Influences of external determinants on the physiological development and composition of the infant microbiome in healthy infants from birth during the first 6 years of life using novel molecular techniques.
Subclinical and clinical disease, transmission, and risk factors of SARS-CoV-2 among children and their families: a household study within the "Microbiome Healthy Infant Study"

The microbiome is defined as the ecological community of commensal, symbiotic,
and pathogenic microbes that literally share our body space. Immediately after
birth the body becomes populated with bacteria in various niches (e.g.
nasopharynx, mouth, skin and gastrointestinal tract) of the human body and in
time the so called human microbiome is being formed. During the first 12 months
of life the composition of the microbiome changes rapidly due to external
influences and host-related determinants like a maturing immune system.
Suggested determinants are delivery mode, respiratory infections, age,
antibiotics, maternal microbiome, nutrition, presence of eczema/ atopic disease
and exposure to others (siblings, day care).
The composition of the microbiome of a niche may be relevant for health or
susceptibility to disease. For instance, acquisition of a viral common cold may
alter bacterial outgrowth of common colonizers of the nasopharynx like S.
pneumoniae. Subsequently these bacteria may spread and cause acute otitis media
or cause pneumonia. This development towards infectious disease by commensals
like S. pneumoniae may however not only depend on viral acquisition, but also
on the total microbial composition of which S. Pneumonia is part, that may
counterbalance outgrowth and spread and this way determine susceptibility to
bacterial infections. It has already been shown that vaginal delivery or
breastfeeding may have a protective role against respiratory infections at
infant age. Vaginal delivery or breastfeeding may favour a microbiome that can
control outgrowth of potential pathogenic bacteria for a long time. Better
knowledge about favouring a protective microbiome for respiratory infections is
relevant since respiratory tract infections are the most frequent infections in
childhood for which physicians are consulted, antibiotics are prescribed and
surgery like ventilator ear tubes and adenoidectomy is performed. Insight in
optimal microbial composition may offer tools for future preventive strategies
e.g. via pre- or probiotics. In a similar way, microbiome compositions of the
oral cavity, skin and gastrointestinal tract may be associated with health or
common diseases in childhood like atopic diseases. For example, atopic disease
seems associated with an altered microbiome of the skin.

Up till now, detailed knowledge about the development of the infant microbiome
after birth in the first year of life is scarce. Relations between the
microbiome of the gastrointestinal tract, oral cavity, nasopharynx and skin are
largely unknown. Most research on the microbiome is cross-sectional, and single
niches have been described in literature. With this longitudinal study of these
four niches, we aim to understand the effects of microbial community shifts and
to relate this to infant*s health and disease (atopic, respiratory,
gastro-intestinal).

Studies of the microbiome have become feasible with the development of
molecular methods, since 40 to 80% of microbiome bacteria cannot be detected by
conventional culture techniques. With novel molecular techniques as 454
pyrosequencing (DNA-sequencing), we can evaluate the bacterial microbiome
composition. In the study we will focus on the development of the microbiome
composition in the first year of life of four relevant niches (1) nasopharynx
(2) oral cavity (saliva) (3) skin and (4) gastro-intestinal tract (faeces)
using 454 pyrosequencing referred to as the infant microbiome. We will study
the influence of external determinants on the infant microbiome. Since every
child will develop at least 6-8 symptomatic respiratory infections that are
likely to be initiated by viral acquisition, we will also study alterations in
the nasopharyngeal and oral niche during a symptomatic clinical respiratory
infection in these otherwise healthy infants.

The study population is observed until the age of 6. When the child nearly
reaches the age of 5, parents receive an invitation to participate in an
intensified protocol, called MUIS-5. In this study, the participants will be
invited to the outpatient clinic for (1) additional microbiome sampling (2)
physical examination, (3) saliva sampling for immunoglobines (4) an extended
questionnaire. Optional tests will be: (1) blood test for immunoglobines, anti
vaccine antibodies and T cell profiling, (2) lung function tests (spirometry
and fractioned exhaled nitrous oxide test) (3, only optional voor
non-follow-up-participants) nasopharyngeal microbiome sampling

