A biomarker for the diagnosis of recent and remote tuberculosis infection

Abstract

The present application belongs to the field of biotechnology, and particularly relates to a biomarker for differential diagnosis of recent and long-term tuberculosis infection. The present application determines the differential proteins in blood of Mycobacterium tuberculosis recent infection and long-term infection through proteomics, and provides a diagnostic biomarker for differential diagnosis of Mycobacterium tuberculosis recent infection and long-term infection. The biomarker provided by the present application has good diagnostic efficiency whether it is used for single diagnosis or combined diagnosis, and the AUC value is greater than or close to 0.8. The biomarker can be used for more rapid, efficient, sensitive and specific identification of Mycobacterium tuberculosis recent infection, and provides a diagnostic basis for early intervention of tuberculosis.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a biomarker for differential diagnosis of recent and long-term tuberculosis infection. BACKGROUND

[0002] Tuberculosis is a chronic respiratory infectious disease caused by Mycobacterium tuberculosis (MTB), and the most common type is pulmonary tuberculosis, with typical symptoms of cough, sputum, fever, night sweats, and blood in sputum. According to the World Health Organization (WHO), there were 1060 million new tuberculosis patients worldwide in 2021, resulting in about 1.4 million deaths. Studies have shown that one-third of people exposed to Mycobacterium tuberculosis will be infected, and the remaining exposed people will clear the pathogen from the body through the body's immune response to the pathogen. Among the infected population, less than 10% of the infected people develop tuberculosis (Bloom BR, Small PM. The evolving relation between humans and Mycobacterium tuberculosis. N Engl J Med, 1998, 338(10): 677-8.). Tuberculosis is not only an infectious disease, but also an immune disease. There are genetic differences in humans' resistance to MTB infection, and the body's different immune responses to MTB ultimately determine the occurrence, development, and outcome of infection. Studies have found that recent Mycobacterium tuberculosis infection is at a higher risk of developing tuberculosis than long-term infection. The high-risk period for tuberculosis progression is within the first 1-2 years after initial infection, but the risk of tuberculosis in long-term infection is low. Therefore, early diagnosis of recent tuberculosis infection and timely intervention are crucial to reduce TB incidence. However, current clinical diagnostic methods can identify whether Mycobacterium tuberculosis is infected, but cannot distinguish between recent and long-term infection. Therefore, there is a need for diagnostic markers to clearly identify recent and long-term Mycobacterium tuberculosis infection in the clinic.

[0003] Proteomics technology can deeply study the structure, function, efficacy and interaction of proteins (Voyer P, Champoux N, Desrosiers J, et al. RADAR: a measure of the sixth vital sign?[J]. Clinical nursing research, 2016, 25(1): 9-29), and the proteome is downstream of the genome and transcriptome. Analysis of it can identify different proteins caused by post-translational modification, changes in biological system activity and function during DNA and RNA translation, thereby discovering the relationship between proteins and diseases (Ouyang H, Fu H, Du Y. Application of omics technology in essential hypertension[J]. Practical Journal of Cardiovascular Disease, 2022, 30(9): 118-123.), and help researchers better understand the function and structure of proteins, and provide valuable information for clinical practice (J. H. Han, E. E. Zimmerman, N. Cutler, et al., Delirium in older emergency department patients: recognition, risk factors, and psychomotor subtypes, Acad. Emerg. Med. 2009, 16(3): 193-200.). Because it reflects different stages of cell differentiation and state, its characteristics are multifaceted and dynamic in time and space, providing opportunities to further elucidate complex biological processes. In the past few years, proteomics has been used to analyze differentially expressed proteins in cancer, diabetes, Alzheimer's disease, true polycythemia, and even animal and plant research. Proteomics has advantages over conventional marker detection methods and provides researchers with a new perspective to explore the network regulation between whole proteins. It has important significance for early diagnosis of postoperative cognitive dysfunction, finding new markers, etc. At present, proteomics is widely used and can be applied to: Proteomics technology includes four clinical applications: (1) identification of proteins in a specific biological sample; (2) detection of protein expression profiles that describe the specific state of an organism or cell; (3) determination of protein interactions in specific functional protein networks; (4) precise localization of post-translational protein modifications (Kosteria I, Anagnostopoulos AK, Kanaka-Gantenbein C, et al. The use of proteomics in assisted reproduction[J]. in vivo, 2017, 31(3): 267-283.).

