Maternal vitamin D supplementation during pregnancy and lactation to prevent acute respiratory infections in infancy in Dhaka, Bangladesh (MDARI trial): protocol for a prospective cohort study nested within a randomized controlled trial

The MDIG trial is a randomized placebo-controlled dose-ranging trial of maternal vitamin D supplementation during pregnancy and lactation to improve infant linear growth in Dhaka, Bangladesh. The detailed methods of the MDIG trial are published elsewhere [30]. The research group is primarily a collaboration between icddr,b (International Centre for Diarrhoeal Disease Research, Bangladesh) and the Hospital for Sick Children (Toronto, Canada). Pregnant women were enrolled beginning on March 18, 2014 and enrolment is expected to proceed until August 2015. Enrolment of infants in the MDARI sub-study began on December 2, 2014 and will continue until all infants born in the MDIG trial have been offered enrolment (expected completion by January 2016).

The single-site trial is being conducted in Dhaka. Microbiologic testing is being conducted at the Virology Laboratory of icddr,b. Enrolment and clinical activities of the MDIG trial is based at the Maternal and Child Health Training Institute (MCHTI), commonly known as Azimpur Maternity Center, a government facility that provides low-cost health care to pregnant women and children in its referral area in central Dhaka. Full details regarding the trial site geography and population are provided in the MDIG trial methods paper [30].

All infants born to women enrolled in the MDIG trial are eligible for the MDARI sub-study. Maternal inclusion criteria for the MDIG trial include being at least 18 years of age, 17 to 24 completed weeks of gestation based on a combination of recalled last menstrual period (LMP) and ultrasound, according to a conventional algorithm (per the Society of Obstetricians and Gynecologists of Canada), and intention to permanently reside in the trial catchment area for the duration of the study. Participants are informed of the ARI sub-study by a research study physician at a clinical visit after enrolment in the main trial. We obtain separate written informed consent for participation in the MDARI sub-study. We aim to enroll at the 30 week gestational age study visit, however, given that the MDIG trial began before MDARI, we also enroll at prenatal visits after 30 weeks, at the time of delivery, and during home visits to infants who were younger than 6 months of age at the time MDARI launched. Participants are free to withdraw from MDARI while remaining in MDIG but withdrawal from MDIG results in automatic withdrawal from MDARI.

A full description of the intervention groups along with the rationale for choice of vitamin D dosages may be found in the methods paper describing the MDIG trial [30]. Three intervention groups are receiving weekly prenatal vitamin D supplementation of 4200 IU/week, 16,800 IU/week, or 28,000 IU/week respectively given as a single weekly dose beginning in the 2^nd trimester of pregnancy. These three groups all receive placebo post-partum. A fourth group is receiving 28,000 IU/week during pregnancy and will continue with this level of supplementation until 26 weeks postpartum. A fifth control group is receiving placebo both pre and post-partum (Fig. 1). Infant dietary patterns are assessed at weekly visits to assess breastfeeding patterns and timing of introduction and frequency of consumption of specific complementary foods.

Most infants born to mothers in the placebo and 4200 IU/week groups are not expected to demonstrate neonatal 25 (OH) D ≥ 50 nmol/L, whereas most infants in the 16,000 IU/week and the 28,000 IU/week groups (including participants that will continue to receive 28,000 IU/week post-delivery) are expected to have 25 (OH) D ≥ 50 nmol/L in early infancy. Although the clinical significance of the 50 nmol/L threshold in early infancy remains to be established, this is the cut-off corresponding to the recommended dietary allowance established by the Institute of Medicine in 2011 for all age groups [31]. To promote optimal regimen adherence, doses are delivered weekly (e.g., 28,000 IU per week to deliver the approximate equivalent of 4000 IU/day) and supplementation is directly observed by study personnel whenever feasible.

