Cambodia has targeted malaria elimination by 2025. As the malaria burden has decreased in Cambodia, transmission has become more focal, and Plasmodium vivax has become the predominant species. The recurrent nature of P. vivax, due to its dormant liver stages causing relapses, is the main obstacle to malaria elimination in Cambodia. In 2021, Cambodia’s National Center for Parasitology, Entomology and Malaria Control (CNM) rolled out low-dose 14-day primaquine (total dose 3.5 mg/kg) supported by point-of-care quantitative testing for glucose-6-phosphate dehydrogenase deficiency. However, this treatment is limited by poor adherence to its prolonged duration and suboptimal efficacy of the low total dose. The EFFORT clinical trial was conducted in four malaria-endemic countries, including Cambodia, to assess the safety and effectiveness of a 7-day unsupervised high-dose course of primaquine (7 mg/kg total dose) and single dose tafenoquine (300 mg) compared to 14-day unsupervised low-dose primaquine for the treatment of patients presenting with P. vivax malaria. In addition, data were collected on the feasibility and cost-effectiveness of these treatment options. CNM organized the national dissemination of the EFFORT study results on March 27, 2025, to inform key stakeholders and discuss the implications of the study findings for policy and practice in Cambodia.

BackgroundBy 2015, the emergence and dissemination of multidrug-resistant Plasmodium falciparum in the Greater Mekong Subregion threatened regional and global malaria control efforts. In response, Greater Mekong Subregion countries committed to malaria elimination by 2030, with strengthened surveillance as a strategic pillar. In 2017, Cambodia introduced an elimination-oriented digital Malaria Information System (MIS). Its health center app enables real-time, geo-located, case-based malaria reporting across primary health centers, and is fully integrated with the MIS. ObjectiveThis study aimed to evaluate the real-world national implementation of Cambodia’s Android-based health center app, considering coverage, fidelity, timeliness, and data use, and their effects on malaria surveillance performance, case management, programmatic response, and public health outcomes. MethodsSystem performance and public health use were assessed using system-generated metadata, national surveillance data, and user surveys. Operational indicators included technical performance, data completeness, and reporting timeliness, alongside surveillance outcomes such as case notification, classification, reactive case detection, and foci investigation. Nationwide user experience was measured via a survey of 761 health centers across 21 provinces, with in-depth structured surveys at 9 health centers in 3 provinces. Descriptive analyses evaluated system functionality, contribution to malaria surveillance and response, and usability among frontline health workers. ResultsThe health center app demonstrated strong technical performance, with rapid loading and resilient data transmission under low-bandwidth conditions, supporting reliable reporting in resource-constrained settings. Integrated real-time dashboards provided analytics for case management, surveillance monitoring, risk stratification, and targeted public health interventions. Data completeness remained high (99%, 89/90 fields in 2024), demonstrating consistent routine use even as case incidence declined. Between January 1, 2025, and July 31, 2025, 69 malaria cases were reported nationally (23 locally acquired, 7 domestically imported, and 39 internationally imported). Of these, 95.7% (66/69) were notified and classified within 1 day. Reactive case detection was completed within 3 days for all 21 eligible cases, and 16 of 19 eligible foci received a response within 7 days, indicating strong operational responsiveness. User surveys showed 96.3% (733/761) of health centers were satisfied or very satisfied, 90.1% (686/761) reported rare or no technical issues, and 91.7% (698/761) found the app easy to navigate. Operational challenges included limited internet connectivity, transport to remote areas, and electricity interruptions. In-depth surveys confirmed high uptake, confidence in reporting, and routine use of surveillance data, although gaps in local analytical capacity were identified. ConclusionsDeveloped and managed locally to enhance sustainability, the MIS drove significant reductions in malaria case incidence, with the health center app contributing timely, complete, structured reporting at the point of care. Public health responses were facilitated by real-time analysis, targeted interventions, and decentralized decision-making. User engagement was sustained as malaria cases declined, and further enhancements are planned to ensure seamless transition to postelimination surveillance, reducing the risk of malaria reestablishment in Cambodia.

