elevated CRP levels," if "the measured concentration is increased above normal," the same applies as described in "1. Microbial blood biomarkers."

[0048] The determination method of the present invention includes, but is not limited to, the following aspects. In other words, this is a method for determining whether a subject with a high CRP level has a bone infection, by measuring the blood concentration of one or more substances selected from the group consisting of sphingosine-1-phosphate, taurocholic acid, glycololic acid, 11a,b-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-dihydroxy-5a-pregnan-20-one-2, aldosterone, bisacron-2, naringenin, and tauro-a-muricholic acid-6, if the measured blood concentration is increased to 200% or more of the normal value.

[0049] Regarding "measuring the blood concentration of one or more substances selected from the group consisting of 1-deoxysphingosine, 12(S)-HETE-2, 2-arachidonoylglycerol, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3b-hydroxy-5-cholestenoic acid, AEA(22:4)-1, AEA(22:4)-2, bilverdin, sphingosine, malic acid, and succinic acid in subjects with high CRP levels," if "the measured concentration is lower than the normal value," the same applies as described in "1. Microbial blood biomarkers."

[0050] The determination method of the present invention includes, but is not limited to, the following aspects. In other words, this is a method for determining whether a subject with a high CRP level has a bone infection, by measuring the blood concentration of one or more substances selected from the group consisting of 1-deoxysphingosine, 12(S)-HETE-2, 2-arachidonoylglycerol, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3b-hydroxy-5-cholestenoic acid, AEA(22:4)-1, AEA(22:4)-2, bilverdin, sphingosine, malic acid, and succinic acid, and determining that the subject has developed a bone infection if the measured blood concentration is reduced to 60% or less of the normal value.

[0051] 4. Plasma biomarker concentration measurement kit for bone infection The present invention relates to a kit for measuring the concentration of a plasma biomarker for bone infection.

[0052] The measurement kit includes a reagent and a standard sample, and may further include other components as required.

[0053] The concentration is measured by at least one of dye colorimetry, ion chromatography, GC-MS, HPLC, immunochromatography, enzyme-linked immunosorbent assay (ELISA), chemiluminescent enzyme immunoassay (CLEIA), chemiluminescent immunoassay (CLIA), fluorescent enzyme immunoassay, aptamer method, immunoturbidimetry, immunonephelometry, latex agglutination, Jaffe method, and colloidal gold colorimetry. Example

[0054] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. Those skilled in the art can easily make modifications and variations to the present invention based on the description in this specification, and such modifications and variations are within the technical scope of the present invention.

[0055] Example 1: Search for new biomarkers using cases in which CRP levels increased again after the third day after surgery In this example, we searched for new biomarkers using cases in which CRP levels re-elevated after three days postoperatively. Specifically, after osteoarticular surgery, C-reacted protein (CRP), an inflammatory response, transiently increased, then decreased to normal levels. Blood samples were collected from six cases in which the CRP levels re-elevated after three days postoperatively. Blood samples were collected using 1 ml vacuum blood collection tubes (containing EDTA2K, TERUMO). Samples were centrifuged (4°C, 1200 rpm x 10 minutes), and 300 μl of plasma was collected and used for analysis. Three patients (control group) recovered naturally without antibiotics, and three patients (infection group) eventually worsened and were diagnosed with bone infection. Blood samples taken at the time of re-elevated CRP were compared using metabolomic analysis. There were no significant differences between the two groups in age, sex, eGFR, CRP, or white blood cell count. Patients with autoimmune disease, diabetes (HbA1c 7 or higher), and impaired renal function (eGFR 50 or lower), which may affect the results, were excluded from this study.

[0056] (Metabolome analysis) In addition to nucleic acids (DNA) and proteins, living organisms contain thousands of small molecules, including sugars, organic acids, amino acids, lipids, and steroid derivatives. Many of these are metabolic products produced by metabolic activity such as enzymes. The term "metabolome" refers to the collection and cataloging of all small molecules in a single tissue, including metabolic intermediates, hormones, signaling molecules, and secondary metabolites. The metabolome changes constantly over time. Metabolomic analysis uses bioinformatics to study the actual state of metabolism and the overall diversity of metabolic pathways, which differ subtly at each level: cell, tissue, organ, individual, and species. It is a method that can comprehensively analyze the types and concentrations of minute substances all at once.

[0057] The collected blood was subjected to comprehensive metabolomic analysis, measuring data on approximately 1,200 types of molecules, including sugars, amino acids, lipids, and steroid derivatives. For the metabolomic analysis in this example, HumanMetabolome's Dual Scan (CE-TOFMS and LC-TOFMS) was used.

[0058] The metabolomic analysis detected 331 candidate compounds as potential biomarkers, and based on reproducibility between samples, 12 compounds that were significantly higher in the group that developed bone infection and 12 compounds that were significantly lower in the group that developed bone infection were identified as biomarkers specific to human bone infection. As can be seen in the figure, the concentrations of each biomarker (the figure shows the percentage when the control group is set to 1) were significantly higher or lower than the control group (all control groups are set to 1 in the figure for relative comparison). [Industrial Applicability]

[0059] The plasma biomarker for bone infection, the method for determining the onset of bone infection, and the kit for measuring plasma biomarker concentrations for bone infection developed in this study will enable early detection of bone infection and are therefore useful in the field of orthopedic disease treatment.

