Development and validation of an efficient nomogram for risk assessment of norovirus infection in pediatric patients.

This study aimed to establish a predictive model and nomogram based on routine laboratory blood indicators and clinical symptoms, subsequently providing a rapid risk assessment of norovirus (NoV) infection in children. This retrospective study enrolled 307 pediatric patients with symptoms of acute gastroenteritis and detected NoV using real-time quantitative polymerase chain reaction. Significant indicators selected by multivariate logistic regression, including routine blood tests and consultation symptoms, were used to develop the nomogram. We divided the sample into training and internal validation sets and performed external validation of the final model. Furthermore, we evaluated the clinical performance using the Akaike information criterion (AIC), area under the curve (AUC), calibration curve, decision curve analysis (DCA), sensitivity, specificity, concordance rate, positive predictive value, and negative predictive value. Overall, 153 cases were NoV-PCR-positive, and 154 were negative. The multivariate logistic regression included five predictors of NoV infection, including symptoms of vomiting, upper respiratory tract infection, and indicators of white blood cells, lymphocyte absolute counts, and platelet counts. The nomogram showed a significant predictive value with overall internal set diagnosis, with an AUC of 0.827 (95% confidence interval (CI): 0.785-0.868), and 0.812 (95% CI: 0.755-0.869) with 0.799 (95% CI: 0.705-0.894) in the training and internal validation sets, respectively. Nevertheless, the AUC in the external validation set was higher (0.915; 95% CI: 0.862-0.968). This nomogram is a useful tool for risk assessment for NoV infection. Moreover, the evaluated indicators are accessible, substantially reducing the time for laboratory testing.

A simple and low-cost paper-based colorimetric method for detecting and distinguishing the GII.4 and GII.17 genotypes of norovirus.

In modern times, viruses still threaten people's lives. Among them, norovirus was the main pathogenic factor in the cause of gastroenteritis and foodborne illness, of which the GII.4 and GII.17 genotypes are prevalent in China and most parts of the world. A simple and low-cost platform for rapid and accurate norovirus detection remains a major challenge. After the cell-free system and paper-based chromogenic system were optimized, a rapid and specific norovirus detection method was established based on norovirus-specific sequences in combination with toehold switch elements. The development of a visible color change during detection eliminates the need for any complicated instruments. We validated this strategy and its specificity in differentiating GII.4, GII.17, Zika virus, and human coronavirus HKU1. The results showed that the optimized detection system not only provided a simple and rapid detection method for the sufficient differentiation of the two norovirus genotypes but also showed high specificity and no cross-reactivity with other viruses. Using nucleic acid isothermal amplification, this assay showed a limit of detection of 0.5 pM for the GII.4 genotype and 2.6 fM for the GII.17 genotype in reactions that could be observed directly with the naked eye. Our results suggested that this paper-based colorimetric method could serve as a simple and low-cost visual detection method for pathogens in clinical samples, especially in remote or rural areas.

Comparing Mouse Health Monitoring Between Soiled-bedding Sentinel and Exhaust Air Dust Surveillance Programs.

To monitor rodent colony health in research facilities, soiled-bedding sentinel (SBS) animals have traditionally been used. SBS can be tested by various methods, which may include serology, PCR analysis, and necropsy. Several pathogens are unreliably detected by using SBS or transmitted poorly through soiled bedding, and collection and evaluation of SBS samples can be time-intensive. Recently, exhaust air dust (EAD) testing through PCR analysis has emerged as an adjunct or replacement method for rodent colony health monitoring. EAD monitoring may provide a more efficient, sensitive, and humane method for monitoring health status. Using both EAD and SBS health monitoring, we evaluated colony health over the course of 1 y in 3 research barrier rooms in which mice were housed exclusively on IVC racks. Three pathogens-Helicobacter spp., Rodentibacter spp. (previously Pasteurella pneumotropica), and murine norovirus (MNV)-were not excluded in 2 of the rooms, and we expected that these mice would test positive with some regularity. EAD monitoring was significantly more sensitive than SBS for detection of the bacterial agents. SBS failed to detect Helicobacter spp. at time points when EAD had 100% detection in the rooms that did not exclude the bacteria. The detection of MNV did not differ between health monitoring systems at any time point. The findings suggest that EAD is especially valuable in detecting bacteria poorly transmitted through soiled bedding. In addition, the corresponding results with MNV detection suggest that EAD surveillance can reliably be implemented as an alternative to SBS monitoring in a facility in which mice are housed exclusively on IVC racks.

