In�ammation-mediated changes in haemostatic variables of pulmonary tuberculosis infected subjects in the course of treatment.

Abstract

Introduction Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb), a bacterium that in majority of cases affect the lungs [1]. It is ranked as second only to Human immunode ciency virus (HIV) infection as the greatest killer worldwide due to a single infectious agent. About one third of the world's population is thought to have been infected with M. tuberculosis. In Nigeria, Tuberculosis is a major public health problem. According to CDC [2], Nigeria is the country worst affected by tuberculosis in Africa and ranks third in the world [2]. Tuberculosis burden in Nigeria is thought to be further compounded by the high prevalence of HIV/AIDS among the general population.
In ammation is part of the complex biological response of body tissues to harmful stimuli, such as pathogens, damaged cells, or irritants [3], and is a protective response involving immune cells, blood vessels, and molecular mediators. In ammation plays a dual role in immune response to mycobacterium infection. On the one part, it is a prerequisite for successful pathogen elimination while on the other part, it mediates tissue injury and disease progression. At the onset of the infection, in ammatory reactions are largely protective; during active disease, the deleterious effect of in ammation prevails, making in ammation a paramount pathogenic factor of TB progression. In ammation in TB infection involves both immune cells such as Neutrophils, lymphocytes and monocytes/macrophages and cytokines such as IL-6, TNF-α, IL-10, TGF-β, IL-1β and IFN-Υ [4]. Hence the measurement of in ammatory response in TB subjects involves the assessment of these cells and cytokines.
In TB patients, severe infection and in ammation are known to lead to haemostatic abnormalities, ranging from insigni cant laboratory changes to severe disseminated intravascular coagulation (DIC) [5].
Haemostasis is a balanced process that halts bleeding after blood vessels have been traumatized and involves the entire process that maintains the owing blood in a uid state and con ned to the circulatory system [6]. Thus while in ammation aims at restoring the integrity of damaged or threatened tissues, most frequently because of injury or infectious pathogens, haemostasis is a physiological defence mechanism to stop bleeding due to vessel wall damage [7].
Quite obviously, haemostasis and in ammation are closely linked, both in health and disease. They are tightly interrelated patho-physiologic processes that considerably affect each other. It is a bidirectional relationship in which in ammation leads to activation of the haemostatic system that in turn in uences in ammatory activity [8]. It is well established that local activation of the haemostatic system is an essential part of the host defenses in both infectious and non-infectious in ammatory states. This implies that the haemostatic system acts in concert with the in ammatory cascade creating an in ammation-haemostasis cycle in which each activated process promotes the other and the two systems function in a positive feedback loop [9]. Thus the failure of the complex balance between proand anti-coagulation, or between pro-and anti-in ammatory reactions because of genetic or acquired disturbances may result in disease. The many established links between in ammation and coagulation help explain the pro-thrombotic tendency observed in patients with acute in ammatory or infectious diseases [8]. The vast crosstalk between in ammatory and haemostatic systems, involves all components of haemostasis such as the endothelium, platelets, clotting and anticoagulant pathways as well as the brinolytic system [10]. Platelets which are blood cells mediate primary haemostasis leading to formation of a platelet plug, while the coagulation factors drive the secondary haemostasis leading to formation of a stable brin clot [6]. The action of platelet in haemostasis involves adhesion, activation and change of shape from discoid to spiky sphere, release of granular content most of which have haemostatic effect and marks the haemostatic process and platelet aggregation. Each of this action is mediated and marked by a number of measurable parameters such as Platelet factor -4, P-selectin, GP IIb/IIIa, and Thrombopoietin that regulates the formation of platelets in the bone marrow. It could be assumed that the general effect of in ammation on haemostasis which is already established due to the cross-talk between them would most likely affect these parameters that mediates and marks the activity of platelet in primary haemostasis. This assumption was put to test by this study as the changes that take place in these parameters with in ammation were monitored in TB infected subjects. From the foregoing, it could be deduced that exploring the already established knowledge of a cross-talk between haemostasis and in ammation will strengthen our understanding of the patho-physiological relationship between in ammation and alterations in haemostatic variables in TB infected individuals. This study therefore monitored the changes in the levels of in ammatory cytokines and correlated it with the alterations in haemostatic variables of individuals infected with Tuberculosis in the course of therapy.

Study setting
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.

Study Design
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.

Study Population
The study population comprised subjects con rmed 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 xed 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 su cient 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.

Inclusion and exclusion criteria
Individuals of both gender con rmed 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 Consideration
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.

