Cryo-EM structure of an essential Plasmodium vivax invasion complex.

Plasmodium vivax is the most widely distributed malaria parasite that infects humans1. P. vivax invades reticulocytes exclusively, and successful entry depends on specific interactions between the P. vivax reticulocyte-binding protein 2b (PvRBP2b) and transferrin receptor 1 (TfR1)2. TfR1-deficient erythroid cells are refractory to invasion by P. vivax, and anti-PvRBP2b monoclonal antibodies inhibit reticulocyte binding and block P. vivax invasion in field isolates2. Here we report a high-resolution cryo-electron microscopy structure of a ternary complex of PvRBP2b bound to human TfR1 and transferrin, at 3.7 Å resolution. Mutational analyses show that PvRBP2b residues involved in complex formation are conserved; this suggests that antigens could be designed that act across P. vivax strains. Functional analyses of TfR1 highlight how P. vivax hijacks TfR1, an essential housekeeping protein, by binding to sites that govern host specificity, without affecting its cellular function of transporting iron. Crystal and solution structures of PvRBP2b in complex with antibody fragments characterize the inhibitory epitopes. Our results establish a structural framework for understanding how P. vivax reticulocyte-binding protein engages its receptor and the molecular mechanism of inhibitory monoclonal antibodies, providing important information for the design of novel vaccine candidates.

High proportions of asymptomatic and submicroscopic Plasmodium vivax infections in a peri-urban area of low transmission in the Brazilian Amazon.

BACKGROUND: Population-based studies conducted in Latin America have shown a high proportion of asymptomatic and submicroscopic malarial infections. Considering efforts aiming at regional elimination, it is important to investigate the role of this asymptomatic reservoir in malaria transmission in peri-urban areas. This study aimed to estimate the prevalence of Plasmodium spp. and gametocyte burden on symptomatic and asymptomatic infections in the Brazilian Amazon. RESULTS: Two cross-sectional household surveys (CS) were conducted including all inhabitants in a peri-urban area of Manaus, western Amazonas State, Brazil. Malaria parasites were detected by light microscopy (LM) and qPCR. Sexual stages of Plasmodium spp. were detected by LM and RT-qPCR. A total of 4083 participants were enrolled during the two surveys. In CS1, the prevalence of Plasmodium vivax infections was 4.3% (86/2010) by qPCR and 1.6% (32/2010) by LM. Fifty percent (43/86) of P. vivax infected individuals (qPCR) carried P. vivax gametocytes. In CS2, 3.4% (70/2073) of participants had qPCR-detectable P. vivax infections, of which 42.9% (30/70) of infections were gametocyte positive. The P. vivax parasite density was associated with gametocyte carriage (P < 0.001). Sixty-seven percent of P. vivax infected individuals and 53.4% of P. vivax gametocyte carriers were asymptomatic. CONCLUSIONS: This study confirms a substantial proportion of asymptomatic and submicroscopic P. vivax infections in the study area. Most asymptomatic individuals carried gametocytes and presented low asexual parasitemia. This reservoir actively contributes to malaria transmission in the Brazilian Amazon, underscoring a need to implement more efficient control and elimination strategies.

Colorimetric Detection of Plasmodium vivax in Urine Using MSP10 Oligonucleotides and Gold Nanoparticles.

Plasmodium vivax is the most prevalent cause of human malaria in the world and can lead to severe disease with high potential for relapse. Its genetic and geographic diversities make it challenging to control. P. vivax is understudied and to achieve control of malaria in endemic areas, a rapid, accurate, and simple diagnostic tool is necessary. In this pilot study, we found that a colorimetric system using AuNPs and MSP10 DNA detection in urine can provide fast, easy, and inexpensive identification of P. vivax. The test exhibited promising sensitivity (84%), high specificity (97%), and only mild cross-reactivity with P. falciparum (21%). It is simple to use, with a visible color change that negates the need for a spectrometer, making it suitable for use in austere conditions. Using urine eliminates the need for finger-prick, increasing both the safety profile and patient acceptance of this model.

[Performance assessment employing slide sets: A tool for the classification of senior microscopists from Colombia´s Malaria Control Program]. / Evaluación del desempeño mediante paneles de láminas: una herramienta para la clasificación de los microscopistas sénior del Programa de Control de la Malaria en Colombia.

INTRODUCTION: One of the most important activities for quality assurance of malaria diagnosis is performance assessment. In Colombia, performance assessment of malaria microscopists has been done through the external performance assessment and indirect external performance assessment programs. OBJECTIVES: To assess the performance of malaria microscopists of public reference laboratories using slide sets, and to describe the methodology used for this purpose. MATERIALS AND METHODS: This was a retrospective study to evaluate the concordance of senior microscopists regarding parasite detection, species identification and parasite count based on the results of the assessment of competences using two sets, one comprising 40 slides, and another one with 17 slides. RESULTS: The concordance for parasite detection was 96.9% (95% CI: 96.0-97.5) and 88.7% (95% CI: 86.6-90.5) for species identification. The average percentage of concordant slides in the group evaluated was 89.7% (95% CI: 87.5-91.6). CONCLUSIONS: Most of the senior microscopists in Colombia were classified in the two top categories in the performance assessment using slide sets. The most common difficulty encountered was the identification of parasite species. The use of this tool to assess individual performance of microscopists in the evaluation of samples with different degrees of difficulty allows for characterizing the members of the malaria diagnosis network and strengthening the abilities of those who require it.

Invasion of erythroblasts by Pasmodium vivax: A new mechanism contributing to malarial anemia.

