Tsin W. Yeo

Population transcriptomics of human malaria parasites reveals the mechanism of artemisinin resistance

Mechanisms propelling drug resistance If it were to spread, resistance to the drug artemisinin would seriously derail the recent gains of global malaria control programs (see the Perspective by Sibley). Mutations in a region called the K13-propeller are predictive for artemisinin resistance in Southeast Asia. Mok et al. looked at the patterns of gene expression in parasites isolated from more than 1000 patients sampled in Africa, Bangladesh, and the Mekong region. A range of mutations that alter protein repair pathways and the timing of the parasites developmental cycle were only found in parasites from the Mekong region. Straimer et al. genetically engineered the K13 region of parasites obtained from recent clinical isolates. Mutations in this region were indeed responsible for the resistance phenotypes. Science, this issue p. 431, p. 428; see also p. 373 Resistance to the primary antimalarial drug lies in mutations in protein repair and developmental pathways. [Also see Perspective by Sibley] Artemisinin resistance in Plasmodium falciparum threatens global efforts to control and eliminate malaria. Polymorphisms in the kelch domain–carrying protein K13 are associated with artemisinin resistance, but the underlying molecular mechanisms are unknown. We analyzed the in vivo transcriptomes of 1043 P. falciparum isolates from patients with acute malaria and found that artemisinin resistance is associated with increased expression of unfolded protein response (UPR) pathways involving the major PROSC and TRiC chaperone complexes. Artemisinin-resistant parasites also exhibit decelerated progression through the first part of the asexual intraerythrocytic development cycle. These findings suggest that artemisinin-resistant parasites remain in a state of decelerated development at the young ring stage, whereas their up-regulated UPR pathways mitigate protein damage caused by artemisinin. The expression profiles of UPR-related genes also associate with the geographical origin of parasite isolates, further suggesting their role in emerging artemisinin resistance in the Greater Mekong Subregion.

Severe Plasmodium knowlesi Malaria in a TertiaryCare Hospital, Sabah, Malaysia

The simian parasite Plasmodium knowlesi causes severe human malaria; the optimal treatment remains unknown. We describe the clinical features, disease spectrum, and response to antimalarial chemotherapy, including artemether-lumefantrine and artesunate, in patients with P. knowlesi malaria diagnosed by PCR during December 2007–November 2009 at a tertiary care hospital in Sabah, Malaysia. Fifty-six patients had PCR-confirmed P. knowlesi monoinfection and clinical records available for review. Twenty-two (39%) had severe malaria; of these, 6 (27%) died. Thirteen (59%) had respiratory distress; 12 (55%), acute renal failure; and 12, shock. None experienced coma. Patients with uncomplicated disease received chloroquine, quinine, or artemether-lumefantrine, and those with severe disease received intravenous quinine or artesunate. Parasite clearance times were 1–2 days shorter with either artemether-lumefantrine or artesunate treatment. P. knowlesi is a major cause of severe and fatal malaria in Sabah. Artemisinin derivatives rapidly clear parasitemia and are efficacious in treating uncomplicated and severe knowlesi malaria.

SeverePlasmodium knowlesiMalaria in a Tertiary Care Hospital, Sabah, Malaysia

The simian parasite Plasmodium knowlesi causes severe human malaria; the optimal treatment remains unknown. We describe the clinical features, disease spectrum, and response to antimalarial chemotherapy, including artemether-lumefantrine and artesunate, in patients with P. knowlesi malaria diagnosed by PCR during December 2007-November 2009 at a tertiary care hospital in Sabah, Malaysia. Fifty-six patients had PCR-confirmed P. knowlesi monoinfection and clinical records available for review. Twenty-two (39%) had severe malaria; of these, 6 (27%) died. Thirteen (59%) had respiratory distress; 12 (55%), acute renal failure; and 12, shock. None experienced coma. Patients with uncomplicated disease received chloroquine, quinine, or artemether-lumefantrine, and those with severe disease received intravenous quinine or artesunate. Parasite clearance times were 1-2 days shorter with either artemether-lumefantrine or artesunate treatment. P. knowlesi is a major cause of severe and fatal malaria in Sabah. Artemisinin derivatives rapidly clear parasitemia and are efficacious in treating uncomplicated and severe knowlesi malaria.

