Characterization of the primary structure of the major silk gene, h-fibroin, across caddisfly (Trichoptera) suborders.

Larvae of caddisflies (Trichoptera) produce silk to build various underwater structures allowing them to exploit a wide range of aquatic environments. The silk adheres to various substrates underwater and has high tensile strength, extensibility, and toughness and is of interest as a model for biomimetic adhesives. As a step toward understanding how the properties of underwater silk evolved in Trichoptera, we used genomic data to identify full-length sequences and characterize the primary structure of the major silk protein, h-fibroin, across the order. The h-fibroins have conserved termini and basic motif structure with high variation in repeating modules and variation in the percentage of amino acids, mainly proline. This finding might be linked to differences in mechanical properties related to the different silk usage and sets a starting point for future studies to screen and correlate amino acid motifs and other sequence features with quantifiable silk properties.

Assessment of exposure to pesticides and the knowledge, attitude and practice among farmers of western Bhutan.

An estimated 69% of the population of Bhutan is engaged in agriculture. Farmers are exposed to a wide variety of pesticides during the preparation, transport, storage, mixing and application of pesticides posing a significant health risk. A controlled cross-sectional study of farmers in selected sites of Bhutan was conducted to characterize the level of exposure to pesticides and assess their knowledge attitude and practice on the safe handling of pesticides. A total of 399 participants were enrolled in the study comprising of 295 exposed farmers and 104 healthy and unexposed controls. A structured investigator administered questionaries was used to assess their Knowledge, Attitude and practice, and their blood samples were taken for measuring Acetyl Cholinesterase enzyme activity level. There was a significant difference between the Acetyl Cholinesterase enzyme inhibition of exposed and non-exposed control groups observed in the study (P < 0.001). Of the total of 295 farmers, 62 (21.01%) had severe enzyme inhibition of >30% as compared to the unexposed group. Safety practices of handling pesticides were low. The most common symptoms self-reported were headache (OR 1.08, 0.60-1.93) and neurological problems like forgetfulness, lack of concentration (OR 1.12, 0.50-2.48) and increased tiredness (OR 1.075, 0.52-2.19) that were significantly associated with the enzyme inhibition. In addition, we record a very low level of knowledge (17.0%), a fair attitude (63.0%) and poor practice (35.0%) on the safe handling and management of pesticides. This pilot study provides indication of exposure to pesticides in the selected sites of the country. Furthermore, it provides evidence for public health interventions by identifying the exposure patterns and pathways of individuals most at risk in the farming communities of the country. Surveillance and bio-monitoring programs are deemed necessary.

Genetic diversity and evolutionary patterns of SARS-CoV-2 among the Bhutanese population during the pandemic.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic, caused by a dynamic virus, has had a profound global impact. Despite declining global COVID-19 cases and mortality rates, the emergence of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants remains a major concern. This study provides a comprehensive analysis of the genomic sequences of SARS-CoV-2 within the Bhutanese population during the pandemic. The primary aim was to elucidate the molecular epidemiology and evolutionary patterns of SARS-CoV-2 in Bhutan, with a particular focus on genetic variations and lineage dynamics. METHODS: Whole-genome sequences of SARS-CoV-2 collected from Bhutan between May 2020 and February 2023 (n=135) were retrieved from the Global Initiative on Sharing All Influenza Database. RESULTS: The SARS-CoV-2 variants in Bhutan were predominantly classified within the Nextstrain clade 20A (31.1%), followed by clade 21L (20%) and clade 22D (15.6%). We identified 26 Pangolin lineages with variations in their spatial and temporal distribution. Bayesian time-scaled phylogenetic analysis estimated the time to the most recent common ancestor as February 15, 2020, with a substitution rate of 0.97×10-3 substitutions per site per year. Notably, the spike glycoprotein displayed the highest mutation frequency among major viral proteins, with 116 distinct mutations, including D614G. The Bhutanese isolates also featured mutations such as E484K, K417N, and S477N in the spike protein, which have implications for altered viral properties. CONCLUSION: This is the first study to describe the genetic diversity of SARS-CoV-2 circulating in Bhutan during the pandemic, and this data can inform public health policies and strategies for preventing future outbreaks in Bhutan.

Knowledge, attitude, and practice on antibiotic use and antibiotic resistance among the veterinarians and para-veterinarians in Bhutan.

