RU2405151C2 - Method for evaluation of risk of progressing latent tuberculous infection in children - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: when converting PPD test, gene NRAMP1 polymorphism is characterised, locus DRB1 alleles of the major histocompatibility complex are typed, and essential cytokine concentrations are determined, including: interferon-gamma (IFNγ), interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-18 (IL-18), and a cytokine index is calculated (IFNγ/IL-4 and IL-18/IL-10). When observing GG genotype in introne 4 of gene NRAMP1, alleles *04 and *16 of gene HLA DRB1, and the decreased cytokine index (less than 2), progressing tuberculous infection is detected.

EFFECT: method allows high-reliable evaluation of perspectives of the disease, enabled well-timed correction of the therapeutic regimen and its length, improves medical and social rehabilitation of children.

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Description

The present invention relates to medicine, namely to phthisiology, and can be used to control the risk of respiratory tuberculosis in children. In our country, tuberculin diagnostics using the Mantoux test with 2 TE PPD-L is regulated as the main way of actively detecting tuberculosis in children in our country, and tuberculin diagnostics in combination with radiation methods in adolescents.

From the level of knowledge of this problem, it is known that from 7 to 70% of children with tuberculosis are detected by the tuberculinodiagnostic method (N.K. Borisova et al., 1995; O.I. Korol et al., 1997). The low quality of tuberculin diagnostics leads to the fact that in every third child with a newly diagnosed local form of tuberculosis, the process is characterized by the presence of calcination, i.e. with a disease duration of at least 2 years, the patient is not treated (Y. V. Lazareva, 2001). Individual differences are manifested quite significantly in the nature of the reactions that occur in response to the same dilution of tuberculin. There are also differences in the rate of evolution of the reaction. As a result, the cited authors come to a very important conclusion: “... tuberculosis infection sensitizes each individual in a different way, either qualitatively or quantitatively. These differences in sensitization, which are detected using tuberculin samples, should likewise exist in the foci of tuberculous lesions when reactions occur due to mycobacteria and their products; we must think that the differences between skin reactions illustrate the differences in allergies that exist more deeply. " This fact suggests that a high incidence of tuberculosis can be associated not only with social and epidemiological, but also with genetic factors.

The influence of genetic factors, in particular genes of the main histocompatibility complex (HLA) of a person, on susceptibility to tuberculosis is indicated in the works of domestic and foreign scientists (Pospelov L.E., 1986; Gergert V.Ya. et al., 2004; Dubaniewicz Z. , 2000). The genes of the HLA complex are involved in almost all stages of the immune response from recognition of the infectious principle to elimination of the pathogen from the body. The susceptibility to tuberculosis is also affected by the Nrampl gene. This gene (according to modern Slcllal nomenclature) encodes a membrane protein, the functional activity of which is manifested in phagolysosomes containing live bacteria. This protein regulates the release of bivalent Fe ²⁺ and Mn ²⁺ cations from the phagolysosomes and thereby inhibits the replication of bacteria, including tuberculosis mycobacteria (Gobin J. et al., 1995; Bellamy R., 1998). Persons susceptible to tuberculosis infection often have a mutation in the gene of this protein (Hellyer J. et al., 1999). It is believed that a mutated version of the Nrampl gene is not able to prevent the free multiplication of mycobacterium tuberculosis inside the macrophage, which ultimately leads , to the death of the phagocyte (Liu J. et al., 1995).

A number of researchers have obtained evidence that the predisposition to diseases in all its polymorphic manifestation is determined by the immunological reactivity of the body. At the same time, the severity of both the humoral and the cellular link is of an individual character (Gergert V.Ya. et al., 2001; Hanferyan R.A. et al., 2002; Bloom B.R. et al., 2002). Accumulated scientific facts about the reactions of the immune system in patients in response to tuberculosis infection made it possible to attribute tuberculosis to interleukin-dependent immunodeficiencies with marked changes in the cytokine network (Poddubnyak, O.P. et al., 2008).

As is known, the population of CD4 + T-lymphocytes includes two subpopulations of cells that differ in the type of cytokines produced by them and in relation to antigen-presenting cells (Street N.S., Mosmann T.R., 1991). During the development of immune responses, T-helpers of the first (Th1) and second (Th2) types exhibit signs of cross-regulation, which contributes to the dominance of one of the subpopulations. Protective immunity is directly related to the proliferation of CD4 + lymphocyte Th1 cells, which correspond to their cytokine profile (interleukin-2, interleukin-12, interleukin-18) and the development of cellular immunity reactions. The immune response due to Th2 cells of CD4 + lymphocytes is accompanied by the production of another spectrum of cytokines: interleukin-4, interleukin-5, interleukin-6, interleukin-10, interleukin-13.

