Changes in the levels of adipokines in newly diagnosed pulmonary tuberculosis patients in case of treatment failure

Матеріали та методи. У проспективне дослідження включили 58 хворих на вперше діагностований туберкульоз легень, яких поділили на 2 групи: у 1 увійшли 28 пацієнтів із неефективно лікованим уперше діагностованим туберкульозом легень; 2 група (порівняння) – 30 хворих, які згодом успішно завершили курс лікування. Контрольна група – 29 здорових донорів-волонтерів. Зміни показників адипокінів вивчали в Навчальному медико-лабораторному центрі Запорізького державного медичного університету шляхом дослідження рівнів лептину й адипонектину в сироватці крові методом твердофазного імуноферментного аналізу на приладі імуноферментний рідер SirioS із застосуванням наборів «Mediagnost» (ФРН), (нг/мл). Співвідношення лептин/адипонектин розраховували математично (ум. од.). Зразки крові хворих збирали вранці натще. Статистичний аналіз виконали, використовуючи програму Statistica for Windows 13.0 (Copyright 1984–2018 TIBCO Software Inc. all rights reserved, ліцензія No JPZ8041382130ARCN10-J).

The World Health Organization's (WHO) Global Report states that 6,4 million of new tuberculosis cases had been diagnosed all over the world in the 2017 [1]. According to the findings of the State Institution "Department of Public Health of the Ministry of Healthcare of Ukraine", the rate of successful treatment of all tuberculosis cases remained the lowest among the countries of the European Region of WHO and was 76.5 %, when WHO's indicator was 85 % [2]. In the Zaporizhzhia region, the indicator was 72.9 %. Through a statistical analysis, it was elucidated that one of the reasons of low treatment efficacy among tuberculosis patients is a treatment failure. The treatment failure indicator for Ukraine was 7.4 %, when the similar indicator for the Zaporizhzhia region went beyond the national one by 1.7 times and was 12.6 %.
The study of adipokines has attracted our attention while we were searching for the identification of pathogenetic mechanisms of treatment failure among patients with pulmonary tuberculosis. So, as it is known, once mycobacterium tuberculosis penetrates a human body, under favourable conditions, they growth and pervade to various organs and body systems. In recent times, researchers suggest that adipocytes, which are influenced by a nutritional status, control metabolism and have much power in the functional activity of the immune system, might be one of the places of mycobacterium tuberculosis in the human body [3]. Also protracted mycobacterium tuberculosis persistence in the adipocytes might lead to a metabolic imbalance [4].
Among all the adipokines, leptin and adiponectin involvement in tuberculosis pathogenesis has been extensively studied nowadays [5,6]. Leptin is a key mediator of an energy metabolism, apart from that it mediates an appetite regulation, adipose tissue redistribution, increase in T-hel pers 1 activity, lymphocytes and peripheral mononuclear cells proliferation and production of γ-interferon [7,8]. Adiponectin promotes an increase in the body tissues irritability and T helper 1 products [9].

