Malnutrition in women of reproductive age remains a public health concern in Sub-Saharan Africa (SSA). Malnutrition during pregnancy affects foetal growth with a tendency of the exposed infants to also develop it. The interaction of the mother with the infant shapes the seeding and the trajectory of the infant intestinal microbiota which is crucial for development of a healthy immune system Malnutrition has been associated with intestinal inflammation, intestinal leakage and reduced calorie absorption. Early life malnutrition and environmental enteric dysfunction (EED) immunopathology remains poorly described in the context of mother-infant dyads. This is essential as malnutrition, poor water, sanitation and hygiene (WASH), including the presence of infectious diseases limit the developmental potential of the exposed infants in SSA, including Zimbabwe. In addition, maternal stress and poor mental health may also affect standard hygiene practices, including how a mother cares for her baby, potentially aggravating EED and the risk of the infant being malnourished. Primary outcomes 1. Infant malnutrition and recovery. 2. Gut dysfunction (gut inflammation, leaky gut, malabsorption, dysbiosis) 3. Diarrhea episodes, defined as any episode of acute diarrhoea (≥3 passages of loose stool within 24 hours as reported by the mother) occurring before the next study visit. Definition of malnutrition outcomes to be assessed in babies born to malnourished women, is a mid- upper arm circumference (MUAC) \<23cm; * MUAC for age: Malnourished defined as those below -2 standard (SD) of the World Health Organisation (WHO) reference * Weight-for-age: Underweight defined as those below -2SD WHO reference * Weight-for-height: Wasted defined as those below -2SD WHO reference * Height-for-age: Stunted defined as those below -2SD WHO reference * Z-scores (as they are i.e. a continuous variable, taking age of infants into account) * A composite variable, any of malnourished, underweight, wasted or stunted.
Background Malnutrition, poor hygiene, lack of access to clean water, infectious diseases, and low access to medicine limit life expectancy and developmental potential of infants in Sub-Saharan Africa (SSA) including Zimbabwe. These conditions severely affect infant growth and neurodevelopment, especially within the first 1000 days of life, and the affected infants might never reach their full development potential in life. The interaction of the mother with the infant shapes the trajectory of the intestinal microbiota and the development of its immune system is considered crucial for a healthy development, yet the therapeutic success remains limited. Malnutrition is a subacute or chronic condition related to insufficient consumption or absorption of nutrients. Malnutrition leads to changes in body composition and diminished functions, exemplified by anthropometric measures of the infant below 2 standard deviations compared to an age-matched control population. Underweight is defined by a low weight-for-age Z-score, wasted by a low weight-for-height Z-score, stunted by a low height-for-age Z-score and malnourished refers to a low mid-upper arm circumference (MUAC) for age. In a systematic review in SSA, risk factors for malnutrition were low maternal education, short birth intervals of \<18 months, being first or second in birth order, male sex, lack of safe drinking water, and economic instability. On the other hand, antenatal health education could reduce the risk for malnutrition. Malnutrition is associated with detrimental infant outcomes including reduced physical growth, lower intellectual quotient, behavioral problems, and deficient social skills. Poor water, sanitation and hygiene (WASH) affects approximately one third of the world's population. For instance, in Africa, approximately 70% of water systems are frequently non-functional and people walk long distances to collect water. On the other hand, in SSA, sewage systems are frequently inefficient with sewage bursts or lacking altogether. Lack of efficient WASH practices has been associated with intestinal infections and malnutrition in infants in SSA. The effects of WASH and malnutrition on infant development are at least partially mediated by the intestinal microbiota and the disruption of interaction networks of intestinal bacteria. From birth throughout life, human microbiome profile is constantly changing either temporarily or permanently due to diet, exposure to microbes, or medications. In birth cohorts from Bangladesh and other continents, an "ecogroup" of bacterial taxa showed consistent co-variation. Variations in this ecogroup concisely reflected microbiota development and the level of microbiota perturbation in infants with severe and moderate acute malnutrition. A causal role of the microbiota is also suggested by animal experiments. Intestinal microbiota transplantation from malnourished infants to germ-free mice resulted in a malnourished phenotype and this phenotype could even be reproduced with two isolated strains (Ruminococcus gnavus and Clostridium symbiosome). Finally, microbiota-directed complementary food corrected the intestinal microbiota of malnourished infants to a composition similar to age-matched controls, and showed superior effects in correcting growth compared to ready-to-use supplementary food. The trajectory of the development of the intestinal microbiota is closely linked to the maturation of the infant intestinal immune system. A critical "window of opportunity" exists from birth until the end of weaning (\~18 months of age), during which environmental influences can prime the immune system. In mice, a decrease in maternal epidermal growth factor intake causes the intestinal barrier to open, resulting in increased antigen passage and a vigorous immune response. This "weaning reaction" is characterized by high expression of interferon-γ (IFN-γ) and tumor necrosis factor in the intestine. Inhibition of this weaning reaction by antibiotics usage results in increased susceptibility to inflammatory conditions such as inflammatory bowel diseases (IBD) and allergies. However, how priming of the intestinal immune system is effected by environmental challenges such as infections, poor hygiene and nutrition remains unknown. The intestinal microbiota has manifold bidirectional interactions with the host immune system. For example, Th17 cells, induced