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Recovered COVID-19 patients may test positive for SARS-CoV-2 for a long time from intermittent shedding of viral fragments. A 36-year-old man who tested positive for SARS-CoV-2 in the Czech Republic and recovered tested positive again in Bhutan, 105 days beyond his first positive test. He experienced minimal symptoms and recovered without complications. Although no virological test was conducted to rule out reinfection, the repeat positive test after initial recovery likely resulted from prolonged shedding of dead viral particles than a reinfection.

This report describes the MalariaGEN Pv4 dataset, a new release of curated genome variation data on 1,895 samples of Plasmodium vivax collected at 88 worldwide locations between 2001 and 2017. It includes 1,370 new samples contributed by MalariaGEN and VivaxGEN partner studies in addition to previously published samples from these and other sources. We provide genotype calls at over 4.5 million variable positions including over 3 million single nucleotide polymorphisms (SNPs), as well as short indels and tandem duplications. This enlarged dataset highlights major compartments of parasite population structure, with clear differentiation between Africa, Latin America, Oceania, Western Asia and different parts of Southeast Asia. Each sample has been classified for drug resistance to sulfadoxine, pyrimethamine and mefloquine based on known markers at the dhfr, dhps and mdr1 loci. The prevalence of all of these resistance markers was much higher in Southeast Asia and Oceania than elsewhere. This open resource of analysis-ready genome variation data from the MalariaGEN and VivaxGEN networks is driven by our collective goal to advance research into the complex biology of P. vivax and to accelerate genomic surveillance for malaria control and elimination.

Since 2014, mountain communities in Ladakh, India have been constructing dozens of Artificial Ice Reservoirs (AIRs) by spraying water through fountain systems every winter. The meltwater from these structures is crucial to meet irrigation water demands during spring. However, there is a large variability associated with this water supply due to the local weather influences at the chosen location. This study compared the ice volume evolution of an AIR built in Ladakh, India with two others built in Guttannen, Switzerland using a surface energy balance model. Model input consisted of meteorological data in conjunction with fountain discharge rate (mass input of an AIR). Model calibration and validation were completed using ice volume and surface area measurements taken from several drone surveys. The model was successful in estimating the observed ice volume evolution with a root mean square error within 18% of the maximum ice volume for all the AIRs. The location in Ladakh had a maximum ice volume four times larger compared to the Guttannen site. However, the corresponding water losses for all the AIRs were more than three-quarters of the total fountain discharge due to high fountain wastewater. Drier and colder locations in relatively cloud-free regions are expected to produce long-lasting AIRs with higher maximum ice volumes. This is a promising result for dry mountain regions, where AIR technology could provide a relatively affordable and sustainable strategy to mitigate climate change induced water stress.

Rabies is one of the most important zoonotic diseases that mostly affect children. We conducted a rabies education among 129 secondary school children (intervention group = 94 students, control group = 35 students) in two schools in southeast Bhutan and evaluated the effectiveness of the lesson by comparing the knowledge, perception and safety behaviour score about rabies before and after education. We also assessed the knowledge retention capacity of the students at three months post intervention. Our findings indicated that short rabies lesson significantly (P

In 2011, Bhutan’s Royal Centre for Disease Control began Japanese encephalitis (JE) surveillance at 5 sentinel hospitals throughout Bhutan. During 2011–2018, a total of 20 JE cases were detected, indicating JE virus causes encephalitis in Bhutan. Maintaining JE surveillance will help improve understanding of JE epidemiology in this country.

ABSTRACTDengue is an important emerging vector-borne disease in Bhutan. This study aimed to quantify the spatial and temporal patterns of dengue and their relationship to environmental factors in dengue-affected areas at the sub-district level. A multivariate zero-inflated Poisson regression model was developed using a Bayesian framework with spatial and spatiotemporal random effects modelled using a conditional autoregressive prior structure. The posterior parameters were estimated using Bayesian Markov Chain Monte Carlo simulation with Gibbs sampling. A total of 708 dengue cases were notified through national surveillance between January 2016 and June 2019. Individuals aged ≤14 years were found to be 53% (95% CrI: 42%, 62%) less likely to have dengue infection than those aged >14 years. Dengue cases increased by 63% (95% CrI: 49%, 77%) for a 1°C increase in maximum temperature, and decreased by 48% (95% CrI: 25%, 64%) for a one-unit increase in normalized difference vegetation index (NDVI). There was significant residual spatial clustering after accounting for climate and environmental variables. The temporal trend was significantly higher than the national average in eastern sub-districts. The findings highlight the impact of climate and environmental variables on dengue transmission and suggests prioritizing high-risk areas for control strategies.

