Inhibition of malaria and babesiosis parasites by putative red blood cell targeting small molecules.

Background: Chemotherapies for malaria and babesiosis frequently succumb to the emergence of pathogen-related drug-resistance. Host-targeted therapies are thought to be less susceptible to resistance but are seldom considered for treatment of these diseases. Methods: Our overall objective was to systematically assess small molecules for host cell-targeting activity to restrict proliferation of intracellular parasites. We carried out a literature survey to identify small molecules annotated for host factors implicated in Plasmodium falciparum infection. Alongside P. falciparum, we implemented in vitro parasite susceptibility assays also in the zoonotic parasite Plasmodium knowlesi and the veterinary parasite Babesia divergens. We additionally carried out assays to test directly for action on RBCs apart from the parasites. To distinguish specific host-targeting antiparasitic activity from erythrotoxicity, we measured phosphatidylserine exposure and hemolysis stimulated by small molecules in uninfected RBCs. Results: We identified diverse RBC target-annotated inhibitors with Plasmodium-specific, Babesia-specific, and broad-spectrum antiparasitic activity. The anticancer MEK-targeting drug trametinib is shown here to act with submicromolar activity to block proliferation of Plasmodium spp. in RBCs. Some inhibitors exhibit antimalarial activity with transient exposure to RBCs prior to infection with parasites, providing evidence for host-targeting activity distinct from direct inhibition of the parasite. Conclusions: We report here characterization of small molecules for antiproliferative and host cell-targeting activity for malaria and babesiosis parasites. This resource is relevant for assessment of physiological RBC-parasite interactions and may inform drug development and repurposing efforts.

Who was the first to visualize the malaria parasite?

Human malaria, an ancient tropical disease, is caused by infection with protozoan parasites belonging to the genus Plasmodium and is transmitted by female mosquitoes of the genus Anopheles. Our understanding of human malaria parasites began officially in 1880 with their discovery in the blood of malaria patients by Charles Louis Alphonse Lavéran (1845-1922), a French army officer working in Algeria. A claim for priority was made by Philipp Friedrich Hermann Klencke (1813-1881) in 1843, who wrote a chapter entitled: "Marvellous parallelism between the manifestations of vertigo and the presence of animalcule vacuoles in living blood." We should not lose sight of this old controversy, which is rarely mentioned in historical reviews on malaria.

Characterization of merozoite-specific thrombospondin-related anonymous protein (MTRAP) in Plasmodium vivax and P. knowlesi parasites.

Plasmodium vivax, the most widespread human malaria parasite, and P. knowlesi, an emerging Plasmodium that infects humans, are the phylogenetically closest malarial species that infect humans, which may induce cross-species reactivity across most co-endemic areas in Southeast Asia. The thrombospondin-related anonymous protein (TRAP) family is indispensable for motility and host cell invasion in the growth and development of Plasmodium parasites. The merozoite-specific TRAP (MTRAP), expressed in blood-stage merozoites, is supposed to be essential for human erythrocyte invasion. We aimed to characterize MTRAPs in blood-stage P. vivax and P. knowlesi parasites and ascertain their cross-species immunoreactivity. Recombinant P. vivax and P. knowlesi MTRAPs of full-length ectodomains were expressed in a mammalian expression system. The MTRAP-specific immunoglobulin G, obtained from immune animals, was used in an immunofluorescence assay for subcellular localization and invasion inhibitory activity in blood-stage parasites was determined. The cross-species humoral immune responses were analyzed in the sera of patients with P. vivax or P. knowlesi infections. The MTRAPs of P. vivax (PvMTRAP) and P. knowlesi (PkMTRAP) were localized on the rhoptry body of merozoites in blood-stage parasites. Both anti-PvMTRAP and anti-PkMTRAP antibodies inhibited erythrocyte invasion of blood-stage P. knowlesi parasites. The humoral immune response to PvMTRAP showed high immunogenicity, longevity, and cross-species immunoreactivity with P. knowlesi. MTRAPs are promising candidates for development of vaccines and therapeutics against vivax and knowlesi malaria.

Revisiting the determinants of malaria transmission.

Malaria parasites have coevolved with humans over thousands of years, mirroring their migration out of Africa. They persist to this day, despite continuous elimination efforts worldwide. These parasites can adapt to changing environments during infection of human and mosquito, and when expanding the geographical range by switching vector species. Recent studies in the human malaria parasite, Plasmodium falciparum, identified determinants governing the plasticity of sexual conversion rates, sex ratio, and vector competence. Here we summarize the latest literature revealing environmental, epigenetic, and genetic determinants of malaria transmission.

