Plasmodium falciparum clearance is rapid and pitting independent in immune Malian children treated with artesunate for malaria

Abstract

Background: In Plasmodium falciparum-infected patients treated with artemisinins, parasitemia declines through so-called pitting, an innate splenic process that transforms infected red blood cells (iRBCs) into once-infected RBCs (O-iRBCs).

Methods: We measured pitting in 83 French travelers and 42 Malian children treated for malaria with artesunate.

Results: In travelers, O-iRBCs peaked at 107.7% initial parasitemia. In Malian children aged 1.5-4 years, O-iRBCs peaked at higher concentrations than in children aged 9-13 years (91.60% vs 31.95%; P = .0097). The parasite clearance time in older children was shorter than in younger children (P = .0001), and the decline in parasitemia in children aged 1.5-4 years often started 6 hours after treatment initiation, a lag phase generally absent in infants and older children. A 6-hour lag phase in artificial pitting of artesunate-exposed iRBCs was also observed in vitro. The proportion of iRBCs recognized by autologous immunoglobulin G (IgG) correlated with the parasite clearance time (r = -0.501; P = .0006) and peak O-iRBC concentration (r = -0.420; P = .0033).

Conclusions: Antimalarial immunity correlates with fast artemisinin-induced parasite clearance and low pitting rates. In nonimmune populations, artemisinin-induced P. falciparum clearance is related to pitting and starts after a 6-hour lag phase. In immune populations, passively and naturally acquired immune mechanisms operating faster than pitting may exist. This mechanism may mitigate the emergence of artemisinin-resistant P. falciparum in Africa.

Keywords: Plasmodium falciparum; acquired immunity; artemisinin; malaria; parasite clearance; pitting; spleen.

© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Figures

Figure 1.

Kinetics of parasite clearance and pitting in French travelers. Mean concentrations (±standard error of the mean) of infected red blood cells (iRBCs; filled circles) and once-infected red blood cells (O-iRBCs; empty circles) were measured over 7 days in peripheral blood specimens from French travelers treated with intravenous artesunate (A; n = 48) or quinine (B; n = 35) for severe Plasmodium falciparum malaria. iRBC and O-iRBC concentrations were estimated by flow cytometry, using the DNA-staining Hoechst or SYBR green I dyes (which detect iRBCs only) and a RESA-staining protocol (which detects both iRBCs and O-iRBCs). iRBCs are identified as DNA-positive/RESA-positive cells, while O-iRBCs are identified as DNA-negative/RESA-positive cells. C, Monitoring of parasite clearance over 3 days in a French traveler. Flow cytometry analysis of blood samples collected 0, 6, and 72 hours after initiating artesunate treatment shows progressive reductions in the concentration of iRBCs (upper right quadrants) and simultaneous increases in the concentration of O-iRBCs (upper left quadrants). In this patient, parasite clearance was completely pitting dependent.

Figure 2.

Kinetics of parasite clearance and pitting in Malian children. A, Mean concentrations (±standard error of the mean [SEM]) of infected red blood cells (iRBCs; filled circles) and once-infected red blood cells (O-iRBCs; empty circles) were measured every 6 hours in peripheral blood specimens from 42 children treated with oral artesunate for uncomplicated Plasmodium falciparum malaria. iRBC and O-iRBC concentrations were estimated by the erythrocyte membrane immunofluorescence method, using the DNA-staining Hoechst dye (which detects iRBCs only) and a RESA-staining protocol (which detects both iRBCs and O-iRBCs). iRBCs are identified as DNA-positive/RESA-positive cells, while O-iRBCs are identified as DNA-negative/RESA-positive cells. These cells were quantified among 10 000 RBCs, and their concentrations were normalized to the initial parasitemia set at 100%. B, iRBC clearance curves for the 42 children in panel A, stratified by age, as follows: 1.5–4 years (solid line), 5–8 years (dashed line), and 9–13 years (dotted line). Mean concentrations (±SEM) of iRBCs over time are shown. C, O-iRBC appearance curves of the 42 children in panel A, stratified by age, as follows: 1.5–4 years (solid line), 5–8 years (dashed line), and 9–13 years (dotted line). Mean concentrations (±SEM) of O-iRBCs over time are shown.

Figure 3.

Artificial pitting in vitro, using microsphiltration. A, Infected red blood cells (iRBCs; containing ring-stage parasites) were left unexposed or exposed to 1 µM artesunate for 2 or 6 hours (i) and then filtered through microsphere layers as described elsewhere (see Methods) [12]. After being stained for DNA (blue) and RESA (green) using the erythrocyte membrane immunofluorescence (EMIF) method, iRBCs (blue and green) and once-infected red blood cells (O-iRBCs; green only [white arrows]) are easily identified by conventional fluorescence microscopy (ii). Some iRBCs in the microsphere layer contained parasite remnants (black arrow) in the process of being pitted (iii). B, Samples upstream (Up) and downstream (Dw) from the filters were stained using the EMIF method, and the concentrations of O-iRBCs were measured. Mean pitting rates (±standard error of the mean) are shown. iRBCs exposed to artesunate for 6 hours were pitted at a significantly higher rate than those exposed to artesunate for 2 hours (P = .0189) or left untreated (P = .0360). Abbreviation: NS, not significant.

Figure 4.

Relationship between age and lag-phase duration in the parasite clearance curve. A, Correlation between age and lag-phase duration was investigated in 215 Malian children treated with oral artesunate for uncomplicated Plasmodium falciparum malaria. The lag phase was defined as the length of time parasitemia remained >75% of its initial level. Using data from all 215 children, no correlation was found (r = −0.097; P = .155; many data points overlap). B, Mean lag-phase durations (±standard error of the mean), stratified by age category, are shown. Lag-phase duration was significantly longer in children aged 2–4 years, compared with children aged 0–1.5 (P = .007), 5–8 (P = .008), and 9–15 (P = .013) years.