Plasmodium vivax in Africa: hidden in plain sight?

People who live in tropical Africa, south of the Sahara, are predominantly negative for the Duffy blood-group antigen, which mediates invasion of reticulocytes by Plasmodium vivax. Recent reports of a parasite that was molecularly diagnosed as P. vivax from populations who are suspected, or known, to be Duffy negative confound a large body of evidence that states that invasion of P. vivax requires the Duffy antigen. If confirmed, one of several possible explanations is that P. vivax, which originated in Asia, is now evolving to exploit alternate invasion receptors in Africa.     

The origins of African Plasmodium vivax; insights from mitochondrial genome sequencing

Plasmodium vivax, the second most prevalent of the human malaria parasites, is estimated to affect 75 million people annually. It is very rare, however, in west and central Africa, due to the high prevalence of the Duffy negative phenotype in the human population. Due to its rarity in Africa, previous studies on the phylogeny of world-wide P. vivax have suffered from insufficient samples of African parasites. Here we compare the mitochondrial sequence diversity of parasites from Africa with those from other areas of the world, in order to investigate the origin of present-day African P. vivax. Mitochondrial genome sequencing revealed relatively little polymorphism within the African population compared to parasites from the rest of the world. This, combined with sequence similarity with parasites from India, suggests that the present day African P. vivax population in humans may have been introduced relatively recently from the Indian subcontinent. Haplotype network analysis also raises the possibility that parasites currently found in Africa and South America may be the closest extant relatives of the ancestors of the current world population. Lines of evidence are adduced that this ancestral population may be from an ancient stock of P. vivax in Africa.     

Host switch leads to emergence of Plasmodium vivax malaria in humans

The geographical origin of Plasmodium vivax, the most widespread human malaria parasite, is controversial. Although genetic closeness to Asian primate malarias has been confirmed by phylogenetic analyses, genetic similarities between P. vivax and Plasmodium simium, a New World primate malaria, suggest that humans may have acquired P. vivax from New World monkeys or vice versa. Additionally, the near fixation of the Duffy-negative blood type (FY x B(null)/FY x B(null)) in West and Central Africa, consistent with directional selection, and the association of Duffy negativity with complete resistance to vivax malaria suggest a prolonged period of host-parasite coevolution in Africa. Here we use Bayesian and likelihood methods in conjunction with cophylogeny mapping to reconstruct the genetic and coevolutionary history of P. vivax from the complete mitochondrial genome of 176 isolates as well as several closely related Plasmodium species. Taken together, a haplotype network, parasite migration patterns, demographic history, and cophylogeny mapping support an Asian origin via a host switch from macaque monkeys.     

Speculations on the origins of Plasmodium vivax malaria

It is likely that Plasmodium vivax diverged approximately 2 million years ago from a group of malaria parasites which are now endemic in monkeys and apes in southern Asia. In those times, primates were spread throughout most of Eurasia and Africa, indicating an Old World location, but nothing more precise, for the place of divergence of P. vivax. From approximately 1 million years ago, the Ice Ages would have isolated human malaria, including P. vivax, into humid temperate or warm climate refuges around the Mediterranean, in sub-Saharan Africa and in south and east Asia. As there appears to be no record of humans in south and east Asia from 100,000 to 60,000 years ago, they might not have passed on their parasites, including P. vivax, to modern humans entering the region after this time. Today, all P. vivax might be descended from parasites which infected human populations in the Mediterranean region and in sub-Saharan Africa during the last Ice Age, between 100,000 and 20,000 years ago. Evidence for the latter is provided by the presence of very high frequency RBC Duffy negativity in sub-Saharan Africa.     

Getting down to malarial nuts and bolts: the interaction between Plasmodium vivax merozoites and their host erythrocytes

Of the four Plasmodium species that routinely cause malaria in humans, Plasmodium falciparum is responsible for the majority of malaria mortality and consequently gets most of the headlines. Outside Africa, however, more malaria cases are caused by its distant cousin Plasmodium vivax, resulting in a daunting morbidity and economic burden for countries across Asia and the Americas. Plasmodium life cycles are complex, but the symptoms and pathology of malaria occur during the blood phase, when merozoites recognize and invade erythrocytes, initiating a developmental programme that culminates in lysis of the erythrocyte and release of multiple daughter merozoites. P. vivax merozoites are dependent on a single host cell receptor for erythrocyte invasion, the Duffy antigen receptor for chemokines, and humans that do not express this receptor on the surface of their erythrocytes are immune to P. vivax infection. This essential receptor-ligand interaction is addressed from both the host and parasite side in two papers in this issue of Molecular Microbiology, with important implications for plans to develop a P. vivax vaccine.     

