Microbiome and Volatile Organic Compounds in Patients With CDH
Condition: Congenital Diaphragmatic Hernia
MeSH terms: Hernias, Diaphragmatic, Congenital; Hernia; Hernia, Diaphragmatic
Study type: Interventional

Summary: 
      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.
    

Detailed description: 
      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.
    

Intervention name(s): initial VOC; initial fecal microbiome; initial pulmonary microbiome; Maximum oxygen uptake; Functional residual capacity; Probiotic treatment; VOC probiotic; Fecal microbiome probiotic; Pulmonary microbiome probiotic
Intervention type(s): Diagnostic Test; Diagnostic Test; Diagnostic Test; Diagnostic Test; Diagnostic Test; Dietary Supplement; Diagnostic Test; Diagnostic Test; Diagnostic Test
Intervention description(s): Difference in VOC profile between patients with CDH and healthy controls (2 samples per patient will be obtained after obtaining informed consent).; Difference of alpha and beta diversity and relative fecal bacterial abundance between patients with CDH and healthy controls (1 stool sample will be taken per patient after obtaining informed consent); Difference of alpha and beta diversity and relative pulmonary bacterial abundance between patients with CDH and healthy controls (1 deep induced sputum sample will be taken per patient after obtaining informed consent); Comparison of the maximum oxygen uptake (corrected for body weight and gender) as determined by bicycle spiroergometry between patients with CDH and healthy controls; FRC will be determined by spirometry, bodyplethysmography and N2-breath wash out method. FRC will be compared between patients after CDH and healthy controls.; CDH patients will receive OmniBiotic 6(R) (Allergosan, Graz, Austria) probiotic supplementation 1 sachet daily for 3 months.; Determination of the VOC profile 3 months after discontinuing probiotic treatment. Comparison to the profiles before the treatment.; Determination of the fecal microbiome from 1 sample per patient (alpha and beta diversity, relative bacterial abundance at the genus level) 3 months after discontinuing probiotic treatment. Comparison to the profiles before the treatment.; Determination of the fecal microbiome from 1 deep induced sputum sample per patient (alpha and beta diversity, relative bacterial abundance at the genus level) 3 months after discontinuing probiotic treatment. Comparison to the profiles before the treatment.
Allocation: Randomized
Intervention model: Parallel Assignment
Masking: Single (Investigator)
Primary purpose: Basic Science
Keywords: congenital diaphragmatic hernia; volatile organic compounds; microbiome; pulmonary function; multiple breath washout