RU2503005C1 - Diagnostic technique for pulmonary tuberculosis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: patient's whole blood is incubated with mycobacterial antigens representing a mixture of the proteins ESAT-6, CFP-10, TB 7.7, centrifuged to separate plasma; the supernatant is examined for the content of antigen-induced gamma-interferon (IFNγ), antigen-induced interleukin-6 (IL-6) and spontaneous production of transforming growth factor-α (TGFα). If observing IFNγ ≥6.4 IU/ml, or IL-6≥2039 pg/ml, or TGFα≥17.0 pg/ml, the active tuberculosis infection is stated. All three values being below the threshold show the latent infection.

EFFECT: using the given technique enables the differential diagnostic between the individuals suffering active tuberculosis and latent tuberculosis infection.

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Description

The invention relates to clinical medicine, more specifically to phthisiology and immunology, and in particular to methods for assessing the condition of patients with pulmonary tuberculosis (TB).

In recent years, in many countries, regardless of their level of economic development, there has been an increase in the incidence and spread of tuberculosis, which is increasingly mentioned among the so-called "resurgent" infections. In order to prevent the spread of tuberculosis, the possibility of diagnosing this disease, in particular, identifying the contingent who is sick with tuberculosis in an active form and is dangerous to others, is of great importance. This is especially true in the context of the widespread latent form of tuberculosis, which does not pose a serious danger to others, and the limited capabilities of medical institutions.

Currently, the most widespread diagnosis of diseases using the Mantoux test and, in the presence of a positive reaction, bacteriological methods of analysis. However, the result of the Mantoux intradermal allergy test is taken into account after 72 hours, and due to the high percentage of infection in the TB population, the frequency of confirming the diagnosis of the disease in different age groups is from 25 to 50% (Borodulina E.A., Borodulin B.E. Differential diagnosis of post-vaccination and infectious tuberculin allergy in children with atopic diseases. // Problems of Tuberculosis and Pulmonary Diseases. - No. 1, 2006, - p. 9-12). The results of the Mantoux test are influenced by previous vaccination, uncontrolled administration of broad-spectrum antibiotics and the presence of concomitant pathology, including allergopathology (Aksenova V.A., Ovsyankina E.S., Aleksandrova T.M. Methods of quality control during mass tuberculin diagnostics. // Problems of tuberculosis. - 2002, No. 2. - C.3-5). The disadvantages of bacteriological methods are the low detection rate of Mycobacterium tuberculosis (MBT) and the long-term growth of MBT in nutrient media (up to 30 days). All of the above determines the complexity of the clinical and laboratory diagnosis of tuberculosis and necessitates the development of faster and more reliable methods of laboratory diagnosis of tuberculosis infection.

Known accelerated method of bacteriological diagnosis of tuberculosis according to the Price method, which allows you to select and identify the office after 7-14 days (Medical Microbiology, Virology and Immunology. / Ed. By A.A. Vorobyov. M .: Medical News Agency, 2004. - 671 p. .), however, these terms are also quite large, and the reliability of the method is rather low.

Enzyme-linked immunosorbent assay (ELISA) is widely used for the diagnosis of tuberculosis, which allows one to determine the presence of tuberculosis infection by the concentration (titer) of specific immunoglobulins of classes A, M, G, E (Avdeenko VG et al. Improving the effectiveness of tuberculosis immunodiagnosis by using monoclonal antibodies against human immunoglobulin G. / Clinical laboratory diagnostics. - 1999, No. 6. - C.22-35; Avdeenko V.G. et al. Anti-tuberculosis IgE antibodies. Immunodominant antigens // Problems of tuberculosis. - 2002, part 1, No. 2. - S.30-33). The disadvantage of this method is the need for re-examination of blood serum after 10-14 days to detect an increase in the titer of antibodies to MBT, indicating an active infectious process in the body.

A common drawback of the above methods is the duration of the analysis, their lack of selectivity, the inability to subdivide the patient population into patients with latent and active forms of tuberculosis, which does not allow for timely activities with patients from the second of the above groups.

