RU2707571C1 - Method for prediction of developing tuberculosis in healthy individuals - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to a method for prediction of developing tuberculosis in healthy individuals. Method for prediction of development of tuberculosis in healthy people, consisting in carrying out a complex of immunological studies in individuals with positive immunological test results, wherein the prepared blood plasma samples are added with the tuberculosis antigens ESAT-6/SFP-1, followed by the dynamic light scattering method, determining and analyzing the formed immune complexes and their isotypes and with the content of IgG3, IgE, IgG1+IgG3, IgG3+IgE, IgG1+IgE higher than 1.0 % shows the risk of developing tuberculosis.

EFFECT: method described above enables higher prognosis of tuberculosis development to 100 % in individuals with positive immunological tests due to sensitivity and specificity.

1 cl, 6 tbl, 2 ex

Description

The invention relates to medicine, namely to TB, and can be used to identify individuals with a high risk of developing tuberculosis.

It is not possible to monitor the spread of tuberculosis infection without early detection of the disease [1, 2, 3].

Latent, or latent, tuberculosis infection is a natural stage in the development of the infectious process in the body, which is accompanied by the persistence of M. tuberculosis in the body and is characterized by a long asymptomatic presence of the pathogen with preservation of its pathogenetic properties, ability to reproduce and reverse, as Dienes L. and Parish wrote about NM back in the middle and end of the 20th century.

In recent years, special attention has been focused on the diagnosis of latent tuberculosis infection (LTI), including through the introduction of various immunological methods for determining the activity of tuberculosis infection [4, 5].

The revolution in the development of new methods of immunological diagnostics was the decoding of the genome of mycobacterium tuberculosis, in which more than 4000 proteins were encoded. A group of proteins expressed during the propagation of mycobacteria encoded in the RDI zone (region of difference) named ESAT-6 and CFP-10 was isolated, which allowed the development of new highly informative immunological tests in vitro (IGRA tests: QuantiFERON (QFT) -TB, T-SPOT.TB test, IP-10) and in vivo (with tuberculin, with a recombinant tuberculosis allergen (test with Diaskintest ^® )) [6, 7].

According to a number of authors, laboratory tests QuantiFERON-TB Gold / QuantiFERON-TB Gold In-Tube and ELISPOT / T-SPOT.TB should complement tuberculin diagnostics, primarily to identify false-negative results of detection of latent tuberculosis infection, active tuberculosis and new cases of tuberculosis in patients undergoing therapy with TNF-α inhibitors, especially in countries with moderate and high prevalence of tuberculosis, can also serve as useful tools for screening and monitoring latent tuberculosis infection in combination with internally rikozhnoy Mantoux tuberculin 2 TE [8, 9, 10]

In Russia, based on the proteins encoded in the RDI zone, in 2006 a new diagnostic drug, Diaskintest ^® , was developed, which is a recombinant protein CFP-10-ESAT-6 produced by Escherichia coli. The main mechanism of action of the test is also the formation of a delayed-type hypersensitivity reaction. In this case, the protein CFP-10-ESAT-6 does not have sensitizing activity and is not toxic. The high diagnostic value of the Diaskintest test (DST) is confirmed in many Russian studies [11, 12, 13, 14].

In the work, which is the prototype of the invention “Immunological tests in the diagnosis of tuberculosis in patients with HIV infection with different levels of immunosuppression” [19], the author investigated the diagnostic capabilities of new immunological tests (a sample with a recombinant tuberculosis allergen (ATP), quantiferon test (CF) , T-SPOT) in determining the activity of tuberculosis infection, including in people with HIV infection. However, the study shows that both patients with tuberculosis and persons with latent tuberculosis infection have positive immunological tests, which does not allow distinguishing between these conditions and highlighting the high-risk group for the development of the disease. The updated WHO recommendations (2018) (http://www.who.int/tb/publications/digitalhealth-TB-agenda/en/) on the management of latent tuberculosis infection states that today there is no universal test - the gold standard, for diagnosis of this condition.

A known method based on the use of dynamic light scattering (DLS), using which it has been proved the possibility of determining free immune complexes in various pathologies, including tuberculosis [20]. The DSR method today has no analogues among immunological methods in determining large particles, which are immune complexes [16, 17, 18]. It is based on the analysis of Rayleigh broadening scattered by a certain angle of the laser beam on diffusers “freely” diffusing in the solution. The advantage of the DSL method is that it is an accurate diffusiometric method that allows one to determine large-sized immune complexes - from units of nanometers to tens of microns.

