Josephine van Dongen

Chapter 4 96 Sample size calculation was based on eight par ticipating hospitals, an assumed VE of at least 60% against severe rotavirus AGE, at a cumulative incidence of four percent until 18 months of age and, a between hospital intra-class correlation of 0.002, as documented in study protocol. 11 Microbiological analysis Fecal samples were tested by multiplex realtime PCR for presence of rota-, noro-, adeno(type 40/41)- and, astrovirus.The laboratory analysis is described more elaborate in the supplement. Definitions AGE was defined as acute diarrhea or looser than normal stools more than three times per 24 hours, and/or forceful vomiting. 12 Repor ts of AGE were derived from three different sources; actively repor ted by parents, repor ted on monthly questionnaire and, retrieved form medical char t review (more elaborate in supplement). Severity was assessed by the Modified Vesikari score. 13,14 The primary endpoints were defined as: 1. The protective effectiveness of at least one dose of HRV against severe rotavirus related AGE from 14 days post-dose one until 18 months of age. 2. The impact of the selective HRV vaccination program defined as the relative reduction in rotavirus related hospitalization (including symptomatic nosocomial infection) during follow- up comparing infants enrolled in the pre-implementation versus post-implementation periods. Pre-specified secondary endpoints included 1) the effectiveness of a full series of HRV against severe rotavirus related AGE and, 2) the protective effectiveness of at least one dose of HRV against rotavirus related AGE of any severity. Next, we performed subgroup analyses for infants born with 32-37 weeks, 30-32 weeks and < 30 weeks of gestation, term infants with congenital disorders and, infants with more than one risk condition. As post-hoc analysis, we added all-cause (severe) AGE as outcome for all specified primary endpoints. This was decided because a stool sample was not obtained from a substantial propor tion of AGE episodes (e.g. without a rotavirus test result) and previous studies have estimated up to 50 percent of severe AGE in infants caused by rotavirus. 15,16 In addition we quantified the probability of incorrectly estimating VE because of sparse confirmed rotavirus events.