Kit for identifying active tuberculosis infection and latent tuberculosis infection and application thereof
Patent Information
- Application Number
- CN202510194697.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-21
- Publication Date
- 2025-05-23
AI Technical Summary
The prior art is difficult to effectively distinguish between active tuberculosis and latent tuberculosis infection, especially in countries with high tuberculosis burden.
A kit, combined with flow cytometry, distinguishes between active and latent tuberculosis infection by detecting the proportion of V1 and V2 cells in tuberculosis-specific CD154+CD4+ T cells.
It achieves a simple and efficient distinction between active tuberculosis and latent tuberculosis infection, has high operational sensitivity, strong specificity, and can be quickly diagnosed.
Smart Images
Figure HDA0005281119110000011 
Figure HDA0005281119110000021 
Figure HDA0005281119110000031
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of medical detection, and in particular to a kit for distinguishing active tuberculosis and latent tuberculosis infection and application thereof. Background Art
[0002] Tuberculosis (TB) remains a major infectious disease that threatens human health. On October 29, 2024, the World Health Organization (WHO) released its latest TB report, which showed that there will be 10.8 million new TB patients and 1.25 million deaths in 2023, with TB returning to the top spot as the single infectious disease cause of death in the world. The estimated number of new TB patients in my country in 2023 is 741,000, ranking third among the 30 countries with a high TB burden. WHO stressed that we are still far from ending the global tuberculosis epidemic.
[0003] Early, efficient, and accurate diagnosis is the key factor in controlling the TB epidemic. However, TB diagnosis still faces many problems that cannot be overcome: (1) Methods based on pathogen detection, such as Mycobacterium tuberculosis (MTB) nucleic acid detection (Xpert MTB / RIF) and MTB culture, have a high proportion of false negatives caused by paucibacillary and difficult-to-obtain MTB infections; (2) Methods based on host markers, such as cytokines, metabolites, and RNA markers, do not meet the requirements for specificity. These substances are also increased in other inflammatory diseases such as bacterial pneumonia; (3) Methods based on detection products after antigen induction cannot distinguish between active tuberculosis (ATB) and latent tuberculosis infection (LTBI), especially in countries with a high burden of tuberculosis. Finding a method to effectively distinguish different tuberculosis infection states, namely ATB and LTBI, is not only useful for differential diagnosis of ATB, which is particularly important in countries with a high burden of tuberculosis, but also the diagnosis and status judgment of LTBI is a prerequisite for early prediction of ATB incidence. Therefore, accurately distinguishing between active tuberculosis and latent tuberculosis, two types of tuberculosis infection, plays an important role in tuberculosis prevention and control in countries with a high tuberculosis burden.
[0004] The IFN-γ release assay (IGRA) is a new technology developed in recent years that can be used for the diagnosis of MTB infection. The principle of this technology is that both ATB and LTBI individuals have tuberculosis-specific T cells. When T cells sensitized by MTB antigens encounter the same antigen again, they can produce high levels of IFN-γ. Therefore, it can be used to diagnose ATB and LTBI individuals by quantitatively detecting the IFN-γ released by the subject's peripheral blood mononuclear cells under the stimulation of MTB-specific antigens. The IGRA test is not affected by BCG vaccination and non-tuberculous mycobacterium infection, and has high sensitivity and specificity for detecting MTB infection. Although IGRA can quickly diagnose MTB infection, the biggest limitation of this method is that it cannot be used to distinguish ATB from LTBI, especially in countries with a high burden of tuberculosis.
[0005] In summary, there is an urgent need to develop a product that can distinguish active tuberculosis from latent tuberculosis infection. Summary of the invention
[0006] The present invention aims to provide a kit for distinguishing active tuberculosis from latent tuberculosis infection and its application. The kit can simply and efficiently distinguish active tuberculosis infection from latent tuberculosis infection in combination with flow cytometry.
[0007] To achieve the above object, the present invention adopts the following technical solution:
[0008] In a first aspect of the present invention, a kit for distinguishing active tuberculosis from latent tuberculosis infection is provided, the kit comprising: fluorescently labeled CD154 antibody and CD4 antibody for multicolor flow cytometry detection.
