Indrani Banerjee

New oligonucleotide primers for P-typing of rotavirus strains: Strategies for typing previously untypeable strains

BACKGROUND The use of molecular methods for rotavirus characterisation provides increased sensitivity for typing, and allows the identification of putative reassortant strains. However, due to the constant accumulation of point mutations through genetic drift; and to the emergence of novel genotypes; and possibly zoonotic transmission and subsequent reassortment, the reagents and methods used for genotyping require close monitoring and updating. OBJECTIVES To design and evaluate a new VP4 consensus oligonucleotide primer pair that provides increased sensitivity and allows typing of strains that were untypeable using available methods. STUDY DESIGN A total of 489 rotavirus-positive faecal specimens from studies conducted between 1996 and 2006 were used for the evaluation of the new VP4 primers which was performed in the WHO Rotavirus Collaborating and Reference centres in the US, Australia, South Africa and the UK. RESULTS The new primer pair allowed P-typing of rotavirus strains and provided increased sensitivity, allowing typing of a significant number of strains that previously could not be P-typed. CONCLUSIONS This study highlights the importance of a constant reconsideration of primer sequences employed for the molecular typing of rotaviruses.

Comparative Study of the Epidemiology of Rotavirus in Children from a Community-Based Birth Cohort and a Hospital in South India

ABSTRACT 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

ABSTRACT 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.

Closing the diarrhoea diagnostic gap in Indian children by the application of molecular techniques.

A large proportion of diarrhoeal illnesses in children in developing countries are ascribed to an unknown aetiology because the only available methods, such as microscopy and culture, have low sensitivity. This study was aimed at decreasing the diagnostic gap in diarrhoeal disease by the application of molecular techniques. Faecal samples from 158 children with and 99 children without diarrhoea in a hospital in South India were tested for enteric pathogens using conventional diagnostic methods (culture, microscopy and enzyme immunoassays) and molecular methods (six PCR-based assays). The additional use of molecular techniques increased identification to at least one aetiological agent in 76.5 % of diarrhoeal specimens, compared with 40.5 % using conventional methods. Rotavirus (43.3 %), enteropathogenic Escherichia coli (15.8 %), norovirus (15.8 %) and Cryptosporidium spp. (15.2 %) are currently the most common causes of diarrhoea in hospitalized children in Vellore, in contrast to a study conducted two decades earlier in the same hospital, where bacterial pathogens such as Shigella spp., Campylobacter spp. and enterotoxigenic E. coli were more prevalent. Molecular techniques significantly increased the detection rates of pathogens in children with diarrhoea, but a more intensive study, testing for a wider range of infectious agents and including more information on non-infectious causes of diarrhoea, is required to close the diagnostic gap in diarrhoeal disease.

Neonatal Infection with G10P[11] Rotavirus Did Not Confer Protection against Subsequent Rotavirus Infection in a Community Cohort in Vellore, South India

BACKGROUND Various observational studies have suggested that neonatal rotavirus infection confers protection against diarrhea due to subsequent rotavirus infection. We examined the incidence of rotavirus infection and diarrhea during the first 2 years of life among children infected with the G10P[11] rotavirus strain during the neonatal period and those not infected with rotavirus. METHODS Children were recruited at birth and were followed up at least twice weekly. Stool samples, collected every 2 weeks for surveillance and at each episode of diarrhea, were screened by enzyme-linked immunosorbent assay and were genotyped by polymerase chain reaction. RESULTS Among 33 children infected neonatally with G10P[11] and 300 children not infected with rotavirus, there was no significant difference in the rates of rotavirus-positive diarrhea (rate ratio [RR], 1.05 [95% confidence interval [CI], 0.61-1.79]), moderate or severe rotavirus-positive diarrhea (RR, 1.42 [95% CI, 0.73-2.78]), or asymptomatic rotavirus shedding (RR, 1.25 [95% CI, 0.85-1.83]). CONCLUSION Neonatal G10P[11] infection with a strain resembling a vaccine candidate did not confer protection against subsequent rotavirus infection or diarrhea of any severity in this setting.

Geographic Information Systems and Genotyping in Identification of Rotavirus G12 Infections in Residents of an Urban Slum with Subsequent Detection in Hospitalized Children: Emergence of G12 Genotype in South India

ABSTRACT Rotavirus infections by G12 strains in several countries have recently been described. In this study, we report the emergence of G12 strains in south India. Fourteen cases of G12 infection were identified between June and September 2005. G12 was seen in combination with P[6], P[8], or nontypeable P type. Nine cases, including five symptomatic infections and four asymptomatic infections, were identified as part of routine surveillance for rotavirus infections in a birth cohort in the community between June and July 2005. Significant temporal and time-space clustering of eight of these cases represents a possible recent introduction of a new rotavirus VP7 genotype. Previous rotavirus infections had been documented for six of the nine children in the community. In the following 2 months, five cases of G12 infection were identified among children presenting to a referral hospital with diarrhea. This is the first description of symptomatic and asymptomatic G12 infections in children in the community. The detection of G12 strains from different parts of the world in recent years suggests the possibility of its emergence as an important global genotype. Monitoring of cocirculating rotavirus strains and detection of emerging strains is important in the context of the availability of rotavirus vaccines.

