Application of FKBP5 and hsa-miR-320a-3p in diagnosis of tuberculosis in HIV infected population

Abstract

The application belongs to the technical field of biological medicine, and specifically discloses application of FKBP5 and hsa-miR-320a-3p in diagnosis of tuberculosis in HIV infected population. It is found that FKBP5 and hsa-miR-320a-3p can be used as biomarkers for diagnosing tuberculosis in HIV / AIDS population, and have the advantages of high sensitivity, high specificity, fast detection speed and the like, and are suitable for screening of tuberculosis in HIV / AIDS population. The application provides a new biomarker and detection method for diagnosis of HIV combined with mycobacterium tuberculosis infection, and is beneficial to early diagnosis and early treatment of HIV combined with mycobacterium tuberculosis infection, prolongs the life of patients, and improves the life quality of patients.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biological medicine, in particular to application of FKBP5 and hsa-miR-320a-3p in diagnosis of tuberculosis in HIV-infected people. BACKGROUND

[0002] Tuberculosis (TB) is caused by Mycobacterium tuberculosis (MTB) infection and is a chronic granulomatous infectious disease.

[0003] Human immunodeficiency virus (HIV) is the pathogen causing acquired immune deficiency syndrome (AIDS). HIV is divided into two types, HIV-1 and HIV-2. HIV-1 is the main pathogen causing AIDS.

[0004] Due to the immunosuppressive effect of HIV on the host, HIV-positive people have a 26-31 times higher risk of developing active tuberculosis than HIV-negative people. Active tuberculosis also accelerates the deterioration of the immune function of HIV-infected people, promotes the progression from HIV infection to AIDS, and increases the mortality rate.

[0005] Due to the huge global economic burden caused by HIV combined with Mycobacterium tuberculosis infection, it is urgent to screen all HIV-positive individuals for Mycobacterium tuberculosis infection in order to facilitate timely anti-tuberculosis treatment and prolong the lives of patients.

[0006] The most widely used laboratory tuberculosis diagnosis at present is based on sputum detection, including sputum smear and sputum culture. However, in HIV patients combined with Mycobacterium tuberculosis infection, the incidence of sputum smear-negative (culture-negative, symptomatic) and subclinical (culture-positive, asymptomatic active) tuberculosis is high. This leads to a decrease in the sensitivity of screening tests and subsequent diagnostic delays. In addition, although pulmonary tuberculosis is the most common active tuberculosis, it is more likely to develop into independent or concurrent extrapulmonary tuberculosis in HIV patients combined with Mycobacterium tuberculosis infection, especially when the CD4 + T cell count is low. The diagnosis of extrapulmonary tuberculosis is more difficult, and sputum-based diagnostic analysis cannot solve this problem.

[0007] Therefore, it is necessary to screen sensitive and convenient biomarkers of HIV combined with Mycobacterium tuberculosis infection for the screening of tuberculosis in HIV / AIDS populations, early diagnosis, early treatment, prolongation of life and improvement of the quality of life of patients with combined infection. SUMMARY

[0008] In view of the above-mentioned disadvantages of the prior art, the purpose of the present application is to provide the application of FKBP5 and hsa-miR-320a-3p in the diagnosis of tuberculosis in HIV-infected populations, to solve the problems of low sensitivity, low specificity or time-consuming in the prior art tuberculosis diagnosis method for HIV-infected populations.

[0009] To achieve the above-mentioned purpose and other related purposes, the first aspect of the present application provides the application of FKBP5 as a biomarker in the preparation of a detection kit for diagnosing tuberculosis in HIV-infected populations, monitoring and evaluating the efficacy of HIV combined with Mycobacterium tuberculosis infection.

[0010] The HIV-infected population in the present application refers to HIV / AIDS patients, including HIV-infected individuals and AIDS patients, wherein the HIV-infected individual refers to an individual who has been infected with HIV but has not yet developed to the AIDS stage, and the AIDS patient refers to a patient who has developed to the AIDS stage after being infected with HIV (see the national health industry standard WS293-2019 "AIDS and HIV infection diagnosis" implemented on July 1, 2019).

[0011] Optionally, the detection kit is used to determine the expression level of FKBP5 in the body fluid sample.

[0012] Optionally, the method for determining the expression level of FKBP5 in the body fluid sample by the detection kit is selected from any one of real-time fluorescent quantitative PCR (q-PCR), Western blot, immunochemiluminescence method, immunoturbidimetry, immunoblotting method, enzyme-linked immunosorbent assay, and preferably real-time fluorescent quantitative PCR (q-PCR).

[0013] Optionally, the body fluid sample is selected from at least one of whole blood nucleated cells, serum, plasma, and cerebrospinal fluid.

