Dietary diversity modification through school-based nutrition education among Bangladeshi adolescent girls: A cluster randomized controlled trial

Objective To measure the efficacy of school-based nutrition education on dietary diversity of the adolescent girls in Bangladesh. Methods A matched, pair-cluster randomized controlled trial was conducted from July 2019 to September 2020. Randomization was done to select intervention and control schools. There were 300 participants (150 in the intervention and 150 in the control arm) at baseline. We randomly selected our study participants (adolescent girls) from grades six, seven, and eight of each school. Our intervention components included parents’ meetings, eight nutrition education sessions, and the distribution of information, education, and communication materials. An hour-long nutrition education session was provided using audio-visual techniques in a class of intervention school once a week by trained staffs of icddr,b for two months. Data on dietary diversity, anthropometry, socio-economic and morbidity status, a complete menstrual history, and haemoglobin status of adolescent girls were collected at recruitment and after five months of intervention. We calculated the mean dietary diversity score of adolescent girls at baseline and at the endline. As the dietary diversity score was incomparable between the control and intervention arm at baseline, we performed the difference-in-difference analysis to assess the effect of the intervention. Results Mean age of the adolescent girls was 12.31 years and 12.49 years in the control and intervention arms respectively. Percentages of consumption of organ meat, vitamin A-rich fruits and vegetables, legumes, nuts, and seeds were higher in the intervention arm than in the control arm at the end-line. The mean dietary diversity score remained unchanged in the control arm at 5.55 (95% CI: 5.34–5.76) at baseline and 5.32 (95% CI: 5.11–5.54) at the endline. After the intervention, mean dietary diversity increased from 4.89 (95% CI: 4.67–5.10) at baseline to this mean was 5.66 (95% CI: 5.43–5.88) at the endline. Result from the difference-in-difference analysis revealed that the mean dietary diversity was likely to increase by 1 unit due to intervention. Conclusion The shorter duration of the intervention in our study could not show whether it could change the behavior of adolescent girls in increasing dietary diversity through school-based nutrition education, but it showed a pathway for increasing dietary diversity at school. We recommend including more clusters and other food environment elements in retesting to increase precision and acceptability. Trial registration This study was registered with ClinicalTrials.gov, trial registration no: NCT04116593. https://clinicaltrials.gov/ct2/show/NCT04116593.

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The above-mentioned project has been discussed and reviewed at the Division level.
Dr. Tahmeed  . However, nutrition module in the curriculum is not compulsory for all the students and 'Krishi Shikhya' that covers agriculture or horticulture is the elective subject for the adolescent boys. Although 'Home Economics' module includes a brief discussion of balanced diet it does not cover dietary diversity focusing on 16 food groups. Moreoevr, 'Home Economics' is also an elective subject; therefore, the likelihood of receiving the messages even on the balanced diet by all the adolescent girls in the schools is uncertain. Therefore, it is very likely that a large number of adolescents girls are not receving the messages on dietary diversity from schools. Except for an intervention based on behaviour change communication, the other interventions aimed at improving the nutritional status of the adolescent girls are expensive and may not be scalable or sustainable at the program level. Keeping the above mentioned context in mind, we aim to deliver school-based nutrition education and assess its impact on dietary diversity of girls who are in their early adolescence period.
Along with that, using a qualitative approach we also aim to identify the barriers that may influence the intake of diversified food among the adolescent girls.

Hypothesis:
We hypothesise that school-based nutrition education will increase dietary diversity among the adolescent girls. Objectives: The objective of this study is to measure the efficacy of school-based nutrition education on dietary diversity of the adolescent girls in Bangladesh. The study will also explore the barriers and facilitators that may influence the intake of diversified food.