Shigella sonnei is one of the main species causing shigellosis worldwide. Here the authors analyse nearly 400 S. sonnei genome sequences and carry out experimental evolution experiments to shed light into the evolutionary processes underlying the recent emergence of fluoroquinolone resistance in this pathogen.

The snow leopard (Panthera uncia) is one of the world's most elusive felids. In Bhutan, which is one of the 12 countries where the species still persists, reliable information on its distribution and habitat suitability is lacking, thus impeding effective conservation planning for the species. To fill this knowledge gap, we created a country-wide species distribution model using “presence-only” data from 420 snow leopard occurrences (345 from a sign survey and 77 from a camera-trapping survey) and 12 environmental covariates consisting of biophysical and anthropogenic factors. We analyzed the data in an ensemble model framework which combines the outputs from several species distribution models. To assess the adequacy of Bhutan's network of protected areas and their potential contribution toward the conservation of the species, we overlaid the output of the ensemble model on the spatial layers of protected areas and biological corridors. The ensemble model identified 7,206 km2 of Bhutan as suitable for the snow leopard: 3,647 km2 as highly suitable, 2,681 km2 as moderately suitable, and 878 km2 as marginally suitable. Forty percent of the total suitable habitat consisted of protected areas and a further 8% of biological corridors. These suitable habitats were characterized by a mean livestock density of 1.3 individuals per hectare, and a mean slope of 25°; they closely match the distribution of the snow leopard's main wild prey, the bharal (Pseudois nayaur). Our study shows that Bhutan's northern protected areas are a centre for snow leopard conservation both at the national and regional scale.

Against the background of climate change-induced glacier melting, numerous glacial lakes are formed across high mountain areas worldwide. Existing glacial lake inventories, chiefly created using Landsat satellite imagery, mainly relate to 1990 onwards and relatively long (decadal) temporal scales. Moreover, there is a lack of robust information on the expansion and the GLOF hazard status of glacial lakes in the Bhutan Himalaya. We mapped Bhutanese glacial lakes from the 1960s to 2020, and used these data to determine their distribution patterns, expansion behavior, and GLOF hazard status. 2,187 glacial lakes (corresponding to 130.19 ± 2.09 km2) were mapped from high spatial resolution (1.82–7.62 m), Corona KH-4 images from the 1960s. Using the Sentinel-2 (10 m) and Sentinel-1 (20 m × 22 m), we mapped 2,553 (151.81 ± 7.76 km2), 2,566 (152.64 ± 7.83 km2), 2,572 (153.94 ± 7.83 km2), 2,569 (153.97 ± 7.79 km2) and 2,574 (156.63 ± 7.95 km2) glacial lakes in 2016, 2017, 2018, 2019 and 2020, respectively. The glacier-fed lakes were mainly present in the Phochu (22.63%) and the Kurichu (20.66%) basins. A total of 157 glacier-fed lakes have changed into non-glacier-fed lakes over the 60 years of lake evolution. Glacier-connected lakes (which constitutes 42.25% of the total glacier-fed lake) area growth accounted for 75.4% of the total expansion, reaffirming the dominant role of glacier-melt water in expanding glacial lakes. Between 2016 and 2020, 19 (4.82 km2) new glacial lakes were formed with an average annual expansion rate of 0.96 km2 per year. We identified 31 lakes with a very-high and 34 with high GLOF hazard levels. These very-high to high GLOF hazard lakes were primarily located in the Phochu, Kurichu, Drangmechu, and Mochu basins. We concluded that the increasing glacier melt is the main driver of glacial lake expansion. Our results imply that extending glacial lakes studies back to the 1960s provides new insights on glacial lake evolution from glacier-fed lakes to non-glacier-fed lakes. Additionally, we reaffirmed the capacity of Sentinel-1 and Sentinel-2 data to determine annual glacial lake changes. The results from this study can be a valuable basis for future glacial lake monitoring and prioritizing limited resources for GLOF mitigation programs.