Abstract Between 2015 and 2025, Cambodia reported a 99.9% decline in the number of cases of malaria. To aid acceleration of elimination, the National Center for Parasitology, Entomology and Malaria Control (CNM) implemented a package of interventions known as the Last Mile (LM) elimination program. The aim of this study was to determine the impact of the LM program on case numbers and evaluate the coverage of interventions. LM was rolled out between November 2020 and December 2023 in villages reporting a locally acquired case of Plasmodium falciparum or mixed infection with P. falciparum and P. vivax and included combinations of targeted drug administration (TDA), intermittent preventative treatment for forest goers (IPTf), active fever screening (AFS), the recruitment of a village or mobile malaria worker (VMW/MMW) and the top-up of insecticide-treated bed nets (ITN) depending on the vulnerability and receptivity of the village. A total of 103 full and 82 partial villages in seven provinces were included. Two rounds of TDA were administered, with a total of 10,678 individuals (67.6%) given during the first round and 9,678 (62.3%) during the second round. Coverage varied by province with none meeting the recommended threshold of 80%. IPTf was implemented each month among 35% (n=35) of full LM villages and 56% (n=42) of partial LM villages. A total of 11.7% (n=12) of full LM villages implemented AFS consistently on a weekly basis. Controlled interrupted time series showed no statistically significant difference in the number of malaria cases before and after the implementation of LM. Although we were unable to prove a statistically significant impact of LM, likely due to the small number of cases prior to LM, it is important to add to the limited evidence-based for Accelerator Strategies in countries approaching the elimination of malaria. Furthermore, findings from the feasibility and impact of individual interventions were used to change policy at the national level.

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.

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.

Cambodia is approaching the final stages of malaria elimination, reporting only 1,384 cases in 2023 and aiming to eliminate all species of human malaria by 2025. Given the importance of surveillance in control and elimination efforts, the National Center for Parasitology, Entomology, and Malaria Control (CNM) elected to assess its malaria surveillance system to identify strengths and gaps and make recommendations. The WHO Malaria Surveillance Assessment Toolkit was utilized to evaluate 68 indicators across four primary objectives: Performance, Context and Infrastructure, Technical and Process, and Behavior. Data from 2021-2023 and activities which took place in 2023 under the 2021 edition of Cambodia's Surveillance for Malaria Elimination Guidelines were utilized. Indicators were evaluated through a survey completed by subnational staff, a field data quality assessment (DQA) at the service delivery level, a national-level DQA, and a national-level desk review including interviews with CNM staff. The assessment demonstrated strong performance of Cambodia's surveillance system. Of the core indicators assessed, 82% of Performance (14/19), 77% of Context and Infrastructure (8/13), 79% of Technical and Process (4/7), and 100% of Behavior indicators (3/3) were met. Notable successes included the improved completeness and timeliness of reporting and case investigation in the previous three years. Key areas for improvement include revising case investigation and updating case classifications, enhancing data cleaning and verification processes to improve data quality, and upgrading the national malaria information system to include unique patient identifiers and enable interoperability with other national information systems. Cambodia's surveillance system is strong overall and CNM is committed to addressing identified areas of improvement. Lessons learned from this assessment informed revisions to the national Surveillance for Malaria Elimination Guidelines, implemented in May 2024. Additionally, the results will be used to guide the design of surveillance and response-related objectives for Cambodia's 2026-2035 National Strategic Plan for malaria.

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.

Responding to the emergence of Plasmodium falciparum partial resistance to artemisinins and partner drugs of artemisinin-based combination therapies in the Greater Mekong subregion (GMS) of southeast Asia, the Regional Artemisinin-resistance Initiative (RAI) was established in 2014 and has made remarkable progress in eliminating falciparum malaria. In Cambodia, Laos, and Viet Nam, the number of malaria cases has declined from hundreds of thousands in 2010 to 2313 cases in 2023, with only 246 caused by falciparum malaria. The key components of this success have been an effective package of interventions curbing malaria transmission, with an emphasis on early diagnosis and treatment in hard-to-reach populations through an extended and well organised network of community and mobile malaria workers; improved surveillance systems; and evidence-driven implementation of intensified approaches such as active case detection, chemoprevention in specific risk groups, and targeted drug administration. The RAI is funded by the Global Fund to Fight AIDS, Tuberculosis and Malaria and governed by a closely collaborating Regional Steering Committee, including technical partners, key development partners, and stakeholders from ministries of health, national malaria control programmes, civil society organisations, the private sector, academia, and regional multilateral organisations. The RAI has brought the countries of the eastern GMS close to eliminating P falciparum, the deadliest malaria-causing Plasmodium species. Nonetheless, a worrying rise in malaria cases in Myanmar with cross-border spillover requires urgent action. Lessons learned from the RAI's approach to antimalarial drug resistance in the GMS can inform countries in sub-Saharan Africa, where artemisinin partial resistance has now also emerged.