Methods:
We propose a matched, pair-cluster randomized controlled trial to measure the efficacy of school-based nutrition education on dietary diversity of the adolescent girls in Bangladesh. Our study will have two arms (one intervention and one control arm). After screening, based on exclusion criteria, we will prepare two separate lists (one for urban and one for rural) of schools in Rangpur district. From each list, clusters (schools) will be paired based on monthly tution fees provided by the students (as a proxy indicator of socio-economic status of the students) and infrastructure of the schools. We will randomly select one pair from each list and within each pair one school will be assigned to intervention arm and another one will be assigned to control arm through randomization. Targeting an effect size of 20 percentage point reduction of inadequate dietary diversity, a minimum of 148 adolescent girls will be required for each arm. Considering this study as a superiority trial and an effect size of 10 percentage point we also estimated sample size using the formula for sample size estimation in superiority trial. It yields the equal sample size, 148 in each arm. Therefore, it will also represent the sample size calculated based on the effect size of 20 percentage point using the formula of sample size estimation for conventional cluster randomized control trial. Eleven to fifteen years old adolescent girls studying in grade six, seven and eight will be recruited from each school in Rangpur as this division has the highest prevalence of inadequate dietary diversity, which is 72% [6]. Regions prone to natural calamities and schools already receiving any kind of intervention from other programs or projects (nutritional education, health messages, mid-day meal etc) will be excluded. To ensure household level participation and support, caregivers will be invited to the school for a discussion at the beginning of the intervention. Nutritional education will be delivered using audio-visual techniques (audio-visual presentation) once in a week for each class, for 3 months. Individual (IDDS) and household dietary diversity scores (HDDS) will be used for measuring dietary diversity at individual and household level, respectively [15]. IDDS and HDDS data will be collected at recruitment, at the end of education intervention and again after 3 months of the completion of intervention. Following national guidelines, weekly iron-folic acid (IFA) supplementation will be provided to both intervention and control arm for 3 months. For identifying the barriers to and facilitators of intake of diversified food, a qualitative research will be conducted after the intervention. Adolescent girls having improved and girls showing no improvement in individual dietary diversity score will be recruited purposively for the qualitative assessment.
Outcome measures/variables: Primary outcome of the intervention trial will be individual level dietary diversity among the adolescent girls. As the secondary outcome of interest we will measure household dietary diversity of the adolescent girls, changes inanthropometry, and anaemia status. We hypothesise that school-based nutrition education will increase the dietary diversity among the adolescent girls.

Specific Objectives:
Describe the specific objectives of the proposed study. State the specific parameters, gender aspects, biological functions, rates, and processes that will be assessed by specific methods.
• To measure the efficacy of school-based nutrition education in improving dietary diversity among Bangladeshi adolescent girls • To explore the barriers and facilitators that may influence the intake of diversified food

Background of the Project including Preliminary Observations:
Provide scientific validity of the hypothesis based on background information of the proposed study and discuss previous works on the research topic, including information on sex, gender and diversity (ethnicity, SES) by citing specific references. Critically analyze available knowledge and discuss the questions and gaps in the knowledge that need to be filled to achieve the proposed aims. If there is no sufficient information on the subject, indicate the need to develop new knowledge. After first year, adolescence (10-19 years) is the second most critical period for physical growth [1]. During this period, adolescents gain up to 50% of their final adult weight, 15% of their final adult height [2] and 45% of the maximal skeletal mass [22]; of this entire growth, 80% is achieved during the early adolescence (10-15 years) [3].
There are some specific reasons for which adolescence is a unique intervention period in the life cycle [23]. Firstly, after adult height is achieved, the accumulation of significant amounts of additional bone mass is implausible [24]. Secondly, peak velocity of linear growth of adolescent girls take place approximately six to twelve months prior to menarche [24]. The current mean age at menarche is 12.8 years in Bangladesh [25]. Hence, any kind of health intervention will be very worthwhile if it is given during the early adolescence. This early adolescence is recognised as a nutritionally vulnerable period because of the sudden growth spurt during adolescence and its close association with cognitive, emotional and hormonal changes [1,2]. Hence, it is essential to ensure proper and adequate nutritional support during adolescence. In Bangladesh, nutritional status of adolescent girls is not satisfactory. Stunting among adolescence is 32% in India, 36% in Bangladesh, and 47% in Nepal and low BMI is 53% in India, 50% in Bangladesh, 36% in Nepal [26]. Study conducted among 12-19 years old post-menarcheal adolescent girls in rural Bangladesh found undernutrition is widespread in this population, with nearly half of the adolescents being stunted and more than 40% underweight [27]. Another study found that, a large number of adolescent girls suffer from various degrees of nutritional disorders [28,29].
Most hazardous impacts of adolescent malnutrition are reduced productivity, poor school performance and adverse reproductive outcomes [30]. A significant association was found between height of the mother and stunting status of the adolescent son or daughter [31]. Results from a multi-country study showed maternal height are a key determinant of childhood nutritional status [10]. Since maternal height cannot be increased, we have to go down the life-cycle and consider increasing height of adolescent girls at the population level. It would be the last opportunity to intervene and break the inter-generational vicious cycle of malnutrition [11,12].
Many studies conducted in both developed and developing countries revealed that nutrient adequacy is strongly associated with dietary diversity [32,33]. Diet containing diverse food items provides wide range of macro and micronutrients and enhances nutritional quality of diet [34]. In contrary, monotonous diets based on starchy staples lack essential nutrients and contribute to the burden of malnutrition [4,35]. Dietary diversity among women of Bangladesh is very low. At the national level, two-thirds (66%) of women consumed inadequately diversified diet [6]. One in every three women and adolescent girls of Bangladesh aged 10-49 years consumes diets with inadequate diversity which was found to be highest in Sylhet and Rangpur division (72%) [6]. Previous study showed a large proportion of the participants of that study consumed meat (62.5%), fish (53.8%) and eggs (58.4%) 3 to 4 times or less in the week preceding the interview. A substantial proportion of the girls did not take milk (46.1%) and liver (64.6%) at all in the week. About 27.7% did not take leafy vegetables; while substantial proportions of the participants had other vegetables (72.2%) 3 to 4 times or more in the week [36].
Lack of knowledge of selecting proper food items increases the burden of malnutrition among adolescent girls [36]. Health and nutrition knowledge and healthy habits of female adolescents have crucial roles in maintaining future family health and nutrition [13]. However, except for an intervention based on behaviour change communication (BCC), the other interventions are expensive and may not be scalable or sustainable at program level. Moreover, there is limited data regarding nutritional status of Bangladeshi adolescent girls who are at their early adolescence. There is no study which has ever assessed the role of nutritional education in improving dietary diversity of adolescent girls of Bangladesh. Keeping this context in mind, we aim to deliver school-based nutrition education and assess its impact on dietary diversity of girls who are in their early adolescent period.
Study conducted among participants more than or equal to eighteen years of age (55% women) in Switzerland revealed that among the factors of healthy eating, evidence points to food price (e.g., healthy foods are too expensive) [37,38], food taste (e.g., healthy foods lack taste) [38,39], time constrains (e.g., lack of time to prepare and cook heathy foods) [38,39] and lack of willingness [40]these factors are impediments to maintain healthy eating behaviour [41]. Another study conducted among the fifth-grade children and adult caregivers identified overarching facilitators of consuming several food groups included emphasizing health benefits increasing availability of and accessibility to foods, affordability and guidance on food preparation [42]. A study conducted in rural Bangladesh among 15-49 years old women identified numerous food security determinants including land tenure, use of vegetable gardens, income generation, women's empowerment, access of market for women, access to media and literacy [43]. However, in case of dietary diversity, they found only literacy to be associated with improved dietary diversity [43]. To our knowledge, there is no study which has ever identified these barriers and facilitators which might influence dietary diversity. Considering mentioned perspective, along with the aim to deliver nutrition education and assess its impact on dietary diversity, we also aim to identify the barriers and facilitators that may influence the intake of diversified food among adolescent girls.