by the intestinal microbiota are a crucial line of defence against external bacteria. Further, short chain fatty acids (SCFA), and aryl hydrocarbon receptor (Ahr) agonists are microbiota products with beneficial immunomodulatory effects. Any interaction of the intestinal immune system with the microbiota should be regarded as an effort to maintain the intestinal barrier ensuring absorption of nutrients while excluding bacteria from the system and maintaining the integrity of the host. WASH, malnutrition and associated microbiota changes lead to changes in intestinal physiology including inflammation, leakage and reduced calory absorption, which can be assessed in infants. Faecal calprotectin (FC) is an established diagnostic marker for intestinal inflammation. In the first year of life, FC values are much higher than in adulthood (277 mg/kg, vs. \<50 in adults), likely related to an immature infant immune system as well as the gut microbiota. FC values decrease during the first 1.5 years of life. Variable results have been observed between 1-4 years, with possibly higher measurements in healthy infants in developing countries (Uganda). Malnutrition has also been associated with intestinal leakage and leaky gut syndrome. Intestinal leakage leads to the loss of small and large molecules including albumin and antibodies from the systemic circulation into the gut lumen, associated with loss of calories and reduced calory absorption in the intestine. On the other hand, permeability of the intestinal wall for bacteria, bacterial toxins and/ or pathogen associated molecular patterns is increased, leading to intestinal inflammation. Intestinal leakage can be non-invasively assessed with the lactulose rhamnose permeability test. Intestinal calory absorption can be non-invasively assessed. Bomb calorimetry can be used to measure the residual chemical energy organic material. To this end, the material will be saturated with oxygen in a sealed container and ignited, leading to the conversion of organic material to carbon dioxide, water and gaseous nitrogen. The method assesses enthalpy changes upon conversion of the organic material such as food or stool into simple gaseous molecules. Upon assessment of total energy intake with detailed food questionnaires and quantification of the residual energy in stool the total absorption capacity for energy, carbohydrates, proteins and fat has been estimated to be 89.4%, 92.5%, 86.9% and 87.3%, respectively in healthy adults. Malnutrition is a complex condition and is also related to oral health. In African countries, inflammatory oral diseases (e.g. periodontal diseases, acute necrotising gingivitis, noma) can aggravate malnutrition due to reduced calory absorption. On the other hand, malnourishment and related conditions can also manifest as oral pathologies. Poor oral health such as missing teeth or dental caries and periodontal disease lead to diminished ability to chew or swallow certain food, leading to poorer nutritional status. Finally, the health of the infant cannot be separated from the well-being of the mother. Mothers in low-resource settings face stress, anxiety and depression and challenges to female empowerment which might negatively impact on mother child bonding. The current project attempts to do a comprehensive analysis of malnutrition in infants from high-density areas in Harare, Zimbabwe considering the infant microbiota, immune system, intestinal function as well as all surrounding factors affecting the health of the mother and the infant. Rationale Gut microbiome characterization remains inadequately described in resource limited settings. In this project, investigators will recruit and follow pregnant women at high risk for subsequent malnutrition of the infant. Investigators will study development of the intestinal microbiota as well as the infant immune system, intestinal inflammation, leakage and calory extraction. This longitudinal study with comprehensive analysis of bio-samples will provide insights to move from correlation to causality. This work will inform future mechanistic studies and downstream translational work such as the development of next-generation probiotics and prebiotics. Main hypothesis Environmental and microbiota factors result in intestinal dysfunction leading to malnutrition and suboptimal infant growth and development.
Study Type
OBSERVATIONAL
Enrollment
368
MUAC ≤23 cm in pregnancy at least 20 weeks gestational age
Poor water (source, quality, access, reliability), sanitation (toilet type, cleanliness, number of people using toilet) and personal (hand wash practices) and household hygiene (dumpster availability and emptying frequency) index scores. WASH Index score ranges; 1.Basic services (76-100%), 2.Semi-basic services (51-75%) 3.Poor services (26-50%) 4.No services (0-25%)
University of Zimbabwe
Harare, Zimbabwe
RECRUITINGAlpha diversity of maternal gut microbiota
Alpha diversity of maternal stool microbiota will be assessed by shotgun metagenomic sequencing, and calculated using the Shannon index. This measure reflects within-sample diversity and evenness of microbial communities. Unit of Measure: Shannon index (unitless; typical range 1-6)
Time frame: Third trimester of pregnancy, week(s) 1, 6, 14, 24, 48, 96, 144 after delivery
Beta diversity of maternal gut microbiota
Beta diversity of maternal stool microbiota will be assessed by shotgun metagenomic sequencing and calculated using Bray-Curtis dissimilarity. This measure reflects between-sample differences in community composition. Unit of Measure: % variance explained
Time frame: Third trimester of pregnancy, week(s) 1, 6, 14, 24, 48, 96, 144 after delivery
Alpha diversity of human breast milk microbiota
Alpha diversity of maternal breast milk microbiota will be assessed by shotgun metagenomic sequencing, and calculated using the Shannon index. This measure reflects within-sample diversity and evenness of microbial communities. Unit of Measure: Shannon index (unitless; typical range 1-6)
Time frame: Week(s) 1, 6, 14, 24, 48, 96, 144 after delivery/ until weaning
Beta diversity of human breast milk microbiota
Beta diversity of breast milk microbiota will be assessed by shotgun metagenomic sequencing, and calculated using Bray-Curtis dissimilarity. This measure reflects between-sample differences in community composition. Unit of Measure: % variance explained.
