Bat coronaviruses related to SARS-CoV-2 and infectious for human cells.

The animal reservoir of SARS-CoV-2 is unknown despite reports of SARS-CoV-2-related viruses in Asian Rhinolophus bats1-4, including the closest virus from R. affinis, RaTG13 (refs. 5,6), and pangolins7-9. SARS-CoV-2 has a mosaic genome, to which different progenitors contribute. The spike sequence determines the binding affinity and accessibility of its receptor-binding domain to the cellular angiotensin-converting enzyme 2 (ACE2) receptor and is responsible for host range10-12. SARS-CoV-2 progenitor bat viruses genetically close to SARS-CoV-2 and able to enter human cells through a human ACE2 (hACE2) pathway have not yet been identified, although they would be key in understanding the origin of the epidemic. Here we show that such viruses circulate in cave bats living in the limestone karstic terrain in northern Laos, in the Indochinese peninsula. We found that the receptor-binding domains of these viruses differ from that of SARS-CoV-2 by only one or two residues at the interface with ACE2, bind more efficiently to the hACE2 protein than that of the SARS-CoV-2 strain isolated in Wuhan from early human cases, and mediate hACE2-dependent entry and replication in human cells, which is inhibited by antibodies that neutralize SARS-CoV-2. None of these bat viruses contains a furin cleavage site in the spike protein. Our findings therefore indicate that bat-borne SARS-CoV-2-like viruses that are potentially infectious for humans circulate in Rhinolophus spp. in the Indochinese peninsula.

How microbiomes influence metazoan development: insights from history and Drosophila modeling of gut-microbe interactions.

Since Metchnikoff developed his views on the intestinal microflora, much effort has been devoted to understanding the role of gut microbiomes in metazoan physiology. Despite impressive data sets that have been generated by associating a phenotype-causing commensal community with its corresponding host phenotype, the field continues to suffer from descriptive and often contradictory reports. Hence, we cannot yet draw clear conclusions as to how the modifications of microbiomes cause physiological changes in metazoans. Unbiased, large-scale genetic screens to identify key genes, on both microbial and host sides, will be essential to gain mechanistic insights into gut-microbe interactions. The Drosophila genome-commensal microbiome genetic model has proven to be well suited to dissect the complex reciprocal cross talk between the host and its microbiota. In this review, we present a historical account, current views, and novel perspectives for future research directions based on the insights gleaned from the Drosophila gut-microbe interaction model.

Detection of pyrethroid resistance mutations and intron variants in the voltage-gated sodium channel of Aedes (Stegomyia) aegypti and Aedes (Stegomyia) albopictus mosquitoes from Lao People's Democratic Republic.

In Lao People's Democratic Republic, Aedes aegypti (Linnaeus 1762) and Aedes albopictus (Skuse 1894) mosquitoes (Diptera: Culicidae) are vectors of arboviral diseases such as dengue. As the treatment for these diseases is limited, control of the vectors with the use of pyrethroid insecticides is still essential. However, mutations in the voltage-gated sodium channel (vgsc) gene giving rise to pyrethroid resistance are threatening vector control programs. Here, we analysed both Ae. aegypti and Ae. albopictus mosquitoes, which were collected in different districts of Laos (Kaysone Phomvihane, Vangvieng, Saysettha and Xaythany), for vgsc mutations commonly found throughout Asia (S989P, V1016G and F1534C). Sequences of the vgsc gene showed that the F1534C mutation was prevalent in both Aedes species. S989P and V1016G mutations were detected in Ae. aegypti from each site and were always found together. In addition, the mutation T1520I was seen in Ae. albopictus mosquitoes from Saysettha district as well as in all Ae. aegypti samples. Thus, mutations in the vgsc gene of Ae. aegypti are prevalent in the four districts studied indicating growing insecticide resistance throughout Laos. Constant monitoring programmes and alternative strategies for controlling Aedes should be utilized in order to prolong the effectiveness of pyrethroids thereby maximizing vector control.

New insights into malaria vector bionomics in Lao PDR: a nationwide entomology survey.

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.

Description of a new species of Dermacentor Koch, 1844 (Acari: Ixodidae) from the mountains of Laos and Thailand.

Dermacentor pasteuri n. sp. (Acari: Ixodidae) is described based on adults ex wild boar and vegetation from Laos and Thailand. Adults of D. pasteuri n. sp. are similar to those of D. compactus Neumann, 1901, but can be distinguished by the shape of conscutum in the male, development of dorsal cornua in the female, size and shape of spurs on coxae and coloration of leg segments in both sexes.

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.

Description of a new species of Dermacentor Koch, 1844 (Acari: Ixodidae) from Laos and Thailand.

Dermacentor laothaiensis n. sp. (Acari: Ixodidae) is described based on adults ex wild boar and vegetation from Laos and Thailand. Adults of D. laothaiensis n. sp. are similar to those of D. bellulus (Schulze, 1935) and D. steini (Schulze, 1933) but can be distinguished by the conscutum shape, colour pattern of the conscutum and scutum, the density of punctations on the pseudoscutum and scutum and the shape of female genital structures.

Hypoglycemic Toxins and Enteroviruses as Causes of Outbreaks of Acute Encephalitis-Like Syndrome in Children, Bac Giang Province, Northern Vietnam.