Research Design and Methods
Describe the research design and methods and procedures to be used in achieving the specific aims of the research project. If applicable, mention the type of personal protective equipment (PPE), use of aerosol confinement, and the need for the use BSL2 or BSL3 laboratory for different part of the intended research in the methods.. Define the study population with inclusion and exclusion criteria, the sampling design, list the important outcome and exposure variables, describe the data collection methods/tools, and include any follow-up plans if applicable. Justify the scientific validity of the methodological approach (biomedical, social, gender, or environmental).
Also, discuss the limitations and difficulties of the proposed procedures and sufficiently justify the use of them.
We propose a matched, pair-cluster randomized controlled trial to measure the efficacy of school-based nutrition education in improving dietary diversity among Bangladeshi adolescent girls. After screeing, based on exclusion criteria (schools having any programme such as nutrition education, health message, mid-day meal), we will prepare two separate lists (one for urban and one for rural) of schools in Rangpur district. From each list, clusters (schools) will be paired based on monthly tution fees provided by the students (as a proxy indicator of socio-economic status of the students) and infrastructure of the schools. We will randomly select one pair from each list and within each pair one school will be assigned to intervention arm and another one will be assigned to control arm through randomization. Randomization will be done by computer-generated random numbers using STATA. Eleven to fifteen years old adolescent girls studying in grade six, seven and eight will be recruited from four selected schools (2 urban and 2 rural schools). Nutrition education will be given in intervention arm and information on water, sanitation and hygiene (WASH) and iron-folic acid will be provided in both the arms. Although we will provide intervention to all the students in each grade, we will select an equal number of gilrs from each grade to assess the outcomes of intervention. We will also conduct surveys at the households of those selected girls to capture data on household level dietary diversity, water, sanitation and hygiene, and socio-economic status. Three surveys will be conducted among the same adolescent girls and at their household level at three different time points-before rolling out of the intervention, after accompolishment of the intervention and after three months of completion of intervention. The required sample size for survey has been mentioned under the section of sample size calculation. At the end of intervention, in-depth interviewes with some purposively selected adolescent girls will be conducted to explore the barriers and facilitators which may influence the intake of diversified food among the adolescent girls.