From the presented it can be concluded that the most important task facing phthisiology, to determine the risk of progression of latent tuberculosis infection and to prevent tuberculosis in children should be solved on the basis of genetic and immunological tests.

A known method for determining the risk of progression of latent tuberculosis infection in the body of a child infected with mycobacterium tuberculosis according to the results of the Mantoux test with 2 TE PPD-L ("Tuberculosis" edited by academician of the Russian Academy of Medical Sciences A.G. Khomenko, Moscow, "Medicine", 1996, p. 95-102). But the sensitivity of the organism of an infected or sick person can be different: pronounced or hyperergic (infiltration of 17 mm in children and more than 21 mm in adults with the development of regional lymphadenitis, lymphangitis and vesiculonecrotic reactions), normergic ( infiltrate 5-16 mm in diameter), doubtful or hypoergic (infiltrate 2-4 mm or hyperemia of any size) and negative (anergy). There is also no clear correlation between the size of the infiltrate according to the Mantoux test and the progression of tuberculosis infection in the body of the infected person, which is A significant drawback of the method: the widespread introduction of intradermal BCG vaccination also reduces the differential diagnostic value of tuberculin samples. T. Loos (1979) adheres to the same point of view, reporting the impossibility of establishing with sufficient accuracy the frequency of infection during BCG vaccination after birth.

The closest analogue is a method for predicting the course of the tuberculous process in the lungs (Bukharin O.V., Usvyatsov B.Ya., Golanov VS, Kudrin A.V., Marshinsky A.V., Ushakova A.G., Andreev L.P. , Meshcheryakov V.G., patent RU (11) 2121302 (13) Cl, 1998). In this work, they solve the problem of predicting the progression of the tuberculous process by microbiological isolation of the L-forms of mycobacterium tuberculosis from the sputum of patients and determining the pathogen of antilysocyme activity in the isolated L-forms. The method consists in the microbiological isolation of L-forms of Mycobacterium tuberculosis from the sputum of patients, in addition, the antilysocyme activity is determined in the isolated L-forms of the pathogen, and an exacerbation of tuberculosis is predicted with a level of severity of this symptom equal to or more than 4 μg / ml.

The method is as follows.

1. Produce microbiological isolation of L-forms of Mycobacterium tuberculosis from the sputum of patients.

2. Determine the antilysocyme activity (ALA) of the isolated L-forms of Mycobacterium tuberculosis according to the method developed by the authors.

3. According to the developed technique, the following components are added successively to the wells of a polyvinyl tablet: a) 0.2 ml of a solution of egg lysozyme preparation in a concentration of 1 μg / ml to 5 μg / ml; b) 0.15 ml of Shkolnikova’s medium with L-forms of mycobacterium tuberculosis isolated from the examined patients after a 4-week incubation at t = 37 ° C; c) 0.15 ml of 0.5 billion suspension of daily agar test culture of M.lysodeikticus in 0.06% meat and peptone agar. The plate is incubated for 24 hours at 37 ° C, after which the result is taken into account. In wells with antilysocimactive L-forms of the pathogen, growth zones of the M.lysodcikticus test culture are observed. A quantitative assessment of ALA is carried out according to the maximum concentration of lysozyme in the well of the plate, which was inactivated by this L-form.

4. Predict the progression of the tuberculosis process in the lungs. When the severity of antilysocyme activity (ALA) is greater than or equal to 4 μg / ml, exacerbation of tuberculosis is predicted.

A limitation of this method is the impossibility of its use in people without local tuberculous changes in the lungs, which does not allow adequate decisions to be taken on planning preventive measures that impede the progression of latent tuberculosis infection, especially in children.

The objective of the claimed method is to increase the accuracy of determining the risk of progression of latent tuberculosis infection by conducting a series of genetic and immunological tests in children with conversion of tuberculin sensitivity.

The essence of the invention is to determine the risk of progression of latent tuberculosis infection in children, including performing a Mantoux test, characterized in that when converting a reaction to a Mantoux test, NRAMP1 gene polymorphism is determined, alleles of the DRB1 locus of the main histocompatibility complex are typed, and concentrations of key cytokines are determined: interferon gamma (IFNγ) , interleukin-4 (IL-4), interleukin-10 (IL-10), inteleukin-18 (IL-18) and calculate the cytokine index (QI: IFNγ / IL-4 and IL-18 / IL-10), which corresponds to a norm of 2 or more. When the GG genotype is detected in the intron 4 of the NRAMP1 gene and the * 04 and / or * 16 alleles of the HLA DRB1 gene, confirming the genetic predisposition to tuberculosis, and a decrease in the cytokine index (QI) of less than 2, the progression of tuberculosis infection in the body is determined, and if the QI value is equal to or more than 2, determine the lack of risk of progression of latent tuberculosis infection in children.

