Prevalence and determinants of anaemia during the second or third trimester of pregnancy in Bangladesh: a cross-sectional study protocol

Objectives and outcomes

The primary objective is to determine the prevalence of anaemia (haemoglobin, Hb <11 g/dL) during the second and third trimester (13–32 weeks) of pregnancy in women from Rupganj, Sonargaon and Bandar Upazilas (sub-districts) of Narayanganj district.

The key secondary objectives are to determine the prevalence of moderate anaemia (Hb <10 g/dL), severe anaemia (Hb <7 g/dL), iron deficiency (ferritin <15 mg/L), and iron deficiency anaemia (concurrent anaemia and iron deficiency). The association between the woman’s drinking water iron level and anaemia status will be assessed, along with the association between the woman’s iron status and anaemia status.

Secondary objectives are to describe women’s sociodemographic characteristics, pregnancy history, antenatal and delivery care seeking practices and challenges, nutritional knowledge and existing practices for anaemia management, delivery preparedness and autonomy, water-sanitation-hygiene practices, mental health status, and COVID-19 history.

Exploratory objectives include investigating the genetic, immune, metabolic and molecular aetiologies related to anaemia. These analyses are beyond the scope of this protocol and will be reported separately.

Study design

This will be a cross-sectional survey to determine the prevalence of anaemia (Hb <11g/dL) in pregnant women during the second and third trimester (13–32 weeks).

Eligibility criteria

Inclusion criteria

Exclusion criteria

Study setting and participants

This will be conducted in three Upazilas (sub-districts) of the Narayanganj district: Rupganj, Sonargaon and Bandar. Each Upazila is subdivided into a number of unions. Each union is subdivided into nine wards. There are six or more Family Welfare Assistant (FWA) units in ward level depending upon the area and size of population. Each Upazila comprises of ~350,000–600,000 people, and estimated pregnancies is ranging from ~8,000 to ~12,000. The study area has low levels of iron in the groundwater^14,15. There is no data on the prevalence of anaemia in pregnant women from this district.

Study procedures

Screening. All women of reproductive age living in the study site are screened regularly for pregnancy (via questioning on last menstrual period (LMP)) by Family Welfare Assistants (FWAs), and pregnant women are identified and recorded in a FWA register. The FWAs work principally in FWA units in rural areas in the Upazila (sub-district) set up under Directorate General of Family Planning (DGFP). Study Field Research Assistants (FRAs) will collect the names with other details of the pregnant women from FWA register to prepare a pregnancy database. Calculated from first day of LMP, they will identify pregnant women in their second or third trimester (13–32 weeks) and arrange a household visit. When LMP dates are missing, FRAs will confirm the date during household visits. During the home visits, FRAs will verify that the women fulfil the eligibility criteria, ensuring that they are 13–32 weeks pregnant and a resident in the study catchment area. Following the acquisition of informed consent, the participants will be asked to provide information on their socio-demographics, household assets, income, smoking status, and autonomy.

Water iron testing

Drinking water iron level will be measured from freshly collected water from the family drinking water source. The HACH iron test kit, Model IR-18B will be used for iron testing.

Enrolment

Physical measures

Following FRAs recommended date, participants will attend a nearby Family Welfare Centre, and a physician will perform a physical examination ensuring privacy of the participants. This will include a general examination: checking for pallor, blood pressure, pulse, oedema, and anthropometric measurements: height, weight and mid-upper arm circumference.

Questionnaires

All participants will be asked to provide information pertaining to their current & previous pregnancy history, including anaemia testing & treatment, iron-folic acid (IFA) use, nutritional knowledge & practice, delivery preparedness & complication readiness, and COVID-19 history. A water, sanitation and hygiene (WASH) questionnaire will also be conducted¹⁶. Finally, women’s mental health will be measured using the Edinburgh Postnatal Depression Scale (EPDS)^17–19.

Blood collection & Haemoglobin (Hb) measurement

After all aseptic precautions, the study physician will collect 5 ml of venous blood sample. Immediately after collection of blood, Hb level will be measured from venous blood using HemoCue® Hb 301 portable haemoglobin analyzer (Hemocue, Inc., Ängelholm, Sweden).

Laboratory procedures

At the field site laboratory, lab technicians will do the initial sample processing. The field site lab will have small laboratory equipment for sample separation, and storage. Lab technicians will use standard operating procedures (SOPs) for all laboratory processing of samples, developed by a senior Laboratory Technologist. Samples will be stored initially at -20°C but periodically will be transferred for long-term storage to International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b) lab where there is a -80°C freezer.

Blood samples: separation and long-term storage

Blood will be centrifuged, and serum will be separated from the blood cells as soon as possible. Blood will also be stored in appropriate preservatives to enable iron and inflammatory biomarker measurements (serum ferritin and CRP) and further analysis of genetic, immune, metabolic and molecular aetiologies related to anaemia. Residual whole blood (EDTA) samples from participants will be stored for future extraction of DNA and/or RNA.