Randomization of schools
Intervention Arm

Intervention component:
At the beginning of the intervention, we will arrange a parents' meeting at the schools. In parents' meeting, we will discuss about the objective of the study, overview of dietary diversity, health benefits of dietary diversity (DD) on adolescent health and parent's role in ensuring intake of diversified food of adolescent girls. An hour-long nutrition education session will be conducted to each class/grade of the schools under intervention arm. Eight such educational discussion sessions will be held in first 2 months and during month three the previous eight education sessions will be repeated. Nutrition education session will be delivered using audio-visual techniques (audio-visual presentation). In addition, we will arrange quizes to inspire them and to reduce monotony of the sessions. We will provide pamphlets for their remembrance. Components of eight educational sessions will include overview of 16 food groups of individual dietary diversity score (IDDS) chart, detail description of 16 food groups presenting dietary diversity, overview of basic food components, importance of consuming diversified foods, strategies to select a diversified meal and remove monotony in food, proper timing and frequency of taking meals during 24 hours, selecting diversified meal in low expense, consequences of absence or scarcity of dietary diversity in meal (Different food items under 16 food groups are given in Appendix-4).
Following national guideline, weekly iron-folic acid (IFA) supplementation will be provided to both intervention and control arm for 3 months. We will provide capsule Ferocit Z (provided by government of Bangladesh in different government medical college and healthcare facilities) as Iron-folic acid (IFA) supplementation. It contains ferrous sulphate -150Mg, folic acid -0.5Mg and zinc sulphate monohydrate -61.8Mg. Iron-folic acid (IFA) supplementation will be provided among the girls of each class/grade (six, seven and eight) of all selected schools regardless of intervention and control arm of the study. In the intervention arm, apart from nutrition education the adolescent girls will also receive messages of water, sanitation and hygiene (WASH). But in control arm, the girls will receive messages on WASH only (along with IFA). The meassage on WASH will include safe source of drinking water, improved toilet faciltity and how to maintain good sanitation and five critical times of handwashing.

Description of field site:
The study will be carried out in 2 urban and 2 rural schools of Rangpur district. Rangpur is situated in the northern part of Bangladesh. Rangpur has been selected as the study site because prevalence of inadequate dietary diversity among adolescent girls is higher (72%) in Rangpur [6]. In addition, Rangpur is one of the areas where adolescent girls with low wealth are food insecure and are more likely to have inadequate dietary diversity, particularly during the post-aus season [44]. Regions which are prone to natural calamities and schools already receiving any kind of interventions (nutritional education, health messages etc.) from other programs or projects will be excluded.

Selection of study participants:
Since the intervention will be given all adolescent girls, the following individual level inclusion and exclusion criteria are for evaluation of process and outcomes only.
a. Inclusion criteria: All of the following criteria will be met for a participant to be enrolled in the study-• Never married adolescent school girls studying in grade six, seven and eight (age range 11-15) at the selected schools • Household(s) belong to selected study participants • Girl(s) willing to give assent b. Exclusion criteria: Adolescent girls will be excluded if they have the following criteria- • Adolescent girls who are < 11 years and > 15 years • Presence of any kind of chronic disease among study participants • Presence of any major psychiatric illness

Enrolment, Screening and Consenting:
Screening and enrollment of the study participants will be done at the study site (selected urban and rural schools of Rangpur). Trained research staff of icddr,b will explain the study in detail, answer any question from the parents, school teachers and adolescent girls.
Since our intervention will be school-based nutrition education, we will obtain initial approval from the school authority and headmaster of the school. If the legal guardians (school headmasters) and adolescent girls are interested to volunteer in the study, the designated staffs will proceed to screening and consenting. Screening will be done based on the inclusion and exclusion criteria. If the adolescent girl is eligible to participate, the next step will be taking consent. As our study participants are <18 years, we will take assent from the adolescent girls and we will also take consent from the mother or caregiver of the adolescent girls before including them in this study. If the mother or caregiver of an adolescent girl refuses to give consent, we will not include that adolescent girl in our study. Prior to signing the consent form, they will have an opportunity to ask any question about the study.

Data collection:
Anthropometry data: Weight of the study participants will be measured by portable electronic weighting scale (TANITA Corporation Japan) and height will be measured by Seca stadiometer. Weight and height will be used for computing BMI and height-for-age z-score. BMI below 18.5 is referred to as underweight, 18.5-24.9 as normal range, 25.0-29.9 as overweight and 30.0 and above as obese [45]. Height-for-age z-score <-2SD will be considered as stunted [13].
Mid upper-arm circumference (MUAC) will be measured at the midpoint of the left upper arm (extended with the palm facing inwards) between the acromion process and the tip of the olecranon, using a plastic nonstretchable tape to the nearest millimetre [46].
Under supervision of research fellow (RF), trained field research assistants (FRA) will take all the measurements and trained health workers (HW) will help in keeping records. All the anthropometry will be measured at recruitment, at the end of the intervention and again after 3 months of the completion of intervention.

Socio-economic and morbidity data:
Socio-economic and morbidity data of adolescent school girls will be collected at recruitment, at the end of education intervention and again after 3 months of the completion of intervention by using pretested, semistructured questionnaire. General morbidity symptoms they have experienced in the previous one week will be recorded. The morbidity data will include fever, cough/cold, diarrhoea/dysentery, stomach ache, respiratory problems, ear and eye problems, skin problems and others. A complete menstrual history will be taken during morbidity related data collection.

