Progress in understanding the phylogeny of the Plasmodium vivax lineage.

There has been some controversy about the evolutionary origin of Plasmodium vivax, particularly whether it is of Asian or African origin. Recently, a new malaria species which closely related to ape P. vivax was found in chimpanzees, in addition, the host switches of P. vivax from ape to human was confirmed. These findings support the African origin of P. vivax. Previous phylogenetic analyses have shown the position of P. vivax within the Asian primate malaria parasite clade. This suggested an Asian origin of P. vivax. Recent analyses using massive gene data, however, positioned P. vivax after the branching of the African Old World monkey parasite P. gonderi, and before the branching of the common ancestor of Asian primate malaria parasites. This position is consistent with an African origin of P. vivax. We here review the history of phylogenetic analyses on P. vivax, validate previous analyses, and finally present a definitive analysis using currently available data that indicate a tree in which P. vivax is positioned at the base of the Asian primate malaria parasite clade, and thus that is consistent with an African origin of P. vivax.

Assessing the in vitro sensitivity with associated drug resistance polymorphisms in Plasmodium vivax clinical isolates from Delhi, India.

The drug resistance of Plasmodium vivax in clinical cases remains largely unknown till date because of the difficulty in diagnosing the resistant P. vivax strains. The present study was undertaken to determine the prevalence of mutant alleles in drug resistance genes viz P. vivax multi-drug resistance (pvmdr-1), chloroquine resistance transporter (pvcrt-o), dihydrofolate reductase (pvdhfr) and dihydropteroate synthase (pvdhps) along with in vitro chloroquine (CQ) sensitivity in P. vivax clinical isolates. During August-October 2017 a total of 86 samples of the febrile patients were screened and 31 samples were found to be positive for P. vivax in Safdarjung hospital, New Delhi. Sequence genotyping of the drug resistance genes was carried out in these P. vivax samples and in vitro CQ susceptibility for 23 isolates was determined by the schizont maturation assay (SMA). The CQ inhibitory concentrations (IC50) for the clinical isolates was found to be in the range of 25.6-176.7 nM. All the 31 clinical isolates analyzed for pvmdr-1 gene, showed mutant alleles and in only two isolates novel mutations at 861 and 898 codons were observed. Sequence analysis of pvcrt-o, pvdhfr and pvdhps genes revealed wild type genotypes in all the 31 studied isolates. The presence of mutations in pvmdr-1 gene and the increase in the CQ IC50 value indicates the possibility of shift in drug tolerance where CQ with primaquine (PQ) is still the first line of treatment for P. vivax malaria in the country. The regular molecular surveillance in P. vivax would provide useful information for the policy makers of the malaria control programme.

Caracterização imunológica da formulação vacinal baseada em VLP (Virus Like Particle) da Proteína Circumsporozoíta de Plasmodium vivax / Immunological characterization of the VLP (Virus Like Particle) vaccine formulation fused to Circumsporozoite Protein of Plasmodium vivax

O Plasmodium vivax é a espécie mais comum de parasita causador da malária humana encontrada fora da África, com maior endemicidade na Ásia, América Central e do Sul e Oceania. Embora o Plasmodium falciparum cause a maioria do número de mortes, o P. vivax pode levar à malária grave e resultar em morbimortalidade significativa. O desenvolvimento de uma vacina protetora será um passo importante para a eliminação da malária. Recentemente, uma formulação contendo as três variantes alélicas da proteína circumsporozoíta de P. vivax (PvCSP - All epitopes) induziu proteção parcial em camundongos após desafio com esporozoíto híbrido Plasmodium berghei (Pb), no qual as repetições centrais do PbCSP foram substituídas por repetições PvCSP-VK210 (esporozoítos Pb/Pv). No presente estudo, a proteína quimérica PvCSP contendo as variantes alélicas (VK210, VK247 e P. vivax-like) fusionadas com a proteína de nucleocapsídeo do vírus da caxumba (formando partículas semelhantes a nucleocapsídeos ou do inglês, NLP - Núcleo Like Particles) na ausência (NLP-CSPR) ou na presença do domínio C-terminal (CT) conservado da PvCSP (NLP-CSPCT). Para a realização do estudo selecionamos os adjuvantes Poly (I:C), um RNA sintético de dupla fita, agonista do receptor Toll do tipo 3 (TLR3) ou o adjuvante Montanide ISA 720, uma emulação óleo em agua. Para obter uma forte resposta imune, a levedura Pichia pastoris foi usada para expressar as proteínas recombinantes na forma de NLPs. Camundongos foram imunizados com cada uma das proteínas recombinantes em combinação com os adjuvantes citados. Embora ambas as NLPs tenham sido capazes de gerar uma forte resposta imune, com altos níveis de títulos e longevidade, apenas a formulação contendo a proteína NLP-CSPCT na presença do adjuvante Poly (I:C) foi selecionada para ser explorada em experimentos futuros. Esta proteína em combinação com o adjuvante Poly (I:C) induziu alta frequência de células secretoras de anticorpos específicas para o antígeno homólogo nos dias 5 e 30, no baço e na medula óssea, respectivamente. Altos títulos de IgG contra as 3 variantes de PvCSP foram detectados nos soros. Posteriormente camundongos imunizados com NLP-CSPCT foram desafiados com esporozoítos Pb/Pv e a parasitemia no 5º dia demonstrou proteção estéril em 30% dos camundongos desafiados. Portanto, a formulação vacinal gerada neste estudo tem potencial para ser explorada no desenvolvimento de uma vacina universal contra a malária causada por P. vivax

