Pulsed stimulated Brillouin microscopy enables high-sensitivity mechanical imaging of live and fragile biological specimens.

Brillouin microscopy is an emerging optical elastography technique capable of assessing mechanical properties of biological samples in a three-dimensional, all-optical and noncontact fashion. The typically weak Brillouin scattering signal can be substantially enhanced via a stimulated Brillouin scattering (SBS) process; however, current implementations require high pump powers, which prohibit applications to photosensitive or live imaging of biological samples. Here we present a pulsed SBS scheme that takes advantage of the nonlinearity of the pump-probe interaction. In particular, we show that the required pump laser power can be decreased ~20-fold without affecting the signal levels or spectral precision. We demonstrate the low phototoxicity and high specificity of our pulsed SBS approach by imaging, with subcellular detail, sensitive single cells, zebrafish larvae, mouse embryos and adult Caenorhabditis elegans. Furthermore, our method permits observing the mechanics of organoids and C. elegans embryos over time, opening up further possibilities for the field of mechanobiology.

Understanding parasite-brain microvascular interactions with engineered 3D blood-brain barrier models.

Microbial interactions with the blood-brain barrier (BBB) can be highly pathogenic and are still not well understood. Among these, parasites present complex interactions with the brain microvasculature that are difficult to decipher using experimental animal models or reductionist 2D in vitro cultures. Novel 3D engineered blood-brain barrier models hold great promise to overcome limitations in traditional research approaches. These models better mimic the intricate 3D architecture of the brain microvasculature and recapitulate several aspects of BBB properties, physiology, and function. Moreover, they provide improved control over biophysical and biochemical experimental parameters and are compatible with advanced imaging and molecular biology techniques. Here, we review design considerations and methodologies utilized to successfully engineer BBB microvessels. Finally, we highlight the advantages and limitations of existing engineered models and propose applications to study parasite interactions with the BBB, including mechanisms of barrier disruption.

Plasmodium vivax spleen-dependent genes encode antigens associated with cytoadhesion and clinical protection.

Plasmodium vivax, the most widely distributed human malaria parasite, causes severe clinical syndromes despite low peripheral blood parasitemia. This conundrum is further complicated as cytoadherence in the microvasculature is still a matter of investigations. Previous reports in Plasmodium knowlesi, another parasite species shown to infect humans, demonstrated that variant genes involved in cytoadherence were dependent on the spleen for their expression. Hence, using a global transcriptional analysis of parasites obtained from spleen-intact and splenectomized monkeys, we identified 67 P. vivax genes whose expression was spleen dependent. To determine their role in cytoadherence, two Plasmodium falciparum transgenic lines expressing two variant proteins pertaining to VIR and Pv-FAM-D multigene families were used. Cytoadherence assays demonstrated specific binding to human spleen but not lung fibroblasts of the transgenic line expressing the VIR14 protein. To gain more insights, we expressed five P. vivax spleen-dependent genes as recombinant proteins, including members of three different multigene families (VIR, Pv-FAM-A, Pv-FAM-D), one membrane transporter (SECY), and one hypothetical protein (HYP1), and determined their immunogenicity and association with clinical protection in a prospective study of 383 children in Papua New Guinea. Results demonstrated that spleen-dependent antigens are immunogenic in natural infections and that antibodies to HYP1 are associated with clinical protection. These results suggest that the spleen plays a major role in expression of parasite proteins involved in cytoadherence and can reveal antigens associated with clinical protection, thus prompting a paradigm shift in P. vivax biology toward deeper studies of the spleen during infections.

Severe adult malaria is associated with specific PfEMP1 adhesion types and high parasite biomass.

The interplay between cellular and molecular determinants that lead to severe malaria in adults is unexplored. Here, we analyzed parasite virulence factors in an infected adult population in India and investigated whether severe malaria isolates impair endothelial protein C receptor (EPCR), a protein involved in coagulation and endothelial barrier permeability. Severe malaria isolates overexpressed specific members of the Plasmodium falciparum var gene/PfEMP1 (P. falciparum erythrocyte membrane protein 1) family that bind EPCR, including DC8 var genes that have previously been linked to severe pediatric malaria. Machine learning analysis revealed that DC6- and DC8-encoding var transcripts in combination with high parasite biomass were the strongest indicators of patient hospitalization and disease severity. We found that DC8 CIDRα1 domains from severe malaria isolates had substantial differences in EPCR binding affinity and blockade activity for its ligand activated protein C. Additionally, even a low level of inhibition exhibited by domains from two cerebral malaria isolates was sufficient to interfere with activated protein C-barrier protective activities in human brain endothelial cells. Our findings demonstrate an interplay between parasite biomass and specific PfEMP1 adhesion types in the development of adult severe malaria, and indicate that low impairment of EPCR function may contribute to parasite virulence.

