Application of extracellular vesicle B and T lymphocyte weakening factor in active tuberculosis diagnosis

Abstract

The invention discloses an application of an extracellular vesicle B and T lymphocyte weakening factor in active tuberculosis diagnosis, relates to the technical field of biomedicine, and particularly discloses an application of the extracellular vesicle B and T lymphocyte weakening factor as a diagnostic marker of active tuberculosis in preparation of an active tuberculosis diagnostic product. The extracellular vesicle B and T lymphocyte weakening factor can effectively distinguish active tuberculosis patients from healthy people, tuberculosis latent infection patients and lung cancer patients, can be used for diagnosing active tuberculosis, and has high sensitivity and specificity.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of an extracellular vesicle B and T lymphocyte weakening factor in the diagnosis of active pulmonary tuberculosis. Background Technology

[0002] Tuberculosis (TB) is a chronic, fatal infectious disease caused by Mycobacterium tuberculosis (Mtb). Rapid and effective screening for active pulmonary TB is a key means of TB prevention and control. Currently, clinical methods for diagnosing active pulmonary TB mainly include bacterial smear microscopy, bacterial culture, imaging examinations, gene diagnosis, tuberculin skin test, Mycobacterium tuberculosis antibody detection, and interferon-γ (INF-γ) release assay. However, these methods cannot meet clinical needs. Among them, bacterial smear microscopy has low sensitivity and specificity, and microscopic examination of sputum smears after acid-fast staining can only detect acid-fast positive bacilli, failing to distinguish between Mycobacterium tuberculosis and non-tuberculous mycobacteria. Bacterial culture requires a long time period and has a low detection rate; moreover, Mycobacterium tuberculosis is a highly pathogenic microorganism, and isolation and culture carry a certain risk of infection, requiring appropriate protective measures. Imaging methods require highly skilled operators and have poor diagnostic specificity, making them difficult to differentiate from diseases such as pneumonia, pulmonary nodules, and lung cancer. Mycobacterium tuberculosis antibody testing has a low level of evidence in assessing disease activity and is prone to false positives or false negatives. The tuberculin skin test cannot distinguish between Mycobacterium tuberculosis infection, non-tuberculous mycobacterial infection, and BCG vaccination, and requires 48–72 hours of follow-up; it can also cause false positives in patients who have received BCG vaccination. Molecular detection methods based on Mycobacterium tuberculosis DNA cannot distinguish between dead and live bacteria, therefore they cannot be used for monitoring treatment efficacy. Furthermore, these methods require high-quality samples; for pediatric patients, invasive procedures are needed to obtain respiratory samples that may contain small amounts of Mycobacterium tuberculosis. Additionally, these methods are susceptible to contamination by nucleic acid products, requiring high standards for laboratory conditions and environment. The interferon-gamma release assay is complex and time-consuming, and its results are easily affected by a weakened immune system. This method can only diagnose whether a patient has been infected with Mycobacterium tuberculosis; it cannot distinguish between past and current infections, nor between latent infections and active pulmonary tuberculosis. Biomarkers for disease screening are widely welcomed in clinical practice due to their simple and easy sampling, rapid results, ease of operation, and cost-effectiveness. They can be used for disease diagnosis, differential diagnosis, and efficacy monitoring. However, there is currently no universally accepted biomarker for the diagnosis of active pulmonary tuberculosis. Therefore, there is an urgent need to find highly specific and sensitive biomarkers for the diagnosis, differential diagnosis, efficacy monitoring, and bacterial load assessment of active pulmonary tuberculosis. Summary of the Invention

[0003] In view of the shortcomings of the prior art, the purpose of this invention is to provide an application of extracellular vesicle B and T lymphocyte attenuating factors in the diagnosis of active pulmonary tuberculosis, aiming to provide a highly specific and sensitive diagnostic biomarker for active pulmonary tuberculosis.