INTRODUCTION: Antimicrobial resistance is (AMR) an emerging global public health problem. Rationale use of antibiotic can prevent the rise of antimicrobial resistance. The objective of this study was to understand the knowledge, attitude and practice on antibiotic usage and AMR among the veterinarians and para-veterinarians in Bhutan. METHOD: A cross-sectional questionnaire survey among the veterinarians and para-veterinarians was conducted from June to July 2020. A score of one to the correct answers and zero for the wrong answers was allotted to each respondent answers. The total score was added and those who scored above the mean was categorized as having good knowledge and favourable attitude. RESULT: A total of 219 animal health workers participated in this study. The mean knowledge score was 12.05 ±1.74 with 38.8% of the respondents having good knowledge on antibiotic use and AMR. Similarly, the mean scores for the attitude level were 8.32±1.61 with 51% them having favorable attitude towards antibiotic usage and AMR. The mean practice score was 3.83±1.06 with 77% of them having good practices on antibiotic use. The respondents who read national plan on AMR were found to have good knowledge on antibiotics and AMR (AOR: 2.39; 95% CI: 1.19-4.82). The female respondents (AOR: 2.16; 95% CI: 1.01-4.61), respondents from the eastern region (AOR: 2.53; 95% CI: 1.18-5.44), west central (AOR: 3; 95% CI: 1.30-6.92), animal health supervisors (AOR: 9.77; 95% CI: 1.98-48.29), and livestock production supervisors (AOR: 2.77; 95% CI: 1.21-6.35) have favorable attitude towards antibiotics and AMR. CONCLUSIONS: Our study identified that most animal health workers in Bhutan had poor knowledge on antibiotics usage and AMR. Therefore, regular awareness education on antibiotics and AMR in the form of refresher course/training must be provided to the animal health workers in the country to avoid inappropriate use of antibiotics.

Beyond Standard Practice in Liquid Biopsy: Selective Venous Sampling.

Liquid biopsy is a molecular diagnostic procedure that aims to provide readily accessible genetic profiling of tumors for primary diagnosis, detection of minimal residual or metastatic disease, and therapeutic decision-making, especially for molecularly targeted treatments. Cancers release various biological markers into the circulation, although the most widely used are cell-free tumor DNA and circulating tumor cells. The paucity of biological material means that laboratory methods mainly based on genetic sequencing expose this innovative diagnostic method to a considerable incidence of false negatives. The 3 cases presented here show how the sensitivity and specificity of liquid biopsy may be improved through selective venous sampling.

A review on foodborne disease outbreaks in Bhutan.

Foodborne diseases (FBDs) are a public health threat that can result in loss of lives and are a hurdle to socioeconomic development. The Foodborne Disease Burden Epidemiology Reference Group estimated that the annual burden of FBDs in the South-East Asia Region was more than 150 million illnesses, causing about 175,000 deaths. This review paper aims to compile evidence from the published articles and gray literatures on food-related disease outbreaks in Bhutan. Information was generated from the annual health bulletin published by the Ministry of Health and data from the National Early Warning Alert Response Surveillance and Information System hosted by Royal Centre for Disease Control. The laboratory confirmation rate of FBD investigation outbreaks increased from 25.5% (2012-2018) to 91.7% in 2020, as per the National Food Safety Indicator (FSI). At least 0.15% of the total population had suffered FBD from 2015 to 2020, and the baseline report shows that most of the FBD events occurred during hot and wet seasons (June and August). The common pathogen causing FBD was Salmonella species accounting for 42.9% of cases, followed by Shigella species (21.4%). Most of the FBD events were reported in schools (46.5%). The frequency of the reported FBD outbreaks had increased from 8 events in 2015 to 15 events in 2017 and 10 events in 2018. The study confirms the improved surveillance for FBDs in Bhutan and highlights the need to raise public awareness on food safety.

Seroprevalence and risk factors of canine distemper virus in the pet and stray dogs in Haa, western Bhutan.

