Abstract
Degradation of the endothelial glycocalyx is associated with mortality in adult falciparum malaria. However, its role in the pathogenesis of non-falciparum malaria is unknown. In Malaysian patients with knowlesi (n = 200) and vivax (n = 61) malaria, and in healthy controls (n = 50), we measured glycocalyx breakdown products plasma syndecan-1 and urinary glycosaminoglycans, and evaluated correlations with biomarkers of disease severity. Urinary glycosaminoglycans were increased in patients with knowlesi and vivax malaria compared to healthy controls, and in knowlesi malaria were highest in those with severe disease. In knowlesi malaria, plasma syndecan-1 was also highest in those with severe disease, and correlated with markers of endothelial activation (angiopoietin-2, osteoprotegerin, ICAM-1), asymmetric dimethylarginine (ADMA) and impaired microvascular reactivity. Syndecan-1 also correlated with endothelial activation (ICAM-1, angiopoietin-2) and ADMA in vivax malaria. In knowlesi malaria increased syndecan-1 was associated with acute kidney injury, after controlling for age and parasitemia. In knowlesi malaria, the difference in median syndecan-1 between severe and non-severe disease was more marked in females than males. Endothelial glycocalyx degradation is increased in knowlesi and vivax malaria, and associated with disease severity and acute kidney injury in knowlesi malaria. Agents that inhibit glycocalyx breakdown may represent adjunctive therapeutics for severe non-falciparum malaria.
Similar content being viewed by others
Introduction
Malaria causes major morbidity and mortality worldwide. While Plasmodium falciparum is the major cause of malaria deaths globally, outside of Africa the non-falciparum species Plasmodium vivax and Plasmodium knowlesi cause significant disease burden, with both species able to cause severe and fatal disease1,2,3,4,5,6. P. knowlesi is a particular health problem in Malaysia, where this species now accounts for > 98% of all malaria cases7, and severe disease occurs in 6–9% of patients8,9. Complications include respiratory distress and acute kidney injury (AKI), with the latter particularly common in older adults3, 9,10,11. Given the long-term consequences of AKI12, adjunctive treatments of severe malaria are warranted to attenuate this complication. Development of adjunctive treatments requires improved understanding of the pathogenic mechanisms of severe disease.
As with falciparum malaria, severe knowlesi and vivax malaria are characterised by microvascular dysfunction, leading to impaired tissue perfusion and organ dysfunction11,13, 14. Contributing factors include intravascular haemolysis, impaired nitric-oxide (NO) bioavailability, and endothelial activation11,13,14. In falciparum malaria, degradation of the endothelial glycocalyx also occurs15,16,17,18, and in adults is associated with disease severity and mortality15. However, the role of the glycocalyx in knowlesi and vivax malaria has not been evaluated.
The endothelial glycocalyx is a gel-like layer that covers the endothelium, and maintains vascular haemostasis, tone and permeability (reviewed in19,20). It regulates endothelial cell NO release, prevents leakage of intravascular fluid and proteins, and attenuates leukocyte- and platelet-binding to endothelial adhesion receptors19. The glycocalyx is comprised of membrane-bound proteoglycans, glycosaminoglycan chains (GAGs), glycoproteins, and adherent plasma proteins (see19,20 for review, images and figures). The major glycosaminoglycans are heparan sulfate, chondroitin sulfate and the non-sulfated hyaluronan, while the proteoglycans include the syndecans and glycipans. When the glycocalyx degrades, a number of these components are shed into the plasma and/or urine and can be measured as biomarkers of glycocalyx breakdown. The best studied of these biomarkers include plasma syndecan-1, plasma heparin sulfate, urinary hyaluronan, and total urinary sulphated GAGs. Glycocalyx degradation has been increasingly recognized to play a key role in the pathogenesis of a number of other conditions, including sepsis20, dengue21 and most recently in severe COVID-1922. In sepsis, breakdown is mediated by glycocalyx “sheddases” such as heparanase, metalloproteinases, and hyaluronidase, activated by reactive oxygen species and proinflammatory cytokines20. Another activator of heparanase is the endothelial NO synthase (eNOS) inhibitor asymmetric dithemethylarginine (ADMA)23. ADMA is known to be elevated in falciparum malaria24,25 and sepsis26, and hence may contribute to glycocalyx breakdown in these conditions. While ADMA has not been found to be increased in vivax malaria14, the role of ADMA in knowlesi malaria has not been evaluated.
In this study, we measured concentrations of the glycocalyx breakdown products plasma syndecan-1 and urinary GAGs, and plasma concentrations of ADMA, in Malaysian adults with severe and non-severe knowlesi and vivax malaria, and in healthy controls. We assessed correlations between the glycocalyx breakdown products and endothelial activation (as measured by angiopoietin-2, osteoprotegerin [OPG] and intercellular adhesion molecule-1 [ICAM-1]), ADMA, microvascular function, and other biomarkers of disease severity. Plasma concentrations of the glycocalyx stabiliser sphingosine-1-phosphate (S-1-P) were also evaluated.