Novel Ion Channel Genes in Malaria Parasites.

Ion channels serve many cellular functions including ion homeostasis, volume regulation, signaling, nutrient acquisition, and developmental progression. Although the complex life cycles of malaria parasites necessitate ion and solute flux across membranes, the whole-genome sequencing of the human pathogen Plasmodium falciparum revealed remarkably few orthologs of known ion channel genes. Contrasting with this, biochemical studies have implicated the channel-mediated flux of ions and nutritive solutes across several membranes in infected erythrocytes. Here, I review advances in the cellular and molecular biology of ion channels in malaria parasites. These studies have implicated novel parasite genes in the formation of at least two ion channels, with additional ion channels likely present in various membranes and parasite stages. Computational approaches that rely on homology to known channel genes from higher organisms will not be very helpful in identifying the molecular determinants of these activities. Given their unusual properties, novel molecular and structural features, and essential roles in pathogen survival and development, parasite channels should be promising targets for therapy development.

The prevalence of simian malaria in wild long-tailed macaques throughout Peninsular Malaysia.

The parasite Plasmodium knowlesi has been the sole cause of malaria in Malaysia from 2018 to 2022. The persistence of this zoonotic species has hampered Malaysia's progress towards achieving the malaria-free status awarded by the World Health Organisation (WHO). Due to the zoonotic nature of P. knowlesi infections, it is important to study the prevalence of the parasite in the macaque host, the long-tailed macaque (Macaca fascicularis). Apart from P. knowlesi, the long-tailed macaque is also able to harbour Plasmodium cynomolgi, Plasmodium inui, Plasmodium caotneyi and Plasmodium fieldi. Here we report the prevalence of the 5 simian malaria parasites in the wild long-tailed macaque population in 12 out of the 13 states in Peninsular Malaysia using a nested PCR approach targeting the 18s ribosomal RNA (18s rRNA) gene. It was found that all five Plasmodium species were widely distributed throughout Peninsular Malaysia except for states with major cities such as Kuala Lumpur and Putrajaya. Of note, Pahang reported a malaria prevalence of 100% in the long-tailed macaque population, identifying it as a potential hotspot for zoonotic transmission. Overall, this study shows the distribution of the 5 simian malaria parasite species throughout Peninsular Malaysia, the data of which could be used to guide future malaria control interventions to target zoonotic malaria.

Malaria infection among adults residing in a highly endemic region from the Democratic Republic of the Congo.

BACKGROUND: Adults infected with Plasmodium spp. in endemic areas need to be re-evaluated in light of global malaria elimination goals. They potentially undermine malaria interventions but remain an overlooked aspect of public health strategies. METHODS: This study aimed to estimate the prevalence of Plasmodium spp. infections, to identify underlying parasite species, and to assess predicting factors among adults residing in an endemic area from the Democratic Republic of Congo (DRC). A community-based cross-sectional survey in subjects aged 18 years and above was therefore carried out. Study participants were interviewed using a standard questionnaire and tested for Plasmodium spp. using a rapid diagnostic test and a nested polymerase chain reaction assay. Logistic regression models were fitted to assess the effect of potential predictive factors for infections with different Plasmodium spp. RESULTS: Overall, 420 adults with an estimated prevalence of Plasmodium spp. infections of 60.2% [95% CI 55.5; 64.8] were included. Non-falciparum species infected 26.2% [95% CI 22.2; 30.5] of the study population. Among infected participants, three parasite species were identified, including Plasmodium falciparum (88.5%), Plasmodium malariae (39.9%), and Plasmodium ovale (7.5%) but no Plasmodium vivax. Mixed species accounted for 42.3% of infections while single-species infections predominated with P. falciparum (56.5%) among infected participants. All infected participants were asymptomatic at the time of the survey. Adults belonging to the "most economically disadvantaged" households had increased risks of infections with any Plasmodium spp. (adjusted odds ratio, aOR = 2.87 [95% CI 1.66, 20.07]; p < 0.001), compared to those from the "less economically disadvantaged" households. Conversely, each 1 year increase in age reduced the risk of infections with any Plasmodium spp. (aOR = 0.99 [95% CI 0.97, 0.99]; p = 0.048). Specifically for non-falciparum spp., males had increased risks of infection than females (aOR = 1.83 [95% CI 1.13, 2.96]; p = 0.014). CONCLUSION: Adults infected with malaria constitute a potentially important latent reservoir for the transmission of the disease in the study setting. They should specifically be taken into account in public health measures and translational research.

