Metabolome marker for differential diagnosis of mycobacterium avium complex or mycobacterium tuberculosis infectious disease

Abstract

The present invention relates to a composition for differential diagnosis of a Mycobacterium avium complex (MAC) or Mycobacterium tuberculosis (MTb) infectious disease using lipid metabolites, amino acids, and / or amino acid derivatives as markers. The present invention applies the metabolites as markers for predicting, with high accuracy, Mycobacterium avium complex and Mycobacterium tuberculosis infection diseases, which are two diseases having similar clinical expression characteristics, thereby enabling rapid discrimination of the diseases. Accordingly, a patient-customized treatment strategy can be established at an early stage, and thus, the present invention can be helpfully used to significantly improve the survival rate of a patient.

Description

Metabolite markers for differential diagnosis of Mycobacterium avium complex or Mycobacterium tuberculosis infections

[0001] The present invention relates to a metabolite marker for the differentiation or diagnosis of patients with nontuberculous mycobacterial infections and patients with tuberculosis infections.

[0002] There are currently over 72 known species of Mycobacterium, including not only species that cause serious diseases in humans and animals, such as tuberculosis, Mycobacterium bovis, and Mycobacterium leprae, but also species called opportunistic infections, and saprophytic species found in the natural environment, of which 25 species are known to be associated with human diseases. These Mycobacterium species are not easily stained with commonly used staining solutions, but once stained, they do not easily decolorize even when treated with alcohol or hydrochloric acid, which is why they are also called acid-fast bacteria. Acid-fast bacteria are divided into nontuberculous mycobacteria (NTM), Mycobacterium tuberculosis complex, and Mycobacterium leprae.

[0003] More than 180 nontuberculous mycobacterial strains have been officially identified as commonly causing lung disease in humans. The Mycobacterium avium complex (MAC) primarily includes M. avium and M. intracellulare, while Mycobacterium abscessus (MAB) primarily includes M. abscessus subspecies abscessus and M. abscessus subspecies massiliense.

[0004] Meanwhile, Mycobacterium tuberculosis (MTB) is a bacterium that causes tuberculosis and belongs to the tubercle bacilli family.

[0005] Despite the recent increase in reports of lung infections caused by mycobacteria worldwide, there is a lack of biomarkers to distinguish patients with lung infections caused by Mycobacterium avium complex (MAC), a nontuberculous mycobacterium that is particularly common in humans and causes lung diseases, from patients with lung infections caused by Mycobacterium tuberculosis, or studies on the pathophysiology of the disease.

[0006] Accordingly, the inventors of the present invention have completed the present invention by discovering a metabolite that can serve as a biomarker that can rapidly differentiate and diagnose two diseases with similar clinical manifestation characteristics by comparing and analyzing the metabolites of serum samples at the time of diagnosis of a patient infected with Mycobacterium tuberculosis (Mtb) and at the time of diagnosis of a patient infected with Mycobacterium avium complex (MAC).

[0007] The present inventors have conducted extensive research to discover objective and reliable diagnostic markers that can rapidly differentiate between Mycobacterium avium complex (MAC) infection and Mycobacterium tuberculosis (Mtb) infection, two diseases with very similar clinical manifestations. As a result, we have discovered 17 polar and lipid metabolites that can be reflected as diagnostic markers with high reproducibility and reliability in differentiating between Mycobacterium avium complex infection and Mycobacterium tuberculosis infection.

[0008] Therefore, the purpose of the present invention is to provide a composition for differentiating between nontuberculous mycobacterial infections and tuberculosis infections.

[0009] Other objects and advantages of the present invention will become more apparent from the detailed description, claims and drawings below.

[0010] According to one aspect of the present invention, the present invention provides a composition for differentiating between nontuberculous mycobacterial infections and tuberculosis infections, comprising as an active ingredient a preparation for measuring one or more metabolites selected from the group consisting of lipid metabolites, amino acids and amino acid derivatives.

[0011] The lipid metabolite is at least one selected from the group consisting of palmitamide, stearamide, oleamide, linoleamide, behenamide, anandamide (arachidoyl ethanolamide), stearamine, behenic acid, lyso phosphatidylethanolamine (Lyso PE), sphinganine, sphingosine, and dimethylsphingosine.

[0012] The composition is characterized in that the amino acid and amino acid derivative are at least one selected from the group consisting of 2-hydroxyglutaric acid, allantoin, choline, and uric acid.

[0013] The present inventors have conducted extensive research to discover diagnostic markers for differentiating patients infected with nontuberculous mycobacteria (NTM), particularly Mycobacterium avium complex (MAC), which is a very common cause of lung disease in humans, and Mycobacterium tuberculosis (MTB), which has very similar clinical characteristics and thus makes the development of objective and reliable differential markers difficult. As a result, we have discovered 31 polar metabolites and 97 lipid metabolites as differential diagnostic markers that can reflect infectious diseases caused by nontuberculous mycobacteria or tuberculosis with high reproducibility and reliability.

[0014] In this specification, the term "nontuberculous mycobacteria" refers to mycobacteria other than tuberculosis bacilli, and specifically includes all mycobacteria that do not cause tuberculosis or leprosy. Unlike general bacteria, mycobacteria refer to strains that have the ability to withstand and not dissolve when acid is added during the staining process. The most representative of these mycobacteria is Mycobacterium tuberculosis, and acid-fast bacteria other than tuberculosis bacilli are called nontuberculous mycobacteria (NTM), and new nontuberculous mycobacteria are discovered almost every year.

[0015] As used herein, the term "tuberculosis bacilli" refers to acid-fast bacilli other than non-tuberculosis bacilli, specifically all mycobacteria that do not cause non-tuberculosis or leprosy. Unlike typical bacteria, acid-fast bacilli are strains that can withstand and remain insoluble even when acid is added during the staining process. A representative example of these acid-fast bacilli is Mycobacterium tuberculosis, which is characterized by high pathogenicity and a high relapse rate after treatment.

[0016] As used herein, the term "differentiation" may refer to the classification and subdivision of a specific disease or condition present in an individual. In the present invention, differentiation refers to the evaluation of a condition based on objective markers that can objectively distinguish and identify multiple diseases with similar clinical characteristics.

[0017] As used herein, the term “differential diagnostic composition” refers to an integrated mixture or device including a means for measuring the concentration of lipid metabolites, amino acids or amino acid derivatives to determine whether a subject is infected with nontuberculous mycobacteria or tuberculosis bacteria, and may be expressed as a “differential diagnostic kit.” Since the differential diagnostic composition of the present invention includes a means for measuring the metabolites discovered in the present invention, the term “differential diagnostic composition” may also be expressed as a “quantitative device” for the metabolites.

[0018] The term "metabolite" herein, also known as a metabolite or metabolite, refers to an intermediate or product of metabolism. These metabolites have diverse functions, including fuel, structure, signaling, enzyme stimulatory and inhibitory effects, their own catalytic activity (typically as a cofactor for enzymes), defense, and interactions with other organisms (e.g., pigments, aromatic compounds, pheromones). Primary metabolites are directly involved in normal growth, development, and reproduction. Secondary metabolites are not directly involved in these processes, but often have important ecological functions.

[0019] According to the present invention, the metabolite refers to a metabolite obtained from a sample of biological origin, i.e., a biological sample, and the biological sample refers to a biological fluid, tissue or cell.

