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BACKGROUND: To interrupt residual malaria transmission and achieve successful elimination of Plasmodium falciparum in low-transmission settings, the World Health Organization (WHO) recommends the administration of a single dose of 0.25 mg/kg (or 15 mg/kg for adults) primaquine (PQ) combined with artemisinin-based combination therapy (ACT), without glucose-6-phosphate dehydrogenase (G6PD) testing. However, due to the risk of haemolysis in patients with G6PD deficiency (G6PDd), PQ use is uncommon. Thus, this study aimed to assess the safety of a single low dose of PQ administered to patients with G6PD deficiency. METHODS: An observational cohort study was conducted with patients treated for uncomplicated P. falciparum malaria with either single-dose PQ (0.25 mg/kg) (SLD PQ) + ACT or ACT alone. Microscopy-confirmed uncomplicated P. falciparum malaria patients visiting public health facilities in Arjo Didessa, Southwest Ethiopia, were enrolled in the study from September 2019 to November 2022. Patients with uncomplicated P. falciparum malaria were followed up for 28 days through clinical and laboratory diagnosis, such as measurements of G6PD levels and haemoglobin (Hb) concentrations. G6PD levels were measured by a quantiative CareSTART™ POCT S1 biosensor machine. Patient interviews were also conducted, and the type and frequency of clinical complaints were recorded. Hb data were taken on days (D) 7, 14, 21, and 28 following treatment with SLD-PQ + ACT or ACT alone. RESULTS: A total of 249 patients with uncomplicated P. falciparum malaria were enrolled in this study. Of these, 83 (33.3%) patients received ACT alone, and 166 (66.7%) received ACT combined with SLD-PQ treatment. The median age of the patients was 20 (IQR 28-15) years. G6PD deficiency was found in 17 (6.8%) patients, 14 males and 3 females. There were 6 (7.2%) and 11 (6.6%) phenotypic G6PD-deficient patients in the ACT alone and ACT + SLD-PQ arms, respectively. The mean Hb levels in patients treated with ACT + SLD-PQ were reduced by an average of 0.45 g/dl (95% CI = 0.39 to 0.52) in the posttreatment phase (D7) compared to a reduction of 0.30 g/dl (95% CI = 0.14 to - 0.47) in patients treated with ACT alone (P = 0.157). A greater mean Hb reduction was observed on day 7 in the G6PDd ACT + SLD-PQ group (- 0.60 g/dL) than in the G6PDd ACT alone group (- 0.48 g/dL); however, there was no statistically significant difference (P = 0.465). Overall, D14 losses were 0.10 g/dl (95% CI = - 0.00 to 0.20) and 0.05 g/dl (95% CI = - 0.123 to 0.22) in patients with and without SLD-PQ, respectively (P = 0.412). CONCLUSIONS: This studys findings indicate that using SLD-PQ in combination with ACT is safe for uncomplicated P. falciparum malaria regardless of the patients G6PD status in Ethiopian settings. Caution should be taken in extrapolating this finding in other settings with diverse G6DP phenotypes.

BACKGROUND: Artemisinin resistance in Plasmodium falciparum threatens global malaria elimination efforts. To contain and then eliminate artemisinin resistance in Eastern Myanmar a network of community-based malaria posts was instituted and targeted mass drug administration (MDA) with dihydroartemisinin-piperaquine (three rounds at monthly intervals) was conducted. The prevalence of artemisinin resistance during the elimination campaign (2013-2019) was characterized. METHODS: Throughout the six-year campaign Plasmodium falciparum positive blood samples from symptomatic patients and from cross-sectional surveys were genotyped for mutations in kelch-13-a molecular marker of artemisinin resistance. RESULT: The program resulted in near elimination of falciparum malaria. Of 5162 P. falciparum positive blood samples genotyped, 3281 (63.6%) had K13 mutations. The prevalence of K13 mutations was 73.9% in 2013 and 64.4% in 2019. Overall, there was a small but significant decline in the proportion of K13 mutants (p

Standard diagnostics used in longitudinal antimalarial studies are unable to characterize the complexity of submicroscopic parasite dynamics, particularly in high transmission settings. We use molecular markers and amplicon sequencing to characterize post-treatment stage-specific malaria parasite dynamics during a 42 day randomized trial of 3- versus 5 day artemether-lumefantrine in 303 children with and without HIV (ClinicalTrials.gov number NCT03453840). The prevalence of parasite-derived 18S rRNA is >70% in children throughout follow-up, and the ring-stage marker SBP1 is detectable in over 15% of children on day 14 despite effective treatment. We find that the extended regimen significantly lowers the risk of recurrent ring-stage parasitemia compared to the standard 3 day regimen, and that higher day 7 lumefantrine concentrations decrease the probability of ring-stage parasites in the early post-treatment period. Longitudinal amplicon sequencing reveals remarkably dynamic patterns of multiclonal infections that include new and persistent clones in both the early post-treatment and later time periods. Our data indicate that post-treatment parasite dynamics are highly complex despite efficacious therapy, findings that will inform strategies to optimize regimens in the face of emerging partial artemisinin resistance in Africa.

Global efforts to eradicate malaria are threatened by multiple factors, particularly the emergence of antimalarial drug resistant strains of Plasmodium falciparum. Heat shock proteins (HSPs), particularly P. falciparum HSPs (PfHSPs), represent promising drug targets due to their essential roles in parasite survival and virulence across the various life cycle stages. Despite structural similarities between human and malarial HSPs posing challenges, there is substantial evidence for subtle differences that could be exploited for selective drug targeting. This review provides an update on the potential of targeting various PfHSP families (particularly PfHSP40, PfHSP70, and PfHSP90) and their interactions within PfHSP complexes as a strategy to develop new antimalarial drugs. In addition, the need for a deeper understanding of the role of HSP complexes at the host-parasite interface is highlighted, especially heterologous partnerships between human and malarial HSPs, as this opens novel opportunities for targeting protein-protein interactions crucial for malaria parasite survival and pathogenesis.

Routine sampling of pregnant women at first antenatal care (ANC) visits could make Plasmodium falciparum genomic surveillance more cost-efficient and convenient in sub-Saharan Africa. We compare the genetic structure of parasite populations sampled from 289 first ANC users and 93 children from the community in Mozambique between 2015 and 2019. Samples are amplicon sequenced targeting 165 microhaplotypes and 15 drug resistance genes. Metrics of genetic diversity and relatedness, as well as the prevalence of drug resistance markers, are consistent between the two populations. In an area targeted for elimination, intra-host genetic diversity declines in both populations (p = 0.002-0.007), while for the ANC population, population genetic diversity is also lower (p = 0.0004), and genetic relatedness between infections is higher (p = 0.002) than control areas, indicating a recent reduction in the parasite population size. These results highlight the added value of genomic surveillance at ANC clinics to inform about changes in transmission beyond epidemiological data.

Malaria elimination requires interventions able to target both the asexual blood stage (ABS) parasites and transmissible gametocyte stages of Plasmodium falciparum. Lead antimalarial candidates are evaluated against clinical isolates to address key concerns regarding efficacy and to confirm that the current, circulating parasites from endemic regions lack resistance against these candidates. While this has largely been performed on ABS parasites, limited data are available on the transmission-blocking efficacy of compounds with multistage activity. Here, we evaluated the efficacy of lead antimalarial candidates against both ABS parasites and late-stage gametocytes side-by-side, against clinical P. falciparum isolates from southern Africa. We additionally correlated drug efficacy to the genetic diversity of the clinical isolates as determined with a panel of well-characterized, genome-spanning microsatellite markers. Our data indicate varying sensitivities of the isolates to key antimalarial candidates, both for ABS parasites and gametocyte stages. While ABS parasites were efficiently killed, irrespective of genetic complexity, antimalarial candidates lost some gametocytocidal efficacy when the gametocytes originated from genetically complex, multiple-clone infections. This suggests a fitness benefit to multiclone isolates to sustain transmission and reduce drug susceptibility. In conclusion, this is the first study to investigate the efficacy of antimalarial candidates on both ABS parasites and gametocytes from P. falciparum clinical isolates where the influence of parasite genetic complexity is highlighted, ultimately aiding the malaria elimination agenda.

Obligate intracellular malaria parasites dramatically remodel their erythrocyte host through effector protein export to create a niche for survival. Most exported proteins contain a pentameric Plasmodium export element (PEXEL)/host-targeting motif that is cleaved in the parasite ER by the aspartic protease Plasmepsin V (PMV). This processing event exposes a mature N terminus required for translocation into the host cell and is not known to occur in non-exported proteins. Here, we report that the non-exported parasitophorous vacuole protein UIS2 contains a bona fide PEXEL motif that is processed in the P. falciparum blood stage. While the N termini of exported proteins containing the PEXEL and immediately downstream ~10 residues are sufficient to mediate translocation into the RBC, the equivalent UIS2 N terminus does not promote the export of a reporter. Curiously, the UIS2 PEXEL contains an unusual aspartic acid at the fourth position, which constitutes the extreme N-terminal residue following PEXEL cleavage (P1', RIL↓DE). Using a series of chimeric reporter fusions, we show that Asp at P1' is permissive for PMV processing but abrogates export. Moreover, mutation of this single UIS2 residue to alanine enables export, reinforcing that the mature N terminus mediates export, not PEXEL processing per se. Prompted by this observation, we further show that PEXEL sequences in the N termini of other non-exported rhoptry proteins are also processed, suggesting that PMV may be a more general secretory maturase than previously appreciated, similar to orthologs in related apicomplexans. Our findings provide new insight into the unique N-terminal constraints that mark proteins for export.IMPORTANCEHost erythrocyte remodeling by malaria parasite-exported effector proteins is critical to parasite survival and disease pathogenesis. In the deadliest malaria parasite Plasmodium falciparum, most exported proteins undergo proteolytic maturation via recognition of the pentameric Plasmodium export element (PEXEL)/host-targeting motif by the aspartic protease Plasmepsin V, which exposes a mature N terminus that is conducive for export into the erythrocyte host cell. While PEXEL processing is considered a unique mark of exported proteins, we demonstrate that PEXEL motifs are present and processed in non-exported proteins. Importantly, we show that specific residues at the variable fourth position of the PEXEL motif inhibit export despite being permissive for processing, reinforcing that features of the mature N terminus, and not PEXEL cleavage, identify cargo for export. This opens the door to further inquiry into the nature and evolution of the PEXEL motif.

