Our findings showed the importance of four soluble receptors including sEPCR, sICAM-1, suPAR, and sTie-2 with its ligand Ang-2, and two other molecules PCT, and PTX3, as informative biomarkers of malaria disease severity, coma and mortality.
The plasma levels of sEPCR, sICAM-1, sTie-2 and suPAR were significantly higher in children with CM or SM compared to children with UM using both logistical regression analysis (Table:3) and non-parametric tests (Figures:1,2,3). These molecules have also been found to be increased in patients with other infectious or chronical diseases such as sepsis, HIV-1-AIDS, tuberculosis, rheumatoid-arthritis and various cancers with suPAR, sEPCR and PTX3, often predicting poor clinical outcome as in our present study [12, 18-20]. Furthermore, sEPCR, suPAR and PTX3 were associated with both deep coma and fatality.
sEPCR, sTie-2/Ang-2 and sICAM-1 are involved in cytoprotective activity to maintain micro-vessels endothelial barrier functions. The soluble as well as the membranous form of EPCR bind with similar affinity to activated C-reactive-protein (APC) to maintain endothelium integrity through the activation of protease-activator-1 [21, 22] promoting anticoagulation and anti-inflammation. During malaria, EPCR has been found to bind the P. falciparum erythrocyte-membrane-protein-1 to the same region as APC does thus, decreasing anticoagulant activity of APC and promoting thrombosis and obstruction of blood circulation [11, 23]. Our data supports the hypothesis that increased sEPCR in severe cases contributes for maintaining APC anticoagulant activity in these patients preventing acute thrombosis.
Similarly, the level of Ang-2 and its receptor sTie-2 was higher in SM or CM groups with sTie-2 highly increased in coma cases suggesting a pathophysiological role during CM. Interestingly, Ang-2 and Ang-2/Ang-1 ratio were shown as independent predictors of metabolic acidosis, coma and mortality [16, 24, 25]. The Ang1/Ang-2-Tie-2 system is a paramount regulator of endothelial integrity with Ang-1 signals through Tie-2 to maintain vascular quiescence. This is antagonized by Ang-2 resulting in endothelial dysfunction [26]. The sTie-2 contains the ligand binding domain that binds angiopoietins and inhibit their interaction with cellular Tie-2. Given the importance of sTie-2 in regulating angiopoietins availability it contributes to vascular pathology when its level is impaired [27]. Therefore, the high production of sTie-2 may thwart the antagonizing effects of Ang-2 which was highly expressed in CM and SM.
Moreover, the enhanced level of Ang-2 and sTie-2 leads to sICAM-1 expression which was significantly higher in children with CM or SM as well as in children with coma (Figure:1 C, D, E) (Figure:2 B, C).
We also found that suPAR level was significantly higher in children with SM or CM compared to those with UM, in children with BCS<3 compared to those with BCS >2 and in children who died from SM or CM making this molecule a potential marker of severity, coma and fatality. Produced by activated monocytes and endothelial cells, suPAR plays several roles in innate immune defence and inflammation. It acts in the recruitment of effector cells (monocytes/macrophages) and clearance of pathogens at infection sites [28]. In addition to the uPA/suPAR binding which triggers the plasminogen activation system, suPAR also binds integrins and other receptors to activate different intracellular signaling pathways implicated in cell proliferation, invasion, angiogenesis, and metastasis. Therefore, suPAR has been described to be increased in cancers, in coronary heart diseases (CHD) and in infectious diseases including malaria [15]. An immunohistochemistry analysis of CM patients brain showed an accumulation of suPAR in macrophages/microglial cells in Durks granuloma adjacent to petechial haemorrhages, as well as in astrocytes, and in endothelial cells while the expression of suPAR was low in normal brains suggesting the association of suPAR expression with tissue damage of the blood brain barrier during CM [14, 29, 30]. High suPAR level was associated with disease severity and poor prognosis in cancers, CHD and infectious diseases including SARS-CoV-2 [31-34].
We found that PCT came out as the best biomarker for diagnosing malaria severity, with an AUROCC of [0.83]. This result is consistent with previous findings [35-37]. This molecule able to discriminate between viral and bacterial, fungi or parasitic infections is used in guiding antibiotic treatments in patients [38].
Interestingly, PCT was higher in children with BSC<3 than those with BCS>2 (P=0.004) and showed moderate predictive value for coma with an AUROCC of [0.65] which is to our knowledge a novel finding. Of note, PCT in combination with CRP, chitinase-3-like-protein, and S100beta was recently described to be a promising biomarker in determining the presence, location, and extent of traumatic intracranial lesions [39]
The level of PTX3 was higher in children with SM or CM compared to children with UM, in children with BCS<3 compared to those with BCS >2 and in children who died compared to those who survived with significant diagnosis performance for severity, coma and mortality as shown by the significance of the AUROCC analyses (Figure:4D,5A,6B).
PTX3 is involved in the humoral innate immunity by recognizing microbial moieties and damaged tissues and in regulating inflammation and autoimmunity. PTX3 is stored in specific granules in neutrophils and is released in response to microbial recognition and inflammatory signals. Increased levels of plasma PTX3 was associated with the clearance of apoptotic cells by dendritic cells resulting from high immune response and endothelial dysfunction in cardiovascular disorders [40, 41]. PTX3 was investigated as biomarker in infectious diseases and has emerged as strong independent predictor of mortality as recently described in SARS-CoV2 syndrome [42]. However, this study is only the second one that investigated PTX3 as potential biomarker during malaria and revealed the potential of this molecule at distinguishing SM and CM from UM cases, deep comas from mild and conscious cases and fatal cases from patients who survived. This result highlights the unique interest of PTX3 as biomarker to assess the progression of the disease and response to treatment in hospitalized patients.
Nine (75%) of the fatal cases among the SM group were children who had consciousness alteration with BCS=3. In these children the levels of sEPCR, sICAM, sTie-2, Ang-2 and suPAR were similar to those with CM BCS<3. This finding suggests that SM children with (BCS=3) who died are probably children with CM misclassified in the SM group for compliance with the WHO definition of CM conditioned by the BCS <3 criterion. It might also reflect an underestimation of the BCS evaluation in these children by the clinicians.
In addition, sEPCR and sICAM -1 levels were higher in children with BCS = 3 who died than in those who survived emphasizing the interest of sEPCR as marker of mortality from malaria supporting earlier findings (Figure: 2 F, G) [12].
The enhanced levels of sTie-2, Ang-2, suPAR and sICAM-1 were significantly associated with reduced hemoglobin with sTie-2 also associated with low platelets count and hypoglycemia and sTie-2, Ang-2, associated with high leucocytes count. These results emphasize a deleterious effect of these analytes during severe malaria as the fall of hemoglobin, platelets count and glycemia are hallmarks of SM and CM pathologies [43].
Beyond the observed association between the elevated levels of the analytes and SM and CM manifestations, these molecules might be implicated in the processes that control blood parameters and deserve to be fully investigated.