Quality assessment of malaria microscopic diagnosis at the Aristide Le Dantec University Hospital of Dakar, Senegal, in 2020.

BACKGROUND: Following WHO guidelines, microscopy is the gold standard for malaria diagnosis in endemic countries. The Parasitology-Mycology laboratory (LPM) is the National Reference Laboratory and is currently undergoing ISO 15189 accreditation. In this context, we assessed the performance of the laboratory by confirming the reliability and the accuracy of results obtained in accordance with the requirements of the ISO 15189 standards. This study aimed to verify the method of microscopic diagnosis of malaria at the LPM, in the Aristide Le Dantec hospital (HALD) in Dakar, Senegal. METHODS: This is a validation/verification study conducted from June to August 2020. Twenty (20) microscopic slides of thick/thin blood smear with known parasite densities (PD) selected from the Cheick Anta Diop University malaria slide bank in Dakar were used for this assessment. Six (6) were used to assess microscopists' ability to determine PD and fourteen (14) slides were used for detection (positive vs negative) and identification of parasites. Four (4) LPM-HALD microscopists read and recorded their results on prepared sheets. Data analysis was done with Microsoft Excel 2010 software. RESULTS: A minimum threshold of 50% concordance was used for comparison. Of the twenty (20) slides read, 100% concordance was obtained on eight (8) detection (positive vs negative) slides. Four (4) out of the six (6) parasite density evaluation slides obtained a concordance of less than 50%. Thirteen (13) out of the fourteen (14) identification slides obtained a concordance greater than 50%. Only one (1) identification slide obtained zero agreement from the microscopists. For species identification a concordance greater than 80% was noted and the microscopists obtained scores between 0.20 and 0.4 on a scale of 0 to 1 for parasite density reading. The microscopists obtained 100% precision, sensitivity, specificity and both negative and positive predictive values. CONCLUSION: This work demonstrated that the microscopic method of malaria diagnosis used in the LPM/HALD is in accordance with the requirements of WHO and ISO 15189. Further training of microscopists may be needed to maintain competency.

Role for gene conversion in the evolution of cell-surface antigens of the malaria parasite Plasmodium falciparum.

While the malaria parasite Plasmodium falciparum has low average genome-wide diversity levels, likely due to its recent introduction from a gorilla-infecting ancestor (approximately 10,000 to 50,000 years ago), some genes display extremely high diversity levels. In particular, certain proteins expressed on the surface of human red blood cell-infecting merozoites (merozoite surface proteins (MSPs)) possess exactly 2 deeply diverged lineages that have seemingly not recombined. While of considerable interest, the evolutionary origin of this phenomenon remains unknown. In this study, we analysed the genetic diversity of 2 of the most variable MSPs, DBLMSP and DBLMSP2, which are paralogs (descended from an ancestral duplication). Despite thousands of available Illumina WGS datasets from malaria-endemic countries, diversity in these genes has been hard to characterise as reads containing highly diverged alleles completely fail to align to the reference genome. To solve this, we developed a pipeline leveraging genome graphs, enabling us to genotype them at high accuracy and completeness. Using our newly- resolved sequences, we found that both genes exhibit 2 deeply diverged lineages in a specific protein domain (DBL) and that one of the 2 lineages is shared across the genes. We identified clear evidence of nonallelic gene conversion between the 2 genes as the likely mechanism behind sharing, leading us to propose that gene conversion between diverged paralogs, and not recombination suppression, can generate this surprising genealogy; a model that is furthermore consistent with high diversity levels in these 2 genes despite the strong historical P. falciparum transmission bottleneck.

Malaria blood stage infection suppresses liver stage infection via host-induced interferons but not hepcidin.

