Cerebral malaria caused by Plasmodium vivax in adult subjects

Cerebral malaria is a diffuse encephalopathy associated with seizures and status epilepticus which can occur in up to one-third of patients with severe malaria, particularly that caused by Plasmodium falciparum.In this article, we report three cases of Plasmodium vivax malaria (all adult male patients) complicated by seizures and symptoms of diffuse meningoencephalitis. Two patients had predominantly meningeal signs, while in the third patient the features were purely of encephalitis All cases were treated with artesunate. Usually, cerebral malaria is caused by P. falciparum, and rarely, cerebral malaria is a presenting complication or occurs during the course of P. vivax infection.     

Epitope-specific humoral immunity to Plasmodium vivax Duffy binding protein

Erythrocyte invasion by Plasmodium vivax is completely dependent on binding to the Duffy blood group antigen by the parasite Duffy binding protein (DBP). The receptor-binding domain of this protein lies within a cysteine-rich region referred to as region II (DBPII). To examine whether antibody responses to DBP correlate with age-acquired immunity to P. vivax, antibodies to recombinant DBP (rDBP) were measured in 551 individuals residing in a village endemic for P. vivax in Papua New Guinea, and linear epitopes mapped in the critical binding region of DBPII. Antibody levels to rDBP(II) increased with age. Four dominant linear epitopes were identified, and the number of linear epitopes recognized by semi-immune individuals increased with age, suggesting greater recognition with repeated infection. Some individuals had antibodies to rDBP(II) but not to the linear epitopes, indicating the presence of conformational epitopes. This occurred in younger individuals or subjects acutely infected for the first time with P. vivax, indicating that repeated infection is required for recognition of linear epitopes. All four dominant B-cell epitopes contained polymorphic residues, three of which showed variant-specific serologic responses in over 10% of subjects examined. In conclusion, these results demonstrate age-dependent and variant-specific antibody responses to DBPII and implicate this molecule in partial acquired immunity to P. vivax in populations in endemic areas.     

Plasmodium vivax malaria vaccine development.

Plasmodium vivax represents the most widespread malaria parasite worldwide. Although it does not result in as high a mortality rate as P. falciparum, it inflicts debilitating morbidity and consequent economic impact in endemic communities. In addition, the relapsing behavior of this malaria parasite and the recent resistance to anti-malarials contribute to making its control more difficult. Although the biology of P. vivax is different from that of P. falciparum and the human immune response to this parasite species has been rather poorly studied, significant progress is being made to develop a P. vivax-specific vaccine based on the information and experience gained in the search for a P. falciparum vaccine. We have devoted great effort to antigenically characterize the P. vivax CS protein and to test its immunogenicity using the Aotus monkey model. Together with other groups we are also assessing the immunogenicity and protective efficacy of the asexual blood stage vaccine candidates MSP-1 and DBP in the monkey model, as well as the immunogenicity of Pvs25 and Pvs28 ookinete surface proteins. The transmission-blocking efficacy of the responses induced by these latter antigens is being assessed using Anopheles albimanus mosquitoes. The current status of these vaccine candidates and other antigens currently being studied is described.     

The immunology of Plasmodium vivax malaria

Plasmodium vivax infection, the predominant cause of malaria in Asia and Latin America, affects ~14 million individuals annually, with considerable adverse effects on wellbeing and socioeconomic development. A clinical hallmark of Plasmodium infection, the paroxysm, is driven by pyrogenic cytokines produced during the immune response. Here, we review studies on the role of specific immune cell types, cognate innate immune receptors, and inflammatory cytokines on parasite control and disease symptoms. This review also summarizes studies on recurrent infections in individuals living in endemic regions as well as asymptomatic infections, a serious barrier to eliminating this disease. We propose potential mechanisms behind these repeated and subclinical infections, such as poor induction of immunological memory cells and inefficient T effector cells. We address the role of antibody-mediated resistance to P. vivax infection and discuss current progress in vaccine development. Finally, we review immunoregulatory mechanisms, such as inhibitory receptors, T regulatory cells, and the anti-inflammatory cytokine, IL-10, that antagonizes both innate and acquired immune responses, interfering with the development of protective immunity and parasite clearance. These studies provide new insights for the clinical management of symptomatic as well as asymptomatic individuals and the development of an efficacious vaccine for vivax malaria.     