[0009] Furthermore, the fluorescent molecules on the fluorescently labeled CD154 antibody and the fluorescent molecules on the fluorescently labeled CD4 antibody can be distinguished when multicolor flow cytometry is performed.
[0010] As a specific embodiment, the CD154 antibody is labeled with PE, and the CD4 antibody is labeled with APC-Cy7.
[0011] Furthermore, the kit also includes:
[0012] AF700 fluorescently labeled FVS antibody, i.e. FVS-AF700.
[0013] The FVS-AF700 is used to distinguish between dead cells and living cells. FVS-AF700 positive cells are dead cells. - The cells were gated as live cells.
[0014] In a second aspect of the present invention, a method for distinguishing active tuberculosis from latent tuberculosis infection is provided, the method comprising:
[0015] Peripheral blood mononuclear cells were induced using ESAT-6 and CFP-10;
[0016] Add the fluorescently labeled antibodies and FVS-AF700 in the kit to the induced peripheral blood mononuclear cells;
[0017] Detection of tuberculosis-specific CD154 by multicolor flow cytometry + CD4 + The proportion of V1 and V2 type cells in T cells.
[0018] Furthermore, the tuberculosis-specific CD154 + CD4+ The proportion of V1 and V2 cells in T cells specifically includes:
[0019] Use flow cytometry to detect lymphocytes, circle the lymphocyte gate based on the forward scatter angle (FSC) and side scatter angle (SSC), and select FVS in the lymphocyte gate. - Cell gate as live cells,
[0020] CD154 in living cells - CD4 + The vertical line at the apex of the T cell cluster is taken as the central axis, and the CD154 + CD4 + The left V-shaped circle gate of T cells is type V1, and the right V-shaped circle gate is type V2;
[0021] The percentage of V1 and V2 cell numbers was analyzed as the measurement result:
[0022] If the proportion of V1 type accounts for more than 50% of the total of V1 and V2, that is, V1 / (V1+V2)>50%, it is active tuberculosis infection;
[0023] If the proportion of V2 accounts for more than 50% of the total of V1 and V2, that is, V2 / (V1+V2)>50%, it is latent tuberculosis infection.
[0024] When and only when V1 / (V1+V2)=50%, it indicates that the test result is in a critical state and needs to be comprehensively judged in combination with other clinical indicators.
[0025] In the third aspect of the present invention, there is provided the use of the kit for distinguishing active tuberculosis from latent tuberculosis infection in the preparation of products for diagnosing active tuberculosis and latent tuberculosis infection.
[0026] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:
[0027] 1. The present invention provides a kit for distinguishing active tuberculosis from latent tuberculosis infection, which detects tuberculosis-specific CD4 + The proportion of V1 and V2 types of T cell CD154 molecules can be manually judged as active or latent tuberculosis infection. The operation is simple and can achieve rapid diagnosis.
[0028] 2. The kit provided by the present invention for distinguishing active tuberculosis from latent tuberculosis infection has strong specificity and high sensitivity: the performance of using the V1 type proportion to distinguish active tuberculosis from latent tuberculosis is good, and the area under the ROC curve reaches 0.991. When the V1 type proportion is greater than 50% as the critical value, the sensitivity of using the V1 type proportion to distinguish active tuberculosis from latent tuberculosis reaches 94.44%, and the specificity reaches 95.24%. BRIEF DESCRIPTION OF THE DRAWINGS
[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.
[0030] Figure 1 Detection of tuberculosis-specific CD4 + Simulation diagram and actual flow cytometry gate diagram of V-shaped expression of CD154 molecule on T cells.
[0031] Figure 2 CD154 of V1 and V2 types in 36 patients with active tuberculosis infection and 21 patients with latent tuberculosis infection + CD4 + The percentage of T cells.
[0032] Figure 3 ROC curve for distinguishing ATB from LTBI using V1 type proportion data. DETAILED DESCRIPTION
[0033] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.
[0034] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.
[0035] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.