[0004] The technical solution of the present invention is as follows:

[0005] In a first aspect, the invention provides the application of extracellular vesicle B and T lymphocyte attenuating factors as diagnostic markers for active pulmonary tuberculosis in the preparation of diagnostic products for active pulmonary tuberculosis.

[0006] In a second aspect, the invention provides the application of extracellular vesicle B and T lymphocyte weakening factors as biomarkers for evaluating the efficacy of treatment for active pulmonary tuberculosis in the preparation of products for evaluating the efficacy of treatment for active pulmonary tuberculosis.

[0007] In a third aspect, the invention provides the application of extracellular vesicle B and T lymphocyte weakening factors as biomarkers for assessing active tuberculosis load in the preparation of products for assessing active tuberculosis load.

[0008] Optionally, the product is a reagent or a kit.

[0009] Optionally, the sample used when applying the product is serum.

[0010] Optionally, the kit includes quality control samples and / or standards.

[0011] Beneficial Effects: In this invention, extracellular vesicle B and T lymphocyte attenuating factors can effectively distinguish between patients with active pulmonary tuberculosis and healthy individuals, patients with active pulmonary tuberculosis and patients with latent tuberculosis infection, and patients with active pulmonary tuberculosis and lung cancer patients. It can be used to diagnose active pulmonary tuberculosis with high sensitivity and specificity. The procedure for diagnosing active pulmonary tuberculosis using this biomarker is simple, sample acquisition is convenient, and the cost is low. It is non-invasive, making it easier, faster, and safer for patients to undergo testing, thus showing broad application prospects. Attached Figure Description

[0012] Figure 1Statistical graphs and ROC curves were generated to analyze the differences in BTLA expression levels in serum of healthy individuals, patients with latent tuberculosis infection, lung cancer patients, and patients with active pulmonary tuberculosis. Graphs A, B, C, D, E, and F were used to analyze the differences in BTLA expression levels in serum of healthy individuals, patients with latent tuberculosis infection, patients with active pulmonary tuberculosis, and patients with lung cancer and active pulmonary tuberculosis, respectively.

[0013] Figure 2 A statistical graph showing the difference in serum extracellular vesicle BTLA expression levels in patients with active pulmonary tuberculosis before treatment and one month after standard drug treatment.

[0014] Figure 3 A statistical graph showing the difference in expression levels of extracellular vesicle BTLA in the serum of patients with active pulmonary tuberculosis who were negative for sputum smear acid-fast staining (sputum negative) and positive for sputum smear acid-fast staining (sputum positive).

[0015] Figure 4 A statistical graph showing the difference in expression levels of extracellular vesicle BTLA in serum samples from patients with negative and positive tuberculosis antibodies. Detailed Implementation