Plasmodium vivax is the most common species of human malaria parasite found outside Africa, with high endemicity in Asia, Central and South America, and Oceania. Although Plasmodium falciparum causes the majority of deaths, P. vivax can lead to severe malaria and result in significant morbidity and mortality. The development of a protective vaccine will be a major step toward malaria elimination. Recently, a formulation containing the three allelic variants of the P. vivax circumsporozoite protein (PvCSP--All epitopes) showed partial protection in mice after a challenge with the hybrid Plasmodium berghei (Pb) sporozoite, in which the PbCSP central repeats were replaced by the VK210 PvCSP repeats (Pb/Pv sporozoite). In the present study, the chimeric PvCSP allelic variants (VK210, VK247, and P. vivax-like) were fused with the mumps virus nucleocapsid protein (assembling into nucleo like particles - NLP) in the absence (NLP-CSPR) or presence of the conserved C-terminal (CT) domain of PvCSP (NLP-CSPCT). To carry out the study, we selected the adjuvants Poly (I:C), a synthetic double-stranded RNA, Toll-like receptor 3 (TLR3) agonist or Montanide ISA 720 adjuvant, an oil-water emulation. To elicit stronger immune response, Pichia pastoris yeast was used to produce the NLPs. Mice were immunized with each recombinant protein in combination with above. Although both NLPs were able to generate stronger immune response, with high antibodies titer levels and longevity, formulation containing NLP-CSPCT in the presence of Poly (I:C) was selected to be explored in future experiments. NLP-CSPCT with Poly (I:C) adjuvant presented a high frequency of antigen-specific antibody-secreting cells (ASCs) on days 5 and 30, respectively, in the spleen and bone marrow. Moreover, high IgG titers against all PvCSP variants were detected in the sera. Later, immunized mice with NLP-CSPCT were challenged with Pb/Pv sporozoites. Sterile protection was observed in 30% of the challenged mice. Therefore, this vaccine formulation use has the potential to be a good candidate for the development of a universal vaccine against P. vivax malaria.

Whole genome sequencing of Plasmodium vivax isolates reveals frequent sequence and structural polymorphisms in erythrocyte binding genes.

