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.

Combination of PURE-DNA extraction and LAMP-DNA amplification methods for accurate malaria diagnosis on dried blood spots.

BACKGROUND: Malaria is one of the most important parasitic infectious diseases for which almost half of the world's population is at risk. Although several diagnostic methods are now available to detect the infection, more sensitive and applicable tests are still required in the field. The loop-mediated isothermal amplification (LAMP) method is a DNA amplification tool in which the DNA amplification can be achieved by incubation at a stable temperature. A malaria detection kit based on this methodology has already been commercialized and is being used in some countries. The kit includes two reaction tubes: one targeting the common Plasmodium genus (Pan tube) and the other specifically targeting Plasmodium falciparum (Pf tube). In parallel, a simple DNA extraction method, the procedure for ultra rapid extraction (PURE), which can produce a DNA solution suitable for the LAMP reaction without the use of a centrifuge, has also become available. In this study, the sensitivity of the combination of the PURE and LAMP methods (PURE-LAMP) was evaluated with archived dried clinical blood samples of imported malaria cases, including P. falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae. RESULTS: Using a nested PCR as the reference, 117 samples including 46 P. falciparum, 7 P. vivax, 9 P. ovale, 4 P. malariae, and 51 negative cases were tested. The PURE-LAMP Pan correctly identified 64 of the 66 positives and the 51 negatives. Among the Pan-positive samples 45 P. falciparum were also detected with the PURE-LAMP Pf. The PURE-LAMP Pan and PURE-LAMP Pf had respective sensitivities of 96.96% (95% CI 89.47-99.63) and 97.82% (95% CI 88.47-99.94) and common specificity of 1. CONCLUSION: The PURE-LAMP system is accurate when used with dried blood spots and extendable to the field.

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.

Household clustering of asymptomatic malaria infections in Xepon district, Savannakhet province, Lao PDR.

BACKGROUND: In the Lao PDR, malaria morbidity and mortality have remarkably decreased over the past decade. However, asymptomatic infections in rural villages contribute to the on-going local transmission. The primary objective of this study was to explore the characteristics of infections in a malaria-endemic district of the Lao PDR. The specific objectives were to investigate the prevalence and species of malaria parasites using molecular methods and to assess individual and household parasite levels and the characteristics associated with malaria infection. METHODS: The study population included 870 participants from 236 households in 10 villages of the Xepon district. Interviews, blood examinations and body temperature measurements were conducted between August and September 2013. A multilevel logistic regression model, with adjustment for clustering effects, was used to assess the association between predictor variables and an outcome variable (malaria infection status as principally determined by PCR). The predictive factors included individual-level factors (age, gender, past fever episode, and forest activity during night time) and household-level factors (household member size, household bed net usage/density and a household with one other malaria-infected member). RESULTS: Fifty-two participants (including 26 children) tested positive (positive rate: 6.0 %): Plasmodium falciparum mono-infection was the most common infection (n = 41, 78.8 %), followed by P. falciparum and Plasmodium vivax mixed infections (n = 9, 17.3 %). The majority of infected participants (n = 42, 80.8 %) had no fever episodes in the two previous weeks or a measurable fever (>37 °C) at the time of survey. Living in a household with one other malaria-infected member significantly increased the odds of infection (odds ratio 24.33, 95 % confidence interval 10.15-58.32). Among the 40 households that had at least one infected member, nine households were responsible for 40.4 % of the total infections. CONCLUSIONS: Plasmodium vivax was detected more frequently than it was reported from the district hospital. Most infections were asymptomatic and sub-microscopic and were highly clustered within households. To further eliminate malaria in Xepon and other similar settings in the country, the National Malaria Control Programme should consider household-based strategies, including reactive case detection targeting the household members of index cases.

First case of Plasmodium knowlesi infection in a Japanese traveller returning from Malaysia.

This is the first case of Plasmodium knowlesi infection in a Japanese traveller returning from Malaysia. In September 2012, a previously healthy 35-year-old Japanese man presented to National Center for Global Health and Medicine in Tokyo with a two-day history of daily fever, mild headaches and mild arthralgia. Malaria parasites were found in the Giemsa-stained thin blood smear, which showed band forms similar to Plasmodium malariae. Although a nested PCR showed the amplification of the primer of Plasmodium vivax and Plasmodium knowlesi, he was finally diagnosed with P. knowlesi mono-infection by DNA sequencing. He was treated with mefloquine, and recovered without any complications. DNA sequencing of the PCR products is indispensable to confirm P. knowlesi infection, however there is limited access to DNA sequencing procedures in endemic areas. The extent of P. knowlesi transmission in Asia has not been clearly defined. There is limited availability of diagnostic tests and routine surveillance system for reporting an accurate diagnosis in the Asian endemic regions. Thus, reporting accurately diagnosed cases of P. knowlesi infection in travellers would be important for assessing the true nature of this emerging human infection.

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.

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.

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.

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.

Health-seeking behaviours in a malaria endemic district in Lao People's Democratic Republic: a mixed methods study.

