Analysis of the genetic variability of PvMSP-3α among Plasmodium vivax in Brazilian field isolates

Reliable molecular markers are essential for a better understanding of the molecular epidemiology of Plasmodium vivax, which is a neglected human malaria parasite. The aim of this study was to analyze the genetic diversity of P. vivax isolates from the Brazilian Amazon using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the highly polymorphic merozoite surface protein-3alpha (PvMSP-3α) gene. To accomplish this, 60 isolates of P. vivax from different endemic areas in the Brazilian Amazon were collected. The PvMSP-3α gene was amplified by nested-PCR. Three major types of the PvMSP-3α locus were detected at different frequencies: type A (68%), B (15%) and C (17%). A single sample showed two PCR fragments, which corresponded to infection with types A and C. PCR-RFLP analysis using the HhaI restriction enzyme for 52 isolates clearly identified 11 haplotypes, eight of which were from type A, two from type B and only one from type C. Seven other isolates did not show a clear pattern using PCR-RFLP. This result might be due to multiple clone infections. This study showed a high diversity of the PvMSP-3α gene among P. vivax isolates from the Brazilian Amazon, but also indicated that the detection performance of PCR-RFLP of the PvMSP-3α gene may not be sufficient to detect multiple clone infections.     

The genetic diversity of Plasmodium vivax populations

Little is known of the genetic diversity and population structure of Plasmodium vivax, a debilitating and highly prevalent malaria parasite of humans. This article reviews the known polymorphic genetic markers, summarizes current data on the population structure of this parasite and discusses future prospects for using knowledge of the genetic diversity to improve control measures.     

A Plasmodium vivax vaccine candidate displays limited allele polymorphism, which does not restrict recognition by antibodies.

BACKGROUND: The 19 kDa C-terminal region of the merozoite surface protein 1 (MSP1(19)) has been suggested as candidate for part of a subunit vaccine against malaria. A major concern in vaccine development is the polymorphism observed in different plasmodial strains. The present study examined the extension and immunological relevance of the allelic polymorphism of the MSP1(19) from Plasmodium vivax, a major human malaria parasite. MATERIALS AND METHODS: We cloned and sequenced 88 gene fragments representing the MSP1(19) from 28 Brazilian isolates of P. vivax. Subsequently, we evaluated the reactivity of rabbit polyclonal antibodies, a monoclonal antibody, and a panel of 80 human sera to bacterial and yeast recombinant proteins representing the two allelic forms of P. vivax MSP1(19) described thus far. RESULTS: We observed that DNA sequences encoding MSP1(19) were not as variable as the equivalent region of other species of Plasmodium, being conserved among Brazilian isolates of P. vivax. Also, we found that antibodies are directed mainly to conserved epitopes present in both allelic forms of the protein. CONCLUSIONS: Our findings suggest that the use of MSP1(19) as part of a subunit vaccine against P. vivax might be greatly facilitated by the limited genetic polymorphism and predominant recognition of conserved epitopes by antibodies.     

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.     

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.     

Variant genes and the spleen in Plasmodium vivax malaria

It is generally accepted that Plasmodium vivax, the most widely distributed human malaria, does not cytoadhere in the deep capillaries of inner organs and thus this malaria parasite must have evolved splenic evasion mechanism in addition to sequestration. The spleen is a uniquely adapted lymphoid organ whose central function is the selective clearance of cell and other particles from the blood, and microbes including malaria. Splenomegaly is a hallmark of malaria and no other disease seems to exacerbate this organ as this disease does. Besides this major selective clearance function however, the spleen is also an erythropoietic organ which, under stress conditions, can be responsible for close to 40% of the RBC populations. Data obtained in experimental infections of human patients with P. vivax showed that anaemia is associated with acute and chronic infections and it has been postulated that the continued parasitemia might have been sufficient to infect and destroy most circulating reticulocytes. We review here the basis of our current knowledge of variant genes in P. vivax and the structure and function of the spleen during malaria. Based on this data, we propose that P. vivax specifically adhere to barrier cells in the human spleen allowing the parasite to escape spleen-clearance while favouring the release of merozoites in an environment where reticulocytes, the predominant, if not exclusive, host cell of P. vivax, are stored before their release into circulation to compensate for the anaemia associated with vivax malaria.     