Vaccination remains a key public health intervention against the COVID-19 pandemic. However, vaccine distribution and coverage are variable between countries due to access and implementation issues. Vaccine inequity was evident with some countries having no access to the vaccines while others have initiated multiple booster doses. We share Bhutan’s approach to COVID-19 vaccination and lessons learned during the successful conduct of a nationwide vaccination program. As of 12 December 2021, 80.3% of the Bhutanese population have received at least one dose of COVID-19 vaccine and 77.0% have received at least two doses. Considering age groups, 97.2% of adults (18 years) have received at least one dose and 93.6% have received at least two doses. The first dose coverage for the adolescents 12–17 years was 99.7% and second dose coverage was 92.3% since some were not yet due for their second dose at the time of writing this report. The well-established existing national immunization program was especially useful in the implementation of the national COVID-19 vaccination program. The Bhutan Vaccine System, a digital platform for registration and monitoring of vaccination, was rapidly developed and extensively utilized during the campaign. The selfless leadership of the king, the government, and prior detailed planning with multi-sectoral collaboration and coordination, was the key in this exemplary vaccination program. Bhutan has successfully vaccinated children between 5–11 years with high coverage and no serious issues. Many adults have also received first and second booster doses, based on their risks and preferences.

Abstract Background Malaria causes a reduction in haemoglobin that is compounded by primaquine, particularly in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. The aim of this study was to determine the relative contributions to red cell loss of malaria and primaquine in patients with uncomplicated Plasmodium vivax. Methods A systematic review identified P. vivax efficacy studies of chloroquine with or without primaquine published between January 2000 and March 2017. Individual patient data were pooled using standardised methodology, and the haematological response versus time was quantified using a multivariable linear mixed effects model with non-linear terms for time. Mean differences in haemoglobin between treatment groups at day of nadir and day 42 were estimated from this model. Results In total, 3421 patients from 29 studies were included: 1692 (49.5%) with normal G6PD status, 1701 (49.7%) with unknown status and 28 (0.8%) deficient or borderline individuals. Of 1975 patients treated with chloroquine alone, the mean haemoglobin fell from 12.22 g/dL [95% CI 11.93, 12.50] on day 0 to a nadir of 11.64 g/dL [11.36, 11.93] on day 2, before rising to 12.88 g/dL [12.60, 13.17] on day 42. In comparison to chloroquine alone, the mean haemoglobin in 1446 patients treated with chloroquine plus primaquine was − 0.13 g/dL [− 0.27, 0.01] lower at day of nadir (p = 0.072), but 0.49 g/dL [0.28, 0.69] higher by day 42 (p 25% to 5 g/dL. Conclusions Primaquine has the potential to reduce malaria-related anaemia at day 42 and beyond by preventing recurrent parasitaemia. Its widespread implementation will require accurate diagnosis of G6PD deficiency to reduce the risk of drug-induced haemolysis in vulnerable individuals. Trial registration This trial was registered with PROSPERO: CRD42016053312. The date of the first registration was 23 December 2016.

The Kangchenjunga Landscape (KL) in the Eastern Himalayas is a transboundary complex shared by Bhutan, India, and Nepal. It forms a part of the ‘Himalayan Biodiversity Hotspot’ and is one of the biologically richest landscapes in the Eastern Himalayas. In this paper, we use secondary information to review and consolidate the knowledge on the flora of the KL. We reviewed 215 journal articles, analysed the history of publications on the flora of the KL, their publication pattern in terms of temporal and spatial distribution and key research areas. Our review shows that the landscape has a long history of botanical research that dates back to the 1840s and progressed remarkably after the 1980s. Most of the studies have been carried out in India, followed by Nepal and Bhutan. The majority of these have been vegetation surveys, followed by research on ethnobotanical aspects and Non-Timber Forest Products (NTFPs). This paper describes the forest types and characteristic species of the KL and details the species richness, diversity and dominant families of seed plants. A total of 5198 species of seed plants belonging to 1548 genera and 216 families have been recorded from the landscape, including 3860 dicots, 1315 monocots and 23 gymnosperms. Among families, Orchidaceae is the most diversely represented family in terms of species richness. This paper also draws attention to the threatened and endemic flora of the KL, including 44 species that are threatened at national and global level and 182 species that are endemic. Finally, the paper reviews the major challenges facing the KL, the conservation efforts and practices that are currently in place and recommends systematic and comprehensive floral surveys, particularly long-term data collection and monitoring and transboundary collaboration, to address the existing knowledge gaps on floral diversity of the KL. Keywords: Floral diversity, Transboundary landscape, Nepal, India, Bhutan, Biodiversity

