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Abstract Background The Lao Government changed the national policy for uncomplicated Plasmodium falciparum malaria from chloroquine to artemether-lumefantrine (AL) in 2005. Since then, no information on AL efficacy has been reported. With evidence of resistance to artemisinin derivatives in adjacent Cambodia, there has been a concern as to AL efficacy. Monitoring of AL efficacy would help the Lao Government to make decisions on appropriate malaria treatment. Methods The efficacy of a three-day, twice daily oral artemether-lumefantrine for the treatment of uncomplicated falciparum malaria in Xepon District, Savannakhet Province, southern Laos was studied over 42 days follow-up. This was part of a trial of thiamin supplementation in falciparum malaria. Results Of 630 patients with P. falciparum enrolled in the trial of thiamin treatment, 549 (87%, 357 children ≤15 years and 192 adults) were included in this study. The per protocol 42-day cure rates were 97% (524/541) [96% (337/352) for children and 99% (187/189) for adults, p = 0.042]. By conventional intention-to-treat analysis, the 42-day cure rates adjusted for re-infection, were 97% (532/549) [96% (342/357) in children and 99% (190/192) in adults, p = 0.042]. The proportion of patients who remained parasitaemic at day 1 after treatment was significantly higher in children [33% (116/356)] compared to adults [15% (28/192)] (p Conclusions The current nationally-recommended anti-malarial treatment (artemether-lumefantrine) remains highly efficacious for the treatment of uncomplicated falciparum malaria five years after introduction in Laos. Regular monitoring is required in case artemisinin-resistant P. falciparum parasites should appear. Trial registration ISRCTN85411059.

Abstract Background Accurate information on the geographical distribution of malaria is important for efficient resource allocation. The Lao People's Democratic Republic has experienced a major decline in malaria morbidity and mortality in the past decade. However, efforts to respond effectively to these changes have been impeded by lack of detailed data on malaria distribution. In 2008, a countrywide survey on Plasmodium falciparum diagnosed in health centres and villages was initiated to develop a detailed P. falciparum risk map with the aim to identify priority areas for malaria control, estimate population at risk, and guide resource allocation in the Lao People's Democratic Republic. Methods P. falciparum incidence data were collected from point-referenced villages and health centres for the period 2006-2008 during a country-wide survey between December 2008 and January 2009. Using the highest recorded annual rate, continuous surfaces of P. falciparum incidence were produced by the inverse distance weighted interpolation technique. Results Incidence rates were obtained from 3,876 villages and 685 health centres. The risk map shows that P. falciparum is highly heterogeneous in the northern and central regions of the country with large areas of no transmission. In the southern part, transmission is pervasive and the risk of P. falciparum is high. It was estimated that 3.4 million people (60% of the population) live at risk of malaria. Conclusions This paper presents the first comprehensive malaria risk map of the Lao People's Democratic Republic based entirely on empirical data. The estimated population at risk is substantially lower than previous estimates, reflecting the presence of vast areas with focal or no malaria transmission as identified in this study. These findings provide important guidance for malaria control interventions in the Lao People's Democratic Republic, and underline the need for detailed data on malaria to accurately predict risk in countries with heterogeneous transmission.

While there has been significant progress in controlling falciparum malaria in the Lao People's Democratic Republic (PDR), sporadic cases persist in southern provinces where the extent and patterns of transmission remain largely unknown. To assess parasite transmission in this area, 53 Plasmodium falciparum (Pf) positive cases detected through active test and treat campaigns from December 2017 to November 2018 were sequenced, targeting 204 highly polymorphic amplicons. Two R packages, MOIRE and Dcifer, were applied to assess the multiplicity of infections (MOI), effective MOI (eMOI), within-host parasite relatedness, and between-host parasite relatedness ([Formula: see text]). Genomic data were integrated with survey data to characterize the temporal and spatial structures of identified clusters. The positive cases were mainly captured during the focal test and treat campaign conducted in 2018, and in the Pathoomphone area, which had the highest test positivity and forest activity. About 30% of the cases were polyclonal infections, with over half of theses (63%) showing within-host relatedness greater than 0.6, suggesting that cotransmission rather than superinfection was primarily responsible for maintaining polyclonality. A large majority of cases (81%) were infected by parasites genetically linked to one or more other cases. We identified five genetically distinct clusters in forest fringe villages within the Pathoomphone district, characterized by a high degree of genetic relatedness between parasites (mean [Formula: see text] = 0.8). Four smaller clusters of 2-3 cases linked Moonlapamok and Pathoomphone districts, with an average [Formula: see text] of 0.6, suggesting cross-district transmission. Most of the clustered cases occurred within 20 km and 2 months of each other, consistent with focal transmission. Transmission clusters identified in this study confirm the role of ongoing focal parasite transmission occurring within the forest or forest-fringe in the highly mobile population., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: The present study aimed to analyze the impact of deforestation on the malaria distribution in the Lao People's Democratic Republic (Lao PDR), with consideration of climate change., Methods: Malaria distribution data from 2002 to 2015 were obtained from the Ministry of Health of Lao PDR and each indicator was calculated. Earth observation satellite data (forested area, land surface temperature, and precipitation) were obtained from the Japan Aerospace Exploration Agency (JAXA). Structured equation modeling (SEM) was conducted to clarify the relationship between the malaria incidence and Earth observation satellite data., Results: As a result, SEM identified two factors that were independently associated with the malaria incidence: area and proportion of forest. Specifically, malaria was found to be more prevalent in the southern region, with the malaria incidence increasing as the percentage of forested land increased (both p < 0.01). With global warming steadily progressing, forested areas are expected to play an important role in the incidence of malaria in Lao PDR. This is believed because malaria in Lao PDR is mainly forest malaria transmitted by Anopheles dirus., Conclusion: To accelerate the elimination of malaria in Lao PDR, it is important to identify, prevent, and intervene in places with increased forest coverage (e.g., plantations) and in low-temperature areas adjacent to malaria-endemic areas, where the vegetation is similar to that in malaria-endemic areas., (© 2023. The Author(s).)

Background: The Lao PDR National Strategic Plan for malaria control and elimination for year 2021-2025 emphasizes the importance of routine entomological surveillance being conducted in areas with high transmission and in active malaria foci in elimination targeted areas. The collection of entomological surveillance data that is closely linked to recent epidemiological data is crucial for improving impact, as it contributes to the evidence package that supports operational and strategic decision-making of national malaria programmes, as they accelerate their last mile of elimination., Methods: The Center for Malariology Parasitology and Epidemiology (CMPE) entomology team conducted entomological surveillance activities at 13 sentinel sites in 8 provinces and at active transmission foci sites from 2018 to 2020. The techniques used for the mosquito collection were indoor and outdoor human landing collections (from houses and from cultivation areas) and cattle baited net trap collections., Results: There were 5601 Anopheles mosquito females captured and identified throughout the study, on both human and cow bait. They represented 15 different species or species complexes. The primary malaria vectors as well as the secondary vectors were present in all collection sites in the south, indicating that people living in these rural areas with high malaria incidence are exposed to the vectors. The vectors were highly zoophilic, but they still bite humans throughout the night with a high peak of activity before midnight, both indoors and outdoors. Overall, 17% of the malaria vectors were collected indoors when the people are sleeping. This confirms the importance of bed net use during the night. Thirty-two percent of primary and secondary vectors were collected outdoors at times when people are usually awake and outdoors, which shows that people are exposed to potentially infectious mosquitoes and the importance of personal protection at these times. The findings showed that residual transmission may occur outdoors in the villages, and outside the villages in cultivation fields and forested areas. Epidemiological data showed that transmission was higher in surveillance sites which were targeted as part of a malaria response rather than sentinel sites., Conclusions: Understanding where and how transmission is persisting, monitoring and mapping vector species distribution in areas with active transmission, monitoring biting trends, and designing evidence based and effective vector control interventions are critical to accelerating progress toward malaria elimination. In this context, the role of entomological surveillance combined with epidemiological data should be considered as a cornerstone in achieving malaria elimination., (© 2023. BioMed Central Ltd., part of Springer Nature.)