Ribonucleoprotein complexes are composed of RNA, RNA-dependent proteins (RDPs) and RNA-binding proteins (RBPs), and play fundamental roles in RNA regulation. However, in the human malaria parasite, Plasmodium falciparum, identification and characterization of these proteins are particularly limited. In this study, we use an unbiased proteome-wide approach, called R-DeeP, a method based on sucrose density gradient ultracentrifugation, to identify RDPs. Quantitative analysis by mass spectrometry identifies 898 RDPs, including 545 proteins not yet associated with RNA. Results are further validated using a combination of computational and molecular approaches. Overall, this method provides the first snapshot of the Plasmodium protein-protein interaction network in the presence and absence of RNA. R-DeeP also helps to reconstruct Plasmodium multiprotein complexes based on co-segregation and deciphers their RNA-dependence. One RDP candidate, PF3D7_0823200, is functionally characterized and validated as a true RBP. Using enhanced crosslinking and immunoprecipitation followed by high-throughput sequencing (eCLIP-seq), we demonstrate that this protein interacts with various Plasmodium non-coding transcripts, including the var genes and ap2 transcription factors.

Malaria sterile immunity has been reproducibly induced by immunization with Plasmodium radiation-attenuated sporozoites (RAS). Analyses of sera from RAS-immunized individuals allowed the identification of P. falciparum antigens, such as the circumsporozoite protein (CSP), the basis for the RTS, S and R21Matrix-M vaccines. Similar advances in P. vivax (Pv) vaccination have been elusive. We previously reported 42% (5/12) of sterile protection in malaria-unexposed, Duffy-positive (Fy +) volunteers immunized with PvRAS followed by a controlled human malaria infection (CHMI). Using a custom protein microarray displaying 515 Pv antigens, we found a significantly higher reactivity to PvCSP and one hypothetical protein (PVX_089630) in volunteers protected against P. vivax infection. In mock-vaccinated Fy + volunteers, a strong antibody response to CHMI was also observed. Although the Fy- volunteers immunized with non-irradiated Pv-infected mosquitoes (live sporozoites) did not develop malaria after CHMI, they recognized a high number of antigens, indicating the temporary presence of asexual parasites in peripheral blood. Together, our findings contribute to the understanding of the antibody response to P. vivax infection and allow the identification of novel parasite antigens as vaccine candidates.Trial registration: ClinicalTrials.gov number: NCT01082341.

Malaria poses an enormous threat to human health. With ever increasing resistance to currently deployed drugs, breakthrough compounds with novel mechanisms of action are urgently needed. Here, we explore pyrimidine-based sulfonamides as a new low molecular weight inhibitor class with drug-like physical parameters and a synthetically accessible scaffold. We show that the exemplar, OSM-S-106, has potent activity against parasite cultures, low mammalian cell toxicity and low propensity for resistance development. In vitro evolution of resistance using a slow ramp-up approach pointed to the Plasmodium falciparum cytoplasmic asparaginyl-tRNA synthetase (PfAsnRS) as the target, consistent with our finding that OSM-S-106 inhibits protein translation and activates the amino acid starvation response. Targeted mass spectrometry confirms that OSM-S-106 is a pro-inhibitor and that inhibition of PfAsnRS occurs via enzyme-mediated production of an Asn-OSM-S-106 adduct. Human AsnRS is much less susceptible to this reaction hijacking mechanism. X-ray crystallographic studies of human AsnRS in complex with inhibitor adducts and docking of pro-inhibitors into a model of Asn-tRNA-bound PfAsnRS provide insights into the structure-activity relationship and the selectivity mechanism.

BACKGROUND: Timely molecular surveillance of Plasmodium falciparum kelch 13 (k13) gene mutations is essential for monitoring the emergence and stemming the spread of artemisinin resistance. Widespread artemisinin resistance, as observed in Southeast Asia, would reverse significant gains that have been made against the malaria burden in Africa. The purpose of this study was to assess the prevalence of k13 polymorphisms in western Kenya and Ethiopia at sites representing varying transmission intensities between 2018 and 2022. METHODS: Dried blood spot samples collected through ongoing passive surveillance and malaria epidemiological studies, respectively, were investigated. The k13 gene was genotyped in P. falciparum isolates with high parasitaemia: 775 isolates from four sites in western Kenya (Homa Bay, Kakamega, Kisii, and Kombewa) and 319 isolates from five sites across Ethiopia (Arjo, Awash, Gambella, Dire Dawa, and Semera). DNA sequence variation and neutrality were analysed within each study site where mutant alleles were detected. RESULTS: Sixteen Kelch13 haplotypes were detected in this study. Prevalence of nonsynonymous k13 mutations was low in both western Kenya (25/783, 3.19%) and Ethiopia (5/319, 1.57%) across the study period. Two WHO-validated mutations were detected: A675V in three isolates from Kenya and R622I in four isolates from Ethiopia. Seventeen samples from Kenya carried synonymous mutations (2.17%). No synonymous mutations were detected in Ethiopia. Genetic variation analyses and tests of neutrality further suggest an excess of low frequency polymorphisms in each study site. Fu and Lis F test statistic in Semera was 0.48 (P > 0.05), suggesting potential population selection of R622I, which appeared at a relatively high frequency (3/22, 13.04%). CONCLUSIONS: This study presents an updated report on the low frequency of k13 mutations in western Kenya and Ethiopia. The WHO-validated R622I mutation, which has previously only been reported along the north-west border of Ethiopia, appeared in four isolates collected from eastern Ethiopia. The rapid expansion of R622I across Ethiopia signals the need for enhanced monitoring of the spread of drug-resistant P. falciparum parasites in East Africa. Although ACT remains currently efficacious in the study areas, continued surveillance is necessary to detect early indicators of artemisinin partial resistance.

In 2021, nationwide malaria molecular surveillance revealed a high prevalence of a validated artemisinin resistance marker, the kelch13 (k13) Arg561His mutation, in the Kagera region of northwestern Tanzania. We aimed to investigate the efficacy of artemether–lumefantrine and artesunate–amodiaquine and to confirm the presence of artemisinin partial resistance (ART-R) in the Karagwe district of this region. This single-arm, therapeutic efficacy study was carried out at the Bukangara dispensary in the Karagwe district of the Kagera region in northwestern Tanzania. Eligible participants were aged between 6 months and 120 months, had confirmed Plasmodium falciparum asexual parasitaemia, and met other inclusion criteria according to WHO's standard protocol. Participants were enrolled, treated sequentially with either artemether–lumefantrine or artesunate–amodiaquine, and assessed clinically and parasitologically for 28 days as per WHO protocol. Parasitaemia was measured every 8 h until day 3, on day 7, and then during weekly follow-up visits until day 28. Mutations in the k13 gene and extended haplotypes with the mutations were analysed, and comparisons were made with previous samples collected in the same region of Kagera and in Rwanda and southeast Asia. The primary endpoint was PCR-corrected cure rate. Between April 29 and Sept 1, 2022, 343 patients were screened, of whom 176 were enrolled: 88 in each treatment group. The PCR-corrected cure rate was 98% (95% CI 91–100) in the artemether–lumefantrine group and 100% (96–100) in the artesunate–amodiaquine group. Persistent parasitaemia on day 3 occurred in 11 (13%) of 88 patients in the artemether–lumefantrine group and 17 (19%) of 88 patients in the artesunate–amodiaquine group. Arg561His mutations on day 0 and parasitaemia on day 3 were reported in eight (9%) of 87 patients in the artemether–lumefantrine group and ten (12%) of 86 patients in the artesunate–amodiaquine group. The median parasite clearance half-life in patients harbouring parasites with Arg561His mutation was 6·1 h in the artemether–lumefantrine group and 6·0 h in the artesunate–amodiaquine group. Parasites with the Arg561His mutation were not similar to those from southeast Asia and Rwanda but had similar haplotypes to parasites reported in the same Tanzanian region of Kagera in 2021. This study confirms the presence of ART-R in Tanzania, although artemether–lumefantrine and artesunate–amodiaquine showed high efficacy. A context-specific response strategy and vigilance to detect the reduced efficacy of current antimalarial treatments and ART-R in other parts of the country are urgently needed. The Bill & Melinda Gates Foundation and the US National Institutes of Health. [ABSTRACT FROM AUTHOR]