The objective of this method was to be able to determine the immune complexes in the studied biological fluids under various pathological conditions and to obtain information about the set of antibody isotypes in biological fluids: whole blood, blood plasma, blood serum, lymph, saliva, sperm, cerebrospinal fluid, milk, colostrum, sputum, amniotic fluid, urine, pus, mucus, effusion, ascitic fluid or pleural fluid in various pathological conditions i.e. determining the diagnostic capabilities of the technique itself. To determine the activity of tuberculosis infection, the DSR method was not used.

The objective of the invention is to increase the effectiveness of predicting the development of tuberculosis in healthy individuals.

The problem is solved due to the fact that in individuals with positive immunological tests, ESAT-6 / SFP-1 tuberculosis antigens are added to the prepared blood plasma samples, then the formed immune complexes and their isotypes are determined and analyzed by dynamic light scattering and with the content of IgG3, IgE , IgG1 + IgG3, IgG3 + IgE, IgG1 + E above 1.0%, establish the risk of developing tuberculosis.

The method is as follows.

All patients who underwent a set of examinations, including: examination, history taking, chest X-ray, chest MSCT (multispiral computed tomography) of the chest, immunological tests (Mantoux tests with 2 TE, tests with Diaskintest, quantiferon test (CF), T -SPOT), bacteriological examination of sputum to determine M. tuberculosis and molecular genetic methods for determining MBT DNA as in sputum, a fence is carried out in a vacuum tube with serum coagulation activator without filler 6 l of venous blood in the morning before meals, to determine the specific immune complexes.

Stage I: preparation of a patient taken for analysis of peripheral venous blood.

The blood plasma obtained from patients is diluted 4 times with a phosphate buffer containing a concentration of ethylenediamine-tetraacetic acid, centrifuged for 15 minutes at 15,000 rpm and filtered through a filter with a pore size of 100 nm to remove all particles and protein aggregates exceeding this the size. Measurement of dynamic light scattering (DLS) of the resulting preparation should show the absence of any formations exceeding 100 nm in size. The measurements are carried out on a laser correlation spectrometer (certificate RU. P. 39.003. A No. 5381) LKS-03 (INTOX-MED, Russia).

Stage II: preparation of the studied blood plasma with the addition of in vitro antigenic material of tuberculous mycobacteria.

In the obtained plasma samples with a volume of 400 μl add 10 μl of the prepared antigen. As a specific antigen, it is necessary to use a complex of tuberculosis antigens ESAT-6 / SFP-10 (recombinant tuberculosis allergen, No: LSR-006435/08 of 03/26/12).

Stage III: Registration of the formation of specific immune complexes.

After 20 minutes of preparation of the samples necessary for the formation of specific immune complexes, the measurement of specific immune complexes is carried out by the DSL method. In the absence of antibodies in the plasma that bind the components of the added antigenic material, the size distribution of plasma particles does not change. In the presence of antibodies, new peaks form on the histogram, which is the size distribution of the main diffusers.

Stage IV: Verification of the nature of the formations recorded by the DLS method.

The appearance of new peaks in the histograms is then added to the measured sample of Sepharose beads bearing on their surface staphylococcal protein A that binds immunoglobulins or mouse antibodies that bind human immunoglobulins correspond to the formed immune complexes. Subsequent removal of the added beads by centrifugation is accompanied by the disappearance in the histograms of the peaks formed by the addition of antigenic material. This allows us to make an unambiguous conclusion that the registered formations really contain human immunoglobulins, that is, they are immune complexes.

A prospective study was conducted from December 2016 to July 2017 with the inclusion of 135 patients with pulmonary tuberculosis (group I) who were treated at the St. Petersburg Research Institute of Phthisiopulmonology, Ministry of Health of the Russian Federation, 28 people with latent tuberculosis infection (LTI) (II group) and 24 people (healthy individuals) - control group (III group). The study was approved by the independent ethical committee of the Federal State Budgetary Institution SPb NIIF of the Ministry of Health of Russia (extract from protocol No. 34.2 of 01/19/2017) and the Federal State Budgetary Educational Institution of Higher Education of St. Petersburg State University (extract from protocol No. 02-126 of July 30, 2017), all study participants signed an informed consent.