Time frame: Week(s) 1, 6, 14, 24, 48, 96, 144 after delivery/ until weaning
Protein content of breast milk
Breast milk samples will be analyzed by photo-spectrometric protein assays, such as the BSA assay. Comparison of milk of mothers of infants with and without clinical signs of malnutrition (defined by WHO growth standards: WLZ or LAZ \< -2), 100 infants per group. Unit of Measure: g/100mL
Time frame: Maternal breast milk samples collected at week(s) 1, 6, 14, 24, 48 post birth.
Fat content of breast milk
Breast milk samples will be analyzed by creamatocrit test. Comparison of milk of mothers of infants with and without clinical signs of malnutrition (defined by WHO growth standards: WLZ or LAZ \< -2), 100 infants per group. Unit of Measure: mL/100mL
Time frame: Maternal breast milk samples collected at week(s) 1, 6, 14, 24, 48 post birth.
Lactose content of breast milk
Breast milk samples will be analyzed by high-performance liquid chromatography (HPLC) Comparison of milk of mothers of infants with and without clinical signs of malnutrition (defined by WHO growth standards: WLZ or LAZ \< -2), 100 infants per group. Unit of Measure: g/100mL
Time frame: Maternal breast milk samples collected at week(s) 1, 6, 14, 24, 48 post birth.
Micronutrient content of breast milk
Breast milk micronutrients (vitamins A, D, B12, folate; trace elements such as iron, zinc, selenium) assessment using ELISA, and colorimetric assays. Comparison of milk of mothers of infants with and without clinical signs of malnutrition (defined by WHO growth standards: WLZ or LAZ \< -2), 100 infants per group. Vit A: µg/L• Vit D: ng/mL• Folate: ng/mL• Vitamin B12: pg/mL• Iron, zinc, selenium;µg/L.
Time frame: Maternal breast milk samples collected at week(s) 1, 6, 14, 24, 48 post birth.
Metabolic composition of breast milk Assessing whether compositional differences in maternal milk are associated with infant intestinal development potential
Breast milk samples will be analyzed by untargeted mass spectrometry. Comparison of milk of mothers of infants with and without clinical signs of malnutrition (defined by WHO growth standards: WLZ or LAZ \< -2), 100 infants per group. Unit of Measure: relative counts of ionized metabolites (mass-to-charge ratio, m/z)
Time frame: Maternal breast milk samples collected at week(s) 1, 6, 14, 24, 48 post birth.
Impact of breast milk on infant intestinal organoid barrier function
Primary infant-derived 2D intestinal epithelial organoids will be generated from patient biopsies and treated with diluted breast milk samples to assess epithelial cell proliferation, barrier maturation, and differentiation. Transepithelial electrical resistance (TEER) will be measured as a read-out of intestinal barrier. Comparison of milk of mothers of infants with and without clinical signs of malnutrition (defined by WHO growth standards: WLZ or LAZ \< -2), 100 infants per group. Unit of Measure: TEER: ohms·cm²
Time frame: Maternal breast milk samples collected at week(s) 1, 6, 14, 24 post birth.
Impact of breast milk on infant intestinal organoid morphology
Primary infant-derived 3D intestinal epithelial organoids will be generated from patient biopsies and treated with diluted breast milk samples for several days. Microscopy will be used to determine the organoid morphology: organoid-to-spheroid ratio. Comparison of milk of mothers of infants with and without clinical signs of malnutrition (defined by WHO growth standards: WLZ or LAZ \< -2), 100 infants per group. Organoid morphology: villus-crypt architecture scores Unit of Measure: qualitative/semi-quantitative
Time frame: Maternal breast milk samples collected at week(s) 1, 6, 14, 24 post birth.
Impact of breast milk on infant intestinal organoid size
Primary infant-derived 3D intestinal epithelial organoids will be generated from patient biopsies and treated with diluted breast milk samples for several days. Microscopy will be used to determine the organoid size. Comparison of milk of mothers of infants with and without clinical signs of malnutrition (defined by WHO growth standards: WLZ or LAZ \< -2), 100 infants per group. Unit of Measure: µm
Time frame: Maternal breast milk samples collected at week(s) 1, 6, 14, 24 post birth.