Malaria-causing Plasmodium parasites first replicate as liver stages (LS), which then seed symptomatic blood stage (BS) infection. Emerging evidence suggests that these stages impact each other via perturbation of host responses, and this influences the outcome of natural infection. We sought to understand whether the parasite stage interplay would affect live-attenuated whole parasite vaccination, since the efficacy of whole parasite vaccines strongly correlates with their extend of development in the liver. We thus investigated the impact of BS infection on LS development of genetically attenuated and wildtype parasites in female rodent malaria models and observed that for both, LS infection suffered severe suppression during concurrent BS infection. Strikingly and in contrast to previously published studies, we find that the BS-induced iron-regulating hormone hepcidin is not mediating suppression of LS development. Instead, we demonstrate that BS-induced host interferons are the main mediators of LS developmental suppression. The type of interferon involved depended on the BS-causing parasite species. Our study provides important mechanistic insights into the BS-mediated suppression of LS development. This has direct implications for understanding the outcomes of live-attenuated Plasmodium parasite vaccination in malaria-endemic areas and might impact the epidemiology of natural malaria infection.

Biomolecular interactions between Plasmodium and human host: A basis of targeted antimalarial therapy.

Malaria is one of the serious health concerns worldwide as it remains a clinical challenge due to the complex life cycle of the malaria parasite and the morphological changes it undergoes during infection. The malaria parasite multiplies rapidly and spreads in the population by changing its alternative hosts. These various morphological stages of the parasite in the human host cause clinical symptoms (anemia, fever, and coma). These symptoms arise due to the preprogrammed biology of the parasite in response to the human pathophysiological response. Thus, complete elimination becomes one of the major health challenges. Although malaria vaccine(s) are available in the market, they still contain to cause high morbidity and mortality. Therefore, an approach for eradication is needed through the exploration of novel molecular targets by tracking the epidemiological changes the parasite adopts. This review focuses on the various novel molecular targets.

Host metabolomic responses in recurrent P. vivax malaria.

Malaria is the leading parasitic disease worldwide, with P. vivax being a major challenge for its control. Several studies have indicated metabolomics as a promising tool for combating the disease. The study evaluated plasma metabolomic profiles of patients with recurrent and non-recurrent P. vivax malaria in the Brazilian Amazon. Metabolites extracted from the plasma of P. vivax-infected patients were subjected to LC-MS analysis. Untargeted metabolomics was applied to investigate the metabolic profile of the plasma in the two groups. Overall, 51 recurrent and 59 non-recurrent patients were included in the study. Longitudinal metabolomic analysis revealed 52 and 37 significant metabolite features from the recurrent and non-recurrent participants, respectively. Recurrence was associated with disturbances in eicosanoid metabolism. Comparison between groups suggest alterations in vitamin B6 (pyridoxine) metabolism, tyrosine metabolism, 3-oxo-10-octadecatrienoate ß-oxidation, and alkaloid biosynthesis II. Integrative network analysis revealed enrichment of other metabolic pathways for the recurrent phenotype, including the butanoate metabolism, aspartate and asparagine metabolism, and N-glycan biosynthesis. The metabolites and metabolic pathways predicted in our study suggest potential biomarkers of recurrence and provide insights into targets for antimalarial development against P. vivax.

Exploiting integrative metabolomics to study host-parasite interactions in Plasmodium infections.

Despite years of research, malaria remains a significant global health burden, with poor diagnostic tests and increasing antimalarial drug resistance challenging diagnosis and treatment. While 'single-omics'-based approaches have been instrumental in gaining insight into the biology and pathogenicity of the Plasmodium parasite and its interaction with the human host, a more comprehensive understanding of malaria pathogenesis can be achieved through 'multi-omics' approaches. Integrative methods, which combine metabolomics, lipidomics, transcriptomics, and genomics datasets, offer a holistic systems biology approach to studying malaria. This review highlights recent advances, future directions, and challenges involved in using integrative metabolomics approaches to interrogate the interactions between Plasmodium and the human host, paving the way towards targeted antimalaria therapeutics and control intervention methods.

Epigenetic regulation as a therapeutic target in the malaria parasite Plasmodium falciparum.

Over the past thirty years, epigenetic regulation of gene expression has gained increasing interest as it was shown to be implicated in illnesses ranging from cancers to parasitic diseases. In the malaria parasite, epigenetics was shown to be involved in several key steps of the complex life cycle of Plasmodium, among which asexual development and sexual commitment, but also in major biological processes like immune evasion, response to environmental changes or DNA repair. Because epigenetics plays such paramount roles in the Plasmodium parasite, enzymes involved in these regulating pathways represent a reservoir of potential therapeutic targets. This review focuses on epigenetic regulatory processes and their effectors in the malaria parasite, as well as the inhibitors of epigenetic pathways and their potential as new anti-malarial drugs. Such types of drugs could be formidable tools that may contribute to malaria eradication in a context of widespread resistance to conventional anti-malarials.