[0020] According to the present invention, the metabolite may be a metabolite obtained from a liquid sample of blood, specifically serum.

[0021] According to a specific embodiment of the present invention, the lysophosphatidylethanolamine (Lyso PE) is at least one lysophosphatidylethanolamine selected from the group consisting of Lyso PE (18:0) and Lyso PE (20:4).

[0022] According to a specific embodiment of the present invention, the sphinganine is sphinganine (d18:0).

[0023] According to a specific embodiment of the present invention, the sphingosine is at least one sphingosine selected from the group consisting of sphingosine (d16:1) and sphingosine (d18:1).

[0024] According to a specific embodiment of the present invention, the dimethylsphingosine is dimethylsphingosine (d18:1).

[0025] According to a specific embodiment of the present invention, the 2-hydroxyglutaric acid, allantoin, choline and uric acid are in L-form.

[0026] According to a specific embodiment of the present invention, if the concentration of palmitamide, stearamide, oleamide, linoleamide or lysophosphatidylethanolamine increases compared to the group of patients with non-tuberculous mycobacterial infection, the patient is determined to have tuberculosis bacteria.

[0027] According to a specific embodiment of the present invention, if the concentration of behenamide, anandamide, stearamine, behenic acid, sphinganine, sphingosine or dimethylsphingosine decreases compared to a group of patients with non-tuberculous mycobacterial infection, the patient is determined to have tuberculosis bacteria.

[0028] According to a specific embodiment of the present invention, if the concentration of 2-hydroxyglutaric acid, allantoin, choline or uric acid increases compared to the group of patients with non-tuberculous mycobacterial infection, it is determined to be tuberculosis bacteria.

[0029] In the present invention, the nontuberculous mycobacterial infection disease or tuberculosis infection disease may be, but is not limited to, pulmonary disease, lymphadenitis, skin / soft tissue / bone infection, or disseminated disease.

[0030] In the present invention, the measured metabolite concentration can be used to diagnose whether the infection is caused by nontuberculous mycobacteria or tuberculosis bacteria. Specifically, the diagnosis can be made by distinguishing between Mycobacterium avium among nontuberculous mycobacteria and Mycobacterium tuberculosis among tuberculosis bacteria, but is not limited thereto.

[0031] The term “increased or high concentration” used when referring to the “differential diagnostic composition” in the composition of the present invention means a case where the concentration of a metabolite in the serum of a patient with tuberculosis infection is significantly higher than that of a patient with a nontuberculous mycobacterial infection, particularly a patient with a Mycobacterium avium complex infection, and specifically means a case where the concentration of the metabolite increases by about 5% or more, about 10% or more, or about 15% or more, and more specifically means a case where the concentration of the metabolite increases by about 20% or more, and does not exclude a range exceeding this.

[0032] The term “reduced or lower concentration” used when referring to the “differential diagnostic composition” in the composition of the present invention means a case where the concentration of a metabolite in the serum of a patient with tuberculosis infection is significantly lower than that of a patient with a non-tuberculous mycobacterial infection, particularly a patient with a Mycobacterium avium complex infection, and specifically means a case where the concentration of the metabolite is reduced by about 5% or more, about 10% or more, about 15% or more, about 20% or more, or about 25% or more compared to a patient with a non-tuberculous mycobacterial infection, and more specifically means a case where the concentration of the metabolite is reduced by about 30% or more, but does not exclude a range exceeding this.

[0033] According to a specific embodiment of the present invention, the lipid metabolites, amino acids and amino acid derivatives are selected from the group consisting of whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, synovial fluid. It is present in fluid, joint aspirate, organ secretions, cells, cell extracts, and cerebrospinal fluid, including, but not limited to, serum.

[0034] Specifically, whole blood, plasma, or serum may be preprocessed to detect the metabolites. This may include, for example, filtration, distillation, extraction, separation, concentration, inactivation of interfering components, and addition of reagents. Furthermore, the metabolites may include substances produced by metabolism and metabolic processes, or substances resulting from chemical metabolism by biological enzymes and molecules.

[0035] According to a specific embodiment of the present invention, the nontuberculous mycobacteria are Mycobacterium avium, Mycobacterium abscessus, Mycobacterium flavescence, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium chelonae, Mycobacterium celatum, Mycobacterium fortuitum, Mycobacterium gordonae, Mycobacterium gastri, Mycobacterium haemophilum, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium The bacterium may be selected from the group consisting of, but is not limited to, M. malmoense, M. marinum, M. szulgai, M. terrae, M. scrofulaceum, M. ulcerans, M. simiae, and M. xenopi.

[0036] According to a specific embodiment of the present invention, the tubercle bacilli may be selected from the group consisting of, but not limited to, Mycobacterium tuberculosis, Mycobacterium bovis, M. bovis BCG, Mycobacterium africanum, M. canetti, M. caprae, M. microti, and Mycobacterium tuberculosis K strain.

[0037] According to the present invention, the non-tuberculous mycobacterial infection disease includes all clinical symptoms exhibited by infection with the non-tuberculous mycobacteria, and the tuberculosis infection disease includes all clinical symptoms exhibited by infection with the tuberculosis bacilli, and the infectious diseases may include lung disease, lymphadenitis, skin / soft tissue / bone infection, or disseminated disease.

[0038]

[0039] According to another aspect of the present invention, the present invention provides a method for providing information for differentiating between a nontuberculous mycobacterial infection disease and a tuberculosis infection disease, comprising the step of measuring one or more metabolites selected from the group consisting of lipid metabolites, amino acids, and amino acid derivatives.

[0040] The lipid metabolite is at least one selected from the group consisting of palmitamide, stearamide, oleamide, linoleamide, behenamide, anandamide (arachidoyl ethanolamide), stearamine, behenic acid, lyso phosphatidylethanolamine (Lyso PE), sphinganine, sphingosine, and dimethylsphingosine.

[0041] The above amino acids and amino acid derivatives are provided as active ingredients comprising a preparation that measures one or more metabolites selected from the group consisting of 2-hydroxyglutaric acid, allantoin, choline and uric acid.

[0042] In the present invention, lipid metabolites, amino acids and amino acid derivatives, which are measurement targets for the differentiation of nontuberculous mycobacteria or tuberculosis bacteria, have already been described above, and therefore, their description is omitted to avoid excessive duplication.

[0043] According to a specific embodiment of the present invention, the step of measuring the concentration of the metabolite uses a quantitative device such as a chromatograph or mass spectrometer.

[0044] The chromatography used in the present invention is high performance liquid chromatography (HPLC), liquid-solid chromatography (LSC), paper chromatography (PC), thin-layer chromatography (TLC), gas-solid chromatography (GSC), liquid-liquid chromatography (LLC), foam chromatography (FC), emulsion chromatography (EC), gas-liquid chromatography (GLC), ion chromatography (IC), gel filtration chromatography (GFC) or gel permeation chromatography (Gel Any quantitative chromatography commonly used in the art may be used, including but not limited to, GPC (Gamma Permeation Chromatography).

[0045] In the present invention, the mass spectrometer may use a conventionally known mass spectrometer without any particular limitation, but specifically, for example, it may be a Fourier transform mass spectrometer (FTMS), a MALDI-TOF mass spectrometer (MALDI-TOF MS), a Q-TOF MS, or a LTQ-Orbitrap MS, but is not limited thereto.

