|Year : 2020 | Volume
| Issue : 4 | Page : 373-379
Comparison of the levels of some hemostatic and inflammatory markers between tuberculosis patients with and without malaria at pretreatment, intensive, and continuation phase treatment
Chizoba Okechukwu Okeke1, Grace I Amilo2, Martin O Ifeanyichukwu1, Chisom M Okeke3
1 Department of Medical Laboratory Science, Faculty of Health Sciences and Technology, College of Health Sciences, Nnamdi Azikiwe University, Awka, Nigeria
2 Department of Haematology, Faculty of Medicine, College of Health Sciences, Nnamdi Azikiwe University, Awka, Nigeria
3 Department of Family Medicine, Nnamdi Azikiwe University Teaching Hospital, Nnewi, Anambra State, Nigeria
|Date of Submission||11-Sep-2020|
|Date of Acceptance||22-Sep-2020|
|Date of Web Publication||15-Dec-2020|
Chizoba Okechukwu Okeke
Department of Medical Laboratory Science, Faculty of Health Sciences and Technology, College of Health Sciences, Nnamdi Azikiwe University, Nnewi Campus P.M.B. 5001, Awka, Anambra State
Source of Support: None, Conflict of Interest: None
Background: Tuberculosis and malaria (TB/MP) co-infection generates severe pathology that affects the levels of cytokines and hemostatic parameters than either disease. Anti-TB treatment regimen involves phases of different drug cocktails that may additionally modulate the levels of inflammatory cytokines and hemostatic parameters. This study investigated the variations in the levels of hemostatic and inflammatory markers when compared between TB patients with and without malaria at pretreatment, intensive, and continuation phase treatment. Method: In this cross-sectional study, 180 patients were recruited comprising; 35 TB-only and 25 TB/malaria patients at pretreatment, 36 TB-only and 24 TB/malaria patients at intensive phase treatment, and 27 TB-only and 33 TB/malaria patients at continuation phase therapy. P-selectin (P-SEL), platelet-activating factor (PAF), platelet factor-4, GP IIb/IIIa complex, tumor necrosis factor-alpha (TNF-α), interleukin (IL)-10, IL-6, IL-2, transforming growth factor (TGF)-β, and thrombopoietin (TPO) were assayed using enzyme-linked immunosorbent assays. Mann–Whitney test and Spearman's rank correlation were applied for statistical test. Results: At pretreatment, the median levels of IL-6 and IL-10 were significantly lowered, while P-selectin (P-SEL), GP IIb/IIIa, and PAF were significantly increased in TB/malaria patients compared to TB patients without malaria. At intensive treatment, TNF-α, IL-6, and IL-2 were significantly higher, while IL-10 and PAF were significantly reduced in TB/malaria patients compared with TB patients without malaria. At continuation phase treatment, TNF-α, IL-6, TGF-β, PF4, GP IIb/IIIa, and TPO were significantly reduced, while P-SEL was significantly increased in TB/malaria patients compared with TB patients without malaria. Conclusion: Differences in the levels of inflammatory cytokines and hemostatic markers between TB patients co-infected with malaria and nonmalaria-infected TB patients vary with anti-TB treatment.
Keywords: Hemostasis, inflammatory cytokines, malaria, tuberculosis
|How to cite this article:|
Okeke CO, Amilo GI, Ifeanyichukwu MO, Okeke CM. Comparison of the levels of some hemostatic and inflammatory markers between tuberculosis patients with and without malaria at pretreatment, intensive, and continuation phase treatment. Int J Mycobacteriol 2020;9:373-9
|How to cite this URL:|
Okeke CO, Amilo GI, Ifeanyichukwu MO, Okeke CM. Comparison of the levels of some hemostatic and inflammatory markers between tuberculosis patients with and without malaria at pretreatment, intensive, and continuation phase treatment. Int J Mycobacteriol [serial online] 2020 [cited 2021 Dec 6];9:373-9. Available from: https://www.ijmyco.org/text.asp?2020/9/4/373/303447
| Introduction|| |
Plasmodium falciparum co-infection with Mycobacterium tuberculosis (MTB) could generate both innate and acquired immunity involving inflammatory responses which could affect the plasma levels of cytokines., Malaria has known immunomodulatory effect and alters the balance of circulating cytokines that are instrumental to the control of mycobacterial infection. Moreover, studies has shown that pro- and anti-inflammatory cytokines are involved in the malarial pathogenesis and the outcome of malaria infection is determined by the balance in induction and counter-regulation of both pro-and anti-inflammatory cytokines., Similarly, aside the role of platelets in hemostasis, its granular content (platelet factor-4 [PF-4]) has been shown to play a major role in malaria parasite killing. These suggest that malaria co-infection with tuberculosis (TB) will have obvious effects on inflammatory, hemostatic, and other markers of human physiological processes.
