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Introduction: Malaria infection during pregnancy increases the risk of low birth weight and infant mortality and should be prevented and treated. Artemisinin-based combination treatments are generally well tolerated, safe and effective; the most used being artemether-lumefantrine (AL) and dihydroartemisinin-piperaquine (DP). Pyronaridine-artesunate (PA) is a new artemisinin-based combination. The main objective of this study is to determine the efficacy and safety of PA versus AL or DP when administered to pregnant women with confirmed Plasmodium falciparum infection in the second or third trimester. The primary hypothesis is the pairwise non-inferiority of PA as compared with either AL or DP., Methods and Analysis: A phase 3, non-inferiority, randomised, open-label clinical trial to determine the safety and efficacy of AL, DP and PA in pregnant women with malaria in five sub-Saharan, malaria-endemic countries (Burkina Faso, Democratic Republic of the Congo, Mali, Mozambique and the Gambia). A total of 1875 pregnant women will be randomised to one of the treatment arms. Women will be actively monitored until Day 63 post-treatment, at delivery and 4-6 weeks after delivery, and infants' health will be checked on their first birthday. The primary endpoint is the PCR-adjusted rate of adequate clinical and parasitological response at Day 42 in the per-protocol population., Ethics and Dissemination: This protocol has been approved by the Ethics Committee for Health Research in Burkina Faso, the National Health Ethics Committee in the Democratic Republic of Congo, the Ethics Committee of the Faculty of Medicine and Odontostomatology/Faculty of Pharmacy in Mali, the Gambia Government/MRCG Joint Ethics Committee and the National Bioethics Committee for Health in Mozambique. Written informed consent will be obtained from each individual prior to her participation in the study. The results will be published in peer-reviewed open access journals and presented at (inter)national conferences and meetings., Trial Registration Number: PACTR202011812241529., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY. Published by BMJ.)

Introduction: With the rising resistance to artemisinin-based combination treatments, there is a need to hasten the discovery and development of newer antimalarial agents. Herbal medicines are key for the development of novel drugs. Currently, herbal medicine usage in communities for treatment of malaria symptoms is common as an alternative to conventional (modern) antimalarial agents. However, the efficacy and safety of most of the herbal medicines has not yet been established. Therefore, this systematic review and evidence gap map (EGM) is intended to collate and map the available evidence, identify the gaps and synthesise the efficacy of herbal antimalarial medicines used in malaria affected regions globally., Methods and Analysis: The systematic review and EGM will be done following PRISMA and Campbell Collaboration guidelines respectively. This protocol has been registered in PROSPERO. Data sources will include PubMed, MEDLINE Ovid, EMBASE, Web of Science, Google Scholar and grey literature search. Data extraction will be done in duplicate using a data extraction tool tailored in Microsoft Office excel for herbal antimalarials discovery research questions following the PICOST framework. The Risk of Bias and overall quality of evidence will be assessed using Cochrane risk of bias tool (clinical trials), QUIN tool (in vitro studies), Newcastle-Ottawa tool (observational studies) and SYRCLE's risk of bias tool for animal studies (in vivo studies). Data analysis will be done using both structured narrative and quantitative synthesis. The primary review outcomes will be clinically important efficacy and adverse drug reactions. Laboratory parameters will include Inhibitory Concentration killing 50% of parasites, IC 50; Ring Stage Assay, RSA 0-3 hou; Trophozoite Survival Assay, TSA 50. ETHICS AND DISSEMINATION: The review protocol was approved by the School of Biomedical Science Research Ethics Committee, Makerere University College of Health Sciences (SBS-2022-213)., Prospero Registration Number: CRD42022367073., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Mounting evidence demonstrates that nutritional environment can alter pathogen drug sensitivity. While the rich media used for in vitro culture contains supraphysiological nutrient concentrations, pathogens encounter a relatively restrictive environment in vivo . We assessed the effect of nutrient limitation on the protozoan parasite that causes malaria and demonstrated that short-term growth under physiologically relevant mild nutrient stress (or "metabolic priming") triggers increased tolerance of a potent antimalarial drug. We observed beneficial effects using both short-term survival assays and longer-term proliferation studies, where metabolic priming increases parasite survival to a level previously defined as resistant (>1% survival). We performed these assessments by either decreasing single nutrients that have distinct roles in metabolism or using a media formulation that simulates the human plasma environment. We determined that priming-induced tolerance was restricted to parasites that had newly invaded the host red blood cell, but the effect was not dependent on genetic background. The molecular mechanisms of this intrinsic effect mimic aspects of genetic tolerance, including translational repression and protein export. This finding suggests that regardless of the impact on survival rates, environmental stress could stimulate changes that ultimately directly contribute to drug tolerance. Because metabolic stress is likely to occur more frequently in vivo compared to the stable in vitro environment, priming-induced drug tolerance has ramifications for how in vitro results translate to in vivo studies. Improving our understanding of how pathogens adjust their metabolism to impact survival of current and future drugs is an important avenue of research to slow the evolution of resistance. IMPORTANCE There is a dire need for effective treatments against microbial pathogens. Yet, the continuing emergence of drug resistance necessitates a deeper knowledge of how pathogens respond to treatments. We have long appreciated the contribution of genetic evolution to drug resistance, but transient metabolic changes that arise in response to environmental factors are less recognized. Here, we demonstrate that short-term growth of malaria parasites in a nutrient-limiting environment triggers cellular changes that lead to better survival of drug treatment. We found that these strategies are similar to those employed by drug-tolerant parasites, which suggests that starvation "primes" parasites to survive and potentially evolve resistance. Since the environment of the human host is relatively nutrient restrictive compared to growth conditions in standard laboratory culture, this discovery highlights the important connections among nutrient levels, protective cellular pathways, and resistance evolution., Competing Interests: The authors declare no conflict of interest.