The diagnosis of pulmonary tuberculosis was established in the presence of clinical manifestations, characteristic radiological changes; positive results of testing for tuberculosis (detection of M. tuberculosis (MBT) and / or MTV DNA in sputum tests according to molecular genetic and bacteriological methods), histological verification of changes in the lungs (detection of epithelioid cell granulomas with areas of caseous necrosis and acid-resistant bacteria) .

The state of latent tuberculosis infection was characterized by the presence of a positive immunological test result (tests with Diaskintest, quantiferon test (CF), T-SPOT) in the absence of clinical, radiological data on active tuberculosis.

The inclusion criteria were: age from 18 to 65 years; in patients with tuberculosis, the presence of bacterial excretion according to laboratory examination; patients with a history of pulmonary sarcoidosis no more than two years old; anti-tuberculosis therapy no more than one month.

The exclusion criteria were: anamnestic data on the use of immunosuppressive therapy, treatment with anti-TB drugs for more than one month, the presence of HIV infection, syphilis, tumor diseases, diabetes mellitus, and the detection of other granulomatous lung diseases.

Inclusion criteria for a group of healthy individuals were: absence of acute and chronic diseases, lack of contact with a patient with tuberculosis.

All patients underwent a set of examinations, including a clinical assessment of the disease, multispiral computed tomography (MSCT) of the chest organs, laboratory blood tests, a standard set of examinations for tuberculosis.

After a comprehensive examination, a blood sampling was performed to conduct QuantiFERON TV Gold (QFT) and ELISPOT, then a Mantoux test with 2TE (PM / TST) and a recombinant tuberculosis allergen (ATP / Diaskintest / DST) test were performed. Over the 5-year follow-up period, interferon-γ release tests (IGRA tests) due to limited capabilities were performed only in a part of the examined patients. Evaluation of the results of immunological tests was carried out taking into account the obtained sample for each test.

A sample with a recombinant tuberculosis allergen (DST) sample is similar to a sample with tuberculin. The injection is carried out intradermally, the result is read after 72 hours by measuring the diameter of the papule at the injection site.

According to the instructions, in the presence of a papule of any size, the DST results were interpreted as positive. The presence of hyperemia in the absence of papules was regarded as a dubious test. In this study, for an objective assessment of the presence of papules, a cut off≥5 mm is defined.

TST in the framework of Russian legislation was carried out using PPD-L tuberculin with 2 tuberculin units (Russia, Pharmstandard JSC). TST results were evaluated as follows: positive - papule 5 mm or more, dubious - papule up to 4 mm inclusive or hyperemia of any size. The absence of papules and hyperemia in both tests was a negative result.

T-SPOT ^® Test. TB was performed in accordance with the manufacturer's instructions (Oxford Immunotek, UK). Purified peripheral blood lymphocytes were incubated with test antigens using GIBCO AIM-V ™ culture medium (Invitrogen, Paisley, UK). The number of spots in each well (representing cells secreting IFN-γ) was evaluated visually using a magnifying glass by two independent observers who did not know the results of QFT. The results were interpreted according to the criteria defined by the manufacturer for the use of the test outside the United States. A positive result was defined as> 6 spots either in the ESAT-6 well or in CFP-10 well after subtracting the number of spots found in the negative control well, where the negative control has 0-5 spots. If the negative control part had ≥6 spots, the ESAT-6 or CFP-10 panel should have at least twice as many spots found on the negative panel for a positive result. The result was uncertain if there were more than 10 spots in the negative control well or less than 20 in the mitogen control (with <6 in the ESAT-6 and CFP-10 wells).

The QuantiFERON-TB Gold (QFT) test was also performed in accordance with the manufacturer's instructions (Cellstys Limited, Australia). Venous blood was collected from each patient from three special evacuated and heparinized blood tubes calibrated to draw 1 ml of blood. The kit included a tube coated with TV-Antigen, a NIL tube (negative control) and a mitogenic (phytohemagglutinin) tube as a positive control. As recommended, the cutoff for a positive test was IFN-γ> 0.35 IU / ml, for TB-Antigen minus NIL. A negative result was recorded if this response was <0.35 IU / ml, and the mitogen control was minus NIL-≥0.5 IU / ml. If the IFN-γ level for both TB-Antigen-NIL and mitogen-NIL was less than their respective cutoffs, the result was interpreted as uncertain. The maximum level of IFN-γ, accurately determined by ELISA with QFT, is 10 IU / ml, and thus, exceeding values are reported as 10 IU / ml.