Plasmodium falciparum adhesion domains linked to severe malaria differ in blockade of endothelial protein C receptor.

Cytoadhesion of Plasmodium falciparum-infected erythrocytes to endothelial protein C receptor (EPCR) is associated with severe malaria. It has been postulated that parasite binding could exacerbate microvascular coagulation and endothelial dysfunction in cerebral malaria by impairing the protein C-EPCR interaction, but the extent of binding inhibition has not been fully determined. Here we expressed the cysteine-rich interdomain region (CIDRα1) domain from a variety of domain cassette (DC) 8 and DC13 P. falciparum erythrocyte membrane protein 1 proteins and show they interact in a distinct manner with EPCR resulting in weak, moderate and strong inhibition of the activated protein C (APC)-EPCR interaction. Overall, there was a positive correlation between CIDRα1-EPCR binding activity and APC blockade activity. In addition, our analysis from a combination of mutagenesis and blocking antibodies finds that an Arg81 (R81) in EPCR plays a pivotal role in CIDRα1 binding, but domains with weak and strong APC blockade activity were distinguished by their sensitivity to inhibition by anti-EPCR mAb 1535, implying subtle differences in their binding footprints. These data reveal a previously unknown functional heterogeneity in the interaction between P. falciparum and EPCR and have major implications for understanding the distinct clinical pathologies of cerebral malaria and developing new treatment strategies.

Demographic and clinical profiles of Plasmodium falciparum and Plasmodium vivax patients at a tertiary care centre in southwestern India.

BACKGROUND: Malaria remains an important cause of morbidity and mortality in India. Though many comprehensive studies have been carried out in Africa and Southeast Asia to characterize and examine determinants of Plasmodium falciparum and Plasmodium vivax malaria pathogenesis, fewer have been conducted in India. METHODS: A prospective study of malaria-positive individuals was conducted at Goa Medical College and Hospital (GMC) from 2012 to 2015 to identify demographic, diagnostic and clinical indicators associated with P. falciparum and P. vivax infection on univariate analysis. RESULTS: Between 2012 and 2015, 74,571 febrile individuals, 6287 (8.4%) of whom were malaria positive, presented to GMC. The total number of malaria cases at GMC increased more than two-fold over four years, with both P. vivax and P. falciparum cases present year-round. Some 1116 malaria-positive individuals (mean age = 27, 91% male), 88.2% of whom were born outside of Goa and 51% of whom were construction workers, were enroled in the study. Of 1088 confirmed malaria-positive patients, 77.0% had P. vivax, 21.0% had P. falciparum and 2.0% had mixed malaria. Patients over 40 years of age and with P. falciparum infection were significantly (p < 0.001) more likely to be hospitalised than younger and P. vivax patients, respectively. While approximately equal percentages of hospitalised P. falciparum (76.6%) and P. vivax (78.9%) cases presented with at least one WHO severity indicator, a greater percentage of P. falciparum inpatients presented with at least two (43.9%, p < 0.05) and at least three (29.9%, p < 0.01) severity features. There were six deaths among the 182 hospitalised malaria positive patients, all of whom had P. falciparum. CONCLUSION: During the four year study period at GMC, the number of malaria cases increased substantially and the greatest burden of severe disease was contributed by P. falciparum.

Protective Effect of Myo-Inositol Hexaphosphate (Phytate) on Abdominal Aortic Calcification in Patients With Chronic Kidney Disease.