Informed Consent
The aim of the research was explained to prospective participants and those who gave oral informed consent were recruited into the study. Con dentiality was ensured according to Helsinki declaration.

Research Questionnaire
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.

Sample Collection
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 ampli cation 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 nal 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 (K 2 -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 [11].

Procedure
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 xation: The slide with the smear uppermost was rapidly passed three times through the ame 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 su ciently 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).

Procedure
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 realtime 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 ampli ed 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 Determine TM 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 [6].
Procedure 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 lled 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 10 6 Volume counted (µl) Measurement of in ammatory 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 speci c 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.

Results
Majority of the participants were males (58.3%), aged 18-30 years (41.7%) and married (60%). The total mean age of the participants was 37.53 ± 15.65 years with an age-range of 18-65 years. The mean age for males was 38.23 ± 16.17 years while that of females was 35.76 ± 14.59 years. Moreover about 63% of the participants had secondary education while only a few (6%) had no educational quali cation (Table 1).
The systolic and diastolic blood pressures were within the normal range at pre-treatment, 2-month and 6month into therapy (Figure 2).
The median levels of TNF-α (pg/ml), IL-6 (pg/ml) and IL-2 (pg/ml) was signi cantly increased at 2-month compared to the pre-treatment value (P<0.001 respectively). However, there was a signi cant decline at 6month into therapy compared to the value at 2-month into therapy (P=0.025, 0.006, <0.001). Furthermore, there was no signi cant change in the median levels of IL-10 (pg/ml) and TGF-β (pg/ml) at 2-month into therapy compared to the level at pre-treatment (P>0.05). However, there was a signi cant increase at 6month into therapy compared to the level at pre-treatment and 2-month into therapy (P=0.020 and 0.001) (See table 2).
There was no signi cant change in the median values of PF-4 (ng/ml) at 2-month into therapy compared to pre-treatment (P>0.05), but there was a signi cant decline at 6-month into therapy (0.35) compared to 2-month into therapy and pre-treatment (P=0.030). Moreover, there was a signi cant increase in the median levels of GP IIb/IIIa (ng/ml) and PSEL (ng/ml) at 2-month into therapy compared with pretreatment (P=0.037 and 0.004) and a signi cant decline at 6-month into therapy compared to the level at 2-month into therapy (P=0.007 and <0.001). Furthermore, there was a signi cant increase in the median values of TPO (pg/ml) at 2-month into therapy compared to pre-treatment (P=0.017). But no signi cant change at 6-month into therapy compared to 2-month into therapy (P>0.05) (See table 3).
Additionally, the Mean ± SD of total platelet count (x10 9 /l) increased signi cantly at 2-month into therapy