This is a prospective cohort study nested within a randomized, placebo-controlled trial with concealment of allocation and blinding throughout intervention and analysis. In the main MDIG trial, a total of 1,300 pregnant women will be randomized, targeting 260 women in each group (Fig. 1). The allocation sequence was generated by the trial statistician using a computer-generated random number sequence, according to a simple randomization scheme. Participants, investigators, field personnel, study lab staff and data analysts are blinded to vitamin D or placebo group allocation. Consent to the MDIG trial is necessary to be eligible for the MDARI study; however, participants in MDIG may choose to decline participation in MDARI.

The primary study outcome is microbiologically confirmed ARI. Main secondary outcomes include ARI with microbiologic confirmation of influenza A, influenza B, or RSV; clinical ARI; and quantitative nasal S. pneumoniae carriage density. The clinical case definitions for ARI, URTI, and LRTI are shown in Table 1. When an infant meets any of the sets of criteria for clinical URTI or LRTI, nasal swab collection is indicated. All nasal swabs are optimally collected within 24 h of notification, however a nasal swab may still be collected within 7 days following clinical case ascertainment (or within 7 days of hospital discharge) [32‐34]. No participant has more than 1 nasal swab collected within 7 days, regardless of the severity of the illness. Swabs are tested by polymerase chain reaction (PCR) for a panel of respiratory viruses consisting of influenza A, influenza B, RSV, parainfluenza 1, 2, and 3, adenovirus, and human metapneumovirus. Swabs are also be tested for quantitative S. pneumoniae carriage density. Full details of microbiologic testing are presented below. To be considered an incident case, there must be either a preceding surveillance week in which a clinical case definition was not met or a worsening of a case from URTI to LRTI or from LRTI to hospitalized LRTI. To avoid including common neonatal problems that may present similarly to ARI (e.g., transient tachypnea of the newborn), case definitions cannot be met in the first 7 days of life.

Active and passive surveillance mechanisms are being utilized to prospectively identify new cases of clinical ARI among infants during the 0–6 month postnatal period.

Active surveillance visits are attempted on a weekly basis during the first 6 months of life and are implemented through the existing design and infrastructure of the MDIG study [30]; the majority of these visits occur in participants’ homes. At each surveillance-visit, study field personnel trained according to the Integrated Management of Childhood Illness (IMCI) program (WHO/UNICEF) [35] administer a standardized questionnaire and conduct an infant-clinical examination. The questionnaire and physical exam have been designed to optimize the collection of maternal-reported and CHW-observed infant morbidity data. Information pertaining to the following ARI-related symptoms is collected: 1) maternal-reported cough, rhinorrhea (i.e., runny nose), nasal congestion (i.e., stuffy nose), difficulty breathing, and infant-hospitalizations related to a breathing problem or chest infection (e.g., pneumonia); and, 2) CHW-observed axillary temperature ≥ 37.5 °C (confirmed with a second measurement), lower chest wall indrawing, and elevated respiratory rate (≥60 breaths per minutes for infants up to 59 days of age or ≥ 50 breaths per minute for infants 60 days of age or older). The routine collection of these data facilitates the weekly identification of new, worsened, or persistent cases of ARI. The identification of any one symptom indicative of a possible ARI during weekly surveillance visits triggers a field worker to notify the Nasal Swab Dispatcher (MDARI study physician). The Nasal Swab Dispatcher assesses the information to determine whether the clinical case definition of URTI and/or LRTI has been met, and whether a nasal swab specimen should be collected (i.e., no nasal swab collected within past 7 days, at least one study visit at which URTI and LRTI were absent or if not, the ARI clinical case definition has worsened since the last nasal swab was collected).