Biochemical, biophysical, and functional characterization of bacterially expressed and refolded receptor binding domain of Plasmodium vivax duffy-binding protein

Invasion of erythrocytes by malaria parasites is mediated by specific molecular interactions. Plasmodium vivax is completely dependent on interaction with the Duffy blood group antigen to invade human erythrocytes. The P. vivax Duffy-binding protein, which binds the Duffy antigen during invasion, belongs to a family of erythrocyte-binding proteins that also includes Plasmodium falciparum sialic acid binding protein and Plasmodium knowlesi Duffy binding protein. The receptor binding domains of these proteins lie in a conserved, N-terminal, cysteine-rich region, region II, found in each of these proteins. Here, we have expressed P. vivax region II (PvRII), the P. vivax Duffy binding domain, in Escherichia coli. Recombinant PvRII is incorrectly folded and accumulates in inclusion bodies. We have developed methods to refold and purify recombinant PvRII in its functional conformation. Biochemical, biophysical, and functional characterization confirms that recombinant PvRII is pure, homogeneous, and functionally active in that it binds Duffy-positive human erythrocytes with specificity. Refolded PvRII is highly immunogenic and elicits high titer antibodies that can inhibit binding of P. vivax Duffy-binding protein to erythrocytes, providing support for its development as a vaccine candidate for P. vivax malaria. Development of methods to produce functionally active recombinant PvRII is an important step for structural studies as well as vaccine development.     

Plasmodium yoelii: a differential fluorescent technique using Acridine Orange to identify infected erythrocytes and reticulocytes in Duffy knockout mouse

Both human malarial parasite Plasmodium vivax and mouse malaria parasite Plasmodium yoelii use Duffy protein as the receptor for invasion and they preferentially invade reticulocytes. Recently, it has been shown that P. yoelii invades mouse reticulocytes by a Duffy independent pathway. Parasite invasion is generally visualized by time consuming staining procedures with dyes like Giemsa or Wright-Giemsa. Fluorochromatic dye like Acridine Orange has been used for instantaneous detection of parasites in RBCs. Acridine Orange binds to both DNA and RNA but with different emission spectra; and the binding can be distinguished with a fluorescent microscope using a green or a red filter, respectively. We have used this differential emission of Acridine Orange to determine P. yoelii invasion into erythrocytes and reticulocytes of Duffy positive and Duffy knockout mice. Moreover, we show that this method can be used to determine the maturity of reticulocytes in the peripheral blood of anemic mice.     

Targeted deletion of Plasmodium knowlesi Duffy binding protein confirms its role in junction formation during invasion

Red cell invasion by Plasmodium merozoites involves multiple steps such as attachment, apical reorientation, junction formation and entry into a parasitophorous vacuole. These steps are mediated by specific molecular interactions. P. vivax and the simian parasite P. knowlesi require interaction with the Duffy blood group antigen to invade human erythrocytes. P. vivax and P. knowlesi Duffy binding proteins (PvDBP and PkDBP), which bind the Duffy antigen during invasion, share regions of sequence homology and belong to a family of erythrocyte binding proteins (EBPs). By deletion of the gene that encodes PkDBP, we demonstrate that interaction of PkDBP with the Duffy antigen is absolutely necessary for invasion of human erythrocytes by P. knowlesi. Electron microscopy studies reveal that PkDBP knockout parasites are unable to form a junction with human erythrocytes. The interaction of PkDBP with the Duffy antigen is thus necessary for the critical step of junction formation during invasion. These studies provide support for development of intervention strategies that target EBPs to inhibit junction formation and block erythrocyte invasion by malaria parasites.     

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.     

Mass blood survey for malaria: pooling and realtime PCR combined with expert microscopy in north-west Thailand

ABSTRACT: BACKGROUND: Asymptomatic carriage of Plasmodium falciparum and Plasmodium vivax is common in both low-and high-transmission settings and represents an important reservoir of infection that needs to be targeted if malaria elimination is to succeed. METHODS: Mass blood examinations (475 individuals) were conducted in two villages in Mae Hong Son, an area of endemic but low-transmission malaria in the north-west of Thailand. The microscopist at the local malaria clinic did not detect any infections. Pools of four samples were screened by real-time PCR; individual members of all of the positive pools were then re-examined by expert microscopy and by a second species-specific PCR reaction. RESULTS: Eight subjects were found to be positive by both PCR and expert microscopy and one was found to be positive by PCR alone. The slides contained asexual stage parasites of P. vivax, P. falciparum and Plasmodium malariae, but no gametocytes. The local clinic was notified within two to eight days of the survey. CONCLUSION: A combination of pooling, real-time PCR and expert microscopy provides a feasible approach to identifying and treating asymptomatic malaria infections in a timely manner.     