[0004] Olink Proteomics relies on its core protein detection patent - Proximity Extension Assay (PEA) to achieve accurate detection of more than 3000 proteins in 1-6 μL of sample. PEA is a dual recognition immunoassay, that is, two matching antibodies bind to a target at the same time and are labeled with unique DNA oligonucleotides. The protein in the solution brings the two antibodies close together, and their DNA oligonucleotides hybridize as a template for a DNA polymerase-dependent extension step. This will create a double-stranded DNA "barcode" that is unique to the specific antigen and is proportional in quantity to the initial concentration of the target protein. PCR amplification is performed immediately after hybridization and extension, and finally the amplicon is quantified by microfluidic qPCR. PEA successfully combines antibody-based immunoassay with the powerful features of quantitative real-time PCR (qPCR) and high-throughput sequencing ("Next-generation" sequencing technology, NGS), thus forming a method with multiple channels and high specificity (Liu H. Plasma immunoproteomics research of Behcet's disease patients based on PEA technology[D]. Henan: Zhengzhou University, 2022.). PEA breaks through the difficulties of traditional proteomics detection techniques such as limited sensitivity, low specificity, limited dynamic range of detection, large sample consumption, and low throughput, and can simultaneously quantify thousands of protein biomarkers to understand complex, real-time human biology, effectively screen markers for disease diagnosis, with high specificity, multiple channels, and help precision medicine, translational medicine, drug development, and other innovative proteomic detection solutions (Hou M. Proteomic analysis of inflammatory factors in the plasma of elderly patients with delirium after general anesthesia[D]. Henan: Henan University, 2023.).

[0005] The immune system of the human body plays an important role in resisting Mycobacterium tuberculosis infection, the onset and outcome of tuberculosis. From the immunological point of view, tuberculosis is an immune disorder disease closely related to immune response. As effectors and mediators secreted by various immune cells, cytokines have the function of transmitting information between cells, and the disorder of their network is related to the onset of tuberculosis (Dong HP, Wang CZ, Liang XC, et al. The significance of plasma cytokines in the recurrence of pulmonary tuberculosis [J]. Modern Hospital, 2019, 19(7): 1015-1018.). Cytokines are low-molecular-weight soluble polypeptides produced by various cells (mainly immune cells) that can participate in immune response, and are divided into interleukins, tumor necrosis factors, interferons, etc., which can mediate natural immunity, specific immune response and other physiological processes, and also participate in the induction of apoptosis, stimulation of hematopoiesis and other processes. In summary, cytokines may have certain auxiliary diagnostic value for pulmonary tuberculosis and lung cancer. However, due to the wide source of cytokines and many influencing factors, there is currently no biomarker that can be used to distinguish between recent and long-term infections of Mycobacterium tuberculosis. SUMMARY

[0006] The present application identifies the differential proteins in the blood of recent and long-term Mycobacterium tuberculosis infected persons through proteomics, and provides a diagnostic biomarker for distinguishing between recent and long-term Mycobacterium tuberculosis infections. The biomarker can be used to more quickly, efficiently, sensitively and specifically distinguish between recent and long-term Mycobacterium tuberculosis infections, and provide diagnostic evidence for early intervention of tuberculosis. Based on this, the present application is completed.

[0007] In a first aspect, the present application provides a biomarker for differential diagnosis of recent and long-term tuberculosis infection, wherein the biomarker is selected from one or more of CCL3, TNFα and / or CCL20; when the protein concentration of the biomarker is lower than the critical value, it indicates recent pulmonary tuberculosis infection.

[0008] Further, when the protein concentration of TNFα in the biological sample of the subject is lower than 93.37 pg / ml, it indicates that the subject is a recent pulmonary tuberculosis infection.

[0009] Further, when the protein concentration of CCL20 in the biological sample of the subject is lower than 149.55 pg / ml, it indicates that the subject is a recent pulmonary tuberculosis infection.

[0010] Further, when the protein concentration of CCL3 in the biological sample of the subject is lower than 170.65 pg / ml, it indicates that the subject is a recent pulmonary tuberculosis infection.

[0011] Further, the biological sample is from whole blood, serum or plasma of the patient.

[0012] Preferably, the biological sample is from the plasma of the patient.

