WANG et al .
coexisting illness (cardiovascular, gastrointestinal, endocrinological, immunological, metabolic, or any condition which contraindicates, in the investigators judgment, entry to the study), (v) usage of antibiot - ics within the past 3 months prior to participation in the study, (vi) enrolment in another clinical trial not less than 60 days prior to this study, and (vii) exposure to any nonregistered drug product within 30 days prior to this study. Fulfilment of inclusion and exclusion cri - teria was validated by the investigator and appointed research nurse.
and written, informed consent was obtained from all parents/ guardians on behalf of the child. The trial was conducted in ac - cordance with the ethical principles set forth in the current ver - sion of the Declaration of Helsinki (seventh revision; October 2013). The study was registered at ClinicalTrials.gov (identifier: NCT03416595).
2.4 | Salivary cortisol analysis
2.3 | Study design
A saliva sample was collected by a research nurse using a cotton swab from the subject's mouth. Samples were centrifuged and stored at −80 ℃ until used. Salivary cortisol was measured using a commercial enzyme-linked immunosorbent assay kit (Enzo Life Sciences).
The study followed the design of a randomized, double-blind, paral - lel, placebo-controlled clinical trial, which involved at least four visits over an 8-week period (Figure 1). Healthy subjects were randomized to two groups and given the probiotic or placebo samples once a day (2 g/day dose) with warm water or milk. The parent/guardian needed to record the number of bowel movements the subjects had each day, the stool consistency, and if they experienced any abdominal pain, bloating, or flatulence (gas). Stool and saliva samples were also collected at the study site during three visits (visits 2, 3, and 4) for analysis of microbiota composition, short-chain fatty acids (SCFAs), and cortisol. The study was approved by the Clinical Research Ethics Committee of the Cork Teaching Hospitals (CREC, Ireland),
2.5 | Fecal microbiota analysis
Fresh fecal samples were collected by parent/guardian for micro - biota and metabolomic analysis. Microbial DNA was extracted using the repeat bead beating plus column method described by Yu and Morrison (2004) with some modifications. Libraries were prepared with Illumina 16S metagenomic sequencing library preparation guide and sequenced on the MiSeq sequencing platform adhering
FIGURE 1 A flow chart showing disposition of subjects. The study involved four visits over an 8-week period. Values are expressed as the number of subjects. SAEs, Serious adverse events; SUSARs, Suspected Unexpected serious adverse reactions
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