Microbiome and Volatile Organic Compounds in Patients With CDH

Despite improved prenatal diagnostics and therapeutic possibilities, congenital diaphragmatic hernia (CDH) represents a cross-disciplinary challenge. With an incidence of 1:2000-1:5000, it is a common disease that effects centres of paediatrics and juvenile medicine. The etiology is still unclear. Patients with this diagnosis are usually affected by other comorbities such as failure to thrive, gastroesophageal reflux, funnel chest, etc. Depending on the extent of CDH, a more or less pronounced lung hypoplasia with functional impairment occurs. The health-relevant importance of the human microbiome is increasingly evident. While it was previously particularly associated with the gastrointestinal tract, other systems such as the pulmonary microbiome have become the focus of scientific interest.

Research into changes in the microbiome and volatile organic compounds (VOCs) could provide new insights into the underlying mechanisms and therapeutic measures of this disease.

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.

Lung function measurement before treatment with a probiotic: Measurement of lung function using spirometry and body plethysmography (Fa Jäger spirometer and body plethysmograph) and nitrogen washout process (N2-multiple breath washout, System Exhalyzer D and Spiroware 3.1, Eco Medics AG, Duernten, Switzerland). Spirometry and body plethysmography are performed according to published ERS/ATS Standards. The "multiple breath washout" method is performed under resting breathing and detects the Ventilation (in)homogeneity at the level of the functional residual capacity (= FRC = that lung volume that is still in the lungs after a calm spontaneous exhalation is left behind). The system consists of a flow meter, a fast analyzing gas measuring system, a gas administration system and the corresponding Computer analysis software. As a "foreign gas" it will be use 78% of the gas in air occurring nitrogen (N2). A flow-volume measurement is performed via an Ultrasonic flowmeter performed directly in the inhalation and exhalation flow of the test person/patient and via a laser O2 sensor using the side current measurement method and an infrared CO2 sensor in the main current measuring procedure (= directly in the patient's respiratory flow) the respective gas concentration. The N2 component is then indirectly measured via the O2 and CO2 concentration (N2 = 1 - O2 - CO2). During calm spontaneous breathing, the Patient on a snorkel mouthpiece via a bacterial filter through the flowmeter 100% oxygen and thus "washes" N2 out ("N2-multiple breath washout"). In doing so, the flow-volume curve of spontaneous breathing "online" is displayed on the screen and the measurement at Reaching a 1/40 (= 2.5%) of the initial nitrogen concentration is completed. After that, wait the subject is safe in the length of twice the duration of the measurement around the oxygen of exhaling. This is followed by the next measurement. A total of 3 measurements whose mean value serves as a result. The so-called "lung clearance index", which indicates the number of functional residual capacity lung volumes, which can be used to reduce the initial nitrogen concentration to a 1/40 after oxygenation was required. (LCI = quotient between exhaled volume and FRC). It expresses how long it takes for the inhaled gas (in our case the physiologically occurring nitrogen in the air) through inhalation of 100% oxygen. For healthy persons, this value is on average 7 and is significantly higher in lung patients. Further measuring parameters, which makes a statement about the peripheral airways proximal to the terminal bronchioles on the one hand and via the more distal azine airways on the other hand are calculated.

Sports medical examination: To exclude contraindications for ergometry, a 12-channel resting ECG and a resting blood pressure measurement (CombynTM Function \& Spaces ECG, Academic Technologies) are performed at the beginning of the examination. After the anthropometric data (height, weight, BMI) have been collected, the muscle mass and fat mass are determined by multi-frequency impedance measurement in six body segments (CombynTM Function \& Spaces ECG, Academic Technologies). The lung function is measured by means of small spirometry at rest and after exercise (Spirometer Oxycon Pro, Reiner). In order to determine cardiopulmonary performance, ergospirometry is performed on a bicycle (Excalibur Sport ergometer, Lode company; Oxycon Pro spiroergometry unit, Reiner company) with a gradual increase in stress up to subjective exhaustion. The evaluation of these data allows conclusions to be drawn about the performance-limiting system (cardiovascular system, lungs, musculature) in addition to the determination of aerobic performance.

Microbiome/VOCs analysis after treatment with a probiotic: After sampling for microbiome and VOC analysis and carrying out lung function measurements and sports medical examination, the participants in the study group will take a probiotic (Omnibiotic 6, purchased from the Allergosan Institute, dietary supplement) for a period of 3 months. Immediately afterwards and another month later, measurements of the pulmonary microbiome and the VOCs in the breath are taken.

Eligibility Criteria:
Inclusion Criteria:

* Age from 6-16 years
* Age 0-6 months at time of CDH-OP (except control group)
* reliable diagnosis of congenital diaphragmatic hernia (except control group)
* surgical occlusion with patch (except control group)
* surgical occlusion without patch (except control group)
* given approval

Exclusion Criteria:

* chronic pulmonary diseases
* Infection within 4 weeks before the test date
* unaccepted consent