Risk Factors for Plasmodium Falciparum Infection in Pregnant Women in Burkina Faso: A Community-Based Malaria Cross-Sectional Survey

Background: Malaria in pregnancy remains a public health problem in sub-Saharan Africa. Identifying risk factors for malaria in pregnancy could assist in developing interventions to reduce the risk of malaria in Burkina Faso and other countries in the region. Methodology: Two cross-sectional surveys were carried out to measure Plasmodium falciparum infection using microscopy in pregnant women in Saponé Health District, central Burkina Faso. Data were collected on individual, household and environmental variables and their association with P. falciparum infection assessed using multivariate analysis. Results: A total of 356 pregnant women were enrolled in the surveys, 174 during the dry season and 182 during the wet season. The mean number of doses of sulphadoxine pyrimethamine for Intermittent Preventive Treatment in pregnancy (IPTp-SP) was 0.4 doses during the rst trimester, 1.1 doses at the second and 2.3 doses at the third. Overall prevalence of P. falciparum infection by microscopy was 15.7%; 17.8% in the dry season and 13.7% in the wet season. 88.2% of pregnant women reported sleeping under an insecticide-treated net on the previous night. P. falciparum infection risk in pregnancy was reduced in those women who reported using an ITN (Odds ratio, OR=0.31, 95% CI 0.12-0.79, p=0.02) and an increasing number of IPTp-SP doses during pregnancy, with each additional dose reducing the odds by 40% (OR=0.59, 95% CI 0.43–0.81, p<0.001). The prevalence of P. falciparum infection among pregnant women remains high in Burkina Faso although use of IPTp-SP and ITNs were found to reduce the odds of infection. Despite this, compliance with IPTp remains far from that recommended by the National Malaria Control Programme and World Health Organization. Behaviour change communication should be improved to encourage compliance with protective malaria control tools during pregnancy. children despite high coverage of insecticide-treated nets (ITNs) and prompt and effective treatment with antimalarials [3]. to identify risk factors for falciparum in including potential socioeconomic and environmental risk factors. Identifying risk factors for in pregnancy could assist in developing interventions to reduce malaria burden in pregnancy in Burkina Faso and other countries in the region.


Introduction
Malaria in pregnancy remains a major public health problem in sub-Saharan Africa [1], despite the decline in malaria transmission observed throughout the region from 2004-2015 [2]. Burkina Faso is a high burden country and is not experiencing declines in malaria and all-cause mortality in children despite high coverage of insecticide-treated nets (ITNs) and prompt and effective treatment with antimalarials [3].
Pregnant women are at high risk from malaria because of their lowered immunity during pregnancy [4] and because they are more attractive to Anopheles gambiae, the most important African malaria vector [5]. Infection with Plasmodium falciparum can lead to poor outcomes for the mother, the foetus and child, resulting in maternal anaemia, low birth weight, preterm delivery and perinatal mortality [6][7][8][9]. Pregnant women, especially those pregnant for the rst time (primigravidae), are at increased risk of more frequent and more severe malaria infections [10][11][12][13]. The World Health Organization (WHO) recommends the use of ITNs (distributed free-of-charge at Antenatal Clinic (ANC) visits), intermittent preventive treatment in pregnancy (IPTp) with sulphadoxine pyrimethamine (SP) and prompt access to diagnosis and effective case management, to prevent and manage malaria risk in pregnancy [14]. According to national guidelines in Burkina Faso, pregnant women are advised to receive at least three doses of IPTp-SP starting from the second trimester, with a minimum interval of one month between doses [15].
The incidence of malaria infections in pregnant women in Burkina Faso in 2014 was 39.2 per 1,000 women-months, and was more than twice as great in primigravids at 88.6 per 1,000 women-months than multigravids [13]. In 2014, another study in the country found that malaria infection was ve-fold greater in primigravids than in multigravids [12]. There have been many studies of risk factors for malaria in pregnancy in sub-Saharan Africa, where increased risk was reported to be associated with younger age in pregnancy, primigravidae, rst trimester of pregnancy infection, nonuse of ITNs, lack of education and HIV co-infection [11][12][13]16]. Few, however, have evaluated socioeconomic and environmental risk factors for malaria in pregnancy. For example, recently a number of studies have shown that malaria in children is associated with poor housing [17][18][19], but it is not known whether this is also true for pregnant women. The goal of the present study was to identify risk factors for P. falciparum infection in pregnancy in Burkina Faso, including potential socioeconomic and environmental risk factors. Identifying risk factors for malaria in pregnancy could assist in developing interventions to reduce malaria burden in pregnancy in Burkina Faso and other countries in the region.

