Kamolrat Silamut

Artemisinin Resistance in Plasmodium falciparum Malaria

BACKGROUND Artemisinin-based combination therapies are the recommended first-line treatments of falciparum malaria in all countries with endemic disease. There are recent concerns that the efficacy of such therapies has declined on the Thai-Cambodian border, historically a site of emerging antimalarial-drug resistance. METHODS In two open-label, randomized trials, we compared the efficacies of two treatments for uncomplicated falciparum malaria in Pailin, western Cambodia, and Wang Pha, northwestern Thailand: oral artesunate given at a dose of 2 mg per kilogram of body weight per day, for 7 days, and artesunate given at a dose of 4 mg per kilogram per day, for 3 days, followed by mefloquine at two doses totaling 25 mg per kilogram. We assessed in vitro and in vivo Plasmodium falciparum susceptibility, artesunate pharmacokinetics, and molecular markers of resistance. RESULTS We studied 40 patients in each of the two locations. The overall median parasite clearance times were 84 hours (interquartile range, 60 to 96) in Pailin and 48 hours (interquartile range, 36 to 66) in Wang Pha (P<0.001). Recrudescence confirmed by means of polymerase-chain-reaction assay occurred in 6 of 20 patients (30%) receiving artesunate monotherapy and 1 of 20 (5%) receiving artesunate-mefloquine therapy in Pailin, as compared with 2 of 20 (10%) and 1 of 20 (5%), respectively, in Wang Pha (P=0.31). These markedly different parasitologic responses were not explained by differences in age, artesunate or dihydroartemisinin pharmacokinetics, results of isotopic in vitro sensitivity tests, or putative molecular correlates of P. falciparum drug resistance (mutations or amplifications of the gene encoding a multidrug resistance protein [PfMDR1] or mutations in the gene encoding sarco-endoplasmic reticulum calcium ATPase6 [PfSERCA]). Adverse events were mild and did not differ significantly between the two treatment groups. CONCLUSIONS P. falciparum has reduced in vivo susceptibility to artesunate in western Cambodia as compared with northwestern Thailand. Resistance is characterized by slow parasite clearance in vivo without corresponding reductions on conventional in vitro susceptibility testing. Containment measures are urgently needed. (ClinicalTrials.gov number, NCT00493363, and Current Controlled Trials number, ISRCTN64835265.)

Artesunate versus quinine in the treatment of severe falciparum malaria in African children (AQUAMAT): an open-label, randomised trial.

Summary Background Severe malaria is a major cause of childhood death and often the main reason for paediatric hospital admission in sub-Saharan Africa. Quinine is still the established treatment of choice, although evidence from Asia suggests that artesunate is associated with a lower mortality. We compared parenteral treatment with either artesunate or quinine in African children with severe malaria. Methods This open-label, randomised trial was undertaken in 11 centres in nine African countries. Children (<15 years) with severe falciparum malaria were randomly assigned to parenteral artesunate or parenteral quinine. Randomisation was in blocks of 20, with study numbers corresponding to treatment allocations kept inside opaque sealed paper envelopes. The trial was open label at each site, and none of the investigators or trialists, apart from for the trial statistician, had access to the summaries of treatment allocations. The primary outcome measure was in-hospital mortality, analysed by intention to treat. This trial is registered, number ISRCTN50258054. Findings 5425 children were enrolled; 2712 were assigned to artesunate and 2713 to quinine. All patients were analysed for the primary outcome. 230 (8·5%) patients assigned to artesunate treatment died compared with 297 (10·9%) assigned to quinine treatment (odds ratio [OR] stratified for study site 0·75, 95% CI 0·63–0·90; relative reduction 22·5%, 95% CI 8·1–36·9; p=0·0022). Incidence of neurological sequelae did not differ significantly between groups, but the development of coma (65/1832 [3·5%] with artesunate vs 91/1768 [5·1%] with quinine; OR 0·69 95% CI 0·49–0·95; p=0·0231), convulsions (224/2712 [8·3%] vs 273/2713 [10·1%]; OR 0·80, 0·66–0·97; p=0·0199), and deterioration of the coma score (166/2712 [6·1%] vs 208/2713 [7·7%]; OR 0·78, 0·64–0·97; p=0·0245) were all significantly less frequent in artesunate recipients than in quinine recipients. Post-treatment hypoglycaemia was also less frequent in patients assigned to artesunate than in those assigned to quinine (48/2712 [1·8%] vs 75/2713 [2·8%]; OR 0·63, 0·43–0·91; p=0·0134). Artesunate was well tolerated, with no serious drug-related adverse effects. Interpretation Artesunate substantially reduces mortality in African children with severe malaria. These data, together with a meta-analysis of all trials comparing artesunate and quinine, strongly suggest that parenteral artesunate should replace quinine as the treatment of choice for severe falciparum malaria worldwide. Funding The Wellcome Trust.