To identify cases of ARI that arise between scheduled visits, a passive surveillance system has been implemented that relies on the report of ARI symptoms by parents/guardians. At enrolment, mothers are instructed to notify the Nasal Swab Dispatcher by telephone if their infant develops any one of the following symptoms: runny nose, nasal congestion, cough, difficulty breathing, feels hot to the touch, or the infant has been hospitalized. An incentive of approximately $0.25 USD of cell phone credit is provided to families who report an ARI-related symptom. The Nasal Swab Dispatcher assesses the information to evaluate whether the ARI clinical case definition has possibly been met (at least one of cough, runny nose, congestion, hot to the touch, difficulty breathing, or ARI-related hospitalization) and whether collection of a nasal swab is possible (i.e., no nasal swab collected within past 7 days, at least one study visit at which URTI and LRTI were absent or if not, a reassessment must be conducted to determine whether the ARI clinical case definition has worsened since the last nasal swab was collected). Caregiver-reported ARI symptoms are verified in-person through a short questionnaire and infant-clinical examination conducted by a trained nasal swab collector; the clinical assessment is typically performed in the participants’ home. A nasal swab specimen will only be collected if the clinical criteria for URTI and/or LRTI are verified. All MDIG-study physicians have been trained to notify the Nasal Swab Dispatcher if any symptom indicative of a possible ARI is detected during an interaction with an enrolled infant. The Nasal Swab Dispatcher assesses the physician-reported information to determine whether the clinical case definition of an ARI has been met. If an infant is hospitalized with a physician diagnosis of pneumonia or bronchiolitis, the nasal swab collector collects a swab during the hospitalization, or if this is not possible, within 7 days of discharge.

Measures of infant peripheral oxygen saturation (SpO₂) are collected for every infant that meets or possibly meets the clinical case definition of an ARI. SpO₂ is measured with a hand-held pulse oximeter device (Rad-5v with LNCS Y-shaped sensor and LNC-04 connector cable, Masimo California, USA). A threshold of SpO₂ < 90 % is used to guide the referral of infants to hospital for further assessment.

Among infants meeting the definition for clinical ARI as described above, the study worker obtains parental consent to perform nasal swabbing on the infant using sterile flocked swabs (Copan Diagnostics, Inc., model number 56780CS01) to obtain nasal epithelial cells for laboratory analysis. Nasal flocked swabs, which are inserted more proximately in the nares than a traditional nasopharyngeal swab, are better tolerated by the child, easier to perform for the health care worker, and reliably provide adequate sample for molecular testing for respiratory viruses [36‐38]. Testing is performed by swabbing the nares to the level of the nasal turbinate to obtain a sample of exfoliated cells from the mucosal lining. The infant’s head is tilted backwards to an angle of 70° and the swab is gently inserted by the study worker horizontally into the nasal passage approximately one-half the distance from the nostril to the earlobe or until the stopper meets the infant’s nose or until resistance is met at the level of the turbinate. The swab is then rotated two to three times and held it place for 5–10 s to absorb sample material. The swab is then removed and immediately placed into a collection vial labeled with the patient’s study number and containing 1–3 ml of universal transport medium (Copan Diagnostics, Inc.). The vial is placed into an insulated cold bag and transported to a −80 °C study freezer for storage until testing is done.

Only one nasal swab will be collected for each episode of ARI and there will not be more than 1 swab collected in a 1-week period. We estimate that most infants will not receive more than about one swab per month until they reach 6 months of age, and typically we expect an infant to have 3–4 swabs during this period. Participant families receive 200 taka (~$2.50 USD) for the time involved in the nasal swab procedure.

Respiratory samples are processed and analyzed in the Virology Lab at icddr,b in Dhaka. For each nasal swab specimen, two aliquots are prepared; one aliquot is processed for real-time assays and one aliquot is stored at −80 °C for future reference. Total nucleic acids are extracted from sample using InviMag Pathogen Kit/KF96 (STRATEC Molecular, Berlin, Germany) to an eluate volume of 200 μl according to the manufacturer’s instructions. The InviMag Pathogen Kit/ KF96 contains the lyophilized lysis component (lysis buffer, Proteinase K and Carrier RNA) for bacterial DNA, viral DNA and viral RNA isolation. Lysed samples are mixed with magnetic beads and nucleic acids are isolated according to the manufacturer’s instructions. All real time PCR tests undergo a visual check; if quality of the amplification is not good the test is repeated and/or considered negative. A house-keeping gene, RNP, is used as an indicator of perfect nucleic acid extraction, to check for epithelial cells, sample quality, quality of RT-PCR, and to monitor for PCR inhibition.