The prevalence of Schistosoma mekongi in humans in the Lao People's Democratic Republic (Lao PDR) has been relatively well monitored and has decreased due to effective interventions such as preventative chemotherapy with mass drug administration of praziquantel and community awareness programs. However, the prevalence among potential domestic reservoir animals remains broadly unclear, except for a few villages in the endemic area. Therefore, we conducted S. mekongi surveys for the domestic animals that had contact with Mekong River water. We conducted a cross-sectional study of the domestic animals in the seven sentinel villages in the Khong and Mounlapamok Districts of Champasak Province in southern Lao PDR in 2018 by random sampling with a statistically reliable sample size. Stool samples of the five predominant domestic animal species, cattle (n = 160), pig (n = 154), buffalo (n = 149), dog (n = 143), and goat (n = 85), were collected and examined using parasitological FECT method and the LAMP technique. The microscopic analysis did not detect any eggs of S. mekongi in the stool samples of any animal species. However, S. mekongi DNA was detected by the LAMP test in dog stool samples (0.7%; 1/143). On the other hand, the prevalence of other helminths was quite high and heterogeneous among animal species and sentinel sites by the microscopic analysis. These findings suggested that an intervention for S. mekongi infection should focus solely on human populations. However, periodic surveillance for S. mekongi infection among dogs should be conducted to monitor a possible resurgence of S. mekongi infection in the domestic animal population.

Increasing attention is being given to the effect of climate change on schistosomiasis, but the impact is currently unknown. As the intermediate snail host ( Neotricula aperta ) of Schistosoma mekongi inhabits the Mekong River, it is thought that environmental factors affecting the area of water will have an impact on the occurrence of schistosomiasis mekongi. The aim of the present study was to assess the impact of precipitation on the prevalence of human schistosomiasis mekongi using epidemiological data and Earth observation satellite data in Khong district, Champasak province, Lao PDR. Structural equation modelling (SEM) using epidemiological data and Earth observation satellite data was conducted to determine the factors associated with the number of schistosomiasis mekongi patients. As a result, SEM identified 3 significant factors independently associated with schistosomiasis mekongi: (1) a negative association with mass drug administration (MDA); (2) negative association with total precipitation per year; and (3) positive association with precipitation during the dry season. Precisely, regardless of MDA, the increase in total yearly precipitation was suggested to decrease the number of schistosomiasis patients, whereas an increase in precipitation in the dry season increased the number of schistosomiasis patients. This is probably because when total precipitation increases, the water level of the Mekong River rises, thus decreasing the density of infected larvae, cercaria, in the water, and the frequency of humans entering the river would also decrease. In contrast, when precipitation in the dry season is higher, the water level of the Mekong River also rises, which expands the snail habitant, and thus water contact between humans and the snails would also increase. The present study results suggest that increasing precipitation would impact the prevalence of schistosomiasis both positively and negatively, and precipitation should also be considered in the policy to eliminate schistosomiasis mekongi in Lao PDR., Competing Interests: The authors declare that they have no competing interests., (© 2023 The Authors.)

Background: In the Greater Mekong Subregion (GMS), forest-going populations are considered high-risk populations for malaria and are increasingly targeted by national control programmes' elimination efforts. A better understanding of forest-going populations' mobility patterns and risk associated with specific types of forest-going trips is necessary for countries in the GMS to achieve their objective of eliminating malaria by 2030., Methods: Between March and November 2018, as part of a focal test and treat intervention (FTAT), 2,904 forest-goers were recruited in southern Lao PDR. A subset of forest-goers carried an "i-Got-U" GPS logger for roughly 2 months, configured to collect GPS coordinates every 15 to 30 min. The utilization distribution (UD) surface around each GPS trajectory was used to extract trips to the forest and forest-fringes. Trips with shared mobility characteristics in terms of duration, timing and forest penetration were identified by a hierarchical clustering algorithm. Then, clusters of trips with increased exposure to dominant malaria vectors in the region were further classified as high-risk. Finally, gradient boosting trees were used to assess which of the forest-goers' socio-demographic and behavioural characteristics best predicted their likelihood to engage in such high-risk trips., Results: A total of 122 forest-goers accepted carrying a GPS logger resulting in the collection of 803 trips to the forest or forest-fringes. Six clusters of trips emerged, helping to classify 385 (48%) trips with increased exposure to malaria vectors based on high forest penetration and whether the trip happened overnight. Age, outdoor sleeping structures and number of children were the best predictors of forest-goers' probability of engaging in high-risk trips. The probability of engaging in high-risk trips was high (~ 33%) in all strata of the forest-going population., Conclusion: This study characterized the heterogeneity within the mobility patterns of forest-goers and attempted to further segment their role in malaria transmission in southern Lao People's Democratic Republic (PDR). National control programmes across the region can leverage these results to tailor their interventions and messaging to high-risk populations and accelerate malaria elimination., (© 2023. The Author(s).)

Background: The amplification of GTP cyclohydrolase 1 (pfgch1) in Plasmodium falciparum has been linked to the upregulation of the pfdhfr and pfdhps genes associated with resistance to the antimalarial drug sulfadoxine-pyrimethamine. During the 1990s and 2000s, sulfadoxine-pyrimethamine was withdrawn from use as first-line treatment in southeast Asia due to clinical drug resistance. This study assessed the temporal and geographic changes in the prevalence of pfdhfr and pfdhps gene mutations and pfgch1 amplification a decade after sulfadoxine-pyrimethamine had no longer been widely used., Methods: A total of 536 P. falciparum isolates collected from clinical trials in Thailand, Cambodia, and Lao PDR between 2008 and 2018 were assayed. Single nucleotide polymorphisms of the pfdhfr and pfdhps genes were analyzed using nested PCR and Sanger sequencing. Gene copy number variations of pfgch1 were investigated using real-time polymerase chain reaction assay., Results: Sequences of the pfdhfr and pfdhps genes were obtained from 96% (517/536) and 91% (486/536) of the samples, respectively. There were 59 distinct haplotypes, including single to octuple mutations. The two major haplotypes observed included IRNI-AGEAA (25%) and IRNL-SGKGA (19%). The sextuple mutation IRNL-SGKGA increased markedly over time in several study sites, including Pailin, Preah Vihear, Ratanakiri, and Ubon Ratchathani, whereas IRNI-AGEAA decreased over time in Preah Vihear, Champasak, and Ubon Ratchathani. Octuple mutations were first observed in west Cambodia in 2011 and subsequently in northeast Cambodia, as well as in southern Laos by 2018. Amplification of the pfgch1 gene increased over time across the region, particularly in northeast Thailand close to the border with Laos and Cambodia., Conclusion: Despite the fact that SP therapy was discontinued in Thailand, Cambodia, and Laos decades ago, parasites retained the pfdhfr and pfdhps mutations. Numerous haplotypes were found to be prevalent among the parasites. Frequent monitoring of pfdhfr and pfdhps in these areas is required due to the relatively rapid evolution of mutation patterns., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Srisutham et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Schistosomiasis mekongi infection represents a public health concern in Laos and Cambodia. While both countries have made significant progress in disease control over the past few decades, eradication has not yet been achieved. Recently, several studies reported the application of loop-mediated isothermal amplification (LAMP) for detecting Schistosoma DNA in low-transmission settings. The objective of this study was to develop a LAMP assay for Schistosoma mekongi using a simple DNA extraction method. In particular, we evaluated the utility of the LAMP assay for detecting S. mekongi DNA in human stool and snail samples in endemic areas in Laos. We then used the LAMP assay results to develop a risk map for monitoring schistosomiasis mekongi and preventing epidemics. A total of 272 stool samples were collected from villagers on Khon Island in the southern part of Laos in 2016. DNA for LAMP assays was extracted via the hot-alkaline method. Following the Kato-Katz method, we determined that 0.4% (1/272) of the stool samples were positive for S. mekongi eggs, as opposed to 2.9% (8/272) for S. mekongi DNA based on the LAMP assays. Snail samples ( n = 11,762) were annually collected along the riverside of Khon Island from 2016 to 2018. DNA was extracted from pooled snails as per the hot-alkaline method. The LAMP assay indicated that the prevalence of S. mekongi in snails was 0.26% in 2016, 0.08% in 2017, and less than 0.03% in 2018. Based on the LAMP assay results, a risk map for schistosomiasis with kernel density estimation was created, and the distribution of positive individuals and snails was consistent. In a subsequent survey of residents, schistosomiasis prevalence among villagers with latrines at home was lower than that among villagers without latrines. This is the first study to develop and evaluate a LAMP assay for S. mekongi detection in stools and snails. Our findings indicate that the LAMP assay is an effective method for monitoring pathogen prevalence and creating risk maps for schistosomiasis.

