The health consequences of Plasmodium infections in wild great apes, particularly in asymptomatic animals, remain poorly understood. This study investigated the hematological and immune impacts of natural malaria infections in 27 semi-wild chimpanzees (Pan troglodytes troglodytes) from Gabon. Using PCR and qPCR to identify infected individuals, and MinION sequencing to determine the Plasmodium species involved, results showed a 48.15% overall Plasmodium infection rate, with frequent multi-species co-infections involving Plasmodium gaboni, Plasmodium reichenowi, and Plasmodium vivax-like parasites. In addition, infected chimpanzees were younger than non-infected individuals, although no significant association was detected between age and parasitemia levels, and this interpretation should be considered cautiously given the limited number of juvenile animals included in the study. Multi-species infections, particularly triple infections involving P. vivax-like parasites, were associated with higher parasitemia levels. Profiling of 15 hematological markers and 8 cytokines/chemokines known to be associated with malarial infections in humans revealed significant alterations in infected chimpanzees, including elevated urea, reduced creatinine, and increased systemic concentrations of pro-inflammatory (TNF, IL-1β, CCL3) and anti-inflammatory (IL-10) cytokines. Ex vivo PBMC stimulation yielded higher IL-10 in infected than non-infected individuals, indicating a regulatory-skewed cytokine response at the time of sampling. These results suggest that malaria in chimpanzees is associated with systemic immune modulation and accompanied by signs of physiological stress, including potential renal dysfunction. This study challenges the assumption that chronic Plasmodium infections are entirely benign in great apes and highlights the need to integrate immunological health indicators into conservation strategies. Broader immune profiling and longitudinal studies will be essential in the future to assess long-term health outcomes and resilience in these endangered populations.

The creation of transgenic Plasmodium falciparum lines with robust fluorescence across the entire life cycle is essential for advancing our understanding of parasite biology, which in turn informs the development of new drugs and vaccines. In this study, we utilized Plasmodium-optimized genome editing to integrate an mCherry expression cassette into a selected intergenic locus without gene disruption. The resulting marker-free line, NF54-mCh, exhibited intense fluorescence throughout all developmental stages, including asexual and sexual blood stages, as well as mosquito (ookinete, oocyst, and sporozoite) and liver stages. NF54-mCh showed normal proliferation, gametocytogenesis, and efficient transmission to mosquitoes. The ultra-high brightness in salivary gland sporozoites allowed for the non-invasive identification of infected mosquitoes. Sporozoites remained highly infectious to humanized mouse livers, thus enabling the completion of the full life cycle. NF54-mCh serves as a parental line for performing additional genetic modifications, because the CRISPR/Cas9-based genome editing method is free of introduced drug resistance markers. The broader applicability of this strategy was validated by generating similar reporter lines in Plasmodium species utilized in rodent malaria models. In summary, NF54-mCh represents a unique, versatile platform that will accelerate fundamental research and support the future development of malaria control strategies, including new vaccines and drugs.

Asymptomatic malaria in pregnancy (AMiP) remains a major public health challenge with important maternal and neonatal consequences. Despite ongoing malaria control efforts, little is known about trimester-specific disparities in AMiP and the extent to which these differences are explained by socioeconomic, clinical, and environmental factors. Most existing studies have focused on overall malaria burden rather than explaining disparities across gestation. This study examined the determinants of trimester disparities in AMiP in the Kintampo South District of Ghana using Oaxaca-Blinder decomposition. A facility-based cross-sectional study was conducted among 462 pregnant women in the Kintampo South District of Ghana. Peripheral blood samples were screened for Plasmodium species using microscopy, while structured questionnaires captured sociodemographic, clinical, and environmental characteristics. Data were analysed with STATA 17 using Oaxaca-Blinder decomposition to partition trimester differences in AMiP into explained(characteristics) and unexplained (coefficients) components. The overall prevalence of AMiP was 13.4%, with a significantly higher burden in the second trimester (χ2 = 10.96, p < 0.001). Plasmodium falciparum accounted for most infections. Oaxaca-Blinder decomposition showed that 73.7% of the trimester disparity in AMiP was explained by differences in observed characteristics (endowments). The major contributors to the explained disparity were suboptimal uptake of intermittent preventive treatment with sulfadoxine-pyrimethamine (IPTp-SP) (71.3%), frequent evening outdoor activity (58.6%), infrequent use of mosquito repellents (50.6%), proximity to overgrown vegetation (42.5%), ≤2 malaria diagnostic tests during antenatal care (ANC) (24.1%), and infrequent iron-folate supplementation (18.4%). Conversely, ownership of long-lasting insecticidal nets (LLINs) reduced the unexplained disparity by 41.9%, highlighting its protective effect. Disparities in AMiP across trimesters are primarily driven by modifiable socioeconomic, programmatic, and environmental factors rather than inherent biological differences in gestation. These highlight the need to strengthen the prioritisation of early initiation of ANC and frequent exposure to provider-supervised IPTp-SP uptake. In addition, promoting sustained use of LLINs and repellents, alongside community-level environmental management, is critical to reducing exposure risk. Finally, embedding these targeted interventions within ANC services, supported by coordinated multisectoral efforts, will be essential to reduce trimester-specific inequities and improve maternal health outcomes.