Results
Patients
A total of 261 patients with malaria were enrolled and had plasma and/or urine available for measurement of glycocalyx breakdown products, including 200 patients with knowlesi malaria (152 non-severe and 48 severe) and 61 with vivax malaria (52 non-severe, 9 severe). Plasma was available for 197 patients with knowlesi malaria (149 non-severe, 48 severe), and 58 patients with vivax malaria (50 non-severe, 8 severe). Urine was available for 126 patients with knowlesi malaria (85 non-severe, 41 severe) and 51 with vivax malaria (43 non-severe, 8 severe). Fifty healthy controls were enrolled; plasma syndecan-1 and S-1-P were measured in 20, and urinary GAGS were measured in 47. Clinical and pathophysiological data from these patients have been previously reported10,11,13,14,27. Baseline demographic, clinical and laboratory features are shown in Table 1. Patients were enrolled a median of 6 h (IQR 1.4–12.0 h) after commencement of antimalarial treatment.
As previously reported10, among the 48 patients with severe knowlesi malaria, WHO severity criteria included hyperparasitemia (n = 24, 50%), jaundice (n = 21, 44%), respiratory distress (n = 14, 29%), severe AKI (creatinine > 265 mmol/L; n = 11, 23%), shock (n = 11, 23%), metabolic acidosis (n = 4, 8%), severe anaemia (n = 5, 10%), and abnormal bleeding (n = 5, 8%). Using Kidney Disease: Improving Global Outcomes (KDIGO) criteria to define AKI, and the Modification of Diet in Renal Disease [MDRD] equation to estimate baseline creatinine, AKI was present on or during admission in 31 (65%) patients with severe malaria, and 14 (9%) patients with non-severe malaria. Of the 9 patients with severe vivax malaria, severity criteria included shock (n = 6, 67%), jaundice (n = 2, 22%), respiratory distress (n = 1, 11%), metabolic acidosis (n = 1, 11%), abnormal bleeding (n = 1, 11%), and convulsions (n = 1, 11%). AKI by KDIGO criteria occurred in 3 (33%) patients with severe vivax malaria, and 3 (6%) with non-severe vivax malaria.
Glycocalyx breakdown products and disease severity in knowlesi and vivax malaria
In patients with knowlesi malaria, urinary concentrations of GAGs were higher in those with severe compared to non-severe disease (median 2.36 [IQR 0.29–4.73] vs. 1.32 [IQR 0.18–2.28] g/mol creatinine, p = 0.016), and were higher in both disease groups compared to controls (0.06 [IQR 0.03–0.12] g/mol creatinine, p < 0.0001 for both comparisons; Table 1 and Fig. 1). Similarly, plasma concentrations of syndecan-1 were higher in patients with severe disease compared to non-severe disease (median 283 [IQR 206–575] vs. 154 [IQR 106–231] ng/ml, p < 0.0001). Surprisingly, concentrations of syndecan-1 were lower in those with non-severe disease compared to healthy controls (median 177 [IQR 130–519 ng/ml]), although this was not statistically significant. Concentrations of the glycocalyx stabilizer S-1-P were lower in patients with severe compared to non-severe knowlesi malaria (median 0.19 [IQR 0.58–1.63] vs. 1.35 [IQR 1.01–1.86] µM, p = 0.003); however, again there was no significant difference between those with non-severe disease and healthy controls (median 1.49 [IQR 1.08–2.44] µM).
In vivax malaria, as with knowlesi malaria, the median urinary GAGs was higher in patients with severe (1.46 [IQR 0.36–3.57] g/mol creatinine) and non-severe (1.12 [IQR 0.23–3.17] g/mol creatinine) disease compared to healthy controls (0.06 [IQR 0.03–0.12] g/mol creatinine, p < 0.001 for both comparisons; Table 1 and Fig. 1). However, there was no difference observed between patients with severe and non-severe vivax malaria. Plasma concentrations of syndecan-1 were again unexpectedly reduced in patients with non-severe disease compared to healthy controls (median 140 [IQR 109–205] vs. 177 [IQR 130–519 ng/ml p = 0.046). Concentrations of plasma syndecan-1 were higher in those with severe (median 180 [IQR 154–326] ng/ml) compared to non-severe disease, although numbers were small and this was not significant. In patients with vivax malaria there was no significant difference in concentrations of S-1-P between disease groups, or between either disease group and controls (Table 1 and Fig. 1).
Plasma concentrations of syndecan-1 were correlated with urinary GAGs in knowlesi malaria (r = 0.21, p = 0.019), although this did not remain significant after controlling for parasitemia. There was no correlation between plasma syndecan-1 and urinary GAGs in vivax malaria. There was also no correlation between S-1-P and either plasma syndecan-1 or urinary GAGs, in either knowlesi or vivax malaria.
Plasma concentrations of syndecan-1 were correlated with duration of fever in knowlesi malaria (r = 0.30, p < 0.0001) and in vivax malaria (r = 0.49, p < 0.001); in both species this remained significant after controlling for parasitemia (knowlesi malaria: p < 0.001; vivax malaria: p = 0.020). No such correlation occurred with urinary GAGs, in either knowlesi or vivax malaria. There was no difference in syndecan-1 or urinary GAGs in malaria patients enrolled before, or after, commencing antimalarial treatment.
