Direct costs of hospitalization for rotavirus gastroenteritis in different health facilities in India.

BACKGROUND & OBJECTIVES: Diarrhoeal disease is the fifth leading cause of all mortality globally. To this burden, rotavirus contributes over half a million deaths annually. This pilot study was conducted to determine the economic burden of diarrhoeal episodes on families from different geographical regions accessing medical facilities in India. METHODS: Participants were enrolled from four study sites with eight reporting hospitals, categorized as non-profit and low cost, private and government facilities between November 2008 and February 2009. Questionnaires detailing healthcare utilization, medical and non-medical expenditure and lost income were completed by families of children < 5 yr of age hospitalized for gastroenteritis. All available faecal samples were tested for rotavirus. RESULTS: A total of 211 patients were enrolled. The mean total cost of a hospitalized diarrhoeal episode was ` 3633 (US$ 66.05) for all facilities, with a marked difference in direct costs between governmental and non-governmental facilities. Costs for rotavirus positive hospitalizations were slightly lower, at ` 2956 (US$ 53.75). The median cost of a diarrhoeal episode based on annual household expenditure was 6.4 per cent for all-cause diarrhoea and 7.6 per cent for rotavirus diarrhoea. Of the 124 samples collected, 66 (53%) were positive for rotavirus. INTERPRETATION & CONCLUSIONS: Data on direct costs alone from multiple facilities show that diarrhoeal disease constitutes a large economic burden on Indian families. Affordable, effective vaccines would greatly reduce the economic burden of severe gastroenteritis on patients, families and the government.

Analysis of genetic diversity and molecular evolution of human group B rotaviruses based on whole genome segments.

Group B rotavirus (GBR) is a rare enteric pathogen that causes severe diarrhoea, primarily in adults. Nearly full-length sequences of all 11 RNA segments were determined for human GBRs detected recently in India (IDH-084 in 2007, IC-008 in 2008), Bangladesh (Bang117 in 2003) and Myanmar (MMR-B1 in 2007), and analysed phylogenetically with the sequence data of GBRs reported previously. All RNA segments of GBR strains from India, Bangladesh and Myanmar showed >95 % nucleotide sequence identities. Among the 11 RNA segments, the VP6 and NSP2 genes showed the highest identities (>98 %), whilst the lowest identities were observed in the NSP4 gene (96.1 %), NSP5 gene (95.6 %) and VP8*-encoding region of the VP4 gene (95.9 %). Divergent or conserved regions in the deduced amino acid sequences of GBR VP1-VP4 and NSP1-NSP5 were similar to those in group A rotaviruses (GARs), and the functionally important motifs and structural characteristics in viral proteins known for GAR were conserved in all of the human GBRs. These findings suggest that, whilst the degree of genetic evolution may be dependent on each RNA segment, human GBR may have been evolving in a similar manner to GAR, associated with the similar functional roles of individual viral proteins.

Characterization of full-length VP4 genes of OP354-like P[8] human rotavirus strains detected in Bangladesh representing a novel P[8] subtype.

The G1 and G9 rotavirus strains MMC71 and MMC38 (subgroup II, NSP4 genogroup B), respectively, isolated from children in Bangladesh, were analyzed genetically. Full-length VP4 genes of these strains had 98.9% identity to each other and showed 83.9-89.4% identity to those of the P[4] and P[8] rotaviruses. Phylogenetic analysis of VP4 nucleotide sequences revealed that strains MMC38 and MMC71 were located in a lineage of P[8] strains. However, the cluster was highly divergent from the previously established P[8] strains. The VP8* portions of strains MMC38 and MMC71 showed more than 93.9% nucleotide sequence identity to OP354-like P[8] strains, and these strains were clustered into the same lineage. These findings indicate that the VP4 of these strains should be classified into a subtype of the P[8] genotype (P[8]b) that is distinct from that of common P[8] rotaviruses (P[8]a).

Full genomic analysis of a human group A rotavirus G9P[6] strain from Eastern India provides evidence for porcine-to-human interspecies transmission.