An update on the search for a Plasmodium vivax vaccine

Although Plasmodium falciparum is the leading cause of morbidity and mortality due to malaria worldwide, nearly 2.5 billion people, mostly outside Africa, are also at risk from malaria caused by Plasmodium vivax infection. Currently, almost all efforts to develop a malaria vaccine have focused on P. falciparum. For example, there are 23 P. falciparum vaccine candidates undergoing advanced clinical studies and only two P. vivax vaccine candidates being tested in preliminary (Phase I) clinical trials, with few others being assessed in preclinical studies. More investment and a greater effort toward the development of P. vivax vaccine components for a multi-species vaccine are required. This is mainly because of the wide geographical coexistence of both parasite species but also because of increasing drug resistance, recent observations of severe and lethal P. vivax cases and relapsing parasite behaviour. Availability of the P. vivax genome has contributed to antigen discovery but new means to test vaccines in future trials remain to be designed.     

Search for malaria parasites by PCR and Southern blot in patients with imported malaria in Italy

The present study evaluates the sensitivity, specificity and usefulness of a PCR method with Southern blot hybridization to detect malaria parasites in blood samples from subjects with a suspect clinical diagnosis of malaria imported to Italy. Plasmodia were detected by PCR using a genus-specific primer-set corresponding to the sequences common to P. falciparum, P. vivax, P. malariae and P. ovale, as described by Arai (Arai et al., Nucleosides Nucleotides, 1994, 13, 1363-1364) and Kimura (Kimura et al., Journal of Clinical Microbiology, 1995, 33, 2342-2346). In addition, four distinct tandemly repetitive species-specific probes, described by Kawai (Kawai et al., Analytical Biochimestry, 1993, 209, 63-69), were synthesized to specifically detect the four malaria parasites species by Southern blot hybridization. Fifteen blood samples from 12 patients (7 with malaria) were tested and the genus-specific PCR method showed a sensitivity of 100% and a specificity of 100%, when compared to microscopy, in detecting malaria parasites in the tested blood samples. Fourteen samples (nine were positive and five negative by PCR) were confirmed by Southern blot, whereas only one P. vivax positive sample was not hybridized with the species-specific probes. We conclude that this PCR method with Southern blot hybridization may be useful in detecting malaria parasites in patients with malaria imported to Italy.     

High rate of detection of mixed infections of Plasmodium vivax and Plasmodium falciparum in South-East of Iran, using nested PCR

Sistan and Baluchestan province, South-East of Iran, has been reported as an endemic area of malaria [Sadrizadeh B. Malaria in the world, in the eastern Mediterranean region and in Iran: Review article. WHO/EMRO Report 2001: 1-13.]. The main objective of this research was to perform rapid and correct diagnoses of malaria infection. Blood specimens were collected from 140 suspected volunteers. The Giemsa-stained slides examination and nested PCR for amplification of the Plasmodium small subunit ribosomal genes (ssrRNA) were utilized. The results demonstrated 118 out of 140 cases (84.3%) positive for malaria parasites, including 60.7%, 20.7% and 2.9% as having Plasmodium vivax (P.v), Plasmodium falciparum (P.f) and mixed infections (P.v+P.f), respectively by microscopy. The nested PCR detected malaria parasites in 134 samples (94.3%), consisting of 51.4% P.v, 12.6% P.f and 29.3% mixed infections. The PCR analysis detected 37 cases of mixed infections more than that of the routine microscopy. These results suggested that there are a considerable number of cases with mixed infections in the study area that mainly remain undiagnosed by microscopy. It is also concluded that the nested PCR is a suitable complement to microscopy for accurate specific diagnosis of malaria species in field.     