Asymmetric dimethylarginine and disease severity in knowlesi malaria
In patients with knowlesi malaria, the median concentration of ADMA was higher in patients with severe compared to non-severe disease (0.60 [IQR 0.48–0.75] vs. 0.49 [IQR 0.43–0.54] µM, p < 0.0001), and higher in those with severe disease compared to controls (0.52 [IQR 0.48–0.58], p = 0.014). As with syndecan-1, concentrations of ADMA were unexpectedly lower in those with non-severe disease compared to controls (p = 0.007). The concentrations of ADMA in patients with severe knowlesi malaria were comparable to those of patients with severe falciparum malaria, enrolled in the same study cohort (median 0.57 [IQR 0.52–0.65] µM, N = 2224).
Glycocalyx breakdown products and biomarkers of severity in knowlesi and vivax malaria
In patients with knowlesi malaria, biomarkers of severity were more closely correlated with syndecan-1 than with urinary GAGs (Table 2). After applying Bonferonni’s correction for multiple comparisons (level of statistical significance, p < 0.0045) and adjusting for parasitemia, syndecan-1 was associated with ADMA (r = 0.33, p < 0.0001), creatinine (r = 0.46, p < 0.0001), lactate (r = 0.25, p < 0.001), angiopoietin-2 (r = 0.48, p < 0.0001), intercellular adhesion molecule 1 (ICAM-1; r = 0.30, p < 0.0001), osteoprotegerin (OPG; r = 0.25, p < 0.001), and inversely with microvascular reactivity (r = − 0.36, p < 0.001). Syndecan-1 was also inversely correlated with albumin (r = − 0.41, p < 0.0001), sodium (r = − 0.24, p < 0.001), bicarbonate (r = − 0.31, p < 0.0001), and chloride (r = − 0.21, p = 0.004).
In patients with knowlesi malaria, in a logistic regression model controlling for age and parasitemia, log-transformed plasma syndecan-1 was associated with severe disease (OR 3.72 [95% CI 2.01–6.89], p < 0.0001); this remained significant after controlling for the other biomarkers of severity listed above. After controlling for age and parasitemia, log-transformed syndecan-1 was also associated with acute kidney injury (OR 3.25, p < 0.0001), again remaining significant after controlling for the other biomarkers listed above.
In vivax malaria (Table 3), urinary GAGs correlated with angiopoietin-2 (r = 0.41, p = 0.003), ADMA (r = 0.40, p = 0.004), and ICAM-1 (r = 0.54, p < 0.0001), with the correlation with ADMA and ICAM-1 remaining significant after adjusting for parasitemia (p = 0.0001). After adjusting for parasitemia, plasma syndecan-1 also correlated with ADMA (r = 0.45, p < 0.001), angiopoietin-2 (r = 0.44, p = 0.002), and ICAM-1 (r = 0.53, p < 0.0001). In vivax malaria, neither urinary GAGs nor plasma syndecan-1 were independently associated with risk for severe disease, or AKI.
Glycocalyx breakdown products and S-1-P, and correlations with haemoglobin and platelets
Red blood cells (RBCs) and platelets are both known to produce the glycocalyx stabiliser S-1-P28,29, and therefore anaemia and/or thrombocytopenia could potentially lead to reduced S-1-P with consequent glycocalyx breakdown. In patients with knowlesi malaria, syndecan-1 was inversely correlated with haemoglobin on enrolment (r = − 0.23, p = 0.001; Table 2), remaining significant after controlling for parasitemia and fever duration (p < 0.0001). However, there was no correlation between haemoglobin and S-1-P (r = 0.09, p = 0.216). In knowlesi malaria, syndecan-1 was also inversely correlated with platelet count on enrolment (r = − 0.23, p = 0.001), again remaining significant after controlling for parasitemia (r = − 0.23, p = 0.001). Platelet count was also correlated with S-1-P (r = 0.36, p < 0.0001), remaining significant after controlling for parasitemia (r = 0.28, p = 0.001).
As with P. knowlesi, in patients with vivax malaria syndecan-1 was inversely correlated with platelet count on enrolment, remaining significant after controlling for parasitemia (r = − 0.50, p = 0.001). Platelet count on enrolment was also correlated with S-1-P (r = 0.29, p = 0.026), remaining significant after controlling for parasitemia (r = − 0.33, p = 0.013). In vivax malaria there was no correlation between either syndecan-1 or S-1-P with haemoglobin.
