Complement activation, placental malaria infection, and birth weight in areas characterized by unstable malaria transmission in central Sudan

A cross-sectional study was conducted during August to December 2011 (the rainy and
post-rainy season) in the labor ward of the Medani Maternity Hospital. The area of
this study is characterized by unstable malaria transmission. P. falciparum is the main malaria parasite species in the area and transmission occurs during the
rainy (July to September) and post-rainy season 17]. Medani Maternity Hospital is a referral tertiary hospital caring for women who receive
antenatal care at the hospital or are referred from other health centers and hospitals,
and women who live close to the hospital facility. High-risk pregnancies are referred
to the hospital. However, many women without a high-risk pregnancy deliver at this
hospital.

The total sample size was calculated to have over 80% power to detect a difference
of 5% at ??=?0.05. We assumed that 10% of women might not respond or have incomplete
data.

After obtaining signed informed consent from the patients, information on socio-demographics,
history of obstetrics, medical history, antennal attendance characteristics, and bed
net use was gathered using structured questionnaires. Body mass index was calculated
by measuring maternal weight and height, which was expressed as weight (kg)/height
(m)². Newborns were weighed immediately following birth using the Salter scale and the
sex of each newborn was recorded.

Giemsa-stained blood smears for light microscopy

Maternal, placental, and cord blood films were prepared. Slides were stained by 10%
Giemsa and the number of asexual parasites was counted per 200 leukocytes, assuming
a leukocyte count of 8000 leukocytes/?l (for thick films) or per 1000 red blood cells
(for thin films). Blood films were considered negative if no parasites were detected
in 100 oil immersion fields of a thick blood film, which was double-checked in a blind
manner by an expert microscopist. Maternal hemoglobin concentrations were estimated
by the HemoCue hemoglobinometer (HemoCue AB, Angelhom, Sweden).

The blood (maternal and cord) was then allowed to clot and centrifuged for 10 minutes
at 3000 rpm and the serum was separated and stored at – 20°C till the analyses.

Placental histology

The details of how placental histology was performed have been mentioned previously
8],18],19]. Briefly, a 3-cm³ sample was obtained from the maternal surface approximately half the distance between
the umbilical cord and the edge of the placenta. Each biopsy sample was immediately
placed in 10% neutral buffered formalin. Buffer was used to prevent formation of formalin
pigment, which has similar optical characteristics and polarized light activity as
malaria pigment 20]. All of the biopsy samples were stored at room temperature until histology was performed.
The placental biopsy samples were then processed and were embedded in paraffin wax,
by standard techniques. In every case, paraffin sections that were 4-mm thick were
stained with hematoxylin-eosin and Giemsa stains. Placental malaria infection was
characterized using histology as previously described by Bulmer et al. as follows
21]: uninfected (no parasites or pigment), acute (parasites in intervillous spaces),
chronic (parasites in maternal erythrocytes and pigment in fibrin, or cells within
fibrin and/or chorionic villous syncytiotrophoblast or strom), and previous (no parasites,
and pigment confined to fibrin or cells within fibrin). The slides were examined by
a pathologist who remained blind regarding the clinical characteristics of these samples.

ELISA for measuring TCC levels

Maternal and cord serum levels were measured using a human TCC ELISA kit (Biotain
Pharma Co., Ltd., Xiamen City, Fujian Province, China) by following the manufacturer’s
protocol.

Statistical analysis

Data were entered into a computer using SPSS for windows (version 16.0). Continuous
data (including TCC levels) were normally distributed and were compared between groups
using Student’s t test. Multivariate analyses were performed using binary models for placental malaria
infection as the dependent variable and linear models with hemoglobin, birth weight,
and TCC (maternal and cord levels) levels as continuous dependent variables. Socio-demographic
characteristics, education, antenatal care, residence, and placental malaria infections
were the independent predictor of interest. Odds ratios (OR) and 95% confidence intervals
(CI) were calculated and a P value of 0.05 was considered significant.

Ethics

The study received ethical clearance from the Research Board at the Faculty of Medicine,
University of Khartoum, Sudan.