RU2498311C2 - Method for assessing tuberculosis activity in children and adolescents - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: specific cell-mediated immunity is assessed by modified lymphocyte blast transformation reaction (mLBTR) with PPD-L tuberculine; the level of interferon-gamma (IFN-γ) with PPD-L is tested with using a small volume (200 mcl) of whole blood and 4 dilutions of the PPD-L antigens. If the cell immunity stimulation index is more than 3.2 and IFN-γ with PPD-L more than 7.0, an active tuberculosis process, while the mLBTR with PPD-L more than 3.2 and IFN-γ with PPD-L less than 7.0 show an inactive tuberculosis infection.

EFFECT: using the given method enables more specific diagnosis of the tuberculosis infection, and assessed effectiveness of the conducted treatment.

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Description

The invention relates to medicine, mainly to phthisiology, can be used to diagnose primary infection, latent activity of tuberculosis in children and evaluate the effectiveness of specific therapy.

Known immunological method for determining anti-tuberculosis antibodies - enzyme-linked immunosorbent assay (ELISA). In the diagnosis of tuberculosis, serological methods, including ELISA, did not live up to all expectations, since it was not possible to detect specific M. tuberculosis antigens, for which most patients develop the corresponding antibodies. Existing enzyme immunoassay systems have a specificity of 90-94% (6-10% false-positive results in healthy individuals or patients with non-tuberculosis diseases), while sensitivity (the number of positive results in patients with active tuberculosis) is, on average, about 70%. Therefore, it is not yet possible to talk about the independent significance of this method for the differential diagnosis of respiratory tuberculosis (Diagnosis and chemotherapy of respiratory tuberculosis. A manual for doctors. - M .: "Medicine and Life", 2003. - 48 pp.), As well as for differential diagnosis of Mycobacterium tuberculosis infection and paraallergy, the sensitivity of ELISA in infected individuals is 23.2% (Nekrasov S.V. Diagnostic and prognostic significance of determining anti-tuberculosis antibodies in case of infection AI disease and pulmonary tuberculosis in children and adults: Avtoref.dis ... Candidate of medical sciences - M., 1999. - 23 c)....

Known immunological method for determining the level of interferon-gamma (IFN-γ) using ELISA. The role of IFN-γ in anti-tuberculosis immunity is confirmed by the severe course of mycobacterial infections in mice - knockouts of the IFN-γ gene (Dalton DK, Pitts-Meek S., Keshav S. et al. Multiple defects of immune cell function in mice with disrupted interferon-gamma genes // Science. 1993. - Vol.259, No. 5102. - P.1739-1742) and the development of severe fatal mycobacteriosis in people with genetically determined complete or partial deficiency of the receptor for IFN-γ (Jouanguy E., Doffinger R ., Dupuis S. et al. IL-12 and IFN-gamma in host defense against mycobacteria and salmonella in mice and men // Curr. Opin. Immunol. 1999. - Vol. 11, No. 3. - P.346-351 ) The course of tuberculosis infection in humans is also accompanied by the production of IFN-γ - antigen-specific cells. The basis for the clinical use of serum IFN-γ was information about its reduced production in tuberculosis, 380 ± 147.8 pg / ml (490 ± 54.9 pg / ml in healthy individuals). Effective chemotherapy contributes to a distinct increase in the level of IFN-γ (p <0.02) compared with the initial data (Kuznetsov V.P., Markelova E.V., Fedyanina L.N. Natural preparations of alpha-interferon and cytokines in the practice of TB specialist: abstract review No. 1. - M., 1999. - 59 pp .; Khasanova PP, Voronkova OV, Urazova OI, et al. Role of cytokines in modulating the subpopulation composition of blood lymphocytes in patients with pulmonary tuberculosis // Probl. Tub. - 2008. - No. 3. - S. 31-34.). At the same time, according to some observations in patients with tuberculosis, especially with a favorable course of the process, a high level of serum IFN-γ can also be noted. Given these facts, it is difficult to talk about the diagnostic significance of the level of serum IFN-γ in assessing the activity of tuberculosis infection.

