John W. Barnwell

Comparative genomics of the neglected human malaria parasite Plasmodium vivax.

The human malaria parasite Plasmodium vivax is responsible for 25–40% of the ∼515 million annual cases of malaria worldwide. Although seldom fatal, the parasite elicits severe and incapacitating clinical symptoms and often causes relapses months after a primary infection has cleared. Despite its importance as a major human pathogen, P. vivax is little studied because it cannot be propagated continuously in the laboratory except in non-human primates. We sequenced the genome of P. vivax to shed light on its distinctive biological features, and as a means to drive development of new drugs and vaccines. Here we describe the synteny and isochore structure of P. vivax chromosomes, and show that the parasite resembles other malaria parasites in gene content and metabolic potential, but possesses novel gene families and potential alternative invasion pathways not recognized previously. Completion of the P. vivax genome provides the scientific community with a valuable resource that can be used to advance investigation into this neglected species.

Plasmodium vivax interaction with the human Duffy blood group glycoprotein: Identification of a parasite receptor-like protein

The interaction between merozoites of the human pathogen, Plasmodium vivax, and the Duffy blood group glycoprotein on the surface of human erythrocytes is essential for the invasion of erythrocytes and the survival of the parasite. We have identified a P. vivax protein of 135 to 140 kDa which binds with receptor-like specificity to the human Duffy blood group glycoprotein. This interaction can be specifically inhibited by purified Duffy glycoprotein and by pretreating erythrocytes with a monoclonal antibody directed against a novel Duffy determinant. A protein with similar specificity for the Duffy glycoprotein from the phylogenetically related simian malaria, P. knowlesi, is shown to be immunologically related by the generation of cross-reactive antibodies. Despite their shared properties, these two Duffy associating proteins from P. vivax and P. knowlesi differ in some aspects of their interaction with the Duffy glycoprotein. The identification of these proteins will help elucidate the molecular mechanisms of erythrocyte invasion by Plasmodium.

A human 88-kD membrane glycoprotein (CD36) functions in vitro as a receptor for a cytoadherence ligand on Plasmodium falciparum-infected erythrocytes.

Plasmodium falciparum-infected erythrocytes (IE) specifically adhere to vascular endothelium in vivo and to human endothelial cells, some human melanoma cell lines, and human monocytes in vitro. The tissue cell receptor for a ligand on the surface of the infected erythrocytes is an Mr 88,000 glycoprotein (GP88) recognized by the MAb OKM5, which also blocks cytoadherence of IE. Isolated, affinity-purified GP88 (CD36) competitively blocks cytoadherence and when absorbed to plastic surfaces, specifically binds P. falciparum IE. Additionally, monoclonal and polyclonal antibodies to GP88 block cytoadherence to both target cells and immobilized GP88. Binding to GP88 by IE is unaffected by the absence of calcium or the absence of thrombospondin, a putative mediator for cytoadherence of P. falciparum IE. Thus, GP88 (CD36), which has been demonstrated to be the same as platelet glycoprotein IV, interacts directly with P. falciparum IE, presumably via a parasite-induced ligand exposed on the surface of the infected erythrocytes. CD36 is shown to be present on brain endothelium in both individuals without malaria and individuals with cerebral malaria. This would suggest that factors other than just cerebral sequestration of IE play an initiating role in the genesis of cerebral malaria.

A reticulocyte-binding protein complex of plasmodium vivax merozoites

Plasmodium vivax merozoites primarily invade reticulocytes. The basis of this restricted host cell preference has been debated. Here we introduce two novel P. vivax proteins that comigrate on reducing SDS-polyacrylamide gels, colocalize at the apical pole of merozoites, and adhere specifically to reticulocytes. The genes encoding these proteins, P. vivax reticulocyte-binding proteins 1 and 2 (PvRBP-1 and PvRBP-2), have been cloned and analyzed. Homologous genes are evident in the closely related simian malaria parasite, P. cynomolgi, which also prefers to invade reticulocytes, but are not evident in the genome of another related simian malaria parasite, P. knowlesi, which invades all red blood cell subpopulations. Native PvRBP-1 is likely a transmembrane-anchored disulfide-linked protein, and along with PvRBP-2 may function as an adhesive protein complex. We propose that the RBPs of P. vivax, and homologous proteins of P. cynomolgi, function to target the reticulocyte subpopulation of red blood cells for invasion.

Ensuring quality and access for malaria diagnosis: how can it be achieved?

The replacement of conventional antimalarial drugs with high-cost, artemisinin-based alternatives has created a gap in the successful management of malaria. This gap reflects an increased need for accurate disease diagnosis that cannot be met by traditional microscopy techniques. The recent introduction of rapid diagnostic tests (RDTs) has the potential to meet this need, but successful RDT implementation has been curtailed by poor product performance, inadequate methods to determine the quality of products and a lack of emphasis and capacity to deal with these issues. Economics and a desire for improved case management will result in the rapid growth of RDT use in the coming years. However, for their potential to be realized, it is crucial that high-quality RDT products that perform reliably and accurately under field conditions are made available. In achieving this goal, the shift from symptom-based diagnosis to parasite-based management of malaria can bring significant improvements to tropical fever management, rather than represent a further burden on poor, malaria-endemic populations and their overstretched health services.