Betulinic and ursolic acids from Nauclea latifolia roots mediate their antimalarial activities through docking with PfEMP-1 and PfPKG proteins.

BACKGROUND: Chemotherapies target the PfEMP-1 and PfPKG proteins in Plasmodium falciparum, the parasite that causes malaria, in an effort to prevent the disease's high fatality rate. This work identified the phytochemical components of Nauclea latifolia roots and docked the chemical compounds against target proteins, and examined the in vivo antiplasmodial effect of the roots on Plasmodium berghei-infected mice. METHODS: Standard protocols were followed for the collection of the plant's roots, cleaning, and drying of the roots, extraction and fraction preparation, assessment of the in vivo antiplasmodial activity, retrieval of the PfEMP-1 and PfPKG proteins, GCMS, ADME, and docking studies, chromatographic techniques were employed to separate the residual fraction's components, and the Swis-ADME program made it possible to estimate the drug's likeness and pharmacokinetic properties. The Auto Dock Vina 4.2 tool was utilized for molecular docking analysis. RESULTS: The residual fraction showed the best therapeutic response when compared favorably to amodiaquine (80.5%) and artesunate (85.1%). It also considerably reduced the number of parasites, with the % growth inhibition of the parasite at 42.8% (D2) and 83.4% (D5). Following purification, 25 compounds were isolated and characterized with GCMS. Based on their low molecular weights, non-permeation of the blood-brain barrier, non-inhibition of metabolizing enzymes, and non-violation of Lipinski's criteria, betulinic and ursolic acids were superior to chloroquine as the best phytochemicals. Hence, they are lead compounds. CONCLUSION: In addition to identifying the bioactive compounds, ADME, and docking data of the lead compounds as candidates for rational drug design processes as observed against Plasmodium falciparum target proteins (PfEMP-1 and PfPKG), which are implicated in the pathogenesis of malaria, the study has validated that the residual fraction of N. latifolia roots has the best antiplasmodial therapeutic index.

Antigenic strain diversity predicts different biogeographic patterns of maintenance and decline of antimalarial drug resistance.

The establishment and spread of antimalarial drug resistance vary drastically across different biogeographic regions. Though most infections occur in sub-Saharan Africa, resistant strains often emerge in low-transmission regions. Existing models on resistance evolution lack consensus on the relationship between transmission intensity and drug resistance, possibly due to overlooking the feedback between antigenic diversity, host immunity, and selection for resistance. To address this, we developed a novel compartmental model that tracks sensitive and resistant parasite strains, as well as the host dynamics of generalized and antigen-specific immunity. Our results show a negative correlation between parasite prevalence and resistance frequency, regardless of resistance cost or efficacy. Validation using chloroquine-resistant marker data supports this trend. Post discontinuation of drugs, resistance remains high in low-diversity, low-transmission regions, while it steadily decreases in high-diversity, high-transmission regions. Our study underscores the critical role of malaria strain diversity in the biogeographic patterns of resistance evolution.

Drug resistance among strains of the parasites that cause malaria is a growing problem for people relying on antimalarial drugs to protect them from the disease. This phenomenon is global yet exactly how resistance emerges, spreads and persists in a population often differs greatly between regions, which can complicate malaria control projects. For example, discontinuing the use of antimalarials can lead to the frequency of resistant strains declining in an area, such as Africa, but persisting at high levels in others, including Asia and South America. Gaining resistance often leads to parasites becoming less transmissible than other strains. When antimalarials are not used, sensitive strains usually outcompete their resistant counterparts. However, prolonged use of antimalarial drugs tends to eliminate susceptible strains, allowing the previously outcompeted resistant strains to dominate. The local dynamics of antimalarial resistance are also shaped by multiple other factors such as transmission levels (how common the disease is in the region), the type of antimalarial measures used (such as drugs and mosquito nets), or previous immunity the population may have developed to specific strains. While many computational models have been developed to capture these dynamics, they usually fail to include strain diversity ­ a parameter reflecting the number of malaria strains the immune system is exposed to. This parameter is important as parasites need to escape both host immunity and drugs in order to be successful. To address this gap, He, Chaillet, and Labbé created a computational model to investigate how strain diversity, transmission levels and other related factors influence antimalarial resistance. The model was used to explore how the frequency of resistant and susceptible strains changes over time once antimalarial drugs are rolled out and then halted. These analyses show that in areas with both low strain diversity and low transmission levels, susceptible parasites are more likely to be wiped out from the population, leading to a high frequency of resistant strains that persist after drugs are discontinued. However, in high diversity and high transmission regions, susceptible strains can remain in the population. Therefore, when drug treatments are stopped, resistance levels are more likely to drop due to these parasites outcompeting the drug-resistant ones. Overall, this work demonstrates how modelling approaches that include strain diversity can help inform public health decisions aimed at reducing antimalarial resistance. In particular, they can provide important insights into the control strategies that are best suited for a specific region, suggesting that in low transmission areas intensive drug treatment may contribute to resistance. Instead, preventative strategies such as eliminating mosquitos and preventing bites with bed nets may prove more beneficial at reducing transmission rates in such areas.