Monitoring telehealth vomiting calls as a potential public health early warning system for seasonal norovirus activity in Ontario, Canada.

Norovirus is a predominant cause of infectious gastroenteritis in countries worldwide [1-5]. It accounts for approximately 50% of acute gastroenteritis (AGE) and >90% of viral gastroenteritis outbreaks [6, 7]. The incubation period ranges between 10 and 48 h and illness duration is generally 1-3 days with self-limiting symptoms; however, this duration is often longer (e.g. 4-6 days) in vulnerable populations such as hospital patients or young children [2, 8]. Symptomatic infection of norovirus presents as acute vomiting, diarrhoea, abdominal cramps and nausea, with severe vomiting and diarrhoea (non-bloody) being most common [2, 5, 9].

Single-step detection of norovirus tuning localized surface plasmon resonance-induced optical signal between gold nanoparticles and quantum dots.

A new method of label free sensing approach with superior selectivity and sensitivity towards virlabel-freeon is presented here, employing the localized surface plasmon resonance (LSPR) behavior of gold nanoparticles (AuNPs) and fluorescent CdSeTeS quantum dots (QDs). Inorganic quaternary alloyed CdSeTeS QDs were capped with L-cysteine via a ligand exchange reaction. Alternatively, citrate stabilized AuNPs were functionalized with 11-mercaptoundecanoic acid to generate carboxylic group on the gold surface. The carboxylic group on the AuNPs was subjected to bind covalently with the amine group of L-cysteine capped CdSeTeS QDs to form CdSeTeS QDs/AuNPs nanocomposites. The fluorescence of CdSeTeS QDs/AuNPs nanocomposite shows quenched spectrum of CdSeTeS QDs at 640 nm due to the close interaction with AuNPs. However, after successive addition of norovirus-like particles (NoV-LPs), steric hindrance-induced LSPR signal from the adjacent AuNPs triggered the fluorescence enhancement of QDs in proportion to the concentration of the target NoV-LPs. A linear range of 10-14 to 10-9 g mL-1 NoV-LPs with a detection limit of 12.1 × 10-15 g mL-1 was obtained. This method was further applied on clinically isolated norovirus detection, in the range of 102-105 copies mL-1 with a detection limit of 95.0 copies mL-1, which is 100-fold higher than commercial ELISA kit. The superiority of the proposed sensor over other conventional sensors is found in its ultrasensitive detectability at low virus concentration even in clinically isolated samples. This proposed detection method can pave an avenue for the development of high performance and robust sensing probes for detection of virus in biomedical applications.

Methods for ascertaining norovirus disease burdens.

Norovirus is the commonest cause of gastrointestinal disease worldwide in. Infections with norovirus occur in all age groups, however, the highest incidence is in children aged less than five years. Surveillance of norovirus is complicated because most people do not contact medical services when they are ill. Nevertheless, Public health laboratory surveillance worldwide has demonstrated the dominance of GII.4 viruses in the population. Better epidemiological surveillance and outbreak investigations, coupled with wider implementation of molecular-based laboratory diagnostics are leading to better estimates of the burden of norovirus infections as well as improved outbreak control. Recent advances in cell culture systems for norovirus and current research investigating the distribution of norovirus-associated disease in the population, for whom the disease burden is greatest, understanding host susceptibility factors, and methodologies for ascertaining cases, are important in increasing our understanding of norovirus. The key to surveillance of norovirus is allying the epidemiology with surveillance of virology. With recent advances in laboratory culture systems for norovirus, next generation sequencing technologies, improved diagnostics and measuring phenotypic characteristics of noroviruses, there are new opportunities to advance understanding of this common and important human pathogen that will help design strategies for vaccine and antiviral development, and how these might be best deployed to control norovirus infection.

Case-Control Assessment of the Roles of Noroviruses, Human Bocaviruses 2, 3, and 4, and Novel Polyomaviruses and Astroviruses in Acute Childhood Diarrhea.

