A biomarker for differential diagnosis of severe tuberculosis

Abstract

The application belongs to the technical field of biotechnology, and particularly relates to a biomarker for differential diagnosis of severe pulmonary tuberculosis. It is found that the XO content in blood of a severe pulmonary tuberculosis patient is significantly lower than that of a non-severe subject, the area (AUC) under the ROC curve in a screening test is 0.898, and the area (AUC) under the ROC curve in a verification test is 0.904. The application is helpful for early assessment of the condition of a pulmonary tuberculosis patient, and provides a basis for early intervention and comprehensive treatment of severe patients.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biotechnology, and particularly relates to a biomarker for differential diagnosis of severe pulmonary tuberculosis. BACKGROUND

[0002] Tuberculosis (TB) is a chronic granulomatous inflammation caused by Mycobacterium tuberculosis (MTB) infection, and pulmonary tuberculosis (PTB) is the most common. According to the report of the World Health Organization, about 1 / 3 of the world's population has been infected with Mycobacterium tuberculosis. After Mycobacterium tuberculosis invades lung tissue, inflammatory cells gather and release a large amount of pro-inflammatory factors, activate effector cells such as polymorphonuclear leukocytes, alveolar macrophages and endothelial factors, release a large amount of oxygen free radicals, induce lipid peroxidation reaction, cause damage to alveolar epithelium, capillary leakage, and digestion of antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GPX), and increase of malondialdehyde (MDA). Therefore, there is an imbalance between oxidation and antioxidant in pulmonary tuberculosis. Antioxidant therapy is beneficial to the recovery of tuberculosis patients in the comprehensive treatment of tuberculosis. At present, drug treatment is mainly used for pulmonary tuberculosis patients in clinic.

[0003] Due to diagnostic delay, dialysis, HIV infection or presence of immunosuppressive state, and multiple drug-resistant diseases, patients can develop more severe forms and need treatment in intensive care units. The most common indication for TB-related admission to intensive care units (ICUs) is acute respiratory failure caused by pneumonia or acute respiratory distress syndrome (ARDS). ARDS is a rare phenomenon of PTB, but it is the most common cause of ICU admission for TB patients. About 3.4% of hospitalized tuberculosis patients need to be admitted to the ICU. Compared with other causes of severe pneumonia, patients who need to be admitted to the ICU have poor prognosis and high mortality (60 vs 25%) (CHAUDHRY D, TYAGI D. Tuberculosis in Intensive Care Unit [J]. Indian J Crit Care Med, 2021, 25 (Suppl 2): S150-S4.). It has been reported that the mortality rate of pulmonary tuberculosis patients depends on the severity of the disease. Early warning, timely diagnosis and targeted treatment of severe pulmonary tuberculosis patients directly affect the mortality rate of patients (Chen HM, Wu XG, Ma LP, et al. Clinical characteristics of 163 hospitalized elderly tuberculosis patients who died [J]. Chinese Journal of Antituberculosis, 2014, 36(01): 55-8.). The research on TB at home and abroad 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 development of effective prevention and control measures have clinical significance for reducing the mortality rate of patients.

[0004] Similar to other lung diseases, oxidative and antioxidant imbalance plays a key role in the development of tuberculosis (LIANG Qingtao, LI Qi, GU Shuxiang, et al. Effect of N-acetylcysteine on oxidative / antioxidant imbalance in patients with pulmonary tuberculosis [J]. Clinical Journal of Pulmonary Medicine, 2013, 18(2): 306-307.). Xanthine oxidase (XO) is a key enzyme in the human body that catalyzes the generation of uric acid from xanthine and hypoxanthine, and is also a stress-induced enzyme, which is the source of host inflammation; while catalyzing the generation of uric acid, it also catalyzes the generation of reactive oxygen species (ROS) (DOEHNER W, LANDMESSER U. Xanthine oxidase and uric acid in cardiovascular disease: clinical impact and therapeutic options [J]. Semin Nephrol, 2011, 31(5): 433-40.), and ROS plays an important role in resisting various intracellular pathogens (MILKOVIC L, CIPAK GASPAROVIC A, CINDRIC M, et al. Short Overview of ROS as Cell Function Regulators and Their Implications in Therapy Concepts [J]. Cells, 2019, 8(8.).). XO expression is regulated by various factors, including hormones, growth factors, inflammatory cytokines, etc. Studies have shown that the XO level of malaria patients is elevated and is related to the inflammatory response (TY M C, ZUNIGA M, A, et al. Malaria inflammation by xanthine oxidase-produced reactive oxygen species [J]. EMBO Mol Med, 2019, 11(8): e9903.). There is no related report on the role of XO in the progress of PTB. SUMMARY

[0005] The present application finds that the XO content in the blood of patients with severe tuberculosis is significantly lower than that of non-severe subjects, and based on this, the present application is completed.