The Duffy binding protein as a key target for a Plasmodium vivax vaccine: lessons from the Brazilian Amazon

Plasmodium vivax infects human erythrocytes through a major pathway that requires interaction between an apical parasite protein, the Duffy binding protein (PvDBP) and its receptor on reticulocytes, the Duffy antigen/receptor for chemokines (DARC). The importance of the interaction between PvDBP (region II, DBPII) and DARC to P. vivax infection has motivated our malaria research group at Oswaldo Cruz Foundation (state of Minas Gerais, Brazil) to conduct a number of immunoepidemiological studies to characterise the naturally acquired immunity to PvDBP in populations living in the Amazon rainforest. In this review, we provide an update on the immunology and molecular epidemiology of PvDBP in the Brazilian Amazon - an area of markedly unstable malaria transmission - and compare it with data from other parts of Latin America, as well as Asia and Oceania.     

The paroxysm of Plasmodium vivax malaria

The paroxysms of Plasmodium vivax malaria are antiparasite responses that, although distressing to the human host, almost never impart serious acute pathology. Using plasma and blood cells from P. vivax patients, the cellular and noncellular mediators of these events have been studied ex vivo. The host response during a P. vivax paroxysm was found to involve T cells, monocytes and neutrophils, and the activity, among others, of the pyrogenic cytokines tumor necrosis factor alpha and interleukin 2 in addition to granulocyte macrophage-colony stimulating factor. However, interferon gamma activity, associated with serious acute pathogenesis in other studies on malaria, was absent. Induction of the cytokines active during a P. vivax paroxysm depends upon the presence of parasite products, which are released into the plasma before the paroxysm. Chemical identification of these natural parasite products will be important for our understanding of pathogenesis and protection in malaria.     

Determination of the Plasmodium vivax schizont stage proteome

With the genome of the malaria parasite Plasmodium vivax sequenced, it is important to determine the proteomes of the parasite in order to assist efforts in antigen and drug target discovery. Since a method for continuous culture of P. vivax parasite is not available, we tried to study the proteome of the erythrocytic stages using fresh parasite isolates from patients. In schizont-enriched samples, 316 proteins were confidently identified by tandem mass spectrometry. Almost 50% of the identified proteins were hypothetical, while other major categories include proteins with binding function, protein fate, protein synthesis, metabolism and cellular transport. To identify proteins that are recognized by host humoral immunity, parasite proteins were separated by two-dimensional gel electrophoresis and screened by Western blot using an immune serum from a P. vivax patient. Mass spectrometry analysis of protein spots recognized by the serum identified four potential antigens including PV24. The recombinant protein PV24 was recognized by antibodies from vivax malaria patients even during the convalescent period, indicating that PV24 could elicit long-lasting antibody responses in P. vivax patients.     

Antibody response of naturally infected individuals to recombinant Plasmodium vivax apical membrane antigen-1