The diagnosis and management of malaria in non-endemic countries presents a continuing challenge. Plasmodium falciparum, which is capable of rapidly inducing severe and life-threatening multiorgan disease, is the species most frequently diagnosed in Europe and North America. To summarise the more relevant diagnostic findings and clinical features of malaria observed in non-endemic settings and to provide an update of the key management decision points using three illustrative clinical scenarios of uncomplicated and severe malaria. The discussion is based on a relevant literature search spanning the last 20 years. Recommendations are based on available clinical guidelines including those of the WHO, observational studies conducted in non-endemic settings, and, when available, extrapolation from randomised studies from malaria-endemic settings. The following topics are covered: diagnosis, including the use of molecular biology; clinical characteristics; management with a specific focus on complicated (severe) and uncomplicated malaria; and areas of resistance to available antimalarial drugs. Malaria imported to non-endemic settings, especially P. falciparum malaria, is sometimes initially overlooked and the delayed diagnosis is responsible for every year of preventable deaths. This review aims to raise awareness of malaria outside endemic countries and to provide clinicians with a practical guide for efficient diagnosis and targeted therapy for the different species involved. [ABSTRACT FROM AUTHOR]

This article presents a novel methodology for the detection of malaria infection in human Red Blood Cells (RBCs) through a capacitive micro-sensor from a known volume (here 400 µL) of infected cells suspended in Phosphate-buffered saline (PBS) solution. It has been studied through a literature survey that when a healthy RBC is infected by a malaria pathogen (i.e. plasmodium falciparum), certain changes like deformation in size/shape, increased membrane rigidity, more adhesiveness to endothelial surface, increased cytoplasm and membrane permittivity occur in the said blood cell. This work, therefore, deals with variation in capacitance due to the change in cytoplasm and membrane permittivity of an RBC caused by the aforementioned reasons. Thus, these changes form the basis of detection for malaria infection. The change in capacitance of an infected RBC leads to an overall change in bio-impedance signature. This is then measured by exciting the blood cell using a 2V p–p signal over a frequency range of 1–100 kHz. The difference in bio-impedance signature obtained for the different concentrations of normal and malaria-infected RBCs is used here as a resolving factor for the identification of the malaria infection. The design of the said micro-sensor and the electrical behavior have been studied using Finite Element Method (FEM) based COMSOL Multiphysics® software. [ABSTRACT FROM AUTHOR]

Background: Pulmonary oedema is a feared and difficult to predict complication of severe malaria that can emerge after start of antimalarial treatment. Proinflammatory mediators are thought to play a central role in its pathogenesis. Methods: An exploratory study was conducted to evaluate the predictive capacity of biomarkers for development of clinical pulmonary oedema in patients with severe falciparum malaria at two hospitals in Bangladesh. Plasma concentrations of interleukin-6 (IL-6), IL-8, tumour necrosis factor (TNF), soluble Receptor of Advanced Glycation End-products (sRAGE), surfactant protein-D (SP-D), club cell secretory protein (CC16), and Krebs von den Lungen-6 (KL-6) on admission were compared with healthy controls. Correlations between these biomarker and plasma lactate and Plasmodium falciparum histidine-rich protein 2 (PfHRP2) levels were evaluated. Receiver Operating Characteristic (ROC) curves were constructed to assess the predictive capacity for clinical pulmonary oedema of the biomarkers of interest. Results: Of 106 screened patients with falciparum malaria, 56 were classified as having severe malaria with a mortality rate of 29%. Nine (16%) patients developed clinical pulmonary oedema after admission. Plasma levels of the biomarkers of interest were higher in patients compared to healthy controls. IL-6, IL-8, TNF, sRAGE, and CC16 levels correlated well with plasma PfHRP2 levels (rs = 0.39; P = 0.004, rs = 0.43; P = 0.001, rs = 0.54; P < 0.001, rs = 0.44; P < 0.001, rs = 0.43; P = 0.001, respectively). Furthermore, IL-6 and IL-8 levels correlated well with plasma lactate levels (rs = 0.37; P = 0.005, rs = 0.47; P < 0.001, respectively). None of the biomarkers of interest had predictive capacity for development of clinical pulmonary oedema. Conclusions: IL-6, IL-8, TNF, sRAGE, SP-D, CC16 and KL-6 cannot be used in predicting clinical pulmonary oedema in severe malaria patients. [ABSTRACT FROM AUTHOR]

Anophelinae mosquitoes are exposed to a variety of microbes including Plasmodium parasites that cause malaria. When infected, mosquitoes mount versatile immune responses, including the production of antimicrobial peptides. Cecropins are one of the most widely distributed families of antimicrobial peptides in insects and all previously studied Anopheles members are playing roles in adult mosquito immunity. We have identified and characterized a novel member of the Anopheles gambiae cecropin family, cecropin D (CecD), that is uniquely expressed and immune-responsive at late larval stages to promote microbial clearance through its broad-spectrum antibacterial activity during larval-pupal developmental transition. Interestingly, Cecropin D also exhibited highly potent activity against Plasmodium falciparum sporozoites, the malaria parasite stage that is transmitted from mosquitoes and infects humans and thereby holds promise as a malaria transmission-blocking agent. Finally, we have defined unequivocal cecropin-specific molecular signatures to systematically organize the diversity of the cecropin family in malaria vectors. Author summary: Anopheles mosquitoes that transmit the deadly malaria are exposed to a variety of microbes in their natural habitats. Mosquitoes use their innate immune system that also comprises antimicrobial peptides to fight infections with these microbes including the Plasmodium malaria parasite. Cecropins are one of the most widely distributed antimicrobial peptides in insects and all previously studied Anopheles cecropins are playing roles in adult mosquito immunity. We have identified and characterized a novel Anopheles gambiae cecropin, cecropin D (CecD), that is uniquely produced and immune-responsive at late larval stages to promote broad spectrum microbial clearance during larval-pupal developmental transition. Interestingly, Cecropin D also shows potent activity against Plasmodium falciparum sporozoites, the malaria parasite stage that is transmitted from mosquitoes to humans and could therefore be developed into a malaria control strategy. [ABSTRACT FROM AUTHOR]

Introduction: Plasmodium falciparum is the most damaging malaria pathogen and brings a heavy burden to global health. Host switching and morphological changes in P. falciparum are dependent on an effective gene expression regulatory system. C5 methylation of cytosines is a common RNA modification in eukaryotes, and the NSUN family are essential m5C modification executors. Currently, little is known about this family in Plasmodium spp. In this study, we focus on exploring the function of PfNSUN1 protein. Methods: An efficient CRISPR/Cas9 gene editing technique was applied to construct the PfNSUN1 knockdown strain. The knockdown efficiency was confirmed by growth curves and western blot experiments. The knockdown transcriptome data was acquired to find differentially expressed genes, and target genes of PfNSUN1 protein were identified by RNA immunoprecipitation and high-throughput sequencing experiments. Results: The efficiency of PfNSUN1 protein down-regulated was about 34%. RNA-seq data revealed that differentially expressed genes were mainly downregulated. And there were 224, 278, 556 genes that were down-regulated with more than 2-fold changes and p-adj<0.05 at ring, trophozoite and schizont stages, respectively. PfNSUN1 protein was significantly enriched on 154 target genes, including 28S ribosomal RNA and pfap2-g5 transcription factor. Discussion: PfNSUN1 is a crucial RNA post-transcriptional modification protein in P. falciparum. It plays a pivotal role in regulating gene expression and parasite growth by targeting 28S ribosomal RNA and pfap2-g5 transcription factor. [ABSTRACT FROM AUTHOR]

Background: Submicroscopic Plasmodium infections can be a source of persistent malaria transmission. The aim of this study was to assess their frequency, distribution, morbidity and associated factors in a pre-elimination malaria setting in sub-Saharan Africa, Guinea-Bissau, where the Plasmodium falciparum is the predominant Plasmodium species. Methods: Dried fingerprick whole blood samples from 601 participants in the 2017 national, household-based, cross-sectional survey to estimate malaria prevalence were subjected to DNA extraction. The DNA was used in nested end-point PCR assays targeting genus- and species-specific regions of the Plasmodium 18S rRNA genes. Statistical analysis of socio-demographic, clinical and molecular data was carried out using the Statistical Package for the Social Sciences, version 29. Factors associated with submicroscopic P. falciparum infections and their magnitude were sought using Chi-square test and multiple logistic regression models, respectively. Statistically significant level was considered at P-value < 0.05. Results: Nested PCR assays detected submicroscopic P. falciparum infections in 20.3% (95% CI = 16.8–23.8) of individuals microscopically negative for Plasmodium species in the general population and in 21.4% (95% CI = 9.9–36.5) of microscopically negative pregnant women. Submicroscopic Plasmodium malariae infections were also detected as co-infections in 3.0% individuals who were microscopically positive only for P. falciparum. Infections with other Plasmodium species were not detected. Submicroscopic P. falciparum infections were not associated with age, sex, or the presence of fever. A logistic regression model adjusted for ethnicity and health region showed that individuals from the Balanta and Bijagos ethnic groups, most of whom live in the low malaria-transmission areas of Quinara and Bissau, and the Bijagos archipelago, respectively, were less likely to have submicroscopic P. falciparum infections than individuals from the large Fula ethnic group, most of whom live in the high malaria-transmission area of Gabu. Submicroscopic P. falciparum infections were not associated with anaemia in children under 5 years of age. Conclusion: The results obtained highlight the contribution of asymptomatic and submicroscopic P. falciparum infections to malaria transmission in high malaria-transmission areas and the need for molecular-based tools to detect submicroscopic Plasmodium species. [ABSTRACT FROM AUTHOR]

