Falciparum Research Articles

Slow clearance of histidine-rich protein-2 in Gabonese with uncomplicated malaria.

Detecting Plasmodium falciparum, the parasite responsible for the severest form of malaria, typically involves microscopy, polymerase chain reaction (PCR), or rapid diagnostic tests (RDTs) targeting the histidine-rich protein 2 or 3 (HRP2/3). While microscopy and PCR quickly turn negative after the infection is cleared, HRP2 remains detectable for a prolonged period. The exact duration of HRP2 persistence had not been well defined. Our study in Gabon tracked HRP2 levels over 4 weeks, resulting in a new model for antigen clearance. We discovered that a two-compartment model accurately predicts HRP2 levels, revealing an initial rapid reduction followed by a much slower elimination phase that can take several weeks. These findings are crucial for interpreting RDT results, as lingering HRP2 can lead to false positives, impacting malaria diagnosis and treatment decisions.

Exploring the Antibacterial and Antiparasitic Activity of Phenylaminonaphthoquinones-Green Synthesis, Biological Evaluation and Computational Study.

Organic compounds with antibacterial and antiparasitic properties are gaining significance for biomedical applications. This study focuses on the solvent-free synthesis (green synthesis) of 1,4-naphthoquinone or 2,3-dichloro-1,4-naphthoquinone with different phenylamines using silica gel as an acid solid support. The study also includes in silico PASS predictions and the discovery of antibacterial and antiparasitic properties of phenylaminonaphthoquinone derivatives 1-12, which can be further applied in drug discovery and development. These activities were discussed in terms of molecular descriptors such as hydrophobicity, molar refractivity, and half-wave potentials. The in vitro antimicrobial potential of the synthesized compounds 1-12 was evaluated against a panel of six bacterial strains (three Gram-positive: Staphylococcus aureus, Proteus mirabilis, and Enterococcus faecalis; and three Gram-negative bacteria: Escherichia coli, Salmonella typhimurium, and Klebsiella pneumoniae). Six compounds (1, 3, 5, 7, 10, and 11) showed better activity toward S. aureus with MIC values between 3.2 and 5.7 μg/mL compared to cefazolin (MIC = 4.2 μg/mL) and cefotaxime (MIC = 8.9 μg/mL), two cephalosporin antibiotics. Regarding in vitro antiplasmodial activity, compounds 1 and 3 were the most active against the Plasmodium falciparum strain 3D7 (chloroquine-sensitive), displaying IC50 values of 0.16 and 0.0049 μg/mL, respectively, compared to chloroquine (0.33 μg/mL). In strain FCR-3 (chloroquine-resistant), most of the compounds showed good activity, with compounds 3 (0.12 μg/mL) and 11 (0.55 μg/mL) being particularly noteworthy. Additionally, docking studies were used to better rationalize the action and prediction of the binding modes of these compounds. Finally, absorption, distribution, metabolism, excretion, and toxicity (ADMET) predictions were performed.

Transcriptomic and Proteomic Insights into Host Immune Responses in Pediatric Severe Malarial Anemia: Dysregulation in HSP60-70-TLR2/4 Signaling and Altered Glutamine Metabolism.

Severe malarial anemia (SMA, Hb < 6.0 g/dL) is a leading cause of childhood morbidity and mortality in holoendemic Plasmodium falciparum transmission zones. This study explored the entire expressed human transcriptome in whole blood from 66 Kenyan children with non-SMA (Hb ≥ 6.0 g/dL, n = 41) and SMA (n = 25), focusing on host immune response networks. RNA-seq analysis revealed 6862 differentially expressed genes, with equally distributed up-and down-regulated genes, indicating a complex host immune response. Deconvolution analyses uncovered leukocytic immune profiles indicative of a diminished antigenic response, reduced immune priming, and polarization toward cellular repair in SMA. Weighted gene co-expression network analysis revealed that immune-regulated processes are central molecular distinctions between non-SMA and SMA. A top dysregulated immune response signaling network in SMA was the HSP60-HSP70-TLR2/4 signaling pathway, indicating altered pathogen recognition, innate immune activation, stress responses, and antigen recognition. Validation with high-throughput gene expression from a separate cohort of Kenyan children (n = 50) with varying severities of malarial anemia (n = 38 non-SMA and n = 12 SMA) confirmed the RNA-seq findings. Proteomic analyses in 35 children with matched transcript and protein abundance (n = 19 non-SMA and n = 16 SMA) confirmed dysregulation in the HSP60-HSP70-TLR2/4 signaling pathway. Additionally, glutamine transporter and glutamine synthetase genes were differentially expressed, indicating altered glutamine metabolism in SMA. This comprehensive analysis underscores complex immune dysregulation and novel pathogenic features in SMA.

