Malarial merozoites use an array of ligands, including members of the Reticulocyte Binding Like (RBL) super-family of invasion proteins, to identify and invade erythrocytes. RBL family members are large Type I membrane anchored proteins expressed at the invasive end of merozoites that share homology with the Reticulocyte Binding Proteins 1 and 2 (PvRBP1 and 2) of Plasmodium vivax. Plasmodium species vary widely both in the number and sequence of their RBL genes, with the recently completed Plasmodium falciparum genome containing five RBL genes. Of these, three encode proteins shown to be involved in erythrocyte invasion, a fourth is a pseudogene, and the role of the fifth is as yet unclear. In order to identify sequence similarities and differences that may have functional implications for erythrocyte invasion as well as to gain insights into the recent evolutionary history of the P. falciparum RBL genes, we have sequenced all five corresponding RBL genes from the chimpanzee parasite Plasmodium reichenowi, which is the closest phylogenetic relative of P. falciparum, yet is unable to invade human erythrocytes. Two of the five P. falciparum RBL genes have highly conserved complete open reading frames in both species, while the other three genes show evidence of gene conversion and rapid evolution. The RBL super-family, therefore, appears to be surprisingly dynamic and divergent, implying that it is involved in species-specific aspects of erythrocyte recognition and invasion.

Recently, a real-time PCR assay known as photo-induced electron transfer (PET)-PCR which relies on self-quenching primers for the detection of Plasmodium spp. and Plasmodium falciparum was described. PET-PCR assay was found to be robust, and easier to use when compared to currently available real-time PCR methods. The potential of PET-PCR for molecular detection of malaria parasites in a nationwide malaria community survey in Haiti was investigated.

We sequenced and annotated the genomes of four P. vivax strains collected from disparate geographic locations, tripling the number of genome sequences available for this understudied parasite and providing the first genome-wide perspective of global variability in this species. We observe approximately twice as much SNP diversity among these isolates as we do among a comparable collection of isolates of P. falciparum, a malaria-causing parasite that results in higher mortality. This indicates a distinct history of global colonization and/or a more stable demographic history for P. vivax relative to P. falciparum, which is thought to have undergone a recent population bottleneck. The SNP diversity, as well as additional microsatellite and gene family variability, suggests a capacity for greater functional variation in the global population of P. vivax. These findings warrant a deeper survey of variation in P. vivax to equip disease interventions targeting the distinctive biology of this neglected but major pathogen.

Previous work suggests that Brazilian Plasmodium falciparum has limited genetic diversity and a history of bottlenecks, multiple reintroductions due to human migration, and clonal expansions. We hypothesized that Brazilian P. falciparum would exhibit clonal structure. We examined isolates collected across two decades from Amapá, Rondônia, and Pará state (n = 190). By examining more microsatellites markers on more chromosomes than previous studies, we hoped to define the extent of low diversity, linkage disequilibrium, bottlenecks, population structure, and parasite migration within Brazil. We used retrospective genotyping of samples from the 1980s and 1990s to explore the population genetics of SP resistant dhfr and dhps alleles. We tested an existing hypothesis that the triple mutant dhfr mutations 50R/51I/108N and 51I/108N/164L developed in southern Amazon from a single origin of common or similar parasites. We found that Brazilian P. falciparum had limited genetic diversity and isolation by distance was rejected, which suggests it underwent bottlenecks followed by migration between sites. Unlike Peru, there appeared to be gene flow across the Brazilian Amazon basin. We were unable to divide parasite populations by clonal lineages and pairwise FST were common. Most parasite diversity was found within sites in the Brazilian Amazon, according to AMOVA. Our results challenge the hypothesis that triple mutant alleles arose from a single lineage in the Southern Amazon. SP resistance, at both the double and triple mutant stages, developed twice and potentially in different regions of the Brazilian Amazon. We would have required samples from before the 1980s to describe how SP resistance spread across the basin or describe the complex internal migration of Brazilian parasites after the colonization efforts of past decades. The Brazilian Amazon basin may have sufficient internal migration for drug resistance reported in any particular region to rapidly spread to other parts of basin under similar drug pressure.

