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Abstract Background Southeast Asia is well known as a hotspot of biodiversity. However, very little is known about cave-dwelling hematophagous insects that are medically important. Taxonomic knowledge and ecology of phlebotomine sand flies are very poorly studied in Laos, as well as in other countries in the region. Herein, we report species diversity data and some notes on the ecology of the detected species from these karstic limestone areas of Laos. Methods Phlebotomine sand flies were collected using Centers for Disease Control and Prevention (CDC) light traps from limestone cave locations in three districts of Vientiane Province, Laos. Both morphological and molecular techniques were used for sand fly identification. Species diversity and abundance were analyzed according to sites, locations, collection seasons, and trapping positions. Results A total of 6564 sand flies, of which 5038 were females and 1526 were males, were morphologically identified into 20 species belonging to five genera (Chinius, Idiophlebotomus, Phlebotomus, Sergentomyia, and Grassomyia). The most abundant species were Chinius eunicegalatiae, Phlebotomus stantoni, Sergentomyia hivernus, Se. siamensis, and Idiophlebotomus longiforceps. Cytochrome b analysis results supported the morphological identification and revealed that Se. siamensis was separated from other members of the Se. barraudi group. Two new species, Se. dvoraki n. sp. and Se. marolii n. sp., were described. Sand fly density was generally high except in a cave in Vangvieng, with species richness ranging from 14 to 18 across different caves. Outside caves had higher species richness (R = 20) and diversity (H = 2.50) than cave entrances (R = 18, H = 2.41) and interiors (R = 16, H = 2.13). Seasonal variations showed high sand fly density in Feung and Hinheup during both dry and rainy seasons, while Vangvieng had a notable decrease in density during the dry season (D = 6.29). Conclusions This study revealed that the diversity of phlebotomine sand fly fauna in Laos, particularly in karstic limestone areas, is greater than previously known. However, the taxonomic status of many species in Laos, as well as Southeast Asia, still needs more in-depth study using both morphological characters and molecular methods. Many species could be found from inside, at the entrance, and outside of caves, indicating a wide range of host-seeking behavior or possible natural breeding in the karstic cave areas. Graphical abstract

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.

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.

Abstract Background In Laos, the malaria burden remains high despite a significant reduction of cases during the last decade. In the context of the disease elimination by 2030, a nationwide entomological survey was conducted to better understand the distribution, abundance and behaviour of major malaria vectors (Anopheles spp.) in the country. Methods Mosquito collections were implemented in ten villages from ten provinces during the rainy and dry seasons of 2014 and 2015 by using human landing catch (HLC) and cow bait collection (CBC) methods. After morphological identification in the field, molecular identification of the sibling species of Anopheles mosquitoes from the Funestus, Leucosphyrus, and Maculatus groups were determined using PCR specific alleles. A screening of Plasmodium falciparum and Plasmodium vivax infections in the vectors was carried out by quantitative PCR assays. Results A total of 14,146 adult mosquitoes representing 25 different Anopheles species were collected and morphologically identified. Molecular identification revealed the presence of 12 sibling species within the main primary vector groups, including Anopheles maculatus, Anopheles rampae, Anopheles sawadwongporni, Anopheles pseudowillmori, Anopheles dravidicus, Anopheles minimus, Anopheles aconitus, Anopheles pampanai, Anopheles harrisoni, Anopheles dirus, Anopheles baimaii, Anopheles nemophilous. Anopheles maculatus and An. minimus were predominant during both the dry and rainy seasons, but showed highly zoophilic preferences (Zoophilic index of 98% and 95%, respectively). Overall, 22% of the total malaria vectors were collected between 10:00 PM and 5:00 AM indoors when people are sleeping. Twenty-seven percent of primary and secondary vectors were collected outdoors before 10:00 PM or after 5:00 AM, times when people are usually awake and outdoors. Only two specimens were positive for P. falciparum, one An. aconitus from Phongsaly and one An. minimus from Vientiane Province Conclusions The results indicate that people living in rural areas in Laos are constantly exposed to malaria vectors throughout the year and specifically outdoors. The use of LLINs/IRS remains important but innovative tools and new strategies are needed to address locally, the early and outdoor malaria transmission. Lack of expertise in general entomological methods may further exacerbate the situation.