Avian malaria in a feral-pet pigeon: a case report

BackgroundAvian malaria is caused by diverse parasite species of the genus Plasmodium, and it affects various bird species. The occurrence of this disease in some wild bird species is sparsely documented due to the scarce availability of samples. Hence the pathogenicity in some hosts is not completely known. In addition, feral birds may act as reservoirs bridging the transmission cycle from wild migratory birds to domestic and zoo-kept bird species.Case presentationAn owner of pigeons adopted a feral pigeon (Columba livia forma domestica) and housed it together with his other pet-pigeons. The bird died unexpectedly a few weeks after a surgical procedure and necropsy revealed a severely anaemic carcass, with pale organs and hydropericardium. Histopathologic analysis revealed inflammatory infiltrates in the lung and liver, and monocytes and Kupffer cells contained haemozoin pigment indicative of phagocytosis of Plasmodium-infected erythrocytes. A high erythrocytic infection rate of 18% was evident in tissues and blood vessels in various organs. Furthermore, the thyroid had masses classified as thyroid carcinomas. Immunohistochemistry with anti- Plasmodium falciparum HSP70 antibody revealed positive signals in erythrocytes and intravascular leucocytes. Further microscopy analysis using a Hemacolor-stained impression smear revealed a high parasitaemia with an asynchronous infection showing all erythrocytic stages. Molecular diagnosis by PCR identified Plasmodium relictum, lineage GRW11 as the aetiological agent. The bird presented died most likely due to an acute infection as evidenced by the high blood parasitaemia, leading to major erythrocyte destruction. Further analyses of feral pigeons (n = 22) did not reveal any additional cases of Plasmodium infections.ConclusionThis study reports the first mortality associated with P. relictum lineage GRW11. The study supports previous studies, suggesting that Plasmodium infections are not frequent in pigeons. Host conditions like immunosuppression due to the tumour may have influenced the infection outcome in this fatal case. Use of anti-P. falciparum HSP70 antibody for detection of P. relictum antigens for immune assays in blood and tissue samples will be a useful tool for future studies.

Antibodies to specific domains of Plasmodium falciparum erythrocyte membrane protein-1 and its relationship with protection from severe malarial anemia: A prospective study among Ghanaian children.

Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP-1) is important in malaria pathogenicity as it mediates Pf-infected erythrocytes cytoadherence to host endothelial microvasculature receptors. Naturally acquired antibodies against specific PfEMP-1 antigens may be beneficial in clinical malaria protection. This study determined antibodies to DBLα2, CIDRα1, DBLβ12, and DBLγ6 domains of PfEMP-1 in children with P. falciparum malaria in Tamale, Ghana. Sixty P. falciparum-infected children, and 30 controls, aged 1-12 years were recruited for this case-control study from April to July 2023 in Northern Ghana. Participants with uncomplicated malaria had asexual P. falciparum in peripheral blood and Hb ≥ 5.0 g/dL, and severe malaria was diagnosed when participants had Hb < 5.0 g/dL in addition to asexual P. falciparum in peripheral blood. Blood cell indices were measured using hematology analyzer, and IgG antibodies to DBLα2, CIDRα1, DBLβ12, and DBLγ6 domains of PfEMP-1 and pro-inflammatory cytokines were detected using enzyme-linked immunosorbent assay. Data were analyzed using SPSS version 26.0. The prevalence of PfEMP-1 IgG antibodies among P. falciparum-infected children and the uninfected group was 65.0% and 6.7%, respectively. PfEMP-1 IgG antibodies were present in 83.3% of uncomplicated malaria cases, and 46.7% in severe malaria subjects. Plasma levels of PfEMP-1 IgG antibodies were elevated in participants with uncomplicated malaria compared to those with severe malaria (p < 0.001). Hemoglobin, RBC, HCT, and platelet were significantly lower among P. falciparum-infected children without PfEMP-1 IgG antibodies than among those with the antibodies. Prevalence of anemia among children with PfEMP-1 IgG antibodies and those without the antibodies were 74.4% and 100%, respectively. The high prevalence of PfEMP-1 IgG antibodies to DBLα2, CIDRα1, DBLβ12, and DBLγ6 domains observed in participants with uncomplicated malaria, and the relationship between PfEMP-1 IgG antibodies and blood cell parameters could indicate that the antibodies may be related to effective erythropoietic response in P. falciparum malaria. Immune antibodies against DBLα2, CIDRα1, DBLβ12, and DBLγ6 domains of PfEMP-1 may suppress the deteriorating effects of PfEMP-1 antigens and provide immune protection against severe malarial anemia in children.

