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The spread of malarial parasites resistant to first-line treatments such as artemisinin combination therapies is a global health concern. Differentiation-inducing factor 1 (DIF-1) is a chlorinated alkylphenone (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl) hexan-1-one) originally found in the cellular slime mould Dictyostelium discoideum. We previously showed that some derivatives of DIF-1, particularly DIF-1(+2) (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl) octan-1-one), exert potent antimalarial activities. In this study, we synthesised DIF-1(+3) (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl) nonan-1-one). We then evaluated the effects of DIF-1(+3) in vitro on Plasmodium falciparum and in vivo over 7 days (50-100 mg/kg/day) in a mouse model of Plasmodium berghei. DIF-1(+3) exhibited a half-maximal inhibitory concentration of approximately 20-30 % of DIF-1(+2) in three laboratory strains with a selectivity index > 263, including in strains resistant to chloroquine and artemisinin. Parasite growth and multiplication were almost completely suppressed by treatment with 100 mg/kg DIF-1(+3). The survival time of infected mice was significantly increased (P = 0.006) with no apparent adverse effects. In summary, addition of an acyl group to DIF-1(+2) to prepare DIF-1(+3) substantially enhanced antimalarial activity, even in drug-resistant malaria, indicating the potential of applying DIF-1(+3) for malaria treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Objectives: Artemisinin-resistant Plasmodium falciparum malaria is currently spreading globally, including in Africa. Artemisinin resistance also leads to resistance to partner drugs used in artemisinin-based combination therapies. Sequencing of kelch13, which is associated with artemisinin resistance, culture-based partner drug susceptibility tests, and ELISA-based growth measurement are conventionally used to monitor resistance; however, their application is challenging in resource-limited settings., Methods: An experimental package for field studies with minimum human/material requirements was developed., Results: First, qPCR-based SNP assay was applied in artemisinin resistance screening, which can detect mutations within 1 h and facilitate sample selection for subsequent processes. It had 100% sensitivity and specificity compared with DNA sequencing in the detection of the two common artemisinin resistance mutations in Uganda, C469Y and A675V. Moreover, in the partner drug susceptibility test, the cultured samples were dry-preserved on a 96-well filter paper plate and shipped to the central laboratory. Parasite growth was measured by ELISA using redissolved samples. It well reproduced the results of direct ELISA, reducing significant workload in the field (Pearson correlation coefficient: 0.984; 95% CI: 0.975-0.990)., Conclusions: Large-scale and sustainable monitoring is required urgently to track rapidly spreading drug-resistant malaria. In malaria-endemic areas, where research resources are often limited, simplicity and feasibility of the procedure is especially important. Our approach combines a qPCR-based rapid test, which is also applicable to point-of-care diagnosis of artemisinin resistance and centralized analysis of ex vivo culture. The approach could improve efficiency of field experiments and accelerate global drug resistance surveillance., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Malaria, which is caused by protozoa of the genus Plasmodium, remains a major endemic public health problem worldwide. Since artemisinin combination therapies are used as a first-line treatment in all endemic regions, the emergence of parasites resistant to these regimens has become a serious problem. Differentiation-inducing factor 1 (DIF-1) is a chlorinated alkylphenone originally found in the cellular slime mold Dictyostelium discoideum. DIF-1 and its derivatives exhibit a range of biological activities. In the present study, we investigated the effects of 41 DIF derivatives on the growth of Plasmodium falciparum in vitro using four laboratory strains and 12 field isolates. Micromolar concentrations of several DIF derivatives strongly suppressed the growth of the four laboratory strains, including strains that exhibited resistance to chloroquine and artemisinin, as well as strains that were susceptible to these drugs. In addition, DIF-1(+2), the most potent derivative, strongly suppressed the growth of 12 field isolates. We also examined the effects of DIF-1(+2) on the activity of the rodent malarial parasite Plasmodium berghei in mice. Intraperitoneal administration of DIF-1(+2) over 4 days (50 or 70 mg/kg/day) significantly suppressed the growth of the parasite in the blood with no apparent adverse effects, and a dose of 70 mg/kg/day significantly prolonged animal survival. These results suggest that DIF derivatives, such as DIF-1(+2), could serve as new lead compounds for the development of antimalarial agents., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In Uganda, artemether-lumefantrine was introduced as an artemisinin-based combination therapy (ACT) for malaria in 2006. We have previously reported a moderate decrease in ex vivo efficacy of lumefantrine in Northern Uganda, where we also detected ex vivo artemisinin-resistant Plasmodium falciparum. Therefore, it is necessary to search for candidate partner alternatives for ACT. Here, we investigated ex vivo susceptibility to four ACT partner drugs as well as quinine and chloroquine, in 321 cases between 2013 and 2018. Drug-resistant mutations in pfcrt and pfmdr1 were also determined. Ex vivo susceptibility to amodiaquine, quinine, and chloroquine was well preserved, whereas resistance to mefloquine was found in 45.8%. There were few cases of multi-drug resistance. Reduced sensitivity to mefloquine and lumefantrine was significantly associated with the pfcrt K76 wild-type allele, in contrast to the association between chloroquine resistance and the K76T allele. Pfmdr1 duplication was not detected in any of the cases. Amodiaquine, a widely used partner drug for ACT in African countries, may be the first promising alternative in case lumefantrine resistance emerges. Therapeutic use of mefloquine may not be recommended in this area. This study also emphasizes the need for sustained monitoring of antimalarial susceptibility in Northern Uganda to develop proper treatment strategies., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

