Plasma biomarkers for bone infections

Abstract

The aim is to provide plasma biomarkers for bone infections in humans. [Solution] A plasma biomarker for bone infection comprising 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-didihydroxy-5a-pregnane-20-one-2, aldosterone, bisacron-2, naringenin, tauro-α-mulicolic acid-6, 1-deoxysphingosine, 12(S)-HETE-2, 2-arachidonoylglycerol, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3b-hydroxy-5-cholestic acid, AEA(22:4)-1, AEA(22:4)-2, bilbertin, sphingosine, malic acid, and succinic acid.

Description

Technical Field

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

Background Art

[0002] With the advent of a super-aged society, in the field of orthopedics, surgeries using biomaterials such as artificial joint replacement, spinal implant surgery, and artificial bone due to degenerative diseases and trauma have become widespread. On the other hand, in open bone and soft tissue injuries and implant surgeries, the frequency of complications of osteomyelitis during the treatment process is not low. In addition, in highly susceptible elderly people, osteomyelitis such as pyogenic spondylitis and pyogenic arthritis may develop hematogenously even without surgery or trauma, and this frequency has been steadily increasing. The therapeutic effect of antibiotics against osteomyelitis is poor in the tissue transferability of drugs from the blood compared to infections in other parts, so it is not uncommon for the treatment to be delayed. Therefore, early diagnosis is important, but in the early stage of infection, there is only a mild inflammatory reaction, and there is no specific infectious disease marker in blood tests, so there are many cases where diagnosis is difficult. Especially in the case of surgical site infections, it is difficult to distinguish between inflammation due to surgical invasion and inflammation due to infectious diseases, making early diagnosis of infectious diseases difficult, and delays in starting treatment often lead to intractable conditions.

[0003] Many studies have been conducted on biomarkers for infectious diseases, but most of them are for the purpose of evaluating the severity of severe sepsis with poor general condition in intensive care areas and the like. These markers often focus on inflammatory cytokines due to their disease background. However, osteomyelitis is a specific infectious disease characterized by a large increase in inflammatory cytokines, not in a state where the general condition deteriorates, but rather with a very high recurrence rate and a tendency to progress to chronic infectious diseases while maintaining the general condition. There are no reports of specific biomarkers for osteomyelitis, and reports focusing on metabolites are also very few even including infections in other parts. Even with procalcitonin, one of the representative biomarkers, the sensitivity and specificity for bone infections have been reported to be very low, at around 40% (Non-Patent Literature 1), so it is clear that biomarker exploration from a new perspective is necessary.

[0004] We have conducted basic research on bone infections and successfully created bacterial strains that stably fluoresce in vivo and in vitro. Using these bacteria, we have successfully created a reproducible osteomyelitis model in mice (Funao H. et al. Infect Immun, 2011, Non-Patent Literature 2) and a soft tissue infection model using the gluteus superficialis muscle (Yoshioka K. et al. PLOS ONE, 2014, Non-Patent Literature 3). Furthermore, we have conducted comprehensive metabolome analysis of metabolites using the blood of these femoral osteomyelitis mice and reported that the increase in sphingolipids sphingosine and sphinganin from the early stages of infection, and the decrease in α-ketoglutarate and succinate in the TCA cycle, may serve as specific biomarkers for the early stages of bone infection (Isogai N et al. Scientific Reports, 2020, Patent Literature 1). The problem here is that in the blood of human bone infection patients, similar results were observed in mice. The point is that it is unclear whether biomarkers are useful. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Republished Patent Publication 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 [Overview of the project] [Problems that the invention aims to solve]

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

[0008] The inventors have diligently researched the above problem and have found that when bone infections develop in humans, 12 specific substances (sphingosine-1-phosphate, taurocholic acid, glycololic acid, 11a,b-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-didihydroxy-5a-pregnane-20-one-2, aldosterone, bisacron-2, naringenin, tauro-α-mulicolic acid-1, tauro-α-mulicolic acid-6) are involved in the development of the disease. We discovered that the blood concentration of certain substances increased compared to cases where the disease was not present, and that the blood concentration of 12 specific substances (1-deoxysphingosine, 12(S)-HETE-2, 2-arachidonoylglycerol, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3b-hydroxy-5-cholestic acid, AEA(22:4)-1, AEA(22:4)-2, bilbertin, sphingosine, malic acid, succinic acid) decreased compared to cases where the disease was not present, which led us to conceive of the present invention. The present invention encompasses the following embodiments, though not limited thereto.

