KR101733864B1 - Biomarker for differentiating between active and latent tuberculosis in conjunction with Interferon gamma release assay and its use - Google Patents

Abstract

The present invention discloses a method for determining an active tuberculosis and a latent tuberculosis by detecting the concentration of VEGF-A in serum by supplementing an interferon gamma release assay such as IGRA and a composition used in the method. The composition or method of the present invention can be used to distinguish between latent tuberculosis and active tuberculosis with high sensitivity and specificity, either alone or in conjunction with conventional culture or QFT-IT tests.

Description

Biomarker for differentiation between active and latent tuberculosis used with interferon gamma release assay and its use {Intermediate gamma release assay and its use for biomarker for differentiating between active and latent tuberculosis}

The present invention relates to a biomarker for use in conjunction with interferon gamma release assay to determine whether it is active or latent tuberculosis and its use.

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (M. tb ), and is the leading cause of death worldwide. Approximately one-third of the world's population is potentially infected with tuberculosis, and in 2012, about 6.6 million new cases were reported to WHO.

Currently, a widely used method for diagnosing tuberculosis is to identify tuberculosis by clinically isolating the tuberculosis (acid-fast bacillus (AFB)) stain or culture. Although the stain dyeing is advantageous in that it rapidly detects a patient with a high infectivity by rapidly detecting the Mycobacterium tuberculosis, the sensitivity is low to 20-80% as compared with the culture test, and the immune system such as a child, an elderly person or acquired immunodeficiency Failure rates are high in injured patients. The incidence of non-tuberculous mycobacterial isolates is increasing in Korea, so smear testing alone is not enough to diagnose active tuberculosis. In addition, even in the case of nontuberculous mycobacteria (NTM) pulmonary disease, sputum is positive in horse test, so it is difficult to diagnose as active tuberculosis only by smear test.

Another method is to detect latent TB infection (LTBI) by immunological methods such as tuberculin skin test (TST) and IGRA (IFN-γ release assay). TST can detect latent tuberculosis, but additional tests such as radiation, sputum, and culture tests are necessary to confirm latent tuberculosis.

TST is a method of measuring the amount of induration at the injection site 48 to 72 hours after injection of tuberculin reagent into the arm. The QuantiFERON ^® -TB (QFT) test, an IGRA, is an in vitro latent tuberculosis test that measures the level of gamma interferon from cells by sensitizing the lymphocytes of the patient sample to a tuberculosis-specific antigen. The purified protein derivative (PPD) Specificity is low. The QuantiFERON ^® -TB Gold (QFT-G) test is an improved method of QFT, in which samples are mixed with heparin coagulant and the synthetic peptides of ESAT-6 and CFP-10, It is used as the leukocyte stimulating antigen of the subject. It is an in vitro test for the diagnosis of Mycobacterium tuberculosis infection in all tuberculosis including active tuberculosis including latent tuberculosis. ^{QuantiFERON ® -TB Gold In-Tube (} QFT-IT) test is biseuthande QFT-G tests, principles, but QFT-G is ESAT-6, but each scan the CFP-10, QFT-IT is TB 7.7 in the same tube , So blood samples are taken directly into the antigen and control containers instead of the heparin tubes during sample collection. The QFT-IT does not distinguish between active and latent tuberculosis, the test results are determined only by positive or negative, not by antigen. In other words, the TST and IGRA tests alone are not enough to distinguish active tuberculosis, latent tuberculosis and NTM disease.

After tuberculosis infection through the respiratory tract, some (10%) of the tuberculosis develops into tuberculosis through latent period, and in most cases, it is latent TB infection (LTBI). However, this latent tuberculosis may progress to active tuberculosis for a lifetime depending on the immunity of the infected person. Therefore, in order to effectively eradicate tuberculosis, active tuberculosis as well as diagnosis and treatment of latent tuberculosis are important for ultimate prevention and prevention of tuberculosis, so it is necessary to diagnose active tuberculosis and latent tuberculosis separately.

International Publication No. 2013-175459 discloses a method of distinguishing between active and inactive tuberculosis but does not disclose the detection of active tuberculosis and latent tuberculosis separately using VEGF-A. Also, International Publication No. 2011-0036590 discloses genes that are differentially expressed during active or inactive tuberculosis, but there is no disclosure of genes expressing VEGF-A.

The present invention provides a biomarker and its use that can be used in the treatment of effective tuberculosis by diagnosing whether human-derived serum and plasma samples determined to be positive in interferon gamma release assay are active tuberculosis or latent tuberculosis.

