Platelets mediate the clearance of senescent red blood cells by forming prophagocytic platelet-cell complexes.

ABSTRACT: In humans, ∼0.1% to 0.3% of circulating red blood cells (RBCs) are present as platelet-RBC (P-RBC) complexes, and it is 1% to 2% in mice. Excessive P-RBC complexes are found in diseases that compromise RBC health (eg, sickle cell disease and malaria) and contribute to pathogenesis. However, the physiological role of P-RBC complexes in healthy blood is unknown. As a result of damage accumulated over their lifetime, RBCs nearing senescence exhibit physiological and molecular changes akin to those in platelet-binding RBCs in sickle cell disease and malaria. Therefore, we hypothesized that RBCs nearing senescence are targets for platelet binding and P-RBC formation. Confirming this hypothesis, pulse-chase labeling studies in mice revealed an approximately tenfold increase in P-RBC complexes in the most chronologically aged RBC population compared with younger cells. When reintroduced into mice, these complexes were selectively cleared from the bloodstream (in preference to platelet-free RBC) through the reticuloendothelial system and erythrophagocytes in the spleen. As a corollary, patients without a spleen had higher levels of complexes in their bloodstream. When the platelet supply was artificially reduced in mice, fewer RBC complexes were formed, fewer erythrophagocytes were generated, and more senescent RBCs remained in circulation. Similar imbalances in complex levels and senescent RBC burden were observed in humans with immune thrombocytopenia (ITP). These findings indicate that platelets are important for binding and clearing senescent RBCs, and disruptions in platelet count or complex formation and clearance may negatively affect RBC homeostasis and may contribute to the known risk of thrombosis in ITP and after splenectomy.

Reduced red blood cell deformability in vivax malaria.

Reduced deformability of both infected and uninfected red blood cells (RBC) contributes to pathogenesis in falciparum malaria. Whole blood RBC-deformability is not well-characterised in vivax malaria. We used a laser-assisted optical rotational cell analyzer to measure the RBC deformability in fresh whole blood from Malaysian patients with vivax malaria (n=25). Deformability of whole blood RBC, the vast majority of which were uninfected, was reduced in vivax malaria compared to controls (n=15), though not to the same degree as in falciparum malaria (n=90). Reduced RBC-deformability may contribute to the pathogenesis of vivax malaria, including splenic retention of uninfected RBC.

Malaria.

Malaria is resurging in many African and South American countries, exacerbated by COVID-19-related health service disruption. In 2021, there were an estimated 247 million malaria cases and 619 000 deaths in 84 endemic countries. Plasmodium falciparum strains partly resistant to artemisinins are entrenched in the Greater Mekong region and have emerged in Africa, while Anopheles mosquito vectors continue to evolve physiological and behavioural resistance to insecticides. Elimination of Plasmodium vivax malaria is hindered by impractical and potentially toxic antirelapse regimens. Parasitological diagnosis and treatment with oral or parenteral artemisinin-based therapy is the mainstay of patient management. Timely blood transfusion, renal replacement therapy, and restrictive fluid therapy can improve survival in severe malaria. Rigorous use of intermittent preventive treatment in pregnancy and infancy and seasonal chemoprevention, potentially combined with pre-erythrocytic vaccines endorsed by WHO in 2021 and 2023, can substantially reduce malaria morbidity. Improved surveillance, better access to effective treatment, more labour-efficient vector control, continued drug development, targeted mass drug administration, and sustained political commitment are required to achieve targets for malaria reduction by the end of this decade.

Retention of uninfected red blood cells causing congestive splenomegaly is the major mechanism of anemia in malaria.