The rapid and aggressive spread of artemisinin-resistant Plasmodium falciparum carrying the C580Y mutation in the kelch13 gene is a growing threat to malaria elimination in Southeast Asia, but there is no evidence of their spread to other regions. We conducted cross-sectional surveys in 2016 and 2017 at two clinics in Wewak, Papua New Guinea (PNG) where we identified three infections caused by C580Y mutants among 239 genotyped clinical samples. One of these mutants exhibited the highest survival rate (6.8%) among all parasites surveyed in ring-stage survival assays (RSA) for artemisinin. Analyses of kelch13 flanking regions, and comparisons of deep sequencing data from 389 clinical samples from PNG, Indonesian Papua and Western Cambodia, suggested an independent origin of the Wewak C580Y mutation, showing that the mutants possess several distinctive genetic features. Identity by descent (IBD) showed that multiple portions of the mutants' genomes share a common origin with parasites found in Indonesian Papua, comprising several mutations within genes previously associated with drug resistance, such as mdr1, ferredoxin, atg18 and pnp. These findings suggest that a P. falciparum lineage circulating on the island of New Guinea has gradually acquired a complex ensemble of variants, including kelch13 C580Y, which have affected the parasites' drug sensitivity. This worrying development reinforces the need for increased surveillance of the evolving parasite populations on the island, to contain the spread of resistance., Competing Interests: The authors have declared that no competing interests exist.

Background: Usage of chloroquine was discontinued from the treatment of Plasmodium falciparum infection in almost all endemic regions because of global spread of resistant parasites. Since the first report in Malawi, numerous epidemiological studies have demonstrated that the discontinuance led to re-emergence of chloroquine-susceptible P. falciparum, suggesting a possible role in future malaria control. However, most studies were cross-sectional, with few studies looking at the persistence of chloroquine recovery in long term. This study fills the gap by providing, for a period of at least 6 years, proof of persistent re-emergence/stable recovery of susceptible parasite populations using both molecular and phenotypic methods., Methods: Ex vivo drug-susceptibility assays to chloroquine (n = 319) and lumefantrine (n = 335) were performed from 2013 to 2018 in Gulu, Northern Uganda, where chloroquine had been removed from the official malaria treatment regimen since 2006. Genotyping of pfcrt and pfmdr1 was also performed., Results: Chloroquine resistance (≥ 100 nM) was observed in only 3 (1.3%) samples. Average IC 50 values for chloroquine were persistently low throughout the study period (17.4-24.9 nM). Parasites harbouring pfcrt K76 alleles showed significantly lower IC 50 s to chloroquine than the parasites harbouring K76T alleles (21.4 nM vs. 43.1 nM, p-value = 3.9 × 10 -8 ). Prevalence of K76 alleles gradually increased from 71% in 2013 to 100% in 2018., Conclusion: This study found evidence of stable persistence of chloroquine susceptibility with the fixation of pfcrt K76 in Northern Uganda after discontinuation of chloroquine in the region. Accumulation of similar evidence in other endemic areas in Uganda could open channels for possible future re-use of chloroquine as an option for malaria treatment or prevention.