Phenotypic variation of Plasmodium falciparum merozoite proteins directs receptor targeting for invasion of human erythrocytes

The members of the phylum Apicomplexa parasitize a wide range of eukaryotic host cells. Plasmodium falciparum, responsible for the most virulent form of malaria, invades human erythrocytes using several specific and high affinity ligand–receptor interactions that define invasion pathways. We find that members of the P.falciparum reticulocyte‐binding homolog protein family, PfRh2a and PfRh2b, are expressed variantly in different lines. Targeted gene disruption shows that PfRh2b mediates a novel invasion pathway and that it functions independently of other related proteins. Phenotypic variation of the PfRh protein family allows P.falciparum to exploit different patterns of receptors on the erythrocyte surface and thereby respond to polymorphisms in erythrocyte receptors and to evade the host immune system.

A Large Proportion of P. falciparum Isolates in the Amazon Region of Peru Lack pfhrp2 and pfhrp3: Implications for Malaria Rapid Diagnostic Tests

Background Malaria rapid diagnostic tests (RDTs) offer significant potential to improve the diagnosis of malaria, and are playing an increasing role in malaria case management, control and elimination. Peru, along with other South American countries, is moving to introduce malaria RDTs as components of malaria control programmes supported by the Global Fund for AIDS, TB and malaria. The selection of the most suitable malaria RDTs is critical to the success of the programmes. Methods Eight of nine microscopy positive P. falciparum samples collected in Iquitos, Peru tested negative or weak positive using HRP2-detecting RDTs. These samples were tested for the presence of pfhrp2 and pfhrp3 and their flanking genes by PCR, as well as the presence of HRP proteins by ELISA. To investigate for geographic extent of HRP-deleted parasites and their temporal occurrence a retrospective study was undertaken on 148 microscopy positive P. falciparum samples collected in different areas of the Amazon region of Peru. Findings Eight of the nine isolates lacked the pfhrp2 and/or pfhrp3 genes and one or both flanking genes, and the absence of HRP was confirmed by ELISA. The retrospective study showed that 61 (41%) and 103 (70%) of the 148 samples lacked the pfhrp2 or pfhrp3 genes respectively, with 32 (21.6%) samples lacking both hrp genes. Conclusions This is the first documentation of P. falciparum field isolates lacking pfhrp2 and/or pfhrp3. The high frequency and wide distribution of different parasites lacking pfhrp2 and/or pfhrp3 in widely dispersed areas in the Peruvian Amazon implies that malaria RDTs targeting HRP2 will fail to detect a high proportion of P. falciparum in malaria-endemic areas of Peru and should not be used. RDTs detecting parasite LDH or aldolase and quality microscopy should be use for malaria diagnosis in this region. There is an urgent need for investigation of the abundance and geographic distribution of these parasites in Peru and neighbouring countries.

Two Nonrecombining Sympatric Forms of the Human Malaria Parasite Plasmodium ovale Occur Globally

BACKGROUND Malaria in humans is caused by apicomplexan parasites belonging to 5 species of the genus Plasmodium. Infections with Plasmodium ovale are widely distributed but rarely investigated, and the resulting burden of disease is not known. Dimorphism in defined genes has led to P. ovale parasites being divided into classic and variant types. We hypothesized that these dimorphs represent distinct parasite species. METHODS Multilocus sequence analysis of 6 genetic characters was carried out among 55 isolates from 12 African and 3 Asia-Pacific countries. RESULTS Each genetic character displayed complete dimorphism and segregated perfectly between the 2 types. Both types were identified in samples from Ghana, Nigeria, São Tomé, Sierra Leone, and Uganda and have been described previously in Myanmar. Splitting of the 2 lineages is estimated to have occurred between 1.0 and 3.5 million years ago in hominid hosts. CONCLUSIONS We propose that P. ovale comprises 2 nonrecombining species that are sympatric in Africa and Asia. We speculate on possible scenarios that could have led to this speciation. Furthermore, the relatively high frequency of imported cases of symptomatic P. ovale infection in the United Kingdom suggests that the morbidity caused by ovale malaria has been underestimated.

The malaria parasite Plasmodium vivax exhibits greater genetic diversity than Plasmodium falciparum

We sequenced and annotated the genomes of four P. vivax strains collected from disparate geographic locations, tripling the number of genome sequences available for this understudied parasite and providing the first genome-wide perspective of global variability in this species. We observe approximately twice as much SNP diversity among these isolates as we do among a comparable collection of isolates of P. falciparum, a malaria-causing parasite that results in higher mortality. This indicates a distinct history of global colonization and/or a more stable demographic history for P. vivax relative to P. falciparum, which is thought to have undergone a recent population bottleneck. The SNP diversity, as well as additional microsatellite and gene family variability, suggests a capacity for greater functional variation in the global population of P. vivax. These findings warrant a deeper survey of variation in P. vivax to equip disease interventions targeting the distinctive biology of this neglected but major pathogen.

The Role of Animal Models for Research on Severe Malaria

In light of the recent controversies over the role of animal models for research into the development of new treatments for severe malaria, particularly cerebral disease, a group of scientists came together to discuss the relative merits of a range of animal models and their overlap with the complex clinical syndromes of human disease. While it was not possible to fully resolve differences over the utility of the Plasmodium berghei ANKA model of experimental cerebral malaria, the meeting did bring the two research communities closer together to identify further work to provide information needed to validate the model and revitalise the development of other animal models displaying features of human pathology. The driving force behind this was the desire to ensure better translation of experimental findings into effective treatments for severe malaria.