BACKGROUND: Canine Distemper Virus (CDV) is a highly contagious virus belonging to family Paramyxovirade, genus Morbillivirus and responsible for high morbidity and mortality in dogs worldwide. Infected domestic dogs can cause spillover infections to wild carnivores that are in contact. We conducted a seroprevalence survey of CDV in domestic dogs in two areas of western Bhutan (Haa district) located at the periphery of the Jigme Khesar Strict Nature Reserve, which is home to several endangered wildlife. A total of 238 serum samples, 119 each from the pet and stray dog, were collected during summer and winter seasons. Samples were tested for CDV antibodies using a sandwich enzyme-linked immune-sorbent assay (ELISA) test. RESULTS: The seroprevalence of CDV was found to be 11.3% (95% CI 6.7-14.2). Dogs sampled during winter were less likely to test seropositive against CDV antibodies than those sampled during summer (adjusted odds ratio: -2.6; 95% CI: - 1.2-6.1). Dogs in good body condition were found to be more likely to test seropositive against CDV than dogs in poor condition and obese dogs (adjusted odds ratio: 2.2; 95% CI: 0.1-5.9). There were no significant differences in the seroprevalence of CDV among different sexes, breeds and age classes, pet and stray dogs and between the two study sites. CONCLUSIONS: Our study indicates that CDV seroprevalence was equally distributed among pet and stray dogs. We suggest strengthening the management practices of dogs through responsible dog ownership, dog population management and waste management to minimize the transmission risk of infectious diseases to wildlife.

Soil-Transmitted Helminth infections reduction in Bhutan: A report of 29 years of deworming.

Soil Transmitted Helminth (STH) infections affect over 1.5 billion people worldwide. Although prevalent in all age groups, school aged children are a high-risk groups for STH infections. In Bhutan, epidemiological data on STH were collected from western Bhutan in 2003, which found a prevalence of 16.5%. However, little evidence is available on the prevalence of infection at national level. Therefore, this study was conducted with the aim to assess the prevalence and intensity of STH infections, and identify significant correlates of STH among students. A school-based survey was conducted in three regions of Bhutan. Two-stage cluster sampling was adopted to select a sample of 1500 students from 24 schools, in equal proportion from three regions of the country. A total of 1456 (97%) students were interviewed and their stool sample examined for the presence of parasites. Mini-FLOTAC technique was used to detect the parasite eggs/ova. The prevalence of any STH infection was 1.4%, with 0.8% Ascaris lumbricoides, 0.5% Trichuris trichiura and 0.2% hookworms. The eastern region had the highest prevalence at 2.3%. Except for one student who had moderate intensity of A. lumbricoides, the rest had light infection. Any STH presence was significantly associated with father's occupation, father's education level, type of house and the flooring of the house in which students reported to live. No significant associations were observed between water, sanitation and hygiene (WASH) variables measured and presence of any STH infection. The prevalence of STH was found to be very low with primarily light intensity in this study. Nonetheless, it was also found that the sanitation situation is not ideal in the country, with several students reporting constant or partial open defecation leading to environmental contamination. Based on this prevalence and in line with the WHO guideline, it is recommended that deworming be reduced to once a year in combination with concerted health education on proper hygiene and sanitation practice.

Genetic diversity and population structure of three traditional horse breeds of Bhutan based on 29 DNA microsatellite markers.

The genetic variability and population structure of three Bhutanese traditional horse breeds were assessed through genotyping of 74 horses (Boeta 25, Sharta 14 and Yuta 35) for 29 microsatellite DNA loci. Altogether, 282 alleles were detected across 29 polymorphic loci. The allelic diversity (NE) (Boeta 4.94; Sharta 4.65; Yuta 5.30) and gene diversities (HE) (Boeta 0.78; Sharta 0.77; Yuta 0.79) were high. None of the breeds deviated significantly from the Hardy-Weinberg equilibrium. There was no sign of significant population bottleneck for all the breeds. The inbreeding estimates (FIS) of the breeds were low (Boeta 0.023; Sharta 0.001; Yuta 0.021). Analysis of molecular variance showed 0.6% of the total genetic variation among breeds, 1.9% among individuals and 97.5% within individuals. The global FIT, FST, and FIS estimates for the population were 0.025, 0.006 and 0.019 respectively. The analysis of population structure failed to distinguish subpopulations in traditional horses and this was supported by a high genetic exchange among the breeds. Overall, the results of this study suggest a rich genetic diversity in the traditional horse despite a very low genetic differentiation among the breeds in Bhutan.