Original research
However, the study results of the leptin and adiponectin level in tuberculosis patients are controversial enough. So, some researchers believe, that the blood levels of leptin in these patients are decreased, while the levels of adiponectin are elevated [10][11][12]. Keicho N. et al. [13] also recommend using a decreased leptin/adiponectin ratio (LAR) as a predictive measure to estimate a progressive course of tuberculosis, alongside the specified measurements.
Perna V. et al. [8] have determined in their study that in diagnosed tuberculosis, the level of leptin is reduced compared to the normal ranges of adiponectin indicators. Other researchers point that leptin levels in tuberculosis patients are not different from stated values [14], or even increased [15].
In the process of considering an adipokines impact on a treatment failure, N. Santucci, et al. [16] found that the levels of leptin are decreased, the levels of adiponectin are increased and particular decreasing in the LAR with tuberculous inflammation progresses.
Thus, improving the treatment efficacy for patients with newly diagnosed tuberculosis by reducing the number of treatment failure cases is very urgent issue nowadays. The literature review shows that adipokines are an important part of tuberculosis pathogenesis associated with the impact on both the metabolism of human body and the cell-mediated immunity.
The results of the studying the leptin and adiponectin involvement, which attract the interest of researchers more than anything else does nowadays, are controversial, especially in predicting/forecasting a newly diagnosed tuberculosis treatment failure. The changes in the adipokines level in patients with pulmonary pathology have been studied only in relation to chronic obstructive pulmonary disease (COPD) in Ukraine [17]. There has been no assessment of these indicators in tuberculosis patients in national literature. According to the literature data on the subject of pathogenetic importance of adipokines indicators in the specific process progression it could be argued that the study of this indicators changes in treatment failure is very important nowadays and also allows developing the case management algorithm to increase the treatment effectiveness among this patient population as a result.

Aim
To define the characteristics of adipokines levels changes in newly diagnosed pulmonary tuberculosis patients in case of treatment failure by examining the levels of leptin, adiponectin and leptin/adiponectin ratio.

Materials and methods
This prospective study included 58 patients with newly diagnosed pulmonary tuberculosis. Patients were divided into 2 groups: the first group was made up of 28 patients with the ineffectively treated newly diagnosed pulmonary tuberculosis; the second group (comparison group) consisted of 30 patients who completed the treatment successfully. The control group was made up of 29 healthy voluntary donors.
There were no significant age and sex differences between the groups ( Table 1).
Changes in the blood serum adipokines levels were determined by leptin and adiponectin levels measurements via enzyme linked immunosorbent assay using immunoassay analyzer Sirio S with "Mediagnost" kit (Germany) (ng/ml) at the Scientific Medical Laboratory Center of Zaporizhzhia State Medical University. LAR was calculated mathematically (relative units). Blood samples were collected after an overnight fasting.
All the patients received an inpatient treatment in the Communal Institution "Zaporizhzhia Regional TB Clinical Dispensary" during 2017-2019. Patients were included in the study after voluntary signing an informed consent. The study was approved by Commission for Bioethics of Zaporizhzhia State Medical University (Protocol No. 1 dated 02.02.2017).
Inclusion criteria: newly diagnosed pulmonary tuberculosis, massive pulmonary tuberculous process (infiltrative and disseminated), pulmonary tissue destructions of over 3 сm in diameter and positive sputum smear.
Exclusionary criteria: other cases of the 2 nd category, chemoresistant tuberculosis, TB/HIV co-infection, comorbidities, age over 70 years, non-compliance with treatment.
Since the massive pulmonary tuberculous process was diagnosed in patients, an intensive phase for patients was 90 doses. Therefore, treatment efficacy was evaluated in 3 months according to the Unified Medical Care Protocol "Tuberculosis" (Order of the Ministry of Healthcare of Ukraine No. 620 dated 04.09.2014) [18]. Assessments of the changes in adipokines levels were performed at the beginning of the treatment by using antituberculosis drugs (0 doses) and after 3 months of the treatment (90 doses).
Statistica for Windows 13.0 (Copyright 1984-2018 TIBCO Software Inc., all rights reserved, License No. JPZ8041382130ARCN10-J) software was used for the statistical analysis. Normality of the quantitative indicators distribution was performed by the Shapiro-Wilk method. The study results were presented in a form of M ± m, where M -the mean value, m -the mean error. A statistical significance of differences between groups was determined using the Student's method (since the normality of the quantitative indicators distribution was performed). The results considered to be significant only if the indicator of P was lower than 0.05.

