Study area
This study was conducted in four adjoining administrative areas, the Kintampo North Municipality, Kintampo South District, Nkoranza South Municipality, and Nkoranza North District, located in the Bono East Region, within the forest-savannah, transitional ecological zone in the middle belt of Ghana (Fig. 1). The Kintampo districts have a surface area of 7162 sq km, an approximate resident population of 150,000 and 4000 births per year [14]. Mean monthly temperature ranges from 18 to 38 °C while rainfall averages 1250 mm per annum. The main vectors for transmission of malaria are Anopheles gambiae and Anopheles funestus and the transmission is perennial, but peaks between April and October. The annual entomological inoculation rate was 269 infective bites per person per year in 2005 [13, 15], but is currently likely to be less. The Nkoranza districts have an approximate surface area of 2400 sq km and resident population of 130,000.
Study design
This was an analysis of baseline data collected from pregnant women enrolled into a cohort study evaluating the effectiveness and safety of four or more doses of IPTp-SP. Eligibility criteria included pregnant women of any gravidity who attended the antenatal care (ANC) clinic for the first time, who had a gestational age 13–22 weeks, were aged 18 years or above, were known negative for human immunodeficiency virus (HIV) infection, and were willing to participate in the study (demonstrated by signing or thumb-printing an informed consent form).
Enrolment of participants
Enrolment of study participants was done at ANC clinics within the study area from July 2017 to March 2019. A general introduction on the nature of the study was given to the pregnant women by the nursing staff at the ANC during their health talks. An initial screening was done to determine eligibility. For those meeting the inclusion criteria, a member of the study team then explained the study further including what an informed consent is, why the study was being done, what it involved in terms of benefits, risks, compensation, confidentiality, right to refuse to be part of the study, and the right to still receive all routine ANC services. Following collection of informed consent, eligible study participants had a questionnaire completed which included information on demographics, obstetric history, socio-economic status (SES), malaria symptoms, anti-malarial drug use, insecticide-treated bed net (ITN) use, and use of other malaria vector preventive measures.
Sample collection
Prior to commencement of IPTp-SP, a blood sample (2 mL) was collected by a trained member of the study team using sterile venepuncture and transferred into a K2-EDTA tube (BD, Berkshire, UK) for a full blood count. Samples were kept in a transport box containing cold packs and transported to the laboratory in Kintampo for analysis.
Haematological analysis
Haemoglobin estimations were performed using K2-EDTA anticoagulated blood on a validated and calibrated Horiba-ABX Micros 60 or Pentra 60 C+ haematology analyser (Horiba-ABX, Montpellier, France) within 8 h of blood draw in the Haematology Unit of the Kintampo Health Research Centre (KHRC) Clinical Laboratory. In addition to regular user maintenance, routine servicing of the analysers was performed quarterly. Three levels of quality control samples (low, normal and high) were tested daily prior to analysis of study samples to ensure the reliability of results. The laboratory also participated in monthly external quality assessment schemes organized by the UK National External Quality Assessment Scheme (UK NEQAS, Watford, UK).
Malaria microscopy
Preparation and reading of malaria blood slides was done in the Parasitology Unit of KHRC Clinical Laboratory, as described by Swysen et al. [16] and WHO guidelines for preparation, staining and reading of malaria blood slides were followed [17]. Briefly, 6 µL and 2 µL of blood was used to prepare a thick smear and a thin smear, respectively, on the same slide using a template and the smears were then air-dried. Thin smears were fixed with absolute methanol, allowed to air-dry and both thick and thin smears were stained with 10% Giemsa stain (BDH Laboratory Supplies, Poole, Dorset, UK) in phosphate-buffered water (pH 7.2) for 10 min. Stained smears were air-dried and thick films were examined using an Olympus CX21 Microscope (Olympus, Tokyo, Japan) with × 10 eyepieces and ×100 oil immersion objectives to determine the presence, species and stages of any parasites that were present. The thin smear was used for confirming species and counting of parasites if more than 100 parasites were seen in the first field in the thick smear. Parasite density (parasites per µL of blood) was calculated using participants’ white blood cell (WBC) or red blood cell (RBC) count determined on an automated haematology analyser. A minimum of 100 fields were examined before a slide was recorded as negative. Each slide was independently examined by two microscopists who were certified by South Africa’s National Institute for Communicable Diseases (NICD) and/or the World Health Organization. The final result was determined as described by Swysen et al. [16]. As part of quality assurance, each batch of the 10% Giemsa stain was quality controlled by staining a known positive and negative smear prior to staining smears of study participants. Only certified malaria microscopists were allowed to read and report results for the study.
Sample size calculation
A total of 1655 pregnant women who were enrolled in the main study designed to evaluate the impact of four or more doses of SP on placental malaria, LBW and anaemia were included in this cross-sectional study. With a malaria prevalence of 17.6% reported among pregnant women at outpatient departments (OPDs) in Ghana by the National Malaria Control Programme [18], the study estimated the prevalence of malaria with a precision of 1.8%.
Outcome definitions
Clinical malaria was defined as parasitaemia and an axillary temperature ≥ 37.5 °C or a history of fever within the past 48 h, while asymptomatic malaria was defined as any level of parasitaemia without fever. The level of parasitaemia was classified as very low (1–199 parasites/µL), low (200–999 parasites/μL), medium (1000–10,000 parasites/μL), and high (> 10,000 parasites/μL) after modification of published cut-offs to create the very low category [19, 20]. Anaemia was defined as a haemoglobin concentration < 11.0 g/dL, and sub-divided as severe (< 7.0 g/dL), moderate (7.0–9.9 g/dL), and mild (10.0–10.9 g/dL) [21]. ITN use was defined as sleeping under a net the previous night, and use of other malaria vector preventive measures defined as usage within the past 7 days.
Data management and statistical analysis
Enrolment, haematology and parasitology data were entered into pre-coded questionnaires and checked for completeness. Completed questionnaires were double-entered and verified using a database designed with CSharp application as front end and SQL Server as back end. Data processing and statistical analysis were carried out using Stata 14 (StataCorp, College Station, USA). Principal components analysis (PCA) was used to derive a wealth index variable (SES) based on each woman’s household assets and characteristics. Socio-demographic, obstetric and malaria vector prevention characteristics of all women enrolled in the study were summarized.
Wilcoxon rank-sum test was used to assess the association between parasite density and age (< 25 or ≥ 25) and gestational age (< 18 weeks or ≥ 18 weeks). The association of gravidity (primagravid, secundigravid, or multigravid) with parasite density was explored using Kruskal–Wallis test. The prevalence of malaria parasitaemia was computed together with its 95% confidence interval. Univariate and multivariate logistic regression was used to study the association between socio-demographic, obstetric and malaria vector prevention characteristics and the risk of malaria parasitaemia. Variables in the univariate analysis with a p-value < 0.15 were included in a multivariate logistic regression model. The interaction of age and gravidity on the risk of malaria parasitaemia was also explored.
Haemoglobin concentration in participants who had malaria parasitaemia was compared to those who did not have malaria parasitaemia using Wilcoxon rank-sum test. The prevalence of anaemia was further classified into mild, moderate and severe, and reported with 95% confidence intervals. Univariate and multivariate logistic regression was used to assess the association of socio-demographic, obstetric and malaria vector prevention characteristics of study participants with the risk of anaemia. Similar to the approach used in the analysis of malaria parasitaemia, variables from the univariate analysis with a p-value < 0.15 were included in a multivariate logistic regression model. The interaction of age and gravidity on the risk of anaemia was also explored. In all multivariate models, significance was established at a 5% level.