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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 FROM AUTHOR]

In this study, a simple, portable, unibody semi-flow injection system was coupled with a screen-printed electrode (SFI/SPE) for the on-site electrochemical screening of sibutramine (SBM) in food supplements. The SFI was fabricated by laser engraving acrylic plastic and was attached to a modified SPE with double-sided adhesive tape. The SPE was modified with a nanocomposite of nitrogen-doped graphene nanoflakes and carbon nanotubes synthesized using hydrothermal and ultrasonic methods. The morphological structure and electrochemical properties of the nanocomposite were thoroughly investigated. The portable SFI-SPE sensing system was designed to be interfaced with a smartphone. Sample solutions can be continuously injected into the sensing system using a plastic syringe and SBM is determined by square wave anodic stripping voltammetry, controlled by means of a smartphone. The developed sensing system displayed linear ranges from 0.010 to 10 μg mL⁻ 1 and 10-60 μg mL⁻ 1 with an LOD of 0.0035 μg mL⁻ 1 . The sensor delivered a response in a short time and demonstrated not only good reproducibility (RSDs ranging from 2.9 % to 4.9 % (n = 10)) and excellent resistance to interference but also high accuracy. The developed sensor was utilized for the screening and quantification of SBM in food supplements but the system could be used in a wider range of electrochemical (bio) sensor applications in the future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objectives: The emergence of multidrug-resistant tuberculosis (MDR-TB) has made the elimination of TB difficult. Currently, there are limited data on MDR-TB and other drug-resistant TB in Bhutan. We aimed to estimate the incidence and explore the potential risk factors associated with MDR/pre-extensively drug-resistant (pre-XDR)-TB using comprehensive national TB data., Methods: We used the data from the Tuberculosis Information Surveillance System of the National Tuberculosis Reference Laboratory to analyze the resistance pattern and epidemiologic data for all TB cases tested for resistance for the year 2018-2021. Multiple logistic regression was performed to identify risk factors associated with MDR/pre-XDR-TB., Results: Of the 2,290 samples tested for drug resistance, 10.6% (n = 243) was MDR-TB, 3.5% (n = 81) was isoniazid-resistant TB, and 0.61% (n = 14) was pre-XDR-TB. A high incidence of MDR/RR-TB/pre-XDR-TB was documented among patients in Thimphu, Samtse, and Sarpang districts. MDR/pre-XDR-TB was more likely documented among patients aged 18-39 years (adjusted odds ratio [aOR] 2.79; 95% confidence interval [CI] 1.46-6.07), female sex (aOR 1.37; 95% CI 1.01-1.86), and patients previously treated for TB (aOR 2.98; 95% CI 1.99-4.42)., Conclusions: Given the high burden of MDR-TB in some districts, decentralization of diagnostic facilities for more rapid characterization could improve early recognition of drug-resistant cases and assist in management. Comprehensive follow-up and monitoring of high-risk groups should be performed., Competing Interests: The authors have no competing interests to declare., (© 2024 The Author(s).)

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., Competing Interests: Authors have declared that no competing interest exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

