Mass drug administration for the acceleration of malaria elimination in a region of Myanmar with artemisinin-resistant falciparum malaria: a cluster-randomised trial.

BACKGROUND: To contain multidrug-resistant Plasmodium falciparum, malaria elimination in the Greater Mekong subregion needs to be accelerated while current antimalarials remain effective. We evaluated the safety, effectiveness, and potential resistance selection of dihydroartemisinin-piperaquine mass drug administration (MDA) in a region with artemisinin resistance in Myanmar. METHODS: We did a cluster-randomised controlled trial in rural community clusters in Kayin (Karen) state in southeast Myanmar. Malaria prevalence was assessed using ultrasensitive quantitative PCR (uPCR) in villages that were operationally suitable for MDA (villages with community willingness, no other malaria control campaigns, and a population of 50-1200). Villages were eligible to participate if the prevalence of malaria (all species) in adults was greater than 30% or P falciparum prevalence was greater than 10% (or both). Contiguous villages were combined into clusters. Eligible clusters were paired based on P falciparum prevalence (estimates within 10%) and proximity. Community health workers provided routine malaria case management and distributed long-lasting insecticidal bed-nets (LLINs) in all clusters. Randomisation of clusters (1:1) to the MDA intervention group or control group was by public coin-flip. Group allocations were not concealed. Three MDA rounds (3 days of supervised dihydroartemisinin-piperaquine [target total dose 7 mg/kg dihydroartemisinin and 55 mg/kg piperaquine] and single low-dose primaquine [target dose 0·25 mg base per kg]) were delivered to intervention clusters. Parasitaemia prevalence was assessed at 3, 5, 10, 15, 21, 27, and 33 months. The primary outcomes were P falciparum prevalence at months 3 and 10. All clusters were included in the primary analysis. Adverse events were monitored from the first MDA dose until 1 month after the final dose, or until resolution of any adverse event occurring during follow-up. This trial is registered with ClinicalTrials.gov, NCT01872702. FINDINGS: Baseline uPCR malaria surveys were done in January, 2015, in 43 villages that were operationally suitable for MDA (2671 individuals). 18 villages met the eligibility criteria. Three villages in close proximity were combined into one cluster because a border between them could not be defined. This gave a total of 16 clusters in eight pairs. In the intervention clusters, MDA was delivered from March 4 to March 17, from March 30 to April 10, and from April 27 to May 10, 2015. The weighted mean absolute difference in P falciparum prevalence in the MDA group relative to the control group was -10·6% (95% CI -15·1 to -6·1; p=0·0008) at month 3 and -4·5% (-10·9 to 1·9; p=0·14) at month 10. At month 3, the weighted P falciparum prevalence was 1·4% (0·6 to 3·6; 12 of 747) in the MDA group and 10·6% (7·0 to 15·6; 56 of 485) in the control group. Corresponding prevalences at month 10 were 3·2% (1·5 to 6·8; 34 of 1013) and 5·8% (2·5 to 12·9; 33 of 515). Adverse events were reported for 151 (3·6%) of 4173 treated individuals. The most common adverse events were dizziness (n=109) and rash or itching (n=20). No treatment-related deaths occurred. INTERPRETATION: In this low-transmission setting, the substantial reduction in P falciparum prevalence resulting from support of community case management was accelerated by MDA. In addition to supporting community health worker case management and LLIN distribution, malaria elimination programmes should consider using MDA to reduce P falciparum prevalence rapidly in foci of higher transmission. FUNDING: The Global Fund to Fight AIDS, Tuberculosis and Malaria.

Prevalence and seroprevalence of Plasmodium infection in Myanmar reveals highly heterogeneous transmission and a large hidden reservoir of infection.

Malaria incidence in Myanmar has significantly reduced over recent years, however, completeness and timeliness of incidence data remain a challenge. The first ever nationwide malaria infection and seroprevalence survey was conducted in Myanmar in 2015 to better understand malaria epidemiology and highlight gaps in Annual Parasite Index (API) data. The survey was a cross-sectional two-stage stratified cluster-randomised household survey conducted from July-October 2015. Blood samples were collected from household members for ultra-sensitive PCR and serology testing for P. falciparum and P. vivax. Data was gathered on demography and a priori risk factors of participants. Data was analysed nationally and within each of four domains defined by API data. Prevalence and seroprevalence of malaria were 0.74% and 16.01% nationwide, respectively. Prevalent infection was primarily asymptomatic P. vivax, while P. falciparum was predominant in serology. There was large heterogeneity between villages and by domain. At the township level, API showed moderate correlation with P. falciparum seroprevalence. Risk factors for infection included socioeconomic status, domain, and household ownership of nets. Three K13 P. falciparum mutants were found in highly prevalent villages. There results highlight high heterogeneity of both P. falciparum and P. vivax transmission between villages, accentuated by a large hidden reservoir of asymptomatic P. vivax infection not captured by incidence data, and representing challenges for malaria elimination. Village-level surveillance and stratification to guide interventions to suit local context and targeting of transmission foci with evidence of drug resistance would aid elimination efforts.