In one embodiment, the present invention provides a method of treating a patient diagnosed with a tuberculin test (TST) or interferon gamma release assay to determine whether a patient is positive for at least one of the above- Providing a serum sample derived from the sample; And detecting the concentration of VEGF-A in the sample, wherein the result of the detection comprises: i) if the concentration of VEGF-A in the sample is higher than that of the control, the sample is used as active tuberculosis; ii) Wherein the sample is identified as latent tuberculosis when the concentration of A is low or low compared to the control.

In another aspect, the disclosure also provides a composition for use in determining or diagnosing whether a patient determined to be positive in an interferon gamma release assay is active or latent tuberculosis, comprising a reagent for detecting VEGF-A. In one embodiment, the sample used with the composition according to the present invention comprises serum.

In one embodiment, a reagent for detecting a VEGF-A marker that may be included in a composition according to the present invention is a polypeptide comprising an antibody that specifically recognizes said VEGF-A, wherein detection using said polypeptide comprises radial immunodiffusion But are not limited to, immunoassay analysis, including radioimmunoassay, Radial Immunodiffusion, immuno-electrophoresis or immuno-electrophoresis, RIA (Radioimmunoassay) or ELISA (Enzyme Linked Immunosorbent Assay).

In another embodiment, detection using a polypeptide comprises the use of a detection antibody, wherein said detection antibody specifically binds to human IgG, said detection antibody comprising a chromophore; Enzymes including alkaline phosphatase, biotin, beta-galactosidase or peroxidase; Radiation material; Or color particles comprising colloidal gold particles or color latex particles.

In another embodiment herein, the invention encompasses a VEGF-A antibody-adsorbed support and a detection antibody, wherein the detection antibody specifically binds to human IgG and comprises a chromophore; Enzymes including alkaline phosphatase, biotin, beta-galactosidase or peroxidase; Radiation material; Or color particles comprising colloidal gold particles or color latex particles, wherein the kit is for detecting or diagnosing active or latent tuberculosis of sputum negative tuberculosis.

In one embodiment, the support included in the kit according to the present invention includes, but is not limited to, microplates, microarrays, chips, glass, beads or particles, or membranes.

In another embodiment, the kit according to the present invention can be used for ELISA, dip stick or lateral flow analysis, but is not limited thereto.

We will be able to diagnose active tuberculosis and latent tuberculosis more accurately by supplementing analysis of interferon gamma release which can not distinguish between active tuberculosis and latent tuberculosis. .

Figure 1 shows the personnel involved in the experiments conducted herein. (N = 86), tuberculosis (n = 51) and healthy controls (n = 133) were enrolled in this study. Patients with tuberculosis (n = 28) who had cancer or diabetes or who took immunosuppressive drugs were excluded. QFT-IT negative tuberculosis contact group (n = 25) were excluded. Among the 133 healthy controls recruited, only those with negative responses in both TST (<10 mm) and QFT-IT tests were included (n = 55). Tuberculosis patients were reevaluated after 2 and 6 months of antituberculous treatment. Serum and QFT-IT plasma samples were collected from each group.
Figure 2 compares serum cytokine levels in tuberculosis, latent tuberculosis and control. The concentration of 17 cytokines in the tuberculosis patients (n = 58), the contact group with latent tuberculosis (n = 26) and the control group (n = 55) Tuberculosis patients showed significantly higher IL-22, CXCL10, and VEGF-A serum levels compared to the control. Only the VEGF-A levels were different between the tuberculosis patient and the latent tuberculosis group (P <0.01).
Figure 3 shows the response of cytokines to tuberculous antigens ESAT-6, CFP, and TB 7.7 in QFT-IT plasma of tuberculosis (n = 21), latent tuberculosis (n = 13), and control will be. In TB-specific reactions, IFN-γ, IL-2 and CXCL10 responses were significantly higher in active and latent tuberculosis than in the control. None of the assays distinguished between tuberculosis and latent tuberculosis. All three groups showed high cytokine levels for PHA. Intermediate reactions were represented by horizontal bars (** P <0.01).
Figure 4 shows the ROC curve of serum VEGF-A to distinguish between active tuberculosis and latent tuberculosis. The AUC was analyzed by the ROC curve of serum VEGF-A detected in patients with tuberculosis (n = 58) and patients with tuberculosis (n = 26) (AUC = 0.7576, P <0.001).