[0014] The second aspect of the present application provides the application of hsa-miR-320a-3p as a biomarker in the preparation of a detection kit for diagnosing tuberculosis in HIV-infected populations, monitoring and evaluating the efficacy of HIV combined with Mycobacterium tuberculosis infection.

[0015] Optionally, the detection kit is used to determine the expression level of hsa-miR-320a-3p in the body fluid sample.

[0016] Optionally, the detection kit determines the expression level of hsa-miR-320a-3p in the body fluid sample by real-time fluorescent quantitative PCR (q-PCR).

[0017] Optionally, the body fluid sample is selected from at least one of whole blood nucleated cells, serum, plasma, cerebrospinal fluid.

[0018] The third aspect of the present application provides that FKBP5 and hsa-miR-320a-3p are used as biomarkers in the preparation of a detection kit for the diagnosis of tuberculosis in HIV infected population, and the efficacy monitoring and evaluation of HIV combined with Mycobacterium tuberculosis infection.

[0019] The fourth aspect of the present application provides a detection kit for the diagnosis of tuberculosis in HIV infected population, and the efficacy monitoring and evaluation of HIV combined with Mycobacterium tuberculosis infection by determining the expression level of FKBP5 and / or hsa-miR-320a-3p in a body fluid sample.

[0020] Optionally, the method for determining the expression level of FKBP5 in the body fluid sample is selected from any one of real-time fluorescent quantitative PCR (q-PCR), Western blot, immunochemiluminescence method, immunoturbidimetry, immunoblotting method, enzyme-linked immunosorbent assay, and preferably real-time fluorescent quantitative PCR (q-PCR).

[0021] Optionally, the body fluid sample is selected from at least one of whole blood nucleated cells, serum, plasma, cerebrospinal fluid.

[0022] As described above, the application of FKBP5 and hsa-miR-320a-3p in the present application for the diagnosis of tuberculosis in HIV infected population has the following beneficial effects:

[0023] The present application provides two new biomarkers with high sensitivity, good specificity and fast detection speed for the diagnosis of HIV combined with Mycobacterium tuberculosis infection. According to the expression level of FKBP5 and hsa-miR-320a-3p in the body of the test subject, it can be evaluated whether there is Mycobacterium tuberculosis infection in the HIV infected population, and the efficacy of HIV combined with Mycobacterium tuberculosis infection can also be monitored and evaluated.

[0024] The technical means provided by the present application is conducive to the early diagnosis and early treatment of HIV combined with Mycobacterium tuberculosis infection, prolongs the life of patients and improves their quality of life. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The scatter plot shows the expression level of FKBP5 in the blood nucleated cells of HIV / AIDS patients and HIV / TB patients in the embodiments of the present application.

[0026] Figure 2A scatter plot showing the expression level of hsa-miR-320a-3p in blood nucleated cells of HIV / AIDS patients and HIV / TB patients in the embodiments of the present application.

[0027] Figure 3 A receiver operating characteristic (ROC) plot showing the expression level of FKBP5 in blood nucleated cells of HIV / AIDS patients and HIV / TB patients in the embodiments of the present application.

[0028] Figure 4 A receiver operating characteristic (ROC) plot showing the expression level of hsa-miR-320a-3p in blood nucleated cells of HIV / AIDS patients and HIV / TB patients in the embodiments of the present application.

[0029] Figure 5 A receiver operating characteristic (ROC) plot showing the expression level of FKBP5 and hsa-miR-320a-3p in blood nucleated cells of HIV / AIDS patients and HIV / TB patients in the embodiments of the present application. DETAILED DESCRIPTION

[0030] Other advantages and effects of the present application can be easily understood by those skilled in the art from the above description of the embodiments of the present application. The present application can also be implemented or applied by other different embodiments, and the details in the present specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present application.

[0031] According to the guidelines of the World Health Organization (WHO), active tuberculosis refers to tuberculosis with clinical symptoms and signs, as well as current pathological, bacteriological and / or radiological evidence. It is defined by one or more of the following diagnostic methods:

[0032] (1) Acid-fast bacilli are identified or detected in culture from any clinical specimen such as bronchoalveolar lavage, sputum, endotracheal aspirate, pleural effusion;

[0033] (2) Histological examination of the specimen shows caseous granulomas;

[0034] (3) Mycobacterium tuberculosis DNA is detected using PCR;

[0035] (4) Chest X-ray examination combined with clinical symptoms.

[0036] Non-active tuberculosis refers to individuals who have had tuberculosis or stable imaging abnormalities without bacteriological evidence, without clinical symptoms and / or radiological evidence, with positive TST test or IGRA detection.

[0037] Laboratory diagnosis of HIV includes antibody detection, etiology detection, nucleic acid detection.

