Glycocalyx Breakdown Is Associated With Severe Disease and Fatal Outcome in Plasmodium falciparum Malaria

Abstract

Background: Interactions between the endothelium and infected erythrocytes play a major role in the pathogenesis of falciparum malaria, with microvascular dysfunction and parasite sequestration associated with worsening outcomes. The glycocalyx is a carbohydrate-rich layer that lines the endothelium, with multiple roles in vascular homeostasis. The role of the glycocalyx in falciparum malaria and the association with disease severity has not been investigated.

Methods: We prospectively enrolled Indonesian inpatients (aged ≥18 years) with severe (SM) or moderately severe (MSM) falciparum malaria, as defined by World Health Organization criteria, and healthy controls (HCs). On enrollment, blood and urine samples were collected concurrently with measurements of vascular nitric oxide (NO) bioavailability. Urine was assayed for glycocalyx breakdown products (glycosaminoglycans) using a dimethylmethylene blue (GAG-DMMB) and liquid chromatography-tandem mass spectrometry (GAG-MS) assay.

Results: A total of 129 patients (SM = 43, MSM = 57, HC=29) were recruited. GAG-DMMB and GAG-MS (g/mol creatinine) were increased in SM (mean, 95% confidence interval: 3.98, 2.44-5.53 and 6.82, 5.19-8.44) compared to MSM patients (1.78, 1.27-2.29 and 4.87, 4.27-5.46) and HCs (0.22, 0.06-0.37 and 1.24, 0.89-1.59; P < 0.001). In SM patients, GAG-DMMB and GAG-MS were increased in those with a fatal outcome (n = 3; median, interquartile range: 6.72, 3.80-27.87 and 12.15, 7.88-17.20) compared to survivors (n = 39; 3.10, 0.46-4.5 and 4.64, 2.02-15.20; P = 0.03). Glycocalyx degradation was significantly associated with parasite biomass in both MSM (r = 0.48, GAG-DMMB and r = 0.43, GAG-MS; P < 0.001) and SM patients (r = 0.47, P = 0.002 and r = 0.33, P = 0.04) and inversely associated with endothelial NO bioavailability.

Conclusions: Increased endothelial glycocalyx breakdown is associated with severe disease and a fatal outcome in adults with falciparum malaria.

Keywords: Plasmodium falciparum; endothelium; glycocalyx; severe malaria.

Figure 1.

A, Plasma syndecan-1 levels measured in Indonesian adults who were enrolled as healthy controls and with moderately severe malaria or severe malaria. B, Urinary glycosaminoglycan levels measured by dimethylmethylene blue colorimetric assay in Indonesian adults who were enrolled as healthy controls and with moderately severe malaria or severe malaria. C, Urinary glycosaminoglycan levels (sum of chondroitin sulfate, dermatan sulfate, and heparan sulfate) measured by a mass spectrometric method assay in Indonesian adults who were enrolled as healthy controls and with moderately severe malaria or severe malaria. Lines indicate mean value. P < .001 by analysis of variance. ○, survivors; ●, nonsurvivors. Abbreviation: DMMB, dimethylmethylene blue.

Figure 2.

A, Urinary chondroitin sulfate levels measured by a mass spectrometric method assay in Indonesian adults who were enrolled as healthy controls and with moderately severe malaria or severe malaria. B, Urinary dermatan sulfate levels measured by a mass spectrometric method assay in Indonesian adults who were enrolled as healthy controls and with moderately severe malaria or severe malaria. C, Urinary heparan sulfate levels measured by a mass spectrometric method assay in Indonesian adults who were enrolled as healthy controls and with moderately severe malaria or severe malaria. Lines indicate mean value. P < .001 by analysis of variance. ○, survivors; •, nonsurvivors.

Figure 3.

A, Nonparametric receiver operating curve to assess urinary glycosaminoglycan levels measured by mass spectrometry as a prognostic indicator for mortality in Indonesian adults with severe malaria. Area under the receiver-operating curve (AUROC), 0.89; 95% confidence interval [CI], 0.76–0.99. B, Nonparametric receiver operating curve to assess plasma syndecan-1 levels as a prognostic indicator for mortality in Indonesian adults with severe malaria. AUROC, 0.78; 95%, CI 0.54–0.99.