Malaria incidence in the Lao People’s Democratic Republic has declined over the past 10 years. There is a continued risk of outbreaks, particularly in the Southern region, due to high-risk behaviors, primarily in remote ethnic communities among forest goers (individuals who regularly work or sleep in the forest), farmers on forest fringes, and vulnerable populations in these highly receptive areas. Conventional malaria control interventions alone in these areas are insufficient to push elimination beyond “the last mile”. In 2022, an innovative approach to accelerate malaria elimination, termed locally as “Accelerator Strategies” was implemented. Activities included targeted drug administration and intermittent preventive treatment for forest goers and mobile populations, specifically farmers on forest fringes, as chemoprevention among individuals at increased risk of malaria irrespective of infectious status. Community engagement approaches were essential to ensure participation and acceptance as the intervention requires individuals without symptoms to take medicine. Three key enablers for community participation were identified as: (1) Service delivery and community engagement by the community members themselves; (2) Strong advocacy and political commitment from senior local political leaders, and village authorities and influencers and (3) Delivering people-centered services beyond the village with granular local data on risk behaviors, population movement and geographic information system mapping. Early and sustained community engagement resulted in high coverage of the interventions and greater acceptance by the community that resulted in a decreased malaria burden.

Artemisinin-based combination therapies (ACTs) were introduced as the standard of care for uncomplicated malaria in Africa almost two decades ago. Recent studies in East Africa have reported a gradual increase in kelch13 (k13) mutant parasites associated with reduced artesunate efficacy. As part of the Community Access to Rectal Artesunate for Malaria project, we collected blood samples from 697 children with signs of severe malaria in northern Uganda between 2018 and 2020, before and after the introduction of rectal artesunate (RAS) in 2019. K13 polymorphisms were assessed, and parasite editing and phenotyping were performed to assess the impact of mutations on parasite resistance. Whole-genome sequencing was performed, and haplotype networks were constructed to determine the geographic origin of k13 mutations. Of the 697 children, 540 were positive for Plasmodium falciparum malaria by PCR and were treated with either RAS or injectable artesunate monotherapy followed in most cases by ACT. The most common k13 mutation was C469Y (6.7%), which was detected more frequently in samples collected after RAS introduction. Genome editing confirmed reduced in vitro susceptibility to artemisinin in C469Y-harboring parasites compared to wild-type controls (P < 0.001). The haplotypic network showed that flanking regions of the C469Y mutation shared the same African genetic background, suggesting a single and indigenous origin of the mutation. Our data provide evidence of selection for the artemisinin-resistant C469Y mutation. The realistic threat of multiresistant parasites emerging in Africa should encourage careful monitoring of the efficacy of artemisinin derivatives and strict adherence to ACT treatment regimens.

BACKGROUND:Plasmodium falciparum is an apicomplexan parasite responsible for lethal cases of malaria. According to WHO recommendations, P falciparum cases are treated with artemisinin-based combination therapy including dihydroartemisinin-piperaquine. However, the emergence of resistant parasites against dihydroartemisinin-piperaquine was reported in southeast Asia in 2008 and, a few years later, suspected in South America. METHODS:To characterise resistance emergence, a treatment efficacy study was performed on the reported patients infected with P falciparum and treated with dihydroartemisinin-piperaquine in French Guiana (n=6, 2016-18). Contemporary isolates collected in French Guiana were genotyped for P falciparum chloroquine resistance transporter (pfCRT; n=845) and pfpm2 and pfpm3 copy number (n=231), phenotyped using the in vitro piperaquine survival assay (n=86), and analysed through genomic studies (n=50). Additional samples from five Amazonian countries and one outside the region were genotyped (n=1440). FINDINGS:In field isolates, 40 (47%) of 86 (95% CI 35·9-57·1) were resistant to piperaquine in vitro; these phenotypes were more associated with pfCRTC350R (ie, Cys350Arg) and pfpm2 and pfpm3 amplifications (Dunn test, p<0·001). Those markers were also associated with dihydroartemisinin-piperaquine treatment failure (n=3 [50%] of 6). A high prevalence of piperaquine resistance markers was observed in Suriname in 19 (83%) of 35 isolates and in Guyana in 579 (73%) of 791 isolates. The pfCRTC350R mutation emerged before pfpm2 and pfpm3 amplification in a temporal sequence different from southeast Asia, and in the absence of artemisinin partial resistance, suggesting a geographically distinctive epistatic relationship between these genetic markers. INTERPRETATION:The high prevalence of piperaquine resistance markers in parasite populations of the Guianas, and the risk of associated therapeutic failures calls for caution on dihydroartemisinin-piperaquine use in the region. Furthermore, greater attention should be given to potential differences in genotype to phenotype mapping across genetically distinct parasite populations from different continents. FUNDING:Pan American Health Organization and WHO, French Ministry for Research, European Commission, Santé publique France, Agence Nationale de la Recherche, Fundação de Amparo à Pesquisa do Estado do Amazonas, Ministry of Health of Brazil, Oswaldo Cruz Foundation, and National Institutes of Health. TRANSLATIONS:For the French and Portuguese translations of the abstract see Supplementary Materials section.