Malaria in the Republic of Ireland; a retrospective review of the clinical epidemiology of malaria between 2016 and 2020

ObjectivesMalaria is a parasitic infection that continues to present significant morbidity and mortality figures throughout the world. In the Republic of Ireland (Ireland), malaria is an imported infection linked most frequently to travel routes from West-Africa. There is a paucity of data on the clinical epidemiology and hospital service utilisation of malaria in non-endemic setting to inform physician practice. This study aims to fill that gap, providing a clinical context to the national picture of both adult and paediatric cases. MethodsA retrospective review of data from all public Irish hospitals was performed from 2016 to 2020, inclusive. Ethical approval was granted following review by the Social Research Ethics Committee (SREC) of University College Cork. Results and ConclusionsDuring the study period, there were 337 cases of malaria presenting to Irish hospitals, 15.1% (51/337) in the paediatric age category. Presentations occurred nationwide with a predictable seasonality. The majority (77.45%; 261/337) were Plasmodium falciparum and 6% (20/337) required critical care admission. Interestingly, females had a higher likelihood of admission to critical care (OR 2.75, 95% CI 1.1 – 6.86). The COVID-19 pandemic saw a 78% reduction in malaria presentations in 2020. A total of 1,166 bed days were utilised by cases of malaria.

Characterization of two Plasmodium falciparum lipid transfer proteins of the Sec14/CRAL-TRIO family

Invasion of human red blood cells by the malaria parasite Plasmodium falciparum is followed by dramatic modifications of erythrocytes properties, including de novo formation of new membrane systems. Lipid transfer proteins from both the parasite and the host cell are most likely an important part of those membrane remodeling processes. Using bioinformatics and in silico structural analysis, we have identified five P. falciparum potential lipid transfer proteins containing cellular retinaldehyde binding – triple functional domain (CRAL-TRIO). Two of these proteins, C6KTD4, encoded by the PF3D7_0629900 gene and Q8II87, encoded by the PF3D7_1127600 gene, were studied in more detail. In vitro lipid transfer assays using recombinant C6KTD4 and Q8II87 confirmed that these proteins are indeed bona fide lipid transfer proteins. C6KTD4 transfers sterols, phosphatidylinositol 4,5 bisphosphate, and, to some degree, also phosphatidylcholine between two membrane compartments. Q8II87 possesses phosphatidylserine transfer activity in vitro. In the yeast model, the expression of P. falciparumQ8II87 protein partially complements the absence of Sec14p and its closest homologue, Sfh1p. C6KTD4 protein can substitute for the collective essential function of oxysterol-binding related proteins. According to published whole genome studies in P. falciparum, absence of C6KTD4 and Q8II87 proteins has severe consequences for parasite viability. Therefore, CRAL-TRIO lipid transfer proteins of P. falciparum are potential targets of novel antimalarials, in search for which the yeast model expressing these proteins could be a valuable tool.