The method is as follows.

Tuberculin sensitivity is determined using a Mantoux test with 2 TE PPD-L.

Peripheral blood samples for genotyping are collected and placed in EDTA tubes. DNA is isolated from mononuclear cells of the peripheral (fresh or frozen at -20 ° C) blood of patients using DNA extraction kits, for example, IzoGen Laboratory LLC (Russia). 200 μl of peripheral blood is placed in a tube with a lysis solution. After lysis, a magnetic nucleus is added to the tube and placed on a magnetic tripod. The next step is three sequential washing of the blood to isolate pure DNA. After that, washing liquid is removed from the test tube with a nucleus with a water-jet spray and an extragen is added. After vortexing and centrifugation, pure DNA is placed in a separate tube.

The principle of genotyping.

The primers and amplification conditions of the polymerase chain reaction for determining the polymorphism of the NRAMP1 gene in intron 4 (INT4) are used according to the method of C. Søborg, et al., 2002.

The test is based on the use of the amplification process (increase, multiplication) of DNA by PCR using sequence specific primers (SSP-PCR). Primers are oligonucleotides ranging in size from 15 to 30 nucleotides corresponding to the regions of the target DNA that will be amplified.

When the sequence of primers with the complementary allele matrix coincides, the selected allele sequence is amplified, and when two or more bases of the nucleotide sequence of the primers do not coincide with the complementary allele matrix, a specific reaction does not pass.

Allele typing is based on the presence or absence of a PCR product that is detected by agarose gel electrophoresis stained with ethidium bromide in UV light at a wavelength of 302 nm (in the form of a luminous band that determines the allelic specificity and hetero- or homozygosity of a given blood sample - genotypes GG, CC, CG).

The presence or absence of a specific amplification fragment of a certain size corresponding to an allele-specific or group-specific mixture of primers is used for typing the HLA-DRB1 locus.

To determine HLA-DR, primers are used, for example, IsoGen Laboratory LLC (Russia) and the GenePak HLA-DR PCR test. The set for 50 studies included 19 master mixes (allelic sets), each master mix contains 50 test tubes with their own allelic variant or combination. 5 μl of DNA, 10 μl of solvent, are added to each test tube of the kit. The polymerase chain reaction was carried out from 15 μl of volume. Amplification is carried out on the TERTIK amplifiers (DNA-Technology, Moscow) under the following conditions: step 1 - 120 seconds at 95 degrees, 60 seconds at 66 degrees, 120 seconds at 74 degrees; step 2 - 60 seconds at 95 degrees, 50 seconds at 64 degrees, 60 seconds at 74 degrees; step 3 - 33 cycles of 60 seconds at 95 degrees, 40 seconds at 62 degrees, 60 seconds at 74 degrees; step 4 - 120 seconds at 74 degrees. The amplification products were detected by gel electrophoresis in 12% agarose gel stained with ethidium bromide.

The concentrations of key cytokines, namely interferon gamma (IFNγ), interleukin-4 (IL-4), 10 (IL-10), 18 (IL-18), are determined in blood serum, which is taken from the subjects on an empty stomach from the cubital vein venipuncture of 2-3 ml of blood in a dry test tube. The blood is kept in an thermostat at + 37 ° C for 60 min, after which the clots are circled with Pasteur pipettes, centrifuged at 1500 rpm and the serum is collected using an automatic dispenser in test tubes. The study is carried out by enzyme-linked immunosorbent assay using, for example, reagent kits LLC "Cytokine" (St. Petersburg). Evaluation of the results was carried out on an immunoenzyme analyzer Sunrise. The cytokine index is determined by the ratio of IFNγ / IL4 and IL18 / IL10.

To accomplish this task, molecular typing of the HLA genes of the DRB1 * locus and intron 4 of the NRAMP1 gene by the polymerase chain reaction (PCR-SSP) method is performed in 72 children with respiratory tuberculosis and 70 healthy children.

Studies of alleles of the INT4 locus of the Nrampl gene showed that the CC genotype was found only in the examined group of healthy children (10%), which allows us to regard this genotype as protective. At the same time, the GG genotype, on the contrary, was much more common in groups of children and adolescents with tuberculosis (55.6%, p <0.05). The frequency of occurrence of the CG genotype in all compared groups did not statistically differ from each other.

