The 2023 Belgian clinical guidance on anticoagulation management in hospitalized and ambulatory COVID-19 patients.

COVID-19 is associated with an increased risk for thrombotic complications. The trials investigating the optimal thromboprophylactic dose are performed in challenging times and seemingly produce conflicting evidence. The burdensome circumstances, divergent endpoints, and different analytical approaches hamper comparison and extrapolation of available evidence. Most importantly, clinicians should provide thromboprophylaxis in hospitalized COVID-19 patients while (re)assessing bleeding and thrombotic risk frequently. The COVID-19 Thromboprophylaxis Working Group of the BSTH updated its guidance document. It aims to summarize the available evidence critically and to guide clinicians in providing the best possible thromboprophylaxis.

Belgian clinical guidance on anticoagulation management in hospitalised and ambulatory patients with COVID-19.

OBJECTIVES: COVID-19 predisposes patients to thrombotic disease. The aim of this guidance document is to provide Belgian health-care workers with recommendations on anticoagulation management in COVID-19 positive patients. METHODS: These recommendations were based on current knowledge and a limited level of evidence. RESULTS: We formulated recommendations for the prophylaxis and treatment of COVID-related venous thromboembolism in ambulatory and hospitalised patients, as well as recommendations for the use of antithrombotic drugs in patients with prior indication for anticoagulation who develop COVID-19. CONCLUSIONS: These recommendations represent an easy-to-use practical guidance that can be implemented in every Belgian hospital and be used by primary care physicians and gynaecologists. Of note, they are likely to evolve with increased knowledge of the disease and availability of data from ongoing clinical trials.

In silico analysis of structural modifications in and around the integrin αIIb genu caused by ITGA2B variants in human platelets with emphasis on Glanzmann thrombasthenia.

BACKGROUND: Studies on the inherited bleeding disorder, Glanzmann thrombasthenia (GT), have helped define the role of the αIIbß3 integrin in platelet aggregation. Stable bent αIIbß3 undergoes conformation changes on activation allowing fibrinogen binding and its taking an extended form. The αIIb genu assures the fulcrum of the bent state. Our goal was to determine how structural changes induced by missense mutations in the αIIb genu define GT phenotype. METHODS: Sanger sequencing of ITGA2B and ITGB3 in the index case followed by in silico modeling of all known GT-causing missense mutations extending from the lower part of the ß-propeller, and through the thigh and upper calf-1 domains. RESULTS: A homozygous c.1772A>C transversion in exon 18 of ITGA2B coding for a p.Asp591Ala substitution in an interconnecting loop of the lower thigh domain of αIIb in a patient with platelets lacking αIIbß3 led us to extend our in silico modeling to all 16 published disease-causing missense variants potentially affecting the αIIb genu. Modifications of structuring H-bonding were the major cause in the thigh domain although one mutation gave mRNA decay. In contrast, short-range changes induced in calf-1 appeared minor suggesting long-range effects. All result in severe to total loss of αIIbß3 in platelets. The absence of mutations within a key Ca2+-binding loop in the genu led us to scan public databases; three potential single allele variants giving major structural changes were identiffied suggesting that this key region is not protected from genetic variation. CONCLUSIONS: It appears that the αIIb genu is the object of stringent quality control to prevent platelets from circulating with activated and extended integrin.

[Is there a place for thrombin generation assay in routine clinical laboratory?]. / Quelle place pour le test de génération de thrombine au sein du laboratoire de biologie clinique ?

Laboratory diagnosis of coagulopathies primarily relies on assays selectively exploring either the extrinsic (PT), the intrinsic (aPTT) or the common (TT) pathway of the coagulation system. Although these tests are very useful to rapidly identify severe coagulation disorders or to monitor anticoagulant therapy, they only poorly correlate with the clinical manifestations. Global assays that evaluate the whole coagulation process could potentially more accurately reflect the hemorrhagic or thrombotic phenotype of an individual. Thrombin generation assay (TGA), first described in the 1950's, has been developed and automated in the 1990's. This technique is widely used in fundamental research but has yet failed to integrate clinical laboratories. In this article, we describe TGA and review its clinical applications. Laboratory aspects and technical issues will also be discussed.