Gain of hTERC: a genetic marker of malignancy in oral potentially malignant lesions.

Oral squamous cell carcinoma (OSCC) is the most common oral cancer, and major efforts is being made to identify molecular markers capable to differentiate oral potentially malignant lesions (OPMLs) with indolent course from lesions with aggressive behavior. We undertook a study to evaluate if gain of the human telomerase RNA component (hTERC) gene in OPMLs could indicate lesions at high risk of developing OSCC. The study was performed on 30 OPMLs with long-term follow-up using a dual-color interphase fluorescence in situ hybridization (FISH) for hTERC status. Progression to malignancy was observed in 9 of 10 cases harboring hTERC gain and in 1 of 20 cases retaining a normal copy number of hTERC (P < .0001). Combining morphological grading and FISH analysis, all the cases with high-grade squamous intraepithelial lesion or carcinoma in situ harboring hTERC amplification progressed to OSCC, whereas none of the low-grade squamous intraepithelial lesions without hTERC gain progressed. Intermediate situations occurred. The data suggest that precise morphological evaluation together with FISH assessment for hTERC gain might pave the way to stratify OPMLs into high-risk and low-risk categories and could be helpful in selecting the most appropriate treatment.

A Prospective Hospital-based Surveillance to Estimate Rotavirus Disease Burden in Bhutanese Children under 5 Years of Age.

As part of efforts to develop an informed policy for rotavirus vaccination, this prospective study was conducted to estimate the burden of rotavirus diarrhea among children less than 5 years old attended to the Department of Pediatrics, Jigme Dorji Wangchuk National Referral Hospital (JDWNRH), Thimphu, Bhutan. The duration of the study was three years, extending from February 2010 through December 2012. We estimated the frequency of hospitalization in the pediatric ward and dehydration treatment unit (DTU) for diarrhea and the number of events attributable to rotavirus infection among children under 5 years of age. During the study period, a total of 284 children (1 in 45) were hospitalized in the pediatric ward, and 2,220 (1 in 6) in the DTU with diarrhea among children residing in the Thimphu district. Group A rotavirus was detected in 32.5% and 18.8% of the stool samples from children hospitalized in the pediatric ward, respectively. Overall, 22.3% of the stool samples were rotavirus-positive, and the majority (90.8%) of them was detected in children under 2 years of age. From this study, we estimated that the annual incidence of hospitalization in the pediatric ward and DTU due to rotavirus diarrhea was 2.4/1000 (95% CI 1.7-3.4) and 10.8/1000 (95% CI 9.1-12.7) children, respectively. This study revealed that rotavirus is a major cause of diarrhea in Bhutanese children in Thimphu district and since no study has been performed previously, represents an important finding for policy discussions regarding the adoption of a rotavirus vaccine in Bhutan.

Introduction and establishment of fluoroquinolone-resistant Shigella sonnei into Bhutan.

Shigella sonnei is a major contributor to the global burden of diarrhoeal disease, generally associated with dysenteric diarrhoea in developed countries but also emerging in developing countries. The reason for the recent success of S. sonnei is unknown, but is likely catalysed by its ability to acquire resistance against multiple antimicrobials. Between 2011 and 2013, S. sonnei exhibiting resistance to fluoroquinolones, the first-line treatment recommended for shigellosis, emerged in Bhutan. Aiming to reconstruct the introduction and establishment of fluoroquinolone-resistant S. sonnei populations in Bhutan, we performed whole-genome sequencing on 71 S. sonnei samples isolated in Bhutan between 2011 and 2013.We found that these strains represented an expansion of a clade within the previously described lineage III, found specifically in Central Asia. Temporal phylogenetic reconstruction demonstrated that all of the sequenced Bhutanese S. sonnei diverged from a single ancestor that was introduced into Bhutan around 2006. Our data additionally predicted that fluoroquinolone resistance, conferred by mutations in gyrA and parC, arose prior to the introduction of the founder strain into Bhutan. Once established in Bhutan, these S. sonnei had access to a broad gene pool, as indicated by the acquisition of extended-spectrum ß-lactamase-encoding plasmids and genes encoding type IV pili. The data presented here outline a model for the introduction and maintenance of fluoroquinolone-resistant S. sonnei in a new setting. Given the current circulation of fluoroquinolone-resistant S. sonnei in Asia, we speculate that this pattern of introduction is being recapitulated across the region and beyond.