A novel cost-effective disposable porous graphene electrode (P-GE) modified with bismuth nanoneedles (nano-BiNDs) is proposed as a "mercury-free" sensor for detecting heavy metals through smartphone-assisted electrochemical sensing. The P-GE was fabricated using screen-printing. Nano-BiNDs were generated on the P-GE by potentiostatic electrodeposition. Using an optimal potential of -1.20 V (vs. pseudo-Ag/AgCl) and a deposition time of 200 s, the nano-BiNDs had an average length and width of 189 ± 5 nm and 20 ± 2 nm, respectively. The analytical performances of the fabricated sensing platform were demonstrated by detecting Cd 2+ and Pb 2+ using square-wave anodic stripping voltammetry (SWASV) under optimized conditions. In the optimal conditions, the fabricated sensor exhibited sharp, well-defined stripping peaks for Cd 2+ and Pb 2+ with excellent peak-to-peak separation. The linear detection ranges were from 0.01 to 50 μg mL -1 for Cd 2+ and 0.006-50 μg mL -1 for Pb 2+ . The detection limits for Cd 2+ and Pb 2+ were 3.51 and 2.10 ng mL -1 , respectively. The developed portable sensor demonstrated high sensitivity, good repeatability, reproducibility, and anti-interference properties. The proposed portable sensor quantified Cd 2+ and Pb 2+ in commercial seaweed products with good accuracy, consistent with the results obtained using the standard ICP-OES method., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Nitrite (NO 2 - S 2 S 3 S 2 S 3 S 2 S 3 MS and Pd 2+ S 2 S 3 MS/SPE was coupled with a smartphone-controlled portable potentiostat and applied to determine nitrite in food samples. The linear range of the sensor was 0.01-500 μM and the limit of detection was 0.0033 μM. The proposed system showed good repeatability, reproducibility, catalytic stability, and immunity to interferences. The proposed electrode material and a smartphone-based small potentiostat created a simple, portable, fast electrochemical sensing system that accurately measured nitrite in food samples., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Key Clinical Message: Rheumatic heart disease is a preventable disease. Patients may not present with a typical history of sore throat and polyarthritis but may present with Sydenham's chorea. We should not rely completely on clinical findings to rule out carditis. Echocardiography should be done to rule out subclinical carditis., Abstract: Sydenham's chorea is a major manifestation of rheumatic fever. It occurs primarily in children and is seen rarely after the age of 20 years. We describe a 16-year-old girl who presented with purposeless involuntary movements of her upper and lower limbs. Laboratory blood reports showed raised erythrocyte sedimentation rate and anti-streptolysin O. 2D Doppler Echocardiography confirmed subclinical carditis, thickened mitral and aortic valve with mild mitral regurgitation. She was managed as Acute Rheumatic Fever with oral Phenoxymethyl penicillin and Carbamazepine. At the latest follow-up interviewing the caregiver, the patient had no sequelae. Early diagnosis is key to preventing late consequences of acute rheumatic fever and rheumatic heart disease. Sydenham's chorea is a rare presentation of acute rheumatic fever. The absence of clinical carditis does not rule out carditis., Competing Interests: There is no any conflict of interest., (© 2024 The Author(s). Clinical Case Reports published by John Wiley & Sons Ltd.)

We present a highly sensitive and selective electrode of laser-induced graphene modified with poly(phenol red) (P(PhR)@LIG) for measuring zinc nutrition in rice grains using square wave anodic stripping voltammetry (SWASV). The physicochemical properties of P(PhR)@LIG were investigated with scanning electron microscopy (SEM), energy-dispersive X-ray (EDX), Fourier infrared spectroscopy (FT-IR) and Raman spectroscopy. The modified electrode demonstrated an amplified anodic stripping response of Zn 2+ due to the electropolymerization of P(PhR), which enhanced analyte adsorption during the accumulation step of SWASV. Under optimized parameters, the developed sensor provided a linear range from 30 to 3000 μg L -1 with a detection limit of 14.5 μg L -1 . The proposed electrode demonstrated good reproducibility and good anti-interference properties. The sensor detected zinc nutrition in rice grain samples with good accuracy and the results were consistent with the standard ICP-OES method., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This work introduces a 3D-printed portable electroplating device for the visualization of latent fingerprints (LFPs) on metallic substrates. An electroplating solution of Ag + -Cu 2+ in a deep eutectic solvent (DES) is used. The electroplating is performed by two electrodes equivalent to an anode (+) and a cathode (-). The cathode is connected to the metal surface with the magnetic or alligator clip for carrying the LFP. The anode is connected to cotton dipped in the electroplating solution. The device was optimized in terms of the electroplating solution composition, and electroplating potential, current, and time. The device produced images with good resolution, revealing LFP ridges in minute detail of more than 12 points. The device also exhibited good repeatability and images were assessed against guidelines from the Centre for Applied Science and Technology (CAST) and the International Fingerprint Research Group (IFRG). The developed device could be applied to visualize LFPs in forensic investigations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Introduction: Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections worldwide, particularly in young children. In Bhutan, respiratory disease continues to be among the top 10 diseases of morbidity for several years. This study aimed to estimate the prevalence of RSV among hospitalized patients with severe acute respiratory infection (SARI) in Bhutan., Method: Respiratory specimens were collected from SARI patients of all ages in 2016 and 2018 following influenza surveillance guidelines. Specimens were tested for influenza and RSV, human metapneumovirus, adenovirus, and human parainfluenza virus types 1, 2, and 3 using real-time reverse-transcription polymerase chain reaction assay. Descriptive statistics were used to analyze the result in STATA 16.1., Result: Of the 1339 SARI specimens tested, 34.8% were positive for at least one viral pathogen. RSV was detected in 18.5% of SARI cases, followed by influenza in 13.4% and other respiratory viruses in 3%. The median age of SARI cases was 3 (IQR: 0.8-21 years) years. RSV detection was higher among children aged 0-6 (Adj OR: 3.03; 95% CI: 1.7-5.39) and 7-23 months (Adj OR: 3.01; 95% CI: 1.77-5.12) compared with the children aged 5-15 years. RSV was also associated with breathing difficulty (Adj OR: 1.73; 95% CI: 1.17-2.56) and pre-existing lung disease, including asthma (Adj OR: 2.78; 95% CI: 0.99-7.8)., Conclusion: Respiratory viruses were detected in a substantial proportion of SARI hospitalizations in Bhutan., Competing Interests: All authors declared that they have no conflict of any financial interest to disclose., (© 2024 The Authors. Influenza and Other Respiratory Viruses published by John Wiley & Sons Ltd.)