Plasmodium vivax malaria is much less common in Africa than the rest of the world because the parasite relies primarily on the Duffy antigen/chemokine receptor (DARC) to invade human erythrocytes, and the majority of Africans are Duffy negative. Recently, there has been a dramatic increase in the reporting of P. vivax cases in Africa, with a high number of them being in Duffy negative individuals, potentially indicating P. vivax has evolved an alternative invasion mechanism that can overcome Duffy negativity. Here, we analyzed single nucleotide polymorphism (SNP) and copy number variation (CNV) in Whole Genome Sequence (WGS) data from 44 P. vivax samples isolated from symptomatic malaria patients in southwestern Ethiopia, where both Duffy positive and Duffy negative individuals are found. A total of 123,711 SNPs were detected, of which 22.7% were nonsynonymous and 77.3% were synonymous mutations. The largest number of SNPs were detected on chromosomes 9 (24,007 SNPs; 19.4% of total) and 10 (16,852 SNPs, 13.6% of total). There were particularly high levels of polymorphism in erythrocyte binding gene candidates including merozoite surface protein 1 (MSP1) and merozoite surface protein 3 (MSP3.5, MSP3.85 and MSP3.9). Two genes, MAEBL and MSP3.8 related to immunogenicity and erythrocyte binding function were detected with significant signals of positive selection. Variation in gene copy number was also concentrated in genes involved in host-parasite interactions, including the expansion of the Duffy binding protein gene (PvDBP) on chromosome 6 and MSP3.11 on chromosome 10. Based on the phylogeny constructed from the whole genome sequences, the expansion of these genes was an independent process among the P. vivax lineages in Ethiopia. We further inferred transmission patterns of P. vivax infections among study sites and showed various levels of gene flow at a small geographical scale. The genomic features of P. vivax provided baseline data for future comparison with those in Duffy-negative individuals and allowed us to develop a panel of informative Single Nucleotide Polymorphic markers diagnostic at a micro-geographical scale.

Population genomics reveals the expansion of highly inbred Plasmodium vivax lineages in the main malaria hotspot of Brazil.

BACKGROUND: Plasmodium vivax is a neglected human malaria parasite that causes significant morbidity in the Americas, the Middle East, Asia, and the Western Pacific. Population genomic approaches remain little explored to map local and regional transmission pathways of P. vivax across the main endemic sites in the Americas, where great progress has been made towards malaria elimination over the past decades. METHODOLOGY/PRINCIPAL FINDINGS: We analyze 38 patient-derived P. vivax genome sequences from Mâncio Lima (ML)-the Amazonian malaria hotspot next to the Brazil-Peru border-and 24 sequences from two other sites in Acre State, Brazil, a country that contributes 23% of malaria cases in the Americas. We show that the P. vivax population of ML is genetically diverse (π = 4.7 × 10-4), with a high polymorphism particularly in genes encoding proteins putatively involved in red blood cell invasion. Paradoxically, however, parasites display strong genome-wide linkage disequilibrium, being fragmented into discrete lineages that are remarkably stable across time and space, with only occasional recombination between them. Using identity-by-descent approaches, we identified a large cluster of closely related sequences that comprises 16 of 38 genomes sampled in ML over 26 months. Importantly, we found significant ancestry sharing between parasites at a large geographic distance, consistent with substantial gene flow between regional P. vivax populations. CONCLUSIONS/SIGNIFICANCE: We have characterized the sustained expansion of highly inbred P. vivax lineages in a malaria hotspot that can seed regional transmission. Potential source populations in hotspots represent a priority target for malaria elimination in the Amazon.

Molecular surveillance over 14 years confirms reduction of Plasmodium vivax and falciparum transmission after implementation of Artemisinin-based combination therapy in Papua, Indonesia.

Genetic epidemiology can provide important insights into parasite transmission that can inform public health interventions. The current study compared long-term changes in the genetic diversity and structure of co-endemic Plasmodium falciparum and P. vivax populations. The study was conducted in Papua Indonesia, where high-grade chloroquine resistance in P. falciparum and P. vivax led to a universal policy of Artemisinin-based Combination Therapy (ACT) in 2006. Microsatellite typing and population genetic analyses were undertaken on available isolates collected between 2004 and 2017 from patients with uncomplicated malaria (n = 666 P. falciparum and n = 615 P. vivax). The proportion of polyclonal P. falciparum infections fell from 28% (38/135) before policy change (2004-2006) to 18% (22/125) at the end of the study (2015-2017); p<0.001. Over the same period, polyclonal P. vivax infections fell from 67% (80/119) to 35% (33/93); p<0.001. P. falciparum strains persisted for up to 9 years compared to 3 months for P. vivax, reflecting higher rates of outbreeding in the latter. Sub-structure was observed in the P. falciparum population, but not in P. vivax, confirming different patterns of outbreeding. The P. falciparum population exhibited 4 subpopulations that changed in frequency over time. Notably, a sharp rise was observed in the frequency of a minor subpopulation (K2) in the late post-ACT period, accounting for 100% of infections in late 2016-2017. The results confirm epidemiological evidence of reduced P. falciparum and P. vivax transmission over time. The smaller change in P. vivax population structure is consistent with greater outbreeding associated with relapsing infections and highlights the need for radical cure to reduce recurrent infections. The study emphasizes the challenge in disrupting P. vivax transmission and demonstrates the potential of molecular data to inform on the impact of public health interventions.