OBJECTIVES: This mixed methods study was conducted to explore the barriers and facilitators for health-seeking behaviours in a malaria endemic district in Lao PDR. DESIGN: A convergent mixed methods design. SETTING: Two malaria endemic villages in Thapangthong district, Savannakhet Province, Lao PDR. PARTICIPANTS: Villagers and healthcare workers in the two villages in Thapangthong district. METHODS: In the quantitative part, a pretested questionnaire was used to identify the health-seeking behaviours of the villagers. In the qualitative part, focus group discussions were employed to explore health-seeking behaviours of the villagers and in-depth interviews were used to explore the perceptions of the healthcare workers. Descriptive statistics were computed and multiple logistic regressions were used to identify the factors associated with perceived severity and perceived susceptibility. Thematic analysis was used to analyse the qualitative data. Quantitative and qualitative results were integrated in joint displays. RESULTS: In the quantitative part, data were collected from 313 villagers from both villages. For malaria, 96.0% and 98.2% of villagers from villages A and B, respectively, would first seek treatment at public health facilities. Villagers who have not experienced malaria before were more likely to perceive that the consequences of malaria were serious compared with those who have experienced malaria before (adjusted OR=1.69, 95% CI: 1.03 to 2.75). However, qualitative data showed that villagers faced problems such as lack of medicines and medical equipment. Healthcare workers also mentioned the lack of manpower and equipment in the in-depth interviews. Nevertheless, villagers still preferred to seek treatment at the health center as the National Health Insurance was introduced. CONCLUSIONS: Public health facility usage was high but barriers existed. Effective policy and enabling environment such as the introduction of the National Health Insurance could help accelerate the progress towards the malaria elimination goal. Moreover, the benefits could go beyond the context of malaria.

Geographical origin of Plasmodium vivax in the Republic of Korea: haplotype network analysis based on the parasite's mitochondrial genome.

BACKGROUND: The Republic of Korea (South Korea) is one of the countries where vivax malaria had been successfully eradicated by the late 1970s. However, re-emergence of vivax malaria in South Korea was reported in 1993. Several epidemiological studies and some genetic studies using antigenic molecules of Plasmodium vivax in the country have been reported, but the evolutionary history of P. vivax has not been fully understood. In this study, the origin of the South Korean P. vivax population was estimated by molecular phylogeographic analysis. METHODS: A haplotype network analysis based on P. vivax mitochondrial (mt) DNA sequences was conducted on 11 P. vivax isolates from South Korea and another 282 P. vivax isolates collected worldwide. RESULTS: The network analysis of P. vivax mtDNA sequences showed that the coexistence of two different groups (A and B) in South Korea. Groups A and B were identical or close to two different populations in southern China. CONCLUSIONS: Although the direct introduction of the two P. vivax populations in South Korea were thought to have been from North Korea, the results of this analysis suggest the genealogical origin to be the two different populations in southern China.

Genetic diversity and population structure of Plasmodium falciparum in the Philippines.

BACKGROUND: In the Philippines, malaria morbidity and mortality have decreased since the 1990s by effective malaria control. Several epidemiological surveys have been performed in the country, but the characteristics of the Plasmodium falciparum populations are not yet fully understood. In this study, the genetic structure of P. falciparum populations in the Philippines was examined. METHODS: Population genetic analyses based on polymorphisms of 10 microsatellite loci of the parasite were conducted on 92 isolates from three provinces (Kalinga, Palawan, and Davao del Norte) with different malaria endemicity. RESULTS: The levels of genetic diversity and the effective population sizes of P. falciparum in the Philippines were similar to those reported in the mainland of Southeast Asia or South America. In the low malaria transmission area (Kalinga), there was a low level of genetic diversity and a strong linkage disequilibrium (LD) when the single-clone haplotype (SCH) was used in the multilocus LD analysis, while in the high malaria transmission areas (Palawan and Davao del Norte), there was a high level of genetic diversity and a weak LD when SCH was used in the multilocus LD analysis. On the other hand, when the unique haplotypes were used in the multilocus LD analysis, no significant LD was observed in the Kalinga and the Palawan populations. The Kalinga and the Palawan populations were, therefore, estimated to have an epidemic population structure. The three populations were moderately differentiated from each other. CONCLUSION: In each area, the level of genetic diversity correlates with the local malaria endemicity. These findings confirm that population genetic analyses using microsatellite loci are a useful tool for evaluating malaria endemicity.

Malaria among foreign migrant workers in Savannakhet Province, Lao People's Democratic Republic.

