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Abstract Background The national malaria control programmes in Cambodia, Nepal, and Bhutan aim to achieve malaria elimination by 2025–2030. While the vivax malaria burden remains challenging, the consistent decline in falciparum malaria in these countries over the last five years suggests that the goal is achievable. However, unexpected cases in previously falciparum malaria-free districts continue to occur. A virtual meeting was convened in May 2024, followed by smaller meetings, to discuss and share experiences among regional partners, focusing on falciparum malaria elimination. Main text The discussion among regional partners from these three countries was prompted by an isolated outbreak of falciparum malaria in April 2022 in a previously malaria-free district in Pursat province, Cambodia. The National Center for Parasitology, Entomology and Malaria Control (CNM) worked with stakeholders to contain the outbreak, which was likely to have originated in forest goers. Community-based village malaria workers (VMWs) were augmented with the additional resources required to test and treat malaria among forest goers under the supervision of health centres and district hospitals. This response rapidly reduced malaria cases in the subsequent months. Regional partners from Nepal and Bhutan, who are also engaged in the final phase of malaria elimination, reported that isolated malaria outbreaks in their countries were not unusual and were mostly imported from a neighbouring country, India. Importation of cases was facilitated by unsupervised transborder travel including the movement of migrant workers. The imported cases were only established during the case investigation such as when responding to the isolated outbreaks. In contrast, in Cambodia, spread of malaria was known to be facilitated by mobile and migrant workers, and forest goers within the country. The specific differences between Nepal and Bhutan (South Asia), and Cambodia (Greater Mekong Subregion) offered insights into strategies for malaria elimination. A relevant component for countries embarking on malaria elimination included strengthening of local malaria surveillance and response in collaboration with the community health workers, and community members targeting the ‘at risk’ population. Conclusion In countries approaching malaria elimination, resourcing community-based health workers could play a critical role in enhancing malaria surveillance, early case detection and treatment, including interventions targeting migrant populations such as forest goers and seasonal migrant workers.

Abstract The emergence of Plasmodium falciparum parasites resistant to artemisinins compromises the efficacy of Artemisinin Combination Therapies (ACTs), the global first-line malaria treatment. Artemisinin resistance is a complex genetic trait in which nonsynonymous SNPs in PfK13 cooperate with other genetic variations. Here, we present population genomic/transcriptomic analyses of P. falciparum collected from patients with uncomplicated malaria in Cambodia and Vietnam between 2018 and 2020. Besides the PfK13 SNPs, several polymorphisms, including nonsynonymous SNPs (N1131I and N821K) in PfRad5 and an intronic SNP in PfWD11 (WD40 repeat-containing protein on chromosome 11), appear to be associated with artemisinin resistance, possibly as new markers. There is also a defined set of genes whose steady-state levels of mRNA and/or splice variants or antisense transcripts correlate with artemisinin resistance at the base level. In vivo transcriptional responses to artemisinins indicate the resistant parasite’s capacity to decelerate its intraerythrocytic developmental cycle (IDC), which can contribute to the resistant phenotype. During this response, PfRAD5 and PfWD11 upregulate their respective alternatively/aberrantly spliced isoforms, suggesting their contribution to the protective response to artemisinins. PfRAD5 and PfWD11 appear under selective pressure in the Greater Mekong Sub-region over the last decade, suggesting their role in the genetic background of the artemisinin resistance.

Abstract Background Southeast Asia is undergoing an epidemiological transition with non-communicable illnesses becoming increasingly important, yet infectious diseases (tuberculosis, HIV, hepatitis B, malaria) remain widely prevalent in some populations, while emerging and zoonotic diseases threaten. There are also limited population-level estimates of many important heath conditions. This restricts evidence-based decision-making for disease control and prevention priorities. Cross-sectional surveys can be efficient epidemiological tools to measure the prevalence of a wide range of diseases, but no systematic assessment of their coverage of different health conditions has been produced for the region. Methods We conducted a systematic search in Medline, Embase, Global Health, CINAHL, Scopus, Web of Science Core Collection, and Global Index Medicus, and additionally Google Scholar. Our inclusion criteria were cross-sectional surveys conducted with community-based recruitment, in Bangladesh, Cambodia, Laos, Myanmar, and Thailand, published between January 1, 2010 and January 27, 2021, and reporting the prevalence of any health condition. Results 542 publications from 337 surveys were included. Non-communicable conditions (n = 205) were reported by more surveys than infectious conditions (n = 124). Disability (n = 49), self-report history of any disease or symptoms (n = 35), and self-perceived health status (n = 34), which reflect a holistic picture of health, were studied by many fewer surveys. In addition, 45 surveys studied symptomatic conditions which overlap between non-communicable and infectious conditions. The most surveyed conditions were undernutrition, obesity, hypertension, diabetes, intestinal parasites, malaria, anemia, diarrhea, fever, and acute respiratory infections. These conditions overlap with the most important causes of death and disability in the Global Burden of Disease study. However, other high-burden conditions (e.g. hearing loss, headache disorder, low back pain, chronic liver and kidney diseases, and cancers) were rarely studied. Conclusion There were relatively few recent surveys from which to estimate representative prevalences and trends of health conditions beyond those known to be high burden. Expanding the spectrum of health conditions in cross-sectional surveys could improve understanding of evolving disease patterns in the region.

Engaging young people in health research has been promoted globally. We explored the outcomes of youth advisory group on health and research engagement (YAGHRE) in rural Cambodia. In May 2021, the Mahidol Oxford Tropical Medicine Research Unit (MORU) partnered with a local health centre and a secondary school to establish a youth engagement group. Ten students underwent training and led health engagement activities in schools and communities. Activities were documented as field notes and audio-visual materials which underwent content analysis using theory of change supplemented by iterative discussions with YAGHRE members and stakeholders. Five major outcomes were identified: 1. Increased respect. Engagement activities developed based on input from students and stakeholders may have fostered greater respect. 2. Built trust and relationships. Frequent visits to MORU’s laboratory and interactions with researchers appeared to contribute to the building of trust and relationship. 3. Improved health and research literacy. Learning new health and research topics, through participatory activities may have improved literacy; 4. Improved uptake of health and research interventions. Health promotional activities and communication with research participants potentially increased the uptake of interventions; 5. Improved community health. YAGHRE’s health promotional interventions may have contributed in enhancing community’s health.