The genome sequence available for different Plasmodium species is a valuable resource for understanding malaria parasite biology. However, comparative genomics on its own cannot fully explain all the species-specific differences which suggests that other genomic aspects such as regulation of gene expression play an important role in defining species-specific characteristics. Here, we developed a comprehensive approach to measure transcriptional changes of the evolutionary conserved syntenic orthologs during the intraerythrocytic developmental cycle across six Plasmodium species. We show significant transcriptional constraint at the mid-developmental stage of Plasmodium species while the earliest stages of parasite development display the greatest transcriptional variation associated with critical functional processes. Modeling of the evolutionary relationship based on changes in transcriptional profile reveal a phylogeny pattern of the Plasmodium species that strictly follows its mammalian hosts. In addition, the work shows that transcriptional conserved orthologs represent potential future targets for anti-malaria intervention as they would be expected to carry out key essential functions within the parasites. This work provides an integrated analysis of orthologous transcriptome, which aims to provide insights into the Plasmodium evolution thereby establishing a framework to explore complex pathways and drug discovery in Plasmodium species with broad host range.

Recent studies in Southeast Asia have demonstrated substantial zoonotic transmission of Plasmodium knowlesi to humans. Microscopically, P. knowlesi exhibits several stage-dependent morphological similarities to P. malariae and P. falciparum. These similarities often lead to misdiagnosis of P. knowlesi as either P. malariae or P. falciparum and PCR-based molecular diagnostic tests are required to accurately detect P. knowlesi in humans. The most commonly used PCR test has been found to give false positive results, especially with a proportion of P. vivax isolates. To address the need for more sensitive and specific diagnostic tests for the accurate diagnosis of P. knowlesi, we report development of a new single-step PCR assay that uses novel genomic targets to accurately detect this infection.

The study of antigenic targets of naturally-acquired immunity is essential to identify and prioritize antigens for further functional characterization. We measured total IgG antibodies to 38 P. vivax antigens, investigating their relationship with prospective risk of malaria in a cohort of 1-3 years old Papua New Guinean children. Using simulated annealing algorithms, the potential protective efficacy of antibodies to multiple antigen-combinations, and the antibody thresholds associated with protection were investigated for the first time. High antibody levels to multiple known and newly identified proteins were strongly associated with protection (IRR 0.44-0.74, p<0.001-0.041). Among five-antigen combinations with the strongest protective effect (>90%), EBP, DBPII, RBP1a, CyRPA, and PVX_081550 were most frequently identified; several of them requiring very low antibody levels to show a protective association. These data identify individual antigens that should be prioritized for further functional testing and establish a clear path to testing a multicomponent P. vivax vaccine.

Mild to severe anaemia is a common complication of malaria that is caused in part by insufficient erythropoiesis in the bone marrow. This study used systems biology to evaluate the transcriptional and alterations in cell populations in the bone marrow during Plasmodium cynomolgi infection of rhesus macaques (a model of Plasmodium vivax malaria) that may affect erythropoiesis.

P. cynomolgi, a malaria-causing parasite of Asian Old World monkeys, is the sister taxon of P. vivax, the most prevalent malaria-causing species in humans outside of Africa. Because P. cynomolgi shares many phenotypic, biological and genetic characteristics with P. vivax, we generated draft genome sequences for three P. cynomolgi strains and performed genomic analysis comparing them with the P. vivax genome, as well as with the genome of a third previously sequenced simian parasite, Plasmodium knowlesi. Here, we show that genomes of the monkey malaria clade can be characterized by copy-number variants (CNVs) in multigene families involved in evasion of the human immune system and invasion of host erythrocytes. We identify genome-wide SNPs, microsatellites and CNVs in the P. cynomolgi genome, providing a map of genetic variation that can be used to map parasite traits and study parasite populations. The sequencing of the P. cynomolgi genome is a critical step in developing a model system for P. vivax research and in counteracting the neglect of P. vivax.

