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Primaquine, the only widely available treatment to prevent relapsing Plasmodium vivaxmalaria, is produced as 15 mg tablets, and new paediatric formulations are being developed. To inform the optimal primaquine dosing regimen for children, we aimed to determine the efficacy and safety of different primaquine dose strategies in children younger than 15 years.

Platelets are understood to assist host innate immune responses against infection, although direct evidence of this function in any human disease, including malaria, is unknown. Here we characterized platelet–erythrocyte interactions by microscopy and flow cytometry in patients with malaria naturally infected with Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, or Plasmodium knowlesi. Blood samples from 376 participants were collected from malaria-endemic areas of Papua, Indonesia, and Sabah, Malaysia. Platelets were observed binding directly with and killing intraerythrocytic parasites of each of the Plasmodium species studied, particularly mature stages, and was greatest in P vivax patients. Platelets preferentially bound to the infected more than to the uninfected erythrocytes in the bloodstream. Analysis of intraerythrocytic parasites indicated the frequent occurrence of platelet-associated parasite killing, characterized by the intraerythrocytic accumulation of platelet factor-4 and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling of parasite nuclei (PF4+TUNEL+ parasites). These PF4+TUNEL+ parasites were not associated with measures of systemic platelet activation. Importantly, patient platelet counts, infected erythrocyte-platelet complexes, and platelet-associated parasite killing correlated inversely with patient parasite loads. These relationships, taken together with the frequency of platelet-associated parasite killing observed among the different patients and Plasmodium species, suggest that platelets may control the growth of between 5% and 60% of circulating parasites. Platelet–erythrocyte complexes made up a major proportion of the total platelet pool in patients with malaria and may therefore contribute considerably to malarial thrombocytopenia. Parasite killing was demonstrated to be platelet factor-4-mediated in P knowlesi culture. Collectively, our results indicate that platelets directly contribute to innate control of Plasmodium infection in human malaria.

Platelets are understood to assist host innate immune responses against infection, although direct evidence of this function in any human disease, including malaria, is unknown. Here we characterized platelet–erythrocyte interactions by microscopy and flow cytometry in patients with malaria naturally infected with Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, or Plasmodium knowlesi. Blood samples from 376 participants were collected from malaria-endemic areas of Papua, Indonesia, and Sabah, Malaysia. Platelets were observed binding directly with and killing intraerythrocytic parasites of each of the Plasmodiumspecies studied, particularly mature stages, and was greatest in P vivaxpatients. Platelets preferentially bound to the infected more than to the uninfected erythrocytes in the bloodstream. Analysis of intraerythrocytic parasites indicated the frequent occurrence of platelet-associated parasite killing, characterized by the intraerythrocytic accumulation of platelet factor-4 and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling of parasite nuclei (PF4+TUNEL+parasites). These PF4+TUNEL+parasites were not associated with measures of systemic platelet activation. Importantly, patient platelet counts, infected erythrocyte-platelet complexes, and platelet-associated parasite killing correlated inversely with patient parasite loads. These relationships, taken together with the frequency of platelet-associated parasite killing observed among the different patients and Plasmodiumspecies, suggest that platelets may control the growth of between 5% and 60% of circulating parasites. Platelet–erythrocyte complexes made up a major proportion of the total platelet pool in patients with malaria and may therefore contribute considerably to malarial thrombocytopenia. Parasite killing was demonstrated to be platelet factor-4-mediated in P knowlesiculture. Collectively, our results indicate that platelets directly contribute to innate control of Plasmodiuminfection in human malaria.

The simian parasite Plasmodium knowlesi can cause severe and fatal human malaria. However, little is known about the pathogenesis of this disease. In falciparum malaria, reduced red blood cell deformability (RBC-D) contributes to microvascular obstruction and impaired organ perfusion. In P knowlesi infection, impaired microcirculatory flow has been observed in Macaca mulatta (rhesus macaques), unnatural hosts who develop severe and fatal disease. However, RBC-D has not been measured in human infection or in the natural host M fascicularis (long-tailed macaques). Using ektacytometry, we measured RBC-D in adults with severe and non-severe knowlesi and falciparum malaria and in healthy controls. In addition, we used micropipette aspiration to determine the relative stiffness of infected RBCs (iRBCs) and uninfected RBCs (uRBCs) in P knowlesi–infected humans and M fascicularis. Ektacytometry demonstrated that RBC-D overall was reduced in human knowlesi malaria in proportion to disease severity, and in severe knowlesi malaria, it was comparable to that of severe falciparum malaria. RBC-D correlated inversely with parasitemia and lactate in knowlesi malaria and HRP2 in falciparum malaria, and it correlated with hemoglobin nadir in knowlesi malaria. Micropipette aspiration confirmed that in humans, P knowlesi infection increased stiffness of both iRBCs and uRBCs, with the latter mostly the result of echinocytosis. In contrast, in the natural host M fascicularis, echinocyte formation was not observed, and the RBC-D of uRBCs was unaffected. In unnatural primate hosts of P knowlesi, including humans, reduced deformability of iRBCs and uRBCs may represent a key pathogenic mechanism leading to microvascular accumulation, impaired organ perfusion, and anemia.

The emergence of human malaria due to the monkey parasite Plasmodium knowlesithreatens elimination efforts in southeast Asia. Changes in land use are thought to be driving the rise in reported P knowlesicases, but the role of individual-level factors is unclear. To address this knowledge gap we assessed human and environmental factors associated with zoonotic knowlesi malaria risk.

