Biomarker for differential diagnosis of active pulmonary tuberculosis

Abstract

Provided in the present invention is a biomarker for the differential diagnosis of active pulmonary tuberculosis, wherein the biomarker is a TRANCE protein, and has an AUC value of >0.8 for the differential diagnosis of patients with active pulmonary tuberculosis, indicating that the biomarker screened in the present invention has a relatively high diagnostic efficacy on active pulmonary tuberculosis.

Description

A biomarker for the differential diagnosis of active pulmonary tuberculosis Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a biomarker for the identification and diagnosis of active pulmonary tuberculosis. Background Technology

[0002] Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis, primarily characterized by respiratory symptoms. In the early stages of tuberculosis infection, most people's immune defense mechanisms play a protective role. However, as an intracellular parasite, Mycobacterium tuberculosis can escape macrophage killing through complex immune escape mechanisms. These mechanisms include altering the way macrophages phagocytose pathogens, inhibiting the acidification of phagosomes and the formation of phagolysosomes, inhibiting macrophage apoptosis, reducing the toxic effects of reactive oxygen and nitrogen products, and affecting the antigen presentation function of macrophages. This allows it to remain in the cell for a long time, forming latent tuberculosis infection (LTBI) (Chen Yanqing, Zhang Linbo. Research progress on the molecular mechanism of immune escape of Mycobacterium tuberculosis [J]. Journal of Immunology, 2014, 30(1): 84-88.). Because LTBI patients usually do not have the clinical symptoms of active tuberculosis infection, it is often overlooked and is generally regarded as a disease state that does not pose a threat of infection. However, according to WHO data, 5%-10% of people with latent tuberculosis infection can develop into active tuberculosis (ATBI) (World Health Organization. Global tuberculosis control: WHO (World Health Organization) report 2019 [R]. Accessed 17 October 2019.). Especially in recent years, the increased incidence of diseases such as AIDS, cancer, and diabetes has led to a decline in the body's immunity, further increasing the risk of LTBI patients developing active pulmonary tuberculosis. Therefore, the diagnosis and regular monitoring of patients with latent tuberculosis infection are crucial for effectively reducing the incidence of tuberculosis and controlling its development and spread (Targeted tuberculin testing and treatment of latent tuberculosis infection [J]. American Thoracic Society. MMWR Recommendations Reports, 2000, 49(RR-6): 151.). However, because the bacteria in LTBI patients are essentially dormant in the body, the diagnosis of latent tuberculosis remains quite difficult.

[0003] Currently, laboratory testing methods for tuberculosis infection mainly include sputum smear for acid-fast bacilli, isolation and culture, Mycobacterium tuberculosis nucleic acid detection, T-SPOT, and the Interferon-Gamma Release Assay (IGRA). Among these, sputum smear for acid-fast bacilli not only has a low positive rate but also cannot differentiate between Mycobacterium tuberculosis and non-tuberculous mycobacteria; while isolation and culture is the gold standard for diagnosing pulmonary tuberculosis, the long culture period hinders timely clinical diagnosis and treatment, and the results are easily affected by the number and viability of Mycobacterium tuberculosis, resulting in low specificity; Mycobacterium tuberculosis nucleic acid detection has high specificity and sensitivity, but it requires sophisticated samples, experimental conditions, and techniques; T-SPOT and IGRA can compensate for these shortcomings to some extent, possessing high sensitivity and specificity, but they cannot distinguish between active and non-active Mycobacterium tuberculosis, and diagnosing different stages of tuberculosis remains very difficult.

[0004] Tumor necrosis factor-associated activator-inducing factor (TRANCE), also known as tumor necrosis factor ligand superfamily member 11 (TNFSF11) or nuclear factor κB receptor activator ligand (RANKL), is a member of the TNF superfamily. It is involved in osteoblasts and CD4+ cell proliferation. + and CD8 + TRANCE can be expressed by T cells, megakaryocytes, bronchial and intestinal epithelial cells, and other cells. Further investigation into whether TRANCE protein is an effective biomarker for diagnosing active and latent pulmonary tuberculosis is well-suited to current clinical testing needs. Summary of the Invention

[0005] This invention has identified a biomarker for the differential diagnosis of active pulmonary tuberculosis. This biomarker exhibits significant changes in the patient's plasma and demonstrates strong diagnostic efficacy for active pulmonary tuberculosis. Based on this, this invention was completed.