The epidemiology of malaria changes as prevalence falls in low-transmission settings, with remaining infections becoming more difficult to detect and diagnose. At this stage active surveillance is critical to detect residual hotspots of transmission. However, diagnostic tools used in active surveillance generally only detect concurrent infections, and surveys may benefit from sensitive tools such as serological assays. Serology can be used to interrogate and characterize individuals' previous exposure to malaria over longer durations, providing information essential to the detection of remaining foci of infection. We ran blood samples collected from a 2016 population-based survey in the low-transmission setting of northern Lao PDR on a multiplexed bead assay to characterize historic and recent exposures to Plasmodium falciparum and vivax . Using geostatistical methods and remote-sensing data we assessed the environmental and spatial associations with exposure, and created predictive maps of exposure within the study sites. We additionally linked the active surveillance PCR and serology data with passively collected surveillance data from health facility records. We aimed to highlight the added information which can be gained from serology as a tool in active surveillance surveys in low-transmission settings, and to identify priority areas for national surveillance programmes where malaria risk is higher. We also discuss the issues faced when linking malaria data from multiple sources using multiple diagnostic endpoints., 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 Byrne, Cramer, Nelli, Rerolle, Wu, Patterson, Rosado, Dumont, Tetteh, Dantzer, Hongvanthong, Fornace, Stresman, Lover, Bennett and Drakeley.)

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

Forest-going populations are key to malaria transmission in the Greater Mekong Sub-region (GMS) and are therefore targeted for elimination efforts. Estimating the size of this population is essential for programs to assess, track and achieve their elimination goals. Leveraging data from three cross-sectional household surveys and one survey among forest-goers, the size of this high-risk population in a southern province of Lao PDR between December 2017 and November 2018 was estimated by two methods: population-based household surveys and capture-recapture. During the first month of the dry season, the first month of the rainy season, and the last month of the rainy season, respectively, 16.2% [14.7; 17.7], 9.3% [7.2; 11.3], and 5.3% [4.4; 6.1] of the adult population were estimated to have engaged in forest-going activities. The capture-recapture method estimated a total population size of 18,426 [16,529; 20,669] forest-goers, meaning 61.0% [54.2; 67.9] of the adult population had engaged in forest-going activities over the 12-month study period. This study demonstrates two methods for population size estimation to inform malaria research and programming. The seasonality and turnover within this forest-going population provide unique opportunities and challenges for control programs across the GMS as they work towards malaria elimination., (© 2021. The Author(s).)

As countries in the Greater Mekong Sub-region (GMS) increasingly focus their malaria control and elimination efforts on reducing forest-related transmission, greater understanding of the relationship between deforestation and malaria incidence will be essential for programs to assess and meet their 2030 elimination goals. Leveraging village-level health facility surveillance data and forest cover data in a spatio-temporal modeling framework, we found evidence that deforestation is associated with short-term increases, but long-term decreases confirmed malaria case incidence in Lao People's Democratic Republic (Lao PDR). We identified strong associations with deforestation measured within 30 km of villages but not with deforestation in the near (10 km) and immediate (1 km) vicinity. Results appear driven by deforestation in densely forested areas and were more pronounced for infections with Plasmodium falciparum ( P. falciparum ) than for Plasmodium vivax ( P. vivax ). These findings highlight the influence of forest activities on malaria transmission in the GMS., Competing Interests: FR, ED, AL, JM, BH, HS, AB No competing interests declared, (© 2021, Rerolle et al.)

Introduction: Novel interventions are needed to accelerate malaria elimination, especially in areas where asymptomatic parasitemia is common, and where transmission generally occurs outside of village-based settings. Testing of community members linked to a person with clinical illness (reactive case detection, RACD) has not shown effectiveness in prior studies due to the limited sensitivity of current point-of-care tests. This study aims to assess the effectiveness of active case finding in village-based and forested-based settings using novel high-sensitivity rapid diagnostic tests in Lao People's Democratic Republic (Lao PDR). Methods and analysis: This study is a cluster-randomized split-plot design trial. The interventions include village-based mass test and treat (MTAT), focal test and treat in high-risk populations (FTAT), and the combination of these approaches, using high-sensitivity rapid diagnostic tests (HS-RDTs) to asses P. falciparum infection status. Within four districts in Champasak province, Lao PDR fourteen health center-catchment areas will be randomized to either FTAT or control; and within these HCCAs, 56 villages will be randomized to either MTAT or control. In intervention areas, FTAT will be conducted by community-based peer navigators on a routine basis, and three separate rounds of MTAT are planned. The primary study outcome will be PCR-based Plasmodium falciparum prevalence after one year of implementation. Secondary outcomes include malaria incidence; interventional coverage; operational feasibility and acceptability; and cost and cost- effectiveness. Ethics and dissemination: Findings will be reported on clinicaltrials.gov, in peer-reviewed publications and through stakeholder meetings with Ministry of Health and community leaders in Lao PDR and throughout the Greater Mekong Subregion. Trial registration: clinicaltrials.gov NCT03783299 (21/12/2018)., Competing Interests: No competing interests were disclosed., (Copyright: © 2019 Lover AA et al.)

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., (Copyright © 2019 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Background: Primaquine is effective against the latent liver stage of Plasmodium vivax. Eliminating the latent liver stage of P. vivax is one of the necessary conditions to achieve the goal of malaria elimination in Lao People's Democratic Republic (PDR) by 2030. However, people with glucose-6-phosphate dehydrogenase (G6PD) deficiency are at risk of haemolysis when ingesting primaquine. The aim of this study was to detect the prevalence of the G6PD Viangchan variant, which is said to be common in Lao PDR and which can result in severe haemolysis in patients exposed to primaquine., Methods: Blood samples were collected from villagers in three malaria endemic provinces: Champasak and Savannakhet in the south, and Phongsaly in the north. Each blood sample was semi-quantitatively assayed for G6PD enzyme activity using the G6PD Assay Kit-WST Lyophilized (DOJINDO Laboratories, Japan). Blood samples that were found to be G6PD deficient were sequenced to detect G6PD Viangchan mutation., Results: In total, 2043 blood samples were collected from Phongsaly (n = 426, 20.9%), Savannakhet (n = 924, 45.2%), and Champasak (n = 693, 33.9%) provinces in Lao PDR from 2016 to 2017. Of these, 964 (47.2%) were taken from male villagers and 1079 (52.8%) were taken from female villagers. G6PD Viangchan mutation was not detected in Phongsaly province in this study. In Savannakhet province, 48 of the 924 samples (45 males, 3 females) had the G6PD Viangchan mutation (n = 48, 5.2%). In Champasak province, 42 of the 693 samples (18 males, 24 females) had the G6PD Viangchan mutation (n = 42, 6.1%)., Conclusions: G6PD Viangchan variant, which can cause severe haemolysis in the carrier when exposed to primaquine, was detected among 6.1% of the villagers in Champasak and 5.2% in Savannakhet but not in Phongsaly in this study. G6PD Viangchan variant might be common in the south of Laos but not so in the north. In the north, other G6PD deficiency variants might be more prevalent. However, in order not to overlook anyone and ensure a safe primaquine therapy for people living in malaria endemic areas in Lao PDR, G6PD testing is necessary.

