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Circulating Endothelial Microparticles in Malawian Children With Severe Falciparum Malaria Complicated With Coma
To the Editor: Malaria remains a major problem of public health in developing countries and is responsible for more than 1 million deaths each year.1 Life-threatening malaria is predominantly a result of Plasmodium falciparum infection, and central nervous system involvement, ie, "cerebral malaria," is observed in approximately 1% of P falciparum infections and is a major cause of death. In P falciparum malaria, parasitized erythrocytes sequester in capillary beds through adhesion to vascular endothelium, a process that occurs predominantly in deep tissues not amenable to direct examination.2 Activation of microvascular endothelium in a variety of disease states causes the release of endothelial microparticles (EMPs) into the circulation.3 Plasma concentrations of tumor necrosis factor (TNF) are increased in patients with malaria,2 and TNF can induce the release of procoagulant and proadhesive microparticles from cultured endothelial cells in vitro.3 Numbers of EMPs in peripheral blood may therefore serve as a marker of endothelial activation in deep tissues. Although increased numbers of EMPs are found in patients with inflammatory disease and severe sepsis,4 EMPs have not previously been studied in human malaria.
Methods
With guardians' informed consent and with the approval of the College of Medicine Research and Ethics Committee, University of Malawi, we studied children admitted to the malaria research ward of the Queen Elizabeth Central Hospital, Blantyre, Malawi. Children aged 6 months to 12 years with P falciparum parasitemia were enrolled and later categorized into 1 of 4 clinical groups: (1) uncomplicated malaria (n = 48; mean [SD] age, 27.8 [23.2] months), (2) cerebral malaria, defined as a Blantyre coma score of 0-2 out of 5 in the absence of another identifiable cause (n = 138; mean [SD] age, 45 [29] months), (3) severe anemia, defined as hematocrit <16% (n = 37; mean [SD] age, 35 [23] months), and (4) both coma and severe anemia (n = 27; mean [SD] age, 37 [27] months). Antimalarial and supportive therapies were given according to standard protocols. Plasma samples from healthy age-matched individuals living in Malawi were used as a further comparison group (n = 60; mean [SD] age, 41.6 [26.5] months). Venous blood was obtained from patients on admission and, in those who survived and attended follow-up, during convalescence 19 to 21 days later. Citrated blood (0.5 mL) was centrifuged at 1500g for 15 minutes, and plasma was then cooled to –20°C before storage at –80°C. Plasma EMPs were quantitated by flow cytometry using a specific endothelial monoclonal antibody (FITC-CD51, Beckman Coulter Immunotech, Fullerton, Calif), and results were expressed as the mean (SD) number of EMPs per microliter of plasma. Groups were compared with the Mann-Whitney U test.
Results
When compared with the numbers in control patients, the mean EMPs per microliter were increased in admission blood samples only in patients with coma (ie, those with cerebral malaria or coma and severe anemia, P = .005 and P<.001, respectively) (Figure 1). When patients in these 2 groups were combined (n = 165), the mean (SD) EMP count was 87 (166) (median [IQR], 24 [7-80]) particles per microliter compared with 14 (23) (median [IQR], 8 [1.5-25.5]) particles per microliter in uncomplicated malaria (P<.001) and 25 (38) (median [IQR], 10 [0-26.5]) particles per microliter in healthy children (P<.001). During convalescence the mean absolute number of EMPs was significantly less than in the acute stage among patients with cerebral malaria or coma and severe anemia (P = .001 and P = .04) (Figure 1). For all patients with coma (ie, those with cerebral malaria plus those with coma and severe anemia), the mean EMP count decreased from 87 (166) (median [IQR], 24 [7-80]) particles per microliter on admission to 30 (55) (median [IQR], 6 [0-34.5]) particles per microliter during convalescence (n = 93, P<.001). In children presenting with uncomplicated malaria (n = 48) or severe anemia without coma (n = 37), numbers of EMPs were not increased, and remained similar in convalescence. Analyses restricted to patients from whom both acute and convalescent samples were available gave similar results (data not shown). Admission numbers of EMPs tended to be lower in comatose patients who died than in those who recovered.
