Levels of E, A, M, G immunoglobulins in children with newly diagnosed tuberculosis at the beginning of antimycobacterial therapy

Tuberculosis in children is a chronic inflammatory process in which immunological mechanisms occupy one of the leading positions in pathogenesis. Therefore, the immune status indicators study among children makes it possible to determine the disease activity, predict its course for the purpose of revealed violations early correction and determine the effectiveness of the treatment. One of the immunological indicators of tuberculosis is immunoglobulins level. The aim of the work – to investigate IgE, A, M, G levels in children’s blood serum with newly diagnosed tuberculosis at the beginning of antimycobacterial therapy for further development of rationally directed immunocorrective therapy. Materials and methods. The study of IgE, A, M, G levels in blood serum was performed in 28 children with newly diagnosed tuberculosis aged 1 to 16 years (average age was 9.2 ± 1.1 years old). The levels of IgE, IgA, IgM and IgG in the blood serum were studied using the method of solid phase enzyme-linked immunosorbent assay on the equipment of the Sirio S immuno-enzyme reader with the use of reagent kits “Granum” LLC (Kharkiv, Ukraine). The results of the study were processed by modern methods of analysis on a personal computer using statistical software package Statistica ® for Windows 6.0 (StatSoft Inc., AXXR712 D833214FAN5). Results. Children with newly diagnosed tuberculosis at the beginning of the intensive phase of antimycobacterial therapy had a significant increase in IgE and A levels in 2.1 and 1.2 times respectively. With an increase in the prevalence of a specific process and the appearance of destruction, there is a significant increase in the IgG level in 1.2 times. Direct correlations between IgE and IgA, IgA and IgM, IgM and IgG in the disseminated process, and IgA, IgM levels in the local process have been found. A reliable correlation between the decrease in the BCG post-vaccination sign size and the increase in IgE content has been revealed, that may be a prognostic factor in the post-vaccination immunity evaluation. Cellular component of immune system activation is determined in the children, as evidenced by a significant increase in albumins level in 1.5 times against the background of the specific inflammatory process low activity (α 1 - and α 2 -globulins levels were within the age norm). There is a significant worsening of dysproteinemia (a decrease in albumin levels by 9 % and an increase in β— and γ-globulins levels in 1.3 times) and specific inflammatory process activity (an increase in α 1 -globulin level in 1.3 times) with the increase in the specific process prevalence and destruction development. It has been established that the decrease in the albumin level is associated with growth in the IgE, A and G levels; growth in the β-globulin level is associated with growth in the IgA and G levels, and with IgE, M, and G – in γ-globulins. It has been found that IgA, M and G, and the protein fractions parameters – albumins, β- and γ-globulins are significant and highly informative for the immune changes diagnosis in children with newly diagnosed tuberculosis. Conclusions. In children with newly diagnosed pulmonary tuberculosis at the beginning of an intensive phase of antimycobacterial therapy with increase in the specific process prevalence and destruction development, significant changes in the levels of immunoglobulins and protein fractions are observed. Data resulting from the study suggest that the humoral component of immune system is also activated on the background of the cellular component of immune system activation with patient’s state aggravation, which is an unfavorable prognosis for patients’ recovery.

Tuberculosis in children is a chronic inflammatory process in which immunological mechanisms occupy one of the leading positions in pathogenesis. Therefore, the immune status indicators study among children makes it possible to determine the disease activity, predict its course for the purpose of revealed violations early correction and determine the effectiveness of the treatment [1,2].
Выводы. У детей, больных впервые диагностированным туберкулезом легких, в начале интенсивной фазы антимикобактериальной терапии при нарастании распространенности специфического процесса и появления деструкции наблюдаются достоверные изменения уровней иммуноглобулинов и белковых фракций. Полученные данные свидетельствуют: на фоне активации клеточного звена иммунной системы с утяжелением состояния происходит активация гуморального звена, что является неблагоприятным прогнозом для выздоровления пациентов. are excreted in a human body: serum and secretory. Serum IgA provides local immunity, and secretory participates in providing protection of mucous membranes (respiratory, urogenital tract and digestive tract) from microorganisms, including mycobacterium tuberculosis (MBT). The decrease in IgA level indicates a lack of humoral local immunity, and in the absence of appropriate treatment it leads to the immune system depletion, which causes infectious process inhibition, etc. Increase in IgA level is observed in infectious diseases, both in acute and chronic exacerbation processes [4]. Under the influence of IgM the compliment system is activated in response to an acute infectious process, providing primary immunity. Serum IgG provides long-term humoral immunity to infectious agents. In chronic infectious and autoimmune diseases an increase in the serum IgG level is detected and its deficiency contributes to the weakening of a body resistance to infectious diseases.