Climate change, increasing population and changes in land use are all rapidly driving the need to be able to better understand surface water dynamics. The targets set by the United Nations under Sustainable Development Goal 6 in relation to freshwater ecosystems also make accurate surface water monitoring increasingly vital. However, the last decades have seen a steady decline in in situ hydrological monitoring and the availability of the growing volume of environmental data from free and open satellite systems is increasingly being recognized as an essential tool for largescale monitoring of water resources. The scientific literature holds many promising studies on satellite-based surface-water mapping, but a systematic evaluation has been lacking. Therefore, a round robin exercise was organized to conduct an intercomparison of 14 different satellite-based approaches for monitoring inland surface dynamics with Sentinel-1, Sentinel-2, and Landsat 8 imagery. The objective was to achieve a better understanding of the pros and cons of different sensors and models for surface water detection and monitoring. Results indicate that, while using a single sensor approach (applying either optical or radar satellite data) can provide comprehensive results for very specific localities, a dual sensor approach (combining data from both optical and radar satellites) is the most effective way to undertake largescale national and regional surface water mapping across bioclimatic gradients.

Glacial lakes pose a serious threat to downstream areas and significantly impact glacier melt. The number and area of lakes has grown in most regions during the last decades due to the ongoing atmospheric warming and retreating glaciers. It is therefore important to identify and monitor these lakes. However, mapping of glacial lakes in alpine regions is challenged by many factors. These factors include the small size of glacial lakes, cloud cover in optical satellite images, cast shadows from mountains and clouds, seasonal snow in satellite images, varying degrees of turbidity amongst glacial lakes, and frozen glacial lake surfaces. In our study, we have developed a fully automated method for mapping glacial lakes across alpine regions including the Python package called “GLakeMap”. The method uses multi-source data such as Sentinel-1 Synthetic Aperture Radar and Sentinel-2 Multi-spectral Instrument data, a digital elevation model, and a random forest classifier model. We use multi-source datasets as inputs for rule-based segmentation of images, mainly aiming at extracting glacial lake objects from satellite images using a set of rules. Segmented objects are then classified either as glacial lake or non-glacial lake objects by the random forest classifier model. The method was tested in eight sites across alpine regions mainly located in High Mountain Asia but also in the Alps and the Andes. We show that the proposed method overcomes a majority of the aforementioned challenges to detect and delineate glacial lakes. The method performs efficiently irrespective of geographic, geologic, and climatic conditions of glacial lakes.

Dengue continues to pose a significant public health problem in tropical and subtropical countries. In Bhutan, first outbreak of dengue fever (DF) was reported in 2004 in a southern border town, followed by sporadic cases over the years. In this study, we analysed DF outbreaks that occurred in 3 different places during the years 2016 and 2017. A total of 533 cases in 2016 and 163 in 2017 were suspected of having of DF, where young adults were mostly affected. A total of 240 acute serum specimens collected and analyzed for serotype by nested RT-PCR revealed predominance of serotypes 1 and 2 (DENV-1 and 2). Phylogenetic analysis using envelope gene for both the serotypes demonstrated cosmopolitan genotype which were closely related to strains from India, indicating that they were probably imported from the neighboring country over the past few years.

Abstract Background Influenza disease burden varies by age and this has important public health implications. We compared the proportional distribution of different influenza virus types within age strata using surveillance data from twenty-nine countries during 1999-2014 (N=358,796 influenza cases). Methods For each virus, we calculated a Relative Illness Ratio (defined as the ratio of the percentage of cases in an age group to the percentage of the country population in the same age group) for young children (0-4 years), older children (5-17 years), young adults (18-39 years), older adults (40-64 years), and the elderly (65+ years). We used random-effects meta-analysis models to obtain summary relative illness ratios (sRIRs), and conducted meta-regression and sub-group analyses to explore causes of between-estimates heterogeneity. Results The influenza virus with highest sRIR was A(H1N1) for young children, B for older children, A(H1N1)pdm2009 for adults, and (A(H3N2) for the elderly. As expected, considering the diverse nature of the national surveillance datasets included in our analysis, between-estimates heterogeneity was high (I2>90%) for most sRIRs. The variations of countries’ geographic, demographic and economic characteristics and the proportion of outpatients among reported influenza cases explained only part of the heterogeneity, suggesting that multiple factors were at play. Conclusions These results highlight the importance of presenting burden of disease estimates by age group and virus (sub)type.

