Long Term Development of Gut Microbiota Composition in Atopic Children: Impact of Probiotics

Introduction Imbalance of the human gut microbiota in early childhood is suggested as a risk factor for immune-mediated disorders such as allergies. With the objective to modulate the intestinal microbiota, probiotic supplementation during infancy has been used for prevention of allergic diseases in infants, with variable success. However, not much is known about the long-term consequences of neonatal use of probiotics on the microbiota composition. The aim of this study was to assess the composition and microbial diversity in stool samples of infants at high-risk for atopic disease, from birth onwards to six years of age, who were treated with probiotics or placebo during the first year of life. Methods In a double-blind, randomized, placebo-controlled trial, a probiotic mixture consisting of B. bifidum W23, B. lactis W52 and Lc. Lactis W58 (Ecologic® Panda) was administered to pregnant women during the last 6 weeks of pregnancy and to their offspring during the first year of life. During follow-up, faecal samples were collected from 99 children over a 6-year period with the following time points: first week, second week, first month, three months, first year, eighteen months, two years and six years. Bacterial profiling was performed by IS-pro. Differences in bacterial abundance and diversity were assessed by conventional statistics. Results The presence of the supplemented probiotic strains in faecal samples was confirmed, and the probiotic strains had a higher abundance and prevalence in the probiotic group during supplementation. Only minor and short term differences in composition of microbiota were found between the probiotic and placebo group and between children with or without atopy. The diversity of Bacteroidetes was significantly higher after two weeks in the placebo group, and at the age of two years atopic children had a significantly higher Proteobacteria diversity (p < 0.05). Gut microbiota development continued between two and six years, whereby microbiota composition at phylum level evolved more and more towards an adult-like configuration. Conclusion Perinatal supplementation with Ecologic® Panda, to children at high-risk for atopic disease, had minor effects on gut microbiota composition during the supplementation period. No long lasting differences were identified. Regardless of intervention or atopic disease status, children had a shared microbiota development over time determined by age that continued to develop between two and six years.

Nature and extent of the burden and risks associated with participation, benefit and group relatedness: Parents and child are asked to fill in a questionnaire, and pay one visit to the out patient department for physical examination, blood sampling and pulmonary function testing with measurement of exhaled nitric oxide. The risks of these procedures are negligible and the burden minimal. It should be carried out specifically in these children, since they have been included in the former study and received placebo or probiotics.

