OBJECTIVES:Artemisinin-resistant Plasmodium falciparum has emerged in several East African countries neighbouring Madagascar. Despite the island's substantial malaria burden, recent data on artemisinin partial resistance are limited, raising concerns about the potential emergence of resistant parasites. This study provides an updated overview of the prevalence and diversity of P. falciparum Kelch13 (pfkelch13) polymorphisms in Madagascar. METHODS:During a nationally representative, cross-sectional survey conducted between January and May 2024, dried blood samples were collected from 4850 febrile patients at 65 health facilities. Pfkelch13 genotyping was performed using a targeted amplicon deep sequencing approach. RESULTS:Of the 1944 P. falciparum-positive samples, 963 (49.5%) pfkelch13 sequences were successfully obtained, and 885 (91.9%) corresponded to the 3D7 wild-type. Non-synonymous and synonymous mutations were detected in 1.8% (17/963) and 6.2% (60/963) of isolates, respectively, whereas one isolate (0.1%) carried double mutations. Of the 18 mutations identified, 5 had not been previously reported. The two most frequent polymorphisms in Madagascar were the synonymous mutations C469C (3.0%, 29/963) and P417P (2.8%, 27/963). None of the WHO-validated artemisinin partial resistance markers were detected. CONCLUSION:This study provides an updated baseline of pfkelch13 polymorphisms in Madagascar, with no evidence of artemisinin partial resistance emergence. Importantly, no parasites harbouring a validated artemisinin resistance marker were detected across the regions sampled, suggesting that resistant parasites have not yet become established. These findings provide a valuable baseline for future genomic surveillance efforts aimed at the early detection of mutations associated with artemisinin partial resistance.

The key ligand involved in Plasmodium vivax reticulocyte invasion is the Duffy Binding Protein (PvDBP) which binds the Duffy receptor on reticulocytes. Anti-PvDBP human monoclonal antibodies can inhibit PvDBP-Duffy receptor binding and neutralize reticulocytes invasion in vitro. However, parasites with multiple copies of the pvdbp gene are protected in vitro against neutralization. Here, we evaluated whether this gene amplification also protects parasites in vivo.We hypothesized that: (i) multi-pvdbp copy parasites are more frequent in areas with a high P. vivax prevalence, (ii) individuals with naturally acquired binding inhibitory anti-PvDBP Abs (BIAbs) are predominantly infected over time by multi-copy parasites and (iii) multi-copy parasites infect asymptomatic carriers more frequently than symptomatic individuals. We analyzed samples from a 2019-2020 longitudinal cohort of individuals living in nine villages in Eastern Cambodia with low (~5%) to high (~30%) P. vivax prevalence. Using a PCR assay targeting the boundaries of the pvdbp duplication, we estimated the frequency of multi-copy parasites over time. Then, using a flow cytometry assay, we determined the presence of naturally acquired BIAbs in 657 participants' plasma. Finally, we compared parasite gene copy number over the 21-month follow-up in cohort participants according to the presence of BIAbs at the start of the study. In parallel, we determined the frequency of pvdbpduplication in samples collected among symptomatic treatment-seeking patients in the same area over the same period. We compared the frequency of infection with multi-copy parasites between asymptomatic cohort members and symptomatic patients. We found a significant association between P. vivax prevalence and the proportion of multi-copy parasites, which ranged from 35% in low prevalence villages to 47% in high prevalence villages (p=0.0246). We also observed that the more inhibitory the Abs in the hosts' plasma, the higher the proportion of multi-copy parasites: 87% (40/46) from individuals with high BIAbs while 38% (193/514) from individuals without any BIAbs (p<0.0001). This association between immunity and infection by multi-copy parasites remained consistent over the 21-month longitudinal follow-up. Finally, we found that the frequency of multi-copy parasites was higher in asymptomatic carriers than in symptomatic individuals. Overall, these results indicate that pvdbp duplication helps the parasites to avoid the hosts' anti-PvDBP immunity in vivo. It warrants further investigations to determine if immunization with a PvDBP vaccine could overcome this immune evasion mechanism.