Purpose
To investigate IgE, A, M, G levels in children's blood serum with newly diagnosed tuberculosis at the beginning of anti-mycobacterial therapy for further development of rationally directed immunocorrective therapy.

Materials and methods
The study of IgE, A, M, G levels in blood serum was performed in 28 children with NDTB aged 1 to 16 years (average age was 9.2 ± 1.1 years old) who were on inpatient treatment in a children's unit of the clinical base of Phthisiology and Pulmonology Department of Zaporizhzhia State Medical University at Communal Institution "Zaporizhzhia Regional Antituberculous Clinical Dispensary" and constituted the main group. There were 16 (57.1 %) girls and 12 (42.9 %) boys. The comparison group included 30 healthy children aged 1 to 16 (average age was 8.9 ± 1.0 years old). The comparison group were comparable to age and gender.
The levels of IgE, IgA, IgM and IgG in the blood serum were studied using the method of solid phase enzyme-linked immunosorbent assay on the equipment of the Sirio S immuno-enzyme reader with the use of reagent kits "Granum" LLC (Kharkiv, Ukraine). Units of measurement: IgE (МU/ml), IgA, М, G (g/l). The analysis of blood serum protein fractions parameters was carried out using rheum sampling electrophoresis method with distribution on acetate-cellulose film on the fraction. The levels of albumins and globulins (α 1 , α 2 , β, γ), (%) were determined [5].
All indicators were evaluated at the beginning of the intensive phase (IF) of antimycobacterial therapy (AMBT). Parents of all sick children signed a patient's written informed consent to participate in the study.
The diagnostic significance of indicators assessment was carried out using the diagnostic utility coefficient (DUC) calculation according to A. M. Zemskova formula [6]: DUC (d 1 2 + d 2   2 )/(М 2 -М 1 ), where d 1 is the mean average deviation of a healthy person's parameters; d 2 is the mean square deviation of children with NDTB parameters; М 1 and М 2 are the average means, respectively. The coefficient which had a value of 1, was considered highly informative, in the range from 1.1 to 10 was medium-informative, exceeding 10 was low informative.
The results of the study were processed by modern methods of analysis on a personal computer using statistical software package Statistica ® for Windows 6.0 (StatSoft Inc., AXXR712 D833214FAN5). The distribution of quantitative characteristics normality was analyzed using the Shapiro-Wild's test. The descriptive statistics for quantitative variables distributed under the normal distribution law included the mean (M), standard deviation (σ). The confidence interval for the mean with confidence probability of 0.95 was built. The reliability of compared values differences was determined by the Student's t-criterion. All tests were bi-directional. The difference for P < 0.05 was considered statistically significant. Correlation analysis was performed using the Pearson correlation coefficient (r).

