Diagnosing Severe Falciparum Malaria in Parasitaemic African Children: A Prospective Evaluation of Plasma PfHRP2 Measurement.

In African children, distinguishing severe falciparum malaria from other severe febrile illnesses with coincidental Plasmodium falciparum parasitaemia is a major challenge. P. falciparum histidine-rich protein 2 (PfHRP2) is released by mature sequestered parasites and can be used to estimate the total parasite burden. We investigated the prognostic significance of plasma PfHRP2 and used it to estimate the malaria-attributable fraction in African children diagnosed with severe malaria. Admission plasma PfHRP2 was measured prospectively in African children (from Mozambique, The Gambia, Kenya, Tanzania, Uganda, Rwanda, and the Democratic Republic of the Congo) aged 1 month to 15 years with severe febrile illness and a positive P. falciparum lactate dehydrogenase (pLDH)-based rapid test in a clinical trial comparing parenteral artesunate versus quinine (the AQUAMAT trial, ISRCTN 50258054). In 3,826 severely ill children, Plasmadium falciparum PfHRP2 was higher in patients with coma (p = 0.0209), acidosis (p<0.0001), and severe anaemia (p<0.0001). Admission geometric mean (95%CI) plasma PfHRP2 was 1,611 (1,350-1,922) ng/mL in fatal cases (n = 381) versus 1,046 (991-1,104) ng/mL in survivors (n = 3,445, p<0.0001), without differences in parasitaemia as assessed by microscopy. There was a U-shaped association between log(10) plasma PfHRP2 and risk of death. Mortality increased 20% per log(10) increase in PfHRP2 above 174 ng/mL (adjusted odds ratio [AOR] 1.21, 95%CI 1.05-1.39, p = 0.009). A mechanistic model assuming a PfHRP2-independent risk of death in non-malaria illness closely fitted the observed data and showed malaria-attributable mortality less than 50% with plasma PfHRP2≤174 ng/mL. The odds ratio (OR) for death in artesunate versus quinine-treated patients was 0.61 (95%CI 0.44-0.83, p = 0.0018) in the highest PfHRP2 tertile, whereas there was no difference in the lowest tertile (OR 1.05; 95%CI 0.69-1.61; p = 0.82). A limitation of the study is that some conclusions are drawn from a mechanistic model, which is inherently dependent on certain assumptions. However, a sensitivity analysis of the model indicated that the results were robust to a plausible range of parameter estimates. Further studies are needed to validate our findings. Plasma PfHRP2 has prognostic significance in African children with severe falciparum malaria and provides a tool to stratify the risk of "true" severe malaria-attributable disease as opposed to other severe illnesses in parasitaemic African children

Antibodies in children with malaria to PfEMP1, RIFIN and SURFIN expressed at the Plasmodium falciparum parasitized red blood cell surface

Abstract Naturally acquired antibodies to proteins expressed on the Plasmodium falciparum parasitized red blood cell (pRBC) surface steer the course of a malaria infection by reducing sequestration and stimulating phagocytosis of pRBC. Here we have studied a selection of proteins representing three different parasite gene families employing a well-characterized parasite with a severe malaria phenotype (FCR3S1.2). The presence of naturally acquired antibodies, impact on rosetting rate, surface reactivity and opsonization for phagocytosis in relation to different blood groups of the ABO system were assessed in a set of sera from children with mild or complicated malaria from an endemic area. We show that the naturally acquired immune responses, developed during malaria natural infection, have limited access to the pRBCs inside a blood group A rosette. The data also indicate that SURFIN4.2 may have a function at the pRBC surface, particularly during rosette formation, this role however needs to be further validated. Our results also indicate epitopes differentially recognized by rosette-disrupting antibodies on a peptide array. Antibodies towards parasite-derived proteins such as PfEMP1, RIFIN and SURFIN in combination with host factors, essentially the ABO blood group of a malaria patient, are suggested to determine the outcome of a malaria infection

Effects of sevuparin on rosette formation and cytoadherence of Plasmodium falciparum infected erythrocytes.

In severe falciparum malaria cytoadherence of parasitised red blood cells (PRBCs) to vascular endothelium (causing sequestration) and to uninfected red cells (causing rosette formation) contribute to microcirculatory flow obstruction in vital organs. Heparin can reverse the underlying ligand-receptor interactions, but may increase the bleeding risks. As a heparin-derived polysaccharide, sevuparin has been designed to retain anti-adhesive properties, while the antithrombin-binding domains have been eliminated, substantially diminishing its anticoagulant activity. Sevuparin has been evaluated recently in patients with uncomplicated falciparum malaria, and is currently investigated in a clinical trial for sickle cell disease. The effects of sevuparin on rosette formation and cytoadherence of Plasmodium falciparum isolates from Thailand were investigated. Trophozoite stages of P. falciparum-infected RBCs (Pf-iRBCs) were cultured from 49 patients with malaria. Pf-iRBCs were treated with sevuparin at 37°C and assessed in rosetting and in cytoadhesion assays with human dermal microvascular endothelial cells (HDMECs) under static and flow conditions. The proportion of Pf-iRBCs forming rosettes ranged from 6.5% to 26.0% (median = 12.2%). Rosetting was dose dependently disrupted by sevuparin (50% disruption by 250 μg/mL). Overall 57% of P. falciparum isolates bound to HDMECs under static conditions; median (interquartile range) Pf-iRBC binding was 8.5 (3.0-38.0) Pf-iRBCs/1000 HDMECs. Sevuparin in concentrations ≥ 100 μg/mL inhibited cytoadherence. Sevuparin disrupts P. falciparum rosette formation in a dose dependent manner and inhibits cytoadherence to endothelial cells. The data support assessment of sevuparin as an adjunctive treatment to the standard therapy in severe falciparum malaria

Median (interquartile range) Pf-iRBCs adhering to HDMECs under shear stress at 0.05 Pa, 0.5% parasitaemia and 1% haematocrit at different concentrations of sevuparin (n = 10).

