Molecular characterization of G11P[25] and G3P[3] human rotavirus strains associated with asymptomatic infection in South India.

Rotaviruses are the major etiological agents of diarrhea in children less than 5 years of age. Two unusual rotavirus strains not previously reported in India, G11P[25] (CRI 10795) and G3P[3] (CRI 33594) were isolated from faecal samples of asymptomatic children in India. The strains were characterized by sequence analysis of the genes encoding the VP7, VP4, VP6, and NSP4. The G11P[25] strain was closely related to the human G11P[25] strains from Bangladesh (with 98% identity at the nucleotide [nt] level and the amino acid [aa] level for the VP7 gene and 96% identity at the nt and 98% at the aa level for the VP4 gene). The G3P[3] strain was found to be related to a G3P[3] strain isolated in Thailand (CMH222; 88% identity at the nt level and 97% at aa level for the VP7 gene and 84% identity at the nt level and 90% at the aa level for the VP4 gene). Phylogenetic analysis of the VP6 and the NSP4 genes revealed that the Vellore G11P[25] strain was of VP6 subgroup II and NSP4 genotype B. The G3P[3] strain was identified as NSP4 genotype C and the VP6 gene showed 97% identity at the deduced amino acid level with strain CMH222 (Thailand) strain but did not cluster with sequences of SGI, SGII, SGI+II or SG-nonI/nonII. Both strains had gene segments of animal rotavirus origin suggesting inter-species transmission of rotavirus, and in the case of G11P[25] possibly underwent reassortment subsequently with human strains resulting in an animal-human hybrid strain.

Modification of rotavirus multiplex RT-PCR for the detection of G12 strains based on characterization of emerging G12 rotavirus strains from South India.

Rotaviruses are the major etiological agents of diarrhea in children less than 5 years of age. The commonest G types in humans are G1-4 and G9. G12 is a rare human rotavirus (HRV) strain first reported in the Philippines. In this study, 13 G12 strains obtained from a community-based cohort and a hospital-based surveillance system in 2005 were characterized by phylogenetic analysis of partial nucleotide sequences of VP7, VP6, and NSP4 genes. Sequence and phylogenetic analysis of VP7 gene sequences showed that these southern Indian strains had the greatest homology with G12 strains recently reported from eastern India (97-99% identity both at the nucleotide level and deduced amino acid level) and less homology with the prototype G12 strain, L26 (89-90% identity at the nucleotide level and 90-94% at the deduced amino acid level). Phylogenetic analysis of the VP6 and the NSP4 genes revealed that the Vellore G12 strains belonged to VP6 subgroup II and NSP4 genotype B. The P types associated with these strains were P[6] and P[8]. A G12 type-specific primer was designed for inclusion in an established VP7 G-typing multiplex RT PCR, and tested against a panel of known G types and untyped samples and was found to detect G12 strains in the multiplex-PCR. Close homology of the South Indian G12 strains to those from Kolkata suggests that G12 HRV strains are emerging in India. Methods for characterization of rotaviruses in epidemiological studies need to be updated frequently, particularly in developing countries.

Human caliciviruses in symptomatic and asymptomatic infections in children in Vellore, South India.

Pediatric gastroenteritis is a major cause of childhood morbidity and mortality worldwide, especially in developing countries. It has been increasingly recognised that human caliciviruses (HuCV), comprising noroviruses (NoV), and sapoviruses (SaV), are important in both outbreak and non-outbreak settings. This study aimed to characterise caliciviruses detected in the faeces of hospitalized children and children in the community in India. This study examined 350 faecal samples from children presenting to the hospital with acute gastroenteritis and 673 samples collected from children in the community, 500 from children with diarrhea, and 173 samples from children without diarrhea. Strain characterisation was performed by reverse transcription-polymerase chain reaction (RT-PCR) and partial sequencing of the gene encoding the RNA-dependent RNA polymerase (RdRp) and/or a region spanning the open reading frames (ORFs) 1 and 2 (ORF1/ORF2) junction. A total of 68 of 350 specimens (19.4%) from hospitalized children were positive, and SaV and NoV accounted for 5.1 and 15.1% of the infections, respectively. Mixed infections of HuCVs with other enteric pathogens were seen in 9.4% of the total children tested. Sixty-eight out of 673 (10.1%) samples collected from children in the community were positive for caliciviruses, and SaV and NoV accounted for 3.4 and 6.6% of the infections. In the community cohort 55/500 (11%) and 13/173 (7.5%) were from symptomatic and asymptomatic children, respectively, and SaVs accounted for 17/500 (3.4%) and NoVs for 38/500 (7.6%) of the symptomatic infections. This is the first report of genotyping of circulating caliciviruses in both hospital and community in India and has increased the evidence for the role of these viruses in pediatric gastroenteritis in India.

