Although the power of genetic surveillance tools has been acknowledged widely, there is an urgent need in malaria endemic countries for feasible and cost-effective tools to implement in national malaria control programs (NMCPs) that can generate evidence to guide malaria control and elimination strategies, especially in the case of Plasmodium vivax. Several genetic surveillance applications (‘use cases’) have been identified to align research, technology development, and public health efforts, requiring different types of molecular markers. Here we present a new highly-multiplexed deep sequencing assay (Pv AmpliSeq). The assay targets the 33-SNP vivaxGEN-geo panel for country-level classification, and a newly designed 42-SNP within-country barcode for analysis of parasite dynamics in Vietnam and 11 putative drug resistance genes in a highly multiplexed NGS protocol with easy workflow, applicable for many different genetic surveillance use cases. The Pv AmpliSeq assay was validated using: 1) isolates from travelers and migrants in Belgium, and 2) routine collections of the national malaria control program at sentinel sites in Vietnam. The assay targets 229 amplicons and achieved a high depth of coverage (mean 595.7 ± 481) and high accuracy (mean error-rate of 0.013 ± 0.007). P. vivax parasites could be characterized from dried blood spots with a minimum of 5 parasites/µL and 10% of minority-clones. The assay achieved good spatial specificity for between-country prediction of origin using the 33-SNP vivaxGEN-geo panel that targets rare alleles specific for certain countries and regions. A high resolution for within-country diversity in Vietnam was achieved using the designed 42-SNP within-country barcode that targets common alleles (median MAF 0.34, range 0.01-0.49. Many variants were detected in (putative) drug resistance genes, with different predominant haplotypes in the pvmdr1 and pvcrt genes in different provinces in Vietnam. The capacity of the assay for high resolution identity-by-descent (IBD) analysis was demonstrated and identified a high rate of shared ancestry within Gia Lai Province in the Central Highlands of Vietnam, as well as between the coastal province of Binh Thuan and Lam Dong. Our approach performed well in geographically differentiating isolates at multiple spatial scales, detecting variants in putative resistance genes, and can be easily adjusted to suit the needs in other settings in a country or region. We prioritize making this tool available to researchers and NMCPs in endemic countries to increase ownership and ensure data usage for decision-making and malaria policy.

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

Despite the scale-up of vector control, diagnosis and treatment, and health information campaigns, malaria persists in the forested areas of South-Central Vietnam, home to ethnic minority populations. A mixed-methods study using an exploratory sequential design was conducted in 10 Ra-glai villages in Bac Ai district of Ninh Thuan province to examine which social factors limited the effectiveness of the national malaria elimination strategy in the local setting. Territorial arrangements and mobility were found to directly limit the effectiveness of indoor residual spraying and long-lasting insectidical treated nets (LLINs). Households (n=410) were resettled in the “new villages” by the government, where they received brick houses (87.1%) and sufficient LLINs (97.3%). However, 97.6% of households went back to their “old villages” to continue slash-and-burn agriculture. In the old village, 48.5% of households lived in open-structured plot huts and only 5.7% of them had sufficient LLIN coverage. Household representatives believed malaria could be cured with antimalarials (57.8%), but also perceived non-malarial medicines, rituals, and vitamin supplements to be effective against malaria. Household members (n = 1,957) used public health services for their most recent illness (62.9%), but also reported to buy low-cost medicines from the private sector to treat fevers and discomfort as these were perceived to be the most cost-effective treatment option for slash-and-burn farmers. The study shows the relevance of understanding social factors to improve the uptake of public health interventions and calls for contextually adapted strategies for malaria elimination in ethnic minority populations in Vietnam and similar settings.

Malaria elimination in the Greater Mekong Sub-Region is challenged by a rising proportion of malaria attributable to P. vivax. Primaquine (PQ) is effective in eliminating the parasite’s dormant liver stages and can prevent relapsing infections, but it induces severe haemolysis in patients with Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency, highlighting the importance of testing enzyme activity prior to treatment. A mixed-method study was conducted in south-central Vietnam to explore the factors that affect acceptability of G6PD testing, treatment-seeking behaviors, and adherence to current regimens. The majority of respondents (75.7%) were unaware of the different parasite species and rather differentiated malaria by perceived severity. People sought a diagnosis if suspected of malaria fever but not if they perceived their fevers as mild. Most respondents agreed to take prescribed medication to treat asymptomatic infection (94.1%) and to continue medication even if they felt better (91.5%). Health professionals did not have G6PD diagnostic tools nor the means to prescribe PQ safely. Adherence to treatment was linked to trust in public providers, who were perceived to make therapeutic decisions in the interest of the patient. Greater focus on providing acceptable ways of assessing G6PD deficiency will be needed to ensure the timely elimination of malaria in Vietnam.