Effectiveness and safety of 3 and 5 day courses of artemether-lumefantrine for the treatment of uncomplicated falciparum malaria in an area of emerging artemisinin resistance in Myanmar.

BACKGROUND: Artemisinin resistance in Plasmodium falciparum has emerged and spread in Southeast Asia. In areas where resistance is established longer courses of artemisinin-based combination therapy have improved cure rates. METHODS: The standard 3-day course of artemether-lumefantrine (AL) was compared with an extended 5-day regimen for the treatment of uncomplicated falciparum malaria in Kayin state in South-East Myanmar, an area of emerging artemisinin resistance. Late parasite clearance dynamics were described by microscopy and quantitative ultra-sensitive PCR. Patients were followed up for 42 days. RESULTS: Of 154 patients recruited (105 adults and 49 children < 14 years) 78 were randomized to 3 days and 76 to 5 days AL. Mutations in the P. falciparum kelch13 propeller gene (k13) were found in 46% (70/152) of infections, with F446I the most prevalent propeller mutation (29%; 20/70). Both regimens were well-tolerated. Parasite clearance profiles were biphasic with a slower submicroscopic phase which was similar in k13 wild-type and mutant infections. The cure rates were 100% (70/70) and 97% (68/70) in the 3- and 5-day arms respectively. Genotyping of the two recurrences was unsuccessful. CONCLUSION: Despite a high prevalence of k13 mutations, the current first-line treatment, AL, was still highly effective in this area of South-East Myanmar. The extended 5 day regimen was very well tolerated, and would be an option to prolong the useful therapeutic life of AL. Trial registration NCT02020330. Registered 24 December 2013, https://clinicaltrials.gov/NCT02020330.

Assessment of household ownership of bed nets in areas with and without artemisinin resistance containment measures in Myanmar.

BACKGROUND: Myanmar lies in the Greater Mekong Subregion where there is artemisinin-resistant Plasmodium falciparum malaria. As the artemisinin compound is the pillar of effective antimalarial therapies, containing the spread of artemisinin resistance is a national and global priority. The use of insecticide-treated bed nets/long-lasting insecticidal nets (ITNs/LLINs) is the key intervention for ensuring the reduction of malaria transmission and the spread of resistant strains, and for eventually eliminating malaria. This study aimed at assessing household ownership of, access to, and utilization of bed nets in areas of Myanmar with and without artemisinin resistance containment measures. METHODS: Secondary data from a nationwide community-based malaria survey conducted by the National Malaria Control Program in 2014 were analyzed. Based on evidence of artemisinin resistance, Myanmar was divided into tiers 1, 2, and 3: townships in tiers 1 and 2 were aggregated as the Myanmar Artemisinin Resistance Containment (MARC) areas and were compared with tier 3 townships, which were defined as non-MARC areas. The chi-square test was used to compare groups, and the level of significance was set at P ≤ 0.05. RESULTS: Of the 6328 households assessed, 97.2% in both MARC and non-MARC areas had at least one bed net (any type), but only 63% of households had ITNs/LLINs. Only 44% of households in MARC areas and 24% in non-MARC areas had adequate numbers of ITNs/LLINs (one ITN/LLIN per two persons, P <  0.001). Nearly 44% of household members had access to ITNs/LLINs. Regarding the utilization of ITNs/LLINs, 45% of household members used them in MARC areas and 36% used them in non-MARC areas (P <  0.001, desired target = 100%). Utilization of ITNs/LLINs among children aged below five years and pregnant women (high malaria risk groups) was low, at 44% and 42%, respectively. CONCLUSIONS: This study highlights the nationwide shortfalls in the ownership of, access to, and utilization of ITNs/LLINs in Myanmar, which is of particular concern in terms of containing the spread of artemisinin resistance. It highlights the need for priority attention to be paid and mobilization of resources in order to improve bed net coverage and utilization through bed net distribution and/or social marketing, information dissemination, and awareness-raising.

