KR102253363B1 - A Novel Metabolite Marker for Tuberculosis and A Method for Diagnosing Tuberculosis Using the Same - Google Patents

Abstract

The present invention relates to a novel metabolite marker for a mycobacterium infected disease and a method for diagnosing a mycobacterium infected disease using the same. The present invention uses the metabolite marker as a marker and rapidly provides reliable information for mycobacterium infection and infection of latent tuberculosis or active tuberculosis which is difficult to distinguish from the prior art. Accordingly, the present invention rapidly and accurately diagnoses the tuberculosis, evaluates reactivity for a tuberculosis medicine, and is usefully used as a screening tool of a medicine candidate material.

Description

A Novel Metabolite Marker for Tuberculosis and A Method for Diagnosing Tuberculosis Using the Same}

The present invention relates to a novel metabolite marker serving as a marker for Mycobacterium infection, and a method for diagnosing Mycobacterium infection, specifically active tuberculosis, by measuring its serum concentration.

Tuberculosis (TB) is the leading cause of death from a single infectious agent, and it is estimated that 10 million people died worldwide in 2017, of which 1.6 million died (World Health Organization, Global tuberculosis report 2018 (WHO/CDS/ TB/2018.20). 2018). Timely diagnosis and treatment are critical to successfully managing TB. Currently, there are two methods for evaluating contact with tuberculosis: tuberculin skin test (TST) and interferon gamma release assay (IGRA). However, these existing diagnostic methods are difficult to distinguish between latent tuberculosis infection (LTBI) and active tuberculosis, because these methods are indirect methods that show immunological responses to Mybacteria Tuberculosis (MTB) antigens ( Targowski, T., S. Chelstowska, and T. Plusa, Lung , 192 (6): 869-874 (2014)). Therefore, the development of a biomarker showing the actual presence of Mycobacterium tuberculosis in the human body is important in establishing a treatment strategy for patients with positive results for TST and IGRA.

Metabolomics is a new technology that enables remarkable advances in the study of new biomarkers by enabling multiplex profiling and comparison of metabolites in biological samples (Isa, F., et al., EBio Medicine , 31 : 157-165 (2018)). Metabolomics is a means of performing non-biased identification and quantification of metabolites (or small molecule compounds) present in biological samples using highly specialized analytical methods along with various computational, statistical and mathematical analyses (Dunn, WB, NJ Bailey). , and HE Johnson, Analyst , 130 (5):606-625 (2005)). Therefore, metabolomics has been successfully used to identify new biomarkers for different disease states (Nagana Gowda, GA and D. Raftery, Curr Metabolomics , 1 (3):227-240 (2013)). New markers can be used to increase sensitivity to disease and host responses and consequently to develop improved diagnostic and therapeutic strategies (Loots, DT, Biomark Med , 10 (10): 1025-1028 (2016)). With regard to tuberculosis, several metabolomics studies are currently being conducted to explore new metabolic markers that may be associated with tuberculosis infections or treatment responses using sputum, blood, breath, and urine, but these metabolite studies are still limited. Is losing.

Throughout this specification, a number of papers and patent documents are referenced and citations are indicated. The disclosure contents of the cited papers and patent documents are incorporated by reference in this specification as a whole, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly described.

Patent Document 1. Korean Application No. 10-2017-0121014

The present inventors have made intensive research efforts to develop a method for quickly and highly accurate judgment of Mycobacterium infection in a subject through a simple process. As a result, by measuring the levels of the eight metabolites listed above in a biological sample isolated from the subject, it was found that direct information on the presence or absence of Mycobacterium in the subject can be obtained in real time with remarkably improved reliability, thereby completing the present invention. Was done.

Accordingly, an object of the present invention is to provide a diagnostic kit for Mycobacterium infection.

Another object of the present invention is to provide a method of providing information necessary for diagnosis of a Mycobacterium infection disease.

Other objects and advantages of the present invention will become more apparent by the following detailed description, claims and drawings.

According to one aspect of the present invention, the present invention is a mycobacterium comprising a quantification device for one or more metabolites selected from the group consisting of glutamate, glutamine, sulfoxy methionine, methionine, aspartate, asparagine, kynurenine and tryptophan. ( Mycobacterium ) Provides a diagnostic kit for infectious diseases.

The present inventors have made intensive research efforts to develop a method for quickly and with high accuracy determining whether Mycobacterium infection in a subject is through a simple process. As a result, it was found that direct information on the presence or absence of Mycobacterium in the subject can be obtained in real time with remarkably improved reliability by measuring the level of all or part of the eight metabolites listed above in the biological sample isolated from the subject. .

