Effectiveness of intermittent preventive treatment with Sulfadoxine-Pyrimethamine in pregnant women in San Pedro, Côte D’Ivoire

ABSTRACT

Malaria remains a major public health issue for pregnant women. Côte d’Ivoire has adopted a series of measures aimed at combatting this plague, and these measures include administering Sulfadoxine-Pyrimethamine (SP) as an intermittent preventive treatment to pregnant women in the second and third terms.

This cross-sectional study included a parturient population after informed written consent. We recruited women from the Terre Rouge maternity ward and the labor room of the Regional Medical Center of San-Pedro. Plasmodial DNA (desoxyribo nucleic acid) was extracted from Whatman filter papers with dried blood samples prepared from the venous, placental, and cord blood, utilizing Chelex 100. The extracts obtained were amplified by nested PCR.

In all, 197 women were included, with an average age of 27-year-old (sd = 6.7 years old). The rates of the placental, venous and cord blood infections were 16, 2%, 15, 2% and 3, 6%, respectively. The women who took three doses of ITP were less infected at the cord (3, 2%), placental (10,6%) and venous level (13,8%). A statistically significant relationship between the number of doses and the rate of placental infection was established (p = 0.042). IPT reduces plasmodial infestation at the placental (OR = 0.4; CI = [0.2–1]), cord (OR = 0.8; CI = [0.2–3.7]) and venous (OR = 0.8; CI = [0.6–2.3]) level.

In conclusion, the low frequency of placental, venous, and cord infestation in pregnant women who consistently followed a preventive treatment strategy clearly showed the efficiency of IPT against malaria during pregnancy.

Introduction

Malaria remains a serious health concern in tropical countries, particularly in children aged between 0 and 5 years and pregnant women who are the most vulnerable group [1]. Plasmodium falciparum infection during pregnancy may result in parasite sequestration in maternal placental vascular space. Interestingly, the extreme sensitivity of pregnant women to this parasite is due to the presence of parasitic strains that specifically adhere to chondroitin sulfate A present in the placental tissue [2–4]; lack of immunity to such parasites, and possibility of these parasites binding to hyaluronic acid in the placenta. This phenomenon often causes adverse outcomes such as intrauterine growth restriction, preterm delivery, low birthweight (LBW), stillbirth, early neonatal death, and maternal anemia. According to the World Health Organization (WHO), approximately 52 million women in Africa are at a risk of P. falciparum infection every year [5]. Thus, gestational malaria is becoming a priority for public health officials in an effort to counter the deleterious effects of this disease in mothers, fetuses, and newborns [6,7]. WHO recommends the administration of intermittent preventive treatment with Sulfadoxine-Pyrimethamine (IPT-SP) during pregnancy as one of the strategies to reduce the burden of malaria and improve birth outcomes [5]. In Cote d’Ivoire, the National Malaria Control Program (NMCP) recommends that SP should be provided at each scheduled focused antenatal care (ANC) visit in the second and third terms [5]. While previous studies have reported that IPT with SP is an effective treatment [8], there are other countrywide reports of a high prevalence of mutations in the DHFR and DHPS genes, leading to SP resistance [9,10]. Notably, conventional microscopy is unable to detect cases with submicroscopic infections. Therefore, PCR analysis is more efficient than conventional microscopy in the diagnosis of weak parasitemia [11–13]. This study investigated the effectiveness of IPT-SP against malaria in pregnant women within Côte d’Ivoire (San-Pedro) following 10 years of inception via molecular methods.

Materials and methods

Study location

This study was conducted in San-Pedro, the administrative center of Bas-Sassandra district in the southwest of Côte d’Ivoire. Despite some economic advantages (presence of forest and seaport), San-Pedro has poor sanitation, which exposes inhabitants to disadvantageous living conditions leading to health problems. These factors, when combined with high levels of rain and a warm climate, favor the proliferation of the female Anopheles mosquitoes and a continuous transmission cycle of malaria; hence, this particular study site was chosen. San-Pedro is the sentinel site for malaria surveillance in Côte d’Ivoire. Moreover, the effects of placental malaria on maternal and newborn outcomes have been poorly documented in this town; however; malaria prevalence is high in the Southwest region (63%) and the proportion of children with hemoglobin levels below 8 g/dL is 25%.