[0009] [1] A plasma biomarker for bone infections comprising one or more substances selected from the group consisting of sphingosine-1-phosphate, taurocholic acid, glycolic acid, 11a,b-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-didihydroxy-5a-pregnane-20-one-2, aldosterone, bisacron-2, naringenin, tauro-α-mulicole-6, 1-deoxysphingosine, 12(S)-HETE-2, 2-arachidonoylglycerol, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3b-hydroxy-5-cholestic acid, AEA(22:4)-1, AEA(22:4)-2, bilbertin, sphingosine, malic acid, and succinic acid. [2] The plasma biomarker described in [1] for determining whether or not the bone infection has occurred. [3] A plasma biomarker according to [1] or [2] for determining the development of bone infection during surgery. [4] A plasma biomarker in which, if the blood concentration of one or more substances selected from the group consisting of sphingosine-1-phosphate, taurocholic acid, glycololic acid, 11a,β-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-didihydroxy-5a-pregnane-20-one-2, aldosterone, bisacron-2, naringenin, and tauro-α-mulicoleic acid-6 increases to 200% or more of the normal value in the blood three days or more after surgery, it is judged that a bone infection has developed. [5] A plasma biomarker in which a bone infection is judged to have developed 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-cholestic acid, AEA(22:4)-1, AEA(22:4)-2, bilbertin, sphingosine, malic acid, and succinic acid decreases to 60% or less of the normal value in the blood three days or more after surgery. [6] A method for determining the development of bone infection in surgery, In subjects 3 days or more post-surgery, the blood concentration of one or more substances selected from the group consisting of sphingosine-1-phosphate, taurocholic acid, glycololic acid, 11a,β-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-didihydroxy-5a-pregnane-20-one-2, aldosterone, bisacron-2, naringenin, and tauro-α-mulicolic acid-6 was measured. If the blood concentration of the above substance is higher than normal, A method for determining whether a bone infection has developed. [7] The determination method according to claim 6, wherein if the blood concentration of the above substance has increased to 200% or more of the normal value, it is determined that a bone infection has developed. [8] A method for determining the development of bone infection in surgery, In subjects 3 days or more post-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-cholestic acid, AEA(22:4)-1, AEA(22:4)-2, bilbertin, sphingosine, malic acid, and succinic acid was measured. If the blood concentration of the above substance is lower than the normal value, A method for determining whether a bone infection has developed. [9] If the blood concentration of the above substance decreases to 60% or less of the normal value, it is determined that a bone infection has developed, according to the determination method described in [8].

[10] A method for determining the onset of bone infection in a subject, In subjects with high CRP levels, 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-didihydroxy-5a-pregnane-20-one-2, aldosterone, bisacron-2, naringenin, and tauro-α-mulicoleic acid-6 is measured. If the blood concentration of the above substance increases 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 bone infection in a subject, In subjects with high CRP levels, 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-cholestic acid, AEA(22:4)-1, AEA(22:4)-2, bilbertin, sphingosine, malic acid, and succinic acid is measured. If the blood concentration of the above substance decreases to 60% or less of the normal value, A method for determining whether a bone infection has developed.

[12] The determination method according to

[10] or

[11] , wherein the subject is a mammal.

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

[14] The test kit described in

[13] wherein the measurement is performed by at least one of the following methods: dye colorimetric method, ion chromatography method, GC-MS method, HPLC method, immunochromatography method, enzyme immunosorbent assay (ELISA), chemiluminescent enzyme immunosorbent assay (CLEIA), chemiluminescent immunosorbent assay (CLIA), fluorescence enzyme immunosorbent assay, aptamer method, immunoturbidimetry, immunohistochemistry, latex agglutination method, Jaffe method, and gold colloid colorimetric method. [Effects of the Invention]

[0010] Because the specific substances described above show a significant difference in their levels—either a significant increase or a significant decrease—in the blood of patients with bone infections, these substances are highly effective as plasma biomarkers for bone infections. Furthermore, they enable detection or diagnosis at an early stage, which is undetectable with conventional markers. Moreover, since the plasma biomarkers of this invention are based on the blood concentrations of these substances, measurement is simple, rapid, and inexpensive.