Background: Malaria is a major health problem in Lao People's Democratic Republic (Lao PDR) with high transmission in remote and forest areas, particularly in the South. The military is at risk of malaria infection especially those deployed in forest areas. This study determined the prevalence of malaria infection and assessed knowledge, perception, and preventive and treatment behavior regarding malaria among military personnel in two southern provinces in Lao PDR., Methods: Quantitative and qualitative approaches were undertaken in Champasak and Attapeu provinces in 2017. From 313 military personnel, quantitative data were collected through questionnaire-based interviews and blood samples used for parasite detection by polymerase chain reaction (PCR). Qualitative data were collected through 7 focus group discussions and 17 in-depth interviews among 49 military personnel. Fisher's exact test and Mann-Whitney U test were used to assess the association between malaria infection and participant characteristics. Content analysis for qualitative data was performed to explore perception and treatment behaviors regarding malaria., Results: The prevalence of malaria infection was 11.2% ( Plasmodium falciparum : 1.3%, Plasmodium vivax : 9.3% and mixed infections: 0.6%). Many participants understood that malaria is transmitted through mosquito bites, although they did not necessarily know the name of vector mosquitoes ( Anopheles ). Surprisingly, more than a half also believed that malaria is transmitted through drinking stream water. One-third of the participants used long-lasting insecticidal nets. Due to limited supply, participants were often unable to use mosquito repellent and coils when necessary. Because participants were unable to receive timely diagnosis and appropriate treatment for malaria in their camps, they commonly practiced self-treatment using antibiotics, painkillers, and/or traditional medicines. They only go to a healthcare facility through their supervisor if their conditions worsen., Conclusions: The prevalence of symptomatic and asymptomatic malaria was conspicuous among military in forest areas. Many participants believed that malaria is transmitted not only by mosquito bites but also from drinking stream water. Preventive equipment was often insufficient. Self-treatment was practiced before referring to healthcare facility. To further prevent military from contracting malaria, the National Malaria Control Program and military body should provide adequate and suitable health education, protective equipment, and on-site malaria case management., Competing Interests: Ethical clearance was obtained from the National Ethics Committee for Health Research, Lao PDR (No. 073/NECHR), and from the Ethics Review Committee for Epidemiology Study, University of the Ryukyus (No. 1172). The permission to conduct the study was given by the General Logistic Department, the Ministry of Defense (No. 358), Lao PDR, and also from the camp leaders. The interviewers and moderator explained the purpose of the study and procedures in Laotian. The participants were also informed that their participation was voluntary, and that the results of the blood analyses and interviews would be treated in confidence. Moreover, they were informed that they can withdraw from the interview and discussion anytime without consequences.Not applicable.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Background: The emergence and transnational spread of artemisinin resistance in Plasmodium falciparum in the Greater Mekong Sub-region (GMS) is a serious threat to malaria elimination in the region and could present a threat to malaria control in Africa. Recently, the Lao Government adopted the goal of malaria elimination by 2030, for which monitoring of artemisinin-resistant malaria within the country is indispensable. This study's objectives were to assess the distribution of k13 mutations in Laos., Methods: Plasmodium falciparum isolates (n = 1151) were collected from five southern provinces in Laos between 2015 and 2016, and three isolates from the northernmost province bordering China in 2017. Polymorphisms of the k13 gene and two flanking regions were analysed to estimate relationship among the isolates., Results: In the five southern provinces, overall 55.5% of the isolates possessed artemisinin-resistant mutations of the k13 gene (C580Y, P574L, R539T, Y493H). The C580Y was the predominant mutation (87.2%). The frequencies of the k13 mutations were heterogeneous in the five southern provinces, but with a clear tendency showing the highest frequency in the south (72.5%) and to a lower degree when moving northward (28.0%). The three isolates from the Lao-Chinese border also possessed the C580Y mutation. Analysis of the flanking loci demonstrated that these three isolates were genetically very close to resistant strains originating from western Cambodia., Conclusions: Artemisinin resistance was observed to be rapidly increasing and spreading northwards through Laos and has now reached the Chinese border. The Lao and Chinese governments, as well as the international community, should make dedicated efforts to contain the spread of k13 mutations within Laos and in the GMS.

Background: Artemisinin resistance in Plasmodium falciparum has been spreading across Southeast Asia. Patients' adherence to artemisinin-based combination therapy (ACT) is critical to avoid expanding this resistance. The objectives of this research were to examine patients' adherence to ACT for the treatment of uncomplicated malaria and to examine the healthcare workers' perception of medication adherence and their dispensing practices for malaria patients in Savannakhet province, Lao PDR., Methods: A prospective observational study of patients and a descriptive study of healthcare workers were conducted in Xepon, Phin, and Nong districts. In the patient study, patients aged 18 years old or older who were prescribed artemether-lumefantrine (AL) at six healthcare facilities between October 2016 and August 2017 were examined. Patient interviews and tablet counts were conducted on the first day of treatment (day 0) and the follow-up day (around day 3). In the healthcare workers study, a self-administered questionnaire survey was conducted., Results: Of the 54 patients examined, 51 (94.4%) were adherent to the AL regimen. The other three patients stopped medication because they felt better, even though the importance of completing the regimen was explained to all patients when it was prescribed. Among 152 healthcare workers who had ever instructed a malaria patient, 74.3% reported that they occasionally saw a malaria patient who adhered poorly to medication instructions. The healthcare workers perceived the major reasons for poor adherence to be illiteracy and poor understanding of medication instructions by patients. In practice, 27.6% of the healthcare workers did not regularly explain the importance of completing the regimen to patients, and 32.2% did not often or always confirm the patients' understanding of medication instructions., Conclusions: Patient adherence to AL was high. The healthcare workers perceived that poor adherence was attributable to the patients, i.e., their poor understanding and illiteracy, which appeared to be related to linguistic differences. However, poor adherence also appeared to be attributable to the healthcare workers, who should tell patients of the importance of completing the AL regimen regardless of their improvement in physical condition and also confirm the patients' understanding of the instructions., Competing Interests: This study was approved by the National Ethics Committee for Health Research, Ministry of Health, Lao PDR (No. 034/NECHR, 2016) and the Ethics Review Committee for Epidemiological Study, University of Ryukyus, Japan (No. 346). Before starting the study, surveyors explained the details of the study to the participants, such as its purpose, voluntary participation, information that would be collected and how the data would be stored and managed. Written informed consent was obtained from each participant. For the healthcare workers study, self-administered anonymous questionnaires were used.Not applicable.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Malaria morbidity and mortality have decreased gradually in the Greater Mekong Subregion (GMS). Presently, WHO sets a goal to eliminate malaria by 2030 in the GMS. However, drug-resistant malaria has been reported from several endemic areas. To achieve the goal of elimination, the status of the emergence and spread of drug resistance should be monitored. In this study, the genotype of the Plasmodium falciparum chloroquine (CQ) resistance transporter gene (pfcrt) and 6 microsatellite DNA loci flanking the gene were examined. P. falciparum isolates (n = 136) was collected from malaria patients in Thailand (n = 50, 2002-2005), Vietnam (n = 39, 2004), Laos (n = 15, 2007) and Cambodia (n = 32, 2009). Amino acid sequences at codons 72-76 on the gene were determined. All of the isolates from Thailand were CQ-resistant (CVIET), as were all of the isolates from Cambodia (CVIET, CVIDT). Thirteen of the 15 isolates (87%) from Laos were CQ-resistant (CVIET, CVIDT), whereas the other 2 (13%) were CQ-susceptible (CVMNK). In contrast, 27 of the 39 isolates (69%) from Vietnam were CQ-susceptible (CVMNK), whereas the other 12 (31%) were CQ-resistant (CVIET, CVIDT, CVMDT) or mixed (CVMNK/CVIDT). The mean of expected heterozygosity of the microsatellite loci was 0.444 in the Thai population, 0.482 in the Cambodian population, and 0.734 in the Vietnamese population. Genetic diversity in the Thai population was significantly lower than that in the Vietnamese population. These results suggested that chloroquine selective pressure on P. falciparum populations is heterogeneous in the GMS. Therefore, further examination to understand the mechanisms behind the emergence and spread of drug-resistant malaria are needed., (Copyright © 2018. Published by Elsevier B.V.)

