Meals, Microbiota & Mental Health of Children & Adolescents. An Observational, Longitudinal Case-control Study.

The current pilot study will be used to test the feasibility of the methods used. If the methodology is found to be feasible, the n=40+ cases and controls/siblings in the pilot study applied for here will be included in the larger study. For those cases who are invited to participate and decline participation, information on final medical diagnosis of the child will be collected from journals, but no other information. In the current pilot study (information from first 20 cases, controls and siblings) the participants will be asked if they are willing give reasons for not participating (while at the same time assured that they absolutely do not need to do so).

Questionnaires on diet, mental health and background data will be web based with help of the research data management company Smart Trial (https://www.smart-trial.co) transforming them into a readable electronic format. This procedure has been thoroughly quality-checked and will secure proper storage of data. Testing the dietary programs for nutrient calculations from food and supplement intake data, based on the Icelandic food composition database (ISGEM)1,2 as well as software for mental health questionnaires analysis.

While on the waiting list to come to the outpatient clinic, the guardians and child will receive invitation letters. If interested in participating after a follow-up telephone call, a home visit follows from the project deputy of UNR, where the informed consent is signed. Contact information to the child´s teacher is also received at this stage. A kit including tools for collecting biological samples with good instructions as well as a booklet for the three-day dietary recording will be provided. The questionnaires collecting additional information such as the short validated frequency on food and supplement intake, the ICEFFQII, designed for the Icelandic food environment3,4, food aversions, physical ailments or diagnosis ROMEIV5-7, atopic diseases iFAAM8 and medication as well as other important background factors are completed online with each participant having codes used for unlocking the questionnaires. The mental health questionnaires used in this study have all been validated internationally as well as in Iceland. These are; 1) Teacher and parent-reports: The ADHD Rating Scales9-11, the Child Behavior Checklist (TRF and CBCL) a family of symptom checklists developed to provide information on a broad range of emotional and behavior problems.12-15; the High-Functioning Autism Spectrum Screening Questionnaire (ASSQ)16 and the Social Responsiveness Scale (SRS)17-21. 2) Self-reports for children >10y: The Children's Depression Inventory (CDI)22-26 and the Multi-Dimensional Anxiety Scale for Children (MASC)27,28-33. Furthermore, if allowed by the guardians, information on prescription medication dispensed for the child from birth to current date will be retrieved from Icelandic Medicine Registry (IMR). Further information on the questionnaires used can be found in Appendix 1.

After the collection of data from the case, a randomly selected child, matched by gender and age, will be found within the same postal area by Social Sciences Research Institute (SSRI) at the University of Iceland and is invited to participate by sending an introduction letter, followed by a phone call. The SSRI will find 5 controls for each case that has finalized the study as participation rate might be low. These will be called in the sequence provided by the SSRI.

The biological samples are preferably to be collected towards the end of the 3-day food registration, with help of guardians, according to standard procedure using dedicated devices, including stabilization factors, and stored cold (4-8°C). The kit is then collected from both cases and controls by the researchers along with the food diary when close Reykjavik, Akureyri and surroundings. Participants can also deliver the samples either to BUGL or the Nutritional Unit, both at Landspitali University Hospital. For other locations the samples will be sent out including guidelines on how to resend them to the researchers after their collection. The urine samples will be aliquoted into smaller tubes for storing at a laboratory unit at the University Hospital or Akureyri Hospital in a -80°C freezer along with the other biological samples. Over the year of recruitment, diagnosis of all patients coming to BUGL will be recorded to be able to detect potential selection biases in data collection.