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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., Competing Interests: CJ, NT, MM, RM, HQ, BH, VV, TN, HR, Rv, Lv, RF, FN, MH, NP, SG, PR, KC, PN, EA, SP, RM, RL, CH, LD, KN, TN, TH, AS, DU, CB, CF, MO, TP, RT, CA, AM, GD, JL, DP, NC, DL, SS, JC, PJ, BH, SP, AP, FS, KL, MT, TH, PS, SS, PB, AT, SB, NV, AA, AU, AF, CK, ED, MK, MA, KL, SR, AJ, KR, CH, MD, RV, NK, PW, JA, RP, CA, TC, CM, TN, JS, BJ, JK, KJ, DM, XC, JS, RA, VS, SG, KR, ND, AD, DK, OM No competing interests declared, HN, NZ, SC is an employee of Vysnova Partners Inc

Background: Artesunate-amodiaquine is a potential therapy for uncomplicated malaria in Cambodia., Methods: Between September 2016 and January 2017, artesunate-amodiaquine efficacy and safety were evaluated in a prospective, open-label, single-arm observational study at health centers in Mondulkiri, Pursat, and Siem Reap Provinces, Cambodia. Adults and children with microscopically confirmed Plasmodium falciparum malaria received oral artesunate-amodiaquine once daily for 3 days plus single-dose primaquine, 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). An amodiaquine parasite survival assay (AQSA) was developed and applied to whole genome sequencing results to evaluate potential amodiaquine resistance molecular markers., Results: In 63 patients, day-28 PCR-adjusted ACPR was 81.0% (95% confidence interval [CI], 68.9-88.7). Day 3 parasite positivity rate was 44.4% (28/63; 95% CI, 31.9-57.5). All 63 isolates had the K13(C580Y) marker for artemisinin resistance; 79.4% (50/63) had Pfpm2 amplification. The AQSA resistance phenotype (≥45% parasite survival) was expressed in 36.5% (23/63) of isolates and was significantly associated with treatment failure (P = .0020). Pfmdr1 mutant haplotypes were N86/184F/D1246, and Pfcrt was CVIET or CVIDT at positions 72-76. Additional Pfcrt mutations were not associated with amodiaquine resistance, but the G353V mutant allele was associated with ACPR compared to Pfmdr1 haplotypes harboring F1068L or S784L/R945P mutations (P = .030 and P = .0004, respectively)., Conclusions: For uncomplicated falciparum malaria in Cambodia, artesunate-amodiaquine had inadequate efficacy owing to amodiaquine-resistant P. falciparum. Amodiaquine resistance was not associated with previously identified molecular markers., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America.)

This single-arm trial ( n  = 104) in western Cambodia showed high efficacy for 3-day treatment with pyronaridine-artesunate plus single-dose primaquine in Plasmodium falciparum malaria. Day 42 PCR-adjusted adequate clinical and parasitological response (ACPR) was 98.3% (58/59) (95% confidence interval [CI], 90.9 to 100.0) in Trapeng Chau in Kampong Speu and 100% (41/41) (95% CI, 91.4 to 100) in Veal Veng in Pursat; 80.6% (83/103) of the patients had P. falciparum with drug resistance molecular markers. For Plasmodium vivax malaria, pyronaridine-artesunate day 28 ACPR was 98.3% (59/60) (95% CI, 91.1 to 100) and 100% (60/60) (95% CI, 94.0 to 100), respectively. (This study is registered in the Australian New Zealand Clinical Trials Registry [ANZCTR] under reference no. ACTRN12618001999224.)., (Copyright © 2019 American Society for Microbiology.)

