Marker composition for diagnosis and prognosis of severe pulmonary tuberculosis

Abstract

The invention provides a marker composition for diagnosis and prognosis of severe tuberculosis. The marker composition comprises sCD25, sTim-3, IP-10, neutrophil count, lymphocyte count and LDH for diagnosis of an AUC value gt of a patient with severe tuberculosis. 0.85, predicting the AUC value gt of the pulmonary tuberculosis prognosis effect; the result shows that the biomarker obtained by screening has relatively high diagnosis efficiency on the pulmonary tuberculosis.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and in particular relates to a marker composition for diagnosing severe pulmonary tuberculosis and prognosis. Background Art

[0002] Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis. The disease is mainly transmitted through droplets. The droplets produced when patients cough, talk or sneeze will fall into the dust and fly in the air after drying. The susceptible people around them will be infected after inhaling the tuberculosis bacteria in the droplets (Guo Gaiyan, Shu Lisha. LncRNA BLACAT1 promotes the proliferation, migration and invasion of cervical cancer cells by inhibiting miR-519d [J]. Chinese Journal of Gerontology, 2021, 41(21): 4784-4788.). The first anti-tuberculosis drug was discovered in the 1940s, marking a new era in tuberculosis treatment. However, factors such as inappropriate drug use and treatment interruption have led to the emergence of multi-drug resistant tuberculosis (MDR-TB). MDR-TB refers to tuberculosis that is resistant to both isoniazid and rifampicin, which means that patients need to use more expensive, longer-term and more side-effect second-line drugs to treat tuberculosis (Lv Hengliang, Zhang Xin, Zhang Wenyi, et al. Research progress on epidemiological characteristics and influencing factors of tuberculosis [J / OL]. Disease surveillance, 1-10 [2024-0314]. http: / / kns.cnki.net / kcms / detail / 11.2928.R.20231211.1517.002.html.). If not treated promptly and effectively, an active tuberculosis patient can infect 10 to 15 people per year on average. According to the World Health Organization (WHO) (World Health Organization. Global tuberculosis report 2022 [R]. Geneva: World Health Organization, 2022.), the global prevalence of tuberculosis has declined in the past few decades due to advances in public health measures and innovations in drug treatment, but in some regions, the prevalence of tuberculosis remains high.

[0003] The symptoms of pulmonary tuberculosis are mainly poisoning symptoms such as low fever, general fatigue, and night sweats. In addition, some patients have cough, sputum, difficulty breathing, chest pain, etc. About 1 / 3 of patients may experience hemoptysis, mainly with blood in the sputum. Chest CT shows that the lesions are mostly located in the apex of the lung, the posterior segment of the upper lobe, and the dorsal segment of the lower lobe, which are prone to form cavities and spread. However, some patients lack specific imaging manifestations, which makes the diagnosis of pulmonary tuberculosis extremely difficult (Villar-Hernández, R., et al., Use of IFN-γ and IP-10 detection in the diagnosis of latent tuberculosis infection in patients with inflammatory rheumatic diseases. J Infect, 2017. 75(4): p. 315-325.). The current gold standard for diagnosing pulmonary tuberculosis is to directly find positive acid-fast staining results of Mycobacterium tuberculosis in sputum smear or bronchoalveolar lavage fluid or positive sputum tuberculosis culture results. However, due to the above detection methods, which are prone to contamination, slow growth of tuberculosis bacteria, and long time consumption, the above diagnosis methods are not suitable for the early diagnosis of tuberculosis (Wang Sen and Zhang Wenhong, New progress in tuberculosis diagnosis technology. Microbiology and Infection, 2016. 11(03): p. 188-192.). At present, it is believed that negative pulmonary tuberculosis refers to pulmonary tuberculosis that is negative in three sputum smears and (or) one sputum culture, but positive in non-bacteriological methods (Li Zhensheng, Li Dexin, Li Chunxia, ​​Research on the value of multi-index combined detection in the diagnosis of negative pulmonary tuberculosis. Chinese Journal of Health Care Medicine, 2017.19(04): p.307-309.). Studies have shown that only about 30% of active pulmonary tuberculosis patients have positive pathogen detection, and more than half of tuberculosis patients find it difficult to directly find Mycobacterium tuberculosis in sputum specimens (Chen Xia, Zhang Jianyong, and Zhao Jianjun, The diagnostic value of TB-LAMP in bronchoalveolar lavage fluid for negative pulmonary tuberculosis. Chinese Journal of Infection Control, 2020.19(07): p.643-647.). Therefore, negative pulmonary tuberculosis has a high missed diagnosis rate and misdiagnosis rate in clinical work, which invisibly increases its transmission intensity and disease risk. Such patients have also become a new source of infection, making management and standardized treatment more difficult. In addition, since the time and probability of disease diagnosis delay in patients with bacteria-negative pulmonary tuberculosis are significantly higher than those in patients with pathogen-positive pulmonary tuberculosis, these characteristics lead to high disability and mortality rates, and poor survival status and quality of life of patients (Roberts, DJ, et al., Factors associated with delay in treatment initiation for pulmonary tuberculosis. ERJ Open Res, 2020.6(1).).Currently, there is a lack of effective biomarkers in clinical practice to predict the severity and prognosis of tuberculosis patients. Summary of the invention