Deduced amino acid sequence and phylogenetic analyses of a group A rotavirus G9P[6] strain (designated as mcs/13-07), detected from a 3-year-old child in Eastern India, revealed a VP8* closely related to porcine P[6] strains (P[6] sublineage 1D), and the VP7 clustered with G9 lineage-III strains. To our knowledge, this is the first report of human P[6] strain clustering in sublineage Id. Thus, to further characterize the evolutionary diversity of strain mcs/13-07, all gene segments were analyzed. VP6 and NSP4 exhibited genetic relatedness to Wa-like human subgroup II strains, while VP1-3, NSP1-3 and NSP5 were closely related to porcine strains. Based on the new classification system of rotaviruses, mcs/13-07 revealed a G9-P[6]-I1-R1-C1-M1-A8-N1-T1-E1-H1 genotype with close similarity to human Wa-like and porcine Gottfried strains. Therefore, considering the porcine-like or porcine origin of multiple gene segments, it might be tempting to assume that strain mcs/13-07 represents a rare instance of whole-virus transmission from pig to human, after which the virus evolved with time. Alternatively, it is possible that strain mcs/13-07 resulted from multiple reassortment events involving human subgroup II and porcine P[6] strains. Nevertheless, detection of strain mcs/13-07 provides further evidence for complex interspecies transmission events, which are frequent in developing countries.

Whole genomic characterization of a human rotavirus strain B219 belonging to a novel group of the genus Rotavirus.

Novel rotavirus strains B219 and ADRV-N derived from adult diarrheal cases in Bangladesh and China, respectively, are considered to belong to a novel rotavirus group (species) distinct from groups A, B, and C, by genetic analysis of five viral genes encoding VP6, VP7, NSP1, NSP2, and NSP3. In this study, the nucleotide sequences of the remaining six B219 gene segments encoding VP1, VP2, VP3, VP4, NSP4, and NSP5 were determined. The nucleotide sequences of the group B human rotavirus VP1 and VP3 genes were also determined in order to compare the whole genome of B219 with those of group A, B, and C rotavirus genomes. The nucleotide and deduced amino acid sequences of all B219 gene segments showed considerable identity to the ADRV-N (strain J19) sequences (87.7-94.3% and 88.7-98.7%, respectively). In contrast, sequence identity to groups A-C rotavirus genes was less than 61%. However, functionally important domains and structural characteristics in VP1-VP4, NSP4, and NSP5, which are conserved in group A, B, or C rotaviruses, were also found in the deduced amino acid sequences of the B219 proteins. Hence, the basic structures of all B219 viral proteins are considered to be similar to those of the known rotavirus groups.

Detection of Genogroup I and II human picobirnaviruses showing small genomic RNA profile causing acute watery diarrhoea among children in Kolkata, India.

Picobirnaviruses (PBVs) with bisegmented small RNA genome profile (1.75 and 1.55kbp for segment 1 and 2, respectively) were detected from 1999 to 2003 in faecal specimens of acute watery diarrhoea cases, largely children (n=20) and an adult in Kolkata, India. Varying degrees of dehydration necessitated their visit to hospital for further treatment and management of acute watery diarrhoea. PBV was associated with rotavirus (n=3) or astrovirus (n=3) and with both in one case. No co-infection with norovirus, sapovirus or adenovirus was detected in the picobirnavirus positive cases. No co-infection with parasites (Cryptosporidium spp., Giardia spp., Entamoeba spp., helminths) or bacteria (Vibrio spp., Shigella spp., Escherichia coli) was detected among the picobirnavirus positive cases. There was a single instance of co-infection with Salmonella spp. (n=1). PBVs not associated with serious diarrhoea illness and showing large genome profile (2.3-2.6 and 1.5-1.9kbp for segment 1 and 2, respectively) have earlier been reported in adult individuals and recently among children from a slum community in Kolkata, India. The short genome profile PBVs associated with acute watery diarrhoea may be another emerging diarrhoeagenic virus in Kolkata, India. Molecular characterization using reported primers PicoB25-PicoB43 for Genogroup I and PicoB23-PicoB24 for Genogroup II in RT-PCR showed the presence of Genogroup I PBVs (n=6) and Genogroup II PBVs (n=5), while some could not be amplified (n=3) with these primers. Sequence analysis of Genogroup I amplicons indicated remarkable sequence heterogeneity. After more than a decade, four PBV positives of Genogroup II were detected during this study. Phylogenetic analysis showed varying degree of genetic diversity amongst PBV strains from Kolkata and other countries.

