Evaluation of the adhesion of pregnant women to intermittent presumptive treatment with Sulfadoxine-Pyrimethamine in a rural area of Gabon

Fleuramie Mirembou-Boukoumba Ecole Doctoral Régionale D’Afrique Centrale En Infectiologie Tropicale Sydney Maghendji-Nzondo Département d’Epidémiologie Biostatistique et Informatique médicale, Université des Sciences de la Santé Pierre Blaise Matsiegui Centre de Recherches Médicale de la Ngounie Irene Pegha-Moukandja Centre de Recherches Médicale de la Ngounie Davy Tanguy Mendene-Abessolo Centre de Recherches Médicale de la Ngounie Jean Bernard Lekana-Douki (  lekana_jb@yahoo.fr ) Unit of Evolution, Epidemiology and Parasitic Resistances (UNEEREP), Centre International de Recherches Médicales de Franceville (CIRMF) B.P. 769 Franceville Gabon. Phone : 00241 06 25 95 90.


Introduction
In 2016, the global count of malaria reached 212 million cases and 429 000 deaths among which more than half were recorded in sub-Saharan Africa [1]. Pregnant women are among the most vulnerable individuals to malaria, with children under ve years old and non-immunized travelers [2].
During pregnancy, women must tolerate the fetus, which carries 50% of the father's genetic material [3], they are therefore prone to immune suppression. This situation increases the risk of susceptibility to infectious pathogens, such as Plasmodium falciparum [4][5][6]. Gestational malaria is often externalized by hyperthermia, which is related to parasitemia. Susceptibility to malaria decreases with the number of pregnancies [7], suggesting the acquisition of a speci c immunity directed against this pathology [8][9][10].
Anemia is common during pregnancy [11,12], it affects primiparae more than multiparae. Cerebral malaria is not frequent among pregnant women in areas of high transmission, both in multiparae and primiparae.
One of the pathophysiological factors of gestational malaria is placental sequestration of parasitized red blood cells [13]. Numerous studies have shown that this preferably occurs through the cytoadherence of parasitized red blood cells on placental syncytiotrophoblates via Chondroitin Sulfate A [14]. Thus, the placenta behaves like a niche concentrating parasitized red blood cells, called CSA cytoadherence phenotypes [15,16]. The consequences of gestational malaria are therefore increased morbidity and mortality in pregnant women [17]. For fetuses, placental infection leads to spontaneous abortions, in utero growth delays, and children born with low weight [18][19][20][21].
To prevent placental malaria, WHO recommends the use of intermittent presumptive treatment with Sulfadoxine-Pyrimethamine (IPT-SP), in addition to the use of insecticide-treated bed nets (ITNs).
However, in 2014, among the 28 million pregnant women registered, 15 million pregnant women had not received IPT-SP [22]. Moreover, among the pregnant women who had received IPT-SP, 50% had received one dose, 35% two doses and only 15% had received at least three doses.
In Africa, the Sulfadoxine-Pyrimethamine (SP) therapeutic combination was adopted in most countries.
In Gabon, IPT based on SP in pregnant women was adopted in 2003. As a result, the prevalence of P.falciparum infection declined among pregnant women both in urban and rural areas [27].
The aim of this study was to determine the level of adhesion of pregnant women to IPT-SP in the rural area of Fougamou, Gabon.

Area and study population
The study was conducted in a Gabonese semi-urban town named Fougamou located in the Ngounié province, in the western central part of Gabon. Fugamou is the chief town of the Tsamba-Magotsi department, as such, its hospital receives patients from villages throughout the department.
The study population was composed of pregnant women who received antenatal care at the maternity of the Fougamou medical center.
The women met the following criteria: they had been seen in consultation at the maternity ward and had been entered in the consultation registers. Women who did not ll the criteria were excluded from the study.

Study Design And Sampling Collection
We conducted a prospective study from February to May 2018 at the maternity of the Fougamou medical center. Retrospective data from 2018 to January 2018 from maternity registries were also included in this study. Blood samples from the pregnant women were collected in ethylene diamine tetra acetic acid (EDTA) tubes during the prenatal consultation after the pregnant women gave their informed consent.

Questionnaire Interview And Ethical Consideration
An interview based on a questionnaire was given to all the participants. The questionnaire included questions on socio-demographic data (age, education and family income/month), medical history and clinical data (the number of IPT-SP doses taken). Data on their use of IPT-SP, their use of ITNs, indoor residual sprays (IRS) and their knowledge, attitudes and practices regarding malaria were collected.
This study was approved by the Gabonese national committee of ethics and registered under PROT 0020/2015/SG/CNE; it was performed in accordance with the principles of Committee. To ensure their voluntary participation, an informed consent form was signed by all the participants.