[0006] In a first aspect, the present application provides a biomarker for differential diagnosis of severe tuberculosis patients, the biomarker is xanthine oxidase (XO), when the XO content in the patient's biological sample is significantly lower than that of non-severe sample, the patient is a severe tuberculosis patient.

[0007] Further, the patient's biological sample is selected from blood.

[0008] Preferably, the biological sample of the patient is at least one of peripheral blood, plasma, and serum.

[0009] In a second aspect, the present application provides a use of a biomarker in the preparation of a reagent for differential diagnosis of a severe tuberculosis patient, wherein the biomarker is XO, and the reagent is a reagent capable of detecting XO; when the content of XO in the blood of a patient is significantly lower than that in a non-severe sample, the patient is a severe tuberculosis patient.

[0010] Further, the biological sample of the patient is selected from blood.

[0011] Preferably, the biological sample of the patient is at least one of peripheral blood, plasma, and serum.

[0012] In a third aspect, the present application provides a kit for predicting a severe tuberculosis patient, wherein the kit contains a reagent for detecting the content of XO; when the content of XO in the biological sample of a patient is significantly lower than that in a non-severe sample, the patient is a severe tuberculosis patient.

[0013] Further, the biological sample of the patient is selected from blood.

[0014] Preferably, the biological sample of the patient is at least one of peripheral blood, plasma, and serum.

[0015] Further, the kit can be an ELISA detection kit and / or a colloidal gold detection kit.

[0016] Still further, the diagnosis method of the kit comprises a direct method, an indirect method, a double-antibody sandwich method, and / or a competition method.

[0017] Advantages

[0018] The present application finds that the content of XO in the blood of a severe tuberculosis patient is significantly lower than that in a non-severe subject, and the area under the ROC curve (AUC) in the screening test is 0.898; the area under the ROC curve (AUC) in the verification test is 0.904. This is helpful for early assessment of the condition of a tuberculosis patient, and in particular provides a basis for early intervention and comprehensive treatment of severe patients. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Figure 2 shows the expression levels of XO in peripheral blood mononuclear cells of NSTB and STB patients and ROC curves; (A) is the expression level of XO in peripheral blood mononuclear cells of 30 NSTB and STB patients; (B) is the ROC curve of the expression level of peripheral blood mononuclear cells of 30 NSTB and STB patients; (C) is the expression level of XO in peripheral blood mononuclear cells of 15 NSTB and STB patients; (D) is the ROC curve of the expression level of peripheral blood mononuclear cells of 15 NSTB and STB patients. DETAILED DESCRIPTION

[0020] The following is a further description of specific embodiments of the present invention. It should be noted that the description of these embodiments is intended to facilitate understanding of the present invention and does not constitute a limitation of the present invention. In addition, the technical features involved in the embodiments described below may be combined with each other as long as they do not conflict with each other.

[0021] 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.

[0022] Example 1 Study Subject Inclusion Criteria

[0023] Patients who visited Beijing Chest Hospital affiliated to Capital Medical University and were clearly diagnosed with pulmonary tuberculosis from February 2024 to June 2024 were divided into severe group and non-severe group. Patients in the severe group of pulmonary tuberculosis had to meet any one of the following four conditions: (1) oxygen saturation ≤93% when breathing air at rest; (2) oxygenation index (arterial oxygen partial pressure / oxygen concentration) ≤300 mmHg, or arterial oxygen partial pressure ≤60 mmHg when breathing air at rest; (3) chest imaging showed that the cumulative range of bilateral tuberculosis lesions exceeded 1 / 2 of the whole lung; (4) patients who met the above-mentioned conditions for severe patients but developed shock or combined with other organ failure and required ICU monitoring and treatment, and those who did not meet the above-mentioned criteria for severe patients during the same period were all included in the non-severe patient group.

[0024] All patients included in the experiment met the above inclusion criteria.

[0025] Experimental set: According to the above criteria, 30 patients with severe tuberculosis (STB) and 30 patients with non-severe tuberculosis (NSTB) were collected, and all included patients were matched by age and gender.

[0026] Validation set: According to the above criteria, 15 patients with severe tuberculosis (STB) and 15 patients with non-severe tuberculosis (NSTB) were collected, and all included patients were matched by age and gender.