[0046]

[0047] According to another aspect of the present invention, the present invention provides a device for differentiating between nontuberculous mycobacterial infections and tuberculosis infections, comprising a preparation for measuring one or more metabolites selected from the group consisting of lipid metabolites, amino acids, and amino acid derivatives.

[0048] The lipid metabolite is at least one selected from the group consisting of palmitamide, stearamide, oleamide, linoleamide, behenamide, anandamide (arachidoyl ethanolamide), stearamine, behenic acid, lyso phosphatidylethanolamine (Lyso PE), sphinganine, sphingosine, and dimethylsphingosine.

[0049] The device is characterized in that the amino acid and amino acid derivative are at least one selected from the group consisting of 2-hydroxyglutaric acid, allantoin, choline, and uric acid.

[0050] In the present invention, lipid metabolites, amino acids and amino acid derivatives, which are measurement targets for the differentiation of nontuberculous mycobacteria or tuberculosis bacteria, have already been described above, and therefore, their description is omitted to avoid excessive duplication.

[0051] The present invention can be used to distinguish between nontuberculous mycobacterial infections and tuberculosis infections with similar clinical manifestations with high accuracy at an early stage by using metabolites of serum samples, thereby establishing a patient-tailored treatment strategy at an early stage, thereby significantly improving the patient's survival rate.

[0052] FIG. 1 is a diagram showing 25 polar metabolites whose expression levels were increased in serum samples from tuberculosis patients compared to patients with Mycobacterium avium complex (MAC) infection lung disease according to one embodiment of the present invention. Specifically, 2-hydroxyglutaric acid, adenosine triphosphate, α-ketoisovalerate, alanine, allantoin, α-oxoglutarate, arginine, betaine, choline, cis-Aconitate, glutamine, glycerol 3-phosphate, isoleucine, kynurenine, lactate, leucine, ornithine, succinate, uric acid, phenylalanine, proline, This is a graph comparing the expression levels of R5-P / Ru5-P / X5-P, serine, tryptophan, or valine.

[0053] Figure 2 is a diagram showing six polar metabolites whose expression levels were reduced in serum samples from tuberculosis patients compared to patients with Mycobacterium avium complex (MAC)-infected lung disease according to one embodiment of the present invention. Specifically, this diagram is a graph comparing the expression levels of citrate, G6-P / F6-P / M6-P, gluconate, pyruvate, sedoheptulose 7-phosphate, or thiamine.

[0054] FIG. 3 is a diagram showing 22 lipid metabolites whose expression levels were increased in serum samples from tuberculosis patients compared to patients with Mycobacterium avium complex (MAC) infection lung disease according to one embodiment of the present invention. Specifically, this is a graph comparing the expression levels of CE(20:3), palmitamide, stearamide, oleamide, linoleamide, Lyso PC(14:0), Lyso PC(16:0), Lyso PC(16:1), lyso PC(17:0), Lyso PC(18:0), Lyso PC(18:1), Lyso PC(20:3), Lyso PC(20:4), Lyso PC(22:5), Lyso PC(22:6), Lyso PC(e16:0), Lyso PC(e18:0), Lyso PE(18:0), Lyso PE(20:4), PG 34:2, DG 36:5, or arachidonic acid.

[0055] FIGS. 4A to 4C are diagrams showing 75 lipid metabolites whose expression levels were reduced in serum samples from tuberculosis patients compared to patients with Mycobacterium avium complex (MAC)-infected lung disease according to one embodiment of the present invention. Specifically, 5-HETE behenic acid, behenamide, anandamide (arachidoyl ethanolamide), stearamine, Cer d40:1, Cer d40:2, Cer d41:1, Cer d42:2, Cer d42:3, SM d34:2, SM d38:2, SM d40:2, SM d41:2, SM d42:3, Sphinganine (18:0), Sphingosine (16:1), Sphingosine (18:1), Dimethylsphingosine (18:0), PS 36:1, PS 36:4, PS 38:4, PS 42:5, Cholesterol, TG 36:0, TG 48:2, TG 49:1, TG 50:4, TG 50:5, TG 52:6, TG 58:8, PC 28:0, PC 30:0, PC 32:0, PC 32:1, PC 32:2, PC 33:1, PC 34:0, PC 34:2, PC 34:3, PC 34:4, PC 36:1, PC 36:2, PC 36:3, PC 36:4, PC 36:5, PC 36:6, PC 38:2, PC 38:4, PC 38:5, PC 38:6, PC 38:7, PC 39:6, PC 40:5, PC 40:6, PC 40:7, PC e34:2, PC e36:3, PC This is a graph comparing the expression levels of e36:5, PC e38:4, PC e38:6, PC e40:6, PE 36:1, PE 36:2, PE 38:6, PE 40:6, PE 40:7, PE 40:8, PE 42:9, PE e36:2, PE e36:4, PE e38:4, PE e38:5, PE e38:6 or PE e40:6.

[0056] Figure 5 is a diagram showing the receiver operating characteristic (ROC) curves of four polar metabolites with an area under the curve (AUC) value (>0.8) for differential diagnosis of tuberculosis patients compared to patients with Mycobacterium avium complex (MAC) infection pulmonary disease, according to one embodiment of the present invention. Specifically, the graph shows the results obtained by combining all four metabolites: 2-hydroxyglutaric acid, allantoin, choline, and uric acid. This is an important combination of polar metabolites that can serve as biomarkers for differential diagnosis.

[0057] FIG. 6 is a diagram showing ROC curves of 13 lipid metabolites with AUC values ​​(0.8 or higher) for differential diagnosis of tuberculosis patients compared to patients with Mycobacterium avium complex (MAC) infection pulmonary disease according to one embodiment of the present invention. Specifically, the results of combining all 13 types of lipid metabolites, including palmitamide, stearamide, oleamide, linoleamide, behenamide, anandamide (arachidoyl ethanolamide), stearamine, behenic acid, lysophosphatidylethanolamine (Lyso PE(20:4)), sphinganine (d18:0), sphingosine (d16:1), sphingosine (d18:1), and dimethylsphingosine (d18:1), are presented in a graph. This is a combination of important lipid metabolites that can serve as biomarkers for differential diagnosis.

[0058] FIG. 7 is a diagram showing ROC curves of 17 polar metabolites and lipid metabolites with AUC values ​​(0.8 or higher) for differential diagnosis of tuberculosis patients compared to patients with Mycobacterium avium complex (MAC) infection lung disease according to one embodiment of the present invention. Specifically, four polar metabolites of 2-hydroxyglutaric acid, allantoin, choline and uric acid, and lipids of palmitamide, stearamide, oleamide, linoleamide, behenamide, anandamide (arachidoyl ethanolamide), stearamine, behenic acid, lysophosphatidylethanolamine (Lyso PE(20:4)), sphinganine(d18:0), sphingosine(d16:1), sphingosine(d18:1) and dimethylsphingosine(d18:1) This graph combines the results of 17 polar metabolites, including 13 metabolites, and all lipid metabolites. This is the most important combination of polar and lipid metabolites that can serve as biomarkers for differential diagnosis.