Malaria caused by Plasmodium falciparum remains a health burden worldwide due to drug resistance and limited treatment options. Calcium-dependent protein kinase 1 (CDPK1) plays a central role in parasite development and invasion, but the downstream molecular alterations that occur upon its disruption remain poorly understood. We present a proteogenomic-based data analysis pipeline for the reanalysis of the publicly available P. falciparum CDPK1 mutant dataset (PRIDE: PXD005207), integrating proteomic and phosphoproteomic data with six-frame genome translation. This led to the discovery of 24 new protein-coding genes, including 17 exonic and 7 intronic peptides, thereby enriching the current genome annotation. Several peptides, such as NILLTFDK, THNNNPQPNPQQK, and EVTSNFGNIR, mapped to previously unannotated genomic regions, which showed orthologous evidence in other Plasmodium species. The reanalysis of phosphoproteomics data identified 37 novel peptides that imply changes in phosphorylation signaling upon CDPK1 knockdown. The identification of conserved peptides like those associated with metacaspase and HSP70, indicates their potential roles in the survival and adaptation of parasites. Overall, this study highlights the potential of proteogenomics to improve genome annotation and reveal hidden coding regions of the P. falciparum genome. This provides new insights into kinase-regulated pathways and potential molecular targets for malaria control.

Despite progress in reducing the burden of malaria, achieving global malaria elimination remains challenging. This review explores recent advances and future directions in malaria vaccine development, highlighting the need for new approaches to achieve sustainable protection against Plasmodium species. Early research emphasized the importance of immunogenic antigens and adjuvants to strengthen immune responses, prompting efforts to develop vaccines targeting multiple stages of the parasite's life cycle. Recent initiatives have showcased diverse vaccine candidates in various trial phases, including pre-erythrocytic, blood stage, and transmission blocking vaccines, reflecting an encouraging shift toward more collaborative research. However key challenges persist, particularly in identifying pre-erythrocytic antigens owing to difficulties in obtaining infectious sporozoites and establishing reliable human hepatocyte models. Nevertheless, advances in mass spectrometry, bioinformatics, and structural biology hold promise in filling these gaps. In Africa, where the burden of malaria is the highest, the implementation of the RTS,S/AS01 and R21/Matrix-M vaccines through national Expanded Program on Immunization schedules is under way, underscoring the importance of community engagement and health system readiness while highlighting the need for next generation vaccines with greater durability and breadth. Achieving elimination of malaria requires improving current vaccine strategies, integrating novel technologies, and strengthening delivery mechanisms in endemic regions to overcome both biological and operational barriers.

A fused dihydropyrrolidino-pyrimidine hit with low lipophilicity and excellent ligand efficiency was identified in a biochemical screen of the Global Health Chemical Diversity Library (GHCDL) against Plasmodium lysyl-tRNA synthetase (KRS). Structure-guided lead optimization delivered analogues with potent parasite growth inhibition, excellent biochemical and cellular selectivity (>1000-fold), and oral efficacy in the malaria NOD-scid-IL2Rγnull (SCID) mouse model. Structural information and computational methods were deployed to identify a potent and selective basic KRS inhibitor (30) with an extended half-life to reduce the dose regimen to a single-dose cure. Compound 30 displayed a long half-life across preclinical species, favorable safety, and activity across Plasmodium species as well as against drug-resistant and sensitive P. falciparum strains and field isolates. Unfortunately, 30 lacked oral bioavailability, which could not be mitigated with a prodrug approach. Nevertheless, learnings from this series will assist future KRS programs in delivering a clinical candidate with this novel mode of action.

