Plasmodium falciparum clearance rates in response to artesunate in Malian children with malaria: effect of acquired immunity

Abstract

Background: Artemisinin resistance, a long parasite clearance half-life in response to artemisinin, has been described in patients with Plasmodium falciparum malaria in southeast Asia. Few baseline half-lives have been reported from Africa, where artemisinins were recently introduced.

Methods: We treated P. falciparum malaria in 215 Malian children aged 0.5-15 years with artesunate (0, 24, 48 hours) and amodiaquine (72, 96, 120 hours). We estimated half-life by measuring parasite density every 6 hours until undetectable and evaluated the effects of age, sex, ethnicity, and red blood cell (RBC) polymorphisms on half-life. We quantified the proportion of parasitized RBCs recognized by autologous immunoglobulin G (IgG).

Results: The geometric mean half-life was 1.9 hours (95% confidence interval, 1.8-2.0) and did not correlate with parasite ex vivo susceptibility to artemisinins. In a linear model accounting for host factors, half-life decreased by 4.1 minutes for every 1-year increase in age. The proportion of parasitized RBCs recognized by IgG correlated inversely with half-life (r = -0.475; P = .0006).

Conclusions: Parasite clearance in response to artesunate is faster in Mali than in southeast Asia. IgG responses to parasitized RBCs shorten half-life and may influence this parameter in areas where age is not an adequate surrogate of immunity and correlates of parasite-clearing immunity have not been identified.

Clinical trials registration: NCT00669084.

Figure 1.

Clearance of ring-stage Plasmodium falciparum parasites from peripheral blood during a parasite clearance rate study. Dihydroartemisinin, the active metabolite of all artemisinins, causes ring-stage parasites to undergo pyknosis (A). These circulating pyknotic forms are eventually “pitted” from red blood cells (RBCs) as they pass through endothelial slits in the spleen, which returns the previously infected, intact RBCs to the peripheral blood. This process occurs in all patients treated with artesunate and is likely the predominant mechanism of parasite clearance in most cases. Depending on the maturity of ring-stage parasites, however, 2 additional processes may also work to reduce parasite density in an artemisinin-independent manner. Ring-stage parasites that are sufficiently mature (indicated by a thicker ring of light blue cytoplasm in the figure) may place PfEMP1-laden knobs on the surface of their host RBCs at the same time artesunate is consumed and begins to kill parasites (B and C). If these PfEMP1-expressing parasitized RBCs are traveling through a microvessel at this time, they may successfully sequester there and disappear from the peripheral blood (C). On the other hand, if these parasitized RBCs are traveling through other blood vessels where sequestration does not occur, they become targets for antibodies against PfEMP1 and other surface antigens (B). By preventing parasitized RBCs from sequestering and opsonizing them for phagocytosis in the spleen, these antibodies may contribute to the clearance of ring-stage parasites. Abbreviation: DHA, dihydroartemisinin.

Figure 2.

In vivo and ex vivo responses of parasites to artemisinin derivatives. A, Distribution of parasite clearance half-lives in 215 children with uncomplicated Plasmodium falciparum malaria in Kenieroba, Mali, compared with that of 168 (mostly adult) patients in Pursat, western Cambodia. The geometric mean half-life in Pursat (5.9 hours) is 3.0 times longer (95% confidence interval [CI], 2.8–3.3; P < .0001) than in Kenieroba (1.9 hours). The geometric mean parasite clearance half-life 95% CIs are indicated by horizontal lines and whiskers. B, Ex vivo responses of P. falciparum isolates to antimalarial drugs. The geometric mean (GM) IC₅₀ values for chloroquine (CQ; n = 87), amodiaquine (AQ; n = 41), monodesethylamodiaquine (MDAQ; n = 57), quinine (QN; n = 27), artesunate (AS; n = 96), and dihydroartemisinin (DHA; n = 87) were 119 nM, 7.79 nM, 16.7 nM, 49.9 nM, 1.17 nM, and 1.50 nM, respectively. The GM IC₅₀ and 95% CIs are indicated by horizontal lines and whiskers. This assay showed that approximately 50% of parasite isolates have reduced susceptibility to CQ (IC₅₀ > 100 nM) but remain susceptible to the other antimalarial drugs tested. These data are consistent with the only other in vitro responses of parasite isolates reported from Mali [36]. The IC₅₀ values for CQ correlated with those for AQ (r = 0.608; P = .0008) and MDAQ (r = 0.707; P < .0001). The only other IC₅₀ correlation among the 6 drugs was that observed for QN and AS (r = 0.585; P = .002). Abbreviations: AQ, amodiaquine; AS, artesunate; CQ, chloroquine; DHA, dihydroartemisinin; IC₅₀, median inhibitory concentration; MDAQ, monodesethylamodiaquine; QN, quinine.

Figure 3.

Influence of host factors on parasite clearance half-life. A, Results from a linear model in which the response is half-life in hours and the independent variables are age, sex (female reference), ethnicity (non-Fulani reference), hemoglobin (Hb) AC, HbAS, G6PD deficiency, α-thalassemia, and log_e-transformed initial parasite density. Each effect is given as the predicted change in half-life while holding all other variables constant. Hemoglobinopathy effects compare heterozygous with wild type (with homozygous and hemizygous having double effects), whereas age and ln (Pf day 0) are compared with adding 1 unit to that variable. Without correcting for multiple comparisons, we see that only the age effect is significant. The age parameter states that for every 1-year increase in age, the half-life changes by −0.0686 hours (95% confidence interval [CI], −.0946 to −.0425). The parameters with 95% CIs (uncorrected for multiple comparisons) are plotted. B, Half-life is inversely correlated with age (r = −0.312; P < .0001), a surrogate of naturally acquired immunity in our study population. The linear model estimates that there is a 4.1-minute (95% CI, 2.6–5.7; P < .0001) shortening of half-life for every 1-year increase in age. Abbreviations: Hb, hemoglobin; HE, heterozygote; Pf day 0, initial P. falciparum density.

Figure 4.

Autologous immunoglobulin G (IgG) responses against the surface of Plasmodium falciparum–infected red blood cells (RBCs) obtained directly from Malian children with malaria and cultured to the trophozoite-stage. A, Correlation between screening and initial parasite densities. Although screening and initial parasite densities correlated (r = 0.736; P < .0001), the parasite densities of many children dropped before the first artesunate dose was taken (data points below the line of equality). This finding may be due to the often-synchronous sequestration of late ring-stage parasitized RBCs in microvessels [26] and opsonic removal of parasitized RBCs, both of which may decrease half-life. B, Representative flow cytometry scatterplot showing that 20.6% of parasitized RBCs in this child were recognized by autologous IgG. C, The median proportions of IgG-positive parasitized RBCs were 21.0% (interquartile range [IQR], 11.7–37.2), 10.8% (IQR, 6.92–23.7), and 5.94% (IQR, 4.04–17.6) for plasma tested at 1:10 (n = 48), 1:20 (n = 48), and 1:40 (n = 22) dilutions, respectively. For each dilution doubling, the proportion of IgG-positive parasitized RBCs changed on average by a factor of 0.61 (95% CI, .54–.69; P < .0001). D, The plot shows an inverse correlation between IgG responses and half-life (r = −0.475; P = .0006), suggesting they are involved in clearing ring-stage parasites from peripheral blood. Abbreviations: IgG, immunoglobulin; FL1-H, fluorescence channel 1-height; FL2-H, fluorescence channel 2-height.