To evaluate intervention impacts, we will assess primary outcomes of child growth along with language, cognitive, motor, and social-emotional development milestones. Secondary outcomes of interest, which are also hypothesized to serve as mediating factors between the intervention and primary outcomes, include indicators for child health and nutrition (bone maturation, brain growth, respiratory or diarrheal infections, dietary intake, nutrient biomarkers), and responsive parenting based on observations of the home environment and parent-child interactions. Table
3 summarizes measures that correspond with outcomes of interest.
Table 3
Grandi Byen study outcomes
Primary outcomes |
Child growth | Anthropometric measures of height and weight | Length-for-age Z score (LAZ) Weight-for-age Z score (WAZ) Weight-for-length Z score (WLZ) | Baseline, 3 mo, 6 mo, 9mo, 12 mo |
Cognitive, Language, Motor and Social Emotional Development | Ages and Stages Questionnaires (ASQ)– Social-Emotional, Communication, Gross Motor, Fine Motor, and Problem-Solving Domains | ASQ scores | Baseline, 6 mo, 12 mo |
Secondary outcomes |
Responsive parenting | Parenting Interactions with Children: Checklist of Observations Linked to Outcomes (PICCOLO); Home Observation Measurement of the Environment (HOME), mother-infant observation | PICCOLO: Scores in Responsiveness, Affection, and Encouragement HOME: environment subscale will be used to measure aspects of the household environment (i.e., availability of toys/items for child to play with) and responsive parenting. | Baseline, 6 mo, 12 mo |
Child dietary intake | 24-hour recall and food frequency questionnaire | Nutrient intakes, dietary diversity, and ASF consumption | Baseline, 6 mo, 12 mo (24-hour recall) Baseline, 3 mo, 6 mo, 9 mo, 12 mo (food frequency questionnaire) |
Child nutrient biomarkers | Blood draw, mass spectrometry (LC-MS-MS, CP-OES) | Plasma concentrations of nutrient biomarkers | Baseline, 6 mo |
Child bone maturation | Ultrasound imaging of hand and wrist | Bone age Z score | Baseline, 6 mo, 12 mo |
Child brain growth | Ultrasound imaging of the head until anterior fontanelle closes | Dimensions of brain regions (gangliothalamic ovoid, biparietal diameter, corpus callosum, other standard measures) | Baseline, 6 mo |
Child morbidities | Blood draw, immulite immunoassay methods Stool samples 2-week morbidity recall by mothers | Plasma concentrations of inflammatory biomarkers (ovomucoid and ovalbumin IgE, C-reactive Protein [CRP], and Insulin-like growth factor 1 [IGF-1]). Diarrhea, respiratory conditions, allergies | Baseline, 6 mo |
Child genetics | Buccal swabs | Targeted genomic analyses | Baseline |
Primary outcomes
Child growth will be evaluated using anthropometric measures. Child weight and length will be measured following the WHO’s guidelines on child growth assessments [
72]. Measurements will be obtained at five time points—baseline and at 3-, 6-, 9-, and 12-month follow-ups—using the Seca Model 874 (Digital) 440 lbs x 0.1-lb. resolution and the ShorrBoard® stadiometer, respectively. All measures will be repeated at least once to ensure precision. Weight and height measures that differ by more than 0.1kg and 0.7 cm, respectively, during the second measurement will be repeated a third time. Measures will be converted to weight-for-age (WAZ), weight-for-height (WHZ), and length-for-age (LAZ) Z-scores using WHO Growth Standards [
73]. Ages will be calculated using the child’s date of birth reported at baseline.
Child development will be assessed using the Ages & Stages Questionnaires (ASQ) at baseline and at 6- and 12-month follow-ups. The ASQ, which has been used widely for developmental screening and studies in low- and middle-income countries [
74], is a parent report measure of several aspects of child development including self-regulation, affect, and social communication in very young children. The Ages & Stages Questionnaires, Third Edition (ASQ-3) will be used to assess developmental milestones along the domains of
communication (language),
problem solving (cognition),
fine motor, and
gross motor development [
75], while the Ages & Stages Questionnaires: Social-Emotional, Second Edition (ASQ:SE-2) will be used to examine changes in child
social-emotional development [
76].
Secondary outcomes
Responsive Parenting will be assessed using the Parenting Interactions with Children: Checklist of Observations Linked to Outcomes (PICCOLO) [
77] and the infant-toddler Home Observation Measurement of the Environment (HOME) [
78]. These assessments will be completed at three time points—at baseline and at 6- and 12-month follow-ups. The validated PICCOLO assessments measure nurturing parenting behaviors, present across diverse cultures worldwide, which determine psychosocial development during a child’s early years and throughout their life course [
77]. For this study, we will specifically assess parenting behaviors along the PICCOLO domains of
responsiveness,
affection, and
encouragement. The environment subscale from the HOME assessment will be used in addition to the three PICCOLO domains to measure aspects of the household environment in relation to child stimulation. Assessments with these instruments will be based on a combination of mother self-report and observations by study enumerators to assess interactions between the mother and child, and the quality and quantity of stimulation in the home environment [
78,
79].
Because observation within the home environment is essential to assessing these constructs, study enumerators will complete the observation items on the HOME during visits to the participants’ homes. For PICCOLO assessments, mothers will be instructed to engage with their children as they typically would for 15 minutes, and their interactions will be videotaped. Independent raters, blind to the dyad’s study condition, will review and code five-minute segments per video, randomly selected from three five-minute segments. Raters will undergo extensive training and procedures will be put in place to ensure interrater reliability.