Abstract Introduction The national malaria programme of Cambodia targets the rapid elimination of all human malaria by 2025. As clinical cases decline to near-elimination levels, a key strategy is the rapid identification of malaria outbreaks triggering effective action to interrupt local transmission. We report a comprehensive, multipronged management approach in response to a 2022 Plasmodium falciparum outbreak in Kravanh district, western Cambodia. Methods The provincial health department of Pursat in conjunction with the Center for Parasitology, Entomology and Malaria Control (CNM) identified villages where transmission was occurring using clinical records, and initiated various interventions, including the distribution of insecticide-treated bed nets, running awareness campaigns, and implementing fever screening with targeted drug administration. Health stations were set up at forest entry points, and later, targeted drug administrations with artesunate–pyronaridine (Pyramax) and intermittent preventive treatment for forest goers (IPTf) were implemented in specific village foci. Data related to adherence and adverse events from IPTf and TDA were collected. The coverage rates of interventions were calculated, and local malaria infections were monitored. Results A total of 942 individuals were screened through active fever surveillance in villages where IPTf and TDA were conducted. The study demonstrated high coverage and adherence rates in the targeted villages, with 92% (553/600) coverage in round one and 65% (387/600) in round two. Adherence rate was 99% (551/553) in round one and 98% (377/387) in round two. The study found that forest goers preferred taking Pyramax over repeated testing consistent with the coverage rates: 92% in round one compared to 65% in round two. All individuals reachable through health stations or mobile teams reported complete IPTf uptake. No severe adverse events were reported. Only six individuals reported mild adverse events, such as loss of energy, fever, abdominal pain, diarrhoea, and muscle aches. Two individuals attributed their symptoms to heavy alcohol intake following prophylaxis. Conclusions The targeted malaria outbreak response demonstrated high acceptability, safety, and feasibility of the selected interventions. Malaria transmission was rapidly controlled using the available community resources. This experience suggests the effectiveness of the programmatic response for future outbreaks.

ABSTRACT Piperaquine (PPQ) is widely used in combination with dihydroartemisinin as a first-line treatment against malaria. Multiple genetic drivers of PPQ resistance have been reported, including mutations in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and increased copies of plasmepsin II/III (pm2/3). We generated a cross between a Cambodia-derived multidrug-resistant KEL1/PLA1 lineage isolate (KH004) and a drug-susceptible Malawian parasite (Mal31). Mal31 harbors a wild-type (3D7-like) pfcrt allele and a single copy of pm2/3, while KH004 has a chloroquine-resistant (Dd2-like) pfcrt allele with an additional G367C substitution and multiple copies of pm2/3. We recovered 104 unique recombinant parasites and examined a targeted set of progeny representing all possible combinations of variants at pfcrt and pm2/3. We performed a detailed analysis of competitive fitness and a range of PPQ susceptibility phenotypes with these progenies, including PPQ survival assay, area under the dose response curve, and a limited point IC50. We find that inheritance of the KH004 pfcrt allele is required for reduced PPQ sensitivity, whereas copy number variation in pm2/3 further decreases susceptibility but does not confer resistance in the absence of additional mutations in pfcrt. A deep investigation of genotype-phenotype relationships demonstrates that progeny clones from experimental crosses can be used to understand the relative contributions of pfcrt, pm2/3, and parasite genetic background to a range of PPQ-related traits. Additionally, we find that the resistance phenotype associated with parasites inheriting the G367C substitution in pfcrt is consistent with previously validated PPQ resistance mutations in this transporter.IMPORTANCEResistance to piperaquine, used in combination with dihydroartemisinin, has emerged in Cambodia and threatens to spread to other malaria-endemic regions. Understanding the causal mutations of drug resistance and their impact on parasite fitness is critical for surveillance and intervention and can also reveal new avenues to limiting the evolution and spread of drug resistance. An experimental genetic cross is a powerful tool for pinpointing the genetic determinants of key drug resistance and fitness phenotypes and has the distinct advantage of quantifying the effects of naturally evolved genetic variation. Our study was strengthened since the full range of copies of KH004 pm2/3 was inherited among the progeny clones, allowing us to directly test the role of the pm2/3 copy number on resistance-related phenotypes in the context of a unique pfcrt allele. Our multigene model suggests an important role for both loci in the evolution of this multidrug-resistant parasite lineage.

Abstract In Southeast Asia malaria elimination is targeted by 2030. Cambodia aims to achieve this by 2025, driven in large part by the urgent need to control the spread of artemisinin-resistant falciparum malaria infections. Rapid elimination depends on sustaining early access to diagnosis and effective treatment. In much of Cambodia, rapid elimination will rely on a village malaria worker (VMW) network. Yet as malaria declines and is no longer a common cause of febrile illness, VMWs may become less popular with febrile patients, as VMWs do not diagnose or treat other conditions at present. There is a risk that VMWs become inactive and malaria rebounds before the complete interruption of transmission is achieved. During 2021–23 a large-scale operational research study was conducted in western Cambodia to explore how a VMW network could be sustained by including health activities that cover non-malarial illnesses to encourage febrile patients to continue to attend. 105 VMWs received new rapid diagnostic tests (including dengue antigen–antibody and combined malaria/C-reactive protein tests), were trained in electronic data collection, and attended health education packages on hygiene and sanitation, disease surveillance and first aid, management of mild illness, and vaccination and antenatal care. In August 2023 the National Malaria Control Programme of Cambodia convened a stakeholder meeting in Battambang, Cambodia. Findings from the study were reviewed in the context of current malaria elimination strategies. The discussions informed policy options to sustain the relevance of the VMW network in Cambodia, and the potential for its integration with other health worker networks. This expansion could ensure VMWs remain active and relevant until malaria elimination is accomplished.

