Plasmodium vivax can cause severe malaria, however its pathogenesis is poorly understood. In contrast to P. falciparum, circulating vivax parasitemia is low, with minimal apparent sequestration in endothelium-lined microvasculature, and pathogenesis thought unrelated to parasite biomass. However, the relationships between vivax disease-severity and total parasite biomass, endothelial autocrine activation and microvascular dysfunction are unknown. We measured circulating parasitemia and markers of total parasite biomass (plasma parasite lactate dehydrogenase [pLDH] and PvLDH) in adults with severe (n = 9) and non-severe (n = 53) vivax malaria, and examined relationships with disease-severity, endothelial activation, and microvascular function. Healthy controls and adults with non-severe and severe falciparum malaria were enrolled for comparison. Median peripheral parasitemia, PvLDH and pLDH were 2.4-fold, 3.7-fold and 6.9-fold higher in severe compared to non-severe vivax malaria (p = 0.02, p = 0.02 and p = 0.015, respectively), suggesting that, as in falciparum malaria, peripheral P. vivax parasitemia underestimates total parasite biomass, particularly in severe disease. P. vivax schizonts were under-represented in peripheral blood. Severe vivax malaria was associated with increased angiopoietin-2 and impaired microvascular reactivity. Peripheral vivax parasitemia correlated with endothelial activation (angiopoietin-2, von-Willebrand-Factor [VWF], E-selectin), whereas markers of total vivax biomass correlated only with systemic inflammation (IL-6, IL-10). Activity of the VWF-cleaving-protease, ADAMTS13, was deficient in proportion to endothelial activation, IL-6, thrombocytopenia and vivax disease-severity, and associated with impaired microvascular reactivity in severe disease. Impaired microvascular reactivity correlated with lactate in severe vivax malaria. Findings suggest that tissue accumulation of P. vivax may occur, with the hidden biomass greatest in severe disease and capable of mediating systemic inflammatory pathology. The lack of association between total parasite biomass and endothelial activation is consistent with accumulation in parts of the circulation devoid of endothelium. Endothelial activation, associated with circulating parasites, and systemic inflammation may contribute to pathology in vivax malaria, with microvascular dysfunction likely contributing to impaired tissue perfusion.
Severe dengue can be lethal caused by manifestations such as severe bleeding, fluid accumulation and organ impairment. This study aimed to investigate the role of dengue non-structural 1 (NS1) protein and host factors contributing to severe dengue. Electrical cell-substrate impedance sensing system was used to investigate the changes in barrier function of microvascular endothelial cells treated NS1 protein and serum samples from patients with different disease severity. Cytokines and metabolites profiles were assessed using a multiplex cytokine assay and liquid chromatography mass spectrometry respectively. The findings showed that NS1 was able to induce the loss of barrier function in microvascular endothelium in a dose dependent manner, however, the level of NS1 in serum samples did not correlate with the extent of vascular leakage induced. Further assessment of host factors revealed that cytokines such as CCL2, CCL5, CCL20 and CXCL1, as well as adhesion molecule ICAM-1, that are involved in leukocytes infiltration were expressed higher in dengue patients in comparison to healthy individuals. In addition, metabolomics study revealed the presence of deregulated metabolites involved in the phospholipid metabolism pathway in patients with severe manifestations. In conclusion, disease severity in dengue virus infection did not correlate directly with NS1 level, but instead with host factors that are involved in the regulation of junctional integrity and phospholipid metabolism. However, as the studied population was relatively small in this study, these exploratory findings should be confirmed by expanding the sample size using an independent cohort to further establish the significance of this study.
Plasma leakage is the main pathophysiological feature in severe dengue, resulting from altered vascular barrier function associated with an inappropriate immune response triggered upon infection. The present study investigated functional changes using an electric cell-substrate impedance sensing system in four (brain, dermal, pulmonary and retinal) human microvascular endothelial cell (MEC) lines infected with purified dengue virus, followed by assessment of cytokine profiles and the expression of inter-endothelial junctional proteins. Modelling of changes in electrical impedance suggests that vascular leakage in dengue-infected MECs is mostly due to the modulation of cell-to-cell interactions, while this loss of vascular barrier function observed in the infected MECs varied between cell lines and DENV serotypes. High levels of inflammatory cytokines (IL-6 and TNF-α), chemokines (CXCL1, CXCL5, CXCL11, CX3CL1, CCL2 and CCL20) and adhesion molecules (VCAM-1) were differentially produced in the four infected MECs. Further, the tight junctional protein, ZO-1, was down-regulated in both the DENV-1-infected brain and pulmonary MECs, while claudin-1, PECAM-1 and VE-cadherin were differentially expressed in these two MECs after infection. Non-purified virus stock was also studied to investigate the impact of virus stock purity on dengue-specific immune responses, and the results suggest that virus stock propagated through cell culture may include factors that mask or alter the DENV-specific immune responses of the MECs. The findings of the present study show that high DENV load differentially modulates human microvascular endothelial barrier function and disrupts the function of inter-endothelial junctional proteins during early infection with organ-specific cytokine production.