Trial design
This is a multiarm community-based randomized controlled, open-label, assessor-blinded superiority trial with a treatment allocation ratio of 1:1:1. A multiarm trial was selected to determine the incremental impact of BEP to the mother, along with oral azithromycin to the infants, on length velocity.
Study setting and study population
The trial is being conducted in the peri-urban communities of Karachi, Pakistan. These are impoverished coastal slums with a population of approximately 250,000 residents, according to the census conducted in 2017. The annual birth cohort is approximately 5,000 each year. The population is multiethnic and includes Sindhi, Pashtun, Punjabi, Bengali, and Urdu-speaking individuals. Through a demographic surveillance system (DSS), bimonthly visits are made to married women of reproductive ages (13-49 years) in the catchment area. During these visits, mortality, pregnancy, in- and outmigration and the current number of children under 5 are recorded. According to previous studies, there is a high prevalence of low birth weight (30%), stunting (52%) and wasting (18%) in the study areas. The study population includes only lactating women of reproductive ages who have recently delivered and their infants.
Participant eligibility criteria
Lactating women between 13 and 49 years of age and their newborns will be enrolled if they fulfill the inclusion and exclusion criteria provided in Table 1. The mid-upper arm circumference (MUAC) is measured routinely during the surveillance rounds, and the women with a MUAC of less than 23 cm in the first week of delivery are screened for eligibility by a research team. For eligible participants, written informed consent is obtained in the local language.
Table 1 Eligibility Criteria
Inclusion
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Exclusion
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Lactating women with a mid-Upper-arm-circumference of < 23.0 cm
Infants with a live birth outcome, captured within 168 hours
Individuals with the intention to stay in the catchment area for entire duration of trial after enrollment
Individuals with the intention to exclusively breastfeed child for at least 6 months of age
Individuals who have provided voluntary written informed consent
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Newborns with a birth weight of less than 1500 grams
Newborns with a known congenital anomaly or other severe illness based on the study physician’s assessment before enrollment.
Lactating women with known allergies to peanut, lentils, chickpea or dairy products
Individuals who were previously enrolled in the trial
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Sample size
There are limited data available on the impact of BEP on length velocity over the first 6 months of life in infants. However, in one of the studies, the mothers received only perinatal supplement, and the overall increase in length (cm/month) was b=3.289 among the LNS group and b= 3.346 among the MMN group. (18) However, the women in this trial received supplements with smaller doses/energy than the women in our study will receive, and the intervention time was also different, i.e., the perinatal period rather than the infancy period. Another study showed that the difference in length velocity (cm/month) was 0.02 between the MMN and IFA groups over the period of 0-18 months. (19) Therefore, in the absence of clear evidence on the impact of these interventions, we hypothesized the effect size to be 0.12 cm/month. This is also based on learning through field experience; when severely malnourished lactating women (MUAC < 19.0 cm) were provided with chickpea-based ready-to-eat supplements (100 gm/day for 3 months), the difference was 0.06 cm/month at 3 months of age between these women and women who did not receive any supplements (unpublished data that was used purely for implementation and was not included in a study).
The null hypothesis for this trial is that the mean difference in length velocity between an infant of a lactating woman receiving standard breastfeeding counseling with BEP alone (Intervention arm 1) for 6 months or in combination with a single prophylactic dose of azithromycin at 42 days of age (Intervention arm 2) and an infant of a lactating woman receiving standard breastfeeding counseling alone (control arm) is less than 0.12 cm/month (primary outcome). Due to the absence of evidence of such interventions on length velocity, we based this hypothesis on our local experience and field data from our field sites. The alternate hypothesis for this trial is that an infant of a lactating woman receiving BEP alone for 6 months or in combination with a single prophylactic dose of azithromycin at 42 days of age has a mean length velocity that is 0.12 cm/month or more higher than that of an infant of a lactating woman not receiving an intervention. The sample size takes multiple comparisons into account and is based on the primary outcome of length velocity with an effect size of at least 0.12 cm per month between the arms, a 1-sided test, and an alpha of 0.025 to account for multiple comparisons (the lower alpha). A drop-out rate of 14% (i.e., 10% loss to follow up and infant mortality rate of 4%) is assumed in the study, so the minimum total sample size required is 957 (319 lactating women in each arm).