[0059] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0060]

[0061] Preparation Example 1. Collection of samples from patients with pulmonary disease infected with Mycobacterium tuberculosis and patients with pulmonary disease infected with MAC.

[0062] The present invention collected 100 serum samples at the time of diagnosis from 100 patients with lung disease infected with Mycobacterium tuberculosis (Mtb) at Seoul National University Hospital over a period of approximately 3 years from October 2010 to June 2013, and 81 serum samples at the time of diagnosis from 81 patients with lung disease infected with Mycobacterium avium complex (MAC) at Seoul Samsung Hospital over a period of approximately 6 years from January 2017 to May 2022.

[0063]

[0064] Preparation Example 2. Pretreatment of the sample

[0065] 2.1 Sample pretreatment for polar metabolite analysis

[0066] For the analysis of polar metabolites in serum, the serum sample obtained in Preparation Example 1 was pretreated. Specifically, 290 μl of 70% ACN (Acetonitrile-H2O, 70:30, v / v) was added to the serum sample (10 μl) for extraction, and at this time, an internal standard (13C-labeled yeast extract; ISO+13C-taurine 1000 ppb, 13C-allantoin 1000 ppb, 13C-hypoxanthine 100 ppb, 13C-2-chloro adenosine 1000 ppb, 13C-Lactate 100 ppb, 13C-Glucose 1000 ppb, 13C-Uric acid 1000 ppb) diluted 1:30 was added to the solvent for quality control of the polar metabolite analysis. Here, the internal standards are shown in Tables 4 and 6 below. After mixing for 30 seconds, extraction was performed on ice for 10 minutes. After centrifugation at 13,000 rpm and 4°C for 10 minutes using a centrifuge, the supernatant, excluding the precipitate, was filtered using a filter tube (Costar 8169) to remove possible impurities, and then polar metabolite analysis was performed using an HPLC-QQQ-MS instrument.

[0067] 2.2 Sample preparation for lipid metabolite analysis

[0068] For analysis of lipid metabolites in serum, the serum sample obtained in Preparation Example 1 was pretreated. Specifically, 300 μl of CHCl3 and 150 μl of MeOH (Chloroform-Methanol, 2:1, v / v, 4°C) were added to the serum sample (40 μl) and mixed for 30 seconds. Additionally, 150 μl of water was added, mixed for 30 seconds, and extracted on ice for 10 minutes. After centrifugation at 13,000 rpm and 4°C for 10 minutes, the lower layer (250 μl) was separated and dried using a liquid nitrogen concentrator (low temperature -195°C, 30 minutes). The supernatant was re-dissolved in 250 μl of IPA / ACN / H2O (Isopropanol-Acetonitrile-Water, 2:1:1, v / v). At this time, the SPLASH_Lipido MIX internal standard was added in a 1:50 dilution to the re-dissolution solvent of the supernatant to control the quality of lipid metabolite analysis. After filtration using a filter tube (Costar 8169) to remove possible impurities, lipid metabolite analysis was performed using a UPLC-Q-Exactive Orbitrap / MS instrument.

[0069] In order to control the quality of the sample and device during the preprocessing of the above serum sample, 20 μl of the entire sample was collected to create a QC, and repeated measurements were made by inserting the sample between batches to measure the difference in the analysis quality of the sample and device within each batch.

[0070]

[0071] Example 1. Metabolite analysis using HPLC-Triple Quad (QQQ)-MS

[0072] To analyze polar metabolites in the processed serum samples, liquid chromatography-tandem mass spectrometry (HPLC-QQQ-MS) was used. The equipment used was an Agilent 1200 HPLC and a SCIEX API4000 triple quadrupole MS. The positive mode analysis conditions for polar metabolites using HPLC were a gradient elution with a solvent at 40 °C using an XBridge BEH Amide (4.6 x 250 mm, 3.5 μm, Waters) column. The mobile phases used were (A) 85% Water + 15% Acetonitrile (30 mM ammonium acetate + 0.2% acetic acid) and (B) 85% Acetonitrile (0.2% acetic acid). The negative mode analysis conditions for polar metabolites were as follows: a Luna PFPP (2.0 x 150 mm, 3 μm, Phenomenex) column was used, and gradient elution was performed at 40 ℃ according to the solvent, and (A) Water (0.1% formic acid) and (B) 100% Acetonitrile were used as the mobile phases. The gradient elution for each condition was performed as shown in Tables 1 and 2 below, and the total analysis time was 17 minutes and 15 minutes, respectively. The mass analysis conditions using MS / MS were as follows: the nebulizer gas (Ion-Source Gas 1 / 2) unit was 50 / 50 arbitrary unit, and the curtain gas unit was set to 25 arbitrary unit. The source temperature was 500 ℃, the ion-spray floating voltage was 5.5 kV (negative -4.5 kV), and the mass range was 50-1000 m / z.Sample injection was performed at 3 μl per injection using the HTC_PAL system / CTC analytics auto-sampler, and the tandem mass spectrometry conditions (scheduled multiple reaction monitoring, sMRM) were performed as shown in Tables 3 to 6 below. The results obtained through sMRM analysis were converted to raw data using SCIEX's Quantitative Analysis Software, and polar metabolites with a relative standard deviation (RSD<20) or less were calculated using the average value of QC data.

[0073] Time (min)Mobile phase A (%)Mobile phase B (%)Flow rate (mL / min)010901.20210901.20550501.20950501.201110901.201710901.20

[0074] Time (min)Mobile phase A (%)Mobile phase B (%)Flow rate (mL / min)010000.35873270.35915850.351010000.351510000.35

