Maternal anemia in pregnancy: Assessing the effect of routine preventive measures in a malaria-endemic area

We investigated the effectiveness of routine preventive measures for anemia in Beninese pregnant women during pregnancy. Anemia (hemoglobin < 110 g/L) was common: 68.3% at first antenatal visit (ANV1), 64.7% at second antenatal visit (ANV2), and 40.6% at delivery. Parasitic infections and nutritional deficiencies were the most preventable causes. After intermittent preventive treatment (IPTp) and antihelminthic treatments, malaria prevalence decreased from 15.1% (ANV1) to 4.0% (ANV2) and increased again to 9.6% at delivery. Helminth infections dropped from 11.1% (ANV1) to 7.2% (ANV2) and 2.4% at delivery. Malaria was associated with lower mean hemoglobin on ANV1 and delivery, and iron deficiency was associated with lower mean hemoglobin on ANV1 and ANV2. IPTp and antihelminthic treatments were efficacious to clear parasitic infections and improve hematologic status, whereas the effectiveness of daily iron and folic acid supplements to correct iron and folate deficiencies and decrease anemia was less marked, possibly because of lack of compliance. Copyright © 2013 by The American Society of Tropical Medicine and Hygiene. We followed a cohort of 1,005 pregnant women participating in Malaria in Pregnancy Preventive Alternative Drugs (MiPPAD; http://clinicaltrials.gov/ct2/show/{"type":"clinical-trial","attrs":{"text":"NCT00811421","term_id":"NCT00811421"}}NCT00811421), a randomized trial of IPTp with either SP or mefloquine (MQ), from early pregnancy until the time of delivery. The study site and population have been described elsewhere.8 Briefly, the study was conducted in the district of Allada, a semirural area located in southern Benin. Malaria is perennial, and Plasmodium falciparum is the most common species. There are two high transmission peaks: from April to July and from October to November. The MiPPAD study population was composed of human immunodeficiency virus (HIV) -negative pregnant women of less than or equal to 28 weeks gestational age who attended one of three study maternity clinics of the area for the first time between January of 2010 and May of 2011. The eligibility criteria included no intake of IPTp, iron, folic acid, vitamin B12, or antihelminthic treatment, which are part of the ANV package in Benin, since the beginning of the pregnancy. Two doses of IPTp (1,500/75 mg SP per dose or 15 mg/kg MQ per dose) were administered on ANVs. The second dose of IPTp was given at least 1 month apart from the administration of the first dose. On the day of inclusion, each woman received a long-lasting insecticide-treated net that was replaced in case of damage or loss during the follow-up. Women were also systematically given 600 mg albendazole to be taken at home (100 mg two times per day for 3 days) according to the guidelines of the Beninese Ministry of Health. In addition, supplements of oral ferrous sulfate (200 mg per day) and folic acid (5 mg per day) were given to the women for home treatment (Figure 1). Pregnant women found to have a Hb concentration below 110 g/L were treated according to the severity of anemia (i.e., 200 mg oral ferrous sulfate two times per day for mild or moderate anemia when Hb was between 70 and 110 g/L) and referred to the tertiary hospital of the district in case of severe anemia (Hb < 70 g/L). All the medications prescribed to the women during their participation in the study were free of charge. Study procedures. At enrolment (ANV1), all pregnant women who attended any of the maternity clinics for ANV were approached to participate in the study. They were screened for inclusion and exclusion criteria, and sociodemographic data, such as age, parity, area of residence, marital status, level of education, occupation, and socioeconomic characteristics (sanitation in the house, personal means of transportation, possession of fridge or television, and connection to electricity), were recorded. ANV1 was also the occasion to administer antiparasitic treatments and nutritional supplements to the women. At ANV1, the woman was examined, and parity, gestational age, middle upper arm circumference, weight, and height were recorded. Medical history, including history of previous pregnancies, history of any known disease (such as high blood pressure, diabetes, or asthma), and information on previous children (birth weight, gestational age at delivery, and notion of anemia during previous pregnancy), was also recorded. At the time of ANV2, at delivery, and during the unscheduled visits, gestational age, middle upper arm circumference, weight, and height were measured again. The second intake of IPTp was also given on ANV2. Weights were measured to the nearest 0.1 kg using an electronic scale (SECA France, Semur-en Auxois, France), and heights were measured to the nearest 0.1 cm with a SECA bodymeter device (SECA France). At ANV1, ANV2, and delivery, 8 mL venous blood were collected from each participant; 4 mL were dispensed into a dipotassium (ethylenedinitrilo)tetraacetic acid (EDTA) tube, and 4 mL were dispersed into a dry iron-free tube. A container was also given to the woman to collect stools in search of intestinal helminths. These containers were collected the next day by the study nurses within the first 6 