Rotavirus Disease Burden and Molecular Epidemiology in Children with Acute Diarrhoea Age Less than 5 Years in Nepal

Introduction: Rotavirus is the most common cause of severe gastroenteritis in infants and young children worldwide. The aim of the study was to determine rotavirus disease burden and distribution of rotavirus genotypes in children less than five years of age. Methodology: Stool samples were collected from 1768 hospital admitted children under 5 years of age with acute watery diarrhea during November 2005 to October 2008. Rotavirus in stool samples was detected by Enzyme Immuno Assay (EIA) and positive specimens for rotavirus positive was genotyped by reverse transcription-polymerase chain reaction (RTPCR) and sequencing methods. Results: The prevalence of rotavirus was 36.59% (647/1768). Detection was higher in male (33.3%) than female (26.9%). The prevalence was higher during the month of January (2005-06) and February in other periods. During 2005-06, high prevalence of rotavirus was noted in children of age between 6-11 months while in 2006-07 and 2007-08 high prevalence was seen in age group 12-23 months. The most common genotyping of rotavirus identified 5 G types and 3 P types. Genotypes G12 and P [8] were most common during both periods studied (G12; 50%, 29% 33.7% in 2005-06, 2006-07 and 2007-08 respectively and P[8]; 47%, 35% and 43.5% in 2005-06, 2006-07 and 2007-08 respectively). Among six combined genotypes, G12P [6] was most prevalent (34%, 24% and 47.5% in 2005-06, 2006-07 and 2007-08 respectively) where as G1P [6] (4%) was seen in year 200708 only. Conclusions: The study demonstrates the burden of rotavirus diarrhea in Nepal. The data on rotavirus genotypes will help inform decisions as to whether rotavirus vaccine should be considered for inclusion in to Nepal’s National Immunization Program.


Introduction
A global estimate suggests that 19% of total child deaths are attributable to diarrhoea and 73% of these deaths are concentrated in 15 developing countries.It is the second commonest cause of death due to infectious diseases in children under fi ve and also a major cause of adult death in such countries [1][2] .R otavirus remains the most common cause of severe, dehydrating diarrhoea among children worldwide [3][4][5] .It is estimated to be responsible for more than 600,000 deaths per year in these children with 85% occurring in low-income countries 3 .
The study done by Nepal Human Development Report published in 1998, 16 to 25% of childhood deaths occur due to diarrhoea.A study of travelers and foreign residents reported an incidence of Rotavirus in 8% of cases.Group C Rotavirus infection was demonstrated from convalescent sera in patients with diarrhoea by others 6 .They primarily spread by the feco-oral route.Symptoms include diarrhoea, vomiting and fever.In severe cases acidosis, electrolyte abnormalities, severe dehydration and death can occur.Infection can be asymptomatic, especially in infants aged less than three months.Diagnosis is usually confi rmed by detection of rotavirus in stool samples 4 .Rotavirus infects the proximal small intestine, where it elaborates an enterotoxin and destroys the epithelial surface, resulting in blunted villi, extensive damage, and shedding of massive quantities of virus in stool.The estimated incubation period for rotavirus diarrheal illness is <48 hours 4 .The virus was discovered 30 years ago.It consists of 11 double-stranded RNA segments encased in a bilayered capsid containing three viral proteins (VP4, VP6 and VP7) 6 .These proteins, which are infective and induce an immune response in the host, are the basis of the classifi cation system used today.VP6 determines the group and subgroup, while VP4 is indicative of type P, and VP7 is associated with type G.The variations in these viral proteins have an impact on immunity, the methods used for detection, and effective vaccine development 7 .
Since, Nepal is one of the developing countries and more people inhabit are illiterate and have poor economical status, children death even today occur due to diarrhoea.The study therefore is aimed to determine rotavirus disease burden and distribution of rotavirus genotypes in hospitalized children less than fi ve years of age.

Detection Rotavirus by EIA
Stool samples were analyzed for rotavirus by using Premier Rotaclone which is Enzyme Immunoassay EIA (Meridian Bioscience, USA) for the detection of rotavirus antigen in human fecal samples according to the instructions of the manufacturer.All EIA positive samples were subsequently confi rmed by Reverse Transcription-Polymerase Chain Reaction (RT-PCR).Rotavirus positive samples were frozen at -70°C until genotyping of rotavirus strains processed.

Rotavirus Genotyping
Detection of rotaviruses in clinical specimens and determination of the G-types and P-types can be accomplished by extraction of the viral RNA from fecal specimens and analysis by reverse-transcription polymerase chain reaction (RT-PCR) with primers specifi c for the VP7 genes of G serotypes 1,2,3,4, 8 and 0 and VP4 genes of P serotypes 4, 6, 8, 9, 10 and 11. in the study for rotavirus strain identifi cation rotavirus positive samples were transported to the Department of Medical Microbiology, Liverpool University where RT-PCR amplifi cation were used according the method for genotyping method described by Gouvea et al and Gentsch et al 7,8 .

Results
A total of 1768 children were enrolled in between three years study (November 2005-2008) period that fulfi ll the enrollment criteria for the study.Of them 647 (36.59%) cases were positive for rotavirus by EIA, among the positive cases, 480 samples were carried out genotyping by RT-PCR.