Splenomegaly frequently occurs in patients with Plasmodium falciparum (Pf) or P. vivax (Pv) malarial anemia, but mechanisms underlying this co-occurrence are unclear. In malaria-endemic Papua, Indonesia, we prospectively analyzed red blood cell (RBC) concentrations in the spleen and spleen-mimetic retention in 37 subjects splenectomized for trauma or hyperreactive splenomegaly, most of whom were infected with Plasmodium. Splenomegaly (median 357 g [range: 80-1918 g]) was correlated positively with the proportion of red-pulp on histological sections (median 88.1% [range: 74%-99.4%]; r = .59, p = .0003) and correlated negatively with the proportion of white-pulp (median 8.3% [range: 0.4%-22.9%]; r = -.50, p = .002). The number of RBC per microscopic field (>95% uninfected) was correlated positively with spleen weight in both Pf-infected (r = .73; p = .017) and Pv-infected spleens (r = .94; p = .006). The median estimated proportion of total-body RBCs retained in Pf-infected spleens was 8.2% (range: 1.0%-33.6%), significantly higher than in Pv-infected (2.6% [range: 0.6%-23.8%]; p = .015) and PCR-negative subjects (2.5% [range: 1.0%-3.3%]; p = .006). Retained RBCs accounted for over half of circulating RBC loss seen in Pf infections. The proportion of total-body RBC retained in Pf- and Pv-infected spleens correlated negatively with hemoglobin concentrations (r = -.56, p = .0003), hematocrit (r = -.58, p = .0002), and circulating RBC counts (r = -.56, p = .0003). Splenic CD71-positive reticulocyte concentrations correlated with spleen weight in Pf (r = 1.0; p = .003). Retention rates of peripheral and splenic RBCs were correlated negatively with circulating RBC counts (r = -.69, p = .07 and r = -.83, p = .008, respectively). In conclusion, retention of mostly uninfected RBC in the spleen, leading to marked congestion of the red-pulp, was associated with splenomegaly and is the major mechanism of anemia in subjects infected with Plasmodium, particularly Pf.

No Association between the Plasmodium vivax crt-o MS334 or In9pvcrt Polymorphisms and Chloroquine Failure in a Pre-Elimination Clinical Cohort from Malaysia with a Large Clonal Expansion.

Increasing reports of resistance to a frontline malaria blood-stage treatment, chloroquine (CQ), raises concerns for the elimination of Plasmodium vivax. The absence of an effective molecular marker of CQ resistance in P. vivax greatly constrains surveillance of this emerging threat. A recent genetic cross between CQ sensitive (CQS) and CQ resistant (CQR) NIH-1993 strains of P. vivax linked a moderate CQR phenotype with two candidate markers in P. vivax CQ resistance transporter gene (pvcrt-o): MS334 and In9pvcrt. Longer TGAAGH motif lengths at MS334 were associated with CQ resistance, as were shorter motifs at the In9pvcrt locus. In this study, high-grade CQR clinical isolates of P. vivax from a low endemic setting in Malaysia were used to investigate the association between the MS334 and In9pvcrt variants and treatment efficacy. Among a total of 49 independent monoclonal P. vivax isolates assessed, high-quality MS334 and In9pvcrt sequences could be derived from 30 (61%) and 23 (47%), respectively. Five MS334 and six In9pvcrt alleles were observed, with allele frequencies ranging from 2 to 76% and 3 to 71%, respectively. None of the clinical isolates had the same variant as the NIH-1993 CQR strain, and none of the variants were associated with CQ treatment failure (all P > 0.05). Multi-locus genotypes (MLGs) at 9 neutral microsatellites revealed a predominant P. vivax strain (MLG6) accounting for 52% of Day 0 infections. The MLG6 strain comprised equal proportions of CQS and CQR infections. Our study reveals complexity in the genetic basis of CQ resistance in the Malaysian P. vivax pre-elimination setting and suggests that the proposed pvcrt-o MS334 and In9pvcrt markers are not reliable markers of CQ treatment efficacy in this setting. Further studies are needed in other endemic settings, applying hypothesis-free genome-wide approaches, and functional approaches to understand the biological impact of the TGAAGH repeats linked to CQ response in a cross are warranted to comprehend and track CQR P. vivax.

Quantification of parasite clearance in Plasmodium knowlesi infections.

