Elevated cell-specific microparticles are a biological marker for cerebral dysfunctions in human severe malaria

Abstract

Cerebral malaria (CM) and severe anemia (SA) are the most severe complications of Plasmodium falciparum infections. Although increased release of endothelial microparticles (MP) correlates with malaria severity, the full extent of vascular cell vesiculation remains unknown. Here, we characterize the pattern of cell-specific MP in patients with severe malaria. We tested the hypothesis that systemic vascular activation contributes to CM by examining origins and levels of plasma MP in relation to clinical syndromes, disease severity and outcome. Patients recruited in Douala, Cameroon, were assigned to clinical groups following WHO criteria. MP quantitation and phenotyping were carried out using cell-specific markers by flow cytometry using antibodies recognizing cell-specific surface markers. Platelet, erythrocytic, endothelial and leukocytic MP levels were elevated in patients with cerebral dysfunctions and returned to normal by discharge. In CM patients, platelet MP were the most abundant and their levels significantly correlated with coma depth and thrombocytopenia. This study shows for the first time a widespread enhancement of vesiculation in the vascular compartment appears to be a feature of CM but not of SA. Our data underpin the role of MP as a biomarker of neurological involvement in severe malaria. Therefore, intervention to block MP production in severe malaria may provide a new therapeutic pathway.

Figure 1. Flow cytometry analysis details and representative cytograms in a CM patient.

MP were discriminated by their size and structures (Fig. 1A ). And events in MP gate were further analyzed to differentiate Annexin V⁺ MP from the background signal. An internal standard, constituted of fluorosphere beads of a known size and at a known concentration was used to help set the MP gate and calculate the MP level. Representative cytograms: 1B to 1H Illustrations of the differences in MP positive events in cytograms representing total MP and cell-derived MP in a CM patient. (B): T-MP:annexin V⁺; (C): P-MP:CD41⁺; (D): E-MP:CD105⁺; (E): R-MP:CD235a⁺; (F): E-MP:CD51⁺; (G): M-MP:CD11b⁺; (H): L-MP:CD3⁺.These MP positive events were used in the calculations of the number of MP/µl of plasma (see the methods section).

Figure 2. Total and cell-specific MP levels per µl of plasma on admission in the different groups.

(A): T-MP:annexin V⁺; (B): P-MP:CD41⁺; (C): E-MP:CD105⁺; (D): R-MP:CD235a⁺; (E): M-MP:CD11b⁺; (F): L-MP:CD3⁺. Dot plots representations showing elevations of total MP and the different cell-specific MP investigated in CM and CM+SA patients. C: Controls; UM: uncomplicated malaria; SA: severe malaria anemia patients on admission; CM: cerebral malaria patients on admission; CM+SA: patients with the combine symptoms of CM and SA on admission; MP levels are given in MP/µl of plasma. *: p<0.05, **: p<0.001, ***: p<0.0001.

Figure 3. Total and cellular MP levels per µl of plasma at follow-up/discharge (7 days after admission:

A) versus levels on admission. (A): T-MP:annexin V⁺; (B): P-MP:CD41⁺; (C): E-MP:CD105⁺; (D): R-MP:CD235a⁺; (E): M-MP:CD11b⁺; (F): L-MP:CD3⁺. Dot plot representations showing how in most of the cases, MP levels decreases in patients with CM at discharge (D). Control group is represented to show that patient levels are returning to control levels. C: Controls; SA: severe malaria anemia patients on admission; CM: cerebral malaria patients on admission; CM+SA: patients with the combine symptoms of CM and SMA on admission; SA(D): severe malaria anemia patients at discharge; CM(D): cerebral malaria patients at discharge; CM+SA(D): patients with the combine symptoms of CM and SMA at discharge; MP levels are given in MP/µl of plasma. The values of the deceased patients are presented as black stars on Figure 3. *: p<0.05, **: p<0.001, ***: p<0.0001.

Figure 4. Correlations between MP and some clinical/biological parameters.

A to E: correlations between P-MP numbers and temperature, fever resolution time (FRT), Blantyre coma score (BCS), coma resolution time (CRT) and platelet counts, respectively. F: correlation between R-MP and hemoglobin levels (Hgb).