Dietary diversity data:
Dietary diversity data will be collected at individual as well as at household level. Data will be collected using 24-hour recall dietary diversity questionnaire. Individual level data will be collected from the adolescent girls and household level data will be collected from the mothers or caregivers of the selected adolescent girls. Individual dietary diversity score (IDDS) and household dietary diversity score (HDDS) will be used for measuring dietary diversity at individual and household level respectively [15]. Our questionnaire regarding dietary diversity will include 16 food groups: cereals; white roots and tubers; vitamin A rich vegetables and tubers; dark green leafy vegetables; other vegetables; vitamin A rich fruits; other fruits; organ meat; flesh meats; eggs; fish and seafood; legumes; nuts and seeds; milk and milk products; oils and fats; sweets; spices, condiments and beverages [15]. The 16 food groups included in dietary diversity questionnaire will be aggregated into 12 food groups and 9 food groups to create household dietary diversity score (HDDS) and individual dietary diversity score (IDDS) respectively [15]. IDDS and HDDS data will be collected at recruitment, at the end of education intervention and again after 3 months of the completion of intervention.

Estimation of haemoglobin level:
Haemoglobin status will be measured from a finger prick blood sample using a HemoCue machine (Hb 301, HemoCue AB, Angelholm, Sweden). We will take the second drop of blood from the study participants as sample. Haemoglobin level will be estimated at recruitment, at the end of educational intervention and again after 3 months of the completion of intervention.

Qualitative data:
To explore the barriers to and facilitators of intake of diversified food among the adolescent girls, in-depth interviews will be conducted among the purposively selected girls. Twelve IDIs will be conducted (3 adolescent girls having improved and 3 adolescent girls showing no improvement in dietary diversity score; from each intervention school). Although we have assumed the estimated number (12) of IDIs will provide us with sufficient information, number of IDIs will depend on data saturation. Keeping the research objective in mind we will develop a flexible guideline (appendix-3) to conduct the interview. It will take about one hour to conduct an in-depth interview.

Sample Size Calculation and Outcome (Primary and Secondary) Variable(s)
Clearly mention your assumptions. List the power and precision desired. Describe the optimal conditions to attain the sample size. Justify the sample size that is deemed sufficient to achieve the specific aims.
The "State of Food Security and Nutrition in Bangladesh, 2015" reports the nationwide prevalence of inadequate dietary diversity among 10-49 years old women to be 66% [6]. Considering this prevalence, we assume that our intervention would reduce 20 percentage points. Based on the assumption and following conditions below, our estimated sample size will be as follows: P1= However, considering this study as a superiority trial and an effect size of 10 percentage point we also estimated sample size using the formula for sample size estimation in superiority trial. It yields the equal sample size, 93 in each arm.
π1= Test value of the population proportion (inadequate dietary diversity) which is 66% π2 = Anticipated proportion in the intervention group which is 56% n = required sample size It will also represent the sample size calculated based on the effect size of 20 percentage point using the formula of sample size estimation for conventional cluster randomized control trial.After considering design effect 1.5 and 5% attrition rate, desired sample size will be 148 in each arm. Since we will have two schools in each arm, we will collect data of 74 girls from each school. Thus, to reach the desired sample size we will collect data of 25 girls from each grade in a school.

Data Analysis
Describe plans for data analysis, including stratification by sex, gender and diversity. Indicate whether data will be analysed by the investigators themselves or by other professionals. Specify what statistical software packages will be used and if the study is blinded, when the code will be opened.
For clinical trials, indicate if interim data analysis will be required to determine further course of the study.
Data entry will be done by using Microsoft access and Data analysis will be done by STATA (version 14) statistical software program.

Quantitative data analysis:
Dietary diversity scores will be calculated by summing up the number of food groups consumed by the individual participant over the last 24 hours. There is no established cut off point in terms of food groups to indicate adequate or inadequate dietary diversity for the IDDS and HDDS. So, it is recommended to use the mean score for analytical purpose [15]. Dietary  will be presented as frequency and percentage. Continuous variables will be presented as mean with standard deviation. To see the relationship with study group, t-test and Mann-Whitney test (skewed data) will be used. Linear mixed effect model-a longitudinal data analysis technique will be used to measure the role of intervention on individual dietary diversity and household level dietary diversity. Intention-to-treat and per protocol analysis will be done to assess the effect of nutritional education on changes in the mean dietary diversity scores among the participants.
We will perform pairwise comparison tests to see whether the baseline survey data are comparable between intervention and control groups. If the baseline survey reports are found to be not comparable, we will adjust the variables using difference-in-differences analysis.

Qualitative data:
The transcripts of the interviews will be read thoroughly for familiarity with the data. Furthermore, the field impression notes or memos will be sent to the investigator by the interviewers in order to get feedback on what issues they need to investigate more in-depth. The investigator will provide feedback on those immediately so that the interviewers can investigate more in-depth on those issues from the next interviews.
In this iterative way, initial analysis will begin during data collection. After data collection, recorded in-depth interviews will be transcribed and read thoroughly for familiarity with the data. Team members will code these data from transcripts based on apriori and inductive code. Apriori code will be based on topic guide considering existing literature. Coded transcripts will be exchanged to check consistency. Common patterns will be identified from coding and data will be analyzed thematically.