[0075] - 99 types of polar metabolites in positive mode sMRM analysis conditions Metabolite type (Compounds) Q1 Q3 RTCE Internal standard 3-Methyl-2-oxovalerate oxovalerate (3-Methyl-2-oxovalerate) 131907713C_Leucine (13C_Leucine)5'-Deoxy-5'-Methylthioadenosine2981364.22513C_2-chloroadenosine5-Methyluridine259723.634313C_2-chloroadenosine6-phosphogluconate2779763761113C_Glutamatea-Aminoadipate162987.52113C_LysineAcetyl CoA CoA)8101843.34513C_Acetyl CoA (13C_Acetyl CoA)Adenine (Adenine)1361195.252413C_Adenine (13C_Adenine)Adenosine (Adenosine)2681365.32713C_2-chloroadenosine (13C_2-chloroadenosine)Adenosine monophosphate (Adenosine monophosphate)3481367.352113C_2-chloroadenosine (13C_2-chloroadenosine)Adenosine triphosphate (Adenosine) triphosphate)508.31843.713313C_2-chloroadenosine(13C_2-chloroadenosine)Agmatine(Agmatine)131.2727.952313C_Arginine(13C_Arginine)a-Ketoisovalerate(a-Ketoisovalerate)117716.692113C_Glutamate(13C_Glutamate)Alanine(Alanine)90447.031713C_Alanine(13C_Alanine)Arginine(Arginine)175707.83513C_Arginine (13C_Arginine)Argininosuccinate (Argininosuccinate)291708.296113C_Argininosuccinate (13C_Argininosuccinate)Asparagine (Asparagine)133747.41613C_Asparagine (13C_Asparagine)Aspartate (Aspartate)134887.681313C_Aspartate (13C_Aspartate)Betaine (Betaine)118566.573413C_Valine (13C_Valine)Biotin (Biotin)2452274.351813C_Glutamate (13C_Glutamate) Cadaverine 10386.17.85 1313C_Arginine 13C_Carnitine 162.210 37.072 313C_Acetyl CoA 13C_Choline 104606.0520 13C_Choline 13C_Cyclic adenosine monophosphate 330.2134.27.72 1313C_2-chloroadenosine 13C_2-chloroadenosine 13C_Cyclic guanosine monophosphate monophosphate)3461526.912013C_2-chloroadenosineCystathionine2231348.21713C_CystathionineCysteine122596.562713C_CysteineCytidine2441126.181213C_2-chloroadenosineCytidine monophosphate3241127.752013C_2-chloroadenosineCytidine triphosphate triphosphate)482.54653.211913C_2-chloroadenosine (13C_2-chloroadenosine)Cytosine (Cytosine)112955.81713C_2-chloroadenosine (13C_2-chloroadenosine)Deoxyadenosine (Deoxyadenosine)25213652013C_2-chloroadenosine (13C_2-chloroadenosine)Deoxyadenosine monophosphate (Deoxyadenosine monophosphate)332.43157.52913C_2-chloroadenosine (13C_2-chloroadenosine)Deoxycytidine monophosphate (Deoxycytidine monophosphate)3081127.531613C_2-chloroadenosine (13C_2-chloroadenosine)Deoxyguanosine Deoxyguanosine monophosphate 348 151.87.617 13C_2-chloroadenosine Deoxyinosine 253 1375.512 13C_2-chloroadenosine Deoxyuridine 229 1135.8111 13C_2-chloroadenosine Diacetylspermine 287.41007.4327 13C_Glutamate Dihydroxyisovalerate 134 747.7419 13C_Glutamate (13C_Glutamate)Folate4422957.21913C_Glutamate (13C_Glutamate)Glucosamine1801627.71013C_Glutamate (13C_Glutamate)Glutamate148847.62313C_Glutamate (13C_Glutamate)Glutamine1471307.341413C_Glutamate (13C_Glutamate)Glutathione(Glutathione, oxidized)6133558.43313C_Glutathione (13C_Glutathione, oxidized)Glutathione (Glutathione, reduced)308767.83913C_Glutathione (13C_Glutathione, oxidized)Glycine76307.232013C_GlycineGuanine1521107.782713C_2-chloroadenosineGuanosine2841526.331713C_2-chloroadenosineGuanosine diphosphate diphosphate)4441528.662013C_2-chloroadenosine(13C_2-chloroadenosine)Guanosine monophosphate(Guanosine monophosphate)3641527.782113C_2-chloroadenosine(13C_2-chloroadenosine)Guanosine triphosphate(Guanosine triphosphate)523.3393.27313C_2-chloroadenosine(13C_2-chloroadenosine)Histidine(Histidine)1561107.741213C_Histidine(13C_Histidine)Histidinol(Histidinol)142957.21813C_Histidine(13C_Histidine)Homocysteine(Homocysteine)136906.451513C_Methionine(13C_Methionine)Homoserine(Homoserine)120567.192713C_Homoserine(13C_Homoserine)Inosine(Inosine)2691375.91413C_2-chloroadenosine(13C_2-chloroadenosine)Inosine Inosine monophosphate 3491377.51713C_2-chloroadenosine 13C_2-chloroadenosine Isoleucine 132866.271413C_Isoleucine Kynurenine 20919261313C_Phenylalanine Leucine 132866.111513C_Leucine (13C_Leucine)Lysine (Lysine)147848.012313C_Lysine (13C_Lysine)Methionine (Methionine)1501046.41113C_Methionine (13C_Methionine)Mevalonate (Mevalonate)14984.87.591913C_Acetyl CoA (13C_Acetyl CoA) N,N-dimethylglycine (N,N-dimehtylglycin) 104586.716 13C_Valine (13C_Valine) N-Acetylornithine (N-Acetylornithine) 175115814 13C_Ornithine (13C_Ornithine) N-Acetylspermidine (N-Acetylspermidine) 1881717.72 113C_Arginine (13C_Arginine) N-Acetylspermidine (N- Acetylspermine)245.31006.612713C_Arginine(13C_Arginine)NAD+6641367.84013C_NAD+NADH6661367.854013C_NADHNADP+744.65753.222913C_NADP+NADPH746.51843.284513C_NADPHNicotinamide)12380.14.222913C_Nicotinamide 13C_Nicotinamide 13C_Ornithine 133708.0425 13C_Ornithine 13C_Phenylalanine 1661206.0117 13C_Phenylalanine 13C_Proline 116706.720 13C_Proline 13C_Pseurouridine 245.314 7.181113C_2-chloroadenosinePutrescine8971.881513C_ArginineRiboflavin3772435.493513C_2-chloroadenosineS-adenosylhomocystine385.31367.42513C_Methionine (13C_Methionine) S-adenosylmethionine (S-adenosylmethionine) 399.32508.22113C_Methionine (13C_Methionine) Sarcosine (Sarcosine) 90727.131613C_Sarcosine (13C_Sarcosine) Serine (Serine) 106607.411513C_Serine (13C_Serine) Spermidine )145.922104.37.081513C_Arginine (13C_Arginine)Spermine (Spermine)203.24112.0568.52513C_Arginine (13C_Arginine)Taurine (Taurine)1261086.622713C_Taurine (13C_Taurine)Thiamine (Thiamine)2651226.581713C_2-chloroadenosine (13C_2-chloroadenosine) Threonine 120747.214 13C Threonine Thymidine 243126.84.413 13C 2-chloroadenosine Thymine 1271104.42 113C 2-chloroadenosine Tryptophan 2051885.9131 3C_Phenylalanine (13C_Phenylalanine)Tyrosine (Tyrosine)1821366.561213C_Phenylalanine (13C_Phenylalanine)Uracil (Uracil)113707.422313C_2-chloroadenosine (13C_2-chloroadenosine)Uridine (Uridine)2451135.41313C_2-chloroadenosine (13C_2-chloroadenosine)Uridine Uridine diphosphate 405174.97.992913C_2-chloroadenosine 13C_2-chloroadenosine Uridine monophosphate 325977.472013C_2-chloroadenosine Valine 118726.571513C_Valine (13C_Valine) Xanthine (Xanthine) 1531105.523 13C_2-chloroadenosine (13C_2-chloroadenosine) Xanthosine (Xanthosine) 2851536.220 13C_2-chloroadenosine (13C_2-chloroadenosine) Malonyl CoA (Malonyl CoA) 8543477.545 13C_Acetyl CoA (13C_Acetyl CoA) Crotonoyl CoA (Crotonoyl CoA) 8363296.843 13C_Acetyl CoA (13C_Acetyl CoA).