In the present specification, the term “diagnosis” encompasses determination of an individual's susceptibility to a specific disease, determination of whether an individual currently has a specific disease, and determination of the prognosis of an individual suffering from a specific disease. It is meaning. According to the present invention, the present inventors determined that the concentrations of the eight metabolites listed above were significantly different in the biological samples of Mycobacterium-infected individuals and normal individuals, specifically, serum. It has been newly discovered that it can function as a marker.

In the present specification, the term “ Mycobacterium infection disease” is meant to encompass all clinical symptoms manifested by infection with pathogens of the genus Mycobacterium including tuberculosis or tuberculosis mycobacteria, lung disease, lymphadenitis, skin It includes, but is not limited to, soft tissue, osteoinfection, and disseminated diseases, and includes all pathological conditions that directly or indirectly cause infection with pathogens of the genus Mycobacterium. According to a specific embodiment of the present invention, the Mycobacterium infection disease diagnosed with the kit of the present invention is tuberculosis, and more specifically, active tuberculosis.

As used herein, the term “quantitative device” refers to a device that provides quantitative numerical information on the relative or absolute amount of a specific metabolite as well as the presence or absence of a specific metabolite in a biological sample. Specifically, the quantification device is a mass spectrometry device. In the present specification, the term mass spectrometry (MS) refers to a process of measuring the mass of a target substance in order to analyze the chemical composition of a sample. Mass spectrometry provides information on mass by generating charged molecules or molecular fragments through ionization of a target substance present in a sample, and measuring the mass-to-charge ratio (m/z) and the abundance ratio of gaseous ions. Such mass spectrometry devices are, for example, Matrix-Assisted Laser Desorption/ Ionization Time of Flight (MALDI-TOF), Surface Enhanced Laser Desorption/Ionization Time of Flight (SELDI-TOF), Electrospray ionization time-of-flight (ESI-TOF). ), liquid chromatography-Mass Spectrometry (LC-MS) and liquid chromatography-Mass Spectrometry/Mass Spectrometry (LC-MS/MS), but are not limited thereto. Specifically, the mass spectrometer that can be used in the present invention is LC-MS/MS.

According to a specific embodiment of the present invention, the concentration of one or more metabolites selected from the group consisting of glutamate, sulfoxy methionine, aspartate and kynurenine is increased, or glutamate/glutamine, aspartate/asparagine, sulfoxy An increase in the ratio between the concentrations of one or more metabolites selected from the group consisting of methionine/methionine and kynurenine/tryptophan indicates an increased risk of Mycobacterium infection disease.

According to the present invention, the concentrations of glutamate, sulfoxy methionine, aspartate, and kynurenine among the metabolites of the present invention were significantly increased in patients with tuberculosis infection, and the ratio between the concentrations of the two metabolites, glutamate/glutamine, as well as Partate/asparagine, sulfoxy methionine/methionine and kynurenine/tryptophan were also significantly increased. Based on these data with statistical significance, it can be seen that these metabolites and the ratio between metabolites function as a highly reliable positive marker for Mycobacterium infection.

The terms "increase in concentration" or "increase in concentration liver ratio" used while referring to "diagnosis kit" or "diagnostic composition" in the constitution of the present invention means the concentration of large cells in the serum compared to normal people who are not infected with Mycobacterium or It means a case in which the ratio of the concentration between the two metabolites is significantly high, and specifically, an increase of about 10% or more, an increase of about 20% or more, an increase of about 30% or more, an increase of about 40% or more compared to the normal person and the control group, It refers to an increase of about 50% or more, or an increase of about 60% or more, but does not exclude a range beyond this.

According to a specific embodiment of the present invention, a decrease in the concentration of one or more metabolites selected from the group consisting of glutamine, methionine and asparagine indicates an increased risk of Mycobacterium infection disease.

According to the present invention, the concentrations of glutamine, methionine, and asparagine in the metabolites of the present invention were significantly reduced in patients with tuberculosis infection, and based on these data with statistical significance, these metabolites were highly reliable against Mycobacterium infection. It can be seen that it functions as a negative marker.

According to another aspect of the present invention, the present invention determines the concentration of one or more metabolites selected from the group consisting of glutamate, glutamine, sulfoxy methionine, methionine, aspartate, asparagine, kynurenine and tryptophan in a biological sample isolated from a subject. It provides a method of providing information necessary for the diagnosis of Mycobacterium infection disease comprising the step of measuring.