BACKGROUND: The etiology of acute childhood diarrhea often eludes identification. We used a case-control study-stool archive to determine if nucleic acid tests for established and newly identified viruses diminish our previously published 32% rate of microbiologically unexplained episodes. METHODS: Using polymerase chain reaction, we sought to detect noroviruses GI and GII, classic and novel astroviruses, and human bocaviruses (HBoVs) 2, 3, and 4 among 178 case and 178 matched control stool samples and St. Louis and Malawi polyomaviruses among a subset of 98 case and control stool samples. We calculated adjusted odds ratios and 95% confidence intervals using conditional logistic regression. RESULTS: Noroviruses were more common in cases (GI, 2.2%; GII, 16.9%) than in controls (GI, 0%; GII, 4.5%) (adjusted odds ratio, 5.2 [95% confidence interval, 2.5-11.3]). Astroviruses and HBoVs 2, 3, and 4 were overrepresented among the cases, although this difference was not statistically significant. Malawi polyomavirus was not associated with case status, and St. Louis polyomavirus was identified in only 1 subject (a control). When identified in cases, HBoVs 2, 3, and 4 were frequently (77%) found in conjunction with a bona fide diarrheagenic pathogen. Thirty-five (20%) case and 3 (2%) control stool samples contained more than 1 organism of interest. Overall, a bona fide or plausible pathogen was identified in 79% of the case stool samples. Preceding antibiotic use was more common among cases (adjusted odds ratio, 4.5 [95% confidence interval, 2.3-8.5]). CONCLUSION: Noroviruses were found to cause one-third of the diarrhea cases that previously had no identified etiology. Future work should attempt to ascertain etiologic agents in the approximately one-fifth of cases without a plausible microbial cause, understand the significance of multiple agents in stools, and guide interpretation of nonculture diagnostics.

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.

Norovirus in a United States virgin islands resort: outbreak investigation, response, and costs.

BACKGROUND: During 8-20 April 2012, an outbreak of gastrointestinal illness occurred among guests and employees of a resort hotel in St. Thomas, US Virgin Islands. We describe outbreak characteristics, and estimate indirect (non-medical) costs to travellers. METHODS: Employees who met the case definition were interviewed and provided stool samples. Samples were tested for norovirus by real-time reverse-transcription polymerase chain reaction. Guests were asked to complete a survey aimed to identify and characterize cases, and to estimate quality adjusted vacation days (QAVD) lost. RESULTS: Overall, 66 persons (20 employees and 46 guests) met the probable case definition. The first reported illness onset occurred in a hotel employee on 8 April, while the first reported onset in a guest occurred on 13 April. An employee suffered a public diarrhoea incident on 13 April in the central kitchen, followed by illness onset in the next day among employees that assisted with the clean-up. On 15 April, after 10 guests reported ill, the hotel implemented an outbreak response protocol instructing ill employees to take a 3-day leave, and obtain medical clearance prior to resuming work. Ill guests were advised to self-isolate, and rapid cleaning of public areas and guest rooms where suspected contamination occurred was implemented. We estimated that 65 QAVDs were lost by 43 guests (1.5 days/guest). Using an approximate cost of $450 per vacation day, we estimated indirect illness cost at $675 per guest case. Seven (64%) of 11 cases' stool specimens were positive for norovirus genotype GII.4 Den Haag. CONCLUSIONS: A norovirus outbreak in a resort hotel resulted in substantial indirect costs and loss of vacation days to ill travellers. We recommend outbreak control measures including exclusion of ill employees, until ≥48-72 h after resolution of symptoms, self-isolation of ill guests and appropriate cleaning in hotel-associated norovirus outbreaks.

Performance of a one-step quantitative duplex RT-PCR for detection of rotavirus A and noroviruses GII during two periods of high viral circulation.

Rotavirus A (RVA) and noroviruses (NoV) are the major viral agents of acute gastroenteritis (AGE) worldwide. In the present study, we aimed to evaluate the performance of a one-step duplex quantitative RT-PCR (dRT-qPCR) assay, established for detection and quantification of RVA and NoV genogroup II (GII) using a single DNA standard curve (SC), as well as to investigate the association between fecal viral load and optical density (OD) values, and viruses' genotyping. The results obtained by dRT-qPCR in 530 fecal samples from AGE cases were compared with methods employed for the diagnosis of those viruses as follows: enzyme immunoassay (EIA) and polyacrylamide gel electrophoresis (PAGE) for RVA; and qualitative PCR for NoV. By using dRT-qPCR, we detected RVA and NoV in 353 (66%), increasing the positivity rate by 22.5% for RVA and 11.5% NoV, comparing the number of positive samples. RVA and NoV GII were detected in a range of 5.17 × 10(3) to 6.56 × 10(9) and 3.76 × 10(3) to 9.13 × 10(10) genome copies per gram of feces, respectively. We observed a significant direct correlation between genome copies values and optical density, using dRT-qPCR and EIA assays, respectively (Spearman ρ=0.41; p<0.0001). Viruses characterization demonstrated a predominance of NoV GII.4 Sidney 2012 variant during October 2013 to February 2014, followed by the emergence of RVA genotype G12P[8] in 2014. The established assay using a single SC provides an early feedback concerning detection and quantification, with the advantage of detecting simultaneously RVA and NoV GII, reducing time and reagent costs.

