Biomarker for differential diagnosis and prognosis of severe tuberculosis and application of biomarker

Abstract

The invention belongs to the technical field of biology, and particularly relates to a biomarker for differential diagnosis and prognosis of severe tuberculosis and application of the biomarker. According to the invention, the expression level of sCD27 in patients with severe pulmonary tuberculosis is obviously increased; when sCD27 in a biological sample of a subject is greater than or equal to 95097 pg / ml, the subject is diagnosed as severe pulmonary tuberculosis; the area under the curve is 0.7674 (95% CI is equal to 0.6158 to 0.9190); the sensitivity is 45.45%, and the specificity is 88.24%. When the sCD27 level is higher than 158510pg / ml, the prognosis is poor, and a subject has a death risk; the area under the curve is 0.7465 (95% CI is equal to 0.5756 to 0.9175); the sensitivity is 57.14%, and the specificity is 83.87%. The sCD27 as a biomarker for distinguishing severe and non-severe pulmonary tuberculosis patients and predicting prognosis effects of the pulmonary tuberculosis patients has relatively high accuracy and specificity, and is helpful for determining clinical diagnosis and treatment schemes.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a biomarker for differential diagnosis of severe tuberculosis and prognosis and an application thereof. Background Art

[0002] Tuberculosis is still one of the infectious diseases with the highest mortality rate in the world. Tuberculosis requires at least 6 months of multi-drug combination therapy. Literature reports that the mortality rate of patients with pulmonary tuberculosis depends on the severity of the disease. Although most people infected with Mycobacterium tuberculosis are asymptomatic (latent tuberculosis infection, TBI), or have mild and moderate tuberculosis, some tuberculosis patients will develop severe disease. In patients with severe tuberculosis, fibrosis, cavitation and necrosis can cause long-term and irreversible damage to lung tissue and function, which may lead to respiratory failure and death. Early warning, timely diagnosis and targeted treatment of patients with severe pulmonary tuberculosis directly affect the mortality rate of patients. Domestic and foreign research on TB mainly focuses on disease susceptibility, early diagnosis, differential diagnosis and other aspects. There are few studies on the progression of pulmonary tuberculosis. Early judgment of the patient's condition and the formulation of effective prevention and control measures are of clinical significance in reducing the mortality rate of patients.

[0003] After Mycobacterium tuberculosis infects the host, it can stimulate T cells to proliferate and differentiate into effector T cells, and some T cell subsets differentiate into memory T cells. Memory T cells can survive for a long time and respond quickly to secondary immunization (Doherty M, Wallis RS, Zumla A. Biomarkers for tuberculosis disease status and diagnosis. Curr Opin Pulm Med. 2009; 15: 181–187.). Anti-tuberculosis protective immunity depends on CD4 + Cells. CD4 + The reduction in the number and dysfunction of T cells is the main mechanism of AIDS, and AIDS patients are highly susceptible to tuberculosis, which proves that CD4 + T cell immunity plays a key role in anti-tuberculosis immunity. Although the resistance of CD4 gene-deficient mice is significantly reduced in the early stage of Mycobacterium tuberculosis infection, their anti-tuberculosis immunity in the middle and late stages is similar to that of normal mice.

[0004] CD27 is a member of the tumor necrosis factor receptor family and plays an important role in the differentiation and function of effector T lymphocytes and memory T cells (Nolte MA, van Olffen RW, van Gisbergen KP, van Lier RA. Timing and tuning of CD27–CD70 interactions: the impact of signal strength in setting the balance between adaptive responses and immunopathology. Immunol Rev. 2009; 229: 216–231.). The development of memory T cells also depends on the CD27 molecule. In CD27 knockout mice, the secondary immune response is significantly reduced.

[0005] CD27 molecules also exist in a soluble form, namely sCD27, which may be released into body fluids through cell surface shedding or protease cleavage. After T cells are activated by the TCR / CD3 complex, not only is the expression of CD27 on the membrane surface enhanced, but sCD27 with a molecular weight of 28-32KD is also released. The release of sCD27 is limited to freshly isolated CD27 + T cells, sCD27 is also present in body fluids. Increased serum sCD27 is seen in the state of immune activation in the body. The level of sCD27 may change under certain pathological conditions. Therefore, the change in the level of sCD27 can reflect the pathogenesis and progression of various diseases. So far, there are no reports on the relationship between sCD27 levels and patients with severe pulmonary tuberculosis and the effect of predicting prognosis. Summary of the invention

[0006] The present invention found that the expression level of sCD27 increased significantly in patients with severe pulmonary tuberculosis; when sCD27 in the biological sample of the subject was ≥95097pg / ml, the subject was diagnosed with severe pulmonary tuberculosis; when the sCD27 level was higher than 158510pg / ml, the prognosis was poor and the subject had a risk of death. Based on this, the present invention was completed.