[0036] Unless otherwise specified, the reagents involved in the embodiments of the present invention are all commercially available products and can be purchased through commercial channels.
[0037] APC-Cy7 labeled CD4 antibody (clone number, SK3; catalog number, 341115), PE labeled CD154 antibody (clone number, 24-31; catalog number, 310806).
[0038] Definition and Use of Terms
[0039] CD154 - CD4 + T cells: a subset of T cells that are CD154 negative and CD4 positive.
[0040] CD154 + CD4 + T cells: T cell subsets that are CD154 antibody positive and CD4 positive.
[0041] Tuberculosis-specific CD4 for identification of tuberculosis infection status + V-shaped expression pattern of CD154 molecules on T cells: including V1 and V2 types, the gating strategy is based on CD154 - CD4 + The vertices in the T cell flow cytometry graph are used as reference to divide the V-shape into two sides. The left V-shape is V1 type, and the right V-shape is V2 type.
[0042] The general idea of the present invention is as follows:
[0043] According to a typical embodiment of the present invention, a kit for distinguishing active tuberculosis from latent tuberculosis infection is provided, the kit comprising: fluorescently labeled CD154 antibody and CD4 antibody for multicolor flow cytometry detection.
[0044] Specifically, for flow cytometry detection of tuberculosis-specific CD4 + Antibodies with a V-shaped expression pattern of CD154 molecules on T cells can be prepared into a kit in a separately packaged manner, such as a kit containing fluorescently labeled antibodies against CD154 and CD4. In some preferred embodiments, the kit also includes fluorescently labeled FVS.
[0045] In some preferred embodiments, antibodies to CD4, CD154 and FVS are fluorescently labeled with APC-Cy7, PE and AF700, respectively. In some other embodiments, other fluorescent molecules may also be linked to the antibodies, in principle so that they can be distinguished during multi-color flow cytometry.
[0046] In some preferred embodiments, the kit may further include antigen peptides for inducing peripheral blood mononuclear cells: ESAT-6 and CFP-10.
[0047] According to another typical embodiment of the present invention, a method for distinguishing active tuberculosis from latent tuberculosis infection is provided, comprising:
[0048] Peripheral blood mononuclear cells were induced using ESAT-6 and CFP-10;
[0049] Add the fluorescently labeled antibodies and FVS-AF700 in the kit to the induced peripheral blood mononuclear cells;
[0050] Detection of tuberculosis-specific CD154 by multicolor flow cytometry + CD4 + The proportion of V1 and V2 type cells in T cells.
[0051] As a specific embodiment, the tuberculosis-specific CD154 + CD4 + The proportion of V1 and V2 cells in T cells includes:
[0052] Use flow cytometry to detect lymphocytes, circle the lymphocyte gate based on the forward scatter angle (FSC) and side scatter angle (SSC), and select FVS in the lymphocyte gate. - Cell gate as live cells,
[0053] CD154 in living cells - CD4 + The vertical line at the apex of the T cell cluster is taken as the central axis, and the CD154 + CD4 + The left V-shaped circle gate of T cells is V1 type, and the right V-shaped circle gate is V2 type.
[0054] The percentage of V1 and V2 cell numbers was analyzed as the measurement result:
[0055] If the proportion of V1 type accounts for more than 50% of the total of V1 and V2, it is active tuberculosis infection;
[0056] If the proportion of V2 type accounts for more than 50% of the total of V1 and V2, it is latent tuberculosis infection.
[0057] The ratio of the number of V1 and V2 cells mentioned above: the number of V1 and V2 cells is automatically counted after the gate on the flow cytometer, and then the V1 ratio and V2 ratio are obtained by calculation.
[0058] The present invention provides a kit for distinguishing active tuberculosis and latent tuberculosis infection, which is combined with flow cytometry to determine the CD154 molecule expression pattern, and the V-shaped expression pattern includes V1 and V2 types. The gate strategy of V1 and V2 types is based on CD154 - CD4+ The vertices in the T cell flow graph are used as references to divide the V-shape into two sides, the left V-shape is V1 type, and the right V-shape is V2 type. If the proportion of V1 type accounts for more than 50% of the total of V1 and V2, it corresponds to an individual with active tuberculosis infection (ATB); if the proportion of V2 type accounts for more than 50% of the total of V1 and V2, it corresponds to an individual with latent tuberculosis infection (LTBI), thus making it easy and efficient to distinguish active tuberculosis from latent tuberculosis infection.