Results
The study results of the adipokines levels are presented in Fig. 1-3.
When comparing the initial levels of the adipokines studied and LAR, it was found that the leptin levels ( Fig. 1) were decreased in both groups of patients in comparison with the control group: in the 1 st group by 12.7 times, in the 2 nd group -by 3.8 times (1.20 ± 0.35 ng/ml and 4.10 ± 1.09 ng/ml, respectively, against 15.40 ± 2.91 ng/ml; P ˂ 0.01). Besides, the level of leptin was 3.3 times lower in the 1 st group compared to the 2 nd group (1.2 ± 0.35 ng/ml against 4.10 ± 1.09 ng/ml; P <0.05). The levels of adiponectin (Fig. 2) were also increased in both groups of patients in comparison to the control: by 1.4 times in the 1 st group and by 1.8 times in the 2 nd group (17.20 ± 1.25 ng/ml and 21.10 ± 1.16 ng/ml, respectively, against 12.00 ± 1.06 ng/ml; P < 0.01). Wherein the levels of adiponectin in the 2 nd group 1,2 times exceeded that in the 1 st group (21.10 ± 1.16 ng/ml against 17.20 ± 1.25 ng/ml; P < 0.05).
According to the data obtained, a decrease in LAR was observed (Fig. 3) in both groups of patients as compared to the control: in the 1 st group by 21 times and in the 2 nd group by 6.1 times (0.07 ± 0.03 relative units and 0.24 ± 0.07 relative units, respectively, against 1.47 ± 0.32 relative units; P < 0.001). In the 1 st group, this indicator was 3.4 times lower (P < 0.05) than that in the 2 nd group.
Assessment of adipokine indicators was performed after 3 months (90 doses) only in the 1 st and 2 nd groups. Analyzing the dynamics of the indicators studied it was found that the levels of leptin (Fig. 1) were elevated in all the patients regardless of the further treatment effect: in the 1 st group by 2 times (2.40 ± 0.46 ng/ml against 1.20 ± 0.35 ng/ml; P < 0.01) and in the 2 nd group by 1,4 times (5.7 ± 1.37 ng/ ml against 4.10 ± 1.09 ng/ml; P < 0.01). The levels of adiponectin (Fig. 2) increased in dynamics, but no significant differences were found. The LAR indicators (Fig. 3) were 1.7 times increased in the 1 st group (0.12 ± 0.03 relative units against 0.07 ± 0.03 relative units; P < 0.05).
Assessing the levels of adipokines in patients treated for 3 months (90 doses), it was found that, despite changes in the dynamics, the levels of leptin (Fig. 1) remained 6.4 times lower in the 1 st group and 2.7 times in the 2 nd group compared to the control (2.40 ± 0.46 ng/ml and 5.7 ± 1.37 ng/ml, respectively, against 15.4 ± 2.91 ng/ml; P < 0.05). No significant differences were found between leptin levels in the 1 st and 2 nd groups.
The levels of adiponectin (Fig. 2) remained elevated in patients of both groups compared to the control group: by 1.6 times in the 1 st group, by 1.8 times in the 2 nd group (18.8 ± 1.76 ng/ml and 22.20 ± 1.35 ng/ml, respectively, against 12.00 ± 1.06 ng/ml; P ˂ 0.01). No significant differences were found between adiponectin levels in the 1 st and 2 nd groups.
The LAR indicators (Fig. 3) remained lower in patients of both groups compared to the that values in the control group: by 12.3 times in the 1 st group and by 3.6 times in the 2 nd group (0.12 ± 0.03 relative units and 0.41 ± 0.14 relative units, respectively, against 1.47 ± 0.32 relative units; P ˂ 0.01). The LAR indicators were significantly 3,4 times lower in the 1 st group compared to the 2 nd group (0.12 ± 0.03 relative units against 0.41 ± 0.14 relative units; P < 0.05).