BACKGROUND: The incidence of zoonotic Plasmodium knowlesi infections in humans is rising in Southeast Asia, leading to clinical studies to monitor the efficacy of anti-malarial treatments for knowlesi malaria. One of the key outcomes of anti-malarial drug efficacy is parasite clearance. For Plasmodium falciparum, parasite clearance is typically estimated using a two-stage method, that involves estimating parasite clearance for individual patients followed by pooling of individual estimates to derive population estimates. An alternative approach is Bayesian hierarchical modelling which simultaneously analyses all parasite-time patient profiles to determine parasite clearance. This study compared these methods for estimating parasite clearance in P. knowlesi treatment efficacy studies, with typically fewer parasite measurements per patient due to high susceptibility to anti-malarials. METHODS: Using parasite clearance data from 714 patients with knowlesi malaria and enrolled in three trials, the Worldwide Antimalarial Resistance Network (WWARN) Parasite Clearance Estimator (PCE) standard two-stage approach and Bayesian hierarchical modelling were compared. Both methods estimate the parasite clearance rate from a model that incorporates a lag phase, slope, and tail phase for the parasitaemia profiles. RESULTS: The standard two-stage approach successfully estimated the parasite clearance rate for 678 patients, with 36 (5%) patients excluded due to an insufficient number of available parasitaemia measurements. The Bayesian hierarchical estimation method was applied to the parasitaemia data of all 714 patients. Overall, the Bayesian method estimated a faster population mean parasite clearance (0.36/h, 95% credible interval [0.18, 0.65]) compared to the standard two-stage method (0.26/h, 95% confidence interval [0.11, 0.46]), with better model fits (compared visually). Artemisinin-based combination therapy (ACT) is more effective in treating P. knowlesi than chloroquine, as confirmed by both methods, with a mean estimated parasite clearance half-life of 2.5 and 3.6 h, respectively using the standard two-stage method, and 1.8 and 2.9 h using the Bayesian method. CONCLUSION: For clinical studies of P. knowlesi with frequent parasite measurements, the standard two-stage approach (WWARN's PCE) is recommended as this method is straightforward to implement. For studies with fewer parasite measurements per patient, the Bayesian approach should be considered. Regardless of method used, ACT is more efficacious than chloroquine, confirming the findings of the original trials.

Plasmodium malariae and P. ovale genomes provide insights into malaria parasite evolution.

Elucidation of the evolutionary history and interrelatedness of Plasmodium species that infect humans has been hampered by a lack of genetic information for three human-infective species: P. malariae and two P. ovale species (P. o. curtisi and P. o. wallikeri). These species are prevalent across most regions in which malaria is endemic and are often undetectable by light microscopy, rendering their study in human populations difficult. The exact evolutionary relationship of these species to the other human-infective species has been contested. Using a new reference genome for P. malariae and a manually curated draft P. o. curtisi genome, we are now able to accurately place these species within the Plasmodium phylogeny. Sequencing of a P. malariae relative that infects chimpanzees reveals similar signatures of selection in the P. malariae lineage to another Plasmodium lineage shown to be capable of colonization of both human and chimpanzee hosts. Molecular dating suggests that these host adaptations occurred over similar evolutionary timescales. In addition to the core genome that is conserved between species, differences in gene content can be linked to their specific biology. The genome suggests that P. malariae expresses a family of heterodimeric proteins on its surface that have structural similarities to a protein crucial for invasion of red blood cells. The data presented here provide insight into the evolution of the Plasmodium genus as a whole.

Identifying Targets of Protective Antibodies against Severe Malaria in Papua, Indonesia, Using Locally Expressed Domains of Plasmodium falciparum Erythrocyte Membrane Protein 1.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), a diverse family of multidomain proteins expressed on the surface of malaria-infected erythrocytes, is an important target of protective immunity against malaria. Our group recently studied transcription of the var genes encoding PfEMP1 in individuals from Papua, Indonesia, with severe or uncomplicated malaria. We cloned and expressed domains from 32 PfEMP1s, including 22 that were upregulated in severe malaria and 10 that were upregulated in uncomplicated malaria, using a wheat germ cell-free expression system. We used Luminex technology to measure IgG antibodies to these 32 domains and control proteins in 63 individuals (11 children). At presentation to hospital, levels of antibodies to PfEMP1 domains were either higher in uncomplicated malaria or were not significantly different between groups. Using principal component analysis, antibodies to 3 of 32 domains were highly discriminatory between groups. These included two domains upregulated in severe malaria, a DBLß13 domain and a CIDRα1.6 domain (which has been previously implicated in severe malaria pathogenesis), and a DBLδ domain that was upregulated in uncomplicated malaria. Antibody to control non-PfEMP1 antigens did not differ with disease severity. Antibodies to PfEMP1 domains differ with malaria severity. Lack of antibodies to locally expressed PfEMP1 types, including both domains previously associated with severe malaria and newly identified targets, may in part explain malaria severity in Papuan adults.