Impact of 6 % hydroxyethyl starch (HES) 130/0.4 on the correlation between standard laboratory tests and thromboelastography (TEG®) after cardiopulmonary bypass.

BACKGROUND: Hydroxyethyl starches (HES) affect the results of thromboelastography (TEG®). We sought to determine whether using HES rather than crystalloids for cardiopulmonary bypass (CPB) prime and intraoperative fluid therapy changes the TEG cutoff values best identifying patients with a low platelet count or a low fibrinogen level after CPB. METHODS: Data from 96 patients who had on-pump cardiac surgery, a TEG® (kaolin-heparinase) and standard investigations of blood clotting performed after separation from CPB and protamine administration were retrospectively reviewed. Patients were assigned to the HES or crystalloid group according to whether balanced 6% HES 130/0.4 or balanced crystalloids were used for intraoperative fluid therapy and pump prime. Mutlivariable linear regression models with computation of the standardized regression coefficients were used to identify independent associations between the four main TEG parameters (R time, alpha angle, K time and MA) and the type of fluid used, the INR, the aPTT, the fibrinogen level and the platelet count. Receiver-operating-characteristic curves were used to assess the effect of HES on the ability of TEG parameters to identify patients with a platelet count<80.000µl(-1) or a fibrinogen level<1.5 gr l(-1) and on the cutoff values best identifying these patients. RESULTS: The type of fluid used significantly affected the MA (P<0.001), the K time (P<0.001) and the alpha angle (P<0.001) regardless of the results of the standard clotting tests. According to standardized ß regression coefficients the platelet count and the type of fluid used were stronger predictors of the MA, the alpha angle and the K time than the fibrinogen level. MA better predicted platelets<80.000µl(-1) than K time and alpha angle (P=0.023). The best cutoff value of MA identifying patients with platelets<80.000µl(-1) was 62mm in the crystalloid group and 53mm in the HES group. MA, K time and alpha angle were poor predictors of the postoperative fibrinogen level. CONCLUSION: HES significantly changes the cutoff value of TEG® MA best identifying patients<80.000µl(-1) after on-pump cardiac surgery.

Low molecular weight poly (2-dimethylamino ethylmethacrylate) polymers with controlled positioned fluorescent labeling: Synthesis, characterization and in vitro interaction with human endothelial cells.

Poly (2-dimethylamino ethylmethacrylate) (PDMAEMA) is an attractive non-degradable polymer studied as nonviral vector for gene delivery but it can be also adopted for delivery of other biopharmaceutical drugs. As a parenteral carrier, the PDMAEMA free form (FF) might interact with tissues and cells. Few data are available on its selective internalization and efflux from cells, while the majority of studies published have followed the distribution of DNA complexed with PDMAEMA. In order to address polycation safety, the first aim was to synthesize by atom transfer radical polymerisation (ATRP) fluorescent labeled PDMAEMA of low molecular weight (Mw) (below 15 kDa), controlling the position and density of fluorescein. The second goal was to analyze the possible difference in uptake and subcellular distribution of this labeled FF polycation between human umbilical vein endothelial cells (HUVEC) and hCMEC/D3 cells. These two cell lines have been chosen in order to detect selectivity towards the blood-brain barrier (BBB). In both cases, polycation was detected along the plasma membrane followed by progressive migration to the peri-nuclear region, where it overlapped with lysosomal structures. The analysis by fluorescence-activated cell sorting (FACS) of the PDMAEMA uptake by hCMEC/D3 cells showed a significant (p<0.05) inhibition (40%) in presence of 2-dexoxy-D-glucose inhibitor, a result supporting an energy-dependence mechanism(s). Cytotoxicity study showed that low Mw PDMAEMA (10 kDa) lead to a minor cytotoxicity compared to the higher ones. As main conclusion this study highlights the similitude in cell trafficking of FF PDMAEMA and data previously reported for PDMAEMA/DNA complexes.

