Human Malaria Research Articles

Recent Progress in Thiazole, Thiosemicarbazone, and Semicarbazone Derivatives as Antiparasitic Agents Against Trypanosomatids and Plasmodium spp.

Neglected tropical diseases (NTDs), including Chagas disease, human African trypanosomiasis (HAT), leishmaniasis, and malaria, remain a major global health challenge, disproportionately affecting low-income populations. Current therapies for these diseases suffer from significant limitations, such as reduced efficacy, high toxicity, and emerging parasite resistance, highlighting the urgent need for new therapeutic strategies. In response, substantial efforts have been directed toward the synthesis of new molecules with improved potency, selectivity, and pharmacokinetic profiles. However, despite many of these compounds exhibiting favorable ADMET (absorption, distribution, metabolism, excretion, and toxicity) profiles and strong in vitro activity, their translation into in vivo models remains limited. Key challenges include the lack of investment, the absence of fully representative experimental models, and difficulties in extrapolating cell-based assay results to more complex biological systems. In this review, we analyzed the latest advancements (2019–2024) in the development of these compound classes, correlating predictive parameters with their observed biological activity. Among these parameters, we highlighted the partition coefficient (LogP), which measures a compound’s lipophilicity and influences its ability to cross biological membranes, and Caco-2 cell permeability, an in vitro model widely used to predict intestinal drug absorption. Additionally, we prioritized the most promising molecules and structural classes for pharmaceutical development, discussing structure–activity relationships (SARs) and the remaining challenges that must be overcome to enable the clinical application of these compounds in the treatment of NTDs.

Defining the genetics of the widely used G3 strain of the mosquito, Anopheles gambiae

Mosquito species in the Anopheles gambiae complex have been referred to as “the deadliest animals in the world” due to their role as vectors of malaria throughout sub-Saharan Africa. Consequently, An. gambiae was among the first species to have its whole genome sequenced in 2002 and it continues to be the subject of intense study. An. gambiae is one member of a nine member species complex and, along with its sister species, An. coluzzii, is among the most important vectors of human malaria. Laboratory research on malaria vectors across a broad range of disciplines utilizes a strain known as G3, which was established in 1975 from mosquitoes collected from McCarthy Island, The Gambia. This strain is well known to be a mongrel strain, nonetheless it is often referred to as An. gambiae, which it is not. The issue with G3 goes far beyond the typical inbreeding associated with long-standing laboratory colonies. G3 is an An. gambiae/An. coluzzii interspecific hybrid. Although these two species are known to hybridize in nature, the pattern of interspecific introgression in G3 we describe in this paper is unlike any observed in natural populations. In this report we provide an in-depth analysis of the genetics of the G3 strain and compare it with natural populations of its two parental species. We discuss potential concerns that results obtained from research using the G3 strain may not apply to populations of these mosquito species as they occur in nature.

Quantification of Plasmodium falciparum: validation of quantitative polymerase chain reaction assays for detection of parasites in controlled human malaria infection studies

BackgroundControlled human malaria infection (CHMI) studies are considered a powerful tool for assessing the efficacy of malaria vaccines and investigating immunity against infection. The monitoring of infection has historically been carried out by microscopy and/or more recently sensitive quantitative polymerase chain reaction (qPCR) based on the 18S ribosomal RNA (rRNA) gene. Here we describe the validation of a molecular assay previously developed to quantify malaria parasites in CHMI.MethodsWe used primers and probes for the 18S rRNA Plasmodium falciparum gene. The validation of the assay was performed using cultured 3D7 parasites to generate standards of known quantities of parasites. We determined the specificity, accuracy, precision, lower limit of detection, linearity, and robustness. We also evaluated the effect of using different volumes of whole blood for DNA extraction on the assay performance.ResultsThe validation revealed: (1) specificity of 100% (n=5 independent experiments); (2) linearity with R2 values ≥ 0.98, a slope of −3.8 to −3.1, and efficiency 89–100%; (3) lower limit of detection of 0.3 parasites/microliter and lower limit of quantification of 2.6 parasites/microliter; (4) precision with both inter-assay repeatability and intra-assay reproducibility with coefficients of variation (CV) values of <10%; and (5) accuracy and good extraction efficiency of >90%. The use of large blood volumes for extraction had an adverse effect on precision.ConclusionWe show that this qPCR method for P. falciparum parasite quantification from whole blood is specific, precise, sensitive, accurate, and robust. However, our whole blood qPCR method is not suitable when DNA extraction is from large blood volumes, where using a larger extraction volume of 1000 µl has a considerable effect on the robustness, reproducibility, and repeatability of qPCR, highlighting the importance of standardized protocols in DNA extraction. Other qPCR methods should be considered where high volumes of blood are required.

