Epidemiology of malaria among pregnant women during their first antenatal clinic visit in the middle belt of Ghana: a cross sectional study

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.

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 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.

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.

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).

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.

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%.

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.

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.

A total of 1655 participants were eligible for inclusion in the study. Malaria microscopy results were available for 1647 (99.5%) participants, while haemoglobin results were available for 1465 participants (88.5%); samples from 182 were not adequate for haemoglobin estimation. Mean age (± SD) of the participants was 27.4 (± 6.2) years, with nearly half (49.6%) of them aged 25–34 years and the mean gestational age at enrolment (± SD) was 16.7 (± 4.3) weeks, with about two-thirds (67.4%) of women having a gestational age ≤ 18 weeks. Primigravidae and secundigravidae formed 21.0% (n = 355) and 23.7% (n = 392) of the participants, respectively. About one-fifth, 20.9% (n = 346) had completed high school or higher education.

Almost 90.0% (1488/1655) of participants reported possessing an ITN, of which 12.1% (180/1488) were reported torn. Reported ITN use among all women and those possessing an ITN was 66.2% (1095/1655) and 73.6% (1095/1488), respectively. About one-fifth of the participants reported using a mosquito coil within the last 7 days (Table 1). The main clinical complaints at enrolment were abdominal pain (7.9%, n = 131), bodily pains (7.9%, n = 130), loss of appetite (6.6%, n = 109), fever (6.2%, n = 106), drowsiness (4.8%, n = 80), vomiting (4.6%, n = 76), and nausea (4.2%, n = 69).

Overall prevalence of malaria parasitaemia was 20.4% (95% CI 18.5–22.4%) and the prevalence of clinical malaria was 1.5% (95% CI 1.0–2.2%). The prevalence of parasitaemia was higher in women aged < 25 years (31.2%; 95% CI 27.6–35.0%) compared to those ≥ 25 years (14.5%, 95% CI 12.5–16.8%), and also higher in primigravidae (31.0%, CI 26.4–36.0%) compared to multigravidae (15.1%, 95% CI 12.9–17.6%). Plasmodium falciparum was the only species identified among the Plasmodium isolates. Overall geometric mean parasite density (GMPD) was 442 parasites per µL (95% CI 380–515). Density was also higher in women aged < 25 years (628 parasites per µL, 95% CI 511–772) compared to those ≥ 25 years (293 parasites per µL, 95% CI 238–362), and in primigravidae (721 parasites per µL, 95% CI 554–938) compared with multigravidae (295 parasites per µL, 95% CI 234–372) (Table 2). Among pregnant women with malaria parasites, more than three-quarters had parasite density below 1000 parasites/µL of blood. The proportion of women with very low, low, medium, and high density parasitaemia were 31.1% (95% CI 26.3–36.2%), 47.0% (95% CI 41.7–52.4%), 18.9% (95% CI 15.1–23.5%), and 3.0% (95% CI 1.6–5.4%), respectively.

In the unadjusted analyses, age, education, household SES, marital status, profession, gravidity, and ITN use were associated with risk of malaria parasitaemia (Table 3). In the multivariate analysis, women who reported using an ITN were 1.43 times likely to have parasitaemia compared to those who did not. Women ≥ 25 years of age were 0.57 times less likely than those < 25 years of age, multigravidae were 0.50 times less likely compared to primagravidae, traders or food sellers were 0.67 times less likely than women with no profession, women educated to high school or above were 0.53 times less likely compared to those with no education and the least poor were 0.34 times less likely compared to the most poor to have malaria parasitaemia (Table 3).

Figure 2 shows the effect of the interaction between age and gravidity on the prevalence of malaria parasitaemia. Prevalence was highest (33.2%) in primi- or secundigravidae aged < 25 years old and lowest (14.1%) in multigravidae aged ≥ 25 years. Compared to multigravidae aged ≥ 25 years, malaria parasataemia prevalence was 3.04 times higher (95% CI 2.31–4.01, p < 0.001) in primigravidae aged < 25 years and 1.89 times higher (95% CI 1.16–3.08, p = 0.011) in those ≥ 25 years, respectively. There was, however, no significant difference between multigravid women aged < 25 years and those ≥ 25 years (OR 1.17; 95% CI 0.80–1.71, p = 0.424).

Mean (± SE) haemoglobin concentration for the pregnant women at first ANC visit was 10.7 g/dL (± 0.04). Among women with malaria parasitaemia, mean haemoglobin concentration was significantly lower than in those without malaria parasitaemia (9.9 g/dL vs 10.9 g/dL; p-value < 0.001) (Fig. 3). Overall prevalence of anaemia (haemoglobin < 11.0 g/dL) and severe anaemia (haemoglobin < 7.0 g/dL) were 56.0% (95% CI 53.4–58.5%) and 1.4% (95% CI 0.9–2.2%), respectively (Table 4).

In the univariate analysis, age, gestational age, education, household SES, marital status, profession, gravidity, and malaria parasitaemia status were associated with risk of anaemia (Table 5). In the multivariate logistic regression analysis, the risk of anaemia was 0.61 times lower in multigravidae compared to primagravidae. Women with gestational age ≥18 weeks were 2.24 times more likely to have anaemia compared to those aged < 18 weeks, and unmarried women were 1.44 times likely to be anaemic compared to the married women. Women who had parasitaemia up to 999 parasites/µL were 3.49 times likely to have anaemia, whiles those with parasitaemia ≥ 1000 parasites/µL were 4.05 times more likely to have anaemia, compared to those without parasitaemia (Table 5).

The effect of the interaction between age and gravidity on prevalence of anaemia is shown in Fig. 4. Prevalence of any anaemia was highest (66.2%) in primi- or secundigravidae aged < 25 years and least (50.0%) in multigravid pregnant women aged ≥ 25 years. Compared to multigravid pregnant women aged ≥ 25 years, anaemia prevalence was significantly higher (p < 0.001) in primi- or secundigravidae aged < 25 years (COR 1.96; 95% CI 1.53–2.51). There was no significant difference in the prevalence of anaemia between multigravidae < 25 years and ≥ 25 years.