Micronutrient status among pregnant women in zinder, niger and risk factors associated with deficiency

Anemia and micronutrient (MN) deficiencies in pregnant women are associated with adverse pregnancy outcomes. In Niger, 58.6% of pregnant women are anemic, however, MN statuses are unknown. The study objectives were to estimate the prevalence of MN deficiencies among pregnant women in Zinder, Niger and explore associated risk factors. Pregnant women living in randomly selected rural villages (n = 88) were included. Capillary and venous blood samples (n = 770) were analyzed for hemoglobin (Hb) and plasma ferritin, soluble transferrin receptor (sTfR), zinc (pZn), retinol binding protein (RBP), folate and vitamin B12. C-reactive protein and alpha-1-acid glycoprotein were measured to adjust for inflammation. The prevalence of MN deficiencies in pregnant woman was high, indicative of a severe public health problem. Prevalence of iron deficiency was 20.7% and 35.7%, by ferritin (<15 μg/L) and sTfR (>8.3 mg/L), respectively. In total, 40.7% of women had low pZn (<50 μg/dL), 79.7% had marginal RBP (<1.32 μmol/L), 44.3% of women had low folate (<10 nmol/L) and 34.8% had low B12 concentrations (<148 pmol/L). Common risk factors associated with MN status included gravidity, mid-upper-arm circumference, geophagy, malaria, and result of the woman’s last pregnancy. Interventions to promote the strengthening of antenatal care, and access and adherence to nutrition and health interventions are critical among pregnant women in this population. This study was a community-based cross-sectional survey among pregnant women in rural communities in the Zinder Region of Niger, and was conducted as a part of the baseline assessment for the Niger Maternal Nutrition (NiMaNu) Project. The NiMaNu project was a programmatic intervention to improve ANC services, designed with a rigorous research component to assess and evaluate the impact of the intervention. The project was implemented March 2014–September 2015, using a continuous enrollment schedule over a period of 18 months to account for potential effects of seasonality in the outcome measures. Pregnant women in rural villages within the catchment areas of 18 out of 45 governmental integrated health centers (CSI) located in two districts (Zinder and Mirriah) of the Zinder Region of Niger were eligible to participate. CSI were selected based on convenience sampling and randomized to the order of participation; criteria for inclusion were determined in collaboration with local governmental health officials and included year-around accessibility, distance to regional capital (Zinder) and a limited number of interventions being implemented in the catchment area. Within each catchment area of each CSI, the village in which the CSI was located was automatically included in the study. In addition, one village with a health post (CS) was randomly selected and included. Finally, from among the remaining villages in the catchment area of the CSI, four communities ≤10 km from the CSI and four communities >10 km from the CSI were randomly selected and randomized to order of participation. Women from the first 4 villages in each CSI catchment area (CSI-village, CS-village, and 2 additional villages) were enrolled with a target of approximately 16–20 women enrolled per village. If the sample size of approximately 77 women per CSI catchment area was not met by the first 4 randomized villages of each CSI, then additional villages on the randomization list of each CSI catchment area were included until the sample size was reached. A total of 88 communities were included in the present survey. Pregnant women were identified using the random walk method [16], with the starting point randomly selected for each village (market, primary school or mosque). Pregnant women at any week of gestation were eligible to participate in the survey if they provided written informed consent, had resided in the village for the previous six months and had no plans to move out of the study area within the coming two months. Women were ineligible if they had an illness warranting immediate hospital referral or were unable to provide consent due to mental disability. The NiMaNu Project was approved by the National Ethical Committee in Niamey (Niger: 005/2013/CCNE) and the Institutional Review Board of the University of California, Davis (USA) (IRB, University of CA, Davis: 447971). Consent materials were presented in both written and oral format, in the presence of a neutral witness. Informed consent was documented with a written signature or a fingerprint prior to enrollment in the study. The study was registered at