Impact of breast milk on infant intestinal organoid gene expression
Primary infant-derived 2D intestinal epithelial organoids will be generated from patient biopsies and treated with diluted breast milk samples for 24 hours. RNA will be isolated and used for reverse transcription quantitative polymerase chain reaction (RT-qPCR). Comparison of milk of mothers of infants with and without clinical signs of malnutrition (defined by WHO growth standards: WLZ or LAZ \< -2), 100 infants per group. Expression of tight junction proteins (e.g., ZO-1, occludin): relative fluorescence intensity Unit of Measure: relative expression of gene of interest in relation to housekeeping gene (2\^(-delta CT)) Unit of Measure: TEER: ohms·cm²
Time frame: Maternal breast milk samples collected at week(s) 1, 6, 14, 24 post birth.
Alpha diversity of infant gut microbiota
Alpha diversity of infant stool microbiota will be assessed by shotgun metagenomic sequencing, and calculated using the Shannon index. This measure reflects within-sample diversity and evenness of microbial communities. Unit of Measure: Shannon index (unitless; typical range 1-6)
Time frame: Week(s) 1, 6, 14, 24, 48, 96, 144 of age
Beta diversity of infant gut microbiota
Beta diversity of infant stool microbiota will be assessed by shotgun metagenomic sequencing and calculated using Bray-Curtis dissimilarity. This measure reflects between-sample differences in community composition. Unit of Measure: % variance explained.
Time frame: Week(s) 1, 6, 14, 24, 48, 96, 144 of age
Relative abundance of dominant bacterial taxa and microbial metabolic pathways
Relative abundance of dominant bacterial taxa in maternal and infant stool, including breast milk, and of microbial metabolic pathways (KEGG modules) in infant stool, will be determined using MetaPhlAn3 and HUMAnN3. Unit of Measure: % of total sequences assigned. Relative abundance (%) of dominant bacterial taxa in maternal and infant stool Relative abundance (%) of microbial metabolic pathways (KEGG) in infant stool.
Time frame: Maternal stool: third trimester, birth, 6, 12, and 24 months postpartum • Breast milk: birth, 6, and 12 months postpartum • Infant stool: birth, 6, 12, and 24 months old.
Maternal serum immunoglobulin concentrations
Total IgG, IgA, and IgM will be quantified in maternal serum using ELISA/Luminex assays as biomarkers of systemic and mucosal barrier function. Unit of Measure: mg/mL
Time frame: From pregnancy and once every year for 3 years
Maternal levels of serum proteins
Albumin, prealbumin, and total protein will be measured in maternal serum using ELISA/Luminex assays to assess intestinal barrier integrity and nutritional status. Unit of Measure: g/dL
Time frame: From pregnancy and once every year for 3 years
Maternal levels of serum inflammatory markers
Myeloperoxidase, neopterin, soluble CD14, soluble CD163 and C-reactive protein will be quantified using ELISA/Luminex assays in maternal serum as biomarkers of intestinal and systemic inflammation. Unit of measurement: µg or ng/mL
Time frame: Once in pregnancy, and once every year for 3 years.
Maternal biomarkers of epithelial and microbial translocation
Intestinal fatty-acid binding protein (I-FABP) and endotoxin core antibodies (EndoCAb) will be measured using ELISA/Luminex assays in maternal serum as biomarkers of epithelial barrier disruption and microbial translocation. Unit of Measure: pg/mL (I-FABP) and EU/mL (EndoCAb).
Time frame: Once in pregnancy, and once every year for 3 years.
Maternal Iron and anabolism-related biomarkers
Soluble transferrin receptor (sTfR), hepcidin, and insulin-like growth factor 1 (IGF-1) will be measured in maternal serum as biomarkers of iron status and systemic growth regulation. Unit of Measure: ng/mL
Time frame: Mother from pregnancy and once every year for 3 years
Maternal serum biochemical indicators
Urea, creatinine, and liver enzyme alkaline phosphatase will be measured in maternal serum to assess renal and hepatic function. Unit of Measure: mg/dL (urea, creatinine) and U/L (alkaline phosphatase)
Time frame: Once in pregnancy, and once every year for 3 years after delivery.
Timing of infant malnutrition and oral health status
The objective is to assess the relationship between the timing of malnutrition in the infant and the development of oral health issues: * Dental caries * Oral infections and other oral manifestations, * Enamel dysplasia) in children from 6 months to 2 years of age. The standardized oral health assessment tool will be used to describe the following; * Dmft index (decayed, missing, filled teeth) for dental caries * Visual examination for enamel dysplasia * Oral infections including other oral manifestations Oral thrush (fungal) Herpetic gingivostomatitis (herpes simplex virus) Tooth abscess (bacterial) Gangrenous disease noma (bacterial) Apthous stomatitis Eruption cysts around teething teeth
Time frame: From 6 months after delivery or birth and once a year for 2 years
Correlation between Oral Health of mother and that of the child
The objective is to evaluate the correlation between the oral health status of mothers and their children, including the presence of dental caries, oral infections, and periodontal disease. Measurement Tool: Standardized oral health assessment tools will be used to define the following * DMFT index (Decayed, Missing, Filled Teeth) for mothers * dmft index for children * Plaque Index (PI) and Gingival Index (GI) for both mothers and children Oral health issues: * Dental caries * Oral infections and other oral manifestations, * Enamel dysplasia) in children from 6 months to 2 years of age. * Plaque/Gingival index score; Total number of surfaces with plaque/gingivitisis divided by total number of observed surfaces.