Malaria Diagnosis
The Optimal-IT® rapid diagnostic test was used. The sensibility and speci city of the tests were 94 and 97 % [28]. Previous work in Gabon showed that this test is a good tool to diagnose malaria [29]. Parasite load was determined on blood smears using the Lambarene method [30]. All blood smears were read by two independent technicians and quality control was done for 10 % of slides by a third reader. Fever and P. falciparum infection (1000 parasites per µl of blood) were considered as malaria.

Statistical analysis
Statistical analysis was performed with the use of Stat view 5.0 1992-1998 (SAS Institute, Cary, USA). A chi-square test was used to compare quantitative variables among groups.
The average values of the qualitative variables were compared by the Student's t-test. Quantitative variables were compared by the chi-square test and when the numbers were lower than 5, Fisher's exact test was used. The threshold of signi cance was established for α = 5%.

Study population
During the study period, 607 pregnant women were received in prenatal consultation in the medical center of Fougamou. The general characteristics of the women are described in Table 1. Unemployed pregnant women were the most numerous (60.9%), then came high school students and employed pregnant women (p < 0.001). The proportion of women aged between 20-25 years (37.7%) was signi cantly higher than the proportion of women of the other age groups (p < 0.001). The proportion of women aged between 20-25 years was similar to the proportion of women aged between 26-30 years (19.9%) and 31-35 years (16.0%) (p > 0.05). Hematological values were normal. The interval between the prenatal consultations was of 1 month.

Level Of Adherence To IPT-SP Treatment
The level of adherence was analyzed from 427 women who gave birth in the Fougamou medical center. Ninety-four percent (94.4%; n = 403) of women included in the study had taken at least one dose of IPT-SP and 3.3% (n = 14) had not taken the treatment. This information was unavailable for 10 women. There was a signi cant difference between women who took the treatment and women who did not take IPT-SP (p < 0.05). The prevalence of women who took 1 dose (16.1%; n = 65) of IPTp-SP was signi cantly lower than the prevalence of women who took 2 doses (29.5%; n = 119) and 3 doses (47.9%; n = 193) of IPT-SP (P < 0.001) (Fig. 2). The prevalence of women who took 2 doses of IPT-SP was signi cantly lower than the prevalence of women who took 3 doses (p < 0.001). Two women (2.1%; n = 2/96) had received IPT-SP between the rst and 15th week of amenorrhea (WA). In addition, the majority of women who had not received a dose of IPT-SP were those with one to 25 WA.

Plasmodium characterization
The overall prevalence of Plasmodium infection was 11.7% upon blood smear examination (n = 67/508).
The mean values of parasitemia were calculated based on the 67 pregnant women with positive thick lms. Mean parasitemia was low at 1052 880) p/lL of blood. We found that 100% of infections were due to P. falciparum. Table 2 shows that Plasmodium-infected women were less old than uninfected women (p < 0.05). Figure 1 shows that the proportion of Plasmodium-infected women aged between 14-19 years (22.8%) was signi cantly higher than the proportion of Plasmodium-infected women aged between 20-25 years (11.0%), 26-30 years (8.8%), 31-35 years (7.6%) and 36-40 years (7.9%) (p < 0.05). The Kruskal-Wallis test showed a signi cant difference in mean parasitemia according to age groups (p = 0.02). The mean values of red blood cell were signi cantly lower in infected women than uninfected women (p < 0.01) whereas white blood cells were higher in infected women than uninfected women (p = 0.0007), In uence of IPT-SP treatment on plasmodial infection.

Preventive Measures
We analyzed the patients' use of preventive measures ( Table 2). The overall coverage of bed nets was 68.2% (n = 413/606). The proportion of insecticide house spray use was 44.7% (n = 268/600) and the proportion of pregnant women showing essential information on malaria was 74.3% (n = 437/588). Univariate and multivariate analysis showed no association between the use of preventive measures and malaria infection (p > 0.05). The use of preventive measures had no preventive effect in pregnant women without and with malaria.