Brief Description of the Drawings

[0011] [Figure 1] Figure 1 is a graph comparing the concentrations in the control group and the infected group for 4 out of 12 substances that show high values in the group with osteomyelitis. [Figure 2] Figure 2 is a graph comparing the concentrations in the control group and the infected group for 4 out of 12 substances that show high values in the group with osteomyelitis. [Figure 3] Figure 3 is a graph comparing the concentrations in the control group and the infected group for 4 out of 12 substances that show high values in the group with osteomyelitis. [Figure 4] Figure 4 is a graph comparing the concentrations in the control group and the infected group for 4 out of 12 substances that show low values in the group with osteomyelitis. [Figure 5] Figure 5 is a graph comparing the concentrations in the control group and the infected group for 4 out of 12 substances that show low values in the group with osteomyelitis. [Figure 6] Figure 6 is a graph comparing the concentrations in the control group and the infected group for 4 out of 12 substances that show low values in the group with osteomyelitis.

Modes for Carrying Out the Invention

[0012] The inventors collected blood samples from patients with osteomyelitis in humans and control patients who had difficulty in clinical differentiation, and performed metabolome analysis on the samples with matched background data. As a result, 331 candidate compounds were identified. All of these are novel plasma biomarker candidates not reported in existing reports.

[0013] Following orthopedic bone and joint surgery, C-reacted protein (CRP), an inflammatory response, transiently increases and then decreases. This study focused on cases where CRP levels rose again during the CRP decrease phase. Three cases that spontaneously resolved without antibiotic administration (control group) and three cases that ultimately worsened and were diagnosed with bone infection (infection group) were compared using metabolome analysis of blood samples collected at the time of CRP recurrence. As a result, 331 samples were detected as candidate biomarkers, but based on reproducibility between samples, the following 24 metabolites were identified as biomarkers specific to bone infections in humans. This invention utilizes these 24 substances as plasma biomarkers for bone infections in humans.

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

[0015] (1) Plasma biomarker substances The blood biomarker of the present invention 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-didihydroxy-5a-pregnane-20-one-2, aldosterone, bisacron-2, naringenin, tauro-α-mulicoleic acid-1, tauro-α-mulicoleic acid-6, 1-deoxysphingosine, 12(S)-HETE-2, 2-arachidonoylglycerol, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3b-hydroxy-5-cholestic acid, AEA(22:4)-1, AEA(22:4)-2, bilbertin, sphingosine, malic acid, and succinic acid.

[0016] The inventors have found that in cases where bone infections develop, compared to cases where they do not develop, sphingosine-1-phosphate, taurocholic acid, glycololic acid, 11a,b-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-didihydroxy-5a-pregnane-20-one-2, aldosterone, bisacron-2, naringenin, and tauro-α-mulicolic acid-1 We discovered that tauro-α-mulicolic acid-6 levels were increasing, and that 1-deoxysphingosine, 12(S)-HETE-2, 2-arachidonoylglycerol, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3b-hydroxy-5-cholestic acid, AEA(22:4)-1, AEA(22:4)-2, bilbertin, sphingosine, malic acid, and succinic acid levels were decreasing, which led us to conceive of the present invention. This invention relates to sphingosine-1-phosphate, taurocholic acid, glycololic acid, 11a,b-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-didihydroxy-5a-pregnan-20-one-2, aldosterone, bisacron-2, naringenin, tauro-α-mulicolic acid-6, 1-deoxysphingosine, 12(S)-HETE-2, 2-arachidonoylglycerol, 3-hydro This plasma biomarker consists of one or more substances selected from the group comprising hydroxide decanoic acid, 3-hydroxytetradecanoic acid, 3b-hydroxy-5-cholestic acid, AEA(22:4)-1, AEA(22:4)-2, bilbertine, sphingosine, malic acid, and succinic acid. Bone infection is diagnosed when the blood concentration of one or more of these substances is increased or decreased compared to normal values ​​(pre-infection).

