Lineage-informative microhaplotypes for recurrence classification and spatio-temporal surveillance of Plasmodium vivax malaria parasites.

Challenges in classifying recurrent Plasmodium vivax infections constrain surveillance of antimalarial efficacy and transmission. Recurrent infections may arise from activation of dormant liver stages (relapse), blood-stage treatment failure (recrudescence) or reinfection. Molecular inference of familial relatedness (identity-by-descent or IBD) can help resolve the probable origin of recurrences. As whole genome sequencing of P. vivax remains challenging, targeted genotyping methods are needed for scalability. We describe a P. vivax marker discovery framework to identify and select panels of microhaplotypes (multi-allelic markers within small, amplifiable segments of the genome) that can accurately capture IBD. We evaluate panels of 50-250 microhaplotypes discovered in a global set of 615 P. vivax genomes. A candidate global 100-microhaplotype panel exhibits high marker diversity in the Asia-Pacific, Latin America and horn of Africa (median HE = 0.70-0.81) and identifies 89% of the polyclonal infections detected with genome-wide datasets. Data simulations reveal lower error in estimating pairwise IBD using microhaplotypes relative to traditional biallelic SNP barcodes. The candidate global panel also exhibits high accuracy in predicting geographic origin and captures local infection outbreak and bottlenecking events. Our framework is open-source enabling customised microhaplotype discovery and selection, with potential for porting to other species or data resources.

The mRNA content of plasma extracellular vesicles provides a window into molecular processes in the brain during cerebral malaria.

The impact of cerebral malaria on the transcriptional profiles of cerebral tissues is difficult to study using noninvasive approaches. We isolated plasma extracellular vesicles (EVs) from patients with cerebral malaria and community controls and sequenced their mRNA content. Deconvolution analysis revealed that EVs from cerebral malaria are enriched in transcripts of brain origin. We ordered the patients with cerebral malaria based on their EV-transcriptional profiles from cross-sectionally collected samples and inferred disease trajectory while using healthy community controls as a starting point. We found that neuronal transcripts in plasma EVs decreased with disease trajectory, whereas transcripts from glial, endothelial, and immune cells increased. Disease trajectory correlated positively with severity indicators like death and was associated with increased VEGFA-VEGFR and glutamatergic signaling, as well as platelet and neutrophil activation. These data suggest that brain tissue responses in cerebral malaria can be studied noninvasively using EVs circulating in peripheral blood.

Relation between the Dantu blood group variant and bacteraemia in Kenyan children: a population-based case control study.

BACKGROUND: The Dantu blood group variant protects against P. falciparum infections but its wider consequences have not been previously explored. Here, we investigate the impact of Dantu on susceptibility to bacteraemia. METHODS: We conducted a case-control study in children presenting with community-acquired bacteraemia to Kilifi County Hospital in Kenya between 1998 and 2010. We used logistic regression to test for associations between the Dantu marker SNP rs186873296 A>G and both all-cause and pathogen-specific bacteraemia under an additive model. We used date of admission as a proxy measure of malaria transmission intensity, given known differences in malaria prevalence over the course of the study. RESULTS: Dantu was associated with protection from all-cause bacteraemia (OR=0.81, p=0.014), the association being greatest in homozygotes (OR=0.30, p=0.013). This protection was shared across the major bacterial pathogens but, notably, was only significant during the era of high malaria-transmission pre-2003 (OR=0.79, p=0.023). CONCLUSIONS: Consistent with previous studies showing the indirect impact on bacteraemia risk of other malaria-associated red cell variants, our study also shows that Dantu is protective against bacteraemia via its effect on malaria risk. Dantu does not appear to be under balancing selection through an increased risk of bacterial infections.

20 years of BioMalPar: Building a collaborative malaria research network.

In 2004 the first annual BioMalPar meeting was held at EMBL Heidelberg, bringing together researchers from around the world with the goal of building connections between malaria research groups in Europe. Twenty years on it is time to reflect on what was achieved and to look ahead to the future.

APEX2-based proximity proteomic analysis identifies candidate interactors for Plasmodium falciparum knob-associated histidine-rich protein in infected erythrocytes.

The interaction of Plasmodium falciparum-infected red blood cells (iRBCs) with the vascular endothelium plays a crucial role in malaria pathology and disease. KAHRP is an exported P. falciparum protein involved in iRBC remodelling, which is essential for the formation of protrusions or "knobs" on the iRBC surface. These knobs and the proteins that are concentrated within them allow the parasites to escape the immune response and host spleen clearance by mediating cytoadherence of the iRBC to the endothelial wall, but this also slows down blood circulation, leading in some cases to severe cerebral and placental complications. In this work, we have applied genetic and biochemical tools to identify proteins that interact with P. falciparum KAHRP using enhanced ascorbate peroxidase 2 (APEX2) proximity-dependent biotinylation and label-free shotgun proteomics. A total of 30 potential KAHRP-interacting candidates were identified, based on the assigned fragmented biotinylated ions. Several identified proteins have been previously reported to be part of the Maurer's clefts and knobs, where KAHRP resides. This study may contribute to a broader understanding of P. falciparum protein trafficking and knob architecture and shows for the first time the feasibility of using APEX2-proximity labelling in iRBCs.

Novel stem cell technologies are powerful tools to understand the impact of human factors on Plasmodium falciparum malaria.

Plasmodium falciparum parasites have a complex life cycle, but the most clinically relevant stage of the disease is the invasion of erythrocytes and the proliferation of the parasite in the blood. The influence of human genetic traits on malaria has been known for a long time, however understanding the role of the proteins involved is hampered by the anuclear nature of erythrocytes that makes them inaccessible to genetic tools. Here we overcome this limitation using stem cells to generate erythroid cells with an in-vitro differentiation protocol and assess parasite invasion with an adaptation of flow cytometry to detect parasite hemozoin. We combine this strategy with reprogramming of patient cells to Induced Pluripotent Stem Cells and genome editing to understand the role of key genes and human traits in malaria infection. We show that deletion of basigin ablates invasion while deletion of ATP2B4 has a minor effect and that erythroid cells from reprogrammed patient-derived HbBart α-thalassemia samples poorly support infection. The possibility to obtain patient-secific and genetically modifed erythoid cells offers an unparalleled opportunity to study the role of human genes and polymorphisms in malaria allowing preservation of the genomic background to demonstrate their function and understand their mechanisms.