This is based on the discovery that VEGF-A may be used as a marker to determine whether a patient judged positive in interferon gamma release assays is an active or latent tuberculosis infection.

In one embodiment, the subject is provided with a serum derived from a patient determined to be positive in the assay, in order to provide information for diagnosing whether a person or patient determined to be positive in the TST or interferon gamma release assay is an active tuberculosis infection or a latent tuberculosis infection ; And detecting the concentration of VEGF-A in the sample, wherein the result of the detection comprises: i) if the concentration of VEGF-A in the sample is higher than that of the control, the sample is used as active tuberculosis; ii) A method for determining whether or not an active tuberculosis infection is detected through VEGF marker detection, comprising the step of identifying or confirming the sample as latent tuberculosis when the concentration is not statistically different from the control group .

According to the method of the present invention, it is possible to diagnose tuberculosis from healthy persons by distinguishing active tuberculosis from latent tuberculosis by supplementing the existing TST or interferon gamma release assay.

The 'active tuberculosis' is a condition in which tuberculosis that can induce tuberculosis causes active proliferation, not latency, and may be positive in sputum test (smear, culture, nucleic acid amplification (PCR)), (Chest x-ray, CT, etc.), and active tuberculosis. "Latent TB infection" refers to a condition that is infected with tuberculosis but is not clinically diagnosed as tuberculosis and is negative in tuberculosis tests such as tuberculosis and radiation, and can not be transmitted to others.

Samples or samples according to the present invention are specimens derived from humans that are judged to be positive in the TST or interferon gamma release assay, particularly serum.

Tuberculin test (TST) is a test for the diagnosis of infection by measuring the magnitude of the injection site 48 to 72 hours after injection of tuberculin reagent into the arm.

Interferon gamma release assay (IGRA) is an in vitro latent tuberculosis test that measures gamma interferon from cells by sensitizing a sample's lymphocytes to a tuberculosis-specific antigen. QuantiFERON ^® -TB (QFT ), QFT-G, and QFT-IT. The QuantiFERON ^® -TB Goild (QFT-G) test is an improvement of QFT. The sample is prepared by mixing the blood collected from the patient with the heparin coagulant and using the synthetic peptides of ESAT-6 and CFP-10, It is used as the leukocyte stimulating antigen of the subject. It is an in vitro test for the diagnosis of Mycobacterium tuberculosis infection in all tuberculosis including active tuberculosis including latent tuberculosis. ^{QuantiFERON ® -TB Goild In-Tube (} QFT-IT) test is biseuthande QFT-G tests, principles, but QFT-G is ESAT-6, but each scan the CFP-10, QFT-IT is TB 7.7 in the same tube And blood samples are taken directly into the antigens and containers for the control rather than the heparin tubes at the time of sample collection. The QFT-IT test is not positive by antigen, but by positive or negative.

However, this method does not distinguish between active tuberculosis and latent tuberculosis.

The method according to the present invention can be used in conjunction with the TST or interferon gamma release assay as described above to determine whether the sample determined to be positive in the assay is an active tuberculosis or a latent tuberculosis.

The marker used in the method according to the present invention is VEGF-A (Vascular endothelial growth factor A), a glycoprotein of dimer and known to play an important role in the growth of vascular cells. The sequence is known, and in human cases, the protein sequence is known as GenBank accession number NP_001020537, and the nucleic acid sequence is known as GenBank accession number NM_001025366.

Such VEGF-A can be detected at the nucleic acid and protein levels, and in one embodiment, particularly at the protein level.

At the time of detection at the protein level, an antibody specifically recognizing it specifically or a polypeptide comprising the fragment is used. For example, an antigen-antibody reaction, a substrate that specifically binds to the marker, a nucleic acid or a peptide aptamer, a receptor or ligand that specifically interacts with the marker, or a cofactor.

Detection at the nucleic acid level can be carried out by conventional methods such as hybridization using a chip method, polymerase chain reaction using a primer or a probe, and Southern blotting. The detection at the mRNA level is performed using a reverse transcription polymerase chain reaction Reaction, RNase protection assay, or Northern blot. In the present invention, detection includes quantitative and qualitative analysis, including detection of presence or absence, or detection of the amount of expression, and such methods are well known in the art, and those skilled in the art can select an appropriate one for the practice of the present invention There will be.