Human Plasmodium vivax diversity, population structure and evolutionary origin.

More than 200 million malaria clinical cases are reported each year due to Plasmodium vivax, the most widespread Plasmodium species in the world. This species has been neglected and understudied for a long time, due to its lower mortality in comparison with Plasmodium falciparum. A renewed interest has emerged in the past decade with the discovery of antimalarial drug resistance and of severe and even fatal human cases. Nonetheless, today there are still significant gaps in our understanding of the population genetics and evolutionary history of P. vivax, particularly because of a lack of genetic data from Africa. To address these gaps, we genotyped 14 microsatellite loci in 834 samples obtained from 28 locations in 20 countries from around the world. We discuss the worldwide population genetic structure and diversity and the evolutionary origin of P. vivax in the world and its introduction into the Americas. This study demonstrates the importance of conducting genome-wide analyses of P. vivax in order to unravel its complex evolutionary history.

Genetic diversity of Plasmodium vivax and Plasmodium falciparum lactate dehydrogenases in Myanmar isolates.

BACKGROUND: Plasmodium lactate dehydrogenase (pLDH) is a major target in diagnosing the erythrocytic stage of malaria parasites because it is highly expressed during blood-stage parasites and is distinguished from human LDH. Rapid diagnostic tests (RDTs) for malaria use pLDH as a target antigen; however, genetic variations in pLDH within the natural population threaten the efficacy of pLDH-based RDTs. METHODS: Genetic polymorphisms of Plasmodium vivax LDH (PvLDH) and Plasmodium falciparum LDH (PfLDH) in Myanmar isolates were analysed by nucleotide sequencing analysis. Genetic polymorphisms and the natural selection of PvLDH and PfLDH were analysed using DNASTAR, MEGA6, and DnaSP ver. 5.10.00 programs. The genetic diversity and natural selection of global PvLDH and PfLDH were also analysed. The haplotype network of global PvLDH and PfLDH was constructed using NETWORK ver. 5.0.0.3. Three-dimensional structures of PvLDH and PfLDH were built with YASARA Structure ver. 18.4.24 and the impact of mutations on structural change and stability was evaluated with SDM ver. 2, CUPSAT and MAESTROweb. RESULTS: Forty-nine PvLDH and 52 PfLDH sequences were obtained from Myanmar P. vivax and P. falciparum isolates. Non-synonymous nucleotide substitutions resulting in amino acid changes were identified in both Myanmar PvLDH and PfLDH. Amino acid changes were also identified in the global PvLDH and PfLDH populations, but they did not produce structural alterations in either protein. Low genetic diversity was observed in global PvLDH and PfLDH, which may be maintained by a strong purifying selection. CONCLUSION: This study extends knowledge for genetic diversity and natural selection of global PvLDH and PfLDH. Although amino acid changes were observed in global PvLDH and PfLDH, they did not alter the conformational structures of the proteins. These suggest that PvLDH and PfLDH are genetically well-conserved in global populations, which indicates that they are suitable antigens for diagnostic purpose and attractive targets for drug development.

Molecular evidence of sustained urban malaria transmission in Amazonian Brazil, 2014-2015.

The relative contribution of imported vs. locally acquired infections to urban malaria burden remains largely unexplored in Latin America, the most urbanised region in the developing world. Here we use a simple molecular epidemiology framework to examine the transmission dynamics of Plasmodium vivax in Mâncio Lima, the Amazonian municipality with the highest malaria incidence rate in Brazil. We prospectively genotyped 177 P. vivax infections diagnosed in urban residents between June 2014 and July 2015 and showed that local parasites are structured into several lineages of closely related microsatellite haplotypes, with the largest genetic cluster comprising 32% of all infections. These findings are very unlikely under the hypothesis of multiple independent imports of parasite strains from the rural surroundings. Instead, the presence of an endemic near-clonal parasite lineage circulating over 13 consecutive months is consistent with a local P. vivax transmission chain in the town, with major implications for malaria elimination efforts in this and similar urban environments across the Amazon.