Correlations with age and sex
In patients with knowlesi malaria, median plasma syndecan-1 was higher in males compared to females (187 [IQR 123–307] vs. 133 [IQR 95–264] ng/ml, p = 0.014). Interestingly however, this difference occurred only in those with non-severe disease (168 [IQR 115–242] ng/ml in males vs. 110 [82–154] ng/ml in females, p < 0.001); in patients with severe knowlesi malaria, median plasma syndecan-1 was as high in females (281 [IQR 234–610] ng/ml) as it was in males (286 [IQR 192–541] ng/ml). Thus, in females, median syndecan-1 was 2.6-fold higher in severe compared to non-severe knowlesi malaria, while in males, the median was only 1.7-fold higher. Median syndecan-1 was also higher in healthy male controls (180 [IQR 143–663] ng/ml, n = 15) compared to healthy female controls (122 [IQR 115–350] ng/ml, n = 5), although numbers were small and this was not statistically significant. There were no significant sex differences in urinary GAGs in patients with knowlesi malaria. Median plasma S-1-P was lower in males compared to females (1.23 [IQR 0.89–1.82] vs. 1.63 [IQR 1.01– 1.91] µM, p = 0.05), with this difference observed in both severity groups.
In patients with vivax malaria there was a trend towards higher median plasma syndecan-1 in males compared to females (158 [IQR 118–229] vs. 109 [77–181] ng/ml, p = 0.068), and lower S-1-P (1.82 [IQR 1.24–2.64] vs. 3.2 [IQR 1.44–3.85] µM, p = 0.07). There were no significant sex differences in urinary GAGs in patients with vivax malaria.
There was a weak correlation between age and syndecan-1 in patients with knowlesi malaria (r = 0.16, p = 0.027); however, this did not remain significant after controlling for parasitemia. There was no correlation between age and syndecan-1 in patients with vivax malaria. There were also no significant correlations between age and S-1-P, in malaria patients or in controls.
Discussion
In this study, we demonstrate that urinary GAGs, markers of glycocalyx breakdown, are increased in knowlesi and vivax malaria, and in knowlesi malaria are increased in proportion to disease severity. In knowlesi malaria the plasma marker of glycocalyx degradation, syndecan-1, was also elevated in severe compared to non-severe disease, and was independently correlated with nearly all biomarkers of severity evaluated, including endothelial activation, microvascular dysfunction, creatinine, and lactate. In knowlesi malaria, in logistic regression models syndecan-1 was also independently associated with both severe disease and AKI. Taken together these findings suggest that glycocalyx breakdown is likely a key contributor to disease pathogenesis in knowlesi malaria.
The finding that urinary GAGs, but not syndecan-1, were increased in non-severe knowlesi malaria, but that syndecan-1 correlated more strongly with biomarkers of severity, suggests that shedding of negatively charged GAG sidechains, which comprise the most superficial layer of the intraluminal glycocalyx, may occur earlier in the disease process. In contrast, shedding of syndecan-1, a deeper core glycocalyx protein attached to endothelial cells through a transmembrane domain, may occur later in disease, reflecting greater glycocalyx damage with greater functional impairment. The correlation of syndecan-1, but not urinary GAGs, with fever duration, is consistent with this hypothesis.
Consistent with previous reports in falciparum malaria15,16, we found that in both knowlesi and vivax malaria, syndecan-1 was associated with the endothelial activation marker angiopoietin-2, likely reflecting the known role of angiopoietin-2 in mediating glycocalyx breakdown30. Syndecan-1 was also associated with the adhesion molecule ICAM-1 in both knowlesi and vivax malaria. ICAM-1 is contained within the glycocalyx, and the correlation between syndecan-1 and ICAM-1 likely reflects shedding of these adhesion molecules. Glycocalyx degradation may also expose adhesion molecules; while this has been shown in vitro to increase endothelial cytoadherence of P. falciparum-infected red cells31,32, the consequences in knowlesi and vivax malaria are less certain, given the apparent paucity of endothelial cytoadherence with these species33,34.
We also found that syndecan-1 was associated with OPG, a soluble decoy receptor of receptor activator of NF-kB (RANK) ligand. In addition to the ability of OPG to bind to syndecan-135, OPG has also been shown to inhibit RANK/RANK ligand-activation of eNOS, and may therefore contribute to glycocalyx breakdown by reducing eNOS-mediated inhibition of heparanase expression23. Increased heparanase expression may lead to glycocalyx degradation not just by the cleavage of heparan sulfate chains from core proteoglycans36, but also by activating metalloproteinases37, which cleave proteoglycans such as syndecan-1 directly from endothelial cells38.
Heparanase expression may also be induced by the eNOS inhibitor ADMA23. Although ADMA is known to be elevated in severe falciparum malaria, an association with glycocalyx breakdown has not previously been evaluated. In this study we found that ADMA was increased in severe knowlesi malaria, and was associated with both syndecan-1 and urinary GAGs. The findings in our study suggest that in addition to the role of angiopoietin-2 in mediating glycocalxy breakdown in malaria, agents that increase heparanase expression such as ADMA and OPG may also contribute. Importantly, ADMA and OPG are both increased in other conditions associated with glycocalyx breakdown, such as sepsis26, 39, and may represent common mechanisms of glycocalyx degradation. It is notable that in vivax malaria, an increase in ADMA is not observed14, and it is possible that this may account for the lack of any observed increase in plasma syndecan-1 in these patients.