BACKGROUND:Artemisinin-based combination therapies (ACTs) have played a crucial role in decreasing the impact of malaria worldwide. Since 2005, artemether-lumefantrine (AL) has been the main first-line treatment for uncomplicated Plasmodium falciparum malaria in Laos. Herein, we aimed to study the efficacy of AL in the context of malaria elimination in Laos. METHODS:Between Aug 1, 2019, and June 11, 2023, AL efficacy was evaluated in four provinces of southern Laos: Attapeu, Champassack, Salavan, and Savannakhet. Adults and children (aged 1-60 years) with microscopically confirmed P falciparum malaria received oral AL twice a day for 3 days, with follow-up on days 7, 14, 21, and 28. The primary outcome was PCR-adjusted adequate clinical and parasitological response (ACPR) by day 28. Resistance to dihydroartemisinin (DHA) and lumefantrine (LM) was assessed by an in vitro phenotypic analysis, and mutations in P falciparum kelch13 (pfkelch13), P falciparum multidrug resistance 1 (pfmdr1), P falciparum plasmepsin 2 (pfpm2), and P falciparum chloroquine resistant transporter (pfcrt) were characterised in parasites collected from enrolled patients. Safety outcomes included the frequency and nature of adverse events and serious adverse events. FINDINGS:A total of 198 patients (median age 16 years [IQR 10-28]; 124 [63%] male and 74 [37%] female) were initially enrolled, of whom three were lost to follow-up, resulting in 195 patients who received the 3-day AL regimen. At day 28, the PCR-adjusted ACPR was 96% (95% CI 92-98), with a treatment failure rate of 2% (1-5) and a reinfection rate of 2% (1-5). Among the four PCR-confirmed recrudescent isolates, one showed markedly reduced LM susceptibility (LM 50% inhibitory concentration [IC50] 59·9 nM, 2·5 times higher than the median IC50 of other isolates) and high artemisinin resistance in vitro (ring-stage survival survival rate 35·8%), which was associated with the pfkelch13 R539T mutation and day-3 microscopy-positive parasitaemia. Among 190 isolates with successfully determined pfkelch13 sequencing, nine (5%) carried the pfkelch13 mutation R539T and 43 (23%) carried the C580Y mutation, and both were associated with day-3 microscopy-positive parasitaemia (p=0·044). No amplification of pfmdr1 or pfpm2, nor any mutations in pfmdr1 and pfcrt, were associated with treatment failure. INTERPRETATION:Our findings indicate the potential emergence of LM resistance in Laos. Although AL remains efficacious, vigilance for decreasing efficacy and close monitoring of LM efficacy should be considered to support the country's goal of eliminating malaria by 2030. Importantly, none of the known pfmdr1 or pfcrt haplotypes were uniquely associated with treatment failure, including the isolate with the highest LM IC50, underscoring the need to identify reliable molecular markers for LM resistance. FUNDING:Bill and Melinda Gates Foundation and The Global Fund.

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

Background Asymptomatic Plasmodium vivax infections undermine malaria control by sustaining transmission. This study aimed to validate an eight-marker serological panel (selected from 14 antigens) of P. vivax exposure to detect individuals likely harbouring hypnozoites in rural Cambodia, and to compare it with molecular surveillance for identifying at-risk populations in low-transmission settings. Methods Two studies were conducted in Mondulkiri, Cambodia. In a longitudinal cohort (N = 471), participants were tested monthly for one year for P. vivax using qPCR. At 12 months, IgG responses to 14 antigens were assessed, and Random Forest models (‘Cambodia-specific’ and ‘global’) were used to classify previous infection, with PCR as the reference standard. In a cross-sectional survey (N = 3538), risk factors for sero- and PCR positivity were assessed using generalized linear mixed-effects models. Findings PCR detected P. vivax in 29.9% (141/471) of participants over the 12-month follow-ups, while 31.8% (150/471) were classified as seropositive at month 12. Of 141 PCR-confirmed cases, 95 were seropositive (67.4%) and 46 seronegative (32.6%) under the 8-antigen panel. The ‘global’ model achieved an AUC of 0.81, with 83.3% specificity and 69.5% sensitivity for infections within nine months, increasing to 82% sensitivity for last-month infections. In the cross-sectional study, serology identified more positives than PCR, reflecting detection of exposure not captured by PCR. Interpretation This study provides the first validation of serological markers of P. vivax exposure in Cambodia, showing that such tools can identify recent and asymptomatic infections, though sensitivity was below target. Serology-based surveillance and strategies such as PvSeroTAT could accelerate malaria elimination. Funding NIH/NIAID, ICEMR Asia-Pacific U19AI129392.