Dengue, Zika and chikungunya viruses cause significant human public health burdens in the world. These arboviruses are transmitted by vector mosquito species notably Aedes aegypti and Aedes albopictus. In the Pacific region, more vector species of arboviruses belonging to the Scutellaris Group are present. Due to the expansion of human travel and international trade, the threat of their dispersal in other world regions is on the rise. Strengthening of entomological surveillance ensuring rapid detection of introduced vector species is therefore required in order to avoid their establishment and the risk of arbovirus outbreaks. This surveillance relies on accurate species identification. The aim of this study was to assess the use of the Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) as a tool for an international identification and surveillance of these mosquito vectors of arboviruses. Field-mosquitoes belonging to 8 species (Ae. aegypti, Ae. albopictus, Aedes polynesiensis, Aedes scutellaris, Aedes pseudoscutellaris, Aedes malayensis, Aedes futunae and Culex quinquefasciatus) from 6 countries in the Pacific, Asian and Madagascar, were included in this study. Analysis provided evidence that a MALDI-TOF database created using mosquitoes from the Pacific region allowed suitable identification of mosquito species from the other regions. This technic was as efficient as the DNA sequencing method in identifying mosquito species. Indeed, with the exception of two Ae. pseudoscutellaris, an exact species identification was obtained for all individual mosquitoes. These findings highlight that the MALDI-TOF MS is a promising tool that could be used for a global comprehensive arbovirus vector surveillance.

In 2012–2013, chikungunya virus (CHIKV) was the cause of a major outbreak in the southern part of Lao People’s Democratic Republic (Lao PDR). Since then, only a few imported cases, with isolates belonging to different lineages, were recorded between 2014 and 2020 in Vientiane capital and few autochthonous cases of ECSA-IOL lineage were detected in the south of the country in 2020. The CHIKV epidemiological profile contrasts with the continuous and intensive circulation of dengue virus in the country, especially in Vientiane capital. The study’s aim was to investigate the ability of the local field-derived Aedes aegypti population from Vientiane capital to transmit the Asian and ECSA-IOL lineages of CHIKV. Our results revealed that, for both CHIKV lineages, infection rates were low and dissemination rates were high. The transmission rates and efficiencies evidenced a low vector competence for the CHIKV tested. Although this population of Ae. aegypti showed a relatively modest vector competence for these two CHIKV lineages, several other factors could influence arbovirus emergence such as the longevity and density of female mosquitoes. Due to the active circulation of CHIKV in Southeast Asia, investigations on these factors should be done to prevent the risk of CHIKV emergence and spread in Lao PDR and neighboring countries.

Zika virus (ZIKV) is a Flavivirus transmitted by Aedes mosquitoes, and was responsible for a worldwide outbreak between 2013 and 2016. However, no ZIKV outbreak has been described in Southeast Asia since 2017. In this study, we report the first microcephaly case with probable ZIKV infection during pregnancy in Lao People’s Democratic Republic.

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.

