Early Life Malnutrition, Environmental Enteric Dysfunction and Microbiome Trajectories

Early Life Diarrhoea Episode(s), Malnutrition, Environmental Enteric Dysfunction and Microbiome Trajectories From Birth Until 3 Years of Life ; The University of Zimbabwe Birth Cohort Study-2 (UZBCS-2)

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.

Inclusion Criteria for Cases:

• MUAC ≤23 cm in pregnancy
• ≥18 years' old
• At least 20 weeks' gestational age
• Height ≥150 cm
• Planning to be staying in the study area for the next 3 years
• Willing to participate and comply with all study requirements and procedures.

Any pregnant woman meeting the above eligible criteria regardless of HIV status who meet the above inclusion criteria will be invited to participate in the study

Inclusion criteria for Controls

• Age, HIV status, gestational age at enrolment, and area residence matched normo-nourished peers with MUAC ≥25 - ≤35 cm
• Haemoglobin level of ≥11g/dL
• ≥18 years' old
• At least 20 weeks' gestational age
• Height ≥150 cm
• Planning to stay in the study area for the next 3 years

Exclusion Criteria

• Acute or chronic conditions in mothers interfering with the study according to the judgment of the investigator (HIV infection is not an exclusion criterion)
• Presence of severe mental health disorders interfering with study procedures according to the judgment of the investigator.