Persistent hypocoagulability in patients with septic shock predicts greater hospital mortality: impact of impaired thrombin generation.

PURPOSE: Sepsis induces hypercoagulability, hypofibrinolysis, microthrombosis, and endothelial dysfunction leading to multiple organ failure. However, not all studies reported benefit from anticoagulation for patients with severe sepsis, and time courses of coagulation abnormalities in septic shock are poorly documented. Therefore, the aim of this prospective observational cohort study was to describe the coagulation profile of patients with septic shock and to determine whether alterations of the profile are associated with hospital mortality. METHODS: Thirty-nine patients with septic shock on ICU admission were prospectively included in the study. From admission to day 7, analytical coagulation tests, thrombin generation (TG) assays, and thromboelastometric analyses were performed and tested for association with survival. RESULTS: Patients with septic shock presented on admission prolongation of prothrombin time, activated partial thromboplastin time (aPTT), increased consumption of most procoagulant factors as well as both delay and deficit in TG, all compatible with a hypocoagulable state compared with reference values (P < 0.001). Time courses revealed a persistent hypocoagulability profile in non-survivors as compared with survivors. From multiple logistic regression, prolonged aPTT (P = 0.007) and persistence of TG deficit (P = 0.024) on day 3 were strong predictors of mortality, independently from disease severity scores, disseminated intravascular coagulation score, and standard coagulation tests on admission. CONCLUSIONS: Patients with septic shock present with hypocoagulability at the time of ICU admission. Persistence of hypocoagulability assessed by prolonged aPTT and unresolving deficit in TG on day 3 after onset of septic shock is associated with greater hospital mortality.

Induction of HIV type 1 expression correlates with T cell responsiveness to mycobacteria in patients coinfected with HIV type 1 and Mycobacterium tuberculosis.

An in vitro mononuclear cell system to model the microenvironment of coinfection with HIV-1 and Mycobacterium tuberculosis (MTB) was developed. This cellular system was used to assess the interaction of MTB-infected monocytes and T cells from dually infected HIV-1/TB patients with pulmonary tuberculosis (TB). Subjects with higher induction of HIV-1 gag/pol mRNA expression after MTB stimulation had increased MTB-specific T cell IFN-gamma and TNF-alpha production. Lack of HIV-1 mRNA induction did not correlate with increased induction of regulatory T cells (T-reg) as measured by MTB-induced Foxp3 mRNA. HIV-1 induction did not significantly correlate with clinical parameters including plasma HIV-1 viral load or CD4(+) T cell count. These data model MTB-induced HIV-1 replication at the microenvironment of MTB reactivation/infection. The data suggest that the magnitude of MTB-specific T cell responses drives local viral pathogenesis regardless of the stage of HIV-1 disease as reflected by plasma viral load or CD4(+) T cell count.

Immune correlates of acute Mycobacterium tuberculosis infection in household contacts in Kampala, Uganda.

To determine immunologic and epidemiologic correlates of acute Mycobacterium tuberculosis infection in household contacts of infectious tuberculosis cases, we performed a prospective, community-based cohort study of index cases and their household contacts in Kampala, Uganda. Contacts were evaluated for tuberculin skin test (TST) conversion over two years. Interferon-gamma expression was measured using a whole blood assay after stimulating with M. tuberculosis culture-filtrate. In 222 contacts with a TST less than 5 mm at baseline, the one-year rate of TST conversion was 27%. The TST conversion was associated with the infectiousness of the index case and proximity of contact. Interferon-gamma levels at baseline were greater among TST converters compared with those who did not convert. The risk of TST conversion increased four-fold as the baseline interferon-gamma increased 10-fold, but only in contacts with BCG vaccination. In household contacts of tuberculosis, interferon-gamma responses to non-specific mycobacterial antigens may be used to make an early diagnosis of tuberculosis infection, especially in resource-limited settings where bacille Calmette-Guérin vaccination is commonly used.