Neglected Tropical Diseases: Current Treatment Challenges and Future Therapeutic Potentials

Neglected tropical diseases (NTDs) predominantly affect developing countries. Human African trypanosomiasis, leishmaniasis, Chagas disease, and malaria are protozoan infections that are endemic in developing countries and for which new drugs are desperately needed. Gaining better control of these diseases requires discovering and developing safe, effective, and affordable new drugs for the populations at risk. Unfortunately, incentives for the research and development of new medicine to combat NTDs are currently insufficient. This paper examines recent efforts to increase R&D investments in the development of new anti-parasitic drugs and provides an overview of the most recent and promising compounds at different development stages for each pathogen. Additionally, information on the development of novel formulations combining existing drugs and delivery systems that can improve therapeutic outcomes as well as recent advances in drug discovery, obstacles to developing new chemical entities (NCEs), and the role of public-private partnerships (PPPs) are discussed. In conclusion, drug discovery for neglected diseases entails a larger challenge of demonstrating translational readiness at an early stage. Classification systems can help identify gaps and focus research and development efforts on candidates with the highest likelihood of becoming clinical options. To overcome funding shortages and reduce the incubation time for promising drug discovery initiatives, it is necessary to harness the power of collaborative networks and use innovative funding models.

Asymptomatic malaria reservoirs are the last challenge in the elimination in Cambodia

BackgroundMalaria, a mosquito-borne disease, is a serious public health issue globally and a leading cause of morbidity and mortality in many developing countries worldwide. Cambodia is in the last stages of malaria elimination and aims to eliminate all species of human malaria by 2025. Despite tremendous progress, eliminating malaria in Cambodia has proven to be challenging due to pockets of residual transmission in high-risk populations sustained by untreated asymptomatic malaria reservoirs. Understanding the extent of asymptomatic malaria reservoirs in ‘last-mile’ communities such as those in Mondulkiri and Kampong Speu, is vital for an effective malaria elimination strategy.MethodsMalaria cross-sectional surveys were conducted in high-risk populations (forest dwellers, forest goers and forest rangers) at three different time points (T0, T1, T2) from October 2022 to February 2023, overlapping the rainy, malaria transmission season and into the dry season. Blood samples (n = 6350) collected on filter paper from participants from all target groups were screened for Plasmodium species using qPCR.ResultsAll qPCR-diagnosed cases were asymptomatic, indicating an untreated parasite reservoir. In Mondulkiri, the prevalence of Plasmodium falciparum was 0.63% at T0, increasing to 0.81% at T1, and decreasing to 0.18% at T2. Plasmodium vivax decreased from 4.80% at T0 to 1.97% at T1 and 1.65% at T2. In Kampong Speu, overall prevalence was 7.06% at T0, declining to 5.19% at T1 and 4.59% at T2. Plasmodium falciparum prevalence was 0.30% at T0, decreasing to 0.09% at T1 and rising slightly to 0.10% at T2. The forest goers showed a prevalence increase to 1.95% at T1 and decrease to 1.46% by T2, while forest dwellers decreased to 3.25% at T1 and further to 3.13% at T2. Passively reported malaria case showed that 1.09% of cases in Mondulkiri and 0.21% of cases in Kampong Speu were rapid diagnostic test (RDT) positive.ConclusionEvidence generated during this study point to the continued presence of an untreated asymptomatic reservoir in high-risk populations. Targeted epidemiological and/or vector-based intervention strategies tailored to specific risk groups may enable a reduction of this sustaining reservoir of parasites, thereby leading to eliminating malaria in Cambodia.