While substantial progress has been made toward malaria elimination in Southeast Asia, major challenges remain. The principal mosquito vectors, Anopheles dirus and Anopheles minimus, inhabit diverse ecological niches and exhibit a wide range of behavioural and physiological insecticide resistance phenotypes, complicating vector control efforts. In Africa, genomic surveillance has transformed our understanding of vector evolution and resistance, supported by open-access tools and data. Comparable resources for Southeast Asian vectors remain limited. Here, we release Adir1.0, a curated, analysis-ready catalog of 540 An. dirus whole-genome sequences, accessible for interactive cloud-based analysis via the malariagen-data-python application programming interface (API). Alongside, we provide an updated Amin1.0 API to enhance functionality and usability, enabling integrated analyses across historical and new datasets. We demonstrate these resources by performing the first exploratory population genomic analysis of An. dirus from Bangladesh, Thailand, and Cambodia, revealing population structure, candidate regions of physiological resistance, and genomic structural variation. Together, these resources provide a foundation for genomic surveillance studies to inform vector control and malaria elimination strategies in Southeast Asia.

To challenge the multidrug resistance of Plasmodium falciparum malaria parasites, new hybrid compounds were synthesized and evaluated against laboratory strains and multidrug-resistant clinical isolates. Among these hybrids, emoquine-1 was the most active on proliferative P. falciparum, with IC50 values in the range of 20-55 nM and a high selectivity index with respect to mammalian cells. This drug retained its activity on several multiresistant field isolates from Cambodia and Guiana, exhibited no cross-resistance to artemisinin, and is also very active against the quiescent stage of the artemisinin-resistant parasites, three features that constitute the gold standard for new antimalarial drugs. In vivo, emoquine-1 is active against Plasmodium vinckei petteri at 25 mg/kg/d per os and by the intraperitoneal route at 1-5 mg/kg/d, with total cure at 10 mg/kg/d, making emoquine-1 an ideal candidate to fight Plasmodium parasites resistant to artemisinin-based combination therapies (ACTs) with a capacity to eliminate persistent parasites.

Radical cure of Plasmodium vivax malaria must include elimination of quiescent ‘hypnozoite’ forms in the liver; however, the only FDA-approved treatments are contraindicated in many vulnerable populations. To identify new drugs and drug targets for hypnozoites, we screened the Repurposing, Focused Rescue, and Accelerated Medchem (ReFRAME) library and a collection of epigenetic inhibitors against P. vivax liver stages. From both libraries, we identified inhibitors targeting epigenetics pathways as selectively active against P. vivax and P. cynomolgi hypnozoites. These include DNA methyltransferase inhibitors as well as several inhibitors targeting histone post-translational modifications. Immunofluorescence staining of Plasmodium liver forms showed strong nuclear 5-methylcystosine signal, indicating liver stage parasite DNA is methylated. Using bisulfite sequencing, we mapped genomic DNA methylation in sporozoites, revealing DNA methylation signals in most coding genes. We also demonstrated that methylation level in proximal promoter regions as well as in the first exon of the genes may affect, at least partially, gene expression in P. vivax . The importance of selective inhibitors targeting epigenetic features on hypnozoites was validated using MMV019721, an acetyl-CoA synthetase inhibitor that affects histone acetylation and was previously reported as active against P. falciparum blood stages. In summary, our data indicate that several epigenetic mechanisms are likely modulating hypnozoite formation or persistence and provide an avenue for the discovery and development of improved radical cure antimalarials.

