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ORIGINAL ARTICLE
Year : 2021 | Volume
: 46
| Issue : 4 | Page : 201-207
Evaluation of plasma protein C and antithrombin levels in patients with tuberculosis
Hagar G Shahin, Dina A Fouad, Mervat A.A Alfeky
Department of Clinical Pathology, Faculty of Medicine, Ain Shams University, Cairo, Egypt
Correspondence Address:
Mervat A.A Alfeky
39 Abdullah Ben Taher, Nasr City, Cairo 11765
Egypt
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/ejh.ejh_55_21
Background Tuberculosis (TB) is a global health problem and thromboembolic complications, when occurring, are often fatal, with no proven markers to predict. The most common type is latent tuberculosis infection (LTBI); however, patients can develop active TB disease and become infectious. Managing LTBI properly can prevent active disease evolution, and exclusion of active TB is the main primary question in LTBI management. Protein C (PC) and antithrombin (AT) are natural anticoagulants with anti-inflammatory properties, so they are suggested to have a role in hypercoagulability due to inflammatory processes.
Aim To correlate PC and AT levels in TB patients with patients’ coagulable and clinical state.
Patients and methods Sixty patients (20 pretreatment, 20 posttreatment active TB, and 20 LTBI) and 20 normal-controls were included. Activity levels of PC and AT were measured and correlated to patients’ coagulable and clinical states, and routine laboratory results.
Results Activity levels of PC and AT are significantly low in active TB, increasing with treatment, and normal in LTBI. No thromboembolic events were detected in all patients included, so correlation with PC and AT could not be verified.
Conclusion Active TB is associated with hypercoagulable state, with low activity levels of PC and AT. Both proteins are suggested to be used as adjuvant markers of activation of LTBI and during their pretreatment assessment, together with monitoring therapeutic response in patients with active TB.
Keywords: antithrombin, protein C, tuberculosis
Tuberculosis (TB), a contagious disease, caused by bacillus Mycobacterium tuberculosis, is the major cause of illness and one of the worldwide top 10 reasons of death. At nationwide level, TB incidence rate diverges from fewer than 5 to more than 500 new and relapse cases per 100 000 population per year and about 25% of the world’s population is infected with M. tuberculosis [1]. In Egypt, TB has middle level of prevalence and death [2]. Postponement in diagnosis or in the start of the right treatment is one of the consequences of poor prognosis in up to a quarter of cases [3].
Most of the infected persons develop latent tuberculosis infection (LTBI) demarcated as having suggestion of M. tuberculosis infection by immunologic tests, transdermal skin test, or interferon-gamma (IFN-γ)-release assay, lacking clinical symptoms or signs of illness with free chest radiograph. Identifying and treating persons with LTBI is imperative to control the disease worldwide [4].
Patients who have active TB are susceptible to develop thrombotic complications, so, careful monitoring and proper treatment for thromboembolism are mandatory [5],[6].
It is uncertain how TB infection is complicated with thrombosis in some patients [7]. It is likely that huge amounts of interleukins (ILs) secreted by the monocyte–macrophage system in the active stage encourage abnormal liver functions and hemostatic changes toward the advance of hypercoagulable states [6].
Protein C (PC) is a natural anticoagulant, vitamin K-dependent serine protease. It is synthesized mainly in the liver, and also identified in the kidney, lung, brain, and male-reproductive organs. Activated PC acts as an anticoagulant, in conjunction with its cofactor protein S, by deactivating factors Va and VIIIa [8]. Additionally, several anti-inflammatory properties have been attributed to activated PC [9].
Antithrombin (AT, formerly AT-III) is a potent natural anticoagulant, 58-kD glycoprotein [10]. It is a serine protease inhibitor, synthesized in the liver and assessed to deactivate several enzymes in the coagulation cascade, nevertheless, thrombin and factor Xa are its primary targets [11],[12]. The AT is not only a potent anticoagulant but also has anti-inflammatory properties, which are accomplished by inhibiting thrombin and other clotting factors, as well as by coagulation-independent effect through direct interaction with cellular mediators of inflammation [13].