Year and month wise distribution of rotavirus positive on hospitalized children:
Though the infection occurred all year round; but the prevalence trend was higher in winter season (Fig 1).The seasonal and monthly frequency of isolation of rotavirus in hospitalized children was found to be highest from December to March which did not differ substantially between the years of the study.A majority of positive cases occurred in January; 57% (61/106), 62.5% (20/32)

Age distribution of rotavirus positive children whom were hospitalized
The highest prevalence was seen in children from 3 to 23 months of age which was shown in below (fi gure 2).In all three years i.

Discussions
Diarrheal disease remains one of the largest health problems in many parts of the world 4,5,6,9 .Studies in developing countries have shown that children in the fi rst 2 years of the life may have up to 10 episodes of diarrheal disease, often with signifi cant mortality 10,11 .Diarrheal disease occupied the second place among the top ten diseases in Nepal 12 .
The prevalence of rotavirus was 36.6% in hospital admitted patients.Fever and vomiting were signifi cantly associated with rotavirus infection compared to other causes of diarrhea.In this study, of the total 1185 cases having abdominal pain with 38.31% rotavirus positive, 873 with fever with 36.42% rotavirus positive, 977 with nausea and vomiting with 41.76% rotavirus positive were seen.It was also known that rotavirus usually dominate in hospital based survey in children 14,15,16 .Rotavirus infection is commonly prevalent between age group 0-5 yrs 6,14,15,17 .Rotavirus was a major cause of pediatric gastroenteritis and responsible for causing half of the cases to be suffered with acute diarrheal illness among hospitalized patients of 6-24 months of age 18 .Similarly, this study showed that the infection rate was found to be higher in age group 3-23 months.In the present study, a clear seasonal pattern in rotavirus diarrhea was seen.Rotavirus infection occurred all year round but peaked during the fall and winter months, from December to March.Our results are similar to those of studies conducted in Korea, China, and Thailand but differed from those of a Japanese study, in which rotavirus was rarely detected from September to December 5,17 .
Three year (2005-2008) data of RT-PCR genotyping showed that G12 strains as the most prevalent Genotype; accounting for one-half of circulating rotavirus detected which was consistent with the preceding studies 9,19,20,21 , where as detection of other G genotypes changed more markedly over time.Similarly, serotype G1 and G9 was the second most common serotype found in the study periods.Serotype G9 has also emerged as one of the common types in other countries during the recent years 22 .Similarly, of the three different P-types, P [8] was predominant in all year followed by P [6].Of combined genotypes, G12P [6] was the most prevalent accounting in all year.This showed that many different rotavirus types circulate simultaneously, particularly in developing countries.In addition, the prevailing types may differ considerably from one season to the next, even within the same geographical area.The type of rotavirus does not usually correlate with the severity of the disease 17,20 .
Despite the overall predominance of G12 strains, there were a total of 13 different G and P genotype combinations observed during 3 years period, of which G12P [6], G1P [8], G2P [4], and G9P [8], together accounted up to 75% of the rotavirus-positive specimens (Table 3).Although the relative frequency of each of the major G and P combinations of rotavirus strains circulating in Nepal varied in the previous two studies 9,19 and the current study, G12P [6], G12P [8], G1P [8], G2P [4], and G9P [6] rotavirus were all detected, indicating that there existed a large genetic pool of rotavirus genotypes in Nepal.In 2008 G1P [6] was found 4% which was not detected in 2006 and 2007.
Of rotavirus-positive samples, 4-9% remained untypeable for G type, 4-8% for P type and 8-12% for both G and P types.This could be due to few virus particles with intact RNA in the stool specimens, or the viruses not belonging to genotypes included in the primer set in RT-PCR.

Conclusion
The study demonstrates that rotavirus is the most prevalent causes of diarrhea in children less than 5 years of age.Rotavirus strain, study is essential for disease control program with continued identifi cation of the most common G and P serotypes circulating causing disease an important priority establish policy regarding future implementation of rotavirus vaccine in Nepal.
e. from November 2005 to October 2006, November 2006 to October 2007 and November 2007 to October 2008.The distribution of rotavirus among various age groups indicated that the highest number was found in age group 3-23 months.Age wise distribution showed that the infection rate was found to be highest in age group 6-11 months holding 51.24%, followed by age group 3-5 months (42.5%) in year 2005-06 similarly 12-23 months (46.4%) followed by age groups 3-5 months (44.4%) in year 2006-07 and in year 2007-08, age group between 6-23 months showed highest rate of infection holding same positive percentage i.e.39 %.This showed that rotavirus positive cases increasing slowly from age 3 months and decreasing after 23 months except in year 2006-07 where rotavirus positive was found to be highest in age group 12-23 months.The percentage of positive cases was shown in fi gure 2. From the fi gure the highest prevalence of rotavirus was seen in children age group between 6-11 months in the year 2006-2007, while the highest prevalence of rotavirus was seen in children age group between 12-23 months in the year 2005-2006 and 2007-2008.Lowest positive cases were seen in children between 0-2 months and 24-60 months in all periods.The positive rate was declined after 12-23 months for all periods.

Table 1 :
Distribution of G types among rotavirus-positive specimens from hospitalized children between 2005 and 2008

Table 2 :
Distribution of rotavirus P types among rotavirus-positive specimens of hospitalized children

Table 3 :
Distribution of major combination of G and P types among rotavirus-positive specimens of hospitalized children *** Strains could not be typed by this method.