Plasmodium knowlesi is a monkey malaria species that is becoming a serious public health concern infecting hundreds and perhaps thousands of humans in Southeast Asia. Invasion of erythrocytes by merozoites entails a cascade of molecular interactions. One step involves the adhesion of Plasmodium reticulocyte binding-like (RBL) proteins. Plasmodium knowlesi merozoites express only two RBL invasion ligands, known as Normocyte Binding Proteins (PkNBPXa and PkNBPXb).

Three members of the Plasmodium vivax merozoite surface protein-3 (PvMSP3) family (PvMSP3-α, PvMSP3-β and PvMSP3-γ) were initially characterized and later shown to be part of a larger highly diverse family, encoded by a cluster of genes arranged head-to-tail in chromosome 10. PvMSP3-α and PvMSP3-β have become genetic markers in epidemiological studies, and are being evaluated as vaccine candidates. This research investigates the gene and protein expression of the entire family and pertinent implications.

Mutation rates vary between species across several orders of magnitude, with larger organisms having the highest per-generation mutation rates. Hypotheses for this pattern typically invoke physiological or population-genetic constraints imposed on the molecular machinery preventing mutations [1]. However, continuing germline cell division in multicellular eukaryotes means that organisms with longer generation times and of larger size will leave more mutations to their offspring simply as a byproduct of their increased lifespan [2, 3]. Here, we deeply sequence the genomes of 30 owl monkeys (Aotus nancymaae) from six multi-generation pedigrees to demonstrate that paternal age is the major factor determining the number of de novo mutations in this species. We find that owl monkeys have an average mutation rate of 0.81 × 10-8 per site per generation, roughly 32% lower than the estimate in humans. Based on a simple model of reproductive longevity that does not require any changes to the mutational machinery, we show that this is the expected mutation rate in owl monkeys. We further demonstrate that our model predicts species-specific mutation rates in other primates, including study-specific mutation rates in humans based on the average paternal age. Our results suggest that variation in life history traits alone can explain variation in the per-generation mutation rate among primates, and perhaps among a wide range of multicellular organisms.

Malaria is a major mosquito transmitted, blood-borne parasitic disease that afflicts humans. The disease causes anaemia and other clinical complications, which can lead to death. Plasmodium vivax is known for its reticulocyte host cell specificity, but many gaps in disease details remain. Much less is known about the closely related species, Plasmodium cynomolgi, although it is naturally acquired and causes zoonotic malaria. Here, a computational model is developed based on longitudinal analyses of P. cynomolgi infections in nonhuman primates to investigate the erythrocyte dynamics that is pertinent to understanding both P. cynomolgi and P. vivax malaria in humans.

Plasmodium falciparum resistance to artemisinin has emerged in the Greater Mekong Subregion and now poses a threat to malaria control and prevention. Recent work has identified mutations in the kelch propeller domain of the P. falciparum K13 gene to be associated artemisinin resistance as defined by delayed parasite clearance and ex vivo ring stage survival assays. Species specific primers for the two most prevalent human malaria species, P. falciparum and P. vivax, were designed and tested on multiple parasite isolates including human, rodent, and non- humans primate Plasmodium species. The new protocol described here using the species specific primers only amplified their respective species, P. falciparum and P. vivax, and did not cross react with any of the other human malaria Plasmodium species. We provide an improved species specific PCR and sequencing protocol that could be effectively used in areas where both P. falciparum and P. vivax are circulating. To design this improved protocol, the kelch gene was analyzed and compared among different species of Plasmodium. The kelch propeller domain was found to be highly conserved across the mammalian Plasmodium species.