Plasmodium vivax Malaria Viewed through the Lens of an Eradicated European Strain.

The protozoan Plasmodium vivax is responsible for 42% of all cases of malaria outside Africa. The parasite is currently largely restricted to tropical and subtropical latitudes in Asia, Oceania, and the Americas. Though, it was historically present in most of Europe before being finally eradicated during the second half of the 20th century. The lack of genomic information on the extinct European lineage has prevented a clear understanding of historical population structuring and past migrations of P. vivax. We used medical microscope slides prepared in 1944 from malaria-affected patients from the Ebro Delta in Spain, one of the last footholds of malaria in Europe, to generate a genome of a European P. vivax strain. Population genetics and phylogenetic analyses placed this strain basal to a cluster including samples from the Americas. This genome allowed us to calibrate a genomic mutation rate for P. vivax, and to estimate the mean age of the last common ancestor between European and American strains to the 15th century. This date points to an introduction of the parasite during the European colonization of the Americas. In addition, we found that some known variants for resistance to antimalarial drugs, including Chloroquine and Sulfadoxine, were already present in this European strain, predating their use. Our results shed light on the evolution of an important human pathogen and illustrate the value of antique medical collections as a resource for retrieving genomic information on pathogens from the past.

Microsatellite analysis reveals connectivity among geographically distant transmission zones of Plasmodium vivax in the Peruvian Amazon: A critical barrier to regional malaria elimination.

Despite efforts made over decades by the Peruvian government to eliminate malaria, Plasmodium vivax remains a challenge for public health decision-makers in the country. The uneven distribution of its incidence, plus its complex pattern of dispersion, has made ineffective control measures based on global information that lack the necessary detail to understand transmission fully. In this sense, population genetic tools can complement current surveillance. This study describes the genetic diversity and population structure from September 2012 to March 2015 in three geographically distant settlements, Cahuide (CAH), Lupuna (LUP) and Santa Emilia (STE), located in the Peruvian Amazon. A total 777 P. vivax mono-infections, out of 3264, were genotyped. Among study areas, LUP showed 19.7% of polyclonal infections, and its genetic diversity (Hexp) was 0.544. Temporal analysis showed a significant increment of polyclonal infections and Hexp, and the introduction and persistence of a new parasite population since March 2013. In STE, 40.1% of infections were polyclonal, with Hexp = 0.596. The presence of four genetic clusters without signals of clonal expansion and infections with lower parasite densities compared against the other two areas were also found. At least four parasite populations were present in CAH in 2012, where, after June 2014, malaria cases decreased from 213 to 61, concomitant with a decrease in polyclonal infections (from 0.286 to 0.18), and expectedly variable Hexp. Strong signals of gene flow were present in the study areas and wide geographic distribution of highly diverse parasite populations were found. This study suggests that movement of malaria parasites by human reservoirs connects geographically distant malaria transmission areas in the Peruvian Amazon. The maintenance of high levels of parasite genetic diversity through human mobility is a critical barrier to malaria elimination in this region.

Genetic Diversity of Plasmodium vivax in Clinical Isolates from Southern Thailand using PvMSP1, PvMSP3 (PvMSP3α, PvMSP3ß) Genes and Eight Microsatellite Markers.

Plasmodium vivax is usually considered morbidity in endemic areas of Asia, Central and South America, and some part of Africa. In Thailand, previous studies indicated the genetic diversity of P. vivax in malaria-endemic regions such as the western part of Thailand bordering with Myanmar. The objective of the study is to investigate the genetic diversity of P. vivax circulating in Southern Thailand by using 3 antigenic markers and 8 microsatellite markers. Dried blood spots were collected from Chumphon, Phang Nga, Ranong and, Surat Thani provinces of Thailand. By PCR, 3 distinct sizes of PvMSP3α, 2 sizes of PvMSP3ß and 2 sizes of PvMSP1 F2 were detected based on the length of PCR products, respectively. PCR/RFLP analyses of these antigen genes revealed high levels of genetic diversity. The genotyping of 8 microsatellite loci showed high genetic diversity as indicated by high alleles per locus and high expected heterozygosity (HE). The genotyping markers also showed multiple-clones of infection. Mixed genotypes were detected in 4.8% of PvMSP3α, 29.1% in PvMSP3ß and 55.3% of microsatellite markers. These results showed that there was high genetic diversity of P. vivax isolated from Southern Thailand, indicating that the genetic diversity of P. vivax in this region was comparable to those observed other areas of Thailand.