[0017] The blood biomarkers of the present invention are sphingosine-1-phosphate, taurocholic acid, glycololic acid, 11a,b-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-didihydroxy-5a-pregnane-20-one-2, aldosterone, bisacron-2, naringenin, tauro-α-mulicolic acid-6, 1-deoxysphingosine, 12(S)-HETE-2, 2-arachidonoylglycerol, 3-hydroxydodecanoic acid, and 3-hydroxytetradecane. The material consists of 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, or 24 substances selected from the group consisting of acids, 3b-hydroxy-5-cholestic acid, AEA(22:4)-1, AEA(22:4)-2, bilbertine, sphingosine, malic acid, and succinic acid.

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

[0019] In this invention, it was revealed that 12 of the 24 substances—1-deoxysphingosine, 12(S)-HETE-2, 2-arachidonoylglycerol, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3b-hydroxy-5-cholestic acid, AEA(22:4)-1, AEA(22:4)-2, bilbertin, sphingosine, malic acid, and succinic acid—were reduced in the group that developed bone infections.

[0020] (2) Bone infection Most orthopedic diseases are benign, and treatments, including surgery, aim to improve daily living function. Postoperative bone infections, unlike sepsis which involves a significant increase in inflammatory cytokines and a poor overall condition, are unique infections characterized by a very high recurrence rate and a tendency to progress to chronic infection, even though the patient's overall condition remains stable. This postoperative infection is a problem that can significantly reduce treatment outcomes, and treatment often requires invasive re-operation to clean the infected site. The therapeutic effect of antibiotics on bone infections is often prolonged compared to infections in other parts of the body, due to the poor tissue penetration of drugs from the bloodstream. Therefore, early diagnosis is crucial, but in the early stages of infection, only a mild inflammatory response is present, and there are no specific infection markers in blood tests, making diagnosis difficult in many cases. In particular, in surgical site infections, it is difficult to distinguish between inflammation caused by surgical trauma and inflammation caused by infection, making early diagnosis of the infection difficult, and delays in initiating treatment often lead to the infection becoming difficult to treat. Furthermore, in immunocompromised elderly individuals, bone infections such as suppurative spondylitis and suppurative arthritis can develop hematogenously even without a triggering surgery or trauma. Bone infections in the elderly without a specific triggering factor are often difficult to diagnose, especially when symptoms are mild, and treatment is frequently delayed. If bone infections can be detected and treated early, and their chronicity can be prevented, it is expected that medical costs will be reduced due to prolonged treatment and daily living abilities that may be impaired will be preserved, thus leading to a significant reduction in medical expenses.

[0021] (3) Blood concentration of plasma biomarkers The present invention determines 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-didihydroxy-5a-pregnane-20-one-2, aldosterone, bisacron-2, naringenin, tauro-α-mulicolic acid-6, 1-deoxysphingosine, 12(S)-HETE-2, 2-arachidonoylglycerol, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3b-hydroxy-5-cholestic acid, AEA(22:4)-1, AEA(22:4)-2, bilbertin, sphingosine, malic acid, and succinic acid as plasma biomarkers (indicators). The development of bone infections can manifest as conditions such as osteomyelitis, pyogenic spondylitis, septic arthritis, and postoperative infections related to bone and joint diseases.

[0022] The "subjects" on which the plasma biomarkers of the present invention can be used are not particularly limited to any organism that may develop bone infections. Preferably, these are mammals such as humans, monkeys, gorillas, orangutans, cattle, horses, dogs, cats, mice, rats, rabbits, sheep, goats, and pigs. More preferably, these are primates such as humans and monkeys. Most preferably, these are humans.

[0023] The inventors have identified sphingosine-1-phosphate, taurocholic acid, glycololic acid, 11a,b-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-didihydroxy-5a-pregnane-20-one-2, aldosterone, bisacroline-2, naringenin, tauro-α-mulicolate-6, 1-deoxysphingosine, 12(S)-HETE-2, 2-arachidonoyl The present invention was conceived based on the discovery that the blood concentration of one or more substances selected from the group consisting of glycerol, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3b-hydroxy-5-cholestic acid, AEA(22:4)-1, AEA(22:4)-2, bilbertine, sphingosine, malic acid, and succinic acid increases or decreases compared to normal values ​​(cases without bone infection) in the event of bone infection. Therefore, if these substances are increased or decreased compared to normal values ​​(cases without bone infection), it is determined that a bone infection has occurred.