[0006] In a first aspect, the present invention provides a biomarker for the differential diagnosis of active pulmonary tuberculosis, wherein the biomarker is TRANCE protein.

[0007] Furthermore, the biomarkers are derived from one or more of the patient's serum, plasma, blood, pleural effusion, and / or cerebrospinal fluid.

[0008] Furthermore, when the concentration of TRANCE protein in serum is ≤4.533 pg / ml, the subject is identified as an active pulmonary tuberculosis patient; when the concentration of TRANCE protein in serum is greater than 4.533 pg / ml and less than 5.391 pg / ml, the subject is identified as a latent pulmonary tuberculosis patient.

[0009] Furthermore, the pulmonary tuberculosis is an infection caused by Mycobacterium tuberculosis.

[0010] Furthermore, the pulmonary tuberculosis mentioned includes primary pulmonary tuberculosis, secondary pulmonary tuberculosis, hematogenous disseminated pulmonary tuberculosis, tracheobronchial tuberculosis, tuberculous pleurisy, and sputum-negative pulmonary tuberculosis.

[0011] Furthermore, the Mycobacterium tuberculosis infection includes: primary infection, secondary infection, and extrapulmonary infection.

[0012] Secondly, the present invention provides the application of a biomarker in the preparation of a reagent for the differential diagnosis of active pulmonary tuberculosis, wherein the biomarker is TRANCE protein, and the reagent is a reagent containing a substance capable of detecting the TRANCE protein content in a patient's biological sample.

[0013] Furthermore, the biological sample is one or more of the following: patient serum, plasma, blood, pleural effusion, and / or cerebrospinal fluid.

[0014] Furthermore, when the concentration of TRANCE protein in serum is ≤4.533 pg / ml, the subject is identified as an active pulmonary tuberculosis patient; when the concentration of TRANCE protein in serum is greater than 4.533 pg / ml and less than 5.391 pg / ml, the subject is identified as a latent pulmonary tuberculosis patient.

[0015] Thirdly, the present invention provides a kit for the differential diagnosis of active pulmonary tuberculosis, the kit comprising the reagents described in the second invention of the present invention.

[0016] 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.

[0017] 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. Beneficial effects

[0018] The TRANCE protein, a biomarker obtained by screening in this invention, can significantly diagnose patients with active pulmonary tuberculosis. The AUC value for differentiating and diagnosing patients with active pulmonary tuberculosis is >0.8, indicating that the biomarker obtained by screening in this invention has high diagnostic efficacy for active pulmonary tuberculosis. Attached Figure Description

[0019] Figure 1 shows the comparison of TRANCE scores among the healthy group, the latent tuberculosis group, and the active tuberculosis group, and their ROC curve analysis.

[0020] Figure 2 shows the comparison of TRANCE screening results between the latent pulmonary tuberculosis group and the active tuberculosis group, and its ROC curve analysis.

[0021] Figure 3 shows the comparison of TRANCE in the validation set among the healthy group, the latent tuberculosis group, and the active tuberculosis group, and its ROC curve analysis.

[0022] Figure 4. Comparison of TRANCE in the validation set between the latent pulmonary tuberculosis group and the active tuberculosis group and its ROC curve analysis. Detailed Implementation

[0023] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0024] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0025] Example 1: Screening of diagnostic markers for active and latent pulmonary tuberculosis

[0026] 1. Study subjects: 48 patients with active tuberculosis (TB), 20 patients with latent tuberculosis infection (LTBI), and 20 healthy individuals (HC group).

[0027] (1) Healthy controls: aged 18-65 years; no clinical symptoms of tuberculosis; negative gamma-interferon release test; no lung lesions found on chest X-ray or CT; no other serious medical history.

[0028] (2) Latent tuberculosis infection: no clinical symptoms of tuberculosis; positive gamma-interferon release test; no history of tuberculosis treatment.

[0029] (3) Patients with active tuberculosis: aged 18 years or older; with clinical symptoms of tuberculosis; and lung lesions found on chest X-ray or CT scan. Patients with active pulmonary tuberculosis included 12 patients with seropositivity pulmonary tuberculosis and 28 patients with seropositivity pulmonary tuberculosis.