Abstract Background Malaria transmission in Southeast Asia is increasingly confined to forests, where marginalized groups are exposed primarily through their work. Anti-malarial chemoprophylaxis may help to protect these people. This article examines the effectiveness and practical challenges of engaging forest-goers to participate in a randomized controlled clinical trial of anti-malarial chemoprophylaxis with artemether-lumefantrine (AL) versus a control (multivitamin, MV) for malaria in northeast Cambodia. Methods The impact of engagement in terms of uptake was assessed as the proportion of people who participated during each stage of the trial: enrolment, compliance with trial procedures, and drug intake. During the trial, staff recorded the details of engagement meetings, including the views and opinions of participants and community representatives, the decision-making processes, and the challenges addressed during implementation. Results In total, 1613 participants were assessed for eligibility and 1480 (92%) joined the trial, 1242 (84%) completed the trial and received prophylaxis (AL: 82% vs MV: 86%, p = 0.08); 157 (11%) were lost to follow-up (AL: 11% vs MV: 11%, p = 0.79); and 73 (5%) discontinued the drug (AL-7% vs MV-3%, p = 0.005). The AL arm was associated with discontinuation of the study drug (AL: 48/738, 7% vs 25/742, 3%; p = 0.01). Females (31/345, 9%) were more likely (42/1135, 4%) to discontinue taking drugs at some point in the trial (p = 0.005). Those (45/644, 7%) who had no previous history of malaria infection were more likely to discontinue the study drug than those (28/836, 3%) who had a history of malaria (p = 0.02). Engagement with the trial population was demanding because many types of forest work are illegal; and the involvement of an engagement team consisting of representatives from the local administration, health authorities, community leaders and community health workers played a significant role in building trust. Responsiveness to the needs and concerns of the community promoted acceptability and increased confidence in taking prophylaxis among participants. Recruitment of forest-goer volunteers to peer-supervise drug administration resulted in high compliance with drug intake. The development of locally-appropriate tools and messaging for the different linguistic and low-literacy groups was useful to ensure participants understood and adhered to the trial procedures. It was important to consider forest-goers` habits and social characteristics when planning the various trial activities. Conclusions The comprehensive, participatory engagement strategy mobilized a wide range of stakeholders including study participants, helped build trust, and overcame potential ethical and practical challenges. This locally-adapted approach was highly effective as evidenced by high levels of trial enrolment, compliance with trial procedures and drug intake.

Abstract CARAMAL was a large observational study which recorded mortality in children with suspected severe malaria before and after the roll-out of rectal artesunate in Nigeria, Uganda and the Democratic Republic of the Congo. The results of CARAMAL have had a huge impact on public health policy leading to a World Health Organization moratorium on the roll-out of rectal artesunate. The conclusion reported in the abstract uses strong causal language, stating that “pre-referral RAS [rectal artesunate suppositories] had no beneficial effect on child survival”. We argue that this causal interpretation of the study results is not justified. Data from the CARAMAL study inform chiefly on the strengths and weaknesses of referral systems in these three countries and do not inform reliably as to the beneficial effect of providing access to a known life-saving treatment.

Abstract Background Quantitative measurement of Glucose-6-Phosphate Dehydrogenase (G6PD) enzyme activity is critical to decide on appropriate treatment and provision of radical cure regimens for vivax malaria. Biosensors are point-of-care semi-quantitative analysers that measure G6PD enzyme activity. The main objective of this study was to evaluate the operational aspects of biosensor deployment in the hands of village malaria workers (VMWs) in Cambodia over a year. Methods Following initial orientation and training at Kravanh Referral Hospital, each VMW (n = 28) and laboratory technician (n = 5) was provided a biosensor (STANDARD SD Biosensor, Republic of Korea) with supplies for routine use. Over the next 12 months VMWs convened every month for refresher training, to collect supplies, and to recalibrate and test their biosensors. A quantitative self-administered questionnaire was used to assess the skills necessary to use the biosensor after the initial training. Subsequently, VMWs were visited at their location of work for field observation and evaluation using an observer-administered questionnaire. All quantitative questionnaire-based data were analysed descriptively. Semi-structured interviews (SSIs) were conducted among all participants to explore their experience and practicalities of using the biosensor in the field. SSIs were transcribed and translated into English and underwent thematic analysis. Results A total of 33 participants completed the training and subsequently used the biosensor in the community. Quantitative assessments demonstrated progressive improvement in skills using the biosensor. VMWs expressed confidence and enthusiasm to use biosensors in their routine work. Providing G6PD testing at the point of first contact avoids a multitude of barriers patients have to overcome when travelling to health centres for G6PD testing and radical cure. Deploying biosensors in routine work of VMWs was also considered an opportunity to expand and strengthen the role of VMWs as health care providers in the community. VMWs reported practical concerns related to the use of biosensor such as difficulty in using two pipettes, difficulty in extracting the code chip from the machine, and the narrow base of buffer tube. Conclusions VMWs considered the biosensor a practical and beneficial tool in their routine work. Providing VMWs with biosensors can be considered when followed by appropriate training and regular supervision. Providing community management of vivax malaria at the point of first contact could be key for elimination.

Abstract Background Microscopic examination of Giemsa-stained blood films remains the reference standard for malaria parasite detection and quantification, but is undermined by difficulties in ensuring high-quality manual reading and inter-reader reliability. Automated parasite detection and quantification may address this issue. Methods A multi-centre, observational study was conducted during 2018 and 2019 at 11 sites to assess the performance of the EasyScan Go, a microscopy device employing machine-learning-based image analysis. Sensitivity, specificity, accuracy of species detection and parasite density estimation were assessed with expert microscopy as the reference. Intra- and inter-device reliability of the device was also evaluated by comparing results from repeat reads on the same and two different devices. This study has been reported in accordance with the Standards for Reporting Diagnostic accuracy studies (STARD) checklist. Results In total, 2250 Giemsa-stained blood films were prepared and read independently by expert microscopists and the EasyScan Go device. The diagnostic sensitivity of EasyScan Go was 91.1% (95% CI 88.9–92.7), and specificity 75.6% (95% CI 73.1–78.0). With good quality slides sensitivity was similar (89.1%, 95%CI 86.2–91.5), but specificity increased to 85.1% (95%CI 82.6–87.4). Sensitivity increased with parasitaemia rising from 57% at 200–200,000 parasite/µL. Species were identified accurately in 93% of Plasmodium falciparum samples (kappa = 0.76, 95% CI 0.69–0.83), and in 92% of Plasmodium vivax samples (kappa = 0.73, 95% CI 0.66–0.80). Parasite density estimates by the EasyScan Go were within ± 25% of the microscopic reference counts in 23% of slides. Conclusions The performance of the EasyScan Go in parasite detection and species identification accuracy fulfil WHO-TDR Research Malaria Microscopy competence level 2 criteria. In terms of parasite quantification and false positive rate, it meets the level 4 WHO-TDR Research Malaria Microscopy criteria. All performance parameters were significantly affected by slide quality. Further software improvement is required to improve sensitivity at low parasitaemia and parasite density estimations. Trial registration ClinicalTrials.gov number NCT03512678.

