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.

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.

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.

Evolution of Influenza A(H3N2) Viruses in Bhutan for Two Consecutive Years, 2022 and 2023.

BACKGROUND: Influenza A viruses pose a significant public health threat globally and are characterized by rapid evolution of the hemagglutinin (HA) gene causing seasonal epidemics. The aim of this study was to investigate the evolutionary dynamics of A(H3N2) circulating in Bhutan during 2022 and 2023. METHODS: We analysed 166 whole-genome sequences of influenza A(H3N2) from Bhutan, obtained from the GISAID database. We employed a Bayesian Markov Chain Monte Carlo (MCMC) framework, with a curated global dataset of HA sequences from regions with significant migration links to Bhutan. Phylogenetic, temporal, and phylogeographic analyses were conducted to elucidate the evolutionary dynamics and spatial dissemination of the viruses. RESULTS: Our phylogenetic analysis identified the circulation of influenza A(H3N2) Clade 3C.2a1b.2a.2 in Bhutan during 2022 and 2023, with viruses further classified into three subclades: 2a.3 (39/166), 2a.3a.1 (58/166) and 2a.3b (69/166). The TMRCA estimates suggest that these viral lineages originated approximately 1.93 years prior to their detection. Phylogeographic analysis indicates introductions from the United States in 2022 and Australia in 2023. The mean evolutionary rate across all gene segments was calculated to be 4.42 × 10-3 substitutions per site per year (95% HPD: 3.19 × 10-3 to 5.84 × 10-3), with evidence of purifying selection and limited genetic diversity. Furthermore, reassortment events were rare, with an estimated rate of 0.045 events per lineage per year. CONCLUSION: Our findings show that primary forces shaping the local evolution of the influenza A(H3N2) in Bhutan are largely stochastic, with only sporadic instances of adaptive change, and thus underscore the importance of continuous surveillance to mitigate the impact of evolving strains.

The occurrence and human health risk assessment of total and aflatoxin B1 in selected food commodities in Bhutan.

Aflatoxins are mycotoxins that contaminate staple foods globally and pose a significant health risk. To the best of our knowledge, information on the occurrence of aflatoxins in Bhutanese diets is scarce. This study aimed to estimate the aflatoxin levels in selected foodstuffs in Bhutan and determine the health risk associated with aflatoxin exposure. Ten different types of food commodities were randomly collected from farmers' markets, shelves of supermarkets, and wholesale and retail shops from 20 districts of the country. The samples were subjected to analysis by an enzyme-linked immunosorbent assay for both total aflatoxins (B1, B2, G1 and G2) and aflatoxin B1. Among the 315 samples included, 48.81% and 79.35% were positive for total aflatoxins and aflatoxin B1, respectively. The overall mean total aflatoxin concentration was 11.49 ± 12.83 µg/kg, and that for B1 was 17.62 ± 23.99 µg/kg. The most prevalent food commodity with the highest aflatoxin contamination was chili products. In addition, the estimated daily intake and margin of exposure to aflatoxin B1 via the consumption of chili products ranged from 0.98 to 5.34 ng kg-1 bw day-1 and from 74.90 to 408.10, indicating a risk for public health. The liver cancer risk was estimated to be 0.01 and 0.007 cancers per year per 100,000 population resulting from the consumption of chili products. The present findings revealed the presence of total aflatoxins and aflatoxin B1 in the selected samples. The margin of exposure values was exorbitant, demanding a stringent public health measure. Notably, these results suggest the need for routine monitoring of aflatoxin contamination in the region and stress rigorous safety management strategies to reduce exposure.

Epidemiology and genetic characterization of influenza viruses circulating in Bhutan in 2022.

INTRODUCTION: Influenza (Flu) causes considerable morbidity and mortality globally, and in Bhutan, Flu viruses are a leading cause of acute respiratory infection and cause outbreaks during Flu seasons. In this study, we aim to analyze the epidemiology and the genetic characterization of Flu viruses circulated in Bhutan in 2022. METHOD: Respiratory specimens were collected from patients who meet the case definition for influenza-like illness (ILI) and severe acute respiratory infection (SARI) from sentinel sites. Specimens were tested for Flu and SARS-CoV-2 viruses by RT-PCR using the Multiplex Assay. Selected positive specimens were utilized for Flu viral genome sequencing by next-generation sequencing. Descriptive analysis was performed on patient demographics to see the proportion of Flu-associated ILI and SARI. All data were analyzed using Epi Info7 and QGIS 3.16 software. RESULT: A weekly average of 16.2 ILI cases per 1000 outpatient visits and 18 SARI cases per 1000 admitted cases were reported in 2022. The median age among ILI was 12 years (IQR: 5-28) and SARI was 6.2 (IQR: 2.5-15) years. Flu A(H3N2) (70.2%) subtype was the most predominant circulating strain. Flu A(H1N1)pdm09 and Flu B viruses belonged to subclades that were mismatched to the vaccine strains recommended for the 2021-2022 season but matched the vaccine strain for the 2022-2023 season with vaccine efficacy 85.14% and 88.07% respectively. Flu A(H3N2) virus belonged to two subclades which differed from the vaccine strains recommended in both the 2021-2022 and 2022-2023 seasons with vaccine efficacy 68.28%. CONCLUSION: Flu virus positivity rates were substantially elevated during the Flu season in 2022 compared to 2021. Flu A(H3N2) subtype was the most predominant circulating strain in the country and globally. Genetic characterization of the Flu viruses in Bhutan showed a close relatedness of high vaccine efficacy with the vaccine strain that WHO recommended for the 2022-23 season.

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.

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.

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.

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.

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.

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.

Extramedullary erythropoiesis in chronic subdural hematoma simulating metastatic small round cell tumor.

We report two cases of extramedullary erythropoiesis within chronic subdural hematoma that caused diagnostic confusion. In both cases, the initial favored diagnosis by the submitting pathologists was that of a metastatic malignant tumor, including lymphoma, carcinoma, and malignant melanoma. In both cases, the subdural chronic hematoma contained cohesive clusters of small round blue cells with scant cytoplasm and round hyperchromatic nuclei. In both cases, some mitotic figures were identified. There was no gross or microscopic evidence of a meningeal mass lesion. The erythroblastic nature of the cells was confirmed using immunohistochemistry for CD43, glycophorin A, and erythropoietin A. It is important for surgical pathologists to be aware of this benign process and not to overinterpret it as either a primary or metastatic malignant tumor.

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.

Spitz nevus of the tongue with pseudoepitheliomatous hyperplasia: report of three cases of a pseudomalignant condition.

Three cases of Spitz nevus of the tongue associated with pseudoepitheliomatous hyperplasia are reported: two occurring in children and one in an adult. The location at an unusual site and the complex pattern resulting from the intimate admixture of the neoplastic melanocytic component and the hyperplastic keratinocytic component led in each case to consider diagnoses such as malignant melanoma and invasive squamous cell carcinoma. Staining for S-100 protein and keratin was useful to identify and separate the two components. Spitz nevus of the tongue carries some intriguing similarities with granular cell tumor, suggesting a possible histogenetic and pathogenetic relationship.