Background: In 2020 Lao PDR had low reported COVID-19 cases but it was unclear whether this masked silent transmission. A seroprevalence study was done August - September 2020 to determine SARS-CoV-2 exposure. Methods: Participants were from the general community (n=2433) or healthcare workers (n=666) in five provinces and bat/wildlife contacts (n=74) were from Vientiane province. ELISAs detected anti- SARS-CoV-2 Nucleoprotein (N; n=3173 tested) and Spike (S; n=1417 tested) antibodies. Double-positive samples were checked by IgM/IgG rapid tests. Controls were confirmed COVID-19 cases (n=15) and pre-COVID-19 samples (n=265). Seroprevalence for the general community was weighted to account for complex survey sample design, age and sex. Findings: In pre-COVID-19 samples, 5·3%, [95% CI=3·1-8·7%] were anti-N antibody single-positive and 1·1% [0·3-3·5%] were anti-S antibody single positive. None were double positive. Anti-N and anti-S antibodies were detected in 5·2% [4·2-6·5%] and 2·1% [1·1-3·9%] of the general community, 2·0% [1·1-3·3%] and 1·4% [0·5-3·7%] of healthcare workers and 20·3% [12·6-31·0%] and 6·8% [2·8-15·3%] of bat/wildlife contacts. 0·1% [0·02-0·3%] were double positive for anti-N and anti-S antibodies (rapid test negative). Interpretation: We find no evidence for significant SARS-CoV-2 circulation in Lao PDR before September 2020. This likely results from early decisive measures taken by the government, social behavior, and low population density. High anti-N /low anti-S seroprevalence in bat/wildlife contacts may indicate exposure to cross-reactive animal coronaviruses with threat of emerging novel viruses. Funding: Agence Française de Développement. Additional; Institut Pasteur du Laos, Institute Pasteur, Paris and Luxembourg Ministry of Foreign and European Affairs (“PaReCIDS II”).

Abstract 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.

Abstract 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.

Abstract 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.

Abstract Background The mosquito Aedes aegypti is the primary vector of several arboviruses, such as dengue, chikungunya and Zika, and represents a major public health problem in Southeast Asia. In Laos, where dengue is reemerging, several Ae. aegypti populations from the capital Vientiane have shown resistance to the organophosphate temephos, a commonly-used larvicide for public health interventions. Methods Here, we tested the insecticide susceptibility of a wild larval population of Ae. aegypti against Bacillus thuringiensis israelensis (Bti), diflubenzuron, pyriproxyfen and spinosad. Residual efficacies of Bti (VectobacWG®), diflubenzuron (Killmos®) and temephos (Abate®) were then evaluated under simulated field conditions against the wild Ae. aegypti population. Results The larval bioassays showed that the wild Ae. aegypti strain was moderately resistant to temephos and spinosad (resistance ratio, RR < 5) and fully susceptible to the other insecticides (RR = 1). The simulated field trial bioassays showed that all of the insecticides tested remained above the WHO acceptable larvicide threshold after 28 weeks. Conclusions These results suggest that Bti and diflubenzuron may be promising alternative larvicides for controlling dengue vectors in water-storage containers in Laos, especially against Ae. aegypti populations, in which resistance to temephos has been detected.

Abstract 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.

Abstract 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.

Abstract Background Protecting people outdoors against mosquito-borne diseases is a major challenge. Here we compared commercially available personal protection methods to identify the most effective method for outdoor use in northern Lao PDR. Methods From June to August 2016 the protective efficacy of treatments were compared in a secondary forest during the afternoon and a village during the evening. Comparisons were made using a replicated Latin square design between: (i) short permethrin-treated overalls; (ii) long permethrin-treated overalls; (iii) short untreated overalls with para-menthane-3,8-diol (PMD) applied topically; (iv) short permethrin-treated overalls plus PMD applied topically; (v) short untreated overalls with metofluthrin coils in a metal casing worn on a belt; and (vi) long untreated overalls. Short untreated overalls served as the control. Cone tests were conducted on the treated and untreated fabric before and after field experiments. A questionnaire survey was used to measure social acceptability. Results Mosquito coils in a metal casing worn on a belt resulted in 92.3% (95% confidence interval, CI: 88.9–94.6%). landing protection from female mosquitoes in the afternoon and 68.8% (95% CI: 41.7–83.3%) protection in the evening compared to short untreated clothing. PMD was protective both when combined with short permethrin-treated overalls (afternoon, 68.2%, 95% CI: 52.6–78.7%; evening, 52.3%, 95% CI: 33.8–65.7%) and when used in combination with short untreated overalls (afternoon, 55.0%, 95% CI: 41.7–65.2%; evening, 25.2%, 95% CI: 9.4–38.2%). Whilst long permethrin-treated overalls were protective (afternoon, 61.1%, 95% CI: 51.4–68.8%; evening, 43.0%, 95% CI: 25.5–56.4%), short permethrin-treated overalls and long untreated overalls were not. Exposure to new permethrin-treated fabric in cone tests resulted in 25.0% (95% CI, 17.8–32.2%) and 26.2% (95% CI 16.7–35.8%) mortality for susceptible Ae. albopictus and susceptible Ae. aegypti, respectively. There was a loss of efficacy of permethrin-treated clothing after use in the field, with 3 min knockdown rates of Ae. albopictus and 1 h knockdown of Ae. aegypti decreasing over time. Participants considered all treatments acceptable. Conclusions The portable mosquito coils were highly protective against outdoor biting mosquitoes, although there are safety concerns related to its use. The combination of permethrin-treated clothing and PMD repellent represent an alternative treatment for protection against outdoor-biting mosquitoes.