[0013] Further, the tuberculosis is an infection caused by the pathogen Mycobacterium tuberculosis.

[0014] Still further, the Mycobacterium tuberculosis infection includes primary infection, secondary infection and extrapulmonary infection.

[0015] Further, the tuberculosis includes primary tuberculosis, hematogenous disseminated tuberculosis, secondary tuberculosis, tracheal or bronchial tuberculosis and tuberculous pleurisy.

[0016] In a second aspect, the present application provides a use of a biomarker in the preparation of a reagent for differential diagnosis of recent and long-term tuberculosis infection, wherein the biomarker is one or more of CCL3, TNFα and / or CCL20, and the reagent is a reagent for detecting the content of CCL3, TNFα and CCL20 in a biological sample of a patient; when the concentration of the biomarker protein is lower than the critical value, it indicates recent tuberculosis infection.

[0017] Further, when the concentration of TNFα protein in the biological sample of the subject is lower than 93.37 pg / ml, it indicates that the subject is a recent tuberculosis infection.

[0018] Further, when the concentration of CCL20 protein in the biological sample of the subject is lower than 149.55 pg / ml, it indicates that the subject is a recent tuberculosis infection.

[0019] Further, when the concentration of CCL3 protein in the biological sample of the subject is lower than 170.65 pg / ml, it indicates that the subject is a recent tuberculosis infection.

[0020] Further, the biological sample is from whole blood, serum or plasma of the patient.

[0021] Preferably, the biological sample is from plasma of the patient.

[0022] Further, the tuberculosis is an infection caused by the pathogen Mycobacterium tuberculosis.

[0023] Still further, the Mycobacterium tuberculosis infection includes primary infection, secondary infection and extrapulmonary infection.

[0024] Further, the tuberculosis includes primary tuberculosis, hematogenous disseminated tuberculosis, secondary tuberculosis, tracheal or bronchial tuberculosis and tuberculous pleurisy.

[0025] In a third aspect, the present application provides a kit for differential diagnosis of recent and long-term tuberculosis infection, wherein the kit comprises a reagent for detecting the content of CCL3, TNFα and CCL20 in a biological sample of a patient; when the concentration of the biomarker protein is lower than the critical value, it indicates recent tuberculosis infection.

[0026] Furthermore, when the concentration of TNFα protein in the subject's biological sample was below 93.37 pg / ml, it indicated that the subject had recently been infected with pulmonary tuberculosis.

[0027] Furthermore, when the concentration of CCL20 protein in the subject's biological sample was below 149.55 pg / ml, it indicated that the subject had recently been infected with pulmonary tuberculosis.

[0028] Furthermore, when the concentration of CCL3 protein in the subject's biological sample was below 170.65 pg / ml, it indicated that the subject had recently been infected with pulmonary tuberculosis.

[0029] Furthermore, the biological sample is derived from the patient's whole blood, serum, or plasma.

[0030] Preferably, the biological sample is derived from the patient's plasma.

[0031] Furthermore, the tuberculosis is an infection caused by the pathogen Mycobacterium tuberculosis.

[0032] Furthermore, the Mycobacterium tuberculosis infection includes primary infection, secondary infection, and extrapulmonary infection.

[0033] Furthermore, the pulmonary tuberculosis includes primary pulmonary tuberculosis, hematogenous disseminated pulmonary tuberculosis, secondary pulmonary tuberculosis, tracheal or bronchial tuberculosis, and tuberculous pleurisy.

[0034] Furthermore, the kit may be one or more of the following: ELISA detection kit, colloidal gold detection kit, immunohistochemistry kit, immunofluorescence kit, and / or in situ hybridization staining kit.

[0035] Furthermore, the diagnostic methods of the kit include one or more of the following: direct method, indirect method, double-antibody sandwich method, and / or competitive method.

[0036] Beneficial effects

[0037] 1. This invention is the first to use TNF-α, CCL3, and CCL20 as differential diagnostic markers for different stages of pulmonary tuberculosis infection, distinguishing between recent and long-term pulmonary tuberculosis infection. Both alone and in combination, they exhibit good diagnostic efficacy, with AUC values ​​greater than or close to 0.8. Among them, TNF-α, when used alone as a diagnostic marker, shows a specificity as high as 100%.