Time frame: From 6 months after delivery or birth and once a year for 2 years
Maternal socio-demographic and the development of infant malnutrition and gut dysfunction and mortality
Determine the correlation between maternal socio-demographic, including household characteristics in pregnancy at least 20 weeks gestational age assessed using a questionnaire administered by a research nurse and, 1. adverse pregnancy outcomes (low birth weight, pre-maturity birth \< 37 weeks gestational age at birth 2. development of infant malnutrition from birth, weeks 6, 24, 24 48, 96 and 144 of age 3. Gut dysbiosis as in objective 1 above Comparing infants born of women in the 4 groups, malnourished versus normo-nourished in pregnancy at least 20 weeks gestational age by poor versus relatively better WASH living conditions Definition of malnutrition outcomes to be assessed * MUAC for age: Malnourished defined as those below -2SD WHO reference * Weight-for-age: Underweight defined as those below -2SD WHO reference * Weight-for-height: Wasted defined as those below -2SD WHO reference * Height-for-age: Stunted defined as those below -2SD WHO reference
Time frame: Mothers; from pregnancy and every 6 months until 3 years after deliveryInfants; from birth and every 6 months until 3 years old
Maternal stress assessment using Cohen Perceived Stress Scale (CPSS)
The CPSS tool measures the perception of stress in individuals and how unpredictable, uncontrollable, and overloaded mothers find their lives. It consists of 10 items with mothers rating their feelings and thoughts during the last month. Each item is scored 0 to 4: 0: Never 1. Almost never 2. Sometimes 3. Fairly often 4. Very often Total Score The total score is calculated by summing the scores of all items, yielding a range from 0 to 40: Low Stress: Scores between 0-13. Indicates low levels of perceived stress. Moderate Stress: Scores between 14-26. Suggests moderate levels of perceived stress. High Stress: Scores between 27-40. Indicates high levels of perceived stress. All the 300 mothers will be assessed.
Time frame: In pregnancy , weeks 6 and 24 months after delivery
Maternal stress measurement using Edinburgh Post Partum Depression Scale (EPDS)
EPDS screening tool will be used to identify mothers who may be experiencing postpartum depression. It consists of 10 multiple-choice questions, each focusing on the presence or absence symptoms experienced in the past week. Scoring Each item on the EPDS is scored from 0 to 3, with higher scores indicating more severe symptoms. The total score can range from 0 to 30. Categorization of Scores 0-9: Low risk of postpartum depression. Indicates minimal or no depressive symptoms. 10-12: Moderate risk of postpartum depression. Suggests the need for further evaluation and possibly monitoring. 13-30: High risk of postpartum depression. Indicates significant depressive symptoms and suggests the need for further assessment and intervention. All the 300 mothers will be assessed.
Time frame: weeks 6 and 24 weeks and 48 weeks after delivery
Maternal sleeping disorders as indicator of stress using Pittsburgh Sleep Quality Index (PSQI)
The PSQI has 7 components: * Subjective Sleep Quality: Self-rated sleep quality. * Sleep latency: Time taken to fall asleep. * Sleep Duration: Total hours of sleep per night. * Habitual Sleep Efficiency: Ratio of actual sleep time to time spent in bed. * Frequency of problems that disrupt sleep. * Use of sleeping medications to aid sleep. * Daytime Dysfunction: Impact of sleepiness or fatigue on daily activities. Scoring Each component is scored from 0 to 3, with higher scores indicating worse sleep quality. Total score ranges from 0 to 21. Interpretation 0-5: Good sleep quality 6-10: Moderate sleep problems 11-21: Severe sleep problems This assessment helps in identifying mothers who may require further evaluation or intervention for sleep-related issues. All the 300 mothers will be assessed.
Time frame: In pregnancy, weeks 6 and 24 months after delivery
Sleeping disorders assessment using Epworth Sleepiness Scale (ESS)
Maternal sleeping disorders will be screened using The ESS tool that measures daytime sleepiness and assesses mothers' propensity to fall asleep in different situations. It consists of eight scenarios where Mothers rate their likelihood of dozing off on a scale from 0 to 3. Each of the eight items is scored as follows: 0: Would never doze 1. Slight chance of dozing 2. Moderate chance of dozing 3. High chance of dozing All the eight scenarios are summed to give a total score ranging from 0 to 24. Interpretation 0-10: Normal daytime sleepiness 11-12: Mild excessive daytime sleepiness 13-15: Moderate excessive daytime sleepiness 16-24: Severe excessive daytime sleepiness This tool helps identifying mothers who may have sleep disorders or excessive daytime sleepiness that could impact their daily functioning, including caring for their babies. All the 300 mothers will be assessed.
