Josephine van Dongen

Chapter 4 110 Supplementary Material Methods Laboratory analyses Fecal samples were placed in biosecurity envelopes and mailed to the study laboratory for PCR testing. RNA and DNA were isolated from the samples using the MagnaPura96® (Roche Diagnostics, Pleasanton, CA USA) and amplified by ABI75000 realtime PCR system® (Thermofisher Scientific, Foster City, CA, USA). Prior to extraction, a non-human internal control was spiked into the lysis buffer of the samples to monitor for sample inhibition. Positive and negative controls for each pathogen were tested in every run. Fecal samples collected within four teen days of symptom onset were defined as AGE samples. Three sources of AGE reporting • Episodes actively repor ted by parents with or without a fecal sample collected and including a daily symptom severity score and healthcare attendance. For these episodes we calculated the modified Vesikari Score (MVS). A score of less than eight was defined mild, nine to ten moderate and a score of eleven and more as severe. 1,2 • Episodes repor ted on the monthly questionnaire for which the study team was not notified. For these episodes information was available on duration of symptoms and healthcare attendance. • Episodes retrieved from medical char t review. These included hospitalized episodes only, with or without diagnostic fecal testing performed. Nosocomial AGE was defined as AGE occurring ≥ 48 hours of admission. We repor ted this cohor t study according to the STROBE checklist. 3 Results Secondary outcomes Analyses for complete series HRV resulted in an univariate HR of 0.68 (95%CI 0.35;1.34) for rotavirus vaccinated versus unvaccinated infants in ATP cohor ts, adjusted for attending daycare and season the HR was 0.62 (95%CI 0.31;1.21). Translating this into a VE of 38% (95%CI -21;69%) after completing the two doses HRV series against severe rotavirus AGE. The incidence rate (IR) of rotavirus AGE of any severity in the at least one dose HRV vaccinated (ATP) cohor t was 67.1 per 1000 person-years (95%CI 51.3;86.1). In the willing to vaccinate (ATP) cohor t, the incidence rate was 61·5 per 1000 person-years (95%CI 44.3;83.1). There was no statistical significant difference, incidence rate ratio (IRR) 1.05 (95%CI 0.72;1.55). The negative binomial model resulted in an adjusted IRR of 1.02 (95%CI 0.69;1.50), corresponding with a VE of -2% (95%CI -50;31%).