Discussion
In ammation and haemostasis are two biological processes that considerably affect each other. Tuberculosis infection is marked by changes in in ammatory markers which could result in alterations in haemostatic parameters. This study assessed in ammation by measuring TNF-α, IL-6, IL-2 which are known pro-in ammatory cytokines as well as IL-10 and Transforming growth factor-beta (TGFβ) which are anti-in ammatory cytokines. Furthermore, the haemostatic variables assessed in this study were Platelet count, P-selectin, Platelet activating factor, Platelet factor-4, GP IIb/IIIa complex and Thrombopoietin hormone.
The nding showed that TNF-α, IL-6 and IL-2 levels signi cantly increased at 2-month into therapy and declined at 6-month into therapy. Since TNF-α and IL-6 are known pro-in ammatory markers this nding gives a strong basis to conclude that 2-month into treatment is the peak of in ammation in TB subjects and that their pro-in ammatory activities are down-regulated at 6-months into therapy as seen in this study. According to Mootoo et al [12], TNF-α is an essential component of the innate defence mechanism of the host against pathogenic challenge and plays a major role in the pathology of Tuberculosis while IL-6 it is a pleitropic pro-in ammatory cytokine with a wide range of biological activities in immune regulation, haematopoiesis, in ammation, oncogenesis and is of critical importance in acquired immunity against M. tuberculosis infection [13]. Thus the increase observed in TNF-α at 2-month into treatment, could be due to its in ammatory role which is coordinated via induction of other cytokines (such as IL-6) and the recruitment of immune and in ammatory cells. This may explain why a similar pattern of response was obtained for IL-6 in this study. Also, IL-2 has been shown to have multiple and sometimes opposing functions during an in ammatory response and in its dual and contrasting functions, it contributes to both the induction and termination of in ammatory immune responses [14]. Thus the signi cant increase at 2-month into treatment could be part of its contribution to the induction of in ammatory response. The haemostatic implication of the increased TNF-α level is an increase in platelet adhesion molecule expression as it has been shown to play a role in the up-regulation of adhesion molecules [15]. While the haemostatic implication of an increased IL-6 is a release of platelet because when IL-6 reaches the bone marrow it promotes megakaryocyte maturation [16]. This agrees with the nding in this study of a persistent increase in platelet count up till 6-month into treatment. The decline of these cytokines at 6-month into treatment could be due to the reduction in in ammation as anti-in ammatory markers predominates. This correlates with our nding of elevated anti-in ammatory cytokines at 6-months which have been reported by Shalev et al. [17] to inhibit the in ammatory roles of TNF-α and IL-6 . It also con rms that the expression of these cytokines is strictly controlled, as their continual production can mediate damaging effects [18].
The IL-10 and TGF-β level was signi cantly lower at pre-treatment and at 2-month into therapy, but increased signi cantly at 6-months into therapy. The signi cant reduction in IL-10 and TGF-β at the early stage (pre-treatment and 2-month) compared to 6-month into therapy is of obvious bene ts to the subjects. It has been found that an increased level or over-production of IL-10 in TB patients has been associated with immune-suppression and greater susceptibility to the disease [19] while an increased TGF-β results in macrophage deactivation and suppression of T-cell responses to Mycobacterium tuberculosis. Similarly, the increase in the level of IL-10 and TGF-β at the later stage of the infection (6-month into therapy) is also bene cial to the subjects as their role in modulation of in ammation and proin ammatory cytokines is necessary to avoid pathologic consequences that will result from an unregulated in ammatory process. This as earlier stated supports the reduction in IL-2, TNF-α and IL-6 observed in this study at 6 months into therapy.
In this study, a signi cant increase in platelet count was found at 2-month into therapy compared to pretreatment and a further signi cant increase at 6-month into therapy. According to Iqbal et al. [20], platelet count has a signi cant role in immune functions and reactive thrombocytosis is generally seen in chronic in ammation. However, the ndings of this study did not agree with that of Koju et al. [21] and Iqbal et al. [20] that discovered a decreased platelet count from diagnosis till completion of initiation phase (at 2month) with resumption of normal platelet count during continuation phase of treatment. They attributed this observed decrease to Rifampicin which is considered as a major drug that decreases platelet count in anti-tuberculosis therapy. However, clinically low platelet counts are often associated with poor prognosis and increased risk for infection. In the light of this, the increased platelet count in our study is a sign of enhanced prognosis with treatment since the platelet count values were within the normal range.
This study also reported a signi cant increase in Thrombopoietin hormone (TPO) from the second month till 6-month into therapy where there was a slight but non-statistically signi cant decline. The reason for the signi cant increase at 2-month into treatment could mean that the hormonal increase is tied to the increase in in ammation and pro-in ammatory cytokines which peaks at 2-month into therapy. This thinking is re-enforced by the nding by Kaushansky [22] that in the liver, the production of TPO is augmented by IL-6. This is supported by the signi cant positive correlation between IL-6 and TPO in our study. Thus it can be safely assumed that an increase in IL-6 at 2-month into treatment resulted to an increased TPO production. The increase could also have a direct relationship with the increased platelet count as it is known to regulate the production of platelets by stimulating the production and differentiation of megakaryocytes. The slight though non-signi cant decline at 6-month into therapy compared to 2-month into therapy may suggest that the process of reduction in TPO has set in with the down-regulation of pro-in ammatory cytokines observed at 6-month into treatment especially IL-6 which as earlier stated plays a role in the production of TPO. If this be the case then the rate of decline of TPO is not proportionate with rate of decline of in ammatory cytokines, thus the non-signi cant decline. It could also be due to the negative feedback resulting from a further signi cant increase in the platelet count at 6-month into therapy, this may have initiated the decline at 6-month into therapy though yet to be statistically signi cant.
This study found a signi cant increase in P-selectin (P-SEL) at 2-month into therapy and then a signi cant decrease at 6-month into therapy. P-selectin functions as a cell adhesion molecule and a marker of platelet activation and degranulation [23]. According Keane [15], TNF-α has been shown to play a role in the up-regulation of adhesion molecules of which P-selectin is one. Thus the signi cant increase in the level of P-selectin at 2-month into therapy which is the peak of in ammatory process in our study, points to the role of in ammation in up-regulation of the activity of P-selectin. This may also be linked to the weak signi cant positive correlation observed between IL-6 and P-selectin as well as the non-signi cant positive correlation between TNF-α and P-selectin. Functionally, P-selectin plays an essential role in the initial recruitment of leukocytes to the site of injury during in ammation and aggregation of platelet at areas of vascular injury [24]. The signi cant decline at 6-month into therapy shows that the activity of P-selectin wears off as the in ammatory cytokines recedes and anti-in ammatory markers dominates.
A signi cant increase in GP IIb/IIIa was seen at 2-months into therapy, followed by a signi cant decline at 6-months of therapy. The increase in GP IIb/IIIa complex at 2-month into therapy could also be a response to in ammation as TNF-α has been earlier stated to play a role in the up-regulation of adhesion molecules [15]. This argument is supported by our nding of a signi cant positive correlation between GP IIb/IIIa and TNF-α. Also based on the signi cant negative correlation between GP IIb/IIIa and TGF-β obtained in this study, the signi cant decline of GP IIb/IIIa at 6-month into therapy could be due to the down-regulation of in ammatory cytokines as earlier established in this study.
The ndings of this study showed that Platelet factor-4 (PF-4) increased non-signi cantly at 2-month into therapy but decreased signi cantly at the 6-month into therapy. An elevated concentration of platelet factor-4 could be used as an indicator for platelet activation since they are usually released from alpha granules of activated platelets. Elshamaa et al. [25] proved that PF-4 plays an important role in in ammation and wound repair and it is a strong chemo-attractant for neutrophils which is very crucial in the in ammatory process. Thus up-regulation and down-regulation of in ammation may be the reason for the non-signi cant increase at 2-month into therapy and decrease at 6-month into therapy. There was also a signi cant weak positive correlation between PF-4 and TNF-α.
Platelet activating factor (PAF) is important to the process of haemostasis because it causes platelets to aggregate and blood vessels to dilate [26]. In this study, there was a signi cant but weak negative correlation between TGF-β (an anti-in ammatory marker) and PAF. High PAF level are associated with variety of conditions involving in ammation and it can trigger in ammatory and thrombotic cascades and mediate molecular and cellular interactions (cross-talks) between in ammation and thrombosis. This link between PAF and in ammation makes it unclear the reason for the non-signi cant decrease in PAF that was observed from the second month till the 6 month into therapy with increase and decrease in in ammation as well as the non-signi cant positive correlations with IL-6 and TNF-α. However, since PAF is synthesized by two different pathways (de novo pathway and remodeling pathway) and the remodelling pathways (which is the primary source of PAF under pathological condition) is activated by in ammatory agents, the non-signi cant change could mean that the threshold or extent of in ammation was not su cient to activate the remodeling pathway for PAF synthesis.