Study design
Putative risk factors for P. falciparum infection were measured during two cross-sectional surveys, one in the dry season and one in the wet season.

Study site
The study was conducted in Saponé Health District, situated in the central region of Burkina Faso, 30 Km south-west of Ouagadougou, the capital of Burkina Faso. In the study area, malaria transmission is intense and highly seasonal [20], with the peak of malaria transmission occurring at the end of the rainy season (June to October) and markedly reduced transmission during the dry season (December to May) [21]. The main vectors are Anopheles gambiae s.s., An. arabiensis and An. funestus, and P. falciparum accounts for more than 95% of all malaria infections [20][21][22]. This is a rural area of open Sudanian savannah, where farming is dominant and the major crops grown are sorghum and millet. Houses in the study area are typically constructed with mud walls and oors, with thatched or metal roofs [23].

Surveys
Two cross-sectional surveys were carried out. One at the beginning of the dry season in December 2018 and the second one at the end of the rainy season from September to October 2019. Pregnant women were enrolled through a Demographic Health Surveillance System (DHSS), with home visits in 21 villages in the study area. All women of child-bearing age in the study area were identi ed and visited at home for pregnancy screening. This approach was adopted, rather than screening at the ANC because ANC attendance is relatively low in the study area, with only 35% of women attending the ANC at least three times [24]. Women thought to be pregnant were referred to the health facilities for a pregnancy test or, if willing to provide urine for a dipstick pregnancy test, eldworkers performed the test at the woman's home. Women identify as pregnant, but who had not visited their ANC, were referred to the local health facilities. At the ANC, the study protocol and procedures were explained by trained staff to the potential participants in French or the main local language of Moore. Pregnant women were enrolled if aged between 15-40 years, provided written informed consent and agreed with the study procedures, including taking of blood. Pregnant women with a known history of SP allergy or any other medical condition that in the opinion of a study physician may be a threat to her or the foetus were not recruited into the study.

Clinical data collection
All study participants completed a questionnaire at enrolment, where demographic data, medical and obstetrical history including previous ANC visits, IPTp doses and use of antimalarials or any other medication within 14 days prior to study enrolment were recorded. Each participant donated a nger prick blood sample (100 µL) for malaria infection detection and characterisation. P. falciparum quantitative sexual and asexual parasite count and qualitative species identi cation was performed by microscopy. Two blood smears were prepared and read by two independent experienced microscopists based at the Centre National de Recherche et de Formation sur le Paludisme (CNRFP) according to established standard operating procedures. In case of fever (axillary temperature ≥37.5°C or reported fever in last 24 h) or other symptoms/signs of clinical malaria, a rapid diagnostic test for malaria (SD BIOLINE Malaria Ag P.f/Pan, Abbott Laboratories, Illinois, USA) was performed. Subjects presenting with clinical malaria were referred to the nearest health facility and treated according to national guidelines [15]. Enrolment procedures were performed at home unless there was a need to check more about the health status of the volunteer. If this was the case, the woman was referred to the health facility to complete the clinical examination.
Risk factor data collection Study participants were visited at home by eldworkers who recorded information about the household, including whether the woman had slept under an ITN the previous night. If the answer was no, the reason why the woman did not use an ITN was recorded. Women sleeping under an ITN were asked about the bed net source and how many times they left their ITN during the previous night. ITN fabric integrity was also assessed by eldworker observation and classi ed as entire/complete, with any hole, or torn. Women were asked to estimate the time they went to bed and the time they get out of bed in the morning. Social and economic risk factors for malaria were recorded, including ethnicity, education level and occupation, ownership of a radio or mobile phone, estimated distance to the nearest health facility, and use of other protective measures, including mosquito coils, insecticide sprays, traditional spatial repellent or commercial topical repellents.
House construction (metal or thatched roof, presence of open eaves, electricity supply to sleeping room), household size (number of persons) and the presence of clothes hanging in the sleeping room were recorded. The presence or absence of big domestic animals (donkey, horses, sheep, cows, goats, dogs) and rubbish within 5 m of each study participant household was recorded.