The Effect of Regularly Dosed Acetaminophen vs No Acetaminophen on Renal Function in Plasmodium knowlesi Malaria (PACKNOW): A Randomized, Controlled Trial.

BACKGROUND: Acetaminophen inhibits cell-free hemoglobin-induced lipid peroxidation and improves renal function in severe falciparum malaria but has not been evaluated in other infections with prominent hemolysis, including Plasmodium knowlesi malaria. METHODS: PACKNOW was an open-label, randomized, controlled trial of acetaminophen (500 mg or 1000 mg every 6 hours for 72 hours) vs no acetaminophen in Malaysian patients aged ≥5 years with knowlesi malaria of any severity. The primary end point was change in creatinine at 72 hours. Secondary end points included longitudinal changes in creatinine in patients with severe malaria or acute kidney injury (AKI), stratified by hemolysis. RESULTS: During 2016-2018, 396 patients (aged 12-96 years) were randomized to acetaminophen (n = 199) or no acetaminophen (n = 197). Overall, creatinine fell by a mean (standard deviation) 14.9% (18.1) in the acetaminophen arm vs 14.6% (16.0) in the control arm (P = .81). In severe disease, creatinine fell by 31.0% (26.5) in the acetaminophen arm vs 20.4% (21.5) in the control arm (P = .12), and in those with hemolysis by 35.8% (26.7) and 19% (16.6), respectively (P = .07). No difference was seen overall in patients with AKI; however, in those with AKI and hemolysis, creatinine fell by 34.5% (20.7) in the acetaminophen arm vs 25.9% (15.8) in the control arm (P = .041). Mixed-effects modeling demonstrated a benefit of acetaminophen at 72 hours (P = .041) and 1 week (P = .002) in patients with severe malaria and with AKI and hemolysis (P = .027 and P = .002, respectively). CONCLUSIONS: Acetaminophen did not improve creatinine among the entire cohort but may improve renal function in patients with severe knowlesi malaria and in those with AKI and hemolysis. CLINICAL TRIALS REGISTRATION: NCT03056391.

Degradation of endothelial glycocalyx in Tanzanian children with falciparum malaria.

A layer of glycocalyx covers the vascular endothelium serving important protective and homeostatic functions. The objective of this study was to determine if breakdown of the endothelial glycocalyx (eGC) occurs during malaria infection in children. Measures of eGC integrity, endothelial activation, and microvascular reactivity were prospectively evaluated in 146 children: 44 with moderately severe malaria (MSM), 42 with severe malaria (SM), and 60 healthy controls (HC). Biochemical measures of eGC integrity included plasma syndecan-1 and total urinary glycosaminoglycans (GAG). Side-stream dark field imaging was used to quantitatively assess integrity of eGC. Plasma angiopoietin-2 (Ang-2) was measured as a marker of endothelial activation and also as a possible mediator of eGC breakdown. Our results show that urinary GAG, syndecan-1, and Ang-2 were elevated in patients with MSM and SM compared with HC. Syndecan-1 and GAG levels correlated significantly with each other and with plasma Ang-2. The eGC breakdown products also inversely correlated significantly with hemoglobin and platelet count. In the MSM group, imaging results provided further evidence for eGC degradation. Although not correlated with markers of eGC degradation, vascular function (assessed by non-invasive near infrared spectroscopy [NIRS]) demonstrated reduced microvascular reactivity, particularly affecting the SM group. Our findings provide further evidence for breakdown of eGC in falciparum malaria that may contribute to endothelial activation and adhesion of parasitized red blood cells, with reduced nitric oxide formation, and vascular dysfunction.

Increased circulating myeloid-derived suppressor cells in vivax malaria and severe falciparum malaria.

BACKGROUND: Circulating myeloid-derived-suppressor-cells (MDSC) with immunosuppressive function are increased in human experimental Plasmodium falciparum infection, but have not been studied in clinical malaria. METHODS: Using flow-cytometry, circulating polymorphonuclear-MDSC were evaluated in cryopreserved samples from patients with uncomplicated Plasmodium vivax (n = 8) and uncomplicated (n = 4) and severe (n = 16) falciparum malaria from Papua, Indonesia. RESULTS: The absolute number of circulating polymorphonuclear-MDSC were significantly elevated in severe falciparum malaria patients compared to controls (n = 10). Polymorphonuclear-MDSC levels in uncomplicated vivax malaria were also elevated to levels comparable to that seen in severe falciparum malaria. CONCLUSION: Control of expansion of immunosuppressive MDSC may be important for development of effective immune responses in falciparum and vivax malaria.