[0007] In a first aspect, the present invention provides a biomarker for differential diagnosis of severe pulmonary tuberculosis, wherein the biomarker is sCD27; when sCD27 in a biological sample of a subject is ≥95097 pg / ml, the subject is diagnosed with severe pulmonary tuberculosis.

[0008] Furthermore, the biological sample of the subject is selected from blood.

[0009] Preferably, the biological sample of the subject is at least one of peripheral blood, plasma and serum.

[0010] In a second aspect, the present invention provides an application of a biomarker in the preparation of a reagent for differential diagnosis of patients with severe pulmonary tuberculosis, wherein the biomarker is sCD27, and the reagent is a reagent that can detect sCD27; when sCD27 in the patient's biological sample is ≥95097pg / ml, the subject is diagnosed with severe pulmonary tuberculosis.

[0011] Furthermore, the biological sample of the subject is selected from blood.

[0012] Preferably, the biological sample of the subject is at least one of peripheral blood, plasma and serum.

[0013] In a third aspect, the present invention provides a kit for differential diagnosis of severe pulmonary tuberculosis patients, wherein the kit contains a reagent for detecting the content of sCD27. When the content of sCD27 in the patient's biological sample is ≥95097 pg / ml, the patient is a severe pulmonary tuberculosis patient.

[0014] Furthermore, the biological sample of the subject is selected from blood.

[0015] Preferably, the biological sample of the subject is at least one of peripheral blood, plasma and serum.

[0016] Furthermore, the kit may be an ELISA detection kit and / or a colloidal gold detection kit.

[0017] Furthermore, the diagnostic method of the kit includes a direct method, an indirect method, a double antibody sandwich method and / or a competitive method.

[0018] In a fourth aspect, the present invention provides a biomarker for the prognosis of patients with pulmonary tuberculosis, wherein the biomarker is sCD27; when the sCD27 level is higher than 158510 pg / ml, the prognosis is poor, indicating that the subject is at risk of death.

[0019] Furthermore, the biological sample of the subject is selected from blood.

[0020] Preferably, the biological sample of the subject is at least one of peripheral blood, plasma and serum.

[0021] In a fifth aspect, the present invention provides an application of a biomarker in the preparation of a reagent for the prognosis of patients with pulmonary tuberculosis, wherein the biomarker is sCD27, and the reagent is a reagent that can detect the level of sCD27 in a biological sample of a patient; when the sCD27 level is higher than 158510 pg / ml, the prognosis is poor, indicating that the subject is at risk of death.

[0022] Furthermore, the biological sample of the subject is selected from blood.

[0023] Preferably, the biological sample of the subject is at least one of peripheral blood, plasma and serum.

[0024] In a sixth aspect, the present invention provides a kit for the prognosis of patients with pulmonary tuberculosis, the kit comprising a reagent for detecting the level of sCD27 in a biological sample of a patient; when the sCD27 level is higher than 158510 pg / ml, the prognosis is poor, indicating that the subject is at risk of death.

[0025] Furthermore, the biological sample of the subject is selected from blood.

[0026] Preferably, the biological sample of the subject is at least one of peripheral blood, plasma and serum.

[0027] Furthermore, the kit may be an ELISA detection kit and / or a colloidal gold detection kit.

[0028] Furthermore, the diagnostic method of the kit includes a direct method, an indirect method, a double antibody sandwich method and / or a competitive method.

[0029] Beneficial Effects

[0030] The present invention found that the expression level of sCD27 increased significantly in patients with severe pulmonary tuberculosis; when sCD27 in the biological sample of the subject was ≥95097pg / ml, the subject was diagnosed with severe pulmonary tuberculosis; the area under the curve was 0.7674 (95%CI=0.6158-0.9190); the sensitivity was 45.45% and the specificity was 88.24%. When the sCD27 level was higher than 158510pg / ml, the prognosis was poor and the subject had a risk of death; the area under the curve was 0.7465 (95%CI=0.5756-0.9175); the sensitivity was 57.14% and the specificity was 83.87%. It shows that sCD27, as a biomarker for distinguishing severe and non-severe pulmonary tuberculosis patients and predicting the prognosis of pulmonary tuberculosis patients, has high accuracy and specificity, which helps to determine the clinical diagnosis and treatment plan. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 11-8 gradient dilution.