[0004] The present invention screens out a marker composition for diagnosing severe pulmonary tuberculosis and predicting the prognosis of pulmonary tuberculosis, the markers show significant changes in the patient's plasma, and have strong diagnostic efficacy for diagnosing severe pulmonary tuberculosis and predicting the prognosis of pulmonary tuberculosis. Based on this, the present invention is completed.

[0005] In a first aspect, the present invention provides a biomarker composition for diagnosing severe pulmonary tuberculosis, wherein the biomarker composition comprises sCD25, sTim-3, IP-10, neutrophil count, lymphocyte count and LDH.

[0006] Furthermore, the marker composition is derived from whole blood, plasma or serum of a patient.

[0007] Further, the immune imbalance score (SIII) is calculated according to the level of the marker. When the SIII index is greater than 0.5535, the patient is judged to be a patient with severe pulmonary tuberculosis. The SIII index is calculated by the following formula:

[0008]

[0009] Among them, sCD25 is the concentration of sCD25 in the specimen, sTIM-3 is the concentration of sTim-3 in the specimen, IP-10 is the concentration of IP-10 in the specimen, Lymphocyte is the lymphocyte count of the specimen, Neulrophil is the neutrophil count of the specimen, and LDH is the LDH level of the specimen.

[0010] Furthermore, the pulmonary tuberculosis is an infection caused by Mycobacterium tuberculosis.

[0011] Furthermore, the pulmonary tuberculosis includes primary pulmonary tuberculosis, secondary pulmonary tuberculosis, blood type disseminated pulmonary tuberculosis, tracheobronchial tuberculosis, tuberculous pleurisy, septic tuberculosis, etc.

[0012] Furthermore, the Mycobacterium tuberculosis infection includes: primary infection, secondary infection, and extrapulmonary infection.

[0013] In a second aspect, the present invention provides an application of a biomarker composition in the preparation of a reagent for diagnosing severe pulmonary tuberculosis, wherein the biomarker composition comprises sCD25, sTim-3, IP-10, neutrophil count, lymphocyte count and LDH, and the reagent contains sCD25, sTim-3, IP-10, neutrophils, lymphocytes and LDH content that can detect the patient's biological sample.

[0014] Furthermore, the biological sample is whole blood, plasma or serum of a patient.

[0015] Further, the immune imbalance score (SIII) is calculated according to the level of the marker. When the SIII index is greater than 0.5535, the patient is judged to have severe pulmonary tuberculosis. The SIII index is calculated by the following formula:

[0016]

[0017] Among them, sCD25 is the concentration of sCD25 in the specimen, sTIM-3 is the concentration of sTim-3 in the specimen, IP-10 is the concentration of IP-10 in the specimen, Lymphocyte is the lymphocyte count of the specimen, Neulrophil is the neutrophil count of the specimen, and LDH is the LDH level of the specimen.

[0018] In a third aspect, the present invention provides a kit for diagnosing severe pulmonary tuberculosis, wherein the kit comprises the reagent as described in the second aspect of the present invention.

[0019] Furthermore, the kit may be one or more of an ELISA detection kit, a colloidal gold detection kit, an immunohistochemistry kit, an immunofluorescence kit and / or an in situ hybridization staining kit.

[0020] Furthermore, the diagnostic method of the kit includes one or more of a direct method, an indirect method, a double antibody sandwich method and / or a competitive method.