Serological markers are a promising tool for surveillance and targeted interventions for Plasmodium vivaxmalaria. P. vivaxis closely related to the zoonotic parasite P. knowlesi, which also infects humans. P. vivaxand P. knowlesiare co-endemic across much of South East Asia, making it important to design serological markers that minimize cross-reactivity in this region. To determine the degree of IgG cross-reactivity against a panel of P. vivaxserological markers, we assayed samples from human patients with P. knowlesimalaria. IgG antibody reactivity is high against P. vivaxproteins with high sequence identity with their P. knowlesiortholog. IgG reactivity peaks at 7 days post-P. knowlesiinfection and is short-lived, with minimal responses 1 year post-infection. We designed a panel of eight P. vivaxproteins with low levels of cross-reactivity with P. knowlesi. This panel can accurately classify recent P. vivaxinfections while reducing misclassification of recent P. knowlesiinfections.

ABSTRACTPlasmodium knowlesicauses severe and fatal malaria in Malaysia. Microscopic misdiagnosis is common and may delay appropriate treatment. P. knowlesican cross-react with “species-specific” parasite lactate dehydrogenase (pLDH) monoclonal antibodies used in rapid diagnostic tests (RDTs) to detect P. falciparumand P. vivax. At one tertiary-care hospital and two district hospitals in Sabah, we prospectively evaluated two combination RDTs for malaria diagnosis by using both a pan-Plasmodium-pLDH (pan-pLDH)/P. falciparum-specific-pLDH (Pf-pLDH) RDT (OptiMAL-IT) and a non-P. falciparumVOM-pLDH/Pf-HRP2 RDT (CareStart). Differential cross-reactivity among these combinations was hypothesized to differentiate P. knowlesifrom other Plasmodiummonoinfections. Among 323 patients with PCR-confirmed P. knowlesi(n= 193), P. falciparum(n= 93), and P. vivax(n= 37) monoinfections, the VOM-pLDH individual component had the highest sensitivity for nonsevere (35%; 95% confidence interval [CI], 27 to 43%) and severe (92%; CI, 81 to 100%) P. knowlesimalaria. CareStart demonstrated a P. knowlesisensitivity of 42% (CI, 34 to 49%) and specificity of 74% (CI, 65 to 82%), a P. vivaxsensitivity of 83% (CI, 66 to 93%) and specificity of 71% (CI, 65 to 76%), and a P. falciparumsensitivity of 97% (CI, 90 to 99%) and specificity of 99% (CI, 97 to 100%). OptiMAL-IT demonstrated a P. knowlesisensitivity of 32% (CI, 25 to 39%) and specificity of 21% (CI, 15 to 29%), a P. vivaxsensitivity of 60% (CI, 42 to 75%) and specificity of 97% (CI, 94 to 99%), and a P. falciparumsensitivity of 82% (CI, 72 to 89%) and specificity of 39% (CI, 33 to 46%). The combination of CareStart plus OptiMAL-IT for P. knowlesiusing predefined criteria gave a sensitivity of 25% (CI, 19 to 32%) and specificity of 97% (CI, 92 to 99%). Combining two RDT combinations was highly specific for P. knowlesimalaria diagnosis; however, sensitivity was poor. The specificity of pLDH RDTs was decreased for P. vivaxand P. falciparumbecause of P. knowlesicross-reactivity and cautions against their use alone in areas where P. knowlesimalaria is endemic. Sensitive P. knowlesi-specific RDTs and/or alternative molecular diagnostic tools are needed in areas where P. knowlesimalaria is endemic.

ABSTRACTPlasmodium knowlesican cause severe and fatal human malaria in Southeast Asia. Rapid diagnosis of all Plasmodiumspecies is essential for initiation of effective treatment. Rapid diagnostic tests (RDTs) are sensitive for detection of uncomplicated and severe falciparum malaria but have not been systematically evaluated in knowlesi malaria. At a tertiary referral hospital in Sabah, Malaysia, we prospectively evaluated the sensitivity of two combination RDTs for the diagnosis of uncomplicated and severe malaria from all three potentially fatal Plasmodiumspecies, using a pan-Plasmodiumlactate dehydrogenase (pLDH)-P. falciparumhistidine-rich protein 2 (PfHRP2) RDT (First Response) and a pan-Plasmodiumaldolase-PfHRP2 RDT (ParaHIT). Among 293 hospitalized adults with PCR-confirmed Plasmodiummonoinfection, the sensitivity of the pLDH component of the pLDH-PfHRP2 RDT was 74% (95/129; 95% confidence interval [CI], 65 to 80%), 91% (110/121; 95% CI, 84 to 95%), and 95% (41/43; 95% CI, 85 to 99%) for PCR-confirmed P. knowlesi, P. falciparum, and P. vivaxinfections, respectively, and 88% (30/34; 95% CI, 73 to 95%), 90% (38/42; 95% CI, 78 to 96%), and 100% (12/12; 95% CI, 76 to 100%) among patients tested before antimalarial treatment was begun. Sensitivity in severe malaria was 95% (36/38; 95% CI, 83 to 99), 100% (13/13; 95% CI, 77 to 100), and 100% (7/7; 95% CI, 65 to 100%), respectively. The aldolase component of the aldolase-PfHRP2 RDT performed poorly in all Plasmodiumspecies. The pLDH-based RDT was highly sensitive for the diagnosis of severe malaria from all species; however, neither the pLDH- nor aldolase-based RDT demonstrated sufficiently high overall sensitivity for P. knowlesi. More sensitive RDTs are needed in regions of P. knowlesiendemicity.