[0024] The blood concentrations of each of these substances can be measured, for example, as follows: In a non-limiting manner, for example, 3 ml of blood can be collected in a human. For example, it can be measured by an enzymatic method, such as the F-Kit series (manufactured by Roche Diagnostics). For example, succinic acid can be measured using a succinic acid assay kit (Succinate Colorimetric Assay Kit, manufactured by BioVision). Alternatively, each substance can be measured by the method described in, for example, Anal. Chem., 2009, 81, 6165-6174 (Non-Patent Literature 4).

[0025] By pre-measuring the blood concentrations (normal values) of each substance in cases that have not developed bone infections, it is possible to compare these concentrations with those after circumstances suggesting the possibility of bone infections have occurred. Circumstances suggesting the possibility of bone infections include, for example, surgical procedures, trauma, contact with infected individuals, and local redness, swelling, and warmth.

[0026] In one embodiment, the plasma biomarker for bone infections of the present invention can be used to determine whether or not a bone infection has developed. Microbial infections can occur due to surgery (during and after surgery), treatment (injections, etc.), trauma, or when the host is an immunocompromised host (e.g., autoimmune diseases such as rheumatoid arthritis, liver dysfunction, kidney dysfunction, immunodeficiency, etc.).

[0027] In one embodiment, the plasma biomarker of the present invention is used to determine microbial infection during surgery. Hereinafter, the embodiments of the present invention will be described, without limitation, using "surgery" as an example of circumstances in which infection is suspected.

[0028] In one aspect of the present invention, for example, the blood of sphingosine-1-phosphate, taurocholic acid, glycolic acid, 11a,b-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-didihydroxy-5a-pregnan-20-one-2, aldosterone, bisacron-2, naringenin, tauro-α-mulicolate-6, 1-deoxysphingosine-1-phosphate, taurocholic acid, glycolic acid, 11a,β-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-didihydroxy-5a-pregnane The blood concentration of one or more substances selected from the group consisting of ngosine, 12(S)-HETE-2, 2-arachidonoylglycerol, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3b-hydroxy-5-cholestic acid, AEA(22:4)-1, AEA(22:4)-2, bilbertin, sphingosine, malic acid, and succinic acid is compared to the blood concentration (normal value) of each substance in cases that have not developed bone infection. Preferably, this comparison is made 1 day after surgery, 2 days after surgery, or 3 days after surgery. On the other hand, it is also made within 30 days, 20 days, 10 days, or 5 days after surgery.

[0029] A comparison of postoperative blood concentrations with the blood concentrations (normal values) of each substance in cases without bone infections indicates that if the blood concentration of one or more substances selected from the group consisting of sphingosine-1-phosphate, taurocholic acid, glycololic acid, 11a,β-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-didihydroxy-5a-pregnane-20-one-2, aldosterone, bisacron-2, naringenin, and tauro-α-mulicoleic acid-6 is elevated compared to the blood concentrations (normal values) of each substance in cases without bone infections, then bone infection is considered to have developed.

[0030] "Increased" means, for example, that when comparing the blood concentration after surgery with the blood concentration (normal value) of a case that has not developed bone infection, 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 that has not developed bone infection. More preferably, it is 300% or more, even more preferably 500% or more, and particularly preferably 1000% or more.

[0031] A comparison of postoperative blood concentrations with the blood concentrations (normal values) of each substance in cases without bone infections indicates that a bone infection has developed 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-cholestic acid, AEA(22:4)-1, AEA(22:4)-2, bilbertin, sphingosine, malic acid, and succinic acid is decreased compared to the blood concentrations (normal values) of each substance in cases without bone infections.

[0032] "When there is a decrease" means, without limitation, for example, when, as a result of comparing the blood concentration after surgery with the blood concentration (normal value) of a case in which a bone infection has not developed, the blood concentration of one or more substances has 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, it is 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. In other words, this plasma biomarker indicates that a bone infection has developed if the blood concentration of one or more substances selected from the group consisting of sphingosine-1-phosphate, taurocholic acid, glycololic acid, 11a,β-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-didihydroxy-5a-pregnane-20-one-2, aldosterone, bisacron-2, naringenin, and tauro-α-mulicoleic acid-6 increases to 200% or more of the pre-operative blood concentration (normal value) of the corresponding substance, three days or more after surgery.