Bhutan successfully introduced multiple vaccines since the establishment of the Vaccine Preventable Disease Program in 1979. Surveillance and subsequent introduction of influenza vaccination became a public health priority for the Ministry of Health following the influenza A(H1N1)pdm09 pandemic. Sentinel surveillance for influenza in Bhutan began in 2008, and a study of severe acute respiratory infection was conducted in 2017, which found the highest influenza burden in children aged <5 years and adults ≥50 years. Following review of surveillance and burden of disease data, the National Technical Advisory Group presented recommendations to Bhutan's Ministry of Health which approved influenza vaccine introduction for all five high-risk groups in the country. Upon the official launch of the program in June 2018, the Vaccine Preventable Disease Program began planning, budgeting, and procurement processes with technical and financial support from the Partnership for Influenza Vaccine Introduction, the United States Centers for Disease Control and Prevention, the Bhutan Health Trust Fund, and the World Health Organization. Influenza vaccination for high-risk groups was integrated into Bhutan's routine immunization services in all health care facilities beginning in November 2019 and vaccinated all populations in 2020 in response to the COVID-19 pandemic. Coverage levels between 2019 and 2022 were highest in children aged 6-24 months (62.5%-96.9%) and lowest in pregnant women (47.7%-62.5%). Bhutan maintained high coverage levels despite the COVID-19 pandemic by continued provision of influenza vaccine services at health centers during lockdowns, conducting communication and sensitization efforts, and using catch-up campaigns. Bhutan's experience with introducing and scaling up the influenza vaccine program contributed to the country's capacity to rapidly deploy its COVID-19 vaccination program in 2021., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: The National Action Plan on Antimicrobial Resistance in Bhutan promotes the rational use of antibiotics. It is important to establish baseline data on the use of antibiotics and the quality indicators of antibiotic prescriptions to identify where improvement efforts may need to be focused., Objectives: To describe the prevalence and patterns of antibiotic prescription and establish baseline data regarding quality indicators of antibiotic prescriptions in four major hospitals in Bhutan., Methods: This was a point prevalence survey of antibiotic use among inpatients in June 2022 conducted using the Australian National Antibiotic Prescribing Survey (NAPS)., Results: There were 314 patients (41.5%) receiving at least one antibiotic on the audit day. Among prescriptions reviewed, 278 (88.5%) had indications for use documented, 102 (32.5%) had a review or stop date documented and 120 (38.2%) had microbiology samples collected prior to antibiotics. Ceftriaxone (68; 21.7%), cefazolin (41; 13.1%) and metronidazole (32; 10.2%), were the common antibiotics prescribed. The most common indications for use were surgical prophylaxis (42; 13.4%), community-acquired pneumonia (39; 12.4%) and sepsis (26; 8.3%). There were 125 prescriptions (39.8%) that were compliant with national/therapeutic antibiotic guidelines and 169 (53.8%) where antibiotic prescriptions were appropriate., Conclusions: This study identified key areas for targeted interventions in antimicrobial stewardship programmes in Bhutan. The prevalence of antibiotic use, indications for use, and drug choices were similar to data from other countries. Documentation plans for durations of use, prolonged surgical prophylaxis and concordance of choices with guideline recommendations present opportunities for improvement., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

