This study was conducted at the TB Clinic of Mile Four Hospital Abakaliki Ebonyi State. This is a well known Catholic mission hospital that is a Special Tuberculosis and Leprosy Referral Centre in the region. It is located in Abakaliki, the capital of Ebonyi State, South-Eastern Nigeria and serves patients referred from nearby states.
This study was a longitudinal cohort study in which blood samples were collected from study subjects before commencement of anti-tuberculosis therapy, 2-months into therapy and at 6-months into therapy.
The study population comprised subjects confirmed to be positive for pulmonary tuberculosis by Sputum-Smear Acid Fast Bacilli by Ziehl Neelsen’s stain and GeneXpert MTB/RIF assay. The baseline samples were collected before commencement of therapy (pre-treatment) and participants followed up in the course of treatment and samples collected after 2 months and 6 months therapy. Tuberculosis treatment regimen involves two months of therapy (Intensive phase) in which the patients are given 4 fixed dose combination (Rifampicin, Isoniazid, Pyrazinamide and Ethambutol hydrochloride) and the Continuation phase in which the subjects are given Rifampicin and Isoniazid only for 4 months. The dosage of therapy is dependent on the body weight of the subjects.
Sample Size Determination
Sample size was calculated using G*Power software version 3.0.10 (Universitat Dusseldorf Germany). Power analysis for a repeated measures ANOVA with three measurements was conducted in G*Power to determine a sufficient sample size using an alpha of 0.05, a power of 0.90 and a medium effect size. Based on these, the calculated sample size was 58.
A total of sixty (60) Tuberculosis subjects aged 18 to 65 years (37.53 ± 15.65 years) were enlisted for this study. They consist of 35 males (38.23 ± 16.17 years) and 25 females (35.76 ± 14.59 years).
Inclusion and exclusion criteria
Individuals of both gender confirmed to be positive for active pulmonary Mycobacterium tuberculosis were included while individuals with any known bleeding disorders or history of bleeding, pregnant women, those that withheld their consent before or in the course of the study, subjects on aspirin and anticoagulant therapy, females on oral contraceptives, smokers, those taking any local herbal concoctions, individuals that have other known clinical diseases such as cancer, HIV, diabetes, chronic infections, chronic kidney and liver diseases were excluded from the study.
Ethical approval was obtained from the Ethics committee of Federal Teaching Hospital Abakaliki (FETHA) Ebonyi State with reference number: FETHA/REC/VOL.2/2018/105 and permission was sought and obtained from the management of Mile four hospital Abakaliki before sample collection.
The aim of the research was explained to prospective participants and those who gave oral informed consent were recruited into the study. Confidentiality was ensured according to Helsinki declaration.
Socio-demographic information such as gender, age, marital and educational status, occupation etc. and clinical information such as symptoms, history of infection, blood transfusion, smoking, alcohol use etc were obtained using a standardized questionnaire.
Sputum for TB diagnosis
Sputum samples consisting of one spot sample and one early morning sample was collected in a wide mouth container from the subjects for Acid fast bacilli (AFB) test as well as for the automated GeneXpert MTB/RIF real-time nucleic acid amplification test for rapid and simultaneous detection of TB and Rifampicin resistance.
Blood sample collection
All the necessary precautions were observed in collecting and processing the blood samples. Eight millilitres (8ml) of blood sample was collected from each subject before commencement of therapy and at 2-months and 6-months into therapy. Three millilitres (3mls) was dispensed into plain sample bottles. Serum was obtained after clotting by spinning at 3000rpm for 10 minutes and used for evaluation of Tumor Necrosis Factor - alpha (TNF-α), IL-10, IL-6, IL-2, Transforming growth factor-beta (TGF-β), Thrombopoietin and HIV screening. Also, two and half millilitres (2.5 ml) of blood were dispensed into 0.28ml (280µl) of 3.2% tri-sodium citrate to give a final blood: tri-sodium citrate ratio of 9:1. The sample was mixed properly by reverse uniform inversion and centrifuged at 3000rpm for 10 minutes at room temperature. The clear plasma was separated into a clean dry plastic container and used for the determination of P-selectin, Platelet activating factor, Platelet factor-4 and Gp IIb/IIIa complex. The remaining two and half millilitres (2.5ml) was dispensed into bottles containing di-potassium salt of Ethylenediamine tetra-acetic acid (K2-EDTA) at a concentration of 1.5mg/ml of blood and used for platelet count.
Methods of Sample Analysis
Ziehl-Neelsen technique for Mycobacterium tuberculosis diagnosis as described by WHO .
Smear preparation: A piece of clean stick was used to transfer and spread sputum materials evenly covering an area of about 15-20mm diameter on a glass slide. The smear was air dried and labelled.
Heat fixation: The slide with the smear uppermost was rapidly passed three times through the flame of a Bunsen burner and allowed to cool.
Ziehl –Neelsen staining: The slide containing the smear was placed on a slide rack and the smear covered with Carbol Fuchsin stain. The stain was heated until vapour just begins to rise. The heated stain was allowed to remain on the slide for 5 minutes. The stain was washed off with clean water and then covered with 3% v/v acid alcohol for 5 minutes or until the smear is sufficiently decolorized, i.e. pale pink. The slide was washed off with clean water. The smear was covered with methylene blue stain for 1-2 minutes and then washed off with clean water. The back of the slide was wiped clean and placed in a draining rack for the smear to air-dry.