Claims

1. A plasma biomarker for bone infection consisting of one or more substances selected from the group consisting of sphingosine-1-phosphate, taurocholic acid, glycololic acid, 11a,b-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-dihydroxy-5a-pregnan-20-one-2, aldosterone, bisacron-2, naringenin, tauro-a-muricholic acid-6, 1-deoxysphingosine, 12(S)-HETE-2, 2-arachidonoylglycerol, 3-hydroxydodecanoic acid, 3b-hydroxy-5-cholestenoic acid, AEA(22:4)-1, AEA(22:4)-2, and bilverdin.

2. The plasma biomarker of claim 1 for determining whether or not a bone infection has developed.

3. The plasma biomarker according to claim 1 or 2 for determining the onset of bone infection during surgery.

4. A plasma biomarker consisting of one or more substances selected from the group consisting of sphingosine-1-phosphate, taurocholic acid, glycololic acid, 11a,b-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-dihydroxy-5a-pregnan-20-one-2, aldosterone, bisacron-2, naringenin, and tauro-a-muricholic acid-6, which is present in the blood on or after three days after surgery, and which indicates the onset of a bone infection when the blood concentration of said one or more substances is increased to 200% or more of the normal value.

5. A plasma biomarker comprising one or more substances selected from the group consisting of 1-deoxysphingosine, 12(S)-HETE-2, 2-arachidonoylglycerol, 3-hydroxydodecanoic acid, 3b-hydroxy-5-cholestenoic acid, AEA (22:4)-1, AEA (22:4)-2, and bilverdin, in the blood three days or later after surgery, wherein a blood concentration of the one or more substances is reduced to 60% or less of the normal value, indicating the onset of a bone infection.

6. 1. A method for classifying a patient as having a bone infection in surgery, comprising: In the subject on or after 3 days after surgery, measuring the blood concentration of one or more substances selected from the group consisting of sphingosine-1-phosphate, taurocholic acid, glycololic acid, 11a,b-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-dihydroxy-5a-pregnan-20-one-2, aldosterone, bisacrone-2, naringenin, and tauro-a-muricholic acid-6; If the blood levels of the above substances are higher than normal, A method for classifying patients as having a bone infection.

7. 7. The method of claim 6, wherein a blood concentration of the substance is classified as having a bone infection if it is increased to 200% or more of normal.

8. 1. A method for classifying a patient as having a bone infection in surgery, comprising: measuring the blood concentration of one or more substances selected from the group consisting of 1-deoxysphingosine, 12(S)-HETE-2, 2-arachidonoylglycerol, 3-hydroxydodecanoic acid, 3b-hydroxy-5-cholestenoic acid, AEA(22:4)-1, AEA(22:4)-2, and bilverdin in the subject on or after 3 days after surgery; If the blood levels of the above substances are lower than normal, A method for classifying patients as having a bone infection.

9. 9. The method of claim 8, wherein a blood concentration of the substance is classified as having a bone infection if it is reduced to 60% or less of normal.

10. 1. A method for classifying a subject as having a bone infection, comprising: In a subject with an elevated CRP level, measuring the blood concentration of one or more substances selected from the group consisting of sphingosine-1-phosphate, taurocholic acid, glycololic acid, 11a,b-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-dihydroxy-5a-pregnan-20-one-2, aldosterone, bisacrone-2, naringenin, and tauro-a-muricholic acid-6; If the blood concentration of the substance is increased to 200% or more of the normal value, A method for classifying patients as having a bone infection.

11. 1. A method for classifying a subject as having a bone infection, comprising: In a subject with an elevated CRP level, measuring the blood concentration of one or more substances selected from the group consisting of 1-deoxysphingosine, 12(S)-HETE-2, 2-arachidonoylglycerol, 3-hydroxydodecanoic acid, 3b-hydroxy-5-cholestenoic acid, AEA(22:4)-1, AEA(22:4)-2, and bilverdin; If the blood concentration of the substance is reduced to 60% or less of the normal value, A method for classifying patients as having a bone infection.

12. 12. The method of claim 10 or 11, wherein the subject is a mammal.

13. A test kit for bone infections configured to measure the concentration of the plasma marker of claim 1 in human blood.

14. The test kit according to claim 13, wherein the measurement is carried out by at least one of dye colorimetry, ion chromatography, GC-MS, HPLC, immunochromatography, enzyme-linked immunosorbent assay (ELISA), chemiluminescent enzyme immunoassay (CLEIA), chemiluminescent immunoassay (CLIA), fluorescent enzyme immunoassay, aptamer method, immunoturbidimetry, immunonephelometry, latex agglutination, Jaffe method, and colloidal gold colorimetry.

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