In contrast to the serum level of IFN-γ, the number of T-lymphocytes containing intracellular IFN-γ in tuberculosis patients is increased compared to healthy donors and carriers of latent infection (Veenstra H., Crous I., Brahmbhatt S. et al. Changes in the kinetics of intracellular IFN-gamma production in TB patients during treatment // Clin. Immunol. 2007. - Vol. 124, No. 3. - P.336-344.). Data on the high content of IFN-γ-producing lymphocytes in patients with active tuberculosis suggest that the number of IFN-γ-producing cells can be a measure of mycobacterial load and used to diagnose tuberculosis. New approaches to the diagnosis of latent and active tuberculosis are based on determining the number (frequency) of cells producing IFN-γ (T-SPOT test) and / or the number of IFN-γ produced by peripheral blood cells after they are stimulated in vitro with M. tuberculosis antigens (Quantiferon Gold) (Lalvani A., Millington KA T Cells and Tuberculosis: Beyond Interferon-gamma // J. Infect. Dis. 2008. - Vol.197. - P.941-3; Pai M., Zwerling A., Menzies D. Systematic Review: T-Cell-Based Assays for the Diagnosis of Latent Tuberculosis Infection: An Update // Annals. Internal, medicine. 2008. - Vol. 149, No. 3; Ferrara G., Lost M. et al. Routine Hospital Use of a New Commercial Whole Blood Interferon-gamma Assay for the Diagnosis of Tuberculosis Infection // Am. J. Respir. Crit. Ca re Med. 2005. - Vol. 172, No. 5. - P.631-5; Ferrara G., Lost M. et al. Use in routine clinical practice of two commercial blood tests for diagnosis of infection with Mycobacterium tuberculosis: a prospective study // Lancet. 2006. - Vol. 367. - P.1328-34.), When setting up the reaction, a short exposure time of 24 hours is recommended, therefore the test is mainly used as a screening test for selection for a more detailed examination for tuberculosis, which does not exclude the receipt of false negative results. In domestic practice, these tests are not used (Kiselev V.I., Baranovsky P.M. et al. New skin test for the diagnosis of tuberculosis based on the recombinant protein ESAT-CFP // Molecular Medicine. - 2008, No. 4. - P.28 -34), therefore, it is not possible to assess the sensitivity and specificity of these tests.

The reaction of blast transformation of lymphocytes (RBTL) with tuberculin (PPD-L) is known to assess the state of specific cellular immunity used in the clinic (especially in combination with a number of other immunological tests) for differential diagnosis of tuberculosis, assessment of its activity and the detection of minimal activity of tuberculous lesions. The mechanism of this reaction is the transformation of lymphocytes into blast cells under the action of a specific mitogen - tuberculin. The reaction consists of 3 stages:

1. the allocation of lymphocytes (or leukocyte mass);

2. cultivation of lymphocytes (leukocytes) with PPD-L;

3. evaluation of the results.

The disadvantages of the method:

- This reaction is often evaluated by the morphological method (visual count in stained smears), which can give subjective errors.

- A low percentage of detection of an increased level of blast formation (positive results) can be explained by the loss of lymphocytes at the stage of their isolation (at fecol-verographin). For the optimal course of RBTL, the presence of macrophages is necessary, therefore, the use of highly purified lymphocytes is not recommended (Lebedev K.A., Ponyakina I.D. Immunogram in clinical practice. - M .: Nauka, 1990. - 224 p.).

The objective of the invention is to improve the quality and reduce the time of diagnosis of active tuberculosis in children and adolescents.