Cerebrospinal Fluid Pterins, Pterin-Dependent Neurotransmitters, and Mortality in Pediatric Cerebral Malaria.

BACKGROUND: Cerebral malaria (CM) pathogenesis remains incompletely understood. Having shown low systemic levels of tetrahydrobiopterin (BH4), an enzymatic cofactor for neurotransmitter synthesis, we hypothesized that BH4 and BH4-dependent neurotransmitters would likewise be low in cerebrospinal fluid (CSF) in CM. METHODS: We prospectively enrolled Tanzanian children with CM and children with nonmalaria central nervous system conditions (NMCs). We measured CSF levels of BH4, neopterin, and BH4-dependent neurotransmitter metabolites, 3-O-methyldopa, homovanillic acid, and 5-hydroxyindoleacetate, and we derived age-adjusted z-scores using published reference ranges. RESULTS: Cerebrospinal fluid BH4 was elevated in CM (n = 49) compared with NMC (n = 51) (z-score 0.75 vs -0.08; P < .001). Neopterin was increased in CM (z-score 4.05 vs 0.09; P < .001), and a cutoff at the upper limit of normal (60 nmol/L) was 100% sensitive for CM. Neurotransmitter metabolite levels were overall preserved. A higher CSF BH4/BH2 ratio was associated with increased odds of survival (odds ratio, 2.94; 95% confidence interval, 1.03-8.33; P = .043). CONCLUSION: Despite low systemic BH4, CSF BH4 was elevated and associated with increased odds of survival in CM. Coma in malaria is not explained by deficiency of BH4-dependent neurotransmitters. Elevated CSF neopterin was 100% sensitive for CM diagnosis and warrants further assessment of its clinical utility for ruling out CM in malaria-endemic areas.

Positron emission tomography and magnetic resonance imaging in experimental human malaria to identify organ-specific changes in morphology and glucose metabolism: A prospective cohort study.

BACKGROUND: Plasmodium vivax has been proposed to infect and replicate in the human spleen and bone marrow. Compared to Plasmodium falciparum, which is known to undergo microvascular tissue sequestration, little is known about the behavior of P. vivax outside of the circulating compartment. This may be due in part to difficulties in studying parasite location and activity in life. METHODS AND FINDINGS: To identify organ-specific changes during the early stages of P. vivax infection, we performed 18-F fluorodeoxyglucose (FDG) positron emission tomography/magnetic resonance imaging (PET/MRI) at baseline and just prior to onset of clinical illness in P. vivax experimentally induced blood-stage malaria (IBSM) and compared findings to P. falciparum IBSM. Seven healthy, malaria-naive participants were enrolled from 3 IBSM trials: NCT02867059, ACTRN12616000174482, and ACTRN12619001085167. Imaging took place between 2016 and 2019 at the Herston Imaging Research Facility, Australia. Postinoculation imaging was performed after a median of 9 days in both species (n = 3 P. vivax; n = 4 P. falciparum). All participants were aged between 19 and 23 years, and 6/7 were male. Splenic volume (P. vivax: +28.8% [confidence interval (CI) +10.3% to +57.3%], P. falciparum: +22.9 [CI -15.3% to +61.1%]) and radiotracer uptake (P. vivax: +15.5% [CI -0.7% to +31.7%], P. falciparum: +5.5% [CI +1.4% to +9.6%]) increased following infection with each species, but more so in P. vivax infection (volume: p = 0.72, radiotracer uptake: p = 0.036). There was no change in FDG uptake in the bone marrow (P. vivax: +4.6% [CI -15.9% to +25.0%], P. falciparum: +3.2% [CI -3.2% to +9.6%]) or liver (P. vivax: +6.2% [CI -8.7% to +21.1%], P. falciparum: -1.4% [CI -4.6% to +1.8%]) following infection with either species. In participants with P. vivax, hemoglobin, hematocrit, and platelet count decreased from baseline at the time of postinoculation imaging. Decrements in hemoglobin and hematocrit were significantly greater in participants with P. vivax infection compared to P. falciparum. The main limitations of this study are the small sample size and the inability of this tracer to differentiate between host and parasite metabolic activity. CONCLUSIONS: PET/MRI indicated greater splenic tropism and metabolic activity in early P. vivax infection compared to P. falciparum, supporting the hypothesis of splenic accumulation of P. vivax very early in infection. The absence of uptake in the bone marrow and liver suggests that, at least in early infection, these tissues do not harbor a large parasite biomass or do not provoke a prominent metabolic response. PET/MRI is a safe and noninvasive method to evaluate infection-associated organ changes in morphology and glucose metabolism.