The recent emergence of artemisinin resistance in Africa is drawing scrutiny toward the use of alternative anti-malarial therapy based on Artemisia annua and Artemisia afra phytotherapies. This study aimed to determine if either A. annua and A. afra extracts are active against artemisinin-resistant Plasmodium falciparum isolates and determine the selectivity of inhibitory phytotherapies. Artemisia extracts were tested in vitro to mimic parasites exposure to extracts in population drinking Artemisia sp. teas. Artemisia extracts were tested in Ring Stage Survival Assays (RSA0−3 h) against Cambodian clinical isolates previously genetically and phenotypically characterized as artemisinin resistant or sensitive. Primary human hepatocytes and a human hepatoblastoma cell line (HepG2 cells) were used to assess the cytotoxicity of Artemisia extracts. The study revealed a substantially decreased in vitro activity of A. annua extracts when tested on artemisinin-resistant parasites mutated in the Pfkelch13 gene (RSA50 0.137–2.56 g.L−1) compared to artemisinin-sensitive parasites (RSA50 0.080 g.L−1). Conversely, the A. afra extracts have a similar activity on the isolates tested whether they are sensitive or resistant to artemisinin (RSA50 0.537–0.758 g.L−1) However, the selectivity index for A. afra extracts was much lower than for A. annua extracts (A. afra: 4.628, 4.305 and 6.076 vs A. annua: 387.625, 226.350 and 12.099, respectively for WT, C580Y and R539T). Artemisia annua activity is driven by artemisinin, implicating the same resistance profiles and concerns associated with semisynthetic artemisinin derivatives. Artemisia afra showed artemisinin-independent antiplasmodial activity. However, the molecular basis of this activity is unknown and may not present a sufficient selectivity, thus further characterization of A. afra is essential.

Background: Artemisinin-based combination therapies (ACTs) have played a crucial role in decreasing the impact of malaria worldwide. Since 2005, artemether-lumefantrine (AL) has been the main first-line treatment for uncomplicated Plasmodium falciparum malaria in Laos. We aimed here to study the efficacy of AL in the context of malaria elimination in Laos. Methods: Between 2019 and 2023, AL efficacy was evaluated in four provinces of southern Laos: Attapeu, Champasack, Salavan and Savannakhet. Adults and children with microscopically confirmed P. falciparum malaria received oral AL once daily for 3 days, with follow-up on days 7, 14, 21 and 28. The primary outcome was day–28 polymerase chain reaction (PCR)-adjusted adequate clinical and parasitological response (ACPR). Resistance to dihydroartemisinin (DHA) and lumefantrine (LM) was assessed by in vitro phenotypic analysis and mutations in P. falciparum kelch13 (pfkelch13), P. falciparum multidrug resistance 1 (pfmdr1), P. falciparum plasmepsin 2 (pfpm2) and P. falciparum chloroquine resistant transporter (pfcrt) were characterized in parasites collected from enrolled patients. Findings: A total of 195 patients were enrolled and treated with a 3-day AL regimen. The day–28 PCR adjusted ACPR was 96·0% (95%CI, 92·1–98·2) with a treatment failure rate of 2.0% (0·6–5·2).Among the four PCR-confirmed recrudescent cases, two isolates were successfully adapted to in vitro culture and subjected to whole-genome sequencing. One of these isolates was confirmed as a true recrudescence and exhibited a LM IC₅₀ of 59·94 nM, which was 2·5 times higher than the median (IQR) IC₅₀ of 22·3 nM (IQR 19·32–28·15) observed in other Laos isolates (n = 8). The same isolate was also resistant to DHA in vitro by showing the highest RSA survival rate (35·84%) associated with pfkelch13 mutation (R539T), as well as a microscopy positive parasitemia on day 3. The pfkelch13 mutations R539T and C580Y were respectively detected at 4·8% (9/189) and 22·8% (43/189) of isolates, and were significantly associated with day-3 microscopy positive parasitemia (p=0.0440). Mutations in pfmdr1 were identified as follows: Y184F in 26·0% (47/181), V1109I in 12·2% (22/181), N1042Y in 3·3% (6/181), and both Y184F-N1042Y and Y184F-F1068L in 0·6% (1/181) of isolates. Two out of the 4 recrudescent isolates carried the NFD (the N86, Y184F, D1246) allele, while the other 2 carried NYD (N86, Y184, D1246) allele. No amplification of pfmdr1 or pfpm2, nor mutations in pfcrt, were associated with treatment failure. Interpretation: Our findings indicate potential emergence of LM resistance in Laos. Although AL remains efficacious, vigilance for decreasing efficacy and close monitoring of LM effectiveness should be considered to support the country’s goal of eliminating malaria by 2030. Importantly, none of the known pfmdr1 or pfcrt haplotypes were uniquely associated with treatment failure, including the isolate with the highest LM IC50, underscoring the need to identify reliable molecular markers for LM resistance.