The anaemia of Plasmodium vivax malaria

Plasmodium vivax threatens nearly half the world’s population and is a significant impediment to achievement of the millennium development goals. It is an important, but incompletely understood, cause of anaemia. This review synthesizes current evidence on the epidemiology, pathogenesis, treatment and consequences of vivax-associated anaemia. Young children are at high risk of clinically significant and potentially severe vivax-associated anaemia, particularly in countries where transmission is intense and relapses are frequent. Despite reaching lower densities than Plasmodium falciparum, Plasmodium vivax causes similar absolute reduction in red blood cell mass because it results in proportionately greater removal of uninfected red blood cells. Severe vivax anaemia is associated with substantial indirect mortality and morbidity through impaired resilience to co-morbidities, obstetric complications and requirement for blood transfusion. Anaemia can be averted by early and effective anti-malarial treatment.

L-arginine and vitamin D adjunctive therapies in pulmonary tuberculosis: a randomised, double-blind, placebo-controlled trial

Background Vitamin D (vitD) and L-arginine have important antimycobacterial effects in humans. Adjunctive therapy with these agents has the potential to improve outcomes in active tuberculosis (TB). Methods In a 4-arm randomised, double-blind, placebo-controlled factorial trial in adults with smear-positive pulmonary tuberculosis (PTB) in Timika, Indonesia, we tested the effect of oral adjunctive vitD 50,000 IU 4-weekly or matching placebo, and L-arginine 6.0 g daily or matching placebo, for 8 weeks, on proportions of participants with negative 4-week sputum culture, and on an 8-week clinical score (weight, FEV1, cough, sputum, haemoptysis). All participants with available endpoints were included in analyses according to the study arm to which they were originally assigned. Adults with new smear-positive PTB were eligible. The trial was registered at ClinicalTrials.gov NCT00677339. Results 200 participants were enrolled, less than the intended sample size: 50 received L-arginine + active vitD, 49 received L-arginine + placebo vit D, 51 received placebo L-arginine + active vitD and 50 received placebo L-arginine + placebo vitD. According to the factorial model, 99 people received arginine, 101 placebo arginine, 101 vitamin D, 99 placebo vitamin D. Results for the primary endpoints were available in 155 (4-week culture) and 167 (clinical score) participants. Sputum culture conversion was achieved by week 4 in 48/76 (63%) participants in the active L-arginine versus 48/79 (61%) in placebo L-arginine arms (risk difference −3%, 95% CI −19 to 13%), and in 44/75 (59%) in the active vitD versus 52/80 (65%) in the placebo vitD arms (risk difference 7%, 95% CI −9 to 22%). The mean clinical outcome score also did not differ between study arms. There were no effects of the interventions on adverse event rates including hypercalcaemia, or other secondary outcomes. Conclusion Neither vitD nor L-arginine supplementation, at the doses administered and with the power attained, affected TB outcomes. Registry ClinicalTrials.gov. Registry number: NCT00677339

Plasmodium knowlesi Malaria in Children

Plasmodium knowlesi can cause severe malaria in adults; however, descriptions of clinical disease in children are lacking. We reviewed case records of children (age <15 years) with a malaria diagnosis at Kudat District Hospital, serving a largely deforested area of Sabah, Malaysia, during January–November 2009. Sixteen children with PCR-confirmed P. knowlesi monoinfection were compared with 14 children with P. falciparum monoinfection diagnosed by microscopy or PCR. Four children with knowlesi malaria had a hemoglobin level at admission of <10.0 g/dL (minimum lowest level 6.4 g/dL). Minimum level platelet counts were lower in knowlesi than in falciparum malaria (median 76,500/µL vs. 156,000/μL; p = 0.01). Most (81%) children with P. knowlesi malaria received chloroquine and primaquine; median parasite clearance time was 2 days (range 1–5 days). P. knowlesi is the most common cause of childhood malaria in Kudat. Although infection is generally uncomplicated, anemia is common and thrombocytopenia universal. Transmission dynamics in this region require additional investigation.