Molecular epidemiology of human picobirnaviruses among children of a slum community in Kolkata, India.

Picobirnaviruses are a group of unclassified, non-enveloped, small spherical viruses, 35-41 nm in diameter without any apparent surface morphology. They have characteristic bisegmented double stranded RNA genome of two types namely large profile (2.3-2.6 kbp for the larger and 1.5-1.9 kbp for the smaller segment, respectively) or small profile (1.75 and 1.55 kbp for segments 1 and 2, respectively). Human picobirnaviruses (n=12 positives; 2/56 diarrhoeic children and 10/607 non-diarrhoeic children) with large (n=11) or small (n=1) genome pattern were observed in faecal specimens of children from a slum community by silver stained PAGE gels. Faecal specimen from four asymptomatic cases (P597_02_IND, K135_02_IND, A373_03_IND, A356_03_IND) and one diarrhoeic case (K135_03_IND) had genogroup I picobirnaviruses (1-CHN-97 like) showing amplicons within the 201 bp region, with primers PicoB25-PicoB43, targeting the conserved domain of RNA-dependent RNA polymerase (RdRp) gene. It was interesting to note that only the PBV strain P597_02_IND from Kolkata with large genome was closely related to a reported strain (similarity with 2-GA-91 from USA was 87% at the nucleotide level and 90% at the amino acid level). Sequence analysis showed three conserved amino acid domains as well as a highly conserved D-S-D motif, characteristic of RNA-dependent RNA polymerase gene of bisegmented, double stranded RNA viruses. Sequence data of the picobirnavirus A356_03_IND indicated strong heterogeneity with all other picobirnavirus strains sequenced till date. After nearly a decade a genogroup II picobirnavirus strain (R227_03_IND) was isolated from a diarrhoea case in the community, with small genome profile and amplified with specific primers PicoB23-PicoB24; but the sequence data showed that it was divergent from the hitherto reported prototype strain 4-GA-91 of genogroup II human picobirnaviruses.

Molecular epidemiology of human astrovirus infections in Kolkata, India.

UNLABELLED: The study is aimed to determine the seasonal distribution and clinical characteristics of astroviruses associated with acute watery diarrhoea among children in Kolkata and characterize them at the molecular level. METHOD OF STUDY: Faecal specimens of acute watery diarrhoea cases (n=857) and non-diarrhoeic samples (n=211) from the hospitals and a nearby field community were screened with IDEIA Astrovirus detection kit; astrovirus co-infections with rotavirus and/or picobirnavirus were detected by RNA-PAGE and silver staining. Further RT-PCR was carried out using specific primers, viz. Mon340 (+) and Mon348 (-) targeting a highly conserved domain of ORF1a (289 bp) of human astroviruses. RESULTS: Astrovirus infection was detected in 50 cases (50/857); astroviruses were detected mostly in children aged 6-12 months (50%); all non-diarrhoeic samples (n=211) were negative for astrovirus. In 52% of astrovirus positive cases, the virus was detected as the sole agent; mixed infections were also detected with other diarrhoeic pathogens such as rotavirus (32%), picobirnavirus (2%), rotavirus and picobirnavirus (2%), picobirnavirus and Enterotoxigenic E. coli (ETEC) (2%), rotavirus and ETEC (2%), rotavirus and Enteroaggregative E. coli (EAEC) (2%), Enteropathogenic E. coli (EPEC) (2%), Shigella flexneri type 3a (2%) and Ascaris (2%). RT-PCR and sequencing of amplicons of astroviruses from Kolkata, with specific primers targeted to the conserved domain of ORF1a (289 bp) of the astrovirus genome, showed maximum homology to the astrovirus strain ("5-158") from Seoul (98%). RESULTS AND CONCLUSIONS: Clinical characteristics of the diarrhoeic children in Kolkata indicated that astrovirus infections were detected throughout the year and were associated with varying degree of dehydration and acute watery diarrhoea. In-depth molecular epidemiological surveillance of astroviruses in Kolkata is essential for better understanding of their overall genetic nature.

Predominance of unusual rotavirus G1P[6] strain in North India: An evidence from hospitalized children and adult diarrheal patients.