[0030] 2. Blood sample collection and storage: Venous blood was collected from the subjects, and plasma was separated and stored for later use.

[0031] 3. Olink proteomics technology detection

[0032] (1) Protein sample preparation

[0033] (2) Incubation

[0034] A. Prepare the incubation mix (400ul): Target 96 Incubation Solution、 Target 96 Incubation Stabilizer, Target 96 A-probes and Target 96 B-probes;

[0035] B. Mix the incubation mixture thoroughly, add it to the incubation plate, and set up control wells and sample quality control wells.

[0036] (3) Extension

[0037] A. Prepare the extension mix: High Purity Water, Target 96 PEA Solution Target 96 PEA Enzyme and Target 96 PCR Polymerase;

[0038] B. Centrifuge the incubation plate and add the mixed amplification mixture to the well plate;

[0039] C. Perform PCR amplification.

[0040] (4) Detection

[0041] A. Injection of 96.96 Dynamic Array TM Integrated fluid circuit (IFC);

[0042] B. Prepare the detection mix: Target 96 Detection Solution, High Purity Water, Target 96 Detection Enzyme and Target 96 PCR Polymerase;

[0043] C. Add the mixed test mixture to the sample plate;

[0044] D. Remove the incubation plate from the PRC instrument and transfer the solution in the wells to the sample plate;

[0045] E. Transfer the solutions from the primer plate and sample plate into the wells of the Dynamic Array IFC;

[0046] F. Place the chip into Q100 and run the program.

[0047] (5) The Q100 files generated by Q100 (qPCR) detection were preprocessed using Olink NPX Signature software.

[0048] (6) Obtain the input file NPX Data required for the bioinformatics analysis process and standardize the NPX data.

[0049] 4. Verification using the double-antibody sandwich method

[0050] TRANCE capture antibody is coated onto an ELISA plate to capture the target in samples and standards. Biotin-labeled antibody is added to bind to the target, followed by the addition of HRP-conjugate to bind to the biotin-antibody, forming an immune complex. Free components are washed away during the detection process. TMB chromogenic solution is added, and under the catalysis of HRP, the reaction wells turn blue if the target is present. Stop solution is added, and the solution turns yellow under acidic conditions. The OD value is measured at 450 nm using an ELISA reader. The color intensity is directly proportional to the TRANCE concentration in the sample. The concentration of the target in the sample is calculated by plotting a standard curve.

[0051] 5. Experimental Results

[0052] 5.1. Differentially expressed protein analysis:

[0053] In a comparative study of three groups—healthy patients, latent infection group, and patients with active pulmonary tuberculosis—47 differentially expressed proteins were identified, including: IL8, MCP-3, CDCP1, CD244, IL7, LAP, TGF-beta-1, IL6, CXCL11, AXIN1, TRAIL, CXCL9, CST5, OSM, CXCL1, CCL4, CD6, SCF, IL18, TGF-alpha, MCP-4, CCL19, PD-L1, Beta-NGF, CXCL5, TRANCE, HGF, IL-24, CCL23, CCL3, Flt3L, CXCL6, CXCL10, 4E-BP1, SIRT2, EN-RAGE, FGF-19, LIF, CASP-8, TNFRSF9, NT-3, TWEAK, ST1A1, STAMBP, CSF-1, DNER, IFN-gamma, and TNF.

[0054] Through further statistical analysis, TRANCE was identified as a significantly differentially expressed biomarker for diagnosing active tuberculosis and latent pulmonary tuberculosis in the healthy group, active tuberculosis group, and latent tuberculosis group.

[0055] When comparing the active tuberculosis group, the latent pulmonary tuberculosis group, and the healthy group, the concentration of TRANCE protein in the serum of patients with active tuberculosis was significantly lower than that in the healthy group and the latent pulmonary tuberculosis group (P < 0.0001), indicating that the concentration of TRANCE protein can be used to diagnose active pulmonary tuberculosis (Figure 1).

[0056] 5.2. ROC analysis of TRANCE protein concentration in serum of TB pulmonary tuberculosis and healthy groups

[0057] ROC curves were plotted based on the concentrations of TRANCE in serum samples from the healthy group, the latent infection group, and the active pulmonary tuberculosis group. The results are shown in Figure 1. The area under the ROC curve was 0.8059, indicating that TRANCE concentration has significant value in the differential diagnosis of active pulmonary tuberculosis.

