Colonization and Persistence Capacity of a Multi-strain Probiotics in Pediatric Food Allergy to Milk or Egg.

This study evaluates the colonization capacity of three probiotics (Bifidobacterium longum, Bifidobacterium breve and Bifidobacterium infantis) in milk and/or egg allergic children. It is expected the recruitment of about 20 egg and/or milk allergic patients aged between 10 and 14 months old confirmed with double-blind oral tolerance test against placebo (Group 1). Furthermore, about 10 patients sensible to milk or egg but not confirmed with double-blind oral tolerance test against placebo (Group 2) will be recruited and about 10 healthy individuals (Group 3). For all 40 patients: 1. Baseline presence of Bifidobacterium longum BB536, Bifidobacterium breve M-16V and Bifidobacterium infantis M-63 will be evaluated; Only for Group 1 children it will be also evaluate: 2. The presence of the same strains during 30 days of administration of multi-strain probiotics containing 3.5 x 109 UFC of Bifidobacterium longum BB536, Bifidobacterium breve M-16V and Bifidobacterium infantis M-63 and after 60 days from the suspension of the administration. Quantification of B. breve, B. longum subsp. longum and B. longum subsp. infantis in faecal samples was carried out by qRT-PCR using the Light Cycler 480 platform (Roche Diagnostics, Mannheim, Germany). The assays were performed with a 20 µl PCR amplification mixture containing: 10 µl LightCycler 480 Probe Master mix (Roche Diagnostics), 2 µl primers and probes (optimized concentrations, 0.5 µM and 0.1 µM, respectively), 3 µl molecular-grade H2O and 5 µl DNA template. Each sample was tested in duplicate to ensure data reproducibility. The RT-PCR temperature profile consisted of an initial denaturation at 95°C for 10 min, 45 amplification cycles at 95°C for 10 sec, 60°C for 30 sec and 72°C for 1 sec followed by a final cooling step at 40°C for 30 sec. Absolute quantification was performed using the "second derivative maximum method" Statistical analyses: Wilcoxon signed-rank test was used to compare probiotic species concentrations during the time-course.