Molecular epidemiology of dengue fever outbreaks in Bhutan, 2016-2017.

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.

Epidemiological and Molecular Characterization of Dengue Virus Circulating in Bhutan, 2013-2014.

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

A Prospective 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.

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

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

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.

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

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

Influenza surveillance from November 2008 to 2011; including pandemic influenza A(H1N1)pdm09 in Bhutan.

OBJECTIVE: Describe the influenza A(H1N1) pandemic in Bhutan. DESIGN: Observational study from sentinel surveillance sites. SETTING: Bhutan remains isolated, with only one to two flights a day at the lone airport, no trains, and only three major roads that enter from India. MAIN OUTCOME MEASURES: PCR positive human respiratory samples. RESULTS: The first case of A(H1N1)pdm09 infection was detected in Bhutan in July 2009, 3 months after the virus was first reported in Mexico in April 2009. During the official WHO pandemic period (11 June 2009 to 8 August 2010), a total of 2149 samples were collected and tested by RT-PCR of which 22.7% (487) were confirmed A(H1N1)pdm09; H3N2, H1N1, and B were positive in 2.2%, 1.1%, and 7.2%, respectively. The highest rate of A(H1N1)pdm09 cases (57.4%) was detected in the 6-20 year-old age group. Importantly, Bhutan increased from 3 sentinel sites in April 2009 to 11 a year later, and in April 2010 established PCR capability for influenza. CONCLUSIONS: Despite relative isolation, the A(H1N1)pdm09 reached Bhutan within 3 months of identification in Mexico. The H1N1 pandemic has made Bhutan more prepared for epidemics in the future.