INTRODUCTION AND RATIONALE
The last decennia a rising incidence of allergy and atopic disorders is seen, especially amongst children. The aim of this study is to investigate possible primary prevention in high risk children by means of adminstration of probiotics from 4-6 weeks prenatally until the age of one year.
The hypothesis is, that early administration of probiotics to pregnant women with allergy and subsequently to their offspring, leads to prevention or reduction of development of sensitisation and allergic disorders in childhood.
Modification of the intestinal flora early in life by administration of probiotic bacteria may be a potential approach to prevent atopic disease. Strachan (1) suggested a decreased occurrence of T helper type 1 (Th1)-associated infections to be a causative factor in the increased prevalence of allergy. More recently, modifications of the hygiene hypothesis have moved away from the impact of Th1-driving infections and towards the influence of the diversity of the microbial exposure in general during establishment of the intestinal flora (2,3).
In industrial countries having a high degree of hygiene, changed microbial exposure might cause an altered composition of the intestinal flora and thus, an altering of the immunomodulatory microbial signalling posed during the critical stages of immune development in infancy (4). Atopic disease is the result of a T helper type 2 (Th2)-deviated immune respons characterized by the production of the cytokines IL-4, IL-5, and IL-13. According to the hygiene hypothesis, insufficient infection-related Th1 responses during the neonatal period and infancy were considered to lead to unrestricted Th2 responses, resulting in allergic diseases. Because the gastro-intestinal (GI) tract encompasses the largest surface of the human body where microbial products interact with the immune system, much attention is focused on the possibility that the intestinal flora may play a role in the development of atopic disease (2). Indeed, different bacterial colonization patterns have been observed in the intestinal flora of allergic children when compared with non-allergic children. Lactobacilli and bifidobacteria are more commonly found in the intestinal flora of non-allergic children, while allergic children seem to harbour higher number of clostridia and staphylococci (5,6). Lactobacilli and bifidobacteria are most frequently investigated probiotic species for possible beneficial effects in prevention and treatment of allergic disease (7). Preliminary data indicate that probiotic bacteria can be effective in prevention and treatment of atopic disease (8)(9)(10)(11)(12)(13). Perinatal administration of probiotic bacteria (Lactobacillus rhamnosus GG) significantly reduced the development of eczema during the first 2-4 years of life in children with a family history of atopic disease (8,9). In addition, specific probiotic strains have been beneficial in the treatment of infants suffering from atopic eczema and cow's milk allergy by significantly decreasing the SCORAD score (10,11,14). The mechanism by which probiotic bacteria may modify immune-mediated diseases such as atopic disease, and which different species or strains may contribute are poorly understood. Niers et al (15,16) showed the ability of live lactic acid bacteria to modulate the immune responses by inducing IL-10 in peripheral blood mononuclear cells, and these effects appeared to be strain specific. Based on this study, they selected B. Bifidum, B. infantis, and Lc Lactis (because of their good IL-10-inducing capacity as well as efficient inhibition of IL-5 and IL-13) to be used as a multi-species probiotics in a clinical trial on primary prevention of allergic diseases by probiotic bacteria. B. Bifidum was most potent to polarize dendritic cells to drive Th1 cell responses involving increased IFN-gamma producing T cells concomitant with reduction of IL-4-producing T cells. L. Lactis was much less effective in this respect.
This study will encompass a follow-up of the previously included children in the above mentioned double blinded randomized clinical trial performed by Niers from 2003 till 2007. It will be carried out when the children have reached the age of 5 years. Since Niers completed follow-up till the age of 2 years, a thorough evaluation of the development of asthma and allergic rhinitis was not possible. The diagnosis of asthma is best made at an age beyond 5 years for reasons of lung function testing, and the prevalence of allergic rhinitis rises from the age of 4. The aim of this follow-up study is to study if administration of probiotics during pregnancy and in the first year of life has any influence on the prevalence of asthma and allergic rhinitis, in Caucasian children at high risk of developing atopy, at the age of 5 or 6 years.
Besides this, we wish to perform a pilot study evaluating the diagnostic value of measurement of nasal nitric oxide (nNO) in the diagnosis of allergic rhinitis. An effect of allergic rhinitis on nNO is not consistently reported. However, most studies suggest that nNO is increased in allergic rhinitis [12][13][14][15][16][17] . Normal values for nNO in children are well defined by Struben et al 18 . De Winter et al will shortly publish an evaluation of methodology for nNOmeasurements, with good reproducibility and applicability, investigated at the Department of Paediatric Pulmonology of our hospital 19 . We do not expect a direct relationship between probiotics and nNO, but want to support the diagnosis of allergic rhinitis. This test is easily available in our hospital, easy to perform and not experienced as invasive by the child. As we expect a direct relationship between the severity of allergic rhinitis and level of nNO, we wish to evaluate the predictive value of the measurement of nNO for the diagnosis of allergic rhinitis.

OBJECTIVES
Primary Objective: The aim of the present study is to determine the effect of probiotics in high-risk infants on the prevalence and severity of asthma and allergic rhinitis, lung function and specific IgE at the age of 5 or 6 years.

STUDY DESIGN
A randomised double-blind placebo-controlled prospective study was carried out in 156 highrisk children. 98 children completed the intervention period and the two years of follow-up.
The present study comprises a follow-up at the age of 5 or 6 years, in the outpatient clinic. We will try to get as little as possible loss to follow-up by trying to find all the former participants of the PANDA-study and to ask for the collaboration.
For the pilot study evaluating the diagnostic value of measurement of nasal nitric oxide, we will evaluate the complete cohort, not considering whether the child has received probiotics. We estimate the prevalence of allergic rhinitis to be 15% in this cohort. To be able to detect a statistical significant difference between nasal NO in the subjects with a diagnosis of allergic rhinitis from the questionnaire and subjects who do not, we will need a minimum of 10-15 participants with allergic rhinitis, and the same amount who do not suffer from allergic rhinitis. In evaluating this, we consider the means for NO in both groups to be 450 and 600 ppb, the standard 6. METHODS

Main study parameter/endpoint
The prevalence of allergic disease, focussing on the prevalence of asthma and allergic rhinitis. Definition of asthma: Periodic wheezing and dyspnoea, combined with a 9% reversibility in FEV1 and/or exhaled NO above 20 ppb. Definition of allergic rhinitis: Periodic or perennial sneezing, runny, or blocked nose when not having a cold or the flu.

