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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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kattenberg, Nguyen, Nguyen, Sauve, Nguyen, Chopo-Pizarro, Trimarsanto, Monsieurs, Guetens, Nguyen, Esbroeck, Auburn, Nguyen and Rosanas-Urgell.)

Background: With the decline in local malaria transmission in Vietnam as a result of the National Malaria Control Program (NMCP) elimination activities, a greater focus on the importation and potential reintroduction of transmission are essential to support malaria elimination objectives., Methods: We conducted a multi-method assessment of the demographics, epidemiology, and clinical characteristics of imported malaria among international laborers returning from African or Southeast Asian countries to Vietnam. Firstly, we conducted a retrospective review of hospital records of patients from January 2014 to December 2016. Secondly, we conducted a mixed-methods prospective study for malaria patients admitted to the study sites from January 2017 to May 2018 using a structured survey with blood sample collection for PCR analysis and in-depth interviews. Data triangulation of the qualitative and quantitative data was used during analysis., Results: International laborers were young (median age 33.0 years IQR 28.0-39.5 years), predominantly male (92%) adults returning mostly from the African continent (84%) who stayed abroad for prolonged periods (median time 13.5 months; IQR 6.0-331.5 months) and were involved in occupations that exposed them to a higher risk of malaria infection. Epidemiological trends were also similar amongst study strands and included the importation of Plasmodium falciparum primarily from African countries and P. vivax from Southeast Asian countries. Of 11 P. malariae and P. ovale infections across two study strands, 10 were imported from the African continent. Participants in the qualitative arm demonstrated limited knowledge about malaria prior to travelling abroad, but reported knowledge transformation through personal or co-worker's experience while abroad. Interestingly, those who had a greater understanding of the severity of malaria presented to the hospital for treatment sooner than those who did not; median of 3 days (IQR 2.0-7.0 days) versus 5 days (IQR 4.0-9.5 days) respectively., Conclusion: To address the challenges to malaria elimination raised by a growing Vietnamese international labor force, consideration should be given to appropriately targeted interventions and malaria prevention strategies that cover key stages of migration including pre-departure education and awareness, in-country prevention and prophylaxis, and malaria screening upon return., (© 2022. The Author(s).)

Background: A key step to advancing the goal of malaria elimination in Viet Nam by 2030 is focusing limited resources for treatment and prevention to groups most at risk for malaria transmission., Methods: To better understand risk factors for malaria transmission in central Viet Nam, a survey of 1000 malaria positive cases and 1000 malaria negative controls was conducted. Cases and controls were matched for age and gender and self-presented at commune health stations (CHS) in Binh Phuoc, Dak Nong and Dak Lak Provinces. Diagnoses were confirmed with microscopy, rapid diagnostic test and PCR. Participants were interviewed about 50 potential risk factors for malaria, which included information about occupation, forest visitation, travel, healthcare-seeking behaviour and prior use of anti-malaria interventions. Participants were enrolled by trained government health workers and the samples were analysed in Vietnamese government laboratories. Data were analysed by univariable, block-wise and multivariable logistic regression., Results: Among cases, 61.8% had Plasmodium falciparum, 35.2% Plasmodium vivax and 3% mixed species infections. Median (IQR) age was 27 (21-36) years and 91.2% were male. Twenty-five risk factors were associated with being a case and eleven with being a control. Multivariable analysis found that malaria cases correlated with forest workers, recent forest visitation, longer duration of illness, having a recorded fever, number of malaria infections in the past year, having had prior malaria treatment and having previously visited a clinic., Conclusions: This study demonstrates the benefits of increased statistical power from matched controls in malaria surveillance studies, which allows identification of additional independent risk factors. It also illustrates an example of research partnership between academia and government to collect high quality data relevant to planning malaria elimination activities. Modifiable risk factors and implications of the findings for malaria elimination strategy are presented., (© 2021. The Author(s).)