For example, the antigen-antibody reaction can be detected by immunoprecipitation assay, radioimmunoassay (RIA) or enzyme linked immunosorbent assay (ELISA), including radial immunodiffusion, immunoelectrophoresis or reverse-flow electrophoresis. In one embodiment according to the present invention, an ELISA method, in particular a sandwich ELISA, is used, in which case the detection antibody described below is also used.

The intensity of the final signal of the sample is analyzed through the detection of the antigen-antibody reaction and compared with the signal of the sample of the normal control group. If it is higher than this, diagnosis of active or latent tuberculosis can be made as described above.

In one embodiment according to the present application, for the detection of an antigen-antibody, the antigen is labeled with a labeling substance described later, or a detection antibody or a secondary antibody is used.

The detection antibody that can be used in the method according to the present invention is specifically bound to a human IgG, and the detection antibody can be labeled with a substance that can be detected using visual or various image detection equipment.

In one embodiment according to the present invention, the marker or detection antibody according to the present invention is a labeling substance which is a labeling substance such as a peroxidase such as horseradish peroxidase, an alkaline phosphatase, a glucose oxidase, Beta-galactosidase, urease, catalase, asparginase, ribonuclease, malate dehydrogenase, starch, and the like. (S) such as staphylococcal nuclease, triose phosphate isomerase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholine Catalyzes a chemical reaction in the presence of a specific substrate such as acetylcholine esterase to form a detectable chromogenic reaction or light But is not limited to, an enzyme capable of releasing the enzyme.

In another embodiment, the marker or detection antibody according to the present invention is a bioluminescence, chemiluminescence, electroluminescence, electrochemiluminescence and / or radioimmunoassay that emits light of a wavelength different from that of the light irradiated by light. Chromophores used in photoluminescence, for example, green fluorescent proteins as proteins; Examples of the organic compounds include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, and fluorecamine. But are not limited to,

In another embodiment, a marker or detection antibody according to the present invention may be labeled with a variety of radioactive isotope materials.

Detection of a labeling substance in the present invention can be performed, for example, by a scintillation counter in the case of a radioactive isotope. For example, in the case where the labeling substance is a fluorescent substance, detection with a spectroscope, a phosphoimaging device, Can be performed by the same method. When labeled with an enzyme, it can be carried out by measuring the chromogenic product which is indicated by the conversion of the chromogenic substrate by the enzyme in the presence of a suitable substrate. It can also be detected as a color comparison of the chromogenic products produced by enzymatic reactions through comparison with appropriate standards or controls.

In one embodiment, the marker or detection antibody according to the present invention is, for example, a chromophore; Enzymes including alkaline phosphatase, biotin, beta-galactosidase or peroxidase; Radiation material; Or nanoparticles such as colloidal gold particles or colored latex particles, and the like.

In another embodiment, the present invention also relates to a composition capable of diagnosing active tuberculosis or latent tuberculosis in a patient determined to be positive in a TST or interferon gamma release assay, comprising a reagent for detecting VEGF-A.

The reagent for detecting VEGF-A according to the present invention may contain a detection reagent at the above-mentioned nucleic acid or protein, especially at the protein level, as described above.

In another embodiment, the disclosure also encompasses a solid support on which a VEGF-A antigen is adsorbed and a detection antibody, wherein the detection antibody specifically binds to human IgG and comprises a chromophore; Enzymes including alkaline phosphatase, biotin, beta-galactosidase or peroxidase; Radiation material; Or colored particles comprising colloidal gold particles or color latex particles. The present invention also relates to a kit for the detection or diagnosis of active tuberculosis or latent tuberculosis of sputum negative tuberculosis.

It is possible to refer to the above description of the configurations included in the kit according to the present application that overlap with those described above.

In the kit according to the present invention, the marker according to the present invention may be a micro well plate such as a 96 well microwell plate, a bead or particle comprising colloidal gold particles or colored latex particles or a cellulosic material such as cellulose, nitrocellulose, polyethersulfone, polyvinylidene, Fluorine, fluoride, nylon, charged nylon and polytetrafluoroethylene, and the like. As a method for adhering or coating, known methods can be used, for example, those described in the Examples herein can be referred to.