Triple artemisinin-based combination therapies (TACTs) have been proposed to delay the emergence of multidrug-resistant Plasmodium falciparum by combining two partner drugs with an artemisinin derivative. Among these, mefloquine-piperaquine (MQ-PPQ) is a leading candidate, based on the assumption that simultaneous resistance to both partner drugs would be difficult to develop. Here, we assess the efficacy and resistance potential of MQ-PPQ using Cambodian clinical isolates with distinct resistance profiles. We find that MQ resistance confers significant cross-tolerance to the MQ-PPQ combination, whereas PPQ-resistant and -sensitive strains remain susceptible. Under repeated MQ-PPQ pressure for four months, parasites rapidly acquire MQ-PPQ tolerance, driven by pfmdr1 amplification. Mechanistic investigations reveal that MQ inhibits PPQ accumulation in a dose-dependent manner, providing a functional explanation for the compromised efficacy of the combination. These findings demonstrate that MQ resistance alone can undermine MQ-PPQ TACT efficacy, calling into question the strategic rationale of this combination and underscoring the need for alternative regimens with a lower risk of resistance selection.

Background:Cambodia is targeting malaria elimination by 2025, aligning with the WHO's Mekong Malaria Elimination program. While elimination of Plasmodium falciparum is nearly achieved, Plasmodium vivax elimination presents challenges inherent to this species due to the occurrence of dormant parasite stages, known as hypnozoites. A new approach has been proposed to serologically identify individuals likely carrying hypnozoites that should receive appropriate antimalarial treatment: P. vivax serological testing and treatment (PvSeroTAT). This study aims to determine the technical feasibility of a PvSeroTAT approach in malaria endemic communities with highly mobile populations in Eastern Cambodia. Methods:From October 24th 2021 to February 26th 2023, two successive rounds of PvSeroTAT were conducted in adult and adolescent males in three villages of Mondolkiri, Eastern Cambodia. At each round, capillary blood samples were collected from consenting participants to be used for P. vivax serology and G6PD activity determination. Seropositive participants, who were G6PD normal, were then recontacted to be provided an anti-hypnozoite primaquine regimen following Cambodian treatment guidelines (0.25 mg/kg for 14 days). Cross-sectional surveys to evaluate P. vivax prevalence were conducted before, during and after the PvSeroTAT interventions in the same three villages and in three additional neighboring control villages where interventions were not implemented. Findings:Participation was high, with 96% (456/477) of eligible individuals enrolled in at least one round of PvSeroTAT. However, only 63% of participants enrolled in the first PvSeroTAT round agreed to participate in the second round. In the first and second round of PvSeroTAT, 31% (101/327) and 30% (98/334) of enrolled participants, respectively, were seropositive and among those, 82% (163/199) were eligible for primaquine treatment. All 163 seropositive eligible individuals could be recontacted and offered a primaquine treatment, this occurred within 10 days for 96% of individuals (157/163). P. vivax prevalence decreased in all villages, including the control ones, after the first round of PvSeroTAT from 7.7% to 2.7% overall. Interpretation:The participation rates and overall technical feasibility of PvSeroTAT in highly mobile individuals living within communities in malaria endemic areas of Cambodia were very promising. PvSeroTAT with a lab-based assay is feasible in Cambodia even if it is logistically more challenging than using point-of-care assays. Further studies to understand community perspectives about test and treat approaches in the absence of clinical symptoms will be important for the development of tailored community education and awareness material to improve participation in multiple rounds of test and treat interventions. Funding:The PvSeroTAT interventions received funding from the Global Fund RAI3 initiative. Cross-sectional surveys were funded by the NIH International Centers of Excellence for Malaria Research (ICEMR) Asia-Pacific (U19AI129392).