Discussion
This paper presents the study results of the adipokines levels (leptin, adiponectin and LAR) in patients with newly diagnosed pulmonary tuberculosis with generalized pulmonary involvement, lung tissue destruction of over 3 cm in diameter and positive sputum smear. Such changes in patients with newly diagnosed pulmonary tuberculosis are the risk factor for ineffective treatment, but determination of adipokines levels in this category of patients is under-reported in the literature available.
As a result of the measurements, it was found that all the patients with newly diagnosed pulmonary tuberculosis presented significantly reduced baseline values of leptin and LAR, and significantly increased levels of adiponectin. The data obtained did not contradict the majority of other researchers' results [11][12][13]16].  *: a significant difference between the levels of indicators relative to the control group, P ˂ 0.05; #: a significant difference between the levels of indicators in the dynamics within one group, P ˂ 0.05; ■: significant difference between the levels of indicators of the 1 st and 2 nd groups, P ˂ 0.05.
It was established that patients with newly diagnosed pulmonary tuberculosis and the risk of ineffective treatment by clinical, radiological and bacteriological indexes, who were further diagnosed with ineffective treatment, presented more profound changes in adipokines levels demonstrating significantly lower levels of leptin, adiponectin and LAR than patients with newly diagnosed pulmonary tuberculosis successfully completed the antimycobacterial chemotherapy later.
In the dynamics, all the indicators of adipokines (leptin, adiponectin and LAR) were found to be increased in all the patients, regardless of further treatment results, that has been confirmed in several works [8,19]. In contrast to our results, Keicho N. et al. found that leptin levels continued to decrease in dynamics and adiponectin levels increased [13].
Such changes occurred as a result of treatment, the number of mycobacteria and their activity decreased, which had an impact on the functional activity of adipocytes changes. In turn, changes in the activity of adipocytes had an effect on the immune cells functional activity, which in certain values of adipokines contributed to the positive course of tuberculous process in some patients (the 2 nd group) and protracted one in others (the 1 st group).
It was found that despite some increase in LAR among patients who subsequently were diagnosed with treatment failure, it remained significantly lower compared to the results of patients who completed the treatment successfully later. That is, we can agree with the data obtained by N. Keicho et al. [13] that the LAR has a great diagnostic value in predicting ineffective treatment.
However, the results showed that in patients with newly diagnosed pulmonary tuberculosis and generalized pulmonary involvement, lung tissue destructions of over 3 cm in diameter and positive sputum smear at the beginning of antimycobacterial therapy, the prognostic value of ineffective changes have divergent changes in adipokine indicators: decreased leptin levels and increased adiponectin levels. The LAR reduction has the prognostic value after 3 months of treatment (90 doses).

Conclusions
In patients with newly diagnosed pulmonary tuberculosis with generalized pulmonary involvement, lung tissue destructions of over 3 cm in diameter and positive sputum smear, who were further diagnosed with an ineffective treatment, the following significant changes in adipokines levels were established: 1. At the beginning of antimycobacterial therapy, there was a significant decrease in the levels of leptin by 12.7 times, an increase in the levels of adiponectin by 1.4 times and a decrease in the LAR by 21 times.
2. The levels of leptin, adiponectin and the LAR were significantly worse than in patients who successfully completed the treatment course.
3. In the process of antimycobacterial therapy, the levels of leptin was slightly increased, but remained significantly reduced, and the levels of adiponectin continued to increase significantly. The LAR was 3.4 times significantly lower than in patients who successfully completed the treatment course.
Thus, patients with newly diagnosed pulmonary tuberculosis with the risk of ineffective treatment were diagnosed with severe impairment of the adipocytes functional activity, when antimycobacterial chemotherapy could not even restore the balance to the adipokine system and smooth down resulting concomitant variations in immune response. The findings showed the prognostic value of the changes in leptin, adiponectin and leptin/adiponectin ratio indicators at the beginning of the treatment in relation to treatment failure among patients with newly diagnosed pulmonary tuberculosis.
Prospects for further scientific research. To develop an algorithm for managing this category of patients in order to reduce the number of treatment failure and increase the effectiveness of antimycobacterial therapy.