In the present invention, since the method of measuring the concentration of the mycobacterium infection disease and amino acid metabolite to provide information necessary for diagnosis has already been described above, description thereof will be omitted in order to avoid excessive redundancy.

In the present invention, the term "biological sample" is any sample containing or likely to contain pathogenic strains of the genus Mycobacterium, obtained from mammals including humans, and includes blood, tissues, organs, cells, or cell cultures, It is not limited thereto. Specifically, the biological sample used in the present invention is serum or plasma.

In the present specification, the term "subject" refers to an individual that provides a sample for investigating whether or not Mycobacterium is infected, and is ultimately an object of analysis for whether or not it is infected by a Mycobacterium strain. Subjects include, without limitation, humans, mice, rats, guinea pigs, dogs, cats, horses, cows, pigs, monkeys, chimpanzees, baboons or rhesus monkeys, specifically humans.

According to a specific embodiment of the present invention, the method of the present invention comprises at least one metabolite selected from the group consisting of glutamate/glutamine, aspartate/asparagine, sulfoxy methionine/methionine and kynurenine/tryptophan from the measured metabolite concentration. It further comprises the step of obtaining a value of the ratio between the concentrations.

Since the positive markers, negative markers, and criteria for determining Mycobacterium infection discovered in the present invention have already been described above, descriptions thereof will be omitted to avoid excessive redundancy.

The features and advantages of the present invention are summarized as follows:

(a) The present invention proposes a concentration ratio between 8 serum metabolites and some of them showing a significant difference in concentration between a patient infected with Mycobacterium and a normal person.

(b) The present invention uses the above-described metabolite concentration or concentration ratio as a marker , as well as the presence of Mycobacterium infection, as well as infection of latent tuberculosis or active tuberculosis, which was difficult to distinguish with the prior art. Quickly provide reliable information on whether or not.

(c) The present invention can be usefully used as a tool for rapid and accurate diagnosis of tuberculosis, as well as a reactivity evaluation for a tuberculosis therapeutic agent and a screening tool for a therapeutic agent candidate.

1 is a diagram showing the results of metabolite profiling in the serum of a normal control group, latent tuberculosis infected patients, and active tuberculosis patients through PCA-DA analysis.
2 is a diagram showing the results of comparing the levels of metabolites that are statistically significantly increased or decreased in active tuberculosis patients through volcano plot analysis.
3 is a normal control group, glutamate, glutamine, glutamate/glutamine, sulfoxy methionine, methionine, sulfoxy methionine/methionine (FIG. 3A ), aspartate, asparagine, aspartate/ It is a figure showing the result of comparing the concentrations of asparagine, kynurenine, tryptophan, and kynurenine/tryptophan (FIG. 3B).
Figure 4 is a glutamate, glutamine, glutamate / glutamine, sulfoxy methionine, methionine, sulfoxy methionine / methionine (Fig. 4a), aspartate, asparagine, as The results of performing ROC analysis on partate/asparagine, kynurenine, tryptophan, and kynurenine/tryptophan (FIG. 4B) are shown, respectively.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Example 1. Identification of metabolites from serum samples using LC-MS/MS

The specimens used in the present invention were serum from 21 active tuberculosis patients, 28 healthy controls, and 20 latent tuberculosis infected patients, along with 21 active tuberculosis patients at Shinchon Severance Hospital.

After diluting 10 μl of serum of each of active tuberculosis patients, latent tuberculosis infected patients, and healthy control groups to 15:1 in 40% methanol, metabolite analysis was performed. The LC-MS/MS analysis procedure was as follows: Separation of amino acids and biological amines was performed using an Agilent 1260 infinity HPLC system (SCIEX, Woodlands Centeral, Singapore) equipped with Waters Acquity. The analyte was separated using a gradient from 0.1% formic acid dissolved in water to 0.1% formic acid dissolved in acetonitrile. The total LC analysis time took about 12 minutes for each sample. Thereafter, FIA-MS/MS was performed to analyze acylcarnitines, glycerophospholipids, and hexose. For peak integration, calibration, and concentration calculation, LC-MS/MS data were processed using the ^{SCIEX application program Analyst™.} The LC-MS/MS data and FIA-MS/MS data analyzed from the SCIEX application program Analyst ^TM were analyzed using the ^{Biocrates MetlDQ TM program.} This strategy can simultaneously quantify 187 metabolites (42 amino acids and binary amines, 40 acylcarnitines, 90 glycerol phospholipids, 14 sphingomyelins, 1 monosaccharide, but 7 metabolites). Was not included in the analysis because the detection limit was too low.