In one embodiment, the methods, compositions or kits according to the present invention can be used in a sandwich immunoassay such as an ELISA (Enzyme Linked Immuno Sorbent Assay), RIA (Radio Immuno Assay) and the like. Such methods include adding a sample to an antigen coupled to a bead, membrane, slide, or microwell plate made of a solid substrate such as glass, plastic (e.g., polystyrene), polysaccharide, nylon or nitrocellulose , A labeling substance capable of direct or indirect detection, for example, a radioactive substance such as 3H or 125I as described above, a fluorescent substance, a chemiluminescent substance, a hapten, a biotin, a digoxigenin or the like, Or can be qualitatively or quantitatively detected through binding of an antibody conjugated with an enzyme such as luminescent horseradish peroxidase, alkaline phosphatase, or malate dehydrogenase. Immunoassay methods are also described in Enzyme Immunoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme-linked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, J.M. ed., Humana Press, NJ, 1984. An ELISA kit can be prepared by mixing a secondary detection antibody labeled with the above substance, such as a reagent capable of detecting the bound antibody, such as chromophores, an enzyme (e. G., Conjugated with an antibody) And the like.

In other embodiments, a method, composition or kit according to the present invention may be used in the form of an array or chip comprising a microarray. Antigens can be attached to the surface of a support such as glass or nitrocellulose, and array fabrication techniques are described, for example, in Schena et al., 1996, Proc Natl Acad Sci USA. 93 (20): 10614-9; Schena et al., 1995, Science 270 (5235): 467-70; And US Pat. Nos. 5,599,695, 5,556,752, or 5,631,734. Fluorescence intensity can be measured using a scanning confocal microscope, for example Affymetrix, Inc. Or from Agilent Technologies, Inc., and the like.

The methods, compositions or kits according to the invention are also used in the form of dip stick rapid kits according to the analytical mode. In the case of dip stick, a technique widely used in the field of POCT (Point of Care Treatment), in which an antigen according to the present invention is bound to a substrate such as nitrocellulose and is contacted with a sample such as serum, When the one end is immersed in a serum sample, the sample is detected in such a manner that the sample migrates the substrate by the capillary phenomenon and develops color when bound to the antibody in the substrate.

The kits herein can also be used for lateral flow analysis according to the analysis mode. A lateral flow assay is a method for quantitatively or qualitatively measuring a specific substance contained in a specimen, for example, a specific nucleic acid or protein, for example, a nitrocellulose membrane (for example, And a specific nucleic acid or protein in the sample is detected through an antigen-antibody reaction by moving the analyte by a chromatographic method using a developing medium.

The kits herein may also include instructions on how to use the biomarkers in accordance with the present disclosure. In addition, a control-specific antibody or a reagent capable of detecting bound antibody may be further included. The intensity of the final signal of the sample is analyzed through the antigen-antibody complex detection process and compared with the signal of the sample of the normal control group. If it is higher than this, it can be diagnosed as active or latent tuberculosis as described above.

Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Example

Materials and Methods

Subjects: From November 2010 to December 2013, 86 subjects who were diagnosed with tuberculosis (mean age 32 years, 44 males and 42 females aged 20 to 76), and those who were exposed to TB A total of 51 subjects (aged 18 to 82 years, mean age 44 years, 13 males and 38 females) were recruited (Fig. 1). 133 healthy healthy subjects (mean age 31 years, aged from 20 to 61 years, 63 males and 70 females) who had never been contacted with tuberculosis patients and who had no tuberculosis symptoms on chest X-ray were recruited (Figure 1) . The diagnosis of active tuberculosis was confirmed by smear / culture of tuberculosis by sputum or radiation. Immunosuppressants were taken, or people with cancer or diabetes were excluded. People with HIV or kidney disease were also excluded. Latency or normal controls were classified based on TST and QFT-IT tests. Twenty-six of 51 TB contacts were positive for latent tuberculosis (LTBI) as compared to 25 patients with tuberculosis who had a negative IFN-γ response, showing a positive IFN-γ response in the QFT-IT test. Of the 133 normal healthy individuals, 55 patients with a negative IFN-γ response in the QFT-IT test and a TST induration size <10 mm were used as controls. Thus, 58 TB patients, 26 TB contacts and 55 healthy controls were included in the experiments of the present invention (FIG. 1). In the QFT-IT plasma sample analysis, there were fewer patients who could not obtain plasma samples and the immune response of 21 patients with tuberculosis was compared with thirteen latency tuberculosis patients and 21 controls (Fig. 1). All of the patients were recruited at Severance Hospital in Seoul, Korea, and the study was explained to the subjects and blood samples were taken for immunoassays such as TST, clinical trials (eg chest X-ray) and QFT-IT I have received a consent to provide information. Ethical approvals for this trial were received at the Severance Hospital Ethics Review Board: The approval number for active pulmonary tuberculosis patients, tuberculosis contact group, healthy controls is 4-2010-0213 and the approval number for NTM patients is 4-2011-0241. Table 1 shows the characteristics of the subjects participating in the experiment at baseline. All 58 patients were diagnosed as pulmonary tuberculosis.