One of the most promising areas for the diagnosis of tuberculosis (TB) is the in vitro analysis using antigens specific for Mycobacterium tuberculosis. As generalized analytical data show, in vitro cell tests are highly specific: 99% of vaccinated individuals have negative reactions, and 78% of tuberculosis patients have positive reactions (Menzies T. Meta-analysis: New tests for the diagnosis of latent tuberculosis infection: areas of uncertainty and recommendations for research march / T. Menzies, M. Pai, G. Comstock // Annals. Intern. Med. - 2007. - Vol. 146 - P.340-354; Pai M. T-cell based assays for the diagnosis of latent tuberculosis infection: an update / M. Pain, A. Zwerling, D. Menzies // Ann. Intern. Med. - 2008. - Vol. 149 (3). - P.177-184).

Currently, the T-SPOT test has been certified, which is an express analog of the standard diagnosis of tuberculosis based on delayed skin reactions (Skin test with the drug "DIASKINTEST" - new possibilities for identifying tuberculosis infection. Edited by Academician of the Russian Academy of Sciences and RAMS M.A. Paltseva The second edition, revised and enlarged. - M .: Publishing house "Chicot", 2011. 256 s). A method for immunological diagnosis and monitoring of T-SPOT tuberculosis infection (Oxford Immunotec, USPTO Applicaton No. 20070196878) is based on determining the number of cells producing interferon-gamma (IFNγ) in the presence of specific inducers such as ESAT-6 proteins (early secretion antigen) and CFP -10 (culture filtrate protein), which are absent in the mycobacteria vaccine strain M. bovis (BCG) and most non-tuberculous environmental mycobacteria, with the exception of Mycobacterium marinum and Mycobacterim kansasii. In this case, preliminary BCG vaccination does not significantly affect the results of in vitro IFNγ induction, and an increase in its number indicates the presence of tuberculosis infection (Mordovskaya L.I. IFNγ induction in whole venous blood samples in vitro is a test for determining tuberculosis infection in children and adolescents / L.I. Mordovskaya, M.A. Vladimirsky, V.A. Aksenova, E.E. Efremov, G.I. Ignashenkova, T.N. Vlasik // Problems of Tuberculosis and Pulmonary Diseases. -2009. -№ 6. - S.19-24).

The main disadvantages of this test are its complexity and complexity, the need to cultivate cells under sterile conditions, and the inability to diagnose on its basis whether the disease is latent or active.

The closest in essence to the method proposed in this invention is a method using the test system "QuantiFERON-TB Gold In-Tube" ("Cellestis", Australia), based on measuring the level of IFNγ production by peripheral blood cells during stimulation of blood samples Mycobacterium antigens. Proteins ESAT-6, CFP-10 and TB 7.7 (p4) are used as antigens (Mordovskaya L.I. Induction of IFNγ in whole venous blood samples in vitro - test for determining tuberculosis infection in children and adolescents. / L.I. Mordovskaya, M.A. Vladimirsky, V.A. Aksenova, E.E. Efremov, G.I. Ignashenkova, T.N. Vlasik // Problems of Tuberculosis and Pulmonary Diseases. - 2009. - No. 6. - P.19-24 ) The diagnostic method includes taking blood from a patient, subsequent cultivation of each sample of whole blood stabilized with heparin at 37 ° C for 18-24 hours in three tubes: the first (antigen-induced production) - in the presence of mycobacterial antigens, the second (spontaneous production) - without antigens, the third (mitogen-induced production) - in the presence of mitogen (the last test is used as a positive control). After cultivation, the tubes are centrifuged, plasma is taken and the IFNγ content in them is determined by enzyme-linked immunosorbent assay (ELISA). Results are evaluated using QFT 2.62 software provided by the manufacturer. The test results are considered positive if the difference between the antigen-induced and spontaneous IFNγ production is more than 0.35 IU / ml and the difference between the mitogen-induced and spontaneous IFNγ production is more than 0.5 IU / ml.