MTB is the bacterium that is known to cause TB which is an infectious disease that generally affects the lungs in the cases of pulmonary TB, and other parts of the body in the cases of extra-pulmonary TB. Africa still has the highest incidence of TB cases at 237 per 100,000 population with 25% of the incident cases globally and more than 95% of deaths occurred in developing countries such as Nigeria. Malaria, on the other hand, is the world's most prevalent parasitic disease and an impediment to economic development. Malaria remains a global problem with 219 million cases reported in 2017, 92% of which were in Africa.
Considerable geographic overlap exists in the distribution of malaria and TB with concentration of both diseases in Africa, Asia, and South America. According to Baluku et al., malaria could impair TB containment and increase mortality of TB patients. Malaria and TB together account for millions of deaths worldwide annually. An attendant problem with malaria among TB patients is the overlap of symptoms, clinical signs, and complications such as anemia, cough, and respiratory distress. This delays the diagnosis and initiation of treatment of either infection, yet malaria has the potential to increase mortality of TB patients. The increase in mortality is plausibly due to deleterious immune interactions between TB and malaria which have not been studied widely in human subjects. Individuals residing in zones of high transmission such as Nigeria may become co-infected with both pathogens, and biological interactions may exist between P. falciparum and MTB.
Studies among malaria/TB co-infected patients suggested protective humoral and cellular immune responses against the complications of each disease, which resulted in an increased production of gamma interferon, tumor necrosis factor alpha (TNF-a), and humoral factors induced by TB infection that are protective against malaria infection. The above studies and that by Baluku et al. have sought to understand the prevalence, clinical factors, and immunologic interaction of malaria and TB as a pathway for early diagnosis, control, and eradication of two notorious diseases that present a global emergency. Our own study focused on the multi-dimensional effect the interaction between malaria and TB infection will have on some hemostatic and inflammatory parameters before treatment and at each phase of anti-TB treatment. It is worthy of note that a number of studies have been done focusing on inflammatory cytokines, but much attention has not been given to the primary hemostatic parameters assessed in this study. Therefore, the emphasis of this study was on investigating how both primary hemostatic parameters and inflammatory cytokines vary in TB patients with malaria compared with TB patients without malaria before and during each phase of treatment.
| Method|| |
This study was carried out at the TB Center of Mile Four Hospital Abakaliki Ebonyi State, Nigeria. Mile 4 hospital is a renowned Catholic mission hospital that is a Special TB and Leprosy Referral Center with isolation wards for admission of TB patients. It is located in Abakaliki, the capital of Ebonyi State, South-Eastern Nigeria.
This is a cross-sectional study designed to ascertain differences in the levels of hemostatic and inflammatory markers when compared between TB patients co-infected with malaria and TB patients without malaria at pretreatment, intensive phase, and continuation phase of treatment.
The patients that met the inclusion criteria were recruited consecutively until the sample size was attained.
Sample size determination
Sample size was calculated using G*Power software version 3.0.10 (Universitat Dusseldorf Germany).
A total of 180 patients were recruited comprising sixty at pretreatment (35 TB infected and 25 TB/malaria infected), sixty patients after the second month (intensive phase) of treatment (36 TB infected and 24 TB/malaria infected), and sixty patients after the 6th month (continuation phase) of treatment (27 TB infected and 33 TB/malaria infected).
Adult individuals positive for active pulmonary MTB co-infected with malaria as well as TB patients not infected with malaria were included.