Group A Rotavirus remains the leading cause of gastroenteritis in children and accounts for 0.2 million fatalities each year; out of which, approximately 47,100 deaths occur in India. In adults also, rotavirus is reported to be responsible for diarrhea severe enough to require hospitalizations. India has recently introduced rotavirus vaccine in the Universal Immunization Programme and Himachal Pradesh became the first Indian state to implement this project. This study is an attempt to provide the pre-vaccination data on rotavirus gastroenteritis burden and circulating genotypes in Himachal Pradesh, India. A total of 607 faecal specimens (247 children ≤5years, 50 older children and 310 adults) from hospitalized diarrheal patients from Himachal Pradesh, India were screened for rotavirus using ELISA and RT-PCR. The positive samples were further G/P genotyped using semi-nested PCR. Rotavirus was detected in 25.2% and 28.3% of samples with ELISA and RT-PCR, respectively. In children, rotavirus frequency was significantly high with positivity in 49.0% cases whereas 14.0% adult samples have rotavirus in them. Genotyping of the positive samples revealed predominance of G1 (66.0%) and P[6] (66.7%) genotypes. The most common G and P combination was G1P[6] (62.8%) followed by G1P[8] (16.5%), G9P[6] (7.4%) and G12P[6] (5.0%). Molecular analysis reveals the belonging of P[6] strains in Lineage 1a. This pre-vaccination data on rotavirus prevalence and diversity would be helpful for assessing the affect of vaccination on the disease burden and its comparison with post-vaccination data of circulating genotypes would help in studying the effect on diversity of rotavirus strains possibly due to vaccine selection pressure.

Novel human astroviruses: challenges for developing countries.

Astroviruses have been gaining widespread importance over the past few decades owing to their detection through advanced molecular techniques. The association of astrovirus-associated enteric infections have been reported from various settings among different age groups. The tremendous efforts of scientists from different countries to detect and characterize these star-like viruses in the course of surveillance has shown the emergence of novel astroviruses from varied host species, necessitating changes in the classification to update their taxonomy. The public health importance of these viruses implies new control measures are essential to reduce disease burden in developing countries.

Novel human astrovirus strains showing multiple recombinations within highly conserved ORF1b detected from hospitalized acute watery diarrhea cases in Kolkata, India.

Human astroviruses (HAstVs) associated with acute watery diarrhea among hospitalized infants, children and adults as sole or mixed infection, were earlier reported from Kolkata, India. Further, novel recombinations have been detected through sequencing of the highly conserved ORF1b (RdRp) region of seven human astrovirus strains in Kolkata, India. Primers were designed and the ORF1b region was amplified by RT-PCR and sequenced. To examine the evolutionary pressures influencing the evolution of human astroviruses we implemented evolutionary genetics analysis. Maximum recombination break points detected in Kolkata strain IDH1300 were 8 and a single break point location was detected at 1205nt position. Partition-wise phylogenetic analyses of the IDH1300 Kolkata strain did not show close homology to the reference strains. Further phylogenetic analyses of full length ORF1b region of the seven human astrovirus strains showed that they formed a close cluster with each other and displayed a separate lineage in comparison to reference human astrovirus strains worldwide. This study shows the emergence of novel recombinant human astrovirus strains in Kolkata, India, warranting stringent surveillance to monitor the genetic diversity of human astrovirus strains infecting different age groups.

Whole genomic analyses of asymptomatic human G1P[6], G2P[6] and G3P[6] rotavirus strains reveal intergenogroup reassortment events and genome segments of artiodactyl origin.