Our finding that glycocalyx breakdown was an independent risk factor for AKI in knowlesi malaria is consistent with the key role that the glycocalyx in known to play in kidney function, with the glomerular glycocalyx regulating exchange of plasma proteins across glomerular capillaries40. Mouse models have demonstrated that glycocalyx degradation increases glomerular permeability to albumin, leading to albuminuria41. We have found that microalbuminuria is common in AKI complicating knowlesi malaria (unpublished), further suggesting that glycocalyx breakdown may be a key mechanism of malaria-associated AKI. Albumin is also able to stabilise glycocalyx42, and hence hypoalbuminemia may in itself exacerbate glycocalyx degradation.
A notable finding is our study was the inverse correlation in knowlesi malaria between syndecan-1 and haemoglobin, with this correlation remaining significant after controlling for parasitemia and fever duration. RBCs are known to be major producers of the glycocalyx stabiliser S-1-P28; however, we did not find a correlation between S-1-P and haemoglobin, and therefore reduced RBC production of S-1-P does not seem a likely explanation for the increased glycocalyx breakdown and elevated levels of syndecan-1. Alternatively, shedding of glycocalyx components from RBCs themselves may lead to increased plasma syndecan-1, given that RBCs, like endothelial cells, are known to contain a glycocalyx43. Shedding of glycocalyx from RBCs could further exacerbate anaemia, due to exposure of phosphatidylserine or other receptors enhancing eryptosis and/or phagocytosis.
We also found independent inverse correlations between platelets and both syndecan-1 and GAGs, in both vivax and knowlesi malaria. As with RBCs, platelets have been shown to produce S-1-P29, and in our study S-1-P was independently associated with platelet count. Thus, it is possible that reduced platelet-derived S-1-P could potentiate glycocalyx breakdown in malaria. Alternatively, glycocalyx breakdown may contribute directly to thrombocytopenia by mediating platelet adhesion and aggregation44,45.
We observed sex differences in glycocalyx degradation products. In patients with knowlesi malaria, syndecan-1 was higher and S-1-P lower, in males compared to females. This difference however was only observed in those with non-severe disease, and is consistent with a previous study in acute coronary syndrome demonstrating that males shed more glycocalyx than females46, and another study demonstrating that males have a thicker glycocalyx than females47. Consistent with this, we also found a trend towards higher syndecan-1 in male controls compared to female controls. Interestingly we found that in severe knowlesi malaria, syndecan-1 was as high in females compared to males, suggesting that severe disease in females may be associated with a greater proportionate loss of glycocalyx. In knowlesi malaria female sex is an independent risk factor for death3. While the cause of this increased risk is not well understood, it is possible that a thinner glycocalyx in females and a greater proportionate loss may contribute. Sex differences in disease severity and outcomes are observed in many acute infections48, 49; whether differences in glycocalyx degradation contribute warrants further evaluation.
Our study had some limitations. First, the small number of cases of severe vivax malaria limited our ability to examine relationships between markers of glycocalyx degradation and disease severity in vivax malaria. However, with larger numbers of cases of severe knowlesi malaria clear associations with disease severity were found. Second, the median age of the control group was higher than that of the patients with vivax malaria; however, given that we did not find significant correlations between age and markers of glycocalyx breakdown, this is unlikely to have affected our results. Third, our study did not have a non-malaria fever comparison group; however, it is well documented that glycocalyx degradation is not specific to malaria fevers, with degradation also found in a number of other conditions including sepsis20, dengue21 and COVID-1922. Finally, although we found associations between markers of glycocalyx degradation and biomarkers of disease severity, the nature of our study does not provide definitive evidence of causation.
In conclusion, we found that glycocalyx breakdown occurs in patients with knowlesi and vivax malaria, and is associated with disease severity and AKI in knowlesi malaria. Plasma syndecan-1 appeared to be a more clinically relevant biomarker than urinary GAGs, and may reflect more severe glycocalyx damage, occurring later in disease. Several agents have been demonstrated to stabilise endothelial glycocalyx, and may be potential agents for adjunctive treatment of severe non-falciparum malaria.
Methods
Ethics statement
The study was approved by the Ethics Committees of the Malaysian Ministry of Health and Menzies School of Health Research. Informed written consent was provided by all adults, and by the parent or guardian of any participant aged < 18 years.
Study site and patients
Patients were enrolled as part of a prospective observational study of all malaria patients admitted to Queen Elizabeth Hospital, an adult tertiary-referral hospital in Sabah, Malaysia11,27. For the current study, patients enrolled between September 2010 and December 2012 were included if they had PCR-confirmed P. knowlesi or P. vivax monoinfection, were non-pregnant, ≥ 12 years old, had no major comorbidities or concurrent illness, were within 18 h of commencing antimalarial treatment, and had stored frozen urine and/or plasma available for measurement of glycocalyx breakdown products. Severe malaria was defined according to modified WHO criteria50, as previously described11. Renal function was further assessed using the Kidney Disease: Improving Global Outcomes (KDIGO) criteria for AKI51. Healthy controls were visitors or relatives of malaria patients, with no history of fever in the past 48 h and with blood film negative for malaria parasites.
