Clinical characteristics of norovirus gastroenteritis among hospitalized children in Japan

Authors


Yoshinori Ito, Department of Pediatrics, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. Tel: 81 52 744 2294; Fax: 81 52 744 2974; e-mail: yoshi-i@med.nagoya-u.ac.jp

ABSTRACT

Acute diarrhea is one of commonest pediatric illnesses worldwide. Although the importance of norovirus as a cause of gastroenteritis outbreaks is well documented, its role in sporadic acute gastroenteritis is not well characterized. The aim of this study was to clarify the prevalence and clinical characteristics of norovirus gastroenteritis among hospitalized children. Between November 2007 and April 2008, inpatients under 12 years of age with acute gastroenteritis in a single hospital in Japan were investigated. A stool sample from each patient was screened for enteropathogenic bacteria and tested by reverse transcription polymerase reaction for norovirus and by an immunochromatographic method for rotavirus and enteric adenoviruses. The clinical features of children with norovirus gastroenteritis were compared with those of children with rotavirus and children without noro- or rotovirus infections. Among 107 patients included in this study, norovirus and rotavirus were detected in 36 (34%) and 37 (35%) patients, respectively. Compared with rotavirus enteritis, the duration of vomiting and diarrhea was significantly longer, and serum C-reactive protein concentrations were higher, in patients with norovirus enteritis. Norovirus was detected as frequently as rotavirus in hospitalized pediatric gastroenteritis patients. Our results suggest that norovirus gastroenteritis among hospitalized young children is not less severe than rotavirus gastroenteritis.

List of Abbreviations: 
ALT

alanine aminotransferase

AST

aspartate aminotransferase

CRP

C-reactive protein

Acute gastroenteritis causes approximately 1.76 million deaths worldwide each year and in the developing world is one of the leading causes of death in children < 5 years of age (1). In developed countries, acute gastroenteritis is rarely fatal, but remains a frequent reason for hospitalization and outpatients visits. Rotavirus, a double-stranded RNA virus within the family Reoviridae, has been considered the most important cause of severe, acute gastroenteritis in infants and young children. It is responsible for approximately 40% of all hospital admissions due to diarrhea (2). On the other hand, noroviruses, positive sense single-strand RNA viruses within the Caliciviridae family, are another major cause of pediatric gastroenteritis (3, 4). Noroviruses have been detected in 3–31% of hospitalized children and in 5–36% of outpatients, and may be responsible for the deaths of up to 200,000 children < 5 years of age annually in developing countries (5).

The global licensure and increasing uptake of rotavirus vaccines into childhood immunization programs offers promise that the global burden of rotavirus disease will be greatly reduced over the next few years (6). On the other hand, the development of vaccines to prevent norovirus gastroenteritis faces substantial challenges because of the short-lived immunity after natural infection, great diversity among circulating strains, and ability of the virus to evade the immune system. Noroviruses comprise five genogroups of which three (most commonly genogroups I and II) are associated with human infection (3). Although noroviruses were the first viruses to be clearly associated with acute gastroenteritis, an inability to culture them has caused underestimation of the disease burden caused by them and hampered epidemiologic studies. Since the introduction of an RT-PCR method for their detection, noroviruses have been increasingly recognized as important causes of gastroenteritis outbreaks and acute sporadic enteric infection in children worldwide (7). The aim of this study was to clarify the prevalence, clinical characteristics and severity of acute norovirus infection among children hospitalized with acute gastroenteritis in Japan. The clinical features of norovirus gastroenteritis were compared to those of rotavirus infection.

MATERIALS AND METHODS

Patients

Between November 2007 and April 2008, episodes of acute gastroenteritis in children under 12 years of age who required hospitalization at Konan Kosei Hospital, Aichi, Japan were prospectively studied. A gastroenteritis episode was defined as at least two looser-than-normal stools within a 24 hr period or an episode of forceful vomiting with any loose stools lasting less than 14 days. Those who had underlying diseases associated with diarrhea or positive stool bacterial cultures were excluded. Data on the patient characteristics, clinical manifestations and laboratory results were analyzed. Informed consent was obtained from each child's parent prior to enrolment.