CoKids:
The SARS-CoV-2 pandemic is a threat to the health and wellbeing of children and
parents. The causes and contributing factors to this are unknown, as is the
role of asymptomatic and symptomatic children in the spread of the virus and
the development of herd immunity. The Ministry of Health, Welfare and Sport and
ZonMw have approached a number of research groups to help answer questions
related to the spread and transmission of the virus in specific groups. The
UMCU/Erasmus MC and Spaarne Gasthuis in collaboration with the RIVM will study
children in various age ranges, who are participating in ongoing cohort
studies, to examine carriership and transmission in children and families.
Given the impact and relevance of findings for public health decisions, we
would like to contribute. Due to its prospective, longitudinal and detailed
data collection in a large population and its existing infrastructure, the
Microbiome Healthy Infant Study represents an excellent basis for studies into
transmission and risk factors of SARS-CoV-2 in children and their families

Primary objectives:
1. To study the development of the infant microbiome, defined as the microbial
communities in the nasopharynx (transnasal), oral cavity (saliva), skin and
gastro-intestinal tract (faeces), in healthy infants born by vaginal delivery
or caesarean section, during the first 12 months of life using novel molecular
techniques.
2. To study changes in the infant nasopharyngeal and oral microbiome during
clinical respiratory infections in the first year of life.
3. To study the long-term effects of the infant respiratory, oral, (saliva)
gut and skin microbiome on the respiratory and gastrointestinal and atopy
health status of children till 6 years of age.
*
Secondary objectives:
1. To study the influence of different determinants on the infant microbiome
such as maternal conditions during labour (prolonged rupture of membranes,
maternal fever (amnionitis) and antibiotics) nutrition (breastfeeding versus
formula feeding), antibiotics, presence of eczema/ atopic disease and exposure
to others (parents, siblings, day care).
2. To study the influence of life style and environmental factors on the human
microbiome and the development of atopic, respiratory, and gastrointestinal
complaints.
3. study the microbiome status (nasopharynx (transnasal), oral cavity
(saliva), skin and gastro-intestinal tract (faeces)), at age 5 to mucosal
immunoglobulins and anti vaccine antibodies.

Explorative:
1. To explore study the microbiome status (nasopharynx (transnasal), oral
cavity (saliva), skin and gastro-intestinal tract (faeces)), at age 5 to serum
immunoglobulins and anti vaccine antibodies and Tcel development.

CoKids:
1 To determine the incidence of overall, asymptomatic, mild and medically
attended SARS-CoV-2 infection in children and their parents
2. To determine transmission patterns of SARS-CoV-2 within households with
young children
3. To describe symptom severity in children

Observational prospective cohort study. The development of the infant
microbiome during the first 12 months of life and the potential differences in
the microbial composition between infants born vaginally and by caesarean
section will be studied. During the follow-up children will be followed til the
age of 6. At the age of 5, all MUIS participants will be invited for a one-off
intensified protocol.

CoKids:
The project will be embedded in the Microbiome Healthy Infant Study, a
population-based prospective study from birth onwards.

Participation in this study holds no more risks than negligible risk and no
benefits to the infant or the mother, except for the venipuncture, were
children could experience anxiety, we try to reduce anxiety by distracting and
comforting the child. With local anaestetic EMLA cream we reduce pain levels.
There is a 2% risk of developing a local hematoma.
We believe that the risk of this study is no more than negligible for all
sampling methods are non-invasive and generally accepted as fully save.
Sampling moments will take place during home visits to minimize burden and time
spent for the study participants. There is no personal benefit for the infant
or the mother.
We will follow the code of conduct relating to expressions of objection by
minors participating in medical research, as stated by the CCMO.
Each sample moment will take 30 minutes of participant*s time. Also signing
informed consents forms will take 30 minutes. Total time required for
participation will be 7.5 hours. During follow-up, 10 visits will be performed
over a time period of 5 years, each visit will take 30 minutes.
If parents participate in the MUIS-5 study, they are invited to visit the
outpatient clinic for the different tests. This visit will take an estimated
two to three hours. Parents receive an appropriate allowance for travelling and
parking at the hospital.

CoKids: We believe that the risk of this study is no more than negligible for
all sampling methods are non-invasive and generally accepted as fully save.
Sampling moments will take place at home. There is no personal benefit for the
families.