Secondary study parameters/endpoints (if applicable)
Specific IgE to allergens.

Other study parameters (if applicable)
Not applicable.

Randomisation, blinding and treatment allocation
The previous study was double-blinded, this was abated at the end of the PANDA study. In this follow-up study, the researcher will be blinded, but parents are not.

Study procedures
The questionnaire is based on the ISAAC questionnaire. It will be used to evaluate the participants for symptoms indicative of asthma, allergic rhinitis, eczema and food allergy at the age of 5 or 6 years, with the most used and best comparable questionnaire of nowadays research in allergy.
The treating physician will be asked for doctor-diagnosed atopic diseases (asthma, allergic rhinitis, food allergy, eczema, urticaria). Physical examination. Baseline pulmonary function (forced expiratory volume in 1 second (FEV1), forced vital capacity (FVC), peak expiratory flow (PEF), and subdivisions of forced expiratory flow) will be measured. Airway reversibility will be measured after administration of 400 microgram of salbutamol. Exhaled nitric oxide will be measured, including nasal nitric oxide (nNO). nNO will be measured during breath holding (nNO-BH) and during humming (nNO-HE). nNO-measurement is not experienced as invasive by the child. nNO-BH is measured during breath hold after inhalation to TLC. The subjects will be asked to hold their breath for 10 seconds while air is sampled from the right nostril.
To measure nNO-HE, subjects will be asked to make a humming noise during nasal exhalation at the frequency of a tuning fork. Before nNO-HE measurements subjects are asked not to speak during 5 minutes. After inhalation of ambient air to TLC subjects exhale to FRC through their left nostril, while air is sampled from the right. The nasal humming procedure will be done at two different frequencies with the assistance of tuning forks of respectively 128 and 440 Hz (nNO-HE 128 and nNO-HE 440 , respectively). Participants will be asked to stop the possible use of nasal corticosteroids for 6 weeks before nNO-measurement, long acting β-mimetica for 48 hours before lung function testing, and short acting β-mimetica for 12 hours before lung function testing, if clinically acceptable for the medical situation of the child. This will be evaluated by the investigator and deliberated with the treating physician. Specific IgE will be determined by Phadiatop assay, concerning the most common allergens, in 1 ml. of blood.

Withdrawal of individual subjects
Subjects can leave the study at any time for any reason if they wish to do so without any consequences. The investigator can decide to withdraw a subject from the study for urgent medical reasons.

Specific criteria for withdrawal (if applicable)
Not applicable.
6.5 Replacement of individual subjects after withdrawal Not applicable.
6.6 Follow-up of subjects withdrawn from treatment Not applicable.

Premature termination of the study
No reason to terminate this study prematurely.

Section 10 WMO event
In accordance to section 10, subsection 1, of the WMO, the investigator will inform the subjects and the reviewing accredited METC if anything occurs, on the basis of which it appears that the disadvantages of participation may be significantly greater than was foreseen in the research proposal. The study will be suspended pending further review by the accredited METC, except insofar as suspension would jeopardise the subjects' health. The investigator will take care that all subjects are kept informed.

Adverse and serious adverse events
No serious adverse effects are to be expected, children will undergo physical examination, capillary blood sampling and lung function testing. Mild adverse effects could be dyspnoea or wheezing during the 2 days before the lung function testing, for which the investigator will provide good accessibility for deliberations of the parents.
Another mild adverse effect could be exacerbation of allergic rhinitis since intranasal corticosteroids will be stopped during 6 weeks before testing. For this, the investigator can be contacted. Restarting the medication is possible at any time by the child, parents or treating physician and discontinuation of the medication can be rejected at any time by the child, parents or treating physician.
Adverse events are defined as any undesirable experience occurring to a subject during a clinical trial, whether or not considered related to the investigational drug. All adverse events reported spontaneously by the subject or observed by the investigator or his staff will be recorded.
A serious adverse event is any untoward medical occurrence or effect that at any dose results in death; is life threatening (at the time of the event); requires hospitalisation or prolongation of existing inpatients' hospitalisation; results in persistent or significant disability or incapacity; is a congenital anomaly or birth defect; is a new event of the trial likely to affect the safety of the subjects, such as an unexpected outcome of an adverse reaction, lack of efficacy of an IMP used for the treatment of a life threatening disease, major safety finding from a newly completed animal study, etc.
All SAEs will be reported to the accredited METC that approved the protocol, according to the requirements of that METC.