ABSTRACT Jingmenvirus is a recently identified group of segmented RNA viruses phylogenetically linked with unsegmented Flaviviridae viruses. Primarily identified in various tick genera originating in China, Jingmenvirus geographical distribution has rapidly expanded to cover Africa, South America, Caribbean, and Europe. The identification of Jingmen-related viruses in various mammals, including febrile humans, opens the possibility that Jingmenviruses may be novel tick-borne arboviruses. In this study, we aimed at increasing knowledge of the host range, genetic diversity, and geographical distribution of Jingmenviruses by reporting for the first time the identification of Jingmenviruses associated with Rhipicephalus microplus ticks originating in the French Antilles (Guadeloupe and Martinique islands), with Amblyomma testudinarium ticks in Lao PDR, and with Ixodes ricinus ticks in metropolitan France, and from urine of Pteropus lylei bats in Cambodia. Analyses of the relationships between the different Jingmenvirus genomes resulted in the identification of three main phylogenic subclades, each of them containing both tick-borne and mammal-borne strains, reinforcing the idea that Jingmenviruses may be considered as tick-borne arboviruses. Finally, we estimated the prevalence of Jingmenvirus-like infection using luciferase immunoprecipitation assay screening (LIPS) of asymptomatic humans and cattle highly exposed to tick bites. Among 70 French human, 153 Laotian human, and 200 Caribbean cattle sera tested, only one French human serum was found (slightly) positive, suggesting that the prevalence of Jingmenvirus human and cattle infections in these areas is probably low. IMPORTANCE Several arboviruses emerging as new pathogens for humans and domestic animals have recently raised public health concern and increased interest in the study of their host range and in detection of spillover events. Recently, a new group of segmented Flaviviridae-related viruses, the Jingmenviruses, has been identified worldwide in many invertebrate and vertebrate hosts, pointing out the issue of whether they belong to the arbovirus group. The study presented here combined whole-genome sequencing of three tick-borne Jingmenviruses and one bat-borne Jingmenvirus with comprehensive phylogenetic analyses and high-throughput serological screening of human and cattle populations exposed to these viruses to contribute to the knowledge of Jingmenvirus host range, geographical distribution, and mammalian exposure.

Abstract 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.

We investigated the cause of seasonal outbreaks of pediatric acute encephalitis-like syndrome associated with litchi harvests (May–July) in northern Vietnam since 2008. Nineteen cerebrospinal fluid samples were positive for human enterovirus B, and 8 blood samples were positive for hypoglycemic toxins present in litchi fruits. Patients who were positive for hypoglycemic toxins had shorter median times between disease onset and admission, more reports of seizures, more reports of hypoglycemia (glucose level

Since its first detection in 1979, dengue fever has been considered a major public health issue in the Lao People’s Democratic Republic (PDR). Dengue virus (DENV) serotype 1 was the cause of an epidemic in 2010–2011. Between 2012 and 2020, major outbreaks due successively to DENV-3, DENV-4 and recently DENV-2 have been recorded. However, DENV-1 still co-circulated in the country over this period. Here, we summarize epidemiological and molecular data of DENV-1 between 2016 and 2020 in the Lao PDR. Our data highlight the continuous circulation of DENV-1 in the country at levels ranging from 16% to 22% among serotyping tests. In addition, the phylogenetic analysis has revealed the circulation of DENV-1 genotype I at least since 2008 with a co-circulation of different clusters. Sequence data support independent DENV-1 introductions in the Lao PDR correlated with an active circulation of this serotype at the regional level in Southeast Asia. The maintenance of DENV-1 circulation over the last ten years supports a low level of immunity against this serotype within the Lao population. Thereby, the risk of a DENV-1 epidemic cannot be ruled out in the future, and this emphasizes the importance of maintaining an integrated surveillance approach to prevent major outbreaks.