Results and discussion
On hospital admission 5 (17.8 %) children with NDTB were diagnosed with extrapulmonary forms of tuberculosis, 3 (10.7 %) of them had tuberculosis of the intracranial lymph glands and 2 (7.1 %) had specific pleurisy. 23 patients (82.2 %) were diagnosed with pulmonary forms: 5 (17.8 %)    Оригинальные исследования had pleurisy, 5 (17.8 %) had primary tuberculosis complex, 11 (39.5 %) had infiltrative form and 2 (7.1 %) -disseminated. Bacterial secretion and destructive process in the lungs were registered in 7 children (25 %). Local forms of tuberculosis without a destructive process in the lungs were diagnosed in children with an average age of 5.4 ± 1.5, and disseminated tuberculosis including a destructive process presence were diagnosed among two times older children with an average age of 12.5 ± 1.1. During blood serum Ig E, A, M, G total levels investigation in children with NDTB at the beginning of the IF AMBT ( Table 1), reliable changes have been found only in the IgE and A. Thus, the level of IgE was within normal range in 13 patients (46.5 %), and it was elevated in 15 (53.5 %). The average IgE level exceeded the age norm by 2.1 times (P < 0.05). IgA levels were high only in 2 (7.1 %) and the level of all the other 26 children (92.9 %) indicators were within the normal range. The mean IgA level exceeded the age-matched norm in 1.2 times (P < 0.05). IgM levels in all 100 % cases were within the age range. IgG level was decreased in 1 patient (7.1 %), and the average level of its content did not exceed the norm. Furthermore the direct correlation of IgE and IgA levels (r = 0.309; P < 0.05), IgA and IgM (r = 0.341; P < 0.05) have been found.
When analyzing the changed levels of Ig depending on tuberculosis prevalence, ( Table 2), the following features have been established. At normal average values the level of IgG content reliably depended on tuberculosis severity, namely its prevalence and destructive process presence. Thus, its level growth in 1.2 times (P < 0.05) was determined when disseminated process in comparison with the local without destructions. The IgE level increase in 1.2 times was also diagnosed in children with a disseminated process. The levels of IgA and IgM did not depend on the prevalence and destructive process presence.
DUCs were calculated for all Ig parameters in the local process without destructions. It has been found that the DUCs for IgA, M, and G values were up to 1.0 (0.05; -0.08 and 0.94 respectively), indicating their high informativeness for diagnosis. IgE informational content was average, since its CG was 14.9. When studying the correlation of Ig levels with the dimensions of BCG post-vaccination signs among vaccinated children with NDTB a direct correlation between the decrease in the sign size and the IgE level increase (r = 0.670; P < 0.001) has been established.
So far as the γ-globulin fraction contains immunoglobulins IgG, IgA, IgM, IgE, the analysis of protein fraction indicators ( Table 3) has been performed. It has been established that children with VDTB at the beginning of IF AMBT had an increase in the albumin level in 1.5 times in comparison with healthy children (P < 0.05). Furthermore, a high level of albumin was diagnosed in 26 children (92.8 %) of the main group. In contrast, an increase in the proteins globulin fraction levels was determined by increasing the β-and γ-globulins levels in 1.5 and 1.3 times (P < 0.05) respectively, which was recorded in 19 patients (67.8 %). The levels of α 1 -and α 2 -globulins were within the age norm, indicating specific inflammatory process low activity in children at the beginning of treatment. The level of α 2 -globulins has been determined in 14 children (50 %).
Analysis of protein fractions altered levels depending on the prevalence of tuberculosis ( Table 4) has showed the following results. Children with a common specific process had an increase in dysproteinemia, which was manifested by a decrease in albumin levels by 9 % (P < 0.05) and an increase in the levels of β-and γ-globulins in 1.3 times (P < 0.05). In this context, an increase in the inflammatory specific process activity was determined, indicating an increase in the level of α 1 -globulin in 1.3 times (P < 0.05).
DUC was also calculated for all protein fractions parameters. It has been established that DUC for albumin indicators, β-and γ-globulin values were up to 1.0 (0.16, 0.13 and 0.37 respectively), indicating their high informativeness for diagnosis. The informativeness of the α 1 -and α 2 -globulin indices was average, since their DUC were 1.11 and 4.49 respectively.
The investigation of correlation between levels of Ig content and protein fractions parameters ( Table 5) made it possible to establish that children with NDTB at the beginning of IF AMBT had a decrease in albumin levels which was associated with an increase in IgE, A, and G levels. The growth in β-globulin levels was associated with an increase in levels of IgA and G, and γ-globulins IgE, M and G.

Conclusions
1. Children with NDTB, at the beginning of the IF AMBT had a significant increase in IgE and A levels in 2.1 and 1.2 times, respectively. With an increase in the specific process prevalence and destruction appearance there was a significant increase in the IgG level in 1.2 times. Direct correlation of IgE and IgA, IgA and IgM, IgM and IgG in the disseminated process, and IgA, IgM levels in the local process has been established. 2. A reliable correlation between the decrease in the BCG post-vaccination sign size and the increase in IgE content has been found which can be a prognostic factor in the evaluation of post-vaccination immunity.
3. The activation of immune system cellular component is determined among the children as evidenced by a significant increase in albumins level in 1.5 times against the background of specific inflammatory process low activity (levels of α 1 -and α 2 -globulins were within the age range). There is a significant increase in dysproteinemia (a decrease in albumin levels by 9 % and an increase in β-and γ-globulins levels in 1.3 times) and specific inflammatory process activity (an increase in α 1 -globulin levels in 1.3 times) with the increase in the specific process prevalence and destruction appearance. 4. It has been established that the decrease in the albumin level is associated with the growth in IgE, A and G levels; the growth in β-globulin levels is associated with the growth in IgA and G levels, and with IgE, M and G -in γ-globulins. 5. It has been found that IgA, M and G and the protein fractions parameters -albumins, β-and γ-globulins are significant and highly informative for the immune changes diagnosis among children with NDTB.
Prospects for further researches. The revealed changes are the basis for the development of rationally directed immunocorrective therapy among children with NDTB, and it will contribute to improvement of treatment effectiveness.