Our earlier research has highlighted a high prevalence of anaemia, micronutrient deficiencies, and impaired infant growth among lactating women and infants in Sumedang district, West Java, Indonesia that were associated with large deficits in their micronutrient intakes. In response to these disturbing findings we have developed the micronutrient- enriched crackers (MEC) based on powdered eggshells and chicken liver, a readily available and affordable, local animal-source food. We will supply these MEC or a placebo, manufactured locally, weekly to a group of women in Sumedang district from 8-14 weeks gestation to 5 months post-partum in an effort to enrich the micronutrient content of the daily diets of the treatment group so they no longer have any dietary shortfalls in micronutrients. Our overall aim is to determine if MEC consumed daily by mothers for 6 months during pregnancy improves the birth length of their infants, and if daily consumption of MEC is continued daily to 5 months post-partum enhances infant linear growth compared to the placebo. This study is funded by the UK Wellcome Trust, the grant reference number is 216447/Z/19/Z.
A double-blind two-phase cluster randomized controlled trial (RCT) will be conducted in West Java, Indonesia to determine whether daily consumption of MEC from 8-14 weeks' gestation to delivery improves birth length compared to placebo (Phase 1) and whether daily consumption of MEC from 8-14 weeks gestation to 5 months post-partum improves attained linear growth of infants at age 5 months and linear growth velocity of infants from birth to age 5 months compared to placebo (Phase 2). A total of 324 pregnant women will be recruited through midwives/public health centre from 28 clusters (villages) in Sumedang district. Clusters will be randomly assigned to receive placebo or micronutrient- enriched crackers (MEC) daily, distributed by cadres from 8-14 weeks gestation to 5 months post-partum. Women who fulfil trial criteria will be invited to participate in the RCT. Placebo and MEC (based on powdered eggshells and chicken liver) will be manufactured locally according to Standard Operational Procedures (SOPs), so they are identical in size, color, and packaging, with a code known only by the local production manager. The code will not be shared until the primary outcome has been analysed statistically or if requested by the Ethics Committee and/or Data Safety Monitoring Board (DSMB).
Study Type
INTERVENTIONAL
Allocation
RANDOMIZED
Purpose
PREVENTION
Masking
TRIPLE
Enrollment
324
MECs are manufactured according to the Standard Operational Procedures which have been developed taking into account the Hazard Analysis and Critical Control Point (HACCP) to maintain quality and safety and ensure the "Ready-to-eat MEC products" are safe. Data on the micronutrient concentrations and any potential bacteriological (Salmonella sp is negative, Bacillus cereus, Enterobacteriaceae, and Coagulase positive streptococci level are considered safe) and chemical contaminants (Pb, Hg, Cd, As, and Sn are not detectable) in the MEC products have been obtained from analysis in the laboratory of Saraswanti Indo Genetech, which has been accredited by KAN LP-184-IDN with SNI ISO/IEC 17025: 2008. Laboratory analysis of MEC products will be repeated at the middle and the end of the RCT to check on the quality and safety of the MEC product.
Placebo crackers are manufactured according to SOPs which have been developed taking into account the HACCP to maintain quality and safety and ensure the "Ready-to-eat placebo products" are safe. Data on the micronutrient concentrations and any potential bacteriological (Salmonella sp is negative, Bacillus cereus, Enterobacteriaceae, and Coagulase positive streptococci level are considered safe) and chemical contaminants (Pb, Hg, Cd, As, and Sn are not detected) in the placebo have been obtained based on the analysis in the laboratory of Saraswanti Indo Genetech, which has been accredited by KAN LP-184-IDN with SNI ISO/IEC 17025: 2008. Laboratory analysis of placebo samples will be repeated at the middle and the end of the RCT to check on the quality and safety of the placebo product.
Tanjungsari, Pamulihan, and Sukasari Subdistrict
Sumedang, West Java, Indonesia
Birth length
All measurements will be taken to the nearest millimetre (mm) using standardized techniques and trained anthropometrists using a portable infantometer (SECA 417, measuring range: 10-100 cm). The measurements will be made on nude infants and will be recorded in duplicate, or triplicate if the difference between the first and second measurement is more than the recommended range (i.e., 7 mm).
Time frame: 24 hours after birth
Attained linear growth and growth velocity
All length measurement will be taken to the nearest millimetre (mm) using standardized techniques by trained anthropometrists using a portable infantometer (SECA 417, measuring range: 10-100 cm) The measurements will be made on nude infants and will be recorded in duplicate, or triplicate if the difference between the first and second measurement is more than the recommended range (i.e., 7 mm).
Time frame: 5 months (+/- 1 week) post partum
Birth weight
All weight measurements will be taken to the nearest 10 grams using standardized protocols by trained anthropometrists using an electronic scale (SECA 334). The measurements will be made on nude infants and will be recorded in duplicate, or triplicate if the difference between the first and second measurement is more than the recommended range (i.e., 100g).
Time frame: 24 hours after birth
Maternal haemoglobin level
Blood will be taken by a trained personnel and hemoglobin concentration (g/dL) in the mother will be analysed by complete blood count in a certified laboratory (Prodia Laboratory; Sysmex XN-1000).