Promoting community malaria control in rural Myanmar through an active community participation program using the participatory learning approach.

INTRODUCTION: Malaria is prevalent in more than 80% of townships in Myanmar. The National Malaria Control Programme (NMCP) has been implementing community-based malaria control programs nationwide. However, these programs are mostly developed and directed by health authorities, while communities are passively involved. This study aimed to increase community participation in malaria control and promote community malaria control knowledge and practice in rural Myanmar. METHODS: A community-based study, which employed a mixed method approach, collecting data quantitatively and qualitatively, was conducted in two rural villages. The study implemented an active community participation program (ACPP) using the participatory learning approach in a village (ACPP village) but only routine malaria control was given in another village (non-ACPP village). All households with 142 and 96 household representatives from ACPP and non-ACPP villages participated in baseline and endline surveys. The ACPP was evaluated by process and outcome indicators. A spider gram analysis using five process indicators was applied to evaluate the process of the ACPP. Community participation status in malaria control activities and level of community malaria knowledge and practice were determined as outcomes of the ACPP. RESULTS: The spider gram analysis showed that three indicators (needs assessment and planning, leadership and resource mobilization) gained a score of 4, the organization indicator a score of 5 and the management and evaluation indicator a score of 3. The outcome indicators of the program at 6 months showed that the community participation in malaria control activities in the ACPP village had significantly increased (6.9% to 49.3%) (p<0.001). The program promoted community malaria control knowledge and practice in the ACPP village. The mean scores of knowledge, perception, preventive behavior and treatment-seeking behavior were increased significantly, from 3.0 to 5.9 (p<0.001), 20.1 to 21.0 (p<0.001), 3.4 to 4.2 (p<0.001) and 3.1 to 5.6 (p<0.001), respectively. However, no significant change of outcome indicators was found in the non-ACPP village. CONCLUSIONS: The ACPP implemented by community volunteers using the participatory learning approach was feasible in community-based malaria control. This study suggests several features in the ACPP model that may be useful strategies for the implementation of the current NMCP programs in similar rural settings; however, the effect of the ACPP over a longer period to ascertain the impact of such community participation has yet to be further studied.

Comparison of microscopy and PCR for the detection of human Plasmodium species and Plasmodium knowlesi in southern Myanmar

Objectives: To determine the distribution of Plasmodium (P) species including Plas-modium knowlesi and to compare the specificity and sensitivity of microscopy with nested PCR in malaria diagnosis. Methods: The study was conducted in Kawthaung, southern Myanmar. Ninety clinically suspected malaria patients were screened for malaria by Giemsa stained microscopy and confirmed by nested PCR. Results: Among the participants, 57 (63.3％) were positive and 33 (36.7％) were negative by microscopy. Of positive samples, 39 (68.4％) were Plasmodium falciparum, 17 (29.8％) Plasmodium vivax and 1 (1.8％) Plasmodium malariae, whereas 59-amplified by PCR were 40 (67.8％), 18 (30.5％) and 1 (1.7％) respectively. PCR amplified 2 mi-croscopy negative samples. Two samples of P. falciparum detected by microscopy were amplified as P. vivax and vice versa. All samples were negative for Plasmodium ovale, P. knowlesi and mixed infections. Microscopy had a very good measure of agreement (k= 0.95) compared to nested PCR. Sensitivity and specificity of microscopy for diag-nosis of P. falciparum were 92.5％(95％CI:79.6–98.4) and 96.0％(95％CI:86.3–99.5) respectively, whereas for P. vivax were 83.3％(95％CI:58.6–96.4) and 97.2％(95％CI:90.3–99.7). Conclusions: P. knowlesi was not detected by both microscopy and PCR. Giemsa stained microscopy can still be applied as primary method for malaria diagnosis and is considered as gold standard. As to the lower sensitivity of microscopy for vivax malaria, those with previous history of malaria and relapse cases should be diagnosed by RDT or PCR combined with microscopy. Inaccuracy of species diagnosis highlighted the requirement of training and refresher courses for microscopists.

Historical environmental change in Africa drives divergence and admixture of Aedes aegypti mosquitoes: a precursor to successful worldwide colonization?