Microscopic examination of Ziehl-Neelsen stained smear: The smear was examined microscopically using the 100x oil immersion objective.
Results: AFB - Red, straight or slightly curved rods, occurring singly or in small groups. Cells and background material appear blue.
Genexpert method for detection of Mycobacterium tuberculosis and rifampicin resistance (GeneXpert MTB/RIF).
The assay consists of a single-use multi-chambered plastic cartridge pre-loaded with the liquid buffers and lyophilized reagent beads necessary for sample processing, DNA extraction, and hemi-nested real-time PCR. Sputum samples were treated with the sample reagent (containing NaOH and isopropanol). The sample reagent was added in the ratio of 2:1 to the sputum sample and the closed specimen container was manually agitated twice during 15 minutes of incubation at room temperature. Two (2) mls of the treated sample was transferred into the test cartridge, the cartridge was loaded into the GeneXpert instrument and an automatic step will complete the remaining assay steps. The assay cartridge also contained lyophilized Bacillus globigii spores which served as an internal sample processing and PCR control. The spores was automatically re-suspended and processed during the sample processing step and the resulting B. globigii DNA was amplified during PCR step. The standard user interface indicates the presence or absence of M. tuberculosis, the presence or absence or Rifampicin resistance and a semi quantitative estimate of M. tuberculosis concentration (high, medium, low and very low). Assays that are negative for M. tuberculosis and also negative for B. globigii internal control was reported as invalid.
Screening for HIV-1 and HIV-2
Subjects were tested for HIV using Inverness DetermineTM 1 and 2 (Inverness Medicals Co. Ltd, Japan) and STAT-PAK (Chembio Diagnostic system, New York, USA). Uni-Gold (Trinity Biotech, Bray, Ireland) was used as the tie-breaker according to the national guidelines for HIV counselling and testing. Tests were carried out according manufacturer’s instructions.
Platelet count as described by Lewis et al .
A 1 in 20 dilution of well mixed blood was made in diluent by adding 20µl of blood to 0.38ml of ammonium oxalate (10g/l). Before the dilution, the blood sample was examined to rule out the presence of any blood clot. The suspension was mixed properly and an Improved Neubauer counting chamber was filled with the suspension using a Pasteur pipette. The counting chamber was placed in a moist Petri dish and left untouched for 20 minutes to give time for the platelets to settle. The preparation was examined using x40 objective.
The platelet count was determined as follows;
Platelet count per litre = No of cells counted x dilution x 106
Volume counted (µl)
Measurement of inflammatory cytokines and haemostatic parameters
TNF-α, IL-10, IL-6, and IL-2 were assayed using enzyme-linked immunosorbent assays (ELISA) test kits from UCyTech Biosciences (Utrecht, Netherlands). The method employs quantitative sandwich enzyme immunoassay. A monoclonal antibody specific for human TNF-α, IL-10, IL-6, and IL-2 has been coated onto a microplate for each cytokine. Subsequently, 100 μL of blank, diluted standard, controls, and samples were added to each well. The plates were sealed and incubated for 2 hours at 37°C and washed six times with the Wash buffer using the automated microplate Washer. Then, 100 μl of the diluted detection antibody solution was added to each well and the plate was sealed and incubated for 1 hour at 37°C. The washing step was repeated, and 100 μL of diluted SPP conjugate was added to each well and the plates sealed and incubated for 1 hour at 37°C. The washing step was repeated, and 100 μL of TMB substrate solution was added into each well and incubated in the dark for 20 min. The reaction was stopped with 100 μL of stop solution. The results were evaluated using a Microplate reader at 450 nm.
Additionally, TGF-β, P-SEL, GP IIb/IIIa complex, TPO, PF-4, and PAF were assayed using ELISA kit from Elabscience Biotechnology Inc., (Wuhan, Hubei). The protocol for each parameter was carried out according to the manufacturer’s instructions. A monoclonal antibody specific for human TGF-β, P-SEL, GP IIb/IIIa complex, TPO, PF-4 and PAF has been coated on the wells. Subsequently, 100 μL of blank, standard, controls, and samples were added to the micro ELISA plate well and incubated for 90 min at 37°C. The liquid was removed without washing, and 100 μL of Biotinylated Detection antibody working solution was immediately added to each well, covered with the Plate sealer, gently mixed, incubated for 1 hour at 37°C, and washed three times using a Microplate washer. 100 μL of horseradish peroxidase conjugate working solution was added to each well and incubated for 30 min at 37°C. The washing step was repeated, and 90 μL of substrate reagent was added and incubated for 15 min at 37°C in the dark. The reaction was stopped with 50 μl of stop Solution. Results were evaluated using a microplate reader at 450 nm.
Statistical Package for Social Sciences software (IBM SPSS, Armonk, NY, USA) version 22 was used in data analysis. A normality test was conducted to assess the distribution of each variable using Kolmogorov–Smirnov statistic. Data were not normally distributed and thus were expressed as median (range), frequencies and percentages in tables, Bar chart and Pie chart.
Friedman ANOVA and post-hoc analysis with Wilcoxon Signed Rank test was applied for comparison of multiple repeated measurements (at Pre-treatment, 2-months and 6-months into therapy) and Spearman Rank Order correlation was used to test relationship between variables. P < 0.05 was considered statistically significant.