The following patients were excluded from the study: patients with any known bleeding disorders or history of bleeding; pregnant women; patients that had blood transfusion in the previous 3 months; patients on aspirin and anticoagulant therapy; females on oral contraceptives; smokers; those taking any local herbs or herbal concoctions; and patients that have other known clinical diseases such as cancer, HIV, diabetes, chronic infections, chronic kidney and liver disease.
Ethical approval was obtained from the Ethics committee of Federal Teaching Hospital Abakaliki (FETHA/REC/VOL. 2/2018/105), and permission was sought and obtained from the management of Mile four hospital Abakaliki before sample collection. The reason for the research was explained to prospective participants and those who gave informed consent were recruited into the study and confidentiality was ensured according to the Helsinki declaration.
Blood sample collection
All the necessary precautions in collection, separation, and processing of blood samples were observed. Eight milliliters of blood was collected from each patients and processed ensuring the integrity of cellular elements and avoiding preanalytical errors arising from sample collection and processing. Three milliliters was dispensed into plain sample bottles. Serum was obtained after clotting by spinning at 3000 rpm for 10 min and used for evaluation of TNF-α, interleukin (IL)-10, IL-6, IL-2, transforming growth factor (TGF)-β, thrombopoietin (TPO), and HIV screening. Also, 2½ mL of blood was dispensed into 0.28 mL (280 μL) 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 3000 rpm for 10 min at room temperature. The clear plasma was separated into a clean dry plastic container and used for the determination of P-SEL, platelet-activating factor (PAF), PF-4 and Gp IIb/IIIa complex. The remaining 2½ mL was dispensed into bottles containing di-potassium salt of Ethylenediamine tetra-acetic acid at a concentration of 1.5 mg/mL of blood and used malaria parasite diagnosis and parasite count.
Sputum samples for tuberculosis diagnosis
Two sputum samples (consisting of one spot sample and one early morning sample) collected in a wide mouth container from the patients was required for acid-fast bacilli 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.
Methods of sample analysis
Diagnosis of tuberculosis
This was done by Ziehl–Neelsen technique for MTB diagnosis as described by WHO and Genexpert method for detection of MTB and rifampicin resistance (GeneXpert MTB/RIF) as described by Blakemore et al.
Diagnosis of malaria
Whole blood was used for the diagnosis of malaria using thick and thin blood smears for microscopic detection as described by Cheesbrough.
Procedure for malaria parasite count
The parasite counts were done using thick blood films as described by the WHO.
Measurement of cytokines and hemostatic parameters
TNF-α, IL-10, IL-6, and IL-2 were assayed using enzyme-linked immunosorbent assays (ELISA) test kits from U-CyTech 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 h 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 h 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 h 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. In addition, TGF-β, P-SEL, GP IIb/IIIa complex, TPO, PF-4, and PAF were assayed using ELISA kit from Elabscience Biotechnology Inc., (Wuhan, Hubei). 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 h 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.
The data were subjected to statistical analysis using Statistical Package for Social Sciences software (IBM SPSS, Armonk, NY, USA) version 22 was used in the statistical 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). Mann–Whitney test was used for comparison between malaria parasite infected and noninfected TB groups and Spearman's rank order correlation was used to test relationship between median parasite count (MPC) and the other variables. P < 0.05 was considered statistically significant.
| Results|| |
At pretreatment, the median values of IL-6 (pg/mL) and IL-10 (pg/ml) were significantly lower in TB patients co-infected with malaria (TB/MP+) compared to TB patients without malaria (TB/MP-) (P = 0.045 and 0.039, respectively). Conversely, the median levels of P-SEL (ng/ml), GP IIb/IIIa (ng/ml), and PAF (pg/ml) was significantly higher in TB/MP+ compared with TB/MP- (P = 0.045, 0.025, and 0.044, respectively) [Table 1].
|Table 1: Comparison of inflammatory and hemostatic markers between treatment naive tuberculosis patients with and without malaria infection, median (range)|
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At intensive phase treatment, the median levels of TNF-α (pg/ml), IL-2 (pg/ml), and IL-6 (pg/ml) was significantly higher in TB/MP + compared to TB/MP- (P = 0.044, 0.045, and 0.027, respectively). Conversely, IL-10 (pg/ml) and PAF (pg/ml) were significantly lower in TB/MP + compared with TB/MP- (P = 0.044 and 0.030, respectively) [Table 2].