[0034] The blood biomarkers of the present invention include, but are not limited to, the following embodiments. In other words, it is a plasma biomarker in which a bone infection is judged to have developed 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-cholestic acid, AEA(22:4)-1, AEA(22:4)-2, bilbertin, sphingosine, malic acid, and succinic acid in the blood three days or more after surgery is reduced to 60% or less of the blood concentration (normal value) of the corresponding substance before surgery.

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

[0036] The determination method of the present invention is In postoperative subjects, the blood concentration of one or more substances selected from the group consisting of sphingosine-1-phosphate, taurocholic acid, glycololic acid, 11a,β-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-didihydroxy-5a-pregnane-20-one-2, aldosterone, bisacron-2, naringenin, and tauro-α-mulicolic acid-6 was measured. If the measured blood concentration is higher than the normal range, it is determined that a bone infection has developed. This process includes the following steps.

[0037] The determination method of the present invention is In postoperative subjects, 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-cholestic acid, AEA(22:4)-1, AEA(22:4)-2, bilbertin, sphingosine, malic acid, and succinic acid was measured. If the measured blood concentration is lower than the normal value, 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-didihydroxy-5a-pregnane-20-one-2, aldosterone, bisacron-2, naringenin, tauro-α-mulicolic acid-6, 1-deoxysphingosine, 12(S)-HETE-2, 2-arachidonoylglycerol, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3b-hydroxy-5-cholestic acid, AEA(22:4)-1, AEA(22:4)-2, bilbertin, sphingosine, malic acid, succinic acid," and "blood concentration" are as described in "1. Plasma Markers for Bone Infection."

[0039] Regarding the "measurement of blood concentrations of one or more substances selected from the group consisting of sphingosine-1-phosphate, taurocholic acid, glycololic acid, 11a,β-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-didihydroxy-5a-pregnane-20-one-2, aldosterone, bisacron-2, naringenin, and tauro-α-mulicoleic acid-6 in postoperative subjects," the date on which measurement should be taken after surgery, and the case where "the measured concentration is higher than the normal value," are the same as described in "1. Plasma biomarkers for bone infections."

[0040] The determination method of the present invention includes, but is not limited to, the following embodiments. In other words, this is a diagnostic method in which, in subjects three days or more after surgery, 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-didihydroxy-5a-pregnane-20-one-2, aldosterone, bisacron-2, naringenin, and tauro-α-mulicoleic acid-6 is measured, and if the measured blood concentration has increased to 200% or more of the normal value, it is determined that a bone infection has developed.

[0041] Regarding the "measurement of 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-cholestic acid, AEA(22:4)-1, AEA(22:4)-2, bilbertin, sphingosine, malic acid, and succinic acid in postoperative subjects," the date on which measurement should be taken after surgery, and the case where "the measured concentration is lower than the normal value," are the same as described in "1. Plasma biomarkers for bone infections."

[0042] The determination method of the present invention includes, but is not limited to, the following embodiments. In other words, this is a diagnostic method in which, in subjects three days or more after surgery, 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-cholestic acid, AEA(22:4)-1, AEA(22:4)-2, bilbertin, sphingosine, malic acid, and succinic acid decreases to 60% or less of the normal value, it is determined that a bone infection has developed.

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

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

[0045] The determination method of the present invention is In subjects with high CRP levels, 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-cholestic acid, AEA(22:4)-1, AEA(22:4)-2, bilbertin, sphingosine, malic acid, and succinic acid is measured. If the measured concentration is lower than the normal value, 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-didihydroxy-5a-pregnane-20-one-2, aldosterone, bisacron-2, naringenin, tauro-α-mulicolic acid-6, 1-deoxysphingosine, 12(S)-HETE-2, 2-arachidonoylglycerol, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3b-hydroxy-5-cholestic acid, AEA(22:4)-1, AEA(22:4)-2, bilbertin, sphingosine, malic acid, succinic acid," and "blood concentration" are as described in "1. Plasma Markers for Bone Infection."