Study type, period, and target population

This cross-sectional study was conducted from June to July 2017. It included a parturient population. Informed written consent was obtained from parturients or their parents/legal guardians in the case of minors (under 18 years of age). We recruited women from Terre Rouge, the maternity ward and the labor room of the Regional Medical Center of San-Pedro. Pregnant women were excluded in this study if they were unable to understand the questionnaires or if sample collection was impossible (for example, in restless women). Furthermore, calculation of the optimal size for our study was made with reference to preliminary survey data from the assessment of the impact of IPT on birth weight.

Sample size

This study was part of a larger project exploring the effectiveness of IPT-SP across the country, encompassing a total of six sentinel sites. Furthermore, the optimal size for our study was calculated with reference to preliminary survey data from the assessment of the impact of IPT on birth weight. The objective of the overall study was to compare the prevalence of low birth weight (LBW) in groups with or without IPT-SP. LBW occurred in 12% of the IPT negative (IPT-) subjects compared to only 3% for the IPT positive (IPT+) subjects. In order to prove a significant difference between these two groups with a power of 90% and a usual percentage of female IPT+ at 30% in the population, it would require approximately 1043 subjects, including 695 IPT- and 348 IPT+. Therefore, a total number of 1100 women (from all six sentinel sites) were selected, with approximately 200 women per site.

Data collection

After obtaining informed consent, each woman included in the study received a questionnaire to collect data such as demographic details, pregnancy history, vector control measures and malarial chemoprophylaxis (long-lasting insecticide-treated mosquito nets (LLITNs), IPT-SP) and newborn information (sex, weight, height, appearance, pulse, grimace, activity, respiration, life/death, and date of death). This information was also obtained from the patients’ medical charts and the consultation notebooks.

A woman was considered to be compliant when she received at least three doses of SP (3 SP tablets taken once a month from the second trimester of pregnancy) orally during pregnancy.

Molecular diagnosis

Plasmodial DNA was extracted from Whatman filter papers (Whatman International Ltd., Maidstone, England) with dried blood samples prepared from the venous, placental, and cord blood, utilizing Chelex 100*(Sigma Aldrich, St. Louis, MA, USA) [14]. The extracts obtained were kept at –20°C until use as the fragment of interest (18S ssrRNA of) for amplification by nested PCR. The 96-well SimpliAmp^™ thermocycler (Thermo Fisher Scientific, Waltham, MA USA) was used for amplification. A primary nonspecific amplification was achieved with the use of primers rPlu5 and rPlu6 (Table l) with 1 μL of the DNA extract, followed by a second specific amplification using 1 μL of the primary PCR product as a matrix with specific primers (Table 1) for use with the four species of Plasmodium (P. falciparum, P. ovale, P. malaria, P. vivax). All amplifications were carried out in a total volume of 25 μL. The DNA fragments amplified by PCR were separated by electrophoresis (Kuro GEL Midi 13 VWR*) on 1.5% agarose gel with EDTA tris-base-borate sorting solution. These fragments were then visualized under UV light using ethidium bromide and photographed.

Table 1.

Primer sequences used for nested PCR

Step	Species	Primers: oligonucleotides sequences (5ʹ-3ʹ)	Size in bp
First amplification	Plasmodium sp.	rPLU5: CCT GTT GTT GCC TTA AAC TTC rPLU 6: TTA AAA TTG TTG CAG TTA AAA CG	1100
Second amplification	P. falciparum	rFAL 1: TTA AAC TGG TTT GGG AAA ACC AAA TAT ATT rFAL 2: ACA CAA TGA ACT CAA TCA TGA CTA CCC GTC	205
	P. vivax	rVIV 1: CGC TTC TAG CTT AAT CCA CAT AAC TGA TAC rVIV 2: ACT TCC AAG CCG AAG CAA AGA AAG TCC TTA	120
	P. malariae	rMAL 1: ATA ACA TAG TTG TAC GTT AAG AAT AAC CGC rMAL 2: AAA ATT CCC ATG CAT AAA AAA TTA TAC AAA	144
	P. ovale	rOVA 1: ATC TCT TTT GCT ATT TTT TAG TAT TGG AGA rOVA 2: GGA AAA GGA CAC ATT AAT TGT ATC CTA GTG	800