Background: A cluster of suspected shigellosis was reported from health center in Pemagatshel district to Royal Center for Disease Control on 14th May 2012. The investigation was done to determine the cause and risk factor for the outbreak so that appropriate control and prevention measures can be implemented. Methods: A descriptive study was used for the outbreak investigation. The food items and drinks served to boarding students were collected from the mess in-charge in order to assess their risk for the outbreak. The kitchen and its premises were inspected to study the likely contamination by rodents and other animals. The water and stool specimens were tested in the laboratory to identify all possible enteric pathogens. Results: 82 boarding students were affected with an overall attack rate of 28% (82/294). Diarrhea was the predominant symptom followed by abdominal pain and headache. The onset date of the cases varied between 11th and 18th May, 2012. Shigella species was isolated from stool specimens that showed resistant to amoxicillin, nalidixic acid, chloramphenicol and sulfamethoxazole. Water specimen collected from source, distribution reservoir and tap water at school were found grossly contaminated. Conclusion: The outbreak was caused by novel strain of Shigella species which was not detected earlier in Bhutan. The promotion and provision of boiled water will greatly reduce the incidence of shigellosis especially in boarding facility.

Abstract The study objectives were to gather feedback and opinions of goat farmers on the Forest and Nature Conservation Rule on goat rearing in Bhutan and identify field constraints arising from the conservation rule. Focus group and individual farmer survey methods were employed, and a semi-structured questionnaire was used to interview 180 goat farmers of six districts. All respondents were aware of the conservation rule. Majority of respondents knew goats as a threat to forest and crops. The vast majority of respondents felt that the conservation rule is not relevant in modern times, and all respondents felt the need to revise the rule. The main problem faced by farmers was difficulty in maintaining their goat numbers to four. The other problems faced were frequent conflicts with Forest personnel and restricted opportunities to earn more income. While the expected changes in the conservation rule included provisions to allow a farmer to rear more number of goats under stall-fed conditions, the additional rule suggested by farmers was allowing goats to browse freely in the forest. Majority of farmers reared goats under stall-fed conditions. The most common practice of managing goat populations was selling goats to fellow farmers. Despite the constraints, a majority of farmers expressed their willingness to continue goat rearing in the future, mainly to generate more income. The study findings suggest revision of the rule on goat rearing, with strong consideration of the needs of the modern farming system and growing economic demands. In revising the rule, the study recommends balanced representation from stakeholders and technical experts from both forest and livestock disciplines.

We describe the epidemiological characteristics, pattern of circulation, and geographical distribution of influenza B viruses and its lineages using data from the Global Influenza B Study. We included over 1.8 million influenza cases occurred in thirty-one countries during 2000-2018. We calculated the proportion of cases caused by influenza B and its lineages; determined the timing of influenza A and B epidemics; compared the age distribution of B/Victoria and B/Yamagata cases; and evaluated the frequency of lineage-level mismatch for the trivalent vaccine. The median proportion of influenza cases caused by influenza B virus was 23.4%, with a tendency (borderline statistical significance, p = 0.060) to be higher in tropical vs. temperate countries. Influenza B was the dominant virus type in about one every seven seasons. In temperate countries, influenza B epidemics occurred on average three weeks later than influenza A epidemics; no consistent pattern emerged in the tropics. The two B lineages caused a comparable proportion of influenza B cases globally, however the B/Yamagata was more frequent in temperate countries, and the B/Victoria in the tropics (p = 0.048). B/Yamagata patients were significantly older than B/Victoria patients in almost all countries. A lineage-level vaccine mismatch was observed in over 40% of seasons in temperate countries and in 30% of seasons in the tropics. The type B virus caused a substantial proportion of influenza infections globally in the 21st century, and its two virus lineages differed in terms of age and geographical distribution of patients. These findings will help inform health policy decisions aiming to reduce disease burden associated with seasonal influenza.