Chemoproteomics-based profiling reveals potential antimalarial mechanism of Celastrol by disrupting spermidine and protein synthesis.

BACKGROUND: Malaria remains a global health burden, and the emergence and increasing spread of drug resistance to current antimalarials poses a major challenge to malaria control. There is an urgent need to find new drugs or strategies to alleviate this predicament. Celastrol (Cel) is an extensively studied natural bioactive compound that has shown potentially promising antimalarial activity, but its antimalarial mechanism remains largely elusive. METHODS: We first established the Plasmodium berghei ANKA-infected C57BL/6 mouse model and systematically evaluated the antimalarial effects of Cel in conjunction with in vitro culture of Plasmodium falciparum. The potential antimalarial targets of Cel were then identified using a Cel activity probe based on the activity-based protein profiling (ABPP) technology. Subsequently, the antimalarial mechanism was analyzed by integrating with proteomics and transcriptomics. The binding of Cel to the identified key target proteins was verified by a series of biochemical experiments and functional assays. RESULTS: The results of the pharmacodynamic assay showed that Cel has favorable antimalarial activity both in vivo and in vitro. The ABPP-based target profiling showed that Cel can bind to a number of proteins in the parasite. Among the 31 identified potential target proteins of Cel, PfSpdsyn and PfEGF1-α were verified to be two critical target proteins, suggesting the role of Cel in interfering with the de novo synthesis of spermidine and proteins of the parasite, thus exerting its antimalarial effects. CONCLUSIONS: In conclusion, this study reports for the first time the potential antimalarial targets and mechanism of action of Cel using the ABPP strategy. Our work not only support the expansion of Cel as a potential antimalarial agent or adjuvant, but also establishes the necessary theoretical basis for the development of potential antimalarial drugs with pentacyclic triterpenoid structures, as represented by Cel. Video Abstract.

Evaluation of the antimalarial and CD4+ T-cell modulatory effects of leaf methanol extract of Phyllanthus muellerianus (Kuntze) Exell (Phyllanthaceae) in Plasmodium berghei-infected mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Phyllanthus muellerianus (Kunze) Exell, a member of the Phyllanthaceae family, is a medicinal plant widely distributed in Africa. Decoctions from the leaves are used in Nigeria to treat fevers, convulsions, some neurological disorders and malaria. AIM OF THE STUDY: This study is to evaluate the anti-malarial properties of methanol extract of Phyllanthus muellerianus (MEPM) leaves and its ethyl acetate fraction using a murine malaria model infected with Plasmodium berghei. Additionally, we seek to investigate the potential modulatory effects of this extract and fraction on CD4+ T-cell populations in the context of malaria infection. MATERIALS AND METHODS: The anti-malarial effects of the leaf methanol extract of Phyllanthus muellerianus (MEPM) were screened using three established in vivo models of anti-plasmodial screening namely the curative, suppressive and prophylactic models. The methanol extract (MEPM) was afterwards fractionated into hexane (HFPM), ethyl acetate (EAFPM), and methanol (MFPM) fractions. In the pilot anti-malarial screening of the fractions, EAFPM exhibited the best antiparasitic activity. Subsequently, EAFPM was screened for anti-malarial activity using the three models above. The effects of the MEPM and EAFPM on haematological indices (Hb and PCV) of the inoculated animals were further screened and the mean survival time (MST) of the animals was monitored. CD4+ T cells of various groups were counted before and after treatment using a flow cytometer. The EAFPM was further subjected to HPLC analysis for identification of its major compounds. RESULTS: The EAFPM (100 and 200 mg/kg) elicited 88% and 93% cure respectively in the curative model, while artesunate (5 mg/kg,- the positive control) gave 87% protection. The MEPM and EAFPM also gave significant suppression of parasitemia in the suppressive model. The treated groups survived beyond 28 days as against 11 days by the control group (infected but not treated). The treated groups also prevented anaemia seen in the negative control. The EAFPM group significantly modulated the CD4+ T cell. Compounds identified were Gallocatechin, Quercetin -3-O-gallate, Ellagic acid, and Methylellagic acid rhamnoside). CONCLUSION: The study established that the leaf of Phyllanthus muellerianus possesses antimalarial activity, thus lending support to its use in the folkloric treatment of malaria.