In the present study, we evaluate the naturally acquired antibody response to the Plasmodium vivax apical membrane antigen 1 (PvAMA-1), a leading vaccine candidate against malaria. The gene encoding the PvAMA-1 ectodomain region (amino acids 43-487) was cloned by PCR using genomic DNA from a Brazilian individual with patent P. vivax infection. The predicted amino acid sequence displayed a high degree of identity (97.3%) with a previously published sequence from the P. vivax Salvador strain. A recombinant protein representing the PvAMA-1 ectodomain was expressed in Escherichia coli and refolded. By ELISA, this recombinant protein reacted with 85 and 48.5% of the IgG or IgM antibodies, respectively, from Brazilian individuals with patent P. vivax malaria. IgG1 was the predominant subclass of IgG. The frequency of response increased according to the number of malaria episodes, reaching 100% in individuals in their fourth malaria episode. The high degree of recognition of PvAMA-1 by human antibodies was confirmed using a second recombinant protein expressed in Pichia pastoris (PV66/AMA-1). The observation that recognition of the bacterial recombinant PvAMA-1 was only slightly lower than that of the highly immunogenic 19kDa C-terminal domain of the P. vivax Merozoite Surface Protein-1 was also important. DNA sequencing of the PvAMA-1 variable domain from 20 Brazilian isolates confirmed the limited polymorphism of PvAMA-1 suggested by serological analysis. In conclusion, we provide evidence that PvAMA-1 is highly immunogenic during natural infection in humans and displays limited polymorphism in Brazil. Based on these observations, we conclude that PvAMA-1 merits further immunological studies as a vaccine candidate against P. vivax malaria.     

Local adaptation and vector-mediated population structure in Plasmodium vivax malaria

Plasmodium vivax in southern Mexico exhibits different infectivities to 2 local mosquito vectors, Anopheles pseudopunctipennis and Anopheles albimanus. Previous work has tied these differences in mosquito infectivity to variation in the central repeat motif of the malaria parasite's circumsporozoite (csp) gene, but subsequent studies have questioned this view. Here we present evidence that P. vivax in southern Mexico comprised 3 genetic populations whose distributions largely mirror those of the 2 mosquito vectors. Additionally, laboratory colony feeding experiments indicate that parasite populations are most compatible with sympatric mosquito species. Our results suggest that reciprocal selection between malaria parasites and mosquito vectors has led to local adaptation of the parasite. Adaptation to local vectors may play an important role in generating population structure in Plasmodium. A better understanding of coevolutionary dynamics between sympatric mosquitoes and parasites will facilitate the identification of molecular mechanisms relevant to disease transmission in nature and provide crucial information for malaria control.     

Genetic diversity of Plasmodium falciparum and its implications in the epidemiology of malaria

Genetic diversity provides Plasmodium falciparum with the potential capacity of avoiding the immune response, and possibly supporting the selection of drug or vaccine resistant parasites. These genetic characters play key roles in the selection of appropriate malaria control measures. Diverse clones of Plasmodium falciparum, often denoted as strains, has been documented, and the degree of genetic diversity supported by several kinds of PCR (polymerase chain reaction) assays. Many studies in different endemic regions with differences in their level of disease transmission have clarified the interactions between the parasite populations and malaria epidemiology. This paper describes recombination events of the malaria parasite life cycle that originate such genetic diversity in P. falciparum, reviewing different studies on this aspect and its implications in the immunity and development of control measures in regions with different degrees of endemicity.     

On cytoadhesion of Plasmodium vivax: raison d'être?

It is generally accepted that Plasmodium vivax, the most widely distributed human malaria parasite, causes mild disease and that this species does not sequester in the deep capillaries of internal organs. Recent evidence, however, has demonstrated that there is severe disease, sometimes resulting in death, exclusively associated with P. vivax and that P. vivax-infected reticulocytes are able to cytoadhere in vitro to different endothelial cells and placental cryosections. Here, we review the scarce and preliminary data on cytoadherence in P. vivax, reinforcing the importance of this phenomenon in this species and highlighting the avenues that it opens for our understanding of the pathology of this neglected human malaria parasite.     

New serological test for malaria antibodies.

In an enzyme-linked immunosorbent assay test for malaria antibodies, antibodies to Plasmodium vivax and P. falciparum in man are detected using a crude antigen prepared from the simian malaria parasite P. knowlesi. The test may be suitable for epidemiological studies.     