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 <3 mmol/L), lower median numbers of leukocytes in cerebrospinal fluid, and higher median serum levels of alanine aminotransferase and aspartate transaminase than did patients who were positive for enteroviruses. We suggest that children with rapidly progressing acute encephalitis-like syndrome at the time of the litchi harvest have intoxication caused by hypoglycemic toxins, rather than viral encephalitis, as previously suspected. These children should be urgently treated for life-threatening hypoglycemia.

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.

Large-Scale Survey for Tickborne Bacteria, Khammouan Province, Laos.

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.

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.

Detection of chikungunya virus antigen by a novel rapid immunochromatographic test.

Chikungunya fever is a mosquito-borne disease of key public health importance in tropical and subtropical countries. Although severe joint pain is the most distinguishing feature of chikungunya fever, diagnosis remains difficult because the symptoms of chikungunya fever are shared by many pathogens, including dengue fever. The present study aimed to develop a new immunochromatographic diagnosis test for the detection of chikungunya virus antigen in serum. Mice were immunized with isolates from patients with Thai chikungunya fever, East/Central/South African genotype, to produce mouse monoclonal antibodies against chikungunya virus. Using these monoclonal antibodies, a new diagnostic test was developed and evaluated for the detection of chikungunya virus. The newly developed diagnostic test reacted with not only the East/Central/South African genotype but also with the Asian and West African genotypes of chikungunya virus. Testing of sera from patients suspected to have chikungunya fever in Thailand (n = 50), Laos (n = 54), Indonesia (n = 2), and Senegal (n = 6) revealed sensitivity, specificity, and real-time PCR (RT-PCR) agreement values of 89.4%, 94.4%, and 91.1%, respectively. In our study using serial samples, a new diagnostic test showed high agreement with the RT-PCR within the first 5 days after onset. A rapid diagnostic test was developed using mouse monoclonal antibodies that react with chikungunya virus envelope proteins. The diagnostic accuracy of our test is clinically acceptable for chikungunya fever in the acute phase.

MALACOLOGICAL INVESTIGATION OF THE FULLY OPERATIONAL NAM THEUN 2 HYDROELECTRIC DAM PROJECT IN KHAMMOUANE PROVINCE, CENTRAL LAO PDR.

We conducted a malacological investigation in four districts of the Nam Theun 2 (NT2) hydroelectric dam project area, Khammouane Province, central Lao PDR (Nakai, Gnommalath, Mahaxai and Xe Bang Fai), after the first and second years of full operation in March 2010 and November 2011 to determine health risks for humans. A total 10,863 snail specimens (10 families/23 species) from 57 sampling stations and 12,902 snail specimens (eight families/21 species) from 66 sampling stations were collected in 2010 and 2011, respectively. Neotricula aperta (gamma race), the intermediate host for Schistosoma mekongi, was found in large numbers (5,853 specimens) in 2010 in Nam Gnom (downstream) at Station 25 (Mueang Gnommalath: Gnommalath District) and in fewer numbers (170 specimens) at Station 26 (Ban Thathod: Gnommalath District). In 2011, significantly fewer numbers (434 specimens) of N. aperta were found at Station 25. No snails were found to be infected with S. mekongi; however, 3.6% and 0.45% of Bithynia (D.). s. goniomphalos specimens collected were found to be infected with Opisthorchis viverrini (human liver fluke) during 2010 and 2011, respectively. Pomacea canaliculata, the rice crop pest, the intermediate host of Angiostrongylus (Parastrongylus) cantonensis, was found in the greatest numbers during 2010 and 2011; the prevalence increased significantly from 1.3% in 2010 to 53.3% in 2011. We also found seasonal variation in snail populations in terms of abundance and diversity. The snail fauna and risk for transmission of parasitic diseases need to be monitored continuously to evaluate the long-term impact of the dam project.

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.

Impact of mosquito bites on asexual parasite density and gametocyte prevalence in asymptomatic chronic Plasmodium falciparum infections and correlation with IgE and IgG titers.

An immunomodulatory role of arthropod saliva has been well documented, but evidence for an effect on Plasmodium sp. infectiousness remains controversial. Mosquito saliva may orient the immune response toward a Th2 profile, thereby priming a Th2 response against subsequent antigens, including Plasmodium. Orientation toward a Th1 versus a Th2 profile promotes IgG and IgE proliferation, respectively, where the former is crucial for the development of an efficient antiparasite immune response. Here we assessed the direct effect of mosquito bites on the density of Plasmodium falciparum asexual parasites and the prevalence of gametocytes in chronic, asymptomatic infections in a longitudinal cohort study of seasonal transmission. We additionally correlated these parasitological measures with IgE and IgG antiparasite and anti-salivary gland extract titers. The mosquito biting density was positively correlated with the asexual parasite density but not asexual parasite prevalence and was negatively correlated with gametocyte prevalence. Individual anti-salivary gland IgE titers were also negatively correlated with gametocyte carriage and were strongly positively correlated with antiparasite IgE titers, consistent with the hypothesis that mosquito bites predispose individuals to develop an IgE antiparasite response. We provide evidence that mosquito bites have an impact on asymptomatic infections and differentially so for the production of asexual and sexual parasites. An increased research focus on the immunological impact of mosquito bites during asymptomatic infections is warranted, to establish whether strategies targeting the immune response to saliva can reduce the duration of infection and the onward transmission of the parasite.