BACKGROUND: Although mobile and migrant populations are considered an important group in malaria elimination settings, there is currently a lack of understanding about foreign migrant workers in the Lao People's Democratic Republic (Lao PDR). The present study aimed to document the migration characteristics, positive rate of malaria infection, and preventive and treatment-seeking behavior for malaria among foreign migrant workers in the malaria-endemic districts of Savannakhet province, Lao PDR. METHODS: A community-based survey was undertaken in four districts of Savannakhet province between February and June, 2015. Questionnaire-based interviews and blood examinations, including rapid diagnostic tests and PCR assays, were conducted with 391 migrant workers who were registered at local police departments. RESULTS: Most of the study participants were men (75.7%) and Vietnamese (92.6%). The median age (interquartile range) was 31 (25 to 41) years old. Most common occupation was factory worker (47.6%), followed by trader/shopkeeper (21.5%) and plantation worker/farmer (16.4%). The median length of stay (interquartile range) in the districts was 405 (183 to 1207) days. The majority of the participants (85.9%) had not worked in a province other than the study province, nor had the majority (92.6%) worked in a foreign country other than the Lao PDR. Although most of the participants (62.7%) reportedly used a bed net daily, these nets were mostly conventional untreated ones. No one tested positive for malaria. However, 10.0% of the participants reported a malaria-like illness episode that had occurred in the Lao PDR. The most common measure taken for the episode was to visit a hospital/health center in the Lao PDR, followed by conducting self-medication alone. Forty-one participants reported an experience of working in the forest while living in the Lao PDR. CONCLUSIONS: Foreign migrant workers who are registered at local police departments are unlikely to play a major role in maintaining local transmissions and spreading drug-resistant malaria in the study province. However, some of them were involved in forest-related activities, suggesting that these workers are potentially at risk of malaria. The Lao National Malaria Control Program should educate foreign migrant workers about the risk of malaria when living in Lao PDR.

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.

Differences in genetic population structures of Plasmodium falciparum isolates from patients along Thai-Myanmar border with severe or uncomplicated malaria.

BACKGROUND: There have been many reports on the population genetic structures of Plasmodium falciparum from different endemic regions, but few studies have examined the characteristics of isolates from patients with different clinical outcomes. The population genetic structures of P. falciparum isolates from patients with either severe or uncomplicated malaria were examined. METHODS: Twelve microsatellite DNA loci from P. falciparum were used to assess the population genetic structures of 50 isolates (i.e., 25 isolates from patients with severe malaria and 25 from patients with uncomplicated malaria) collected in the Thai-Myanmar border area between 2002 and 2005. RESULTS: Genetic diversity and effective population sizes were greater in the uncomplicated malaria group than in the severe malaria group. Evidence of genetic bottlenecks was not observed in either group. Strong linkage disequilibrium was observed in the uncomplicated malaria group. The groups demonstrated significant genetic differentiation (P < 0.05), and allele frequencies for 3 of the 12 microsatellite loci differed significantly between the two groups. CONCLUSION: These findings suggest that the genetic structure of P. falciparum populations in patients with severe malaria differs from that in patients with uncomplicated malaria. The microsatellite loci used in this study were presumably unrelated to antigenic features of the parasites, but, these findings suggest that some loci may influence the clinical outcome of malaria.

Phylogenetic relationship of Plasmodium falciparum populations in the Philippines.

Malaria is one of the major infectious diseases in the Philippines. It is being targeted for control through sustained early diagnosis, treatment and mosquito control. It is in this light that understanding the genetic background of the parasite population is important not only for basic biology of the organism but also for epidemiology and control of the disease. In the present study, molecular phylogenetic relationships of the 3 Plasmodium falciparum populations in the Philippines with the other populations in the world were inferred based on polymorphisms of 9 highly polymorphic microsatellite DNA loci in the parasite genome. A total of 92 P. falciparum isolates collected from 3 provinces (Kalinga, Palawan and Davao del Norte) in the Philippines, and 8 from other populations (3 African, 2 South American, 2 Papua New Guinean, and 1 Thai) that were previously reported, were used for the analysis. The phylogenetic tree showed that the 3 Philippine populations were genetically divergent from each other as compared to the other populations. The branching pattern of the tree suggests that the 3 Philippine populations were relatively close to the Thai population, rather than the Papua New Guinean populations, indicating that the ancestor of the 3 Philippines populations were introduced from Indochina peninsula, and not from countries located south of the Philippines such as Papua New Guinea or Indonesia.

Patients' adherence to artemisinin-based combination therapy and healthcare workers' perception and practice in Savannakhet province, Lao PDR.

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.

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.

Case Report: Reversible Cerebral Vasoconstriction Syndrome in Cerebral Malaria.

Cerebral malaria is a severe complication of falciparum malaria that occurs infrequently in adults. Here, we describe the case of a 21-year-old man who presented with fever and headache 13 days after returning from a 12-day trip to Kenya and was subsequently diagnosed with falciparum malaria. Complications of cerebral malaria developed within 1 day after the initiation of therapy with intravenous quinine, and the patient entered a deep coma. Magnetic resonance angiography (MRA) revealed multiple vasoconstrictions in his brain. The resulting neurocognitive disorders that persisted after parasite clearance improved gradually, as confirmed by MRA, enabling the patient to perform activities of daily living upon discharge. In this case of cerebral malaria, the MRA findings indicated the involvement of reversible cerebral vasoconstriction syndrome.

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.