STUDY DESIGN. Study description:
This observational study will involve the analysis of clinical data, stool, urine and serum
(if available) samples that will be collected from infants with suspected or confirmed
non-IgE-mediated CMPA. Clinical data will be collected from the medical notes of the patients
with the written consent of the parents/carers. Dirty and wet (stools and urine samples)
nappies from infants with possible non-IgE-mediated CMPA before dairy exclusion at time of
enrolment, will be collected during acute and recovery phase according to the following
schedule:
visit 1: recruitment day (two stool samples on two consecutive bowel motions), ideally before
introduction of dairy free diet; visit 2: end of week 1; visit 3: end of week 4; visit 4:
week 5, challenge to Cow's Milk Proteins (CMP) (two stool samples on two consecutive bowel
motions, 6 hours up to 24 hours post challenge); visit 5: within a week post CMP challenge;
visit 6: week 6. Samples from infants with possible non-IgE-mediated CMPA while on dairy free
diet at enrolment will be collected at baseline (2 samples on 2 consecutive bowel motions),
week 1, week 4 and week 6. If these patients are challenged to CMP within the 6 weeks of the
study, faecal and urine samples will also be collected before (last bowel movement before
challenge) and after challenge to CMP (two stool samples on two consecutive bowel motions, 6
hours up to 24 hours post challenge). For the IgE-mediated CMPA infants and healthy
participants, faecal and urine samples will only be collected at 3 time points: on
recruitment day, on week 4 and on week 6.
Infants presenting to primary and secondary care within Greater Glasgow & Clyde health board
with symptoms suggesting possible non-IgE-mediated CMPA will be treated according to the
local non-IgE-mediated CMPA guideline (for the formula fed infants a hypoallergenic formula
milk will be offered; extensively hydrolyzed (eHF) or amino acid (AAF) formula, based on
severity of the symptoms and history, while those on breast milk the mother will be advised
to go on a dairy free diet). As per local guidelines, after 4 weeks of elimination diet
(cow's milk from infant's and mother's if breast fed), infants with mild to moderated
non-IgE-mediated CMPA will be offered an unsupervised oral food challenge (OFC) at home under
the guidance of the community dietitian while more severe cases (such as CMPIE) will be
offered an OFC under physician's supervision. In case of a convincing history such as
repeated exposure to the incriminated food eliciting repetitive vomiting and/or diarrhoea
within 24h, without any other cause for symptoms and absence of symptoms after elimination of
cow's milk, the OFC will be omitted. Confirmed cases (following positive challenge or
convincing history) will return to the exclusion diet (a hypoallergenic formula milk or
breastfeeding with mother's dairy free diet) as per local guideline.
A follow up study will be set up to follow the subjects during their further development at 3
and 5 years of age as it will be scientifically interesting to evaluate specific outcomes at
a later stage. Therefore, the participant information sheet and consent form will already
address this option and ask parents/carers of the participants whether the parents/carers
agree to be contacted in the future. Informed consent for participation in a follow up study
will be requested at that time.
Subject discontinuation during the study:
Should the participant's parents/carers decide to withdraw their consent, samples already
acquired will be kept for the study unless the participant's parents/carers tell us not to do
so, in which case the samples will be destroyed.
Samples will be disposed of as per Greater Glasgow & Clyde NHS policy. Should participants'
parents/carers loose capacity to consent during the study, the infant's participation to the
study will be withdrawn and identifiable data or samples collected with consent will be
retained and used for the study. No further data or samples will be collected or any other
research procedures carried out on or in relation to the participant.