Malaria is a deadly disease caused by Plasmodium parasites. While potent drugs are available in the market for malaria treatment, over the years, Plasmodium parasites have successfully developed resistance against many, if not all, front-line drugs. This poses a serious threat to global malaria eradication efforts, and the continued discovery of new drugs is necessary to tackle this debilitating disease. With recent unprecedented progress in machine learning techniques, single-cell transcriptomic in Plasmodium offers a powerful tool for identifying crucial proteins as a drug target and subsequent computational prediction of potential drugs. In this study, We have implemented a mutual-information-based feature reduction algorithm with a classification algorithm to select important proteins from transcriptomic datasets (sexual and asexual stages) for Plasmodium falciparum and then constructed the protein-protein interaction (PPI) networks of the proteins. The analysis of this PPI network revealed key proteins vital for the survival of Plasmodium falciparum. Based on the function and identification of a few strong binding sites on a couple of these key proteins, we computationally predicted a set of potential drug molecules using a deep learning-based technique. Lead drug molecules that satisfy ADMET and drug-likeliness properties are finally reported out of the generated drugs. The study offers a general computational pipeline to identify crucial proteins using scRNA-seq data sets and further development of potential new drugs. [ABSTRACT FROM AUTHOR]

Malaria remains a key health and economic problem, particularly in sub-Saharan Africa. The emergence of artemisinin drug resistance (ART-R) parasite strains poses a serious threat to the control and elimination of this scourge. This is because artemisinin-based combination therapies (ACTs) remain the first-line treatment in the majority of malaria-endemic regions in Sub-Saharan Africa. Certain single-nucleotide polymorphisms in the propeller domains of Plasmodium falciparum Kelch 13 protein (K13) have been associated with delayed parasite clearance in vivo and in vitro. These mutations serve as vital molecular markers for tracking the emergence and dispersion of resistance. Recently, there have been increasing reports of the emergence and spread of P. falciparum ART-R parasites in the Eastern Africa region. This necessitates continued surveillance to best inform mitigation efforts. This study investigated the presence of all reported mutations of K13 propeller domains in the parasite population in Busia County, Kenya, a known malaria-endemic region. Two hundred twenty-six participants with microscopically confirmed uncomplicated malaria were recruited for this study. They were treated with artemether–lumefantrine under observation for the first dose, and microscopic examination was repeated 1 day later after ensuring the participants had taken the second and third doses. P. falciparum DNA from all samples underwent targeted amplification of the K13 gene using a semi-nested PCR approach, followed by Sanger sequencing. The recently validated ART-R K13 mutation C469Y was identified in three samples. These three samples were among 63 samples with a low reduction in parasitemia on day 1, suggesting day 1 parasitemia reduction rate is a useful parameter to enrich the ART-R parasites for further analysis. Our findings highlight the need for continuous surveillance of ART-R in western Kenya and the region to determine the spread of ART-R and inform containment. [ABSTRACT FROM AUTHOR]

Seasonal Malaria Chemoprevention consisting of monthly administration of amodiaquine/sulfadoxine-pyrimethamine to children aged 3–59 months during the transmission season could promote SP-resistance. Mutations in dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes were assessed before and after SMC adoption in Burkina Faso. A total of 769 dried blood spots were selected from studies conducted in Nanoro, Burkina Faso, between 2010 and 2020. Of those, 299 were pre-SMC (2010–2012) and 470 were post-SMC-samples. Pfdhps and Pfdhfr genes were PCR-amplified and sequenced. A systematic review/meta-analysis of published studies conducted in Burkina Faso (2009–2023) was additionally performed. In Nanoro, the prevalence of Pfdhfr triple mutations (CIRNI) rose from 43.6% pre-SMC to 89.4% post-SMC (p < 0.0001). There was no mutation in Pfdhfr 164 and Pfdhps 540; Pfdhps A437G mutation increased from 63.9% (2010–2012) to 84.7% (2020) (p < 0.0001). The VAGKGS haplotype was 2.8% (2020). Pfdhfr/Pfdhps quintuple mutant IRN-436A437G rose from 18.6% (2010–2012) to 58.3% (2020) (p < 0.0001). Meta-analysis results from Burkina Faso showed an increase in mutations at Pfdhfr N51I, C59R, S108N, and Pfdhps A437G after SMC adoption. Post-SMC, the pyrimethamine-resistance marker prevalence increased, while the sulfadoxine-resistance marker prevalence remained stable. Detection of emerging PfdhpsVAGKGS haplotypes in 2020 underscores the importance of continuous SP-resistance monitoring. [ABSTRACT FROM AUTHOR]

Background: Malaria remains a significant public health concern, especially for the deadliest parasite, Plasmodium falciparum. During acute malaria, various cytokines, including osteopontin (OPN), regulate the immune response. OPN has been shown to be protective against malaria in mice. Nonetheless, its precise function and potential ability to control parasites during acute malaria in humans remain poorly understood. Results: Blood samples were collected from Swedish adults with imported malaria, Ugandan children and adults with symptomatic malaria (including follow-up after 42 days), Ugandans with non-malarial fever and healthy individuals from both Uganda and Sweden. Parasitemia was determined by microscopy. Malaria-negative samples were verified by LAMP. OPN and interferon-γ (IFN- γ) levels were measured using ELISA. In children, OPN levels were significantly higher during acute infection compared to levels after 42 days, whereas Ugandan adults showed no difference. Swedish adults with imported malaria had elevated OPN levels compared to both Swedish controls and Ugandan adults with malaria. Parasitemia was significantly correlated with both OPN and IFN-γ levels across the entire cohort. While a significant correlation between OPN and IFN-γ was evident overall, it remained statistically significant only in Ugandan adults when analyzed by subgroups. This suggests that OPN is not just a general marker of inflammation but may be regulated differently during the development of malaria immunity. Conclusions: In acute malaria, elevated OPN levels showed a stronger correlation with lack of immunity than age. These findings underscore the potential importance of OPN in malaria, particularly in non-immune individuals. [ABSTRACT FROM AUTHOR]

Conditional regulation is a highly beneficial system for studying the function of essential genes in Plasmodium falciparum and dimerizable Cre recombinase (DiCre) is a recently adapted conditional regulation system suitable for this purpose. In the DiCre system, two inactive fragments of Cre are reconstituted to form a functionally active enzyme in the presence of rapamycin. Different loci have been targeted to generate parasite lines that express the DiCre enzyme. Here, we have used marker-free CRISPR-Cas9 gene editing to integrate the DiCre cassette in a redundant cg6 locus. We have shown the utility of the newly generated ∆cg6DC4 parasites in mediating robust, rapid, and highly specific excision of exogenously encoded gfp sequence. The ∆cg6DC4 parasites are also capable of conditional excision of an endogenous parasite gene, PF3D7_1246000. Conditional deletion of PF3D7_1246000 did not cause any inhibition in the asexual proliferation of the parasites. Furthermore, the health and morphology of the mutant parasites were comparable to that of the control parasites in Giemsa smears. The availability of another stable DiCre parasite strain competent for conditional excision of target genes will expedite functional characterization and validation of novel drug and vaccine targets against malaria. [ABSTRACT FROM AUTHOR]

The malaria vaccination landscape has seen significant advancements with the recent endorsement of RTS,S/AS01 and R21/Matrix-M vaccines, which target the pre-erythrocytic stages of Plasmodium falciparum (Pf) infection. However, several challenges remain to be addressed, including the incomplete protection afforded by these vaccines, their dependence on a single Pf antigen, and the fact that they were not designed to protect against P. vivax (Pv) malaria. Injectable formulations of whole-sporozoite (WSpz) malaria vaccines offer a promising alternative to existing subunit vaccines, with recent developments including genetically engineered parasites and optimized administration regimens. Clinical evaluations demonstrate varying efficacy, influenced by factors, such as immune status, prior exposure to malaria, and age. Despite significant progress, a few hurdles persist in vaccine production, deployment, and efficacy in malaria-endemic regions, particularly in children. Concurrently, transgenic parasites expressing Pv antigens emerge as potential solutions for PvWSpz vaccine development. Ongoing clinical studies and advancements in vaccine technology, including the recently described PfSPZ-LARC2 candidate, signify a hopeful future for WSpz malaria vaccines, which hold great promise in the global fight against malaria. This Review discusses challenges in developing malaria vaccines and promising recent developments in whole-sporozoite vaccines using genetically engineered parasites with a perspective on future directions of these vaccines for the prevention of malaria. [ABSTRACT FROM AUTHOR]

Three new steroids, turranin M, N and O (1-3), together with four known limonoids, nymania 1 (4), rubralin B (5), aphapolynin C (6) and Trichillia substance Tr B (7), were isolated from the leaves of Turraea obtusifolia. Their chemical structures were elucidated using NMR and MS. Rubralin B (5) displayed good activity against the asexual parasites from the drug sensitive Plasmodium falciparum NF54 strain with an IC50 value of 3.47 µg/mL (4.57 µM), nymania 1 (4) showed a weak activity (IC50 13.36 µg/mL (19.40 µM)) and the rest of compounds had IC50 > 20 µg/mL. [ABSTRACT FROM AUTHOR]