The characteristics of patients used from Shinchon Severance Hospital are summarized in Table 1 below. The sample composition in this study was 28 normal control subjects (average age 28 years, 10 males (35.7%)), latent tuberculosis infection 20 subjects (mean age 48.5 years old, 6 males (30.0%)), active tuberculosis patients 21 People (average age 27 years, 10 males (47.6%)).

Clinical information results of normal control group, latent tuberculosis infection, and active tuberculosis patient Active tuberculosis patients
(N=21) People with latent tuberculosis
(N=20) Normal control
(N=28) Median Age (Range) 27 (20-50) 48.5 (23-69) 28 (22-57) Gender, male 10 (47.6) 6 (30.0) 10 (35.7) BMI, kg/m ² , median (IQR) 20.0 (18.6-21.9) 21.7 (20.9-23.9) 22.1 (20.6-23.4) Past TB history 0 (0) 2 (10.0) 0 (0) BCG scars present 16 (76.2) 17 (85.0) 19 (67.9) Comorbid disease High blood pressure 0 (0) 2 (10.0) 0 (0) diabetes 0 (0) 1 (5.0) 0 (0) Other ^b 1 (4.8) 3 (15.0) 0 (0) Diagnosis of pulmonary TB Sputum AFB smear, positive 1 (4.8) Sputum AFB culture, positive 19 (90.5) Chest imaging range ^e Less than a third 17 (81.0) Less than two-thirds 4 (19.0) More than two-thirds 0 (0) Extrapulmonary injury combination 2 (9.5) TST positive 2 (100) 16 (80.0) 0 (0) TST hardened, mm, median (range) 15 (12-18) 14 (0-25) 0 (0-4) QFT-GIT training 19 (100) 20 (100) 0 (0)

Example 2. Differences in serum metabolite profiles of active tuberculosis patients and healthy controls and latent tuberculosis infected patients using PCA-DA

The metabolites derived from Example 1 were subjected to metabolite profiling using PCA-DA (Principal component analysis and discriminant analysis). As shown in FIG. 1, it was confirmed that the profiling of metabolites in the serum of active tuberculosis patients, healthy controls, and latent tuberculosis infected patients clearly differed.

Example 3. Selection of biomarker metabolites specific to active tuberculosis patients

In order to find a biomarker that was specifically increased or decreased in active tuberculosis patients, the fold change, p-value, which affects the difference in metabolite profiling derived from Example 2 from each metabolite was calculated. . Volcano based on p-value (<0.00001; -log ₁₀ (0.00001)=5) or fold change (>1.414; log ₂ (1.414)=0.5 or <0.707; log ₂ (0.707)=-0.5) as a reference (cutoff). Through plot analysis, it was confirmed that 8 metabolites are biomarkers suitable for diagnosing active tuberculosis (FIG. 2).

Example 4. Analysis of the concentration and ratio of 8 metabolites for the diagnosis of active tuberculosis through a serum sample

Eight metabolites selected from Example 3, glutamate, glutamine, sulfoxy methionine, methionine, aspartate, asparagine, kynurenine, tryptophan, glutamate/glutamine, sulfoxy methionine/methionine, which are the ratio of concentration and 4 , Aspartate/asparagine, and kynurenine/tryptophan ratios were analyzed. As a result, glutamate, glutamate/glutamine, sulfoxy methionine, sulfoxy methionine/methionine, aspartate, aspartate/asparagine, kynurenine and kynurenine/tryptophan increased statistically significantly in active tuberculosis patients, while glutamine , It was confirmed that the concentration of methionine and asparagine was statistically significantly reduced (FIG. 3).

Example 5. ROC (receiver operating characteristic) analysis of metabolite biomarkers for diagnosis of active tuberculosis using serum samples

ROC was analyzed to test the usefulness of diagnosis of active tuberculosis using 12 biomarkers in the serum sample selected through Example 4 (FIG. 4). As a result, the AUC values of glutamate, glutamine, glutamate/glutamine, sulfoxy methionine, methionine, sulfoxy methionine/methionine, aspartate, asparagine, aspartate/asparagine, kinurenin, tryptophan and kinurenin/tryptophan were 0.998. It was confirmed that they were 1.0000, 1.0000, 1.0000, 0.9916, 1.0000, 0.9732, 0.9325, 0.9980, 0.6329, 0.4807 and 0.6379 (Table 2).