Detection of viruses

Fecal specimens were prepared as 10% (w/v) suspensions in sterile distilled water and clarified by centrifugation. The supernatants were collected and stored at –20°C until before testing. Norovirus was tested for by RT-PCR using a norovirus GI and GII RNA detection kit (Shimazu, Japan) according to the manufacturer's instructions (8). Briefly, 1 μL of supernatant from each fecal suspension was mixed with 19 μL of the sample treatment regent and treated at 85°C for 1 min, then stored on ice. For the RT reaction, 25 μL of RT master mixture was added to treated fecal supernatant. Reverse transcription was carried out at 37°C for 30 min, followed by inactivation at 95°C for 5 min. PCR was carried out in 51 μL of SYBR Green PCR master mix containing 1 μL of template and each primer using Mx3000P (Stratagene, La Jolla, CA, USA) under the following conditions: initial denature at 95°C for 10 min, followed by 45 cycles at 95°C for 10 sec, 56°C for 30 sec, and 72°C for 60 sec, and finished with a final extension at 72°C for 7 min. Norovirus positive samples were determined by examining the melt temperature for PCR amplicons. Compared to standard real time RT-PCR, the sensitivity and specificity of this assay are 94% and 75%, respectively (8). Rotavirus and adenovirus antigen were detected in stool supernatants using an enzyme immunoassay (Orion Diagnostica Oy, Espoo, Finland) according to the manufacturer's instructions.

Statistical comparisons of clinical characteristics and laboratory data were evaluated using the Mann-Whitney U test. Probability values < 0.05 were considered statistically significant.

RESULTS

One hundred and seven hospitalized patients under 12 yrs of age with acute gastroenteritis with a median age of 2.8 years and male/female ratio of 59/48 (55% male) were studied. Stool specimens were collected from all patients. Eighty-four (78%) were less than 3 years old and 17 (16%) 4–6 years old. Overall, the detection rate of rotavirus was 34.6% (37/107) and that of norovirus 33.6% (36/107). Among patients positive for norovirus, the norovirus belonged to group II in 35 (97.2%) and to group I in 1 (2.8%). Adenovirus was detected in 1 (0.9%) patient. In 33 (31%) patients, all tests for pathogens were negative. Dual infections were not identified in any patients. As to seasonal distribution, the peak incidences of rotavirus and norovirus were in February and December, respectively.

The clinical characteristics of patients with norovirus, rotavirus and “non-noro/non-rota” gastroenteritis (1 adenovirus and 33 no pathogens detected) are shown in Table 1. Norovirus-infected patients had a greater frequency of vomiting than did those with non-noro/non-rota gastroenteritis (34/35 vs 29/34; P < 0.01). Compared to the patients with rotavirus gastroenteritis, patients with norovirus gastroenteritis had a longer duration of vomiting and diarrhea (3.6 days vs 2.6 days; P= 0.03 and 6.0 days vs 4.4 days; P < 0.01, respectively). On the other hand, peak body temperature was significantly higher in patients with rotavirus gastroenteritis than in patients with norovirus gastroenteritis (38.8°C vs 38.4°C; P < 0.01).

Table 1.  Comparison of clinical characteristics of norovirus, rotavirus and non-noro/non-rota gastroenteritis in hospitalized children
 Norovirus (n = 36)Rotavirus (n= 37)Non-noro/rota (n= 34) P
Noro versus rotaNoro versus non-noro/rota
  1. Open spaces in fourth and fifth columns: not significant

  2. Bold, statistically significant data with P < 0.05

Age (yrs); mean ± SD2.4 ± 1.82.8 ± 2.13.2 ± 2.8  
Vomiting no. (%) 34 (94) 35 (95)29 (85) <0.01
 Duration (days); mean ± SD 3.6 ± 2.3 2.6 ± 1.81.8 ± 1.2 0.03<0.01
 Freq. ≧ 10 episodes/day, no. (%)10 (29)11 (31)5 (17)  
Diarrhea, no (%)30 (83)35 (95)25 (73)  
 Duration (days); mean ± SD 6.0 ± 2.5 4.4 ± 2.55.7 ± 3.9<0.01 
 Freq.≧10 episode/day, no. (%)6 (20)8 (23)3 (12)  
Fever > 38°C, no. (%)26 (72)29 (78)24 (71)  
 Duration (days); mean ± SD1.7 ± 1.41.9 ± 0.92.2 ± 1.6  
 Maximum (°C); mean ± SD38.4 ± 0.5  38.8 ± 0.738.7 ± 0.9 <0.01 
Seizures, no. (%)3 (8)7 (19)4 (12)  

Comparisons of laboratory findings for norovirus, rotavirus and other gastroenteritis are shown in Table 2. Increased serum AST and ALT concentrations were frequently observed in patients with norovirus and rotavirus gastroenteritis (56% and 46%, respectively). The numbers of the patients with high concentrations of AST (>50 IU/L) and ALT (>50 IU/L) were significantly higher in both the norovirus and rotavirus patients than in the non-noro/non-rota patients (20/36 vs 7/34; P < 0.01 and 18/37 vs 7/34; P= 0.01, respectively). Compared to the patients with rotavirus gastroenteritis, serum Na and CRP concentrations were significantly higher in patients with norovirus gastroenteritis (138 vs 136; P < 0.01 and 1.2 vs 0.6; P < 0.05, respectively).