Novel schiff base as potent antimalarial: Synthesis, single crystal x-ray structure, spectroscopic analysis, theoretical investigation and molecular docking studies

The title compound N’-[(E)-(2-Hydroxy-1-naphthyl)methylene]benzohydrazide was compounded completely by the condensation reaction between 2-hydroxynaphthaldehyde and benzhydrazide. The Schiff base obtained was attributed by single-crystal X-ray diffraction (SCXRD), FT-IR, UV–Visible spectroscopy, and mass spectrometry. Computational validation was performed using DFT- B3LYP/6–311 + G(d,p) theory to ascertain the optimal geometry of the compound. Thermodynamic parameters and nonlinear optical (NLO) plots were also investigated. The HOMO-LUMO energy gap was found to be 3.6380 eV. Mulliken charges were found and electrostatic field surface mapping conducted to explore various properties. Theoretical findings were consistent with experimental results. Molecular docking studies revealed that the Schiff base exhibits antimalarial activity against Plasmodium falciparum, with the LibDock score and binding energy assessed for the malaria-causing protein 4WZ3. ADMET analysis indicated favorable pharmacokinetic properties of the synthesized ligand compared to current antimalarial drugs, highlighting its potential for malaria treatment.

Virtual Screening of Flavonoids against Plasmodium vivax Duffy Binding Protein Utilizing Molecular Docking and Molecular Dynamic Simulation.

Plasmodium vivax (P. vivax) is one of the highly prevalent human malaria parasites. Due to the presence of extravascular reservoirs, P. vivax is extremely challenging to manage and eradicate. Traditionally, flavonoids have been widely used to combat various diseases. Recently, biflavonoids were discovered to be effective against Plasmodium falciparum. In this study, in silico approaches were utilized to inhibit Duffy binding protein (DBP), responsible for Plasmodium invasion into red blood cells (RBC). The interaction of flavonoid molecules with the Duffy antigen receptor for chemokines (DARC) binding site of DBP was investigated using a molecular docking approach. Furthermore, molecular dynamic simulation studies were carried out to study the stability of top-docked complexes. The results showed the effectiveness of flavonoids, such as daidzein, genistein, kaempferol, and quercetin, in the DBP binding site. These flavonoids were found to bind in the active region of DBP. Furthermore, the stability of these four ligands was maintained throughout the 50 ns simulation, maintaining stable hydrogen bond formation with the active site residues of DBP. The present study suggests that flavonoids might be good candidates and novel agents against DBP-mediated RBC invasion of P. vivax and can be further analyzed in in vitro studies.

The Inhibitory Potentials of the Root Extract and Fractions of Brenania brieyi (De Wild) (Rubiaceae) on Lactate Dehydrogenase, Phospholipase A2, and α- Amylase in Plasmodium falciparum - Infected Persons

Medicinal plants have been used as an alternative in malaria chemotherapy. This study aimed to determine the in vitro inhibitory potentials of the extract and fractions of Brenania brieyi on Lactate Dehydrogenase (LdH), Phospholipase A2 (PLA2), and α-Amylase enzyme extracted from Plasmodium falciparum. Cold maceration and Liquid-Liquid partitioning were used to obtain the extract and fractions respectively, using different solvent of varying polarity. Phytochemical analysis was done using Gas Chromatography-Flame Ionization detector (GCFID) method to identify and quantify different phytoconstituents. In vitro enzyme assay (α-Amylase, LdH and PLA2) were performed using standard procedure. The extract contained Alkaloids such as Spartein (5.05 µg/ml), Dihydrocytisine (23.12 µg/ml), Aphyllidine (5.25 µg/ml), Ribalinidine (2.65 µg/ml), Epihedrine (1.91 µg/ml), Saponins such as sapogenin (5.70 µg/ml), Tannins (16.25 µg/ml), Flavonoids such as Kaempferol (4.59 µg/ml), Catechin (5.50 µg/ml), Proanthocyanidin (9.81 µg/ml), Anthocyanin (3.22 µg/ml), Narigenin (3.62 µg/ml), Flavonones (8.73 µg/ml), Steroids (8.58 µg/ml), Polyphenols such as Reservatrol (20.68 µg/ml) and Glycosides such as cardiac glycoside (4.42 µg/ml), Cyanogenic glycosides (17.72 µg/ml) while Terpenoids are absent. The percentage inhibition of the enzyme (PLA2, LdH and α-amylase) at 100 mg/ml of extract showed the highest inhibition (37 %, 0 % and 38.33 %) when compared to controls, while ethyl acetate fraction has the highest inhibition (98 %, 34 % and 92 %) at P ˂ 0.05 at 100 mg/kg respectively. This study justifies the use of ethnomedicinal remedies (B. brieyi) in treating malaria in infected individuals. The study also suggests that the extract and fractions may act through the inhibition of plasmodial enzymes in infected persons.