A Quantitative Analysis of the Microvascular Sequestration of Malaria Parasites in the Human Brain

Microvascular sequestration was assessed in the brains of 50 Thai and Vietnamese patients who died from severe malaria (Plasmodium falciparum, 49; P. vivax, 1). Malaria parasites were sequestered in 46 cases; in 3 intravascular malaria pigment but no parasites were evident; and in the P. vivax case there was no sequestration. Cerebrovascular endothelial expression of the putative cytoadherence receptors ICAM-1, VCAM-1, E-selectin, and chondroitin sulfate and also HLA class II was increased. The median (range) ratio of cerebral to peripheral blood parasitemia was 40 (1.8 to 1500). Within the same brain different vessels had discrete but different populations of parasites, indicating that the adhesion characteristics of cerebrovascular endothelium change asynchronously during malaria and also that significant recirculation of parasitized erythrocytes following sequestration is unlikely. The median (range) ratio of schizonts to trophozoites (0.15:1; 0.0 to 11.7) was significantly lower than predicted from the parasite life cycle (P < 0.001). Antimalarial treatment arrests development at the trophozoite stages which remain sequestered in the brain. There were significantly more ring form parasites (age < 26 hours) in the cerebral microvasculature (median range: 19%; 0-90%) than expected from free mixing of these cells in the systemic circulation (median range ring parasitemia: 1.8%; 0-36.2%). All developmental stages of P. falciparum are sequestered in the brain in severe malaria.

Estimation of the Total Parasite Biomass in Acute Falciparum Malaria from Plasma PfHRP2

Background In falciparum malaria sequestration of erythrocytes containing mature forms of Plasmodium falciparum in the microvasculature of vital organs is central to pathology, but quantitation of this hidden sequestered parasite load in vivo has not previously been possible. The peripheral blood parasite count measures only the circulating, relatively non-pathogenic parasite numbers. P. falciparum releases a specific histidine-rich protein (PfHRP2) into plasma. Quantitative measurement of plasma PfHRP2 concentrations may reflect the total parasite biomass in falciparum malaria. Methods and Findings We measured plasma concentrations of PfHRP2, using a quantitative antigen-capture enzyme-linked immunosorbent assay, in 337 adult patients with falciparum malaria of varying severity hospitalised on the Thai–Burmese border. Based on in vitro production rates, we constructed a model to link this measure to the total parasite burden in the patient. The estimated geometric mean parasite burden was 7 × 1011 (95% confidence interval [CI] 5.8 × 1011 to 8.5 × 1011) parasites per body, and was over six times higher in severe malaria (geometric mean 1.7 × 1012, 95% CI 1.3 × 1012 to 2.3 × 1012) than in patients hospitalised without signs of severity (geometric mean 2.8 × 1011, 95% CI 2.3 × 1011 to 3.5 × 1011; p < 0.001). Parasite burden was highest in patients who died (geometric mean 3.4 × 1012, 95% CI 1.9 × 1012 to 6.3 × 1012; p = 0.03). The calculated number of sequestered parasites increased with disease severity and was higher in patients with late developmental stages of P. falciparum present on peripheral blood smears. Comparing model and laboratory estimates of the time of sequestration suggested that admission to hospital with uncomplicated malaria often follows schizogony—but in severe malaria is unrelated to stage of parasite development. Conclusion Plasma PfHRP2 concentrations may be used to estimate the total body parasite biomass in acute falciparum malaria. Severe malaria results from extensive sequestration of parasitised erythrocytes.