Introduction: Genomic data constitute a valuable adjunct to routine surveillance that can guide programmatic decisions to reduce the burden of infectious diseases. However, genomic capacities remain low in Africa. This study aims to operationalise a functional malaria molecular surveillance system in Mozambique for guiding malaria control and elimination., Methods and Analyses: This prospective surveillance study seeks to generate Plasmodium falciparum genetic data to (1) monitor molecular markers of drug resistance and deletions in rapid diagnostic test targets; (2) characterise transmission sources in low transmission settings and (3) quantify transmission levels and the effectiveness of antimalarial interventions. The study will take place across 19 districts in nine provinces (Maputo city, Maputo, Gaza, Inhambane, Niassa, Manica, Nampula, Zambézia and Sofala) which span a range of transmission strata, geographies and malaria intervention types. Dried blood spot samples and rapid diagnostic tests will be collected across the study districts in 2022 and 2023 through a combination of dense (all malaria clinical cases) and targeted (a selection of malaria clinical cases) sampling. Pregnant women attending their first antenatal care visit will also be included to assess their value for molecular surveillance. We will use a multiplex amplicon-based next-generation sequencing approach targeting informative single nucleotide polymorphisms, gene deletions and microhaplotypes. Genetic data will be incorporated into epidemiological and transmission models to identify the most informative relationship between genetic features, sources of malaria transmission and programmatic effectiveness of new malaria interventions. Strategic genomic information will be ultimately integrated into the national malaria information and surveillance system to improve the use of the genetic information for programmatic decision-making., Ethics and Dissemination: The protocol was reviewed and approved by the institutional (CISM) and national ethics committees of Mozambique (Comité Nacional de Bioética para Saúde) and Spain (Hospital Clinic of Barcelona). Project results will be presented to all stakeholders and published in open-access journals., Trial Registration Number: NCT05306067., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ.)

Introduction: Drivers of lower vaccine efficacy and impaired vaccine-specific immune responses in low-income versus high-income countries, and in rural compared with urban settings, are not fully elucidated. Repeated exposure to and immunomodulation by parasite infections may be important. We focus on Plasmodium falciparum malaria, aiming to determine whether there are reversible effects of malaria infection on vaccine responses., Methods and Analysis: We have designed a randomised, double-blind, placebo-controlled, parallel group trial of intermittent preventive malaria treatment versus placebo, to determine effects on vaccine response outcomes among school-going adolescents (9 to 17 years) from malaria-endemic rural areas of Jinja district (Uganda). Vaccines to be studied comprise BCG vaccine on day 'zero'; yellow fever, oral typhoid and human papilloma virus vaccines at week 4; and tetanus/diphtheria booster vaccine at week 28. Participants in the intermittent preventive malaria treatment arm will receive dihydroartemisinin/piperaquine (DP) dosed by weight, 1 month apart, prior to the first immunisation, followed by monthly treatment thereafter. We expect to enrol 640 adolescents. Primary outcomes are BCG-specific interferon-γ ELISpot responses 8 weeks after BCG immunisation and for other vaccines, antibody responses to key vaccine antigens at 4 weeks after immunisation. In secondary analyses, we will determine effects of monthly DP treatment (versus placebo) on correlates of protective immunity, on vaccine response waning, on whether there are differential effects on priming versus boosting immunisations, and on malaria infection prevalence. We will also conduct exploratory immunology assays among subsets of participants to further characterise effects of the intervention on vaccine responses., Ethics and Dissemination: Ethics approval has been obtained from relevant Ugandan and UK ethics committees. Results will be shared with Uganda Ministry of Health, relevant district councils, community leaders and study participants. Further dissemination will be done through conference proceedings and publications., Trial Registration Number: Current Controlled Trials identifier: ISRCTN62041885., Competing Interests: Competing interests: AE reports a grant from the Medical Research Council, UK (POPVAC programme funding)., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY. Published by BMJ.)

The protozoan parasite Plasmodium falciparum causes the most severe form of human malaria and is estimated to kill 400,000 people a year. The parasite infects and replicates in host red blood cells (RBCs), where it expresses an array of proteases to carry out multiple essential processes. We are investigating the function of falcilysin (FLN), a protease known to be required for parasite development in the RBC. We previously developed a piperazine-based hydroxamic acid scaffold to generate the first inhibitors of FLN, and the current study reports the optimization of the lead compound from that series. A range of substituents were tested at the N1 and N4 positions of the piperazine core, and inhibitors with significantly improved potency against purified FLN and cultured P. falciparum were identified. Computational studies were also performed to understand the mode of binding for these compounds, and predicted a binding model consistent with the biochemical data and the distinctive SAR observed at both the N1 and N4 positions., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Despite an unprecedented 2 decades of success, the combat against malaria - the mosquito-transmitted disease caused by Plasmodium parasites - is no longer progressing. Efforts toward eradication are threatened by the lack of an effective vaccine and a rise in antiparasite drug resistance. Alternative approaches are urgently needed. Repurposing of available, approved drugs with distinct modes of action are being considered as viable and immediate adjuncts to standard antimicrobial treatment. Such strategies may be well suited to the obligatory and clinically silent first phase of Plasmodium infection, where massive parasite replication occurs within hepatocytes in the liver. Here, we report that the widely used antidiabetic drug, metformin, impairs parasite liver stage development of both rodent-infecting Plasmodium berghei and human-infecting P. falciparum parasites. Prophylactic treatment with metformin curtails parasite intracellular growth in vitro. An additional effect was observed in mice with a decrease in the numbers of infected hepatocytes. Moreover, metformin provided in combination with conventional liver- or blood-acting antimalarial drugs further reduced the total burden of P. berghei infection and substantially lessened disease severity in mice. Together, our findings indicate that repurposing of metformin in a prophylactic regimen could be considered for malaria chemoprevention.