Preferential transmission of minority and drug-resistant clones in polyclonal infections in Mali

BackgroundIn polyclonal human malaria infections, the roles of individual clones in human-to-mosquito transmission and their relative transmissibility remain poorly understood. In addition, mutations conferring drug resistance can result in a transmission advantage or disadvantage.MethodsAmplicon sequencing of complexity of infection and drug resistance markers was used to analyse post-treatment stage-specific malaria parasite dynamics in human blood infections and in the midguts of mosquitoes that became infected after direct membrane feeding assays (DMFAs). Blood samples originated from 50 asymptomatic Plasmodium falciparum gametocyte-carrying participants. These were collected prior to treatment and at five timepoints over 28 days following a three-day artemisinin-based combination therapy (ACT) regimen of dihydroartemisinin-piperaquine or pyronaridine-artesunate at the Ouélessébougou Clinical Research Unit of the Malaria Research and Training Centre of the University of Bamako (Bamako, Mali). At each study visit, DMFAs were conducted.ResultsA total of 57 Pfcsp haplotypes and 53 Pftrap haplotypes were identified, indicating high genetic diversity among parasite clones. Prior to treatment, human infections were more often polyclonal and had a higher median multiplicity of infection (MOI; 3 (IQR 2–5)), compared to mosquito midgut infections (1 (IQR 1–2)). At this timepoint, it is likely that some clones detected in human blood are not producing gametocytes and are, therefore, not contributing to mosquito transmission. Minority clones preferentially transmitted, and these same clones often persisted in the human blood samples post-treatment. These observations mirror the rapid decline in asexual parasite density that occurs after ACT initiation, and the more persistent circulation of gametocytes. The data, therefore, suggests that asexual gametocyte-non-producing clones outnumber the gametocyte-producing clones at baseline, yet it is these gametocyte-producing minority clones that are transmitted to and surviving in mosquitoes. Certain haplotypes were also found to be more prevalent in human samples compared to mosquito infections, and vice versa, with 12.6% of haplotypes at baseline exclusively observed in mosquitoes. Along with this, varying odds of transmission for different parasite clones were observed, indicating that there are inherent clonal differences in gametocyte productivity or viability. To assess the transmission of drug-resistant clones, the overall prevalence of molecular markers of drug resistance was determined in both human and mosquito hosts, followed by a pairwise comparison between human blood infections and paired infected midguts. This showed that Asn51Ile in Pfdhfr and Lys540Glu in Pfdhps may have a transmission advantage under ACT, while Ala613Ser in Pfdhps may confer a transmission disadvantage.ConclusionsOverall, these findings indicate that parasite dynamics and clonal transmissibility are highly complex, even after ACT. This complexity may have important epidemiological implications, as it suggests the transmission of minority clones and highlights the impact of drug resistance markers on transmissibility.

Deep learning image analysis for continuous single-cell imaging of dynamic processes in Plasmodium falciparum-infected erythrocytes

Continuous high-resolution imaging of the disease-mediating blood stages of the human malaria parasite Plasmodium falciparum faces challenges due to photosensitivity, small parasite size, and the anisotropy and large refractive index of host erythrocytes. Previous studies often relied on snapshot galleries from multiple cells, limiting the investigation of dynamic cellular processes. We present a workflow enabling continuous, single-cell monitoring of live parasites throughout the 48-hour intraerythrocytic life cycle with high spatial and temporal resolution. This approach integrates label-free, three-dimensional differential interference contrast and fluorescence imaging using an Airyscan microscope, automated cell segmentation through pre-trained deep-learning algorithms, and 3D rendering for visualization and time-resolved analyses. As a proof of concept, we applied this workflow to study knob-associated histidine-rich protein (KAHRP) export into the erythrocyte compartment and its clustering beneath the plasma membrane. Our methodology opens avenues for in-depth exploration of dynamic cellular processes in malaria parasites, providing a valuable tool for further investigations.