Standardized history and physical examination were documented. Haematology, biochemistry, and lactate (by bedside blood analysis; iSTAT system) were obtained on enrolment. Parasite counts were determined by microscopy, and parasite species identified by PCR52,53. Patients with severe disease were treated with intravenous artesunate, while those with non-severe disease received oral artemisinin combination treatment (and 14 days of primaquine for those with vivax malaria), as previously described27.
Laboratory assays and measurements of microvascular function
Venous blood collected on enrolment in lithium heparin and citrate tubes was centrifuged within 30 min of collection and plasma stored at – 70 °C. Urine was also collected on enrolment and stored within 30 min at – 70 °C. ELISA was used to measure heparanized plasma concentrations of syndecan-1 (Abcam); sphingosine-1-phosphate (S-1-P; Echelon Biosciences); angiopoietin-2, ICAM-1 and E-selectin (Quantikine R&D Systems) and osteoprotegerin (DuoSet R&D Systems). Cell free haemoglobin was measured on citrated platelet-free plasma by ELISA (Bethyl Laboratories). Heparanized plasma concentrations of the endothelial nitric oxide synthase (eNOS)-inhibitor asymmetric dimethylarginine (ADMA) were measured by HPLC54. Total urinary sulfated GAGs were measured on a subset by the dimethylmethylene blue (DMMB) colorimetric assay as previously described15,55. Urinary creatinine was measured by the alkaline picrate method, and the urine GAG concentrations were normalised to creatinine levels. All samples were diluted as per manufacturers’ recommendations but were adjusted if the resulting OD was outside the standard curve. Microvascular function was assessed using Near Infrared Spectroscopy (InSpectra 650, Hutchinson, MN), as previously described56. These data have been previously reported11, and are included here to assess correlation with glycocalyx degradation.
All methods were carried out in accordance with relevant guidelines and regulations.
Statistics
Statistical analysis was performed with STATA software (v15). Student’s T-test or Wilcoxon-Mann–Whitney tests were used for two-group comparisons, depending on distribution. Categorical variables were compared using χ2 or Fisher’s exact tests. Associations between continuous variables were assessed using Spearman’s correlation coefficient. Partial correlation was used to evaluate associations between continuous variables after adjusting for parasitemia. For this analysis, non-normally distributed variables were log-transformed to a normal distribution. Since correlations between glycocalyx breakdown products (urinary glycosaminoglycans and syndecan-1) and 11 variables were assessed, Bonferroni’s correction was used to adjust for multiple correlations, with correlations considered statistically significant if p < 0.0045 (p = 0.05/11).
Backward stepwise regression was used to evaluate factors associated with severe malaria and AKI, with variables removed at a significance level of > 0.05; for this analysis, patients with hyperparasitemia as a sole severity criterion were reclassified as having non-severe malaria.
Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request and with an appropriate data sharing agreement.
References
Lacerda, M. V. G. et al. Postmortem characterization of patients with clinical diagnosis of Plasmodium vivax malaria: to what extent does this parasite kill?. Clin. Infect. Dis. 55, 67–74 (2012).
Douglas, N. M. et al. Mortality attributable to Plasmodium vivax malaria: a clinical audit from Papua, Indonesia. BMC Med. 12, 217 (2014).
Rajahram, G. S. et al. Deaths from Plasmodium knowlesi malaria: case series and systematic review. Clin. Infect. Dis. 69, 1–36 (2019).
Siqueira, A. M. et al. Characterization of Plasmodium vivax-associated admissions to reference hospitals in Brazil and India. BMC Med. 13, 57 (2015).
Cox-Singh, J. et al. Plasmodium knowlesi malaria in humans is widely distributed and potentially life threatening. Clin. Infect. Dis. 46, 165–171 (2008).
Tjitra, E. et al. Multidrug-resistant Plasmodium vivax associated with severe and fatal malaria: a prospective study in Papua, Indonesia. PLoS Med. 5, e128 (2008).
Cooper, D. J. et al. Plasmodium knowlesi malaria in Sabah, Malaysia, 2015–2017: ongoing increase in incidence despite near-elimination of the human-only Plasmodium species. Clin. Infect. Dis. 70, 361–367 (2019).
Daneshvar, C. et al. Clinical and laboratory features of human Plasmodium knowlesi infection. Clin. Infect. Dis. 49, 852–860 (2009).
Grigg, M. J. et al. Age-related clinical spectrum of Plasmodium knowlesi malaria and predictors of severity. Clin. Infect. Dis. 67, 350–359 (2018).
Barber, B. E. et al. Intravascular haemolysis in severe Plasmodium knowlesi malaria: association with endothelial activation, microvascular dysfunction, and acute kidney injury. Emerg. Microb. Infect. 7, 106 (2018).
Barber, B. E. et al. Effects of aging on parasite biomass, inflammation, endothelial activation and microvascular dysfunction in Plasmodium knowlesi and P. falciparum malaria. J. Infect. Dis. 215, 1908–1917 (2017).
Chawla, L. S., Eggers, P. W., Star, R. A. & Kimmel, P. L. Acute kidney injury and chronic kidney disease as interconnected syndromes. N. Engl. J. Med. 371, 58–66 (2014).