Recruitment
Because married women are included in the surveillance system established by Department of Pediatrics and Child Health at Aga University, VITAL has access to a list of all pregnant women in the catchment area. Using this list, the research team will visit these households, connect with the pregnant women, and leave our contact number so that they can notify us of birth-related events. During each pregnancy touchpoint, the research team will provide standard antenatal and nutrition counseling to each pregnant woman and encourage them to seek proper care. At the time of birth, the randomization/enrollment team will receive a birth notification so that the team can visit the household for an eligibility assessment.
Informed consent procedure
For the eligible participants, written informed consent will be obtained by the same team in a local language (mostly Urdu and where required, in the languages Sindhi and Pashto). The team members will explain the details of the trial, including the purpose, follow-up procedures, specimen collections, and other related processes. If a participant is eligible and agrees to undergo the procedure as explained, the research team will give the consent form to the participant or decision maker, of if they cannot read it, a team member will read it word-by-word for them in Urdu or the local language. The participants will be allowed to ask any questions related to the consent form and trial procedure. If the participant/decision maker requires additional time to make more informed decisions, the team will also allow this opportunity and wait until the final voluntary decision is made. If the participant voluntarily agrees to participate, the participant will sign the consent form in the presence of a witness; either the form is duly signed, or a thumb impression is provided by participant and the witness. The ethics committee approved the use of a thumb impression by the participant and witness if they cannot read or write. Only the designated study team will be involved in obtaining written informed consent. A copy of informed consent will be provided to all participants and attached in the file with the study ID. Additional analyses are planned, which may involve sending samples and data abroad. The consent document covers all the aspects of these procedures, and the participants have an opportunity to opt out from biobanking or from participating in future research at any point of the trial. Nevertheless, any future secondary analysis will require institutional and national ethics committee approval.
Randomization and allocation concealment
After written informed consent is obtained, randomization will be performed by the team. Stratified block randomization with blocks of sizes 3, 6, and 9 will be used. Sequence generation will be performed by an independent statistician using a random selection method before the beginning of the trial. Self-adhesive, precoded sticky labels with unique identification numbers will be applied to sealed opaque envelopes containing the coded randomization identification number and intervention name to ensure that the randomization process and allocation are blinded. Baseline information regarding nutrition and exclusive breastfeeding will be recorded. Anthropometry measurements of both the mother and newborn will be performed, and follow-up procedures will be explained. Figure 1 shows the trial processes in detail.
Blinding
The outcome assessors will be blinded, responsible only for the anthropometry measurements, and will be assigned a schedule that does not overlap with those of the follow-up teams. All investigators will also be blinded to group allocation throughout the period of the study. Furthermore, a statistician will independently perform the interim analysis for the Data safety and monitoring board (DSMB) blinded by arm. Furthermore, the data analyst who will perform the final analysis will be blinded, and the code will eventually be revealed after the blinded results are shared with DSMB and investigators in a final review meeting.
Interventions
In the control arm, the lactating women will receive standard nutritional counseling and promotional messages of exclusive breastfeeding as well as standard-of-care by a trained research team. In ‘intervention arm 1’, the lactating women receive, in addition to the above, 2 sachets of BEP supplements per day until the infant reaches 6 months of age, and the sachets will be distributed by a trained research team at enrollment and each follow-up visit. BEP is a certified product of the World Food Program and is locally produced by Ismail Industries in Karachi. The manufacturers do not and will not play a role in any part of the study. Each sachet contains a caloric value of 400 kilocalories per 75 grams and approximately 10.5 grams of protein. The sources of protein are mainly chickpea, peanuts, lentils, legumes, and skimmed milk. In the ‘intervention arm 2’, the lactating women receive standard nutritional counseling and promotional messages of exclusive breastfeeding along with BEP supplements (same as intervention arm 1), but in addition, their infants will also receive a single prophylactic dose of azithromycin oral suspension, 20 mg/kg, at day 42 of life (window period of an additional 7 days). All participants in the arms will receive routine care, including newborn care, immunization, counseling regarding newborn and infant care at home and timely referral to health facilities in cases of urgent need. All other nonstudy treatments, such as medications, formula milk for infants, etc. will be recorded at each follow-up. In the case of any severe illness or adverse event (reported or observed), the intervention may be paused or stopped for a limited period after the investigators and the Data Safety and Monitoring Board are consulted.