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Figure. Numbers of Endothelial Microparticles (EMPs) in Plasma of Patients With Malaria, at Admission and at Follow-up Days 19-21
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Comment
We found increased numbers of circulating EMPs in children with coma and severe malaria. In patients with fatal malaria, endothelial activation is evidenced by up-regulation of intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 in the brain.2, 5 Our findings suggest that increased numbers of circulating EMPs could be another indicator of endothelial activation in patients with malaria, but that this increase is restricted to malaria complicated by coma. The high concentrations of plasma TNF2 in patients with cerebral malaria suggest that TNF may enhance endothelial vesiculation in vivo, as it does in vitro.3 EMPs shed from cultured endothelial cells have both proinflammatory and prothrombotic properties.3 Our finding of abundant circulating EMPs in patients with severe malaria complicated by coma, but not in those with malaria complicated by anemia alone, suggests that EMPs may play a part in the pathogenesis of the widespread deposition of fibrin and platelets observed in fatal cases of cerebral malaria.6 Apart from endothelium, other cell types including platelets, monocytes, and erythrocytes can release microparticles. Further studies are in progress to investigate microparticles of these additional origins in patients with malaria.
Funding/Support: This investigation was supported by the French Ministry of Research, the PAL+ Programme, and the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR).
Acknowledgment: Drs Grau and Molyneux contributed equally to the study.
Valéry Combes, PhD
Experimental Parasitology Unit
Faculty of Medicine
Université de la Méditerranée
Marseille, France
Terrie E. Taylor, DO
Malawi-Liverpool-Wellcome Trust Clinical Research Programme and Blantyre Malaria Project
Blantyre, Malawi
Irène Juhan-Vague, MD, PhD
Haematology Laboratory
Faculty of Medicine
Université de la Méditerranée
Jean-Louis Mège, MD, PhD
Rickettsia Unit
Faculty of Medicine
Université de la Méditerranée
James Mwenechanya, MB;
Madalitso Tembo, MB
Malawi-Liverpool-Wellcome Trust Clinical Research Programme and Blantyre Malaria Project
Georges E. Grau, MD, PhD
georges.grau{at}medecine.univ-mrs.fr
Experimental Parasitology Unit
Faculty of Medicine
Université de la Méditerranée
Malcolm E. Molyneux, MD, FRCP
Malawi-Liverpool-Wellcome Trust Clinical Research Programme and Blantyre Malaria Project
1. Beales PF, Brabin B, Dorman E, et al. Severe falciparum malaria. Trans Roy Soc Trop Med Hyg. 2000;94:S1-S90.
2. Lou J, Lucas R, Grau GE. Pathogenesis of cerebral malaria: recent experimental data and possible applications for humans. Clin Microbiol Rev. 2001;14:810-820.
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3. Combes V, Simon AC, Grau GE, et al. In vitro generation of endothelial microparticles and possible prothrombotic activity in patients with lupus anticoagulant. J Clin Invest. 1999;104:93-102.
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4. Freyssinet JM. Cellular microparticles: what are they bad or good for? J Thromb Haemost. 2003;1:1655-1662.
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5. Turner GD, Morrison H, Jones M, et al. An immunohistochemical study of the pathology of fatal malaria: evidence for widespread endothelial activation and a potential role for intercellular adhesion molecule-1 in cerebral sequestration. Am J Pathol. 1994;145:1057-1069.
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6. Grau GE, Mackenzie CD, Carr RA, et al. Platelet accumulation in brain microvessels in fatal pediatric cerebral malaria. J Infect Dis. 2003;187:461-466.
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Letters Section Editor: Stephen J. Lurie, MD, PhD, Senior Editor.
JAMA. 2004;291:2542-2544.
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