Iron and Prebiotics Fortification in Kenyan Infants
Condition: Anemia; Iron Deficiency; Diarrhea; Malaria; Respiratory Tract Infections (RTI); Antibiotics; Gut Inflammation; Prebiotics; Vaccine Response
MeSH terms: Respiratory Tract Infections; Iron Deficiencies; Inflammation; Diarrhea
Study type: Interventional

Summary: 
      Iron deficiency and anemia are health issues affecting mainly infants and women in developing
      countries. Iron deficiency in infancy can have long-lasting impact on cognitive and motor
      development of the child. Iron fortification has shown to be effective against anemia.
      However, in areas with a high burden of infectious diseases iron may increase the risk of
      unfavorable gut microbiota composition possibly influencing diarrhea prevalence. Therefore we
      want to assess the effects of home fortification of complementary food with two
      iron-containing micronutrient powders (MNPs) with and without the addition of a prebiotic
      (7.5 g of galactooligosaccharides as GOS-75) compared to a control on the composition of the
      gut microbiota of Kenyan infants. In addition, iron deficiency may iimpair adaptive immunity.
      Following Kenyan Minstry of Health guidelines, infants receive their first measles vaccine at
      9 months. In this study we will use an MNP with a moderate iron dose of 5 mg, with 2.5 mg of
      Fe as NaFeEDTA and 2.5 mg of Fe as ferrous fumarate (+Fe). There will be 3 study groups MNP,
      MNP+Fe and MNP+Fe+GOS. The infants will be enrolled in the study at the age of 6-10 months
      and will consume a home-fortified maize porridge for four months. At baseline and endpoint
      (after 4 months of intervention), we will collect blood samples of the infants in order to
      assess anemia, iron status, and inflammation. In addition, we will assess the effect of iron
      supplementation on measles vaccine response. Fecal samples (from child and mother) will be
      collected at baseline, 3 weeks and at endpoint in order to evaluate the changes in gut
      microbiota and gut inflammation.

      During the intervention, in a sub-group of children who receive broad-spectrum antibiotics,
      we will compare how the three different interventions modify the effect of antibiotics on the
      infant gut microbiota. We will opportunistically select children that are enrolled in the
      study and who become ill, and who are prescribed antibiotics by the local health care team,
      according to the local standard of care in the study area. Five additional stool samples from
      these children will be collected (day 0 (before the first antibiotic dose), 5, 10, 20 and 40)
      to evaluate the changes in the gut microbiota and gut inflammation.

      Three years after the study end, we would like to collect a blood and stool sample from the
      children and examine the iron status and gut microbiome respectively.
    

Detailed description: 
      Fifty infants per group will be enrolled (n=150) and randomly divided in three groups, from
      those identified in selected villages in the catchment area of the Kikoneni Health Clinic in
      southern coastal Kenya, about 2 hours south of Mombasa. All children whose caregivers agree
      to participate in the study will have their iron status assessed by measurement of hemoglobin
      (Hb), erythrocyte zinc protoporphyrin (ZPP), serum ferritin (SF) and serum transferrin
      receptor (TfR). Enrolled subjects will be randomized into three groups. Group 1 will receive
      the MNP alone, group 2 will receive an identical MNP with iron (2.5 mg Fe as NaFeEDTA and 2.5
      mg as ferrous fumarate), and group 3 will receive the MNP with iron (2.5 mg Fe as NaFeEDTA
      and 2.5 mg as ferrous fumarate) and 7.5 mg of galactooligosaccharides as GOS-75. Each
      household will receive 2 kg of maize meal per week and the caregivers of the participating
      children will be shown how to prepare a maize porridge, which is the most widely-used local
      complementary food. This maize meal will be provided free-of-charge to all participating
      families. The caregiver will add one MNP sachet each day to a portion of maize porridge, and
      feed the entire portion to the infant. The Kikoneni Clinic will serve as the collection point
      for the MNPs, the monitoring center for surveillance of infant health, as well as the blood
      and stool collection point.

      At baseline and at endpoint of the intervention (after 4 months), a blood sample will be
      collected from the infants for measurement of Hb, SF, TfR and ZPP to define anemia and iron
      status and C-reactive protein (CRP) to define systemic inflammation status. In addition,
      anti-measles Serum IgG will be measured, as infants receive their first measles vaccination
      at 9 months, following Kenyan Ministry of Health guidelines. At baseline, after 3 weeks and
      after 4 month, a stool sample will be collected from both the infant and his/her mother, for
      measurement of the gut microbiota and gut inflammation.The mothers will be trained to collect
      the stool samples at home in a provided container with a tight, screw-top lid, that includes
      an Anerocult® sachet to create an anaerobic environment. At these 3 timepoints (baseline,
      after 3 weeks and after 4 month) the mother will bring in the two stool samples of the same
      day, where they will be labeled and kept at 4°C. The samples will be then transferred to the
      central lab, filled in 2 ml Eppendorf tubes, labelled and frozen at -20°C.

      In addition, compliance and morbidity will be assessed weekly during the distribution of the
      MNPs. The children will be checked and referred to the health center clinicians whenever
      indicated by the clinical history. In the case of fever, a rapid malaria test (RTD) kit will
      be performed according to local guidelines. If the test results positive, the child will be
      treated for presumptive malaria, mild malaria cases will be treated at Kikoneni clinic as per
      WHO Integrated management of childhood illness (IMCI) guidelines. Cases of diarrhea will be
      treated according to the local standard of care including oral rehydration salt and, if
      necessary, antibiotics. If deemed necessary by the Kikoneni clinical management, the study
      team will support the clinic in re stocking ORS, iron supplements and antimalaric drugs for
      the study duration.

      Anthropometry (weight, height, age and sex) will be recorded at baseline and endpoint using
      standardized procedures to calculate the prevalence of child stunting. The measurements will
      be done twice and the average used for data analysis. The data analysis software WHO Anthro
      (WHO, Geneva Switzerland) will be used to calculate the prevalence of stunting among this
      infant population.

      To investigate the effect of oral antibiotics on the infant gut microbiota we will select the
      first 12 infants of each of the three study groups that become ill during the study and
      receive broad-spectrum antibiotic treatment. We will also select 6 non-treated controls from
      each study group matched for sex and weeks of MNP consumption. A total of n=54 children will
      be included in this sub study and we will collect five additional stool samples from the
      selected children to determine the gut microbiota composition. The first stool sample will be
      collected before starting the antibiotic treatment (day 0). Thereafter, a stool sample will
      be collected at day 5, 10, 20 and 40. Stool samples will be collected as described above.
      Besides these additional stool samples the children continue the intervention according to
      the main protocol.

      A breast milk sample will be collected from 90 mothers at two time points (week 3 and week
      16). These samples will be analyzed for the concentration of human milk oligosaccharides, a
      potential source of natural prebiotics.

      Three years after the end of the study, we would like to examine iron status and gut
      microbiota of these children and determine whether the differences observed after the
      intervention persist or if they all converge and have a similar microbiome profile. In
      addition, we will measure anti-measles serum IgG, as infants received their second measles
      vaccine at 18 months following Kenyan Ministry of Health guidelines.
    

Intervention name(s): Fortified maize porridge
Intervention type(s): Dietary Supplement
Intervention description(s): Maize porridge will be home-fortified with either A) MNP, B) MNP+Fe, C) MNP+Fe+GOS
Allocation: Randomized
Intervention model: Parallel Assignment
Masking: Quadruple (Participant, Care Provider, Investigator, Outcomes Assessor)
Primary purpose: Basic Science