Members of the haloacid dehalogenase (HAD) family of metabolite phosphatases play an important role in regulating multiple pathways in Plasmodium falciparum central carbon metabolism. We show that the P. falciparum HAD protein, phosphoglycolate phosphatase (PGP), regulates glycolysis and pentose pathway flux in asexual blood stages via detoxifying the damaged metabolite 4-phosphoerythronate (4-PE). Disruption of the P. falciparum gene caused accumulation of two previously uncharacterized metabolites, 2-phospholactate and 4-PE. 4-PE is a putative side product of the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase, and its accumulation inhibits the pentose phosphate pathway enzyme, 6-phosphogluconate dehydrogenase (6-PGD). Inhibition of 6-PGD by 4-PE leads to an unexpected feedback response that includes increased flux into the pentose phosphate pathway as a result of partial inhibition of upper glycolysis, with concomitant increased sensitivity to antimalarials that target pathways downstream of glycolysis. These results highlight the role of metabolite detoxification in regulating central carbon metabolism and drug sensitivity of the malaria parasite. pgp gene caused accumulation of two previously uncharacterized metabolites, 2-phospholactate and 4-PE. 4-PE is a putative side product of the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase, and its accumulation inhibits the pentose phosphate pathway enzyme, 6-phosphogluconate dehydrogenase (6-PGD). Inhibition of 6-PGD by 4-PE leads to an unexpected feedback response that includes increased flux into the pentose phosphate pathway as a result of partial inhibition of upper glycolysis, with concomitant increased sensitivity to antimalarials that target pathways downstream of glycolysis. These results highlight the role of metabolite detoxification in regulating central carbon metabolism and drug sensitivity of the malaria parasite. IMPORTANCE The malaria parasite has a voracious appetite, requiring large amounts of glucose and nutrients for its rapid growth and proliferation inside human red blood cells. The host cell is resource rich, but this is a double-edged sword; nutrient excess can lead to undesirable metabolic reactions and harmful by-products. Here, we demonstrate that the parasite possesses a metabolite repair enzyme (PGP) that suppresses harmful metabolic by-products (via substrate dephosphorylation) and allows the parasite to maintain central carbon metabolism. Loss of PGP leads to the accumulation of two damaged metabolites and causes a domino effect of metabolic dysregulation. Accumulation of one damaged metabolite inhibits an essential enzyme in the pentose phosphate pathway, leading to substrate accumulation and secondary inhibition of glycolysis. This work highlights how the parasite coordinates metabolic flux by eliminating harmful metabolic by-products to ensure rapid proliferation in its resource-rich niche., (Copyright © 2019 Dumont et al.)

We investigated the effect in vitro and in vivo of doxycycline hyclate (Dx), a broad-spectrum antibiotic inhibitor of matrix metaloproteinases (MMPs), on adult Schistosoma mansoni worms and granulomatous liver inflammation in infected mice. Adult S. mansoni worms in culture treated with different concentrations of Dx (50-180 μg/mL) were studied for eight days to assess its morphology, eggs production, and mortality. Uninfected mice and those infected with S. mansoni, untreated and treated with praziquantel (Pz; 200 mg/kg) or Dx (50 mg/kg), were evaluated for 60 days. Our results indicated that Dx induced dose-dependent integumentary lesions (bubbles, tubercle collapse, spicule disappearance, peeling, and erosion), reduced mating rate and eggs-laying in adult S. mansoni worms. The effective lethal dose required to kill 50% of worms was 112.0 μg/mL Dx (DL 50 ). In mice, S. mansoni infection induced hepatomegaly, intense IL-4, IL-10, TNF-α and TGF-β production, granulomatous inflammation and hepatic glycogen depletion. The number and size of the granulomas was similar in untreated and Dx-treated animals. Untreated animals showed a predominance of productive granulomas, and intense MMP-2 and MMP-9 activities. Dx-treated mice exhibited a significant increase in IL-4 levels, tissue inflammation, proportion of involutive granulomas, and hepatic collagenogenesis, as well as attenuated MMP-2 and MMP-9 activities. Our findings indicated that Dx is toxic to adult S. mansoni worms in vitro. However, in vitro beneficial effects were not reproduced in vivo, since Dx treatment increased liver granulomatous inflammation and collagenogenesis in S. mansoni-infected mice by a process potentially associated with Dx-mediated hepatic MMP-2 and MMP-9 inhibition., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The phylum Apicomplexa contains a group of protozoa causing diseases in humans and livestock. Plasmodium spp., the causative agent of malaria, contains a mitochondrion that is very divergent from that of their hosts. The malarial mitochondrion is a clinically validated target for the antimalarial drug atovaquone, which specifically blocks the electron transfer activity of the bc 1 complex of the mitochondrial electron transport chain (mtETC). Most mtETC proteins are nuclear-encoded and imported from the cytosol, but three key protein subunits are encoded in the Plasmodium mitochondrial genome: cyt b , COXI, and COXIII. They are translated inside the mitochondrion by mitochondrial ribosomes (mitoribosomes). Here, we characterize the function of one large mitoribosomal protein in Plasmodium falciparum , PfmRPL13. We found that PfmRPL13 localizes to the parasite mitochondrion and is refractory to genetic knockout. Ablation of PfmRPL13 using a conditional knockdown system (TetR-DOZI-aptamer) caused a series of adverse events in the parasite, including mtETC deficiency, loss of mitochondrial membrane potential (Δψ m ), and death. The PfmRPL13 knockdown parasite also became hypersensitive to proguanil, a drug proposed to target an alternative process for maintaining Δψ m Surprisingly, transmission EM revealed that PfmRPL13 disruption also resulted in an unusually elongated and branched mitochondrion. The growth arrest of the knockdown parasite could be rescued with a second copy of PfmRPL13, but not by supplementation with decylubiquinone or addition of a yeast dihydroorotate dehydrogenase gene. In summary, we provide first and direct evidence that mitoribosomes are essential for malaria parasites to maintain the structural and functional integrity of the mitochondrion., (© 2018 Ke et al.)

Objective: To evaluate whether patient age has a significant impact on mefloquine concentrations in the plasma and erythrocytes over the course of treatment for uncomplicated falciparum malaria., Methods: A total of 20 children aged between 8 and 11 years and 20 adult males aged between 22 and 41 years with uncomplicated falciparum malaria were enrolled in the study. Mefloquine was administered to patients in both age groups at a dose of 20mgkg(-1). The steady-state drug concentrations were measured by reversed-phase high performance liquid chromatography., Results: All patients had an undetectable mefloquine concentration on day 0. In adults, the plasma mefloquine concentrations ranged from 770 to 2930ngmL(-1) and the erythrocyte concentrations ranged from 2000 to 6030ngmL(-1). In children, plasma mefloquine concentrations ranged from 881 to 3300ngmL(-1) and erythrocyte concentrations ranged from 3000 to 4920ngmL(-1). There was no significant correlation between mefloquine concentrations in the plasma and erythrocytes in either adults or children., Conclusion: In the present study, we observed no effect of patient age on the steady-state concentrations of mefloquine in the plasma and erythrocytes. We found that the mefloquine concentration in the erythrocytes was approximately 2.8-times higher than in the plasma. There were no significant correlations between mefloquine concentrations in the erythrocytes and plasma for either age group., (Copyright © 2016 Sociedade Brasileira de Infectologia. Published by Elsevier Editora Ltda. All rights reserved.)