Human infections with simian parasites such as Plasmodium inui, P. cynomolgi, and P. coatneyi have been reported in several countries within Southeast Asia. Though infections caused by these parasites are mostly asymptomatic and mild, this could hamper the efforts to eradicate malaria in this region. In this report, we outlined the development and optimisation of new TaqMan-based real-time PCR assays to accurately identify and quantify P. inui, P. cynomolgi, and P. coatneyi. Species-specific hydrolysis probes were designed for real-time PCR assays targeting variable regions within the 18S rRNA genes of P. inui, P. cynomolgi and P. coatneyi. The assays were tested against plasmid DNA containing the target 18S rRNA gene as well as plasmid DNA harbouring 18S rRNA gene fragments of other human and simian Plasmodium species. The assays were further tested using genomic DNA samples from macaques and Anopheles mosquitoes that were previously confirmed to be positive for simian Plasmodium by nested PCR assays. The developed assays demonstrated high specificity, amplifying only the target 18S rRNA gene of their respective Plasmodium species. The assays were also able to detect and quantify as few as 10 copies of template DNA per PCR reaction, which is equivalent to approximately five parasites, with amplification efficiencies of > 95%. The generated standard curves for each assay showed linear relationship between the copy numbers (10-106 copies) and the cycle threshold (Ct) values. The accuracy of the assays was further supported by their ability to produce results equivalent to those obtained using conventional nested PCR assays. The newly developed assays can accurately detect and quantify P. inui, P. cynomolgi and P. coatneyi, with limit of detection of approximately five parasites. Given the streamlined nature of real-time PCR, these assays could be implemented for routine malaria surveillance in areas where zoonotic malaria is prevalent. In addition, these assays are suitable for the detection of submicroscopic infections and in mosquito samples, which often have low parasite densities.

The present study was conducted to investigate the clinical profile, hematobiochemical alterations, and confirmation of Plasmodium bubalis infection by PCR and sequence analysis, as well as to detect Plasmodium bubalis and human Plasmodium species using a lateral flow assay targeting the Plasmodium lactate dehydrogenase (pLDH) antigen in domestic Indian water buffaloes (Bubalus bubalis). Out of 56 suspected cases, five (8.93%) buffaloes were found positive for P. bubalis infection by PCR, while blood smear examination could detect only three (5.36%) buffaloes positive for P. bubalis infection. Moreover, the human rapid diagnostic test (RDT) targeting the pLDH antigen showed negative results for both P. bubalis and human Plasmodium species. The infected buffaloes showed clinical signs such as fever, tachycardia, tachypnoea and icteric or pale conjunctival mucous membranes. The important hematobiochemical changes observed in infected buffaloes includes marked anaemia, thrombocytopenia, hyperbilirubinemia and hypoalbuminemia. Sequence and evolutionary phylogenetic analyses revealed, for the first time, the prevalence of Plasmodium bubalis Type I infection in domestic Indian water buffaloes. The characteristic clinical signs, haemato-biochemical alterations, microscopic and molecular findings will be useful for early diagnosis and therapeutic management of bubaline malaria.

Malaria remains a serious global health problem, particularly in areas where drug-resistant Plasmodium species and the expanded geographical distribution of malaria caused by climate change are of concern. In response to the pressing need for a rapid and highly sensitive malaria diagnostic method, this paper proposes a novel surface plasmon resonance (SPR) biosensor design with a multilayer N-FK51a+SiO2+Cu+HfO2+BP structure. The biosensor performance is analyzed using the transfer matrix method (TMM) and finite element method (FEM), and further verified by finite difference time domain (FDTD) simulations. The inclusion of SiO2, Cu, HfO2, and black phosphorus (BP) enhances the coupling, confinement, and interaction of the plasmonic field with the analyte. The biosensor design exhibits a maximum angular sensitivity of 541.42deg/RIU, a narrow FWHM of 1.98deg, a detection accuracy of 0.502 deg-1, and a quality factor of 272.21 RIU-1, which are outstanding, particularly for ring-stage malaria diagnosis. To further enhance the biosensor reliability, the sensor responses are modeled using ANN, ANFIS, and RBFNN models, which can predict the stages correctly. The comparative analysis proves the enhanced sensitivity and accuracy compared to the previously published SPR sensors and highlights the robustness of the sensor for real-time malaria diagnosis and broader biomedical application.

