Complicated norovirus infection and assessment of severity by a modified Vesikari disease score system in hospitalized children

Background Norovirus (NoV) GII.4 is the most common genotype for norovirus gastroenteritis worldwide. New variants or subgenotypes are continuously emerging, thus posing a serious threat to child health. Methods We compared retrospectively the clinical manifestations and complications of norovirus gastroenteritis in children from April, 2004 through December, 2012. NoV variants were analyzed to investigate the association of circulating viral strains with the complications. A modified disease severity score system based on Vesikari score system was devised and to evaluate disease severity. Results Compared to the outbreak in 2004/2005 winter, significant higher incidence of complications in the later periods are: convulsive disorder (p < 0.001) in 2006/2007 winter gastrointestinal hemorrhage (p = 0.047) and severe abdominal pain or irritability (p = 0.033) in 2008/09/10 winter; gastrointestinal hemorrhage (p = 0.030), severe abdominal pain or irritability (p = 0.014), and prominent hyperthermia (fever >39 °C, p = 0.001) in 2011/2012 winter. GII.4 Den_Haag_2006b, GII.4 2010, GII.4 Sydney 2012, and GII.4 2012b were the predominant strains in the outbreaks after 2006. By the modified severity score system, severe norovirus disease occurred in 28.5 %, 32 %, 33.3 %, and 30.2 % of the patients in the four periods. A longer duration of hospitalization (p = 0.02) were found in those with high score irrespective of the year of admission. Conclusions Our study demonstrated NoV outbreaks in northern Taiwan caused by different GII.4 variants that were associated with specific complications and uncommon clinical presentations. A modified severity score system first proposed in this study was able to identify severe cases with a longer hospital stay in NoV-infected children.

needs hospitalization. Complications of norovirus AGE in children, such as convulsion, severe dehydration, malnutrition, bowel obstruction and even death were sporadically reported [5,6]. Significant clinical features correlated with different viral strains has been documented recently, and after implementing rotavirus vaccination, norovirus infection has become one of the most important enteric pathogens threatening human health [3,7]. Our recent study showed that in Taiwan under suboptimal use of rotavirus vaccines, a significant increase of norovirus infection was observed in the postrotavirus vaccine era [8].
Disease severity score system for AGE was established previously [9]. The severity score for different clinical features and complications caused by emerging enteric viruses such as NoV is lacking. The purpose of this study was to investigate the clinical features and complications with norovirus infection in hospitalized pediatric patients supposed to have moderate to severe illness, compared to children treated in the community setting, and furthermore, we proposed a disease scoring system modified from Vesakari score system for evaluation of norovirus disease with variable severity in hospitalized patients.

Patient selection and identification of norovial infection
From April of 2004 through December of 2012, pediatric patients with the diagnosis of AGE hospitalized in the Division of Pediatric Gastroenterology, Chang Gung Children's Hospital (CGCH) were randomly enrolled. Their fecal specimens were collected in a clean container within 3 days of hospitalization with guardian's informed consent in this study with exclusion of those with major underlying diseases. Norovirus infection was confirmed by the detection of NoV using RT-PCR from fecal specimens of the patients with diarrheal disease [10]. The patient enrollment was irrespective of age, sex, ethnicity, and hospitalization wards. The symptoms were determined and documented in electronic medical records to determine how patient-reported symptoms were addressed by clinicians and clinical data of the patients enrolled were collected retrospectively from the chart records. These studies were approved by the Institutional Review Board of the Chang Gung Memorial Hospital.

Norovirus genome sequencing and genotyping
Viral nucleic acid extraction from fecal samples was performed using a kit according to the manufacturer's recommendations (QIAamp Viral RNA Mini Kit; Qiagen, Hilden, Germany). The PCR primers and conditions used for determining NoV genotypes were described previously [10]. The cDNA products were cloned into a plasmid (pCR-XL-TOPO® vector; Invitrogen), and the recombinant plasmid was transferred into competent Escherichia coli (Topo® XL PCR Cloning kit; Invitrogen). The sequences of different PCR products from the same specimen were used to reconstruct the near-full-length norovirus genome using the Vector NTi software package (Invitrogen). The reference genome sequences of NoV used for comparison were all obtained from the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov/).

