Serum Profiles of Pro-Inflammatory Cytokines and Some Other Biomarkers of Inflammation in Nigerian Tuberculosis Patients Undergoing Standard Chemotherapy at Enugu

Abstract

Introduction: Tuberculosis (TB) is a communicable disease that has afflicted man for centuries. It is one of the commonest causes of death from infectious diseases in the world. Nigeria is ranked among the 22 countries with the highest TB burden in the world. The discovery of host or pathogen specific biomarkers of TB for diagnosis, treatment, and prognosis has of recent become top research priority globally. The pathogenesis of TB involves cell-mediated immune response against Mycobacterium tuberculosis and different cytokines have been found to be specifically involved in the pathogenesis of pulmonary TB. Cytokines which are a group of regulatory molecules that permit contact-independent communication between the cells of the human body have been considered to be candidate biomarkers for TB. There has been a lot of TB- related research but to the best of our knowledge no prospective study has concurrently and intermittently measured the blood levels of Tumor necrosis factor alpha (TNFα), Interleukin 1 beta (IL-1 β) and Interleukin 6 (IL-6) in Nigerian TB patients from commencement of TB treatment to the end of short course chemotherapy. This study aims at documenting the serum concentrations of these three cytokines (IL-1 β; IL-6; and TNFα) in eligible TB patients using human ELISA kits, from the time of diagnosis to completion of six months short course chemotherapy of TB. This prospective study is expected to yield useful information that would be utilized in the quest for better biomarkers to improve TB management. Methodology: The study was a prospective observational study carried out at the Chest Clinic of the University of Nigeria Teaching Hospital (UNTH) Enugu with the written permission of UNTH Management. The research proposal was approved by the Research Ethics Committee of UNTH Enugu. Freshly diagnosed drug-naive adult TB patients of both genders who met the inclusion criteria were recruited after giving informed consent. Uniform standard WHO - approved treatment was prescribed for and supplied to the patients. Each subject was followed up for six months starting from their respective times of recruitment into the project. Healthy volunteers were also recruited. Blood samples were taken at zero time (i.e. after diagnosis and enrolment into study but before ingestion of TB drugs); at 2 months of treatment; and also at 6months of treatment. Serum concentrations of Interleukin 1 beta (IL-1 β); Interleukin 6 (IL-6); and Tumor necrosis factor alpha (TNFα) were measured using human ELISA kits (e-Bioscience, USA). Erythrocyte Sedimentation Rate (ESR) was measured by Westergren method and Haemoglobin concentration was estimated by the Cyanmethaemoglobin method. Total leukocyte count and differential counts for the percentage counts of Neutrophils, Lymphocytes, Monocytes, Basophils, and Eosinophils were done by routine manual microscopy. Results were analysed using Graphpad prism and SPSS computer soft-wares and displayed as tables, pie, and bar charts.. Results: Forty two tuberculosis patients and thirty one healthy volunteers were able to complete the study. The mean serum concentration of Interleukin 1 beta (IL-1 β) for the TB patients prior to treatment was abnormally raised (30.0 ± 2.0 pg/ml). As treatment progressed there was statistically significant progressive reduction in the mean IL-1 β serum concentration at 2months as well as at 6 months of treatment (22.0±1.1pg/ml and 17.0 ± 1.3 pg/ml respectively). There was a significant difference between the mean IL-1beta obtained after 6months of treatment and for the healthy volunteers (13.0 ± 1.3pg/ml). A similar trend was observed for both Il-6 and TNFα with values obtained at 6months of treatment approximating those of healthy volunteers. TNFα mean serum concentration at zero time was 40.7±2.4pg/ml and 23.1±1.4 pg/ml after 6 months of treatment. For haemoglobin the trend was different. The mean Haemoglobin concentration appreciated from 11.4±0.2g/dl prior to treatment to 11.8±0.2g/dl after 2 months of treatment, and to 13.2±0.1g/dl four months later. Compared to healthy volunteers (14.4±0.2g/dl) the mean haemoglobin concentration of the TB patients was significantly lower at zero time and at 2 months of treatment. The profile of mean Erythrocyte Sedimentation Rate (ESR) documented in the study mirrored those of the cytokines. The mean Monocyte count, Eosinophil count, and Basophil count obtained before commencement of treatment and during the course of treatment all ranged from 0 to 3% and exhibited no significant difference between TB patients, and healthy volunteers on one hand and between zero time values and mean counts made at 2months and 6months of treatment respectively on the other hand. However, both the mean total leukocyte count and the percentage Neutrophil count in the peripheral blood increased during the course of treatment, while lymphocyte count decreased over time during the corresponding time intervals. The mean total leukocyte count was 4785.0 ± 158.0/mm3 at zero time, 4805.0 ± 106/mm3 and 4835.0 ± 143.0/mm3 at six months of treatment. Conclusion: This study shows that in Nigerian adult patients erythrocyte sedimentation rate (ESR) as well as the three cytokines assayed in this study were all very high in the serum of drug-naïve TB patients compared to those of the healthy volunteers but as TB chemotherapy continued over the period of six months there was progressive reduction in the serum levels of both ESR and the 3 cytokines. At the completion of TB treatment, the serum concentrations of the cytokines were comparable to those of the healthy human controls. The serum profiles therefore indicate that these cytokines are good candidate biomarkers for Nigerian TB patients. Larger funded prospective studies would be needed to confirm and elucidate the suitability of these cytokines singly or in combination to serve as diagnostic and/or prognostic host biomarker(s) for tuberculosis.