Increasing globalization has promoted the spread of exotic species, including disease vectors. Understanding the evolutionary processes involved in such colonizations is both of intrinsic biological interest and important to predict and mitigate future disease risks. The Aedes aegypti mosquito is a major vector of dengue, chikungunya and Zika, the worldwide spread of which has been facilitated by Ae. aegypti's adaption to human-modified environments. Understanding the evolutionary processes involved in this invasion requires characterization of the genetic make-up of the source population(s). The application of approximate Bayesian computation (ABC) to sequence data from four nuclear and one mitochondrial marker revealed that African populations of Ae. aegypti best fit a demographic model of lineage diversification, historical admixture and recent population structuring. As ancestral Ae. aegypti were dependent on forests, this population history is consistent with the effects of forest fragmentation and expansion driven by Pleistocene climatic change. Alternatively, or additionally, historical human movement across the continent may have facilitated their recent spread and mixing. ABC analysis and haplotype networks support earlier inferences of a single out-of-Africa colonization event, while a cline of decreasing genetic diversity indicates that Ae. aegypti moved first from Africa to the Americas and then to Asia. ABC analysis was unable to verify this colonization route, possibly because the genetic signal of admixture obscures the true colonization pathway. By increasing genetic diversity and forming novel allelic combinations, divergence and historical admixture within Africa could have provided the adaptive potential needed for the successful worldwide spread of Ae. aegypti.

Malaria community health workers in Myanmar: a cost analysis.

BACKGROUND: Myanmar has the highest malaria incidence and attributed mortality in South East Asia with limited healthcare infrastructure to manage this burden. Establishing malaria Community Health Worker (CHW) programmes is one possible strategy to improve access to malaria diagnosis and treatment, particularly in remote areas. Despite considerable donor support for implementing CHW programmes in Myanmar, the cost implications are not well understood. METHODS: An ingredients based micro-costing approach was used to develop a model of the annual implementation cost of malaria CHWs in Myanmar. A cost model was constructed based on activity centres comprising of training, patient malaria services, monitoring and supervision, programme management, overheads and incentives. The model takes a provider perspective. Financial data on CHWs programmes were obtained from the 2013 financial reports of the Three Millennium Development Goal fund implementing partners that have been working on malaria control and elimination in Myanmar. Sensitivity and scenario analyses were undertaken to outline parameter uncertainty and explore changes to programme cost for key assumptions. RESULTS: The range of total annual costs for the support of one CHW was US$ 966-2486. The largest driver of CHW cost was monitoring and supervision (31-60% of annual CHW cost). Other important determinants of cost included programme management (15-28% of annual CHW cost) and patient services (6-12% of annual CHW cost). Within patient services, malaria rapid diagnostic tests are the major contributor to cost (64% of patient service costs). CONCLUSION: The annual cost of a malaria CHW in Myanmar varies considerably depending on the context and the design of the programme, in particular remoteness and the approach to monitoring and evaluation. The estimates provide information to policy makers and CHW programme planners in Myanmar as well as supporting economic evaluations of their cost-effectiveness.

Mobility dynamics of migrant workers and their socio-behavioral parameters related to malaria in Tier II, Artemisinin Resistance Containment Zone, Myanmar.

BACKGROUND: Areas with dynamic population movements are likely to be associated with higher levels of drug-resistant malaria. Myanmar Artemisinin Resistance Containment (MARC) Project has been launching since 2012. One of its components includes enhancing strategic approaches for mobile/migrant populations. We aimed to ascertain the estimated population of mobile migrant workers and their families in terms of stability in work setting in townships classified as tier II (areas with significant inflows of people from areas with credible evidence of artemisinin resistance) for Artemisinin resistance; to identify knowledge, attitudes and practices related to prevention and control of malaria and to recommend cost-effective strategies in planning for prevention and control of malaria. METHODS: A prospective cross-sectional study conducted between June to December 2013 that covered 1,899 migrant groups from 16 tier II townships of Bago Region, and Kayin and Kayah States. Trained data collectors used a pre-tested and subsequently modified questionnaire and interviewed 2,381 respondents. Data of migrant groups were analyzed and compared by category depending upon the stability of their work setting. RESULTS: The estimated population of the 1,899 migrant groups categorized into three on the nature of their work setting was 56,030. Bago region was the commonest reported source of origin of migrant groups as well as their transit. Malaria volunteers were mostly within the reach of category 1 migrant groups (43/66, 65.2 %). Less stable migrant groups in category 3 had limited access to malaria information (14.7 %) and malaria care providers (22.1 %), low level of awareness and use of long-lasting insecticide-treated nets (46.6 and 38.8 %). Also, they had poor knowledge on malaria prevention on confirming suspected malaria and on using artemisinin combined therapy (ACT). Within two weeks prior to the survey, only 16.5 % of respondents in all categories combined reported acute undifferentiated fever. DISCUSSION AND CONCLUSIONS: Mobility dynamics of migrant groups was complex and increased their vulnerability to malaria. This phenomenon was accentuated in less stable areas. Even though migrant workers were familiar with rapid diagnostic tests for malaria, ACT still needed wide recognition to improve practices supportive of MARC including the use of appropriate personal protection. High mobility calls for re-designation of tier II townships to optimize ACT resistance containment.

