This systematic review of studies that used RT-PCR for detection of NoVs in fecal specimens clearly indicates that these viruses play an important role in the cause of both mild and severe gastroenteritis worldwide. In 1979, Greenberg et al. demonstrated that virtually all children in various countries worldwide acquired antibodies to NoVs by 5–15 years of age (Technical Appendix 2
). However, despite strong evidence that NoV infection was ubiquitous, detection rates in children hospitalized with diarrhea were low in early studies that used electron microscopy and antigen assays (4
). The advent of conventional RT-PCR for the diagnosis of NoVs has substantially changed our understanding of their epidemiology. Among all reported studies that used conventional RT-PCR, NoVs were detected in ≈12% of children <5 years of age with severe diarrhea, which suggests that these viruses are the second most common cause of severe childhood gastroenteritis, following rotavirus. In addition, although some studies suggest that NoV infections in the community are slightly less severe than rotavirus infections, data also exist to suggest that these childhood infections may be similar in severity, which may particularly apply to hospitalized children (Technical Appendix 2
). On the basis of the pooled detection rates of NoV in our review, we would estimate that in the United States alone NoVs may account for >235,000 clinic visits, 91,000 emergency room visits, and 23,000 hospitalizations among children <5 years of age (10
). Limited data from developing countries are available to make firm estimates, but NoV disease may cause >1 million hospitalizations and 200,000 deaths each year among children <5 years of age.
Although these figures provide a preliminary indication of the substantial magnitude of illness from NoV disease, they may underestimate the true extent of disease. Evidence suggests that detection of NoVs in fecal specimens by conventional RT-PCRs may be limited by factors such as low virus concentrations in feces, improper specimen storage, inefficient viral RNA extraction, presence of fecal reverse transcriptase inhibitors, and use of different primers (Technical Appendix 2
). In addition, NoVs are extremely genetically diverse and none of the reported conventional RT-PCR assays is able to detect all strains (Technical Appendix 2
). These hypotheses are supported by the findings of an evaluation of children with gastroenteritis in Peru in which both RT-PCR testing of fecal specimens and serologic assays were used to assess NoV infection, and serologic testing was found to increase the rates of NoV detection from 35% with fecal testing alone to 55% by use of either assay (28
). A recent validation study comparing state-of-the-art real-time RT-PCR with conventional RT-PCR found that the sensitivity of real-time RT-PCR was greater than that of the conventional method, especially for samples containing low NoV concentrations or RT-PCR inhibitors (Technical Appendix 2
). The broader application of real-time RT-PCR assays to diagnose NoV among children hospitalized with gastroenteritis should provide better estimates of the true prevalence of disease.
Some factors could have led us to overestimate the extent of sporadic NoV disease. Our estimates of sporadic NoV disease prevalence are based on a review of active surveillance data of all diarrhea cases and do not exclude cases originating from an outbreak. In a few studies, NoVs were detected in patients who were co-infected with another pathogen, and only a limited number of studies enrolled concurrent healthy controls, thus making it difficult to determine the fraction of diarrhea cases truly attributable to NoV. Among all studies testing for co-infections, however, the median rate of detecting another pathogen in addition to NoV was low (2%). This finding, combined with the fact that most studies only assessed for NoV among samples that previously tested negative for other bacterial and viral pathogens, suggests that our overall pooled proportion attributable to NoVs is unlikely to be much lower. In addition, for the studies that enrolled diarrhea-free controls, we subtracted control prevalence from the case prevalence of NoV disease when calculating the overall pooled estimate. Lastly, other unmeasured factors underestimating disease prevalence that could not be accounted for, such as inefficient primers, low virus shedding, delays in specimen collection, and lack of a sensitive case-definition are also likely to exist in these studies.
The heterogeneity in the NoV literature is evident and should be considered when interpreting the results of this review. Our systematic approach and strict inclusion criteria likely reduced heterogeneity but do not eliminate biases in the original studies, diversity in study design and population, and publication bias. Nonetheless, the findings of this review suggest that NoVs are a frequent cause of mild and severe sporadic gastroenteritis among children in high- and middle-income countries. In addition, hospital studies in our review only assessed patients admitted with diarrhea. Because of the highly infectious nature of NoVs, substantial additional health and economic effects would also occur from nosocomial disease and outbreaks in healthcare facilities, as previously identified by Lopman et al (7
). NoVs are also a frequent cause of severe illness and death from diarrhea among children in developing countries, although firm conclusions cannot be made because of limited data. Systematic evaluations that use broadly reactive, state-of-the-art diagnostic assays, with concurrent evaluation of healthy controls and examination of potential co-infection, are needed to fully understand the role of NoVs in the etiology of sporadic childhood gastroenteritis. These evaluations are especially necessary in developing countries, where diarrhea remains a leading cause of childhood death, causing >1.8 million annual deaths (1
The increasing evidence documenting the magnitude of the NoV disease prevalence provides support for considering targeted interventions, such as vaccines, for reducing the extent of this illness among young children. However, if one considers that NoV frequently causes both sporadic and epidemic gastroenteritis and can affect all age groups, some other potential targets for vaccination may include elderly persons in nursing homes, who are vulnerable to severe complications, and military recruits, in whom sporadic and epidemic NoV disease is known to incur substantial illness and financial costs from work disruption (7
; Technical Appendix 2
). The development of vaccines against NoVs will likely be challenging because the immunity to these viruses and the diversity and evolution of circulating strains are incompletely understood (Technical Appendix 2
). However, genotype II, cluster 4 NoV strains appeared to be by far the most prevalent strains among the studies we reviewed, and these strains may be the primary targets for vaccine development.
Carefully designed epidemiologic studies that evaluate NoV prevalence in children with diarrhea and a suitable comparison group and that use sensitive molecular assays will help further define target groups that would benefit from vaccines and other interventions. A particularly pressing need exists for better quantifying the extent of severe norovirus disease among children in developing countries and identifying prevention strategies to help reduce the prevalence of deaths from diarrhea in the poorest countries.