The recent emergence of artemisinin resistance in the Greater Mekong Subregion poses a major threat to the global effort to control malaria. Tracking the spread and evolution of artemisinin-resistant parasites is critical in aiding efforts to contain the spread of resistance. A total of 417 patient samples from the year 2007, collected during malaria surveillance studies across ten provinces in Thailand, were genotyped for the candidate Plasmodium falciparum molecular marker of artemisinin resistance K13. Parasite genotypes were examined for K13 propeller mutations associated with artemisinin resistance, signatures of positive selection, and for evidence of whether artemisinin-resistant alleles arose independently across Thailand. A total of seven K13 mutant alleles were found (N458Y, R539T, E556D, P574L, R575K, C580Y, S621F). Notably, the R575K and S621F mutations have previously not been reported in Thailand. The most prevalent artemisinin resistance-associated K13 mutation, C580Y, carried two distinct haplotype profiles that were separated based on geography, along the Thai-Cambodia and Thai-Myanmar borders. It appears these two haplotypes may have independent evolutionary origins. In summary, parasites with K13 propeller mutations associated with artemisinin resistance were widely present along the Thai-Cambodia and Thai-Myanmar borders prior to the implementation of the artemisinin resistance containment project in the region.

Plasmodium vivax is a complex protozoan parasite with over 6,500 genes and stage-specific differential expression. Much of the unique biology of this pathogen remains unknown, including how it modifies and restructures the host reticulocyte. Using a recently published P. vivax reference genome, we report the proteome from two biological replicates of infected Saimiri boliviensis host reticulocytes undergoing transition from the late trophozoite to early schizont stages. Using five database search engines, we identified a total of 2000 P. vivax and 3487 S. boliviensis proteins, making this the most comprehensive P. vivax proteome to date. PlasmoDB GO-term enrichment analysis of proteins identified at least twice by a search engine highlighted core metabolic processes and molecular functions such as glycolysis, translation and protein folding, cell components such as ribosomes, proteasomes and the Golgi apparatus, and a number of vesicle and trafficking related clusters. Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8 enriched functional annotation clusters of S. boliviensis proteins highlighted vesicle and trafficking-related clusters, elements of the cytoskeleton, oxidative processes and response to oxidative stress, macromolecular complexes such as the proteasome and ribosome, metabolism, translation, and cell death. Host and parasite proteins potentially involved in cell adhesion were also identified. Over 25% of the P. vivax proteins have no functional annotation; this group includes 45 VIR members of the large PIR family. A number of host and pathogen proteins contained highly oxidized or nitrated residues, extending prior trophozoite-enriched stage observations from S. boliviensis infections, and supporting the possibility of oxidative stress in relation to the disease. This proteome significantly expands the size and complexity of the known P. vivax and Saimiri host iRBC proteomes, and provides in-depth data that will be valuable for ongoing research on this parasite's biology and pathogenesis.

Vaccination with Plasmodium falciparum MSP1(42)/complete Freund's adjuvant (FA) followed by MSP1(42)/incomplete FA is the only known regimen that protects Aotus nancymaae monkeys against infection by erythrocytic stage malaria parasites. The role of adjuvant is not defined; however complete FA cannot be used in humans. In rodent models, immunity is strain-specific. We vaccinated Aotus monkeys with the FVO or 3D7 alleles of MSP1(42) expressed in Escherichia coli or with the FVO allele expressed in baculovirus (bv) combined with complete and incomplete FA, Montanide ISA-720 (ISA-720) or AS02A. Challenge with FVO strain P. falciparum showed that suppression of cumulative day 11 parasitemia was strain-specific and could be induced by E. coli expressed MSP1(42) in combination with FA or ISA-720 but not with AS02A. The coli42-FVO antigen induced a stronger protective effect than the bv42-FVO antigen, and FA induced a stronger protective effect than ISA-720. ELISA antibody (Ab) responses at day of challenge (DOC) were strain-specific and correlated inversely with c-day 11 parasitemia (r = -0.843). ELISA Ab levels at DOC meeting a titer of at least 115,000 ELISA Ab units identified the vaccinees not requiring treatment (noTx) with a true positive rate of 83.3% and false positive rate of 14.3 %. Correlation between functional growth inhibitory Ab levels (GIA) and cumulative day 11 parasitemia was weaker (r = -0.511), and was not as predictive for a response of noTx. The lowest false positive rate for GIA was 30% when requiring a true positive rate of 83.3%. These inhibition results along with those showing that antigen/FA combinations induced a stronger protective immunity than antigen/ISA-720 or antigen/AS02 combinations are consistent with protection as ascribed to MSP1-specific cytophilic antibodies. Development of an effective MSP1(42) vaccine against erythrocytic stage P. falciparum infection will depend not only on antigen quality, but also upon the selection of an optimal adjuvant component.