TRNA regulation and amino acid usage bias reflect a coordinated metabolic adaptation in Plasmodium falciparum

tRNA regulation and amino acid usage bias reflect a coordinated metabolic adaptation in Plasmodium falciparum

Cryo-EM structures of the Plasmodium falciparum apicoplast DNA polymerase

The apicoplast DNA polymerase (apPol) from Plasmodium falciparum is essential for the parasite’s survival, making it a prime target for antimalarial therapies. Here, we present cryo-electron microscopy structures of the apPol in complex with DNA and incoming nucleotide, offering insights into its molecular mechanisms. Our structural analysis reveals that apPol contains critical residues for high-fidelity DNA synthesis, but lacks certain structural elements to confer processive DNA synthesis during replication, suggesting the presence of additional accessory factors. The enzyme exhibits large-scale conformational changes transitioning upon DNA and nucleotide binding, particularly within the fingers and thumb subdomains. These movements reveal potential allosteric sites that could serve as targets for drug design. Our findings provide a foundation for advancing the understanding of apPol’s unique functional mechanisms and potentially offering new avenues for the development of novel inhibitors and therapeutic interventions against malaria.

Frequency of chloroquine-resistant haplotype of Plasmodium falciparum (CVIET) in Ibadan, Southwest Nigeria 17 years post-chloroquine withdrawal

The replacement of chloroquine with artemisinin-based combination therapies (ACTs) for over a decade has had varying impacts on the ability of the malaria parasite to sustain its chloroquine resistance prowess in different malaria-endemic regions. We evaluated the frequency of Plasmodium falciparum chloroquine resistance transporter (PfCRT) mutations in Ibadan, Nigeria 17 years after the replacement of chloroquine with ACTs for malaria treatment. Fragments of PfCRT gene from genomic DNA of microscopically confirmed P. falciparum-infected patients were amplified and sequenced. There were 19% CVIET mutant and 81% CVMNK wild-type haplotypes on residues 72–76. A220S change were found in 16.7% of samples occurring concurrently with the CVIET haplotype, while a Q271E mutation occurred in a PfCRT wild-type isolate. The reduced prevalence of the PfCRT mutant alleles in this study compared to previous reports suggests a gradual disappearance of chloroquine-resistant malaria parasites following reduced drug pressure. It may also be a result of fitness demand on the parasites in attempts to evolve resistance against the current first-line regimen. However, evaluating the prevalence of other chloroquine resistance markers such as Plasmodium falciparum multidrug resistance 1 gene mutations in this population, and a more robust sample size will help to consolidate these findings.

Experimental and Computational Insights Into Organotellurium (IV) Complexes as Potent PfNDH2 Inhibitors: Synthesis, Characterization, and Antimalarial Evaluation

ABSTRACTThe study examines the binding affinities and pharmacological potential of synthesized metal compounds with PfNDH2 in Plasmodium falciparum. So as to identify the combatting agent for malaria, six new organotellurium (IV) complexes (8a–8f) were prepared through a condensation reaction of 3‐methyl‐2‐thiophene carboxaldehyde and p‐nitroaniline. The synthesized compounds were inclusively characterized through physical and spectral techniques signifying a distorted octahedral geometry. The reported complexes were screened for antimalarial and antioxidant activities and analyzed using micro assays and DPPH assays, respectively. Using docking interactions, the research evaluates the binding energies, hydrogen bonds, and steric interactions of various compounds with PfNDH2, aiming to identify promising candidates for antimalarial drug development. Among the compounds, 8f showed the strongest binding affinity, with the lowest binding energy of −135.626 kcal/mol, and diverse hydrogen bonds with the target enzyme. Structure‐based pharmacophore modeling revealed critical interaction profiles and selectivity scores for the compounds, with 8f emerging as a promising candidate. Molecular electrostatic potential (MESP) analysis offered insights into the distribution of electron density within the compounds, influencing their reactivity and potential as antimalarial agents. ADMET analysis highlighted the pharmacokinetic and toxicological profiles of the compounds, revealing critical aspects for optimizing safety and efficacy. Overall, the findings provide valuable information for further development and optimization of potential antimalarial drugs.

