Surveillance of norovirus among children with diarrhea in four major hospitals in Bhutan: Replacement of GII.21 by GII.3 as a dominant genotype

BACKGROUND Diarrhea is a major cause of morbidity and mortality among Bhutanese children. The etiology of diarrhea is not well known due to the challenges of conducting routine surveillance with Bhutan's modest research facilities. Establishing an etiology is crucial toward generating evidence that will contribute to policy discussions on a diarrheal disease control program. Our previous study, during 2010-2012, revealed that norovirus (NoV) is an important cause of diarrhea among Bhutanese children, and that GII.21 was the major genotype circulating at that time. In other countries, GII.4 is the major genotype responsible for NoV infections. In this update report, we provide new prevalence data to describe the progression of the transformation and distribution of the NoV genotype among Bhutanese children. METHODS From June 2013 through May 2014, diarrheal stool samples were collected at one national referral hospital in Thimphu, two regional referral hospitals in the eastern and central regions, and one general hospital in the western region of Bhutan. NoV was detected by reverse transcription-polymerase chain reaction (RT-PCR), by amplifying the capsid gene. The RT-PCR results were confirmed by nucleotide sequencing of the amplicons. RESULTS The proportion of NoV-positive stool samples was 23.6% (147/623), of which 76.9% were NoV GII and the remainders were NoV GI. The median age of infected children was 15.5 months, with a fairly balanced female: male ratio. NoV GII was most prevalent in the colder months (late November-mid April) and NoV GI had the highest prevalence in the summer (mid April-late September). Nucleotide sequencing was successful in 99 samples of GII strains. The most common genotypes were GII.3 (42.6%), GII.4 Sydney 2012 (15.8%), and GII.4 unassigned (11.9%). No GII.21 was found in any child in the present study. Phylogenetic analysis showed that GII.3 strains in the present study belonged to an independent cluster in lineage B. These strains shared an ancestor with those from different countries and Bhutanese strains circulating during 2010. CONCLUSION NoV remains an important cause of diarrhea among Bhutanese children. Genotype GII.3 from a single ancestor strain has spread, replacing the previously circulating GII.21. Current NoV genotypes are similar to the strains circulating worldwide but are primarily related to those in neighboring countries. NoV GII is prevalent during the cold season, while GI is prevalent during the summer. To develop a NoV infection control policy, further studies are needed.