Cerebral malaria is the most deadly manifestation of infection with Plasmodium falciparum. The pathology of cerebral malaria is characterized by the accumulation of infected erythrocytes (IEs) in the microvasculature of the brain caused by parasite adhesins on the surface of IEs binding to human receptors on microvascular endothelial cells. The parasite and host molecules involved in this interaction are unknown. We selected three P. falciparum strains (HB3, 3D7, and IT/FCR3) for binding to a human brain endothelial cell line (HBEC-5i). The whole transcriptome of isogenic pairs of selected and unselected parasites was analyzed using a variant surface antigen-supplemented microarray chip. After selection, the most highly and consistently up-regulated genes were a subset of group A-like var genes (HB3var3, 3D7_PFD0020c, ITvar7, and ITvar19) that showed 11-to >100-fold increased transcription levels. These var genes encode P. falciparum erythrocyte membrane protein (PfEMP)1 variants with distinct N-terminal domain types (domain cassette 8 or domain cassette 13). Antibodies to HB3var3 and PFD0020c recognized the surface of live IEs and blocked binding to HBEC-5i, thereby confirming the adhesive function of these variants. The clinical in vivo relevance of the HBEC-selected parasites was supported by significantly higher surface recognition of HBEC-selected parasites compared with unselected parasites by antibodies from young African children suffering cerebral malaria (Mann-Whitney test, P = 0.029) but not by antibodies from controls with uncomplicated malaria (Mann-Whitney test, P = 0.58). This work describes a binding phenotype for virulence-associated group A P. falciparum erythrocyte membrane protein 1 variants and identifies targets for interventions to treat or prevent cerebral malaria.adherence | pathogenesis | sequestration

A simplified protocol for the identification of Plasmodium species by semi-nested multiplex polymerase chain reaction (SnM-PCR) in human blood samples is compared with microscopical examination of thin and thick blood films in 2 field trials in Côte d'Ivoire and Cameroon. Also, dried blood spots or liquid blood collected from Dutch soldiers returning from Goma, Zaire (n = 141), Angola (n = 40), and from Marechaussee (Dutch border police) returning from various parts of the world (n = 161) were examined, together with miscellaneous other material obtained from laboratories and hospitals. The method is based on features of the small subunit nuclear ribosomal ribonucleic acid (RNA) gene (ssrDNA), a multicopy gene which possesses both highly conserved domains and domains characteristic for each of the 4 human malaria parasites. The first reaction of the SnM-PCR includes a universal reverse primer with 2 forward primers specific for Plasmodium and mammals, respectively. The mammalian-specific primer was included as a positive control to distinguish uninfected cases from simple PCR failures. The second PCR reaction includes a Plasmodium-specific forward primer plus species-specific reverse primers for P. vivax, P. ovale, P. falciparum and P. malariae. The technique worked better with samples collected in the field as dried blood spots on filter paper and heparinized blood rather than with frozen pelleted blood; it was more sensitive and more specific than the standard microscopical examination.

SignificanceSequestration of Plasmodium falciparum-infected erythrocytes (IEs) in the brain microvasculature underlies the pathology of cerebral malaria. Parasites that express P. falciparum erythrocyte membrane protein 1 of domain cassette (DC) 8 and DC13 types bind to brain endothelial cells. Recent studies, largely based on recombinant proteins, have identified endothelial protein C receptor (EPCR) as the key receptor for endothelial cell binding. Using DC8- and DC13-expressing IEs, we show that binding of DC13 IEs to brain endothelial cells is not EPCR-dependent and that cytoadhesion of EPCR-binding DC8 IEs to brain endothelial cells is blocked by human serum. This study highlights differences between recombinant protein and native protein in EPCR-binding properties and suggests that other receptors are also required for sequestration in cerebral malaria.

BackgroundCOVID-19 is associated with a dysregulated immune response but it is unclear how immune dysfunction contributes to the chronic morbidity persisting in many COVID-19 patients during convalescence (long COVID).MethodsWe assessed phenotypical and functional changes of monocytes in COVID-19 patients during hospitalization and up to 9 months of convalescence following COVID-19, respiratory syncytial virus (RSV) or influenza A (flu). Progressive fibrosing interstitial lung disease (PFILD) patients were included a positive control for severe, ongoing lung injury.ResultsMonocyte alterations in acute COVID-19 patients included aberrant expression of leucocyte migration molecules, continuing into convalescence (n=142) and corresponding to specific symptoms of long COVID. Long COVID patients with unresolved lung injury, indicated by sustained shortness of breath and abnormal chest radiology, were defined by high monocyte expression of chemokine receptor CXCR6 (p<0.0001) and adhesion molecule PSGL-1 (p<0.01), alongside preferential migration of monocytes towards CXCR6 ligand CXCL16 (p<0.05) which is abundantly expressed in the lung. Monocyte CXCR6 and lung CXCL16 were heightened in PFILD patients (p<0.001) confirming a role for the CXCR6-CXCL16 axis in ongoing lung injury. Conversely, monocytes from long COVID patients with ongoing fatigue exhibited sustained reduction of the prostaglandin-generating enzyme COX-2 (p<0.01) and CXCR2 expression (p<0.05). These monocyte changes were not present in RSV or flu convalescence.ConclusionsOur data define unique monocyte signatures that define subgroups of long COVID patients, indicating a key role for monocyte migration in COVID-19 pathophysiology. Targeting these pathways may provide novel therapeutic opportunities in COVID-19 patients with persistent morbidity.