Improving uptake and use of malaria rapid diagnostic tests in the context of artemisinin drug resistance containment in eastern Myanmar: an evaluation of incentive schemes among informal private healthcare providers.

BACKGROUND: As efforts to contain artemisinin resistance and eliminate Plasmodium falciparum intensify, the accurate diagnosis and prompt effective treatment of malaria are increasingly needed in Myanmar and the Greater Mekong Sub-region (GMS). Rapid diagnostic tests (RDTs) have been shown to be safe, feasible, and effective at promoting appropriate treatment for suspected malaria, which are of particular importance to drug resistance containment. The informal private sector is often the first point of care for fever cases in malaria endemic areas across Myanmar and the GMS, but there is little published information about informal private provider practices, quality of service provision, or potential to contribute to malaria control and elimination efforts. This study tested different incentives to increase RDT use and improve the quality of care among informal private healthcare providers in Myanmar. METHODS: The study randomized six townships in the Mon and Shan states of rural Myanmar into three intervention arms: 1) RDT price subsidies, 2) price subsidies with product-related financial incentives, and 3) price subsidies with intensified information, education and counselling (IEC). The study assessed the uptake of RDT use in the communities by cross-sectional surveys of 3,150 households at baseline and six months post-intervention (6,400 households total, 832 fever cases). The study also used mystery clients among 171 providers to assess quality of service provision across intervention arms. RESULTS: The pilot intervention trained over 600 informal private healthcare providers. The study found a price subsidy with intensified IEC, resulted in the highest uptake of RDTs in the community, as compared to subsidies alone or merchandise-related financial incentives. Moreover, intensified IEC led to improvements in the quality of care, with mystery client surveys showing almost double the number of correct treatment following diagnostic test results as compared to a simple subsidy. CONCLUSIONS: Results show that training and quality supervision of informal private healthcare providers can result in improved demand for, and appropriate use of RDTs in drug resistance containment areas in eastern Myanmar. Future studies should assess the sustainability of such interventions and the scale and level of intensity required over time as public sector service provision expands.

Spread of artemisinin-resistant Plasmodium falciparum in Myanmar: a cross-sectional survey of the K13 molecular marker.

BACKGROUND: Emergence of artemisinin resistance in southeast Asia poses a serious threat to the global control of Plasmodium falciparum malaria. Discovery of the K13 marker has transformed approaches to the monitoring of artemisinin resistance, allowing introduction of molecular surveillance in remote areas through analysis of DNA. We aimed to assess the spread of artemisinin-resistant P falciparum in Myanmar by determining the relative prevalence of P falciparum parasites carrying K13-propeller mutations. METHODS: We did this cross-sectional survey at malaria treatment centres at 55 sites in ten administrative regions in Myanmar, and in relevant border regions in Thailand and Bangladesh, between January, 2013, and September, 2014. K13 sequences from P falciparum infections were obtained mainly by passive case detection. We entered data into two geostatistical models to produce predictive maps of the estimated prevalence of mutations of the K13 propeller region across Myanmar. FINDINGS: Overall, 371 (39%) of 940 samples carried a K13-propeller mutation. We recorded 26 different mutations, including nine mutations not described previously in southeast Asia. In seven (70%) of the ten administrative regions of Myanmar, the combined K13-mutation prevalence was more than 20%. Geospatial mapping showed that the overall prevalence of K13 mutations exceeded 10% in much of the east and north of the country. In Homalin, Sagaing Region, 25 km from the Indian border, 21 (47%) of 45 parasite samples carried K13-propeller mutations. INTERPRETATION: Artemisinin resistance extends across much of Myanmar. We recorded P falciparum parasites carrying K13-propeller mutations at high prevalence next to the northwestern border with India. Appropriate therapeutic regimens should be tested urgently and implemented comprehensively if spread of artemisinin resistance to other regions is to be avoided. FUNDING: Wellcome Trust-Mahidol University-Oxford Tropical Medicine Research Programme and the Bill & Melinda Gates Foundation.

Linkage disequilibrium network analysis (LDna) gives a global view of chromosomal inversions, local adaptation and geographic structure.