Although P[6] group A rotaviruses (RVA) cause diarrhoea in humans, they have been also associated with endemics of predominantly asymptomatic neonatal infections. Interestingly, strains representing the endemic and asymptomatic P[6] RVAs were found to possess one of the four common human VP7 serotypes (G1-G4), and exhibited little antigenic/genetic differences with the VP4 proteins/VP4 encoding genome segments of P[6] RVAs recovered from diarrhoeic children, raising interest on their complete genetic constellations. In the present study, we report the overall genetic makeup and possible origin of three such asymptomatic human P[6] RVA strains, RVA/Human-tc/VEN/M37/1982/G1P2A[6], RVA/Human-tc/SWE/1076/1983/G2P2A[6] and RVA/Human-tc/AUS/McN13/1980/G3P2A[6]. G1P[6] strain M37 exhibited an unusual genotype constellation (G1-P[6]-R1-C1-M1-A1-N1-T2-E1-H1), not reported previously, and was found to originate from possible intergenogroup reassortment events involving acquisition of a DS-1-like NSP3 encoding genome segment by a human Wa-like RVA strain. On the other hand, G2P[6] strain 1076 exhibited a DS-1-like genotype constellation, and was found to possess several genome segments (those encoding VP1, VP3, VP6 and NSP4) of possible artiodactyl (ruminants) origin on a human RVA genetic backbone. The whole genome of G3P[6] strain McN13 was closely related to that of asymptomatic human Wa-like G3P[6] strain RV3, and both strains shared unique amino acid changes, which might have contributed to their attenuation. Taken together, the present study provided insights into the origin and complex genetic diversity of P[6] RVAs possessing the common human VP7 genotypes. This is the first report on the whole genomic analysis of a G1P[6] RVA strain.

Diversity of circulating rotavirus strains in children hospitalized with diarrhea in India, 2005-2009.

BACKGROUND: India accounts for 22% of the 453,000 global rotavirus deaths among children <5 years annually. The Indian Rotavirus Strain Surveillance Network provides clinicians and public health partners with valuable rotavirus disease surveillance data. Our analysis offers policy-makers an update on rotavirus disease burden with emphasis on regional shifts in rotavirus strain epidemiology in India. METHODS: Children <5 years requiring hospitalization for acute gastroenteritis were selected from 10 representative hospitals in 7 cities throughout India between November 2005 through June 2009. We used a modified World Health Organization protocol for rotavirus surveillance; stool specimens were collected and tested for rotavirus using enzyme immunoassay and reverse-transcription polymerase chain reaction. RESULTS: A total of 7285 stool specimens collected were tested for rotavirus, among which 2899 (40%) were positive for rotavirus. Among the 2899 rotavirus detections, a G-type could not be determined for 662 (23%) and more than one G type was detected in 240 (8%). Of 1997 (69%) patients with only one G-type, the common types were G1 (25%), G2 (21%), G9 (13%), and G12 (10%). The proportion of rotavirus infections attributed to G12 infections rose from 8% to 39% in the Northern region and from 8% to 24% in the Western region. CONCLUSIONS: This study highlights the large, ongoing burden of rotavirus disease in India, as well as interesting regional shifts in rotavirus strain epidemiology, including an increasing detection of G12 rotavirus strains in some regions. While broad heterotypic protection from rotavirus vaccination is expected based on pre- and post-licensure data from other settings, effectiveness assessments and rotavirus strain monitoring after vaccine introduction will be important.

Emerging trends in the epidemiology of human astrovirus infection among infants, children and adults hospitalized with acute watery diarrhea in Kolkata, India.

Human astroviruses (HAstVs) have now emerged as another common cause of non-bacterial acute gastroenteritis (AGE) in humans worldwide. This study investigated the epidemiology and genetic diversity of human astrovirus strains circulating among infants, younger children (up to 6 years), older children and adolescents (>6-17 years) and adults (18 years and above) hospitalized for diarrhea and their role in AGE in Kolkata, India. A total of 2535 fecal samples were screened for the presence of known enteric viral, bacterial and parasitic etiologies by conventional microbiological assays and molecular methods. The overall incidences of sole or mixed infection of HAstV with known enteric viral, bacterial and parasitic pathogens were detected in 60 cases (2.4%) among all age groups. The clinical symptoms of astrovirus-associated acute watery diarrhea cases were recorded for all sole and mixed infection cases. A high number of sole (n = 13/60 [21.7%]) and mixed infection cases (n = 22/60 [36.7%]) were observed in adults (18 years old or more). Considering all age groups, 18 sole infection cases (n = 18/60 [30%]) and 42 mixed infection cases (n = 42/60 [70%]) with Rotavirus (n = 11/25 [44%]), Vibrio cholerae O1 (n = 6/24 [25%]) Cryptosporidium spp and Giardia lamblia (n = 5/13 [38.4%]) were observed. Further, eleven HAstV samples from infants and children (up to 6 years), children and adolescents (>6-17 years) and adults (18 years and above) were analyzed for their sequences of overlap region between ORF1b (RdRp) and ORF2 (capsid). Among these, ten strains were found to have close genetic relatedness to the Japanese strain HAstV_G1 [AB009985]. Additionally, the IDH2211 Kolkata strain showed a close genetic match with the Thai HAstV_G3 strain [EU363889]. Our study reports show that HAstVs as the sole agent and as mixed infection with other known enteric viral, bacterial, parasitic pathogens are also responsible for AGE among infants, children, adolescents and adults in Kolkata, India.