Mutations in the "chloroquine resistance transporter" (PfCRT) are a major determinant of drug resistance in the malaria parasite Plasmodium falciparum. We have previously shown that mutant PfCRT transports the antimalarial drug chloroquine away from its target, whereas the wild-type form of PfCRT does not. However, little is understood about the transport of other drugs via PfCRT or the mechanism by which PfCRT recognizes different substrates. Here we show that mutant PfCRT also transports quinine, quinidine, and verapamil, indicating that the protein behaves as a multidrug resistance carrier. Detailed kinetic analyses revealed that chloroquine and quinine compete for transport via PfCRT in a manner that is consistent with mixed-type inhibition. Moreover, our analyses suggest that PfCRT accepts chloroquine and quinine at distinct but antagonistically interacting sites. We also found verapamil to be a partial mixed-type inhibitor of chloroquine transport via PfCRT, further supporting the idea that PfCRT possesses multiple substrate-binding sites. Our findings provide new mechanistic insights into the workings of PfCRT, which could be exploited to design potent inhibitors of this key mediator of drug resistance., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Malaria is a global health challenge with endemicity in sub-Saharan Africa, where there are multiple drug-resistant strains and limited access to modern health care facilities, especially in rural areas. Studies indicate that African traditional medicine could make a substantial contribution to the reduction of malaria-related deaths and achievement of universal health coverage (UHC), particularly in these regions. Thus, this study evaluated the curative antimalarial effects of Chromolaena odorata leaf extract using mouse model. Forty-five (45) albino mice weighing between 18 and 22 g were grouped into nine groups of 5 animals each. Animals in groups 2–9 were infected with the chloroquine-resistant strain of Plasmodium berghei, while animals in groups 3–9 were subsequently treated with 10 mg/kg chloroquine, a combination of 1.4 mg/kg artemether and 8.75 mg/kg lumefantrine (Coartem), and varying concentrations of the fraction from the aqueous leaf extract of C. odorata at day 3 post-infection. The findings from this study indicate that treatment with 400 mg/kg of the ethanolic fraction of the crude extract resulted in a significant decrease in parasite load (97.6%), which was comparable to the activities of the conventional drugs chloroquine (98.6%) and Coartem (98.8%). The ethyl acetate and ethanolic fractions at 400 mg/kg also ameliorated the significant alterations in the red blood cells, white blood cells, and platelets of the infected animals. The high antimalarial activity displayed by the ethanolic fraction could be due to the presence of quercetin and kaempferol, as detected by high performance liquid chromatography (HPLC) analysis. The findings suggest that the fractions from C. odorata could serve as an alternative source of malaria therapy, particularly in sub-Saharan Africa. [ABSTRACT FROM AUTHOR]

Malaria, a disease caused by the Plasmodium parasite, accounts for more than 450 000 deaths annually. The devastating impact of this disease is compounded by the emergence or risk of widespread resistance to current antimalarial drugs, underscoring the need to develop new therapies. Australian scientists are at the forefront of fundamental, clinical and surveillance research, and have made significant contributions to advancing the field of malaria research. A significant component of this research has been directed toward the development of new antimalarial therapies. This perspective summarises the recent endeavours by Australian researchers in chemistry and drug discovery sciences in the identification and development of new antimalarial therapies in the global challenge to treat and eliminate malaria. Malaria is a devasting disease that causes significant morbidity and mortality worldwide. New therapies are urgently needed, because of the emergent resistance against current frontline therapies. This perspective highlights the contributions of Australian chemists and drug discovery experts to the field of antimalarial development. [ABSTRACT FROM AUTHOR]

Background: Malaria is one of the leading causes of death worldwide caused by parasites of the genus Plasmodium. The reduced efficacy of the mainstay antimalarial drugs due to the widespread of drug-resistant Plasmodium falciparum (P. falciparum) necessitates an effort to develop novel antimalarial drugs with new targets. The effects of a phenolic compound, ellagic acid, against the malaria parasite have previously been reported. This present study aimed to evaluate the effect of ellagic acid on pH of the P. falciparum digestive vacuole. Methods: The antimalarial potential of ellagic acid against the chloroquine-sensitive strain (3D7) of P. falciparum was assessed by using a malarial SYBR Green 1 fluorescence-based (MSF) assay. The effect of different concentrations of ellagic acid on the pH of the parasite's digestive vacuole at mid-trophozoite stage was examined by using a ratiometric pH indicator, fluorescein isothiocyanate (FITC)-dextran on the flow cytometry. Results: The result of the MSF assay showed that ellagic acid has an antimalarial activity (half-maximal inhibitory concentration [IC50] = 1.85 ± 4.57 nM) at par with a standard drug, artemisinin (IC50 = 1.91 ± 5.41 nM). The pH of the digestive vacuole of ellagic acid-treated parasites was significantly changed (pH values ranged from 6.11 to 6.74) in a concentration-dependent manner as compared to untreated parasites (P < 0.001). A similar effect was shown by the parasites treated with a standard proton pump inhibitor, concanamycin A. Conclusion: These findings suggest that ellagic acid might have altered the digestive vacuole pH through the inhibition of proton pumps that regulate the acidification of this organelle. Overall, this study provides a valuable insight into the potential of ellagic acid as a promising antimalarial candidate with a novel mechanism of action. [ABSTRACT FROM AUTHOR]

Background Cipargamin (KAE609) is a potent antimalarial in a phase II trial. Here we report efficacy, pharmacokinetics, and resistance marker analysis across a range of cipargamin doses. These were secondary endpoints from a study primarily conducted to assess the hepatic safety of cipargamin (hepatic safety data are reported elsewhere). Methods This phase II, multicenter, randomized, open-label, dose-escalation trial was conducted in sub-Saharan Africa in adults with uncomplicated Plasmodium falciparum malaria. Cipargamin monotherapy was given as single doses up to 150 mg or up to 50 mg once daily for 3 days, with artemether-lumefantrine as control. Key efficacy endpoints were parasite clearance time (PCT), and polymerase chain reaction (PCR)–corrected and uncorrected adequate clinical and parasitological response (ACPR) at 14 and 28 days. Pharmacokinetics and molecular markers of drug resistance were also assessed. Results All single or multiple cipargamin doses ≥50 mg were associated with rapid parasite clearance, with median PCT of 8 hours versus 24 hours for artemether-lumefantrine. PCR-corrected ACPR at 14 and 28 days was >75% and 65%, respectively, for each cipargamin dose. A treatment-emerging mutation in the Pfatp4 gene, G358S, was detected in 65% of treatment failures. Pharmacokinetic parameters were consistent with previous data, and approximately dose proportional. Conclusions Cipargamin, at single doses of 50 to 150 mg, was associated with very rapid parasite clearance, PCR-corrected ACPR at 28 days of >65% in adults with uncomplicated P. falciparum malaria, and recrudescent parasites frequently harbored a treatment-emerging mutation. Cipargamin will be further developed with a suitable combination partner. Clinical Trials Registration ClinicalTrials.gov (NCT03334747). [ABSTRACT FROM AUTHOR]