[0032] Figure 2 Flow cytometry was used to detect the plasma sCD27 levels in healthy people, non-severe and severe pulmonary tuberculosis patients in the screening group.

[0033] Figure 3 This is the ROC curve for judging severe and non-severe pulmonary tuberculosis based on the sCD27 level of patients in the screening group.

[0034] Figure 4 Flow cytometry was used to detect the plasma sCD27 levels in the validation group of healthy subjects, non-severe and severe pulmonary tuberculosis patients.

[0035] Figure 5 This is the ROC curve for judging severe and non-severe pulmonary tuberculosis based on sCD27 levels in the validation group.

[0036] Figure 6 Flow cytometry was used to detect the plasma sCD27 levels in the surviving and deceased pulmonary tuberculosis patients in the screening group.

[0037] Figure 7 ROC curve analysis of sCD27 level in the screening group for predicting death from pulmonary tuberculosis.

[0038] Figure 8 Flow cytometry was used to detect the plasma sCD27 levels in the surviving and non-surviving pulmonary tuberculosis patients in the validation group.

[0039] Fig. 9 ROC curve analysis of sCD27 level in the validation group to predict the death of patients with pulmonary tuberculosis. DETAILED DESCRIPTION

[0040] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, 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.

[0041] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0042] Example

[0043] Example 1 Sample inclusion criteria

[0044] The screening group consisted of 10 healthy controls, 17 patients with non-severe pulmonary tuberculosis, and 22 patients with severe pulmonary tuberculosis, and was used to preliminarily evaluate the correlation between plasma sCD27 levels and disease status.

[0045] The validation group consisted of 10 healthy controls, 17 patients with non-severe pulmonary tuberculosis, and 22 patients with severe pulmonary tuberculosis, and was used to further verify the results of the screening group.

[0046] Healthy controls: aged ≥18 years; no clinical symptoms of tuberculosis; negative IGRA test; no lung lesions found in chest X-ray or CT; no other serious medical history.

[0047] Non-severe pulmonary tuberculosis: age ≥ 18 years; clinical symptoms of tuberculosis; lung lesions found on chest X-ray or CT, but the extent of lung lesions does not reach the level of severe pulmonary tuberculosis.

[0048] Severe pulmonary tuberculosis: active pulmonary tuberculosis patients aged ≥18 years, with unilateral dense lung lesions ≥1 / 3 of the lung field on one side, or bilateral lesions involving ≥1 / 2 of the whole lung, or cavity diameter >4cm, or with large pleural effusion, and serum CRP ≥75mg / L and PO2<80mmHg without oxygen inhalation or oxygenation index <300 with oxygen inhalation.

[0049] Exclusion criteria: patients receiving immunosuppressive drugs or hormone therapy; patients with autoimmune diseases; patients with viral hepatitis; patients with incomplete clinical data, patients transferred to other hospitals or patients who gave up the treatment; patients with cancer and active infection.

[0050] Example 2 Detection of sCD27 concentration in plasma samples by flow cytometry

[0051] 2.1 Sample preservation

[0052] Take 2 mL of EDTA anticoagulant, centrifuge at 3000 rpm for 10 min, and take out plasma for cryopreservation.

[0053] 2.2 Detection of sCD27 concentration in plasma by cytometric bead array (CBA)

[0054] 1) Prepare standards:

[0055] Prepare the test according to the instructions of the kit. The standard is lyophilized powder. Centrifuge before use, add diluent to mix, and ice bath. Take 8 EP tubes, mark 1-8, transfer the standard after ice bath to tube 1, and dilute it in tubes 2-8 (such as Figure 1 shown);

[0056] 2) Prepare 1× buffer: Calculate the total amount of 1.0 mL / tube according to the number of experimental tubes to prepare 1× buffer;

[0057] 3) Calculate the amount of microspheres and secondary antibodies according to the number of experimental samples (n+1)*10uL, add the experimental diluent and mix well;

[0058] 4) Add 40 μL of microsphere-antibody mixture;

[0059] 5) Add 20 μL of standard or sample to be tested, mix well, and incubate;

[0060] 6) Add 20 μL SA-PE and incubate;

[0061] 7) Add buffer, centrifuge, and discard the supernatant;

[0062] 8) Repeat step 7;

[0063] 9) Add 1× buffer, mix well and then test.