SAMPLE COLLECTION FOR LABORATORY MEASUREMENTS. Faecal and urine samples:
Nappies will be collected by the researcher at a time and place which is convenient for the
participant's parents/carers, including the hospital ward or clinic or the participant's
home. If participant's parents/carers would find it helpful, courtesy calls will be made a
few days before collection and gentle reminders via text, phone or email will be given a few
days before and/or during sample collection to assist with any queries the participant may
have. When a dirty and wet nappy becomes available, the participant's parents/carers will
call/text the researcher (study's direct line number) immediately for collection and
transportation of the samples to the laboratory; the sample will be either collected by a
pre-paid taxi or by the researchers themselves. For non-IgE-mediated CMPA participants, stool
and urine samples will be collected at baseline (visit 1: recruitment day, two samples on 2
consecutive bowel motions), week 1 (visit 2, day 7), week 4 (visit 3, day 28) and week 6
(visit 6: day 37). If an OFC is performed, additional faecal and urine samples will be
collected prior to milk reintroduction (last bowel movement before OFC), on food challenge
day (2 samples on 2 consecutive bowel motions, 6 hours up to 24 hours post challenge) and
post food challenge (within one week post challenge). 3 stool and urine samples will be
collected from the IgE-mediated CMPA and healthy control groups, one at baseline (recruitment
day), one on week 4 (day 28) and a last one on week 6 (day 37). Attempts should be made to
schedule visits (assessments) on the exact date. If this is not possible these should be
scheduled within a 48-hour window.
Microbiota analysis:
Bacterial diversity: Diversity, richness and relative abundance of all microbial genera will
be explored with Next Generation sequencing of the 16S rRNA gene on the IlluminaMiSeq®
platform. Bacterial functional capacity: Whole genome sequencing (shotgun metagenomics) will
be performed on an IlluminaHiSeq® platform. Shotgun metagenomics will generate functional
profiles of the microbiota and metabolic pathways by mapping unassembled reads to the KEGG
database of metabolic pathways using HUMAnN. Bacterial metabolic activity: Metabolic
phenotyping of samples will be performed with NMR/LC/GC-FID/MS and other assays as described
previously. Faecal metabolomics analysis will enable correlations of the metabolic signatures
directly with the functional profiles and the annotated bacterial abundances and hence,
determination of the metabolic roles of the key community members. Disease markers &
Immunophenotyping: Faecal calprotectin will be measured from faecal samples in order to
identify possible colonic inflammation in these patients.
Upon study completion all samples will be stored and may be used to address the same research
questions as this study as new lab methods arise. This is particularly relevant for
microbiological analysis, as novel methods may offer us a better understanding of the role of
the microbiota in gut inflammation. Permission to store samples and use in future research
will be gained by written consent.
Blood samples:
With regards to venous blood samples collection, this will apply only for any infant included
in the study who will undergo blood testing for clinical reasons (no additional needle
insertion is required). Blood samples collected for clinical purposes which are no longer
needed (redundant samples), will be requested from the haematology lab of the Royal Hospital
for Children and transferred to Professor Milling's lab at the University of Glasgow where
the samples will be prepared and stored for the purpose of this study. Collected blood
samples will be used to measure blood inflammatory and immune markers, and to assess the
immunophenotype, by techniques that may include conventional enzyme-linked immunosorbent
assay (ELISA), cytokine multiplex analysis, flow cytometry, and gene expression analysis.
Inflammatory analysis:
Various immune cells and cytokines will be measured in stool supernatants, urine and blood.
The investigators will look for the following cytokines including the following: Th1
cytokines: Interleukin (IL) 12, Interferon gamma (IFN-γ), Th2 cytokines: IL-5, IL-13, IL-4,
IL-9, Th17 cytokines: IL17, IL22, Treg cytokines: IL-10, Transforming growth factor beta
(TGF-β) and inflammatory cytokines: Tumor necrosis factor alpha (TNF-α), IL-1β, IL-2, IL-6,
and IL-8. The humoral immune response will be assessed by quantification of antigen-specific
and total secretory Immunoglobulin A (sIgA), IgG, IgM and IgE, calprotectin and
Eosinophil-derived neurotoxin (EDN). The above inflammatory markers in stools, urine and
serum may be assessed by ELISA, cytometric bead array (CBA) assay, multiplex protein
analysis, or flow cytometry.