Background: Opisthorchis viverrini (Ov) infection is one of the foodborne trematodiases, which is highly endemic in Lao People's Democratic Republic (PDR). The infection occurs especially when people eat raw fish containing Ov metacercariae. As eating raw fish is a traditional culture in Lao PDR, changing this behavior is difficult. A new approach is necessary to control Ov infection because people easily get re-infected even after taking praziquantel unless they change their behaviors. This study aimed to explore factors associated with Ov infection among children and to identify the existing behaviors and perception that might contribute to the control of Ov infection in Lao PDR. We conducted a cross-sectional study in Yommalath district, Khammouane province, in Lao PDR in August and September 2015. In this cross-sectional study, we used a semi-structured questionnaire and interviewed 348 mothers who had a child aged 5-15 years. We also collected the fecal samples from each mother-child pair and used the Kato-Katz method (three slides/sample) to detect Ov eggs., Results: Of 284 children, 82.8% were infected with Ov. The children were more likely to be infected with Ov when their mothers were infected with Ov (adjusted odds ratio [AOR] 10.45, 95% confidence interval [CI] 3.13-34.86) or when their mothers liked raw fish dishes (AOR 2.47, 95% CI 1.07-5.69). Even though most mothers are primarily in charge of cooking family meals, fathers were also involved in the preparation of raw fish dishes., Conclusion: This study suggests that a new approach to control Ov infection should target families or communities, rather than children only. Cooking or food preparation behaviors should be investigated in more depth., Competing Interests: This study is one of the epidemiological studies conducted by the Science and Technology Research Partnership for Sustainable Development (SATREPS) project supported by the Japan Agency for Medical Research and Development and the Japan International Cooperation Agency. The SATREPS project obtained the ethical approval from the National Ethics Committee for Health Research, National Institute of Public Health, Ministry of Health, in Lao PDR (reference number 049NIOPH/NECHR) in 2014. We conducted this study under this project. The Research Ethics Committee of the University of Tokyo also approved this study in 2015(reference number 10928). We strictly protected the privacy of the participants. After fecal examination, the results were sent to Yommalath district hospital and announced to village leaders or health workers so that the mothers would know the results from them. If they were Ov positive, they could select either to wait for the next mass drug administration of praziquantel, which was planned to start in October 2015, or to go to the district hospital to get praziquantel free of charge.Not applicable.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Background: Lao People Democratic Republic (PDR; Laos), a landlocked country in Southeast Asia, has made important progress in reducing malaria morbidity and mortality in the past 5-6 years, and the northern provinces have very low reported incidence. To support national progress towards elimination, it is critical to verify and understand these changes in disease burden., Methods: A two-stage cluster cross-sectional survey was conducted in four districts within four northern provinces (Khua, Phongsaly Province; Paktha, Bokeo Province; Nambak, Luang Prabang, and Muang Et, Huaphanh Province). During September and October 2016, demographics and malaria risk factors were collected from a total of 1492 households. A total of 5085 persons consented to collection of blood samples for testing, by rapid diagnostic test (RDT) and polymerase chain reaction (PCR)-based testing. Risk factors for infection were examined using logistic regression; and a randomized subset of males was tested for glucose-6-phosphate dehydrogenase (G6PD) deficiencies using a combined PCR and sequencing approach., Results: There were zero positives by RDT, and PCR detected Plasmodium infections in 39 (0.77%; 95% CI 0.40-1.47%) of 5082 analysable samples. The species distribution was Plasmodium vivax (28 total); Plasmodium falciparum/P. vivax (5); P. falciparum (3), Plasmodium malariae (2), and P. vivax/P. malariae (1). In multivariable analysis, the main risk factors included having any other cases within the household [aOR 12.83 (95% CI 4.40 to 37.38), p < 0.001]; and lack of bed net ownership within the household [aOR 10.91 (95% 5.42-21.94), p < 0.001]; age, sex and forest-travel were not associated with parasitaemia. A total of 910 males were tested for the six most common G6PDd in SE Asia; and 30 (3.3%; 95% CI 2.1-5.1%) had a G6PD variant allele associated with G6PD deficiency, with the majority being the Union (14) and Viangchan (11) polymorphisms, with smaller numbers of Canton and Mahidol., Conclusion: This is the first rigorous PCR-based population survey for malaria infection in Northern Lao PDR, and found a very low prevalence of asymptomatic Plasmodium infections by standard PCR methods, with P. vivax predominating in the surveyed districts. Clustering of cases within households, and lack of a bed nets suggest reactive case detection, and scale-up of coverage should be prioritized. The predominance of infections with P. vivax, combined with moderate levels of serious G6PD deficiencies highlight the need for careful rollout of primaquine towards elimination goals.

Background: Although the malaria burden in the Lao PDR has gradually decreased, the elimination of malaria by 2030 presents many challenges. Microscopy and malaria rapid diagnostic tests (RDTs) are used to diagnose malaria in the Lao PDR; however, some studies have reported the prevalence of sub-microscopic Plasmodium infections or asymptomatic Plasmodium carriers in endemic areas. Thus, highly sensitive detection methods are needed to understand the precise malaria situation in these areas., Methodology/principal Findings: A cross-sectional malaria field survey was conducted in 3 highly endemic malaria districts (Xaysetha, Sanamxay, Phouvong) in Attapeu province, Lao PDR in 2015, to investigate the precise malaria endemicity in the area; 719 volunteers from these villages participated in the survey. Microscopy, RDTs and a real-time nested PCR were used to detect Plasmodium infections and their results were compared. A questionnaire survey of all participants was also conducted to estimate risk factors of Plasmodium infection. Numbers of infections detected by the three methods were microscopy: P. falciparum (n = 1), P. vivax (n = 2); RDTs: P. falciparum (n = 2), P. vivax (n = 3); PCR: Plasmodium (n = 47; P. falciparum [n = 4], P. vivax [n = 41], mixed infection [n = 2]; 6.5%, 47/719). Using PCR as a reference, the sensitivity and specificity of microscopy were 33.3% and 100.0%, respectively, for detecting P. falciparum infection, and 7.0% and 100.0%, for detecting P. vivax infection. Among the 47 participants with parasitemia, only one had a fever (≥37.5°C) and 31 (66.0%) were adult males. Risk factors of Plasmodium infection were males and soldiers, whereas a risk factor of asymptomatic Plasmodium infection was a history of ≥3 malaria episodes., Conclusions/significance: There were many asymptomatic Plasmodium carriers in the study areas of Attapeu province in 2015. Adult males, probably soldiers, were at high risk for malaria infection. P. vivax, the dominant species, accounted for 87.2% of the Plasmodium infections among the participants. To achieve malaria elimination in the Lao PDR, highly sensitive diagnostic tests, including PCR-based diagnostic methods should be used, and plans targeting high-risk populations and elimination of P. vivax should be designed and implemented.

Background: In the Lao PDR, the incidence of malaria greatly differs among villages even within a subdistrict, and the reasons for this difference are poorly understood. The objective of this study was to identify differences in villagers' behavior and the household environment between villages with high incidences and those with low incidences of malaria in a rural district of the Lao PDR., Methods: A case-control study was conducted in Xepon district, Savannakhet province. Case villages were defined as those with a high incidence (> 10 cases per 1000 population per year), and control villages were those with a low incidence (0-10 cases per 1000 population per year). Data were collected from 178 households in the six case villages and six control villages between December 2016 and January 2017. The data collection consisted of an interview survey with the heads of households and an observational survey in and around the house. Logistic regression was used to assess the association between the case-control status and individual-level behavioral factors and household-level environmental factors adjusted for socio-demographic and economic factors., Results: Compared to the household members in the control villages, household members in the case villages were significantly more likely to work at night in the forest (adjusted odds ratio 1.95; 95% confidence interval 1.28 to 2.98) and more likely to sleep overnight in the forest (adjusted odds ratio 1.94; 95% confidence interval 1.13 to 3.33). Additionally, compared to the households in the control villages, households in the case villages were significantly more likely to have an open space on the house surface (adjusted odds ratio 3.64; 95% confidence interval 1.68 to 7.84)., Conclusions: There were significant differences in nighttime working and sleeping behaviors in the forest and the presence of an open space on the house surface in the case versus control villages. These differences can partly explain the difference in the incidences of malaria among the villages. The Lao National Malaria Control Program should recommend that villagers use personal protection when working and sleeping in the forest and to reduce any open space on the house surfaces.

