Table 1

Primary and secondary objectives with related outcome measures

DomainData sourceTechniqueOutcome measuresBetween group analyses*Within group analyses†
Primary objectives
(1) Intestinal microbiome(1) Stool sampleBacterial communities (16S rRNA (V4)13or MSS)Alpha diversity (richness and Shannon index)Student’s t-test or Wilcoxon signed-rank testPearson or Spearman correlations with:
  • Gastrointestinal microbiome

  • Respiratory microbiome

  • Diet

  • Secondary objectives


Descriptive
Beta diversity (UniFrac) distances46PERMANOVA73
Relative abundances of bacteriaANCOM74
Viral communities (metagenomic sequencing49Alpha diversity (richness and Shannon index)Student’s t-test or Wilcoxon signed-rank test
Beta diversity (Bray-Curtis dissimilarities)PERMANOVA73
(2) Respiratory microbiome(2) Oropharyngeal swab or sputum sampleRelative abundances of virusesANCOM74
Proteomics (LC-MS)21Protein z-score normalised LFQ intensitiesStudent’s t-test
Pathway/network upregulation or downregulationCondition/HC ratio
Metabolomics (UHPLC-MS/MS)20Metabolite normalised abundanceStudent’s t-test
Pathway/network upregulation or downregulationCondition/HC ratio
(3) Diet(i) ACAES (ages 2 to 18 year)
(ii) 24 hours food recall
(ages 0 up to 2 year)
Energy intakeStudent’s t-test, Wilcoxon signed-rank test or Fisher’s exact test
Percent energy from core foods
Macronutrient intake
Micronutrient intake
Diet quality score‡
Secondary objectives
(1) BiomarkersStool, oropharyngeal swab or sputum sampleELISAInflammation (calprotectin, M2-PK, CRP and interleukins)Student’s t-test, Wilcoxon signed-rank test or Fisher’s exact testDescriptive
(2) Symptomatology and HRQOLPedsQL Infant Scales (0–2 year) and gastrointestinal symptoms module (2–18 year)54–56HRQOL and gastrointestinal symptomsStudent’s t-test, Wilcoxon signed-rank test or Fisher’s exact testDescriptive
Rome IV Questionnaire57 58Gastrointestinal symptoms
Spence Children’s Anxiety Scale59 60Anxiety symptoms
Short Mood and Feelings Questionnaires61 62Depressive symptoms
(3) Phenotypic and clinical informationAnthropometricsZ-scores; weight, length/height, weight-for-length (ages 0 to 2 year) and BMI (ages 2 to 20 year)Student’s t-test, Wilcoxon signed-rank test or Fisher’s exact testDescriptive
Clinical presentationsNumber and length of hospitalisations, emergency department presentations, medications, vaccinations
ResultsBiochemistry, microbiology and imaging results
Perinatal factorsMode of delivery, feeding during infancy
(4) Socio-demographic factorsEthnicityDescriptiveDescriptive
SEIFA code63DescriptiveDescriptive
  • All samples, questionnaires and data will be collected from all participants at each time point.

  • *Between group analyses describe comparisons between a condition and healthy control groups.

  • †Within group analyses describe analyses of two outcome measures within subjects of the same condition group.

  • ‡ACAES only.

  • ACAES, Australian Child and Adolescent Eating Survey; ANCOM, analysis of composition of microbiomes; BMI, body mass index; CRP, C-reactive protein; HC, healthy control; HRQOL, health-related quality of life; LC-MS, liquid chromatography-mass spectrometry; LFQ, label-free quantification; M2-PK, M2 pyruvate kinase; MSS, metagenomic shotgun sequencing; PERMANOVA, permutational multivariate analysis of variance; rRNA, Ribosomal nucleic acid; SEIFA, socio-economic indexes for areas (a measure of relative socio-economic advantage and disadvantage in Australia); UHPLC-MS/MS, ultra-high performance liquid chromatography-tandem mass spectrometry; V4, Hypervariable region V4.