Rajesh Arumugam

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.

Rotavirus antigenemia in Indian children with rotavirus gastroenteritis and asymptomatic infections

BACKGROUND Rotavirus gastroenteritis results in significant morbidity and mortality in Indian children. Although there are numerous studies on rotavirus diarrhea, there are few reports on antigenemia and extraintestinal presentations in these populations. METHODS Following screening for rotavirus antigen of stool samples from children with and without acute gastroenteritis with a commercial enzyme immunoassay (EIA), a total of 199 stool and serum sample pairs were identified for additional testing. All EIA-positive stool samples were genotyped, and viral load estimated by real-time reverse-transcriptase polymerase chain reaction (RT-PCR). Serum samples were tested for rotavirus antigen by an in-house EIA, and antigen was quantified by optical density. Scoring of disease severity was performed for all hospitalized children. Data on extra-intestinal presentations were collected if available. RESULTS Based on screening of stool samples by EIA, the study population could be divided into 3 groups, including 111 children with rotavirus diarrhea, 44 children with diarrhea and no rotavirus detected in stool specimens, and 44 children with asymptomatic rotavirus infection. Antigenemia was significantly higher among children with rotavirus diarrhea (50.4%) than among children with non-rotaviral diarrhea (16%) or asymptomatic infections (2.3%) (P < .001). Low copies of rotavirus were detected by RT-PCR in all 7 children with EIA-negative stool specimens and antigenemia. Presence and levels of rotavirus antigen in serum specimens correlated with stool viral load. Children with antigenemia had significantly more-severe disease but not more extraintestinal presentations than did children without antigenemia. CONCLUSIONS Antigenemia occurs frequently in rotavirus infection and correlates with virus replication in the gut but not with extra-intestinal presentations.

Investigation of the environment and of mothers in transmission of rotavirus infections in the neonatal nursery.

A distinct feature of neonatal rotavirus infection is the association of unusual strains that appear to be prevalent only in neonatal units and persist for long periods of time. The main aims of this study were to determine if rotavirus can be detected on environmental surfaces in the neonatal nursery and whether the infection occurs in mothers of infected and uninfected neonates. Thirty rotavirus positive neonates and an equal number of negative neonates were enrolled in this study. Stool samples from 15 mothers in each group and environmental swabs collected from the bed and surfaces around neonates were tested for rotavirus using single round and nested PCR for the VP6 gene. Rotavirus could be detected in environmental swabs using single round PCR for VP6 gene in 40% of neonates positive for rotavirus antigen by enzyme immunoassay (EIA) and 33.3% of EIA negative neonates. The detection rate was almost 100% using the nested VP6 PCR. Rotavirus was detected in maternal samples only if the nested VP6 PCR was used, with no significant difference between rates of rotavirus detection in maternal fecal samples of infected and uninfected neonates (p‐0.4). Sequence analysis of nested VP6 amplicons from two environmental swabs revealed them to be closest in identity to G10P[11], the most common genotype causing infections in neonates in this setting. Interestingly, sequences of amplicons from maternal stool samples did not cluster with G10P[11] or other VP6 subgroup I strains but showed clustering with human strains of VP6 subgroup II. J. Med. Virol. 80:1099–1105, 2008.

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.

Multi-center surveillance of rotavirus diarrhea in hospitalized children <5 years of age in India, 2009–2012

Diarrheal disease due to Group A rotaviruses continues to be an important cause of morbidity in the developing world and India contributes significantly to the disease burden. Surveillance carried out between July 2009 and June 2012 at two medical centers in south India and one center in north India estimated 39% of all diarrheal admissions to be due to rotavirus. The most prevalent genotype isolated was G1P[8](33%) followed by G2P[4](17%). G9P[4] has also emerged as a significant cause of rotavirus diarrhea. No seasonal variation was noticed from the centers in south India, whereas we observed increased rotavirus diarrhea in the center in north India during March and April.

Approach to molecular characterization of partially and completely untyped samples in an Indian rotavirus surveillance program

Surveillance networks for rotavirus document the burden of the disease using the proportion of children hospitalized with gastroenteritis positive for rotavirus by enzyme immunoassay. They also describe genotypes of circulating viruses by polymerase chain reaction for the VP7 and VP4 genes, which determine G and P types, respectively. A proportion of samples cannot be genotyped based on initial testing and laboratories need to assess further testing strategies based on resources and feasibility. To 365 samples obtained from an Indian rotavirus strain surveillance program, we applied an approach to determine the G and P types in antigen positive samples that failed to type initially with the standard laboratory protocol. Fifty-eight samples (19%) were negative for the VP6 gene, indicating that the antigen test was likely to have been false positive. Alternative extraction and priming approaches resulted in the identification of G and P types for 264 strains. The identity of one strain was determined by sequencing the first-round amplicons. Thirty-five strains were partially typed and seven strains could not be typed at all. The distribution of G and P types among strains that had initially failed to type, except one strain, did not differ from that in strains that were typed using the standard laboratory protocol.