Profile of molecular markers of Sulfadoxine-Pyrimethamine-resistant Plasmodium falciparum in individuals living in southern area of Brazzaville, Republic of Congo

BackgroundAlthough the seasonal and perennial malaria chemopreventions are not implemented in the Republic of Congo, resistance to Sulfadoxine-pyrimethamine (SP) threatens the intermittent preventive treatment during pregnancy (IPTp-SP) and others treatments using the drug. The objective of this study was to determine the prevalence of molecular markers of P.falciparum resistance to SP in individuals with microscopic malaria infection in the south of Brazzaville. MethodsTwo parallel surveys (health facilities and community-based cross sectional studies) were carried out in urban and rural areas in southern Brazzaville. Between March and October 2021, blood samples were collected from 328 P. falciparum microscopic positive individuals (1- 83 years old, and sex ratio female/male of 1.1) to characterize dhfr and dhps genes involved in the P.falciparum resistance to SP. Restriction Fragment Length Polymorphism PCR was used for the detection of mutations within these parasite genes. ResultsHigh prevalence of mutations was reported within Pfdhfr gene: N51I; 328/328 (100%) ratio (prevalence) [95 CI uncertainty], C59R; 317/328 (96.6 %) [94.1-98.1%], S108N; 326/326 (100%), N164L; 3/326 (0.9%) [0.3-2.7%], and Pfdhps gene: A437G; 292/327 (89.3%) [85.5-92.2%], K540E; 140/327(42.8 %) [37.6-48.2%], A581G; 136/325 (41.8%) [36.6-42.3%]. The quintuple mutant (N51I+ C59R + S108N + A437G + K540E) and sextuple mutant haplotypes (N51I+ C59R + S108N + A437G + K540E + A581G) were reported for 11/144 (7.6%) [4.3-13.2%] and 5/144 (3.4% ) [1.5-7.9%]) of the participants respectively. The K540E and A437G mutants were more prevalent in the rural community; 81/139 (58.3%) [50.0-66.1%] and 135/139 (97.1%) [92.8-98.9%] respectively) than in the urban community; 21/50 (46.3%) [33.7-59.4%] and 47/54(87.0%) [75.6-93.6%] (p= 0.004 and p˂0.0001 respectively) ConclusionThese results indicate high prevalence of SP resistance mutations within the dhfr and dhps genes of P. falciparum isolates circulating in study sites, which may limit the efficacy of treatments using SP in these settings.

A New Isoflavone Xylopyranoside and Isoflavone Derivatives from the Roots of Ochna integerrima and Their DPPH Radical Scavenging Activity.

O chna integerrima (Louri) Merr. is one of the two species of the genus Ochna (family Ochnaceae) found in Thailand. Its bark is used in traditional Thai medicine to treat digestive disorders. Phytochemical investigation of the crude MeOH extract of the root woods of O. integerrima furnished an unreported isoflavone glycoside, gerontoisoflavone A-4'-O-β-D-xylopyranoside (1), together with the previously described irisolone methyl ether (2), iriskumaonin methyl ether (3), iriskumaonin (4), gerontoisoflavone A (5), isoprunetin (6), a flavone glycoside, vitexin (7), and a chromone derivative, lophilone A (8). The structure of 1 was elucidated by 1D and 2D NMR spectral analysis as well as HRMS data. Compounds 1-7 were evaluated for their 2,2-diphenyl-1-picrylhydrazil radical(DPPH●) scavenging activity and antiplasmodial activity against a chloroquine- and pyrimethamine-resistant strain of Plasmodium falciparum (K1). Compounds 5 exhibited strongest scavenging activity, with a scavenging concentration at 50% (SC50) of 137.7 mM, while 4 and 6 displayed weak scavenging activity, with SC50 values of 4 and 5 times higher than that of 5. None of the tested compounds showed antiplasmodial activity against P. falciparum (K1) at a concentration of 5 mg/mL.

Identification of the drug/metabolite transporter 1 as a marker of quinine resistance in a NF54×Cam3.II P. falciparum genetic cross.