To detect and quantitate pneumococcal copy number, quantitative real-time PCR (qPCR) assays are performed to amplify lytA, a single-copy autolysin gene that is specific to all S. pneumoniae strains (Table 2). This method is based on the U.S. Centers for Disease Control and Prevention (CDC) method as described by Carvalho et al., and adapted from the National Institute of Communicable Diseases (NICD) in South Africa. The qPCR assays are carried out in a final reaction volume of 25 μl, each containing 10 μl of extracted total nucleic acid. The lytA primers and probes are added to a final concentration of 10 μM. DNA is amplified in a ABI 7500 (Applied Biosystem, USA) using the following cycling parameters: 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. All standards are performed in triplicate and a negative (no template) and positive (lytA plasmid control) control is included in every run along with the samples. lytA amplification data is analyzed using 7500 software version 2.0.6 (Applied Biosystem, USA). The baseline and threshold are manually set at >0.05, the slope of the standard curve is between −3.1 to −3.6 and correlation should be >0.9. The cutoff value CT < 35 is considered as positive. A CT value of ≤38 showing appropriate amplification on visual check is also considered a positive result.

Individual real-time one step reverse transcriptase-PCR (qRT-PCR) assays are being used to detect influenza A and B viruses. Primer and probe sequences were designed on the basis of CDC recommendations (Table 2). The qRT-PCR assays are carried out in a final reaction volume of 25 μl, each containing 5.0 μl of extracted nucleic acid. The specific influenza A and B primers and probes are added to a final concentration of 20 μM and 5.0 μM respectively. The qRT-PCR reaction is carried out with any of the following machines; ABI 7500 (Applied Biosystem, USA), CFX-96 (Bio-Rad, USA) or LightCycler 480 (Roche, Switzerland) using the following cycling parameters: 50 °C for 30 min, 95 °C for 5 min, 46 cycles at 95 °C for 15 s and 55 °C for 1 min. A negative control (no template) and a CDC provided positive control is included in every run along with the samples. The amplification data is analyzed using machine specific softwares. The cutoff value CT < 35 is considered positive. A CT value of ≤38 showing appropriate amplification on visual check is also be considered a positive result.

Individual qRT-PCR assays for 6 viruses (adenovirus, human metapneumovirus [HMPV], human parainfluenza viruses 1–3 and respiratory syncytial virus [RSV]) are being carried out according to the procedure described by Weinberg et al. [39]. Primer and probe sequences were designed on the basis of CDC recommendations (Table 2). All individual qRT-PCR assays are performed with any of the following machines; ABI 7500 (Applied Biosystem, USA), CFX-96 (Bio-Rad, USA) or LightCycler 480 (Roche, Switzerland), using following cycling parameters; 45 °C for 10 min, 95 °C for 10 min, and 45 cycles of 95 °C for 15 s and 55 °C for 1 min. A negative control (no template) and CDC provided specific positive control is included in every run along with the samples. The amplification data is analyzed using machine specific softwares. The cutoff value CT < 35 is considered positive. A CT value of ≤38 showing appropriate amplification on visual check is also considered a positive result.

A site supervisor reviews all forms for completeness and protocol deviations before sending forms to the data management center at icddr,b. Electronic (scanned) versions of all forms are also regularly saved for long-term storage. The database was designed using SQL Server 2008 and data are entered using Visual Studio 2010. A set of range and consistency checks are built into the data capture system to provide immediate feedback to data entry personnel regarding errors or inconsistent data. Double data entry is used to further reduce the rate of date entry errors.