www.clinicaltrials.gov as {“type”:”clinical-trial”,”attrs”:{“text”:”NCT01832688″,”term_id”:”NCT01832688″}}NCT01832688. As part of the baseline survey, each enrolled woman participated in two study visits, spaced approximately one month apart. The first visit occurred in the home, and the follow-up visit (i.e., visit 2) was conducted in a central village location. Information on socio-economic status (SES) and demographic characteristics of the woman and her household, pregnancy and health status, dietary practices, food security, and knowledge, attitudes and practices (KAP) pertaining to ANC and nutrition were collected via structured interviews by trained female fieldworkers at both study visits. Woman’s weight, height, mid-upper arm circumference (MUAC) and symphysis-fundal height (SFH) were measured in duplicate at both visits, to 50 g and 0.1 cm precision, respectively [17,18]. If the 2 measurements were >0.2 kg (weight) or >0.5 cm apart (height, MUAC, and SFH), a third measurement was taken; results represent the mean of the two closest measurements. Low MUAC was defined as <23 cm [19]. Dietary MN adequacy was assessed using the Minimum Dietary Diversity for Women (MDD-W) as a proxy indicator; the population of women who reported consuming at least five of ten defined food groups in the previous 24 h (using a list-based food frequency questionnaire) was considered to have a higher likelihood of MN adequacy [20]. Household SES was estimated using three proxy indices (housing quality, household assets, and household livestock), as previously described in detail elsewhere [21]. Household food insecurity, defined as the limited or uncertain access to adequate food of sufficient quality, was assessed using the Household Food Insecurity Access Scale (HFIAS) of the Food and Nutrition Technical Assistance/USAID [22]. IFA coverage was defined as the individual having received IFA supplements at any point during her current pregnancy. IFA adherence was defined as the individual having consumed IFA daily in the previous week. Gestational age was estimated as a weighted average of the following obtained information: reported last menstrual period (LMP; by estimated number of months, lunar cycles and/or proximity to a religious or cultural event), time elapsed since quickening, and two fundal height measurements taken approximately one month apart [23,24]. Trimester of pregnancy was defined as follows: 1st trimester, <13 weeks; 2nd trimester: ≥13 weeks to <27 weeks; and 3rd trimester, ≥27 weeks. Capillary blood samples were collected at visit 2 for the measurement of Hb in all women. In a sub-set of women, 7.5 mL venous blood samples were collected at the same time point for measurement of plasma ferritin, soluble transferrin receptor (sTfR), zinc (pZn), retinol binding protein (RBP), α-1-acid glycoprotein (AGP), C-reactive protein (CRP), vitamin B12, folate and histidine-rich protein II (HRP2) concentrations. According to procedures recommended by the International Zinc Nutrition Consultative Group (IZiNCG) [25,26], blood was drawn from the antecubital vein and collected in an evacuated, trace element free polyethylene tube containing lithium heparin (Sarstedt AG & Co., Numbrecht, Germany). In the field, samples were stored immediately between 4 and 8 °C until separation. Plasma was separated from heparinized blood at the Regional Hospital of Zinder ≤8 h from collection, by centrifuging at 3000 rpm for 10 min. Plasma was aliquoted in clear or amber (B12 and folate) microcentrifuge tubes and stored at −20 °C until shipment to the United States and Germany on high-salinity ice packs for laboratory analyses. Hb concentration was measured immediately after capillary blood collection with a HemoCue 201+ photometer (Hemocue AB, Angelholm, Sweden). Plasma ferritin, sTfR, RBP, CRP, and AGP concentrations were measured using a combined sandwich ELISA technique and pZn was measured colormetrically (WAKO Chemicals GmbH, Neuss, Germany) at the VitMin Lab (Willstaett, Germany) [27]. All samples were measured in duplicate and the inter-assay CV for the control sample ranged from 3.0 to 9.2%. Plasma B12 and folate concentrations were measured by enzyme-based immunoanalysis using a Cobas(R) e41 analyzer (Roche Diagnostics, Switzerland) and the reagents vitamin B12 (theoretical normality values 95% CI: 191–663 pg/mL) and folate III (4.6–18.7 ng/mL). Plasma HRP2 concentrations (indicative of current or recent malaria parasitemia) were measured using a commercially available CELISA kit (Cellabs Pty Ltd., Brookvale, Australia), following the manufacturer’s instructions. Plasma samples were measured in singlicate on plates with positive and