The genetic basis of Plasmodium falciparum resistance to quinine (QN), a drug used to treat severe malaria, has long been enigmatic. To gain further insight, we used FRG-NOD human liver-chimeric mice to conduct a P. falciparum genetic cross between QN-sensitive and QN-resistant parasites, which also differ in their susceptibility to chloroquine (CQ). By applying different selective conditions to progeny pools prior to cloning, we recovered 120 unique recombinant progeny. These progeny were subjected to drug profiling and QTL analyses with QN, CQ, and monodesethyl-CQ (md-CQ, the active metabolite of CQ), which revealed predominant peaks on chromosomes 7 and 12, consistent with a multifactorial mechanism of resistance. A shared chromosome 12 region mapped to resistance to all three antimalarials and was preferentially co-inherited with pfcrt. We identified an ATP-dependent zinc metalloprotease (FtsH1) as one of the top candidates and observed using CRISPR/Cas9 SNP-edited lines that ftsh1 is a potential mediator of QN resistance and a modulator of md-CQ resistance. As expected, CQ and md-CQ resistance mapped to a chromosome 7 region harboring pfcrt. However, for QN, high-grade resistance mapped to a chromosome 7 peak centered 295kb downstream of pfcrt. We identified the drug/metabolite transporter 1 (DMT1) as the top candidate due to its structural similarity to PfCRT and proximity to the peak. Deleting DMT1 in QN-resistant Cam3.II parasites significantly sensitized the parasite to QN but not to the other drugs tested, suggesting that DMT1 mediates QN response specifically. We localized DMT1 to structures associated with vesicular trafficking, as well as the parasitophorous vacuolar membrane, lipid bodies, and the digestive vacuole. We also observed that mutant DMT1 transports more QN than the wild-type isoform in vitro. Our study demonstrates that DMT1 is a novel marker of QN resistance and a new chromosome 12 locus associates with CQ and QN response, with ftsh1 is a potential candidate, suggesting these genes should be genotyped in surveillance and clinical settings.

A promising class of Antiprotozoal agents, design and synthesis of novel Pyrimidine–Cinnamoyl Hybrids

Malaria, caused by parasitic protozoans of the Plasmodium genus, continues to be one of the greatest global health crises, especially in Africa. The emergence of antimalarial drug resistance continues to be a health problem necessitating an urgent need for alternative and cost-effective antimalarials. Using a molecular hybridization approach, we report the design and synthesis of an efficacious novel class of antiprotozoal agents; (E)-1-(4-(4,6-diphenylpyrimidin-2-yl)piperazin-1-yl)-3-phenyl prop-2-en-1-one derivatives (8a-r). The in vitro inhibitory activity of the synthesized compounds was evaluated against the NF54 chloroquine-sensitive strain of Plasmodium falciparum. From the antiprotozoal screening, three compounds displayed propitious activity with IC50 values (0.18-0.21μM), using quinine and chloroquine as standard antimalarials. Compounds 8o and 8l emerged as the most potent candidates with IC50 values of 0.18 ± 0.02 μM and 0.21 ± 0.001 μM with an associated good safety index of 18.59 and 16.75 to human kidney epithelial (HEK293) cells, respectively. The synthesized analogues present a new chemical architecture structurally unrelated to the current regime of antimalarial drugs, representing a valid strategy to combat resistance in P. falciparum species to current commercial drugs. We further investigated the binding affinities of the compounds against recombinant forms of two P. falciparum heat shock protein 70 homologues; PfHsp70-1 and PfHsp70-z, both of which are essential and promising druggable candidates. Compound 8l exhibited the highest binding affinity for both PfHsp70s. Furthermore, molecular docking revealed that compounds 8k, 8l, 8m, and 8o exhibited better fitness to PfHsp70-1, with compounds 8l and 8o showing the highest binding affinity of 10.5 kcal/mol and 10.1 kcal/mol, respectively. Therefore, it can be speculated that PfHsp70-1 may be a possible target of some of the inhibitors tested in this study. The presence of electron-donating groups on the phenyl ring of 4,6-pyrimidine moiety and cinnamoyl group demonstrated a positive correlation between the observed computational data and the biological activity. Taken together, this paper demonstrates the importance of using the molecular hybridization approach in the development of newer cinnamoyl clubbed with 4,6-diphenyl pyrimidine hybrids as potential antiprotozoal agents.

