Recurrent Wheezing in Infants: Risk Factors and Prevention With Probiotics.
Condition: Respiratory Tract Diseases; Wheezing
MeSH terms: Respiratory Tract Diseases; Respiratory Sounds
Study type: Interventional
Phase: Phase 3

Summary: 
      The purpose of this study is to evaluate whether Probiotics promote reduction of recurrent
      wheezing in infants, stimulating the immune system to Th1 response.
    

Detailed description: 
      OBJECTIVES

      GENERAL

        -  Assess whether probiotic supplementation reduces the frequency of recurrent wheezing in
           infants .

      SPECIFIC

        -  Identifying the presence of environmental and genetic factors to recurrent wheezing in
           infants, which may serve as major confounders of the effect of probiotics in the sample
           studied risk factors .

        -  Check the effect of probiotics as the recurrence of wheezing in infants.

        -  Evaluate the effects of probiotics on the immune response of the infant wheezing ,
           through the measurement of IL - 10 , IL - 12 and Interferon-gama before and after
           intervention with the use of probiotics .

      DESIGN RESEARCH

      Study Design

        -  Inferential, the type longitudinal, randomized trial, double-blind, parallel,
           placebo-controlled study.

      Intervention to be held:

        -  Patients attended at the Infant Hospital of the Federal University of Pernambuco will
           undergo clinical investigation and completion of baseline examinations is eligible to
           participate in the study after informed consent from parents or guardians. Infants with
           recurrent wheezing, six months to two years of age, will be divided into two groups -
           one receiving the formula with probiotics (treatment group) and the other formula with
           similar organoleptic characteristics, placebo (control group).

      Flowchart of randomization

        -  Patients assessed for eligibility verification will be randomized and follow the CONSORT
           flowchart second Guide - 2011.

      IMPLEMENTATION OF RESEARCH

      Local and survey period

        -  Hospital das Clinicals, Federal University of Pernambuco - UFPE, Brazil. Period April
           2014 to Abril 2015.

      Population Research

        -  Method of Selection of Sample

        -  Sample selection of recurring wheezing infants attended at Infant Hospital das Clinicas
           UFPE, to be considered eligible for admission to the study and meet the criteria for
           inclusion and exclusion. The wheezing infants selected as eligible for the study
           remained with wheezing even using inhaled corticosteroids.

      Type of Randomization and Allocation Mechanism

        -  A simple randomization of patients will be by draw of numbers 1 - 60.

      Randomization was performed by a third person who used a randomization program on the
      computer.

      The sequence of random numbers, recruitment and allocation of patients to the intervention by
      the principal investigator of the study will be conducted.

      Sample Size

        -  The sample size calculation was based on two studies on probiotics and asthma, whereas a
           level of less than 5% , a power study of 80% and an acceptable error of up to 20%, 60
           patients significance can be sufficient to analyze the number of desired effect
           evaluation of wheezing in infants and reject or accept the null hypothesis.

      Operationalization Steps

        -  The infants were considered eligible for the study, will have the free and informed
           consent to participate in the study signed by their parents and or guardians.

      These patients initially attended, shall undergo a medical examination protocol with
      questions and answers QE - EISL (technical interview with the parents or guardians) and
      achievement of immediate skin test sensitivity to major aeroallergens and food allergens
      (mites, cat and dog epithelium, molds, feathers, cockroaches, egg yolk, egg white and cow's
      milk proteins) through the skin prick test and chest radiography.

      Patients before starting probiotics or placebos, will the dosage of the cytokines IL-10,
      IL-12 and INF-gamma, IgE and eosinophils.

      The wheezing infants will be using the inhaled corticosteroid beclomethasone 50mcg - 02 jets
      per day (100mcg - dia), found this medication for free, brand name and specifications: Clenil
      HFA 50mcg spray 200 doses as initial and fundamental treatment for wheezing this age group,
      at doses considered minimal.

      The formula with probiotics containing 10 CFU (Colony Forming Unit)/g consortium of probiotic
      strains of lactobacilli, lactococcus, streptococcus, yeasts and acetobacter type in
      appropriate concentrations per gram. It will be supplied in lyophilized form, in each sachet
      containing 1 g of the probiotic, to be used in addition to milk, juice or yoghurt in the
      first morning meal once daily. The placebo will be supplied in the same way with the same
      organoleptic characteristics of formula with probiotics.

      During this period, the infant wheezing present crisis, parents or guardians will note the
      day of wheezing attacks and treatment used in a standardized form with open answers, which
      will be provided, and will contact you by telephone, with the researcher. Parents or
      guardians will receive a daily record of control, to assess the occurrence of wheezing
      attacks.

      After eight weeks of using probiotics, infants will undergo new measurement of cytokines IL -
      10 , IL - 12 and Interferon - gama, with the objective to evaluate the immune response of
      probiotics in both groups.