[0059] The present application will be described in detail below with reference to embodiments and experimental data.
[0060] Example 1. Kit for distinguishing active tuberculosis from latent tuberculosis infection
[0061] The embodiment of the present invention provides a kit for distinguishing active tuberculosis and latent tuberculosis infection, comprising:
[0062] ESAT-6 and CFP-10;
[0063] PE-labeled CD154 antibody;
[0064] APC-Cy7 labeled CD4 antibody;
[0065] FVS-AF700.
[0066] Example 2: Method for distinguishing active tuberculosis from latent tuberculosis infection
[0067] A total of 57 samples were collected, including 36 patients with clinical active tuberculosis infection and 21 patients with latent tuberculosis infection. The inclusion criteria for active tuberculosis infection were positive Mycobacterium tuberculosis PCR or culture in the patient's clinical samples, and the patient had clinical symptoms and imaging manifestations related to active tuberculosis infection. The inclusion criteria for latent tuberculosis infection were T-SPOT.TB positive but the infected person had no clinical symptoms and imaging manifestations related to active tuberculosis infection.
[0068] 1. Cell separation:
[0069] 4 mL of heparin-anticoagulated peripheral blood was collected from each test subject, and peripheral blood mononuclear cells (PBMC) were separated by density gradient centrifugation. The cells were resuspended in 1640 cell culture medium containing 10% calf serum for later use.
[0070] 2. Cell culture:
[0071] Use two flow cytometry tubes. The first tube is a negative control tube. Add 1×10 6 PBMC, the final volume is 500mL; the second tube is the assay tube: add 1×10 6PBMC, the final volume is 500mL, ESAT-6 and CFP-10 are added for induction, so that the concentration of ESAT-6 and CFP-10 in the culture medium is 2μg / mL respectively; PE-labeled CD154 antibody is added to each tube for pre-labeling before the start of culture; the negative control tube and the assay tube are placed at 37℃, 5% CO 2 Incubate in an incubator for 24 hours.
[0072] 3. Multicolor flow cytometry detection:
[0073] After the culture was completed, 350 mL of supernatant was discarded, 0.5 mL of AF700 fluorescently labeled FVS and 5 mL of APC-Cy7 labeled CD4 antibody were added to each flow tube, and the mixture was gently shaken and kept at room temperature and protected from light for 20 min; 2 mL of PBS washing solution was added and shaken to mix, and the supernatant was discarded after centrifugation; 500 mL of PBS was added to resuspend the cells and the flow cytometer was used for detection, and the lymphocyte gate was circled in the forward scatter angle (FSC) and side scatter angle (SSC), and FVS was placed in the lymphocyte gate. - Cells were gated as live cells and CD154 - CD4 + The vertices in the T cell flow cytometry graph were used as references, and CD154 + CD4 + The left V-shaped circle gate of T cells is V1 type, and the right V-shaped circle gate is V2 type, and the proportion of V1 and V2 types is analyzed as the measurement result.
[0074] TB-specific CD154 + CD4 + T cell flow cytometry V-circle gate Figure 1 shown.
[0075] 4. Test results
[0076] The results of the proportion of V1 and V2 cells in the total V1 and V2 of 36 cases of active tuberculosis infection and 21 cases of latent tuberculosis infection are as follows Figure 2 shown.
[0077] Example 3: Sensitivity and specificity analysis
[0078] The ROC curve was drawn using the V1 type proportion data to analyze the performance of V1 type proportion in distinguishing ATB from LTBI. The results are as follows: Figure 3 shown.