[0038] (1) Antibody detection: mainly includes enzyme-linked immunosorbent assay, chemiluminescence or immunofluorescence test, simple test (gelatin particle agglutination test), rapid test (dot-ELISA and dot immunogold or colloidal selenium), HIV self-test, etc. HIV antibody confirmation test methods include Western blotting (Western blotting, WB), strip immunoblot assay (strip immunoblot assay, SIA; linear immunoblot assay, LIA), immunofluorescence assay (immunofluorescence assay, IFA) and radio-immuno-precipitation assay (radio-immuno-precipitation assay, RIPA).

[0039] (2) Etiological detection: including HIV p24 antigen detection, virus culture.

[0040] (3) Nucleic acid detection: including quantitative detection and qualitative detection.

[0041] In the population infected with HIV and Mycobacterium tuberculosis, it is more likely to develop independent or concurrent extrapulmonary tuberculosis, and the detection means for active pulmonary tuberculosis is not suitable for detecting extrapulmonary tuberculosis in the population infected with HIV and Mycobacterium tuberculosis, or the diagnosis is more difficult.

[0042] Based on this, the application provides two high-sensitivity, high-specificity, rapid and convenient biomarkers for HIV and Mycobacterium tuberculosis infection, which are FKBP5 and hsa-miR-320a-3p.

[0043] FKBP5 gene encodes FKBP5 protein, which is also called FKBP51 protein, belongs to immunophilin family, and is composed of FKBP and tetrapeptide repeat domain (TPR). One of the main functions of FKBP5 is to participate in the regulation of the function of steroid receptors (including progesterone, androgen and glucocorticoid receptors) by forming a complex with heat shock proteins Hsp90 / Hsp70.

[0044] Hsa-miR-320a-3p is a microRNA that targets and regulates FKBP5 gene, can target 3'UTR region of gene, and regulates gene expression at post-transcriptional level by recognizing homologous sequences and interfering transcription, translation or epigenetic process.

[0045] The research of the embodiment of the application includes two groups of participants. The control group is HIV-positive adults (over 18 years old), 18 people; the experimental group is HIV-positive adults (over 18 years old) combined with active tuberculosis, 17 people, and the active tuberculosis is diagnosed according to the Tuberculosis Diagnosis Standard (WS288-2017). The collected samples are the remaining blood of the patients, which is provided by the Chongqing Public Health Medical Treatment Center (in accordance with the requirements of the Chongqing Public Health Medical Treatment Center Ethics Committee).

[0046] The researcher characteristics are shown in Table 1.

[0047] Table 1. Researcher characteristics

[0048]

[0049] The research process of the embodiment is as follows:

[0050] I. Detection of blood cell FKBP5 and hsa-miR-320a-p expression in HIV-infected patients with tuberculosis

[0051] The whole blood of 18 HIV / AIDS patients and 17 HIV / TB patients (i.e. HIV patients combined with Mycobacterium tuberculosis infection) was collected, the serum was discarded after centrifugation, and Beyotime red blood cell lysis solution (red blood cell lysis solution to red blood cell ratio was 10:1) was added, mixed and lysed for 10 min, centrifuged at 400g for 5 min, the supernatant was discarded, and the above steps were repeated once again. After the second lysis, the nucleated cells were resuspended with PBS, centrifuged at 400g for 5 min, the supernatant was discarded, and finally the nucleated cells of the blood were obtained. The total RNA was extracted from each sample using Invitrogen TRIzol, and its concentration and purity were determined using IMPLEN spectrophotometer. The mRNA was reverse transcribed using Takara reverse transcription kit, and the reaction conditions were 37℃ for 15 min, 85℃ for 5 s, and 4℃ for ending. The microRNA was reverse transcribed using Accurate Biotechnology miRNA 1st strand cDNA synthesis kit, and the reverse transcription conditions were 37℃ for 60 min, 85℃ for 5 min, and 4℃ for ending.

[0052] Next, PCR was performed using SYBR® Premix ExTaq™ Kit (Takara) based on LightCycler® 480 II real-time PCR system (Roche). The q-PCR program was set to pre-denature at 95°C for 30 s, incubate at 95°C for 5 s, incubate at 60°C for 30 s, and cycle 40 times. GAPDH was used as the mRNA internal control, and U6 was used as the miRNA internal control. The relative expression of each selected mRNA and miRNA between cases and controls was determined using the 2-ΔΔCt method. The primer sequences used in this example are shown in Table 2.

[0053] Table 2. Primer sequences

[0054]

[0055] Figure 1 A scatter plot showing the expression level of FKBP5 in the blood nucleated cells of HIV / AIDS patients and HIV / TB patients.

[0056] As can be seen from FIG. 1, when HIV / AIDS patients are complicated with active tuberculosis, the expression level of FKBP5 is significantly increased, and the difference is statistically significant, Mann-Whitney test p = 0.0055.