Near-field communication (NFC) was used to control a portable glucose biosensor for diabetes diagnosis. The system comprised a smartphone and an NFC potentiostat connected to a screen-printed carbon electrode (SPCE) modified with Prussian blue-graphene ink and functionalized with gold nanoparticles-embedded poly (3,4ethylene dioxythiophene):polysulfonic acid coated with glucose oxidase (GOx-AuNPs-PEDOT:PSS/PB-G). GOx catalyzed the glucose redox reaction while the conductivity and sensitivity of the AuNPs-PEDOT:PSS composite enhanced electron transfer to the PB-G, which was used as a mediator. The fabrication process was characterized by scanning electron microscopy (SEM) with energy dispersibe x-ray analysis (EDX). The platform was electrochemically characterized by electrical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The NFC biosensing device was then applied to quantify glucose in human blood serum by amperometry. The linear concentration range and detection limit for glucose were 0.5-500 μM and 0.15 μM, respectively. The accuracy of the device was good and results were in agreement with the results obtained from the standard hospital method. This NFC glucose sensing device can be a simple, sensitive, selective and portable platform for medical diagnosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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<0.001) improved the mean knowledge score from 19.98(±2.72) to 26.96(±2.24) in the intervention group. Similarly, mean scores for perception and safety behaviour improved significantly (P<0.001) from 10.77 (±1.89) to 13.95 (±1.36) and 9.809 (±1.85) to 12.97 (±1.54), respectively. Although the scores have reduced significantly (P<0.001) at three months post intervention, most of the rabies information was largely retained by the students. In control group, significant increase in mean scores were also observed for perception from 10.17 (±2.38) to 11.2 (±2.44) and safety behaviour from 9.14(±1.44) to 10.74 (±1.95) after 3 months of education. The finding demonstrate that a short rabies lesson is effective in improving knowledge, perceptions and understanding of dog bites safety behaviour among the school children. However, there is a need for a frequent awareness program, at least quarterly or half yearly. Rabies education should focus on critical points such as dog bites being the main source of rabies and the importance washing a dog/animal bite wound with soap and water, and visiting the hospital for medical advice following animal bites., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Lungten et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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.

Background: Bhutan is no exception to the rising global threat of drug resistance tuberculosis (TB), particularly multidrug-resistant (MDR) TB. Although drug resistance surveillance has been carried out in Bhutan since 2010, limited analysis reports are available. Therefore, we looked at data from 2015-2019 to understand patient characteristics., Method: To obtain data for MDR-TB from the past 5 years, we looked at manual registers and laboratory worksheets for all samples received at National TB Reference Laboratory. Epidemiological factors and laboratory variables were analyzed using descriptive statistics., Result: Among 304 patients with MDR-TB, 85.20% (n=259) are new cases with no previous history of treatment. Those aged 16-25 years from both genders are affected more (46.05%, n=140) than other age groups. The majority (94.62%, n=264) of rifampicin resistance was found in the MUT 3 rpoB gene. For Isoniazid, 97.13% (n=271) resistance was seen in the MUT1 band of the katG gene., Conclusion: A high number of MDR-TB cases among new patients and little variation in the resistance band pattern over 5 years could indicate uncontrolled ongoing transmission. Whole-genome sequencing for the samples is required to further understand the epidemiology of the resistance pattern., Competing Interests: No conflict of interest to declare., (© 2022 The Author(s).)

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., Competing Interests: No competing interests were disclosed., (Copyright: © 2022 MalariaGEN et al.)