The transboundary landscape approach builds on principles of integrated social-ecological systems with conservation and development perspectives at a transboundary level. The evolution of one such transboundary landscape in the Hindu Kush Himalaya (HKH) from the 1990's to the present is discussed through a phase-wise process. Both global and regional discourses have been influential in designing the transboundary Kangchenjunga Landscape which is shared by Bhutan, India and Nepal. The 25,085 sq.km landscape ranges in elevation from 40 to 8,586 m asl and is home to more than seven million people, while hosting more than 4,500 species of plants and at least 169 mammal and 618 bird species. With the aim of conserving and managing the landscape for sustained ecosystem services that ultimately contribute to the livelihoods of the women and men residing therein, the Kangchenjunga Landscape Conservation and Development Initiative was implemented since 2016. Lessons from the Kangchenjunga Landscape indicate that participatory and iterative boundary delineation, transboundary cooperation, identification of a lead institution, ensuring the global-local-global feedback cycle, and regional data sharing are key components for implementing transboundary landscape programmes in the region. Keywords: Landscape approach, Ecosystem management, Participatory planning, Regional cooperation, Theory of change

Background: A suspected peripheral neuropathy outbreak was reported from Dechentsemo Central School, Thinleygang, Punakha, following which the investigation team was immediately dispatched in the field. Objective: The aim of investigation was to ascertain the cause and risk factor for the outbreak in order to implement control measures. Methods: A case control study was devised for the investigation to study about the past exposure or deficiencies in order to find out the suspected cause and risk factors. A semi-quantitative food frequency questionnaire was administered to both cases and controls to collect information on the type of food they have consumed. The information garnered was analyzed using Chi-Square or Fischer Exact test for categorical variables and Man-Whitney U-test for quantitative variables. Results: All 17 cases were females with mean age of 13 years (SD 2.7 years). The average daily amount of thiamine intake was 0.6 mg/day for case and 0.8 mg/day for controls against the recommended daily allowance (RDA) of 1.2 mg/day. Case and control patients differed significantly with respect to fat intake (p-value = 0.02), more strongly with folate and iron intake (p-value < 0.01). Conclusion: The outbreak of peripheral neuropathy in Dechentsemo Central School appears to be linked to reduced dietary intake rich in vitamin B1 coupled with low intake of folate and iron in their diet.

Background: A suspected peripheral neuropathy outbreak was reported from Dechentsemo Central School, Thinleygang, Punakha, following which the investigation team was immediately dispatched in the field. Objective: The aim of investigation was to ascertain the cause and risk factor for the outbreak in order to implement control measures. Methods: A case control study was devised for the investigation to study about the past exposure or deficiencies in order to find out the suspected cause and risk factors. A semi-quantitative food frequency questionnaire was administered to both cases and controls to collect information on the type of food they have consumed. The information garnered was analyzed using Chi-Square or Fischer Exact test for categorical variables and Man-Whitney U-test for quantitative variables. Results: All 17 cases were females with mean age of 13 years (SD 2.7 years). The average daily amount of thiamine intake was 0.6 mg/day for case and 0.8 mg/day for controls against the recommended daily allowance (RDA) of 1.2 mg/day. Case and control patients differed significantly with respect to fat intake (p-value = 0.02), more strongly with folate and iron intake (p-value < 0.01). Conclusion: The outbreak of peripheral neuropathy in Dechentsemo Central School appears to be linked to reduced dietary intake rich in vitamin B1 coupled with low intake of folate and iron in their diet.

Introduction: The Widal test is widely used in hospitals in Bhutan for diagnosis of typhoid fever. The right test with high sensitivity and specificity supplements clinical judgement and contributes to correct diagnosis of disease. This study focuses on the contribution of the Widal test in the diagnosis of typhoid fever. Methods: Data was collected from records of patients who presented to Damphu hospital from March 2011 to June 2012 with clinical suspicion of typhoid fever. Blood samples were collected from patients and tested at Damphu Hospital, Tsirang and the Royal Centre for Disease Control, Thimphu. Seventy records were used for the study. Results: There was no growth of Salmonella typhi on blood cultures from patients who had tested positive in the Widal test. There were 20 (28.57%) samples which tested positive for scrub typhus; among these Widal test was positive in 10 (50%) samples. Thirty four out of 36 (94.44%) patients had duration of illness less than seven days and among them 26 (74.47%) had positive Widal test results. Conclusion: None of the samples that tested positive by Widal test gave a definite diagnosis of typhoid fever with blood culture. Clinical judgement may be more challenging because patients with other febrile illnesses like Scrub typhus also have positive Widal test result. We conclude that it is best not to rely on the Widal test alone for the diagnosis of typhoid fever and this test should be replaced by more accurate ones.

