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Detection, genetic characterization, and quantification of norovirus RNA from sera of children with gastroenteritis

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Abstract

Background

Norovirus (NoV) infection is thought to be confined to the intestines, whereas many reports suggest antigenemia and viremia occur during rotavirus gastroenteritis.

Objectives

To detect NoV RNA in sera and cerebrospinal fluids (CSF) from NoV-infected children, and to quantify and genetically characterize the NoV found in these compartments.

Study design

Semi-nested PCR was conducted on stool, serum and CSF samples from 56 patients with acute gastroenteritis. Positive samples for NoV were analyzed further by sequencing and real-time PCR.

Results

From 39 patients with NoV RNA in stools, 6 also had NoV RNA in sera and none had NoV RNA in CSF. Genotypes of the NoV in stool and serum from the same patient matched completely. The strains in this study had high homology (98.1–100%) with registered strains in the database. The median viral load in stools of the serum-positive patients was greater than that of the serum-negative patients, but this difference was not statistically significant (9.8 × 109 copies/g versus 1.1 × 109 copies/g (p = 0.117)).

Conclusions

NoV RNA appeared in the blood stream in 15% of the patients of NoV gastroenteritis. Although the viral load in stool was not statistically correlated with NoV appearance in serum, genetic analysis indicated that NoV RNA in sera originated from the NoV gastroenteritis.

Introduction

Norovirus (NoV), a member of the family Caliciviridae in the genus Norovirus, is a major cause of non-bacterial acute gastroenteritis all over the world.1, 2, 3 The main symptoms of NoV infection are diarrhea and vomiting, which are usually mild and self-limiting. However, a recent case report demonstrated that a patient suffered from disseminated intravascular coagulation during a NoV outbreak, in association with obtundation, headache and photophobia4 and Ito et al.5 reported NoV-associated encephalopathy with altered consciousness. These reports indicate a potential spread of NoV to organs other than the intestines.

Many studies have been conducted seeking evidence of extra-intestinal manifestations of rotavirus infection. These included detection of rotavirus RNA in blood6, 7, CSF7, 8, 9 and throat swabs.7 Although early works suggested that this was due to unusual rotavirus strains or rare host genetic or immunologic defects in the infected child,10 recent analysis revealed that rotavirus antigen is commonly detected in sera of immunocompetent children with rotavirus diarrhea (43–67%).11, 12, 13

Human NoV, unlike rotavirus, is not capable of growing in cell lines and has no animal model available, thus hindering study of systemic spread after intestinal infection. Detection of NoV RNA from specimens other than stools has been limited to one case in which NoV was present in serum and CSF from a previously healthy NoV-infected girl with encephalopathy.5

In this study, we sought to detect NoV RNA in blood and CSF from patients with NoV gastroenteritis. Genetic analyses and quantification of NoV RNA were undertaken on positive samples.

Section snippets

Sample collection

From the diarrheal patients who attended Department of Pediatrics in Teikyo University Hospital, Eijudo clinic, and Red Cross Society Wakayama Medical Center from December 2005–2006, 56 cases who needed venepuncture for examination or infusion were recruited into this study. Stool samples were collected from 56 cases only once, while several blood samples were taken from patients who were required additional venepuncture in later course (total = 90). The interval between stool and the first blood

RT-PCR and patients’ characteristics

Among the 56 stool samples collected, 26 were positive for NoV GII by first round PCR and 13 were positive by second round. Among the 90 serum samples collected from the 56 patients, 6 were positive for NoV GII by second round PCR and were confirmed by sequence analysis to be NoV. Neither of the two CSF samples contained NoV RNA even by second round PCR, although stool samples from these patients contained NoV GII RNA by first round PCR. All of the samples tested were negative for NoV GI.

Nucleotide sequence and phylogenetic analysis of NoV GII

The

Discussion

Potential extra-intestinal spread is an important issue in understanding the pathogenesis of viral gastroenteritis. In this study, we observed that 15% of the NoV gastroenteritis patients (6/39) had NoV RNA in serum and could not detect NoV RNA in either of two CSF samples.

Our genetic analysis showed a very high homology between strains found in stool and serum, indicating that the viral RNA in blood had originated from the intestinal tract. The high homology between the strains in this study

Acknowledgements

This study was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Sciences and Technology and the Ministry of Health, Labor and Welfare, Japan.

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