Background:The WHO malaria treatment guidelines recommend a total dose in the range of 3·5 to 7·0 mg/kg of primaquine to eliminate Plasmodium vivax (P. vivax) hypnozoites and prevent relapses. There are however indications that for tropical P. vivax isolates, notably from Southeast Asia, the lower dose of 3·5 mg/kg is insufficient. Determining the most effective regimen to eliminate P. vivax hypnozoites is needed to achieve elimination of this malaria parasite. Methods:We conducted an open-label randomised controlled trial in Kampong Speu province, Western Cambodia. P. vivax infected patients with uncomplicated malaria, diagnosed at the community level or in health centres of the province, were offered to participate. Patients aged less than 15 years old, and pregnant or breastfeeding women were excluded. Enrolled patients were treated with a blood schizonticidal artesunate regimen of 2 mg/kg/day for 7 days. Upon enrolment, patients' glucose-6-phosphate dehydrogenase (G6PD) activity was determined. G6PD normal patients were randomly assigned (2:2:1) to receive either (i) 3·5 mg/kg (low dose as 0·25 mg/kg/day) or (ii) 7·0 mg/kg (high dose as 0·5 mg/kg/day) of primaquine over 14 days or (iii) no primaquine as comparator arm. G6PD deficient patients were assigned to the no-primaquine comparator arm. Randomisation was done by blocks of 5 using sealed envelopes. Upon enrolment, patients were relocated to the study site in Aoral town where no malaria transmission occurs to ensure that they were not reinfected during their 90-day follow-up. After 90 days of relocation, G6PD normal patients in the no-primaquine arm were provided 3·5 mg/kg for 14 days of primaquine to be taken unsupervised. At day 90, all the patients returned home and they were further followed monthly for three months until day 180. The primary outcome was the treatment failure rate defined as the proportion of patients with at least one P. vivax recurrence within 90 days of relocated follow-up. All patients that completed treatment and complied with relocation without interruption before any recurrence was detected were included in the primary efficacy analysis. All patients enrolled and assigned to an intervention arm were included in the safety analysis. The study is registered on ClinicalTrials.gov (NCT04706130). Findings:Between Nov 10, 2021, and Feb 10, 2024, a total of 160 patients were enrolled and 156 were allocated to one of the three study arms. Of these, 37 G6PD deficient patients were assigned to the no primaquine arm and 119 G6PD normal patients were randomised: 24 in the no primaquine arm, 49 in the primaquine 3·5 mg/kg arm, and 46 in the primaquine 7·0 mg/kg arm. The proportion of participants with at least one P. vivax recurrence within 90 days in the no primaquine arm was 81·4% (95% CI 69·6-89·2). The proportion of participants with recurrence was higher in the low dose primaquine arm (24·4%, 95% CI 14·2-38·7) compared to the high primaquine arm (4·7%, 95% CI 0·8-15·5, p=0·0141) resulting in a hazard ratio of high dose primaquine compared to low dose of 0·17 (95% CI 0·04-0·79, p=0·0229). Both primaquine arms were well tolerated. Interpretation:Not providing primaquine to patients led to a considerable rate of P. vivax recurrence. The risk of P. vivax recurrence was 5·9 times lower for the 7·0 mg/kg of primaquine treatment compared to 3·5 mg/kg. Tolerability and safety of both primaquine regimens in G6PD normal individuals was comparable. Policy makers in Cambodia and most likely in other Southeast Asian countries should endorse the 7·0 mg/kg of primaquine regimen to reduce the risk of P. vivax relapses. Funding:National Institutes of Health (R01AI146590).

