The risk factors for maternal anemia (hemoglobin level less than 110 g/L) were studied in human immunodeficiency virus-negative pregnant women in Benin at the time of first antenatal visit and prior to any prevention. Data for the first 1,005 pregnant women included in a multicentre randomized controlled trial were analyzed. Anemia was common (68.3%), and malaria and helminth infestations were prevalent in 15.2% and 11.1% of the women. A total of 33.3%, 31.3% and 3.6% of the women were iron, folic acid and vitamin B12 deficient, respectively. These parasitic infections and nutrient deficiencies were associated with a high risk of anemia. Twenty-one percent, 15%, 12%, 11% and 7% of anemia were attributable to malnutrition, malaria, iron, folic acid deficiencies, and helminth infestations, respectively. Most anemia was caused by factors that could be prevented by available tools, stressing the need to reinforce their implementation and to evaluate their effectiveness throughout the course of the pregnancy. Copyright © 2012 by The American Society of Tropical Medicine and Hygiene.
The study was a cross-sectional survey conducted at the inclusion of the first 1,005 pregnant women participating in the MiPPAD trial. The study was conducted in the district of Allada, a semi rural area located 50 km north of Cotonou, the economic capital of Benin. The entire district is made of 12 sub-districts, 84 villages, and a total of 91,778 inhabitants. The study participants were recruited in three maternity clinics in three sub-districts: Allada, Attogon, and Sékou. There are several ethnicities living in the district of Allada, the most important being Aïzo, a local ethnic group. Malaria is perennial and Plasmodium falciparum is the most common species. There are two high transmission peaks from April though July and October through November. Transmission is low during the rest of the year. The study population was composed of human immunodeficiency virus (HIV)–negative pregnant women (less than 28 weeks of gestational age) residing in the district of Allada, who attended the ANV at any of the three maternity clinics for the first time during January 2010–May 2011. The eligibility criteria included no intake of IPTp, iron, folic acid, vitamin B12, or anti-helminthic treatment, which are part of the ANV package in Benin, since the beginning of the pregnancy. All women were offered confidential pre-test HIV counseling and thereafter informed consent was obtained for blood sample collection. All pregnant women who attended any of the three maternity clinics for ANV were approached to participate in the study. After informed consent was obtained, they were screened for inclusion and exclusion criteria and socio-demographic data such as age, parity, area of residence, marital status, level of education, occupation, and information useful to determine the socioeconomic level were recorded. They were clinically examined and gestational age (assessed by measuring the fundal height), mid upper-arm circumference, weight, and height were evaluated. Weights were measured to the nearest 0.1 kg by using an electronic scale (to ± 100 grams; Seca Corp., Hanover, MD) and heights to the nearest 0.1 cm by using a bodymeter device (Seca® 206 Bodymeter; Seca Corp.). Weights and heights were measured by two nurses, and the mean of the two measurements was calculated for each participant. Information on previous pregnancies and children and history of chronic diseases were also recorded. Eight milliliters of of venous blood was obtained from each participant, of which 4 mL was collected into a tube containing dipotassium EDTA and 4 mL was collected into an iron-free dry tube. Blood samples were collected before the administration of hematinics, folic acid, IPTp, or antihelminthic drugs as part of antenatal prophylaxis. Containers were given to the women to collect feces for intestinal helminth infestations. These containers were collected the next day within the first six hours after defecation. The hemoglobin level was measured with a Hemo-Control photometer (EKF Diagnostics, Barleben/Magdeburg, Germany) device with 10 μL of blood. Daily calibration of the Hemo-Control device was performed by laboratory technicians. In addition, an external quality control was made by sending one of 10 consecutive samples to the Allada Central Hospital laboratory, where dosages were determined by using a hematology analyzer (Erma Laboratory, Tokyo, Japan). Women with a hemoglobin concentration less than 110 g/L were treated according to the national guidelines, i.e., 200 mg of iron twice a day for mild and moderate anemia (hemoglobin levels = 70 g/L and less than 110 g/L, respectively), and treated locally or referred to the tertiary hospital of the district in case of severe anemia (hemoglobin level less than 70 g/L). Furthermore, women were advised to consume iron-rich foods, such as beef, eggs, and green leafy