Computational Analysis of Plasmodium falciparum DNA Damage Inducible Protein 1 (PfDdi1): Insights into Binding of Artemisinin and its Derivatives and Implications for Antimalarial Drug Design.

Human malaria remains a global health challenge, with Plasmodium falciparum responsible for the most severe cases. Despite global efforts, eradicating malaria has proven difficult, mainly because of the rise in drug resistance, particularly against artemisinin and its derivatives. One possible cause of this resistance is the activation of the unfolded protein response (UPR), which helps maintain cellular balance under stress. In P. falciparum, the UPR operates through the ubiquitin-proteasome system (UPS), which involves proteins such as Dsk2, Rad23, and Ddi1. Among these, Plasmodium falciparum DNA-damage-inducible protein 1 (PfDdi1) plays a crucial role in DNA repair and is present throughout the parasite life cycle, making it an attractive drug target. However, there is limited research on PfDdi1 as a therapeutic target. Recent in vitro studies have indicated that artemisinin (ART) and dihydroartemisinin (DHA) inhibit PfDdi1 activity. Building on this, we investigated whether ART and its derivatives could serve as inhibitors of PfDdi1 using computational modeling. Our study included clinically relevant ART derivatives such as artemether (ARM), arteether (AET), artemiside (AMD), and artesunate (ATS). All these compounds showed strong binding to PfDdi1, with free binding energies ranging from -20.75 kcal/mol for AET to -34.24 kcal/mol for ATS. ARM increased PfDdi1's structural rigidity and hydrophobic stability, whereas AMD improved its kinetic stability, resulting in the least residue motion. Unlike AET and AMD, the other ligands destabilize the PfDdi1 structure. Importantly, three key binding regions-Loop 1 (GLN 266 - ILE 269), Loop 2 (ILE 323 - TYR 326), and Loop 3 (ALA 292 - GLY 294)-were identified as potential targets for new antimalarial drugs against PfDdi1. This study highlights the potential of ART derivatives as PfDdi1 inhibitors, paving the way for further experimental validation.

Anti-Trypanosomatid and Antiplasmodial Activities of Alkaloids From Hippeastrum Species.

Diseases caused by trypanosomatid parasites like human African trypanosomiasis (HAT), Chagas disease (CD), leishmaniasis, and malaria are persistent health problems in developing countries that still demand new drug development. The species of the Amaryllidoideae subfamily (Amaryllidaceae) represent a vast source of alkaloids with a wide range of bioactive properties, including antiparasitic effects. The aim of this study was to evaluate the antiparasitic activity of the alkaloids hamayne, 7-hydroxyclivonine, 4-O-methylnangustine, and candimine against Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani, and Plasmodium falciparum parasites. The alkaloids were isolated from the leaves of Hippeastrum argentinum and Hippeastrum escoipense using several chromatographic techniques and then identified by GC-MS, UPLC-MS/MS, and NMR data. The compounds were assessed against different life cycle stages of these four parasites. Furthermore, the cytotoxic activity of the alkaloids against L6 rat skeletal myoblast cells was tested. P. falciparum was very sensible to 7-hydroxyclivonine. Candimine showed significant antiparasitic activity against all the evaluated parasites, especially T. b. rhodesiense. Candimine merits deeper research regarding its effect against trypanosomatid parasites as a lead compound for the development of alternative treatments for HAT, CD, and malaria.

