Development and Evaluation of Molecular Methods for Characterization of Enteric Caliciviruses in Food Borne Disease and Human Infections

Bindhu Monica, S (2009) Development and Evaluation of Molecular Methods for Characterization of Enteric Caliciviruses in Food Borne Disease and Human Infections. Doctoral thesis, The Tamilnadu Dr. M.G.R. Medical University, Chennai.


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Pediatric gastroenteritis is a major cause of childhood morbidity and mortality worldwide. In developing countries, diarrhea remains the second most common cause of death among children under 5 years of age, with the majority of disease occurring in infants under 1 year of age. Infection rates in young children and infants living in developing countries may have been underestimated in previous studies due to the lack of sensitive diagnostic techniques for identification of viral particles in stool specimens. The role of HuCVs in causing acute gastroenteritis in young children has not been comprehensively studied in India due to the lack of sensitive and specific diagnostic techniques. This is clear by the limited number of published data (n=4) from India. The present study, for the first time in India, has developed and standardized methods for molecular characterization of HuCVs, and estimated their prevalence in a large cohort of symptomatic and asymptomatic children in both the community (n = 559 and n= 210, respectively) and hospital settings (n= 350 and n=100, respectively). The relative contributions of HuCVs in hospitalized children (n= 350) were evaluated for an extended time period (December 2001 to December 2004) and the genotype of the strains circulating in the hospital population was also investigated. A prevalence of 19.4% of HuCV was detected among this group. Although it is lower than 25-30% incidence of rotavirus gastroenteritis in hospitalized children in this region of India, it indicates that HuCVs are also an important cause of sporadic cases of acute gastroenteritis in infants and young children in India. There is little information on the asymptomatic carriage among children and on the implication in the transmission of HuCVs from these individuals. The application of a RT-PCR assay to test asymptomatic fecal specimens collected from children <5 years age without gastroenteritis presenting to the Child Health out patient service at the Christian Medical College (CMC) hospital was undertaken to ascertain if HuCVs were present in those populations. The detection of 11% 131 HuCV positives among a total of 100 children without gastroenteritis indicates the asymptomatic carriage of HuCVs in the community. Our findings on asymptomatic HuCVs suggest that young children could be an important reservoir for the transmission of HuCVs and their surveillance may be required to to control HuCV outbreaks. The data from the community based studies shows that there is a significant burden of illness due to HuCV related sporadic gastroenteritis in India, although the proportion of HuCV associated disease is less than in hospitalized children in the same town. The incidence of sapovirus and norovirus gastroenteritis in the community was 3.5 % (17/500) and 6.8% (38/500), respectively. These figures are is relatively lower when compared to other community based studies done in developed countries, and may be reflective of the fact that in developed countries, gastroenteritis in children is less common, but mainly contributed to by viral infections, whereas in developing countries, gastroenteritis occurs more frequently in young children, and bacteria and parasites form a significant proportion of the aetiologic agents. It is also interesting to note that in 8 of 9 repeat infections identified in the community, the genotypes seen in the second infection were different from those first encountered. This highlights the high incidence and diversity of HuCVs in our setting. Immune responses to homotypic and heterotypic viruses have been insufficiently studied in Calicivirus infection, in part due to the diversity of the agents and the lack of appropriate assays. A prospective cohort study aimed at genotyping infecting strains in conjunction with measurement of immune responses would allow dissection of the immune response and the presence or lack of protection from infection and/or disease, but no such studies have been conducted. Only a few past studies have focused on asymptomatic HuCV infection in the community. Our present study showed a high rate of asymptomatic infection (9.5%) in children living in an urban community in Vellore, South India. The living conditions and poor hygienic measures could facilitate the transmission of enteric infections Higher rates could be caused by other factors, such as the season or year of the study, or be related to the sensitive RT-PCR methods used. Detection of HuCVs in asymptomatic children signifies that these viruses can circulate in the general population in the absence of disease in young children. Asymptomatic infection may be widespread in young children as, although immunity is of short duration, the frequency of infection in this age group may result in fewer symptomatic infections. These asymptomatic infections however constitute a significant reservoir for infection in the community, and may act as a source of both endemic and epidemic disease. Future studies are needed to understand the importance of asymptomatic HuCV infection in children as it relates to the transmission of infection and gastroenteritis. Recent