In 2006, Senegal adopted artemisinin-based combination therapy (ACT) as first-line treatment in the management of uncomplicated malaria. This study aimed to update the status of antimalarial efficacy more than ten years after their first introduction. This was a randomized, three-arm, open-label study to evaluate the efficacy and safety of artemether-lumefantrine (AL), artesunate-amodiaquine (ASAQ) and dihydroartemisinin-piperaquine (DP) in Senegal. Malaria suspected patients were screened, enrolled, treated, and followed for 28 days for AL and ASAQ arms or 42 days for DP arm. Clinical and parasitological responses were assessed following antimalarial treatment. Genotyping (msp1, msp2 and 24 SNP-based barcode) were done to differentiate recrudescence from re-infection; in case of PCR-confirmed treatment failure, Pfk13 propeller and Pfcoronin genes were sequenced. Data was entered and analyzed using the WHO Excel-based application. A total of 496 patients were enrolled. In Diourbel, PCR non-corrected/corrected adequate clinical and parasitological responses (ACPR) was 100.0% in both the AL and ASAQ arms. In Kedougou, PCR corrected ACPR values were 98.8%, 100% and 97.6% in AL, ASAQ and DP arms respectively. No Pfk13 or Pfcoronin mutations associated with artemisinin resistance were found. This study showed that AL, ASAQ and DP remain efficacious and well-tolerated in the treatment of uncomplicated P. falciparum malaria in Senegal. [ABSTRACT FROM AUTHOR]

Artemisinin resistance is threatening global efforts for malaria control and elimination. Primaquine (PQ) and methylene blue (MB) are gametocytocidal drugs that can be combined with artemisinin-based combination therapy (ACT) to reduce malaria transmission, including resistant strains. Children (6–59 months) with uncomplicated falciparum malaria in Burkina Faso were treated with artesunate-amodiaquine (AS-AQ) and randomized to MB (15 mg/kg/day for 3 days) or PQ (0.25 mg/kg at day 2) with the aim to show non-inferiority of the MB regimen with regard to haematological recovery at day 7 (primary endpoint). MB-AS-AQ could not be shown to be non-inferior to PQ-AS-AQ (mean Hb difference between treatment groups on day 7 was -0.352, 95% CI -0.832–0.128, p = 0.0767), however, haemoglobin recovery following treatment was alike in the two study arms (day 7: mean 0.2±1.4 g/dl vs. 0.5±0.9 g/dl, p = 0.446). Occurrence of adverse events was similar in both groups, except for vomiting, which was more frequent in the MB than in the PQ arm (20/50 vs 7/50, p = 0.003). Adequate clinical and parasitological response was above 95% in both groups, but significantly more asexual parasites were cleared in the MB arm compared to the PQ arm already on day 1 (48/50, 96%, vs 40/50, 80%, p = 0.014). Moreover, P. falciparum gametocyte prevalence and density were lower in the MB arm than in the PQ arm, which reached statistical significance on day 2 (prevalence: 2/50, 4%, vs 15/49, 31%, p<0.001; density: 9.6 vs 41.1/μl, p = 0.024). However, it should be considered that PQ was given only on day 2. MB-ACT appears to be an interesting alternative to PQ-ACT for the treatment of falciparum malaria. While there is a need to further improve MB formulations, MB-ACT may already be considered useful to reduce falciparum malaria transmission intensity, to increase treatment efficacy, and to reduce the risk for resistance development and spread. Trial registration: ClinicalTrials.gov . [ABSTRACT FROM AUTHOR]

Background: Artemisinin-based combination therapy (ACT) is recommended for uncomplicated Plasmodium vivax malaria in areas of emerging chloroquine resistance. We undertook a systematic review and individual patient data meta-analysis to compare the efficacies of dihydroartemisinin-piperaquine (DP) and artemether-lumefantrine (AL) with or without primaquine (PQ) on the risk of recurrent P. vivax.Methods and Findings: Clinical efficacy studies of uncomplicated P. vivax treated with DP or AL and published between January 1, 2000, and January 31, 2018, were identified by conducting a systematic review registered with the International Prospective Register of Systematic Reviews (PROSPERO): CRD42016053310. Investigators of eligible studies were invited to contribute individual patient data that were pooled using standardised methodology. The effect of mg/kg dose of piperaquine/lumefantrine, ACT administered, and PQ on the rate of P. vivax recurrence between days 7 and 42 after starting treatment were investigated by Cox regression analyses according to an a priori analysis plan. Secondary outcomes were the risk of recurrence assessed on days 28 and 63. Nineteen studies enrolling 2,017 patients were included in the analysis. The risk of recurrent P. vivax at day 42 was significantly higher in the 384 patients treated with AL alone (44.0%, 95% confidence interval [CI] 38.7-49.8) compared with the 812 patients treated with DP alone (9.3%, 95% CI 7.1-12.2): adjusted hazard ratio (AHR) 12.63 (95% CI 6.40-24.92), p < 0.001. The rates of recurrence assessed at days 42 and 63 were associated inversely with the dose of piperaquine: AHRs (95% CI) for every 5-mg/kg increase 0.63 (0.48-0.84), p = 0.0013 and 0.83 (0.73-0.94), p = 0.0033, respectively. The dose of lumefantrine was not significantly associated with the rate of recurrence (1.07 for every 5-mg/kg increase, 95% CI 0.99-1.16, p = 0.0869). In a post hoc analysis, in patients with symptomatic recurrence after AL, the mean haemoglobin increased 0.13 g/dL (95% CI 0.01-0.26) for every 5 days that recurrence was delayed, p = 0.0407. Coadministration of PQ reduced substantially the rate of recurrence assessed at day 42 after AL (AHR = 0.20, 95% CI 0.10-0.41, p < 0.001) and at day 63 after DP (AHR = 0.08, 95% CI 0.01-0.70, p = 0.0233). Results were limited by follow-up of patients to 63 days or less and nonrandomised treatment groups.Conclusions: In this study, we observed the risk of P. vivax recurrence at day 42 to be significantly lower following treatment with DP compared with AL, reflecting the longer period of post-treatment prophylaxis; this risk was reduced substantially by coadministration with PQ. We found that delaying P. vivax recurrence was associated with a small but significant improvement in haemoglobin. These results highlight the benefits of PQ radical cure and also the provision of blood-stage antimalarial agents with prolonged post-treatment prophylaxis. [ABSTRACT FROM AUTHOR]

