Descriptive and analytical studies
The outbreak occurred during the Easter holidays, between March 31st and April 7th 2002, in the province of Bari (Puglia region, South Italy). In Italy, at Easter many people eat in restaurants and the day after Easter most people go picnicking. In the province of Bari seafood (especially mussels) are often consumed on these occasions. The Regional Epidemiological Office, alerted by the Local Health Unit, performed a field study, collecting data from all General Practitioners and all Emergency Units in the province. Active surveillance was conducted until April 15th 2002 (one week after the last case onset). The aim of the investigation was to describe the outbreak and to identify the etiologic agent, the source of infection and the means of transmission.
A case was defined as illness in a resident in the area during the period March 31st – April 7th, who had diarrhoea (three or more loose stools in any 24 hour period) or vomiting (at least one episode). Fever greater than or equal to 38°C, abdominal pain or nausea were considered additional symptoms. A "probable" secondary case was defined as illness in a household with onset of symptoms more than 24 hours after the primary case.
A retrospective cohort study was performed in order to assess risk factors associated with illness.
All households where a case occurred were included in the study.
Information, collected by means of a standardised questionnaire, included: 1) demographic individual characteristics such as age, gender, occupation; 2) type of food consumed during the last 72 hours before onset of symptoms; 3) if ill, type, date and time of onset of symptoms.
To assess the association between food consumption and disease, relative risks (RR) and 95% confidence intervals (95% CI) were calculated. Age and gender were compared between ill and unaffected individuals by the chi squared and Mann-Whitney tests. A p-value less than 0.05 was considered significant. Variables associated to the illness in the univariate analysis were included in the stratified analysis; summary RR and 95% CI were calculated from the formulas of Greenland and Robins . Collected data were analysed by using Epi Info 6.04 (Centres for Disease Control and Prevention, Atlanta, GA) and Statview 4.0 (Sas Institute Inc., Cary, NC) software.
Faecal specimens were collected from ill individuals. Part of the specimens was refrigerated and processed within 12 hours to detect ova and parasites by direct microscopy and common bacteria by standard methods. The rest was stored at -20°C until examination by NV-specific reverse transcription-polymerase chain reaction (RT-PCR).
Viral nucleic acid extraction and purification from stool specimens was performed as previously reported . RT-PCR was carried out with the primers JV12-SM31 specific for the polymerase gene of NV .
Eleven samples of mussels were collected from two fish-markets from which the cases had bought the shellfish they consumed. Mussel samples were also processed within 12 hours to detect common bacteria by standard methods.
Then a nested PCR was used. The procedure for mussel processing as well as viral RNA extraction and purification has been previously described in full .
The primers used for first round PCR were JV12 and SM31. Nested PCR was carried out with the use of the primers SR33 for negative strand DNA synthesis and SR48 and SR46 for positive strand synthesis of genogroup I (GI) and genogroup II (GII) sequences, respectively .
The 333-bp or 123-bp amplification products from cases and from mussels were subjected to sequencing with PCR primers. When required, cloning was carried out on PCR products. Sequences obtained were aligned with those available in the GenBank.