Changing epidemiology of malaria in Sabah, Malaysia: increasing incidence of Plasmodium knowlesi

BackgroundWhile Malaysia has had great success in controlling Plasmodium falciparum and Plasmodium vivax, notifications of Plasmodium malariae and the microscopically near-identical Plasmodium knowlesi increased substantially over the past decade. However, whether this represents microscopic misdiagnosis or increased recognition of P. knowlesi has remained uncertain.MethodsTo describe the changing epidemiology of malaria in Sabah, in particular the increasing incidence of P. knowlesi, a retrospective descriptive study was undertaken involving a review of Department of Health malaria notification data from 2012–2013, extending a previous review of these data from 1992–2011. In addition, malaria PCR and microscopy data from the State Public Health Laboratory were reviewed to estimate the accuracy of the microscopy-based notification data.ResultsNotifications of P. malariae/P. knowlesi increased from 703 in 2011 to 815 in 2012 and 996 in 2013. Notifications of P. vivax and P. falciparum decreased from 605 and 628, respectively, in 2011, to 297 and 263 in 2013. In 2013, P. malariae/P. knowlesi accounted for 62% of all malaria notifications compared to 35% in 2011. Among 1,082 P. malariae/P. knowlesi blood slides referred for PCR testing during 2011–2013, there were 924 (85%) P. knowlesi mono-infections, 30 (2.8%) P. falciparum, 43 (4.0%) P. vivax, seven (0.6%) P. malariae, six (0.6%) mixed infections, 31 (2.9%) positive only for Plasmodium genus, and 41 (3.8%) Plasmodium-negative. Plasmodium knowlesi mono-infection accounted for 32/156 (21%) and 33/87 (38%) blood slides diagnosed by microscopy as P. falciparum and P. vivax, respectively. Twenty-six malaria deaths were reported during 2010–2013, including 12 with ‘P. malariae/P. knowlesi’ (all adults), 12 with P. falciparum (seven adults), and two adults with P. vivax.ConclusionsNotifications of P. malariae/P. knowlesi in Sabah are increasing, with this trend likely reflecting a true increase in incidence of P. knowlesi and presenting a major threat to malaria control and elimination in Malaysia. With the decline of P. falciparum and P. vivax, control programmes need to incorporate measures to protect against P. knowlesi, with further research required to determine effective interventions.

Parasite biomass-related inflammation, endothelial activation, microvascular dysfunction and disease severity in vivax malaria

