An open dataset of Plasmodium falciparum genome variation in 7,000 worldwide samples.

MalariaGEN is a data-sharing network that enables groups around the world to work together on the genomic epidemiology of malaria. Here we describe a new release of curated genome variation data on 7,000 Plasmodium falciparum samples from MalariaGEN partner studies in 28 malaria-endemic countries. High-quality genotype calls on 3 million single nucleotide polymorphisms (SNPs) and short indels were produced using a standardised analysis pipeline. Copy number variants associated with drug resistance and structural variants that cause failure of rapid diagnostic tests were also analysed.  Almost all samples showed genetic evidence of resistance to at least one antimalarial drug, and some samples from Southeast Asia carried markers of resistance to six commonly-used drugs. Genes expressed during the mosquito stage of the parasite life-cycle are prominent among loci that show strong geographic differentiation. By continuing to enlarge this open data resource we aim to facilitate research into the evolutionary processes affecting malaria control and to accelerate development of the surveillance toolkit required for malaria elimination

Pf7: an open dataset of Plasmodium falciparum genome variation in 20,000 worldwide samples

We describe the MalariaGEN Pf7 data resource, the seventh release of Plasmodium falciparum genome variation data from the MalariaGEN network.  It comprises over 20,000 samples from 82 partner studies in 33 countries, including several malaria endemic regions that were previously underrepresented.  For the first time we include dried blood spot samples that were sequenced after selective whole genome amplification, necessitating new methods to genotype copy number variations.  We identify a large number of newly emerging crt mutations in parts of Southeast Asia, and show examples of heterogeneities in patterns of drug resistance within Africa and within the Indian subcontinent.  We describe the profile of variations in the C-terminal of the csp gene and relate this to the sequence used in the RTS,S and R21 malaria vaccines.  Pf7 provides high-quality data on genotype calls for 6 million SNPs and short indels, analysis of large deletions that cause failure of rapid diagnostic tests, and systematic characterisation of six major drug resistance loci, all of which can be freely downloaded from the MalariaGEN website

PfEMP1 A-Type ICAM-1-Binding Domains Are Not Associated with Cerebral Malaria in Beninese Children

International audiencePfEMP1 is the major antigen involved in Plasmodium falciparum-infected erythrocyte sequestration in cerebrovascular endothelium. While some PfEMP1 domains have been associated with clinical phenotypes of malaria, formal associations between the expression of a specific domain and the adhesion properties of clinical isolates are limited. In this context, 73 cerebral malaria (CM) and 98 uncomplicated malaria (UM) Beninese children were recruited. We attempted to correlate the cytoadherence phenotype of Plasmodium falciparum isolates with the clinical presentation and the expression of specific PfEMP1 domains. Cytoadherence level on Hbec-5i and CHO-ICAM-1 cell lines and var genes expression were measured. We also investigated the prevalence of the ICAM-1-binding amino acid motif and dual receptor-binding domains, described as a potential determinant of cerebral malaria pathophysiology. We finally evaluated IgG levels against PfEMP1 recombinant domains (CIDRα1.4, DBLβ3, and CIDRα1.4-DBLβ3). CM isolates displayed higher cytoadherence levels on both cell lines, and we found a correlation between CIDRα1.4-DBLβ1/3 domain expression and CHO-ICAM-1 cytoadherence level. Endothelial protein C receptor (EPCR)-binding domains were overexpressed in CM isolates compared to UM whereas no difference was found in ICAM-1-binding DBLβ1/3 domain expression. Surprisingly, both CM and UM isolates expressed ICAM-1-binding motif and dual receptor-binding domains. There was no difference in IgG response against DBLβ3 between CM and UM isolates expressing ICAM-1-binding DBLβ1/3 domain. It raises questions about the role of this motif in CM pathophysiology, and further studies are needed, especially on the role of DBLβ1/3 without the ICAM-1-binding motif.IMPORTANCE Cerebral malaria pathophysiology remains unknown despite extensive research. PfEMP1 proteins have been identified as the main Plasmodium antigen involved in cerebrovascular endothelium sequestration, but it is unclear which var gene domain is involved in Plasmodium cytoadhesion. EPCR binding is a major determinant of cerebral malaria whereas the ICAM-1-binding role is still questioned. Our study confirmed the EPCR-binding role in CM pathophysiology with a major overexpression of EPCR-binding domains in CM isolates. In contrast, ICAM-1-binding involvement appears less obvious with A-type ICAM-1-binding and dual receptor-binding domain expression in both CM and UM isolates. We did not find any variations in ICAM-1-binding motif sequences in CM compared to UM isolates. UM and CM patients infected with isolates expressing the ICAM-1-binding motif displayed similar IgG levels against DBLβ3 recombinant protein. Our study raises interrogations about the role of these domains in CM physiopathology and questions their use in vaccine strategies against cerebral malaria. Copyrigh

Geographic origin of the samples used for genomic studies of <i>PFI1785w</i>.

