The study was carried out in Yemetu-Adeoyo, a semi-urban community in Ibadan in south-west Nigeria where transmission of malaria is perennial. The hospital in this community, Adeoyo Maternity Hospital (AMH), is the oldest maternity hospital in Nigeria, dating from 1927. There are over 4,000 deliveries annually. Ethical approval for the study was obtained from the joint University of Ibadan/University College Hospital ethical committee and the University of Manchester Ethics committee.
Study procedures, follow-up, delivery and recruitment of babies
Healthy pregnant women aged 18-45 years presenting at AMH before 36 weeks' gestation and all babies born ≥ 37 weeks' gestation were eligible. Women who were HIV positive or had sexually transmitted infections at booking, those with pre-term deliveries, as well as those with multiple pregnancies or with chronic diseases, such as hypertension and diabetes were excluded. Babies with known syndromes, metabolic defects, major congenital abnormalities or severe birth trauma were also excluded.
Standard operating procedures (SOP) were developed and women were enrolled over one year to cover both wet and dry seasons. The study protocol and the rationale for the study were explained carefully in appropriate language, most commonly Yoruba or English, with questions answered as needed and written informed consent was obtained from all participants. After the delivery of their babies, another written informed consent was also obtained for the participation of the babies in the study.
Information on socio-demographic, obstetric, family, and health history including malarial frequency and use of anti-malarial drugs, was collected. All participants were issued prescriptions of sulphadoxine-pyrimethamine for intermittent preventive therapy (IPT) for malaria according to standard hospital practice.
Antenatal visits followed routine practice with frequency of attendance determined by gestational age. Standardized measures of anthropometry were carried out on all women at every visit until delivery. Maternal weight was measured to the nearest 0.1 kg on a SECA scale, and height on a stadiometer, both without shoes according to the SOP.
Visit schedule and blood measurements
At booking, 2 ml of blood was collected into an EDTA tube for full blood count and blood films for malaria parasites (MP) were prepared, stained with 3% Giemsa at pH 7.2. Repeat thick blood films for MP were obtained at every subsequent visit and at delivery. In addition, the placenta was weighed, turned to the maternal surface, cotyledons exposed and 1 ml of blood obtained from the inter-villous space for a placental malarial blood smear.
Blood films were examined for MP under light microscopy and recorded as negative only after 100 or more high-power microscope fields had been scanned. In those with malaria, parasite densities were determined by counting the number of parasites (np) among 200 leucocytes on the thick film using the following equation: Absolute parasite counts = (np x TLC)/200 where TLC = subject's total leucocyte count. For quality control, 30% of negative samples and 40% of positive samples were re-examined by two different, trained microscopists.
At the second antenatal visit, of 624 healthy pregnant women enrolled, 467 women gave their consent for fasting blood to be taken for biochemical markers while 396 of them had samples for malaria parasites. At delivery, 27 were excluded due to four maternal deaths (0.9%), 11 still births (2.4%), five miscarriages (1.1%) and seven neonatal deaths (1.5%), leaving 436 mother-baby pairs.
Cord blood was collected from the umbilical vein on the foetal surface of the placenta. Plasma was separated by centrifugation at 3,000 rpm and 4°C for 10 minutes and aliquoted into microtubes and stored at -80°C prior to lipid, glucose, insulin, TNF and IGF-I (cord blood only) assays. Cord glucose could not be measured immediately and so was not included in the assays.
Based on availability of complete maternal and cord blood samples, 187 mother-baby pairs were selected. There were no significant differences in the socio-demographic and clinical data of excluded women.