Data collection and data management
Case report forms (CRFs) are designed to capture details on screening, eligibility, randomization, household demography, newborn assessment, danger signs, severe adverse events, compliance with interventions, exclusive breastfeeding, 24-hour food recall (to estimate usual intake as well as diversity on a monthly basis) and anthropometry. The data will be collected on tablets with built-in logic checks and skip patterns by a trained team and updated on secure servers in real-time using digital applications, which are designed and built in-house. Auto alerts will be used to remind the participants about the follow-up schedule, and the data will be tracked according to key indicators. All the data will be collected in a real-time manner and uploaded on a cloud server, which is password protected and only accessible to the trial data management team and manager. Participant confidentiality will be maintained through a unique ID system, and the participant identification information will not expose to anyone outside the trial team. The tablets that will be used will be password protected and only accessible to the study team. Deidentified data will be used for analysis purposes.
Follow-ups
Follow-up teams will perform the home visits to provide counseling for all arms. BEP will also be provided (intervention arm 1), and compliance will be measured by logging the number of empty sachets since the last visit. Azithromycin 20 mg/kg stat oral dose (intervention arm 2) will be given to infants at day 42 of life. Compliance with exclusive breast feeding will be assessed for all arms since the last visit. Follow-ups will be performed daily for the first 15 days on alternate days for 2 weeks, every 72 hours for two weeks and finally, weekly until the child reaches the age of 6 months. Monthly 24-hour food recall data will also be collected. At each visit, counseling will be provided to the participants to reinforce adherence to the BEP and exclusive breast feeding protocols. For participants who move out of the catchment area, the team has developed a system to follow them at the new location, if possible. There is no plan of retention for these participants once their 6-month follow-ups are completed. However, through our existing free-of-cost primary health care facilities, standard of care is available to all participants, even after the completion of the trial. After enrollment (within 168 hours of birth), the total follow-up duration of each participant in the trial will be 6 months. Figure 2 shows the schedule of enrollment, interventions, and assessments.
Anthropometry
The teams will be trained to perform anthropometry measurements for the infants and mothers by a master trainer using the INTERGROWTH-21st standards, and they will attend monthly refreshers. The measurements will include the infant length, weight, MUAC and head circumference and the maternal MUAC and weight. Two team members will measure the infant and mother, and the team members will be blinded to the other member’s measurements; the data will be entered in digitally in the system, which will calculate the average value automatically. For the infant weight, we will use the Laica weighing scale model PS3001, whereas the SECA adult weighing scale model 874 will be used to assess the maternal weight. We imported MUAC tape from UNICEF. The SECA scale, model 417, and SECA scale, model 213, will be used to measure the infant length and maternal height, respectively. The allowable difference between the two measures according to the study standard procedure is ± 0.5 cm for maternal MUAC, ±0.2 kg for maternal weight, ±0.5 cm for maternal height, ±20 gm for infant weight, and ±0.4 cm for infant length, infant MUAC and infant head circumference. To record growth longitudinally, the same anthropometry measurements will be taken at days 27, 56, 85, 114, 143 and 179 of the infant’s life.
Primary outcome
The primary outcome of interest will be the length-velocity (cm/month) at 6 months. This will be defined as the mean difference in length velocity measured at birth (or baseline) and at 6 months of age, expressed as the change in cm/month.