Relation of the stage of parasite development in the peripheral blood to prognosis in severe falciparum malaria

Admission blood films from 72 patients who died of severe falciparum malaria (50 Thai adults, 22 Gambian children) were matched retrospectively for parasitaemia with equal numbers of survivors. The peripheral blood parasites from fatal cases were more mature than those from survivors. Tiny rings (TR) comprised > 50% of parasites in 47/72 (65%) survivors but only 12/72 (17%) of fatal cases (P < 0.001). Parasites containing visible pigment (MTS: mature trophozoites and schizonts) comprised < 20% of the total parasite count in 10/72 (14%) survivors compared with 31/72 (43%) fatal cases (P < 0.001). Of the 39 patients with > 10(4) MTS/microL, 30 (81%) died. These findings were confirmed in a prospective study of 279 adult Thai patients admitted sequentially with acute falciparum malaria. Only 4 of the 19 fatal cases (21%) had > 50% TR, compared with 130 of 260 (50%) survivors, whereas > 20% MTS were found in 10/19 (53%) fatal cases, compared with 28/108 (27%) severe malaria survivors, and 26/155 (17%) patients with moderately severe malaria (P = 0.001). As a predictor of fatal outcome, the finding of either > 10(4) MTS/microL or > 5 x 10(5) parasites/microL in severe malaria had a sensitivity of 90% (95% confidence interval [CI] = 75-97%) and a specificity of 72% (95% CI = 59-86%). These observations are consistent with the hypothesis that a predominance of mature parasites in the peripheral blood reflects a greater sequestered biomass, and thus more severe disease. Simple microscopical assessment of parasite maturity on an admission blood slide provides important pathophysiological and prognostic information in severe falciparum malaria.

The Deformability of Red Blood Cells Parasitized by Plasmodium falciparum and P. vivax

Red blood cells (RBCs) must deform considerably during their multiple passages through the microvasculature and the sinusoids of the spleen. RBCs infected with Plasmodium falciparum (Pf-IRBCs) become increasingly rigid as they mature but avoid splenic clearance by sequestering in venules and capillaries. In contrast, RBCs infected with P. vivax (Pv-IRBCs) do not sequester. We compared the effects of P. vivax and P. falciparum infection on RBC deformability in a laminar shear flow system. Pf-IRBCs became more rigid as the parasite matured, but equivalent maturation of Pv-IRBCs resulted in a doubling of flexibility. Coincidentally, the IRBC surface area increased from 56.7+/-1.3 microm2 to 74.7+/-0.6 microm2 to 90.9+/-1.1 microm2 in ring-, trophozoite-, and schizont-stage Pv-IRBCs, respectively, whereas Pf-IRBCs did not increase in size. P. vivax increases the deformability of IRBCs and thereby avoids splenic entrapment.

Predicting the Clinical Outcome of Severe Falciparum Malaria in African Children: Findings From a Large Randomized Trial

Four predictors were independently associated with an increased risk of death: acidosis, cerebral manifestations of malaria, elevated blood urea nitrogen, or signs of chronic illness. The standard base deficit was found to be the single most relevant predictor of death.

Randomized comparison of artesunate and quinine in the treatment of severe falciparum malaria.