Recent advances in sequencing allow population-genomic data to be generated for virtually any species. However, approaches to analyse such data lag behind the ability to generate it, particularly in nonmodel species. Linkage disequilibrium (LD, the nonrandom association of alleles from different loci) is a highly sensitive indicator of many evolutionary phenomena including chromosomal inversions, local adaptation and geographical structure. Here, we present linkage disequilibrium network analysis (LDna), which accesses information on LD shared between multiple loci genomewide. In LD networks, vertices represent loci, and connections between vertices represent the LD between them. We analysed such networks in two test cases: a new restriction-site-associated DNA sequence (RAD-seq) data set for Anopheles baimaii, a Southeast Asian malaria vector; and a well-characterized single nucleotide polymorphism (SNP) data set from 21 three-spined stickleback individuals. In each case, we readily identified five distinct LD network clusters (single-outlier clusters, SOCs), each comprising many loci connected by high LD. In A. baimaii, further population-genetic analyses supported the inference that each SOC corresponds to a large inversion, consistent with previous cytological studies. For sticklebacks, we inferred that each SOC was associated with a distinct evolutionary phenomenon: two chromosomal inversions, local adaptation, population-demographic history and geographic structure. LDna is thus a useful exploratory tool, able to give a global overview of LD associated with diverse evolutionary phenomena and identify loci potentially involved. LDna does not require a linkage map or reference genome, so it is applicable to any population-genomic data set, making it especially valuable for nonmodel species.

Mosquito genomics. Highly evolvable malaria vectors: the genomes of 16 Anopheles mosquitoes.

Variation in vectorial capacity for human malaria among Anopheles mosquito species is determined by many factors, including behavior, immunity, and life history. To investigate the genomic basis of vectorial capacity and explore new avenues for vector control, we sequenced the genomes of 16 anopheline mosquito species from diverse locations spanning ~100 million years of evolution. Comparative analyses show faster rates of gene gain and loss, elevated gene shuffling on the X chromosome, and more intron losses, relative to Drosophila. Some determinants of vectorial capacity, such as chemosensory genes, do not show elevated turnover but instead diversify through protein-sequence changes. This dynamism of anopheline genes and genomes may contribute to their flexible capacity to take advantage of new ecological niches, including adapting to humans as primary hosts.

Molecular assessment of artemisinin resistance markers, polymorphisms in the k13 propeller, and a multidrug-resistance gene in the eastern and western border areas of Myanmar.

BACKGROUND: As K13 propeller mutations have been recently reported to serve as molecular markers, assessment of K13 propeller polymorphisms in multidrug-resistant gene in isolates from Myanmar, especially the eastern and western border areas, is crucial if we are to understand the spread of artemisinin resistance. METHODS: A 3-day surveillance study was conducted in the eastern and western border areas in Myanmar, and K13 propeller and Plasmodium falciparum multidrug resistance-associated protein 1 (pfmrp1) mutations were analyzed. RESULTS: Among the 1761 suspected malaria cases screened, a total of 42 uncomplicated falciparum cases from the eastern border and 49 from the western border were subjected to 3 days of surveillance after artemether-lumefantrine treatment. No parasitemic case showing positivity on day 3 was noted from the western border, but 26.2% (11/42) of cases were positive in the eastern border. Although we found no marked difference in the prevalence of the pfmrp1 mutation in the eastern and western borders (36% vs 31%, respectively), K13 mutations were more frequent in the eastern border area (where the 3-day persistent cases were detected; 48% vs 14%). C580Y, M476I, A481V, N458Y, R539T, and R516Y accounted for 68.9% of all K13 mutations significantly associated with day 3 parasitaemia. CONCLUSIONS: The K13 mutations were significantly associated with day 3 parasitaemia, emphasizing the importance of K13 surveillance. The low prevalence of K13 mutations and the absence of day 3 parasitaemic cases indicate that artemisinin resistance may not have spread to the western Myanmar border region. Although analysis of multiple K13 mutations is challenging, it should be done at various sentinel sites in Myanmar.

Challenges in universal coverage and utilization of insecticide-treated bed nets in migrant plantation workers in Myanmar.