Safety, tolerability and immunogenicity of a condensed, multi-dose prime regimen of PfSPZ Vaccine for the prevention of Plasmodium falciparum malaria infection

BackgroundThe World Health Organization (WHO) has called for new malaria vaccines with > 90% efficacy against Plasmodium falciparum infection to expand the anti-disease benefit provided by the RTS,S/AS01 and R21/Matrix M subunit vaccines currently administered to infants and young children in sub-Saharan Africa. Attenuated P. falciparum sporozoites (PfSPZ) are being developed as a traveller’s vaccine and to fulfill WHO’s call for high-level efficacy in endemic countries to support malaria elimination.MethodsPfSPZ Vaccine, comprised of radiation-attenuated PfSPZ, was compared with normal saline placebo in a randomized, double-blind trial targeting 60 malaria-naive US adults to assess safety, tolerability, immunogenicity, and efficacy against heterologous controlled human malaria infection three and twelve weeks after immunization. Pharmacists provided syringes to blinded clinicians using 3:1 (vaccine:placebo) blocked randomization, for administration by direct venous inoculation on days 1 and 8 (multidose prime) and day 29 (boost), a condensed regimen with superior efficacy. Primary outcomes included adverse events and antibody responses to the P. falciparum circumsporozoite protein (PfCSP).Results31 participants were screened, randomized and immunized twice (V1, V2) 5–7 days apart, with one withdrawal after an intercurrent adverse event. A vial issue, later traced to the vial manufacturer, halted further immunizations. Solicited local and systemic adverse events recorded for 2 and 7 days after immunizations, respectively, occurred with equal frequency and severity in the 23 vaccinees and 7 controls receiving two immunizations, as did unsolicited adverse events recorded for 28 days and laboratory abnormalities 1 and 5 weeks after V2. Four of 23 vaccinees and one of 7 controls (p = 1.00) developed grade 2 adverse events including subjective fever, headache, malaise, fatigue, rigors, arthralgia and myalgia after V2 but not V1, these symptoms generally resolving within 24 h. Twenty-two of 23 (96%) vaccinees developed IgG (median 99-fold increase over baseline) and IgM (median 1,110-fold increase) antibodies to PfCSP one week after V2. Antibody responses were not associated with reactogenicity.ConclusionsThe two-dose priming immunization regimen was safe, well tolerated and highly immunogenic. Larger studies may better define the adverse event profile of condensed regimens of PfSPZ Vaccine in malaria-naive adults.Trial registration number: clinicaltrial.gov NCT05604521.

Development and Assessment of a Multiple-Analysis System for Diagnosing Malaria and Other Blood Parasite Infections in Humans and Non-Human Primates

Background/Objectives: Many tropical diseases such as malaria, Chagas, human African Trypanosomiasis, and Lymphatic filariasis coexist in endemic countries, affecting more than 1 billion people worldwide, and are recognised as major global vector-borne diseases. Tackling this disease requires an accurate diagnosis that is sensitive, specific, and rapid. This study aimed to describe and validate a new highly sensitive and specific multiple-analysis system that can effectively detect numerous etiological agents in a single test. Methods: A total of 230 human blood samples were assessed retrospectively for parasite characterisation, as well as 58 stool samples from non-human primates. Primers and probes were designed in the small subunit ribosomal RNA gene, except for Plasmodium spp., for which the novel target was Cytochrome Oxidase Subunit 1. Results: The analytical specificity of the presented method was 100%, with no unspecific amplifications or cross-reactions with other blood parasitic diseases. The detection limit obtained was between 0.6 and 3.01 parasites/µL for Plasmodium species, 1.8 parasites/mL for Trypanosomatidae, and 2 microfilariae/mL in the case of Filariae. The sensitivity, specificity, predictive values, and kappa coefficient reached almost 100%, except for Filariae, whose sensitivity dropped to 93.9% and whose negative predicted value dropped to 89.5%. The operational features described a turnaround and a hands-on time shorter than the compared methods with a lower cost per essay. Conclusions: This work presents a cost-effective and highly sensitive multiplexed tool (RT-PCR-bp) capable of performing simultaneous detection for blood parasitic diseases using specific fluorescence probes, enabling the diagnosis of low parasite loads and coinfections.

Antiprotozoal Aminosteroids from Pachysandra terminalis.