[0047] Regarding the "measurement of blood concentrations of one or more substances selected from the group consisting of sphingosine-1-phosphate, taurocholic acid, glycololic acid, 11a,β-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-didihydroxy-5a-pregnane-20-one-2, aldosterone, bisacron-2, naringenin, and tauro-α-mulicoleic acid-6 in subjects with high CRP levels," the case where "the measured concentration is higher than the normal value" is the same as described in "1. Blood biomarkers in microorganisms."

[0048] The determination method of the present invention includes, but is not limited to, the following embodiments. In other words, this is a diagnostic method in which, in individuals with high CRP levels, 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-didihydroxy-5a-pregnane-20-one-2, aldosterone, bisacron-2, naringenin, and tauro-α-mulicoleic acid-6 is measured, and if the measured blood concentration is increased to 200% or more of the normal value, it is determined that a bone infection has developed.

[0049] Regarding the "measurement of 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-cholestic acid, AEA(22:4)-1, AEA(22:4)-2, bilbertin, sphingosine, malic acid, and succinic acid in subjects with high CRP levels," the case where "the measured concentration is lower than the normal value" is the same as described in "1. Blood biomarkers in microorganisms."

[0050] The determination method of the present invention includes, but is not limited to, the following embodiments. In other words, this is a diagnostic method in which, in individuals with high CRP levels, 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-cholestic acid, AEA(22:4)-1, AEA(22:4)-2, bilbertin, sphingosine, malic acid, and succinic acid is measured, and if the measured blood concentration is reduced to 60% or less of the normal value, it is determined that a bone infection has developed.

[0051] 4. Plasma biomarker concentration measurement kit for bone infections This invention relates to a kit for measuring plasma biomarker concentrations in bone infections.

[0052] The measurement kit includes reagents and standard samples, and may also include other components as needed.

[0053] Concentration measurements are performed by at least one of the following methods: dye colorimetric method, ion chromatography, GC-MS, HPLC, immunochromatography, enzyme immunosorbent assay (ELISA), chemiluminescent enzyme immunosorbent assay (CLEIA), chemiluminescent enzyme immunosorbent assay (CLIA), fluorescence enzyme immunosorbent assay, aptamer method, immunoturbidimetric method, immunohistochemistry, latex agglutination method, Jaffe method, and gold colloid colorimetric method. Examples

[0054] The present invention will be described in detail below 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 herein, and such modifications fall within the technical scope of the present invention.

[0055] Example 1: Search for novel biomarkers using cases in which CRP levels rose again from the 3rd day post-surgery onward. In this study, we investigated novel biomarkers using cases in which CRP levels rose again from the third day postoperatively onward. Specifically, we collected blood samples from six cases in which C-reacted protein (CRP), an inflammatory response, transiently increased after bone and joint surgery, then decreased to normal levels, and then rose again from the third day postoperatively onward. Blood was collected at the time of the CRP re-elevation. Blood was collected using 1 ml vacuum blood collection tubes (containing EDTA2K, TERUMO Co., Ltd.). The samples were centrifuged (4°C, 1200 rpm x 10 minutes), and 300 μl of plasma was collected and used for analysis. We compared blood samples taken at the time of recurrence of elevated CRP levels in three cases (control group) that spontaneously resolved without antibiotic administration and three cases (infected group) that eventually worsened and were diagnosed with bone infection. There were no significant differences between the two groups in age, sex, eGFR, CRP, or white blood cell count. Cases with autoimmune diseases, diabetes (HbA1c ≥ 7), and impaired renal function (eGFR ≤ 50), which are thought to affect the results, were excluded from this study.

[0056] (Metabolome analysis) In addition to nucleic acids (DNA) and proteins, living organisms contain many small molecules, including sugars, organic acids, amino acids, lipids, and steroid derivatives, totaling thousands of different types. Many of these are metabolites produced by metabolic activity such as enzymes. The collective term for all small molecules in a living organism, including metabolic intermediates, hormones, signaling molecules, and secondary metabolites contained in a single tissue, is "metabolome." The metabolome changes constantly over time. Metabolome analysis is a study based on bioinformatics methods that examines the reality of metabolism and the totality of the diversity of metabolic pathways that subtly differ at each level—cell, tissue, organ, individual, and species—and is a method that can comprehensively analyze the types and concentrations of minute substances all at once.