Data Safety Monitoring Plan (DSMP)
All clinical investigations (research protocols testing biomedical and/or behavioural intervention(s)) should include the Data and Safety Monitoring Plan (DSMP). The purpose of DSMP is to provide a framework for appropriate oversight and monitoring of the conduct of clinical trials to ensure the safety of participants and the validity and integrity of the data. It involves involvement of all investigators in periodic assessments of data quality and timeliness, participant recruitment, accrual and retention, participant risk versus benefit, performance of trial sites, and other factors that can affect study outcome.
1. Study data collection and administrative forms will be identified by coded number to maintain participants' confidentiality and to enable tracking throughout the study.
2. All information regarding study subjects will be kept in password-protected computer files or in locked file cabinets that can be accessed only by authorized study personnel. Chart information and information from study records will not be released without written permission from the participant's legal guardian (school headmaster). These records will be kept in locked file cabinets. However, records may be reviewed by representatives from the Research Review Committee and Ethical Review Committee of icddr,b.
3. All study related documents will be kept in locked cabinets in locked rooms with limited access. Information in the electronic database established at icddr,b will be password protected and access will be available only to authorized research team members, any information printed from the database will be stored in locked files until its use is complete and then shredded.
4. The study investigators will be responsible for ensuring complete and accurate documentation for the study and for each subject including records detailing each participant's progress through the study, signed informed consent forms, correspondence with IRB(s), adverse event reports and information regarding participant's discontinuation and completion of the study.
5. Data entry and cleaning will be done at icddr,b.

Ethical Assurance for Protection of Human rights
Describe the justifications for conducting this research in human participants. If the study needs observations on sick individuals, provide sufficient reasons for using them. Indicate how participants' rights will be protected, and if there would be benefit or risk to each participants of the study. Discuss the ethical issues related to biomedical and social research for employing special procedures, such as invasive procedures in sick children, use of isotopes or any other hazardous materials, or social questionnaires relating to individual privacy. Discuss procedures safeguarding participants from injuries resulting from study procedures and/or interventions, whether physical, financial or social in nature.
[Please see Guidelines] Justification for conducting this research in human subject: We will provide nutritional education as intervention to improve dietary diversity among Bangladeshi adolescent school girls aged 11-15 years. To do so, recruitment of human subject (adolescent girls) is a must. Therefore, we propose this study involving human participants (adolescent school girls).
Steps to ensure participant's right: Each participant will be treated according to what is morally right and proper. Fair subject selection will be done to ensure validity and reliability. Participants will be recruited after proper screening and taking consent as well as assent. Autonomy and justice will be ensured by-• Taking well-informed consent from the legal guardian of the participant (We will take approval from the headmaster of the schools. Moreover, we will organize a parents' meeting at the school before rolling out the intervention. We will take consent from them on that day.) • Taking assent from participant (our study participants will be 11-15 years old adolescent girls. As they are below 18 years, so they are not eligible for providing consent. This is why, we will take assent from them) • Ensuring privacy and confidentiality • Ensuring right to withdrawal at any stage • Delivering adequate information in an understandable, clear, unambiguous, effective manner and in non-technical language • Negating coercion, undue induction, unduly influence or intimidation

Study benefits:
We will provide intervention (nutritional education) to all the students of intervention arm. Additionally, all adolescent girls (both in intervention and in control arm) will receive weekly Iron-folic acid (IFA) supplementation for 3 months following national guideline and information on water, sanitation and hygine (WASH).

Significant risks / adverse events:
Our intervention (nutrition education) is not related to any health hazard of study participants. Haemoglobin will be measured from a finger prick sample using a HemoCue machine (Hb 301, HemoCue AB, Angelholm, Sweden) and trained staff will be recruited to take blood samples from the adolescent girls. So, we do not envision any significant risks related to participation in this study.

Ethical issues related to social or biomedical research:
Our research project does not include any steps which may result stigmatization or social exclusion of any of the participant.

Procedures safeguarding participants from risks resulting from study procedures and/or interventions:
The principle investigator and the team are obliged to secure the wellbeing and beneficence of the study participants.

Use of Animals
Describe if and the type and species of animals to be used in the study. Justify with reasons the use of particular animal species in the research and the compliance of the animal ethical guidelines for conducting the proposed procedures.

Collaborative Arrangements
Describe if this study involves any scientific, administrative, fiscal, or programmatic arrangements with other national or international organizations or individuals. Indicate the nature and extent of collaboration and include a letter of agreement between the applicant or his/her organization and the collaborating organization.