[Table 1]

TST: Tuberculin PPD solution (0.1 ml of RT-23, Statens Serum Institute, Copenhagen, Denmark) was injected intradermally into tuberculosis patient, tuberculosis contact group and normal healthy control group. Induration size was measured at 48 and 72 hours, and those measured at 10 mm or more were classified as positive. QFT-IT test Serum samples were obtained from 4 ml of blood (VACUETTE ^® serum tube, Greiner Bio-One GmbH, Frickenhausen, Germany) and total 3 ml of blood was transferred to three QFT-IT tubes [Nil, M. tb Ag tube -6, CFP-10, and TB7.7 peptide antigens), and mitogen tubes; PHA, Cellestis, CA, USA]. The QFT-IT tubes were incubated in the uptake at 37 ° C for 24 hours, then the plasma was harvested and divided into aliquots for IFN-y ELISA and multiplex bead arrays. IFN-? ELISA was performed according to the manual (QuantiFERON-TB Gold, Cellestis) and the data were analyzed using QFT-IT analysis software (Cellestis).

Cytokine concentration assays: Multiplex bead arrays with 17 different assays containing cytokines, chemokines, and growth factors were purchased from BD FACSVerse (BD Biosciences, San Jose, CA, USA) using serum and QFT- Respectively. IL-1b, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-13, IL- ?, TNF- ?, IFN- ?, sCD40L, CXCL10 (IP-10), and vascular endothelial growth factor A (VEGF-A). The multiplex bead array was analyzed according to the manual (eBioscience, San Diego, Calif., USA). The concentration of each analyte was calculated with FlowCytomix Pro software (eBioscience), and the values outside the standard curve range were adjusted by setting the minimum and maximum values. The values of the 17 analytes of the QFT-IT plasma were corrected by subtracting the negative control value (nil tube) to the background level. In order to attenuate false positives, respondents defined responder as higher than twice the detection limit of the standard curve: 5.5 pg / mL for IL-9, 27 pg / mL for IL-17A, 34.5 pg IL-10, IL-12, IL-13, IFN- ?, TNF- ?, IL- IFN- ?, VEGF-A, 110 pg / mL for sCD40L, and 220 pg / mL for IL-22.

Statistical analysis: The differences in concentration of 17 analytes from serum and QFT-IT plasma samples obtained from active TB patients, contact groups with latent tuberculosis and normal healthy controls were analyzed by Kruskal-Wallis and Dunn multiple comparison tests. The Mann-Whitney test was used to analyze the concentration differences of 17 analytes of active tuberculosis and NTM disease. P values were adjusted to Bonferroni correction to account for the various comparisons. Diagnostic values of 17 analytes of serum and QFT-IT plasma were analyzed by AUC (area under the receiver operating characteristic (ROC) curves).

Example 1. Classification of active tuberculosis, latent tuberculosis, and NTM patients through analysis of biological characteristics of 17 analytes obtained from serum and QFT-IT plasma

1-1 Analysis of analytes obtained from serum

In order to differentiate between active TB and latent tuberculosis and to monitor the therapeutic effect, the biological characteristics of 17 analytes obtained from the sera obtained from the active tuberculosis group, the latent tuberculosis group and the normal healthy control group in this example were examined.

The median concentrations of serum IL-22, CXCL10, and VEGF-A were significantly higher in 58 TB patients (P <0.05) than in the 55 control group, whereas only VEGF-A concentrations differed between active and latent tuberculosis P < 0.01) (Figure 2A). AUC analysis shows that serum VEGF-A may be a good biomarker to distinguish active tuberculosis from latent tuberculosis (AUC = 0.7576, P <0.001, Fig. 4).

1-2 Analysis of analytes from QFT-IT plasma

In contrast to 1) above, the biological characteristics of 17 analytes were investigated in plasma samples (QFT-IT plasma) stimulated with tuberculous antigens obtained from the active tuberculosis group, the latent tuberculosis group and the normal healthy control group.