Introduction: To achieve malaria elimination in the Greater Mekong Subregion (GMS) by 2030, proper case management is necessary. 8-aminoquinolines, such as primaquine, are the only available medicines effective in preventing relapse of the hypnozoite stage of Plasmodium vivax , as well as the onward transmission of Plasmodium falciparum . However, primaquine can cause haemolysis in individuals who have glucose-6-phosphate dehydrogenase deficiency (G6PDd). We conducted a systematic review on the reported clinical manifestations of G6PDd to provide a comprehensive overview of the situation in the GMS., Methods: The protocol for this systematic review was registered on PROSPERO: International prospective register of systematic reviews (CRD42016043146). We searched the PubMed/MEDLINE, CINAHL, and Web of Science databases for published articles describing the clinical manifestations of G6PDd in the GMS. We included articles of all study designs from inception until 31 July 2016, reporting the clinical manifestations of G6PDd. We then performed a narrative synthesis of these articles., Results: We included 56 articles in this review, 45 of which were from Thailand. Haemolysis in G6PD-deficient individuals was caused not only by primaquine but also by other medicines and infections. Other clinical manifestations of G6PDd that were found were favism, neonatal jaundice and chronic non-spherocytic haemolytic anaemia. G6PDd also influenced the clinical presentations of genetic disorders and infections, such as thalassemia and typhoid fever., Conclusion: As G6PDd also affects the clinical presentations of other infections, the benefits of G6PD testing and proper record keeping transcend those of malaria case management. Therefore, healthcare workers at the community level should be made familiar with complications resulting from G6PDd as these complications extend beyond the scope of malaria., Competing Interests: Competing interests: None declared.

Although the Lao People's Democratic Republic (Lao PDR) is comparatively small landlocked country with patterns of both in- and out-migration, its human migration situation has been poorly studied. This is despite all of the country's 18 provinces sharing both official and unofficial border checkpoints with neighboring countries. Economic reforms in the last decade have seen a gradual increase in the promotion of foreign investment, and main towns and transportation networks have been expanding thus offering new opportunities for livelihoods and economic activities.In the last decade, there has also been a significant reduction of reported malaria cases in Lao PDR and while this is an important prerequisite for eliminating malaria in the country, malaria outbreaks reported in the last four years suggest that population mobility, particularly in the south, is an important factor challenging current control efforts.Bolder investment in social sector spending should be geared towards improving health service provision and utilization, ensuring equitable access to primary health care (including malaria) through efforts to achieve universal health coverage targets. This should be extended to populations that are mobile and migrants. The local government plays a critical role in supporting policy and enforcement issues related to private sector project development in the provinces. Cross-border initiatives with neighboring countries, especially in terms of data sharing, surveillance, and response, is essential. Mechanisms to engage the private sector, especially the informal private sector, needs to be explored within the context of existing regulations and laws. Existing and new interventions for outdoor transmission of malaria, especially in forest settings, for high-risk groups including short- and long-term forest workers and their families, mobile and migrant populations, as well as the military must be combined into integrated packages with innovative delivery mechanisms through social marketing approaches. This should happen at multiple points in the mobility pathway and involve the private sector rather than being fully reliant on the national malaria vertical programThis article based on the review of existing literature from abstracts and full texts, includes published, peer-reviewed English language literature sourced through PubMed and grey literature sources through Google and Google Scholar. The review included also case reports, sector reports, conference proceedings, research reports, epidemiology studies, qualitative studies, and census reports in both Lao and English languages. The authors used the search terms: malaria and mobile populations, malaria control program and elimination, health system performance, malaria outbreak, Lao PDR; and included articles published until June 2015.

Background: There are no data on the burden of malaria in pregnancy (MiP) in Laos, where malaria still remains prevalent in the south., Methods: Two cross-sectional surveys were conducted in 2014 to assess the prevalence of MiP in Vapi District, Salavan Province, southern Laos: the first consisted of screening 204 pregnant women during pregnancies [mean (95 % CI) gestational age: 23 (22-25) weeks] living in 30 randomly selected villages in Vapi District; the second was conducted among 331 pregnant women, who delivered during the study period in Vapi and Toumlane District Hospitals and in Salavan Provincial Hospital. Peripheral and placental malaria was detected using rapid diagnostic tests (RDT), thick blood smears (TBS) and real-time quantitative polymerase chain reactions (RT-qPCR). Factors associated with low birth weight (LBW) and maternal anaemia were assessed., Results: In the villages, 12/204 women (5.9 %; 95 % CI 3.1-10.0) were infected with malaria as determined by RT-qPCR: 11 were Plasmodium vivax infections and 1 was mixed Plasmodium vivax/Plasmodium falciparum infection, among which 9 were sub-microscopic (as not detected by TBS). History of malaria during current pregnancy tended to be associated with a higher risk of MiP (aIRR 3.05; 95 % CI 0.94-9.88). At delivery, two Plasmodium falciparum sub-microscopic infections (one peripheral and one placental) were detected (4.5 %; 0.6-15.5) in Vapi District. In both surveys, all infected women stated they had slept under a bed net the night before the survey, and 86 % went to the forest for food-finding 1 week before the survey in median. The majority of infections (94 %) were asymptomatic and half of them were associated with anaemia. Overall, 24 % of women had LBW newborns. Factors associated with a higher risk of LBW were tobacco use (aIRR 2.43; 95 % CI 1.64-3.60) and pre-term delivery (aIRR 3.17; 95 % CI 2.19-4.57). Factors associated with a higher risk of maternal anaemia were no iron supplementation during pregnancy, Lao Theung ethnicity and place of living., Conclusions: The prevalence of MiP in this population was noticeable. Most infections were asymptomatic and sub-microscopic vivax malaria, which raises the question of reliability of recommended national strategies for the screening and prevention of MiP in Laos.

Background: Recent gains in reducing the global burden of malaria are threatened by the emergence of Plasmodium falciparum resistance to artemisinins. The discovery that mutations in portions of a P. falciparum gene encoding kelch (K13)-propeller domains are the major determinant of resistance has provided opportunities for monitoring such resistance on a global scale., Methods: We analyzed the K13-propeller sequence polymorphism in 14,037 samples collected in 59 countries in which malaria is endemic. Most of the samples (84.5%) were obtained from patients who were treated at sentinel sites used for nationwide surveillance of antimalarial resistance. We evaluated the emergence and dissemination of mutations by haplotyping neighboring loci., Results: We identified 108 nonsynonymous K13 mutations, which showed marked geographic disparity in their frequency and distribution. In Asia, 36.5% of the K13 mutations were distributed within two areas--one in Cambodia, Vietnam, and Laos and the other in western Thailand, Myanmar, and China--with no overlap. In Africa, we observed a broad array of rare nonsynonymous mutations that were not associated with delayed parasite clearance. The gene-edited Dd2 transgenic line with the A578S mutation, which expresses the most frequently observed African allele, was found to be susceptible to artemisinin in vitro on a ring-stage survival assay., Conclusions: No evidence of artemisinin resistance was found outside Southeast Asia and China, where resistance-associated K13 mutations were confined. The common African A578S allele was not associated with clinical or in vitro resistance to artemisinin, and many African mutations appear to be neutral. (Funded by Institut Pasteur Paris and others.).

We report a large multicenter genome-wide association study of Plasmodium falciparum resistance to artemisinin, the frontline antimalarial drug. Across 15 locations in Southeast Asia, we identified at least 20 mutations in kelch13 (PF3D7&#95;1343700) affecting the encoded propeller and BTB/POZ domains, which were associated with a slow parasite clearance rate after treatment with artemisinin derivatives. Nonsynonymous polymorphisms in fd (ferredoxin), arps10 (apicoplast ribosomal protein S10), mdr2 (multidrug resistance protein 2) and crt (chloroquine resistance transporter) also showed strong associations with artemisinin resistance. Analysis of the fine structure of the parasite population showed that the fd, arps10, mdr2 and crt polymorphisms are markers of a genetic background on which kelch13 mutations are particularly likely to arise and that they correlate with the contemporary geographical boundaries and population frequencies of artemisinin resistance. These findings indicate that the risk of new resistance-causing mutations emerging is determined by specific predisposing genetic factors in the underlying parasite population.