negative controls provided by the manufacturer; samples with an absorbance value greater than the OD of the negative control +0.1 unit were considered positive for malaria antigen. Assuming a 50% prevalence of deficiency, the total sample size required to estimate the prevalence of each MN deficiency ±3.5% (95% CI) was n = 784. Venous blood for assessment of MN concentrations was collected from all eligible pregnant women participating in visit 2 of the NiMaNu Project until the required sample size was obtained. The study had a power of 80% to detect regression coefficients between continuous variables when the absolute value of the standardized coefficient was ≥0.122, assuming an intra-cluster correlation coefficient of 0.01. Descriptive statistics were calculated for all variables. Variables not normally distributed (ferritin, sTfR, folate, B12) were log transformed, and characterized by parametric distribution, prior to subsequent analysis. Biomarkers considered to be acute phase proteins or reactants (ferritin, pZn, RBP) were adjusted for elevated CRP and AGP to remove the confounding effect of sub-clinical infection and inflammation on the assessment of nutritional status and deficiency. A continuous regression method was used to adjust MN biomarker concentrations to the 10th percentile of CRP and AGP concentrations for the study-specific population (CRP = 0.38 mg/L; AGP = 0.23 g/L) [28,29]. MN deficiency cut-offs in pregnant women are not well established, especially in a setting with a high prevalence of sub-clinical infection and inflammation. Therefore, the following cut-offs were used for descriptive purposes only; all analyses were conducted using continuous MN concentrations to retain the maximum information available. Anemia was defined as Hb < 11.0 g/dL in the first and third trimesters and Hb < 10.5 g/dL in the second trimester [13]. Iron deficiency was variously defined as plasma ferritin <15 µg/L (unadjusted and adjusted) and sTfR > 8.3 mg/L [27,30]. Zinc deficiency was defined as pZn < 50 µg/dL [26]. Marginal vitamin A status was defined as RBP < 1.32 µmol/L [31]. Low and marginal B12 concentrations were defined as plasma B12 < 148 pmol/L and 148–221 pmol/L, respectively. Low folate was defined as plasma folate concentration < 10 nmol/L [32,33]. Multiple micronutrient (MMN) deficiency was calculated for each woman as the sum total number of micronutrients [iron (ferritin and/or sTfR), zinc, vitamin A (RBP), B12 and/or folate] for which she was categorized as deficient (range = 0–5). Elevated CRP and AGP were defined as concentrations >5 mg/L and >1 g/L, respectively. Associations between independent risk factors and outcome variables (Hb, plasma ferritin, sTfR, pZn, RBP, B12, and folate) were evaluated using bivariate generalized linear mixed models (SAS PROC GLIMMIX). The association between independent risk factors and MMN deficiency was evaluated using mixed-model logistic regression (SAS PROC GLIMMIX). Hierarchical clustering models used a random effect of village nested within CSI (i.e., cluster) and a fixed effect of region. Potential predictors included SES and demographic factors, obstetric history, KAP concerning pregnancy, and current nutritional and health status. All bivariate models were reconsidered including trimester of pregnancy as an additional independent variable to assess whether any of the significant associations in the bivariate models were attributable to variation in gestational age. Although all pregnant women in the survey catchment area were eligible for participation, only one participant was in her first trimester at visit 2; therefore models controlling for trimester of pregnancy are restricted to women in their second and third trimesters. Hypothesis driven multivariable generalized linear mixed models were constructed to explore relationships between malaria antigenemia, adolescence or primigravidae, and biomarkers of anemia and iron status. All statistical analyses were completed with SAS System software for Windows release 9.4 (SAS Institute, Cary, North Carolina, United States). Data are presented as mean (95% CI), geometric mean (95% CI), or β (95% CI) unless otherwise noted. β-coefficients from analyses using log transformed outcome variables are exponentiated regression coefficients and interpreted as percent increase/decrease (multiplicative); β coefficients from analyses using non-transformed outcome variables are interpreted as unit increase/decrease (additive). A p value < 0.05 was considered statistically significant. All models were evaluated for model assumptions, including normality of residuals, linearity and observations with undue leverage.