Statistical analyses
The reproducibility of the assay was assessed from sera of four
individuals, which were measured in each analysis run (thus providing
six replicates), by assessing the coefficient of variation (CV) and
intraclass correlation coefficient (ICC) for each antigen.
To meet the assumptions of regression-based methods, RSV and RV-A/B/C
were normalized by using the Ordered Quantile Normalization method (21)
in the pooled EGEA1 and EGEA2 measured samples.
To assess the associations between potential determinants and IgG
responses to RSV and RV-A/B/C, multiple mixed linear regression models
considering age, sex, BMI, tobacco smoking (passive in children, active
in adults) and season of blood sampling were performed, adjusted for
ever-asthma and allergic sensitization. Family correlation was
considered as a random effect. The analyses were performed separately in
the EGEA1 and EGEA2 populations to assess these associations in children
and in adults, respectively.
Interaction between age and sex was tested by adding an interaction
age*sex term to the previous models.
The main analysis and the analysis testing the age*sex interaction were
then performed with stratification on asthma status. Indeed, due to the
case-control design of EGEA cohort, collider bias can be an issue when
investigating, as in our study, associations between factors (some of
the personal factors and RSV and RV-specific IgG levels) that may both
influence asthma. Results from separate analyses among participants with
and without asthma may inform about the presence of such a bias.
Finally, among individuals with RSV and RV-specific IgG responses
measured at both time points, associations between time elapsed during
the two follow-ups and the evolution of IgG level over time was assessed
using linear mixed models to account for repeated data, adjusted for age
at first survey, sex, BMI, exposure to tobacco, season of blood
sampling, allergic sensitization and ever-asthma.