Data analysis

Statistical analysis was carried out using SPSS version 21 software (IBM; Chicago, IL, USA). The chi-square and Fisher tests were used to determine the association between proportions at a 5% level of significance. Plasmodium indices were determined, and SP treatment was considered effective in the absence of Plasmodium spp.

Ethical considerations

This study was approved by the National Research Ethics Committee (CNER, 041/MSLS/CNER-kp) and conducted in accordance with the Helsinki Declaration adopted during the 18^th World Medical Assembly in 1964, and the International Conference on Harmonization recommendations. It is consistent with good clinical practices and all applicable regulatory requirements for clinical studies as well as Côte d’Ivoire’s national laws and regulations.

Results

Sociodemographic characteristics

In the one-month duration of this study, 200 parturients were visited at the maternity wards of the Regional Hospital Center and Terre-Rouge in San-Pedro city. Dried blood spot samples collected from three of these women were rejected because of poor quality. The average age of the participating women was 27.07 (standard deviation (SD) = 6.7 years) ranging from 15 to 45 years. The 18–40-year age range was the most represented (86.2%) (Table 2). The majority of the women were living with a partner (88.5%). Analysis of the professional characteristics of the study participants showed that among the 197 women, 55.3% were housewives, and 28% were traders (The remaining proportions are in Table 2). However, traders received more doses (three doses) of IPT-SP than the housewives, as recommended by the WHO. Most women were illiterate (56.9%); among these, 48 (43%) followed the IPT-SP correctly (Table 2).

Table 2.

Characteristics of the study participants

Characteristics	Global frequency (N = 197)	Dose of IPT		p-Value
		≤2	>2
Age (years)
<18	18 (9.1%)	12 (66.7%)	6 (33.3%)
[–18–40]	171 (86.2%)	86 (50.1%)	85 (49.7%)	0.416
>40	4 (2%)	2 (50%)	2 (50%)
Profession
Housewife	109 (55.3%)	63 (57.8%)	46 (42.2%)
Trader	55 (28%)	23 (41.8%)	32 (58.2%)	0.048
Hairdresser/seamstress	20 (10.1%)	10 (50%)	10 (50%)
Student	5 (2.5%)	5 (100%)	0 (0%)
Civil servant	8 (4.1%)	2 (0.25%)	6 (0.7%)
Education
None	112 (56.9%)	64 (57%)	48 (43%)
Primary	56 (28.4%)	29 (51.8%)	27 (48.2%)	0.191
Secondary	26 (13.2%)	9 (34.6%)	17 (65.7%)
Higher	3 (1.5%)	1 (33.3%)	2 (67.7%)
Gravidity
Primigravidity	40 (20.3%)	23 (57.5%)	17 (42.5%)
Secundigravidity	48 (24.4%)	26 (54.2%)	22 (45.8%)	0. 659
Multigravidity	109 (55.3%)	54 (49.5%)	55 (50.5%)
Parity
Nulliparity	47 (23.8%)	24 (51.1%)	23 (48.9%)
Primiparity	50 (25.4%)	28 (56%)	22 (44%)	0. 721
Secondiparity	38 (19.3%)	17 (16.5%)	21 (22.3%)
Multiparity	62 (31.5%)	34 (33%)	28 (28%)
ANC visits
<2	49 (24.9%)	45 (91.8%)	4 (8.2%)
[3–5]4,5	132 (67%)	55 (41.7%)	77 (58.3%)	<0,0001
>6	16 (8.1%)	(18.7%)	13 (81.3%)

Obstetric history and use of LLITNs

The average number of pregnancies were three, with eight pregnancies being the maximum. Multigravida and multiparous women represented 55.3% and 31.5% of the population, respectively. The pregnant women were using at least one method of prevention against malaria, with insecticide-impregnated mosquito netting being the most common (49%). More than two-thirds of the women (67%) had attended three to five prenatal consultations and most of the women gave birth easily (90%). Results showed that no LBWs were observed in the newborns.