Modified score system for severity of norovirus infection
We evaluated the disease severity of norovirus infection by the Vesikari score system (total score < 7, mild; 7-≦10, moderate, and >10, severe. total maximum score 20) [9] and a modified score system based on clinical information from the hospitalized patients (Table 1). The modified severity score system has a 0-24 point numerical score, including the assessment for the presence of unusual clinical features, such as gastrointestinal hemorrhage (gross bloody stool or occult blood ≧ 2+ in stool), convulsion, and additional presentations such as abdominal pain or flatulence and prolonged or high fever. The modified system has a more simple stratification of general AGE symptoms from 3 points to 2 points hence with a total maximum score of 24. In our 24 points score system, mild degree is as less than 1/3 maximum score (8) and severe degree is half score or higher (12), that is score, < 8, mild; 8-≦11, moderate, and >11 (12 or higher) severe. The differences of the two score systems are listed in Table 1.

Statistical analysis
The chi-squared test or Fisher's exact test was applied to dichotomous variable analysis, and the unpaired-sample student's t-test was used for continuous variable analysis. A p value of < 0.05 was considered statistically significant. All the tests were analyzed using SAS system software version 8 for Windows.      Disease severity assessment by a modified score system compared to Vesikari score system

Discussion
NoV is one of the emerging pathogens causing enteric infections in humans, and the associated clinical presentations are diverse [11][12][13][14]. Other than gastrointestinal symptoms, norovirus infection is associated with several extraintestinal or unusual complications, including benign infantile convulsion [5], necrotizing enterocolitis [15], and exacerbations of inflammatory bowel disease [16], as well as chronic, serious outcomes in immunocompromised patients [17,18] [5,15]. Although the specific NoV subgenotypes related to severe infection and complications was only sporadically reported, the rapid genetic evolution of NoV is believed to be the main factor driving the changing clinical manifestations in the infected patients. On the other hand, there is an unmet need that we need an objective disease severity score system specifically for the assessment of the severity of norovirus infection, as the Vesikari score used in rotavirus vaccine trials [19,20].
Recently, a modified Vesikari score system for severity assessment of AGE in children in the emergency departments has been devised [21]. This is a simplified version of Vesikari score system by the deletion of dehydration status as a score item. Nevertheless, the evidence-based guidelines in Europe highlighted that enteric infectious agent is frequently associated with dehydration which reflects severity of disease and should be monitored by established severity score systems [22]. Complications of AGE and dehydration usually require aggressive management in norovirus infection. Thus, the addition of clinical complications as score items to form a modified system for severity assessment of norovirus infection is necessary. Based on this, we constructed such a modified system and verified its performance in assessing norovirus infection in different outbreaks in this study. A longer hospitalization in patients classified into mild severity in Veskari score system could be due to a prolonged course of fever, and presence of complications, abdominal pain and flatulence not included in Vesikari score system. These symptoms may cause poor appetite and delayed recovery of activity that require intravenous hydration. Furthermore, a significantly longer hospital stay in the higher-scored patients was found as practically expected because it is complicationweighted. Thus, the newly devised modified score system could be a useful tool in clinical practice. There are several limitations in our study. First of all, our study enrolled hospitalized children usually having a moderate or severe illness rather than children with mild illness treated in a community setting. Secondly, with regards to the NoV subgenotypes, we could only verify the major strains circulating in each outbreak. Thirdly, although all the fecal samples were tested for bacterial pathogens and viruses, and NoV was found the only agent positive in these specimens, still other pathogens that may be associated with the clinical manifestations are not included in the panel. Fourthly, in spite of the presence of the complications in norovirus AGE, the mechanisms for the occurrence of such intestinal or extra-intestinal complications remain to be determined.

Conclusions
Vaccine development for the prevention of norovirus infection is currently under way and a more objective assessment of the infection is of paramount importance [23][24][25][26][27]. This study demonstrated that different circulating NoV subgenotypes were associated with different complications and uncommon presentations in a series of outbreaks over time in northern Taiwan. The stratification of hospitalized patients by assessment of the severity of their disease severity using a modified severity score system is possible, suggesting that the system is useful for future outbreak investigation. NoV is a rapidly evolving virus with the circulating genotypes or subgenotypes varying over time.