“Novel chemo-enzymatic synthesis, structural elucidation and first antiprotozoal activity profiling of the atropoisomeric dimers of trans-8-Hydroxycalamenene”

Leishmaniasis and malaria are two debilitating protozoan diseases affecting millions globally, particularly in tropical and subtropical regions. Current therapeutic options face significant challenges due to emerging drug-resistant strains, necessitating the discovery of novel antiprotozoal agents. This study explores, for the first time, the antiprotozoal potential of calamenenes and their dimers, naturally occurring sesquiterpenes found in essential oils, through a novel chemo-enzymatic synthesis approach. Using the laccase from Trametes versicolor, atropoisomeric dimers of (−)- and (+)-8-trans-hydroxycalamenene were synthesized from commercially available (−)- and (+)-menthol. Structural elucidation was achieved via 2D-NMR spectroscopy, electronic circular dichroism, and DFT calculations. In vitro profiling against Leishmania spp and drug-resistant Plasmodium falciparum revealed that calamenene dimers exhibited significantly higher antiprotozoal activity compared to their monomeric counterparts, highlighting the potential of dimeric terpenoids as promising antiprotozoal agents. This work lays the foundation for developing novel antimalarial drugs based on calamenene scaffolds, encouraging further interactome studies to optimize their pharmacological properties.

Genotyping methods to distinguish Plasmodium falciparum recrudescence from new infection for the assessment of antimalarial drug efficacy: an observational, single-centre, comparison study

Distinguishing Plasmodium falciparum recrudescence from new infections is crucial for the assessment of antimalarial drug efficacy against Pfalciparum. We aimed to compare the efficacy of different genotyping methods to assess their effect on drug efficacy estimates, particularly in patients from high-transmission settings with polyclonal infections. In this head-to-head comparison study, we compared five different genotyping methods currently used: fast capillary electrophoresis (F-CE) using msp1, msp2, and glurp; high-resolution capillary electrophoresis (H-CE) using msp1, msp2, and glurp; H-CE using microsatellites; targeted amplicon deep sequencing (TADS) using single nucleotide polymorphism (SNP)-rich markers; and high-resolution melting (HRM) analysis using msp1 and msp2. We assessed their sensitivity in detecting minority clones in polyclonal infections, their reproducibility, and the genetic diversity of the markers used. Our study used four well characterised Pfalciparum laboratory strains mixed in varying ratios, and 20paired samples collected from an in-vivo clinical trial. The experiments were performed at the Swiss Tropical and Public Health Institute in Basel, Switzerland between May 5, 2020, and Aug 23,2021. H-CE using msp1 and msp2 and TADS revealed the highest sensitivity in detecting minority clones (up to ratios of 1:100 for H-CE and 50:1:1:1 for TADS in the FCB1:HB3 and 3D7:K1:HB3:FCB1 laboratory strain mixtures, respectively), highest reproducibility (intra-assay: 99% and 91% for H-CE and TADS, respectively; inter-assay: 98% and 92% for H-CE and TADS, respectively), and highest genetic diversity in the used markers (up to 36and32unique genotypes in 20paired samples for H-CE using msp2 and TADS using cpmp, respectively). Microsatellites assessed by H-CE had a lower genetic diversity compared with msp1, msp2, and glurp assessed by H-CE and the SNP-rich markers assessed by TADS, with a maximum of 13unique genotypes, and some genotypes having allelic frequencies larger than 30%.Markers used by TADS gave the most consistent results in distinguishing recrudescence from new infection across all methods (in 18of 20pairs of samples vs 15of 20pairs for H-CE). WHO currently recommends replacing glurp with microsatellites. However, in this study, the replacement of glurp with microsatellites did not change the genotyping outcome, probably due to the lower genetic diversity of microsatellites. More studies with large sample sizes are required to identify the most suitablemicrosatellites that could replace glurp. Our study indicates that TADS should be considered the gold standardfor genotyping to distinguish recrudescence from new infection, and that it should be used to validate other methods. Swiss Tropical and Public Health Institute.