Assessment of safety

COVID-19 precautions. Considering the global pandemic of COVID-19, the icddr,b has its own guidelines and SOPs regarding infection prevention and control (IPC) to be implemented. All contacts with participants during data collection or physical measurements procedures will strictly follow IPC SOPs. For bio-sample collection and processing in the field and storage in the lab we will follow Bio-Safety Level (BSL) guidelines. The central biosafety team of icddr,b is in charge of the compliance management. All study staff will receive training on IPC and necessary personal protective equipment prior to commencement of field activities.

Data handling and record keeping

Data will be collected using smart phones (7 inch) with internet enabled SIM cards for data collection and transmission from the community to a central database server at icddr,b. The data management system is designed to run on an android platform and configured based on the working areas of data collectors. Data will be stored locally in the device during interview, and uploaded to icddr,b servers on a regular basis. The databases will be password protected and only members of the study team and investigators will have access. Only deidentified data (study identification numbers but no names) will be stored on the database.

Database lock. We will declare and document the data as being clean only once the validation process is concluded. The database will be locked only after all decisions on the evaluability of the data from each individual subject have been made and documented.

Statistical considerations

Sample size calculations. We will screen 1,500 pregnant women (500 in each Upazila (sub-district)). Overall, 40% will be recruited from each of the second and third trimesters respectively and the remaining 20% will be recruited on a competitive basis. With a minimum sample size of 600 women across all Upazilas (sub-districts) per trimester, the precision of the two-sided 95% confidence interval of the true underlying prevalence is at most ±4% (assuming prevalence of 50%) using the Wald method to measure the two-sided 95% confidence interval. The expected precision is ±3.8% assuming the prevalence of anaemia in pregnancy is (210/600) 35% (i.e., 95% CI 31.2% to 38.8%), ±2.4% assuming the prevalence of moderate/severe anaemia in pregnancy is (60/600) 10% (i.e., 95% CI 7.6% to 12.4%), and ±3.9% assuming the prevalence of iron deficiency in pregnancy is (240/600) 40% (i.e., 95% CI 36.1% to 43.9%).

Data analysis. The proportion of women with anaemia, moderate anaemia, severe anaemia, iron deficiency, and iron deficiency anaemia will be obtained per trimester, along with two-sided 95% confidence intervals. Women’s sociodemographic characteristics, antenatal and delivery care seeking practices and challenges, nutritional knowledge and existing practices for anaemia management, preparedness and autonomy, water-sanitation-hygiene practices, mental health status, COVID-19 history, and laboratory outcomes will be summarised per trimester.

Univariable and multivariable regression models will be used to explore the association between potential determinants of anaemia and anaemia collected in the study. A causal diagram informed by clinical expertise will be developed to help determine which factors to include in the multivariable models. The association between the woman’s ferritin, iron deficiency status, haemoglobin, anaemia status and her family’s drinking water iron level will be explored similarly. We will present the estimate and two-sided 95% confidence interval of each association alongside two-sided p-values. Data will be analysed using STATA (StataCorp, Statistical Software: College Station, TX).

Study oversight

Quality control and quality assurance. Authorised representatives of the sponsor, an Institutional Ethics Committee/Review Board (IRB) or regulatory authority may visit the study site to perform audits or inspections, including source data verification.

Ethics and dissemination

Ethical considerations

Research ethics approval

This protocol, the informed consent forms and any subsequent modifications will be reviewed and approved by the Institutional Review Board (IRB) of icddr,b as well as by the human research ethics committee of WEHI, Melbourne, Australia. Approval from the ethics committee at icddr,b and WEHI was obtained on the 29^th of March, 2021 (PR-20125) and on the 2^nd of June, 2021 (21/5) subsequently prior to the commencement of the study.

Ethical review

The IRB of icddr,b will review the study activities anytime during the study period and will provide feedback.

Informed consent

Informed written consent will be obtained from each participant before conducting any study related procedure. The PI will ensure that the participant (and the subject's parent/guardian in case of assent) is given full and adequate oral and written information about the study. Information will be provided in the local language of the participant. The participant will be given the opportunity to ask questions and allowed time to consider the information provided.

Risks to the study participants

Blood sampling. A total of 5 ml of blood will be collected through a venepuncture. A small bruise or mild pain on the venepuncture site from where the blood is taken may develop. All efforts will be made to minimise pain, discomfort, and fear for pregnant women. Only well-trained and fully qualified staff will be hired for this project. Only new disposable needles and lancets are used for the blood taking procedures, and these will be discarded immediately after their use.

Subject data protection

Participant confidentiality is strictly held in trust by the participating investigators, research staff, and the sponsoring institution and their agents. This confidentiality is extended to cover testing of biological samples and genetic tests in addition to the clinical information relating to participating participants. The study protocol, documentation, data and all other information generated will be held in strict confidence. No information concerning the study, or the data, will be released to any unauthorised third party, without prior written approval of the sponsoring institution. All laboratory specimens, evaluation forms, reports and other records that leave the site will be identified only by the Participant’s Unique Identification Number to maintain participant confidentiality. Clinical information will not be released without written permission of the participant, except as necessary for monitoring by regulatory agencies.