[0021] In a fourth aspect, the present invention provides a biomarker composition for predicting the prognosis of pulmonary tuberculosis, wherein the biomarker composition comprises sCD25, sTim-3, IP-10, neutrophil count, lymphocyte count and LDH.

[0022] Furthermore, the marker composition is derived from whole blood, plasma or serum of a patient.

[0023] Further, the immune imbalance score (SIII) is calculated according to the level of the marker. When the SIII index is greater than 0.7580, the patient is judged to have a risk of death. The SIII index is calculated by the following formula:

[0024]

[0025] Among them, sCD25 is the concentration of sCD25 in the specimen, sTIM-3 is the concentration of sTim-3 in the specimen, IP-10 is the concentration of IP-10 in the specimen, Lymphocyte is the lymphocyte count of the specimen, Neulrophil is the neutrophil count of the specimen, and LDH is the LDH level of the specimen.

[0026] In a fifth aspect, the present invention provides an application of a biomarker composition in a reagent for predicting the prognosis of pulmonary tuberculosis, wherein the marker combination includes sCD25, sTim-3, IP-10, neutrophil count, lymphocyte count and LDH, and the reagent contains a reagent that can detect the content of sCD25, sTim-3, IP-10, neutrophils, lymphocytes and LDH in a patient's biological sample.

[0027] Furthermore, the biomarkers are derived from whole blood, plasma or serum of the patient.

[0028] Further, the immune imbalance score (SIII) is calculated according to the level of the marker. When the SIII index is greater than 0.7580, the patient is judged to have a risk of death. The SIII index is calculated by the following formula:

[0029]

[0030] Among them, sCD25 is the concentration of sCD25 of the sample, sTIM is the concentration of sTim-3 of the sample, IP is the concentration of IP-10 of the sample, Lymphocyte is the lymphocyte count of the sample, Neulrophil is the neutrophil count of the sample, and LDH is the LDH level of the sample.

[0031] In a sixth aspect, the present invention provides a kit for predicting the prognosis of pulmonary tuberculosis, wherein the kit comprises the reagent as described in the fifth aspect of the present invention.

[0032] Furthermore, the kit may be one or more of an ELISA detection kit, a colloidal gold detection kit, an immunohistochemistry kit, an immunofluorescence kit and / or an in situ hybridization staining kit.

[0033] Beneficial Effects

[0034] The immune imbalance score (SIII) calculated by the biomarker composition of the present invention can significantly distinguish patients with severe pulmonary tuberculosis from patients with non-severe pulmonary tuberculosis and predict the prognosis of pulmonary tuberculosis. The AUC value for diagnosing severe pulmonary tuberculosis patients is >0.85, and the AUC value for predicting the prognosis of pulmonary tuberculosis is >0.75, indicating that the biomarkers screened by the present invention have a high diagnostic efficacy for pulmonary tuberculosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 To screen the levels of SIII in severe and non-severe pulmonary tuberculosis;

[0036] Figure 2 ROC curve analysis for screening SIII in the diagnosis of severe pulmonary tuberculosis;

[0037] Figure 3 is the level of SIII in surviving and dying pulmonary tuberculosis;

[0038] Figure 4 ROC curve analysis for SIII in predicting death from tuberculosis;

[0039] Figure 5 To verify the concentration of SIII in severe and non-severe pulmonary tuberculosis;

[0040] Figure 6 To verify the ROC curve analysis of SIII in the diagnosis of severe pulmonary tuberculosis;

[0041] Figure 7 To verify the level of SIII in the living and dead pulmonary tuberculosis in the set;

[0042] Figure 8 ROC curve analysis of SIII in the validation set for predicting tuberculosis mortality. DETAILED DESCRIPTION

[0043] 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.

[0044] 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.

[0045] Example 1 Screening of SIII for judging the severity of pulmonary tuberculosis and predicting the prognosis

[0046] 1. Obtaining plasma samples

[0047] Plasma samples from subjects: 34 patients with non-severe pulmonary tuberculosis and 44 patients with severe pulmonary tuberculosis.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 2. Detect the concentrations of sCD25, sTim-3, and IP-10 in plasma samples; obtain neutrophil counts, lymphocyte counts, and LDH levels

[0053] (1) Sample storage: plasma is packaged and frozen;

[0054] (2) Detection of plasma concentrations of sCD25, sTim-3, and IP-10 using flow cytometric microsphere immunoassay system

[0055] 1) Prepare the standard: dilute according to the kit instructions;

[0056] 2) Prepare buffer solution;

[0057] 3) Add microsphere-antibody mixture;

[0058] 4) Add standard or sample to be tested, mix well and incubate;

[0059] 5) Add SA-PE and incubate;

[0060] 6) Add buffer, vortex, centrifuge, and discard the supernatant;

[0061] 7) Repeat step 6.