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

Abstract 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.

We screened 768 tick pools containing 6,962 ticks from Khammouan Province, Laos, by using quantitative real-time PCR and identified Rickettsia spp., Ehrlichia spp., and Borrelia spp. Sequencing of Rickettsia spp.–positive and Borrelia spp.–positive pools provided evidence for distinct genotypes. Our results identified bacteria with human disease potential in ticks in Laos.

Dengue outbreaks have regularly been recorded in Lao People’s Democratic Republic (PDR) since the first detection of the disease in 1979. In 2012, an integrated arbovirus surveillance network was set up in Lao PDR and an entomological surveillance has been implemented since 2016 in Vientiane Capital. Here, we report a study combining epidemiological, phylogenetic, and entomological analyzes during the largest DENV-4 epidemic ever recorded in Lao PDR (2015–2019). Strikingly, from 2015 to 2019, we reported the DENV-4 emergence and spread at the country level after two large epidemics predominated by DENV-3 and DENV-1, respectively, in 2012–2013 and 2015. Our data revealed a significant difference in the median age of the patient infected by DENV-4 compared to the other serotypes. Phylogenetic analysis demonstrated the circulation of DENV-4 Genotype I at the country level since at least 2013. The entomological surveillance showed a predominance of Aedesaegypti compared to Aedesalbopictus and high abundance of these vectors in dry and rainy seasons between 2016 and 2019, in Vientiane Capital. Overall, these results emphasized the importance of an integrated approach to evaluate factors, which could impact the circulation and the epidemiological profile of dengue viruses, especially in endemic countries like Lao PDR.

Since the end of the 1990s, unexplained outbreaks of acute encephalitis in children coinciding with litchi harvesting (May–July) have been documented in the Bac Giang Province in northern Vietnam. A retrospective ecologic analysis of data for 2004–2009 involving environmental, agronomic, and climatic factors was conducted to investigate the suspected association between the outbreaks and litchi harvesting. The clinical, biological, and immunologic characteristics of the patients suggested a viral etiology. The ecologic study revealed an independent association between litchi plantation surface proportion and acute encephalitis incidence: Incidence rate ratios were 1.52 (95% CI 0.90–2.57), 2.94 (95% CI 1.88–4.60), and 2.76 (95% CI 1.76–4.32) for second, third, and fourth quartiles, respectively, compared with the lowest quartile. This ecologic study confirmed the suspected association between incidence of acute encephalitis and litchi plantations and should be followed by other studies to identify the causative agent for this syndrome.

The “Programme Meso-Vietnam” was implemented in Vietnam from October 2007 to December 2010 to reduce dengue and dengue hemorrhagic fever incidence and to improve the quality of life in the four project communes. This dengue control project was based on biological control using Mesocyclops, a larvivorous micro-crustacean, as well as on the establishment of a strong community involvement to educate the population on dengue transmission and to reduce Aedes breeding by removal of containers. During three years, a network of collaborators was responsible for introducing Mesocyclops in all the containers defined as key breeding-sites, and regular activities such as community training workshops, school programmes, clean-up campaigns and health promotion through IEC programmess were performed. To make this programme sustainable, local leadership has been strengthened as well. The use of these larvivorous micro-crustaceans as a biological control strategy against dengue over the medium and long term, when combined with community participation and effective health promotion, demonstrated a significant reduction in dengue vector populations and dengue cases. In Vietnam, this study reconfirms that there is an ecological alternative to systematic use of insecticides to control dengue vector population. (Abstract word count: 186)