[0058] 5.3. ROC analysis of TRANCE protein concentration in serum of patients with active pulmonary tuberculosis and latent tuberculosis.

[0059] As shown in Figure 2, the concentration of TRANCE protein in the serum of patients with active pulmonary tuberculosis was significantly lower in the latent infection group than in the latent infection group (P < 0.0001), indicating that the concentration of TRANCE protein can be used to diagnose different stages of pulmonary tuberculosis: the latent infection stage and the active pulmonary tuberculosis stage. ROC curves were plotted based on the concentrations of TRANCE in the serum of patients with active tuberculosis and those with latent pulmonary tuberculosis. The area under the ROC curve was 0.8250, the sensitivity was 75%, and the specificity was 80%, indicating that the concentration of TRANCE has important value in the differential diagnosis of active tuberculosis and latent pulmonary tuberculosis.

[0060] 5.4. Result Interpretation in Practical Applications of TRANCE Protein

[0061] If the concentration of TRANCE protein in the serum of the test subject is lower than 5.391 pg / ml (sensitivity 100%, confidence interval 86.20%–100.00%; specificity 10.00%, confidence interval 51.29%–40.42%), the test subject has or is suspected of having active pulmonary tuberculosis; if the concentration of TRANCE protein in the serum of the test subject is equal to or greater than 5.391 pg / ml, the test subject does not have or is suspected of not having active tuberculosis.

[0062] Example 2: Validation of diagnostic markers for active and latent pulmonary tuberculosis

[0063] 1. Study subjects: 48 patients with active tuberculosis (TB), 20 patients with latent tuberculosis infection (LTBI), and 20 healthy individuals (HC group).

[0064] (4) Healthy controls: aged 18-65 years; no clinical symptoms of tuberculosis; negative gamma-interferon release test; no lung lesions found on chest X-ray or CT; no other serious medical history.

[0065] (5) Latent tuberculosis infection: no clinical symptoms of tuberculosis; positive gamma-interferon release test; no history of tuberculosis treatment.

[0066] (6) Patients with active tuberculosis: aged 18 years or older; with clinical symptoms of tuberculosis; and lung lesions found on chest X-ray or CT scan. Patients with active pulmonary tuberculosis included 12 patients with seropositivity pulmonary tuberculosis and 28 patients with seropositivity pulmonary tuberculosis.

[0067] 2. Blood sample collection and storage: 5 ml of venous blood was collected from all eligible subjects using EDTA anticoagulant blood collection tubes, and the plasma was separated and stored at -80℃ for later use.

[0068] 3. Olink proteomics technology detection

[0069] (1) Protein sample preparation

[0070] (2) Incubation

[0071] A. Prepare the incubation mix (400ul): Target 96 Incubation Solution、 Target 96 Incubation Stabilizer, Target 96 A-probes and Target 96 B-probes;

[0072] B. Mix the incubation mixture thoroughly, add it to the incubation plate, and set up control wells and sample quality control wells.

[0073] (3) Extension

[0074] A. Prepare the extension mix: High Purity Water, Target 96 PEA Solution Target 96 PEA Enzyme and Target 96 PCR Polymerase;

[0075] B. Centrifuge the incubation plate and add the mixed amplification mixture to the well plate;

[0076] C. Perform PCR amplification.

[0077] (4) Detection

[0078] A. Injection of 96.96 Dynamic Array TM Integrated fluid circuit (IFC);

[0079] B. Prepare the detection mix: Target 96 Detection Solution, High Purity Water, Target 96 Detection Enzyme and Target 96 PCR Polymerase;

[0080] C. Add the mixed test mixture to the sample plate;

[0081] D. Remove the incubation plate from the PRC instrument and transfer the solution in the wells to the sample plate;

[0082] E. Transfer the solutions from the primer plate and sample plate into the wells of the Dynamic Array IFC;

[0083] F. Place the chip into Q100 and run the program.

[0084] (5) The Q100 files generated by Q100 (qPCR) detection were preprocessed using Olink NPX Signature software.