Background: Countries of the greater Mekong subregion have made a transition from malaria control to an aim for falciparum and vivax malaria elimination. The elimination of falciparum malaria will have to be achieved against a background of increasing artemisinin and multi-drug resistance. This ambitious goal requires an operational research (OR) agenda that addresses the dynamic challenges encountered on the path to elimination, which will need to be flexible and developed in close relation with the cambodian national programme for parasitology, entomology and malaria control (CNM). In Cambodia, a number of meetings with stakeholders were convened by the CNM and emergency response to artemisinin resistance (ERAR) hub, producing an initial list of priority OR topics. The process and outcome of these meetings are described, which could serve as a template for other countries in the region., Methods: A landscaping exercise was conducted to gather all past, on-going and planned malaria focussed OR activities conducted by the cambodian research consortium in Cambodia and categorized according to research theme. The six themes included (1) malaria epidemiology, surveillance and response, (2) malaria case management, (3) malaria vector control, (4) malaria behavioural issues, (5) malaria clinical studies, and (6) other vector-borne diseases (dengue, neglected tropical diseases, soil-transmitted helminths). The different themes were discussed in small focus groups, which made an initial prioritization list which was then presented to a plenary group for further discussion. This produced a list of research questions ranked according to priority., Results: OR priorities produced by the thematic groups were discussed in the plenary meeting and given a priority score by group voting. A list of 17 OR questions were developed, finalized and listed, which included questions on surveillance, active case detection and treatment efficacy., Conclusion: This paper describes ERAR's work on supporting Cambodia's transition to malaria elimination by identifying national operational research priorities. ERAR has initiated and currently plays a critical role in the development of country specific research agendas for malaria elimination. The first example of this has been the described exercise in Cambodia, which could serve a template for setting OR priorities in the wider region.

This commentary offers insight into how to best address barriers that may hinder the translation of malaria research findings into policy. It also proposes viable methods of implementing these policies in Cambodia. Currently, a wide range of malaria research is being conducted by in-country stakeholders, including Cambodia's National Programme for Parasitology, Entomology and Malaria Control's (CNM), non-governmental organizations, and academic institutions. Coordinating research amongst these partners, as well as within the Ministry of Health, is a challenge. Results are rarely disseminated widely and seldom inform programme and policy decisions. CNM and its research partners have severely limited access to each other's databases. This lack of accessibility, timeliness, engagement and cooperation between CNM and its partners greatly impacts overall research efficiency in this field, and is stifling innovation both within and beyond CNM. Cambodia has set a goal to eradicate all forms of malaria by 2030. As countries approach the elimination phase, there is a greater need for sharing research-generated evidence amongst partners, in order to ensure that appropriate and impactful activities are conducted. The Cambodian Research Consortium was established to serve as a framework for partners, stakeholders and researchers to share research projects, information and results, and to promote the goals of CNM. The sharing of malaria research results will help to inform prevention, control and elimination activities in the country. It will also determine and address the country's operational research needs, and could potentially become a framework model to be used in other countries aiming to transition from malaria control to elimination.

BackgroundNational 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.MethodsSamples 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.ResultsGenRe-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.ConclusionsGenRe-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.FundingThe 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.

Southeast Asia (SEA) emerged relatively unscathed from the first year of the global SARS-CoV-2 pandemic, but as of July 2021 the region is experiencing a surge in case numbers primarily driven by Alpha (B.1.1.7) and subsequently the more transmissible Delta (B.1.617.2) variants. While initial disease burden was mitigated by swift government responses, favorable cultural and societal factors, the more recent rise in cases suggests an under-appreciation of prior prevalence and over-appreciation of possible cross-protective immunity from exposure to endemic viruses, and highlights the effects of vaccine rollout at varying tempos and of variable efficacy. This burgeoning crisis is further complicated by co-existence of malaria and dengue in the region, with implications of serological cross-reactivity on interpretation of SARS-CoV-2 assays and competing resource demands impacting efforts to contain both endemic and pandemic disease.