Sample size
The sample size was estimated based on the sample size for frequency of the disease in a population (https://www.openepi.com/SampleSize/SSCohort.htm). To determine malaria parasite prevalence, we assumed a population prevalence of 2.5% in the study area based on previous data of the frequency of malaria in pregnant women in the study area recorded in 2017 [25]. We assumed a 2% precision, with 5% level of signi cance and 95% con dence limits. Considering housing type as major risk factor (improved housing reduces risk of malaria prevalence by~50%, Odds ratio = 2) [26] and 10% non-response, a sample size of 175 pregnant women was considered necessary for each cross-sectional survey.

Data management and statistical analysis
Data were collected on Android personal digital assistants programmed using Open Data Kit (https://getodk.org/) and included drop down boxes and consistency checks to reduce data entry errors. Following cleaning, the dataset was locked and saved in Microsoft Access and analysed with Stata 15 (Statacorp, Texas, USA).
The primary outcome measure was the prevalence of microscopically con rmed P. falciparum infection in pregnant women during each crosssectional survey. Logistic regression was used to investigate the association between predictor variables and the primary outcome, adjusting for clustering by village. The multivariable model was constructed using a forwards stepwise process and models were compared using a Wald test.

Results
A total of 356 pregnant women were enrolled in the surveys, 182 during the wet season and 174 during the dry season ( Table 1). The mean age of the study participants was 26.9 years, ranging from 15 to 40 years old, and was similar in both surveys. Of these women, 78 (21.9%) were in their rst pregnancy, 74 (20.8%) in their second and 204 (57.3%) in their third pregnancy or more. Most women were enrolled in their second and third trimester of pregnancy (37.1% and 30.3% of women where gestational age was recorded). Fewer were enrolled at their rst trimester: only 5/78 (6.4%) of primigravidae, 11/74 (14.9%) of secundigravidae and 20/204 (9.8%) of multigravidae were enrolled in their rst trimester of pregnancy. 59.0% of women were illiterate and most were farmers (69.9%) or traders (22.1%). 73.1% of primigravidae were literate compared to only 42.5% of those on their second pregnancy and 27.6% of women with two or more pregnancies. 97.5% of study participants were from the Mossi ethnic group. Most women lived in households with three or fewer people 57.9% (206/356). Only 46.3% of women reported having an electricity supply in the sleeping room. Most houses were constructed with metal roofs (95.5%) with 64.6% of houses having hanging clothes inside. Large domesticated animals were common near the house (78.9%), with 45.8% of participants reporting rubbish within 5m of their households.  where CI = con dence interval, GMPD = geometric mean parasite density, IPTp-SP = intermittent preventive treatment in pregnancy with sulphadoxine pyrimethamine, ITN = insecticide treated net, SD = standard deviation  [12]) and other high burden countries in sub-Saharan Africa e.g. 20.1% in Kenya [27] and 21.6% in Ghana [28]. These results suggest that P. falciparum malaria infection is common in pregnant women in the community and the burden of P. falciparum infection in pregnancy remains high despite the use of standard malaria control interventions. The overall geometric mean of parasites density in the study area was 777.3/µl (95% CI 496.0-1218.2). Fana and coworkers from Nigeria, another high burden country, recorded a similar mean parasite density of 800/µl [16]. The high parasite densities found in pregnant women results from their decreased immune competence [4,29]. As expected, the parasite density was higher in primigravidae and secundigravidae compared to those women multigravide and in younger women compared to older women, since younger women are more likely to be primigravid. There was no signi cant difference in P. falciparum prevalence between the wet season (13.7%) and dry season (17.8%). This may be because we conducted the dry season survey in the early stages of the dry season when infections from the end of the rains may still be present. Parasite density was, however, higher in the wet season compared to dry season, showing that even in this high burden area, malaria is seasonal in pregnancy [20]. P. falciparum gametocyte carriage was low in this study (1.7%). Low parasites density may result in lower gametocyte identi cation by microscopy.