"The Primate Malarias" book has been a uniquely important resource for multiple generations of scientists, since its debut in 1971, and remains pertinent to the present day. Indeed, nonhuman primates (NHPs) have been instrumental for major breakthroughs in basic and pre-clinical research on malaria for over 50 years. Research involving NHPs have provided critical insights and data that have been essential for malaria research on many parasite species, drugs, vaccines, pathogenesis, and transmission, leading to improved clinical care and advancing research goals for malaria control, elimination, and eradication. Whilst most malaria scientists over the decades have been studying Plasmodium falciparum, with NHP infections, in clinical studies with humans, or using in vitro culture or rodent model systems, others have been dedicated to advancing research on Plasmodium vivax, as well as on phylogenetically related simian species, including Plasmodium cynomolgi, Plasmodium coatneyi, and Plasmodium knowlesi. In-depth study of these four phylogenetically related species over the years has spawned the design of NHP longitudinal infection strategies for gathering information about ongoing infections, which can be related to human infections. These Plasmodium-NHP infection model systems are reviewed here, with emphasis on modern systems biological approaches to studying longitudinal infections, pathogenesis, immunity, and vaccines. Recent discoveries capitalizing on NHP longitudinal infections include an advanced understanding of chronic infections, relapses, anaemia, and immune memory. With quickly emerging new technological advances, more in-depth research and mechanistic discoveries can be anticipated on these and additional critical topics, including hypnozoite biology, antigenic variation, gametocyte transmission, bone marrow dysfunction, and loss of uninfected RBCs. New strategies and insights published by the Malaria Host-Pathogen Interaction Center (MaHPIC) are recapped here along with a vision that stresses the importance of educating future experts well trained in utilizing NHP infection model systems for the pursuit of innovative, effective interventions against malaria.

Plasmodium vivax infections in humans or in new world monkeys pose research challenges that necessitate the use of alternative model systems. Plasmodium cynomolgi is a closely related species that shares genetic and biological characteristics with P. vivax, including relapses. Here, the haematological dynamics and clinical presentation of sporozoite-initiated P. cynomolgi infections in Macaca mulatta (rhesus macaques) are evaluated over a 100-day period.

Isothermal nucleic acid amplification assays such as the loop mediated isothermal amplification (LAMP), are well suited for field use as they do not require thermal cyclers to amplify the DNA. To further facilitate the use of LAMP assays in remote settings, simpler sample preparation methods and lyophilized reagents are required. The performance of a commercial malaria LAMP assay (Illumigene Malaria LAMP) was evaluated using two sample preparation workflows (simple filtration prep (SFP)) and gravity-driven filtration prep (GFP)) and pre-dispensed lyophilized reagents. Laboratory and clinical samples were tested in a field laboratory in Senegal and the results independently confirmed in a reference laboratory in the U.S.A. The Illumigene Malaria LAMP assay was easily implemented in the clinical laboratory and gave similar results to a real-time PCR reference test with limits of detection of ≤2.0 parasites/μl depending on the sample preparation method used. This assay reliably detected Plasmodium sp. parasites in a simple low-tech format, providing a much needed alternative to the more complex molecular tests for malaria diagnosis.