Most malaria rapid diagnostic tests (RDTs) detect Plasmodium falciparum histidine-rich protein 2 (PfHRP2) and PfHRP3, but deletions of pfhrp2 and phfrp3 genes make parasites undetectable by RDTs. We analyzed 19,313 public whole-genome-sequenced P. falciparum field samples to understand these deletions better. Pfhrp2 deletion only occurred by chromosomal breakage with subsequent telomere healing. Pfhrp3 deletions involved loss from pfhrp3 to the telomere and showed three patterns: no other associated rearrangement with evidence of telomere healing at breakpoint (Asia; Pattern 13-TARE1); associated with duplication of a chromosome 5 segment containing multidrug-resistant-1 gene (Asia; Pattern 13-5++); and most commonly, associated with duplication of a chromosome 11 segment (Americas/Africa; Pattern 13-11++). We confirmed a 13--11 hybrid chromosome with long-read sequencing, consistent with a translocation product arising from recombination between large interchromosomal ribosome-containing segmental duplications. Within most 13-11++ parasites, the duplicated chromosome 11 segments were identical. Across parasites, multiple distinct haplotype groupings were consistent with emergence due to clonal expansion of progeny from intrastrain meiotic recombination. Together, these observations suggest negative selection normally removes 13-11++pfhrp3 deletions, and specific conditions are needed for their emergence and spread including low transmission, findings that can help refine surveillance strategies. [ABSTRACT FROM AUTHOR]

Background: The ability to predict and comprehend molecular interactions offers significant insights into the biological functions of proteins. The interaction between surface protein 47 of Plasmodium falciparum (Pfs47) and receptor of the protein 47 (P47Rec) has attracted increased attention due to their role in parasite evasion of the mosquito immune system and the concept of geographical coevolution between species. The aims of this study were as follows: to apply a bioinformatics approach to investigate the interaction between Pfs47 and P47Rec proteins and to identify the potential binding sites, protein orientations and receptor specificity sites concerning the geographical origins of the vectors and the parasite. Methods: Public sequences of the pfs47 and p47rec genes were downloaded and subsequently filtered to predict functional and structural annotations of the Pfs47-P47Rec complex. Phylogenetic analyses of both proteins were carried out. In addition, the p47Rec gene was subjected to sequencing and subsequent analysis in 2 distinct Anopheles species collected in Honduras. Results: The examination of motifs reveals a significant degree of conservation in pfs47, suggesting that Pfs47 might have undergone recent evolutionary development and adaptation. Structural models and docking analyses supported the theory of selectivity of Plasmodium falciparum strains towards their vectors in diverse geographical regions. A detailed description of the putative interaction between the Pfs47-P47Rec complex is shown. Conclusions: The study identifies coevolutionary patterns between P47Rec and Pfs47 related to the speciation and geographic dispersion of Anopheles species and Plasmodium falciparum, with Pfs47 evolving more recently than P47Rec. This suggests a link between the parasite's adaptability and existing anopheline species across different regions. P47Rec likely has a cytoplasmic localization due to its lack of membrane attachment elements. However, these findings are based on simulations and require validation through methods like cryo-electron microscopy. A significant limitation is the scarcity of sequences in global databases, which restricts precise interaction modelling. Further research with diverse parasite isolates and anopheline species is recommended to enhance understanding of these proteins' structure and interaction. [ABSTRACT FROM AUTHOR]

Asexual replication of Plasmodium falciparum in the human blood results in exponential parasite growth and causes all clinical symptoms of malaria. However, at each round of the replicative cycle, some parasites convert into sexual precursors called gametocytes, which develop through different stages until they become infective to mosquito vectors. The genome-wide distribution of heterochromatin, a type of chromatin generally refractory to gene expression, is identical at all asexual blood stages, but is altered in stage II/III and more mature gametocytes. However, it is not known if these changes occur concomitantly with sexual conversion or at a later time during gametocyte development. Using a transgenic line in which massive sexual conversion can be conditionally induced, we show that the genome-wide distribution of heterochromatin at the initial stages of sexual development (i.e., sexual rings and stage I gametocytes) is almost identical to asexual blood stages, and major changes do not occur until stage II/III. However, we found that at loci with heterochromatin alterations, transcriptional changes associated with sexual development typically precede, rather than follow, changes in heterochromatin occupancy. [ABSTRACT FROM AUTHOR]

Background/Objectives: YAT2150 is a first-in-class antiplasmodial compound that has been recently proposed as a new interesting drug for malaria therapy. Methods/Results: The fluorescence of YAT2150 rapidly increases upon its entry into Plasmodium, a property that can be of use for the design of highly sensitive diagnostic approaches. YAT2150 blocks the in vitro development of the ookinete stage of Plasmodium and, when added to an infected blood meal, inhibits oocyst formation in the mosquito. Thus, the compound could possibly contribute to future transmission-blocking antimalarial strategies. Cell influx/efflux studies in Caco-2 cells suggest that YAT2150 is internalized by endocytosis and also through the OATP2B1 transporter, whereas its main export route would be via OSTα. YAT2150 has an overall favorable drug metabolism and pharmacokinetics profile, and its moderate cytotoxicity can be significantly reduced upon encapsulation in immunoliposomes, which leads to a dramatic increase in the drug selectivity index to values close to 1000. Although YAT2150 binds amyloid-forming peptides, its in vitro fluorescence emission is stronger upon association with peptides that form amorphous aggregates, suggesting that regions enriched in unstructured proteins are the preferential binding sites of the drug inside Plasmodium cells. The reduction of protein aggregation in the parasite after YAT2150 treatment, which has been suggested to be directly related to the drug's mode of action, is also observed following treatment with quinoline antimalarials like chloroquine and primaquine. Conclusions: Altogether, the data presented here indicate that YAT2150 can represent the spearhead of a new family of compounds for malaria diagnosis and therapy due to its presumed novel mode of action based on the interaction with functional protein aggregates in the pathogen. [ABSTRACT FROM AUTHOR]

Dobinin K is a novel eudesmane sesquiterpenoids compound isolated from the root of Dobinea delavayi and displays potential antiplasmodial activity in vivo. Here, we evaluate the antiplasmodial activity of dobinin K in vitro and study its acting mechanism. The antiplasmodial activity of dobinin K in vitro was evaluated by concentration-, time-dependent, and stage-specific parasite inhibition assay. The potential target of dobinin K on Plasmodium falciparum was predicted by transcriptome analysis. Apoptosis of P. falciparum was detected by Giemsa, Hoechst 33258, and TUNEL staining assay. The reactive oxygen species (ROS) level, oxygen consumption, and mitochondrial membrane potential of P. falciparum were assessed by DCFH-DA, R01, and JC-1 fluorescent dye, respectively. The effect of dobinin K on the mitochondrial electron transport chain (ETC) was investigated by enzyme activity analysis and the binding abilities of dobinin K with different enzymes were learned by molecular docking. Dobinin K inhibited the growth of P. falciparum in a concentration-, time-dependent, and stage-specific manner. The predicted mechanism of dobinin K was related to the redox system of P. falciparum. Dobinin K increased intracellular ROS levels of P. falciparum and induced their apoptosis. After dobinin K treatment, P. falciparum mitochondria lost their function, which was presented as decreased oxygen consumption and depolarization of the membrane potential. Among five dehydrogenases in P. falciparum ETC, dobinin K displayed the best inhibitory power on NDH2 activity. Our findings indicate that the antiplasmodial effect of dobinin K in vitro is mediated by the enhancement of the ROS level in P. falciparum and the disruption of its mitochondrial function. [ABSTRACT FROM AUTHOR]

In this paper, we describe our discovery of burnettiene A (1) as an antimalarial compound from the culture broth of Lecanicillium primulinum (current name: Flavocillium primulinum) FKI-6715 strain utilizing our original multidrug-sensitive yeast system. This polyene-decalin polyketide natural product was originally isolated as an antifungal active compound from Aspergillus burnettii. However, the antifungal activity of 1 has been revealed in only one fungal species, and the mechanism of action of 1 remains unknown. After the validation of mitochondrial function inhibitory of 1 , we envisioned a new antimalarial drug discovery platform based on mitochondrial function inhibitory activity. We evaluated antimalarial activity and 1 showed antimalarial activity against Plasmodium falciparum FCR3 (chloroquine sensitive) and the K1 strain (chloroquine resistant). Our study revealed the utility of our original screening system based on a multidrug-sensitive yeast and mitochondrial function inhibitory activity for the discovery of new antimalarial drug candidates. [ABSTRACT FROM AUTHOR]

Background and Objectives: Plasmodium species are naturally transmitted by Anopheles mosquitos. The parasite infects red blood cells (RBCs) and can be transfused with blood products. In non‐endemic areas, the main risk of infection arises from travellers coming back and people immigrating from malaria‐endemic regions. Endemic countries face a permanent risk of infection from transfusion‐transmitted malaria (TTM). TTM may cause life‐threatening complications in patients dependent on blood donations. This study aimed to investigate the efficacy of Plasmodium falciparum inactivation in RBC units by treatment with short‐wavelength ultraviolet C (UVC) light in the absence of photochemical additives. Materials and Methods: RBC units were spiked with P. falciparum to a parasite density of 0.1%–1% and irradiated with up to 4.5 J/cm2 UVC. The parasite density of UVC‐treated dilution series and untreated controls were compared over 3 weeks after irradiation. Results: The lowest dose of 1.5 J/cm2 UVC led to a 3.1 log reduction in parasite load compared with the untreated control. The inactivation capacity was dose‐dependent. Strikingly, 4.5 J/cm2 led to ≥5.3 log unit reduction, which was equivalent to a complete inactivation in two out of three experiments. Conclusion: Pathogen reduction with UVC light was previously shown to be effective for different bacteria and viruses, but the inactivation of parasites in RBC concentrates was not addressed until now. The present study provides evidence for significant inactivation of P. falciparum‐infected RBCs by UVC light. [ABSTRACT FROM AUTHOR]