Conclusion
Pro-in ammatory response in TB subjects is highest at 2-month into therapy as indicated by an increase in TNF-α, IL-6 and IL-2 and is down-regulated at 6-month into therapy as indicated by a decrease in these cytokines, while anti-in ammatory response were highest at 6 month into therapy as indicated by an increase in IL-10 and TGF-β. The levels of P-selectin, GP IIb/IIIa, and TPO (haemostatic variables) are in uenced by in ammation because they change accordingly with changes in pro-in ammatory cytokines in TB subjects. Thus it can be deduced that in ammation modulates the levels of these haemostatic variables in TB subjects.

Declarations
Grant for this research was provided by Tertiary Education Trust Fund (TETFUND), Nigeria.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Authors' contributions COO conceived the study, collected data, carried out ELISA analysis, participated in statistical analysis and drafted the rst manuscript. GIA Participated in the design of the study, data collection and manuscript revision and coordination of the study. POM participated in the study design, and manuscript revision. JOA contributed to the design of the study, data interpretation and critically revised the manuscript. NCI participated in data interpretation and manuscript revision. All authors read and approved the nal manuscript.
Ethics approval and consent to participate The study was approved by the Ethics committee of Federal Teaching Hospital Abakaliki (FETHA) (Ref: FETHA/REC/VOL.2/2018/105). The subjects gave informed consent before they were recruited into the study and con dentiality was ensured according to Helsinki declaration.
1 vs 3 = Comparison of parameters at pre-treatment and 6-months into therapy.
2 vs 3 = Comparison of parameters at 2-months and 6-months into therapy. 1 vs 3 = Comparison of parameters at Pre-treatment and 6-months into therapy.
2 vs 3 = Comparison of parameters at 2-months and 6-months into therapy.   A bar chart showing the blood pressure values (mm/Hg) at pre-treatment, 2-month and 6-month into therapy.