OBJECTIVE: The aim of this study was to evaluate the relationship between physiological levels of myo-inositol hexaphosphate (phytate) and cardiovascular (CV) calcification in patients with chronic kidney disease (CKD). DESIGN AND METHODS: This was a prospective cross-sectional study conducted from December 2012 to June 2013. SUBJECTS: Sixty-nine consecutive patients with CKD who were not undergoing renal replacement therapy. INTERVENTION: All subjects were given lateral lumbar X-rays to quantify abdominal aortic calcification (AAC). Clinical laboratory analyses and phytate food frequency questionnaires were also performed. MAIN OUTCOME MEASURE: Phytate urinary excretion, estimated phytate consumption (based on food frequency questionnaire) and AAC score. Patients were divided into two groups based on median abdominal aortic calcification (AAC) score: no/mild AAC (AAC ≤ 6, n = 35) and moderate/severe AAC (AAC > 6, n = 34). RESULTS: Patients with no/mild AAC were younger, had lower pulse pressure, greater dietary intake of phytate, greater urinary phytate and the prevalence of prior CV disease was significantly lower compared to patients with moderate/severe AAC. Among the top 10 phytate-rich foods, lentil consumption was significantly greater in patients with no/mild AAC than in those with moderate/severe AAC. Multivariate logistic regression analysis indicated that age, prior CV disease, urinary phytate (or lentil consumption) were independently associated to AAC. CONCLUSION: Our results suggest that adequate consumption of phytate can prevent AAC in patients with CKD. Further prospective studies must be performed to elucidate the benefits of a phytate-rich diet and the associated risk of phosphorus bioavailability in these patients.

Biosynthesis of GDP-fucose and other sugar nucleotides in the blood stages of Plasmodium falciparum.

Carbohydrate structures play important roles in many biological processes, including cell adhesion, cell-cell communication, and host-pathogen interactions. Sugar nucleotides are activated forms of sugars used by the cell as donors for most glycosylation reactions. Using a liquid chromatography-tandem mass spectrometry-based method, we identified and quantified the pools of UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, GDP-mannose, and GDP-fucose in Plasmodium falciparum intraerythrocytic life stages. We assembled these data with the in silico functional reconstruction of the parasite metabolic pathways obtained from the P. falciparum annotated genome, exposing new active biosynthetic routes crucial for further glycosylation reactions. Fucose is a sugar present in glycoconjugates often associated with recognition and adhesion events. Thus, the GDP-fucose precursor is essential in a wide variety of organisms. P. falciparum presents homologues of GDP-mannose 4,6-dehydratase and GDP-L-fucose synthase enzymes that are active in vitro, indicating that most GDP-fucose is formed by a de novo pathway that involves the bioconversion of GDP-mannose. Homologues for enzymes involved in a fucose salvage pathway are apparently absent in the P. falciparum genome. This is in agreement with in vivo metabolic labeling experiments showing that fucose is not significantly incorporated by the parasite. Fluorescence microscopy of epitope-tagged versions of P. falciparum GDP-mannose 4,6-dehydratase and GDP-L-fucose synthase expressed in transgenic 3D7 parasites shows that these enzymes localize in the cytoplasm of P. falciparum during the intraerythrocytic developmental cycle. Although the function of fucose in the parasite is not known, the presence of GDP-fucose suggests that the metabolite may be used for further fucosylation reactions.

Broadly inhibitory antibodies against severe malaria virulence proteins.

Plasmodium falciparum pathology is driven by the accumulation of parasite-infected erythrocytes in microvessels. This process is mediated by the parasite's polymorphic erythrocyte membrane protein 1 (PfEMP1) adhesion proteins. A subset of PfEMP1 variants that bind human endothelial protein C receptor (EPCR) through their CIDRα1 domains is responsible for severe malaria pathogenesis. A longstanding question is whether individual antibodies can recognize the large repertoire of circulating PfEMP1 variants. Here, we describe two broadly reactive and binding-inhibitory human monoclonal antibodies against CIDRα1. The antibodies isolated from two different individuals exhibited a similar and consistent EPCR-binding inhibition of 34 CIDRα1 domains, representing five of the six subclasses of CIDRα1. Both antibodies inhibited EPCR binding of both recombinant full-length and native PfEMP1 proteins as well as parasite sequestration in bioengineered 3D brain microvessels under physiologically relevant flow conditions. Structural analyses of the two antibodies in complex with two different CIDRα1 antigen variants reveal similar binding mechanisms that depend on interactions with three highly conserved amino acid residues of the EPCR-binding site in CIDRα1. These broadly reactive antibodies likely represent a common mechanism of acquired immunity to severe malaria and offer novel insights for the design of a vaccine or treatment targeting severe malaria.

A new computational approach redefines the subtelomeric vir superfamily of Plasmodium vivax.