Zoonotic malaria, particularly Plasmodium knowlesi, has emerged as a growing public health concern across Southeast Asia, with increasing incidence reported in multiple countries. The recent detection of additional zoonotic Plasmodium species further complicates the epidemiological landscape, underscoring the expanding interface among humans, wildlife reservoirs, and mosquito vectors. Despite this trend, progress towards effective control and elimination remains limited, largely due to substantial gaps in entomological knowledge across many endemic regions. Currently, only a narrow range of vector control approaches is available, and these strategies are often ill-suited to the ecological and behavioural characteristics of zoonotic malaria transmission. A major challenge lies in the complexity of vector species assemblages in Southeast Asia. Some of the vectors belong to species complexes with high morphological similarity, making accurate taxonomic identification difficult. Misidentification of mosquito species can distort assessments of vector competence, biting behaviour, and ecological niche, leading to inaccurate inferences about transmission dynamics and potentially resulting in ineffective surveillance and misdirected vector control interventions. Compounding this issue are rapid landscape changes driven by deforestation, agricultural expansion, and habitat fragmentation, which alter mosquito bionomics. Growing evidence suggests that such environmental disturbances may promote outdoor and forest-associated biting, enhance vector adaptability, and shift transmission risk toward human settlements. Thus, this review synthesises current knowledge on the ecological, behavioural, and taxonomic complexities of zoonotic malaria vectors within a rapidly changing landscape. By critically examining challenges in vector identification, surveillance, and control, we highlight key gaps that impede effective intervention. Finally, we discuss pathways forward, emphasising the need for integrated, context-specific, and One Health-oriented strategies to address the unique challenges posed by zoonotic malaria transmission in Southeast Asia.

In their vertebrate hosts, malaria parasites typically inhabit erythrocytes where they harvest the cell's abundant supply of hemoglobin as a nutrient source. A byproduct of hemoglobin digestion is free heme, which the parasites detoxify by converting it to a brown crystal known as hemozoin. Hemozoin is a hallmark of Plasmodium infection, and this enzymatic pathway is well studied in mammalian Plasmodium species. Despite their evolutionary relatedness to mammalian Plasmodium, wildlife malaria parasites, particularly those that infect birds and lizards, are understudied, leaving their vast genetic diversity to be explored. Plasmodium floridense, Plasmodium azurophilum, and Plasmodium leucocytica infect Anolis lizards throughout the Caribbean islands, including the endemic anole on the island of Saba. Like other Plasmodium species, P. floridense infects red blood cells and produces hemozoin. P. azurophilum also infects red blood cells, however, its sister species, P. leucocytica, infects white blood cells. This is atypical for Plasmodium parasites and represents an expansion into a new cellular niche. Two of these three parasites (P. azurophilum and P. leucocytica) also do not produce hemozoin and have seemingly evolved alternative mechanisms of hemoglobin digestion for nutrient acquisition. To investigate this, we assembled parasite transcriptomes from infected Saban anole blood samples and analyzed them for hemoglobin digestion transcripts. The transcriptome results indicate that all three lizard parasites transcribe the genes canonically involved in the hemoglobin digestion pathway. This is the first evidence that these parasites possess genes for the same digestive enzymes as the better characterized mammalian Plasmodium, indicating conservation of this pathway across the Plasmodium tree. However, there is evidence for shifts in selective pressure on some of these proteins in all three lizard-infective species. These genes may not be as functionally important relative to other Plasmodium species. Since the genes are not yet pseudogenes, however, there remains the alternative hypothesis that these genes also play additional roles in malaria parasite biology.