BACKGROUNDS:The therapeutic efficacy studies of DHA-PIP for uncomplicated Plasmodium falciparum patients were implemented from 2012 to 2016 along China (Yunnan province)-Myanmar border, which verified the high efficacy of DHA-PIP. With the samples collected in these studies, the genetic characteristics of P. falciparum parasites based on in vivo parasite clearance time (PCT) was investigated to explore if these parasites had developed resistance to DHA and PIP at molecular level. METHODS:The genetic characteristics were investigated based on K13 genotypes, copy numbers of genes pfpm2 and pfmdr1, and nine microsatellite loci (Short Tandem Repeats, STR) flanking the K13 gene on chromosome 13. The PCT 50s were compared based on different K13 genotypes, sites, periods and copy numbers. RESULTS:In the NW (North-West Yunnan province bordering with Myanmar) region, F446I was the main K13 genotype. No significant differences for PCT 50s presented among three K13 genotypes. In SW (South-West Yunnan province bordering with Myanmar) region, only wild K13 genotype was detected in all parasite isolates whose PCT 50s was significantly longer than those in NW region. For the copy numbers of genes, parasite isolates containing multiple copies of pfmdr1 gene were found in both regions, but only single copy of pfpm2 gene was detected. Though the prevalence of parasite isolates with multiple copies of pfmdr1 gene in SW region was higher than that in NW region, no difference in PCT 50s were presented between isolates with single and multiple copies of pfmdr1 gene. The median He values of F446I group and Others (Non-F446I K13 mutation) group were 0.08 and 0.41 respectively. The mean He values of ML group (Menglian County in SW) and W (wild K13 genotype in NW) group were 0 and 0.69 respectively. The mean Fst values between ML and W groups were significantly higher than the other two K13 groups. CONCLUSIONS:P. falciparum isolates in NW and SW regions had very different genetic characteristics. The F446I was hypothesized to have independently appeared and spread in NW region from 2012 and 2016. The high susceptibility of PIP had ensured the efficacy of DHA-PIP in vivo. Multiple copy numbers of pfmdr1 gene might be a potential cause of prolonged clearance time of ACTs drugs along China-Myanmar border. TRIAL REGISTRATION:Trial registration: ISRCTN, ISRCTN 11775446. Registered 17 April 2020-Retrospectively registered, the registered name was Investigating resistance to DHA-PIP for the treatment of Plasmodium falciparum malaria and chloroquine for the treatment of Plasmodium vivax malaria in Yunnan, China. http://www.isrctn.com/ISRCTN11775446.

We describe the MalariaGEN Pf7 data resource, the seventh release of Plasmodium falciparum genome variation data from the MalariaGEN network. It comprises over 20,000 samples from 82 partner studies in 33 countries, including several malaria endemic regions that were previously underrepresented. For the first time we include dried blood spot samples that were sequenced after selective whole genome amplification, necessitating new methods to genotype copy number variations. We identify a large number of newly emerging crt mutations in parts of Southeast Asia, and show examples of heterogeneities in patterns of drug resistance within Africa and within the Indian subcontinent. We describe the profile of variations in the C-terminal of the csp gene and relate this to the sequence used in the RTS,S and R21 malaria vaccines. Pf7 provides high-quality data on genotype calls for 6 million SNPs and short indels, analysis of large deletions that cause failure of rapid diagnostic tests, and systematic characterisation of six major drug resistance loci, all of which can be freely downloaded from the MalariaGEN website.