BACKGROUND: Subtelomeric multigene families of malaria parasites encode virulent determinants. The published genome sequence of Plasmodium vivax revealed the largest subtelomeric multigene family of human malaria parasites, the vir super-family, presently composed of 346 vir genes subdivided into 12 different subfamilies based on sequence homologies detected by BLAST. RESULTS: A novel computational approach was used to redefine vir genes. First, a protein-weighted graph was built based on BLAST alignments. This graph was processed to ensure that edge weights are not exclusively based on the BLAST score between the two corresponding proteins, but strongly dependant on their graph neighbours and their associations. Then the Markov Clustering Algorithm was applied to the protein graph. Next, the Homology Block concept was used to further validate this clustering approach. Finally, proteome-wide analysis was carried out to predict new VIR members. Results showed that (i) three previous subfamilies cannot longer be classified as vir genes; (ii) most previously unclustered vir genes were clustered into vir subfamilies; (iii) 39 hypothetical proteins were predicted as VIR proteins; (iv) many of these findings are supported by a number of structural and functional evidences, sub-cellular localization studies, gene expression analysis and chromosome localization (v) this approach can be used to study other multigene families in malaria. CONCLUSIONS: This methodology, resource and new classification of vir genes will contribute to a new structural framing of this multigene family and other multigene families of malaria parasites, facilitating the design of experiments to understand their role in pathology, which in turn may help furthering vaccine development.

Plasmodium vivax malaria in Mali: a study from three different regions.

BACKGROUND: Plasmodium vivax has traditionally been considered virtually absent from Western and Central Africa, due to the absence of the Duffy blood group in most of the population living in these areas. Recent reports, however, suggest the circulation of P. vivax in sub-Saharan Africa. METHODS: Giemsa/Field-stained smears from febrile patients recruited in five different cities (Goundam, Tombouctou, Gao, Bourem and Kidal) pertaining to three regions from Northern Mali were examined. Nested-PCR and DNA sequence analyses of selected samples were performed to fully confirm the presence of P. vivax infections. RESULTS: Results demonstrated the presence of P. vivax infections in close to 30% of the cases as detected by Giemsa/Field-stained smears and nested-PCR and DNA-sequence analyses of selected samples unequivocally confirmed the presence of P. vivax. CONCLUSIONS: The diagnostics of this human malaria parasite should be taken into account in the context of malaria control and elimination efforts, not only in Mali, but also in sub-Saharan Africa.

Binding of Plasmodium falciparum-Infected Red Blood Cells to Engineered 3D Microvessels.

P. falciparum-infected red blood cell (iRBC) sequestration in the microvasculature is a pivotal event in severe malaria pathogenesis. In vitro binding assays using endothelial cell monolayers under static and flow conditions have revealed key ligand-receptor interactions for iRBC sequestration. However, mechanisms remain elusive for iRBC sequestration in specific vascular locations, which prevents further development of effective therapies. New models are needed to better recapitulate the complex geometry of blood flow in human blood vessels and organ-specific vascular signatures. Recent advances in engineering 3D microvessels in vitro have emerged as promising technologies to not only model complex human vascular structures but also allow for precise and step-wise control of individual biological and biomechanical parameters. By designing networks with different branching structures and change of vessel diameter along the flow path, these models recapitulate pressure and flow changes occurring in vivo. Here, we describe the methodology employed to build 3D microvessels using soft lithography and injection molding techniques, as well as the protocol to fabricate capillary-size vessels through collagen photoablation. Furthermore, we describe the methodology of using these models to study malaria and narrate necessary steps for perfusion of P. falciparum through 3D microvessels and different options to quantify P. falciparum-iRBC binding.

Biophysical Tools and Concepts Enable Understanding of Asexual Blood Stage Malaria.