[0079] Depend on Figure 3It can be seen that the use of V1 type proportion to distinguish active tuberculosis from latent tuberculosis has a good performance, and the area under the ROC curve reaches 0.991. When the V1 type proportion is greater than 50% as the critical value, the sensitivity of using V1 type proportion to distinguish active tuberculosis from latent tuberculosis reaches 94.44%, and the specificity reaches 95.24%. Application example: tuberculosis-specific CD4 + Application of V-shaped expression pattern of CD154 molecule on T cells
[0080] Peripheral blood mononuclear cells of the sample to be tested were obtained, ESAT-6 and CFP-10 were used to induce the peripheral blood mononuclear cells, PE-labeled CD154 antibody was added to the culture medium for pre-labeling, the induced peripheral blood mononuclear cells were treated with FVS-AF700 and APC-Cy7 labeled CD4 antibodies, and the cell surface markers CD4 and CD154 were determined by flow cytometry.
[0081] If CD4 + If the T cells do not express CD154, then the cells are not tuberculosis-specific CD154 + CD4 + T cells, i.e., samples from people with inactive or latent tuberculosis infection;
[0082] If CD4 + If the T cells express CD154, then the cells are tuberculosis-specific CD154 + CD4 + T cells from people with active or latent tuberculosis infection.
[0083] For tuberculosis-specific CD154 + CD4 + T cells, the left V-shaped circle gate is V1 type, the right V-shaped circle gate is V2 type, and the V1 and V2 type ratios are analyzed as the measurement results. If the individual V1 type ratio accounts for more than 50% of the total V1 and V2, the individual is an active tuberculosis infected person; if the individual V2 type ratio accounts for more than 50% of the total V1 and V2, the individual is judged to be a latent tuberculosis infected person.
[0084] Individual 1, female, 50 years old, basic information of individual 1: no clinical manifestations of active tuberculosis such as cough and fever, normal lung CT, positive IGRA, in line with the diagnostic criteria for latent tuberculosis. + CD4 + The T cell ratio result was 0.85, which can be interpreted as tuberculosis infection. + CD4 + In the T cell V-circle gate, the V1 type accounted for 21.6% and the V2 type accounted for 78.4%, and the individual was subsequently diagnosed as a latent tuberculosis infected person.
[0085] Individual 2, male, 55 years old, basic information of individual 2: cough and sputum for more than 3 months, low-grade fever in the afternoon, night sweats, lung CT showed patchy shadows and cavitation, bronchoscopic washing fluid Xpert MTB / RIF positive, meeting the diagnostic criteria for active tuberculosis. + CD4 + The T cell ratio result was 1.21, which can be interpreted as tuberculosis infection. + CD4 + In the T cell V-circle gate, the V1 type accounted for 96.6% and the V2 type accounted for 3.4%, and the individual was subsequently diagnosed as having active tuberculosis infection.
[0086] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.
[0087] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.
[0088] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.
Claims
1. A kit for distinguishing active tuberculosis from latent tuberculosis infection, characterized in that: The kit comprises: CD154 antibody and CD4 antibody with fluorescence labeling for multicolor flow cytometry detection.
2. A kit for distinguishing active tuberculosis and latent tuberculosis infection according to claim 1, characterized in that: The fluorescent molecules on the fluorescently labeled CD154 antibody and the fluorescent molecules on the fluorescently labeled CD4 antibody can be distinguished when multicolor flow cytometry is performed.
3. A kit for distinguishing active tuberculosis and latent tuberculosis infection according to claim 1, characterized in that: The CD154 antibody was labeled with PE, and the CD4 antibody was labeled with APC-Cy7.
4. A kit for distinguishing active tuberculosis and latent tuberculosis infection according to claim 1, characterized in that: The kit also includes: AF700 fluorescently labeled FVS antibody, i.e. FVS-AF700.
5. A kit for distinguishing active tuberculosis and latent tuberculosis infection according to claim 1, characterized in that: The kit also includes: Antigenic peptides for induction of peripheral blood mononuclear cells: ESAT-6 and CFP-10.
6. Use of the kit for distinguishing active tuberculosis and latent tuberculosis infection according to any one of claims 1 to 5 in the preparation of products for diagnosing active tuberculosis and latent tuberculosis infection.