Time frame: In pregnancy, weeks 6 and 24 months after delivery
Maternal assessment of life stressful events
The Life Stressful Events Questionnaire tool will be used to assess the impact of stressful life events (frequencies and severity) on the mothers. Categories of stressful events: Personal events: * Death of a loved one * Divorce or separation * Serious illness or injury * Job loss * Family Events: * Family member illness * Marital problems * Birth of a child * Changes in family structure Social Events: * Moving to a new location * Change in social status * Major life transitions (e.g retirement) Economic Events: * Financial difficulties * Significant changes in income or employment status Environmental Events: * Natural disasters * Major accidents Higher scores or frequency of reported stressful events are typically associated with increased levels of stress and potential negative health outcomes. Tool helps identify individuals who may benefit from further support or intervention based on their stress levels.
Time frame: In pregnancy, weeks 6 and 24 months after delivery
Maternal sleep quality measurements
Sleep quality will be measured as * Total sleep duration (time in hours from sleep onset to final waking) * Sleep onset latency (time in minutes from bedtime to the first 20-minute period of sleep) * Sleep efficiency (ratio of total sleep duration to total time spent in bed (hours) Classification into 3 groups by duration of sleeping; -Short sleep duration: Generally defined as less than 6 hours per night. Associated with increased risks for health issues, including fatigue, cognitive impairment, and mood disorders. -Adequate sleep duration: Defined as 7-9 hours per night. Considered optimal for good health, cognitive function, and overall well-being. -Long sleep duration: Defined as more than 9 hours per night. Should also be considered together with other factors such as sleep efficiency, sleep disturbances etc for a more comprehensive assessment. All the 300 mothers will be assessed.
Time frame: In pregnancy, weeks 6 and 24 months after delivery
Maternal empowerment status using SWPER tool
The empowerment status of women may influence how they care for their infants, ultimately affecting infant mortality and morbidity. Domains to be assessed; * Social independence * Decision making in house hold matters * Attitude towards violence A total of 14 items in the above SWPER 3 domains will be assessed. Standard cut points to categorize scores into low, medium, and high levels of empowerment will be used and correlated with infant health. All the 300 mothers will be assessed.
Time frame: In pregnancy, weeks 6 and 24 months after delivery
Women's marital satisfaction status using Kansas tool
Marital satisfaction status of women may influence how they care for their infants, ultimately affecting infant mortality and morbidity. The main question; 'How satisfied are you with your marriage?' Two more related questions are also asked. The responses to all the 3 questions are a seven-point Likert scale (1 = 'Extremely dissatisfied'; 2 = 'Very dissatisfied'; 3 = 'Somewhat dissatisfied'; 4 = 'Mixed'; 5 = 'Somewhat satisfied'; 6 = 'Very satisfied'; 7 = 'Extremely satisfied'). Score will range from 3 to 21, with a higher score indicating greater marital satisfaction and will be correlated with infant health. All the 300 mothers will be assessed.
Time frame: In pregnancy, weeks 6 and 24 months after delivery
Concentrations of infant gut intestinal inflammation
Infant stool will be quantified using ELISA or multiplex immunoassay for the following platforms: * Fecal calprotectin (intestinal inflammation) Comparison between infants born to mothers with different nutritional statuses (MUAC ≤23 cm vs. 25-35 cm) and exposed to either improved or poor WASH environments during pregnancy. These comparisons aim to characterize the biological signatures of environmental enteric dysfunction (EED), dysbiosis, and malnutrition, in the 4 groups. Unit of mesurement: Fecal calprotectin: µg/g stool•
Time frame: Stool samples at 12, 24, and 36 months of age
Concentrations of infant intestinal permeability
Infant stool will be quantified using ELISA or multiplex immunoassay for the faecal α1-antitrypsin and albumin. Biomarker concentrations will be compared between infants born to mothers with different nutritional statuses (MUAC ≤23 cm vs. 25-35 cm) and exposed to either improved or poor WASH environments during pregnancy. These comparisons aim to characterize the biological signatures of environmental enteric dysfunction (EED), dysbiosis, and malnutrition, in the 4 groups. Unit of measurements: Fecal α1-antitrypsin, albumin: mg/dL or µg/g.
Time frame: Stool samples at 12, 24, and 36 months of age •
Infant fecal pH and fecal fat content
Infant stool will be assessed for biomarkers of malabsorption and digestive efficiency. The van de Kamer test and/or non extractive method of nuclear magnetic resonance or infrared spectroscopy will be used measure fat in dried stool samples. A nitrazine paper strip will be used to measure pH of the stool samples Comparison between infants born to mothers with different nutritional statuses (MUAC ≤23 cm vs. 25-35 cm) and exposed to either improved or poor WASH environments during pregnancy. These comparisons aim to characterize the biological signatures of environmental enteric dysfunction (EED), dysbiosis, and malnutrition, in the 4 groups. Units of measurement; Fecal pH: pH units (1-7 scale) Fecal fat: % of stool mass.