According to WHO, between 2000 and 2021, there were approximately 247 million malaria cases and 627,000 deaths globally, with the majority of cases occurring in sub-Saharan Africa. In Turkey, indigenous P. vivax malaria was a major public health problem until its eradication was achieved in 2010. Although indigenous malaria transmission has been significantly reduced since 2010, the country is challenged with imported malaria due to increasing global travel and migration from endemic regions. In this study, all imported malaria cases admitted to Dr. Sadi Konuk Research and Training Hospital, Istanbul, between 2018 and 2023 were included. DNA extraction was performed using archived slides and EDTA blood samples. Real-time PCR was performed to identify samples at the species level using previously reported primers and probes. In addition, all available patient demographics are presented. During the six years between 2018 and 2023, 157 patients were diagnosed with imported malaria. According to the real-time PCR results, 149 cases were P. falciparum (94.9%), five cases were P. vivax (3.2%), two cases were P. ovale (1.3%), and one case was P. malariae (0.6%). The male/female ratio among diagnosed patients was 2.34 (110♂/47♀) among diagnosed patients. Plasmodium falciparum was detected in patients from all African regions, whereas P. vivax was detected only in patients from Liberia and Djibouti. Although malaria cases in Turkey have significantly decreased due to elimination efforts and effective public health interventions, the recent increase in both imported and indigenous cases, as well as the presence of suitable vector species in the country, indicates that malaria still remains a serious public health problem for Turkey. [ABSTRACT FROM AUTHOR]

[Display omitted] • A NanoLuc reporter allows rapid quantification of Plasmodium falciparum transfection outcome. • Optimized hypotonic lysis and resealing improves erythrocyte plasmid loading. • Improved plasmid loading increases malaria parasite transfection efficiency. • The hypotonic loading method is broadly accessible and less effort-intensive. • Improved transfection outcomes will accelerate antimalarial therapy research. In vitro modification of Plasmodium falciparum genes is the cornerstone of basic and translational malaria research. Achieved through DNA transfection, these modifications may entail altering protein sequence or abundance. Such experiments are critical for defining the molecular mechanisms of key parasite phenotypes and for validation of drug and vaccine targets. Despite its importance, successful transfection remains difficult and is a resource-intensive, rate-limiting step in P. falciparum research. Here, we report that inefficient loading of plasmid into erythrocytes limits transfection efficacy with commonly used electroporation methods. As these methods also require expensive instrumentation and consumables that are not broadly available, we explored a simpler method based on plasmid loading through hypotonic lysis and resealing of erythrocytes. We used parasite expression of a sensitive NanoLuc reporter for rapid evaluation and optimization of each step. Hypotonic buffer composition, resealing buffer volume and composition, and subsequent incubation affected plasmid retention and successful transfection. While ATP was critical for erythrocyte resealing, addition of Ca++ or glutathione did not improve transfection efficiency, with increasing Ca++ concentrations proving detrimental to outcomes. Compared with either the standard electroporation method or a previously reported hypotonic loading protocol, the optimized method yields greater plasmid loading and higher expression of the NanoLuc reporter 48 h after transfection. It also produced significantly faster outgrowth of parasites in transfections utilizing either episomal expression or CRISPR-Cas9 mediated integration. This new method produces higher P. falciparum transfection efficiency, reduces resource requirements and should accelerate molecular studies of malaria drug and vaccine targets. [ABSTRACT FROM AUTHOR]

Non‐canonical nucleotides, generated as oxidative metabolic by‐products, significantly threaten the genome integrity of Plasmodium falciparum and thereby, their survival, owing to their mutagenic effects. PfHAM1, an evolutionarily conserved inosine/xanthosine triphosphate pyrophosphohydrolase, maintains nucleotide homeostasis in the malaria parasite by removing non‐canonical nucleotides, although structure–function intricacies are hitherto poorly reported. Here, we report the X‐ray crystal structure of PfHAM1, which revealed a homodimeric structure, additionally validated by size‐exclusion chromatography–multi‐angle light scattering analysis. The two monomeric units in the dimer were aligned in a parallel fashion, and critical residues associated with substrate and metal binding were identified, wherein a notable structural difference was observed in the β‐sheet main frame compared to human inosine triphosphate pyrophosphatase. PfHAM1 exhibited Mg++‐dependent pyrophosphohydrolase activity and the highest binding affinity to dITP compared to other non‐canonical nucleotides as measured by isothermal titration calorimetry. Modifying the pfham1 genomic locus followed by live‐cell imaging of expressed mNeonGreen‐tagged PfHAM1 demonstrated its ubiquitous presence in the cytoplasm across erythrocytic stages with greater expression in trophozoites and schizonts. Interestingly, CRISPR‐Cas9/DiCre recombinase‐guided pfham1‐null P. falciparum survived in culture under standard growth conditions, indicating its assistive role in non‐canonical nucleotide clearance during intra‐erythrocytic stages. This is the first comprehensive structural and functional report of PfHAM1, an atypical nucleotide‐cleansing enzyme in P. falciparum. [ABSTRACT FROM AUTHOR]

Plasmodium falciparum is the pathogen responsible for 90 % of all malaria cases and is associated with severe complications and the highest fatality rate. Plasmodium falciparum lactate dehydrogenase (PfLDH) is a useful biomarker for the treatment and diagnosis of malaria; however, its use is limited by low production yield and functional activity. In this study, using Escherichia coli Rosetta(DE3) strain specialized for eukaryotic protein expression, we successfully expressed and purified PfLDH without a codon optimization process, which has previously been considered essential for protein expression in E. coli strains. The induction temperature and time were optimized for the overexpression of PfLDH using the transformed strain, and 31.3 mg/L of PfLDH protein was successfully purified using immobilized metal affinity chromatography. The physical properties of the purified PfLDH were assessed by verifying tetramer formation, and the functional properties of PfLDH were assessed with a colorimetric assay using a substrate that reacts with PfLDH. Subsequently, the binding of PfLDH with a DNA aptamer, which is known to specifically bind to PfLDH, was verified. These results are expected to provide important suggestions for research on obtaining large amounts of PfLDH from bacteria, thereby facilitating the treatment and diagnosis of malaria. [Display omitted] • PfLDH was successfully purified from an E. coli Rosetta(DE3) strain. • Expression conditions for PfLDH purification were optimized • PfLDH was obtained in a yield of 31.3 mg/L. • Purified PfLDH maintained its structural and functional characteristics. [ABSTRACT FROM AUTHOR]

The Apicomplexa phylum encompasses numerous obligate intracellular parasites, some associated with severe implications for human health, including Plasmodium, Cryptosporidium, and Toxoplasma gondii. The iron-sulfur cluster [Fe-S] biogenesis ISC pathway, localized within the mitochondrion or mitosome of these parasites, is vital for parasite survival and development. Previous work on T. gondii and Plasmodium falciparum provided insights into the mechanisms of [Fe-S] biogenesis within this phylum, while the transporter linking mitochondria-generated [Fe-S] with the cytosolic [Fe-S] assembly (CIA) pathway remained elusive. This critical step is catalyzed by a well-conserved ABC transporter, termed ATM1 in yeast, ATM3 in plants and ABCB7 in mammals. Here, we identify and characterize this transporter in two clinically relevant Apicomplexa. We demonstrate that depletion of TgATM1 does not specifically impair mitochondrial metabolism. Instead, proteomic analyses reveal that TgATM1 expression levels inversely correlate with the abundance of proteins that participate in the transfer of [Fe-S] to cytosolic proteins at the outer mitochondrial membrane. Further insights into the role of TgATM1 are gained through functional complementation with the well-characterized yeast homolog. Biochemical characterization of PfATM1 confirms its role as a functional ABC transporter, modulated by oxidized glutathione (GSSG) and [4Fe-4S]. Author summary: The Apicomplexa phylum includes harmful parasites like Plasmodium falciparum and Toxoplasma gondii, posing significant threats to human health. Understanding their survival mechanisms is crucial for developing effective treatments. Here, our focus is on iron-sulfur cluster [Fe-S] synthesis pathways found in the mitochondrion and cytosol, essential for the function of many enzymes. Although the transporter responsible for moving sulfur-containing compounds from mitochondria to the cytosol is known in other organisms, it remains unidentified in apicomplexans. Our study identifies PfATM1 and TgATM1 as critical transporters in the mitochondrial membrane of these parasites. Downregulation or overexpression of TgATM1 levels do not hinder mitochondrial functions but affect the level of other proteins, suspected to donate [Fe-S] to cytosolic proteins. We further confirm the transporter's role by rescuing TgATM1 depleted parasites with the yeast homolog ScATM1. Additionally, we identify glutathione-coordinated [4Fe-4S] as a probable substrate for PfATM1. These findings provide valuable insights into how parasites manage essential resources, potentially opening new avenues for targeted drug development. [ABSTRACT FROM AUTHOR]