Background In early 2016, in Preah Vihear, Northern Cambodia, artesunate/mefloquine was used to cope with dihydroartemisinin/piperaquine-resistant Plasmodium falciparum parasites. Following this policy, P. falciparum strains harbouring molecular markers associated with artemisinin, piperaquine and mefloquine resistance have emerged. However, the lack of a viable alternative led Cambodia to adopt artesunate/mefloquine countrywide, raising concerns about a surge of triple-resistant P. falciparum strains. Objectives To assess the prevalence of triple-resistant parasites after artesunate/mefloquine implementation countrywide in Cambodia and to characterize their phenotype. Methods For this multicentric study, 846 samples were collected from 2016 to 2019. Genotyping of molecular markers associated with artemisinin, piperaquine and mefloquine resistance was coupled with phenotypic analyses. Results Only four triple-resistant P. falciparum isolates (0.47%) were identified during the study period. These parasites combined the pfk13 polymorphism with pfmdr1 amplification, pfpm2 amplification and/or pfcrt mutations. They showed significantly higher tolerance to artemisinin, piperaquine and mefloquine and also to the mefloquine and piperaquine combination. Conclusions The use of artesunate/mefloquine countrywide in Cambodia has not led to a massive increase of triple-resistant P. falciparum parasites. However, these parasites circulate in the population, and exhibit clear resistance to piperaquine, mefloquine and their combination in vitro. This study demonstrates that P. falciparum can adapt to more complex drug associations, which should be considered in future therapeutic designs.

BACKGROUND Although the clinical efficacy of antimalarial artemisinin-based combination therapies in Africa remains high, the recent emergence of partial resistance to artemisinin in Plasmodium falciparum on the continent is troubling, given the lack of alternative treatments. METHODS In this study, we used data from drug-efficacy studies conducted between 2016 and 2019 that evaluated 3-day courses of artemisinin-based combination therapy (artesunate-amodiaquine or artemether-lumefantrine) for uncomplicated malaria in Eritrea to estimate the percentage of patients with day-3 positivity (i.e., persistent P. falciparum parasitemia 3 days after the initiation of therapy). We also assayed parasites for mutations in Pfkelch13 as predictive markers of partial resistance to artemisinin and screened for deletions in hrp2 and hrp3 that result in variable performance of histidine rich protein 2 (HRP2)-based rapid diagnostic tests for malaria. RESULTS We noted an increase in the percentage of patients with day-3 positivity from 0.4% (1 of 273) in 2016 to 1.9% (4 of 209) in 2017 and 4.2% (15 of 359) in 2019. An increase was also noted in the prevalence of the Pfkelch13 R622I mutation, which was detected in 109 of 818 isolates before treatment, from 8.6% (24 of 278) in 2016 to 21.0% (69 of 329) in 2019. The odds of day-3 positivity increased by a factor of 6.2 (95% confidence interval, 2.5 to 15.5) among the patients with Pfkelch13 622I variant parasites. Partial resistance to artemisinin, as defined by the World Health Organization, was observed in Eritrea. More than 5% of the patients younger than 15 years of age with day-3 positivity also had parasites that carried Pfkelch13 R622I. In vitro, the R622I mutation conferred a low level of resistance to artemisinin when edited into NF54 and Dd2 parasite lines. Deletions in both hrp2 and hrp3 were identified in 16.9% of the parasites that carried the Pfkelch13 R622I mutation, which made them potentially undetectable by HRP2-based rapid diagnostic tests. CONCLUSIONS The emergence and spread of P. falciparum lineages with both Pfkelch13-mediated partial resistance to artemisinin and deletions in hrp2 and hrp3 in Eritrea threaten to compromise regional malaria control and elimination campaigns. (Funded by the Bill and Melinda Gates Foundation and others; Australian New Zealand Clinical Trials Registry numbers, ACTRN12618001223224, ACTRN12618000353291, and ACTRN12619000859189.).