Viral gastrointestinal outbreaks in residential care facilities: an examination of the value of public health unit involvement.

OBJECTIVE: To evaluate the value of notification to public health units (PHUs) in the management of viral gastrointestinal outbreaks in residential care facilities (RCFs) in Queensland. METHODS: Routine outbreak characteristics and control measures were collected at notification and conclusion of outbreaks notified between May and August 2008. This was supplemented with detailed interviews with RCFs and PHUs, including perceptions of the value of the RCF-PHU interaction. RESULTS: Norovirus was the confirmed aetiological agent in 39 (68%) of 60 outbreaks. A significant association was found between increased outbreak duration and longer (≥4 days) time to outbreak notification (p<0.01) and larger facility size (≥150 people; p<0.05). Increased attack rate was not significantly associated with either time to outbreak notification or facility size, but was significantly associated with difficulty in cohorting (p<0.05). Most (92%) RCFs considered that outbreak notification was important and that PHU support was useful (97%). Most PHUs (77%) also considered that outbreak notification was important. CONCLUSIONS: This study demonstrates an association between prompt notification of viral gastroenteritis outbreaks to PHUs and shorter duration of outbreaks. IMPLICATIONS: This study provides evidence to support recommendations in current Australian guidelines that notification of outbreaks should occur promptly.

Assessment of gastroenteric viruses frequency in a children's day care center in Rio De Janeiro, Brazil: a fifteen year study (1994-2008).

This 15-year study aimed to determine the role of the main viruses responsible for acute infantile gastroenteritis cases in a day care center in the city of Rio de Janeiro, Brazil. From 1994 to 2008, 539 fecal samples were obtained from 23 outbreaks as well as sporadic cases that occurred in this period. The detection of Rotavirus group A (RVA), norovirus (NoV) and astrovirus (AstV) was investigated both by classical and molecular methods of viral detection. RVA was detected by enzymatic immune assay and/or polyacrylamide gel electrophoresis and genotyped by using semi-nested multiplex PCR. NoV and AstV were subsequently tested by real time PCR in all RVA-negative samples and genotyped throughout genome sequencing. Three protocols for molecular characterization of NoV nucleotide sequencing were performed with the partial nucleotide sequencing of genomic regions known as region B (polymerase gen), C and D (capsid gen).Viruses were identified in 47.7% (257/539) of the cases, and the detection rates of RVA, NoV and AstV in16.1% (87/539), 33.4% (151/452), and 6.3% (19/301), respectively. Most gastroenteritis cases were reported in autumn and winter, although NoV presented a broader monthly distribution. Viruses' detection rates were significantly higher among children aged less than 24 months old, although NoV cases were detected in all age groups. RVA genotypes as G1P[8], G9P[8], G2P[4], G3P[8] and G1+G3P[8] and RVA was no longer detected after 2005. NoV characterization revealed genotypes variability circulating in the period as GI.2, GI.3, GI.8 GII.2, GII.3, GII.4, GII.4 variants 2001 and 2006b, GII.6, GII.7, GII.12 and GII.17. AstV genotypes 1, 2, 4 and 5 were also characterized. Those data demonstrate the impact of NoV infection in cases of infantile gastroenteritis, surpassing RVA infection responsible for high morbidity rate in children under five years old.

Assessment of a rapid immunochromatographic test for the diagnosis of norovirus gastroenteritis.