[0076] - Positive mode polar metabolite internal standard sMRM analysis conditions Internal standard type Q1Q3RTCE13C_2-chloroadenosine3021704.292513C_acetyl CoA833.6151893.34913C_Adenine141.132123.95.32913C_Alanine9345.87.031713C_Arginine181747.93613C_Argininosuccinate301748.295513C_Asparagine137767.41213C_Aspartate138767.681313C_Choline10962.962513C_Cystathione2301388.191913C_Cysteine125616.563213C_Glutamate153887.561213C_Glutamine1521357.292313C_Glutathione, oxidized6332398.574513C_Glycine78317.22013C_Histidine1621157.841213C_Homoserine12477.17.162713C_Isoleucine138916.211513C_Leucine13891.16.041513C_Lysine1538982313C_Methionine1551086.41113C_NAD+685.21417.86913C_NADH687.21417.844013C_NADP+765.624189.13.354913C_NADPH 767.623189.23.34713C_Nicotinamide129.02854.22913C_Ornithine138748.042313C_Phenylalanine1751285.941713C_Proline121746.641613C_Sarcosine93757.131013C_Serine109627.371513C_Taurine12845.96.622713C_Threonine124597.151613C_Tryptophan2161995.91313C_Tyrosine1911446.481213C_Valine123766.5615

[0077] - sMRM analysis conditions for 47 polar metabolites in negative mode Metabolite type (Compounds) Q1Q3RTCE Internal standard 2-hydroxyglutaric acid 147 129 2.15-14 13C_2-hydroxyglutaric acid 2-PG / 3-PG 185 97 1.8-22 13C_2-PG / 3-PG 3-hydroxybutyric acid acid)103413.8-3213C_Citrate (13C_Citrate) Acetoacetate (Acetoacetate)101576.6-2113C_Citrate (13C_Citrate) Acetylphosphate (Acetylphosphate)139791.6-2213C_Citrate (13C_Citrate) Adenosine diphosphate (Adenosine diphosphate)426792.8-8813C_Hypoxanthine(13C_Hypoxanthine)Allantoate(Allantoate)1751321.21-1213C_Allantoin(13C_Allantoin)Allantoin(Allantoin)1571141.4-2013C_Allantoin(13C_Allantoin)a-Oxoglutarate(a-Oxoglutarate)1451011.2-1413C_a-Oxoglutarate(13C_a-Oxoglutarate)Carbamoyl phosphate(Carbamoyl phosphate)140791.23-3013C_Citrulline(13C_Citrulline)cis-Aconitate(cis-Aconitate)173855.5-1813C_Citrate(13C_Citrate)Citrate(Citrate)1911112.84-19.113C_Citrate(13C_Citrate)Citrulline(Citrulline)1741311.34-1813C_Citrulline(13C_Citrulline)Deoxyribose 1-phosphate213791.6-3313C_G6-P / F6-P / M6-PDihydroxyacetone phosphate(Dihydroxyacetone phosphate)169971.51-513C_Dihydroxyacetone phosphate (13C_Dihydroxyacetone phosphate) Erytrhose 4-phosphate 4-phosphate)199971.43-1413C_G6-P / F6-P / M6-P Fructose-1,6-bisphosphate)3392411.9-1213C_G6-P / F6-P / M6-P Fumarate)115711.9-1213C_Fumarate)G6-P / F6-P / M6-P 259971.4-2213C_G6-P / F6-P / M6-PGluconate)19578.61.3-3613C_Glucose)Glucose, fructose, galactose, mannose mannose)179891.21-1213C_Glucose)Glutamylcysteine)24996.41.33-1413C_a-Oxoglutarate)Glycerate)105751.21-1513C_Lactate)Glycerol 3-phosphate)171791.68-2213C_Sedoheptulose 7-phosphate)Guanosine diphosphate)4423441.4-1613C_Hypoxanthine)Hydroxyphenyl Hydroxyphenylpyruvate 1791071.2-1113C_Lactate 13C_Hypoxanthine 135923.5-2213C_Hypoxanthine Inosine triphosphate 5074091.6-1913C_Hypoxanthine Isocitrate 191731.9-29.413C_Citrate Itaconate 129856.3-1413C_Citrate (13C_Citrate) Lactate (Lactate) 89431.9-1813C_Lactate (13C_Lactate) Malate (Malate) 1331152-1613C_Malate (13C_Malate) Mannitol (Mannitol) 181711.2-2013C_Glucose (13C_Glucose) Myo-inositol (my o-Inositol)1791611.22-1513C_Glucose (13C_Glucose)Oxaloacetate (Oxaloacetate)131872.6-12.813C_Malate (13C_Malate)Phosphoenolpyruvate (Phosphoenolpyruvate)167792.1-1613C_Malate (13C_Malate)Phosphoribosyl Phosphoribosyl pyrophosphate 3892911.43-18 13C_Sedoheptulose 7-phosphate p-Hydroxybenzoate 137939.27-21 13C_Sedoheptulose 7-phosphate Pyruvate 87432.16-12 13C_Lactate R5-P / Ru5-P / X5-P 229791.4-48 13C_Sedoheptulose 7-phosphate Sedoheptulose 7-phosphate 7-phosphate)289971.4-2213C_Sedoheptulose 7-phosphate)Shikimate)173931.94-1613C_Sedoheptulose 7-phosphate)Succinate)117733.8-1813C_Succinate)Trehalose)341942.1-3013C_Glucose)Uric acid)1671242.8-2213C_Uric acid (13C_Uric acid) Uridine triphosphate (Uridine triphosphate) 483791.46-10013C_Hypoxanthine (13C_Hypoxanthine) Malonic acid (Malonic acid) 103591.88-1413C_Lactate (13C_Lactate).

[0078] - 2PG, 3PG; 2-phosphoglycerate, 3-phosphoglycerate

[0079] - G6-P, F6-P, M6-P; Glucose 6-phosphate, Fructose 6-phosphate, Mannose 6-phosphate

[0080] - R5-P, Ru5-P, X5-P; Ribose 5-phosphate, Ribulose 5-phosphate, Xylulose 5-phosphate

[0081]

[0082] - Negative mode polar metabolite internal standard sMRM analysis conditions Internal standard type Q1Q3RTCE 13C_2-Hydroxyglutarate 151.889.22.15-20 13C_2-PG / 3-PG 187.778.81.4-34 13C_Allantoin 159 116 1.4-20 13C_a-Oxoglutarate 150 132 1.2-16 13C_Citrate 196.8115.72.9-18 13C_Citrulline 180 136 1.2-18 13C_Dihydroxyacetone 13C_Dihydroxyacetone phosphate 171.878.81.51-40 13C_Fumarate 119741.9-9 13C_G6-P / F6-P / M6-P 265971.4-24 13C_Glucose 180901.2-12 13C_Hypoxanthine 140963.5-22 13C_Lactate 92451.9-18 13C_Malate 1371192-16 13C_R5-P / Ru5-P / X5-P 234793-56 13C_Sedoheptulose 7-phosphate 7-phosphate)29696.61.4-3813C_Succinate(13C_Succinate)121763.8-1813C_Trehalose(13C_Trehalose)353.196.62.1-2813C_Uric acid(13C_Uric acid)1711262.8-22

[0083] - Q1 refers to the precursor ion with a mass to charge ratio (m / z) value for the molecular ion.

[0084] - Q3 represents a product ion with the m / z value of a fragment ion for the molecular ion.

[0085] - RT stands for Retention time.

[0086] - CE stands for Collision energy.