Background: The emergence of artemisinin-resistant Plasmodium falciparum in Southeast Asia threatens malaria treatment efficacy. Mutations in a kelch protein encoded on P. falciparum chromosome 13 (K13) have been associated with resistance in vitro and in field samples from Cambodia., Methods: P. falciparum infections from artesunate efficacy trials in Bangladesh, Cambodia, Laos, Myanmar, and Vietnam were genotyped at 33 716 genome-wide single-nucleotide polymorphisms (SNPs). Linear mixed models were used to test associations between parasite genotypes and parasite clearance half-lives following artesunate treatment. K13 mutations were tested for association with artemisinin resistance, and extended haplotypes on chromosome 13 were examined to determine whether mutations arose focally and spread or whether they emerged independently., Results: The presence of nonreference K13 alleles was associated with prolonged parasite clearance half-life (P = 1.97 × 10(-12)). Parasites with a mutation in any of the K13 kelch domains displayed longer parasite clearance half-lives than parasites with wild-type alleles. Haplotype analysis revealed both population-specific emergence of mutations and independent emergence of the same mutation in different geographic areas., Conclusions: K13 appears to be a major determinant of artemisinin resistance throughout Southeast Asia. While we found some evidence of spreading resistance, there was no evidence of resistance moving westward from Cambodia into Myanmar., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Evidence of severe hepatobiliary morbidity associated with Opisthorchis viverrini liver fluke infection including cholangiocarcinoma (CCA) is scarce in Laos although O. viverrini infection is highly prevalent. We assessed hepatobiliary morbidity using abdominal ultrasonography (US) in O. viverrini adult patients in Saravan province, Southern Laos. A random sample of 431 O. viverrini patients from 10 villages underwent abdominal US. Mild, moderate and markedly advanced periductal fibrosis was diagnosed in 7.0%, 66.5%, and 17.0% of patients, respectively. Normal liver parenchyma was seen in only 9.5% of patients. Presence of gall stones (13.2%), sludge (1.4%), gall wall thickening (1.2%), bile duct dilatation (1.6%), fatty liver (12.0%), kidney stones (8.6%) and cysts (7.9%) were diagnosed in considerable frequencies. In five patients (1.2%) hepatobiliary lesions suggesting CCA were diagnosed. Tumour markers, i.e. Interleukin-6, plasminogen activator inhibitor and carbohydrate antigen 19-9 were within normal range. The number of CCA suspected liver masses and hepatobiliary morbidity diagnosed among clinically asymptomatic adult patients in O. viverrini endemic area presents a major public health concern in Laos. However, definitive diagnosis of Opisthorchis-related severe sequelae including CCA is urgently needed to gauge the burden of this deadly disease in Laos., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Artemisinin resistance in Plasmodium falciparum has emerged in Southeast Asia and now poses a threat to the control and elimination of malaria. Mapping the geographic extent of resistance is essential for planning containment and elimination strategies., Methods: Between May 2011 and April 2013, we enrolled 1241 adults and children with acute, uncomplicated falciparum malaria in an open-label trial at 15 sites in 10 countries (7 in Asia and 3 in Africa). Patients received artesunate, administered orally at a daily dose of either 2 mg per kilogram of body weight per day or 4 mg per kilogram, for 3 days, followed by a standard 3-day course of artemisinin-based combination therapy. Parasite counts in peripheral-blood samples were measured every 6 hours, and the parasite clearance half-lives were determined., Results: The median parasite clearance half-lives ranged from 1.9 hours in the Democratic Republic of Congo to 7.0 hours at the Thailand-Cambodia border. Slowly clearing infections (parasite clearance half-life >5 hours), strongly associated with single point mutations in the "propeller" region of the P. falciparum kelch protein gene on chromosome 13 (kelch13), were detected throughout mainland Southeast Asia from southern Vietnam to central Myanmar. The incidence of pretreatment and post-treatment gametocytemia was higher among patients with slow parasite clearance, suggesting greater potential for transmission. In western Cambodia, where artemisinin-based combination therapies are failing, the 6-day course of antimalarial therapy was associated with a cure rate of 97.7% (95% confidence interval, 90.9 to 99.4) at 42 days., Conclusions: Artemisinin resistance to P. falciparum, which is now prevalent across mainland Southeast Asia, is associated with mutations in kelch13. Prolonged courses of artemisinin-based combination therapies are currently efficacious in areas where standard 3-day treatments are failing. (Funded by the U.K. Department of International Development and others; ClinicalTrials.gov number, NCT01350856.).

Background: In a recent study one third of Lao patients presenting with uncomplicated Plasmodium falciparum malaria had biochemical evidence of thiamin deficiency, which was associated with a higher incidence of adverse events. Thiamin supplementation might, therefore, reduce adverse events in this population., Methods: An exploratory, double-blind, parallel group, placebo-controlled, superiority trial of thiamin supplementation in patients of all ages with uncomplicated and severe falciparum malaria was conducted in Xepon District, Savannakhet Province, southern Laos. Patients were randomly assigned to either oral thiamin 10 mg/day for 7 days immediately after standard anti-malarial treatment then 5 mg daily until day 42, or identical oral placebo., Results: After interim analyses when 630 patients (314 in thiamin and 316 in placebo groups) had been recruited, the trial was discontinued on the grounds of futility. On admission biochemical thiamin deficiency (alpha ≥ 25%) was present in 27% of patients and 9% had severe deficiency (alpha > 31%). After 42 days of treatment, the frequency of thiamin deficiency was lower in the thiamin (2%, 1% severe) compared to the placebo (11%, 3% severe) groups (p < 0.001 and p = 0.05), respectively. Except for diarrhoea, 7% in the placebo compared to 3% in the thiamin group (p = 0.04), and dizziness on day 1 (33% vs 25%, p = 0.045), all adverse events were not significantly different between the groups (p > 0.05). Clinical, haematological, and parasitological responses to treatment did not differ significantly between the two groups., Conclusion: Thiamin supplementation reduced biochemical thiamin deficiency among Lao malaria patients following anti-malarial drug treatment, but it did not reduce the frequency of adverse events after anti-malarial therapy or have any detected clinical or parasitological impact., Trial Registration: ISRCTN 85411059.

Background: Because of reductions in the incidence of Plasmodium falciparum malaria in Laos, identification of the causes of fever in people without malaria, and discussion of the best empirical treatment options, are urgently needed. We aimed to identify the causes of non-malarial acute fever in patients in rural Laos., Methods: For this prospective study, we recruited 1938 febrile patients, between May, 2008, and December, 2010, at Luang Namtha provincial hospital in northwest Laos (n=1390), and between September, 2008, and December, 2010, at Salavan provincial hospital in southern Laos (n=548). Eligible participants were aged 5-49 years with fever (≥38°C) lasting 8 days or less and were eligible for malaria testing by national guidelines., Findings: With conservative definitions of cause, we assigned 799 (41%) patients a diagnosis. With exclusion of influenza, the top five diagnoses when only one aetiological agent per patient was identified were dengue (156 [8%] of 1927 patients), scrub typhus (122 [7%] of 1871), Japanese encephalitis virus (112 [6%] of 1924), leptospirosis (109 [6%] of 1934), and bacteraemia (43 [2%] of 1938). 115 (32%) of 358 patients at Luang Namtha hospital tested influenza PCR-positive between June and December, 2010, of which influenza B was the most frequently detected strain (n=121 [87%]). Disease frequency differed significantly between the two sites: Japanese encephalitis virus infection (p=0·04), typhoid (p=0·006), and leptospirosis (p=0·001) were more common at Luang Namtha, whereas dengue and malaria were more common at Salavan (all p<0·0001). With use of evidence from southeast Asia when possible, we estimated that azithromycin, doxycycline, ceftriaxone, and ofloxacin would have had significant efficacy for 258 (13%), 240 (12%), 154 (8%), and 41 (2%) of patients, respectively., Interpretation: Our findings suggest that a wide range of treatable or preventable pathogens are implicated in non-malarial febrile illness in Laos. Empirical treatment with doxycycline for patients with undifferentiated fever and negative rapid diagnostic tests for malaria and dengue could be an appropriate strategy for rural health workers in Laos., Funding: Wellcome Trust, WHO-Western Pacific Region, Foundation for Innovative New Diagnostics, US Centers for Disease Control and Prevention