The main disadvantage of this method is that it does not allow differential diagnosis of active TB and latent tuberculosis infection (LTBI), which seems relevant in conditions of widespread (according to various estimates up to 80%) spread of tuberculosis infection.

The problem solved by the authors was the development of a fairly quick and reliable method for the diagnosis of tuberculosis, allowing differential diagnosis of active tuberculosis and latent tuberculosis infection. This problem was solved by establishing a circle of proteins produced by blood cells in patients infected with tuberculosis in the presence of Mycobacterium tuberculosis antigens, selecting specific cytokines and determining threshold values that would make it possible to diagnose the patient with active tuberculosis and latent tuberculosis infection.

The technical result was achieved by culturing the blood of patients with suspected tuberculosis in the presence of a commercially available mixture of proteins ESAT-6, CFP-10, TB 7.7 (from the QuantiFERON-TB Gold In-Tube kit) as antigens, followed by determination of interferon gamma (IFNγ), interleukin-6 (IL-6) and transforming growth factor alpha (TGFα), and diagnosing the active form of tuberculosis when at least one of the cytokines reaches a threshold value that is for: IFNγ _AG - 6.4 IU / ml, for TGFα _NIL - 17.0 pg / ml and for IL-6 _AG - 2039 pg / ml, and at values below the threshold for all three indicators - latent tuberculosis infection.

The proposed criteria are selected on the basis of clinical studies of 53 patients who have established the presence of infection with the office. The group included 27 patients with newly diagnosed, mainly infiltrative pulmonary tuberculosis, confirmed by the cultural method before initiating specific anti-TB treatment (13 women and 14 men aged 18 to 64 years, average age 36 years), and 26 people with latent infection (LTBI), which were employees of hospitals of TB facilities (n = 26).

We studied the spontaneous (NIL) and antigen-induced (AG) production of various cytokines, in particular epidermal growth factor (EGF), macrophage inflammatory protein - 1β (MIP-β), endothelial growth factor (VEGF), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-1alpha (IL-1α), interferon-alpha2 (IFN-α2), transforming growth factor alpha (TGFα), tumor necrosis factor-alpha (TNFα), as well as the soluble interleukin-2-alpha receptor (sIL-2Rα) and the soluble form of the CD40 ligand (sCD40L). The studies were performed using Milliplex Mag magnetic particles and a MagPix analyzer (Millipore, USA) using xMar technology.

For statistical calculations, the values of spontaneous (NIL) production of the studied biomarkers, antigen-induced (AG) products and the difference between them (AG-NIL) were used. Statistical processing was performed using the SPSS program (version 15.0). Mann-Whitney test was used to compare groups.

Table 1 shows the indicators characterizing the differences in the concentrations of cytokines for the compared groups.

Table 1 Comparative characteristics of indicators of spontaneous and stimulated secretion of cytokines in patients with active and latent forms of TB Indicator Patients with active TB (n = 27), Me (min, max) Persons with latent TB (n = 26), Me (min, max) R* Threshold value Sensitivity% Specificity,% AUC ** IFNγ _AG , IU / ml 6.4 (1.6-10.2) 1.7 (1.0-3.6) 0.008 4.3 56 81 0.71 IFNγ _AG-NIL
IU / ml 5.3 (1.2-10.0) 1.6 (0.7-3.5) 0.014 3.9 56 81 0.70 TGFα _NIL, pg / ml 12.7 (10.0-18.4) 9.6 (5.6-11.7) 0.002 12.2 60 81 0.75 TGFα _AG , pg / ml 17.3 (15.1-20.5) 12.5 (8.0-15.1) 0.000 15.6 67 81 0.80 IFNα2 _AG , pg / ml 7.4 (3.0-11.5) 3.0 (3.0-3.0) 0.041 6.0 56 81 0.66 TNFα _AG , pg / ml 141.7 (86.0-360.2) 94.9 (39.7-195.8) 0.039 223.2 37 81 0.67 sIL2-Rα _NIL , pg / ml 18.6 (3.0-46.9) 3.0 (3.0-11.0) 0.006 13.7 63 81 0.72 sIL2-Rα _AG , pg / ml 30.0 (5.9-76.7) 3.0 (3.0-13.2) 0.005 26.3 56 81 0.73 sIL2-Rα _AG-NIL , pg / ml 6.7 (0.0-13.0) 0.0 (0.0-13.1) 0.041 4.2 52 81 0.66 IL- _NIL , pg / ml 627.7 (214.8-1640.8) 212.9 (125.3-827.2) 0.037 887.2 45 81 0.67 IL- _AG , pg / ml 1463.0 (650.7-2973.6) 604.7 (306.3-1023.4) 0.018 257.0 56 81 0.69 Note: * p - significance of the difference between the group of patients and contact persons, calculated using the Mann-Whitney criterion. ** AUC is the area under the characteristic ROC curve.