In response to the TB challenge, QFT-IT plasma IFN-α, IL-2, and CXCL10 responses were significantly higher in the active and latent tuberculosis groups than in the control group (P <0.01, FIG. 3). Although not significant, IL-13 levels were also higher in tuberculosis patients than in controls (P> 0.05). QFT-IT Plasma VEGF-A was not different between active and latent groups, unlike serum VEGF-A, and none of the 17 analytes in the two groups differed in response to tuberculosis antigen (FIG. 3). Analysis of cytokines for mitogens (PHA) showed no significant difference between the two groups (P> 0.05), indicating that all of the measured cytokine responses were not affected by nonspecific immunosuppression (Data not shown ). Plasma cytokine responses to ESAT-6, CFP, and TB 7.7 antigens used in IGRA do not distinguish between active and latent tuberculosis, not only IFN-y, but also other cytokines. Therefore, the method according to the present invention can be used to supplement the IGRA test because it distinguishes between active and latent tuberculosis infection using serum VEGF-A.

1-3 Diagnosis of active tuberculosis and latent tuberculosis

The results of the analytes obtained from the sera can be used to distinguish between active and latent tuberculosis by examining the serum VEGF-A concentration in addition to the tuberculosis test to confirm tuberculosis by culture test. In addition, the levels of IFN-γ, IL-2, and CXCL10 in the QFT-IT supernatant were significantly higher in patients with tuberculosis than in the normal control, Can increase the sensitivity of IGRA, which measures only the IFN-γ level, by measuring the combination of IL-2 and CXCL10 and diagnosing tuberculosis infection. On the other hand, as previously reported, none of the above three analytes can clearly distinguish between active tuberculosis and latent tuberculosis.

The serum VEGF-A concentration can be used as a biomarker to distinguish between active tuberculosis and latent tuberculosis. In addition, the combination of IFN-γ, IL-2, and CXCL10, Can be improved.

In conclusion, serum VEGF-A is the most useful marker to distinguish active tuberculosis from latent tuberculosis. Measuring the response of tuberculous antigen-specific IFN-γ, IL-2 and CXCL-10 may improve the sensitivity and specificity of IGRA have. Therefore, measuring a large number of analytes in serum or QFT-IT plasma can speed up the diagnosis and can be used as a secondary marker, and can be very useful for diagnosing differential diagnosis between active and latent tuberculosis.

Claims (7)

In order to provide information to diagnose patients with positive tuberculin test (TST) and interferon gamma release assays as positive for active tuberculosis infection,
Providing a non-sensitized serum sample from a patient determined to be positive in said assay; And
Detecting the concentration of VEGF-A in the sample,
If the concentration of VEGF-A in the sample is higher than that of the control, the sample is used as active tuberculosis. Ii) When the concentration of VEGF-A is not different from that of the control group, Determining the presence of active tuberculosis infection.
A composition for determining the presence of active tuberculosis tuberculosis using a non-sensitized serum sample derived from a patient determined to be positive in a tuberculin test (TST) and interferon gamma release assay, comprising a reagent for detecting VEGF-A.
The method according to claim 2, wherein the reagent for detecting the VEGF-A marker is a polypeptide comprising an antibody that specifically recognizes the VEGF-A, and the detection using the polypeptide includes Radial Immunodiffusion, (Immunoassay Analysis, RIA (Radioimmunoassay) or ELISA (Enzyme Linked Immunosorbent Assay), including electrophoresis or reverse-flow electrophoresis.
4. The method of claim 3, wherein detection with the polypeptide comprises the use of a detection antibody, wherein the detection antibody specifically binds to a human IgG, wherein the detection antibody comprises a chromophore; Enzymes including alkaline phosphatase, biotin, beta-galactosidase or peroxidase; Radiation material; Or colored particles comprising colloidal gold particles or color latex particles.
A support on which the VEGF-A antibody is adsorbed, and a detection antibody,
Wherein said detection antibody specifically binds to human IgG and comprises a chromophore; Enzymes including alkaline phosphatase, biotin, beta-galactosidase or peroxidase; Radiation material; (TST) and interferon gamma release assays, which are labeled with a material comprising colloidal gold particles or color particles comprising colloidal gold particles or colloidal latex particles. Kits for determining the presence of active tuberculosis. 6. The kit of claim 5, wherein the support is a microplate, microarray, chip, glass, bead or particle, or membrane.
7. The kit of claim 6, wherein the kit is for ELISA, dip stick or lateral flow analysis.

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