The novel antimalarial ganaplacide combined with lumefantrine solid dispersion formulation (LUM‐SDF) was effective and well tolerated in the treatment of uncomplicated falciparum malaria in adults, adolescents, and children in a multinational, prospective, randomized, active‐controlled Phase II study conducted between August 2017 and June 2021 (EudraCT 2020‐003284‐25, Clinicaltrials.gov NCT03167242). Pharmacokinetic data from that study are reported here.The trial comprised three parts: a run‐in part in 12 adult/adolescent patients treated with a single dose of ganaplacide 200 mg plus LUM‐SDF 960 mg assessed potential pharmacokinetic (PK) interactions between ganaplacide and lumefantrine; in Part A, adult/adolescent patients received one of the six ganaplacide‐LUM‐SDF regimens or artemether‐lumefantrine; and in Part B, three dose regimens identified in Part A, and artemether‐lumefantrine, were assessed in children aged 2 to <12 years, with body weight ≥10 kg. A rich blood sampling schedule was used for all 12 patients in the PK run‐in part and a subset of patients (N = 32) in Part A, with sparse sampling for remaining patients in Parts A (N = 275) and B (N = 159). Drug concentrations were determined by a validated protein precipitation and reverse phase liquid chromatography with tandem mass spectrometry detection method. Parameters including AUCinf, AUClast, AUC0–t, Cmax, and tmax were reported where possible, using non‐compartmental analysis.In the PK run‐in part, there was no notable increase in ganaplacide or lumefantrine exposure when co‐administered. In Parts A and B, ganaplacide exposures increased with dose, but lumefantrine exposure was numerically under dose‐proportional. Lumefantrine exposure was higher with ganaplacide‐LUM‐SDF than with artemether‐lumefantrine, although high variability was observed. Ganaplacide and lumefantrine exposures (Cmax and AUC0–24 h) were comparable across age and body weight groups. Drug exposures needed for efficacy were achieved using the dose regimen 400 mg ganaplacide plus lumefantrine 960 mg once daily for 3 days under fasted conditions. [ABSTRACT FROM AUTHOR]

Background: Malaria is a disease transmitted by vectors and caused by unicellular Plasmodium parasites. Malaria pathogenesis is associated with the ABO phenotype. However, there is little information on the frequency of malaria disease and its relationship with the ABO blood group in the study area. Therefore, the purpose of this study was to determine the prevalence of malaria infection and its association with the ABO blood group at Woldia Comprehensive Specialized Hospital. Method: An institutional‐based cross‐sectional study was conducted from December 3, 2022, to February 30, 2023. Convenient sampling was used for selecting the study participants. To identify malaria parasites, thick and thin blood films were made. Additionally, blood was drawn to identify the ABO blood group type. Before being analyzed with SPSS software Version 27, the data was coded and entered into EpiData Version 3.1. To ascertain the variable's association, a logistic regression was done. Results: Out of 192 patients that attended Woldia Comprehensive Specialized Hospital, 16 (8.3%) were found to be infected with Plasmodium parasites using microscopy. Among them, 9 (4.7%), 5 (2.6%), and 2 (1.0%) had Plasmodium falciparum, Plasmodium vivax, or mixed infections, respectively. As a result, 30.7%, 25.5%, 24.5%, and 19.3% of the participants had blood types A, B, AB, and O, respectively (AOR = 2.359, 95% CI: 1.03–12.289, p = 0.03). Conclusion and Recommendation: The total number of microscopically confirmed malaria parasites was 8.3%. P. falciparum was dominant over P. vivax. Individuals with blood group O were less likely to get severe malaria than those with other blood groups. Based on the findings of this study, we recommend that additional studies investigate the probable relationship between the ABO blood group phenotype and malaria infection. [ABSTRACT FROM AUTHOR]

Background: Malaria continues to cause unacceptably high levels of disease and death despite increased global efforts and is still significant public health problem. African countries are disproportionately affected by malaria. The objective of this study was to describe a rare case of haemorrhagic stroke as a possible complication of malaria in a 26-year-old male patient. Case presentation: A 26-year-old male from southwest Ethiopia presented with complaint of loss of consciousness (LOC) of 12 h duration. He had fever, headache, vomiting, chills, rigors and shivering three days prior to the loss of consciousness. On physical examination, pulse rate 116 beats/min, blood pressure of 120/90 mmHg, respiratory rate was 24 breaths/min, a temperature of 38.9◦C and oxygen saturation of 94%. Nervous system examination; stuporous with Glasgow Coma Scale (GCS) 10/15(M5, E3, V2). Blood film and RDT confirmed a Plasmodium falciparum infection and a non-contrast CT scan found a right cerebral parenchymal haemorrhage. Discussion and conclusion: The presented case described a very rare case of a 26-year-old male patient who was diagnosed with left side hemiparesis secondary to a haemorrhagic stroke, associated with P. falciparum malaria. This report highlights the fact that malaria with stroke should be considered a differential diagnosis in a patient presenting with body weakness in a malaria endemic area and in individuals who had travel history to malaria endemic areas. [ABSTRACT FROM AUTHOR]

Background: In recent years, there has been an increasing trend in the number of imported Plasmodium falciparum cases in Turkey. To improve treatment success and to better understand malaria epidemiology among imported cases, it is necessary to determine anti-malarial drug resistance. This study aimed to survey polymorphisms of resistance genes in imported P. falciparum patients using archived thin smear preparations and EDTA blood samples. Methods: A total of 100 imported P. falciparum patients admitted to Bakırköy Dr. Sadi Konuk Research and Training Hospital between 2017 and 2022 were included in this study. DNA extraction was performed using an archived slide and EDTA blood samples that were microscopically diagnosed. After confirming the samples by real-time PCR, the pfmdr1, pfcrt, and pfk13 genes were amplified and sequenced. Single nucleotide polymorphisms (SNPs) were screened using Geneious R9 software, with the reference P. falciparum clone 3D7 isolate. Results: All studied samples were confirmed to be P. falciparum using real-time PCR. Nested PCR was conducted and the pfcrt (92 samples), pfmdr1 (91 samples), and pfk13 (93 samples) genes were successfully amplified. Sequence analysis revealed the highest mutation rate in the pfmdr1 (74.5%) gene, with the identification of five haplotypes: NYSND (wild-type, 23%), NFSND (56%), NYSDD (2.2%), NFSDD (15.4%), and YFSND (3.4%)]. The pfcrt mutation was identified in 11 samples (12.2%), whereas the pfk13 mutation was found in only two samples. Conclusion: This study is the first molecular survey of anti-malarial drug resistance genes in Turkey. With the increasing number of imported Plasmodium malaria cases and recent reports of sporadic indigenous P. falciparum cases, malaria is becoming a growing concern in Turkey. Although molecular screening for resistance markers in P. falciparum malaria is not routinely conducted, the data from this study will enhance treatment success rates and contribute to global malaria elimination. [ABSTRACT FROM AUTHOR]

Background: Malaria and hepatitis B are significant public health infections in Nigeria. Coinfection with both pathogens is common where both diseases are endemic. Epidemiological surveys are essential for determining the burden of diseases and possible coinfection with multiple pathogens in vulnerable populations. There has been a lack of reports on HBV/malaria coinfection, particularly among marginalized groups in Ondo State. Thus, we used malaria microscopy and the HBsAg serological test to examine the prevalence of asymptomatic malaria parasitemia and HBV infections respectively among inmates at the Nigerian Correctional Center in Owo, Ondo State, Nigeria. Results: Out of the 126 prisoners and staff members who were evaluated, 20.6% and 7.9% tested positive for malaria and HBV infections, respectively. It was discovered that 1.6% of the individuals were coinfected with malaria and HBV. Plasmodium falciparum was the only malaria species recovered in malaria-infected individuals. Except for HBV, where gender was found to differ considerably with the proportion of HBV infection, variations in single infections with either pathogen did not vary with demographic characteristics. Conclusion: We suggest that the prison system should be considered in healthcare programs to improve the health of inmates. [ABSTRACT FROM AUTHOR]

Background: The World Health Organization 2022 malaria chemoprevention guidelines recommend providing a full course of antimalarial treatment at pre-defined intervals, regardless of malaria status to prevent illness among children resident in moderate to high perennial malaria transmission settings as perennial malaria chemoprevention (PMC) with sulfadoxine-pyrimethamine (SP). The dhps I431V mutation circulating in West Africa has unknown effect on SP protective efficacy. Methods: This protocol is for a three-arm, parallel, double-blinded, placebo-controlled, randomised trial in Cameroon among children randomly assigned to one of three directly-observed treatment groups: (i) Group 1 (n = 450) receives daily artesunate (AS) placebo on days − 7 to -1, then active SP plus placebo amodiaquine (AQ) on day 0, and placebo AQ on days 1 and 2; (ii) Group 2 (n = 250) receives placebo AS on days − 7 to -1, then active SP and AQ on day 0, and active AQ on days 1 and 2; and (iii) Group 3 (n = 200) receives active AS on days − 7 to -1, then placebo SP on day 0 and placebo AQ on days 0 to 2. On days 0, 2, 5, 7, and thereafter weekly until day 28, children provide blood for thick smear slides. Dried blood spots are collected on the same days and weekly from day 28 to day 63 for quantitative polymerase chain reaction (qPCR) and genotype analyses. Discussion: Our aim is to quantify the chemopreventive efficacy of SP, and SP plus AQ, and measure the effect of the parasite genotypes associated with SP resistance on parasite clearance and protection from infection when exposed to SP chemoprevention. We will report unblinded results including: (i) time-to-parasite clearance among SP and SP plus AQ recipients who were positive on day 0 by qPCR and followed to day 63; (ii) mean duration of SP and SP plus AQ protection against infection, and (iii) mean duration of symptom-free status among SP and SP plus AQ recipients who were parasite free on day 0 by qPCR. Our study is designed to compare the 28-day follow-up of the new WHO malaria chemoprevention efficacy study protocol with extended follow-up to day 63. Trial registration: ClinicalTrials.gov NCT06173206; 15/12/2023. [ABSTRACT FROM AUTHOR]

