The Ethics Committee of S. Martino Hospital (Genoa, Italy) approved the study protocol and the written informed consent form (N° 17/2010).

Study design

A matched case-control study was performed during the 2010-11 influenza season in Genoa (Italy). Both cases and controls were recruited by 4 General Practitioners (GPs), each of whom has about 1,200 patients aged over 18 years. Cases and controls were matched in a 1:1 ratio on the basis of gender, age (+/- 3 years) and socio-economic status (evaluated on the basis of educational level and the district of residence). Each case and matched control had the same GP.

Case definition and selection

The potential cases were all subjects who had had at least one episode of ILI during the study period (December 2010-March 2011). Only subjects who communicated the disease to their GP were recruited. The case-definition of ILI was: presence of fever >38°C (100.4°F) and at least one other systemic symptom (headache, malaise, myalgia, chills or sweats, retrosternal pain, asthenia) and at least one respiratory symptom (cough, sore throat, nasal congestion or runny nose) during the study period [23, 24].

The exclusion criteria for cases were: refusal to participate in the study and inability to provide informed consent.

Control definition and selection

The controls were all subjects who had not had ILI during the study period (December 2010-March 2011).

At the moment of recruitment of each case, GP identified potential control subjects corresponding to matching criteria from among the patients registered in his databases. At the end of the study period (31 March 2011) controls were entered into the study by randomly selecting each control from within the group of potential candidates identified at the time of recruitment of the cases. Each GP then contacted the chosen control by telephone in order to ascertain that he/she had not had ILI during the study period. To minimize the possibility of enlisting a false negative control, the GP read the definition of ILI and provided any information necessary. In the event of refusal to participate (no signed informed consent), the GP excluded the subject and contacted the next subject on the list of potential controls.

Study period (recruitment)

The diagnosis of ILI was based on clinical definition. In order to improve the specificity of diagnoses, the study period was therefore limited to the weeks of the influenza peak (December 2010-March 2011) according to the Italian Influenza Surveillance Network (INFLUNET) [25].

Data collection

All GPs and researchers who carried out the study strictly complied with Italian regulations on privacy [26]. GPs administered an ad hoc written questionnaire to both cases and controls. The questionnaire was administered either by telephone or by face-to-face interview, after written informed consent had been obtained. The following data were recorded: personal habits (smoking, alcohol consumption [more than 2 glasses of wine or more than one glass of spirits a day]) and the presence of underlying diseases, which were classified in accordance with the International Statistical Classification of Diseases and Related Health Problems (ICD-10) [27]. Specifically, cardiovascular diseases (ICD-10 codes: I00-I02; I05-I09; I20-I99), hypertension (ICD-10 codes: I10 – I15), respiratory diseases (ICD-10 codes: J00 – J99 excluding J09 – J18), kidney diseases (ICD-10 codes: N00 – N99), diabetes (ICD-10 codes: E10-E14), cancer (ICD-10 codes: C00-D48), dyslipidemia (ICD-10 code: E78), gastric ulcer (ICD-10 code: K-25) and gastric diseases (ICD-10 codes K-21; K28-K31) were recorded. Furthermore, GPs collected data on influenza vaccination and therapy with OFC (omeprazole, esomeprazole, lansoprazole, pantoprazole, rabeprazole), fibrates (bezafibrate, ciprofibrate, clofibrate, gemfibrozil, etc), statins (atorvastatin, fluvastatin, lovastatin, simvastatin, etc) and antibiotics. OFC were administered at a dosage of 20-40 mg/day for a period of at least 2 weeks before enrolment, and this therapy was continued for at least 8 weeks. With regard to fibrates, these had to be assumed by at least 30 days before the moment of enrolment; Gemfibrozil, for instance, was administered at a dosage of between 900 and 1200 mg/day, preferably 1200 mg, and was continued for long periods of time. Finally, with regard to statins, these also had to be assumed by at least 30 days before the moment of enrolment. As an example of the dosage of statins, atorvastatin was administered at dosages of 10-80 mg/day for long periods.

All data collected from questionnaires regarding the anamnesis, therapy and vaccination status were verified by patients’ electronic records.

Statistical analysis

The characteristics of the study population are reported as means and standard deviation (SD) for continuous variables and as proportions for categorical variables. Differences between cases and controls were analysed by means of McNemar’s test for matched case-control studies (two-tailed p-value).

A multivariable conditional logistic regression model was used to evaluate the effectiveness of OFC use preliminarily and to estimate the adjusted odds ratio and its 95% Confidence Interval (CI). In the model, the binary variables “ILI” and “OFC” were designated as the “outcome” and “main exposure”, respectively. The decision regarding which factors to include in the multivariable model was based on a conceptual framework describing the hierarchical relationships [28] between exposures (Table 1), with the interaction term (OFC & Influenza vaccination) being entered at a separate level. The results of the interaction analyses are presented both as the separate effects of the two exposures and as their joint effects (Table 2). In accordance with the general consensus in the epidemiological community [29, 30], we presented the interaction effects on the additive scale, as this approach is the most appropriate for public health purposes. To calculate the measure of interaction, the two exposures “OFC” and “Influenza vaccination”, as preventive factors, were recoded “in such a way that the stratum with the lowest risk, when both factors are considered jointly, becomes the reference category” [31]. The measure of interaction RERI (Relative Excess Risk due to Interaction) was then calculated (Table 2) in order to examine the presence of interaction by using measurements derived from the logistic regression [31–34]. In order to estimate all three ORs that are needed to assess biological interaction, the model was set up in such a way as to include terms for three of the four possible combinations of exposure, while the fourth category served as a reference category.

Table 1 Hierarchical theoretical model of potential proactive action of the omeprazole family compounds against influenza viruses Full size table

Table 2 Separate and joint effects of the preventive exposures “Influenza vaccination” and “OFC” on the risk of ILI after recoding and results of analyses of interaction Full size table

Analyses were performed by means of SPSS vers.16.0 for Windows, EpiInfo vers. 3.5.3, Graph-Pad software and an Excel sheet available at: http://www.epinet.se for the assessment of biological interaction. The coefficients estimated by the conditional logistic regression were obtained by using the procedure of SPSS COXREGR; this is equivalent to the conditional logistic regression when there is only one case and one or more controls in each layer.