Time frame: Stool samples at 12, 24, and 36 months of age •
Infant systemic endotoxemia from gut leakage
Infant serum lipopolysaccharide (LPS) will be measured using ELISA or multiplex immunoassay. Biomarker concentrations will be compared between infants born to mothers with different nutritional statuses (MUAC ≤23 cm vs. 25-35 cm) and exposed to either improved or poor WASH environments during pregnancy. These comparisons aim to characterize the biological signatures of environmental enteric dysfunction (EED), dysbiosis, and malnutrition, in the 4 groups. Unit of measurement; Serum LPS: ng/mL
Time frame: Stool samples at 12, 24, and 36 months of age •
Percentages of circulating T and B lymphocyte subsets and natural killer
PBMCs will be isolated from peripheral blood samples of infants at 6, 12, 24, and 36 months of age. Multicolor flow cytometry will be used to quantify the percentages of : * CD4⁺ and CD8⁺ T lymphocytes * Regulatory T cells (CD4⁺CD25⁺FOXP3⁺) * CD19⁺ B cells * CD56⁺ natural killer (NK) cells Comparisons will be stratified by maternal nutritional status (MUAC ≤23 cm vs. MUAC 25-35 cm) and household water, sanitation, and hygiene (WASH) exposure (better vs. poor WASH conditions). Arm/Group title: Children will be assigned to 4 groups defined by maternal nutritional status (MUAC ≤23 cm vs. 25-35 cm) and household WASH conditions (improved vs. poor) at the time of pregnancy. Overall number of participants: 300 children
Time frame: Infant blood samples collected at 12, 24, and 36 months of age. Expected measures/ Unit of measures: Percentage (%) of total PBMCs per cell subset (e.g., % CD4⁺ T cells, % CD56⁺ NK cells)
Metabolic dysregulation and vaccine immune responses in infants presenting with malnutrition
Investigators hypothesise that there is presence of metabolic dysregulation in malnourished infants further exacerbating this condition. Peripheral blood mononuclear cells of malnourished babies, including their matched normo-nourished counterparts will analysed using flow cytometry to characterise natural killer (NK) cells important in early life immunity and measure fatty acid uptake (BODIPY FL C16), glucose uptake and mitochondrial mass \[MitoTracker Green (MTG)\]. Granzyme B and perforin levels will be correlated with mean fluorescence intensity (MFI) MTG of NKG2A+/-CD56 dim and bright NK cells. All malnourished babies and theirs controls will be assessed, these results will be correlated with humoural standard EPI vaccine immune responses (titres).
Time frame: weeks 48, 96 and 144 old.
Short and long term relationship between presence and frequency of early life diarrhea episodes and infant malnutrition, neurodevelopment and mental health
The prevalence and incidence of diarrhea, including recovery cycles will be determined in infants born of malnourished /normonourished women in pregnancy living relatively better/poor WASH conditions. Diarrhea episodes will be defined as any episode of acute diarrhoea (≥3 passages of loose stool for 24 hours as reported by the mother) occurring before the next study visit In stool samples we will assess intestinal pathogens (viruses, parasites, bacterial pathogens). Plating of stool samples on McConkey agars and Enterobacteria will be further characterized using standard and advanced microbiology tools including full-length sequencing if possible. Intestinal helminthes ova will be assessed using Kato Katz smear of 41.7mg stool and calculate the number of egg count per 1gram stool.
Time frame: From birth and followed up to 3 years of age
Intestinal leakage of severely malnourished babies
In all severely malnourished 50% of matched infants without malnutrition, a lactulose/ rhamnose permeability test will be done. Weight-for-length/height below -3 standard deviations from the median with bilateral pitting edema and mid-upper arm circumference (MUAC) measurement below 110 mm in children aged 12-36, according to the World Health Organization definition of severe malnourished. Following drinking the sweet solution the urine will be periodically collected by the Study Gastroenterologist, and samples will stored appropriately. Mass spectrometry assessments of urine will be done in Switzerland.
Time frame: From 1 years old up until 3 years of age
Intestinal calory extraction in severely malnourished babies
In a subset of randomly chosen infants (20 with malnutrition and 20 matched infants without malnutrition), total calory extraction will be assessed longitudinally. Following consuming a specified diet, where the weight of the food will be accurately measured mothers will collect total infant stool for 24 hours on two subsequent days, using specific diapers. Analysis of remaining calories in infant stool samples will be done in Bern, Switzerland where a bomb calorimetry is available.
Time frame: Weeks 48, 96 and 144 old regardless of whether or not the babies recover from malnutrition.
Colonisation patterns of Clostridioides difficile in Zimbabwe and Switzerland maternal-infant dyads
To compare the differential impact of infant intestinal Clostridioides difficile colonization in Zimbabwe and Switzerland maternal-infant dyads.