Child dietary intake and levels of different
nutrient biomarkers will be evaluated as secondary nutrition outcomes. All caregivers will undergo a 24-hour dietary recall using the standardized multi-pass interview approach [
22,
80] at baseline and at 6- and 12-month follow-ups. In the first pass, caregivers will be asked to list everything the child ate and drank in the last 24 hours. In the second pass, they will report the time of consumption, ingredients, and brands of each item listed in pass one. In the third pass they will be asked to give the closest estimated amounts the child consumed using measuring cups and graduated cylinders. Culturally appropriate visual aids such as cups, dishes, and pictures of foods are also provided in order to obtain the most accurate food measurements. Lastly, the items reported are all reviewed with the mother to ensure that the history is complete. Additional information, such as current breastfeeding status will also be obtained. The goal of the 24-hour recall interview is to capture the child’s usual daily dietary intake in order to correctly estimate nutrient intakes. Mothers will therefore be asked whether the dietary intake reported represents the child’s daily usual intake. A small subset of participants will undergo repeat 24-hour recalls to assess for intra-individual variability [
22]. Records from the 24-hour recall will be entered into the
NutriSurvey software (EBISpro, Germany) [
81] which will analyze and estimate nutrient intakes for each infant. Data from the 24-hour recall interviews will also enable us assess child dietary diversity as well as changes in the consumption of Animal Source Foods (ASFs). Food frequency questionnaires will also be administered at baseline and at 3-, 6-, 9- and 12-month follow-ups.
To delineate the effect of the egg intervention relative to the multicomponent intervention, we will assess different biological indicators including levels of blood biomarkers of nutritional status at baseline and 6 months post-enrollment. We will examine changes in plasma concentrations of a suite of minerals—iron, zinc, iodine, and selenium. Plasma concentrations of vitamins (choline and vitamin B12), choline-related markers (methionine and betaine), and DHA will also be assessed. A trained phlebotomist will collect 3-4 ml of venous blood from the children using trace element-free sample collection methods and processing. Blood specimens will be aliquoted and processed using sterile methods to prevent environmental contamination and subsequently stored in a -80°C freezer. Plasma mineral concentrations will be measured using inductively coupled plasma mass spectrometry (ICP-MS; PerkinElmer NexION 2000) and inductively coupled plasma optical emission spectrometry (ICP-OES: PerkinElmer Optima 7300DV). For DHA and choline-related markers, liquid chromatography-tandem mass spectrometry (LC-MS/MS) will be applied to quantify them. Vitamin B12 levels, as well markers of inflammation, will be measured using Immulite immunoassay and other methods.
Inflammatory biomarkers will be assessed in relation to caregiver reports on child morbidities surveys during biweekly home visits. Specific biomarkers of interest are ovomucoid and ovalbumin immunoglobulin E (IgE) to evaluate risk of egg allergies, C-reactive Protein (CRP) as a marker of systemic inflammation, and Insulin-like growth factor 1 (IGF-1) as an indicator of growth.
A standard two-week recall on a range of infectious illnesses will supplement CRP data. Additionally, questions to assess diarrheal severity will include the frequency of diarrhea in the children, number of sick contacts within the household, presence of blood or fever, use of anti-microbials, and requirement for additional medical care at a clinic or local provider. A similar assessment for respiratory infections including cough, rhinorrhea, fever, rash, and lethargy will be obtained through the same two-week recall survey.
Stool samples and fecal swabs will also be essential for assessments on diarrheal and infectious outcomes, future assessment of microbiome changes, and relative biomarker changes. Samples collected at baseline and at 6- and 12-month follow-ups will be associated with assessments of current or recent (past 3 days) diarrheal disease. Our pilot data coupled with national data suggests a diarrheal prevalence of 30%, supporting adequate sampling at these intervals to estimate pathogen burden (unpublished observations—F.M. Kuhlmann, Washington University School of Medicine). Stool samples will be collected from children using fecal swabs and fresh stool samples will be obtained, if available, from the child’s diaper. Should an unanticipated increase in diarrheal incidence be noted in the intervention arms, intensified efforts to identify pathogens associated with eggs (Campylobacter and Salmonella) will be pursued to determine if the egg preparations are increasing the risk of infectious diarrhea.
Ultrasound methods will be used to assess
bone maturation at baseline and at 6- and 12-month follow-ups.
Brain growth will also be assessed at baseline and 6 months post-enrollment using ultrasound
. For bone maturation, ultrasound images of the child’s left hand and wrist will be obtained to assess maturity of the ossification centers. Following protocols used in our previous study in Ecuador [
82], image findings will be adjusted for the child’s age and sex and indicated by the bone age Z-score (BAZ) using standards of Greulich and Pyle [
83]. Additionally, brain ultrasound images will be obtained through the anterior fontanelle at baseline and 6 months. Images will assess the size of the gangliothalamic ovoid, corpus callosum, as well as ventricular diameter and extra axial space. Closure of the fontanelle will be noted, and in such cases, brain images will not be obtained. Our group conducted a feasibility study in Ecuador using a similar method [
84], which will be implemented in the present study. Assessing intervention effects on child brain growth using these methods may support evidence related to DHA and choline’s role in brain development. All images will be obtained using the Mindray M7 Premium portable ultrasound with the 7L4s Linear Array Probe (Mindray North America, Mahwah, NJ).
The last biological indicator that will be evaluated is
child genetics using buccal samples that will be obtained at the first study visit only. Buccal swab collection requires wiping the inside of the cheek with a nylon swab and placing the swab into stabilization solution. This minimally invasive and standardized technique allows for collection and preservation of genetic material and kept at room temperature for extended periods of time. Of note, interactions between host genetics, enteric pathogens, gut microbiota, and cognitive development are recently being recognized as an important component of child development [
85‐
87]. The interpretation of the study results will almost certainly be impacted by the foresight to collect samples for retrospective targeted genomic analysis in association with the study findings.