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Real time RT-PCR is the gold standard for laboratory diagnosis

Noroviruses are the most common cause of viral gastroenteritis in humans. In recent years diagnostic methods for Noroviruses, especially real-time reverse transcription-polymerase chain reaction (RT-PCR) for the detection of Norovirus-RNA, have been improved and become more widely available.

by Dr Christoph Metzger-Boddien

Noroviruses are transmitted by fecally contaminated food or water, by person-to-person contact, and via aerosolization of the virus and subsequent contamination of surfaces. They are the most common cause of viral gastroenteritis in humans [15]. Symptoms include nausea, vomiting, diarrhea, and stomach cramping. Additional symptoms are fever, chills, headache, muscle aches and a general sense of tiredness. The onset of symptoms can begin quickly and an infected person may feel sick after a very short period of time. In most people, the illness lasts for about one or two days. People with Norovirus illness are contagious from onset of symptoms until at least three days after recovery. Some people may be contagious for even longer. Noroviruses are highly contagious. The estimated dose is as low as 18 viral particles. Approximately 5 billion infectious doses can be present in each gram of feces during peak shedding [16]. Infection can be more severe in young children and elderly people. Dehydration can occur rapidly and may require medical treatment or hospitalization [10].

Sporadic disease
In recent years diagnostic methods for Noroviruses, especially real-time reverse transcription-polymerase chain reaction (RT-PCR) for the detection of Norovirus-RNA, were improved and became more widely available. Subsequently, it became obvious that Noroviruses are the leading cause of sporadic gastroenteritis in all age groups. In Germany, since the implementation of the notification requirement according to §§6 and 7 of the infection protection act (Infektionsschutzgesetz, IfSG) a rise of reported cases can be observed with a seasonal accumulation during the winter months from October to March (2001: 9,223 cases, 2004: 64,973 cases, 2007: 201,242 and 2008: 212,769 cases, source: Robert-Koch-Institute, RKI, Berlin), but still a high estimated number of unreported cases remain.

Noroviruses are the predominant cause of gastroenteritis outbreaks worldwide. Data from the United States and European countries show that Norovirus is responsible for approximately 50% of all reported gastroenteritis outbreaks (range: 36%–59%) [12]. Periodic increases in Norovirus outbreaks are associated with the emergence of new GII.4 strains. These emergent GII.4 strains are rapidly replacing existing strains predominating in circulation and sometimes cause seasons with high Norovirus activity, as in 2002–2003 and 2006—2007 [17, 20]. Genetic drift successfully promotes the re-emergence of GII-4 variants in the population [13]. Because the virus can be transmitted by food, water and contaminated environmental surfaces as well as directly from person to person, and because there is no long-lasting immunity to Noroviruses, outbreaks can occur in a variety of institutional settings (e.g. nursing homes, hospitals, and schools) and affect people of all ages. Multiple routes of transmission can occur within an outbreak; for example, point-source outbreaks from a food exposure often result in secondary person-to-person spread within an institution or community [4]. Of the 1,518 Norovirus outbreaks in the USA, during 2010 – 2011, laboratory confirmed by the CDC, 59% were from long-term care facilities (889 outbreaks); 8% were from restaurants (123 Outbreaks); 7% were from parties & events 7% (99 outbreaks); 4% were from hospitals (65 outbreaks); 4% were from schools (64 outbreaks); 4% were from cruise ships (55 outbreaks); and 14% were from other and unknown events (223 outbreaks) [10].

Foods that are commonly involved in outbreaks of Norovirus infection are e.g. leafy greens, fresh fruits, and shellfish. However, any food that is served raw or is being handled after cooking can get contaminated.

In Germany, according to data published by the RKI, the number of Norovirus outbreaks has increased by 20% between 2009 and 2010. Recently, the RKI published the final report of a huge outbreak of acute gastroenteritis in five Eastern German federal states. The source of the outbreak was a batch of deep-frozen strawberries. In total, over 11,000 cases of disease occurred. It was Germany’s largest foodborne outbreak of gastroenteritis, with several hundred institutions affected. In a considerable proportion of tested patients, Noroviruses were found [4].