Table 2.  Comparison of laboratory findings in norovirus, rotavirus and non-noro/non-rota gastroenteritis in hospitalized children.
 Norovirus (n = 36)Rotavirus (n = 37)Non-noro/rota (n = 34) P
Noro versus rotaNoro versus non-noro/rota
  1. BUN, blood urea nitrogen; WBC, white blood cell

  2. Open spaces in fourth and fifth columns, not significant

  3. Bold, statistically significant data with P < 0.05

AST (IU/L); mean ± SD 65 ± 46 65 ± 5044 ± 15   0.03
ALT (IU/L); mean ± SD 45 ± 51 51 ± 8526 ± 18 <0.01
AST or ALT > 50 IU/L (%) 20 (56) 18 (49)7 (21) <0.01
BUN (mg/dL); mean ± SD15 ± 6 18 ± 9 14 ± 7   
>20 mg/dL (%)8 (22)9 (24)7 (21)  
Glucose (mg/dL); mean ± SD88 ± 2880 ± 2695 ± 30  
< 50 mg/dL (%)3 (8)4 (11)1(3)  
Na (mEq/L); mean ± SD 138 ± 4  136 ± 3  138 ± 4  <0.01 
WBC (cells/μL); mean ± SD11100 ± 500  10000 ± 400  13300 ± 700    
CRP (mg/dL); mean ± SD 1.2 ± 2.1 0.6 ± 0.81.6 ± 3.2 0.04 

DISCUSSION

Noroviruses are recognized as the leading cause of epidemics of gastroenteritis and an important cause of sporadic gastroenteritis in both children and adults (3). However, because routine diagnostic methods for noroviruses have not been available to most clinicians, the clinical features of norovirus gastroenteritis are less well-established than those of rotavirus gastroenteritis. Although norovirus gastroenteritis is generally mild and short in duration, new evidence suggests that this illness can be severe and sometimes fatal, especially among vulnerable populations (young children and the elderly) and is a common cause of hospitalization for gastroenteritis (3). To clarify the clinical significance and etiologic impact of norovirus in Japanese children, we investigated 107 children hospitalized with acute, mostly moderately severe or severe, gastroenteritis.

Norovirus is reportedly the second most common cause of viral gastroenteritis after rotavirus, the rate of norovirus detection among hospitalized children in previous reports being 10–20% (9–12). In Japan, Sakai et al. have reported a detection rate of norovirus in hospitalized children of 18% (13). Compared to previous reports, in this study the detection rate of norovirus was high; we detected it as frequently as rotavirus (34% vs 35%). The reasons for this high frequency of norovirus gastroenteritis are unclear. Because this is a single center study, epidemics of norovirus gastroenteritis in areas covered by our hospital could have had a large impact on the results. Another possibility is that the RT-PCR assay used to detect norovirus in this study is more sensitive than the RT-PCR or other assays used in previous studies. On the other hand, we detected adenovirus in only one patient in this study. The limited observation period of this study (November to April) provides a possible explanation for this low frequency because adenovirus gastroenteritis occurs throughout the year. We limited the observation period to the cold months because we designed this study to clarify the clinical characteristics of norovirus gastroenteritis. In Japan, more than 80% of cases of norovirus and rotavirus gastroenteritis occur from November to April (14).

Gastroenteritis caused by norovirus is considered to be a less severe infection than that due to rotavirus (3). However, in this study inpatients with norovirus gastroenteritis had longer durations of diarrhea and vomiting than did those with rotavirus infection. These findings suggest that the norovirus gastroenteritis among hospitalized children is not less severe than rotavirus gastroenteritis. This finding may be because the present study did not include outpatients with gastroenteritis. Additionally, one previous study reported that durations of vomiting and diarrhea in norovirus gastroenteritis were longer than in rotavirus gastroenteritis among hospitalized patients (13), consistent with our results. Another possibility is that epidemic viruses may have different pathogenicity in different geographical regions. A recent study reported no significant difference in duration of diarrhea, vomiting, and hospitalization between norovirus and rotavirus gastroenteritis of inpatient children in Libya (9). Concerning laboratory findings, we observed increases in serum AST and ALT concentrations in patients with norovirus gastroenteritis as frequently as in patients with rotavirus gastroenteritis. These results indicate that gastroenteritis caused by norovirus is difficult to distinguish clinically from that caused by rotavirus.

Our findings suggest that the clinical burden of norovirus gastroenteritis in pediatric patients is equivalent to that of rotavirus gastroenteritis. Because a routine assay for diagnosis of norovirus infection has not been available, many clinicians have probably underestimated the importance of norovirus gastroenteritis. With the increasing use of rotavirus vaccine, understanding other enteric viruses, especially norovirus, which cause viral gastroenteritis has become increasingly important.

DISCLOSURE

The authors have no commercial or other associations that might pose a conflict of interest.

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