studies suggest that human histo-blood group antigens, such as secretor factor or blood type, could determine the human susceptibility to Norovirus [150, 151]. Blood group analysis on the HuCV positive children in the community showed no association, but this was a limited assessment.Previous published reports [146] have shown that B Rh positive individuals are more resistant and O Rh positive individuals are more susceptible to norovirus infections. A significantly larger number of children (n=68/350, Vs n=58/559, p<0.001) in the hospital cohort had HuCVs when compared to the symptomatic children in the community. Analysis of clinical disease through severity scores and of sequence diversity showed that disease in the community associated with HuCVs was mild or moderate whereas disease severity ranged from moderate to severe among the hospitalized children. Differences in the distribution of genotypes of HuCVs between children with diarrhea in the community and those presenting to hospital were also observed in the present series, indicating different reservoirs acting as the source of infection. This study has also carried out molecular typing of HuCVs for the first time from India by DNA sequencing. Phylogenetic analysis of the regions of the genes encoding the RdRp, capsid, and/or the region spanning the ORF1/ORF2 of the genome of selected sapovirus and norovirus strains has been performed to classify the strains co-circulating in the study populations during a defined surveillance period. As reported from other parts of the world, genogroup II Norovirus were more prevalent in children in the hospital and the community, and belonged to multiple genotypes. It is instructive to note that the norovirus GII 4 strains are widely distributed in the world. This strain was seen in both the community (39.5 %) and the hospital (18.5%) in this study, but in the hospital cohort GII-4 was not the most prevalent genotype. Among the sapoviruses, genogroup II strains were more common than genogroup I and this finding differs from reports from other parts of the world where genogroup I sapoviruses are found to be the predominant strain. These data confirm that different strains of HuCV may predominate in different parts of the world as environmental factors may influence the transmissibility of HuCVs. Thus there is also a need for any preventive strategy (i.e, vaccine development) to be tailored to different geographical and demographic requirements. In order to compare methods for the molecular detection of HuCVs, the detection technique was cross validated between two centers, the Christian Medical College (CMC, Vellore) and All India Institute of Medical Sciences (AIIMS, New Delhi). RT-PCR assay sensitivity evaluated in the present study included primers that target the RNA polymerase gene (CMC) and 3′-end of ORF1 (AIIMS). A sensitivity of 37% by RT-PCRs was observed in CMC detection method and 23.5% by the AIIMS method. This data suggests that primers targeting the RNA polymerase gene are more sensitive than the primers that target 3′-end of ORF1. Several factors can affect the sensitivity of RT-PCR assays, including the viral RNA extraction method, the primers used in amplification, and the methods used for confirmation of test results. Differing efficacy of RNA extraction and reverse transcription might also explain concordance discrepancies seen in these validation experiments that could be addressed with viral cDNA as the starting material. Noroviruses are the commonest cause of food borne gastroenteritis worldwide. In this study, a precipitation technique using polyethylene glycol was used for concentration of norovirus in food samples. Norovirus could be recovered from all spiked food samples except oysters, mussels and clams. None of the test samples were positive of GII norovirus. Detection of norovirus in food samples is an important part of outbreak investigations as well as for routine testing of food samples. To the best of our knowledge, this is the first report of testing for norovirus in food samples in India. In summary, we have described the prevalence of co-circulating HuCVs in children with or without gastroenteritis in Vellore, southern India using sensitive and validated molecular diagnostic tools. The public health impact of HuCVs will not be fully appreciated, nor will interventions be completely evaluated, until methods to detect these viruses are more routinely used. Frequent symptomatic HuCV infections in infants and young children living in developing countries may impact quality of life substantially and contribute to dehydration and malnutrition. These studies have provided valuable insights to the current understanding of the burden of HuCV related diarrheal disease in India and will form the base for further elucidation of the epidemiology and pathogenesis of HuCV infections. Further information on the molecular epidemiology of HuCV infections, transmission patterns, and immunity is needed to support the development of effective public health interventions to reduce the burden of pediatric HuCV disease in this part of the world.

Item Type: Thesis (Doctoral)
Uncontrolled Keywords: Development, Evaluation, Molecular Methods, Characterization, Enteric Caliciviruses, Food Borne Disease, Human Infections.
Subjects: Respiratory Medicine > Gastroenterology
> Respiratory Medicine > Gastroenterology
Depositing User: Subramani R
Date Deposited: 19 Aug 2017 08:46
Last Modified: 27 Oct 2022 02:35

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