Plasmodium vivax: Merozoites, invasion of reticulocytes and considerations for malaria vaccine development

Several Plasmodium vivax merozoite proteins have been characterized over the past few years, including two that bind specifically to reticulocytes. Here, Mare Galinski and John Barnwell examine P. vivax merozoites and constituent molecules that are involved in host cell selection and invasion, and that also are viewed as malaria vaccine candidates. They also discuss how knowledge of the reticulocyte-binding proteins furthers the development of a conceptual framework for malaria merozoite invasion at the molecular level, not only for P. vivax, but for all species of the parasite.     

Mitochondrial genome sequences support ancient population expansion in Plasmodium vivax

Examination of nucleotide diversity in 106 mitochondrial genomes of the most geographically widespread human malaria parasite, Plasmodium vivax, revealed a level of diversity similar to, but slightly higher than, that seen in the virulent human malaria parasite Plasmodium falciparum. The pairwise distribution of nucleotide differences among mitochondrial genome sequences supported the hypothesis that both these parasites underwent ancient population expansions. We estimated the age of the most recent common ancestor (MRCA) of the mitochondrial genomes of both P. vivax and P. falciparum at around 200,000-300,000 years ago. This is close to the previous estimates of the time of the human mitochondrial MRCA and the origin of modern Homo sapiens, consistent with the hypothesis that both these Plasmodium species were parasites of the hominid lineage before the origin of modern H. sapiens and that their population expansion coincided with the population expansion of their host.     

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.     

The epidemiology of malaria in Papua New Guinea

Papua New Guinea (PNG) is a patchwork of different ecological zones, inhabited by human populations of exceptional cultural and linguistic diversity. This results in complex variations in vector ecology and malaria epidemiology. Malaria is the main cause of morbidity in many health facilities in lowland areas, but it is absent in much of the highlands. All four human malaria species occur, but endemicity varies widely, with Plasmodium falciparum locally reaching holo-endemic levels that are rarely found outside sub-Saharan Africa. The high frequency of Plasmodium vivax is an important difference to most African situations. PNG is therefore a prime location for studies of interactions between different parasite species, and of the biology of local human genetic adaptation and its implications for malaria morbidity and mortality.     

The baseline distribution of malaria in the initial phase of elimination in Sabang Municipality, Aceh Province, Indonesia

ABSTRACT: BACKGROUND: Sabang Municipality, in Aceh Province, Indonesia, plans to initiate a malaria elimination programme in 2013. A baseline survey of the distribution of malaria in the municipality was conducted to lay the foundations for an evidence-based programme and to assess the island's readiness to begin the elimination process. METHODS: The entire population of the municipality was screened for malaria infection and G6PD deficiency. Specimens collected included blood slides, blots and tubes for selected households. Results and Discussion Samples were collected from 16,229 residents. Microscopic examination of the blood smears revealed 12 malaria infections; 10 with Plasmodium falciparum and 2 with Plasmodium vivax. To confirm the parasite prevalence, polymerase chain reaction (PCR) diagnosis was performed on the entire positive cases by microscopy and randomized 10% of the microscopically negative blood samples. PCR revealed an additional 11 subjects with malaria; one P. falciparum infection from the village of Paya Keunekai, and nine P. vivax infections and one mixed P. falciparum/P. vivax infection from the village of Batee Shok. The overall slide positivity rate was 0.074% (CI 95%: 0.070 - 0.078) and PCR corrected prevalence 0,590% (CI 95%: 0.582 - 0.597). Analysis of 937 blood samples for G6PD deficiency revealed two subjects (0.2%) of deficient G6PD. Analysis of several genes of the parasite, such as Pfdhfr, Pfdhps, Pfmdr1, Pfcrt, Pfmsp1, Pfmsp2, Pvdhfr, Pvdhps, Pvmdr1 and host gene, such as G6PD gene revealed that both P. falciparum and P. vivax carried the mutation associated with chloroquine resistance. CONCLUSION: Malariometric and host genetic analysis indicated that there is a low prevalence of both malaria and G6PD deficiency in the population of Sabang Municipality. Nevertheless, malaria cases were clustered in three rural villages and efforts for malaria elimination in Sabang should be particularly focused on those three villages.