Background: Chloroquine treatment for Plasmodium falciparum has been discontinued in almost all endemic regions due to the spread of resistant isolates. Reversal of chloroquine susceptibility after chloroquine discontinuation has been reported in dozens of endemic regions. However, this phenomenon has been mostly observed in Africa and is not well documented in other malaria endemic regions. To investigate this, an ex vivo study on susceptibility to chloroquine and lumefantrine was conducted during 2016-2018 in Wewak, Papua New Guinea where chloroquine had been removed from the official malaria treatment regimen in 2010. Genotyping of pfcrt and pfmdr1 was also performed., Results: In total, 368 patients were enrolled in this study. Average IC 50 values for chloroquine were 106.6, 80.5, and 87.6 nM in 2016, 2017, and 2018, respectively. These values were not significantly changed from those obtained in 2002/2003 (108 nM). The majority of parasites harboured a pfcrt K76T the mutation responsible for chloroquine resistance. However, a significant upward trend was observed in the frequency of the K76 (wild) allele from 2.3% in 2016 to 11.7% in 2018 (P = 0.008; Cochran-Armitage trend test)., Conclusions: Eight years of chloroquine withdrawal has not induced a significant recovery of susceptibility in Papua New Guinea. However, an increasing tendency of parasites harbouring chloroquine-susceptible K76 suggests a possibility of resurgence of chloroquine susceptibility in the future.

Because ≈90% of malaria cases occur in Africa, emergence of artemisinin-resistant Plasmodium falciparum in Africa poses a serious public health threat. To assess emergence of artemisinin-resistant parasites in Uganda during 2014-2016, we used the recently developed ex vivo ring-stage survival assay, which estimates ring-stage-specific P. falciparum susceptibility to artemisinin. We conducted 4 cross-sectional surveys to assess artemisinin sensitivity in Gulu, Uganda. Among 194 isolates, survival rates (ratio of viable drug-exposed parasites to drug-nonexposed controls) were high (>10%) for 4 isolates. Similar rates have been closely associated with delayed parasite clearance after drug treatment and are considered to be a proxy for the artemisinin-resistant phenotype. Of these, the PfKelch13 mutation was observed in only 1 isolate, A675V. Population genetics analysis suggested that these possibly artemisinin-resistant isolates originated in Africa. Large-scale surveillance of possibly artemisinin-resistant parasites in Africa would provide useful information about treatment outcomes and help regional malaria control.