AimConsidering the role of PC and AT in hemostatic and inflammatory processes, we aimed to assess the plasma levels of PC and AT in patients with pulmonary tuberculosis (PTB) (active either pretreatment or posttreatment, and latent) to correlate their levels with patients’ coagulable and clinical states and other laboratory markers.
Patients and methodsPatients
The present study was conducted on 80 adult patients, including group I: 20 newly diagnosed active PTB patients, they were 17 males and three females, with age range from 28 to 50 years (mean: 40.2±7.466 years). Group II: 20 active PTB patients on treatment for a month (treated with rifampicin, isoniazid, pyrazinamide, and streptomycin), they were 18 males and two females. Their ages ranged from 29 to 52 years (mean: 39.5±6.977 years). Group III: 20 patients with LTBI, they were 12 males and eight females. Their ages ranged from 18 to 48 years (mean: 31.55±8.501 years). In addition to 20 healthy age-matched and sex-matched controls, all patients were recruited from Abbasia Chest Hospital within a period of 1 year.
The study had been approved by the Ethical Committee of the Clinical Pathology Department of the Faculty of Medicine, Ain Shams University, and was carried out according to the standards of the Declaration of Helsinki. Informed verbal consent was obtained from all participants to be included in the study.
Exclusion criteria
Patients were excluded if they had chronic liver or kidney diseases, pregnant and lactating females, severe trauma or burn, or taking oral contraceptive or anticoagulant therapy.
The patients were comprehensively subjected to:
Clinical evaluation: complete history taking and thorough clinical examination laying stress on symptoms and signs of any thrombotic complications.
Diagnostic workup of tuberculosis
Sputum examination: smears prepared, ZN stained and examined microscopically from three successive morning specimens.Radiological examination: chest radiograph.Diagnosis of LTBI:QuantiFERON-TB Gold (QFT): positive test.Excluding active TB: careful history taking with the absence of active TB symptoms and signs, and free chest radiograph.Laboratory investigations
Complete blood counts, using Sysmex XN-1000 cell counter (Sysmex Europe, Hamburg, Germany; GmbH); erythrocyte-sedimentation rate (ESR); blood chemistry (aspartate aminotransferase, alanine aminotransferase, urea, and creatinine), using UniCel DxC 800 Synchron Clinical Systems (Beckman Coulter Inc., Brea, California, USA); and prothrombin time (PT) and activated partial thromboplastin time (aPTT), together with evaluation of PC and AT activity levels, using Sysmex CA 1500 (Siemens Healthcare Products GmbH, Marburg, Germany).
Methods
Specimen collection and preparation
Ten-milliliters venous blood samples were collected and handled following the standard precautions for each test.
For PC and AT, platelet-poor plasma was prepared using citrated blood by centrifugation at 1500 g for at least 15 min and the supernatant plasma was tested rapidly for PT and PTT, then aliquoted, and frozen in a well-closed plastic tube container at −20°C for subsequent analysis.
Sample collection in QFT blood-collection tubes (only for patients suspected to have LTBI).
Measurement of protein C and antithrombin activity
Quantitative determination of PC activity was measured by chromogenic assay using Berichrom PC and AT reagents manufactured by Dade Behring Marburg GmbH (Germany). In brief, PC in the sample is activated by specific snake venom activator, the resulting PC is assayed in kinetic test by measuring the increase in absorbance at 405 nm.
The AT in the sample is converted by heparin into an immediate inhibitor and inactivates the thrombin present. The residual thrombin content is determined in a kinetic test measuring the increase in absorbance at 405 nm. The absorbance change is inversely correlated to the AT activity in the sample.
Reference values
PC: 70–140%; AT: 75–125%.
QuantiFERON-TB Gold ELISA
QFT is a test for cell-mediated immune responses to antigens that simulate mycobacterial proteins. Lymphocytes in the blood of infected persons recognize these antigens and then generate and secrete IFN-γ. The detection and quantification of IFN-γ forms the base of this test [14].