[0062] 8) Add buffer, mix well and wait for detection.

[0063] (3) Neutrophil count, lymphocyte count, and LDH level were measured using an automatic blood cell counter and an automatic biochemical analyzer. The specific values ​​of neutrophil count, lymphocyte count, and LDH were obtained from the patients' routine blood and biochemical test results.

[0064] 3. Calculation of immune imbalance score (SIII)

[0065] SIII was calculated according to the following formula: |

[0066]

[0067] Among them, sCD25 is the concentration of sCD25 in plasma samples, sTIM-3 is the concentration of sTim-3 in plasma samples, IP-10 is the concentration of IP-10 in plasma samples, Lymphocyte is the lymphocyte count, Neulrophil is the neutrophil count, and LDH is the LDH level.

[0068] 4. Experimental Results

[0069] 4.1.SIII is used to determine the severity of pulmonary tuberculosis

[0070] The level of SIII in patients with severe pulmonary tuberculosis was significantly higher than that in patients with non-severe pulmonary tuberculosis (see Figure 1 ). According to ROC curve analysis, when the critical value of SIII for judging patients as severe pulmonary tuberculosis was 0.5535, the area under the curve (AUC) was 0.9278 (95% CI: 0.8500-1.000), the sensitivity was 86.36%, and the specificity was 82.35% (see Figure 2 ).

[0071] 4.2.SIII for predicting the prognosis of pulmonary tuberculosis

[0072] The level of SIII in non-surviving patients was significantly higher than that in surviving patients (see Figure 3 ). According to ROC curve analysis, when the critical value of SIII for predicting patient death was 0.7580, the area under the curve (AUC) was 0.9278 (95% CI: 0.7646-0.9943), the sensitivity was 85.71%, and the specificity was 87.50% (see Figure 4 ).

[0073] Example 2 Validation of SIII for determining the severity of pulmonary tuberculosis and predicting prognosis

[0074] 1. Obtaining plasma samples

[0075] Plasma samples from subjects: 17 patients with non-severe pulmonary tuberculosis and 22 patients with severe pulmonary tuberculosis.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 2. Detect the concentrations of sCD25, sTim-3, and IP-10 in plasma samples; obtain neutrophil counts, lymphocyte counts, and LDH levels

[0081] (1) Sample storage: plasma is packaged and frozen;

[0082] (2) Detection of plasma concentrations of sCD25, sTim-3, and IP-10 using flow cytometric microsphere immunoassay system

[0083] 1) Prepare the standard: dilute according to the kit instructions;

[0084] 2) Prepare buffer solution;

[0085] 3) Add microsphere-antibody mixture;

[0086] 4) Add standard or sample to be tested, mix well and incubate;

[0087] 5) Add SA-PE and incubate;

[0088] 6) Add buffer, vortex, centrifuge, and discard the supernatant;

[0089] 7) Repeat step 6.

[0090] 8) Add buffer, mix well and wait for detection.

[0091] (3) Neutrophil count, lymphocyte count, and LDH level were measured using an automatic blood cell counter and an automatic biochemical analyzer. The specific values ​​of neutrophil count, lymphocyte count, and LDH were obtained from the patients' routine blood and biochemical test results.

[0092] 3. Calculation of immune imbalance score (SIII)

[0093] SIII was calculated according to the following formula: |

[0094]

[0095] Among them, sCD25 is the concentration of sCD25 in plasma samples, sTim-3 is the concentration of sTim-3 in plasma samples, IP-10 is the concentration of IP-10 in plasma samples, Lymphocyte is the lymphocyte count, Neulrophil is the neutrophil count, and LDH is the LDH level.

[0096] 4. Experimental Results

[0097] 4.1.SIII is used to determine the severity of pulmonary tuberculosis

[0098] The level of SIII in patients with severe pulmonary tuberculosis was significantly higher than that in patients with non-severe pulmonary tuberculosis (see Figure 5 ). When the critical value of SIII for judging patients as having severe pulmonary tuberculosis was 0.5535, the area under the curve (AUC) was 0.8930 (95% CI: 0.7875-0.9986), the sensitivity was 81.82%, and the specificity was 82.35% (see Figure 6 ).