The emergence of artemisinin-resistant parasites since the late 2000s at the border of Cambodia and Thailand poses serious threats to malaria control globally, particularly in Africa which bears the highest malaria transmission burden. This study aimed to obtain reliable data on the current state of the kelch13 molecular marker for artemisinin resistance in Plasmodium falciparum in Cameroon. DNA was extracted from the dried blood spots collected from epidemiologically distinct endemic areas in the Center, Littoral and North regions of Cameroon. Nested PCR products from the Kelch13-propeller gene were sequenced and analyzed on an ABI 3730XL automatic sequencer. Of 219 dried blood spots, 175 were sequenced successfully. We identified six K13 mutations in 2.9% (5/175) of samples, including 2 non-synonymous, the V589I allele had been reported in Africa already and one new allele E612K had not been reported yet. These two non-synonymous mutations were uniquely found in parasites from the Littoral region. One sample showed two synonymous mutations within the kelch13 gene. We also observed two infected samples with mixed K13 mutant and K13 wild-type infection. Taken together, our data suggested the circulation of the non-synonymous K13 mutations in Cameroon. Albeit no mutations known to be associated with parasite clearance delays in the study population, there is need for continuous surveillance for earlier detection of resistance as long as ACTs are used and scaled up in the community. [ABSTRACT FROM AUTHOR]

Introduction: Plasmodium falciparum synthesizes phosphatidylcholine for the membrane development through serine decarboxylase–phosphoethanolamine methyltransferase pathway for growth in human host. Phosphoethanolamine-methyltransferase (PfPMT) is a crucial enzyme for the synthesis of phosphocholine which is a precursor for phosphatidylcholine synthesis and is considered as a pivotal drug target as it is absent in the host. The inhibition of PfPMT may kill malaria parasite and hence is being considered as potential target for rational antimalarial drug designing. Methods: In this study, we have used computer aided drug designing (CADD) approaches to establish potential PfPMT inhibitors from Asinex compound library virtually screened for ADMET and the docking affinity. The selected compounds were tested for in-vitro schizonticidal, gametocidal and cytotoxicity activity. Nontoxic compounds were further studied for PfPMT enzyme specificity and antimalarial efficacy for P. berghei in albino mice model. Results: Our results have identified two nontoxic PfPMT competitive inhibitors ASN.1 and ASN.3 with better schizonticidal and gametocidal activity which were found to inhibit PfPMT at IC50 1.49μM and 2.31μM respectively. The promising reduction in parasitaemia was found both in orally (50 & 10 mg/kg) and intravenous (IV) (5& 1 mg/kg) however, the better growth inhibition was found in intravenous groups. Conclusion: We report that the compounds containing Pyridinyl-Pyrimidine and Phenyl-Furan scaffolds as the potential inhibitors of PfPMT and thus may act as promising antimalarial inhibitor candidates which can be further optimized and used as leads for template based antimalarial drug development. [ABSTRACT FROM AUTHOR]

Drug-resistant strains of malaria parasites have emerged for most of antimalarial medications. A new chemotherapeutic compound is needed for malarial therapy. Antimalarial activity against both drug-sensitive and drug-resistant P. falciparum has been reported for an isocryptolepine derivative, 8-bromo-2-fluoro-5-methyl-5H-indolo[3,2-c]quinoline (ICL-M), which also showed less toxicity to human cells. ICL-M has indoloquinoline as a core structure and its mode of action remains unclear. Here, we explored the mechanisms of ICL-M in P. falciparum by assessing the stage-specific activity, time-dependent effect, a proteomic analysis and morphology. Since human topo II activity inhibition has been reported as a function of isocryptolepine derivatives, malarial topo II activity inhibition of ICL-M was also examined in this study. The ICL-M exhibited antimalarial activity against both the ring and trophozoite stages of P. falciparum. Our proteomics analysis revealed that a total of 112 P. falciparum proteins were differentially expressed after ICL-M exposure; among these, 58 and 54 proteins were upregulated and downregulated, respectively. Proteins localized in the food vacuole, nucleus, and cytoplasm showed quantitative alterations after ICL-M treatment. A bioinformatic analysis revealed that pathways associated with ribosomes, proteasomes, metabolic pathways, amino acid biosynthesis, oxidative phosphorylation, and carbon metabolism were significantly different in P. falciparum treated with ICL-M. Moreover, a loss of ribosomes was clearly observed by transmission electron microscopy in the ICL-M-treated P. falciparum. This finding is in agreement with the proteomics data, which revealed downregulated levels of ribosomal proteins following ICL-M treatment. Our results provide important information about the mechanisms by which ICL-M affects the malaria parasite, which may facilitate the drug development of isocryptolepine derivatives. [ABSTRACT FROM AUTHOR]

In epidemiological surveys and surveillance the application of molecular tools is essential in detecting submicroscopic malaria. A genus-specific conventional cytochrome b (cytb) PCR has shown high sensitivity in field studies, detecting 70% submicroscopic malaria. The main objective of this study was to assess the conversion from conventional to real-time PCR testing both SYBR and probe protocols, and including quantitative (q) PCR. The protocols were assessed applying well-defined clinical patient material consisting of 33 positive and 80 negative samples. Sequencing of positive PCR products was performed. In addition, a sensitivity comparison of real-time PCR methods was done by including five relevant assays investigating the effect of amplification target and platform. Sensitivity was further examined using field material consisting of 111 P.falciparum positive samples from Tanzanian children (< 5 years), as well as using related patient data to assess the application of q-PCR with focus on low-level parasitaemia. Both the cytb SYBR and probe PCR protocols showed as high sensitivity and specificity as their conventional counterpart, except missing one P. malariae sample. The SYBR protocol was more sensitive and specific than using probe. Overall, choice of amplification target applied is relevant for achieving ultra-sensitivity, and using intercalating fluorescence dye rather than labelled hydrolysis probes is favourable. Application of q-PCR analysis in field projects is important for the awareness and understanding of low-level parasitaemia. For use in clinical diagnosis and epidemiological studies the highly sensitive and user-friendly cytb SYBR q-PCR method is a relevant tool. The genus-specific method has the advantage that species identification by sequencing can be performed as an alternative to species-specific PCR. [ABSTRACT FROM AUTHOR]