Principles and procedure of the assay:
The QFT system uses specialized collection tubes that include Nil tubes and TB antigen tubes containing the mycobacterial antigens, which are used to collect whole blood. The tubes were incubated at 37°C as soon as possible for 16–24 h, then centrifuged, and the plasma is removed. Plasma samples were stored at −20°C till the time of testing for IFN-γ. The amount of IFN-γ was measured by ELISA using QuantiFERON-TB Gold ELISA manufactured by Cellestis Ltd (Carnegie, Victoria, Australia); and has been read at 450 nm using Fax 2100 microplate reader (Awareness Technology, Palm City, Florida, USA).
A test is considered positive if the IFN-γ response to the TB antigen tube is significantly above the Nil IFN-γ (by ≥0.35 IU/ml and ≥25% of Nil tube).
Patients suspected to have LTBI were excluded if the level of Nil sample was more than 8.0 IU/ml.
Statistical analysis
Data were collected, revised, coded, and entered to the Statistical Package for Social Science (IBM SPSS), version 20 (IBM SPSS Statistics for Windows, Version 20.0. Armonk, NY: IBM Corp). Qualitative data were described in the form of numbers and percentages. Quantitative data were described in the form of means (SD), and ranges. χ2 test and Fisher exact test was used to compare between two independent groups regarding qualitative data. While the comparison between two independent groups regarding quantitative data with parametric distribution was done by using independent t test.
Spearman correlation coefficients were used to assess the correlation between PC (%) and AT (%) with the other studied parameters in each group.
The confidence interval was set to 95% and the margin of error accepted was set to 5%. So, the probability of being by chance (P) value was calculated for all parameters and was evaluated as follows: P value more than 0.05: nonsignificant; P value less than 0.05: significant; P value less than 0.01: highly significant.
ResultsClinical features of active pulmonary tuberculosis patients
Thorough history revealed the following ordered symptoms: cough, fatigue, weight loss/anorexia, fever, night sweats, hemoptysis, and chest and back pain.No thromboembolic events were detected in any of the patients.Laboratory data
Group-I and group-II patients had highly significant lower PC activity levels (means: 35.88±7.53 and 65.79±11.18%, respectively) compared with the control group (mean: 85.51±7.45%).
Patients of group I had a highly significant lower AT activity level (mean: 68.22±25.10%) compared with the control group (mean: 93.34±9.43%). Interestingly, group-II patients had a significantly higher AT activity level (mean: 100.70±13.11%) in comparison to that of controls.
Group III showed no significant difference comparing with the controls regarding both PC and AT activity levels (means: 83.47±8.96 and 89.85±10.18%).
Intergroup comparison revealed that patients of group I had significantly lower PC and AT activity levels compared with group II and group III. Patients of group II had lower PC and higher AT activity levels compared with group III.
Comparisons are summarized in [Table 1] and [Table 2].
Table 1 Comparison between group I and group II as regards the studied parameters
Table 2 Comparison between group I and group III as regards the studied parametersCorrelations between AT and PC activity levels and other studied parameters in the different patients’ groups are summarized in [Table 3],[Table 4],[Table 5].
Table 3 Correlation between protein C and antithrombin activity levels and the other studied parameters in group I
Table 4 Correlation between protein C and antithrombin activity levels and the other studied parameters in group II
Table 5 Correlation between protein C and antithrombin activity levels and the other studied parameters in group III Discussion Hemostatic discrepancies and stimulation of inflammatory pathways during active PTB are evident [15]. As most infected individuals have LTBI [16], managing LTBI properly can prevent evolution to active TB; and exclusion of active disease is the main primary question in LTBI management [17].
From these points of view, we aimed to evaluate the activity levels of PC and AT in active and latent TB patients to correlate their levels with other laboratory findings and patients’ clinical and coagulable states.
Group-I patients had significantly lower PC and AT in comparison with controls defining the hypercoagulable state, which is consistent with previous studies that reported hypercoagulability in active TB, presented as low levels of PC and AT, higher fibrin-degradation products, tissue plasminogen activator, plasminogen activator inhibitor 1, and D-dimer [18],[19],[20].