[0087]

[0088] Example 2. Lipid metabolite analysis using UPLC-MS (Q-Exactive Ortbitrap Plus)

[0089] To analyze lipid metabolites in the analytical samples isolated from serum, chromatography-tandem mass spectrometry (UHPLC-MS) was used. The equipment used was Thermo Scientific's Ultimate 3000RS pump UHPLC and Q-Exactive Orbitrap Plus MS. The chromatographic conditions for hydrophilic interactions were an Acquity UPLC BEH C18 (2.1 x 100 mm, 1.7 μm, Waters) column and gradient elution at 35 °C to separate lipid metabolites. The mobile phases used were (A) 10 mM ammonium formate in 50% ACN + 0.1% formic acid (v / v) and (B) 2 mM ammonium formate in ACN / IPA / Water 10:88:2 + 0.02% formic acid (v / v). The gradient elution of the following mobile phase was performed in the same manner as in Table 7 below, with a total analysis time of 28 minutes. Electrospray ionization (ESI) was performed with two ionization modes: positive and negative. The full scan mass range was 250-1200 m / z, with a resolution of 70,000. The automatic gain control (AGC) target was 1x106 ions, and the maximum injection time (IT) was 100 ms. The collision energy (CE) was 30, the source ionization spray voltage was 3.0 kV, and the capillary temperature was 370 ℃. The results obtained through the analysis were analyzed using Thermo Scientific's analysis software (Compound Discoverer 3.2) Raw data was produced through alignment and normalization, and lipid metabolites with a relative standard deviation (RSD<20) or less were produced after statistical processing.

[0090] Time (min)Mobile phase A (%)Mobile phase B (%)Flow rate (mL / min)065350.30440600.301215850.302101000.302401000.302865350.30

[0091]

[0092] Example 3. Results of metabolite analysis in serum samples from patients infected with Mycobacterium tuberculosis and MAC infection.

[0093] In order to differentially diagnose two diseases with similar clinical manifestations, the identification and concentration of polar and lipid metabolites in serum samples from patients with MTB and MAC infection were compared using the following statistical test methods. For the statistical test methods, Metaboanalyst, metilin, lipidmaps and hdmb (statistics site, Database) and Quantitative Analysis Software of SCIEX, Compound Discoverer of Thermofisher, SPSS (statistics program) were used to derive polar and lipid metabolites with high significance (p<0.05, FC≤-0.12, 0.12≤FC) between the two groups. Based on the results, a total of 128 metabolites, including 31 polar metabolites and 97 lipid metabolites that could be indicators for differential diagnosis of the two diseases, were selected based on their respective p-values ​​and Fold change values, and the results are shown in Tables 8 and 9 and Figures 1 to 4 below. Among them, the four polar metabolites and 13 lipid metabolites with high AUC values ​​(AUC>0.8 or higher) were each represented by ROC curves, and a total of 17 metabolites including all four polar metabolites and 13 lipid metabolites were represented by ROC curves. The results are shown in Figures 5 to 7. At this time, significance means *P<0.05; **P<0.01; ***P<0.001 in unpaired Welch's t-test.

[0094]

[0095] 25 Polar Metabolites Increased in Serum of Tuberculosis Patients Compared to MAC Lung Disease Patients Metabolite Type (Compounds) Significance (p-value) Log2FC (Mtb / MAC) AUC 2-hydroxyglutaric acid < 0.0001 2.15 0.966 Adenosine triphosphate < 0.0001 0.49 0.786 a-Ketoisovalerate 0.009 0.20 0.610 Alanine < 0.0001 0.23 0.680 Allantoin < 0.0001 1.26 0.876 a-Oxoglutarate 0.01 20. 230.631 Arginine < 0.0001 0.24 0.715 Betaine 0.0001 0.25 0.674 Choline < 0.0001 0.99 0.885 cis-Aconitate < 0.0001 0.43 0.772 Glutamine 0.001 2 0.21 0.656 Glycerol Glycerol 3-phosphate <0.00010.370.696 Isoleucine <0.00010.380.740 Kynurenine 0.00010.350.644 Lactate <0.00010.300.655 Leucine <0.00010.280.712 Ornithine <0.00010.290.716 Succinate 0.00010.320.329 Uric acid Acid)<0.00010.840.844Phenylalanine0.0190.130.597Proline0.0220.150.606R5-P / Ru5-P / X5-P0.040.130.571Serine0.0040.120.634Tryptophan0.0050.170.621Valine0.0010.160.646MAC Six polar metabolites decreased in the serum of tuberculosis patients compared to patients with lung disease Citrate0.0006-0.160.644G6-P / F6-P / M6-P<0.0001-0.540.763Gluconate0.011-0.200.615Pyruvate0.014-0.490.556Sedoheptulose 7-phosphate0.0008-0.820.598Thiamine0.003-0.410.654.

[0096] 22 lipid metabolites increased in serum of tuberculosis patients compared to MAC lung disease patients Metabolite type (Compounds) Significance (p-value) Log2FC (Mtb / MAC) AUCCE (20:3) 0.025 0.33 0.59 4 Palmitamide < 0.000 12.89 0.99 9 Stearamide 0.024 1.67 0.90 5 Oleamide < 0.000 14.86 1.00 13.75 0.99 9 Lyso PC (14:0) 0.000 7 0.30 0.68 9 Lyso PC (16:0) < 0.000 10.35 0.75 Lyso PC(16:1)<0.00010.370.745Lyso PC(17:0)<0.00010.390.715Lyso PC(18:0)<0.00010.390.693Lyso PC(18:1)<0.00010.370.691Lyso PC(20:3)0.00010.330.675Lyso PC(20:4)0.00020.290.66Lyso PC(22:5)0.0010.330.668Lyso PC(22:6)0.0280.200.600Lyso PC(e16:0)<0.00010.440.765Lyso PC(e18:0)<0.00010.470.727Lyso PE(18:0)0.00030.250.666Lyso PE(20:4)<0.00017.010.999PG 34:2<0.00010.380.702DG 36:50.00050.440.659Arachidonic acid0.0250.230.632MAC 75 lipid metabolites decreased in serum of tuberculosis patients compared to patients with lung disease5-HETE0.04-0.630.543Behenic acid<0.0001-0.820.968Behenamide<0.0001-0.740.960Anandamide Ethanolamide)<0.0001-0.770.988Stearamine<0.0001-0.760.966Cer d40:10.034-0.170.612Cer d40:2<0.0001-0.410.720Cer d41:10.0027-0.270.615Cer d42:2<0.0001-0.550.701Cer d42:30.0006-0.310.732SM d34:20.001-0.200.656SM d38:20.008-0.210.605SM d40:20.003-0.170.625SM d41:2<0.0001-0.420.696SM d42:30.008-0.140.613 Sphinganine (d18:0) < 0.0001-0.740.941 Sphingosine (d16:1) < 0.0001-0.770.923 Sphingosine (d18:1) < 0.0001-0.740.977 Dimethylsphingosine (d18:1) < 0.0001-0.690.977 PS 36:10.014-0.140.591 PS 36:40.045-0.820.586 PS 38:40.002-0.210.651 PS 42:5<0.0001-0.340.691 Cholesterol 0.018-0.150.626 TG 36:0<0.0001-0.850.780 TG 48:20.017-0.380.583 TG 49:10.036-0.430.526 TG 50:40.044-0.350.555 TG 50:50.044-0.290.557 TG 52:60.028-0.350.588 TG 58:80.044-0.200.621 PC 28:00.038-0.510.600 PC 30:00.0001-0.530.683 PC 32:00.002-0.170.657PC 32:10.0046-0.350.615PC 32:2<0.0001-0.650.745PC 33:10.037-0.200.605PC 34:00.0003-0.220.645PC 34:2<0.0001-0.170.733PC 34:3<0.0001-0.480.714PC 34:4<0.0001-0.760.766PC 36:1<0.0001-0.380.671PC 36:20.0007-0.130.651PC 36:30.0089-0.200.614PC 36:4<0.0001-0.260.729PC 36:50.0015-0.430.669PC 36:6<0.0001-0.720.731PC 38:20.009-0.180.593PC 38:40.0007-0.200.661PC 38:50.0010-0.420.667PC 38:6<0.0001-0.310.715PC 38:7<0.0001-0.780.750PC 39:60.009-0.270.634PC 40:50.010-0.290.607PC 40:60.0009-0.270.653PC 40:7<0.0001-0.330.684PC e34:20.021-0.260.588PC e36:30.0024-0.240.640PC e36:50.033-0.150.601PC e38:4<0.0001-0.370.688PC e38:60.0006-0.250.658PC e40:60.033-0.150.615PE 36:10.049-0.260.558PE 36:20.036-0.130.605PE 38:60.034-0.270.612PE 40:6<0.0001-0.530.697PE 40:70.045-0.330.608PE 40:8<0.0001-0.400.698PE 42:90.016-0.420.588PE e36:20.0019-0.360.639PE e36:4<0.0001-0.440.685PE e38:4<0.0001-0.670.744PE e38:5<0.0001-0.560.720PE e38:6<0.0001-0.430.679PE e40:6<0.0001-0.650.739.