BACKGROUND:Mosquitoes are important drivers of infectious diseases transmission, with Anopheles mosquitoes being responsible of malaria transmission. In Cambodia, where malaria is prevalent in forested regions, understanding the ecology of these vectors is crucial. This study aimed to investigate the abundance, distribution, seasonal patterns, biting behaviour of Anopheles mosquitoes, and prevalence of Plasmodium, in Mondulkiri province, Northeastern Cambodia. METHODS:Conducted in 9 sites, seven in forested and two in neighbouring villages, over one year, the collection of Anopheles mosquitoes was made hourly for a 72-h period every month, using a human-baited double net trap. Each mosquito was collected using a mouth-aspirator and identified morphologically, and screened for the presence of Plasmodium. RESULTS:Primary vectors, including Anopheles baimaii, Anopheles dirus, Anopheles maculatus, and Anopheles minimus, constituted 11.1% of all female mosquitoes, while 12 secondary vector species represented 29.4% of the overall collection. Anopheles species were more prevalent during the late rainy season (August to November), with year-round activity observed. Primary vectors were predominantly found in forest sites, while other vector species were found in both village and forest environments. Notably, primary vectors exhibited a preference for nocturnal biting, yet a significant proportion (19.2%) displayed daytime activity, highlighting a potential risk of daytime malaria transmission. Among 5,056 Anopheles specimens tested, only 36 Plasmodium spp.-infected samples were detected, mainly in forest sites (94%), and in specimens collected at night. This study provides essential insights into the ecology of Anopheles in Mondulkiri Forest. CONCLUSIONS:The identification of primary and secondary vectors, their seasonal dynamics, and biting behaviour contribute to enhances our understanding of malaria transmission risks in these areas, guiding future strategies toward effective and context-specific control measures, while stressing the need for individual protection during daytime.

Plasmodium vivax malaria remains a significant global health challenge, complicated by the parasite's ability to form dormant liver stages (hypnozoites) that cause relapses. Radical cure of P. vivax malaria requires administration of a hypnozoitocidal drug, such as primaquine or tafenoquine. However, these drugs can cause severe haemolysis in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency. G6PD deficiency is caused by more than 230 different variants at the gene level that confer different degrees of deficiency phenotypically. Understanding the distribution of different G6PD variants in affected populations is essential to inform safer antimalarial treatment strategies. This study aimed to develop a cost-effective sequencing assay targeting key regions of the G6PD gene, suitable for field deployment. A novel assay based on Nanopore technology was designed to amplify two amplicons covering exon 3 to exon 13, focusing on known variants associated with enzyme deficiency. A total of 79 samples from individuals in Cambodia, Vietnam, Afghanistan, and China were sequenced, and a bioinformatics pipeline was created for the targeted variant calling of 192 G6PD SNP mutations. The assay demonstrated reliable detection of known variants, with high concordance between runs, within runs, and with Sanger sequencing. The Nanopore MinION long-amplicon sequencing assay offers a robust and portable solution for large-scale G6PD genotyping in low-resource settings, that will improve malaria control and elimination strategies by enabling safer antimalarial treatment.