Prevalence of Plasmodium falciparum Pfcrt and Pfmdr1 alleles in settings with different levels of Plasmodium vivax co-endemicity in Ethiopia.

Plasmodium falciparum and P. vivax co-exist at different endemicity levels across Ethiopia. For over two decades Artemether-Lumefantrine (AL) is the first line treatment for uncomplicated P. falciparum, while chloroquine (CQ) is still used to treat P. vivax. It is currently unclear whether a shift from CQ to AL for P. falciparum treatment has implications for AL efficacy and results in a reversal of mutations in genes associated to CQ resistance, given the high co-endemicity of the two species and the continued availability of CQ for the treatment of P. vivax. This study thus assessed the prevalence of Pfcrt-K76T and Pfmdr1-N86Y point mutations in P. falciparum. 18S RNA gene based nested PCR confirmed P. falciparum samples (N = 183) collected through community and health facility targeted cross-sectional surveys from settings with varying P. vivax and P. falciparum endemicity were used. The proportion of Plasmodium infections that were P. vivax was 62.2% in Adama, 41.4% in Babile, 30.0% in Benishangul-Gumuz to 6.9% in Gambella. The Pfcrt-76T mutant haplotype was observed more from samples with higher endemicity of P. vivax as being 98.4% (61/62), 100% (31/31), 65.2% (15/23) and 41.5% (22/53) in samples from Adama, Babile, Benishangul-Gumuz and Gambella, respectively. However, a relatively higher proportion of Pfmdr1-N86 allele (77.3-100%) were maintained in all sites. The observed high level of the mutant Pfcrt-76T allele in P. vivax co-endemic sites might require that utilization of CQ needs to be re-evaluated in settings co-endemic for the two species. A country-wide assessment is recommended to clarify the implication of the observed level of variation in drug resistance markers on the efficacy of AL-based treatment against uncomplicated P. falciparum malaria.

Origin of the New World Plasmodium vivax: Facts and New Approaches

Malaria is one of the most important human diseases throughout tropical and sub-tropical regions of the world. Global distribution and ample host range have contributed to the genetic diversity of the etiological agent, Plasmodium. Phylogeographical analyses demonstrated that Plasmodium falciparum and Plasmodium vivax follow an Out of Africa (OOA) expansion, having a higher genetic diversity in African populations and a low genetic diversity in South American populations. Modeling the evolutionary rate of conserved genes for both P. falciparum and P. vivax determined the approximate arrival of human malaria in South America. Bayesian computational methods suggest that P. falciparum originated in Africa and arrived in South America through multiple independent introductions by the transatlantic African slave trade; however, in South America, P. vivax could have been introduced through an alternate migratory route. Alignments of P. vivax mitogenomes have revealed low genetic variation between the South American and Southeast Asian populations suggesting introduction through either pre-Columbian human migration or post-colonization events. To confirm the findings of these phylogeographical analyses, molecular methods were used to diagnose malaria infection in archeological remains of pre-Columbian ethnic groups. Immunohistochemistry tests were used and identified P. vivax but not P. falciparum in histologically prepared tissues from pre-Columbian Peruvian mummies, whereas shotgun metagenomics sequencing of DNA isolated from pre-Columbian Caribbean coprolites revealed Plasmodium-homologous reads; current evidence suggests that only P. vivax might have been present in pre-Columbian South America

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Induction and Kinetics of Complement-Fixing Antibodies Against Plasmodium vivax Merozoite Surface Protein 3α and Relationship With Immunoglobulin G Subclasses and Immunoglobulin M.