To assess the diagnostic value of the analyzed cytokines using the SPSS 15.0 program, a characteristic curve (receiver-operating-characteristic curve-ROC) was analyzed at a fixed specificity value (81%). Graphical images of the characteristic curves and the area under the curve were considered - AUC (Area Under Curve). The analysis data showed that independently none of the selected indicators has high sensitivity, which excludes the possibility of their isolated use for diagnostic purposes. As a result, we applied the method of constructing decision trees in the JMP 9.0 program, based on the results of which it was found that the joint determination of three markers: IFNγ _AG , TGFα _NIL and IL-6 _AG at threshold values of 6.4 IU / ml, respectively; 17.0 pg / ml and 2039 pg / ml is sufficient for selecting individuals in the group of patients with active TB and latent tuberculosis infection (LTBI). Using this combination of biomarkers makes it possible to achieve 96.3% (26 of 27) of the selectivity of detecting cases of active tuberculosis (the value of the area under the ROC curve in this case reaches 0.9.).

Figure 1 shows the curve of the operational characteristics when using a combination of three biomarkers (IFNγ _AG , TGFα _NIL and IL-6 _AG ) to separate individuals infected with MBT with active and latent tuberculosis infection.

The proposed method is as follows. Samples of whole blood of individuals (1000 μl) infected with heparin-stabilized MBT (50 units / ml) were cultured at 37 ° C for 18-24 hours in the presence of a mixture of mycobacterial antigens - ESAT-6, CFP-10, TB 7.7 and in a zero control tube (without antigens). After cultivation, the samples are centrifuged, the supernatant is separated and the amount of IFNγ _AG , TGFα _NIL and IL-6 _AG is determined using multiplex or enzyme immunoassay according to standard methods. Then, the obtained cytokine contents are compared with the threshold ones: 6.4 IU / ml for IFNγ _AG-NIL , 17.0 pg / ml for TGFα _NIL and 2039 pg / ml for IL-6 _AG . When the threshold values are reached by at least one of the indicators, a conclusion is drawn about the presence of an active form of TB, and if the values are below the threshold in three indicators at the same time, a latent tuberculosis infection is detected.

The essence and advantages of the method are illustrated by the following examples.

Example 1. Patient F. Diagnosis: infiltrative pulmonary tuberculosis in the decay phase. The result of sputum culture is positive. M. tuberculosis infection test result (QuantiFERON-TB Gold in-tube): positive.

During the research, the following concentrations of biomarkers were obtained:

IFNγ _AG - 24 IU / ml, TGFα _NIL - 14 pg / ml and IL-6 _AG - 598 pg / ml.

An increase in the concentration level of IFNγ in comparison with

threshold value (6.4 IU / ml).

Conclusion: active tuberculosis process, which confirms the clinical diagnosis.

Example 2. Patient T. Diagnosis: infiltrative tuberculosis of the upper lobe of the left lung in the decay phase. The result of sputum culture is positive. M. tuberculosis infection test result (QuantiFERON-TB Gold in-tube): positive.

During the research, the following concentrations of biomarkers were obtained:

IFNγ _AG - 22.6 IU / ml, TGFα _NIL - 9.75 pg / ml and IL-6 _AG - 10000 pg / ml.

Compare the obtained values with the threshold :.

