Sample size We estimated that we would require 51 households (average size 3.8) in the control arm to allow determination of a secondary attack ratio of approximately 24% [21] to within +/27%. Allowing for potential dropout, we therefore planned to recruit at least 60 households in the control arm, and a further 25–30 households to each of the face mask and hand hygiene arms to evaluate the feasibility of the interventions and allow a preliminary albeit imprecise estimate of efficacy. This pilot study was not powered to detect small or moderate efficacies of the interventions with statistical significance. We did not specify any early stopping rules or interim analyses. Randomization Randomization lists were prepared by a biostatistician (B.J.C.). Eligible study participants were randomly allocated to three groups. The first 100 households were randomized in the ratio 2:1:1 and subsequent households were randomized in the ratio 8:1:1 using a random number generator (R software). The rationale for changing the randomization ratio was to allow us to gather maximum information about the natural characteristics of influenza transmission in households in the absence of control measures, after evaluating the feasibility of each of the interven- tions in at least 25 households. Interventions were assigned to households by the study manager (R.O.P.F.) based on the randomization sequence. The allocation to specific intervention arms was concealed to recruiting doctors/clinics throughout. Blinding Participants and those administering interventions were not blinded to the interventions, but participants were not informed of the specific nature of the other interventions applied to other participating households. Laboratory methods Nasal swabs were collected by inserting and rotating a sterile swab (Collection swab; EUROTUBO, Madrid, Spain) into the anterior nares. Throat swabs were collected by rubbing a second sterile swab against the tonsillar fossa. Both swabs were snapped off into a tube containing viral transport medium (5% bovine serum albumin in Earle’s balanced salt solution with antibiotic). At recruitment, additional nose and throat swabs were collected using sterile foam swabs and then combined and tested by the QuickVue Influenza A+B rapid diagnostic test. Specimens collected from index subjects at recruitment were stored in a 2–8uC refrigerator (overnight, if required). Specimens collected during home visits were stored in a cool box with at least two icepacks immediately after collection. Before the end of the day of a home visit, study nurses took samples to the nearest collection point for storage in a 2–8uC refrigerator (overnight, if required) or directly to the central testing laboratory. Samples stored at 2–8uC were delivered to the central testing laboratory by courier in cool boxes en route. Samples were eluted and cryopreserved at 270uC immediately after receipt. All clinical specimens were cultured on Madin-Darby canine kidney cells with exogenous trypsin (2 ug/ml) added. In households which were successfully followed up with home visits, the clinical specimens collected from index subjects at the recruiting clinic and during the first home visit were additionally tested by reverse transcription polymerase chain reaction (RT- PCR) for influenza A and B viruses if both specimens were negative by viral culture. For household contacts who reported symptoms during the follow-up but whose corresponding clinical Effectiveness of NPIs for Flu specimens (collected within +/22 days of self-reported fever or other respiratory symptoms) were negative by viral culture, those specimens were additionally tested for influenza A and B by RT- PCR. Additional technical details of the laboratory procedures employed in viral culture and RT-PCR testing are given in Text S1. Statistical methods To evaluate the SAR and to compare between groups we used exact binomial 95% confidence intervals, and x2 tests and multivariable logistic regression models adjusting for potential within-household correlation [22,23], with a 5% type I error rate. We estimated the intra-cluster correlation coefficient from the mean squared errors in the SAR between and within households [22]. All analyses were by intention-to-treat. We evaluated the three definitions of clinical influenza described above using receiver operating characteristic (ROC) analysis to determine the clinical definition that corresponds most closely to the laboratory outcome measure [24]. All analyses were conducted in R version 2.4.1 [25]. Results Nine hundred and forty-four subjects were initially recruited to the study between February 24 and September 14, 2007. Figure 1 shows the progress of subjects and household contacts through the study. Overall, and in each intervention arm, the median household size was 4. Both the recruitment rate of subjects and the percentage of positive rapid influenza test results among recruited subjects increased in line with other measures of influenza activity including sentinel outpatient visits and labora- tory isolations in mainly inpatient specimens during the periods of peak influenza activity in February and June (Figure S1). Of the 944 recruited subjects, 198 met the criteria for randomization and further follow-up. In a protocol deviation we randomized 9 subjects who had symptoms for (slightly) more than 48 hours; these 9 subjects were retained in the analyses. Baseline data Characteristics of the 198 subjects are shown in Table 1 according to intervention arm. In general the groups were well- matched. After randomization 70 (35%) of the households declined any home visits or could not be contacted after numerous repeated attempts. Proportionally more of these dropouts were in households where the index was a young adult, whereas there were few dropouts when the index subject was a child. Dropout was higher in households of index subjects who had a negative result on the rapid influenza test (25/44, 57%) compared to those who had a positive result (45/154, 29%). We implemented the interventions in the remaining 128 households, and 127 (99%) were successfully followed for all four home visits; one household completed three home visits. (Table 1) The median household sizes were 4 in all intervention arms. We were typically able to apply the intervention within 1–2 days of symptom onset in the index case (Figure 2). Delays between symptom onset and intervention did not significantly differ between study arms (data not shown). Numbers analyzed Influenza could not be confirmed by viral culture or RT-PCR in the index subjects in 6 of the 128 households; therefore we only retained 122 households for analysis of crude SARs. Five household contacts had missing data on age, and these were further excluded for the multivariable regression analyses. PLoS ONE | www.plosone.org 3 May 2008 | Volume 3 | Issue 5 | e2101