<p>Median (interquartile range) Pf-iRBCs adhering to HDMECs under shear stress at 0.05 Pa, 0.5% parasitaemia and 1% haematocrit at different concentrations of sevuparin (n = 10).</p

Effects of sevuparin on rosette formation and cytoadherence of <i>Plasmodium falciparum</i> infected erythrocytes - Fig 2

<p>(A) The effects of sevuparin on Pf-iRBC adherence (n = 28). Median (interquartile range) adherence number of Pf-iRBCs binding per 1000 HDMECs at each concentration of sevuparin. (B) The inhibition of sevuparin on cytoadherence of <i>P</i>. <i>falciparum</i> (n = 28). Median (interquartile range) inhibition effect on cytoadhesion. The cytoadherence of patient isolates was significantly inhibited by sevuparin at concentrations ≥ 100 μg/mL (all p < 0.05).</p

The correlation between the adherent number of Pf-iRBCs binding per 1000 HDMECs and rosetting number (%) of <i>P</i>. <i>falciparum</i> (n = 28), (r_s = 0.152, p = 0.439).

<p>The correlation between the adherent number of Pf-iRBCs binding per 1000 HDMECs and rosetting number (%) of <i>P</i>. <i>falciparum</i> (n = 28), (r_s = 0.152, p = 0.439).</p

Effects of sevuparin on rosette formation and cytoadherence of <i>Plasmodium falciparum</i> infected erythrocytes - Fig 1

<p>(A) Effect of sevuparin on rosetting (%) of <i>P</i>. <i>falciparum</i> (n = 47) is dose dependent. The data show the median with interquartile range of rosettes formed at each concentration of sevuparin. (B) Median (interquartile range) % disruption of rosetting at each concentration of sevuparin. Sevuparin significantly disrupted rosette formation, p < 0.001.</p

Effects of sevuparin on rosette formation and cytoadherence of <i>Plasmodium falciparum</i> infected erythrocytes

<div><p>In severe <i>falciparum</i> malaria cytoadherence of parasitised red blood cells (PRBCs) to vascular endothelium (causing sequestration) and to uninfected red cells (causing rosette formation) contribute to microcirculatory flow obstruction in vital organs. Heparin can reverse the underlying ligand-receptor interactions, but may increase the bleeding risks. As a heparin-derived polysaccharide, sevuparin has been designed to retain anti-adhesive properties, while the antithrombin-binding domains have been eliminated, substantially diminishing its anticoagulant activity. Sevuparin has been evaluated recently in patients with uncomplicated <i>falciparum</i> malaria, and is currently investigated in a clinical trial for sickle cell disease. The effects of sevuparin on rosette formation and cytoadherence of <i>Plasmodium falciparum</i> isolates from Thailand were investigated. Trophozoite stages of <i>P</i>. <i>falciparum</i>-infected RBCs (Pf-iRBCs) were cultured from 49 patients with malaria. Pf-iRBCs were treated with sevuparin at 37°C and assessed in rosetting and in cytoadhesion assays with human dermal microvascular endothelial cells (HDMECs) under static and flow conditions. The proportion of Pf-iRBCs forming rosettes ranged from 6.5% to 26.0% (median = 12.2%). Rosetting was dose dependently disrupted by sevuparin (50% disruption by 250 μg/mL). Overall 57% of <i>P</i>. <i>falciparum</i> isolates bound to HDMECs under static conditions; median (interquartile range) Pf-iRBC binding was 8.5 (3.0–38.0) Pf-iRBCs/1000 HDMECs. Sevuparin in concentrations ≥ 100 μg/mL inhibited cytoadherence. Sevuparin disrupts <i>P</i>. <i>falciparum</i> rosette formation in a dose dependent manner and inhibits cytoadherence to endothelial cells. The data support assessment of sevuparin as an adjunctive treatment to the standard therapy in severe <i>falciparum</i> malaria.</p></div

Demographic, clinical, and laboratory characteristics of children diagnosed with severe falciparum malaria according to outcome.

<p>BCS, Blantyre coma scale; BE, base excess; GCS, Glasgow coma scale, Hb, haemoglobin.</p

Plasma PfHRP2 according to clinical and laboratory features of severe malaria.

<p>BCS, Blantyre coma scale; BE, base excess; GCS, Glasgow coma scale, Hb, haemoglobin.</p>a<p>Data are geometric mean (95% CI).</p>b<p>BE available for n = 3,075 and Hb available for n = 3,264 due to missing i-STAT values.</p>c<p>Compensated and decompensated shock combined.</p