.The problem is solved in that in a method for assessing the activity of tuberculosis in children and adolescents, which includes, at the first stage, assessing the state of specific cellular immunity using a modified reaction of blast transformation of lymphocytes (mRTBL) with tuberculin (PPD-L), using four dilutions, the level of specific cell response by determining the stimulation index. Additionally, after stimulation with PPD-L, the amount of INF-γ in supernatants is estimated using an enzyme immunoassay. MRBTL with PPD-L is performed using a small volume (200 μl) of whole blood culture without isolation of lymphocytes. Heparinized blood (25 IU of heparin per 1 ml of blood) 0.02 ml is cultured in a humid chamber of a thermostat or CO2 incubator under sterile conditions, in round-bottom immunological. tablets, in 0.120 ml of complete growth medium (ORS) (medium 199 containing 10% fetal calf serum, L-glutamine (2mM), HEPES (10mM), gentamicin sulfate 50 μg / ml) - control 3 blood samples. Test samples of 0.02 ml blood were cultured in 0.120 ml of ORS containing LPD-L as an inducer of IFN-γ in four dilutions (60, 30, 15, 7.5 μg / ml) for 96 hours. 18 hours before the end of cultivation, 1 MKCi ³ Hi-thymidine is introduced into the cell cultures (control and experimental). At the end of the cultivation, blood cells are harvested by a semi-automatic cell collector on glass fiber filters and dried. The count of radioactivity in imp / min is carried out in scintillation fluid using a "Betta-2" camera. The assessment of the level of specific cellular response is carried out by determining the stimulation index (I.S.), the calculation of I.S. according to the formula:

.

It was established that the result of a lymphocyte stimulation index of up to 3.2 is negative, more than 3.2 is positive (indicates a sensitization of the body of the office). 0.05 ml supernatants (supernatant) were taken from the obtained control cultures and cell cultures with PPD-L, combined into a common control (0.15 ml) and a common experimental sample (0.20 ml), which are then used for quantitative determination gamma interferon spectrophotometrically using ELISA using test systems designed to determine gamma interferon in supernatants. The assessment of the level of IFN-γ is carried out by determining the stimulation index. With a stimulation index of less than 7.0, the absence of active tuberculosis infection is diagnosed, with a stimulation index of more than 7.0, an active tuberculosis process is diagnosed.

The developed reaction based on RBTL (modified RBTL with PPD-L) made it possible to increase the informativeness of this immunological test and the informativeness of ELISA when evaluating IFN-γ. When performing the test, the loss of T-lymphocytes and other blood cells participating in the reaction (macrophages) is excluded, several doses of antigen (tuberculin) are used, in parallel, to determine the activity of the tuberculosis process, the maximum amount of gamma-interferon secreted by the cells is determined. Distinctive features of the proposed method is: the use of a culture supernatant, the possibility of using a small volume of blood (200 μl) and the exposure time of 96 hours.

In the studies, the specificity and sensitivity of mRBTL with PPD-L was determined. High specificity was established - 97.9% and method sensitivity - 97.8%.

We studied the informative value of the proliferation rate of lymphocytes during stimulation with tuberculin (mRBTL with PPD-L) in 279 children. Among them, in 117 children (41.9%) of 2-17 years old, respiratory tuberculosis was confirmed radiologically, including in the infiltration phase in 72 (61.5%), in the decay phase in 6 (5.1%), in the phase resorption and compaction in 11 (9.4%), calcifications in 16 (13.7%), or extrapulmonary tuberculosis. Infection was diagnosed in 107 children (38.4%) of preschool and primary school age. There were 31 children (11.1%) of preschool age who were not responsive to tuberculin or with questionable results of the Mantoux test with 2 TE PPD-L, of which 9 children were examined in dynamics after 6-12 months.

The results are shown in table 1. As can be seen from the table, the level of proliferative response of lymphocytes to tuberculin in children not infected, infected (including during the “turn” period) and patients with tuberculosis, significantly (p = 0,000) differed.

When examining children with tuberculosis in the infiltration phase before treatment, proliferation of lymphocytes for tuberculin in all (100%) was expressed, while the results for the phases of the tuberculosis process before treatment did not differ significantly (p = 0.302).

Table 1 The level of stimulation index in MRBT with PPD-L in children with tuberculosis, infected with MBT and not infected with MBT Observation groups n The result of MRTL with PPD-L, stimulation index M ± m Confidence interval Non-infected MBT 29th 2.1 ± 0.2 * 1.1-3.1 Patients with tuberculosis in the infiltration phase before treatment 25 16.6 ± 2.0 * 6.7-26.5 Patients with tuberculosis, regardless of the phase of the process before treatment 42 14.6 ± 1.4 * 5.3-23.9 Mycobacterium tuberculosis infected (tuberculin-positive) 107 7.4 ± 0.4 * 3,5-11,3 The period of early tuberculosis infection ("turn") 71 7.7 ± 0.5 * 3.3-12.1 Mycobacterium tuberculosis infected for over a year 36 6.7 ± 0.4 4.1-9.3 * p = 0,000