BACKGROUND: High coverage of the bed nets can reduce mortality and morbidity of mosquito-borne diseases including malaria. Although the migrant workers are at high risk of malaria, there are many hidden challenges in universal coverage and utilization of the insecticide-treated nets (ITNs) in this populations. METHODS: Cross sectional study was conducted in 170 migrant workers in palm oil plantation sites in Tanintharyi Region and 175 in rubber plantation sites in Mon State. A multistage stratified cluster sampling was applied to select the participants. During household visit, face-to-face interviews using structured pre-coded, pre tested questionnaires and direct observation on installation of the bed nets was conducted. Two focus group discussions in each site were done by sample stratified purposive sampling method mainly focused on effective utilization of bed nets. RESULTS: Among them, 332 (96.2%) had a bed net and 284 (82.3%) had an ITN, while 204 (59.1%) had unused extranets. Among the ITNs users, 28.9% reported problems including insecticide smell (56.9%), dizziness (20.2%), headache (12.8%) and itchiness (9.2%). More than 75% received ITNs from health authorities and NGOs free-of-charge. More than 70% wanted to buy a net but they were unaffordable for 64% of them. On observation, only five families could show no bed net, but 80% showed 1-3 ITNs. Consistent utilization in all seasons was noted in 189 (53.1%), that was higher in palm oil plantation than rubber plantation workers (p = 0.0001) due to the nature of the work at night. Perceived malaria risk was also significantly higher ITNs consistent users than non-users (p = 0.0004) and better willingness to buy an ITN by themselves (p = 0.0005). They said that effectiveness of the ITNs was reduced after 6 months and 2-3 times washing. They wished to receive more durable smooth nets with small holes in lace. Misuses of the ITNs such as use the nets for animals and fishing, were also noted. CONCLUSION: There should be efforts to improve effective utilization of ITNs by continuous mass free distribution, durability monitoring, surveillance of insecticide resistance of the vector and behaviour change interventions in migrant plantation workers.

Repeated landmass reformation limits diversification in the widespread littoral zone mosquito Anopheles sundaicus sensu lato in the Indo-Oriental Region.

Southeast Asia harbours abundant biodiversity, hypothesized to have been generated by Pliocene and Pleistocene climatic and environmental change. Vicariance between the island of Borneo, the remaining Indonesian archipelago and mainland Southeast Asia caused by elevated sea levels during interglacial periods has been proposed to lead to diversification in the littoral zone mosquito Anopheles (Cellia) sundaicus (Rodenwaldt) sensu lato. To test this biogeographical hypothesis, we inferred the population history and assessed gene flow of A. sundaicus s.l. sampled from 18 populations across its pan-Asian species range, using sequences from mitochondrial cytochrome c oxidase subunit 1 (CO1), the internal transcribed spacer 2 (ITS2) and the mannose phosphate isomerase (Mpi) gene. A hypothesis of ecological speciation for A. sundaicus involving divergent adaptation to brackish and freshwater larval habitats was also previously proposed, based on a deficiency of heterozygotes for Mpi allozyme alleles in sympatry. This hypothesis was not supported by Mpi sequence data, which exhibited no fixed differences between brackish and freshwater larval habitats. Mpi and CO1 supported the presence of up to eight genetically distinct population groupings. Counter to the hypothesis of three allopatric species, divergence was often no greater between Borneo, Sumatra/Java and the Southeast Asian mainland than it was between genetic groupings within these landmasses. An isolation-with-migration (IM) model indicates recurrent gene flow between the current major landmasses. Such gene flow would have been possible during glacial periods when the current landmasses merged, presenting opportunities for dispersal along expanding and contracting coastlines. Consequently, Pleistocene climatic variation has proved a homogenizing, rather than diversifying, force for A. sundaicus diversity.

Containing artemisinin resistance of Plasmodium falciparum in Myanmar: achievements, challenges and the way forward.

Artemisinin resistance is a major threat to global malaria control and elimination efforts. Myanmar detected the first indication of the resistance in 2009 in the eastern part of the country, bordering Thailand. Since 2010, WHO has played a vital role in ensuring that a comprehensive programme on the containment of the resistance is in place. This paper documents achievement made in terms of output, outcomes and early impact on malaria from July 2011 to December 2013. It also identifies enabling factors to success and, most importantly, challenges awaiting the national programme and its partners.

Comparative phylogeography reveals a shared impact of pleistocene environmental change in shaping genetic diversity within nine Anopheles mosquito species across the Indo-Burma biodiversity hotspot.