[0057] Metabolome analysis was performed on collected blood samples by comprehensively measuring data for approximately 1200 molecules, including sugars, amino acids, lipids, and steroid derivatives. In this example, HumanMetabolome's Dual Scan (CE-TOFMS and LC-TOFMS) was used for the metabolome analysis.

[0058] Metabolome analysis revealed 331 candidate compounds as potential biomarkers. Based on reproducibility across samples, 12 compounds were identified as significantly higher in the group that developed bone infection, and 12 compounds were identified as significantly lower in the group that developed bone infection, as biomarkers specific to bone infection in humans. As shown in the figure, the concentrations of each biomarker (shown as a percentage relative to the control group, which is set to 1 in the figure for relative comparison) were significantly higher or lower in the group that developed bone infection. [Industrial applicability]

[0059] The plasma biomarkers for bone infections, the method for determining the onset of bone infections, and the plasma biomarker concentration measurement kit for bone infections developed in this study are useful in the field of orthopedic disease treatment because they enable the early detection of bone infections.

Claims

1. A plasma biomarker for bone infections comprising 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-didihydroxy-5a-pregnane-20-one-2, aldosterone, bisacron-2, naringenin, tauro-a-mulicoleic acid-6, 1-deoxysphingosine, 12(S)-HETE-2, 2-arachidonoylglycerol, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3b-hydroxy-5-cholestic acid, AEA(22:4)-1, AEA(22:4)-2, bilbertin, sphingosine, malic acid, and succinic acid.

2. A plasma biomarker according to claim 1 for determining whether or not the aforementioned bone infection has occurred.

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

4. A plasma biomarker in which bone infection is diagnosed if 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-didihydroxy-5a-pregnane-20-one-2, aldosterone, bisacron-2, naringenin, and tauro-α-mulicoleic acid-6 increases to 200% or more of the normal value in the blood three days or more after surgery.

5. A plasma biomarker in which a decrease in 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-cholestic acid, AEA(22:4)-1, AEA(22:4)-2, bilbertin, sphingosine, malic acid, and succinic acid to 60% or less of the normal value, three days or more after surgery, indicates the development of a bone infection.

6. A method for determining the occurrence of bone infection during surgery, In subjects three days or more post-surgery, 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-didihydroxy-5a-pregnane-20-one-2, aldosterone, bisacron-2, naringenin, and tauro-a-mulicolic acid-6 was measured. If the blood concentration of the above substance is higher than normal, A method for determining whether a bone infection has developed.

7. The determination method according to claim 6, wherein if the blood concentration of the above substance has increased to 200% or more of the normal value, it is determined that a bone infection has developed.

8. A method for determining the occurrence of bone infection during surgery, In subjects three days or more post-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-cholestic acid, AEA(22:4)-1, AEA(22:4)-2, bilbertin, sphingosine, malic acid, and succinic acid was measured. If the blood concentration of the above substance is lower than the normal value, A method for determining whether a bone infection has developed.

9. The determination method according to claim 8, wherein if the blood concentration of the above substance decreases to 60% or less of the normal value, it is determined that a bone infection has developed.

10. A method for determining the onset of bone infection in a subject, In subjects with high CRP levels, 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-didihydroxy-5a-pregnane-20-one-2, aldosterone, bisacron-2, naringenin, and tauro-a-mulicolic acid-6 is measured. If the blood concentration of the above substance increases 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 bone infection in a subject, In subjects with high CRP levels, 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-cholestic acid, AEA(22:4)-1, AEA(22:4)-2, bilbertin, sphingosine, malic acid, and succinic acid is measured. If the blood concentration of the above substance has decreased to 60% or less of the normal value, A method for determining whether a bone infection has developed.

12. The determination method according to claim 10 or 11, wherein the subject is a mammal.

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

14. The test kit according to claim 13, wherein the measurement is performed by at least one of the following methods: dye colorimetric method, ion chromatography method, GC-MS method, HPLC method, immunochromatography method, enzyme immunoassay (ELISA), chemiluminescent enzyme immunoassay (CLEIA), chemiluminescent immunoassay (CLIA), fluorescence enzyme immunoassay, aptamer method, immunoturbidimetry, immunowax method, latex agglutination method, Jaffe method, and gold colloid colorimetric method.

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