[0064] Example 3 sCD27 differential diagnosis of severe pulmonary tuberculosis

[0065] 3.1 Screening test

[0066] There was no significant difference in sCD27 between non-severe pulmonary tuberculosis patients and healthy controls in the screening group, but the level of sCD27 in severe pulmonary tuberculosis was significantly higher than that in non-severe pulmonary tuberculosis patients (such as Figure 2 sCD27 concentration for judging severe pulmonary tuberculosis was 95097pg / ml, and the area under the ROC curve was 0.7674 (95%CI=0.6158-0.9190); the sensitivity was 45.45% and the specificity was 88.24% (as shown in Figure 2 ). Figure 3 shown).

[0067] 3.2 Verification test

[0068] In the validation group, there was no significant difference in sCD27 between patients with non-severe pulmonary tuberculosis and healthy controls, but the level of sCD27 in patients with severe pulmonary tuberculosis was significantly higher than that in patients with non-severe pulmonary tuberculosis (e.g. Figure 4 As shown); when the concentration of sCD27 for judging patients with severe pulmonary tuberculosis was 89812pg / ml, the area under the ROC curve was 0.7353 (95%CI=0.5712-0.8993); the sensitivity was 45.45% and the specificity was 88.24% (as shown); Figure 5 shown).

[0069] Example 4sCD27 Prognosis of Pulmonary Tuberculosis Patients

[0070] 4.1 Screening test

[0071] In the screening group, the level of sCD27 in non-surviving patients was significantly higher than that in surviving patients (e.g. Figure 6 As shown); According to the ROC curve, the concentration of sCD27 for predicting death from pulmonary tuberculosis was 147317 pg / ml, and the area under the curve was 0.8304 (95% CI = 0.7024-0.9584); the sensitivity was 42.86% and the specificity was 84.38% (as shown); Figure 7 shown).

[0072] 4.2 Verification test

[0073] In the validation group, the level of sCD27 in patients who died was significantly higher than that in patients who survived (e.g. Figure 8 As shown); when the concentration of CD27 for predicting death from pulmonary tuberculosis was 158510 pg / ml, the area under the ROC curve was 0.7465 (95% CI = 0.5756-0.9175); the sensitivity was 57.14% and the specificity was 83.87% (as shown); Fig. 9 shown).

Claims

1. A biomarker for differential diagnosis of severe pulmonary tuberculosis, wherein the biomarker is sCD27; when sCD27 in the biological sample of the subject is ≥95097pg / ml, the subject is diagnosed with severe pulmonary tuberculosis.

2. The use of a biomarker in the preparation of a reagent for differential diagnosis of severe pulmonary tuberculosis patients, wherein the biomarker is sCD27, and the reagent is a reagent that can detect sCD27; when sCD27 in the patient's biological sample is ≥95097pg / ml, the subject is diagnosed with severe pulmonary tuberculosis.

3. A kit for differential diagnosis of severe pulmonary tuberculosis patients, the kit containing a reagent for detecting the sCD27 content. When the sCD27 content in the patient's biological sample is ≥95097pg / ml, the patient is a severe pulmonary tuberculosis patient.

4. A biomarker for the prognosis of patients with pulmonary tuberculosis, wherein the biomarker is sCD27; when the sCD27 level is higher than 158510 pg / ml, the prognosis is poor, indicating that the subject is at risk of death.

5. Use of a biomarker in the preparation of a reagent for the prognosis of patients with pulmonary tuberculosis, wherein the biomarker is sCD27, and the reagent is a reagent that can detect the level of sCD27 in a biological sample of a patient; when the sCD27 level is higher than 158510pg / ml, the prognosis is poor, indicating that the subject is at risk of death.

6. A kit for prognosis of pulmonary tuberculosis patients, the kit comprising a reagent for detecting the level of sCD27 in a biological sample of a patient; when the sCD27 level is higher than 158510 pg / ml, the prognosis is poor, indicating that the subject is at risk of death.

7. The kit as claimed in claim 3 or 6 can be an ELISA detection kit and / or a colloidal gold detection kit.

8. The diagnostic method of the kit according to claim 3 or 6 comprises a direct method, an indirect method, a double antibody sandwich method and / or a competitive method.

9. The biological sample of the subject according to any one of claims 1 to 6 is selected from blood.

10. The biological sample of the subject according to any one of claims 1 to 6 is at least one of peripheral blood, plasma, and serum.

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