[0038] 2. This invention applies proteomics technology to detect differentially expressed protein biomarkers related to pulmonary tuberculosis. It has high sensitivity and specificity, is suitable for the detection of pulmonary tuberculosis, and provides high accuracy in the detection results, which helps to improve detection efficiency.

[0039] 3、The application of the differential protein markers related to pulmonary tuberculosis to the detection related to pulmonary tuberculosis has the advantages of simple sample processing, easy acquisition, small sample amount, good repeatability, etc. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 CCL3 protein expression levels in the three groups of people.

[0041] Figure 2 CCL20 protein expression levels in the three groups of people.

[0042] Figure 3 TNF-α protein expression levels in the three groups of people.

[0043] Figure 4 CCL3 protein expression levels in the recent pulmonary tuberculosis infection group and the remote pulmonary tuberculosis infection group.

[0044] Figure 5 CCL20 protein expression levels in the recent pulmonary tuberculosis infection group and the remote pulmonary tuberculosis infection group.

[0045] Figure 6 TNF-α protein expression levels in the recent pulmonary tuberculosis infection group and the remote pulmonary tuberculosis infection group.

[0046] Figure 7 ROC analysis results in the screening test.

[0047] Figure 8 Differences in TNF-α, CCL3 and CCL20 protein expression between the recent pulmonary tuberculosis infection group and the remote pulmonary tuberculosis infection group in the verification test.

[0048] Figure 9 ROC analysis results in the verification test. DETAILED DESCRIPTION

[0049] The specific embodiments of the application will be further described below. It should be noted that the description of the embodiments is used to help understand the application, but does not constitute a limitation on the application. In addition, the technical features involved in the following described embodiments can be combined with each other as long as they do not conflict with each other.

[0050] The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all commercially available unless otherwise specified.

[0051] Example 1 Screening of differentially expressed proteins between recent pulmonary tuberculosis infection and remote pulmonary tuberculosis infection based on proteomics technology 1.1 Research object and grouping

[0052] Inclusion criteria: age 30-60 years, recent TB infection, long-term TB infection, healthy controls from Beijing Chest Hospital, Capital Medical University. The specific grouping is as follows:

[0053] (1) Recent TB infection group (15 people): IGRA (γ-interferon release test) result is negative at baseline, 12 months later, or IGRA is negative 12 months ago, 24 months later, individuals who are defined as recent infection. For these individuals, the first positive sample after the negative sample is used for analysis.

[0054] (2) Long-term TB infection group (15 people): doctors and nurses working in TB hospitals for at least 2 years, IGRA positive at baseline and 24 months, indicating infection for at least 2 years.

[0055] (3) Healthy control group (15 people): no history of active TB, no clinical symptoms, normal IGRA test.

[0056] 1.2 Blood collection, preservation and IGRA test

[0057] Anticoagulated venous blood was collected from all study subjects and divided into "N", "T" and "P" culture tubes. "N" tube contains Tris, "T" tube contains Mycobacterium tuberculosis specific antigen, and "P" tube contains phytohemagglutinin (PHA). After mixing and incubating, centrifugation was performed, and the supernatant was collected and divided into new centrifuge tubes for ELISA detection and proteomics detection. The reagents for IGRA were purchased from Red Biopharm Company and strictly followed the manufacturer's instructions.

[0058] 1.3 Screening of differential proteins by proteomics technology

[0059] (1) Data preprocessing: According to the manufacturer's instructions, Olink Proteomics AB, Uppsala, Sweden was used to quantify protein levels. Olink panel is based on proximity extension assay (PEA) technology. PEA allows simultaneous analysis of 92 analytes. In short, oligonucleotide-labeled antibody probes are allowed to bind to target proteins in the sample. When two antibodies are closely bound, DNA oligonucleotides hybridize and extend by DNA polymerization to form a polymerase chain reaction (PCR) reporter sequence. Microfluidic real-time PCR instrument is used for detection, amplification and quantification of sequences. Protein abundance is reported as normalized protein expression (NPX), which is an arbitrary unit in Log2 scale. High NPX value indicates high protein concentration. However, NPX values cannot be compared between different proteins.