Time frame: 35-36 gestational age and 5 months (+/- 1 week) post partum
Maternal food intake
Weighed records will be collected by the mothers on 3 non-consecutive days: 2 weekdays and 1 weekend day using a digital scale with precision of 4 grams (Kitchen Scale EK3131). On the next day after each weighed food record has been completed by the mother, an experienced research assistant will conduct a 24-hour recall on the mother using telephone/online communication. Results from the weighed record and recall will be combined to determine maternal food intake, which will be converted into nutrient intakes based on the weight and nutritional value of each food using the Indonesian Food Composition Table. The Multiple Source Method (MSM) program will be applied to estimate individual usual intakes of energy and nutrients as well as the usual intake distributions for the study population.
Time frame: 35-36 weeks gestational age, 2 months (+/- 1 week) and 5 months (+/- 1 week) post partum
Status of breastfed infants (exclusive vs. non-exclusive) and breast milk volume
The deuterium dose-to-the mother technique will be performed to measure the volume of breast milk consumed by the infants and the status of breastfed infants. As much as 30 grams of Deuterium Oxide (D2O) are taken by the mother orally, which is then excreted into the breast milk consumed by the infant. Saliva sampling will be carried out according to protocol guidelines established by the International Atomic Energy Agency (IAEA; Human Health Series No. 7) with some adjustments to the number of days of sampling. Maternal and infant saliva samples will be taken on the 0th day before the mother is given D2O dose, and subsequently post-dose on day 2 or 3, day 8 or 9, and on day 13 or 14. A total of 2 mL of maternal and 1 mL of infant saliva will be collected on each sampling day. Saliva will be analyzed using Fourier Transform Infrared Spectrometry (FTIR), from which exclusive breastfeeding categorization and breast milk volume will be calculated using the Microsoft Excel Spreadsheet.
Time frame: 5 months (+/- 1 week) post partum
Breast milk micronutrient concentration
Full breast milk samples will be collected by mothers from one breast (washed with distilled, deionised water) using acid washed breast milk pumps (Medela Harmony Manual Breast Pump) into acid washed glass bottles. Mothers will be requested to refrain from breastfeeding on one breast at least 2 hours before the scheduled breast milk collection to allow for the collection of fore- and hindmilk. Breast milk micronutrient concentrations, including Na, Mg, P, K, Ca, Fe, Cu, Zn, Se will be analysed using ICP- MS Agilent 7900; Free Thiamine, TMP, and TPP using HPLC-FLD Agilent 1200; Cobalamin using Competitive Chemiluminescent Enzyme Immunase IMMULITE 1000; Riboflavin, FAD, FMN, NAD, panthothenic acid, pyridoxal, pyridoxine, biotin using UPLC-MS with a Waters ACQUITY UPLC I-Class system coupled to a Sciex 4500 triple quadrupole mass spectrometer; Performed retinol, Alpha-carotene, Beta-carotene, Beta-cryptoxanthin, Alpha- tocopherol, and Gamma-tocopherol using HPLC Agilent 1260.
Time frame: 5 months (+/- 1 week) post partum
Infant breast milk micronutrient intake
Data generated from the volume of infant's breast milk intake using Deuterium Oxide Dose-to- Mother Technique and analyzed breast milk micronutrient concentrations will be used to calculate breast milk micronutrient intakes of the infants at 5 months post-partum.
Time frame: 5 months (+/- 1 week) post partum
Maternal micronutrient status
Data generated from maternal micronutrient concentrations in serum (Ferritin, Soluble Transferrin Receptor (sTfR), Retinol-Binding Protein (RBP), Zn, and Se) and adjusted for inflammation status, where appropriate, using C-reactive protein and Alpha-1-acid glycoprotein will be used to assess maternal micronutrient status. The cutoffs used to define deficiency for each maternal biomarker will be as follows: adjusted serum ferritin, \< 15 μg/L; adjusted sTfR, \> 8.3 mg/L; adjusted zinc \< 10.7 μmol/L; adjusted RBP \< 1.20 μmol/L; and adjusted selenium \< 0.82 μmol/L.
Time frame: 35-36 weeks gestational age and 5 months (+/- 1 week) post partum
Infant micronutrient status
Data generated from infant's serum micronutrient concentrations (Ferritin, sTfR, RBP, Zn, and Se) at 5 months post- partum, adjusted for inflammation status, where appropriate using C-reactive protein and Alpha-1-acid glycoprotein will be used to assess infant micronutrient status at 5 months post-partum. The cutoffs used to define deficiency for each biomarker among the infants will be as follows: adjusted ferritin, \< 12 μg/ L, adjusted sTfR, \> 8.3 mg/L; adjusted zinc \< 9.9 μmol/L; adjusted RBP \< 0.83 μmol/L (27); adjusted selenium \< 0.82 μmol/L; vitamin B12 \< 148 pmol/L.
Time frame: 5 months (+/- 1 week) post partum
Morbidity incidence rate of mothers and infant
The morbidity calendar for both mother and infant will be recorded by respondents independently and checked by research cadres once a week. The maternal morbidity calendar will be recorded over one month (for seizure, fever, headache, diarrhea, vaginal bleeding, cough, sore throat) at 14 weeks and 31-32 weeks of gestation, and 1 month and 4 months post-partum (for diarrhea, fever, cold, cough, vomit, ear infection, sore throat). The infant morbidity calendar will also be recorded over one month when the infant is 1 month and 4 months post-partum (for diarrhoea, fever, cold, cough, vomit, ear infection, sore throat). Digital thermometers will be provided to the respondents for self assessment of maternal and infant fever.
Time frame: 14 weeks and 31-32 weeks gestational age, 1 month (+/- 1 week) and 4 months (+/- 1 week) post partum
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