Sulphated tyrosines mediate association of chemokines and Plasmodium vivax Duffy binding protein with the Duffy antigen/receptor for chemokines (DARC)

Plasmodium vivax is one of four Plasmodium species that cause human malaria. P. vivax and a related simian malaria parasite, Plasmodium knowlesi, invade erythrocytes by binding the Duffy antigen/receptor for chemokines (DARC) through their respective Duffy binding proteins. Here we show that tyrosines 30 and 41 of DARC are modified by addition of sulphate groups, and that the sulphated tyrosine 41 is essential for association of the Duffy binding proteins of P. vivax (PvDBP) and P. knowlesi (PkDaBP) with DARC-expressing cells. These sulphated tyrosines also participate in the association of DARC with each of its four known chemokine ligands. Alteration of tyrosine 41 to phenylalanine interferes with MCP-1, RANTES and MGSA association with DARC, but not with that of IL8. In contrast, alteration of tyrosine 30 to phenylalanine interferes with the association of IL8 with DARC. A soluble sulphated amino-terminal domain of DARC, but not one modified to phenylalanine at residue 41, can be used to block the association of PvDBP and PkDaBP with red blood cells, with an IC50 of approximately 5 nM. These data are consistent with a role for tyrosine sulphation in the association of many or most chemokines with their receptors, and identify a key molecular determinant of erythrocyte invasion by P. vivax.     

Unusual plasmodium malariae-like parasites in southeast Asia

During malaria surveys in Myanmar, 2 peculiar forms of Plasmodium malariae-like parasites were found. The morphologies of their early trophozoite stages were distinct from that of the typical P. malariae, resembling instead that of Plasmodium vivax, var. minuta, reported by Emin, and Plasmodium tenue, reported by Stephens, both in 1914. Two polymerase chain reaction (PCR)-based diagnoses, which target the same regions in the small subunit ribosomal RNA (SSUrRNA) genes, indicated that these parasites were new variant forms of P. malariae and that they could be separated into 2 genetic types that correlated with the 2 morphological types. Sequence analysis of the SSUrRNA and the circumsporozoite protein genes revealed that they were distinct both from each other and from other known P. malariae isolates and that the P. tenue-like type was closer to a monkey quartan malaria parasite, Plasmodium brasilianum. These results illustrate that the microscopic appearance of human P. malariae parasites may be more varied than previously assumed and suggest the value of molecular tools in the evaluation of malaria morphological variants.     

Species-specific features of DARC, the primate receptor for Plasmodium vivax and Plasmodium knowlesi

The DARC (Duffy antigen/receptor for chemokines) gene, also called Duffy or FY, encodes a membrane-bound chemokine receptor. Two malaria parasites, Plasmodium vivax and Plasmodium knowlesi, use DARC to trigger internalization into red blood cells. Although much has been reported on the evolution of DARC null alleles, little is known about the evolution of the coding portion of this gene or the role that protein sequence divergence in this receptor may play in disease susceptibility or zoonosis. Here, we show that the Plasmodium interaction domain of DARC is nearly invariant in the human population, suggesting that coding polymorphism there is unlikely to play a role in differential susceptibility to infection. However, an analysis of DARC orthologs from 35 simian primate species reveals high levels of sequence divergence in the Plasmodium interaction domain. Signatures of positive selection in this domain indicate that species-specific mutations in the protein sequence of DARC could serve as barriers to the transmission of Plasmodium between primate species.     

Clinical proteomics of the neglected human malarial parasite Plasmodium vivax

Recent reports highlight the severity and the morbidity of disease caused by the long neglected malaria parasite Plasmodium vivax. Due to inherent difficulties in the laboratory-propagation of P. vivax, the biology of this parasite has not been adequately explored. While the proteome of P. falciparum, the causative agent of cerebral malaria, has been extensively explored from several sources, there is limited information on the proteome of P. vivax. We have, for the first time, examined the proteome of P. vivax isolated directly from patients without adaptation to laboratory conditions. We have identified 153 proteins from clinical P. vivax, majority of which do not show homology to any previously known gene products. We also report 29 new proteins that were found to be expressed in P. vivax for the first time. In addition, several proteins previously implicated as anti-malarial targets, were also found in our analysis. Most importantly, we found several unique proteins expressed by P. vivax.This study is an important step in providing insight into physiology of the parasite under clinical settings.     

Duffy-null promoter heterozygosity reduces DARC expression and abrogates adhesion of the P. vivax ligand required for blood-stage infection

The Duffy blood group antigen is an essential receptor for Plasmodium vivax entry into erythrocytes in a process mediated by the parasite ligand, the Duffy binding protein (DBP). Recently, individuals living in a malaria endemic region of Papua New Guinea were identified as heterozygous for a new allele conferring Duffy negativity, which results in 50% less Duffy antigen on their erythrocytes. We demonstrate that DBP adherence to erythrocytes is significantly reduced for erythrocytes from heterozygous individuals who carry one Duffy antigen negativity allele. These data provide evidence that emergence of this new allelic form of Duffy negativity is correlated with resistance against vivax malaria.