Facilities Available
Describe the availability of physical facilities at site of conduction of the study. If applicable, describe the use of Biosafety Level 2 and/or 3 laboratory facilities. For clinical and laboratory-based studies, indicate the provision of hospital and other types of adequate patient care and laboratory support services. Identify the laboratory facilities and major equipment that will be required for the study. For field studies, describe the field area including its size, population, and means of communications plus field management plans specifying gender considerations for community and for research team members.
We will set up a field office in an urban area of Rangpur where logistics will be temporarily stored. Projectors, laptops, sound systems, iron-folic acid supplementation will be carried from field office to schools by health workers. Staff members of the study will use rickshaw or autorickshaw to go from filed office to schools. Iron-folic acid (IFA) supplements will also be carried by health workers. As our study participants are adolescent school girls, we will recruit female staffs as field research assistants (FRA) and health workers (HW) as they will be involved in taking face-to-face interview of adolescent girls, measuring anthropometry, provision of iron-folic acid (IFA) to the adolescent girls.

Literature Cited
Identify all cited references to published literature in the text by number in parentheses. List all cited references sequentially as they appear in the text. For unpublished references, provide complete information in the text and do not include them in the list of Literature Cited. There is no page limit for this section, however, exercise judgment in assessing the "standard" length.  (5)

Reviewer comment:
In page 15 the flowchart shows that both intervention and control group will receive IFA and WASH, but in page 16 it is stated that in the control groups girls will only receive WASH only. Please address the inconsistency.
Response: Thanks for your observation. We have made correction at page 16, according to your suggestion.
Reviewer comment: Consent from headmaster, considering him as a legal guardian, may not be accepted. Please check with lawyer, whether headmaster are eligible to become legal guardian.
(page 17) Response: Thanks for your comments. Since our intervention will be school-based nutrition education, we will obtain initial approval from the school authority and headmaster of the school. We will take assent from the adolescent girls and we will also take consent from the mother or caregiver of the adolescent girls before including them in this study. As per your suggestion, we have mentioned it at page 17.

Other Support
Describe sources, amount, duration, and grant number of all other research funding currently granted to PI or under consideration.

Gender Analysis Tool:
In May not be applicable

No information available
We do not have any reference to assume any difference * Of different classes, ethnic groups, ages or other relevant differences between women and between men: Evidence shows that higher income groups are more likely to take preventive action; though also more likely to show resistance because of incorrect use of drugs.

Purpose of the research
After first year, adolescence (10-19 years) is the second most critical period for physical growth. Of the entire growth of the body, 80% is achieved during the early adolescence (10-15 years). It was revealed from the previous studies that this sudden growth spurt makes early adolescence a nutritionally vulnerable period and inadequate dietary diversity is one of the major causes of adolescent nutritional deficiency. As an adolescent girl is a future mother and offsprings growth is directly related to mother's proper growth. For this, proper nutrition of a mother is very crucial. Adult weight & height gain are ultimately ceased after early adolescence. So, adolescence is the last opportunity to intervene and break the vicious cycle of inter-generational malnutrition. Keeping all these contexts in mind, we aim to conduct a research in Rangpur district. We will select two urban and two rural schools from Rangpur district and from each school we will select three grades which will be grade six, seven and eight. We will provide eight nutrition education sessions regarding dietary diversity to adolescent girls. Each session will be conducted in one grade once a week and our total duration of the intervention will be three months. Along with the nutrition education session, we will give iron-folic acid supplementation to all the students under the selected grades, once a week for three months. The result of the study will generate knowledge on how to improve dietary diversity among the adolescent girls and thereby improve their nutritional status.

Why invited to participate in the study?
We are trying to provide nutrition education to adolescent girls to measure the improvement of dietary diversity. You are an adolescent girl studing in a school selected for our study and this is why we are inviting you to help us by participating in this study.

Methods and procedures
Our research will be conducted for 14 months and we will provide nutrition education as intervention for three months to the selected schools. Our skilled female research staffs will collect data at three different time points: before rolling out of the intervention, after completion of the intervention and after three months of intervention.
Our female staffs will collect data regarding demographic information, your dietary diversity, menstrual history and health as well as nutritional status (height, weight, MUAC and hemoglobin concentration). It will take about an hour to collect data.

Risk and benefits
Apart from spending of valuable time, you will not face any risk due to participation in the study. We will provide our intervention (nutrition education sessions) using projector and laptop by skilled research staffs. We will provide these sessions to all of the students of grade six, seven and eight. In addition, our female staffs will provide iron-folic acid tablets to all students of these mentioned classes once a week (it is the recommended dose of Bangladesh Government). Your information will be collected by our skilled female staffs and they will maintain confidentiality, so we would expect that it will not be a cause of your embarrassment. During investigation, if we find that you have been suffering from severe anaemia, we will refer you to the nearest health care centre.
Your participation in this study will help the adolescent girls to achieve appropriate growth and we will mention your contribution in our research.

Principle of compensation
You will not be given any financial or other compensation for the time you spend as a result of participating in the study.