Background: Cambodia is the epicentre of resistance emergence for virtually all antimalarial drugs. Selection and spread of parasites resistant to artemisinin-based combination therapy (ACT) is a major threat for malaria elimination, hence the need to renew the pool of effective treatments.Objectives: To determine whether ACT resistance haplotypes could have an effect on ferroquine in vitro antimalarial activity.Methods: In vitro susceptibility to ferroquine was measured for 80 isolates from Cambodia characterized for their molecular resistance profile to artemisinin, piperaquine and mefloquine.Results: Among the 80 isolates tested, the overall median (IQR) IC50 of ferroquine was 10.9 nM (8.7-18.3). The ferroquine median (IQR) IC50 was 8.9 nM (8.1-11.8) for Pfk13 WT parasites and was 12.9 nM (9.5-20.0) for Pfk13 C580Y parasites with no amplification of Pfpm2 and Pfmdr1 genes. The median (IQR) IC50 of ferroquine for Pfk13 C580Y parasites with amplification of the Pfpm2 gene was 17.2 nM (14.5-20.5) versus 9.1 nM (7.9-10.7) for Pfk13 C580Y parasites with amplification of the Pfmdr1 gene.Conclusions: Ferroquine exerts promising efficacy against ACT-resistant isolates. Whereas Pfpm2 amplification was associated with the highest parasite tolerance to ferroquine, the susceptibility range observed was in accordance with those measured in ACT resistance-free areas. This enables consideration of ferroquine as a relevant therapeutic option against ACT-resistant malaria. [ABSTRACT FROM AUTHOR]

Background: In 2012, the World Health Organization recommended the addition of single low-dose primaquine (SLDPQ, 0.25 mg base/kg body weight) to artemisinin combination therapies to block the transmission of Plasmodium falciparum without testing for glucose-6-phosphate dehydrogenase deficiency. The targeted group was non-pregnant patients aged ⩾ 1 year (later changed to ⩾ 6 months) with acute uncomplicated falciparum malaria, primarily in countries with artemisinin-resistant P. falciparum (ARPf). No dosing regimen was suggested, leaving malaria control programmes and clinicians in limbo. Therefore, we designed a user-friendly, age-based SLDPQ regimen for Cambodia, the country most affected by ARPf. Methods: By reviewing primaquine's pharmacology, we defined a therapeutic dose range of 0.15-0.38 mg base/kg (9-22.5 mg in a 60-kg adult) for a therapeutic index of 2.5. Primaquine doses (1-20 mg) were tested using a modelled, anthropometric database of 28,138 Cambodian individuals (22,772 healthy, 4119 with malaria and 1247 with other infections); age distributions were: 0.5-4 years (20.0 %, n = 5640), 5-12 years (9.1 %, n = 2559), 13-17 years (9.1 %, n = 2550), and ⩾ 18 years (61.8 %, n = 17,389). Optimal age-dosing groups were selected according to calculated mg base/kg doses and proportions of individuals receiving a therapeutic dose. Results: Four age-dosing bands were defined: (1) 0.5-4 years, (2) 5-9 years, (3) 10-14 years, and (4) ⩾15 years to receive 2.5, 5, 7.5, and 15 mg of primaquine base, resulting in therapeutic doses in 97.4 % (5494/5640), 90.5 % (1511/1669), 97.7 % (1473/1508), and 95.7 % (18,489/19,321) of individuals, respectively. Corresponding median (1st-99th centiles) mg base/kg doses of primaquine were (1) 0.23 (0.15-0.38), (2) 0.29 (0.18-0.45), (3) 0.27 (0.15-0. 39), and (4) 0.29 (0.20-0.42). Conclusions: This age-based SLDPQ regimen could contribute substantially to malaria elimination and requires urgent evaluation in Cambodia and other countries with similar anthropometric characteristics. It guides primaquine manufacturers on suitable tablet strengths and doses for paediatric-friendly formulations. Development of similar age-based dosing recommendations for Africa is needed. [ABSTRACT FROM AUTHOR]