The results of IGRA tests of the border line were classified as negative due to the uncertain probability of TB infection.

The plasma of all included patients was studied at the St. Petersburg Institute of Nuclear Physics. B.P. Konstantinova "with the determination of the in vitro immune complexes (IR) formed by dynamic light scattering (DLS) according to the proposed method (patent application No. 2015149694; publication date May 24, 2017, Filatov M.V., Landa SB). The measurements were carried out on a laser correlation spectrometer (certificate RU. P. 39.003. A No. 5381) LKS-03 (INTOX-MED, Russia) [19, 20].

The DSR method allows one to determine the constituent IR components without isolating complexes from the plasma and to determine large particles, which are IR. In addition, the method allows you to work with native samples in physiological conditions and has a narrow range of necessary preanalytical procedures: dilution, centrifugation, and filtering.

The blood plasma obtained from patients is diluted 4 times with phosphate buffer containing a 10 mM concentration of ethylenediamine-tetraacetic acid, centrifuged for 15 minutes at 15 thousand rpm and filtered through a filter with a pore size of 100 nm to remove all particles and protein aggregates, exceeding this size. The DLS measurement of the resulting preparation should show the absence of any formations exceeding 100 nm in size.

In the obtained plasma samples with a volume of 400 μl add 10 μl of the prepared antigen. ESAT-6 / SFP-10 (Generium, Russia) was used as a specific tuberculosis antigen.

For statistical data analysis, the methods available in the Statistica 7.0 program were used. When processing the results, there are also methods of descriptive statistics characterizing the subjects included in the study. For quantitative parameters, the arithmetic mean (Mean) was evaluated; standard deviation (SD); 95% confidence interval (CI) for the average. Differences in the compared groups were considered significant at the level of statistical differences p <0.05.

Indicators of the diagnostic significance of the methods were also evaluated: diagnostic sensitivity (PM); diagnostic specificity (DS); diagnostic effectiveness (DE). The odds ratio (OR) indicator was calculated using the formula (a / c) / (b / d) = (a⋅d) / (b⋅c) (a is a truly positive and b is a positive result; c is a false negative and d is a true negative result). Significant was the value of the relative risk of more than 1.0.

Patient examination results.

Positive results of the examination according to clinical, radiological and bacteriological methods in patients with tuberculosis (group I) are presented in table 1.

The data presented in table 1 demonstrate the possibility of obtaining a positive result according to the methods on the basis of which the diagnosis of tuberculosis was carried out. In all patients, only the presence of radiological changes was determined, while clinical symptoms could be determined in 70% of cases, and bacteriological confirmation of the diagnosis was obtained only in 45.2% of cases.

Thus, the examination complex carried out clearly demonstrates the lack of verification of the diagnosis of pulmonary tuberculosis in half the cases, while in the presence of appropriate radiological changes, clinical symptoms are recorded in 60% of cases. At the same time, immunological tests show positive results in 85% of cases, which leaves 15% of negative results. In the absence of bacterial excretion, the indicators of positive immunological tests can not exceed 79%.

Registration of latent tuberculosis infection is possible only with a positive immunological test. In this study, only the results of new immunological tests (T-SPOT, QTF and DST) were taken into account in the absence of clinical and radiological manifestations of active tuberculosis. Obviously, in patients with LTI in 100% of cases there is a positive immunological test, while a positive test in patients with tuberculosis was in 69.4% and 90.6% of cases, depending on the diagnostic capabilities of the test used. The data obtained demonstrate the inability to confirm tuberculosis on the basis of bacteriological methods and to distinguish LTI and active tuberculosis in the absence of differences in the results of existing immunological methods, which requires the development of new criteria for determining the activity of tuberculosis infection on the basis of specifically immunological methods that allow ascertaining the immune response to the pathogen the presence of mycobacterium tuberculosis in the human body.

Next, an analysis was made of the level of immunoglobulins, which were determined in three observation groups using the method of dynamic light scattering. Moreover, the determination of immune complexes (IR) was carried out in patients with tuberculosis with a verified diagnosis (n = 50, Ia).