South-East Asia is one of the world's richest regions in terms of biodiversity. An understanding of the distribution of diversity and the factors shaping it is lacking, yet essential for identifying conservation priorities for the region's highly threatened biodiversity. Here, we take a large-scale comparative approach, combining data from nine forest-associated Anopheles mosquito species and using statistical phylogeographical methods to disentangle the effects of environmental history, species-specific ecology and random coalescent effects. Spatially explicit modelling of Pleistocene demographic history supports a common influence of environmental events in shaping the genetic diversity of all species examined, despite differences in species' mtDNA gene trees. Populations were periodically restricted to allopatric northeastern and northwestern refugia, most likely due to Pleistocene forest fragmentation. Subsequent southwards post-glacial recolonization is supported by a north-south gradient of decreasing genetic diversity. Repeated allopatric fragmentation and recolonization have led to the formation of deeply divergent geographical lineages within four species and a suture zone where these intraspecific lineages meet along the Thai-Myanmar border. A common environmental influence for this divergence was further indicated by strong support for simultaneous divergence within the same four species, dating to approximately 900 thousand years ago (kya). Differences in the geographical structuring of genetic diversity between species are probably the result of varying species' biology. The findings have important implications for conservation planning; if the refugial regions and suture zone identified here are shared by other forest taxa, the unique and high levels of genetic diversity they house will make these areas conservation priorities.

Spatial genetic structure of Aedes aegypti mosquitoes in mainland Southeast Asia.

Aedes aegypti mosquitoes originated in Africa and are thought to have spread recently to Southeast Asia, where they are the major vector of dengue. Thirteen microsatellite loci were used to determine the genetic population structure of A. aegypti at a hierarchy of spatial scales encompassing 36 sites in Myanmar, Cambodia and Thailand, and two sites in Sri Lanka and Nigeria. Low, but significant, genetic structuring was found at all spatial scales (from 5 to >2000 km) and significant F IS values indicated genetic structuring even within 500 m. Spatially dependent genetic-clustering methods revealed that although spatial distance plays a role in shaping larger-scale population structure, it is not the only factor. Genetic heterogeneity in major port cities and genetic similarity of distant locations connected by major roads, suggest that human transportation routes have resulted in passive long-distance migration of A. aegypti. The restricted dispersal on a small spatial scale will make localized control efforts and sterile insect technology effective for dengue control. Conversely, preventing the establishment of insecticide resistance genes or spreading refractory genes in a genetic modification strategy would be challenging. These effects on vector control will depend on the relative strength of the opposing effects of passive dispersal.

Mitochondrial pseudogenes in the nuclear genome of Aedes aegypti mosquitoes: implications for past and future population genetic studies.

BACKGROUND: Mitochondrial DNA (mtDNA) is widely used in population genetic and phylogenetic studies in animals. However, such studies can generate misleading results if the species concerned contain nuclear copies of mtDNA (Numts) as these may amplify in addition to, or even instead of, the authentic target mtDNA. The aim of this study was to determine if Numts are present in Aedes aegypti mosquitoes, to characterise any Numts detected, and to assess the utility of using mtDNA for population genetics studies in this species. RESULTS: BLAST searches revealed large numbers of Numts in the Ae. aegypti nuclear genome on 146 supercontigs. Although the majority are short (80% < 300 bp), some Numts are almost full length mtDNA copies. These long Numts are not due to misassembly of the nuclear genome sequence as the Numt-nuclear genome junctions could be recovered by amplification and sequencing. Numt evolution appears to be a complex process in Ae. aegypti with ongoing genomic integration, fragmentation and mutation and the secondary movement of Numts within the nuclear genome.The PCR amplification of the putative mtDNA nicotinamide adenine dinucleotide dehydrogenase subunit 4 (ND4) gene from 166 Southeast Asian Ae. aegypti mosquitoes generated a network with two highly divergent lineages (clade 1 and clade 2). Approximately 15% of the ND4 sequences were a composite of those from each clade indicating Numt amplification in addition to, or instead of, mtDNA. Clade 1 was shown to be composed at least partially of Numts by the removal of clade 1-specific bases from composite sequences following enrichment of the mtDNA. It is possible that all the clade 1 sequences in the network were Numts since the clade 2 sequences correspond to the known mitochondrial genome sequence and since all the individuals that produced clade 1 sequences were also found to contain clade 2 mtDNA-like sequences using clade 2-specific primers. However, either or both sets of clade sequences could have Numts since the BLAST searches revealed two long Numts that match clade 2 and one long Numt that matches clade 1. The substantial numbers of mutations in cloned ND4 PCR products also suggest there are both recently-derived clade 1 and clade 2 Numt sequences. CONCLUSION: We conclude that Numts are prevalent in Ae. aegypti and that it is difficult to distinguish mtDNA sequences due to the presence of recently formed Numts. Given this, future population genetic or phylogenetic studies in Ae. aegypti should use nuclear, rather than mtDNA, markers.