Background Despite freely distributed insecticide-treated nets (ITNs) and health information campaigns to increase their use among populations at risk, malaria transmission persists in forested areas in Vietnam, especially among ethnic minority communities. A mixed-methods study was conducted in four villages of Ca Dong and M’nong ethnicity in Central Vietnam between 2009 and 2011 to assess factors limiting the uptake of ITNs. Methods The mixed-methods research design consisted of a qualitative study to explore the context and barriers to ITN use, and a cross-sectional household survey (n = 141) to quantify factors for limited and appropriate net use. Results The Ca Dong and M’nong’s livelihood was dependent on swidden farming in the forest. Poverty-related factors, including the lack of beds, blankets, the practice of sleeping around the kitchen fire and deteriorated ITNs due to open housing structures, were reasons for alternative and non-use of ITNs. When household members stayed overnight in plot huts at fields, ITNs were even more unavailable and easily deteriorated. 72.5% of households reported having received one net for every two persons, and 82.2% of participants reported to have used ITNs the night before the survey. However, only 18.4% of participants were estimated to be effectively protected by ITNs after accounting for the availability of torn ITNs and the way ITNs were used, for example as blankets, at both village and fields. Multi-variable logistic regression showed the effect of four significant factors for appropriate ITN use: i) being female (AOR = 8.08; p = 0.009); ii) aware of mosquito bites as the sole cause of malaria (AOR = 7.43; p = 0.008); iii) not sleeping around the kitchen fire (AOR = 24.57; p = 0.001); and iv) having sufficient number of ITNs in the household (AOR = 21.69; p = 0.001). Conclusion This study showed how social factors rooted in poverty and swidden agriculture limited the effective use of ITNs, despite high coverage, among ethnic minority populations in Central Vietnam. An in-depth understanding of the local context is essential to develop specific indicators for measuring ITN use.

Background: Despite free distribution of insecticidal-treated nets (ITNs) and long-lasting insecticidal nets (LLINs) to populations at-risk, malaria transmission persists in forested areas in Vietnam especially amongst ethnic minority communities. A study was conducted between April 2009 and December 2010 in four villages of Ca Dong and M’nong ethnicity located in Central Vietnam with persistent malaria transmission. The objective was to assess the feasibility of bed net use in forest settings with a specific emphasis on contrasting people’s willingness/choice to use nets versus structural limitations to the uptake of the intervention. Methods: The study used an exploratory mixed-method design for complementarity. The qualitative strand included participant observation, informal interviews, in-depth interviews, and group discussions to explore contextual barriers to bednet use. The quantitative strand consisted of a cross sectional household survey (n=141) in a selected village with direct access to bed nets and the health centre. The main outcome was bednet use and related risk factors (using survey logistic regression), and secondary outcomes included the quantification of contextual factors identified during the qualitative strand. Results: Despite self-reported bednet coverage (72.5% of households had sufficient net coverage) and appropriate betnet use the night before the survey (82.2% of HHMs), only 18.4% of HHMs were effectively protected by nets in both the village home and the forest field. The distribution of LLINs and ITNs was not accounted for multiple residency and sleeping places linked to slash-and-burn agriculture, and contextual factors including open-housing structured, high poverty, and animistic beliefs contributing to inappropriate use and shorter lifespan of nets. Health messages promoting bednet use were provided to the population but only 19.8% of respondents considered mosquito bites as the sole cause of malaria. The multi-variable logistic regression showed the effect of four significant factors to appropriate bednet use: being female (AOR=8.08; p=0.009); having knowledge of mosquito bites and malaria link (AOR=7.43; p=0.008); not sleeping in the kitchen (AOR=24.57; p=0.001); and, having sufficient number of bednets in the household (AOR=21.69; p=0.001). Conclusion: This study identified contextual factors that constrains effective bednet use amongst ethnic minority populations living in forested areas of Central Vietnam and calls for multi-sectoral interventions to address the complexity of residual malaria transmission and reach the malaria elimination goal of 2030.