Discussion
Gestational malaria remains a major public health problem since it leads to severe risks for the mother, the fetus or the newborn child. This study investigated the epidemiology of malaria in Gabon and estimated the level of adherence to IPT-SP in pregnant women. We noted an average of about 200 deliveries per year, which represents between four and ve deliveries per week and is representative of the low demography of Fougamou.
The mean age of the pregnant women in our study was about 25.8 ± 6.7 years. This result is consistent with previous data from rural areas in Burkina Faso, in Benin and in the capital of Gabon, Libreville, and its surroundings, in 2011 [31][32][33]. In Gabon, this data highlights the fact that there does not seem to be a signi cant difference in the distribution of age of the pregnancy between urban and rural areas.
In this work, we noticed that nearly half of the women included in the study lived in villages. The rural context of Fougamou is con rmed by the socio-demographical factors (a majority of women were unemployed).
However, the analysis of the hematological parameters revealed a good general health state, suggesting an e cient medical follow-up of these women.
The excellent level of adhesion to IPT-SP consolidates this hypothesis. Previous data from Gabon showed that the level of adherence to IPT-SP was very high, resulting in a minimization of the consequences of malaria associated with pregnancy [34]. In other African countries such as Kenya or Burkina Faso, similar results were reported [35][36][37]. The level of adherence to IPT-SP in our study was 94.4%. This proportion is higher than the value previously reported in Libreville and in Burkina Faso [31,37]. This result in Fougamou ts the goal set by OMS, which aimed to have 80% of women receive at least 2-doses of IPTp-SP during pregnancy.
Although the national directive indicates that pregnant women have to receive at least 3 doses of IPT-SP, in this study, we found that women received a maximum of 3 doses, which is an erroneous reading of the national directive. In this study, the proportion of women having received IPT-SP during the rst quarter of pregnancy is similar to that observed in Libreville where 5.9 % of the women had received IPT-SP in the rst quarter. After delivery, half the women had received 3 doses of IPT-SP.
Data showed that the majority of women without IPT-SP were between one and 25 weeks of amenorrhea, which could suggest a late start in their antenatal care. This observation is revealing of the rural context of Fougamou. The time between consultations con rms the high attendance of women to prenatal visits in Fougamou, which ranged from one to nine visits.
The low prevalence of plasmodial infection observed in our study could be a consequence of the adherence of women to IPT-SP. Moreover, nulliparae (primigravidea), without IPT or under IPT, remain the most exposed to malaria. Similar results were observed in Burkina Faso, in Benin, in Malawi and in Gabon [31,33,[38][39][40][41]. This could be explained by the fact that multigravidae develop, over subsequent pregnancies, a protective immunity against placental malaria infection. These women develop antibodies which are able to speci cally inhibit the cytoadhesion on Chondroitin Sulfate A [42][43][44]. The more women age and have parity, the more they are protected from malaria.
The overall malaria rate in women having bene ted from IPT-SP obtained in our study is similar to the rates of malaria infections reported in Burkina Faso, in Mali, in Kenya and in Malawi [39,41,[45][46][47].
The low prevalence of plasmodial infection observed in this study could also be a consequence of adhesion to other recommended malaria prevention measures. Indeed, an overall coverage rate in bed nets of about 70 % was observed. This very high coverage rate could have reduced the development of plasmodial infection. Several studies reported the association of IPT-SP and the use of bed nets to protect women against plasmodial infection during their pregnancy [48,49]. However, this rate contrasts the women's knowledge on malaria because during the questionnaire only 74.3 % of the women answered the three questions which de ne the level of knowledge on malaria correctly (What is malaria? How can you avoid it? How does it manifest itself?). These data suggest that women follow IPT without a clear understanding of its role and might indicate that the impact of public health messages is erroneous.
The positive impact of IPT-SP in pregnant woman is con rmed in this study by the fact that all the pregnant women infected by P. falciparum were asymptomatic and that the average parasitemia was low. This is consistent with previous data indicating that in areas of stable transmission, plasmodial infection in pregnant women is often asymptomatic [50].

Conclusion
This study shows that almost all women giving birth in the Fougamou medical center receive at least one dose of IPT-SP during their pregnancy. About 80% of them were 14 to 30 years old with a peak between 20 and 25 years. We also con rm the e ciency of IPT-SP in association with preventive measures against malaria. It would be necessary to pursue these malaria control strategies in pregnant woman by looking at the impact of IPT-SP on the genetics of the parasite.

Declarations
Ethics approval and consent to participate: This study was approved by the Gabonese national Committee of ethics and registered under PROT 0020/2015/SG/CNE; it was performed in accordance with the principles of Committee. To ensure their voluntary participation, an informed consent form was signed by all the participants.
Consent for publication: All authors read the last le of this paper and agree the submission for publication Availability of data and materials: The authors declare that the data will be made available after the article's acceptance and publication