Abstract Background Artesunate–amodiaquine (ASAQ) and Artemether–lumefantrine (AL) are the recommended treatment for uncomplicated Plasmodium falciparum malaria in Liberia. Intermittent preventive treatment with sulfadoxine/pyrimethamine is also recommended for pregnant women. The therapeutic efficacy of Artesunate–amodiaquine and Artemether–lumefantrine, and the frequency of molecular markers associated with anti-malarial drug resistance were investigated. Methods The therapeutic efficacy of ASAQ and AL was evaluated using the standard World Health Organization protocol (WHO. Methods for Surveillance of Antimalarial Drug Efficacy. Geneva: World Health Organization; 2009. https://www.who.int/malaria/publications/atoz/9789241597531/en/ ). Eligible children were recruited and monitored clinically and parasitologically for 28 days. Polymorphisms in the Pfkelch 13, chloroquine resistance transporter (Pfcrt), multidrug resistance 1 (Pfmdr-1), dihydrofolate reductase (Pfdhfr), and dihydropteroate synthase (Pfdhps) genes and copy number variations in the plasmepsin-2 (Pfpm2) gene were assessed in pretreatment samples. Results Of the 359 children enrolled, 180 were treated with ASAQ (89 in Saclepea and 91 in Bensonville) and 179 with AL (90 in Sinje and 89 in Kakata). Of the recruited children, 332 (92.5%) reached study endpoints. PCR-corrected per-protocol analysis showed ACPR of 90.2% (95% CI: 78.6–96.7%) in Bensonville and 92.7% (95% CI: 83.4.8–96.5%) in Saclepea for ASAQ, while ACPR of 100% was observed in Kakata and Sinje for AL. In both treatment groups, only two patients had parasites on day 3. No artemisinin resistance associated Pfkelch13 mutations or multiple copies of Pfpm2 were found. Most samples tested had the Pfcrt 76 T mutation (80/91, 87.9%), while the Pfmdr-1 86Y (40/91, 44%) and 184F (47/91, 51.6%) mutations were less frequent. The Pfdhfr triple mutant (51I/59R/108 N) was the predominant allele (49.2%). For the Pfdhps gene, it was the 540E mutant (16.0%), and the 436A mutant (14.3%). The quintuple allele (51I/59R/108 N-437G/540E) was detected in only one isolate (1/357). Conclusion This study reports a decline in the efficacy of ASAQ treatment, while AL remained highly effective, supporting the recent decision by NMCP to replace ASAQ with AL as first-line treatment for uncomplicated falciparum malaria. No association between the presence of the mutations in Pfcrt and Pfmdr-1 and the risk of parasite recrudescence in patients treated with ASAQ was observed. Parasites with signatures known to be associated with artemisinin and piperaquine resistance were not detected. The very low frequency of the quintuple Pfdhfr/Pfdhps mutant haplotype supports the continued use of SP for IPTp. Monitoring of efficacy and resistance markers of routinely used anti-malarials is necessary to inform malaria treatment policy. Trial registration ACTRN12617001064392.

Background and Objective: In 2005, Chad, like several other WHO countries, withdrew chloroquine as a first-line treatment for Plasmodium falciparum malaria in response to WHO recommendations related to the reason for the increase in treatment failures and the global spread of chloroquine resistance. Artemisinin-based combination therapy (ACTs), Artemether-lumefantrine, has replaced chloroquine as the first-choice treatment for malaria. The present study assessed pfcrt polymorphism in Plasmodium falciparum isolates in Massakory. Methodology and Results: Blood samples for PCR analysis were collected on Whatman 3MM filter paper in Massakory during a therapeutic efficacy study (TES) conducted from December 14, 2019 to March 14, 2020. Genomic DNA was extracted from 113 dried blood spots with the QIAamp DNA Micro Kit (Qiagen, Valencia, CA) as per manufacturer’s protocol and amplified by nested-PCR with pfcrt specific primer. The amplification products were revealed by electrophoresis on 2% agarose gel and then sequenced according to Sanger method. A total of 71 sequences were readable. The pfcrt analysis showed that of the 71 readable sequences, high mutation prevalence: 66 (92.96%) IET, 2 (4.22%) IDT and 3 (4.22%) MNK wild pfcrt isolates. Conclusion: These results challenge the highest health authorities in the country. The government, through the Ministry of Public Health and National Solidarity and the National Malaria Control Program, must raise awareness for the effective withdrawal of chloroquine. This action will promote on the one hand the re-emergence of parasites sensitive to chloroquine, and on the other hand make possible the reintroduction of chloroquine in the treatment of simple malaria after the suppression of drug pressure.