We additionally studied the informative value of the IFN-γ indicator with PPD-L: in 42 children and adolescents with tuberculosis, the average level of the indicator was 624.6 ± 205.2 pg / ml; in 30 healthy children and adolescents, it was 147.6 ± 16.2 pg / ml, while these indicators had significant differences, p = 0,000 (Mann-Whitney test). In primary forms of tuberculosis (n = 20), a higher level of IFN-y synthesis after stimulation of PPD-L (846.0 ± 291.6 pg / ml) was recorded than in secondary (n = 22, 403.2 ± 291.6 pg / ml), but no significant differences were found (p = 0.288). At the same time, a weak inverse correlation was found between the level of IFN-γ synthesis after stimulation of PPD-L and the disease genesis (r = -0.17, p <0.05), which confirms the tendency of a more pronounced cellular response in the primary genesis of tuberculosis. At the same time, a moderately pronounced direct relationship was established between mRBTL with PPD-L and IFN-γ with PPD-L (g = 0.61, p <0.05 according to Spearman) in patients with tuberculosis and a moderately pronounced direct relationship in healthy (g = 0.43, p <0.05 according to Spearman). Estimating the degree of correlation between the parameters (mRBTL with PPD-L and IFN-γ with PPD-L) in all examined children and adolescents, a moderately expressed level of correlation dependence was established (r = 0.67, p <0.05 according to Spearman). An evaluation of IFN-γ with PPD-L was performed according to the stimulation index, so the average stimulation index in patients with tuberculosis was 12.2 ± 2.6, in healthy children 2.2 ± 0.3. Determining the level of IFN-γ without stimulation and with stimulation with a non-specific mitogen (PHA) in patients with tuberculosis and in healthy children, no significant differences were established (p <0.05), therefore, these indicators of diagnostic value in determining the activity of tuberculosis infection do not (Table 2 ) In addition, the specificity and sensitivity of IFN-γ with PPD-L for tuberculosis patients were determined. The specificity level was determined at 70% and the sensitivity of the method at 97%. Analyzing the obtained false-negative results of IFN-γ with PPD-L in children suffering from tuberculosis, it was found that these indicators were noted when the tuberculosis process was completed (a decrease in activity was combined with a low level of lymphocyte proliferation for tuberculin), in one case, the development of negative anergy during generalization of tuberculosis was recorded .

table 2 The level of IFN-γ with stimulation by various mitogens and without stimulation in patients with tuberculosis and healthy children and adolescents Indicators Not infected with MBT Patients with respiratory tuberculosis before treatment IFN-γ with PPD-L: M ± m, pg / ml 147.6 ± 16.2 * 624.6 ± 205.2 * Confidence Interval, pg / ml 115.2-181.8 208.8-1040.4 M ± m, I.S. 2.2 ± 0.3 12.2 ± 2.6 Confidence Interval, i.s. 1.6-2.9 7.0-17.4 IFN-γ spontaneous: M ± m, pg / ml 71.1 ± 7.2 82.8 ± 14.4 Confidence Interval, pg / ml 57.6-64.8 55.8-111.6 IFN-γ with PHA: M ± m, pg / ml 9397.8 ± 1155.6 7189.2 ± 1517.4 Confidence Interval, pg / ml 7021.8-11775.6 3756.6-10621.8 * p = 0,000

The obtained results of studies of mRBTL with PPD-L and IFN-γ with PPD-L suggest that in children with a positive lymphocyte stimulation index (more than 3.2) and a positive result of the level of IFN-γ after stimulation with tuberculin (more than 209 pg / ml or IS more than 7.0) there is an active tuberculosis process. This allows this immunological test to be used both in the diagnosis of the primary specific process and in the development of tuberculosis of secondary origin, including extrapulmonary localization.

The following extracts from case histories can serve as examples of informativeness in determining the level of specific cellular response in primary tuberculosis infection in children.