A study of the typing results of the HLA gene and the inherent genetic characteristics of allelic specificities in healthy and sick children - carriers of the C / G genotype of the NRAMP1 gene showed that sick children are 3.3 times more likely (p <0.05) to have HLA antigens with a genetic predisposition sign to the disease - * 04, * 16 (table 1).

Among children with the G / G genotype in the INT 4 locus of the NRAMP 1 gene, there is a significant increase in patients, compared with healthy ones, in the number of specificities * 04 and * 16 associated with a predisposition to tuberculosis - 2.2 times, p <0.05 (table 2).

Also a reliable indicator of insufficient resistance to the progression of tuberculosis infection is a low level of the cytokine index (CI <2.0), which reflects an increase in the Th-2-mediated immune response in terms of the ratio of key cytokines: IFN-γ / IL-4, IL-18 / IL110, found in children with tuberculosis (table 3).

Table 3 Cytokine index in children with limited and common forms of tuberculosis, n = 72 Clinical forms of tuberculosis The nature of cytokines and their indicators of IFNγ / IL4 QI cytokine index The nature of cytokines and their indicators of IL18 / IL10 QI cytokine index Limited uncomplicated 51.4 / 18.7 2.7 36.6 / 16.5 2.2 TVGLU, PTK, focal, tuberculoma complicated 42.6 / 19.5 2.1 22.1 / 17.0 1.8 Common uncomplicated 44.6 / 26.2 1.7 28.6 / 16.9 1,6 Infiltrative disseminated complicated 39.5 / 23.4 1,5 12.0 / 21.1 0.6 Healthy uninfected children (control) 9.6 / 4.1 2.6 8.4 / 3.7 2.2

The risk of progression of latent tuberculosis infection is determined by the presence of factors of a genetic predisposition to tuberculosis and a decrease in the cytokine index - CI <2.

A mandatory indicator of sufficient resistance to the progression of mycobacterial infection in children with a genetic predisposition to tuberculosis should be considered to achieve a positive shift in the ratio of Th1 / Th2 towards T-helpers of the first type (Th1), which is determined by an increase in the cytokine index: CI> 2.

The reaction of the children's immune system to infection with mycobacterium tuberculosis is individual in nature, genetically determined, which should be taken into account when planning preventive measures to prevent cases of tuberculosis.

Technical result: by increasing the information content of the identified indicators characterizing the risk of progression of the disease with tuberculosis, a high degree of reliability of the prospects for the development of the disease is achieved, which allows you to timely adjust the treatment regimen and its timing, improve medical and social rehabilitation of children

Examples

Ivan S., 4 years old.

Born on March 30, 2002, vaccinated according to the calendar with BCG vaccine. Contact with a tuberculosis patient has not been established. For the first time, the reaction to tuberculin in the Mantoux test with 2TE PPD-L was positive in March 2006, the infiltrate was 10 mm in diameter. Symptoms of intoxication were absent, the general development was without features. Well-being did not suffer. Peripheral lymph nodes are slightly enlarged in groups I-II-III, painless. Isoniazid was prescribed as a "chemoprophylaxis", which the child took 3 months. After 6 months (20 / VIII-2006) - repeated staging of the Mantoux test with 2TE RRD-L, infiltrate - 15 mm. It continues to be taken into account in the VIA group of DNs, an R-gram of the chest organs and TG of the mediastinum has been performed, tuberculosis of the intrathoracic lymph nodes (TBHLU), uncomplicated course has been diagnosed.

Additionally identified in the immunogram: CD3 ⁺ - 49.8%; CD4 ⁺ - 40.4%; CD8 ⁺ - 31.7%; CD4 ⁺ / CD8 ⁺ - 1.27; CD16 ⁺ - 16.8%; CD19 ⁺ - 19.7%; AgE - 278 ME / m1; NBTspon - 13.4%; NBTstim - 21.3; IgM - 1.56 g / l; IgG - 16.4 g / l; IgA - 1.91 g / l.

Genotyping revealed the GG genotype of the NRAMP1 gene and the * 8 and * 16 alleles of the HLA gene of the DRB1 locus.

The concentration of key cytokines in serum: IFNγ - 38.4 pq / m1; IL4 22.4 pq / m1; IL18 - 22.0 pq / m1; IL 10 - 14.6 pq / ml.

The cytokine index - IFNγ / IL4 - 1.7; IL18 / IL10 - 1.5.