Malaria was defined as the presence of asexual blood stages of Plasmodium falciparum in peripheral blood. There were two definitions of malaria:
a) 'Malaria at recruitment' = Malaria at second antenatal visit when blood for biochemical markers was also obtained. This was only used in analyses of effects on maternal biochemical markers at the same time-point. There were 72 blood smears positive for malaria parasites out of the 396 samples giving the prevalence of malaria as 18%.
b) 'Maternal malaria' = Malaria parasitaemia present at least once during pregnancy and/or at delivery (n = 211). These included (i) malaria parasitaemia in peripheral blood at least once during pregnancy (n = 138); and (ii) at delivery and/or in the placenta (n = 73). This was used in analyses of relationships with cord parameters and effects on birth indices.
Out of 187 mother-baby pairs with complete maternal and cord blood samples, 97 had malaria giving a prevalence of 52%.
Anaemia was defined as a packed cell volume (PCV) < 30%.
Total cholesterol (TC), High-density lipoprotein-cholesterol (HDL-C) and triglyceride concentrations were determined by using standard enzymatic procedures on an automatic analyser (COBAS MIRA/HITACHI 704 - Roche Diagnostics, Germany). The inter- and intra-assay coefficients of variation (CVs) for all parameters were < 5%. Low-density lipoprotein- cholesterol (LDL-C) was calculated using the Friedewald formula .
The normal values of lipids (mmol/L) in adult women are TC 3-5, HDL-C 1.2-2.2, LDL-C 2-3 and TG 0.6-1.68.
Maternal glucose was measured by the glucose oxidase method using a commercial kit (Randox, Crumlin, UK) on a YSI 2300 stat plus analyser (YSI, Farnborough, Hants, UK). The intra-assay CV was 1.5% at 4.1 mmol/L, and inter-assay CVs were 2.8% and 1.7% at 4.1 and 14.1 mmol/L respectively. Normal fasting glucose values are 3.9-6 mmol/L.
Insulin was measured by ELISA, using a commercial kit (Mercodia, Uppsala, Sweden). Assay sensitivity was 1 mU/L. Intra-assay CVs were 3.4% and 3.2% at 11 and 154 mU/L, and equivalent inter-assay CVs were 3.6% and 2.9%.
IGF-I and TNF were measured using Immulite 2000 assays (DPC, Lumigen Inc, Southfield, UK). Respective assay sensitivities were 25 μg/L and < 0.09 ng/L. Inter-assay CV values for IGF-1 at 48.9 and 158.5 μg/L were 7.6 and 9.2%. For TNF, intra-assay CVs were 6.7% and 5.3% at 6.3 and 19 ng/L, and the inter-assay CVs at 6.1 and 18.6 ng/L were 8.2 and 9.7% respectively.
Babies were measured within 72 hours of birth. They were weighed naked to the nearest 0.1 kg and length measured on an infant stadiometer from crown to heel to the nearest 0.1 cm. Occipito-frontal circumference (OFC) was taken around the widest circumference of the head using a non-stretchable tape. Skinfold thicknesses (triceps, biceps, sub-scapular, and suprailiac) were measured using Holtain calipers on the left side to the nearest 0.1 mm. Measurements were obtained in duplicate or triplicate if disagreeing by > 15%.
Validity of anthropometric measurements
Based on the WHO Manual (1995), three nurses, already proficient in paediatric venipuncture, were trained in anthropometry methods. They carried out all measurements on the same equipment throughout the study. They also had three-monthly protocol-refresher training sessions and used training videos to minimize inter-observer and within-observer errors.
Data were analysed using SPSS version 14 (SPSS Inc, Chicago, IL). Results were expressed as mean (SD) or median (IQ range), using Student t/Mann Whitney and Chi square tests for associations between maternal and infant clinical characteristics and malaria. Levels of insulin, IGF-I and TNF were skewed and, therefore, tested non-parametrically, while lipids and glucose levels were normally distributed and tested parametrically. Correlations were assessed by Spearman's test. All tests were two-sided and p values < 0.05 were considered significant.
Simple linear regression was used to assess the determinants of infant size, indicating those with p < 0.001, p
< 0.01 and < 0.05. Due to high co-linearity between biochemical parameters, a stepwise method of model selection was used to identify the key variables.