Persistent replication of human immunodeficiency virus type 1 despite treatment of pulmonary tuberculosis in dually infected subjects.

Tuberculosis (TB) is the most common life-threatening infection in human immunodeficiency virus (HIV)-infected persons and frequently occurs before the onset of severe immunodeficiency. Development of TB is associated with increased HIV type 1 (HIV-1) viral load, a fall in CD4 lymphocyte counts, and increased mortality. The aim of this study was to examine how treatment of pulmonary TB affected HIV-1 activity in HIV-1/TB-coinfected subjects with CD4 cell counts of >100 cells/mul. HIV-1/TB-coinfected subjects were recruited in Kampala, Uganda, and were monitored over time. Based upon a significant (0.5 log10 copies/ml) decrease in viral load by the end of treatment, two patient groups could be distinguished. Responders (n = 17) had more rapid resolution of anemia and pulmonary lesions on chest radiography during TB treatment. This group had a significant increase in viral load to levels not different from those at baseline 6 months after completion of TB treatment. HIV-1 viral load in nonresponders (n = 10) with TB treatment increased and at the 6 month follow-up was significantly higher than that at the time of diagnosis of TB. Compared to baseline levels, serum markers of macrophage activation including soluble CD14 decreased significantly by the end of TB treatment in responders but not in nonresponders. These data further define the impact of pulmonary TB on HIV-1 disease. HIV-1 replication during dual HIV-1/TB infection is not amenable to virologic control by treatment of TB alone. Concurrent institution of highly active antiretroviral treatment needs to be evaluated in patients dually infected with pulmonary TB and HIV-1.

Mechanisms of apoptosis of T-cells in human tuberculosis.

The role of TGF-beta TNF-alpha FasL and Bcl-2 in apoptosis of CD4 T-cells during active TB was studied. Coculture of PBMC from TB patients with neutralizing antibodies to TGF-beta or TNF-alpha decreased spontaneous (P < or = 0.05) and MTB-induced (P < or = 0.02) T-cell apoptosis by 50-90%, but effects were not additive. Interestingly, only levels of TGF-beta in supernatants correlated with rates of spontaneous and MTB-induced apoptosis. FasL surface and mRNA expression were higher in unstimulated and MTB-stimulated PBMC from patients than controls, and neutralization of FasL abrogated apoptosis of T-cells from patients only. Intracellular Bcl-2 protein was lower among unstimulated CD4 T-cells from patients than those from controls (P < or = 0.02), and MTB stimulation reduced intracellular Bcl-2 content in CD4 T-cells from patients only (P < or = 0.001). These findings may indicate that, during TB, predisposition of CD4 T-cells to apoptosis may involve both low expression of Bcl-2, and excessive expression of TGF-beta TNF-alpha and FasL.

Heritability analysis of cytokines as intermediate phenotypes of tuberculosis.

Numerous studies have provided support for genetic susceptibility to tuberculosis (TB); however, heterogeneity in disease expression has hampered previous genetic studies. The purpose of this work was to investigate possible intermediate phenotypes for TB. A set of cytokine profiles, including antigen-stimulated whole-blood assays for interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta, and the ratio of IFN to TNF, were analyzed in 177 pedigrees from a community in Uganda with a high prevalence of TB. The heritability of these variables was estimated after adjustment for covariates, and TNF-alpha, in particular, had an estimated heritability of 68%. A principal component analysis of IFN-gamma, TNF-alpha, and TGF-beta reflected the immunologic model of TB. In this analysis, the first component explained >38% of the variation in the data. This analysis illustrates the value of such intermediate phenotypes in mapping susceptibility loci for TB and demonstrates that this area deserves further research.