Follow-up of adverse events
Adverse events are not expected to occur during this study.
All adverse events will be followed until they have abated, or until a stable situation has been reached. Depending on the event, follow up may require additional tests or medical procedures as indicated, and/or referral to the general physician or a medical specialist.

Data Safety Monitoring Board (DSMB)
Not applicable.

Descriptive statistics:
For the main endpoint we will calculate the relative risks with 95% confidence intervals.
For the secondary endpoints we will apply parametric and non-parametric tests, depending on the distribution of the parameter.
For the pilotstudy evaluating the diagnostic value of nNO, we will calculate sensitivity, specificity, positive and negative predictive values for nNO in the diagnosis of allergic rhinitis.
All analysis will be performed with the SPSS/PC+ package.

Regulation statement
The study will be conducted according to the principles of the Declaration of Helsinki

(version 2004) and in accordance with the Medical Research Involving HumanSubjects
Act (WMO).

Recruitment and consent
Subjects will be recruited by drs. D.M.W. Gorissen among the participants of the previous PANDA-study. Parents will be asked if their child is willing to participate. They will receive printed information and will be contacted by phone after one week by the investigator. If they decide to participate, they are asked to return the informed consent form attached to the printed information.

Objection by minors or incapacitated subjects (if applicable)
Subjects can abandon the research any time. Medical staff, the investigator and the parents will observe the child during the examination, blood sampling and lung function testing to make sure the child is not being forced to co-operate. The Dutch Association of Pediatrics has edited a Behavioural Code concerning Resistance of minors in case of medical scientific research with children (11). This will be applied.

Benefits and risks assessment, group relatedness
No direct benefit can be expected for the participants, except for a thorough screening for the presence of atopic diseases with if indicated an appropriate reference to a specialist.
The risk of blood sampling is negligible, and the same applies to pulmonary function testing including exhaled nitric oxide measurement. For the need of stopping the inhaled beta-mimetics, a risk of dyspnoea and wheezing exists, which will be treated with readministration of beta-mimetics. The same applies to discontinuation of nasal corticosteroids, which carries the risk of exacerbation of allergic rhinitis. If the condition of the child does not allow stopping the above mentioned medication or the parents are not willing to do so, they can still fullfill the questionnaires but they cannot perform lungfunction testing in case of beta-mimetics, or nasal NO measurement in case of nasal corticosteroids.
We are convinced that the yield of the study for addressing the effect of probiotics on the development of atopic disease exceeds the burden of one visit to the hospital with the above mentioned diagnostic procedures and the risks.
This follow-up study can only be carried out on these subjects, since they have participated in the primary intervention (the PANDA-study) with either probiotics or placebo.

Compensation for injury
We will insure for the risks of adverse effects of the study to the participants, by use of the general insurance of the University Medical Center of Utrecht.

Incentives (if applicable)
Subjects will be able to obtain a travelling allowance for the visit to the outpatient department..

Handling and storage of data and documents
The handling of personal data complies with the Dutch Personal Data Protection Act.
They will be handled confidentially and anonymously, provided with a code, kept by the investigators drs. D.M.W. Gorissen and dr. C.K. van der Ent.
The sera will be kept for the timespan of the study, which is estimated on 2 years.
Thereafter they will be destroyed.

Amendments
Amendments are changes made to the research after a favourable opinion by the accredited METC has been given. All amendments will be notified to the METC that gave a favourable opinion.
All substantial amendments will be notified to the METC and to the competent authority.
Non-substantial amendments will not be notified to the accredited METC and the competent authority, but will be recorded and filed by the sponsor.
< Examples of non-substantial amendments are typing errors and administrative changes like changes in names, telephone numbers and other contact details of involved persons mentioned in the submitted study documentation.>

Annual progress report
The investigator will submit a summary of the progress of the trial to the accredited METC once a year. Information will be provided on the date of inclusion of the first subject, numbers of subjects included and numbers of subjects that have completed the trial, serious adverse events/ serious adverse reactions, other problems, and amendments.

End of study report
The investigator will notify the accredited METC of the end of the study within a period of 8 weeks. The end of the study is defined as the last patient's last visit.
In case the study is ended prematurely, the investigator will notify the accredited METC, including the reasons for the premature termination.
Within one year after the end of the study, the investigator/sponsor will submit a final study report with the results of the study, including any publications/abstracts of the study, to the accredited METC.

Public disclosure and publication policy
The previous trail has been registrated at ClinicalTrials.gov.