This was a case control study comparing different fetal outcomes and maternal characteristics of parturients with positive parasitemia (cases) and those that have negative parasitemia (controls).
Study population were booked parturients at 36 weeks gestation who had their antenatal care at the Alex Ekwueme Federal University Teaching Hospital, Abakaliki (AEFUTHA). The institution is a tertiary hospital in Ebonyi state and has delivery rate of about 3000 per annum. The study was carried out over a six months period between 1 June, 2018 and 30 November, 2018. As part of antenatal care education, booked women are counselled on the need to take their routine drugs and to prevent malaria using IPT and LLITNs. Women who have symptoms of malaria are actively treated as per departmental protocol.
Parturients who met the inclusion criteria (booked parturients with singleton pregnancy at 36 weeks gestation, asymptomatic of malaria infestation, and willing to participate in the study) were recruited by consecutive sampling technique. They were followed up till they presented in labour. A parturient was booked if she attended antenatal care in the study centre and had been reviewed with her routine antenatal investigations and also received at least one dose of IPT. Booked parturients receive IPT as a major component of their antenatal care. Exclusion criteria include unbooked women, women with symptoms and signs of malaria infection, multiple gestation, sickle cell disease, and retroviral disease, pregnancy complications like diabetes mellitus, hypertensive disorders of pregnancy, antepartum haemorrhage, and parturients who declined consent to the study.
The minimum sample size was determined using the formula for calculating sample size for case control studies 53
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n = minimum sample size
Zα = standard normal variate corresponding to 1.96 (at 5% type 1 error, and p\(cript>\) 0.05)
Zβ \(=\) standard normal variate for power or type 2 error = 0.84
m = number of control subjects per experimental subject = 1: 1
P1 = probability of event in experimental subjects = 18.2% (based on previous study8)
P0 = probability of event in controls = 37%8
p = (P1+mPo ) /( m+1)
Using a previously reported prevalence of placental parasitemia of 18.2% by Ibanga GI et al 8 ,
\(n=\frac{0.96\sqrt{(1 + /1)0.28(1-0.28)}}{}\frac{+ 0.84\sqrt{0.37 (1- 0.37 )/1 + 0.18 (1-0.82) }}{}\) = 86
To allow for attrition rate of 10%, the minimum sample size for this study was 95 on each arm.
The participants were counselled on the study and its objectives. Informed Consent was obtained and participants were recruited by consecutive sampling technique. Blood films for malaria parasite were performed for each participant. Based on the results, cases of parasitemia positive and controls of parasitemia negative women were raised. Their case notes were appropriately marked and they were followed up in the clinic till they presented in labour.
A designed proforma was used to collect information including maternal age, parity, gestational age, IPT usage, and use of LLITNs. The social class of the parturients was determined using the classification system proposed by Olusanya, Okpere, and Ezimokhai54. Birth weight was measured within one hour of delivery using an electronic weighing scale accurate to ± 10 gramme. Newborn was classified as normal birth weight (\(\ge\) 2.5kg) or LBW (\(\le\) 2.5 kg) according to WHO guidelines55. APGAR score of ≥ 7 in the 5th minute was taken as normal while a score of < 7 in the 5th minute was regarded as perinatal asphyxia. Perinatal asphyxia was classified as mild, if the APGAR score was 6, moderate if the score was between 4–5, whereas a score of 0–3 signified severe perinatal asphyxia56.
Pre-delivery, about 3ml of blood was collected from the peripheral vein of each participant, after aseptic procedure, into a pre-labelled sample bottle containing ethylene diamine tetra-acetic acid (EDTA) for estimation of packed cell volume (PCV) and for determination of intrapartum peripheral parasitemia. The PCV was done using micro-haematocrit method. Two non-heparinised capillary tubes for each subject, each with three- quarter of the tubes filled with blood taken from the 3ml of peripheral blood already obtained by venepuncture, were sealed at the distal end with plasticine. The capillary tubes containing blood samples were placed in a Hawksley microhematocrit centrifuge and spun at 3000 revolutions per minute for 5 minutes. The PCV was determined using a Hawksley hematocrit reader. The mean of the two values of the PCV was recorded and used. Subjects with PCV below 30% were regarded as being anaemic. This was based on the finding by Lawson that, in African women, no adverse effect occurred in them or their babies at a hematocrit level of 30% and above, hence his advocating for that value57. The WHO however defines anaemia in pregnancy as hematocrit of less than 33%58.
After delivery of the baby, 3 ml of blood was collected from the placenta end of the cord into an EDTA sample bottle for determination of malaria parasite (congenital malaria). After delivery of the placenta, a section of it measuring about 2cm × 2cm × 1cm was biopsied from the maternal surface using a scalpel. The specimen was put in a plane bottle, fixed in 10ml neutral buffered formalin, and sent to the histology laboratory for processing. The blood specimens were properly labelled and sent to the laboratory for analysis.
Thick and thin blood films preparation and staining were achieved with a drop of cord or peripheral blood placed on the middle portion of a clean 76mm × 25mm microscope slide. A thick film was made by dispersing the drop to fill a large circle at the middle of the slide. A thin film was prepared to be thinner than thick film, in such a way that newsprint could be read through it. The smears were allowed to air-dry. The thin blood smears were fixed in absolute ethanol for 5 seconds before being placed on the rack for 2 minutes. The slides were stained in a trough containing 3% Giemsa stain for 30 seconds and thereafter rinsed in a buffer solution to maintain a PH of 7.2. The slides were allowed to air-dry before microscopic examination. The stained smears were examined under ×100 oil immersion lens of a binocular light microscope. The diagnosis of malaria was based on identification of asexual stages of Plasmodium species on the thick blood film, while thin film was used for species identification. Parasite density was determined by counting the number of parasites per High Power Field (HPF) and ranged from + (1–10 parasites per 100 HPF), ++ (> 10 parasites per 100 HPF), +++ (1–10 parasites in one HPF), and ++++ (> 10 parasites in one HPF). The slides were reported as negative if no parasite was identified per 100 power fields3.
The placental biopsies were processed using standard techniques. Sections about 5µm thick were made and stained with haematoxylin and eosin (H \(\infty\) E) and then examined using light microscopy by the histopathologists for the presence of malaria parasites, malaria pigments, placental morphology, and signs of infections other than malaria. Placental malaria was defined as the presence of parasites and/or pigments in the monocytes in the intervillous space of the placenta as described by Bulmer et al22.
Primary outcome measure was prevalence of placental malaria and the association between placental malaria and maternal anaemia, while the secondary outcomes were:
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Association between placental malaria and dose of IPT
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Relation between placental malaria and fetal outcomes like birth weight, NICU admission, stillbirth, and congenital malaria.
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Relation between parity and placental malaria
STATISTICAL ANALYSIS
Data was collected, tabulated, and then statistically analysed using Statistical Product and Service Solutions (SPSS) software (version 20, Chicago IL, USA). Numerical variables were presented as mean and standard deviation (Mean ± SD), while categorical variables were presented as numbers and percentages. Statistical comparisons and test of significance between positive and negative groups were calculated using Chi-square test (X2) for categorical variables. The strength of association between variables was quantified using Odds ratio (OR) and Confidence (CI). Logistic regression was used to analyze factors associated with the main outcome variables. A p-value \(cript>\) 0.05 was considered statistically significant.