Background: Rab5 GTPase regulates membrane trafficking between the plasma membrane and endosomes and harbours a conserved C-terminal isoprenyl modification that is necessary for membrane recruitment. Plasmodium falciparum encodes three Rab5 isotypes, and one of these, Rab5b (PfRab5b), lacks the C-terminal modification but possesses the N-terminal myristoylation motif. PfRab5b was reported to localize to the parasite periphery. However, the trafficking pathway regulated by PfRab5b is unknown., Methods: A complementation analysis of Rab5 isotypes was performed in Plasmodium berghei. A constitutively active PfRab5b mutant was expressed under the regulation of a ligand-dependent destabilization domain (DD)-tag system in P. falciparum. The localization of PfRab5b was evaluated after removing the ligand followed by selective permeabilization of the membrane with different detergents. Furthermore, P. falciparum N-terminally myristoylated adenylate kinase 2 (PfAK2) was co-expressed with PfRab5b, and trafficking of PfAK2 to the parasitophorous vacuole membrane was examined by confocal microscopy., Results: PfRab5b complemented the function of PbRab5b, however, the conventional C-terminally isoprenylated Rab5, PbRab5a or PbRab5c, did not. The constitutively active PfRab5b mutant localized to the cytosol of the parasite and the tubovesicular network (TVN), a region that extends from the parasitophorous vacuole membrane (PVM) in infected red blood cells (iRBCs). By removing the DD-ligand, parasite cytosolic PfRab5b signal disappeared and a punctate structure adjacent to the endoplasmic reticulum (ER) and parasite periphery accumulated. The peripheral PfRab5b was sensitive to extracellular proteolysis after treatment with streptolysin O, which selectively permeabilizes the red blood cell plasma membrane, indicating that PfRab5b localized on the iRBC cytoplasmic face of the TVN. Transport of PfAK2 to the PVM was abrogated by overexpression of PfRab5b, and PfAK2 accumulated in the punctate structure together with PfRab5b., Conclusion: N-myristoylated Plasmodium Rab5b plays a role that is distinct from that of conventional mammalian Rab5 isotypes. PfRab5b localizes to a compartment close to the ER, translocated to the lumen of the organelle, and co-localizes with PfAK2. PfRab5b and PfAK2 are then transported to the TVN, and PfRab5b localizes on the iRBC cytoplasmic face of TVN. These data demonstrate that PfRab5b is transported from the parasite cytosol to TVN together with N-myristoylated PfAK2 via an uncharacterized membrane-trafficking pathway.

Although artemisinin combination therapies have been deployed as a first-line treatment for uncomplicated malaria in almost all endemic countries, artemisinin-resistant parasites have emerged and have gradually spread across the Greater Mekong subregions. There is growing concern that the resistant parasites may migrate to or emerge indigenously in sub-Saharan Africa, which might provoke a global increase in malaria-associated morbidity and mortality. Therefore, development of molecular markers that enable identification of artemisinin resistance with high sensitivity is urgently required to combat this issue. In 2014, a potential artemisinin-resistance responsible gene, Plasmodium falciparum kelch13, was discovered. Here, we review the genetic features of P. falciparum kelch13 and discuss its related resistant mechanisms and potential as a molecular marker.

Atovaquone, a coenzyme Q analogue has been indicated to specifically target the cytochrome bc1 complex of the mitochondrial respiratory chain in the malarial parasite and other protozoan. Various mutations in the quinone binding site of the cytochrome b gene of Plasmodium spp. such as M133I, L144S, L271V, K272R, Y268C, Y268S, Y268N, and V284F are suggesting to associate with resistance to atovaquone. There is no direct evidence of relation between the mutations and resistance to atovaquone in Plasmodium parasite that has been available. Technical difficulties in isolating active assayable mitochondria in the malarial parasite hinder us to obtain direct biochemical evidence to support the relation between the mutations and drug resistance. The establishment of a mitochondrial isolation method for the malaria parasite has allowed us to test the degree of resistance of Plasmodium berghei isolates to atovaquone directly. We have tested the activity of dihydroorotate (DHO)-cytochrome c reductase in various P. berghei atovaquone resistant clones in the presence of a wide concentration range of atovaquone. Our results show the IC(50) of P. berghei atovaquone resistant clones is much higher (1.5 up to 40 nM) in comparison to the atovaquone sensitive clones (0.132-0.465 nM). The highest IC(50) was revealed in clones carrying Y268C and Y268N mutations (which play an important role in atovaquone resistance in Plasmodium falciparum), with an approximately 100-fold increase. The findings indicate the importance of the mutation in the quinone binding site of the cytochrome b gene and that provide a direct evidence for the atovaquone inhibitory mechanism in the cytochrome bc1 complex of the parasite., (Copyright © 2014. Published by Elsevier Ireland Ltd.)