In Kayin State (Myanmar), Plasmodium falciparum incidence declined substantially between 2014 and 2020 following intensive elimination efforts, leaving P. vivax as the predominant species. Elimination of P. vivax is challenging due to relapses, presence of low density reservoirs and a high prevalence of glucose-6-phosphate dehydrogenase enzyme (G6PD) deficiency in the area, which constrains the use of 8-aminoquinoline drugs for radical cure. This study assessed the prevalence of low-density, sub-RDT (rapid diagnostic test) P. vivax infections and G6PD deficiency in 23 villages in Kayin State from 2020 to 2021. Participants were screened for malaria using malaria RDT (mRDT), a reverse transcriptase real-time polymerase chain reaction (rRT-PCR), an enzyme-linked immunosorbent assay (ELISA) and their G6PD status was assessed using a quantitative point-of-care test. Prevalence estimates with 95% confidence intervals were calculated and stratified by sex and geographic area and group differences were compared using chi-square or Fisher's exact tests. The mRDT detection rate was very low (0.5%), with 26 P. vivax and 1 P. falciparum infections detected among 5509 individuals. Positivity by rRT-PCR for P. vivax was 14.3% (317/2219) followed by P. falciparum 8.3% (185/2219) and unidentified Plasmodium species infections 0.8% (18/2219). The prevalence of sub-RDT P. vivax infections (defined as rRT-PCR positive and mRDT negative) was 14.1% (311/2211). The corresponding figure for P. falciparum was 8.3% (184/2218). Median village prevalence of sub-RDT P. vivax infections was 15.2% [IQR 4.0-23.6, range 0-40.4] and 6.0% [IQR 2.1-16.0, range 0-27.0] for P. falciparum. Hpapun township (north) had a sub-RDT P. vivax prevalence six times higher than Myawaddy township (south). The overall proportion of G6PD deficiency among males was 21.7% and 10.6% in females (P < 0.001). G6PD deficiency showed village-level variation, with a median of 14.4% (IQR 12.3-14.8) in Myawaddy and 17.3% (IQR 14.0-23.1) in Hpapun (P = 0.05). No association was observed between G6PD status and sub-RDT P. vivax infection in either males or females across townships. The coexistence of sub-RDT P. vivax reservoirs and a high proportion of G6PD deficiency pose a major barrier to elimination. These findings highlight the need for high-sensitivity diagnostics to detect low-density infections and reliable point-of-care G6PD testing to effectively target P. vivax malaria in Kayin State, Myanmar.

Malaria is caused by apicomplexan parasites of the genus Plasmodium, with all malaria symptoms and pathology caused by parasite stages that develop within, or transit between, host erythrocytes. The ability of Plasmodium cells to parasitise erythrocytes depends on distinctive intracellular compartments associated with invasion, as well as the development of unique cellular niches within the infected host cell. However, our understanding of the biology of the malaria parasite is limited by the fact that a large proportion of the parasite's proteome has no known cellular location or function. To address this problem, we have generated comprehensive high-resolution maps of protein subcellular localisation for the invasive stage of the erythrocytic life cycle of Plasmodium falciparum, the major cause of malaria mortality. Using the spatial proteomics technique hyperplexed Localisation of Organelle Proteins by Isotopic Tagging (hyperLOPIT) we generated data for 3000 P. falciparum proteins expressed in late schizont stages. Our hyperLOPIT data resolve 24 distinct cellular niches, and using supervised machine-learning we can classify 1646 proteins into one of these compartments including exported sites within the host cell. Through comparative genomic analyses our data resolve the spatial patterns of cell evolution that have shaped the development of Plasmodium species and ongoing adaptive pressures and responses that challenge our efforts to manage these major disease-causing organisms.

Medicinal plants have long been used as a primary healthcare resource for treating malaria and other diseases globally. Malaria, caused by Plasmodium species, remains a serious health threat, with the World Health Organization reporting that over 40 % of the global population lives in malaria-endemic regions. The rise of drug-resistant strains has further complicated treatment efforts, necessitating the search for new therapeutic agents. This review emphasizes the antimalarial potential of plant-derived secondary metabolites, which exhibit diverse pharmacological activities. Several compounds, such as ceramicine R (IC50=2.80 μM), knipholone clooxanthrone (IC50=14.58 μM for D6 and 9.42 μM for W2), joziknipholone A (IC50=0.17 μM), joziknipholone B (IC50=0.26 μM), and cajachalcone (IC50=7.4 μM), have shown potent activity against Plasmodium falciparum. Data were collected from Web of Science, PubMed, Scopus, Google Scholar, and Science Direct. A total of 34 medicinal plants were identified, along with details on extraction techniques and factors influencing metabolite efficacy. Key secondary metabolites include alkaloids, flavonoids, terpenoids, and their derivatives (e.g., quinine, artemisinin, quercetin, chalcones). This review highlights their mechanisms of action and their promise as templates for developing novel drugs to combat both drug-sensitive and drug-resistant malaria strains.