Transcriptomic analysis of isolates from the malaria parasite (Plasmodium falciparum) in the Greater Mekong Subregion of Southeast Asia identifies gene expression patterns that are correlated with resistance to a common anti-malaria drug, artemisinin.

Summary: In Cambodia, malaria cases are on a trajectory towards the goal of malaria elimination by 2025. Vivax malaria is difficult to eliminate because of hypnozoites that can cause relapse. Primaquine, an 8-aminoquinoline, clears hypnozoites but requires testing for glucose-6-phosphate dehydrogenase (G6PD) deficiency. Routine primaquine treatment of vivax malaria has recently been implemented in Cambodia in which Village Malaria Workers (VMWs) diagnose vivax malaria by rapid diagnostic test and refer patients to health centres for G6PD testing and further treatment. Patients are referred back to the VMWs for monitoring adverse symptoms and treatment adherence. This article explores how VMWs’ roles might be optimized for the community-based management of vivax malaria. With sufficient training and supervision, the role of VMWs might be expanded to include G6PD testing, making referral to the health centre superfluous. Community-based management of vivax malaria could increase the coverage of radical cure and accelerate vivax malaria elimination.

Abstract Background In the Greater Mekong Subregion, adults are at highest risk for malaria, particularly those who visit forests. The absence of effective vector control strategies and limited periods of exposure during forest visits suggest that chemoprophylaxis could be an appropriate strategy to protect forest goers against malaria. Methods Alongside a clinical trial of anti-malarial chemoprophylaxis in northern Cambodia, qualitative research was conducted, including in-depth interviews and observation, to explore the acceptability of malaria prophylaxis for forest goers, the implementation opportunities, and challenges of this strategy. Results Prophylaxis with artemether–lumefantrine for forest goers was found to be acceptable under trial conditions. Three factors played a major role: the community’s awareness and perception of the effectiveness of prophylaxis, their trust in the provider, and malaria as a local health concern. The findings highlight how uptake and adherence to prophylaxis are influenced by the perceived balance between benefits and burden of anti-malarials which are modulated by the seasonality of forest visits and its influence on malaria risk. Conclusions The implementation of anti-malarial prophylaxis needs to consider how the preventive medication can be incorporated into existing vector-control measures, malaria testing and treatment services. The next step in the roll out of anti-malarial prophylaxis for forest visitors will require support from local health workers.

Abstract Background In many endemic areas, Plasmodium vivax malaria is predominantly a disease of young adults and children. International recommendations for radical cure recommend fixed target doses of 0.25 or 0.5 mg/kg/day of primaquine for 14 days in glucose-6-phosphate dehydrogenase normal patients of all ages. However, for many anti-malarial drugs, including primaquine, there is evidence that children have lower exposures than adults for the same weight-adjusted dose. The aim of the study was to develop 14-day weight-based and age-based primaquine regimens against high-frequency relapsing tropical P. vivax. Methods The recommended adult target dose of 0.5 mg/kg/day (30 mg in a 60 kg patient) is highly efficacious against tropical P. vivax and was assumed to produce optimal drug exposure. Primaquine doses were calculated using allometric scaling to derive a weight-based primaquine regimen over a weight range from 5 to 100 kg. Growth curves were constructed from an anthropometric database of 53,467 individuals from the Greater Mekong Subregion (GMS) to define weight-for-age relationships. The median age associated with each weight was used to derive an age-based dosing regimen from the weight-based regimen. Results The proposed weight-based regimen has 5 dosing bands: (i) 5–7 kg, 5 mg, resulting in 0.71–1.0 mg/kg/day; (ii) 8–16 kg, 7.5 mg, 0.47–0.94 mg/kg/day; (iii) 17–40 kg, 15 mg, 0.38–0.88 mg/kg/day; (iv) 41–80 kg, 30 mg, 0.37–0.73 mg/kg/day; and (v) 81–100 kg, 45 mg, 0.45–0.56 mg/kg/day. The corresponding age-based regimen had 4 dosing bands: 6–11 months, 5 mg, 0.43–1.0 mg/kg/day; (ii) 1–5 years, 7.5 mg, 0.35–1.25 mg/kg/day; (iii) 6–14 years, 15 mg, 0.30–1.36 mg/kg/day; and (iv) ≥ 15 years, 30 mg, 0.35–1.07 mg/kg/day. Conclusion The proposed weight-based regimen showed less variability around the primaquine dose within each dosing band compared to the age-based regimen and is preferred. Increased dose accuracy could be achieved by additional dosing bands for both regimens. The age-based regimen might not be applicable to regions outside the GMS, which must be based on local anthropometric data. Pharmacokinetic data in small children are needed urgently to inform the proposed regimens.