The decrease in activity levels of AT and PC in inflammatory situations was previously explained by their sensitivity to downregulation by inflammatory mediators [21]. At the same point of view, it was suggested that the decrease in plasma levels of AT and the fact that PC is a negative acute-phase protein may be interpreted as participating in the host-defense mechanisms [22]. In addition, the overstimulation of IL secretions by the monocyte–macrophage system in the active stage of TB is suggested to cause abnormal liver functions and hypercoagulable state [6].
Although group-I patients had significantly lower levels of PC and AT, which signifies hypercoagulable state, they did not suffer any thromboembolic manifestations. Hence, we suggest that other factors than PC and AT might play more significant roles in thromboembolic manifestations in patients with active TB. Our suggestion is strengthened by similar studies and reports [5],[23],[24].
Group-II patients had significant lower levels of PC and higher levels of AT when compared with the control group and group III. However, levels of both proteins showed a highly significant increase when compared with group I. This result is in agreement with a study by Turken et al. [6], who found that PC and AT levels returned to normal levels in active PTB patients after 4 weeks of treatment. Elevated AT levels were reported in patients on coumarin anticoagulants, presence of vitamin-K deficiencies or antagonist, and acute hepatitis [25],[26]. It was previously reported that rifampicin used in TB treatment can cause vitamin-K deficiency [27], the finding that may explain the elevation in AT level detected in our study.
No significant difference was found between patients of group III and controls as regards PC and AT activity levels. To our knowledge, no previously published study was conducted on LTBI regarding PC and AT levels and we suggest that the normal levels are due to low levels of inflammatory mediators and normal liver functions. Shitrit et al. [28] demonstrated normal level of D-dimer in cases with LTBI, which encourage suggestion of low inflammatory activity.
Mild anemia was observed in almost all patients of group I and a highly significant difference in hemoglobin (HB) concentration was found between group I and both group II and group III, and no significant difference between group II and group III was detected. The finding was reported by previous studies [6],[29].
Leukocytosis was observed mostly in all patients of group I, while in group II, the total leukocyte count was lower; and in group III, it was almost normal in all patients. Leukocytosis is believed to be resulted from immune reaction taking place in response to foreign antigen (M. tuberculosis) that also causes increase in cytokine levels. These cytokines include IFN-γ and IL-1, which in turn cause further proliferation of leukocytes [30]. This finding agrees with a similar study [31], however, different results were previously reported [29],[32].In accordance to our results, a significant difference between active PTB patients before treatment and normal-controls as regards platelet count was reported [6]. However, Yaranal et al. [32] reported normal platelet count in most of TB patients.
Thrombocytosis with active PTB may be a result of the release of inflammatory cytokines and mediators that are involved in the formation of granulomatous lesions encountered in TB, among them, IL-6 has been reported to promote platelet production [33].
Many studies reported the high predictive value of ESR in monitoring PTB activity [32],[34],[35]. In accordance, the present study showed that almost all patients of group I and group II had increased ESR with a significant higher level in group I.
We found that group I had prolonged PT and aPTT. No significant difference between group II and group III was detected, which is in accordance with other studies [18],[33].
The increase in PT in active PTB patients may be explained by the associated abnormal liver functions, while aPTT prolongation may be a consequence of the repressive effects on thrombin generation by increased fibrinogen concentrations in TB patients as a result of acute-phase response [18].
Group I showed a significant positive correlation between AT activity levels and HB concentration; group II showed a significant positive correlation between PC activity levels and both HB concentration and platelet count and a significant negative correlation between PC activity levels and ESR.
These results can be interrelated to the presence of high inflammatory and acute-phase reaction mediators, those that can affect all mentioned parameters in the detected manner.
ConclusionAlthough the low activity levels of PC and AT in active PTB describing hypercoagulable state, no thromboembolic manifestations were detected in the studied patients, which may suggest additional roles of other factors in thromboembolic events. However, the small sample size of our study together with the absence of thrombotic manifestations in the studied patients’ groups makes an enquiry. Yet, PC and AT are suggested to be used as adjuvant markers in LTBI to detect activation and during pretreatment assessment, as well as monitoring of therapeutic response in patients with active PTB. Comprehensive studies using additional thrombophilia markers on larger sample size and including TB patients with thromboembolic events, though might necessitate long-term studies to achieve, are recommended.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
References
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