Forces and mechanical properties of cells and tissues set constraints on biological functions, and are key determinants of human physiology. Changes in cell mechanics may arise from disease, or directly contribute to pathogenesis. Malaria gives many striking examples. Plasmodium parasites, the causative agents of malaria, are single-celled organisms that cannot survive outside their hosts; thus, thost-pathogen interactions are fundamental for parasite's biological success and to the host response to infection. These interactions are often combinations of biochemical and mechanical factors, but most research focuses on the molecular side. However, Plasmodium infection of human red blood cells leads to changes in their mechanical properties, which has a crucial impact on disease pathogenesis because of the interaction of infected red blood cells with other human tissues through various adhesion mechanisms, which can be probed and modelled with biophysical techniques. Recently, natural polymorphisms affecting red blood cell biomechanics have also been shown to protect human populations, highlighting the potential of understanding biomechanical factors to inform future vaccines and drug development. Here we review biophysical techniques that have revealed new aspects of Plasmodium falciparum invasion of red blood cells and cytoadhesion of infected cells to the host vasculature. These mechanisms occur differently across Plasmodium species and are linked to malaria pathogenesis. We highlight promising techniques from the fields of bioengineering, immunomechanics, and soft matter physics that could be beneficial for studying malaria. Some approaches might also be applied to other phases of the malaria lifecycle and to apicomplexan infections with complex host-pathogen interactions.

On cytoadhesion of Plasmodium vivax: raison d'être?

It is generally accepted that Plasmodium vivax, the most widely distributed human malaria parasite, causes mild disease and that this species does not sequester in the deep capillaries of internal organs. Recent evidence, however, has demonstrated that there is severe disease, sometimes resulting in death, exclusively associated with P. vivax and that P. vivax-infected reticulocytes are able to cytoadhere in vitro to different endothelial cells and placental cryosections. Here, we review the scarce and preliminary data on cytoadherence in P. vivax, reinforcing the importance of this phenomenon in this species and highlighting the avenues that it opens for our understanding of the pathology of this neglected human malaria parasite.

Bioengineered 3D Microvessels for Investigating Plasmodium falciparum Pathogenesis.

Plasmodium falciparum pathogenesis is complex and intimately connected to vascular physiology. This is exemplified by cerebral malaria (CM), a neurovascular complication that accounts for most of the malaria deaths worldwide. P. falciparum sequestration in the brain microvasculature is a hallmark of CM and is not replicated in animal models. Numerous aspects of the disease are challenging to fully understand from clinical studies, such as parasite binding tropism or causal pathways in blood-brain barrier breakdown. Recent bioengineering approaches allow for the generation of 3D microvessels and organ-specific vasculature that provide precise control of vessel architecture and flow dynamics, and hold great promise for malaria research. Here, we discuss recent and future applications of bioengineered microvessels in malaria pathogenesis research.

Determinants of brain swelling in pediatric and adult cerebral malaria.

Cerebral malaria (CM) affects children and adults, but brain swelling is more severe in children. To investigate features associated with brain swelling in malaria, we performed blood profiling and brain MRI in a cohort of pediatric and adult patients with CM in Rourkela, India, and compared them with an African pediatric CM cohort in Malawi. We determined that higher plasma Plasmodium falciparum histidine rich protein 2 (PfHRP2) levels and elevated var transcripts that encode for binding to endothelial protein C receptor (EPCR) were linked to CM at both sites. Machine learning models trained on the African pediatric cohort could classify brain swelling in Indian children CM cases but had weaker performance for adult classification, due to overall lower parasite var transcript levels in this age group and more severe thrombocytopenia in Rourkela adults. Subgrouping of patients with CM revealed higher parasite biomass linked to severe thrombocytopenia and higher Group A-EPCR var transcripts in mild thrombocytopenia. Overall, these findings provide evidence that higher parasite biomass and a subset of Group A-EPCR binding variants are common features in children and adult CM cases, despite age differences in brain swelling.

Biophysical and biomolecular interactions of malaria-infected erythrocytes in engineered human capillaries.

Microcirculatory obstruction is a hallmark of severe malaria, but mechanisms of parasite sequestration are only partially understood. Here, we developed a robust three-dimensional microvessel model that mimics the arteriole-capillary-venule (ACV) transition consisting of a narrow 5- to 10-µm-diameter capillary region flanked by arteriole- or venule-sized vessels. Using this platform, we investigated red blood cell (RBC) transit at the single cell and at physiological hematocrits. We showed normal RBCs deformed via in vivo-like stretching and tumbling with negligible interactions with the vessel wall. By comparison, Plasmodium falciparum-infected RBCs exhibited virtually no deformation and rapidly accumulated in the capillary-sized region. Comparison of wild-type parasites to those lacking either cytoadhesion ligands or membrane-stiffening knobs showed highly distinctive spatial and temporal kinetics of accumulation, linked to velocity transition in ACVs. Our findings shed light on mechanisms of microcirculatory obstruction in malaria and establish a new platform to study hematologic and microvascular diseases.

