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.

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.

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.

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.