Abstract Background Malaria reactive case detection is the testing and, if positive, treatment of close contacts of index cases. It is included in national malaria control programmes of countries in the Greater Mekong Subregion to accelerate malaria elimination. Yet the value of reactive case detection in the control and elimination of malaria remains controversial because of the low yield, limited evidence for impact, and high demands on resources. Methods Data from the epidemiological assessments of large mass drug administration (MDA) studies in Myanmar, Vietnam, Cambodia and Laos were analysed to explore malaria infection clustering in households. The proportion of malaria positive cases among contacts screened in a hypothetical reactive case detection programme was then determined. The parasite density thresholds for rapid diagnostic test (RDT) detection was assumed to be > 50/µL (50,000/mL), for dried-blood-spot (DBS) based PCR > 5/µL (5000/mL), and for ultrasensitive PCR (uPCR) with a validated limit of detection at 0.0022/µL (22/mL). Results At baseline, before MDA, 1223 Plasmodium infections were detected by uPCR in 693 households. There was clustering of Plasmodium infections. In 637 households with asymptomatic infections 44% (278/637) had more than one member with Plasmodium infections. In the 132 households with symptomatic infections, 65% (86/132) had more than one member with Plasmodium infections. At baseline 4% of households had more than one Plasmodium falciparum infection, but three months after MDA no household had more than one P. falciparum infected member. Reactive case detection using DBS PCR would have detected ten additional cases in six households, and an RDT screen would have detected five additional cases in three households among the 169 households with at least one RDT positive case. This translates to 19 and 9 additional cases identified per 1000 people screened, respectively. Overall, assuming all febrile RDT positive patients would seek treatment and provoke reactive case detection using RDTs, then 1047 of 1052 (99.5%) Plasmodium infections in these communities would have remained undetected. Conclusion Reactive case detection in the Greater Mekong subregion is predicted to have a negligible impact on the malaria burden, but it has substantial costs in terms of human and financial resources.

Abstract Cambodia targets malaria elimination by 2025. Rapid elimination will depend on successfully identifying and clearing malaria foci linked to forests. Expanding and maintaining universal access to early diagnosis and effective treatment remains the key to malaria control and ultimately malaria elimination in the Greater Mekong Subregion (GMS) in the foreseeable future. Mass Drug Administration (MDA) holds some promise in the rapid reduction of Plasmodium falciparum infections, but requires considerable investment of resources and time to mobilize the target communities. Furthermore, the most practical drug regimen for MDA in the GMS—three rounds of DHA/piperaquine—has lost some of its efficacy. Mass screening and treatment benefits asymptomatic P. falciparum carriers by clearing chronic infections, but in its current form holds little promise for malaria elimination. Hopes that “highly sensitive” diagnostic tests would provide substantial advances in screen and treat programmes have been shown to be misplaced. To reduce the burden on P. falciparum and Plasmodium vivax infections in people working in forested areas novel approaches to the use of malaria prophylaxis in forest workers should be explored. During an October 2019 workshop in Phnom Penh researchers and policymakers reviewed evidence of acceptability, feasibility and effectiveness of interventions to target malaria foci and interrupt P. falciparum transmission and discussed operational requirements and conditions for programmatic implementation.

In rural areas of South and Southeast Asia malaria is declining but febrile illnesses still account for substantial morbidity and mortality. Village health workers (VHWs) are often the first point of contact with the formal health system, and for patients with febrile illnesses they can provide early diagnosis and treatment of malaria. However, for the majority of febrile patients, VHWs lack the training, support and resources to provide further care. Consequently, treatable bacterial illnesses are missed, antibiotics are overused and poorly targeted, and patient attendance wanes along with declining malaria. This Open Letter announces the start of a new initiative, the Rural Febrile Illness (RFI) project, the first in a series of projects to be implemented as part of the South and Southeast Asian Community-based Trials Network (SEACTN) research programme. This multi-country, multi-site project will run in Bangladesh, Cambodia, Lao PDR, Thailand, and Myanmar. It will define the epidemiological baseline of febrile illness in nine remote and underserved areas of Asia where malaria endemicity is declining and access to health services is limited. The RFI project aims to determine the incidence, causes and outcomes of febrile illness; understand the opportunities, barriers and appetite for adjustment of the role of VHWs to include management of non-malarial febrile illnesses; and establish a network of community healthcare providers and facilities capable of implementing interventions designed to triage, diagnose and treat patients presenting with febrile illnesses within these communities in the future.

Vivax malaria can relapse after an initial infection due to dormant liver stages of the parasite. Radical cure can prevent relapses but requires the measurement of glucose-6-phosphate dehydrogenase enzyme (G6PD) activity to identify G6PD-deficient patients at risk of drug-induced haemolysis. In the absence of reliable G6PD testing, vivax patients are denied radical curative treatment in many places, including rural Cambodia. A novel Biosensor, ‘G6PD Standard’ (SD Biosensor, Republic of Korea; Biosensor), can measure G6PD activity at the point of care. The objectives of this study were to compare the G6PD activity readings using Biosensors by village malaria workers (VMWs) and hospital-based laboratory technicians (LTs), and to compare the G6PD deficiency categorization recommended by the Biosensor manufacturer with categories derived from a locally estimated adjusted male median (AMM) in Kravanh district, Cambodia. Participants were enrolled between 2021 and 2022 in western Cambodia. Each of the 28 VMWs and 5 LTs received a Biosensor and standardized training on its use. The G6PD activities of febrile patients identified in the community were measured by VMWs; in a subset, a second reading was done by LTs. All participants were tested for malaria by rapid diagnostic test (RDT). The adjusted male median (AMM) was calculated from all RDT-negative participants and defined as 100% G6PD activity. VMWs measured activities in 1344 participants. Of that total, 1327 (98.7%) readings were included in the analysis, and 68 of these had a positive RDT result. We calculated 100% activity as 6.4 U/gHb (interquartile range: 4.5 to 7.8); 9.9% (124/1259) of RDT-negative participants had G6PD activities below 30%, 15.2% (191/1259) had activities between 30% and 70%, and 75.0% (944/1259) had activities greater than 70%. Repeat measurements among 114 participants showed a significant correlation of G6PD readings (rs = 0.784, p < 0.001) between VMWs and LTs. Based on the manufacturer’s recommendations, 285 participants (21.5%) had less than 30% activity; however, based on the AMM, 132 participants (10.0%) had less than 30% activity. The G6PD measurements by VMWs and LTs were similar. With the provisions of training, supervision, and monitoring, VMWs could play an important role in the management of vivax malaria, which is critical for the rapid elimination of malaria regionally. Definitions of deficiency based on the manufacturer’s recommendations and the population-specific AMM differed significantly, which may warrant revision of these recommendations.