Privacy, anonymity and confidentiality
According to the laws of this country, we do hereby affirm that privacy; anonymity and confidentiality of the information provided by you will strictly be maintained. Information provided by you will be kept confidential and will only be used for research purposes in this study with various segments. We will not mention your name or your identity or any kind of information by which you could be recognized.

Storage of data
Even after completion of the research, your shared information will be stored for three years which will be used for research report or publications.

Future use of information
Collected information of this research could be shared by other researchers but we will maintain confidentiality in case of your identity and address. Moreover, if you give us permission, we will use your name and address to make contact with you for participating in another research in future.

Right not to participate and withdraw
Your participation in this study is voluntary. You may not answer any questions if you want. You can withdraw yourself from the study even after giving informed written consent or any time during interview session. It is entirely your right whether you will participate in this research or not and there will be no negative impact of your decision on taking service from icddr,b in future. You will receive nutrition education, message on water, sanitation and hygiene, and IFA supplementation just like other participants.

Communication:
If you have any question, you can ask me right now or at any time later on. If you want to know anything about rights and benefits for participation in this study you can contact in the following addresses: If you agree to our proposal for enrolling you/your patient in our study, please put  mark on appropriate box(es) of the following and finally sign on the specified place for you.

Purpose of the research
After first year, adolescence (10-19 years) is the second most critical period for physical growth. Of the entire growth of the body, 80% is achieved during the early adolescence (10-15 years). It was revealed from the previous studies that this sudden growth spurt makes early adolescence a nutritionally vulnerable period and inadequate dietary diversity is one of the major causes of adolescent nutritional deficiency. As an adolescent girl is a future mother and offsprings growth is directly related to mother's proper growth. For this, proper nutrition of a mother is very crucial. Adult weight & height gain are ultimately ceased after early adolescence. So, adolescence is the last opportunity to intervene and break the vicious cycle of inter-generational malnutrition. Keeping all these contexts in mind, we aim to conduct a research in Rangpur district. We will select two urban and two rural schools from Rangpur district and from each school we will select three grades which will be grade six, seven and eight. We will provide eight nutrition education sessions regarding dietary diversity to adolescent girls. Each session will be conducted in one grade once a week and our total duration of the intervention will be three months. Along with the nutrition education session, we will give iron-folic acid supplementation to all the students under the selected grades, once a week for three months. The result of the study will generate knowledge on how to improve dietary diversity among the adolescent girls and thereby improve their nutritional status.

Why invited to participate in the study?
We are trying to provide nutrition education to adolescent girls to measure the improvement of dietary diversity. We are inviting you to our study because you are a mother/legal guardian of the adolescent girl (please mention the name of the girl) who is studing in the selected school under our research.

Methods and procedures
The duration of our research will be fourteen months. We will provide nutrition education in two urban and two rural secondary high schools of Rangpur district. For intervention, we will select the girls from sixth, seventh and eighth grade of each school. We will provide one nutrition session in one grade in a week. Along with this, we will give iron-folic acid (IFA) supplementation once a week for three months. We will collect data at three different time points: at the beginning of the intervention, at the end of the intervention and again after three months of the intervention. Our total intervention period will be three months. Our skilled research staffs will collect data from you regarding socio-economic condition, water, sanitation and hygiene and dietary diversity of your family.
The information collected from you will never be used for any purpose other than analysis. It will take about an hour to complete data collection. Moreover, we will arrange a parents' meeting at the schools before intervention. We will highly appreciate your presence in the parents' meeting. If you agree to our proposal for enrolling you/your patient in our study, please put  mark on appropriate box(es) of the following and finally sign on the specified place for you. • Atleast 15 decimal cultivable Land?

W1
What is the main source of drinking water for you and your girl?

S1
What kind of toilet facility do members of your household usually use?

H2
Record if soap or detergent or anything is present at the specific place for hand washing. • None ন েু ই িো

H3
Request the mother/caregiver to show Yes / িযো = 1 Vitamin A rich fruits ripe mango, cantaloupe, apricot (fresh or drie), ripe papaya, dried peach, and 100% fruit juice made from these + other locally available vitamin A rich fruits 7 Other fruits other fruits, including wild fruits and 100% fruit juice made from these 8 Organ meat liver, kidney, heart or other organ meats or blood-based foods 9 Flesh meat beef, pork, goat, lamb, chicken, duck, rabbit, game, other birds, insects 10 Eggs eggs from chicken, duck, guinea fowl or any other egg 11 Fish and sea foods fresh or dried fish or shell fish

12
Legumes, nuts and seeds dried beans, dried peas, lentils, nuts, seeds or food made from thses (e.g. peanut butter)? 13 Milk and milk products milk, cheese, yogurt or other milk products? 14 Oils and fats Oil, fats or butter added to food or used for cooking 15 Sweets sugar, honey, sweetened soda or sweetened juice drinks, sugary foods such as chocolates, candies, cookies, cakes etc?