For two indicators (IFNγ _AG , IL-6 _AG ), threshold values are exceeded (IFNγ - 6.4 IU / ml, 2039 pg / ml for IL-6).

Conclusion: active tuberculosis process.

Example 3. Patient C. Diagnosis: infiltrative pulmonary tuberculosis in the phase of decay and contamination. The result of sputum culture is positive. M. tuberculosis infection test result (QuantiFERON-TB Gold in-tube): positive.

During the research, the following concentrations of biomarkers were obtained:

IFNγ _AG - 6.89 IU / ml, TGFα _NIL - 22.2 pg / ml and IL-6 _AG - 755 pg / ml

For two indicators (IFNγ _AG , TGFα _NIL ) we observe an excess of the threshold

values (IFNγ - 6.4 IU / ml, 17.0 pg / ml for TGFα).

Conclusion: active tuberculosis process.

Example 4. An employee of the TB dispensary T. The test result for infection with M. tuberculosis (QuantiFERON-TB Gold in-tube): positive.

The results of x-ray examination: changes in the lung tissue were not detected. There are no complaints. Clinical signs of tuberculosis are absent. During the research, the following concentrations of biomarkers were obtained:

IFNγ _AG - 4.9 IU / ml, TGFα _NIL - 11.6 pg / ml and IL-6 _AG - 220 pg / ml.

Compare the obtained values with the threshold ones: IFNγ - 6.4 IU / ml, 17.0

pg / ml for TGFα and 2039 pg / ml for IL-6.

Conclusion: latent infection of M. tuberculosis without signs of disease.

Example 5. An employee of the TB dispensary M. The result of the M. tuberculosis infection test (QuantiFERON-TB Gold in-tube): positive. The results of x-ray examination: changes in the lung tissue were not detected. There are no complaints. Clinical signs of tuberculosis are absent.

During the research, the following concentrations of biomarkers were obtained:

IFNγ _AG - 3.1 IU / ml, TGFα _NIL - 0.1 pg / ml and IL-6 _AG - 162 pg / ml.

The obtained values are below the threshold for all three indicators.

Conclusion: latent infection of M. tuberculosis without signs of disease.

Example 6. Patient T. Diagnosis: infiltrative tuberculosis of the upper lobe of the right lung. The result of sputum culture is positive. M. tuberculosis infection test result (QuantiFERON-TB Gold in-tube): positive.

During the research, the following concentrations of biomarkers were obtained:

IFNγ _AG - 2.22 IU / ml, TGFα _NIL - 25.28 pg / ml and IL-6 _AG - 3998 pg / ml.

Two indicators (TGFα _NIL , IL-6 _AG ) exceeded the threshold values (TGFα _NIL - 17 pg / ml, 2039 pg / ml for IL-6).

Conclusion: active tuberculosis process.

The advantages of the proposed method compared to traditional methods is that when analyzing a small amount of peripheral blood, a high diagnostic significance is achieved in the differentiation between people with active tuberculosis and people with latent tuberculosis infection. The risk for the patient from using this method is minimized, since it does not require the introduction of foreign agents into the patient's body. The method does not affect the health of patients and allows you to speed up, standardize and automate the process of obtaining the final diagnostic result. An undoubted advantage is the convenience for the patient, since a second visit to the doctor is not required to evaluate the results.

Claims (1)

A method for the diagnosis of pulmonary tuberculosis, including taking the patient’s peripheral blood, incubating whole blood with mycobacterial antigens, which are a mixture of ESAT-6, CFP-10, TB 7.7 proteins, and without them, centrifuging the sample with plasma separation, determining gamma in supernatants interferon, characterized in that in the supernatants of individuals infected with mycobacteria, in addition to the antigen-induced production of gamma-interferon, the content of antigen-induced interleukin-6 and the spontaneous production of transforming growth factor alpha and at a level of gamma interferon ≥6.4 IU / ml, or interleukin-6 ≥2039 pg / ml, or transforming growth factor alpha ≥17.0 pg / ml, active tuberculosis infection is detected, and at values lower threshold for all three indicators - latent infection.

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