Immunity against sexual stage Plasmodium falciparum and Plasmodium vivax parasites

The efficient spread of malaria from infected humans to mosquitoes is a major challenge for malaria elimination initiatives. Gametocytes are the only Plasmodium life stage infectious to mosquitoes. Here, we summarize evidence for naturally acquired anti-gametocyte immunity and the current state of transmission blocking vaccines (TBV). Although gametocytes are intra-erythrocytic when present in infected humans, developing Plasmodium falciparum gametocytes may express proteins on the surface of red blood cells that elicit immune responses in naturally exposed individuals. This immune response may reduce the burden of circulating gametocytes. For both P. falciparum and Plasmodium vivax, there is a solid evidence that antibodies against antigens present on the gametocyte surface, when co-ingested with gametocytes, can influence transmission to mosquitoes. Transmission reducing immunity, reducing the burden of infection in mosquitoes, is a well-acknowledged but poorly quantified phenomenon that forms the basis for the development of TBV. Transmission enhancing immunity, increasing the likelihood or intensity of transmission to mosquitoes, is more speculative in nature but is convincingly demonstrated for P. vivax. With the increased interest in malaria elimination, TBV and monoclonal antibodies have moved to the center stage of malaria vaccine development. Methodologies to prioritize and evaluate products are urgently needed.     

Molecular markers for detection and differentiation of Plasmodium falciparum and Plasmodium vivax in human blood samples by pyrosequencing

PCR amplification coupled with pyrosequencing was used to measure molecular markers that could be used to detect and differentiate Plasmodium falciparum and Plasmodium vivax in human blood samples. The detection rates were in agreement with the results of Giemsa-stained film microscopy, which is the current gold standard for detection. This method provides an exciting alternative for malaria diagnosis.     

Stage-specific and species-specific antigens of Plasmodium vivax and Plasmodium ovale defined by monoclonal antibodies.

Monoclonal antibodies (MAbs) were produced against the asexual blood stages of Plasmodium vivax and Plasmodium ovale and used to define antigens of plasmodial parasites in an indirect fluorescent antibody assay. The anti-P. vivax MAbs produced two distinct patterns in the indirect fluorescent antibody assay. Four patterns were found with the anti-P. ovale MAbs. Species-specific epitopes were defined for P. vivax and P. ovale; epitopes shared among all four species of human malaria parasites were also defined. Some of the anti-P. vivax MAbs reacted only with mature stages, and others reacted with all asexual stages. No asexual blood-stage specificity could be found with the anti-P. ovale antibodies. Five of the anti-P. vivax MAbs and three of the anti-P. ovale MAbs also reacted with sporozoites.     

Plasmodium vivax malaria: An unusual presentation

Acute renal failure, disseminated intravascular coagulation (DIC), acute respiratory distress syndrome (ARDS), hypoglycemia, coma, or epileptic seizures are manifestations of severe Plasmodium falciparum malaria. On the other hand, Plasmodium vivax malaria seldom results in pulmonary damage, and pulmonary complications are exceedingly rare. We report the case of a 42-year-old male living in a malaria-endemic area who presented with ARDS and was diagnosed as having Plasmodium vivax malaria. A diagnosis of Plasmodium vivax malaria was established by a positive Plasmodium LDH immunochromatographic assay while a negative PfHRP2 based assay ruled out P. falciparum malaria. After specific anti-plasmodial therapy and intensive supportive care, the patient recovered and was discharged from hospital. The use of NIPPV in vivax-malaria related ARDS was associated with a good outcome.     

Single-chain antibody fragment specific for Plasmodium vivax Duffy binding protein

Phage display of single-chain variable fragment (scFv) antibodies is a powerful tool for selecting important, useful, and specific human antibodies. We constructed a library from three patients infected with Plasmodium vivax. Panning on recombinant PvRII enriched a population of scFvs that recognized region II of the P. vivax Duffy binding protein (DBP). Three clones of scFvs that reacted with PvRII were selected, and their biological functions were analyzed. These scFvs inhibited erythrocyte binding to DBP. Clone SFDBII92 had the greatest affinity (dissociation constant = 3.62 x 10(-8) M) and the greatest inhibition activity (50% inhibitory concentration approximately 2.9 microg/ml) to DBP. Thus, we demonstrated that human neutralizing antibody could be made from malaria patients using phage display and that these neutralizing scFvs should prove valuable for developing both passive and active immunization strategies based on DBP.     

Evaluation of the OptiMAL Test for Rapid Diagnosis of Plasmodium vivax and Plasmodium falciparum Malaria