Noroviruses are the leading cause of acute gastroenteritis in people of all ages. Since the viruses are highly infectious, rapid and early diagnosis is important to prevent and control the disease. The present study aimed to evaluate the commercial immunochromatographic test RIDA® QUICK Norovirus for the detection of norovirus in stool samples from patients with acute gastroenteritis in Thailand. As compared with reference RT-PCR results, the RIDA® QUICK Norovirus assay provided a sensitivity of 48.2 and 83.3% with a specificity of 87.5%. False positive results were observed in 12.5% of norovirus-negative stool samples. Based on commercial quantitative real-time RT-PCR, the RIDA® QUICK Norovirus assay revealed a highly significant association, p-value <0.001, and good agreement (kappa = 0.6). The assay could detect norovirus in stool samples ranging from 3.22 × 10(6) to 3.26 × 10(8) copies/ml. False negative results occurred in the stool samples containing 5.9 × 10(6) copies/ml of norovirus GI or 1.85 × 10(4) - 4.28 × 10(5) copies/ml of GII. The immunochromatographic RIDA® QUICK Norovirus assay may be useful for rapid screening of norovirus infections in patients with acute gastroenteritis in both developed and developing countries where the RT-PCR method has not been established for routine diagnosis.

Updated norovirus outbreak management and disease prevention guidelines.

Noroviruses are the most common cause of epidemic gastroenteritis, responsible for at least 50% of all gastroenteritis outbreaks worldwide, and a major cause of foodborne illness. In the United States, approximately 21 million illnesses attributable to norovirus are estimated to occur annually. Since 2001, when the most recent norovirus recommendations were published (CDC. "Norwalk-like viruses." Public health consequences and outbreak management. MMWR 2001;50[No. RR-9]), substantial advances have been made in norovirus epidemiology, immunology, diagnostic methods, and infection control. As molecular diagnostic techniques have improved in performance and become more widely available, detection and reporting of norovirus outbreaks have increased. Although the inability to culture human noroviruses in vitro has hampered progress, assessment of the performance of disinfectants has been facilitated by the discovery of new, cultivable surrogates for human noroviruses. In addition, the periodic emergence of epidemic strains (from genogroup II type 4, GII.4) and outbreaks in specific populations (e.g., the elderly in nursing homes) have been characterized. This report reviews these recent advances and provides guidelines for outbreak management and disease prevention. These recommendations are intended for use by public health professionals investigating outbreaks of acute gastroenteritis, including state and local health authorities, as well as academic and research institutions.

SYBR Green based real-time RT-PCR assay for detection and genotype prediction of bovine noroviruses and assessment of clinical significance in Norway.

A novel SYBR Green based real-time RT-PCR assay for detection of genogroup III bovine noroviruses (BoNoV) was developed and the assay applied to 419 faecal samples from calves with and without diarrhoea. The samples were obtained from 190 Norwegian dairy and beef herds. BoNoV was detected in 49.6% of the samples from 61.1% of the herds indicating that BoNoV is ubiquitous in Norway. The overall prevalence was not significantly different in diarrhoea and non-diarrhoea samples. Analyses of polymerase gene sequences revealed both genotype III/1 and III/2 with genotype III/2 (Newbury2-like) being the most prevalent. Detected capsid sequences were restricted to Newbury2-like and the chimeric Bo/Thirsk10/00/UK strain. The RNA polymerase genotypes of the circulating BoNoVs in Norway were predicted by melting temperature analysis. Additional data from a challenge experiment suggest that a high proportion of young calves are shedding low levels of BoNoV for a prolonged time after recovering from the associated diarrhoea. The findings may explain some of the discrepancies in detection rates from previous studies and explain why some studies have failed to detect significant prevalence differences between calves with and without diarrhoea. It may also shed new light on some epidemiological aspects of norovirus infections.

[Assessment of viral gastroenteritis diagnosis using an immunochromatography-based method].

We evaluated the norovirus (NoV) detection reagent kit, which uses immunochromatography (IC), against outbreaks of food-borne acute viral gastroenteritis having occurred in Osaka Prefecture between November 2008 and March 2009. A total of 33 outbreaks, RT-PCR identified 27 NoV-positive cases, whereas the IC kit identified 26 cases. Out of 103 specimens positive for NoV using RT-PCR, the IC kit identified 68 positive cases, with a positive conformity ratio of 66%. The mean copy number of NoV was approximately 10(7.1) per 10 mg feces for IC kit positive samples, and approximately 105(5.8) per 10 mg for negative samples. Although the NoV genogroup (G) II/4 was associated with 18 outbreaks and a total of 8 different genotypes were identified in NoV positive samples, G I/7 was not detected using the IC kit. Our results suggest that the IC kit, which detected 96% of the outbreaks of food-borne acute viral gastroenteritis by NoV, facilitates the diagnosis of outbreaks.