Secondary outcomes
The other outcomes of interest will be weight velocity (gm/kg/day), length-for-age z-score (LAZ), weight-for-age z-score (WAZ), and weight-for-length z-score (WLZ). Weight velocity (gm/kg/day) will be defined as the mean difference in weight velocity measured at birth (or baseline) and at 6 months of age, expressed as the change in gm/kg/day. Furthermore, the mean differences in the specific z-score indicators (LAZ, WAZ and WLZ) measured at birth (or baseline) and at 6 months of age will be assessed. Furthermore, the anthropometry measurements of the mother will also be assessed, i.e. height (cm), weight (kg), and MUAC (cm), and the mean change in each of these indicators between different arms will be assessed.
Biomarker assessment (other secondary outcomes)
All laboratory specimens from the mothers and infants will be collected on days 40 and 56 of the infant’s life, i.e., two different time-points, which were selected based on the administration of oral azithromycin to the infants in intervention 2 at day 42. Therefore, the first time point (day 40) of specimen collection will be the before the azithromycin dose, and the second time point (day 56) will be at 14 days after the azithromycin dose. A window period of an additional 7 days will be allowed for specimen collection, depending on the delay, if any, in the administration of the azithromycin dose to the infant. To standardize the arms, the same time points for specimen collection will be used for all the arms. Furthermore, to complete the infant-mother dyad, the same mother’s specimens will also be collected at the same time points. in the differences in the mean values (all continuous variables) of the specimens collected at the second time point (day 56) are of primary interest, and comparisons will be made with the values of specimens collected at the first time point, i.e., the baseline measurement. Further, comparisons will be made within the arm (second time point compared to baseline) between the arms as well as the mother-infant dyad. Furthermore, we will also calculate the interquartile ranges for similar comparisons as mentioned above.
For the laboratory procedures, blood samples will be collected from all infants whose parents/caregivers have provided consent for hemoglobin (gm/dl), ferritin (ng/ml), transferrin (mg/dl), C-reactive protein (CRP) (mg/l), and alpha1-acid glycoprotein (AGP) (mg/ml) tests. The rationale for assessing the levels of hemoglobin, ferritin and transferrin is to determine whether there are any differences in the markers for iron deficiency anemia in the infants across arms. Hemoglobin assessment will be performed with Hemacue equipment, while ferritin and transferrin will be assessed using an immune-turbidimetric assay with a Roche Cobas c-311 automated clinical chemistry analyzer. AGP and CRP will be analyzed using the same assay and equipment to observe differences in these inflammatory biomarkers among the three arms. To complement and link the findings of the infant biomarkers with the maternal intervention, 50 lactating women from each arm will also be approached at the same time point to collect blood specimens for the same biomarkers for the mother-infant dyad analysis. These assays will be performed at the Nutrition Research Laboratory (NRL) at Aga Khan University. Using the same subsample dyad, we are also planning to perform plasma proteomics to assess the element of antibiotic resistance.
Furthermore, breast milk from the same lactating women will be collected to assess the quality of breast milk composition (macro- and micronutrients), human milk oligosaccharides (HMOs), immunoglobulins and microbiome analysis. The analysis of breastmilk specimens will be performed in the Azad Lab at the University of Manitoba. A material transfer agreement (MTA) will be developed with the University of Manitoba for the shipment of the specimens.
We will also collect stool specimens at the same time points from the same women and their infants, i.e., 50 pairs per arm. We will assess inflammatory biomarkers in the stool, such as calprotectin (ug/gm), lipokalin-2 (pg/ml), and myeloperoxidase (MPO), using ELISA. Furthermore, the stool samples will also be analyzed to detect enteropathogens using the TaqMan Array Card (TAC) system for polymerase chain reaction (PCR), which will be performed in the Infectious Disease Research Lab (IDRL). Moreover, we will perform targeted Bifidobacterium identification using real-time PCR at IDRL. We will also send the stool samples to the University of Stanford after signing the MTA for additional metagenomic analyses.
The specimens included for further analysis and for future research (indefinite time) will be stored at the IDRL and NRL storage areas at Aga Khan University in -80-degree Celsius freezers. The samples will be deidentified with barcodes, specific IDs for different time points and mother-infant dyad information and color coded by the type of specimen as part of a unique identification system. All the ethical aspects pertaining to the storage of these samples have been approved by the Ethics Review Committee at Aga Khan University.