Comparative study of the epidemiology of rotavirus in children from a community-based birth cohort and a hospital in South India.

Rotavirus gastroenteritis is the major cause of severe dehydrating diarrhea in children worldwide. This study compares rotavirus diarrhea in 351 children in a community-based cohort and 343 children admitted to a hospital during the same period. Clinical information and fecal specimens were obtained during diarrheal episodes. Fecal samples were screened for VP6 antigen, and the positive samples were G and P typed by reverse transcription-PCR. Rotavirus was detected in 82/1,152 (7.1%) episodes of diarrhea in the community and 94/343 (27.4%) cases in the hospital. The median age of affected children (7.5 versus 10.5 months) and the mean severity of symptoms (Vesikari score, 7.6+/-3.4 versus 11+/-2.5) were lower in the community. A larger proportion of children in the community were breast-fed than were children admitted to the hospital (73% versus 34.8%). In the community, the genotypes identified in symptomatic patients, in order of frequency, were G1 (36.5%), G10 (17.1%), G2 (15.9%), and G9 (7.3%) and mixed infections (7.3%). The most common G-P combinations were G1P[8], G2P[4], G1P[4], and G10P[11]. The distribution of G types from hospitalized children was G1 (46.8%), G9 (19.1%), G2 (8.5%), G10 (1.1%), and 4.3% mixed infections. The most common G-P combinations were G1P[8] and G9P[8]. This study documents significant genetic heterogeneity of rotaviruses in the community and the hospital. G10P[11] strains resembling a vaccine candidate strain caused disease in the community, indicating the need for careful epidemiological studies as well as safety studies for the vaccine candidates.

Multilocus genotyping of Cryptosporidium sp. isolates from human immunodeficiency virus-infected individuals in South India.

This study characterized cryptosporidial infections in 48 human immunodeficiency virus-infected individuals in India by multilocus genotyping. Cryptosporidium hominis, C. parvum, C. felis, C. muris, and C. meleagridis were identified. Cpgp40/15 PCR-restriction fragment length polymorphism identified six subgenotypes. Cryptosporidial diarrhea was associated with decreased CD4 counts, below 200 (P = 0.009), but not high viral loads.

Water contamination in urban south India: household storage practices and their implications for water safety and enteric infections.

Water contamination, at source and during household storage, is a major cause of enterically transmitted infections in developing countries. This study assessed contamination of the municipal water in a south Indian town, which obtains its water intermittently from a surface lake and by pumping subsurface water from a dry river bed, and monitored microbial contamination during household storage. All samples of the 'treated' municipal water were contaminated when freshly pumped, and on household storage, 25/37 (67%) showed increased contamination during storage periods from 1 to 9 days. Household storage in brass, but not in containers of other materials significantly decreased contamination of water (p = 0.04). This was confirmed in the laboratory by testing water seeded with 10(3) to 10(5) Escherichia coli per 100 ml stored in containers of different materials (p < 0.01). Despite the requirements for provision of safe drinking water in municipal areas, in practice the water supplied in Vellore is contaminated and current household storage practices increase the level of contamination in at least two-thirds of households. The implementation of locally appropriate point-of-use disinfection and safe household storage practices in developing countries is an urgent need to ensure a safe, reliable year-round supply in areas where clean water is not available.

Quantitation of group A rotavirus by real-time reverse-transcription-polymerase chain reaction: correlation with clinical severity in children in South India.

The epidemiology and pathogenesis of rotaviruses are not completely understood, although recent developments in polymerase chain reaction (PCR) techniques now make it possible to quantify the viral load during an infective episode and investigate its relevance to clinical features of the disease. We studied rotavirus-positive stool samples collected from 10 children without symptoms of gastroenteritis and from 81 children with acute gastroenteritis and in whom the clinical severity of disease was recorded. A semi-quantitative real-time reverse-transcription (RT)-PCR was used to estimate the rotavirus load and to assess its correlation with the Vesikari score for severity of diarrhoea. There was a significant negative correlation (r = -0.80, P < 0.001) between severity and the PCR cycle at which the PCR amplicons were detectable (crossing point) on the assay, indicating that children with more severe diarrhoea excrete more virus than children with less severe disease.