<p>Geographic origin of the samples used for genomic studies of <i>PFI1785w</i>.</p

Skyline output and calibration range for GHTVDSELTTEDPVIQKK.

<p>Retention time is displayed on x axis and intensity for each ion or fragment is displayed on y axis. Each parent ion and fragment is shown in a different colour. AUC corresponds to the integration of signal under the curve (A). Regression line is obtained using AUC from each concentration point.</p

Linear regression line obtained with Skyline integration of raw files.

<p>Linear regression line obtained with Skyline integration of raw files.</p

Secondary structure of PFI1785w predicted using Phyre 2 and predictive 3D structure of amino-acid 224–360.

<p>Secondary structure of PFI1785w predicted using Phyre 2 and predictive 3D structure of amino-acid 224–360.</p

Peptide concentration measured within calibration range of each peptide.

<p>Peptide concentrations calculated by linear regression for each studied protein, y axis presents peptide concentration. Each dot represents one sample, and reference strains are displayed separately on the right of each plot (CSA+: CSA adhesion selected parasite strain, CSA-: non selected parasite strain). PAM samples and CSA+ parasite strains display a placental malaria like binding phenotype (in red), and UM samples and CSA- parasite strain display a uncomplicated malaria phenotype (in blue). PF14_0018, PFA_0410w, PFB0115w and PFI1785w are preferentially found in PAM like samples.</p

From genomic to LC-MS/MS evidence: Analysis of PfEMP1 in Benin malaria cases

International audienceBackground PfEMP1 is the major protein from parasitic origin involved in the pathophysiology of severe malaria, and PfEMP1 domain subtypes are associated with the infection outcome. In addition, PfEMP1 variability is endless and current publicly available protein repositories do not reflect the high diversity of the sequences of PfEMP1 proteins. The identification of PfEMP1 protein sequences expressed with samples remains challenging. The aim of our study is to identify the different PfEMP1 proteins variants expressed within patient samples, and therefore identify PfEMP1 proteins domains expressed by patients presenting uncomplicated malaria or severe malaria in malaria endemic setting in Cotonou, Benin. Methods We performed a multi-omic approach to decipher PfEMP1 expression at the patient's level in different clinical settings. Using a combination of whole genome sequencing approach and RNA sequencing, we were able to identify new PfEMP1 sequences and created a new custom protein database. This database was used for protein identification in mass spectrometry analysis. Results The differential expression analysis of RNAsequencing data shows an increased expression of the var domains transcripts DBL alpha 1.7, DBL alpha 1.1, DBL alpha 2 and DBL beta 12 in samples from patients suffering from Cerebral Malaria compared to Uncomplicated Malaria. Our approach allowed us to attribute PfEMP1 sequences to each sample and identify new peptides associated to PfEMP1 proteins in mass spectrometry. Conclusion We highlighted the diversity of the PfEMP1 sequences from field sample compared to reference sequences repositories and confirmed the validity of our approach. These findings should contribute to further vaccine development strategies based on PfEMP1 proteins

Identification of Plasmodium falciparum and host factors associated with cerebral malaria: description of the protocol for a prospective, case-control study in Benin (NeuroCM)

International audienceIn 2016, an estimated 216 million cases and 445 000 deaths of malaria occurred worldwide, in 91 countries. In Benin, malaria causes 26.8% of consultation and hospitalisation motif in the general population and 20.9% in children under 5 years old.The goal of the NeuroCM project is to identify the causative factors of neuroinflammation in the context of cerebral malaria. There are currently very few systematic data from West Africa on the aetiologies and management of non-malarial non-traumatic coma in small children, and NeuroCM will help to fill this gap. We postulate that an accurate understanding of molecular and cellular mechanisms involved in neuroinflammation may help to define efficient strategies to prevent and manage cerebral malaria.Ethics approval for the NeuroCM study has been obtained from Comité National d'Ethique pour la Recherche en santé of Benin (n°67/MS/DC/SGM/DRFMT/CNERS/SA; 10/17/2017). NeuroCM study has also been approved by Comité consultatif de déontologie et d'éthique of Institut de Recherche pour le Développement (IRD; 10/24/2017). The study results will be disseminated through the direct consultations with the WHO's Multilateral Initiative on Malaria (TDR-MIM) and Roll Back Malaria programme, through scientific meetings and peer-reviewed publications in scientific or medical journals, and through guidelines and booklets