Evaluation of splenic accumulation and colocalization of immature reticulocytes and Plasmodium vivax in asymptomatic malaria: A prospective human splenectomy study.

BACKGROUND: A very large biomass of intact asexual-stage malaria parasites accumulates in the spleen of asymptomatic human individuals infected with Plasmodium vivax. The mechanisms underlying this intense tropism are not clear. We hypothesised that immature reticulocytes, in which P. vivax develops, may display high densities in the spleen, thereby providing a niche for parasite survival. METHODS AND FINDINGS: We examined spleen tissue in 22 mostly untreated individuals naturally exposed to P. vivax and Plasmodium falciparum undergoing splenectomy for any clinical indication in malaria-endemic Papua, Indonesia (2015 to 2017). Infection, parasite and immature reticulocyte density, and splenic distribution were analysed by optical microscopy, flow cytometry, and molecular assays. Nine non-endemic control spleens from individuals undergoing spleno-pancreatectomy in France (2017 to 2020) were also examined for reticulocyte densities. There were no exclusion criteria or sample size considerations in both patient cohorts for this demanding approach. In Indonesia, 95.5% (21/22) of splenectomy patients had asymptomatic splenic Plasmodium infection (7 P. vivax, 13 P. falciparum, and 1 mixed infection). Significant splenic accumulation of immature CD71 intermediate- and high-expressing reticulocytes was seen, with concentrations 11 times greater than in peripheral blood. Accordingly, in France, reticulocyte concentrations in the splenic effluent were higher than in peripheral blood. Greater rigidity of reticulocytes in splenic than in peripheral blood, and their higher densities in splenic cords both suggest a mechanical retention process. Asexual-stage P. vivax-infected erythrocytes of all developmental stages accumulated in the spleen, with non-phagocytosed parasite densities 3,590 times (IQR: 2,600 to 4,130) higher than in circulating blood, and median total splenic parasite loads 81 (IQR: 14 to 205) times greater, accounting for 98.7% (IQR: 95.1% to 98.9%) of the estimated total-body P. vivax biomass. More reticulocytes were in contact with sinus lumen endothelial cells in P. vivax- than in P. falciparum-infected spleens. Histological analyses revealed 96% of P. vivax rings/trophozoites and 46% of schizonts colocalised with 92% of immature reticulocytes in the cords and sinus lumens of the red pulp. Larger splenic cohort studies and similar investigations in untreated symptomatic malaria are warranted. CONCLUSIONS: Immature CD71+ reticulocytes and splenic P. vivax-infected erythrocytes of all asexual stages accumulate in the same splenic compartments, suggesting the existence of a cryptic endosplenic lifecycle in chronic P. vivax infection. Findings provide insight into P. vivax-specific adaptions that have evolved to maximise survival and replication in the spleen.

Novel RNA viruses associated with Plasmodium vivax in human malaria and Leucocytozoon parasites in avian disease.

Eukaryotes of the genus Plasmodium cause malaria, a parasitic disease responsible for substantial morbidity and mortality in humans. Yet, the nature and abundance of any viruses carried by these divergent eukaryotic parasites is unknown. We investigated the Plasmodium virome by performing a meta-transcriptomic analysis of blood samples taken from patients suffering from malaria and infected with P. vivax, P. falciparum or P. knowlesi. This resulted in the identification of a narnavirus-like sequence, encoding an RNA polymerase and restricted to P. vivax samples, as well as an associated viral segment of unknown function. These data, confirmed by PCR, are indicative of a novel RNA virus that we term Matryoshka RNA virus 1 (MaRNAV-1) to reflect its analogy to a "Russian doll": a virus, infecting a parasite, infecting an animal. Additional screening revealed that MaRNAV-1 was abundant in geographically diverse P. vivax derived from humans and mosquitoes, strongly supporting its association with this parasite, and not in any of the other Plasmodium samples analyzed here nor Anopheles mosquitoes in the absence of Plasmodium. Notably, related bi-segmented narnavirus-like sequences (MaRNAV-2) were retrieved from Australian birds infected with a Leucocytozoon-a genus of eukaryotic parasites that group with Plasmodium in the Apicomplexa subclass hematozoa. Together, these data support the establishment of two new phylogenetically divergent and genomically distinct viral species associated with protists, including the first virus likely infecting Plasmodium parasites. As well as broadening our understanding of the diversity and evolutionary history of the eukaryotic virosphere, the restriction to P. vivax may be of importance in understanding P. vivax-specific biology in humans and mosquitoes, and how viral co-infection might alter host responses at each stage of the P. vivax life-cycle.