The intraerythrocytic form of the human malaria parasite Plasmodium falciparum relies on glycolysis for its energy requirements. In glycolysis, lactate is an end product. It is therefore known that lactate accumulates in in vitro culture; however, its influence on parasite growth remains unknown. Here we investigated the effect of lactate on the development of P. falciparum during in vitro culture under lactate supplementation in detail. Results revealed that lactate retarded parasite development and reduced the number of merozoites in the schizont stage. These findings suggest that lactate has the potential to affect parasite development., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Malaria parasites in human hosts depend on glycolysis for most of their energy production, and the mitochondrion of the intraerythrocytic form is acristate. Although the genes for all tricarboxylic acid (TCA) cycle members are found in the parasite genome, the presence of a functional TCA cycle in the intraerythrocytic stage is still controversial. To elucidate the physiological role of Plasmodium falciparum mitochondrial complex II (succinate-ubiquinone reductase (SQR) or succinate dehydrogenase (SDH)) in the TCA cycle, the gene for the flavoprotein subunit (Fp) of the enzyme, pfsdha (P.falciparum gene for SDH subunit A, PlasmoDB ID: PF3D7_1034400) was disrupted. SDH is a well-known marker enzyme for mitochondria. In the pfsdha disruptants, Fp mRNA and polypeptides were decreased, and neither SQR nor SDH activity of complex II was detected. The suppression of complex II caused growth retardation of the intraerythrocytic forms, suggesting that complex II contributes to intraerythrocytic parasite growth, although it is not essential for survival. The growth retardation in the pfsdha disruptant was rescued by the addition of succinate, but not by fumarate. This indicates that complex II functions as a quinol-fumarate reductase (QFR) to form succinate from fumarate in the intraerythrocytic parasite., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Cerebral malaria (CM) is the most severe complication of Plasmodium infection. Although inappropriate immune responses to Plasmodium falciparum are reported as the major causes of CM, the precise mechanisms for development remain unclear. IL-23 and IL-17 have critical roles in the onset of autoimmunity and inflammatory diseases triggered by microbial infections. Thus, we investigated the influence of IL-23 and IL-17 on experimental CM (ECM) using Plasmodium berghei ANKA infection of C57BL/6 mice. Both IL-23 deficient mice and wild-type (WT) mice developed ECM. IL-17 deficient mice also developed ECM, while IL-17 producing cells other than CD4(+) T cells (Th17) were increased in WT mice that developed ECM. In conclusion, this study showed that IL-23 and IL-17 are not involved in ECM development., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The nuclear genome of the human malaria parasite Plasmodium falciparum encodes a homolog of the bacterial HU protein (PfHU). In this study, we characterised PfHU's physiological function. PfHU, which is targeted exclusively to the parasite's plastid, bound its natural target--the plastid DNA--sequence-independently and complemented lack of HU in Escherichia coli. The HU gene could not be knocked-out from the genome of Plasmodium berghei, implying that HU is important for the parasite's survival. As the human cell lacks the HU homolog, PfHU is a potential target for drugs to control malaria.

Recent studies show that some human malaria parasite species Plasmodium falciparum and P.vivax parasitize erythroblasts; however, the biological and clinical significance of this is unclear. To investigate further, we generated a rodent malaria parasite (P. yoelii 17XNL) expressing GFP-ovalbumin (OVA). Its infectivity to erythroblasts was confirmed and parasitized erythroblasts were capable of initiating malaria infections. Experiments showed that MHC class I molecules were highly expressed on parasitized erythroblasts. As CD8+ T cells recognize MHC class I and peptide complexes on target cells and are involved in protection or pathology against malaria, we examined whether erythroblasts are targeted by CD8+ T cells. Purified non-parasitized erythroblasts pulsed with OVA peptides were recognized by OVA-specific CD8+ T cells. Crucially, parasitized erythroblasts isolated from GFP-OVA-, but not GFP- infected-mice, activated OT-I CD8+ T cells, indicating that CD8+ T cells recognize parasitized erythroblasts in an antigen-specific manner. [ABSTRACT FROM AUTHOR]

