1), by means of computer generated random numbers, printed and placed in opaque envelopes, sealed and numbered. After signing the consent form the envelopes were opened in the order of presentation of the volunteers. Randomization used permutation blocks of size 6, ratio of 1:1. The codes were opened after statistical analysis. Each vial of vaccine was used in only one participant. The MMR vaccine was administered according to routine immunization services, selleck kinase inhibitor without interference
from the study. The number of participants was calculated using the following parameters: beta = 0.2, alpha = 0.05 (two-tailed test), 90% seroconversion in one group (p1), and minimum difference between the groups (p1 − p2) of 5 percentage points [11]. The sample size with a 20% correction for loss of follow up was 1740 children, 870 in each comparison group. A questionnaire was administered before vaccination with items on age, sex, birth weight and weight at vaccination, immunization history and history
of allergies to food and drugs. We asked the children’s parents to record daily, in a diary, during the 10 days after the vaccination, the adverse events expected for the yellow fever find more vaccine (fever, vomiting, pain and redness at the injection site and irritability) and any health problems observed in that period. The clinical events occurring after this period were recorded on a postvaccination questionnaire. Samples of 4 mL of blood were collected on the day of MMR vaccination and 30 days after yellow fever vaccination to titrate antibodies against yellow Urease fever, rubella, measles and mumps. Thus, subgroups
defined by the interval between the vaccines also differed in the interval between post-vaccination blood collection and MMR: 30 days in those who received the vaccines on the same day and 60 days in those who received YFV 30 days after. The titration of antibodies against yellow fever and the antibodies against measles was performed at Virologic Technology Laboratory of Bio-Manguinhos (LATEV, FIOCRUZ, Rio de Janeiro) with Plaque Reduction Neutralization Test (PRNT). PRNT was conducted in serial twofold dilutions starting at 1:5, in 50 μL aliquots of heat inactivated (at 56 °C for 30 min) serum, in 96-well tissue culture plates. A positive monkey serum sample with yellow fever antibody content calibrated by a WHO International Reference Preparation, with 1115 mIU/mL was the standard serum for each set of tests [12]. For measles the standard serum contained 3000 mUI/mL [13]. The log10 dilution of the test sera and the standard serum, which reduced the plaque numbers by 50% relative to the virus control, was determined by linear interpolation. To convert reciprocal dilutions into mIU/mL a unitage constant was calculated for each assay run, dividing the antibody concentration in the standard serum by the reciprocal dilution of the standard serum in that assay run.