Abstract Background Trials to assess the efficacy of the radical cure of Plasmodium vivax malaria with 8-aminoquinolines require that most post-treatment relapses are identified, but there is no consensus on the optimal duration of follow-up in either symptomatic or asymptomatic vivax malaria. The efficacy of a 14-day course of primaquine on the cumulative incidence of recurrent asymptomatic P. vivax infections detected by ultrasensitive quantitative PCR (uPCR) as a primary endpoint was assessed. Methods A randomized, placebo-controlled, single-blind trial was conducted in four villages of the Lao PDR during 2016–2018 nested in a larger project evaluating mass drug administrations (MDA) with dihydroartemisinin-piperaquine (DP) and a single low-dose primaquine to clear Plasmodium falciparum infections. In the nested sub-study, eligible participants with mono- or mixed P. vivax infections detected by uPCR were randomized to receive either 14 days of primaquine (0.5 mg/kg/day) or placebo during the last round of MDA (round 3) through directly observed therapy. Participants were checked monthly for 12 months for parasitaemia using uPCR. The primary outcome was cumulative incidence of participants with at least one recurrent episode of P. vivax infection. Results 20 G6PD-normal participants were randomized in each arm. 5 (29%) of 20 participants in the placebo arm experienced asymptomatic, recurrent P. vivax infections, resulting in a cumulative incidence at month 12 of 29%. None of the 20 participants in the intervention arm had recurrent infections (p = 0.047 Fisher’s exact test). Participants with recurrent P. vivax infections were found to be parasitaemic for between one and five sequential monthly tests. The median time to recurrence of P. vivax parasitaemia was 178 days (range 62–243 days). Conclusions A 14-day course of primaquine in addition to a DP-MDA was safe, well-tolerated, and prevented recurrent asymptomatic P. vivax infections. Long follow-up for up to 12 months is required to capture all recurrences following the treatment of asymptomatic vivax infection. To eliminate all malarias in settings where P. vivax is endemic, a full-course of an 8-aminoquinolines should be added to MDA to eliminate all malarias. Trial registration This study was registered with ClinicalTrials.gov under NCT02802813 on 16th June 2016. https://clinicaltrials.gov/ct2/show/NCT02802813

Abstract Background Adding 8-aminoquinoline to the treatment of falciparum, in addition to vivax malaria, in locations where infections with both species are prevalent could prevent vivax reactivation. The potential risk of haemolysis under a universal radical cure policy using 8-aminoquinoline needs to be weighed against the benefit of preventing repeated vivax episodes. Estimating the frequency of sequential Plasmodium vivax infections following either falciparum or vivax malaria episodes is needed for such an assessment. Methods Quarterly surveillance data collected during a mass drug administration trial in the Greater Mekong Subregion in 2013–17 was used to estimate the probability of asymptomatic sequential infections by the same and different Plasmodium species. Asymptomatic Plasmodium infections were detected by high-volume ultrasensitive qPCR. Quarterly surveys of asymptomatic Plasmodium prevalence were used to estimate the probability of a P. vivax infection following Plasmodium falciparum and P. vivax infections. Results 16,959 valid sequential paired test results were available for analysis. Of these, 534 (3%) had an initial P. falciparum monoinfection, 1169 (7%) a P. vivax monoinfection, 217 (1%) had mixed (P. falciparum + P. vivax) infections, and 15,039 (89%) had no Plasmodium detected in the initial survey. Participants who had no evidence of a Plasmodium infection had a 4% probability to be found infected with P. vivax during the subsequent survey. Following an asymptomatic P. falciparum monoinfection participants had a 9% probability of having a subsequent P. vivax infection (RR 2.4; 95% CI 1.8 to 3.2). Following an asymptomatic P. vivax monoinfection, the participants had a 45% probability of having a subsequent P. vivax infection. The radical cure of 12 asymptomatic P. falciparum monoinfections would have prevented one subsequent P. vivax infection, whereas treatment of 2 P. vivax monoinfections may suffice to prevent one P. vivax relapse. Conclusion Universal radical cure could play a role in the elimination of vivax malaria. The decision whether to implement universal radical cure for P. falciparum as well as for P. vivax depends on the prevalence of P. falciparum and P. vivax infections, the prevalence and severity of G6PD deficiency in the population and the feasibility to administer 8-aminoquinoline regimens safely. Trial registration ClinicalTrials.gov Identifier: NCT01872702, first posted June 7th 2013, https://clinicaltrials.gov/ct2/show/NCT01872702. This study was registered with ClinicalTrials.gov under NCT02802813 on 16th June 2016. https://clinicaltrials.gov/ct2/show/NCT02802813

Abstract Background Sample size calculations for cluster randomized trials are a recognized methodological challenge for malaria research in pre-elimination settings. Positively correlated responses from the participants in the same cluster are a key feature in the estimated sample size required for a cluster randomized trial. The degree of correlation is measured by the intracluster correlation coefficient (ICC) where a higher coefficient suggests a closer correlation hence less heterogeneity within clusters but more heterogeneity between clusters. Methods Data on uPCR-detected Plasmodium falciparum and Plasmodium vivax infections from a recent cluster randomized trial which aimed at interrupting malaria transmission through mass drug administrations were used to calculate the ICCs for prevalence and incidence of Plasmodium infections. The trial was conducted in four countries in the Greater Mekong Subregion, Laos, Myanmar, Vietnam and Cambodia. Exact and simulation approaches were used to estimate ICC values for both the prevalence and the incidence of parasitaemia. In addition, the latent variable approach to estimate ICCs for the prevalence was utilized. Results The ICCs for prevalence ranged between 0.001 and 0.082 for all countries. The ICC from the combined 16 villages in the Greater Mekong Subregion were 0.26 and 0.21 for P. falciparum and P. vivax respectively. The ICCs for incidence of parasitaemia ranged between 0.002 and 0.075 for Myanmar, Cambodia and Vietnam. There were very high ICCs for incidence in the range of 0.701 to 0.806 in Laos during follow-up. Conclusion ICC estimates can help researchers when designing malaria cluster randomized trials. A high variability in ICCs and hence sample size requirements between study sites was observed. Realistic sample size estimates for cluster randomized malaria trials in the Greater Mekong Subregion have to assume high between cluster heterogeneity and ICCs. This work focused on uPCR-detected infections; there remains a need to develop more ICC references for trials designed around prevalence and incidence of clinical outcomes. Adequately powered trials are critical to estimate the benefit of interventions to malaria in a reliable and reproducible fashion. Trial registration: ClinicalTrials.govNCT01872702. Registered 7 June 2013. Retrospectively registered. https://clinicaltrials.gov/ct2/show/NCT01872702

