BackgroundTreatment failure of chloroquine for P. vivax infections has reached high levels in the eastern provinces of Indonesia, however, in vitro characterization of chloroquine resistance and its associated molecular profile have yet to be determined.MethodsUsing a modified schizont maturation assay we investigated the in vitro chloroquine susceptibility profile and molecular polymorphisms of P. vivax isolates collected from Papua, Indonesia, where high levels of clinical chloroquine treatment failure have been reported, and from Thailand, where chloroquine treatment is generally effective.ResultsThe geometric mean chloroquine IC50 for P. vivax isolates from Papua (n = 145) was 312 nM [95%CI: 237–411 nM] compared to 46.8 nM [95%CI: 34.7–63.1 nM] from Thailand (n = 81); p<0.001. Correlating with the known clinical efficacy of the area, a cut off for chloroquine resistance was defined as 220nM, a level exceeded in 13.6% (11/81) of Thai isolates and 65% (94/145) of Papuan isolates; p<0.001. Several sequence polymorphisms in pvcrt-o and pvmdr1, and difference in pvmdr1 copy number were identified. A Y976F mutation in pvmdr1 was present in 96% (123/128) of Papuan isolates and 25% (17/69) of Thai isolates; p<0.001. Overall, the geometric mean chloroquine IC50 in isolates with the Y976F mutation was 283 nM [95%CI: 211–379], compared to 44.5 nM [95%CI: 31.3–63.4] in isolates with the wild type; p< 0.001. Pvmdr1 amplification occurred in 23% (15/66) of Thai isolates compared to none (0/104) of Indonesian isolates (p<0.001), but was not associated with increased chloroquine resistance after controlling for geographical location.Conclusions In vitro susceptibility testing of P. vivax discriminates between populations with differing levels of clinical efficacy of chloroquine. The pvmdr1 polymorphism at Y976F may provide a useful tool to highlight areas of emerging chloroquine resistance, although further studies defining its clinical correlates are needed.

Adherence of parasitized erythrocytes to activated endothelium causes microvascular obstruction, tissue ischemia, and clinical complications in severe malaria (SM); however, the mechanisms leading to endothelial activation remain unclear. The angiogenic factors, angiopoietin-2 (Ang-2) and vascular endothelial growth factor (VEGF) are modulators of endothelial activation, with Ang-2 release from Weibel-Palade bodies (WPBs) being regulated by endothelial nitric oxide (NO). We explored the relationships between endothelial NO bioavailability, Ang-2, VEGF, tissue perfusion, and clinical outcomes in SM. We measured plasma Ang-2 and VEGF, together with biomarkers of severity from 146 adults with and without SM, in parallel with longitudinal measures of endothelial function by using reactive hyperemia peripheral arterial tonometry (a measure of endothelial NO bioavailability). Regression was used to relate concentrations of Ang-2/VEGF with malaria disease severity, biomarkers of perfusion, endothelial activation, and parasite biomass. The longitudinal relationship between Ang-2 and endothelial function was assessed by using a mixed-effects model. Ang-2 concentrations were elevated in SM and associated with increased venous lactate, plasma intercellular cell adhesion molecule-1 concentrations, parasite biomass, and mortality. In contrast, VEGF concentrations were inversely associated with these biomarkers. Ang-2 concentrations were significantly better predictors of death than venous lactate (P ‫؍‬ 0.03). Recovery of endothelial function was associated with falling concentrations of Ang-2. Ang-2 release from endothelial cells with reduced NO bioavailability is likely to contribute to endothelial activation, sequestered parasite biomass, impaired perfusion, and poor outcome in severe falciparum malaria. Agents that improve endothelial NO, reduce WPB exocytosis, and/or antagonize Ang-2 may have therapeutic roles in SM.Plasmodium falciparum ͉ VEGF ͉ Weibel-Palade bodies ͉ endothelial function

In Papua, Indonesia, the antimalarial susceptibility of Plasmodium vivax (n ‫؍‬ 216) and P. falciparum (n ‫؍‬ 277) was assessed using a modified schizont maturation assay for chloroquine, amodiaquine, artesunate, lumefantrine, mefloquine, and piperaquine. The most effective antimalarial against P. vivax and P. falciparum was artesunate, with geometric mean 50% inhibitory concentrations (IC 50 In vitro drug susceptibility assays assess antimicrobial activity in the absence of the confounding effects of the host. Although such assays have become useful for monitoring the antimalarial resistance of Plasmodium falciparum, the assay has been of limited use with P. vivax. This is in part a consequence of a perception of the importance of antimalarial drug resistance with P. vivax, compounded by difficulties in standardizing a field-based assay. Over the last decade, a number of clinical studies have demonstrated the emergence of highgrade chloroquine resistance in Papua, Indonesia, and Papua, New Guinea (1, 18, 21), and its spread to other regions of Asia (6) and South America (20). However, assessment of the clinical efficacy of antimalarial drugs against P. vivax infection is confounded by the occurrence of both reinfections and relapses, making the attributable fraction of recurrent infections due to intrinsic parasite resistance difficult to gauge (2, 3, 10). To confirm the emergence of the spread of antimalarial drug resistance of P. vivax and to investigate alternative antimalarial drugs, it is critical that a standardized in vitro assay be developed and validated. The aim of this study was to define the in vitro susceptibility profiles of a range of antimalarial drugs and to investigate the confounding factors that modulate the derived estimate of drug efficacy. MATERIALS AND METHODS Field location and sample collection. Between March 2004 and May 2007,Plasmodium isolates were collected from patients attending malaria clinics in Timika, located in the southern part of Papua province, Indonesia. Timika is a region of endemicity for multidrug-resistant strains of P. vivax and P. falciparum, with a risk of treatment failure of 65% within 28 days after chloroquine monotherapy for P. vivax malaria and 48% failure after multidrug therapy with chloroquine-sulfadoxine-pyrimethamine for P. falciparum malaria (16). In 2004, treatment guidelines were changed accordingly to recommend an artemisinin combination therapy for both P. falciparum and P. vivax infection, precluding further clinical studies of the use of chloroquine monotherapy in this region (15). Patients with symptomatic malaria who presented to an outpatient facility were recruited into the study if they were singly infected with P. falciparum or with P. vivax, with a parasitemia of between 2,000 l Ϫ1 and 80,000 l Ϫ1 . Patients treated with antimalarials in the previous 3 weeks were excluded from this study. Venous blood (5 ml) was collected by venipuncture and, after the host white blood cells were removed using a CF11 column, 2 ml of packed infected red bl...