[0016] This invention provides an application of extracellular vesicle B and T lymphocyte attenuating factors in the diagnosis of active pulmonary tuberculosis. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0018] Extracellular vesicles (EVs), including exosomes and microvesicles, are nanoscale particles released from cells into the extracellular environment. They possess a lipid bilayer structure and contain various components such as lipids, proteins, mRNAs, and non-coding RNAs. Widely distributed in human body fluids, they participate in various physiological processes, including intercellular communication and immune regulation. EVs play a complex and crucial role in the pathogenesis of tuberculosis. After Mycobacterium tuberculosis infects the body, it releases EVs containing lipoproteins, lipids, and nucleic acids to suppress the host's immune response and regulate autophagy or apoptosis in host cells, enabling them to survive long-term in macrophages. Mycobacterium tuberculosis EVs can also activate the NLRP3 inflammasome, exacerbating lung inflammation. On the other hand, after Mycobacterium tuberculosis enters the body, host cells also respond to infection by secreting EVs, such as enhancing the bactericidal ability of macrophages and promoting granuloma formation and caseous necrosis. Therefore, EVs play a key role in the development and progression of tuberculosis. Extracellular vesicles are widely distributed in various bodily fluids of tuberculosis patients, including blood, urine, saliva, sputum, and pleural effusion, facilitating non-invasive or minimally invasive sampling and providing a basis for liquid biopsy. These extracellular vesicles in bodily fluids carry various tuberculosis-related biomolecules, including proteins, lipids, mRNA, miRNA, and DNA. These contents are protected from degradation by the lipid bilayer structure of the extracellular vesicles and remain stable even after ex vivo, ensuring the accuracy of detection. Furthermore, these contents possess unique disease expression profiles, comprehensively reflecting the disease state. In addition, extracellular vesicle separation, such as ultracentrifugation, can achieve enrichment, increasing the concentration of extracellular vesicle components and thus improving the sensitivity and reliability of disease diagnosis. Therefore, extracellular vesicles show great potential as new diagnostic biomarkers for tuberculosis. Current research has also identified some extracellular vesicle components that are likely to become diagnostic biomarkers for tuberculosis, but these biomarkers are mainly miRNAs, and despite the protection of the lipid bilayer structure, their stability is still not as good as that of protein components. In their previous research, the inventors discovered that the immune negative regulatory molecule B and T lymphocyte attenuator (BTLA) plays a crucial role in mediating the immune escape of Mycobacterium tuberculosis. They then used ELISA to detect the expression level of BTLA in serum extracellular vesicles of patients with active pulmonary tuberculosis (APT) and compared it with the expression levels in serum extracellular vesicles of healthy individuals, patients with latent tuberculosis infection, and lung cancer patients. This comparative analysis confirmed that BTLA can serve as a diagnostic marker for active pulmonary tuberculosis. Furthermore, extracellular vesicle BTLA can also be used to observe the treatment efficacy and assess bacterial load in patients with active pulmonary tuberculosis.Currently, there are no universally accepted biomarkers for the diagnosis of active pulmonary tuberculosis in clinical practice, and patients with active pulmonary tuberculosis are difficult to distinguish from patients with latent tuberculosis infection and lung cancer patients. Therefore, this invention provides the application of extracellular vesicle BTLA as a diagnostic biomarker for active pulmonary tuberculosis in the preparation of diagnostic products for active pulmonary tuberculosis.

[0019] In this invention, extracellular vesicle BTLA can effectively distinguish between patients with active pulmonary tuberculosis and healthy individuals, patients with active pulmonary tuberculosis and patients with latent tuberculosis infection, and patients with active pulmonary tuberculosis and lung cancer patients. It can be used to diagnose active pulmonary tuberculosis with high sensitivity and specificity. The procedure for diagnosing active pulmonary tuberculosis using this biomarker is simple, sample acquisition is convenient, and the cost is low. It is non-invasive, making it easier, faster, and safer for patients to undergo testing, thus showing broad application prospects.

[0020] This invention also provides the application of extracellular vesicle BTLA as a biomarker for evaluating the efficacy of treatment for active pulmonary tuberculosis in the preparation of products for evaluating the efficacy of treatment for active pulmonary tuberculosis.

[0021] This invention also provides the application of extracellular vesicle BTLA as a biomarker for assessing active tuberculosis load in the preparation of products for assessing active tuberculosis load.

[0022] In some embodiments, the product is a reagent or a kit.

[0023] In some embodiments, the sample used when applying the product is serum.

[0024] In some embodiments, the kit includes quality control materials and / or standards.

[0025] The present invention will be further described below through specific embodiments.

[0026] Example 1

[0027] The study compared and analyzed serum extracellular vesicle BTLA levels in healthy individuals, patients with active pulmonary tuberculosis, patients with latent tuberculosis infection, patients with active pulmonary tuberculosis, and patients with lung cancer and active pulmonary tuberculosis, and evaluated the diagnostic efficacy of extracellular vesicle BTLA as a biomarker for active pulmonary tuberculosis. The specific steps included:

