All children aged £59 months who presented to a participating CRS with acute gastroenteritis (³3 loose stools in a 24 hour period for 5 or less days), and required hospitalization for diarrhea management, were enrolled after obtaining informed and written consent from the accompanying parent or guardian. Study medical officers enrolled eligible children and collected clinical and demographic details on standardized clinical recruitment forms (CRF).

Whole stool specimens (~5 mL) were collected and transported within 2 hours to the testing laboratory or stored in a refrigerator at 40C until transportation. Samples stored at 40C were transported in boxes with ice packs at weekly or fortnightly intervals to the testing laboratories. All stool samples were subjected to rotavirus screening using commercial enzyme immunoassay (Premier Rotaclone, Meridian Biosciences) kits following the manufacturer’s instructions. Rotavirus positive samples were subjected to rotavirus genotyping for VP7 (G-typing) and VP4 (P-typing) by Reverse-transcription polymerase chain reaction (RT-PCR) [5,6]. Initially all positives were genotyped but subsequently it was decided to restrict genotyping to every third positive sample at each testing laboratory. There were no gender, age, region-wise or seasonal considerations for choosing samples for genotyping. Aliquots of all samples were stored at -700C.

Data captured on the CRF were entered at each of the regional and referral laboratories using the online data capture portal hosted on the NIE web server. Site- specific summary data on diarrheal hospital admissions and rotavirus positivity data from laboratories was received at the Coordination Center (NIE) on a monthly basis.

Statistical analysis: Data were analyzed to assess the proportions of rotavirus-positive cases in terms of demographic factors, symptoms, disease severity (Vesikari score), median duration of hospitalization, genotype status and also by season and regions. Proportion ratios (PR) were calculated to compare the strength of association of severe infection in rotavirus infected and uninfected children with seasonal rotavirus burden and length of hospitalization. Analyses were carried out using MS Excel 2007, SPSS v. 17.0 and Stata v 10.0.

Results

Details of case enrolment and testing are provided in Fig. 1. Rotavirus was detected in 39.6% (4042/10207) of diarrheal cases. Rotavirus infection was detected throughout the year in all CRS. The detection rates were higher during December- February (56.4%; 1668/2959).

Fig. 1 Flow diagram summarizing case enrollment and laboratory testing.

Most cases of rotavirus infection (41.5%; 3404/8206) occurred among children less than 2 years of age (Table I). The highest positivity (46.7%) was observed among children between 12 and 23 months of age. Among infants aged <3 months and <6 months, the proportion of rotavirus positivity was 17.4% (127/729) and 27.4% (497/1814) respectively. There were 118 neonates, and 22.9% (27/118) of them were rotavirus positive. The median (IQR) age for rotavirus infection was 12 (8,18) months, which was not significantly different from that seen among cases of non-rotavirus gastroenteritis [median 12 mo. IQR (6,21) mo, P=0.51]. Hospital admissions due to rotavirus gastroenteritis among boys (2520/6363) outnumbered that among girls (1522/3844) with no significant difference in rotavirus positivity rates between the two genders (39.6% vs 39.6%; P=0.992).

Burden of rotavirus varied significantly across regions and seasons (Table I). Rotavirus infections usually occurred more commonly during the cooler months of December - February (56.4%), followed by September -November (38.4%).

TABLE I	Rotavirus Positivity in Children Hospitalized with Acute Gastroenteritis 

Variables	Levels	N	Rotavirus
			positivity, n (%)
Age (mo)	0-2	729	127 (17.4)
	3-5	1085	370 (34.1)
	6-11	3148	1393 (44.3)
	12-23	3244	1514 (46.7)
	≥24	2001	638 (31.9)
	Median (IQR)		12 (8 – 18)
Gender	Male	6363	2520 (39.6)
	Female	3844	1522 (39.6)
Disease	Mild [0-5]	433	123 (28.4)
severity	Moderate [6-10]	3779	1334 (35.3)
	Severe [11-15]	5509	2394 (43.5)
	Very Severe [16-20]	484	191 (39.5)
Treatment	Oral	1987	788 (39.7)
	Intravenous	8220	3254 (39.6)
Season	Dec-Feb	2959	1668 (56.4)
	Mar-May	1838	694 (37.8)
	Jun-Aug	2239	461 (20.6)
	Sep-Nov	3171	1219 (38.4)
Region	East	2032	827 (40.7)
	West	1755	689 (39.3)
	South	4080	1521 (37.3)
	North	2340	1005 (42.9)
Oral RV vaccination	Yes	340	111 (32.6)