IPT compliance

Coverage and compliance rates of IPT in the case of the recruited women in this study were 93.5% and 47.7%, respectively (Table 2). The compliance rate of three doses of IPT-SP was higher in the multigravida (50.5%) group than in the multipara group; this could be because of better level of education in the former (Table 2). We could not establish a significant relation between the compliance and gravidae (p = 0.659) and the level of education (p = 0.191). However, the number of prenatal consultations influenced the observance of IPT-SP (p= 0.0001) (Table 2).

Plasmodium infection

In this study, signs of Plasmodium infection indices were as follows: 16.2% women showed infection in placental tissues, 15.2% in the venous blood, and 3.6% in the umbilical cord detected by PCR (Table 3). Notably, 2.5% of the women were infected in all three levels: venous, placental, and cord levels (Table 4). P. falciparum was the only species found based on molecular analysis. The average parasite densities in the placental, venous, and cord blood were 4311.7 ± 23,128.2, 2837.2 ± 19,001, and 100.8 ± 950.3 parasites μL⁻¹, respectively. Out of 197 women, approximately 6.5% confirmed that they had malaria during pregnancy.

Table 3.

Distribution of plasmodial infection detected by PCR in study participants

Variable	Frequency (N = 197)	Percentage (%)
Peripheral parasitemia
Positive	30	15.2
Negative	167	84,8
Placental parasitemia
Positive	32	16.2
Negative	165	83.8
Cord parasitemia
Positive	7	3.6
Negative	190	96.4

Table 4.

Relationships between placental, venous and cord plasmodial infestation

CORD			Venous		Total
			Negative	Positive
Negative	Placental	Negative	159	4	163
		Positive	6	21	27
	Total		165	25	190
Positive	Placental	Negative	2	0	2
		Positive	0	5	5
	Total		2	5	7

Influence of SP chemoprophylaxis on malaria

Women who took three doses of IPT had less infection at the placental (10/94), venous (13/94), and cord (3/94) levels. However, a statistically significant relationship was established between the number of doses and the rate of placental infection only (p = 0.042; Table 5). IPT reduces plasmodial infestation at the placental (odds ratio; OR = 0.4; confidence interval; CI = [0.2–1]), cord (OR = 0.8; CI = [0.2–3.7]), and venous (OR = 0.8; CI = [0.6–2.3]) level.

Table 5.

Association between plasmodial infection, obstetrical history and dose of IPT

Plasmodial infestation		Parity		p-Value	Dose of IPT		p-Value
		≤2	>2		≤2	>2
Venous	Positive	22	8		17	13
	Negative	113	54	0.010	86	81	0.602
Placental	Positive	24	8	0.05	22	10	0.042
	Negative	111	54		81	84
Cord	Positive	6	1	0.593	4	3	0.793
	Negative	129	61		99	91

Relation between parity and parasite infestation

Multiparous women showed less infestation; however, the differences were statistically significant only at the venous level (p = 0.010), and not at the placental and cord levels (Table 5).

Discussion

The detection of asexual parasites by light microscopy of Giemsa-stained thick and thin films remains the standard laboratory method for the diagnosis of malaria in studies that evaluate the effectiveness of IPT-SP. However, PCR is more sensitive and specific than examination of thick or thin blood smears, particularly in cases with low parasite rates or mixed infections. This study used molecular methods to investigate the effectiveness of IPT-SP against malaria in pregnant women in San-Pedro, Côte d’Ivoire following 10 years of inception. Despite the high rates of SP resistance [11], we show a continued benefit of providing IPTp-SP to both primigravid and multigravida Ivoirian women. The average age of the women was 27 (range 18–40) years which is similar to that in previous reports in Burkina Faso [15], Benin [16], and Senegal [17]. Thus, the women who gave birth were between 15 and 45 years of age, irrespective of the region to which they belonged. Notably, 9.1% of participants were young women under 18 years of age, and this could be due to a lack of sex education and due to deleterious traditional practices.