vegetables. Hemoglobin genotypes were determined by using alkaline electrophoresis on cellulose acetate (Helena Laboratories, Beaumont, TX) with 50 μL of blood. Serum ferritin, folic acid, and vitamin B12 concentrations were measured by using an AxSym Immuno-Assay Analyzer (Abbott Laboratories, Abbott Park, IL) with 500 μL of serum. C-reactive protein (CRP) concentrations were determined by using a rapid slide test (CRP Latex; Cypress Diagnostics Inc., Campbellville, Ontario, Canada) to correct for increased ferritin levels associated with inflammatory syndromes.13 Detection of HIV detection is part of the first ANV package in Benin. The Determine (HIV 1 and 2 Kit; Abbott Laboratories) and Bioline (HIV 1 and 2 3.0 Kit; Bioline, Taunton, MA) rapid tests were used to detect HIV infections by using a serial testing algorithm. HIV-positive tests were sent to the District of Allada Central Hospital for confirmation by using an enzyme-linked immunosorbent assay. When an HIV-positive result was confirmed, she was treated according to the Benin National Program against HIV and Acquired Immunodeficiency Syndrome Guidelines (Program National de Lutte Contre le VIH/SIDA). The Lambaréné technique was used to assess malarial infection. Ten microliters of blood was spread on a rectangular area of 1.8 cm2 (1.8 cm × 1 cm) of a slide. The slide was stained with Giemsa and read at a magnification of 1,000 × with an oil immersion lens. A multiplication factor was applied to the average parasitemia/field to determine the number of parasites/microliter, The Lambaréné method detection threshold has been estimated to be 5 parasites/μL.14 Infestations by helminths were assessed by using the Kato-Katz concentration method15 (Vestergaard Frandsen, Lausanne, Switzerland). Because fecal samples must be processed and examined extemporaneously, no external control was used, but the slides were read by two laboratory technicians independently. For the definition of severe anemia, we preferred to use a more discriminating 80 g/L threshold than the 70 g/L recommended by the Beninese Ministry of Health. Anemia was defined as a hemoglobin concentration less than 110 g/L. Severe, moderate, and mild anemia were defined as hemoglobin concentrations less than 80 g/L, 80–99 g/L, and 100–109 g/L, respectively. Iron deficiency was defined as a serum ferritin concentration less than 12 μg/L or as serum ferritin concentration of 12–70 μg/L in the context of inflammatory syndrome.16 Iron deficiency anemia was defined a hemoglobin concentration less than 110 g/L with ID. Inflammation was defined as positive CRP result, i.e., CRP concentration less than 5 mg/mL.17 Folic acid deficiency was defined as a serum folic acid concentration less than 6 ng/mL. Vitamin B12 deficiency was defined as a vitamin B12 serum concentration less than 150 pg/mL. Intestinal helminth infestations were diagnosed by the presence of intestinal helminth eggs in the fecal sample. Eggs were counted (number of eggs/gram of feces). From the end of the first trimester of gestation, pregnant women gain 1 kg per month until delivery.18 We used the gestational age at inclusion to estimate the weight (in kilograms) that women were supposed to gain since the beginning of the pregnancy. This amount was then subtracted from the weight on the day of inclusion to obtain a rough estimate of the weight before pregnancy (Table 1). Estimation of body mass index at beginning of pregnancy in Benin* Data were collected in parallel to the MiPPAD study by using the source documents from the trial. They were double-entered into an Microsoft (Redmond, WA) Access database and analyzed by using with Stata Software for Windows version 11.0 (StataCorp LP, College Station, TX). We first described the baseline characteristics of the women and the factors potentially influencing their hemoglobin levels. Means of hemoglobin concentrations were computed and compared by using the Students t-test. Relationships between anemia and risk factors were studied by using univariate logistic regression. A multiple logistic regression was performed that took into account all factors with P values < 0.20 by univariate analysis. Population-attributable risks were also calculated to estimate the proportion of anemia that could be prevented by the elimination of each of the assessed risk factors. A P value below 0.05 was considered statistically significant. This study was approved by the Ethics Committee of the Faculty of Medicine of Cotonou, Bénin. Before each inclusion, the study was explained in local language to the participant and her voluntary consent was obtained. In case the woman could not read, an impartial witness was involved in the process. In addition to the assent of minors, consent was obtained from the parents or legal guardians. Women were free to interrupt their participation at any time during the study.
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