Determining the Effect of Probiotics on Microbiome and Volatile Organic Compounds in Patients After Surgical Repair of Congenital Diaphragmatic Hernia

Aim: The aim of this study is to evaluate children who have been enrolled on the basis of a hernia, the pulmonary microbiome and the volatile organic compounds (VOCs) in the exhaled air and to compare them with a control group which does not have a chronic or acute lung disease. Furthermore, the lung function by spirometry, whole body plethysmography and "multiple breath washout" procedures be precisely recorded and a sports medical examination, including ergospirometry. In addition, any influence of probiotics on the pulmonary microbiome in children with diaphragmatic hernia will be investigated.

Study design: This is a prospective study in children who were treated surgically in early childhood for congenital diaphragmatic hernia. After an initial determination of the microbiome and the composition of volatile organic substances in breathing air and feces and a lung function measurement as well as sports medical examination, the study group receive a probiotic (Omnibiotics 6, obtained from the Allergosan Institute, food supplements) for 3 months. The microbiome and VOCs are observed afterwards. The subjects are divided according to age, gender, care with or without patch. In addition, a comparison with a control group that does not show any chronic or acute lung disease is made according to age and gender of the test group. In addition, in cooperation with the Division of Pediatric Pulmonology and Allergology of the Department of Pediatrics and Adolescent Medicine at the Medical University of Graz, the lung function of former diaphragmatic hernias patients (before administration of probiotics) and a control group is to be measured. In cooperation with the Medical Center of Sports Medicine the investigator also carry out a sports medical examination including ergospirometry. The duration of the study is set at 12 months.

Study participants: Study group: Children between 6-16 years of age, who are enrolled at the Department of Pediatric and Adolescent Surgery at the Medical University of Graz between 2000 and 2010 due to a congenital diaphragmatic hernia have received an operative closure with or without patch. Control group: Children between 6-16 years who do not have pulmonary disease. Recruited from the outpatient area at the Department of Pediatric and Adolescent Surgery at the Medical University of Graz. The aim is to establish contact with the above-mentioned patients and their parents and to achieve willingness to participate in this study by means of an information letter. The control group should come from the area of outpatient area in the Department of Pediatric and Adolescent Surgery, Medical University of Graz, after appropriate information and possible consent.

Microbiome analysis before treatment with a probiotic: Collection of sample material - in this case sputum - from the deep respiratory tract by induced sputum after inhalation of hypersaline saline solution with resulting provocation of cough. The sample is then deep-frozen. The Microbiome measurement is performed as a comparative 16S rDNA-based profile via chip-based next-generation sequencing as already published, analyzed using SnowMAn, Qiime and MOTHUR as well as the own "R"-based software. A sequencing depth of 5,000-10,000 reads per sample.

VOCs analysis before treatment with a probiotic: I) Taking of the exhaled gas samples: One sample from inspiration and two from expiration is taken from each subject (n = 3). For sampling investigator use an automatic sampling system that is directly connected to a capnometer. This system contains a so-called needle-trap microextraction (NTME) as a microextraction technique and meets the requirements of an optimal sampling on currently technically highest level. The exhaled gas samples obtained in this way are then sent to our cooperation partner, to the Institute for Breathing Gas Analysis at the University of Rostock for analysis. II) Analysis of exhaled gas samples: There, the exhaled gas samples are thermally transferred into the inert carrier gas stream (He) in an injector of a chromatograph. The substances are assigned according to their retention time in the chromatogram and their mass spectrum. Unknown Compounds in the exhaled gas are identified by comparison with a reference database based on the mass spectrum. Vital and laboratory data, as well as microbiological information, are taken from the patients' findings. III) Identification of biomarkers of exhaled gas samples: From the results of the patient measurements, those substances and substance concentrations are determined which are specific for study group 1 and group 2, i.e. compounds which are not present in the comparison group or only in significantly lower or higher concentrations. The selected volatile markers, as well as any volatile contaminants that may have been detected in the environment, are stored in an analytical reference database and, after elimination of the contamination, bundled into possibly disease-specific marker profiles. IV) Analysis of fecal samples: The fecal samples are also sent to the Institute for Breathing Gas Analysis of the University of Rostock for analysis and analyzed there after appropriate preconcentration by solid phase microextraction (SPME). V) Identification of biomarkers of fecal samples: This is done in analogy to the exhaled gas samples.