Imported malaria in Trinidad, West Indies: Detection of multiple non-endemic Plasmodium species.

Vector control remains a key strategy in reducing mosquito-borne disease transmission. Understanding mosquito species distribution, diversity, and breeding habitat ecology is crucial for effective surveillance and to define targeted vector control interventions. We conducted a study to understand the diversity and habitat preferences of mosquito larvae across Cambodia during the rainy season from July to September 2024. Mosquito larvae were collected from a variety of breeding habitats located around households across all 25 provinces. The national sampling was conducted once during the rainy season in urban (city) and rural (village) areas within each province. Collected larvae were reared to adult emergence in the insectarium for morphological identification, further confirmed with molecular techniques. We found 37 mosquito species in the households, of which 12 are vectors of pathogens such as dengue and Japanese encephalitis viruses, and Plasmodium species, representing 93% of all collected mosquitoes. Larvae were predominantly found in anthropized artificial breeding habitats, accounting for 98% of all larvae collected. Notably, the two primary dengue vectors, Aedes aegypti and Ae. albopictus, were recorded from all 52 sampling locations. In addition, our study identified the presence of Aedes vittatus in 12 provinces, a new confirmed vector of dengue in Cambodia. We also recorded eight Japanese encephalitis vectors, with at least one species from all sampling sites. There were no statistically significant differences in larval mosquito biodiversity (relative abundance, number of species, Shannon and Simpson diversity indices) between cities and villages, with 15 species occurring in both environments, representing 41% of the species and 99% of all mosquitoes collected. The widespread and predominant presence of dengue and Japanese encephalitis vectors in every household confirms the endemic circulation of these diseases in Cambodia.

Cory's Shearwater (Calonectris borealis), a migratory seabird from the Procellariidae family, is widely distributed across the Atlantic Ocean. It breeds on islands of Azores, Madeira, Canary, and Berlengas, and winters in coastal areas of South America and Africa. This species' wide habitat range contributes to pathogen exchange across global regions. Plasmodium, a genus of apicomplexan parasites causing avian malaria, affects many wild bird species worldwide. Although rare in seabirds, Plasmodium infections can significantly impact these hosts. We identified and characterized a new Plasmodium species in an adult male C. borealis found live-stranded on the northern coast of São Paulo, Brazil, during the daily activities of the Santos Basin Beach Monitoring Project (PMP-BS). The bird was rescued and kept in rehabilitation for 61 days but was euthanized due to poor prognosis. Blood smears revealed parasitemia varying from 0.04% until 2.09%. Blood and tissue samples were tested using PCR targeting a cytochrome b (cytb) gene fragment. The obtained sequence was 100% similar to Plasmodium lineage pLK06. Phylogenetic analysis placed the parasite within the Novyella clade, with morphological features closely related to P. vaughani. Necropsy showed hepato- and splenomegaly, while histopathology confirmed hepatitis and pulmonary hemorrhage. These findings, combined with clinical relapse despite anti-malaria treatment, indicate the parasite's pathogenicity in C. borealis. This represents the first haemosporidian infection in this seabird and the first identification of the pLK06 lineage in Procellariiformes, resulting in the description of Plasmodium borealis sp. nov. These findings expand knowledge of parasite diversity in seabirds and highlight the need for continued surveillance in understudied avian groups.