Binding Heterogeneity of Plasmodium falciparum to Engineered 3D Brain Microvessels Is Mediated by EPCR and ICAM-1.

Cerebral malaria is a severe neurological complication associated with sequestration of Plasmodium falciparum-infected erythrocytes (IE) in the brain microvasculature, but the specific binding interactions remain under debate. Here, we have generated an engineered three-dimensional (3D) human brain endothelial microvessel model and studied P. falciparum binding under the large range of physiological flow velocities that occur in both health and disease. Perfusion assays on 3D microvessels reveal previously unappreciated phenotypic heterogeneity in parasite binding to tumor necrosis factor alpha (TNF-α)-activated brain endothelial cells. While clonal parasite lines expressing a group B P. falciparum erythrocyte membrane protein 1 (PfEMP1) present an increase in binding to activated 3D microvessels, P. falciparum-IE expressing DC8-PfEMP1 present a decrease in binding. The differential response to endothelium activation is mediated by surface expression changes of endothelial protein C receptor (EPCR) and intercellular adhesion molecule 1 (ICAM-1). These findings demonstrate heterogeneity in parasite binding and provide evidence for a parasite strategy to adapt to a changing microvascular environment during infection. The engineered 3D human brain microvessel model provides new mechanistic insight into parasite binding and opens opportunities for further studies on malaria pathogenesis and parasite-vessel interactions.IMPORTANCE Cerebral malaria research has been hindered by the inaccessibility of the brain. Here, we have developed an engineered 3D human brain microvessel model that mimics the blood flow rates and architecture of small blood vessels to study how P. falciparum-infected human erythrocytes attach to brain endothelial cells. By studying parasite lines with different adhesive properties, we show that the malaria parasite binding rate is heterogeneous and strongly influenced by physiological differences in flow and whether the endothelium has been previously activated by TNF-α, a proinflammatory cytokine that is linked to malaria disease severity. We also show the importance of human EPCR and ICAM-1 in parasite binding. Our model sheds new light on how P. falciparum binds within brain microvessels and provides a powerful method for future investigations of recruitment of human brain pathogens to the blood vessel lining of the brain.

Meta-analysis of Plasmodium falciparum var Signatures Contributing to Severe Malaria in African Children and Indian Adults.

The clinical presentation of severe Plasmodium falciparum malaria differs between children and adults, but the mechanistic basis for this remains unclear. Contributing factors to disease severity include total parasite biomass and the diverse cytoadhesive properties mediated by the polymorphic var gene parasite ligand family displayed on infected erythrocytes. To explore these factors, we performed a multicohort analysis of the contribution of var expression and parasite biomass to severe malaria in two previously published pediatric cohorts in Tanzania and Malawi and an adult cohort in India. Machine learning analysis revealed independent and complementary roles for var adhesion types and parasite biomass in adult and pediatric severe malaria and showed that similar var profiles, including upregulation of group A and DC8 var, predict severe malaria in adults and children. Among adults, patients with multiorgan complications presented infections with significantly higher parasite biomass without significant differences in var adhesion types. Conversely, pediatric patients with specific complications showed distinct var signatures. Cerebral malaria patients showed broadly increased expression of var genes, in particular group A and DC8 var, while children with severe malaria anemia were classified based on high transcription of DC8 var only. This study represents the first large multisite meta-analysis of var expression, and it demonstrates the presence of common var profiles in severe malaria patients of different ages across distant geographical sites, as well as syndrome-specific disease signatures. The complex associations between parasite biomass, var adhesion type, and clinical presentation revealed here represent the most comprehensive picture so far of the relationship between cytoadhesion, parasite load, and clinical syndrome.IMPORTANCEP. falciparum malaria can cause multiple disease complications that differ by patient age. Previous studies have attempted to address the roles of parasite adhesion and biomass in disease severity; however, these studies have been limited to single geographical sites, and there is limited understanding of how parasite adhesion and biomass interact to influence disease manifestations. In this meta-analysis, we compared parasite disease determinants in African children and Indian adults. This study demonstrates that parasite biomass and specific subsets of var genes are independently associated with detrimental outcomes in both childhood and adult malaria. We also explored how parasite var adhesion types and biomass play different roles in the development of specific severe malaria pathologies, including childhood cerebral malaria and multiorgan complications in adults. This work represents the largest study to date of the role of both var adhesion types and biomass in severe malaria.