Polymerase chain reaction in the detection of an ‘outbreak’ of asymptomatic viral infections in a community birth cohort in south India

Asymptomatic enteric infections are important where sequelae or protection from subsequent illness is an outcome measure. The use of reverse transcription-polymerase chain reaction (RT-PCR) to identify asymptomatic enteric infections in a birth cohort followed for rotaviral infections in a south Indian urban slum is reported. Of 1191 non-diarrhoeal samples from 371 children collected in May-June 2003, 22 (1.9%) were positive by ELISA. A total of 147 (40.6%) of 362 samples tested by VP6 RT-PCR were positive. In those samples that could be typed, a high diversity of G types including G1, G2, G4, G8, G9 and G10, and a high proportion (34.4%) of mixed infections were detected. Noroviruses were identified in 6/28 (21.4%) samples tested. The identification of infections undetectable by conventional techniques indicates the importance of the use of sensitive diagnostic techniques in research studies. Asymptomatically infected children may also act as a source of infection for other susceptible hosts.

Comparison of viral load and duration of virus shedding in symptomatic and asymptomatic neonatal rotavirus infections

A single rotavirus strain causing asymptomatic infections as well as severe gastrointestinal disease has been described in the neonatal nurseries of the Christian Medical College, Vellore. In this study, quantitative real‐time RT‐PCR was used to determine the association of viral load with the presence of gastrointestinal symptoms in neonates. Viral load was estimated in terms of the crossing point [C(t) value] at which the amplicon could be detected in the real‐time PCR assay. The study was carried out on 103 neonates, including 33 asymptomatic neonates and 70 neonates with different gastrointestinal symptoms. The duration of virus shedding was also compared between five symptomatic and four asymptomatic neonates using real‐time RT‐PCR. There was no significant difference in viral load between symptomatic and asymptomatic neonates (P = 0.087). Among neonates with different gastrointestinal symptoms, those presenting with feed intolerance and abdominal distension had a significantly higher viral load than those with other gastrointestinal symptoms (P = 0.02). For the study on virus shedding, nine neonates were followed up for a median duration of 53 days, with a median of 31 samples tested per child. Extended shedding of low copies of rotavirus was found, with no significant differences in pattern of shedding between symptomatic and asymptomatic neonates. The lack of correlation between viral load and gastrointestinal disease demonstrates yet another difference between neonatal rotavirus infection and infection in older children where higher viral load correlates with severe disease. J. Med. Virol. 82:1803–1807, 2010.

Assignment of the group A rotavirus NSP4 gene into genotypes using a hemi-nested multiplex PCR assay: a rapid and reproducible assay for strain surveillance studies.

The rotavirus non-structural protein NSP4 has been implicated in a number of biological functions during the rotavirus cellular cycle and pathogenesis, and has been addressed as a target for vaccine development. The NSP4 gene has been classified into six genotypes (A-F). A semi-nested triplex PCR was developed for genotyping the major human NSP4 genotypes (A-C), which are common in human rotavirus strains but are also shared among most mammalian rotavirus strains. A total of 192 previously characterized human strains representing numerous G and P type specificities (such as G1P[8], G1P[4], G2P[4], G3P[3], G3P[8], G3P[9], G4P[6], G4P[8], G6P[4], G6P[9], G6P[14], G8P[10], G8P[14], G9P[8], G9P[11], G10P[11], G12P[6] and G12P[8]) were tested for NSP4 specificity by the collaborating laboratories. An additional 35 animal strains, including the reference laboratory strains SA11 (simian, G3P[2]), NCDV (bovine, G6P[1]), K9 and CU-1 (canine, G3P[3]), together with 31 field isolates (canine, G3P[3]; feline, G3P[9]; porcine, G2P[23], G3P[6], G4P[6], G5P[6], G5P[7], G5P[26], G5P[27], G9P[6] and G9P[7]) were also successfully NSP4-typed. Four human G3P[9] strains and one feline G3P[9] strain were found to possess an NSP4 A genotype, instead of NSP4 C, suggesting a reassortment event between heterologous strains. Routine NSP4 genotyping may help to determine the genomic constellation of rotaviruses of man and livestock, and identify interspecies transmission of heterologous strains.

Evidence of intrafamilial transmission of rotavirus in a birth cohort in South India

Transmission of rotavirus infection was studied in a birth cohort of children based in an urban slum in Vellore and their familial contacts. Contemporaneous samples from index patients and their familial contacts were collected for analysis in three different settings. Firstly, samples were collected from familial contacts during a period of rotavirus infection in children from the cohort. Secondly, on occasions when a family member had rotavirus diarrhea, samples from the cohort child were taken for analysis. Lastly, asymptomatic surveillance samples collected at predetermined time points from both the cohort child and familial contacts were analyzed. From 560 samples collected from family members during symptomatic and asymptomatic rotavirus infections in these children, three rotavirus transmissions were identified, accounting for a secondary attack rate of 0.54%. In four instances of rotavirus diarrhea in a family member, one infection was transmitted to the cohort child. Nucleotide sequence and phylogenetic analysis demonstrated a high degree of similarity in all these pairs ranging between 99% and 100% at both the nucleotide and the deduced amino acid levels, highly suggestive of person‐to‐person transmission of rotavirus infection. There was complete concordance of rotavirus genotyping between these pairs. No transmission events were noted from 14 asymptomatic rotavirus infections identified during routine surveillance of family members. This study is the first to use phylogenetic analysis to study the intrafamilial spread of rotavirus infection. J. Med. Virol. 80:1858–1863, 2008.