A randomized, open-label comparison of artesunate and quinine was conducted in 113 adults with clinically severe falciparum malaria in western Thailand. Mortality was 12% with artesunate and 22% with quinine treatment (relative risk, 0.53; 95% confidence interval, 0.23-1.26; P=.22). Multiple logistic regression analysis found admission plasma lactate level, Glasgow Coma Scale score, and total serum bilirubin level to be independent risk factors for death. Coma recovery and times to normalize plasma lactate levels were similar, but the parasite clearance time was much shorter among artesunate-treated patients (P=.019). Fewer patients became hypoglycemic during artesunate therapy (10%) than during quinine therapy (28%) (P=.03). Artesunate is at least as effective as quinine in the treatment of adults with severe malaria. Larger trials are required to determine whether mortality is reduced among patients treated with artesunate.

Febrile temperatures induce cytoadherence of ring-stage Plasmodium falciparum-infected erythrocytes

In falciparum malaria, the malaria parasite induces changes at the infected red blood cell surface that lead to adherence to vascular endothelium and other red blood cells. As a result, the more mature stages of Plasmodium falciparum are sequestered in the microvasculature and cause vital organ dysfunction, whereas the ring stages circulate in the blood stream. Malaria is characterized by fever. We have studied the effect of febrile temperatures on the cytoadherence in vitro of P. falciparum-infected erythrocytes. Freshly obtained ring-stage-infected red blood cells from 10 patients with acute falciparum malaria did not adhere to the principle vascular adherence receptors CD36 or intercellular adhesion molecule-1 (ICAM-1). However, after a brief period of heating to 40°C, all ring-infected red blood cells adhered to CD36, and some isolates adhered to ICAM-1, whereas controls incubated at 37°C did not. Heating to 40°C accelerated cytoadherence and doubled the maximum cytoadherence observed (P < 0.01). Erythrocytes infected by ring-stages of the ICAM-1 binding clone A4var also did not cytoadhere at 37°C, but after heating to febrile temperatures bound to both CD36 and ICAM-1. Adherence of red blood cells infected with trophozoites was also increased considerably by brief heating. The factor responsible for heat induced adherence was shown to be the parasite derived variant surface protein PfEMP-1. RNA analysis showed that levels of var mRNA did not differ between heated and unheated ring-stage parasites. Thus fever-induced adherence appeared to involve increased trafficking of PfEMP-1 to the erythrocyte membrane. Fever induced cytoadherence is likely to have important pathological consequences and may explain both clinical deterioration with fever in severe malaria and the effects of antipyretics on parasite clearance.

Erythrocyte survival in severe falciparum malaria

Erythrocyte survival was studied in 17 Thai patients (10 males, 7 females; aged 13-57 years) with severe falciparum malaria. To ensure radioisotopic labelling of cells before bone marrow recovery and survival analysis under near-steady state conditions, 51Cr labelling of autologous erythrocytes was performed at the time of admission (0 h) and calculation of mean cell lifespan (MCL) was based on semilogarithmic plots of corrected counts from 60 h onwards. Five patients received blood transfusions, all within 48 h of admission. The overall mean (+/- S.D.) MCL was short (44.1 +/- 21.7 days). Nontransfused patients had similar MCL values (43.6 +/- 20.4) to those of transfused patients (45.5 +/- 27.3 days, p greater than 0.8). Patients with and without palpable splenomegaly had MCL values which were not significantly different (54.1 +/- 28.8 vs. 37.2 +/- 12.3 days respectively, p greater than 0.1). There was no association between admission haematocrit or peripheral parasitaemia and MCL (p greater than 0.2 in each case), but there was an inverse correlation between total serum bilirubin and MCL (r = -0.49, p less than 0.025). There is accelerated destruction of non-parasitised erythrocytes in severe malaria resulting in a mean MCL that is half that found previously in healthy Thai volunteers (89.6 +/- 13.1 days, p less than 0.001) and significantly shorter than that reported previously in Thai patients with uncomplicated P. falciparum infections studied after parasite clearance (56.8 +/- 10.2 days, p less than 0.05).