Diarrhea is a major cause of morbidity and mortality among Bhutanese children. The etiology of diarrhea is not well known due to the challenges of conducting routine surveillance with Bhutan's modest research facilities. Establishing an etiology is crucial toward generating evidence that will contribute to policy discussions on a diarrheal disease control program. Our previous study, during 2010-2012, revealed that norovirus (NoV) is an important cause of diarrhea among Bhutanese children, and that GII.21 was the major genotype circulating at that time. In other countries, GII.4 is the major genotype responsible for NoV infections. In this update report, we provide new prevalence data to describe the progression of the transformation and distribution of the NoV genotype among Bhutanese children.From June 2013 through May 2014, diarrheal stool samples were collected at one national referral hospital in Thimphu, two regional referral hospitals in the eastern and central regions, and one general hospital in the western region of Bhutan. NoV was detected by reverse transcription-polymerase chain reaction (RT-PCR), by amplifying the capsid gene. The RT-PCR results were confirmed by nucleotide sequencing of the amplicons.The proportion of NoV-positive stool samples was 23.6% (147/623), of which 76.9% were NoV GII and the remainders were NoV GI. The median age of infected children was 15.5 months, with a fairly balanced female: male ratio. NoV GII was most prevalent in the colder months (late November-mid April) and NoV GI had the highest prevalence in the summer (mid April-late September). Nucleotide sequencing was successful in 99 samples of GII strains. The most common genotypes were GII.3 (42.6%), GII.4 Sydney 2012 (15.8%), and GII.4 unassigned (11.9%). No GII.21 was found in any child in the present study. Phylogenetic analysis showed that GII.3 strains in the present study belonged to an independent cluster in lineage B. These strains shared an ancestor with those from different countries and Bhutanese strains circulating during 2010.NoV remains an important cause of diarrhea among Bhutanese children. Genotype GII.3 from a single ancestor strain has spread, replacing the previously circulating GII.21. Current NoV genotypes are similar to the strains circulating worldwide but are primarily related to those in neighboring countries. NoV GII is prevalent during the cold season, while GI is prevalent during the summer. To develop a NoV infection control policy, further studies are needed.

In 2012, chikungunya virus (CHIKV) was reported for the first time in Bhutan. IgM ELISA results were positive for 36/210 patient samples; PCR was positive for 32/81. Phylogenetic analyses confirmed that Bhutan CHIKV belongs to the East/Central/South African genotype. Appropriate responses to future outbreaks require a system of surveillance and improved laboratory capacity.

BackgroundAntimicrobial resistance is a major issue in the Shigellae, particularly as a specific multidrug-resistant (MDR) lineage of Shigella sonnei (lineage III) is becoming globally dominant. Ciprofloxacin is a recommended treatment for Shigella infections. However, ciprofloxacin-resistant S. sonnei are being increasingly isolated in Asia and sporadically reported on other continents. We hypothesized that Asia is a primary hub for the recent international spread of ciprofloxacin-resistant S. sonnei.Methods and findingsWe performed whole-genome sequencing on a collection of 60 contemporaneous ciprofloxacin-resistant S. sonnei isolated in four countries within Asia (Vietnam, n = 11; Bhutan, n = 12; Thailand, n = 1; Cambodia, n = 1) and two outside of Asia (Australia, n = 19; Ireland, n = 16). We reconstructed the recent evolutionary history of these organisms and combined these data with their geographical location of isolation. Placing these sequences into a global phylogeny, we found that all ciprofloxacin-resistant S. sonnei formed a single clade within a Central Asian expansion of lineage III. Furthermore, our data show that resistance to ciprofloxacin within S. sonnei may be globally attributed to a single clonal emergence event, encompassing sequential gyrA-S83L, parC-S80I, and gyrA-D87G mutations. Geographical data predict that South Asia is the likely primary source of these organisms, which are being regularly exported across Asia and intercontinentally into Australia, the United States and Europe. Our analysis was limited by the number of S. sonnei sequences available from diverse geographical areas and time periods, and we cannot discount the potential existence of other unsampled reservoir populations of antimicrobial-resistant S. sonnei.ConclusionsThis study suggests that a single clone, which is widespread in South Asia, is likely driving the current intercontinental surge of ciprofloxacin-resistant S. sonnei and is capable of establishing endemic transmission in new locations. Despite being limited in geographical scope, our work has major implications for understanding the international transfer of antimicrobial-resistant pathogens, with S. sonnei acting as a tractable model for studying how antimicrobial-resistant Gram-negative bacteria spread globally.