[0027] Example 2 Test method

[0028] 2.1 Blood sample collection and processing

[0029] (1) Collect 10 ml of peripheral blood from the subjects using EDTA anticoagulant vacuum blood collection tubes;

[0030] (2) Add human peripheral blood lymphocyte separation buffer (TBD) of equal volume to the blood sample, mix well, and then centrifuge;

[0031] (3) After centrifugation, the second white cell layer was transferred to a new centrifuge tube, and PBS was added and centrifuged;

[0032] (4) After centrifugation, discard the supernatant, add red blood cell lysis solution to resuspend the cells, lyse, add buffer to terminate lysis, centrifuge;

[0033] (5) Discard the supernatant, and add the obtained cells to FreeZol Reagent for subsequent RNA extraction.

[0034] 2.2 RNA extraction

[0035] 1) Extract total RNA from peripheral blood mononuclear cells, mix the cells with FreeZol Reagent, and stand still;

[0036] 2) Add Dilution Buffer, mix well and stand still, and centrifuge;

[0037] 3) Absorb the supernatant, add isopropanol, mix well and stand still, and centrifuge;

[0038] 4) Discard the supernatant, add ethanol, resuspend the precipitate, and centrifuge; repeat the operation, dry, and resuspend the RNA precipitate;

[0039] 5) Measure the concentration of the RNA sample, and use a UV spectrophotometer to measure the total RNA concentration and A260 / 280

[0040] ratio of all samples;

[0041] 6) This study selects RNA samples with an A260 / 280 ratio of about 2 for subsequent experiments, and when all samples meet the standard, subsequent experiments are performed.

[0042] 2.3 Reverse transcription to synthesize cDNA

[0043] Use Hifar II first-strand cDNA synthesis kit (Yesen) for reverse transcription, the total system is 20 μL, and the configuration method is as follows:

[0044] Table 1. Reverse transcription system preparation

[0045]

[0046] After the reaction system is prepared, mix well, and perform the reaction on the PCR instrument according to the conditions shown in Table 2. After the reaction is completed, the cDNA sample synthesized by reverse transcription is frozen.

[0047] Table 2. Reverse transcription program settings

[0048]

[0049] 2.4 RT-qPCR

[0050] 2.4.1 Primer design and synthesis

[0051] The primer sequences of human GAPDH and differential genes were searched on the Primer Bank website and verified by Primer-Blast on the NCBI website. The primers were synthesized by Beijing Ruibo Biological Technology Co., Ltd. The primer sequences are shown in Table 3.

[0052] Table 3. Primer sequences

[0053]

[0054] 2.4.2 RT-qPCR detection of mRNA expression levels of each gene

[0055] (1) The expression levels of each gene to be detected in the two groups were detected by qPCR SYBR green master mix (Low Rox) kit; (2) Each sample was added to the corresponding 96-well PCR reaction plate according to the reaction system shown in Table 4 below for detection;

[0056] Table 4. RT-qPCR reaction system

[0057]

[0058] (3) The reaction was performed on the PCR system, and the conditions were 95℃ for 5min, 95℃ for 10s and 60℃ for 30s;

[0059] (4) The internal reference was GADPH, and the relative expression level of the gene was based on 2 -ΔΔCt Method normalization.

[0060] 2.5 Data processing and statistical analysis

[0061] The experimental results were statistically analyzed using GraphPad Prism 8 software. The difference between groups was analyzed by unpaired T test, and the statistical difference was considered significant when P value was less than 0.05. The asterisk indicates the degree of significance (****, P<0.0001).

[0062] Example 3 test results

[0063] 3.1 Screening test

[0064] Clinical collection of 30 cases of STB patients and 30 cases of NSTB patients, isolation of peripheral blood mononuclear cells of patients, extraction of total RNA reverse transcription, RT-qPCR, found that the XO level of STB patients was reduced (A), and the area under the ROC curve (AUC) was 0.898 (B).

[0065] Figure 1 Figure 1 ​​​

[0066] 3.2 Verification test

[0067] According to the inclusion criteria, 15 STB and 15 NSTB patients were continuously included, and the expression of XO in peripheral blood mononuclear cells was detected according to the above operation steps. It was found that the lower results of XO in STB patients were consistent with the previous results Figure 1 C), and the AUC was 0.904 Figure 1 D), with high prediction performance.

[0068] Therefore, XO can be used as a biomarker for differential diagnosis of PTB patients.

Claims

1. Use of a reagent for detecting XO in the preparation of a kit for distinguishing patients with severe pulmonary tuberculosis from patients with non-severe pulmonary tuberculosis.

2. A kit for distinguishing between severe pulmonary tuberculosis patients and non-severe pulmonary tuberculosis patients, the kit comprising a reagent for detecting XO content.

3. The kit according to claim 2, wherein the kit is selected from an ELISA detection kit or a colloidal gold detection kit.

4. The kit according to claim 3, wherein the diagnostic method of the kit comprises a direct method, an indirect method, a double antibody sandwich method or a competitive method.

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