Background: The declining efficacy of dihydroartemisinin-piperaquine against Plasmodium falciparum in Cambodia, along with increasing numbers of recrudescent cases, suggests resistance to both artemisinin and piperaquine. Available in vitro piperaquine susceptibility assays do not correlate with treatment outcome. A novel assay using a pharmacologically relevant piperaquine dose/time exposure was designed and its relevance explored in retrospective and prospective studies.Methods: The piperaquine survival assay (PSA) exposed parasites to 200 nM piperaquine for 48 hours and monitored survival 24 hours later. The retrospective study tested 32 culture-adapted, C580Y-K13 mutant parasites collected at enrolment from patients treated with a 3-day course of dihydroartemisinin-piperaquine and having presented or not with a recrudescence at day 42 (registered ACTRN12615000793516). The prospective study assessed ex vivo PSA survival rate alongside K13 polymorphism of isolates collected from patients enrolled in an open-label study with dihydroartemisinin-piperaquine for uncomplicated P. falciparum malaria in Cambodia (registered ACTRN12615000696594).Results: All parasites from recrudescent cases had in vitro or ex vivo PSA survival rates ≥10%, a relevant cut-off value for piperaquine-resistance. Ex vivo PSA survival rates were higher for recrudescent than non-recrudescent cases (39.2% vs. 0.17%, P <1 × 10(-7)). Artemisinin-resistant K13 mutants with ex vivo PSA survival rates ≥10% were associated with 32-fold higher risk of recrudescence (95% CI, 4.5-224; P = 0.0005).Conclusion: PSA adequately captures the piperaquine resistance/recrudescence phenotype, a mainstay to identify molecular marker(s) and evaluate efficacy of alternative drugs. Combined ex vivo PSA and K13 genotyping provides a convenient monitor for both artemisinin and piperaquine resistance where dihydroartemisinin-piperaquine is used. [ABSTRACT FROM AUTHOR]

Background: Cambodia stopped using co-blistered, non-fixed, artesunate-mefloquine (ASMQ) in 2008 when treatment failure rates approximated 20%. Fixed dose combination (FDC) ASMQ is efficacious against acute uncomplicated, drug resistant Plasmodium falciparum malaria in Southeast Asia but has not been tested in Cambodia.Methods: A 42-day WHO therapeutic efficacy study (TES) was conducted in 2010 in Oral, Kampong Speu province, southwest Cambodia, in patients with acute uncomplicated P. falciparum. Daily administered FDC ASMQ for three days was dosed by age. Genotyping of isolates at day 0 and day of recrudescence by polymerase chain reaction (PCR) classified posttreatment recurrent falciparum parasitaemia. Ex vivo drug sensitivity testing ([3H] hypoxanthine method) was performed on baseline parasites and reported as the drug concentration inhibiting 50% parasite growth vs no drug (IC50). Results: Recruited patients numbered 45; five aged <15 years. On day 3, five of 45 [11.1 (3.7-24.05)] % patients were still parasite-positive; one of whom later failed treatment on day 21. There were 5/45 (11.1%) late treatment failures on day 21,28 and 35; all were PCR diagnosed recrudescent infections. The day 0 MQ IC50s ranged from 11.5-238.9 (median 58.6) nM. Conclusions: This TES demonstrated reasonable efficacy in an area of possible reduced artemisinin sensitivity and high MQ IC50s. Efficacy testing of FDC ASMQ should continue in Cambodia and be considered for reintroduction if efficacy returns. [ABSTRACT FROM AUTHOR]