Trypanosoma brucei rhodesiense (Tbr) and Plasmodium falciparum (Pf) are protozoan parasites that cause severe diseases, namely, Human African Trypanosomiasis (HAT) and Malaria. Due to limited treatment options, there is an urgent need for new antiprotozoal drugs. Pachysandra terminalis (P. terminalis), a plant belonging to the family Buxaceae, is known as a rich source of aminosteroid alkaloids, and a previous study of our working group already showed that the alkaloid-enriched fraction of P. terminalis aerial parts showed promising activity against protozoan parasites. In the present study, the alkaloid-enriched fraction obtained from a 75% ethanol extract of aerial parts was separated to isolate a chemically diverse array of Pachysandra alkaloids for assessment of their antiprotozoal activity and later structure-activity studies. This work yielded a new megastigmane alkaloid (1), 7 new aminosteroids (2, 7, 16, 17, 18, 19, 20), along with 10 known aminosteroids (3-5, 8, 10-15) and 2 artifacts (6, 9) that were formed during the isolation process. The structures were elucidated by UHPLC/+ESI-QqTOF-MS/MS, as well as extensive 1- and 2D-NMR measurements. The extract and its fractions, as well as the isolated compounds, were tested in vitro against Tbr and Pf, as well as cytotoxicity against mammalian cells (L6 cell line). The activity (IC50 values) of the isolated alkaloids ranged between 0.11 and 26 µM (Tbr) and 0.39 and 80 µM (Pf). 3α,4α-diapachysanaximine A (7) showed the highest activity against Tbr (IC50 = 0.11 µM) with a selectivity index (SI) of 133 and was also quite active against Pf with IC50 = 0.63 µM (SI = 23). This compound is, therefore, a promising new antiprotozoal target for further investigations.

Deletion of the chloroquine resistance transporter gene confers reduced piperaquine susceptibility to the rodent malaria parasite Plasmodium berghei.

Malaria parasites acquire drug resistance through genetic changes, the mechanisms of which remain incompletely understood. Understanding the mechanisms of drug resistance is crucial for the development of effective treatments against malaria, and for this purpose, new genetic tools are needed. In a previous study, as a forward genetic tool, we developed the rodent malaria parasite Plasmodium berghei mutator (PbMut) line, which has a greatly increased rate of mutation accumulation and from which we isolated a mutant with reduced susceptibility to piperaquine (PPQ). We identified a mutation in the chloroquine resistance transporter (PbCRT N331I) as responsible for this phenotype. In the current study, we generated a marker-free PbMut to enable further genetic manipulation of the isolated mutants. Here, we screened again for PPQ-resistant mutants in marker-free PbMut and obtained a parasite population with reduced susceptibility to PPQ. Of five isolated clones, none had the mutation PbCRT N331I; rather, they possessed a nonsense mutation at amino acid 119 (PbCRT Y119*), which would truncate the protein before eight of its ten predicted transmembrane domains. The PbCRT orthologue in the human malaria parasite Plasmodium falciparum, PfCRT, is an essential membrane transporter. To address the essentiality of PbCRT, we successfully deleted the full PbCRT gene [PbCRT(-)] from wild-type parasites. PbCRT(-) parasites exhibited reduced susceptibility to PPQ, along with compromised fitness in mice and following transmission to mosquitoes. Taken together, our findings provide the first evidence that P. berghei can acquire reduced PPQ susceptibility through complete loss of PbCRT function.

Allelic variations in putative antimalarial drug resistance and vaccine candidate genes among Plasmodium vivax reference genomes