INTRODUCTION:Determining the optimal time to vaccinate is important for influenza vaccination programmes. Here, we assessed the temporal characteristics of influenza epidemics in the Northern and Southern hemispheres and in the tropics, and discuss their implications for vaccination programmes. METHODS:This was a retrospective analysis of surveillance data between 2000 and 2014 from the Global Influenza B Study database. The seasonal peak of influenza was defined as the week with the most reported cases (overall, A, and B) in the season. The duration of seasonal activity was assessed using the maximum proportion of influenza cases during three consecutive months and the minimum number of months with ≥80% of cases in the season. We also assessed whether co-circulation of A and B virus types affected the duration of influenza epidemics. RESULTS:212 influenza seasons and 571,907 cases were included from 30 countries. In tropical countries, the seasonal influenza activity lasted longer and the peaks of influenza A and B coincided less frequently than in temperate countries. Temporal characteristics of influenza epidemics were heterogeneous in the tropics, with distinct seasonal epidemics observed only in some countries. Seasons with co-circulation of influenza A and B were longer than influenza A seasons, especially in the tropics. DISCUSSION:Our findings show that influenza seasonality is less well defined in the tropics than in temperate regions. This has important implications for vaccination programmes in these countries. High-quality influenza surveillance systems are needed in the tropics to enable decisions about when to vaccinate.

We identified a new genotype of bufavirus, BuV3, in fecal samples (0.8%) collected to determine the etiology of diarrhea in children in Bhutan. Norovirus GII.6 was detected in 1 sample; no other viral diarrheal pathogens were detected, suggesting BuV3 as a cause of diarrhea. This study investigates genetic diversity of circulating BuVs.

Dengue is one of the most significant public health problems in tropical and subtropical countries, and is increasingly being detected in traditionally non-endemic areas. In Bhutan, dengue virus (DENV) has only recently been detected and limited information is available. In this study, we analyzed the epidemiological and molecular characteristics of DENV in two southern districts in Bhutan from 2013-2014. During this period, 379 patients were clinically diagnosed with suspected dengue, of whom 119 (31.4%) were positive for DENV infection by NS1 ELISA and/or nested RT-PCR. DENV serotypes 1, 2 and 3 were detected with DENV-1 being predominant. Phylogenetic analysis of DENV-1 using envelope gene demonstrated genotype V, closely related to strains from northern India.

A prospective study was performed to determine the molecular characteristics of rotaviruses circulating among children aged

To determine the serotype and genotype of dengue virus (DENV) in Bhutan, we conducted phylogenetic analyses of complete envelope gene sequences. DENV-2 (Cosmopolitan genotype) predominated in 2004, and DENV-3 (genotype III) predominated in 2005–2006; these viruses were imported from India. Primary dengue infections outnumbered secondary infections, suggesting recent emergence.

ABSTRACT In 2018, Bhutan reported 54 cases of malaria, of which six were indigenous, 14 introduced and 34 imported. Considering the continuous reduction in the number of indigenous cases, Bhutan plans to eliminate malaria by 2025 under the Bhutan Malaria Elimination Strategy. The study was conducted to assess the presence of asymptomatic plasmodial infection in both, Bhutanese population living in malaria-risk areas and in migrant workers to guide the elimination strategies. A cross-sectional study was conducted from April to May 2016 in 750 Bhutanese people and 473 migrant workers. Plasmodium falciparum and Plasmodium vivax infections were investigated by using a rapid diagnostic test (RDT) and the polymerase chain reaction (PCR). Prevalence of asymptomatic plasmodial infection based on PCR was 0.27% (95% CI: 0.05–1.07%) among Bhutanese people with a mean age of 43 years old. The proportions of males and females were 45% and 55%, respectively. Among migrant workers, the prevalence of asymptomatic plasmodial infection was 0.42% (95% CI: 0.07–1.69%) with a mean age of 30 years old. The majority of migrant workers were from the neighboring Indian State of West Bengal (57.51%), followed by Assam (12.26%). RDT in both study groups did not detect any plasmodial infection. The presence of a low prevalence of asymptomatic plasmodial infection indicates that the current elimination strategies and interventions are effective.