[0028] Peripheral venous blood was collected from healthy individuals (15 cases, denoted as the HC group, i.e., the healthy control group), patients with latent tuberculosis infection (20 cases, denoted as the LTBI group, i.e., the latent tuberculosis infection group), lung cancer patients (20 cases, denoted as the LC group, i.e., the lung cancer group), and active pulmonary tuberculosis patients (33 cases, denoted as the APT group, i.e., the active pulmonary tuberculosis group). Extracellular vesicles were isolated, and the expression level of BTLA in serum extracellular vesicles in each group was detected by ELISA (i.e., the concentration of BTLA in serum extracellular vesicles in each group was detected). The differential expression of BTLA in serum extracellular vesicles was compared and analyzed between the healthy control group and the active pulmonary tuberculosis group, between the latent tuberculosis infection group and the active pulmonary tuberculosis group, and between the lung cancer group and the active pulmonary tuberculosis group. Then, the diagnostic efficacy of serum extracellular vesicle BTLA as a biomarker for active pulmonary tuberculosis was analyzed using ROC (receiver operating characteristic) curves, including area under the curve, specificity, and sensitivity. The results are as follows: Figure 1 As shown (where *** represents p<0.001, and ** represents p<0.01).

[0029] Depend on Figure 1 As shown in A and D, the concentration of extracellular vesicle BTLA in the serum of the active pulmonary tuberculosis group (i.e., the APT group) was significantly higher than that of the healthy control group (i.e., the HC group). The ROC curve analysis showed that the diagnostic efficacy of extracellular vesicle BTLA in diagnosing active pulmonary tuberculosis was 0.83, the cutoff value was 3.442, the sensitivity was 78.79%, and the specificity was 80%.

[0030] Depend on Figure 1 As shown in B and E, the concentration of extracellular vesicle BTLA in the serum of the active pulmonary tuberculosis group (i.e., the APT group) was significantly higher than that of the latent tuberculosis infection group (i.e., the LTBI group). ROC curve analysis showed that the diagnostic efficacy of extracellular vesicle BTLA in diagnosing active pulmonary tuberculosis was 0.74, the cutoff value was 2.494, the sensitivity was 90.91%, and the specificity was 50%.

[0031] Depend on Figure 1 As shown in C and F, the serum extracellular vesicle BTLA concentration in the active pulmonary tuberculosis group (i.e., APT group) was significantly higher than that in the lung cancer group (i.e., LC group). ROC curve analysis showed that the diagnostic efficacy of extracellular vesicle BTLA in diagnosing active pulmonary tuberculosis was 0.72, the cutoff value was 3.775, the sensitivity was 75.76%, and the specificity was 65.4%.

[0032] The above results demonstrate that extracellular vesicle BTLA can serve as a diagnostic marker for active pulmonary tuberculosis.

[0033] Example 2

[0034] Serum extracellular vesicle BTLA was used to evaluate the efficacy of treatment in patients with active pulmonary tuberculosis, and the specific steps included:

[0035] Peripheral venous blood samples were collected from 8 patients with active pulmonary tuberculosis (different from the active pulmonary tuberculosis patients in Example 1) before treatment. After one month of standard anti-tuberculosis drug treatment, peripheral venous blood was collected again, extracellular vesicles were isolated, and the expression level of extracellular vesicle BTLA was detected by ELISA. The difference in serum extracellular vesicle BTLA concentration between patients with active pulmonary tuberculosis before and after treatment was compared to evaluate the efficacy of extracellular vesicle BTLA in active pulmonary tuberculosis.

[0036] The results are as follows Figure 2 As shown in the figure (where ** indicates p<0.01), it can be seen that the expression level of extracellular vesicle BTLA in the serum of patients with active pulmonary tuberculosis was significantly lower than that before treatment after one month of short-term drug treatment. Extracellular vesicle BTLA can be used to evaluate the efficacy of treatment for active pulmonary tuberculosis.