In a child with a identified genetic predisposition, immunological parameters indicate a decrease in immunity with a pronounced prevalence of Th2 - mediated response, judging by the low cytokine index (1.7 and 1.5). The treatment was prescribed: the intensive phase of chemotherapy for 4 months with the antibacterial drugs isoniazid + pyrazinamide in combination with immunomodulators and a diet was carried out in a climatic anti-tuberculosis sanatorium.

In II-2007, the control formulation of the Mantoux test with 2TE PPD-L, infiltrate - 8 mm. The boy is healthy. Key cytokine concentrations: IFNγ - 41.3 pq / mL, IL4 - 12.2 pq / ml, IL18 - 28.1 pq / mL, IL10 - 9.1 pq / ml. Cytokine index: IFN _γ / IL4 = 3.3, IL18 / IL10 = 3.0. Recommended deregistration in a TB dispensary.

Valeria N., 4 years old.

Born on time, 03/02/2003, vaccinated with the BCG vaccine according to the calendar. Contact with a mother with fibro-cavernous pulmonary tuberculosis. There is no data on the sensitivity of MVT. Mother died when the girl was 2 years 1 month old. The reaction to tuberculin according to the Mantoux test with 2TE PPD-L: 03/14/2004 - infiltrate 7 mm; 06/26/2005 - infiltrate 10 mm; September 26, 2005 - an infiltrate of 12 mm; January 22, 2006 - an infiltrate of 17 mm. An increase in tuberculin sensitivity was observed. One course of treatment with isoniazid in 2005 (3 months) and two drugs: isoniazid + pyrazinamide was also carried out for 3 months in 2006. The girl often has acute respiratory infections, underwent bilateral focal pneumonia, and is considered in the group of frequently ill children in a children's clinic. Peripheral lymph nodes are isolated in groups I-II, painless. In January 2007, the response to 2TE PPD-L was 16 mm Mantoux test. On the R-gram of the chest and TG mediastinum pathology was not detected.

Genotyping revealed the GG genotype of the NRAMP1 gene and the * 4 and * 16 alleles of the HLA gene of the DRB1 locus.

Immunological research: CD3 ⁺ - 49.8%; CD4 ⁺ - 36.2%; CD8 ⁺ - 24.1%; CD4 ⁺ / CD8 ⁺ - 1.5; CD16 ⁺ - 21.4%; CD19 ⁺ - 17.1%; NBTspon - 9.6%; NBTstim - 18.4; IgE - 299 ME / m1; IgM - 1.56 g / l; IgG - 16.4 g / l; IgA - 1.91 g / l.

Serum cytokine concentrations: IFNγ - 34.2 pq / ml; IL10 - 18.7 pq / ml; IL4 - 15.5 pq / ml; IL18 - 14.1 pq / ml. Cytokine index: IFNγ / IL4 - 1.9; IL18 / IL10 - 1.6.

The child was prescribed treatment with three antibacterial drugs, since the mother's mycobacterial section could not be established, and immunological parameters with a genetic predisposition to tuberculosis indicated a clear prevalence of the Th2-mediated response: a low cytokine index, insufficient production of IL18 and IFN _γ .

These data allowed us to substantiate the treatment within 6 months.

After 6 months, the reaction to 2TU PPD-L according to the Mantoux test - 9 mm infiltrate.

Immunogram at the end of treatment: the cytokine index increased significantly - IFNγ / IL4 = 4.2; IL18 / IL10 = 3.8. It is recommended that a TB specialist observe at least two years and control studies of the cytokine profile once every 6 months.

Thus, children with conversion of tuberculin sensitivity need to study the NRAMP1 gene polymorphisms, the allelic specificities of the main histocompatibility complex DRB1 and the cytokine index in order to establish a genetic predisposition to tuberculosis and to determine the risk of progression of latent tuberculosis infection

Claims (1)

A method for determining the risk of progression of latent tuberculosis infection in children, including performing a Mantoux test, characterized in that when converting the reaction to a Mantoux test, the NRAMP1 gene polymorphism is determined, alleles of the DRB1 locus of the main histocompatibility complex are typed, and the concentrations of key cytokines are determined: interferon-gamma (IFN _γ ) , interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-18 (IL-18) and calculate the QI - cytokine index (IFN _γ / IL-4 and IL-18 / IL-10) and when the QI value is less than 2, the identification of the GG genotype in intron 4 of the NRAMP1 gene and alleles * 04 and * 16 ge and HLA DRB1, confirming the genetic susceptibility to tuberculosis, determine the progression of tuberculosis infection in the body, and a value of TI equal to or greater than 2 determine the absence of risk of progression of latent TB infection in children.