Severe malarial anemia causes considerable mortality and morbidity in endemic areas. Possible mechanisms underlying the anemia include lysis of parasitized and nonparasitized red cells as well as parasite product-mediated effects on erythropoiesis. The latter include suppression of erythropoiesis, dyserythropoiesis, and ineffective erythropoiesis. Present transmission electron microscope data in two cases of Pasmodium vivax malaria show a hitherto undescribed mechanism contributing to malarial anemia, namely, infection of erythroblasts by parasites and their subsequent degradation. No parasites were detected in the peripheral blood but parasites were found in the bone marrow. These findings emphasise the value of bone marrow examination in the diagnosis and eradication of malaria.

Plasmodium vivax: impaired escape of Vk210 phenotype ookinetes from the midgut blood bolus of Anopheles pseudopunctipennis.

The site in the midguts of Anopheles pseudopunctipennis where the development of Plasmodium vivax circumsporozoite protein Vk210 phenotype is blocked was investigated, and compared to its development in An. albimanus. Ookinete development was similar in time and numbers within the blood meal bolus of both mosquito species. But, compared to An. pseudopunctipennis, a higher proportion of An. albimanus were infected (P=0.0001) with higher ookinete (P=0.0001) and oocyst numbers (P=0.0001) on their internal and external midgut surfaces, respectively. Ookinetes were located in the peritrophic matrix (PM), but neither inside epithelial cells nor on the haemocoelic midgut surface by transmission electron microscopy in 24h p.i.-An. pseudopunctipennis mosquito samples. In contrast, no parasites were detected in the PM of An. albimanus at this time point. These results suggest that P. vivax Vk210 ookinetes cannot escape from and are destroyed within the midgut lumen of An. pseudopunctipennis.

The Plasmodium vivax homolog of the ookinete adhesive micronemal protein, CTRP.

The Plasmodium circumsporozoite protein/thrombospondin-related anonymous protein-related protein (CTRP) is expressed at the mosquito midgut ookinete stage and is considered to be a transmission-blocking vaccine candidate. CTRP is composed of multiple von Willebrand factor A (vWA) and thrombospondin type 1 domains in the extracellular portion of the molecule, and a short acidic cytoplasmic domain that interacts with the actomyosin machinery. As a means to predict functionally relevant domains within CTRP we determined the nucleotide sequences of CTRP from the Plasmodium vivax Sall and the Plasmodium yoelii 17XL strains and characterized the conservation of domain architectures and motifs across Plasmodium genera. Sequence alignments indicate that the CTRP 1st to 4th vWA domains exhibit greater conservation, and thereby are perhaps functionally more important than the 5th and 6th domains. This point should be considered for the development of a transmission-blocking vaccine that includes CTRP recombinant subunit. To complement previous cellular studies on CTRP, we further determined the expression and cellular localization of CTRP protein in P. vivax and P. yoelii.

The Plasmodium vivax rhoptry-associated protein 1.

Rhoptries are cellular organelles localized at the apical pole of apicomplexan parasites. Their content is rich in lipids and proteins that are released during target cell invasion. Plasmodium falciparum rhoptry-associated protein 1 (RAP1) has been the most widely studied among this parasite species' rhoptry proteins and is considered to be a good anti-malarial vaccine candidate since it displays little polymorphism and induces antibodies in infected humans. Monoclonal antibodies directed against RAP1 are also able to inhibit target cell invasion in vitro and protection against P. falciparum experimental challenge is induced when non-human primates are immunized with this protein expressed in its recombinant form. This study describes identifying and characterizing RAP1 in Plasmodium vivax, the most widespread parasite species causing malaria in humans, producing more than 80 million infections yearly, mainly in Asia and Latin America. This new protein is encoded by a two-exon gene, is proteolytically processed in a similar manner to its falciparum homologue and, as observed by microscopy, the immunofluorescence pattern displayed is suggestive of its rhoptry localization. Further studies evaluating P. vivax RAP1 protective efficacy in non-human primates should be carried out taking into account the relevance that its P. falciparum homologue has as an anti-malarial vaccine candidate.

Novel Plasmodium vivax dhfr alleles from the Indonesian Archipelago and Papua New Guinea: association with pyrimethamine resistance determined by a Saccharomyces cerevisiae expression system.

In plasmodia, the dihydrofolate reductase (DHFR) enzyme is the target of the pyrimethamine component of sulfadoxine-pyrimethamine (S/P). Plasmodium vivax infections are not treated intentionally with antifolates. However, outside Africa, coinfections with Plasmodium falciparum and P. vivax are common, and P. vivax infections are often exposed to S/P. Cloning of the P. vivax dhfr gene has allowed molecular comparisons of dhfr alleles from different regions. Examination of the dhfr locus from a few locations has identified a very diverse set of alleles and showed that mutant alleles of the vivax dhfr gene are prevalent in Southeast Asia where S/P has been used extensively. We have surveyed patient isolates from six locations in Indonesia and two locations in Papua New Guinea. We sequenced P. vivax dhfr alleles from 114 patient samples and identified 24 different alleles that differed from the wild type by synonymous and nonsynonymous point mutations, insertions, or deletions. Most importantly, five alleles that carried four or more nonsynonymous mutations were identified. Only one of these highly mutant alleles had been previously observed, and all carried the 57L and 117T mutations. P. vivax cannot be cultured continuously, so we used a yeast assay system to determine in vitro sensitivity to pyrimethamine for a subset of the alleles. Alleles with four nonsynonymous mutations conferred very high levels of resistance to pyrimethamine. This study expands significantly the total number of novel dhfr alleles now identified from P. vivax and provides a foundation for understanding how antifolate resistance arises and spreads in natural P. vivax populations.