Analysis of outbreak costs
In fact there is a huge socio-economic impact of Norovirus-associated diseases. A study of Johnston et al. 2007, showed the costs of an outbreak including the estimated loss of revenue because of unit closures, sick leave and cleaning expenses [7]. Because of the high contagiousness of Noroviruses early diagnosis in order to set up appropriate hygiene interventions is the most useful measure. In 2004, Lopman et al. showed, that diagnosis of the first case within three days instead of four reduces the duration of an outbreak by seven days [5, 8].

Diagnostic methods
The clinical specimens used for Norovirus diagnosis in most cases are stool and vomit samples. There is no cell culture method for the isolation of Noroviruses from clinical specimens available. Therefore, the majority of clinical virology laboratories perform RT-PCR assays for Norovirus detection. Additionally, for preliminary identification of Norovirus as the cause of gastroenteritis outbreaks, there are enzyme immunoassays (EIA) and rapid tests available. However, these kits are not recommended for individual diagnosis.

Real-time RT-PCR assays
The region between ORF1-ORF2 is the most conserved region of the Norovirus genome, with a high level of nucleotide sequence identity across strains within a genogroup [6]. This region is ideal for designing broadly reactive primers and probes for real-time RT-PCR (RT-qPCR) assays for high throughput screening in clinical diagnostic laboratories and for the detection of Norovirus RNA in
environmental samples (e.g. food and water).

The quality of the real time RT-PCR results is dependent on the quality of template RNA-extraction from clinical and environmental samples. The implementation of extraction controls in commercial RT-PCR duplex assays (e.g. Control-RNA in MutaREX Norovirus Kit, Immundiagnostik AG, Bensheim, Germany) minimizes the risk of false negative results due to inhibition or partial inhibition of the reverse transcription step and/or the PCR and due to processing errors during the extraction of RNA. Control RNA is added to a sample before RNA extraction with a commercial kit (e.g. High pure viral RNA Kit, Roche Diagnostics GmbH, Mannheim, Germany;  or intron viral gene spin, gerbion, Kornwestheim, Germany) and its recovery is measured subsequently in the duplex real time RT-PCR. The latest generation of commercially available Norovirus real time RT-PCR Kits is extremely sensitive and specific [18]. Therefore such tests have become the gold standard for Norovirus laboratory diagnosis in the past few years.

Enzyme immunoassays
For detection of Norovirus antigen in clinical samples, rapid assays (e.g. EIA) offer an alternative to real time RT-PCR assays. However, the development of a broadly reactive EIA for Noroviruses has been challenging because of the number of antigenically distinct Norovirus strains and the high viral load required for a positive signal in these assays. Commercial kits include pools of cross-reactive monoclonal and polyclonal antibodies. In evaluation studies, the sensitivity of these kits ranged from 36% to 80%, and specificity has ranged from 47% to 100% compared with real time RT-PCR [1, 2, 3, 9, 11, 14, 19].

Norovirus real time RT-PCR Kits offer a sensitive, specific, fast and cost effective diagnosis. Results can be generated within one hour. But clearly only real time RT-PCR Kits containing control RNA used as extraction control for process monitoring produce feasible and reliable results. RNA extraction from clinical specimens and the reverse transcription of RNA to cDNA are the most crucial steps in Norovirus RT-PCR procedures. Errors in sample preparation and/or RT-reaction can lead to false negative results in conventional RT-PCRs as well as real time RT-PCRs when internal controls (RNA or DNA) are already added to the PCR master-mix. Laboratories performing in-house RT-PCR for Noroviruses should critically evaluate their tests with regard to these high quality standards. Because of the modest performance of Norovirus Enzyme Immunoassays, particularly their poor sensitivity, they are not recommended for clinical diagnosis of Norovirus infection in sporadic cases of gastroenteritis. Negative samples will have to be confirmed by real time RT-PCR in outbreaks as well as in sporadic cases.

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The author
Christoph Metzger-Boddien, PhD
gerbion GmbH & Co. KG
Remsstr. 1, D-70806 Kornwestheim, Germany