[Assessment of two methods of antigenic detection by ELISA for the diagnosis of norovirus outbreaks]. / Evaluación de dos métodos de detección antigénica por ELISA para el diagnóstico de brotes causados por norovirus.

INTRODUCTION: The aim of this study was to assess two ELISA techniques for the detection of outbreaks of norovirus. METHOD: One-hundred and sixty-five fecal samples from patients affected in 30 outbreaks were studied. RESULTS: On the basis of a specific consensus criterion between techniques for confirming outbreaks, the sensitivity and specificity was respectively 80% and 90% for RIDASCREEN, and 80% and 100% for IDEIA. CONCLUSION: These techniques could be useful for rapid detection of norovirus outbreaks in laboratories that lack molecular confirmation techniques.

Multiplex real time RT-PCR for the detection and quantitation of norovirus genogroups I and II in patients with acute gastroenteritis.

BACKGROUND: Conventional reverse transcription-polymerase chain reaction (Con RT-PCR) assay to detect norovirus is a complex multi-step procedure that requires gel electrophoresis as well as hybridization or sequencing to confirm the results. OBJECTIVE: To develop and evaluate a multiplex real time RT-PCR (Mrt RT-PCR) assay that detect and quantify norovirus GI and GII with a single amplification and detection step. STUDY DESIGN: The primers and TaqMan probes for the Mrt RT-PCR were selected from the ORF1-ORF2 junction region. A total of 97 stools from 41 gastroenteritis outbreaks and 726 stools from children with sporadic diarrhoea were used for this study. RESULTS: For the 97 outbreak samples, norovirus were detected in 61 of the 69 previously tested positive and 11 of the 28 previously tested negative samples. Eight samples that tested positive for GII by Con RT-PCR but negative by the Mrt RT-PCR also tested negative by a Light Cycler RT-PCR assay. Eighty-two GII and two GI were detected in the 726 sporadic samples. Random primers were more sensitive than specific primers in the cDNA synthesis. The two-step assay using the random primers in RT reaction was 100 times more sensitive than the one-step assay. The Mrt RT-PCR had the same sensitivity as that using two real time RT-PCR for separate detection of GI and GII. A wide dynamic range was obtained with the two-step assay, detecting from 3000 to 3x10(11) of copies RNA/g stool. Very good precision was observed with no cross-reaction with other enteric viruses. The new assay is able to detect both GI and GII in one reaction and brings a cost reduction of approximately 40% compared to separate reactions for GI and GII. CONCLUSIONS: The assay has good precision, sensitivity and specificity and is cost-effective as a routine diagnostic test.

Foodborne viruses.

Foodborne and waterborne viral infections are increasingly recognized as causes of illness in humans. This increase is partly explained by changes in food processing and consumption patterns that lead to the worldwide availability of high-risk food. As a result, vast outbreaks may occur due to contamination of food by a single foodhandler or at a single source. Although there are numerous fecal-orally transmitted viruses, most reports of foodborne transmission describe infections with Norwalk-like caliciviruses (NLV) and hepatitis A virus (HAV), suggesting that these viruses are associated with the greatest risk of foodborne transmission. NLV and HAV can be transmitted from person to person, or indirectly via food, water, or fomites contaminated with virus-containing feces or vomit. People can be infected without showing symptoms. The high frequency of secondary cases of NLV illness and - to a lesser extent - of hepatitis A following a foodborne outbreak results in amplification of the problem. The burden of illness is highest in the elderly, and therefore is likely to increase due to the aging population. For HAV, the burden of illness may increase following hygienic control measures, due to a decreasing population of naturally immune individuals and a concurrent increase in the population at risk. Recent advances in the research of NLV and HAV have led to the development of molecular methods which can be used for molecular tracing of virus strains. These methods can be and have been used for the detection of common source outbreaks. While traditionally certain foods have been implicated in virus outbreaks, it is clear that almost any food item can be involved, provided it has been handled by an infected person. There are no established methods for detection of viruses in foods other than shellfish. Little information is available on disinfection and preventive measures specifically for these viruses. Studies addressing this issue are hampered by the lack of culture systems. As currently available routine monitoring systems exclusively focus on bacterial pathogens, efforts should be made to combine epidemiological and virological information for a combined laboratory-based rapid detection system for foodborne viruses. With better surveillance, including typing information, outbreaks of foodborne infections could be reported faster to prevent further spread.