BACKGROUND:Most malaria-endemic countries, including Cambodia, use a total dose of 3·5 mg/kg of primaquine to eliminate Plasmodium vivax hypnozoites and prevent relapses. There are, however, indications that the lower dose of 3·5 mg/kg is insufficient for tropical P vivax isolates, particularly in southeast Asia, and WHO now recommends a total dose of 7·0 mg/kg in most countries. We aimed to determine the most effective regimen to eliminate P vivax hypnozoites to support elimination efforts of this malaria parasite. METHODS:We conducted an open-label, randomised controlled trial in Kampong Speu province, western Cambodia. Patients infected with P vivax aged at least 15 years were offered to participate. Exclusion criteria were severe malaria or other diseases requiring treatment, low haemoglobin (<8·0 g/dL), pregnancy or breastfeeding, sensitivity to study drugs, and use of antimalarials in the preceding month. Enrolled patients were treated with an artesunate regimen of 2 mg/kg per day for 7 days. Patients with normal glucose-6-phosphate dehydrogenase (G6PD) levels were randomly assigned (2:2:1) to receive 3·5 mg/kg (low dose [0·25 mg/kg per day]), 7·0 mg/kg (high dose [0·5 mg/kg per day]), or no primaquine for 14 days. Patients with deficient G6PD levels were assigned to the no primaquine comparator arm. Patients were relocated to the study site in Aoral town where no malaria transmission occurs to ensure that they were not reinfected during their 90-day follow-up. After 90 days of relocation, G6PD-normal patients in the no primaquine arm were provided 3·5 mg/kg of primaquine for 14 days to be taken unsupervised. At day 90, relocation was terminated, and patients were followed up monthly for 3 months until day 180. The primary outcome was P vivax recurrence within 90 days of relocated follow-up, assessed in all patients who completed treatment and complied with relocation without interruption. All patients enrolled and assigned to an intervention arm were included in the safety analysis. The study is registered on ClinicalTrials.gov and recruitment is completed (NCT04706130). FINDINGS:Between Nov 10, 2021, and Feb 10, 2024, 160 patients were enrolled and 147 were included in the primary analysis-59 were assigned to the no primaquine arm (37 assigned as G6PD deficient [median age 22 years, IQR 18-28]; 22 randomly assigned [18, 17-25]), 45 to the low-dose primaquine arm (23, 19-30), and 43 to the high-dose primaquine arm (22, 18-25). Participants were mostly male (135 [92%] of 147) and all Cambodian. 48 (81% [95% CI 69·6-89·2]) participants in the no primaquine arm had at least one P vivax recurrence within 90 days, as did 11 (24%, 14·2-38·7) in the low-dose group and two (5%, 0·8-15·5) in the high-dose group (p=0·0141 for high vs low). After imputation for missing data, low-dose primaquine remained associated with more recurrences than high-dose primaquine (hazard ratio 0·17 [95% CI 0·04-0·79], p=0·0229). Both primaquine regimens were well tolerated with no serious adverse events reported. INTERPRETATION:Not providing primaquine to patients led to a considerable rate of P vivax recurrence. The risk of P vivax recurrence was substantially lower for 7·0 mg/kg primaquine treatment compared with 3·5 mg/kg. Tolerability and safety of both primaquine regimens in G6PD normal individuals was comparable. FUNDING:US National Institutes of Health (R01AI146590).

Trichothecenes (TCNs) are a large group of tricyclic sesquiterpenoid mycotoxins that have intriguing structural features and remarkable biological activities. Herein, we focused on three TCNs (anguidine, verrucarin A, and verrucarol) and their ability to target both the blood and liver stages of Plasmodium species, the parasite responsible for malaria. Anguidine and verrucarin A were found to be highly effective against the blood and liver stages of malaria, while verrucarol had no effect at the highest concentration tested. However, these compounds were also found to be cytotoxic and, thus, not selective, making them unsuitable for drug development. Nonetheless, they could be useful as chemical probes for protein synthesis inhibitors due to their direct impact on parasite synthesis processes.