Altered Subpopulations of Red Blood Cells and Post-treatment Anemia in Malaria

Abstract

In acute malaria, the bulk of erythrocyte loss occurs after therapy, with a nadir of hemoglobin generally observed 3-7 days after treatment. The fine mechanisms leading to this early post-treatment anemia are still elusive. We explored pathological changes in RBC subpopulations by quantifying biochemical and mechanical alterations during severe malaria treated with artemisinin derivatives, a drug family that induce "pitting" in the spleen. In this study, the hemoglobin concentration dropped by 1.93 G/dl during therapy. During the same period, iRBC accounting for 6.12% of all RBC before therapy (BT) were replaced by pitted-RBC, accounting for 5.33% of RBC after therapy (AT). RBC loss was thus of 15.9%, of which only a minor part was due to the loss of iRBC or pitted-RBC. When comparing RBC BT and AT to normal controls, lipidomics revealed an increase in the cholesterol/phosphatidylethanolamine ratio (0.17 versus 0.24, p < 0.001) and cholesterol/phosphatidylinositol ratio (0.36 versus 0.67, p = 0.001). Using ektacytometry, we observed a reduced deformability of circulating RBC, similar BT and AT, compared to health control donors. The mean Elongation Index at 1.69Pa was 0.24 BT and 0.23 AT vs. 0.28 in controls (p < 0.0001). At 30Pa EI was 0.56 BT and 0.56 AT vs. 0.60 in controls (p < 0.001). The retention rate (rr) of RBC subpopulations in spleen-mimetic microsphere layers was higher for iRBC (rr = 20% p = 0.0033) and pitted-RBC (rr = 19%, p = 0.0031) than for healthy RBC (0.12%). Somewhat surprisingly, the post-treatment anemia in malaria results from the elimination of RBC that were never infected.

Keywords: P. falcipaprum; RBC deformability; artemisinin derivatives; malaria anemia; membrane lipid balance; pitted or once infected RBC; spleen filtering funcion.

FIGURE 1

Hemoglobin decline and RBC clearance. Hemoglobin concentration for the 13 patients with Hb data available both before and after therapy. Paired t-test, p value for hemoglobin comparison = 0.0003. Para: mean parasitemia for the same patients before treatment with AD.; pit: mean pittemia for the same patient after treatment with AD.

FIGURE 2

Alterations of cholesterol and phospholipid RBC membrane composition before and after antimalarial therapy in severe malaria. (A). Lipid content of RBC membranes using HPLC and MS/MS on samples from 10 healthy donors (CTL), patients sampled before (N = 10) and after (N = 12) antimalarial therapy. (B). Cholesterol/Phosphatidylethanolamine (ratio Chol/PE) and Cholesterol/Phosphatidylinositol (ratio Chol/PI). Stars indicate significant differences.

FIGURE 3

Altered functional properties of circulating RBCs. (A). Elongation index at different shear stresses of RBC from patients before antimalarial therapy (n = 12) or upon follow-up (n = 16), compared to RBCs from healthy donors (CTL, n = 27). (B). Retention of glutaraldehyde-fixed RBC (Rigidified RBC, n = 8), healthy RBC (n = 8), patient iRBC (n = 6), and pitted RBC (n = 7). Negative values reflect retention in microspheres; positive values reflect enrichment.