• PfDHPS-A437G is known as one of sulfadoxine-resistant mutations in P. falciparum. • PbDHPS-A394G in P. berghei corresponds to PfDHPS-A437G. • We generated a transgenic PbDHPS-A394G parasite and investigated the developments. • PbDHPS-A394G parasite grew in mice and produced oocysts with similar efficiency to wild-type. • The PbDHPS-A394G mutation may not impose fitness costs on the parasite during the examined stages. Genetic changes conferring drug resistance are generally believed to impose fitness costs to pathogens in the absence of the drug. However, the fitness of resistant parasites against sulfadoxine/pyrimethamine has been inconclusive in Plasmodium falciparum. This is because resistance is conferred by the complex combination of mutations in dihydropteroate synthase (dhps) and dihydrofolate reductase (dhfr), which makes it difficult to separately assess the extent and magnitude of the costs imposed by mutations in dhps and dhfr. To assess the fitness costs imposed by sulfadoxine resistance alone, we generated a transgenic rodent malaria parasite, P. berghei clone harboring an A394G mutation in dhps (PbDHPS-A394G), corresponding to the causative mutation for sulfadoxine resistance in P. falciparum (PfDHPS-A437G). A four-day suppressive test confirmed that the PbDHPS-A394G clone was resistant to sulfadoxine. PbDHPS-A394G and wild-type clones showed similar growth rates and gametocyte production. This observation was confirmed in competitive experiments in which PbDHPS-A394G and wild-type clones were co-infected into mice to directly assess the survival competition between them. In the mosquitoes, there were no significant differences in oocyst production between PbDHPS-A394G and wild-type. These results indicate that the PbDHPS-A394G mutation alters the parasites to sulfadoxine resistance but may not impose fitness disadvantages during the blood stages in mice and oocyst formation in mosquitoes. These results partly explain the persistence of the PfDHPS-A437G mutant in the natural parasite populations. [Display omitted] [ABSTRACT FROM AUTHOR]

The serine repeat antigen gene family of Plasmodium falciparum (Pf-SERA) consists of nine gene members. By sequence similarity search, 45 genes were identified to be homologous to the Pf-SERA genes in the ongoing seven Plasmodium genome sequencing project databases for the species: P. reichenowi, P. vivax, P. knowlesi, P. yoelii, P. berghei, P. chabaudi, and P. gallinaceum. In combination with additional PCR-based sequencing, we found that almost all SERA genes in each species were aligned in a tandem cluster and sandwiched between two conserved hypothetical protein genes, except for P. reichenowi, which could not be confirmed. The minimum and maximum numbers of clustered genes were 2 and 12 for P. gallinaceum and P. vivax, respectively. The best tree of the maximum likelihood analysis demonstrated that all Plasmodium SERA homologues, except for SERA1 of P. gallinaceum (Pg-SERA1), can be classified into four groups, represented by Pf-SERA5, Pf-SERA6, Pf-SERA7, and Pf-SERA8. Genes in the Pf-SERA8 group, although highly divergent and distantly related to the sequences of other groups, were not pseudogenes. P. berghei SERA5, the counterpart of Pf-SERA8, was expressed in the mosquito stage. P. gallinaceum lacks the orthologues to Pf-SERA5, Pf-SERA6, and Pf-SERA7, suggesting that P. gallinaceum diverged from a common ancestor of all eight Plasmodium species examined before gene duplication(s) occurred to generate these paralogous groups. Here, we reveal an evolutionary trail of SERA gene cluster in the genus Plasmodium and discuss a phylogeny of Plasmodium species from the viewpoint of the evolution of a multigene family. [ABSTRACT FROM AUTHOR]

Gastrointestinal symptoms, such as abdominal pain and diarrhea, are frequently observed in patients with Plasmodium falciparum malaria. However, the correlation between malaria intestinal pathology and intestinal microbiota has not been investigated. In the present study, infection of C57BL/6 mice with P. berghei ANKA (PbA) caused intestinal pathological changes, such as detachment of epithelia in the small intestines and increased intestinal permeability, which correlated with development with experimental cerebral malaria (ECM). Notably, an apparent dysbiosis occurred, characterized by a reduction of Firmicutes and an increase in Proteobacteria. Furthermore, some genera of microbiota correlated with parasite growth and/or ECM development. By contrast, BALB/c mice are resistant to ECM and exhibit milder intestinal pathology and dysbiosis. These results indicate that the severity of cerebral and intestinal pathology coincides with the degree of alteration in microbiota. This is the first report demonstrating that malaria affects intestinal microbiota and causes dysbiosis. [ABSTRACT FROM AUTHOR]