[0097] - CE; cholesterol ester

[0098] - Lyso PC; lyso-phosphatidylcholine

[0099] - PC; phosphatidylcholine

[0100] - ePC; ether-phosphatidylcholine

[0101] - Lyso PE; lyso-phosphatidylethanolamine

[0102] - PE; phosphatidylethanolamine

[0103] -ePE; ehter-phosphatidylethanolamine

[0104] - PS; phosphatidylserine

[0105] - PG; phosphatidylglycerol

[0106] - SM; sphingomyelin

[0107] - Cer; Ceramide

[0108] - DG; dicylglyceride

[0109] - TG; triacylglyceride

[0110]

[0111] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred implementation examples and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A composition for differentiating between nontuberculous mycobacterial infections and tuberculosis infections, comprising as an active ingredient a preparation for measuring one or more metabolites selected from the group consisting of lipid metabolites, amino acids and amino acid derivatives. The above lipid metabolite is at least one selected from the group consisting of palmitamide, stearamide, oleamide, linoleamide, behenamide, anandamide (arachidoyl ethanolamide), stearamine, behenic acid, lyso phosphatidylethanolamine (Lyso PE), sphinganine, sphingosine, and dimethylsphingosine. A composition characterized in that the amino acid and amino acid derivative are at least one selected from the group consisting of 2-hydroxyglutaric acid, allantoin, choline, and uric acid.

2. In paragraph 1, A composition characterized in that the above lysophosphatidylethanolamine (Lyso PE) is at least one lysophosphatidylethanolamine selected from the group consisting of Lyso PE (18:0) and Lyso PE (20:4).

3. In paragraph 1, A composition characterized in that the above sphinganine is sphinganine (d18:0).

4. In paragraph 1, A composition characterized in that the above sphingosine is at least one sphingosine selected from the group consisting of sphingosine (d16:1) and sphingosine (d18:1).

5. In paragraph 1, A composition characterized in that the above dimethylsphingosine is dimethylsphingosine (d18:1).

6. In paragraph 1, A composition characterized in that when the concentration of the above palmitamide, stearamide, oleamide, linoleamide or lysophosphatidylethanolamine increases compared to a group of patients with nontuberculous mycobacterial infection, the patient is diagnosed as having tuberculosis.

7. In paragraph 1, A composition characterized in that when the concentration of the above behenamide, anandamide, stearamine, behenic acid, sphinganine, sphingosine or dimethylsphingosine decreases compared to a group of patients with nontuberculous mycobacterial infection, the patient is diagnosed as having tuberculosis.

8. In paragraph 1, A composition characterized in that when the concentration of the above 2-hydroxyglutaric acid, allantoin, choline or uric acid increases compared to the group of patients with nontuberculous mycobacterial infection, the patient is diagnosed as having tuberculosis.

9. In paragraph 1, The above metabolites were obtained from whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, trachea. A composition characterized by its presence in organ secretions, cells, cell extracts and cerebrospinal fluid.

10. In paragraph 1, The above nontuberculous mycobacteria are Mycobacterium avium, Mycobacterium abscessus, Mycobacterium flavescence, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium chelonae, Mycobacterium celatum, Mycobacterium fortuitum, Mycobacterium gordonae, Mycobacterium gastri, Mycobacterium haemophilum, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium malmoense, A composition characterized in that the composition is selected from the group consisting of Mycobacterium marinum, Mycobacterium szulgai, Mycobacterium terrae, Mycobacterium scrofulaceum, Mycobacterium ulcerans, Mycobacterium simiae, and Mycobacterium xenopi.

11. In paragraph 1, A composition characterized in that the above tubercle bacilli are selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovis BCG, Mycobacterium africanum, Mycobacterium canetti, Mycobacterium caprae, Mycobacterium microti, and Mycobacterium tuberculosis K strain.

12. In paragraph 1, A composition characterized in that the nontuberculous mycobacterial infection disease or tuberculosis infection disease is a pulmonary disease, lymphadenitis, skin / soft tissue / bone infection, or disseminated disease.

13. A method for providing information for differentiating between a nontuberculous mycobacterial infection disease and a tuberculosis infection disease, comprising the step of measuring one or more metabolites selected from the group consisting of lipid metabolites, amino acids and amino acid derivatives, The above lipid metabolite is at least one selected from the group consisting of palmitamide, stearamide, oleamide, linoleamide, behenamide, anandamide (arachidoyl ethanolamide), stearamine, behenic acid, lyso phosphatidylethanolamine (Lyso PE), sphinganine, sphingosine, and dimethylsphingosine. A method characterized in that the above amino acid and amino acid derivatives contain as an active ingredient a preparation that measures one or more metabolites selected from the group consisting of 2-hydroxyglutaric acid, allantoin, choline and uric acid.

14. In paragraph 13, A method characterized in that the step of measuring the concentration of the above metabolite is performed using a quantitative device such as a chromatograph or a mass spectrometer.

15. A device for differentiating between nontuberculous mycobacterial infections and tuberculosis infections, comprising a preparation for measuring one or more metabolites selected from the group consisting of lipid metabolites, amino acids and amino acid derivatives. The above lipid metabolite is at least one selected from the group consisting of palmitamide, stearamide, oleamide, linoleamide, behenamide, anandamide (arachidoyl ethanolamide), stearamine, behenic acid, lyso phosphatidylethanolamine (Lyso PE), sphinganine, sphingosine, and dimethylsphingosine. A device characterized in that the amino acid and amino acid derivative are at least one selected from the group consisting of 2-hydroxyglutaric acid, allantoin, choline, and uric acid.

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