Background In the Republic of the Congo, malaria represents a major public health problem affecting all age groups. A regular surveillance of the current efficacy of first-line anti-malarial drugs is required in the face of possible emergence and spread of artemisinin-resistant Plasmodium falciparum strains in Africa. The purpose of this study was to determine the prevalence of malaria among febrile patients of all ages and assess the efficacy of artemether-lumefantrine (AL) and artesunate-amodiaquine (ASAQ) in Congolese children. Methods Febrile patients of all ages were initially screened for malaria by both rapid diagnostic test (RDT) and microscopy. Patients less than 12 years of age, with parasitaemia ≥ 1000 asexual parasites of P. falciparum /µL of blood, without any signs of severity, were enrolled in a therapeutic efficacy study and treated after obtaining their parents' (or legal guardian’s) informed consent in two health centres in Dolisie. The patients were followed for 28 days in accordance with the 2009 World Health Organization standard protocol. If parasitaemia reappeared on or after day 7, the genetic profiles (genes expressing merozoite surface protein-1 [ msp1 ], merozoite surface protein-2 [ msp2 ], and glutamine-rich protein [ glurp ]) of pre-treatment and post-treatment isolates were compared by nested polymerase chain reaction (PCR) followed by capillary electrophoresis to make a distinction between recrudescence and re-infection. The clinical and parasitological outcome was analysed by the per-protocol method and Kaplan–Meier survival curves. Results A total of 994 febrile patients of all ages were screened by RDT and microscopy. Of 994 patients, 323 (32.5%) presented a positive RDT, and 266 (26.8%) were microscopy-positive. Based on microscopy as the reference diagnostic method, the sensitivity and the specificity of the RDT were 98.9 and 91.8%, respectively. The Cohen’s kappa coefficient was 0.86. A total of 121 children aged less than 12 years (61 in AL treatment group and 60 in ASAQ treatment group) were included in therapeutic efficacy study. Before PCR correction, the proportions of adequate clinical and parasitological response were 96.6% for AL and 86.0% for ASAQ in the per-protocol population ( P < 0.05). The PCR-corrected efficacy rates were 98.2% and 94.2% for AL and ASAQ, respectively ( P > 0.05). Both treatments were well tolerated. Conclusions AL and ASAQ remain highly effective for the first-line treatment of uncomplicated P. falciparum malaria in Dolisie. Despite high efficacy of first- and second-line treatment, there is a continuing need to scale up effective malaria preventive interventions and vector control strategies in the country. Trial Registration Number: ACTRN12616001422415.

Additional file 2. Methodological quality assessment of interventional studies.

Background: National Malaria Control Programmes (NMCPs) currently make limited use of parasite genetic data. We have developed GenRe-Mekong, a platform for genetic surveillance of malaria in the Greater Mekong Subregion (GMS) that enables NMCPs to implement large-scale surveillance projects by integrating simple sample collection procedures in routine public health procedures. Methods: Samples from symptomatic patients are processed by SpotMalaria, a high-throughput system that produces a comprehensive set of genotypes comprising several drug resistance markers, species markers and a genomic barcode. GenRe-Mekong delivers Genetic Report Cards, a compendium of genotypes and phenotype predictions used to map prevalence of resistance to multiple drugs. Results: GenRe-Mekong has worked with NMCPs and research projects in eight countries, processing 9623 samples from clinical cases. Monitoring resistance markers has been valuable for tracking the rapid spread of parasites resistant to the dihydroartemisinin-piperaquine combination therapy. In Vietnam and Laos, GenRe-Mekong data have provided novel knowledge about the spread of these resistant strains into previously unaffected provinces, informing decision-making by NMCPs. Conclusions: GenRe-Mekong provides detailed knowledge about drug resistance at a local level, and facilitates data sharing at a regional level, enabling cross-border resistance monitoring and providing the public health community with valuable insights. The project provides a rich open data resource to benefit the entire malaria community. Funding: The GenRe-Mekong project is funded by the Bill and Melinda Gates Foundation (OPP11188166, OPP1204268). Genotyping and sequencing were funded by the Wellcome Trust (098051, 206194, 203141, 090770, 204911, 106698/B/14/Z) and Medical Research Council (G0600718). A proportion of samples were collected with the support of the UK Department for International Development (201900, M006212), and Intramural Research Program of the National Institute of Allergy and Infectious Diseases.