BACKGROUND: Complement-fixing antibodies are important mediators of protection against Plasmodium falciparum malaria. However, complement-fixing antibodies remain uncharacterized for Plasmodium vivax malaria. P. vivax merozoite surface protein 3α (PvMSP3α) is a target of acquired immunity and a potential vaccine candidate. METHODS: Plasma from children and adults with P. vivax malaria in Sabah, Malaysia, were collected during acute infection, 7 and 28 days after drug treatment. Complement-fixing antibodies and immunoglobulin M and G (IgM and IgG), targeting 3 distinctive regions of PvMSP3α, were measured by means of enzyme-linked immunosorbent assay. RESULTS: The seroprevalence of complement-fixing antibodies was highest against the PvMSP3α central region (77.6%). IgG1, IgG3, and IgM were significantly correlated with C1q fixation, and both purified IgG and IgM were capable of mediating C1q fixation to PvMSP3α. Complement-fixing antibody levels were similar between age groups, but IgM was predominant in children and IgG3 more prevalent in adults. Levels of functional antibodies increased after acute infection through 7 days after treatment but rapidly waned by day 28. CONCLUSION: Our study demonstrates that PvMSP3α antibodies acquired during P. vivax infection can mediate complement fixation and shows the important influence of age in shaping these specific antibody responses. Further studies are warranted to understand the role of these functional antibodies in protective immunity against P. vivax malaria.

Estimating Relatedness Between Malaria Parasites.

Understanding the relatedness of individuals within or between populations is a common goal in biology. Increasingly, relatedness features in genetic epidemiology studies of pathogens. These studies are relatively new compared to those in humans and other organisms, but are important for designing interventions and understanding pathogen transmission. Only recently have researchers begun to routinely apply relatedness to apicomplexan eukaryotic malaria parasites, and to date have used a range of different approaches on an ad hoc basis. Therefore, it remains unclear how to compare different studies and which measures to use. Here, we systematically compare measures based on identity-by-state (IBS) and identity-by-descent (IBD) using a globally diverse data set of malaria parasites, Plasmodium falciparum and P. vivax, and provide marker requirements for estimates based on IBD. We formally show that the informativeness of polyallelic markers for relatedness inference is maximized when alleles are equifrequent. Estimates based on IBS are sensitive to allele frequencies, which vary across populations and by experimental design. For portability across studies, we thus recommend estimates based on IBD. To generate estimates with errors below an arbitrary threshold of 0.1, we recommend ∼100 polyallelic or 200 biallelic markers. Marker requirements are immediately applicable to haploid malaria parasites and other haploid eukaryotes. C.I.s facilitate comparison when different marker sets are used. This is the first attempt to provide rigorous analysis of the reliability of, and requirements for, relatedness inference in malaria genetic epidemiology. We hope it will provide a basis for statistically informed prospective study design and surveillance strategies.

Molecular epidemiology of residual Plasmodium vivax transmission in a paediatric cohort in Solomon Islands.

BACKGROUND: Following the scale-up of intervention efforts, malaria burden has decreased dramatically in Solomon Islands (SI). Submicroscopic and asymptomatic Plasmodium vivax infections are now the major challenge for malaria elimination in this country. Since children have higher risk of contracting malaria, this study investigated the dynamics of Plasmodium spp. infections among children including the associated risk factors of residual P. vivax burden. METHODS: An observational cohort study was conducted among 860 children aged 0.5-12 years in Ngella (Central Islands Province, SI). Children were monitored by active and passive surveillances for Plasmodium spp. infections and illness. Parasites were detected by quantitative real-time PCR (qPCR) and genotyped. Comprehensive statistical analyses of P. vivax infection prevalence, molecular force of blood stage infection (molFOB) and infection density were conducted. RESULTS: Plasmodium vivax infections were common (overall prevalence: 11.9%), whereas Plasmodium falciparum infections were rare (0.3%) but persistent. Although children acquire an average of 1.1 genetically distinct P. vivax blood-stage infections per year, there was significant geographic heterogeneity in the risks of P. vivax infections across Ngella (prevalence: 1.2-47.4%, p < 0.01; molFOB: 0.05-4.6/year, p < 0.01). Malaria incidence was low (IR: 0.05 episodes/year-at-risk). Age and measures of high exposure were the key risk factors for P. vivax infections and disease. Malaria incidence and infection density decreased with age, indicating significant acquisition of immunity. G6PD deficient children (10.8%) that did not receive primaquine treatment had a significantly higher prevalence (aOR: 1.77, p = 0.01) and increased risk of acquiring new bloodstage infections (molFOB aIRR: 1.51, p = 0.03), underscoring the importance of anti-relapse treatment. CONCLUSION: Residual malaria transmission in Ngella exhibits strong heterogeneity and is characterized by a high proportion of submicroscopic and asymptomatic P. vivax infections, alongside sporadic P. falciparum infections. Implementing an appropriate primaquine treatment policy to prevent P. vivax relapses and specific targeting of control interventions to high risk areas will be required to accelerate ongoing control and elimination activities.