[0060] (2) Quality control analysis: Data quality control (QC) was performed in two steps: First, the standard deviation of each run for detection control, incubation control, and both was determined. Only runs with a standard deviation of less than 0.2 for each control passed quality control. Second, each sample was quality checked using both detection control and incubation control 2. All samples in a run were compared to the calculated run median for each control. If a sample deviated from the plate median of both control groups by more than 0.3 NPX, it failed QC and a QC warning was issued in the data output file. All samples were measured successfully.

[0061] 1.4 Statistical analysis

[0062] The experimental data was expressed as mean ± standard deviation. The normality analysis was determined by skewness coefficient, kurtosis coefficient and Shapiro-Wilk test. For data conforming to normal distribution, single factor analysis of variance was used for comparison, and Tukey test was used for comparison between two groups and more data. For non-normal distribution data, Nemenyi test was used for comparison between two groups of data and Kruskal-Wallis test. P<0.05 indicated statistically significant difference. Thus, potential differential protein markers were screened.

[0063] 1.5 Results

[0064] 1.5.1 Screening of differentially expressed proteins

[0065] By comparing the protein expression levels of the three groups of people, namely, recent tuberculosis infection, long-term tuberculosis infection, and healthy controls, the significantly differentially expressed markers were screened, and the distribution of the three groups of people on the proteomics data was significantly different. Further analysis selected the most significant differentially expressed markers (TNF-a, CCL3, CCL20), and through graphpad software, one-way ANOVA was used to determine the statistical significance between the three groups (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0066] Figure 1 It is shown that the protein expression levels of TNF-a, CCL3, and CCL20 in the three groups of people are significantly different. Among them, CCL3, TNF-a, and CCL20 are all lowly expressed in the recent tuberculosis infection group compared with the other two groups.

[0067] Figure 2 It is shown that the three differentially expressed proteins have significant differences between the recent tuberculosis infection and long-term tuberculosis infection groups. Among them, TNF-a, CCL3, and CCL20 are highly expressed in the long-term tuberculosis infection group and lowly expressed in the recent tuberculosis infection group.

[0068] 1.5.2 Diagnostic performance analysis of differentially expressed markers

[0069] For the screened differential proteins, SPSS software was used for receiver operating curve (ROC) analysis based on logistic model.

[0070] The results showed that, in the 95% confidence interval, the AUC of the diagnosis of 3 protein biomarkers alone or in combination was between 0.800-0.929, as shown in Table 1. It was shown that the 3 differential protein markers screened had high accuracy in differentiating recent and long-term tuberculosis infection, and had important significance for the diagnosis of tuberculosis.

[0071] Table 1 ROC data of differential proteins

[0072]

[0073]

[0074] Example 2 Verification of CCL3, CCL20 and TNF-α expression levels in plasma of patients with different infection states of pulmonary tuberculosis

[0075] Patients aged 30-80 years old with recent infection of pulmonary tuberculosis and long-term infection of pulmonary tuberculosis treated in Beijing Chest Hospital, Capital Medical University were included. The specific grouping was as follows:

[0076] (4) Pulmonary tuberculosis recent infection group (33 people): IGRA result was negative at baseline, and turned positive after 12 months, or IGRA was negative 12 months ago, and turned positive after 24 months. Individuals were defined as recent infection. For these individuals, the first positive sample after the negative sample was used for analysis.

[0077] (5) Pulmonary tuberculosis long-term infection group (45 people): doctors and nurses and other staff working in tuberculosis hospital for at least 2 years, individuals with positive IGRA at baseline and positive for 24 months, indicating infection for at least 2 years.

[0078] 2.2 Blood collection, preservation and IGRA test

[0079] Collect all the study objects lithium heparin anticoagulation of venous blood, divided into "N" "T" "P" three kinds of culture tube, wherein "N" tube contains Tris, "T" tube contains Mycobacterium tuberculosis specific antigen, "P" tube contains phytohemagglutinin (PHA), after mixing, centrifugation, collection of supernatant, take 3 tube supernatant each into new centrifuge tube, for ELISA detection and proteomics detection, frozen to use. IGRA related reagents are purchased from Red Biopharm Co., Ltd., and the operation steps are strictly in accordance with the instructions.

[0080] 2.3 Plasma sample analysis

[0081] The above collected plasma samples were analyzed by Luminex using Bio-Rad's commercially available typing combination (TNF-α, CCL3, CCL20) to identify differentially expressed markers related to recent infection and long-term infection of tuberculosis. According to the manufacturer's instructions, the specific operation steps are as follows:

[0082] 2.3.1 Sample preparation and standard preparation

[0083] Plasma samples: centrifugation, take supernatant, dilute with Sample Diluent, finally take diluted sample for detection.