Abstract Background Over the last 20 years, malaria incidence has decreased across the Greater Mekong Sub-region (GMS) and the emergence of artemisinin resistance has stimulated efforts to accelerate regional elimination. In the GMS, the malaria transmission is focused increasingly in forested zones. This article describes forest-going activities and examines forest workers’ attitudes to and experiences of malaria prevention and control in north-eastern Cambodia. Methods In Stung Treng Province, Cambodia, 19 in-depth interviews were conducted in villages with participants recently diagnosed with uncomplicated falciparum malaria who reported working in forests. Two focus group discussions with respondents’ forest-working peers were held. Interviews and focus groups were audio-recorded transcribed, and translated for thematic analysis. Results Forest work is an essential source of income for respondents. Many combine it with farming, which influences the timing and duration of forest visits. Forest activities include logging and collecting other forest products, particularly malva nuts. Men log year-round, whereas gathering forest products is seasonal and can involve entire families. Forest workers sleep chiefly in unimpregnated hammock nets in make-shift encampments. Respondents are concerned about symptomatic malaria, but unfamiliar with the concept of asymptomatic infection. They view the forest as an area of potential malaria infection and seek to protect themselves from mosquito bites through wearing long-sleeved clothes, using repellents, and lighting fires. Forest workers express a willingness to self-test and self-administer anti-malarials. Conclusions Forest workers’ behaviour and perceptions of risk indicate that improvements are needed to current control measures. There is potential to: better target distribution of impregnated hammock nets; offer curative or presumptive treatment while in forests; and expand access to screening. Establishing the efficacy and feasibility of prophylaxis for forest workers in the GMS is a priority.

Abstract Background Mutations in Pfkelch13 and Pfplasmepsin2/3 gene amplification are well-established markers for artemisinin and piperaquine resistance in Plasmodium falciparum, a widespread problem in the Greater Mekong Subregion (GMS). The Plasmodium vivax parasite population has experienced varying drug pressure dependent on local drug policies. We investigated the correlation between drug pressure from artemisinins and piperaquine and mutations in the P. vivax orthologous genes Pvkelch12 and Pvplasmepsin4 (Pvpm4), as candidate resistance markers. Methods Blood samples from 734 P. vivax patients were obtained from Thailand (n = 399), Lao PDR (n = 296) and Cambodia (n = 39) between 2007 and 2017. Pvkelch12 and Pvpm4 was amplified and sequenced to assess gene mutations. To assess PvPM4 gene amplification, a Taqman® Real-Time PCR method was developed and validated. Selection of non-synonymous mutations was assessed by its ratio with synonymous mutations (Ka/Ks ratios). Mutation rates were compared to the estimated local drug pressure. Results Polymorphisms in Pvkelch12 were rare. Pvkelch12 mutations V552I, K151Q and M124I were observed in 1.0% (7/734) of P. vivax samples. V552I was the most common mutation with a frequency of 0.7% (5/734), most of which (4/5) observed in Ubon Ratchathani, Thailand. Polymorphisms in Pvpm4 were more common, with a frequency of 40.3% (123/305) in 305 samples from Thailand, Lao PDR and Cambodia, but this was not related to the estimated piperaquine drug pressure in these areas (Pearson’s χ2 test, p = 0.50). Pvpm4 mutation V165I was most frequent in Tak, Thailand (40.2%, 43/107) followed by Pailin, Cambodia (43.5%, 37/85), Champasak, Lao PDR (40.4%, 23/57) and Ubon Ratchathani, Thailand (35.7%, 20/56). Pvpm4 amplification was not observed in 141 samples from Thailand and Cambodia. For both Pvkelch12 and Pvpm4, in all areas and at all time points, the Ka/Ks values were

Background: Across the Greater Mekong Sub-region, malaria persists in isolated communities along international borders. Arts and drama have been used to reach to communities in Cambodia to engage them in malaria research, prevention and control. The “Village Drama Against Malaria” (VDAM) project was conducted in north eastern and western Cambodia: Stung Treng; Battambang and Pailin provinces during 2016 to 2019. In total, VDAM reached 55 rural villages, 2,378 student participants and 43,502 audience members. Methods: This article presents the results of two stakeholder-led evaluation workshops in which participants collaboratively developed theories of change to better understand the potential and actual impact of arts and drama-based activities on malaria in these communities. The workshops had a particular focus on identifying areas for monitoring and evaluation so that impact can be measured. Workshop participants included village malaria workers, community leaders, professional and student drama performers, and representatives from the local health authorities and the national malaria control programme. Results: Five broad areas were identified as relevant for monitoring and evaluation: logistical and practical challenges; embeddedness and reach of engagement; health knowledge and confidence of young people; effectiveness of communications; impact on malaria. These areas align well with the monitoring and evaluation conducted to date and point to additional opportunities for data collection. Conclusions: The findings from these workshops will inform future engagement strategies, for example, we may engage a smaller number of young people but over a longer period and more in-depth.