Dominance of emerging G9 and G12 genotypes and polymorphism of VP7 and VP4 of rotaviruses from Bhutanese children with severe diarrhea prior to the introduction of vaccine.

A prospective study was performed to determine the molecular characteristics of rotaviruses circulating among children aged <5 years in Bhutan. Stool samples were collected from February 2010 through January 2011 from children who attended two tertiary care hospitals in the capital Thimphu and the eastern regional headquarters, Mongar. The samples positive for rotavirus was mainly comprised genotype G1, followed by G12 and G9. The VP7 and VP4 genes of all genotypes clustered mainly with those of neighboring countries, thereby indicating that they shared common ancestral strains. The VP7 gene of Bhutanese G1 strains belonged to lineage 1c, which differed from the lineages of vaccine strains. Mutations were also identified in the VP7 gene of G1 strains, which may be responsible for neutralization escape strains. Furthermore, we found that lineage 4 of P[8] genotype differed antigenically from the vaccine strains, and mutations were identified in Bhutanese strains of lineage 3. The distribution of rotavirus genotypes varies among years, therefore further research is required to determine the distribution of rotavirus strain genotypes in Bhutan.

Investigation and control of anthrax outbreak at the human-animal interface, Bhutan, 2010.

In 2010, we investigated anthrax outbreak in Bhutan. A total of 43 domestic animals died, and cutaneous anthrax developed in 9 persons, and 1 died. All affected persons had contact with the carcasses of infected animals. Comprehensive preparedness and response guidelines are needed to increase public awareness of anthrax in Bhutan.

Novel human bufavirus genotype 3 in children with severe diarrhea, Bhutan.

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.

Molecular characterization and PCR-based replicon typing of multidrug resistant Shigella sonnei isolates from an outbreak in Thimphu, Bhutan.

BACKGROUND: Shigella species are an important cause of diarrhea in developing countries. These bacteria normally acquire their antibiotic resistance via several different mobile genetic elements including plasmids, transposons, and integrons involving gene cassettes. During a diarrhea surveillance study in Thimphu, Bhutan in June and July, 2011, Shigella sonnei were isolated more frequently than expected. This study describes the antibiotic resistance of these S. sonnei isolates. METHODS: A total of 29 S. sonnei isolates from Thimphu, Bhutan was characterized for antimicrobial susceptibility by disc diffusion assay and minimum inhibitory concentration (MIC) assay. All isolates were tested by pulsed-field gel electrophoresis (PFGE) with restriction enzyme XbaI and were tested for plasmid. The plasmid patterns and the PFGE patterns were analyzed by Bionumerics software. DNA sequencing was performed on amplified products for gyraseA gene and class 1 and class 2 integrons. S. sonnei isolates were classified for incompatibility of plasmids by PCR-based replicon typing (PBRT). RESULTS: These S. sonnei were resistant to multiple drugs like ciprofloxacin, nalidixic acid, trimethoprim-sulfamethoxazole, streptomycin, and tetracycline but susceptible to azithromycin. All isolates had class 2 integrons dfrA1, sat1 and aadA1 genes. Two point mutations in Gyrase A subunit at position Ser83Leu and Asp87Gly were detected in these quinolone resistant isolates. The plasmid and PFGE patterns of S. sonnei isolates suggested a clonal relationship of the isolates. All isolates carried common ColE plasmid. ColE plasmid co-resided with B/O plasmid (nine isolates) or I1 plasmid (one isolate). CONCLUSIONS: The characteristics of 29 S. sonnei isolates from Thimphu, Bhutan in June and July, 2011 are identical in PFGE, plasmid and resistance pattern. This study suggests that these recent S. sonnei isolates are clonally related and multidrug-resistant.

Chikungunya fever outbreak, Bhutan, 2012.

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.

Complete Genome Sequences of Two Astrovirus MLB1 Strains from Bhutanese Children with Diarrhea.

In addition to the eight genotypes of classic human astroviruses, seven new genotypes have been reported from two novel clades, MLB and VA. However, the epidemiology of these highly diverse astroviruses remains largely unknown. We report here the complete genome sequences of two MLB1 strains from Bhutanese children with diarrhea.