[0084] 2.3.2 Standard preparation

[0085] Add Standard Diluent to the standard bottle, vortex, ice bath, then dilute the standard curve S1-S8.

[0086] 2.3.3 Sample incubation

[0087] Take the microbeads and shake them, dilute the microbeads with Assay Buffer;

[0088] Diluted microbeads are shaken again and added to the well plate and washed.

[0089] Take the prepared standard, sample, reference and blank and add them to the well plate, seal with sealing film, shake, avoid light, incubate at room temperature.

[0090] 2.3.4 Incubation of detection antibody

[0091] Discard the sample and wash it;

[0092] Dilute the Detection Antibody with Antibody Diluent according to the instructions;

[0093] Add the diluted Detection Antibody to each well, seal with sealing film, shake, avoid light, incubate at room temperature.

[0094] 2.3.5 Color Development

[0095] Discard the antibody test result and wash.

[0096] Dilute Streptavidin-PE using Assay Buffer as per the instructions.

[0097] Add diluted Streptavidin-PE to each well, seal with film, shake, protect from light, incubate at room temperature;

[0098] washing;

[0099] Add Assay Buffer to each well to resuspend, cover with sealing film, incubate at room temperature, away from light, and shake.

[0100] The value is read from the calibrated Luminex 200 machine.

[0101] 2.3.6 Data Analysis

[0102] The fluorescence of the samples and standards tested in this experiment was automatically calculated and optimized by software after being detected by the Luminex 200 detector.

[0103] 2.4 Data Analysis

[0104] Statistical analysis was performed using GraphPad Prism and MedCalc-version software. For normally distributed data, t-tests were used for comparison; for non-normally distributed data, the Mann-Whitney test was used to compare the two groups. Statistical significance was determined by P < 0.05, indicating a statistically significant difference. This process was used to screen for potential differentially expressed protein biomarkers and to validate biomarkers for the diagnosis of recent and long-term pulmonary tuberculosis infections.

[0105] 2.5 Results

[0106] like Figure 4 As shown, there were significant differences in plasma expression levels of CCL3, CCL20, and TNF-α between the group of patients recently infected with pulmonary tuberculosis and the group of patients infected in the long term with pulmonary tuberculosis.

[0107] Further ROC-based comparisons were used to validate the diagnostic efficacy of CCL3, CCL20, and TNF-α in patients with recent and long-term pulmonary tuberculosis infection. Figure 5The AUC value of CCL3 was 0.859 (95% confidence interval, 0.760-0.928; sensitivity, 86.36%; specificity, 75.00%); the AUC value of CCL20 was 0.783 (95% confidence interval, 0.674-0.870; sensitivity, 68.18%; specificity, 93.75%); and the AUC value of TNF-α was 0.851 (95% confidence interval, 0.751-0.922; sensitivity, 56.82%; specificity, 100.00%).

[0108] The diagnostic efficiency of the combination of CCL3, CCL20 and TNF-α was evaluated. A combination diagnostic model was constructed by multivariate logistic regression analysis, and a ROC curve of the combination diagnosis was drawn. The AUC values of each combination are shown in Table 2, in which the diagnostic efficiency of CCL3+CCL20 in the combination marker was the largest, with an AUC value of 0.863. The results verified the diagnostic efficiency of the marker combination in expanding the clinical sample, and determined the feasibility of its application in actual clinical diagnosis. When the concentration of TNFα protein was lower than 93.37 pg / ml, it was a recent infection of pulmonary tuberculosis; when the concentration of CCL20 protein was lower than 149.55 pg / ml, it was a recent infection of pulmonary tuberculosis; and when the concentration of CCL3 protein was lower than 170.65 pg / ml, it was a recent infection of pulmonary tuberculosis.

[0109] Table 2 ROC data of differential proteins

[0110]

Claims

1. Use of a reagent for detecting a biomarker, which is one or more of CCL3, TNF alpha and / or CCL20, in the manufacture of a kit for differentiating between recent and remote tuberculosis infection, the reagent being a reagent for detecting the level of CCL3, TNF alpha and CCL20 in the plasma of a patient.

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