Our study was carried out in 2017, a period in which the use of two doses of SP constituted the first strategy to fight against malaria in terms of chemoprophylaxis and could be the reason for the low observance (47.7%) of three doses of SP (SP3) in our study. The distribution of SP throughout Côte d’Ivoire and sensitization of women to the problems associated with gestational malaria, initiated by the NMCP through information, education and communication campaigns, will allow the achievement of the NMCP objective, namely, at least 80% of pregnant women should receive SP3 by 2020 [18]. A poorer rate of SP3 observance was reported in Burkina Faso when compared with that observed in this study [15].

The minimum number of antenatal care (ANC) visit in Côte d’Ivoire is four [19]. In our study, the few women who opted for more than three prenatal consultations were the most observant of the three doses. Thus, regular consultation at the health center can lead to a better prenatal follow-up and prevention of disease.

Moreover, vector control is one of the essential means of reducing the transmission of malaria if it is well conducted. In our study, each woman used at least one method of prevention against malaria, in particular MILDA. The insecticide-impregnated mosquito net and other prevention methods during pregnancy can have an advantage on the pregnancy outcome in malaria-endemic zones, particularly if IPT is strictly observed [5].

The relatively higher prevalence of parasite infestation in this study than that reported by Famanta et al. [20] could be explained by the endemicity level of the study area (tropical region) and the resurgence of malaria transmission during rainy (June–July) seasons in San-Pedro.

In this study, women who took three doses of IPT-SP were the least infected. These results were consistent with that of a previous Ivorian study [8]. Thus, IPT-SP is efficient and can be used as a practical strategy to reduce the risk of placental infection by Plasmodium in malaria-endemic zones. Similar results were also reported in West Africa, namely in Burkina Faso [21], Mali [22], Benin [16], Nigeria [23], Ghana [24], and in East Africa in Kenya [25], Malawi [26], Tanzania [27] and Mozambique [28]. Previous studies have shown that parasitemia is more recurrent in pregnant women globally because of pregnancy-induced immunosuppression. Educating pregnant women on the role of IPT in preventing malaria will have a positive impact on reducing the prevalence of malaria. Furthermore, the government should intensify efforts in ensuring that IPTs are readily available at all health facilities and IPT should be distributed to all pregnant women, especially primigravida, during ANC visits in order to encourage its utilization.

Multiparous women were significantly less exposed to malaria. Maximum malaria susceptibility observed during the first pregnancy decreased with subsequent pregnancies although multigravidity and/or multiparity lessen the risk of Plasmodium infections [29]. We also noticed that primiparous women and those with second pregnancies were the most exposed to or at the highest risk of malarial infection, even when they had taken three doses of IPT-SP. The same tendencies were observed in Burkina Faso [15] and in Benin [16]. This can be explained by the fact that multiparous women developed protective immunity in the previous pregnancies against placental malaria infestation [30]. LBW is an indicator of the consequences of malaria, and in our study, no cases of LBW were observed among the newborns in women who received IPT-SP or no.

In conclusion, through molecular analysis, this study revealed a relatively high prevalence of Plasmodium in pregnant women. However, the low frequency of placental, venous, and funicular infestation in pregnant women who consistently followed a preventive treatment strategy clearly showed the efficiency of IPT against malaria during pregnancy. As SP is currently the only antimalarial drug used for malaria prevention in pregnant women, pharmacovigilance, particularly for resistance signs in combination therapy, is essential. Furthermore, a large sample will allow a better assessment of the effectiveness of this combination in preventing malaria. It will therefore be interesting to extend this study to several sites and also to look for markers of resistance to SP.

Disclosure statement

The authors declare that they have no competing interests.