      Before and at the end of each period, the patient will undergo physical examination and
      parents or guardians to an interview, to assess the occurrence of wheezing attacks.

      These data will be analyzed to assess whether supplementation with probiotics reduces the
      frequency of wheezing in infants.

      Masking and Allocation Mechanism

      A third member of the study will assign a number to probiotics and placebo recipients in
      accordance with the numbers of randomization, without the knowledge of the investigator and
      the patient. Containers should be equal and contain substances of the same organoleptic
      characteristics. The containers will be provided in numerical order, as service and previous
      randomization.

      Instruments Measurement and Assessments

      QE- EISL: written questionnaire (International Study of Wheezing in Infants) - see Appendix
      01, which is an instrument consisting of 45 questions on demographic characteristics,
      wheezing and its potential risk factors. Originally developed in Spanish, was translated into
      Portuguese (Brazilian) and validated. This protocol will be used with closed questions with
      answers, using the technical interview, conducted by the researcher, the parents or
      guardians.

      Statement of Informed Consent Form (ICF) - see apêncice 02 - after approval thereof in having
      their children participating in the study.

      Control plug - clinical - see Appendix 03: will be used by parents or guardians to describe
      the date, " wheezing " and perceived treatment need, while using the probiotic or placebo (08
      weeks) and 08 weeks more.

      Prick Test: standardized extracts dihiclorada histamine (10mg/ml) and 0.9% saline (FDA -
      Immunotech , Rio de Janeiro , Brazil) will be used. The extracts will be dripped on the volar
      surface of the forearm and then performed one puncture with sterile lancet. As a positive
      control we histamine and negative saline control. The diameter of each reaction will be
      evaluated after 15 minutes and the result expressed as positive if wheal ≥ 3mm negative
      control.

      Eosinophil count and total serum IgE Dosage: will be held in peripheral blood collection
      vessel with qualified professional lab, in the amount of 7.0 ml. The reference values are
      appropriate for age and standardization of laboratory.

      Chest radiography: held at the radiology department of the Hospital das Clinicals - UFPE.

      Dose of IL - 10 , IL - 12 and Interferon-gama: will be held in peripheral blood collection
      vessel with qualified professional lab in the amount of 5.0 ml. This blood will be taken to
      the Laboratory Ageu Magalhães, which will be submitted to centrifugation and performed by the
      supernatant of peripheral blood mononuclear cells (PBMC), the count of cytokines in serum.
      Then the cultivation of these cells for 48 hours and subsequent counting of cytokine IL - 10
      , IL - 12 and Interferon - gama is performed.

      The peripheral blood mononuclear cells at a concentration of 1 x 106 cells/mL are collected
      from the patients of the treatment group and the placebo group and are incubated with
      phytohemaglutinin (PHA ; 10μg/ml) for 48 hours. Production of IL - 10, IL - 12 and Interferon
      - gama in culture supernatant will be analyzed using enzyme-linked immunosorbent assay
      (ELISA) kits. The detection limit for these cytokines will be 5ρg/ml .

      STATISTICAL ANALYSIS

      Statistical Methods for Data Analysis

        -  For statistical analysis Initially , a probability distribution (percent analysis) is
           performed. Subsequently, the techniques of normal distribution and homogeneity of
           variances by means of ShapiroWillks and bartlett, respectively, tests will be applied to
           characterize the use of the parametric Student's t tests or Manny Whitney for
           independent samples. For categorical variables the chi- square test for association with
           Yates correction will be used. Data are expressed as mean and standard deviation (
           parametric test ) or median and standard error ( nonparametric test ) with a
           significance level of p < 0.05 . 21 for Windows 2011 - to generate the analyzes , the
           statistical package SPSS will be used.
    

Intervention name(s): Beclomethasone; Probiotics; Placebo
Intervention type(s): Drug; Drug; Drug
Intervention description(s): The wheezing infants will be using the inhaled corticosteroid beclomethasone 50mcg - 02 jets per day (100mcg/dia).; Lyophilized form, each sachet containing 1 g of the probiotic, to be used in addition to milk, juice or yoghurt in the first morning meal once daily for 60 days.; The placebo will be supplied in the same way with the same organoleptic characteristics of formula with probiotics, 1 g sachet for 60 days, in the juice, milk or yoghurt.
Other intervention name(s): Clenil; Lactobacillus; Bifidobacterium; Leveduras; Acetobacter; Maltodextrose
Intervention MeSH terms: Beclomethasone
Allocation: Randomized
Intervention model: Parallel Assignment
Masking: Double (Participant, Investigator)
Primary purpose: Treatment
Keywords: wheezing, immune system, cytokines, probiotics, infants.