Introduction: Asymptomatic carriage of P. falciparum is frequent in areas endemic for malaria and individual diagnosis of clinical malaria attacks is still difficult. We investigated the impact of changes in malaria endemicity on the diagnostic criteria for malaria attacks in an area of seasonal malaria transmission. Methods: We analyzed the longitudinal data collected over 20 years from a daily survey of all inhabitants of Ndiop, a rural community in central Senegal, in a logistic regression model to investigate the relationship between the level of Plasmodium falciparum parasitemia and the risk of fever, with the aim of determining the best parasitemia thresholds for attributing to malaria a fever episode. Results: A total of 34,136 observations recorded from July 1993 to December 2013 from 850 individuals aged from 1 day to 87 years were included. P. falciparum asymptomatic carriage declined from 36% to 1% between 1993 and 2013. A total of 9,819 fever episodes were associated with a positive blood film for P. falciparum. Using age-dependent parasitemia thresholds for attributing to malaria a fever episode, we recorded 6,006 malaria attacks during the study period. Parasitemia thresholds seemed to be lower during the low-to-zero transmission season and tended to decrease with changes in control policies. The number of clinical malaria attacks was overestimated for all age groups throughout the study when all fever episodes associated with P. falciparum parasitemia were defined as malaria attacks. Conclusion: Pyrogenic thresholds are particularly sensitive to changes in malaria epidemiology and are therefore an interesting tool to accurately assess the burden of malaria in the context of declining transmission. [ABSTRACT FROM AUTHOR]

Background: Mass azithromycin distributions have been shown to reduce mortality in preschool children, although the factors mediating this mortality reduction are not clear. This study was performed to determine whether mass distribution of azithromycin, which has modest antimalarial activity, reduces the community burden of malaria.Methods and Findings: In a cluster-randomized trial conducted from 23 November 2014 until 31 July 2017, 30 rural communities in Niger were randomized to 2 years of biannual mass distributions of either azithromycin (20 mg/kg oral suspension) or placebo to children aged 1 to 59 months. Participants, field staff, and investigators were masked to treatment allocation. The primary malaria outcome was the community prevalence of parasitemia on thick blood smear, assessed in a random sample of children from each community at study visits 12 and 24 months after randomization. Analyses were performed in an intention-to-treat fashion. At the baseline visit, a total of 1,695 children were enumerated in the 15 azithromycin communities, and 3,029 children were enumerated in the 15 placebo communities. No communities were lost to follow-up. The mean prevalence of malaria parasitemia at baseline was 8.9% (95% CI 5.1%-15.7%; 52 of 552 children across all communities) in the azithromycin-treated group and 6.7% (95% CI 4.0%-12.6%; 36 of 542 children across all communities) in the placebo-treated group. In the prespecified primary analysis, parasitemia was lower in the azithromycin-treated group at month 12 (mean prevalence 8.8%, 95% CI 5.1%-14.3%; 51 of 551 children across all communities) and month 24 (mean 3.5%, 95% CI 1.9%-5.5%; 21 of 567 children across all communities) than it was in the placebo-treated group at month 12 (mean 15.3%, 95% CI 10.8%-20.6%; 81 of 548 children across all communities) and month 24 (mean 4.8%, 95% CI 3.3%-6.4%; 28 of 592 children across all communities) (P = 0.02). Communities treated with azithromycin had approximately half the odds of parasitemia compared to those treated with placebo (odds ratio [OR] 0.54, 95% CI 0.30 to 0.97). Parasite density was lower in the azithromycin group than the placebo group at 12 and 24 months (square root-transformed outcome; density estimates were 7,540 parasites/μl lower [95% CI -350 to -12,550 parasites/μl; P = 0.02] at a mean parasite density of 17,000, as was observed in the placebo arm). No significant difference in hemoglobin was observed between the 2 treatment groups at 12 and 24 months (mean 0.34 g/dL higher in the azithromycin arm, 95% CI -0.06 to 0.75 g/dL; P = 0.10). No serious adverse events were reported in either group, and among children aged 1 to 5 months, the most commonly reported nonserious adverse events (i.e., diarrhea, vomiting, and rash) were less common in the azithromycin-treated communities. Limitations of the trial include the timing of the treatments and monitoring visits, both of which took place before the peak malaria season, as well as the uncertain generalizability to areas with different malaria transmission dynamics.Conclusions: Mass azithromycin distributions were associated with a reduced prevalence of malaria parasitemia in this trial, suggesting one possible mechanism for the mortality benefit observed with this intervention.Trial Registration: The trial was registered on ClinicalTrials.gov (NCT02048007). [ABSTRACT FROM AUTHOR]

Therapeutics with novel modes of action and a low risk of generating resistance are urgently needed to combat drug-resistant Plasmodium falciparum malaria. Here, we report that the peptide vinyl sulfones WLL-vs (WLL) and WLW-vs (WLW), highly selective covalent inhibitors of the P. falciparum proteasome, potently eliminate genetically diverse parasites, including K13-mutant, artemisinin-resistant lines, and are particularly active against ring-stage parasites. Selection studies reveal that parasites do not readily acquire resistance to WLL or WLW and that mutations in the β2, β5 or β6 subunits of the 20S proteasome core particle or in components of the 19S proteasome regulatory particle yield only hundred-fold decreases in susceptibility. We observed no cross-resistance between WLL and WLW. Moreover, most mutations that conferred a modest loss of parasite susceptibility to one inhibitor significantly increased sensitivity to the other. These inhibitors potently synergized multiple chemically diverse classes of antimalarial agents, implicating a shared disruption of proteostasis in their modes of action. These results underscore the potential of targeting the Plasmodium proteasome with covalent small molecule inhibitors as a means of combating multidrug-resistant malaria. [ABSTRACT FROM AUTHOR]