Emergence of novel Norovirus recombinants with NVGII.1/NVGII.5 RdRp gene and NVGII.13 capsid gene among children and adults in Kolkata, India.

Norovirus (NoV) is a leading cause of non bacterial acute gastroenteritis in human beings. Molecular characterization of NoVs following continuous, stringent surveillance had earlier shown that novel strains representing an intergenogroup as well as GII NoV intergenotype recombinants were in circulation among acute watery diarrhoea cases in Kolkata, India. The present study documents characterization of two recombinant NoV strains (Hu/NoV/ IDH1501/2009/IND and Hu/NoV/IDH1873/2009/IND) along with other interesting GII NoV strains. Similarity plot and phylogenetic analysis confirmed the strain Hu/NoV/IDH1501/2009/IND as a NoV recombinant strain with genes for RNA dependent RNA polymerase (RdRp) GII.1-like and capsid GII.13-like; the strain Hu/NoV/IDH1873/2009/IND was a NoV recombinant strain with its RdRp gene GII.5-like and capsid gene being GII.13-like. Clinical symptoms chiefly associated with the cases that had NoV infection was varying duration of diarrohea and vomiting with some dehydration.

Emergence of unique variants and inter-genotype recombinants of human astroviruses infecting infants, children and adults in Kolkata, India.

Two conserved genomic fragments viz. 289bp of ORF1a and 449bp of ORF2 amplified by RT-PCR showed emergence of interesting recombinant strains representing new and novel genetic variants (n=5) within eight different genotypes of astroviruses known to date. HAstV-positive cases with ORF1a [HAstV genotype G2 or G8] and ORF2 [HAstV genotype G1, G2, or G3] were detected as sole or mixed infection among infants, children and adults in Kolkata with severe illness owing to acute gastroenteritis that required hospitalization for treatment between 2007 and 2009. The twelve interesting recombinants were of type HAstV _ ORF1a _ ORF2 as HAstV _ G8_ G2 (n=1), HAstV _ G8_ G1 (n=10) and HAstV _ G2_ G3 (n=1).

Detection and molecular characterization of multiple strains of Picobirnavirus causing mixed infection in a diarrhoeic child: Emergence of prototype Genogroup II-like strain in Kolkata, India.

BACKGROUND: Picobirnaviruses (PBVs) associated with viral gastroenteritis were reported from humans and several animal species to date. PBVs belonging to family Picobirnaviridae under proposed order Diplornavirales are small, non-enveloped, with bisegmented dsRNA genome. METHODS: PBV was detected by polyacrylamide gel electrophoresis (PAGE) and silver staining. Confirmatory RT-PCR using primer pair PicoB25 (+) and PicoB43 (-) for genogroup I PBV and PicoB23(+) and PicoB24(-) for genogroup II PBV, resulted in amplicons of 201bp and 369bp respectively. The amplicons of genogroup I PBV were cloned and sequenced; amplicon of genogroup II PBV was directly sequenced. Further, the phylogenetic relationship and genetic diversity of strains from Kolkata was compared with hitherto reported PBV strains. RESULTS: In PAGE, a faecal specimen showed three sets of PBV with large profile bisegmented genomic RNA with slight variation in migration pattern. Molecular cloning experiments confirmed that PBV/ Human/INDIA/GPBV6/2007 had mixed infection comprising four different strains of PBV genogroup I [GPBV6C1P-GPBV6C4P] and one PBV genogroup II strain [GPBV6G2P]. CONCLUSION: Sequence comparison and phylogenetic analysis of gene segment 2 of GPBV6 clones (C1, C2, C3 and C4) revealed low nucleotide identities (59-63%) and distant genetic relatedness to other human and porcine genogroup I picobirnaviruses. The strain GPBV6G2P represents another PBV genogroup II strain after prototype strain 4-GA-91/USA as genogroup II PBVs have seldom been reported to date, except from Kolkata, India and Netherlands. We are reporting the first incidence of detection of multiple strain (mixed) infection of picobirnavirus [genogroups I and II] from a diarrhoeic child in a slum community of Kolkata, India.