Origin of the New World Plasmodium vivax: Facts and New Approaches.

Malaria is one of the most important human diseases throughout tropical and sub-tropical regions of the world. Global distribution and ample host range have contributed to the genetic diversity of the etiological agent, Plasmodium. Phylogeographical analyses demonstrated that Plasmodium falciparum and Plasmodium vivax follow an Out of Africa (OOA) expansion, having a higher genetic diversity in African populations and a low genetic diversity in South American populations. Modeling the evolutionary rate of conserved genes for both P. falciparum and P. vivax determined the approximate arrival of human malaria in South America. Bayesian computational methods suggest that P. falciparum originated in Africa and arrived in South America through multiple independent introductions by the transatlantic African slave trade; however, in South America, P. vivax could have been introduced through an alternate migratory route. Alignments of P. vivax mitogenomes have revealed low genetic variation between the South American and Southeast Asian populations suggesting introduction through either pre-Columbian human migration or post-colonization events. To confirm the findings of these phylogeographical analyses, molecular methods were used to diagnose malaria infection in archeological remains of pre-Columbian ethnic groups. Immunohistochemistry tests were used and identified P. vivax but not P. falciparum in histologically prepared tissues from pre-Columbian Peruvian mummies, whereas shotgun metagenomics sequencing of DNA isolated from pre-Columbian Caribbean coprolites revealed Plasmodium-homologous reads; current evidence suggests that only P. vivax might have been present in pre-Columbian South America.

Genomic Analysis of Plasmodium vivax in Southern Ethiopia Reveals Selective Pressures in Multiple Parasite Mechanisms.

The Horn of Africa harbors the largest reservoir of Plasmodium vivax in the continent. Most of sub-Saharan Africa has remained relatively vivax-free due to a high prevalence of the human Duffy-negative trait, but the emergence of strains able to invade Duffy-negative reticulocytes poses a major public health threat. We undertook the first population genomic investigation of P. vivax from the region, comparing the genomes of 24 Ethiopian isolates against data from Southeast Asia to identify important local adaptions. The prevalence of the Duffy binding protein amplification in Ethiopia was 79%, potentially reflecting adaptation to Duffy negativity. There was also evidence of selection in a region upstream of the chloroquine resistance transporter, a putative chloroquine-resistance determinant. Strong signals of selection were observed in genes involved in immune evasion and regulation of gene expression, highlighting the need for a multifaceted intervention approach to combat P. vivax in the region.

Asymptomatic Natural Human Infections With the Simian Malaria Parasites Plasmodium cynomolgi and Plasmodium knowlesi.

BACKGROUND: In Southeast Asia, Plasmodium knowlesi, a parasite of long-tailed macaques (Macaca fascicularis), is an important cause of human malaria. Plasmodium cynomolgi also commonly infects these monkeys, but only one naturally acquired symptomatic human case has been reported previously. METHODS: Malariometric studies involving 5422 subjects (aged 6 months to 65 years) were conducted in 23 villages in Pailin and Battambang, western Cambodia. Parasite detection and genotyping was conducted on blood samples, using high-volume quantitative PCR (uPCR). RESULTS: Asymptomatic malaria parasite infections were detected in 1361 of 14732 samples (9.2%). Asymptomatic infections with nonhuman primate malaria parasites were found in 21 individuals living close to forested areas; P. cynomolgi was found in 11, P. knowlesi was found in 8, and P. vivax and P. cynomolgi were both found in 2. Only 2 subjects were female, and 14 were men aged 20-40 years. Geometric mean parasite densities were 3604 parasites/mL in P. cynomolgi infections and 52488 parasites/mL in P. knowlesi infections. All P. cynomolgi isolates had wild-type dihydrofolate reductase genes, in contrast to the very high prevalence of mutations in the human malaria parasites. Asymptomatic reappearance of P. cynomolgi occurred in 2 subjects 3 months after the first infection. CONCLUSIONS: Asymptomatic naturally acquired P. cynomolgi and P. knowlesi infections can both occur in humans. CLINICAL TRIALS REGISTRATION: NCT01872702.