Shigella flexneri serotype 6 is an understudied cause of diarrhoeal diseases in developing countries, and has been proposed as one of the major targets for vaccine development against shigellosis. Despite being named as S. flexneri , Shigella flexneri serotype 6 is phylogenetically distinct from other S. flexneri serotypes and more closely related to S. boydii . This unique phylogenetic relationship and its low sampling frequency have hampered genomic research on this pathogen. Herein, by utilizing whole genome sequencing (WGS) and analyses of Shigella flexneri serotype 6 collected from epidemiological studies (1987-2013) in four Asian countries, we revealed its population structure and evolutionary history in the region. Phylogenetic analyses supported the delineation of Asian Shigella flexneri serotype 6 into two phylogenetic groups (PG-1 and -2). Notably, temporal phylogenetic approaches showed that extant Asian S. flexneri serotype 6 could be traced back to an inferred common ancestor arising in the 18 th century. The dominant lineage PG-1 likely emerged in the 1970s, which coincided with the times to most recent common ancestors (tMRCAs) inferred from other major Southeast Asian S. flexneri serotypes. Similar to other S. flexneri serotypes in the same period in Asia, genomic analyses showed that resistance to first-generation antimicrobials was widespread, while resistance to more recent first-line antimicrobials was rare. These data also showed a number of gene inactivation and gene loss events, particularly on genes related to metabolism and synthesis of cellular appendages, emphasizing the continuing role of reductive evolution in the adaptation of the pathogen to an intracellular lifestyle. Together, our findings reveal insights into the genomic evolution of the understudied Shigella flexneri serotype 6, providing a new piece in the puzzle of Shigella epidemiology and evolution.

Background: Respiratory syncytial virus (RSV) infections are one of the leading causes of lower respiratory tract infections and have a major burden on society. For prevention and control to be deployed effectively, an improved understanding of the seasonality of RSV is necessary., Objectives: The main objective of this study was to contribute to a better understanding of RSV seasonality by examining the GERi multi-country surveillance dataset., Methods: RSV seasons were included in the analysis if they contained ≥100 cases. Seasonality was determined using the "average annual percentage" method. Analyses were performed at a subnational level for the United States and Brazil., Results: We included 601 425 RSV cases from 12 countries. Most temperate countries experienced RSV epidemics in the winter, with a median duration of 10-21 weeks. Not all epidemics fit this pattern in a consistent manner, with some occurring later or in an irregular manner. More variation in timing was observed in (sub)tropical countries, and we found substantial differences in seasonality at a subnational level. No association was found between the timing of the epidemic and the dominant RSV subtype., Conclusions: Our findings suggest that geographical location or climatic characteristics cannot be used as a definitive predictor for the timing of RSV epidemics and highlight the need for (sub)national data collection and analysis., (© 2021 The Authors. Influenza and Other Respiratory Viruses published by John Wiley & Sons Ltd.)

Background: Respiratory syncytial virus (RSV) is one of the leading causes of acute respiratory tract infections. To optimize control strategies, a better understanding of the global epidemiology of RSV is critical. To this end, we initiated the Global Epidemiology of RSV in Hospitalized and Community care study (GERi)., Methods: Focal points from 44 countries were approached to join GERi and share detailed RSV surveillance data. Countries completed a questionnaire on the characteristics of their surveillance system., Results: Fifteen countries provided granular surveillance data and information on their surveillance system. A median (interquartile range) of 1641 (552-2415) RSV cases per season were reported from 2000 and 2020. The majority (55%) of RSV cases occurred in the <1-year-olds, with 8% of cases reported in those aged ≥65 years. Hospitalized cases were younger than those in community care. We found no age difference between RSV subtypes and no clear pattern of dominant subtypes., Conclusions: The high number of cases in the <1-year-olds indicates a need to focus prevention efforts in this group. The minimal differences between RSV subtypes and their co-circulation implies that prevention needs to target both subtypes. Importantly, there appears to be a lack of RSV surveillance data in the elderly., (© The Author(s) 2021. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Bhutan experienced its largest and first nation-wide dengue epidemic in 2019. The cases in 2019 were greater than the total number of cases in all the previous years. This study aimed to characterize the spatiotemporal patterns and effective reproduction number of this explosive epidemic. Weekly notified dengue cases were extracted from the National Early Warning, Alert, Response and Surveillance (NEWARS) database to describe the spatial and temporal patterns of the epidemic. The time-varying, temperature-adjusted cohort effective reproduction number was estimated over the course of the epidemic. The dengue epidemic occurred between 29 April and 8 December 2019 over 32 weeks, and included 5935 cases. During the epidemic, dengue expanded from six to 44 subdistricts. The effective reproduction number was <3 for most of the epidemic period, except for a ≈1 month period of explosive growth, coinciding with the monsoon season and school vacations, when the effective reproduction number peaked >30 and after which the effective reproduction number declined steadily. Interventions were only initiated 6 weeks after the end of the period of explosive growth. This finding highlights the need to reinforce the national preparedness plan for outbreak response, and to enable the early detection of cases and timely response.