[0099] 4.2.SIII for predicting the prognosis of pulmonary tuberculosis

[0100] The level of SIII in non-surviving patients was significantly higher than that in surviving patients (see Figure 7 ); when the critical value of SIII for predicting patient death was 0.7580, the area under the curve (AUC) was 0.9330 (95% CI: 0.8477-1.000), the sensitivity was 85.71%, and the specificity was 84.38% (see Figure 8 ).

[0101] The results verified the diagnostic efficacy of the marker composition of the present invention in determining the severity of pulmonary tuberculosis and predicting the prognosis of pulmonary tuberculosis, and confirmed the feasibility of its application in actual clinical diagnosis.

Claims

1. A biomarker composition for diagnosing severe pulmonary tuberculosis, the biomarker composition comprising sCD25, sTim-3, IP-10, neutrophil count, lymphocyte count and LDH.

2. The composition according to claim 1, wherein the immune imbalance score (SIII) is calculated according to the level of the marker, and when the SIII index>0.5535, the patient is judged to be a patient with severe pulmonary tuberculosis; the SIII index is calculated by the following formula: in, sCD25 is the concentration of sCD25 in the specimen, sTIM-3 is the concentration of sTim-3 in the specimen, IP-10 is the concentration of IP-10 in the specimen, Lymphocyte is the lymphocyte count in the specimen, Neulrophil is the neutrophil count in the specimen, and LDH is the LDH level in the specimen.

3. Use of a biomarker composition in the preparation of a reagent for diagnosing severe pulmonary tuberculosis, the biomarker composition comprising sCD25, sTim-3, IP-10, neutrophil count, lymphocyte count and LDH, and the reagent containing sCD25, sTim-3, IP-10, neutrophils, lymphocytes and LDH content in a patient's biological sample.

4. The use according to claim 3, wherein the immune imbalance score (SIII) is calculated according to the level of the marker, and when the SIII index>0.5535, the patient is judged to have severe pulmonary tuberculosis; the SIII index is calculated by the following formula: in, sCD25 is the concentration of sCD25 in the specimen, sTIM-3 is the concentration of sTim-3 in the specimen, IP-10 is the concentration of IP-10 in the specimen, Lymphocyte is the lymphocyte count in the specimen, Neulrophil is the neutrophil count in the specimen, and LDH is the LDH level in the specimen.

5. A kit for diagnosing severe pulmonary tuberculosis, comprising the reagent as claimed in claim 3.

6. A biomarker composition for predicting the prognosis of pulmonary tuberculosis, the biomarker composition comprising sCD25, sTim-3, IP-10, neutrophil count, lymphocyte count and LDH.

7. The composition according to claim 6, wherein the immune imbalance score (SIII) is calculated according to the level of the marker, and when the SIII index>0.7580, the patient is judged to have a risk of death; the SIII index is calculated by the following formula: in, sCD25 is the concentration of sCD25 in the specimen, sTIM-3 is the concentration of sTim-3 in the specimen, IP-10 is the concentration of IP-10 in the specimen, Lymphocyte is the lymphocyte count in the specimen, Neulrophil is the neutrophil count in the specimen, and LDH is the LDH level in the specimen.

8. Use of a biomarker combination in a reagent for predicting the prognosis of pulmonary tuberculosis, wherein the marker combination includes sCD25, sTim-3, IP-10, neutrophil count, lymphocyte count and LDH, and the reagent is a reagent that can detect the content of sCD25, sTim-3, IP-10, neutrophils, lymphocytes and LDH in a patient's biological sample.

9. The use according to claim 8, wherein the immune imbalance score (SIII) is calculated according to the level of the marker, and when the SIII index>0.7580, the patient is judged to have a risk of death; the SIII index is calculated by the following formula: in, sCD25 is the concentration of sCD25 of the sample, sTIM is the concentration of sTim-3 of the sample, IP is the concentration of IP-10 of the sample, Lymphocyte is the lymphocyte count of the sample, Neulrophil is the neutrophil count of the sample, and LDH is the LDH level of the sample.

10. A kit for predicting the prognosis of pulmonary tuberculosis, comprising the reagent according to claim 8.

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