Plasmodium vivax causes the majority of malaria outside Africa, but is poorly understood at a cellular level partly due to technical difficulties in maintaining it in in vitro culture conditions. In the past decades, drug resistant P. vivax parasites have emerged, mainly in Southeast Asia, but while some molecular markers of resistance have been identified, none have so far been confirmed experimentally, which limits interpretation of the markers, and hence our ability to monitor and control the spread of resistance. Some of these potential markers have been identified through P. vivax genome-wide population genetic analyses, which highlighted genes under recent evolutionary selection in Southeast Asia, where chloroquine resistance is most prevalent. These genes could be involved in drug resistance, but no experimental proof currently exists to support this hypothesis. In this study, we used Plasmodium knowlesi, the most closely related species to P. vivax that can be cultured in human erythrocytes, as a model system to express P. vivax genes and test for their role in drug resistance. We adopted a strategy of episomal expression, and were able to express fourteen P. vivax genes, including two allelic variants of several hypothetical resistance genes. Their expression level and localisation were assessed, confirming cellular locations conjectured from orthologous species, and suggesting locations for several previously unlocalised proteins, including an apical location for PVX_101445. These findings establish P. knowlesi as a suitable model for P. vivax protein expression. We performed chloroquine and mefloquine drug assays, finding no significant differences in drug sensitivity: these results could be due to technical issues, or could indicate that these genes are not actually involved in drug resistance, despite being under positive selection pressure in Southeast Asia. These data confirm that in vitro P. knowlesi is a useful tool for studying P. vivax biology. Its close evolutionary relationship to P. vivax, high transfection efficiency, and the availability of markers for colocalisation, all make it a powerful model system. Our study is the first of its kind using P. knowlesi to study unknown P. vivax proteins and investigate drug resistance mechanisms. [ABSTRACT FROM AUTHOR]

Background: Malaria control activities can have a disproportionately greater impact on Plasmodium falciparum than on P. vivax in areas where both species are coendemic. We investigated temporal trends in malaria-related morbidity and mortality in Papua, Indonesia, before and after introduction of a universal, artemisinin-based antimalarial treatment strategy for all Plasmodium species.Methods and Findings: A prospective, district-wide malariometric surveillance system was established in April 2004 to record all cases of malaria at community clinics and the regional hospital and maintained until December 2013. In March 2006, antimalarial treatment policy was changed to artemisinin combination therapy for uncomplicated malaria and intravenous artesunate for severe malaria due to any Plasmodium species. Over the study period, a total of 418,238 patients presented to the surveillance facilities with malaria. The proportion of patients with malaria requiring admission to hospital fell from 26.9% (7,745/28,789) in the pre-policy change period (April 2004 to March 2006) to 14.0% (4,786/34,117) in the late transition period (April 2008 to December 2009), a difference of -12.9% (95% confidence interval [CI] -13.5% to -12.2%). There was a significant fall in the mortality of patients presenting to the hospital with P. falciparum malaria (0.53% [100/18,965] versus 0.32% [57/17,691]; difference = -0.21% [95% CI -0.34 to -0.07]) but not in patients with P. vivax malaria (0.28% [21/7,545] versus 0.23% [28/12,397]; difference = -0.05% [95% CI -0.20 to 0.09]). Between the same periods, the overall proportion of malaria due to P. vivax rose from 44.1% (30,444/69,098) to 53.3% (29,934/56,125) in the community clinics and from 32.4% (9,325/28,789) to 44.1% (15,035/34,117) at the hospital. After controlling for population growth and changes in treatment-seeking behaviour, the incidence of P. falciparum malaria fell from 511 to 249 per 1,000 person-years (py) (incidence rate ratio [IRR] = 0.49 [95% CI 0.48-0.49]), whereas the incidence of P. vivax malaria fell from 331 to 239 per 1,000 py (IRR = 0.72 [95% CI 0.71-0.73]). The main limitations of our study were possible confounding from changes in healthcare provision, a growing population, and significant shifts in treatment-seeking behaviour following implementation of a new antimalarial policy.Conclusions: In this area with high levels of antimalarial drug resistance, adoption of a universal policy of efficacious artemisinin-based therapy for malaria infections due to any Plasmodium species was associated with a significant reduction in total malaria-attributable morbidity and mortality. The burden of P. falciparum malaria was reduced to a greater extent than that of P. vivax malaria. In coendemic regions, the timely elimination of malaria will require that safe and effective radical cure of both the blood and liver stages of the parasite is widely available for all patients at risk of malaria. [ABSTRACT FROM AUTHOR]

Background: In Vietnam, the importance of vivax malaria relative to falciparum during the past decade has steadily increased to 50%. This, together with the spread of multidrug-resistant Plasmodium falciparum, is a major challenge for malaria elimination. A 2-year prospective cohort study to assess P. vivax morbidity after radical cure treatment and related risk factors was conducted in Central Vietnam.Methods and Findings: The study was implemented between April 2009 and December 2011 in four neighboring villages in a remote forested area of Quang Nam province. P. vivax-infected patients were treated radically with chloroquine (CQ; 25 mg/kg over 3 days) and primaquine (PQ; 0.5 mg/kg/day for 10 days) and visited monthly (malaria symptoms and blood sampling) for up to 2 years. Time to first vivax recurrence was estimated by Kaplan-Meier survival analysis, and risk factors for first and recurrent infections were identified by Cox regression models. Among the 260 P. vivax patients (61% males [159/260]; age range 3-60) recruited, 240 completed the 10-day treatment, 223 entered the second month of follow-up, and 219 were followed for at least 12 months. Most individuals (76.78%, 171/223) had recurrent vivax infections identified by molecular methods (polymerase chain reaction [PCR]); in about half of them (55.61%, 124/223), infection was detected by microscopy, and 84 individuals (37.67%) had symptomatic recurrences. Median time to first recurrence by PCR was 118 days (IQR 59-208). The estimated probability of remaining free of recurrence by month 24 was 20.40% (95% CI [14.42; 27.13]) by PCR, 42.52% (95% CI [35.41; 49.44]) by microscopy, and 60.69% (95% CI [53.51; 67.11]) for symptomatic recurrences. The main risk factor for recurrence (first or recurrent) was prior P. falciparum infection. The main limitations of this study are the age of the results and the absence of a comparator arm, which does not allow estimating the proportion of vivax relapses among recurrent infections.Conclusion: A substantial number of P. vivax recurrences, mainly submicroscopic (SM) and asymptomatic, were observed after high-dose PQ treatment (5.0 mg/kg). Prior P. falciparum infection was an important risk factor for all types of vivax recurrences. Malaria elimination efforts need to address this largely undetected P. vivax transmission by simultaneously tackling the reservoir of P. falciparum and P. vivax infections. [ABSTRACT FROM AUTHOR]