Abstract Background Despite decreases in incidence and related mortality, malaria remains a major public health challenge in the Greater Mekong Sub-region (GMS). The emergence of artemisinin resistance threatens these gains and has prompted efforts to accelerate elimination in the region. In the GMS, transmission now clusters in hotspots along international borders and among high-risk populations, including forest-goers. To eliminate malaria in the region, interventions must target such hard-to-reach populations. This review provides a comprehensive overview of the qualitative research on behaviours and perceptions that influence uptake of and adherence to malaria interventions among forest-goers in the GMS. Methods A systematic search strategy was used to identify relevant sources, including database (OVID SP, PubMed, ISI Web of Knowledge) and bibliographic searches. Relevant findings from qualitative research methods were extracted and thematic analysis undertaken. Results Of 268 sources retrieved in searches twenty-two were reviewed. Most reported studies were conducted in Cambodia (n = 10), and were published after 2014 (n = 16). Four major themes emerged that are particularly relevant to the design of intervention packages targeted at forest-goers: (1) understanding of malaria and perceived risk; (2) preventive measures used when visiting the forest; (3) behaviours that put forest-goers at risk of infection; and, (4) malaria-related treatment seeking. There were notable differences across the reviewed articles that suggest the need for a locally tailored approach. Conclusion A more detailed characterization of forest activities is needed but research on this topic raises methodological challenges. Current vector control measures have limitations, with use of insecticidal-treated nets, hammocks and repellents influenced by the type of forest activities and the characteristics of these measures. In contrast, anti-malarial drugs, for example, as chemoprophylaxis, hold promise but require further evaluation.

Abstract Background Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy worldwide. Primaquine is the only licensed drug that effectively removes Plasmodium vivax hypnozoites from the human host and prevents relapse. While well tolerated by most recipients, primaquine can cause haemolysis in G6PD deficient individuals and is, therefore, underused. Rapid diagnostic tests (RDTs) could permit ascertainment of G6PD status outside of laboratory settings and hence safe treatment in remote areas. The performance of the fluorescent spot test (Trinity, Ireland; FST) and a G6PD RDT (Carestart, USA) against spectrophotometry were assessed. Methods Participants were enrolled during cross-sectional surveys in Laos and by purposive sampling in Cambodia. FST and RDT were performed during village surveys and 3 mL of venous blood was collected for subsequent G6PD measurement by spectrophotometry. Results A total of 757 participants were enrolled in Laos and 505 in Cambodia. FST and RDT performed best at 30% cut-off activity and performed significantly better in Laos than in Cambodia. When defining intermediate results as G6PD deficient, the FST had a sensitivity of 100% (95%CI 90–100) and specificity of 90% (95%CI 87.7–92.2) in Laos and sensitivity of 98% (94.1–99.6) and specificity of 71% (95%CI 66–76) in Cambodia (p

Abstract Two mass drug administrations (MDA) against falciparum malaria were conducted in 2015–16, one as operational research in northern Cambodia, and the other as a clinical trial in western Cambodia. During an April 2017 workshop in Phnom Penh the field teams from Médecins Sans Frontières and the Mahidol-Oxford Tropical Medicine Research Unit discussed lessons for future MDAs.

Abstract Background Mass anti-malarial administration has been proposed as a key component of the Plasmodium falciparum malaria elimination strategy in the Greater Mekong sub-Region. Its effectiveness depends on high levels of coverage in the target population. This article explores the factors that influenced mass anti-malarial administration coverage within a clinical trial in Battambang Province, western Cambodia. Methods Qualitative data were collected through semi-structured interviews and focus group discussions with villagers, in-depth interviews with study staff, trial drop-outs and refusers, and observations in the communities. Interviews were audio-recorded, transcribed and translated from Khmer to English for qualitative content analysis using QSR NVivo. Results Malaria was an important health concern and villagers reported a demand for malaria treatment. This was in spite of a fall in incidence over the previous decade and a lack of familiarity with asymptomatic malaria. Participants generally understood the overall study aim and were familiar with study activities. Comprehension of the study rationale was however limited. After the first mass anti-malarial administration, seasonal health complaints that participants attributed to the anti-malarial as “side effects” contributed to a decrease of coverage in round two. Staff therefore adapted the community engagement approach, bringing to prominence local leaders in village meetings. This contributed to a subsequent increase in coverage. Conclusion Future mass anti-malarial administration must consider seasonal disease patterns and the importance of local leaders taking prominent roles in community engagement. Further research is needed to investigate coverage in scenarios that more closely resemble implementation i.e. without participation incentives, blood sampling and free healthcare.

Background: The malaria burden is decreasing throughout the Greater Mekong Subregion, however transmission persists in some areas. Human movement, subclinical infections and complicated transmission patterns contribute to the persistence of malaria. This research describes the micro-geographical epidemiology of both clinical malaria and subclinical Plasmodium infections in three villages in Western Cambodia. Methods: Three villages in Western Cambodia were selected for the study based on high reported Plasmodium falciparum incidence. A census was conducted at the beginning of the study, including demographic information and travel history. The total population was 1766. Cross-sectional surveys were conducted every three months from June 2013 to June 2014. Plasmodium infections were detected using an ultra-sensitive, high-volume, quantitative polymerase chain reaction (uPCR) technique. Clinical episodes were recorded by village health workers. The geographic coordinates (latitude and longitude) were collected for all houses and all participants were linked to their respective houses using a demographic surveillance system. Written informed consent was obtained from all participants. Results: Most clinical episodes and subclinical infections occurred within a single study village. Clinical Plasmodium vivax episodes clustered spatially in each village but only lasted for a month. In one study village subclinical infections clustered in geographic proximity to clusters of clinical episodes. The largest risk factor for clinical P. falciparum episodes was living in a house where another clinical P. falciparum episode occurred (model adjusted odds ratio (AOR): 6.9; CI: 2.3–19. 8). Subclinical infections of both P. vivax and P. falciparum were associated with clinical episodes of the same species (AOR: 5.8; CI: 1.5–19.7 for P. falciparum and AOR: 14.6; CI: 8.6–25.2 for P. vivax) and self-reported overnight visits to forested areas (AOR = 3.8; CI: 1.8–7. 7 for P. falciparum and AOR = 2.9; CI: 1.7–4.8 for P. vivax). Discussion: Spatial clustering within the villages was transient, making the prediction of spatial clusters difficult. Interventions that are dependent on predicting spatial clusters (such as reactive case detection) would only have detected a small proportion of cases unless the entire village was screened within a limited time frame and with a highly sensitive diagnostic test. Subclinical infections may be acquired outside of the village (particularly in forested areas) and may play an important role in transmission.

Following publication of the original article [1], it was brought to the authors’ attention that one of the names in the author list had been provided with the incorrect spelling.