Analysis of diarrheal disease severity showed that the proportion rotavirus positive (64%) was greater among children with very severe or severe disease compared to children with mild to moderate infection (36%). A proportion ratio analysis revealed that proportion of severe rotavirus gastroenteritis was more during December-February (ratio: 1.75; CI: 1.65- 1.85), and these children were likely to stay in the hospital for ³3 days (ratio: 1.72; CI: 1.60-1.85) (Table II).

TABLE II Proportion Ratio Analysis

Only 3.3% (340/10206) of enrolled children had a history of rotavirus vaccination, and among them 111 children (32.6%) were rotavirus positive. Majority of vaccinated children were seen at private hospitals (87.9 %).

During the reporting period, 15 (0.15%) enrolled children died during their hospital stay. Rotavirus antigen was detected in stool from four children. Twelve children were below one year of age with a median (IQR) age of 5 (1,7) months. On admission, nine children had severe to very severe diarrhea (median (IQR) Vesikari score 15 (14,15)). Thirteen children with severe diarrheal disease (median (IQR)) Vesikari score 14 (10,15) had received intravenous fluids and the remaining two children with mild to moderate diarrhea had received oral rehydration. Analysis of the cause of death revealed that the children had died due to serious complications viz. sepsis and shock (10 cases), sepsis and meningitis (3 cases), bronchopneumonia (1 case), and milk-aspiration (1 case).

Analysis of overall distribution of various rotavirus genotypes showed the preponderance of G1P[8] strains (62.7%) followed by G2P[4] strains (7.6%) (data not shown). Among neonates hospitalized with gastroenteritis, eight genotypes were observed with G1P[8] (45.5%; 10/22) as the commonest strain.

Using a standardized approach for patient enrollment and testing in country-wide surveillance, rotavirus was detected in 39.6% of children admitted with diarrhea. This is consistent with findings from the earlier phase of rotavirus surveillance in India [4-6]. Our report highlights the substantial rotavirus disease burden in India. In the present round of surveillance, although most cases of rotavirus gastroenteritis were seen among children between 12 and 23 months of age, it was documented in all age sub-groups, including neonates. These rates are; however, higher than previously reported from India [5-7]. Neonatal infections which are usually mild or asymptomatic are caused by different nursery rotavirus strains, but in this study, nearly half the neonates (10/22 genotyped) had gastroenteritis due to G1P[8], which was also the predominant genotype circulating among older children. These findings are in agreement with data from the previous surveillance that reported early incidence of rotavirus infection in India [6].

Analysis of diarrheal disease severity showed that children with rotavirus infection have severe disease and the occurrence of severe rotavirus gastroenteritis was more commonly observed during December – February period that represents cooler season in India. The seasonal pattern observed in this surveillance period, with more infections occurring during cooler months was similar to the observation during the previous iteration of the NRSN, and is consistent with reports from most parts of the world [6,8,9].

The reported low mortality among hospitalized children with acute gastroenteritis could not be attributed to rotavirus infection. This probably reflects that an effective diarrheal disease management protocol is practiced in the health care facilities participating in this surveillance. It would be necessary to study the community burden of rotavirus gastroenteritis and related morbidity and mortality outcomes to know the true burden in the community settings and non-hospitalized children. The limitation of the present analysis is that the findings that have been presented in this paper represent interim analysis of data.

The data from the expanded NRSN was already available with policy makers before the recent decision to introduce rotavirus vaccine in the UIP [10]. Continued surveillance and studies among vaccinated children will generate evidence regarding impact of rotavirus vaccine rolled out through the national program on morbidity and mortality due to rotavirus infections in young Indian children.

The successfully implemented NRSN surveillance platform will continue to generate data on trends in rotavirus disease burden and its correlates. It will also contribute significantly in the assessment of the impact of the rotavirus vaccine after implementation.