Barber, B. E. et al. Parasite biomass-related inflammation, endothelial activation, microvascular dysfunction and disease severity in vivax malaria. PLoS Pathog. 11, e1004558 (2015).
Barber, B. E. et al. Nitric oxide–dependent endothelial dysfunction and reduced arginine bioavailability in Plasmodium vivax malaria but no greater increase in intravascular hemolysis in severe disease. J. Infect. Dis. 214, 1557–1564 (2016).
Yeo, T. W. et al. Glycocalyx breakdown is associated with severe disease and fatal outcome in Plasmodium falciparum malaria. Clin. Infect. Dis. 69, 1712–1720 (2019).
Lyimo, E. et al. In vivo imaging of the buccal mucosa shows loss of the endothelial glycocalyx and perivascular hemorrhages in pediatric Plasmodium falciparum malaria. Infect. Immun. 88, e00679-e1619 (2020).
Georgiadou, A. & Cunnington, A. J. Shedding of the vascular endothelial glycocalyx: a common pathway to severe malaria?. Clin. Infect. Dis. 39, 1720–1723 (2019).
Yeo, T. W. et al. Glycocalyx breakdown is increased in African children with cerebral and uncomplicated falciparum malaria. FASEB J. 33, 14185–14193 (2019).
Pillinger, N. & Kam, P. Endothelial glycocalyx: basic science and clinical implications. Anaesth. Intensive Care 45, 295–307 (2017).
Uchimido, R., Schmidt, E. P. & Shapiro, N. I. The glycocalyx: a novel diagnostic and therapeutic target in sepsis. Crit. Care 23, 16 (2019).
Lam, P. K. et al. Visual and biochemical evidence of glycocalyx disruption in human dengue infection, and association with plasma leakage severity. Front. Med. 7 (2020).
Rovas, A. et al. Microvascular dysfunction in COVID-19: the MYSTIC study. Angiogenesis, 1–13 (2020).
Garsen, M. et al. Endothelial nitric oxide synthase prevents heparanase induction and the development of proteinuria. PLoS ONE 11 (2016).
Barber, B. E. et al. Asymmetric Dimethylarginine (ADMA) in adult falciparum malaria: relationships with disease severity, antimalarial treatment, haemolysis and inflammation. Open Forum Infect. Dis. ofw027 (2016).
Yeo, T. et al. Increased asymmetric dimethylarginine in severe falciparum malaria: association with impaired nitric oxide bioavailability and fatal outcome. PLoS Pathog. 6, 214–217 (2010).
Davis, J. S. et al. Asymmetric dimethylarginine, endothelial nitric oxide bioavailability and mortality in sepsis. PLoS ONE 6, e17260 (2011).
Barber, B. E. et al. A prospective comparative study of knowlesi, falciparum and vivax malaria in Sabah, Malaysia: high proportion with severe disease from Plasmodium knowlesi and P. vivax but no mortality with early referral and artesunate therapy. Clin. Infect. Dis. 56, 383–397 (2013).
Pappu, R. et al. Promotion of lymphocyte egress into blood and lymph by distinct sources of sphingosine-1-phosphate. Science 316, 295–298 (2007).
Gazit, S. L. et al. Platelet and erythrocyte sources of S1P are redundant for vascular development and homeostasis, but both rendered essential after plasma S1P depletion in anaphylactic shock. Circ. Res. 119, e110–e126 (2016).
Lukasz, A. et al. Endothelial glycocalyx breakdown is mediated by angiopoietin-2. Cardiovasc. Res. 113, 671–680 (2017).
Introini, V., Carciati, A., Tomaiuolo, G., Cicuta, P. & Guido, S. Endothelial glycocalyx regulates cytoadherence in Plasmodium falciparum malaria. J. R. Soc. Interface 15, 20180773 (2018).
Hempel, C., Wang, C. W., Kurtzhals, J. A. L. & Staalsø, T. Binding of Plasmodium falciparum to CD36 can be shielded by the glycocalyx. Malar. J. 16, 1–11 (2017).
Carvalho, B. O. et al. On the cytoadhesion of Plasmodium vivax–infected erythrocytes. J. Infect. Dis. 202, 638–647 (2010).
Govindasamy, G. et al. Retinal changes in uncomplicated and severe Plasmodium knowlesi malaria. J. Infect. Dis. 213, 1476–1482 (2015).
Arnold, N. D. et al. A therapeutic antibody targeting osteoprotegerin attenuates severe experimental pulmonary arterial hypertension. Nat. Commun. 10, 1–18 (2019).
Schmidt, E. P. et al. The pulmonary endothelial glycocalyx regulates neutrophil adhesion and lung injury during experimental sepsis. Nat. Med. 18, 1217–1223 (2012).
Purushothaman, A., Chen, L., Yang, Y. & Sanderson, R. D. Heparanase stimulation of protease expression implicates it as a master regulator of the aggressive tumor phenotype in myeloma. J. Biol. Chem. 283, 32628–32636 (2008).
Endo, K. et al. Cleavage of syndecan-1 by membrane type matrix metalloproteinase-1 stimulates cell migration. J. Biol. Chem. 278, 40764–40770 (2003).