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.

As the COVID-19 pandemic continues, there is growing concordance and persisting conflicts on the virus and the disease process. We discuss limited transmissibility of the virus by asymptomatic and mild cases of COVID-19 patients in Bhutan. We followed up the secondary transmission of SARS-CoV-2 in the contacts of asymptomatic and mild COVID-19 patients in Bhutan. Bhutan had 33 confirmed COVID-19 cases in the country as of May 29, 2020. Of these, 22 (67%) were females. Except the first two cases (American tourists), the rest were Bhutanese living outside the country. The mean age of the Bhutanese patients was 26.3 (range 16-33) years. Close contacts of 27 of the 33 cases were followed up for signs and symptoms and COVID-19 positivity. The first two cases had 73 and 97 primary contacts, respectively, and equal number of secondary contacts (224). From the third case, a mandatory 21-day facility quarantine was instituted, all primary contacts were facility quarantined, and there were no secondary contacts. In total, the 27 cases had 1,095 primary contacts and 448 secondary contacts. Of these, 75 individuals were categorized as definite high-risk contacts. Secondary transmission occurred in seven high-risk contacts. Therefore, the overall secondary transmission was 9.0% (7/75) and 0.6% (7/1,095) among the high-risk and primary contacts, respectively. No transmission occurred in the secondary contacts. In contrast to several reports indicating high transmissibility of SARS-CoV-2 in contacts of confirmed cases, the mostly young, asymptomatic, and mild cases of COVID-19 in Bhutan showed limited secondary transmission.

Dengue 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.

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.

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., Competing Interests: The authors have declared that no competing interests exist.

Threat assessment is critical to species conservation and management planning, because prior identification and assessment of key threats to conservation planning can assist in developing appropriate interventions or strategies. Comprehensive threat assessments are currently lacking for many threatened primates. In this paper, we classify and rank all direct threats to the endangered golden langur (Trachypithecus geei) in Bhutan in order to provide a practical guide to future conservation of the species. Information on threats was based on interviews with local people, discussion with field forestry staff, and social media interaction. We classified threats to golden langur habitats and populations, and ranked them using Miradi™, an analytical software for the adaptive management of conservation projects. We identified five habitat threats: (1) hydropower development, (2) road development, (3) housing development, (4) resource extraction, and (5) agricultural expansion. We also identified seven population threats: (1) electrocution, (2) road kill, (3) road injury, (4) dog kill, (5) retaliatory killing, (6) illegal pet keeping, and (7) hybridization with capped langurs. We rated the overall threat to golden langurs in Bhutan as 'medium'. Hydropower, road, and housing development constituted 'high' impact, while agricultural expansion, resource extraction, electrocution, and road kill had 'medium' impact; the remaining threats had 'low' impact. To immediately mitigate threats to golden langurs, we recommend: (a) installing speed limit signage and speed breakers with strict enforcement of speed limits; (b) installing insulated electric cables and fencing around power transformers; and (c) reducing and restraining domestic dog populations.

Background Marked differences among genotype frequencies (Caucasians, Asians, and Africans) have been observed in cytochrome P450 (CYP) genes. Data on the frequency of pharmacogenetic relevant polymorphisms in Bhutanese population is absent. This study aimed to investigate the frequencies of pharmacogenetic relevant polymorphisms of CYP2C9 (*2 and *3), CYP2C19 (*2 and *3), CYP2D6 (*10), and CYP3A5 (*3) in Bhutanese population. Methods Genotyping was performed in 443 DNA samples using polymerase chain reaction-restriction fragment length polymorphism. Results For CYP2C9, allele frequencies of *2 and *3 variants were 0.339% and 0%, respectively. For CYP2C19, frequencies of *2 and *3 variants were 30.135% and 15.689%, respectively. Allele frequencies of CYP2D6*10 and CYP3A5*3 were 21.332% and 77.314%, respectively. Allele frequencies of CYP2C9*2 are similar to most Asians while CYP2C9*3 was absent. CYP2C19*2 showed a close resemblance to Japanese and Burmese, while CYP2C19*3 is near to Japanese and Korean. CYP2D6*10 is noticeably lower than other Asians. CYP3A5*3 is similar to East Asians (Chinese, Japanese, and Korean). Conclusions The Bhutanese population is polymorphic for these CYP genes, except for CYP2C9*3. Similar to other populations, genetic testing for these genes may, therefore, be helpful to obtain the benefit from pharmacological treatments and prevent adverse drug reactions.