Identification of P[8]b subtype in OP354-like human rotavirus strains by a modified RT-PCR method.

In our previous study, a novel P[8] subtype, i.e., P[8]b was identified for human rotavirus strains MMC38 and MMC71 detected in Bangladesh, of which the P types could not be determined by conventional RT-PCR genotyping methods. In the present study, a modified multiplex RT-PCR method was developed to detect P[8]b as well as common human rotavirus P types. With this method, P[8]b was detected in three strains among the 26 rotavirus specimens which had been judged as mixed P types in the previous study in Bangladesh. The VP4 nucleotide sequences of these strains showed more than 98.9% identities to those of strains MMC38 and MMC71. The newly designed RT-PCR method was considered as useful for identifying P[8]b and avoiding misclassification by the conventional RT-PCR genotyping methods.

Emerging trends in the etiology of enteric pathogens as evidenced from an active surveillance of hospitalized diarrhoeal patients in Kolkata, India.

BACKGROUND: This study was conducted to determine the etiology of diarrhoea in a hospital setting in Kolkata. Active surveillance was conducted for 2 years on two random days per week by enrolling every fifth diarrhoeal patient admitted to the Infectious Diseases and Beliaghata General Hospital in Kolkata. RESULTS: Most of the patients (76.1%) had acute watery diarrhoea in association with vomiting (77.7%) and some dehydration (92%). Vibrio cholerae O1, Rotavirus and Giardia lamblia were the important causes of diarrhoea. Among Shigella spp, S. flexneri 2a and 3a serotypes were most predominantly isolated. Enteric viruses, EPEC and EAEC were common in children <5 year age group. Atypical EPEC was comparatively higher than the typical EPEC. Multidrug resistance was common among V. cholerae O1 and Shigella spp including tetracycline and ciprofloxacin. Polymicrobial infections were common in all age groups and 27.9% of the diarrhoea patients had no potential pathogen. CONCLUSIONS: Increase in V. cholerae O1 infection among <2 years age group, resistance of V. cholerae O1 to tetracycline, rise of untypable S. flexnerii, higher proportion of atypical EPEC and G. lamblia and polymicrobial etiology are some of the emerging trends observed in this diarrhoeal disease surveillance.

Detection of closely related Picobirnaviruses among diarrhoeic children in Kolkata: evidence of zoonoses?

The genus, Picobirnavirus (PBV), Spanish 'pico'='small', birna for 'bipartite RNA' genome, belongs to the family Picobirnaviridae under the proposed order Diplornavirales. PBV infections have been reported from diarrhoeic animal species and humans as well as from asymptomatic cases. The detection of Picobirnaviruses (PBVs) in diarrhoeic faecal specimens from children aged <5 years, suggestive of zoonotic transmission is being reported. 23 Picobirnavirus positive faecal specimens were detected by polyacrylamide gel electrophoresis (PAGE) and silver staining from a set of 1112 faecal specimens collected from an urban slum community in Kolkata between July and October 2007. The Picobirnaviruses showed either large profile (n=22) or small profile (n=1) for their bisegmented genomic double-stranded RNA (dsRNA). 13/23 positives were amplified by reverse transcription polymerase chain reaction (RT-PCR) as 201bp amplicon with genogroup I primers [PicoB25(+) and PicoB43(-) specific for RNA dependent RNA polymerase (RdRp) gene fragment encoded by genomic segment 2] and seven amplicons were sequenced [GPBV1-5, 7 and 8]. Sequence analyses showed that four PBV strains [GPBV1-3 and 8] resembled different clones of porcine PBV strains (D4, D6 and C10) reported in 2008 from Hungary and two PBV strains [GPBV4 and 7] resembled human PBV strains (P597, Kolkata and 2-GA-91, USA) with the maximum nucleotide (nt) identity ranging from 78% to 92%. One strain GPBV5 clustered with human PBVs and porcine PBVs that were reported from Hungary, Venezuela and Argentina showing close homology to human-like PBVs. Therefore, the close monitoring of their global spread as well as in-depth molecular characterization is essential for better understanding of emerging PBV strains.