Deforestation inhibits malaria transmission in Lao PDR: a spatial epidemiology using Earth observation satellites.

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.

Bionomics of malaria vectors in Lao PDR, 2018-2020: entomological surveillance as a key tool for malaria elimination.

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.

Low Prevalence of Schistosoma mekongi Infection and High Prevalence of Other Helminth Infections among Domestic Animals in Southern Lao People's Democratic Republic.

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.

Impact of precipitation on the prevalence of schistosomiasis mekongi in Lao PDR: Structural equation modelling using Earth observation satellite data.

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.

Characterizing mobility patterns of forest goers in southern Lao PDR using GPS loggers.

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.

Detection of Schistosoma mekongi DNA in Human Stool and Intermediate Host Snail Neotricula aperta via Loop-Mediated Isothermal Amplification Assay in Lao PDR.

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.

Ten-year persistence and evolution of Plasmodium falciparum antifolate and anti-sulfonamide resistance markers pfdhfr and pfdhps in three Asian countries.

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.

Characterizing the spatial distribution of multiple malaria diagnostic endpoints in a low-transmission setting in Lao PDR.

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.

Genetic surveillance in the Greater Mekong subregion and South Asia to support malaria control and elimination.

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.

Population size estimation of seasonal forest-going populations in southern Lao PDR.

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.

Spatio-temporal associations between deforestation and malaria incidence in Lao PDR.

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.

Biting mosquitos spread the malaria parasite to humans. Along the Mekong River in Southeast Asia, spending time in the surrounding forest increases a person's risk of malaria. This has led to a debate about whether deforestation in this area, which is called the Greater Mekong Sub-region (GMS), will increase or decrease malaria transmission. The answer to the debate is not clear because some malaria-transmitting mosquitos thrive in heavily forested areas, in particular in the GMS, while others prefer less forested areas. Scientists studying malaria in the Amazon in South America suspect that malaria transmission increases shortly after deforestation but decreases six to eight years later. Some studies have tested this 'frontier malaria' theory but the results have been conflicting. Fewer studies have tested this theory in Southeast Asia. But deforestation has been blamed for recent malaria outbreaks in the GMS. Using data on malaria testing and forest cover in the GMS, Rerolle et al. show that deforestation around villages increases malaria transmission in the first two years and decreases malaria rates later. This trend was driven mostly by a type of malaria called Plasmodium falciparum and was less strong for Plasmodium vivax. The location of deforested areas also mattered. Deforestation within one to 10 kilometer of villages did not affect malaria rates. Deforestation further away in about a 30 kilometer radius did affect malaria transmission. Rerolle et al. suggest this may be because villagers have to spend longer times trekking through forests to hunt or harvest wood when the wider area is deforested. Currently, National Malaria Control Programs in the GMS focus their efforts on reducing forest-related transmission. This study strengthens the evidence supporting this approach. The results also suggest that different malaria elimination strategies may be necessary for different types of malaria parasite. Using this new information could help malaria control programs better target resources or educate villagers on how to protect themselves. The innovative methods used by Rerolle et al. reveal a more complex role of deforestation in malaria transmission and may inspire other scientists to think more carefully about environmental drivers of malaria.

Evolution and expansion of multidrug-resistant malaria in southeast Asia: a genomic epidemiology study.

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.

Prevalence of G6PD Viangchan variant in malaria endemic areas in Lao PDR: an implication for malaria elimination by 2030.

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.

Malaria prevalence, knowledge, perception, preventive and treatment behavior among military in Champasak and Attapeu provinces, Lao PDR: a mixed methods study.

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.

Study protocol for a cluster-randomized split-plot design trial to assess the effectiveness of targeted active malaria case detection among high-risk populations in Southern Lao PDR (the AcME-Lao study).

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).

Heterogeneous distribution of k13 mutations in Plasmodium falciparum in Laos.

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.

Pfcrt genotypes and related microsatellite DNA polymorphisms on Plasmodium falciparum differed among populations in the Greater Mekong Subregion.

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.

Mothers' Opisthorchis viverrini infection status and raw fish dish consumption in Lao People's Democratic Republic: determinants of child infection status.

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.

Prevalence and risk factors for asymptomatic malaria and genotyping of glucose 6-phosphate (G6PD) deficiencies in a vivax-predominant setting, Lao PDR: implications for sub-national elimination goals.

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.