The malaria parasites Plasmodium falciparum and Plasmodium vivax differ in key biological processes and associated clinical effects, but consequences on population-level transmission dynamics are difficult to predict. This co-endemic malaria study from Guyana details important epidemiological contrasts between the species by coupling population genomics (1396 spatiotemporally matched parasite genomes, primarily from 2020–21) with sociodemographic analysis (nationwide patient census from 2019). We describe how P. falciparum forms large, interrelated subpopulations that sporadically expand but generally exhibit restrained dispersal, whereby spatial distance and patient travel statistics predict parasite identity-by-descent (IBD). Case bias towards working-age adults is also strongly pronounced. P. vivax exhibits 46% higher average nucleotide diversity (π) and 6.5x lower average IBD. It occupies a wider geographic range, without evidence for outbreak-like expansions, only microgeographic patterns of isolation-by-distance, and weaker case bias towards adults. Possible latency-relapse effects also manifest in various analyses. For example, 11.0% of patients diagnosed with P. vivax in Greater Georgetown report no recent travel to endemic zones, and P. vivax clones recur in 11 of 46 patients incidentally sampled twice during the study. Polyclonality rate is also 2.1x higher than in P. falciparum, does not trend positively with estimated incidence, and correlates uniquely to selected demographics. We discuss possible underlying mechanisms and implications for malaria control. P. falciparum and vivax are responsible for most cases of malaria but are not genetically closely related and differ in their clinical and epidemiological impacts. In this study, the authors investigate the genomic and epidemiological characteristics of the two parasites in a co-endemic setting of Guyana. [ABSTRACT FROM AUTHOR]

While there has been significant progress in controlling falciparum malaria in the Lao People's Democratic Republic (PDR), sporadic cases persist in southern provinces where the extent and patterns of transmission remain largely unknown. To assess parasite transmission in this area, 53 Plasmodium falciparum (Pf) positive cases detected through active test and treat campaigns from December 2017 to November 2018 were sequenced, targeting 204 highly polymorphic amplicons. Two R packages, MOIRE and Dcifer, were applied to assess the multiplicity of infections (MOI), effective MOI (eMOI), within-host parasite relatedness, and between-host parasite relatedness (rb^). Genomic data were integrated with survey data to characterize the temporal and spatial structures of identified clusters. The positive cases were mainly captured during the focal test and treat campaign conducted in 2018, and in the Pathoomphone area, which had the highest test positivity and forest activity. About 30% of the cases were polyclonal infections, with over half of theses (63%) showing within-host relatedness greater than 0.6, suggesting that cotransmission rather than superinfection was primarily responsible for maintaining polyclonality. A large majority of cases (81%) were infected by parasites genetically linked to one or more other cases. We identified five genetically distinct clusters in forest fringe villages within the Pathoomphone district, characterized by a high degree of genetic relatedness between parasites (mean rb^ = 0.8). Four smaller clusters of 2–3 cases linked Moonlapamok and Pathoomphone districts, with an average rb^ of 0.6, suggesting cross-district transmission. Most of the clustered cases occurred within 20 km and 2 months of each other, consistent with focal transmission. Transmission clusters identified in this study confirm the role of ongoing focal parasite transmission occurring within the forest or forest-fringe in the highly mobile population. Author summary: In low-transmission settings, integrating genomic data could help capture fine-scale dynamics in circulating parasites that may not be easily detected by traditional surveillance. Our work demonstrates that even with very few malaria cases, incorporating active surveillance and genomic analysis can help track residual transmission pockets and routes, including among highly mobile populations whose infection sources are historically difficult to trace. Transmission clusters identified in this study provided clear evidence of ongoing focal transmission of Plasmodium falciparum in southern Laos. The addition of high-resolution genomic data to active surveillance can aid in targeting and assessing interventions in similar areas approaching elimination. [ABSTRACT FROM AUTHOR]

Plasmodium falciparum undergoes sequestration within deep tissues of the human body, spanning multiple organ systems with differing oxygen (O2) concentrations. The parasite is exposed to an even greater range of O2 concentrations as it transitions from the human to the mosquito host, suggesting a high level of plasticity as it navigates these different environments. In this review, we explore factors that may contribute to the parasite's response to different environmental O2 concentrations, recognizing that there are likely multiple pieces to this puzzle. We first review O2-sensing mechanisms, which exist in other apicomplexans such as Toxoplasma gondii and consider whether similar systems could exist in Plasmodium. Next, we review morphological and functional changes in P. falciparum's mitochondrion during the asexual-to-sexual stage transition and discuss how these changes overlap with the parasite's access to O2. We then delve into reactive oxygen species (ROS) as ROS production is influenced by O2 availability and oxidative stress impacts Plasmodium intraerythrocytic development. Lastly, given that the primary role of the red blood cell (RBC) is to deliver O2 throughout the body, we discuss how changes in the oxygenation status of hemoglobin, the RBC's O2-carrying protein and key nutrient for Plasmodium, could also potentially impact the parasite's growth during intraerythrocytic development. This review also highlights studies that have investigated P. falciparum biology under varying O2 concentrations and covers technical aspects related to P. falciparum cultivation in the lab, focusing on sources of technical variation that could alter the amount of dissolved O2 encountered by cells during in vitro experiments. Lastly, we discuss how culture systems can better replicate in vivo heterogeneity with respect to O2 gradients, propose ideas for further research in this area, and consider translational implications related to O2 and malaria. [ABSTRACT FROM AUTHOR]

Artemisinin-based treatments (ACTs) are the first therapy currently used to treat malaria produced by Plasmodium falciparum. However, in recent years, increasing evidence shows that some strains of P. falciparum are less susceptible to ACT in the Southeast Asian region. A data reanalysis of several omics approaches currently available about parasites of P. falciparum that have some degree of resistance to ACT was carried out. The data used were from transcriptomics and metabolomics studies. One mitochondrial carrier of the parasite possibly involved in the mechanisms of tolerance to oxidative stress was modeled and subjected to molecular dockings with citrate and oxoglutarate. An increase in glutathione production was detected, changing the direction of the flux of metabolites in the tricarboxylic acid cycle and boosting the glucose consumed. The models of the mitochondrial carrier, called PfCOCP, show that it may be important in transporting citrate and oxoglutarate from the mitochondrial matrix to the cytosol. If so, it may allow the parasite to tolerate the oxidative stress produced by artemisinin. This insilico analysis shows that P. falciparum may tolerate artemisinin's oxidative stress through metabolic changes not reported before, showing the need for further experimental research on the many metabolic aspects linked to this phenotype. [ABSTRACT FROM AUTHOR]

Background: Artemether–lumefantrine (AL) has been the primary anti-malarial drug used to treat uncomplicated Plasmodium falciparum malaria in Ethiopia since 2004. However, there have been recent reports of AL resistance mutations in different African countries, including Ethiopia. This is concerning and requires periodic monitoring of anti-malarial drug resistance. Therefore, the current study aimed to evaluate the therapeutic efficacy of AL in treating uncomplicated P. falciparum malaria in the Arba Minch Zuria District, Gamo Zone, Southwest Ethiopia. Methods: A single-arm prospective study with a 28-day follow-up period was conducted from July to October 2022. Capillary blood samples were collected for RDT and microscopic examination. The study enrolled monoinfected P. falciparum patients aged ≥ 18 years at Ganta Sira Health Post. Sociodemographic and clinical data were recorded, and a dried blood spot (DBS) was prepared for each participant. Nested polymerase chain reaction (nPCR) genotyping of the msp-1 and msp-2 genes was only performed for recurrent cases to distinguish between recurrence and reinfection. Data entry and analysis were performed using the WHO Excel spreadsheet and SPSS version 26. Results: A total of 89 patients were enrolled, and 67 adequately completed the 28-day follow-up period. AL showed a 100% clearance rate for fever on day 2 and asexual parasites on day 3. Gametocytes were detected in 13.5% (12/89) of the participants. The gametocyte clearance rate was 58.3% (7/12) until day 7 and 100% (12/12) until day 14. Five participants developed recurrent malaria, three of whom experienced relapse and two of whom experienced reinfection. Based on the Kaplan–Meier survival analysis, the PCR-uncorrected and PCR-corrected cumulative incidence of success were 93.7% (95% CI 85.5–97.3) and 96.2% (95% CI 85.5–98.7), respectively. Conclusion: AL was efficacious in treating uncomplicated P. falciparum malaria in the study area. However, the detection of recurrent patients highlights the need for continuous efficacy studies in this area. [ABSTRACT FROM AUTHOR]