[0037] Example 3

[0038] The assessment of serum extracellular vesicle BTLA for bacterial load analysis in patients with active pulmonary tuberculosis includes the following steps:

[0039] Peripheral venous blood was collected from patients with active pulmonary tuberculosis who were sputum smear negative for acid-fast bacilli (8 cases, denoted as sputum-negative group) and patients with active pulmonary tuberculosis who were sputum smear positive for acid-fast bacilli (17 cases, denoted as sputum-positive group). Extracellular vesicles were isolated, and the expression level of BTLA in extracellular vesicles was detected by ELISA. The difference in BTLA expression level between the sputum-negative group and the sputum-positive group was compared to assess whether BTLA in extracellular vesicles can serve as a marker of bacterial burden in patients with active pulmonary tuberculosis.

[0040] The results are as follows Figure 3 As shown in the figure (where * indicates p<0.05), the expression level of extracellular vesicle BTLA in the serum of patients with active pulmonary tuberculosis who are sputum-positive is significantly higher than that of patients with sputum-negative sputum. Extracellular vesicle BTLA can be used to assess the bacterial load in patients with active pulmonary tuberculosis.

[0041] Example 4

[0042] Serum extracellular vesicle BTLA is used for disease course analysis in patients with active pulmonary tuberculosis, and the specific steps include the following:

[0043] In the early stages of pulmonary tuberculosis, tuberculosis antibodies may be negative. As the disease progresses, patients with pulmonary tuberculosis will gradually become positive for tuberculosis antibodies. Peripheral venous blood was collected from 18 patients with active pulmonary tuberculosis who were negative for tuberculosis antibodies (labeled as antibody-negative group) and 7 patients with active pulmonary tuberculosis who were positive for tuberculosis antibodies (labeled as antibody-positive group). Extracellular vesicles were isolated, and the expression level of BTLA in extracellular vesicles was detected by ELISA. The difference in BTLA expression levels in extracellular vesicles between the antibody-negative and antibody-positive groups was compared to assess whether BTLA in extracellular vesicles can serve as an indicator of the course of active pulmonary tuberculosis.

[0044] The results are as follows Figure 4 As shown, there was no significant difference in the expression level of extracellular vesicle BTLA in serum between patients with negative and positive tuberculosis antibodies and active pulmonary tuberculosis. Therefore, extracellular vesicle BTLA cannot be used to reflect the course of the disease.

[0045] Therefore, based on the results of Examples 1 to 4, serum extracellular vesicle BTLA can be used as a diagnostic marker for active pulmonary tuberculosis, and as a biomarker for observing the efficacy of treatment and bacterial load in active pulmonary tuberculosis, but it cannot be used to reflect the course of the disease.

[0046] In summary, there are currently no universally accepted biomarkers for the diagnosis of active pulmonary tuberculosis (TB), and patients with active TB are difficult to distinguish from those with latent TB infection or lung cancer. Proteins, due to their stability outside the body, are among the most widely used biomarkers in clinical practice. The inventors discovered that the expression level of the protein BTLA molecule in the serum extracellular vesicles of patients with active TB is significantly higher than that of healthy individuals, and also significantly higher than that of patients with latent TB infection and lung cancer, with an area under the ROC curve greater than 0.7, indicating high diagnostic efficacy. Therefore, serum extracellular vesicle BTLA can serve as a diagnostic biomarker for active TB. Further analysis shows that serum extracellular vesicle BTLA can also serve as a biomarker for monitoring treatment efficacy and assessing bacterial load.

[0047] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. Application of extracellular vesicle B and T lymphocyte attenuating factors as diagnostic markers for active pulmonary tuberculosis in the preparation of diagnostic products for active pulmonary tuberculosis.

2. Application of extracellular vesicle B and T lymphocyte attenuating factors as biomarkers for evaluating the efficacy of treatment for active pulmonary tuberculosis in the preparation of products for evaluating the efficacy of treatment for active pulmonary tuberculosis.

3. Application of extracellular vesicle B and T lymphocyte weakening factors as biomarkers for assessing active tuberculosis load in the preparation of products for assessing active tuberculosis load.

4. The application according to any one of claims 1-3, characterized in that, The product is a reagent or kit.

5. The application according to claim 4, characterized in that, The product is applied using serum as the sample.

6. The application according to claim 4, characterized in that, The kit includes quality control products and / or standards.

Images