Experimental studies have indicated that low serum zinc levels affect immune responses. However, few studies have evaluated the impact of serum zinc levels on antibody responses in the field in developing countries. We investigated an association between the anti-Plasmodium falciparum (Pf) antibody (immunoglobulin G) titer and serum zinc concentration among villagers in rural areas of the Lao People's Democratic Republic. Blood samples were collected to detect Pf infection. An enzyme-linked immunosorbent assay (ELISA) was used to measure the anti-PfIgG antibody titer. Each serum sample was assayed to measure the concentration of zinc. Pearson's correlation coefficient was applied to the association between zinc concentration and anti-PfIgG antibody titers. Multiple linear regression analysis was used to assess the association between zinc concentration and anti-PfIgG antibody titers, controlling for age and albumin level. Of 71 blood samples, 40 were Pf positive and 31 were Pf negative. The median serum zinc concentrations were 56.0 μg/dl in the Pf-positive group and 62.5 μg/dl in the Pf-negative group. The median anti-Pf titers were 833.4 in the positive group and 1237.2 in the negative group. Unexpectedly, there was a negative correlation between serum zinc and anti-Pf IgG antibody titers; the correlation coefficient were -0.453 and (p=0.003) in the positive group and -0.461 (p=0.009) in the negative group. The results of this study indicated sustained antibody responses among the villagers, who had likely been exposed to malaria periodically throughout their lives. Further studies are necessary to determine the conditions in which zinc could be effective against malaria., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Malaria morbidity and mortality have been significantly reduced through the proper use of insecticide-treated mosquito nets, but the extra protection afforded by the insecticide diminishes over time. The insecticide depletion rates vary according to location where wash frequency and wear are influenced by cultural habits as well as the availability of water. Monitoring of available insecticides on the net surface is essential for determining the effective life of the net. Therefore, a rapid and inexpensive colorimetric field test for cyanopyrethroids (Cyanopyrethroid Field Test or CFT) was used to measure surface levels of deltamethrin on insecticide-coated polyester nets (PowerNets™) in rural Lao PDR over a two-year period., Methods: Net surface levels of deltamethrin were measured by wiping the net with filter paper and measuring the adsorbed deltamethrin using the CFT. A relationship between surface levels of deltamethrin and whole net levels was established by comparing results of the CFT with whole levels assayed by high-performance liquid chromatography (HPLC). An effective deltamethrin surface concentration (EC80) was determined by comparing mosquito mortality (WHO Cone Test) with CFT and HPLC results. Five positions (roof to bottom) on each of 23 matched nets were assayed for deltamethrin surface levels at 6, 12, and 24 months. Mosquito mortality assays (WHO Cone Tests) were performed on a subset of eleven 24-month old nets and compared with the proportion of failed nets as predicted by the CFT., Results: At six months, the nets retained about 80% of the baseline (new net) levels of deltamethrin with no significant differences between net positions. At 12 months, ~15-40%, and at 24 months <10% of deltamethrin was retained on the nets, with significant differences appearing between positions. Results from the CFT show that 93% of the nets failed (deltamethrin surface levels

We conducted an open-label, randomized clinical trial to assess parasite clearance times (PCT) and the efficacy of 4 mg/kg (group 1, n = 22) and 2 mg/kg (group 2, n = 22) of oral artesunate for three days followed by artemether-lumefantrine in patients with uncomplicated Plasmodium falciparum malaria at Xepon Interdistrict Hospital, Savannakhet Province in southern Laos. Slides were read in duplicate. The overall mean (95% confidence interval; range) PCT in hours was 23.2 (21.2-25.3; 12-46) and 22.4 (20.3-24.5; 12-46) for the first and second microscopists, respectively (P = 0.57). Ten (23%) patients remained parasitemic on day 1 after treatment (4 [18%] in group 1 and 6 [27%] in group 2; P = 0.47). No patient had patent asexual parasitemia on the second and third days of treatment. The 42-day polymerase chain reaction-corrected cure rates were 100% in both treatment groups. Serious adverse events did not develop during or after treatment in any patients. In conclusion, no evidence of P. falciparum in vivo resistance to artesunate was found in southern Laos.

Background: Accurate information on the geographical distribution of malaria is important for efficient resource allocation. The Lao People's Democratic Republic has experienced a major decline in malaria morbidity and mortality in the past decade. However, efforts to respond effectively to these changes have been impeded by lack of detailed data on malaria distribution. In 2008, a countrywide survey on Plasmodium falciparum diagnosed in health centres and villages was initiated to develop a detailed P. falciparum risk map with the aim to identify priority areas for malaria control, estimate population at risk, and guide resource allocation in the Lao People's Democratic Republic., Methods: P. falciparum incidence data were collected from point-referenced villages and health centres for the period 2006-2008 during a country-wide survey between December 2008 and January 2009. Using the highest recorded annual rate, continuous surfaces of P. falciparum incidence were produced by the inverse distance weighted interpolation technique., Results: Incidence rates were obtained from 3,876 villages and 685 health centres. The risk map shows that P. falciparum is highly heterogeneous in the northern and central regions of the country with large areas of no transmission. In the southern part, transmission is pervasive and the risk of P. falciparum is high. It was estimated that 3.4 million people (60% of the population) live at risk of malaria., Conclusions: This paper presents the first comprehensive malaria risk map of the Lao People's Democratic Republic based entirely on empirical data. The estimated population at risk is substantially lower than previous estimates, reflecting the presence of vast areas with focal or no malaria transmission as identified in this study. These findings provide important guidance for malaria control interventions in the Lao People's Democratic Republic, and underline the need for detailed data on malaria to accurately predict risk in countries with heterogeneous transmission.

The usefulness of rapid diagnostic tests (RDT) in malaria case management depends on the accuracy of the diagnoses they provide. Despite their apparent simplicity, previous studies indicate that RDT accuracy is highly user-dependent. As malaria RDTs will frequently be used in remote areas with little supervision or support, minimising mistakes is crucial. This paper describes the development of new instructions (job aids) to improve health worker performance, based on observations of common errors made by remote health workers and villagers in preparing and interpreting RDTs, in the Philippines and Laos. Initial preparation using the instructions provided by the manufacturer was poor, but improved significantly with the job aids (e.g. correct use both of the dipstick and cassette increased in the Philippines by 17%). However, mistakes in preparation remained commonplace, especially for dipstick RDTs, as did mistakes in interpretation of results. A short orientation on correct use and interpretation further improved accuracy, from 70% to 80%. The results indicate that apparently simple diagnostic tests can be poorly performed and interpreted, but provision of clear, simple instructions can reduce these errors. Preparation of appropriate instructions and training as well as monitoring of user behaviour are an essential part of rapid test implementation.

A malariometric survey was conducted in the Keoudom District, in the northern part of Vientiane Province, Laos, where an artificial dam-lake on the Nam River is located. The parasite rate of the whole cohort representing 1,105 subjects was 2.44% with the predominance of Plasmodium vivax (70%), while P. falciparum represented 30% with the average parasite density index 3. The low spleen rate (2.3%) characterized the study area as a hypoendemic zone. IFAT antibodies were examined in 419 subjects. The seropositivity of 195 persons < 15 years was 13.7% while in > 15 year old subjects seropositivity was 61.6% with a low GMRT in both groups (140:148). Automedication with aminoquinoline was assayed by urinary analysis in 125 outpatients. Of these, 36 (28.8%) were positive, 89 (71.2%) negative. The frequency of positive blood films for P. falciparum was higher in subjects with aminoquinoline in the urine (36.1%) than in those without (10.1%). Chloroquine sensitivity assay of 15 strains of P. falciparum displayed resistance in 39.3%.