ObjectivesPlasmodium vivax is the most widespread Plasmodium spp. globally and the second most common cause of human malaria. However, very little is known about its biology due to the lack of continuous in vitro/ex vivo culture. In such circumstances, genomic studies provide an alternative for understanding the biology of P. vivax parasites. To date, most of the genomic studies on P. vivax have been based on Sal-I as a reference genome; however, with the recent release of the PvP01 and PvW1 reference genomes with higher quality and improved assemblies following continual improvements in annotation, the adoption of these genomes as a reference for genomic studies appears more advantageous. In this study, allelic differences in putative antimalarial drug resistance genes and vaccine candidate genes with assembly features of these three genomes are analyzed and summarized.MethodsThe nucleotide and amino acid sequences of five P. vivax putative antimalarial drug resistance genes and three vaccine candidate genes for all three reference genomes (i.e., Sal-I, PvP01, and PvW1) were retrieved from PlasmoDB and aligned together using MEGA11. Any differences in the nucleotide and codon sequences between the genomes were recorded.ResultsVarious allelic differences in the putative antimalarial drug resistance and vaccine candidate genes between the three reference genomes were observed, which included the positions previously identified as candidate markers for these gene variants.ConclusionsAs antimalarial drug resistance genotyping studies rely on candidate genetic markers to classify resistant or sensitive parasites, knowledge of the allelic differences among references is important. Furthermore, the heterogeneity of the vaccine candidate genes should be taken into account when designing vaccines for P. vivax.

Identifying malaria risks amongst forest going populations in Mondulkiri province and Kampong Speu province, Cambodia: a large cross-sectional survey

BackgroundCambodia strives to eliminate all species of human malaria by 2025, requiring that foci among forest-exposed populations in remote settings be addressed. This study explores malaria risks amongst forest-exposed groups in Mondulkiri and Kampong Speu Provinces, Cambodia as part of a multi-stage study on novel mosquito bite prevention tools (Project BITE).MethodsA serial cross-sectional survey explored the demographics, housing structure openness, mosquito bite prevention habits, and protection from malaria amongst three target groups: forest goers who work in the forest, forest dwellers who live in the forest, and forest rangers who patrol forested regions. Malaria prevalence data was collected at three time points using rapid diagnostic tests (RDTs) for febrile individuals and qPCR for all participants. Infection locations and travel patterns of Plasmodium falciparum-infected individuals were analysed for clustering and the potential movement of infections.Results2935 participants were enrolled between October 2022 and February 2023, consisting of 1093 (37%) forest goers and 1787 (61%) forest dwellers across both provinces, and 55 (5%) forest rangers in Mondulkiri province. Most worked outdoors as farmers, day labourers, and forest collectors, and reported going to the forest five to seven days a week. For housing, 29% and 39% of participants reported living in partially open primary and secondary structures, respectively. The main methods of mosquito bite protection used were insecticide-treated nets, wearing long sleeves, and burning mosquito coils, with limited protection during the daytime and outside at night. All febrile individuals had negative RDT test results. For qPCR, 24 P. falciparum infections (< 1%) were detected among forest goers and dwellers, clustered in Pu Trom and Pu Nhav villages in Mondulkiri Province, and Banteay Roka and Banteay Roka Kirisenchey (M) villages in Kampong Speu Province. Plasmodium vivax cases were detected (216 cases, 5%) across all enrolled villages. Only two infections were found in forest rangers.ConclusionMalaria elimination strategies for forest-exposed populations in Cambodia should focus on vector intervention strategies that offer protection during the day and outside at night, and drug-based strategies to clear subpatent infections, targeting forest goers and dwellers in villages where cases are detected.

Maintenance of pyrophosphate homeostasis in multiple subcellular compartments is essential in Plasmodium falciparum.

Pyrophosphate is a byproduct of numerous cellular reactions that use ATP or other nucleoside triphosphates to synthesize DNA, RNA, protein, and other molecules. Its degradation into monophosphate is thus crucial for the survival and proliferation of all life forms. The human malaria parasite Plasmodium falciparum encodes two classes of pyrophosphatases to hydrolyze pyrophosphate. The first consists of P. falciparum proton pumping vacuolar pyrophosphatases (PfVP1 and PfVP2), which localize to the parasite's subcellular membranes and work as proton pumps. The second includes P. falciparum soluble pyrophosphatases (PfsPPases), which have not been well characterized. Interestingly, the gene locus of PfsPPase encodes two isoforms, PfsPPase1 (PF3D7_0316300.1) and PfsPPase2 (PF3D7_0316300.2). PfsPPase2 contains a 51- amino acid organellar localization peptide that is absent in PfsPPase1. Here, we combine reverse genetics and biochemical approaches to identify the localization of PfsPPase1 and PfsPPase2 and elucidate their individual functions. We show that PfsPPases are essential for the asexual blood stage. While PfsPPase1 solely localizes to the cytoplasm, PfsPPase2 exhibits multiple localizations including the mitochondrion, the apicoplast, and, to a lesser extent, the cytoplasm. Our data suggest that P. falciparum has taken a unique evolutionary trajectory in pyrophosphate metabolism by utilizing a leader sequence to direct sPPases to the mitochondrion and apicoplast. This differs from model eukaryotes as they generally encode multiple sPPases at distinct genetic loci to facilitate pyrophosphate degradation in cytosolic and organellar compartments. Our study highlights PfsPPases as promising targets for the development of novel antimalarial drugs.