The Genome of Plasmodium gonderi: Insights into the Evolution of Human Malaria Parasites.

Plasmodium species causing malaria in humans are not monophyletic, sharing common ancestors with nonhuman primate parasites. Plasmodium gonderi is one of the few known Plasmodium species infecting African old-world monkeys that are not found in apes. This study reports a de novo assembled P. gonderi genome with complete chromosomes. The P. gonderi genome shares codon usage, syntenic blocks, and other characteristics with the human parasites Plasmodium ovale s.l. and Plasmodium malariae, also of African origin, and the human parasite Plasmodium vivax and species found in nonhuman primates from Southeast Asia. Using phylogenetically aware methods, newly identified syntenic blocks were found enriched with conserved metabolic genes. Regions outside those blocks harbored genes encoding proteins involved in the vertebrate host-Plasmodium relationship undergoing faster evolution. Such genome architecture may have facilitated colonizing vertebrate hosts. Phylogenomic analyses estimated the common ancestor between P. vivax and an African ape parasite P. vivax-like, within the Asian nonhuman primates parasites clade. Time estimates incorporating P. gonderi placed the P. vivax and P. vivax-like common ancestor in the late Pleistocene, a time of active migration of hominids between Africa and Asia. Thus, phylogenomic and time-tree analyses are consistent with an Asian origin for P. vivax and an introduction of P. vivax-like into Africa. Unlike other studies, time estimates for the clade with Plasmodium falciparum, the most lethal human malaria parasite, coincide with their host species radiation, African hominids. Overall, the newly assembled genome presented here has the quality to support comparative genomic investigations in Plasmodium.

Correlative light-electron microscopy methods to characterize the ultrastructural features of the replicative and dormant liver stages of Plasmodium parasites.

BACKGROUND: The infection of the liver by Plasmodium parasites is an obligatory step leading to malaria disease. Following hepatocyte invasion, parasites differentiate into replicative liver stage schizonts and, in the case of Plasmodium species causing relapsing malaria, into hypnozoites that can lie dormant for extended periods of time before activating. The liver stages of Plasmodium remain elusive because of technical challenges, including low infection rate. This has been hindering experimentations with well-established technologies, such as electron microscopy. A deeper understanding of hypnozoite biology could prove essential in the development of radical cure therapeutics against malaria. RESULTS: The liver stages of the rodent parasite Plasmodium berghei, causing non-relapsing malaria, and the simian parasite Plasmodium cynomolgi, causing relapsing malaria, were characterized in human Huh7 cells or primary non-human primate hepatocytes using Correlative Light-Electron Microscopy (CLEM). Specifically, CLEM approaches that rely on GFP-expressing parasites (GFP-CLEM) or on an immunofluorescence assay (IFA-CLEM) were used for imaging liver stages. The results from P. berghei showed that host and parasite organelles can be identified and imaged at high resolution using both CLEM approaches. While IFA-CLEM was associated with more pronounced extraction of cellular content, samples' features were generally well preserved. Using IFA-CLEM, a collection of micrographs was acquired for P. cynomolgi liver stage schizonts and hypnozoites, demonstrating the potential of this approach for characterizing the liver stages of Plasmodium species causing relapsing malaria. CONCLUSIONS: A CLEM approach that does not rely on parasites expressing genetically encoded tags was developed, therefore suitable for imaging the liver stages of Plasmodium species that lack established protocols to perform genetic engineering. This study also provides a dataset that characterizes the ultrastructural features of liver stage schizonts and hypnozoites from the simian parasite species P. cynomolgi.