The development of rapid and specific diagnostic tests to identify individuals infected with malaria is of paramount importance in efforts to control the severe public health impact of this disease. This study evaluated the ability of a newly developed rapid malaria diagnostic test, OptiMAL (Flow Inc., Portland, Oreg.), to detect Plasmodium vivax and Plasmodium falciparum malaria during an outbreak in Honduras. OptiMAL is a rapid (10-min) malaria detection test which utilizes a dipstick coated with monoclonal antibodies against the intracellular metabolic enzyme parasite lactate dehydrogenase (pLDH). Differentiation of malaria parasites is based on antigenic differences between the pLDH isoforms. Since pLDH is produced only by live Plasmodium parasites, this test has the ability to differentiate live from dead organisms. Results from the OptiMAL test were compared to those obtained by reading 100 fields of traditional Giemsa-stained thick-smear blood films. Whole-blood samples were obtained from 202 patients suspected of having malaria. A total of 96 samples (48%) were positive by blood films, while 91 (45%) were positive by the OptiMAL test. The blood films indicated that 82% (79 of 96) of the patients were positive for P. vivax and 18% (17 of 96) were infected with P. falciparum. The OptiMAL test showed that 81% (74 of 91) were positive for P. vivax and 19% (17 of 91) were positive for P. falciparum. These results demonstrated that the OptiMAL test had sensitivities of 94 and 88% and specificities of 100 and 99%, respectively, when compared to traditional blood films for the detection of P. vivax and P. falciparum malaria. Blood samples not identified by OptiMAL as malaria positive normally contained parasites at concentrations of less than 100/μl of blood. Samples found to contain P. falciparum were further tested by two other commercially available rapid malaria diagnostic tests, ParaSight-F (Becton Dickinson, Cockeysville, Md.) and ICT Malaria P.f. (ICT Diagnostics, Sydney, Australia), both of which detect only P. falciparum. Only 11 of the 17 (65%) P. falciparum-positive blood samples were identified by the ICT and ParaSight-F tests. Thus, OptiMAL correctly identified P. falciparum malaria parasites in patient blood samples more often than did the other two commercially available diagnostic tests and showed an excellent correlation with traditional blood films in the identification of both P. vivax malaria and P. falciparum malaria. We conclude that the OptiMAL test is an effective tool for the rapid diagnosis of malaria.     

Identification of proteins from Plasmodium falciparum that are homologous to reticulocyte binding proteins in Plasmodium vivax

Plasmodium falciparum infections can be fatal, while P. vivax infections usually are not. A possible factor involved in the greater virulence of P. falciparum is that this parasite grows in red blood cells (RBCs) of all maturities whereas P. vivax is restricted to growth in reticulocytes, which represent only approximately 1% of total RBCs in the periphery. Two proteins, expressed at the apical end of the invasive merozoite stage from P. vivax, have been implicated in the targeting of reticulocytes for invasion by this parasite. A search of the P. falciparum genome databases has identified genes that are homologous to the P. vivax rbp-1 and -2 genes. Two of these genes are virtually identical over a large region of the 5' end but are highly divergent at the 3' end. They encode high-molecular-mass proteins of >300 kDa that are expressed in late schizonts and localized to the apical end of the merozoite. To test a potential role in merozoite invasion of RBCs, we analyzed the ability of these proteins to bind to mature RBCs and reticulocytes. No binding to mature RBCs or cell preparations enriched for reticulocytes was detected. We identified a parasite clone that lacks the gene for one of these proteins, showing that the gene is not required for normal in vitro growth. Antibodies to these proteins can inhibit merozoite invasion of RBCs.     

Individual Plasmodium vivax msp1 variants within polyclonal P. vivax infections display different propensities for relapse

Using a newly developed Plasmodium vivax merozoite surface protein 1 gene (Pvmsp1) heteroduplex tracking assay, we genotyped 107 P. vivax infections in individuals from Cambodia, 45 of whom developed recurrent parasitemia within 42 days. The majority of isolates were polyclonal, but recurrent parasitemias displayed fewer variants compared to initial parasitemias. Two Pvmsp1 gene variants occurred more frequently in the initial genotypes of those who developed recurrent parasitemia, representing the first time P. vivax variants associated with a higher risk of relapse have been described.     

Cloning of the Plasmodium vivax Duffy receptor.

Plasmodium vivax and Plasmodium knowlesi merozoites invade only Duffy blood group-positive human erythrocytes. Soluble P. vivax and P. knowlesi merozoite proteins of 135 kDa bind specifically to Duffy blood group determinants. The gene encoding a member of the Duffy receptor gene family of P. knowlesi has been cloned. We report here the molecular cloning of the presumptive Duffy receptor gene of P. vivax, using the P. knowlesi gene as a probe. There is a single gene in P. vivax which codes for a protein of 1115 amino acids. The deduced amino acid sequence predicts a putative signal sequence at the amino-terminus and a transmembrane region followed by 45 amino acids at the carboxy-terminus. The three introns found at the 3' end of the P. knowlesi gene were conserved in P. vivax, including high homology for the sequences of the introns. Comparison of the portion of the proteins amino to the transmembrane region between P. vivax and the partial sequence of P. knowlesi indicated at least three domains. Two homologous regions were separated by a non-homologous region. The cysteines in the homologous regions were conserved in number and position, indicating that the folding is similar and suggesting that these regions may be the Duffy blood group binding domains. In both P. vivax and P. knowlesi, the non-homologous region is hydrophilic and proline-rich, although the position of the prolines is not conserved. As prolines tend to stiffen a protein, this region may act as a 'hinge region' similar to those in the immunoglobulin gene family.     

Plasmodium vivax ookinetes in human peripheral blood.

Microscopic examination of a peripheral blood smear revealed ookinetes of Plasmodium vivax. This unusual finding was probably due to an excessive delay between blood collection and smear preparation. Ookinete formation normally occurs in the mosquito gut. When seen in blood smears, it can cause confusion and misidentification of the parasite.