Synthesis and SAR Studies of Acyl-Thiourea Platinum(II) Complexes Yield Analogs with Dual-Stage Antiplasmodium Activity.

Mixed-ligand platinum(II) complexes incorporating bipyridine and acyl-thiourea ligands were synthesized and evaluated for their in vitro growth inhibitory activity against the human malaria parasite Plasmodium falciparum (Pf). The substituents at four distinct sites were varied to identify structure-activity relationships for this series. Most complexes displayed potent PfNF54 activity with IC50 values in the nanomolar range and favorable cytotoxicity profiles. Five complexes (C1, C11, C12, C15, and C17) exhibited activity against both the asexual blood and sexual (gametocyte) stage parasites, with another complex (C8) exhibiting activity against late-stage gametocytes only. In addition, the complexes showed comparable ABS potency against the PfK1 multidrug-resistant strain. The pharmacokinetic parameters of one analog (C6), which displayed good solubility and mouse microsomal metabolic stability, were measured. This work demonstrates the potential of acyl-thiourea platinum(II) complexes as selective, multistage-active antiplasmodium compounds as part of the search for new antimalarial agents.

Molecular Docking and Pharmacokinetic Properties of Chrysin, Indole-3-carbinol, and Curcumin in the Biological Context of Plasmodium falciparum 3D7.

Plasmodium falciparum is a protozoan parasite that causes malaria in humans. It is considered the most deadly species of Plasmodium that infects humans. Research continues to focus on understanding the biology and genetics of Plasmodium falciparum to develop new tools, vaccines, and treatments aimed at reducing the global burden of malaria caused by this deadly parasite. In this study, three active compounds-chrysin, indole-3-carbinol, and curcumin-were optimized using Gaussian16, and a molecular docking study was conducted against Plasmodium falciparum 3D7, along with an evaluation of pharmacokinetic properties. Molecular docking studies indicated that the chrysin compound exhibits a stronger binding affinity to selected receptors of Plasmodium falciparum compared to the other compounds. Additionally, in-silico ADME screening was used to predict the pharmacological characteristics of acetyl-CoA reductase inhibitors and their metabolites. The findings predict that the chrysin compound exhibits better affinity with Plasmodium falciparum proteins, suggesting that this compound can effectively interact with specific targets within the parasite. It also highlights the favorable electrostatic interactions that contribute to its pharmacological effectiveness as an acetyl-CoA reductase inhibitor.

Anopheles gambiae phagocytic hemocytes promote Plasmodium falciparum infection by regulating midgut epithelial integrity

For successful transmission, the malaria parasite must traverse tissue epithelia and survive attack from the insect’s innate immune system. Hemocytes play a multitude of roles in mosquitoes, including defense against invading pathogens. Here, we show that hemocytes of the major malaria vector Anopheles gambiae promote Plasmodium falciparum infection by maintaining midgut epithelial integrity by controlling cell proliferation upon blood feeding. The mosquito’s hemocytes also control the midgut microbiota and immune gene expression. Our study unveils novel hemocyte functions that are exploited by the human malaria parasite to evade the mosquito’s immune system.

Meeting summary: Global vaccine and immunization research forum, 2023.

Meeting summary: Global vaccine and immunization research forum, 2023.