Development of a real-time RT-PCR and Reverse Line probe Hybridisation assay for the routine detection and genotyping of Noroviruses in Ireland

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Abstract

Background

Noroviruses are the most common cause of non-bacterial gastroenteritis. Improved detection methods have seen a large increase in the number of human NoV genotypes in the last ten years. The objective of this study was to develop a fast method to detect, quantify and genotype positive NoV samples from Irish hospitals.

Results

A real-time RT-PCR assay and a Reverse Line Blot Hybridisation assay were developed based on the ORF1-ORF2 region. The sensitivity and reactivity of the two assays used was validated using a reference stool panel containing 14 NoV genotypes. The assays were then used to investigate two outbreaks of gastroenteritis in two Irish hospitals. 56 samples were screened for NoV using a real-time RT-PCR assay and 26 samples were found to be positive. Genotyping of these positive samples found that all positives belonged to the GII/4 variant of NoV.

Conclusion

The combination of the Real-time assay and the reverse line blot hybridisation assay provided a fast and accurate method to investigate a NoV associated outbreak. It was concluded that the predominant genotype circulating in these Irish hospitals was GII/4 which has been associated with the majority of NoV outbreaks worldwide. The assays developed in this study are useful tools for investigating NoV infection.

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Most cited references 15

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Molecular and epidemiologic trends of caliciviruses associated with outbreaks of acute gastroenteritis in the United States, 2000-2004.

H Blanton, Sandra N. Bulens, Stephan S. Monroe … (2006)

Between July 2000 and June 2004, fecal specimens from 270 outbreaks of acute gastroenteritis were sent to the Centers for Disease Control and Prevention by local or state health departments for calicivirus testing. Of the 226 outbreaks that met the criteria for inclusion in the present study, caliciviruses were detected in 184 (81%) by reverse-transcription polymerase chain reaction and nucleotide sequencing. Nursing homes, retirement centers, and hospitals were the most frequently reported settings, and person-to-person contact was the most common mode of transmission, followed by foodborne spread. Overall, genogroup II norovirus (NoV) strains were the most abundant (79%), followed by genogroup I NoV strains (19%) and sapovirus (2%). Nucleotide-sequence analysis indicated a great diversity of NoV strains and implicated the emergence of one particular sequence variant in outbreaks occurring between July 2002 and June 2003. The public health impact of caliciviruses will not be fully appreciated, nor will interventions be completely evaluated, until methods to detect these viruses are more routinely used.

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The Discovery of the 27-nm Norwalk Virus: An Historic Perspective

Albert Z Kapikian (2000)

Abstract In 1972, a 27-nm virus-like particle was discovered by use of immune electron microscopy (IEM) in an infectious stool filtrate derived from an outbreak of gastroenteritis in an elementary school in Norwalk, Ohio. IEM enabled the direct visualization of antigen-antibody interaction, as the particles were aggregated and coated by specific antibodies. This allowed the recognition and identification of a 27-nm virus-like particle that did not have a distinctive morphology, was low-titered, and was among the smallest viruses known. Serum antibody responses to the 27-nm particle were demonstrated in key individuals infected under natural or experimental conditions; this and other evidence suggested that this virus-like particle was the etiologic agent of the Norwalk gastroenteritis outbreak. The fastidious 27-nm Norwalk virus is now considered to be the prototype strain of a group of noncultivatable viruses that are important etiologic agents of epidemic gastroenteritis in adults and older children.

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Surveillance for foodborne-disease outbreaks--United States, 1998-2002.

Michael Lynch, Christopher Braden, Rachel S. Y e l k Woodruff … (2006)

Since 1973, CDC has maintained a collaborative surveillance program for collection and periodic reporting of data on the occurrence and causes of foodborne-disease outbreaks (FBDOs) in the United States. 1998-2002. The Foodborne Disease Outbreak Surveillance System reviews data on FBDOs, defined as the occurrence of two or more cases of a similar illness resulting from the ingestion of a common food. State and local public health departments have primary responsibility for identifying and investigating FBDOs. State, local, and territorial health departments use a standard form to report these outbreaks to CDC. In 1998, CDC implemented enhanced surveillance for FBDOs by increasing communication with state, local, and territorial health departments and revising the outbreak report form. Since 2001, reports of FBDOs are submitted through a web application on the Internet called the electronic Foodborne Outbreak Reporting System (eFORS). During 1998-2002, a total of 6,647 outbreaks of foodborne disease were reported (1,314 in 1998, 1,343 in 1999, 1,417 in 2000, 1,243 in 2001, and 1,330 in 2002). These outbreaks caused a reported 128,370 persons to become ill. Among 2,167 (33%) outbreaks for which the etiology was determined, bacterial pathogens caused the largest percentage of outbreaks (55%) and the largest percentage of cases (55%). Among bacterial pathogens, Salmonella serotype Enteritidis accounted for the largest number of outbreaks and outbreak-related cases; Listeria monocytogenes accounted for the majority of deaths of any pathogen. Viral pathogens, predominantly norovirus, caused 33% of outbreaks and 41% of cases; the proportion of outbreaks attributed to viral agents increased from 16% in 1998 to 42% in 2002. Chemical agents caused 10% of outbreaks and 2% of cases, and parasites caused 1% of outbreaks and 1% of cases. Following implementation of measures to enhance outbreak surveillance, the annual number of FBDOs reported to CDC increased during this period compared with previous years. Viral pathogens accounted for an increased proportion of outbreaks each year during this reporting period and a higher proportion of outbreaks of known etiology during this reporting period than preceding reporting periods, probably reflecting the increased availability of improved viral diagnostic tests. S. Enteritidis continued to be a major cause of illness and L. monocytogenes was a major cause of death. In addition, multistate outbreaks caused by contaminated produce and outbreaks caused by Escherichia coli O157:H7 remained prominent. Methods to detect FBDOs are improving, and several changes to improve the ease and timeliness of reporting FBDO data have been implemented (e.g., a revised form to simplify FBDO reporting by state health departments and improved electronic reporting methods). State and local health departments continue to investigate and report FBDOs as part of efforts to better understand and define the epidemiology of foodborne disease in the United States. At the regional and national levels, surveillance data provide an indication of the etiologic agents, vehicles of transmission, and contributing factors associated with FBDOs and help direct public health actions to reduce illness and death caused by FBDOs.

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Author and article information

Journal

Journal ID (nlm-ta): Virol J

Title: Virology Journal

Publisher: BioMed Central (London )

ISSN (Electronic): 1743-422X

Publication date Collection: 2007

Publication date (Electronic): 6 September 2007

Volume: 4

Page: 86

Affiliations

[1 ]Lab 439, Food Science Building, Department of Microbiology, University College Cork, Cork, Republic of Ireland

Article

Publisher ID: 1743-422X-4-86

DOI: 10.1186/1743-422X-4-86

PMC ID: 2008195

PubMed ID: 17822552

SO-VID: 1462c054-716f-4973-92b5-ff6794f997ab

License:

This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Development of a real-time RT-PCR and Reverse Line probe Hybridisation assay for the routine detection and genotyping of Noroviruses in Ireland

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Abstract

Background

Results

Conclusion

Related collections

Point-of-Care Testing for Infectious Diseases Super Collection

Most cited references 15

Molecular and epidemiologic trends of caliciviruses associated with outbreaks of acute gastroenteritis in the United States, 2000-2004.

The Discovery of the 27-nm Norwalk Virus: An Historic Perspective

Surveillance for foodborne-disease outbreaks--United States, 1998-2002.

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