Background: Glucose-6-phosphate-dehydrogenase deficiency (G6PDd) rates are unknown in malaria-infected Cambodian patients. These data are key to a rational drug policy for malaria elimination of Plasmodium falciparum and Plasmodium vivax. Methods: From September 2010-2012, a two-year survey of G6PDd and haemoglobinopathies assessed by quantitative enzyme activity assay and haemoglobin electrophoresis, respectively, was conducted in malaria-infected patients presenting to 19 health centres throughout Cambodia. Results: A total of 2,408 confirmed malaria patients of mean age 26.7 (range 2-81) years were recruited from mostly western Cambodia (n = 1,732, 71.9%); males outnumbered females by 3.9:1. Plasmodium falciparum was present in 1,443 (59.9%) and P. vivax in 965 (40.1%) patients. Mean G6PD activity was 11.6 (CI 95%: 11.4-11.8) U/g Hb, G6PDd was present in 13.9% of all patients (335/2,408) and severe G6PDd (including WHO Class I and II variants) was more common in western (158/1,732, 9.1%) versus eastern (21/414, 5.1%) Cambodia (P = 0.01). Of 997/2,408 (41.4%) had a haemoglobinopathy. Mean haemoglobin concentrations were inversely related to age: 8.1 g/dL < five years, 8.7 g/dL five to 14 years, and 10.4 g/dL >15 years (P <0.001). Conclusions: G6PDd prevalence, anaemia and haemoglobinopathies were common in malaria-infected patients. The deployment of primaquine in Cambodia should be preceded by primaquine safety studies paralleled with evaluations of easy to use tests to detect G6PDd. [ABSTRACT FROM AUTHOR]

Background: A multidrug-resistant co-lineage of Plasmodium falciparum malaria, named KEL1/PLA1, spread across Cambodia in 2008-13, causing high rates of treatment failure with the frontline combination therapy dihydroartemisinin-piperaquine. Here, we report on the evolution and spread of KEL1/PLA1 in subsequent years.Methods: For this genomic epidemiology study, we analysed whole genome sequencing data from P falciparum clinical samples collected from patients with malaria between 2007 and 2018 from Cambodia, Laos, northeastern Thailand, and Vietnam, through the MalariaGEN P falciparum Community Project. Previously unpublished samples were provided by two large-scale multisite projects: the Tracking Artemisinin Resistance Collaboration II (TRAC2) and the Genetic Reconnaissance in the Greater Mekong Subregion (GenRe-Mekong) project. By investigating genome-wide relatedness between parasites, we inferred patterns of shared ancestry in the KEL1/PLA1 population.Findings: We analysed 1673 whole genome sequences that passed quality filters, and determined KEL1/PLA1 status in 1615. Before 2009, KEL1/PLA1 was only found in western Cambodia; by 2016-17 its prevalence had risen to higher than 50% in all of the surveyed countries except for Laos. In northeastern Thailand and Vietnam, KEL1/PLA1 exceeded 80% of the most recent P falciparum parasites. KEL1/PLA1 parasites maintained high genetic relatedness and low diversity, reflecting a recent common origin. Several subgroups of highly related parasites have recently emerged within this co-lineage, with diverse geographical distributions. The three largest of these subgroups (n=84, n=79, and n=47) mostly emerged since 2016 and were all present in Cambodia, Laos, and Vietnam. These expanding subgroups carried new mutations in the crt gene, which arose on a specific genetic background comprising multiple genomic regions. Four newly emerging crt mutations were rare in the early period and became more prevalent by 2016-17 (Thr93Ser, rising to 19·8%; His97Tyr to 11·2%; Phe145Ile to 5·5%; and Ile218Phe to 11·1%).Interpretation: After emerging and circulating for several years within Cambodia, the P falciparum KEL1/PLA1 co-lineage diversified into multiple subgroups and acquired new genetic features, including novel crt mutations. These subgroups have rapidly spread into neighbouring countries, suggesting enhanced fitness. These findings highlight the urgent need for elimination of this increasingly drug-resistant parasite co-lineage, and the importance of genetic surveillance in accelerating malaria elimination efforts.Funding: Wellcome Trust, Bill & Melinda Gates Foundation, UK Medical Research Council, and UK Department for International Development. [ABSTRACT FROM AUTHOR]