Membrane Attack Complex/Perforin (MACPF) domain proteins are β-pore forming toxins (β-PFTs) involved in the pathogenesis of various organisms. Among them, Perforin-like proteins (PLPs), produced by Plasmodium species, play essential roles in parasite invasion and egress. Due to increasing drug resistance in Plasmodium, PLPs represent promising but underexplored therapeutic targets, largely due to the lack of structural and mechanistic data. This study investigates the binding and pore formation mechanism of the Plasmodium falciparum PLP1 (PfPLP1), which is expressed during the human life cycle of the parasite. We modeled PfPLP1 structure and performed both all-atom and coarse-grained molecular dynamics simulations in soluble and membrane-associated states. PfPLP1 comprises two domains, a canonical MACPF domain and a β-pleated sheet domain- apicomplexan perforin β-domain (APCβ). Initial membrane binding is mediated by cationic residues at the base of the APCβ domain, which interact with the polar headgroups of the lipids from the host cell membrane. We analyzed the membrane-inserted tetrameric form where water molecules were observed to penetrate between the tetramer and the lipid bilayer, initiating pore opening. During this process, lipids reorganize into a toroidal edge to shield their hydrophobic tails, while water mixes with lipid headgroups in a disordered, heterogeneous fashion. Larger oligomeric assemblies show lateral displacement of lipids and a clear tendency to form pore-like structures. This study provides molecular insights into PfPLP1's membrane binding and pore-forming behavior in both monomeric and oligomeric forms. The outcome of this study would be applicable in understanding pore formation mechanism in other PLPs and similar toxins.

The emergence of artemisinin resistance in Plasmodium falciparum threatens the sustainability of malaria control programs and underscores the need for new therapeutic strategies. Recently, two proteins, RAD5 (PfRAD5) and WD40-repeat protein 11 (PfWD11), have been implicated in parasite survival and drug resistance. Their druggability, however, remains underexplored. We integrated computational and experimental approaches to assess PfRAD5 and PfWD11 as antimalarial targets. Structural models were retrieved from AlphaFold and aligned with orthologues across Plasmodium species. A library of 216 antibiotics was screened by molecular docking, and molecular dynamics simulations. Top-ranked compounds were tested against P. falciparum 3D7 cultures invitro, alone and in combination, and gene expression changes in PfRAD5 and PfWD11 were quantified by qPCR. Docking identified strong binders to PfRAD5 (talampicillin, dicloxacillin, raltegravir) and PfWD11 (cervinomycin A2 monoacetate, eAmSPC 2593, puromycin). Molecular dynamics confirmed the stability of protein-ligand complexes. Invitro, puromycin exhibited the highest inhibition (85% at 100 μg/mL), while combinations enhanced activity. The triple combination (puromycin + raltegravir + dicloxacillin) achieved complete inhibition, and puromycin + chloroquine exhibited synergistic effects at in a concentration-dependent manner. qPCR showed consistent downregulation of PfRAD5 and PfWD11 following puromycin-based treatments, whereas chloroquine alone upregulated PfRAD5 expression. PfRAD5 and PfWD11 represent novel antimalarial targets. Repurposed antibiotics, particularly puromycin in synergistic regimens, offer a cost-effective strategy to counteract emerging resistance and accelerate therapeutic development.

Xenomonitoring is an approach of epidemiological infection risk assessment addressing vector-transmitted infections like malaria in hematophagous arthropods. Standardization and automation can facilitate its use even in remote areas. In this study, the suitability of an automated commercial loop-mediated isothermal amplification (LAMP) assay, originally designed for the detection of malaria parasite DNA in human blood, was assessed for its applicability for xenomonitoring purposes. An automated generic LAMP assay for malaria detection in human blood was applied with Anopheles spp. The results were compared with commercial generic as well as species-specific real-time PCR. LAMP identified 15/43 (34.9%, 95% CI (20.1%; 50.9%)) mosquitoes artificially exposed to Plasmodium berghei, while this was the case for 22/43 (51.2%, 95% CI (35.5%; 66.7%)) samples applying real-time PCR. Considerably less discrepancy was observed with Anopheles spp. imported to Germany from field studies with 12/172 pools (7.0%, 95% CI (3.7%; 11.9%)) for LAMP and 13/172 pools (7.6%, 95% CI (4.1%; 12.6%)) for real-time PCR. Single testing and typing of DNA isolates indicated an overall infection rate of 1.2% (20/1711) with Plasmodium falciparum, P. malariae and P. ovale. Low rates of invalid results during a field exercise proved the general suitability of the LAMP approach for use at tropical settings. Imperfect sensitivity in case of artificial exposure of mosquitoes to non-human plasmodial species was detected for both compared molecular approaches with slightly lower sensitivity of the LAMP approach. For Anopheles spp. infected with Plasmodium spp. causing human malaria, comparable diagnostic reliability could be shown for both approaches and the LAMP assay was shown to be suitable for application under tropical field conditions.