Rotavirus surveillance at a who-coordinated invasive bacterial disease surveillance site in Bangladesh: A feasibility study to integrate two surveillance systems

The World Health Organization (WHO) currently coordinates rotavirus diarrhea and invasive bacterial disease (IBD) surveillance at 178 sentinel sites in 60 countries. However, only 78 sites participate in both surveillance systems using a common sentinel site. Here, we explored the feasibility of extending a WHO-IBD surveillance platform to generate data on the burden of rotaviral diarrhea and its epidemiological characteristics to prepare the countries to measure the impact of rotaviral vaccine. A six-month (July to December, 2012) surveillance, managed by IBD team, collected stool samples and clinical data from under-five children with acute watery diarrhea at an IBD sentinel site. Samples were tested for rotavirus antigen by ELISA and genotyped by PCR at the regional reference laboratory (RRL). Specimens were collected from 79% (n = 297) of eligible cases (n = 375); 100% of which were tested for rotavirus by ELISA and 54% (159/297) of them were positive. At RRL, all the cases were confirmed by PCR and genotyped (99%; 158/159). The typing results revealed the predominance of G12 (40%; 64/159) genotype, followed by G1 (31%; 50/159) and G9 (19%; 31/159). All in all, this exploratory surveillance collected the desired demographic and epidemiological data and achieved almost all the benchmark indicators of WHO, starting from enrollment number to quality assurance through a number of case detection, collection, and testing of specimens and genotyping of strains at RRL. The success of this WHO-IBD site in achieving these benchmark indicators of WHO can be used by WHO as a proof-of-concept for considering integration of rotavirus surveillance with WHO-IBD platforms, specifically in countries with well performing IBD site and no ongoing rotavirus surveillance.

Molecular characterisation and clinical correlates of rotavirus in children and adults in a tertiary care centre, Chennai, South India

Aims: This study was undertaken to determine the rate of detection of rotavirus causing diarrhoea among children and adults, identify the common genotypes circulating and determine clinical correlates. Settings and Design: This is a cross-sectional study in a tertiary care centre. Materials and Methods: Stool samples were collected from adults and children, transported on ice, aliquoted and stored at − 80°C. Rotavirus antigen detection enzyme-linked immunosorbent assay was performed on all samples. Representative samples were typed by conventional hemi-nested VP7 and VP4 reverse transcription-polymerase chain reaction. Statistical Analysis Used: Test of proportion, Students t-test and Chi-square test were used for statistical analysis. Results: A total of 444 stool samples were collected and tested over 14 months. Among these, 116 were paediatric with a rate of positivity of 36.21% and 328 were adults with rate of positivity of 20.73%. Among children under 5 years (n = 90), the rate of positivity was 41.11%. Vesikari scale was used for clinical assessment. The mean ± standard deviation Vesikari score in rotavirus-infected children and rotavirus-uninfected children was 11.2 ± 3.2 and 8.9 ± 3.6, respectively, and the difference was statistically significant. Nineteen samples were genotyped in children < 5 years, 94.7% were of G1P[8] and 5.3% were of G9P[4] genotype. Genotyping of 14 adult samples, G1P[8](85.7%) was found as the predominant genotype, two samples (14.3%) were partially typed (G9PUT and G12PUT). Conclusions: The rate of positivity of rotavirus in children under 5 years was 41.11%. G1P[8] is the most common strain circulating across all age groups.

Molecular epidemiology of rotaviruses in the south-east Asian region from 2009 to 2015

BACKGROUND In Asia, rotavirus accounts for approximately 45% of admissions due to acute gastroenteritis in children <5 years, and causes about 145,000 deaths every year. We studied the distribution of rotavirus strains from Myanmar, Sri Lanka, and Nepal during 2009-2015. METHODS Stool samples collected from children <5 years of age hospitalized with acute diarrhea in the three sites and positive for rotavirus antigen by enzyme immunoassay (EIA) were sent to the Christian Medical College, Vellore from 2009 to 2015. G and P typing of rotavirus strains were performed using reverse-transcription polymerase chain reaction (RT-PCR). RESULT Of the 2354 EIA positive samples tested, G12P[8] (36.8%), G1P[8] (30.1%), and G12P[6] (41.3%) were the most common strains isolated from Myanmar, Sri Lanka, and Nepal respectively. CONCLUSION There was substantial diversity of rotavirus genotypes, and continued surveillance in developing countries of Asia will help in understanding the epidemiology of rotavirus before and after introduction of vaccines.

Genotype distribution of Group A rotavirus from southern India, 2005–2016

Diarrheal disease due to Group A rotaviruses remain a leading cause of mortality and morbidity in the less developed parts of the world. India has started a phased roll out of rotavirus vaccine in the national immunization program. This analysis summarizes the rotavirus genotype strain distribution pre-vaccine introduction in Vellore, India from December 2005 to June 2016. Rotavirus was responsible for 32% of all diarrheal admission to the hospital. G2P[4] was the predominant strain in the initial years and was gradually replaced by G1P[8]. The emergence of G9P[4] replacing G9P[8], and the detection of G12 strains over several years were documented. There was no clear seasonality of disease. These data form the baseline to monitor genotype distribution post-vaccine introduction in Tamil Nadu.