Time frame: From birth and followed up every 6 months up until 3 years of age
Intestinal inflammation assessment via endoscopy
All infants with severe malnutrition and 50% randomly selected infants with moderate malnutrition will be offered endoscopy. Gastroscopy/rectosigmoidoscopy with general anesthesia will be done by the study Gastroenterologist. During endoscopy, biopsies will be acquired for histopathological assessment by the histopathologist, as well as assessment of inflammation and the intestinal microbiota.
Time frame: Any affected babies from one year of age
Concentrations of anti-Gal natural antibodies and memory B cell frequencies in infant blood and stool across early life
Anti-Gal natural antibody levels and memory B cell responses will be characterized in infant serum, stool, and peripheral blood mononuclear cells (PBMCs) over the first three years of life. Anti-Gal IgG, IgA, and IgM concentrations will be quantified using ELISA in both serum and stool. Additionally, memory B cells specific for the α-Gal epitope will be identified and quantified by B cell ELISpot and/or flow cytometry with α-gal-conjugated tetramers or labeled glycoconjugates. This outcome will assess the ontogeny and maturation of the anti-Gal antibody response and its relationship with microbial colonization, nutritional status, and WASH exposure. Functional immune memory will be evaluated in the context of microbial antigen exposure and early-life health outcomes. Arm/Group title: Children will be assigned to one of the four groups defined by maternal nutritional status (MUAC ≤23 cm vs. 25-35 cm) and household WASH conditions (improved vs. poor) at the time of pregnancy.
Time frame: Infant blood, serum, and stool samples at 3, 6, 12, 24, and 36 months of age.• Serum anti-Gal IgG, IgA, IgM: µg/mL• Stool anti-Gal IgA, IgM: µg/g• Memory B cell frequency specific to α-gal: % of total B cells or spot-forming units (SFU) per 10⁶ PBMCs
Serum concentrations of anti-Gal antibodies
Concentrations of anti-Gal IgG, IgA, and IgM will be quantified in infant serum using ELISA to characterize the ontogeny of the anti-Gal response and its relationship with microbial colonization, nutritional status, and WASH exposure. These measurements will provide insight into the systemic development of natural antibody repertoires across early life. Unit of Measure: µg/mL
Time frame: Infant serum samples at 3, 6, 12, 24, and 36 months of age.
Stool concentrations of anti-Gal antibodies
Concentrations of anti-Gal IgA and IgM will be quantified in infant stool using ELISA to assess mucosal anti-Gal antibody responses. These data will reveal how gut-associated natural antibodies develop in relation to early microbial colonization and nutritional status. Unit of Measure: µg/g
Time frame: Infant serum samples at 3, 6, 12, 24, and 36 months of age.
Frequency of anti-Gal-specific memory B cells
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Frequencies of memory B cells specific to the α-Gal epitope will be quantified in infant PBMCs using B cell ELISpot and/or flow cytometry with α-Gal-conjugated tetramers or labeled glycoconjugates. This measure captures the functional development of immune memory directed against microbial glycans in early life. Unit of Measure: % of total B cells or spot-forming units (SFU) per 10⁶ PBMCs
Time frame: Infant PBMCs at 12, 24, and 36 months of age.
Correlation of neonate mortality/morbidity and the presence of group B streptococcus (GBS)
Maternal under-nutrition is generally associated low birth weight which at least in murine models increases the shedding of GBS faecal load. PCR on neonate stool samples using relevant primers. Comparison GBS shedding in neonates born to malnourished women in pregnancy vs those born to their normo-nourished peers Unit of measurement: Copies GBS/gram of stool
Time frame: Neonates from pregnancy up to 6 weeks of age
Levels of environmental toxins, agrochemicals, microplastics, antibiotics, antiretrovirals in drinking water and soils (heavy metals)
To determine the levels of environmental toxins, agrochemicals, microplastics, antibiotics, antiretrovirals in drinking water and soils (heavy metals) and mycotoxins in foods (fermented cow milk), their impact on infants' health and development
Time frame: 5 years (1.5 years of recruitment plus about 3 years of follow up after birth.
Maternal breast milk including including urinary versus fermented cow milk aflatoxin levels
Aflatoxin B1 (AFB1) and fumonisin B1 (FB1) are toxic secondary products of fungi that frequently contaminate staple crops in resource-limited settings. Urinary aflatoxin M1 (AFM1) and FB1, biomarkers of AFB1 and FB1 exposure, respectively, will be quantified in random antenatal urine via ELISA/Luminex and correlated with adverse birth outcomes and infant growth. AFB1and ochratoxin A will be tested in breastmilk around delivery and once a year for 2 years/ until weaning in all the 300 women, 4 groups. Unit of measurements ng/mL. Aflatoxin levels will also be determined in fermented cow milk sampled during the same period.
Time frame: From pregnancy and 24 months after delivery (post-lactation)