[0057] Figure 2 A scatter plot showing the expression level of hsa-miR-320a-3p in the blood nucleated cells of HIV / AIDS patients and HIV / TB patients.

[0058] As can be seen from FIG. 2, when HIV / AIDS patients are complicated with active tuberculosis, the expression level of hsa-miR-320a-3p is significantly decreased, and the difference is statistically significant, Mann-Whitney test p < 0.0001.

[0059] II. ROC curve analysis of FKBP5 and hsa-miR-320a-3p for the diagnostic efficiency of AIDS infection related tuberculosis

[0060] The expression data obtained in Step 1 above was subjected to ROC curve drawing. The R (version 3.6.3) software was used, and the statistical analysis and visualization methods were as follows: the logistics model was constructed using the glm function, the ROC analysis was performed using the pROC package, and the visualization was performed using the ggplot2 package.

[0061] Figure 3 A receiver operating characteristic (ROC) curve graph showing the expression level of FKBP5 in the blood nucleated cells of HIV / AIDS patients and HIV / TB patients.

[0062] As can be seen from FIG. 3, when HIV / AIDS patients are complicated with active tuberculosis, the expression level of FKBP5 is significantly increased, and the difference is statistically significant, Mann-Whitney test p = 0.0055.Figure 3 It can be known that the area under the curve is 0.770, which proves that the use of FKBP5 to diagnose HIV / TB patients has better diagnostic efficiency.

[0063] Figure 4 The receiver operating characteristic (ROC) curve is shown according to the expression level of hsa-miR-320a-3p in blood nucleated cells of HIV / AIDS patients and HIV / TB patients.

[0064] From Figure 4 It can be known that the area under the curve is 0.889, which proves that the use of hsa-miR-320a-3p to diagnose HIV / TB patients has better diagnostic efficiency.

[0065] Figure 5 The receiver operating characteristic (ROC) curve is shown according to the expression level of FKBP5 and hsa-miR-320a-3p in blood nucleated cells of HIV / AIDS patients and HIV / TB patients.

[0066] From Figure 5 It can be known that the area under the curve is 0.886, which proves that the use of FKBP5 and hsa-miR-320a-3p to diagnose HIV / TB patients has better diagnostic efficiency.

[0067] In summary, the present application verifies by experiment that FKBP5 and hsa-miR-320a-3p can be used as biomarkers for diagnosing tuberculosis in HIV / AIDS population, and have the advantages of high sensitivity, high specificity, fast detection speed, etc., and are suitable for screening tuberculosis in HIV / AIDS population, which is conducive to early diagnosis and early treatment of HIV patients infected with Mycobacterium tuberculosis, prolongs the life of patients and improves their quality of life.

[0068] The above examples only exemplarily illustrate the principles and effects of the present application, and are not used to limit the present application. Any person skilled in the art can modify or change the above examples without departing from the spirit and scope of the present application. Therefore, all equivalent modifications or changes completed by those skilled in the art without departing from the spirit and technical thought disclosed by the present application should be covered by the claims of the present application.

Claims

1. Use of FKBP5 as a biomarker for the preparation of a test kit for the diagnosis of active tuberculosis in HIV-infected populations.

2. Use according to claim 1, characterized in that: The test kit is used to determine the expression level of FKBP5 in a body fluid sample.

3. Use according to claim 2, characterized in that: The method for determining the expression level of FKBP5 in a body fluid sample by the test kit is selected from any one of real-time fluorescent quantitative PCR, immunoblotting test, immunochemiluminescence method, immunoturbidimetry, enzyme-linked immunosorbent assay.

4. Use according to claim 2, characterized in that: The body fluid sample is selected from at least one of whole blood nucleated cells, serum, plasma, cerebrospinal fluid.

5. Use of hsa-miR-320a-3p as a biomarker for the preparation of a test kit for the diagnosis of active tuberculosis in HIV-infected populations.

6. Use according to claim 5, characterized in that: The test kit is used to determine the expression level of hsa-miR-320a-3p in a body fluid sample.

7. Use according to claim 6, characterized in that: The test kit determines the expression level of hsa-miR-320a-3p in a body fluid sample by real-time fluorescent quantitative PCR.

8. Use according to claim 6, characterized in that: The body fluid sample is selected from at least one of whole blood nucleated cells, serum, plasma, cerebrospinal fluid.

9. Use of FKBP5 and hsa-miR-320a-3p in combination as biomarkers for the preparation of a test kit for the diagnosis of active tuberculosis in HIV-infected populations.

10. A test kit comprising: The test kit comprises test reagents for the expression levels of FKBP5 and hsa-miR-320a-3p, and is used for the diagnosis of active tuberculosis in HIV-infected populations by determining the expression levels of FKBP5 and hsa-miR-320a-3p in a body fluid sample.

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