Example 1. Alexey R., 15 years old, hospital patient card No. 68, was admitted to the hospital of the Omsk Region Budgetary Healthcare Institution of the Specialized Children's Tuberculosis Clinical Hospital (BUZ OO SDTKB) on April 30, 2009 for examination and treatment. Clinical diagnosis: Exudative pleurisy, left-sided, tuberculous etiology, MBT-. A preventive examination in April 2009 revealed changes in the fluorogram. Antibacterial therapy carried out in an institution of a common treatment network did not produce an effect.

Upon receipt of complaints was not noted, but the manifestation of intoxication in the form of pale skin, slight sweating was observed. For other organs and systems without features. Clarifying the epidemiological history, family contact was established. A bacteriological study on the office was not informative. They refused to conduct bronchoscopy. Upon admission, the level of specific cellular response in mRTBL with PPD-L was determined; the stimulation index was 23.5. The level of interferon-gamma, after stimulation with tuberculin was 763.2 pg / ml (IS = 7.5). An X-ray examination against the background of anti-tuberculosis therapy revealed a positive radiological dynamics in the form of resorption of pleural effusion at 3 months of chemotherapy. Thus, the etiology of pleurisy and the activity of tuberculosis infection were confirmed using mRBTL with PPD-L and IFN-γ with PPD-L.

Example 2. Valikhan A., 14 years old, hospital patient card No. 51, was admitted to the hospital BUZ OO SDTKB 04/07/2009 in order to assess the activity of the tuberculosis process and prepare for surgical treatment.

Diagnosis at admission: Multiple tuberculomas of the left lung, decay and seeding phase, MBT +, exacerbation. Tuberculosis of the left bronchus. Urinary tuberculosis, MBT-.

Family contact, prolonged, multidrug resistance. Since 2006, a child has expressed and hyperergic tuberculin tests. By contact I received courses of anti-tuberculosis therapy.

Upon admission to the hospital manifestations of tuberculous intoxication, bronchopulmonary syndrome was not observed. Examination of organs and systems revealed no pathological changes. When conducting bronchoscopy, pathology was not detected. In general blood and urine tests without pathology. According to the results of tuberculin diagnostics in January 2009, an infiltrate of 16 mm (pronounced positive reaction). X-ray determined 2 rounded shadows up to 2 cm in diameter, of a homogeneous structure without a zone of perifocal inflammation and foci of screening in the surrounding tissue. According to the results of bacterioscopy, acid-resistant mycobacteria were detected, when sowing, the result was negative (after 3 months). The level of specific cellular response in mRTTL with PPD-L was determined, the stimulation index was 3.2, which corresponds to the level of infection with mycobacterium tuberculosis, without the activity of a specific process. The level of IFN-γ with PPD-L was determined, which amounted to 144.9 pg / ml (I.s. = 0.7), which also confirmed the absence of activity of a specific process.

Thus, on the basis of the data obtained, it was possible to talk about the lack of progression of the tuberculosis process and recommend surgical treatment in a planned manner after 3 months against the background of anti-tuberculosis therapy, taking into account the possible risk of drug resistance. On 04.08.2009, the child underwent resection of C2, C6 of the left lung; there were no complications in the postoperative period.

In our opinion, given the high specificity and sensitivity of the method, the use of MRBTL with PPD-L in clinical practice in combination with IFN-γ with PPD-L will improve the quality of diagnosis of specific inflammation, evaluate the effectiveness of specific treatment, shorten the diagnosis of active tuberculosis infection, therefore to conduct timely anti-tuberculosis therapy.

Claims (1)

A method for assessing tuberculosis activity in children and adolescents, including assessing the state of specific cellular immunity using a modified response of the blast transformation of lymphocytes (mRTL) with tuberculin (PPD-L) and enzyme-linked immunosorbent assay (ELISA) with determination of interferon-gamma (IFN-γ) level PPD-L, characterized in that mRBTL and ELISA are performed using a small volume (200 μl) of whole blood with a tuberculosis allergen (PPD-L), 4 dilutions of antigen are used, the level of specific cellular response and IFN-γ are evaluated stimulation according to the definition of the stimulation index and with indicators of the index of stimulation of the cellular response of more than 3.2 and IFN-γ with PPD-L more than 7.0 diagnose active tuberculosis process, and when the rate of MRBT with PPD-L is more than 3.2 and IFN-γ PPD-L less than 7.0 is not an active tuberculosis infection.