The Plasmodium falciparum transcriptome in severe malaria reveals altered expression of genes involved in important processes including surface antigen-encoding var genes.

Within the human host, the malaria parasite Plasmodium falciparum is exposed to multiple selection pressures. The host environment changes dramatically in severe malaria, but the extent to which the parasite responds to-or is selected by-this environment remains unclear. From previous studies, the parasites that cause severe malaria appear to increase expression of a restricted but poorly defined subset of the PfEMP1 variant, surface antigens. PfEMP1s are major targets of protective immunity. Here, we used RNA sequencing (RNAseq) to analyse gene expression in 44 parasite isolates that caused severe and uncomplicated malaria in Papuan patients. The transcriptomes of 19 parasite isolates associated with severe malaria indicated that these parasites had decreased glycolysis without activation of compensatory pathways; altered chromatin structure and probably transcriptional regulation through decreased histone methylation; reduced surface expression of PfEMP1; and down-regulated expression of multiple chaperone proteins. Our RNAseq also identified novel associations between disease severity and PfEMP1 transcripts, domains, and smaller sequence segments and also confirmed all previously reported associations between expressed PfEMP1 sequences and severe disease. These findings will inform efforts to identify vaccine targets for severe malaria and also indicate how parasites adapt to-or are selected by-the host environment in severe malaria.

Reduced circulating dendritic cells in acute Plasmodium knowlesi and Plasmodium falciparum malaria despite elevated plasma Flt3 ligand levels.

BACKGROUND: Plasmodium falciparum malaria increases plasma levels of the cytokine Fms-like tyrosine kinase 3 ligand (Flt3L), a haematopoietic factor associated with dendritic cell (DC) expansion. It is unknown if the zoonotic parasite Plasmodium knowlesi impacts Flt3L or DC in human malaria. This study investigated circulating DC and Flt3L associations in adult malaria and in submicroscopic experimental infection. METHODS: Plasma Flt3L concentration and blood CD141+ DC, CD1c+ DC and plasmacytoid DC (pDC) numbers were assessed in (i) volunteers experimentally infected with P. falciparum and in Malaysian patients with uncomplicated (ii) P. falciparum or (iii) P. knowlesi malaria. RESULTS: Plasmodium knowlesi caused a decline in all circulating DC subsets in adults with malaria. Plasma Flt3L was elevated in acute P. falciparum and P. knowlesi malaria with no increase in a subclinical experimental infection. Circulating CD141+ DCs, CD1c+ DCs and pDCs declined in all adults tested, for the first time extending the finding of DC subset decline in acute malaria to the zoonotic parasite P. knowlesi. CONCLUSIONS: In adults, submicroscopic Plasmodium infection causes no change in plasma Flt3L but does reduce circulating DCs. Plasma Flt3L concentrations increase in acute malaria, yet this increase is insufficient to restore or expand circulating CD141+ DCs, CD1c+ DCs or pDCs. These data imply that haematopoietic factors, yet to be identified and not Flt3L, involved in the sensing/maintenance of circulating DC are impacted by malaria and a submicroscopic infection. The zoonotic P. knowlesi is similar to other Plasmodium spp in compromising DC in adult malaria.

Reduced Cardiac Index Reserve and Hypovolemia in Severe Falciparum Malaria.