hours after emission. At delivery, a placental blood smear was performed to look for placental malaria (Figure 1). The study sample examination techniques have been described elsewhere.8 Hb rate was measured with a Hemo_Control photometer (EKF Diagnostics, Magdeburg, Germany) on 10 μL blood. Hb genotypes were determined by alkaline electrophoresis on cellulose acetate (Helena Laboratories, Mount Waverley, Victoria, Australia) on 50 μL blood. Serum ferritin and vitamin B12 concentrations were measured using a microparticle enzyme immunoassay (MEIA) method. A fluorescence polarization immunoassay (FPIA) technique was used to determine folic acid concentrations with an AxSym Immuno-Assay Analyzer (Abbott Diagnostics, Frankfurt, Germany). C-reactive protein (CRP) concentrations were determined with a rapid slide test (Cypress Diagnostics, Langdorp, Belgium). HIV detection is part of the first ANV package in Benin. Determine Kit HIV 1 and 2 package insert (Alere Orgenics, Paris, France) and SD Bioline Kit HIV 1 and 2 3.0 package insert (Umhlanga, Kwazulu Natal, South Africa) rapid tests were used to detect HIV infections with a serial testing algorithm. Lambaréné technique was used to assess malarial infection. It consists of spreading 10 μL blood on a slide's rectangular area of 1.8 cm2 (1.8 × 1 cm). The slide is stained with Giemsa and read at 100× oil immersion. To assess parasite density, a multiplication factor is applied to the average parasitemia per field to get a number of parasites per microliter. Lambaréné method detection threshold has been estimated to 5 parasites/μL. Infestations by helminths were assessed using the Kato–Katz concentration method (Vestergaard Frandsen, New Delhi, India). Anemia was defined as Hb below 110 g/L. Severe, moderate, and mild anemia were defined as Hb concentrations less than 80 g/L, between 80 and 99 g/L, and between 100 and 109 g/L, respectively. Iron deficiency (ID) was defined as serum ferritin < 12 μg/L or serum ferritin between 12 and 70 μg/L in the context of inflammation defined as a positive CRP (i.e., CRP concentration > 5 mg/mL). Iron deficiency anemia (IDA) was defined as Hb < 110 g/L with ID. Folic acid deficiency was defined as a serum concentration below 6 ng/mL. Vitamin B12 deficiency was defined as a serum concentration below 150 pg/mL. Intestinal helminth infestations were diagnosed by the presence of intestinal helminth eggs in the stool sample. Eggs were counted as number of eggs per 1 g stool. Data were entered and analyzed with ACCESS 2003 and STATA 11.0 Softwares for Windows (Stata Corp, College Station, TX). We first described the baseline and general characteristics of the women at each IPTp administration and delivery. The variations of mean Hb between ANV1, ANV2, and delivery were assessed by a Kruskal–Wallis test. We compared the variation of the proportions between ANV1 and ANV2 or between ANV1 and delivery with a McNemar test. The effectiveness of preventive measures was assessed at ANV2 and delivery by studying the variations of the risk factors found at ANV1 before any prevention. At each time point (ANV1, ANV2, and delivery), we estimated the association of the risk factors with the Hb concentration. Means were compared with Student or Mann–Whitney non-parametric tests as appropriate. All variables with P values less than 0.2 were then included in a multilinear regression. The impact of preventive measures on the risk of maternal anemia was appreciated by using a univariate logistic regression. Thereafter, all variables with P values below 0.2 were included in a multivariate logistic regression for each ANV. The previous analyses investigated the association between different risk factors and Hb concentrations on each visit and at the time of delivery, but they did not take into account the evolution of Hb with time throughout pregnancy. Assuming that successive Hb measurements in the same individual are correlated and dependent on gestational age, the data presented a hierarchical two-level structure, where Hb measurements (level 1) were clustered within women (level 2). We then analyzed our data using a linear mixed model with a random intercept and a random slope, which is specified in the equation Hemoglobin (ij) is the ith Hb measurement of woman j. β0 is the intercept. Xqj is the q explicative variables of woman j with their associated coefficients βq. μ0j is the random intercept corresponding to the woman-to-woman variation in Hb level [μ0j − N (0, π00)]. μ1j is the random slope corresponding to the variation in Hb level throughout time (gestational age in weeks), and ∈(ij) is the residual variation [∈(ij) − N (0, σ2)]. We assumed that random effects [μ0j and ∈(ij)] were independent. Fixed effects parameters were estimated using the maximum likelihood method, and variance components were estimated using the restricted maximum likelihood method. All variables with P < 0.20 in the univariate analyses were included in the model. Statistical significance was set at P < 0.05. This study was approved by the Ethics Committee of the Faculty of Medicine of Cotonou, Benin. Before each inclusion, the study was explained in the local language to the participant, and her voluntary consent was obtained. In the case that 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 in the study.