Monitoring and quality assurance
There will be a specific team from Aga Khan University with expertise in data management and trial implementation working with the investigators, and this team will be responsible for general trial processes, such as auditing the trial data and processes to ensure the completeness and accuracy of the protocols, and training the research staff and outcome assessors. Furthermore, there will be independent experts who will make visits for monitoring. A mechanism has also been developed for the research teams to report weekly progress on key progress indicators with the investigators at the VITAL Pakistan Trust and Aga Khan University. Furthermore, the astringent quality assurance mechanism was developed through 10% of the data being checked by trial supervisors and associates. All research teams have received Good Clinical Practice certifications. Comprehensive training and refreshers will be conducted on a routine basis. The anthropometric measurements will be standardized, and the team members will be trained by WHO-trained master trainers.
Data safety and monitoring board
The independent group of experts, comprising 5 members, constitute the Data safety and monitoring board (DSMB) for the trial and are responsible for monitoring safety indicators, adverse events, and the results of the interim analysis. The interim analysis (blinded by arm) is scheduled for when 50% of the enrolled participants complete the six-month follow-up. Only DSMB members will have access to the results of interim analysis, which will be shared by the independent statistician. Data on severe adverse events will be shared with the board on a monthly basis in the form of a progress report.
Participant safety
Close follow-up will be performed to ensure participant safety, and both the lactating women and the infants will be referred if needed to physicians at the primary health care clinic, with facilitated referral to tertiary hospitals when required. An independent DSMB will monitor the safety of the trial participants and provides trial oversight. Monthly reports on severe adverse events will be shared with the DSMB, and when a safety signal is observed, the DSMB may stop this trial prior to the completion of recruitment. The safety net, including facilitated referral and reporting, is believed to minimize the chances of harm to participants.
Possible risks
There may be diarrhea, nausea, vomiting, skin rash, or abdominal distension after the use of BEP. Similarly, the infants may develop diarrhea, nausea, vomiting, skin rash, and abdominal distension after the azithromycin dose. We will systematically collect information on all adverse events at each follow-up by asking about the history of any illness since the past visit and assessing signs of concern at each visit. If the mother and infant are considered to have any sign or symptom that is concerning, there is a referral mechanism in place. A 24/7 phone number is provided, and the participant can call this number for any kind of illness; immediate referral will be arranged. This information is well documented and recorded under adverse event reporting. For reporting purposes to the ethics committee and DSMB, we specifically divided ‘severe adverse events’ into two main categories. The first category is ‘fatal events”, which includes all fatal events, regardless of the underlying cause, occurring among participants (mother and infant) during the follow-up period, and the second category is ‘nonfatal events’, where study participants (mother and infant) require hospitalization or receive injectable therapy for any illness or diagnosis. Risk management includes prevention through rigorous follow-ups, continuous monitoring of danger signs, documentation and prompt referrals. Every illness or danger sign reported or identified is addressed through facilitated referral for both women and infants.
Statistical analysis
Statistical analysis will be performed by Stata, version 15. The baseline characteristics will be assessed by arm. The primary analysis will be intention-to-treat (ITT) analysis. We will compare the mean length velocity (cm/month) (primary outcome), weight velocity (gm/kg/day), LAZ, WLZ, and WAZ between the two intervention arms and the control arm using repeated measures ANOVA, with the model adjusted for the birth weight and age of the infants at enrollment. If an outcome is missing for the intention-to-treat infants, means from that group will be imputed. Subgroup analysis will be performed according to the maternal BMI and MUAC categories and compliance with the intervention.
Participants and public involvement
The investigators have extensive experience working with the community and their representatives/elders. During the protocol development phase, the team discussed and received feedback on the research question and trial design from the community representatives. Furthermore, community perspectives about the trial procedures, especially the frequency and duration of follow-up and biospecimen collection procedure, were also collected. Additionally, during the pilot phase, the aim was not only to test the consent and questionnaires but also to understand how the community responds to different questions and how sensitive information regarding the antenatal and postnatal periods can be collected in a receptive and profound manner.