|Table 2: Comparison of inflammatory and hemostatic markers between tuberculosis patients with and without malaria infection after 2-month (intensive phase) treatment, median (range)|
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At continuation phase treatment, the median levels of TNF-α (pg/ml), IL-6 (pg/ml), TGF-β (pg/ml), PF-4 (ng/ml), GP IIb/IIIa (ng/ml), and TPO (pg/ml) was significantly lower in TB/MP+ compared with TB/MP- (P = 0.047, 0.046, 0.023, 0.024, 0.047, and 0.044, respectively). Conversely, the median levels of P-SEL (ng/mL) was significantly higher in TB/MP + compared with TB/MP- (P = 0.048) [Table 3].
|Table 3: Comparison of inflammatory and hemostatic markers between tuberculosis patients with and without malaria infection after 6-month (continuation phase) treatment, median (range)|
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There was a weak positive correlation between MPC and PF4 (r = 0.480; P = 0.046) and a moderate positive correlation with PSEL (r = 0.589; P = 0.008) [Table 4].
|Table 4: Correlation of median parasite count with hemostatic and inflammatory parameters in the tuberculosis/malaria co-infected patients|
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| Discussion|| |
In developing nations such as Nigeria, there is a co-endemicity of TB and malaria infection which may alter the levels of physiological parameters differently in treated and treatment-naive patients. This study explored these alterations in primary hemostatic and inflammatory parameters between TB patients with and without malaria before and during anti-TB treatment.
In TB patients that are yet to start treatment (pretreatment), there was a significant reduction in IL-6 (pro-inflammatory cytokine) and IL-10 (anti-inflammatory cytokine) in TB patients co-infected with malaria compared to TB patients not infected with malaria. This seems to suggest that in treatment naive TB patients co-infected with malaria, the levels of both pro-inflammatory and anti-inflammatory cytokines are lowered. According to Robinson et al., an increased plasma IL-6 level is associated with fatal outcomes in malaria infection, thus the reduction in the levels of IL-6 in TB patients co-infected with malaria could be a measure to minimize the fatality of the condition and enhance the prognosis of the infected patients. However, this disagrees with the findings of previous studies in which there was an increase in pro-inflammatory response with malaria infection and malaria infection was associated with high circulating levels of inflammatory cytokines such as TNF-α, IL-1, and IL-6. The reduction in IL-10 in TB patients co-infected with malaria in this study disagrees with the findings of Chukwuanukwu et al. that TB and malaria co-infection caused a marked increase in the production of the anti-inflammatory cytokines IL-10 and IL-4 in response to TB antigen suggesting that malaria co-infection promotes an anti-inflammatory response against MTB. Similarly, Olaniyan et al. stated that there was a significantly higher plasma IL-10 in MTB and P. falciparum co-infected patients than MTB mono-infected patients.
This current study revealed that before treatment, in TB patients with malaria the levels of adhesion molecules, P-SEL, and GP IIb/IIIa as well as PAF levels was significantly higher compared with TB patients without malaria. According to Chang et al., there is a marked upregulation of P-SEL during malaria infection and this increased P-SEL expression which may be due to increased endothelial cell expression, activated platelet expression, or both contributes to malarial pathogenesis. The finding of this study re-emphasizes the indispensable role of P-SEL in malaria parasite infection as an important adhesion molecule allowing parasitized erythrocytes to roll and adhere in vitro to endothelial cells.