Overall, 91.3% of pregnant women owned an ITN, with 88.2% reporting using an ITN the night before the survey. This is similar to other surveys from Burkina Faso; in the Banfora Region, 80.6 % of surveyed children reported sleeping under an ITN the previous night [18]. The high reported ITN use is encouraging, although accurately determining net use is challenging and reporting can be susceptible to response bias [30].
The study found that IPTp-SP and ITNs are highly effective interventions for preventing malaria infection during pregnancy. For each additional dose of IPTp reported as being received by women, the odds of malaria infection fell by 40%. At the time of the survey, relatively few women, had however taken three or more doses of IPTp-SP (20.2%) which is recommended by the NMCP and WHO [1,15]. We also found lower use of IPTp-SP among women aged 20-30 compared to other age groups and among secundigravidae compared to other women. This suggests that women in their second pregnancy may be more compliant with ANC attendance and malaria prevention than women in their rst pregnancy or later pregnancies. This nding contrasts with another study in Burkina Faso that found compliance with IPTp-SP in adolescent women to be more problematic due to structural constraints (e.g. social position and household labour requirements) and needs (e.g. anonymity in the health encounter) [31]. Numbers of secundigravidae women were relatively small and so this nding requires further exploration.
ITNs were associated with 69% reduction in the odds of P. falciparum infection, which is higher than other studies have found [32]. This indicates that ITNs are protective against malaria in pregnancy despite high levels of insecticide resistance present in Burkina Faso [33,34]. This contrasts with ndings from a cohort study in children aged 5-15 years in south-west Burkina Faso which showed no difference in malaria risk between ITN users and non-users [18], and in all age in a community-wide survey in Banfora region (Yaro et al, unpublished). Women reported going to bed at 20.21 h during the dry season and 20.13 h during the rainy season. This nding contrasts with a study by Guglielmo and co-workers who reported that 100% of females in south-west Burkina Faso (sample of 211 and 695 females observed in two villages) were outdoors until 22.00 h, after which point women started to move indoors to bed [35]. It may be that pregnant women tend to go to bed earlier and so those using ITNs are more likely to be protected from vectors biting during the early evening which has been observed in Burkina Faso [35]. Increased malaria risk in human including pregnant women who go to bed later has been observed in other studies in sub-Saharan Africa [36][37][38].
Our study has a number of limitations. Firstly, the sample size was probably not large enough to identify minor risk factors in this study. Secondly, ITN ownership and use was self-reported and subject to social desirability bias and we lack objective tools for measuring bednet use in this study.
What are the implications of this research for control of malaria in pregnancy in Burkina Faso? Behaviour change communication is necessary to ensure high ANC attendance and compliance with IPTp-SP and ITN use. Messages need to be tailored to the different vulnerable groups of women.
For example, we found lower IPTp-SP compliance among women aged 20-30 than the other age groups. As it this common in sub-Saharan Africa, pregnant women are often unaware that they are pregnant and so do not attend or are unwilling to attend an ANC in the early stages of pregnancy. An association between early ANC attendance and a higher average number of IPTp-SP doses has been demonstrated in several studies [39][40][41]. One option to increase IPTp-SP coverage is community delivery by community health workers, rather than ANC. This delivery route has been shown in a clinical trial in Burkina Faso to increase IPTp-SP compliance from 2.1 to 2.8 doses in the community delivery study arm with no apparent decrease in ANC attendance [42].

Conclusion
The prevalence of P. falciparum infection among pregnant women remained high despite wide deployment of ITNs and access to IPTp-SP. Nonetheless, women who took IPTp-SP and use ITNs during their pregnancy were at much reduced risk of being infected by malaria. These ndings suggest that IPTp-SP and ITNs use are effective at reducing malaria infection in pregnant women living in malaria high burden countries, but that research is needed to increase uptake of IPTp-SP.

Declarations
Ethics approval and consent to participate