Median and diagnostic value of each metabolite biomarker Metabolites and ratios Active tuberculosis patients Healthy people and people with latent tuberculosis infection P-value ^b AUC ^c (N=21) (N=48) Glu (nmol/L) 386000 [344000-428000] 100200 [70900-150000] <0.001 0.9980 [0.9933-1.0000] Gln (nmol/L) 180000 [136000-257000] 638000 [596000-715000] <0.001 1.0000 [NA] Glu/Gln 1.7650 [1.5341-2.9930] 0.1506 [0.1133-0.2144] <0.001 1.0000 [NA] MetSO (nmol/L) 23500 [19800-27200] 1485 [1210-2030] <0.001 1.0000 [NA] Met (nmol/L) 3400 [1530-7350] 28250 [24900-31850] <0.001 0.9916 [0.9770-1.0000] MetSO/Met 7.4359 [3.5374-22.3197] 0.0516 [0.0424-0.0763] <0.001 1.0000 [NA] Asp (nmol/L) 83200 [72400-94200] 41050 [31750-47400] <0.001 0.9732 [0.9430-1.0000] Asn (nmol/L) 30900 [28300-42900] 51800 [46450-58500] <0.001 0.9325 [0.8704-0.9947] Asp/Asn 2.3108 [2.1264-2.9890] 0.7557 [0.6119-0.9315] <0.001 0.9980 [0.9933-1.0000] Kyn (nmol/L) 2170 [1810-2720] 1995 [1680-2275] 0.082 0.6329 [0.4719-0.7940] Trp (nmol/L) 62400 [56000-71100] 60700 [56050-73500] 0.804 0.4807 [0.3230-0.6383] Kyn/Trp 0.0329 [0.0282-0.0412] 0.0309 [0.0271-0.0350] 0.071 0.6379 [0.4756-0.8002]

As described above, specific parts of the present invention have been described in detail, and it is obvious that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereto for those of ordinary skill in the art. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (16)

A diagnostic kit for Mycobacterium infection disease, including a quantification device for methionine sulfoxide.
The kit according to claim 1, wherein the Mycobacterium infection disease is tuberculosis.
The kit according to claim 2, wherein the tuberculosis is active tuberculosis.
The kit according to claim 1, wherein the kit further comprises a quantitative device for one or more metabolites selected from the group consisting of glutamate, glutamine, methionine, aspartate and asparagine.
The method of claim 4, wherein the quantification device is MALDI-TOF (Matrix-Assisted Laser Desorption/ Ionization Time of Flight), SELDI-TOF (Sulface Enhanced Laser Desorption/Ionization Time of Flight), ESI-TOF (Electrospray ionization time-of). -flight), liquid chromatography-mass spectrometry (LC-MS), and LC-MS/MS (liquid chromatography-Mass Spectrometry/ Mass Spectrometry). Kit to do.
The kit according to claim 5, wherein the mass spectrometry device is LC-MS/MS.
The method of claim 4, wherein the concentration of one or more metabolites selected from the group consisting of glutamate, methionine sulfoxide and aspartate is increased, or the group consisting of glutamate/glutamine, aspartate/asparagine and methionine sulfoxide/methionine A kit, characterized in that it indicates an increased risk of Mycobacterium infection disease when the ratio between the concentration of one or more metabolites selected from is increased.
The kit according to claim 4, wherein a decrease in the concentration of one or more metabolites selected from the group consisting of glutamine, methionine and asparagine indicates an increased risk of Mycobacterium infection.
A method of providing information necessary for diagnosis of a Mycobacterium infectious disease comprising the step of measuring the concentration of methionine sulfoxide in a biological sample isolated from a subject.
The method of claim 9, wherein the Mycobacterium infection disease is tuberculosis.
11. The method of claim 10, wherein the tuberculosis is active tuberculosis.
The method of claim 9, wherein the biological sample is serum or plasma.
The method of claim 9, wherein the method further comprises measuring the concentration of one or more metabolites selected from the group consisting of glutamate, glutamine, methionine, aspartate and asparagine.
The method of claim 13, wherein the method further comprises obtaining a ratio value between the concentrations of one or more metabolites selected from the group consisting of glutamate/glutamine, aspartate/asparagine and methionine sulfoxide/methionine from the measured metabolite concentration. Method comprising the.
The method of claim 14, wherein the concentration of one or more metabolites selected from the group consisting of glutamate, methionine sulfoxide and aspartate is increased, or the group consisting of glutamate/glutamine, aspartate/asparagine and methionine sulfoxide/methionine When the ratio between the concentration of one or more metabolites selected from increases, it is determined that the risk of Mycobacterium infection is increased.
14. The method of claim 13, wherein when the concentration of one or more metabolites selected from the group consisting of glutamine, methionine and asparagine decreases, it is determined that the risk of mycobacterium infection is increased.

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