In TB patients that have completed 2 months of intensive phase therapy (with rifampicin, isoniazid, pyrazinamide, and ethambutol), there was a significant increase in the levels of TNF-α, IL-6, and IL-2 in TB patients co-infected with malaria compared with nonmalaria-infected TB patients. This may suggest an upregulation in pro-inflammatory cytokines in malaria-infected TB patients after intensive-phase therapy. This aligns with the finding that malaria has long been associated with high circulating levels of inflammatory cytokines such as TNF-α, IL-1, and IL-6. In line with our findings, Olaniyan et al. found significantly higher plasma TNF-alpha in MTB and P. falciparum co-infected patients than MTB mono-infected patients. TNF-α has also been shown to have potent anti-parasitic activity and high levels is associated with malaria pathology such as fever while a sustained high levels was associated with rapid clearance of fever and parasites. This study also discovered that the level of IL-10 was also reduced in TB patients co-infected with malaria after the intensive phase therapy. This suggests that whether in treatment naive TB patients or in those that has been treated for 2 months, there is reduction in the levels of anti-inflammatory cytokine, IL-10 in TB patients when co-infected with malaria. However, there was a reduction in the level of PAF in TB patients co-infected with malaria as against the increase seen in treatment-naive TB patients co-infected with malaria when compared with nonmalaria-infected TB patients. This seems to suggest that the initiation of the intensive-phase therapy resulted in the suppression of PAF levels in TB patients co-infected with malaria which is at variance with the trend in treatment-naive patients.
In TB patients that have completed 6 months of anti-TB therapy wherein the patients have been treated with rifampicin and isoniazid for 4 months (continuation-phase therapy), there was a significant reduction in the levels of TNF-α, IL-6, TGF-β, PF4, GP IIb/IIIa, TPO as well as an increase in the level of P-SEL in TB patients co-infected with malaria compared with TB patients not infected with malaria. This suggests that there was a significant downregulation of pro-inflammatory cytokines in TB patients co-infected with malaria after continuation phase therapy similar to what was obtained before treatment was initiated. However, the level of the anti-inflammatory cytokine, TGF-β aligned with the reduction observed in TB patients co-infected with malaria treatment naive and after 2-months intensive-phase treatment compared with TB patients not infected with malaria. This corroborates the finding of Hansen and Schofield that low levels of regulatory cytokines, such as TGF-β and IL-10, have been correlated with malaria infection. These findings put together imply that anti-TB treatment results in significant variations in inflammatory cytokines in TB patients co-infected with malaria.
There was also a significant reduction in the levels of PF-4, GP IIb/IIIa, and TPO in TB patients co-infected with malaria compared to those without malaria after continuation-phase therapy. PF-4 belongs to growing list of chemokine molecules called kinocidins, which have a remarkable capacity to function as both chemotactic and antimicrobial molecules. They have been shown to have an important in vivo malaria-protective role that involves control of parasite growth during the early stage of erythrocytic malarial infection. This mechanism of platelet-associated parasite killing involves the binding of platelets preferentially to parasite-infected red cells and release of PF4 from the platelets leading to intra-erythrocytic accumulation of PF4 which are subsequently absorbed into the parasite where it lyses the parasite food vacuole and kills the parasite. It can thus be adduced that the reduction in PF4 in TB patients co-infected with malaria compared to TB patients without malaria could be due to its role in the process of platelet-associated parasite killing in which it is used up leading to a reduction in the level of PF4 available in circulation. However, this finding does not align with that of Aisha et al. that found a significant increase in PF4 in malaria parasite-infected patients. Our finding also shows a significant moderate positive correlation between MPC and PF4. TPO is the hormone that regulates the production of platelets. Thus, the implication of a reduction in the level of TPO hormone in malaria parasite infected TB patients compared to the malaria noninfected TB patients at the continuation phase of treatment is a likely reduction in platelet count. This aligns with findings in literature that malaria infections are commonly accompanied by a thrombocytopenia or loss of platelets, the severity of which closely mirrors the increasing parasite burden. The significant increase in P-SEL after continuation-phase therapy in TB patients with malaria in comparison to those without malaria aligns with the significant moderate positive correlation found between MPC and P-SEL in TB patients co-infected with malaria which further confirms the relationship between malaria infection and P-SEL activity.
Conclusively, the levels of inflammatory and hemostatic markers vary in TB patients co-infected with malaria in comparison to nonmalaria infected TB patients at different phases of anti-TB treatment (pretreatment, intensive phase and continuation phase). Our study has the limitation that it was a cross-sectional study in which the patients were not followed up in the course of treatment. Therefore, a longitudinal study that will also consider the specific contributions of the anti-TB medications to the observed variations is suggested for further study.
Financial support and sponsorship
Financial support was provided by Tertiary Education Trust Fund (TETFUND), Nigeria. The content is solely the responsibility of the authors and does not necessarily represent the official views of TETFUND.
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4]