Shigella sonnei increasingly dominates the international epidemiological landscape of shigellosis. Treatment options for S. sonnei are dwindling due to resistance to several key antimicrobials, including the fluoroquinolones. Here we analyse nearly 400 S. sonnei whole genome sequences from both endemic and non-endemic regions to delineate the evolutionary history of the recently emergent fluoroquinolone-resistant S. sonnei. We reaffirm that extant resistant organisms belong to a single clonal expansion event. Our results indicate that sequential accumulation of defining mutations (gyrA-S83L, parC-S80I, and gyrA-D87G) led to the emergence of the fluoroquinolone-resistant S. sonnei population around 2007 in South Asia. This clone was then transmitted globally, resulting in establishments in Southeast Asia and Europe. Mutation analysis suggests that the clone became dominant through enhanced adaptation to oxidative stress. Experimental evolution reveals that under fluoroquinolone exposure in vitro, resistant S. sonnei develops further intolerance to the antimicrobial while the susceptible counterpart fails to attain complete resistance.

Background: In 2017, measles elimination was verified in Bhutan, and the country appears to have sufficiently high vaccination coverage to achieve rubella elimination. However, a measles and rubella serosurvey was conducted to find if any hidden immunity gaps existed that could threaten Bhutan's elimination status., Methods: A nationwide, three-stage, cluster seroprevalence survey was conducted among individuals aged 1-4, 5-17, and >20 years in 2017. Demographic information and children's vaccination history were collected, and a blood specimen was drawn. Serum was tested for measles and rubella immunoglobulin G (IgG). Frequencies, weighted proportions, and prevalence ratios for measles and rubella seropositivity were calculated by demographic and vaccination history, taking into account the study design., Results: Of the 1325 individuals tested, 1045 (81%, 95% CI 78%-85%) were measles IgG seropositive, and 1290 (97%, 95% CI 95%-99%) were rubella IgG seropositive. Rubella IgG seropositivity was high in all three age strata, but only 47% of those aged 5-17 years were measles IgG seropositive. Additionally, only 41% of those aged 5-17 years who had documented receipt of two doses of measles- or measles-rubella-containing vaccine were seropositive for measles IgG, but almost all these children were rubella IgG seropositive., Conclusions: An unexpected measles immunity gap was identified among children 5-17 years of age. It is unclear why this immunity gap exists; however, it could have led to a large outbreak and threatened sustaining of measles elimination in Bhutan. Based on this finding, a mass vaccination campaign was conducted to close the immunity gap., (Copyright © 2019. Published by Elsevier Ltd.)

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.

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., Competing Interests: Clotilde El-Guerche Séblain is an employee of Sanofi Pasteur. She was the coordinator of the research project at Sanofi Pasteur, she helped define the study objectives, and critically revised the manuscript. When reviewing the manuscript, the revisions concerned the epidemiological findings of the study and not the public health findings or conclusions. This does not alter our adherence to PLOS ONE policies on sharing data and materials. Cheryl Cohen has received grant support from Sanofi Pasteur, Advanced Vaccine Initiative, US Centers for Disease Control and Prevention, and payment of travel costs from Parexel. All of the other authors declare that they have no conflict of interests to disclose.

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 < 0.001). On day 42, patients with recurrent parasitaemia had a mean haemoglobin concentration - 0.72 g/dL [- 0.90, - 0.54] lower than patients without recurrence (p < 0.001). Seven days after starting primaquine, G6PD normal patients had a 0.3% (1/389) risk of clinically significant haemolysis (fall in haemoglobin > 25% to < 7 g/dL) and a 1% (4/389) risk of a fall in haemoglobin > 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.