Kemperman, H. et al. Osteoprotegerin is higher in sepsis than in noninfectious SIRS and predicts 30-day mortality of SIRS patients in the intensive care. J. Appl. Lab. Med. 3, 559–568 (2019).
Salmon, A. H. & Satchell, S. C. Endothelial glycocalyx dysfunction in disease: albuminuria and increased microvascular permeability. J. Pathol. 226, 562–574 (2012).
Desideri, S. et al. A novel assay provides sensitive measurement of physiologically relevant changes in albumin permeability in isolated human and rodent glomeruli. Kidney Int. 93, 1086–1097 (2018).
Becker, B. F., Chappell, D., Bruegger, D., Annecke, T. & Jacob, M. Therapeutic strategies targeting the endothelial glycocalyx: acute deficits, but great potential. Cardiovasc. Res. 87, 300–310 (2010).
Oberleithner, H. Vascular endothelium leaves fingerprints on the surface of erythrocytes. Eur. J. Physiol. 465, 1451–1458 (2013).
Chappell, D. et al. Protection of glycocalyx decreases platelet adhesion after ischaemia/reperfusion: an animal study. Eur. J. Anaesthesiol. 31, 474–481 (2014).
Chappell, D. et al. Sevoflurane reduces leukocyte and platelet adhesion after ischemia-reperfusion by protecting the endothelial glycocalyx. J. Am. Soc. Anesthesiol. 115, 483–491 (2011).
Miranda, C. H., de Carvalho Borges, M., Schmidt, A., Marin-Neto, J. A. & Pazin-Filho, A. Evaluation of the endothelial glycocalyx damage in patients with acute coronary syndrome. Atherosclerosis 247, 184–188 (2016).
Valerio, L., Peters, R. J., Zwinderman, A. H. & Pinto-Sietsma, S.-J. Sublingual endothelial glycocalyx and atherosclerosis. A cross-sectional study. PLoS ONE 14 (2019).
Garenne, M. Demographic evidence of sex differences in vulnerability to infectious diseases. J. Infect. Dis. 211, 331–332 (2015).
Docherty, A. B. et al. Features of 20 133 UK patients in hospital with covid-19 using the ISARIC WHO Clinical Characterisation Protocol: prospective observational cohort study. BMJ 369 (2020).
World Health Organization. Severe Malaria. Trop. Med. Int. Health 19, 7–131 (2014).
Khwaja, A. KDIGO clinical practice guidelines for acute kidney injury. Nephron Clin. Pract. 120, c179–c184 (2012).
Padley, D., Moody, A., Chiodini, P. & Saldanha, J. Use of a rapid, single-round, multiplex PCR to detect malarial parasites and identify the species present. Ann. Trop. Med. Parasitol. 97, 131–137 (2003).
Imwong, M. et al. Spurious amplification of a Plasmodium vivax small-subunit RNA gene by use of primers currently used to detect P. knowlesi. J. Clin. Microbiol. 47, 4173 (2009).
Jones, C. E., Darcy, C. J., Woodberry, T., Anstey, N. M. & McNeil, Y. R. HPLC analysis of asymmetric dimethylarginine, symmetric dimethylarginine, homoarginine and arginine in small plasma volumes using a Gemini-NX column at high pH. J. Chromatog. B 878, 8–12 (2010).
Schmidt, E. P. et al. Urinary glycosaminoglycans predict outcomes in septic shock and acute respiratory distress syndrome. Am. J. Respir. Crit. Care Med. 194, 439–449 (2016).
Yeo, T. W. et al. Impaired skeletal muscle microvascular function and increased skeletal muscle oxygen consumption in severe falciparum malaria. J. Infect. Dis. 207, 528–536 (2013).
Acknowledgements
We thank all the study participants in Sabah, Malaysia; the Sabah malaria research team nursing and laboratory staff; and the Malaysian Ministry of Health hospital director and clinical staff at Queen Elizabeth Hospital. We also recognise the support of the Clinical Research Centre, Sabah, and the Director General of Health, Malaysia, for permission to publish this study.
Funding
This work supported by the National Health and Medical Research Council of Australia (Program Grants 1132975, Project Grant 1098334 and fellowships to BEB [1088738], MJG [1138860], and NMA [1135820]), and the National Institutes of Health (Grant R01 HL130763-01). The Sabah malaria research program is supported by US National Institutes of Health (R01 AI116472-03).
Author information
Authors and Affiliations
Contributions
B.E.B., J.B.W., T.W.Y. and N.M.A. conceived and designed the study. B.E.B. and M.J.G. collected data. K.A.P., Y.C. and J.B.W. performed assays. T.W. facilitated and supervised patient enrolments. B.E.B. analysed the data and wrote the first draft of the manuscript. All authors reviewed and approved the final draft of the manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Barber, B.E., Grigg, M.J., Piera, K.A. et al. Endothelial glycocalyx degradation and disease severity in Plasmodium vivax and Plasmodium knowlesi malaria. Sci Rep 11, 9741 (2021). https://doi.org/10.1038/s41598-021-88962-6
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41598-021-88962-6
- Springer Nature Limited