The epidemiological and clinical features identified (symptoms, incubation period, disease duration), together with the laboratory results, were consistent with the initial hypothesis of a foodborne norovirus outbreak, fulfilling Kaplan criteria [9
As in other outbreaks [10
], the statistical analysis did not establish any food as the vehicle of infection, because almost all diners had eaten all the foods. However, two facts suggest that the food vehicle was probably the round of beef (Table ). Firstly, everyone with symptoms of gastroenteritis had eaten round of beef (both paella and round of beef or only beef). Secondly, the four people who became ill after eating beef but not paella were: a child in whom norovirus was detected, the symptomatic food hander (FH A) and two children from whom clinical samples could not be obtained.
Although laboratory analysis of the suspected foods did not find norovirus, this in no way discards the possible implication of the round of beef, since it is known that, in foods other than shellfish, the sensitivity of the analysis is very low due to the low levels of the virus normally present in contaminated foods, but enough to cause infection, and to the presence of RT-PCR inhibitors [2
The most important finding of the investigation was the presence of norovirus in stool samples from an asymptomatic food handler (FH B), who denied having had symptoms before or after the suspected lunch. She had not consumed the suspected round of beef, but she had prepared, cut and served it to the diners (including FH A). Although vomiting may be a source of infection [13
], this possibility may be discarded, as very little more than 24 hours had passed between the appearance of the first cases and the taking of stool samples from FH B. Phylogenetic analysis confirmed that the sequence of the virus detected in FH B was identical to that detected in cases (Genotype GII.2). The region of the genome chosen for sequencing (the RNA-dependent RNA polymerase) is regarded as a good target to discriminate different norovirus genotypes, although it has also been reported that the hypervariable region encoding the P2 domain in the capsid gene can be helpful in tracking transmission events within outbreaks [15
]. Unlike GII.4, GII.2 is not a prevalent genotype, meaning that a possible chance association could be ruled out. In addition, children and monitors lived in different towns than FH B. Therefore, our results suggest that the source of the infection was probably an asymptomatic food handler.
Asymptomatic excretion of norovirus is recognized in facilities with outbreaks [16
] and without outbreaks [18
]. Ozawa et al
. showed that the prevalence of norovirus detection in food handlers was 19% in different food-catering settings in Japan, and that detection of norovirus in asymptomatic food handlers associated with outbreaks was 7%, a substantial proportion.
Transmission by a food handler may be difficult to prove and there is little information about their implication in the transmission of the virus, even though it has been shown that GII-norovirus infected asymptomatic individuals have mean viral loads similar to those of symptomatic subjects (3.31 × 108
versus 5.53 × 108
copies/g of stool), showing the importance of transmission by people who are infected but not ill [17
]. In addition, a very low dose of virus is needed to infect (10-100 viral particles). Although there are not sufficient studies to show the role of asymptomatic food handlers, our study suggests that asymptomatic carriers may be infectious.
Daniels et al
. were the first authors to demonstrate the implication of an asymptomatic food handler in an outbreak [20
], when they found the same genomic sequence of norovirus in the implicated food and in relatives of a food handler not exposed to the food. Molecular analysis of the genomic sequences of the virus detected in the food and in food handlers would be desirable in this kind of outbreak, but due to the difficulty in detecting the virus in foods the association is usually established either by identifying the same viral genome in the food handler and in affected people or through analytical epidemiological studies. Therefore, stool samples from affected people are essential since it allows the common origin of the cases to be shown. Simultaneously, stool samples from all food handlers should be analyzed, together with the epidemiological information available. Molecular epidemiology analysis of norovirus detected in patients and food handlers should be extended when epidemiological information suggests that the source may be a food handler.
Monitoring of safe practices in the food chain [9
] and the recommendation that food handlers with diarrhoea should not work until 48-72 hours after becoming asymptomatic [7
] are the current strategies employed to prevent foodborne infections. Several studies have demonstrated that viral shedding of norovirus may last longer [24
]. In one study in which the virus was inoculated in fifty volunteers, the results showed that specimens collected 7 days after inoculation remained positive [24
]. In another human experimental norovirus infection model, the virus was detected in stool samples for a median of 4 weeks and for up to 8 weeks after virus inoculation [27
]. Although virus detection in faeces does not mean the virus is infectious, the low infective dose and prolonged shedding of norovirus makes transmission almost certain. Therefore, the recommendation of exclusion of workers for 48-72 hours after disappearance of symptoms may not be sufficient to avoid the transmission of infection.
Moreover, a recent study evaluating the effectiveness of control measures during a norovirus outbreak showed that the implementation of recommended strategies reduced transmission of the virus by 85% (95% predictive interval: 81-87%), but could not contain the outbreak [28
]. The use of protective barriers such as gloves, aprons, facemasks and hairnets seems not to be effective against norovirus.
In an investigation of a gastroenteritis outbreak, accurate diagnosis and early identification of the origin of contaminated food is essential to prevent new cases, since it enables control measures to be implemented. However, it is not established which measures should be introduced - hand washing, use of barriers or correct cleaning of environmental surfaces [2
]. At present, we do not know how to stop norovirus transmission. Strategies focus on correct hand washing as a preventive action and assessing the efficacy of disinfectants in eliminating the virus from the environment (water and surfaces). Effective prevention strategies should be determined and implemented according to scientific evidence.