The results of determining the level of specific IR in blood plasma in the comparison groups (Ia and II), as well as in the control group (III) are presented in table 2.

* - the differences are significant (p <0.001 for all indicators) compared with group II.

** - differences are significant (p <0.001 for all indicators) compared with the control group;

According to the data presented in Table 2, the total IRs were determined in all groups in the same percentage of cases, but in groups II and III they were recorded at a reliably low level, just like IgG1. It should be noted that in groups II and III, IgG3 and IgE, as well as isotypes IgG1 + IgG3, IgG1 + IgE and IgG3 + IgE.

Based on the data obtained, a calculation was made of indicators of the diagnostic significance of the method, which are presented in table 3.

As presented in table 4, the determination of total IR stimulated by a specific antigen does not have a high diagnostic value in determining tuberculosis infection, but the determination of IgG3 and IgE, as well as IgG1 + IgG3, IgG1 + IgE and IgG3 + IgE isotypes, demonstrate high specificity and sensitivity.

Determination of a low level of immune complexes by dynamic scattering can determine the development of tuberculosis infection more effectively than standard immunological methods. Moreover, the results obtained make it possible to identify a high-risk group for the development of the disease among healthy individuals already with positive immunological tests.

The general tactics for determining the activity of tuberculosis infection with the determination of immune complexes determined by dynamic light scattering are presented in table 4.

Clinical example 1.

Patient B., 38 years old, is an employee of a TB institution, works in contact with patients with tuberculosis for 8 years. An x-ray examination is annually carried out, pathological changes in the lungs are not revealed. Test with Diaskintest - p 20 mm. After consent to participate in the study and a set of measures, an analysis of the immune complexes in the patient's blood plasma was carried out using the method of dynamic light scattering. The results are presented in table 5.

As can be seen from table 5, the formation of immune complexes after stimulation with specific antigens ESAT-6 / SFP-10 is registered at a low diagnostic level. The immune complexes IgG3 and IgE, as well as their isotypes (IgG1 + IgG3, IgG1 + IgE, IgG3 + IgE) were not determined. The results clearly demonstrate that there is no risk of developing tuberculosis disease when a positive immunological test is detected, which indicates the absence of the need for additional diagnostic measures and preventive treatment.

Clinical example 2.

Patient S., 42 years old, is an employee of a TB institution, has been in contact with patients with tuberculosis for more than 10 years. It is known that an X-ray examination of the chest organs takes place every six months, the last one that does not reveal pathological changes in the lungs. Test with Diaskintest - p 15 mm. After consent to participate in the study and a set of measures, an analysis of the immune complexes in the patient's blood plasma was carried out using the dynamic light scattering method (table 6).

As can be seen from table 6, the formation of immune complexes on specific peptides ESAT-6 / SFP-10 was recorded. At the same time, IgG3, IgE and their isotypes make a greater contribution to the forming immune complexes. The results clearly demonstrate the risk of developing active tuberculosis. The high level of immune complexes demonstrates the need for an in-depth examination and the appointment of a preventive course of therapy for six months, given the high risk of developing the disease.

Thus, the determination of specific immune complexes made it possible in 100% of cases to determine the risk of developing tuberculosis infection in healthy individuals with positive immunological tests. The study was able to obtain data that allows to identify a group of special risk for the development of tuberculosis infection among people with positive immunological tests, where the level of immunoglobulins and their isotypes (IgG3, IgE, IgG1 + IgG3, IgG3 + IgE, IgG1 + E) is less than 1.0 % indicates the absence of the development of tuberculosis, and, conversely, at a level of more than 1% - the risk of developing tuberculosis. Currently, such a forecast cannot be made only with the use of immunological tests used in clinical practice.

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Claims (1)

A method for predicting the development of tuberculosis in healthy individuals by carrying out a complex of immunological studies, characterized in that in individuals with positive immunological tests, ESAT-6 / SFP-1 tuberculosis antigens are added to the prepared blood plasma samples, then the formed immune methods are determined and analyzed by dynamic light scattering complexes and their isotypes and with the content of IgG3, IgE, IgG1 + IgG3, IgG3 + IgE, IgG1 + IgE above 1.0% establish the risk of developing tuberculosis.