[0085] (6) Obtain the input file NPX Data required for the bioinformatics analysis process and standardize the NPX data.

[0086] 4. Verification using the double-antibody sandwich method

[0087] TRANCE capture antibody is coated onto an ELISA plate to capture the target in samples and standards. Biotin-labeled antibody is added to bind to the target, followed by the addition of HRP-conjugate to bind to the biotin-antibody, forming an immune complex. Free components are washed away during the detection process. TMB chromogenic solution is added, and under the catalysis of HRP, the reaction wells turn blue if the target is present. Stop solution is added, and the solution turns yellow under acidic conditions. The OD value is measured at 450 nm using an ELISA reader. The color intensity is directly proportional to the TRANCE concentration in the sample. The concentration of the target in the sample is calculated by plotting a standard curve.

[0088] 5. Experimental Results

[0089] 5.1 Differentially expressed protein analysis

[0090] The concentration of TRANCE protein in the serum of patients with active tuberculosis was significantly lower than that in the healthy group and the group with latent tuberculosis (P < 0.0001) (Figure 3).

[0091] 5.2. ROC analysis of TRANCE protein concentration in serum of TB pulmonary tuberculosis and healthy groups

[0092] ROC curves were plotted based on the concentrations of TRANCE in serum samples from the healthy group, the latent infection group, and the active pulmonary tuberculosis group. The results are shown in Figure 3. The area under the ROC curve in the validation set was 0.7256, indicating that TRANCE concentration has significant value in the differential diagnosis of active pulmonary tuberculosis.

[0093] 5.3. ROC analysis of TRANCE protein concentration in serum of patients with active pulmonary tuberculosis and latent tuberculosis.

[0094] As shown in Figure 4, the concentration of TRANCE protein in the serum of patients with active pulmonary tuberculosis was significantly lower in the latent infection group than in the latent infection group (P < 0.0001), indicating that the concentration of TRANCE protein can be used to diagnose different stages of pulmonary tuberculosis: the latent infection stage and the active pulmonary tuberculosis stage. ROC curves were plotted based on the concentrations of TRANCE in the serum of patients with active tuberculosis and latent pulmonary tuberculosis. In the validation group, the area under the ROC curve was 0.8750, the sensitivity was 95.83%, and the specificity was 80%, indicating that the concentration of TRANCE has important value in the differential diagnosis of active tuberculosis and latent pulmonary tuberculosis.

[0095] 5.4 Interpretation of results in practical applications of TRANCE protein

[0096] If the concentration of TRANCE protein in the serum of the test subject is lower than 4.533 pg / ml (sensitivity 95.83%, confidence interval 79.76%–99.79%; specificity 80.00%, confidence interval 49.02%–96.45%), the test subject has or is suspected of having active pulmonary tuberculosis; if the concentration of TRANCE protein in the serum of the test subject is equal to or greater than 4.533 pg / ml, but less than or equal to 5.391 pg / ml, the test subject has latent pulmonary tuberculosis.

[0097] The results verified the diagnostic efficacy of the biomarker composition of the present invention for diagnosing active tuberculosis and confirmed its feasibility for application in actual clinical diagnosis.

Claims

1. The application of a biomarker in the preparation of a reagent for the differential diagnosis of active pulmonary tuberculosis, characterized in that, The biomarker is TRANCE protein, and the reagent is a reagent containing TRANCE protein content in patient serum samples. When the concentration of TRANCE protein in serum is ≤4.533 pg / ml, the subject is judged to be an active pulmonary tuberculosis patient; when the concentration of TRANCE protein in serum is greater than 4.533 pg / ml and less than 5.391 pg / ml, the subject is judged to be a latent pulmonary tuberculosis patient.

2. The application of a biomarker in the preparation of a kit for the differential diagnosis of active pulmonary tuberculosis, characterized in that, The biomarker is TRANCE protein, and the kit is a kit for detecting the TRANCE protein content in a patient's serum sample. When the concentration of TRANCE protein in serum is ≤4.533 pg / ml, the subject is determined to be a patient with active pulmonary tuberculosis; when the concentration of TRANCE protein in serum is greater than 4.533 pg / ml and less than 5.391 pg / ml, the subject is determined to be a patient with latent pulmonary tuberculosis.

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