BACKGROUND: Impaired microvascular perfusion is central to the development of coma and lactic acidosis in severe falciparum malaria. Refractory hypotension is rare on admission but develops frequently in fatal cases. We assessed cardiac function and volume status in severe falciparum malaria and its prognostic significance. METHODS: Patients with severe (N = 101) or acute uncomplicated falciparum malaria (N = 83) were recruited from 2 hospitals in India and Bangladesh, and healthy participants (N = 44) underwent echocardiography. RESULTS: Patients with severe malaria had 38% shorter left ventricular (LV) filling times and 25% shorter LV ejection times than healthy participants because of tachycardia; however, stroke volume, LV internal diameter in diastole (LVIDd), and LV internal diameter in systole (LVIDs) indices were similar. A low endocardial fraction shortening (eFS) was present in 17% (9 of 52) of severe malaria patients. Adjusting for preload and afterload, eFS was similar in health and severe malaria. Fatal cases had smaller baseline LVIDd and LVIDs indices, more collapsible inferior vena cavae (IVC), and higher heart rates than survivors. The LVIDs and IVC collapsibility were independent predictors for mortality, together with base excess and Glasgow Coma Scale. CONCLUSIONS: Patients with severe malaria have rapid ejection of a normal stroke volume. Fatal cases had features of relative hypovolemia and reduced cardiac index reserve.

Cell-Free Hemoglobin Is Associated With Increased Vascular Resistance and Reduced Peripheral Perfusion in Severe Malaria.

BACKGROUND: In severe falciparum malaria, unlike sepsis, hypotension on admission is uncommon. We hypothesized that low nitric oxide bioavailability due to the presence of cell-free hemoglobin (CFH) increases vascular tone in severe malaria. METHODS: Patients with severe malaria (n = 119), uncomplicated malaria (n = 91), or suspected bacterial sepsis (n = 56), as well as healthy participants (n = 50), were recruited. The systemic vascular resistance index (SVRI) was estimated from the echocardiographic cardiac index and the mean arterial pressure. RESULTS: SVRI and hematocrit levels were lower and plasma CFH and asymmetric dimethylarginine levels were higher in patients with malaria, compared with healthy participants. In multivariate linear regression models for mean arterial pressure or SVRI in patients with severe malaria, hematocrit and CFH but not asymmetric dimethylarginine were significant predictors. The SVRI was lower in patients with suspected bacterial sepsis than in those with severe malaria, after adjustment for hematocrit and age. Plasma CFH levels correlated positively with the core-peripheral temperature gradient and plasma lactate levels and inversely with the perfusion index. Impaired peripheral perfusion, as reflected by a low perfusion index or a high core-peripheral temperature gradient, predicted mortality in patients with severe malaria. CONCLUSIONS: CFH is associated with mean arterial pressure, SVRI, and peripheral perfusion in patients with severe malaria. This may be mediated through the nitric oxide scavenging potency of CFH, increasing basal vascular tone and impairing tissue perfusion.

Early Endothelial Activation Precedes Glycocalyx Degradation and Microvascular Dysfunction in Experimentally Induced Plasmodium falciparum and Plasmodium vivax Infection.

Endothelial activation and microvascular dysfunction are key pathogenic processes in severe malaria. We evaluated the early role of these processes in experimentally induced Plasmodium falciparum and P. vivax infection. Participants were enrolled in induced blood-stage malaria clinical trials. Plasma osteoprotegerin, angiopoietin-2, and von Willebrand Factor (vWF) levels were measured as biomarkers of endothelial activation. Microvascular function was assessed using peripheral arterial tonometry and near-infrared spectroscopy, and the endothelial glycocalyx was assessed by sublingual videomicroscopy and measurement of biomarkers of degradation. Forty-five healthy, malaria-naive participants were recruited from 5 studies. Osteoprotegerin and vWF levels increased in participants following inoculation with P. vivax (n = 16) or P. falciparum (n = 15), with the angiopoietin-2 level also increasing in participants following inoculation with P. falciparum For both species, the most pronounced increase was seen in osteoprotegerin. This was particularly marked in participants inoculated with P. vivax, where the osteoprotegerin level correlated with the levels of parasitemia and the malaria clinical score. There were no changes in measures of endothelial glycocalyx or microvascular function. Plasma biomarkers of endothelial activation increased in early P. falciparum and P. vivax infection and preceded changes in the endothelial glycocalyx or microvascular function. The more pronounced increase in osteoprotegerin suggests that this biomarker may play a role in disease pathogenesis.