Plasmodium vivax can cause severe malaria, however its pathogenesis is poorly understood. In contrast to P. falciparum, circulating vivax parasitemia is low, with minimal apparent sequestration in endothelium-lined microvasculature, and pathogenesis thought unrelated to parasite biomass. However, the relationships between vivax disease-severity and total parasite biomass, endothelial autocrine activation and microvascular dysfunction are unknown. We measured circulating parasitemia and markers of total parasite biomass (plasma parasite lactate dehydrogenase [pLDH] and PvLDH) in adults with severe (nu200a=u200a9) and non-severe (nu200a=u200a53) vivax malaria, and examined relationships with disease-severity, endothelial activation, and microvascular function. Healthy controls and adults with non-severe and severe falciparum malaria were enrolled for comparison. Median peripheral parasitemia, PvLDH and pLDH were 2.4-fold, 3.7-fold and 6.9-fold higher in severe compared to non-severe vivax malaria (pu200a=u200a0.02, pu200a=u200a0.02 and pu200a=u200a0.015, respectively), suggesting that, as in falciparum malaria, peripheral P. vivax parasitemia underestimates total parasite biomass, particularly in severe disease. P. vivax schizonts were under-represented in peripheral blood. Severe vivax malaria was associated with increased angiopoietin-2 and impaired microvascular reactivity. Peripheral vivax parasitemia correlated with endothelial activation (angiopoietin-2, von-Willebrand-Factor [VWF], E-selectin), whereas markers of total vivax biomass correlated only with systemic inflammation (IL-6, IL-10). Activity of the VWF-cleaving-protease, ADAMTS13, was deficient in proportion to endothelial activation, IL-6, thrombocytopenia and vivax disease-severity, and associated with impaired microvascular reactivity in severe disease. Impaired microvascular reactivity correlated with lactate in severe vivax malaria. Findings suggest that tissue accumulation of P. vivax may occur, with the hidden biomass greatest in severe disease and capable of mediating systemic inflammatory pathology. The lack of association between total parasite biomass and endothelial activation is consistent with accumulation in parts of the circulation devoid of endothelium. Endothelial activation, associated with circulating parasites, and systemic inflammation may contribute to pathology in vivax malaria, with microvascular dysfunction likely contributing to impaired tissue perfusion.

Epidemiology of Plasmodium knowlesi malaria in north-east Sabah, Malaysia: family clusters and wide age distribution

BackgroundThe simian parasite Plasmodium knowlesi is a common cause of human malaria in Malaysian Borneo, with a particularly high incidence in Kudat, Sabah. Little is known however about the epidemiology in this substantially deforested region.MethodsMalaria microscopy records at Kudat District Hospital were retrospectively reviewed from January 2009-November 2011. Demographics, and PCR results if available, were recorded for each positive result. Medical records were reviewed for patients suspected of representing family clusters, and families contacted for further information. Rainfall data were obtained from the Malaysian Meteorological Department.Results“Plasmodium malariae” mixed or mono-infection was diagnosed by microscopy in 517/653 (79%) patients. Of these, PCR was performed in 445 (86%) and was positive for P. knowlesi mono-infection in 339 (76%). Patients with knowlesi malaria demonstrated a wide age distribution (median 33, IQR 20–50, range 0.7-89 years) with P. knowlesi predominating in all age groups except those <5 years old, where numbers approximated those of Plasmodium falciparum and Plasmodium vivax. Two contemporaneous family clusters were identified: a father with two children (aged 10–11 years); and three brothers (aged one-11 years), all with PCR-confirmed knowlesi malaria. Cases of P. knowlesi demonstrated significant seasonal variation, and correlated with rainfall in the preceding three to five months.ConclusionsPlasmodium knowlesi is the most common cause of malaria admissions to Kudat District Hospital. The wide age distribution and presence of family clusters suggest that transmission may be occurring close to or inside people’s homes, in contrast to previous reports from densely forested areas of Sarawak. These findings have significant implications for malaria control. Prospective studies of risk factors, vectors and transmission dynamics of P. knowlesi in Sabah, including potential for human-to-human transmission, are needed.

Asymptomatic and Submicroscopic Carriage of Plasmodium knowlesi Malaria in Household and Community Members of Clinical Cases in Sabah, Malaysia

Although asymptomatic carriage of human malaria species has been widely reported, the extent of asymptomatic, submicroscopic Plasmodium knowlesi parasitemia is unknown. In this study, samples were obtained from individuals residing in households or villages of symptomatic malaria cases with the aim of detecting submicroscopic P. knowlesi in this population. Four published molecular assays were used to confirm the presence of P. knowlesi. Latent class analysis revealed that the estimated proportion of asymptomatic individuals was 6.9% (95% confidence interval, 5.6%–8.4%). This study confirms the presence of a substantial number of asymptomatic monoinfections across all age groups; further work is needed to estimate prevalence in the wider community.